A Spec iaIist Period icaI Report
Terpenoids and Steroids Volume 4
A Review of the Literature Published between September 1972 and August 1973
Senior Reporter
K. H.
Overton, Department of Chemistry.
University of Glasgow Reporters C. Altona, Rijksuniversiteit, Leiden D. V. Banthorpe, University College, London G . Britton, University of Liverpool B. V. Charlwood, King's College, University of London J. D. Connolly, University of Glasgow R. A. G . de Graaff, Rijksuniversiteit, 1 eiden J. R . Hanson, University of Sussex H. J. C. Jacobs, Rijksuniversiteit, Leiden D. N. Kirk, Westfield College, University of London R. W. Mills, University of British Columbia T. Money, University of British Columbia C . Romers. Rijksuniversiteit, Leiden L. L. Smith, University of Texas, Medical Branch A. F. Thomas, Firmenich et Cie., Geneva
0 Copyright 1974
The Chemical Society Burlington House, London, W I V OBN
ISBN: 0 85186 2861 Library of Congress Catalog Card No. 74-615720
Set in Times on Monophoto Filmsetter and printed offset by J. W. Arrowsmith Ltd., Bristol, England Made in Great Britain
In tr oduc t ion
While following the pattern established in previous Reports of this series, we have this year added two chapters of a new kind. It seemed to us that areas of terpenoid and steroid chemistry not normally included in our coverage should be reviewed in occasional chapters. These will cover periods of about five years while otherwise preserving the detailed character of our annual Reports. The first results of this decision are Chapters 2 and 3 of Part I1 of this volume. In ‘Microbiological Reactions with Steroids’ Professor L. L. Smith gives a comprehensive account of the very large amount of work that has appeared in this field since the last review was published in 1967. The chapter ‘Steroid Conformations from X-Ray Analysis Data’ by Professor C. Romers and Dr. C Altona and their colleagues goes somewhat beyond the usual scope of Specialist Periodical Reports. The authors have in part used data recorded in the literature to recalculate equilibrium geometries for a large number of steroids. In their critical survey based on these geometries, they provide a most illuminating analysis of the way in which steroid conformations respond to functionality and configuration. This will be of interest to organic chemists and biochemists alike. Because of a change in authorship which we were not able to implement in time for this Report, the chapter on Steroid Synthesis has regretfully been held over for Volume 5, which will cover a two-year period. Finally, we have included a list of selected reviews of terpenoid chemistry that have appeared in the period 1968-1973, to fill what to us seemed a regrettable gap in the tertiary literature.
K. H. OVERTON
Contents Part 1 Terpenoids Chapter 1 Monoterpenoids By A. F. Thomas
3
1 Physical Measurements: Spectra etc.; Chirality
3
2 General Chemistry
6
3 Blogenesis, Occurrence, and Biological Activity
8
4 Acyclic Monoterpenoids Terpene Synthesis from Isoprene 2,6-Dimethyloctanes Artemisyl, Santolinyl, Lavandulyl, and Chrysanthemyl Derivatives
10 10 11
5 Monocyclic Monoterpenoids Cyclobutane Cyclopentanes, including Iridoids p-Menthanes rn-Menthanes Tetramethylcyclohexanes 1,4-Dimethyl-1-ethylcyclohexanes Cycloheptanes
22 22 23 26 37 37 40 41
6 Bicyclic Monoterpenoids Bicyclo[3,1,O]hexanes Bicyclo[2,2, llheptanes Bicyclo[3,1, llheptanes Bicyclo[4,1 ,O]heptanes
42 42 43 56 63
7 Furanoid and Pyramid Monoterpenoids
66
8 Canrtabidds and other Phenolic Monoterpenoids
69
Chapter 2 Sesquiterpenoids By R. W. Mills and T. Money 1 Introduction
19
77 77
V
vi
Terpenoids and Steroids 2 Famesane
77
3 Bisabolanes
87
4 Sesquicarane, Carotane, etc.
89
5 Cuparane, Laurane, Trichothecane,efc.
90
6 Acorane, Bazzanane, Cedrane,Zizaane, etc.
93
7 Chamigrane, Widdrane, and Thujopsane
96
8 Sesquipinane, Sesquifenchane, and Fumagillane
97
9 Sesquicamphane, &Santdane, a-Santdane, etc.
99
10 Amorphane, Cadinane, Cubebaw, Copaane, Copocamphane,
Ylangocamphane, Sativane, etc.
101
11 Himachalane, Longipinane, Longicamphane, Longifolane,
and Cyclolongicamphane
108
12 Humulane, Caryophyllane, etc.
112
13 Germacrane, Eudesmane, Eremophilane, etc.
114
14 Guaiane, Cyperane,Seychellane, Aromadendrane, and
Bourbonane 15 Mono- and Bi-cyclofarnesanes
Chapter 3 Diterpenoids By J. R. Hanson
133 139 145
1 Introduction
145
2 Bicyclic Diterpenoids
145
3 Tricyclic Diterpenoids Naturally Occurring Substances Chemistry of the Tricyclic Diterpenoids
150 150 154
4 Tetracyclic Diterpenoids
157 157
The Kaurene-Phyllocladene Series Beyeranes Gibberellins Grayanotoxins Diterpenoid A1kaloids
160 161 163 164
5 Macrocyclic Diterpenoids and their Cyclization Products
164
6 Miscellaneous Diterpenoid Substances
166
7 Diterpenoid Synthesis
167
Contents
vii
Chapter 4 Sesterterpenoids By J. R. Hanson
171
1 Introduction
171
2 Acyclic and Furanoid Sesterterpenoids
171
3 Gascardic Acid
174
4 ophiobori
176
5 Substances of Miscellaneous Structure
181
Chapter 5 Triterpenoids By J. D. Connolly
183
1 Reviews
183
2 Squalene Group
183
3 Fusidane-Lanostane Group
188
4 Dammarane-Euphane Group Tetranortriterpenoids Bicyclononanolides Quassinoids
197 202 204 206
5 Shionane-Baccharane Group
206
6 LupaneGroup
207
7 Oleanane Group
209
8 UrsaneGroup
218
9 HopaneGroup
219
10 Stictane-Flavicane Group
219
11 Serratane Group
220
Chapter 6 Carotenoids and Polyterpenoids By G.Britton
22 1
1 Introduction
22 1
2 Physical Metbods
22 1
3 New Natural Carotenoids Acyclic Carotenoids Monocyclic Carotenoids Bicyclic Carotenoids Aromatic Carotenoids
226 226 228 229 230
...
Terpenoids.and Steroids
Vlll
Carotenoid Glycosides Car0tenoproteins
23 1 23 1
4 Carotenoid Chemistry
23 1
5 Degraded Carotenoids Retinol and Derivatives Other Degraded Carotenoids Model Cyclizations
234 234 238 245
6 Polyterpenoids and Quinones Polyterpenoids Isoprenylated Quinones
246 246 247
Chapter 7 Biosynthesis of Terpenoids and Steroids By 0.V. Banthorpe and B. V. Charlwood
250
1 Introduction
250
2 Acyclic Precursors
25 1
3 Hemiterpenoids
260
4 Monoterpenoids
260
5 Sesquiterpenoids
264
6 Diterpenoids
267
7 Sesterterpenoids
27 1
8 Steroidal Triterpenoids
272
9 Further Metabolism of Steroids
282
10 Non-steroidal Triterpenoids
287
11 Carotenoids
288
12 Meroterpenoids
29 1
13 Polyterpenoids
296
14 Methods
296
15 Reviews
298
Reviews o n Terpenoid Chemistry
30 1
ix
Contents
Part // Steroids
Chapter 1 Steroid Properties and Reactions By D. N. Kirk
31 1
1 Conformational Analysis, Stereochemistry, and Spectroscopic Methods Spectroscopic and Chiroptical Methods N.M.R. Spectroscopy Mass Spectra
31 1 312 313 314
2 Alcohols and their Derivatives, Halides, and Epoxides Substitution and Elimination Ring-opening of Epoxides Esters, Ethers, and Related Derivatives of Alcohols Oxidation and Reduction Miscellaneous
315 315 322 325 327 329
3 Unsaturated Compounds Addition Reactions Epoxidation Reduction Oxidation Aromatic Compounds Alkynes and Cyclopropanes
330 331 332 338 340 343 345
4 Carbonyl Compounds Ketones : Reactions at the Carbonyl Group Reactions involving Enols and Enolate Ions Enolic Derivatives, Enamines, and their Reactions Reactions involving Oximes, Hydrazones, and Related Derivatives Carboxylic Acids, their Derivatives, and Aldehydes
348 348 354 359
5 Compounds of Nitrogen and Sulphur
367
6 Molecular Rearrangements Contraction and Expansion of Steroid Rings Backbone Rearrangements Aromatization of Steroid Rings Miscellaneous Rearrangements
369 369 373 376 379
7 Functionalhation at Non-activated Positions
383
8 Photochemical Reactions
386
9 Miscellaneous
392
362 364
Terpenoids and Steroids
X
Chapter 2 Microbiological Reactions with Steroids By L. L. Smith
394
1 Introduction
394
2 Hydroxylation Reactions
395
3 Hydroxy-steroid-Oxo-steroid Interconversions
453
4 Dehydrogenationand the Reduction of Carbon-Carbon
Double Bonds
470
5 Olefinic Bond Isomerization
486
6 Esterase, Amidase, and Hydrolase Reactions
489
7 Reactions involving Heteroatoms
494
8 Steroid Degradation Reactions
497
9 MiscellaneousMicrobial Reactions
523
Chapter 3 Steroid Conformations from X - Ray Analysis Data By C. Romers, C. Altona, H. J. C. Jacobs, and R. A. G. de Graaff
53 1
1 Introduction
53 1
2 Presentation of Results
534
3 The Perhydro-1,kyclopentanophenanthreneSkeleton
534
4 The Geometry of Ring A in 3-Oxo-A4-steroids
548
5 The AS3'-System
558
6 The Conformation of Ring B in Oestranes and A5-Compounds
562
7 Five-membered (D) Rings
565
8 Six-membered Boat Conformations
570
9 The Conformation of the Side-chain at C-17
573
10 Biological Activity at the Molecular Level
576
11 summary
578
12 Appendix
578
Errata Author Index
584 585
Part I TERPENOIDS
1 Monoterpenoids ~~
BY A. F. THOMAS
This year, the section on general chemistry has been enlarged, and some reactions that are not specific to monoterpenoids have been included. Physical methods are given a separate section. Unfortunately it must be noted that Chemicd Abstracts contains an increasing number of errors, as well as frequently citing insufficient information for the abstract to be useful. So far as possible, attention has been drawn to these points in each individual case. The abstracts of the Proceedings of the 4th Congress on Essential Oils (Tbilisi, 1968) have appeared, but much of this work is now out of date. 1 Physical Measurements: Spectra etc., Chirality The 3C n.m.r. spectra of citronellol, citronellal, and related substances have been discussed,' and a study of the shifts of the alkene signals induced by Ag' in the I3C n.m.r. spectra of a number of substances including the pinenes has been made.2 A very full discussion of the effect of shift reagents on the 'H and 13C n.m.r. spectra of borneol and isoborneol has shown that the complexes formed with the reagents are effectively axially symmetric, the magnetic axis being practically collinear with the oxygen-metal bond ;3 an estimate of the contact contribution has been made.4 Coupling constants in 7,7-dimethylnorborneols have been examined using the [Eu(dpm),] shift agent.5 In a study of the U.V.spectra of the complexes between boron trifluoride and unsaturated ketones, monoterpenoids are particularly unlucky : piperitone (1) does not fit the attempted correlation, and carvone (2) polymerizes under the conditions of measurement !6 The mass spectra of monoterpenoids have been di~cussed,~ and the loss of EtCONH, in the mass spectrum of (3) (a retro-Ritter reaction) has given rise to
'
A. K. Bose and R. J. Brambilla, J . Agric. Food Chem., 1972, 20, 1013.
' C. D. M . Beverwijk and J. P. C. M. van Dongen, Tetrahedron Letters, 1972, 4271.
'
G. E. Hawkes, D. Leibfritz, D. W. Roberts, and J. D. Roberts, J. Amer. Chem. Soc., 1973,95, 1659. G. E. Hawkes, C. Marzin, S. R. Johns, and J . D. Roberts, J. Amer. Chem. SOC.,1973, 95, 1661. K.-T. Liu, Tetrahedron Letters, 1973, 2747. J. Torri and M. Azzaro, Tetrahedron Letters, 1973, 3251. M. Sakaguchi, A. Hirakata, and H . Yamada, Koryo, 1972, No. 102, p. 41 (Chem. A h . , 1973, 78, 84 555).
3
Terpenoids and Steroids
4
speculations, without the support of labelling studies.' The Raman 'circular dichroism' of a number of optically active monoterpenoids has been examined. Circular intensity differentials (CID), A, = I! - I,"/(lt + I,"), where I!, I," are the scattering intensities with a-polarization in right and left circularly polarized incident light, have been measured in the low-frequency Raman spectra of (+)and ( -)-a-pinene, ( - )-P-pinene, (-)-borneol, and carvone.' The circular differential Raman spectrum of carvone has been reported elsewhere. *
Me
(4)
Monoterpenoids are the most common of the chiral agents used for inducing asymmetry. Measurement of the n.m.r. spectra of esters 'of camphanic acid, such as (4),has been used to find the enantiomeric purity and absolute configuration of a-deuteriated primary alcohols,' and separations of various alcohols and amines using esters of chrysanthemic acid are reported.I2 An interesting mutual resolution can be effected with ( k )-camphorsulphonic acid and a-( k )Me2NCH,CHMeCPh(OH)CH2Ph.13 ( )-Carvomenthol and chloroacetic acid give carvomethylacetic acid (9,which is useful for resolving alanine.14 Mislow et al. have used menthyl methylphenylthioarsenite (6) in an extension to arsenic of their earlier method (see Vol. 2, p. 28) of making optically active phosphine oxides.' Probably the most interesting work taking advantage of the chirality of monoterpenoids has involved the attempts to induce asymmetry in organic
+
* lo
I' l2
l3 l4
l5
S. Blum and S. Sarel, J . Org. Chem., 1972, 37, 3121. L. D . Barron and A. D. Buckingham, J . C . S . Chem. Comm., 1973, 152. M. Diem, J . L. Fry, and D . F. Burrow, J . Amer. Chem. SOC.,1973, 95, 253. H. Gerlach and B. Zagalak, J.C.S. Chem. Comm., 1973, 274. C. J . W. Brooks, M. T. Gilbert, and J. D. Gilbert, Anafyt. Chem., 1973, 45, 896. W. E. Thompson and-A. Pohland, Ger. Offen. 2 230 838. F. Rulko, K. Witkiewicz, and Z. Chabudzinski, Diss. Pharm. Pharmacol., 1972, 24, 297. J. Stackhouse, R. J. Cook, and K. Mislow, J . Amer. Chem. SOC.,1973, 95, 953.
Mono terpenoids
5
Me
’0-c
‘.-
“OCOCH,CC02 Et
I
OH (7)
(8)
(9)
synthesis. As a simple example, the rate of esterification of D-amino-acids with (-)-menthol is greater than that of L-acids, and this has led to a proposal for menthyl ester formation.16 The anion (8), obtained when menthyl acetate (7) is metallated, reacts with ethyl pyruvate to yield the menthyl ester of (S)-citramalic acid (9) in 26 % optical yield.I7 Kergomard et a/. found no asymmetric induction in the reaction between styrene, t-butyl hypobromite, and menthol [leading to (lo)].’* Oxidation of (+)-borne01 with (R)-(+)-menthy1 p-tolyl sulphoxide and dicyclohexylcarbodi-imide in the presence of phosphoric acid in benzene gave (-)-camphor in 7 % optical yield, l 9 and the cyclization of homogeranic ( -)menthyl ester with stannic chloride to cis-tetrahydroactinidiolide(1 1) occurred with only ca. 12 % optical yield, although this rose to 20.8% when the 1,2:5,6-di0-isopropyhdene-or-D-glucofuranoseester was used.*O Asymmetric reductions of diphenylmethyl alkyl ketones by complexes of lithium aluminium hydride and cis-pinane-2,3-diol and benzyl alcohol gave up to 20% optical yields,*’ but far more successful was the reaction of ethylene and cyclo-octa-1,3-diene [to (12)], catalysed by certain n-ally1 complexes of nickel where one ligand is a monoterpenoid phosphine, in which 70 % optical purity was achieved.’
King and Sim have described a useful method for demonstrating the presence of a reactive intermediate in reactions involving chiral diastereomeric transition l6
‘’ l9 2o 21
’*
T. Hayakawa, H. Yamamoto, Y. Murakami, Y.Yobiko, and S. Mitani, Bull. Chem. SOC.Japan, 1972, 45, 3556; see also Vol. 3, p.6. S. Brandange, S. Josephson, and S. Valltn, Acta Chem. Scand., 1973, 27, 1084. G . Dauphin, A. Kergomard, and A. Scarset, Bull. SOC.chim. France 11, 1973, 1104. M. H. Benn, P. Christensen, D . Kjersgaard, and C. Watanatada, Canud. J . Chem., 1973,51, 1977. T. Kato, S. Kumazawa, and Y. Kitihara, Synthesis, 1972, 573. R. Haller and H. J. Schneider, Chem. Ber., 1973, 106, 1312. B. BogdanoviC, B. Henc, B. Meister, H. Pauling, and G. Wilke, Angew. Chem. Znrernar. Edn., 1972, 11, 1023.
6
Terpenoids and Steroids
states; it provided a new piece of evidence for the intermediacy of a sulphene in the reaction between camphor-10-sulphonyl chloride and menth~lamine.,~ The Reporter is ill-placed to criticize a chapter on the synthesis of monoterpenoids in a recently published book on the total synthesis of natural products.23” However, a delay of three years between the latest reference quoted and publication of a book is deplorable. 2 General Chemistry
Sukh Dev has reviewed alumina- and silica gel-induced rearrangements, many of which involve m o n ~ t e r p e n o i d s .The ~ ~ Prins reaction of monoterpenoid hydrocarbons has also been reviewed.2s Microwave discharge of carbon dioxide can function as a singlet oxygen source ;photo-oxygenation by this means has been accomplished using limonene and y-terpinene as substrates.26 A two-phase solvent system is useful for epoxidizing sensitive olefins (e.g.6-methylhept-5-en-2-one) with rn-chloroperbenzoic acid, but limonene gave the same epoxide in the same yield as with the single-phase system., Several novel methods for the reduction and oxidation of oxygenated terpenoids have appeared. Potassium metal in graphite can be used to reduce camphor (a 60:40 exo:endo mixture is obtained), and oxidations of primary alcohols are effected by chromic oxide in graphite (citronellol yields 90 % of the aldehyde in 24h),28 but the preparation of the reagent can be dangerous.,’ Potassium metal in hexamethylphosphoramide, with or without a co-solvent, has also been used to reduce terpenoid ketones; with camphor, more endoproduct is formed than in the potassium-graphite reduction.,’ Hindered saturated secondary alcohols are oxidized by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; thus borneol and isoborneol are 96% and 95% oxidized in 8 h and neoisomenthol (i.e.the all-cis-isomer) and neoisocarvomenthol are 48 % and 40% oxidized in the same time, whereas the all-equatorial alcohols menthol and carvomenthol are hardly affected in this time.31 Reduction of camphor with various silanes (Ph,SiH,, PhSiH,, PhMeSiH,, and Et,SiH,) in the presence of tris(tripheny1phosphine)chlororhodiumgives 73-90 % of isoborneol ( e m ) , but triethylsilane gives only 30 % of isoborneol and phenyldimethylsilane does not reduce. Analogous results were obtained for m e n t h ~ n e , ~but , pulegone (13) presented some irregularities, mixtures of menthone (14) and pulegol (15) being J . F. King and S. K . Sim, J . Amer. Chem. SOC., 1973, 95, 4448. ”‘A. F. Thomas, in ‘The Total Synthesis of Natural Products’, ed. J. W. ApSimon, Wiley, New York, 1973, Vol. 2, p. 1 . 2 4 Sukh Dev. J. Sci. Ind. Res., 1972, 31, 60. 2 s J . Chlebicki, Wiadomosci Chem., 1972, 26, 629. 26 K. Gollnick and G . Schade, Tetrahedron Letters, 1973, 857. ” W. K . Anderson and T. Veysoglu, J . Org. Chem., 1973, 38, 2267. 2 8 J.-M.. Lalancette, G. Rollin, and P. Dumas, Cunud. J . Chem., 1972, 50, 3058. 2 9 Communication from the manufacturers, Ventron Corp., Beverly, Mass. 3 0 M. Larcheveque and T. Cuvigny, Bull. SOC.chim. France I I , 1973, 1145. * J. Iwamura and N . Hirao, Tetrahedron Letters, 1973, 2447. 3 2 I . Ojima, M. Nihonyanagi, and Y . Nagai, Bull. Chem. SOC.Japan, 1972,45, 3722. ”
Mono terpeno ids
c
7
/
+
/
OSi -
OSi -
\
\
P
+
0
(15)
(14)
produced in different amounts depending on the reagent.33 The rate of MeerweinPonndorf reduction (propan-2-01-aluminium isopropoxide) for a variety of terpenoid ketones is unexpectedly high. The half-life of camphor, for example, (the slowest of those measured) was 145.8min at 82 0C.34Triphenyltin hydride reduces the conjugated double bond of unsaturated aldehydes ; thus citral gives citronellal, but in the case of /?-cyclocitral(16),the reaction works less specifically, leading to a 1:l mixture of the saturated aldehyde (17) and the unsaturated alcohol (18).3 H + ~ O
~
(16)
H
+
a~;"" O
(17)
(18)
4-Dimethylaminopyridine is a useful catalyst in acylations ; an 80 % yield of linalyl acetate can be obtained (without rearrangement-see Vol. 3, p. 15) with its aid, using triethylamine as solvent and (presumably, for it is omitted from the experimental details!) acetic anhydride at room temperature for 14 h. Only catalytic amounts are needed, as was demonstrated by the preparation of menthyl m ~ n o p h t h a l a t e . ~Reaction ~ of aminomethylene ketones with 4-aminouracil (19; X = 0),the thio-analogue (20;X = S), or 2,4-diamino-6-hydroxypyrimidine (the enolized imino-analogue), yields '5-deazapteridines' ; those corresponding to menthone (20)and camphor (21) have been reported37 (see Vol. 3, p. 42).
(19) 33 34
3s
36 37
(20) X = 0,S, or NH
(21) X = S or NH
I. Ojima, T. Kogure, and Y. Nagai, Tetrahedron Letters, 1972, 5035. V. Hach, J . Org. Chem., 1973,38, 293. H. R. Wolf and M. P. Zink, Helv. Chim. Acta, 1973, 56, 1062. G. Hofle and W. Steglich, Synthesis, 1972, 619. E. Stark and E. Breitmaier, Tetrahedron, 1973, 29, 2209.
8
Terpenoids and Steroids
The preparation of monoterpenoid aldehydes from ketones (R,CHCHO in place of R,C=O) using the Grignard reagent EtOCH,MgCl is discussed.38 The Kondakov reaction is the reaction of crotonic anhydride with an olefin in the presence of zinc chloride. A number of monoterpenoid hydrocarbons react at their trisubstituted double bonds ; thus 2,6-dimethylocta-2,7-dienegives the ketone (22), car-3-ene gives (23), and menth-1-ene gives both cis- and transisomers.39 Double bonds react with chlorosulphonyl isocyanate to give compounds containing a four-membered heterocyclic ring ; camphene yields (24), and the products from a- and p-pinene and car-3-ene have also been de~cribed.~'
The reaction of vinylmagnesium bromide with unsaturated esters gives the corresponding divinylcarbinol ; ethyl mentha- 1,8-diene-7-carboxylate and ethyl pin-2-en-10-carboxylate have been treated in this way.41 A convenient method for the separation of terpenoid alcohols from mixtuies via the carbarnates is described.
3 Biogenesis, Occurrence, and Biological Activity A brief section on monoterpenoids is included in a review of biogenetic-like syntheses of t e r p e n ~ i d s .For ~ ~ the biosynthesis of monoterpenoids see Section 4 of Chapter 7, p. 260. Granger and Passet have carried out a chemotaxonomic study on Thymus uulgaris, L.44 This plant gives very diverse essential oils, and analysis of the monoterpenoids permits the assignment of a plant to its chemotype. Somewhat ~ of Juniperus similar is the approach of Banthorpe et aE. in an e ~ a m i n a t i o nof~oils and Thuja species. The juniper leaf oils consist of two types characterized by the presence of either predominantly pinene derivatives or thujane derivatives. 38
3y
40
41
42
43 44
45
M . de Botton, Bull. SOC.chim. France Ii, 1973, 2472. E. Klein and W. Rojahn, Dragoco Ber., 1972, 19,239; further examples of this reaction are in E. Klein, Ger. Offen., 2 120413 [Chem. Abs., 1973, 78, 84 555 has formula (22) incorrect]. T. Sasaki, S. Eguchi, and H. Yamada, J . Org. Chem., 1973,38, 679. S. Watanabe, K. Suga, and T. Fujita, Israel J . Chem., 1973, 11, 71. R. C . Gueldner, F. Y. Hutto, A. C. Thompson, and P. A. Hedin, Analyt. Chem., 973, 45, 376. T. Money, Progr. Org. Chem., 1973, 8, 29. R. Granger and J. Passet, Phytochemistry, 1973, 12, 1683. D. V. Banthorpe, H. ff. S . Davies, C . Gatford, and S . R. Williams, Plunta Med., 973, 23, 64.
Mono terpeno ids
9
Blue spruce (Picea pungens) can be identified by analysis of the cortical oleoresin m o n ~ t e r p e n o i d s . The ~ ~ genesis of monoterpenoids in the wood of common Russian conifers has been followed by direct analy~is.~’ Attention is drawn to the remarks on straightforward chemical analysis of plant and animal material made in Volume 3 (p. 8). Among analyses that are of interest for the monoterpenoid chemist are the following : Carphephorus odoratissimus (‘deertongue’, a tobacco additive),48 Cinnamomum reticulatum from Taiwan [containing a remarkable 96.8 % of ( - ) - l i n a l ~ o l ] , ~Crocus ~ s~tivus,’~ PassijZora edulis f. Jlauicarpa (passion fruit),’ Pelargonium tomentosum C86.9% ( - )-isomenthone, for which various possible stereochemical biogenetic routes and Pogostemon plectr~ntoides?~ are discussed],’ various Pinus spp. needle A very complete analysis of certain fractions of burley tobacco has given a plethora of substances, including many 1,1,3-trimethyl- and 1,1,2,3-tetramethyl-hexane derivatives and the novel isoprenoid (25).”
Secretions from the endocrine glands of staphylinid beetles, Bledius mandibularis and B. spectabilis, contain small amounts of citral and nera1.56 The full papers describing the preparation of the hypoglycaemically active arylsulphonylureido- and arylsulphonylamido-acyl derivatives of borneol and isoborneol (see Vol. 2, p. 7) have a ~ p e a r e d . ’ ~ Details of the preparation of the juvenile hormone compounds mentioned in Vol. 3, p. 10 have been p~blished,’~ and some more geranylanilines (with heterocyclic substituents in the aromatic
46
41
48 49 50
51
52 53
54
55 56
51
58
B. A. Rottink and J. W. Hanover, Phytochemistry, 1972, 11, 3255. Yu. A. Poltavchenko and G. A. Rudakov, Izvest. Nauch.-Issled. I n s t . Nefte-Uglekhim. Sin. Irkutsk. Univ., 1969, I 1 (part l), 39 (Chem. Abs., 1973, 78, 2056). K. Karlsson, I. Wahlberg, and C. R. Enzell, Acta Chem. Scand., 1972, 26,2837. Y. Fujita and S. Fujita, Bull. Chem. SOC.Japan, 1972, 45, 1243. A. I. Akhmedov, M . I. Goryaev, Sh. K. Chogovadze, and A. D . Dembitskii, Izoest. Akad. Nauk kaz. S.S.R., Ser. khim., 1972, 22, 56; see also the section on trimethylhexanes. M. Winter and R. Kloti, Helv. Chim. Acta, 1972, 55, 1916. F. W. Hefendehl, Planta Med., 1972, 22, 378. N . M. Joye, jun., A. T. Proveaux, and R. V. Lawrence, J . Chromatog. Sci., 1972, 10, 590. S. S. Nigam and M. Ramaiah, Riechstofle, Aromen, Korperpjlegem., 1972, 22, 378 el seq. E. Demole and D. Berthet, Helv. Chim. Acta, 1972, 55, 1866, 1898. J. W. Wheeler, G. M. Happ, J. Araujo, and J. M. Pasteels, Tetrahedron Letters, 1972, 4635. H. Bretschneider, K. Hohenlohe-Oehringen, and K. Grassmayr, Monarsh., 1972, 103, 1523; K. Hohenlohe-Oehringen, ibid., p. 1531 ; K. Hohenlohe-Oehringen, K. zur Nedden, and H. Bretschneider, ibid., p. 1534. C.-F. Chang and S. Tamura, Agric. and Biol. Chem. (Japan), 1972, 36, 2405.
10
Terpenoids and Steroids
part of the molecule) having juvenile hormone activity have been made.59 The section on chrysanthemic acid includes other compounds having juvenile hormone activity. Isobornyl chloroformate (26; R = COC1) is prepared from isoborneol and phosgene, and can be used as a protecting group for amino-acids which is removed by trifluoroacetic acid.60 Combined with propylenediamines, the amines (26 ; R = CONHCH,CH,CH,NR’R2) can be made which have local anaesthetic properties.6 I
0,’””
4 Acyclic Monoterpenoids
Terpene Synthesis from Isoprene.-The oligomerization of isoprene catalysed by nickel naphthenate and isoprenemagnesium in the presence of various phosphites as electron donors, known to give cyclic dimers (see Vol. 3, p. 12), has been reexamined.62 Oligomerization with cobalt chloride, sodium borohydride, and tripenylphosphine gives (27)as the main product when the ratio Ph,P :CoC1, < 1, but when this ratio is > 1 the tail-to-tail linked isoprenoid (28) and the 2,6-dimethyloctatriene (29) are the main products.63 Telomerization of isoprene by hydrogen chloride in the presence of stannic chloride is reported.64 Anionic telomerization with secondary amines in the presence of alkali-metal catalysts yields dimers having as their main components the ‘lavandulyl’ (30) and the ‘geranyl’ (31) structure^.^^ A similar report elsewhere66contains what appears
(27) 59
6o
61
62
6.3 64
65
“
(28)
(30)
(31)
Z . Arnold, J. Kahovcova, M. Pankova, M . Svoboda, M . Tichy, and F. Sorm, Coll. Czech. Chem. Comm., 1973, 38, 261; J. Kahovcova, Z. Arnold, and F. Sorm, ibid. p. 1165. M. Fujino, S. Shinagawa, 0. Nishimura, and T. Fukuda, Chem. and Pharm. B U N . (Japan), 1972, 20, 1017. G. Jaeger, R. Geiger, and R. Muschaweck, Ger. Often. 2 102 741. K. Suga, S . Watanabe, T. Fujita, and T. Shimada, J . Appl. Chem. Biotechnol., 1973, 23, 131. K. Takabe, K. Uata, T. Katagiri, and J. Tanaka, Nippon Kagaku Kaishi, 1972, 1695. K . Laats, T. Kaal, I. Kalja, I. B. Kudryavtsev, E. Miks, M . Tali, S . Teng, and A. Erm, Eesti N . S . V . Teadirste Akad. Toimetised, Keem., Grol., 1972, 21, 305; K. Laats and S . Teng, ibid., p. 314 (Chem. Abs., 1973, 7 8 , 97 819 is probably faulty). K. Takabe, T. Katagiri, and J. Tanaka, Tetrahedron Letters, 1972, 4009; Bull. Chem. SOC.Japan, 1973,46,218, 222. T . Fujita, K. Suga, and S . Watanabe, Chem. and Ind., 1973, 231.
Mono terpeno ids
11
to be the incorrect structure for geranyldiethylamine. The isoprene hydrochloride dimer [(32) or (33)j can be reduced with magnesium in tetrahydrofuran containing ethyl bromide ; treatment of the mixture with dry oxygen then yields lavandulol(34) and its isomers in >40% yield.67 The ‘regular’ geranyl skeleton is produced when isoprene is allowed to react with alcohols over a PdC1,-PhCN catalyst together with triphenylphosphine and sodium alkoxide. The main ethers thus formed have the skeleton (35).@‘ The geranyl triene (36)is also the main com-
ponent of the complex mixture obtained by treating isoprene and phenol with a sodium phenate-[PdBr,L,] (L = Ph,PCH,CH,PPh,) catalyst, the amount of phenol determining the composition of the mixture of produ~ts.~’With sodium hydride at 40°C under pressure isoprene yields myrcene (37) and the trimer (38).’* Hydrative dimerization of isoprene using a cation-exchange resin catalyst is described,71 and a review (in Japanese) on the oligomerization catalysed by lithium naphthalene has appeared.’,
e:
I:‘yMe Dienes react with B-keto-esters in the presence of P-phenyl-l-phospha-3methylcyclopent-3-ene and palladium chloride, and the addition product (39) from isoprene and methyl acetoacetate can be readily converted into methylheptenone (40).73
2,6-Dimethyloctanes.-The isovalerate of dehydronerol (41) has been isolated from the roots of Anthemis montana, L. ; this is the first report of a dehydronerol 67
68
69 ’O
”
’* 73
T. Kaal and K. Laats, Eesti N . S . V . Teaduste Akad. Toimetised, Kecm., Geol., 1973, 22, 180 (Chem. Abs., 1973,79, 32 133). W. Hoffmann, F. J. Muller, and K. von Fraunberg, Ger. Offen. 2 154 370. K. Takahashi, G. Hata, and A. Miyake, Bull. Chem. SOC.Japan, 1973,46, 2600. K. Takabe, T. Katagiri, and J. Tanaka, Bull. Chem. SOC.Japan, 1972, 45, 2662. T. Katagiri, 0. Nakachi, T. Suzuki, K. Takabe, and J . Tanaka, Bull. Inst. Chem. Res., Kyoto Univ., 1972, SO, 363. K. Suga, S. Watanabe, andT. Fujita, Koryo, 1972, N o . 102, p. 19. S. Watanabe, K. Suga, andT. Fujita, Canad. J . Chem., 1973, 51, 848.
12
Terpenoids and Steroids
derivative in nature.74 The digestive gland of the sea hare, Aplysia californica, contains brominated and chlorinated monoterpenoids characterized by the presence of a terminal vinyl bromide group, e.g. (42) and (43). These compounds and other halogenated monoterpenoids have been found in the red algae, Plocarniurn coccineum, on which the sea hare is known to graze.75The structure of one compound (44)has been fully established by X-ray d i f f r a ~ t i o n .The ~~ p
.
B
r
/
y Br
Br
0
/
Br
c T l \ Br
\
c1
CI
c1 (42)
(41)
CHBr,
’*
*
CHBrz
(44)
(43)
three trienes (49, (46), and (47) have been isolated from Ledum p a l ~ s t r e ; ~ ~ one of them (46)has been previously identified in Pinus p o n d e r ~ s a A . ~nylnber ~ of
(47)
(46)
(45)
cppx$
bifunctional carbonyl compounds (48)-(53) have been isolated from lavandin oil. They all [excepting the aldehyde acetate (48)] can be obtained by the photooxygenation of linalyl acetate, and, apart from (48), they may well be artefact^.^'
OH CHO (481
\
‘0 (49)
(50)
(51)
(52)
(53)
An attempt to prepare photochemically mixtures of allo-ocimenes (54) with exclusive 2-configuration about the central double bond [(54a), (54b)l failed l4 ’5 ’6
”
’13
l9
F. Bohlmann and H. Kapteyn, Tetrahedron Letters, 1973, 2065. D. J . Faulkner and M. 0. Stallard, Tetrahedron Letters, 1973, 1171. D. J. Faulkner, M. 0. Stallard, J. Fayos, and J. Clardy, J . Amer. Chem. SOC.,1973, 95, 3413. M . von Schantz, K.-G. Widen, and R. Hiltunen, Acta Chem. Scand., 1973, 27, 551. R. M. Silverstein, J. 0. Rodin, D. L. Wood, and L. E. Browne, Tetrahedron, 1966, 22, 1929. B. D. Mookherjee and R. W. Trenkle, J . Agric. Food Chem., 1973, 21, 298.
Mono terpeno ids
13
with a variety of sensitizers, although some enrichment was noted.80 Vig et al. have synthesized myrcene (37) from the known ester ( 5 5 ; R = C0,Et) via the corresponding aldehyde (55 ; R = CHO),by a vinyl Grignard reaction, oxidation, and Wittig reaction.81 The pyrolytic conversion of a-pinene into allo-ocimene (54) is well known ;in order to trap the intermediate ocimene, it is necessary to cool the pyrolysate very rapidly.82
(54)
(55)
One method used to introduce oxygen into terpenoid hydrocarbons is by direct, acid-catalysed addition of water. With myrcene, water addition in the presence of Amberlite IR-120 gives a complex reaction mixture, consisting mostly of cyclized components; the hydrated products are mainly 1,8-cineol(56), mentha-1(7),2-dien-8-01 (57), and 2,6-dimethylocta-5,7-dien-2-01 (58).83 Acidcatalysed addition of acetic acid to ( + )-2,6-dimethylocta-2,7-diene [( + )-(59)] gives the tertiary acetate (60) initially, but refluxing for 6-43 h causes stereospecific cyclization to (61),together with formation of the two tetrahydroeucarvols (62).84 The rhodium(rI1) chloride-catalysed addition of ethanol to myrcene (37)
QoAc
'' 82 83 84
V. Ramamurthy, Y. Butt, C. Yang, P. Yang, and R. S. H. Liu, J. Org. Chem., 1973, 38, 1241. 0.P.Vig, M. S. Bhatia, A. S. Dhindsa, and 0. P. Chugb, IndianJ. Chem., 1973,11, 104. G. Rice and J. F. Pollock, U.S.P. 3 714 283. J. Tanaka, T. Katagiri, K. Takabe, and 0. Nakachi, Nippon Kagaku Kaishi, 1972, 1203. H. R. Ansari, Tetrahedron, 1973,29, 1559.
Terpenoids and Steroids
14
leads to oligomerization and isomerization, together with a mixture of the ethyl ethers [(63), (64),(65), and (66)] but none of the derivatives corresponding to those from the palladium-catalysed addition of methanol (see Vol. 2, p. 10).
Addition of acetic acid was also studied, but the mixture of acetates is more complex. Copper salts were less effective catalyst^.^^ Rienacker has used the octadienol ( - )-(59) and its antipode to make a-citronellol(67) by initially isomerizing the double bond to the terminal position (68),then hydroxyalumination, followed by oxygenation (air) and hydrolysis. Some of the isomer (69) is also formed.86
(-
1459)
-
p-r+F
A preliminary note has described the interesting double photo-oxygenation of 2,6-dimethylocta-2,6-diene (70) ; after reduction, two glycols [(71) and (72)] were obtained.87 OH
(70)
(71)
(72)
A new, mild method for making ally1 alcohols from epoxides has been applied to myrcene epoxide (73),thereby synthesizing the natural product (E)-2-methyl6-methyleneocta-3,7-dien-2-01(46) (Scheme 1). Other methods of base-catalysed epoxide-ring opening yield products resulting from attack on the methyl proton.88 85
86
88
R . J. H. Duprey, W . D . Fordham. J. F. Janes, D. V. Banthorpe, and M. R. Young, Chem. and Ind., 1973, 847. For comments on the unsatisfactory name ‘rhodinol’ used here, see ref. 230, p. 15. R. Rienacker, Chimia ( S w i t z . ) , 1973, 27, 97. J. Chaineaux and C. Tanielian, L’ActualirP chimique, 1973, 88. K. B. Sharpless and R . F. Lauer, J . Amer. Chem. Soc., 1973. 95, 2697.
Monot erpenoids
$ (73)
15
1L
Gh
-
(46)
LIH$OH] 0's
Ph
Reagents: i, Ph,Se, in abs. EtOH, then NaBH,; ii, H 2 0 2 .
Scheme 1
The ally1 mesitoate coupling reaction (see Vol. 3, p. 18)has been used to make the pheromone (74) of Ips confusus, and although the yield was only 10% using
lithium in tetrahydrofuran it rose to 52% when the lithium was replaced by zinc.89 Hotrienol (75) has been made from methylheptenone as in Scheme 2."
Reagents: i, KMnO,; ii, Pb(OAc),; iii, Ph,P=CHCMe=CH,;
iv, HC1; v, CH,=CHMgBr.
Scheme 2
Although circuitous, this type of route could be useful for isotopic labelling. Geranic acid (76), together with a small amount of lavandulic acid isomers (77), has been made by condensing prenyl bromide with an ethyl tiglate activated at the terminal methyl group by the presence of a phenylsulphone group (Scheme 3).'l Synthesis of the 2$-dimethyloctane system has also been achieved by reaction of methylmagnesium bromide with cyclopropylmethyl ketone and 89 90
9L
J. A. Katzenellenbogen and R. S. Lenox, J. Org. Chem., 1973, 38, 326. 0. P. Vig, J. Chander, and B. Ram, J. Indian Chem. SOC., 1972, 49, 793. M. Julia and D. Arnould, Bull. SOC.chim. France II, 1973, 743.
Terpenoids and Steroids
16
\
+
SO,Ph
1"
+Co2Et \
U
C
0
,
E
t
+SO,Ph C0,Et
++
C0,Et (76)
(77)
Reagents: i, KOBu'; ii, Na-Hg.
Scheme 3
condensation of the resulting bromide (78; X = Br) with methylated ethyl acetoacetate. Hydrolysis gave 'P-elgenone' (79).92 C0,Et
P C O M e
MeMgBr)
LcH,x UCO MeCO!;C02;I
1
(78)
U
C
O
M
,
(79)
A new synthesis of ally1 alcohols has been employed in the preparation of geraniol (80). The anion from the sulphoxide (81) reacts with alkyl halides, in particular with the iodide (78 ; X = I), to yield a derivative of the linalool type (82), which undergoes a [2,3] sigmatropic rearrangement to yield a 9 : 1 mixture of geraniol(80) and n e r 0 1 . ~The ~ stereoselectivity is reported to be high compared with other [2,3] rearrangements of this kind. (Further sigmatropic rearrangements in the 2,6-dimethyloctane series are discussed below.)
92
93
J . Kulesza and J . Gora, Mezhdunar 4th Kongr. Efirnym. Maslam, 1968 (published 1971), p. 48 (Chem. Abs., 1973, 78, 124 722 is incorrect; the synthesis described leads to a product lacking a methyl group). D. A. Evans, G . C. Andrews, T. T. Fujimoto, and D. Wells, Tetrahedron Letrers, 1973, 1385, 1389.
Monoterpenoids
17
The reduction of citral or citronella1 to the corresponding alcohols has been further examined. A culture medium or cell suspension of various microorganisms reduces (+)-citronella1 to a mixture containing 82 % of (-)-citronello1 and 18 % of ( + ) - c i t r ~ n e l l o l .Citral ~ ~ is reduced ‘quantitatively’at the aldehyde group (without attack on the conjugated double bond) by hydrogen over an iridium ~atalyst,~’and continuous hydrogenation of citral to citronellol, geraniol, and nerol has been de~cribed.’~ The deamination of geranylamine (31 ; R = H) and nerylamine has been compared with the hydrolysis of the corresponding chlorides, phosphates, and pyrophosphates ; the deamination reaction gives less cyclization in the case of the neryl compounds and less isomerization to linalyl compounds in the case of geranylamine.
’
NMe,
a
CH,SH NaOH-MeOH
There is little novelty in the discovery that geranyl dimethylthiocarbamate (83) rearranges on heating to linalyl dimethylthiocarbamate (84) or that the latter gives exclusively geraniol thiol (85) with methanolic base, linalol thiol requiring lithium aluminium hydride reducti~n.’~A mixture of digeranyl selenide and digeranyl diselenide (86) is formed when geranyl chloride reacts with sodium sulphide. The diselenide (86), on treatment with triphenylphosphine, has been found to undergo a [2,3] sigmatropic rearrangement [to geranyl linalyl selenide (87)] more rapidly than the analogous disulphide. The action of hydrogen peroxide on either the mono- or the di-selenide should lead to the selenic acid (88), but this rearranges directly, since only linalool (89) is obtained from this reaction.” (+)-Citronella1 can be converted into its dihydro-compound (go), whose methylation (uia the enamine) leads to 2,3,7-trimethyloctanal (91). The corresponding amine (92) on treatment with nitrous acid leads to various products (by migration of bonds a, b, or c). Migration of bond a gives the alcohol (93) (the formula of which is misprinted in the publication) without loss of optical 94
95
96
91 98
99
Takasago Perfumery Co., Jap. P. 16 191/1973. E. N. Bakhanova, A. S. Astakhova, Kh. A. Brikenshtein, V. G. Dorokhov, V. I. Savchenko, and M. L. Khidekel’, Izvest. Akad. Nauk S.S.S.R., Ser. khim., 1972, 1993. M. M. Paulose, A. J. Pantulu, K. V. Raghavan, and K . T. Achaya, Res. Ind. (New Delhi), 1972, 17, 1 1 . C. A. Bunton, D . L. Hachey, and J.-P. Leresche, J . Org. Chem., 1972, 37, 4036. R. E. Hackler and T. W. Balko, J . Org. Chem., 1973, 38, 2106. Closely similar work, together with relevant literature, unquoted by Hackler and Balko, may be found in V. Rautenstrauch, Helv. Chim. Acra, 1971, 54, 739. K. B. Sharpless and R. F. Lauer, J . Org. Chem., 1972,37, 3973; J . Amer. Chem. Soc., 1972,94,7 154.
Terpenoids and Steroids
18
(89)
(88)
activity, as shown by preparation of the ketone (94) from the alcohol (93) and from oxidation of the Grignard product of dihydrocitronellal(90). The retention of optical activity shows that partial bonding must exist during the migration of bond a.'" H LW
oxirne
QOMe
A (94)
A.
(93)
Oxidation of ( - )-(R)-dihydrolinalool (95) with potassium permanganate leads to a lactone (96), which has been used to prepare (-)-(S)-4-methylhexane1,4-diol (97).' O' Oxymercuration of geraniol results in cyclization to the tetrahydrofurans (100) (98) and (99), and oxymercuration of 2-methyl-6-methyleneoct-7-en-2-01 also gives cyclized products (101) and (102).'02 loo
lo' lo*
T. Shono, K. Fujita, and S . Kumai, Tetrahedron Letters, 1973, 3123. J . Jacobus, J . O r g . Chem., 1973, 38, 402. G . Brieger and E. P. Burrows, J . Agric. Food Chem., 1972, 20, 1010.
Mono t erpenoids
19 HO
Cyclization of cironellal and related substances is treated in the section on p-ment hanes. Artemisyl, Santolinyl, Lavandulyl, and Chrysanthemyl Derivatives.-An excellent review of this series, especially regarding the important biogenetic considerations, has been published by Epstein and P ~ u l t e r . " ~In this it is implied that 'isoartemisia ketone' [i.e. the unconjugated isomer (103)] is a natural product, but there is, in fact, no proof of this.'04 The similarity-between chrysanthemic acid (104) and presqualene alcohol also extends to their absolute configurations, which, contrary to earlier reports, are the same, i.e. R,R.loS The lavandulyl acetate aldehyde (105) has been identified in lavandin and a new santolinyl ester (106) in Artemisia tridentada.'06
Io3
lo4
lo'
Io6
W. W. Epstein and C. D. Poulter, Phytochemislry, 1973, 12, 737. There is also some confusion in other reviews about artemisia and isoartemisia ketone; see, e.g. D. V. Banthorpe, B. V. Charlwood, and M. J. 0. Francis, Chem. Rev., 1972, 72, 101. Much of this confusion arises because literature published prior to 1957 named the natural product, i.e. the conjugated ketone, 'isoartemisia ketone'. G. Popjak, J. Edmond, and S.-M. Wong, J. Amer. Chem. SOC.,1973,95, 2713. W. W. Epstein and J. Shaw, 166th A.C.S. Meeting, August, 1973, Abstracts AGFD, No. 5 5 .
20
Terpenoids and Steroids
Feeding experiments of labelled mevalonic acid to Chrysanthemum cinerariaefoliurn have been carried out, and preliminary analysis of the results indicates that the chrysanthemic acid (104) isolated is labelled only in the cyclopropane half of the molecule (see also the similar result with artemisia ketone, Vol. 2, p. 13).'07 The formation of the dicarboxylic acid, pyrethric acid, was shown to occur by oxidation of the chrysanthemic acid.'08
Rautenstrauch has extended earlier work on the sigmatropic rearrangements of dimethylallyl ethers and has shown that the corresponding quaternary ammonium salts (107)rearrange at - 73 "Cwith sodium amide in liquid ammonia, giving 80 % of the artemisia skeleton (108),together with some of the head-to-head and tail-to-tail linked isomers.'09 BogdanoviC has obtained both citral (109) and lavandulal (110) by reaction of prenyl bromide with the anion (111); the proportion of the products depends on solvent, ether favouring (110) and tetrahydrofuran favouring (109).1'0 Reaction of the precursor (112) of the anion (1 11) with the phosphorane (113)' yields, after hydrolysis, chrysanthemyl aldehyde ( 1 14) (Scheme 4). A new synthesis of yomogi alcohol (115) consists of addition of the lithium salt of 3,3-dimethylpent-l-en-4-yne(1 16) to acetone and reduction of the resulting acetylenic alcohol (117) with lithium aluminium hydride." The mass spectra of the pyrethrins [including that of chrysanthemic acid i104) and pyrethric acid (118)] have been discussed. All the derivatives of chrysanthemic acid, as well as the acid itself, produce a fragment at m/e 123,corresponding to the ion (119).' l 3 The lanthanide-shifted 'H n.m.r. spectra of chrysanthemic The absolute configuration esters and alcohols have also been discussed.' of the cyclopropane part of the natural pyrethrins was already known ; now an X-ray study of the six pyrethrins has established the absolute configuration of the whole molecules.'
''
'
'
lo' Io8
'09 lo
I I I
'I3 II4
I
G . Pattenden and R. Storer, Tetrahedron Letters, 1973, 3473. S. Abou-Donia, C. F. Doherty, and G. Pattenden, Tetrahedron Letters, 1973, 3477. V. Rautenstrauch, Helo. Chim. Acta, 1972, 55, 2233. B. Bogdanovic, personal communication. B. Bogdanovic and S. KonstantinoviC, Synthesis, 1972, 481. A. W. Burgstahler and H. W. Kroeger, Synth. Comm., 1973, 3, 21 I. G. Pattenden, L. Crombie, and P. Hemesley, Org. Mass Spectrometry, 1973, 7 , 719. L. Crombie, D. A. R. Findley, and D. A. Whiting, Tetrahedron Letters, 1972, 4027; T. Sugiyama, A. Kobayashi, and K. Yamashita, Agric. and Biol. Chem. (Japan), 1973, 37, 1497. M. J. Begley, L. Crombie, D. J. Simmonds, and D. A. Whiting, J . C . S . Chem. Comm., 1972, 1276.
Monoterpenoids
21
1 %CHO
U
C
H
O
Reagents: i, Bui,AIH; ii, LiNEt,; iii, Me,C=CHCH,Br; iv, H,O.
Scheme 4
Terpenoids and Steroids
22
Certain chrysanthemic phenyl esters carrying a methyl ester group in the p position (120) possess juvenile hormone activity in the pyrrhocorid bug (Dysdercus) only.'16 Because the main point of metabolic attack in both insects and mammals is on the E-methyl group of the isobutenyl side-chain of the chrysanthemates, Elliott et al. prepared pyrethrins where this group was not present, some of which (notably one with a butadienyl side-chain in place of isobutenyl) were much more potent insecticides,as well as being less toxic to mammals.'" Other modifications to pyrethrins to evaluate insecticidal activity have been made. l 8 Synthetic work in the chrysanthemic acid field includes the publication in full of the Glasgow synthesis (see Vol. 2, p. 14).11' A one-pot method for converting dihydrochrysanthemolactone (121) into ethyl cis-chrysanthemate with acidic ethanol, removing the water with molecular sieves or azeotropically, has been described.' 2 o Pyrolysis of pyrethrin-I (122) at 400°C yields chrysanthemic acid (104) and pyrocin (123),in addition to a hydrindanone (124) from the non-isoprenoid part of the molecule.
HA+
oco
-+ (104)
+
+ 0 (123)
(124)
Sugiyama et al. have published further work on the synthesis of allethrin metabolites (see Vol. 3, p. 23), in connection with the detoxication process.'22 5 Monocyclic Monoterpenoids
Cyc1obutane.-A monocyclic cyclobutane monoterpenoid (125) (not a conventionally head-to-tail linked isoprenoid) has been isolated from Juniperus
11'
11*
'I9 lZo
I2l 122
N . Punja, C. N. E. Ruscoe, and C. Treadgold, Nature New Biol.,1973, 242, 94. These authors d o not consider chrysanthemic acid derivatives to be terpenoid; neither does a reporter in Nature, 1973, 242, 159! In compensation Chemical Abstracts persistently classes compounds of the jasmone type (i.e. related to the non-isoprenoid part of the pyrethrins) as terpenoid! M. Elliott, A. W. Farnham, N. F. James, P. H. Needham, and D. A. Pulman, Nature, 1973, 244, 456. K. Sota, T. Amano, M. Aida, A. Hayashi, and I. Tanaka, Agric. and Biol. Chem. (Japan), 1973, 37, 1019. R. W. Mills, R. D. H. Murray, and R . A. Raphael, J . C . S . Perkin I , 1973, 133. A. Higo, N. Itaya, H. Hirai, and H. Yoshioka, Ger. Offen. 2 159 882. The preparation of the lactone and its conversion into chrysanthemic acid is described by H . Yoshioka, M. Matsui, Y. Yamada, and H. Sakimoto, Ind. Chim. (Bruxelles), 32 (Special number), Comptes rendus 36kme Congr. Internat. de Chimie Industrielle, 1967, Vol. 111, p. 890. Y. Nakada, Y. Yura, and K. Murayama, Bull. Chem. SOC. Japan, 1972,45,2243. T . Sugiyama, A. Kobayashi, K. Yamashita, and T. Suzuki, Agric. and Biol. Chem. (Japan), 1972.36, 2275.
Mono terpenoids
23
communis oil and synthesized from caryophyllene.’ 23 This substance (‘junionone’) is clearly derivable from a pinene skeleton. A stereoisomer of grandisol (126), called ‘fragranol’, with trans-substitution on the cyclobutane ring, has been isolated from Artemisia fragrens. A new synthesis of grandisol (126) constructs the cyclobutane ring by the dimerization of isoprene on a catalyst of bis-(1,5-cyclo-octadiene)nickeland tris-(2-biphenylyl) phosphite. The cyclobutane (127) formed (in 12-1 5 % yield, together with other compounds), was hydroborated with disiamylborane and alkaline peroxide then gave the monoterpenoid (126). 2 5 Mention is also made of a synthesis of (126) from P-pinene,’26 and another from eu~arvone,’~’ but details are not yet available.
& l
+ Isoprene-[Ni(cod),] TBP
1
i, (Siaj,BH-THF, 0 “C ii, H,O,-OH-
Cyclopentanes, including 1ridoids.-An account of work on the biosynthesis of iridoid and secoiridoid glucosides, and other aspects of iridoids, that was presented at the First International Congress of Pharmacognosy and Phytochemistry has appeared in print.’** A new cyclopentane terpenoid (25) from tobacco has been mentioned above.55
124
12’
28
A. F. Thomas and M. Ozainne, J.C.S. Chem. Comm., 1973, 746. F. Bohlmann, C. Zdero, and U. Faass, Chem. Ber., 1973, 106, 2904. W. E. Billups, J. H. Cross, and C. V. Smith, J. Amer. Chem. SOC., 1973, 95, 3438. P. D. Magnus, N. Bosworth, and P. D. Hobbs, 166th A.C.S. Meeting, August, 1973, Abstracts A G F D , No. 12. W. A. Ayer and L. M. Browne, 166th A.C.S. Meeting, August, 1973, Abstracts A G F D , No. 41. H. Inouye, in ‘1st International Congress o n Pharmacognosy and Phytochemistry’, ed. H. Wagner and L. Horhammer, Springer-Verlag, Berlin, 1971, p. 290; P. W. Thies, ibid., p. 41.
Terpenoids and Steroids
24
From Mentzelia decapetala, mentzeloside (128)"' and decaloside ( 129)130 are reported. H
(128) Glu
= P-glucose
(129)
Two biosynthetic studies are reported : the first deals with the incorporation of
4C02 into cispans-nepetalactone in Nepeta cataria.' 31 The other concerns the biogenetic route to genepin (130) in Genipa arnericana, and supports Inouye's postulate132 of the intermediacy of geniposidic acid (131) in the biosynthesis of iridoid glucosides having a hydroxylated C-10 group, the glucoside then being converted into the aglucone by methylation and deglu~osylation.'~~
(132) R = CH,CHMe,
(133)
Alcoholysis of didrovaltratum (132), isolated from Valerianu wallichii, in the presence of one equivalent of a hydrogen halide, HX, leads to compounds with the structure (133).'34 There is not complete agreement about the products from the treatment of the lactone (134) with potassium t-butoxide in dimethylformamide 129
130
'"
'
s4
T. J. Danielson, E. M. Hawes, and C. A. Bliss, Canad. J . Chem., 1973, 51, 760. T. J . Danielson, E. M. Hawes, and C. A. Bliss, Canad. J . Chem., 1973, 51, 1737. E. D. Mitchell, M. Downing, and G . R. Griffith, Phytochemistry, 1972, 11, 3193. H. Inouye, S. Ueda, and Y. Takeda, Tetrahedron Letters, 1970, 3351. R. Guarnaccia, K. M . Madyastha, E. Tegtmeyer, and C. J. Coscia, Tetrahedron Letters, 1972, 5 125. P. W. Thies and A. Asai, Chem. Ber., 1972, 105, 3491.
Mono terpenoids
25
____+
0 (135) 25xofeach isomer
( 1 34)
(136) 25xofeach isomer
followed by lithium aluminium hydride reduction. Andersen and Uh find approximately equal amounts of all the isomers [(135) and (136)],13' whereas Wolinsky and Eustace' 36 find primarily the all-cis isopropenyl isomer [cis-( 136)].
b \
/ + (+)-di-3-pinanylborane -D
HO
(138)
(139)
li
H-OH
H H
Me0,C
I!
'IJR)
CHO
(141) Aco-isa
H
OAc
C0,Me
(140)
1
ii
AcO--
+
+ AcO--
H C0,Me
H
C0,Me
-
(143) I iii
(142)
- - OAC
CH,OAc
AcO - H
C0,Me
Reagents : i, Et,NOAc; ii, h v ; iii. 2,3,4,6-tetra-0-acetyl-~-~-glucose.
Scheme 5 135 136
N. H. Andersen and H. Uh, Tetrahedron Letters, 1973, 2079. J . Wolinsky and E. J. Eustace, personal communication to the authors of ref. 1 3 5 ; see also, Vol. 2, p. 19 of these Reports.
26
Terpenoids and Steroids
The full account of Buchi’s loganin synthesis (cf:Vol. 1, p. 20) has appeared.I3’ Partridge et al. have described a short asymmetrically induced synthesis of loganin penta-acetate (137) from methylcyclopentadiene (138). The latter can be hydroborated in at least 95 optical purity, (+)-di-3-pinanylborane giving the (R,R)-isomer (139), which is converted into the (S,R)-cyclopentenyl acetate (140). Irradiation of the latter in the presence of methyl diformylacetate (141) gives the loganin aglycone derivative (142) regioselectively (Scheme 5)’ 38 Both papers discuss the difficulty of glucosylating the aglycone; this was effected in the presence of boron trifluoride, oia the unstable oxonium ion (143). p-Menthanes.-This year, synthesis of the skeleton will be treated first, followed by the properties, first of the hydrocarbons and halides and then of the oxygenated p-menthanes. New modifications of the Diels-Alder reaction for making p-menthanes continue to appear. The reactioq of chloroprene with methyl vinyl ketone was mentioned before (see Vol. 3, p. 49); the resulting ketone (144) has now been
6 $A 4
Q
>
+(8%Q
COMe (144)
jIV,.
Q (145)
Reagents:
I,
MeMgI; ii, Wittig; iii, Al,O,-py; iv, Li; v, Me,NCHO.
Scheme 6
converted (Scheme 6) into perilla aldehyde (143.‘ 39 Simple a-butenolides are not dienophiles, but the /I-carboxylated derivatives are ; thus with isoprene the butenolide (146) reacted in benzene after 3 days at 140-150°C to give the cisfused lactone (147), which was converted specifically into either (* )-menthone (148) or (+)-isomenthone (149) (Scheme 7).I4O 137
138
I39 140
G. Buchi, J . A. Carlson, J. E. Powell, jun., and L.-F. Tietze, J . Amer. Chem. SOC., 1973, 95, 540. J . J . Partridge, N . K . Chadha, and M . R. Uskokovic, J . Amer. Chem. SOC.,1973, 95, 532. Yu. S. Tsizin and A. A. Drabkina, Zhur. obshchei Khim., 1972, 42, 1852. S. Torii, T. Oie, H. Tanaka, J . D. White, and T. Furuta, Terrahedron Letters, 1973, 247 1 . The formula of one of the saturated lactones is misprinted in this paper.
Mono terpeno ids
27
j--Jco2H + 1 9 1 J$. --T -yo 0
H(147) l i , ii
19 %
75 % Jiii
1 v i . vii
I
b i , vii
1
b i i i , ix
Q, Reagents: i, H,-Pd; ii, distil; iii, LiAlH,; iv, Ac,O, 130°C; v, 14O-15O0C, PtO,; vii, NaOH; viii, pyrolysis of xanthate; ix, 0,. Scheme 7
3 h ; vi, H,-
Terpenoids and Steroids
28
The synthesis of menthone by Conia et al. depends on the fact that b-ethyleneketones cyclize thermally to cyclohexanones. Thus the P-keto-ester (150), readily obtained from ethyl acetoacetate, gives the cyclized P-keto-ester (151) as the main product after 16 h at 300 "C,whereas after 2 h at 350 "C, 50% of the product is a mixture of (+)-menthone and (-t-)-isomenthone (7 : 3). Cyclization of the ketone (152) without the ester group also gives the same mixture, but in lower yield, and under more vigorous conditions. 14' i, CH,=CHCH,CH,Br
MeCOCH ,CO,Et
-:1
ii, Me,CHI ________,
/
300°C. I6 h
C0,Et
In the preparation of isopulegols from (+)-citronella1 (153), the main product using tris(tripheny1phosphine)chlororhodium is ( + )-neoisopulegol (154). Stannic chloride cyclization, on the other hand, yields mostly (-)-isopulegol (155).142 Cyclization occurs when 3,7-dimethyloctane-1,7-diolis heated above 200 "C with an acid anhydride. The product is the pulegol ester (156).143The full paper
''I
'42 143
G. Moinet, J . Brocard, and J.-M. Conia, Tetrahedron Letters, 1972, 4461; J . Brocard, G. Moinet, and J.-M. Conia, Bull. SOC.chim. France II, 1973, 171 1 . K. Sakai and 0. Oda, Tetrahedron Letters, 1972, 4375. W. Hoffmann and W . Reif, Ger. Offen. 2 1 15 130.
Monoterpenoids
29
of Sukh Dev's preparation of the 1-vinylisopulegol (157) from geraniol vinyl ether (158) has appeared. 144
Formation of menthanes by ring-opening of pinanes is discussed in the section on bicyclo[3,l,l]heptanes. Addition of N,03 to ( - )-a-phellandrene (159)yields a crystalline nitro-nitrosocompound, now shown to be the dimer of (2R,4S,5S)-5-nitroso-2-nitromenth-6ene (160).145
(159)
It has been suggested that limonene might be used as a measure of the protonremoving power of 'superbases' ; for example, N-kalioet hylenediamine aromatizes limonene in 5 min at 25 "C (to p-cymene), whereas the lithium analogue requires > 2 h at 90°C.146 Further uses of metallated limonene include the reaction with formaldehyde, which leads to the alcohol (161 ; n = 1),147 and the homologue (161; n = 2) with ethylene oxide.'48 A study of the autoxidation of 2-substituted p-cymenes, in particular whether they react at C-7 or C-8,I4' includes previously published material.'50 The oxidation of limonene with hydrogen peroxide and catalytic amounts of selenium dioxide gives (162) as the main product. The structure of (162) was confirmed by catalytic reduction to a glycol (163). Several likely intermediates in the oxidation were examined, including the epoxide (164),with the results shown in Scheme 8.15' 145 146
14*
151
N. P. Damodaran and Sukh Dev, Tetrahedron, 1973,29, 1209. C. H. Brieskorn and H. H. Frohlich, Chem. Ber., 1972, 105, 3676. C. A. Brown, J . Amer. Chem. SOC.,1973,95, 982. R. J. Crawford, J . Org. Chem., 1972, 37, 3543. R. J. Crawford, U.S. P. 3 676 505. G. Bourgeois and R. Lalande, Bull. SOC.chim. France, 1972, 4324. A. F. Thomas, Helv. Chim. Acta, 1965,48, 1057. C. W. Wilson, tert. and P. E. Shaw, J . Org. Chem., 1973, 38, 1684.
30
Terpenoids and Steroids
(162)
1
( 164)
ii
decomposed Reagents: i , Pd:C - H 2 ;i i , H 2 0 , - S e 0 , .
Scheme 8
Epoxidation of y-terpinene (165) with peroxybenzimidic acid yields two epoxides (166) and (167) in 3 : 1 ratio, which are readily separable by d i ~ t i l l a t i o n . ' ~ ~
(165)
A
A
( 1 66)
(167)
Pure cis-menth-l-ene epoxide (168) was made by separation of the menthane1,2-diols (169)and (1 70), obtainable on treatment of menth-l-ene with performic acid, and hydrolysis of the 2-to~ylate.''~Both pure cis-menth- l-ene epoxide
'
57
A
A
A
(169)
(170)
(168)
S. A . Kozhin and E. I. Sorochinskaya, Zhur. obshchei Khim., 1973,43, 671. K . Piatkowski and A. Siemieniuk, Pracenauk. Inst. Chem. Org. i Fiz. Politech. Wroclawskiej, 1970,7 1 .
31
Monoterpenoids
(168) and the truns-isomer have also been made by the method used by Wylde and Teulon (cf Vol. 1, p. 26) for the limonene epoxides, separating the chlorohydrins through their p-nitrobenzoates. ''4 Continuing their work on brominated menthanes, Carman and Venzke have shown that bromination in light of trans-1,2,8-tribromo-cis-menthane (17 1) (see Vol. 3, p. 32) involves a bridged bromine radical and leads to a rearranged tribromide (172) and the two tetrabromides (173) and (174). The rearranged tribromide (172) is also formed in the dark reaction, which leads in addition to bromination in the 9-position (175).' " Br
I
Br
Br
Br
Br
Br
(174) Br
Buchi and Vederas have developed a method foF converting an unsaturated carbonyl compound into the allylic isomer that involves formation of an isoxazole (by using iodine oxidation of the oxime), followed by reductive ringopening. Whereas the technique worked well in the case of fl-ionone, the yield of carvone (176) from perilla aldehyde oxime (177) was only a few percent, because the isoxazole (178), even with sodium bicarbonate, yields the keto-nitrile (179) which undergoes further transformations.' 5 6 If the carbonyl group of cuminaldehyde (180) is protected as the imidazoline derivative (181), reduction with lithium in liquid ammonia readily gives the dihydro-compound from which the aldehyde (182), responsible for the flavour of cumin seeds, is obtained.I5' A molecular orbital study of the reduction mechanism ofp-mentha- 173-dien-7-al (183) with sodium in liquid ammonia has led to the conclusion that reduction 154
D. Sedzik-Hibner, H . Weinert-Orlik, and Z. Chabudzinski, Roczniki Chem., 1973, 47, 1249.
155
IS6 15'
R. M. Carman and B. N. Venzke, Austral. J . Chem., 1973, 26, 571. G . Buchi and J. C. Vederas, J . Amer. Chem. SOC.,1972, 94, 9128. A. J . Birch and K. P. Dastur, Austral. J . Chem., 1973, 26, 1363.
Terpenoids and Steroids
32
A n
n. .
proceeds through 1,6-biradical addition to the a/?,yd-unsaturated system.’ 5 8 An examination of the reduction of carvone over palladium or platinum catalysts has once again shown how platinum catalysts are more selective than palladium catalysts, platinum black yielding only carvomenthone (184), carvotanacetone (185), and carvomenthol (186); far more intermediates are found using 10% palladium on charcoal, whereas with palladium black, isomerization to carvacrol (187) occurs. 5 9 Wallach’s ‘dicarvelones’,formed by reduction of carvone (176)
(183) 158
i184)
(185)
(186)
(187)
H. Kayahara, Shinshu Daigaku Nogakubu Kiyo, 1972, 9, 83 (Chem. Abs., 1973, 79, 18 855).
E. I . Klabunovskii, L. F. Godunova, and L. K . Maslova, Izvest. Akad. Nauk S.S.S.R. Ser. khim., 1972, 1063.
Monoterpenoids
33
with zinc and have been shown to have the structures Wailach ascribed to them [(188),(189), and (190)].'61
i, 2HBr ii, KOH-MeOH
(176) +
(188) a
O W (190) 1'
Addition of bromine to carvone (176) gives, first, two tetrabromides (191) and (192); further bromination of the crystalline P-tetrabromide (191) yields a pentabromide (193). A discussion of the structures, and much information about other halogenated carvones, has been given by Carman and Venzke.'62 The Br
Br
Br
BrH,C'Br
0
CH,Br Br
0
Br Br
0
addition of bromine to carvone oxime occurs initially to the 8,9-double bond, then to the conjugated double bond, and both the dibromide (from the first addition) and the tetrabromide (from the second) can be reduced back to the oxime with zinc in alkali. Replacement of the tertiary bromine atoms by methoxy (with methanol) and replacement of the 8-bromo-atom by chlorine with nitrosyl chloride are also d i ~ c u s s e d . ' ~When ~ the carvone tribromides (194) and (195) react with methoxide or hydroxide anions (cf. Vol. 1, p. 31), methoxide gives I6O 161
'62 163
0. Wallach and H . Schrader, Annufen, 1894, 279, 377. R. M. Carman, G . N. Saraswathi, and J. Verghese, Austral. J . Chem., 1973,26, 883. R. M. Carman and B. N. Venzke, Austral. J . Chem., 1973, 26, 1283. E. G . Bozzi, C. Shiue, and L. B. Clapp, J . O r g . Chem., 1973, 38, 56.
Terpenoids and Steroids
34
products from the Favorskii rearrangement, trans-tribromocarvomenthone (195) yiclding the epoxide (196) in addition. Hydroxide gives fragmentation products, but the reactions depend on the nature of the base, the solvent, and the configuration of the bromine atom at C-6.1G4
(194) R'
=
R'
=
(195)
Br, R 2 = H H, R 2 = Br
(196)
Metal hydride reduction of carvone epoxide (197) is reported by Zaitsev and Kozhin to yield practically only trans-p-menth-8-ene-trans-2,6-diol (198)? The Russians used lithium aluminium hydride in ether, and their finding does not completely agree with the work of Piqtkowski, who reported a mixture of four glycols and isocarveol epoxide (199).'" The same author has examined the reduction of dihydrocarveol e p ~ x i d e 'and ~ ~ the diepoxide (200), the latter reportedly giving the glycol (201).16' The inaccessibility of the journal and the absysmal quality of the abstracts preclude full assessment of this interesting work. Glycols of the type (201), acetylated on the tertiary hydroxy-group,
A (200) 164
Ih5 Ihh
16'
16'
(201)
J . Wolinsky and R. 0. Hutchins, J . Org. Chem., 1972, 37, 3294. V. V. Zaitsev and S. A. Kozhin, Zhur. org. Khim., 1972, 8, 1841. K . Piatkowski. Pruce nuuk. Inst. Chem. Org. i Fiz. Politrch. Wvoduwskiej, 1970, 3. (Them. A h . , 1973, 78, 97 814); K. Piatkowski and A. Siemieniuk, ibid., p. 53 (Chem. A h . , 1973, 78, 97 820); see also Vol. 3, p. 37 of these Reports. K . Piatkowski and D. Mrozinska, Pruce nauk. Inst. Chem. Org. i Fiz. Politech. Wroduwskiej, 1970, 25 (Chem. Abs., 1973, 78, 97815). K . Piatkowski and D. Mroziriska, Prace nauk. Inst. Chem. Org. i Fiz. Politech. Wroduwskiej, 1970, 37 (Chem. Abs., 1973, 78, 97816).
Mono terpenoids
35
undergo transposition of the acetyl group to the secondary hydroxy when heated in sodium acetate.' 6 9 An introductory stereochemistry experiment for students involves determination of the relative and absolute configurations of (-)-menthol (202) and (+)neomenthol (203) by ( a ) equilibration and (b) reduction of menth~ne.'~' A discussion of columns for the separation of menthol and menthones by gas chromatography includes mention of the problem of interconversion of menthone and isomenthone on various columns. The classical Meerwein-PonndorfVerley reduction of pulegone (204) is known to be inefficient ;however, reduction occurs readily in the presence of a strong base (KOH) in propan-2-01 to yield a 1 : 1 mixture of menthol [( +)-(202)] and neomenthol [( +)-(203)].'72
'
Biological oxidation of piperitone (205) using a Fusarium species, Protoactinomyces roseus, or a local (Australian) species of Aspergillus niger yields in all cases mixtures in which the major component is 7-hydroxymenth- 1-en-3-one (206), accompanied by varying amounts of the 6-hydroxylated compound (207) (particularly with the P. roseus oxidation), and traces of an 8-hydroxylated piperitone (208)' 7 3
A publication by Nagell and Hefendehl 'establishes' the known structure of diosphenolene' 7 4 using much the same reasoning as the unacknowledged original work by Naves.' 7 5 W . Gary, U S . P. 3 676 487. J . Barry, J . Chem. Educ., 1973, 50, 292. D. G . Gillen and J. T. Scanlon, J . Chromatog. Sci., 1972, 10, 729. M . Calas, B. Calas, and L. Giral, Bull. Soc. chim. France II, 1973, 2079. 1 7 3 E. V . Lassak, J. T. Pinhey, B. J . Ralph, T. Sheldon, and J. J . H . Simes, Austral. J. Chem., 1973, 26, 845. "'A. Nagell and F. W. Hefendehl, Phytochemistry, 1972, 11, 3359. Y.-R. Naves, Helv. Chim. Acta, 1966, 49, 2012. lh9
36
Terpenoids and Steroids
Treatment with bromine of the product (209) from the reductive dimerization of (+)-pulegone results in aromatization of one of the cyclohexane rings (210); the stereochemistry of the various isomers has been discussed.' 7 6
(210) X = Br or H
(209)
A study of the alkylation of menthones has been p~blished.'~'It was known that pulegone (204)methylates predominantly in the 4-position, giving (21l), with sodium t-amylate and methyl iodide,'78 but it has now been found that if lithiumsecondary amide bases are used, the major product (212) is derived from the crossconjugated enol (Scheme 9)."'. The full paper on the methylation of (+)pulegone by Cox et a / . 8 o (preliminary communication ref. 178) has confirmed
Reagents: i,
56 "/,
23 %
(212)
(211)
(214)
N(Li)CHMe,-THF, 0 "C; ii, Me1 (excess), 25 " C .
Scheme 9
that ( - )-methylisopulegone (214) has the (1R,4S) configuration* in agreement with findings in the thujane series.'81 A comparison of the c.d. of (-)-methylisoJ. M. Font Cistero, Rev. Real Acad. Cienc. exactas,fis. natur. Madrid, 1972, 66, 455 [Chem. Abs., 1973,78,84 542 includes a n incorrect formula for (150)] Bull. Soc. chim. France I I , 1973, 1049. C. Djerassi, J. Osiecki, and E. J. Eisenbraun, J . Amer. Chem. Soc., 1961, 83, 4433; M. R. Cox, H. P. Koch, W. B. Whalley, H. B. Hursthouse, and D. Rogers, Chem. Comm., 1967,212. R. A. Lee, C. McAndrews, K. M. Patel, and W. Reusch, Tetrahedron Letters, 1973,
"' C. Metge and C. Bertrand,
17'
965.
M. R.Cox, H. P. Koch, and W. B. Whalley, J.C.S. Perkin I, 1973,174. T.Norin, Acta Chem. Scand., 1962,16,640.
* These publications do not use a nomenclature based on the menthane skeleton; this Report uses consistent numbering: thus (2 14) is (1 R,4S)-4-methylmenth-8-en-3-one.
Monot erpenoids
37
pulegone (214) with those of isopulegone and of saturated analogues includes a discussion about the relative dispositions of the double bond and the carbonyl group.*' 8 2 The n-allylpalladium compounds of piperitone (205) and pulegone (204) give the same mixtures of cyano-ketones (e.g. 1-cyanomenth-3-ones from piperitone) as are obtained from the parent crp-unsaturated ketones under similar conditions (KCN-NH,Cl in dimethylformamide at 100"C). The stereochemistry of the piperitone product was not assigned.l 8 Beckmann rearrangement of the oximes of menthone, carvone, and reduced carvones occurs with toluene-p-sulphonyl chloride in aqueous acetone containing sodium hydroxide. The structures and conformations of the lactams obtained have been discussed in the light of their n.m.r. spectra.' 84 Thymoquinone (215 ) reacts with piperylene in the Diels-Alder reaction, but the adduct (216) is not that of a naturally occurring sesquiterpenoid skeleton.'85
rn-Menthanes.-5-Hydroxy-rn-mentha-1,8-diene(217) (among other monoterpenoids) is reported as a constituent of Cannabis sativa resin. The study was an analytical attempt to determine what attracts dogs trained to detect hashish.' 86 However, the compound may not be a naturally occurring rn-terpenoid, but an artefact formed from a carene.
Tetramethylcyc1ohexaes.-The configuration at C-3 of picrocrocin (2 18; R = fl-glucose), the bitter substance of saffron (Crocus sativus) has now been established by correlation with the carotenoid xanthophyll, which was degraded S. Watanabe, Bull. Chem. SOC.Japan, 1973, 46, 1546. C. W. Alexander and W. R. Jackson, J.C.S. Perkin ZZ, 1972, 1601. "'A. Zabia, C. Wawrzenczyk, and H. Kuczydski, Bull. Acad. polon. Sci., Skr. Sci. chim., 1972,20,631 (Chem. Abs., 1972,77, 140 307). J. J . Sims and V. K. Honwad, Tetrahedron Letters, 1973, 2155. E. Stahl and R. Kunde, Tetrahedron Letters, 1973, 2841.
* See footnote o n previous page.
38
Terpenoids and Steroids
to ( - )-(R)-3-methoxy-P-ionone (219). The latter was obtained from picrocrock by saponification to the aglucone (218: R = H), methylation (Ag,O and MeI), and condensation with acetone to (219).18' Sandy sage (Arternisia Jil$olia) has been found to contain two lactones, (220) and (221).lgg Two other compounds, described as 'new' in the abstract, are, in fact, well known (filifolone and 2,6,6trimet hylcyclohex-2-ene-1,4-dione).
A novel cyclization procedure allows exclusively a-cyclocitral(222)to be made in 41 "/;) yield, The enamhe (223) (a mixture of cis- and trans-isomers), prepared from a mixture of the two citral isomers (109a)and pyrrolidine using a molecular sieve, is treated with 90 04 sulphuric acid at 0 "C and the resulting iminium salt (224) is converted (without isolation) into a-cyclocitral (222) by refluxing at pH 3---4.'89 When the same series of reactions is carried out on optically active enamines [ e g (225), obtainable from proli line], an optically active cyclocitral is obtained [(S)-a-cyclocitral (222a) from (2291.' Another type of cyclization
1 /
UHO
R. Buchecker and C. H. Eugster. H r l r . Chirn. Actu. 1973, 56, 1121. S. Torrance and C . Steelink, 166th A.C.S. Meeting, August, 1973, Abstracts A G F D , No. 54. I R 9S. Yamada, M . Shihasaki, and S . Terashima, Tetrahedron Letters, 1973, 377. 19" S. Yamada, M . Shibasaki. and S . Terashima, Tetrahedron Letters, 1973. 381.
I*'
39
Mono terpenoids
involves treatment of methyl geranate (226) with benzenesulphenyl chloride in nitromethane containing silver hexafluoroantimonate, and yields methyl a-cyclogeranate (227)
PhSC1-MeN0,-AgSbF,
In the hope of converting ethyl safranate (228) into a versatile intermediate for the synthesis of carotenoids, Biichi et al. have investigated its oxidation under various conditions. Selenium dioxide in acetic acid causes aromatization with migration of a methyl group to (229), but with dioxan as solvent the skeleton is maintained affording (230). Oxidation of the anion (231) with oxygen yields the mixture shown (Scheme
(231)
(230)
43 %
34 %
3%
Reagents: i, Se0,-HOAc; ii, Se0,-dioxan; iii, KOBu'; iv, 0,; v, heat.
Scheme 10
As usual, the C, substances related to isophorone (232) are included in this Report ; many such substances occur naturally together with safranate-like compounds. The epoxy-alcohol (233)derived from isophorone does not undergo the same ring contraction as unsubstituted cyclohexenol epoxide with lithium bromide in hexamethylphosphoramide.193Of the three possible enol acetates [(234), (235), and (236)] formed from isophorone with isopropenyl acetate, only
19'
192
193
M. T. Mustafaeva, M. Z . Krumer, V. A. Smit, A. V. Semenovskii, and V. F. Kucherov, Izvest. Akad. Nauk S . S . S . R . , Ser. khim., 1972, 2632. G. Buchi, W. Pickenhagen, and H. Wuest, J . Org. Chem., 1972, 37, 4192. G . Magnusson and S. Thoren, J . O r g . Chem., 1973, 38, 1380.
Terpenoids and Steroids
40
two, (234)and (235),undergo a Diels-Alder reaction with a-chloroacrylonitrile to yield bicyclo[2,2,2]octenes, (237) and (238).'94
CI 38 % (237)
62 % (238)
Reduction of the diketone (239)over platinum leads to reduction of the carbonyl group having adjacent methyl groups (240); this paper describes the preparation of a derivative of the other ketol(241; R = tetrahydropyranyl), related to grasshopper ketone.' 9 5
Cr EH
0
0
('39)
1,4-Dimethyl-l-ethylcyclohexanes.-A member of this class of compounds (242) has been found in the oil of Juniperus cornrnunis,but it is not certain that it is truly isoprenoid, since it could arise by biological methylation of a reduced p-methylacetophenone (although the latter could be a degraded isoprenoid, too!).'96
(242)
Iy4 I95
J. Daminao, S . Geribaldi, G . Torri, and M . Azzaro, Tetrahedron Letters, 1973, 2301. K . Mori, Tetrahedron Letters, 1973, 723. A . F. Thomas, Helc. Chim. Acta, 1973, 56, 1800.
Mono terpenoids
41
Cyc1oheptanes.-The Beckmann rearrangement of tetrahydroeucarvone oxime (243) has been di~cussed.'~'Treatment of karahanaenone epoxide (244) with sodium ethoxide results in ring contraction to the cyclohexanol (245), in which assignment of the endo configuration to the hydroxy-group is based on the
assumption supported by n.m.r. of a backside displacement of the epoxide oxygen at C-5 by the anion created at C-7, with inversion at C-5. The dehydration of the alcohol (245) (to the ketones in Scheme 1l), together with other reactions of the same alcohol, are described.' 98
Q
+
y-p=,b, 170
0
OH
(244)
(245)
-
180T
/
OH
KHSO, 170-I80
"C
Scheme 11
Conversion of eucarvone epoxide (246) into the two 1,1,4-trimethylcycloheptane-3,4-diols (247),with a view to examining the properties of the latter, has been described.' 99
197
198
199
A. Zabza, H . Kuczynski, Z . Chabudzinski, and G . Piotrowska, Bull. Acad. polon. Sci., SPr. Sci. chim., 1973, 21, 1 . Y. Gaoni, Tetrahedron, 1972, 28, 5533. Z. Chabudzinski, M. Skwarek, P. Molin, and I. Mielczarek, Roczniki Chem., 1 9 7 3 , 4 7 , 1407.
42
Terpenoids and Steroids
6 Bicyclic Monoterpenoids Bicyclo[3,1,0]hexanes.-Artemi.~iu herbu ulba, a Moroccan species, contains over 70 thuj-3-one (248) in its essential oil. This is the less commonly occurring isomer, called P-thujone by the authors, although there is no doubt which it is.2oo The epoxide of 4-isopropylidenecyclohexanone (249),0” treatment with alcoholic sodium hydroxide, yields a bicyclo[3,1,0]hexane(2%) in over 90 ”/, yield, which
(248)
(249)
(250)
is in principle readily convertible into thujane derivatives.201 Another synthesis of the .ring system involves preparation of the cyclopentanonylmethanol (251) in seven steps from (252); it can then be cyclized with dicyclohexylcarbodi-imide to sabinaketone (253).202
The acid-catalysed hydration of sabinene (254)and a-thujene (255)(Scheme 12) proceeds through a common carbonium ion (256); this work is similar to that of Norin (see Vol. 3, p. 57), but with a slight difference in reaction rate and formation of more menth-l-en-4-01 (257).203 Hach has examined the Meerwein-Ponndorf-Verley reduction of thujone (258) to the thujanols (259),204and converted the latter into the ~innamates.”~ Homoallylic alcohols can be readily dehydrated by photolysis of their thio2oo
202
*04
205
A. Cohen, J.-P. Lavergne, A. Leblanc, and Ph. Viallefont, Bull. S O C .Sci. nut. phys. Maroc, 1972, 52. 1 . Y . Gaoni, Tetrahedron, 1972, 28, 5525. C. Alexandre and F. Rouessac, Bull. Chem. SOC.Japan, 1972, 45, 2241. M . A. Cooper, C . M . Holden, P. Loftus, and D. Whittaker, J.C.S. Perkin If. 1973, 665. V. Hach, J . Org. Chem., 1973, 38, 293. V. Hach and H . G . Higson, Canad. P. 914 214 (the formulae are incorrect in Chem. Abs., 1973, 78,43 770); see also U.S. P. 708 521.
Monoterpeno ids
43
1; (254)
9 (255)
Scheme 12
I
(G93
benzoic 0-esters, although with two of the thujol isomers complex products were obtained.206
Bicyclo[2,2,l]heptanes.-In this Report, after the chemistry of monoterpenoids with this ring system, a section concerning the various rearrangements between bicyclo[2,2,l]heptanes and bicyclo[3,l,l]heptanes is included. The absolute configuration of ( )-fenchone (260) and ( + )-dehydrofenchone (261) using 0.r.d. has been discussed.207 A 3C-labelledcamphene (262)has been made conventionally in order to study its racemization in acid. It was found that there is little endo-methyl migration and tricyclene formation, Wagner-Meerwein 2,6-hydride rearrangement is ca.
+
’06
20’
D. H . R. Barton, M. Bolton, P. D. Magnus, K. G. Marathe, G . A . Poulton, and P. J. West, J . C . S . Perkin I , 1973, 1514. J. Korvola and P. J. Malkonen, Suornen Kern., 1972, B45, 381.
Terpenoids and Steroids
44
41%, and exo-3,2-migration is ca. 53%.208 Results from the dehydration of the camphanols (263) and related primary alcohols with polyphosphoric acid at 185 "C correspond to those from the acid isomerization of camphene, since the latter constitutes 46.5% of the products of the rapid dehydration of the cam-
p h a n o l ~ . ~A' ~kinetic study of the rearrangement in formic acid of cyclofenchene (264) and a-fenchene (265) has been shown to be'very complex (Scheme 13), but a-fenchene (265) is the most important early intermediate from cyclofenchene (264).21 Paasivirta has also examined the reaction of formic acid with tricyclene . , again.21
& (264)
Further products Scheme 13
In the publication about the reaction of phenol with a-fenchene, Gavrilova et al., having come close to plagiarism in earlier papers (see Vol. 2, p. 47), now reproduce exactly the work of Demole.21 208
209 210 21
'12
C . W. David, B. W. Everling, R. J. Kilian, J. B. Stothers, and W. R. Vaughan, J . Amer. Chem. SOC.,1973,95, 1265. E. Degny, F. Petit, M . Evrard, and M. Blanchard, Bull. SOC.chim. France, 1972, 4770. J . Paasivirta and P. Hirsjarvi, Acra Chem. Scand., 1973, 27, 1098. J. Paasivirta, Acra Chem. Scand., 1973, 27, 374. T. F. Gavrilova, I. S. Aul'chenko, and L. Kheifits, Zhur. org. Khim., 1973, 9, 89; see E. Demole, H e h . Chim. Acra, 1964, 47, 1766.
Mono t erpenoids
45
Acid-catalysed rearrangements of the anisyl compounds (266)2l 3 and (267),2l4 together with some other related have been discussed. The competition between Wagner-Meerwein rearrangement and intramolecular electrophilic substitution in the 3-diphenylmethyleneisobornyl system (268) is such that dehydration of the alcohol (268 ; R = H) with toluene-p-sulphonic acid below 60 "C gives only the Wagner-Meerwein products [(269)and (270)]whereas with potassium bisulphate, 2,6-hydrogen shifts and Nametkin rearrangement occur up to 130°C, with further complications at higher temperatures. The acetate (268 ; R = Ac) epimerizes at under 60 "C in acid.2'
P
O
M
e
Q
Earlier work216 on the reaction of N-bromosuccinimide with camphene (271) reported only an inseparable mixture in which the bromocamphene (272) was identified. Now Jefford and Wojnarowski have deduced (based on work with 2-methylnorbornene) that both geometrical isomers of (272)are formed, together with (probably) the tricyclene (273) and (possibly) (274).2l 7 Electrochemical
lL3 'I4 'I5
'I6 'l'
D. L. Adams and W. R. Vaughan, J . Org. Chem., 1972,37,3906. D. W. Kuehl, J. D. Nelson, and R. Caple, J . Org. Chem., 1973, 38, 2723 J. P. Morizur, B. Furth, and J. Kossanyi, J . Org. Chem., 1973, 38, 2698. J. D. Roberts and E. R. Trumbull, J . Amer. Chem. Soc., 1949,71, 1630. C. W. Jefford and W. Wojnarowski, Helv. Chim. Acta, 1972, 55, 2244.
46
Terpenoids and Steroids
reduction of the dibromobornanes (275)and (276)leads, in the case of the di-endoproduct (275), to tricyclene (277) as the primary product and bornane (278) as a secondary product, obtained uia the monobromide (279), which appears in the early stages of the reaction. The em-dibromide (276),formed by bromination of camphene, gives camphene (271) on electrochemical reduction [by solvolysis of the monobromide intermediate (280)], together with bornane (278), by stepwise
Br
Br
reduction of the bromine atoms.218 Formation of a tricyclene derivative (281) also occurs when 3,3-dibromocamphor (282) is reduced with diethylzinc, the initial camphor carbenoid formed (283)acting as an efficient internal trap (Scheme 14).2l 9 The reaction of the tricyclenone (281) with lithium dialkylcuprates leads to addition (284; R = Me) in the case of the dimethylcuprate, and reduction (284 ; R = H) with the di-n-butylcuprate.220
Scheme 14 2'k 'Iy
22v
Azizullah and J . Grimshaw, J.C.S. Perkin I , 1973, 425. L. T. Scott and W. D. Cotton, J. Amer. Chem. Soc., 1973, 95, 2708. L. T. Scott and W . D. Cotton, J.C.S. Chem. Comm., 1973, 320.
Monot erpenoids
47
Some further comments about the nitrous acid deamination of endo-fenchylamine have appeared.221 1 -NN-Dichloroaminoapocamphane (258),prepared by the action of t-butyl hypochlorite on 1-aminoapocamphane, yields the ringopened compounds of Scheme 15 on reaction with aluminium chloride at -75 "C, with only 10 % of the expected chloroamine (286).222
Scheme 15
The mechanism of the oxygen scrambling in the pyrolysis of benzoyl l-apocamphyl carbonate (287) has been discussed.223
H (289)
Borneo1 reacts with chlorodiphenylmethylium ion in t-butyl cyanide to give, by quenching after 5 min reaction time, the t-butyl amide (288 ; R = But). If, on the other hand, acetonitrile is added, the acetamide (288 ; R = Me) is obtained in addition, these reactions occurring via the oxonium ion (289).224The rearrangements occurring on pyrolysis of 10-isobornyl sultone (290), leading to endo- (291) and exo- (292) camphene sultones have been investigated.225 Pyrolysis of
*" 222
223
224 225
C. J. Collins and B. M. Benjamin, J. Org. Chem., 1972, 37, 4358; CJ W. Huckel and H.-J. Kern, Annalen, 1969, 728, 49. R. D. Fisher, T.D. Bogard, and P. Kovacic, J. Amer. Chem. SOC.,1972,94, 7599. S. Oae, K. Fujimori, and Y. Uchida, Tetrahedron, 1972,28, 5321 ; S. Oae, K. Fujimori, and S. Kozuka, ibid., p. 5321. D. H. R. Barton, P. D.Magnus, and R. N.Young, J.C.S. Chem. Comm., 1973, 331. D. R. Dimmel and W. Fu, 165th A.C.S. Meeting, April, 1973, Abstracts O R G N , No. 92. From this abstract, it is difficult to see the novelty beyond J. Wolinsky, D. R. Dimmel, and T. W. Gibson, J . Org. Chem., 1967, 32, 2087.
Terpenoids and Steroids
48
2-exo-bromo-2-endo-nitrobornane (293) gives 15% of the cyclopentene (294) and ca. 2 % of endo-bromocamphor (295).226 The proposed mechanisms, i.e. initial formation of campholenic nitrile (296) [the main product from pyrolysis at 300 “ Cin a sealed tube for 2 h of 2,2-dinitrobornane(297)],followed by bromination, would benefit from additional support.
15
+
Br
+ CN
H
Camphor deuteriated in the 9-methyl group (298) has been prepared.227The + )-camphorato]lanthanide(~r~)agents chiral tris-[3-t-butylhydroxymethylene-( LnT, are dimeric in dry CC14 solution for Ln = Pr, Nd, and Sm, at concentrations commonly used in n.m.r. shift work, unlike the later members of the series and unlike the dpm and fod shift agents.228
(298) R = CD, (299) R = Me
(300)
(3011
(302)
(303)
If any one of the three ketones camphor (299), endo-isocamphanone (300), or exo-isocamphanone (301) is heated at high temperatures with potassium tbut oxide in t-butyl alcohol all three are obtained, because of homoenolization [via the enolate (302)l. Homoenolization is also responsible for the fact that strong base-catalysed exchange of fenchone (303) (t-butyl [2H]alcohol and 226 ”’ 228
S. Ranganathan and H . Raman, Tetrahedron Letters, 1973, 41 1 . R. N. McCarty, Diss. Abs. ( B ) , 1973, 33, 3558. R. G . Denning, F. J. C. Rossotti, and P. J . Sellars, J . C . S . Chem. Comm., 1973, 381.
Mono terpenoids
49
potassium t-butoxide at 185°C) results in exchange of the protons at C-6 and
c-8.229 The long-known rearrangement of camphor to 3,4-dimethylacetophenone in sulphuric acid has received fresh attention. When camphor was specifically labelled at C-8 and C-9, the distribution of label in the acetophenone was as shown in Scheme 16.230 These findings were explained by a series of complex rearrangements, including an endo-2,3 hydroxyl shift, which the authors were reluctant to accept. Using a computer programme allowing enumeration of all possible intermediates in this reaction, it has been found that the observed results can be explained without this unprecedented rearrange men^'^
76 %
\J*
FOMe
0 Scheme 16
Reduction of (lR)-3-endo-aminocamphor(304) with aluminium chloride and tri-isobutylaluminium (i.e. AlC1,H) gives a 74.5 % yield of the endo-aminoborneol (305)rather than the i ~ o b o r n e o l ,reduction ~~~ from the em-side being the usual mode in the presence of the a m i n o - g r ~ u p .The ~ ~ ~mechanism of the
229
230 231
232 233
D . H . Hunter, A. L. Johnson, J . B. Stothers, A. Nickon, J. L. Lambert, and D. F. Covey, J . Amer. Chem. SOC.,1972, 94, 8582. 0. R. Rodig and R. J. Sysko, J . Amer. Chem. SOC.,1972,94,6475. C. J. Collins and C. K. Johnson, J . Amer. Chem. SOC.,1973, 95, 4766. H. Pauling, Ger. Offen. 2 153 819 (Chem. Abs., 1972, 7 7 , 114 599). A. H. Beckett, N . T. Lan, and G . R. McDonough, Tetrahedron, 1969, 25, 5689; A. Daniel and A. A. Pavia, Bull. SOC.chim. France, 1971, 1060.
50
Terpenoids and Steroids
dehalogenation of 3-bromocamphor by dimethylaniline has been shown to involve abstraction of a Br+ ion rather than thermal formation of bromine In order to prepare a molecule in which optical activity would arise only by virtue of replacement of l6O by l8O, and thereby shed light on the argument concerning the sense of twist in a-diketones (see Vol. 3, p. 68), Kokke and Oosterhoff have carried out a series of reactions on fenchone, shown in Scheme 17. The compound obtained showed a small but measurable effect in the c.d. of both low-intensity absorption bands in the region 25&520 nm.235
liv
Reagents: i , NH,OH,HCl; ii, Na-EtOH; iii, HNO,; iv, RuO,; v, N , H , ; vi, H , 1 8 0 - H + ; vii, Se0,-Ac,O.
Scheme 17
The arylidefle-epicamphors (306) exist in both stereoisomeric forms, the amount of each depending on the aryl substituent. Thus when aryl = Ph, only the E-isomer is present (see Vol. 3), but an o-chloro-substituent results in 10%of the 2-isomer and p-chloro in 80% of the Z - i ~ o r n e r . ’ ~ The ~ action of diazonium salts on 3-acyl- or 3-aroyl-camphors (307) results in initial endo attack (308), the other isomer (309) being formed on heating the first (308) in The action of phenylmagnesium bromide on camphor oxime (310) results in the cyclohexenylnitrile (311) (the nomenclature of which is incorrect in the paper) ; the mechanism is deduced to be as shown, since one deuterium atom from 3,3-dideuteriocamphor is removed in the reaction.238 The well-known Beckmann rearrangement of camphor oxime (310) has been re-examined using sodium hydroxide in aqueous acetone and toluene-p-sulphonyl chloride as catalyst. Camphenylone oxime (312) behaves in a similar way and yields ring-opened nitriles, together with ca. 30% of the amide (313). Fenchone oxime [probably having the oxime group syn to the gern-dimethyl group (31411 did not rearrange 234
2’5
236 23’
’”
A. G. Giumanini, ‘Proceedings of the International Symposium on Gas Chromatography Mass Spectrometry’, 1972, ed. A. Frigerio, p. 377 (Chem. Abs., 1973, 78, 124 736). W . C. M . C. Kokke and L. J . Oosterhoff, J . Amer. Chem. Soc., 1972,94, 7583; W. C. M . C. Kokke, J . Org. Chem., 1973, 38, 2989. F. Labruyere and C. Bertrand, Compr. rend., 1972, 275, C , 673. J.-C. Guillaumon, F. Labruyere, C. Metge, and C. Bertrand, Compt. rend., 1973, 276, c, 1 1 1 1 . R. Chaabouni and A. Laurent, Tetrahedron Letters. 1973, 1061.
Mono terpenoids
&
qR2 (306) R'
R'
R' aryl, R 2 = H = H, R 2 = aryl
COAr'
&COAr
Ar'N,;
OH
(307)
=
51
N=N \ Ar2
0 (308)
A-EtOY
v
Ar
/
0
AH
3PhMgB4 NOH
--+
&H CN
O
C
N
N-bOMgBr
(3 10)
(31 1)
& -+Go& N
NOH
'OH (312)
(313)
(314)
under these conditions.239 Beckmann rearrangements of the oxime (3 15) (available from camphor) and its syn- and anti-toluenesulphonates have been discussed by Fleming and Woodward, who used the lactam (316) to prepare methyl cis-P-(3-diazo-1,2,2-trimethylcyclopentyl)acrylate (317).240 The latter compound is of interest because in a dilute solution of sodium methoxide in methanol the major product is the bicyclo[2,2,l]heptene ester (318),241 but the reaction does not appear to be
239
240 241 242
A. Zabza, C. Wawrzenczyk, and H. Kuczynski, Bull. Acad. polon. Sci. SPr. Sci.chim., 1972, 20, 623. I. Fleming and R. B. Woodward, J.C.S. Perkin I , 1973, 1653. E. H. Billett and I. Fleming, J.C.S. Perkin I , 1973, 1658. E. H. Billett, I. Fleming, and S. W. Hanson, J.C.S. Perkin I , 1973, 1661.
52
Terpenoids and Steroids
3-Dimercaptomethylenecamphor (319) can be converted into a series of polysulphides (320), the configurations of which have been studied using dipole moment
SH (319)
6)" (320)
Irradiation of thiofenchone (321) or thiocamphor (322) gives the sulphur analogues of homoenols, (323) and (324), desulphurization with Raney nickel yielding the corresponding tricyclenes. Action of heat on the 'homothioenols' (323) and (324) results in homoketonization back to the starting materials (321) and (322), accompanied in the thiofenchone (323) case by 40% of endo-isothiofenchone (325).244 The action of Grignard reagents on thiofenchone (321) pro-
h
S
(322)
duces the radical (326), which is visible spectrally for several hours (the thiogroup is apparently endo);the products of the reaction are then the thiol(327) and the thioether (328). Thiocamphor (322) behaves similarly (although the radical is less stable) with higher Grignard reagents, but the thioenol ether (329) is formed additionally, and is, indeed, the major product with methylmagnesium bromide.245 A somewhat similar difference between thiofenchone (321) and the enolizable thiocamphor (322) is manifest in their behaviour with dimethyl-
SR (326) 243 244 245
SH (327)
SR (328)
3. Sotiropoulos and A.-M. Lamazouere, Compt. rend., 1973, 276, C,1 1 15. D. S . L. Blackwell and P. de Mayo, J.C.S. Chem. Comm., 1973, 130 M . Dagonneau, D. Paquer, and J . Vialle, Bull. SOC.chim. France I I , 1973, 1969; M. Dagonneau and J. Vialle, Tetrahedron Letters, 1973, 3017.
Mono terpeno ids
53
sulphoxonium methylide, thiofenchone giving the thiirans (330), and thiocamphor the S-methyl-enethiol (329; R = Me).246 The thiirans (330) are 65% of the S-endo-isomer and 35 % of the S-exo-isomer, 2-Diazopropane yields only the thermal decomposition product (33 l).247
The reaction between diazomethane and the intermediate sulphene produced by treatment of (1s)-camphor-10-sulphonyl chloride with triethylamine gives a thiiran dioxide;248 both isomers of this compound (332) have now been ~haracterized.~~’ Notwithstanding the work of Kirmse (see Vol. 3, p. 60), the conversion of bornanes into pinanes has not been easy. Following Scheme 18, Paukstelis and Macharia have converted camphor (299) into nopinone (333). The vital step, rearrangement of the mesylate (334), depends on the fact that the migrating bond must be anti-periplanar to the mesylate group. The scheme shows the best conditions found for making nopinone [in 40% yield from the known 1-trichloroacetoxycamphene (335)], but several other routes to (334) were considered and two others were used.250 The same authors have converted camphor (299) via the known chloro-alcohols (336) into the bicyclo[2,l,l]hexane system (337). 2 5 1 As usual, most of the literature on pinane-bornane rearrangements concerns passage from pinanes to bornanes. The Wagner-Meerwein rearrangement finds new examples every year ; thus verbenone (338) yields 6-chloroepicamphor (339) with gaseous hydrogen chloride, and the chloroepicamphor can be converted into bornane-2,Sdione (340) ; 2 5 2 chrysanthenone (341) behaves ~imilarly.~ 53 Nopadiene (342) or its isomer homoverbenene (343) gives a variety of dichlorohomobornanes (Scheme 19), but it is curious that the maleic anhydride adduct of despite the fact that it nopadiene (344) does not react with hydrochloric does react with hydrogen bromide (see Vol. 1, p. 46),255and the corresponding ester (345 ; R = C0,Et) and alcohol (345 ; R = CH,OH) react normally.254 Wolinsky has already reported the reaction of camphene with sulphur trioxide (see above), and now finds that a-pinene reacts with sulphur trioxide to yield a 246 247
248
249 250 251 252
25 254 255
D . Lecadet, D . Paquer, and A. Thuillier, Compt. rend., 1973, 276, C , 875. J . M. Beiner, D . Lecadet, D . Paquer, A. Thuillier, and J. Vialle, Bull. SOC.chirn. France ZI, 1973, 1979, 1983. N. Fisher and G . Opitz, Org. Synth., 1968, 48, 106. T. Kempe and T. Norin, Acta Chern. Scand., 1973,27, 1452. J. V. Paukstelis and B. W. Macharia, Tetrahedron, 1973, 29, 1955. J. V. Paukstelis and B. W. Macharia, J . Org. Chem., 1973, 38, 646. 0. A. Arpesella, D . I. A. De Iglesias, and J. A. Retamar, Essenze Deriu. Agrurn., 1972, 42, 48 (Chem. Abs., 1972, 77, 164 852). D. J. Merep and J. A. Retamar, Anales SOC.cient. Argentina, 1972, 193,3. B. Bochwic and S. Markowicz, Roczniki Chem., 1973,47, 1083. C. Arcupas and W. S. Roach, Chem. Comm., 1969, 1468.
Terpenoids and Steroids
54
&+&
c1,ccoo
PhC'H,O
,O
I
P h C H&H ,O
0
OH
ii
-t- P h C H & Hl O
OH
(334)
(333)
Reagents: i, NaH-PhCh,Cl; ii, NaBH,; iii, MsC1-py; iv, Pd/C-H,; v, Bu'OK-Bu'OH.
Scheme 18
(299)
5
jf$)
++ $ - $H
+ Hof/$ H
HO (336)
~N~oH-DMF
CHo (337)
(338)
(339)
CO,H
(340)
(341)
55
Mono terpeno idi
(343)
6 'Q"1 n
/ HCI
+ HCI
+ cl..&H2c'
(342)
R
R (344)
(345)
Scheme 19
so2-0 (346)
bornane derivative (3461, albeit in only 7.3 % yield.256 The reaction of chlorosulphonyl isocyanate with a-pinene (347)4072 leads ultimately to a similarly bridged bornane (348) at room temperature, the four-membered-ring adduct (349)being formed at - 70 "C.
''
4
SO2CI
CISO,NCO+ - 70 "C
&O
+ 4
(347)
(349)
&
0
/
CISO,
(348) 256
*"
J . Wolinsky, R. L. Marhenke, and E. J. Eustace, J . Org. Chem., 1973, 38, 1428. G . T. Furst, M . A . Wachsman, J. Pieroni, J. G. White, and E. J. Moriconi, Tetrahedron, 1973,29, 1675.
Terpenoih and Steroids
56
The conversion of the pinane ether (350) into fenchane glycol derivatives (351 ; = Ac or Ts, R2 = Ts or Ac) in the presence of the mixed toluene-p-sulphonicaceticanhydride reagent(see Vol. 3,p. 64)has been thesubject o f c o n t r ~ v e r s y ? ~ ~ ~ ~ ~ ~ Bosworth and Magnus have further developed the products from this type of reaction for the preparation of cyclopentanes suitably substituted for possible sesquiterpenoid syntheses [e.g. (352)].260
R'
Bicyclo[3,1,l]heptanes.-For the first time trans-chrysanthenyl acetate (353) has been described ; it was isolated, together with chrysanthenone (353), from Chrysanthemum shiwogiku, 2 6 1 and also from C .japonese var. debile.262
(353)
(354)
(355)
(356)
The structure of /3-pinene (354), as determined by electron diffraction, agrees with the results of conformational calculations.263 The acid-catalysed rearrangement of the pinenes is thought to start by proton attack on the double bond rather than on the cyclobutane ring,264 and a mechanism for the dimerization of a-pinene with phosphoric acid to give (355) starts similarly.265 A study of the catalytic hydrogenation of pinene derivatives has been made.266 The hydroboration of a-pinene to isopinocampheol(356) in 85 % yield has now appeared in Organic Syntheses.267 Oxidation of P-pinene with oxygen in the
'" 259
O'' 16'
262
263 264
265
266 267
N. Bosworth and P. D. Magnus, J . C . S . Perkin I, 1972,943. C. Grison and Y . Bessiere-Chretien, Bull. Soc. chirn. France, 1972, 4570. N . Bosworth and P. D . Magnus, J . C . S . Perkin I, 1973, 76. A. Matsuo, Y . Uchio, M. Nakayama, and S. Hayashi, Bull. Chem. SOC.Japan, 1973,46, 1565. A. Matsuo, M . Nakayama, T. Nakamoto, Y . Uchio, and S. Hayashi, Agric. and Biol. Chem. (Japan), 1973, 37, 925. V . A. Naumov and V. M. Bezzubov, Zhur. strukt. Khirn., 1972,13,977. G. A . Rudakov and L. S. Ivanova, ref. 92, p. 285 (Chern. Abs., 1973,78, 124 737). N . K . Roy, B. S. Rathore, and G. B. Butler, J . Indian Chem. Soc., 1972, 49, 1221. W. A. Boyd, D i s s . Abs. (B), 1973, 33,4185. G . Zweifel and H . C . Brown, Org. Synth., 1972,52, 59.
Mono terpeno iak
57
presence of t-butyl nitrite gives a mixture of pinocarvone (357) and the pinocarveols (358),this reaction being effected by the peroxynitrite radical, Bu'N
/ \
0 0-0
not by singlet oxygen.268 Using t-amyl hydroperoxide in the presence of hexacarbonylmolybdenum, the oxidation of a-pinene yields epoxides at 40 "C, and a
(357)
(358)
mixture of hydroxypinocamphone (359) and campholenic aldehyde (360) (presumably from an a-pinene epoxide intermediate) at 80 0C.269 Chromyl chloride oxidation of or-pinene yields270a complex mixture containing products of acid-catalysed rearrangement (bornyl chloride, borneol, dipentene), allylic
(359)
(360)
oxidation (myrtenol, myrtenal, verbenone), and oxidative addition and rearrangement [pinocamphone, pinocarveol, campholenic aldehyde, and pinol (36 l)]. Anodic oxidation of a- and fl-pinenes in acetic acid or methanol, with tetraethylammonium toluene-p-sulphonate as the supporting electrolyte, yields p-menthane
derivative^.^^ '
Ozonolysis of a-pinene gives a small amount of isomeric crystalline diperoxides, one isomer of which (362) is readily purified.272 Sabinene (254) gives a similar diperoxide of m.p. 154 0C.273
268
269 270
2'2
273
J . A. Maassen and Th. J. de Boer, Rec. Trau. chim., 1972, 91, 1329. G. A. Tolstikov, U . M. Dzhemilev, and V. P. Yur'ev, Zhur. org. Khim., 1972,8, 1190. F. W. Bachelor and U. 0. Cheriyan, Canad. J . Chem., 1972,50,4022. T. Shono and I. Ikada, J . Amer. Chem. SOC.,1972,94,7892. K . H. Overton and P. Owen, J . C . S . Perkin I , 1973, 226. A. F. Thomas, unpublished work.
Terpenoih and Steroids
58
Further work about the radical additions to p-pinene includes the use of ethylene glycol derivatives. Ethylene glycol itself does not react, possibly because of poor miscibility, but the diacetate and other derivatives give the usual 7substituted m e n t h - l - e n e ~ , ~as’ ~do d i ~ x a l a n s . ~The ~ ’ main products from the radical-initiated addition of t-butyl hypochlorite to a-pinene are myrtenyl (363) and verbenyl (364) chlorides, together with 10% of 2,6-dichlorobornane and about 10% of other d i ~ h l o r i d e s . ~ ~ ~ A conventional method277for the preparation of 6-pinene (365) in 36 % overall yield from cr-pinene requires the preparation, first, of apopinene (366), then reaction of the latter with N-bromosuccinimide, followed by Grignard coupling of the bromide (367). MeMgBr
I LI 2cucI,)
+
(366)
(367)
@.. .
(365)
The reaction between the pinenes and iodine azide has been published once by Bochwic and O l e j n i c ~ a k ,and ~ ~ twice (almost identical papers) by Ranganathan et a1.279 Using acetonitrile as solvent, a-pinene yields a mixture of ring-opened
Scheme 20 273 275 276
2”
278 2’9
M. Cazaux, B. Maillard, and R. Lalande, Compt. rend., 1972, 275, C , 1133. B. Maillard, M. Cazaux, and R . Lalande, Bull. SOC.chim. France I I , 1973. 1368. I. Uzarewicz and A. Uzarewicz, Roczniki Chem., 1973, 47, 921. D. Joulain, C. Moreau, and M. Pfau, Tetrahedron, 1973, 29, 143. The preliminary communication was submitted one month later than the full paper, to J.C.S. Chem. Comm., 1972, 1 1 10. B. Bochwic and B. Olejniczak, Roczniki Chem., 1973,47, 315. S . Ranganathan, D. Ranganathan, and A. K. Mehrotra, Tetrahedron Letters, 1973, 2265 : Synthesis, 1973. 356.
Mono terpeno ids
59
products, particularly the tetrazole (368), the acetonitrile taking part in the reaction as in Scheme 20. In addition to giving the 7-azido-isomer of the tetrazole (368), fi-pinene yields bornanes ; in dimethylformamide solution, the bornane (369) is the sole product in the reaction of c r - ~ i n e n e . ~Azides ’~ are also produced in the reaction of the pinenes with lead acetate-azide, the initial product (370) from pinene rearranging at 6 0 ° C in acetic acid to the verbenyl azide (371).280 Y
N3 I (369)
I370)
(37 1)
(372)
\\
tY
N-N
(373)
With this reagent, /?-pinene always gives skeletal-rearrangement products, 45 % being the azide (372), with 14 % of the tetrazole (373).280
(374)
(375)
COR (376)
0-
0-
Further work on the addition of carbenes to pinenes includes the full paper of an earlier note (see Vol. 2, p. 50) that reported that trans-6-pinene (365) does not react with carbene, whereas the cis-isomer gives only insertion to (374); orthodene (375) and apopinene (366) behave normally.28 The dibromocarbene reaction products with the pinenes have been described for the fourth (?) time!282 Hatem and Waegell have succeeded in removing a single chlorine atom from the dichlorocarbene adduct of a-pinene without the earlier ring-opening (see Vol. 2, p. 50).283 280 281 282
283
A. Stiitz and E. Zbiral, Annalen, 1972, 765, 34. See also the earlier reports quoted in Vol. 3, Part I, Ch. 1 , refs. 273 and 355. D. Joulain and F. Rouessac, Bull. SOC.chim. France I I , 1973, 1428. G . Mehta and S. C. Narang, Indian J . Chem., 1972, 10, 1057. J. Hatem and B. Waegell, Tetrahedron Letters, 1973, 2019, 2023.
Terpenoidr and Steroids
60
The reductive cyclopropane ring-opening of carbene addition products (376) of the pinene ketones occurs with fission of the ring in such a way that there is maximum overlap with the carbonyl n-orbitals, and, in the absence of steric factors, the most stable carbanion is formed. Thus with (376; R = Me), there is 25% formation of (377; R = Me) and 75% of (378; R = Me), but the proportion changes with increasing steric requirements of R until with (376; R = But) the sole product is (377; R = B u ‘ ) . ~ ~ ~ The reaction of P-pinene with an enophile should be stereoselective if it occurs via a cyclic transition state, approach from the methylene-bridge side being strongly favoured. In the reaction with benzene, 2-a-deuterio-P-pinene (379) transfers deuterium to phenyl (380) to the extent of at least 95%.285
(380)
(379)
(38 1)
The stereochemistry of a number of oxygenated pinanes has been discussed in the light of n.m.r. measurements286and 0.r.d. and ~ . d . ” Thioverbenone ~ (381) can be prepared in 5 5 % yield by treating verbenone (338) with a mixture of hydrogen sulphide and hydrogen chloride at 0 0C.288Electrochemical reduction of apoverbenone (382) leads to dimerization, the dimer formed by reaction on the less hindered side of both halves (383) predominating; some dimer (384) is formed from a less hindered-more hindered combination, but none where both halves involve the more hindered side.*”
+y]=t
?-iJ O H
(382)
(383)
(384)
The selenium dioxide oxidation of chrysanthenone (341) leads to chrysanthenonal (385),253the vinyl methyl group being oxidized as in the case of ver284 285
286 2a’ 288 z89
M. M. El Gaied and Y. Bessiere-Chretien, Bull. Soc. chim. France 11, 1973, 1351. R. T. Arnold, D. Koster, and V. Garsky, 165th A.C.S. Meeting, April, 1973, Abstracts ORGN No. 6. T. Hirata, Bull. Chem. SOC.Japan, 1972, 45, 3169. T. Hirata, Bull. Chem. SOC.Japan, 1972, 45, 3458. P. Metzner and J. Vialle, Bull. SOC.chim. France, 1972, 3138. J. Grimshaw and H. R. Juneja, J . C . S . Perkin I , 1972, 2529.
Mono terpenoids
61
benone (338) (see above). Per-acid oxidation of chrysanthenone (341) takes place from the side opposite to the gem-dimethyl group, leading to the trans-epoxide (386).290 The earlier preparation of this epoxide did not specify the stereochemistry.291 In view of these recorded precedents, it is surprising that Bachelor and Cheriyan had hoped to cleave the cyclobutane ring in a Baeyer-Villiger reaction of ~ h r y s a n t h e n o n e . ~ ~ ~ When pinocarveol (358) is heated with ethyl vinyl ether in the presence of phosphoric acid, the rearrangement product (387) is isolated directly.293 CHO
(385)
/
(386)
F C H O
(387)
Pyrolysis of 2-oxygenated pinanes is well known to give a variety of products, mostly ring-opened, but if the acetate is pyrolysed in pyridine, only c(- and ppinenes are The full paper on the pyrolysis of verbanone has appeared.295 A novel synthesis of the bicyclo[3,3,l]nonane system involves a Cope reaction of the enol of the piny1 ketone (388 ;R = Me). This reaction occurs when the ketone (388; R = Me) is heated above 15OoC,but at higher temperatures (above 200°C) a retro-Claisen rearrangement leads to the vinyl ether (389) (Scheme 21). The corresponding aldehyde (388; R = H) gives only the retro-Claisen reaction. 96 A reinvestigation of the reaction of pinane-2,3-dio1(390)with acetic anhydride has shown that the products are all ring-opened, trans-carveyl acetate (391) and trans-sobrerol mono- and di-acetates (392) constituting the majority.297 on the photoisomerization of cisSome anomalies in the earlier verbanone (393)were the incentive for a study which confirmed that there are two products, (394) and (395), formed in the ratio of 7 : l.299
290
291 292
293 294
295
296
297 298 299
B. A. Arbuzov, A. N. Vereshchagin, N. I. Gubkina, I . M. Sadykova, and S. G. Vul'fson, Izvest. Akad. Nauk S . S . S . R . , Ser. khim., 1972, 1288 (Chem. Abs. 1972, 77, 101 911 gives an incorrect structure for the epoxide). Y . Bessiere-Chretien and J.-A. Retamar, Bull. SOC.chim. France, 1963, 884. F. W. Bachelor and U . 0. Cheriyan, 166th A.C.S. Meeting, August 1973, Abstracts AGFD, No. 30. J. B. Hall, Ger. Offen. 2 065 172. T. N. Pisareva, L. S. Ivanova, A. G . Borovskaya, and G. A. Rudakov, Izvesf. nauch.issled. Inst. Nefte-Uglekhim. Sin. Irkutsk. Uniu., 1969, 11, 41 (Chem. Abs., 1972, 77, 152 361). J. M. Coxon, R. P. Garland, and M. P. Hartshorn, Austral. J. Chem., 1972, 25, 2409. Y . Bessitre-Chrktien and C. Grison, J.C.S. Chem. Comm., 1973, 549. There is a precedent for Cope rearrangement of an enol: S. J. Rhoads and C. F. Brandenburg, J . Amer. Chem. SOC.,1971,93, 5805. Z. Rykowski and M. Skwarek, Roczniki Chem., 1973, 47, 1555. T. Matsui, Tetrahedron Letters, 1967, 3761. A. G . Fallis, Tetrahedron Letters, 1972, 4573.
62
Terpenoids and Steroids OH e
\
OH
1
0
A (390)
(391)
koR (392)
R
=
H or Ac
,CHO
(393)
(394)
(395)
Treatment of pinonic acid (396) with 50% sulphuric acid leads to the lactone (397).300 Other pinonic acid derivatives (398) have been treated with methyllithium, and a number of other substituted cyclobutanes were prepared from the alcohols (399) thus obtained.301 300
F. Avotins and I . E. Savochkina, Laro. P . S . R . Zinat. Akad. Vestis, Kim. Ser., 1972, 483 (Chem. Abs., 1973,78,4558).
301
K . P. Sivaramakrishnan, L. H. Brannigan, and C. S. Marvel, J . Org. Chern., 1972, 37, 4206.
Mono terpeno ids
63 ,COMe
Work already well documented that has received further attention includes the rearrangement of ( + )-2-hydroxypinocamphone in oxalic acid302 and the ring contraction of cis-pinane-cis-2,3-di013-tosylate~~~ (see Vol. 2, p. 54 for both these reactions).
Bicycl~4,1,O]heptanes.--The incorrect numbering of the carene skeleton perpetrated until recently by Russian authors and frequently by Chemical Abstracts (both following logical rather than international numbering) penetrated into Volume 3 (see Errata). The reaction of car-3-ene (400) with lead tetra-acetate leads to a mixture of ringopened products, mainly (4O1J3O4
Further investigation (see Vol. 2, p. 56) of the ring-opening of car-3-ene a-epoxide (402)with hydrogen chloride shows that the stepwise low-temperature reaction gives the two chlorohydrins (403) and (404),which subsequently undergo cyclopropane ring-opening to give the corresponding menth-8-ene chlorohydrins, 302
303
304
T. Hirata, J . Sci. Hiroshima Univ., Ser. A-2, 1971,35, 239; T. J. De Pascual, I . Sanchez Bellido, and M . Grande Benito, Anales de Quim., 1973,69, 217. T. Hirata and T. Suga, Yuki Gosei Kagaku Kyokai Shi, 1972, 30, 1050. A publication omitted from earlier Reports is R. C. Carlson and J. K. Pierce, Tetrahedron Letters, 1968, 621 3 , who described this ring contraction independently of the Japanese work. B. A. Arbuzov, V . V. Ratner, and Z . G . Isaeva, Izvest. Akad. Nauk S . S . S . R . , Ser. khim., 1973, 45.
Terpenoids and Steroids
64
together with some m-menthene c h l ~ r o h y d r i n . ~Ring-opening ~' of the epoxide (402) with acetyl bromide in acetic anhydride gives mostly the cis-adduct (405), with minor amounts of trans-adduct (406).305 Treatment of this cis-adduct (405) with alcoholic potassium hydroxide yields the three substances (407), (408),and (409) in the ratio 3 : 1 : 5,305 whereas similar treatment of the trans-adduct gives car-3-ene fl-epoxide (410).307These reactions are summarized in Scheme 22.
+
..OH
+
4--
Scheme 22
Acid-catalysed hydration of car-3-ene a-epoxide (402) yields a diol of m.p. 137 " C ,previously assigned the structure caran-3P,4fl-diol (411). The latter has now been made by several routes, e.g. mercuration-demercuration of the acetates of alcohols (408)308or (409),309or directly from car-3-ene by treatment with silver 305
306
307
308
3 09
B. A. Arbuzov, Z. G. Isaeva, G. Sh. Bikbulatova, and N. I . Semakhina, Doklady Akad. Nauk S.S.S.R., 1972,207,853. I n this paper, Arbuzov draws attention to the I.U.P.A.C. convention for numbering the carane ring. B. A. Arbuzov, E. Kh. Kazakova, and Z. G. Isaeva, Izvest. Akad. Nauk S.S.S.R., Ser. khim., 1972, 1681. Z . G. Isaeva, E. Kh. Kazakova, and R. R. D'yakonova, Sbornik Nekot. Probl. Org. Khim., Muter. Ncuch. Sess., Inst., org.$z. Khim. Akad. Nauk S . S . S . R . ,ed. A. N. Vereshchagin, 1972, p. 40 (Chem. Abs., 1973, 7 8 , 30007). B. A. Arbuzov, Z. G. Isaeva, R. R. D'yakonova, V. A. Shaikhutdinov, and E. Kh. Kazakova, Izvest. Akad. Nauk S . S . S .R., Ser. khim., 1972, 1680. B. A. Arbuzov, V. A. Shaikhutdinov, and Z. G. Isaeva, Izvest. Akad. Nauk S.S.S.R., Ser. khim., 1972, 2124.
65
Monoterpenoids
acetate and iodine in aqueous acetic acid (followedevery time by hydrolysis of the acetates); the diol (411) has m.p. 42 0C.308 The structure was confirmed by inverting the configuration at C-4 of the diol of m.p. 89-90 "C (412) by acetolysis of the tosylate of (412), giving (411) and the bicyclo[3,1,0]hexane (413).309 Car3-ene epoxides (402) and (410) are also opened by sodium bisulphate or sodium
sulphate. Bisulphate yields only glycols, but sulphate yields a mixture containing 27 % of the sulphonate (414) from the or-epoxide (402) and 64 % of the sulphonate (415) from the P-epoxide (410).310
+ (412)
+
+
(411)
A further communication about the stereochemical course of the photolysis of 3-methylcar-4-en-2-one (416) (see Vol. 3, p. 83) in the presence of methanol, leading to (417),confirms that the earlier course311is correct, but only because the stereochemicalassignments for the methyl groups in both educt and product must be rever~ed.~ * Beckmann rearrangement of the caranone oximes (418) and (419) leads, respectively, to the amides (420) and (421), using toluene-p-sulphonyl chloride in basic 310
311
*
E. MySliriski and E. Michal'ek, Roczniki Chem., 1973, 47, 2 8 5 . These authors seem to have been unaware of Arbuzov's work308 and have the configurations of two of the carandiols (of m.p. 137 and 30 "C-the latter probably the same as Arbuzov's glycol of m.p. 42 "C) opposite to those of the Soviet workers. J. E. Baldwin and S. M. Krueger, J . Amer. Chem. Soc., 1969, 91, 2396. A. J. Bellamy and W. Crilly, J . C.S. Perkin 11, 1973, 122.
Terpenoids and Steroids
66
aqueous acetone, but the unsaturated oxime (422: R = H) gives only the tosylate (422; R = Ts) under these condition^.^'^
(420)
I
(422)
A synthesis of chamic acid methyl ester (423) and chaminic acid (424) starts from dimethyl 5-hydroxyisophthalate (425) (Scheme 23). The key step is the cyclization of the chloro-ketone (426) to give 42% of the trans-ester (427) and 28 7; of the cis-ester (428). The double bond is then introduced into (427) under conditions which do not epimerize the ester group.314 A further double-bond isomer of these acids was synthesized as the ester (429) by allowing the cyclohexadiene ester (430) to react with diphenylsulphonium isopropylide (431), when the esters (429) and (432) were obtained, in amounts that varied with the solvent used. Conformations and hydrogen-bonding in the carene derivatives (433) and (434) have been discussed in terms of their i.r. spectra.316
'
7 Furanoid and Pyranoid Monoterpenoids A new synthesis of -?-substituted furans has been applied to making perillene (435). Methylheptenone is converted into the n-butylthiomethylene derivative (436) of its a-formyl derivative and this is then treated with dimethylsulphonium
"' 'I s 'I5 'I6
A . Zabia. C. Wawrzenczyk, and H . Kuczynski, Bull. Acad. polon. Sci., SPr. Sci. chim., 1972, 20, 521.
W. J. Gensler and P. H . Solomon, J. O r g . Chern., 1973, 38, 1726. C. S. F. Tang and H. Rapoport, J. Org. Chern., 1973,38,2806. I . P. Povodyreva, R. R. Shagidullin, and T. N . Ivaseva, Izvest. Akad. Nauk S . S . S . R . , S r r . kkim., 1972, 2618. The numbering is incorrect here, and also in Chern. A h . , 1973, 78. 8 4 5 5 2 .
Mono t erpeno ids
vi, vii
67
1
Reagents: i, Rh/AI,O,-H,; ii, hydrolysis; iii, AcCl; iv, MeMgCl; v, c. HC1; vi, esterify; vii, oxidize; viii, KOBu'; ix, [PhNMeJBr,; x, NaBH,; xi, Zn-MeOH; xii, alkali.
Scheme 23
Terpenoids and Steroids
68
(433)
(434)
methylide, when a 55 % yield of perillene (435)is obtained, together with recovered n-butylthiomethylene derivative (436).3l 7
The absolute configuration at C-2 of the oxides (437)--(440) from Liliurn rnakinoi is the same as that of the congeneric linalool, which is therefore presumed to be a biosynthesic p r e c u r ~ o r .*~
A new synthesis of the lilac alcohols (441) as an isomeric mixture has been carried out by Vig et al. by cyclizing the ester (442)with sodium hydride in benzene, then reducing the ester group with lithium aluminium h ~ d r i d e . ~ ”
(442)
”’ 3‘8 319
(44 11
M . E. Garst and T. A. Spencer, J . Amer. Chem. SOC.,1973, 95, 250. T. Okazaki, A. Ohsuka, and M . Kotake, Nippon Kakaku Kaishi, 1973,359 (Chem. Abs., 1973, 7 8 , 136 423). 0. P. Vig, R. S. Bhatt, J . Kaur, and J . C. Kapur, J . Indian Chem. SOC.,1973, 50, 37.
Mono terpenoids
69
8 Cannabinoidsand other Phenolic Monoterpenoids The name ‘meroterpenoid’has been suggested for compounds arising from mixed bi~genesis,~ 2o and phenolic terpenoids belong to this class. The isolation of alliodorin (443) from Cordia alliodora lends some support to occurring in C. millenii arise by the idea321that the cordiachromes [e.g. (a)] condensation of a benzenoid precursor with geranyl pyrophosphate and subsequent oxidative cyclization of an ally1 methyl group. Alliodorin (443) represents a stage along such a
OH
I
I
0
(443)
(444)
Linalool or myrcene produce the cation (445), which reacts with hydroquinones to produce a mixture of chromans (Scheme 24).323 OH
OH
(445)
R’ = H, RZ = Me, Bu‘, or t-octyl, R3 = H ; or R’
=
RZ = R3 = Me
Scheme 24 320 J21
322 323
J. W. Cornforth, Chem. in Britain, 1968, 4, 102. M. Moir, R. H. Thomson, B. M. Hausen, and M. H. Simatupang, J.C.S. Chem. Comm., 1972, 363. K. L. Stevens, L. Jurd, and G. Manners, Tetrahedron Letters, 1973, 2955. M. H. Stern, T. H. Regan, D. P. Maier, C. D. Robeson, and J. G. Thweatt, J. Org. Chem., 1973, 38, 1264.
Terpenoids and Steroids
70
Sukh Dev’s group has isolated the phenolic terpenoid (446)from Psoralea corylifolia and has established the absolute configuration I
I
Another review of the cannabinoids has a ~ p e a r e d . ~ ’ It has been stated that thin-layer chromatography is preferable to gas chromatography in the analysis of the constituents of cannabis;326 nevertheless gas chromatography takes a prominent place in much of the work described below. An analysis of the headspace over cannabis essential oil [using gas chromatography-mass spectrometry (g.c.-m.s.)] yielded only common monoterpenoids as positively identified.327 One report3’* stated that it was not yet possible to measure the level of tetrahydrocannabinol (THC) in blood using g.c.-m.s. coupling, at about the same time that a prominent manufacturer of g.c.-m.s. equipment was advertising just that ! 3 2 9 A new cannabidiol monomethyl ether (447) has been isolated from hemp leaves of a Japanese variety,330 and from South African Cannabis satiua a new acid, A’-tetrahydrocannabivarolicacid (448).33
’
1’
3 24
325
326 327
328 329 330
33 1
I
G . Mehta, U. R . Nayak, and Sukh Dev, Tetrahedron, 1973,29, 1 1 19; A. S. C. P. Rao, V . K. Bhalla, U. R. Nayak, and Sukh Dev, ibid., p. 1127. R. K. Razdan in ‘Progress in Organic Chemistry’, Vol. 8, ed. W. Carruthers and J. K. Sutherland, Butterworths, 1973. M. R. Paris and R . R. Paris, Bull. SOC.chirn. France 11, 1973, 118. L. V. S. Hood, M. E. Dames, and G. T. Barry, Nature, 1973,242,402; see also ref. 186 for analytical work on hashish smell. Nature, 1973, 244, 3 . Advertisement for LKB in Chem. and Ind., 1973, July 7, p. vi. Y. Shoyama, K. Kuboe, I . Nishioka, and T. Yamauchi, Chem. and Pharm. Bull. (Japan), 1972,20,2072. M. Paris, C. Ghirlanda, M. Chaigneau, and L. Giry, Compr. rend., 1973, 276, C , 205.
71
Monoterpenoids
The metabolism of A'-THC [(449), using normal monoterpenoid numbering] by a liver microsomal fraction of the squirrel monkey, Saimiri sciureus, has resulted in the isolation of two new metabolites, the 6-ketone (450) and the epoxide (451) (the configuration of the latter was not established on the isolated material, but synthetic work referred to below has shown it to be as drawn), and three new metabolitites (5-0x0-A"-THC, 5p- and Sa-hydroxy-A6-THC) were identified from A6-THC metabolism.332 After oral administration of A'-THC (449), the following were isolated from human blood plasma: A'-THC, 7-hydroxy-A1-THC (452), and 6~,7-dihydroxy-A'-THC, together with tentative
identification of 6p- and 6 a - h y d r o ~ y - A l - T H C .Some ~ ~ ~ of these metabolites show activity, and there are suggestions that some cannabis effects are due to them.334 Tested separately, 7-hydroxy-A1-THC has about the same potency as A1-THC, GP-hydroxy-A'-THC is less potent, and the 6a-hydroxylated material is inactive.33 Cannabidiols (453) and cannabinol (454)are inactive in man.336 The marihuana activity of various A'-THC and A6-THC substituted in the aromatic ring at C-3' and C-5', and having various side-chains at C-4', has been examined.337
ho
OH
(453) 332 333
334
335
336 337
(454)
(455)
0. Gurny, D. E. Maynard, R. G . Pitcher, and R. W. Kierstead, J . Amer. Chem. SOC.. 1972, 94, 7928. M . E. Wall, D. R. Brine, C. G . Pitt, and M . Perez-Reyes, J . Amer. Chem., SOC.,1972, 94, 8579. L. Lemberger, J. L. Weiss, A. M . Watanabe, I . M. Galanter, R. J. Wyatt, and P. V. Cardon, New England J . Med., 1972, 286, 685. Many other papers attest to this, including all those on the synthesis of metabolites quoted below. M . Perez-Reyes, M. C. Timmons, M . A. Lipton, H . D. Christensen, K. H . Davies, and M . E. Wall, Experientia, 1973,29, 1009. L. E. Hollister, Experientia, 1973, 29, 825. H. Edery, Y . Grunfeld, G . Porath, 2. Ben-Zvi, A. Shani, and R. Mechoulam, Arzneim.Forsch., 1972, 22, 1995.
Terpenoidr and Steroids
72
(456)
(460)I %
(461) 14%
Reagents: i, SOC1,-py, 0 " C , 16 h ; ii, SO,Cl,; iii, AgOAc.
Scheme 25
Much synthetic work this year has concerned the metabolites of A'-THC. Oxidation with selenium dioxide of A'-THC gives the 6-ketone (450),and epoxidation of A'-THC yields the epoxide (451),the configuration of which was established by conversion with boron trifluoride into the ketone (455), in which the methyl group has to have the same configuration as the epoxide, and which was shown to be in the unfavourable cis-configuration by isomerization with base to the trans-isomer. Finally the 5-oxygenated A6-THC compounds were made by t-butyl chromate oxidation of A6-THC acetate to give the 5-oxo-compound, which was reduced with lithium aluminium hydride to the corresponding alcohols.33* A convenient synthesis of the 7-hydroxy-metabolite from A1(')THC (450), announced briefly last year (see Vol. 3, p. 87), has been improved in the full paper by carrying out an osmium tetroxide oxidation on the acetate of (456) and dehydrating the product (457) with thionyl chloride in pyridine, when the two THC acetates (458; R = Ac) and (459; R = Ac) are obtained in the 378
R. Mechoulam, H. Varconi, Z. Ben-Zvi, H. Edery, and Y. Grunfeld, J . Arner. Chern. S O C . ,1972. 94, 7930.
Mono t erpenoids
73
ratio of 2 : More directly, 7-acetoxy-A1-THC(459; R = H) can be made by treatment of A’-THC with sulphuryl chloride followed by silver acetate, the 6-acetoxy-compounds (460; R = Ac) and (461; R = Ac) being formed at the same time (Scheme 25).340 The same paper reports a new route to 7-hydroxy-A1-THC (452) from the ketone (462), which is outlined in Scheme 26.340
l i i . iii
A (4 52)
A
Reagents: i, 0
NH-Cl,CCO,H ; ii, HC1-ZnC1,,2H20-CHC1,; iii, Et,COK-toluene; L J iv, EtOH-KOH; v, LiAIH,.
Scheme 26
Another paper has been published about the preparation of tritium-labelled cannabinoids from labelled olivetol;341it includes a very similar route to that mentioned earlier (see Vol. 3, p. 88). Some reactions of A6-THC have been carried out by Mechoulam’s group. The epoxide (463), on treatment with boron trifluoride etherate, gives two compounds, the ketone (464) and the ring-contracted aldehyde (465), depending on whether bond a or b migrates (see formula). Hydroboration of A6-THC leads to a mixture of the alcohols (466) and (467)and the B-oriented alcohol (467)can be 339 340
341
R. K. Razdan, D. B. Uliss, and H. C. Dalzell, J . Amer. Chem. SOC.,1973,95, 2361. C. G. Pitt, F. Hauser, R. L. Hawks, S. Sathe, and M. E. Wall, J . Amer. Chem. SOC., 1972,94, 8758. S. Agurell, B. Gustafsson, T. Gosztonyi, K. Hedman, and K. Leander, Acra Chem. Scand., 1973, 27, 1090.
Terpenoids and Steroids
74
dehydrated (uiu the tosylate) to As-THC (468), which is devoid of marihuana
C5H1
1
Some analogues of A6-THC with nitrogen in the pentyl side-chain have been prepared,343and some of A’-THC with nitrogen in the menthane ring.344 There are some papers on more general aspects of reactions of terpenoids with phenols. The full paper of the citric acid-catalysed condensation of orcin and s42
743
344
R . Mechoulam, Z. Ben-Zvi, H . Varconi, and Y . Samuelov, Tetrahedron, 1973, 29, 1615. T. Petrzilka and W . G. Lusuardi, Helu. Chim. Acta, 1973, 56, 510: T. Petrzilka, M . Demuth, and W. G. Lusardi, ibid., p. 519. M . Cushman and N. Castagnoli, jun., J . Org. Chem., 1973, 38, 440. We hope that this paper, using a more appropriate numbering system, presages a change of heart by the American Chemical Society!
75
Mono t erpenoids
monoterpenoid alcohols (see Vol. 3, p. 88) contains a discussion about the formation of the dihydrobenzofurans (469) and (470), formed respectively from menth4-en-3-01 (471) and pulegol (15).345 Crombie’s group has suggested how the
H
I
(15) --+ -+
, o orientation of chromenylation in reactions between phenols and certain aldehydes (catalysed by pyridine) may be governed by the possibility for chelation throughout the reaction sequence, a simple example being illustrated in Scheme 27.
xt:
-0
Scheme 27
For examples of such reactions, see the literature and also Vol. 2, p. 62. A condensation of this nature, between citral (109) and pinocembrene (472), was already known to occur in the presence of pyridine (see Vol. 2, p. 63). Now Montero and Winternitz have effected347a kind of biological synthesis 345 346 347
B. Cardillo, L. Merlini, and S. Stefano, Gazzerra, 1973, 103, 127. D. G. Clarke, L. Crombie, and D. A. Whiting, J.C.S. Chem. Comm., 1973, 580, 582. J. L. Montero and F. Winternitz, Tetrahedron, 1973,29, 1243.
76
Terpenoids and Steroids
by carrying out the reaction in the presence of a pseudo-alkaloid,anibine (derived from nicotinic acid), when they obtained ( & )-rubranine (473) in higher yield than when pyridine was the catalyst. -
HO
Ph (472)
+
--*
k WPh (473)
2 Sesq u iterpenoids BY
R. W. MILLS AND
T. MONEY
1 Introduction The large number of publications which appear each year on the structure, synthesis,’ biosynthesis,2 and natural occurrence of sesquiterpenoids reflects the widespread interest in this group of natural products. This high level of research activity has been maintained during the period covered by the present Report. The excellence of previous Reports3 in this series was largely due to the organized way in which new information was presented. Sesquiterpenoids were classified into structural groups based on biosynthetic consideration^^"-^ and, to some extent, on the number of carbocyclic rings in the basic structure. The present Reporters have adopted a similar classification with minor modifications and, for convenience, skeletal representations of the various structure types are shown in Table 1. Most of the biosynthetic investigations described in the present Report make excellent use of substrates specifically labelled at an appropriate prochiral centre and rely heavily on the pioneering, authoritative studies of the CornforthPopjak group. A new review describing some of these studies has appeared recently. 2 Farnesanes
Current biosynthetic theory assumes that the biosynthesis of all sesquiterpenoids4” involves appropriate modification of the pyrophosphate esters of trans,-
’ C. H. Heathcock, in ‘Total Synthesis of Natural Products’, ed. J. W. ApSimon, Wiley, New York, 1973, Vol. 2. J. R. Hanson, in ‘Biosynthesis’, ed. T. A. Geissman (Specialist Periodical Reports), The Chemical Society, London, 1972, Vol. 1, p. 41. J. S. Roberts, in ‘Terpenoids and Steroids’, ed. K. H. Overton (Specialist Periodical Reports), The Chemical Society, London, ( a ) 1971, Vol. 1 , p. 5 1 : (b) 1972, Vol. 2, p. 65 ; ( c ) 1973, Vol. 3, p. 92. ( a ) W. Parker, J. S. Roberts, and R. Ramage, Quart. Rev., 1967,21, 31 1 and references cited; (6) L. Ruzicka, Experientia, 1953, 9, 357; (c) J. H. Richards and J. B. Hendrickson, ‘Biosynthesis of Steroids, Terpenes, and Acetogenins’, Benjamin, New York, 1964, p. 225; (d)T. A. Geissman and D. H. G . Crout, ‘Organic Chemistry of Secondary Plant Metabolism’, Freeman, Cooper and Co., San Francisco, 1969, p. 269; ( e ) J. W. Cornforth, Angew. Chem. Internat. Edn., 1968, 7 , 903; Quarr. Rev., 1969, 23, 125; (f)W. B. Turner, ‘Fungal Metabolites’, Academic Press, 1971, p. 219. J. W. Cornforth, Chem. SOC.Rev., 1973,2, 1 .
77
Terpenoids and Steroids
78
/-+
Ill
W
E
79
Sesquiterpenoids
>h Ek
“6
Terpenoids and Steroids
80
?d
k‘ Y’
d
.-
-Y
LL
Ill
I
3
t
Monocyclofarnesane
Bicy clofarnesane (Drimane)
Bourbonane
Aromadendrane
a-,
Pseudoguaiane
/
Guaiane
Maaliane
CYPerane
lshwarane
Pre-Seychellane
Aristolane
Seychellane
82
Terpenoids and Steroids
trans-farnesol (l),cis,trans-farnesol(3),or nerolidol(2). The formation of cis,transfarnesol could involve direct biosynthesis from mevalonic acid or isomerization of the trans,trans-isomer (1) (e.g. via nerolidol). Recent studies using [ l,l-2H2]trans,trans-farnesol or [1,l -2H2]-trans,trans-2,3-epoxyfarnesolhave shown that Helrninthosporiurn satiuurn can convert these compounds into the corresponding cispans-compounds via the aldehyde intermediates (4) and (5) (Scheme 1).6
A (31
E
C
H
O
(4)
Scheme 1
(5)
A similar study, using a cell-free system from Andrographis panicdata and appropriately labelled mevalonate as substrate, has also demonstrated that the isomerization of trarqtrans- (1) to cis,trans-farnesol (3) involves aldehyde inter(6) and mediates.' A total loss of 3H label from [2-'4C,(4S)-4-3Hl]mevalonate complete retention from [2-14C,(4R)-4-3H,]mevalonate (9) was found in both (1) and ( 3 ) (Scheme 2).7*8Using [2-14C,5,5-3H,]mevalonate there was total retention of tritium in trans,truns-farnesol (1) but loss of one-sixth of the tritium in the cis,trans-isomer (3) (cf. p. 92). It has also been shown' that cell-free extracts of Citrus sinensis (orange) flavedo can convert geranyl (1 1) and isopentenyl pyrophosphate ( 7 ) into a mixture of farnesols and farnesals. A redox Y . Suzuki and S . Marumo, Tetrahedron Letters, 1972, 1501.
' K. H. Overton and F. M. Roberts, J.C.S. C h m Cornrn., 1973, 378. Cf. E. Jedlicki, G . Jacob, F . Faini, and 0.Cori,.Arch. Biochem. Biophys., 1972,152,590. L. Chayet, R. Pont-Lezica, C. George-Nascimento, and 0. Cori, Phytochemistry, 1973, 12, 95.
Sesquiterpenoids
83
mechanism (cf. Scheme 1) for the isomerization of the farnesols is supported by the fact that farnesyl pyrophosphate could not be isomerized by the cell-free extract. It is also significant that NAD+ favours the isornerization and that neryl pyrophosphate (12) cannot substitute for the trans-isomer (1 1) in the biosynthetic process.' For further discussion of biosynthetic studies see Chapter 7, p. 254.
-
T H
HT y HOH,C
q C0,H
--+
(1)
+ (3)
Scheme 2
Further studies on the sesquiterpenoid constituents of the stock-poisoning shrub, Myoporium deserti A. Cunn, have revealed the presence of (-)-epingaione (1 3), ( - )-dehydrongaione (14), ( - )-dehydroepingaione (1 5), and ( - )-deisopropylngaione (16)" The same group of investigators has also established the stereochemistry of ( - )-myoporone (17) and ( - )-dehydromyoporone (18), which occur in various myoporium and eremophila species' (monocyclic metabolites of this plant are described on p. 143). Dendrolasin (21) has been syn-
'
lo
"
W. D. Hamilton, R. J. Park, G. J . Perry, and M. D. Sutherland, Austral. J . Chern., 1973, 26, 375. I . D. Blackburne, R. J. Park, and M . D. Sutherland, Austral. J . Chem., 1972, 25, 1787.
84
R
3
Terpenoids and Steroih
2
0
(13) (15 ) 2,3-dehydro-( 1 3)
0
(14) R = CH,COCH=CMe,
(17)
(16) R = CH,COMe
(18) 2,3-dehydro-( 17)
Po+ Q?@
thesized from ketone (19)by a synthetic route which should be generally applicable to a variety of furanoid sesquiterpenoids. l 2
0
S(CH d3Me (19)
(20)
(21)
A recent structural elucidation'3.'4 of gyrinal (27), a nor-sesquiterpenoid isolated from the 'whirligig' beetle (cf: p. 143) has been followed by two stereowhich are virtually identical (Scheme 3). The synthesisI6 selective syntheses' ' 9
'
OHC O -R
&OR (22) R
=
0
COMe
(23) R = CO
(24) ki,
111
J L . A - b * R "
OAc
(25)
0
CHO 0 (27) Reagents: i, SeO,; ii, MeCH(0Li)C GCLi-THF; iii, Ac,O-py; iv, Na-NH,; v, LiAlH,; vi, MnO,.
Scheme 3
l 3
l4
''
l6
M. E. Garst and T. A. Spencer, J. Amer. Chem. SOC., 1973,95,250. H. Schildknecht, H. Neumaier, and B. Tauscher, Annalen, 1972, 756, 155. J. Meinwald, K. Opheim, and T. Eisner, Proc. Nat. Acad. Sci. U . S . A . , 1972, 69, 1208. J. Meinwald and K. Opheim, Tetrahedron Letters, 1973, 281. C. H. Miller, J. A. Katzenellenbogen, and S. B. Bowlus, Tetrahedron Letters, 1973,285.
85
Sesquiterpenoidr
which uses geranyl mesitoate (23) as starting material and LiA1H4 to cleave the ester function and reduce the triple bond is reputed to be more efficient. Considerable effort has been expended on the structural elucidation, synthesis, and physiological properties of the juvenile hormone (28) of Hyulophora ce~ropiu.~"' A new stereoselective synthesis (Scheme 4) of this compound
i, iii
lv lviii
J
\
0
Reagents: i, BuLi-DABCO; ii, SOC1,-py; iii, +OTHP;
iv, BuLi-DABCO-/(1
;
c1 v, hydrolysis, Li-EtNH,, -70 " C ;vi, Li-EtNH,, -20 "C;vii, Jones oxidation; viii, acetylation, Ra-Ni; ix, (EtO),PCH ,CO,Me.
It
0
Scheme 4
Reviews: B. M. Trost, Accounts Chem. Res., 1970, 3, 120; J . B. Sidall, 'Chemical Energy', ed. E. Sondheimer and J . B. Simeone, Academic Press, New York, 1970, p. 282.
Terpenoids and Steroids
86
employs an elegant reaction sequence based on the condensation of dihydrothiopyrans.'* An alternative synthesis, based on the Claisen rearrangement of vinyl ally1 etherslg has also been described (Scheme 5).20 A probable biosyn-
r
I
I A
v
C
0
2
1 OH
0
OH Reagents: i,
OMe ; ii, TsC1-py; iii, NaOMe-MeOH.
Scheme 5
thetic precursor of juvenile hormone (28) is bishomofarnesyl pyrophosphate (32), and the formation of this compound by incubating cis-3-methylpent-2-enyl pyrophosphate (29), 3-ethylbut-3-enyl pyrophosphate (30), and isopentenyl pyrophosphate (31)with farnesyl pyrophosphate synthetase has been reported.21
Lop, + + L
(39)
18
2o
''
O
(30)
P
2
L
O P (31)
,
K. Kondo, A . Negishi, K . Matsui, D. Tunemoto, a n d S. Masamune, J . C . S . Chem. Comm., 1972, 1311. W . S. Johnson, T . J . Brocksom, P. Loew, D. H . Rich, L. Werthermann, R. A. Arnold, T. Li, a n d D. J. Faulkner, J . Amer. Chem. SOC.,1970, 92, 4463. D. J . Faulkner and M . R. Petersen, J . Amer. Chem. SOC.,1973.95, 553. T. Koyama. K . Ogura, and S . Seto, Chem. Letters, 1973, 401.
87
Sesquiterpenoids
3 Bisabolanes A recent general method**for the synthesis of bisabolane-type sesquiterpenoids has been used in the synthesis of (-)-cryptomerion (34) from (-)-carvone (33).23 It has also been notedz3that irradiation of cryptomerion provides photocryptomerion (35) in a reaction which is analogous to the photocyclization of carvone (33) to carvonecamphor (36) (Scheme 6). The [2,3]-sigmatropic rearrangement of
0 (33) 1vii
J
(36)
(34)
(35)
Reagents: i, Zn-NaOH; ii, (HOCH,),CMe,-H ; iii, BuLi-TMEDA-Me,C =CHCH ,C1; iv, H+-Me,CO; v, [PhN+Me,]Br,-; vi, py. A ; vii, hv. +
Scheme 6
I
i-0-
BPLi ___+
p \p -
PhS--MeOH
Ar\ OL/
CH,OSAr
(37)
(38)
1
'* 23
(39) R. J. Crawford, W. F. Erman. and C. D. Broaddus, J. Amer. ChPm. SOC.,1972, 94, 4298. G. L. Hodgson, D. F. MacSweeney. and T. Money, J.C.S. Chern. Cornrn., 1973, 236.
Terpenoids and Sterolds
88
allylic sulphoxides [e.g. (37)] to allylic sulphenates [e.g.(38)] forms the basis of a new synthesis of ( )-(E)-nuciferal (39)240(natural nuciferal possesses the 2 configuration). The same group has also described a two-step synthesis (Scheme 7) of (-t )-ar-turmerone (42) using regiospecific alkylation of the dianion (41) of the P-ketophosphonate ester (40).24b 0
0
P
0
A (42)
Reagents: i , N a H - T H F ; ii, BuLi; iii, NaH-dimethoxyethane; iv, M e 2 C 0 .
Scheme 7
Pseudotsugonal (43), ar-pseudotsugonal (44),( + )-methyl todomatuate (45),2 and ( - )-dihydroepitodomatuic acid (46)24 have been isolated from Douglas fir grown in British Columbia and assigned the R,R configuration. The biological activity of these compounds may be interesting since similar metabolites of balsam fir, juvabione (47) and (+)-dehydrojuvabione (48), have been shown to possess insect juvenile hormone activity. Spectroscopic evidence has been provided for the structure of deodarone (49), a component of the essential oil of Cedrus deodoru Loud.”
24 25 26
2,
P. A. Grieco and R . S. Finkeihor, ( a )J. O r g . Chem., 1973, 38, 2245; ( b ) ibid.,p. 2909. T. Sakai and Y . Hirose, Chem. Letters, 1973, 491. I . H . Rogers and J. F . Manville, Canad. J . Chem., 1972,50, 2380. R . Shankaranarayanan, S. Krishnappa, S. C. Bisarya. and S. Dev, Tetrahedron Letters, 1973. 427.
Sesquiterpenoih
89 H
0P
C
O
2
0@C02Me
H
A (47)
0@C02Me
4 Sesquicarane, Carotane, etc.
A new sesquiterpenoid diol, jaeschkeanadiol (50), has been isolated from the roots of Ferula jaeschkeana Vatke.28 Sequential ring-expansion and ring-
(50)
contraction techniques have been used in a recent synthesis of (+)-daucene (51),29and the low-yield conversion of this compound into (+)-carotol(52) and (-)-daucol(53) has also been described (Scheme 8).29
Scheme 8 (continued overleaf) 28
M . C. Sriraman, B. A. Nagasampagi, R. C. Pandey, and S. Dev, Tetrahedron, 1973,29, 885.
29
H. de Broissia, J . Levisalles, and H. Rudler, Bull. Soc. chim. France, 1972, 4314.
Terpenoids and Sterolds
90
iii,
iv
Reagents: i, C H , N , ; ii, PCl,; iii, per-acid; iv, Li-EtNH,; v, p-O,NC,H,CO,H.
Scheme 8
5 Cuparane, Laurane, Trichothecane, etc. Detailed descriptions of recent studies on the biosynthesis of the trichothecane group of sesquiterpenoids have been provided.2,30-34,36-40 Th e structures of these compounds are based on the 12,13-epoxytrichothec-9-enenucleus (62) and the cumulative efforts of various research groups have provided evidence which supports the proposed biosynthetic route shown in Scheme 9. Incorporation experiments, using mevalonic acid (54),geranyl pyrophosphate ( 5 3 , and farnesyl pyrophosphate (56) specifically labelled with 14Cand/or 3H, have shown that trichothecin (65) is derived from three molecules of mevalonic and that cispans-farnesyl pyrophosphate (56)30 is an intermediate on the biosynthetic pathway. Incorporation of tritium from [2-3H]geranyl pyrophosphate (55) into position 2 of the trichothecane nucleus3’ and an e ~ a m i n a t i o nof~the ~ precursor activity of y-bisabolene (57)exclude the latter compound as a precursor of trichothecin (65).* Further support for the biosynthetic proposals outlined in Scheme 9 has been provided by the isolation of trichodiene (59),36trichodiol (60),3“,? 12,13-epoxytrichothec-9-ene(62),3 and 4,8-dihydroxy-12,13-epoxytrichothec-9-ene (68)3 from Trichothecium roseum and by the demonstration that radioactivity from tritiated trichodiene (59) can be incorporated into t richot hecolone (66).
’
”
B. A. Achilladelis, P. M. Adams, and J. R. Hanson, J.C.S. Perkin I , 1972, 1425 and references cited therein.
.’’ Y . Machida and S. Nozoe, Tetrahedron, 1972, 28, 5 1 13. ’’ E. R. H. Jones and G . Lowe, J. Chem. S O C . ,1960, 3959.
R. Achini, B. Muller, and Ch. Tamm, Chem. Comm., 1971, 404. J . M. Forrester and T. Money, Canad. J . Chem., 1972, 50, 3310. 1 5 P. M. Adams and J . R. Hanson, J . C . S . Perkin I , 1972, 586; S. Nozoe, M. Morisaki, and H. Matsumoto, Chem. Comm., 1970, 926. 3 6 S. Nozoe and Y . Machida, Tetrahedron Letters, 1972, 28, 5105. ” Y. Machida and S. Nozoe, Tetrahedron Letters, 1972, 1969. ’* R. Evans, A. M. Holtom, and J. R. Hanson, J . C . S . Chem. Comm., 1973, 465. ’’ S. Nozoe and Y . Machida, Tetrahedron Letters, 1970, 1177. 4 o P. M. Adams and J. R. Hanson, Chem. Comm., 1970, 1569. 3 S
* y-Bisabolene (57) has also been excluded as an intermediate in the biosynthesis of the cuparane-type sesquiterpenoid helicobasidin (7 I). t The ‘trichodiol’ (61) previously described39 is an artifact produced from trichodiol during saponification. 3 6
91
Sesquiterpenoids OH
(63) R = M (64) R = COMe
(65) R (66) R
= =
COCH=CHMe H
(67) R = COCH=CHMe
Scheme 9
Trichodiene (59) and cis,trans-farnesol [c$ (5611 are also formed when trans, trans-[1,1,5,5,9,9-3H,,4,8,12-'4C3]farnesyl pyrophosphate is incubated with a cell-free extract of T. r ~ s e u r n The . ~ ~ loss of one tritium label during the formation of (59) and (56) is readily explained by isomerization of the precursor to the cis,
Terpenoids and Steroids
92
trans-form (56) by a redox mechanism (cf: p. 82), followed by cyclization to trichodiene (59). Comparative experiments using [2,2-3H, ,2-' 4C]mevalonate (54) as precursor demonstrated that trichothecolone (66) (from T. roseurn) and trichodermol(63) (from Trichoderrna sporulosurn)contained four and five [2-3H]mevalonoid labels re~pectively.~'One of the incorporated tritium atoms in trichothecolone (66) was located at position 7 (exchanged with base) and this has prompted the suggestion3' that crotocin (67),which co-occurs with trichothecin, may be an intermediate in the biosynthetic route. Rearrangement of crotocin (67) to trichothecin (65) could involve a 1,2-hydride shift, and this would explain the labelling results described above. When trichodermol (63), containing five [2-3H]mevalonoid labels (positions 4,8, and 14), was fed to T. roseurn the trichothecin (65)and trichothecolone (66)isolated contained only three tritium atoms.40 This evidencejustifies the inclusion of trichodermol(63) as an intermediate in the biosynthetic route to trichothecin etc. Studies on the stereochemistry of trichodermol(63) have shown that epoxidation occurs on the p-face of the m~lecule.~'With verrucarol (15-hydroxytrichodermol) some reaction occurs on the a-face owing to the directing effect of the C- 15 hydroxy-group. Two new trichothecanes, calonectrin (69) and 15-deacetylcalonectrin (70), have been isolated from culture filtrates of Calonectria n i u ~ E i s . ~ ~ The absence of oxygen functionality at C-4 differentiates these compounds from other members of the trichothecane group of fungal sesquiterpenoids.
(69) R = COMe (70) R = H
Gymnomitrol (72 ; R = OH) and its congeners, (72 ; R = OAc), (73 ; R = H2), and (73 ; R = H, P-OAc), have been isolated from the liverwort Gyrnnornitrion
obtusum (Lindb) Pears.43 The structural assignments are based on chemical and spectroscopic evidence and it has been suggested that the biosynthesis of this 41
42 43
P. M . Adarns and J. R. Hanson, J.C.S. Perkin I, 1972, 2283. D. Gardner, A . T. Glen, and W. B. Turner, J.C.S. Perkin I, 1972, 2576. J . D. Connolly, A . E. Harding, and I . M. S. Thornton, J.C.S. Chem. Comm., 1972,1320.
Sesquiterpenoids
93
interesting group of tricyclic sesquiterpenoids could involve cyclization of trichodiene (59) or a closely related compound. The co-occurrence of the tricyclic hydrocarbon (72; R = H) provides indirect support for this proposal.43 Methylenation of the known bicyclic intermediate (74)44is the key feature of a recent synthesis of ( & )-debromolaurinterol (77).45 The 3 : 1 mixture of epilaurinterol methyl ether (76; R = Me) and laurinterol methyl ether (75; R = Me) obtained in this reaction could not be demethylated without prior removal of the bromine substituents. Debromolaurinterol (77) has previously been converted into laurinterol (75 ; R = H), aplysin (79), and d e b r ~ m o a p l y s i n . ~ ~
B
f
l
’
OMe
(74)
6 Acorane, Bazzanane, Cedrane, Zizaane, etc.
A stereospecific synthesis of (-)-cr-acorenol (81) and (+)-P-acorenol (82) from ( + )-(3R)-methylcyclohexanone(80)has been described47 and is shown in Scheme 10. These compounds should be of interest in studies associated with the biosynthesis of the cedrane, allocedrane, and zizaane classes of sesquiterpenoids. ( + )-2,5-Diepi-B-cedrene (83) has been isolated from Sciadopitys verticillata Sieb. et Zucc. and its absolute configuration established by X-ray crystallographic analysis.48 The key feature of an elegant new synthetic route (Scheme to (+)-cedrol(86) and (-t-)-cedrene (87) is a cation-olefin cyclization process 44
4s 46 47
48
K. Yamada, H. Yazawa, D. Vemcera, M. Toda, and Y. Hirata, Tetrahedron, 1969, 25, 3509. R. J . Fentrill, R. N . Mirrington, and R. J. Nicholls, Austral. J . Chem., 1973, 26, 345. T. hie, M. Suzuki, E. Kurosawa, and T . Masamune, Tetrahedron Letters, 1965, 3619. I . G . Guest, C. R. Hughes, R. Ramage, and A. Sattar, J . C . S . Chem. Comm., 1973, 526. T. Norin, S. Sundin, B. Karlsson, P. Kierkegaard, A.-M. Pilotti, and A.-C. Wiehager, Tetrahedron Letters, 1973, 17.
Terpenoidr and Steroids
94
Jvi-viii
1 1
Reagents: i, CH, =CHCN-Triton B; ii, hydrolysis and esterification; iii, Na-C,H,; iv, LiI-DMF; v, Ph,P=CH,-BuOH; vi, p-MeC,H,.SO3H-Me2CO; vii, Ph,CLi-C5H, ,ONO; viii, chloramine; ix, hv.
Scheme 10
involving cyclopropyl ketone (84).49 The cationic enol ester intermediate (85) was implicated in a previous cedrene synthesis." Complete details of the isolation of ( + )-do-cedrol (89) from Juniperus rigida and evidence supporting its structure and absolute configuration have been 49 50
E. J . Corey and R. D. Balanson, Tetrahedron Letters, 1973,9153. E. J. Cotey, N. N. Girotra, and C. T. Mathew, J . Amer. Chem. Soc., 1969,91, 1557
95
Sesquiterpenoidr
a i?r"
H
c-
H
H
r e p ~ r t e d . ~ "The ? ~ cyclization of P-acoradiene (88) to (+)-allo-cedrol(89) and the subsequent rearrangement of the brosylate of (89)to ( - )-prezizaene (90)were also described (Scheme 12). Similar transformations in the antipodal series have been
H
A
iii,
i, i:
,
~;"r
H
Reagents: i, H C 0 , H ; ii, KOH; iii, BrC,H,.SO,Cl-py.
Scheme 12
postulated to account for the biosynthesis of (+)-prezizaene (91)and (+)-zizaene (92).52 An interesting epimerization occurs when (+)-prezizaene (91), (+)zizaene (92), or epizizaene (93) is treated with formic acid.53 The same mixture of alkenes [(94) and (96)] is obtained in each case and intermediate (95) has been proposed to account for the epimerization occurring at a centre remote from the original alkene linkage. 5 1
52
"
B. Tomita and Y . Hirose, Phytochemistry, 1973, 12, 1409. N. H. Andersen and M. S . Falcone, Chem. and Ind., 1971, 61 and references cited; N. H. Andersen and D. D. Syrdal, Tetrahedron Letters, 1972, 899. N . H. Andersen, S. E. Smith, and Y. Ohta, J.C.S. Chem. Comm., 1973,447.
Terpenoidr and Sterozih
96
t-
Experimental details have been provided for the ~ y n t h e s i sof ~ ,epizizanoic ~~ acid (98) and for the isolation55 of this compound and zizanoic acid (97) from vetiver oil.
R'
&
(97) R' (98) R'
= =
C 0 2 H , R2 = H H, R2 = C 0 2 H
7 Chamigrane, Widdrane, and Thujopsane
Interest continues to be focused on the occurrence of novel halogenated sesquiterpenoids in marine algae. The isolation of prepacifenol (99) from Laurencia jifiIiforrnis has been reported,56 and its conversion into pacifenol (100) demonstrated in the laboratory. The original claim that pacifenol (100) occurs in L. pacijica has been retracted since silica gel chromatography employed in the initial separation was shown to produce (100) as an artifact from prepacifenol (99). More cautious separation techniques led only to the isolation of the latter compound. Pacifenol (loo), however, occurs naturally in L. tasrnanica whereas caespitol (101), a possible biosynthetic precursor of prefacifenol (99), has been isolated recently from L. c ~ e s p i t o s a . ~ ~ In the course of studying the behaviour of the cyclopropylmethyl cation in non-aqueous acidic media, the action of anhydrous hydrogen chloride on (-)thujopsene (102) at temperatures between - 10 and + 40"Chas been investigated 54 55
56 57
F. Kido, H. Uda, and A. Yoshikoshi, J . C . S . Perkin I , 1972, 1755. N. Hanayama, F. Kido, R. Tanaka, H. Uda, and A. Yoshikoshi, Tetrahedron, 1973, 29, 945. J . J. Sims, W. Fenical, R. M. Wing, and P. Radlick, J . Amer. Chem. SOC.,1973,95,972. A. G. Gonzalez, J. Darias, and J. D . Martin, Tetrahedron Letters, 1973, 2381.
Sesquiterpenoiak
97
Br (99)
using n.m.r. s p e c t r o ~ c o p y .The ~ ~ initially formed product (103), on warming to 20°C, rearranged to the 1,4-addition product (104). Further heating to 40°C afforded compound (105) whose structure was readily comparable with that of widdrol (106). The final thermodynamic product (107) was obtained after 20 h at 40°C.
8 Sesquipinane, Sesquifenchane, and Fumagillane The structures9and synthesis6' of sesquifenchene (114), the root oil hydrocarbon of Valerianu waalichi, have been reported. Treatment of the cyclic ether (108)61 with acetic anhydride and boron trifluoride gave a mixture of hydroxy-acetates 58
59
6o 61
A. R. Hochstetler and G. C. Kitchens, J . Org. Chem., 1972,37, 2750. S. K. Paknikar and J. K. Kirtany, Chem. and Ind., 1972,803 ; cf. E. J. Corey, D. E. Cane, and L. Libit, J . Amer. Chem. SOC.,1971,93, 7016. Y. Bessiere-Chretien and C. Grison, Compt. rend., 1972, 275, C , 503; Bull. SOC.chim. France, 1972,4570. T. W. Gibson and W. F. Erman, J . Amer. Chem. SOC.,1969,91,4771.
98
Terpenoids and Steroids
(109) and (1 10). Rearrangement of the tosylate (1 11) provided 9-acetoxy-afenchene ( I 12), and the corresponding iodide (1 13) was converted into sesquifenchene (1 14) using the Corey coupling procedure.62 In an attempt to effect a simple synthesis of a-cis-bergarnotene (1 16), it was found that treatment of the ether (108) with acetic anhydride and pyridine chlorohydrate gave a 70% yield of 9-acetoxy-a-pinene (1 15);6 the latter compound, however, could not be converted into a-cis-bergamotene (1 16) using the procedure described above.
1
Ac,O BF, Lt,O
(109) R' = OAC,R 2 = OH (110) R ' = OH, R 2 = OAC ( I 11) R' = OTS,R 2 = OAC
Ovalicin, a fumigallane-type sesquiterpenoid isolated from Pseudeurotium ovalis, has been assigned structure (1 17) on the basis of chemical and spectroscopic evidence.63
'' ''
E. J . Corey and M. F. Semmelhack, J . Amer. Chem. SOC.,1967, 89, 2756. P. Bollinger, H . - P . Sigg, a n d H.-P. Weber, H e f v . Chim.A c t a , 1973, 56, 819.
Sesquiterpeno ids
99
9 Sesquicamphane, fMhtalane, or-Santalane, etc. The synthesis (Scheme 13) of (+)-campherenone (118) and its use as a key intermediate in a general synthetic route to a group of structurally related sesquiterpenoids have been Compounds included within the scope of the general scheme are divided into groups whose members are sesquiterpenoid
1.
/JyC’ “1,--x
,
iv, H ’ ; v , isopropenyl acetate-pvii, (CH,OH),-H ; viii, Nal-Me,CO;
Reagents: i, BuLi-TMEDA; ii, L O ; iii, CC1,-Ph,P; MeC,H,.SO,H; vi, BF,-dH,Cl2; ix, Ph,P; x, dimsylsodium-Me,CO.
+
Scheme 13
analogues of camphor, borneol (isoborneol), camphene, and tricyclene. The success of the general scheme has been illustrated recently by the total synthesis of ( f)-campherenone (118), ( f)-epicampherenone (119), ( f)-p-santalene (12l), ( f)-a-santalene (123), and ( f)-epi-8-santalene (125) (Scheme 14). Furthermore, the synthesis of ( -t)-copacamphor, (+)-ylangocamphor, and ( k)-sativene has also been ~ o m p l e t e dand ~ ~ is, ~described ~ later in this Report (p. 106). A related study6’ has resulted in the synthesis of (-)-campherenone (118), (-)-p-santalene (121),(+)-epicampherenone (1 19),and (+)-epi-p-santalene (125) from ( + )-camphor (1 26) and provides evidence to establish or confirm the absolute 64 65
66 6’
T. Money, Progr. Org. Chem., 1973,8,29. G . L. Hodgson, D. F. MacSweeney, and T. Money, J.C.S. Perkin I, 1973, 2113. G . L. Hodgson, D. F. MacSweeney, and T. Money, Tetrahedrori Letters, 1972, 3683. G. L. Hodgson, D. F. MacSweeney, R. W. Mills, and T. Money, J.C.S. Chem. Cornm., 1973, 235.
Terpenoids and Steroid\.
100
Reagents: i, LiAl(OMe),H; ii, p-MeC,H.SO,Cl-py; iii, HgO-MeOH, A.
Scheme 14
configuration of these naturally occurring enantiomers. In the original report6' describing the synthesis of (-)-campherenone (118) etc., the conversion of (+)camphor (126) into the acetal(131) of ( -)-8-iodocamphor was accomplished in 12 steps using a combination of known reaction sequences. Recent investigations, however, have resulted in the development of a simple stereospecific conversion of camphor into 8-bromocamphor which can be accomplished in three steps.68 These syntheses are summarized in Scheme 15. Thus, the synthesis of (-)- or (+)-campherenone can be accomplished in a simple and'efficient manner from ( -)- or ( + )-camphor respectively and the sesquiterpenoids derived from campherenone (Schemes 14 and 19) are now readily available in either enantiomeric form. C. R . Eck, R. W . Mills, and T. Money, J.C.S. Chem. Comm., 1973,911.
Sesquiterpenoa
1-111
...
I
101
(119) -+ (124) -+
(125)
/
o&Br
Reagents i, Br, (1 mole); ii, Br,-CIS0,H;
iii, Zn-HBr; iv, Br, (2 moles);
10 Amorphane, Cadinane, Cubebane, Copaane, Copacamphane, Ylangocamphane, Sativane, eic. The original structure (132)69assigned to khusinol [a metabolite of Vetiveria zizanoides (L) Nash] was recently revised to ( 133),70and subsequent synthetic studies have confirmed that neither of the C-3 epimers of (132)is identical with the natural product7' The C-2 epimer of khusinol has also been isolated recently 69
70
71
A. A. Rao, K. L. Surve, K. K. Chakravarti, and S. C. Bhattacharyya, T e t r a h e d r o n , 1963, 19, 233. S. V. Tirodkar, S. K. Pakinkar, and K. K. Chakravarti, Science a n d C u l t u r e , 1969, 35, 27. R. B. Kelly and J. Eber, Canad. J . Chern., 1972, 50, 3272.
Terpenoih and Steroids
102
from vetiver C.d. measurements and chemical condensation with known amorphenes have established the structure and absolute configuration of zonarene (134) and l o - e ~ i z o n a r e n e .A~ ~new sesquiterpenoid lactone, arteannuin B, has been isolated from Artemisia annua L. and assigned structure (135) on the basis of spectroscopic evidence.74
Hofi & "i HO
10
3 \
\
#
\
8
H :
\
O.'O'
H :
A
A
\ O (135)
( 134)
(133)
(132)
established the structure of cubebol (138), the Although previous stereochemistry at C-4 and C-10 remained in doubt. A recent synthesis of cubebol (138), a-cubebene (139), and p-cubebene (140) from ( -))-trans-caran-2-one (136) has established the absolute configuration of these The key intermediate, norcubebanone (137), was synthesized by an intramolecular carbene insertion process and its stereochemistry deduced from c.d. data.
5. (F --+
'P
t 136)
A (137 72 7 .3
74 1 5 76
-.+
'P
&IOH 'P I
A t 138)
(139)
(140)
P. S. Kalsi, J . C. Kohli, and M . S. Wadia, Indian J . Chem., 1972, 10, 1127. N. H. Andersen, D. D. Syrdal, B. M . Lawrence, S. J. Terhune, and J . W. Hogg, Phytochemistry, 1973, 12, 827. D. Jeremic, A. Jokic, A. Behbud, and M . Stefanovic, Tetrahedron Letters, 1973, 3039. F. Vanasek, V. Herout, and F. Sorm, Coll. Czech. Chem. Comm., 1960, 25, 919. A. Tanaka, R. Tanaka, H. Uda, and A. Yoshikoshi, J.C.S. Perkin i, 1972, 1721.
I03
Sesquiterpenoids
Some interesting transformations of santonic acid (141) have been discovered as a result of attempts to utilize this compound as starting material in a projected synthesis of copaene (142).77 A summary of the various rearrangements is shown in Scheme 16. An alternative synthesis of ( +)-a-copaene (142) and ( +)-a-ylangene
Me0,C
:
CO,H
+
CO,H
0
(141)
Reagents: i, H,O,-OH-; ii, Na-Hg; iii, Ac,O; iv, KOBr.
Scheme 16
(143) has been reported,78 and although the synthetic plan is similar to that previously used7' a notable feature of this synthesis is the selectivity of the initial Diels-Alder reaction (cf. occidentalol synthesis p. 119). P-Copaene (145) and P-ylangene (146) were also synthesized from the intermediate dienone (144). These syntheses are summarized in Scheme 17. An elegant stereoselective total synthesis of (- )-ylangocamphor (153), ( - )ylangoborneol (154), and ( -)-ylangoisoborneol (155) has been reported and is shown in Scheme 18.80 Octalone (147) was converted into the keto-ester (149) 77
78 79
8o
A. G . Hortmann and D. S. Daniel, J . Org. Chem., 1972, 37, 4446. E. J . Corey and D . S. Watt, J . Amer. Chem. SOC.,1973,95, 2303. C. H . Heathcock, R. A. Badger, and J . W. Patterson, J . Amer. Chem. SOC.,1967, 89, 41 13. E. Piers, M. B. Geraghty, F. Kido, and M. Soucy, Synth. Comm., 1973, 3 , 39.
I 04
Terpenoids and Steroids
1
C0,Me
CH,OR
I
vii, viii
Reagents: i, LiAlH,; ii, Ac,O-DMSO; iii, Li-NH,; iv, TsC1-py; v, H + ;vi, MeSOCH,Na; vii, Mg-MeOH: viii, LiAIH,Bu';; ix, TsNHNH,; x, LiAlH.; xi, Zn-HOAc; xii, Na-Me,CHOH; xiii, TsCI-py; xiv, H +; xv, MeSOCH,Na.
Scheme 17
105
Sesquiterpenoids
(147) R = H, (148) R = CHOH
AcOCH
0 Jiv-vi
(' I 50)
MeOCH+
(149)
H
(153) R', R2 = 0 (154) R' = H, R2 = OH (155) R' = O H , R 2 = H Reagents : i, DDQ; ii, Me,CuLi, iii, MeCOCl; iv, 0,; v, H,O,-OH-; vi, CH,N,; vii, NaN(SiMe,),; viii, NaN(SiMe,),-Me,CHBr; ix, MeOCH=PPh,; x, H +-H,O; xi, K,CO,-MeOH; xii, CH, =PPh,; xiii, disiamylborane-THF; xiv, MsCl; xv, NaN(SiMe,),-(MeOCH,), . Scbeme 18
which underwent intramolecular Claisen condensation in the presence of sodium bis(trimethylsily1)amideto provide (150). Selective formation of the monoenol ether (15 l), followed by appropriate modification of the bridged ketone, provided a mixture of aldehydes, from which the desired keto-mesylate (152) could be obtained. The final cyclization step in the synthesis was accomplished in 84% yield by treating the keto-mesylate (152) with sodium bis(trimethylsily1)amidein dimethoxyethane. Conversion of (- )-ylangocamphor(153) into the correspond-
106
Terpenoih and Steroids
ing alcohols (154) and (155) was accomplished using Ca-liquid NH3 and LiAlH, respectively. Copacamphor ( 1 56), ylangocamphor (1 53), sativene (1 58), and copacamphene (159) have also been synthesized (Scheme 19) as part of a general synthetic
;Scheme
1
I
B...f (155)
(158) R' = H , R 2 = Pr' (159) R' = Pr', R2 = H
(1 57)
Reagents: i, Bu'OK-Bu'OH; ii, SOC1,-py; iii, Pt-H,; iv, LiAlH,; v, MeS0,Cl-py.
Scheme 19
approach to s e s q ~ i t e r p e n o i d s ~(cf: ' . ~p. ~ 99). Since the synthetic route illustrated in Scheme 15 (p. 101)provides the key intermediate, campherenone (118), in either enantiomeric form,67,68the synthesis of any of the enantiomers of copacamphor,
107
Sesquiterpenoids
ylangocamphor, copacamphene, and sativene is now a comparatively simple procedure. The structure of v.ictoxinine (164), a metabolite of Helrninthosporiurn oictoriae and H . satiuurn, has been confirmed by synthesis from prehelminthosporol (160) (Scheme 20)." Other metabolites of H. victoriae include (162) and the trycyclic ether (163),which can also be obtained from (162)by treatment with acid.
Reagents: i, H + ; ii, MeS0,Cl; iii, H,NCH,CH,OH.
Scheme 20
The total synthesis of dendrobine (165), a picrotoxane-type alkaloid isolated ~~,~~ from Dendrobiurn species, has been reported by two research g r o ~ p s . One synthesis,8 involving the separation of isomers at several stages, is summarized in Scheme 21. In the other synthesiss3 (Scheme 22) the tricyclic intermediate (1 67) was formed from (1 66) by intramolecular Michael additions4 followed by aldol condensation. 'I
'* 83
84
F. Dorn and D. Arigoni, J.C.S. Chem. Comm., 1973, 1342. Y. Inubushi, T. Kikuchi, T. Ibuka, K. Tanaka, 1. Saji, and K. Tokane, J.C.S. Chern. Comm., 1972, 1252. K. Yamada, M. Susuki, Y. Hayakawa, K. Aoki, H. Nakamura, H . Nagase, and Y . Hirata, J. Amer. Chem. SOC.,1972,94, 8278; Tetrahedron Letters, 1973, 331. W. S. Johnson, S. Shulman, K. I. Williamson, and R. Pappo, J . Org. Chem., 1962,27, 201 5 .
Terpenoih and Steroidr
108
11 Himachalane, Longipinane, Longicamphane, Longifolane, and Cyclolongicamphane
Acid-catalysed cyclization of the keto-triene (168) to the bicyclic keto-alkene (169) is the key feature of a recent synthesis of a- (170) and P-himachalene (172) (Scheme 23).85 A new synthesis of (+)-a- and (k)-P-longipinene, (177) and (178), has been achieved by photocyclization of the triene (173) followed by ring expansion of the derived ketone (175) (Scheme 24).86 A second total synthesis of ( )-longifolene (184) has been reported.87 Cyclization of the keto-epoxide (179) yielded a tricyclic ketol (180) which was converted into (182) by treatment with dibromocarbene followed by silver ion-assisted ring enlargement. Reductive
lxv
-
xix, x x
xvi-xviii
I
CN
$5
'r
0
0
0
(165) Reagents: i, TsCl; ii, NaCN-DMSO; iii, H,-5% Pd/SrCO,; iv, Br,; v, -HBr; vi, (CH ,OH),-H ; vii, KOH-(CH ,OH) ,-H ,O; viii, HCl-H ,O ; ix, MeNH,HCl; x, Pr'MgBr; xi, KHSO,,A; xii, I,-AgOAc; xiii, KOH-MeOH; xiv, Cr0,-py; xv, EtzAICN; xvi, NaBH,; xvii, KOH-H20; xviii, HCl-H,O; xix, Et,O+BF,- ; xx, NaBH,-glyme. +
Scheme 21
85
B6
''
E. Wenkert and K. Naemura, Synth. Comm., 1973, 3, 45. M. Miyashita and A. Yoshikoshi, J.C.S. Chem. Comm., 1972, 1173. J . E. McMurry and S. J. Isser, J. Amer. Chem. SOC.,1972, 94, 7132.
Sesquiteipeno ib
109
Reagents: i, K0Bu'-HOBu'; ii, CH,N,; iii, Ac,O-H'; iv, 0,;v, NN-carbonyldi-imidazole; vi, MeNH 2-H20-glyme; vii, pyridine bromide perbromide; viii, NaH-glyme ; ix, (CO,H),; x, NaH-glyme; xi, NaBH,; xii, H'; xiii, Et,O+BF,-; xiv, NaBH,glyme.
Scheme 22
(171) Reagents: i, AlCl,, A; ii, Ph,P+CH2Br--PhLi; iii, MeLi; iv, POC1,-py.
Scheme 23
( 1 72)
Terpenoih and Sterods
110
( 1 73)
( 1 74)
6% iii. iv
'ii
1
VI,
vii
( 1 77)
(178)
Reagents: i, hv; ii, Me,S=CH,; iii, NaN,-DMF; iv, Pt-H,-HOAc; v, NaN0,-H,O-H+ ; vi, MeLi; vii, POCl,-py.
Scheme 24
elimination of bromine followed by Collins oxidation provided the tricyclic ene-dione (183), which was converted into longifolene (184) by the sequence of reactions shown in Scheme 25. Recent studiess8 on isolongifolene epoxide (186) and the derived ketone (187) have provided additional support for the revised configurations assigned to these corn pound^.^^ Deuteriated samples of these compounds were prepared from longifolene, and n.m.r. evidence established the stereochemistry of ketone (187) and indicated that epoxidation had occurred at the or-face of isolongifolene. Additional support for these stereochemical assignments has been provided by the results of hydrogenation studiesg0 which show that hydrogenation of (190)and (191)occurs at the ct-face (endo)to yield (192)and (193) respectively. The rearrangement of isolongifolene to the substituted tetralin (194) occurs in trifluoroacetic acid at room temperature." A total " '9
''
G. Mehta and S. K . Kapoor, Tetrahedron Letters, 1973, 497. E. H . Eschinasi, G. W. Shaffer, and A. P. Bartels, Tetrahedron Letters, 1970, 3523. D . V. Banthorpe, A. J. Curtis, and W. D. Fordham, Tetrahedron Letters, 1972, 3865. G . Mehta, Chern. and Ind., 1972, 766.
Sesquiterpenoidr
111
Reagents: i, MeSOCH,Na+-DMSO; ii, H 2 S 0 4 ; iii, Br,CH-KOBu'; iv, AgClO,; v, Na-NH,; vi, Cr0,-py; vii, Me,CuLi; viii, NaBH,-MeS0,CI-NEt,; ix, KOBu'; x, [(Ph,P),RhCl]-H,; xi, MeLi; xii, SOC1,-py.
Scheme 25
&
'0 +-
-go
t
112
Terpenoids and Steroid3
synthesis of (+)-longicyclene (197) has finally been achieved by a route (Scheme 26) in which the construction of the cyclopropane unit was achieved by an intramolecular carbene insertion process [(195)-+( 196)].92 An unsuccessful attempt to use the homo-Diels-Alder reaction in the construction of longicyclene has also been described. 12 Humulane, Caryophyllane, erc.
As part of an intensive study of the biosynthesis of mono- and sesqui-terpenoid constituents of peppermint (Mentha piperita L.) it has been shown94 that caryophyllene (201) is biosynthesized from three molecules of mevalonic acid (MVA) (198) in accordance with biosynthetic theory (Scheme 27).4 Although caryophyllene and other sesquiterpenoids constitute less than 2 % of peppermint oil they incorporate label from [2-I4C]MVA (198) much more efficiently than the monoterpenoids which constitute the bulk of the oil. This low incorporation of label from MVA into monoterpenoid constituents of peppermint oil is probably characteristic of plants with distinct oil gland^.^^*^^ Degradation of labelled caryophyllene (201) demonstrated that the distribution of radioactivity could be 92 93 94 95
96
S. C. Welch and R. L. Walters, Synth. Comm., 1973, 3, 15. T. R . Kelly, Tetrahedron Letters, 1973, 437. R. Croteau and W. D. Loomis, Phyrochemisfry, 1972, 11, 1055. D. V. Banthorpe, B. V. Charlwood, and M . J . 0. Francis, Chem. Rev., 1972, 7 2 , 115. W . D. Loomis, in 'Terpenoids in Plants', ed. J. B. Pridham, Academic Press, New York, 1967, p. 59.
113
Sesquiterpenoih
1
v-viii
(195)
Reagents: i, BuiAlH-THF; ii, HC1; iii, MsCI-NEt,; iv, collidine, A ; v, Ph,P=CHOMeDMSO; vi, HCI0,-H,O-Et,O; vii, K,CO,-MeOH; viii, Cr0,-Me,CO; ix, (COCI),; x, CH,N,; xi, Cu-THF, A; xii. BuiAIH-THF; xiii, MsCI-NEt,; xiv, LiAlH,.
Scheme 24
represented by the asterisks shown in structure (201),and it is interesting to note that the gem-dimethyl group, containing label from dimethylallyl pyrophosphate (200), is considerably less active than the positions derived from isopentenyl pyrophosphate (199).94 A similar effect has been noted in the monoterpenoid s e r i e ~ ~and ~ * may ~ ’ be due to an endogenous dimethylallyl pyrophosphate pool in the plant or to compartmentalization effects. The biosynthesis of illudin M (204) in CIytocybe illudens has been studied using doubly labelled m e v a l o n a t e ~ . ~ ~ Incorporation of three [2,2-3H,]- and one [4(R)-4-3H,I-mevalonoid labels and the loss of a [2,2-3H,] label from the cyclopropane ring suggests that humulene (202) cyclizes to the illudene skeleton (203) in a non-concerted manner (Scheme 27). An interesting new sesquiterpenoid, velleral (206), has been isolated from Lactarius vellerens and L.pergamenus.” It is presumably related biosynthetically to isovelleral(205),which is found in the same source. 97
98 99
D . V. Banthorpe, B. V. Chariwood, and M. R. Young, J.C.S. Perkin I , 1972, 1532 and references cited therein. J. R. Hanson and T. Marten, J.C.S. Chem. Comm., 1973, 171. G. Magnusson, S. Thoren, and T. Drakenberg, Tetrahedron, 1973, 29, 1621.
Terpenoids and Steroids
114
1
*";
(202)
0
Scheme 27
13 Germacrane, Eudesmane, Eremophilane, etc. Considerable interest is being shown in the conformational analysis of sesquiterpenoids containing ten-membered rings (germacranes, germacranolides). The variable-temperature n.m.r. spectrum of deuteriated hedycaryol (206) has been used to demonstrate the existence of specific conformers of this molecule, and
Sesquiterpenoids
115
has provided experimental basis for current biosynthetic theory4 associated with the cyclization of germacradiene-type intermediates.'" A similar study' on germacra-l(10),4,7(1l)-trienyl-9-acetate (208) has shown that in solution at 30 "C this compound exists as two n.m.r.-distinguishable conformers in a ratio of 92 : 8. The interpretation of intramolecular nuclear Overhauser effects (NOE) (cf: ref. 3) indicates that the major conformer is represented by (209). NOE's have also been used to demonstrate that the major conformers of bicyclogermacrene and isobicyclogermacrene in solution are represented by (210) and (211) respectively.lo2 OAc
H
1
The structures of many new germacranolides have been determined recently ; these include cupaserrin (212) and deacetylcupaserrin (213) (Eupatorium semiserratum DC),'03 eupacunin (214), eupacunoxin (215), eupatocunin (216), eupatocunoxin (217), and eupatocunolin (218) (Eupatorium cuneifolium),'O4 provincialin (219) (Liatrisprovincialis Godfrey),'05 deacetyl-laurenobiolide (220) and specioformin (221) (Arternisia tridentata),' O6 alatolide (222) (Jurinea d a t a Cass.),'07 lipiferolide (223) (Liriodendron tulipifera L.),'08 punctatin (224)(Liatris lo' lo*
Io4
lo6
lo'
P. S. Wharton, Y . - C .Poon, and H. C. Kluender, J. Org. Chem., 1973, 38, 735. 1. Horibe, K. Tori, K. Takeda, and T. Ogina, Tetrahedron Letters, 1973, 735. K. Nishimura, I. Horibe, and K. Tori, Tetrahedron, 1973, 29, 271. S. M. Kupchan, T. Fujita, M. Maruyama, and R. W. Britton, J. Org. Chem., 1973, 38, 1260. S. M. Kupchan, M. Maruyama, R. J. Hemingway, J. C. Hemingway, S. Shibuya, and T. Fujita, J. Org. Chem., 1973, 38, 2189. W. Herz and I. Wahlberg, J. Org. Chem., 1973, 38, 2485. F. Shafizadeh and N. R. Bhadane, Phyfochemistry, 1973, 12, 857; cf. R . G . Kelsey, M. S. Morris, N. R. Bhadane, and F. Shafizadeh, ibid., 1973, 12, 1345. D. Drozdz, Z. Samek, M. Holub, and V. Herout, Coll. Czech. Chern. Comm., 1973,38, 727. R. W. Doskotch, S. L. Keely, jun., and C. D. Hufford, J.C.S. Chem. Comm., 1972, 1137; cf. R. W. Doskotch, C. D. Hufford, and F. S. El-Feraly, J. Org. Chem., 1972, 37, 2740.
Terpenoids and Steroids
116
p u n c t ~ t a ) , 'novanin ~~ (225) (Artemisia nova),' l o molephantin (226) (Elephantopus mollis),' and liatrin (227) (Liatris chaprnanii).' l 2 Many of the compounds listed
'
above exhibit interesting antileukaerni~'~~*' l 2 or cytotoxic proper tie^.'^^^'^^^'^^
HO..
w'
(OR OQOR
3
0
0
(214) R' = H, R 2 = Ac,
(212) R = COMe (213) R = H
R3 = COCMe=CHMe 0
(215) R 1 = H, R2 = Ac, R3 = COCL'CHMe
1
Me R2
OR' 0 (216) R' = COCMe=CHMe, R2 = H
AcOQ
CH2R1
R2CH2
(218) R'
=
0 H, R2 = OH
0 (217)
R'
= H, R 2 = COCcCHMe
I
Me
Ace*
OR
k---!+
0
CH,OH (219) R
= COC-CH20C0
II
CHCH,OH log 'lo
'I2
I
'CHMe
W. Herz and I. Wahlberg, Phytochemistry, 1973, 12, 1421. M. A. Irwin and T. A. Geissman, Phytochemistry, 1973, 12, 875. K.-H. Lee, H. Furukawa, M. Kozuka, H.-C. Huang, P. A. Luhan, and A. T. McPhail, J.C.S. Chem. Comm., 1973, 476. S. M. Kupchan, V. H . Davies, T. Fujita, M. R. Cox, R. J. Restivo, and R. F. Bryan, J. Org. Chem., 1973, 38, 1853.
Sesquiterpenoids
117
Wo
&,
OCOCH Me,
,
OH
qc 0
(223)
HOCH, QR 0
Ro
(224) R
AcO
0 =
COC=CHMe
I
CH,OH
(225)
7H2OH OCOC=CHMe
X-Ray crystal-structure analysis has shown that the configurations of mikanolide (228) and miscandenin (230)are in accordance with their postulated formation from the epoxy-diene (229).'l 3 A [3,3] sigmatropic rearrangement can be invoked to account for the formation of miscandenin (230). I
' ''
P.J.
Cox, G . A. Sim,
J. S. Roberts, and W. Herz, J.C.S. Chem. Comm., 1973, 428.
Terpenoih and Steroids
118
According to biosynthetic theory4 the eudesmane group of ssquiterpenoids is derived by cyclization of a germacrane derivative (232) formed from trans,transfarnesyl pyrophosphate (231). The successful duplication of the second step in the sequence has been elegantly demonstrated in the laboratory' l 4 and recent reports represent further examples of these cyclization processes.' 5 , 1 l 6 Thermal
'
cyclization of elemol (235) produces a mixture of hedycaryol (236) and starting material which undergoes silver ion-catalysed rearrangement to a- (237) and Elemol and the eudesmols co-occur in the citronella oil P-eudesmane (238).
q OH
(Ceylon variety) and this may indicate that a biosynthetic relationship exists between them. fl-Elemen-9fl-ol (240) and a new germacrane derivative, agerol (239), have been shown to co-occur in Achillea ageratum L., and the proposed
I'
'I5
T. W. Sam and J . K . Sutherland, Chem. Comm., 1971, 970 and references cited therein. T. C. Jain and J. E. McCloskey, Tetrahedron Letters, 1972, 5139.
Sesquiterpenoids
119
biosynthetic relationship between them is supported by the thermal conversion (239)+(240).’l 6 Similarly acoragermacrone (241), which co-occurs with shyobunone (242) in Acorus calarnus L., can be cyclized to (242) and (243).”’ OH
OH
The obvious difficulties associated with attempts to duplicate the first step, (231)+ (232), in the proposed biosynthetic route to eudesmanes prompted an alternative proposal involving a six-membered-ring intermediate (233).3c,’ A biogenetic-type synthesis of (+)-junenol (244) was based on this hypothesis.3c.’l 8 Acolamone (245) and isoacolamone (246), which are closely related to junenol(244), have recently been isolated from Acorus calarnus L.’
Several synthetic routes to (*)-occidentalol (252) have been reported recently.3c”20’121 In one of these (Scheme 28) a cis-fused decalin system (249) containing the required homoannular 1,3-diene functionality was constructed in 2 5 - 4 0 % yield by Diels-Alder reaction between 3-methoxycarbonyl-2-pyrone (247) and the enone (248)l2’ (cf. Scheme 17, p. 104). An additional feature of this synthesis was the use of an appropriate thioether (250) to construct the inter116
117
118 119
120
R. Grandi, A. Marchesini, U. M . Pagnoni, and R. Trave, Tetrahedron Letters, 1973, 1765. M. Iguchi, M . Niwa, A. Nishiyama, and S. Yamamura, Tetrahedron Letters, 1973, 2759. M. A. Schwartz, J. D. Crowell, and J. H. Musser, J. Amer. Chem. SOC.,1972,94,4361. M. Niwa, A . Nishiyama, M . Iguchi, and S. Yamamura, Chem. Letters, 1972, 8 2 3 . D. S. Watt and E. J . Corey, Tetrahedron Letters, 1972, 4651.
Terpenoidrs and Steroid
[& C02Me
Reagents: i, LiAlH,-THF; MeLi.
ii, (EtO),P(O)CH(SMe)Me-HMPA;
iii, HgC1,-H,O;
iv,
Scheme 28
mediate ketone (25 1) which was subsequently converted into ( +)-occidentalol (252). The other synthesis12' which appeared during the period under review was based on the suggestion'22 that the biosynthesis of (+)-occidentalol (257)'23 and related compounds involves cyclization of dehydrohedycaryol (259) (cf: Scheme 30). Thus irradiation of the trans-decalin derivative (254) (Scheme 29) provided an intermediate cyclodecatriene (255)which underwent thermal cyclizaI
I
I 22
A . G. Hortmann, D. S. Daniel, and J. E. Martinelli, J. Org. Chem., 1973, 38, 728. A. G. Hortmann, Tetrahedron Letters, 1968, 5785; cJ E. J. Corey and A. G. Hortmann, J. Amer. Chem. SOC.,1965,87, 573'6. Further support for the stereochemistry of (+)-occidentalol (257) has been provided by T. Suga, K. lnamura, T. Shishibori, and E. von Rudloff, Bull. Chem. SOC.Japan, 1972,45, 3502; CJ A. G. Hortmann and J. B. DeRoos, J. Org. Chem., 1969,34, 736.
Sesquiterpenoib
121
Reagents: i, hv; ii, MeLi; iii, 250°C; iv, h v ; v, -70°C.
Scheme 29
tion to the synthetic precursors of 7-epi-(-)-occidentalol(256) and ( + )-occidentalol(257). Using (- )-trans-occidentalol(258) instead of (254)in the photofissionthermal cyclization sequence gave (256) and (257) directly and provided experimental support for the suggested intermediacy of a suitably substituted cyclodecatriene [e.g. (259)] in the biosynthesis of ( + )-occidentalol. The relative yield (3 : 2) of (256) and (257) in the thermal cyclization of (259) is similar to the relative abundance of ( + )-occidol(260)and (+)-occidentalol(257) in T.occidenta!is and T. koraiensis and it has been suggestedI2l that 7-epi-(-)-occidentalol(256)
122
Terpenoids and Steroih
the biosynthetic precursor of ( +)-occidol (260). The epoxide (261) of triene (259) has been proposed'24 as a probable intermediate in the biosynthesis (Scheme 30) of ( + )-occidenol (262) [cf. (229)J and the stereochemistry of 7-epi(-)-occidental01 (256) has led to its implication in the biosynthesis of dehydrochamaecynene (264) and chamaecynone (263)' IS
i
T
OH
(264)
Scheme 30
124
B. Tomita and Y . Hirose, Tetrahedron Letters, 1970, 2 3 5 .
Sesquiterpenoic?..
123
Recent synthetic studies and appropriate spectral comparisons have shown that paridisiol, a component of grapefruit oil, is identical to intermedeol (265). 2 5 Related compounds, longlilobol(266) and pygmol(267) have been isolated from Artemisin longilohn' 2 6 and A . p v g m ~ e u 'respectively. ~~ The isolation and structural elucidation of several eudesmanolides have been described recently : these iriclude arbusculin-B (268) and isomers (269) and (270) (Frullania tamarisci),' 28 iasolide (271) (Luser trilohum),*2 9 graveolide (272) (Inula gruveolens).' 3 0 arbusculin-D (273) (Artemisia arbus~ulu),'~~ and ridentin-B (274) ( A . tripnrtita).132
'
H0
125
126 121 128
I29 130 13 I
132
J. W. Huffman and L. H . Zalkow, Tetrahedron Letters, 1973, 751. F. Shafizadeh and N. R. Bhadane. Tetrahedron Letters, 1973, 2171. M. A. Irwin and T. A. Geissman, Phytochemistry, 1973, 12, 849. J. D. Connolly and I . M . S. Thornton, Phytochemistry, 1973, 12, 631. M . Holub and Z. Samek, Coll. Czech. Chem. Comm., 1973, 38, 1428. G. S. d'Alcontres, M . Gattuso, M . C. Aversa, and C. Caristi, Gazzetta, 1973, 103, 239. M. A. Irwin and T. A. Geissman, Phytochemistry, 1973, 12, 8 5 3 . M . A. Irwin and T. A . Geissman, Phytochemistry, 1973, 12, 871.
124
Terpenoih and Steroids
Naturally occurring eudesmanolides having cis- or trans-fused cc-methylene, y-butyrolactone rings have been made more accessible as a result of recent synthetic studies.'33 In the cis-fused bromination and dehydrobromination steps convert cis-cc-methyl-lactones [e.g. (275)] into cis-fused CImethylene-lactones (276) (Scheme 31). Benzoylation followed by thermal
(275) Reagents: i, Ph,CLi-dimethoxyethane;
ii, BrCH,CH,Br ; iii, DBN-MePh, A.
Scheme 31
elimination of benzoic acid produces the corresponding conversion in the trans series, and the use of this in the synthesis of (+)-arbusculin-B (268) (Scheme 32)
q., QOCOPh
O
O
n o
0 (268)
Reagents: i, Ph,CLi-dimethoxyethane;
ii, (PhCO),-dimethoxyethane, 5 "C; iii, -450 "C.
Scheme 32
has been described.'33b The nature of the trichloride (279),formed when santonin (277) is treated under a variety of conditions (PC1,-CHCl,, PC1,-HOAc, or SOCl,), has been determined and the monochloride (278) has been shown to be an intermediate in this t r a n s f ~ r m a t i o n . 'The ~ ~ change in reduction state accompanying formation of (279) is very similar to that previously noted in the conversion of pyrosantonin (280) into (28l).' s
,
0 (277)
'33
134
13*
(278)
(279)
A. E. Green, J.-C. Muller, and G. Ourisson, ( a ) Tetrahedron Letters, 1972, 2489; ( b ) ibid., p. 3375. A. Frohlich, K. Ishikawa, and T. B. H. McMurry, Terrahedron Lerters, 1973, 995. T. B. H . McMurry and D . F. Rane, J . Chem. SOC.( C ) , 1971, 3851.
Sesquiterpenoids
125
0 (280)
(281 )
Further studies on the structural elucidation and reactions of eudesmane-type alkaloids have been Wilfordine (282) and alatamine (283) are components of Euonymus a l u m forma striutus (thumb.) ma kin^,'^^ and the corresponding desoxy-compounds, euonine (284), evonimine (285), and evonine (286), have been isolated from seeds of Euonymus Sieboldiana B l ~ m e . ' ~ ' - ' ~All ~ of these alkaloids may be regarded as derivatives of a- and P-agarofuran (287). The synthesis of (+)-norketoagarafuran (288) has been de~cribed.'~'A full account of the structural elucidation and synthesis of rishitinol (289) [cf: (+)occidol(260)], a metabolite of diseased white potato tubers, has been r e ~ 0 r t e d . I ~ ~ This compound is presumably derived from a eudesmane precursor by 1,2-methyl shift and it is interesting to note that it co-occurs with the noreudesmane sesquiterpenoid rishitin (290). A new method for the synthesis of spiroannelated cyclohexenones from enol ethers of cyclic 1,3-diketones has been elegantly used in a simple stereospecific OAc
PhCOO X*..
AcO
(282) X
I oAc
=
<
OAc
, R'
=
OH, R 2 = H
H
CH
(283) X = 0, R' = OH, R 2 = H OAc (284) X = , R' = H, R 2 = H H (285) X = 0, R' = H, RZ = H (286) X = 0, R' = I4,R2 = Me
<
R2
(287)
(288)
(289)
(290)
Y. Shizuri, K. Yamada, and Y. Hirata, Tetrahedron Letters, 1973, 741. K. Sugiura, K. Yamada, and Y. Hirata, Tetrahedron Letters, 1973, 113. 1 3 8 Y.Shizuri, H. Wada, K. Sugiura, K. Yamada, and Y . Hirata, Tetrahedron, 1973, 29, 1773. L 3 9 Y. Shizuri, H. Wada, K. Yamada, and Y. Hirata, Tetrahedron, 1973, 29, 1795. I4O T. R. Kelly, J . Org. Chem., 1972, 37, 3393. 14' N. Katsui, A. Matsunaga, K. Imaizumi, T. Masamune, and K . Tomiyama, Bull. Chem. SOC.Japan, 1972,45, 2871. 136
13'
126
Terpenoids and Steroids
synthesis of ( & )-vetivone (296).'42 Spiroannelation was accomplished by treating the lithium enolate of enol ether (291) with the dichloride (292) at room temperature and the stereospecificity of the process was rationalized by invoking initial formation of (293) followed by completion of the ring trans to the methyl group as shown in (294). A similar approach to the 0-vetivone system has been described by another group and differsmainly in the sequence of alkylation steps associated with the construction of the spiro system.'42a In the latter process the final step involved intramolecular alkylation of allylic alcohol (293a), and the formation of ( 3- )-10-epi-P-vetivone is consistent with the rationale provided above. These syntheses are illustrated in Scheme 33. 0
P O
(291a)
r-c c%
+ PhCH,O
--+
SPh
PhCH,O
(292a)
Y 10-epimer of (296)
Reagents: i, LiNPr', ( 1 equiv.); ii, LiNPr; (2 equivs.); iii, MeLi; iv, HC1; v, Li-EtNH,, - 7 8 "C;vi, 70% H C 0 , H .
Scheme 33 G . Stork, R . L. Danheiser, and B. Ganem, J . Amer. Chem. SOC.,1973,95, 3414. 1 4 2 a PM . . McCurry, R. K. Singh, and S. Link, Tetrahedron Letters, 1973, 1155. 142
Sesquit erpeno ids
127
Considerable interest is still being shown in the structure, synthesis, biosynthesis, and physiological activity of the eremophilane group of sesquiterpenoids. The structures of capsidiol (297)'"3 and a variety of new furanoeremophilene derivatives, (298 --300),'44 (301-316),'45 (317-321),'46 and (322),14' have been elucidated by chemical and spectroscopic means.
(297)
(298) R = COCMe=CHMe
(299) K
=
COCMe=CHMe
RO
(300) R = COCMe=CHMe
OR (301) R
=
COCMe=CHMe
m OR
(302) R = COCMe=CHMe (303) R = COMe
HO'. OR (304) R (305) R 14' 144 145 146
14'
= =
COCMe=CHMe COCH=CMe2
(307) R (308) R
= =
COCH2CHMe2 COCHMe,
M. Gordon, A . Stoessl, and J. B. Stothers, Cunad. J. Chem.. 1973.51, 748. F. Bohlmann and N. Rao, Tetrahedron Letters, 1973, 613. F. Bohlmann, C. Zdero, and N. Rao, Chem. Ber., 1972, 105, 3523. L. Novotny, M. Krojidlo, Z. Samek, J . Kohoutova. and F. Sorm, CON. Czech. Chem. Comm., 1973, 38, 739. C. Kabuto, N. Takada, S. Maeda, and Y. Kitahara, Chern. Letters, 1973, 371.
Terpenoih and Steroih
128
rn OR
(309) R = COMe (310) R = COCMe=CHMe (311) R = COCH,CHMe, (312) R = COCHMe,
0
H
(313) R = COCH,CHMe, (314) R = COCHMe, (315) R = COC(CH,OH)=CHMe
(316) R
=
COCH,CHMe,
R'O
0RZ (317) R' = H, R2 = COCMe=CHMe (318) R' = H, R2 = COCH(Me)CH,Me (319) R' = H, RZ = COCHMe, (320) R' = COMe, R2 = COCHMe,
OR (321) R = COCMe=CHMe
It is commonly assumed that the erernophilane skeleton (324) is derived in Nature by rearrangement of a eudesmane derivative (323a or b). A biogenetictype transformation based on the proposal is illustrated by the acid-catalysed rearrangement of dihydroalantolactone epoxide (326) to (327).'48 14*
I . Kitigawa, Y. Yamazoe, R. Takeda, and I. Yosioka, Tetrahedron Letters, 1972,4843.
Sesquiterpenoids
129
R (323a)
(323b)
A more conventional approach to the eremophilane system is described in a recent report dealing with the conversion of the ketone (328)into (+)-nootkatone (329)14' and (*)-7-epinootkatone (330). The major product from annelation of ketone (328) is (330) and the amount of (+)-nootkatone formed depends on reaction conditions. Conversion of 11,12-dihydronootkatone (331) into dienone (332) demonstrated that ( + )-nardostachone is not correctly represented by this ~ t r u c t u r e . ' This ~ ~ synthetic result, coupled with a re-appraisal of the published spectroscopic data,'" has resulted in an alternative structural assignment (333)
to (+)-nardo~tachone.'~~ Complete details of a previously reported synthesis of ( f)-eremophilenolide (336), ( k )-tetrahydroligularenolide (337), and ( +_ )-aristolochene (338) have been reported.3b91 A prominent feature of the synthesis was 149
H. C. Odom, jun. and A. R. Pinder, J.C.S. Perkin I, 1972,2193. S. D. Sastry, M . L. Maheswari, K. K. Chakravarti, and S. C. Bhattacharyya, Tetrahedron, 1967, 23, 2491. E. Piers and M . B. Geraghty, Cunud. J . Chem., 1973,51,2166.
Terpenoids and Steroids
130
the use of a common synthetic intermediate (335)derived from octalone (334)by a completely regioselective route (Scheme 34).
(334)
iY 'C0,Me
&> (335)
C0,Me
I 4
C02Me x ---xii
H
0 ---. (338)
0 --.. (336)
(337)
Reagents: i, DDQ; ii, Ag,O; iii, Ag,O-MeI; iv, NaBH,--py; v, NaH-BrCH,CO,Me; vi, NaOH-EtOH-H ? O ;vii, H,-Rh/C-NaOH-EtOH; viii, p-MeC,H,SO,ClPhMe; ix, Ph,CNa-MeI; x, Ra-Ni-EtOH ; xi, MeLi-Et,O; xii, SOCl 2-py.
Scheme 34
As a result of recent synthetic studies the absolute configuration of furfagin A (341) has been established.' 5 2 This information has prompted the suggestion 15*
M. Tada, Y. Moriyama, Y. Tanahashi, and T. Takahasi, Tetrahedron Letters, 1972, 525 1 , 5255, and references cited therein.
Sesquiterprnoids
131
that the formation of this compound from furanoeremophilane-6P,1O~-diol(338) involves a spiro-carbonium ion intermediate (340). Furfagin B (342) and benzofuran (343) are other products formed in the skeletal rearrangement of diol(338).
0€1 (338)
(339)
(342)
(343)
A detailed study of the biosynthesis of petasin (346) has been described. 5 3 Administration of [2- ''C-3R,S]mevalonolactone (344) to growing leaves or flowers of Petasites hybridus L. and the subsequent location of radioactive carbon atoms in petasin (346) by degradative reactions has provided results consistent with current biosynthetic t h e ~ r y . Further ~ support for the rearrangement mechanism shown in Scheme 35 was obtained when [2-' 4C-3R,S,4R-4-3H]mevalonolactone (345) was used as a precursor. Subsequent degradation experiments and spectroscopic data demonstrated that a tritium atom was specifically located at position 4 in petasin (346). The absence of tritium at C-1 and its presence at C-7 was not clearly established by these experiment^.'^^ The same group has recently used the technique of deuterium labelling in transitu in the g.1.c. column to identify fukinone (347) in leaf extracts of Petasites hybridus.'54 Two new C I z ketones (348) and (349), possessing degraded eudesmane and eremophilane structures respectively, have been isolated from Reunion vetiver ' ~ ~ the structure of (349) has been conoil [Vetiveria zizanoides (L.) N a ~ h ] , and firmed by synthesis from (+)-or-vetivone (350). A full account of the previously 15' 154
L55
C . J . W . Brooks and R. A. B. Keates, Phytochemistry, 1972, 11, 3235. R . A. B. Keates, G. M. Anthony, and C. J. W. Brooks, Phytochemistry, 1973, 12, 879. B. Maurer, M . Fracheboud, A. Grieder, and G. Ohloff, Helv. Chim. Acra, 1972.55, 237.
Terpenoids and Steroih
132
T HOCH,
CO,H
(344)
--+
--P
& /*
(345)
OPP
H I
I *
J
Scheme 35
reported3 synthesis of (k)-ishwarane (351) has been published' warane (352) has been isolated from Aristolochia debelis.' 5 7
56
and 3-oxoish-
(351)
R
=
H,
(352) R = 0 156
15'
R. B. Kelly, J. Zamecnik, and B. A. Beckett, Canad. J. Chem., 1972, 50, 3455. R. Nishida and Z. Kumazawa, Agric. and Biol. Chem. (Japan), 1973, 37, 341.
Sesquiterpenoids
133
The fukinane group of sesquiterpenoids is probably derived in Nature from eremophilane precursors, and the numbering system used is based on this presumption. Fukinolide (353), dihydrofukinolide (354), homofukinolide (355), S-fukinolide (356), and fukinanolide (357) are new representatives of this class which have been isolated from the flower stalks of Petasitesjaponicus Maxim.' 5 8
(353) R' = COMe, R2 = COCMe=CHMe (354) R' = COMe, R2 = COCH(Me)CH,Me (355) R' = R2 = COCMe=CHMe (356) R' = COMe, R2 = COCMe=CHSMe
(357)
14 Guaiane, Cyperane, Seychellane, Aromadendrane, and Bourbonane Many sesquiterpenoid lactones (cf. Section 13) have been shown to have interesting anti-tumour or cytotoxic activity and the search for new sesquiterpenoids of this type has resulted in recent report^^^^-'^^ describing the isolation and structural elucidation of several new guaianolides [(358-370) ; see Table 21. Table 2 Guaianolides Compound
Viscidulin A (358) Viscidulin B (359) Visciddin C (360) Rupicolin A (361a) Rupicolin B (361b) Rupin A (362) Rupin B (363) Cumambrin-B oxide (364) Christinine (365) Archangelolide (366) Trilobolide (367) Euparotin (368) Carolenalin (369) Carolenin (370) 15'
Source
Reference
Artemisia cana Pursh ssp. viscidula
159
Artemisia tripartita ssp. rupicoIa
160
Stevia serrata Cav. Laserpitum archengelica Wulf. Laser trilobiurn (L.) Borkh. Eupatorium rotundifolium L.
161 162 163 164
Helenium autumnale
165
K. Naya, M. Hayashi, I. Takagi, S. Nakamura, and M. Kobayashi, Bull. Chem. SOC. Japan, 1972,45, 3673.
159 161
16' 163
164
16s
F. Shafizadeh and N . R. Bhadane, J . Org. Chem., 1972, 37, 3168. M. A. Irwin and T. A. Geissman, Phytochemistry, 1973, 12, 863. M. Salmon, E. Diaz, and A. Ortega, J . Org. Chem., 1973,38, 1759. M. Holub and Z. Samek, Coll. Czech. Chem. Comm., 1973,38, 731. M. Holub, Z. Samek, R. deGroote, V. Herout, and F. Sorm, Coll. Czech. Chem. Comm., 1973,38, 1551. A. T. McPhail and G . A. Sim, Tetrahedron, 1973,29, 1751. H. Furukawa, K.-H. Lee, T. Shingu, R. Meck, and C. Plantadosi, J . Org. Chem., 1973, 38, 1722.
Terpenoids and Steroids
! 34
OR
0
a
0
(361a and b)
(359) R = H (360) R = COMe
(358)
OR
OH
o w o A O?
0
(362) R (363) R
=
=
H COMe
0
(365)
OAc
0 (366) R ' = COCMe=CHMe R 2 = COCH(Me)CH,Me
(367) R' = COCMe=CHMe R 2 = COCH(Me)CH,Me
0 (368) R = COCMe=CHMe
(369) R (370) R
= =
COCMe=CHMe H
Sesquiterpenoids
135
A recent X-ray crystallographic analysis has shown that pseudoivalin has the absolute configuration shown in (373).le6 Pseudoivalin (373)is a rare e ~ a m p l e ' ~ ' of a guaianolide co-occurring with a eudesmanolide, microcephalin (372), and it has been suggested4" that these compounds could be derived from a common germacradiene derivative [cf. (371)l. It is also conceivable that a direct biosynthetic link exists between these compounds. The absolute configurations of three novel cyclopropanoid guaianolides, axivalin (374), ivaxillarin (375), and anhydroivaxillarin (376), have been determined by X-ray analysis.'68 Myliol (377),a constituent of the liverwort Mylia taylorii (Hook.)Gray, has been assigned OH
AcO
;g. % 0
HO
&$ I
HO
0 0
0
0
(377)
an interesting new tetracyclic structure on the basis of chemical and spectroscopic e~idence.'~' Pseudoguaianolides have evoked considerable interest3 and a G . D . Anderson, R. Gitany, R. S . McEwen, and W. Herz, Tetrahedron Letters, 1973, 2409. '67
CJ S. Inayama, T. Kawamata, and M. Yanagita, Phytochemistry, 1973, 12, 1741. G . D. Anderson, R. S. McEwen, and W. Herz, Tetrahedron Letters, 1972, 4423. V . Benesova, P. Sedmera, V. Herout, and F. Sorm, Coll. Czech. Chem. Comm., 1973,
38, 1084.
136
Terpenoids and Steroids
oq
recent report has shown that the modified pseudoguaianolide cordilin (378) is the C-5 epimer of psilostachyin. Both compounds co-occur with psilostachyin C (379) in Ambrosia cordifoh (Gray) Payne. O'
'
p Q ;
0 40'.
0'
HO
0
0
0
0 (379)
(378)
A recent synthesis of guaipyridine (381) and 10-epiguaipyridine (383) from guaiol (380) and a-gurjunene (382) respectively (Scheme 36) has established the absolute configuration of these sesquiterpenoid alkaloids.' Guaipyridine (38 1) and guaiol(380) are components of patchouli oil and it has been suggested that the former compound was previously isolated from this source and incorrectly identified as 10-epiguaipyridine (383).172 A new synthetic route (Scheme 37) 1 1
(9
I
--*
(380)
'
OH
'bH
1
iii
1
1
Reagents: i, H,-PdlC; ii, 0,; iii, NH,OH,HCl; iv, SOCl,-py.
Scheme 36
"' W. Herz, D. Raulais, and G. D. Anderson, Phytochemisrry, 1973, 12, 1415. "'
A. Van der Gen, L. M. Van der Linde, and J. G. Witteveen, Rec. Trav. chim., 1972,91, 1433. G. Buchi, J. M . Goldman, and D. W. Mayo, J. Amer. Chem. SOC.,1966,88, 3109.
137
Sesquiterpmnoids
cn c
a
T f f
gg
Y
0
Y
z'
0
J!J --z 3=
'r' g: 0
138
Terpenoids and Steroids
t
0 3:
t
I
I
Sesquiterprtioids
139
to bulnesol (589)17' and guaiol (3801'74 has been described. A key feature of each synthesis i s the cyclization of epimeric aldehydes, (384) and (385), and a stereospecific hydride shift apparently occurs during the formation of the bicyclic enol acetates (386)and (388). The guaiane skeleton can also be formed by cyclization of appropriate germacrane derivatives, and a recent report' 7 5 describes the conversion of germacrone (390) into the guaiane derivative (391) in 50 "/, yield. Some interesting rearrangements of cyperene epoxide (392)have been recorded On treatment with formic acid the major products are the allylic alcohol (393) and the diol (394).'76 The latter compound is produced by a multistep rearrangement in which the first bond migration is not concerted with ringopening of the epoxide. In contrast, treatment of cyperene epoxide (392) with stannic chloride results in alternate ring-opening/bond-migrationsteps and provides a mixture (7 : 1) of tricyclic (395) and bicyclic ketones (396).'77 These rearrangements are shown in Scheme 38. An attractive feature of recent synthetic routes to ( & )-patchouli alcohol (399)'78" (Scheme 39) and (-t )-seychellene (400)'78b(Scheme 40) is the use of a common bicyclic intermediate (398). The latter compound was obtained from the ketone (397) derived by Diels-Alder reaction between 1,3-dimethyl-1,3cyclohexadiene and methyl vinyl ketone. The value of photochemical procedures in organic synthesis has been demonstrated in the reported synthesis of (-)-cyclocolorenone (402)from (-)-maalione (401) (Scheme 41).'79
15 Mono- and Bi-cyclofarnesanes that the absolute configuration (403) previously Previous indications' assigned to (+)-cis-abscisic acid may require revision have been fully confirmed by recent investigations. The revised configuration (406)*is supported by the conversion of (+)-cis-abscisicacid into the acetoxy-diester (405) derived from S-malic acid (404).182 Additional proof has been provided by the fact that (+)-dehydrovomifoliol (407), whose absolute configuration has been established by spectros ~ o p i c and ' ~ ~synthetic'84 means, can be converted into a mixture of (+)-cisN. H. Anderson and H.-S. Uh, Synth. Comm., 1973, 3, 1 1 5. N. H. Anderson and H.-S. Uh, Tetrahedron Letters, 1973, 2079. '" M. Iguchi, M. Niwa, and S . Yamamura, Tetrahedron Letters, 1973, 1687. L. Bang, M. A. Diaz-Parra, and G. Ourisson, Tetrahedron, 1973, 29, 2087. ''' L. Bang and G . Ourisson, Tetrahedron, 1973,29. 2097. 17' R. N. Mirrington and K. J. Schmalzl ( a )J . Org. Chem., 1972,37,2871 ; ( 6 )ibid., p. 2877. D. Caine and P. F. Ingwalson, J . Org. Chem., 1972,37, 3751. R. S. Burden and H. F. Taylor, Tetrahedron Letters, 1970, 4071. R. S. Cahn, C. K. Ingold, and V. Prelog. Angew. Chem. Internat. Edn., 1966, 5 , 385. l E 2G. Ryback, J . C . S . Chem. Comm., 1972, 1190. M. Xoreeda, G . Weiss, and K . Nakanishi, J . Amer. Chem. Soc., 1973, 95, 239; cf N . Harada and K. Nakanishi, Accounts Chem. Res., 1972,5, 257. lE4 K. Mori, Tetrahedron Letterr., 1973, 2635. 173
I"
* It should be noted that the chiral centre at C-1' in the previous assignment of absolute configuration (403) was designated S. According to the modified Cahn-Ingold-Prelog rules,181this designation now applies to C-1' in the revised formulation (406).
Terpenoids and Steroids
140
liv
v, vi
C0,Et
I
H
(399) Reagents: i, Zn-BrCH ,CO,Et ; ii, MeCOC1-PhNMe, ; iii, NaOEt; iv, Li-NH,-EtOH; v, Ph,CCI-py; vi, B,H,, Cr0,-py; vii, KCPh,-MeI; viii, H,-Pd/C; ix, TsCl; x, NaI; xi, Na-THF.
Scheme 39
Sesquiterpenoids
&
OH
H
141 I
i-iii
,
iv,v
*
H
&
(398)
oc Ph,
tI hi-viii
Reagents: i, Ac,O-py; ii, HC1-Et,O; iii, NaOEt; iv, Ph,CCI-py; v, Os0,-NaIO,; vi, KCPh,-MeI; vii, H,-Pd/C; viii, TsC1; ix, KCPh,.
Scheme 40
H
Reagents: i, HC0,Et-NaOEt; ii, DDQ; iii, Cr0,-Me,CO; iv, hv; v, H,-Pd/C.
Scheme 41
142
Terpenoih and Steroids
abscisic acid (406) and ( + )-trans-abscisic acid (408). A quantitative calculation based on molecular exciton theory and using 0.r.d. data supports the absolute configuration (408) shown for the trans-acid.18'
Interest continues in the biological activity of (+)-cis-abscisic acid and a recent report has shown that excised axes of Phaseolus vulgaris can convert it into 4'-dihydrophaseic acid (410) and a second compound tentatively identified as phaseic acid (409). These metabolites are physiologically inactive and since it may 4'-dihydrophaseic acid (410) is a constituent of whole bean represent the major abscisic acid-deactivation product. For biosynthetic studies of abscisic acid see Chapter 7, p. 266. Chemical and spectroscopic evidence has been provided for the structure of pleraplysillin (41 l)."* This compound co-occurs with dehydrodendrolasin (412) in the sponge PIeraplysilla spinifera and represents a new type of monocyclic sesquiterpenoid. The toxicity of the essential oil of Nyoporium deserti A. Cunn 186
'
87
N. Harada, J. Amer. Chem. SOC.,1973.95, 240. E. T. Tinelli, E. Sondheimer, D. C. Walton, P. Gaskin, and J. MacMillan, Tetrahedron Letters, 1973, 139. M . Takasugi, M. Anetai. N. Katsui, and T. Masamune, Chem. Letters, 1973, 245. G . Cimino, S. Ile Stefano, L. Minale, and E. Trevillone, Tetrahedron, 1972, 28, 4761.
143
Sesquiterpenoids
(Theodore variety) is due to the presence of dehydromyodesmone (413) and Related studies on other varieties of this plant dehydroisomyodesmone (414).lE9 have revealed the presence of other toxic sesquiterpenoids (cc p. 83).
'
'
(413)
(414)
Because of their repellent action on fish the complex mixture of compounds secreted by the abdominal glands of the 'whirligig' beetle (family Gyrinidae) have received considerable a t t e n t i ~ n . ' ~ - ' ~ ,Two ' ~ ~ major compounds of this secretion are the nor-sesquiterpenoids gyrinal (415)I3-l6 (cf: p. 84) and gyrinidone (416),lgoand it is interesting that these compounds have a structural (and presumably biosynthetic) relationship reminiscent of that between citral (417) and the iridoids [e.g.iridodial (418)l.
(415)
(4 16)
0
OH L
CHO H
lS9
190
I. D. Blackburne and M . D. Sutherland, Austral. J . Chem., 1972,25, 1779. J. W. Wheeler, S . K. Oh, E. F. Benfield, and S. E. Neff, J . Amer. Chem. SOC.,1972,94, 7589.
Terpenoih and Steroids
144
The structure of futronolide (420) has been established by synthesis from bicyclofarnesic acid methyl ester (419) (Scheme 42).19' A related compound, purpuride (421), has been isolated from the mycelium of P. purpurogenurn Stoll and its structure has been determined by X-ray a n a 1 y ~ i s . l ~ ~
(419)
Reagents: i, hv, 0,; ii, (CH,OH),-H+; iii, NBS-H,O; iv, NaBH,; v, PBr,; vi, H,Odioxan.
Scheme 42
19'
T. Kato, T. Iida, T. Suzuki, Y .Kitahara, and K. H. Overton, Tetrahedron Letters, 1972,
19'
4257. T. J. King, J. C. Roberts, and D. J. Thompson, J . C . S . Perkin I , 1973, 78.
3 D i ter peno ids ~~~~
~
~
BY J. R . HANSON
1 Introduction
This chapter follows the pattern of the previous Reports, with sections based on the major skeletal types of diterpenoid. The literature that has been covered was that available to August, 1973. An interesting feature of the diterpenoids which has become more apparent during the past year is the wide range of biological activity that these substances show - an aspect which may merit more systematic investigation. The biological activity ranges from tumour-inhibitory substances, antileukaemic compounds, and novel antibiotics through insecticidal compounds to both plant-growth inhibitors and plant-growth hormones. In the structural studies reported during the year, the physical methods that have been used now include I3C n.m.r. spectroscopy. Undoubtedly a number of useful I3C n.m.r. correlations with structure will emerge in this series. 2 Bicyclic Diterpenoids
A review has appeared’ describing recent advances in the chemistry of the bicyclic diterpenoids. A number of 4-hydroxy-l8(or 19)-nor-diterpenoidshave been found in plant extracts. Their probable formation by the autoxidation of the corresponding 4-aldehydes has now been in~estigated.~.’The autoxidation of torulosal to 4-hydroxy-norditerpenoidsand thence to the corresponding olefins has been studied. In the tetracyclic kauran-19-a1 series, the reactions have been shown to occur readily in benzene solution, when autoxidation products can be detected after 3 h and very little starting material is left after 2 days. The oxidation of sclareol has been investigated on a number of occasions in the search for perfumery products with an ambergris odour. The oxidation of manoyl oxide has been studied4 with a similar objective. Some acetals such as (1) and the internal acetals (2), (3), and (4) have been prepared. In this work to prepare cyclic ethers, the peracid epoxidation of the enol ether (5) was shown to give the acetoxy-acid (7), possibly by fragmentation of (6)and further oxidation.
’
J. R. Hanson, Progr. Phytochem., 1972,3, 231. 0 .Tanaka, S. Mihashi, I. Yanagisawa, T. Nikaido, and S. Shibata, Tetrahedron, 1972, 28, 4523. R. Caputo, L. Mangoni, L. Previtera, and R. Iaccarino, Tetrahedron, 1973, 29, 2047. R. C. Cambie, K. N. Joblin, and A. F. Preston, Austral. J. Chem., 1972, 25, 1767.
145
146
Terpenoids and Steroids
The allylic acetylation of labd-8(17)-en-13-01 was examined5 in an effort to introduce C-'7 substituents. The unusual tribromo-orthoester (8) was formed on oxidation with lead tetra-acetate and N-bromosuccinimide.
(8)
A group of double-bond isomers (9) epimeric at C-13 with cupressic acid have been isolated6 from Agathis robusta. The compounds have been interconverted using lithium in diaminoethane and related to 13-epimanool. 13-Epi-isomanool (lo), with the 13s configuration, has been prepared7 from 13-epimanool.
Marine natural products represent a small but interesting group of compounds. Concinndiol (11) is a bromoditerpenoid which was isolated' from the red alga
'
P. K. Grant, R. T. Weavers, and C. Huntrakul, Tetrahedron, 1973, 29, 245. R. M . Carman, W. J . Craig, and I. M . Shaw, A u r r a l . J. Chem., 1973, 26, 209. R. M. Carman, W. J . Craig, and I . M . Shaw, Austral. J. Chem., 1973, 26, 215. J. J. Sims, G . H. Y . Lin, R. M. Wing, and W. Fenical, J.C.S. Chem. Comm., 1973, 470.
Diterpenoids
147
Laurencia concinna. This compound is interesting from the biosynthetic point of view in that the bromine atom might represent the residue of a cyclizing species. The full paper describing the structure and synthesis of taondiol has a ~ p e a r e d . ~ The resin acids from two Hymenaea species, H . oblongifolia and H . paruifolia, have been examined. Enantiopinifolic acid [ent-labd-8(17),ene-l5,18-dioic acid], which has also been found in the related Trachylobium uerrucosum, and entlabd-8(17),13-diene-15,18-dioic acid (guamaic acid) were isolated from the former whilst enantio-13-epilabdanolicacid was isolated from the latter. Succinite (Baltic amber) has been shown' to contain abietic acid, isopimaric acid, agathic acid, and dihydroagathic acid. Sideritis canariensis (Labiatae) is a good source of diterpenoids, containing kauranoid and trachylobane diterpenes. The bicyclic ether ribenol, 3a-hydroxy( - )- 13-epimanoyl oxide (12), and 7P-acetoxy-18-hydroxytrachylobane have now been isolated from this source.' Ribenol was converted into 13-epimanoyl oxide whilst the additional oxygen function was located on ring A through the formation of an ab-unsaturated ketone containing two vinylic protons. Tiganone (13) is a related A-norditerpenoid which has also been isolated' from the same plant.
'
o& HO" '%%
H
'%\
H
(12)
(13)
(14)
(15)
The unsaturated lactone (14)is a minor diterpenoid constituent of Andrographis paniculata.14 It showed spectral similarity to andrographolide. A benzylidene lo
l2
l4
A. G. Gonzalez, J. Darias, J. D. Martin, and C. Pascual, Telrahedron, 1973, 29, 1605. A. Cunningham, S. S. Martin, and J. H. Langenheim, Phytochemistry, 1973, 12, 6 3 3 . L. J. Gough and J. S. Mills, Nature, 1972, 239, 527. A. G. Gonzalez, B. M. Fraga, M. G. Hernandez, and J. G. Luis, Phytochemisfry, 1973, 12, 1113. A. G. Gonzalez, B. M. Fraga, M. G . Hernandez, and J. G . Luis, Anales de Quim., 1971, 67,1245. A. Balmain and J. D. Connolly, J.C.S. Perkin I , 1973, 1247.
Terpenoids and Steroidr
148
compound was formed linking the two hydroxy-groups. Surprisingly, reduction of the 11-ketone with sodium borohydride also resulted in reduction of the double bond of the unsaturated lactone. Other minor diterpenoids isolated from this source were the diene (15) and its dihydro-derivative. The number of known diterpenoids possessing the clerodane skeleton has increased considerably. Two series are apparent - those which possess a cis and those which possess a trans A/B fusion. Haplopappic acid, isolated from Huplopappus foliosus, has been shown15 to have the structure (16) and to be related to cistodioic acid. Solidago species have been a fruitful source of diterpenoids. A furanoid acid, junceic acid (17; R = C0,H) and its related 3a,4a-epoxide have been isolated along with some abietanes, the junceanols, from Solidago juncea. Their relationship to hardwickiic acid, with which they co-occur, was established by conversion into a common compound (17; R = Me). The unusual position of the carboxy-group was established from n.m.r. data. The solidagoic acids A (18; R' = Me, R2 = C02H)and B (18; R1 = CH,OAng, R2 = CO,H), which were isolated" from Solidago giganteu var. serotina, are, on the other hand, cisclerodanes. The furanoid side-chain gives rise to a characteristic A4 - 95 peak in the mass spectrum of these substances. The angular carboxy-group was readily lost on heating, thus establishing its relationship to the ring A double bond. A range of non-acidic diterpenoids representing different oxidation states at C-18 and (2-19 has bcen isolated'* from the same source. They have been assigned the structures (18 ; R' = CHO, R2 = Me), (18 ; R' = R2 = CHO), (18 ; R' = CH,OH, R2 = Me), (18; R' = Me, R2 = CH,OH), (18; R 1 = R2 = CH20H), (19), (20), and (21). The substances were inter-related through their
0
l5 lh
I'
M . Silva and P. G . Sammes, Phytochemistry, 1973, 12, 1755. M. S. Henderson, R. D. H . Murray, R. McCrindle, and D. McMaster, Cunud.J . Chem., 1973, 51, 1322. T. Anthonsen, M. S. Henderson, A. Martin, R. D. H. Murray, R. McCrindle, and D. McMaster, Canad.J. Chem.,1973, 51, 1332. M. S. Henderson, R. McCrindle, and D. McMaster, Cunud. J . Chem., 1973, 51, 1346.
Diterpenoids
149
various lithium aluminium hydride reduction products. The allylic oxidation at C-2 with chromium trioxide-pyridine reagent has been studied with these compounds. Some diterpenoid diol mono-esters (22; R = CH2CHMe2), (22; R = CHMeCH,Me), and (22; R = CMe=CHMe) have been i ~ o l a t e d ' ~from Hinterhubera imbricata. Clerodendrin A [23; R = Me(Et)(OAc)CCO] is a bitter principle and insect antifeeding substance which has been isolated from Clerodendron tricotomum. The assignment of its structure rests on a combination of a chemical2*and an X-ray study.21 Rather surprisingly it is antipodal to clerodin, which is found in related species.
CH,OCOR
OMe
A reinvestigation of the chemistry of the diosbulbins A, B, and C and a reinterpretation of the previous structural evidence has been presentedz2 in terms of the structures which were obtained by X-ray analysis. The full paper of the structures of nepetaefolin, nepetaefuran, and nepetaefuranol has appeared.23 Methoxynepetaefolin (24) is24a minor constituent of Leonotis nepetaefolia. l9
2o
22
23 24
F. Bohlmann, M. Grenz, and H. Schwarz, Chem. Ber., 1973,106, 2479. N. Kato, M. Shibayama, and K. Munakata, J . C . S . Perkin I , 1973, 712. N. Kato, K. Munakata, and C. Katayama, J.C.S. Perkin II, 1973, 69. T. Komori, M. Arita, Y . Ida, T. Fujikura, T. Kawasaki, and K. Sekine, Annafen, 1973, 978. J. D. White and P. S. Manchard, J . Org. Chem., 1973, 38, 720. P. S. Manchard, Tetrahedron Letters, 1973, 1907.
Terpenoids and Steroids
150
3 Tricyclic Diterpenoids Naturally Occurring Substances.--1sopimara-9( 11), 15-diene-3P,I9-diol (25) has been isolated" from Newcustlia viscidu (Verbenacdae). It was converted into isopimara-8(9),15-diene. The stereochemistry of the diol followed from a chemical and spectroscopic examination of the 1la,l6-diol which was formed by hydroboronation of the 3,19-ethylidene derivative. The virescenosides are fungal glycosides of diterpenoid alcohols. Virescenosides F and G are26unusual glycosides in which the sugar is altruronic acid and the aglycones are the previously described virescenols A and B. A group of derivatives (26) and (27) of sandaraR2
HO'
HOCH, (25)
:--+.
8 \II
HOC~H,
(26) R' = H, a-OH, R2 = OAC,R3 = H R' = H, u-OAC,R 2 = OH, R3 = H R ' = 0,R2 = a-OH, R3 = H R ' = H,, R2 = H, R3 = OH or OAc R' = H, a-OH, R 2 = H, R 3 = OH or OAc R' = H, U-OH,R2 = R3 = H R ' = H,, R2 = OAC,R3 = H
H
(27)
copimar- 15-en-8b-01 have been isolated2' from Garuleurn and Osteospermum species (Calendulaceae). They are interesting in that they possess oxygenation on ring c, a feature which is becoming increasingly common in the tricyclic compounds. Oxidation and dehydration served to distinguish those compounds which possess a C-1 1 hydroxy-group. The mass-spectral fragmentation pattern arising from the cleavage of ring c also provided useful structural information. Two fungal diterpenoid antibiotics, LL-S491p and y, (28; R = 0) and (28; R = H,b-OH), have been isolated" from Aspergillus chevalieri. Both possess strong antiprotozoal activity against Tetrahyrnena pyriformis whilst LL-S491y also exhibits antiviral activity against Herpes simplex. LL-S491y was also formed from LL-S491P by reduction with sodium borohydride. The n.m.r. spectra were 2 5
P. R. Jefferies and T. Ratajczak, Austral. J . Chem., 1973, 26, 173.
27
Ceccherelli, N. Cagnoli-Bellavita, J . Polonsky, and Z. Baskevitch, Tetrahedron, 1973, 29, 449. F. Bohlmann, G . Weickgenannt, and C . Zderq, Chem. Ber., 1973, 106, 826. G . A. Ellestad, M. P. Kunstmann, P. Mirando, and G . 0. Morton, J . Amer. Chem. Soc., 1972, 94, 6204.
'' P. 2H
151
Diterpenoids
indicative of a pimarane structure. The presence of vicinal functionality on ring B was revealed by the formation of ortho-catechols on treatment with methanolic hydrogen chloride. Cleavage of ring B with periodate gave the lactone (29). The stereochemistry at C-9 was related to the A/B stereochemistry by the solvent shift of the C-5 proton in the n.m.r. spectrum. The absolute stereochemistry was assigned on the basis of the c.d. curve of the C-7 ketones.
_-
.
OH
.
0
A group of C-13-hydroxylated ent-abietanes have been isolated from Solidago species. Their biogenesis may be visualized in terms of the hydration of the carbonium ion formed during the rearrangement of the methylvinyl group to an isopropyl group. The structures of missourienol A (30; R = 0),B (30; R = H,/?-OH),and C (31), which were isolated from S. missouriensis, were determined2' by dehydration to form the 7,13-dienes. Removal of the C-3 oxygen function via the ketone and a Wolff-Kishner reduction gave ent-abieta-7,13-diene. The positions of the oxygen functions on ring A were assigned from the n.m.r. spectra and the relevant solvent-shift data. The junceanols W (32; R' = OH, R2 = OCOC4H,), X (32 ; R' = OCOC,H, ,R2 = OH), and Y (32 ;R1 = H, R2 = OH) are relatedI6 compounds which were isolated from Solidagojuncea. Coleus species have been the source of some highly oxygenated abietanes. The isolation from Coleus barbathus of barbatusin, whose structure (33) was established3' by an X-ray analysis of its p-bromobenzoyl ester, is interesting since it contains a rare cyclopropane ring. There is the possible implication that the simple methyl-group shift between the pimaranes and the abietanes may not be 29
30
T. Anthonsen and G . Bergland, Acta Chem. Scand., 1 9 7 3 , 2 7 , 1073. A. H. J. Wang, I. C. Paul, R. Zelnik, K. Mizuta, and D. Lavie, J. Amer. Chem. Soc., 1973, 95, 598.
Terpenoids and Steroids
152
a single-step process. Coleon F (34), which is a very labile quinone methide, has been obtained3’ from the same source. Its. structure rests on n.m.r. evidence and an inter-relationship with a reduction product of coleon E. The formation of the propenyl side-chain by fragmentation of the methylcyclopropyl acetate in a relative of barbatusin can be readily envisaged. OAc
0
OAc (33)
(34)
Triptolide (35; R = H) and triptodiolide (35; R = OH) are two novel antileukaemic diterpenoid triepoxides which have been isolated3’ along with a cytotoxic ketone triptonide (36) from Tripterygiurn wilfordii (Celastraceae). Their structures were determined by the direct X-ray method.33 These compounds are unusual both in containing triepoxide functions and in possessing the 18(4 + 3)abeo-abietane skeleton. Podocarpus species contain a number of insect toxins such as nagilactone C. Two further insect toxins, hallactones A (37) and B (38), have been isolated34 from Podocarpus hallii. Another lactone, sellowin A (39), which was also isolated3’ from this source, strongly inhibits the growth of pea-stem segments. Rimuene, nagilactone C, and nubilactone A (40) have been obtained36 from Podocarpus nubigena. The n.m.r. spectra of these compounds are very characteristic. ” 32
33 34
I5 36
P. Reudi and C. H. Eugster, Helv. Chim. Acta, 1973,56, 1129. S. M . Kupchan, W. A. Court, R. G. Dailey, C. J . Gilmore, and R. F. Bryan, J . Amer. Chem. Soc., 1972,94, 7194. C. J. Gilmore and R. F. Bryan, J . C . S . Perkin 11, 1973, 816. G. B. Russell, P. G . Fenemore, and P. Singh, J.C.S. Chem. Comm., 1973, 166. R . C. Cambie and G. B. Russell, Phytochemistry, 1973, 12, 2057. M. Silva, M. Bittner;and P. G. Sammes, Phytochemistry, 1973, 12, 883.
153
Diterpenoicls
J75
0
0
co-0
HO
,-
co-0
(37)
0
0
o&oH ,=
c0-0
CO-0 (39)
Strobic acid (41) is an unusual diterpenoid possessing a seven-membered ring c. The full paper presenting evidence for its structure has appeared.37 The corresponding alcohol and aldehyde, strobol and strobal, have now been isolated38from Pinus strobus cortex tissue along with manoyl oxide and the cisand trans-abienols.
e& :
'\,
H
C02H
(42)R' = R2 = OAc, R3 = Me, R4 = H R' = H,R2 = OAc, R3 = Me, R4 = H R' = R2 = OH, R3 = H, R4 = C 0 2 H R' = R2 = OH,R3 = H,R4 = C 0 , M e R' = OH, R2 = OAc, R3 = H, R4 = C0,Me R 1 = R2 = R4 = OH, R 3 = Me R' = R2 = OAC,R3R4= :CH2
(41)
&To',' H
'
0
OAc (43)
'' D. F. Zinkel and B. P. Spalding, 38
Tetrahedron, 1973, 29, 1441. D. F. Zinkel and B. B. Evans, Phytochemistry, 1972, 11, 3387.
Terpenoids and Steroids
154
Eight new diterpenoids have been isolated3’ from Pterodon ernarginatus, which was investigated because the oil from its fruits provides some protection against infection by Schistosomu rnansoni. The diterpenoids are the vouacapane derivatives (42) and (43). 7fl-Acetoxyvouacapane was converted into the parent hydrocarbon. The N-methylethanolamine cassaic acid esters readily rearrange to amides under conditions that are used in their isolation from Erythrophleum species. Thus some compounds which were originally described4* as esters are in fact the a m i d e ~ . ~The l isolation of norcassamidide and the authentic ester norcassamidine from E . chlorostachys has now been described. The bacterial degradation of dehydroabietic acid by Flavobacterium resinouorurn has been described42in detail. Attack occurs first on ring A and then on the aromatic ring to give the fragments (44) and (45). On the other hand a Pseudornonas species and an Alcaligenes species attack C-7 and then degrade the aromatic ring to give bicyclic products such as (46).43
Me0,C
C0,Me
Me0,C
C0,Me
(47)
lVlC
Me0,C (49)
Chemistry of the Tricyclic Diterpenoids-Work has continued on the structural modification of abietic acid and its relatives, with the partial synthesis of the 39 40 41
4L 43
J. R. Mahajan and M. B. Monteiro, J.C.S. Perkin I , 1973, 520. J. Friedrich-Fiechtli and G. Spiteller, Chem. Ber., 1971, 104, 3548. J. W. Loder, C . C . J. Culvenor, R. H. Nearn, G . B. Russell, and D. W . Stanton, Terrahedron Letters, 1972, 5069. J . F. Biellmann, G . Branlant, M. Gero-Robert, and M. Poiret, Tetrahedron, 1973, 29, 1227. J. F. Biellmann, G . Branlant, M. Gero-Robert, and M. Poiret, Tetrahedron, 1973, 29, 1237.
155
Diterpenoids
gibberellins as an objective. Access to ring A in the hydrofluorenes obtained from dehydroabietic acid has now been achieved44by oxidation of the alcohol (47) with iodine and lead tetra-acetate to give the iodo-ether (48) and thence the ring A olefin (49). Studies have also been carried out on the modification of ring c. Thus nitration of (50) led4’ to substitution at both C-12 and C-13. Hydration via oxymercuration of a 12,13-double-bond has also been studied.46 Attack occurs at both centres. Further oxidation of ring B can occur as a major sidereaction in the nitration of perhydrophenanthrenes. Thus nitration of (51 ; R = H) gave4’ not only the expected substitution product (51; R = NO2) but also the formyl-lactone (52). In some circumstances ring B is also readily autoxidized. Thus the olefin (53) is completely converted48into the unsaturated ketone (54) after 5 weeks‘ aerial exposure.
CHO
A number of naturally occurring tricyclic diterpenoids contain a C-11 oxygen function. A number of successful attempts have been reported of introducing groups at this centre. The r o ~ t e involves ~ ~ ~ ~conversion * of a C-7 ketone into the lactone (55) followed by hydrolysis and methylation to form the methoxyester (56), in which rotation about the C-9--C-10 bond is possible. Ring closure then affords the C-11-methoxy-derivative (57). 11-Methoxydeoxypodocarpic 44
45 46 41
48
49
50
A. Tahara and T. Nakata, Tetrahedron Letters, 1972, 4507. A. Tahara and Y. Ohtsuka, Chem. and Pharm. Bull. (Japan), 1972,20, 1637. A. Tahara and Y. Ohtsuka, Chem. and Pharm. Bull. (Japan), 1972,20, 1648. R. C. Cambie, T. J. Fullerton, R. C. Hayward, J . L. Roberts, and P. S. Rutledge, Austral. J . Chem., 1972, 25, 2279. T. Ohsawa, M . Kawahara, and A. Tahara, Chem. and Pharm. Bull. (Japan), 1973, 21, 487. T. Matsurnoto, S. Irnai, M. Aizawa, H. Kitagawa, and K. Fukui, Chem. Letters(Japan), 1972, 581. Y . Ohtsuka and A. Tahara, Chem. and Pharm. Bull. (Japan), 1973,21, 643.
Terpenoih and Steroids
156
acid" and 1l-methoxydehydroabietic acid5' have been prepared by this route, although in the latter case the acylation reaction is accompanied by some attack from C-18.
A number of N-substituted C- 12-sulphonamides of methyl dehydroabietate, dehydroabietinol, and dehydroabietamides have been prepared for pharmacological The rearrangement of the rings c of abietic acid, levopimaric acid, and neoabietic acid to cyclopentene (59)through a common cation (58)has been ~tudied.5~ N.m.r. evidence was presented for the formation of the cation in 96% sulphuric acid at 0 0C.54 Rearrangement occurred at 25 "C. Levopimaric acid underwent decarbonylation and rearrangement with methyl migration when in chlorosulphonic acid solution to give products such as (60)52which are reminiscent of the reactions of the podocarpic acid series with phosphoryl chloride. The cassane group of diterpenoids, represented by the Erythrophleum alkaloids, the caesalpins, vinhatacoic acid, and voucapenic acid, may be derived by a C-13-C-14 methyl migration in a pimarane. Since C-7 is oxygenated in many of these substances, the rearrangements of derivatives of the ketone (61) which was prepared from isopimaric acid have been studied.55 Although the reactions were unsuccessful in inducing rearrangement, the products were of
' 52
Y . Ohtsuka and A. Tahara, Chem. and Pharm. Bull. (Japan), 1973,21, 653. G. Ntokos, D. Theodoropoulos, P. Catsoulacos, and C. Kokkinas, Bull. Sor. chim. France, 1973 I I , 991.
'' G. Mehta, N. Pattnaik, and S. K. Kapoor, Tetrahedron Letrers, 1972, 4947. G. Mehta and S. K. Kapoor, Tetrahedron Letters, 1973, 2385. *'J. P. Johnston and K. H . Overton, J . C . S . Perkin I, 1973, 853. 54
Diterpenoids
157
interest. For example, treatment of the epoxy-ketone (62) gave the stable complex of boron difluoride with P-diketone (63)and as a minor product the y-lactone (64). Migration of the vinyl substituent can lead to the cleistanthane ring system. A rearrangement of this type has been reporteds6 in the reaction of the pimarane antibiotic LL-S491P (28 ; R = 0)with acetic anhydride and toluene-p-sulphonic acid, which affords products such as (65).
4 Tetracyclic Diterpenoids
The KaurenePhyllocladene Series-The number of known kauranoid diterpenoids, particularly with an oxygenation pattern related to possible gibberellin intermediates, continues to expand ent-Kaur-16-en- 19-oic acid has been isolated" from Mikunia mongenansis (Compositae). ent-Kaur- 16-en-19-01, its acetate, and the corresponding aldehyde along with ent-17-hydroxykauran-19-al and its 17-acetate have been isolated58 from Annona squamosa. An interesting group of 1lp-hydroxylated ent-kaur-16-enes has been isolated from the liverwort 56 57
'*
G. A. Ellestad, M. P. Kunstman, and G. 0. Morton, J.C.S. Chem. Comm.,1973, 312. S. B. Mathur and C. M. Fermin, Phytochernistry, 1973, 12, 226. F. Bohlmann and N. Rao, Chem. Ber., 1973, 106,841.
158
Terpenoidr and Steroidr
Solenostoma t r i ~ t e . ~The ” C-15 acetate (66) was related to (16R)-ent-kauran-15one via the 1 1,12-olefin. Other products were the 11,15-diol, the llp-hydroxyI5-ketone, and the ent-1l~-hydroxy-(l6R)-kauran-15-one. The n.m.r. spectra of these compounds suggested that ring c is in a chair conformation. Vierol (67), whose structure was proven by a partial synthesis from epicandidiol, and powerol (68), related both to ent-kauranol and to the acetate of ent-kaur- 16-en-7P-01, have been isolated6’ from Sideritis canariensis. The partial synthesis of ent-kaur-l6-ene-l5p,18-diol and ent-kaur-16-ene-7a,lSP,18-triol has been described.6 Sideridiol (ent-kaur-15-ene-7aJ8-diol) was converted into ent-kaur-15-en-18-oic acid and thence via the epoxide into ent-kaur-16-ene15P,I 8-diol (candidiol). This compound was also formed by photo-oxygenation of ent-kaur-15-en-18-01. Rearrangement of sideroxol, ent-l5/3,16P-epoxykaurane7a,l8-diol, with boron trifluoride etherate in dimethyl sulphoxide gave ent-kaur16-ene-7cr,15P,18-triol. Previously a number of plant gibberellins had been isolated from Calonyction aculeatum. Some kauranoic acids have now been isolated6 from the same source. These include ent-7a,l6a,l7-trihydroxykauran-19-oic acid (69; R = H) and the corresponding 6/?,7fl-diol(69; R = OH). The stereochemistry of the 16,17-glycol is opposite to that obtained by osmylation of the corresponding olefin. The stereochemistry of the 6fl,7B-glycolwas established by osmylation of the A6*7-16,17-acetonide.Although 6B,7P-glycols do not appear to be the gibberellin intermediates in the fungal system, nevertheless the isolation of this compound from a higher plant is interesting.
59
6o 6 1
62
J. D. Connolly and I . M. S. Thornton, J.C.S. Perkin I, 1973, 736. A . G. Gonzalez, B. M. Fraga, M . G . Hernandez, and J. G . Luis, Tetrahedron, 1973, 29, 561. F. Piozzi. P. Venturella, A. Bellino, and M . L. Marino, J.C.S. Perkin I , 1973, 1164. N . Murofushi, T. Yokota, and N . Takahashi, Tetrahedron Letters, 1973, 789.
159
Diterpenoid.7
The rhizomes of Atruct ylis gunirnif'era contain a toxic glycosicle atractyloside in which the norditerpenoid atractyligenin (70) forms the aglycone. Carboxyatractyloside, which retains a geminal dicarboxylic acid at C-4. has now been isolated63 from this source. Pyrolysis of carboxyatractyligenin gave predominantly atractyligenin with an axial carboxy-group - presumably by a kinetically controlled protonation of the intermediate enol from the less-hindered equatorial face of the molecule. Isoatractyligenin (71), which is formed by the addition of
C0,H (70)
C0,H (71)
bromine to atractyligenin followed by treatment with zinc and alkali, has been shown64 to possess an a-oriented oxetan ring. The exchange of a C-13 bridgehead deuterium atom in t-butyl alcohol containing potassium t-butoxide at 172"C for 72 h from ent-17-norkauran-16-0ne has been quoted65as evidence for bridghead enolization in this series. During the past few years lsodon species have been the source of a number of hydroxylated and ring B seco-kauranoids. The structure and stereochemistry of lasiokaurin (72) and lasiodonin (73), which were isolated66 from Isodon
lusiocarpus, have been described. The structures, mainly based on n.m.r. evidence, were supported by correlation with oridonin diacetate on the one hand and sodoponin on the other. Shikokianidin (74) is a minor component of I. shikokianus and it has been related to shikokianin which was previously described 63
B. Danielli, E. Bombardelli, A. Bornati, and B. Gabetta, Phytochemistry, 1972, 11, 3501.
64 65 66
F. Piozzi, G . Savona, and M. L. Marino, Gazzetta, 1973, 103, 21 1 . D. H. Bowen and J . MacMillan, Tetrahedron Letters, 1972,4111. E. Fujita and M. Taska, Chem. and Pharm. Bull. (Japan), 1972, 20, 1752.
160
Terpenoids and Steroids
from the same plant.67 A full paper has appeared68 on the isolation of isodonal and epinodosin from I . japonicus and on the structural elucidation of sodoponin and epinodosinol. Kauranoid structures were originally proposed for the diterpenoid constituents of Callicarpa mcrophylla. However, calliterpenone (75)has been degraded69
to phyllocladane-16,17-diol and to 17-norphyllocladan-l6-one, and hence calliterpenone must possess the most unusual 13p-kaurane (phyllocladane) skeleton. The microbiological transformation of kauranoid diterpenoids has been described. Thus Aspergillus niger hydroxylates7' 17-norkauran-16-oneand 17norphyllocladan-16-one in the 3-position. In a more extensive study7' the transformation of a group of kauranoid compounds by Aspergihs ochraceus, Calonectria decora, and Rhizopus nigricans has been described. For example, ent-kaur-16-en-19-oic acid gave ent-16a,17-dihydroxykauranoicacid (20% conversion)with A. ochraceus, ent-7a,l5a-dihydroxykaurenoicacid (30 % conversion) with C. decora, and ent-7/3-hydroxykaurenoic acid (25 % conversion) with R. nigr icans. Beyeranes.-A group of beyerenes has been from Erythroxylon australe. They contain a C-1 carbonyl group, the reactions of which are subject 67
68
69
'* 'I
' 2
T. Isobe, T. Kamikawa, I . Kubo, and T. Kubota, Bull. Chem. SOC.Japan, 1973,46,583. E. Fujita, T. Fujita, M. Taoka, H. Katayama, and M . Shibuya, Chem. artd Pharm. Bull. (Japan), 1973,21, 1357. S. A. Ahmad and A. Zaman, Tetrahedron Letters, 1973, 2179. A. B. Anderson, R. McCrindle, and J. K. Turnbull, J . C . S . Chem. Comm., 1973, 143. J. P. Beilby, E. L. Ghisalberti, P. R. Jefferies, M. A . Sefton, and P. N. Sheppard, Tetrahedron Letters, 1973. 2589. J . D. Connolly and A. E. Harding, J.C.S. Perkin I , 1971, 1996.
Diterpenoids
161
to a marked degree of steric hindrance. The compounds include the C-1 ketone, its 15,16-epoxide,the 2-hydroxy-l-ketone, and the related diosphenol, together with the 19-hydroxy-1-ketone. la-Hydroxyerythroxydiol-X and la-acetoxy1lp-hydroxyerythroxydiol-Xwere also isolated from this source. The ent-beyer15-ene-12-toluene-p-sulphonylhydrazone(76) undergoes7, an interesting rearrangement on treatment with hydride to form the novel ent-(16S)-atis-l3-ene system (77). The marked lack of reactivity of the olefin in this system and some anomalous features of the n.m.r. spectrum led to a confirmation of the structure by X-ray analy~is.’~ The structure revealed strong methyl interactions with the double bond.
Gibberellins.-Gas chromatography-mass spectrometry is a powerful combination for the identification of gibberellins. Gibberellin Azo, abscisic acid, and phaseic acid have been identified7, in flowering Bryuphyllurn diagrernuntianurn by this method. Gibberellins A, and A, have been detected76in germinating barley (Hordeurn distachurn), whilst gibberellins A, and A13 have been recorded77 in Enhydrajuctuans. A gradient-elution silica-gelpartition-chromatograph y system has been described78 for 33 gibberellins which has the advantage of resolving some of the more polar ones. The interconversion of gibberellins A, and A3 in dwarf pea (Pisurn satiuurn) has been dem~nstrated.~,The biological activity of some of the more recently isolated gibberellins has been described.80 In general, gibberellins A30, A,, , and A3, were quite active in the bioassays that were used whereas A,, ,A,, , and A,, glucoside were only slightly active. Gibberellin A,, (78 ; R = OH) was as active as gibberellic acid (78 ; R = H). Allogibberic acid has been shown’’ to be the active decomposition product of gibberellic acid in inhibiting the flowering of the duckweed Lernna perpusilla. 73
74 7s
7’
78
79 8o 81
K. H. Pegel, L. P. L. Piacenza, L. Phillips, and E. S. Waight, J.C.S. Chem. Comm., 1973, 552. M. Laing, K. H. Pegel, and L. P. L. Piacenza, Tetrahedron Letters, 1973, 2393. P. Gaskin, J. MacMillan, and J. D. Zeevart, Planta, 1973,111, 347. G. J. P. Murphy and D. E. Briggs, Phytochemistry, 1973, 12, 1299. S. N. Ganguly, T . Ganguly, and S. M. Sircar, Phytochemistry, 1972, 11, 3433. R. C. Durley, A. Crozier, R . P. Pharis, and G. E. McLaughlin, Phytochernistry, 1972, 11, 3029. R. C. Durley, I. D . Railton, and R. P. Pharis, Phyfochemistry, 1973, 12, 1609. H. Yamane, I. Yamaguchi, T. Yokota, N. Murofushi, N. Takahashi, and M. Katsumi, Phytochemistry, 1973, 12, 2 5 5 . R. J. Pryce, Phytochemistry, 1973, 12, 1745.
162
Terpenoids and Steroidr
[14C]Gibberellic acid (78; R = H) has been prepareds2 by a combination of chemical and biosynthetic methods. The [17-'4C]-hydroxy-acid (79) was prepared from 7-hydroxykaurenolide and then transformed by Gibberella fujikuroi into gibberellic acid. Gibberellin glucosides have been isolated from plants and via gibberellin the partial synthesis of gibberellin A, 0-3-~-~-glucopyranoside A , methyl ester and a-acetobromoglucose has been recorded.83 The stereochemistry at C-16 of dihydrogibberellin A , methyl ester and its 16-epimer have been determineds4 by studying the effect of shift reagents on the n.m.r. spectra of the epimers. Gibberellic acid is unstable in aqueous solution, and autoclaving these solutions produces a marked drop in gibberellin-like biological activity and an increase in other activities. The decomposition of gibberellic acid (78; R = H) on autoclaving has been carefully studied.85 Isogibberellic acid (go), allogibberic acid (8 l), and epiallogibberic acid were detected, accompanied by dehydroallogibberic acid and gibberellenic acid.
In view of the interesting biological activity of fluorogibberellins, the fluorination of methyl gibberellate by fluoramine was investigated.86 The C-1 and C-3 fluoro-olefins (82) and (83) and their 13-chlorofluoroacetates were isolated. The
'*
83 84
85
86
(82) (83) J. R. Hanson and J. Hawker, Phytochemistry, 1973, 12, 1073. G . Schneider, Tetrahedron Letters, 1972, 4053. K . Mori, Agric. and Biol. Chem. (Japan), 1972, 36, 2519. R. J . Pryce, Phytochemistry, 1973, 12, 507. J. H. Bateson and B. E. Cross, Tetrahedron Letters, 1973, 1779.
163
D iterpenoids
full paper" describing the rearrangements of the gibbane skeleton on dehydrogenation with DDQ has appeared. The reaction of methyl dihydroallogibberate (84) involves rearrangement through the allylic carbonium ion (85) to form (86).
WOH .~
C0,Me (84)
0
_-
C0,Me (86)
(85)
Grayanotoxins.-A number of new diterpenoids possessing this carbon skeleton have been described during the past year. Lyoniol D (87 ; R' = OH, R 2 = H) has been isolated88from Lyonia oualifolia var. elliptica and the rhodojaponins V (88) and VII (87; R' = H, R2 = OAc) have been isolated89 from Rhododendron japonicum. The grayanotoxins G-I, -11, and -111 have been isolatedg0 as the toxic principles from Agauria polyphylla (Ericaceae). Lyoniol A (lyoniatoxin) has now been inter-relatedg1 with grayanotoxin G-I, whose structure rests on an X-ray determination. This relationship also provided confirmatory evidence for the structures of lyoniol B and lyoniol D. The leucothols A, B, and D, which were isolated from Leucothoe grayana, have been assigned the related structures (89; R' = 0, R 2 = R3 = H), (89; R' = H, P-OH, R2 = R3 = OH), and (89;
HO OAc
OH
OAc
(89)
R' = 0, R2 = R3 = OH) using a combination of 'H and 13Cn.m.r. spectros c o ~ y The . ~ ~interesting suggestion has been made in order to account for the 87 88 89
90 91 92
B. E. Cross and R. E. Markwell, J.C.S. Perkin I , 1973, 1476. J. Sakakibara, K. Ikai, and M. Yasue, Chem. and Pharm. Bull. (Japan), 1972, 20, 861. H . Hikino, T. Ohta, Y. Hikino, and T. Takemoto, Chem. and Pharm. Bull. (Japan), 1972,20, 1090. I . Loriaux, P. Boiteau, and H. P. Husson, Phyrorhemisto, 1973, 12, 1500. J . Sakakibara, K. Ikai, and M . Yasue, Chem. and Pharm. Bull. (Japan), 1973,21, 1395. H. Hikino, S. Koriyama, and T. Takemoto, Tetrahedron, 1973, 29. 773 ; Tetrahedron Letters, 1912, 383 1.
Terpenoich and Steroidr
164
stereochemistry of these compounds that they may be derived from a 9/?,13/3kaurane (98-phyllocladane). However, a compound of the grayanotoxin skeleton has been converted93 into an ent-20-norkaurane derivative. The 1,lO-diol (90) was converted into its methanesulphonate, which underwent rearrangement to the kaurane series (91). Rearrangement of the corresponding 9,lO-diols gave an A-nor-c-homo kaurane.
Diterpenoid Alkaloids.-The 13C n.m.r. spectra of a number of the lycoctonine bases have been assigned94 and these data have been used to determine the structure of two new bases (92) and (93) isolated from Delphinium bicolor.
/-
-N
OH
I
OCOMe
1, OH
OMe
5 Macrocyclic Diterpenoids and their Cyclization Products
Neocembrene A (94; R = H) has been isolated95 as a termite trail pheromone. This work described some micro-scale ozonolysis experiments. Cembrene-A and a related alcohol mukulol (94; R = H) have been isolatedg6 from Commiphora mukul, a plant which is used in Ayurvedic folk medicine. Duva-4,8,13trien- 1-01-3-one (95) and the possible retro-aldol cleavage products 1l-isopropyl4,8-dimethylpentadeca-3,7,12-triene-2,14-dione (A3-cis- and -trans-isomers) have been isolated9’ from Nicotiana tabacum. 93 9J
95
96
’’
S . Gasa, N. Hamanaka, T. Okuno, J. Omi, M . Watanabe, and T. Matsumoto, Tetruhedron, 1972, 28, 4905. A . J . Jones and M. H . Benn, Tetrahedron Letters, 1972, 4351; Cunad. J . Chrm., 1973, 51, 486. A . J. Birch, W. V. Brown, J . E. T. Corrie, and B. P. Moore, J . C . S . Perkin I , 1972, 2653. V. D . Patil, U . R. Nayak, and S. Dev, Tetrahedron, 1973, 29, 341. A. Zane, Phytochernisrry, 1973, 12, 731.
Diterpenoids
165
R
I
The Euphorbiaceae and the related Thymelaceae produce a number of novel diterpenoids, including the phorbol group, the lathyrols, jatrophone, and bertyadionol (96). The full paper detailing the evidence for the structure and
stereochemistry of the latter has appeared.98 Two interesting facets of the chemistry of bertyadionol are the isomerization of the 4(15)-double-bond to give a A'(') unsaturated ketone and the chromium(r1)acetate reduction of the 4-ene3J4-dione to give a C-14 alcohol. The driving force for these is the relief of transannular strain within the eleven-membered ring.
a!* - _ _
J---
H --
OR OH
CH,OH
R=O
/
(97)
Milliamine C is an irritant substance which has been isolated99from Euphorbia rniIlii. It has been shown to be ingenol(97) esterified at C-3 with the anthranilic acid derivative (98). Ingenol esterified at C-3 with tetradeca-2,4,6,8,10-pentaenoic acid has been isolated from E. jolkini.
98
E. L. Ghisalberti, P. R. Jefferies, T. G . Payne, and G . K . Worth, Terrahedron, 1 9 7 3 , 2 9 , 403.
99
D. Uemura and Y . Hiraka, Tetrahedron Letters, 1973, 881.
166
Terpenoids and Steroids 6 Miscellaneous Diterpenoid Substances
Pachydictyol (99) is a diterpenoid alcohol of unique structure with antibiotic activity which has been isolated"' from the brown marine alga Puchydictyon coriaceum. The structure was determined by an X-ray analysis of its p-bromophenylurethane. The perhydroazulene skeleton is well-known in the sesquiterpenoid series and the possibility has been raised that pachydictyol is indeed a sesquiterpenoid which has been isoprenylated after cyclization. The antibiotic aphidicolin has been shown"' by X-ray analysis to possess the unusual tetracyclic structure (100). ,
OH \,.CH20H
Fusicoccin H (101)is one of a group of phytotoxic metabolites which have been isolated'01*'02from Fusicoccurn umygdali. Fusicoccin was shown'03 to have the structure (102), which was closely related to that of the sesterterpenoid ophioHO HO--
\
loo
lo
Io2
Io3
CH20H
I
D. R. Hirshfeld, W. Fenical, G. H . Y . Lin, R. M . Wing, P. Radlick, and J. J. Sims, J . Amer. Chem. Soc., 1973, 95, 4049. K. M . Brundret, W. Dalziel, B. Hesp, J. A. J. Jarvis, and S. Neidle, J . C . S . Chem. C o m m . , 1972, 1027. K . D. Barrow, D . H . R. Barton, Sir E. Chain, U. F. W. Ohnsorge, and R . P. Sharma, J . C . S . Perkin I, 1973, 1590. K. D. Barrow, D. H . R. Barton, Sir E. Chain, U. F. W. Ohnsorge, and R. Thomas, J . Chem. Soc. ( C ) ,1971, 1265.
D iterpeno ids
167
bolins, and since there was an isoprene unit attached to the sugar moiety, it was possible that fusicoccin was a degraded sesterterpenoid. However, fusicoccin H, which is of a lower oxidation level than fusicoccin and lacks the isoprene residue, is an efficient precursor of fusicoccin,strongly suggesting that these substances are diterpenoids. The structure of fusicoccin H rests on oxidative degradation, acetonide formation, and the stepwise removal of the oxygen functions.
7 Diterpenoid Synthesis The synthesis of the bicyclic furanoid diterpenoid lambertianic acid (105) has been described.lo4 Podocarpic acid (103)was converted into the keto-acid (104). The exocyclic methylene group was then introduced under conditions based on those for a Reformatski reaction which were designed to avoid isomerization of the double bond. Finally the furan ring was added. A total synthesis of the tricyclic diterpenoid quinone miltirone (108) has been reported. l o 5 p-Bromoanisole was converted into the tetralone (106) and then ring A was added via the unsaturated ester (107). OH
q9 @: CO,H
@
C0,Me
'
C0,H
A formal total synthesis of enmein has been achieved.lo6 The relay (109) has been converted into the olefin (110) and thence into the ring B seco-ester (111). This was converted into the d-lactone (112), which was in turn transformed into enmein (113). The total synthesis has been de~cribed'~'of some kaurane deriva-
lo'
lo'
R. A. Bell, M. B. Gravestock, and V. Y. Taguchi, Canad. J. Chem., 1972,50, 3749. D. Nasipuri and A. K. Mitra, J.C.S. Perkin I, 1973, 2 8 5 . E. Fujita, M. Shibuya, f . Nakamura, Y. Okada, and T. Fujita, J.C.S. Chem. Comm., 1972, 1107. M. Shiozaki, K. Mori, and M. Matsui, Agric. and B i d . Chem. (Japan), 1972, 36, 2539.
Terpenoids and Steroids
168
tives such as ( 1 14)with oxygen functions on ring A suitably placed for conversion into the grayanotoxins.
Me0 OH
0
(113)
The partial synthesis of kaurene and phyllocladene from abietic acid has been reported.'08 The key step involves the cyclization of an olefinic diazo-ketone (115) to form the cyclopropyl ketones (116) and (117). The cyclopropane rings were cleaved with lithium in liquid ammonia to afford ( 1 18) and (119),respectively, which were then converted into the phyllocladene and kaurene nor-ketones. A similar intramolecular keto-carbenoid addition has been described"' in a synthesis of a gibbane system [(120)+ (121)l. l 1 The The synthesis of racemic epiallogibberic acid has been reported.' key step in the formation of ring D"' was the use of a magnesium alkoxide to hold the molecule in such a manner as to favour the intramolecular aldol conA. Tahara, M . Shimagaki, s. Ohara, and T. Nakata, Tetrahedron Letters, 1973, 1701. U . R . Ghatak, P. C. Chakraborti, B. C. Ranu, and B. Sanyal, J.C.S. Chem. Comm., 1973, 548. ' l oH . 0. House, D. G . Melillo, and F. J. Sauter, J . Org. Chem., 1973,38, 741. I ' I H . 0. House and D. G. Melilio, J. Org. Chem., 1973,38, 1398. lo'
lo'
Diterpenoids
do +
169
@FO
'. H C0,Me
k4
&I0 ',
C0,Me
ri
C0,Me
COCHN2
0
densation (122) to (123). The tetracyclic product was then transformed into the olefin (124) and thence' into epiallogibberic acid.
The construction of the B/C/D ring system of the lycoctonine and delphinine alkaloids has continued to attract attention. In one approach'12 the methoxyK. Wiesner, T. Y .R. Tsai, H. Huber, and S . Bolton, Tetrahedron Letters, 1973, 1233.
170
Terperzoids and Steroids
ketone (125) underwent an oxidative rearrangement in the presence of t-butyl perbenzoate and cuprous bromide to form the compound (126), a step which parallels a proposal in delphinine biogenesis. The solvolysis of methyl 15btoluene-p-sulphonyloxy- 13-isopropyl-17-noratis- 13-en-18-oate (127), which has been prepared from levopimaric acid, has also been studied' as a model for the proposed biosj-nthetic transformation of atisine-tyr". alkaloids into this series. Whereas this solvolysis gave a product (128) containing this carbon skeleton, diazotization of the corresponding amine gave' l 4 a complex mixture of products including nitrated products and some in which the isopropylidene group had been lost. A transformation of the podocarpic acid skeleton i n v ~ l v e d " ~cleavage of the olefin (129) via the glycol to form a diketone which then underwent condensation to the unsaturated ketone (130).
'I'
'I4 'Is
W . A . Ayer and P. D . Deshpande, Canad. J . Chem., 1973,51, 77. W . A . Ayer and P. D . Deshpande, Canad. J . Chem., 1973,51, 2323. A. Tahara and T. Ohsawa, Chem. and Pharm. Bull. (Japan), 1973, 21, 483.
4 Sest ert erpeno ids BY J. R. HANSON
1 Introduction The sesterterpenoids are a novel class of pentaprenyl terpenoids derived from geranylfarnesyl pyrophosphate. The structure of the first representative of this class, gascardic acid, was described in 1965. Subsequently sesterterpenoids have been isolated from insect waxes, fungi, higher plants, and marine organisms. Both X-ray analysis and a wide range of physical and chemical techniques have been used to determine the structures of these substances. A number of key stages in the biosynthesis of the fungal sesterterpenoids have been defined. Very recently biosynthetic work has been reported which shows that fusicoccin, which is structurally very similar to the ophiobolins, is in fact a diterpenoid.
2 Acyclic and Furanoid Sesterterpenoids Both geranylfarnesol’ and geranylnerolidol’ have been isolated, the former from the insect wax of Ceroplustesufbofineatusand the latter from the phytopathogenic fungus Cochliobolus heterostrophus. Comparison3 of the n.m.r. spectra of the natural geranylfarnesol with synthetic all-trans-geranylfarnesol suggested that the material isolated from the insect wax was the 2-cis-isomer. However, alltrans-geranylfarnesyl pyrophosphate (1) is converted into ophiobolin F (2) by a cell-free system from Cochliobolus heterostrophus. H
I
’
T. Rios and C. S. Perez, Chem. Comm., 1969, 214. S . Nozoe, M. Morisaki, K. Fukushima, and S. Okuda, Tetrahedron Letters, 1968,4457. S. Nozoe and M. Morisaki, Chem. Comm., 1969, 1319.
171
Terpenoids and Steroids
172
A series of branched-chain isoprenoid hydrocarbons from C,, to CZ5have been isolated4 from an African Cretaceous shale which was about 120 x lo6 years old. The regular methylation pattern of these hydrocarbon chains suggested that some might have had a sesterterpenoid origin. Since 1965 several groups of phosphorus-containing antibiotics have been isolated from Streptomycetes species. These antibiotics possess a long duration of action against Gram-positive bacteria. Hydrolysis of the antibiotics with hydrochloric acid affords a lipid fraction containing C,, alcohols with a partially isoprenoid skeleton. Whether or not these are sesterterpenoid or whether they represent the methylation of a fatty acid remains to be seen. These compounds include moencinol (3),v6 and diumycinol (4).7 In both cases the corresponding unsaturated tertiary alcohols and hydrocarbons were also obtained.
OH
(3)
,
A number of linear furanosesterterpenoids and their C2 degradation products have also been isolated from marine sponges. They are of interest in that they contain a tetronic acid moiety, which is an unusual terpenoid oxidation state. Their structures rest on a combination of spectral studies and identification of the fragments from careful ozonolysis experiments. Nitenin (5) and the saturated
G. Spyckerelle, P. Arpino, and G. Ourisson, Tetrahedron, 1972, 28, 5703. R. Tschesche, F. X. Brock, and I. Duphorn, Tetrahedron Letters, 1968, 2905; Annalen, 1968, 720, 58.
’
W. A. Slusarchyk and F. L. Weisenborn, Tetrahedron Letters, 1969, 659. W. A. Slusarchyk, J. A. Osband, and F. L. Weisenborn, J . Amer. Chem. SOC.,1970,92, 4486; Tetrahedron, 1973, 29, 1465.
173
Sesterterpenoids
lactone dihydronitenin were isolated8 from Spongia nitens. Furospongin 1 (6) and a group of related C,, furanoterpenoids have been isolated' from the sponges Spongia oflcinalis and Hippospongia cornrnunis. Anhydrofurospongin- 1 has been assigned the structure (7)and furospongin-2 is (8),whilst isofurospongin-2 is the 12,13-isomer of (8). Dihydrofurospongin-2 lacks the 12,13-double-bond and tetrahydrofurospongin-2 lacks both the 7,8- and 12,13-double-b0nds.
21
0
Ircinin-1 (9) and ircinin-2 (9) are and A13*15double-bond isomers which occur in the sponge Zrcinia O T O S . ' ~ Ircinin-3 (10) and ircinin-4 are the related C, furanoterpenoids which have been found in the same sponge.' ' Fasciculatin (11) has been isolated'2 from Ircinia fasciculata and variabilin (12) has been obtainedi3 from 1. variabilis. OH
/ /
0
/
... ,--
I
\
\
(9)
O
O
C02H
(10)
' E. Fattorusso, L. Minale, G . Sodano, and E. Trivellone, Tetrahedron, 1971, 27, 3909. lo
l3
G . Cimino, S. de Stefano, L. Minale, and E. Fattorusso, Tettahedron, 1972, 28, 267. G . Cimino, S . de Stefano, L. Minale, and E. Fattorusso, Tetrahedron, 1972, 28, 333. G . Cimino, S. de Stefano, and L. Minale, Tetrahedron, 1972, 28, 5983. F. Cafieri, E. Fattorusso, C. Santacroce, and L. Minale, Tetrahedron, 1972, 28, 1579. D. J . Faulkner, Tetrahedron Letters, 1973, 3821.
Terptwoids und Steroids
174
OH
3 Gascardic Acid Gascardic acid was isolatedL4from the lac of the insect Gascardia madagascariensis. It was the first sesterterpenoid to be formulated. The structure (13) rests'' on a number of degradative sequences. The structure of the side-chain and its point of attachment to a five-membered ring were established in the first of these. The carboxylic acid was reduced to a methyl group and then, on ozonolysis, a keto-ester (14) was obtained. A Wolff-Kishner reduction followed by further stepwise degradation of the side-chain afforded the cyclopentanone (15), which was shown to contain an a-methylene group by deuteriation. The environment of the rb-unsaturated carboxylic acid was established by decarboxylation of the dihydro-acid with lead tetra-acetate to an olefin and the corresponding alcohol. The latter was converted into a ketone possessing two a-methy!ene groups and which, by bromination and dehydrobromination, afforded an Lmsst:.;ated ketone (16). This underwent an anomalous ozonolysis with decarboxylation to afford the unsaturated esters (17). The third group of degradations was based on the 19
(15) l4
'
(16)
(17)
G. Borchcre and J. Polonsky, Bull. SOC.chim.France, 1960, 963. D. Arigoni, Lecture, Chemical Society, Sept. 1965, Nottingham; R. Scartazzini, Diss. Nr. 3899, E.T.H., Zurich, 1966; D. Arigoni, J. Polonski, R. Scartazzini, G. Settim, and G. Wolff, unpublished work.
Sesterterpenoids
175
exocyclic methylene group. The keto-ester (18), derived by ozonolysis, could be converted on the one hand into an P-unsaturated ketone (19) and on the other into a lactone (20). The oxidation of methyl hexahydrogascardate with potassium dichromate gave an unsaturated keto-ester (21), ozonolysis and hydrolysis of which afforded a dilactone (22), thus establishing a relationship between the carboxylic acid and the methylene group. Further information on the structure of the six-membered ring came from the oxidation of (21) with selenium dioxide, in which case the enedione (23 ; R = H) and a hydroxy-enedione (23; R = OH) were isolated. The ene-dione was converted into an a-acetoxydienone (24). These degradations led to partial structures for dihydrogascardic acid from which the total structure (13)was then derived. The position of the double bond conjugated with the carboxylic acid was established by the formation of acidcatalysed cyclization products of the general structure (25). The stereochemistry
Me0,C (20)
176
Terpenoids and Steroids
of gascardic acid also followed from these cyclizations. Biogenetically, gascardic acid might arise from geranylfarnesyl pyrophosphate by the cyclization shown in Scheme 1. A minor isomeric sesterterpenoid of unknown structure has also been isolated from this source.
Scheme 1
4 Ophiobolins
Most of the sesterterpenoids that have been isolated from natural sources possess this carbon skeleton. A number of the fungal metabolites were studied independently by different groups, who assigned them various trivial names. The identity of these compounds was subsequently established and consequently some of these trivial names have been abandoned. The acceptedI6 names are set out in the Ophiobolin A was isolated from Ophiobolus miyabeanus. Its structure (26) rests on the one hand on the X-ray analysis” of a bromo-derivative and on the other on a chemical degradation. 18v1’ The spectral characteristics of ophiobolin A defined the nature of the oxygen functions and the double bonds. The relationship between the aldehyde and ketonic functions was revealed in a number of reactions. Thus reduction of ophiobolin A gave two stereoisomeric triols, one of which was re-oxidized to ophiobolin A whereas the other on oxidation gave a y-lactone. In tetrahydro-ophiobolin A the aldehyde existed predominantly in
23
K . Tsuda, S. Nozoe, M. Morisaki, K. Harai, A. Itai, S. Okuda, L. Canonica, A. Fiecchi, M. Galli Kienle, and A. Scala, Tetrahedron Letters, 1967, 3369. S . Nozoe, M . Morisaki, K. Tsuda, Y . Iitaka, N. Takahashi, S. Tamura, K. Ishibashi, and M . Shiraska, J. Amer. Chem. SOC.,1965,87, 4968. L. Canonica, A. Fiecchi, M. Galli Kienle, and A. Scala, Tetrahedron Letters, 1966, 1211. L. Canonica, A. Fiecchi, M. Galli Kienle, and A. Scala, Tetrahedron Letters, 1966, 1329. K. Ishibashi, J . Agric. Chem. SOC.Japan, 1961, 35, 323. M . Ohkawa and T . Tamura, Agric. and Biof. Chem. (Japan), 1966, 30, 285. S. Nozoe, K. Hirai, and K. Tsuda, Tetrahedron Letters, 1966, 221 1. A. Itai, S. Nozoe, K. Tsuda, S. Okuda, Y . Iitaka, and Y. Nakayama, Tetrahedron Letters,
l4
1967, 41 11. S. Nozoe, A. Itai, K. Tsuda, and S. Okuda, Tetrahedron Letters, 1967, 41 13.
l6
l9
2o 2’
22
177
Ses ter terpeno ids
Table 1 Nomenclature of sesterterpenoids Literature N m t s
Trivial Names
Systematic Names
Ophiobolin’ 14aJ7-oxide
Cochliobolin A19 Zizanin” Ophiobolin B (27j
Ophiobola-7,l8-dien-21-al-3a,14ac-diol5-one
Zizanin A’9*22 Ophiobolin C (28) Cephalonic acid’ 3 7 2 4 Ophiobolin D (29)
Ophiobola-7,18-dien-21 -al-3a-ol-5-one Ophiobola-3,6,18-trien-8~-ol-21-oic acid
Zizanin B’‘ Cochliobolin B t 9
H
the enolic form, possibly stabilized by hydrogen-bonding with the cyclopentanone. This compound underwent a curious reaction with oxygen to form the cyclic peroxide (30). Hydrolysis of this gave an enolic P-diketone. With hydrazine a pyridazine was formed between these two centres. The structure of the side-chain followed from spectral evidence and several degradations. For example, oxidation of tetrahydro-ophiobolin A with nitric acid afforded the heptanoic acid lactone (31). Ophiobolin B (zizanin B) (27), which was isolatedz2 from the culture filtrate of Hefrninthosporiurn zizaniae or Ophiobofus heterostrophus, lacks the side-chain ether group, possessing in its place a C-14 hydroxy-group. The relationship between ophioboliris A and B was established in one instance” by the formation
Terpenoih and Steroids
178
OFo1 HO
A
of a common hydrogenolysis product (32)and in another by the partial synthesis22 duphiobolin B. Thus ophiobolin A was reduced with lithium aluminium li ydride to the corresponding triols. Hydrogenolysis of the allylic ether with lithium in liquid ammonia generated the tertiary C- 14 alcohol whilst re-oxidation with chromium trioxide in pyridine gave ophiobolin B. Qphiobolin C (zizanin A isolated from Helrninthosporiurn zizaniae) (28) lacked22 the C- 14 hydroxy-group but otherwise underwent the typical dehydration of the C-3 hydroxy-group to form a cyclopentenone. Qphiobolin D (Cephalonic acid) (29) was produced by Cephalosporium caerulens. Its structure was determined23 by X-ray analysis of the bromo-acetate. A number of chemical transformation^,^^ including the ready lactonization between C-21 and C-5 and a correlation with ophiobolin C, confirmed this structure. Qphiobolin F, isolated from Cochliobolus heterostrophus, was assigned2 the structure (2) on the basis of its n.m.r. spectra. The ophiobolin carbon skeleton may arise by a cyclization of geranylfarnesyl pyrophosphate in the manner shown in Scheme 2. In support of this scheme, the
HO-
Scheme 2
J
179
Sesterterpeno ids
biosynthesis of the ophiobolins from m e ~ a l o n a t eand ~ ~ from geranylfarnesyl pyrophosphate3 and the origin of the oxygen a t o m ~ ~have ~ ' ~been ' studied. At one time fusicoccin A, because of its structural similarity, was considered to be a relative of the ophiobolins. Evidence has recently been presented to suggest that it is diterpenoid. It will, however, be briefly described here as it was not treated in the relevant diterpenoid chapter. Fusicoccin A (33) is a highly phyto-
CH,OMe (33)
toxic glycoside which was isolated2* from the culture filtrate of Fusicoccurn amygdali. Its structure rests on independent chemical evidence2' and two X-ray analy~es.~'.~ The aglycone showed reactions typical of an a-glycol. Thus it formed an acetonide and underwent oxidation with sodium periodate to form an unstable dialdehyde. A detailed analysis of the n.m.r. spectrum of the tetrabenzoate led to the identification of a number of major structural fragments. The C-8 hydroxy-group, one of the hydroxy-groups of the a-glycol, formed a C-8-C-2 transannular ether which permitted further degradation, particularly one which established a relationship between the C-19 hydroxy-group and C-9. The massspectral fragmentation pattern of the triacetate of this ether was particularly informative regarding the structure of ring c. Fusicoccin H, a minor metabolite of the fungus, has been shown3' to have the structure (34). Again the formation of a C-8-C-2 ether via a mercuribromide derivative gave useful structural 25
L. Canonica, A. Fiecchi, M. Galli Kienle, G . M. Ranzi, and A. Scala, Tetrahedron Letters, 1966, 3035.
26 2'
'*
S. Nozoe, M. Morisaki, K. Tsuda, and S. Okuda, Tetrahedron Letters, 1967, 3365.
L. Canonica, A. Fiecchi, M. Galli Kienle, G. M. Ranzi, A. Scala, T. Salvatori, and E. Pella, Tetrahedron Letters, 1967, 3371. A. Ballio, E. B. Chain, P. De Leo, B. F. Erlanger, M. Mauri, and A. Tonolo, Nature, 1964,203,297.
29
30
31
K. D. Barrow, D. H. R. Barton, E. B. Chain, U. F. W. Ohnsorge, and R. Thomas, Chem. Comm., 1968, 1198; K. D. Barrow, D. H. R. Barton, E. B. Chain, C . Conlay, T. C. Smale, R. Thomas, and E. S . Waight, J . Chem. SOC.(0,1971,1259; K. D. Barrow, D. H. R. Barton, E. B. Chain, U. F. W. Ohnsorge, and R. Thomas, ibid., 1971, 1265. A. Ballio, M. Brufani, C. G. Casinovi, S. Cerrini, W. Fedeli, R. Pellicciari, B. Santurbano, and A. Vaciago, Experientia, 1968, 24, 631; M. Brufani, S. Cerrini, W. Fedeli, and A. Vaciago, J . Chem. SOC.(B), 1971, 2021. K. D. Barrow, D. H. R. Barton, Sir Ernst Chain, U. F. W. Ohnsorge, and R. P. Sharma, J . C . S . Perkin I, 1973, 1590.
Terpenoidr and Steroids HO HO--
CH,OH
CH,OH (34)
information. This C , , glycoside acted as a precursor of fusicoccin A. A number of isomerization products of fusicoccin A in which the acetoxy-groups of the sugar residue have migrated or have been hydrolysed have been isolated3, from the culture broth. They can be formed non-enzymatically at the pH of the fermentation and are probably artefacts (see also Part I, Ch. 7, p. 271). Cotylenol A is a fungal metabolite which is the aglycone of the cotylenins, a group of substances affecting leaf growth. It has been shown33 to have the structure (35) and is clearly related to the fusicoccins. HO
O H b
(35)
The scale insect Ceroplastes albolineatus secretes a wax as a protection against desiccation. Saponification and extraction of this wax gave ceroplastol I (36),34 ceroplastol I1 (38),3s ceroplasteric acid (37),36 and albolic acid (39).3’ The structure of ceroplastol 1 was determined by X-ray analysis of its p-bromobenzoate. Although closely related to the ophiobolins, these insect substances differ in the stereochemistry of the A/B ring fusion, which is trans. ”
33 34
35 36
3’
A. Ballio, C . G. Casinovi, G. Randazzo, and C . Rossi, Experientia, 1970, 26, 349; A. Ballio, C. G. Casinovi, M. Framon-dino, G. Grandolini, F. Merichini, G . Randazzo, and C. Rossi, ibid., 1972, 28, 128. T. Sassa, Agric. and Biol. Chem. (Japan), 1972, 36, 2037. T. Rios and F. Colunga, Chem. and Ind., 1965, 1184. T. Rios and L. Quijano, Tetrahedron Letters, 1969, 1317. Y. Iitaka, 1. Watanabe, I. T. Harrison, and S. Harrison, J . Amer. Chem. SOC.,1968,90, 1092. T. Rios and F. Gomez, Tetrahedron Letters, 1969, 2929.
181
Sesterterpenoids
(36) R (37) R
(38) R = CH,OH (39) R = C 0 , H
= CH,OH = C02H
5 Substances of Miscellaneous Structure Cheilanthatriol (40),which was isolated38 from the fern Cheilanthanes farinosa, represents a cyclization pattern that is typical of the triterpenoids. Its structure rests on spectral studies of the triol, its acetates, and selective oxidation products, whilst dehydrogenation gave 1,7-dirnethylphenanthrene and 1,7,8-trirnethylphenanthrene. The disposition of the side-chain followed from the mass-spectral fragmentation pattern. OH
/ /
OH
CH,OH
-
C0,H
Retigeranic acid (41) was isolated from the lichens of the Lobaria retigera group and its structure determined by X-ray analysis. Biogenetically it might 38
H. Khan, A. Zaman, G . L. Chetty, A. S. Gupta, and Sukh Dev, Tetrahedron Letters, 1971,4443.
182
Terpenoids and Steroids
arise from geranylfarnesyl pyrophosphate as in Scheme 3.39 Scalarin (42) was isolated from the sponge Cacospongia scalaris. Its structure rests on spectral evidence and on a pyrolytic degradation of the oxidation product (43) to give the known bicyclic compound (44)."'
4 Scheme 3
j9
40
M. Kaneda, R. Takahashi, Y. Iitaka, and S. Shibata, Tetrahedron Letters, 1972, 4609. E. Fattorusso, S. Magno, C. Santacroce, and D. Sica, Tetrahedron, 1972, 28, 5993.
5 Triterpenoids BY J. D. CONNOLLY
1 Reviews
Reviews have appeared on quassinoid bitter principles' and P-amyrin triterpenoids. * 2 Squalene Group The highlight of the year in triterpenoid chemistry is undoubtedly the series of papers3-' by van Tamelen and his colleagues on the biogenetic-type total syntheses of several tetra- and penta-cyclic triterpenoids by Lewis acid-induced cyclization of modified squalene 2,3-oxide derivatives with preformed D or D and E rings. The totally synthetic epoxide precursor (1) was obtained3 as a mixture of epimers. One epimer cyclizes, presumably in the all-chair conformation (2), to isoeuphenol (3), and in the chair-boat-chair conformation (4) to 24,25dihydro-A' 3(1 7)-protostan-3/3-ol (5) and 24,25-dihydroparkeol (6). A further product of the reaction is (-)-isotirucallenol, the enantiomer of the natural compound (7),which is formed from the other epimer. Since 24,25-dihydroparkeol has been converted into 24,25-dihydrolanosterol this work also represents a formal synthesis of the latter. The corresponding 24,25-dehydro-epoxide (8) undergoes cyclization to parkeol(9) and ( - )-isotirucallol. In an extension of this work to pentacyclic systems, the epoxide (10) was converted4 into racemic A' 2-dehydrotetrahymano1 (11). Trifluoroperacetic acid oxidation of the acetate of (ll), followed by Wolff-Kishner reduction of the ketone formed, afforded racemic tetrahymanol(l2). A different approach, which more closely follows the established biological pathway, involved the polyene (13). Treatment of (13) with boron trifluoride etherate or acetic acid-sulphuric acid led to racemic A9(' ')-dehydrotetrahymanol(14). Another pentacyclic triterpenoid, racemic 6-amyrin (15), was successfully synthesized' from the bicyclic epoxide (16). The route to (16) also involved a
'
J . Polonsky, Fortschr. Chem. org. Naturstoffe, 1973, 30, 101. R. B. Boar and J. Allen, Phytochemistry, 1973, 12, 2571. E. E. van Tamelen and R . J. Anderson, J . Amer. Chem. SOC.,1972,94, 8225. ' E. E. van Tamelen, R . A . Holton, R. E. Hopla, and W. E. Konz, J . Amer. Chem. SOC., 1972,94, 8228. E. E. van Tamelen, M. P. Seiler, and W. Wierenga, J . Amer. Chem. SOC.,19?2,94, 8229.
Terpenoids and Steroids
184
PJ
y
0
(1)
(8) 24,25-dehydro
/
* H O (2) H
HO
H
HO
HO (6) (9) 24,25-dehydro
185
Triterpenoids
(12) (14)9,ll-dehydro
HO (15) (19) A12-isomer;18P-H
polyene cyclization step, (17 + (18). The synthesis of 6-amyrin formally embraces /3-amyrin (19) and germanicol (20). The latter is formed from 6-amyrin by a photochemical rearrangement.
EtO,C Eto&
Et0,C Et02@
\
I
HO
Terpenoids and Steroids
186
Arigoni and his colleagues have independently synthesized6 the bicyclic epoxide (16), specifically tritiated at C-1 1. Enzyme-catalysed cyclization, using the homogenate of Pisum satiuum, afforded /?-amyrin (19), without randomization of the label. This provides a further example of the versatility of the cyclase enzyme system. the results of model solvolysis experiments aimed Two groups have at elucidating the details of the transformation of presqualene alcohol into squalene. Reaction of the cyclobutyl tosylate (21) and the cyclopropylmethyl p-nitrobenzoate (22) under solvolytic conditions afforded the acyclic dienols (23) and (24).
A further investigation of model systems pertinent to triterpenoid biogenesis concerned' the solvolysis of 2,2,4aa-trimethyl-l-decalyl methanesulphonate (25). The ring-contracted product (26) was obtained, but deuterium-labelling studies indicated that there was scrambling of the geminal methyl groups, and hence the bridged ion (27) cannot be the sole intermediate. It seems likely that the classical ion (28) intervenes.
+ dH (25)
(26)
' H. Horan, J . P. McCormick, and D. Arigoni, J . C . S . Chem. Comm., 1973, 73. ' C. D. Poulter, 0.J . Muscio, C. J . Spillner, and R. G. Goodfellow, J . Amer. Chem. SOC., 1972,94, 5921. R. M. Coates and W. H. Robinson, J . Amer. Chem. SOC.,1972, 94, 5920. R. M. Coates and S. K . Chung, J . Org. Chem., 1973,38, 3677.
187
Triterpenoids
The absolute configuration of presqualene alcohol has received further attention." Comparison of the c.d. curves of the benzoates of presqualene alcohol (29)and (lR,3R)-chrysanthemum alcohol (30)and their corresponding ozonolysis products (31) and (32) indicates that presqualene alcohol is (lR,2R,3R). This reverses the previous assignment. CH,OH
CH,OH
(29) R = (30) R = H
R = CH2CH20H (32) R = H
(31)
The practical details of the partial synthesis of 1-'4C-labelled 2,3-epoxysqualene, of high specific activity, from the aldehyde of trisnorsqualene have been described. An investigation' of the properties of the squalene-2(3),22(23)di-epoxide-a-onocerin cyclase enzyme system from Ononis spinosa root has provided evidence for the formation of an intermediate, pre-onocerin, on the pathway to a-onocerin from squalene diepoxide. Pre-onocerin is tentatively assigned the structure (33) (see also Chapter 7, p. 287). Further details of the cyclization of 2,3-oxidosqualene by the microsomal fraction of Cephalosporium caerulens have appeared13 (see Vol. 1, p. 163). The enzyme system converts 2,3-oxidosqualene into lanosterol and 3P-hydroxyprotosta-17(20)(16,21-cis),24-diene (34) but no activity appears in the doublebond isomer (35).
(33) lo
'
l 3
(34)
(35)
G. Popjak, J. Edmond, and S.-M. Wong, J. Amer. Chem. SOC.,1973, 95, 2713. J . Bascoul, D. Nikolaidis, A. Crastes de Paulet, and L. Pichat, Bull. SOC.chim. France IZ, 1973, 2318. M. G . Rowan and P. D. G. Dean, Phytochemistry, 1972, 11, 31 1 1 . A. Kawaguchi, H. Kobayashi, and S. Okuda, Chem. and Pharm. Bull. (Japan), 1973, 21, 577.
Terpenoids and Steroids
188
The unusual tetramethylated acyclic triterpenoid botryococcene (36) is producedI4 by the resting state of the green alga Botryococcus braunii. The structural assignment rests on 13Cn.m.r., oxidative degradation, and biosynthetic inference. Permanganate-periodate oxidation affords (37).
Several paped5-” have appeared on the structures and biosynthesis of a group of Daphniphyllum alkaloids, e.g. daphniphyllidone (38), which are formed via a squalene-like intermediate.
3 Fusidane-Lanostane Group
TII, tetracyclic compound (39), a degradation product of fusidic acid, has been synthesized’8 from the bicyclic enone (40) (Scheme 1). The structure and stereochemistry of racemic (39) were confirmed by an X-ray analysis. A model synthesis of the trans-syn-transsystem of fusidic and helvolic acids utilize^'^ the boron l4
l6
l7
l9
R. E. Cox, A. L. Burlingame, D. M. Wilson, G. Eglinton, and J. R. Maxwell, J.C.S. Chem. Comm., 1973,284. M. Toda, H. Niwa, Y. Hirata, and S. Yamamura, Tetrahedron Letters, 1973, 797. K . Suzuki, S. Okuda, H. Niwa, M. Toda, Y. Hirata, and S. Yamamura, Tetrahedron Letters, 1973, 799. H. Niwa, Y. Hirata, K. Suzuki, and S. Yamamura, Tetrahedron Letters, 1973, 2129. W. G. Dauben, G. Ahlgren, T. J. Leitereg, W. C. Schwarzel, and M. Yoshioko, J . Amer. Chem. SOC.,1972, 94, 8593. R . E. Ireland and U. Hengartner, J . Amer. Chem. Soc., 1972,94, 3652.
189
Triterpenoidr
OBu'
t
0
(39) Rezwnts: i, NaH-DMSO; ii, KOBul-Mel; iii, aq. HOAc; iv, Triton B; v, KOH-Bu'OHMeI; vi, NaOH; vii, (COCI),; viii, LiCuEt,; ix, Triton B; x, m-ClC,H,CO,H; xi, BF,,Et,O ;xii, p-MeC,H,-S0,H-C6H,; xiii, Li-NH,-Bu'OH.
Scheme 1
trifluoride-induced rearrangement of the epoxide (41) to give the trans-syn-transperhydrophenanthrene(42). A (2)-17(20) double bond, a necessary feature for the biological activity of compounds like fusidic acid, has been successfully introduced" into the 'O
I. L. Batey, J. T. Pinhey, B. J. Ralph, J. J. H. Simes, and M. Wootton, J.C.S. Perkin I , 1972,2260.
190
Terpenoids and Steroids
tumulosic acid derivative (43)by refluxing in benzene with toluene-p-sulphonic acid to give (44).
Further investigation of the bark of white pine, Pinus rnonticola,has yielded2' two new lanostanes (45) and (46).The stereochemistry at C-24follows from the
R = Me (46) R = H (45)
c.d. curve of the [Pr(dpm),] complex and from nuclear Overhauser effects. Seven new related compounds have been isolated22from the fruit bodies of the fungus Echinodontium tsugicola. They are deacetyl-3-epiechinodol(47),echinodone (48), deacetylechinodone (49), 3-epiechinodol (50), deacetoxy-3-epjechinodol (5l), deacetoxyechinodol (52), and deacetoxyechinodone (53). Further compounds from Neolitsea pulchella include23 3j?,24~-dimethoxy-24,25-dimethyl-lanost9(11)-ene (54), 245-methoxy-24,25-dimethyl-lanost-9( 1l)-en-3-one (59, and the known 24,24-dimethyl-lanosta-9(11),25-dien-3j?-ol.
22
23
J . P. Kutney, G. Eigendorf, R. B. Swingle, G. D. Knowles, J. W. Rowe, and B. A. Nagasampagi, Tetrahedron Letters, 1973, 3 1 1 5. A. Kanematsu and S. Natori, Chem. and Pharm. Bull. (Japan), 1972, 20, 1993. W.-S. Chan qnd W.-H. Hui, J . C . S . Perkin I, 1973, 490.
191
Triterpenoids R2
1
(47) R' = H,a-OH; (48) R' = 0, (49) R' = 0, (50) R 1 = H,a-OH, (51) R' = H,a-OH, (52) R' = H,/?-OH, (53) R' = 0,
(54) R = H, /?-OMe
R2 = OH R2 = OAC R~ = OH R2 = OAc R2 = H R2 = H R2 = H
(55) R
=
0
3-a-Car'boxyacetoxystowardolicacid (56) is another member of the small group of naturally occurring malonate esters. It was isolatedz4 from the fungus Tranetes stowardii. The corresponding dihydro-derivative(57) was converted into the same mixture of butenolides (58) (C-23 epimers) as is obtained from the known fungal metabolite carboxyacetoxyquercinicacid (59).
>
(57) (2-24 and/or (59) C-25 isomers
H I
:
(58)
Separation of the diastereoisomeric 24,25-epoxides of lano~ta-8~24-dien-3P-yl acetate was effected2' by utilizing their differing solubilities in methanol. The absolute configurations of the individual diastereoisomers were established by use of the Horeau method on the corresponding C-24 hydroxy-derivatives. Two 24
25
H. T. Cheung, J. C. F. Seeto, and T. R. Watson, Austral. J . Chern., 1973,26, 609. R. B. Boar, D. A. Lewis, and J. F. McGhie, J.C.S. Perkin I , 1972, 2231.
192
Terpenoids and Steroih
groups of workers have r e p ~ r t e d the ~ ~characterization .~~ of the C-8 epimers of 3P-acetoxylanost-9(1l)-en-7-one (60). The 8P-isomer is one of the products of
the oxidation of 3B-acetoxylanost-8-ene with chromium trioxide in acetic acid whereas the 8cc-isomer is formed, together with the corresponding 7,9(1l)diene, by the action of 30% hydrogen peroxide in acetic acid at 20 "C (see Vol. 3, p. 200). (61) in ethanol Acid rearrangement of (23S)-lanosta-8,24-diene-38,23diol the corresponding 25-ethoxy-derivaaffords2*lanosta-8,23diene-3~,25-diol(62), tive, and a mixture of the epimeric 23-ethoxylanosta-8,24-dien-3/l-ols.
An improved method2' of isolating lanosterol from 'isocholesterol', the nonsaponifiable fraction ofwool fat, involvesbrominationoftheacetylated mixtureand fractionation of the C-24 diastereoisomeric dibromides (63). These compounds Br
" 27
29
R. B. Boar, J. F. McGhie, and D. A. Lewis, J.C.S. Perkin I , 1972, 2590. E. V. Lassak, J. T. Pinhey, and J. J. H. Simes, Ausrrul. J . Chem., 1973, 26, 1051. N. Entwistle and A. D. Pratt, J.C.S. Perkin Z, 1973, 1235. R. B. Boar, D. A. Lewis, and J. F. McGhie, J.C.S. Perkin I , 1973, 1583.
Triterpenoih
193
are useful synthetic intermediates and allow modification of the more hindered A*-double bond followed by facile regeneration of the A24-do~ble bond. Both of the diastereoisomeric dibromides were converted, in high yield, into agnosterol (64) via the 8a,9a-epoxides.
An improved route to triterp-2-enes has been de~cribed.~'Treatment of a triterpenoid 3-ketone with toluene-a-thiol and boron trifluoride etherate in acetic acid affords the 3-benzylthio-2-ene (65) which can be desulphurized in excellent yield by Raney nickel or nickel boride to give the corresponding 2-ene. Desulphurization of 3,3-ethylenedithiolanost-8-ene(66) with nickel boride in
ethanol-boric acid affords lanosta-2,8-diene and lanost-8-ene in the ratio 4 : 1. Nickel boride is more conveniently prepared and handled than is Raney nickel. A number of ring B diosphenol derivatives of lanosterol have been r e p ~ r t e d . ~ The 13C resonances of lanosterol, euphadienol, and euphenol have been assigned.32The assignments for C-6, C-11, C-19, and C-21 of lanosterol are at variance with those reported last year (see Vol. 3, p. 203). The full details of the degradation of the lanosterol side-chain have a ~ p e a r e d . ~ A new sapogenin from the sea cucumber, Stichopus chloronotus, has the structure 23~-acetoxy-l7deoxy-7,8dihydroholothurinogenin (67).34 It is unusual in having a 23-acetoxy-group and lacking a 12-alkoxy- or hydroxy-group. Two new &-methyl CJ1sterols are cyclofuntumienol(68) from the bark and leaves of Funturnia e l ~ s t i c aand ~ ~ cydotrichosantol (69) from the leaves of 30
R. B. Boar, D. W. Hawkins, J. F. McGhie, and D. H. R. Barton, J.C.S. Perkin I , 1973,654.
' W. Kreiser and W. Ulrich, Annafen, 1972, 761, 121. 32 33 34
3s
S. A. Knight, Tetrahedron Letters, 1973, 83. L. H. Briggs, J. P. Bartley, and P. S. Rutledge, J.C.S. P erkin I, 1973, 806. I. Rothberg, B. M. Tursch, and C. Djerassi, J . Org. Chem., 1973,38, 209. L. Mukam, G. Charles, J. Hentchoya, Th. Njimi, and G. Ourisson, Tetrahedron Lefters, 1973,2779.
194
Terpenoids and Steroids OAc
Trichosantes p ~ l r n a t a . ~?le structure of cyclofuntumienol was confirmed by a partial synthesis from cycloeucalenol. The full details of the structures and absolute configurations of cyclograndisolide and epicyclograndisolide from Abies grandis have a ~ p e a r e d . ~An X-ray analysis has confirmed that cyclograndisolide is (23R)-3a-methoxy-9,19-cyclo-9~-lanost-24-ene-26,23-olide (70).
MeO-
Cyclolaudanol (71) has been converted3* into (24S)-9fl,19-cyclo-14a,24dimethylcholest-4-en-3-one (72). The first step in the modification of ring A involved heating cyclolaudanol tosylate in pyridine to give the ~-nor-3-isopropenyl derivative (73). The latter was transformed (Scheme 2) into (72). In all the C-3-substituted A,B-trans-fused A-nOr-9fl,19-cyclolanostane derivatives an
’‘
M . Kocor and J. St. Pyrek, J . Org. Chem., 1973, 38, 3688. J . P. Kutney, D . S. Grierson, G. D . Knowles, N. D. Westcott, and I. H. Rogers, Tetrahedron, 1973, 29, 13. A. S. Narula and Sukh Dev, Tetrahedron, 1973, 29, 569.
-”
.”
195
Triterpertoids
P iii -\I
__*
0
(73)
(71) R = H,P-OH (74) R = 0; AZ5
1
vii
ix, x
+-
8 H
Reagents: i, TsC1-py; ii, py, A ; iii, 0,; iv, CF,CO,H; v, KOH; vi, CrO,; vii, Ph,PMel; viii, Li-H,NCH,CH,NH,; ix, 0,; x, KOH.
Scheme 2
abnormally high chemical shift of one of the cyclopropyl methylene protons (6 -0.11 to -0.33) was observed. This is probably due to a shielding effect of the C-5-C-6 and C-6-C-7 bonds. ~~ The photochemistry of cyclolaudenone (74) has been i n ~ e s t i g a t e d .Ultraviolet irradiation of an aqueous dioxan solution of (74) affords the expected seco-acid (75). In the presence of methanol or cyclohexylamine the corresponding ester or amide is formed. Labelling studies support the intervention of a keten intermediate. A minor product is the ring-expanded alcohol (76).
Datiscacin (77), a cucurbitacin 20-acetate, is another tumour-inhibitory principle from Datisca glomerata.4' Fabacein, a bitter principle from Echinocystis 39 40
C. Ouannes and R. Beugelmans, Bull. SOC.chim. France, 1972, 4275. S. M . Kupchan, G. Tsou, and C. W. Sigel, J. Org. Chem., 1973, 38, 1420.
196
Terpenoids and Steroids
fubacea,41 is the 16-acetate (78) of cucurbitacin B. Fabacein has a much lower cytotoxic activity than cucurbitacin B and this suggests that the 16-hydroxygroup of the latter may hydrogen-bond to the C-22 carbonyl group and assist in activating the ctQ-unsaturated ketone to attack by a biological nucleophile.
23,24-Dihydrocucurbitacin F (79) has been isolated42 from Crinodendron hookerianum together with cucurbitacins D, F, and H. This is the first report of cucurbitacins in the Eleaocarpaceae.
A model synthesis of the cucurbitacin side-chain has been achieved43 starting from the aldehyde (80) (Scheme 3). 41
42
43
S. M . Kupchan and G . Tsou, J. Org. Chem., 1973,38, 1055. M . Bittner, K. A. Poyser, J. P. Poyser, M. Silva, E. Weldt, and P. G. Sammes, Phyiochemistry, 1973, 12, 1427. F. de Reinach-Hirtzbach and G. Ourisson, Tetrahedron Letters, 1973, 1363.
197
Triterpeno idr
{p {pH /
VI, vli
OH 0
OH
+
Reagents: i, Ac,O; ii, p-NO,-C,H,.CO,H; iii, THPO-3v, Na,CO,; vi, LiAlH,; vii, Ag,CO,.
--MgBr;
iv, TsOH;
Scheme 3
Hydrogen peroxide oxidation of hydroxymethyleneanhydrodihydrolitsomentone (81) affords44mainly the ring-contracted acid (82). The presence of the As-double bond appears to favour the formation of this product.
4 Dammarme-Euphane Group
Betulafolientetraol A (83) and B (84) are two new dammaranes from Betula p l ~ t y p h y l l a . ~ ’Photo-oxidation of betulafolientriol (85) afforded a mixture of (83) and (84). 44
T. R. Govindachari, N. Viswanathan, and A . R. Sidhaye, Indian J . Chem., 1972, 10,
45
N . Ikekawa, A. Ohta, M. Seki, and A. Takahashi, Phytochemisrry, 1972,11,3037.
786.
198
Terpenoids and Steroid
The structure (86) has been proposed46 for bacogenin A, which was obtained by acid hydrolysis of bacoside A, a saponin from Bacopa monniera. Mild acidic hydrolysis of the glycosidic fraction of Colletia spinosissima yielded47 the two homologous sapogenins (87) and (88). Mild base treatment of (87) leads to the cyclopentanone (89) by a retro-Aldol process and reaction with acid results in an interesting condensation to give (90).
(87)
R
(88) R 46
"
= =
Me H
(89)
(90)
D. K. Kulshreshtha and R. P. Rastogi, Phytochemistry, 1973, 12, 887. P. Pacheco, M. Silva, P. G. Sammes, and T. W. Tyler, Phytochemistry, 1973, 12, 893.
199
Triterpenoids
Alnincanone (91) and its three diastereoisomers at C-20 and (2-24 have been s y n t h e ~ i z e dfrom ~ ~ dipterocarpol. The configurations of the four isomers have been assigned by several methods. Natural alnincanone is (20S,24R)and has the same C-20 configuration as dipterocarpol and related dammaranes.
(91)
The (24s) configuration of the epoxide of aglaiol(92) was conveniently established4’ by acid-catalysed methanolysis followed by application of the Horeau method to the methoxy-alcohol(93). Aglaiol may arise in vivo by cyclization of
(3S)2,3-(24S)24,25-diepoxysqualene.
HO (92)
(93)
The full papers on the chemistry of the side-chain of dammarane sapogenins from gingseng” and the application of soil bacterial-hydrolysis to the gingseng root saponinsS1 have appeared. The latter provides further evidence for the genuine nature of the sapogenin (20S)-protopanaxadiol (94) and also describes OH
50
R. Labriola and G. Ourisson, Tetrahedron, 1973, 29, 2105. R. B. Boar and K. Damps, J.C.S. Chem. Comm., 1973, 115. T. Ohsawa, N. Tanaka, 0. Tanaka, and S. Shibata, Chem. and Pharm. Bull. (Japan),
51
I. Yosioka, T. Sugawara, K. Imai, and I. Kitagawa, Chem. and Pharm. Bull. (Japan),
48
49
1972,20, 1890. 1972,20,2418.
Terpenoidr and Steroids
200
the characterization of a prosapogenol, 20-0-#?-~-glucopyranosyl-(20S)-protopanaxadiol. Tissue cultures of Panax gingseng produce’ gingsenosides Rb, and Rg,. Further work on the triterpenoid constituents of the galls of Pistacia species has resulted in the isolation of the following new tirucallanes : 26-hydroxytirucallone (95) from P. t e r e b i n t h ~ sand ~ ~ 24,25-dihydromasticadienonicacid (96), 24,25-dihydromasticadienolic acid (97), and 24,25-dihydro-3-epimasticadienolic acid (98) from P . l e n t i s ~ u s .Gluanone ~~ (99) has been reportedJ5 as a natural product from Canscora decussata. It occurs with a second compound, canscoradione, which has been tentatively assigned the structure (100).
*
H
CH,OH
I\t-”\
CO,H
(96) R = 0 (97) R = H,B-OH (98) R = H,a-OH
(99)
Corollatadiol (101) is a new compound from Euphorbia ~ o r o l l a t a .The ~ ~ full details of the structural elucidation of kulinone and kulactone, two euphane derivatives from the bark of Melia azedarach, have appeared.” Two minor components of the extract are kulolactone (102) and methyl kulonate (103). It has been shown58 that tirucall-7-ene derivatives can be rearranged to the corresponding apotirucall-14-enes under the influence of bromine. Thus methyl 52
s3 54
s5 56
” 58
T. Furuya, H. Kojima, H . Syono, T. Ishii, K. Uotani, and M. Nishio, Chem. andPharm. Bull. (Japan), 1973, 21, 98. P. Monaco, R. Caputo, G. Palumbo, and L. Mangoni, Phytochemistry, 1973, 12, 939. P. Monaco, R. Caputo, G . Palumbo, and L. Mangoni, Phytochemisrry, 1973,12,2534. S . Ghosal, R. K. Chaudhuri, and A. Nath, Phyrochemistry, 1973,12, 1763. D. M. Piatak and K. A. Reimann, Tetrahedron Letters, 1972, 4525. C. Chiang and G . C. Chang, Tetrahedron, 1973, 29, 191 1. T. G. Halsall and R. J. Weston, J.C.S. Chem. Comm., 1972, 1212.
201
Triterpeno ids
i?
I
C0,Me
3a~-acetoxytirucalla-7,24-dien-2l-oate is transformed into (104) on treatment with bromine in chloroform. The reaction presumably proceeds via a bromonium ion. Br
( 104)
A new class of cyclopropane-containing triterpenoids has been reporteds9 from Guarea glubra (Meliaceae). The novel carbon skeleton was established by X-ray analysis and is exemplified by glabretal (105). The related 3-ketone, 3a-(ahydroxyvalerate), 3a-angelate, 3a-tiglate, 3a-acrylate, and a seventh compound, tentatively assigned the structure (106),were also isolated. The presence of the C-7axial acetoxy-group suggests that these compounds are formed in viuo by the capture of the C-13methyl group by a C-14cation as a subsequent step to a tirucallol-apotirucallol rearrangement. 5q
G . Ferguson, P. A. Gunn, W. C. Marsh, R. McCrindle, R. Restivo, J. D. Connolly, J. W. B. Fulke, and M. S. Henderson, J.C.S. Chem. Comm., 1973, 159.
202
Terpenoids and Steroids
H O H , C y
Tetranortriterpen0ids.-An interesting group of compounds, which neatly demonstrates the various stages of oxidative modification of tetranortriterpenoids, has been isolated from Carapa guianensis.60 Included in this group are three new compounds, 6a-acetoxyepoxyazadiradione(107), 6a-acetoxygedunin (108), and 6a-hydroxygedunin (109). The structure of nyasin, from Khayu
0
*
OAc
OR
OAc
(108) (109)
R R
= AC =H
nyasica, has been confirmed6' as (110) by its preparation from llb-acetoxykhivorin by mild hydrolysis. Two further examples of the occurrence of ring
0 ACO"
6o 61
'
OAc
D. Lavie, E. C. Levy, and R . Zelnik, Bioorganic Chemistry, 1972, 2, 59. J . D. Connolly and D. A. H . Taylor, J . C . S . Perkin I , 1973, 686.
203
Triterpenoids
A-cleaved tetranortriterpenoids in the Meliaceae have been reported. 68Acetoxy-7a-obacunol (111) and 6P-acetoxy-7a-obacunyl acetate (1 12) were obtained62from Trichilia trifolia together with trifolin (113). The full paper on havanensin and trichilenone has been published.63
0
0
H O * ’ F * OCOFHCHMe, , OAc (111) (112)
R R
OH
=
H
=
AC
(113)
17-Dehydrolimonoate A-ring lactone (1 14) has been detected64 in citrus tissue and juice. It is non-bitter and may be an intermediate in a ‘debittering’ pathway in citrus fruits. Another product of the pyrolysis of methyl angoiensate (1 15) has been characterized as (1 16).65
P
0%
62 63
’‘
65
O
C0,Me
D. R. Taylor, Rev. latinoamer. Quim., 1971, 87. W. R. Chan, J. A. Gibbs, and D. R. Taylor, J.C.S. Perkin I, 1973, 1047. A. C. Hsu, S. Hasegawa, V. P. Maier, and R . D. Bennet, Phytochemistry, 1973, 12, 563. K . Jewers, A . H. Manchanda, and D. A. H . Taylor, Chem. and Ind., 1972,976.
Terpenoids and Steroids
204
Bicyclononano1ides.-The controversy which surrounds the structure of fissinolide (117) has been resolved66 in the following way. Treatment of fissinolide (117) with N-bromosuccinimide in refluxing carbon tetrachloride under U.V. irradiation afforded the diene (118), which resisted hydrogenation, and angustadienolide (119). The amended structure of the latter follows from its U.V.spectrum and the presence of two vinyl protons in the n.m.r. In the original work the signal for H-30 was overlooked. Hydrogenation of angustadienolide gave fissinolide, which is therefore (117), as originally proposed. The H-17 chemical-shift differences discussed earlier (see Vol. 1, p. 177)must be due to factors other than the position of the double bond. The structures of fissinolide and angustadienolide are supported by their I3Cn.m.r. spectra.
I
OAc
I
OAc
A number of base-induced fragmentations of swietenine derivatives have been d e ~ c r i b e d .The ~ ~ diketo-aldehyde (120) is transformed into (122) via the #Idicarbonyl cleavage product (121). Treatment of the sodium salt of (122) with oxalyl chloride affords the y-lactone (123). The triketo-ester (124) undergoes an interesting rearrangement to (125). The reaction of the corresponding hydroxydiketone (126) takes a different course to yield the cyclo-octenone (127). 66 67
D. A. H. Taylor and F. W. Wehrli, J.C.S. Perkin I, 1973, 1599. J . D. Connolly, R. Henderson, R. McCrindle, K. H. Overton, and N. S. Bhacca, J . C . S . Perkin I , 1973, 865.
Triterpenoids
205
Chemical and spectroscopic studies have shown68 that bussein, a complex tetranortriterpenoid from Entandrophragmu bussei and E. caudaturn, is a mixture of bussein A (128) and B (129),both of which contain an unusual enolized P-ketolactone system. The functional groups of azadirachtin, C35H44016, from the seeds of Azadirachta indica, have been disc~ssed.6~ Azadirachtin has also been detected in the fruit of Melia a~edarach.~' 68 69 'O
R. Hanni and Ch. Tamm, J . C . S . Chem. Comm., 1972, 1253. J. H. Butterworth, E. D. Morgan, and G. R. Perey, J.C.S. Perkin I , 1972, 2445. E. D. Morgan and M. D. Thornton, Phytochemistry, 1973,12, 391.
Terpenoids und Steroids
206
I
,Me OCOCH, R (128) (129)
R R
= =
Et Me
Quassinoids.-Bruceantin (1 30) and bruceantarin (13 1) have been isolated from Rruceu ~ntidysentericu.'~ Bruceantin is a potent antileukaemic tumour inhibitor and its activity may be associated with the afl-unsaturated ester function. The full paper on nigakilactones J, K, L, M, and N has been published.72
H0
,A,,CO
Me
I
(130) R = OC (131) R = OCPh
5 Shionane-Baccharane Group Treatment of 3a,4a-epoxyshionane (132) with boron trifluoride etherate followed by oxidation results73 in the formation of bacchar-12-en-3-one (133) and the related 8-ene (134). This confirms the nature of the baccharane skeleton and supports the close biogenetic relationship between baccharis oxide and shionone. Bacogenin A,, from acidic hydrolysis of bacoside A, a saponin of Bacopa monnieru, is identical with ebelin lactone ( 135).74 N.m.r. evidence suggests that S. M. Kupchan, R . W. Britton, M. F. Ziegler, and C . W. Sigel, J . Org. Chem., 1973,38, 178. T. Murae, A . Sugie, T. Tsuyuki, S. Masuda, and T. Takahashi, Tetrahedron, 1973,29, 1515. S. Yamada, S. Yamada, Y. Moriyama, Y. Tanahashi, and T. Takahashi, Tetrahedron Letters, 1972, 5043. D. K. Kulshreshtha and R. P. Rastogi, Phytochemistry, 1973, 12, 2074.
207
Triterpenoids
.
(134)
.
(135)
saponins which produce ebelin lactone on hydrolysis have a cyclopropane ring in the side-chain. 6 Lupane Group
The structure of alphitexolide (136), from the bark of Alphitonia excels^,^^ was confirmed by a partial synthesis from ceanothic acid (1 37). The details of the chemistry of jingullic acid have been published.76
(137)
New lupane natural products include glochilocudiol (138) from Gluchidiun m ~ l t i l o c u l a r e , ~lup-20(29)-ene-3b, ~ 1 1b-diol (139) from Dodonuea attenuata,78 lupeol octacosanoate from the roots of Hemidosmus i n d i c ~ s , ~ 3-epibetulinic ’ 75
76 77
78 79
G . B. Branch, D . V. Burgess, P. J. Dunstan, L. Y. Foo, G . H. Green, J. P. G . Mack, E. Ritchie, and W. C. Taylor, Austral. J . Chem., 1972, 25, 2209. R . A. Eade, J. Ellis, P. Harper, and J. J. H. Simes, Austral. J . Chem., 1973, 26, 831. S. K. Talapatra, S. Bhattacharya, B. C. Maiti, and B. Talapatra, Chem. andInd., 1973, 1033. E. L. Ghisalberti, P. R. Jefferies, and M. A. Sefton, Phytochemistry, 1973, 12, 1125. S. N. Padhy, S. B. Mahato, and N. L. Dutta, Phytochemistry, 1973, 12, 217.
Terpenoidrs and Steroids
208
R'
HO (138) (139)
R' R'
= =
OH, R 2 = H H, R2 = OH
(140) (141)
R' R'
=
=
H,a-OH, RZ = H 0, RZ = Me
acid (140) from Picrarnnia pentandra,80and methyl betulonate (141) from Betula utilixgl The steric course of the thionyl chloride-induced dehydration of the epimeric 20-hydroxy-30-norlupane derivatives (142) and (143) has been considered in details2 The results confirm the previous assignments of configuration at C-20. The (20R)-isomer(142) affords mainly the olefin (144) with a minor amount of the chloro-compound (149, whereas the (20s)-isomer (143) yields the chloroderivative (146) and the olefins (147) and (148).
Hod.
b
\
CH,OAc
-i!
\
CH,OAc
Boron trifluoride-catalysed reaction of iso-y-lupene oxide (149) gives (150) and a series of backbone-rearranged d i e n e ~ .The ~ ~ conformation of ring A of la,3a-
83
W. Herz, P. S. Santhanam, and 1. Wahlberg, Phyrochemisrry, 1972, 11, 3061. A. K. Batta and S. Rangaswami, Phytochemisrry, 1973, 12, 214. E. Klinotova, N. Hovorkova, J. Klinot, and A. Vystrcil, Coil. Czech. Chem. Comm., 1973,38, 1 179. G . Berti, A. Marsili, I. Morelli, and A. Mandelbaum, Guzzetru, 1972, 102, 793.
Triterpenoids
209
I-..,
dihydroxylupane, la,3B-dihydroxylupane, and related compounds is a deformed presumably as a result of 1,3-diaxial interactions.
7 Oleanane Group A proposal that the C-16 configuration of a large group of 16-hydroxylated
oleananes should be reversed8' is untenable. It was based on the observation that the tetraol(l51) obtained by reduction of the methyl ester of quillaic acid readily formed a diacetonide. Formally this requires the involvement of two transdiaxial hydroxy-substituents. It is apparent, however, that ring D cannot maintain a normal chair conformation. A recent-reports6 indicates that it is possible to form acetonides from both the 16a,28 and 16/3,28 glycol systems. Thus longispinogenin (152) and primulagenin A (153) are both converted into the corresponding acetonides on treatment with 2,2-diethoxypropane in acetone in the presence of toluene-p-sulphonic acid. N.m.r. evidence suggests a boat conformation of ring D in primulagenin A acetonide.
HO CH,OH (152) R = H,B-OH (153) R = H,a-OH
The presence of a 16ar-hydroxy-group in primulagenin A (153), echinocystic acid, and related compounds has been clearly established by an X-ray analysis 84
K. Waisser, M. Budesinsky, A. Vitek, and A. Vystrcil, Coll. Czech. Chem. Comm., 1972,37, 3652.
86
R. Segal and A. Taube, Tetrahedron, 1973,29, 675. J. St. Pyrek, J.C.S. Chem. Comm., 1973, 787.
Terpenoidr and Steroids
210
of the echinocystic acid derivative (154)87 and by synthesis of grimulagenin A (153) and echinocystic acid diacetate (155)88 (Scheme 4).
I
ii, i i i
Scheme 4 continued o n p . ZII
Scheme 4 ”
R . B. Boar, D. H. R. Barton, C. H. Carlisle, and J . F. McGhie, personal communication. J . Allen, R . B. Boar, J . F. McGhie, and D. H. R . Barton, J.C.S. Perkin I , 1972, 2994.
Trit erpenoids
21 1
j, xi
Reagents: i, Pb(OAc),-I,-hv;ii, LiAlH,; iii, Ac,O-py; iv, CrO,; v, Li-NH,; vi, Ac,O-py; vii, POC1,-py; viii, Br,; ix, MeC0,H; x, Zn; xi, Li-EtNH,; xii, Ac,O-py; xiii, basic alumina; xiv, Br,; xv, CrO,; xvi, Zn.
Scheme 4 (continued)
212
Terpenoids and Steroih
The sapogenin mixture from the wood of Castanosperum australe contains the known 2fl,3fl-dihydroxyolean-12-en-28-oic acid and 3b-hydroxyolean- 12-ene23,28-dioic acid. The former was identified” by an unambiguous synthesis from methyl crategolate (156) and the latter by synthesis from dimethyl medicagenate (157).
Treatment of 19fl-hydroxyolean-l1,13(18)-dien-3fl-ylacetate (158) with boron trifluoride etherate affords” 11-0x0-a-amyrin acetate (159), olean-9(1l), 12) 8trien-3fl-yl acetate, and an unidentified trienyl acetate. This is the first example of the transformation of an oleanane into the thermodynamically less stable ursane series. The preparation of [22,23-2H2]-P-amyrin(160) has been reported.”
( 160)
*’ 90
91
C. D. Bannon, R . A. Eade, P. James, G. P. McKenzie, and J. J. H. Simes, Austral. J . Chem., 1973?26, 629. R. Leonard and J . B. Thomson, J.C.S. Chem. Comm., 1972, 1282. T. Kikuchi, hl. Niwa, and T. Yokoi, Chem. and Pharm. Bull. (Japan), 1973, 21, 1378.
Triterpenoids
213
Three new 6P-hydroxyoleananes have been isolated. They are daturadiol (161) and daturaolone (162) from the seeds of Datura innoxiag2 and astilbic acid (163), which co-occurs with the corresponding 6-deoxy-derivative, P-peltoboykinolic acid, in the roots of Astilbe th~nbergii.’~ Oleanonic aldehyde (164) and 28-hydroxy-fi-amyrone (165) have been obtained from the galls of P istacia terebinth~s.’~
OH (161) R’ = H,B-OH, R2 = Me (162) R’ = 0, R2 = Me (163) R’ = H,B-OH, RZ = CO,H
(164) R (165) R
= =
CHO CHzOH
The structure of barrinic acid from Barringtonia acutangula has been established as (166).94 Full papers have appeared on the sapogenol constituents of the leaves of Pittosporum tobira?’ the triterpenoids from the resins of Shorea species,96the sapogenols of five Primulaceous plants, including papers on the structure of protoprimulagenin A (167)” and the stereochemistry of polygalacic
HO
92
93 94 95
96
97
M. Kokor, J. St. Pyrek, C. K. Atal, K. L. Bedi, and B. R.Sharma, J. Org. Chem., 1973, 38, 3688. K. Takahashi, K. Kanayama, Y. Tanabe, and M. Takani, Chem. and Pharm. Bull. (Japan), 1972, 20, 2106. A. K. Barua, S.K. Pal, S . P. Dutta, J. Indian Chem. SOC.,1972,49, 518. I. Yosioka, K. Hino, A. Matsuda, and I. Kitagawa, Chem. and Pharm. Bull. (Japan), 1972,20, 1499. H. T. Cheung and T. C. Yan, Austral. J. Chem., 1972,25,2003. I. Kitagawa, A. Matsuda, and I. Yosioka, Chem. and Pharm. Bull. (Japun), 1972, 20, 2226.
214
Terpenoih and Steroids
acid,98 and the structure of platy~odigenin~~ from Platycodon grandijlorum. A prosapogenin from this source has been characterized'" as 3-O-fi-~-glucopyranosylplatycodigenin (168). One of the major molluscicidal saponins from the fruit of Phytolacca dodecundra has been shown to be 3-[2,4-di-O-(fl-~-glucopyranosyl)-~-~-glucopyranosyl]-olean-12-en-2~-oic acid.
The structure of dendropanoxide (campanulin, epoxyglutinane), a triterpenoid oxide from Rhododendron macrophyllum, has been revised to (169) on the basis of an X-ray analysis102and n.m.r. evidence.lo3
Many new friedelane derivatives have been reported this year. Six new compounds, octandrolal(170), octandrolol(171), octandrolic acid (172), octandronal (173), octandronol (174), and octandronic acid (175) were foundlo4 in the bark of Hydnocarpus octandra. Six 1,3-diketofriedelanes(176)-(18 1) were i~olated''~ from the root bark of Salacia prinoides. The structure of the 25,26-oxide (181) 98
99
100
101
I02 I03 I04 10.5
T. Akiyama, 0. Tanaka, and S. Shibata, Chem. and Pharm. Bull. (Japan), 1972, 20, 1945. T. Akiyama, 0. Tanaka, and S. Shibata, Chem. and Pharm. Bull. (Japan), 1972, 20, 1952. T . Akiyama, 0. Tanaka, and S. Shibata, Chem. and Pharm. Bull. (Japan), 1972, 20, 1957. K. M . Parkhurst, D. W. Thomas, W. A. Skinner, and L. W. Cary, Phytochemistry, 1973,12, 1437. J . D. White, J. Fayos, and J . Clardy, J.C.S. Chem. Comm., 1973, 357. J . H. Block and G . H. Constantine, jun., Phytochemistry, 1972, 11, 3279. S. P. Gunasekera and M. U. S. Sultanbawa, Chem. and Ind., 1973, 790. B. S. Joshi, V. N. Kamat, and N. Viswanathan, Tetrahedron, 1973, 29, 1365.
215
Triterpenoids R2
R' (170) (171) (172) (173) (174) (175)
R' R'
R' R'
R' R'
= H,a-OH, = H,a-OH, = Hp-OH, = 0, = 0, = 0,
R2 = CHO R2 = C H 2 0 H R2 = COZH RZ = CHO R2 = C H 2 0 H R2 = C 0 2 H
(176) R' = Me, R2 = H R2 = H (177) R' = CHO, (178) R' = C H 2 0 H , R2 = H (179) R' = CO,H, R2 = H Rz = OH (180) R' = Me,
(182) (183) (184) (185)
R
=0 R = H,a-OH R = H,a-OAc R = H,fi-OAc
was established by X-ray analysis. The bark of Trichadenia zelanica producedio6 trichadonic acid (182), trichadenic acid A (183), the corresponding acetate (184), and the epimeric acetyltrichadenic acid B (185). An X-ray analysis confirms the 16P-hydroxy-group configuration in pachysonol and pachysandiol (see Vol. 2, p. 173 and Vol. 3, p. 223) and also reveals that in the presence of a 16P-substituent the molecule adopts a chair-chair-chairboat-boat c o n f o r r n a t i ~ n . ' ~This ~ contrasts with the all-chair conformation which obtains in friedelin itself and in 16-epimeric derivatives. A mixture of putranjivic acid (186) and the corresponding saturated compound is formedlo8 by irradiation of friedelin in benzene in an oxygen atmosphere. The related putranjic acid (187) has been synthesized (see Vol. 3, p. 223) from friedelane-2,3-dione and the C-2 configuration confirmed as (S).' O9 The full Io6 lo'
Io9
S. P. Gunasekera and M. U. S. Sultanbawa, Tetrahedron Letters, 1973, 2837. T. Kikuchi, M. Niwa, and N . Masaki, Tetrahedron Letters, 1972, 5249. R. Aoyagi, T. Tsuyuki, and T. Takahashi, Bull. Chem. SOC.Japan, 1973,46, 692. R. Aoyagi, Y . Moriyama, T. Tsuyuki, and T. Takahashi, Bull. Chem. SOC.Japan, 1973,
46. 569.
Terpenoids and Steroids
216
(186) R = H (187) R = OH; dihydro
details of the preparation of 4-epifriedelin(188)and 4-epishionone (189),by photoisomerization of friedelin and shionone respectively, have appeared.' The conformational reasons for the instability of the epi-derivatives are discussed.
''
Acid-induced rearrangement of 16-ketofriedel-3-eneaffords 16-keto-olean-12ene and two other products."' The structure of one of these has been shown to be (190) by application of the homonuclear INDOR technique. The third compound is tentatively assigned the structure (191). The same technique has been applied112 to 16a- and
[email protected] results suggest that ring D
(191) 'lo
R. Aoyagi: S. Yamada, T Tsuyuki, and T. Takahashi, Buff. Chem. SOC.Japan, 1973, 46, 959. T. Kikuchi, M. Niwa, M. Takayama, T. Yokoi, and T. Shingu, Tetrahedron Letters, 1973, 1987. T. Kikuchi, T. Shingu, M. Niwa, and T. Yokoi, Chem. and Pharm. Bull. (Japan), 1973, 21, 1396.
21 7
Triterpenoiak
adopts a boat conformation in the presence of a l6g-substituent. This agrees with the X-ray results discussed above. Partially relaxed Fourier-transform 13C n.m.r. made a major contribution to the structural elucidation of tingenin A (192) and tingenin B (193), from the stem bark of Euonymus tingens. l 3 Pristimerin (194) served as a reference compound. Tingenin A is probably identical with the antitumour agent maitenin (see Vol. 3, p. 222). Polpunonic acid (195), a possible precursor of maitenin, occurs with it in the roots of Plenckia polpunea.' l 4 It has antibacterial activity.
(192) (193)
R R
= =
H OH
(194)
(195)
Taraxer-14-en-3a-01 has been converted into 14a-taraxerane (196).' methyl resonances of this reference triterpane have been assigned.
'I3
l4 l5
The
K. Nakanishi, V. P. Gullo, I. Miura, T. R. Govindachari, and N. Viswanathan, J. Amer. Chem. SOC.,1973, 95, 6473. F. Delle Monache, J. F. de Mello, G. B. Marini-Bettblo, 0. Goncalves de Lima, and I. L. d'Albuquerque, Carzettu, 1972, 102, 636. R. E. Corbett, S. D . Cumming, and E. V. Whitehead, J.C.S. Perkin I , 1972, 2827.
218
Terpenoids and Steroids
Genuine sapogenols have been obtained by photolytic cleavage of several oleanane saponins.'16,' l 7 A necessary feature for the success of this reaction is the presence of an uronic acid in the glycoside."7 Several 3,4-seco-acids in the 18~-oleananeseries' and a number of derivatives of 18P-glycyrrhetaldehyde' l 9 have been described.
8 Ursane Group The previously assigned structure of lantic acid (197) has been confirmed12' by its conversion into methyl 3-ketoursolate. Ursadiol (198)12' and the trio1 (199)"* are two related ursanes from the flowers of Calendula oficinalis.
HO - '
HO (197)
(198) R = H (199) R = OH
The structures of faradiol (200) and arnidiol (20l), the principal triterpenoids of yellow Compositae flowers, have been revised.'23
'l 6
''
'la
'
l9
2o
' l 22 12'
I. Kitagawa, M. Yoshikawa, Y. Imakura, and I. Yosioka, Chem. and Ind., 1973, 276. I. Kitagawa, M. Yoshikawa, and I. Yosioka, Tetrahedron Letters, 1973, 3997. J. Klinot, E. Ulehlova, R. Straka, and A. Vystrcil, Coff.Czech. Chem. Comm., 1973, 38, 2648. S. Rozen, I . Shahak, and E. D. Bergmann, Tetrahedron, 1973, 29, 2327. A. K. Barua, P. Chakrabarti, P. K. Sanyal, K. Basu, and K. Nag, J . Indian Chem. SOC., 1972,49, 1063. J . Sliwowski, K. Dziewanowska, and 2. Kasprzyk, Phyrochemistry, 1973, 12, 157. Z . Kasprzyk and B. Wilkomirski, Phytochemistry, 1973, 12, 2299. J. St. Pyrek and E. Baranowska, Tetrahedron Letters, 1973, 809.
Trit erpeno ids
219
9 Hopane Group The results of a fuller investigation of the triterpenoids produced by the prokaryotic organism Bacillus acidocaldarius indicate' 24 that hop-22(29)-ene (202) 1
r;'
is the major component, with hop-l7(21)-ene and hopane present in minor amounts. In addition, all-trms-squalene was detected. Experiments with labelled acetate and mevalonate clearly demonstrate that the triterpenoids are synthesized de nouo by the organism.
10 Stictane-FIavicane Group Ten new compounds (203)--(212),derivatives of a novel triterpane stictane (213), have been isolated'25 from the lichens Sticta coronata, S . colensoi, and S.Javicans. Wolff-Kishner reduction of the diketone (214) afforded the parent hydrocarbon (213), which was not identical with any known triterpane. Dehydration of stictan-22a-01 (215) with phosphoryl chloride in pyridine to the corresponding
(203) R' (204) R' (205) R' (206) R' (207) R' (208) R' (209) R' (210) R' (211) R' (212) R' (213) R' (214) R' (215) R' 124
H,B-OH, R2 = H,a-OH, R2 = H,a-OAc, = H,P-OAc, R2 = H,a-OAc, = H,P-OH, R2 = H,a-OAc, = H,B-OAc, RZ = H,a-OH, = H,B-OAc, R2 = H,a-OAc, = H,B-OH, R2 = H,H = H,B-OAc, R2 = H,H, = H,B-OAc, R2 = H,H, = 0, R2 = H,H, = R2 = R3 = H,H = R3 = 0, R2 = H,H = R2 = H,H, R3 = H,a-OH =
= H,B-OAc,
R 3 = H,a-OH R3 = H,a-OAc R 3 = H,a-OH R 3 = H,a-OH R 3 = H,a-OH R3 = 0 R 3 = H,a-OH R 3 = H,a-OAc R 3 = H,a-OH R3 = H,a-OH
M. De Rosa, A. Gambacorta, L. Minale, and J. D. Bu'Lock, Phytochernistry, 1973, 12, 1 1 17. W. J . Chin, R. E. Corbett, C. K. Heng, and A. L. Wilkins, J.C.S. Perkin I , 1973, 1437.
220
Terpenoids and Steroids
ring E-contracted product (216) followed by hydrogenation yielded another new triterpane flavicane (217). Both stictane and flavicane can be derived by cyclization of squalene oxide in a chair-boat-chair-chair-boat conformation via the classical ion (218). Until now arborinol(219) was the only representative of this group and the appearance of others was to be expected.’26
(2 16) (21 7) 22,29-dihydro
11 Serratane Group
New natural products in this group include 2lcr-methoxyserrat-14-en-3-one (220) from the bark of the common spruce, Picea ubies,’*’ and 3a,2la-dimethoxyserrat1Cene (221) from the bark of the Scots pine, Pinus syluestris.’28
(220) R = 0 (221) R = H,a-OMe
* z6 Iz7
J. D. Connolly and K. H. Overton, ‘The Triterpenoids’ in ‘Chemistry of Terpenes and Terpenoids’, ed. A. A. Newman, Academic Press, London, 1972, p. 227. T. Norin and B. Winell, Acta Chem. Scand., 1972, 26, 2289. T. Norin and B. Winell, Acta Chem. Scand., 1972, 26, 2297.
6 Carotenoids and Polyterpenoids BY G. BRITTON
1 Introduction
The 3rd. I.U.P.A.C. meeting on ‘Carotenoids other than Vitamin A’ was held at Cluj, Romania in September 1972. The texts of the plenary lectures have been published, and include thorough surveys of recent progress in carotenoid chemistry by Weedon and by Liaaen-Jensen.’ The latter author has also published an outline of the state of knowledge in the carotenoid field, ‘anno 1972’,2and a comprehensive review of microbial carotenoids3 to supplement her earlier coverage of this topic.* A monograph on the chemistry of plant pigments’ includes chapters on the structures of citrus carotenoid~,~”yeast carotenoid~,’~and isoprenylated c a r ~ t e n o i d s ,and ~ ~ a survey of the excited states of plant pigments, including carotenoids.5d Other topics to have been reviewed are the chemistry and biochemistry of the carotenoids of blue-green algae6 and the chemistry and biochemistry of abscisic acid. 2 WysicaiMetbods
The separation and purification of carotenoids, especially when labelled with radioisotopes, continues to cause problems. The chromatography of carotenoids and chlorophylls-especially its historical development-has been reviewed,* and new chromatographic methods for the separation of carotenoid glycosidesg
’
Pure Appl. Chem., 1973, 35, 1-130: B. C. L. Weedon, ‘Some Recent Studies on Carotenoids and Related Compounds’, pp. 1 13-1 30; S. Liaaen-Jensen, ‘Structural Elucidation of Carotenoids-a Progress Report’, pp. 81-1 12. S. Liaaen-Jensen, Planta Medica, 1973, 23, 251-268. S. Liaaen-Jensen and A. G. Andrewes, Ann. Rev. Microbiol., 1972, 26, 225-248. S. Liaaen-Jensen, Ann. Rev. Microbiol., 1965, 19, 163-182. C. 0.Chichester, Ado. Food Res., Suppl. 3, ‘The Chemistry of Plant Pigments’, Academic Press, New York, 1972: ( a ) H. Yokoyama, H. C. Guerrero, and H. Boettger, pp, 1-7; ( b ) K. L. Simspon, pp. 9-22; (c) 0. B. Weeks and A. G. Andrewes, pp. 23-32; (4P.-S. Song, T. A. Moore, and M. Sun, pp. 33-74. V. V. Pinevich and V. E. Vasileva, Vestnik Leningrad. Univ., Biol., 1972, 105. D. Gross, Pharmazie, 1972, 27, 619. H. H. Strain and J. Sherma, J. Chromatog., 1972,73, 371. H. Kleinig and H. Reichenbach, J. Chromatog., 1972, 68, 270.
22 1
222
Terpenoids and Steroids
and of apocarotenals, apocarotenoic acids, apocarotenyl acetates, and retinol derivatives'O have been described. Gas chromatography has been used to separate hydrogenated retinol and derivatives,' and the gas chromatographic retention data for carotenoids and hydrogenated carotenoids12 have been tabulated.' The separation of /3-carotene (p,P-carotene*) (1) and its epoxides and hydroxy-derivatives by counter-current distribution has been r e p ~ r t e d . ' ~
'
This method is considered to show promise for the separation of natural extract^.'^ High-pressure liquid chromatography has been used to separate synthetic blumenol A (2) diastereoisomers' as the ( + )-ct-methoxy-ct-trlfluoromethylphenylacetyl (MTPA) esters (3), but this powerful technique has not yet been applied to carotenoids. The optical resolution of (+)-cis-abscisic acid (4) is more readily accomplished by isomerization to the (+)-trans-isomer, resolution of this as the brucine salt,16 and isomerization of the resolved trans-enantiomers to the cis-form, than by direct resolution of the cis-form.'
(2) R = H ( 3 ) R = MTPA
The use of a diffusion-zone process for growth of large crystals of p-carotene has been reported, and the physical properties of the crystals are described.18 10
'' l3
'' l6
''
H. Singh, J. John, and H. R. Cama, J. Chromatog., 1973, 7 5 , 146. T. W. Fenton, H. Vogtmann, and D. R. Clandinin, J. Chromatog., 1973, 77, 410. R. F. Taylor and M. Ikawa, Analyt. Biochem., 1971, 44, 623. Chromatographic Data, J. Chromatog., 1973, 77, D36. H. Pfander, F. Haller, K . Bernhard, and H. Thommen, Chimiu (Switz.), 1973, 27, 103. G. Weiss, M. Koreeda, and K. Nakanishi, J.C.S. Chem. Comm., 1973, 565. J. W. Cornforth, W. Draber, B. V. Milborrow, and G. Ryback, Chem. Comm., 1967, 114. J.-C. Bonnafous, J.-C. Mani, J.-L. Olive, and M. Mousseron-Canet, Tetrahedron Letters, 1973, 1 1 19. A. R . Vaala, A. H. Madjid, and M. T. Torrado, J. Crystal Growth, 1973, 18, 39.
* When a carotenoid is referred to by its trivial name, the semi-systematic name according to the tentative I.U.P.A.C. rules will be given, in parentheses, at the first mention.
223
Carotenoids and Polyterpenoidr
The solubility of 8-carotene and retinol (5) in aqueous solutions containing mixed micelles has been s t ~ d i e d . ’ ~ The 250 MHz ‘H n.m.r. spectra of 15-cis- (6)and all-trans-phytoene (7,8,11,12, 7’,8’,11’,12’-octahydro-I,b,I,b-carotene) (7)” showed good agreement with previous results at 2 2 0 M H ~ . ~The l 13C n.m.r. spectra of these phytoene isomers were also determined ; assignments were made by comparison with other terpenoid compounds,22 and for the central part of the molecule (C-13, C-14, C-15) by comparison with the available data for 15-cis- (11) and all-tr~ns-~-carotene.~~ Data for 13-cis-phytoene (8) are also presented. Theoretical studies of the ‘H n.m.r. spectrum indicate the possibility of an equilibrium between the 12-s-cis and 12-s-trans conformations of 11-cjs-retinal (9).24
(5) R (9) R
= =
CH,OH CHO; 1 1 4 s
R= (6) a,c trans, b cis (7) a,b,c trans ( 8 ) a cis, b,c trans
Details of the n.m.r. and mass spectra of a series of model, carotene-like, aryl polyenes have been d i s c ~ s s e d . ~The mass spectra of fully deuteriated or-carotene (B,&-carotene)(10) and 8-carotene have been compared with those of the normal carotenes.26 l9 2o
21
22
23
24
25 26
M. El-Gorab and B. A. Underwood, Biochim. Biophys. Acta, 1973, 306, 58. P. Granger, B. Maudinas, R. Herber, and J . Villoutreix, J . Magn. Resonance, 1973, 10, 43. N. Khatoon, D. E. Loeber, T. P. Toube, and B. C. L. Weedon, J.C.S. Chem. Comm., 1972,996. M. Jautelat, J. B. Grutzner, and J. D. Roberts, Proc. Nut. Acad. Sci.,U.S.A., 1970, 65, 288; M. W. Duch and D. M. Grant, Macromolecules, 1970, 3, 165. W. Vetter, G . Englert, N. Rigassi, and U. Schwieter, in ‘Carotenoids’, ed. 0. Isler, Birkhauser Verlag, Basel, 197 1, pp. 189-266. J. Langlet and C. Giessner-Prettre, J . Mol. Structure, 1972, 13, 317. G. W. Francis, Acta Chem. Scand., 1972, 26, 2969. W. A. Svec, A. L. Harkness, H . H . Strain, and J. J . Katz, Org. Mass Spectrometry, 1972, 6, 843.
224
Terpenoicls and Steroids
b
0
C
d
e
f
(10)R’ = a, R2 = g (12) R’ = CH=CHC02H, R2 (13) R’ = R 2 = C 0 2 H (15)R’ = R 2 = d (17) R’ = R2 = b (19) R’ = a, R2 = C 0 2 M e (21)R’ = g, R2 = h (23) R’ = g, R2 = C H 2 0 H
(11)R’ = R2 = a; 15,lS-cis 9-cis (14) R’ = b, R2 = c (16) R’ = R2 = e (18) R’ = a, R2 = CHO (20)R’ = b, R2 = f (22) R’ = g, R2 = CHO
= CH=CHCO,Me;
In addition to the widespread use of absorption spectroscopyfor the identification and quantitative estimation of carotenoids and retinol derivatives, several detailed spectroscopic studies have been reported. The vibrational resonance Raman spectra of /3-carotene and its synthetic homologues C,,H,, , C,c,Hc,o, C,,H,, , and C,,H,, and of bixin (methyl hydrogen 9’-ci~-6,6’-diapocarotene6,6’-dioate)( 12), crocetin (8,8’-diapocarotene-8,8’-dioic acid) (13), and capsanthin (3,3’-dihydroxy-#l,~-caroten-6-one) (14) are all similar, suggesting that the conjugated polyene chain is the main contributor to the resonance Raman spectra of
Carotenoids and Poiyterpenoids
225
all car~tenoids.~’ A higher degree of apparent bond alternation is indicated than that inferred from the absorption spectra. The properties of the triplet state of p-carotene and its synthetic C3,-, C35,Cso, and C60 homologues have been compared by flash spectroscopy.28Flash spectroscopy in the presence of anthracene as photosensitizer has been used to determine the triplet-triplet absorption spectra of 15-cis-p-carotene (1 l), 7,7’-dihydro-fl-carotene(7,8,7’,8’-tetrahydroB,p-carotene) (1 5), canthaxanthin (B,/?-carotene4,4‘-dione)(16), zeaxanthin (&/3-carotene-3,3‘-diol) (1 7),j3-apo-8’-carotenal (8’-apo-p-caroten-8’-al)(1 8), and methyl fi-apo-8’-carotenoate (methyl 8’-apo-~-caroten-8’-oate)(19).29 The triplet-triplet absorptions of these molecules (except zeaxanthin) and of retinal and retinol have also been studied by pulse r a d i o l y ~ i s .A ~ ~theoretical basis for the linear relationship observed between the triplet and singlet absorption maxima of these compounds has been provided by Huckel theory calculations30 and possible mechanisms for oxygen quenching of the triplet states have been disc~ssed.~’The caIcuIation of the energy levels and oscillator strengths of fi-carotene has been improved and extended to include bands other than the main absorption band. Stable mixed monolayers of /%caroteneand arachidic acid may be transferred uniformly to glass slides. The spectrum of the fl-carotene layers shows less fine structure in the region of maximum absorption (450-490 nm) than does that of p-carotene in hexane s ~ l u t i o n . ~The electrochromic spectrum of lutein (/I,&carbtene-3,3‘-diol)(20) has been described.33The 77 K optical absorption and emission spectra of trans-retinal, anhydrovitamin A (24), and axerophtene (26),
in a rigid glass matrix, have been determined.34 The 77 K emission spectrum of trans-retinal indicates an aggregation to form tail-to-tail d i m e r ~ . ~ Studies of optical properties have allowed the absolute configurations of more carotenoids and derivatives to be assigned or confirmed. Cotton effects of carotenoids have been measured for the first time in the visible region;36 all carotenoids examined with (6R) chirality showed a positive effect at their longest waveband. This work showed that the absolute configuration at C-6 of a27
29
30
*
32 33 34
35
36
L. Rimai, M. E. Heyde, and D. Gill, J . Amer. Chem. SOC.,1973,95, 4493. P. Mathis and J . Kleo, Photochem. and Photobiol., 1973, 18, 343. T. G. Truscott, F. J. Land, and A. Sykes, Photochem. and Photobiol., 1973, 17,43. 0. L. J. Gijzeman and A. Sykes, Photochem. and Photobiol., 1973, 18, 339. E. A. Castro and 0. M. Sorarrain, 2.phys. Chem., 1972, 250,230. M. Pincus, S. Windreich, and I. R. Miller, Biochim. Biophys. Acta, 1973, 311, 317. S. Schmidt and R. Reich, Ber. Bunsengesellschaftphys. Chem., 1972, 76, 1202. R. L. Christensen and B. E. Kohler, Photochem. and Photobiol., 1973, 18, 293. T. A. Moore and P.-S.Song, Nature New Biol., 1973, 243, 30. R. Buchecker and C. H. Eugster, Helu. Chim. Acta, 1973, 56, 1124.
226
Terpenoids and Steroids
zeacarotene (7’,8’-dihydro-&~-carotene)(2 1)and its derivatives a-apo-8’-carotenal (E’-apo-&-caroten-S‘-al) (22), and u-apo-E’-carotenol (8’-apo-&-caroten-8’-01)(23) is the same as in natural (+)-a-carotene, i.e. (R). C.d. comparison has also been used to show that picrocrocin (25) has the (3R) c~nfiguration.~ Calculations (c.d.) show that specific twisted conformers of 11-cis-retinal (9) make a strong contribution to the optical activity of the visual pigment rhod~ p s i n . ~The ’ 9 4 s (27), 13-cis (28), and all-trans-isomers of retinal each exhibit a
’
(26) R (27) R (28) R
= = =
Me CHO; 9-cis CHO; 1 3 4 s
unique linear dichroism spectrum; linear dichroism spectra can thus be used for quantitative determination of the components of a mixture of retinal isorner~.~’ The sign of the main m* c.d. Cotton effect of blumenol B (29) is opposite to that of blumenol A (2), showing that the effect of the homoconjugated 7-ene outweighs that of the y-hydroxy-gro~p.~~ The nuclear Overhauser effect and optical properties of natural and synthetic theaspirone have been used to show that the natural (-)-theaspirone (30) is the (6S,9S)-isomer.” Application of the exciton chirality method to abscisic acid derivatives has confirmed the (6s) configuration4’ and is in agreement with the conclusions obtained from a theoretical treatment, involving quantitative application of exciton chirality, of the 0.r.d. data for ( +)-trans-abscisic acid.41 A general theoretical discussion of the c.d. of cxP-unsaturated ketones includes a consideration of loliolide (3 1).42
3 New Natural Carotenoids Acyclic Carotenoids.-Prephytoene pyrophosphate ( 32), first isolated from a Mycobacterium p r e p a r a t i ~ n ,is~ ~a new biosynthetic intermediate between geranylgeranyl pyrophosphate (38) and phytoene, analogous to the sterol
’’ R. Buchecker and C. H. Eugster, Helv. Chim. Acfa, 1973, 56, 1121. ’* B. Honig, P. Kahn, and T. E. Ebrey, Biochemistry, 1973, 12, 1637. 39 40
41
‘’ 43
J. Horwitz and J. Heller, J. Biol. Chem., 1973, 248, 1051. M. Koreeda, G. Weiss, and K. Nakanishi, J. Amer. Chem. Sac., 1973, 95, 239. N . Harada, J. Amer. Chem. SOC.,1973, 95, 240. A. F. Beecham, Tetrahedron, 1972, 28, 5543. L. J. Altman, L. Ash, R . C. Kowerski, W. W. Epstein, B. R. Larsen, H . C. Rilling, F. Muscio, and D. E. Gregonis, J. Amer. Chem. Soc., 1972, 94, 3257.
227
Curotenoids and Polyterpenoids
intermediate presqualene pyrophosphate (33).44 Cleavage of the pyrophosphate group with LiAlH4 gave prephytoene alcohol (34), which after ozonolysis and acetylation gave the derivative ( 3 9 , thus confirming the presence of the cyclopropane ring. This intermediate (termed prelycopersene pyrophosphate) has also been isolated from incubations with a carotenoid-synthesizing tomato enzyme system and characterized by mass spectrometry of the pyrophosphate, the alcohol, and the TMS ether of the alcoh01.~' Prelycopersene pyrophosphate
H
Me
R'
RZ
a
b
(32) (33) (34) (35) (36)
a, R2 = b, R 3 = CHZO-@---8 R2 = d, R3 = CH20-@-@ = a, R2 = h, R 3 = CH,OH = R 3 = CH,OAc, R2 = CH2CH2CH2OAc = a, R2 = b, R3 = CHO (37) R' = a, R2 = b, R3 = C 0 2 M e R' R' R' R' R'
=
= C,
(38) R = CH20-@-@ (40) R = C H 2 0 H (42) R = C 0 , M e
-0-0 44
45
(39) R = CHO (41) R = CH2S02Ph
represents a pyrophosphate group
W. W. Epstein and H. C. Rilling, J . Biol. Chem., 1970,245,4597; J. Edmond, G. Popjak, S. M. Wong, and V. P. Williams, ibid., 1971, 246, 6254. A. A. Qureshi, F. J. Barnes, and J. W. Porter, J . Biol. Chem., 1972, 247, 6730; F. J. Barnes, A. A. Qureshi, E. J. Semmler, and J. W. Porter, ibid., 1973, 248, 2768.
Terpenoids and Steroids
228
( s prephytoene pyrophosphate) is produced by incubation of yeast squalene
synthetase with geranylgeranyl pyrophosphate, thus confirming the analogy with presqualene p y r ~ p h o s p h a t e .The ~ ~ absolute configuration of prephytoene pyrophosphate has not been established, but is likely to be similar to that recently deduced4’ from c.d. data for presqualene pyrophosphate, i.e. ( R ) at each chiral centre. Prephytoene alcohol has been synthesized. Addition of the allylic diazocompound derived from geranylgeranial(39) to a solution of geranylgeraniol(40) and ZnI in ether gave the aldehyde (36) which was reduced with LiAlH, (or LiAI3H4) to give prephytoene A second synthesis48 involved the conversion of geranylgeraniol into the sulphone (41), which was condensed with methyl geranylgeranoate (42) to give two stereoisomers of the cyclopropane ester (37). Reduction of the ‘low Rf’isomer with LiAlH, (or LiA13H,) afforded prephytoene alcohol. Lycopersene (43) has again been isolated from a carotenogenic system and suggested to be an intermediate in phytoene f~rmation.~’
Monocyclic Carotenoids.-A new carotenoid isolated from certain Fluuobacterium strains has been identified by n.m.r. and mass spectroscopy as 3hydroxy-/?-zeacarotene (7’,8’-dihydro-/?,/?-caroten-3-01) (45). This compound could be of biosynthetic significance as an intermediate in zeaxanthin forma(44) was also identified. A new t i ~ n .3-Hydroxy-l2’-apo-/?-caroten-12’-al ~ ~
H
O
U
monocyclic carotenoid (designated rhodoauranxanthin) has been isolated from the yeast Rhodotorulu uuruntiacaSo and identified by mass spectrometry as a hydroxy-derivative of plectaniaxanthin (3’,4‘-didehydro-l’,2’-dihydro-/?,$-carotene- 1’,2’-diol)(46), N.m.r. data indicate the third hydroxy-group to be located l’, 2’-triol) (47). at C-2 (i.e. 3’,4-didehydro-l’,2’-dihydro-/?,$-carotene-2, 46
47 48 49
A. A. Qureshi, F. J. Barnes, E. J. Semmler, and J. W. Porter, J . Biol. Chem., 1973,248, 2755. G. Popjak, J. Edmond, and S.-M. Wong, J . Amer. Chem. SOC.,1973, 95, 2713. L. Crombie, D. A. R . Findlay, and D. A. Whiting, J . C . S . Chem. Comm., 1972, 1045. F. J. Leuenberger, A. J. Schocher, G. Britton, and T. W. Goodwin, F.E.B.S. Letters, 1973, 33, 205. K . L. Simpson, I. S. Liu, and C. 0. Chichester, Fed. Proc., 1973, 32, 521.
Carotenoids and Polyterpenoids
HO
229
W'..
b
a
d
HO
e
B'..
1
k
J
(45) R' = a, R2 = b (47) R' = e, R2 = d (49) R' = e, R2 = f (51) R' = g, R2 = a
(53) R' = k, R2 = J
R' R' R' (52) R' (54) R' (46) (48) (50)
R2 = = e, R2 = = R2 = e = C,
d c
= h, R2 = j = 1, R2 = j
Bicyclic Carotenoids.-Several carotenoids containing a P-ring hydroxylated at C-2 have been reported. In addition to a- and j?-carotenes, the green alga Trentepohlia iolithus contains 2-hydroxy-#?-carotene(#?,P-caroten-2-01)(48), 2-hydroxya-carotene (#?,&-caroten-2-01) (49), and 2,2'-dihydroxy-#?-carotene(p,p-carotene2,2'-diol) (50), all as fatty acid esters5' Of their spectroscopic properties only the n.m.r. data are sufficiently distinct from those of the related carotenoids with 3- or 4-hydroxy-#?-ringsto allow conclusive identification. A double doublet (.I= 17 Hz, J , = 4.5 Hz) at 3.55 p.p.m. is characteristic of the 2-hydroxy-#?-ring system with the hydroxy-group equatorial. The gem-methyl groups (1.03, 1.09 p.p.m.) are magnetically non-equivalent, although this non-equivalence is rather smaller than might be expected. The 2-hydroxy-fi-ring can readily be distinguished from the 3- and 4-hydroxy-B-rings by the addition of [Eu(dpm),J.
''
H. Kjmen, N. Arpin, and S. Liaaen-Jensen, Acta Chem. Scand., 1972, 26, 3053.
230
Terpenoids and Steroids
From c.d. data, and from the application of Mills’ rule, the (2R)configuration (55) has tentatively been assigned to the 2-hydroxy-carotenoids, and like other configuration. A natural cx-carotene derivatives, &+caroten-2-01 has the (6’R) carotenoid isolated from Anacystis nidulans has been identifieds2 as P,P-carotene2,3,3’-trio1(51) from its mass spectrum and from the formation of an acetonide with acetone-CuS0, .’3
“ H‘ O
Y
Y
The structure of heteroxanthin from Euglena gracilis was confirmed as 7’,8’didehydro-5,6-dihydro-~,fi-carotene-3,5,3’-triol(52) by its formation from diadinoxanthin (5,6-epoxy-7’,8’-didehydro-5,6-dihydro-~,~-carotene-3,3’-diol) (53) by LiAlH4 reduction, and by its conversion into diadinochrome (5,8-epoxy-7’,8’didehydro-5,8-dihydro-P,P-carotene-3,3’-diol) (54) by treatment with acidified acetone.54 It is considered that heteroxanthin is identical to a ‘trollein-like’ pigment previously reported’ in E. gracilis. Aromatic Carotenoids-Two minor pigments from the sea sponge Renieru japonica have been identifieds6 by n.m.r. and mass spectrometry as 7,8-didehydro$,X-carotene (56) and 7,8-didehydro-4,$-carotene(57), the mono-acetylenic analogues of the aromatic carotenoid hydrocarbons renieratene ($,X-carotene) (58)and isorenieratene ($,@carotene) (59). The proximity of the C-7,8 acetylene group causes significant deshielding of two aromatic methyl substituents. These compounds are the first naturally occurring acetylenic carotenoid hydrocarbons to be described.
a (56) R’ = a, R2 = b (58) R’ = C, R2 = b 52
53 54 55 56
b
C
d (57) R’ = a, R2 = d (59) R’ = C, R2 = d
R. L. Smallidge and F. W. Quackenbush, Phytochemistry, 1973, 12,2481. J. A. McCloskey and M. McClelland, J. Amer. Chem. SOC.,1965, 87, 5090. H. Nitsche, Arch. Mikrobiol., 1973, 90,151. B. P. Schimmer and N . I. Krinsky, Biochemistry, 1966, 5, 1814. T. Hamasaki, N. Okukado, and M. Yamaguchi, Bull. Chem. SOC. Japan, 1973, 46, 1884.
Caroten0 ids and Po lyt erpeno ids
23 1
Carotenoid G1ycosides.-A new carotenoid glycoside from a myxobacterium, Chondrornyces aplicatus, has been identified by its chromatographic and chemical properties and by its n.m.r. and mass spectra as l’-~-~-glucosyloxy-3’,4’-didehydro-l’,T-dihydro-$,$-carotene monoester This carotenoid also accumulated in cultures of Myxococcusfulvus grown in the presence of nicotine.58
(60) R’ = esterified glucosyl, RZ = a (61) R’ = Me, R2 = b
Carotenopr0teins.-A spirilloxanthin [ 1,l’-dimethoxy-3,4,3‘,4-tetradehydro1,2,1’,2’-tetrahydro-$,$-carotine(61)]-containing carotenoprotein, molecular weight ca. 12 OOO, isolated from Rhodospirillurn rubrurn chromatophores, has an absorption spectrum entirelj different from the usual three-peaked spirilloxanthin spectrum, with only one major peak, at 370 nm.59 4 Carotenoid Chemistry A simple method has been described6* for the preparation of apo-carotenoic acids by silver oxide oxidation of the corresponding apo-carotenals. A range of 3-hydroxy- and 3-acetoxy-apo-~-carotenalshas been prepared in low yield by cleavage of zeaxanthin diacetate with potassium permanganate-hydrogen peroxide.61 Treatment of P-apo-8’-carotenyl acetate (8’-apo-a-caroten-8’-yl acetate) (62) with monoperphthalate gave the 5,6-epoxide (5,6-epoxy-5,6-dihydro-8’-apo-P-caroten-8’-yl acetate) (63).62 Dehydration of zeaxanthin and + lutein with MeO,CN--SO,NEt, gave mainly 3,4,3’,4-tetradehydro-P,P-carotene (64).63 Zeaxanthin also gave two minor products, identified as 2,3,3’,4’-tetradehydro-P,p-caro tene (65) and 2,3,2’,3‘-tetradehydro-fl,a-caro tene (66). Products of 6oCoy-irradiation of p-carotene in tributyrin in the presence of oxygen were p-carotene-5,6-epoxide (5,6-epoxy-5,6-dihydro-p,p-carotene) (67), P-carotene-5,6,5‘,6’-diepoxide (5,6,5’,6’-diepoxy-5,6,5’,6’-tetrahydro-a,~-carotene) (68), 4-keto-4’-hydroxy-P-carotene (4-hydroxy-fi,/?-caroten-4-one) (69), and 5’
58
59
6o 61
h2
63
H. Kleinig and H. Reichenbach, Phytochemistry, 1973, 12, 2483. H. Kleinig and H. Reichenbach, Biochim. Biophys. Acta, 1973, 306, 249. U. Schwenker and G. Gingras, Biochem. Biophys. Res. Comm., 1973,51, 94. H. Singh, J. John, and H. R. Cama, Internat. J. Vit. Nutr. Res., 1973, 43, 147. J. John, H. Singh, and H. R. Cama, Internat. J. Vit. Nutr. Res., 1973, 43, 70. H. Singh and H. R. Cama, Internat. J. Vit. Nutr. Res., 1972, 42, 379. S. Takimoto, K. Chin, N. Okukado, and M. Yamaguchi, Mem. Fac. Sci., Kyushu Univ., Ser. C , 1973, 8, 197 (Chem. Abs. 1973,79,42 706).
232
Terpenoids and Steroids
a
b
C
0 d
HO
OH f
e
@.
(62) R’ = a, R2 = CH20Ac (63) R’ = b, R2 = CH20Ac (64) R’ = R2 = c (65) R’ = d, R2 = c (66) R’ = R2 = d
(67) R’ = b, R2 = a (68) R’ = R2 = b (69) R’ = e, R2 = f (70) R’ = g, R2 = h (71) R’ = R2 = J
retin01.~~These products, and p-apo-10’-carotenal (lO’-apo-P-caroten-lO’-al) (72), B-apo-l2’-carotenal (1 2’-apo-p-caroten-l2’-al) (73), and 3,3’,6’-trihydroxy-
(72) R = CH=CHCHO (73) R = CHO
z-carotene-5,8 epoxide (5,8-epoxy-5,8-dihydro-B,E-carotene-3,3’,6’-triol) (70) were also obtained by similar irradiation in light petroleum or n-hexane. Photobleaching of p-carotene in hexane or hexan-1-01solution, especially in the presence of chlorophyll, yields a range of cis-isomers and epoxides, especially aurochrome 64
E. Bancher, J. Washuettl, and P. Riederer, Internat. J. Vit. Nutr. Res., 1972,42,355,362.
Car0ten0 ids and Po lyterpeno ids
233
(5,8,5’,8’-diepoxy-5,8,5’,8‘-tetrahydro-~,/?-carotene) (71).65 At least ten volatile products, including toluene, xylene, ionene (74), and dihydroactinidiolide (75) were obtained by aerobic pyrolysis (180 “C)of B-carotene.66
Two syntheses of prephytoene alcohol have been reported (see previous section). The yield of /?-carotene obtained by condensation of all-trans-retinyl isocyanide (76) with retinal in the presence of butyl-lithium was only slightly less than that obtained in the corresponding Wittig reaction.67 Autoxidation of axerophtylidenetriphenylphosphorane (77) also produced /?-carotene, in up to 35 % yield; when the oxidation was allowed to take place in tritiated ethanol, [15,15’-3H,]-fl-carotene was produced.68 Treatment of axerophtyltriphenylphosphonium periodate (78) with lithium ethoxide also led to /?-carotene.68
(76) R = CH,NC (77) R = CH=PPh, (78) R = CHz;Ph310,-
Full details of the total synthesis of lycoxanthin ($,$-caroten-l6-01) (79) and lycophyll(~,11/-carotene-16,16’-diol) (80) have been r e p ~ r t e d . ~Wittig ’ condensation of the key intermediate (7-methoxycarbonyl-3-methylocta-2,6-dienyl)triphenylphosphonium bromide (87) (synthesized in nine steps from acrolein) (81) gave the C40 diester with crocetin dialdehyde (8,8’-diapocarotene-8,8’-dial) (82) and a C,, monoaldehyde (83); the latter afforded further C40 diester and a C40 monoester (84). The properties of these new methyl carotenoates, including n.m.r. data, are given. LiA1H4 reduction of the esters yielded lycoxanthin and 1ycophyll. Racemic /?,y-carotene (85) and optically inactive y,y-carotene (86) have been ~ y n t h e s i z e dby ~ ~the C,, + Cl0 + CIS route7’ via the phosphonium salt (88). 65
66 67
68 69
C. Zinsou and C. Costes, Physiol. Vc!gkrale, 1973, 1 1 , 191. K. Kawashima and T. Yamanishi, Nippon Nogei Kagaku Kaishi, 1973,47, 79. F. Kienzle, Helv. Chim. Acra, 1973, 56, 1671. H. J. Bestmann, 0. Kratzer, R. Armsen, and E. Maekawa, Annalen, 1973, 760. H. Kjersen and S. Liaaen-Jensen, Acta Chem. Scand., 1972,26,4121. A. G. Andrewes and S. Liaaen-Jensen, Acta Chem. Scand., 1973, 27, 1401. H. Mayer and 0. Isler, ref. 23, pp. 325-575.
234
Terpenoids and Steroids
b
'1
(79) (80) (81) (82)
I Me02C
e
d
C
R' = a, R2 = b R 1 = R2 = a R' = R2 = CHO R' = R2 = c
,
I
(83)
R'
(84)R'
= C, = C, = d,
R2 = CHO R2 = b
(85) R' R2 = e (86) R' = R2 = e
+
CH,PPh,Br
Several carotenoid-like model aryl polyenes have been synthe~ized.'~A series of yellow-orange polymers has been prepared by modification of polymers such as poly-(p-vinylaniline) with fl-apo-8'-carotenal (18) and diamines, or by copolymerization with P-carotene-5,6,5',6'-diepoxide(68).72 5 Degraded Carotenoids
Retinol and Derivatives.-A retinol glycolipid, considered to be a retinyl phosphate-mannose, is produced by a rat liver enzyme p r e p a r a t i ~ nand , ~ ~the formation of a retinyl phosphate-galactose compound has been reported.74Compounds tentatively identified as retinyl glucoside, retinyl mannoside, and retinyl galactoside have been produced by enzymic incubations of retinol and the corresponding UDP-~ugar.~~ Diels-Alder condensation between retinyl acetate and methyl trans-fl-formylcrotonate gave three adducts ( 8 9 H 9 1). Correlation between derivatives of these and degradation products of perhydrokitol has allowed the relative stereochemistry (92) to be assigned to the natural retinol dimer k i t 0 1 . ~ ~ l 2
l 3
l4 i 5
''
H. Kamogawa, J. Polymer Sci., Part B, Polymer Letters, 1972, 10, 929. L. De Luca, N. Maestri, G . Rosso, and G. Wolf, J. Bid. Chem., 1973, 248, 641. T. Helting and P. A. Peterson, Biochem. Biophys. Res. Comm., 1972, 46, 429. P. Rodriguez, 0. Bello, and K. Gaede, F.E.B.S. Letters, 1972, 28, 133. B. V. Burger and C. F. Garbers, J.C.S. Perkin I , 1973, 590.
Curo ten0ids and Poiy terpenoids
235
CHO
C0,Me
Me0,C (89)
(91)
(90)
Oxidation of retinol with peracetic acid gave 1lY12-epoxyretinal(93) in 45 % yield.77 Treatment of methyl retinoate (94) with manganese dioxide in light petroleum gave methyl 4-oxoretinoate (95), from which 4-oxoretinoic acid (96), methyl 4-hydroxyretinoate (97), methyl 3,4-didehydroretinoate (loo), and 3,4didehydroretinol(lO1) were prepared.78All-trans-retinylamine (98), p-ionylamine (102), and related amides have been prepared7' by hydrazine treatment of the corresponding phthalimides (99) and (103) (obtained by the azocarboxylate method). The preparation of all-trans-retinyl isocyanide (76) and fl-ionyl isocyanide (104) has also been reported.67
R1 (94) (95) (96) (97) (98)
R' = H,H, R 1 = 0, R' = 0, R' = H,OH, R' = H,H,
(99) R' = H,H,
R2 = C 0 2 M e R2 = C 0 , M e R2 = C 0 2 H R2 = C 0 , M e R2 = CH2NH2 0 R2 = C
H
2
N
D
0 77
78
l9
Y. Ogata, K . Tomizawa, and K. Takagi, Tetrahedron, 1973, 29,47. A. B. Barua and M. C. Ghosh, Tetrahedron Letters, 1972, 1823; M. S. S. Rao, J. John, and H. R. Cama, Internat. J . Vit. Nutr. Res., 1972, 42, 368. F. Kienzle, Helv. Chim. Acta, 1973, 56, 1662.
Terpenoidr and Steroids
236
(100) R = C 0 , M e
(101) R
=
(102) R = NH,
CH,OH (103) R = -N
0 (104) R
=
NC
Two new syntheses of Vitamin A derivatives have been reported. One (Scheme l),involving condensation of them-anion of sodium 3-methyl-2-butenoate
4
(y-bCN
*[uO] 0
Reagents: i, LiCH,CN-THF; ii, I,; iii, Bu',AIH; iv,
v, Ac,O-py; vi, B u ' O - K + ; vii, Ac,O-NaOAc; viii, Bu'O-K+.
Scheme 1
Na+ *
Li+'
Carotenoids and Polyterpenoids
237
with B-ionylidene acetaldehyde (107), followed by basic elimination, yielded exclusively 13-cis-retinoicacid (108).The fl-ionylidene acetaldehyde was prepared by a new method from /3-ionone (105) and lithioacetonitrile.*' In the second synthesis (Scheme 2),81 retinoic acid was prepared from P-ionylidene bromide
Ii Reagents: i, PBr,; ii, Bu'K-
C 0 , M e ( 1 1 1 ) ; iii, KOH; iv, PhS0,Na; SOlPh
Br
Scheme 2
(1 10) and the phenyl sulphone (111) obtained from methyl y-bromosenecioate (113), or from methyl y-bromosenecioate and the phenyl sulphone (112) obtained from vinyl-&ion01 (109).
G . Cainelli, G . Cardillo, M. Contento, P. Grasselli, and A. U. Ronchi, Guzzetru, 1973, 103, 117. M. Julia and D. Amould, Bull. SOC.chim.France 11, 1973, 7'46.
238
Terpenoids and Steroids
Other Degraded Carotenoids.-Many well known compounds are structurally related to carotenoids and could be formed as carotenoid metabolites, e.g. abscisic acid (4).Some new naturally occurring compounds have been characterized that may also be in this category. Five isomeric megastigmatrienes isolated from tobacco have been identified' as 6c-megastigma-4,7E,9-trien-3-one (114) and four stereoisomers [(62,82),(62,8E), (6E,82), and (6E,8E)] of megastigma4,6,8-trien-3-one( I 15). Their structures were determined by g.c.-m.s. and by their formation by dehydration of 3-0x0-a-ion01 ( 1 16). Amongst the large number of volatile compounds obtained from Curphephorus sp. were several [including /I-ionone (105), dihydroactinidiolide (75), ,!l-cyi.ocitral (117), 2,2,6-trimethylcyclohex-Senone (1 18), damascenone (1 19),and isophorone (12O)J that are clearly related structurally and possibly biogenetically to carotenoids and other higher terpenoid~.~~
A carotenoid-like compound obtained from the hair-pencil secretion of the monarch butterfly is reported to have pheromone activity.84 Th& compound has tentatively been assigned the structure cis-l,3,7,7-tetramethyl-2-oxabicyclo[4,4,0]dec-9-en-8-one(1 21), but details of this characterization have not yet been described. Three novel lipids, diumycinol (122), isodiumycinol (123), and diumycene (124), from the antibiotic diumycin have a carotenoid-like ring with an apparently non-isoprenoid side-chain. Interestingly the absolute configuration R2
''
A. J . Aasen, B. Kimland, S. 0. Almqvist, and C. R. Enzell, Acra Chem. Scand., 1972,
26, 2573. K . Karlsson, I . Wahlberg, and C. R. Enzell, Acta Chem. Scand., 1972, 26, 2837, 3839. J . Meinwald and J . P. Morizur; R. M. Silverstein; cited by J. G . MacConnell and R . M . Siherstein, Angrw. Chem. Internat. Edn., 1973, 12, 644.
Carotenoids and Polyterpenoids
239
(122) R = a (123) R = b (124) R = c
C
( S ) at C-13 ( = C-6, carotene numbering) is opposite to that at C-6' of a-carotene and related c a r o t e n o i d ~ . ~ ~ The chemistry and biochemistry of abscisic acid have been re~iewed.~ Further confirmation of the absolute configuration ( S ) of natural abscisic acid has been obtained by theoretical consideration of its optical properties41 and by syn( + )-a-methoxy-a-trfiuoromethyiphenylthe~is.'~~ The ~ ~ diastereomeric ~*~ acetates (MTPA esters) (125) of the isomeric cis-diols (126) were separated by liquid chromatography, and after hydrolysis the cis-a-diol (127) was converted (Scheme 3) into a product identical with natural (+)-abscisic acid. Consideration
(125) R = MTPA (126) R = H
(4)
+ 0
CO,Et
Reagents: i, Jones oxidation; ii, Wittig reaction.
Scheme 3
of optical properties established the ( S ) configurationat C-6 (carotene numbering) of the intermediate diol(l27) and bisenone (128), and thus also of abscisic acid.40 The absolute configuration at C-6 has been shown to be identical in the natural 86
W. A. Slusarchyk, J. A. Osband, and F. L. Weisenborn, Tetrahedron, 1973, 29, 1465. M . N. Galbraith and D. H. S. Horn, J.C.S. Chem. Comm., 1973, 566.
240
Terpenoids and Steroih
products (+)-abscisic acid, (-)-theaspirone (30), and blumenols A (2) and B (29).’5,86 C.d. and the nuclear Overhauser effect were used to establish the absolute configuration at C-9 of blumenols A and B as (R). Syntheses of optically active ‘grasshopper ketone’ ( 129)87 and dehydrovomifoliol ( 128)88 from the conversion optically active ketone (130) are reported. The well-documented1 of ( + )-dehydrovomifoliolinto ( + )-abscisic acid thus establishes a direct correlation between the latter and grasshopper ketone. 5789
HO
THPO
Excised axes of Phaseolus vulgaris metabolize abscisic acid into two physiologically inert compounds, phaseic acid (131) and 4-dihydrophaseic acid (132),* the latter identified” by n.m.r. and mass spectra of its methyl ester. Metabolism of an abscisic acid precursor, ( & )-5-( 1,2-epoxy-2,6,6-trimethylcyclohexyl)-3methylpenta-cis-2-trans-4-dienoic acid (133),* by tomato shoots and avocado produced ( - )-epi-(1’R,2’S,4S)-2-cis-xanthoxin acid (134): which, unlike its 1,2-epimer (136),* was not further metabolized.” A method for the synthesis of (f)-xanthoxin [(135) + (137)], not by degradation of xanthophylls, is briefly
CO, H
R
(131) R = 0 (132) R = H,OH
HO
HO (134) (135) 87 88
09 90
91
(133)
R R
= C0,H =
CHO
(136) R = C 0 , H (137) R = CHO
K. Mori, Tetrahedron Letters, 1973, 723. K. Mori, Tetrahedron Letters, 1973, 2635. M. Takasugi, M. Anetai, N. Katsui, and T. Masamune, Chem. Letters, 1973, 245. E. T. Tinelli, E. Sondheimer, D. C. Walton, P. Gaskin, and J. MacMillan, Tetrahedron Letters, 1973, 139. B. V. Milborrow and M. Garmston, Phytochemistry, 1973, 12, 1597.
* The abscisic acid numbering system is used for these derivatives; otherwise the carotenoid numbering system is used throughout for possible degraded carotenoids.
24 1
Caroteno id and Po ly terpenoids
rep~rted.’~A series of 3-methylpentadienoic acids structurally related to abscisic acid but with cyclohexenyl, methylcyclohexenyl, furyl, phenyl, hydroxyphenyl, or methylhydroxyphenyl groups in place of the usual abscisic acid ring system has been synthesi~ed.~~
Several syntheses of /?-damascone (138) and /?-damascenone (119) have been achieved, Diels-Alder addition of penta- 1,3-diene (139) to 3-bromo-4-methylpent3-en-2-one (140) yields the bromo-ketone (141) which is readily converted into /I-damascenoneg4(overall yield 30-40 %) (Scheme 4). Similar addition of penta-
14)
vi or vii
p
Reagents : i, AIC13; ii, LiF-Li,C03; iii, NBS; iv, Zn-HOAc; v, N-methylanilino-MgBrMeCHO; vi, TsOH; vii, NaOAc-Ac,O.
Scheme 4
1,3-diene to mesityl oxide gave an adduct which was converted into &damascone (143) via the ketone (142). Damascones and /I-damascenone have been prepared
92
93
94
T. Oritani and K. Yamashita, Agric. and Biol. Chem. (Japan), 1973,37, 1215. T. Oritani, T. Matsunaga, and K. Yamashita, Agric. and Biol. Chem. (Japan), 1973, 37,261. K. S. Ayyar, R. C. Cookson, and D. A . Kagi, J.C.S. Chem. Comm., 1973, 161.
Terpenoids and Steroidr
242
from the readily accessible ionone i s o x a ~ o l e s ,e.g. ~ ~ P-damascone from (144). In the presence of acids, 7,8-didehydro-P-ionol (145) or the related diol (146) is converted into a mixture of P-damascone and 7&didehydrotheaspirane (147),and 6-hydroxy-7,8-didehydro-cx-ionol (148) gives a mixture of 8-damascenone and 8-oxotheaspirane (149). This method also provides access to a-damascone (150) and several naturally occurring damascone derivatives ( 151x154).96 - n e
preparations of P-damascenone from the acetylenic diol(l48) and of P-damascone from P-ionol (155) via an allenic intermediate (156) have suggested a possible mechanism for the biosynthesis of damascenone from neoxanthin ( 157).97
W
95 96 97
O
H
aOyoH
K . H . Schulte-Elte, B. L. Muller, and G. Ohloff, Helv. Chim. Acta, 1973, 56, 310. G. Ohloff, V. Rautenstrauch, and K. H . Schulte-Elte, Helv. Chim. Acta, 1973,56, 1503. S. Isoe, S. Katsumura, and T. Sakan, Helv.Chim. Acta, 1973, 56, 1514.
Carotenoids and Polyterpenoids
243
Eight ionone analogues have been prepared by condensation of hydroaromatic aldehydes with mesityl oxide.98A mild and very selective method for reduction of @-unsaturated carbonyl products by hydrosilane-rhodium(1) complexes has been used to reduce a-ionone (158) and p-ionone (Scheme 5). a-Ionone gave
Reagents: i, Et,SiH-[(Ph JP)3RhC1];ii, K,CO,-Me,CO-MeOH-H,O.
Scheme 5
almost exclusively dihydro-a-ionone (159). In the case of p-ionone, the ratio of dihydro-/?-ionone(160)to the alternative product B-ionol(l55)was very dependent on the hydrosilane used, but with phenyldimethylsilane, (160) formed over 90 of the product.99 Similar speclfic reduction of p-ionone and dehydro-P-ionone (161) has been achieved by use of triphenyltin hydride; use of triphenyltin deuteride afforded deuterium-labelled products."*
The preparation of 2-cis,4-trans- and 2,4-trans-a-ionylidene[2-' 4C]acetic acid [(162) and (163)] by Reformatskii reaction of a-ionone with ethyl bromo[2-14C]acetate has been reported."' Some acyclic analogues of trisporic acid (164) have been prepared.'02
:yf-J-(,: /
(162) R' = H, R2 = C 0 2 H (163) R' = C 0 2 H , R2 = H 98
H
o
0
z
z
o
H
( 164)
E. T. Suleimanova, M. R. Musaev, S. D. Mekhtiev, L. I. Kasumov, and M. I. Mirgasanova, Doklady Akad. Nauk Azerb. S . S . R . , 1971, 21, 26 (Chem. Abs., 1972, 77, 34 720).
99
loo lo' lo'
I. Ojima, T. Kogure, and Y . Nagai, Tetrahedron Letters, 1972, 5035. H . R. Wolf and M. P. Zink, Helv. Chim. Acra, 1973, 56, 1062. H . Lehmann, D. Gross, and H. R. Schuette, Z . Chem., 1972, 12, 416. L. N . Polyachenko, L. P. Davydova, V. V. Mishchenko, and G . I. Samokhvalov, J . Gen. Chem. U.S.S.R., 1973,43, 413.
Terpenoids and Steroids
244
The degraded carotenoid glucoside picrocrocin (28) has been shown to have the ( 3 R ) configuration, by correlation with ( - )-3-methoxy-/?-ionone(165).3
Quantitative preparations of several sterically hindered olefins, especially 7-cis-ionyl and -ionylidene derivatives have been achieved by selective triplet sen~itization.''~ Although in this work fi-ionol and p-ionone were isomerized to the 7-cis-isomers, and later showed also the formation of the 7-cisisomer of a dehydrotetraene ester (166), dehydro-p-ionol (167) and dehydro-& ionone (161) did not isomerize. Direct irradiation of dehydro-j-ionone led to the formation of the 7-cis-isomer (168) and of ionene (74) via (169). Direct
irradiation of 7-trans-P-ionylideneacetonitrileisomers (170) and (171) led105 to facile geometrical isomerization into the 7-cis-isomers (172) and (173) with concurrent accumulation of the irreversibly formed products (174) and (175) and of the reversibly formed product (176). The isomeric (177) was not present in the
(170) R' (171) R' lo'
'04 lo'
= =
H, R2 = CN CN, R2 = H
(172) R' = H, R2 = CN (173) R' = CN, R2 = H
V. Ramamurthy, Y. Butt, C. Yang, P. Yang, and R. S. H. Liu, J . Org. Chern., 1973, 38, 1247. V. Ramamurthy and R. S. H. Liu, Tetrahedron Letters, 1973, 441. V. Ramamurthy and R. S. H. Liu, Tetrahedron Letters, 1973, 1393.
Curoten0ids and Polyterpenoids
R' = H, R2 = CN (175) R' = CN, R2 = H (174)
245
R'
=
(177) R'
=
(176)
H, R2 = CN CN, RZ = H
irradiation mixture but was obtained by thermal cyclization of (172). Direct irradiation of (2)-retro-y-ionone (178) in ethanol gave the bicyclo-octene (179) whereas (E)-retro-y-ionone(180) gave the oxetan (181).lo6 0
Model Cyc1izations.-A subject that continues to arouse considerable interest is the cyclization of acyclic terpenoids, studied as a model of the cyclization reaction of carotenoid biosynthesis. Treatment of methyl truns-geranoate (182) with PhSCl and AgSbF, in nitromethane gave the a-cyclogeranoate derivative (188) in 3 5 4 5 % yield.'" Fluorosulphonic acid also proved to be an efficient cyclizing agent,"' again producing mainly a-rings ;+-ionone (183) gave a-ionone (84 %) and b-ionone (16 %), methyl geranoate gave a-cyclogeranoic acid (189) (72 %), and geraniol(l84) gave a-cyclogeraniol(l90) (62%). The (truns- or cis-) gc-anylacetone cyclic ethylene acetal (185), however, yielded 65% of the pderivative (192). Mercury salts, e.g. Hg(NO& and especially Hg(OCOCF3)2, are also efficient initiators of electrophilic cyclization of isoprenoids. The intermediate mercury compound (193) is reduced with NaBH,, thus giving a way of introducing deuterium or tritium into the cyclic molecule.'0g The conversion of citral (186) into almost exclusively a-cyclocitral (191) via the pyrrolidine amine (194)has been reported.' l o In the presence of concentrated D,S04, deuterium replaces the olefinic hydrogen of a-cyclocitral,presumably via the conjugated imminium salt (187). By use of optically active pyrrolidines, this reaction has led to the preparation of optically active (R)-(+)-rx-cyclocitral(195) '06
lo'
lLo
A. van Wageningen and H. Cerfontain, Tetrahedron Letters, 1972, 3679. M. T. Mustafaeva, M. Z. Krumer, V: A. Smit, A. V. Semenovskii, and V. F. Kucherov, Izvest. Akad. Nauk S.S.S.R., Ser. khim.,197. M. Kurbanov, A. V. Semenovskii, and V. A. Smit, Izvest. Akad. Nauk S.S.S.R., Ser. khim., 1973, 390.
M. Kurbanov, A. V. Semenovskii, V. A. Smit, L. V. Shmelev, and V. F. Kucherov, Tetrahedron Letters, 1972, 2 175. S. Yamada, M. Shibasaki, and S. Terashima, Tetrahedron Letters, 1973, 377.
Terpenoids and Steroids
246
. .+- . (182) (183) (184) (185) (186) (187)
R R R R R R
= = =
C02Me CH=CHCOMe CH,OH
=
a
=
CHO b
=
0
.-c) b
(189) R = C 0 2 H (190) R = C H 2 0 H (191) R = CHO
(195) R (196) R
= =
CHO COCH=CHMe
(27 "/, optical purity), which was in turn converted into (R)-(+)-a-damascone (196).' [The absolute configuration of these compounds is wrongly described" as (S).I
''
'
6 Polyterpenoids and Quinones Po1yterpenoids.-The lipid intermediate in peptidoglycan biosynthesis in E. coli has been characterized as C5,-isopreny1-pyrophosphory1-N-acety1muramy1(pentapeptide)-N-acetylglucosamine.l* The C5 isoprenoid alcohol from Streptococcus fuecalis has a ratio of internal cis to trans double bonds of 8 : 2 . I l 3 A series of fully hydrogenated isoprenoid hydrocarbons isolated from a geological deposit may have been formed from polyisoprenoid alcohols, or in some cases lycopene. The structures of these hydrocarbons were confirmed by synthesis.' l4
'
I"
l3
'I4
S. Yamada, M . Shibasaki, and S. Terashima, Tetrahedron Letters, 1973, 381. J . N. Umbreit and J. L. Strominger, J . Bacteriol., 1972, 112, 1306. J. N. Umbreit, K. J. Stone, and J. L. Strominger, J . Bacteriol., 1972, 112, 1302. C. Spyckerelle, P. Arpino, and G. Ourisson, Tetrahedron, 1972, 28, 5703.
247
Curotenoids und Polyterpenoids
A method has been developed for the synthesis of pyrophosphodiesters of carbohydrates and isoprenoid alcohols." Phosphorylation of ficaprenol(l97 ; mainly C55 ) with o-phenylene phosphochloridate gave the o-hydroxyphenyl phosphate (198),which was converted into ficaprenyl phosphate (199).The latter, with diphenyl phosphochloridate, gave the diphenyl pyrophosphate (200), and this, with 2,3,4,6-tetra-O-acetyl-a-~-galactopyranosyl phosphate (203) gave a product (201) which on deactylation gave the pyrophosphodiester of ficaprenol and D-galactose (202).
(197) R (198) R (199) R (200) R (201) R (202) R
=
OH
0
I1 -o-p-o-...
=a
b c = d, R' = d, R' = =
I
0-
AC =H =
b
0
0
II
I1
PhO-P-0-P-0-I I
OPh
0-
d
C H ,OAc
AC%L-) 7 o-p-oOAc 0 -
Isoprenylated Quinones.-A method for the visualization of isoprenoid quinones on reversed-phase thin-layer chromatograms has been described.' ' 2-Tetraprenyl-l,4-benzoquinone (204), the corresponding quinol (205), and 4-hydroxyl o
'I6
C. D. Warren and R. W. Jeanloz, Biochemistry, 1972, 11, 2565. J . A. S. Rokos, J . Chromatog., 1972, 74, 357.
248
Terpenoids and Steroids (204) R = a, n (205) R = b, n (206) R = c, n (207) R = d, n (208) R = d, n (209) R = e, n (210) R = f, n (211) R = d, n
= = = = = = = =
4 4 4 4 9 9 1 1
OCH,OMe
Me0
0 d
OH e
OCH,OMe f
3-tetraprenylbenzoic acid (206) have been isolated from a marine sponge, Ircinia muscarurn,’ suggesting that these compounds could be intermediates in ubiquinone (207) biosynthesis. A new intermediate in ubiquinone-9 (208) biosynthesis in rat liver has been identified by its n.m.r. and mass spectra as 6-methoxy-
2-n0naprenylphenol(209).~ In a new synthesis of ubiquinone, addition of the isoprenoid side-chain was achieved by condensation of 6-bromo-2,3-dimethoxy-5-methylhydroquinone bis(methoxymethyl) ether (212) with 1,l-dimethyl-n-allylnickelbromide (213) in ?CH,OMe
I
’ G . Cimino, S. De Stefano, and L. Minale, Experientiu, 1972, 28, 1401.
‘ I 8
H. G. Nowicki, G. H . Dialameh, and R. E. Olson, Biochemistry, 1972, 11, 896
Carotenoitls und Polyterpenoids
249
hexamethylphosphoramide. Hydrolysis of the product (210)followed by oxidation gave ubiquinone-l(211). This should provide a good general method for synthesis of higher ubiquinone homologues.' l 9 Other new methods reported for the addition of the prenyl side-chain of benzo- and naphtho-quinones involve condensation of the hydroquinones with isoprenoid alcohols with the use of N-sulphinylamines' 2o or with isoprenyl bromides in the presence of amalgamated zinc or palladium.' 2 1 The phylloquinone from Anacystis nidulans and Euglena gracilis has been characterized by mass spectrometry as 5'-monohydroxyphyl1oquinone(214).122 0
'I9
K. H. H. A.
Sato, S. Inoue, and R. Yamaguchi, J . Org. Chem., 1972, 37, 1889. Sugihara, M. Sasaki, Y . Kawamatsu, and H. Morimoto, Annalen, 1972, 763, 121. Sugihara, Y. Kawamatsu, and H. Morimoto, Annalen, 1972, 763, 128. Law, G. Thomas, and D. R. Threlfall, Phytochemistry, 1973, 12, 1999.
7 Biosynthesis of Terpenoids and Steroids BY D. V. BANTHORPE AND B. V. CHARLWOOD
1. Introduction A large number of reports have appeared of the isolation and characterization of new terpenoids from natural sources, and often hypothetical biogenetic schemes have been appended for novel (and sometimes not so novel) structures. Such schemes usually fall within the accepted framework of theory, or form trivial or readily predictable extensions of it. These are normally not discussed in this chapter, although a few speculations of this kind, together with some biogenetictype syntheses and experiments with model systems, which seemed novel or otherwise important, have been included. The appropriate chapters in Part I of this Report should be consulted for additional information. References are not provided to work carried out in previous years, even when such work is briefly cited to provide relevant background, as these may generally be found in the papers under discussion. It is a matter of concern to note how frequently results and discussions previously recorded either by the writers or by other authors are reiterated either verbatim or with minor modifications. Where the Reporters were unable to consult the primary journals, references are supplemented with those to Chemical Abstracts. Incorporations of mevalonic acid ( 1 ) are given in terms of those of the (3R)isomer, which is presumed to be that component of the (3RS)-racemate normally
HA = (2R) hydrogen H, = (2s)hydrogen H, = (4R) hydrogen H, = (4s) hydrogen HE = (5R)hydrogen H, = (5s)hydrogen
H (1)
fed that is metabolized in terpenoid biosynthesis. Such incorporation figures are quoted only if they differ appreciably from the range of values typically found for the type of organism and feeding experiment under consideration, or if some other point of note is involved. 250
Biosynthesis of Terpenoids and Steroids
25 1
2 Acyclic Precursors
The generally accepted pathway from acetyl coenzyme A to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) (2) involves formation of acetoacetyl coenzyme A (3), and this route has been firmly established to occur in yeast. Some years ago an alternative pathway via malonyl coenzyme A (4)was postulated to occur in pigeon-liver preparations'in which a key difference was the occurrence of proteinbound intermediates (5)+7). The generality and relative importance of the two
routes have been the subject of some controversy. A very thorough study' of cell-free preparations from yeast, rat and pigeon liver, and the latex of Hecea brasiliensis has shown that in all cases labelled terpenoids were obtained after administration of ['4C]malonyl coenzyme A, but the incorporation of acetyl coenzyme A was neither stimulated by bicarbonate nor inhibited by avidin, as would have been expected if carboxylation of acetyl coenzyme A were an obligatory stage in the production of HMG. Furthermore, the pattern of tracer in HMG-CoA biosynthesized from [1,3-14C]- and [2-'4C]-malonyl coenzyme A in liver systems and that in ergosterol formed by yeast from the latter indicated that degradation of the precursors to acetyl coenzyme A occurred before incorporation ;also uptake of tracer into product was related to the amount of malonyl coenzyme A decarboxylase in the preparations. The conclusion that the 'malonyl coenzyme A pathway' to terpenoids is of little importance was confirmed by studies on isolated perfused rat liver : 2 kynurenate, a known inhibitor of acetyl coenzyme A carboxylase, blocked up to 80 % of the fatty acid synthesis but did not significantly affect the rate of formation of cholesterol. The regulation of the biosynthesis of so-called 'active isoprene' has been reviewed; and the properties and subcellular distribution of enzymes required for
'
M . J. P. Higgins and R. G . 0. Kekwick, Biochem. J., 1973, 134, 295. C. A. Barth, J. Hackenschmidt, E. E. Weis, and K . F. A. Decker, J . Biof. Chem., 1973, 248, 738. D. J. Namara and V. W. Kodwell, 'Biochemical Regulatory Mechanisms in Eukaryotic Cells', ed. E. Kun and S. Grisolia, Interscience, New York, 1972, p. 205.
252
Terpenoids and Steroids
the biosynthesis of acetoacetate in chick liver have been r e p ~ r t e d .Two ~ quite different forms of HMG-CoA synthetase have been isolated in homogeneous states from chicken liver:5 one was a single polypeptide (ca. 52000 dalton) whereas the other comprised two identical or similar sub-units (cu. 55 000 each). Also, a cytosolic and two mitochondria1 forms of acetoacetyl coenzyme A thiolase have been purified from avian liver; with HMG-CoA synthetase they rapidly converted acetyl coenzyme A into HMG-COA.~In both chicken2 and rat liver,5feeding of cholesterol lowered the activity of both HMG-CoA synthetase and cytosolic thiolase, and feedback control at this step is presumed. Fatty acid synthesis was unaffected under these conditions. The levels of these enzymes also fell during starvation but returned to supranormal levels during feeding regimes involving high carbohydrate intake.. This apparent induction was blocked by cy~loheximide,~ an inhibitor of protein synthesis. The loss of HMG-CoA synthetase activity resulted from increased activity of HMG-CoA hydroxylase,8 and the HMG released competitively inhibited HMG-CoA reductase. HMG-CoA reductase, which catalyses the essentially irreversible step to form MVA that is unique to terpenoid biosynthesis and which is probably rate-limiting for the sequence as well as being subject to feedback contr01,~has been studied in detail and a simplified assay has been developed." The hepatic enzyme was solubilized from the microsomes and partially purified'',' and possessed the curious property of being cold-sensitive :chilling to 4 "Ccaused rapid inactivation. Previous reports that the enzyme from rat liver was almost entirely bound to the rough endoplasmic reticulum have been contradicted by the demonstration that the bulk (ca. 80 %) of the activity was associated with the smooth membranes of the Golgi apparatus, smooth endoplasmic reticulum, and plasma membrane.' No explanation could be given of the discrepancy but it is known that treatments (e.g.feeding ethanol or barbiturate) known to increase the smooth reticulum also increase the level of hepatic cholesterologenesis. Diurnal variations in activity of the enzyme from rat liver were controlled by the feeding regirnel4 and resulted from changes in the rate of formation: the rate of breakdown was essentially unaltered over the periods studied. Actinomycin D and cycloheximide blocked activity in cultured hepatoma cells' and the latter also prevented the induction of activity in yeast cultures (up to eight times the normal levels) caused by addition J. B. Allred, Biochim. Biophys. Acta, 1973, 297, 22. Moss, and H. D. Lane, Biochem. Biophys. Res. Comm., 1972,48, 255. K . D. Clinkenbeard, T. Sugiyama, J . Moss, W. D. Reed, and H. D. Lane, J . Biof. Chem., 1973,24a,2275. Z . H. Beg and D. M. Gibson, Fed. Proc., 1973,32, 480 (abs.). M. Saleemuddin and M. Siddiqi, Lipids, 1972, 7, 631. V . W. Rodwell, D . J. McNamara, and D. J. Shapiro, Adv. Enzymol. 1973,38, 373. F. H. Hulcher and W. H. Oleson, Fed. Proc., 1972, 32, 480 (abs.). R. A. Heller and R . G. Gould, Biochem. Biophys. Res. Comm., 1973,50, 859. M. S. Brown, S. E. Dana, J. M. Dietschy, and M. D . Siperstein, J . Biol. Chem., 1973, 248,4731. S. Goldfarb, F.E.B.S. Letters, 1972, 24, 153. R. E. Dugan, L. L. Slakey, A. V. Briedis, and J . W. Porter, Arch. Biochem. Biophys., 1972, 152, 21. J. A. Watson, Fed. Proc., 1973, 32, 480 (abs.).
' T. Sugiyama, K . D. Clinkenbeard, J . ' lo
" l2
l3
l4
Biosynthesis of Terpenoids and Steroids
253
of glucose to the medium.16 This again supports the idea that de novo synthesis of the enzyme had been involved. The activity of the hepatic enzyme was also stimulated by insulin" and inhibited by succinate and glutarate," and in these and similar experiments enzyme activity was paralleled by synthesis of cholesterol and bile acids. The two-step reduction of HMG-CoA to MVA mediated by the above reductase involves the notional formation of mevaldic acid (S), and this may be formed in vivo probably by salvage breakdown of the enzyme-bound analogues. (3RS)Mevaldate was reduced with (4R)-[4-2Hl]NADHand hepatic mevaldate reductase to form (3Rs)-[S2H1]MVA,and after this had been resolved into (3R)- and (3s)-components it was proved by means of 'H n.m.r. spectroscopy that the two [5-2H,]-lactones were diastereoisomers and both had the (5R)configuration.20 This confirmed previous conclusions, arrived at indirectly, concerning the stereospecificity of reduction and also implies that both (3R)- and (3s)-mevaldates bind to an identical enzymic site such that the orientations of the substituents at C-3 are immaterial to this binding. The hemithioacetal of mevaldate and coenzyme A (9) is also a substrate for hepatic HMG-CoA reductase and the stereospecificityof reduction of this using (4R)-[4-2H,]NADPHas coenzyme is the same as that above.2
Mevalonate kinase from leaves and etiolated cotyledons of Phaseolus vulgaris had similar properties to enzymes previously purified from plants and mammalian liver, but contrary to some previous results there was no evidence for isoenzymic forms.22 However, extracts of Pinus pinaster and Agave americana yielded, in each case, two fractions with MVA-kinase activity. Both were equally and optimally active at pH 7.9 (the optimum pH for the plant enzyme is usually ca. 6.5) and they could represent isoenzymes, sub-units, or species of intra- or extrachloroplastic origin.23 Less detailed studies of MVA-activating enzymes from cell-free extracts of various plants are a ~ a i l a b l e . ~ ~ $ ~ l6
'' l9
21
22
23 24
25
J . Berndt, M. Boll, M. Lowell, and R. Gaumert, Biochem. Biophys. Res. Comm., 1973, 51, 843. M. R. Lakshmarnan, C. M. Nepokroeff, G . C. Ness, R. E. Dugan, and J. W. Porter, Biochem. Biophys. Res. Comm., 1973, 50, 704. M. R. Boots, S. G . Boots, C. M. Noble, and K. E. Guyer, J . Pharm. Sci., 1973,62,952. H. Danielsson, Steroids, 1972, 20, 63. H. L. Ngan and G. Popjak, Fed. Proc., 1973,32, 628 (abs.). A. S. Beedle, K. A . Munday, and D. C. Wilton, F.E.B.S. Letters, 1972, 28, 13. J . C. Gray and R. G . 0. Kekwick, Biochem. J., 1973,133, 335. E. Garcia-Peregrin, M. D. Suarez, and F. Major, F.E.B.S.Letters, 1973, 30, 15. E. Garcia-Peregrin, M. D . Suarez, M. C. Aragon, and F. Major, Phytochemistry, 1972, 11, 2495. E. D . Mitchell and M. R . Downing, Fed. Proc., 1973, 32, 521 (abs.).
254
Terpenoids and Steroids
Geranyl, farnesyl, geranylgeranyl, and phytyl pyrophosphates are inhibitory, at low concentrations, to MVA kinase from Heuea brasiliensis, Cucumis melo, Phaseolus uufgaris, and Saccharornyces cerevisiae.26 Although feedback inhibition has been demonstrated for mevalonate-5-pyrophosphatedecarboxylase and prenyltransferase, the levels of inhibitor required in these cases are of the same order as the K , of the enzyme. Thus it appears that in vivo the inhibition o f MVA kinase by prenyl pyrophosphates is likely to be of importance in the control of terpenoid biosynthesis. Isopentenyl isomerase in a cell-free preparation from Pinus radiata or Citrus sinensis eliminated the pro-4S hydrogen of mevalonate in the conversion of isopentenyl pyrophosphate (IPP) into its 3,3-dimethylallyl analogue (DMAPP)27 and thus has a similar specificity to the enzyme isolated from yeast and liver. The same result was also deduced from the labelling patterns in geraniol and nerol and their B-D-glucosides formed in vivo by Pelargonium graueolens and Rosa dilecta2' and in a-pinene from several Pinus species29after administration of [2-'4C,4R-4-3H,]MVA and its (4s)-isomer.* The isomerase from P . radiata was completely inhibited by iodoacetate under conditions where the co-occurring prenyl transferase was unaffected. The situation for the Citrus enzyme was similar but the situation for the transferase was less clear-cut.27 A full account has appeared ofthe elegant work (summarized in Scheme 1) in which recovery of
OPP = pyrophosphate Scheme 1
chiral acetate, after degradation of biosynthesized farnesyl pyrophosphate (FPP), made it possible to deduce the stereospecificity of hydrogen transfer to C-4 of 2h
"
28
29
.I. C. Gray and R . G . 0. Kekwick, Biochim. Biophys. Acta, 1972,279, 290. E. Jedlicki, G. Jacob, F. Faini, 0. Cori, and C..A. Bunton, Arch. Biochem. Biophys.,
1972, 152, 590. D. V. Banthorpe, G . N. J. Le Patourel, and M. J. 0. Francis, Biochem. J., 1972,130, 1045. D. V. Banthorpe and G . N. J . Le Patourel, Biochem. J . , 1972,130, 1055.
* In different investigations of prenol biosynthesis, either the free alcohols or their pyrophosphates were recovered for characterization and assay. Presumably in all cases the latter were initially formed in t+vo or in vitro.
Biosynthesis of Terpenoids and Steroids
255
IPP in the enzyme-catalysed interconversion of the C-5 pyroph~sphates.~The stereochemistry of addition and abstraction of hydrogen is consistent with .a concerted mechanism, but a two-step process cannot be excluded ; previous claims of evidence for the latter process have been strongly ~riticized.~ Incorporation studies on Rosa, Pelargonium, and Pinus species using [2-14C,4R-4-3H1]MVAand its (4s)-isomer showed that the pro@ hydrogen of the precursor was retained and the pro-4S hydrogen was eliminated in the formation of geraniol and nerol, their fl-D-glucosides,and ~ t - p i n e n e . These ~ ~ , ~ results ~ indicate that the correlation of retention of the pro-4s mevalonoid hydrogen with formation of a cis-substituted double bond, such as had been found in rubber and certain higher prenols and which had been presumed to be general, may not apply to monoterpenoid biosynthesis. The same stereochemistry of elimination occurred in vitro in systems from Pinus and Citrus species that synthesized geranyl, neryl, and 2-trans-6-trans- and 2-cis-6-trans-farnesyl pyr~phosphate.~’ In the latter case previous work had ruled out the possibility of interconversion of the 2-cis and 2-trans C,, and C15 pyrophosphates. These sets of rather unexpected results can be interpreted in two ways. There is no purely chemical reason why the formation of a given geometrical isomer should be linked to the loss of a particular pro-chiral hydrogen, and the strict stereospecificity could result from the spatial arrangement of the substrates on the enzyme surface during a direct coupling of the C , (or C,, + C5) precursors to give products, viz. (10) --+ trans-A2-prenol and (11) + cis-A2-prenol, both with loss of the pro4S
hydrogen.28 An equivalent view involving bonded carbonium ions as intermediates has been put f ~ r w a r d . ~Alternatively, ’ the prenol pyrophosphates may be isomerized directly or through a dephosphorylation-redox-phosphorylation sequence (Scheme 2).28 There is some evidence for the latter scheme occurring in
R
R
R
CHO
w
OH
Scheme 2 30 31
J. W. Cornforth, K . Clifford, R. Mallaby, and G. T. Phillips, Proc. R o y . SOC.,1972, B182,277. J. W. Cornforth, Chem. SOC.Rev., 1973, 2, 1.
256
Terpenoih and Steroids
petals of Rosa species,32and cell-free systems from Mentha piperita and Daucus carota were reported to convert geraniol or its phosphate (sic)into nerol. These isomerases were soluble and were claimed to require the presence of a flavin, a thiol, and light, but no experimental or mechanistic details were given.33 The possible independence of routes to geranyl and neryl pyrophosphates has its counterpart in sesquiterpenoid biosynthesis, where many skeletal classes can be formally derived from 2-cis-6-trans-FPP. The synthesis of this and its 2-trans-isomer from [1-3H]geranyl pyrophosphate and IPP by a cell-free extract from orange flavedo was demonstrated together with the occurrence of the two corresponding aldehydes. Passage of tracer into products was consistent with a redox scheme similar to that suggested for the m o n ~ t e r p e n o i d s .No ~ ~ direct interconversion of the two F P P isomers could be detected. A redox scheme for the isomerization is also favoured by more detailed labelling studies. Soluble enzyme systems from tissue cultures of Andrographis paniculata incorporated six atoms of tracer (as revealed by measurement of 3H/14Cratios in precursors and products) from [2-'4C,5-3H2]MVAinto 2-trans-6-trans-FPP but only five atoms into the 2-cis-i~omer.~2-trans- 6-trans-FPP was converted into the 2-cis-6-trans-isomer in cell-free systems of the fungus Trichotheciurn roseurn with the loss of one [5-3H,]mevalonoid hydrogen,36 and the conversion of the 2-tran~-[l-~H,]epoxyfarnesol (12) into the 2-cis-isomer by the fungus Helrninthosporiurn satiuurn in-
k volved a similar, but less clear-cut pattern of isotope The last workers briefly reported, with no supporting details, that 'similar' isomerizations occurred with 2-trans-6-trans-fdrnesol and with geraniol. An enzyme able to transfer the terminal phosphate from ATP to trans-FPP to give trans-farnesyl triphosphate has been partially purified from extracts of Gibbereila fujikuroi ;38 the enzyme has an absolute specificity for trans-FPP. Farnesyl triphosphate was not a direct substitute for FPP in the formation of 32 33
34
35 36 31
38
P. J . Dunphy and C. Allcock, Phptochemistry, 1972, 11, 1887. A. J . Burbott, R. Croteau, W. E. Shine, and W. D. Loomis, Plant Physiol., 1973, 51, .suppl., 49. L. Chayet, R. Pont-Lezica, C. George-Nascimento, and 0. Cori, Phytochemistry, 1973, 12, 95. K . H. Overton and F. M. Roberts, J.C.S. Chem. Comm., 1973, 378. R. Evans, A. M . Holton, and J . R . Hanson, J.C.S. Chem. Comm., 1973,465. Y. Suzuki and S. Marumo, Tetrahedron Letters, 1972, 5101. I. Shechter, Biochim. Biophys. Acta, 1973, 316, 222.
257
Biosynthesis of Terpenoids and Steroids
higher terpenoids in the fungus or in yeast, but possibly has a role in the control of terpenoid biosynthesis.
3-Ethylbut-3-enyl pyrophosphate (13; R = Et) acted as a substrate for FPP synthetase from pig liver in place of IPP.39 This is important not only from the viewpoint of enzyme mechanism but also in relation to the biosynthesis of insect juvenile hormones. Other homologues (13 ;R = H, Pr", or Bun)were not effective substrates. Sequences in Scheme 3 using cis-3-methylpent-2-enylpyrophosphate
OPP
OPP
Scheme 3
(14)as a 'starter' provided enzymatic routes to tris-homo-FPP (1 5) and the parent skeleton of certain juvenile hormones (16). This is a good model system but it is not known if it is the pathway whereby the latter are biosynthesized in U ~ U O . ~ ~ The substrate specficity of FPP-synthetase from pumpkin fruit has also been established for the reaction (17)- (18); the upper size limit for R2 (with R'
R' R ' b O P P -
L
O
P
P
-2R
\
\
OPP
R' = Me or Et) was n-CgHI9 and branching of either alkyl group caused a marked decrease in r e a ~ t i v i t y .A~ ~comparison of the substrate specificities of 39 40 41
K . Ogura, T. Koyama, and S. Seto, J.C.S. Chem. Comm., 1972, 881. T. Koyama, K. Ogura, and S. Seto, Chem. Leriers, 1973, 401. T. Nishino, K. Ogura, and S. Seto, J . Amer. Chem. SOC.,1972, 94, 6849.
258
Terpenoids and Steroih
prenyltransferase from pig liver and pumpkin has been made;42 seven allylic pyrophosphates that could not be utilized by the pumpkin enzyme were active for the liver enzyme system. Presqualene alcohol pyrophosphate ( 19)[now known to have the configuration (1R,2R,3R) in the C3 ring43*44] has been generally assumed to be an obligate
R
H R
precursor of squalene in yeast and liver tissue, but a critical discussion31 emphasizes the difficulty of deciding what are 'normal' pathways and whether precursors lie on paths of restricted access, sidetracks, blind alleys, or alternative routes in this type of situation. The pyrophosphate was incorporated in excellent (ca. 1174) yield into squalene in cell-free systems from germinating peas whereas an epimer (R and Me interchanged) was not m e t a b ~ l i z e d .The ~ ~ cautious conclusion was reached that this tissue contained the same enzyme machinery to deal with the same isomer that is metabolized in yeast and animals but (rightly) no claim was made for (19) as an obligatory intermediate. The important role of the latter was further revealed by the formation of squalene and presqualene alcohol (incorporations 2 and 7 respectively)after incubations of [2-I4C]MVA with extracts from tissue cultures of bramble in the absence of NADH : in the presence of the cofactor incorporations were 85 and 304,.46 Studies on model systems for the reductive ring-opening of (19) suggest that enzymic involvement in the process may be chiefly to prevent the thermodynamically favourable mode of ring-opening which leads to artefacts that do iiot occur in N a t ~ r e . ~ ~ . ~ * Squalene synthetase appears to be the first enzyme of the terpenoid pathway to be membrane-bonded. The enzyme from pig liver was partially purified without solubilization by sonication of microsomes, and a detailed kinetic analysis of the conversion of FPP into squalene suggested a 'ping-pong' mechanism. No soluble or enzyme-bonded C30 compounds could be detected as interm e d i a t e ~ .The ~ ~ enzyme from yeast was also purified to near homogeneity and 42
43
" 45 4h
'' 4R ")
T. Nishino. K . Ogura, and S. Seto, Biochim. Biophys. Acra, 1973, 302, 33. L. Crombie, D . A . R . Findley, and D . A . Whiting, Tetrahedron Letters, 1972, 4027. G . Popjak, J . Edmond, and S. Wong, J. Amer. Chem. SOC.,1973, 95, 2713. G . H. Beastall, H . H . Rees, and T. W. Goodwin, F.E.B.S. Letters, 1972,28,243. R. Heintz, P. Benveniste, W. H . Robinson, and R . M. Coates, Biochem. Biophys. Res. Comm., 1972,49, 1541. C. D . Poulter, 0.J . Muscio, C. J . Spillner, and R. G . Goodfellow, J. Amer. Chem. SOC., 1972,94, 592 1 . R . M . Coates and W. H . Robinson, J. Amer. Chem. SOC.,1972, 94, 5920. R. E. Dugan and J . W. Porter, Arch. Biochem. Biophys., 1972,152,28.
Biosynthesis o j Terpenoids and Steroids
259
was released from the particulate fraction by treatment with sucrose and stabilized by glycer01.~' In the absence of the latter the high molecular weight species (ca 450 OOO dalton) so obtained reversibly dissociated into a component of lower (unspecified) molecular weight. The larger species converted FPP into squalene, but the other only formed presqualene pyropho~phate.~The general properties, cofactor requirements, and mechanism of action appear similar for the synthetases from yeast or liver.52 Geranylgeranyl pyrophosphate (GGPP) synthetase has been isolated from extracts of pumpkin fruit ;the enzyme catalyses the condensation of FPP with IPP and also the condensation of DMAPP and G P P with IPP.53 Thus it appears that one enzyme is able to synthesize Cl0, C I S ,and Cz0 precursors from C, intermediates. The activities of the individual enzymes on the pathway of terpenoid synthesis have been measured in homogenates of pea seeds at different stages of germination up to 32 h. All activities increased sharply between 6 and 12 h after germination and, with the exception of prenyltransferase, remained relatively constant. The activities at 16 h were MVA kinase > MVAP kinase > MVAPP kinase > MVAPP decarboxylase IPP isomerase > squalene synthetase > prenyltransferase. The last was rate-limiting throughout the period and the activities of the other enzymes were such as to produce rapidly a pool of C5 pyrophosphates. It seems likely that transferase plays some role in the regulation of the whole sequence.54 The subcellular distributions of enzymes responsible for catalysing the reactions in the conversion of acetyl CoA to squalene in hog aorta have been reported.,' A study of the distribution of enzymes responsible for the synthesis of ergosterol in yeast has led to the suggestion that MVA is formed from acetyl CoA in the mitochondria, whereas the steps from MVA to F P P take place in the cytoplasm ; the microsomal fraction had the ability to synthesize ergosterol from the C , , pyr~phosphate.~ The sites of biosynthesis of triterpenoids in Mentha piperita are completely separate from those of mono- and sesqui-terpenoids, and are readily accessible to exogenous MVA.57 More than 30% of activity from [2-14C]MVA was incorporated into squalene within 4 h, and the labelled h1,drocarbon was rapidly turned over (80 % turn-over in 8 h). The tracer pattern in squalene between 1 and 7 h after feeding showed that the IPP- and DMAPP-derived portions were equally labelled.
-
50 51
52
53 54
55 56 57
A. A, Qureshi, E. D. Beytia, and J . W. Porter, J. Biol. Chem., 1973, 248, 1848. A. A. Qureshi, E. D. Beytia, and J . W. Porter, Biochem. Biophys. Res. Comm., 1972, 48, 1123. E. D. Beytia, A. A. Qureshi, and J. W. Porter, J . Biol. Chem., 1973, 248, 1856. K. Ogura, T. Shinka, and S. Seto, J. Biochem. (Japan), 1972,72, 1101.
T. R. Green and D. J. Baisted, Biochem. J., 1972, 130, 983. L. L. Slakey, G . C. Ness, N. Qureshi, and J . W. Porter, J. Lipid Res., 1973, 14,485 I. Shimizu, J . Nagai, H . Hatanaka, and H . Katsuki, Biochim. Biophys. Acta, 1973, 296, 310. R . Croteau and W. D. Loomis, Phytochemistry, 1973, 12, 1957.
260
Terpenoids and Steroids
For prelycopersene pyrophosphate ( = prephytoene pyrophosphate) see Part I, Chapter 6, p. 226.
3 Hemiterpenoids For a long period isoprene was believed not to be a naturally occurring plant product, but it is now known to be emitted by leaves under certain conditions, although its route of formation is unknown. Leaf discs of Hamamelis jelena liberated the compound and the emission rate responded within 1 min to initiation and cessation of i l l ~ m i n a t i o n .Poplar ~~ leaves also generated isoprene on exposure to [I 3C]C02 and n.m.r. studies revealed that preferential labelling at C-1 and C-5 occurred,59i.e. the presence of distinct acetate pools was indicated.
4 Monoterpenoids The incorporation of [2-' 4C]MVA into monoterpenoids using intact plants is always low (typically in the region 0.001-4.1 %). It has been suggested that the sites of synthesis are compartmentalized and that in the unperturbed plant MVA is synthesized there from translocated photosynthetate (mainly sugars) whereas exogenous MVA has difficulty in penetrating to the sites. Feeding of unlabelled sucrose together with [2-' 4C]MVA to Mentha piperita increased incorporation of tracer into both mono- and sesqui-terpenoids by three- to ten-fold compared with controls in which the sucrose was absent and the period of metabolic turnover of monoterpenoids was markedly increased.60 Administration of [2-14C]MVA to plants in an atmosphere enriched (ca. 5 % v/v) in carbon dioxide had the same effect. The details of this and earlier work have been rationalized in terms ofa general model for terpenoid biosynthesis.60 It is proposed that multiple, isolated biosynthetic sites produce terpenoids when more acetyl coenzyme A is available than can be aerobically oxidized and that this condition could result from a deficiency of oxygen, a lack of functional mitochondria, or an abundance of carbohydrate. These ideas elaborate earlier views that production of essential oils is linked to a form of fermentation. Electron microscopy has revealed the morphology of the oil glands, and has made it possible to correlate the state of degeneracy of the organelles within with the ability to synthesize monoterpenoids.61 . 6 2 Up to 1 % incorporation of [2-I4C]MVA into free monoterpenoids and their ~ ~it may be general glucosides occurred in flowerheads of Tanacetum ~ u l g a r eand that exogenous MVA can penetrate to the active site in petals more easily than in leaf tissue. The catabolism of monoterpenoid precursors has also been followed by determining the time-course of evolution of carbon dioxide. 5R 5y
'O
''
62 63
R. A. Rasmussen and C . A. Jones, Phytochemistry, 1973, 12, 15. G. A. Sanadze and G. Dzhaiani, Fiziol. Rastenii, 1972, 19, 1082 (Chem. Abs., 1973, 7 8 , 2079). R. Croteau, A. J. Burbott, and W. D. Loomis, Phytochemistry, 1972, 11, 2937. G. Heinrich, Planta Med., 1973, 23, 201. G. Heinrich, Planta Med., 1973, 23, 154. D. V. Banthorpe and J. Mann, Phytochemistry, 1972, 11, 2589.
26 1
Biosynthesis of Terpenoids and Steroids
[14C]Geraniol and C5 intermediates derived from [2-14C]MVA were not significantly degraded, but acetate and 3,3-dimethylacrylate were broken down extensively.6 4 a-Pinene in various Pinus species29 and geraniol in Pelargoniurn r ~ s e u r n ~ ~ are new examples of asymmetric labelling whereby the moiety derived from IPP predominantly and specifically carried the bulk ( > 70 "/, ; total incorporation ca. 0.054.001 %) of the tracer incorporated from [2-14C]MVA. This phenomenon is generally accepted to be due to the existence of a pool of DMAPP or its biogenetic equivalent in uiuo. However, linalool in Cinnarnornurn ~ a r n p h o r a , ~ ~ geraniol and nerol and their glucosides in rose petals,28 and geraniol in Pelargoniurn graueolensZ8 were formed from [2-14C]MVA with the portions derived from IPP and DMAPP about equally (and position-specifically) labelled. Up to 10.6 % incorporation was obtained in the petal system and these results are the best demonstration to date of the mevalonoid origin of monoterpenoids of the conventional type in plants. A priori it would be expected that uptake of [14C]C0, would lead to equivalent labelling of the C5portions of monoterpenoids. Nevertheless, between 2 and 12 h after pulse feeding P . graueolens, the label in the IPP-derived moiety of geraniol increased up to 78% of the total (incorporation 0.0034.08%) but declined towards 50 % in the next 12 h period.67 A parallel study68showed that up to 12 h after exposure ofMentha piperita to a pulse of ['4C]C0, over 90 % tracer occurred in a C, fragment containing the IPP-derived portion of pulegone (20 ;a), whereas
(20)
the isopropylidene side-chain containing three carbons hypothetically derived from DMAPP was essentially unlabelled. Further degradations to determine if the more heavily labelled C5 moiety was randomly labelled were not carried out in either of these studies. Both glucose and carbon dioxide were up to ten times more effective precursors of monoterpenoids in M. piperita than was MVA, whereas the reverse occurred for sesquiterpenoids. The simplest explanation was that separate and largely isolated biosynthetic sites were involved for each class. Degradation of a-pinene biosynthesized in the above mentioned experiments indicated that essentially only one carbon was labelled [cf. (21)], as expected if 64 65
66 67
6*
D. V. Banthorpe and B. V. Charlwood, Planta Med., 1972,22, 428. T. Suga and T. Shishibori, Chem. Letters, 1972, 1093. T. Suga, T. Shishibori, and M. Bukeo, Bull. Chem. SOC.Japan, 1972,45, 1480. T. Wuu and D. J. Baisted, Phytochemistry, 1973, 12, 1291. R. Croteau, A. J. Burbott, and W. D. Loomis, Phytochemistry, 1972,11,2459.
Terpenoids and Steroids
262
bicyclization of asymmetrical labelled neryl pyrophosphate (22) occurred by the generally accepted hypothetical p a t h ~ a y . ~This ' pattern contradicted previous work where incomplete degradation of a-pinene indicated a 'symmetrical' and different pattern of labelling (23). Further evidence for the accepted pathway to the pinane skeleton as well as the routes to sabinene (24) and 1,4-cineole has come from the time-course of the composition of the oil of Juniperus c o r n r n ~ n i s . ~ ~
Routes to menthane derivatives have been investigated similarly7' and production of menthol or menthofuran in various Mentha species was controlled by a single dominant gene, whereas another prevented conversion of either a-terpineol into terpinolene or limonene into i~opiperitone.~','~ Studies on the oil of M . uquatica-M. longifolia hybrids showed their taxonomic similarity to M . durnentorurn, which some authorities consider to be a natural hybrid.73 As the start of an ambitious plan to investigate regio- and stereo-specific labelling of monoterpenoids derived from appropriately labelled precursors, [2-'4C]geraniol was proved (Scheme 4) to be incorporated specifically into
Scheme 4 camphor (25) and borneol in Salvia ~ f i c i n a l i s .This ~ ~ implies that a geraniolnerol isomerase (cf.Section 2) is present in vivo. This work encountered some of the most common problems in this field : plants often did not produce the desired monoterpenoids when grown in non-natural habitats, and yields of these
'' H. Horster, Planta Med., 1973, 23, 3 5 3 . 'O F. W. Hefendehl, Planra Med., 1973, 23, 301. '' M. J. Murray and F. W. Hefendehl, Phytochemistry, i2
'3 '4
1972, 11, 2469.
M. J. Murray and F. W. Hefendehl, Phytochemistry, 1973, 12, 1875. M. J. Murray and D. E. Lincoln, Euphytica, 1972,21, 337. A . R . Battersby, D. E. Laing, and R. Ramage, J.C.S. Perkin I, 1972, 2743.
263
Biosynthesis of Terpenoids and Steroids
compounds varied widely and suddenly during the growing (and I4C-feeding) season. Model systems for the formation of terpenoid alcohols in Tanecetum vulgare and Juniperus sabina suggest that chloroplasts in vivo promoted oxidation of sabinene (24) by radical-type processes involving ground-state oxygen. The initial products were then converted into either thujane or sabinane derivatives in different species.75 A detailed review, with some novel proposals, covers the biosynthesis of irregular monoterpenoids of the artemisyl, santolinyl, and lavandulyl series.7 6 Nepetalactone (26) was labelled significantly within three minutes of exposure of Nepeta cataria to [14C]C02 but the intermediates of the Calvin cycle were labelled within six S-Skytanthine (27) and its A5-derivative were labelled after uptake of [2-l4C]MVA by Tecomu stuns whereas [3H]loganin and actinidine were claimed not to be in~orporated.’~The conclusion that these cyclopentane monoterpenoids were of mevalonoid origin and that the [3H]compounds were not precursors, although probably correct, was unjustified as no degradation was carried out nor were the possibilities of compartmentation or failure of translocation to active sites considered. The N-methyl group of (27) was derived as expected from methi~nine.’~
(26)
(27)
Despite considerable efforts little success attends attempts to obtain stable cell-free systems from higher plants that can effectively sustain monoterpenoid synthesis. A preparation from Mentha piperita converted neryl pyrophosphate into a-terpineol in 4 % yield7’ and interconverted geraniol and n e r 0 1 . ~Similar ~ extracts were obtained from leaves of M . spicata and the root of Daucus carota, in which limonene was also formed. The cyclases and dehydratases were membranebound and the preparations were very unstable.33 Other systems that methylated loganic acid (28; R’= 1-D-glucose, R2 = H) to form loganin (28; R’ = p-Dglucose, R2 = Me)80,81and hydroxylated geraniol or nerol at C-1082*83 were isolated from Vinca rosea, and systems that methylated geniposidic acid (29; R’ = fl-D-glucose,R2 = H) to yield geniposide (29 ; R’ = fl-D-glucose,R2 = Me) 75
D. V. Banthorpe and D. Baxendale, Planta Med., 1973, 23, 239.
76
W.W. Epstein and C. D. Poulter, Phytochernistry, 1973, 12, 737.
”
“ 79
82
83
E. D. Mitchell, M. Downing, and G . R. Griffith, Phytochemistry, 1972, 11, 3193. D. Gross, W. Berg, and H. R. Schuette, Biochem, Physiol. Pflanz., 1972, 163, 576. R. Croteau, A. J. Burbott, and W. D . Loomis, Biochem. Biophys. Res. Comm., 1973, 50, 1006. K. M. Madyastha, R . Guarnaccia, and C. J. Coscia, Biochem. J . , 1972, 128,34P. K. M. Madyastha, R. Guarnaccia, C. Baxter, and C. J . Coscia, J. Biol. Chem., 1973, 248, 2497. T. D . Meehan and C. J. Coscia, Biochem. Biophys. Res. Comm., 1973,53, 1043. T. D. Meehan and C. J. Coscia, Fed. Proc., 1973, 32, 521 (abs.)
264
Ho"G
Terpenoids and Steroids
H
C
)
.
OR' . q
0R'
/
,
H
/
C02R2
,
/
H C02R2
were prepared from Genipa ~ r n e r i c a n a .As ~ ~in past years much of this type of work has appeared with sparse experimental detail. The novel compound (30), a presumed monoterpenoid, occurs in Juniperus communis and could be derived either from artemisia ketone (31) or, more probably, from (32),which co-occurs in the plant Other new types of compound are ( 3 3 H 3 5 ) from Ledum palustre.86 These are presumably formed in viuo, or during extraction, by photo-oxygenation of the appropriate A6-compound. The evolution of terpenoids, including that of monoterpenoids in fossil fuels, has been discussed.8
'
5 Sesquiterpenoids
Biosynthesis of the following sesquiterpenoids is covered in detail in Chapter 2: trichothecane group (p. 90); illudin M (p. 113); petasin (p. 131). Unsymmetrical labelling, analogous to that established for monoterpenoids (cJ Section 4), occurred in caryophyllene biosynthesized from [2-14C]MVA by Mentha piperita. The DMAPP-derived portion of the molecule contained 12% of the incorporated tracer with no randomization, after a metabolism period which left the corresponding moiety in the monoterpenoid pulegone unlabelled.88 These results were explained on the basis of an endogenous pool of DMAPP (not necessarily of mevalonoid origin) and different sites for the synthesis of mono- and sesqui-terpenoids. Extensive labelling of a-guaiene, a-patchoulene, caryophyllene, a-bulnesene, and patchouli alcohol (total incorporation 0.14 %) occurred after uptake of ['4C]glucose by leaf discs of Pogostemon ~ a b / i n . * ~ 84
86
88 B9
R. Guarnaccia, K. M. Madyastha, E. Tegtmeyer, and C. J. Coscia, Tetrahedron Letters, 1972, 5125. A. F. Thomas, Helv. Chim. Acta, 1973, 56, 1800. M. von Schantz, K . G. Widen, and R. Hiltunen, Acta Chem. Scand., 1973,27, 5 5 1 . G . Ourisson, Pure Appl. Chem., 1973, 33, 7 3 . R. Croteau and W . D. Loomis, Phyfochemistry, 1972, 11, 1055. M. J. 0. Francis, Planta Med., 1972, 22, 201.
265
Biosynthesis of' Terpenoids and Steroids
[14C]-Leucine,-acetate, -bicarbonate, or -MVA gave negligible or poor incorporations. A novel seco-illudoid (36), isolated from a species of fern, may be a precursor of pterosins (37) in uiuo, as on acid treatment the latter were formed in good yield, possibly as indicated in Scheme 5.90
Scheme 5
Attempts to demonstrate in model systems the methyl shift involved in the transformation of theeudesmane into the eremophilane skeletons [e.g.(38) -+(39)]
-
----*
Hoe*% (39)
(38)
failed,g1although such a shift would be expected to be facile. On the other hand, biogenetic-type syntheses of occidentalol (40),a eudesmane found in Thuja occidentalis that possesses a cis-ring junction, were successful (cf Scheme 6).92
Scheme 6
91 92
(40)
Y. Hayashi, M. Nishizawa, and T. Sakan, Chem. Letters, 1973, 63. J. W. Huffman, J. Org. Chem., 1972, 37, 2736. A. G. Hortmann, D. S. Daniel, and J. E. Martinelli, J. Org. Chem., 1973,38, 728.
266
Terpenoids and Steroids
An interesting series of biogenetic speculations concerning the order of ring oxidation, ring closure, and lactonization has been proposed for formation of eudesmane-type lactones of Avtemisia and other specie^.^^-^^ Double-label studies have shown that the hydrogens at C-3’ and C-4 of abscisic acid (41) formed in avocado fruit are both derived from the pro-2R hydrogen of MVA, whereas the hydrogen at C-5 originated from the pru-SS hydrogen (and is the only such hydrogen retained).97 The epoxy-compound (42) was converted into abscisic acid by tomato and avocado mesocarp although it is not a natural biosynthetic intermediate.98 (- )-Epi-(l’R,2’R,4‘S)-2-cis-xanthoxinacid is a metabolite of (42), but there is a stereospecific block to its further metabolic conversion into abscisic acid. ( + )-(l‘S,2’S,4’S)-2-cis-Xanthoxin acid is readily converted into (41), but trapping experiments indicate that it is probably not a natural intermediate on the pathway to abscisic acid. Unidentfied metabolites of abscisic acid in lettuce have been isolated.99 See also Part I, Chapter 2, p. 142.
A cell-free system from Helminthosporium siccans formed siccanochromen A (43) from MVA or FPP and orsellinic acid. trans-y-Monocyclofarnesol was probably a precursor and other intermediates were characterized.100 Other papers have dealt with theoretical aspects of the cyclizations and formation of short-lived intermediates in sesquiterpenoid biosynthesis’ O’ and the formation of the juvenile hormone of Hyaluphora cecropia.’ O 2
’’ M. A. Irwin and T. A. Geissman, Phytochemistry, 1973, 12, 849, 853, 863, 871, 875. 94
95
” ”
98
’’ loo
lol lo*
S. Inayama, T. Kawamata, and M. Yanagita, Phytochemistry, 1973, 12, 1741. G. D. Anderson, R. Gitany, R. S. McEwen, and W. Herz, Tetrahedron Letters, 1973, 2409. F. Shafizadeh and N. R. Bhadane, Tetrahedron Letters, 1973, 2171. B. V. Milborrow, Biochem. J . , 1972, 128, 1135. B. V. Milborrow and M. Garmston, Phytochemistry, 1973. 12, 1597. J . A. McWha and J. R. Hillman, Planta, 1973, 110, 345. K. T. Suzuki and S. Nozoe, J.C.S. Chem. C o m m . , 1972, 1166. K . E. Harding, R. C. Ligon, T. Wu, and L. Rode, J. Amer. Chem. SOC.,1972,94,6245. K. E. Opheim, Diss. Abs. ( B ) , 1973, 33, 4200.
Biosynthesis of Terpenoids and Steroids
267
6 Diterpenoids
A cell-free preparation from Gibberella fujikuroi converted [2-14C]MVA into kaurene (44) in up to 74% yield.'03 Incorporation of ten deuterium atoms into (44) biosynthesized from [6-2H3,2-14C]MVA by this system excluded the pimaradiene (45) as an intermediate although an enzyme-bound pimarane could not be
ruled out. The mass-spectral fragmentation of kaurene and 17-norkauren-16-one derived from it revealed a pattern of deuteriation consistent with direct cyclization of copalyl pyrophosphate (46), Scheme 7. [2-14C]MVA was incorporated into
Scheme 7
kaurene and squalene in a preparation from pea shoots although the enzymic activity was much less than with seeds.lo4 A similar system from Cucurbita p e p formed kaurene, kaurenol, and kaurenoic acid in excellent yield (the proportions depending on the presence of pyridine nucleotides)lo5 and kaurenoic acid was further converted into intermediates on the pathway to gibberellins by a preparation from Fusariurn rnoniliforrne.'06 Kaurene synthetase was purified from the last source'o6 and was shown to mediate two separate processes, conversion of Io3 lo4
lo5
Io6
R. Evans and J. R. Hanson, J.C.S. Perkin I, 1972, 2382. R. C . Coolbaugh, T. C. Moore, S. A. Barlow, and P. R . Ecklund, Phytochemistry, 1973,12, 1613. J . E. Graebe, Proceedings of the 7th International Conference on Plant Growth Substances, 1970, ed. D. J . Carr, Springer, New York, 1972, p. 151. C. A. West and R. R. Fall; ref. 105, p. 133.
268
Terpenoids and Steroids
geranylgeranyl pyrophosphate into copalyl pyrophosphate and of the latter into kaurene. The former step was inhibited by several commercially available inhibitors of terpenoid biosynthesis and both were blocked by limonene derivatives. Some properties of the enzyme from various higher plant sources have been briefly recorded.'" A non-catalytic carrier protein (analogous to sterol carrier protein ;see Section 8) appears to be involved in the metabolism of kaurene in pea extracts. Kaurene formed from [2-14C]MVA in a soluble extract of immature seeds was bound to a large protein and the combination reacted with mixed-function oxidases present in the microsomal fraction to give oxygenated derivatives :lo* this observation accounts for previous reports that soluble extracts from pea could synthesize kaurene but not metabolize it further. Further investigations have been made on gibberellic acid, mainly using the fungus Gibberella fujikuroi. Kaurene (47) prepared enzymically from [ 5 S - S 3H '3MVA was incorporated into this (48) with the retention of 5-pro-S mevalonoid hydrogens, two of which were localized at C-10 and C-11 respectively, the latter in the p-orientation:'09 this contrasts with the incorporation of only two 3 H labels when gibberellic acid was formed from [5R-5-3Hl]MVA. These results support a previous conclusion that contraction to form ring B involves retention of the 5-pro-S mevalonoid atom at C-10. Incorporation of 7p-hydro~y[6P-~H ,17''C]kaur-16-en-19-oic acid (49) into gibberellic acid occurred with loss of the
6p hydrogen, consistent with the above,' l o . but labelled 6P,7/?-dihydroxykaur16-en-19-oic acid was not a precursor. A suggestion that the 6 p hydrogen migrated to C-7 (kaurene numbering) during ring contraction was supported by using [l-3H2,l-'4C]geranyl pyrophosphate as a precursor, which labelled the C-6 position of kaurene. Although gibberellin A, anhydride (50) was incorporated into gibberellic acidinO.l4"/,yield,theparentgibberellinA13(5l)wasnotdetectablysoutilized." This low (for the system) incorporation was accounted for by the instability of (51) in the fermentation medium. The loss of the substituent at C-4a from the C,, lo'
lo9 lo 'I'
R. G. Frost, Diss. Abs. (B), 1973, 33, 4649. T. C. Moore, S. A. Barlow, and R . C. Coolbaugh, Phytochemistry, 1972, 11, 3225. R . G. Evans, J. R . Hanson, and L. J . Mulheirn, J.C.S. Perkin I, 1973, 753. J . R . Hanson, J . Hawker, and A. F. White, J.C.S. Perkin I , 1972, 1892. J . R . Hanson and J . Hawker, Tetrahedron Letters, 1972, 4299.
Biosynthesis of Terpenoids and Steroids
269
molecule may involve formation of an aldehyde group at this site."2 Other transformations of gibberellin in vivo have been reported,' l 3 and the metabolism and biosynthesis of gibberellic acid in germinating barley have been studied.' 1 4 s 1 [17-14C]Kaurene was incorporated into oridonin (52) and enmein (53) in
(44)
OH
HO OH
yields of ca. 0.004 yg in Isodon japonicus without prior degradation.'16 Kaurenoic acid was converted into 7a-hydroxykauren- 19-oic acid in various fungi and the introduction of a 15p- and a 6a-hydroxy-group in the former paralleled similar hydroxylations in higher plants. The hydroxylation of 16-norkauran-19-oic acid at C-13 provides a pathway to steviol (54).l17
CO,H (54)
' l 1
J . R. Bearder and J . Macmillan, Agric. and Biol. Chem. (Japan), 1972,36, 342. R. C. Durley, I. D. Railton, and R . P. Pharis, Phytochemistry, 1973, 12, 1609. R. Nadeau, L. Rappaport, and C. T. Stolp, Planru, 1972,107, 315. G. J. P. Murphy and D. E. Briggs, Phytochemistry, 1973, 12, 1299. T. Fujita, S. Takao, and E. Fujita, J . C . S . Chem. Comm., 1973,434. J. P. Beilby, E. L. Ghisalberti, P. R. Jefferies, M . A. Sefton, and P. M. Sheppard, Tetrahedron Letters, 1973, 2589.
270
Terpenoids and Steroids
Pachydictyol A (55) has been isolated from an alga.' l 8 The perhydroazulene ring is novel for diterpenoids, but as it is well documented in sesquiterpenoids, this may indicate a novel pathway from (56). However, direct cyclization of geranylgeranyl pyrophosphate (57) seems more likely. The cassane diterpenoids
?OPP
\
OH
\
(58) can be hypothetically derived from the pimarane skeleton (59) by a shift of a methyl from C-13 to C-14. No biochemical studies have been made on this system but a model compound (60) could not be induced to rearrange under a variety of R
treatments to give the cassane skeleton.''' In contrast, migration of a C-13 vinylidene group to C-14 in a pimarane derivative has been demonstrated in similar experiments12' that act as analogues for the suggested biogenesis of the clejstanthane skeleton. 'I8
lly
I2O
D. R. Hirschfeld, W. Fenical, G. H. Y. Lin, R. M. Wing, P. Radlick, and J. J. Sims, J . Amer. Chem. SOC.,1973, 95, 4049. J . P. Johnston and K. H . Overton, J.C.S. Perkin I, 1973, 853. G . A. Ellestad, M . P. Kunstmann, andG. 0. Morton, J.C.S. Chem. Comm., 1973, 312.
27 1
Biosynthesis of Terpenoids and Steroids
Incorporation (2 % yield) of the toxic glucoside fusicoccin H (61) into the more highly hydroxylated fusicoccin in the fungus Fusicoccum amygdali without scrambling of tracer suggests a precursor relationship.121 As it seems likely on biogenetic grounds that fusicoccin H is a diterpenoid, the conclusion is that fusicoccin is not a degraded sesterterpenoid, as had been considered possible. The tetramethylpentadecane (62) occurs in shark liver and is probably derived from zooplanktonic metabolism of phytol (63).122
7 Sesterterpenoids The subcellular distribution of enzymes responsible for the formation of ophiobolin F (64) and of lanosterol in the fungus Cochliobolus heterostrophus has been studied. The synthesis of F P P and (64) from MVA occurred in the 105 000 g H
supernatant, whereas the steps from F P P to the triterpenoid were carried out by the 105 OOO g pellet. Both the supernatant and pellet were required for the epoxidation of ~ q u a 1 e n e . lPreliminary ~~ experiments indicated that there may be two kinds of prenyl transferase in the fungus which provide precursors for sesterterpenoids and triterpenoids respectively. The structure of retigeranic acid (65) from a lichen has been established and the biogenetic scheme shown (Scheme 8) has been
’” 12’
12’ 124
K. D. Barrow, D. H. R. Barton, E. Chain, U. F. W. Ohnsorge, and R. P. Sharma, J.C.S. Perkin I , 1973, 1590. R. E. COX,J. R. Maxwell, R. G. Ackman, and S. N . Hooper, Canad. J . Biochem., 1972, 50, 1239. A. Kawaguchi, S. Nozoe, and S. Okuda, Biochim. Biophys. Acra, 1973,296, 615. M. Kaneda, R. Takahashi, Y. Iitaka, and S. Shibata, Tetrahedron Letters, 1972,4609.
Terpenoids and Steroids
272 n
Scheme 8
8 Steroidal Triterpenoids As in previous years this section will deal with the biosynthesis of cholesterol, related steroids, and phytosterols, whereas the following sections will cover the further metabolism of these classes and the remaining triterpenoid systems. 2,3-Oxidosqualene (66) has been isolated from rat liver in vivo after treatment with the known hypocholesterolaemic agent l-dodecylimidazole.'25 This provides another demonstration of the intermediacy of this epoxide on the pathway to sterols. Cyclization of the same precursor with microsomal fractions from Cephalosporum caerulens yielded lanosterol (67) and the diene (68), and the
enzyme systems responsible could be solubilized with deoxycholate.' 2 6 The proof that aglaiol (69), the major triterpenoid of Aglaia ordorata had the (24s) configuration at the C-24,25 epoxy-group [steroid numbering in (70)] supported lZ5 126
S. D. Atkin, B. Morgan, K . H. Baggaley, and J . Green, Biochem. J., 1972,130, 153. A . Kawaguchi, H. Kobayashi, and S. Okuda, Chem. and Pharm. Bull. (Japan), 1973, 21, 577.
273
Biosynthesis of Terpenoids and Steroids
14
the generally accepted (but experimentally unsupported) view that it is the (3s)isomer of 2,3-oxidosqualene that is utilized in biosynthesis, for whether (69) is formed from either (24S)-24,25-epoxidationof an immediate precursor or (22s)22,23-epoxidation of (3S)-2,3-oxidosqualene,both processes are compatible with the conversion of squalene into its (3S)-2,3-oxido-derivativeand provide evidence for the existence of an enzyme system promoting this stereochemical process.127 Time incorporations of tracer from labelled MVA and carbon dioxide into sapogenins' 2 8 and sterols and their derivatives' 29-' 3' have been recorded and conclusions (of varying worth) about biosynthetic schemes and rates of turn-over have been drawn. As expected, pathways to sterols in certain algae resemble those in higher plants rather than in animals.'32 Identification of phytosterols formed from [l-'4C]acetate by Jerusalem artichokes incubated in dark and redlight regimes suggested that 24-methylene- and 24-ethylidene-cholesterol were precursors of sitosterol, campesterol, and stigmasterol and the regulation of the processes was concluded to be phytochrome-mediated.' MVA was converted into 24-methylenecycloartenol (yield 1.2%) by a cellfree preparation from the endosperm of Pinus pinea seeds.'34 Squalene and cycloartenol (71) (yield 32 % and 0.6 %) were also formed, but lanosterol, its
130 131 13'
133 134
R. B. Boar and K. Damps, J.C.S. Chem. Comm., 1973, 115. F. J. Evans and P. S. Cowley, Phytochemistry, 1973, 12, 791. J . M. C. Geuns, Phytochemistry, 1973, 12, 103. P. B. Bush and C. Grunwald, Pfunt Physiology, 1972,51, 110. R. Hardman and F. R. Y . Fazli, Pfunta Med., 1972,21, 188. M. Rohmer and R. D. Brandt, European J . Biochem., 1973,36,446. M. A. Hartmann, P. Benveniste, and F. Durst, Phytochemistry, 1972, 11, 3003. H . C. Malhotra and W. R. Nes, J . Biol. Chem., 1972, 247, 6243.
274
Terpenoids and Steroids
24-methylene derivative, and 4-monomethyl and 4-desmethyl sterols were absent. This confirms that non-photosynthetic tissue from higher plants uses (as does photosynthetic tissue) the cycloartenol route rather than the lanosterol route to sterols, and this work incidentally involved one of the most effective cell-free preparations yet made from higher plants. 24-Methylenelanosterol and 24methylenecycloartenol were also produced in preparations from rat liver and from higher plants. '3 s A sterol carrier protein (SCP) was discovered in 1971 which possesses a great affinity for cholesterol and its water-insoluble precursors and stimulates synthesis of sterols from inactivated (by washing or purification) enzyme systems. SCP from rat liver also increased squalene synthesis in yeast microsomes threefold,I3 although no effect occurred when purified squalene synthetase from the latter source was used.s2 SCP from rat liver microsomes was purified as a protomer (ca. 16 000 dalton) that, in the presence of phospholipids, aggregated to form a larger species (ca. 150 000 dalton) which was responsible for binding sterols,'37 and preparations from other mammalian sources gave essentially similar resUlts.'38-'40 There is controversy about the distribution of the protein : some'39 assert that it occurs widely in extrahepatic cells, others141 that it is localized in the liver and that other carrier proteins are involved in sterol synthesis in other tissues.'42 SCP also binds lipid components of lipoprotein, and as the aminoacid sequence of a particular preparation closely resembled that of the protein moiety of serum low-density lipoprotein (LDL), the suggestion has been made that SCP acts also as the latter moiety and specifies the assembly of LDL itself.I3* Fatty acids are also bound and the factor may be a general lipid carrier.'37 Inhibition of sterol synthesis in various preparations by c - A M P , ' ~a~lipoprotein fraction,'44 and cholesterol esters14' and stimulation by addition of mitochondria (which contain no enzymes implicated in the pathway to sterols)'46 have also been reported. Several significant papers dealing with the later stages in the formation of cholesterol (74) have appeared. The sequence (72)+ (73) -+ (74) involving a NADPH-linked reductase has been suggested to occur in rat liver homogenate. However, incubation of (72) with [4-3H]NADPH under anaerobic conditions led to the incorporation of tracer at C-14a of (73): the proposal that (75) was an
13' 13'
"'
'" 13'
I4O 14'
14' 143 14' '45
14'
C. Anding, R . Heintz, and G. Ourisson, Compt. rend., 1973, 276, D , 205. H. C. Rilliny, Biochem. Biophys. Res. Comm., 1972, 46, 470. M . C . Ritter and M . E. Dempsey, Proc. Nut. Acad. Sci. U . S . A . , 1973,70, 265. T. J . Scallen, M. V . Srikantaiah, H . B. Skralant, and E . Hansbury, F.E.B.S. Letters, 1972, 25, 227. K . E. McCoy, D. F. Kohler, and J . P. Carlson, Fed. Proc., 1973,32, 519 (abs.). T. Calimbras, Fed. Proc., 1973, 32, 519 (abs.). K . L. Garey, Fed. Pror., 1973, 32, 519 (abs.). R. C . Johnson and S . N. Shah, Fed. Proc., 1973,32, 519 (abs.). L. A. Bricker and G. S. Levey, J . Biol. Chem., 1972, 247, 4914. J. A. Watson, Fed. Proc., 1973, 32, 520 (abs.). D. S. Harry, M. Dini, and N. Mclntyre, Biochim. Biophys. Acta, 1973, 296, 209. S. Ranganathan and T. Ramasarma, Biochem. J., 1973, 134, 737.
Biosynthesis of' Terpenoids and Steroids
11V
27 5
H (73)
R
R=H (76) a; R = Me b;R=Et
(75)
intermediate was confirmed by trapping.'47 The two new sterols (76a and b) containing a A14-bond were isolated from Chlorella species grown in a medium containing the drug AY-9944,'489' 49 which was unexpected as the additive had been considered a rather specfic inhibitor for A7-reductases. The conversion of (73) into cholesterol (74) seems nevertheless well established and implies the existence of cholesta-5,7-dien-3P-o1 (77) as an intermediate. A hypothetical route (Scheme9) was tested by determining the incorporation of various epoxides, diols, triols, ketones, etc. with the cholestane skeleton into the steroids produced by 14'
148
14'
M . Akhtar, C. W. Freeman, A. D . Rahimtula, and D . C. Wilton, Biochem. J . , 1972, 129, 225. L. G . Dickson, G . W. Patterson, C. F. Cohen, and S. R. Dutky, Phytochemistry, 1972, 11. 3473. L.'G. Dickson and G. W. Patterson, Lipids, 1972, 7, 6 3 5 .
276
HO
Terpenoids and Steroids
H
HO
H
HO H
J
(73)
(77)
Scheme 9
rat liver homogenates : l 5 0 most were efficiently converted into cholesterol under aerobic conditions but could not be considered obligatory intermediates, as under anaerobic conditions (73) accumulated. Lanosterol and dihydrolanosterol were the main labelled sterols, formed in systems in which synthesis of cholesterol from [2-14C]MVA was inhibited by carbon monoxide, whereas after incubation under nitrogen the 4,4-dimethyl-sterols contained much less tracer and the label mainly resided in 14-demethyl-lanosterol and 14-demethyldihydrolanosterol. No radioactive 4,4-dimethyl-A8('4)-sterolswere detected under either set of conditions. 5 1 Some more details of studies on the reduction of the A24-bond oflanosterol in rat liver systems are availableand cis-addition ofthe two hydrogens is deduced (cf ref. 163).152Speculations as to the role of a 3-oxonium ion, rather than a 3-oxo-steroid, in the decarboxylation of 3P-hydroxycholest-7-ene-4acarboxylic acid (to yield cholesterol) have been made.' 53 Trapping experiments with suspected intermediates have shown that the conversion of episterol (78) into ergosterol (79) in aerobically grown yeast
I5O 15' 15*
A. Fiecchi, M. Kienle, A . Scala, G . Galli, R. Paoletti, and E. G . Paoletti, J. Biol. Chem.,
1972,247, 5898. G. F. Gibbons and K . A . Mitropoulos, Biochem. J., 1973,132,439. M . G. Kienle, R . K. Varma, L. J. Mulheirn, B. Yagen, and E. Caspi, J. Amer. Chem. SOC.,1973, 95, 1996. I. Kim, Daehan Hwahak Hwoejee, 1972,16,257 (Chem. Abs., 1972,77, 149075).
Biosynthesis of Terpenoids and Steroids
277
involves introduction of A22, then A5, and finally reduction of the 24-methylene (80) was derived group, as a main route.' 5 4 Ergosta-5,7,22,24(28)-tetraen-3P-ol from [3H]ergosterol in yeast in very low (ca. 0.05 %) yield, but no reverse conversion could be dete~ted.'~'The novel product (81) from yeast cultures could be derived from the epidioxide (82). 14C-Labelled (82) and related diols, triols, ene-diols, etc. were all incorporated ,in excellent (> 16%) yields into (81) but although the ergostane skeleton was incorporated intact no unambiguous direct pathway could be picked out: rather a grid of multiple pathways was indi~ a t e d . 'Non-artefactual ~~ (82) was detected in the culture media'56 but it has been suggested elsewhere that this is formed by pigment-sensitized photooxidation of ergosterol.' 5 7
Two mechanisms are extant for the introduction of A5 into ergosterol : one involves direct elimination of the hydrogens from C-5 and C-6, and the other requires a hydroxylation-dehydration.Previous tracer work had favoured the former, but evidence for the latter is the incorporation of [28-14C]-(78) into specifically labelled (83) by aerobically growing yeast.lS8 It seems likely that both routes can co-occur or that either can predominate in different conditions. Other studies pertaining to the transformations of ergosterol and its near relatives have been reported.159y160 154
M . Freyberg, A . 0. Oehlschlager, and A. M. Unrau, Biochem. Biophys. Res. Comm., 1972,48, 593.
155 156
Is' 15'
15'
L. Atherton, J . M . Duncan, and S. Safe, J.C.S. Chem. Comm., 1972, 882. J. D. White, D. W. Perkins, and S. 1. Taylor, Bioorg. Chem., 1973, 2, 163. J. Arditti, R. E. M. H. Fish, and B. H. Flick, J.C.S. Chem. Comm., 1973, 1217. M. Freyberg, A. 0: Oehlschlager, and A. M. Unrau, Biochem. Biophys. Res. Comm., 1973, 51, 219. C. G. Anderson, Diss. A h . (B), 1973, 33, 4643. J . R. Lenton, L. J. Goad, and T. W. Goodwin, Phytochemistry, 1973, 12, 1135.
278
Terpenoids and Steroids
One pathway to phytosterols in higher plants requires the opening of the cyclopropyl ring of the precursor skeleton at the stage of either cycloartenol(71), its 24-methylene derivative, or cycloeucalenol (84), all of which are ubiquitous plant products. An enzyme from cultures of bramble so cleaved the last substrate, but was much less effective with the 4,4-dimethyl-~terols.~~~ Demethylation of the latter sterols is known to proceed via loss of the &-methyl group with the 4P-methyl epimerizing into the 4cx-position in the product. The fate of the 4p-hydrogen during the following final demethylation has been ascertained in the conversion of [2,2,4-3H,]obtusifoliol (85), enzymically derived from (84), into poriferasterol(86). About 30 % of the axial 4P-hydrogen was inverted to the 4%-positionin (86), and the low retention may either represent an experimental artefact in the preparation of labelled precursor or be the result of exchange of the C-4 hydrogens (via enolization of a 3-0x0-compound) during demethylation. h 2
(85) ‘‘I
162
(86)
E. Heintz, T. Bimpson, and P. Benveniste, Biochem. Biophys. Res. Comm., 1972, 49, 820. F. F. Knapp, L. 3. Goad, and T. W. Goodwin, J.C.S. Chem. Comm., 1973, 149.
Biosynthesis of Terpenoids and Steroids
279
Studies on the functionalization of the side-chain of sterols continue. One important finding is that a pro4R hydrogen of MVA occupied the pro-24S position in tigogenin (87) biosynthesized by Digitalis lanata. 163 Unless some
unlikely stereochemical change occurred after saturation of the A24-bondof the precursor, this implies that reduction occurred with trans stereospecificity (cf ref. 152). The 27-methylene group of the related convallamarogenin from Conuallama majalis was derived from C-2 of MVA,’ 6 4 i.e. stereospecific oxidation of one of the terminal methyls occurred. Administration of [2-14C, 4R-4-3H,]MVA (3H : 14C = 1 : 1) to the alga Ochrimonas malhamensis gave cycloartenol (3H : 14C = 1 : 1) and poriferasterol (3H : 14C = 3 : 5 ) with the patterns in (88) and ( 89)165that are consistent with a hydride shift from C-24 to C-25 duringalkyla-
represents 14C; T represents 3 H
tion of the side-chain. [Me-2H3]Methionine was incorporated into poriferasterol to give four atoms of tracer per molecule and this implicated a 24-ethylidene intermediate (90) and was consistent with the previously proposed scheme, Such a scheme had not been experimentally demonstrated in Scheme higher plants, and feeding Larix decidua with the same doubly labelled precursor and measuring the isotope ratio in sitosterol (91) and 28-isofucosterol(92) indicates that although a A24(25)-intermediatemay have been involved, a shift of lh3
h4 Ih5
L. Canonica, F. Ronchetti, and G . Russo, J.C.S. Chem. Comm., 1972, 1309. F. Ronchetti and G . Russo, J.C.S. Chem. Comm., 1973, 184. A. R. H . Smith, L. J . Goad, and T . W. Goodwin, Phytochemistry, 1972,11, 2775.
Terpenoids and Steroids
280
L
f Scheme 10
r-
/
hydrogen from C-24 to C-25 did not now occur.166 Studies on a Trebouxia algal species using [Me-2H,]methionine and labelled phytosterols as precursors suggested that side-chain alkylation to form poriferasterol and related compounds could involve a A25-intermediateas outlined in Scheme 1 l.'67 The A24(28)-bond
Scheme I1 J. Randall, H. H. Rees, and T. W. Goodwin, J.C.S. Chem. Comm., 1972, 1295. ' ' P. L. J . Goad, F. F. Knapp, J . R. Lenton, and T. W. Goodwin, Biochem. J., 1972,129,2 19.
Ih6
Biosynthesis of Terpenoids and Steroids
28 1
of 24-methylenecholesterol was not reduced in uivo as in liver preparations from rats, although the A24(25)-~onds of other sterols were reduced. Hence rats lack the appropriate reductase that is widely distributed in plants. 16' Many phytophagous and omnivorous insects can dealkylate C28 and C29 sterols to obtain cholesterol. The hydrogen at C-25 of 28-isofucosterol was retained during such a process in a mealworm species, and the route in Scheme 12
Scheme 12
(93)
168
,'
(94)
:-I:::: (95)
W. R.Nes, J . W. Cannon, N. S . Thampi, and P. A. G. Malya, J . Biol. Chem., 1973,248,
484.
Terpenoids and Steroids
282
has been ~uggested."~This process is basically the reverse of the route of biosynthesis of the phytosterol, but a more complex situation obtained in a silkworm. Here fucosterol-24,28-epoxide(95)was detected as a possible intermediate in the degradation of sitosterol (93) and this, fucosterol (94), and 24-methylenecholesterol (97) were converted into cholesterol in yields of 15, 10, and 3",/, respectively. Fucosterol and desmosterol(96) were known from previous work to be intermediates en roule from sitosterol to cholesterol and the route shown in Scheme 13 was deduced in which the direct dealkylation route was of minor importance.' 'O 9 Further Metabolism of Steroids
An interesting review' ' discusses the implications from recent work which indicates that certain phytosterols that have been considered secondary metabolites actually may play some fundamental physiological role. This conclusion is based on analyses of complex mixtures which enable non-random metabolic processes (i.e. those that can selectively involve particular components of the mixture) to be identified and assigned either as specifically co-ordinated reactions or as involving metabolic grids. The nature and significanceof these non-random processes is, however, quite obscure. Previous work had indicated that tumour-bearing rats formed 'phytosterols' as judged by incorporation of deuterium from [,Me-2H,]methionine into the components of a certain fraction that was absent in controls. More rigorous studies have disproved this ; the 'phytosterol' fraction was shown to be mainly cholesterol, and incorporation of deuterium into this occurred in both experimental animals and in controls, presumably as a consequence of degradation of the p r e c ~ r s o r . ' ~ ~ Much, mainly inconclusive, work has been concerned with the degradation of the cholesterol side-chain and modification of the resulting products. Although it is established that such degradation to form bile acids commences with C-26-hydroxylation, the stage at which this occurs was not clear. It is now claimed' 7 3 that 5,!?-cholestane-3a,7cr,12cr-trio1 and 5~-cholestane-3a,7a-diolare the main substrates for C-26-hydroxylases of rat liver microsomes and mitochondria and minor pathways involving other substrates have been outlined. On the other hand, the intermediacy of 26-hydroxycholestero1 on the pathway from cholesterol to lithocholic acid in rat liver mitochondria has been proposed on the basis of tracer incorporation and the effects of added quantities of cold 24-hydroxycholestero1on the specific activities of products formed from labelled Ih"
'" I"
'" ".'
P. J . Randall, J . G . Lloyd-Jones, I . F. Cook, H . H . Rees, and T. W . Goodwin, J.C.S. Chem. Comm., 1972, 1296. M. Morisaki, H. Ohtaka, M. Okubayashi, N. Ibekawa, Y . Horie, and S. Nakasone, J.C.S. Chem. Comm., 1972, 1275. B. A . Knight, Chem. in Britain, 1,973, 9, 106. J . G. Lloyd-Jones, P. Heidel, B. Yagen, P. J. Doyle, G. H. Friedell, and E. Caspi, J . Biol. Chem., 1972, 247, 6347. I . Bjorkhem and J. Gustafsson, European J . Biochem., 1973,36, 201.
Biosynthesis o j Terpenoids and Steroids
283
precursors ;l 7 4 alternatively 7a-hydroxylation of cholesterol has been considered as a rate-limiting step for the sequence.' 7 5 The cholesterol-7a-hydroxylaseof rat liver has been purified and shown to be a typical mixed-function oxidase with requirement for NADPH, cytochrome C-reductase, and cytochrome P45O.I 6 , Sterol carrier protein (see Section 8) from liver also stimulated cleavage of the side-chain of cholesterol to form steroid hormones'88 and bile acids.'79 A similar, closely related factor that is either a protein or is associated with one occurs in bovine adrenal mitochondria and increased the formation of pregnenolone up to ten-fold. This factor was heat-stable and remained active after heating had inactivated the cytochrome P450 enzyme system: it was thought to be implicated in the transport of cholesterol within the cell as well as in the cleavage reaction and it may act as a solubilizing agent by the formation of cholesterolphospholipid micelles.' A similar factor isolated from serum proteins stimulated the formation of progesterone and deoxycortisone by solubilized enzymes from adrenal microsomes.'8 ' [4-14C,17a-3H]Pregnen~lone (98) yielded progesterone (99), 17-hydroxyprogesterone, 17-hydroxypregnenolone, and testosterone (100) on incubation 799180
(98)
(99) R (100) R
= =
COMe OH
erepresents 14C
with minced ovarian tissue.'82 The 3H tracer was retained at C-17 in (99) but the other compounds were devoid of this activity and it was concluded that testosterone was formed along established pathways involving C-17-hydroxylation of C , , precursors rather than by direct insertion of oxygen between C-17 K. A. Mitropoulos, M. D . Avery, N . B. Myant, and G . F. Gibbons, Biochem. J . , 1972, 130, 363. S. Balasubramaniam, K. A. Mitropoulos, and N. B. Myant, European J . Biochem., 1973, 34, 77. l i 6 G. S. Boyd, A. M. Grimwade, and M. E. Lawson, European J. Biochem., 1973,37,334. '" J. Robinson and P. M. Stevenson, F.E.B.S. Letters, 1972, 23, 327. K . W. Kan and F. Ungar, J. Bioi. Chem., 1973, 248, 2868. G . A. Grabowski, M. E. Dempsey, and R . F. Hanson, Fed. Proc., 1973,32, 520 (abs.). K . W. Kan, F. Ungar, J . C. Y. Hsiao, R. Gunville, and D. T. Maghane, Fed. Proc., 1973, 32, 519 (abs.). M. A. Hamilton, R. W. McCune, and S. Roberts, J . Endocrinoi., 1972,54, 297. l E 2 L. Milewich and L. R. Axelrod, Arch. Biochem. Biophys., 1972,153, 188. lid
Terpenoids and Steroids
284
and C-20. A number of other studies of the metabolism of pregnenolone, testosterone, and their derivatives in animals and plants have a ~ p e a r e d . ' ~ ~ - ' ~ ~ A large number of studies have also been made on enzymic modifications to the steroid skeleton, although many are fragmentary, some follow well-trodden pathways, and others are inconclusive. However, recent work on ecdysterone (101 ; R = OH), an insect moulting hormone, certainly falls into none of these classes, for, contrary to the situation in animals and some plants, it was demonstrated that oxidation of C-3 is not obligatory for formation of the Cis-A-B ring junction. Administration of [4-'4C,3-3H]cholesterol to seedlings of Tuxus baccatu yielded [14C]ecdysterone containing at least 70% of the 3H tracer retained at C-3.' 9 7 The presumed precursor( 101 ;R = H) was sequentially hydroxylated at the time of puparium formation in a blow-fly species to give ecdy~ t e r 0 n e . I In ~ ~another double-labelling study it was demonstrated that the hydrogens at C-2 of MVA were incorporated with retention of configuration at C-22 of fusidic acid (102), and so the intermediate formation of a product with a
R
183 1n 4
I85 I86 187 188
I89 190 191 1Y2
1q.3
I94
19s 196
197 198
S. J . Stohs and M. M. El-Olemy, Phytochemistry, 1972, 11, 2409. T. Furuya, K. Kawaguchi, and M. Hirotani, Phytochemistry, 1973, 12, 1621. B. P. Lisboa, H. Brewer, and E. Witschi, Z . physiol. Chem., 1972, 353, 1907. B. P. Lisboa, and J. C. Plasse, Steroids Lipids Res., 1972, 3, 142. P. V. Maynard and E. H . D. Cameron, Biochem. J., 1973, 132, 283. W. R. Mayle, Y . C. Kong, and J. Ramachandran, J . Biol. Chem., 1973, 248, 2409. H. J . Lee and C . Monder, Fed. Proc., 1973, 32, 479 (abs.). W. C . Schwarzel, W. C. Krugyel, and H. J . Brodie, Endocrinology, 1973, 92, 866. T. Tabei and W. L. Heinrichs, Endocrinology, 1972, 91, 969. G. J. Van der Vusse, M . L. Kalkman, a n d H. J . Van der Molen, Biochim. Biophys. Acta, 1973, 297. 173. T. A. Van der Hoeven, Diss. Abs. ( B ) . 1972, 33, 1934. T. Katkov, W . D. Booth, and D. B. Gower, Biochim. Biophys. Acta, 1972, 270, 546. I . H . White and J. Jeffery, Biochim. Biophys. Acta, 1973, 296, 604. H . Oshima, K . Ochiai, N. Niizato, and A . Tamaki, Biochim. Biophys. Acta, 1973, 306, 227. J . G. Lloyd-Jones, H. H. Rees, and T . W. Goodwin, Phytochcmistry, 1973, 12, 569. M. N . Galbraith, D. H. S. Horn, E. J. Middleton, and J . A. Thomson, J . C . S . Chem. Comm., 1973, 203.
285
Biosynthesis of Terpenoids and Steroids
A20(22)-bondwas excluded. 199 When digitoxigenin and digoxigenin were formed in Digitalis lanata from [8-3H,4-14C]cholesterol,all the tritium was retained : this could indicate that neither A7-, A8-,nor A8(14)-intermediate~ were involved in cardenolide biosynthesis2" if the assumption is made that no migration of hydrogen occurred during this process. The route from cholesterol (103) to 5a-cholest-7-en-3P-01 (105) in mammals has been shown to involve cholest-4-en-3-one (106). A similar pathway was claimed to exist in two species of starfish from the observation that (104)and (105) together with (103) were labelled when [4-I4C]-(106) was fed, and from the loss of most of the 3H tracer when (104) was biosynthesized from [4-'4C,3a-3Hl]-(103): this was consistent with the route shown in Scheme 14.''' (106) is also thought to be an intermediate in the conversion of cholesterol into its 5P-epimer by intestinal micro-organisms, and the 3-0x0-A4-steroid SP-reductase of such a system has been partially purified and its mode of action investigated.202
(105)
Scheme 14
Several studies of the enzyme systems involved in these and related reactions have appeared. The 3-oxo-A4-steroid 5P-reductase of yeast transfers hydrogen from the A-position of NADPH to the SP-position of the steroid whereas the corresponding 5a-reductase involves hydride shift from the B-position of the coenzyme.203 The reduction of the 3-0x0-group of various steroids by cortisone reductase always resulted in transfer from the B-position of NADH whether the final product was 3a- or 3/l-hydro~ylated,2~'and this same hydrogen was involved when the same enzyme reduced the 20-0x0-group of pregn-4-ene-3,20lg9
201
2oz 203 204
E. Caspi, R. C. Ebersole, W. 0. Godtfredsen, and S . Vangedal, J.C.S. Chem. Comm., 1972, 1191. D. J . Aberhart, J . G. Lloyd-Jones, and E. Caspi, Phytochemistry, 1973,12, 1065. A . G . Smith, R . Goodfellow, and L. J. Goad, Biochem. J . , 1972,128, 1371. I. Bjorkhem, J . Gustafsson, and 0. Wrange, European J. Biochem., 1973, 37, 143. Y. J. Abul-Hajj, Steroids, 1972, 20, 215. W. Gibb and J. Jeffery, European J. Biochem., 1973,34, 395.
286
Terpenoids and Steroids
dione. This presumably means that the reductase can only bind NADH in such a manner that the 4B-hydrogen is available for transfer. Several different 3a- and 3p-hydroxy-steroid dehydrogenases in rat liver also showed this same stereospecificity towards NADH.205 The properties of other A4- and Asreductases,206-’ O8 As -+A3-keto-steroid is om erase^,^^^.^'^ 17p- and 7ahydroxy-steroid dehydrogenases,2 l 4 7a-hydroxylases,’ and other modih ave been studied, as have products of metabolism of fying enzymes2 6-2 steroids with mixed enzyme systems in v i t r ~ . ’ ~ ~ - ’ ~ ’ Evidence for the intermediate formation of hydroperoxides in the 17a-hydroxylation of progesterone has been obtained.228 Cytochrome P450 acted as a microsomal peroxidase in rat adrenal and as this was inhibited by several steroids this system may contribute to the regulation of hydroperoxide decomposition. Pregnan-l7a-hydroperoxidewaz also implicated in the formation of other adrenocortico-hormones.229~230 Preparations of rat liver microsomes converted various neutral 16-dehydro-C19-steroids into 16,17-dihydroxy metabolites, possibly uia a 16,17-epoxide,again with the involvement of cytochrome P450.23 Conversion of taurodeoxycholic acid into cholic acid via 7a-hydroxylation in rat
’-’
I . Bjorkhem, H. Danielsson, and K. Wikvall, European J . Biochem., 1973,36, 8. H. J . Eyssen, G. G. Parmentier, F. C. Compernolle, G . DePauw, and M. PiessensDenef, European J . Biochem., 1973,36, 41 1. ’Oi I . Bjorkhem and I . Holmberg, European J . Biochem., 1973, 33, 364. * 0 8 P. Germain, G . Lefebvre, B. Bena, and R. Gay, Compt. rend. Soc. Biol., 1972, 166, 1123. 2 ” y J . B. Jones and K. D. Gordon, Biochemistry, 1973, 12, 71. 2 1 0 R. J. Martyr m d W. F. Benisek, Biochemistry, 1973, 12, 2172. R. Ghraf, M . Raible, and H . Schriefers, Z . physiol. Chem., 1973, 354, 299. * I 2 J . J. O’Rangers, Diss. Abs. ( B ) , 1972, 33, 1004. 2 L 3 M. A. Chernyavskava, G . M. Segal, and I . V. Torgov, Izuest. Akad. Nauk S . S . S . R . , Ser. biol., 1972, 588 (Chem. Abs., 1972, 77, 123298). ‘I5 I . A. MacDonald, C. N. Williams, and D. E. Mahony, Biochim. Biophys. Acta, 1973, 309, 243. 2 1 5 D. Mayer, F. W. Koss, and A. Glasenapp, Z . physiol. Chem., 1972, 353,921. ‘I6 P. Geynet, J . Gallay, and A. Alfsen, European J . Biochem., 1972,31, 464. ’ ’ W. Voigt and S. L. Hsia, J . Biol. Chem., 1973, 248, 4280. 2 1 8 J. W. Chu and K . Kimura, J . Biol. Chem., 1973,248, 5183. 2 1 q C. Monder and P. T. Wang, J . Steroid Biochem., 1973, 4, 153. 2 2 0 L. A. R. Sallam, A. H . El-Refai, S. Nada, and A. F. Abdel-Fattah, J . Gen. Appl. Microbiol., 1973, 19, 155. *” J . Ramseyer and B. W. Harding, Biochim. Biophys, Acta, 1973, 315, 306. 222 M . Maugra, P. Savigny, and J. LeMatre, Compt. rend., 1973, 276, D,3221. * 1 3 A . S. Goldman and K. Sheth, Biochim. Biophys. Acta, 1973, 315, 233. 2 2 4 J. Van Cantfort, Life Sci., 1972, 11, 773. 2 2 5 1. Bjorkhem, K. Einarssonn, J. A. Gustafsson, and A. Somell, Acta Endocrin., 1972,71, 569. 2 2 6 W. E. Braselton, J . C. Orr, and L. L. Engel, Analyt. Biochem., 1973,53, 64. 227 R . Tschesche and J. Leinert, Phytochemistry, 1973,12, 1619. 2 2 8 E. Hrycay and P. J. O’Brien, Arch. Biochem. Biophys., 1972, 158, 480. *” E. Hrycay, P. J. O’Brien, J . E. Vahnier, and G . Kan, Arch. Biochem. Biophys., 1972, 153, 495. 2 3 0 E. Hrycay and P. J. O’Brien, Arch. Biochem. Biophys., 1973, 157, 7. 231 C. von Bahr, K. Brandt, and J . A. Gustafsson, F.E.B.S. Letters, 1972,25, 65. ’05 20h
287
Biosynthesis o j Terpenoids and Steroids
liver microsomes was also deduced to involve cytochrome P450, and the kinetics of the process were a n a l y ~ e d . ~ ~ ~ Contrary to previous reports, the vitamin D, -C-25-hydroxylase of the chick is not restricted to the liver, but also occurs in the kidney and intestine: neither is it strongly subject to product inhibition, nor affected by the vitamin D status of the bird.233 Since the kidney appears to be the unique site of C-25-hydroxyvitamin D,-1-hydroxylase, renal homogenates are capable of completely converting vitamin D, into its 1,25-dihydroxy-derivative,which is the hormonal form. The latter enzyme is affected by the vitamin D and calcium status and may be involved in the regulation of the hormonal form. All the oxygen utilized in la-hydroxylation of 25-hydroxy-vitamin D, by chick kidney arose from the air, not from water : consequently the enzyme is a mixed-function o x i d a ~ e . ~ , ~ An attempt has been made to elucidate the stereochemistry at C-1 of wortmannin (107), a metabolite of Peniciflium wortmannii, by feeding [2-’4C,2S-3HI]0
MVA and its (2R)-isomer to the fungal cultures. Although incorporations of 1 4 % were achieved, the results were not conclusive as substantial and unexplained loss of tritium (relative to I4C) occurred from both precursors. Nevertheless, the greater retention of tritium from the (2R)-precursor suggested a stereochemistry (as shown) opposite to that deduced from crystallographic st~dies.’~~.~~~ 10 Non-steroidal Triterpenoids
A full account of the cyclase from Ononis spinosa root that converted 2(3),22(23)dioxidosqualene into a-onocerin ( 108) has appeared.23 The properties differed in several respects from those of other oxidosqualene c y ~ I a s e s and ~ ~ *the enzyme may be the first truly soluble enzyme of the class ; consequently differences in susceptibility to inhibitors may be due to differences in structural organization
’
232
233 234 235
236 237
238
D. Trulzsch, H. Greirn, P. Czygan, F. Hutterer, F. Schattner, H . Popper, D. Y . Cooper, and 0. Rosenthal, Biochemistry, 1973, 12, 76. G . Tucker, R. E. Gagnon, and M. R. Haussler, Arch. Biochem. Biophys., 1973,155,47. J. C. Ghazarian, H. K. Schnoes, and H . F. DeLuca, Biochemistry, 1973, 12,2555. J . MacMillan, T. J. Simpson, and S. K. Yeboah, J . C . S . Chem. Comm., 1972, 1063. T. J. Petcher, H . P. Weber, andZ. Kis, J.C.S. Chem. Comm., 1972, 1061. M. G. Rowan and P. D. G. Dean, Phytochemistry, 1972,11, 31 1 1 . P. D. G . Dean, Steroidologia, 1972, 2, 143.
288
Terpenoids and Steroids
rather than in mode of action. The cyclase required both epoxide groups of the substrate to effect cyclization and there was some evidence for an intermediate product in which only one 'end' of the substrate was cyclized. Under conditions where 2,3-oxidosqualene was converted into P-amyrin (109) in 13 yield, an homogenate of pea tissue converted (1 10) into the same It seems very compound in 0.0060~yield without randomization of the unlikely that (110) is a mandatory intermediate for P-amyrin, but the result ()/;
illustrates the ability of the cyclase(s) to generate a normal product from an artificial substrate via a short-cut process which reduces its function to the formation of only three rings. Although tissue cultures of Tylophora indica had lost their ability to produce alkaloids they could synthesize P-amyrin and phyto~terols.~~' A novel product, lup-20(29)-ene-3P,llg-diol (111) was synthesized de nouo in the shrub Dodonea ~ t t e n u a t a .Pentacyclic ~~~ triterpenoids of this type are very rare in bacteria but (112)was formed from [l-'4C]acetate and [2-I4C]MVA in Bacillus acidocaldarius : however, no degradations to locate tracer were carried 11 Carotenoids
Earlier work on the stereochemistry of elimination during double-bond formation in carotenoids was hampered by loss of stereospecificity of label during the 13'
140
2J2
M. Horan, J . P. McCormick, and D. Arigoni, J.C.S. Chem. Comm.,1973, 73. B. D . Benjamin and N . B. Mulchandani, Planta Med., 1 9 7 3 , 2 3 , 394. E. L. Ghisalberti, P. R. Jefferies, and M. A. Sefton, Phytochemistry, 1973, 12, 1125. M. DeRosa, A . Gambacorta, L. Minale, and J . D . Bu'lock, Phytochemistry, 1973, 12, 1 1 17.
Biosynthesis of Terpenoids and Steroids
289
&
HO
long incubation times that were necessary. This has been overcome by employing a preparation from Flauobacterium capable of efficiently synthesizing lycopene, ruboxanthin, zeaxanthin, and others without randomization of tracer.243 The hydrogens lost from C-7, C-11, C-7', and C-11' of phytoene during its conversion into the unsaturated carotenoids arose from the 5-pro-R hydrogen of MVA, whereas those from C-8, C-12, C-8', and C-12' came from the 2-pro-S hydrogen. This corresponds to trans-elimination in the formation of each double bond. The inhibition of carotenogenesis has received considerable attention. Rhodopseudomonas species grown in the presence of nicotine accumulated neurosporene (113) at the expense of spheroidene (116) and hydroxyspheroidene (117): on removal of the inhibitor, the pool of (1 13) rapidly diminished and (116) and (117) were formed.244 These results were held to provide evidence for the pathway in Scheme 15 involving chloroxanthin (114) and demethylated spheroidene (1 15). R2
(113) R' = a , R 2
=
L. L. G.
e
h...
(114) (115) R' R 1 = b, c, R2 R2 = = ee
(116) R' (117) R '
= =
d, R2 d,R2
= =
e f
a
b
..
:
d
C
.*.-
-:.
(113)-
(114)-
(115)-
(116)-
(117)
e
f
OH
Scheme 15 243 244
J. C. B. McDermott, G. Britton, and T. W. Goodwin, Biochem. J . , 1973,134, 1 1 15. R. K . Singh, A . Ben-Aziz, G. Britton, and T. W. Goodwin, Biochem. J . , 1973, 132, 649.
290
Terpenoids and Steroids
Nicotine also inhibited cyclization of carotenoid glucoside esters in Myxococcus fulcus, and the acyclic ester that accumulated underwent monocyclization on removal of the alkaloid from the culture medium. Inhibition of hydroxylation at C-1' occurred at higher concentrations of nicotine,245and 2-(4-chlorophenylthio)triethylamine h y d r o ~ h l o r i d e , ~ ~ ~amongst - ~ ~ * other corn pound^,^^^-^^ also prevented cyclization of acyclic carotenoids :in all these experiments lycopene and its derivatives accumulated, and removal of inhibitor caused cyclization. Carotenogenesis was completely inhibited in other micro-organisms by substituted benzophenones,2" and diphenylamine stopped the oxidation of hydroxygroups to carbonyl.2'2 It is generally accepted that hydroxylation occurs at a late stage in carotenoid biosynthesis ; however, the isolation of 3-hydroxy-Pzeacarotene from the green alga Scenedesmus obliquus indicates that cyclization and hydroxylation can occur at the neurosporene level of d e s a t ~ r a t i o n . ' ~ ~ Several mutants of tomatoes are known in which the carotenoid content of the fruit is markedly altered from that in the standard varieties in that lycopene or 6, b, and 5-carotenes are present. Use of I4C-labelled IPP, phytoene, and lycopene as additives for soluble enzyme systems revealed some unexpected enzymic activities : systems from all mutants had synthetase activity for phytoene and for the formation of p-carotene from lycopene. The apparent lack of some of these activities in the various fruits may be due to the presence of specific inhibitor^.^'^ Other studies of the formation of carotenoids in bacteria, algae, and other organisms are a ~ a i l a b l e . ~ The " ~ ~fossil ~ ~ spore-wall components sporapollenins, which are polymers of units such as (1 18) and (119),appear to be formed by
L4h 247
248
H. Kleinig and H. Reichenbach. Biochim. Biophys. Acta, 1973, 306, 249. P. P. Batra, R . M. Gleason, and J . W. Louda, Phytochemistry, 1973,12, 1309. W. J . Hsu, H . Yokoyama, and C. W. Coggins, Phytochemistry, 1972, 11, 2985. M . Elahi, T. H. Lee, K . L. Simpson, and C. 0.Chichester, Phytochemistry, 1973, 12, 1633.
249 250 251
'
253
M . Elahi, C. 0. Chichester, and K. L. Simpson, Phytochemistry, 1973, 12, 1627. N. F. Lewis and U . S. Kumta, F.E.B.S.Letters, 1973, 30, 144. R . Herber, B. Maudinas, and J. Villoutreix, Phytochemistry, 1972, 31, 3461. 0. G. Sassu, Phytochemistry, 1972, I I , 3 195. F. J. Levenberger, A. J. Schocher, G. Britton, and T. W. Goodwin, F.E.B.S. Letters, 1973, 33, 205.
254
255
15'
C. Papastephanou, F. J . Barnes, A. V. Briedis, and J . W. Porter, Arch. Biochem. Biophys., 1973, 157, 415. G. N. Lutsenko and V . S. Saakov, Fiziol. Biokhim. Kul't. Rast., 1972, 4, 608 (Chem. Abs., 1973, 79, 50 769). M . B. Gochnauer, S. C. Kushwaha, M. Kates, and D. J . Kushner, Arch. Mikrobiol., 1972, 84, 339 (Chem. A h . , 1972, 7 7 , 149 484).
Biosynthesis of Terpenoids and Steroids
29 1
with in vitro model oxidative polymerization of c a r ~ t e n o i d s . ~Experiments ~ systems support the hypothesis that damascones [e.g. P-damascone (1 20)] are derived in vivo by degradation of c a r ~ t e n o i d s . ~ ~ ~ . ~ ’ ~
Carotene 15,15’-dioxygenase, which oxidizes carotene to retinal, has been purified 200-fold from rabbit intestine preparations and the kinetics and parameters of the enzyme have been reported.260 12 Meroterpenoids
This convenient term (Cornforth, 1968) covers compounds of partly terpenoid origin. Only topics in which the biosynthesis of the terpenoid moiety is involved are included. Mycophenolic acid ( I 21) has a terpenoid side-chain. Isolation of 6-farnesyl-5,7dihydroxy-4-methylphthalide from the culture medium of Penicillium brezlicompactum and the high incorporation of this into (121) indicate that the most important route for the formation of the side-chain is the introduction of a C I S chain followed by oxidative degradation at the appropriate double bond.26
The stereochemistry of the A-B ring junction in a group of phytotoxic diterpenoid alkaloids occurring in Aconitum and Delphinium species is similar to that in gibberellins. This suggested that these compounds might inhibit the action or biosynthesis of gibberellic acid but a detailed study showed that although the alkaloids inhibited certain gibberellin-mediated responses (a-amylase synthesis and stem-node elongation) they did not consistently mimic the effect of either abscisic acid or the drug AMO-1618 (known inhibitors of gibberellin action and 257 256
259
260 261
G . Shaw, Proceedings of the Symposium on Sporapollenins, 1971 (publ. 1972), p. 305. G. Ohloff, V. Rautenstrauch, and K. H. Schulte-Elte, Helv. Chim. Acta, 1973, 56, 1503. S. h o e , S. Katsumura, a n d T . Sakan, Helv. Chim. Acta, 1973,56, 1514. M. R. Lakshmanan, H. Chansang, and J . A. Olson, J . Lipid Res., 1972,13,477. L. Canonica, W. Kroszczynski, B. M. Ranzi, B. Rindone, E. Santaniello, and C. Scolastico, J . C . S . Pirkin I , 1972, 2639.
292
Terpenoids and Steroids
biosynthesis respectively). Effects of the alkaloid on gibberellin transport or on uptake by target cells seemed more likely.262 Feeding experiments with [2-14C]-,[5-14C]-,and [4-3H,]-MVA have shown263 that daphniphylline (122) is formed in Daphniphyllurn macropodurn from six MVA molecules oia a squalene-like intermediate, and [14C]squalene was incorporated in low (O.OOSO,,) yield. Degradation made it possible to suggest a biosynthetic pathway. Partial degradation of daphniiactone-B (l24), biosynthesized in fruit of Daphniphyllunz teijsmunni, indicated that four MVA moieties were incorporated per molecule and a plausible route was suggested whereby (123) was an intermediate which was degraded with loss of eight carbon atoms to yield the product.’ h 4
Tomatine [a glucoside of tomatidine (1 25)] was formed in excised tomato root cultures but addition of MVA or the steroid inhibitor SKF-7997-A3 caused
(1Xj 262 263
264
R . H. Laurence and G. R. Waller, Fed. Proc., 1973, 32, 521 (abs.). K . T. Suzuki, S. Okudo, H . Niwa, M . Toda, Y . Hirata, and S. Yamamura, Tetrahedron Letters, 1973, 799. H. Niwa, Y .Hirata, K. T. Suzuki, and S. Yamamura, Tetrahedron Letters, 1973, 2129.
Biosynthesis of Terpenoids and Steroids
293
reduction in the rate of its synthesis and also of root Tomatine was broken down by tomato fruits to form (126) in a combined (probably glucoside)
H
from,266although tomatidine was metabolized by Nocardia restrictus such that 174-dien-3-oneswere formed and the side-chain was left intact.267 [2-14C]MVA was reported to be incorporated intact by Claviceps purpurea into the ergoline moiety of ergotamine.268 Feeding experiments with (5R)-and (5S)-[5-3H,]MVA have confirmed (see Vol. 2, p. 198) that elymoclavine, agroclavine, and chanoclavines are all formed in Claviceps strains with loss of pro-5R and retention of pro-5S hydrogen of m e ~ a l o n a t e . ~ ~Thermodynamic ' considerations of the biosynthesis of the ergot alkaloids are available.270 The enzyme from Aspergillus oirnstelodarni that prenylates cyclo-L-alanyl-Ltryptophanyl(l27) to yield (128)has been partially p ~ r i f i e d . ~The ~ ' ,product ~ ~ ~ is believed to be an intermediate on the pathway to echinulin (129)and its [3H , 14C]0.
(127) R' = RZ = H (128) R' = H , R 2 = CMe,CH=CH, (129) R' = CH,CH=CMe,, RZ = CMe,CH=CH,
labelled form was incorporated into the latter by the mould in 5-14% yield. Echinulin was not degraded to establish the tracer pattern but the preservation of the isotope ratio of precursor in the product implied that intact uptake had 265
266
'" 268 269
270 27'
'"
J. G . Roddick and D. N. Butcher, Phytochemistry, 1972, 11, 2991. E. Heftmann and S. Schwimmer, Phytochemistry, 1972, 11, 2783. I. Belic and H. Sock, J . Steroid Biochem., 1972, 31, 843. R. A. Bassett, E. B. Chain, and K. Corbett, Biochem. J., 1973,134, I . C. I. Abou-Chaar, H. F. Guenther, M. F. Manuel, J . E. Robbers, and H . G . Floss, LIoydia, 1972, 35, 272. Z. Rehacek, P. Sajdl, and A. Kremen, Biotechnol. Bioeng., 1973, 15, 207. C. M. Allen, Biochemistry, 1972, 11, 2154. C. M. Allen, J . Amer. Chem. SOC.,1973, 95, 2386.
294
Terpenoids and Steroids
occurred. The pathways from p-hydroxybenzoate to the ubiquinones in bacteria are well established, where the first step is the formation of 4-carboxy-2-polyprenylphenol. In higher plants the situation is less certain but the appropriate transferase activity has been demonstrated in preparations from bean root and yeast. Such cell-free systems synthesized ( I 30) from p-hydroxybenzoate and either CO,H I
I PP or protein-bound polyprenyl pyrophosphate. The mitochondria contained all of the transferase activity but were unable to construct the side-chain from IPP, possibly owing to damage of the organelles during p r e p a r a t i ~ n . ~ ’ ~ The transferase from E . Coli catalysing coupling of DMAPP to RNA to form a N6-(A2-isopentenyljadenosinelink in the latter has been purified 500-fold, and was found only to accept substrate that lacked the isoprenyl modification normally present in ~ i u o The . ~ enzyme ~ ~ had a molecular weight of 55000 dalton and required reduced SH groups and bivalent metal ions for full Labelled farnesol, geranylgeraniol, dolichols, and ubiquones were isolated after administration of [2-14C]MVA to aerated cultures of the fungus Phytophthoru cactorum whereas the last were not formed in the absence of aeration. Studies with [4R-4-3H,]MVA and its (4s)-isomer gave the stereochemistry of hydrogen loss expected from previous work on these classes of compound: formation of trans-A-bonds in all products except the dolichols resulted from stereospecific loss of the pro-4S hydrogen, whereas the latter contained several cis-A-linkages formed with the loss of the epimeric hydrogen.276 Transmethylation from [Me-2H3]methioninehas been demonstrated into isoprenoid quinones (ubiquinones, rhodoquinones, phytoquinones, etc.) of Euglena g r a ~ i l i s and ,~~~ a pathway for the biosynthesis of ubiquinones in E . coli has been proposed based on genetic analysis and isolation of intermediates (such as 2-octaprenyl-6methoxyphenol and 2-octaprenylphenol) that accumulated in mutants with blocked pathways.278 The proposal that cardiachromes, a novel group of quinones [e.g.(131j], arise by condensation of a benzenoid precursor with geranyl
zi3 2’4
275 276
”’ 278
G. Thomas and D. R.Threlfall, Biochem. J., 1973, 134, 81 1 . N . Rosenbaum and M. L. Gefter, J. Biol. Chem., 1972,247, 5675. J. K . Bartz and D. Soll, Biochemie, 1972, 54, 31. J . B. Richards and F. W. Hemming, Biochem. J., 1972, 128, 1345. D. R. Threlfall, Biochim. Biophys. Acta, 1972, 280, 472. I. G . Young, P. Stroobant, C. G. MacDonald, and F. Gibson, J. Bacteriol., 1973, 114, 42.
Biosynthesis of Terpenoids and Steroids
29 5
0
pyrophosphate and oxidative cyclization is supported by the isolation of alliodorin (132) from heart wood of Cordia alliodora.279~280 OH
Other oxygenated meroterpenoids are cochlioquinones A and B from Cochliobolus miyabeanus, which are formed by the introduction of a C unit [rings A, B, and c in (133)] into an aromatic precursor.281 A group of interesting furanoterpenoids with C, , C,, , C,, , and C, linear chains or truncated C, and C, chains occurs in marine sponges.282 Linear C Z 1compounds closely related to the C,, class occur in the same source and this suggests that the former are degraded sesterterpenoid~.~
,
The role of long-chain isoprenols in the formation of peptidoglycan of bacterial cell walls has been investigated284and reviewed.28 27q
280
281
282
283 284
285
K. L. Stevens, L. Jurd, and G. Manners, Tetrahedron Letters, 1973, 2955. M. Moir, R. H. Thomson, B. M. Hausen, and M. H. Simatupang, J.C.S. Chem. Comm., 1972, 363. L. Canonica, B. M. Ranzi, B. Rindole, A. Scala, and C. Scolastico, J.C.S. Chem. Comm., 1973,213. G. Cimino, S. De Stefano, L. Minale, and E. Trivellone, Tetrahedron, 1972,28,4761. G. Cimino, S. De Stefano, and L. Minale, Tetrahedron, 1972, 28, 5983. J. L. Strominger, Y. Higashi, H. Sandermann, K. J. Stone, and E. Willoughby, Proceedings of the Symposium on the Biochemistry of the Glucosidic Linkage, 1971, ed. R. Piras and H. G. Portis, Academic Press, New York, p. 135. J. Baddiley, ref. 284, p. 337.
296
Terpenoids and Steroids
13 Polyterpenoids Derivatives of C, , - i ~ o p r e n o are l ~ ~of~ importance as lipid-bound intermediates that carry activated sugar fragments for the biosynthesis of peptidoglycan of bacterial cell walls. Kinetic analyses had revealed that the activity of CSsisoprenol phosphokinase, a butanol-soluble enzyme involved in this process, could be manifested if the aqueous interface of mixed micelles consisted of the lecithin cofactor, the prenol, and the apoprotein of the enzyme. Preparation of such a complex and addition of ATP indeed generated full enzymic The phosphokinase was isolated from Staphylococcus aureus288 and the C-55isoprenyl phosphate ester itself accumulated in Micrococcus lysodeikticus cells that had been treated with bacitracin before harvesting.289 The dephosphorylating enzyme for the substrate was located in the membrane of the latter bacteria290and purified.291 Reviews of this work are a ~ a i l a b l e . ~ ~ ~ ~ ~ ~ ’ 14 Methods
Useful methods for the preparation of [1-3H]DMAPP,292[1-’4C]-2,3-oxido~ q u a l e n e and , ~ ~[’~4C]gibberellic have appeared. A timely warning has been given concerning the widespread tendency to assume chemical and radiochemical purity of compounds isolated by g.1.c. or t.1.c. after feeding of 14C-labelled precursors to plant systems, and using the apparent incorporations to construct biosynthetic schemes. For example, camphor was isolated by g.1.c. from various plant species with apparently very significant radioactivity after feeding [2-14C]MVAbut the tracer content decreased to zero when the product was repeatedly re~rystallized.~~ Similar observations have been made on several other occasions (but have been ignored by the majority of workers in the field) in studies involving the isolation of a wide range of terpenoids. It seems that background activity is spread over practically the whole range of the g.1. or t.1. chromatograin in products obtained from both in vivo and in vitro systems. The simplest explanation is probably that highly radioactive products (polyols, epoxides?) are formed (perhaps by salvage mechanisms) on feeding a non-physiological excess of the precursor and break down during the process of separation to give very highly labelled moieties which contaminate any fractions that are collected. Another factor that may introduce artefacts is the much higher labelling of squalene’ (or sesquiterpenoids and carotenoids) than of monoterpenoids biosynthesized by the same in vivo systems. Thus contamination of the monoterpenoid fraction by components of these classes 286
J. N . Umbreit and J. L. Strominger, J . Bucteriol., 1972, 112, 1306.
’” H . Sandermann, F.E.B.S. Letters, 1973, 29, 256. 288 289
290 291 292 293
294
H . Sandermann and J. L. Strominger, J . Biol. Chem., 1972, 247, 5123. K. L. Stone and J. L. Strominger, J . Biol. Chem., 1972, 247, 5107. E. Willoughby, Y. Highasi, and J . L. Strominger, J . Biol. Chem., 1972, 247, 5113. R. Goldman and J. L. Strominger, J . Biol. Chem., 1972, 247, 5116. E. Cardemil and 0. Cori, J . Labelled Compounds., 1973, 9, 15. J . Bascoul, D. Nikolaidis, A. Crastes de Paulet, and L. Pichat, Bull. SOC.chim. France I I , 1973, 2318. J. R. Hanson and J. Hawker, Phytochemistry, 1973, 12, 1073.
Biosynthesis of Terpenoids and Steroids
297
may cause spuriously high incorporation of tracer, and the widespread practice (especially in studies using cell-free systems) of assaying the tracer in the 'hexanesoluble' fraction and considering this fraction equivalent to the tracer in monoterpenoids is absolutely unjustified. An excellent review of the methods (and the difficulties) involved in the study of biosynthesis of terpenoids in higher plants is available295and an interesting discussion (applied to the incorporation of amino-acids into alkaloids but applicable to the field under consideration) concerns the use of D- and L-precursors as metabolites;296it is suggested that the efficienciesof incorporation of each isomer are not per se necessarily suitable measures as to which isomer is metabolized, and the incorporation of doubly labelled enantiomorphs is favoured. A magisterial essay on the logic of working with enzymes has a ~ p e a r e d . ~ ' A study of the incorporation of late intermediates into the meroterpenoid strychnine has application in a wider field. It was shown that although insignificant incorporation of tetra- and hexa-cyclic intermediates occurred in 5 days after administration to Strychnos nux vornica, feeding, repotting, and assaying after 100 days led to 0.2--1.6 incorporation of the presumed intermediate without significant degradation : 2 9 7 incorporations were also detected at shorter times by autoradiographic t.1.c. of the plant extracts. Mevalonate was little catabolized to carbon dioxide (< 0.05 %) after administration for 160 h to various plants where de nouo synthesis of monoterpenoids from exogenous precursors had occurred. Geraniol and C, precursors derived from MVA were degraded to only a small extent (ca. 3 %) whereas acetate and 3'3-dimethylacrylate were extensively (ca. 20 %) broken down.64 An approach to chemical phylogeny of plants based not on the isolation of individual components but upon branching of pathways from particular precursors has been outlined.298and biogenetic speculations based on variations of the monoterpenoid composition and content of various organs during development have been made.299 Reviews and articles on instrumental methods in biosynthetic st~dies,~" the application of 13Cn.m.r.301 and 'H n.m.r.302 to such problems, counting the techniques for I4C and 3H,303-308 general methods of inve~tigation,~'~ solubilization of membrane-enzymes and other aspects of their p~rification,~
''
295 296 297 298
299 '0° 301 302 '03 '04
305 '06
307
308 '09
310 3"
S. A. Brown and L. R. Wetter, Progr. Phytochem., 1972,3, 1 . E. Leistner, R . N. Gupta, and I. D. Spenser, J. Amer. Chem. SOC., 1973,95,4040. S. I. Heimberger and A. I. Scott, J.C.S. Chem. Comm., 1973, 217. A. J . Birch, Pure Appl. Chem., 1973, 33, 17. Y . A. Poltavchenko and G . A. Rudakov, Biol. Nauki, 1972,15,95. H. G . Floss, Lloydia, 1972, 35, 399. J. B. Grutzner, Lloydia, 1972, 35, 375. L. J. Mulheirn, Tetrahedron Letters, 1973, 3175. R. Tykva, Coll. Czech. Chem. Comm., 1973,38, 503. R. M . McKenzie and R. K. Gholson, Analyt. Biochem., 1973,54, 17. R. L. Boeckx, D. J. Protti, and K . Dakshinamurti, Analyt. Biochem., 1973,53, 491. L. Csernay, Acta Med. Acad. Sci. Hung., 1973, 29, 131. W. E. Braselton, J. C. Orr, and L. L. Engel, Analyt. Biochem., 1973,53, 64. A. G . Lacko, H. L. Rutenberg, and L. A. Soloff, Clin. Chim. Acta, 1972,39, 506. J. R. Quayle, Methods Microbiol., 1972, 6B,157. J. L. Gaylor, Adv. Lipid Res., 1972, 10, 89. A. Szewczuk, Wiad. Chem., 1973, 27, 289 (Chem. Abs., 1973,79, 62 972).
298
Terpenoids and Steroids
tissue-culture technique^,^', and the extraction of enzymes from plants in the presence of endogenous phenols3 have appeared.
15 Reviews This section contains a list of reviews that have been published in the year August 1972 to August 1973 (Part A) concerning terpenoid and steroid biosynthesis, and details of the more important reviews in the field published since 1967 (Part B). A. Reviews Published August 1972-August
1973
Reviews are available on : General. The biogenetic isoprene rule ;314 the stereochemical aspects of enzyme action ;3 1 , 3 1 5 the stereochemistry of biogenetic-type cyclizations of olefins ;316 chemistry and biosynthesis of insect attractant^,^'^ terpenoid pheremones and hormones ;318,319the C,--C,, terpenoids ; 3 2 0 the triterpenoids, steroids, and car~tenoids.~~' Methods. General methods employed in the determination of biosynthetic pathways ; 2 9 5 * 3 2 2 , 3 2 3 for details of specific methods applicable see Section 14. Acyclic Precursors. The properties and kinetics of I P P - i s ~ m e r a s e . ~ ~ ~ Monoterpenoids. The chemistry and biosynthesis, in part, of thujane and its derivat i ~ e s . ~ Sesquiterpenoids. Biosynthetic pathways to t ~ t i n and ~ ' ~abscsiic acid."' Diterpenoids. Biosynthesis and chemistry of the cyclic d i t e r p e n ~ i d sand ~ ~the ~.~~~
gibber ell in^.^ 3 0
G. Teuscher, Pharmazie, 1973, 28, 6. G. A. Buzan, Uspeki, biol. Khim., 1972, 13, 102. '14 L. Ruzicka, Ann. Rec. Biochem., 1973, 42, 7. 'I5 J. W. Cornforth, in 'Biosynthesis and its Control in Plants', ed. B. V. Milborrow, Academic Press, 1973, p. 17 1. ' I h K. E. Harding, Bioorg. Chem., 1973, 2, 248 (36 references). 3 1 ' D. A. Evans and C. L. Green, Chem. SOC.Rev., 1973, 2 , 75 (72 references). 3 1 8 H. Z. Levinson, Naturwiss., 1972, 59, 477 (126 references). '19 J. G. MacConnell and R. M. Silverstein, Angew. Chem. Internat. Edn., 1973, 12, 644 (208 references). 32 J. R. Hanson, in 'Biosynthesis', ed. T. A. Geissman (Specialist Periodical Reports), The Chemical Society, London, 1972, Vol. 1, p. 41 (104 references). 32 H. H. Rees and T. W. Goodwin, ref. 320, p. 59 (302 references). 3 2 2 S. A. Brown, ref. 320, p. 1 (162 references). 3 2 3 E. G . Paoletti, A n n . Isr. Super. Sanira., 1972, 8, 244. 3 2 4 P. W. Hollaway in 'The Enzymes-, ed. P. D. Boyer, 3rd. edn., Academic Press, 1972, p. 565. j 2 ' D. Whittaker and D. V. Banthorpe, Chem. Rev., 1972, 72, 305 (1 15 references). 3 2 6 G. Jommi, Cron. Chim., 1972, 20. j Z 7 D. Gross, Pharmazie, 1972, 27, 619. 3 2 8 J. R. Hanson, Fortschr. Chem. org. Naturstofle, 1971, 29, 395 (64 references). 3 2 9 J. R. Hanson, Progr. Phytochem., 1972, 3, 231 (214 references). 3 3 0 C. A. West in ref. 315, p. 143. 312
'13
Biosynthesis oj. Terpenoids and Steroids
299
Triterpenoids and Steroids. Biosynthesis of cholesterol and enzyme systems associated therewith;331- 3 3 3 biosynthesis of plant sterols;334*335 steroidal sapogenins and pentacyclic triterpenoid sapogenins ; 3 3 6 , 3 3 7 the biosynthesis of steroids in fungi;338,339 the properties of As-3-keto-steroid i~omerase.~~’ Carotenoids. Recent advances in the study of the biosynthesis of carotenoids ;341,342 studies of the biosynthesis of carotenoids in micro-organisms in genera1,343,344 in b a ~ t e r i a , ~and ~ ’ ,in~ ~ ~ Meroterpenoids. Biosynthesis of various terpenoid alkaloids ;348-3 biosynthesis of ~biquinones.~ ”, 3 5 2 Two books have appeared which include aspects of these t o p i ~ s . ~ ’ ~ , ~ ~ ~
B. Reviews Published 1967-1972 Amongst the numerous reviews available are : General. The alkylation of olefins in biosynthe~is~~’ and the biosynthesis of insect hormones.3s6 Reviews of the biosynthesis of the various classes of terpenoids are to be found in the Proceedings of conferences held under the auspices of the Phytochemical and Biochemical S~cieties.~ 5 9
”-’
331 332
333 334 335 336
337
338 339 340 341
342
343 344
345 346
347 348 349
350 351
352 3s3 354
355
356 357
”
359
J. W. Cornforth, Labo-Pharma-Probl. Tech., 1972, 20, 51. R. B. Ramsey, Biochem. SOC.Trans., 1973, 1, 341 (58 references). J. L. Gaylor and C. V. Delwiche, Ann. New York Acad. Sci., 1973, 212, 122. L. J. Goad and T. W. Goodwin, Progr. Phytochem., 1972,3, 113 (448 references). L. J. Mulheirn and P. J. Ramm, Chem. SOC.Rev., 1972, 1, 259 (99 references). K. Takeda, Progr. Phytochem., 1972, 3, 287 (147 references). R. Tshesche and G. Wulff, Fortschr. Chem. org. Naturstofle, 1973,30, 461. J. D. Weete, Phytochemistry, 1973, 12, 1843 (181 references). J. D. Bu’Lock, Pure Appl. Chem., 1973, 34, 435 (77 references). P. Talalay and A. M.Benson, ref. 9, p. 591. T. W. Goodwin, Biochem. J., 1972, 128, 11P (7 references). T. W. Goodwin, in ‘Phytochemistry’, ed. L. P. Miller, Van Nostrand-Reinhold, New York, 1973, Vol. 1, p. 112. S. Liaaen-Jensen, Ann. Rev. Microbiol., 1972, 26, 225 (1 56 references). E. P. Feofilova, Uspekhi Mikrobiol., 1972, 8, 159. J. C. B. McDermott, A. Ben-Aziz, R. K. Singh, G. Britton, and T. W. Goodwin, Pure Appl. Chem., 1973, 35, 29 (48 references). T. W. Goodwin, ‘Proceedings of the 2nd International Congress on Photosynthesis’, ed. G . Forti, Junk N.V., The Hague, 1971, Vol. 3, p. 2437. B. H. Davies, Pure Appl. Chem., 1973, 35, 1 (69 references). E. Leete, ref. 320, p. 158 (103 references). R. Thomas and R. A. Bassett, Progr. Phytochem., 1972, 3, 47 (215 references). R. Gabelta, Fitoterapia, 1973, 44,3. F. Gibson, Biochem. SOC. Trans., 1973, 1, 317 (20 references). W. Yamamoto, Farumashia, 1973, 9, 29. H. Metzner, ‘Biochemie der Pflanzen’, Enke, Stuttgart, 1973, 376 pp. N. M. Packter, ‘Biosynthesis of Acetate-derived Compounds’, Wiley, Chichester, 1973, 203 pp. J. W. Cornforth, Angew. Chem. Internat. Edn., 1968, 7, 903 (19 references). C. E. Berkoff, Quart. Rev., 1969, 23, 372 (115 references). ‘Perspectives in Phytochemistry’, ed. J. B. Harborne and T. Swain, Academic Press, 1969. ‘Aspects of Terpenoid Chemistry and Biochemistry’, ed. T. W. Goodwin, Academic Press, 1971. ‘Natural Substances Formed Biologically from Mevalonic Acid., ed. T. W. Goodwin, Academic Press, 1970.
300
Terpenoids and Steroids
Methods. The preparation of biosynthetic intermediates and of enzyme systems of terpenoid biosynthesis ; 3 6 0 the use of asymmetrically labelled and multiply labelled substrates in enzyme studies ; 3 6 1 ? 3 6 2techniques of plant tissue culture.363 Monoterpenoids. The biosynthesis of r n o n o t e r p e n o i d ~ . ~ ~ ~ Sesquiterpenoids. Biosynthetic pathways to sesq~iterpenoids.~~ Diterpenoids. The biosynthesis of the gibber ell in^.^^^ Triterpenoids and Steroids. The biosynthesis of limonoids and quassinoids ;36 the chemistry of sterol and acylic terpenoid epoxides ; 3 6 8 biosynthesis and biochemistry of sterol^,^^^-^^* pregnane derivatives,373 steroidal oestrogens, adrenal corticosteroids ;376376 bile Carotenoids. Biosynthesis of carotenoids and vitamin A.378 Meroterpenoids. Terpene alkaloid biosynthesis ; 3 7 9 , 3 8 0 biosynthesis of vitamins E and K ;381 the distribution, function, and biosynthesis of p h y t o q ~ i n o n e s . ~ ~ ~ Other Terpenoids. The biosynthesis of r ~ b b e r8 .3 ~
36" 361 362 363 364
365 366 367
368
369 370 371 372
373 374
375
376
378 379
380 381 382
383
'Methods in Enzymology', ed. R. B. Clayton, Academic Press, 1969, Vol. 15. J. W. Cornforth, Quart. Reu., 1969, 23, 125 (26 references). J . R. Hanson, Ado. Steroid Biochem. Pharm., 1970, 1, 51 (82 references). E. J. Staba, Adv. Steroid Biochem. Phurm., 1970, 1,75 (274 references). D. V. Banthorpe, B. V. Charlwood, and M. J. 0. Francis, Chem. Retl., 1972, 72, 115 (537 references). W. Parker, J. S. Roberts, and R. Ramage, Quart. Rev., 1967, 21, 331 (203 references). B. E. Cross, Progr. Phytochem., 1968, 1, 195 (78 references). J. D. Connolly, K. H. Overton, and J. Polonsky, Progr. Phytochem., 1970, (146 references). E. E. van Tamelen, Accounts Chem. Res., 1968, 1, 1 1 1 (37 references). I. D. Frantz and G . J. Schroepfer, Ann. Rev. Biochem., 1967,36, 691 (181 references). J. L. Gaylor, Adv. Lipid Res., 1972, 10, 89 (128 references). E. Lederer, Quart. Reu., 1969, 23, 453 (1 18 references). C. J. Sih and H. W. Whitlock, Ann. Rev. Biochem., 1968, 37, 661 (196 references). S. Burstein and M. Gut, Adv. Lipid Res., 1871, 9, 291 (302 references). P. Morand and J. Lyell, Chem. Rev., 1968, 68, 85 (472 references). B. W. Harding, J. J. Bell, L. D. Wilson, and J. A. Whysner, Adu. Enzyme Regulut., 1969, 7, 237 (51 references). E. Griffiths and E. H. D. Cameron, Adu. Steroid Biochem. Pharm., 1970, 2, 223 (132 references). 'Bile Acids', ed. P. P. Nair, Plenum, New York, 197 1 . 'Carotenoids', ed. 0. Isler, Birkhaeuser, Basel, 1971. A. 1. Scott, Accounts Chem. Res., 1970, 3, 151 (33 references). E. Leete, Adv. Enzymol., 1969, 32, 373 (279 references). D. R. Threlfall, Vitamins and Hormones, 1971, 29, 153 (213 references). J. C. Wallwork and F. L. Crane, Progr. Phytochem., 1970, 2, 267 (198 references). B. L. Archer and B. G . Audley, Ado. Enzymol., 1967, 29, 221 (130 references).
Reviews on Terpenoid Chemistry
The following list of reviews on terpenoid chemistry covers the period 1968-1973 and is arranged according to the chapter titles of Part 1. 1 Monoterpenoids
General. A. R. Pinder, ‘The Chemistry of the Terpenes’, Wiley, New York, 1970. T. K. Devon and A. I. Scott, ‘Handbook of Naturally Occurring Compounds’, Vol. 2, ‘Terpenes’, Academic Press, New York, 1971, p. 3. D. Whittaker, in ‘Chemistry of Terpenes and Terpenoids’, ed. A. A. Newman, Academic Press, London, 1972, p. 11. A. F. Thomas, in ‘The Total Synthesis of Natural Products’, ed. J. W. ApSimon, Wiley, New York, 1973, Vol. 2, p. 1. J. Verghese, ‘Chemistry ofa-Terpinene’, Flavour Ind., 1972,3,252 (109 references). D. Whittaker and D. V. Banthorpe, ‘The Chemistry of Thujane Derivatives’, Chem. Reo., 1972, 72,305 (115 references). W. Cocker, ‘A Review of Some Investigations of the Chemistry of Carene’, J . Soc. Cosmetic Chemists, 1971, 22, 249 (63 references). N. E. Bean, ‘Camphora-Curriculum Vitae of a Perverse Terpene’, Chem. in Britain, 1972, 8, 386. Iridoids. J. M. Bobbitt and K.-P. Segebarth, in ‘Cyclopentanoid Terpene Derivatives’, ed. W. I. Taylor and A. R. Battersby, Marcel Dekker, New York, 1970, p. 1. G. W. K. Cavill, in ‘Cyclopentanoid Terpene Derivatives’, ed. W. I. Taylor and A. R. Battersby, Marcel Dekker, New York, 1970, p. 203. V. Plouvier and J. Favre-Bonvin, ‘Les Iridoides et Seco-iridoides : Repartition, Structure, Proprietes, Biosynthese’, Phytochernistry, 1971, 10, 1697 (317 references). H. Inouye, in ‘1st International Congress on Pharmacognosy and Phytochemistry’, ed. H. Wagner and L. Horhammer, Springer-Verglag, Berlin, 1971, p. 290. R. Hegnauer, ‘Pflanzenstoffe und Pflanzensystematik’, Naturwiss., 1971, 58, 585 (88 references).
30 1
302
Terpenoids and Steroids
P. W. Thies, in ‘1st International Congress on Pharmacognosy and Phytochemistry’, ed. H. Wagner and L. Horhammer, Springer-Verlag, Berlin, 1971, p. 41 (iridoid alkaloids). Cannabinoids. R. Mechoulam, ‘Marihuana Chemistry’, Science, 1970,168, 1 159 (87 references). T. Petrzilka, ‘Chemistry of Synthetic Hashish Derivatives’, Bull. Schweiz. Akad. Med. Wiss., 1971, 27, 22 (20 references). C . R. B. Joyce and S. H. Curry, ‘Botany and Chemistry of Cannabis’, Churchill, London, 1970. H. G. Pars and R. K. Razdan, ’Tetrahydrocannabinol and Synthetic Analogs’, Ann. New York Acad. Sci., 1971, 191, 15 (22 references). R. K. Razdan, in ‘Progress in Organic Chemistry’, ed. W. Carruthers and J. K. Sutherland, Butterworths, London, 1973, Vol. 8. W. D. M. Paton and J. Crown, ‘Cannabis and its Derivatives’, Oxford University Press, London, 1972. R. Mechoulam, ‘Marijuana Chemistry, Pharmacology, Metabolism, and Clinical Effects’, Academic Press, New York, 1973. Biogenesis. D. V. Banthorpe, B. V. Charlwood, and M. J. 0 . Francis, ‘The Biosynthesis of Monoterpenes’, Chem. Rev., 1972, 72, 115 (527 references). W. W. Epstein and C. D. Poulter, ‘A Survey of Some Irregular Monoterpenes and their Biogenetic Analogies to Presqualene Alcohol’, Phytochemistry, 1973, 12, 737 (30 references). Mass Spectrometry. C. R. Enzell, R. A. Appleton, and I. Wahlberg, in ‘Biochemical Applications of Mass Spectrometry’, Wiley, New York, 1972, p. 351. Photochemistry. P. G. Sammes, ‘Photochemical Reactions in Natural Product Synthesis’, Quart. Rev., 1970, 24, 37 (120 references). M. Pfau. ‘Photochemistry in the Field of Monoterpenes and Related Compounds’, Flavour Ind., 1972, 3, 89 (87 references). Perfumes and Flavours. Y.-R. Naves, ‘Progress in the Synthesis of Perfumes Based on Pinenes’, Rum. Chem. Rev., 1968, 37, 779 (specifically pinenes) (1 19 references). G. Ohloff, ‘Chemistry of Odoriferous and Flavouring Substances’, Fortschr. chem. Forsch., 1969, 12, 185 (418 references). Resolution. P. H. Boyle, ‘Methods of Optical Resolution’, Quart. Rev., 1971, 25, 323 (132 references).
Reviews on Terpenoid Chemistry
303
2 Sesquiterpenoids General. J. S. Roberts, in ‘Chemistry of Terpenes and Terpenoids’, ed. A. A. Newman, Academic Press, New York, 1972, p. 88. R. Bryant, ‘Mono- and Sesqui-terpenoids’, Interscience, New York, 1969. T. K. Devon and A. 1. Scott, ‘Handbook of Naturally Occurring Compounds’, Vol. 2, ‘Terpenes’, Academic Press, New York, 1971, p. 55. Sesquiterpenoid Lactones. J. Romo, ‘Recent Studies on Sesquiterpenes’, Pure Appl. Chem., 1970, 21, 123 (33 references). T. A. Geissman and M. A. Irwin, ‘Chemical Contributions to Taxonomy and Phylogeny in the Genus Artemisia’, Pure Appl. Chem., 1970, 21, 167 (54 references). S. M. Kupchan, ‘Recent Advances in the Chemistry of Terpenoid Tumor Inhibitors’, Pure Appl. Chem., 1970, 21, 227 (24 references). F. Sorm, ‘Advances in Terpene Chemistry’, Pure Appl. Chem., 1970, 21, 263 (20 references). S. C. Bhattacharyya, ‘Some Interesting Sesquiterpene Lactones’, J. Indian Chem. SOC.,1970, 47, 299 (19 references). W. Herz, ‘Recent Advances in Phytochemistry’, ed. T. J. Mabry, North-Holland Publishing Co., Amsterdam, 1968, Vol. 1, p. 229. W. Herz, G. Anderson, S. Gibaja, and D. Raulais, ‘Sesquiterpene Lactones of Some Ambrosia Species’, Phytochemistry, 1969, 8, 877 (12 references). Sesquiterpenoid Ethers. K. Takeda, ‘Sesquiterpenes Having a Five-membered Ether-ring in the Molecule’, Pure Appl. Chem., 1970, 21, 181 (26 references). Insect Juvenile Hormone. B. M. Trost, ‘The Juvenile Hormone of Hyalophora cecropia’, Accounts Chem. Res., 1970, 3, 120 (29 references). Y. S . Tsizin and A. A. Drabkina, ‘The Juvenile Hormone of Insects and its Analogues’, Russ. Chem. Rev., 1970, 39, 498 (134 references). Biosynthesis. J. R. Hanson, ‘Biosynthesis of Terpenoid Compounds: C,-C,, Compounds’, in ‘Biosynthesis’, ed. T. A. Geissman (Specialist Periodical Reports), The Chemical Society, London, Vol. 1, 1972, p. 41 (104 references); Vol. 2, 1973, p. 1 (77 references). G. P. Moss, ‘The Biogenesis of Terpenoid Essential Oils’, J. SOC.Cosmetic Chemists, 1971, 22, 231 (67 references). Synthesis. C. H. Heathcock, in ‘Total Synthesis of Natural Products’, ed. J. W. ApSimon, Wiley, New York, 1973, Vol. 2, p. 197.
304
Terpenoids and Steroid
S. E. Danishefsky and S. Danishefsky, ‘Progress in Total Synthesis’, AppletonCentury-Crofts, Meredith Corporation, New York, 1971, Vol. 1, p. 1 10.
3 Diterpenoids General. R. McCrindle and K. H. Overton, in ‘Rodd’s Chemistry of Carbon Compounds’, 2nd Edn., Elsevier, Amsterdam, 1969, Vol. IIC, Ch. 14. E. Fujita, ‘The Chemistry on Diterpenoids in 1966’, Bull. Inst. Chem. Res., Kyoto Univ., 1967, 45, 229 (181 references); ‘The Chemistry on Diterpenoids 1967’, ibid., 1969, 47, 522 (180 references); ‘The Chemistry on Diterpenoids in 1968’, ibid., 1970,48, 11 1 (200 references); ‘The Chemistry on Diterpenoids in 1969’, ibid., 1970, 48, 294 (220 references). J. R. Hanson, in ‘Chemistry of Terpenes and Terpenoids’, ed. A. A. Newman, Academic Press, New York, 1972. T. Devon and A. I. Scott, ‘Handbook of Naturally Occurring Compounds’, Vol. 2, ‘Terpenes’, Academic Press, New York, 1972, p. 185. Nomenclature. J. W. Rowe, ‘The Common and Systematic Nomenclature of Cyclic Diterpenes’, U.S. Dept. of Agriculture, Forest Products Laboratory, Madison, Wisconsin, 1968. Bicyclic Diterpenoids. J. R. Hanson, ‘The Bicyclic Diterpenes’, Progr. Phytochem., 1972, 3, 231 (214 references). Resin A cids. D. F. Zinkel, L. C. Zank, and M. F. Wesolowski, ‘Diterpene Resin Acids’ (a compilation of i.r., m.s., n.m.r., and U.V.spectra), U.S. Dept. of Agriculture, Forest Products Laboratory, Madison, Wisconsin, 1971. Tetracy clic Diterpeno ids. J. R. Hanson, ‘The Chemistry of the Tetracyclic Diterpenes’, Pergamon Press, Oxford, 1968. J. R. Hanson, ‘Recent Advances in the Chemistry of the Tetracyclic Diterpenes’, Progr. Phytochem., 1968, 1, 161 (1 27 references).
Gibberellins. B. E. Cross, ‘Biosynthesis of the Gibberellins’, Progr. Phytochem., 1968, 1, 195 (78 references). A. Lang, ‘Gibberellins: Structure and Metabolism’, Ann. Rev. Plant Physiol., 1970, 21, 537 (205 references). C. A. West, M. Oster, D. Robinson, F. Lew, and P. Murphy, ‘Biochemistry and Physiology of Plant Growth Substances’, ed. F. Wightman and G. Setterfield, Runge Press, Toronto, 1970, p. 313. J. MacMillan, in ‘Advances in the Chemistry and Biochemistry of Terpenoid Substances’, ed. T. W. Goodwin, Academic Press, London, 1971, p. 153.
Reviews on Terpenoid Chemistry
305
J. MacMillan, in ‘Phytochemistry’, ed. L. P. Miller, Reinhold, New York, 1971, Ch. 6. Diterpene Alkaloids. S. W. Pelletier and K. H. Lawrence, in ‘Chemistry of the Alkaloids’, ed. S. W. Pelletier, van Nostrand-Reinhold, New York, 1970. Biosyn thesis. J. R. Hanson and B. Achlladelis, ‘The Biosynthesis of the Diterpenes’, Perfumery and Essential Oil Record, 1968, 59, 802 (31 references). J. R. Hanson, ‘The Biosynthesis of the Diterpenes’, Fortschr. Chem. org. Naturstofle, l971,29, 395 (64 references). Chemotaxonomy. R. C. Cambie and R. J. Weston, ‘Chemotaxonomy of the New Zealand Podocarpaceae’, Chem. in New Zealand ( J . New Zealand Inst. Chem.), 1968, 32, 105. T. Norin, ‘Some Aspects of the Chemistry of the Order Pinales’, Phytochemistry, 1972, 11, 1231 (69 references).
4 Sesterterpenoids L. Canonica and A. Fiecchi, ‘Structure and Biosynthesis of Ophiobolins’, Rec. Progr. Org. Biol. Med. Chem., 1970, 2, 51.
5 Triterpenoids General. T. K. Devon and A. I. Scott, ‘Handbook of Naturally Occurring Compounds,’ Vol. 2, ‘Terpenes’, Academic Press, New York, 1972, p. 281. M. J. Kulshreshtha, D. K. Kulshreshtha, and R. P. Rastogi, ‘The Triterpenoids’, Phytochemistry, 1972, 11, 2369 (217 references). J. D. Connolly and K. H. Overton, in ‘Chemistry of Terpenes and Terpenoids’, ed. A. A. Newman, Academic Press, London, 1972, p. 207. Limonoids and Quassinoids. D. L. Dreyer, ‘Limonoid Bitter Principles’, Fortschr. Chem. org. Naturstofle, 1968, 26, 190 (127 references). J. D. Connolly, K. H. Overton, and J. Polonsky, ‘The Chemistry and Biochemistry of the Limonoids and Quassinoids’, Progr. Phytochem., 1970, 2, 385 (146 references). J. Polonsky, ‘Quassinoid Bitter Principles’, Fortschr. Chem. org. Naturstofe, 1973, 30, 101 (82 references). Cucurbitacins. D. Lavie and E. Glotter, ‘The Cucurbitanes: a Group of Tetracyclic Triterpenes’, Fortschr. Chem. org. Naturstofe, 1971, 29, 307 (136 references).
306
Terpenoids and Steroids
Holothurinogenins. E. Premuzic, ‘Chemistry of Natural Products Derived from Marine Sources,’ Fortschr. Chem. org. Nuturstofle, 1971, 29, 417 (338 references). J. S. Grossert, ‘Natural Products from Echinoderms’, Chem. SOC.Rev., 1972, 1, 1 (98 references). Triterpenoids in Mineral Sources. P. Albrecht and G. Ourisson, ‘Biogenetic Substances in Sediments and Fossils’, Angew. Chem. Znternat. Edn., 1971, 10, 209 (114 references). E. V. Whitehead, ‘Chemical Clues to Petroleum Origin’, Chem. and Ind., 1971, 1 1 16 ( I reference). J. R. Maxwell, C. T. Pillinger, and G. Eglinton, ‘Organic Geochemistry’, Quart. Rev., 1971 , 25, 57 1 (296 references).
6 Carotenoids and Polyterpenoids Caro tenoids-Gene ra 1.
J. B. Davis, ‘The Carotenoid Group’, in ‘Rodd’s Chemistry of Carbon Compounds’, Vol. IIB, ed. S. Coffey, Elsevier, Amsterdam, 1968, pp. 231-346 (ca. 900 references). C. Bodea, ‘Cyclization Reactions of Carotenoids’, Pure Appl. Chem., 1969, 20, 5 17 (16 references). B. H. Davies, ‘Structural Studies on Bacterial Carotenoids and their Biosynthetic Implications’, Pure Appl. Chem., 1969, 20, 545 ( I 8 references). S. Liaaen-Jensen, ‘Selected Examples of Structure Determination of Natural Carotenoids’, Pure Appl. Chern., 1969, 20, 421 (96 references). B. C. L. Weedon, ‘Some Recent Advances in the Synthesis of Carotenoids’, Pure Appl. Chem., 1969, 20, 531 (42 references). T. W. Goodwin and L. J. Goad, ‘Carotenoids and Triterpenoids’, in ‘Biochemistry of Fruits and their Products’, ed. A. C. Hulme, Academic Press, London, 1970, pp. 305-368 (404 references). S. Liaaen-Jensen, ‘Developments in the Carotenoid Field’, Experientia, 1970, 26, 697 (55 references). B. C. L. Weedon, ‘Allenic and Acetylenic Carotenoids’, Rev. Pure Appl. Chern. (Australia), 1970. 20, 51 (106 references). G. Britton and T. W. Goodwin, ‘Biosynthesisof Carotenoids’, Methods Enzymol., 1971, 18C. 654 (101 references). T. W. Goodwin. ‘Algal Carotenoids’, in ‘Aspects of Terpenoid Chemistry and Biochemistry’, ed. T. W. Goodwin, Academic Press, London, 1971, pp. 315-356 (175 references). ‘Carotenoids’, ed. 0. Isler, Birkhauser, Basel, 1971 ; Contents: 0. Isler, ‘Introduction’. pp. 11-27 (44 references); B. C. L. Weedon, ‘Occurrence’, pp. 29-59 (283 references); s. Liaaen-Jensen, ‘Isolation, Reactions’, pp. 61 -188 (550 references); W. Vetter, G. Englert. N. Rigassi, and U. Schwieter, ‘Spectroscopic Methods’, pp. 189--266 (65 references); B. C. L. Weedon, ‘Stereochemistry’, pp. 267-323 (184 references); H. Mayer and
Reviews on 7tJrpenoidCtlemis t rj,
307
0. Isler, ‘Total Syntheses’, pp. 325-575 (451 references); T. W. Goodwin, ‘Biosynthesis’, pp. 577-636 (350 references); H. Thommen, ‘Metabolism’, pp. 637-668 (124 references); N. I. Krinsky, ‘Function’, pp. 669-716 (266 references); G. A. J. Pitt, ‘Vitamin A’, pp. 717-742 (173 references); J. C. Bauernfeind, G. B. Brubacher, H. M. Klaui, and W. L. Marusich, ‘Use of Carotenoids’, pp. 743-770 (266 references); 0. Straub, ‘Lists of Natural Carotenoids’, pp. 771-850 (815 references); Appendix, ‘Tentative Rules for the Nomenclature of Carotenoids’, pp. 851-864. B. Ke, ‘Carotenoproteins’, Methods Enzymol., 1971, 23, 624 (39 references). S. Liaaen-Jensen, ‘Recent Progress in Carotenoid Chemistry’, in ‘Aspects of Terpenoid Chemistry and Biochemistry’, ed. T. W. Goodwin, Academic Press, London, 1971, pp. 223-254 (1 11 references). S . Liaaen-Jensen and A. G. Andrewes, ‘Microbial Carotenoids’, Ann. Reu. Microbiol., 1972, 26, 225 (156 references). S . Liaaen-Jensen, ‘Structural Elucidation of Carotenoids-A Progress Report’, Pure Appl. Chem., 1973, 35, 81 (76 references). B. C. L. Weedon, ‘Some Recent Studies on Carotenoids and Related Compounds’, Pure Appl. Chem., 1973, 35, 113 (25 references). Carotenoids-Physical Methods. L. Bartlett, W. Klyne, W. P. Mose, P. M. Scopes, G. Galasko, A. K. Mallams, B. C. L. Weedon, J. Szabolcs, and G. Toth, ‘Optical Rotatory Dispersion of Carotenoids’, J. Chem. SOC.(C), 1969, 2527 (32 references). C. R. Enzell, ‘Mass Spectrometric Studies of Carotenoids’, Pure Appl. Chem., 1969, 20, 497 (1 1 references). C. R. Enzell, G. W. Francis, and S . Liaaen-Jensen, ‘Mass Spectrometric Studies of Carotenoids’, Acta Chem. Scand., 1969, 23, 727 (8 references). U. Schwieter, G. Englert, N. Rigassi, and W. Vetter, ‘Physical Organic Methods in Carotenoid Research’, Pure Appl. Chem., 1969, 20, 365 (34 references). B. C. L. Weedon, ‘Sectroscopic Methods for Elucidating the Structures of Carotenoids’, Fortschr. Chem. org. Naturstofle, 1969, 27, 81 (82 references). H. Budzikiewicz, H. Brzezinka, and B. Johannes, ‘Mass Spectrometric Investigation of Carotenoids’, Monatsh., 1970, 101, 579 (48 references). Degraded Carotenoids. R. Hubbard, P. K. Brown, and D. Bownds, ‘Methodology of Vitamin A and Visual Pigments’, Methodr Enzymol., 1971, 18C, 615 (39 references). B. V. Milborrow, ‘Abscisic Acid’, in ‘Aspects of Terpenoid Chemistry and Biochemistry’, ed. T. W. Gbodwin, Academic Press, London, 1971, pp. 137-151 (25 references). G. A. J. Pitt, ‘Vitamin A’, in ‘Carotenoids’, ed. 0. Isler, Birkhauser, Basel, 1971, pp. 717-742 (173 references). H. F. Taylor and R. S . Burden, ‘Xanthoxin, a Recently Discovered Plant Growth Inhibitor’, Proc. Roy. Soc., 1972, B180,317 (33 references).
308
Terpenoids and Sleroids
Polyterpenoids and isoprenylutrd Quinones. F. W. Hemming, ‘Poiyprenols’, in ‘Substances Formed Biologcally from Mevalonic Acid’, ed. T, W. Goodwin, Academic Press, London, 1970, pp. 105-1 17 (29 references). 0. Wiss and U. Gloor, ‘Nature and Distribution of Terpene Quinones’, in ‘Substances Formed Biologically from Mevalonic Acid’, ed. T. W. Goodwin, Academic Press. London, 1970, pp. 79-87 (20 references). R. Barr and F. L. Crane, ‘Quinones in Algae and Higher Plants’. Methods En:
Part 11 STEROIDS
1 Steroid Properties and Reactions BY D. N. KIRK
The first volume on steroids in the MTP International Review of Science has been published during the year the literature on steroids covering the years 1970 and 1971 has been reviewed, selectively rather than comprehensively, in ten chapters by authors of international repute.
:’
1 Conformational Analysis, Stereochemistry, and Spectroscopic Methods A significant development is reported in the unravelling of mechanisms of transmission of effects of remote substituents.2 ‘Long-range’ effects have, in the past, been discussed in terms of one of or a combination of the following mechanisms : (a) inductive transmission through intervening bonds ; (b) field effects Calculations operating through space ; and ( c ) conformational tran~mission.~ (CND0/2) of electron density distribution in cortisol and its 6a- and 9a-fluoroderivatives, based upon X-ray structures, indicate that inductive effects fall off rapidly beyond the carbon atom bearing the substituent, and that field effects due to polar but non-ionized groups are also small.2 Conformational transmission remains as the main mechanism for long-range influences, but is now divided into two parts. Steric effects due solely to molecular deformation are termed ‘conformational steric transmission’ (CST),which corresponds essentially to the concept of conformational transmission as it was originally envisaged. The novel feature in the present work is the recognition that alterations in molecular geometry may also cause variations in electron distribution, a phenomenon now termed ‘conformational electronic transmission’ (CET). The CET mechanism is regarded as an important contributor to long-range effects, with probable significance in the interpretation of both kinetic and biological data for variously substituted steroids. Further development of this interesting concept will be keenly awaited. ‘Steroids’, ed. W. F. Johns, MTP International Review of Science, Organic Chemistry, Series 1, Vol. 8, Butterworths, London, 1973. P. A. Kollman, D. D. Giannini, W. L. Duax, S. Rothenberg, and M. E. Wolff, J. Amer. Chem. SOC.,1973, 95, 2869. D. N. Kirk and M. P. Hartshorn, ‘Steroid Reaction Mechanisms’, Elsevier, Amsterdam, 5968, p. 16.
31 1
312
Terpenoids and Steroids
Studies of the kinetics of oxidation (Cr"') of the ring D hydroxy-group in oestradiol and D-homo-oestradiol derivatives (1 ; n = 1 or 2, respectively), including compounds of the unnatural 8a-series, have established a long-range A long-range effect operating in the reverse effect of the C-3 sub~tituent.~ direction causes alterations in the acidic dissociation constants of the free phenolic steroids when the size of ring D, or its substitution, is varied. A roughly linear correlation has been demonstrated between the magnitudes of the two transmitted effects, which are considered to stem from a combination of inductive and conformational transmission. Enzymic oxidation of the 17B-OH group shows kinetic variations paralleling those found in chromium(v1)oxidation. OH
(I) R
=
H, OH, OMe, OAc, or OTs
Equilibration of cis- and trans-2-decalones has given thermodynamic parameters, including an enthalpy preference for the truns-isomer amounting to 2.51 kcal mol-', which is slightly smaller than the accepted value (2.7 kcal mol- ') for the isomeric decal in^.^ The data will be useful in considering equilibria of Sa- and 5fl-steroids.Recent force-fieldcalculations6 extending to olefins, including cycloalkenes and methylene-cycloalkanes, should have relevance to the stabilities of unsaturated steroids. An ingenious modification of Horeau's method permits the determination of absolute configurations of chiral alcohols on a 10pmol scale.7 The alcohol is allowed to react with ( f)-a-phenylbutyric anhydride (1.0 molar excess); the residual anhydride is then converted into diastereoisomeric amides by adding ( + )-(R)-a-phenylethylamine and the amides are determined by gas chromatography. An excess of amide derived from (R)-a-phenylbutyric acid generally implies an alcohol of the ( S ) configuration, and vice versa. The method produced correct conclusions for some pairs of steroid alcohols.
Spectroscopic and Chiroptical Methods.-U .v. spectra reveal a difference between the extent of conjugative overlap of the cyclopropane ring and the carbonyl group in 3sr,5a-cyclo-6-oxo-and 3~,S~-cyclo-6-oxo-steroids. Precise geometries of these systems, determined by X-ray analysis, suggest differing p-character in the 3,s-bonds as the cause of the spectroscopic distinction.* 1-0x0-A2-and V. V. Egorova, A. V. Zakharychev, and S. N. Ananchenko, Tetrahedron, 1973,29, 301.
' N. L. Allinger and J. H. Siefert, J. Amer. Chem. SOC.,1972, 94, 8082.
N. L. Allinger and J. T. Sprague, J. Amer. Chem. Soc., 1972, 94, 5734.
J. W. Brooks and J. D. Gilbert, J.C.S. Chenr. Cnntm., 1973, 194. '' C. R. C. Pettersen, 0. Kennard, and W. G . Dauben, J.C.S. Perkin I , 1972, 1929.
Steroid Properties and Reactions
313
3-oxo-A’-steroids show consistent differences in electronic and vibrational spectra and in M, values, which serve to distinguish between these two isomeric types.’ Steroid hydrocarbons exhibit the beginning of a c.d. band below 190 nm.” The sign is apparently associated mainly with the configurations at ring junctions, the cis-fused 5P- or 14P-hydrocarbons giving the largest effects, which are of opposite signs. C.d. data are reported for oestradiol, oestriol, 6-oxo-oestriol and its triacetate, and 6a- and 6P-hydroxyoestriols. The configuration of the 6-hydroxy-substituent has a profound effect upon the shape of the c.d. curve. A study of c.d. properties of nitrate esters includes the nitrates of a series of steroidal alcohols. A planar symmetry rule is proposed, on the basis of the preferred conformation of the nitrato-group.’2 C.d. data are also reported for a variety of pregnan-20-ones with thio-groups (SH, SAC,SCN, etc.) and other substituents at the C-17 p0siti0n.l~ The rules relating the signs of the Cotton effects (n -P TC*)of @-unsaturated lactones to ring geometry have been reexamined in the light of structures based on X-ray analy~is.’~ The earlier rule for butenolides is now reversed. C.d. data are reported and discussed for a variety of steroids oriented in stretched polyethylene films.’ New terminology is proposed for discussing the signs of chiroptical properties. With the dissymmetric molecule in an agreed co-ordinate frame, the effect of a perturbing group at a point P is said to be consignate if the sign of the effect is the same as the sign of the product of the co-ordinates of P ; if these signs differ, the effect is dissignate.I6 For the carbonyl group, the chromophore most thoroughly studied in steroids ( n - + n* transition), the term ‘consignate’ is synonymous with ‘octant ’ behaviour, and ‘dissignate’ with ‘anti-octant’ behaviour.
N.M.R. Spectroscopy.-The equilibrium constants K for complexation of the and with shift reagent [Eu(dpm),] with 3cr,4,4-trirnethyl-5a-cholestan-3~-01 4,4-dimethyl-5a-cholestan-3-oneshow wide variations according to solvent ( K increases in the order CDCl, < C& < Cs, < eel, < C,Hl,). Calculated limiting shifts for methyl signals (corresponding to total formation of a 1 : 1 complex) show only slight dependence on solvent.” The magnetic axis of a lanthanide-alcohol complex is probably collinear, or nearly so, with the O-M axis.’* Uncertainty on this point has been regarded as a major complication in the interpretation of lanthanide shifts. J. J. Schneider, J.C.S. Perkin Z, 1973, 1361. D . N . Kirk, P. M. Scopes, and B. M. Tracey, Tetrahedron Letters, 1973, 1355. l 1 E. P. Burrows, D. L. Di Pietro, and H. E. Smith, J. Org. Chem., 1972, 37, 4000. l 2 R. E. Barton and L. D. Hayward, Canad. J. Chem., 1972,50, 1719. l 3 A. A. Akhrem, A. M. Turuta, G . A. Kogan, I. S. Kovnazkaja, and Z . I. Istomina, Tetrahedron, 1973, 29, 1433. l 4 A. F. Beecham, Tetrahedron, 1972, 28, 5543. B. Norden, Acta Chem. Scand., 1972, 26, 1763. l h W. Klyne and D. N. Kirk, Tetrahedron Letters, 1973, 1483. l 7 J. Bouquant and J. Chuche, Tetrahedron Letters, 1973, 493. l 8 G . E. Hawkes, D. Leibfritz, D. W. Roberts, and J. D. Roberts, J. Amer. Chem. Soc., 1973,95, 1659. lo
3 14
Terpenoids and Steroids
7a- and 7P-substituted A5-steroids are more reliably distinguished by their n.m.r. spectra (chemical shift of C-6 proton, and 6-H,7-H coupling) than by the earlier method of molecular rotation differences.” The n.m.r. method can also be used for analysis of mixtures. Some new observations are reported on the various routes available for the preparation of 7a-hydroxy- and 7P-hydroxyA’-~teroids.’~The resonance due to methyl protons in partial structures C
I
CH3-C-OH
I
can be recognized by the downfield shift accompanying 0-
C formylation : after reaction with acetic-formic anhydride the tertiary methyl protons are deshielded by 0 . 2 5 4 . 3 5 p.p.m.” N.m.r. spectra have been used to determine configurations at C-22 in 22hydroxycholesterols and related 20,22-diols.’ The 220 MHz spectra of 24alkylated hydrocarbons of the cholestane series exhibit non-equivalence of signals due to the terminal methyl groups (C-26 and C-27), which have been correlated with configurations at C-24, and provide the first direct method for the study of configurations and the detection of inhomogeneity at C-24 in sterols of natural origin.22 The 3C spectra of lanost-8-en-3P-01, euphadienol, and euphenol have been analysed by the study of derivatives, the use of shift reagents, and other techn i q u e ~ .Assignments ~~ of 26 of the 30 carbon-atom signals from lanosterol agree with a previous publication, but the C-6, C-11, C-19, and C-21 signals appear to have been wrongly identified in the earlier work. I3C spectra are reported for ten cardenolides, with signal assignments of all carbon atoms,24 and for oestra-l,3,5(1O)-trieneand a wide variety of its derivatives, where substituent effects on the chemical shifts of each carbon atom are reported.25 Mass Spectra.-Mass spectra of steroid hydrocarbons of the 5a- or 5P-series include a fragment ion with m/e 149, but the 5P-isomers with a C-17 side-chain also exhibit an ion at m/e 151, which appears to have diagnostic value. The nature of the fragmentation and hydrogen transfers leading to this ion has been examined by use of deuterium-labelled hydrocarbons. Cleavage of the 8-14 and 9-11 bonds appears to be implicated.26 The mass spectra of 5-hydroxy-steroids (2), including those with a substituent X in ring A, give a characteristic ion (3),resulting from the loss of carbon atoms 1 4 , together with any attached s u b s t i t u e n t ~ . ~ ~
l9
’O 21 22 23 24
25
” ”
P. Morand and A. Van Tongerloo, Steroids, 1973, 21, 65. R. Misra and C. Dev, Tetrahedron Letters, 1972, 4865. T. A. Wittstruck, J. Org. Chem., 1973, 38, 1426. L. J. Mulheirn, Tetrahedron Letters, 1973, 3175. S. A. Knight, Tetrahedron Letters, 1973, 83. K . Tori, H. Ishii, Z. W. Wolkowski, C. Chachaty, M. Sangare, F. Piriou, and G . Lukacs, Tetrahedron Letters, 1973, 1077. T. A. Wittstruck and K. I. H . Williams, J. Org. Chem., 1973, 38, 1542. L. Tokes and B. A. Amos, J. Org. Chem., 1972, 37, 4421. A. Rotman, A. Mandelbaum, and Y. Mazur, Tetrahedron, 1973, 29, 1303.
Steroid Properties and Reactions
315
R
X OH (2)
(3) R
=
R
=
O A c ; mle 278 C8H1,;mle 332
Mass spectra are reported for the trimethylsilyl ethers of 1lp-hydroxysteroids,2* a series of 20-chloro-pregnanes, including some with substituents also at C-16,29 and a number of sterols, including some A14-unsaturated compound~.~~
2 Alcohols and their Derivatives, Halides, and Epoxides Substitution and Elimination.-A smooth reaction between a steroid alcohol, triphenylphosphine, diethyl azodicarboxylate, and a carboxylic acid, in THF, leads to the corresponding carboxylic ester of the alcohol of inverted conjguration., The reaction is at present limited to sterically unhindered alcohols, but it is to be hoped that conditions will be found to broaden its scope. Examples include the conversion of 5a-cholestan-3p-01 into the formate, benzoate, or phenylacetate of the 3a-alcohol in nearly quantitative yield, but the axial 3aalcohol completely failed to react. Cholesterol gave epi-(3a-OH)-cholesteryl benzoate, unlike most substitutions with 3P-hydroxy-A5-steroids,which proceed with retention of configuration or give 3a,5a-cyclo-6P-alcohols or their derivat i v e ~ .Methyl ~ ~ cholate reacted selectively at the equatorial 3a-hydroxy-group, giving a 3-mono-ester of the 3P,7a,12a-triol. A 16a-alcohol gave an ester of the 16P-alcoho1, but the rather more hindered 17P-alcohol was unreactive. Sa-Androstan-1 la-01 reacts with SOC1,-pyridine to give 1la-chloro-Saandrostane, together with 5a-androst-9(11)-ene. Elimination was the only reaction when PCl, was used or when the llp-alcohol was treated with either reagent.33 The powerful nucleophilicity of azide ion has been used in syntheses of three isomeric 2,3-diaminocholestanes (Scheme 1). The trans-diaxial azidoalcohols, as their mesylates (4)and (5) react with NaN,-DMSO to give the 2p,3p- and 2a,3a-diazido-compounds (6) and (7),which afforded the corresponding diamines (8) and (9) on reduction. Ring-opening of either the 2a,3a- (10) or 2p,3/3-aziridines with HN, in chloroform gave the trans-diaxial amino-azide [e.g. (1l)], which was reduced to the 2P,3a-diamine (12).34 28
29
30 31
32 33 34
P. Vouros and D. J. Harvey, J.C.S. Perkin I, 1973, 727. G. Adam, D. Voigt, K. Schreiber, M. von Ardenne, R. Tummler, and K. Steinfelder, J. prakt. Chem., 1973,315, 125. H. Schiller, Fette, Seifen, Anstrichm., 1973, 75, 145. A. K. Bose, B. Lal, W. A. Hoffman, and M. S. Manhas, Tetrahedron Letters, 1973, 1619. Ref. 3, p. 236. C. W. Shoppee and J. Nemorin, J.C.S. Perkin I, 1973, 542. K. Ponsold and D. Klemm, Chem. Ber., 1972,105,2654.
Terpenoids and Steroids
316
(4)
- H2Nxx H2N'
H
(9)
- N3a H
H2N'
(1 1)
Scheme 1
Treatment of a 7or-bromo-5a-hydroxy-6-ketone (13) with lithium carbonate in refluxing DMF gives the oxetanone (14), as well as the A7-compound (15).35
(1 3 )
(15)
(14)
The oxetanone is surprisingly stable. A superior yield of the oxetanone from the 7P-bromo-isomer and the detection of this isomer when the 7a-bromocompound was subjected to brief reaction indicate that the main reaction scheme 1s : 7B-Br
7cr-Br
-€
* oxetanone
*7
A novel route from alkyl halides or tosylates to carbonation products [acids, esters (17), or amides] is illustrated for the steroidal bromo-ester (16). The choice of substrate demonstrates the stability of 0x0- and ester groups to the unusual reagent system {Na,[Fe(CO),] in THF-HMPA, followed by I,-MeOH}. Use of an amine instead of an alcohol for the second stage gives the amide directly.36 The 5a,6~-dibromo-compound(18) reacted with sulphuryl chloride in the presence of dibenzoyl peroxide (free-radical mechanism) to give the 35 .3b
R . Hanna, G . Maalouf, and B. Muckensturm, Tetrahedron, 1973, 29, 2297. J. P. Collman, S. R . Winter, and R. G . Komoto, J. Amer. Chem. Soc., 1973, 95, 249.
Steroid Properties and Reactions
317
OCO(CH,),Br OCO(CH,),CO,Me
(17)
0
X (18) X (19) X
= =
Br C1
5cr-bromo-6~-chloro-analogue( 19).37 When a 7a-acetoxy-6P-bromo-4-en-3-one (20) was treated with NaN, in DMF or DMSO, in order to prepare a 6-azidoderivative, the major product was instead found to be the 4-azido-4,6-dien-3-one (21). KSCN similarly afforded the 4-thiocyanato-derivative (22). The reactions apparently proceed through an allylic substitution (S,2') mechanism [e.g. (20)]. A possible alternative mechanism involving allylic rearrangement of an initially formed 6P-azido- or 6P-thiocyanato-4-en-3-one was excluded by the observations (23) rearranged on heating to that the 7a-hydroxy-6~-thiocyanato-compound give the 4-isothiocyanate (24) rather than the 4-thiocyanate, and also that a
-@ 0
3'
V. Hach, Steroids, 1973, 21, 245
318
Terpenoids and Steroids
7a-acetoxy-6p-azide gave only the 6-azido-4,6-dien-3-one under the reaction conditions. The epoxide rings in 4~,5a-,4j3,5p-, and 5~,6a-epoxycholestan-3P-olshave been opened with sodium azide and acid to give a ~ i d o h y d r i n s .Dehydration ~~ (25) gave a mixture of the 4P-azido-5-ene (26) of the 3~-acetoxy-4~-azido-5~-01 and the 6p-azido-4-ene (27), indicating that an intramolecular allylic migration of the azido-group does occur in this case, in contrast to the related examples above.
AcO
@ N 3
AcO
&
AGO
The (23S)-hydroxylanost-24-ene side-chain (28) reacted with acidified ethanol to give the allylic rearrangement product (29), as well as the ethyl ethers of the (23R)- and (23S)-alcoholsand of the 25-alcohol. More vigorous acidic treatment gave the 22,24-diene (30).40
Methyltriphenoxyphosphonium iodide in hexamethylphosphoramide provides a very mild reagent for the selective dehydration of secondary alcohols, giving Saytzeff-type products with little evidence for rearrangement^.^' Primary alcohols afford the alkyl iodide under the same conditions, and tertiary alcohols appear to be too hindered to react at all. Phosphoryl chloride-pyridine is a useful alternative to previously known reagents for the generation of olefins from iodohydrins. The 3ct-hydroxy-2~-iodo-steroid(3l), prepared from the 2a,3~-epoxide,gave the A*-olefin (33) in 94 % yield.42 The bromohydrin was less reactive, but afforded the olefk in low yield. The mechanism indicated [(31) (32) (33)] is proposed.
-
38
’’ 40 41
42
-
H. L. Herzog, J. Korpi, E. L. Shapiro, G. Teutsch, and L. Weber, J.C.S. Chern. Comm., 1973, 72. S. Julia and R. J. Ryan, Compt. rend., 1973, 276, C , 1565. N. Entwistle and A. D. Pratt, J.C.S. Perkin I, 1973, 1235. R. 0. Hutchins, M. G. Hutchins, and C. A. Milewski, J . Org. Chem., 1972, 37, 4190. A. Guzman, P. Ortiz de Montellano, and P. Crabbe, J.C.S. Perkin I , 1973, 91.
319
Steroid Properties and Reactions
A further study43 of the acetolysis of the mesylates of 5a,6a- and 5p,6/3methanocholestan-3a- and 3 p - 0 1 has ~ ~ ~afforded mixtures of products derived from each of the four isomers. Scheme 2 indicates the main product or products from each mesylate. Mechanisms are discussed in detail. 3a-AcO (16 %) 3P-AcO (13 %) K
80% from 3a-MsO 50%from 3/3-MsO
1’1 % from 3a-MsO 44%from 3P-Ms0
Scheme 2
The acetolysis of methoxy-steroids with BF,-acetic anhydride is liable to give products of both rearrangement and elimination. 2P-Methoxy-5a-cholestane gave 5a-cholest-2-ene and also the 3a-acetoxy-compound, derived uia a hydride 43
44
L. Kohout and J. FajkoS, COIL Czech. Chem. Comm., 1973, 38, 913. ‘Terpenoids and Steroids’, ed. K. H. Overton (Specialist Periodical Reports), The Chemical Society, London, 1973, Vol. 3, pp. 375-6.
Terpenoids and Steroids
320
shift from C-3 to a C-2 carbonium ion : only small amounts of 2-acetoxycholestanes were found. 2a-Methoxy-5a-cholestane gave the same set of products, but with the 2a-acetate predominating; only a little of the 3a-acetate was formed. 6a-Methoxy-5a-cholestane gave the 6a-acetoxy-compound, probably resulting from nucleophilic attack at the methoxy carbon atom, which is less hindered than C-6. Cholest-5-ene was virtually the only product from the 68-methoxyderivative, which allows a concerted diaxial elimination with relief of In a continued examination of A-homo-steroids, the 4(4a)-unsaturated compound (35) has been obtained by regiospecific dehydration of the 4aa-alcohol (34) with thionyl chloride, and also by reductive elimination from the bromo-mesylate (36) with zinc. The bromo-alcohol (37) unexpectedly gave the 4-ketone (38)
@ HO
(34)
Zn
__+
‘(R
S0Cl2
=
Ms)
@
Ro
OAc
Br
OAc
(35) (37) R
=
H
(R = H)
under similar conditions. The olefin (35) afforded the 4a,4aa-epoxide, which undergoes ring-opening reactions with participation of the 6B-acetoxy-group (Scheme 3).46 The conjugated and non-conjugated dienones, (41) and (42) respectively, were the major products when attempts were made to replace 6a-substituents by fluorine in the ~-nor-4-en-3-ones(40).47 Solvolysis of the 5P-substituted 6a-bromo-compounds (43) proceeded with participation of the 58-substituent, forming the 5p,6P-epoxide or derived diols or their mono-esters. The S-methylxanthates (44) and (46) derived from cholest-4-en-3P-01 and cholesterol undergo thermal rearrangements to give the respective dithiolcarbonates (45)and (47),with relatively minor amounts of the elimination product, the 3,5-diene (48). The A4-unsaturated dithiolcarbonate (45) rearranges further 45 46 47
S . E. Bruce and R. E. Gall, J.C.S. Perkin I, 1972, 2319. H. Velgova and V. Cemy, Coll. Czech. Chem. Comm., 1973, 38, 575. A. Kasal, Coll. Czech. Chem. Comm., 1972, 37, 3095.
Steroid Properties and Reactions
_1
(39)
32 1
@-
-HO'
;@ AHO' H O y O
-
Br
OAc
Reagents : i, H ; ii, H ,-Pt ; iii, HBr; iv, Br -. +
Scheme 3
OH
(40)X
= Br
or OH
(41)
AcO
X (43) X = Br, OH, o r OAc
(42)
Terpenoids and Steroids
322
I MeS
0
/c=s (46)
(47)
(48)
when chromatographed on silica gel, forming 3P-methylthiocholest-4-ene (49), with extrusion of SC0.48 Ringapening of Epoxides-Acid-catalysed hydrolysis of the 3P-methoxy-4a,5aepoxide (50) gives the 5-en-4a-01 (51), and not the 4P,Sa-diol (52) as reported previously. The authentic 4P,5a-diol has been obtained by reduction of the 4fi-hydroxy-3/?-methoxy-5a,6a-epoxide (53). Some transformations of the 5-en4cr-01 are described.49 Cholestane
A novel development in organoselenium chemistry provides a simple preparation of allylic alcohols from epoxides (Scheme 4). The phenylselenide ion is a powerful nucleophile, which readily opens an epoxide to given the hydroxy~elenide.~'Oxidation in situ with H,O, then generates the labile selenoxide, which is eliminated along with a vicinal proton under very mild conditions, often at room temperature, to give phenylseleninic acid and the allylic alcohol. Examples include the preparation of allylic alcohols (54)and ( 5 5 ) from 2a,3aand 2P,3P-epoxy-5a-cholestanes,respectively. This reaction sequence provides a useful route to compounds which may not be readily accessible by other methods. The practical procedure starts from the stable, odourless diphenyl 48 49
K . Harano and T. Taguchi, Chem. and Pharm. Bull. (Japan), 1972, 20, 2357. T. H. Campion and G . A. Morrison, Tetrahedron, 1973, 29, 239. K. B. Sharpless and R. F. Lauer, J . Amer. Chem. SOC.,1973,95, 2697.
Steroid Properties and Reactions
323
i ,
Ho'
H
\t:
H
(55)
(54)
Reagents: i, PhSeNa-EtOH-THF; ii, H 2 0 2 . Scheme 4
diselenide, and appears to avoid the obnoxious features generally associated with selenium chemistry ; the selenium compound is readily recovered and recycled. During an investigation of reactions of 1,2-epoxy-3-oxo-steroids (56),sodium borohydride in methanol was found to afford the diaxial methoxyhydrin (59) as one p r ~ d u c t . ' ~The mechanism is not understood, but appears to involve the trans-epoxy-alcohol (58) as the intermediate. The cis-epoxy-alcohol (57)
B,
&?
+
0
H
HO
*'
(60) 51
+ H
HO
H
(59)
M . Weissenberg, D. Lavie, and E. Glotter, Tetrahedron, 1973, 29, 353
324
Terpenoids and Steroids
was not affected by the reagent. Ring cleavage of the 3-tosylhydrazone of the epoxy-ketone (56) in the presence of borohydride proceeded normally to give the seco acetylenic alcohol (60). The 4-methylene derivative reacted similarly. The ~-nor-Sa,6a-epoxide(61) gave the 5P,6a-diol (62) as major product with perchloric acid,52 although acidic reagents usually give the rearranged compound (63).53 Some transformations of the 5P,6a-diol are outlined in Scheme 5.
Scheme 5
6a,7a-Epoxyoestradiol diacetate (64) reacted slowly with HF to give the expected trans-diaxial fluorohydrin (65).54 Fluorine could be introduced at C-7 by treating 6-dehydro-oestradiol diacetate with perchloryl fluoride in the presence of water, which afforded the 7a-fluoro-6-ketone (66) directly in modest yield. Reduction with NaBH, gave the 7a-fluoro-6a-hydroxy-derivative (67), which was transformed into 7a-fluoro-oestradiol (68) by reducing the 6-mesylate with LiAlH,. OAc
(65)
52
53
54
(66) R' = Ac, R 2 = 0 (67) R' = Ac, R 2 = a-OH (68) R' = RZ = H
I . Joska, J. FajkoS, and F. Sorm, CON.Czech. Chem. Comm., 1972, 37, 4091. W. G. Dauben, G. A. Boswell, W. Templeton, J . W. McFarland, and G. H. Berezin, J. Amer. Chem. Soc., 1963, 85, 1672; J . Joska, J. FajkoS, and F. Sorm, Coll. Czech. Chem. Comm., 1963, 28, 82; J. Joska and J. FajkoS, ibid., p. 2605. M . Neeman, Y . Osawa, and T. Mukai, J.C.S. Perkin I, 1973, 1462.
325
Steroid Properties and Reactions
A 19-acetoxy-3p-fluor0-5-ene is hydroxylated normally at the 4,!l-position by SeO,, or can be oxidized to give the 5-en-7-one by t-butyl chromate, but the 19-acetox y-function interferes with the normal course of acid-catalysed hydrolysis of the 5a,6a-epoxide (69j, giving the diequatorial 5,!l,6a-diol (71j instead of the usual Sa,6P-diol. The 19-acetoxy-group presumably affords an intermediate 5P,19-acetoxonium ion (70).55
Esters, Ethers, and Related Derivatives of Alcohols.-The major product when a 21-hydroxypregnan-20-onereacted with an excess of phosgene was the novel 20-chloro-20,21-cyclic carbonate (72) (an epimeric mixture), along with the 21-chloro-ketone (73). The relative yields were reversed in presence of an 110x0-function. Reactions of the 20-chloro cyclic carbonate include dehydrochlorination to give a mixture of the 17,20- and 20,21-unsaturated carbonates (74) and (75). Several other reactions of these interesting systems are also described. H,C-O
I
\
/c=o
c1-c-0
CH,CI
I
co
CH-0
II
\
C-0
(74) (cis55 56
+ trans-isomers)
(75)
A. Guida and M. Mousseron-Canet, Bull. SOC.chim. France I I , 1973, 1098. M. L. Lewbart, J. Org. Chem., 1973, 38, 2328, 2335.
Terpenoids and Steroids
326
Cortisone has been converted with an excess of phosgene into the 17a,21carbonate (76), though in low yield. The 21-chlorocarbonate (77) was also formed, and could be converted in situ into the 21-methoxycarbonyl derivative (78) with methanol. The 17~,21-carbonate was converted by refluxing methanol (79),along with the 21-methoxycarbonyl into the 20~-methoxy-l7a,20cr-carbonate derivative (78). The general reaction when phosgene reacted with an excess of 17a,2l-dihydroxy-20-oxo-steroids was the formation of the bisbregnan-21-yl) carbonates (80),which are stable to acids but sensitive to weak alkalkS7 CH,O-COX
CH,OH
I
co
I
(77) x = Cl (78) X = OMe
(79)
0
II
CHl-0-C-0-CH,
I
I
Further studies are reported" on the rates and selectivity of acetylation of OH groups in compounds of the bile acid type.59 The side-chain terminal group has little effect, but C-3 substituents influence the reactivity of the 1 2 ~ - O Hgroup in 7a, 12a-dihydroxy-compounds. Differences in the reactivities of hydroxygroups have allowed the selective introduction of both trimethylsilyl and perdeuteriotrimethylsilyl groups into the same molecule, as an aid to the interpretation of mass ~pectra.~'Cholesterol reacts with trimethyl or triethyl phosphites to give dicholesteryl phosphonate and mixed alkyl cholesteryl ' pho~phonates.~ 18,21-Diesters of aldosterone are deacylated selectively at C-18 to give 21-mono-esters under acid catalysis, but basic reagents cause an initial hydrolysis at C-21 followed by a rapid intramolecular transfer of the 18-ester group to 57 58
59
6o 61
M. L. Lewbart, J . Org. Chem., 1972, 37, 3892. G . C. Wolf, E. L. Foster, and R. T. Blickenstaff, J . Org. Chem., 1973, 38, 1276. 'Terpenoids and Steroids', ed. K. H. Overton (Specialist Periodical Reports), The Chemical Society, London, 1972, Vol. 2, p. 246. P. Vouros and D. J . Harvey, Analyt. Chem., 1973,45, 7. C. Benezra, J.-L. Bravet, and J. Riess, Canad. J. Chem., 1972, 50, 2264.
Steroid Properties and Reactions
327
C-21.62 The selectivity is dramatically demonstrated by the 18-benzoate 21acetate (81), which gave the 21-acetate (82) in aqueous solvents at pH < 3, but afforded the 21-benzoate (83), via transient formation of the 18-benzoate, in methanol containing sodium methoxide. R’O I
(81)
CH,0R2
I
R’ = Bz,R2 = AC
(82) R’ = H, R2 = AC (83) R’ = H, R2 = BZ
Reaction of aldosterone 21-acetate with methyl ‘acetobromoglucuronate’ gives a mixture of glycosiduronate derivatives, isomeric at C-18, where the sugar moiety is linked via the hydroxy-oxygen of the hemiacetal form of a l d ~ s t e r o n e . ~ ~ Methyl ‘acetobromoglucuronate’ reacts with 3~,2l-dihydroxy-5j?-pregnane11,20-dione in the presence of Ag,CO, to give the 3- and 21-monoglucosiduronates, the 3,21-diglucosiduronate, and the orthoacetate (84),resulting from acetoxy-group p a r t i ~ i p a t i o n .Steroid ~~ glucosiduronates have been permethylated by dimethylsulphinylmethide ion and methyl iodide, as an aid to mass spectrometric re~ognition.~’ C02Me
I
Me (84)
Oxidation and Reduction.-The Fetizon reagent (Ag,CO, on celite) is thought to oxidize alcohols by co-ordination of the hydroxy-group with an Ag’ cation, 62
63 64 65
R. Vitali and R. Gardi, Chem. and Ind., 1973, 584. R. H. Underwood and N. L. Frye, Steroids, 1972, 20, 515. V. R. Mattox and W. D. Vrieze, J . Org. Chem., 1972, 37, 3990. R. M. Thompson and D. M. Desiderio, Biochem. Biophys. Res. Comm., 1972,48, 1303.
328
Terpenoids and Steroids
followed by rate-controlling homolysis of the carbinol C-H bond (Scheme 6).66 This mechanism is consistent with observed solvent, isotope, and steric effects on oxidation rates for a variety of steroidal alcohols. The unhindered steroidal 3p-alcohols are oxidized more rapidly than hindered 6p- and 17~-alcohols; 6u- and 7a-alcohols are resistant to oxidation with this reagent, apparently because of the inaccessibility of the 68 and 78 C-H bonds.
H
\
/’
C’
\o/
H
\
I’
C’
\H
i’
-+
+ H-O=C< 2Ag,C0,’-,Hi
0-
\ / C
0-
co,2-
II
0 Scheme 6
Oxidation with the Collins’ reagent (py2Cr0, in CH,C12) is said to provide the most successful conversion of 3P-hydroxy-As-steroids into the 5-en-3-0nes.~~ Other oxidation procedures gave impure products which were difficult to purify because of the lability of the 5-en-3-one system. When 5u-lanost-8-en-3P-01 is oxidized with the usual chromium(v1) reagents the 3-ketone is invariably contaminated by products of allylic oxidation at C-7 and C-11. The Moffatt reagent (DMSO4icyclohexylcarbodi-imide) is recommended for preparation of the 3-ketone with no detectable contaminant.68 t-Amy1 hydroperoxide and MoCI, in benzene is a novel system for oxidizing secondary alcohols to ketones. Examples include 3-hydro~y-steroids.~~ Lithium dimethylcuprate, an effective reagent for conjugate alkylation of up-unsaturated ketones, is thought to initiate its reactions by electron donation from the anion. The presumption that it should therefore show reducing properties for suitable substrates has been confirmed by the rapid conversion of some a$-epoxy-ketones into b-hydroxy-ketones [e.g. (85).--) (86)].70 Yields
(85) 66
”
h9
’O
(86)
M . Fetizon, M. Golfier, and P. Mourgues, Tetrahedron Letters, 1972, 4445. J. B. Jones and K. D . Gordon, Canad. J. Chem., 1972,50, 2712. D . C. Wigfield, S. Feiner, and D. J . Phelps, Steroids, 1972, 20, 435. G. A. Toistikov, U. M. Dzhemilev, and V. P. Yurev, Zhur. obshchei Khim., 1972, 42, 1611. J. R. Bull and H. H. Lachman, Tetrahedron Letters, 1973, 3055.
329
Steroid Properties and Reactions
may be comparable to those obtained by reduction. with lithium in liquid ammonia, and the dimethylcuprate offers the advantage of being unreactive towards carbonyl groups elsewhere. Preliminary results indicate that a-halogenoand a-acyloxy-ketones are also reduced to the parent ketones by the dimethylcuprate. Low-valency tungsten derivatives obtained by reaction of WCI, with alkyllithium, lithium metal, or certain alkali-metal halides are effective reagents for deoxygenation of epoxides to give alkenes. The 5,6 : 22,23-bis-epoxide derived from stigmasteryl acetate was reduced selectively to give the 22,23-monoe p ~ x i d e . ~Some model aldehydes and ketones provided the olefinic products of reductive coupling (e.g. benzaldehyde -P stilbene) ; the composition of these potentially useful reagents is not yet known. An ally1 carbonium ion has been implicated in the reduction of 3-hydroxycholest-4-enes with AICl,-LiAID, to give mixed 3a- and 3P-deuteriocholest-4-enes. The isomer ratios obtained under different conditions suggest that the 3a-alcohol reacts by the S,1 mechanism, but that the 3P-alcohol is partly reduced by a mechanism not involving a carbonium ion.72 Pentacarbonyliron is a selective reducing agent for enol acetates, vinyl chlorides, ap-unsaturated aldehydes, and acetoxy- ketone^.^ Some examples are illustrated in Scheme 7 ; simple ketones, olefins, esters, and phenolic ethers are unaffected.
Scheme 7
Reduction of 8P,19-oxidoandrost-l4-enes(87) with zinc proceeds through selective attack on the allylically activated 8P C-0 bond, giving the 19-hydroxy14-ene (SS).74 (The method of preparation of the 8/3,19-oxido-14-enesis promised for a paper to be published.) Miscellaneous.-The steroidal ylide (89) reacted with hexafluoroacetone to give the hexafluorodesmosterol side-chain (90).75 The Az4-olefinicbond is unreactive to hydrogenation or hydroboronation.
'' 72 73 74 75
K. B. Sharpless, M. A. Umbreit, M. T. Nieh, and T. C. Flood, J . Amer. Chem. SOC., 1972,94,6538. M. P. Paradisi and A. Romeo, J.C.S. Perkin I , 1972, 2010. S. J . Nelson, G. Detre, and M. Tanabe, Tetrahedron Letters, 1973, 447. C. Kruger and G. I. Birnbaum: Tetrahedron Letters, 1973, 1501. J . E. Herz and S. C. Montalvo, J.C.S. Perkin I , 1973, 1233.
330
Terpenoids and Steroids OR
RO
3 Unsaturated Compounds Addition Reactions.-The reaction between 5a-cholest-7-ene (9 1) and iodinesilver acetate in aqueous acetic acid gives isomeric 8-ene-7,ll-diol derivatives, which afforded the 8-ene-7,ll-dione (92) on oxidation (Scheme 8).76 This finding contradicts a recent claim that the 8-en-7-one (93) and 8(14)-en-7-one (94) were prepared by this route. The reagent system appears, like some other oxidants, to convert the 7-ene initially into the 7,9(11)-diene, which is then attacked further at the C-7 and C-11 positions to give derivatives of the 8-ene7,ll-diols. Authentic 8-en-7-one (93) and 8(14)-en-7-one (94) were obtained by the alternative route outlined in Scheme 8.76 Thallium(1) carboxylates are efficient alternatives to silver salts for preparing cr-iodocarboxylates in a modified Prevost reaction : solvolysis of the initial products provides derivatives of cis- or trans-glycols, according to the conditions chosen.77 A study of the reaction78 of hypobromous or hypochlorous acids with cholest-4-en-3P-01 or its esters extends earlier in this field. Acyl groups migrate from C-3 to C-4, giving 4-acylates of the 5a-halogeno-3P,4P-diol. HOCl converts the 3P-acetate (95) into a complex mixture of products, including Acyloxononium ions (97) the 3-acetate (96) of 4a-chloro-5~-cholestane-3/3,5-diol. are implicated (Scheme 9).7 x The factors which influence the stereochemistry of electrophilic additions to olefins are still imperfectly understood. Although most reagents add stereospecifically trans, chlorine often gives some cis-product," and CF,OF adds I . Midgley and C. Djerassi, J.C.S. Perkin I, 1972, 2771. R. C . Cambie, R . C. Hayward, J. L. Roberts, and P. S. Rutledge, J.C.S. Chem. Comm., 1973, 359. R. Lorne and S. Julia, Bull. Soc. chim. France ZI, 1973, 1357. 'Terpenoids and Steroids', ed. K. H. Overton (Specialist Periodical Reports), The Chemical Society, London, 1971, vol. 1, p. 300. P. 9. D. de la Mare and R. Bolton, 'Eiectrophilic Additions to Unsaturated Systems', Elsevier, London. 1966; see also ref. 3, p. 90.
33 1
Steroid Properties and Reactions
@I
+ H
'OH
H
(91)
@I
H (94)
(93) Reagents: i, I,-AgOAc-aq. v, O H - .
HOAc; ii, 0 0 , - p y ; iii, m-CIC,H,CO,H (2 mol); iv, H + ;
Scheme 8
a? c1.9
O
AcO
(95)
\. + ./ C
I
Me (97)
332
Terpenoids and Steroids
predominantly A correlation of cis addition with high electronegativity, and with the conformational preference of 1,2-disubstituted ethanes, is now suggested : 8 0 b pairs of substituents with highest electronegativity prefer the gauche to the trans conformation. Epoxidation.-Iron( 11) ions accelerate the reaction of hydrogen peroxideacetic acid with cholesterol to give a mixture of acetates of Sa-cholestane3/L5,6fi-tri01.~ The reaction appears to proceed through the 5,6-epoxideformed by the action of peracetic acid. The metal ions catalyse both the formation of peracetic acid and the ring-opening of the epoxide. Steroidal 5-enes are oxidized by t-amyl hydroperoxide with added MoCl, or Mo(CO), to give first the 5a,6aand 5p,6P-epoxides: excess of oxidant gives the 5a-hydroxy-6-ketone as major product.82 MOO, oxidizes a Sene only as far as the 5a,6a-epoxide, but in good yield.83 The two epimeric 24,25-epoxides (98)derived from 5a-lanosta-8,24-dien3p-yl acetate have been separated and their configurations determined.84 The molecular rotations of the derived 24-alcohols and application of the Horeau method (esterification of the 24-alcohol with an excess of racemic a-phenylbutyric anhydride and measurement of the small optical rotation of recovered a-phenylbutyric acid) each indicated the same stereochemical assignments. Epoxidation of 5,7~-cyclo-5~-cholest-l-en-3-one (99) with H202-OH -, or of the derived 3a-01 (100) or 3p-acetoxy-derivative (101)with per-acid, gave the corresponding la,2a-epoxide (102). The 3/3-01 (103) afforded mainly the 1/3,2j?-epo~ide.*~
(99) R (100) (101) (103)
R R R
=0 = a-OH = B-OAC =
( 102)
P-OH
Alkaline hydrogen peroxide converted a 4-en-6-one into the 4/3,5P-epoxy-6ketone ; the reaction is facilitated by a 3P-OH group.86 Ref. 79, p. 296. 'ObL. Phillips and V. Wray, J.C.S. Chem. Comm., 1973, 90. M. Kimura, M. Tohma, andT. Tomita, Chem. andPharm. Bull. (Japan), 1972,20,2185. 8 2 G . A. Tolstikov, U. M. Dzhemilev, and V . P. Yurev, Zhur. org. Khim., 1972, 8, 1190. '' G. A. Tolstikov, U . M. Dzhemilev, and V. P. Yurev, Zhur. org. Khim., 1972, 8, 2204. 8 4 R. B. Boar, D. A. Lewis, and J. F. McGhie, J.C.S. Perkin I, 1972, 2231. S T J. FajkoS, M. BudeSinsky, and J. Joska, ColI. Czech. Chem. Comm., 1973, 38, 1406. 8h D. Baldwin and J. R . Hanson, J.C.S. Perkin I, 1972, 2051.
Steroid Properties and Reactions
333
Hydroxylation of either progesterone or 6/?-hydroxyprogesterone with osmium tetroxide gave mixtures of the 4a,5a- and the 4P,S,!I-dihydroxy-derivatives." Hydroxylation of 4-ene-3,6-diones with Os0,-H,O, gives a 4,5-diol of unknown stereochemistry, which is dehydrated by acids to give the 4-hydroxy4-ene-3,6-dione (104) as major product, although derivatives of isomeric enols were also formed, depending upon the reaction conditions. Methanolic HCl gave the 3-methoxy-2,4-diene derivative (105), which afforded stable iron(rr1) and copper(I1)complexes of the enolized b-diketone system."
@Meom'
0
OH
0
OH
0
The mercuration of a 1,4,6-trien-3-one(106) by Hg(OAc), was recently shown to give the 2-mercuriacetate derivative (108).*' Further study shows 1,4-dien-3ones and 1-en-3-ones to be much less reactive, although still inclined to react at C-2, if at all.90 Kinetic data, and effects of varying the solvent and anion, are interpreted in terms of a simple two-step mechanism, comprising electrophilic attack on the A'-olefinic bond (107),followed by loss of the C-2 proton, although it is not clear which is the slow step. OAc
Selective oxymercuration [Hg(OAc),-aq. THF] of the A24-olefinic bond in desmosterol acetate, and demercuration with NaBH, , provides an efficient synthesis of 25-hydroxycholesterol (see also p. 392).91 Pregn-l7(20)-enes [( 109)
89
90 91
A . D. Tait, Steroids, 1972, 20, 531. A. D.Tait, Steroids, 1972, 20, 417. Ref. 79, p. 306. R. G.Smith, H. E. Ensley, and H. E. Smith, J . Org. Chem., 1972, 37,4430. M. Morisaki, J. Rubio-Lightbourn, and N. Ikekawa, Chem. and Pharm. Bull. (Japan), 1973, 21,457.
Terpenoids and Steroids
334
or (1lo)] are unsuitable for the acid-catalysed Ritter reaction (addition of acetonitrile), because the conditions leading to a carbonium ion at C-17 result in Wagner-Meerwein migration of the C-18 methyl group from C-13 to C-17. Mercury@) dinitrate, however, is reported to promote reaction of pregn-17(20)enes with acetonitrile : demercuration of the products in situ with NaBH, gave a mixture of the four acetamidopregnenes ( t l l H 1 1 4 ) , the 16a-acetamidoderivatives predominating by a ratio of 4 : 1. Hydrogenation gave the saturated aceta midopregnanes9
Some new reactions are reported with the reagent system Pb(OAc),-Me,SiN,, which generates mixed PbIV acetate azides [P~(OAC),_~(N,),] (Scheme
(40 %)
Scheme 10 92 93
B. Delpech and Q. Khuong-Huu, Tetrahedron Letters, 1973, 1533. H. Hug1 and E. Zbiral, Tefrahedron, 1973, 29, 753.
Steroid Properties and Reactions
335
Further examples of the cleavage of A5-steroids by the reagentg4 include the formation of keto-a-azidonitriles (115),as well as keto-nitriles (116).95 Catalytic hydrogenation of the a-azidonitriles led to cyclization to give novel steroidal heterocycles of the types (117) and (1 18).
RO N
I
H
0 2
The A9(' ')-unsaturated steroid (119) gave the 12a-hydroxy- (120), 12-keto(12l), and 12a-azido-derivatives (122), as well as the 9a,11a-epoxide (123), each in low yield.96
(119) (120) (121) (122)
94
95 96
R R R R
=
H LX-OH
= =
0 a-N,
=
Ref. 79, p. 302.
H.Hugl and E. Zbiral, Tetrahedron, 1973,29,759. E. Zbiral and H.Hugl, Tetrahedron, 1973, 29, 769.
Terpenoids and Steroids
336
Hydroboronation-oxidation of a 1,5-dien-3-01 (124) with limited reagent gave a mixture of the 5-en-la,3/3-diol (125) and 5-en-2a,3/3-diol (126), each in 15% yield.97 The reaction is of interest in connection with the synthesis of 1,2S-dihydroxycholecalciferol.
Hydroboronation ofa A14-unsaturated steroid occurs on the a-face (Scheme 1l), so that direct oxidation of the steroidal borane affords 1Sa-hydroxy- or 15-0x0steroids of the 14a configuration. If the steroidal borane is heated in situ it isomerizes to give the less strained 14P,15/3-adduct, which is oxidized to give 15P-hydroxy- or 15-0x0-14b-steroids. The synthetic applications are obvious.98
bxidation
l o r idat ion
Scheme 11
Full details are now available99 on the Simmons-Smith methylenation of cholest-5-ene derivatives. Last year's Report' O0 mentioned contradictory statements concerning the reactivity of such compounds; there now is no doubt that 5,6-methano-derivatives of both a- and /3-configurationsare readily prepared by suitable procedures. N.m.r. data are supplied for a variety of these products. Methylene addition is similarly non-stereospecific in the androstane series.l o ' 9'
98
99 loo lo'
C. Kaneko, S. Yamada, A. Sugimoto, and M . Ishikawa, Tetrahedron Letters, 1973, 2339. E. Mincione and D. Iocco, Ann. Chim. (Italy), 1972, 62, 285. L. Kohout and J . FajkoS, Coll. Czech. Chem. Comm., 1972, 37, 3490. Ref. 44, p. 318. L. Kohout and J. FajkoS, Coil. Czech. Chem. Comm., 1973, 38, 1415.
Steroid Properties and Reactions
337
An attempted methylene addition to the 4cc-hydroxy-~-nor-5-ene(127) gave the 5P,7P-cyclosteroid (128) in very low yield. Acetolysis of the 4cc-mesylate (129) caused the expected cleavage of the adjacent 5,6-bond, leading to the 6a-acetoxymethyl-B-nor-olefin(130).'02
OR
OH (127)
(128) R (129) R
= =
H MS
( 130)
Methylene addition to pregn-15-ene-17a,20-diolsis sterically controlled to give the 15a,l6a-adduct 13I), which was degraded by periodate to give the cyclopropyl ketone (132).Io3 Me
I
8: _ _ _ . _ I
(131)
The addition of dihalogenocarbenes to 5,6-unsaturated B-nor-steroids (133) is more complicated than Simmons-Smith methylene addition, which gave only 5a,7a- and 5/?,7P-cyclosteroids. Dichlorocarbene, generated *from phenyl(trichloromethyl)mercury, added to the olefin (133), but the resulting dichlorobicyclo[3,1,0] hexane system (134) rearranged spontaneously to give the 6,7ccdichloro-5-ene (135). Dichlorocarbene from other sources failed to react.
PhHgCCl, ___)
AcO
AcO
& A'
CI
( 1 33)
c1
(134)
C1
(1 35) '02
'03
J. FajkoS and J. Joska, Coll. Czech. Chem. C o m m . , 1972, 37, 3483. R . Wiechert, D . Bittler, and G.-A. Hoyer, Chem. Ber., 1973, 106, 888.
338
Terpenoidsand Steroids
Difluorocarbene gave a stable adduct analogous to (134), whereas chlorofluorocarbene gave C-6-isomeric adducts of type (134; F and C1 at C-6) and the rearranged 7a-chloro-6-fluoro-5-ene. The rearrangement is rationalized as an electrocyclic ring-opening of the dihalogenocyclopropane, with specific ionization of a 6a C-C1 bond. The 6a-F lacks this propensity for ioni~ation."~ Conjugate addition of nitromethane to a 1,4,6-trien-3-one,in the presence of t-butoxide, gave the la-nitromethyl derivative (136) in good yield. Nitropropane reacted only with difficulty, giving two isomeric products in low yield.lo5 Pyridazine-3,6-dione (137)'06 is less convenient than 4-phenyl-1,2,4-triazoline3,5-dione (t38)lo7as a dienophile for protection of the steroidal 5,7-diene system. The reaction of ergosteryl acetate with tetracyanoethylene"* has yielded some new products.'0g
Reduction.-The hydrogenation of androsta-1,4-diene-3,17-dione with some soluble catalysts has been studied in detail. The catalysts were dichlorotris(triarylphosphine)ruthenium, including phenyl and p-substituted phenyl derivatives ; the methoxy-compound provides the fastest reaction, with excellent selectivity for the A'-olefinic bond. The influence of added bases has also been explored ; Et,N and Et,NH are effective promoters of the hydrogenation.' l o Reduction of 17P-hydroxyoestra-4,6-dien-3-one with tritium and tris(tripheny1phosphine)rhodium chloride in dioxan gave the [6p,7p-3H2]-derivative with high specificity. ' Selective hydrogenation of 17-methyleneandrost-5-en-3~-ol over Pd-C catalyst gave 17P-methylandrost-5-en-3P-01, although other catalysts gave 17a + 17p mixtures. The products formed in the catalytic hydrogenation of 6P-acetoxy-steroidal 4-enes (139) depend upon the catalyst used.' l 2 Platinum causes preferential saturation of the olefinic bond, although some hydrogenolysis of the C-6 substituent also occurs, giving a mixture of the 5P-compounds (140) and (141); similar reactions occur in the absence of the C-1 substituent. When palladium
'
I04 105 106 107 108 109 110
111 112
P. Rosen and R. Karasiewicz, J . Org. Chem., 1973, 38, 289. M . Kocor, M . Gumulka, and T. Cynkowski, Tetrahedron Letters, 1972, 4625. P. E. Georghiou and G . Just, J . C . S . Perkin I , 1973, 888. Ref. 79, p. 305. Ref. 59, p, 261. A. L. Andrews, R. C. Fort, and P. W. Le Quesne, J . Org. Chem., 1973, 38, 237. S. Nishimura, T. Ichino, A. Akimoto, and K. Tsuneda, Bull. Chem. SOC.Japan, 1973, 46, 279. H. J . Brodie, C. E. Hay, and T. A. Wittstruck, J . Org. Chem., 1972, 37, 3361. K. Annen, R . Tschesche, and P. Welzel, Chem. Ber., 1973, 106, 576.
Steroid Properties and Reactions
339
is used, hydrogenolysis of the acetoxy-group affords a mixture of the A4- and As-olefinic products (143) and (144), probably through an intermediate n-ally1 palladium complex (142).
+
I
1
@ OAc
OAc (1 39)
HZ-Pt
Benzyl alcohol was the most effective of several alcohols examined as hydrogen donors for transfer-hydrogenation of unsaturated steroids with a Pd catalyst.' Quantitative yields were obtained in the following reductions : 17fl-hydroxy-5a; 17a-ethynylandrost-5androst-1-en-3-one -P 17fl-hydroxy-5a-androstan-3-one ene-3fl,l7p-diol--* 17wethylandrost-5-ene-3P,17P-diol ; and 3P-hydroxypregna5,16-dien-20-one-+ 3P-hydroxypregn-5-en-20-one.The resistance of 5,6-unsaturation to reduction is noteworthy. A 1,4-dien-3-one was reduced mainly at the A'-bond, and a 4,6-dien-3-one mainly at the A6-bond.'I3 Attempts to effect conjugate addition of benzyl alcohol to a 1-en-3-one (145) under alkaline conditions to give a 1-benzyloxy-3-ketone gave instead the 3-benzylidene-3-ketone Ph
'I4
R. Vitali, G . Caccia, and R. Gardi, J . Org. Chem., 1972, 37, 3745. R. Vitali, G . Caccia, and P. P. Castelli, Ann. Chim. (Italy), 1972, 62, 315.
340
Terpenoids and Steroids
(147).'14 It seems that the benzyloxide ion functions as a hydride-transfer reagent, reducing the olefinic bond in the 1-en-3-one: the benzaldehyde formed in the process then condenses in the usual way with the 3-ketone. The selective reduction of pregna- 14,16-dien-20-ones(148) to give 14-en-20ones (149) with triethylsilane is inconvenient, requiring a sealed tube. The reagents Et,(EtO)SiH or (Me,SiH),O avoid this problem, readily giving the 14-en-2Cbone in high yield. Two convenient routes from the 14-ene to the 14P-hydroxy-derivative are described.'
'
COMe
COMe
Reduction of 5a-cholest-8(14)-en-7-one(150) with Li-NH, or Zn-HOAc gave the new 5a,l4P-cholestan-7-one (151).' l 6 The result is attributed to control by orbital overlap'" rather than by the stability of the product, in view of earlier indications that a bulky 17P-alkyl group destabilizes the 148- more than the 14a-isomer, and so reverses the usual stereochemical preference.
H
Oxidation.-Marker's oxidation of Sa-lanost-8-en-3P-yl acetate (1 52) with chromic acid to give two ketonic products was recently re-examined, and was reported to afford three products, the 8-en-7-one (157), 9(1l)-en-7-one (154), and 8-ene-7,ll-dione (155)."* A new report"' now shows that the 8-en-7-one (157) is not a primary product, but results from isomerization of the 9(1l)-en-7-one (154) during chromatography. Oxidation of the 7,9(11)-diene (153) with HOAcH , 0 2 is shown to give the Scc-isomer (156) of the 9(11)-en-7-one, and not the 8p-isomer (154) as was thought previously. The unstable 8a-configuration is supported by epimerization to give the 8P-isomer in contact with acidic alumina, and was confirmed by an alternative synthesis involving rearrangement of the
E. Yoshii, H. Ikeshima, and K. Ozaki, Chem. and Pharm. Bull. (Japan), 1972,20, 1827. I. Midgley and C. Djerassi, J.C.S. Perkin I, 1973, 155. l L 7 Ref. 3, p. 197. Ref. 44, p. 328. 11)
Steroid Properties and Reactions
34 1
7P,SP-epoxy-9(11)-eneon silica gel.' l 9 Other authors'20 report that the oxidation of the 8-ene (152) is very sensitive to the reaction conditions; it was found difficult to reproduce earlier experiments. The major product with CrO, in CHC1,-HOAc at room temperature for 7 days was the 9(11)-en-7-one (154), but the only product detected after brief reaction was the 7,9(11)-diene (153). Interpreting the results of these and related experiments, including oxidations at 50 or 60°C, the reaction sequence in Scheme 12 is proposed to account for the observed products. The 8-ene-7,ll-dione (155) may arise by an alternative route at 90 "C, the conjugated 8-en-7-one (157) being a possible intermediate. The diene (153) can be prepared in high yield by the action of H,O,-HOAc on the 8-ene (152), providing support for a proposed 8,9-epoxy intermediate (158). These authors also report the characterization of the 8or-lanost-9(1l)-en-7-one (156).'
i
qt
or t-
Scheme 12 R. B. Boar, J . F. McGhie, and D. A. Lewis, J . C . S . Perkin I , 1972, 2590. E. V. Lassak, J. T. Pinhey, and J. J. H. Simes, Austral. J . Chem., 1973, 26, 1051.
342
Terpenoids and Steroids
The pathways of oxidation of cholesterol by oxygen have been clarified by recent work.12' Formation of the 5a-hydroperoxy-6-ene (159) occurs only with excited singlet molecular oxygen, whether this is produced by chemical or by photosensitization methods. Ground-state (triplet) molecular oxygen will react with cholesterol under a variety of conditions, always giving the 7P-hydroperoxyderivative (160) as the major initial product. The oxidation at C-7 probably involves attack of oxygen on an allylic free radical, which may be generated either thermally or by irradiation with daylight, u.v., or 6oCoy-radiation, whether the cholesterol is in the crystalline state, in solution, or dispersed in aqueous sodium stearate solutions. The extreme ease of auto-oxidation of cholesterol is dramatically demonstrated by the detection (g.1.c.) of traces of the 7P-hydroperoxide after mere recrystallization of highly purified cholesterol, without deliberate irradiation.' 2 1 The 7P-hydroperoxide is found to be derived in part from epimerization of the 7a-hydroperoxy-derivative, a most unusual reaction ; 6~-hydroperoxycholest-4-en-3-one has also been identified among the autooxidation products.' 2 2 The oxygenation of cholesterol by several different enzymes has been shown to give 7-hydroperoxy-derivatives, the normal products of radical-induced oxygenation. 2 3 The possibility that the 5a-hydroperoxy-6-ene (159) might be an intermediate in the enzymic process was ruled out by separate experiments, so it seems unlikely that singlet excited oxygen is implicated in the enzyme reactions. An improved procedure is described for preparing 7-dehydrocholesterol uia 7-bromocholesteryl benzoate. 24
HO
OOH
A broad survey125of the oxidative rearrangement of olefins with thalliurn(~rr) salts mentions the reaction between 5a-cholest-2-ene and thallium trifluoroacetate, which formed the A-nor-aldehyde (161) in only 15 % yield. The trinitrate gave higher yields with simple cycloalkenes, but is not mentioned in connection with steroid olefins. An unexpected oxidation of steroidal 14-enes (162) with two equivalents of mercury@) acetate gave the 8( 14)-en-15-ones(163)' 26 12'
' 2 5
L. L. Smith, J. I. Teng, M. J. Kulig, and F. L. Hill, J. Org. Chem., 1973, 38, 1763. J. I. Teng, M. J . Kulig, L. L. Smith, G . Kan, and J. E. van Lier, J. Org. Chem., 1973, 38, 119. J. I. Teng and L. L. Smith, J . Amer. Chem. SOC.,1973, 95, 4060. V. P. Vendt, R. I. Yakhimovich, V. A. Boguslavsky, Y. F. Yaroshenko, N. V. Lopotko, and N. V. Kukharenko, Khim. Farmatsvet. Zhur., 1973, 7 , 45. A. McKillop, J. D. Hunt, F. Kienzle, E. Bigham, and E. C. Taylor, J. Amer. Chem. Soc., 1973,95, 3635. E. C. Blossey and P. Kucinski, J . C . S . Chem. Comm., 1973, 56.
343
Steroid Properties and Reactions
Aromatic Compounds.-Oestradiol reacts with one molar equivalent of benzoyl peroxide in refluxing benzene to give the 2-monobenzoate (164) of 2-hydroxyoestradiol in 60 % yield.’27 The benzoate was hydrolysed quantitatively by methanolic HCl to give 2-hydroxyoestradiol (165), providing the simplest synthesis so far reported for this important oestrogen metabolite. Fremy’s salt [ON(SO,K),] in acetone-aqueous acetic acid oxidizes oestrone or oestradiol to give the o-quinones (166) and (167), which are reduced by KI-acetic acjd to give the separable catechols (165) and ( 168).’28 2-Hydroxyoestrogens [e.g. (165)] are difficult to handle, and like other catechols suffer oxidation so readily that ordinary chromatographic procedures often lead to total loss. Impregnation of paper or silica gel with ascorbic acid, an antioxidant, provides excellent protection for the steroid, without significantly altering the chromatographic characteristics.
’
0 O
l
d
?
HO (164) R (165) R
HO
0
0
=
=
BZ H
e.:”l:
The behaviour of steroids with an aromatic ring A towards RuO, depends upon the aromatic substituents present. A 3-acyloxy-group alone appears to protect the aromatic ring against oxidation : the main reaction is then benzylic 12’
lZ9
I. Yoshizawa, M. Tamura, and M. Kimura, Chem. and Pharm. Bull. (Japan), 1972, 20, 1842. H. P. Gelbke, 0. Haupt, and R. Knuppen, Steroids, 1973, 21, 205. H. P. Gelbke and R. Knuppen, J. Chromatog., 1972, 71, 465.
344
Terpenoids and Steroids
oxidation, giving the 9cr-hydroxy-6-0x0-derivative (169). Each of several compounds with more than one substituent in ring A suffered cleavage of the aromatic ring to give a diacid (170).l3O
HO,C
AcO
0
HO,C
The novel reagent tribenzenesulphenamide [N(SPh),] reacts with phenols, including oestrone and oestradiol, to give quinone phenylthioimines (17 1) and (1 72). The reaction is believed to involve the free radical (PhS),N.
The 3-(trans-2’-butenyl) ether (173) of oestradiol, obtained by alkylating oestradiol with trans-Zbutenyl chloride, undergoes Claisen rearrangement at 200 “C to give both 2- and 4-(l’-methylallyl)oestradiols.’32These products (174) and (175) were 1 : 1 mixtures of isomers at the 1’-position, showing that the chiral steroid framework does not cause any asymmetric induction in the migration of the alkenyl group from oxygen to carbon. A modified alkylation procedure converted oestradiol in low yield into the C-alkylated product, 1Ofi-(trans-2’butenyl)-l7P-hydroxyoestra-l,4-dien-3-one (176). The dienone (176) rearranged at 120°C by a [3s, 3s] sigmatropic process to give almost equal proportions of the 2- and 4-(1’-methylallyl)derivatives (174) and (175). In this case, however, the 1’-methylallylgroups were introduced with 88 : 12 preference for a particular C-1‘ configuration, as revealed by the optical activity of samples of 2-methylbutyric acid obtained by an oxidative degradation of the steroid to isolate C-2 with the attached carbon chain. The stereochemistry of the alkylated oestradiols is used to infer the configurations of transition states in the sigmatropic rearrangements by which they were formed.’32 D. M. Piatak and 0. Ekundayo, Sreroids, 1973, 21, 475. Sir D. H . R. Barton, I. A. Blair, P. D. Magnus, and R. K . Norris, J.C.S. Perkin I , 1973, 1031. A . W underli, J. Zsindely, H.-J. Hansen, and H . Schmid, Helu. Chim. Acta, 1973, 56, 989.
lJo
132
Steroid Properties and Reactions
345
Alkynes and Cyc1opropanes.--The recent literature has contained several examples of the cleavage of 17a-ethynyl-17j3-alcohols to give 17-ketones. The reaction occurs with strong bases or with the Fetizon oxidant (Ag,CO,-celite).' 3 3 A systematic study now shows that either Ag2C0, or A g 2 0 reacts with the steroid in DMSO to give the 17-0x0-compound in excellent yield, but other solvents are less efficient in promoting the cleavage reaction. A mechanism involving the formation and bimolecular decomposition of the silver acetylide derivative (177) is proposed.' 34
Some novel cyclopropenyl and allenic derivatives have been prepared at the C-3 and C-17 positions, by adding difluorocarbene to suitable ethynyl derivatives, with subsequent reactions of the type illustrated in Scheme 13 for the C-3
derivative^.'^^ 133 134
135
Ref. 44,p. 314. R. Vitali, S. Gladiali, and R. Gardi, Gazzetta, 1972, 102, 673. P . Crabbe, H. Carpio, E. Velarde, and J. H. Fried, J . Org. Chem., 1973, 38, 1478.
346
Terpenoids and Steroids
AcO
I_____i a C
H
O K I1 CH
CO,H
Reagents: i.:CF,; ii, Et,NCF,CHCIF; iii, HCO,H, r.t.; iv, HCO,H, reflux
Scheme 13
The ethynyl-glycol (178) afforded the D-homo-ketol (1 80) on reaction with alkali.136 The hydroxy-aldehyde (179), which is known to rearrange to the D-homo-ketol, seems a likely intermediate which could result from cleavage of the ethynyl compound by base.
PH
H. Chwastek, R. Epsztein, and N. Le Goff, Tetrahedrori Letters, 1973, 179.
347
Steroid Properties and Reactions
The reaction between fi-0x0-cyclopropanes in the steroid series and phenyltrimethylammonium tribromide (Jacques' reagent) leads to cyclopropyl ring cleavage whenever the a-position is the favoured site for bromination of the ketone. Complex reaction mixtures generally result, but major products from a selection of reactions of this type are illustrated in Scheme 14.' The generalized diagram (181) illustrates two possible mechanisms (a and b ) ; a third variation has been described for 4a,5-cyclo-~-homo-5a-cholestan-2-one (p. 352), another fi-oxocyclopropane analogue.
Scheme 14
Significant differences have been demonstrated between the reactions of 11-oxygenated 9P,19-cyclo-steroids and analogous 4,4,14a-trialkylated (triterpenoid) compounds derived from Buxus alkaloids. The triterpenoid cyclopropyl alcohol (182) reacted with acid to give the 9(10-+ 19)-abeo-diene(183), but a related 9,!?,19-~yclopregnan11-01 derivative gave instead the rearranged alcohol (184) and the diene-dione (185). The results are rationalized on the basis of conformational differences. 38
'38
V . &my, Coll. Czech. Chem. Comm., 1973, 38, 1563. S. M. Kupchan, J . W. A. Findlay, P. Hackett, and R. M . Kennedy, J . Org. Chem., 1972,31, 2523.
348
Terpenoids and Steroids
4 Carbonyl Compounds
Ketones: Reactions at the Carbonyl Group.-Lithium tri-s-butylborohydride reduces cyclic ketones with high stereoselectivity (9Ck99.5 %) for formation of the less stable alcohol, and should offer an excellent alternative to known methods for preparing steroidal axial alcohols. The reagent is easily prepared by mixing lithium trimethoxyaluminohydride and tri-s-butylborane in THF.' 3 9 The 15-0x0-group in 14-deoxycardenolides (186) is reduced by NaBH, to give the 15a-hydroxy-derivative in the 14/?-series,or the 15/?-hydroxy-derivative in the l4a-series.' 40 The stereochemistry of reduction is not significantly dependent upon the configuration at C- 17. The deuterium-labelled (19R)-19-hydroxy-19methyl-5a-androstane-3,17-dione (187) is transformed by base into the 3pdeuterio-3a-hydroxy- 19-ketone (189), showing that the mechanism of this oxidation-reduction process involves an internal hydride transfer from C-19 to C-3.141 A boat conformation of ring A appears to be necessary in the transition state (188).
Me
D HO
139
140 14'
H
H
H . C. Brown and S. Krishnamurthy, J. Arner. Chern. SOC.,1972,94,.7159. M. Okada and Y. Saito, Chern. and Pharm. Bull. (Japan), 1973,21, 388. J. Wicha and E. Caspi, J . Org. Chem., 1973, 38, 1280.
Steroid Properties and Reactions
349
Extensive data on the hydrogenation of alkylcyclohexanones over various catalysts 142 indicate useful possibilities for stereoselection, which may find applications in steroid chemistry. The reaction of 4-en-3-ones with diborane, followed by acetic anhydride, is known as a useful route to 5a-steroidal 3-enes. Detailed studies, using perdeuteriodiborane, have shown that the carbonyl group is first reduced, hydroboronation of the olefinic bond following (Scheme 15).
Ac ii
1
&
0D..
'B /
liii
Ac
Reagents: i, B,D, (one mole); ii, Ac,O; iii, B,D, (excess).
Scheme 15
It is suggested that acetic anhydride functions through cyclic transition states, in the manner illustrated. An 8(14)-en-7-one (190) reacted with diborane only at the 7-oxo-group, the olefinic bond being too hindered for attack: the endproduct after reaction with acetic anhydride was the 7,14-diene (191).143 142
143
S. Mitsui, H. Saito, Y. Yamashita, M. Kaminaga, and Y. Senda, Tetrahedron, 1973, 29, 1531. I. Midgley and C. Djerassi, Tetrahedron Letters, 1972, 4673.
350
Terpenoids and Steroids
(191)
A mixture of a 3-0x0-steroid (192) and an ammonium salt is reduced selectively by sodium cyanoborohydride (NaBH,CN) to give the corresponding 3-aminosteroid (194), the 3P-isomer predominating. 144 The reaction occurs through the iminium ion (193),which is readily reduced, although the cyanoborohydride is unreactive towards ketones. Suitable ammonium salts include NH4+ OAc-, MeNH,' C1-, and Me,NH,+ C1-, which give respectively the 3-amino-, 3-methylamino-, and 3-dimethylamino-compounds in high total yields. 17-0x0and 20-0x0-steroids are inert under the conditions used, permitting selective amination at C-3 in 3,17-or 3,20-diketones.
Grignard reactions (MeMgI)of the ~-nor-Sfl-and ~-nor-Sa-steroidal6-ketones (195) and (196) gave mainly the 6a-hydroxy-6,!3-methyl(197) and 6p-hydroxy-6amethyl (198) derivatives, respectively. Both isomeric products, as 3-acetates, were dehydrated by SOCl, to give the 6-methyl-5-ene (199). The Oppenauer oxidation product of the corresponding unsaturated alcohol was the 6P-methyl-4en-3-one (200),which appears to be the stable isomer in the ~-nor-series.'~' The 21 -hydroxypregnan-20-one side-chain, as the 21-mesylate (201), reacts with Me,S(:CH,jO to give the cyclopropane aldehyde (202). The likely '41 145
M.-H. Boutigue and R . Jacquesy, Bull. SOL..chim. Frunce 11, 1973, 750. J. Joska, J. FajkoS, and F. Sorm, Coll. Czech. Chem. Comm., 1973, 38, 2121.
35 1
Steroid Properties and Reactions
(195) 5P-H
/
mechanism is illustrated (Scheme 16). By reduction of the aldehyde, and dehydration of the resulting alcohol (203), a typical cyclopropylmethyl ring expansion led to the 17~-cyclobutenylandrostane derivative (204).
(201)
0
II
I
Me,SLCH2-C-O-
CHO
0
CH pOMs
I
I1
C=CH2
Me2S3
/Y
Me,S'-CH,
(204)
(203)
Scheme 16 146
R. Wiechert, M. Maikowski, G.-A. Hoyer, and H. Laurent, Chem. Ber., 1973,106, 882.
Terpenoidsand Steroids
352
Homologation of 3a,5-cyclo-5a-cholestan-2-one (205) with CH,N,-AlCl, gave the 4a75a-cycloketone(206), which afforded the 5P-methyl-2-ketone (207) on hydr~genation.'~'Other reactions of this novel system include the formation of the dienones (208)and (209) with phenyltrimethylammonium perbromide.
[w;]-Rm (205)
Br,
(206)
(208) R = H
(209)
R
=
Br
Ethyl cyanoacetate readily condensed with 5a-cholestan-3-one to give a mixture of geometrically isomeric cyano-esters (210), which were not separated. Catalytic hydrogenation gave the 3a- and 3P-cyanoacetates (21 l), in which the former predominated by a ratio of 5 : 1; alkaline hydrolysis of each isomer, with decarboxylation, gave 5a-cholestan-3a-yl- and -3P-yl-acetic acids (212).'48
Pregnan-20-ones of the normal or 17a-series react with ethoxyacetylene and BF, to give ethyl norchoL20(22)-enates (213), which may be hydrogenated to afford a mixture of norcholanic acids, epimeric at C-20.'49
(2 13) 147
'" 14'
V. Cerny, M. BudeSinsky, and F. Sorm, Coll. Czech. Chem. Comm., 1973, 38, 565. D. Kontonassios, C. Sandris, and G. Tsatsas, Bull. SOC.chim. France I I , 1973, 622. U. Valcavi and S. Innocenti, Farmaco, Ed. x i . , 1972, 27, 955.
Steroid Properties and Reactions
353
5-0xo-3,5-seco-~-norcholestan-3-ol (214) exists largely as a mixture of the hemiacetals (215 ) and (216). Methanol or ethanol afforded the corresponding 5-alkoxy-compounds (217) and (218), which were readily equilibrated in the presence of the merest trace of acid. The oxonium ion (219) is considered to be the intermediate in these transformations.' 5 0
@ @ OR
(215) R = H (217) R = Me or Et
OR
(216) R (218) R
=
=
H Me or Et
Two further mild procedures have been described for the regeneration of ketones from t h i ~ a c e t a l s . ' ~The ~ thioacetal may be stirred with silver oxide in aqueous methanol under reflux, liberating the ketone in 73-85 % yields,'52 or may be treated with copper(r1) chloride in aqueous a ~ e t 0 n e . lTwo ~ ~ novel ' ~ ~ can be employed as protection for carbonyl groups classes of a ~ e t a l , which and removed under non-acidic and aprotic conditions, seem likely to find uses in steroid chemistry. These are the mono- or di-2,2,2-trichloroethyl acetals (220), removed by zinc dust in ethyl acetate or THF, and the bromomethylethylene acetals (221), cleaved almost quantitatively by zinc in refluxing methanol to regenerate the carbonyl group. Chemists who work with corticosteroids should
(R = Et or CH,CCl,)
150
151 152
153
154
J. T. Edward, M. Kaufman, R. K. Wojtowski, D. M . S. Wheeler, and T. M. Barrett, Cunud. J. Chem., 1973,51, 1610. Ref. 44, pp. 340-341. D. Gravel, C. Vaziri, and S. Rahal, J.C.S. Chem. Comm., 1972, 1323. K. Narasaka, T. Sakashita, and T. Mukaiyama, Bull. Chem. SOC.Japan, 1972, 45, 3724. J. L. Isidor and R. M. Carlson, J. Org. Chem., 1973, 38, 554; E. J. Corey and R. A. Ruden, ibid., p. 834.
354
Terpenoidsand Steroids
note a possible health hazard in the use of formaldehyde and hydrochloric acid to protect the dihydroxyacetone side-chain as its bismethylenedioxy (BMD) derivative. The reagent mixture is reported to generate detectable quantities of bichloromethyl ether, a volatile and particularly potent carcinogen. 5 5 Oxalyl chloride converted a 3P-acetoxy-5-en-7-one (222) into the 7-chloro5,7-diene (223),or the 3-deoxy-5-en-7-one (224)into the 7-chloro-4,6-diene (225). Some further transformations of the 7-chloro-5,7-diene are described.' 5 6
(222) R (224) R
= =
(223)
P-OAc
H
Reactions involving Enols and Enolate Ions.-Lithium hexamethyldisilazane is a convenient basic reagent for rapid generation of the unstable A2*4-dienolateion (226) from A4-3-oxo-steroids, under kinetic control.'57 The A2-4-dienolatehas 278 nm), or methylated been trapped as the t-butyldimethylsilyl ether (227 ; A,, (MeI-HMPA) to give a mixture of the 2a- and 2p-methyl-A4-3-ketones(288).
-0
But- Si- 0
The lithium cation is more effective than other alkali metals in preserving the unstable A2.4-enolate,'5 8 which is prone to isomerization to give the thermodynamically favoured A3,5-enolate. Other authors' 5 9 report the related observation that the site of methylation of cholest-4-en-3-one depends upon the base used to generate the dienolate ion. Although t-alkoxides are well known to lead to C-4 methylation, lithium dialkylamides with methyl iodide afford predominant alkylation at C-2 (mainly 2P-Me) through methylation of the kinetically favoured A234-dienolateion. Trityl-lithium, however, caused formation of 4,4-dimethylcholest-5-en-3-one.' 5 9 155
I56
157 158 159
B. R. T. Keene, Chem. in Britain, 1973, 9, 424. R. W. Guthrie, A. Boris, F. A. Mennona, J. G. Mullin, and R. W. Kierstead, J. Medicin. Chem., 1973, 16, 65. M. Tanabe and D. F. Crowe, J.C.S. Chem. Comm., 1973, 564. Cj: ref, 3, p. 199. R. A. Lee, C. McAndrews, K. M. Patel, and W. Reusch, Tetrahedron Letters, 1973, 965; R. A. Lee and W. Reusch, ibid., p. 969.
355
Steroid Properties and Reactions
The stability of metal enolates towards LiAlH, provides a novel means for effecting certain selective reductions of carbonyl functions. The 1,3,5-trien-3olate (229), obtained from prednisone-BMD by reaction with sodium or lithium bistrimethylsilylamide, or with trityl-lithium, can be reduced in situ with LiAlH, to give good yields of prednisolone-BMD (230). Trityl-lithium offers the advantage of a visible colour change, and gives better yields. Pregn-4-ene-3,11,20trione similarly gave 11(3-hydroxyprogesteroneafter the 3- and 20-oxo-functions had been protected as lithium enolates. The enolates decay rather rapidly, so reactions were carried out at - 78 "C in THF, under argon.16'
(229) R (230) R
= =
0 fi-OH
Oxygenation of a solution of an oestr-4-en-3-one derivative (231)in anhydrous D M F or DMSO containing potassium acetate at 120 "C leads to the 3-hydroxyoestra- 1,3,5(lO)-trien-6-one (233), probably via the 4-ene-3,6-dione (232) as an intermediate. Jones oxidation of the 4-en-3-one (231) gave the 4-ene-3,6-dione in low yield.'6 1
\ 0o & -& + H 'o *
0
(231)
(232)
0
(233)
Oxygenation of 3/3-hydroxy-Sa-lanost-8-ene-7,1 l-dione (234), in the presence of potassium t-butoxide to enolize the 7-oxo-function, gave the diosphenol(235) in quantitative yield. The 3,6-diacetate of the diosphenol could be oxidized further by SeO, to afford the 7,11,12-trione (236).162 160
Sir D. H. R. Barton, R. H. Hesse, M. M. Pechet, and C. Wiltshire, J.C.S. Chem. Comm.,
16'
1972, 1017. H. Hofmeister, H. Laurent, and R. Wiechert, Chem. Ber., 1973, 106, 723. W. Kreiser and W. Ulrich, Annulen, 1972, 761, 12 1.
62
356
Terpenoids and Steroids
e
l
7
&jXpl7
AcO
HO OH
OAc
As a key step in the synthesis of lu-hydroxy-vitamin D,, la-hydroxycholesta4,6-dien-3-one (237) was reduced with lithium-ammonia, with added ammonium chloride as a proton source, to give la-hydroxycholesterol(238) directly in 60 yield. Rapid alternating reductions and protonations account for the success of this p r 0 ~ e d u r e . IThe ~ ~ isomerization of cholest-5-en-3-one to give the conjugated 4-en-3-one by the simultaneous action of an acid and a base showed a small but significant difference between rates of reactions promoted by the (R)- and (S)-isomers of NN-dimethyl-1-phenylethylamine, with rn-nitrophenol as the acid.'64
<:
(237)
Ih3
lh4
1
Sir D. H. R. Barton, R. H . Hesse, M. M . Pechet, and E. Rizzardo, J . Amer. Chem. Soc., 1973, 95, 2748. A. Fauve, A. Kergomard, and M. F. Renard, Tetrahedron Letters, 1973, 607.
Steroid Properties and Reactions
357
Thallium(II1)compounds have recently found wide use in organic chemistry as oxidants for unsaturated compounds, but few uses have yet been found in steroid chemistry. Saturated and ap-unsaturated 3-oxo-steroids are now reported to react with thallium triacetate in 95 % acetic acid :16' 5a-cholestan-3-one isolated in underwent ring contraction to give 2a-carboxy-~-nor-5a-cholestane, useful overall yield as the methyl ester. 1,4-Dien-3-oneswere obtained in acceptable yields (43-78%) from a variety of l-en-3-ones and 4-en-3-ones. The detailed mechanism of dehydrogenation is not yet known, but presumably involves attack of the reagent on an enolic derivative of the enone. There is no mention of the formation of 1,4,6-trien-3-ones as by-products, although these are always formed to some extent in similar dehydrogenations with DDQ.'66 Common procedures for dehydrogenating 4-en-3-ones to give 4,6-dien-3-ones were inefficient with oestr-4-en-3-one derivatives, but heating at 50 "C for 2-3 h with chloranil in an alkanol gave a good yield of the required dienone; the byproducts were phenolic derivatives resulting from 1,2-dehydrogenation." The side-chain a-ketol system of 21-hydroxypregn-4-ene-3,20-dione (239) forms a complex with cobalt(I1) ions in solution, particularly in solvents of low water content. The complex is thought to involve the ene-diol structure (240); its formation confers acidic properties on the solution, and has been detected by spectroscopic methods.' ti
'
CHOH
I
CHZOH I
"(y+
......
2,4,4,6-Tetrabromocyclohexa-2,5-dienone (241) selectively brominates afiunsaturated ketones at the a'-position. Cholest-4-en-3-one gave the 2a-bromoderivative (242), in which ring A is considered to be in the chair conformation, and a second product which, in the authors' opinion, is also the 2a-bromoderivative but with ring A in a boat conformation.'68 The latter product was 0 8
Rr --
Br
(241) 165
lh6 lb7 lh8
(242)
A. Romeo and G. Ortar, Tetrahedron, 1972, 28, 5337. Ref. 3, p. 189. C. H. Eger, C. Yarborough, M. Greiner, and D. A. Norton, Steroids, 1972,20, 349,361. V . Calo, L. Lopez, G . Pesce, and P. E. Todesco, Tetrahedron, 1973, 29, 1625.
358
Terpenoids and Steroids
slowly transformed into the former in chloroform solution. The structure of the second compound was assigned on the basis of its n.m.r. spectrum and its negative Cotton effect at 243nm, presumed to be controlled by the bromosubstituent according to the axial halogeno-ketone rule.'68 The Reporter suggests that the 2P-bromo configuration, with ring A in the inverted form (243)
and the Cotton effect controlled by the chirality of the enone system'69 rather than by the bromo-substituent, is equally compatible with the data cited, and more probable than the existence of separable conformers of the 2a-isomer. Isomerization of the 28- into the 2a-bromo-derivative in solution seems reasonable. This interpretation is similar to the situation found for 2-acetoxy-4-en-3ones. Full details have appeared of a six-step degradation of the lanosterol side-chain (244) to give 3~-acetoxy-4,4,14a-trimethyl-5a-pregn-8-en-2O-one (247) (Scheme 17).' '' The 24-0x0-compound (245), on bromination under kinetic control,
'
--<
I -Iw
(245) liii
fi0 VI
t
YHO -
IV
t
(247)
Reagents: i, Monoperphthalic acid; ii, BF,; iii, 2Br,-H ; iv, LiBr-Li,CO,-DMF; v, KMnO,; vi, 02--[2,2'-dipyCu"(OAc),I-py-DM F. +
Scheme 17 169
Ref. 44, p. 288: ref. 59, p. 234. S. L. Patashnik, S. Burstein, and H. L. Kimball, Steroids, 1963, 2, 19; see also ref. 44, p. 281 and ref. 59, p. 228; R. D. Burnett and D. N. Kirk, J . C . S . Perkin I , 1973, 1830. I . H. Briggs, J . P. Bartley, and P. S. Rutledge, J . C . S . Perkin I, 1973, 806.
359
Steroid Properties and Reactions
gives a mixture of the 23- and 25-bromo-ketones and the 23,23-dibromo-ketone : dibromination under thermodynamic control, however,gives the 23,25-dibromide (246). The subsequent dehydrobromination and oxidation steps employ wellknown reagents. Glyoxylic acid can be used in place of formaldehyde in a Mannich reaction. A 3-0x0-steroid reacts with glyoxylic acid and two moles of morpholine in ethanol to give the y-morpholinobutenolide (248), which can be hydrolysed and acetylated to give the y-acetoxybutenolide (249).17* The isomeric 3-benzylidene~-nor-5cc-cholestan-2-ones(250) and (251), as well as the four derived epoxyketones, have been prepared and characterized.' 7 3
o&
H
(248) R = -N (249) R
=
A 0 U
H
Ph H
(250)
OAC
Enolic Derivatives, Enamines, and their Reactions.-The enol esterification of testosterone acetate with heptafluorobutyric anhydride in acetone gives the ;: of 2,4-dienol ester. Both products have 3,5-dienol derivative with less than 1 been fully characterized, and their sensitivities to electron capture detection have been compared. 174 Experiments with 6,7-ditritiated oestr-4-en-3-ones indicate that enol ether formation occurs mainly by loss of the 6fl-hydrogen (or tritium) atom, although there was evidence in one case for slight loss of tritium also from C-7, a reaction which is not readily explained. The results indicate that the tritium atoms, introduced by catalytic tritiation of 6-dehydrooestrone, are mainly in the 6cr,7a-~onfigurations.~ ' An unexpected solvolysis was observed when a 6P-fluoroprogesterone derivative (252) was treated with triethyl orthoformate and an acid catalyst. Instead of the required 6-fluoro-3,5dien-3-01 ether (253),the 3,6-diethoxy-diene (254) resulted, although the isomeric
(252) 17' 173 74 175
J. Schreiber, C.-G. Wermuth, and A. Meyer, Bull. Soc. chim. France I l , 1973, 6 2 5 . J. Muzart and J . P. Pete, Bull. SOC.chim. France 11, 1973, 1376. L. Dehennin and R. Scholler, Tetrahedron, 1973, 29, 159 1. E. J. Merrill and G. G . Vernice, J . Labelled Compounds, 1 9 7 3 , 9 , 4 3 .
360
Terpenoids and Steroids
6a-fluoro-compound gave the 6-fluoro-dienol ether (253). The difference is associated with the axial character of 6P-F, facilitating its ionization and replacement by ethoxide.' 7 6
x (253) X (254) X
=
=
F OEt
Attempts to repeat the preparation of the la,5a-cyclo-6-ketone (255),reported in 1968,' 7 7 have been unsuccessful.' 7 8 Treatment of the 5P-hydroxy-6-ketone (256) with thionyl chloride gave an unstable enol sulphite (257) which decomposed spontaneously with evolution of SO,. Chromatography on alumina converted the enol sulphite into the 4-en-6-one (258), the 5a-hydroxy-6-ketone (259), and the dienone (260). Hydrolysis with aqueous acid gave similar products as well as the 7a-hydroxy-ketone (262). The 5a-hydroxy- and 7a-hydroxy-ketones are thought to arise from an intermediate zwitterion (261) formed by loss of SO, from the sulphite, a reaction which has precedent.' 79
\
\\ AcO
AcO
AcO
0 (255)
OH0 (256) 5p-OH (259) 5u-OH
* \
0
(257)
0
0-
(258)
11'
"*
I No
//s
(260)
F. S. Alvarez and A. Prince, J . Org. Chem., 1972, 37,2920. S. B. Laing and P. J. Sykes, J . Chem. SOC.(C), 1968, 937. P. E. Georghiou and G . Just, J . C . S . Perkin I , 1973, 70. J. Levisalles, E. Rose, and I . Tkatchenko, Chem. Comm., 1969, 445
Steroid Properties and Reactions
36 1
The reaction of 3-acetoxycholesta-3,5-diene(263) with bromine or hypobromous acid has been assumed to be a simple reaction, leading to 6P-bromocholest-4-en-3-one, which can isomerize slowly under suitable conditions to give the 6a-bromo-derivative. Initial 6P-attack is stereoelectronically controlled. A new and detailed study has revealed unexpected complexities (Scheme 18).
Scheme 18
Depending upon reaction conditions, the first-formed intermediate, probably the 6P-bromo allylic cation (264), may lose Ac’ to give the 4-en-3-one (265), or may add a nucleophile at C-3 or C-5. The suggested reaction paths are too many and complex to detail here, but Scheme 18 indicates some of the possibilities. Side-reactions lead to 2a,6a- and 2a,6~-dibromocholest-4-en-3-ones and aromatized products, especially when the reactions are allowed extended time. The side-reactions seem best avoided by use of a molar proportion of bromine in acetic acid containing collidine, to suppress acidity.’ 8o The Simmons-Smith reagent (CH,I,-Zn) converts en01 trimethylsilyl ethers into cyclopropanol derivatives, which are rearranged by bases to give a-methylated ketones. The reaction has been applied to the A’,’-dienol trimethylsilyl ether (266) derived from testosterone, giving the 3,4-methylene derivative (267), and thence 4-methyltestosterone (268). Methanolysis of the cyclopropyl trimethylsilyl ether (267) affords the free cyclopropanol analogue.I8 Similar 180 181
P. B. D. de la Mare and B. N. B. Hannan, J.C.S. Perkin II, 1973, 1086. J. M. Conia and C. Girard, Tetrahedron Letters, 1973, 2767.
362
Terpenoids and Steroids
reactions were reported for smaller alicyclic molecules.'82 Enamines [e.g. (269), (27l)] derived from steroid ketones are similarly converted into cyclopropylamines by methylene addition, using either diethylzinc and di-iodomethane or diazomethane-copper(1) chloride. 8 3 The cyclopropylamines are cleaved to give ketones by heating in aqueous alcohols, although attempts at acid- or basecatalysed cleavage were unrewarding. Two useful alkylations by this method are illustrated (Scheme 19). Many similar alkylations of alicyclic systems are
'
Me
(266) R = Me,SiO (269) R =
c
N-
c
(267) R = Me,SiO (270) R =
(268).
N-
Scheme 19
described, some accompanied by ring homologation through incorporation of the methylene group of the cyclopropane ring into the main ring system. The bicyclic analogue of the 3,4-methylene-3-pyrrolidino-Assteroid (270) gave the 4-methyl-4-en-3-one and the A-homo-ketone (272), in ca. 4 : 1 ratio.'83 Reactions involving Oximes, Hydrazones, and Related Derivatives.-The reductive elimination of a 15~,16~-epoxyandrostan-17-one (273) with hydrazine to give the 16-en-15P-01 (274) is an unreliable route to 15-oxygenated derivatives, so a new variant of the reaction is welcome. The epoxy-ketone reacts with hydrazine and toluene-p-sulphonic acid in air to give the saturated 15P-alcohol (275) G . M . Rubottom and M. I . Lopez, J . Org. Chem., 1973, 38, 2097. M . E. Kuehne and J. C. King, J . Org. Chem., 1973, 38, 304.
363
Steroid Properties and Reactions
d i r e ~ t 1 y . IDi-imide, ~~ resulting from atmospheric oxidation of hydrazine, is suggested as the reducing agent for the olefinic bond ; yields are good. Several other routes to 15-oxygenated androstanes were also explored, but could not compete with the modified epoxy-ketone-hydrazine route. 184
2?
OH
Attempted preparation of Sa-androstan-11-one from the 3,11,17-trione under Huang-Minlon conditions gave the 11-one in only 20 % yield. The main product was the 11-hydrazone, isolated as its N-isopropylidene derivative (azine) after treatment with acetone., (The Reporter has found that ~-homo-Sa-androstane3,11,17a-trione is also more reactive at C-11 than expected, although in this case ordinary Huang-Minlon conditions led to total reduction, giving D-homo5a-androstane as the major product.) The formation of oximes from steroid ketones and hydroxyammonium salicylate in CHC1,-MeOH (2 : 1) is kinetically a second-order reaction. Comparison of rates of reactions for related series of steroidal ketones reveals a pronounced sensitivity to long-range electronic effects of substituents. The 17-0x0-group in free oestrone, for example, reacts at only half the rate found for oestrone 3-methyl ether, and the 3-acetate is even more reactive. Cholest-4en-3-one exhibits a lower enthalpy of activation than oestrone, but a 17phydroxy-group (in testosterone) or its ester approximately doubles the enthalpy. These effects are consistent with rate-determining nucleophilic attack on the carbonyl group, facilitated by electron withdrawal by remote electronegative substituents.' 8 5 Nitrous acid is sometimes employed to recover aldehydes or ketones from oximes (e.g. in the Barton synthesis of aldosterone),'86 although little is known of the mechanism of reaction. A mechanistic scheme explaining ' stable anti-oxime the evolution of N,, N,O, and N O is now p r ~ p o s e d . ' ~The (276) from 4-methylcholest-4-en-3-one rearranged with polyphosphoric acid (Beckmann reaction) to give keto-amides (278)and (279),the products of hydrolysis of the enamine-lactam (277).188The reaction is a further illustration of migration of unsaturated carbon, once thought to be ~ n r e a c t i v e . ' ~ ~ A full report has appeared of the deoxygenation of ketones and aldehydes by reduction of their tosylhydrazones in acidified DMF-sulpholan with NaBH,CN. I. M. Clark, W. A. Denny, Sir E. R. H. Jones, G . D. Meakins, A. Pendlebury, and J. T. Pinhey, J . C . S . Perkin I , 1972, 2765. I. Gaal, B. Matkovics, and M. Marik, Acta Chim. Acad. Sci. Hung., 1973, 75, 171. In6 Sir D. H. R. Barton and J. M. Beaton, J. Amer. Chem. SOC.,1960,82, 2641. la' J. M. Kleigman and R. K. Barnes, J . Org. Chem., 1972, 37, 4222. la' M. Kobayashi and H. Mitsuhashi, Chem. and Pharm. Bull. (Japan), 1972, 20, 1567. '13' Ref. 44, p. 356.
lS4
Terpenoids and Steroids
364
Me
Me (278) 5a-H (279) Sfl-H
Yields are high, and esters, amides, CN, etc., are not attacked.’” The Schmidt reaction (NaN,-polyphosphoric acid) of cholesta-2,4-dien-6-one caused enlargement of ring B to give lactams, as well as oxidation in ring A to give 3-0x0derivatives. The reactions gave a complex mixture of products, from which three were isolated in low yield.’” The Schmidt reaction converted the lahydroxy-5P-steroidal 3-ketone (280) into the la-hydroxy-lactams (281) and (282).’ 92
Carboxylic Acids, their Derivatives, and Aldehydes-Pyridine containing iodine acid (283)with dehydrogenareadily decarboxylates 3,17-dioxo-oestr-4-en-l9-oic tion to give oestra-4,9-diene-3,17-dione (284). Continued reaction with pyridine-
.F----
PY
@
PY,
H
0 T (283)
190
’” 19’
J
R. 0. Hutchins, C. A. Milewski, and B. F. Maryanoff, J . Amer. Chem. Soc., 1973,95, 3662. M. S. Ahmad and N. K . Pillai, Austral. J . Chern., 1973, 26, 603. M. H. Benn and R . Shaw, J.C.S. Chern. Cornm., 1973,288.
Steroid Properties and Reactions
365
(287)
(288)
iodine effected fiirther dehydrogenation to give 9(11)-dehydro-oestrone. The same reagents converted oestra-4,6-diene-3,17-dione(285) into 6-dehydrooestrone (286), and 3,17-dioxo-5a-androstan-19-oic acid (287) into the 2,19lactone (288).Ig3 Hunsdiecker reactions of steroidal 7a- or 7fl-carboxylic acids each gave 1 : 1 mixtures of the 7a- and 7P-bromo-steroids, which were also obtained from the 7-alcohols with PBr, . The epimeric bromides were separable by chromatography. The 6P-carboxylic acid gave only the 6a-bromo-compound. The 6- and 7-cyano-steroids were prepared either from the corresponding carboxylic acids, via dehydration of the acid amides, or from the 6- and 7-ketones, by dehydration of the derived cyanohydrins, followed by catalytic hydrogenation of the resulting olefinic bond. N.m.r. characteristics of these derivatives are reported. 94 Reactions and n.m.r. spectra are reported for some 14a,17a-ethanopregnan-20-one derivatives (289)-(291). A modified Curtius rearrangement transformed the 16a-carboxylic acid (290) into the urethane (291), and the derived N-nitrosourethane decomposed spontaneously to give the 15-ene (292) and the 16,16cyclo (nortricyclene) derivative (293). The same 16,16’-cyclo-compoundresulted as a by-product from Hunsdiecker degradation of the carboxylic acid (290).19’
(289) X = H (290) X = CO,H (291) X = NHC0,Me 19’ 194
lg5
(292)
(293)
F. S. Alvarez and A. N . Watt, J . Org. Chem., 1972, 37, 1972. A. Kurek, L. Kohout, J. FajkoS, and F. Sorm, Coll. Czech. Chem. Cornm., 1973, 38, 583. A. J. Solo, S. Eng. and B. Singh, J . Org. Chem., 1972, 37, 3542.
366
Terpenoids and Steriods
The stereochemistry of ( + )-cis-doisynolic acid has been confirmed, after almost forty years of uncertainty, by stepwise formation of its 3-methyl ether (294) from 14p-oestrone methyl ether. 96 3P-Acetoxyeti-5-enic acid has been used as a resolving agent for the racemic ketol (295).Ig7
Me0
'
H
An 18-methoxycarbonyl group will react intramolecularly with a variety of C-20 substituents to form heterocycles. Some examples are illustrated in Scheme 20.1~8
H
Reagents: i, LiBH,; ii, Zn-HOAc; iii, LiAlH,.
Scheme 20
Reduction of a mixture of the enol-lactones (296) with di-isobutylaluminium hydride gave the keto-aldehyde (297),which reacted with hydroxylamine to give the steroidal pyridine analogue (298).1 9 9 i-Butylidenetriphenylphosphoraneconverted a bisnorcholan-22-a1 (299) into a separable mixture of the cis- and trans-olefins (300) and (301), the former predominating.''' 19' lq7
2oo
J . Iriarte and P. Crabbe, J.C.S. Chem. Comm., 1972, 1 1 10. K. Mori, Tetrahedron Letters, 1973, 723. J. Einhorn, C. Monneret, and Q. Khuong-Huu, Bull. Soc. chim. France li, 1973. 303. A. Frankowski and J. Streith, Compt. rend., 1973, 276, C , 959. A. Metayer and M. Barbier, J.C.S. Chem. Comm., 1973, 424.
Steroid Properties and Rcmtions
367
&
0
0
(296) A5('*'
+ A5(6)
YHO (299)
i?
5 Compounds of Nitrogen and Sulphur
The differences between the first and second basic dissociation constants of the four isomeric 3,20-diaminopregn-5-enes have been correlated with the various distances between the two amino-groups, showing that the diammonium ions are destabilized by the electrostatic interactions between the two charged centres.2c* A novel method for methylating steroidal primary or secondary amines-comprises heating with methanolic formaldehyde, followed by reduction with NaBH,.202 The preparation of 2,3-diamino-steroids is described on p. 315, and the reductive amination of 3-oxo-steroids on p. 350. The 21-hydroxy-20oxo-system (302) of corticosterone reacted with formaldehyde and ammonia, in the presence of copper(I1)diacetate, to give the imidazole (303).203
(302)
(303)
2-Diazo-5ci-cholestan-3-one (304), among other diazoketones, reacted with acetylenedicarboxylic ester to give the N-acylpyrazole (306); the initial product (305)of 1,3-dipolar addition rearranges spontaneously to enlarge ring A . ~ 201 202
'03 '04
J.-P. Mazaleyrat, A . Tchapla, and Q. Khuong-Huu, Cumpt. rend., 1973, 276, C , 61 I . B. L. Sondengam, J. H. Hemo, and G . Charles, Tetrahedron Letters, 1973, 261. J. P. Guthrie, Canad. J . Chem., 1972, 50, 3993. M. Franck-Neumann and C. Buchecker, Angew. Chem. Internat. Edn.. 1973, 12, 240.
~
~
368
Terpenoids and Steroids C0,Me I C //I
0 (304)
3-Tosylazocholesta-3,5-diene(307) reacts with acetic acid to give in high (the yield a product which is thought to be 3-acetoxy-6~-tosylcholest-4-ene configuration at C-3 is not stated). Use of MeC0,D gave a product (308) specifically deuteriated at C-3, suggesting the mechanism illustrated in Scheme 21.,05 A similar reaction with the good dienophiles tetracyanoethylene or
AcO
@
HOAc
I
(308)Ts
N NC NC
CN
Ts
Ts
CN
(309)
Scheme 21 205
L. Caglioti, F. Gasparrini, G. Paolucci, G. Rosini, and P. Masi, J . Org. Chem., 1973, 38, 920.
Steroid Properties and Reactions
369
e.g. (309), N-phenylmaleimide gave 3-spirocyclopropano-6-tosylcholest-4-enes, with evolution of nitrogen.206 Scheme 21 illustrates the probable mechanism, involving a 1,3-dipolar addition to the 3-diazo-derivative. 'Nickel boride', obtained by reducing Ni" salts with borohydride, will desulphurize dithioacetals to give a mixture of saturated and unsaturated hydrocarbons, but is less effective than Raney nickel unless optimum conditions are chosen. The lanost-8-ene derivative ( 3 10) afforded 5a-lanost-8-ene (31 1) and 5cl-lanosta-2,8-diene (312) in good total yield when the solvent was either diethylene glycol or ethanol-boric New methods for the regeneration of ketones from dithioacetals are mentioned on p. 353.
(3 11) 2,3-saturated (312) A'
(310)
6 Molecular Rearrangements
Contraction and Expansion of Steroid Rings.-Silver carbonate on celite (Fetizon's reagent) is exceptionally effective for the semipinacolic rearrangement of bromohydrins.208 The diequatorial bromohydrin (313) was converted almost quantitatively into the A-nor-aldehyde (314), and the isomeric 2a-bromo-3cl-alcohol (315) give the same aldehyde (45%)' accompanied by the 3-ketone (55%). 38-Methyl or 3P-phenyl derivatives (316) and (317) gave the corresponding A-nor-ketones (318) and (319), respectively, in excellent yields. Only a diaxial
R- CO - -
\ &r B R OH H (315) R = H (316) R = Me (317) R = Ph *07 208
cir"j" H
(318) R (319) R
= =
Me Ph
L. Caglioti, G. Rosini, P. Masi, and A. Vigevani, Gazzetta, 1972, 102, 631. R. B. Boar, D. W. Hawkins, J. F. McGhie, and Sir D. H. R. Barton, J.C.S. Perkin I , 1973, 654. M. Fetizon, M. Golfier, and J.-M. Louis, Tetrahedron Letters. 1973, 1931.
370
Terpenoids and Steroids
bromohydrin (2P-Br, 3a-OH) failed to rearrange, because of its propensity to give the 2a,3~-epoxide.The contraction of ring A to give the ~-nor-2a-carboxylic acid is mentioned on p. 342. The well-known ring contraction of 4,4-dialkyl-5a-steroidal 3/?-alcohols (320) with PCI, to give A-nor-olefins (321) has been shown to proceed in the expected stereochemical sense, the 4P-alkyl group appearing in the exo position in the alkylidene group. The 4/?-CD, compound (320a) gave the product (321a) in 70 "/, yield ;the 4P-ethyl analogue (320b) behaved similarly, although the product (321b) was accompanied by significant amounts of double-bond isomers.209
(320) a ; R b; R
= =
CD, Et
*J H
... ....... :. Me
-& R
A
Previous conclusions2 concerning supposed long-range effects of C- 17 su bstituents on the Tiffeneau-Demjanov and diazomethane homologations of 3-0x0-5a-steroids are incorrect, having been based upon the supposition that the ~-homo-Sa-cholestanones(322) and (323) were separable. Most of the data in the literature concerning these A-homo-ketones actually refer to mixtures. The only reliable method of analysis of such mixtures appears to be the measurement of 0.r.d. or c.d. curves. Recent results2 establish the virtual independence of product ratios upon C-17 substitution. Diazomethane reacts mainly (ca.75 %) via approach to give the 38 (equatorial) adduct (324), which rearranges to give a characteristic mixture of ketones (322)and (323). Homologation of a 3,5-cyclo-20x0-steroid with diazomethane is described on p. 352.
''
(322) *09 210
''
(323)
(324)
S. Iwasaki, K. Okaniwa, and S. Okuda, Tetrahedron Letters, 1972, 4601. Ref. 79, p. 353. J. B. Jones and P. Price, Tetrahedron, 1973, 29, 1941.
37 1
Steroid Properties and Reactions
Favorskii rearrangement of the isomeric 6a-bromo- and 7aa-bromo-~-homo5a-cholestan-7-ones (325) and (326) afforded the same set of three carboxylic acid esters (328H330),which were characterized as the corresponding hydroxymethyl steroids after reduction. Common cyclopropanone intermediates (327) are invoked to explain the product distribution.2 Examples of the expansion of ring B appear on pp. 319 and 347.
(325) R' (326) R '
= =
Br, R2 = H H, R 2 = Br
(327)
C0,Me (328)
(3 29)
r
1
(330)
The formolysis of a pregnan-20P-yl tosylate (331) or the derived 17a-methyl~-homoandrostan-l7a~-yl tosylate (332) was reported some years ago to give the 17ap-ylformate (333)as the major product.21 A minor product has now been identified as having the novel 17p-methyl-~-homo13a-androstan-17aa-yl formate structure (334).214The formation of the product (334) is remarkable in that inversion occurs not only at the 17a-position, but also at both the adjoining sites, C- 13 and C- 17. Unless the methyl groups originally at the 13p- and 17a-positions both undergo unprecedented 1,3-shifts,effectively changing places with retention of configuration, it seems necessary to invoke a multiple Wagner-Meerwein 212 *13
*lo
A. Kurek, L. Kohout, J. FajkoS, and F. Sorm, Coll. Czech. Chem. Comm., 1973,38,279. H. Hirschmann, F. B. Hirschmann, and A. P. Zala, J. Org. Chem., 1966, 31, 375; see also ref. 79, p. 36 1 . F. B. Hirschmann and H . Hirschmann, J . Org. Chem., 1973, 38, 1270.
372
Terpenoids and Steroids
OR
OCHO
1
(331)
( 3 3 2 ) R = TS (333) R = CHO
(334)
mechanism, involving a series of 1.2-shifts of uncertain nature. The authors offer some tentative suggestions, but further work is needed to establish the detailed mechanism of this curious reaction.2l 4 A similar rearrangement has been observed in the acetolysis of a 17ab-tosylate corresponding to (332), but lacking the 17a-methyl substituent.21 The formolysis of a 20a-tosyloxypreganane (335) is more complex than that of the 20P-isomer. The reaction was recently reported to give a mixture of products, five of which were identified. One of these was the 17P-methyl-18nor-17a-pregn-13-ene (336). Isotopic labelling of the tosylate (335) at the 17a-H has revealed retention of the labelled atom in the 17a-ethyl group, showing that hydride migration (17 -+ 20) is involved rather than elimination to give an intermediate A' 7(20)-olefin.2 I' Kinetic data suggest that hydride migration is concerted with ionization of the tosylate. Another product was the D-homoandrostane derivative (337), now found to retain the isotopic hydrogen at the 17aa-position. The formation of this product seems to imply a single-step migration of the C-16-C-17 bond (338) despite the expected preference for C- 13-C- 17 bond migration.2 The explanation is still being sought. ,OTs H.y,.Me
(335)
(336)
(337)
(338)
Epoxide ring cleavage in the pregnane derivative (339), with HBr-acetic acid, was accompanied by D-homo rearrangement, giving the 17a-oxo-~-homoandrostane derivative (341).217The structure of the product (341j corresponds to that of other D-homo-ketols formed by acid-catalysed rearrangements involving migration of the C-16-C-17 bond. The facile reaction is probably promoted '15
'" 21h
I . Khattak, D. N. Kirk, C. M . Peach, a n d M. A. Wilson, J . C . S . Chem. Comm., 1973, 341. S. S. Deshmane a n d H . Hirschmann, J . O r g . Chrm., 1973, 38, 748. V. Schwarz, Coil. Czech. Chem. Cnmm., 1972, 37, 3909.
Steroid Properties and Reactions
373
by the 168-bromomethyl substituent in the intermediate bromohydrin (340), in agreement with earlier observations of accelerated D-homo rearrangement when a 168-methyl substituent is present.218 Me
I
r
Me
I
Backbone Rearrangements.-The hyperacidic reagent HF-SbF, causes rapid isomerization of oestr-4-ene-3,17-dione (342) at C-14, giving the more stable 148-isomer (343).2 The same reaction in DF-SbF, caused extensive incorporation of deuterium (up to 12 atoms), mainly located along the ‘backbone’ of the molecule. Evidently the isomerization involves migration of the olefinic bond in the usual way as far as C-14, followed by a reversal of the rearrangement to regenerate the conjugated enone. 14P-Oestr-4-ene-3,17-dione represents the most stable structure accessible by a sequence of protonation-deprotonation steps.
(342) 1 4 ~ - H (343) 14B-H
The ‘Westphalen’diacetate (344)reacts with anhydrous DF-CHC1, at -60 “C to give the 9cr-fluoro (trans addition) product (346)’ the 9(1l)-ene (347), and a complex mixture of other products resulting from backbone rearrangements.220
”* ’I9 ”O
Ref. 3, p. 301. J. C. Jacquesy, R. Jacquesy, and G . Joly, Tetrahedron Letters, 1972, 4739. J. Barbier, C. Berrier, J. C. Jacquesy, and R. Jacquesy, Tetrahedron, 1973, 29, 1047.
Terpenoids and Steroids
374
The 9( 11)-ene (347) afforded the 9a-fluoro-compound (346) in much higher yield than did the original 9-ene (344). Use of DF instead of HF established that addition to the 9(11)-ene occurs in the cis sense, giving the l l a - D derivative (348): preferential cis addition of HF to olefinic bonds is well known. Mass spectrometric analysis of the products of backbone rearrangement showed the incorporation of a large number (up to 19) deuterium atoms, implying that the rearrangements in this medium involve a sequence of addition and elimination steps, rather than multiple hydride shifts which have been invoked in other backbone rearrangements.”’
f344)
AcO OAc
OAc (i45)
(346)
11F
AcO OAc (347)
(148)
The reactions of 5,6-epoxy-steroids with BF, show considerable variations in product type according to the substitution at C-3 and C-17. Electronegative substituents tend to suppress the usual C-5 carbonium ion rearrangements in favour of fluorohydrin formation. The most recent results extend earlier findings in this field and include compounds of the androstane and pregnane series as 12’
Ref. 59, p. 3 0 5 .
375
Steroid Properties and Reactions
well as cholestanes. Inductive effects of substituents show increasing attenuation with distance from the epoxide, but have not yet been fully rationalized. One previously thought rearrangement product of a 3~-methoxy-5a,6a-epoxy-steroid, to be the 6a,8a-ether (349), is now re-formulated, largely on n.m.r. evidence, as the C-5-spiro-6a,9a-ether (350).222 -.
JJ?$
Me0
Me0
(349)
(350)
The behaviour of 3P-methyl- and 3~-isopropy~-3a,~-epoxy-~-nor-~a-~holestanes (351) with acids depends upon the reagent system. HCl-ethanol gives the 3(5),6-dienes (352), but pyridine hydrochloride gives dienes (353) and/or
R
(352)
R
=
&
PI'
O
R
R
8
R' R
(355)
222
I . G . Guest and B. A. Marples, J . C . S . Perkin I , 1973, 900.
376
Terpenoids and Steroids
(354) resulting from 1,2-elimination without rearrangement. BF,-ether gives dienic products of more extensive rearrangement, e.g. (355), accompanied by ketones (356).22 The steroid alkaloid methylparavallarine (357)undergoes a backbone rearrangement similar to that of a con-5-ene derivative,224giving the 14P-A8(9)-isomer (358).225
Aromatization of Steroid Rings.-Although recent work on the 1,4-dien-3-one rearrangement to give phenolic derivatives [(360) and/or (362)] has seemed to clarify some of the factors involved in deciding the reaction path,226a new study of bicyclic analogues with different C-10 alkyl groups (R) has re-opened the question of the solvent Aqueous acid converts 1P-dienones (359a) mainly into the rn-phenolic compounds (361a),whereas acidified acetic anhydride gives mainly the p-phenolic compound (360a). Each of the higher homologues, however, failed to show this solvent effect, (359b), (359c),and (359d) each giving a large preponderance of the corresponding rn-phenolic product (361) with either type of reagent system. The role of the migrating alkyl group is not yet understood, but must be included in any explanation of these reactions.
R
(359) a ; R = Me b; R = E t
(360)
(361)
,
c ; R = Pr d; R = B u b, c, and d are bicyclic structures only
Further examples of HBr-catalysed aromatization of trifunctional steroids228 are reported. Several ene-diols and triols, with OH groups variously sited at 223 224
225
226 227 228
I . Morelli and S. Catalano, Gazzetra, 1972, 102, 572. Ref. 44, p. 381. J. Thierry, F. Frappier, M. Pais, F. X. Jarreau, R. Goutarel, and A. Montagnac, Bull. SOC.chim. France, 1972, 4753. Ref. 59, p. 309; ref. 79, p. 376; ref. 3, p. 277. H. J. Shine and C. E. Schoening, J . Org. Chem., 1972, 37, 2899. Ref. 44, p. 391 ; ref. 59, p. 309.
377
Steroid Properties and Reactions
C-3, C-4, C-5, and C-6, gave 4-methyloestra-1,3,5(10)-trienes in yields ranging was similarly the main up to 40 %.229 4-Methyloestra-1,3,5(lO)-trien-l7-one and product of reactions between 3-substituted-5a,6a-epoxyandrostan-17-ones HBr-acetic The 1,5-dien-3-one (362) in the 19(10+9/l)-abeo-lOa-pregnane series is rearranged by alkalis to give the 1(10),5-dien-3-one(363), or by HCl-MeOH to give (363) and its trienyl ether (364). More vigorous acidic treatment gave the aromatic product (365),whereas acetyl bromide afforded the alternative aromatic compound (366). Possible mechanisms are suggested.23 COMe
0
Me0
\
Trifluoroacetic acid converted the 7-hydroxy-3~-(N-methylacetamido)-A5steroid (367) into the dihydro-oxazinium ion (368), which broke down with rearrangement to give the anthrasteroid (369) when the mixture was heated at 60 "C. The same anthrasteroid resulted when the 4-en-3-one (370) reacted with Mazur's reagent, acetyl bromide-propan-2-01. The 14p configuration in the product (369) implies mobility of unsaturation during the process (cf:backbone rearrangements, p. 373).232 The adducts (37l), obtained from 4,4-dimethyl steroidal 5,7-dien-3-ones and dimethyl azodicarboxylate, rearranged smoothly with BF, to give the anthrasteroids (372),whereas the 3P-alcohol corresponding to the ketone (371) gave the A-seco-ketone (373). An isomeric A-seco-ketone was formed from the 3-acetate. Reasonable mechanisms are proposed.233 22q 230 231
232 233
J . R. Hanson, Tetrahedron Letters, 1972, 4501. A. G. Ogilvie and J. R. Hanson, J.C.S. Perkin I , 1972, 1981. J. R. Bull and A. J. Hodgkinson, Tetrahedron, 1973, 29, 1109. G. Massiot, A. Cavt, H.-P. Husson, and P. Potier, Tetrahedron Letters, 1973, 29. H. de Nijs and W. N. Speckamp, Tetrahedron Letters, 1973, 813.
Terpenoids and Steroids
378
(371)
HN-C0,Me
R
R
(373)
Ring c was readily aromatized by heating 17a-methyl-l7P-hydroxyandrosta4,6,9(1l)-trien-3-one (374)with formic The thermodynamically favourable enol(375) seems a likely intermediate, allowing migration of a second double bond into ring c. Dehydrogenation of the product (376) with chloranil gave 234
A. B. Turner, Chem. and Ind., 1972, 932.
379
Steroid Properties and Reactions
the pentaene (377). 17a-Methyl-9(1l)-dehydrotestosteroneafforded the same pentaene in low yield after a similar reaction sequence, although a dehydrogenation step is required in the aromatization, suggesting a disproportionation mechanism.
L
(374)
(375)
1
(376) 6,7-saturated (377) A6
Although a A’(’ ‘)-unsaturated deoxycholic acid analogue is converted merely into the 12a-methoxy-derivative by methanolic HCl,235 the corresponding 7a-acetoxy- (cholic acid) derivative (378) has been found to afford the rearranged c-aromatic product (380) with the same reagent.236 The 17a configuration is considered to arise from a dehydration step with methyl migration to give the A13(17)-01efin(379), followed by elimination of the 7a substituent and migration of unsaturation into ring c. There is no conclusive evidence, however, as to the precise sequence of steps comprising the overall process. Miscellaneous Rearrangements.-Some further features of the long-debated Kober reaction237of steroidal oestrogens have been elucidated.238 A solution of 3-methoxyoestra-1,3,5(lO)-trien-l7a-ol (381) in 97.2 % sulphuric acid rapidly developed colour, I,,, 372 nm, which appears to be due to a cation of the type (382). The configurations at C-8, C-13, (2-14, and C-17 have not yet been established. Dilution with water and extraction of the product after only brief reaction gave the 8-ene (383), I,,, 273nm, in high yield. Dissolution of the 8-ene in 235 236 237 230
L. F. Fieser and M. Fieser, ‘Steroids’, Reinhold, New York, 1959, p. 644. J. Meney, Y.-H. Kim, R. Stevenson, and T. N. Marguilis, Tetrahedron, 1973, 29, 21. Ref. 59, p. 327; ref. 79, p. 401. M. Kimura, K. Akiyama, and T. Miura, Chem. and Pharm. Bull. (Japan), 1972, 20, 2511.
Terpenoids and Steroids
380 Ht
R
1
C02Me
?&I
* AcO*.
H
H
(379)
1
(378)
Me0 (382)
(381)
Cation, A,,
515 nm
(385)
Dehydrogenation +
Cation, A,,
(384)
465 nm
+
H
k
’
Steroid Properties and Reactions
38 1
97.2 % H 2 S 0 4 caused it to revert to the benzylic cation, A,, 372 nm. Clearly the cation (382) is the first stable species formed under Kober conditions. If the reaction was prolonged, however, particularly in the presence of an oxidant, progressive shifts in U.V.absorption maximum were observed, first to 465 nm and later to 515 nm. These changes correspond to the appearance of dehydrogenated cationic species of uncertain structures, although the corresponding compounds obtained by deprotonation of the cations appear to be the dihydrophenanthrene and phenanthrene derivatives (384) and (385), respectively.238 The colorimetric determination of testosterone by reaction with strong acids depends similarly upon dehydration with Wagner-Meerwein migration of the 18-methylgroup to C-17, followed by a combination of oxidation and protonation leading to chromophoric species of various types, such as (386) and (387).239 The phenol4ienone isomerization of oestrone in HF-SbF, , reported briefly last year,240has now been described in full.241 An n.m.r. study of the reacting solution has given indications of cationic intermediates.
(386)
(387)
The 8a,9a-epoxide (388) derived from dihydrolanosteryl acetate failed to give any products of alkylation with methyl-lithium (no reaction), methylmagnesium iodide, or allylmagnesium bromide. The Grignard reagents gave, respectively, the 7,9(11)-diene and a product believed to be the 7-en-9a-01 (389).242 Acid treatment of the crude mixture of epoxy-alcohols from 5a-cholest-7-ene,followed by chromatography, allowed the separation of the 8(14)-en-7-one,the 9a-hydroxy7-one, and a small amount of the 8(14)-en-15-one.'Full details are reported243 of the fragmentation of a 24,28-epoxystigmastane derivative with BF, .244
239 240 24' 242 243 244
M. Kimura, K. Harita, and T. Miura, Chem. and Pharm. Bull. (Japan), 1972,20, 1829. J. P. Gesson, J. C. Jacquesy, and R. Jacquesy, Tetrahedron Letters, 1971, 4733. J. P. Gesson, J. C. Jacquesy, and R. Jacquesy, Bull. SOC.chim. France II, 1973, 1433. G. R. Petit and W. R. Jones, J. Org. Chem., 1972, 37, 2788. H . Ohtaka, M. Morisaki, and N. Ikekawa, J. Org. Chem., 1973,38, 1688. Ref. 44, p. 389.
382
Terpenoids and Steroids
The thermal rearrangement of 17P-bromo- or 17P-chloro-16a,17a-epoxyandrostanes (390) gives mixtures of 16a- and 16P-halogeno-17-ketones (392). The reaction is faster in polar (protic) than in non-polar solvents, suggesting an ion-pair intermediate (391).245
(390) X = C1 or Br
The adducts (394) and (399, obtained by difluorocarbene addition to the 17P-acetoxy-1701-ethynyl steroid (393), with rearrangement, have now been found to undergo a variety of further rearrangements on reaction with alkali. Some of the novel reactions and products are illustrated in outline for one of the tetrafluorocyclopropyl products in Scheme 22.246
3 8; F
OAc
Ac
/fi?GCH
+
(393)
t ,
,
(394)
Z
A
C
(395)
I
2 F
F
1 Reagents: i, :CF,; ii, NaOH-Me,CO; iii, NaOH-MeOH.
Scheme 22 245 246
P. Catsoulacos and A. Hassner, Bull. SOC.chim. France II, 1973, 717. P. Crabbe, E. Velarde, L. Tokes, and M. L. Maddox, J . Org. Chem., 1972, 37,4003.
383
Steroid Properties and Reactions
Alumina catalyses the conversion of 3a,5-cycl0-5a-cholestan-6/?-01into cholesteryl acetate in refluxing acetic Iodine has been used to catalyse the isomerization of vitamin D3 and its 25-hydroxy-derivative to their 5,6trans-isomers, which exhibited unexpected biological
7 Functionalization at Non-activated Positions In using the hypoiodite route [Pb(OAc),-I, ,hv] for the conversion of a pregnan2Op-01 into C-18-substituted compounds, it has been customary to avoid isolation of the 18-iodo-20~-(20R)-alcohol [e.g. (396)], and to proceed via oxidation and Ag +-assistedsolvolysis to the hemiacetal(397) of the 18-hydroxypregnan-2O-one, and other products. The iodo-alcohol(396) is now found to be reasonably stable, and easily isolated in 60% yield.249 Oxidation and reaction with AgOAcmethanol then provides an advantageous route to a mixture of the C-20-isomeric acetals (397). The iodo-alcohol(396) is converted by mild bases into the 18,20Rether (398), whereas catalytic hydrogenation of the mixed acetals (399) with Pt-HOAc gives the 18,2OS-ether (400).
(396)
(397) R = H (399) R = Me
(398)
(400)
The hypoiodite reaction has been employed to convert a 20(R)-hydroxycholestane (401) into the 18 ---* 20-lactone (402).250
Irradiation of the amide (403) of an etianic acid in the presence of iodine and lead tetra-acetate gave the lactone (404)and the acid anhydride (405),in 247 248 249
A. Mktayer and M. Barbier, Bull. SOC.chim. France, 1972, 3625. M. F. Holick, D. Garabedian, and H. F. DeLuca, Biochemistry, 1972, 11, 2715. P. Choay, C. Monneret, and Q. Khuong-Huu, Bull. SOC.chim. France II, 1973, 1456. G. Habermehl and K. P. Swidersky, Naturwiss., 1972,59, 648.
Terpenoids and Steroids
384
(403)
’
proportions which could be varied by suitable choice of reaction condition^.^^ The mechanism of such reactions was investigated some years ago.’ 5 2 9,lO-Epoxides (406) derived from 3P-hydroxy-5P-methyl 19-nor-compounds (‘Westphalen’ series) are converted by lead tetra-acetate into the cyclic ethers (407) without damage to the epoxide ring. Photolysis of the 3-nitrites gave complex mixtures which included the 3-ketones, 3-alcohols, and the expected oximes (408)of the 5P-formyl derivatives. The ‘backbone-rearranged’A’ 3(1’)-ene (409)readily formed an ether bridge similar to that in (407) on reaction with lead tetra-a~etate.~~
A 17a-methyl substituent is close to a 12a-hydroxy-group (410), permitting functionalization of the methyl group either by the action of lead tetra-acetate, which gave the cyclic ether (41l), or by photolysis of the 12-nitrite, leading to the aldehyde hemiacetal(412) via a nitroso-dimer and ~ x i m e5 4. ~Various transformations of the functionalized 17u-substituent are described. 25‘
*” 253 254
LJ.Valcavi and S. Innocenti, Farmaco, Ed. xi., 1972, 21, 876. D. H . R. Barton, A . L. J . Beckwith, and A. Goosen, J . Chem. Soc., 1965, 181. J . G. L1. Jones and B. A. Marples, J.C.S. Perkin I , 1973, 1143. Ch. R. Engel, D. Mukherjee, and G . J . Beaudoin, Steroids, 1973, 21, 857.
Steroid Properties and Reactions
385
(411) (412)
R R
= =
H OH
Bromine and silver salts react with suitable alcohols to give either tetrahydrofuran derivatives (e.g. 6P,19-epoxy-steroids) or ketones,*” but the controlling factors and mechanisms of these competing reactions are not fully understood. A systematic study of different silver carboxylates with model alcohols showed that silver salts of strong acids (e.g. trifluoroacetic) favour the ketone as product, whereas silver acetate leads mainly to the tetrahydrofuran.*5 6 These observations permit selection of reaction conditions favouring either product, with obvious applications in steroid chemistry. Several unsuccessful attempts have been made to achieve nitrene insertion into a C-H bond by photolysis of a steroidal a ~ i d e . ~ ’ ~ Such a reaction has at last been successful, though in low yield (6 %). 6B-Azido5a-pregnane (413) was irradiated in cyclohexane, and gave three major products (Scheme 23), accompanied by the pyrrolidine (414).258
a ‘ +
NH
& + H&
1
H
SO, column
@
+& H (414)
0
Scheme 23
Several papers published since 1954 have described the hydroxylation of steroids by reagent systems based upon iron@) ions, in solutions containing various combinations of buffers, ethylenediaminetetra-acetic acid, and ascorbic 255 256 257 258
Ref. 59, p. 316; ref. 79, p. 387. N. M. Roscher and E. J. Jedziniak, Tetrahedron Letters, 1973, 1049. Ref. 59, p. 323; ref. 79, p. 399. A. Pancrazi, Q. Khuong-Huu, and R. Goutarel, Tetrahedron Letters, 1972, 5015.
386
Terpenoids and Steroids
acid, with hydrogen peroxide or atmospheric oxygen as o ~ i d a n t . ~ ”Deoxycholic acid (415) is now found to afford the 15a-hydroxy-derivative (416; 10%) and the 14P-15-ketone (417; 8%) when a buffered solution containing iron@) is oxygenated at 70 “C, the other additives being unnecessary.260 The mechanism of this curious reaction and the reason for selective oxidation at C-15 are not yet understood. ,$C02H
14;“H .1,_.
. . R
H
o
HO ’’
H (415) (416)
R R
=
H
(417)
= OH
Hydroxy-substitution occurred at the unactivated 5a- and 14a-positions when Sa-androstane-3P,17fi-dioldiacetate or the 17p-01 acetate was irradiated with peracetic acid in t-butyl The reaction is regarded as proceeding via hydrogen-atom abstraction by methyl radicals, generated from the peracetic acid. Attack at both C-5 and C-14, in contrast to reaction only at C-14 by photochemically generated bromine atoms,262is attributed to the higher reactivity, implying lower selectivity,of methyl radicals, but it is not clear why a 9a-hydroxyderivative was not also obtained. The ‘biomimetic’ introduction of functional groups into various sites in the steroid nucleus by irradiation of esters containing benzophenone moieties, and by various free-radical reactions, has been re~ i e w e dti. ~
,
8 Photochemical Reactions Some photochemical reactions are included in the preceding section. Six products have been found in irradiated solutions of vitamin D, in ethanol. Two of these are the known ‘ s u p r a ~ t e r o l s ’ .The ~ ~ ~others have been identified as the isomeric pair of allenes (418) and (419), and the cyclobutenes (420) and (421).2 259 260
261
262 263 264
265
Ref. 79, p. 391. M. Kimura, M. Kawata, M. Tohma, A. Fujino, K. Yamakazi, and T. Sawaya, Chem. and Pharm. Bull. (Japan), 1972,20, 1883. A. Rotman and Y. Mazur, J . Amer. Chem. SOC., 1972,94, 6228. Ref. 79, p. 397. R. Breslow, Chem. SOC.Rev., 1972, 1, 5 5 3 . W. G. Dauben and P. Baumann, Tetrahedron Letters, 1961, 5 6 5 ; W. G. Dauben, in R. B. Woodward and R. Hoffmann, ‘The Conservation of Orbital Symmetry’, Verlag Chemie, Weinheim, 1970, p. 80. S. A. Bakker, J. Lugtenburg, and E. Havinga, Rec. Trav. chim., 1972, 91, 1459.
387
Steroid Properties and Reactions
@ C
w
R
C
HO..
H-YYH H2*-xYoH Me
10
Me
(420) 6 ~ - H (421) 6fl-H
(419)
The steroidal 5-methyl-1,3-dienes (422) undergo photochemical ring-opening and re-closure in conformity with the orbital-symmetry rules, which uniquely determine the stereochemical possibilities among the products. The principal reactions are illustrated in Scheme 24; they exhibit similarities to the well-known
Scheme 24
photochemistry of 5,7-dienes. Sa-Cholesta-l,3-diene was partially converted by irradiation into the 5P-isomer; the presumed intermediate seco-triene could not be detected in this case.266 U.V. irradiation of a 14P-hydroxypregn-16-en-20-0ne(423) in iso-octane or t-butyl alcohol gave the 14,15-seco-diketone (424). An aldol condensation between the 14-0x0-group and C-21, under basic conditions, gave the pregna14,17(20)-dien-16-one(425).267 266
267
W. G . Dauben, R. G . Williams, and R. D. McKelvey, J . Amer. Chem. SOC.,1973,95, 3932. F. Marti, H. Wehrli, and 0. Jeger, Helo. Chim. Acta, 1973, 56, 1078.
388
Terpenoids and Steroids Me
(423)
(424)
(425)
The 9a,lOa-epoxy-6-ketone (426) in the ‘Westphalen’ series, on irradiation in ether, gave the decarbonylation product (427). With methanol as solvent, the main product was the methyl ester (428).268The latter reaction, which has several precedents, probably involves a keten intermediate.269 Formation of the unsaturated epoxide (427) includes a hydrogen-atom transfer from C-7 to C-5 and a subsequent or synchronous C-8 --+ C-7 hydrogen shift (429), as revealed by use of the [7,7-2H2]epoxy-ketone.268
0
(429)
Triplet-sensitized irradiation of 6,6-dimethylcholest-4-ene-3,7-dione (430) caused contraction of ring B to give the cyclopropyl diketone (431), although the reaction was slow and ineffi~ient.~ ’O Use of deuterium-labelled 4,4-dimethylcholest-5-en-3-one has confirmed the stereochemistry of its photoisomerization to give the cyclopropyl ketone (432). Possible mechanisms of these reactions are discussed.270
2b9
R. J. Chambers and B. A. Marples, J.C.S. Chem. Comm., 1972, 1122. Ref. 3, pp. 4 2 2 4 2 3 ; ref. 59, p. 322. H. Sato, K. Nakanishi, J. Hayashi, and Y. Nakadaira, Tetrahedron, 1973, 29, 275.
389
Steroid Properties and Reactions
Irradiation (366 nm) of 4-methoxycholest-4-en-3-one(433) caused intramolecular cyclization to give the methylene-oxetanols (435) and (436), with a little of the keto-oxetan (437). All three products can arise via hydrogen transfer to give the biradical (434), followed by cyclization either at C-3 or at C-5. The oxetan rings of (435)and (436)areopened by methoxide ion to give the corresponding 3-hydroxy-3-methoxymethyl-4-ketones (438).2
’
A linear free-energy relationship has been demonstrated between the half-wave reduction potentials and the lowest n,n* triplet states of a series of enones: the compounds include steroidal 4-en-3-ones, and their 4-acetoxy-, 4-chloro-, and 4-methyl derivatives.27 2 U.V. irradiation of 2-(N-methylanilino)-5a-cholestan-3-one (439) caused cyclization to give the azetidinol (440). The reaction is a general one for 2(N-alkyl-N-arylamino)cyclohexanones.2 73 Ph
\
\
Me
0 H (439)
’
OH
H
(440)
A. Feigenbaum and J. P. Pete, Tetrahedron Lerters, 1972, 2767.
”’ R. 0. Loutfy and R. 0. Loutfy, Canad. J. Chem., 1972,50,4050. 273
J . Hill and J . Townend, J.C.S. Chem. Comm., 1972, 1108.
390
Terpenoids and Steroids
4-Phenyl-4-aza-steroidal 5-en-3-ones (441) give the novel products (442) on irradiation, resulting from rupture of the N-CO bond, and attack of the carbonyl radical on an ortho position of the phenyl ring.274 The 4-benzyl-4-am-steroid (443), in contrast, rearranges on irradiation to give the 6-benzyl derivative (444).2 This reaction presumably involves homolytic fission of the reactive N-benzyl bond.
R = Ph (443) R = C H , P h
(441)
(442)
(444)
The hydrazone (445)of androsterone afforded a complex mixture on photolysis : two of the products were the 17-ketone and the di-steroidal azine. The N acetylhydrazone (446) gave a different mixture of products, which included the isomeric (cis) acetylhydrazone (447) and the lactams (448).276The formation of both the C-13-isomeric lactams (448) in low yields by photolyses of a 17-oximinosteroid seems to imply a mechanism in which the C-13-C-17 bond is ruptured.277 R
I
N
(445)R = H (446) R = AC
AcNH \
N
(447)
(448) 1 3 ~ + - 13fi-isomers
Irradiation of the imide (449) gave the unsaturated amide (451), probably by 17,17a-bond cleavage and loss of CO to give the biradical (450), followed by hydrogen transfer from the 18-methyl group to nitrogen.278 2’4
275
2’6 277
278
R. P. Gandhi, M. Singh, Y. P. Sachdeva, and S. M. Mukherji, Tetrahedron Letters, 1973,661. R. P. Gandhi, M. Singh, Y. P. Sachdeva, and S. M. Mukherji, Chem. and Ind., 1973, 382. H. Suginome and T. Uchida, Tetrahedron Letters, 1973, 2289. H. Suginome and T. Uchida, Tetrahedron Letters, 1973, 2293. R. P. Gandhi, M. Singh, T. D. Sharma, and S. M. Mukherji, Tetruhedron Letters, 1973, 657.
Steroid Properties and Reactions
39 1
The rigidity of steroids has been exploited in a study of transition-state geometry in the hydroperoxidation of olefins with singlet oxygen.279 Careful analysis of the products obtained by photosensitized oxygenation of the 2- and 3-methyl5cc-cholest-2-enes (452) and (456), followed by reduction of hydroperoxides to alcohols, gave the proportions indicated in Scheme 25. The formation of both
(452)
(453) (57%)
Me
Me O H
(454) (13 %)
(455) (30%)
H
epimeric 2-methylene-3-alcohols (453)and (454) implies at most a modest stereoelectronic preference for formation of the axial alcohol, a reaction which is entirely inhibited in the reaction of the 3-methyl-3-ene by the 2p-lOp diaxial interaction. Related studies with simple olefins carrying deuterium labels showed a rather small kinetic isotope effect (kH/kD generally ca. 1-2). The results are interpreted as evidence for a reactant-like transition state, which explains several earlier observations, viz. that thermodynamic stability of the final double bond has little effect on the reaction ;that a requirement of conformational inversion does not prevent reaction ; and that the reactivity of the allylic C-H bond is not inherently related to its substitution pattern (primary, secondary, or t e r t i a r ~ 7) 9. ~ 279
A. Nickon, V. T. Chuang, P. J. L. Daniels, R. W. Denny, J. B. DiGiorgio, J. Tsunetsuga, H. G. Vilhuber, and E. R. Werstiuk, J . Amer. Chem. SOC.,1972, 94, 5517.
392
Terpenoids and Steroids
A recent report280 that ‘ergosterol peroxide’ is actually a mixture of the 5a,8a- and SP,8,8-endo-peroxides(459)and (460)has been amended. The supposed SP,8P-peroxide is now shown to be known 9,l l-dehydro-5a,8a-peroxide (461).281
(459) 9,ll-saturated (461) 9,ll-ene
Sensitized photo-oxygenation of desmosterol acetate (462) and reduction of hydroperoxides with NaBH, gave a mixture containing the 25-hydroxy-23-ene (463)and the 24-hydroxy-25-ene(464).91Hydrogenation of the former compound gave 25-hydroxycholesterol, a suitable precursor for the chemical synthesis of the biologically important 25-hydroxycholecalciferol (se also p. 333).
OH
9 Miscellaneous The phase-transition temperatures have been measured for binary mixtures of cholesteryl esters with liquid-crystalline properties. Most mixtures show depression of the solid 6 mesomorphic transition temperature compared with the pure components, but a linear relationship between the mesomorphic isotropic transition temperature and mixture composition. The consequence is an
*
280 281
J. Arditti, R. Ernst, M. H. Fisch, and B. H. Flick, J.C.S. Chem. Cornm., 1972, 1217. M. H . Fisch, R. Ernst, B. H. Flick, J. Arditti, Sir D. H. R. Barton, P. D. Magnus, and I. D. Menzies, J.C.S. Chem. Comm., 1973, 5 3 0 .
Steroid Properties and Reactions
393
expansion of the mesomorphic range for mixtures compared with the generally narrow ranges for pure esters.282 Molten cholesteryl hydrogen phthalate, alone or mixed with other cholesteryl esters, supercools readily to give glassy solids which display the iridescence of cholesteric liquid-crystalline materials, yet are stable at room temperature over a period of years. The colours cover the entire visible spectrum.283 Crystalline samples of 2oOr- and 2OP-hydroperoxypregn-5-en-3~-01~ have been isolated from cholesterol after ageing in air at 70°C in the absence of light. The origin and thermal decomposition of these products have been discussed in relation to the biogenesis of steroid hormones.284 The auto-oxidation of cholesterol is reported to give a complex mixture of hydroperoxides, with attack at several sites in the side-chain and in ring B (see p. 342). G.1.c. systems are. described for the analysis of the ring B-substituted systems.285 Cholanic and bisnor-cholanic acids have been detected among the thousands of acidic components in California petroleum. Their probable origins in living organisms are discussed.286 Sephadex LH-20 reacted with 23,24-epoxy-5Pcholane and BF, to give a gel with useful modified chromatographic properties, although it failed to separate enantiomeric compounds, as had been hoped.287 G.1.c. has been used to separate the (E)- and (2)-isomers of 24-ethylidene sterols (465) : the (&isomer was always eluted first. Mass spectral data are reported.288
(465)
Exploration of the topography of the steroid binding site in a 20P-hydroxysteroid dehydrogenase, with studies of the binding or 2a-, 601-, and 6P-bromoprogesterones, has given evidence of a cysteine residue close to the C-6 position of the bound ~ t e r o i d . ~ ”
282
283 284 285 286
287
288
289
S. G. Frank and B. G. Byrd, J . Pharm. Sci., 1972,61, 1762. W. Mahler and M. Panar, J. Amer. Chem. SOC.,1972,94, 7195. J. E. van Lier, G . Khan, and R. Langlois, Steroids, 1973, 21, 521. J. 1. Teng, M. J. Kulig, and L. L. Smith, J. Chromatog., 1973, 75, 108. W. K. Seifert, E. J. Gallegos, and R. M. Teeter, J . Amer. Chem. Soc., 1972, 94, 5880. R. A. Anderson, C. J. W. Brooks, and B. A. Knights, J . Chromatog., 1973,75, 247. C. J. W. Brooks and B. A. Knights, Steroids, 1972, 20, 487. C.-C. Chin and J. C. Warren, Biochemistry, 1972, 11, 2720.
2 Microbiological Reactions with Steroids BY
L. L.
SMITH
1 Introduction
The use of microbiological reactions for the transformation of steroids, dating from 1937, proliferated extensively in the period 1952-1958, during which time several microbial hydroxylations and dehydrogenations became industrially important for the production of steroid hormones and their analogues. The extensive literature which developed has been covered heretofore by three major monograph^,'-^ two of which’*3 are encyclopaedic in nature. Several other monograph^^-^ and are devoted to various aspects of the topic. A. A. Akhrem and Yu. A. Titov, ‘Mikrobiologicheskie Transformatsii Steroidov’, U.S.S.R. Academy of Sciences, Moscow, 1965. A. Capek, 0. H a d , and M. Tadra, ‘Microbial Transformations of Steroids’, Publishing House of the Czechoslovak Academy of Sciences, Prague, 1966. W. Charney and H. L. Herzog, ‘Microbial Transformations of Steroids. A Handbook’, Academic Press, New York-London, 1967. L. L. Wallen, F. H. Stodola, and R. W. Jackson, ‘Type Reactions in Fermentation Chemistry’, ARS-7 1- I 3, Agricultural Research Service, U.S. Dept. of Agriculture, Peoria, Ill., 1959. R. I. Dorfman and F. Ungar, ‘Metabolism of Steroid Hormones’, Academic Press, New York-London, 1965, pp. 224-288. ’ H. Iizuka and A. Naito, ‘Microbial Transformation of Steroids and Alkaloids’, Uniof Tokyo Press, Tokyo, and University Park Press, State College, Pa., 1967. ’ versity G. S. Fonken and R. A. Johnson, ‘Chemical Oxidations with Microorganisms’, Marcel Dekker, New York, 1972. P. H. Goll, Process Biochem., 1966, 1, 201. R. Muller and K. Kieslich, Chem.-Ing.-Tech., 1966, 38, 813; Angew. Chem. Internat. Edn., 1966,5, 653. l o G. K. Skryabin and L. M. Kogan, Izvesr. Akad. Nauk S.S.S.R., Ser. biol., 1967, 742. I I C. Vezina, S. N. Sehgal, and K. Singh in ‘Advances in Applied Microbiology’, ed. W. W. Umbreit and D. Perlman, Academic Press, New York-London, 1968, Vol. 10, pp. 221-268. * J. de Flines in ‘Fermentation Advances’, ed. D. Perlman, Academic Press, New YorkLondon, 1969, pp. 385-390. l 3 V. Petrow, Chem. and Ind., 1969, 18. l 4 C. Vezina, S. N. Sehgal, K. Singh, and K. Kluepfel in ‘Progress in Industrial Microbiology’, ed. D. J. D. Hockenhull, Churchill Livingstone, Edinburgh-London, 197 1 , VOl. 10, pp. 1-47. l 5 W. J. Marsheck, ref. 14, pp. 49-103. l 6 R. Beukers, A. F. Marx, and M. H. J. Zuidweg in ‘Drug Design’, ed. E. J. Ariens, Academic Press, New York, Vol. 3, 1972, pp. 1-13]. A. Akhrem and Yu. Titov, Soviet Sci. Rev., 1972, 3, 232; Ideen exakt. Wissens, 1972, No. 1 , p. 37. K . Kieslich, Synthesis, 1969, 120, 147. Charney, New Scientist, 1969, 43, No. 668, p. 10. I
394
Microbiological Reactions with Steroids
395
Another extensivemonograph on the matter (unavailableto the present reviewer) appears to have been issued.Ig Much of the recent interest centres on the industrial topics of process development for improved dehydrogenations,for resolution of racemic steroid intermediates from total synthesis, and for transformation of sterols to oestrone, and on topics of mechanism of hydroxylation, of hydroxy-steroid dehydrogenation, and of degradation of the steroid molecule into fragments which enter the general metabolism of the organism. The present Report will attempt to cover these aspects together with others of chemical interest since about 1966, when the major reviews previously quoted'-3 appeared. The literature has been examined for chemical interest, and only those papers which deal with steroid substrates nominally alien to the micro-organism involved and which are added as xenobiotic substrates to a controlled culture of a single specified micro-organism are included. Thus, with few exceptions, a substantial amount of literature which deals with biosynthesis steps involving nominally endogenous steroid substrates, with undefined microbial cultures such as intestinal microflora, or with nutritional aspects of steroids and micro-organismshas not been reviewed. In a few instances steroid transformations by cultures of cells from higher plants have been included where such cultures may be performed in fermentors or shake-flasks in a manner similar to that used for micro-organisms. The patent literature has not been reviewed. An arbitrary division of the Report into eight major reaction-type categories has been made : hydroxylation ; hydroxy-steroid/oxo-steroiddehydrogenases ; carbon-arbon bond dehydrogenases and reductases ; olefinic bond isomerization ;ester, amide, ether, acetal, etc. reactions ;hetero-atom reactions ;degradation reactions ;and other reactions.
2 Hydroxylation Reactions Of the several microbial transformations to be reviewed, hydroxylation reactions are unique in their diversity and importance. Hydroxylation reactions offer access to otherwise inaccessible sites in the steroid molecule and as such are of great commercial interest, particularly 11- and 16a-hydroxylations leading to adrenal cortex hormones and their analogues. Hydroxylations depend less directly on the structure of the substrate molecule than do dehydrogenation, reductase, esterase, and other reactions, which by their nature require the presence of a specific functional group for the reaction to proceed. A rich variety of steroid substrates has been examined over the past few years. A representative selection of reported hydroxylations is given in the Table beginning on p. 397. It may be seen that the A4-3-0x0-steroid so much under study in earlier work is now matched in numbers by substrates not bearing this function. Many substrates bearing exotic structural features have been examined. However, there has been a tendency to limit the types of micro-organismemployed l9
A. A. Akhrem and Yu. A. Titov, 'Steroidy i Mikroorganizmy', Nauka Publishing House, Moscow, 1970 (Chern. A h . , 1972, 76, 83 472y, 110 226e).
396
Terpenoids and Steroids
in these studies. Hydroxylative attack at the llcc-position remains the most commonly encountered transformation. Most of the sites of hydroxylation previously reported'-3 have received continuing attention, and several sites of attack not previously observed have now been encountered. Thus 3p- and 4-hydroxylations not heretofore reported have been demonstrated with appropriate substrates, as have 9p- and 14P-hydroxylations of appropriate 9p- and 14P-H steroids. However, hydroxylations at the 13a- (in 18-nor-steroids),the unnatural 8a- and 1Oa- (in 19-nor-steroids),and 17psites have yet to be recorded. Specific hydroxylations at sterol side-chain sites C-22 to C-25 have not been demonstrated, but hydroxylation at the terminal carbon atom of the cholesterol side-chain, yielding 26-hydroxy-(25S)-cholest-4-en-3-one, has been reported.20 A prior assignment of the (25R)-configuration2' to the product has been revised in the light of X-ray diffraction analysis of the 26-p-bromobenzoate ester.22 The microbial hydroxylations listed in the Table involve vegetative cell cultures (and/or spores as noted).
2o 21
22
M. Galli Kienle, R. K. Varma, L. J. Mulheirn, B. Yagen, and E. Caspi, J . Amer. Chem. Suc., 1973, 95, 1996. E. €aspi, M. Galli Kienle, K. R. Varma, and L. J. Mulheirn, J . Amer. Chem. SOC.,1970, 92, 2161. D. J. Duchamp, C. G. Chidester, J. A. F. Wickramasinghe, E. Caspi, and B. Yagen, J . Amer. Chem. SOC.,1971,93, 6283.
Steroid substrute
24
23
Nocardia corallina
Nocardia corallina
Nocardia corallina
Penicillium sp. ATCC 12556 Nocardia corallina
Micro-organism
24
23
Reference
9a-Fluoro-la,2a,l 1~,17P-tetrahydroxy- 24 17a-methylandrost-4-en-3-one 9a-Fluoro-17a-methyl-1 lP,17Pdihydrox yandrosta- 1.4-dien-3-one I7a-Ethynyl-la,2a,17P-trihydroxy24 androst-4-en-3-one 16a,17a-Isopropylidenedioxy24 1a,2a,118,21-tetrahydroxypregn4-ene-3,20-dione 16a,l7a-Isopropylidenedioxy-2 1hydroxypregna-l,4-diene-3,11,20trione 16a,l7a-Isopropylidenedioxy-llP,21dihydroxypregna- 1,4-diene-3,2O-dione
la,3P-Dihydroxy-5a-androstan17one 9a-Fluoro-la,2a,l lP,l7/?-tetrahydroxy17a-methylandrost-4-en-3-one
Products
S. Noguchi and D. K. Fukushima, J . Org. Chem., 1965,30, 3552. K. J. Sax, C. E. Holmlund, L. I. Feldman, R. H. Evans, R. H. Blank, A. J. Shay, J. S. Schultz, and M . Dann, Sreroids, 1965, 5 , 345.
C,,H3,0,
4-en-3-one 16a,17a-lsopropylidenedioxy1lS,21-dihydroxypregn-4-ene3,20-dione
17a-Ethynyl-17P-hydroxyandrost-
C, H,,02
C,,H, ,FO,
9a-Fluoro- 11P, 17P-dihydroxy17a-methylandrosta- 1,4dien-3-one 9a-Fluoro-l l P, 17P-dihydroxy-17amethylandrost-4-en-3-one
C2,H2, F 0 3
la-Hydroxylution C, ,H3,02 3P-Hydroxy-5a-androstan-17-one
Molecular formula of substrate
Table Microbial hydroxylations of steroids
Steroid substrate
Sa-Androstan-16-one 5a-Androstan- 17-one
Sa-Androstane-6,17-dione 5P-Androstane-B,17-dione 5a-Androstane-7,17-dione rac- 13P-Ethyl- 17P-hydroxygon4-en-3-one 5a-Androstan-1 l-one 5a-Androstan-12-one
rac- 17p- Hydroxyoestr-4-en- 3-one
17P-Hydroxyoestr-4-en-3-one
Calonectria decora Calonectria decora
Botryodiplodia malorum CBS 134.50 Botryodiplodia malorum CBS 134.50 Asperg illus ochraceus NRRL 405 Calonectria decora Calonectria decora Calonectria decora Aspergillus ochraceus NRRL 405 Calonectria decora Calonectria decora
Micro-organism
25,28
25-27
Reference
4-en-3-one 1&6a-Dihydroxy-5a-androstan-11-one 32 1&6a,15a-Trihydroxy-5n-androstan32 12-one 1~,6a-Dihydroxy-5a-androstan-16-one32,33 1~,6a-Dihydroxy-5a-androstan-17-one32,33
lfl-Hydroxy-5a-apdrostane-6,17-dione 30 1~-Hydroxy-5/?-androstane-6,17-dione3 1 l~-Hydroxy-5a-androstane-7,17-dione 30 ent-13~-Ethyl-l~,l7fl-dihydroxygon-29
ent- lfl,l7P-Dihydroxyoestr-4-en-3-one 29
lfl,l7P-Dihydroxyoestr-4-en-3-one
l~-Hydroxyoestr-4-ene-3,17-dione
Products
”
H. J. Brodie, C. E. Hay, and J. D. Townsley, Biochim. Biophys. Acta, 1971, 239, 103. 2 6 H. J . Brodie and C. E. Hay, Biochem. J., 1970, 120, 667. ’’ I. Kim, C. E. Hay, and H. J. Brodie, J. Biol. Chem., 1973,248, 2134. ” C. C. Bolt, W. J. Mijs, F. J. Zeelen, S. A. Szpilfogel, J. de Flines, and W. F. Van der Waard, Rec. Trau. chim., 1965, 84, 626. ’’ L. L. Smith, G. Greenspan, R. Rees, and T. Foell, J. Amer. Chem. Soc., 1966,88, 3120. 30 A. M. Bell, W. A. Denny, E. R. H. Jones, G. D. Meakins, and W. E. Muller, J.C.S. Perkin I, 1972, 2759. * J . E. Bridgeman, P. C. Cherry, E. R. H. Jones, and G. D. Meakins, Chem. Comm., 1967,482. 3 2 A. M. Bell, P. C. Cherry, I. M. Clark, W. A. Denny, E. R. H. Jones, G. D. Meakins, and P. D. Woodgate, J.C.S. Perkin I, 1972, 2081. 3 3 J. E. Bridgeman, J. W. Browne, P. C. Cherry, M. G. Combe, J. M. Evans, E. R. H. Jones, A. Kasal, G. D. Meakins, Y. Morisawa, and P. D. Woodgate, Chem. Comm., 1969,463.
C19H30O
C19H,,0,
C18H2602
1P- Hydroxylation C 8 H,,O, Oestr-4-ene-3,17-dione
Molecular formula of substrate
Table-continued
w
d
I?
3
5
Q
g
3 d 9
00
Calonectria decora Calonectria decora
Fusarium sp. S33
3/3,14-Dihydroxy-5B,14j-card20(22)-enolide
36
35
34
5a-Androstan-15-one Calonectria decora 9a-Fluoro-l1~,17~-dihydroxyNocardia corallina 17a-methylandrosta-1,4-dien-3-one 9a-Fluoro-ll~,l7/?-dihydroxyNocardia corallina 17a-methylandrost-4-en-3-one
36
2a,12B-Dihydroxy-5a-androstan15-one 32 9a-Fluoro- la,2a,ll /?, 178-tetrahydroxy- 24 17a-methylandrost-4-en-3-one 9a-Fluoro- la,2a,l1/?,17/3-tetrahydroxy- 24 17a-methylandrost-4-en-3-one
17a-Methyloestra-l,3,5( 10)triene-2,3,17B-triol
1P,3a,6a-Trihydroxy-5a-androstan30 17-one 1&3a,l 5a-Trihydroxy-5a-androstan17-one 5a-Androstane- 18,6a,178-triol 32 32 lB,6a-Dihydroxy-3-rnethylene-5aandrostan-17-one ent-lj?,17/3-Dihydroxy-l3~-propylgon-29 4-en-3-one 18,78,1Sa-Trihydroxy-5a-~32 homoandrostan-17-one 32 3-Methylene-5a-androstanel#l,6a,l7#?-triol 1/3,6a-Dihydroxy-3a-methoxy-5a30 androstan-17-one 34 1B,3&1la-Trihydroxy-5a-pregnan20-one 1/3,1la-Dihydroxy-5a-pregnane3,20-dione 18,38,78,14-Tetrahydroxy-S/?,14835 card-20(22)-enolide
A. S. Clegg, E. R. H. Jones, G. D. Meakins, and J. Pinhey, Chem. Comm., 1970, 1029. M. Schemer-Gervai, L. Gsell, and C. Tamm, Helv. Chim. Acta, 1969, 52, 142. K. Schubert. H. Groh, and C. Horhold, J. Steroid Biochem., 1971, 2, 281.
C20H,9F03
CloH, , F 0 3
C19H300
2a-Hydroxylation
2-Hydroxylation C,9H,,02 17a-Methyloestra-l,3,5(10)-triene3,178-diol
C23H3404
Aspergillus J a w s
Aspergillus ochraceus
3B-Hydroxy-Sa-pregnan-2O-one
2 ‘
lH3,02
Calonectria decora
3a-Methoxy-5a-androstan-17-one
Calonectria decora
rac-17/3-Hydroxy-13fi-propylgon- Aspergillus ochraceus NRRL 405 4-en-3-one Calonectria decora Sa-~-Homoandrostan-17-one
5a-Androstan-178-01 3-Methylene-5a-androstan-17-one
Calonectria decora
C20H3202
C20H320
‘20H3002
C20H300
C19H320
C19H3002
17a-Ethynyl-17fl-hydroxyandrost4-en-3-one 16a,17a-Isopr opylidenedioxy1lP,21-dihydroxypregn-4-ene3,20-dione
Steroid substrate
39
38
37
Calonectria decora Saccharomyces cerevisiae (cell-free)
Lactarius quietus (Fr.)
Nocardia corallina
Nocardia corallina
M icro-organism
D. H . R. Barton and G . P. Moss, Chem. Comm., 1966,261.
Z. Prochazka and V. SaSek, CON.Czech. Chem. Comm., 1967, 32, 610. P. C. Cherry, E. R. H. Jones, and G . D. Meakins, Chem. Comm., 1966, 5 8 7 .
38-Hydroxylation Ci 9H2n0 Androst-5-en-7-one C30H30 Lanosta-8,24-diene
28-Hydroxylation C21 H 3 0 0 4 17a,21-Dihydroxypregn-4-ene3,20-dione
C,,H 406
C, 1HZnO2
Molecular formula of substrate
Table-continued
37
24
24
Reference
3p,12/?-Dihydroxyandrost-5-en-7-one 32, 38 Lanosta-8,24-dien-38-01 39
2p, 17a,21-Trihydroxypregn-4-ene3,20-dione
9a-Fluoro- 11p, 17fi-dihydroxy-17amet hylandrosta- 1,4-dien-3-0ne 17a-Ethynyl-la,2a,17ptrihydroxyandrost-4-en-3-one 16a,17a-Isopropylidenedioxy1a,2a, 118,2l-tetrahydroxypregn-4ene-3,20-dione 16a,17a-Isoprop ylidenedioxy-21hydroxypregna-l,4-diene-3,11,20trione 16a,17a-Isopropylidenedioxy-1 1p,2 1dihydroxypregna-1,4-diene3,20-dione
Products
R
9
P 0
Cholest-5-en-3P-01
17a-Methyloestra-l,3,5(10)-triene3,17P-diol 17P-Hydroxy-17a-met hyl~-norandrost-4-en-3-one
3B-Hydroxy-~-norandrost-4-en17-one 17a-Methyloestra-1,3,5(10)-triene3,178-diol 9p,1Oa-Androst-4-ene-3,17-dione
C, ,H2,0,
"
44
43
42
41
49
Curvularia lunata NRF2L 2380
Asperg illus Javus
Rhizopus nigricans
Absidia orchidis
Cercospora (Corynespora) melonis CKe Cercospora (Corynespora) melonis CKe
Rhizopus nigricans
Calonectria decora
Streptomyces sp.
Protaminobacter ruber
Asperg illus flavus
41
40
36
6a-Hydroxy-~-norandrost-4-ene3,17-dione 3B,5,6a-Trihydroxy-5p-~norandrostan-17-one l7a-Methyloestra- 1,3,5(10)-triene3,6a,17p-triol 6a-Hydroxy-9/3,10a-androst-4-ene3,17-dione
45
36
42
42
3fl,5,6a-Trihydroxy-Sfi-~-norandrostan42 17-one 3a,S-Dihydroxy-SP,14P-androstan43 17-one 3a,5-Dihydroxy-SP-androstan-17-one 44
4P,12~-Dihydroxyandrost-5-en-7-one 32, 38
17a-Methyloestra-l,3,5(10)-triene3,4,17/?-triol 4,9~-Epoxy-l7a-methyl-9,1O-secoB-norandrosta- 1,3,5(lo)-triene3,9(,17P-triol 4-Hydroxycholest-4-en-3-one
K. G . Holden, L. R. Fare, and J. R. Valenta, J . U r g . Chem., 1967, 32, 960. R. L. Brown and G. E. Peterson, J . Gen. Microbiol., 1966, 45, 441. J. Joska, 2. Prochazka, J. FajkoS, and F. Sorm, Coll. Czech. Chem. Comm., 1973,38, 1398. F. Mukawa, Chem. Comm., 1971, 1060. E. Kondo and T. Mitsugi, Tetrahedron, 1967, 23, 2.153. D. Van der Sijde, J. de Flines, W. F. Van der Waard, and A. Smit, Rec. Trav. chim., 1969,88, 1437.
C, ,H,,O,
~-Norandrost-4-ene-3,17-dione
3a-Hydroxy-SP-androstan-17-one
C,,H,,O,
ba-Hydrox ylation
C,,H,,O,
5p- Hydroxylation C, ,H,,O, 3P-Hydroxy-~-norandrost5-en-17-one C, ,H2,O2 Sp,14,8-Androstane-3,17-dione
4B-H ydroxylat ion C,,H*,O Androst-5-en-7-one
C27H46O
C H, 0,
,, ,
C, ,H,QO,
4-Hydroxylation
Calonectria decora
Calonectria decora Calonectria decora Calonectria decora Calonectria decora Calonectria decora Calonectria decora Calonectria decora
Sa-Androstane-2,17-dione 5a-Androstane-11,l’l-dione 5a-Androstan-Zone
5a-Androstan-3-one
5a-Androstan-1 1-one 5a-Androstan- 12-one
Sa-Androstan-15-one
C19H300
Calonectria decora
5a-Androst- 1-en-3-one
5a-Androstane-2,16-dione
Calonectria decora
Micro-organism
5a-Androst-Zen- 1-one
Steroid substrate
C ,,H2 802
C19H280
Molecular formula of substrate
TablHontinued
30
32
32
Reference
128-Hydroxy-5a-androstane2,6,16-trione 6a-Hydroxy-5a-androstane-2,17-dione 30 6a-Hydroxy-5a-androstane-l1,17-dione 30 6a,l la-Dihydroxy-5a-androstan-2-one 32, 33 6a,12fl-Dihyroxy-5a-androstan-2-one 6a,128,1Sa-Trihydroxy-5a-androstan32 3-one 6a-Hydroxy-5a-androstan- 11-one 32 6 4 15a-Dihydroxy-5a-androstan- 12-one 32 18,6a,15a-Trihydroxy-5a-androstan12-one 6a,12P-Dihydroxy-Sa,14p-androstan32 15-one
6a-H ydroxy-5a-androst-2-ene1,16-dione 6a, 168-Dihydroxy-5a-androst2-en- 1-one 6a,l la-Dihydroxy-5a-androst1-en-3-one 6a,128-Dihydrox y-Sa-androstane2,16-dione
Products
*H2@
48
47
46
rue- 17fl-Hydroxyoestr-4-en-3-0ne
17&Hydroxyoestr-4-en-3-one
Oestr-4-en-3-one
rac-6j?,178-Di hydroxyoestr-4-en-3-one
6/?,17p-Dihydroxyoestr-4-en-3-one
6,5,11a-Dihydroxyoestr-4-en-3-one 68,l la-Dihydroxyoestr-4-en-3-one
48
47
29
46 32 47
25-27
1/?,6a-Dihydroxy-Sa-androstan16-one 32,33 6a,l la-Dihydroxy-5a-androstan-16-one l/l,6a-Dihydroxy-5a-androstan17-one 32,33 6a, 17p-Dihydroxy-5a-androstan-3-one 30 6a,15a-Dihydroxy-5a-androstan- 12-one 30 30 1j3,3a,6a-Trihydroxy-5a-androstan17-one 32 5a-Androstane- 1p,6a,17p-trio1 Sa-Androstane-6a, 1la, 17j3-triol 32 3-Methylene-5a-androstane1p,6a, 17p-trio1 6a,l la-Dihydroxy-5a-~32 homoandrostan-17-one 30 1/3,6a-Dihydroxy-3a-methoxy-5aandrostan-17-one
A. M. Bell, J. W. Browne, W. A. Denny, E. R. H. Jones, A. Kasal, and G. D. Meakins, J.C.S. Perkin I , 1972, 2930. Y. Y. Lin, M. Shibahara, and L. L. Smith, J. Org. Chem., 1969, 34, 3530. H.-J. Koch, G. Schulz, and K. Kieslich, Chem. Ber., 1970, 103, 603.
CI8Hz6O2
c1
Bot ryodiplod ia malorum CBS 134.50 Aspergillus ochraceus Calonectria decora Curvularia lunata NRRL 2380 Aspergillus ochraceus NRRL 405 Curvularia lunata NRRL 2380 Aspergillus ochraceus
Calonectria decora
Calonectria decora
Sa-~-Homoandrostan-17-one
3a-Methoxy-5a-androstan-17-one
Calonectria decora
3-Methylene-5a-androstan-17p-01
Calonectria decora
Calonectria decora Calonectria decora Calonectria decora Calonectria decora
5a-Androstan-17-one 17p-Hydroxy-5a-androstan-3-one 15a-Hydroxy-5a-androstan-12-one 3a-Hydroxy-5a-androstan- 17-one
68-Hydrox ylation C,8H2402 Oestr-4-ene-3,17-dione
C20H32O2
C20H320
C19H3002
Calonectria decora
5a-Androstan-16-one
M icro-organism
AspergHlus ochruceus Aspergillus tamarii Kita Q M 1223 Aspergilltls ochraceus Asperg illus flavus
Androst-4-en-3-one Sa-Androstane-3,17-dione
Cl9H28O
C, 9 H 2 8 0 2
55
53
52
5 1
50
49
Reference
46
52
51 45
46 6P-Hydroxy-5a-androstane53 3,17-dione 68,l la-Dihydroxyandrost-4-en-3-one 46 6/3-Hydroxyandrost-4-ene-3,17-dione 54 Androst-4-ene-3,17-dione 3-0~0-13,17-secoandrost-4-eno17,13a-lactone
6B-Hydroxyandrost -4-ene-3,17-dione 6P-Hydroxy-9& 1Oa-androst-4-ene3,17-dione 3-Methoxyoestra-l,3,5(10)-triene68,17P-diol -en6P,11a-Dihydroxy-5a-androst-1 3-one 6p,1 la-Dihydroxyandrost-4-en-3-one
46 6fl-Hydroxyandrost-4-ene-3,17-dione 49 6fl-Hydroxyandrost-4-ene-3,17-dione 50
Products
68,l la-Dihydroxy-5a-oestran-3-one
G. Ambrus and G. Wix, Acta Chim. Acad. Sci.Hung., 1968,55, 99. B. J. Auret and H. L. Holland, Chem. Comm., 1971, 1157. L. A. R. Sallam, A . - M . H. El-Refai, and M. H. K. Kinawy, Pakistun J . Biochprn., 1971, 4, 19. C. Casas-Campillo and M. Bautista, Appl. Microbiol., 1965, 13, 977. D, R. Brannon, F. W. Parrish, B. J. Wiley, and L. Long, J . Org. Chem., 1967, 32, 1521. K . Schubert, J. Schlegel, H. Groh, G . Rose, and C. Horhold, Endokrino/ogie, 1972, 59, 99.
11a-Hydroxyandrost-4-en-3-one 17fl-Hydroxyandrost-4-en-3-one
Fusurium monilforme I H4 Aspergillus ochraceus
3-Methoxyoestra-l,3,5(lO)-trien17fl-01 Scc-Androst-1-en-3-one
,
Sa-Oestran-3-one Androst-4-ene-3,17-dione
Aspergillus ochracrus Absidiu orchidis Aspergillus niger ATCC 9142 Penicillium sp. 9fl,10zc-Androst-4-ene-3,17-dione Curtlularia lunata
Steroid substrcitr
C ,H2,0,
c,sH2,O
Molecular jormulu 0: substratr
Tablwontinued
0 P P
1la-Hydroxypregn-4-ene3,20-dione
REF 129
Rhizopus nigricans
No. 663
Beauveria bassiana
Aspergillus Jischeri
No. 663
Beauveria bassiana
Actinomyces sp. 3375 Aspergillus Jischrri
Stachylidium theobromae
17P-Hydroxy-18-methylandrost-
4-en-3-one Pregn-4-ene-3,20-dione
Aspergillus ochraceus Aspergillus ochraceus Aspergillus jlavus
5a-Andros tan-3-one 1la-Hydroxy-5a-androstan-3-one 17P-Hydroxy-17a-methylandrost4-en-3-one
NRRL 2380
Curvularia lunata
NRRL 405
Aspergillus ochraceus
13B-Ethyl-6P,178-dihydroxygon29 4-en-3-one rac- 13P-Ethyl-6P, 17B-dihydroxygon47 4-en-3-one 13P-Ethyl-6P,l08, 178-trihydroxygon4-en-3-one 6P,1 la-Dihydroxy-5a-androstan-3-one 46 68,l la-Dihydroxy-5a-androstan-3-one 46 54 6~,17~-Dihydroxy-17a-methylandrost4-en-3-one 68-Hydroxy- 18-methylandrost55 4-ene-3,17-dione 6P-Hydroxypregn-4-ene-3,20-dione 56 57 68,l la-Dihydroxypregn-4-ene3,20-dione 68,l la-Dihydroxypregn-4-ene58 3,20-dione 68,l la-Dihydroxypregn-4-ene57 3,20-dione 6P,1 la-Dihydroxypregn-4-ene58 3,20-dione 68,1 la-Dihydroxypregn-4-ene59,60 3,20-dione 1la-Hydroxy-5a-pregnane-3,20-dione
56
55
K. Kieslich, H.-D. Berndt, R. Wiechert, U. Kerb, G . Schulz, and H.-J. Koch, Annalen, 1969, 726, 161. L. M. Kogan, I. M. Volkova, M. I. Bukhar, N. S. Agre, I. S . Zvyagintseva, V. 1. Tsurkova, M. V. Torgov, and G . K. Skryabin, Mikrobiologiya, 1968, 37, 628. ’’ L. A. R. Sallam, A.-M. El-Refai, S. Nada, and A. F. Abdel-Fattah, J. Gen. Appl. Microbiol., 1973, 19, 155. 5 a A. Capek, 0. HanE, M. Tadra, and J. Thma, Folia Microbiol., 1966, 11, 159. 5 9 L. A. R. Sallam, A.-M. H. El-Refai, and I. A. El-Kady, J . Gen. Appl. Microbiol., 1970, 16, 409. A.-M. H. El-Refai, L. A. R. Sallam, and I . A. EI-Kady, Bull. Chem. SOC.Japan, 1970,43,2878. 6 o A.-M. H. El-Refai, L. A. R. Sallam, and I. A. El-Kady, J . Gen. Appl. Microbiol., 1970, 16, 137.
C,, H 3 , 0 3
C, H,,O,
C ,H,,O, C, ,H ,O,
C19H300
rac-13P-Ethyl- 17P-hydroxygon4-en-3-one
P
s
s
E.
Cephalosporium sclerotigenum 31C Verticillium aphidum
Bacillus cereus
M icro-organism
Rhizopus nigricans 17a,21-Dihydroxy- 16met hylenepregn-4-ene-3,20-dione Rhizopus arrhizus 22-Hydroxy-23,24-bisnorchol4-en-3-one Chaetomium cochloides 3-(7a-Methylthio-3-oxoandrostQM624 4-en- 17a-y1)propionic acid y -lactone
21-Hydroxypregn-4-ene3,20-dione 17a,21-Dihydroxypregn-4-ene3,20-dione
Steroid substrate
62
61
Products
6p,2 1-Dihydroxypregn-4-ene3,20-dione 6p, 17a,21-Trihydroxypregn-4-ene3,20-dione 6p, 17a,2 1-Trihydroxypregn-4-ene3,20-dione 6p,17a,21-Trihydroxy-16met hylenepregn-4-ene-3,20-dione 6p,1 la,22-Trihydroxy-23,24- bisnorchol4-en-3-one 3-(6P-Hydroxy-7a-met hy It hio-3oxoandrost-4-en-17a-yl)propionic acid y-lactone 3-(6fi-Hydroxy-3-oxo-7amet hylsulphinylandrost-4-en- 17a-y1)propionic acid y-lactone 3-(3-0~0-7cr-methylsulphinylandrost4-en- 17a-y1)propionic acid y -1actone
J. E. Wilson, R. E. Ober, and C. S . Vestling, Arch. Biochem. Biophys., 1966, 114, 166. J. L. Sardinas and M. A. Pisano, Appl. Microbiol., 1967, 15, 277. 6 3 V. Schwarz, J. Protiva, and J. Martinkova, Coll. Czech. Chem. Comm., 1971, 36, 3455. 6 4 D. S. H. Smith, N. J. Poole, and W. F. A. Jowett, Phytochemistry, 1973, 12, 561. 64a W. J. Marscheck and A. Karim, Appl. Microbiol., 1973, 25, 647.
C,,H,,O,S
C, 2 H, 4 0 2
C , , H3OO4
C 21 H , o 0 4
Molecular formula of substrate
Tabl-ontinued
64a
64
63
62
62
61
Reference
8 a
3aJ4-Dihydroxy- 14P-bufa4,20,22-trienolide
C24H32O4
17P-H ydrox yandrost-4-en-3-one
C ,H, 0,
69
'*
6 '
66
6s
Y . Y. Lin and L. L. Smith, Biochim. Biophys. Acta, 1970, 210, 319. K. Singh, S. N. Sehgal, and C. Vkzina, Canad. J. Microbiol., 1967, 13, 1271
B. Gorlich and J. Wolter, Annalen, 1971, 753, 106. M.J. Frey, H. Jirku, and M. Levitz, J. Labelled Compounds, 1970, 4,
355.
Curvularia lunata NRRL 2380 Absidia orchidis Mucor griseo-cyanus ATCC 1207 (and spores) Aspergillusjlavus
Clomerellafusarioides
Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg Rhizopus arrhizus Fischer or Rhizopus aigricans Ehrenberg Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg
B. Gorlich, F. H. Durr, and J. Wolter, Annalen, 1971, 753, 116.
C,,H,,O,
rac-17P-Hydroxyoestra4,8( 14)-dien-3-one Androst-4-ene-3,17-dione
C, *H2,O2
7a-Hydroxylation C,,H,,O, 3-Hydroxyoestra-1,3,5(10)trien- 17-one
3P,14-Dihydroxy-14B-bufa4,20,22-trienolide (scillarenin)
14-Hy droxy -3-0x0-14P-bufa4,20,22-trienolide
C24H ,04
3,7a-Dihydroxyoestra- 1,3,5(10)trien-17-one Oestra-1,3,5(lO)-triene-3,7a,l7P-triol rac-7a, 17P-Dihydroxyoestra4,8( 14)-dien-3-one 7a-Hydroxyandrost-4-ene-3,17-dione 7a-Hydroxyandrost-4-ene-3,17-dione
3P,6P,14-Trihydroxy-148-bufa4,20,22-trienolide
3a,6&14-Trihydroxy-14P-bufa4,20,22-trienolide
6/?,14-Dihydroxy-3-0~0-14P-bufa4,20,22-trienolide
54
49 69
68
67
65,66
66
65
E
s.
H32°2
1 Fo2
74
73
i2
7o
Mucor griseo-cyanus ATCC 1207 (and spores) Circinella muscae Mucor griseo-cyanus ATCC 1207 (and spores) Mucor griseo-cyunus ATCC 1207 (and spores) Curvularia lunata
17P-Hydroxy-l7a-methylandrost4-en-3-one 3fl-Hydroxypregn-5-en-20-one 21-Fluoro-l7a-methylpregna1,4-diene-3,20-dione
21 -Fluoro-l7a-methyIpregn-4-ene3,20-dione 2 1-Hydroxy- 16a-met hylpregn4-ene-3,20-dione 21-Acetoxy-17a-hydroxy-18methylpregn-4-ene-3,20-dione 3P-Dimet hylaminocon-5-enine (conessine)
Gloeosporium fructigenum
Curvulariu lunata
Gibberella saubinetti (Mont.) Sacc.
M icro-organism
3P-Hydroxyandrost-Sen- 1”-one
Steroid substrate
Products
Reference
3j9,7a-Dihydroxyandrost-5-en-l7-one 70, 7 1 3fl,7a,15a-Trihydroxyandrost-Sen- 17-one 3b-Hydroxyandrost-5-ene-7,17-dione 3P,17P-Dihydroxyandrost-5-en-7-0ne Androst-5-ene-3fl,7a,17P-triol 7a,17P-Dihydroxy-17a-methylandrost- 69 4-en-3-one 3P,7a,9a-Trihydroxypregn-5-en-20-one 72 69 21-Fluoro-7a-hydroxy-17amethylpregna-1,4-diene-3,20-dione 21 -Fluoro-7a,14a-dihydroxy-17amethylpregna- 1,4-diene-3,20-dione 21-Fluoro-7a-hydroxy-17~~69 methylpregn-4-ene-3,20-dione 7a,2 1-Dihydroxy- 16a-methylpregn73 4-ene-3,20-dione 7a,14a,17a,21-Tetrahydroxy-1855 met hylpregn-4-ene-3,20-dione 74 3fl-Dimethylaminocon-5-enin-7a-ol
M. Okuda and Y . Saito, Steroids, 1965, 6 , 651. M . Okada, A. Yamada, and M. Ishitdate, Yakugaku Zasshi, 1965, 85, 816. M. B. Gorovits, A. D. Koveshnikov, and N. K. Abubakirov, Khitn. prirod. Soedinenii, 1969, 125 K . Kieslich, H. Wieglepp, K. Petzoldt, and F. Hill, Tetrahedron, 1971, 27, 445. A. F. Marx, H. C . Beck, W. F. Van der Waard, and J. de Flines, Steroids, 1966, 8, 421.
C24H40N2
C24H3405
c2.2 H32 O3
C22H3
C22H29FO2
‘21
‘20H3002
Molecular formula of substrate
Tablwontinued
8 cx,
3P-Hydroxypregn-5-en-20-one
17a,21-Dihydroxy- 16met hylenepregn-4-ene-3,20-dione
C, H3,OZ
C22H3004
Rhizopus nigricans
Beauueria globulifera
76
Beauveria bassiana
la
l7
76
V. Schwarz, J. Martinkova, J. Protiva, and K. Syhora, Coil. Czech. Chem. Comm., 1966, 31, 4703. J. Protiva, V. Schwarz, J. Martinkova, and K. Syhora, Folia Microbiol., 1968, 13, 146. J. Protiva, V. Schwarz, and J. Martinkova, Folia Microbiol., 1968, 13, 139.
77,78 16a,17a-Epoxy-7&1la-dihydroxy16fi-rnethylpregn-4-ene-3,20-dione 16a,17a-Epoxy-7/3,1la-dihydroxy16fi-methy1-5/?-pregnane-3,20-dione 7j3,17a,21-Trihydroxy-1663 methylenepregn-4-ene-3,20-dione
16/3-methyIpregn-4-ene-3,20-dione
30
3,20-dione 16a,17a-Epoxy-78,1la-dihydroxy-
32
7/3,12/3,15a-Trihydroxypregn-5-ene-
homoandrostan- 17-one
7/3,12&15a-Trihydroxy-5a-~-
15-one 7P,12~,14-Trihydroxy-Sa,148androstan-l5-one 18,78,1Sa-Trihydroxy-Sa-~homoandrostan- 17-one
6'
7~-Hydroxy-5j?-androstane-3,17-dione 7~,12/3-Dihydroxy-5a,14~-androstan- 30
E
5
7/3,1la-Dihydroxy-5a-androstan-17-one 46 3~,7/3-Dihydroxy-SjI-androstan17-one 23
7/3-Hydroxyoestr-4-ene-3,17-dione 25-27 78,l l~-Dihydroxyoestr-4-ene-3,17-dione [4-14C]-7b-Hydroxyoestr-4-ene75 3,17-dione 7fl-Hydroxy-5/?-androstane-3,17-dione 53
Calonectria decora
Calonectria decora
Aspergillus tamarii Kita QM 1223 Aspergillus ochraceus Penicillium sp. ATCC 12556 Calonectria decora
Botryodiplodia malorum CBS 134.50 Botryodiplodia malorum
'' H. J. Brodie, C. E. Hay, and T. A. Wittstruck, J. Org. Chem., 1972, 37, 3361.
Sa-~-Homoandrostan17-one
C20H320
12P-Hydroxy-Sa,148-androstan15-one
5a-Androstan-17-one
3/3-Hydroxy-5/3-androstan-17-one
C19H300
5B-Androstane-3,17-dione
C19H3002
C, 9Hzs0,
[4-'4C,7-3H]Oestr-4-ene-3,1 7-dione
7b-Hydroxylation C,,HZ4O2 Oestr-4-ene-3,17-dione
79
14,l Sfi-Epoxy-3fl-hydroxy5fl, 14fl-bufa-20,22-dienolide (resibufogenin) 3aJ4-Dihydroxy- 14P-bufa4,20,22-trienolide
14-Hydroxy-3-oxo-14fl-bufa4,20,22-trienolide
Products
Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg
3a,7/3,14-Trihydroxy-l4/?-bufa4,20,22-trienolide
3fi-Allyloxy-5a-androstane7/?,12p,lSa-triol Cunninghamella blakesleeana 3p,7p,14-Trihydroxy-SP,14fi-card20(22)-enolide Fusarium sp. S33 38,7p,14-Trihydroxy-5/3,14P-card20(22)-enolide 1P,3fl,7p,14-Tetrahydroxy-5P914pcard-20(22)-enolide 3p,7P,1la,l4-Tetrahydroxy-5/3,14pcard-20(22)-enolide Rhizopus nigricans 3p,7p, 14-Trihydroxy-5P,14~-cardATCC 6227b 20(22)-enolide Rhizopus arrhizus 7fl,14-Dihydroxy-3-oxo-l4fl-bufaFischer 4,20,22-trienolide or Rhizopus nigricans Ehrenberg Absidia orchidis 14,l SP-Epoxy-3p,7P-dihydroxy(Vuill.) Hagem. 5fl, 14P-bufa-20,22-dienolide
Calonectria decora
Micro-organism
M . Okada and M . Hasunuma, Yakugaku Zasshi, 1966,86, 67. E. G . Gros and E. Leete, J . Amer. Chem. SOC.,1965, 87, 3479. L. Gsell and C. Tamm, Helv. Chim. Acfa, 1969, 52, 150.
C24H3204
c2 qH 3 0°4
3fl,14-Dihydroxy-Sfl,14fl-card20(22)-enolide (digitoxigenin)
5a-Androstan-3p-01 3-ally1 ether
c 22 H3 6 0
C23H3404
Steroid substrate
Molecular formula of substrate
Table--continued
65,66
81
65
80
35
79
32
Reference
R
n
f: 0
20,22-dienolide (bufalin) 3P- Acet oxy- 14,15P-epoxy-5P, 14Pcard-20(22)-enolide
3P-Dimethylaminocon-5-enine
(conessine) (25S)-Spirost-5-en-3P-o1 (diosgenin)
3~,l4-Dihydroxy-5P714~-card-
C, 5 H 4 05
Cz4H4,N,
Cz,H4203
C4,H,,0,,
84
83
"
(25S)-Spirost-5-ene-3P,7P,1 la-trio1 (25S)-Spirost-5-ene-3/?,7P,12$-triol 3P,7P,14-Trihydroxy-5P, 14P-card20(22)-enolide3P-tridigitoxoside
(25S)-Spirost-5-ene-3P,7P-diol
3P-Dimethylcon-5-enin-7P-01
3P,7p71 4-Trihydroxy-SP714P-bufa20,22-dienolide 14,l SB-Epoxy-3P,7P-dihydroxy5/3,14fl-card-20(22)-enolide
5.7P, 14-Trihydroxy-5/3,14,4-bufa3,2O722-trieno1ide
3P,7P,14-Trihydroxy-14P-bufa4,20,22-trienolide
Aspergillus nidulans
88,17~-Dihydroxy-9P710a-androst4-en-3-one
Streptomyces griseojavus ATCC 12269, Streptomyces cellulosae ATCC 3313, Chaetomium globosum MN-21 I , or Chrysosporium merdarium MN-72
Cunninghamella blakesleeana
Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg Absidia orchidis (Vuill.) Hagem. Absidia orchidis (Vuill.) Hagem. Gloeosporium fructigenum
K . Kaneko, H. Mitsuhashi, and K . Hirayama, Chem. and Pharm. Bull. (Japan), 1969,17, 2031. Y. Nazaki, M. Mayama, K. Akaki, and D. Satoh, Agric. and Biol. Chem. (Japan), 1965, 29, 783. H. Els, G . Englert, A. Furst, P. Reusser, and A. J. Schocher, Helv. Chim. Acra, 1969, 52, 1157.
8P-H ydroxylation C, ,H2 ,02 17P-Hydroxy-9/3,10a-androst-4-en3-one
20(22)-enolide3p-tridigitoxoside (digitoxin)
3P,14-Dihydroxy-SP,14P-bufa-
C24H3404
5,14-Dihydroxy-5P,14P-bufa3,20,22-trienolide
3P,14-Dihydroxy-14P-bufa4,20,22-trienolide (scillarenin)
84
83
82
74
81
81
65
65,66
s
F -.
9B,lOa-Pregn-4-ene-3,20-dione
C, lH,oOz
3a-Hydroxy-SP-androstan- 17-one
Cercospora (Corynespora) melonis CKe
Protaminobacter ruber
Corynespora melonis CBS Cercospora (Corynespora) melonis CKe
3,20-dione 8fi-Hydroxy-9p,l0a-pregn-4-ene3,20-dione
Curuularia lunata
9a-Hydroxy-Sp,14fl-androstane43 3,17-dione 3a,9a-Epoxy-3p-hydroxy-5B,14Bandrostan- 17-one 9a,17p-Dihydroxy- 17a-methyl40 ~-norandrost-4-en-3-one 3,17P-Dihydroxy-17a-methyl-9,1O-secoB-norandrosta- 1,3,5(lO)-trien-g-one 4,9t-Epoxy- 17a-methyl-9,lO-seco~-norandrosta-1,3,5( lO)-triene3,9(,17@-trioI 44 3a,9a-Epoxy-3a-hydroxy-5pandrostan-17-one 3~,9a-Epoxy-3~, 14a-dihydroxy5P-androstan-17-one
85
84
8j-Hydroxy-9/3,1Oa-pregna-4,6-diene- 84
Helicostylium piriforme
84
Reference
8/?-Hydroxy-9#?,10a-pregna* 4,6-diene-3,20-dione
Products
Nocardia brasiliensis
Micro-organism
*’S. C. Pan, J. Semar, B. Junta, and P. A. Principe, Biotechnol. and Bioeng., 1969, 1 1 , 1 183.
Cl 9H,00,
17P-Hydroxy-17a-met hyl~-norandrost-4-en-3-one
9a-Hydroxylation C H2,02 Oestr-4-ene-3,17-dione
9&10a-Pregna-4,6-diene3,20-dione
Steroid substrate
C, H 2 8 0 2
Molecular formula of substrate
Table-continued
,
3P-Dimethylaminocon-5-enine (conessine)
1 1 P, 18-Epoxy-21 -hydroxypregn-4ene-3,20-dione 6n-Fluoro-21-hydroxy-16amethylpregn-4-ene-3,20-dione 3P-Hydroxypregn-5-en-20-one 21-Hydroxy-6a,16a-dimethylpregn4-ene-3,20-dione 21-Acetoxy-6a-fluoro-16amet hylpregn-4-ene-3,20-dione
"
87
86
Curvularia lunata NRRL 2380
Nocardia lurida
Curvularia lunata (many strains), Curvularia andropogonis CBS 18649, Curvularia falcata CBS 18648, Curvularia maculans CBS 10861, Helminthosporium buchloes CBS 24649, or Helminthosporium macrocarpumloryzae CBS 31064 Botryodiplodia theobromae
Circinella muscae Curvularia Iunata
Corynespora cassicola IMI 56007 Curvularia lunata
9/3,17~-Dihydroxy-9~,10a-androsta- 84 4,6-dien-3-one 9P-Hydroxy-9P,lOa-androst-4-ene45 3,17-dione 9~,17P-Dihydroxy-9P,lOa-androst4-en-3-one
3~-Dimethylaminocon-5-enin-9a-ol 88
11/3,18-Epoxy-9a-hydroxyandrost86 4-ene-3,17-dione 6a-Fluoro-9a,2 1-dihydroxy-16a73 methylpregn-4-ene-3,20-dione 3p,7a,9a-Trihydroxypregn-5-en-20-one 72 9a,21-Dihydroxy-6a,l6a-dimethylpregn-73 4-ene-3,20-dione 6a-Fluoro-9a,21-dihydroxy16a87 methylpregn-4-ene-3,20-dione
E. Kondo, T. Mitsugi, and K . Tori, J . Amer. Chem. Soc., 1965, 87, 4655. W. Koch, K . Kieslich, H. Kosmol, and K . Petzoldt, Arch. Mikrobiol., 1969, 65, 2 2 8 . A. F. Marx, H. C . Beck, W. F. Van der Waard, and J. de Flines, Steroids, 1966, 8, 391.
9p-Hydroxylation C ,,H,,O, 17P-Hydroxy-9P,l0a-androsta4,6-dien-3-one 9~,lOa-Androst-4-ene-3,17-dione
C24H40NZ
C,,H3,F04
C, H3,0, C23H340,
C,,H,,FO,
C, H z s 0 4
21-Hydroxy-9P,lOc+pregn-4-ene3.20-dione
9P,lOa-Pregna-4,6-diene3,20-dione 9/?,10a-Pregn-4-ene-3,20-dione
C , ,H,,02
rac- 17P-Hydroxyoestr-4-en-3-0ne
17B-Hydroxyoestr-4-en-3-one
1Og-Hydroxy lation C18H2402 rac-17~-Hydroxyoestra-4,8(14)dien-3-one Oestr-4-ene-3,17-dione
C , H,,O,
,
C, lH,OO,
C, ,H,,O,
17P-Hydroxy-9P,lOa-androst-
C,9H2802
4-en-3-one
Steroid substrate
Molecular formula of substrate
Tabie-continued
Aspergillus ochraceus NRRL 405
Curvularia lunata NRRL 2380 Corynespora melonis CBS Curvularia lunata NRRL 2380
Curvularia brachyspora Boedijn No. P263 Curvularia lunata NRRL 2380 Mucor corymbifer
Cephalothecium roseum
Cephalothecium roseum
Curvulariu lunata NRRL 2380
Choanephora circinans
Micro-organism
85
68
84
45
45
84
84
45
84
Reference
29 ent-10~,17j?-Dihydroxyoestr-4-en-3-one
%one
47 1OP, 17P-Dihydroxyoestr-4-en-3-one lO~,ll~,l7~-Trihydroxyoestr-4-en-
rac- 1Og, 17P-Dih ydrox yoestra4,8(14)-dien-3-0ne 1OP-Hydroxyoestr-4-ene-3,17-dione
9P, 17P-Dihydroxy-9P,l0a-androst4-en-3-one 9P,17/3-Dihydroxy-9/?,l0a-androst4-en-3-one 9P-Hydroxy-9/?,lOa-androst4-ene-3,17-dione 9P-Hydroxy-9P,lOa-pregna4,6-diene-3,20-dione 9P-Hydroxy-9P,1Oa-pregn-4-ene3,20-dione 9P-Hydroxy-9j?,1Oa-pregn-4-ene3,20-dione 9&21-Dihydroxy-9j?,lOa-pregn4-ene-3,20-dione 98,2 1-Dihydroxy-9P, 1Oa-pregn-4-ene3,20-dione
Products
90
89
Botryodiplodia malorum CBS 134.50
K. Schubert, G. Rose, and C. Horhold, J. Steroid Biochem., 1973,4, 283. K. Kieslich and G. Schulz, Annalen., 1969, 726, 152.
Oestr-4-ene-3,17-dione
Absidia orchidis
Aspergillus f l a w s 98/13 Aspergillus flavus 98/13 Aspergillus flavus 98/13 Aspergillus f l a w s 98/13 Curvularia lunata
17a-Ethynyloestra- 1,3,5(10)-triene3,17B-diol 17a-Ethynyl-17B-hydroxyoestr4-en-3-one 17a-Ethynyl-17B-hydroxyoestr5( lO)-en-3-one 17a-Ethynyloestr-4-en-17B-01
2l-Acetoxy-5-bromo-6#l-Fluoro3B-hydroxy-16a-methyl-5apregnan-20-one
Aspergillus ochraceus NRRL 405 Curvularia lunata NRRL 2380
rac-13/3-Ethyl-17fl-hydroxygon4-en-3-one
11a-Hydroxylation C , 8H2402 ~-Norandrost-4-ene-3,17-dione
C24H36BrF04
c 2OH2 8 0
c 2OH2 60 2
C20H2402
c l 9H2802
Curvularia lunata NRRL 2380
Oestr-4-ene-3,11,17-trione
1 la-Hydroxy-~-norandrost-4-ene3,17-dione 1la-Hydroxyoestr-4-ene-3,17-dione
25,27
42
47 1Og, 17P-Dihydroxyoestr-4-en-3-one rac- lO~,l1~,17~-Trihydroxyoestr4-en-3-one ent- lOg,l l#l,l7B-Trihydroxy-5aoestran-3-one 29 13/3-Ethy1-10~,17/3-dihydroxygon4-en-3-one rac-13~-Ethyl-l0~,l7~-dihydroxygon-47 4-en-3-one 13fl-Ethyl-6B,lOfl-dihydroxygon-4-en3-one 17a-Ethynyl-l0/3,l7~-dihydroxyoestr- 89 4-en-3-one 89 17a-Ethynyl-lop, 17B-dihydroxyoestr4-en-3-one 17a-Ethynyl-l0/3,17j!?-dihydroxyoestr- 89 4-en-3-one 17a-Ethynyl-l0#l,l7~-dihydroxyoestr- 89 4-en-3-one 90 6/?-Fluoro-3~,10#l,21-trihydroxy5,16a-dimethyl-SQ-19-norpregn9(1l)-en-20-one
3-
5.
3fi-Hydroxy-~-norandrost5-en- 17-one
C,XH2602
91
Absidia orchidis Calonectria decora Aspergillus ochraceus
3-one Androst-.l-ene-3,17-dione Sa-Androst- 1-en-3-one
Androst-4-en-3-one
S. N. Sehgal, K. Singh, and C. Vezina, Canad. J . Microbiol., 1967, 14, 529.
C, ,H,,O, C19H280
17a-Hydroxyandrosta-1,4,6-trien-
Androsta- 1,4-diene-3,17-dione
C, 9H2402
Aspergillus ochraceus NRRL 405 Aspergillus ochraceus
Calonectria decoru Rhizopus nigricans
Aspergillus ochruceus
Micro-organism
Calonectria decora Asperg ill us tamarii Kita Q M 1223 Aspergillus ochraceus
5a-Oestran-3-one
C18H28O
ruc- 17P-Hydroxyoestr-4-en-3-one
Oestr-4-en-3-one
Steroid substrate
Cl8H2.50
Molecular formula of substrate
Tablwontinued Reference
29
1la-Hydroxy-5a-oestran-3-one 46 68,l la-Dihydroxy-5a-oestran-3-one 1la, 1Sa-Dihydroxy-5a-oestran-3-one 32 53 1la-Hydroxyandrosta-I ,4-diene3,17-dione 91 1la,l7a-Dihydroxyandrosta1,4,6-trien-3-one 1la-Hydroxyandrost-4-ene-3,17-dione 49 32 6a,l la-Dihydroxy-5a-androst1-en-3-one 46 1la-Hydroxyandrost-4-en-3-one 6fi,11a-Dihydroxyandrost-4-en-3-one
17-one 1l a , 17P-Dihydroxyoestr-4-en-3-one
3P,5,6a-Trihydroxy-5P-~-norandrostan-
1 la-Hydroxyoestr-4-en-3-one 46 6 j , l la-Dihydroxyoestr-4-en-3-one 32 6p,1 la-Dihydroxyoestr-4-en-3-one 3P,1la-Dihydroxy-~-norandrost-5-en- 42 17-one 5,6a-Epoxy-3fi,1 1a-dihydroxy-5a-~norandrostan-17-one 5,6a-Epoxy-3@-hydroxy-5a-~norandrostan-17-one
Products
Y
93
92
Aspergillus ochraceus Aspergillus ochraceus Asperg ill us ochraceus Aspergillus ochraceus Aspergillus ochraceus NRRL 405 (spores) Mucor griseo-cyanus ATCC 1207 (and spores) Aspergillus ochraceus NRRL 405
5a-Androstane-3.16-dione
17B-Hydroxy-9& 10a-androst4-en-3-one
5a-Androstan-4-one 5a-Androstan- 16-one 5a-Androstan- 17-one
Aspergillus ochraceus Aspergillus ochraceus NRRL 405 Calonectria decora Aspergillus ochraceus Rhizopus nigricans Calonectria decora Calonectria decora Aspergillus ochraceus
Asperg ill us ochraceus
Sa-Androstane-3,7-dione
3b-Hydroxyandrost-5-en- 17-one rac-l3/.?-Ethyl-l7~-hydroxygon4-en-3-one 5a-Androstan-2-one 5a-Androstan-3-one
Aspergillus ochraceus
Aspergillus ochraceus
Aspergillus ochraceus
5a-Androstane-3,6-dione
Sa-Androstane-2,16-dione
11a,17fi-Dihydroxy-9~,10a-androst- 91,92 4-en-3-one 1la-Hydroxy-9~,10a-androst4-ene-3,17-dione 38,ll a-Dihydroxyandrost-Sen- 17-one 46 29 rac- 13B-Ethyl- 11a, 17P-dih ydroxygon4-en-3-one 6a, 1la-Dihydroxy-5a-androstan-2-one 32,33 6fi,1la-Dihydroxy-5a-androstan-3-one 46 1 la,l6fi-Dihydroxy-5a-androstan-3-one93 32, 33 11cc,l Sa-Dih~droxy-5a-androstan-4-one 6a,l la-Dihydroxy-5a-androstan-16-one32,33 46 11a-Hydroxy-5a-androstan-17-one 78,ll a-Dihydroxy-5a-androstan- 17-one
1la,l7B-Dihydroxyandrost-4-en-3-one 69
46 1la-Hydroxy-5a-androstane2,16-dione 46 1 1a-Hydroxy-5a-androstane2,17-dione 1 la-Hydroxy-Sa-androstane-3,6-dione 46 38,l la-Dihydroxy-5a-androstan-6-one 1la-Hydroxy-5a-androstane-3,7-dione 46 3p, 1la-Dihydroxy-5a-androstan-7-one 1la-Hydroxy-5a-androstane-3,16-dione 46 38,l la-Dihydroxy-5a-androstan-16-one 1la-Hydroxy-5a-androstane-3,17-dione 46 6p,1 la-Dihydroxyandrost-4-en-3-one 46 11a, 17P-Dihydroxyandrost-4-en-3-one 46 1la,l7/3-Dihydroxyandrost-4-en-3-one 91
D. Van der Sijde, J. de Flines, and W. F. Van der Waard, Rec. Trau. chim., 1966, 85, 721. J. M. Evans, E. R. H. Jones, A. Kasal, V. Kumar, G . D. Meakins, and J. Wicha, Chem. Comm., 1969, 1491.
Cl,H,OO
C, ,H, O2
9H3002
q5
94
17P-Hydroxy- 17a-rnethyl-5Pandrostan-3-one
3D-Hydroxy-~-norpregn5-en-20-one Beauveria globuliferu
Rhizopus nigricans
Curvularia lunata
Aspergillus ochraceus
Aspergillus ochraceus Aspergillus ochraceus Aspergillus ochraceus Aspergillus tamarii Kita Q M 1223 Aspergillus ochraceus Calonectria decoru Beauveria globuliJeru
17P-Hydroxy-Sa-androstan-3-0ne 3p-H ydroxy-Sa-androstan- 16-one 3a-Hydroxy-5a-androstan- I 7-one
3P-Hydroxy-5a-androstan-17-one 5a-Androstan-l7p-01 17~-Hydroxy-17a-methylandrost4-en-3-one
Aspergillus ochraceus Aspergillus ochraceus Aspergillus ochraceus Rhizopus nigricuns
Micro-organism
17P-Hydroxy-Sa-androstan-2-0ne 6/3-Hydroxy-5a-androstan-3-one 16P-Hydroxy-5a-androstan-3-one
Steroid substrate
Reference
38,l la-Dihydroxy -5a-androstan- 17-one 5a-Androstane-6q 1la, l7P-trio1 1la,17P-Dihydroxy-l7amet hylandros t-4-en-3-one 11a, 17P-Dihydroxy-17a-rnethyl-5Pandrostan-3-one 1la-Hydroxy-3~-methoxyandrost5-en- 17-one 3P,l la-Dihydroxy-5,6a-epoxy-5a~-norpregnan-20-one 3P,1 la-Dihydroxy-B-norpregn5-en-20-one 1 la,17P-Dihydroxy-17a-methyl5P-androstan-3-one
78
95
95
46
46 32 78
1la,l7P-Dihydroxy-5a-androstan-2-one46 6P, 1la-Dihydroxy-5a-androstan-3-one 46 1la,16P-Dihydroxy-5a-androstan-3-one 46 1la,16P-Dihydroxy-5a-androstan-3-one 93 Sa-Androstane-3P,ll a, 16P-trio1 1la,l7P-Dihydroxy-Sa-androstan-3-one46 3P,1 la-Dihydroxy-5a-androstan-16-one46 3a,l la-Dihydroxy-5a-androstan-17-one 46,94 394 1la-Dihydroxy-5a-androstan17-one 53
Products
J. W. Blunt, I. M . Clark, J. M. Evans, E. R. H. Jones, G . D. Meakins, and J . T. Pinhey, J . Chem. Soc. (C), 1971, I 136. V. Sanda, J . FajkoS, and F. Sorm, CoII. Czech. Chem. Comm., 1970.35, 3445.
C,,H,,O,
C20H3002
G9H32O
C
Molecular formula of’ substrate
Table-continued
P
13,
n
00
c
loo
99
98
97
96
Aspergillus niger
Aspergillus ochraceus NRRL 405 (spores) Absidiu orchidis Aspergillus fischeri
Rhizopus nigricans
Beauveria bassianu
16a~7a-Epoxy1lor-hydroxypregn91 4-ene-3,20-dione 16a,17a-Epoxy-11a-hydroxypregn77 4-ene-3,20-dione 16a,17a-Epoxy-I la-hydroxypregn77 4-ene-3,20-dione 16a,l 7a-Epoxy-1 la-hydroxypregn77 4-ene-3,20-dione 6a-Fluoro-l1a,16a,17a-trihydroxypregn-9 1,97,98 4-ene-3,20-dione 1 la-Hydroxypregn-4-ene-3,20-dione 77 1la-Hydroxy-5a-pregnane-3,20-dione 99 1la,] 7a-Dihydroxypregn-4-ene3,20-dione 1la-Hydroxypregn-4-ene-3,20-dione 57 6/3,11a-Dihydroxypregn-4-ene3,20-dione 1 1a-Hydroxypregn-4-ene-3,20-dione I00 6p,1 la-Dihydroxypregn-4-ene3,20-dione 1 1a, 17a-Dihydroxypregn-4-ene3,20-dione 1la,l7a,21-Trihydroxypregn-4-ene3,20-dione
96 4-ene-3,20-dione 1la-Hydroxyandrost-4-ene-3,17-dione 91 1 la-Hydroxy-14~,18-cyclopregn-
2. Prochazka and F. Sorm, Cull. Czech. Chem. Comm., 1965, 30, 1874. R. Deghenghi, M. Boulerice, J. G . Rochefort, S . N. Sehgal, and D. J. Marshall, J . Medicin. Chem., 1966,9, 513 K . Singh, S . N. Sehgal, and C . Vezina, Appl. Microbiol., 1968, 16, 393. A.-M. H. El-Refai, L. A . R. Sallam, and I. A. El-Kady, 2. alfg. Mikrohiol., 1970, 10, 183. L. A. R. Sallam, A.-M. H. El-Refai, and I. A. El-Kady, Bull. Chem. Soc. Japan, 1970, 43, 1239.
6a-Fluoro- 16a,l7~-dihydroxypregn4-ene-3,20-dione Pregn-4-ene-3,20-dione
16a,17a-Epoxypregn-4-ene3,20-dione
Aspergillus ochraceus NRRL 405 (spores) Aspergillus ochraceus NRRL 405 (spores) Absidia orchidis
14p,18-Cyclopregn-4-ene-3,20-dione Rhizopus nigricuns
%
\o
Y
P
3 2 2 5
f -. 3
z
56'
5
9
E.
%
5. p. 0 5
loonM.
lo'
REF 129
Rhizopus nigricans
Cladosporium cladosporioides
Aspergillus ochraceus NRRL 405 (spores) Beauveria bassiana No. 663
Micro-organism
9p,1Oa-Pregn-4-ene-3,20-dione
Asperg ill us ochraceus NRRL 405 3a,5-Cyclo-5a-pregnane-6,20-dione Aspergillus ochraceus NRRL 405
Steroid substrate
58, 77
1 la-Hydroxypregn-4-ene-3,20-dione *
3,20-dione 1 1~,17~-Dihydroxyandrost-4-en-3-one 1 la-Hydroxypregn-4-ene-3,20-dione 99 1 la,l7a,21-Trihydroxypregn-4-ene3,20-dione 1la-Hydroxypregn-4-ene-3,20-dione 60, 100, lOOa 1 la-Hydroxy-5a-pregnane-3,20-dione 6p,lla-Dihydroxypregn-4-ene3,20-dione 1 la,l7a-Dihydroxypregn-4-ene3,20-dione 1 la,l7a,21-Trihydroxypregn-4-ene3,20-dione 1 la-Hydroxy-9p, 1 Oa-pregn-4-ene91,92 3,20-dione 1 la-Hydroxy-3a,5-cyclo-5a101 pregnane-6,20-dione
6p,1 la-Dihydroxypregn-4-ene-
98
Reference
1 la-Hydroxypregn-4-ene-3,20-dione
Products
E.Abd-Elsamie,M. B. E.Fayez,H.G.Osman, and L.A. R.Sallam,Z . a&. Mikrobiol., 1969, 9, 173. L. Tan and L. L. Smith, Biochim. Biophys. Aczu, 1968,164, 389.
Molecular formula of substrate
Table-continued
P
N 0
Aspergillus ochraceus NRRL 405 (spores) Rhizopus nigricans E.
14a,17a-Dihydroxypregn-4-ene3,20-dione 17a,21-Dihydroxypregn-4-ene3,20-dione
17a-Hydroxy-9P,l0a-pregn-4-ene3,20-dione
Aspergillus ochraceus NRRL 405 (spores) Aspergillus ustus
Aspergillus ochraceus
Rhizopus nigricans REF 129 Asperg ill us ochraceus NRRL 405
Methyl 3-oxoandrost-4-ene17P-carboxylate Aspergillus ochraceus 3P-Acetoxyandrost-5-en-17-0ne 17a-Hydroxypregn-4-ene-3,2O-dione Aspergillus ochraceus NRRL 405 Beauveria bassiana No. 663 Rhizopus nigricans REF 129 2 1-Hydroxypregn-4-ene-3,20-dione Beauveria bassiana No. 663
16a,17a-Epoxy-3P-hydroxypregn5-en-20-one
Io3
91
102 Methyl 1la-hydroxy-3-oxandrost4-ene-17P-carboxylate 3a,l la-Dihydroxyandrost-5-en-17-one 46 1la,l7a-Dihydroxypregn-4-ene103 3,20-dione 58 1la,l7a-Dihydroxypregn-4-ene3,20-dione 60 1la,l7a-Dihydroxypregn-4-ene3,20-dione 58 1la,21-Dihydroxypregn-4-ene3,20-dione 6a, 1la,21-Trihydroxypregn-4-ene3,20-dione 1la,l7a-Dihydroxyandrost-4-en-3-one 60 1la,21-Dihydroxypregn-4-ene3,20-dione 92 1la,l7a-Dihydroxy-9P,lOa-pregn4-ene-3,20-dione 1 1a-Hydroxy-9P,lOa-androst-4-ene3,17-dione 104 11a,14cq17a-Trihydroxypregn-4-ene3,20-dione 91, 98 1 1a,l7a,21-Trihydroxypregn-4-ene3,20-dione 104a 1 la,l7a,21-Trihydroxypregn-4-ene3,20-dione
16a,17a-Epoxy-3/3,1ladi hydroxypregn-5-en-20-one
A. Schubert and S . Schwarz, Experientia, 1965, 21, 688. L. Tan and P. Falardeau, Biochem. Biophys. Res. Comm., 1970,41, 894. D. Van der Sijde, H. J. Kooreman, K. D. Jaitly, and A. F. Marx, J . Medicin. Chem., 1972, 15, 909. l o C s L .Sedlaczek, H. Pajkowska, A. Jaworski, L. Zych, and E. Czerniawski, Actu Microbiol. Polon., 1973, 5 , 103.
C , H,,O,
C, H,,O,
3
5.
'OS
Aspergillus ochraceus NRRL 405
3 p- H ydroxypregn- 5-en-20-one
6p-Hydroxy-3a,5-cyclo-5a-pregnan- Aspergillus ochraceus NRRL 405 20-one
Aspergillus ochraceus NRRL 405 Aspergillus ochraceus NRRL 405 Aspergillus ochraceus
Verticillium glaucum
Rhizopus nigricans
Monosporium olivaceum
Beauveria bassiana No. 663 Fusarium graminearum
Absidia orchidis
Micro-organism
17a,2l-Dihydroxy-9P,lOa-pregn4-ene-3,20-dione 17a-Hydroperoxypregn-4-ene3,20-dione 3P-Hydroxy-5a-pregn-16-en-20-one
Steroid substrate
A. Smit and J. Bakker, Rec. Trav. chim., 1966,85, 731.
C2lH32O2
Molecular formula of substrate
Tablwontinued
1la, 17c,21-Trihydroxypregn-4-ene3,20-dione 1la,l7a,21-Trihydroxypregn-4-ene3,20-dione 1la,l7a,21-Trihydroxypregn-4-ene3,20-dione 1la, 17a,21-Trihydroxypregn-4-ene3,20-dione 11a,17a,21-Trihydroxypregn-4-ene3,20-dione 11a,l7a,21-Trihydroxypregn-4-ene3,20-dione 1la,17cr,21-Trihydroxy-9p, l0a-pregn4-ene-3,20-dione 11a,l7a-Dihydroxypregn-4-ene3,20-dione lP,3p, 1la-Trihydroxy-5a-pregn16-en-20-one Pregn-4-ene-3,20-dione 11a-Hydroxypregn-4-ene-3,20-dione 6pJl a-Dihydroxypregn-4-ene3,20-dione 6P,l la-Dihydroxy-3a,5-cyclo5a-pregnan-20-one
Products
101
101
34
103
91,92, 105
104a
60,77
104a
104a
58,77
77
Reference
P h) h)
lo’
Aspergillus ochraceus
Rhizopus nigricans
Beauveria globulifera
Beauveria bassiana
Absidia orchidis
Beauveria globulifera
Rhizopus nigricans
Rhizopus nigricans
Aspergillus ochraceus
77, 107
77,78
76
77
20-one
methylpregn-4-ene-3,20-dione 3P,11a-Dihydroxy-5a-pregnan-20-one 34 18,38,1la-Trihydroxy-5a-pregnan-
16a,l7a-Epoxy- 1la-hydroxy- 168met hylpregn-4-ene-3,20-dione 16a,17a-Epoxy-7P,lla-dihydroxy-16Pmet hylpregn-4-ene-3,20-dione 16417a-Epoxy-7P,11a-dihydroxy-16Pmet hylpregn-4-ene-3,20-dione 16a,17a-Epoxy-7P,lla-dihydroxy-16Pmet hyl-5P-pregnane-3,20-dione 16a,l7a-Epoxy-l la-hydroxy-16P-
methylpregn-4-ene-3,20-dione
91 6P,1la-Dihydroxy-3a,5-cyclo5a-pregnan-20-one 95, 106 68,l la-Dihydroxy-3a,5-cyclo5a-pregnan-20-one 16a,17a-Epoxy-l/3,3P,1la34 t ri hydroxy-5a-pregnan-2O-one 106 16a,17a-Epoxy-6j3,lla-dihydrox y- 168methyl-3a,5-cyclo-5a-pregnan20-one 106 6~,11a,17a-Trihydroxy-3a,5-cyclo5a-pregnan-20-one 1la,l7P-Dihydroxy-l6-methylenepregn- 78 4-ene-3JO-dione 1la, 17B-Dihydroxy-16-methylene5B-pregnane-3,20-dione 77 16a,17a-Epoxy-1la-hydroxy-16P-
J. Protiva, V. Schwarz, and K. Syhora, Coll. Czech. Chem. Comm., 1968, 33, 83. Y.-C. Chen and M . Huang, Yao Hsueh Pao, 1964, 11, 587; Scientia Sinica, 1965, 14, 803.
3P-Hydroxy-Sa-pregnan-20-one
C, H 3 4 0 2
lo6
16a,l7a-Epoxy-3P-hydroxy5a-pregnan-20-one 16a,l7a-Epoxy-6P-hydroxy-l6flmethyl-3a,5-cyclo-5a-pregnan20-one 6P,17a-Dihydroxy-3sr,5-cyclo-5apregnan-20-one 17P-Hydroxy-16-methylenepregn4-ene-3,20-dione
C,1H3203
Aspergillus ochraceus NRRL 405 (spores) Rhizopus nigricans
P w h ,
f. 3
Micro-organism
108
108
Reference
G. Greenspan, R. Rees, L. L. Smith, and H . E. Alburn, J . Org. Chem., 1965, 30,4215.
6a,2l-Difluoro-l la-hydroxy-17~91 methylpregna-l,4-diene-3,20-dione 21-Fluoro-1 la-hydroxy-17a91 met hylpregna- 1,4-diene-3.2O-dione 6a-Fluoro-1 la,21-dihydroxy-I7a91 methylpregna- 1,4-diene-3,2O-dione 1 1aJ7a-Dihydroxy- 16-methylenepregn- 77 4-ene-3,20-dione 1la,l7a-Dihydroxy-16-methylenepregn- 63,77 4-ene-3,20-dione 1la, 17a-Dihydroxy-l6-methylenepregn- 63,77 4-ene-3,20-dione 1la,l7a,21-Trihydroxy-l663 met hylenepregn-4-ene-3,20-dione 6a-Fluoro-1 la-hydroxy-17a91 met hylpregn-4-ene-3,20-dione 68,l la,22-Trihydroxy-23,2464 bisnorchol-4-en-3-one
lo8
17a,21-Dihydroxy-16Beauveria bassiana met hylenepregn-4-ene-3,20-dione 6a-Fluoro- 17a-met hylpregn-4-eneAspergillus ochraceus 3,20-dione NRRL 405 (spores) 22-Hydroxy-23,24-bisnorcholRhizopus arrhizus 4-en-3-one
Rhizopus nigricans
Products
1 b,1lr-Dihydroxy-5a-pregnane3,20-one 3a-Amino-1la-hydroxy-5%-pregnan20-one 3a-Amino-Sa-pregnane-1 1a,20a-diol
H30°3
3r-Amino-5%-pregnan-20-one Aspergillus ochruceus (funtumine) NRRL 40.5 3a-Amino-Sa-pregnan-2Or-01 Aspergillus ochraceus (funt umidine) NRRL 405 6a,21-Difluoro- 17a-methylpregnaAspergillus ochraceus 1,4-diene-3,20-dione NRRL 40.5 (spores) 21-Fluoro-l7a-methylpregnaAspergillus ochraceus 1,4-diene-3,20-dione NRRL 405 (spores) 6a-Fluoro-21-hydroxy-17aAspergillus ochraceus methylpregna-1,4-diene-3,20-dione NRRL 405 (spores) 17a-Hydroxy-16-methylenepregnAbsidia orchidis 4-ene-3,20-dione Beauueria bassiana
Steroid substrate
c.?Z
c2 2 H Z gF03
c 2 2 Hz 9 F O 2
C22H28F2OZ
c2 1 H3’7N0
C21H35N0
Molecular formula of substrate
Table-continued
R
a
'I3
' ''
I"
'lo
'09
BHMX
Rhizopus nigricans
Aspergillus ochraceus
Aspergillus ochraceus
(25S)-Spirost-5-en-3P-o1 (diosgenin) Solanidine
Cunninghamella blakesleeana Helicostylium pir forme ATCC 8992
(VNIKHFI-7) 21-Hydroxy-6a,l6a-dimethylpregn- Aspergillus ochraceus 4-ene-3,20-dione 17a,21-Dihydroxy-6a,16aGlomerella cingulata dimethylpregn-4-ene-3,20-dione 17a,21-Dihydroxy-6a,l6~Glomerella cingulata dimethylpregn-4-ene-3,20-dione 2 1,21-Ethylenedioxy-3/?-hydroxyRhizopus nigricans 2 1-methyl-16P-~-norpregnATCC 6227b 5-en-20-one 21-Acetoxy-5-bromo-6/l-fluoro-16a-Aspergillus ochraceus methyl-5a-pregnane-3,20-dione 21-Acetoxy-5-bromo-6/l-fluoro-3~- Aspergillus ochraceus hydroxy-16a-methyl-5a-pregnan20-one 3fi-Dimethylaminocon-5-enin Gloeosporium fructigenum (conessine) Stachybotrys parvispora
3/?-Hydroxy-l6/l-methyl-5apregnan-20-one 14a,l7a-Ethylidenedioxypregn4-ene-3,20-dione 16a,17a-Dimethylpregn-4-ene3,20-dione 34
113
1 1a-Hydroxysolanidine
74 74
90
112
111
110
110
82
73
I lu,2 l-Dihydroxy-6a,1 6 ~ dimethylpregn-4-ene-3,20-dione 1la,l7a,21-Trihydroxy-6a,l4adimethylpregn-4-ene-3,20-dione 1 la,l7a,21-Trihydroxy-6c(,16Pdimethylpregn-4-ene-3,20-dione 21,21-Ethylenedioxy-3P,1ladihydroxy-21-methyl-16P~-norpregn-5-en-20-one 6P-Fluoro-1 la,21-dihydroxy- 16amet hylpregn-4-ene-3,20-dione 6P-Fluoro-3/?,1la,21-trihydroxy5,16a-dirnethyl-5P,19-norpregn9-en-20-one 3P-Dimethylaminocon-5-enin-1 la-ol 1la-Hydroxycon-4-enin-3-one Con-4-enin-3-one (25S)-Spirost-5-ene-3P,7P,1la-trio1
4-ene-3,20-dione
14a,17a-Ethylidenedioxy-l la104 hydroxypregn-4-ene-3,20-dione 1la-Hydroxy-l6a,l7a-dimethylpregn- 109
5a-pregnan-20-one
1 /?,38,1la-Trihydroxy- l6fi-methyl-
L. S. Morozova, K. N. Gabinskaya, and G . S . Grinenko, Khim. prirod. Soedinenii, 1973,46. J. N. Gardner, F. E. Carlon, C . H. Robinson, and E. P. Oliveto, Steroids, 1966,7, 234. I. BeliE, E. Ghera, E. Pertot, and H. SoEiE, Steroids Lipids Res., 1972, 3, 201. K. Kieslich, K. Petzoldt, H. Kosmol, and W. Koch, Annalen, 1969,726, 168. Y . Sato, Y . Sato, and K. Tanabe, Steroids, 1967, 9, 553.
C , H,, NO
C27 H,,O,
C,,H,,N,
C,,H3,BrFo,
C,,H,,BrFO,
C, 3H3404
C23H3403
C23E l 3 4 0 2
C,3 H3204
C, 2 H 3 6 0 2
R v,
Steroid substrate
'I4
rac-17p-Hydroxyoestr-4-en-3-one
Oestra- 1,3,5(lO)-triene-3,17P-diol 17p-Hydroxyoestr-4-en-3-one
A spergill us tamarii Kita Q M 1223
Curvularia lunata NRRL 2380 Curvularia lunata NRRL 2380
Neurospora crassa Aspergillus tamarii Kita 1005
Curvularia lunata NRRL 2380 Botryod iplodia malor um CBS 134.50
Micro-organism
M. Maugras, P. Savigny, and J. Lematre, Compt. rend., 1973, 276, D , 3221. J . T. McCurdy and R. D. Garrett, J . Org. Chern., 1968, 33, 660.
C H2,02 C ,8 H 2 , 0 2
11p-Hydroxylation C18H2402 rac-l7P-Hydroxyoestra-4,8( 14)dien-3-one Oestr-4-ene-3,17-dione
Molecular formula of substrate
Table--continued Reference
rac-llp,l7~-Dihydroxyoestr-4,8( 14)68 dien-3-one 7p, 1lp-Dihydroxyoestr-4-ene25 3,17-dione Oestr-4-ene-3,1l117-dione Oestra-1,3,5(10)-triene-3,1lp,l7p-triol 114 1l/?-Hydroxyoestr-4-ene-3,17-dione 115 1lp-Hydroxy-3-0x0- 13,17-secooestr-4-eno- 17,13a-lactone 1 1pIl7P-Dihydroxyoestr-4-en-3-one 1 I~,l7/3-Dihydroxyoestr-4-en-3-one 47 lop, I 1/?,17P-Trihydroxyoestr-4-en-3-one rac-1 lp, 17P-Dihydroxyoestr-4-en-3-one 47 rac- 1Op,1lp, 17P-Trihydroxyoestr4-en-3-one ent- 1Op, 1lp, 17P-Trihydroxy-5aoestran- 3-one 1 lp-Hydroxyandrosta-l,4-diene53 3,17-dione 1lp, 17P-Dihydroxyandrosta- 1,4-dien3-one
Products
n
%
P N m
O3
I”
‘I6
17~,21-Dihydroxypregn-4-ene3,20-dione
5,6a-Epoxy-Sa-androstane3,17a-diol 5,6a-Epoxy-Sa-androstane3P,17P-diol 5,6a-Epoxy-17a-methyl-5aandrostane-3P, 17P-diol Pregn-4-ene-3,20-dione
Curvularia lunata (many strains)
Cladosporium cladosporioides Penicillium oxalicum Absidia orchidis
Aspergillus tamarii
Curvularia lunata
Curvularia lunata
Aspergillus tamarii Kita QM 1223 Aspergillus tamarii Kita QM 1223 Curvularia lunata
Aspergillus tamarii Kita QM 1223 Aspergillus tamarii Kita QM 1223
Asperg illus tamarii Kita QM 1223 Curvularia lunata
K. Kieslich and H. Wieglepp, Chem. Ber., 1971, 104, 205. D. R. Brannon, J. Martin, A . C . Oehlschlager, N. N. Durham, and L. H . Zalkow, J .
c, l H 3 0 O 4
c2 1 H30°2
C20H32
C19H30O3
3P-Hydroxy-5a-androstan- 17-one
3a-Hydrox y-Sa-androstan- 17-one
17P-Hydroxy-Sa-androstan-3-one
C19H3002
C19H280
C19H2802
17B-Hydrox yandrosta-1,4-dien3-one
5,6a-Epoxy-3P-hydroxy-5aandrostan-17-one Sa-Androstane-3,17-dione
c 1 9 H 26 0 2
116
116
116
53
53
Urg. Chem., 1965, 30, 760.
11~,17~-Dihydroxyandrost-4-en-3-0ne117 3-0xo-13,17-secoandrost-4-eno17,13a-lactone (testololactone) 11/3,17a,21-Trihydroxypregn-4-ene99 3,20-dione 11P-Hydroxypregn-4-ene-3,20-dione 1OOa 1lp,l7a,21-Trihydroxypregn-4-ene77 3,20-dione 118,l 7a,21-Trihydroxypregn-4-ene87 3,20-dione
17-one 3P,1ID-Dihydroxy-5a-androstan17-one 5,6a-Epoxy-5a-androstane3/3,1lflJ7a-triol 5,6a-Epoxy-5a-androstane3fi,llP,17fl-triol 5,6a-Epoxy-l7a-methyl-5aandrostane-3j3,l lP,l7B-triol
Sa-Androstane-3,17-dione 3r,l lp-Dihydroxy-5a-androstan-
11P, 17P-Dihydrox yandrosta- 1,4-dien53 3-one 5,6a-Epoxy-3/?,1lp-dihydroxy-5a116 androstan-17-one 1 l~-Hydroxy-3-oxo-l3,17-seco-5a- 53 androstano-l7,13a-lactone 1lP,l7/3-Dihydroxy-5a-androstan53 3-one 1l~-Hydroxy-5a-androstane-3,17-dione
'I8
Curvularia lunata
P. B. Raman and F. G . Peron, Steroids, 1965, 5, 249. G. Cleve, G.-A. Hoyer, K. Kieslich, and H. Wieglepp, Chem. Ber., 1972, 105, 6 5 8 .
pregnan-20-one
Curvularia lunata
6a-Fluoro-21-hydroxy-16~methylpregn-4-ene-3,20-dione, 5,6a-Epoxy-3/3-hydroxy-5a-
C, , H 3,FO,
C2 H3,03
Curvularia lunata
Curvularia penniseti CBS 33964 Helminthosporium sp. No. 873 Cunninghamella blakesleeana Curvularia lunata
Micro-organism
5,6a : 17a,20a-Bisepoxy-5~pregnane-3P,21-diol
18,21-Dihydroxypregn-4-ene3,20-dione 5,6a :16~,17a-Bisepoxy-3Phydroxy-5a-pregnan-2O-one
Steroid substrate
C, H,,O,
Molecular formula of substrate
Table-continued
pregnane-l1,20-dione 5,6a-Epoxy-3P,17~-dihydroxy-5apregnane-3,20-dione
5,6cc-Epoxy-3~,14a-dihydroxy-5ct-
1 lp,17a,21-Trihydroxypregn-4-ene3,20-dione 1 lp,l7a,21-Trihydroxypregn-4-ene3,20-dione 1 lp,l8,21-Trihydroxypregn-4-ene3,20-dione 5,6a : 16a,17a-Bisepoxy-3~,11~dihydroxy-5a-pregnan-20-one 16a,17a-Epoxy-1l fl-hydroxy-5apregnane-3,6,20-trione 16a,17a-Epoxy-3p,1lp-dihydroxy5a-pregnane-6,20-dione 5,6a : 17a,20a-Bisepoxy-5apregnane-3/?,1lp,21-triol 6a-Fluoro-1 Ifl,21-dihydroxy-l6amethylpregn-4-ene-3,20-dione 5,6a-Epoxy-3p,l lp-dihydroxy-5~pregnan-20-one
Products
116, 119
73
116
119
118
87
87
Reference
C , H 340
C,,H,,O,
C, 2 H 3 4 0 3
C, H 3 0,
, ,
C, H 3 , 0 4
C, H,,O,
C, lH3,O4
Curvularia lunata
Curvularia lunata
Curvularia lunata
Curvularia lunata
Curvularia lunata
Curvularia lunata
Curvularia lunata
Curvularia lunata
21-Hydroxy-6a,l6a-dimethylpregn- Curcularia lunatu 4-ene-3,20-dione 2 1- Acetoxy-5,6a-epoxy-3& Curuularia lunata hydroxy-5a-pregnan-2O-one
5,6a-Epoxy-3P,17p-dihydroxy5a,17a-pregnan-20-one 5,6a-Epoxy-Sa-pregnane3P,20a-diol 5,6a-Epoxy-5a-pregnane3p,20P-diol 5,6a-Epoxy-Sa-pregnane3p,21-diol 5,6a-Epoxy-5a-pregnane3P,20P,21-triol 5,6a-Epoxy-Sa-pregnane3p,17a,20~-triol 2 l-H ydroxy- 16~-methylpregn4-ene-3,20-dione 5,6a-Epoxy-3P-hydroxy-1601methyl-5a-pregnan-20-one '
3p,5,14a-Trihydroxy-6Pmethoxyacetoxy-5a-pregnane11,20-dione 1lP,l4a-Dihydroxy-5a-pregnane3,20-dione 14a-Hydroxy-5r-pregnane3,11,20-trione 5,6a-Epoxy-3&1lp,l7j?-trihydroxy116 5a, 17a-pregnan-20-one 116 5,6a-Epoxy-Sa-pregnane3PJl P,20a-triol 5,6a-Epoxy-Sa-pregnane116 3p,1lp,20P-triol 5,6a-Epoxy-Sa-pregnane116 38,l lP,21-triol 5,6a-Epoxy-Sa-pregnane116 3P,llB,2Op,21-tetra01 116 5,6-Epoxy-Sa-pregnane3P,1l~,l7a,20a-tetraol 1 1/?,21-Dihydroxy-l6a-methylpregn- 73 4-ene-3,20-dione 5,6a-Epoxy-3P,l la-dihydroxy-16a119 met hyl-5a-pregnan-20-one 5,6a-Epoxy-3P-hydroxy-16a-methylSa-pregnane- 11,20-dione 5,6a-Epoxy-3&15P-dihydroxy-16amethyl-5a-pregnan-2O-one 5,6a-Epoxy-3~,15~-dihydroxy-l6amethyl-5a-pregnane-ll,20-dione 11~,21-Dihydroxy-6a,16~73 dimethylpregn-4-ene-3,20-dione 5,6a-Epoxy-3p,l lp,21-trihydroxy116 5a-pregnan-20-one
P
\o N
C2,H, 0,
C24H3605
C24H33F04
C23H3406
M a lecular formula of substrate
Curvularia uncinata No. 899 Helminthosporium sp.
Curvularia lunata (many strains) Curvularia penniseti
Curvularia lunata
Micro-organism
21-Acetoxy-5,6a-epoxy-3Phydroxy-16a-methyl-5apregnan-20-one 3P,21-Diacetoxy-5,6a-epoxy-1601methyl-5a-pregnan-20-one
Curvularia lunata
Curvularia lunata
-Acetoxy-6Q-fluoro-1601 Curvularia lunata methylpregn-4-ene-3,20-dione -Acetoxy-5-bromo-6~-fluoro-l601- Curvularia lunata met hyl-5a-pregnan-3,20-dione Curvularia lunatu -Acetoxy-5-bromo-6~-fluoro3P-hydroxy-16a-methyl-5apregnan-20-one
21-Acetoxy-5,6a-epoxy-3fl,l7adihydroxy-5a-pregnan-20-one 21-Acetoxy-6a-fluoro-16amet hylpregn-4-ene-3,20-dione
Steroid substrate
5,6a-Epoxy-3/?,1lP,1701,21tetrahydroxy-5a-pregnan-20-one 6a-Fluoro-llQ,21-dihydroxy-1601met hylpregn-4-ene-3,20-dione 6a-Fluoro-l1~,21-dihydroxy-16amethylpregn-4-ene-3,20-dione 6a-Fluoro- 11P,2 1-dihydrox y- 16amet hylpregn-4-ene-3,20-dione 6a-Fluoro-ll~,21-dihydroxy-16amet hylpregn-4-ene-3,20-dione 6P-Fluoro-1 lj,21-dihydroxy-l6amethylpregn-4-ene-3,20-dione 6P-Fluoro-1 lP,21-dihydroxy-l6amethylpregn-4-ene-3,20-dione 6P-Fluoro-3P, 1 1$,21-trihydroxy16a-methylpregn-4-en-20-one 6~-Fluoro-3/3,10~,21-trihydroxy5,16a-dimethyl-.5P-l9-norpregn9(1 l)-en-20-one 5,6a-Epoxy-3j?,ll P,21-trihydroxy16a-methyl-5a-pregnan-20-one
Products
116
116
90, 112
112
112
87
87
87
87, 116
116
Rejerencr
n
s.
0
W P
rac-13P-Ethyl-17p-hydroxygon4-en-3-one 14,l5B-Epoxy-3P-hydroxy5&14fi-bufa-20,22-dienolide (resibufogenin) 3B-Dimethylaminocon-5-enine (conessine) 3B-Acetoxy-14,lSB-epoxy-5P,14/3card-20(22)-enolide
C, 9Hz80,
c 19Hz8 0
C19H260
C18H280
88 81
3/3-Dimethylaminocon-5-enin12~-01 14,lSB-Epoxy-3pJ2a-dihydroxy5B, 14B-card-20(22)-enolide
Botryodiplodia theobromae Absidia orchidis (Vuill.) Hagem.
Calonectria decora
Calonectria decora Calonectria decora
Andr ost-4-en-3-one Androst-5-en-7-one
Sa-Androstane-2,16-dione
Calonectria decora Calonectria decora Calonectria decora
Calonectria decora
81
12/3,15a-Dihydroxy-5a-~norandrostan-Zone 12P,15a-Dihydroxy-5a-oestran-3-one 12g,l Sa-Dihydroxyoestr-4-en-3-one 12p, 15a-Dihydroxy-5a-androst- 1-en3-one 12~,15a-Dihydroxyandrost-4-en-3-one 12P-Hydroxyandrost-5-en-7-one 3/3,12B-Dihydroxyandrost-5-en-7-one 4p,12fi-Dihydroxyandrost-5-en-7-one 12/3-Hydroxy-Sa-androstane2,6,16-trione 6a,l2B-Dihydroxy-Sa-androstane2,16-dione
30
32 32, 38
32 32 32
32
ent-13P-Ethyl-l2a,l7#?-dihydroxygon- 47 4-en-3-one 14,15#?-Epoxy-3B,12a-dihydroxy5& 14/?-bufa-20,22-dienolide
ent-12a,l7~-Dihydroxyoestr-4-en-3-one47
Curvularia lunata NRRL 2380 Curvularia lunata NRRL 2380 Absidia orchidis (Vuill.) Hagem.
5a-Oestran-3-one Oestr-4-en-3-one 5a-Androst- 1-en-3-one
12g-Hydroxylation 5a-~-Norandrostan-2-one
C,, H 3 4 0 5
C24H4,Nz
C24H32 0 4
C19HzgO,
12a-Hydroxylation C,,HZ6O2 rac-17/3-Hydroxyoestr-4-en-3-one
p
s
B k?
5.
C,,H,OO
Molecular formula of substrate
Calonectria decora Calonectria decora Calonectria decora
Calonectria decora
Calonectria decora Calonectria decora
5a-Androstan-Cone
5a-Androstan-7-one 5a-Androstan-15-one
5a,l4P-Androstan- 15-one
5B-Androstan-1?'-one
5a-Androst- 1-en-3B-ol
Calonectria decora
Calonectria decora
Calonectria decora
M icro-organism
Calonectria decora Calonectria decora
Steroid substrate
5a-Androstan-2-one Sa-Androstan-3-one
Tablwontinued Reference
12~,15a-Dihydroxy-5a-androstane- 30 3,6-dione 3p,12B-Dihydrox y -Sa-androstan30 ?'-one 128,l Sa-Dihydroxy-5a-androstane3,17-dione 6a,12/3-Dihydroxy-5a-androstan-2-one 32, 33 12P,15a-Dihydroxy-5a-androstan-3-one32, 33 5a-Androstane-3/3,12/3,15a-triol 6a,12fl,15a-Trihydroxy-5aandrostan-3-one 12jl,15a-Dihydroxy-5a-androstan32, 33 4-one 12/3-Hydroxy-5a-androstan-7-one 32 6a, 12P-Dihydroxy-5a,l4P-androstan32 15-one 2a,12P-Dihydroxy-5a-androstan15-one 7/3,12/?-Dihydroxy-5a,14P-androstan32 15-one 7P,12P,14-Trihydroxy-5a,14Pandrostan-15-one 128,l5a-Dihydroxy-SP-androstan32 17-one 12/?,15a-Dihydroxy-Sa-androst-l-en- 32 %one
Products
&
a
h,
w P
3a-Amino-5a-pregnan-20-one (funtumine) 3a-Amino-5a-pregnan-20a-01 (funtumidine) 14-Hydroxy-3-oxo-l4/3-bufa4,20,22-trienolide
Aspergillus ochraceus NRRL 405 Aspergillus ochraceus NRRL 405 Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg
Calonectria decora ATCC 14767 Calonectria decora
Pregn-4-ene-3,20-dione
38-Hydrox y pregn-5-en-20-one
Calonectria decora
Sa-~-Homoandrostan-17-one
P. J. Ramm and E. Caspi, J . Biol. Chem., 1969, 244, 6064. '"'E. Caspi, P. J. Ramm, and R. E. Gain, J . Amer. Chem. Soc., 1969, 91,4012.
' 19'
Calonectria decora Calonectria decora
5a-Androstan-3P-01 3p-Methoxy-5a-androstan-17-one
Calonectria decora
Penicillium sp. ATCC 12556 Calonectria decora
3a-H ydroxy-Sa-androstan- 17-one
3P-Hydroxy-5a-androstan-17-one
Calonectria decora
Androst -4-en-38-01
30
128,15a-Dihydroxy-Sa-androstane-
12P,14-Dihydroxy-3-0~0-14P-bufa4,20,22- trienolide
65
108
108
30
119a. 119b
32
30
32 30
23
3-one 3a,12P-Dihydroxy-Se-androstan17-one 3,17-dione 3p, 128,l 5a-Trihydroxy-5a-androstan17-one 5a-Androstane-3P,12fl,l5a-triol 12P,1Sa-Dihydroxy-3P-methoxy-5aandrostan-17-one 17P-Methoxy-5a-androstane3P,12P,15a-trio1 78,128,l Sa-Trihydroxy-Sa-~homoandrostan- 17-one 128,lSa-Dihydroxypregn-4-ene-3,20dione 3P,128,15a-Trihydroxypregn-Sen20-one 7P, 12P,15a-Trihydroxypregn-5-ene3,20-dione 3a-Amino- 128-hydroxy-5a-pregnan20-one 3a-Amino-5a-pregnane- 128,20a-diol
32
128,15a-Dihydroxyandrost-4-en-
P
W w
7’H4203
C41H64O13
‘2
C24H3404
C24H3204
Molecular formula of substrate
Steroid substrate
3P,l4-Dihydroxy-Sp, l4P-bufa20,22-dienolide (bufalin) (25S)-Spirost-5-en-3P-o1 (diosgenin) 38,14-Dihydroxy-SP,14P-card20(22)-enolide3b-tridigitoxoside (digitoxin)
3P,14-Dihydroxy-14P-bufa4,20,22-trienolide (scillarenin)
14,l SP-Epoxy-3P-hydroxy-5P,14Pbufa-20,22-dienolide (resibu fagenin) 3a,14-Dihydroxy-l4P-bufa4,20,22-trienolide
Table-con t in ued
Cunninghamella blakesleeana Streptomyces owasiensis, Streptomyces diastatochromogeneses s-59, Mortierella isabellina MN-64, Trichocladium asperurn MN-64, Trichocladium asperum MN-37, or Circinella muscae MN- 120
3a,12/?,14-Trihydroxy-14P-bufa4,20,22-trienolide
Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg Rhizopus arrhizus Fischer or Rhizopus nigricans Ehrenberg Gibberella saubinetti
20,22-dienolide (25S)-Spirost-5-ene3,8,7,8,12,8-triol 3/3,12P,14-Trihydroxy-5B,14B-card20(22)-enolide3P-tridigitoxoside
3P,12&14-Trihydroxy-5B,l4P-bufa-
4,20,22-trienolide
3b,12b,14-Trihydroxy-l4P-bufa-
14,l 5b-Epoxy-3/3,12P-dihydroxy5P, 14/?-bufa-20,22-dienolide
Products
Gibberella saubinetti
Micro-organism
83
82
120
65,66
65,66
120
Reference
&
P
Icl 3
w P
IH3O0z
c2 1 H30°3
2‘
C20H3002
C19H3002
C19H2802
C19H2602
C18H2602
21-Hydroxypregn-4-ene-3,20-dione
17/3-Hydroxy-18-methylandrost4-en-3-one
rac-13P-Ethyl-17P-hydroxygon4-en-3-one 3a-Hydroxy-5P-androstan- 17-one
Androst-4-ene-3J7-dione
rac-17P-Hydroxyoestr-4-en-3-one
rac-l7P-Hydroxyoestra4,8(14)-dien-3-one 17P-Hydroxyoestr-4-en-3-0ne
14a-Hydroxylation Oestr-4-ene-3J7-dione
c1BH2402
Mucor griseo-cyanus ATCC 1207 (and spores) Bacillus cereus
Curvularia lunata
Corynespora melonis CBS Curvularia lunata NRRL 2380 Curvularia lunata NRRL 2380 Curvularia lunata NRRL 2380 Absidia orchidis Mucor griseo-cyanus ATCC 1207 (and spores) Mucor griseo-cyanus ATCC 1207 (and spores) Curvularia lunata NRRL 2380 Cercospora (Corynespora) melonis CKe
47
14417j-Dihydroxyoestr-4-en-3-one
47
14a,21-Dihydroxypregn-4-ene3,20-dione
3a,l4a-Dihydroxy-5P-androstan-
61
44 17-one 14a-Hydroxy-5P-androstane-3,17-dione 3a,9a-Epoxy-3P,14a-dihydroxy-5jandrostan-17-one 55 14a,l7fl-Dihydroxy-18methylandrost-4-en-3-one 14a-Hydroxy-18-methylandrost4-ene-3.17-dione . 14a-Hydroxypregn-4-ene-3,2O-dione 69
4-en-3-one
13P-Ethyl-14a,l7P-dihydroxygon-
14a,17j-Dihydroxyandrost-4-en-3-0ne 69
49 69
47
rac- 14a,17P-Dihydroxyoestra4,8( 14)-dien-3-one 14a,17P-Dihydroxyoestr-4-en-3-one
68
85
W P VI
5-
CI
5.
C, H, ,03
C, H3,,04
forrnulu of substrate
Molecular
Steroid substrate
Micro-orgun ism
Products
Reference
14a,21-Dihydroxypregn-4-ene69 Mucor griseo-cyunus ATCC 1207 3,20-dione (and spores) 87 Curvufaria lunata 14a,i7r,2 1-Trihydroxypregn-4-ene17a,21-Dihydroxypregn-4-ene(many strains), 3,20-dione 3,20-dione Curvulariu eragrostidis CBS 21565, Curvufaria inaequalis CBS 11622, Curvularia penniseti CBS 33964, Curcularia protuberuta CBS 37665, Curvularia siddiquii, Curvularia trfolii CBS 34064, or Helrninthosporiurn bucliloes CBS 24649 119 5,6a-Epoxy-3P, 14a-dihydroxy-5a5,6a-Epoxy-3a-hydroxy-5a-pregnan- Curvularia lunata pregnane-l1,20-dione 20-one 3~5,14a-Trihydroxy-6~-methoxyacetoxy5a-pregnane-11,2O-dione 1I~,l4a-Dihydroxy-5a-pregnane3,20-dione 14a-Hydroxy-5u-pregnane3,11,20-trione
Table--continued
a
&
Y
o\
w P
6a-Fluoro-21-hydroxy-16amethylpregn-4-ene-3,20-dione 21-Hydroxy-l6a-methylpregn-
CZ2H, FO,
”‘
U. Vaicavi, Farmaco, Ed. xi.,1971, 26, 105.
CZ4H3,O5
C,,H,,BrFO,
Curvularia lunata
CBS 31064
Helminthosporium macrocarpum/oryzae
CBS 18648, or C . inaequalis CBS 11622
C . falcata
CBS 16460,
C . fallax
CBS 18649,
C . andropogoniis
(many strains),
Curvularia lunata
Mucor griseo-cyanus
Curvularia lunata
Curvularia lunata
(and spores)
ATCC 1207
Mucor griseo-cyanus
methylpregn-4-ene-3,20-dione Curvularia lunata 21-Acetoxy-5-bromo-6~-fluoro-l6amethyl-5a-pregnan-3,20-dione Curvularia lunata 21-Acetoxy-17a-hydroxy1Smethylpregn-4-ene-3,20-dione
21-Acetoxy-6P-fluoro-l6a-
4-ene-3,20-dione C,,H,,04 20,20-Ethylenedioxy-21hydroxy pregn-4-en-3-one C24H33F04 2l-Acetoxy-6a-fluoro-16amethylpregn-4-ene-3,20-dione
C22H3203
21-Fluoro-17a-methylpregna1,4-diene-3,20-dione
C,,H,,FO,
methylpregn-4-ene-3,20-dione ’ 14a,l7cc,21-Trihydroxy-18methylpregn-4-ene-3,20-dione 7a,14a,17a,21-Tetrahydroxy-18methylpregn-4-ene-3,20-dione
6/3-Fluoro-14a,21-dihydroxy-16amethylpregn-4-ene-3,20-dione 6~-Fluoro-14a,21-dihydroxy-16a-
6a-Fluoro-14a,21-dihydroxy16amethylpregn-4-ene-3,20-dione
55
112
112
87
2l-Fluoro-6<,14a-dihydroxy69 17a-methylpregna-1,4-diene3,20-dione 2l-Fluoro-7a,14a-dihydroxy-17amethylpregna-1,4-diene-3,20-dione 73 6a-Fluoro-14a,21-dihydroxy-l6amethylpregn-4-ene-3,20-dione 14a,21-Dihydroxy- 16a-methylpregn73 4-ene-3,20-dione 20,20-Ethylenedioxy14a,21121 dihydroxypregn-4-en-3-one 6a-Fluoro-14a,21-dihydroxy-l6a87,112 methylpregn-4-ene-3,20-dione
Steroid substrnte
C,aH,aO
C18H260
C1,H,,O,
Cercospora (Corynespora) melonis CKe
M icro-organism
Oestra-4-en-3-one 5a-Oestran-3-one
Oestra- 1,3,5(lO)-triene-3,17P-diol (oestradiol)
Calonectria decora
Fusarium monil.$orme (many strains) Gibberella fujikuroi (many strains) Caloiiectria decora Calonectria decora
Fusarium moniliforme (many strains) Gibberella fujikuroi (many strains) Glomerella fusarioides
Calonectria decora 5/7’,14P-Androstane-3,17-dione 12~-Hydroxy-5a,14P-androstan-15- Calonectria decora one
15a-Hydroxylation C 8H220, 3-Hydroxyoestra-1,3,5(lO)-triene-l7one (oestrone)
C, 9 H 3 0 0 2
C19H300
14p-Hvdroxylation C,,H,a02 5fl,l4P-Androstane-3,17-dione
Molecular formula of substrate
Table-ontinued
32
43
Reference
52
52
67
52
52
32 12p,1Sa-Dihydroxyoestr-4-en-3-one 1 la, 1Sa-Dihydroxy-5a-oestran-3-one 32 12,415a-Dihydroxy-5a-oestran-3-one 12p,1Sa-Dihydroxy-Sa-~-norandrostan-32 2-one
3,l Sa-Dihydroxyoestra-l,3,5(10)-triene17-one 3,15a-Dihydroxyoestra-1,3,5(lO)-triene17-one 3,lSa-Dihydroxy oestra- 1,3,5(10)-triene17-one Oestra- 1,3,5(lO)-triene-3,1501,17P-triol Oestra- 1,3,5( lO)-triene-3,15a,178trio1 Oestra- 1,3,5(lO)-triene-3.15~(, 17B-triol
15-one
12P,l4-Dihydr~xy-Sa,l4fi-androstan- 30
14-Hydroxy-SP,14B-androstane-3,17dione 3~,14-Dihydroxy-SP,I4p-androstan-1 7one 14P-Hydroxy-Sa,14B-androstan-15-one
Products
s.
n
Y
00 w
P
lZ2
Calonectria decoru Penicillium urticae Bain Calonectria decora Calonectria decora Calonectria decora Gibberella saubinetti (Mont.) Sacc. Calonectria decora Calonectria decora Calonectria decora
Calonectria decora
Sa-Androstane-3.11-dione
Sa-Androstane-3.12-dione
Sa-Androstane-3,17-dione
17p-Hydroxyandrost-4-en-3-one
3P-Hydroxyandrost-S-en-17-0ne
Andros t-4-en- 3p-01 Sa-Androstan-3-one
Sa-Androstan-4-one
Sa-Androst- 1-en-3p-01
Calonectria decora Calonectria decora
Androst -4-en-3-one Sa-Androstane-3,6-dione
3-0xo-13,17-secoandrosta-1,4-dieno-Penicillium sp. 17,13a-lactone ATCC 11598 Androst-3-ene-3,17-dione Calonectria decora Sa-Androst-1-en-3-one Calonectria decora
15a-Hydroxy-3-0x0- 13,17-secoandrosta- 122 1,4-dieno-17,13a-lactone 1Sa-Hydroxyandrost-4-ene-3,17-dione 30 12p,1Sa-Dihydroxy-Sa-androst-1-en-3- 32 one 12p,1Sa-Dihydroxyandrost-4-en-3-one 32 1Sa-Hydroxy-Sa-androstane-3,6-dione 30 128,lSa-Dihydroxy-Sa-androstane-3,6dione 1Sa-Hydroxy-Sa-androstane-3,ll-dione 30 38,l Sa-Dihydroxy-Sa-androstan-1 1-one 15a-Hydroxy-5a-androstane-3,ll-dione94 3p, 15a-Dihydroxy-Sa-androstan11-one 15a-Hydroxy-Sa-androstane-3,12-dione 30 3p,1Sa-Dihydroxy-Sa-androstan-12-one 12/3,15a-Dihydroxy-5a-androstane-3,17-30 dione 1Sa, 17P-Dihydroxyandrost-4-en-3-one 30 17/?-Hydroxyandrost-4-ene-3,6-dione 70,71 3/?,7a,1SLY-Trihydroxyandrost-Sen17one 12p,1Sa-Dihydroxy-Sa-androst-1-en-3- 32 one 1 2p, lSa-Dihydroxyandrost-4-en-3-one 32 12fl,15a-Dihydroxy-Sa-androstan-3-one32,33 Sa-Androstane-3& 12p,1Sa-trio1 6a,l2p,1Sa-Trihydroxy-Sa-androstan-3one 1la,l5a-Dihydroxy-Sa-androstan-4-one 32, 33 12~,15a-Dihydroxy-Sa-androstan-4-one
S. L. Neidleman, P. A. Diassi, B. Junta, R. M. Palmere, and S. C . Pan, Tetrahedron Letters, 1966, 5337.
C,9H,OO
C, ,H2 802
C,,H,,O, CI9H280
C, 9 H 2 4 0 2
\o
w P
C,,H,,O,
C20H320
C, 0 H 3 0,
C,oH,9C10,
C,,H,,O,
C19H32O C,,H,,ClO,
C1,H300,
Molecular formula of substrate
Calonectria decora Calonectria decora
3P-Methoxy-Sa-androstan-17-one
17P-Methoxy-5a-androstan-3-one
Calonectria decora
Aspergillus fiavus
Aspergillus flavus
Aspergillus fiavus
Calonectria decora Aspergillus fiavus
Calonectria decora
3P-Hydroxy-Sa-androstan- 17-one
Sa-Androstan-3P-01 4-Chloro-17P-hydroxy-17amethylandrosta-1,4-dien-3-one 17P-Hydroxy-17a-methylandrosta1,4-dien-3-one 4-Chloro-17P-hydroxy-17amet hylandrost -4-en-3-one 17P-H ydrox y- 17amethylandrost-4-en-3-one Sa-~-Homoandrostan-17-one
Calonectria decora
3a-Hydroxy-Sa-androstan-17-one
Calonectria decora
5a-Androstan-17-one
M icro-organism
Calonectria decora
Steroid substrate
5a-Androstan- 12-one
Table-continued
32 lP,Ga,lSa-Trihydroxy-Sa-androstan-12one 12P,1Sa-Dihydroxy-5a-androstan- 1732 one lfi,3a,1Sa-Trihydroxy-Sa-androstan- 17- 30 one 12P, 15a-Dihydroxy-Sa-androstan-3,17- 30 dione 3P712P,1Sa-Trihydroxy-5a-androstan17-one Sa-Androstane-3~,12~,1Sa-triol 32 4-Chloro-15a,l7~-dihydroxy-l7a54 methylandrosta-1,4-dien-3-one lSa,l7P-Dihydroxy-l7a54 met hylandrosta- 1,4-dien-3-one 4-Chloro- 15a717P-dihydroxy-17a54 methylandrost-4-en-3-one 15a,17/?-Dihydroxy-l7a54 methylandrost-4-en-3-one lP,7/?,1Sa-Trihydroxy-Sa-~32 homoandrostan-17-0ne 7P, 128,lSa-Trihydroxy-Sa-~homoandrostan- 17-one 12P,1Sa-Dihydroxy-3~-methoxy-Sa- 30 androstan-17-one 17P-Methoxy-handrostane30 3P,12P,lSa-triol
Reference
6a,1Sa-Dihydroxy-Sa-androstan- 12-one
Products
%
n
21-Acetoxy-17a-hydroxy18methylpregn-4-ene-3,20-dione
'24
123
5,6a-Epoxy-3B-hydroxy-l6amethyl-5a-pregnan-20-one
Curvularia lunata
4-Chloro-17P-hydroxy-17aAspergillus jlavus methylandrosta-1,4-dien-3-one 17P-Hydroxy-17a-methylandrost-4-enAspergillus f l a w s en-3-one 21-Hydroxypregn-4-ene-3,20-dione Bacillus cereus
Curvularia lunata
methyl-5a-pregnane-11,20-dione
5,6a-Epoxy-3j?, l5P-dihydroxy-16a-
methyl-5a-pregnan-20-one
4-Chloro-l5P,17~-dihydroxy17a54 methylandrosta-1,bdien-3-one 15P,17~-Dihydroxy-l7a-methylandrost-54 4-en-3-one 1SP,21-Dihydroxypregn-4-ene-3,2061 dione 5,6a-Epoxy-3P,15P-dihydroxy-l6a119
dione 15a,21-Dihydroxypregn-4-ene-3,20-dione6 1 15a,l7a,21-Trihydroxy-9P,lOa-pregn-4- 92 ene-3,20-dione 38,128,l5a-Trihydroxypregn-5-en-20- 30 one 78,128,l5a-Trihydroxypregn-5-ene-3,20dione 15a,17a,21-Trihydroxy-l855 methylpregn-4-ene-3,20-dione
1Sa-Hydroxy-9/3,lOa-pregn-4-ene-3,20-124
I. Belie,E, Pertot,H. SozliC, and T. Suhadolic,J . Steroid Biochem., 1971, 2, 105. J. de Flines,D. Van der Sijde,and W.F.Van der Waard, Rec. Trau. chim., 1966,85, 701.
C,,H,,O,
C , H,oO,
C,,H,,O,
C,,H,,C10,
1 5P-Hydroxylution
C,,H,,O,
C,,H,,O,
C,1 H,,O, C,,H,,O,
ATCC 11598
Penicillium sp.
Colletotrichum gloeosporioides 21-Hydroxypregn-4-ene-3,20-dione Bacillus cereus 17a,21-Dihydroxy-9P,lOa-pregn-4- Aspergillus ochruceus ene-3,20-dione NRRL 405 3P-Hydroxypregn-5-en-20-one Calonectria decora
9P,lOa-Pregn-4-ene-3,20-dione
15a,17fi-Dihydroxyandrost-4-en-3-one 15a-Hydroxyandrost-4-ene-3,17-dione 15a-Hydroxypregn-4-ene-3,20-dione 1 19a
56 123
15a-Hydroxypregn-4-ene-3,20-dione 15a-Hydroxypregn-4-ene-3,20-dione
Actionmyces sp.3375 Fusarium argillaceum
Pregn-4-ene-3,20-dione
C, H, , 0,
5
6'
I?
8
kl
Steroid substrate
M icro-orgunism
12'
'''
Products
Reference
123 125
dione
[4-14C]-16a-Hydr~xypregn-4-ene-3,20-128
16a-Hydroxypregn-4-ene-3,20-dione 16a-Hydroxypregn-4-ene-3,2O-dione
1 1P, 16a-Dihydroxy-5a-androstan-3-one93 16a-Hydroxy-9/3,10a-pregna-4,6-diene- 125, 127 3.20-dione
17-one 3P-sulphate
[7a-3H]-3P,16a-Dihydroxyandrost-5-en126
3-one 3P, 16a-Dihydroxyandrost-5-en-17-one 126 3P-sulphate
16s17/3-Dihydroxy-9P, l0a-androst-4-en- 125
W. F. Van der Waard, D. Van der Sijde, and J . de Flines, Rec. Trau. chim., 1966, 85, 712. E. Younglai and S. Solomon, Endocrinology, 1967, 80, 177. W . C. McGregor, B. Tadenkin, E. Jenkins, and R. E p p s , Biotechnol. and Bioeng., 1972, 14, 831. A. H. Janoski, G . J . Doellgast, and W. G. Kelly, Steroids, 1969, 13, 179.
16a-Hydroxylution Sepedonium ampullosporum C,,H,,O, 17P-Hydroxy-9~,1Oa-androst-4-en-3Damon CBS one Streptomy ces C,,H,,O,S 3P-Hydroxyandrost-5-en-17-one roseochromogenus 3P-sulphate ATCC 3347 [7a-3H]-3/?-Hydroxyandrost-5-en-17- Streptomjces roseochromogenus one 3P-sulphate ATCC 3347 Rhizopus nigricans C, ,H3,02 1 l~-Hydroxy-5a-androstan-3-one C, H, ,O, 9P, lOa-Pregna-4,6-diene-3,20-dione Sepedonium ampu1losporum ATCC 18217 and CBS 392.52 Fusarium urgillaceum C, H , , 0 2 Pregn-4-ene-3,20-dione Sepedonium ampullosporum Damon CBS Streptomyces roseochromogenus ATCC 3347
Molecular formula of substrate
Tabl-ontinued
Sa-Androstan-3,ll-dione 5a-Androstan-3-one 1 la-Hydroxy-5a-androstan-3-one
C, HZ8O4
C19H,,02
Aspergillus niger Cladosporium cladosporioides
AspergillusJischeri
Rhizopus nigricans Rhizopus nigricans Rhizopus nigricans
Calonectria decora
Rhizopus nigricans 5,6a-Epoxy-3p-hydroxy-5a-pregnan- Curvularia lunata 20-one
Aspergillus niger ATCC 9142 11~,18-Epoxy-2l-hydroxypregn-4-eneCorynespora cassicola 3,20-dione IMI 56007
18-Hydroxylation Androst-4-ene-3,17-dione
C, 1H3203
C, H3,,02
17a-Hydroxylation Pregn-4-ene-3,20-dione
C19H3002
C19H300
C,9 H 2 8 0 2
C19H280
16p-Hydroxylation Scr-Androst-2-en-1-one
C, H, 0,
Sepedonium ampullosporum Damon C B S [7a-3H]-3fi-Hydroxypregn-5-en-20- Streptomyces roseochromogenus one ATCC 3347
9fl,1Oa-Pregn-4-ene-3,20-dione
1 lp,l8-Epoxy18,21-dihydroxypregn-4- 86 ene-3,20-dione 1 lp,2l-Dihydroxy-3,20-dioxopregn-4eno-18,llp-lactone
18-Hydroxyandrost-4-ene-3,17-dione 50
1 la,l7a-Dihydroxypregn-4-ene-3,20- 99 dione 17a-Hydroxypregn-4-ene-3,2O-dione 100 17a-Hydroxypregn-4-ene-3,20-dione 99 1 la,l7a,21-Trihydroxypregn-4-ene-3,20dione 1 1/3,17a,21-Trihydroxypregn-4-ene-3,20dione 17a-Hydroxypregn-4-ene-3,20-dione 100 5,6a-Epoxy-3~,17a-dihydroxy-5a119 pregnan-20-one
6a,16/?-Dihydroxy-5a-androst-2-en-l-one 32 6a-Hydrox y-Sa-androst-2-ene- 1,16-dione 16~-Hydroxy-5a-androstane-3,1 I-dione 93 1 la,l6/3-Dihydroxy-5a-androstan-3-one93 1 la,l6P-Dihydroxy-5a-androstan-3-one 93 5a-Androstane-3fl, 11a,l6p-triol
[7~1-~ H]-3/?,16a-Dihydroxypregn-5-en- 128 20-one
16cr-Hydroxy-9P,lOa-pregn-4-ene-3,20- 125 dione
% w
3
5.
Products
Reference
Rhizopus nigricans
Cholest-4-en-3-one
26-Hydroxycholest-4-en-3-one 130 26-Hydroxy-(25S)-cholest-4-en-3-one 20
dione 11/3,17a,2l-Trihydroxypregn-4-ene-3,20dione 21 -Hydroxypregn-4-ene-3,20-dione 100
Mycobacterium sp. 2104 Mycobacterium smegmatis SG 346
rac-21-Hydroxy-18-methyl-19129 norpregn-4-ene-3,11,2O-trione 21-Hydroxypregn-4-ene-3,20-dione 99 2 1-Hydroxypregn-4-ene-3,20-dione 100 11a,l7a,21-Trihydroxypregn-4-ene-3,20-99
Aspergillus niger NRRL 599 Aspergillus fischeri Aspergillus niger Cladosporium cladosporioides
B. Gadsby, M. R. G . Leeming, G . Greenspan, and H. Smith, J. Chem. SOC.(0,1968,2647. I. I. Zaretskaya, L. M. Kogan, 0. B. Tikhomirova, J. D. Sis, N. S. Wulfson, V. I. Zaretskii, V. G. Zaikin, 'G. R. Skryabin, and I. V. Torgov, Tetrahedron, 1968, 24, 1595.
26-Hydroxylation C27H440 Cholest-4-en-3-one C*,H460 Cholest-5-en-3/3-01 (cholesterol)
I3O
M icro-organ ism
8,2 1-Dihydroxypregn-4-ene-3,20- Corynespora cassicola 1ljl,l8,21-Trihydroxypregn-4-ene-3,20- 86 dione IMI 56007 dione dimer or Cercospora (Corynespara) melonis CKe
Steroid substrate
21-Hydroxylation C2 H,,O, rac-18-Methyl- 19-norpregn-4-ene3,11,20-trione C, lH,,O, Pregn-4-ene-3,20-dione
c2 lH3004
Molecular formula ,of substrate
Table-continued
&
k?3
E
Microbiological Reactions with Steroids
A
m
4
d
2 N
f
.% L
0
9 U
445
446
Terpenoids and Steroids
Chromatographic and spectral methods have figured largely in most of the work reported in the Table. Only a few specific analytical methods for use in microbial hydroxylation studies have appeared. ' However, systematic spectral studies designed to facilitate early recognition of the structures of monohydroxylated steroid products obtained from microbial fermentations have been reported. Among these are studies of U.V.absorption,' 3 4 , 1 3 5 i.r. absorpti01-1,'~~ and 'H n.m.r.137-139 Using proton spectra a corrected structure of 128,15a-dihydroxypregn-4-ene-3,20-dione has been assigned to several pregn-4ene-3,20-dionemetabolites previously thought to be 6P,15cx-, 1la,15/3-,or 128,15Pdihydroxypregn-4-ene-3,20-dione. 40 The structures of several 'retro' 9P, 1Oasteroid derivatives hydroxylated by Aspergillus ochraceus have been established from detailed 0.r.d. and 'H n.m.r. data.140a Adequate studies of mechanism of hydroxylation by microbial systems have not been made. Mono-oxygenase (mixed-function oxidase) enzymes are implicated by the obligate use of molecular oxygen and the stereospecificity of hydroxy-group introduction. Other aspects of the hydroxylase system remain obscure despite their fundamental interest and industrial importance. Except for the one case of study of intact cells of Bacillus cereus,61the participation of cytochrome P-450 and other electron-transport proteins has not been examined, nor have cofactor requirements been broadly established. Only in the case of the Curvularia lunata NRRL 2380 cell-free 11P-hydroxylase has a NADPH requirement been demonstrated. l 4 ' '42 NADPH-Stimulation of hydroxylation has been shown for two other cell-free hydroxylase systems, h ~ w e v e r . ' ~ ~ ~ ' ~ ~ A few instances of microbial dihydroxylation are difficult to reconcile with sequential single mono-oxygenase hydroxylases. The la,2a-dihydroxylation of several A4-0x0-steroids and of one A ',4-3-oxo-steroid by Nocardia ~ o r a l l i n a , ~ ~ the 1 O&l18-dihydroxylation of 17~-hydroxyoester-4-en-3-one and the 6fl,lO/?dihydroxylation of rac- 13P-ethyl-17P-hydroxygon-4-en-3-one by Curvularia l u n ~ t u ,the ~ ~ lP,l la-dihydroxylation of 3P-hydroxy-Sa-pregnan-2O-oneby Aspergillus o c h r u c e u ~and , ~ ~ the dihydroxylations of 5a-androstane ketones by 9
E. Ivashkiv, Appl. Microbiol., 1970, 20, 251. L. L. Smith, Steroids, 1963, 1, 544, 570. 13' L. L. Smith, Texas Reports Biol. Med., 1966, 24, 674. 1 3 6 A. D. B o d , J . W. Blunt, J . W. Browne, V. Kumar, G. D. Meakins, J. T. Pinhey, and V. E. M. Thomas, J. Chem. Soc. ( C ) , 1971, 1 1 30. 13' L. L. Smith, Steroids, 1964, 4, 395. 13' K. Tori and E. Kondo, Steroids, 1964; 4, 713; Nippon Kagaku Zasshi, 1966,87, 1 1 17. 1 3 9 J. E. Bridgeman, P. C. Cherry, A. S. Clegg, J. M. Evans, E. R. H. Jones, A. Kasal, V. Kumar, G . D . Meakins, Y. Morisawa, E. E. Richards, and P. D. Woodgate, J. Chem. Soc. (0, 1970, 250. '41 R. M . Dodson, G. Langbein, R. D. Muir, A. Schubert, R. Siebert, C. Tamm, and E. Weiss-Berg, Helo. Chim. Acta, 1966, 48, 1933. 1400G.Saucy, H. Els, F. Miksch, and A. Furst, Helv. Chim. Acta, 1966, 49, 1529. l4I M. H. J . Zuidweg, W. F. Van der Waard, and J. de Flines, Biochim. Biophys. Acta, 1962, 58, 131. 14* M . H . J . Zuidweg, Biochim. Biophys. Acta, 1968, 152, 144. 1 4 3 F. N. Chang and C. J. Sih, Biochemistry, 1964, 3, 1551. ' 4 4 J. E. Wilson and C. S. Vestling, Arch. Biochem. Biophys., 1965, 110, 401.
133 134
Microbiological React ions with Steroids
447
Calonectria suggest possible participation of microbial dioxygenases also. Very recently reported monohydroxylation and dihydroxylation patterns of Rhizopus nigricans on 5a-androstane monoketones and diketones provide additional experimental support on this point. Dihydroxylations were suggested as probably being concerted and involving a single enzyme-substrate complex with a three-point attachment.'44a Dual-purpose sites in which the 7a and l l a positions of 5a-androstane diketones were equivalent were suggested for monohydroxylations. 144b In the case of 5P,6a-dihydroxylation of the ~-nor-A~-3P-alcohol(l) by Rhizopus nigricans both the 3P,SP,6a-triol(2a)and the 5a,6a-epoxide(2b) were formed, thus suggesting an alternative hydroxylation pathway of initial epoxidation of the A5-double bond followed by hydration of the e p ~ x i d e The . ~ ~hydroxylation by Rhizopus nigricans of a number of uncommon steroids has been r e ~ i e w e d . ' ~ ~ '
w
HO
The hydroxylation of pregn-4-ene-3,20-dione by several species of basidiomycetes has been reported.'44d An accumulation of evidence suggests that the steroid hydroxylases of microbial cells are inducible. Studies with intact resting cells of Curvularia lunata NRRL 144sJ. W. Browne, W. .4. Denny, E. R. H. Jones, G. D. Meakins, Y . Mdrisawa, A. Pendlebury, and J. Pragnell, J . C . S . Perkin I, 1973, 1493. lQ4*V.E. M. Chambers, W. A. Denny, J. M. Evans, E. R. H. Jones, A. Kasal, G . D. Meakins, and J. Pragnell, J.C.S. Perkin I , 1973, 1500. 1 4 4 c Z . Prochazka, Abhandl. deutsch. Akad. Wiss.Berlin, Klasse Med., 1968, NO.2 , p. 131. 44d E. C. Schuytema, M. P. Hargie, I. Merits, J. R. Schenk, D. J. Siehr, M. S. Smith, and E. L. Varner, Biotechnol. and Bioeng., 1966, 8, 275.
Terpenoids and Steroids
448
from a 238014571 46 and with cell-free hydroxylase variety of procaryotic and eucaryotic cells support the inducible nature of these hydroxylases. Only for the procaryote Bacillus megateriurn strain KM is induction not i n d i ~ a t e d . ' The ~ ~ matter of 1la-hydroxylase induction in Rhizopus nigricans has been questioned, however.' 52 The preparation of cell-free hydroxylases has received some attention, but the difficulty of this work has limited success. None of the cell-free hydroxylases reported to date is free from other enzyme activities. The following cell-free hydroxylations have been reported : 11P-hydroxylation by Curvularia lunata NRRL 2380 of 17a,21-dihydro~ypregn-4-ene-3,2O-dione~~~*' 42 and of 178hydroxyoestr-4-en-3-0ne'~~.'~~~'~~ as well as lop- and 14a-hydroxylation of 17P-hydroxyoestr-4-en-3-one ;14'*' 429149 9whydroxylation by Nocardia restrictus of androst-4-ene-3,17-dione, pregn-4-ene-3,20-dione, and 21-hydroxypregn-4ene-3,20-dione; 1 4 3 l5P- (and 6P-, 1la-, and 15a-) hydroxylation by Bacillus megaterium strain KM of 21-hydroxypregn-4-ene-3,20-dione ;144 16a-hydroxylation by Actinomyces (Streptomyces) roseochromogenes ATCC 3347 of 1la-hydro~ypregn-4-ene-3~2O-dione ;147 3P-hydroxylation by Saccharomyces cerevisiae of lanosta-8,24-diene ;39 1la-hydroxylation by Aspergillus ochraceus NRRL 405 5 0 and of 17P-hydro~yoestr-4-en-3-one,'~~ as of pregn-4-ene-3,20-di0ne'~~*' well as 68-hydroxylation of 11a-hydroxypregn-4-ene-3,20-dione ;148 1la-hydroxylation by Aspergillus niger of pregn-4-ene-3,20-dione and 6p-, 11a-, 17a-, and 21-hydroxylations by Rhizopus nigricans REF 129 of pregn-4-ene-3,20-di0ne."~ Failures to obtain cell-freehydroxylases from Sepedonium ampullosporiurn' and have ~ ~ been reported. from Botryodiplodia m ~ l o r u m In addition to the previously cited cell-free hydroxylase studies, hydroxylation mechanisms have been examined by kinetics measurements on cell-free systems'43 and on intact resting cells,'49 and by studies in which the patterns of hydroxylated products from many different, related substrates have been determined.30332 , 3 3 . 4 6 3 1 4 7 In certain microbial fermentations a single steroid substrate may be transformed into several individual monohydroxylated products, but several discrete hydroxylases do not appear to be involved. A generalized hydroxylase reminiscent of mammalian hepatic detoxification systems may be more the case. Study of the mechanisms involved has been approached using racemic steroid s u b s t r a t e ~ and ~ ~ ' ~specifically ~ labelled isotope-substituted steroid^.^^,^^ A
;' '
14' 146
14'
14* '49
150
Y . Y . Lin and L. L. Smith, Biochim. Biophys. Acta, 1970, 218, 526. B. K . Lee, W. E. Brown, D. Y . Ryu, and R. W. Thoma, Biotechnol. and Bioeng., 1971, 13, 503. E. A. Elin and L. M. Kogan, Doklady Akad. Nauk S . S . S . R . , 1966, 167, 1175; Doklady Biochem., 1966, 167, 143. M. Shibahara. J. A. Moody, and L. L. Smith, Biochim. Bioph-vs. Acta, 1970, 202, 172. Y. Y . Lin and L. L. Smith, Biochim. Biophys. Acta., 1970, 218, 515. L. Tan and P. Falardeau, J . Steroid Biochem., 1970, 1, 221. T. Nguyen-Dang, M . Mayer, and M.-M. Janot, Compt. rend., 1971, 272, D , 2032. T. Nguyen-Dang, Compt. rend., 1969, 269, D , 520; 1970, 270, D,2035. L. A. R. Sallam, A.-M. H. El-Refai, and I. A. El-Kady. Z . allg. Mikrobiof., 1971, 1 1 , 325.
Microbiological Reactions with Steroids
449
simulation of multiple discrete monohydroxylations has been examined with an analogue computer program.' 5 4 The systematic examination of hydroxylation by Calonectria decora and Aspergillus ochraceus of a large number of mono- and di-oxygenated 5a-androstane derivatives has been r e p ~ r t e d . ~ ' , ~The ~,~~,~~ predominant pattern of dihydroxylations obtained with Calonectria decora involved formation of diequatorial diols (1p,6a-, 6a,l la-, 11a,15a-, and 12P,15a-) in which the carbon atoms hydroxylated were separated in each case by ca. 0.4nm.32 Some speculative ideas involving different modes of steroid substrate binding to Aspergillus tamarii hydroxylases have been given.5 Additional screening of yeasts, actinomyces, and fungi for 1la- and 11phydroxylating capacities has been r e p ~ r t e d . ' ~ The ~ , ' ~effects ~ of a variety of enzyme inhibitors on respiration and 1la-hydroxylation by Monosporium oliuaceum, Aspergillus ustus, Verticillium glaucum, and Fusarium graminearum have been e~amined.''~" The 1la-hydroxylation of pregn-4-ene-3,20-dione by Rhizopus nigricans with very high substrate charges (5 mg ml- ') has been reported, but formation of a mixed crystal of product and substrate diminished the availability of substrate such that about 14 % of substrate was not hydroxylated.'56" The general matter of performing microbial fermentations with high levels of substrate charged as a solid has been reviewed.' 56b Use of microbial transformations of steroids for the preparation of pharmacologically active agents has been reviewed. The utility of microbiological hydroxylations providing access to otherwise difficultly accessible structural features has recently been emphasized. Easy access to 1la-hydroxy-5a-A '-3ketones via Aspergillus ochraceus hydroxylation of 36-hydroxy-5a-pregnan-20and to 15-oxygenated-5a-androstanederivatives via Penicillium urticae hydroxylationg4,'57 constitute good examples of the interplay between chemical and microbiological methods. Data in the Table attest to many successful attempts at synthesis of potentially active steroid hormone analogues and intermediates. Microbiological hydroxylation has been of some use in resolution of racemic 19-nor-steroids obtained by total synthesis. Hydroxylations in the lp-, lop-, and 1lor-positions by Aspergillus ochraceus NRRL 40529and in the lop-, 1I/?-, 12a-, and 14a-positions by Curvularia Eunata NRRL 23804' afforded resolved derivatives of natural or enantiomeric configurations. 21-Hydroxylation by Aspergillus 154
J. Riemann, H. Ropke, K. Kieslich, H.-J. Koch, and H. Gibian, European J . Biochem.,
155
A.-M. H. El-Refai, L. A. R. Sallam, and I. A. El-Kady, J . Gen. Appl. Microbiol., 1969,
ls6
A.-M. H. El-Refai, L. A. R. Sallam, and H. Geith, Acra Microbiol. Polon., Ser. B,
1968, 6, 60. 15, 301.
1972, 4, 31.
W. D. Maxon, J . W. Chen, and F. R. Hanson, Ind. andEng. Chem. (Process Design), 1966, 5, 285. 5 6 b R .Steel, in 'Fermentation Advances', ed. D. Perlman, Academic Press, New YorkLondon, 1969, pp. 491-514. 15' 1. M. Clark, W. A. Denny, E. R. H. Jones, G . D. Meakins, A. Pendlebury, and J. T. Pinhey, J.C.S. Perkin I, 1972, 2765.
1560
Terpenoids and Steroids
450
I
R2 (3) a ; R' = R2 = H b;R' = H , R 2 = O H C ; R ' = OH,R2 = H
niger NRRL 599129failed to yield resolved products, however, as did hydroxylation of the des-D-ring 19-nor-steroid (3a) by Cunninghamella bainieri ATCC 9244. Racemic 6p- and lop-hydroxylated derivatives (3b) and (3c) were ~ b t a i n e d . ' ~ ~ " Attempts to separate the vegetative cell culture aspect from the steroidtransformation aspect of microbial hydroxylations include, besides previously mentioned studies with cell-free hydroxylases, studies with intact resting cells. Successful 1ID-hydroxylations with Curvularia lunata NRRL 2380145,'49and 16a-hydroxylations with Sepedonium ampullosporium' 27 have been reported. lntact cells of Curtlularia lunata embedded in cross-linked polyacrylamide gel retain their useful 1lp-hydroxylating capacity with 17~,21-dihydroxypregn-4ene-3,20-dione.' 58 Gel granules which lost part of their 11P-hydroxylaseactivity could be reactivated by exposure to adequate nutritional components and to 17cr,21-dihydroxypregn-4-ene-3,20-dione.It could not be determined whether reactivation derived from reinduction of 11/?-hydroxylase or from growth of embedded cells. 5 8 Similarly entrapped fungal spores also retained hydroxylase activities, the 11a-hydroxylase of Aspergillus niger and 16a-hydroxylase of Streptomyces roseochromogenus being examples.' Use of fungal spores for hydroxylation also permits a separation between spore production and spore utilization. Furthermore, spores can be re-used, aseptic incubation conditions need not be employed, and industrial economies may be possible. Hydroxylations with Aspergillus ochraceus9 ' * 9 7 * 9 8 and Mucor g r i s e o - ~ v a n u shave ~ ~ been of particular interest, and successful pilot-plant 1l a hydroxylation of 1 kg charges of 17a, 21-dihydroxypregn-4-ene-3,20-dioneand of 6a-fluoro- 16a,17a-dihydroxypregn-4-ene-3,2O-dione in 200 gallon stainless steel vessels has been achieved.98 Spore processes have been reviewed.' It is possible to conduct microbial hydroxylations and microbial A'-dehydrogenations in mixed cultures of two organisms. Simultaneous hydroxylations using Absidia coerulea Squibb No. 5471 (1la- and 1lp-hydroxylations), Aspergillus ochraceus Squibb No. 4057 (1 la-hydroxylation), Curuularia lunata ATCC 12017 (11p-hydroxylation), or Streptomyces roseochromogenes ATCC 13400 (16a-
'
'
1 5 7 a SJ. . Daum, M . M . Riano, P. E. Shaw, and R. L. Clarke, J. Org. Chem., 1967,32, 1435. "* K . Mosbach and P . - 0 . Larsson, Biotechnol. and Bioeng., 1970, 12, 19. H. Hafez-Zedan and R . Plourde, Appl. Microbiol., 1971, 21, 815.
Microbiological Reactions with Steroids
45 1
hydroxylation) with A'-dehydrogenation with Arthrobacter simplex ATCC 6946 have been recorded,' 60-' 6 2 as have 11a-hydroxylations with Aspergillus ochraceus and A' -dehydrogenations with Bacillus l e n t ~ s3 *. 1~63 Sequential hydroxylations followed by dehydrogenations offer another possibility for process improvement~.'~~.'~~ Attempts to direct hydroxylations to certain sites in the steroid molecule or to eliminate unwanted hydroxylated side-products have received attention. Complete blocking of hydroxylation or other metabolic disposition can also be achieved by variation of substrate structure. Whereas 1la-hydroxylation by Aspergillus ochraceus of 17/3-hydroxyandrost-4-en-3-one, 17P-hydroxyoestr-4en-3-one, and 17P-hydroxy-17a-methylandrost-4-en-3-one proceeds readily, 64 and rac-13P-ethyl-17P-hydroxygon-4-en-3-one is hydroxylated in other positions exten~ively,~'no transformations of any sort were obtained with l7a-ethynyl17P-hydroxyoestr-4-en-3-oneor with rac-17a-ethynyl-l3~-ethyl-17P-hydroxygon-4-en-one as substrates, whether vegetative cell culture' 64 or cell-free systems' 5 0 were used. The 17a-ethynyl group interfered sterically with hydroxylation, for in incubations where the lla-hydroxylase had been fully induced prior to addition of substrate there was no hydroxylation. The orientation of an 11-hydroxy-group influences the course of 16-hydroxylation by Rhizopus nigricans, 1la-hydroxy-Sa-androstan-3-one giving high yields of 16P-hydroxylated products and the epimeric 11P-hydroxy-Sa-androstan-3one giving a 56 % yield of 1lP,l6a-dihydroxy-Sa-androstan-3-0ne.~~ Homologation of the 13P-alkyl group affects sites of hydroxylation and proportions of hydroxylated products. Hydroxylation products from rac-13P-ethyl17P-hydroxygon-4-en-3-one using Aspergillus ochraceus or Curuularia lunata are essentially of analogous structure (but widely different proportions) to those from the parent 17P-hydroxyoestr-4-en-3-one, optical configurations not con~ i d e r e d . ~However, ~ . ~ ~ 1lp-hydroxylation of the ruc- 13P-ethyl homologue did not occur with Curvularia l u n ~ t a , 4nor ~ did 1ID-hydroxylation of 17P-hydroxy18-methylandrost-4-en-3-one or 21-acetoxy-17a-hydroxy-18-methylpregn-4-ene3,20-dione.' The 13P-ethyl group clearly sterically hindered 1lp-hydroxylation in these cases. Hydroxylation at other sites took p l a ~ e . ~ ~ , ~ ~ Subtle influences on hydroxylated product structure and proportion are also achieved by the introduction of additional carbonsarbon double bonds into the steroid substrate molecule. rac- 17B-Hydroxyoestra-4,8(14)-dien-3-one with Curuularia lunata affords ruc-7a-, - lop-, and - 11P-monohydroxylated products6' gave resolved lop-, 12a-, and 14awhereas ruc-17~-hydroxyoestr-4-en-3-one monohydroxy- and racemic 6P- and 11P-monohydro~y-derivatives.~~ The state
'
Ibo
D. Y . Ryu, B. K. Lee, R. W. Thoma, and W. E. Brown, Biutechnol. and Bioeng., 1969, 11, 1255.
B. K . Lee, D. Y . Ryu, R. W. Thoma, and W. E. Brown, J . Gen. Microbiol., 1969, 55, 145. Ib2
lb3
lh4
B. K. Lee, W. E. Brown, D. Y . Ryu, H. Jacobson, and R. W. Thoma, J . Gen. Microbiul., 1970, 61, 97. K. Petzoldt,' Chem.-Ing.-Tech., 1971, 43, 78. L. Tan and L. L. Smith, Biochirn. Biophys. Acta, 1968, 152, 758.
452
Terpenoids and Steroids
of C- 17 oxidation in substituted C ,8 steroids also affects the direction of hydroxylation, 17p-hydroxyoestr-4-en-3-one giving almost exclusively lp,l7P-dihydroxyoestr-4-en-3-one with Botryodiplodia malorum and oestr-4-ene-3,17-dione giving mainly 7P-hydroxyoestr-4-ene-3,17-dione. The many directing effects of oxygen functions at different positions in the steroid nucleus on hydroxylations by Calonectria d e ~ o r a , ~Aspergillus ~ * ~ ~ , ~ ~ o c h r a ~ e u s ,and ~ ~ Rhizopus .~~ nigri~ans,'~as well as other micro-organisms, may be discerned from the Table. In the case of Calonectria decora a directing effect on hydroxylation was found, in which the nature of the oxygen substituent (carbonyl, hydroxy, methoxy) was less important than its spatial position relative to the steroid molecule as a whole.30 Attempts to reduce or obviate the unwanted 7a-, 9a-, or 14a-monohydroxylation of substrate by Curuularia lunata attendant on commercial use of the organism in llp-hydroxylations have been directed to the use of substrates bearing an a-face substituent (5a-bromo-,' l 2 16a-rneth~1,'~'1 7 a - a ~ y l o x y - ,or '~~ 5a,6a-epoxy-groups 6 ) , which appear to decrease unwanted side-reactions. afford an 11pFermentations of 17a-acetoxy-21-hydroxypregn-4-ene-3,20-dione hydroxylated product in excellent yield.
The transformation of steroids not oxygenated in the C-3 position into 3-0x0steroids may involve mono-oxygenase hydroxylation and subsequent dehydrogenation. Conversion of the 3,5-diene (4)into the 4,6-dien-3-one ( 5 ) by Rhizopus nigricans or by Rhizopus arrhizus has been reported,65 as has conversion of
165 166
K. Kieslich and G. Raspe, Ger. P. 1 226 575/1966 (Chem. A h . , 1967,66, 38 165h). J . de Flines and F. W. Van der Waard, Dutch P. 6605514/1967 (Chem. Abs., 1968, 69, 77 624p).
Microbiological Reactions with Steroids
453
androsta-3,5-dien-l7-one(6) into androsta-4,6-diene-3,17-dione (7) by Corynebacterium species.' 67 Oxidation of 5a-androst-3-en-17-one and androst-4-en-3one by Corynebacterium species gave the corresponding A4-3-ketone,' whereas 17a-ethynyloestr-4-en-17p-01 (8) was oxidized by Aspergillus JEavus to the lophydroxy-A4-3-ketone (9).*'
The viewpoint that microbial hydroxylations represent a detoxification of the steroids by increasing their water solubility has been put forth.'67" Some additional details of microbial hydroxylations of steroids have been reviewed.'
3 Hydroxy-steroid-Oxoteroid Interconversions Hydroxy-steroid-0x0-steroid interconversions, as indicated in the formal equation R2CH20H
+ NAD' + Enz
R,C=O
+ NADH + H + + Enz
involve NAD+ as cofactor. The enzymes are properly termed hydroxy-steroid : NAD oxidoreductases, no distinction being made whether the reaction proceeds as a dehydrogenation or as a reduction. Microbial oxidoreductases act on steroidal alcohols and ketones but they may also act on enols, the reduction of the 2-hydroxymethylene-3-ketone (10) to the 2a-hydroxymethyl-3-ketone (1 1) by
Ho#o-HC
lh7'
ECH
K . Schubert, W. Schumann, and C. Horhold, Abhandl. deutsch. Akad. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 135; French P. 2 030 35011970 (Chem. Abs., 1571, 75, 128 499k). 0. Hanl! and A. Cizinska, Abhandl. deursch. Akad. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 89.
Terpenoids and Steroids
454
Rhizopus stolonfer having been reported.' 6 8 However, given microbial oxidoreductases are generally best known for either their hydroxy-steroid dehydrogenase or their 0x0-steroid reductase actions. Interconversions of 3-, 17-, and 20-hydroxy-steroids and their corresponding ketones are common microbial reactions often found in combination with other transformations. Dehydrogenations of As-3P-alcohols (regularly accompanied by double-bond isomerization to give A4-3-ketones)are commercially important in the production of 17a,21dihydroxypregn-4-ene-3,20-dione (Reichstein's Substance S) and 17P-hydroxy17cx-methylandrosta-1,4-dien-3-one. 20-0x0-steroid reductions occur as unwanted side-reactions in important hydroxylation and A '-dehydrogenation reactions. Many examples of hydroxy-steroid-0x0-steroidinterconversions may be taken from the Table of hydroxylations. Dehydrogenations of 38-,20,30,32,34,41,89,116,119 &-,30 751-,70,7111a-,25,27 11P-,24,116,11 9 16P-,32and 17p_45,53,54,55,91,92 hydroxysteroids and reductions of 3-0x0-steroids to 3a-41and 3p-30*32333i46393,95 alcohols s ~ ~ the ~ ~broad ~ ~ ~distribution ~ ~ ~ ~ , ~ and of 17-0x0-steroids to 1 7 ~ - a l ~ o h o l suggest of dehydrogenases and reductases in organisms generally recognized for their hydroxylation capacities. Of particular interest in this regard are the 3/?-alcohol3-ketone interconversions of Aspergillus o c h r a c e ~ sl o~' ~and * ~ ~Calonectria ~ decors 30.3 2.33 and 17~-alcohol-l7-ketoneinterconversions of Curvularia l ~ n a t a ~ ~ and Aspergillus t ~ m a r i i as , ~ well ~ as 3P-hydroxy-steroid dehydrogenations by Curvularia lunata' 6 , and 17P-hydroxy-steroid dehydrogenations by Aspergillus o c h r ~ c e u s . ~ ~ ~ ~ ~ In addition to the examples in the Table, many reports of dehydrogenations and reductions in vegetative cell cultures have appeared. The greater availability of cell-free oxidoreductases (compared with cell-free hydroxylases) has also generated many reports of mechanism of action studies using partially purified enzymes. Hydroxy-steroid dehydrogenases inadvertently contaminate certain cell-free hydroxylase preparations, the 9a-hydroxylase of Nocardia restrictus having 9a- and 17P-hydroxy-steroid dehydrogenase capacity' 43 and the lop, 1 1P-,and 14a-hydroxylase of Curvularia lunata NRRL 2380 having an associated 17P-hydroxy-steroidd e h y d r o g e n a ~ e ' ~and ~ , 'a~20-0x0-steroid ~ red~ctase.'~~.'~~ The 16a-hydroxylase from Actinomyces roseochromogenes ATCC 3347 was accompanied by a 178-hydroxy-steroid dehydrogenase.' 47 Takadiastase, a commercial diastatic powder from Aspergillus oryzae, has been shown to retain 17P-hydroxy-steroid dehydrogenase activity. 7 0 Three microbial oxidoreductases have been prepared in states of high purity. The 20-0x0-steroid reductase (20P-hydroxy-steroid : NAD oxidoreductase) (E.C. 1.1.1.53)from Streptomyces hydrogenans ATCC 19631 has been obtained crystalline. The 3a-hydroxy-steroid dehydrogenase (3a-hydroxy-steroid : NAD oxidoreductase) (E.C. 1.1.1SO) from Pseudomonas testosteroni ATCC 11996 has
'
' ''
lh9
A. J. Manson, R. E. Sjogren, and M. M. Riano, J . Org. Chem., 1965, 30, 307. L. L. Smith and Y. Y. Lin, Acta Cientifica VenPzoluna, 197, 22, S u p ~ l No. . 2, R-60.
''O
S. L. Emerman and M . Levitz, Sreroids, 1964, 3, 351.
'68
Microbiological Reactions with Steroids
455
been purified and separated from the 38- and 17P-hydroxy-steroid dehydrogenase (3a- and 17P-hydroxy-steroid : NAD oxidoreductase) (E.C. 1.1.1.51) from the same organism. Additional hydroxy-steroid dehydrogenases of several mutant strains of Pseudomonas testosteroni have been classified into four groups, according to substrate-specificity studies, as follows : (i) a ring ~ / ~ - t r a n s - 3 p hydroxy-steroid dehydrogenase, (ii) a 3cr-hydroxy-steroid dehydrogenase unable to oxidize ~/~-cis-3a-hydroxy-steroids with an 11-0x0-group, (iii) ring AIB-trUnS3a-hydroxy-steroid dehydrogenase, and (iv) an axial 3-hydroxy-steroid dehydrogenase. The well-known hydroxy-steroid dehydrogenase of Pseudomonas testosteroni has been still further purified and characterized, and the enzyme has been applied (androsterone).' 7 2 to an assay for 3a-hydroxy-5a-androstan-17-one Elegant studies of substrate selectivity and steric specificity for the 3a-hydroxyThe steroid dehydrogenase of Pseudomonas testosteroni have been reported. 3a-hydroxy-steroid dehydrogenase will oxidize cyclohexanol and a variety of other cyclic and polycyclic alcohols, including adamantanol,' 74 the rate of catalysis increasing with increasing molecular complexity up to the tetracyclic steroid 3a-alcohol structures. Addition of the ring D to synthetic substrates resulted in a great increase in specificity of enzyme action. Differential dehydrogenation of enantiomeric substrates also increased with increasing molecular complexity, 19-nor-steroid-3a-alcohols of natural configuration being greatly favoured for reaction over their enantiomers. The steroid oxidoreductases of Pseudomonas testosteroni are inducible enzymes whose induction has been related to de nouo protein biosynthesis.'75 The effectiveness of different steroids as inducers of the 3a-hydroxy-steroid dehydrogenase has been examined. A planar molecule with an unobstructed a-face appears to be important.' 7 6 The 3a-hydroxy-steroid dehydrogenase is preferentially induced over the 3p- and 17b-hydroxy-steroid dehydrogenase by several synthetic androstane derivatives of the type (12), where R may be H or Me. The derivative (13) with a fully methylated side-chain suppressed all induction effects.' 7 7 The 3-hydroxy-steroid dehydrogenases of Pseudonomas testosteroni are inhibited by the anti-androgen cyproterone (14) and by 17P-hydroxy-17a-methylandrost-4-en-3-0ne,'~~ as well as by a number of oestrogenic and progestational
''
'
''I
'75
177
17*
C . R. Roe and N. 0. Kaplan, Biochemistry, 1969, 8, 5093. J . Boyer, D. N. Baron, and P. Talalay, Biochemistry, 1965,4, 1825. H . J. Ringold, J. M. H. Graves, A. Clark, and T. Bellas, Proc. Nur. Acad. Sci. U . S . A . , 1966, 56, 255; Recent Progr. Hormone Res., 1967, 23, 349; 'Proceedings of the Second International Congress on Hormonal Steroids', Milan, May 23-28, 1966, ed. L. Martini, F. Fraschini, and M. Motta, Excerpta Medica Foundation, Amsterdam, 1967, pp. 2 19-226. H. J. Ringold, T. Bellas, and A. Clark, Biochem. Biophys. Res. Comm., 1967, 27, 361. A. Wacker, J. Drews, W. B. Pratt, H. Laurent, and K. Petzoldt, 2. Narurforsch., 1965, 20b, 547. Y. Michel-Briand, European J . Biochem., 1969,10, 133. Y. Michel-Briand, G . Balavione, H. Kagan, and J.-P. Vigneron, European J . Biochem., 1971, 19, 218. A. Wacker, L. Trager, M. Maturova, and H. Beckmann, Narurwiss., 1967, 54, 90.
Terpenoids and Steroids
456
R
&o: :
H-CCO,H I ;
I
H
H
CO,H
(12)
(13)
steroids and the enzyme inhibitors 2a-cyano-l7P-hydroxy-4~,4P, 17a-trimethylandrost-5-en-3-one and 17P-hydroxy-2-hydroxymethylene- 17a-methyl-5a-androstan-3-one. 9*18o A protein fraction from Pseudomonas testosteroni uninduced by steroids has been shown to inhibit more effectively the microbial DNAdependent RNA-polymerase implicated in de nouo enzyme biosynthesis than did protein isolated from Pseudornonas testosteroni induced by steroids." 82 The 3p- and l7p-hydroxy-steroid dehydrogenase (E.C. 1.1.1.51)from Pseudornonas testosteroni co-occurs with the 3a-hydroxy-steroid dehydrogenase from which it is fully separable. The enzyme acts on both 3p- and 17P-hydroxy-steroids, and a concept of binding of the 3p- or 17/?-alcoholsat a common site has been advanced.' 83 The participation of a secondary active site was also suggested. The active site of the enzyme has been probed with a variety of substrates, and Michaelis constants K , have been determined. The enzyme is inducible.' 8 3 Dehydrogenation of 17p-hydroxy-steroids by the 3p- and 17p-dehydrogenase of Pseudornonas testosteroni is sensitive to isotopic substitution of the 17ahydrogen, 17c1-[~H]oestra-l,3,5( lO)-triene-3,17P-diolbeing oxidized at only about 35 the rate of the 17a-['HI-compound. 184 The 38- and 17P-hydroxy-steroid dehydrogenase attacked not only unconjugated 17P-hydroxy-steroidsbut also 38sulphate and 3P-glucuronide conjugates. Kinetic studies suggest that the conjugates have diminished affinities for the enzyme (as determined by higher Michaelis constants K,). '8 5 The 3p- and 17p-hydroxy-steroid dehydrogenase
'
','
A. S. Goldman, J . Clin. Endocrinol. Metabolism, 1967, 27, 320; 1968, 28, 1539. A. M. Neville and L. L. Engel, J . Clin. Endocrinol. Metabolism, 1968, 28, 49. la A. Wacker, J . Drews, W. B. Pratt, and P. Chandra, Angew. Chem., 1965,77, 172. 18' J. Drews and A. Wacker, Acta Endocrinol., 1965, Suppl. 100, 123. l R 3M. Fosset and A. Crastes de Paulet, Bull. Soc. Chim. biol., 1967, 49, 1083. J. A. Adams, H. I. Jacobson, H. R. Levy, and P. Talalay, Steroids, 1965, Suppl. I, 75. '*' E. Miigrom and E.-E. Baulieu, Steroids, 1970, 15, 563. 179
Microbiological Reactions with Steroids
457
was inhibited by excess substrates, the A4,'v1 '-3-ketone (15) being more effective than the A4-3-ketone (16), which was more effective than the A496-3-ketone(17). 17Q-Hydroxy-androsta-1,4-dien-3-one did not inhibit the enzyme.'8 6
The purified hydroxy-steroid dehydrogenases from Pseudomonas testosteroni have been used in assay procedures for steroids. Total bile acids in bile'87 and in serum188,189have been measured using the 3a-hydroxy-steroid dehydrogenase, and an ultramicro method (picomole range) for 17P-hydroxyandrost-4en-3-one (16) has been devised with the 3P- and 17Q-hydroxy-steroiddehydrogenase. 90 The 3a-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni and the 20-0x0-steroid reductase from Streptomyces hydrogenans have been used to examine the structure of a 5t-pregnane-3t,20t-diol metabolite from pregn-4ene-3,20-dione.' ' Microbial 3-hydroxy-steroid dehydrogenations frequently have synthetic utility. Flavobacteriurn dehydrogenuns is a useful organism for such purposes, as evinced by the following cases. Dehydrogenation of the ~-nor~A~-3Q-alcohol (18) yielded the corresponding A4-3-ketone.' 9 2 Dehydrogenation of the WagnerMeerwein-rearrangement product (19) or its 16Q-epimer yielded the corresponding A4-3-ketones.'9 3 Ester hydrolysis and dehydrogenations of the C-17 epimeric bromo-steroids (20)and (21) gave the corresponding 17-bromo-A4-ketones.'94
'
I9O 19' lg2
193
194
M. Mousseron-Canet, J.-L. Borgna, Y. Beziat, and C. Chavis, Compt. rend., 1968, 267, D, 797. L. A. Turnberg and A. Anthony-Mote, Clin. Chim. Acta, 1969, 24, 253. G. M. Murphy, B. H. Billing, and D. N . Baron, J . Clin. Pathol., 1970, 23, 594. R. H. Palmer, in 'Methods in Enzymology. Volume XV. Steroids and Terpenoids', ed. R. B. Clayton, Academic Press, New York-London, 1969, pp. 280-288. H. Carstensen, Nature, 1966, 212, 1604. J. H. H. Thijssen and J. Zander, Acta Endocrinol., 1966, 51, 563. H. Reimann, 0. Z . Sarre, and E. P. Oliveto, Steroids, 1966, 7, 5 0 5 . H. L. Herzog, 0. Gnoj, L. Mandel, G. G . Nathansohn, and A. Vigevani, J . Org. Chem., 1967, 32,2906. H. Reimann and 0. Z . Sarre, J. Org. Chem., 1967, 32, 2321.
Terpenoids and Steroids
458
s
C
MeCO,
o
f
:
9: Me COMe
MeC02
Use of the 3P-hydroxy-steroid dehydrogenase of Pseudornonas testosteroni for synthesis of 19-hydroxy-5a-androstane-3,17-dionefrom 3P,19-dihydroxy-5~ androstan-17-one on a micro-scale (50 p g ) has been described.lg4" (Dianabol) (23) Synthesis of 17P-hydroxy-17a-methylandrosta-1,4-dien-3-one by microbial dehydrogenation of the A5-3P-alcohol intermediate (22) appears
&O-HMe
o#oHMe
HO
to be commercially operable. Flauobacteriurn peregrinurn and Mycobacteriurn $ a ~ u r n ' as ~ ~well as Mycobacteriurn r n u c ~ s u r nhave ~ ~ ~been used to advantage for such synthesis. Dehydrogenations of less well-known 3P-hydroxy-steroids by a variety of micro-organisms have been reported. Dehydrogenation of the 5r,6aepoxy-3P-alcohol (24) by Flauobacteriurn dehydrogenans ATCC 13930 gave the 6a-hydroxy-A4-3-ketone (25) whereas Arthrobacter simplex and Bacillus lentus ATCC 13805 gave the 6a-hydro~y-A'>~-3-ketone (26).The epimeric SP,Gp-epoxy3P-alcohol gave the epimeric 6P-hydroxy-A4-3-ketone with Flauobacteriurn dehydrogenans ATCC 13930 and the epimeric 6/J-hydro~y-A'.~-3-ketone with Arthrobactrr simplex. 1 9 7 Tomatanin-3P-01 (tomatidine) (27) is dehydrogenated
1y40J.-A.Gustafsson and J. Sjovall, European J . Biochem., 1')5
''
1968, 6 , 2 2 1 . J. Protiva and V. Schwarz, Ceskoslor. Farm., 1966, 15, 237. A. A. Akhrem, N. E. Voishvillo, and L. E. Kulikova, Izcest. Akad. Nauk S.S.S.R., Ser. khim., 1969, 2625. K. Kieslich, Tetrahedron, 1969, 25, 5863.
Microbiological Reactions with Steroids
fl
0
459
I
Me
OH
to the A'-, A4-, and A1'4-3-ketone derivatives by Nocardia r e s t r i ~ t u s . ' ~Fusidic ~ acid (28) is oxidized by Corynebacteriurn simplex ATCC 6946 to the 3-ketone derivative.
HO-'
Dehydrogenations of a variety of substituted 3P-hydroxypregn-5-en-20-one derivatives by Flavobacteriurn buccalis lead to the corresponding A'-3-ketones. For this organism a 21-hydroxy- or 21-acetoxy-group was necessary for dehydrogenation.' O6 The hydrocarbon-assimilating organism Corynebacteriurn hydrocarboclastus strain 272-9 dehydrogenates a variety of sterols, including cholesterol (cholest-5en-3P-ol), sitosterol (24R-ethylcholest-5-en-3P-ol), campesterol (24R-methylcholest-5-en-3P-ol), and stigmasterol (24S-ethylcholesta-5,trans-22-dien-3P-ol), as well as several of their 19-hydroxylated derivatives, to the corresponding A43-ket0nes.~" '98 199
2oo
I. Belie and H . S d l i E , Experientia, 1971, 27, 626; J. Steroid Biochem., 1972, 3, 843. W. Dvonch, G. Greenspan, and H. E. Alburn, Experientia, 1966, 22, 517. H. Iizuka, M . lida, S. Teshima, and Y . Minemura, Z . allg. Mikrobiol., 1969, 9, 443.
460
Terpenoids and Steroids
3P-Hydroxyandrost-5-en-17-onewas oxidized by Pediococcus cerevisiae ATCC 8081 to androst-4-ene-3,17-dione, which was in turn reduced to 17P-
hydroxyandrost-4-en-3-0ne.~~' 3P-Hydroxy-sterol dehydrogenases are very common among micro-organisms which degrade sterols, cholest-5-en-3P-01being converted into cholest-4-en-3-one (thence to C , 9-A4-and -A'.4-3-ketones) by Streptomyces sp. 14PH8,39Penicillium S P . , ~ ' Streptomyces ~ ~Zivaceus,~'~ various Mycobacterium species, including chiefly Mycobacterium phlei,204,205Mycobacterium r u b r ~ m , ~ ~a~variety , ~ " of Actinomycetes,208 by many species from the genera Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and S t r e p t ~ m y c e s09,2 , ~ l o and by Proactinomyces asteroides 43fL2' Initial 3P-hydroxy-steroid dehydrogenation appears to be present in the microbial degradation of sapogenins also, (25R)-spirost-5-en-3P-o1 (diosgenin) being converted into (25R)-spirost-4-en-3-one by Mycobacterium phlei2' and by Fusarium solani.21 Optimum conditions for transformation by M ycobacterium species of C , and C2, A5-3P-alcohols to the corresponding A'- and A'*4-3-ketones have been sought.214 9 2l 5 Several thermophylic bacteria oxidize 3/3-hydroxypregn-5-en-20one to pregn-4-ene-3,2O-dione.' The reduction of cholest-5-en-3P-01 to 5Pcholestan-3/3-01by intestinal bacteria appears to involve an initial 3P-hydroxysteroid dehydrogenation. Cholest-4-en-3-one is then reduced to 5P-cholestan3fi-01.~' Random microbial reductions have also been reported. Both stereospecific reductions, such as Actinomyces kanamyceticus reduction of pregn-4-ene-3,20dione to 3P-hydroxy-Sa-pregnan-20-0ne,~and non-stereospecific reductions,
''
''I
'''
R. L. Brown and R. C. Wood, Texas Reports Biol. Med., 1967, 25, 65. A. It6 and C. Chihara, J. Ferment. Technol., 1967, 45, 719.
K. Schubert, G . Rose, and C. Horhold, Biochim. Biophys. Acta, 1967, 137, 168. G. Ambrus, E. Tomorkeny, and K. G. Buki, Experientia, 1968, 24, 432. ' 0 5 G. Wix, K. G. Buki, E. Tomorkeny, and G . Ambrus, Steroids, 1968, 11, 401. ' 0 6 A. A. Imshenetskii and L. A. Mavrina, Mikrobiologiya, 1968, 37, 620; 1972,41, 399. 2 0 7 A. A. Imshenetskii, E. F. Efimochkina, L. E. Nikitin, and T. S . Nazarova, Doklady Akad. Nauk. S.S.S.R., 1969, 186, 205. 2 0 8 V. A. Zanin, Mikrobiologiya, 1968, 37, 919. 2 0 9 K. Arima, M. Nagasawa, ,M. Bae, and G. Tamura, Agric. and Biol. Chem. (Japan), 1969,33, 1636. 'lo M. Nagasawa, M. Bae, G. Tamura, and K. Arima, Agric. and Biol. Chem. (Japan), 1969,33, 1644. 'I1 Zh. D. Lebedeva, 0. B. Tikhomirova, L. M. Kogan, I. I. Zaretskaya, G . K. Skryabin, and I. V. Torgov, Izuest. Akad. Nauk S.S.S.R., Ser. biol., 1972, 529. "' G. Ambrus and K. G. Buki, Steroids, 1969, 13, 623. 'I3 E. Kondo and T. Mitsugi, J . Amer. Chem. SOC.,1966, 88, 4737. 'I4 V. N. Shaposhnikov, A. A. Akhrem, and N. E. Voishvillo, Vestnik Moskou. Univ., Biol. Pochvoved, 1969, 24, 45; Izvest. Akad. Nauk S.S.S.R., Ser. khim., 1969, 197. 2 1 5 V. N. Shaposhnikov, A. A. Akhrem, L. E. Kulikova, and N. E. Voishvillo, Izuest. Akad. Nauk S . S . S . R . , Ser. biol., 1969, 770. 2 1 6 I . Bjorkhem and J.-w. Gustafsson, European J . Biochem., 1971, 21, 428. '" L. M. Kogan and I. M. Volkova, Izuest. Akad. Nauk S.S.S.R., Ser. biol., 1971, 146. 203
'04
Microbiological Reactions with Steroids
46 1
such as that of androst-4-ene-3,17-dioneby the yeast Rhodotorula mucilaginosa, which results in a mixture of epimeric 3-alc0hols,~'* have been reported. Microbial reduction of the 3-ketone group of a number of A5('0)-19-nor-steroid 3-ketones to mixtures of the corresponding 3a- and 3P-alcohols has been reported for a variety of yeasts and for Aspergillus fischeri ATCC 1020.48 Preferential reductions by the yeasts Pichia fermentans NCYC 246 and Candida tropicalis strain Monilia (IfG) were used to prepare a variety of A5('0)-3P-alcohols. Reduction of the conane derivative (29) by Penicillium atramentosum Thom to the 3fi-alcohol has been r e p ~ r t e d . ~ '
Cell-free 7a-hydroxy-steroid : NAD(P) oxidoreductases acting on bile acids have been prepared from intestinal anaerobes and enterobacteria. The 7ahydroxy-steroid dehydrogenase was induced by 3a,7a, 12a-trihydroxy-SP-cholan24-oic (cholic) acid and required molecular oxygen. Enzyme prepared from Gram-positive bacteria required NADP as cofactor, whereas dehydrogenases from Gram-negative organisms required NAD.220 The properties of a crude 7a-hydroxy-steroid dehydrogenase from Escherichia coli B have also been described.221 In this case the enzyme differed from the 7a-hydroxy-steroid dehydrogenases previously mentioned220and also from the hydroxy-steroid dehydrogenases of Pseudornonas testosteroni by being constitutive rather than inducible by nature.22 A cell-free inducible 12a-hydroxy-steroid dehydrogenase acting on bile acids has also been prepared from intestinal anaerobes.220 The enzyme required molecular oxygen and NAD (from Gram-negative organisms) or NADP (from Gram-positive organisms) as cofactor.220 17~-Hydroxy-steroid-17-oxo-steroid interconversions are regularly encountered. Interconversion of 3-hydroxyoestra-1,3,5(lO)-trien-l7-one and oestra1,3,5(lO)-triene-3,17P-diol by Takadiastase from Aspergillus oryzae has been m e n t i ~ n e d . 'The ~ ~ same two steroids are interconverted by Neurospora crassa' l 4 and by species of thermophylic micro- organism^.^ 218
219
220
221
J . Dmochowska, S. Siewinski, and A. Nespiak, Bull. Acad. polon. Sci., Str. Sci. biol., 1969, 17, 133. T. Nguyen-Dang, M. Fonquernie, and M.-M. Janot, Ann. Pharm. franc., 1966, 24, 523. V . Aries and M . J. Hill, Biochim. Biophys. A d a , 1970, 202, 535. I. A. MacDonald, C. N . Williams, and D. E. Mahony, Biochim. Biophys. Acta, 1973, 309, 243.
462
Terpenoids and Steroids
Androst-4-ene-3,17-dione and 17P-hydroxyandrost-4-en-3-0neare intercon,~~ verted by Rhodotorulu mucilaginosa2 * and by Aspergillus , f i ~ c h e r iwhereas androst-4-ene-3,11,17-trione and androsta-1,4-diene-3,11,17-trione were reduced Pseudomonas to the corresponding 17P-alcohols by Aspergillus tarnarii. testosteroni has been used for synthesis of the 9a-hydroxy- 17-ketone (31) from the 3,9~,17P-triol(30).222
One of the most interesting developments involving microbial 17-dehydrogenases and reductases has been in the resolution of racemic steroids synthesized by various total-synthesis schemes. Resolution of racemic 17-oxygenatedoestrane derivatives by microbial interconversions of the 17P-alcohol and 17-ketone groups has long been known, and resolution of the racemic gonanes rac-14Pgona-1,3,5(10),8-tetraene-l1,17-dione(32) and rac-14P-gona-1,3,5(10),6,8-pentaene- 11,17-dione (33) by reduction of the carbonyl groups by Rhodotowla mucilaginosa yielded resolved 17-alcohols and 1 1 , 1 7 - d i o l ~ . ~ ~ ~ ~ ~ ~ ~ H
H
Resolution of the racemic des-A-ring analogue (34) by yeast reduction to the dextrorotatory alcohol (35) (further converted into natural steroids) has also
222 223
224
C. J. Sih, S. S. Lee, Y . Y. Tsong, and K. C. Wang, J . Biol. Chem., 1966, 241, 540. A. Siewinski, J. Dmochowska, and S. Mejer, Bull. Acad. polon. Sci., S i r . Sci. chim., 1969, 17, 151. A. Siewinski, Bull. Acad. polon. Sci., SPr. Sci. chim., 1969, 17, 469, 475.
Microbiological Reactions with Steroids
463
been reported.225 However, direct resolution of the 13P-ethyl- and 13P-propylhomologues (36)-(39) by such means cannot be achieved in proper yields. Yeast reduction of the 17-ketone group did not occur, and bacterial dehydrogenation of the 17P-alcohol group gave poor conversions.226 Dehydrogenation of rac-(36) with Corynebacterium simplex ATCC 6946 gave the 17-ketone (38) of natural configuration ; Flauobacteriurn dehydrogenans gave the racemic 17by ketone (38). Dehydrogenations of rac- 13P-ethyl-17fl-hydroxygon-4-en-3-one Corynebacterium simplex ATCC 6946 and by Corynebacterium hoagii ATCC 7005 also yielded (38) of natural configuration, but very little 17-ketone product (39) was obtained with Y U C - l7P-hydroxy- 13P-propylgon-4-en-3-one as substrate. Steric interference of the 13P-ethyl and 13P-propyl groups probably accounted for the diminished enzyme action.226
(36) R (37) R
= =
(38) R = Et (39) R = Pr
Et Pr
By contrast, utilization of microbial 0x0-steroid reductases for resolution of racemic 8,14-seco-steroids obtained by total synthesis has been remarkably successful. Selective stereospecific reduction of only one of the ketone groups by a variety of micro-organisms of the racemic 8,14-seco-steroid intermediate (40) leads to the 8,14-seco-steroid hydroxy-ketone (42), which on cyclization
(40) R (41) R
= =
(42) R (43) R
Me Et
= =
Me Et
(44) R = Me (45) R = Et 225
226
Y. Kurosawa, H. Shimojima, and Y. Osawa, Steroids, 1965, Suppl. I, 185; 1965, 6, x. G . Greenspan, L. L. Smith, R. Rees, T. Foell, and H. E. Alburn, J. Org. Chern., 1966,31, 2512.
464
Terpenoids and Steroids
affords 3-methoxyoestra- 1,3,5(10),8,14-pentaen-17P-01 (44) of natural configuration. Numerous species of moulds, actinomycetes, bacteria, and yeasts performed the required selective reduction, but the yeast Saccharomyces uvarum CBS 1508 gave the best yields (as high as 75 %).227-229 The enantiomeric product ent-3-methoxyoestra-1,3,5(10),8,14-pentaen-17~-01 (or 3-methoxy-13cr-oestra-1,3,5(10),8,14-pentaen-17P-ol)(46) was selectively available by reduction of racemic (40) with Bacillus thuringensis to give a 85% yield of the 13a-17P-alcohol (47), which on cyclization as the l7P-acetate gave (4Q2 7-2 Attempted cyclization of (47) as the free alcohol gave the 9cr,14aepoxy-ketone (48).227,230
(47)
A similar reduction of the racemic 13P-ethylhomologue (41) by Saccharomyces uuarum CBS 1508 gave the ketol (43) which on cyclization gave the tetracyclic ~ ' 17-alcohols formed by these 17P-alcohol (45) of natural c ~ n f i g u r a t i o n . ~The yeast reductases all had the (1 7s)-configuration, as established by application of the Horeau method. The cisltrans relationships of the 13P-alkyland 17-hydroxygroups were established by 'H n.m.r. ~ p e ~ t r a . ~ A similar ~ ~ - resolution ~ ~ ~ , ~ ~ ~ of the racemic B-nor-steroid intermediate (49) afforded the hydroxy-ketone (50) of natural configuration.23 2
' H. Gibian, K . Kieslich, H.-J. Koch, H. Kosmol, C. Rufer, E. Schroder, and R. Vossing, 228
229 "O
231
232
Tetrahedron Letters, 1966, 2321. H. Kosmol, K. Kieslich, R. Vossing, H.-J. Koch, K. Petzoldt, and H. Gibian, Annalen, 1967,701, 198. C. Rufer, E. Schroder, and H. Gibian, Annalen, 1967, 701, 206. T. Asako, K. Hiraga, and T. Miki, Chem. Comm., 1969, 1011; Chem. and Pharm. Bull. (Japan), 1973, 21, 703. C. Rufer, H. Kosmal, E. Schroder, K. Kieslich, and H. Gibian, Annalen, 1967,702, 141. H. Heidenpriem, C . Rufer, H. Kosmol, E. Schroder, and K. Kieslich, Annalen, 1968, 712. 155.
Microbiological Reactions with Steroids
465
Selective stereospecific reduction of one carbonyl group of the racemic Dhomo-l7a-ketone analogue (5 1) with yeasts was reported to yield optically active 17a-alcohols. Saccharomyces carlbergensis and Saccharomyces cereuisiae gave the 17ap-alcohol (52),233-235whereas Candida robusta gave both the 17apalcohol (52) and the 14a,l7ap-diol (53).234 Saccharomyces carlbergensis var. ualdensis gave the cyclic 9a,14a-ether (54).234 In contrast to the five-membered D-ring series, in which only the trans- 13a-17p-alcohol (47) readily cyclized to (48), both the D-homo-hydroxy-ketone (52) and the diol(53) cyclized readily to their corresponding cyclic 9 a , 1 4 ~ - e t h e r s . ~ ~ ~
Selective stereospecific reduction of only one carbonyl group of the racemic ester triketone (55) by Rhizopus arrhizus Fischer, yielding the hydroxy-diketone ester (56) in over 70 % yield, permits total synthesis of oestra-1,3,5(10)-triene3,17P-diol of natural configuration via the bicyclic intermediate (57).236 233 234
235 236
L. M. Kogan, V. E. Gulaya, and I. V. Torgov, Tetrahedron Letters, 1967,4673. V. E. Gulaya, L. M. Kogan, and I. V. Torgov, Izvest. Akad. Nauk S . S . S . R . , Ser. biol., 1970, 1811. L. M. Kogan, V. E. Gulaya, and 1. V. Torgov, Khim.-Farm. Zhur., 1971, 5,No. 12,20. P. Bellet, G . Nomine, and J. Mathieu, Compt. rend., 1966, 263, C,88.
Terpenoids and Steroids
466
MeO,C,)
MeOC , /,.
MeO,C
Reduction of the 17a-ketone group of rac-3-methoxy-8-aza-~-homo-oestra1,3,5(lO)-trien-l7a-one (58) by Aspergillus ochraceus I.C.I. 1357 gave the resolved 17aa-alcohol (59). However, concomitant non-stereospecific reduction also occurred, giving the racemic 17ap-alcohol (60). No hydroxylations were ob~ e r v e d37. ~
(59) R' (60) R'
=
=
OH, R2 = H H, R2 = OH
The action of microbial 20-hydroxy-steroid : NAD oxidoreductases generally is as a reductase, and both 20a- and 20b-alcohols may result. Commercial bakers' yeast reduces pregn-4-ene-3,20-dione to the 20a-alcoh01,~38 whereas a variety of mushrooms (Basidiornycetes) reduce 17a,21-dihydroxypregn-4-ene-3,20-dione to the 20P-alcoh01.~~ Thermophylic organisms of genera Actinornyces, Therrnopolyspora, Micromonospora, and Actinobzfida also reduce C2I 20-0x0-steroids to their 2 0 P - a l ~ o h o l s . ~Screening ~ operations on many Cephalosporiurn, Geotrichum, and Paecilomyces species suggest that the 20-0x0-steroids 17c(,21-dihydroxypregn-4-ene-3,20-dione and llB,l7a,21-trihydroxypregn-4-ene3,20-dione are reduced to the corresponding 2 0 P - a l ~ o h o l s .Reduction ~~ of the by mycelium or spores 20-ketone group of 16a,17a-epoxypregn-4-ene-3,20-dione of Fusariurn solani and of Septomyxa ufJinis ATCC 6737 and by vegetative cells of Bacillus sphaericus ATCC 7 0 S 23 9 or Bacillus rneg~teriurn~~' gave the 20aalcohol (61). Reduction of the 20-ketone group of 17a-hydroxy-3,20-dioxopregn-4-en-21-a1 (62) with the 20-0x0-steroid reductase of Streptornyces hydvogenans proceeded probably to the 20fi-hydroxy-21-aldehyde(63).241Reduction of the 20,21*" P. J. Curtis, Biochem. J., 1965, 97, 148. 238 239 240
24'
Z . ProchAzka and N. G . Chan, Coll. Czech. Chem. Comm., 1970, 35, 2209. R. Plourde, 0. M . El-Tayeb, and H. Hafez-Zedan, Appl. Microbiol., 1972, 23, 601. L. A. Krasnova, L. V. Sokolova, V. M. Ryzhkova, and N. N. Suvorov, Priklad. Biokhim. i Mikrobiol., 1969, 5 , 260. E. S. Szymanski, C. S. Furfine, and C. F. Hammer, Steroids, 1972, 19, 243.
467
Microbiological React ions with Steroids Me I
CHO
COCHO
d-I
- -OH
diketone (64) by vegetative cell cultures of Streptomyces flauus IF0 3359 or Streptomyces lauendulae IF0 3361 or of the yeast Rhodotorula glutinis IF0 071 1 proceeded at the 21-ketone, yielding the 21aaF-hydroxy-20-ketone(65).242 Me
I
COMe
I
H-C-OH
I
Many C , 20-0x0-steroids are vulnerable to reduction during 16a-hydroxylation by Actinomyces (Streptomyces) roseochromogenus ATCC 3347. The 20-0x0steroid reductase may be constitutive in nature243,244 and has been obtained as a soluble 20a-hydroxy-steroid : NADP oxidoreductase free from 16a-hydroxylase, 242
243
244
S. Noguchi, H. Otsuka, M. Obayashi, M. Imanishi, and T. Takahashi, Steroids, 1968, 12, 9. L. M. Kogan and E. A. Elin, Abhandl. deutsch. Akad. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 195. E. A. Elin, L. M. Kogan, 0. S. Tarasov, and I. V. Torgov, Khim. prirod. Soedinenii, 1970,47.
468
Terpenoids and Steroids
A4-reductase, and 17P-hydroxy-steroid oxidoreductase a ~ t i v i t i e s . ~ 17a~~,~~~
Hydroxypregn-4-ene-3,20-dione and 16a, 17a-epoxypregn-4-ene-3,20-dione were both reduced to the corresponding 2 0 a - a l ~ o h o l s .Broad ~ ~ ~ screening of Actinomyces species which 16a-hydroxylated pregn-4-ene-3,20-dione established that many species reduced substrate 17u-hydroxypregn-4-ene-3,2O-dione to the 17a, 20a-diol whereas other species gave the epimeric 17c(,20P-dioland still others gave 16u,17a-Epoxypregn-4-ene-3,20mixtures of 17a,20sr- and dione was also reduced by many Actinomyces species to the corresponding 20aalcohol (6 The 20-0x0-steroid reductase of Streptomyces roseochromogenes Squibb No. 6186 was greatly diminished in effect by growing the organism in mixed culture with Arthrobacter simplex. 9a-Fluoro-1 1p, 17a,21-trihydroxypregn-4-ene-3,20dione (66) was thereby converted in one fermentation step into 9a-fluoro-llp,16a, 17a,21-tetrahydroxypregna-1,4-diene-3,2O-dione(triamcinolone) (67) with minimum 20-ketone reduction.’ 6 1 1).2433247
O -*H
COCH,OH
~
c
o
~ --OH
o
/
o
/
Unwanted 20-ketone reduction is also a problem for certain A‘ -dehydrogenations. Studies attempting to regulate and reverse the 20-ketone reduction of Mycobacterium globiforme strain 193 have been reported, and pH, culture age, and aeration effects appear to influence the direction of the reaction.248 The 20-0x0-steroid reductase (20P-hydroxysteroid :NAD oxidoreductase) of Streptomyces hydrogenans has been crystallized, but the crystalline enzyme retains 3a-hydroxy-steroid (but not 3p- and 17P-hydroxy-steroid)dehydrogenase activity with several C,, and C, 5a,3a-alcohols and 3-0x0-steroid reductase activity with 5a-androstane-3,16-dioneand 5~-androstane-3,17-dione.~~~-~~ Comparative rate measurements with the crystalline enzyme and a variety of 20-0x0steroids gave the kinetic parameters K , and V,,,, which were widely different 245
246
247
248
249
250
25 I
L. M. Kogan, E. A. Elin, M. Krishnamurti, and I. V. Torgov, Khim. prirod. Soedinenii, 1970, 38. E. A. Elin, L. M. Kogan, N. T. Zelkova, and I. V. Torgov, Khim. prirod. Soedinenii, 1969, 145. L. M. Kogan, E. A. Elin, V. I. Mel’nikova, and I. V. Torgov, Khim. prirod. Soedinenii, 1969, 149. M. B. Kupletskaya, L. V. Sokolova, N. F. Kovylkina, N . N . Suvorov, and G. K. Skryabin, Izvest. Akad. Nauk S . S . S . R . , Ser. b i d . , 1969, 406. T. Pocklington and J. Jeffery, Biochem. J., l968,110,28P; European J . Biochem., 1968, 7, 63. T. Pocklington, J. Jeffery, B. S. Middleditch, and C. J . W. Brooks, Biochem. J., 1970, 119, 803. W. Gibb and J. Jeffery, Biochim. Biophys. Acta, 1972, 268, 13.
o
Microbiological Reactions with Steroids
469
for the various s ~ b s t r a t e s . ~ ’ ~It *was ~ ’ ~suggested that the ring A of substrates was situated at an extremity from the active site,253and that interactions between the enzyme and the region around ring B were important, with hydrophobic interactions involving the p-face of the B-ring likely.254 Introduction of 6p-, 11P-, or 17a-hydroxylatio11, of A’-unsaturation, or of 16a-methyl substitution reduced the reduction rate.252 Kinetic studies show that the 20-0x0-steroid reduction proceeds by an Ordered BiBi mechanism in which the cofactor NADH binds initially.255Some reduction may proceed with the substrate binding first, h ~ w e v e r5 6, ~ Hydrogen transfer from the P-side of the pyridine ring of the cofactor NADH to the 20~-position in the steroid molecule has been dernon~trated.~’The 20-0x0-steroid reductase acts on both 3P-hydroxypregn-5-en-20-one and on its 3/3-sulphate, possibly at the same active ~ i t e . ~ ” , ~ ’ ~ The 20-0x0-steroid reductase of Streptomyces hydrogenans is induced by a variety of steroids.258 Pregn-4-ene-3,20-dioneY17p-hydroxy-5a-androstan-3-one, and 11~,21-dihydroxypregna-4,17(20)-dien-3-one are good inducers ; 17P-hydroxyandrost-4-en-3-one, 17fi-hydroxy-5Q-androstan-3-one, and androst-4-ene3,17-dione are weak inducers. New enzyme synthesis, demonstrated by isotopeincorporation studies, was inhibited by actinomycin D and by chloramphenicol. Inducer steroids appear to act as derepressors of an R N A - p o l y r n e r a ~ e . ~ ’ ~ ~ ~ ~ ~ The anti-androgen cyproterone (14), 17P-hydroxy-l7a-methylandrost-4-en-3one, and 17P-hydroxy-7~, 17a-dimethylandrost-4-en-3-one inhibited induction of the 20-0x0-steroid reductase, as did diethylsilbestrol, reserpine, and ergocornin.’ 7 8 , 2 5 8 , 2 6 1 , 2 6 2 A 20-0x0-steroid reductase which reduced 17a,21-dihydroxypregn-4-ene-3,20-dioneto 17a,208,21-trihydroxypregn-4-en-3-onehas been prepared from Curuularia Iunatu. In contrast to that from Streptomyces hydrogenans, which utilized NADH as cofactor, the reductase from Curuularia lunata used NADPH.263 to the 208Reduction of 21-acetoxy-17a-hydroxypregn-4-.ene-3,11,20-trione dihydro-derivative 17or,20/3,21-trihydroxypregn-4-ene-3,1l-dione has been reported for Bacillus meguterium, Bacillus mesentericus, and Bacillus subtilis
fermentation^.^^' In addition to oxidoreductase actions of vegetative cell cultures and purified enzymes obtained therefrom, a variety of fungal spores exhibit hydroxy-steroid dehydrogenase and 0x0-steroid reductase activities.’ 5 9 Suspension cultures of 252 253 254 255 256
”’ 258
259 “O 261
262
263
E. Gerhards, G. Raspe, and R. Wiechert, Arzneim.-Forsch., 1967, 17,431. I. H. White and J. Jeffery, European J. Biochem., 1972, 25, 409. 1. H. White and J. Jeffery, Biochim. Biophys. Acta, 1973,296, 604. G. Betz and J . C. Warren, Arch. Biochem. Biophys., 1968, 128, 745. G . Betz and P. Taylor, Arch. Biochem. Biophys., 1970, 137, 109. T. Pocklington and J. Jeffery, European J. Biochem., 1969,9, 142. L. Trager and A. Wacker, Abhandl. deutsch. Akad. Wiss. Berlin, Klasse, Med., 1968, No. 2, p. 185. A. Wacker, L. Trager, P. Chandra, and H. Feller, Biochem. Z . , 1965, 342, 108. A. Wacker, E. Hoffman, and H. Feller, Biochem. Z . , 1965, 342, 236. A. Wacker, B. Bauer, and L. Trager, 2.physiol. Chem., 1970, 351, 320. L. Trager and A. Wacker, Z . physiol. Chem., 1970,351, 329. J. D. Townsley, H. J. Brodie, M. Hayano, and R. I. Dorfman, Steroids, 1964, 3, 341.
470
Terpenoids and Steroids
cells of the higher plant Cioscorea deltoidea have been shown to possess 17p- and 20P-hydroxy-steroid dehydrogenases ;264,2 6 5 3P-hydroxy-steroid dehydrogenase, 3-0x0-steroid reductase, and 20-keto-steroid reductase activities have also been demonstrated for a variety of cultured plant cells.266 4 Dehydrogenation and the Reduction of CarbowCarbon Double Bonds This section considers the introduction of carbon-carbon double bonds into the A'- and A4-positions of steroids oxygenated at C-3 and the reduction of A'-, A4-, A'-? A7-, and At6-doublebonds by microbial enzymes. The enzymes are oxidoreductases which may catalyse both dehydrogenase and reductase reactions, but relatively few vegetative cell cultures have been shown to operate in both modes. The A'-dehydrogenation of 11~,17a,21-trihydroxypregn-4-ene-3,20-dione by Mycobacteriurn globijorrne strain 193 may be reversed by aeration and medium composition control.267 A cell-free A'-oxidoreductase from Mycobacteriurn gfobzfbrrnehas been prepared which also may operate as a A'-dehydrogenase or as a A '-reductase depending on aeration and on the oxidation-reduction potential state of the system.268 However, distinct A'-dehydrogenase and A'-reductase enzymes are indicated, for the enzymes had different induction and inhibition characteristics and could be separated from one another.269 More recently a partially purified C 2 -steroid-A'-reductase from Mycobacteriurn globijorrne has been described.270 Cell-free A '-oxidoreductases from BacilZus cyclo-oxydans ATCC 1267327 and from Arthrobacter simplex ATCC 6946272 have also been described which function as A '-dehydrogenases under aerobic conditions and as A'-reductases under anaerobic conditions. Much of the progress on these matters derives from prior commercial interests in A ' -dehydrogenations of steroid hormone analogues, but fundamental studies of double-bond dehydrogenations and reductions also figure prominently in microbial metabolism of steroids concerned with other transformations. Dehydrogenations of ring A frequently accompany other reactions involved in degradation of the steroid, both by side-chain scission and B-ring cleavage. Some
*'' 2hh
2h'
'(''
'" 2'' L'2
S. J. Stohs and M. M. El-Olemy, Phytochemistry, 1972, 11, 1397. S. J. Stohs and M. M. El-Olemy, Lloydia, 1972, 35, 81. J. M. H. Graves and W. K. Smith, Nature, 1967, 214, 1248. G. K. Skryabin, I. S. Zvyagintseva, and I. Sokolova, Izvest. Akad. Nauk S . S . S . R . , Ser. h i d . , 1965, 715; G . K. Skryabin, I . S. Zvyagintzeva, M. I . Nazaruk, and L. V. Sokolova, Doicludy Akad. Nauk S.S.S.R., 1965, 161, 472. N. N. Lestrovaya, M. 1. Nazaruk, and G . K. Skryabin, Doklady Akad. Nauk S.S.S.R., 1965, 163, 768. N. N . Lestrovaya and G. K. Skryabin, Abhandl. deutsch. Akad. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 213. N. N. Lestovaya and M. I. Bukhar, Biokhimiya, 1970, 35, 1182. M. Iida, J. D. Townsley, M. Hayano, and H. J. Brodie, Steroids, 1965, Suppl. I, 159. L. Penasse and M. Peyre, Steroids, 1968, 12, 525; L. Penasse and E.-E. Baulieu, Abhatidl. deutsch. Akad. Wiss. Berlin, Klasse M e d . , 1968, No. 2, p. 201.
Microbiological Reactions with Steroids
47 1
examples of A' -dehydrogenations accompanying 3-hydroxy-steroid dehydrogenations and further degradations were mentioned in Section 3. The association of both A'- and A4-dehydrogenase activities with 3P-hydroxy-steroid dehydrogenase, A5-A4-double-bond isomerase, and esterase activities is typified in the action of Alcaligenes fuecalis on appropriate substrate^.^^ Dehydrogenations of the A-ring of 3-oxygenated steroids may proceed to saturated 3-ketone, A'-3-ketone, A4-3-ketone, and/or A'*4-3-ketone stages, as typified in the dehydrogenation of Sa-tomatanin-3P-o1(27) by Nocardia restrict ~ s . ' ~ 'Although A-ring dehydrogenations are associated only with steroid substrates oxygenated at C-3, double-bond reductase action may occur at double bonds conjugated with carbonyl (A1- and A4-3-ketones or Al6-20ketones), at terminal double bonds (A6) of conjugated dienones (A4.6-3-ketones), or at isolated double bonds (A'). In distinction to hydroxy-steroid dehydrogenases, dehydrogenases which introduce carbon+arbon double bonds into steroids do not co-occur regularly with microbial hydroxylases. With the exception of the special cases of Nocardia restrictus, which may A' -dehydrogenate and 9a-hydroxylate a steroid, 143 of Nocardia corulfina, which may A' -dehydrogenate or la,2a-dihydroxylate steroids,24and of Mycobacterium rnucosurn 1210, which may A4-dehydrogenate and 9a-hydroxylate 3a,l2a-dihydroxy-5fLcholan-24-oic a ~ i d , *75~ no ~,~ other examples of dehydrogenation and hydroxylation are found in the Table. Carbon-carbon double-bond reductases occur more commonly along with hydroxylases. Prominent examples from the Table include A4-5a-reduction with O0 A4-5P-reduction and 1la-hydroxylation by Rhizopus nigricans REF 129,59*60,1 1lx-hydroxylation by Beauveria g l ~ b u l f e r a , ~aromatic ~,~' A-ring reduction with 10P-hydroxylation by Aspergillus flavus 98/13,89and A4-5a-reduction with lop, 11P-dihydroxylation by Curvularia lunatn NRRL 2380.47 The latter reduction example involved rac- 17P-hydroxyoestr-4-en-3-one as substrate, and although both enantiomers were lOP,llP-dihydroxylated, only the enantiomeric 1OP,11Pdiol was reduced, yielding the enantiomeric product (68).47
(68)
'-
The A and A4-dehydrogenases are probably flavoproteins which directly dehydrogenate appropriate 3-0x0-steroid substrates. Separate A1-5a- and 273
274
275
G. Wix, K. Albrecht, G. Ambrus, and A. Szabo, Acta Microbiol. Acad. Sci. Hung., 1968, 15, 239. L. 0. Severina, I. V. Torgov, and G. K. Skryabin, Doklady Akad. Nauk S . S . S . R . , 1969, 184, 963. L. 0. Severina, I. V. Torgov, G. K. Skryabin, V. 1. Zaretskii, N. S. Wulfson, and I. B. Papernaja, Tetrahedron, 1969, 25, 5617.
472
Terpenoids and Steroids
A -5P-dehydrogenases, separate A4-501- and A4-5P-dehydrogenases, and individual A'-dehydrogenases acting on A4-3-ketones may exist. The A' -oxidoreductases of Bacillus cyclo-oxydans ATCC 12673271 and Arthrobacter silitplex ATCC 6946272 are inducible enzymes. The Bacillus cyclo-oxydans dehydrogenase was stimulated by menadione and by NAD and NADP. Its reductase activity was markedly stimulated by NADH. The dehydrogenase and reductase activities could not be separated from one another.271The Arthrobacter simplex dehydrogenase was inhibited by Hg2 Cu2 iodoacetate, and arsenite. Michaelis constants K , for several steroid substrates have been reported for the Arthrobacter simplex enzyme.272 The c,ell-free enzyme273as well as vegetative cell cultures of Arthrobacter simplex276 were inhibited by +
+
dicortinone (69), a C40H5008dimer bis-[ 17a-hydroxy-3,1l-dioxoandrost-4-ene17P-yll diketone derived from alkaline oxidation of 17cx,21-dihydroxypregn-4ene-3,11,20-tri0ne.~~~ The inhibition of the cell-free enzyme was determined by kinetic measurements to be n o n - ~ o m p e t i t i v e . ~ ~ ~ The partially purified A '-dehydrogenase from Corynebacterium simplex retained A '-dehydrogenation capacity after being entrapped in a cross-linked polyacrylamide gel. An assay procedure for the inducible A' -dehydrogenase of Bacillus sphaericus ATCC 7055 has been described.277 A soluble A4-5P-dehydrogenase which introduces the A4-double bond into 5P-3-oxo-steroids has been obtained from Pseudomonas testosteroni ATCC 11196. 5a-Steroids were not dehydrogenated. Further purification of acetone powders gave a purified enzyme with a specific activity of 6.5 pmol min- (mg protein)-', using 5P-androstane-3,17-dione(70) as substrate.278 The stoicheiometry of the dehydrogenation was examined using phenazine methosulphate as an artificial electron acceptor. The reduction of two equivalents of cytochrome c was indicated. It was suggested that the partially purified enzyme was a flavoprotein
'
276
277
'''
L. Velluz, L. Penasse, G. Nomine, G. Amiard, P. Berthelemy, and V. Torelli, Compt. rend., 1966, 262, C , 120. J. B. Jones, V. Vlasinich, and J. D. Leman, Cunud. J. Biochem., 1966, 44, 1183. S. J . Davidson and P. Talalay, J . Biol. Chem., 1966, 241, 906.
*
Microbiological Reactions with Steroids
0
H
473
+ 2 Cytochrome (Fe3') c
and that flavin mononucleotide (FMN) was the primary hydrogen acceptor in the oxidation of the steroid substrate.278 Details of an assay procedure for the A4-5P-steroid dehydrogenase from Pseudomonas testosteroni have been published. In order to measure the A4dehydrogenase activity in the presence of A'-dehydrogenase activity the A'-5fl3,17-diketone (72) was used as s~bstrate.~"
A cell-free A4-5/?-dehydrogenase(E.C. 1.3.99.5) and a A'-SP-dehydrogenase (E.C. 1.3.99.4)have been prepared from a strain of Clostridiurn paraputrijicum. The A4-5/?-enzymetransformed the 5P-3,17-dione (70)into the A4-3,17-dione(71) as well as 3-oxo-5/?-cholan-24-oicacid (73) into 3-oxochol-4-en-24-oic acid (74). The dehydrogenases were inducible. Phenazine methosulphate or menaphthone were used as artificial electron acceptors in transformations with the cell-free enzymes2*' Washed intact cells of Clostridiurn paruputrijicurn transformed substrates (70) and (73) to the A4-3-ketones (71) and (74) (using molecular oxygen as electron acceptor), but on disruption of the cells a Ah'-dehydrogenaseactivity was disclosed, with formation of the A'*4-3-ketone(75).280 The mechanism by which the Ah'-doublebond is introduced into appropriate 3-0x0-steroid substrates was suggested some time ago as involving enolization of the 3-carbonyl group in association with hydride-ion abstraction, with stereospecific trans removal of the la- and 2P-hydrogens. Extensive kinetic studies with 279 280
S. J. Davidson, ref. 189, pp. 656-666. V. C . Aries, P. Goddard, and M.J. Hill, Biochim. Biophys. Acta, 1971, 248, 482.
Terpenoids and Steroids
474
i
(74)
(75)
a cell-free A'-dehydrogenase from Bacillus sphaericus ATCC 7055 are in accord with this formulated mechanism.28' The trans removal of the l a - and 28hydrogens has been further supported by studies with vegetative cells of Bacillus sphaericus and labelled 0estr-4-ene-3,17-dione.~~~ Furthermore, cell-free preparations of Bacillus sphuericus acting on [ la-2H]pregn-4-ene-3,20-dione gave pregna- 1,4-diene-3,20-dione with an 88 7; loss of deuterium, thus supporting removal of the l ~ h y d r o g e n So .~~ ~ accepted is this mechanism for Bacillus well sphaericus that the organism may be used to determine the specific location of tritium introduced by synthesis into the 1- or 2-positions of certain A4-3ketone^.^^^,^" Specifically labelled 19-hydroxy [l-3H (83 %-P)]androst-4-ene-3,17-dione(76) was converted by Pseudomonas sp. ATCC 13262,286by Nocardiu restrictus,286 and by Bacillus sphaericus ATCC 7055287into 3-hydroxyoestra-1,3,5(10)-trien17-one (77), in which 22-28% of the tritium had been lost, thus suggesting stereospecific removal of the 1a-hydrogen in the transformation. A similar
2R'
282 283 284 285
28h
R. Jerussi and H. J. Ringold, Biochemistry, 1965, 4, 21 13. T. Anjyo, M. Ito, H . Hosada, and T . Nambara, Chem. and ind., 1972, 384. Y. J. Abul-Hajj, J . B i d Chcm., 1972, 247, 686. B. E. Edwards and P. N. Rao, Biochim. Biophys. Acta, 1966, 115, 518. H. J. Brodie, K . Raab, G. Possanza, N. Seto, and M. Gut, J . Org, Chem., 1969, 34, 2697. H. J. Brodie, G. Possanza, and J. D. Townsley, Biochim. BiophyJ. Acta, 1968, 152, 770.
Microbiological Reactions with Steroids
475
result was obtained using a 6OOO x g supernatant preparation from Nocardia restrictus.28 h Dehydrogenation of [ la-2H]- and [ la,2w2H]-5P-pregnane-3,1 1,20-trione by Septomyxa slfJinisATCC 6737 vegetative cell cultures demonstrated the stereospecific removal of the diequatorially oriented la- and 2p-hydr0gens.'~~Specifically labelled [2-2H]- and [4-2H]-5P-androstane-3,17-diones prepared for the purpose2 were dehydrogenated by cell-free preparations of Nocardia restrictus ATCC 14887, and isotope analyses of the resultant androsta-1,4-diene-3,17-dione suggested stereospecific removal of the 2 P - h y d r 0 g e n . ~ ~ ~ The same experiments established the stereospecificremoval of the 4a-hydrogen ~~~ preparations of by the A4-dehydrogenase of Nocardia r e s t r i ~ t u s .Cell-free Nocardia restrictus have been studied further in regard to their A4-dehydrogena(78) and (79), 4Ption of 4a-bromo- and 4a-methyl-5a-androstane-3,17-dione rnethyl-Sa-androstane-3,17-dione (80), and 4P-bromo- and 4P-rnethyl-SB-androstane-3,17-dione (81)and (82). Product analysis suggested that cis removal of the 4a- and Sa-hydrogens from 5a-3-ketones and trans removal of the 4%-and 5Bhydrogens from SP-3-ketones occurred.290
(78) R = Br (79) R = Me
(81) R (82) R
= =
Br Me
Washed cells of several micro-organisms may be used in lieu of vegetative cell cultures for A'-dehydrogenations. Washed cells of Mycobacterium mucosum '&'
288
H. J. Brodie and P. A. Wang, Tetrahedron, 1967, 23, 5 3 5 . T. Nambara, H. Hosada, T. Anjyo, and S. Ikegawa, Chem. and Pharm. Bull. (Japan),
la9
290
1972,20,2256. S. Ikegawa and T. Nambara, Chem. and Ind., 1973, 230. Y . J. Abul-Hajj, Biochem. Biophys. Res. Comm., 1971, 43, 766.
476
Terpenoids and Steroids
dehydrogenate 17~-hydroxy-l7a-methylandrost-5-en-3~-ol to 17P-hydroxy-17amethylandrost- 1,4-dien-3-one effectively.' 96 A mathematical model for the dehydrogenation of the A4-3-ketone intermediate (83) using washed cells of Septornyxa a . n i s ATCC 13425 (previously induced with 3-oxobisnorchol-4-en2 1-al) has been published. The dehydrogenation involved introduction of the substrate (83) at levels greater than its nominal water solubility.291 FH,OH
(83)
Freeze-dried or acetone-dried cells may also be used effectively for dehydrogenations. Acetone-dried cells of Corynebacteriurn (Arthrobacter) simplex dehydrogenate a variety of steroids when incubated with menadione as an artificial electron a c ~ e p t o r . * ~Dried , ~ ~ *mycelium from Cephulosporium sp. 1 0 7 , 2 9 3 and freeze-dried cells from Bacillus lentus7 are also effectivefor A'-dehydrogenations. Fungal spores may also be utilized for steroid A'-dehydrogenations. Of prime interest in this regard is the dehydrogenation of 17a,2l-dihydroxypregn-4-ene3,20-dione by spores of Septornyxa ufinis ATCC 6737,294 the commercial feasibility of which has been suggested.98 Dehydrogenations by spores of Septomyxa afJinis have been reviewed. Fungal spores from several organisms adsorbed on to silica gel thin-layer chromatoplates retain their A'-dehydrogenase activities.'59 The steroid A'-dehydrogenase of Fusariurn solani spores appears to be bound to or embedded in the plasma membrane.295 Examples of the A'-dehydrogenation of steroids by vegetative cell cultures for synthesis purposes include dehydrogenations by Corynebacteriurn (Arthrobacter) simplex of the 6a,16P-dimethyl steroid (84)' l o and the 6a-fluoro-16cc-rnethyl
'
COCH,OH
0
291
292
293 294 295
J. W. Chen, F. J. Hills, H. J. Koepsell, and W. D. Maxon, Znd. andEng. Chem. (Process Design), 1965, 4, 421. R. C. Erickson, W. E. Brown, and R. W. Thoma, U.S. P. 3 360439/1967. Z. Zakrzewski, Acta Polon. Pharm., 1969, 26, 242. K. Singh, S. N. Sehgal, and C. Vkzina, Canad. J. Microbiol., 1965, 11, 351. R. Plourde and H. Hafez-Zedan, Appl. Microbiof., 1973, 25, 650.
Microbiological Reactions with Steroids
477
steroid (85).' l 2 Arthrobacter simplex has also been used for A'-dehydrogenation of the 6a-hydroxy-~-ringlactone (25) and its 6P-epimer (86).19'
o f l
2oH ;*co;
0
OH
F
(86)
(85)
The 7P-hydroxy-A4-3-ketone (87) was dehydrogenated by Mycobacteriurn ~ a v u r nas , was the A4*6-3-ketone(88).296 COMe
(87)
(88)
4,5-Epoxides may be transformed by Mycobacteriurn phlei into A4- and A',4-3ketones. A variety of C I 9 ,C,,, and C,, 4a,5a- and 4P,SP-epoxides of partial structures (89)and (90) were dehydrogenated via intermediate A4-3-ketonesto the A',4-3-ketones.297
(89)
(90)
The 5a-3-ketone (91) was dehydrogenated by Bacillus sphaericus and by Septornyxa a@nis to the A1-5a-3-ketone,but by Proactinornyces globerulus to the A4-3-ketone and the A' '4-3-ketone. 98 OH
0#--Me 296 29'
298
(91)
J. Protiva, J. Martinkova, and V. Schwarz, Folia Microbiol., 1968, 13, 7 . E. Tomorkeny, K . Albrecht, and L. Ila, Acta Microbiol. Acad. Sci. Hung., 1969,16,261. J . Protiva and V. Schwarz, Folia Microbiol., 1970, 15, 319.
478
Terpenoids and Steroids
Dehydrogenation of the steroid (92) bearing an additional E-ring by several Mycobacteriurn species gave the corresponding A”4-3-ketone.299
(92)
Fermentation of the 6-methyl-A5-3-ketone(93) with Flavobacterium dehydrogenans failed to yield a A ‘-derivative, but the 6P-hydroperoxide (94)was formed, apparently by auto-oxidation during the fermentation.’
’
@ o-Ho*c;o:
COMe
0 Me OOH
Me (93)
(94)
Dehydrogenations of common A4-3-0x0-steroids to the corresponding A .4-3-oxo-steroids have been variously found for Cephalosporium sp. 27C,62for Penicillium S P . , ~for~ several thermophyllic bacteria,56 and for both S- and Rforms of Mycobacteriurn globforme 193.300 A‘ -Dehydrogenations have been carried out by Avthrobacter simplex in mixed cultures with the hydroxylating organisms Absidiu coerulea, Aspergillus ochraceus, Curvularia lunata, and Streptomyces roseochromogenes. 60-1 6 2 Sequential transformations of the 16~,17a-cycloborateester (95), in which no intermediate was isolated, have also been conducted with Aspergillus ochraceus for 1 la-hydroxylation, and with acetone-dried cells of Arthrobacter simplex for A’ -dehydrogenation to yield the product cycloborate ester (96).146 Mixed
’
(95)
2q9
3n0
A. A. Akhrem, N . E. Voishvillo, L. E. Kulikova, and Yu. A. Titov, I z t w t . Akad. Nauk S.S.S.R., Ser. khim., 1972, 2570. I . S . Zvyagintseva and G. K. Skryabin, Mikrobiologiya, 1965, 34, 461.
Microbiological Reactions with Steroids
479
fermentations involving Aspergillus ochraceus and Bacillus lentus have also been r e p ~ r t e d . ~The effects of different crystalline forms of substrate 21-acetoxy-l7ahydroxypregn-4-ene-3,11,2O-trioneon A'-dehydrogenations by Arthrobacter simplex ATCC 6946 have been r e p ~ r t e d . ~ " Technical developmental studies of the A'-dehydrogenation of C, steroids by Mycobacterium globiforme 193 include adsorption effects between the microbial cells and the steroid substrate^,^'^ substrate i n h i b i t i ~ n , ~possibilities '~ of continuous f e r m e n t a t i ~ n , ~synthetic '~ medium studies,305and effects of surfactants. 306 The A '-dehydrogenase of Mycobacterium globiforme 193 is induced differentially from accompanying 21-esterase and 20-0x0-steroid reductase activities. 17a,2l-Dihydroxypregn-4-ene-3,11,20-trione is a powerful inducer of the A'dehydrogenase but not of the 21-esterase or 20-0x0-steroid reductase, which are induced differentially by pregn-4-ene-3,20-dione. Surface-active agents (Tweens) enhance the induction effect of the weak inducer 1lp, 17a,21-trihydroxypregn-4ene-3,20-dione.306a A'-Dehydrogenation of 19-nor-A4-3-ketones or of 19-hydroxy-A4-3-ketones results in aromatization of the A-ring. Introduction of the A'-double bond occurs with stereospecific removal of the la-hydrogen in the same manner as occurs with log-methyl steroids.28 Aromatization of the A-ring also occurs associated with 19-hydroxylation and C-1@-C-19 bond scission and with 9ahydroxylation and C-9-C- 10 bond scission following A '-dehydrogenation as a matter of general degradation, which is covered in Section 8 of this Report. and of its degradation product Fermentation of 17P-hydroxyoestr-4-en-3-one (97) with Cylindrocarpon radicicola gave the A-ring-aromatic products 3-hydroxyoestra-1,3,5(10)-trien-17-one(77) and (99) re~pectively.~'~ The C,, lactone (98) gave the corresponding A '-derivative with Cylindrocarpon radi~icola.~'~
(97) R = H (98) R = Me
(99)
G. Catroux, J.-C. Fournier, and H. Blachere, Canad. J . Biochem., 1968,46, 537. I . S. Zvyagintseva and D. G. Zvyagintseva, Mikrobiologiya, 1969, 38, 816. 3 0 3 G. B. Ksandopulo, Mikrobiologiya, 1970, 39, 792. 3 0 4 V. E. Sterkin, G. R. Morozova, A. M. Zyakin, A. G. Chigaleichik, and G. K. Skryabin, Izvest. Akad. Nauk S.S.S.R., Ser. biol., 1973, 233. 3 0 5 N. N. Klimontovich, E. V. Markova, and V. M. Ryzhkova, Khim.-Farm. Zhur., 1973,7, No. 1, p. 20. 3 0 6 V. M. Ryzhkova, L. V. Sokolova, G. A. Klubnichkina, A. S. Amelina, and N. N. Suvorov, Khim.-Farm. Zhur., 1973, 7, NO. 5, p. 33. 306aV.A. Gotovtseva, A. S. Korovkina, and E. G. Gusakova, Mikrobiologiya, 1973, 42, 434. 3 0 7 E. J . Kusner and R. D. Garrett, Steroids, 1971, 17, 521. 301 302
Terpenoids and Steroids
480
A'-Dehydrogenation of 9a-hydroxyoestr-4-en-3,17-dione(100) by either vegetative cell cultures or acetone-dried cells of Arthrobacter simplex gave 3,9a-dihydroxyoestra-1,3,5(10)-trien-17-one (31) and 3-hydroxy-9,10-seco-oestra1,3,5(lO)-triene-9,17-dione (lOl)? However, the cell-free A'-dehydrogenase preparation from Arthrobacter simplex previously mentioned27 2 failed to dehydrogenate the 9a-hydroxy-derivative (100).
Resolution of racemic 19-nor-steroids obtained by total synthesis may be obtained by microbial A'-dehydrogenations in select cases. Dehydrogenations of the racemic 13P-homologues(102)and (103)of 17P-hydroxyoestr-4-en-3-one by Corynebacterium simplex ATCC 6946 yielded A-ring-aromatic derivatives (104) and (105) of natural configuration. Unaltered substrate (102) was of negative
R
=
(103) R
=
(102)
Et Pr
(104) R = Et (105) R = Pr
specific rotation and therefore of the enantiomeric configuration.226The specificity of Arthrobacter simplex for A4-3-ketones of natural configuration [( 10R)chirality] was further demonstrated by the failure of the cell-free A'-dehydrogen[of (10s)-chirality] ase preparation to act on ent- 17P-hydroxyoestr-4-en-3-0ne~~ or of acetone-dried cells to act on the (10s)-enantiomer of a variety of bi-, tri-, and tetra-cyclic steroid analogues. Racemic 17P-hydroxy-9P,lOa-androst-4-en-3one (106)gave the A'-derivative (107) and unaltered enantiomeric substrate. The
Microbiological Reactions with Steroids
48 I
tetracyclic analogues rac-(108), rac-( log), and rac-(110) each gave a A'-derivative from the antipode of (l0R)-configuration. Racemic tricyclic (111) likewise yielded a A'-product from the antipode of (10R)-chirality. The racemic bicyclic (112) gave a resolved A'-product of (10s)-chirality [corresponding to (10R)chirality of the previous examples]. From these several results it was suggested that the dehydrogenase of Arthrobacter (Corynebacterium) simplex acted on those A4-3-ketones (113) of natural (10R)-configuration but not on those (114) of unnatural (10s)-configuration. 3 0 8 , 3 O9
By contrast fermentations of rue( 102)with Corynebacterium hoagii ATCC 7055 gave the phenol (104) of natural configuration and the phenolic 17-ketone (115)
308 309
J. Fried, M. J. Green, and G. V. Nair, J . Amer. Chem. SOC., 1970, 92, 4136. M. J. Green, N. A. Abraham, E. B. Fleischer, J. Case, and J. Fried, Chem. Comm., 1970, 234.
482
Terpenoids and Steroids
of enantiomeric configuration. Clearly the A'-dehydrogenase of this organism acted on both antipodes of rac-( 102), but a 17P-hydroxy-steroid dehydrogenase of Corynebacteriurn hougii acted only on the enantiomeric substrate en?-(102) or product ent-( 104).226Where the 17P-hydroxy-steroid dehydrogenase could not act, as in fermentation of rac-l3j3,17ct-diethyl-17~-hydroxygon-4-en-3-one, a racemic product m c - 13p,17wdiethylgona- 1,3,5(lO)-triene-3,17P-diol was recovered.2 A spectrophotometric assay for following the progress of the microbial A'dehydrogenation of %-fluoro- 11/3,16a,17~,1 l-tetrahydroxypregn-4-ene-3,20dione has been reported. ' In addition to carbon-carbon double-bond reductase activities associated with hydroxylase activities and those of select cell-free systems previously mentioned, distinct reductase activities have been displayed by several organisms. A A4-reductase acting on 17j-hydroxyandrost-4-en-3-0ne (16) and on androst-4ene-3,17-dione (71) has been reported for the yeast Rhodotorula rnucilaginosa,2' * and Rhodotorulu glutinis reduces (16) and 17/3-hydroxy-l7a-methylandrost-4-en3-one to 3,5a-tetrahydro-deri~atives.~~ Rhodotorula glutinis reduces 4-chloro17p-hydroxy-17c~~methylandrosta1,4-dien-3-one (116) to the 4a-chloro-5adihydro-A '-3-ketone (117) in low yield and to the 4ct-chloro-5a-A'-3/3-alcohol ( 1 18) in 65 :/: yield.5 4 A A'-5x-reductase acting on pregn-4-ene-3,20-dione has been demonstrated for Streptomyces kanamyceticus 690.2
'
'
dl
H
( 1 18)
A A4-Sa-reductase from Nocardiu corallina ATCC 13259 acting on cholest-4en-3-one311has been prepared in cell-free form.312 In contrast to A'- and A4dehydrogenases from the organism, the A4-5a-reductasewas not ind~cible.~' 1,31 'lo 3'1
'
E. Ivashkiv, Biorechnol. mrid Bioeng., 1971, 13, 561. G. Lefebvre, P. Germain, and R. Gay, Cornpt. rend., 1972, 274, D , 449. P. Germain, G. Lefebvre, B. Bena, and R. Gay, Compt. rend.. 1972,274, D , 600; Compt. retid. SOC. B i d . , 1972, 166, 1123.
Microbiological Reactions with Steroids
483
A A4-5a-reductase from Penicillium decumbens NRRL 742 which readily reduced androst-4-ene-3,17-dione (71) and pregn-4-ene-3,20-dione also exhibited some A'-reductase activity against andro~ta-1,4-diene-3,17-dione.~' A A4-5a-reductase acting on 1la-hydroxypregn-4-ene-3,20-dione has been demonstrated for Rhizopus nigricans REF f 29.59,60,'00Methylene blue and potassium ferricyanide both inhibited the A4-5c(-reductase.59 Reductions by Mycobacterium smegmatis SG 98 of 6P-hydroxyandrost-4-ene3,17-dione and of 6P, 17P-dihydroxyandrost-4-en-3-0negave the Sa-dihydroderivative 6P-hydroxy-Sa-androstane-3,17-dione(119), which was further (120).3l 4 dehydrogenated to 5a-androstane-3,16,17-trione
Reduction of 17P-hydroxyoestr-4-en-3-0neby Mycobacterium smegmutis gave the three 5a-derivatives 17P-hydroxy-5a-oestran-3-one, 5a-oestrane-3c(,l7/3diol, and Sa-oe~trane-3P,17P-diol.~' By contrast the enantiomeric substrate ent- 17P-hydroxyoestr-4-en-3-onegave the ent-58-derivatives ent-5P-oestrane3,17-dione and ent-5P-oe~trane-3a,l7P-diol.~~ A4-5a-Reductases acting on pregn-4-ene-3,20-dione have been demonstrated in incubations of cultured cells from the higher plants Dioscorea d e l t ~ i d e a , ~ ~ ~ , * ~ ~ from Nicotiana tabacum and Sophora ang~stifolia,~~ and from several other plants.266 Examples of A4-reductions to the SP-dihydro-derivative include actions of the 1la-hydroxylating fungus Beauveria globulifera on several C19 and C, A4-3ketone^.^^,^^ Clostridium paraputriJicum reduces C19 A4-3-ketones to the 5Pdihydro-derivatives. Reduction of the 3-ketone to the 3a-alcohol also occurred. After relatively short times in the reduction of 17~-hydroxy-17a-methylandrosta1,4-diene-3,17-dione(23), the A4-5P-reduction product 17fl-hydroxy-17a-rnethyl5P-androst-1-en-3-one (121) could be obtained.54 The anaerobic action of Clostridium paraputrijicum on A4- and A1,4-3-ketones gave SP-derivatives. Androst-4-ene-3,17-dione (71) gave the 3a,5P-tetrahydro-derivative (122)whereas androsta-l,4-diene-3,17-dione gave the A1-5P-3-ketone(72).317 * 3 1 '
313 3'4
T. L. Miller and E. J . Hessler, Biochim. Biophys. Acta, 1970,202, 354. K. Schubert, J . Schlegel, K.-H. Bohme, and C. Horhold, Biochim. Biophys. Acta, 1967, 144, 132.
315
316 317
K . Schubert and G. Hobe, Steroids, 1969, 14, 297. T. Furuya, M. Hirotani, and K. Kawaguchi, Phytochemistry, 1971, 10, 1013. K . Schubert, J. Schlegel, and C. Horhold, Steroids, 1965, Suppl. I, 175. J. Schlegel, Abhandl. deutsch. Akad. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 1 1 I .
Terpenoids and Steroids
484
HO”
&
Reductions by Clostridium paraputriJicum H2 of the epimeric 6-hydroxypregn4-en-3,20-diones gave the corresponding 3cc,SP-tetrahydro-derivatives.3 1 A similar 3a,SP-tetrabydro-derivativewas formed from 6p-hydroxyandrost-4-ene3,17-dione. Some details of the microbial reduction of cholest-5-en-3P-01 (cholesterol) ( 1 23) to 5P-cholestan-3P-01 (coprosterol) (125) have been re~iewed.~”Although formation of the 5P-dihydro-derivative (125)by intestinal microflora is formally a A5-reduction, such does not appear to be the case. Rather, prior dehydrogenation and bond isomerization occur to give cholest-4-en-3-one (124)as an intermediate
’
(124)
which is then reduced by A4-5P-reductases.216~320 In contrast to mammalian liver 5p-reductases, which use NADPH as cofactor, NADH was used in the microbial reduction. Furthermore, the 4B-hydrogen of the cofactor was transferred, whereas other A4-reductases utilize the 4A-hydr0gen.~~’The reduction by intestinal microflora of the A5-double bond of cholesta-5,7-dien-3P-o1to give 5pcholest-7-en-3p-01has been reported.32 ’I9
320
’‘
S. Carini, M . C. Cocucci, and A. Ferrari, Progr. Biochem. Pharmacol., 1967, 2, 6 2 . 1. Bjorkhem, J.-hi. Gustafsson, and 6. Wrange, European J. Biochem., 1973, 37, 143. C. F. Cohen, S. J. Louloudes, and M. J. Thompson, Steroids, 1967, 9, 591.
Microbiological React ions with Steroids
485
The A6-double bond of several C19,C21,C27,and CZ8A4,6-3-ketoneswas reduced selectively by Mycobacterium phlei as an aspect of degradation of the substrates by this organism. Androsta-4,6-diene-3,17-dione (7) was transformed into androst-4-ene-3,17-dione (71) and androsta-1,4-diene-3,17-dione ( 126).204
Likewise, 17P-hydroxyandrosta-4,6-dien-3-one (17) was reduced by Nocardia restrictus to the corresponding A4-3-ketone (16). Further degradation of the steroids also occurred.32 However, Clostridium paraputrificum did not reduce (7) or that of androstathe A6-double bond of androsta-4,6-diene-3,17-dione 1,4,6-triene-3,17-dione (127), both of which were reduced to the A6-3s(,5ptetrahydro-derivative ( 128).317
(128)
The isolated A7-double bonds of 5a-cholest-7-en-3P-01 and 5cr-ergosta-7,22dien-3P-01 were reduced by Mycobacterium phlei concomitantly with further degradation.204 A A5-5a-reductase from mammalian intestinal microflora is implied in the (129) into the dihydro-A5transformation of 3P-hydroxypregna-5,16-dien-20-one
1) derivative^.^ 2 3 By perform3/?-alcoho1(130)and tetrahydro-5a-3/3-alcohol(l3 ing the incubations in deuteriated water, incorporation of deuterium into the 322
323
Y. Y. Tsong, K. C. Wang, and C. J. Sih, Biochim. Biophys. Acta, 1964, 93, 398. H. Eriksson, J.-A. Gustafsson, and J. Sjovall, European J . Biochem., 1968, 6, 219.
Terpenoids and Steroids
486
( 130)
(131)
16a- and 17P-positions was demonstrated, thus establishing a trans addition of
hydrogens.3 2 4 Reduction of the aromatic A-ring of 17a-ethynyloestra-1,3,5( 10)-triene-3,17Pdiol (132) with accompanying 10P-hydroxylation to yield (9) is reported for Aspergillus Jauus 98/13.89 The transformation may be regarded as separate A'reductase and 10P-hydroxylaseactions, for (132) under anaerobic conditions was (133).89 converted into 17r-ethynyl-17P-hydroxyoestr-4-en-3-one
( 132)
(133)
Reduction of the A20'22)-double bond of the lactone ring of lanatoside C by intestinal microflora has been reported.325 5 Olefmic Bond Isomerization
Isomerization of the A5-double bond of A5-3P-hydroxy-steroids generally accompanies dehydrogenation of the 3-alcohol so as to yield the corresponding A4-3-0x0-steroid. This has already been mentioned in Section 3 of this Report in connection with 3-hydroxy-ster oid dehydrogenations associated with degradation reactions of sterols and other A5-3P-alcohols. The A5-double-bond isomeriration is a separate reaction from the hydroxy-steroid dehydrogenation, and the A5-isomerasefrom Pseudomonas testosteroni ATCC 11996has been prepared in a crystalline state and studied extensively. Reported double-bond isomerizations catalysed by microbial enzymes are almost entirely confined to cases involving As-double-bond isomerization into the A4-position. However, isomerizations of the A5(l')-double bond into the A4lO)-en-3-one was position are also known. 17a-Ethynyl-17/3-hydroxyoestr-5( isomerized and 10P-hydroxylated by Aspergillus JEauus 98/13 to yield the 324
325
I. Bjorkhem, H. Eriksson, and J . A. Gustafsson, European J . Biochem., 1971, 20, 340. I. Hermann and K . Repke, Ahhandl. deursch. Akad. Wiss. Berlin,.Klasse Med., 1968, No. 2, p. 1 1 5 .
Microbiological Reactions with Steroids
487
10P-hydroxy-A4-3-ketone(9).89 Isomerization of a A3-steroid to a A4-steroid has been reported in association with microbial oxidations at C-3,167 and A3,5derivatives are isomerized to A4*6-derivativesin association with C-3 oxidat i o n ~ . A~ specific ~ , ~ ~listing of other isolated examples of A5-isomerization will not be attempted here. The A5-3-0x0-steroid isomerase from Pseudomonas testosteroni ATCC 11996 has been extensively studied as an enzyme and for its mechanism of action. The steroid-induced enzyme catalyses isomerization of the A5 - and A5('0)-do~ble bonds of A5- and A5(10)-3-oxo-steroidsto the A4-position in conjugation with the 3-ketone group. The biochemistry of the enzyme has been r e v i e ~ e d . The ~~~,~~~ isomerase is the most active enzyme known. Refinements in the process for preparation of the crystalline isomerase have been r e p ~ r t e d . ~ Details ~ ~ , ~ ~of' (134) an assay procedure for the isomerase acting on androst-5-ene-3,17-dione
to yield the A4-3-ketone (71) have been published.327 Crystalline A5-isomerase has been prepared labelled with 14C by growing Pseudomonas testosteroni in media containing 4C-amino-acids.329The crystalline A5-3-0x0-steroid isomerase from Pseudomonas testosteroni exists as a trimer of identical subunits containing 125 amino-acid residues. The amino-acid sequence of the subunit has been determined.330,33 As suggested by c.d. studies the A5-isomeraseis well ordered and has a very high percentage of p-structure as pleated sheets.332 Induction of the A5-isomerase of Pseudomonas testosteroni by steroids was inhibited strongly by reserpine and by Vinca and Ergot alkaloids333and by 6-chloro-8-aza-9-cyclopentylpurineand related deoxyribonucleoside anal o g u e ~34. ~The kinetics of A5-isomerase induction have been examined. Nutritiocal factors profoundly affect the kinetics of induction and the absolute yield of enzyme.329 32b 32i
328 329
330 33'
332 333 334
P. Talalay, Ann. Rev. Biochem., 1965, 34, 347. R. Jarabak, M. Colvin, S. H. Moolgavkar, and P. Talalay, ref. 189, pp. 642-651. P. Talalay and J. Boyer, Biochim. Biophys. Acta, 1965, 105, 389. M. Shikita and P. Talalay, in 'Biogenesis and Action of Steroid Hormones', ed. R . I. Dorfrnan, Y. Yamasaki, and M. Dorfman, Geron-X Inc., Los Altos, Calif., 1968, pp. 41-64. J. Boyer and P. Talalay, J. Biol. Chem., 1966, 241, 180. A. M. Benson, R. Jarabak, and P. Talalay, J. Biol. Chem., 1971, 246, 7514. F. Vincent, H. Weintraub, and A. Alfsen, F.E.B.S. Letters, 1972, 22, 319. M. Maturova, H. Beckmann, and A. Wacker, Z. Naturforsch., 1967, 22b, 621. M . S. Zedeck, A . C. Sartorelli, P. K. Chang, K. Raska, R. K. Robins, and A. D . Welch, Mol. Pharmacol., 1967, 3, 386.
488
Terpenoids and Steroids
Androst-4-ene-3,17-dione,17P-hydroxyoestr-4-en-3-one,pregn-4-ene-3,20dione, and 19-norpregn-4-ene-3,20-dione inhibit the isomerase reaction competitively. 3 5 The As-3-0x0-steroid isomerase of Pseudornonas testosteroni was inhibited competitively by 2cr-cyano-l7~-hydroxy-4a,4~,17a-trimethylandrost5-en-3-one and 17fl-hydroxy-2-hydroxymethylene17a-methyl-5a-androstan-3one1*' and by cyproterone and several other steroid hormone ana10gues.l'~ In short-term experiments 17~-acetoxy-6~-bromoandrost-4-en-3-one acted as a competitive inhibitor of the isomerase, but on prolonged incubations a time- and concentration-dependent irreversible inactivation occurred. Using 3H-acetoxylabelled inhibitor it was demonstrated that tritium was incorporated into the isomerase protein, probably covalently.336 2a-Bromo- 17P-hydroxy-5aandrostan-3-one and 17~-acetoxy-2a,4~t-dibromo-5a-androstan-3-one were good competitive inhibitors of the i s o m e r a ~ e . ~ ~ ~ Formation of the enzyme-substrate complex between the As-isomerase and suitable As-3-0x0-steroids has been compared with an extraction process in which the steroid substrate is removed from the aqueous environment by hydrophobic parts of the p r ~ t e i n . ~ ~ ~ , ~ ~ * The mechanism suggested some time ago for As-double-bond isomerization by Pseudornonas testosteroni involved enolization of the 3-carbonyl group with specific transfer of the 4b-hydrogen to the 6P-position. Enolization was initiated by protonation of the 3-carbonyl group by a proton donor, with loss of the 4Pproton to a basic group following as the rate-limiting step. Reprotonation by the protonated basic group at the GP-position followed by proton removal from the 3carbonyl group thengave the product A4-3-ketoneandthe regenerated enzyme.339 The participation of histidine and tyrosine residues in the isomerase action has been suggested. The tyrosine residue affords an acidic site putatively involved in initiating enolization of the 3-carbonyl group. The histidine residue acts as a basic site involved in the transfer of the 4P-hydrogen to the 6P-po~ition.~~' Although large differences in rates obtain for acid-, base-, and enzyme-catalysed reactions the differences in entropy of activation for the three processes are not great.341 The AS* value of - 16.8 cal deg-I mol-' for the enzyme reaction341 supports the concept of charge separation in the transition state as previously suggested.339 An enthalpy of activation of the enzyme reaction of 4.6-5.0 kcal mol- has been r e p ~ r t e d . ~ ~ ' , ~ ~ ~ Extensive kinetic studies of the isomerization, including use of inhibitors, suggest the participation of the solvent in the enzyme reaction. The affinity of steroid substrates for the enzyme and the dielectric constant of the medium decreased as the concentration of organic solvent in the aqueous medium was 335 336 337
338 339 340 341
342
F. Falcoz-Kelly, E.-E. Baulieu, and A . Alfsen, Biochemistry, 1968, 7, 41 19. K. G . Biiki, C. H. Robinson, and P. Talalay, Biochim. Biophys. Acta, 1971, 242, 268. J. B. Jones and S. Ship, Biochim. Biophys. Acta, 1972, 258, 800. A. Alfsen, E.-E. Baulieu, M. J. Claquin, and F. Falcoz-Kelly, ref. 173, p. 508. S. K. Malhotra and H. J. Ringold, J. Amer. Chem. SOC.,1965, 87, 3228. J. B. Jones and D. C. Wigfield, Canad. J . Chem., 1969,47,4459. J. B. Jones and D. C. Wigfield, J . Amer. Chem. SOC.,1967, 89, 5294. H . Weintraub, A. Alfsen, and E.-E. Baulieu, European J . Biochem., 1970, 12, 217.
Microbiological Reactions with Steroids
489
Attention has been called to the importance of substrate micelle formation in the dilute methanol solutions used for isomerase assay. Androst-5ene-3,17-dione (134)and cholest-5-en-3-one aggregated in aqueous systems such that their rates of isomerization were substantially reduced. 344 A systematic study of the isomerization of As-3-0x0-steroids bearing C-17 substituents of varying polarities has been made.345 The photoinactivation of the A5-isomerase has been examined. Irradiation of the enzyme bearing a 3-0x0-steroid substrate bound at the active site inactivates the enzymes uia intermediary excitation of the bound ketone followed by interaction between the excited 3-0x0-steroid and the enzyme.346 In conjunction with the 313-hydroxy-steroid oxidoreductase of Pseudornonas testosteroni the A5-3-0x0-steroid isomerase can operate in the reverse direction, converting the conjugated A4-3-ketone (71) into androst-5-ene-313,17P-diol (135).347 Conversion of the A‘-3-ketone (71) into the As-3-ketone (134) in the absence of 3P-hydroxy-steroid dehydrogenase did not occur. It was suggested that the shift of the A4-double bond to the As-position preceded 3-ketone reduction and that the double-bond isomerization was rate-limiti~~g.~~’
A5-Double-bond isomerases have been demonstrated in cultured cells from higher plants.266 6 Esterase, Amidase, and Hydrolase Reactions This section of the Report considers those enzymic modifications of steroids which involve esterification and de-esterification, amide hydrolysis, glycoside synthesis and hydrolysis, and methyl ether cleavage. Although esterase activity, particularly as regards hydrolysis of steroid esters, is widely reported for vegetative cell cultures of fungi, actinomycetes, and bacteria, little systematic study or use of microbial esterases has developed. Esterification of steroids by microbes is a relatively rare matter. Fatty-acid esters of cholest-5en-313-01 (123) and of other sterols have been reported formed by Mycobacteriurn
343 344 345
346 347
H. Weintraub, E.-E. Baulieu, and A. Alfsen, Biochim. Biophys. Acta, 1972, 258, 655. J. B. Jones and D. C. Wigfield, Canad. J. Chem., 1968,46, 1.459. J. B. Jones and K. D. Gordon, Biochemistry, 1973, 12, 71. R. J. Martyr and W. F. Benisek, Biochemistry, 1973, 12, 2172. M. G. Ward and L. L. Engel, J. Biol. Chem., 1966,241, 3154.
490
Terpenoids and Steroids
smegmatis in conjunction with 26-hydroxylation and A-ring dehydrogenations.’ 3 1 , 3 4 8 , 3 4 9 Esterification of cholest-5-ene-3P,7P-dioland 5P-cholestan-3/3-01 was less complete, whereas cholest-5-ene-3P,25-diol, 6P-hydroxycholest-4-en-3one, 3P-hydroxypregn-5-en-20-one, and 3P-bydroxyandrost-5-en-17-one were not esterified.’ Under certain conditions the mixed succinate esters of (123) and cholest-4-en-3P-01 and of (123) and cholesta- 1,4-dien-3P-ol were also formed. A 6p-methoxyacetate ester of 3P,5,6P,14a-tetrahydroxy-Sa-pregnane-l1,20dione was formed by the action of Curvularia lunata on 5,6a-epoxy-3P,14adihydroxy-5a-pregnane- 11,20-dione.’ The origin of the methoxyacetic acid was thought to be from oxidation during fermentation of the solvent 2-methoxyethanol used to dissolve the steroid substrate for addition to the transformaation system.’ Steroid alcohol esterifications have been reported for suspension cultures of callus tissues of the higher plants Nicotiana tabacum and Sophora angusti$olia. 3P-Palmitate esters of 3P-hydroxy-Sa-pregnan-2O-one and of 3P-hydroxypregn-5en-20-one were f ~ r m e dl 6. ~ De-esterification of steroid esters is frequently encountered in a variety of microbial fermentations. Several examples of adventitious de-esterification of 313- and 21-acetate esters are to be found in the Table of microbial hydroxylations. Deacetylation of 21-acetate esters in connection with 7a-,1lb-, 14a-,,and 15ahydroxylations by Curvularia lunata55~87~90” exemplify such esterase action. Ester hydrolysis also frequently occurs in conjunction with A’ -dehydrogenations. Deacetylation of 3P-, 17a, and 21-acetate estez of C19 and CZ1 steroids by Proactinomyces turbata, for example, is accompanied by hydroxysteroid dehydrogenations and A -dehydrogenations. Similar 38-hydroxysteroid dehydrogenations and A ‘-dehydrogenation have been reported for many Mycobacterium species acting on C19and CZ1~teroids.~” Esterase activity in association with 20-carbonyl reduction is also known, Bacillus megaterium, Bacillus mesentericus, and Bacillus subtilis being reported to convert 21-acetoxy-17a-hydroxypregn-4-ene-3,11,20-trione (136) into 17a,20P,21trihydroxypregn-4-ene-3,ll -trione (13 7).240
’
’
’
CH,OH
( 136) 348 349
35” I
( 137)
K. Schubert and G. Kaufmann, Biochim. Biophys. Acta, 1965, 106, 592. K. Schubert, G. Kaufmann, and C. Horhold, Abhandl. deutsi,h. Akud. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 47. I. I. Zaretskaya, L. M . Kogan, Zh. D. Sys, 0. B. Tikhomirova, G. K. Skryabin, and I. V. Torgov, Mikrobzologiyu, 1968, 37, 430. A. A. Akhrem and N. E. Voishvillo, Zzvest. Akad. Nuuk S . S . S .R . , Ser. biol., 1971, 7 6 8 .
Microbiological Reactions with Steroids
49 1
The microbial hydrolysis of the 21-acetate (136) has received considerable attention. Screening for 21-ester eserase activity among Mycobacteria has been r e p ~ r t e d . ~ ’Mycobacterium album elaborates an exoesterase active in hydrolysis of (136).353 Screening of many species of Actinomyces for esterase activity as regards hydrolysis of the 21-acetate ester (136) has been reported. Actinomyces pheochromogenes 3338 and Actinomyces flavus 3369 were preferred organisms for hydrolysis without associated 20-carbonyl group r e d ~ c t i o n5.4~The deacetylation of (136) by Actinomucor corymbosus 259 has a pH optimum near 7.5 and depends on the age of the culture. Neither characteristic was exhibited by Mycobacterium album 726, which also deacetylates (136).355The deacetylation of (136) by a variety of Mucor, Cunninghamella, Aspergillus, and Rhizopus species has been r e p ~ r t e d5 .6~The esterase of Sclerotinia libertiana, which converts (136) into the corresponding 21-alcohol, has been studied as a cell-free system.357 Some selectivity in ester hydrolysis may be encountered in certain cases. The 17p-acetate and 17P-propionate esters of (16) were hydrolysed negligibly, and the 17P-benzoate ester was not hydrolysed at Differential deacetylation of 20Pacetates by Bacillus megaterium in comparison with the epimeric 20a-acetates has been reported. Bacillus megaterium also hydrolyses 21-acetates more rapidly esterase from Bacillus megaterium which than 3p- or 1l a - a ~ e t a t e s A . ~cell-free ~~ hydrolyses steroid 3-, 17-, and 21-acetate esters but not llcr- or 17a-acetate esters has been described. The hydrolysis of 6a-fluoro-, 6a,9a-difluoro-, and 6a-fluoro-9a-chloro-l1~,21dihydroxypregna- 1,4-diene-3,20-dione 21-trimethylacetate esters to the corresponding 21-alcohols was achieved with several Corticium, Helminthosporiurn, and Curvularia species. Curvularia lunuta Schering strain No. 755 gave rapid conversions in 5 6 7 4 % yields.360 Saponification of the 21-succinate ester of 11~,17a,2l-trihydroxy-6a-methylpregna1,4-diene-3,20-dione by Bacillus pyocyaneus is indicated.361 Deacetylation of the P-D-monodigitoxoside diacetate and P-D-tridigitoxoside tetra-acetate of 14,15~-epoxy-3~-hydroxy-5~,14~-card20(22)-enolide by Absidia hyalospora and Cunninghamella echinulata respectively has been reported.362
352 3s3
3s4
356 357
358 59
3h0 36
362
K. A . Koshcheenko and G. K. Skryabin, Mikrobiologiya, 1966, 35, 1017. N. N. Lestrovaya, K. A. Koshcheenko, and G. B. Ksandopulo, Izuest. Akad. Nauk S.S:S.R., Ser. biol., 1969, 773. G . K. Skryabin, K. A. Koshcheenko, R. N. Meremkulova, and V. T. Sapleva, Priklad. Biokhim. i Mikrobiol., 1965, 1, 513. K. A. Koshcheenko and G. K. Skryabin, Izvest. Akad. Nauk S . S . S .R . , Ser. biol., 1967, 443. K. A. Koshcheenko, and G. K. Skryabin, Mikrobiologiya, 1965, 34, 252. G. B. Ksandopulo and S. V. Shilova, Izvest. Akad. Nauk S . S . S . R . , Ser. biol., 1972, 520. V. M. Ryzhkova, L. V. Sokolova, and N. N. Suvorov, Mikrobiologiya, 1965, 34, 407. N. N. Lestrovaya, L. M. Savko, and I. M. Tsfasman, Zzuest. Akad. Nauk S.S.S.R., Ser. biol., 1966, 301. H. Kosmol, F. Hill, U . Kerb, and K. Kieslich, Tetrahedron Letters, 1970, 641. W. Raab and J. Windisch, Arch. Klin. Exp. Dermatol., 1969, 235, 234. D. Satoh and M. Horie, Chem. and Pharm. Bull. (Japan), 1966, 14, 1 1 33.
Terpenoids and Steroids
492
A steroid esterase from Corynebacterium sp. NRRL B-3791 bound to porous glass retained its ability to hydrolyse the 17P-acetate ester of (16).363 Other enzymes modifying the substrate were also bound to the porous glass beads. Esterase activity towards 3P-acetoxy-substituted steroids by thermophyllic bacteria has been d e m ~ n s t r a t e d . ~ ~ Previous studies of microbial steroid esterases have been reviewed and assay methods described in An example of sulphate ester formation by microbial systems has been reported. Oestra-1,3,5(lO)-triene-3,17P-diol was converted by Aspergillus j a m s in 10 % yield into its 3-sulphate ester and in 60 % into the 3-sulphate ester of 3-hydroxyoestra- 1,3,5(lO)-trien-17-one (77).364 However, oestra- 1,3,5(lO)-triene-3,16a,17Ptrio1 was not esterified. The amide bond of bile acid conjugates with glycine or taurine is hydrolysed by mammalian intestinal microflora. Strict anaerobes are reported to hydrolyse both the glycine conjugates (138)and (1 39) of 3a,7a,l2a-trihydroxy-5P-cholan-24oic and 3a,l2a-dihydroxy-5~-cholan-24-oicacids as well as their taurine conjugates (140) and (141) and conjugates with alanine, aspartic acid, and t y r ~ s i n e .A~ ~ ~ H?
I
H (138) R = OH (139) R = H
(140) R = OH (141) R = H
hydrolase from CIostridium perjiingens which cleaves the amide bond of the glycine conjugates (138) and (139) has been partially purified. The enzyme was by inhibited by the substrate analogue 3,7,12-trioxo-5~-cholan-24-oylglycine, 3,7,12-trioxo-5~-cholan-24-oic acid, and by 3a,7a,l2a-trihydroxy-5P-cholan-2463 3h3a
364 365
M . J. Grove, G . W. Strandberg, and K . L. Smiley, Biotechnol. and Bioeng., 197 1,13,709. M. A. Rahim and C. J. Sih, ref. 189, pp. 675-684. K . Schubert and H. Groh, J . Steroid Biochem., 1971, 2, 387. M . J. Hill and B. S. Drasar, Biochem. J., 1967, 104, 55P.
Microbiological Reactions with Steroids
493
oic acid, which exhibited competitive product inhibition.366 Intestinal microorganisms hydrolysing bile acid conjugates in the human are also strict anaerob e ~ , and~ the ~ etiology ~ ~ of , cancer ~ ~ of~ the~ large bowel has been given speculative consideration in relation to microbial deconjugation and degradation reactions possibly leading to polycyclic hydrocarbon carcinogen^.^ 66c Bile acid conjugate hydrolases have been found in anaerobic faecal bacteria (Enterococcus, Clostridiurn, BiJidobacteriurn,and Bacteroides species)under anaerobic conditions at intracellular, wall-membrane, and extracellular sites.367 Some additional aspects of the microbiological modification of bile acid conjugates have been reviewed.368,369 Formation and hydrolysis of steroid glycosides by microbial enzymes has received very little attention. A glucosyltransferase system using uridine diphosphate glucose has been demonstrated in crude sonicated extracts of the yeast Candida bogoriensis, the product using cholest-5-en-3P-o1(123)as substrate being a cholest-5-en-3P-01 g l u c o ~ i d e . ~ ~ ' Examples of microbial hydrolysis of steroid glucosides include the hydrolysis of proscillaridin A, the 3P-~-rhamnosideof 3j?,14-dihydroxy-l4P-bufa-4,20,22trienolide, by a Penicilliurn species to the corresponding A4-3p-al~oho1.66Both glucoside and acetate ester groups were hydrolysed from lanoside C by mammalian intestinal mi~roflora.~"'Hydrolysis of the glycoside saponin of Dioscorea tokoro M. with Aspergillus terreus gave a 9&95 % recovery of spirost-5-en-3P-01 ( d i o ~ g e n i n ') . ~ ~ Microbial cleavage of the methyl ether bond of a variety of steroidal methyl enol ethers with concomitant reduction of the 3-keto-steroid initially formed has been reported. Several species of bacteria, fungi, yeasts, and actinomyces were effective in converting A2,5('0)-3-methoxyethers into product A'(' ')-3-alcoh ~ l s Fungal . ~ ~ enzymes ~ led to formation of o)-3cealcohols,whereas yeast enzymes generally afforded the epimeric A5(l')-3~-alcohols. The reaction presumably passes through the A5(")-3-ketone (143) as intermediate. Using preferred yeast strains oestr-5( lO)-diene-3/?,17P-diol (144) was prepared in 70 % yield from the A2~'-2-methylether (142) in 30 1 ferment01-s.~~~ 3-Methoxyoestra-2,5(10)-diene-16~,17fl-dio1, 2-methoxy-7cr-methyloestra2,5(10)-dien-17fl-01, 13P-ethyl-3-methoxygona-2,5( 10)-dien-17P-01, 3-methoxyoestra-2,5(10)-dien-1?'-one, and 3-methoxy-17a-methyloestra-2,5( lO)-dien-17/?-01 were all converted into their corresponding 8'~''~-3/l-a1coho1 derivatives, as was the 3-ethyl ether 3-ethoxyoestra-2,5(lO)-dien-17/l-01.~~~ Demethylation of P. P. Nair, M. Gordon, and J. Reback, J. Biol. Chem., 1967, 242, 7. 3 h h o B .S. Drasar, M. J. Hill, and M. Shiner, Lancet, 1966-1, 1237. "'*V. Aries, J. S. Crowther, B. S. Drasar, and M. J. Hill, Gut, 1969, 10, 575. 3 6 h c M. J. Hill, B. S. Drasar, V. Aries, J. S. Crowther, G. Hawksworth, and R. E. D. Williams, Lancet, 1971-1, 95. 3 h 7 V. Aries and M. J. Hill, Biochim. Biophys. Acta, 1970, 202, 526. 3 h 8 R. Lewis and S. Gorbach, Arch. Intern. Med., 1972, 130, 545. 3 6 9 T. F. Kellogg, Fed. Proc., 1971, 30, 1808. 3 7 0 T. W. Esders and R. J. Light, J. Biol. Chem., 1972, 247, 7494. 3 7 1 Y . Nagai, M . Sawai, and Y. Kurosawa, Nippon Nogei Kagaku Kaishi, 1970, 44,15. 3 7 2 K. Kieslich and H.-J. Koch, Chem. Ber., 1970, 103, 610. 3hh
Terpenoids and Steroids
494
(142)
3-methoxy-8-aza-~-homo-oestra-l,3,5( lO)-trien-17a-one ( 5 8 )by Aspergillus j a w s gave the corresponding 3-alcohol, and demethylation of the 8-aza-~-homo-3methyl ether (60) by Cunninghamella blakesleeana ATCC 8688b likewise gave the corresponding 3-alcoh01.~ 37 7 Reactions involving Heteroatoms Inasmuch as steroids bearing heteroatoms are of limited interest, their transformations by microbial agents have been few. Formation of sulphate or phosphate esters and transformations of bile acid conjugates are outside of the present interest but have been already reported in Section 6. Microbial transformations directly involving nitrogen, sulphur, and halogen atoms are reported herein. No recent examples of the introduction of nitrogen into steroid substrates such as was reported some time have appeared, but the deamination of conessine (145)concomitant with 1 1%-hydroxylationby Gloeosporiumfructigenum to give con-4-enin-3-one (146) and the 1la-hydroxy-derivative (147) has been reported.74 Me
Me
I
I
(1 45)
(146) R = H
(147) R
=
OH
’” L. L. Smith, M. Marx, H. Mendelsohn, T. Foell, and J. J. Goodman, J. Amer. Chem. SOC.,1962, 84, 1265.
Microbiological Reactions with Steroids
495
Incorporation of nitrogen into steroids partially degraded by micro-organisms has been reported and is covered in Section 8 of this Report. In analogy with prior results reported some time ago374 a stereospecific oxidation of a 7a-thiomethyl ether to but one of the possible epimeric sulphoxides has been reported. Oxidation of the thiomethyl ether (148) by Chaetomium cochloides QM 624 in association with 6P-hydroxylation give the sulphoxides (149) and (150).64"
0d 3 'SMe 0 o ?@o R
s,
Me
(149) R = H (150) R = OH
Microbial transformations dealing directly with halogens have now been described. The introduction both of bromine and of chlorine into unhalogenated steroids has been achieved, and the dehydrobromination of bromo-steroids has also been described. The mould Caldariornyces fumago ATCC 16373 has a powerful halogenoperoxidase system which can be used in cell-free transformations to halogenate certain steroidal P-diketones and olefins. Using the cell-free enzyme with KBr as source of halogen bromo-steroids were formed. With KCl, corresponding chloro-steroids were obtained. The P-diketone (15.1) was converted in 50 % yields into its corresponding 17a-bromo-derivative (152) and into its 17a-chloroderivative (153), as was ~-norpregn-4-ene-3,16,20-trione.~~~ The 13,17-secosteroid lactone (154) was converted into the 16a,16P-dibromo-derivative (155).
R =H (152) R = Br (153) R = C1 (151)
374
(154) R = H (155) R = Br
C. E. Holmlund, K. J. Sax, B. E. Nielsen, R. E. Hartman, R. H. Evans, and R. H. Blank, J. O r g . Chem., 1962,27, 1468.
496
Terpenoids and Steroids
17P- H ydroxy- 2 - h ydroxymethylene- 5a- androstan- 3-one, 178- hydroxy - 2hydroxymethylene-5P-androstan-3-one,and 17P-hydroxy-2-hydroxymethylene-
androst-4-en-3-one were converted into 2-bromo-derivatives with concomitant loss of the 2-formyl group. The process was recommended as a practical route to 2~-brom0-5P-androstan-3-ones.~~ Steroidal olefins also served as substrates of the halogenoperoxidase, pregna-4,9(1l)-diene-3,20-dione (156) giving 9a-bromo1lP-hydroxypregn-4-ene-3,20-dione (157) in 48 % yield.376 The enol ester (158) gave the 16a-bromo-ketone (159).377However, A5-steroids such as 3fi-hydroxy-
gave the corresponding pregn-5-en-20-one and 3P-acetoxypregn-5-en-20-one 5P,6P-epoxides, presumably following initial 5,6-bromohydrin formation.376 In that halogen ions undergo an overall two-electron reduction in forming the active species in the halogenoperoxidase reaction378 and an ionic electrophilic substitution reaction is indicated, these halogenations of selected steroids may not in fact involve a reaction between the steroid substrate and the microbial enzyme. Dehydrobromination of the 5a-bromo-steroid (160) occurred in
73
376 37’ 378
S. D. Levine, S. L. Neidleman, and M. A. Oberc, Tetrahedron, 1968, 24, 2979. S. L. Neidleman and S. D. Levine, Tetrahedron Letters, 1968, 4057. S. L. Neidleman and M. A. Oberc, J . Bacteriol., 1968, 95, 2424. , F. S. Brown and L. P. Hager, J . Amer. Chem. SOC.,1967,89, 719.
497
Microbiological Reactions with Steroids
conjunction with 1lcc-hydroxylation by Aspergillus ochruceus and 11P-hydroxylation by Curcularia lunata, which gave, for instance, the A4-3P-alcohol( 161).90*'l Z COCH,OH
,.--i-;:..'L 8 Steroid Degradation Reactions
The microbial degradation of steroids leading to their ultimate conversion into CO, and H,O involves many sequential reactions, several of which have now been elucidated. Work on steroid degradation has been stimulated by commercial interests in possible microbial conversions of sterols, sapogenins, bile acids, etc., into useful intermediates for steroid hormone analogue manufacture. 3 7 9 Several reviews of the microbial degradation of steroids have been p ~ b l i s h e d , ~ * ~ - ~ ~ ~ as has a review of the biochemistry of sterol degradation by Nocardia species.383 Depending on the steroid, microbial degradative attack may initiate at the terminus of the steroid side-chain or at the nuclear A-ring (or @+rings) site. In the absence of a C-17 side-chain, attack in the D-ring and/or in the A/B-ring system is recognized. The Report is presented in the order: A-ring reactions, B-ring reactions. C-17 side-chain reactions, and D-ring reactions. Steroids, particularly cholest-5-en-3P-o1(123), may serve as sole carbon sources for micro-organisms. General oxidation of (123) in such cases has been reported for various Nocardiu and Streptomyces species: for Fzisarium diversi~porum,~*'for Actinomyces streptomycini strain 2195,385for Mycobucterium rubrum,20 6 , 2 0 ? , 3 8 6 , 38 ? and for several Pseudomonas species.388-390 Screening operations have shown that numerous species of Actinomyces and Mycobacteria can utilize bile acids and sterols as sole carbon sources.391
'
379
380 381
382
383 384
385 386
387
388 389
390 39'
R. Wiechert, Angew. Chem., 1970, 82, 331; Angew. Chem. Internat. Edn., 1970, 9, 321. K. Schubert, 2. Chem., 1967, 7 , 289. C. Horhold, K.-H. Bohme, and K. Schubert, 2. aflg. Mikrobiol., 1969, 9, 235. K. Schubert, C. Horhold, K.-H. Bohme, H. Groh, F. Ritter, and W. Schumann, Steroidologia, 1970, 1, 201. R. L. Raymond and V. W. Jamison, Adv. Appl. Microbiol., 1971, 14, 93. R. G. Strobel, H. Quinn, and W. Lange, Canad. J. Microbiol., 1967, 13, 121. A. I. Zhukova and V. Kh. Kozlava, Mikrobiologiya, 1968, 37, 223. A. A. Imshchenetskii, E. F. Efimochkina, V. A. Zanin, and L. E. Nikitin, Mikrobioligiya, 1968, 37,31. A. A. Imshchenetskii and L. A. Mavrina, Mikrobiologiya, 1972, 41, 399. V. Kh. Kozlova and N. A. Fonina, Mikrobiofogiya, 1971, 40, 275. A. A. Imshchenetskii and L. A. Mavrina, Mikrobiologiya, 1972,41, 598. V. Kh. Kozlova and N. A. Fonina, Mikrobiologiya, 1972. 41, 602. L. 0. Severina, D. Sys, and G. S. Lenskaya, Mikrobiologiya, 1967, 36, 435.
498
Terpenoids and Steroids
Cell-free extracts of Mycobacterium sp 297 and lyophilized cells retained the ability to decompose c h o l e s t e r 0 1 . ~ ~ ~ * ~ ~ ~ The A-ring is quite commonly the initial point of degradative attack. A5-3PHydroxy-steroids are dehydrogenated to A4- and A' -4-3-oxo-steroids as mentioned in Section 3 of this Report. Cholest-4-en-3-one (124) has been recognized as an early product in cultures of organisms known for their ability to degrade Powerful esterases may act on sterol 3-esters sterols totally.4',202,206-208,214,393 in this connection. In the case of Mycobacterium mucosum A5-3P-esters were converted into A ',4-3-ketones more rapidly than the corresponding A5-3,!lalcohols.2l4 Substrate selectivity may be encountered in certain cases : the Penicillium sp. A- 16 which oxidized cholest-5-en-3P-o1(123)and cholest-4-en-3~-01to cholest4-en-3-one ( 124) and 5cx-cholestan-3P-01 to 5a-cholestan-3-one was unable to dehydrogenate 5a-cholestan-3a-01, 58-cholestan-3a-01, or 5P-cholestan-3P-01 ( 125).202 A second oxidative mode of degradation of the A-ring is 19-hydroxylation (or other oxidation). 19-Hydroxylation in conjunction with A'-dehydrogenation results in cleavage of the C-19 carbon atom and aromatization of the A-ring. The strict anaerobes Escherichiu coli EBC 417 and Clostridium paraputr$cum CC 493 aromatize C, A4-3-ketones.394 Androsta-l,4,7-triene-3,17-dione (162) was aromatized to 3-hydroxyoestra-1,3,5(10),7-tetraen-17-one(163)by several Actinomycetales and Eubacteriales. However, androsta- 1,4-diene-3,17-dione(126) was not a r ~ m a t i z e d An . ~ ~agar-plate ~ method for rapid screening of micro-organisms performing A-ring aromatization has been described.396
Aromatiration of 19-hydroxy-A4~'-3-ketoneshas been examined as a suitable means of synthesis of (1 63). The conversion of 19-hydroxyandrosta-4,7-diene3,17-dione (164) into (163) by Nocurdia restrictus partially poisoned with potassium cyanide397or with Nocardia r ~ b r led a ~to~yields ~ of (163) as high as 40% at 1 g 1- ' substrate A'-Dehydrogenation of 19-hydroxypregn-4-ene3y2
3'J3
3')4 3q5
3y6 3y7 3y8
A. A. Irnshchenetskii, E. F. Efimochkina, L. E. Nikitin, and V. A. Zanin, Doklady Akad.
Nauk S . S . S . R . , 1965, 161, 701. A. A. Irnshchenetskii, E. F. Efimochkina, L. E. Nikitin, and T. S. Nazarova, Doklady Akad. Nauk S . S . S .R . . 1966, 170, 960. P. Goddard and M. J . Hill, Biochim. Biophys. Acta, 1972, 280, 336. D. Kluepfel and C . Vezina, Appl. Microbiol., 1970, 20, 5 1 5 . C . Vezina, K. Singh, and S . N. Sehgal, Appl. Microbiol., 1969, 18, 270. J. F. Bagli, P. F. Morand, K. Wiesner, and R. Gaudry, Tetrahedron Letters, 1964, 387. S. N. Sehgal and C . Vezina, Appl. Microbiol., 1970, 20, 875.
Microbiological Reactions with Steroids
499
3,20-dione (165), 3~-acetoxy-19-hydroxypregn-5-en-20-one, and 3B-acetoxypregn-5-ene-19,20P-diol by Nocardia species was accompanied in each case by A-ring aromatization to give (166).399 Aromatization of the A-ring of 19hydroxylated steroids in conjunction with side-chain degradations to give (77) will be covered in a later section of this Report. The general topic of aromatization of steroids has been extensively reviewed.14*400
&OMe
Ho#coMe
0
Aromatization of steroids is not confined to the A-ring, the aromatization of the w i n g of A5.7-sterolsby Acantharnoeba castellanii (Neff) having been reported. The sterols (167)--(170) were transformed by homogenates of the amoeba to the corresponding B-ring aromatic derivatives (171H174) in which the ZOP-methyl group migrated to the 6- or 7 - p o ~ i t i o n . ~ ~ ’
Me (167) R = Me, A’’ (168) R = Et, A2’ (169) R = Me (170) R = Et
399 400
401
(171) R = Me,A2’ (172) R = Et, AZ2 (173) R = Me (174) R = Et
K. Singh, D. J. Marshall, and C. Vezina, Appl. Microbiol., 1970, 20, 23. K. Schubert, in ‘Biosynthesis of Aromatic Compounds’, ed. G . Billek, PergamonMacmillan, Oxford, Vol. 3, 1968, pp. 81-89. E. D. Korn, A. G. Ulsamer, R. R. Weihing, M. G. Wetzel, and P. L. Wright, Biochim. Biophys. Acta, 1969, 187, 555.
Terpenoids and Steroids
500
A-Ring aromatization and associated B-ring scission are regularly encountered in further degradation of steroids. Arornatization of the A-ring of 9cr-hydroxy19-nor-A4-3-ketones or of 9a,l9-dihydroxy-A4-3-ketones may be accompanied by scission of the 9,lO-bond as well. The 9a-hydroxy-19-nor-A4-3-ketone (100) with either vegetative cells or acetone-dried cells of Arthrobacter simplex gave both the A-ring-aromatic 3,ga-diol (31) and also the 9,lO-seco-steroid (101).85 9r,I9-Dihydroxyandrost-4-ene-3,17-dione (1 75) was converted by Nocardia
restrictus ATCC 14887 in the presence of phenazine methosulphate into the 9,lO-seco-phenol ( 176)."02 Degradation of androst-4-ene-3,17-dione(71) and androsta-1,4-diene-3,17dione (126) involves the same reaction sequences of A-ring aromatization and 9,lO-bond scission. The degradation is considered to involve associated A'dehydrogenation and 9x-hydroxylation steps, with the 9,lO-seco-steroid (177) as product. Although the 9.10-seco-9,17-dione (177) is the usual intermediate of degradation by Nocardia restrictus, the 9,l O-seco-9~-alcohol(178) as well as
(176) R (177) R
= =
CH,OH Me
(178)
( I 77) was obtained with 17[&hydroxyandrosta-4,6-dien-3-0ne( 1 7) as substrate.322 Degradation of androst-4-ene-3,11,17-trione by Proacrinomyces ruber gave the 9,IO-seco-derivative( 1 79).40"
( 179)
jo3
C . J. Sih, S. S. Lee. Y . Y . Tsong. and K . C. Wang, J . Amer. Chem. Soc., 1965, 87, 1385. A . Capek, 0. HanE, and M. Tadra, N u t i i r w o s . , 1967, 54, 70.
Microbiological Reactions with Steroids
501
Scission of the A-ring without aromatization or wring scission has also been reported. Eburicoic acid (180) was degraded by Glomerella fusarioides ATCC 9552 to the 3,4-seco-diacid (181), which exhibited antibacterial activity as the methyl ether.404 CO,H I
Scission of the A-ring m’oiety of the aromatic 9,lO-seco-steroid (177), with formation of the hexahydroindanedione (182), representing the c- and D-rings, and of low molecular weight compounds which enter the genera1 metabolism of the micro-organism, is the next phase of degradation. Studies with Nocardia restrictus ATCC 14887, Pseudornonas testosteroni ATCC 11996, Bacterium cyclooxydans ATCC 12673, Mycobacterium rhodochrous ATCC 13808, and Nocardia corallina ATCC 13259 support these generalizations and suggest that the next degradation step is 4-hydroxylation of the 9,lO-seco-steroid (177) to give the catechol( 183).222*402 The chemical and physical properties of the 4,5-dioxygenase which cleaves (177)have been de~cribed,~” as have kinetic
Additional indication of the importance of 4-hydroxylation in the degradative metabolism of steroids is given by the aromatization of the 2-metho~y-A’>~-3ketone (184) by Nocardia corallina ATCC 13259 to give the 4-hydroxylated 404
405 ‘Oh
A. I. Laskin, J. Fried, C. de L. Meyers, and P. Grabowich, Bacteriol. Proc., 1964, 25; A. I. Laskin, P. Grabowich, C. de L. Meyers, and J. Fried, J . Medicin. Chem., 1964, 7 , 406. H. H . Tai and C. J. Sih, J . Biof. Chem., 1970, 245, 5062. H. H. Tai and C. J. Sih. J . Biol. Chem., 1970,245, 5072.
Terpenoids and Steroids
502
9,10-seco-steroid (1 85)."02 Incubation of the 4-hydroxy-A4-3-ketone (I 86) with frozen cells of Nocurdia restrictus in the presence of phenazine methosulphate gave the 4-hydroxylated 9.1 O-seco-steroid (183).
(184)
0
OH
OH (185)
(186)
An alternative degradation pathway for A-ring-aromatic steroids does not involve scission of the 9,lO-bond or the intermediate (101). 3-Hydroxyoestra1,3,5(IO)-trien-17-one (77) was oxidized by Nocardia sp. (E110) to give the three metabolites (187), (188), and (189). In that 3,4-dihydroxyoestra-l,3,5(lO)-trien17-one (190) was also oxidized but 2,3-dihydroxyoestra- 1,3,5(lO)-trien-l7-one was not. it was suggested that the degradation of (77) proceeded by 4-hydroxylation Ljiu (190) to the bis-seco-steroid ( 187) with loss of the C-4 carbon 2tom. Further degradation of (187) led to (188). The third metabolite, the 4-aza-steroid (189),
(187) R = CH=CHCH,CO,H (188) R = CH,COMe
(189)
503
Microbiological React ions with Steroids
was considered to be formed in a non-enzymic side-reaction between an initially formed intermediate and ammonia.407 The 4-hydroxy-9,10-seco-steroid(183) was readily degraded by cell-free extracts of Nocardia restrictus via the 4,5 :9, 10-bis-seco-derivative408to the hexahydroindane derivative (182).2227402,409Treatment of the 4,5 :9,lO-bisseco-steroid intermediate (191) with ammonia gave ( 192),408the 9,lO-secoanalogue of the 4-aza-steroid (189) recovered from fermentation of (77) with Nocardia sp. (El
The remaining six carbon atoms of the A-ring were retained as 2-oxohex-cis-4enoic acid (193), whose hydration product (194) was further metabolized to propionic a c d (199, and pyruvic acid (196),which entered the general metabolism HO C0,H
1
yH2 Me
y 2 H
co 1
Me
of the o r g a n i ~ m . 0~t ~ her~ experiments ,~~~ with Pseudornonas testosteroni ATCC 11996 established the same points. Extracts of Pseudomonus testosteroni which oxidized [4-I4C]-(71) to CO, also accumulated [1-l4C]-DL-alanine (197) and [ 1-14C]-~-2-aminohex-cis-4-enoic acid (198) in the presence of ethylenediaminetetra-acetic acid.410 2-Oxohex-cis-4-enoic acid (193) is efficiently converted into (197) and (198) by Pseudomonas testosteroni and appears to be their common CO, H
I
THNH, I
Me (197)
H,N*CO,H (198)
40 7
R.G . Coombe, Y. Y. Tsong, P. B. Hamilton, and C. J. Sih, J . Biol. Chem., 1966, 241,
408
D. T . Gibson, K . C. Wang, C. J. Sih, and H. W. Whitlock, J . Biol. Chem., 1966, 241,
1587. 409
410
551. C. J. Sih, K. C. Wang, D. T. Gibson, and H. W. Whitlock, J . Amer. Chem. Soc., 1965, 87, 1386. D. A . Shaw, L. F. Borkenhagen, and P. Talalay, Proc. Nut. Acad. Sci. U.S.A., 1965,54, 837.
Terpenoids and Steroids
504
p r e c ~ r s o r .'~ ' Transamination of the 0x0-acid (193) and of [l-'4C]pyruvate (196)formed from (194) via an aldolase action would afford the observed products (198) and (197) respectively. It may be presumed that the pathway (71) 4(126) -+(177) +(183) *(182) is a general one for many steroids. In this formulation the C-17 side-chain and other c- or D-ring substitution remains unaltered whereas the A- and B-rings are degraded. Nocardia species degrade several sterol derivatives without degradation of the side-chain features. Cholest-5-ene-3/?,25-diol(l99)fermented with Nocardia opaca gave the degraded product (201) with the side-chain intact. Similarly, the 3P-acetate of the 27-nor-ketone (200) was degraded to (202).412
(199) R = CMe,OH (200) R = COMe
(201) R = CMe,OH (202) R = COMe
?*
I
Degradation of 3a,7a,l2a-trihydroxy-5/?-cholan-24-oic acid (203) by Streptomyces rubescens gave the hexahydroindane derivative, (204) with the side-chain i n t a ~ t .3*4 ~ 'l 4 In this instance several further degraded derivatives were formed which also incorporated nitrogen into the degraded fragments. Thus, the amide (205) and the lactams (206) and (207) retaining the C-17 side-chain were formed, together with the lactams (208), (209), and (210) degraded in the sidehai in.^'^.^'^ The 17-0x0-lactam (210) could be formed from (182) by Streptomyces rubescens. It was uncertain whether the other lactams (206)-(209) found in the fermentations were formed en~ymatically.~ l4 The dicarboxylic acid (204)
412
413
414
A. W. Coulter and P. Talalay, Biochem. Biophys. Res. Comm., 1967, 29, 413; J. Biol. Chem., 1968,243, 3238. C. J. Sih, H. H. Tai, and Y. Y. Tsong, J. Amer. Chem. Soc., 1967, 89, 1957; C. J. Sih, H. H. Tai, S. S. Lee, and R. G . Coombe, Biochemistry, 1968, 7, 808. S. Hayakawa, S. Hashimoto, T. Fujiwara, and T . Onaka, Abhandl. deutsch. Akad. Wiss. Berlin, Klasse Med., 1968, No. 2, p. 63. S. Hayakawa, S. Hasimoto, and T. Onaka, Lipids, 1969, 4, 224.
Microbiological Reactions with Steroids
I
R
H O Z C d (204) k (205) R
505
= =
OH NH,
(206) R (207) R (208) R (209) R
= CH,CHzCOzH = CH,CH,CONH, = COMe = COzH
HN
was also obtained from the C,, acid (203) fermented with Corynebacteriurn (Arthrobacter) Corynebacteriurn equi converted (204) into aminoacid conjugate^.^' Pregn-4-ene-3,20-dione (21l), 17a-hydroxypregn-4-ene-3,20-dione (212), 1 7 ~ acetoxypregn-4-ene-3,2Odione (213), and 21-hydroxypregn-4-ene-3,20-dione (214) were transformed by Nocardiu opaca SG 98 into the corresponding degraded derivatives (216)-(219) with their side-chains intact.41 1la-Hydroxypregn-4ene-3,20-dione (215 ) was transformed by Proactinomyces ruber into (220).,03
'
COCH 2R I
#--Rz
R3*J33 0
- -R2
HO,C,/
0
(211) R' = RZ = R3 = H (212) R' = R3 = H, R2 = OH (213) R' = R3 = H, R2 = OCOMe (214) R' = OH, RZ = R3 = H (215) R' = R2 = H, R3 = OH 415
COCH,R'
(216) R' = RZ = R3 = H (217) R' = R3 = H, RZ = OH (218) R' = R3 = H, RZ = OCOMe (219) R' = OH, R2 = R3 = H (220) R' = RZ = H, R3 = OH
S. Hayakawa, Y. Kanematsu, and T. Fujiwara, Nature, 1967, 214, 5 2 0 ; Biochem. J., 1969, 115, 249.
416
417
S . Hayakawa, T. Fujiwara, and H. Tsuchikawa, Nature, 1968, 219, 1160. K. Schubert, K.-H. Bohrne, F. Ritter, and C. Horhold, Biochim. Biophys. Acra, 1968, 152, 401.
506
Terpenoids and Steroids
Degradation of (211) and the epimeric pregn-5-ene-3/?,20-diols(221) and (222) by a Nocardia species to their corresponding hexahydroindane derivatives (216), (223), and (224) has been reported.418 Degradation of the 3/3,20/3-diol(222)was stimulated by calcium ions.4'
Me
Me +C-R2
R',
I
HO2C
HO (221) R' = H, R2 = O H (222) R' = OH, R2 = H
R',
oaC-R2
I
4
(223) R' (224) R'
= =
H, R2 = O H OH, R2 = H
Further degradation of the perhydroindane derivatives (182)and (216)retaining the c- and D-ring features of the parent steroids has been reported. The C1, fragment (216) was reduced by Mycobacteriurn srnegrnatis SG 98 to a variety of alcohols (225)-(230) formed by reduction of both ketone groups and of the carboxylic By contrast (182) was oxidized by Nocardia opaca to aoxoglutaric acid (231) and succinic acid (232).417 * 4 2 1 Me
R3\ 1
C-R4
M eoJ - - JJ IR R2'
(225) R' = H, R2 = O H (226) R' = OH, R2 = H
418
419
420 421
(227) R' = R3 = H, R2 = R4 = O H (228) R' = R4 = H, R2 = R3 = OH (229) R' = R3 = OH, R2 = R4 = H (230) R' = R4 = OH, R2 = R3 = H
A. Strijewski, T.-L. Tan, G. Bozler, W. Zahn, and F. Wagner, 2. physiol. Chem., 1972,353, 1440. T.-L. Tan, A. Strijewski, and F. Wagner, Arch. Mikrobiol., 1972, 87, 249. K. Schubert, K.-H. Bohme, and C. Horhold, Biochim. Biophys. Acra, 1965, 111, 529. K. Schubert, K.-H. Bohme, and C. Horhold, Acfu Biol. Med. Ger., 1967, 18, 295; Abhandl. deursch. Akad. Wiss. Berlin, Klasse Med., 1968, NO.2, p. 81.
507
Microbiological Reactions with Steroih
The 17-ketone degradation fragment (182) was metabolized by Nocardia corallina to (233) and (234) by oxidoreductases. Pseudomonas testosteroni acting on (233) gave the lactone (235).422 Studies with additional hexahydroindanepropionic acid derivatives suggested that the next step in the degradation of (182) was via P-oxidation of the propionic acid side-chain to give the hexahydroindane-4-carboxylic acid (236), which was then further degraded by scission of the six-membered ring.422
HO,C,/ (234)
Tetrahydroindane derivatives have also been obtained in certain cases from microbial degradation of (211). Mycobacterium smegmtis gave the alcohol (237),42 whereas Nocardia rhodochrous 7022 gave the acid (238).41
(237) R = C H 2 0 H (238) R = C 0 2 H
Further degradation of the hexahydroindanedione (182) by Streptomyces acid (239) reprerubescens afforded ( + )-(5R)-methyl-4-oxo-octane-1,8-dioic senting the C-9 and C - 1 1 4 - 1 7 carbon atoms of the original steroid C- and Me
422 423
S. S. Lee and C. J. Sih, Biochemistry, 1967, 6, 1395. K. Schubert, K.-H. Bohme, and C. Horhold, Acfu Biol. Med. Ger., 1967, 18, 291.
508
Terpenoids and Steroids
rings.^^^ The same C9 acid (239) was recovered in fermentations of the parent bile -acid (203) with Arthrobacter simplex, but the acid was not optically active, probably having been racemized during isolation.425 Degradation of steroids may also be initiated at points in the C-17 side-chains and C-3a positions (C-17 and C-14 by steroid D-ring numbering) to [1-14C]succinic acid (232) was obtained in Nocardia opaca fermentation^.^^^ Formation of [l-'4C]succinic acid from labelled (238) is reconciled with scission of the 1,7a- and 3a,4-bonds (13,17- and 8,14-bonds by steroid numbering). Degradation of steroids may also be initiated at points in the C-17 side-chains independently of alterations which may occur in the A-ring. Degradation of the sterol side-chain may begin by attack at the terminal isopropyl group by 26hydroxylation as has been demonstrated for several Mycobacterium species recognized as degrading s t e r ~ l s . ~ ~3~2 ' H ~ owever, ~-' Nocardia species fail to 27-norcholest-5-en-3P-01, attack the side-chains of cholest-5-ene-3P,25-diol(199), 3~-hydroxy-27-norcholest-5-en-24-one, or the 3P-acetate of the 27-nor-25ketone (200).412 Degradation reactions of the sterol side-chain appear to involve conventional fatty-acid oxidation pathways, one equivalent of propionic acid (195) being derived from the terminal*isopropyl group in the formation of C24 acids and one equivalent of acetic acid being derived from the C-23 and C-24 atoms in the formation of CZz Oxidations by Nocardia restrictus of [26,27-14C](123) gave propionic acid (195) labelled only at C-1 and C-3.41 Incubation of [4-14C]-(123) with Nocardia restrictus in the presence of 3-oxochol-4-enic acid (74) and o-phenanthroline led to recovery of 14C-labelled (74), thus suggesting that some (74) was formed from (123).412 The ultimate reaction in side-chain cleavage is transformation to the 17-ketone. Degradation of the 19-hydroxy-A4-3-ketone (240) by Nocardia species gave the four acids (242)-(245) as products, clearly demonstrating C,,-acid formation both with and without attendant A-ring a r ~ m a t i z a t i o n .The ~~~ 6j3,lPepoxy-acid (246) was degraded by Nocardia restrictus to the bisnorcholenic acid (247) and the 17-ketone (248).412 Tracer experiments suggested that the C3 side-chain of bisnorcholenic acid substrates was cleaved to propionic acid and to a 17-0x0steroid.427 The stepwise cleavage of the sterol side-chain via C24 and/or C,, acids to a 17-0x0-steroid is further exemplified by the action of Nocardia restrictus ATCC 14887 and Nocardia corallina ATCC 13259 on the seco-nor-steroid (249). The seco-nor-dicarboxylic acid (250), the 17P-alcohol (25l), and the 17-ketone (252) were also
42'
S. Hayakawa and S. Hashimoto, Biochem. J., 1969, 112, 127. S. Hayakawa and T. Fujiwara, F.E.B.S. Letters, 1969, 4, 288. K . Schubert, F. Ritter, T. Sorkina, K.-H. Bohme, and C. Horhold, J . Steroid Biochem., 1969, 1, 1 . C. J. Sih, K. C. Wang, and H. H. Tai, J . Amer. Chem. SOC.,1967,89, 1956; Biochemistry,
428
1968,7, 796. G. Lefebvre, H. Matringe, M. Maugras, and R. Gay, Compt. rend., 1968,266, D, 1196.
424
425 426
Microbiological Reactions with Steroids
509
(240) R = H (241) R = Et
&
HO,C
(249)
H
0
2
(250) C
G
H
Terpenoids and Steroids
510
Stepwise degradation of the sterol side-chain of (123) to C22intermediates of lower oxidation state by Mycobacterium sp. NRRL B-3683 and NRRL B-3805 is implied by the isolation of the C,, 22-alcohol (253) along with (71) and (126).429 Cleavage of the C-20-C-22 bond of 6p,1lcr,22-trihydroxy-23,24-bisnorchol-4en-3-one by Rhizopus arrhizus to provide 6P,1lcr-dihydroxypregn-4-ene-3,20dione has also been
Complete scission of the sterol side-chain with 17-ketone formation but without or B-ring degradation has been achieved in several ways. Selection of microorganism has received much attention, and many organisms previously mentioned in Section 3 of this Report cleave the sterol side-chain of (123) in conjunction with A-ring dehydrogenation but with no other d e g r a d a t i ~ n . ~ ' , ~"' ~Pilot-~ plant (2501) conversion of (123) into (71) and (126) has been achieved using Mycobacterium sp. NRRL B-3683.429 In other cases inhibition of nuclear degradation by the addition of specific inhibitors has permitted recovery of (126) from sterols. Screening of Mycobacterium species inhibited by 8-hydroxyquinoline resulted in selection of a Mycobacterium phlei strain for conversion of the sterol (123) into the A'74-3,17dione (126).205 Mycobacteriurn phlei inhibited by 8-hydroxyquinoline also acted on C2', C 2 8 ,and C29 steroids to give (126).204 The organism also converted 4~,5-epoxy-5~-cholestan-3-one (254) and 4P,S-epoxy-5#3-pregnane-3,20-dione (255) into (126).297 A-
(254) R = C,H1, (255) R = COMe
Nuclear degradation by species of Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptornyces was inhibited by a,a-bipyridyl and by arsenite ion,209.2 and cleavage of the side-chain without nuclear degradation was achieved with (123) as substrate. 429
W. J. Marsheck, S. Kraychy, and R. D. Muir, Appl. Microbiol., 1972, 23,72.
Microbiological Reactions with Steroids
511
The nuclear degradation of cholest-5-en-3P-o1(123)by Mycobacterium species is inhibited by Ni2+,Co2+,Pb2+,and Se032- ions.430 A similar inhibition of a Mycococcus species by Ni2+ has been as has the inhibition of Proactinomyces asteroides 438 by Co2 ions.21 Nuclear degradations of (123) by Arthrobacter simplex IAM 1660 were inhibited by chelating agents, by a variety of heavy-metal ions, and by redox dyes, with accumulation of the A ' T ~ - ~ ketone (126).432Side-chain cleavage of 19-norcholesta-1,3,5(lO)-trien-3-01 (256) by Nocardia restrictus ATCC 14887gives in 8 % yield the 17-ketone(77)dire~tly.4~ Degradation of the side-chain only of the 3~,5a-cyclosteroids(257), (258), and (259) by a Mycococcus species has been reported. The product was the 3a75acyclosteroid 17-ketone (260).431 +
I
OH (257) R = H (258) R = Et
(259) R
=
Et, AZ2
Degradation of the sapogenin side-chain to give (126) is also reported. Mycobacterium phlei inhibited with 8-hydroxyquinoline converted (25R)-spirost-5-en3P-01(261)into (25R)-spirost-4-en-3-oneand (25R)-spirosta-1,4-dien-3-one(262) but also into the 17-ketones (71) and (126).212 Sapogenins of both the (25R)and (25S)-serieswere also degraded :(25R)-Sa-spirostan-3P-o1,(25S)-Sa-spirostan3p-01, (25R)-S/?-spirostan-3/3-01,and (25S)-SP-spirostan-3P-olwere all converted into (126) in 2.5-6.5 % yields.212 However, (25R)-spirost-4-en-3-one was degraded by Fusarium solani No. 101 in 65 % yield to androsta-1,4-diene-3,16430
431
432
433
W. F. Van der Waard, J. Doodewaard, J. de Flines, and C. Van der Weele, Abhandl. deutsch. Akad. Wiss.Berlin, Klasse Med., 1968, No. 2, p. 101. A. F. Marx, J. Doodewaard, W. F. Van der Waard, and J. de Flines, Rev. SOC.quim. MPxico, 1969, 13, 72A. M. Nagasawa, N . Watanabe, H. Hashiba, M. Murakami, M. Bae, G . Tamura, and K. Arima, Agric. and Biol. Chem. (Japan), 1970, 34, 838. A. Afonso, H. L. Herzog, C. Federbush, and W. Charney, Steroids, 1966, 7, 429.
Terpenoids and Steroids
512
dione (126) with 16a- and 16~-hydroxyandrosta-1,4-dien-3-one each formed in 5 % yield.21 These same 16-oxygenated products were also obtained from (261) but in lower yields. From fermentations using a variety of other sapogenins and 16,20-dioxygenatedpregnane derivatives the course of degradation of (261) was suggested to proceed via (262)-(267).21
0
0
(265) R = COCH,CH,CHMe, (266) R = H
Microbiological Reactions with Steroids
513
Fermentation of (25R)-spirost-4-en-3-one with Verticilliurn theobromae or (268) and with Stachylidiurn bicolor afforded 20cr-hydroxypregn-4-ene-3,16-dione 16-one (269).434 3a,ll P,20cr-trihydroxy-Sc-pregnan-
The ease of side-chain cleavage by Arthrobacter simplex IAM 1660 appeared to diminish with increasing length for Czl to C24 to C27steroids.435 The action of Arthrobacter simplex IAM 1660 on (123) gave, in addition to the expected A4-3-ketone (71) and A's4-3-ketone (126) degradation products, the additional products (16), 17P-hydroxyandrosta-1,4-dien-3-one, 17/?-hydroxy-5/?-androst-len-3-one, and 5P-androstane-3~t,l7fi-diol.~~~ Microbial degradations of bile acids include both minor alterations in the A-, B-, or c-rings and also side-chain scission. Degradation by Mycobacteriurn rnucosum 1210involvesmainly 3a-, 7a-,and 12a-hydroxy-steroiddehydrogenations coupled with A4-dehydrogenation and side-chain scissions. The A4-3-ketone acids (270) and (271) are among the degradation products of the common bile
(270) R = CH2CH2C02H (271) R = C 0 2 H
acid (203).437*4 Further alterations include 12a-hydroxy-steroid dehydrogenations to give the 12-ketones (272)-(276) and transformations to give the 434
435
436
437
E. Kondo and T. Mitsugi, Tetrahedron, 1973, 29, 823. M. Nagasawa, N. Watanabe, H. Hashiba, G. Tamura, and K. Arima, Agric. and Biol. Chem. (Japan), 1970,34, 798. M. Nagasawa, H. Hashiba, N. Watanabe, M. Bae, G. Tamura, and K. Arima, Agric. and Biol. Chem. (Japan), 1970, 34, 801. L. 0. Severina, I. V. Torgov, and G. K. Skryabin, Doklady Akad. Nauk S.S.S.R., 1968, 181,488.
438
L. 0. Severina, I. V. Torgov, G. K. Skryabin, N. S. Wulfson, V. I. Zaretskii, and I. B. Papernaja, Tetrahedron, 1969, 25, 485.
Terpenoids and Steroids
514
(272) R = CH2CH,C02H (273) R = C 0 2 H
0
I
Ly?+02H
0
0+co2H (274)
(275)
(277) R = CH2CH2C02H (278) R = C 0 2 H
B-ring-unsaturated derivatives (274)-(278).439*440 Formation of the A8derivative (275) is unprecedented, but the formation of the A6-derivatives(274), (277), and (278) may represent 7a-hydroxyl dehydrations, whereas formation of the 7-hydro~y-A~>~-3-ketone (276) probably represents 7a-hydroxy-steroid dehydrogenation with concomitant enolization. A cell-free preparation from Mycobacterium rnucosum 1210 performing these transformations has been described.441 Degradation of 3a, 12a-dihydroxy-5P-cholan-24-icacid by Mycobacteriurn rnucosurn 1210 gave the bisnorcholenic acids (279) and (280).274.275The 43q
440
44'
L. 0. Severina, I . V. Torgov, and G. K. Skryabin, Doklady Akad. Nauk S.S.S.R., 1967, 173, 1200; Izuest. Akad. Nauk S . S . S . R . , Ser. biol., 1968, 912; Doklady Akad. Nauk S.S.S.R., 1968, 181, 488. L. 0. Severina, I. V. Torgov, G. K . Skryabin, N. S. Wulfson, V. 1. Zaretskii, and 1. B. Papernaja, Tetrahedron, 1968, 24, 2145. S. M. Shust, L. 0. Severina, and E. L. Ruban, Izoest. Akad. Nauk S . S . S . R . , Ser. biol., 1973. 267.
Microbiological Reactions with Steroids
515
0
I
0' (279) R (280) R
= =
H OH
metabolism of bile salts and acids by mammalian intestinal microflora has been discussed in several monograph^.^^^.^^^ The microbial degradation of the side-chain of C,, steroids such as (211) has also received attention even through C2 steroids are not attractive raw materials for commercial syntheses. Degradation occurs by at least two distinct pathways, one giving the corresponding C l g 17-ketone directly as product, the other giving a 17P-acetoxy-steroid in an enzymic reaction analogous to the Baeyer-Villiger chemical oxidation. From the 17P-acetoxy-product esterase and 17P-hydroxysteroid dehydrogenase actions may then give the 17-ketone as product. Scission of the 17,20-bond of C2 20-ketones to give Clg 17-ketonesbut without nuclear degradation is regularly encountered. Minor nuclear alterations may co-occur, such as A'-dehydrogenation by Fusariurn solani acting on (211) to give Likewise, side-chain cleavage to the 17p(126) in yields exceeding acetoxy- or 17P-alcohol products may be accompanied by minor nuclear alterations, such as lla-hydroxylations by Beauueria bassiana strain 66358 or by Aspergillus ochraceus NRRL 405,'64 and 1% and 16a-hydroxylations by Fusarium a r g i l l a c e ~ m . ~ Chromatographic ~ methods designed specifically for monitoring the microbial degradation of the 17p-acetyl side-chain of (211) have been reported.445 The mechanism by which the 17P-acetyl side-chain is cleaved to a C19 17ketone directly does not appear to have been clarified. However, the enzymic version of the Baeyer-Villiger oxidation has received considerable attention. ,~~~ Degradation of the 3,20-diketone (211) by Aspergillus J E a ~ u s Aspergillus 64 Cladosporium h e r b ~ r u m , ~ Cladosporium ~' resiochraceus NRRL 405,' nae,448*449Cylindrocarpon radicicola, Septomyxa ~ f J i n i s , ~and ~ ' Streptomyces "9
442 443
444
445
446 447
448
449
450
'
G. A. D. Haslewood, 'Bile Salts', Methuen, London, 1967, pp. 50-58. H. Van Belle, 'Cholesterol, Bile Acids and Atherosclerosis', North-Holland Publishing Co., Amsterdam, 1965, pp. 84-89. I. Belie, E. Pertot, and H. SoeiE, Mikrobiologiya, 1968, 5, 127; Mycopathol. Mycol. Applicata, 1969, 38, 225. H. SoEiE and I. Belie, Z . analyt. Chem., 1968, 243, 291. K. Carlstrom, Acta Chem. Scand., 1967, 21, 1297. P. H. Cox and B. A. Sewell, J . SOC.Cosmetic Chemists, 1968, 19, 461. H. Nakano, H. Sato, and B.-I. Tamaoki, Biochim. Biophys. Acta, 1968, 164, 585; Steroids, 1968, 12, 291. H. Nakano, C. Takemoto, H. Sato, and B. Tamaoki, ref. 329, pp. 252 291. K. Singh and S. Rakhit, Biochim. Biophys. Acta, 1967, 144, 139.
Terpenoids and Steroids
516
grise~s~ ~’ proceeds via such a mechanism to give the 17P-acetate (281) as initial product.
16) Support for the mechanism formulated includes the report that [17B-’ 80]-( was formed in incubations of (211) with Cladosporium resinae under an I8O2 atmo~phere.~~ Furthermore, ~,~~’ the action of Septomyxa afinis ATCC 6737 spores on [17a-*H]-(211) yielded [17a-2H]-(16).450 The enzymes involved in the transformation of (211) to (16) by Cylindrocarpon radiciola ATCC 11011 have been resolved and characterized as a steroid-inducible oxygenase utilizing molecular oxygen and NADPH and an e~terase.~” The scission of the 17P-acetyl side-chain of (211) via the Baeyer-Villiger-type mechanism by Aspergillus ochraceus NRRL 405 demonstrated for vegetative cells’ 6 4 has also been demonstrated for cell-free preparations of the organism.150 The side-chain cleavage reaction was the predominant one observed in incubations of cell-free preparations. Spores of Aspergillus ochraceus NRRL 405 degrade pregna4,16-diene-3,20-dione (282) to 1la-hydroxyandrost-4-ene-3,17dione (283), which conversion is viewed as having passed through the putative Baeyer-Villiger-type intermediate enol acetate (284).” Degradation of the 16a,17a-epoxy-20-ketone (285) by Fusarium solmi spores was likewise thought
’
45’
452
K. Carlstrom, Acta Chem. Scand., 1966, 20, 2620. M. A. Rahim and C. J. Sih, J . Biol. Chem., 1966, 241, 3615.
517
Microbiological Reactions with Steroids
to pass through the 17P-acetate and 17P-alcohol intermediates (286)and (287) to the a-ketol (288).239The degradation enzymes of Fusarium solani spores appear to be bound to or imbedded in the plasma membrane.29s
(286) R = COMe (287) R = H
Whereas the degradation of 21-hydroxypregn-4-ene-3,20-dione(214) by CIadosporium herbarurn, yielding the 17P-acetate (28l), is claimed to proceed via the Baeyer-Villiger-type mechanism, the requisite initial 17P-hydroxyacetyl intermediate (289)was not observed. However, only chromatographic evidence was adduced, and several other prominent products of the degradation were not identified.447 OCOCH20H
0
Steroids of the pregnane series with more complicated structures may also be cleaved without major nuclear alterations to CI9 ketones. The 1lp,l8-epoxysteroid (290)was oxidized by Corynespora cassiicola IMI 56007 at the C-18 position to give (291)and (292)and was 9a-hydroxylated and degraded to the
(290) R = H
(291) R
=
OH
9a-hydroxy- 17-ketone (293). 118,17a,21-Trihydroxypregn-4-ene-3,20-dione (294)was transformed by Cladosporium herbarurn to 1lp-hydroxyandrost-4-ene3,17-dione (295).447
518
Terpenoids and Steroids
(293)
COCH,OH
-0
-0
(295)
('94)
Side-chain cleavage to C, 9- 17 ketones has been observed for synthetic steroids of altered nuclear shape also. 9P, 10a-Pregn-4-ene-3,20-dione (296) and its 1% hydroxylated derivative (297) were transformed by Helicosporium lumbricopsis into the corresponding 9P,lOa-17-ketones (300) and (301).124 Mastigosporiurn heterosporurn also cleaved (296) to the 17-ketone (300).12' 9-Hydroxy-9fi,lOapregn-4-ene-3,20-dione(298), on the other hand, was transformed by Gliocladium roseurn into the 17P-acetate (302) and the 9P,17/?-diol (303).84 1la-Hydroxy9P, lOa-pregn-4-ene-3,20-dione (299)was transformed by Sporomina pollaccii into the corresponding 17p-acetate (304) and 17P-alcohol (305) derivative^.'^
(296) (297) (298) (299)
R' R' R' R'
= R2 = R3 = H = R3 = H, R2 = OH = OH, R2 = R3 = H = R2 = H, R3 = OH
(302) (303) (304) (305)
(300) R = H (301) R = OH
= OH, p 2 = H, R3 = COMe R' = OH, R2 = R3 = H R' = H, R2 = OH, R3 = COMe R' = R3 = H, R2 = OH
R'
Microbiological Reactions with Steroids
519
Pregn-4-ene-3,20-dione (211) induces an enzyme for its own degradation to the 17-ketone (71) by Penicilliurn lilacinurn NRRL 895.453 The mechanism by which (211) is degraded remains obscure despite attentions paid to the fermentation. No Baeyer-Villiger-type product 17P-acetate (281) was detected even in the presence of di-isopropyl fluorophosphate to inhibit esterase action. The 17-ketone (71) was produced nonetheless.446 A pathway involving initial 1 7 ~ h y d r ~ x y l a t i o ndoes ~ ~ ~ not appear to operate.453 More recently, use of the experiments in which di-isopropyl fluorophosphate had been used as an inhibitor of the microbial esterase as an argument about the mechanism of side-chain cleavage446 has been criticized on the basis that the esterase of Penicilliurn lilacinurn was probably insensitive to the inhibitor.454 The epimeric 20-hydroxypregn-4-en-3-one derivatives (306) and (307) formed by 20-hydroxy-steroid oxidoreductases of Penicilliurn lilacinurn NRRL 895 acting on the 20-ketone (211) are both degraded by the organism. The 20P-alcohol (307) was demonstrably dehydrogenated back to the 20-ketone (211) prior to degradation to the 17-ketone (71),but the 2Oa-alcohol(306)was degraded directly to the 17-ketone (71) without prior dehydrogenation to (211).453 Different fermentation kinetics were also observed for the epimeric 20-alcohols (306) and (307), and two distinct modes of degradation were A cell-free extract of Penicilliurn lilacinurn NRRL 895 has been prepared which converts the 20-ketone (211) into the epimeric 20-alcohols (306) and (307) and into the 178-
(306) R' (307) R'
= =
H, R2 = OH OH, RZ = H
alcohol (16), 17-ketone (71), and D-ring lactone (98) degradation Treatment of D-ring lactone formation is deferred until after complete discussion of selective side-chain and A-ring degradations. An equally complicated system, which includes epimeric 20-alcohols as well as degraded CI9 derivatives, was obtained in the incubation of Fusariurn solani spores on the 16a,l7a-epoxy20-ketone (285).2 The degradation of steroids by Cladosporiurn resinae could be changed by control of aeration. Under aerated conditions (211) was transformed to the Baeyer-Villiger-type product 17P-acetate (28l), but under partially anaerobic 453
454 4s5
K. Carlstrom, Actb Chem. Scand., 1970, 24, 1759. T. L. Miller, Biochim. Biophys. Acta, 1972, 270, 167. K. Carlstrom, Actu Chem. Scand., 1972, 26, 1718.
520
Terpenoids and Steroids
conditions the 20a-alcohol (306) was obtained instead, with no side-chain cleavage.448 Degradative metabolism of the epimeric 20-alcohols (306) and (307) by Septomyxa afinis ATCC 6737 spores was of two sorts. The 20b-alcohol (307) was A’-dehydrogenated to (308) but not degraded. However, the 20a-alcohol (306) was both A‘-dehydrogenated and degraded to the A1*4-3,17-dione(126) and to the A‘-lactone (309).4’0 It was concluded that the degradation of the 20a-alcohol (306) by spores of Septornyxa afinis proceeded by a mechanism different from that involved in the degradation of the 20-ketone (21l).450 Me
Degradation processes may take place on both the A- and wrings and on the side-chain at the same time, with formation of a variety of doubly degraded steroid derivatives. The most common pattern includes A-ring aromatization with Cz2-and Cz4-acid formation, A-ring aromatization with 17-ketone formation [a sought-after process to obtain (77) and (163)], and A-ring aromatization with 9,lO-bond scission and 17-ketone formation. Aromatization of the A-ring and C,, acid formation by Nocardia species has been mentioned already in the conversion of (240) into (243), (244), and (245).427 A-Ring aromatization and 17-ketone formation is exemplified in the transformation of 19-hydroxypregn-4ene-3,20-dione (165) by Septomyxu ufinis to (77).399 The controlled degradation of the sterol side-chain together with A-ring aromatization to give (77) in good yield has been achieved with 19-hydroxylated (240) gave substrates and Nocardia restrictus. 19-Hydroxycholest-4-en-3-one 30% yields of (77),456and the 3P-acetate of (311) gave (77) in 72% yield.457 Conversion of other 19-oxygenated derivatives, including the 6Q,19-epoxide (312) was also reported.457 Screening of numerous Mycobacterium species known to aromatize (76) for their ability to degrade the sterol side-chain and form (77) from the 19-hydroxy-steroid substrates (240), (241), (310), and (311) and from the 6fl,19-epoxy-steroids (312) and (313) has been reported. Mycobacterium phlei W-32 and Mycobacterium Jlavescens D-50 were preferred organi s m ~ . ~ ’ Screening of Proactinomyces and Mycobacterium species for the 45h
C. J . Sih and K. C. Wang, J. Amer. Chem. SOC.,1965,87, 1387.
4s8
1965,87, 2765. E. Denot, C. Casas-Campillo, and P. Crabbe, European J . Sreroids, 1967, 2, 495.
‘” C. J. Sih, S. S. Lee, Y. Y. Tsong, K. C. Wang, and F. N. Chang, J. Amer. Chem. SOC.,
521
Microbiological Reactions with Steroids
(310) R = H (311) R = Et
(312) R (313) R
=H = Et
transformation of the 3P-acetate of (310)into (77) led to Proactinomyces asteroides 438 and Mycobacterium sp. 202 as best producers.459 The A7-17-ketone(163) was likewise formed by the action of Mycobacterium sp. (RMTP) on 19-hydroxycholesta-4,7-dien-3-one (314). By contrast Nocardia sp. ATCC 19170 transformed (314) into (77) with reduction of the A'-double bonds46o
Degradation of the side-chain, A-ring aromatization, and scission of the B-ring is a commonly encountered process. Cholest-5-en-3P-01 (123) was cleaved by Mycobacterium smegmatis to the 9,lO-seco-steroid(1 77),461previously discussed in connection with the degradation of the A'-4-3,17-dione(126). Degradation of the D-ring may occur as previously reported as part of the metabolism of the hydroindanediones (182) and (238) but also as a process entirely separate from degradations involving the A-, B-, and c-rings. 178Alcohols and 17-ketones and steroids which can be degraded to 17P-alcohols and/or 17-ketones are oxidized enzymatically in a process analogous to the chemical Baeyer-Villiger reaction so as to cleave the 13,17-bond and yield the D-ring lactone (98). Examples of D-ring lactone formation include the transformation of the 17ketone (71) into the lactone (98) by Aspergillus tamarii.' 17P-Hydroxyandrost4-en-3-one (16) was transformed by Aspergillus JEavus into the D-ring lactone (98) in 40 % yield.5 4 Cylindrocarpon radicicola, Fusarium solani, and Septornyxa afJinis acting on (16) gave the A'-dehydrogenated lactone (309).'59 The 19-norA4-3-ketones (315), (316), and (317) were oxidized to the corresponding D-ring 459
460 461
Zh. D. Lebedeva, 0. B. Tikhomirova, L. M. Kogan, I. I. Zaretskii, G . K. Skryabin, and I. V. Torgov, Izuesr. Akad. Nauk S . S . S . R . , Ser. bid., 1970, 781. R. Deghenghi, S. Rakhit, K. Singh, C. Vezina, and C. J. Sih, Steroids, 1967, 10, 313. K. Schubert, H. Groh, and C. Horhold, Naturwiss., 1965, 52, 20.
522
Terpenoids and Steroids
lactones (97), (318), and (319) by Aspergillus tararnii. 1lp-Hydroxylation cooccurred with (315) or (97) as substrate to give (319).1'5,462In the case of the
SH
R '.
0 ' (315) R' = RZ = H (316) R' = Me, RZ = H (317) R' = H, R2 = OH
(318) R' = Me, R2 = H (319) R' = H, R2 = OH
17fl-hydroxy-17cc-methylderivative (91) fermentation with Arthrobacter simplex or with Mycobacteriumjavurn gave the lactol (320).298
H
Among reports of lactone formation from C, substrates are the transformations of the 20-ketone (211) into the D-ring lactone (98) by AspergillusJis~heri,'~ by Aspergillus tamarii,' ' by a Penicilliurn species,' by Penicillium chrysog e n ~ r n ,and ~ ~ by Penicilliurn lilacinurn cell-free extract^.^' Septomyxa afinis transformed (211) and the 2Oa-alcohol(306) into the A'-lactone (309),450,454and Aspergillus tarnarii converted 5a-pregnane-3,20-dione (321) into the saturated lactone (322).53 16cc,l7a-Epoxypregn-4-ene-3,20-dione (285) was oxidized by
'
'
Cylindrocarpon radicicola, Fusarium solani, or Septomyxa a@nis to the 16ahydroxy-A'-lactone (323).'59 11-Hydroxylated C, and C , steroids were not substrates for D-ring lactone formation with Aspergillus tarnarii. 3,
''
462
R. D. Garett and J. T. McCurdy, J . Medicin. Chem., 1968, 11, 194.
Microbiological Reactions with Steroids
523
The mechanism by which Septomyxa affinis degrades the C2 20-ketone (211) to the D-ring lactone (98) has been viewed as a process in which the same enzyme functions twice, first to convert (211) into the 17b-acetate (281), which is then hydrolysed to the 17/3-alcoholand dehydrogenated, and secondly by lactonization of the 17-ketone (71). Both reactions in which a carbon+arbon bond is cleaved are formally analogous to the chemical Baeyer-Villiger oxidation.454 Furthermore, the degradation of the 17-ketone (71) to the lactone (98) is inhibited by the 20-ketone (21 l), the substrate for the initial degradation reaction. Indeed, the 20-ketone (211) appeared to be the preferred substrate for the degradation to the lactone (98) rather than the 17-ketone (71).454 Ring-D lactone formation and concomitant degradation of the A- and wrings also has been observed. The A1l4-3,17-dione(126) was converted into the lactone carboxylic acid (324) by Aspergillus f l a t ~ u s The . ~ ~ same ~ lactone (324) was also obtained by the action of Nocardia restrictus on the lactone (98) as
HO,C 'J (324)
9 Miscellaneous Microbial Reactions In this section several microbial reactions of steroids will be reported which do not fall properly into one of the earlier categories but which are regularly observed. Among these miscellaneous reactions are epoxidations and epoxide transformations, dehydrations of alcohols to olefins, reductive removal of hydroxy- and carbonyl groups, epimerizations and inversions of stereochemical centres, and allylic rearrangements and methyl migrations. Epoxidation of olefinic double bonds is a well-known transformation, but relatively few have been reported recently. Epoxide formation with associated A-ring dehydrogenation of (325) by Corynebacterium simplex ATCC 6946 has been reported, the A' p4-9a,11a-Epoxide (326) being formed.464 The action of K. Schubert, K.-H. Bohme, F. Ritter, and C. Hiirhold, J . Steroid Biochem., 1971, 2, 245. C. Corelli, D. Kluepfel, and P. Sensi, Experienfia, 1964, 20, 208.
lh3
4h4
&
Terpenoids and Steroids
524
COCH,OCOMe H
& o
:'OH
Me
n 1
Me /
H
Rhizopus nigricans on the ~-nor-A'-steroid(1) gave the 5a,6a-epoxide(2b) among the A few epoxide transformations have also been reported. The 4a,5a- and 4P,5Pepoxides of several C,,, C, and C , , 3-ketones were transformed by Mycobacterium phlei into the corresponding A4- and A '*4-3-ketones.297The B-nor5a76a-epoxide(2b)obtained from the ~-hor-d'-steroid(1)by the action of Rhizopus nigricans was also hydrated to give the 5&6a-diol (2a).42 Both the 5a,6a-epoxide (24) and its 5fi,6P-epimer were transformed into the 6a- and 6P-hydroxy-A4-3ketones (25) and (86) respectively.' 9 7 The 5a,6a : 16a,17a-bisepoxide(327) was transformed by Curuularia lunata in prolonged fermentations into the 5a-6-ketones (328)and (329). The 5a,6a-epoxide
11,20-dione was opened and ring of 5,6a-epoxy-3P,14a-dihydroxy-5a-pregnaneconverted into the 6P-methoxyacetate ester of 3P75,6P,14a-tetrahydroxy-5apregnane-11,2O-dione. The 5a,6~-epoxidering of 5,6a-epoxy-3B-hydroxy-5apregnan-20-one was completely reductively removed by Curuularia lunata, giving 7a,14a-dihydroxy-5a-pregnane-3,20-dione, 11P, 14a-dihydroxy-5a-pregnane-3,20dione, and 14a-hydroxy-5a-pregnane-3,11,20-trione.
'
525
Microbiological Reactions with Steroids
Dehydration of alcohols to olefins has been reported in several cases. Dehydration of the epimeric 3-alcohols (330)and (331)to the A3,5-derivative(4)by Rhizopus arrhizus Fischer or by Rhizopus nigricans Ehrenberg occurred when the culture medium became acid (pH < 4.5).66 Similarly, the 7P-hydroxy-group of (332)
(330) R' = OH, R2 = H (331) R' = H, R2 = OH
(332)
was dehydrated to the A4v6-3-ketone(5) in acid medium conditions by the same Rhizopus species.66 By contrast dehydration of the 7P-hydroxy-A4-3-ketone(87) by Mycobacterium flavum occurred at neutral pH to give the A476-3-ketone(88). A'-Dehydrogenation occurred also to give the A'34-3-ketone(333)from substrate (87). The A4,6-3-ketone(88) could be further A'-dehydrogenated to the A1*4,6derivative (334).296 Notably, the 7P,lla-diacetate of (87) was not attacked by
(333)
.
(334)
Mycobacteriurn flavurn even on prolonged fermentation.296 A cell-free extract of Saccharornyces cerevisiae dehydrated the A7-3P,5a-diol(335) to the A597-3/3alcohol (336).465
465
R. W. Tophan and J. L. Gaylor, Biochem. Biophys. Res.
Comm., 1972,47, 180.
526
Terpenoids and Steroids
Dehydration of the 9a-hydroxy-phenol (31) by Arthrobacter simplex acetonedried cells gave the A9(' ')-product 3-hydroxyoestra-l,3,5(10),9(1l)-tetraen-17-one. Although spontaneous dehydration also occurred, it was concluded that an enzyme catalysed the dehydration in incubations of dried cells.85 Dehydration of the 16a-hydroxy-group of (337) to a AI6-derivative by mammalian intestinal microflora is suggested to account for loss of the hydroxy-group and subsequent formation of 17a-pregnane derivatives.323
(337)
The reductive removal of the 7a-hydroxy-group from the common bile acid (203) is accomplished by strictly anaerobic mammalian intestinal and faecal microflora. The 7-deoxy-steroid (338) is formed. 7a-Dehydroxylation does not occur with the bile acid conjugates (138) or (140). Several screening operations seeking intestinal micro-organisms which 7a-dehydroxylate (203) have been reported.365,466,467 The reductive removal of the 7a-hydroxy-group has been reported for Bacteroides (Zuberella) strain 28s from human faeces,468for a mixed culture of human faecal m i c r o - o r g a n i ~ m s for , ~ ~an ~ obligate anaerobe ,~~ isolated from rabbit faeces,47ofor members of the tribe L a c t ~ b a c i l l e a e1474 for the type cultures Clostridium bifermentans ATCC 9714, Clostridium biferrnentus NCIB 506, and Clostridium sordeEZii NCIB 6929,475and, for the first time, for cell-free preparations of Clostridium welchii, Eschereschia coli, and Streptococcus faecalis.220 7cc-Dehydroxylation of [24-l4C]-(2O3) was demonstrated in germ-free rats whose intestinal tract had been contaminated with a member of the tribe Lactobacilleae which 7a-dehydroxylated (203) in in uitro incubations.474 Neomycin administration reduced the amount of 7a-dehydroxylation which occurred in uiuo.470,476 Strictly anaerobic intestinal micro-organisms also 7a4hh
4h7
468 4h9
470 471
472
473 474
475 4i6
M . J. Hill and B. S. Drasar, Gut, 1968, 9, 22. T. Midtvedt and A. Norman, Acta Pathol. Microbiol. Scand., 1968, 72, 337. T. Hattori and S. Hayakawa, Microbios, 1969, 3, 287. L. Canonica, A. Ferrari, B. Rindone, G . RUSSO,and C. Scolastico, Ann. Chim. (Italy), 1971, 61, 695. V. Bokkenheuser, T. Hoshita, and E. H. Mosbach, J. Lipid Res., 1969, 10, 421. T. Midtvedt, Acta Pathol. Microbiol. Scand., 1967, 71, 147. T. Midtvedt and A. Norman, Acta Pathol. Microbiol. Scand., 1967, 71, 629. T. Midtvedt and A. Norman, Acta Pathol. Microbiol. Scand., 1968, 7 2 , 313. B. E. Gustafsson, T. Midtvedt, and A. Norman, Acta Pathol. Microbiol. Scand., 1968, 72, 433. S. Hayakawa and T. Hattori, F.E.B.S. Letters, 1970, 6 , 131. E. H . Mosbach, V. Bokkenheuser, A. F. Hofmann, T. Hoshita, and S. Frost, Circulation, 1967, 35/36, Suppl. 11, 29.
527
Microbiological Reactions with Steroids
dehydroxylate 3a,7a-dihydroxy-5~-cholan-24-oic acid (339) to (340)4719473*477 and 3a,7a,l2a-trihydroxy-5a-cholan-24-oic acid (341) to (342).470 Likwise 78acid (343) to (338) by dehydroxylation of 3a,7~,12cr-trihydroxy-5~-cholan-24-oic a mixed culture of human faecal micro-organisms has been reported.469 Much of the earlier work dealing with the microbial conversion of (203) into the 7deoxy-derivative(338) has been r e ~ i e w e d . ~ ’ ~ * ~ ’ * The enzymes which are responsible for 7a-dehydroxylation of (203) appear to be inducible.220 7a-Dehydroxylation has been viewed as involving sequential dehydration to the A6-steroid (344) and A6-reduction. However, no recent evidence for this mechanism has been reported. The requisite A6-steroid (344) could not be detected when sought in incubations of (203) with Bacteroides (Zuberella) strain 28S.468
uo2H
HO’*
HO” @02H
H
(338) R’ = H, R2 = OH (339) R’ = OH, R2 = H (340) R’ = R2 = H
HO**=02H H
’R
(341)R (342)
H (343)
ri
2
HO** 0 :
R
= =
OH H
H (344)
Reductive removal of the 12a-hydroxy-group by strict anaerobes has also been reported.365 Formation of the degradation fragment (204) from the bile Conversion of the 3flY16a-diol(337) acid (203) implies a 12~t-dehydroxylation.~~~ by rat caecal microflora to a variety of 16-deoxy-steroidsestablished the reductive removal of the 16a-hydroxy-group also. In this case, the formation of an intermediate A’ 6-derivative was suggested, with subsequent reduction giving both 17a- and 17gpregnane derivatives as products.32 Reductive removal of the 477
478
B. E. Gustafsson, T. Midtvedt, and A. Norman, J . Exp. Med., 1966, 123,413. H. Danielsson and T. T. Tchen, in ‘Metabolic Pathways’, ed. D. M. Greenberg, 3rd E X . , Academic Press, New York, Vol. 2, 1968, p. 11 7.
Terpenoids and Steroids
528
21-hydroxy-group of 3P,21-dihydroxy-Sa-pregnan-20-one by rat caecal microflora, yielding 3P-hydroxy-Sa-pregnan-20-one and Sa-pregnane-3P,20,21-triols, has also been Reductive removal of the 7-ketone function of 3P-hydroxyandrost-S-ene-7,17dione (345)by Mycobacteriurn srnegrnatis has been reported. Among the products are the A4-3,17-dione (71), the A',4-3,17-dione (126), and 5a-androst-l-ene-3,17which may be an artifact, however.480 dione, as well as androsta-3,5-dien-7-one,
Epimerization of 3-alcohols by micro-organisms has been reported. The A4-3a-alcohol (331) was reversibly epimerized to (330) by Rhizopus arrhizus Fischer or by Rhizopus nigricans Ehrenberg when fermentation conditions became acid (pH < 5.0).66Mammalian intestinal microflora epimerize the 38hydroxy-group of 3P,16a-dihydroxy-5a-pregnan-20-one(337) to give the 3aalcohols (346) and (347), among other products.32 The epimerization involves a mixed culture of micro-organisms and probably occurs by dehydrogenation and subsequent 3a-reduction. Likewise, formation of the 3a-hydroxy-6-ketone (329) from the 5a,6a-epoxy-3P-alcohol (327) by Curvularia Iunata probably proceeds via the 3,6-diketone (328)which was also a product ofthe fermentation.' l9 Epimerization of the 17P-acetyl side-chain of (337) occurs, and the 16a-hydroxygroup is also removed. The reaction sequence probably involves 16a-hydroxy dehydration to a putative AI6-intermediate which is then reduced to give the 17a-pregnane derivatives (346) and (348) as well as the epimeric 17P-steroids (347) and (349).323
(346) R' (347) R'
= =
H, RZ = COMe COMe, R2 = H
(348) R' = H, RZ = COMe (349) &' = COMe, R2 = H
Inversion of the stereochemistry at C-5 has been demonstrated for the metaacid (338) by intestinal microflora bolism of 3a,l2a-dihydroxy-SP-cholan-24-i~ of the rat. The SP-cholanic acid (338) was converted into the Sa-isomer during 479 480
H. Eriksson, J.-A. Gustafsson, and J. Sjovall, European J . Biochem., 1969, 9, 550. K. Schubert and K . Wehrberger, Narurwiss., 1965, 52, 431.
529
Microbiological Reactions with Steroids
entero-hepatic circulation in the rat, most probably by intestinal m i ~ r o f l o r a . ~ ~ The reverse isomerization of the Sa-acid (342) to the 5P-acid (338) was demonstated following intracaecal administration of the steroid to the rat.482 A few rearrangements of steroids have been reported for microbial fermentations. Both the 3a- and 3P-alcohols (331) and (330) respectively were rearranged to the A3-5/?-alcohol(350) in fermentations involving Rhizopus species in which acidic conditions prevailed.66 Fermentation of the 5a-bromo-6P-fluoro-steroid (160) with Curuularia lunata gave the A4-118-alcohol (161) which underwent Westphalen-LettrC rearrangement in acidified culture medium to give the A'(' I ) - 10P-alcohol (351). Fermentation of (160) with Aspergillus ochraceus, presumably via the A4-l la-alcohol intermediate (352), gave the A9-l la-alcohol (353).90
3. COCH,OH
HO
+*
Me
F
COCH,OH
'Me
HO
COCH,OH
Me
HO*-*
F
(352)
F
(353)
A few microbial reactions of steroid hydroperoxides and peroxides have been reported. Peroxidase action of Aspergillus ochraceus N R R L 405 on the 17ahydroperoxide (354) gave the corresponding 17a-alcohol which was then 1lahydroxylated to the 1la,l7a-diol (355).l o 3 The 5ct,8a-peroxide (356) was
(354) R' = OH,R2 = H (355) R' = H,R2 = OH 4B1 482
A. Kallner, Acta Chem. Scand., 1967, 21, 87. A. Kallner, Acra Chem. Scand., 1967, 21, 315.
5 30
Terpenoids and Steroids
isomerized by Mycobacterium crystallophagurn to the isomeric epoxy-diols 5,6aepoxy-5a-ergosta-8,22-diene-3P,7a-diol(357) and 5,6a-epoxy-5a-ergosta-8(14),22diene-3B77a-diol(358). The A8-derivative (357) is also a major thermal decomposition product of the 5a,8a-peroxide (356).48 The 5a,8a-peroxides (356)and (360). (359) were transformed by Penicilfium rubrum into the A4*6*8('4)-3-ketone The same product was obtained using ergosta-5,7,22-trien-3fi-01(336) and 5a,8aergosta-6,22-diene-3P,5,8-trioI (361) as substrates.484 A cell-free extract of
HO
j ,% '\
0/
(356)
(358)
HO
0 -'
'OH
(357)
(359)
Succharornyces cereuisiae transformed the 5a78a-peroxide (356) into the AsV73P-alcohol (336).465
483 484
K. Petzoldt and K. Kieslich, Annufen, 1969, 724,194. J. D. White and S. I. Taylor, J . Amer. Chem. Soc., 1970, 92, 581 I .
3 Steroid Conformations from X - Ray Analysis Data ~
BY C. ROMERS, C. ALTONA, H. J. C. JACOBS, AND R. A. G. DE GRAAFF
1 Introduction
A'-Ray diffraction analysis of steroids started in the early thirties with the work of Bernal,' who examined six steroids, including cholesterol, ergosterol, and calciferol. From unit-cell dimensions and space-group considerations he arrived at the conclusion that these molecules are elongated and have dimensions of about 4, 7, and 20 A. This view led to the proposal of the perhydro-l,2-~yclopentanophenanthrene ring system associated with the names of Rosenheim and King2 and Wieland and Dane.3 An extended survey of unit-cell dimensions of some eighty steroids by Bernal, Crowfoot, and Fankuchen4 confirmed this conclusion in 1940. In this survey the identified' exceptions were precalciferol and calciferol, which had different structures and dimensions. These early diffraction studies and later investigations around 1950 into the crystal structures of cholesteryl iodide6 and the 4-iodo-3-nitrobenzoyl esters of calciferol' and lumisterol* by Crowfoot et al. provided highly important information on the overall molecular structure and the position of functional groups. They did not, however, offer a detailed quantitative description in terms of bond lengths and valency angles-not to mention torsion angles, a concept that was not introduced into the crystallographic literature until 19649710and which received wider attention only some years later.' Generally, the introduction of heavy atoms to overcome the phase problem and a lack of computing facilities for the acquisition of accurate data meant that the geometrical information could not be other than crude. The use of digital computers and evaluation of complete three-dimensional photographic data by '7"
J. D. Bernal, Nature, 1932, 129, 277. 0. Rosenheim and H. King, Chem. and Ind., 1932, 51, 464. H. Wieland and E. Dane, 2. physiol. Chem., 1932, 210, 268. J . D. Bernal, D . Crowfoot, and I. Fankuchen, Phil. Trans., 1940, A239, 135. D. Crowfoot and J . D . Bernal, Chem. Weekbl., 1937, 34, 19. ' C. H. Carlisle and D . Crowfoot, Proc. Roy. SOC.,1944, A184,64. ' D. C. Hodgkin, B, M . Rimmer, J . D . Dunitz, and K. N. Trueblood, J . Chem. Sac., 1963,977,4945. * D . C. Hodgkin and D . Sayre, J . Chem. SOC.,1952,4561. ' H . J . Geise, Thesis, Leiden, 1964. l o C. Altona, Thesis, Leiden, 1964. I H. J. Geise, C. Altona, and C. Romers, Tetrahedron, 1967, 23, 439. ' * C. Altona, H. J . Geise, and C. Romers, Tetrahedron, 1968, 24, 13.
53 1
532
Terpenoids and Steroids
Romers and co-workers' 3-'5 substantially added to accuracy, resulting in standard deviations of 0.02 8, for carbon-carbon bond distances. However, the first truly accurate structure determinations of steroids without heavy atoms were accomplished in 1965 by Huber and HoppeI6 and in 1966 by High and Kraut, who claimed standard deviations of ca. 0.006A for bond distances in androsterone.' Simultaneously the first successful attempts, by Bucourt and Hainaut,' 8 * 1 to predict conformational features of steroids by simple force-field calculations of cyclohexanes became available and induced Altona, Geise, and Romers' to carry out a comparative study of eleven steroids. The results of these considerations concerning the overall shape, i.e. the convex bending of the carbon skeleton towards the r-side, the mean staggering (ca.56") of the A, B, and c rings, and the conformational decription of ring D in terms of a phase angle of pseudorotation, A, and maximum torsion angle, @ ,, were promising, but no quantitative treatment of the bond distances could be given. A very large number of X-ray structure determinations of steroids (ca. 150) comprising oestranes, androstanes, cholestanes, ergostanes, pregnanes, and compounds on the borderline with alkaloids and triterpenoids have been published since. The use of automatic diffractometers permitting accurate diffraction intensities (accuracy ca. 2 %) to be obtained within a rather short period of time ( 2-3 weeks) and the application of digital computers for direct methods2'Y2' or vector coincidence program^,"^'^ as well as the increased interest in the biochemical understanding of steroid hormones, greatly enhanced the output of published work. It is tempting to reconsider the available data for a more detailed analysis and survey of the conformational features of steroids than was possiblel1>" six years ago. Applying computer programs, the Reporters have calculated weighted mean values and population variances for bond distances and valency and torsion angles, taking into account the configurations at carbon atoms 5, 9, 10, and 13, functional groups at C-3 and C-17, and the presence of double bonds in the carbon skeleton. In addition the presence of carbonyl groups at other places in the skeleton has been accounted for. However, the effect of substituents such as hydroxy-groups or halogen atoms at positions other than C-3 and C-17 has been ignored. It is hoped that their influence on the calculated mean values will be balanced, i.e. that neglect of such groups does not introduce a bias. A careful
'
" 3 '
-
lJ
l4 l 5 l6
I*
2o 2 L
22
C. Romers, B. Hesper, E. van Heykoop, and H. J. Geise, Acta Crysr., 1966, 20, 363. H. J. Geise and C. Romers, Acta Crysr., 1966, 20, 257. H. J. Geise, C. Romers, and E. W. M. Rutten, Acta Cryst., 1966, 20, 249. R. Huber and W. Hoppe, Chem. Ber., 1965,98,2403. D. F. High and J . Kraut, Acta Crysr., 1966, 21, 88. R. Bucourt and D. Hainaut, Bull. SOC.chim. France, 1965, 1366. R . Bucourt, Bull. SOC.chim. France, 1964,2080. I . L. Karle and J . Karle, A c f a Crysr., 1964, 17, 8 3 5 . G. Germain, P. Main, and M. M. Woolfson, Acta Crysr., 1971, A27, 368. P. B. Braun, J. Hornstra, and J . I . Leenhouts, Philips Res. Report, 1969, 24, 85. R. A. Jacobsen, 'Crystallographic Computing', Proceedings of the 1969 International Summer School on Crystallographic Computing, Munksgaard, Copenhagen, p. 83.
533
Steroid Conformationsfrom X-Ray Analysis Data
comparison of the individual steroid geometry with the mean values seems to confirm this thesis. Special attention is paid to the conformation of ring A in 3-0x0-A4-steroids, since several sex hormones, drugs, and hormones of the corticoid group have this structural feature in common. Where our data are at variance with those originally published, the difference is the result of a recalculation of the geometricalentities from the published crystallographic co-ordinates. Simultaneously,we have carried out valence force (VF)calculations (‘molecular mechanics’)of equilibrium geometries and corresponding energiesfor a number of key steroid structures, adopting the ‘full relaxation’ approach. The first exploration into this area was reported in 1970,24and apparently VF calculations are rapidly reaching the stage where they can compete with diffraction techniques in accurately describing the molecular architecture of steroids. An up-to-date review of the VF approach has recently a~peared.’~ Our VF calculations serve several purposes : (i) As an aid in understanding unusual geometrical features observed, such as ‘long’ carbon-carbon bonds, abnormal bond angles, etc., i.e. the distribution of ‘strain’ over the steroid nucleus. (ii) The high accuracy of the weighted mean geometries provides an ideal testing ground for comparison of the relative merits of various force fields employed in the literature ;in some cases ‘better’VF parameters can be extracted from this work. (iii) VF calculations, by making it possible to constrain a selected geometrical feature, for instance by forcing a given torsion angle to adopt a non-optimal value, make it possible to study the ‘flexibility’ of a molecule or part of a molecule. We propose to show that ‘flexibility’, loosely defined as being inversely proportional to the price in kcal mol- a molecule has to pay for certain deformations, may well turn out to have great biological significance. Some comments have been made in this Report on the relationship between chiroptical properties (0.r.d. and c.d.) and the spatial structures of steroidal molecules. The terms for conformations such as chair, half-chair, and their abbreviations are given in the Appendix, which also contains the mathematical formulae and force-field parameters. Five-membered (D) and six-membered rings with boat conformations were analysed in terms of the rotation phase angle and the maximum torsion angle. Some remarks have been ventured on the overall shape of steroid molecules in relation to their biological activity. With the exception of functional groups at positions 3 and 17 and the 17P-alkyl side-chain occurring in cholestanes, ergostanes, and lanostanes, no attention is paid to the configurations of substituents in steroids. We are aware that some contributions to structure determinations have not been mentioned and others may have been overlooked. The literature has been
’
24 25
C. Altonaand H . Hirschmann, Tetrahedron, 1970, 26, 2173. C. Altona and D. H. Faber, Fortschr. chem. Forschung, 1974,45,1.
534
Terpenoids and Steroids
covered up to May 1st 1973. For a more exhaustive survey the reader is referred to the ‘Atlas of Steroid Structure’ by Duax and Norton.26 2 Presentation of Results The steroids under consideration are listed in Table 1 ;the abbreviations used for substituents are explained in Section (vi) of Table 1. A number of more intricate steroids such as aldosterone [see structure (70)] are reproduced separately in Section (v) of Table 1. For crystallographic details such as space groups and unitcell dimensions the reader is referred to the original literature or to the ‘Atlas of Steroid Structure’.26 The presence of solvent molecules, H,O, MeOH, etc., in some crystal structures is ignored in Table 1,since their possible influence on the structure (e.g. by hydrogen bonds) has not been taken into account in this Report. Each steroid has been allotted a number which has been used as a code for computational purposes. A steroid carrying two or more numbers indicates that two or more symmetry-independent molecules are present in one unit cell, resulting in two or more geometrical structures for such molecules. The space groups, unit-cell constants, and positional parameters of each steroid have been processed by a computational program, which computes bond distances, bond angles, and torsion angles as well as least-squares planes. As stated before the program also calculates the weighted mean value (usually called the standard value), the standard error, and the estimator of the standard deviation of the mean of these entities. This estimator gives some information on the scatter of individual points and informs us whether or not the calculated mean values are associated with one population. It also indicates that in most cases the experimental standard deviations of the mean values are greatly underestimated and should be multiplied by a factor of 3 or 4. Details of the expressions used to calculate these values are given in the Appendix, Sections (i)-(iv).
3 The Perhydro-l,2-~yclopentanophenanthreneSkeleton About eight years ago it was generally held that sp3-sp3-hybridized carboncarbon bond distances in steroid-like molecules should be equal and this assumption was used by crystallographers to estimate standard deviations of bond lengths between ‘chemically equivalent’ sp3-hybridized carbon atoms. This in turn supported the assumption, since the outcome of such estimations agreed rather well with machine-computed standard errors resulting from the least-squares refinement of crystal structures. However, standard deviations determined by least-squares methods always were somewhat lower (ca. 50 %), which should have served as a warning against the assumed equality of C-C single bond lengths. Recently obtained values of highly increased accuracy usually yield standard errors of only 0.003--0.006 8, in heavy-atom-free steroid molecules, however, the scatter of values in the range 1.51-1.57 A is about the same as was found in earlier structure determinations with standard deviations of 0.02-4.03A. This 26
D . A. Norton and W. L. Duax, ‘Atlas of Steroid Structure’, 1973.
Steroid Conformationsfrom X-Ray Analysis Data
535
Table 1 List of steroids referred to in the discussion
R' R' a
RZa
- H Designation
(i) Saturated steroids (excluding oxo-steraids) a-OH OH a-OH OH OH a-CH(0H)Me (OMe), OTs 19-nor a-OH a-Me 2a-Br C8H 1 7 2a-Br Br Cl C8H 1 7 2a-C1 a-C1 C8H17 2P-Cl ?-OH a-OH OH
H OH
C,H8CO,H C,H8COAnBrd Card Br C8H 1
,ON
OBzBr
CHClMe
H H
C8H17
C8H1,
l2a-OH 12a-OH 14P-OH 16P-Br 16a-OH 16P-OBzBr 3a,5a-cyclo, 6P-OAcCl 3a,5a-cyclo, 6P-OAcBr
Conjiguration
No.c
R ej'.
5a
5P 5a 5a 5a,14P 5a 5a 5a 5P
5P 58,148 5a 5a 5a 5a 5a
Unless stated otherwise the orientation of R ' and/or R 2 is 8. 'The configurations at carbon atoms 8,9,10,13, 14, and 17 are denoted only if different from the natural configuration (i.e.88,91, log, 138, 14a, 178). Two or more code numbers for one compound indicate For symbols used for functional groups two or more independent molecules per unit cell. see Part (vi) of this Table.
21
28
29
G. Precigoux, B. Busetta, C. Courseille, and M. Hospital, Cryst. Structure Comm., 1965, 1, 265. C. M. Weeks, A. Cooper, D. A. Norton, H. Hauptman, and J. Fisher, Acta Crysr., 1971, B27, 1562. R. A. G. de Graaff, C. A. M. van der Ende, and C. Romers, to be published in Acra Crysr.
30
31
32
33 34
35
36
37
A. Chiaroni and C. Pascard-Billy, Acra Crysr., 1972, B28, 1085. S. Candeloro de Sanctis, E. Giglio, V. Pavel, and C. Quagliata, Acra Crysr., 1972, B28,3656. J. P. Schaefer and L. L. Reed, Acfa Crysr., 1972, B28, 1743. I . L. Karle and J. Karle, Acra Cryst., 1969, B25,434. N. Mandel and J. Donohue, Acra Crysr., 1972, B28,308. E. Hoehne, I. Seidel, G. Adam, D. Voigt, and K. Schreiber, J. prakt. Chem., 1971, 313, 51. H. H. Worch, E. Hoehne, G. Adam, and K . Schreiber, J. prakt. Chem., 1970, 312, 1043. H. R. Harrison, D. C. Hodgkin, E. M. Maslen, and W. D. S. Motherwell, J. Chem. SOC.(0,1971, 1275.
Terpenoids and Steroids
536 Table l-continued R'
R'
OMe a-OMe OAc
HzBsAc C8H1,(OBzBr), Cdl,
a-OBzBr
=C(MC)C,H
Designation
Conjiguration
5P,19-cyclo, 6/?-OMe 5P 9P,19-cyclo, 4,4,14a-Me3 5a,9p 4,4,14a-Me3, 7a,1 la-Br,, 5a,8a 8a,9a-0 18-nor, 4a,8a,14/3-Me3, 5a,8a,9/?, 1 1a-OH, 16fi-OAc 13a,14fi
No. (19) (20) (21) (22)
Ref. 38 39
40 41
[For compounds (23), (24), a n d (25), see Part (v) of this Table]
(ii) Saturated 0x0-steroids 3-0xO0 xo OH OX0 OTs OX0 OAcI OX0 OAcI OX0 OHla-Me 4-0xOEOT C,H,, C8H17 H
5a
4,4-Me, 19-nor, 4,4-Me2 4-oxa, 6a-Br
4-OXO A-homo, 3-aza, 4-0x0,
5a 5a 5a 5a
42 156 43 43
44
5a 5P
45 46
5a 5P
47 47
5a 5a
28 17
5a 5a 5a
48 49 50
5a
51
4a,4a-C1
6-0x0-
H H 17-0x0OH a-OH
OBzBr
OAc
3a,5a-cyclo, 6-0x0 3fl,SP-cyclo, 6-0x0
OX0
OX0
I1,20-di0~0-
OH OAc OH
Ac/a-OH Ac/a-OH AcBr/a-OH
16/?-Br 16P-Br
(iii) Conjugated and homoconjugated 0x0-steroids 1-0x0-A2-
H 38
40 41 42
" 44
45
46 47 48 49
50 51
OX0
1-0x0-A', 4a-Br
G. A. Sim and C. Tamura, J. Chem. SOC.( B ) , 1968,8. F. H. Allen and J. Trotter, J. Chem. SOC.( B ) , 1971, 1079. J. K.Fawcett and J. Trotter, J. Chem. SOC.( B ) , 1966, 174. A. Cooper and D. C. Hodgkin, Tetrahedron, 1968,24, 909. B. Busetta, C. Courseille, J. M. Formes-Marquina, and M. Hospital, Cryst. Structure Comm., 1972, 1. 43. G . Ferguson, E. W. Macaulay, J. M. Midgley, J. M. Robertson, and W. B. Whalley, Chem. Comm., 1970, 954. J. S. McKechnie, L. Kubina, and I. C. Paul, J. Chem. SOC.( B ) , 1970, 1476. A. Cooper and D. A. Norton, J. Org. Chem., 1968,33,3535. H.Altenburg, D. Mootz, and B. Berking, Acfa Cryst., 1972,B28, 567. R. C. Pettersen, 0. Kennard, and W. G. Dauben, J.C.S. Perkin I I , 1972, 1929. J. M. Ohrt, A. Cooper, and D. A. Norton, Acra Cryst., 1969,B25,41. J. M. Ohrt, B. Haner, A. Cooper, and D. A. Norton, Acfa Cryst., 1968,B24, 312. J. M. Ohrt, A. Cooper, G. Kartha, and D. A. Norton, Acfa Cryst.,.1968,B24, 824. J. R. Hanson, T. D. Organ, G. A. Sim, and D . N . J. White, J. Chem. SOC.(0,1970, 2111.
Steroid Conformationsfrom X-Ray Analysis Data
537
Table l+ontinued R2
R’
Designation
Conjiguration
Ref.
3-oxo-A4 OX0 OX0
Ac AcOHja-OH
OX0
OX0
OX0
OH OH Acja-0Dec OSil Ac/a-OH AcOH OH a-OH CH(0H)Me OAc OBs OAc OAcCl AcO H/u-OH Ac OAc
OX0 OX0
OX0 OX0
OX0 OX0 OX0 OX0 OX0
OX0 OX0
OX0 OX0
OX0 OX0 OX0
C2H4C02
OX0 OX0 OX0
AcOAC/U-OH AcOH/a-OH AcOH/a-OH Ac Ac AcOH/a-OH
OX0
OX0 OX0
A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4 A4, 2,2,68-C13 19-nor, A4, 2P-Me A4, 2P-OAc A4, 2B-OAc A4, 4-CI,ll-oxo A4, 6P-Br A4, 6/3,7/3-CHz A4, 7a-SAC A4, 11-0x0 A4, 11P-OH A4, 11-0x0 A4, llB-OH, 12a-Br A 4 * I 5 , 14a,17a-C,Hz, 15,16-(CF,), A4, 6a-F, 11P-OH
52 53 54 55
56 57 58 59 60 61 62 163 63 64 138 138 65 66 67 68 69 70 71 72 73 70
H. Campsteyn, L. Dupont, and 0. Dideberg, Acta Cryst., 1972, B28, 3032. L. Dupont, 0.Dideberg, and H. Campsteyn, Acta Cryst., 1973, B29, 205. 5 4 B. Busetta, G. Comberton, C. Courseille, and M. Hospital, Cryst. Structure Comm., 1972, 1, 129. 5 s A. Cooper, E. M. Gopalakrishna, and D. A. Norton, Actu Cryst., 1968, B24, 935. 5 6 A. Cooper, G. Kartha, E. M. Gopalakrishna, and D. A. Norton, Acta Cryst., 1969, B25,2409. 5 7 W. H. Watson, Kuan Tee Go, and R. H. Purdy, Actu Cryst., 1973, B29, 199. C. M. Weeks, H. Hauptman, and D. A. Norton, Crysf. Structure Cumm., 1972, 1, 79. 5 9 J. P. Declercq, G. Germain, and M. van Meerssche, Cryst. Structure Comm., 1972, 1,9. 6 o 0. Dideberg, H. Campsteyn, and L. Dupont, Actu Cryst., 1973, B29, 103. 6 1 B. Busetta, C. Courseille, F. Leroy, and M. Hospital, Acra Cryst., 1972, B28, 3293. 6 2 N. W. Isaacs, W. D. S. Motherwell, J. C. Coppola, and 0. Kennard, J.C.S. Perkin 11, 1972,2335. W. L. Duax, Y. Osawa, A. Cooper, and D. A. Norton, Tetrahedron, 1971,27,331. 6 4 V. Cody and W. L. Duax, Cryst. Structure Comm., 1972, 1, 439. 6 5 W. L. Duax, A. Cooper, and D. A. Norton, Acta Cryst., 1971, B27, 1 . 6 6 E. M. Gopalakrishna, A. Cooper, and D. A. Norton, Actu Cryst., 1969, B25, 639. 6’ P. B. Braun, J. Hornstra, and J. I. Leenhouts, Actu Cryst., 1970, B26, 352. 0. Dideberg and L. Dupont, Actu Cryst., 1972, B28, 3014. 6 9 J. P. Declercq, G. Germain, and M. van Meerssche, Cryst. Structure Comm., 1972,1,59. 7 0 L. Dupont, 0. Dideberg, and H. Campsteyn, Cryst. Structure Comm., 1972, 1, 177. ’*J. P. Declercq, G. Germain, and M. van Meerssche, Cryst. Structure Cumm., 1972,1, 13. ” A. Cooper and D. A. Norton, Actu Cryst., 1968, B24, 81 1 . 7 3 G. I. Birnbaum, Acta Cryst., 1973, B29, 54. 52
”
‘’
538
Terpenoids and Steroids
Table l-continued
R'
R2
Designat ion
AcOH/a-0 H OH/a-Me
OX0 OX0
OAcBr OAc Ac OAc OAc
OX0
OX0 OX0 OX0 OX0 OX0 OX0 OX0 OX0
OX0
Ac a-OBzBr 'gH1
7Br2
A4, 9a-F, 11P-OH A,' 9a-Br, 1l-0x0 See Part (v) of this Table See Part (v) of this Table A4 19-nor, A4 A,' 6a-Me A4, 6a-Me, 6P-F A4, 6P-Me, 6a-F A4, 9P-OH A4, 1la-OH 19-nor, A4, 7,7-Me,
A4
See Part (v) of this Table
Conjiguration
90,lOa 9P,1Oa 9P,lOa 9P,lOa 9P,lOcr 9/?,10a 9P,1Oa 8u,14P 9P
Ref: 74 75 157 76 77 78 79 79 79 79 79 80 81 82
3-oxo-A5AcOAc/a-OH ~-OXO-L\~('~)OX0 OH OX0 OAcI 3-0X0-A''4OX0 OH OX0 AcOH/a-OH 3-0~0-A~~~OX0
OX0
OX0
Ac Ac 3-0xo-A~~~OX0 OH/a-CrCH 6-0x0-A~C8H 17O2 OH OH C8H1703 17-oxo-A 4OX0 OX0
c1
14
75
76
'I 78
''
'O 81
82
83 84 85
86
*'
88
OX0
A5, 4a,6,7a-C13, 1l-0x0 lg-nor, A5('O) 19-nor, A 5 [ l 0 ) ~ 1 . 4
A1,', 6a-Me, 11P-OH ~ 4 . 6
162
83 84 133 85
A4s6,4-Br
79 79 13
19-nor, A4v9,1l-0x0
86
6-oxo-A7,2P,14a-(OH), 6-0x0-A7,2P,14a-(OH),
16 87
4,5-seco, A2,8,14,5-0x0
88
~ 4 . 6
L. Dupont, 0. Dideberg, and H. Campsteyn, Acta. Cryst., 1972, B28, 3023. A.Cooper, C. T. Lu, and D. A. Norton, J. Chem. SOC.( B ) , 1968, 1228. G . W. Krakower, B. T. Keeler, and J. Z. Gougoutas, Tetrahedron Letters, 1971, 291. W. E. Oberhansli and J. M. Robertson, Helo. Chim. Acta, 1967, 50, 53. B. Busetta, C. Courseille, and M. Hospital, Cryst. Structure Comm., 1972, 1, 235. P. B. Braun, J. Hornstra, and J. I. Leenhouts, Philips Res. Report, 1969, 24,427. D. Lednicer, D. E. Emmert, C. G. Chidester, and D. J. Duchamp, J . Org. Chem., 1971, 36, 3260. B. Hesper, H. J. Geise, and C. Romers, Rec. Truo. chim., 1969, 88,871. M. 0. Chaney and N. D. Jones, Cryst. Structure Comm., 1972,1, 197. R. R. Sobti, S. G. Levine, and J. Bordner, Acta Cryst., 1972, B28,2292. R. R. Sobti, J. Bordner, and S. G. Levine, J. Amer. Chem. SOC.,1971,93, 5588. J. P. Declercq, G. Germain, and M. van Meerssche, Cryst. Structure Comm., 1972,1, 5 . G. Lepicard, M. J. Delettre, and M. J. P. Morman, to be published in Acta Cryst. B. Dammeier and W. Hoppe, Chem. Ber., 1971,104, 1660. C. S. Yoo, J. Pletcher, and M. Sax, Acta Cryst.. 1972, B28,2838.
539
Steroid Conformationsfrom X-Ray Analysis Data
Table l-continued R’ R2 17-0Xo-A8(14)OX0
Designation 10-aza
OX0
Configuration
5b9P
No.
Ref:
(96)
89
(iv) Unsaturated steroids Monoenes
A4 A’ OAc a-I A5 OBzI C10H19 A5 OBzBr 0x0 A5 OBzBr 0x0 A’ OBzI CIlH21 A’, 9,1l-seco, 9-0x0, 11-OAc H OAcI 19-nor, A5(’O) OBzBr C9H,, A6, 5a,8a-cyclo OAcI C8H17 A’, 4,4,14a-Me3 OAcI a-C8H15 A’, 4,4,14p-Me3 [For compound (109), see Part (v) of this Table] H
90 91 92 93 94 95 96
OBs OH
c1
Dienes ED0
(105) 5a,8a,9p (106) 5a (107) 5a,13a,14p (108)
(110) 9p,lOa (111) OBzIN C,Hl, A5.7 9/I,lOa (112) OBzDN C,H,, ~ 5 . 7 (113) 1Oa [For compounds (114), (115), (116), (117), and (118), see Part (v) of this Table] OH
C(ED0)Me
Oestra-1,3,5(10)-trienes OH OH OH OH
OH OH 89
90
91
92 93 94
” 96 97
A’,’
A5,7
OH OX0
A-ar. A-ar. A-ar. A-ar.
(119) (120) (121) (122)-(125)
97 98 99 100
101 149 8 102
103 104 105 106
J. G. H. de Jong, C. J. Dik-Edixhoven, and H. Schenk, Cryst. Structure Comm., 1973,2. 33. J. Bordner, S. G. Levine, Y.Mazur, and L. R. Morrow, Cryst. Structure Comm., 1973,2, 59. C . M. Weeks, A. Cooper, and D. A. Norton, Acta Cryst., 1971,B27,531. H. C. Mez and G. Rihs, Helu. Chim. Actu, 1972,55,375. I. Nan Hsu and D. van der Helm, Rec. Trau. chim., 1973,92, 1134. J. C.Portheine, C. Romers, and E. W. M. Rutten, Acta Cryst., 1972,B28, 849. J. C. Portheine and C. Romers, Acta Cryst., 1970,BM, 1791. E. L. Enwall and D. van der Helm, Rec. Trau. chim.. 1974,93,53. J. Bordner, R. L. Greene, S.G. Levine, and R. R. Sobti, Cryst. Structure Comm., 1973,2, 55.
98 99 loo
lo’
lo’
‘06
G. L. Hardgrove, R. W. Duerst, and L. D. Kispert, J. Org. Chem., 1968,33,4393. J. Fridrichsons and A. McL. Matthieson, J. Chem. SOC.,1953,2159. C. H. Carlisle and M. F. C. Ladd, Acta Cryst., 1966,21,689. P.B. Braun, J. Hornstra, C. Knobler, E. W. M. Rutten, and C. Romers, Acta Cryst., 1973,B29,463. A. J. de Kok, E. W. M. Rutten, and C. Romers, to be published in Acta Cryst. B. Busetta and M. Hospital, Compt. rend., 1969, 268, C,1300. W. L. Duax, Acta Cryst., 1972,B28, 1864. B. Busetta, C. Courseille, S. Geoffre, and M. Hospital, Acta Cryst., 1972,B28, 1349. B. Busetta, C. Courseille, and M. Hospital, Actu Cryst., 1973,B29, 298.
Terpenoidsand Steroids
540
Table 1-ontinued
R'
RZ
OH OH OH OH OMe OMe OMe OMe OMe OMe OMe OMe
OX0
OH OH
OH CH,OBs a-CH,OBs OAcBr OAcBr ED0 OAc H OX0
Other trienes OMe OBzBr C(E D 0 ) M e CgHl,
No.
Ref: 107 108 109 110 111 111 112 113 114 114 115 116
A-ar. A-ar., 4-Br A-ar., 2,4-Br2 A-ar., 16u-OH A-ar., D-nor A-ar., D-nor A-ar., A6, 8u-Me A-ar., 7a,8u-CH2 A-ar., 6,7,8-CH A-ar., 7-thia A-ar., 8-aza, 12-0x0 A-at., A", 11,13-diaza, 12-NH2 (141)
19-nor, m(l0a)-homo, ~
ED0 OBzIN
Conjiguration
Designat ion
117
1 10).2,4 (
9,10-seco, A5*7,'( 9,1O-seco, A 5 * 7 * 1'(
9, 19)
(v) Formulae Me
1
.N-CN ,H
H
BrBzO'
H (23) ref. 118
lo'
Io8 lo9 ' l o
I
*
I I I l 3
'I5 'I6 'I7 l8
H (24) ref. 119
T. D. J. Debaerdemaeker, Cryst. Structure Comm., 1972, 1, 39. D. A. Norton, G. Kartha, and C. T. Lu, Acra Cryst., 1964,17, 77. V. Cody, F. DeJarnette, W. L. Duax, and D. A. Norton, Acta Crysr., 1971, B27, 2458. A. Cooper, D. A. Norton, and H. Hauptman, Acra Cryst., 1969, B25, 814. P. Coggon, A. T. McPhail, S. G. Levine, and R. Misra, Chem. Comm., 1971, 1133. H . P.Weber and E. Galantay, Helv. Chirn. Acta, 1972, 55, 544. C. M. Weeks and D. A. Norton, J. Chem. Soc. ( B ) , 1970, 1494. C. F. W. van de Ven and H. Schenk, Cryst. Structure Comm., 1972, 1, 121. J . N. Brown, R. L. R. Towns, and L. M. Trefonas, J. Hererocyclic Chem., 1971,8, 273. A . H. Joustra and H. Schenk, Rec. Trav. chim., 1970, 89, 988. H . Hope and A. T. Christensen, Acta Cryst., 1968, B24, 375. J . S. McKechnie and I. C. Paul, J . Amer. Chem. SOC.,1968,90, 2144. J. Guilhem, Acta Cryst., 1972, B28, 291.
54 1
Steroid Conformationsfrom X-Ray Analysis Data
Table l-continued Me
.OH ‘CH,OH
(25) ref. 120
(70) ref. 157
OH
CH,Br
______
& o y e
0
/
0
/
(83) ref. 82
(71) ref. 76 Me
Me
Me
CN BrBzO
BrAcO (114) ref. 122
(109) ref. 121
Me
(115) ref. 124
Me
(116), (117) (bromide)ref. 123
(118) (iodide) ref. 123
’” ”*
R. F. Bryan, R. J . Restivo, and S. M. Kupchan, J.C.S. Perkin II, 1973, 386. E. Thorn and A. T. Christensen, Acra Crysf., 1971, B27, 794. D. R. Pollard and F. R . Ahmed, Acra Crysf., 1971, B27, 1976. R. T. Puckett, G. A. Sim, and M. G. Waite, J . Chem. SOC.( B ) , 1971, 935. I. L. Karle and J. Karle, Acra Cryst., 1969, B25,428.
542
Terpenoids and Steroids
Table l-continued
(vi) Symbols for functional groups Halogenoacetyl (COCH,X) AcX Halogenobenzoyl (COC,H,X) BzX 3,S-Dinitrobenzo y l BzDN BzIN 4-Iodo-3-nitrobenzoyl p-Bromobenzenesulphonyl (brosyl) Bs HzAcBs p-Bromobenzenesulphonyl-N-acetylhydrazono [=N-N(COMe)SO,C,H,Br] Toluene-p-sulphonyl (tosyl) Ts Methoxycarbonyl MC Ethylenedioxy (-OCH,CH ,O-) ED0 Ethyleneoxythio (-OCH ,CH , S - ) EOT Sil Trimethylsily1 AnBr p-Bromoanilino Card Cardenolide side-chain Dec COC,H ,,COCH ,C1
means that the various sp3-sp3 distances found do not belong to the same population; in other words they are chemically non-equivalent. In Table 2 we have tabulated the average bond lengths (standard values) that we have obtained for (i) sp3-sp3 single bonds, (ii) saturated compounds having the SP-configuration, and (iii) compounds having the corresponding Scc-configuration(Figure 1). If
Figure 1 The SLY- and Si?-conjigurations of saturated steroids, (a) and (b) respectively. The numbering of atoms is indicated in (a)
Steroid Conformations from X- Ray Analysis Data
543
the observed differences between the lengths of the various sp3-sp3 bonds were random, or more precisely not systematic, their mean values should be about equal, in striking contrast with the values listed in column 2 of Table 2. Note also the small standard errors (-0.001 A) and small estimator values (-0.003 A) in columns 3 and 4. The same trend is observed if we discriminate between 5a- and 5P-configurations and only consider compounds without double bonds. The particularly small C-2-C-3 and C-3-C-4 bond lengths (1.510 and 1.513A for 5a-configuration) are biased, since nearly all inspected steroids contain electrondonating substituents (halogen atoms, hydroxy-groups) at position 3. Ignoring the C-2-C-3 and C-3-C-4 bonds, the C-8-C-14 bond is smallest, while the C-5-C-10, C-9-C-10, C-13-C-17, and C-16-C-17 bonds are ‘long’.
Table 2 Standard bond lengths r, standard errors CT, and estimators S (all in for sp3-sp3 hybridization and steroids with 5a- and 5P-conjigurations
A)
sp3-sp 3-hybrid” 5B-conjigurationa 5a-conjigurat iona 103(r - 1) 1040b 104Sb 103(r - 1) 1040 104s 103(r - 1) 1 0 4 ~ 104s 22 77 542 7 20 549 45 537 17 29 535 16 530 7 23 528 45 150 74 513 17 92 527 45 513 14 69 70 511 18 180 51 14 57 527 516 49 533 20 100 535 19 92 543 51 36 549 17 543 46 110 549 16 44 523 16 40 97 55 525 16 43 531 28 528 16 536 48 52 524 7 20 43 529 16 530 7 22 531 48 140 72 35 544 16 552 48 545 7 19 553 17 91 18 564 45 560 7 22 29 544 16 97 534 45 542 7 18 26 537 16 120 537 7 19 530 45 529 17 49 98 530 45 529 7 26 544 16 95 551 45 35 54 1 7 26 519 16 26 130 523 7 25 537 48 535 16 75 534 7 19 531 45 23 44 543 130 16 536 45 545 7 26 150 44 550 18 46 8 28 546 544 120 553 19 65 46 8 31 545 553 140 537 16 43 538 45 537 7 29 110 541 17 37 539 46 545 8 21 sp3-sp3-hybrid : a variable number (20-60) of steroids were involved 5P-configuration: compounds (2), (lo), (1 l), (12), (19), (39, (93), (94) 5a-configuration: compounds ( l ) , (3), (4),(5), (61, (8), (9), (17), 18), (26), (23, (31), (3% (36), (37h (381, (1 14).
Bond 1-2 1-10 2-3 3-4 4-5 5-10 5-6 6-7 7-8 8-9 9-10 9-11 11-12 12-13 13-14 8-14 14-15 15-16 16-17 13-17 13-18 10-19
a Single bonds without sp3-sp3 hybridization were not taken into account. tions, see Appendix, Sections (ii) and (iii).
For defini-
One might argue that the increased lengths of the C-5-C-10, C-9-C-10, and C-16-(2-17 bonds are due to overcrowding effects such as intramolecular repulsion between hydrogen atoms connected to carbon atoms 1,11, and 19 and
Terpenoids and Steroids
544
the (partial) eclipsing of those attached to C-15, C-16, and C-17. Force-field calculations (see Appendix) take account of these and other interactions. Depending on the 'softness' of the parameters for the different interactions in the fields proposed by Altona (hereafter AL),12' by Warshel and Lifson (LW),'26 by Boyd et al. (B),' 2 7 and by Allinger et al. (A),' 2 8 the various types of calculation reproduce properly either bond and torsion angles or bond length^.^' Table 3 indicates that the methods AL and A give the best agreement with the observed average distances for steroids with the 5u-configuration. Curiously, method AL
Table 3 Comparison of experimentally determined standard bond lenghts r for saturated Su-steroih and theoretical values for 20-methyl-5a-pregnane computed from various force fields. The values listed are 103(r- 1) in units of A. The agreement index s is deJined in Section (iv) of the Appendix Bond
1-2 1-10 2-3 3-4 4-5 5-10 5-6 6-7 7-8 8-9 9- 10 9-11 11-12 12-13 13-14 8-14 14-15 15-16 16-17 13-17 13-18 10-19
5a-Configuration (see Table 2) 537 535 513 511 533 549 523 528 529 544 553 544 537 529 544 519 535 543 550 553 537 54 1
104s:
AL 534 549 525" 530" 538 548 528 531 532 545 571 543 544 531 54 1 532 523 546 554 558 545 549
LW 523 544 521" 523" 529 544 528 520 533 549 54 1 533 533 515 563 502 535 547 567 540 522 525
B 542 552 538" 539" 543 558 540 537 54 1 556 567 553 545 543 538 543 536 545 550 550 554 553
78
114
117
A 532 539 523" 529" -
541 528 527 527 536 548 539 534 525 5 29 528 533 544 512b 5 14b 538 538 61
Excluded from the calculation of the agreement index, since the experimentally determined structures contain substituents at C-3. Excluded since in this calculation C-17 was substituted.
'" 12'
C. Altona, unpublished work; see also Appendix. A. Warshel and S . Lifson, J. Chem. Phys., 1970,53, 582. S. Chang, D. McNally, S. S.Tehrany, M. J. Hickey, and R. H. Boy& J. Arner. Chern. SOC.,1970, 92, 3109.
12*
N. L. Allinger and F. Wu, Tetrahedron, 1971, 27, 5093.
Steroid Conformations from X-Ray Analysis Data
545
overestimates the length of the C-9-C-10 bond (calculated value 1.571 A). This A5,and A5*7bond is indeed the longest in many SB-steroids, and in 3-0xo-A~~ compounds, but not in saturated 5a-steroids. Standard bond angles for 5a- and 5P-compounds are listed in Table 4. In this case the estimator is about five times the standard deviation, implying that in steroid molecules the degree of variation of valency angles is somewhat larger than the corresponding degree of variation in bond lengths. This is in agreement
Table 4 Standard bond angles 8, standard errors 0 , and estimators S (decimal degrees) for rings A, B, and c in 5u- and 5p-steroids Angle 10-1-2 1-2-3 2-3-4 3-4-5 4-5-10 5-10-1 4-5-6 1-1&9 1-10-19 9-10-19 9-10-5 10-5-6 5-6-7 6-7-8 7-8-9 8-9-10 7-8-14 10-9-1 1 8-14-13 14-8-9 8-9-1 1 9-1 1-12 11-1 2-1 3 12-1 3-14 15-14-8 12-1 3-17 12-1 3-1 8 14-1 3-1 8
e
5a-Configuration
113.3 112.2 111.4 112.0 112.1 107.3 111.8 110.9 109.3 111.2 107.6 112.6 111.0 112.5 110.8 112.6 111.5 113.8 114.8 109.0 111.2 113.4 110.9 108.2 119.5 117.1 111.0 112.5
io20 10 10 11 11 10 10 11 13 11 11 10 10 10 10 10 10 10 11 10 10 10 10 10 10 10 13 10 13
102S 51 69 76 85 39 50 59 56 59 28 43 50 32 38 30 31 52 27 68 33 36 62 49 58 76 53 52 61
5/?-Conjiguration 102S io2a 110 113.8 27 110.6 27 120 110.8 27 93 111.7 27 140 114.4 24 110 108.1 24 80 112.3 21 93 110.9 21 89 108.2 21 88 111.4 21 68 108.7 21 71 111.8 24 69 111.8 24 65 111.7 24 39 110.9 21 88 111.5 21 51 62 111.7 21 113.8 21 82 117.4 22 140 M8.6 22 160 111.7 22 52 112.6 22 61 111.3 22 76 41 107.8 22 118.9 22 91 113.9 22 150 120 111.2 22 114.1 22 63
e
,
with the various V F calculations since a change in bond length requires considerably more energy than an equivalent change in bond angle. We note that the endocyclic bond angles about quaternary carbon atoms 10 and 13 have small values in the range 107-109", in accord with earlier predictions by B u ~ o u r t ' ~ * ' ~ and observations by Geise et al.' Table 5 lists a comparison of the standard bond angles of 5a-steroids with theoretical values resulting from the fields AL, LW, and B. With the exception of
Terpenoids and Steroids
546
Table 5 Comparison of standard bond angles (decimal degrees) of rings A, B, and c in Sa-steroids and the corresponding theoretical values calculated ,from the force$elds AL, L W , and B Angle 1&1-2 1-2-3 2-34 34-5 4-5-10 5-10-1 4--5 -6 1--10-9 1-10- 19 9-10-19 9-10-5 10-5-6 5-6-7 6-7-8 7-8-9 8-9- 10 7-8-14 10-5-6 8-14-13 14-8- 9 8-9-1 1 9-11-12 11-12-13 12-1 3-14 15-14-8 12- 13- 17 12-13-18 14-1 3-1 8
5a-Conjguration ( S P P Table 4) 113.3
AL 115.0 111.6 110 7 111.4 115.0 106.1 112.2 111.9 108.0 109.7 108.4 112.4 110.8 112.4 112.4 114.0 112.1 117.7 115.9 111.3 110.7 114.5 113.5 106.2 119.9 117.4 109.9 113.8
112.2 111.4 112.0 112.1 107.3 111.8 110.9 109.3 111.2 107.6 112.6 111.0 112.5 110.8 112.6 111.5 113.8 114.8 109.0 111.2 113.4 110.9 108.2 119.5 117.1 111.0 112.5 s:
1.38
LW 113.6 111.9 112.0 111.3 112.8 107.1 111.3 110.1 108.8 111.2 107.9 111.9 110.8 112.4 110.3 112.1 111.3 113.8 113.6 109.8 110.7 113.0 112.1 106.9 118.8 116.8 110.5 112.4
B 113.0 11 1.4 111.6 111.0 112.8 107.4 111.1 110.3 108.8 110.8 107.6 111.8 110.5 111.6 110.7 112.0 111.1 113.5 113.3 109.0 110.8 112.5 111.1 107.7 117.4 116.5 110.0 113.3
0.62
0.74
angle 9-10-5, for which B produces too large a value, the fields B and LW predict better bond angles than the field AL. Table 6 contains the standard endocyclic torsion angles of 5cr-steroids together with theoretical values predicted by the field LW. The discrepancy between CT and S is even larger, in agreement with the fact that the amount of energy required for a change of one degree in the torsion angle is smaller than for the corresponding change of valency angles (‘Pitzer’ strain is ‘softer’ than ‘Baeyer’ strain). In Table 6 are also listed the overall average endocyclic values of rings A, B, and c according to experiment and to the four quoted fields as well as those of Qav of cyclohexane,’2 9 methylcyclohexane,’Z9 1,l-dimethylcyclohexane,’30 and 1,ldicarboxycyclohexane. Ignoring 1,l-dimethylcyclohexane the experimental
’
130 13’
H . J . Geise, H . R . Buys, and F. C. Mijlhoff, J . Mol. Srrucrure, 1971, 9, 447. H. J. Geise, F. C. Mijlhoff, and C. Altona, J . Mol. Srructure, 1972, 13, 21 1 . C. Pedone, E. Benedetti, and G. Allegra, Acra Crysr., 1970, B26, 933.
547
Steroid Conformationsfrom X-Ray Analysis Data
Table 6 Comparison of average endocyclic torsion angles @ for 5a-steroids (excluding 3-oxo-compounds), their standard errors, and estimators (decimal degrees) and theoretical values of jield L W . Included are average (Dav values of experiment and fields AL, L W , A , and B Ring A 1-10 1-2 2-3 3 4 4-5 5-10 Ring
0
55.0 - 55.7 53.0 - 53.7 56.8 - 55.6
0.2 0.2 0.2 0.2 0.2 0.2
S 0.6 1.3 2.1 2.0 1.4 0.9
58.6
- 56.9
0.2 0.2 0.2 0.2 0.2 0.2
0.6 1.1 1.2 0.5 0.6 0.8
52.6 52.8 - 54.4 55.7 - 59.6 57.6
0.2 0.2 0.2 0.2 0.2 0.2
0.5 3.4 0.7 0.9 1.1 1.o
LW 55.7" - 54.7 51.7 - 52.8 56.9 - 56.6
Average value Exp. AL LW A B C6H12
@'av
55.0 f 0.1 54.2 54.7 55.0 55.5 55.9
B
5-10 5-6 6-7 7-8 8-9 9-10 Ring c 8-9 9-1 1 11-12 12-13 13-14 8-14
a
CD
- 58.2
54.2 - 52.5
55.0
-
58.6 - 57.6
54.5 - 53.4
55.8 - 57.6 - 52.9
52.1 - 55.7 56.2 - 59.7 58.6
Exp. AL LW A B C6H11Me
55.9 53.8 56.3 55.4 56.8 55.3
*
0.1
55.4 & 0.1 53.1 55.9 53.7 56.6 51.7 55.9
Standard deviation 1.4".
values for rings A, B, and c are in close agreement with those observed for the cyclohexane compounds. It can also be seen that the best agreement is obtained with the LW field. Introduction of two 1,l -methyl groups into cyclohexane induces a considerable flattening of the ring system. The axial methyl groups 18 and 19, however, do not cause a corresponding flattening of the steroid nucleus. Close inspection of Table 6 reveals that the largest staggering is encountered in the region of the quaternary atoms C-10 and C-13* while puckering is smallest about the bonds C-2-C-3, C-3-C-4, C-6-C-7, C-7-C-8, and C-9-C-11. The total effect is a bending of the skeleton (see Figure 2) toward the a-side of the steroid molecule. This phenomenon was previously observed by Geise et a2.9,11,132 and has been mentioned subsequently by several workers, in particular by the Buffalo
* This effect is, of course, related to the small inner valency angles about (2-10 and C-13, since the mean torsion angle is related" to the mean valency angle ma, by the equation cos ma, = -cos ma,/( 1 + cos a,,). 13''
13*
C. K. Johnson, Chemical Division Annual Progress Report, Oak Ridge National Laboratory, 1967, N o . 4164, p. I16 H. J. Geise, A. Tieleman, and E. Havinga, Tetrahedron, 1966, 22, 183.
548
Terpenoids and Steroids
Figure 2 ORTEP-projections' la of saturated 5a-steroids : (a) 5a,17a-pregnane-3B,20adiol as found in the crystal structure determination by Romers et al. '5 6 Hydrogen atoms are deleted; (b) 5a,17B-pregnane-3B,20B-diolas resulting from VF calculations by Altona and H i r ~ c h m a n n ~ ~
group' 3 3 , 1 3 4 in their studies of 3-oxo-A4- and -A'i4-compounds [(58), (63), (67), (87), and (88)l. The same effect is also observed for other unsaturated steroids with normal configurations. We will return to this feature in Section 10 and mention here that an analogous effect exists for vitamin A-like compounds, the conjugated doublebond chains of which are bent by the repulsive action of protruding methyl groups.'35 It seems very likely that the preference for the bent form in all these cases is somehow connected with the biological activity. 4 The Geometry of Ring A in 3-Oxo-A4-steroids
The 3-oxo-A4 moiety is the most interesting feature of the important class of corticoid steroid hormones and of many steroid sex hormones. The geometry of ring A in these compounds can be described in terms of the various possible 133
L34
135
W. L. Duax, D. A. Norton, S. Pokrywiecki, and C. Eger, Sreroids, 1971, 18, 525. P. A. Kollman, D. D. Giannini, W. L. Duax, S. Rothenberg, and M. E. Wolff, J . Amer. Chem. Soc., 1973,95, 2869. J. C . J. Bart and C. H. MacGillavry, Acta Crysf., 1968, B24, 1587 and various papers cited therein.
Steroid Conformationsfrom X-Ray Analysis Data
549
conformations of cyclohexane, in addition to a specification of the endocyclic torsion angles. The relevant data are collected in Table 7. The calculation of a least-squares plane through atoms 3,4,5,and 10 indicates that this system is almost invariably planar to within 0.02 A. Frequently carbon atoms 1 and 2 and the oxygen atom at C-3 are at much larger distances, I,, i,, and I,, from this plane. We use the designation half-chair ( H C ) if I, and 1, are approximately equal but opposite in sign ;identity of sign implies a boat conformation (B). For a perfect sofa conformation ( S ) either I , or I, is zero. Intermediate forms are termed S-HC or S-B. The crossing-over points are rather arbitrariIy set at ll2/II[ (or \!,/I21) equal to and 4. Table 7 reveals that in most 3-0x0-A4-steroids ring A adopts an S or S-HC conformation. When the configuration at C-10 is P, the S(1)a form (atom 1 at the a-side of the plane of atoms 2, 3,4, 5, and 10; see also the Appendix) prevails and vice uersu. However, within the same designation appreciable variations in torsion angle and ratio !,/I1 occur. In the group of compounds (42)-(53), having no substituents in rings A, B, or c, I,/], ranges from -0.18 to -0.62 and torsion angle 3-4 varies from + 3" to - 8". We further note that in these compounds the formal double bond C-4-C-5 is always twisted in the same (negative) sense. A perfect or nearly perfect H C conformation is realized in the c-ring-substitued analogues (64)and (65). In other c-ring-substituted steroids [(62),(63),and (7011, however, the geometry of ring A is very much like that in unsubstituted specimens [note that (62) differs from (64)only in the acetoxylation of the 21hydroxy-group]. Additional introduction of fluorine in the 6a-position [cJ (63) and (67)]leaves the form of ring A unaltered. In the 6P-bromo-steroid (59) the distortion of ring B, caused by the 1,3-diaxial interaction of the 10-methyl group and the bulky 6/?-substituent, is transmitted to ring A ;the overall shape may still be called S( l)a but, as a result of changes in torsion angles, the ratio lJI1 has become positive. Curiously, no such effect is found in (60),where the presence of the 6,7P-methylene bridge profoundly affects the conformation of ring B. Introduction of an axial 7or-substituent (61) apparently does not disturb the form of ring A. In contrast, rather large changes occur on 9a-substitution. The effect of 9a-halogenation on the molecular structure of corticoid steroids has recently been discussed by Weeks, Duax, and W 0 1 f f . l ~As ~ far as ring A is concerned, owing to the interaction of the axial hydrogen atom at C-1 and the 9asubstituent, C- 1 is tilted upwards, thereby decreasing torsion angles 1-10 and 5-10 and increasing the angles 2-3 and 3-4. As a result the overall shape of ring A tends to or reaches the S(2)P conformation* [compounds (68) and (69)]. Major changes in conformation may also occur on substitution in ring A itself [steroids (54)--(58)]. In (58) the interaction of the 4-chloro-substituent with the
* As judged by the bond lengths in ring A the data of compound (71) are less reliable. In addition, in view of the rather large torsion angle 4-5, ring A can hardly be treated as a substituted cyclohexene. A distorted boat form seems to be the most appropriate designation. 136
C. M . Weeks, W. L. Duax, and M . E. Wolff, J . Amer. Chem. SOC.,1973,95, 2865.
1-10
1-2
21.9 28.9 38.9 30.6 27.6
D
(iii) Substituents in rings B, c, and -50.7 52.6 -51.9 45.9 -59.4 45.6 45.6 -53.9 48.5 -53.9
(59) (60) (61) (62) (63)
29.5 33.5 -42.4 -45.7 25.4
-51.9 -58.4 58.3 60.0 -53.2
(ii) Substituents in ring (54) 49.8 (55) 51.2 (56) - 40.9 (57) -41.9 (58) 54.8
A
-53.4 -53.0 -54.4 -58.0 -56.1 -54.5 -56.3 -56.1 -52.2 -57.5 -57.6 -55.8
28.7 27.9 29.9 31.0 34.8 32.6 35.6 35.1 33.1 37.6 36.2 36.8
2- 3
47.1 45.6 47.2 49.0 46.1 45.4 45.5 44.0 42.0 45.9 44.7 43.6
(42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) (53)
(i) N o substituents in rings A , B, and c
Compound
3.7 -1.7 -5.6 -1.0 2.7
-2.3 12.1 18.1 -0.3
- 8.3
0.5 3.0 0.3 2.0 - 4.0 - 2.4 - 4.8 - 3.4 - 6.8 -8.1 - 4.0 -8.1
34
0.7 -3.8 -8.6 -7.0 -7.1
9.0 -6.2 4.7 - 1.1 4.0
-6.0 -9.4 -6.4 -12.0 -5.8 -6.9 -6.1 -8.7 -2.9 -3.2 -9.5 -4.0
4-5
-28.7 -18.4 - 12.5 -16.1 - 18.7
-29.0 -18.8 9.7 11.8 -31.0
-18.1 -15.2 - 17.6 - 13.0 - 16.5 -15.3 - 14.4 -12.1 -14.5 - 16.2 -11.4 -14.1
5-10
-0.10 0.11 0.29 0.14 -
-
0.04 0.17 -0.38 -0.42 -0.07
0.10 0.10 0.1 1 0.15 0.19 0.17 0.22 0.23 0.21 0.24 0.26 0.26
12
-0.70 -0.51 -0.46 -0.52
-0.58 -0.56 0.32 0.27 -0.70
-0.55 -0.54 -0.55 -0.55 -0.53 - 0.48 - 0.47 - 0.46 -0.41 - 0.46 - 0.45 - 0.42
11
0.12 0.04 -0.02 -0.02 -
0.01 -0.03 0.30 0.30 0.07
0.01 0.05 0.03 0105 -0.13 0.02 - 0.03 - 0.06 -0.04 -0.08 -0.04 -0.12
13
-
0.14 -0.21 -0.62 -0.28
-0.06 -0.30 -1.19 -1.54 0.10
-0.18 -0.18 - 0.20 - 0.28 - 0.36 -0.36 - 0.47 -0.51 -0.51 -0.53 -0.58 -0.62
lJll
S(1)a S(1)a S(1)a-HC S(l)a Sub
S(1)a S(1)a HC S(2)a-HC S(1)a
Conformation
Table 7 Conformation of ring A for 3-0x0-A4-steroids. The endocyclic torsion ungles (decimal degrees) are listed in columns 2-7; I,, l,, and I, (A) w e distances of atoms C-I, C-2, and 0 - 3 from the leust-squares plane through atoms C-3, C-4, (2-5, and C-10
$
&
Q
si%
2
ul
- 59.3 - 44.0
- 58.7
52.1 51.2 49.3 54.9 57.1 56.6 58.0 51.2 54.0
(iv) 9P,lOa-steroids (72) -45.7 (73) -48.3 (74) - 53.8 (75) - 48.4 (76) -47.7 (77) -43.3 (78) -46.5 (79) -51.7 (83) - 52.9
(v) Other configurations (80) 52.3 (81) 46.3 (82) 52.0
- 52.9 - 55.6 - 55.9 - 54.8 - 53.3
- 56.7 - 53.9 - 53.9
41.2 39.6 42.9 47.4 38.5 33.3 50.4 39.9
34.1 39.4 11.0
- 38.0 -21.4 - 25.0
- 29.5 - 24.8 - 19.3 - 30.0 - 34.1 - 39.2
40.8 39.4 34.5 27.9 43.0 49.5 26.9 28.3
13.0
- 8.7
0.4
2.2 -4.1 - 3.7 0.1 5.6 12.3 10.2 - 5.2 - 3.3
- 6.3 0.3 - 14.9 - 20.9 3.4 11.4
- 11.7 - 15.2
- 2.6 - 3.0
- 6.4
5.0 6.3 - 3.8 6.7 3.3 0.4 0.2 2.6 I .5
2.0 - 4.4 -4.3 - 3.5 - 4.0 - 6.5 - 22.2
- 4.8
- 20.4 - 17.0 - 30.0
16.9 20.4 31.8 17.4 17.2 15.1 17.8 25.3 27.8
- 10.5 - 12.1 - 14.1 - 20.5 - 8.7 - 2.8 - 20.6 - 5.6 0.40 0.55 0.04 0.12
- 0.28 -0.14 -0.63 - 0.58
- 0.49 - 0.75
- 0.62
0.12 0.23 - 0.24
-0.14 - 0.02 0.16 -0.13 -0.19 - 0.29 - 0.24 0.07 0.05
-
-
0.50 0.6 1 0.72 0.55 0.49 0.39 0.45 0.67 0.70
0.35 0.32 0.23
-0.35 - 0.28 - 0.43
0.07 0.14 + 0.24
0.07 - 0.08 -0.11 - 0.06 0.10 0.21 0.14 - 0.14 - 0.05
- 0.25 - 0.03 0.09 0.02
-
-0.23 -0.11 -0.11
+0.32
- 0.48
-0.19
- 0.27 - 0.03 0.22 - 0.23 - 0.39 -0.76 - 0.54 0.11 0.07
- 1.42 - 3.84 - 0.06 -0.21
-
-0.53
- 1.01 - 1.16
HC HC S( 1)a-H C S( 1)a HC-S(2)/? S(2)P S( 1)a B(dist.)?
s
b
x $3 4
552
Terpenoids and Steroids
equatorial 6a-hydrogen leads to virtually the same shape of ring A as noted in the 6~-bromo-compound(59). The effect of the bulky 2p-acetoxy-groups in (56) and (57) is even more drastic, leading to inversion of sign of torsion angles 5-10, 1-10, 1-2, and 2-3, i.e. to an inverted H C or S(2)ct-HC conformation, in which atoms 1 and 2 are at p- and a-positions respectively. A similar inversion of ring A is observed in the 9,10-seco-compound (142) (vitamin D anal~gue).’~’The effect has been discussed extensively by Duax et in connection with the biochemical activity (see Section 10)and by Jacobs,’39 who draws attention to the unusual type of ring junction in these compounds, where the torsion angles about the common bond C-5-C-10 (junction of rings A and B) have identical signs. The 2P-methyl-19-nortestosterone derivative (55) shows the “normal” S( 1)ct conformation. The same overall shape is also found in the 2,2,6~-trichloro-compound (54); note, however, the inversion of sign of torsion angle 4-5 and the enlargement of angle 5-10. Analogous observations may be made in the less numerous family of 9p,lOasteroids [(72H79),(83)]. The shape of ring A in the 6a-methyl compound (74), in which ring B has a twist-boat conformation, is very similar to that of the 6pbromo-compound (59). In the 6P-fluoro-6a-methyl derivative (75), however, the strain in ring B is transmitted to ring c, which also adopts a boat form, and not to ring A, which is essentially an undisturbed sofa form. Introduction of a 9psubstituent does have some effect on the torsion angles of ring A [(77)and (78)],but clearly not as much as 9a-substitution in 9a,lOP-steroids [(68) and (69)]. The difference in behaviour is probably related to the difference in orientation of the 9-substituent, which is axial to both rings B and c in 9a,lO~-compoundsbut axial to B and equatorial to c in steroids with 9P,lOct-configuration. The positive l J l 1 ratio in (79)should be ascribed to the non-bonded interaction of the equatorial lla-hydroxy-group and the la-hydrogen, as a result of which C-1 is pushed upwards, accompanied by an increase of torsion angles 1-10 and 5-10 and a decrease of angles 2-3 and 3-4. The compound with 9P-configuration (82) is very remarkable in that it shows the largest 1,/11 ratio in ring A yet observed, in addition to twist-boat forms in both rings B and c. In the past decade several attempts have been made to correlate the chiroptical properties (0.r.d and c.d.) of ap-unsaturated ketones with the molecular geometry. A survey has been published by Crabbe.’40 Although useful generalizations have been found, the subject does not seem to be closed. It seems worthwhile, therefore, to compare the available information on the conformation of 3-oxo-A4steroids in the crystal with the chiroptical properties displayed in solution. The signs of the Cotton effects associated with the n-n* transition (A ca. 335 nm) and longest-wavelength n-n* transition (A ca. 240 nm) have been correlated 13’
’
38 3q
C. Knobler, C. Romers, P. B. Braun, and J . Hornstra, Acta Cryst., 1972, B28, 2097. W. L. Duax, C. Eger, S. Pokrywiecki, and Y. Osawa, J . Medicin. Chem., 1971, 14, 295. H . J. C. Jacobs, Thesis, Leiden, 1972. P. Crabbe, ‘O.R.D. and C.D. in Chemistry and Biochemistry. An Introduction’, Academic Press, New York, 1972.
553
Steroid Conformations,from X-Ray Analysis Data
with the chirality of the C=C-C=O chromophore. From the data in Table 7 on unsubstituted 3-0x0-A4-steroidsit is clear that even in the solid state the shape of ring A is rather flexible, giving rise to torsion angles 3-4 of different sign, as well as to enone chiralities of different sign (not listed in Table 7). This flexibility, therefore, precludes the possibility of confirming or disproving the enone chirality rule from X-ray crystal data. However, it is reasonable to assume that the flexibility is sufficiently restricted not to induce major differences in the orientation of (pseudo) axial or equatorial hydrogen atoms or substituents in the immediate vicinity of the enone chromophore. This being the case one may try to correlate the nature and relative disposition of substituents on carbon atoms 2,6, and 10 with the sign and intensity of the Cotton effects. This is essentially the approach advanced by Burg~tahler'~' and by H ~ d e c . According '~~ to this approach the Cotton effect in the band at ca. 215 nm, the electronic nature of which is not yet fully known, is dominated by the chirality contribution of the pseudoaxial bond at C-2, whereas the chirality of the 240 nm n-n* transition is controlled by the axial substituent at C-6; the Cotton effect of the 335 nm n-n* transition depends on the nature of the axial substituents at C-6, C-2, and C- 10.
Table 8 Correlation between geometry and chiroptical properties of 3-oxo-A4steroids Axial substituent at c-2" C-6b
H+
c1+ Me
+
H H H H H H H H H H H
+ + + + +
Cotton efSect 335nm'
Hc1 HH Br CH, H H H
H F F H
-
+ (-15
-
+
+ +
d
-
+ + - 7 - 7
+ +
+
215nm
+
-d -d
+d
+
+ + H +
240nm
+-
+ + -
+d
-
(230) -d
-
" The sign refers to the torsion angle with the C=O bond. * The sign refers to the torsion angle with the C=C bond. 'In the case of a double-humped c.d. curve the sign of the longest-wavelength lobe is given first, brackets indicating a lobe of minor intensity. Inferred from 0.r.d. curve.
For a number of compounds of Table 7 we have indicated in Table 8 the nature of the (pseudo) axial substituent at C-2 and C-6, in addition to the sign of the torsion angle with the C=O and C=C bond, respectively. With the exception of compound (55), where it is hydrogen, the substituent at C-10 is invariably a 14' 14'
A. W. Burgstahler and R. C. Barkhurst, J . Amer. Chem. SOC.,1970, 92, 7601. R. N. Totty and J. Hudec, Chem. Comm., 1971, 785.
554
Terpenoids and Steroids
methyl group and has therefore been omitted. The Cotton effect data are in part from the literat~re,'~'in part from our measurements. The Table shows a one-to-one correspondence of the 215 nm c.d. band to the orientation of the C-2 substituent. However, the examples are admittedly rather small in number and not very varied in nature. As to the 240nm band, if we assume that the chirality contributions of hydroger, dnd fluorine are opposite in sign to those of other substituents (cf. cc-axially substituted saturated ketones), then nearly all these compounds show a correlation between the sign of the 240 nm Cotton effect and the nature and orientation of the axial substituent at C-6. The 2P-substituted compounds (55), (56), and (57) are exceptions; it should be noted, however, that the sign of the Cotton effect in these compounds has been inferred from the tailing of the 0.r.d. curve at shorter wavelengths. The possibility that a weak Cotton effect in the 3'1 ,,ln region has been obscured by a strong Cotton effect of opposite sign at shorter wavelengths should not be ignored. Subject to the same assumptions concerning the contribution of fluorine and hydrogen, we find the same correspondence between the sign of the Cotton effect at ca.335 nm and the substituent at C-6. Again the 2P-substituted steroids are exceptional ; here, however, the contribution of the axial substituents at C-2 has to be taken into account. In conclusion, it is clear that many more data are required to establish beyond doubt the type of relationship discussed above. The limited evidence available seems to indicate that the approach may be promising. Table 9 contains the standard bond lengths, standard errors, and estimators of 3-0x0-A4-steroidswith normal (9a,10P) and retro (9B,10a)configurations. Bonds C-8-C-9 and C-13-C-17 excepted, the agreement between the corresponding average bond lengths of the 901,108-and 9,!I,lOa-systems is surprisingly good. We note only a slight discrepancy between the values of the standard deviations and the estimator S . Although ring A occurs in a variety of conformations there is only a slight or no effect on the bond lengths, which may be considered to belong to the same statistical population. Note that the bond C-9-(2-10 is longest. Furthermore it can be seen that the regular sp3-sp3 bonds C-6-C-7, C-7-C-8, and C-8C-14 are rather small. A first requirement of V F calculations is a reproduction of these striking features. The field AL (Table 10) applied to 20-methylpregn-4-en3-one meets this condition.* Standard bond angles, standard errors, and estimators of 9a,lOB- and 9p,10~-30x0-A4-steroids are tabulated in Table 11. With few exceptions (notably angles 9-1&19, 6-7-8, 148-9, and 9-11-12) reversal of the configurations at C-9 and C-10 has little effect on the bond angles throughout the skeleton. The agreement between calculated and standard bond angles in the case of the 9a,lOP-configuration (force field AL) (Table 12) is less satisfying than for the corresponding bond lengths. Nevertheless the overall trend for calculated and observed values is the same.
* The other force fields used in this work cannot handle the 3-0x0-A4-system without the addition of new parameters. Therefore we decided to use only field AL, despite its shortcomings as far as prediction of angles is concerned. Similar remarks pertain to Table 12.
555
Steroid Conformations from X-Ray Analysis Data
Table 9 Standard bond lengths (A), standard deviations, and estimators 9a,lOB- and 9/?, l0a-3-oxo-A4-steroids Bond 1-2 1-10 2-3 3-4 4-5 5-10 5-6 6-7 7-8 8-9 9-10 9-11 11-12 12-13 13-14 8-14 14- 15 15-16 16-17 13-17 13-18 10-19
9a, 1Op 1.541 1.531 1.493 1.457 1.339 1.523 1.497 1.523 1.529 1.542 1.563 1.538 1.539 1.528 1.541 1.526 1.534 1.545 1.541 1,554 1.534 1.543
104g 11 11 11 11 11 11 11 12 12 12 12 12 12 12 11 12 11 11 11 11 11 12
104s 36 28 59 37 36 30 32 32 38 37 39 20 43
44 21 23 41 34 46 40 42 35
9p, 1Oa 1.543 1.526 1.492 1.454 1.340 1.525 1.508 1.513 1.538 1.563 1.572 1.547 1.540 1.535 1.536 1.522 1.537 1.546 1.541 1.534 1.540 1.550
104~ 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18
of
1045 40 48 91 74 59 32 96 92 47 51 35 45 60 27 41 59 58 89 19 99 75 52
Table 10 Comparison of standard bond lengths (A)of 3-oxo-A4-steroids (9a,lOflconfiguration) and calculated values (forcefield A L ) of 20-methylpregn4-en-3-one Bond 1-2 1-10 2-3 3-4 4-5 5-10 5-6 6-7 7-8 8-9 9- 10
Exp. 1.541 1.531 1.493 1.457 1.339 1.523 1.497 1.523 1.529 1.542 1.563
Calc. 1.531 1.552 1.498 1.458 1.332 1.530 1.506 1.525 1.531 1.544 1.569
Bond 9-11 11-12 12-13 13-14 8-14 14-15 15-16 16-17 13-17 13-18 10-19
Exp. 1.538 1.539 1.528 1.541 1.526 1.534 1.545 1.541 1.554 1.534 1.543
Calc. 1.540 1.544 1.530 1.541 1.531 1.523 1.546 1.554 1.558 1.545 1.542
Terpenoids and Steroids
556
Table 11 Standard bond angles (decimal degrees) of 9a,lOB-and 9~,lOa-3-0~0A4-steroids, their standard errors, and estimators Angle 1@-1-2 1-2-3 2-34 3-4-5 4-5-10 5-10-1 4-5-6 1-10-9 1-10-19 9-10-19 9-10-5 1&5-6 5-6-7 6-7-8 7-8-9 8-9-10 7-8- 14 10-9-1 1 8-14-13 148-9 8-9-1 1 9-1 1-12 11-1 2-1 3 12-13-14 15-14-8 12-1 3-1 7 12-1 3-1 8 14-1 3-1 8
9a, 1Op 113.2 111.4 116.4 123.3 123.2 109.8 120.2 108.6 109.9 112.1 108.4 116.7 112.4 111.7 110.1 113.7 111.4 113.2 113.5 108.8 111.8 113.2 110.5 108.6 119.3 116.9 109.9 112.4
lO20
7 7 7 7 7 7 8 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 8 9
102s 38 35 36 36 24 42 36 39 32 24 37 32 41 27 29 33 37 33 19 17 34 43 38 22 28 34 48 30
9B,lOa ii3.9 111.3 117.5 123.6 122.7 108.3 119.3 108.3 108.6 114.0 110.0 117.9 114.2 115.0 111.9 115.4 111.8 114.4 113.3 112.1 110.5 116.7 110.9 108.2 118.3 116.1 110.9 113.6
lO20 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8
102S 31 52 41 24 33 40 38 26 24 29 45 32 200 180 34 43 86 66 43 35 46 58 34 41 30 35 35 28
Table 12 Comparison of standard bond angles (decimal degrees) of9a, lop-3-0x0A4-steroids and calculated values (force Jield AL) of 20-methylpregn4-en-3-one Angles 10-1-2 1-2-3 2-34 34-5 4-5-10 5-10-1 4-5-6 1-10-9 1-10-19 9-10-19 9-10-5 10-5-6 5-6-7 6-7-8
Exp. 113.2 111.4 116.4 123.3 123.2 109.8 120.2 108.6 109.9 112.1 108.4 116.7 112.4 111.7
Calc. 113.6 109.7 116.8 124.6 122.5 107.7 121.4 110.0 108.9 109.1 109.1 115.4 110.7 110.7
Angles 7-8-9 8-9-10 7-8-14 10-9-1 1 8-14-13 14-8-9 8-9-1 1 9-1 1-12 11-12-13 12-1 3-14 15-14-8 12-1 3-1 7 12-1 3-1 8 14-13-18
Exp. 110.1 113.7 111.4 113.2 113.5 108.8 111.8 113.2 110.5 108.6 119.3 116.9 109.9 112.4
Calc. 110.7 114.7 112.2 116.7 115.7 111.4 110.7 114.5 113.5 106.2 120.1 117.0 109.8 113.8
Exp.
44.7 -54.9 34.2 - 3.7 - 4.5 -14.8
Bond
1-10 1-2 2-3 3-4 4-5 5-10
Ring
A
1.2 0.4 1.2 1.4 1.2 1.2
S
50.4 -57.2 31.3 - 1.7 - 4.2 -19.8
Calc. 5-10 5-6 6-7 7-8 8-9 9-10
Bond 49.5 -49.5 50.3 -53.9 56.6 -52.2
Exp.
Ring B
1.1 1.8 1.8 1.3 0.7 0.7
S
57.7 -55.8 51.4 - 45.8
- 55.7
48.1
Calc. 8-9 9-11 11-12 12-13 13-14 8-14
Bond
-51.2 51.8 -54.5 56.8 -60.6 57.2
Exp.
Ring c
0.6 1.o 1.0 0.7 0.4 0.5
S
-48.2 48.4 - 53.8 54.4 -57.3 56.1
Calc.
Table 13 Comparisonof standard torsion angles (decimal degrees) of 3-oxo-A4-steroia!swith 9a,I Ob-configuration,their estimators S , and the calculated values (force field A L ) of 20-methylpregn-4-en-3-one.Compounds (57), (56), (80),and (81) were not included in the calculation. The standard deviations are 0.1"for all angles
wl wl 4
3
b
2;.
9
b
~
9
F !XI
6s
'
3s.
s
b'
n
558
Terpenoids and Steroids
Finally, standard endocyclic torsion angles of rings A, B, and c (9a,lOP-configuration) are compared with the calculated values (field AL) of 20-methylpregn4-en-3-one (Table 13). Not included in the statistical calculation are the compounds (57) and (56) (inverted A-rings) and (80) and (81) (8a,l4/3-configuration). The rather poor agreement is hardly suprising in view of the several different conformations of ring A and the variety of substituents at positions 6,7,9, 11, 12, 16, and 17. Nevertheless the VF calculations predict the correct signs of all torsion angles and the correct order of magnitude. Ring c is most staggered about bonds C-12-C-13, C-13-C-14, and C-8-C-14 and sqmewhat flattened about C-8C-9 and C-9-C-11. This effect is also reprodcced by the calculations. The agreement is less satisfying for ring B where the forcefield predicts large puckering about bonds C-5-C-6 and C-6-C-7, whereas the standard torsion angles are largest for bonds C-7-C-8 and C-8-C-9. Since the estimator values are about ten times as large as the corresponding standard deviations, the comparison with standard torsion angles is hardly justified. The calculation of torsion angles is best tested with standard values from non-substituted compounds, but even then the agreement is rather poor. 5 The A5.'-System
The number of A5-7-steroidsanalysed by X-ray diffraction methods is very limited. This conjugated double-bond system occurs in (analogues of) ergosterol, lumisterol, pyrocalciferol, and calciferol, e.g. the compounds (1 lo), (11l), (112), (113), (142), and (143). These isomers, and others such as tachysterol and precalciferol, belong to the vitamin D series. Their chemistry and stereochemistry have been discussed extensively by Sanders, Pot, and H a ~ i n g a . 'The ~ ~ first three compounds are 9,10-stereoisomers, and calciferol is a seco-steroid, having lost the bond between atoms 9 and 10. For this reason no statistical treatment of the experimental geometrical entities has been applied. The 9a,lOp-, 9P,lOa-, and 9or,10a-A5~7-systems have been inspected by V F calculations (field AL) using the molecules 17p-methylandro~ta-5~7-diene and 9p, 1Ocr- and 9c(,lOa-androsta-5,7-dien-3/3-01,respectively. The results of these calculations are listed in Tables 14,15, and 16, which also present the experimental bond distances, valency, and torsion angles of compounds (1lo), (11l), (1 13),and (142). In view of the uncertainties and the approximate nature of the force field used for this conjugated double-bond system the agreement seems quite satisfactory. A serious discrepancy, however, is bond length C-13-C-14 (see Table 14). The assumption of combined large standard errors in observation and calculation is not sufficient to explain the observed differences for this particular bond, and we cannot offer an adequate explanation for this discrepancy. It reminds us that the calculations must be considered as a first step to our goal, an accurate prediction of the geometry of this class of compound.
* 43
G . M. Sanders, J. Pot, and E. Havinga, Fortschr. Chem. org. Naturstoffe, 1969, 27, 131.
559
Steroid Conformationsfrom X-Ray Analysis Data
Table 14 Comparison of observed and calculated bond distances (A) in ergosterol (1lo), lurnisterol(l1l),pyrocaIciferol(113),and calciferol(142). Calculated and values (Jield AL) are derived from 17fl-methylandrosta-5,7-diene 98,l Oa- and 9a, 1Oa-androsta-5,7-dien-3#I-o1. Standard deviations are 0,014, 0.006,0.004, and 0.004 A, respectively Bond 1-2 1-10 2-3 3-4 4-5 5-10 5-6 6-7 7-8 8-9 9-10 9-11 11-12 12-13 13-14 8-14 14- 15 15-16 16-17 13-17 13-18 10-19
(110) Exp. Calc. 1.550 1.531 1.530 1.541 1.523 1.500 1.523 1.520 1.510 1.512 1.540 1.530 1.340 1.343 1.452 1.450 1.340 1.342 1.520 1.519 1.540 1.567 1.547 1.550 1.536 1.560 1.525 1.540 1.527 1.580 1S O 5 1.500 1.510 1.532 1.550 1.547 1.560 1.544 1.540 1.560 1.544 1.540 1.550 1.545
(111) Exp. Calc. 1.530 1.531 1.545 1.543 1.523 1.521 1.520 1.523 1.507 1.508 1.530 1.535 1.345 1.344 1.461 1.450 1.337 1.341 1.523 1.512 1.554 1.561 1.537 1.538 1.542 1.544 1.561 1.539 1.548 1.531 1.494 1.496 1.531 1.534 1.547 1.547 1.560 1.545 1.548 1.532 1.537 1.547 1.545 1.539
(113) Exp. Calc. 1.539 1.531 1.545 1.534 1.512 1.528 1.522 1.514 1.511 1.515 1.522 1.524 1.338 1.343 1.456 1.449 1.330 1.341 1.519 1.515 1.567 1.570 1.555 1.547 1.541 1.550 1.529 1.530 1.567 1.522 1.500 1.505 1.524 1.535 1.552 1.549 1.554 1.548 1.532 1.563 1.527 1.545 1.561 1.550
(142) Exp. 1.516 1.499 1.500 1.510 1.536 1.494 1.323 1.442 1.359 1.485
-
1.529 1.551 1.535 1.566 1.490 1.522 1.533 1.554 1.559 1.539 1.317
In view of the results with saturated and 3-0x0-A4-steroidsthe agreement for valency and torsion angles is surprisingly good. Considering the torsion angles (Table 16) we note that the A-rings of (1lo), (11l), and (113) are distorted chairs. The puckering about the bonds C-2-C-3 and C-1-C-2 is large. The VF calculations (Tables 14, 15, and 16) nicely reproduce this feature. In agreement with our conjecture that the moieties C-10-C-5-C-6-C-7 and C-6-C-7-C-8-C-9 should be planar, ring B is a 1,3-diplanar ring with very small (but not zero) torsion angles about the bonds C-5-C-6 and C-7-C-8. The shape of this ring type was first discussed by Bucourt.' More recent calculations by Favini et ~ 1 . have l ~ resulted ~ in the values 0, - 17.5,0, 31.2, -44.7, and 31.2" for the torsion angles of unsubstituted 1,3-cyclohexadiene. Electrondiffraction investigations of this molecule confirm the predicted conformation.145-147 Traetteberg'46 reported torsion values of 0, - 18, 0, 32, -46, and L44 14'
146 14'
G . Favini, F. Zuccarello, and G. Buemi, J . Mol. Structure, 1969, 3, 385. G. Dallinga and L. H. Toneman, J . Mol. Structure, 1967, 1, 11. M. Traetteberg, Acta Chem. Scand., 1968, 22, 2305. H . Oberhammer and S. H . Bauer, J . Amer. Chem. Soc., 1969, 91, 10.
Terpenoids and Steroids
560
Table 15 Valency angles (decimal degrees) of compounds (1 lo), (1 1l), (1 13), and (142) and corresponding calculated values (142)
Angle
Exp.
Calc.
Exp.
Calc.
Exp.
Calc.
Exp.
1&1-2 1-2-3 2-3-4 3 4 5 4-5-10 5-10-1 4-5-6 1-10-9 1-10-19 9-1&19 9-10-5 10-5-6 5-6-7 6-7-8 7-8-9 8-9-10 7-8- 14 1&9-11 8-14-13
115 111 111 114 118 112 123 110 108 112 110 119 123 121 119 113 124 113 115 116 114 116 110 106 119 116 112 109
115.8 109.9 109.7 113.0 118.1 109.8 120.7 109.5 110.4 106.9 109.1 119.3 121.6 121.4 118.9 111.5 120.5 115.2 112.2 116.6 114.2 114.2 111.1 108.4 122.4 115.4 110.1 109-6
114.0 110.9 110.5 113.6 117.9 110.8 121.1 107.8 110.6 111.3 109.2 120.4 121.5 120.8 118.7 112.6 126.5 114.5 112.6 114.8 109.7 112.1 113.4 108.5 119.8 116.0 110.4 110.4
115.8 109.8 108.8 112.2 117.5 109.8 121.4 109.5 110.6 110.2 107.8 119.8 121.3 120.4 118.6 111.1 124.8 118.3 111.1 116.3 111.2 112.9 115.3 109.9 123.3 116.0 109.6 111.2
114.2 110.9 111.4 113.3 118.2 111.3 120.4 110.9 109.3 106.8 112.4 120.7 122.1 121.4 121.1 113.0 125.8 117.3 110.2 111.5 106.2 111.9 112.9 107.6 122.1 117.1 110.9 111.4
114.9 109.6 110.0 113.4 117.0 109.2 120.8 109.8 110.6 109.0 110.5 120.1 121.5 121.3 120.0 111.4 123.0 122.7 110.2 114.6 107.7 112.2 114.0 109.2 126.3 115.7 109.7 111.5
110.9 112.3 111.5 113.9 110.7 114.0 121.8
148-9 8-9-1 1 9-1 1-12 11-12-13 12-13-14 15-14-8 12-1 3-1 7 12- 13-1 8 14-1 3-1 8
-
122.3
-
127.5 127.3 126.5 124.3 -
122.0
-
113.2 113.4 112.6 113.1 110.4 107.8 -
115.1 111.0 109.5
32”.* The cited and experimental values roughly agree for the endocyclic torsion angles of ring B in compounds (1lo), (1 1l), and (113). Apparently only small distortions are required to fit the 1,3-diplanar ring in the A5g7-steroidskeleton. The observed torsion angles about the bonds C-5-C-6 and C-7-C-8 are small (7”) but not zero, indicating that the butadiene moieties are not quite planar and that small distortions about the double bonds are possible. A recent electron-diffraction study of tetrameth~lethene’~~ seems to corroborate this conclusion. It can be seen that the force field predicts these features and reproduces the correct signs for the torsion angles about C-5-C-6 and C-7-C-8. From mechanical-model considerations (planar double bonds) two ring B conformations can be envisaged, characterized by right-handed (+) and lefthanded (-) chirality about the C-=-7 bond. The question is whether or not 148
S. W. Eisma, C. Altona, H. J. Geise, F. C. Mijlhoff, and G. H. Renes, J . Muf. Structure, 1974, 20, 25 I .
* Inversion of ail signs of the torsion angles in 1,3-cyclohexadiene of course yields the energetically equivalent enantiomer, whereas two different conformations are envisaged in ring B of A5+’-steroids.
56 1
Steroid Conformationsfrom X-Ray Analysis Data
Table 16 Endocyclic torsion angles (decimal degrees) of compounds (1 lo), (1 1l), (1 13), and (142) and corresponding calculated values Bond Ring A 1-10 1-2 2-3 3-4 4-5 5-10 Ring B 5-10 5-6 6-7 7-8 8-9 9-10 Ring c 8-9 11-12 12-13 13-14 13-14 8-14
Exp.
Calc.
(142) Exp.
40.3
-43.6 55.9 -60.7 57.9 -49.4 40.3
-44.3 54.9 - 57.6 51.8 - 43.7 39.1
- 49.7 58.4 - 57.5 52.9 - 47.6 43.9
- 55.0 55.1 - 53.3 50.8 -49.1 51.6
-33.1 -0.8 18.5 3.1 -38.3 50.6
- 24.8 2.7 9.5 3.5 - 25.7 34.5
- 31.5 3.3 12.9 3.4 -31.9 43.8
-
33.3 - 47.9
-29.9 -0.4 15.5 4.0 -35.4 45.9
-36.1 37.6 - 51.3 61.5 - 59.6 47.9
17.7 -59.9 40.1 18.3 -61.1 42.3
3.7 -49.4 41.4 11.9 -57.7 50.8
- 62.2
- 57.6 50.9 -51.8 52.8 - 55.7 61.5
- 49.4 49.6 - 54.5 56.5 - 57.8 54.8
Exp.
Calc.
40 -55 59 - 54 42 - 35
44.6 - 56.3 59.1 - 54.7 46.6 - 39.6
34 -6 -11 -2 30 -44
- 32 36 - 53 62 - 60 46
36.2 - 6.2 - 13.3 - 2.2
-46.0 56.5 -57.9 51.8 -44.5
56.6 - 55.6 53.5 - 57.5 64.9
-
two energy minima ( = stable conformers) exist side by side in solution. In order to settle this point, computer experiments were carried out on the 9a,lOP-system, forcing ring B to adopt right-handed chirality. The resulting molecular model was unfavourable by several kcal mol- compared with the left-handed system. Moreover, when all co-ordinates were allowed to ‘relax’ the model flipped over into the low-energy conformer. We conclude that probably but one stable minimum exists. Ring c of compound (110)is a quite distorted chair. The large puckering about bonds C- 12-C- 13 and C- 1 3 4 -14 and small puckering about bonds C - 8 4 - 9 and C-9-(2-11 are neatly reproduced by the VF calculation. The agreement is less satisfying for (113), where theory predicts a flatter ring than the experiment indicates. Nonetheless theory and experiment agree that ring c of (113) is most staggered about bond C-8-C-14. It can be seen that the V F calculations reproduce the correct boat conformation for ring c in lumisterol(l1l).149 The iteration converged to a relatively steep potential well for the correct conformation. From a comparison of the torsion angles in the three configurational isomers (1lo), (11l), and (113) it is clear that ring B in the syn-isomer (113) is much flatter than in the two anti-isomers (1 10)and (111)(although considerably more puckered than suggested by Dreiding models with planar ethylene moieties). This is no doubt connected with the conflicting demands imposed on ring B by the 149
A. J . de
Kok and C. Romers, to be published in Acra Crysr.
562
Terpenoids and Steroids
a-junction to ring A as well as to ring c . This situation has been discussed extensively elsewhere.' 39 Suffice it here to mention that the B/C junction in compound (1 13) is another example of the rather unusual type of ring junction [also noted in compounds (56) and (57) ;see Section 41 involving one sp3 and one sp2 bridgehead carbon atom, with torsion angles of the same sign about the common bond. In all three 5,7-dienes the sign of torsional angle 6-7, as deduced from the observed Cotton effects on the basis of the skewed-diene rule' 5 0 accords with the results obtained from the X-ray analysis. However, an attempt to correlate the intensity of the Cotton effects with the magnitude of the torsion angles meets with considerable difficulties. In a recent detailed analy~is'~'it is suggested that in addition to torsion of the single bond C-6-C-7 the twisting of the formal double bonds C-5-C-6 and C-7-C-8 might play a role. Possibly the influence of extrachromophoric groups, e.g. allylic axially disposed groups, should also be taken into account. A theoretical evaluation of the relative importance of these three factors in the determination of sign and intensity of the Cotton effect would be most welcome.
6 The Conformation of Ring B in Oestranes and A5-Compounds In Table 17 are listed the torsion angles of ring B of As('')- and A'~3~5('0)-oestranes and A'-compounds. The experimental values of gaseous cyclohexene'5 are included at the bottom of the list. Since an approximately zero torsion angle would be expected about bond C - 5 4 - 1 0 for oestranes and about C - 5 4 - 6 for A'-steroids, it is not surprising that the half-chair form is predominant among a family of distorted chairs, sofas, in-between forms, and even nearly ideal boat conformations. The diversity of forms is much smaller for A'-steroids than for oestranes, but a close inspection of Part (iii) of Table 17 reveals that even for this class of compound the shape of ring B is not constant. It seems tempting to attribute the diversity of conformations to the direct influence of substituents. It can, however, be surmised that substituents governing the gross shape of molecules are a dominant factor in the ultimate crystal structure, thereby indirectly fixing the conformation. It is uncertain whether a flexible molecule in other surroundings (e.g.in solvent or on substrate) adopts the same ultimate shape. On the other hand, crystal-structure analyses of a large variety of substituted steroids indicate which conformations are at least possible. One might even conjecture that flexibility is an essential requirement for activity of steroid hormones. Whereas flexibility of ring A seems to be vital to the biological activity of testosterone, progesterone, and corticosteroids, it is the variable shape of ring B which conditions the action of the female sex hormones oestradiol and oestrone and their derivatives.
'" 15'
H . J . C. Jacobs and E. Havinga, Rec. Trau. chim., 1965,84,932. J. F. Chiang and S. H. Bauer, J . Amer. Chem. Soc., 1969,91, 1898.
6-7 47.8 43.1 43.1 45.4 48.3 39.5 33.7 42.9 36.3 48.0 56.3 33.8 54.0 60.5 44.3 43.0 41.0 45.6 48.2 39.3 46.7 47.6 48.0
5-6
- 15.5 - 10.7
- 9.3 - 17.0 - 14.3 - 5.2 - 3.0 - 14.7 - 6.4 - 18.7 - 24.2 - 32.9 - 23.0 - 36.4 -11.9 - 14.0 - 18.0 - 14.7 - 15.3 - 8.7 - 15.0 - 15.8 55.0 -69.1 - 66.6 - 62.5 - 62.5 - 64.0 - 64.8 -60.1 -1.9 - 64.0 - 62.2 - 65.3 - 66.0 - 62.0 - 65.7 - 67.8 - 64.5 -66.1 - 67.9 - 6.0
- 65.1 - 64.2
- 62.6 - 63.7
7-8
52.0 52.5 53.5 59.7 59.5 53.7 59.9 51.1 28.3 -32.4 41.0 36.4 52.1 58.0 57.0 52.2 52.1 57.6 50.9 53.1 56.0
43.3 51.0
8-9
- 31.2 - 26.7 - 30.8 - 22.5 4.2 35.2 - 10.0 - 2.3 - 20.0 -31.0 - 34.0 - 23.2 - 19.9 - 28.5 - 20.1 - 20.3 - 58.0
- 19.4 - 25.1 - 20.7 - 26.5
- 20.1
-11.1
9-10
9P
9PJ4P
9P
Conjiguration
HC-S(8)/? HC-C (distorted) HC S(8M S(8)P HC-C (distorted) S(8)8 HC S(7)a B (flattened) HC-S(7)a S(7b HC HC-C (distorted) C (distorted) HC HC HC-S( 8)P HC HC B
HC HC-S(S)P
Conformation
Table 17 Conformation of ring B of oestranes and A5-compounds. The angles quoted in columns 2-7' are in decimal degrees
o w \
ul
b 3
E.
9
b
5-10
19.0 14.4 16.6 13.1 7.1 20.8 11.0 14.0
15.2
(iv) Cyclohexene (ref:151)
(101)
(loo)
(102) (103) (98) (99) (109) (104) (84)
(iii) A5-Compounds 12.0
Compound
Table 17-continued
0
5.0 0 1.3 0.6 3.5 6.4 - 2.6 3.0 1.o
5-6
15.2
12.0 11.0 16.1 12.3 11.5 14.6 12.8 17.0 13.0
6-7
- 44.9
- 48.0 - 42.0
- 40.8
- 46.1
-42.5
- 44.0 - 39.0 - 46.8 - 40.6
7-8
60.2
62.0 60.0 66.0 60.1 60.6 63.9 60.7 64.0 59.0
8-9
- 44.9
- 45.0 - 50.0 - 47.5 - 46.8 - 44.1 - 42.7 - 48.7 - 44.0 - 43.0
9-10
Configuration
HC (ideal)
HC HC HC HC HC S(8)P HC HC HC
Conformation
Y
Steroid Conformations from X-Ray Analysis Data
565
7 Five-membered (D) Rings It has been shown earlier' 2 , 4 5 , 1 5 2 that five-memberedD-rings can be characterized by the maximum angle of puckering amand the phase angle of 'pseudo-rotation', which are related to the endocyclic torsion angle Q j by the equations
aj = omcos where
j = 0, 1,2,3, or 4
(; +
j6)
6
and
=
144",
and
fl
The adopted numbering of torsion angles in ring D as well as in the other rings is indicated in Figure 3. 2
5 05
3
4
1
43
2
Figure 3 Convention for the numbering of endocyclic torsion angles in rings A,
B,
c, and
D
The configurations and angles A and a,,,of 129 D-moieties (and corresponding to 118 different compounds; see note c to Table 1) are tabulated in Table 18. The following conclusions can be drawn : (i) Irrespective of the configurations of atoms 5 , 8 , 9 , and 10, the phase angles of steroids with normal 13p,14a-configuration are confined to a range of values between + 38.0" and - 42.8", corresponding with D-ring conformations between C,(13)P and C,(14)a. Exceptions to this rule are compounds (70) and (83), having phase angles of - 74.6" and 60.1". They will be discussed below. (ii) The A values of 17-0x0-compounds obeying the first rule always are negative; the lower limit is -42.8" for compound (41). It has been shown earlier12 that the carbonyl group at C-17 requires small torsion angles about bonds C-13C-17 and C-16-C-17. This demand constrains the range of possible conformations to the conformation range between C,(16) and Cs(14)a. This rule also applies when there is an exocyclic double bond C=C or C=N at position 17. An examples is compound (19). (iii) The phase angle of the remaining steroids obeying the first rule is usually confined to the positive part of the quoted range of values. Exceptions to this rule are observed for the compounds (12) and (115) with unusual 5p,14Pconfigurations, the 8-aza-compound (139) with 9P-configuration, and the steroid 5a,17a-pregnane-3P,20-diol(3,4) bearing a 17a-CHOHMe group. (iv) The remaining steroids with A values outside the range + 38 to - 43" have the unusual configurations 13a,14a, 13P,14P, or 13a,14#3,i.e. compounds with cis or inverse trans couplings between rings c and D. H. J . Geise, C . Altona, and C . Romers, Tetrahedron Letters, 1967, 1383.
566
Terpenoids and Steroids
Table 18 Conformation of ring D. The angles are in decimal degrees Compound A @In Conjigurat ion (i) 17B-Hydroxy-compounds(oestranes) 45.9 (119) 20.9 49.1 (120) 22.3 49.8 (127) 21.9 (128) 19.7 46.8 49.8 (129) 22.0 48.5(130) 22.0 (131) 29.6 50.0 50.0 (132) 28.7 46.3 (137) -7.2 52.3 8a (135) 11.5 (121) 17.4 47.2 (ii) 17~-Hydroxy-cornpounds(androstanes) (2) 24.6 47.3 5a 46.5 (85) 21.3 (45) 13.1 47.0 46.3 (46) 17.5 47.1 (98) 27.2 (99) 32.8 49.3 47.7 (87) 17.5 47.3 (92) 13.8 47.0 (51) 19.2 45.9 (83) 60.1 46.6 (26) 15.7 (1) 19.7 48.5 (30) 22.0 57.5 44.5 (52: 17a) 9.2 48.5 (69) 24.1 (iii) 17-Oxo-compounds(androstane and oestranes)
(36) (37) (102) (103) (77) (78) (89) (95) (140) (41) (122) (123) (124) (125) (126) (44) (96)
153
- 11.2
-7.5 - 10.2 327.0 -32.4 -24.1 -7.5 450.0 274.2 -42.8 -19.4 -16.0 - 5.9 -37.1 -18.2 -23.8 -22.3
45.2 45.1 43.0 40.2 44.6 43.8 44.6 24.9 35.0 39.5 42.3 43.2 40.9 42.4 43.1 41.7 30.9
-
-
1301 9P,lOa 9P,lOa 9P,lOa 13a 9P914P -
5b9B
"The nomenclature has been adopted from ref. 153. For instance C,(14) means an envelope with 14 as flap, C2(16) means a half-chair with a two-fold axis running through 16; C,( 1 3)-C2(16) indicates a conformation between C,(13) and C,(16). C. Romers, C. Altona, H. R. Buys, and E. Havinga, Topics Stereochem., 1969, 4, 39.
567
Steroid Conformations from X-Ray Analysis Data
Table 18-continued Compound A @In Conjiguration (iv) 3-Oxo-A4-compounds(pregnanes and androstanes) 19.8 47.6 (60) 24.1 48.5 (69) 9P,10a 19.4 48.8 (76) 9,!?,lOu 19.7 48.0 (75) 12.0 45.5 (59) 46.5 (65) 11.4 11.7 47.8 (58) 4.2 45.6 (67) (74) 0.1 47.6 9&10a (79) 22.7 49.5 9P,lOa (68) 15.2 47.9 1.7 46.2 (42) (61) 18.5 46.3 46.8 (53) 23.1 24.1 49.7 (57) 11.7 46.0 (56) 23.8 46.9 (54) (72) 28.7 49.0 9P,lOa 31.9 47.7 (71) 9P 25.4 45.2 (82) 26.6 48.0 (63) 33.3 8a,14P 129.5 (80) 8a,14p 134.0 36.0 (81) 35.2 58.6 (66) 13.4 45.0 (50) 23.0 47.7 (47) 38.0 46.9 (43) 5.1 45.2 (49) (64) 12.6 46.7 (62) 28.4 50.2 (48) 22.4 49.3 2.7 47.5 9p, 1Oa (73) (55) 33.9 50.0 (70) -74.6 40.8 (v) Remaining compounds -23.3 47.4 (139) 15.8 47.6 (136) 21.8 48.7 (141) 24.3 50.4 (5) 24.6 47.5 (62) 15.5 42.5 (114) (19) -20.8 48.7 (90) 4.6 47.6 2.0 46.7 (91) (3) -37.1 42.1 (4) -4.6 46.1 3.7 45.6 (142) 3.7 42.2 (143) (110) 21.6 47.2 5.0 46.2 (38)
9P 8a --
5P 9P,lOa 9P,lOa -
-
Conformat ion"
Terpenoids and Steroids
568
Table l k o n t i n u e d Compound
A 4.0 21.2 9.0 16.5 8.6 16.1 351.3 11.5 - 22.9 32.2 399.5 7.8 15.1 14.0 6.4 12.0 28.0 6.1 7.5 7.2 26.6 14.4 8.5 - 30.9 27.8 9.0 - 9.2 16.6 20.8 2.3 33.7 22.9 23.8 440.1 25.9 26.5 18.1
@Ill
48.5 45.9 47.9 50.2 46.8 46.6 41.7 48.3 18.8 47.1 41.1 43.3 48.7 48.6 51.7 53.8 44.2 52.1 56.1 50.3 44.1 46.6 45.2 35.7 52.2 49.7 48.3 46.8 48.8 47.1 48.1 44.5 47.1 43.6 49.1 49.8 48.0
(v) The mean (Dmvalue of steroids belonging to the third category is ca. 48". For
17-oxo-compounds we observe the somewhat lower value of 43". Interestingly, the puckering in gaseous cyclopentane' 5 4 amounts to 42.5", whereas the value 37.2" is observed for gaseous cyclopentanone.*' 5 5 L54 55 ' 5 6
W. J. Adams, H. J. Geise, and L. S. Bartell, J . Amer. Chern. Soc., 1970, 92, 5013. H. J. Geise and F. C. Mijlhoff, Rec. Trau. chim.,1971, 90,577. R. A. G . de Graaff and C. Romers, to be published in Acta Cryst.
* This reduction of Om is a phenomenon which has also been encountered in ring A of the saturated 3-oxo-compounds (27), (28), and (29). Although steric hindrance of axial methyl groups at positions 4 and 10 can be invoked for compound (28) (mean puckering angle 49.4") such an explanation holds neither for the serious flattening156 observed in ring A of compound (27) (O,,, = 48") nor for the smaller flattening of ring A of compound (29) ( Oav = 53.6") and the flattening of ring D of 17-oxo-compounds.
Steroid Conformations from X-Ray Analysis Data
569
OH 0
2.2
3r9
26.9
A
20.6 47.0
B
E
28.1
12
-
F
35.3
T3
CH 20H
15
8
56.5
(a) 4
6
y43 12
25.0
0
A 4
27.8 2 r 9
B 6
I
.OH
26.8
(b)
Figure 4 Endocyclic torsion angles of compounds (70)(a)'and ( 8 3 )(b)
The compounds (70)and (83)(Figure 4) deserve special attention. Aldosteronet (70)is highly strained by the presence of two additional five-memberedrings E and F, which results in unusually large torsion angles about bonds C-9-C- 11, C- 11C-12, C-12-C-13, and C-13-C-14 in ring c. Instead of its usual value of ca. -40" the torsionangle@(14-13-17-16)is reduced to - 10.6",and@(17-13-14-15) is reduced from 47" to 31.7". It is difficult to surmise why, in particular, torsion angle @( 14-13-17-16) has suffered this reduction, and possibly future force-field calculations can pinpoint which interaction terms are responsible for the observed change. The total effect is, however, a rather low Om value (40.8"),a very low A value (- 74.6"), and the unusual C2(17)-C,(15) conformation. W . L. Duax and H. Hauptman, J . Amer. Chem. SOC., 1972,94, 5467. The torsion angles of ring D given in the original paper' 5 7 should be shifted clockwise by one bond. 157
570
Terpenoids and Steroids
Compound (83) contains a seven-membered ring c with @(12-13-14-8) = The sum rule' demands a value of ca. 109" for the sum of@(12-13-14-8) and (D(17-13-14-15). The corollary that q17-13-14-15) should be reduced to a lower value (observed 39.3') in order to relieve (at least partially) the strain in the junction of rings c and D is inescapable. Since in a 'strain-free' ring D amamounts to ca. 47", it can be deduced that its conformation shoots through the 'magical' barrier A = + 36" to the value A = 60.1" in which @(14-13-17-16) now acquires the largest value ( - 45.6"). Note that the molecules (3) and (4) are 17a-pregnane derivatives. They are tightly hydrogen-bonded with a water molecule in the crystal lattice.' 5 8 The V F calculations of Altona and HirschmannZ4indicate that the phase angle A of 17a-pregnane-20-diols adopts a value of ca. -36", in contrast to the positive value (&-36") found for 17P-pregnanes. In view of these calculations 17apregnane-3P,20-diol(3) has a 'normal' A value ( - 37.1'), whereas its companion molecule (4) (A = -4.6') behaves differently. The occurrence of two molecules in the asymmetric unit [the pairs (77,78), (131, 132), (3,4), (98,99), (32,33), (15, 16), (116, 117),and (80, Sl)] or polymorphy [the quadruplet (122, 123, 124, 125)] offers a good opportunity to study the flexibility of ring D of the same compound in diflerent surroundings. Taking into account a standard deviation o(A) = 1" the observed differences of A values are insignificant for the pairs (131,132),(32,33),and (15,16), small for the pairs(80,81) and (98,99),but large for the pairs (77,78), (116,117),and (3,4)and the quadruplet (122-125). For (3) and (4) the observed conformations are the envelope C,(14) and the half-chair C,( 16),respectively. The quadruplet displays the forms C,( 14), C,(16), and the in-between forms C,(14)-C2(16) and C2(16)-C,(14). Since it is well-known that packing forces (van der Waals type, hydrogen bonds) are relatively weak we can accept the observed conformational differences in these examples as experimental evidence for the concept of flexibility of steroidal D-rings. - 82.9".
8 Six-membered Boat Conformations Since boats or twist-boats are less stable than chairs (the difference amounts to 5.6 kcal mol- for cyclohexane) we can easily understand why they rarely occur in steroids and other biologically important molecules containing six-membered rings. So far only 13 boat/twist-boat conformations (Table 19) have been encountered in 11 sterojds.* It has been shown by Buys and G e i ~ e that ' ~ ~six-membered boats display the same sort of pseudorotation as five-membered rings.' 5 2 Their presence introduces a certain amount of flexibility comparable with the variable shape of ring A in 3-0x0-A4-steroids and ring B in oestranes and A5- and A5*7-compounds.Its
'
Is*
159
C. Romers, R. A. G. de Graaff, F. J. M. Hoogenboom, and E. W. M. Rutten, Acta Cryst., 1974, B30, 1063. H. R. Buys and H. J. Geise, Tetrahedron Letters, 1968, 5619,
* Another example of a boat conformation in ring A can be found in: D. S. Savage, A. F. Cameron, G. Ferguson, C. Hannaway, and 1. R. Mackay, J. Chem. SOC.( B ) , 1971, 410.
Chd
=
a0
-40.5 25 42 42.3 -27.2 - 48.7 46.6 58.6 - 19 45.9 26.8 39.7 66.9 0
@I
-11.6 30 19 17.7 -27.8 11.2 13.6 10.5 - 32.9 -28.1 -60.1 19.6 -37.7 55.0
cyclohexane-1 ,Cdione.
Compound
Chd" (82) (82) (111) (74) (75) (75) (115) t 136) (22) (139) (24) (84) (138)
52.5 - 58 - 58 - 59.9 58.7 42.5 - 58.5 - 66.6 33.8 - 30.0 29.0 -56.9 - 17.5 - 48.0
a2
27 23 18.3 - 25.5 18.7 17.8 9.2 - 32.4 - 49.1 - 63.6 33.2 - 10.7 56.0
29 35 40.1 - 31.6 - 57.7 40.9 55.2 - 1.9 67.0 30.0 29.5 ' 43.0 - 6.0
a4
- 12.4
@3
- 39.9 53.3 - 55 - 66 -61.1 54.0 31.5 - 64.4 - 66.0 35.6 - 5.4 32.0 - 68.4 -41.1 - 58.0
@,
B
A
B B C D
A
C
B
B C C B
Ring
Table 19 Boat conformations in steroids. The angles are in decimal degrees
137 - 60 - 49 - 47 122 156 - 45 - 37 93 11 61 - 59 -3 267
A 55 56 62 61 55 75 62 73 39 57 60 61 58 63
@In
Configuration Conformation TBB TB 9PJOP TBB 9PJOP 9P,lOa TBB 98,lOa TB 9p, 1Oa B (distorted) 98,lOa TBB 58,148 B 8a B (flattened) 8a,9P,13a,148 TB TB 9B TB 13a,14a T B (distorted) B 9B
E
b
9G.
3b
e2a
F
3
6'
E
$
$
K
5 2
572
Terpenoids and Steroids
pseudorotation can be described by the equations = @, cos (A
j = 0, I, 2,3,4, or 5
and
+ jS)
(3)
6 = 120",
and tan(A
+ jd) =
@j+2
- @j+l
m j f i
where Qrn and A have meanings similar to those stated before. Values A = 0" & (n x 60") indicate twist-boat forms ( T B ) with C2 (222) symmetry. Values A = 30" k ( n x 60") refer to ideal boat forms ( B ) with Czc (mm2) symmetry. Other A values reveal the in-between forms (TBB) with C 2 (2) symmetry in which only the non-intersecting dyad perpendicular to the average plane of the ring is retained. Using equations (3) and ( 5 )we have analysed the boat conformations occurring in compounds (22), (24), (74), ( 7 3 , (82), (84), ( l l l ) , (115), (136), (138), and (139). The numbering of angles $ j is indicated in Figure 3. Whereas equations (1) and (2) usually hold well within 0.5" for five-membered wrings, the application of (3) and ( 5 )to six-membered rings is questionable. Equation (4) holds nicely for cycloh e ~ a n e - 1 , 4 - d i o n e ' ~and ~ * ' to ~ ~a lesser degree also for compounds (82), (1 1 l), (24), (115), (139), and (138), but is not satisfactory for the remaining compounds. Nevertheless the analyses give information on the conformation and the degree of deformation. With the exception of (84)the occurrence of boat conformations is related to unusual configurations. In (84)the presence of two sp2-hybridized carbon atoms in ring A as well as of three chlorine atoms (see Figure 5) is responsible for the excep-
Figure 5 Torsion angles in rings A and I6O 16'
B
of (84)
A . Mossel and C . Romers, Acta Cryst., 1964, 17, 1217. P. Groth and 0. Hassel, Acta Chem. Scand., 1961, 18, 923.
Steroid Conformations from X-Ray Analysis Data
573
tional behaviour. The 4a-chlorine atom, already hindered by the presence of 6and 7a-chlorine atoms, would have too small an intramolecular contact'62 with 0 - 3 if ring A were to assume its normal chair form. This and similar conclusions are usually deduced ad hoc from Dreiding models. However, not too much credit should be given to these or other models. For example, on the basis of a model of a BP,lOa-steroid one might suppose that the short intramolecular distance between the angular 10-methylgroup and the axial hydrogen atoms at c-12 and C-14 should lead to a (twist) boat conformation of either ring B or ring c ; the steric relationship is analogous to that in the axial conformer of t-butylcyclohexane. In actual fact [see (72), (90), and (91)] the steric interactions are relieved by a considerable decrease of torsion angles 148-9-1 1 and 8-9-1 1-12, thereby turning the 10-methyl group away from the a-side of ring c and leaving ring c in a distorted chair conformation.
9 The Conformation of the Side-chain at C-17 It is obvious that 17P-side-chains play an important role in the activity of pregnanes, cholestanes, ergostanes, and cholic acids. A characterization of the conformation and the designation of the chirality of the asymmetrically substituted carbon atoms may, therefore, contribute to a better description and understanding of these functional groups. A number of dihedral angles about the bonds C- 17-C-20, C-20-C-22, C-22-C-23, C-23-C-24, and C-24-C-25 are listed in Table 20. In view of the large torsion angles (ca. 180")it can be concluded that the normal cholestane side-chain, 21
20
22
23
CH, -CH-CH2-CH2-CH2
24
25
-CH-(CH,),
is fully stretched in compounds (11l), (7), (8), (9), (32), (33), (20), and (18). This is also true for the ergostane compound (82). The ergostane derivative lumisterol (113)and the gorgosterol compounds (101)and (104)(steroids extracted from soft corals), calciferol (143), and compound (113) exhibit a folded chain with a sharp bend about bond C-20-C-22. Although the seco-compound (104) has an open ring c and (101) has an extra C-30, forming a cyclopropane ring with C-22 and C-23, their side-chain conformations are quite similar. The insect-moulting hormones (93) and (94) are also folded about bond C-20-C-22. The side-chain of cholic acid (11) is stretched, but the tail of (10) is folded, again about C-20c-22. With the exception of the lanostane compound (108) (having the 13a,14ficonfiguration) methyl group 21 invariably is oriented towards the a-side of the molecule. From the value of the dihedral angle (D(13-17-20-21) (see Table 20 and Figure 6) we note that this &-orientation is nearly perfect for the cortico= -89') but gauche for the remaining steroids (58), (67), (63)J and (88) (aav 162
R. W. Kierstead, J. Blount, K. E. Fahrenholtz, A. Faraone, R. A. LeMahieu, and P. Rosen, J . Org. Chern., 1970, 35,4141.
Terpenoids and Steroids
574
Table 20 Dihedral angles (decimal degrees) in the side-chain. The column numbers refer to the following angles: 2 : @( 13-1 7-2&2 1) 3: @(16--17-2&21) 4 @( 13-1 7-2&22) 5 : @( 17-2&22-23) Compound 2 (8) -59 (9) -57 (7) -55 (17) -55 (18) -50 (20) -58 (32) -51 (33) -60 (111) -54 (82) -55 (113) -57 (143) -49 (104) -53 (101) -54 (93) -56 (94) -53 (108) 194 (10) -59 (11) -63 (53) -57 (90) -70 (74) -76 (79) -82 (58) -88 (88) -89 (67) -89 (63) -89
3 182 182 184 183 192 183 189 181 184 182 182 186 185 183 187 181 -45 185 177
4 5 181 204 180 195 183 206 177 181 183 187 181 197 183 200 180 185 182 179 172 184 181 236 190 -119 189 -104 185 -91 180 74 184 71 57 70 185 62 176 188
6 q20-22-23-24) 7 @(22-23-24-25) 8 : q23-24-25-26) 9 @(23-24-25-27) 6 7 8 9 181 179 173 -62 176 184 164 -73 63 13 181 179 130 174 177 -67 184 163 191 -73 183 176 175 -60 196 194 176 -57 61 177 175 185 72 195 176 184 175 177 184 -57 184 111 60 -65 175 111 -55 65 188 145 149 -42 65 191 148 147 -54 181 189 -100 194 -88 -39 192 173 -38 181 174
Type Cholestane Cholestane Cholestane Cholestane Cholestane Cholestane Cholestane Cholestane Cholestane Ergostane Ergostane Ergostane Ergostane Ergostane Ergostane Ergostane Lanostane Cholic acid Cholic acid 20-OH-Pregnane 20-0x0-Pregnane Pregnane Pregnane Corticosteroid Corticosteroid Corticosteroid Corticosteroid
H
I
21,
Ha
(a)
I Ha
(b)
(Cl
Figure6 Newman projections along the bond C- 17-C-20 for normal pregnanes, cholestanes, ergostanes,.and cholic acids (a), corticosterozds (b) and retro-steroids (c)
Steroid Conformations from X-Ray Analysis Data
575
= - 56"). The retrosteroids, including 20-hydroxyprogesterone (53) (aav (74), and (79) are an intermediate group between the cholestanes and steroids (W), corticosteroids, with 0 values between - 70" and - 82". The chirality of the side-chain is given in Table 21. First turning our attention to 20-hydroxyprogesterone (53), we note that the chirality of centre 20 is
Table 21 Chirality of the side-chin according to the Cahn-lngold-Prelog rules'64 c-20 R R R R R R R R R R R R R R R R R R S R
This molecule is the 2Oa-epimer. VF calculation^^^ indicate that this epimer in the gauche conformation is, indeed, most stable, but that in the liquid state the anticonformation with CD ca. 190" may play a role for some derivatives, such as c-18 ~ x i m e s Nonetheless .~~ the C-21 methyl group is always oriented towards the aside of the molecule, even in the case of the 2 0 P - e ~ i m e r having ' ~ ~ R chirality. Although ( 108) deviates completely from the regular dihedral-angle pattern, it has the usual R chirality at centre 20. Table 21 may be useful rather for establishing relationships between possible epimers than for reading the conformation. The conformation of corticosteroids about bond C - 1 7 4 - 2 0 has been discussed extensively by Duax and collaborator^.^^^^^^^+^^^ All these studies indicate that the constant C - 1 7 4 - 2 0 conformation in the solid (crystalline) state may also be dominant when acting on biological receptors. 16'
N. W. Isaacs, W. D. S . Motherwell, J. C. Coppola, and 0. Kennard, J.C.S. Perkin ZZ, 1972, 2331.
16'
166
R. S . Cahn, C. Ingold, and V. Prelog, Angew. Chem., 1966,78,413. H. Lee, V. S. Bhaca, and M. E. Wolff, J. Org. Chem., 1966, 31, 2692. A. Cooper and W. L. Duax, J. Pharm. Sci., 1969, 58, 1159.
Terpenoids and Steroids
576
10 Biological Activity at the Molecular Level It is with reserve that we comment on the physiological properties of hormonal steroids in correlation with molecular geometry, because their biological activity is one of the most important reasons for crystallographic research into this class of compound. Unfortunately, most steroid hormones have more than one function and some play an intermediary role between two or more organs. Whereas enzymes have a straightforward function in the living cell or in the digestive tract, hormones usually work on special organs (glands) or membranes. The diversity of their functions is also reflected in synthetic drugs, which have the disadvantage of sometimes showing unwanted side effects. The arguments in favour of their non-specificity are : (i) Their diverse functions. For example, adrenocorticosteroids are important as (a)regulators of electrolyte excretion (membranes in kidneys),(hj participants of glucometabolism (liver), and ( c ) agents for anti-inflamatory action (mucous membranes). (ii) The introduction of substituents such as methyl groups or fluorine-atoms (usually at positions 6, 9, 11, or 16) gradually changes their properties. (iii) Takeover of some of the functions by chemically quite different drugs. For example, the molecule diethylstilbestrol' 67-169 is a potent agent for uterus oestrogen receptors, competing in activity with natural oestradiol. '
(ivj Experimental evidence for quantitatively different effects at different concentrations, say at moll- (inhibitory) and at 10- moll- or lower (stimula t ing). The most favoured view'70 of the mechanism of action is embodied in the receptor theory involving a combination of steroid (substrate) with protein (receptor). The interaction may be direct, but, more likely, is indirect via a third substance, such as a pyridine nucleotide, or via lipids in membranes. The globular protein surface (usually an enzyme) largely consists of a fl-pleated structure,17' although the hydrophobic a-helix sometimes occurs at the outside of the molecule. The 0-3-0-17 separation in oestranes and testostereone' l 2 and the 0-3-0-20 16'
16' '69
''I
M. Hospital, B. Busetta, and C. Courseille, Communication on the Stockholm Symposium on the Structure of Biological Molecules, July 9-12, 1973. E. C. Dodds, L. Goldberg, W. Lawson, and R. Robinson, Nature, 1938, 141, 247. C. M. Weeks, A. Cooper, and D. A. Norton, Actu Cryst., 1970, B26,429. ( a ) H. G. Williams-Ashman and A. H. Reddi, Ann. Rev. Physiol., 1971, 33, 3 1 ; ( h ) E.-E. Baulieu, A. Alberga, I . Jung, M.-C. Lebeau, C. Mercier-Bodard, E. Milgrom, J.-P. Reynaud, C . Raynaud-Jammet, H . Rochefort, H. Truong, and P. Robel, Rec. Progr. Hormone Res., 1971, 27, 3 5 1 ; ( c ) E. V. Jensen and E. R. DeSombre, Ann. Rev. Biochem., 1972, 41, 203. R. E. Marsh, R . B. Corey, and L. Pauling, Actu Cryst., 1955, 8, 710.
Steroid Conformations from X-Ray Analysis Data
577
separation in corticosteroids and progesterone"* are in the range 1l . G l 1 . 8 A. These distances roughly correspond both with two turns of the protein a-helix (10.76 A) and with a few interchain 0-.-N distances occurring in antiparallel chain-pleated protein sheets. It is difficult, however, to envisage a close approach of the steroid molecule to the innate hydrogen bonds which support the backbone of the protein structure. The bulky side-groups would prevent such an attack. It is more likely that hydrophilic side-groups of the enzyme become hydrogenbonded to the substrate molecule, e.g. to hydroxy- or 0x0-groups at C-3, C-17, and C-20. In this view the steroid probably does not act on the active side (cleft) of the enzyme but on its surface. The disruption of the tertiary protein structure might enhance or block the biological activity. The flexibility of ring A (progesterone, testosterone, and corticosteroids) and/or ring B (oestranes and A5*7-steroidsof the vitamin D group), together with the -flexibility of ring D, also fits into this picture. This flexibility is a prerequisite for arranging the steroid functional groups in the proper directions for the formation of hydrogen bonds with the receptor groups. It had been thought earlier' that the bend of the steroid molecules towards the a-side might be correlated with activity. The hydrophobic P-side with protruding methyl groups 18 and 19 might be complementary to the receptor surface. The optimum oestrogenic activity of oestradiol with a 13P-Me group relative to the 13P-H and 13P-Et analogues' supports this idea. The P-side hydrogenation of 17/3-hydroxy-l,4-androstadien-3-one by heterogeneous catalysis,' 3 3 as well as the optimum anti-inflammatory activity of 9a-fluorocortisol with maximally bent surface in comparison with also seems to agree with this view. Serious objections against the complementarity theory can be put forward. It does not give an account of the role of the hydrophilic functions of 0 - 3 and the 17P-side-group. It is necessary to presuppose either (a) a ternary complex of a steroid attached with its P-side to the receptor and hydrogen-bonded to a third body by means of its hydrophilic side groups ( b )consecutive action by means of its P-side with a complementary receptor and of its hydrophilic side-groups with a third body, or (c) oice versa. Such operations seem to be quite artificial and unnecessarily complicated. Moreover, the presence of a hydrophilic 11-0x0- or 11-hydroxy-group in corticosteroids contradicts the complementary theory, since surface interactions cannot be both hydrophobic and hydrophilic within short distances of, say, 3.5 A. It is more probable that the presence of the 10-Me group contributes to the overall bent shape, thereby arranging the C-3-0-3 and C-20-0-20 vectors in the appropriate directions. These directions' 7 2 are 138" for cortisol, 145" for progesterone, and 160" for cortisone. It is clear that rather than further diffraction analyses of steroid crystal structures, future crystallographic work should be focused on investigations into steroid-receptor complexes.
'
172
0. Dideberg, L. Dupont, and H. Campsteyn, Communication on the Stockholm Symposium on the Structure of Biological Molecules, July 9-12, 1973.
578
Terpenoids and Steroids 11 Summary
Some 140 steroid crystal structures have been subjected to statistical and conformational analysis, in which weighted average experimental values for bond distances and valency and torsion angles are compared with values obtained by valence force calculations. The analysis takes into account the configuration, the hybridization, and the character of substituents bonded to carbon atoms 3 and 17. Special attention is drawn to 3-0x0-A4-steroids for which the conformation and the Cotton effect are discussed at some length. The data, compiled in several tables, underline the flexibility of biologically active steroids. With rare exceptions nearly all steroids have flexible five-membered D-rings. The flexibility at the other end of the molecule can be assessed in terms of the sofa and/or halfchair conformation (and in-between forms) of ring A in 3-0x0-A4-steroids,and of the same conformations of ring B in oestranes and A5-steroids. Six-membered boat. forms sometimes occurring in steroids with abnormal configurations also display a large degree of flexibility. It is speculated that flexibility at both ends of the steroid nucleus is a necessary condition for the formation of hydrogen bonds with receptor molecules. The authors wish to thank Miss S. Amadio and Mrs. H. Bavelaar for respectively their linguistic and typewriting help in preparing this Report. They are indebted to Messrs. D. van Ingen Schenau, H. de Sitter, H. van der Lee, and J. M. G. Bonfrer for computational aid in producing some of the data used in this Report. 12 Appendix
(i) Weighted Average Value or Standard Value qav. q,, =
E
i:
qi.;'/
i= 1
a;2
i= 1
qi is the individual observation, bi its standard deviation, and n the number of observations. (ii) Standard Error (Deviation) 0.
. [i =
.;2]-"'
i= 1
(iii) Estimator of the Standard Deviation S.
ij is the unweighted average of entity qi (assuming weights 1).
(iv) Agreement Zndex s. s = [(n-l)-'
i
(qyb
- qf"'")2
i= 1
q'b and q;alc are observed and calculated values of q i .
1
lI2
579
Steroid Conformationsfrom X-Ray Analysis Data
chair,
C
4,
boat,
B
c,,,
twist-boat,
TB
D,
sofa,
S(5b
c,
sofa,
S(5W
c,
half-chair,
HC
C,
1,3-diplanar,
C,
half-chair,
C,!4)
c,
envelope,
CS(4b
c,
1
4
2
5
5 5
3
5 3
5 4
Figure 7 Five- and six-membered ring conformations
580
Terpenoids and Steroids
(v) Geometrical Forms qj' Rings used in the Discussion. The observed geometrical forms are depicted in Figure 7. The first column shows the ring in a projection perpendicular to the bond C-1-C-2. The plane of projection is perpendicular to a dyad (two-fold rotational axis) for the six-membered chair, twist-boat, halfchair, 1,3-diplanar chair, and the five-membered half-chair. The projection is perpendicular to the plane of atoms 1,2,4, and 5 (boat), 1,2,3,4, and 6 (sofa),and 1,2,3, and 5 (five-memberedenvelope). The second column presents the ring with signs of endocyclic torsion angles, the third the name, the fourth the abbreviation, and finally the fifth the symmetry in Schoenflies notation. (vi) Valence Force Culculutions und Parameters. The basic ideas and principles of the V F method have been reviewed r e ~ e n t l y . ~ ~In" 'all ~ V F calculations the total strain energy is written as the sum of several types of energy contribution, as follows :
V,,,
=
V(r)+ V ( 0 ) + V ( 4 ) + V ( 6 ) + V(1,3) + V(nb) + V(cou1) + cross terms
where V(r),V ( @ ,V($),and V ( S )represent the total energies of bond length, bond angle, torsional, and out-of plane deformations, respectively, and V (1,3), V(nb), and V(coul) stand for Urey-Bradley, non-bonded, and coulomb interaction energy contributions. The computer program package used in the present work has recently been ~ u t l i n e d . ' The ~ central routine UTAH is a modification of the program devised by Boyd.' 74 In addition to a set of interactions describing the structural parameters and the constants for the chosen potential expressions the input consists of the Cartesian co-ordinates of the trial model. The program collects the appropriate interactions for the participating kinds of atoms and the first and second derivatives of the energy with respect to the deformations, i.e. the potential energy surface is expanded in a truncated Taylor series around the trial structural parameters. The resulting set of simultaneous equations is solved directly. Because of thc approximations involved in the various transformations' 7 4 the calculated shifts of the atomic co-ordinates will not minimize the potential energy in a single calculation. Therefore, the new model is automatically used as input for another cycle of computations until the co-ordinate shifts are smaller than a prespecified value (a convergence limit of 0.001 8, was used). The output consists of a list of energies, geometrical properties, and Cartesian co-ordinates of the refined model. The program user may choose between the force fields ,41,,'".'7"77 B,'27 and LW.'26 We wish to stress that the calculated geometry in no way depends on the input co-ordinates (except by being biased toward a particular conformer because during the iteration process the structure J. E. Williams, P. Stang, and P. von R. Schleyer, Ann. Rev. Phys. Chern., 1968, 19,
'" l i b
''-
531. R. H. Boyd, J . Chem. Phys., 1968, 49, 2574. C. Altona and M. Sundaralingam, J . Amer. Chem. Soc., 1970, 92, 1995. C . Altona and M. Sundaralingam, Tetrahedron, 1970, 26, 925. N. L. Allinger, J. A. Hirsch, M . A. Miller, I. J. Tyminski, and F. A. Van-Catledge, J . Amer. Chem. Soc., 1968, 90, 1199; N. L. Allinger, J. A. Hirsch, M . A. Miller, and I. J. Tyminski, ibid.. 1968, 90, 5773; ibid., 1969, 91, 337.
58 1
Steroid Conformations from X-Ray Analysis Data
cannot 'jump' over an energy barrier separating two conformers) but only on the force-field equations and parameters ~ h o s e n . ~A more detailed discussion concerning the a priori calculation of steroid molecules is given elsewhere.24 In the calculations with force field AL the same parameters were used as reported earlier ; l 75,1 7 6 since the original set did not contain constants appropriate for the calculation of structures containing one or more double bonds these are shown in Table 22. In our earlier report24 the non-bonded interaction list had to be cut
Table 22 Additional force-field parameters used in the valence .field A L for the calculation of steroids containing one or more carbon-carbon double bonds (i) Bond stretch: V
=
ik,(r, - r J 2
-
Bond" C-=-C --= =-H =-C
kk(ri - ro)
ki
kr
9.7 1.56 5.3 1.56
ro 1.336 1.370 1.087 1.424
0 - 0.077 0 - 0.045
(ii) Angle bend: see ref. 177; the k, values shown were multiplied by 1.15 throughout the calculations. Angle H
4
k,
0.66
80
119.1"
122.6"
=q"
0.66
I 17.8"
0.55
122.4"
1.1
122.6"
1.1
I 16.9"
2.1
121.6"
H
c
4'
C
582
Terpenoids and Steroids
Table 22 (continued) (iii) Torsions:’
V
=
+K{ I
Torsion angle
R
R
R
R
h=&
‘/
f cos ( n $ ) }
V,
n
Sign
R
40.0
1
-
H or C
1.5
3
-
3
+
3
-
0.49
(iv) Out-of’-plane bending: V
=
3
+
-
-
C H
3
+
HorC
3
+
H or C
0.5k,h2
R
=3 R
k,
R
0.4
H or C
(v) Non-bonded interacfions: the Hill exponential equation’
Atoms C(sp2).. C(sp2) C(sp2).-C(sp3) C(sp2)** .H C(spZ)-..o (vi) 1,3-lnteractions: the Hill 6/12 equation‘” Atoms C(sp2).. C ( s p 2 ) C(sp2).. C(sp3) C ( s p 2 ) -.H .
i
1.85 1.75 1.65 1.70
77
was used. E j
0.0200 0.0283 0.0447 0.0374
was used. r* 1.39 1.39
1.215
E
0.22 0.22 0.09
” C stands for C ( s p 3 ) unless indicated otherwise. ‘ I n m d y n k ’ . In mdyn. “n A. In mdyn A rad - *. The torsional functions and parameters are chosen to reproduce the curvature at the bottom of the potential well and arc not intended to reproduce the entire torsion potential. In kcal mol-’. In mdynA r a d - *. ‘ In A. ’In kcal mol-’.
short at a given distance for practical reasons. In the present work all nonbonded interactions were included in the calculations. All calculations were carried out on an IBM 360/65 computer of the University of Leiden.
Steroid Conformations from X-Ray Analysis Data
583
Note added in proof. Recently it was found that certain slight modifications of field B'27 led to significant improvements of calculated bond lengths, bond angles, and torsion angles of the saturated steroid skeleton (C. Altona et al., unpublished work).
ERRATA Vol. 3, 1973 Page 72, line 5 up. Delete the name ‘orthodene’ as applied for formula (344). ‘Orthodene’ is the trivial name for 3,7,7-trimethylnorpin-2-ene. Page 82, line 5 up. For ‘p-caran-4-01’ read ‘p-caran-3-01’. Page 86. Formula (425) lacks a methyl group at C-2 of the chromene ring.
584
Author Index
Aasen, A. J., 238 Abdel-Fattah, A. F., 286, 405 Abd-Elsamie, M. E., 420 Aberhart, D. J., 285 Abon-Chaar, C. I., 293 Abou-Donia, S., 20 Abraham, N. A., 481 Abubakirov, N. K., 408 Abul-Hajj, Y. J. 285,474, 475 Achaya, K. T., 17 Achilladelis, B. A., 90, 305 Achini, R., 90 Ackman, R. G., 271 Adam, G.,3 15,535 Adams, D. L., 45 Adams, J. A., 456 Adams, P. M., 90, 92 Adams, W. J., 568 Afonso, A., 5 11 Agre, N. S., 405 Agurell, S., 73 Ahlgren, G., 188 Ahmad, M. S., 364 Ahmad, S. A., 160 Ahmed, F. R., 541 Aida, M., 22 Aizawa, M., 155 Akaki, K., 41 1 Akhmedov, A. I., 9 Akhrem, A. A., 3 13,394, 395,458,460,478,490 Akhtar, M., 275 Akimoto, A., 338 Akiyama, K., 379 Akiyama, T., 2 14 Alberga, A., 576 Albrecht, K., 471, 477 Albrecht, P., 306 Alburn, H. E., 424, 459, 463 Alexander, C. W., 37 Alexandre, C., 42 Alfsen, A., 286,487,488, 489 Allcock, C., 256 Allegra, G., 546 Allen, C. M., 293 Allen, F. H., 536
Allen, J., 183, 210 Allinger, N. L., 312, 544, 580 Allred, J. B., 252 Almqvist, S. O., 238 Altenburg, H., 536 Altman, L. J., 226 Altona, C., 531,533,544, 546,560,565,566,580 Alvarez, F. S., 360, 365 Amano, T., 22 Ambrus, G., 404, 460, 47 1 Amelina, A. S., 479 Amiard, G., 472 Amos, B. A., 314 Ananchenko, S. N., 312 Andersen, N. H., 25, 95, 102, 139 Anderson, A. B., 160 Anderson, C. G., 277 Anderson, G. D., 135, 136,266,303 Anderson, R. A., 393 Anderson, R. J., 183 Anderson, W. K., 6 Anding, C., 274 Andrewes, A. G., 221, 233,307 Andrews, A. L., 338 Andrews, G. C., 16 Anetai, M., 142, 240 Anjyo, T., 474, 475 Annen, K., 338 Ansari, H. R., 13 Anthonsen, T., 148, 151 Anthony, G. M., 131 Anthony-Mote, A., 457 Aoki, K., 107 Aoyagi, R.. 215, 216 Appleton, R. A., 302 Aragon, M. C., 253 Araujo, J., 9 Arbuzov, B. A., 61,63,64 Archer, B. L., 300 Arditti, J., 277, 392 Aries, V. C., 461, 473, 493 Arigoni, D., 107, 174, 186,288 Arima, K., 460, 51 1, 513
585
Arita, M., 149 Armsen, R., 233 Arnold, R. A., 86 Arnold, R. T., 60 Arnold, Z., 10 Arnould, D., 15, 237 Arpesella, 0. A., 53 Arpin, N., 229 Arpino, P., 172, 246 Asai, A., 24 Asako, T., 464 Ash, L., 226 Astakhova, A. S., 17 Atal, C. K., 213 Atherton, L., 277 Atkin, S. D., 272 Audley, B. G., 300 Aul’chenko, I. S., 44 Auret, B. J., 404 Aversa, M. C., 123 Avery, M. D., 283 Avotins, F., 62 Axelrod, L. R., 283 Ayer, W. A., 23, 170 Ayyar, K. S., 241 Azizullah, 46 Azzaro, M., 3, 40 Bachelor, F. W., 57, 61 Baddiley, J., 295 Badger, R. A., 103 Bae, M., 460, 511, 513 Baggaley, K. H., 272 Bagli, J. F.. 498 Baisted, D. J., 259, 261 Bakhanova, E. N., 17 Bakkei, J., 422 Bakker, S. A., 386 Balanson, R. D., 94 Balasubramaniam, S., 283 Balavione, G., 455 Baldwin, D., 332 Baldwin, J. E., 65 Balko, T. W., 17 Ballio, A., 179, 180 Balmain, A., 147 Bancher, E., 232 Bang. L., 139 Bannon, C. D., 212
Author Index
5 86 Banthorpe. D. V., 8, 14, 19, 110, 112, 113, 254, 260, 261, 263, 298, 300,301, 302 Baranowska, E., 218 Barbier, M., 366, 373, 383 Barkhurst, R. C., 553 Barlow, S. A., 267, 268 Barnes, F. J., 227, 228, 290 Barnes, R. K., 363 Baron, D. N., 455, 457 Barr, R., 308 Barrett, T. M., 353 Barron, L. D., 4 Barrow, K. D., 166, 179, 27 1 Barry, G. T., 70 Barry, J., 35 Bart, J. C. J., 548 Bartell, L. S., 568 Bartels, A. P., 110 Barth, C. A., 251 Bartlett, L., 307 Bartley, J. P., 193, 358 Barton, D. H. R., 43, 47, 166, 179, 193, 210, 271, 344, 355, 356, 363,369,384,392,400 Barton, R. E., 313 Bartz, J. K., 294 Barua, A. B., 235 Barua, A. K., 213, 218 Bascoul, J., 187, 296 Baskevitch, Z., 150 Bassett, R. A., 293, 299 Basu, K., 2 18 Bateson, J. H., 162 Batey, I. L., 189 Batra, P. P., 290 Batta, A. K., 208 Battersby, A. R., 262 Bauer, B., 469 Bauer, S. H., 559, 562 Bauernfeind, J. C., 307 Raulieu, E.-E.: 456, 470, 488,489, 576 Baumann, P., 386 Rautista, M., 404 Baxendale, D., 263 Baxter, C., 263 Bean, N. E., 301 Bearder, J. R., 269 Beastall, G. H., 258 Beaton, J. M., 363 Beaudoin, G. J., 384 Beck, H. C., 408, 413 Beckett, A. H., 49 Beckett, B. A., 132 Beckmann, H., 455,487
Beckwith, A. L. J., 384 Bedi, K. L., 213 Beecham, A. F., 226,313 Beedle, A. S., 253 Beg, Z. H., 252 Begley, M. J., 20 Behbud, A., 102 Beilby, J. P., 160, 269 Beiner, J. M., 53 BeliC, I., 293, 425, 441, 459, 515 Bell, A. M., 398, 403 Bell, J. J., 300 Bell, R. A., 167 Bellamy, A. J., 65 Bellas, T., 455 Bellet, P., 465 Bellino, A., 158 Bello, O., 234 Bena, B., 286,482 Ben-Aziz, A., 289, 299 Benedetti, E., 546 Benesova, V., 135 Benezra, C., 326 Benfield, E. F., 143 Benisek, W. F., 286, 489 Benjamin, B. D., 288 Benjamin, B. M., 47 Benn, M. H., 5,164,364 Bennet, R. D., 203 Benson, A. M., 299,487 Benveniste, P.. 258, 273, 278 Ben-Zvi, Z., 71, 72, 74 Berezin, G. H., 324 Berg, W., 263 Bergland, G., 151 Bergmann, E. D., 218 Berking, B., 536 Berkoff, C. E., 299 Bernal, J. D., 531 Berndt, H.-D., 405 Berndt, J., 253 Bernhard, R., 222 Berrier, C., 373 BerthCICmy, P., 472 Berthet, D., 9 Berti, G., 208 Bertrand, C., 36, 50 Bessiere-ChrCtien, Y., 56, 60, 61, 97 Bestmann, H. J., 233 Betz, G., 469 Beugelmans, R., 195 Beukers, R., 394 Beverwijk, C. D. M., 3 Beytia, E. D., 259 Beziat, Y., 457 Bezzubov, V. M., 56 Bhaca, V. S., 575 Bhacca, N. S., 204
Bhadane, N. R., 115,123, 133,266 Bhalla, V. K., 70 Bhatia, M. S., 13 Bhatt, R. S., 68 Bhattacharya, S., 207 Bhattacharyya, S. C., 101, 129,303 Biellmann, J. F., 154 Bigham, E., 342 Bikbulatova, G. Sh., 64 Billett, E. H., 51 Billing, B. H., 457 Billups, W. E., 23 Bimpson, T., 278 Birch, A. J., 31, 164, 297 Birnbaum, G. I., 329,537 Bisarya, S. C., 88 Bittler, D., 337 Bittner, M., 152, 196 Bjorkhem, I., 282, 285, 286,460,484,486 Blachkre, H., 479 Blackburne, I. D., 83,143 Blackwell, D. S. L., 52 Blair, I. A., 344 Blanchard, M., 44 Blank, R. H., 397,495 Blickenstaff, R. T., 326 Bliss, C. A., 24 Block, J. H., 214 Blossey, E. C., 342 Blount, J., 573 Blum, S., 4 Blunt, J. W., 418, 446 Boar, R. B., 183, 191, 192, 193, 199, 210, 273,332,341,369 Bobbitt, J. M., 301 Bochwic, B., 53, 58 Bodea, C., 306 Boeckx, R. L., 297 Bohme, K.-H., 483, 497, 505,506,507,508,523 Boettger, H., 221 Bogard, T. D., 47 BogdanoviC, B., 5 , 20 Boguslavsky, V. A., 342 Bohlmann, F., 12, 23, 127,149,150,157 Boiteau, P., 163 Bokkenheuser, V., 526 Boll, M., 253 Bollinger, P., 98 Bolt, C. C., 398 Bolton, M., 43 Bolton, R., 330 Bolton, S., 169 Bombardelli, E., 159 Bonnafous, J.-C., 222 Booth, W. D., 284
Author Index Boots, M. R., 253 Boots, S. G., 253 Borchere, G., 174 Bordner, J., 538, 539 Borgna, J.-L., 457 Boris, A., 354 Borkenhagen, L. F., 503 Bornati, A., 159 Borovskaya, A. G., 61 Bose, A. K., 3, 315 Boswell, G. A., 324 Bosworth, N., 23, 56 Boul, A. D., 446 Boulerice, M., 419 Bouquant, J., 313 Bourgeois, G., 29 Boutigue, M.-H., 350 Bowen, D. H., 159 Bowlus, S. B., 84 Bownds, D., 307 Boyd, G. S., 283 Boyd, R. H., 544, 580 Boyd, W. A., 56 Boyer, J., 455, 487 Boyle, P. H., 302 Bozler, G., 506 Bozzi, E. G., 33 Brambilla, R. J., 3 Branch, G. B., 207 Brandange, S., 5 Brandenburg, C. F., 61 Brandt, K., 286 Brandt, R. D., 273 Branlant, G., 154 Brannigan, L. H., 62 Brannon, D . R., 404, 427 Braselton, W. E., 286, 297 Braun, P. B., 532, 537, 538,539,552 Bravet, J.-L., 326 Breitmaier, E., 7 Breslow, R., 386 Bretschneider, H., 9 Brewer, H., 284 Bricker, L. A., 274 Bridgeman, J. E., 398, 446 Briedis, A. V., 252, 290 Brieger, G., 18 Brieskorn, C.H., 29 Briggs, D. E., 161, 269 Briggs, L. H., 193, 358 Brikenshtein, Kh. A., 17 Brine, D. R., 71 Britton, G., 228, 289, 290,299,306 Britton, R. W., 115, 206 Broaddus, C. D., 87 Brocard, J., 28
587 Brock, F. X., 172 Brocksom, T. J., 86 Brodie, H.. J., 284, 338, 398, 409, 469, 470, 474,475 Brooks, C. J. W., 4, 131, 312,393,468 Brown, C. A., 29 Brown, F. S., 496 Brown, H. C., 56, 348 Brown, J. N., 540 Brown, M. S., 252 Brown, P. K., 307 Brown, R. L., 401,460 Brown, S. A., 297, 298 Brown, W. E., 448, 451, 476 Brown, W. V., 164 Browne, J. W., 398, 403, 446,447 Browne, L. E., 12 Browne, L. M., 23 Brubacher, G. B., 307 Bruce, S. E., 320 Brufani, M., 179 Brundret, K. M., 166 Bryan, R. F., 116, 152, 541 Bryant, R., 303 Brzezinka, H., 307 Buchecker, C., 367 Buchecker, R., 38, 225, 226 Buckingham, A. D., 4 Bucourt, R., 532 BudCSiriskjr, M., 209,332, 352 Budzikiewicz, H., 307, 445 Buchi, G., 26, 31, 39, 136 Buki, K. G., 460,488 Buemi, G., 559 Bukeo, M., 261 Bukhar, M. I., 405, 470 Bull, J. R., 328, 377 Bu’Lock, J . D., 219, 288, 299 Bunton, C. A., 17, 254 Burbott, A. J., 256, 260, 261,263 Burden, R. S., 139, 307 Burger, B. V., 234 Burgess, D. V., 207 Burgstahler, A. W., 20, 553 Burlingame, A. L., 188 Burnett, R. D., 358 Burrow, D. F., 4 Burrows, E. P., 18, 313 Burstein, S., 300, 358
Busetta, B., 535, 536, 537,538,539,576 Bush, P. B., 273 Butcher, D. N., 293 Butler, G. B., 56 Butt, Y., 13, 244 Butterworth, J. H., 205 Buys, H. R., 546, 566, 570 Buzan, G. A., 298 Byrd, B. G., 393 Caccia, G., 339 Cafieri, F., 173 Caglioti, L., 368, 369 Cagnoli-Bellavita, N., 150 Cahn, R. S., 139, 575 Caine, D., 139 Cainelli, G., 237 Calas, B., 35 Calas, M., 35 Calimbras, T., 274 Caln, V., 357 Cama, H. R., 222, 231, 235 Cambie, R. C., 145, 152, 155,305,330 Cameron, A. F., 570 Cameron, E. H. D., 284, 300 Campion, T. H., 322 Campsteyn, H., 537, 538, 577 Candeloro de Sanctis, S., 535 Cane, D. E., 97 Cannon, J . W., 281 Canonica, L., 176, 179, 279,291,295,305,526 b p e k , A., 394,405,500 Caple, R., 45 Caputo, R., 145, 200 Cardemil, E., 296 Cardillo, B., 75 Cardillo, G., 237 Cardon, P. V., 71 Carini, S., 484 Caristi, C., 123 Carlisle, C. H., 210, 531, 539 Carlon, F. E., 425 Carlson, J. A., 26 Carlson, J. P., 274 Carlson, R. C., 63 Carlson, R. M, 353 Carlstrom, K., 515, 516, 5 19 Carman, R. M., 31, 33, 146 Carpio, H., 345
Author Index
588 Carstensen, H., 457 Cary, L. W., 214 Casas-Campillo, C., 404, 520 Case, J., 481 Casinovi, C. G., 179, 180 Caspi, E., 276, 282, 285, 348,396,433 Castagnoli, N., jun., 74 Castelli, P. P., 339 Castro, E. A., 225 Catalano, S., 376 Catroux, G., 479 Catsoulacos, P., 156, 382 Cave, A., 377 Cavill, G. W. K., 301 Cazaux, M., 58 Ceccherelli, P., 150 Cerfontain, H., 245 cerny, V., 320, 347, 352 Cerrini, S., 179 Chaabouni, R., 50 Chabudzinski, Z., 4, 31, 41 Chachaty, C., 314 Chadha, N. K., 26 Chaigneau, M., 70 Chain, E. B., 166, 179, 271,293 Chaineaux, J., 14 Chakkaborti, P. C., 168 Chakrabarti, P., 218 Chakravarti, K. K., 101, 129 Chambers, R. J., 388 Chambers,'V. E. M., 447 Chan, N. G., 466 Chan, W. R., 203 Chan, W.-S., 190 Chander, J., 15 Chandra, P., 456,469 Chaney, M. O., 538 Chang, C.-F., 9 Chang, F. N.. 446, 520 Chang, G. C., 200 Chang, P. K., 487 Chang, S., 544 Chansang, H., 291 Charles, G., 193, 367 Charlwood, B. V., 19, 112,113,261,300,302 Charney, W., 394,5 11 Chaudhuri, R. K., 200 Chavis, C., 457 Chayet, L., 82, 256 Chen, J. W., 449,476 Chen, Y.-C., 423 Cheriyan, U. O., 57, 61 Chernyavskava, M. A., 286
Cherry, P. C., 398, 400, 446 Chetty, G. L., 181 Cheung, H. T., 191,213 Chiang, C., 200 Chiang, J. F., 562 Chiaroni, A., 535 Chichester, C. O., 221, 228,290 Chidester, C. G., 396, 538 Chigaleichik, A. G., 479 Chihara, C., 460 Chin, C.-C., 393 Chin, K., 231 Chin, W. J., 219 Chlebicki, J., 6 Choay, P., 383 Chogovadze, Sh. K., 9 Christensen, A. T., 540, 54 1 Christensen, H. D., 71 Christensen, P., 5 Christensen, R. L., 225 Chu, J. W., 286 Chuang, V. T., 391 Chuche, J., 313 Chugh, 0. P., 13 Chung, S. K., 186 Chwastek, H., 346 Cimino, G., 142, 173, 248,295 Cizinska, A., 453 Clandinin, D. R., 222 Clapp, L. B., 33 Claquin, M. J., 488 Clardy, J., 12, 214 Clark, A., 455 Clark, I. M., 363, 398, 418,449 Clarke, D. G., 75 Clarke, R. L., 450 Clegg, A. S., 399, 446 Cleve, G., 428 Clifford, K., 255 Clinkenbeard, K. D., 252 Coates, R. M., 186, 258 Cocker, W., 301 Cocucci, M. C., 484 Cody, V., 537,540 Coggins, C. W., 290 Coggon, P., 540 Cohen, A., 42 Cohen, C. F., 275,484 Collins, C. J., 47, 49 Collman, .I. P., 316 Colunga, F., 180 Colvin, M., 487 Combe, M. G., 398 Comberton, G., 537 Compernolle, F. C., 286
Conia, J.-M., 28, 361 Conlay, C., 179 Connolly, J. D., 92, 123, 147, 158, 160, 201, 204,220,300,305 Constantine, G. H., jun., 214 Contento, M., 237 Cook, I. F., 282 Cook, R. J., 4 Cookson, R. C., 241 Coolbaugh, R. C., 267, 268 Coombe, R. G., 503,504 Cooper, A., 535, 536, 537, 538, 539, 540, 575,576 Cooper, D. Y., 287 Cooper, M. A., 42 Coppola, J. C., 537,575 Corbett, K., 293 Corbett, R. E., 217, 219 Corelli, C., 523 Corey, E. J., 94, 97, 98, 103, 119, 120,353 Corey, R. B., 576 Cori, O., 82, 254, 256, 296 Cornforth, J. W., 69, 77, 222,255,298,299,300 Corrie, J. E. T., 164 Coscia, C. J., 24,263,264 Costes, C., 233 Cotton, W. D., 46 Coulter, A. W., 504 Courseille, C., 535, 536, 537: 538, 539, 576 Court, W. A., 152 Covey, D. F., 49 Cowley, P. S., 273 Cox, M. R., 36, 116 Cox, P. H., 515 Cox, P. J., 117 Cox, R. E., 188,271 Coxon, J. M.. 61 Crabbe, P., 3 18,345,366, 382,520,552 Craig,'W. J:, 146 Crane, F. L., 300, 308 Crastes de Paulet, A., 187, 296,456 Crawford, R. J., 29, 87 Crilly, W., 65 Crombie, L., 20, 75, 228, 258 Cross, B. E., 162, 163, 300,304 Cross, J . H., 23 Croteau, R., 112, 256, 259,260,261,264 Crout, D. H. G., 77
Author Index Crowe, D. F., 354 Crowell, J. D., 119 Crowfoot, D., 531 Crown, J., 302 Crowther, J. S., 493 Crozier, A., 161 Csernay, L., 297 Culvenor, C. C. J., 154 Cumming, S. D., 217 Cunningham, A., 147 Cupas, C. A., 53 Curry, S. H., 302 Curtis, A. J., 110 Curtis, P. J., 466 Cushman, M., 74 Cuvigny, T., 6 Cynkowski, T., 338 Czerniawski, E., 421 Czygan, P., 287 Dagonneau, M., 52 Dailey, R. G., 152 Dakshinamurti, K., 297 d’albuquerque, I. L., 217 d’Alcontres, G. S., 123 Dallinga, G., 559 Dalzell, H. C., 73 Dalziel, W., 166 Dames, M. E., 70 Damiano, J., 40 Darnmeier, B., 538 Damodaran, N. P., 29 Damps, K., 199, 273 Dana, S. E., 252 Dane, E., 531 Danheiser, R. L., 126 Daniel, A., 49 Daniel, D. S., 103, 120, 265 Danielli, B., 159 Daniels, P. J. L., 391 Danieison, T. J., 24 Danielsson, H., 253, 286, 527 Danishefsky, S., 304 Danishefsky, S. E., 304 Dann. M., 397 Darias, J., 96, 147 Dastur, K. P., 31 Dauben, W. G., 188,312, 324,386,387,536 Daum, S. J., 450 Dauphin, G., 5 David, C. W., 44 Davidson, S. J., 472, 473 Davies, B. H., 299, 306 Davies, V. H., 116 Davies, H. Ff. S., 8 Davies, K. H., 71 Davis, J. B., 306 Davydova, L. P., 243
589 Dean, P. D. G., 187, 287 Debaerdemaeker, T. D. J., 540 de Boer, Th. J., 57 de Botton, M., 8 de Broissia, H., 89 Decker, K. F. A., 251 Declercq, J. P., 537, 538 de Flines, J., 394, 398, 401, 408, 413, 417, 441,442,446,452,5 11 Deghenghi, R., 419,521 Degny, E., 44 de Graaf, R. A. G., 535, 568,570 de Groote, R., 133 Dehennin, L., 359 De Iglesias, D. I. A., 53 De Jarnette, F., 540 de Jong, J. G. H. 539 de Kok, A. J., 539, 561 de la Mare, P. B. D., 330, 361 De Leo, P., 179 Delettre, M. J., 538 Delle Monache, F., 2 17 de L. Meyers, C., 501 Delpech, B., 334 De Luca, H. F., 287, 383 De Luca, L., 234 Delwiche, C. V., 299 de Mayo, P., 52 Dembitskii, A. D., 9 de Mello, J. F., 2 17 Demole, E., 9, 44 Dempsey, M. E., 274,283 Demuth, M., 74 de Nijs, H., 377 Denning, R. G., 48 Denny, R. W., 391 Denny, W. A., 363, 398, 403,447,449 Denot, E., 520 De Pascual, T. J., 63 De Pauw, G., 286 de Reinach-Hirtzbach, F., 196 De Roos, J. B., 120 De Rosa, M., 219, 288 Deshmane, S. S. 372 Deshpande, P. D., 170 Desiderio, D. M., 327 De Sombre, E. R., 576 De Stefano, S., 142, 173, 248,295 Detre, G., 329 Dev, C., 314 Dev, S., 6, 29, 70, 88, 89, 164, 181,194 Devon, T. K., 301, 303, 304,305
Dhindsa, A. S., 13 Diamlarneh, G. H., 248 Diassi, P. A., 439 Diaz, E., 133 Diaz-Parra, M. A., 139 Dickson, L. G., 275 Dideberg, O., 537, 538, 577 Diem, M., 4 Dietschy, J. M., 252 Di Giorgio, J. B., 391 Dik-Edixhoven, C. J., 539 Dimmel, D. R., 47 Dini, M., 274 Di Pietro, D. L., 3 13 Djerassi, C., 36, 193,330 340, 349 Dmochowska, J., 461 462 Dodds, E. C., 576 Dodson, R. M., 446 Doellgast, G. J., 442 Doherty, C. F., 20 Donohue, J., 535 Doodewaard, J., 51 1 Dorfman, R. I., 394, 469 Dorn, F., 107 Dorokhov, V. G., 17 Doskotch, R. W., 115 Downing, M., 24, 253, 263 Doyle, P. J., 282 Draber, W., 222 Drabkina, A. A., 26, 303 Drakenberg, T., 113 Drasar, B. S., 492, 493, 526 Drews, J., 455, 456 Dreyer, D. L., 305 Drozdz, D., 115 Duax, W. L., 311, 534, 537, 540, 548, 549, 552,569,575 Duch, M. W., 223 Ducharnp, D. J., 396,538 Durr, F. H., 407 Duerst, R. W., 539 Dugan, R. E., 252, 253, 258 Dumas, P., 6 Duncan, J. M., 277 Dunitz, J. D., 531 Dunphy, P. J., 256 Dunstan, P. J., 207 Duphorn, I., 172 Dupont, L., 537,‘538,577 Duprey, R. J. H., 14 Durham, N. N., 427 Durley, R. C., 161, 269 Durst, F., 273 Dutky, S. R., 275
590 Dutla, N. L., 207 Dutta, S. P., 213 Dvonch, W., 459 D'yakonova. R. R., 64 Dzhaiani, G., 260 Dzhemilev, U. M., 57, 328,332 Dziewanowska, K., 2 18 Eade, R. A., 207, 212 Eber, J., 101 Ebersole, R. C., 285 Ebrey, T. E., 226 Eck, C. R., 100 Ecklund, P. R., 267 Edery, H., 71, 72 Edmond, J., 19, 187,227, 228, 258 Edward, J. T., 3 5 3 . Edwards, B. E., 474 Efimochkina, E . F., 460, 497,498 Eger, C., 548, 552 Eger, C. H., 357 Eglinton. G., 188, 306 Egorova, V. V., 3 12 Eguchi, S., 8 Eigendorf, G., 190 Einarssonn, K., 286 Einhorn, J., 366 Eisenbraun, E . J., 36 Eisma, S. W., 560 Eisner, T., 84 Ekundayo, O., 344 Elahi, M., 290 El-Feraly, F. S., 115 El Gaied, M. M., 60 El-Gorab, M . , 223 Elin, E. A., 448,467,468 El-Kady, I. A., 405, 419, 448,449 Ellestad, G. A., 150, 157, 270 Elliott, M., 22 Ellis, J., 207 El-Olemy, M. M., 284, 470 El-Refai, A.-M. H., 286, 404,405,419,448,449 Els, H., 41 1, 446 El-Tayeb, 0. M., 466 Emerman, S. L., 454 Emmert, D. E., 538 Eng, S., 365 Engel, Ch. R., 384 Engel, L. L., 286, 297, 456,489 Englert, G., 223, 306, 307,411 Ensley, H. E., 333 Entwistle, N., 192, 3 18
Author Index Enwall, E . L., 539 Enzell, C. R., 9,238,302, 307 Epps, R., 442 Epstein, W. W., 19, 226, 227,263,302 Epsztein, R., 346 Erickson, R. C., 476 Eriksson, H., 485, 486, 528 Erlanger, B. F., 179 Erm, A., 10 Erman, W. F., 87, 97 Ernst, R., 392 Eschinasi, E . H., 110 Esders, T. W., 493 Eugster, C. H., 38, 152, 225,226 Eustace, E. J., 25, 55 Evans, B. B., 153 Evans, D. A., 16, 298 Evans, F. J., 273 Evans, J. M., 398, 417, 418,446,447 Evans, R., 90, 256, 267, 268 Evans, R. H., 397,495 Everling, B. W., 44 Evrard, M., 44 Eyssen, H. J., 286 Faass, U., 23 Faber, D. H., 533 Fahrenholtz, K. E., 573 Faini, F., 82, 254 FajkoS, J., 319, 324, 332, 336, 337, 350, 365, 371,401,418 Falardeau, P., 421, 448 Falcone, M. S., 95 Falcoz-Kelly, F.. 488 Fall, R. R., 267 Fallis, A. G., 61 Fankuchen, I., 531 Faraone, A., 573 Fare, L. R., 401 Farnham, A. W., 22 Fattorusso, E., 173, 182 Faulkner, D. J., 12, 86, 173 Fauve, A., 356 Favini, G., 559 Favre-Bonvin, J., 301 Fawcett, J. K., 536 Fayez, M . B. E., 420 Fayos, J., 12, 214 Fazli, F. R. Y., 273 Fedeli, W., 179 Federbush, C., 51 1 Feigenbaum, A., 389 Feiner, S., 328
Feldman, L. I., 397 Feller, H., 469 Fenemore, P. G., 152 Fenical, W., 96, 146, 166, 270 Fenton, T. W., 222 Fentrill, R. J., 9 3 Feofilova, E. P., 299 Ferguson, G., 201, 536, 570 Fermin, C. M., 157 Ferrari, A., 484, 526 Fetizon, M., 328, 369 Fiecchi, A., 176, 179, 276,305 Fieser, L. F., 379 Fieser, M., 379 Findlay, J. W. A., 347 Findley, D. A. R., 20, 228,258 Finkelhor, R. S., 8 8 Fisch, M. H., 392 Fish, R. E . M. H., 277 Fisher, J., 535 Fisher, N., 5 3 Fisher, R. D., 47 Fleischer, E. B., 481 Fleming, I., 51 Flick, B. H., 277, 392 Flood, T. C., 329 Floss, H. G., 293, 297 Foell, T., 398, 463, 494 Fonina, N. A., 497 Fonken, G. S., 394 Fonquernie, M., 461 Font Cistero, J. M., 36 Foo, L. Y., 207 Fordham, W. D., 14, 110 Formes-Marquina, J. M., 536 Forrester, J. M., 9 0 Fort, R. C., 338 Fosset, M., 456 Foster, E. L., 326 Fournier, J.-C., 479 Fracheboud, M., 1 31 Fraga, B. M., 147, 158 Framon-dino, M., 180 Francis, G. W., 223, 307 Francis, M. J. O., 19, 112, 254,264,300,302 Franck-Neumann, M., 367 Frank, S. G., 393 Frankowski, A., 366 Frantz, I. D., 300 Frappier, F., 376 Freeman, C. W., 275 Frey, M. J., 407 Freyberg, M., 277 Fridrichsons, J., 539
Author Index Fried, J., 481, 501 Fried, J. H., 345 Friedell, G. H., 282 Friedrich-Fiechtli, J., 154 Frohlich, H. H., 29 Frohlich, A., 124 Frost, R. G., 268 Frost, S., 526 Fry, J. L., 4 Frye, N. L., 327 Fu, W., 47 Fiirst, A., 41 1, 446 Fujikura, T., 149 Fujimori, K., 47 Fujimoto, T. T., 16 Fujino, A., 386 Fujino, M., 10 Fujita, E., 159, 160, 167, 269,304 Fujita, K., 18 Fujita, S., 9 Fujita, T., 8, 10, 11, 115, 116,160,167,269 Fujita, Y., 9 Fujiwara, T., 504, 505, 508 Fukuda, T., 10 Fukui, K., 155 Fukushima, D. K., 397 Fukushima, K., 171 Fulke, J. W. B., 201 hllerton, T. J., 155 Furfine, C. S., 466 Furst, G. T., 55 Furth, B., 45 Fuhkawa, H., 116 133 , Furuta, T., 26 Furuya, T., 200,284,483 GaB1, I., 363 Gabelta, R., 299 Gabetta, B., 159 Gabinskaya, K. N., 425 Gadsby, B., 444 Gaede, K., 234 Gagnon, R. E., 287 Gain, R. E., 433 Galantay, E., 540 Galpnter, 1. M., 71 Galasko, G., 307 Galbraith, M. N., 239, 2 84 Gall, R. E., 320 Gallay, J., 286 Gallegos, E. J., 393 Galli, G., 276 Galli Kienle, M., 176, 179,396 Gambacorta, A., 219,288 Gandhi, R. P., 390 Ganem, B., 126
59 1 Ganguly, S. N., 161 Ganguly, T., 161 Gaoni, Y., 41, 42 Garabedian, D., 383 Garbers, C. F., 234 Garcia-Peregrin, E., 253 Gardi, R., 327, 339, 345 Gardner, D., 92 Gardner, J. N., 425 Garey, K. L., 274 Garland, R. P., 61 Garmston, M., 240, 266 Garrett, R. D., 426, 479, 522 Garsky, V., 60 Garst, M. E., 68, 84 Gary, W., 35 Gasa, S., 164 Gaskin, P., 142, 161, 240 Gasparrini, F., 368 Gatford, C., 8 Gattuso, M., 123 Gaudry, R., 498 Gaurnert, R., 253 Gavrilova, T. F., 44 Gay, R., 286,482,508 Gaylor, J. L., 297, 299, 300,525 Gefter, M. L., 294 Geiger, R., 10 Geise, H. J., 531, 532, 538, 546, 547, 560, 565,568,570 Geissman, T. A., 77, 116, 123,133,266, 303 Geith, H., 449 Gelbke, H. P., 343 Gensler, W. J., 66 Geoffre, S., 539 George-Nascimento, C., 82,256 Georghiou, P. E., 338, 360 Geraghty, M. B., 103,129 Gerhards, E., 469 Geribaldi, S., 40 Gerlach, H., 4 Germain, G., 532, 537, 538 Germain, P., 286, 482 Gero-Robert, M., 154 Gesson, J. P., 381 Geuns, J. M. C., 273 Geynet, P.,286 Ghatak, U. R., 168 Ghazarian, J. C., 287 Ghera, E., 425 Ghirlanda, C., 70 Ghisalberti, E. L., 160, 165,207,269,288 Gholson, R. K., 297
Ghosal, S., 200 Ghosh, M. C., 235 Ghraf, R., 286 Giannini, D. D., 311, 548 Gibaja, S., 303 Gibb, W., 285, 468 Gibbons, G. F., 276, 283 Gibbs, J. A., 203 Gibian, H., 449, 464 Gibson, D. M., 252 Gibson, D. T., 503 Gibson, F., 294, 299 Gibson, T. W., 47,97 Giessner-Prettre, C., 223 Giglio, E., 535 Gijzeman, 0. L. J., 225 Gilbert, J. D., 4, 312 Gilbert, M. T., 4 Gill, D., 225 Gillen, D. G., 35 Gilmore, C. J., 152 Gingras, G., 231 Giral, L., 35 Girard, C., 361 Girotra, N. N., 94 Giry, L., 70 Gitany, R., 135, 266 Giumanini, A. G., 50 Gladiali, S., 345 Glasenapp, A., 286 Gleason, R. M., 290 Glen, A. T., 92 Gloor, U., 308 Glotter, E., 305, 323 Gnoj, O., 457 Goad, L. J., 277, 278, 279,280,285,299,306 Gochnauer, M. B., 290 Goddard, P., 473, 498 Godtfredsen, W. O., 285 Godunova, L. F., 32 Gorlich, B., 407 Goldberg, L., 576 Goldfarb, S., 252 Goldman, A. S., 286,456 Goldrnan, J. M., 136 Goldman, R., 296 Golfier, M., 328, 369 Goll, P. H., 394 Gollnick, K., 6 Gomez, F., 180 Goncalves de Lima, O., 217 Gonzalez, A. G., 96, 147, 158 Goodfellow, R., 285 Goodfellow, R. G., 186, 258 Goodman, J. J., 494
A uthot Index
592 Goodwin, T. W., 228, 258, 277, 278. 279. 280, 282,' 284, 289, 290,298,299,306,307 Goosen, A., 384 Gopalakrishna, E. M., 537 Gora, J., 16 Gorbach, S., 493 Gordon, K. D., 286, 328, 489 Gordon, M., 127, 493 Gorovits, M. B., 408 Goryaev, M. I., 9 Gosztonyi, T., 7 3 Gotovtseva, V. A., 479 Gough, L. J., 147 Gougoutas, J. Z., 538 Gould, R. G., 252 Goutarel, R., 376, 385 Govindachari, T. R., 197, 217 Gower, D. B., 284 Grabowich, P., 501 Grabowski, G. A., 283 Graebe, J. E., 267 Grande Benito, M., 6 3 Grandi, R., 119 Grandolini, G., 180 Granger, P., 223 Granger, R., 8 Grant, D. M., 223 Grant, P. K., 146 Grasselli, P., 237 Grassmayr, K., 9 Gravel, D., 353 Graves, J. M. H., 455,470 Gravestock, M. B., 167 Gray, J. C., 253, 254 Green, A. E., 124 Green, C. L., 298 Green, G. H., 207 Green, J., 272 Green, M. J., 481 Green, T. R., 259 Greene, R. L., 539 Greenspan, G., 398, 424, 444,459,463 Gregonis, D. E., 226 Greim, H., 287 Greiner, M., 357 Grenz, M., 149 Grieco, P. A., 8 8 Grieder, A., 131 Grierson, D. S., 194 Griffith, G . R., 24, 263 Griffiths, E., 300 Grimshaw, J., 46, 6 0 Grimwade, A. M., 283 Grinenko, G. S., 425 Grison, C . , 56, 61, 97
Groh, H., 399, 404, 492, 497,521 Gros, E. G., 410 Gross, D., 221, 243, 263, 298 Grossert, J. S., 306 Groth, P., 572 Grove, M. J., 492 Grunfeld, Y., 71, 72 Grunwald, C., 273 Grutzner, J. B., 223, 297 Gsell, L., 399, 410 Guarnaccia, R., 24, 263, 264 Gubkina, N. I., 61 Gueldner, R. C., 8 Guenther, H. F., 293 Guerrero, H. C., 221 Guest, I. G., 93, 375 Guida, A., 325 Guilhem, J., 540 Guillaumon, J.-C., 5 0 Gulaya, V. E., 465 Gullo, V. P., 2 17 Gumulka, M., 338 Gunasekera, S. P., 214, 215 Gunn, P. A., 201 Gunville, R., 283 Gupta, A. S., 181 Gupta, R. N., 297 Gurny, O., 71 Gusakova, E. G., 479 Gustafsson, B., 73 Gustafsson, B. E., 526, 527 Gustafsson, J.-A., 282, 285, 286, 458, 460, 484,485,486,528 Gut, M., 300, 474 Guthrie, J. P., 367 Guthrie, R. W., 354 Guyer, K. E., 253 Guzman, A., 318 Habermehl, G., 383 Hach, V., 7, 42, 317 Hachey, D. L., 17 Hackenschmidt, J., 25 1 Hackett, P., 347 Hackler, R. E., 17 Hafez-Zedan, H., 450, 466,476 Hagef, L. P., 496 Hainaut, D., 532 Hall, J. B., 6 1 Haller, F., 222 Haller, R., 5 Halsall, T. G., 200
Hamanaka, N., 164 Hamasaki, T., 230 Hamilton, M. A., 283 Hamilton, P. B., 503 Hamilton, W. D., 8 3 Hammer, C. F., 466 Hanayama, N., 96 Hank, O., 394, 405, 453, 5 00 Haner, B., 536 Hanna, R., 316 Hannan, B. N. B., 361 Hannaway, C., 570 Hanni, R., 205 Hanover, J. W., 9 Hansbury, E., 274 Hansen, H.-J., 344 Hanson, F. R., 449 Hanson, J. R., 77, 90,92, 113, 145, 162, 256, 267, 268, 296, 298, 300, 303, 304, 305, 332,377,536 Hanson, R. F., 283 Hanson, S. W., 51 Happ, G . M., 9 Harada, N., 139,142,226 Harano, K., 322 Hardgrove, G. L., 539 Harding, A. E., 92, 160 Harding, B. W., 286, 300 Harding, K. E., 266, 298' Hardman, R., 273 Hargie, M. P., 447 Harita, K., 381 Harkness, A. L., 223 Harper, P., 207 Harrison, H. R., 535 Harrison, I. T., 180 Harrison, S., 180 Harry, D. S., 274 Hartman, R. E., 495 Hartmann, M. A., 273 Hartshorn, M. P., 6 1 , 3 1 1 Harvey, D. J., 315, 326 Hasegawa, S., 203 Hashiba, H., 511, 513 Hashimoto, S., 504, 508 Haslewood, G . A. D., 515 Hassel, O., 572 Hassner, A., 382 Hasunuma, M., 410,434 Hata, G., 11 Hatanaka, H., 259 Hatem, J., 59 Hattori, T., 526 Haupt, O., 343 Hauptman, H., 535, 537, 540,569 Hausen, B. M., 69, 295 Hauser, F., 73
Author Index Haussler, M. R., 287 Havinga, E., 386, 547, 558, 562, 566 Hawes, E. M., 24 Hawker, J., 162,268,296 Hawkes, G. E., 3, 313 Hawkins, D. W., 193,369 Hawks, R. L., 73 Hawksworth, G., 493 Hay, C . E., 338,398,409 Hayakawa, S., 504, 505, 508, 526 Hayakawa, T., 5 Hayakawa, Y., 107 Hayano, M., 469,470 Hayashi, A., 22 Hayashi, J., 388 Hayashi, M., 133 Hayashi, S., 56 Hayashi, Y., 265 Hayward, L. D., 313 Hayward, R. C., 155, 330 Heathcock, C. H., 77, 103, 303 Hedin, P. A., 8 Hedman, K., 73 Hefendehl, F. W., 9, 35, 262 Heftmann, E., 293 Hegnauer, R., 301 Heidel, P., 282 Heidenpriem, H., 464 Heimberger, S. I., 297 Heinrich, G., 250 Heinrichs, W. L., 284 Heintz, E., 278 Heintz, R., 258, 274 Heller, J., 226 Heller, R. A., 252 Helting, T., 234 Hemesley, P., 20 Hemingway, J. C., 115 Hemingway, R. J., 115 Heniming, F. W., 294, 308 HCmo, J. H., 367 Henc, B., 5 Henderson, M. S., 148, 20 1 Henderson, R., 204 Hendrickson, J. B., 77 Heng, C. K., 219 Hengartner, U., 188 Hentchoya, J., 193 Herber, R., 223, 290 Hermann, I., 486 Hernandez, M. G., 147, 158 Herout, V., 102, 115, 133,135 Herz, J. E., 329
593 Herz, W., 115, 116, 117, 135,136,208,266,303 Herzog, H. L., 318, 394, 457, 511 Hesp, B., 166 Hesper, B., 532, 538 Hesse, R. H., 355, 356 Hessler, E. J., 483 Heyde, M. E., 225 Hickey, M. J., 544 Higashi, Y . , 295, 296 Higgins, M. J. P., 251 Higk, D. F., 532 Higo, A., 22 kiigson, H. G., 42 Hikino, H., 163 Hikino, Y., 163 Kill, F., 408, 491 Hill, F. L., 342 Hill, J., 389 Hill, M. J., 461,473,492, 493,498,526 Hillman, J. R., 266 Hills, F. J., 476 Hiltunen, R., 12, 264 Hino, K., 2 13 Hiraga, K., 464 Hirai, H., 22 Hirai, K., 176 Hiraka, Y., 165 Hirakata, A., 3 Hirao, N., 6 Hirata, T., 60, 63 Hirata, Y., 93, 107, 125, 188,292 Hirayama, K., 41 1 Hirose, Y., 88, 95, 122 Hirotani, M., 284, 483 Hirsch, J. A., 580 Hirschfeld, D. R., 166, 270 Hirschmann, F. B., 371 Hirschmann, H., 37 1, 372,533 Hirsjarvi, P., 44 Hobbs, P. D., 23 Hobe, G., 483 Hochstetler, A. R., 97 Hodgkin, D. C., 531,535, 536 Hodgkinson, A. J., 377 Hodgson, G. L., 87, 99 Hofle, G., 7 Hoehne. E., 535 Horhold, C., 399, 404, 415, 445, 453, 460, 483, 490, 497, 505, 506,507,508,521,523 Hoffman, E., 469 Hoffman, W. A., 315 Hoffmann, R., 386
Hoffmann, W., 11,28 Hofmann, A. F., 526 Hofmeister, H., 355 Hogg, J. W., 102 Hohenlohe-Oehringen, K., 9 Holden, C. M., 42 Holden, K. G., 401 Holick, M. F., 383 Holland, H. L., 404 Hollaway, P. W., 298 Hollister, L. E., 71 Holmberg, I., 286 Holmlund, C. E., 397, 495 Holtom, A. M., 90, 256 Holton, R. A., 183 Holub, M., 115, 123, 133 Honig, B., 226 Honwad, V. K., 37 Hood, L. V. S., 70 Hoogenboom, F. J. M., 570 Hooper, S. N., 271 Hope, H., 540 Hopla. R. E., 183 Hoppe, W., 532, 538 Horan, H., 186 Horan, M., 288 Horibe, I., 115 Horie, M., 491 Horie, Y., 282 Horn, D. H. S., 230, 284 Hornstra, J., 532, 537, 538,539,552 Horster, H., 262 Hortmann, A. G., 103, 120,265 Horwitz, J., 226 Hosada, H., 474,475 Hoshita, T., 526 Hospital, M., 535, 536, 537,538,539,576 House, H. O., 168 Hovorkova, N., 208 Hoyer, G.-A., 337, 351, 428 Hsia, S. L. 286 Hsiao, J. C. Y., 283 Hsu, A. C., 203 Hsu, W. J., 290 Huang, H.-C., 116 Huang, M., 423 Hubbard, R., 307 Huber, H., 169 Huber, R., 532 Hudec, J., 553 Huckel, W., 47 Huffman, J. W., 123,265 Hufford, C. D., 115
Author Index
5 94 Hughes, C. R., 9 3 Hugl, H., 334, 335 Hui, W.-H., 190 Hulcher, F. H., 252 Hunt, J. D., 342 Hunter, D. H., 49 Huntrakul, C., 146 Hursthouse, H. B., 36 Husson, H.-P., 163, 377 Hutchins, M. G., 318 Hutchins, R. O., 34, 318, 3 64 Hutterer, F., 287 Hutto, F. Y., 8 Hrycay, E., 286 Iaccarino, R., 145 Ibekawa, N., 282 Ibuka, T., 107 Ichino, T., 338 Ida, Y., 149 Iguchi, M., 119, 139 Iida, M., 459, 470 Iida, T., 144 Iitaka, Y., 176, 180, 182, 27 1 Iizuka, H., 394, 459 Ikada, I., 57 Ikai, K., 163 Ikawa, M., 222 Ikegawa, S., 475 Ikekawa, N., 197, 333, 381 Ikeshima, H., 340 Ila, L., 477 Imai, K., 199 Imai, S., 155 Imaizumi, K., 125 Imakura, Y., 218 Imanishi, M., 467 Imshchenetskii, A. A., 460,497,498 Inamura, K., 120 Inayama, S., 135, 266 Ingold, C. K., 139, 575 Ingwalson, P. F., 139 Innocenti, S., 352, 384 Inoue, S., 249 Inouye, H., 23, 24, 301 Inubushi, Y., 107 Iocco, D., 336 Ireland, R. E., 188 Iriate, J., 366 hie, T., 9 3 Irwin, M. A., 116, 123, 133,266, 303 Isaacs, N. W , 537, 575 Isaeva, Z. G., 63, 64 Ishibashi, K., 176 Ishidate. M., 408
Ishii, H., 314 Ishii, T., 200 Ishikawa, K., 124 Ishikawa, M., 336 Isidor, J. L., 353 Isler, O., 233, 306, 307, 308 Isobe, T., 160 Isoe, S., 242, 291 h e r , S. J., 108 Istomina, Z. I., 3 13 Itai, A., 176 Itaya, N., 22 Ito, A., 460 Ito, M., 474 Ivanova, L. S., 56, 61 Ivaseva, T. N., 66 Ivashkiv, E., 446, 482 Iwamnra, J., 6 Iwasaki, S., 370 Jackson, R. W., 394 Jackson, W. R., 37 Jacob, G., 82, 254 Jacobs, H. J. C., 552,562 Jacobsen, R. A., 532 Jacobson, H., 451 Jacobson, H. I., 456 Jacobus, J., 1 8 Jacquesy, J. C., 373, 381 Jacquesy, R., 350, 373, 381 Jaeger, G., 10 Jain, T. C., 118 Jaitly, K. D., 421 James, N. F., 22 James, P., 212 Jamison, V. W., 497 Janes, J. F., 14 Janoski, A. H., 442 Janot, M.-M., 4 4 8 , 4 6 1 Jarabak, R., 487 Jarreau, F. X., 376 Jarvis, J. A. J., 166 Jautelat, M., 223 Jaworski, A., 421 Jeanloz, R. W., 247 Jedlicki, E., 82, 254 Jedziniak, E. J., 385 Jefferies, P. R., 150, 160, 165,207,269,288 Jeffery, J., 284, 285, 468, Jefford, C. W., 4 5 Jeger, O., 387 Jenkins, E., 442 Jensen, E. V., 576 Jeremic, D., 102 Jerussi, R., 474 Jewers, K., 203 Jirku, H., 407 Joblin, K. N., 145
Johannes, B., 307 John, J., 222, 231, 235 Johns, S. R., 3 Johnson, A. L., 4 9 Johnson, C. K., 49,547 Johnson, R. A., 394 Johnson, R. C., 274 Johnson, W. S., 86, 107 Johnston, J. P., 156, 270 Jokic, A., 102 Joly, G., 373 Jommi, G., 298 Jones, A. J., 164 Jones, C. A., 260 Jones, E. R. H., 90, 363, 398, 399, 400, 403, 417,418,446,447,449 Jones, J. B., 286, 328, 370,472,488,489 Jones, J. G. Li., 384 Jones, N. D., 538 Jones, W. R., 381 Josephson, S., 5 Joshi, B. S., 214 Joska, J., 324, 332, 337, 350,401 Joulain, D., 58, 59 Joustra, A. H., 540 Jowett, W. F. A., 406 Joyce, C. R. B., 302 Joye, N. M., jun., 9 Julia, M., 15, 237 Julia, S . . 318, 330 Juneja, H. R.,6 0 Jung, I., 576 Junta, B., 412, 439 Jurd, L., 6 9 , 2 9 5 Just, G., 338, 360 Kaal, T., 10, 11 Kabuto, C., 127 Kagan, H., 455 Kagi, D. A., 241 Kahn, P., 226 Kahovcova, J., 10 Kalja, I., 10 Kalkman, M. L., 284 Kallner, A., 529 Kalsi, P. S., 102 Kamat, V. N., 214 Kamikawa, T., 160 Kaminaga, M., 349 Kamogawa, H., 234 Kan, G., 286,342 Kan, K. W., 283 Kanayama, K., 2 13 Kaneda, M., 182,271 Kaneko, C., 336 Kaneko, K., 411 Kanematsu, A., 190 Kanematsu, Y ., 505
Author Index Kaplan, N. O., 455 Kapoor, S. K., 110, 156 Kapteyn, H., 12 Kapur, J. C., 68 Karasiewicz, R., 338 Karim, A., 406 Karle, I. L., 532,535,541 Karle, J., 532, 535, 541 Karlsson, B., 93 Karlsson, K., 9, 238 Kartha, G., 536, 537,540 Kasal, A., 320, 398, 403, 417,446,447 Kasprzyk, Z., 218 Kasumov, L. I., 243 Katagiri, T., 1 0 , 1 1 , 13 Katayama, C., 149 Katayama, H., 160 Kates, M., 290 Katkov, T., 284 Kato, N., 149 Kato, T., 5 , 144 Katsui, N., 125, 142, 240 Katsuki, H., 259 Katsumi, M., 161 Katsumura, S., 242, 291 Katz, J. J., 223 Katzenellenbogen, J. A., 15,84 Kaufman, M., 353 Kaufmann, G., 445,490 Kaur, J., 68 Kawaguchi, A., 187, 271, 272 Kawaguchi, K., 284,483 Kawahara, M., 155 Kawamata, T., 135, 266 Kawamatsu, Y., 249 Kawasaki, T., 149 Kawashima, K., 233 Kawata, M., 386 Kayahara, H., 32 Kazakova, E. Kh., 64 Ke, B., 307 Keates, R. A. B., 131 Keeler, B. T., 538 Keely, S. L., jun., 115 Keene, €3. R. T., 354 Kekwick, R. G. O., 251, 253,254 Kellogg, T. F., 493 Kelly, R. B., 101, 132 Kelly, T. R., 112, 125 Kelly, W. G., 442 Kelsey, R. G., 115 Kempe, T., 53 Kennard, O., 312, 536, 537,575 Kennedy, R. M., 347 Kerb, U., 405,491 Kergomard, A., 5, 356
595 Kern, H.-J., 47 Khan, G., 393 Khan, H., 181 Khatoon, N., 223 Khattak, I., 372 Kheifits, L., 44 Khidekel', M. L., 17 Khuong-Huu, Q., 334, 366,367,383,385 Kido, F., 96, 103 Kienle, M. G., 276 Kie'nzle, F., 233,235, 342 Kierkegaard, P., 93 Kierstead, R. W., 71,354, 573 Kieslich, K., 394, 403, 405, 408, 413, 415, 425, 427, 428, 449, 452, 458, 464, 491, 493,530 Kiruchi, T., 107, 212, 215,216 Kilian, R. J., 44 Kim, I., 276,398 Kim, Y.-H., 379 Kimball, H. L., 358 Kimland, B., 238 Kimura. K., 286 Kimura, M., 332, 343, 379,381, 386 Kinawy, M. H. K., 404 King, H., 531 King, J. C., 362 King, J. F., 6 King, T. J., 144 Kirk, D. N., 311, 313, 358,372 Kirtany, J. K., 97 Kis, Z., 287 Kispert, L. D., 539 Kitagawa, H., 155 Kitagawa, I., 199, 213, 218 Kitahara, Y., 127, 144 Kitchens, G. C., 97 Kitigawa, I., 128 Kitihara, Y., 5 Kjersgaard, D., 5 KjBsen, H., 229, 233 Klabunovskii, E. I., 32 Klaui, H. M., 307 Kleigman, J. M., 363 Klein, E., 8 Kleinig, H., 22 1,23 1,290 Klemm, D., 315 Kleo, J.. 225 Klimontovich, N. N., 479 Klinot. J., 208, 218 Klinotovoa, E., 208 Kloti, R., 9 Klubnichkina, G. A., 479
Kluender, H. C.. 115 Kluepfel, D., 498, 523 Kluepfel. K., 394 Klyne, W., 307, 313 Knapp, F. F., 278, 280 Knight, S. A., 193, 314 Knights, B. A., 282, 393 Knobler, C., 539, 552 Knowles, G. D., 190, 194 Knuppen, R., 343 Kobayashi, A., 20, 22 Kobayashi, H., 187, 272 Kobayashi, M., 133, 363 Koch, H.-J., 403, 405, 449,464,493 Koch, H. P., 36 Koch, W., 413,425 Kocor, M., 194, 338 Koepsell, H. J., 476 Kogan, G. A., 313 Kogan, L. M., 394, 405, 444, 448, 460, 465, 467,468,490,521 Kogure, T., 7, 243 Kohler, B. E., 225 Kohler, D. F., 274 Kohli. J. C., 102 Kohout, L., 319, 336, 365,371 Kohoutova, J.. 127 Kojima, H.. 200 Kokke, W. C. M. C., 50 Kokkinas, C., 156 Kokor, M., 2 13 Kollman, P. A., 311, 548 Komori, T., 149 Komoto, R. G., 316 Kondo, E., 401,413,446, 460,513 Kondo, K., 86 Kong, Y. C., 284 Konstantinovii, S., 20 Kontonassios, D., 352 Konz, W. E., 183 Kooreman, H. J., 421 Koreeda, M., 139, 222, 226 Koriyama, S., 163 Korn, E. D., 499 Korovkina, A. S., 479 Korpi, J., 318 Korvola, J., 43 Koshcheenko, K. A., 491 Kosmol, H., 413, 425, 464,491 Koss, F. W., 286 Kossanyi, J., 45 Koster, D., 60 Kotake, M., 68 Kovacic, P., 47
Author Index
596 Koveshnikov, A. D., 408 Kovnazkaja, I. S., 313 Kovylkina, N. F., 468 Kowerski, R. C., 226 Koyama, T., 86, 257 Kozhin, S. A., 30, 34 Kozlava, V. Kh., 497 Kozuka, M., 116 Kozuka, S., 47 Krakower, G. W., 538 Krasnova, L. A., 466 Kratzer, O., 233 Kraut, J., 532 Kraychy, S., 510 Kreiser, W., 193, 355 Kremen, A., 293 Krinsky, N. I., 230, 307 Krishnamurthy, S., 348 Krishnamurti, M., 468 Krishnappa, S., 88 Kroeger, H. W., 20 Krojidlo, M., 127 Kroszczynski, W., 29 1 Krueger, S . M . , 65 Kruger, C., 329 Krugyel, W. C., 284 Krumer, M. Z., 39, 245 Ksandopula, G. B., 479, 49 1 Kuan Tee Go, 537 Kubina, L., 536 Kubo, I., 160 Kuboe, K., 70 Kubota, T., 160 Kucherov, V. F., 39, 245 Kucinski, P., 342 Kuczynski, H., 3 7 , 4 1 , 5 1 , 66 Kudryavtsev, I. B., 10 Kuehl, D. W., 4 5 Kuehne, M. E., 362 Kukharenko, N. V., 342 Kulesza, J., 16 Kulig, M. J., 342, 393 Kulikova, L. E., 458,460, 478 Kulshreshtha, D. K., 198, 206,305 Kulshreshtha, M. J., 305 Kumai, S., 18 Kumar, V., 417, 446 Kumazawa, S., 5 Kumazawa, Z., 132 Kumta, U. S., 290 Kunde, R., 37 Kunstmann, M. P., 150, 157,270 Kupchan, S. M., 115,116, 152, 195, 196, 206, 303,347,541 Kupletskaya, M. B,, 468
Kurbanov, M., 245 Kurek, A., 365, 371 Kurosawa, E., 93 Kurosawa, Y., 463, 493 Kushner, D. J., 290 Kushwaha, S. C., 290 Kusner, E. J., 479 Kutney, J. P., 190, 194 Laats, K.. 10, 11 Labriola, R., 199 Labruykre, F., 50 Lachman, H. H., 328 Lacko, A. G., 297 Ladd, M. F. C., 539 Laing, D. E., 262 Laing, M., 161 Laing, S. B., 360 Lakshmanan, M. R., 253, 29 1 Lal, B., 315 Lalancette, J.-M., 6 Lalande, R., 29, 58 Lamazoukre, A.-M., 52 Lambert, J. L., 49 Lan, N. T., 49 Land, E. J., 225 Lane. H. D., 252 Lang, A., 304 Langbein, G., 446 Lange, W., 497 Langenheim, J. H., 147 Langlet, J., 223 Langlois, R., 393 Larcheveque, M., 6 Larsen, B. R.,226 Larsson, P.-O., 450 Laskin, A. I., 501 Lassak, E. V., 35, 192, 341 Lauer, R. F., 14, 17, 332 Laurence, R. H., 292 Laurent, H., 351, 355, 455 Lavergne, J.-P., 4 2 Lavie, D., 151, 201, 305, 323 Law, A., 249 Lawrence, B. M., 102 Lawrence, K. H., 305 Lawrence, R. V., 9 Lawson, M. E., 283 Lawsan, W., 576 Leander, K., 7 3 Lebeau, M.-C., 576 Lebedeva, Zh. D., 460, 521 Leblanc, A., 42 Lecadet, D., 5 3 Lederer, E., 300 Lednicer, D., 538
Lee, B. K., 448,451 Lee, H., 575 Lee, H. J., 284 Lee, K.-H., 116, 133 Lee, R. A., 36, 354 Lee, S. S., 462, 500, 504, 507,520 Lee, T. H., 290 Leeming, M. R. G., 444 Leenhouts, J. I., 532,537, 538 Leete, E., 299, 300, 410 Lefebvre, G., 286, 482, 508 Le Goff, N., 346 Lehmann, H., 243 Leibfritz, D., 3, 313 Leinert, J., 286 Leistner, E., 297 Leitereg, T . J., 188 Leman, J. D., 472 LeMathieu, R. A., 573 LeMatre, J., 286, 426 Lemberger, L., 71 Lenox, R. S., 1 5 Lenskaya, G. S., 497 Lenton, J. R., 277, 280 Leonard, R., 212 Le Patourel, G. N. J., 254 Lepicard, G., 538 Le Quesne, P. W., 338 Leresche, J.-P., 17 Leroy, F., 537 Lestrovaya, N. N.,. 470, 491 Leuenberger, F. J., 228, 290 Levey, G. S., 274 Levine, S. D., 496 Levine, S. G., 538, 539, 540 Levinson, H. Z., 298 Levisalles, J., 89, 360 Levitz, M., 407, 454 Levy, E. C., 201 Levy, H. R., 456 Lew, F., 304 Lewbart, M. L., 325, 326 Lewis, D. A., 191, 192, 332,341 Lewis, N. F., 290 Lewis, R., 493 Li, T., 86 Liaaen-Jensen, S., 22 1, 229,233,299,306,307 Libit, L., 97 Lifson, S., 544 Light, R. J., 493 Ligon, R. C., 266 Lin, G. H. Y., 146, 166, 270
597
Author Index Lin, Y. Y., 403,407,448, 454 Lincoln, D. E., 262 Link, S., 126 Lipton, M. A., 71 Lisboa, B. P., 284 Liu, I. S., 228 Liu, K.-T., 3 Liu, R. S. H., 13, 244 Lloyd-Jones, J. G., 282, 284,285 Loder, J. W., 154 Loeber, D. E., 223 Loew, P., 86 Lowell, M., 253 Loftus, P., 42 Long, L., 404 Loomis, W. D., 112, 256, 259,260,261,263,264 Lopez, L., 357 Lopez, M. I., 362 Lopotko, N. V., 342 Loriaux, I., 163 Lorne, R., 330 Louda, J. W., 290 Louis, J.-M., 369 Louloudes, S. J., 484 Loutfy, R. O., 389 Lowe, G., 90 Lu, C. T., 538, 540 Lugtenburg, J., 386 Luhan, P. A., 116 Luis, J. G., 147;158 Lukacs, G., 314 Lusuardi, W. G., 74 Lutsenko, G. N., 290 Lyall, J., 300 Maalouf, G., 316 Maassen, J. A., 57 McAndrews, C., 36, 354 Macaulay, E. W.,536 McCarty, R. N., 48 McClelland, M., 230 McCloskey, J. A., 230 McCloskey, J. E., 118 MacConnell, J. G., 238, 298 McCormick, J. P., 186, 288 McCoy, K. E., 274 McCrindle, R., 148, 160, 201,204,304 McCune, R. W., 283 McCurdy, J. T., 426, 522 McCurry, P. M., 126 McDermott, J. C. B., 289, 299 MacDonald, C. G., 294
MacDonald, I. A., 286, 46 1 McDonough, G. R.,49 McEwen, R. S., 135,266 McFarland, J. W., 324 McGhie, J. F., 191, 192, 193,210,332,341,369 MacGillavry, C. H., 548 McGregor, W. C., 442 Macharia, B. W., 53 Machida, Y.,90 McIntyre, N., 274 Mack, J. P. G., 207 Mackay, I. R., 570 McKechnie, J. S., 536, 540 McKelvey, R. D., 387 McKenzie, G. P., 212 McKenzie, R. M., 297 McKillop, A., 342 McLaughlin, G. E., 161 McMaster, D., 148 MacMillan, J., 142, 159, 161, 240, 269, 287, 304,305 McMurry, J. E., 108 McMurry, T. B. H., 124 McNally, D., 544 McNamara, D. J., 252 McPhail, A. T., 116, 133, 540 MacSweedey, D. F., 87, 99 McWha, J. A., 266 Maddox, M. L., 382 Madjid, A. H., 222 Madyastha, K. M., 24, 263,264 Maeda, S., 127 Maekawa, E., 233 Malkonen, P. J., 43 Maestri, .M., 234 Maghane, D. T., 283 Magno, S., 182 Magnus, P. D., 23,43,47, 56,344,392 Magnusson, G., 39, 113 Mahajan, J. R., 154 Mahato, S. B., 207 Maheswari, M. L., 129 Mahler, W., 393 Mahony, D. E., 286,461 Maier, D. P., 69 Maier, V. P., 203 Maikowski, M., 351 Maillard, B., 58 Main, P., 532 Maiti, B. C., 207 Major, F.,253 Malhotra, H. C., 273 Malhotra, S. K., 488
Mallaby, R., 255 Mallams, A. K., 307 Malya, P. A. G., 281 Manchanda, A. H., 203 Manchard, P. S., 149 Mandel, L., 457 Mandel, N., 535 Mandelbaum, A., 208, 3 14 Mangoni, L., 145,200 Manhas, M. S., 315 Mani, J.-C., 222 Mann, J., 260 Manners, G., 69,295 Manuel, M. F., 293 Manville, J. F., 88 Manson, A. J., 454 Marathe, K. G., 43 Marchesini, A., 119 Margulis, T. N., 379 Marhenke, R. L., 55 Marik, M., 363 Marini-Bettolo, G. B., 217 Marino, M. L., 158, 159 Markova, E. V., 479 Markowicz, S., 53 Markwell, R. E., 163 Marples, B. A., 375, 384, 388 Marsh, R. E., 576 Marsh, W. C., 201 Marshall, D. J., 419, 499 Marsheck, W. J., 394, 406,510 Marsili, A., 208 Marten, T., 113 Marti, F., 387 Martin, A., 148 Martin, J., 427 Martin, J. D., 96, 147 Martin, S. S., 147 Martinelli, J. E., 120,265 Martinkova, J., 406, 409, 477 Martyr, R.J., 286,489 Marumo, S., 82, 256 Marusich, W. L., 307 Maruyama, M., 115 Marvel, C. S., 62 Marx, A. F., 394, 408, 413,421,511 Marx, M., 494 Maryanoff, B. F., 364 Marzin, C., 3 Masaki, N., 215 Masamune, S., 86 Masamune, T., 93, 125, 142,240 Masi, P., 368, 369 Maslen, E. M., 535
Author Index
598 Maslova, L. K., 32 Massiot, G., 377 Masuda, S., 206 Mathew, C. T., 94 Mathieu, J., 465 Mathis, P., 225 Mathur, S. B., 157 Matkovics, B., 363 Matringe, H., 508 Matsuda, A., 213 Matsui, K., 8 6 Matsui, M., 22, 167 Matsui. T., 61 Matsumoto, H., 9 0 Matsumoto, T., 155, 164 Matsunaga, A., 125 Matsunaga. T., 24 1 Matsuo, A., 56 Matthieson, A. McL., 539 Mattox, V. R., 327 Maturova, M., 355, 487 Maudinas, B., 223, 290 Maugras, M., 286, 426, 508 Maurer, B., 131 Mauri, M., 179 Mavrina, L. A., 460, 497 Maxon, W. D., 449,476 Maxwell, J . R., 188, 271, 306 Mayama, M., 41 1 Mayer, D., 286 Mayer, H., 233, 306, 308 Mayer, M., 448 Mayle, W. R., 284 Maynard, D. E., 71 Maynard, P. V., 284 Mayo, D. W., 136 Mazaleyrat, J.-P., 367 Mazur, Y., 314. 386, 539 Meakins, G. D., 363,398, 399, 400, 403, 417, 418,446,447,449 Mechoulam, R., 71, 72, 74,302 Meck, R., 133 Meehan, T. D., 263 Mehrotra, A. K., 58 Mehta, G., 59, 70, 110, 156 Meinwald, J., 84, 238 Meister, B., 5 Mejer, S., 462 Mekhtiev, S. D., 243 Melillo, D. G., 168 Mel’nikova, V. I., 468 Mendelsohn, H., 494 Meney, J., 379 Mennona, F. A., 354 Menzies, I. D., 392 Mercier-Bodard, C., 576
Meremkulovq, K. N., 49 1 Merep, D. J., 5 3 Merichini, F., 180 Merits, I., 447 Merlini, L., 75 Merrill, E. J., 359 Mktayer, A., 366, 383 Metge, C., 36, 50 Metzner, H., 299 Metzner, P., 6 0 Meyer, A., 359 Mez, H. C., 539 Michatek, E., 65 Michel-Briand, Y., 455 Middleditch, B. S., 468 Middleton, E. J., 284 Midgley, I., 330, 340,349 Midgley, J. M., 536 Midtvedt, T., 526, 527 Mielczarek, I., 41 Mihashi, S., 145 Mijlhoff, F. C., 546, 560, 568 Mijs, W. J., 398 Miki, T., 464 Miks, E., 10 Miksch, F., 446 Milborrow, B. V., 222, 240, 266, 307 Milewich, L., 283 Milewski, C. A., 3 18, 364 Milgrom, E., 456, 576 Miller, C. H., 84 Miller, I. R., 225 Miller, M. A., 580 Miller, T. L., 483, 5 19 Mills, J. S., 147 Mills, R. W., 22, 99, 100 Minale, L., 142, 173,219, 248, 288, 295 Mincione, E., 336 Minemura, Y., 459 Mirando, P., 150 Mirgasanova, M. I., 243 Mirrington, R. N., 93, 139 Mischenko, V. V., 243 Mislow, K., 4 Misra, R., 314, 540 Mitani, S., 5 Mitchell, E. D., 24, 253, 263 Mitra, A. K., 167 Mitropoulos, K. A., 276, 283 Mitsugi, T., 401, 413, 460, 5 13 Mitsuhashi, H., 363, 41 1 Mitsui, S., 349 Miura, I., 217 Miura, T., 379, 381 Miyake, A., 11
Miyashita, M., 108 Mizuta, K., 151 Moinet, G., 28 Moir, M., 69, 295 Molin, P., 4 1 Monaco, P., 200 Monder, C., 284,286 Money, T., 8, 87, 90, 99, 100 Monneret, C., 366, 383 Montagnac, A., 376 Montalvo, S. C., 329 Monteiro, M. B., 154 Montero, J. L., 75 Moody, J. A., 448 Mookherjee, B. D., 12 Moolgavkar, S. H., 487 Moore, B. P., 164 Moore, T. A., 221, 225 Moore, T. C., 267, 268 Mootz, D., 536 Morand, P., 300, 314 Morand, P. F., 498 Moreau, C., 58 Morelli, I., 208, 376 Morgan, B., 272 Morgan, E. D., 205 Mori, K., 40, 139, 162, 167,240, 366 Moriconi, E. J., 5 5 Morimoto, H., 249 Morisaki, M., 90, 171, 176,179,282,333,381 Morisawa, Y., 398, 446, 447 Moriyama, Y., 130, 206, 215 Morizur, J. P., 45, 238 Morman, M. J. P., 538 Morozova, G. R., 479 Morozova, L. S., 425 Morris, M. S., 115 Morrison, G. A., 322 Morrow, L. B.,539 Morton, G. O., 150, 157, 270 Mosbach, E. H., 526 Mosbach, K., 450 Mose, W. P., 307 Moss, G. P., 303, 400 Moss, J., 252 Mossel, A., 572 Motherwell, W. D. S., 535,537,575 Mourgues, P., 328 Mousseron-Canet, M., 222,325,457 Mrozinska, D., 34 Muckensturm, B., 316 Muller, B. L., 242 Muller, F. J., 11
5 99
Author Index Muller, R., 394 Muir, R. D., 446, 510 Mukai, T., 324 Mukaiyama, T., 353 Mukam, L., 193 Mukawa, F., 401 Mukherjee, D., 384 Mukherji, S. M., 390 Mulchandani, N. B., 288 Mulheirn, L. J., 268, 276, 297, 299, 314,396 Muller, B., 9 0 Muller, J.-C., 124 Muller, W. E., 398 Mullin, J. G., 354 Munakata, K., 149 Munday, K. A., 253 Murae, T., 206 Murakami, M., 5 11 Murakami, Y., 5 Murayama, K., 22 Murofushi, N., 158, 161 Murphy, G. J. P., 161,269 Murphy, G. M., 457 Murphy, P., 304 Murray, M. J., 262 Murray, R. D . H., 22,148 Musaev, M. R., 243 Muschaweck, R., 10 Muscio, F., 226 Muscio, 0. J., 186, 258 Musser, J. H., 119 Mustafaeva, M. T., 39, 245 Muzart, J., 359 Myant, N. B., 283 MySlinski, E., 65 Nada, S., 286, 405 Nadeau, R., 269 Naemura, K., 108 Nag, K., 218 Nagai, J., 259 Nagai, Y., 6, 7, 243, 493 Nagasampagi, B. A., 89, 190 Nagasawa, M., 460, 511, 513 Nagase, H., 107 Nagell, A., 35 Nair, G. V., 481 Nair, P. P., 493 Naito, A., 394 Nakachi, O., 11, 13 Nakada, Y., 22 Nakadaira, Y., 388 Nakamoto, T., 56 Nakamura, H., 107 Nakamura, S., 133, 167 Nakanishi, K., 139, 217, 222,226,388
Nakano, H., 515 Nakasone, S., 282 Nakata, T., 155, 168 Nakayama, M., 56 Nakayama, Y., 176 Namara, D. J., 251 Nambara, T., 474, 475 Nan Hsu, I., 539 Narang, S. C., 5 9 Narasaka, K., 353 Narula, A. S., 194 Nasipuri, D., 167 Nath, A., 200 Nathansohn, G. G., 457 Natori, S., 190 Naumov, V. A., 56 Naves, Y.-R., 35, 302 Naya, K., 133 Nayak, U. R., 70, 164 Nazaki, Y., 41 1 Nazarova, T. S., 460,498 Nazaruk, M. I., 470 Nearn, R. H., 154 Needham, P. H., 22 Neeman, M., 324 Neff, S. E., 143 Negishi, A., 86 Neidle, S., 166 Neidleman, S. L., 439, 496 Nelson, J. D., 45 Nelson, S. J., 329 Nemorin, J., 315 Nepokroeff, C. M., 253 Nes, W. R., 273, 281 Nespiak, A., 461 Ness, G. C., 253,259 Neumaier, H., 8 4 Neville, A. M., 456 Ngan, H. L., 253 Nguyen-Dang, T., 448, 46 1 Nicholls, R. J., 9 3 Nickon, A., 49, 391 Nieh, M. T., 329 Nielsen, B. E., 495 Nigam, S. S., 9 Nihonyanagi, M., 6 Niizato, N., 284 Nikaido, T., 145 Nikitin, L. E., 460, 497, 498 Nikolaidis, D., 187, 296 Nishida, R., 132 Nishimura, K., 115 Nishimura, O., 10 Nishimura, S., 338 Nishino, T., 257, 258 Nishio, M., 200 Nishioka, I., 7 0 Nishiyama, A., 119
Nishizawa, M., 265 Nitsche, H., 230 Niwa, H., 188,292 Niwa, M., 119, 139, 212, 215,216 Njimi, Th., 193 Noble, C. M., 253 Noguchi, S., 397, 467 Nomink, G., 465,472 Norden, B., 3 13 Norin,T., 36,53,93,220, 305 Norman, A., 526,527 Norris, R. K., 344 Norton, D . A., 357, 534, 535, 536, 537, 538, 539,540,548,576 Novotny, L., 127 Nowicki, H. G., 248 Nozoe, S., 90, 171, 176, 179,266,271 Ntokos, G., 156 Oae, S., 47 Obayashi, M., 467 Ober, R. E., 406 Oberc, M. A., 496 Oberhansli, W. E., 538 Oberhammer, H., 559 O’Brien, P. J., 286 Ochiai, K., 284 Oda, O., 28 Odom, H. C., jun., 129 Oehlschlager, A. C., 277, 427 Ogata, Y., 235 Ogilvie, A. G., 377 Ogino, T., 115 Ogura, K., 86, 257, 258, 259 Oh, S. K., 143 Ohara, S., 168 Ohkawa, M., 176 Ohloff, G., 131,242,291, 302 Ohnsorge, U. F. W., 166, 179,271 Ohrt, J. M., 536 Ohsawa,T., 155,170,199 Ohsuka, A., 68 Ohta, A., 197 Ohta, T., 163 Ohta, Y., 9 5 Ohtaka, H., 282, 381 Ohtsuka, Y., 155, 156 Oie, T., 26 Ojima, I., 6, 7, 243 Okada, M., 348,408,410, 434 Okada, Y., 167 Okaniwa, K., 370
Author Index
600 Okazaki, T., 68 Okubayashi, M., 282 Okuda, S., 171, 176, 179, 187, 188, 271, 272, 292,370 Okukado, N., 230,231 Okuno, T., 164 Olejniczak, B., 58 Oleson, W. H., 252 Olivk, J.-L., 222 Oliveto, E. P., 425, 457 Olson, J. A., 291 Olson, R. E., 248 Omi, J., 164 Onaka, T., 504 Oosterhoff, L. J., 50 Opheim, K. E., 84, 266 Opitz, G., 53 O'Rangers, J. J., 286 Organ, T. D., 536 Oritani, T., 241 Orr, J. C., 286, 297 Ortar, G., 357 Ortega, A., 133 Ortiz de Montellano, P., 3 18 Osawa, Y., 324,463,537, 552 Osband, J . A., 172, 239 Oshima, H., 284 Osiecki, J., 36 Osman, H. G., 420 Oster, M., 304 Otsuka, H., 467 Ouannes, C., 195 Ourisson, G., 124, 139, 179, 193, 196, 199, 246,264,274,306 Overton, K. H., 57, 82, 144, 156, 204, 220, 256,270,300,304,305 Owen. P., 57 Ozainne, M., 23 Ozaki, K., 340 Paasivirta, J., 44 Pacheco, P., 198 Packter, N. M., 299 Padhy, S. N., 207 Pagnoni, U. M., 119 Pais, M., 376 Pajkowska, H., 421 Paknikar, S. K., 97, 101 Pal, S. K., 213 Palmer, R. H., 457 Palmere, R. M., 439 Palumbo, G., 200 Pan, S. C., 412, 439 Panar, M., 393 Panctazi, A., 385
Pandey, R. C., 89 Pankova, M., 10 Pantulu, A, J., 17 Paoletti, E. G., 276, 298 Paoletti, R., 276 Paolucci, G., 368 Papastephanou, C., 290 Papernaja, I. B., 471,5 13, 514 Pappo, R., 107 Paquet, D., 52, 53 Paradisi, M. P., 329 Paris, M. R., 70 Paris, R. R., 70 Park, R. J., 83 Parker, W., 77, 300 Parkhurst, R. M., 214 Parmentier, G. G., 286 Parrish, F. W., 404 Pars, H. G., 302 Partridge, J . J., 26 Pascard-Billy, C., 535 Pascual, C., 147 Passet, J., 8 Pasteels, J. M., 9 Patashnik, S. L., 358 Patel, K. M., 36, 354 Patil, V. D., 164 Paton, W. D. M., 302 Pattenden, G., 20 Patterson, G. W., 275 Patterson, J. W., 103 Pattnaik, N., 156 Pankstelis, J. V., 53 Paul, I. C., 151, 536, 540 Pauling, H., 5, 49 Pauling, L., 576 Paulose, M. M., 17 Pavel, V., 535 Pavia, A. A., 49 Payne, 'T. G., 165 Peach, C. M., 372 Pechet, M. M., 355, 356 Pedone, C., 546 Pegel, K. H., 161 Peila, E., 179 Pelletier, S. W., 305 Pellicciari, R., 179 Penasse, L., 470, 472 Pendlebury, A., 363,447, 449 Perey, G. R., 205 Perez, C. S., 171 Perez-Reyes, M., 71 Perkins, D. W., 277 Peron, F. G., 428 Perry, G. J., 83 Pertot, E., 425, 441, 515 Pesce, G., 357 Petcher, T. J., 287
Pete, J. P., 359, 389 Petersen, M. R., 86 Peterson, G. E., 401 Peterson, P. A., 234 Petit, F., 44 Petit, G. R., 381 Petrow, V., 394 Petrzilka, T., 74, 302 Pettersen, R. C., 312,536 Petzoldt, K., 408, 413, 425,451,455,464,530 Peyre, M., 470 Pfander, H., 222 Pfau, M., 58,302 Pharis, R. P., 161, 269 Phelps, D. J., 328 Phillips, G. T., 255 Phillips, L., 161, 332 Piacenza, L. P. L., 161 Piatak, D. M., 200, 344 Piatkowski, K., 30, 34 Pichat, L., 187, 296 Pickenhagen, W., 39 Pierce, J. K., 63 Pieroni. J., 55 Piers, E., 103, 129 Piessens-Denef, M., 286 Pillai, N. K., 364 Pillinger, C. T., 306 Pilotti, A.-M., 93 Pincus, M., 225 Pinder, A. R., 129, 301 Pinevich, V. V., 221 Pinhey, J. T., 35, 189, 192, 341, 363, 399, 418,446,449 Piotrowska, G., 41 Piozzi, F., 158, 159 Piriou, F., 314 Pisano, M. A., 406 Pisareva, T. N., 61 Pitcher, R. G., 71 Pitt, C. G., 71, 73 Pitt, G. A. J., 307 Plantadosi, C., 133 Plasse, J. C., 284 Pletcher, J., 538 Plourde, R., 450, 466, 476 Plouvier, V., 301 Pocklin ton, T.. 468,469 Pohlancf A., 4 Poiret, M., 154 Pokrywiecki, S., 548, 552 Pollard, D. R., 541 Pollock, J . F., 13 Polonsky, J., 150, 174, 183,300,305 Poltavchenko, Yu. A., 9, 297 Polyachenko, L. N., 243
Author Index Ponsold, K., 315 Pont-Lezica, R., 82, 256 Poole, N. J., 406 Poon, Y.-C., 115 Popjik; G., 19, 187, 227, 228,253,258 Popper, H., 287 Porath, G., 71 Porter, J. W., 227, 228, 252,253,258,259,290 Porthheine, J. C., 539 Possanza, G., 474 Pot, J., 558 Potier, P., 377 Poulter, C. D., 19, 186, 258,263,302 Poulton, G. A., 43 Povodyreva, I. P., 66 Powell, J. E., jun., 26 Poyser, J. P., 196 Poyser, K. A., 196 Pragnell, J., 447 Pratt, A. D., 192, 318 Pratt, W. B., 455, 456 Precigoux, G., 535 Prelog, V., 139, 575 Premuzic, E., 306 Preston, A. F., 145 Previtera, L., 145 Price, P., 370 Prince, A., 360 Principe, P. A., 412 ProchBzka, 400, 401, 419,447,466 Protiva, J., 406,409,423, 458,477 Protti, D. J., 297 Proveaux, A. T., 9 Pryce, R. J., 161, 162 Puckett, R. T., 541 Pulman, D. A., 22 Punja, N., 22 Purdy, R. H., 537 Pyrek, J. St., 194, 209, 213, 218,
z.,
Quackenbush, F. W., 230 Quagliata, C., 535 Quayle, J. R., 297 Quijano, L., 180 Quinn, H., 497 Qureshi, A. A., 227, 228, 259 Qureshi, N., 259 Raab, K., 474 Raab, W., 491 Radlick, P., 96, 166, 270 Raghavan, K. V., 17 Rahal, S., 353
601 Rahim, M. A., 492,516 Rahimtula, A. D., 275 Raible, M., 286 Railton, I. D., 161, 269 Rakhit, S., 515, 521 Ralph, B. J., 35, 189 Ram, B., 15 Ramachandran, J., 284 Ramage, R., 77, 93, 262, 300 Ramaiah, M., 9 Ramamurthy, V., 13,244 Raman, H., 48 Raman, P. B., 428 Ramasarma, T., 274 Ramm, P. J., 299,433 Ramsey, R. B., 299 Ramseyer, J., 286 Randall, P. J., 280, 282 Randazzo, G., 180 Rane, D. F., 124 Ranganathan, D., 58 Ranganathan, S., 48, 58, 274 Rangaswami, S., 208 Ranu, B. C., 168 Ranzi, B. M., 179, 291, 295 Rao, A. A., 101 Rao, A. S. C. P., 70 Rao, M. S. S., 235 Rao, N., 127, 157 Rao, P. N., 474 Raphael, R. A., 22 Rapoport, H., 66 Rappaport, L., 269 Raska, K., 487 Rasmussen, R. A., 260 RaspC, G., 452, 469 Rastogi, R. P., 198, 206, 305 Ratajczak, T., 150 Rathore, B. S., 56 Ratner, V. V., 63 Raulais, D., 136, 303 Rautenstrauch, V., 17, 20,242,291 Raymond, R. L., 497 Raynaud-Jammet, C., 576 Razdan, R. K., 70, 73, 302 Reback, J., 493 Reddi, A. H., 576 Reed, L. L., 535 Reed., W. D., 252 Rees, H. H., 258, 280, 282,284,298 Rees, R., 398,424, 463 Regan, T. H., 69 Rehacek, Z., 293
Reich, R., 225 Reichenbach, H., 221, 231, 290 Reif, W., 28 Reimann, H., 457 Reimann, K. A., 200 Renard, M. F., 356 Renes, G. H., 560 Repke, K., 486 Restivo, R. J., 116, 201, 54 1 Retamar, J. A., 53, 61 Rendi, P., 152 Reusch, W., 36, 354 Reusser, P., 41 1 Reynaud, J.-P., 576 Rhoads, S. J., 61 Riano, M. M., 450,454 Rice, G., 13 Rich, D. H., 86 Richards, E. E., 446 Richards, J. B., 294 Richards, J. H., 77 Riederer, P., 232 Riemann, J., 449 Rienacker, R., .14 Riess, J., 326 Rigassi, N., 223,306,307 Rihs, G., 539 Rilling, H. C., 226, 227, 274 Rimai, L., 225 Rimmer, B. M., 531 Rindole, B., 295 Rindone, B., 291,526 Ringold, H. J., 455, 474, 488 Rios, T., 171, 180 Ritchie, E., 207 Ritter, F., 497, 505, 508, 523 Ritter, M. C., 274 Rizzardo, E., 356 Roach, W. S., 53 Robbers, J. E., 293 Robel, P., 576 Roberts, D. W., 3, 313 Roberts, F. M., 82, 256 Roberts, J. C., 144 Roberts, J. D., 3,45,223, 313 Roberts, J. L., 155, 330 Roberts, J. S., 77, 117, 300,303 Roberts, S., 283 Robertson, J. M., 536, 538 Robeson, C. D., 69 Robins, R. K., 487 Robinson, C. H., 425,488 Robinson, D., 304
Author Index
602 Robinson, J.. 283 Robinson, R., 576 Robinsr>i~,W . H . , 186,
25s Rcxhcfort, [I.? 576 Rochefort, J . G., 419 Roddick, J. G., 293 Rode, L.. 266 Rodig, 0. R., 49 Rodin, J . O., 12 Rodriguez, P., 234 Rodwell. V. W., 251,252 Roe. C. R., 455 Ropke, H., 44'9 Rogers, D., 36 Rogers, I. H., 88, 194 Rohmer. M., 273 Rojahn, W., 8 Rokos, J. A . S., 247 Rollin, G., 6 Romeo, A., 329. 357 Romers, C., 531, 532, 535, 538. 539, 552. 561, 565, 566, 568, 5 7 0 , 572 Rorno, J . , 303 Ronchetti, F., 279 Ronchi, A. U., 237 Roscher, N . M.. 385 Rose. E., 360 Rose. G., 404, 41 5, 460 Rosen, P., 338, 573 Rosenbaum, N., 294 Rosenheim. O., 531 Rosenthal, O . , 287 Rosini. G., 368, 369 Rossi, C., 180 Rosso, G., 234 Rossotti, F. J. C . , 48 Rothberg, I.. 193 Rothenberg, S., 3 11, 548 Rotman, A , , 314. 386 Kottink, R . A., 9 Rouessac, F., 42. 59 Rowan, M. G . , 187, 287 Rowe, J . W.. 190, 304 Roy, N. K.. 56 Rozen, S.. 218 Ruban. E. L., 514 Rubio-Lightbourn, J., 333 Ruhottom. G. M., 362 Kudakov, G . A.,9,56.61, 297 Ruden, R . A . . 353 Rudler, H., 89 Rufer. C., 464 Kulko. F.. 4 Ruscoe, C . N. E., 22 Kussell, G. B., 152, 154 Russo, G., 279, 526
Rutenberg, H. L., 297 Rutledge, P. S., 155, 193, 330,358 Rutten, E. W. M., 532, 539, 570 Ruzicka, L., 77, 298 Ryan, R. J., 318 Ryback, G., 139,222 Rykowski, Z., 6 1 Ryu, D. Y., 448,451 Ryzhkova, V. M., 466, 479,491 Saakov, V. S., 290 Sachdeva, Y. P., 390 Sadykova. I. M.. 6 1 Sate, S., 277 Saito, H., 349 Saito, Y., 348, 408, 434 Sajdl, P., 293 Saji, I., 107 Sakaguchi, M., 3 Sakai, K., 28 Sakai, T., 88 Sakakibara, J.. 163 Sakan, T., 242, 265, 291 Sakashita, T., 353 Sakimoto. €I., 22 Saleemuddin, M., 252 Sallam, L. A. R., 286, 404, 405, 419, 420, 448,449 Salmon, M., 133 Salvatori, T., 179 Sam, T. W., 118 Samek, Z.. 115, 123, 127, 133 Sammes, P. G., 148, 152. 196, 198, 302 Samokhvalov, G. I., 243 Samuelov, Y., 74 Sanadze, G. A , , 260 zanchez Bellido, I., 63 Sanda, V., 418 Sandermann, H., 295, 296 Sanders, G. M., 558 Sandris, C., 352 Sangare, M., 314 Santacroce, C., 173, 182 Santaniello, E., 291 Santhanam, P. S., 208 Santurbano, B., 179 Sanyal, B., 168 Sanyal, P. K., 218 Sapleva, V. T., 491 Saraswathi. G. N., 33 Sardinas, J. L., 406 Sarel, S., 4 Sarre, 0. 2..457
Sartorelli, A. C., 487 Sasaki, M., 249 Sasaki, T., 8 SaSek, V., 400 Sassa, T., 180 Sassu, 0. G., 290 Sastry, S. D., 129 Sathe, S., 73 Sato, H., 388, 515 Sato, K., 249 Sato, Y., 425 Satoh, D., 41 1, 491 Sattar, A., 93 Saucy, G., 446 Sauter, F. J., 168 Savage, D. S., 570 Savchenko, V. I., 17 Savigny, P., 286, 426 Savko, L. M., 491 Savochkina, I. E., 62 Savona, G., 159 Sawai, M., 493 Sawaya, T., 386 Sax, K. J., 397, 495 Sax, M., 538 Sayre, D., 531 Scala, A., 176, 179, 276, 295 Scallen, T. J., 274 Scanlon, J . T., 35 Scarset, A., 5 Scartazzini, R., 174 Schade, G., 6 Schaefer, J. P., 535 Schattner, F., 287 Schenck, J. R., 447 Schenk, H., 539, 540 Scherrer-Gervai, M., 399 Schildknecht, H., 84 Schiller, H., 3 15 Schimmer, B. P., 230 Schlegel, J., 404, 483 Schleyer, P. von R., 580 Schmalzl, K. J., 139 Schmid, H., 344 Schmidt, S., 225 Schneider, G., 162 Schneider, H. J., 5 Schneider, J. J., 3 13 Schnoes, H. K., 287 Schocher, A. J., 228,290, 41 1 Schoening, C. E., 376 Scholler, R., 359 Schrader, H., 33 Schreiber, J., 359 Schreiber, K., 315, 535 Schriefers, H., 286 Schriider, E., 464 Schroepfer, G . J., 300 Schubert, A., 421, 446
603
Author Index Schubert, K., 399, 404, 415, 445, 453, 460, 483, 490, 492, 497, 499, 505, 506, 507, 508, 521,523, 528 Schuette, H. R., 243, 263 Schulte-Elte, K. H., 242, 29 1 Schultz, J. S., 397 Schulz, G., 4 0 3 , 4 0 5 , 4 1 5 Schumann, W., 453,497 Schuytema, E. C., 447 Schwartz, M. A., 119 Schwarz, H., 149 . Schwarz, S., 421 Schwarz, V., 372, 406, 409,423,458,477 Schwarzel, W. C., 188, 284 Schwenker, U., 231 Schwieter, U., 223, 306, 307 Schwimmer, S., 293 Scolastico, C., 291, 295, 526 Scopes, P. M., 307, 313 Scott, A. I., 297,300,301, 303,304, 305 Scott, L. T., 46 Sedlaczek, L., 421 Sedmera, P., 135 Sedzik-Hibner, D., 31 Seeto, J. C. F., 191 Sefton, M. A., 160, 207, 269,288 Segal, G. M., 286 Segal, R., 209 Segebarth. K.-P., 301 Sehgal, S. N., 394, 407, 416,419,476,498 Seidel, I., 535 Seifert, W. K., 393 Seiler, M. P., 183 Seki, M., 197 Sekine, K., 149 Sellars, P. J., 4 8 Semakhina, N. I., 6 4 Semar, J., 41 2 Semenovskii, A. V., 39, 245 Semmelhack, M. F., 98 Semmler, E. J., 227, 228 Senda, Y., 349 Sensi, P., 523 Seto, N., 474 Seto, S., 86,257,258,259 Settim, G., 174 Severina, L. O., 471,497, 513, 514 Sewell, B. A., 515 Shaffer, G. W., 110
Shafizadeh, F., 115, 123, 133,266 Shagidullin, R. R., 66 Shah, S. N., 274 Shahak, I., 218 Shaikhutdinov, V. A., 6 4 Shani, A.,,71 Shankaranarayanan, R., 88 Shapiro, D. J., 252 Shapiro, E . L., 3 18 Shaposhnikov, V. N., 460 Sharma, B. R., 213 Sharma, R. P., 166, 179, 271 Sharma, T. D., 390 Sharpless, K. B., 14, 17, 322,329 Shaw, D. A., 503 Shaw, G., 291 Shaw, 1. M., 146 Shaw, J., 19 Shaw, P. E., 2 9 , 4 5 0 Shaw, R., 364 Shay, A. J., 397 Shechter, I., 256 Sheldon, T., 35 Sheppard, P. N., 160, 269 Sherma, J., 221 Sheth, K., 286 Shibahara, M., 403, 448 Shibasaki, M., 38, 245, 246 Shibata, S., 145,182,199, 214, 271 Shibayarna, M., 149 Shibuya, M., 160, 167 Shibuya, S., 115 Shikita, M., 487 Shilova, S. V., 491 . Shimada, T., 10 Shimagaki, M., 168 Shimizu, I., 259 Shimoiima. H.. 463 Shinaiaawa; S.,’10 Shine, H . J., 376 Shine, W. E., 256 Shiner, M., 493 Shingu, T., 133, 2 6 Shinka, T., 259 Shiozaki, M., 167 Ship, S., 488 Shirasaka, M., 17( Shishibori, T., 120, 261 Shiue. C., 33 Shizuri, Y., 125 Shmelev, L. V., 245 Shono, T., 18, 57 Shoppee, C. W., 3 15 Shoyama, Y., 70
Shulman, S., 107 Shust, S. M., 514 Sica, D., 182 Sidall, J. B., 8 5 Siddiqi, M., 252 Sidhaye, A. R., 197 Siebert, R., 446 Siefert, J. H., 3 12 Siehr, D. J., 447 Siemieniuk, A., 30, 34 Siewinski, S., 461, 462 Sigel, C. W., 195, 206 Sigg, H.-P., 9 8 Sih, C. J., 300, 446, 462, 485, 492, 500, 501, 503, 504, 507, 508, 516, 520,521 Silva, M., 148, 152, 196, 198 Silverstein, R. M., 12, 238,298 Sim, G. A., 117,133,536, 54 1 Sirn, S. K., 6 Simatupang, M. H., 69, 295 Simes, J. J. H., 35, 189, 192, 207,212, 341 Simmonds, D. J., 20 Simpson, K. L., 228, 290 Simpson, R. L., 221 Simpson, T. J., 287 Sims, J. J., 37, 96, 146, 166,270 Singh, B., 365 Singh, H., 222, 231 Singh, K., 394, 407, 416, 419, 476, 498, 499, 515, 521 Singh, M., 390 Singh, P., 152 Singh, R. K., 126, 289, 299 Siperstein, M. D., 252 Sircar, S. M., 161 Sis, J. D., 444, 490, 497 Sivaramakrishnan, K. P., 62 Sjovall, J., 458, 485, 528 Sjogren, R. E., 454 Skinner, W. A., 2 14 Skralant, H. B., 274 Skryabin. 6.K., 394,405, 444, 460, 468, 470, 471, 478, 479, 490, 491, 513, 514, 521 Skwarek, M., 41, 61 Slakey, L. L., 252, 259 Sliwowski, 3 . . 218 Slusarchyk, W. A., 172, 239
604 Smale, T. C., 179 Smallidge, R. L., 230 Smiley, K. L., 492 Smit, A., 401,422 Smit, V. A., 39, 245 Smith, A. G., 285 Smith, A. R. H., 279 qmith, C. V., 23 Smith, D. S. H., 406 Smith, H., 444 Smith, H. E., 313, 333 Smith, L. L., 342, 393, 398, 403, 407, 420, 424, 446, 448, 451, 454,463,494 Smith, M. S., 447 Smith, R. G., 333 Smith, S. E., 95 Smith, W. K., 470 Sobti, R. R., 538, 539 Sotif, H., 293, 425, 441, 459,515 Sodano, G., 173 SON, D., 294 Sokolova, I., 470 Sokolova, L. V., 466,468, 479,491 Solo, A. J., 365 Soloff, L. A., 297 Solomon, P. H., 66 Solomon, S., 442 Somell, A., 286 Sondengam, B. L., 367 Sondheimer, E., 142, 240 Song, P.-S., 221, 225 Sorarrain, 0. M., 225 Sorkina, T., 508 Sorm, F., 10, 102, 127, 133, 135, 303, 324, 350, 352, 365, 371, 401,418,419 Sorochinskaya, E. I., 30 Sota, K., 22 Sotiropoulos, J., 52 Soucy, M., 103 Spalding, B. P., 153 Speckamp, W. N., 377 Spencer, T. A., 68, 84 Spenser, I. D., 297 Spillner, C. J., 186, 258 Spiteller, G., 154 Sprague, J. T., 3 12 Spycherelle, C., 172, 246 Srikantaiah, M. V., 274 Sriraman, M. C., 89 Staba, E. J., 300 Stackhouse, J . . 4 Stahl, E., 37 Stallard, M. O., 12 Stang, P., 580
Author Index Stanton, D. W., 154 Stark, E., 7 Steel, R., 449 Steelink, C., 38 Stefano, S., 75 Stefanovic, M., 102 Steglich, W., 7 Steinfelder, K., 3 15 Sterkin, V. E., 479 Stern, M. H., 69 Stevens, K. L., 69, 295 Stevenson, P. M., 283 Stevenson, R., 379 Stodola, F. H., 394 Stoessl, A., 127 Stohs, S. J., 284, 470 Stolp, C. T., 269 Stone, K. J., 246,295,296 Storer, R., 20 Stork, G., 126 Stothers, J. B., 44,49,127 Strain, H. H., 221, 223 Straka, R., 218 Strandberg, G. W., 492 Straub, O., 307 Streith, J., 366 Strijewski, A., 506 Strobel, R. G., 497 Strominger, J. L., 246, 295,296 Stroobant, P., 294 Stutz, A., 59 Suarez, M. D., 253 Suga, K., 8, 10, 11 Suga, T., 63, 120,261 Sugawara, T., 199 Sugie, A., 206 Sugihara, H., 249 Sugimoto, A., 336 Suginome, H., 390 Sugiura, K., 125 Sugiyama, T., 20,22,252 Suhadolic, T., 441 Sukh Dev, 6, 29, 70, 88, 89, 164, 181, 194 Suleimanova, E. T., 243 Sultanbawa, M. U. S., 214,215 Sun, M., 221 Sundaralingam, M., 580 Sundin, S., 93 Surve, K. L., 101 Susuki, M., 107 Sutherland, J. K., 118 Sutherland, M. D., 83, 143 Suvorov, N. N., 466,468, 479,491 Suzuki, K., 188 Suzuki, K. T., 266, 292 Suzuki, M., 93
Suzuki, T., 11, 22, 144 Suzuki, Y., 82, 256 Svec, W. A., 223 Svoboda, M., 10 Swidersky, K. P., 383 Swingle, R. B., 190 Syhora, K., 409,423 Sykes, A., 225 Sykes, P. J., 360 Syono, H., 200 Syrdal, D. D., 95, 102 Sys, D., 497 Sys, Zh. D., 444, 490 Sysko, R. J., 49 Szabo, A., 471 Szabolcs, J., 307 Szewczuk, A., 197 Szpilfogel, S. A., 398 Szymanski, E. S., 466 Tabei, T,, 284 Tada, M., 130 Tadenkin, B., 442 Tadra, M., 394, 405, 5 00 Taguchi, T., 322 Taguchi, V. Y., 167 Tahara, A., 155, 156, 168, 170 Tai, H. H., 501, 504 SO8 Tait, A. D., 333 Takabe, K., 10, 11, 13 Takada, N., 127 Takagi, I., 133 Takagi, K., 235 Takahashi, A., 197 Takahashi, K., 11, 213 Takahashi, N., 158, 161, 176 Takahashi, R., 182, 271 Takahashi, T., 130, 206 215,216,467 Takani, M., 213 Takao, S., 269 Takasugi, M., 142, 240 Takayama, M., 216 Takeda, K., 115,303 Takeda, R., 128 Takeda, Y., 24,299 Takemoto, C., 5 15 Takemoto, T., 163 Takimoto, S., 23 1 Talalay, P., 299,455,456, 472,487,488,503,504 Talapatra, B., 207 Talapatra, S. K., 207 Tali, M., 10 Tamaki, A., 284 Tamaoki, B.-I., 515
Author Index Tamm, C., 90, 205, 399, 410,446 Tamura, C., 536 Tamura, G., 460, 511, 5 13 Tamura, M., 343 Tamura, S., 9, 176 Tamura, T., 176 Tan, L., 420, 421, 448, 451 Tan, T.-L., 506 Tanabe, K., 425 Tanabe, M., 329,354 Tanabe, Y., 213 Tanahashi, Y., 130, 206 Tanaka, A., 102 Tanaka, H., 26 Tanaka, I., 22 Tanaka, J., 10, 11, 13 Tanaka, K.,107 Tanaka, N., 199 Tanaka, O., 145,199,214 Tanaka, R., 96, 102 Tang, C. S. F., 66 Tanielian, C., 1 4 Taoka, M., 160 Tarasov, 0. S., 467 Taska, M., 159 Taube, A., 209 Tauscher, B., 8 4 Taylor, D. A. H., 201, 203, 204 Taylor, D. R., 203 Taylor, E. C., 342 Taylor, H. F., 139, 307 Taylor, P., 469 Taylor, R. F., 222 Taylor, S. I., 277, 530 Taylor, W. C., 207 Tchapla, A., 367 Tchen, T. T., 527 Teeter, R. M., 393 Tegtmeyer, E., 24, 264 Tehrany, S. S., 544 Templeton, W., 324 Teng, J. I., 342, 393 Teng, S., 10 Terashima, S., 38, 245, 246 Terhune, S. J., 102 Teshima, S., 459 Teuscher, G., 298 Teutsch, G., 318 Thampi, N. S., 281 Theodoropoulos, D., 156 Thierry, J., 376 Thies, P. W., 23, 24, 302 Thijssen, J. H. H., 457 Thom, E., 541 Thoma, R. W., 448, 451, 476
605 Thomas, A. F., 6, 23, 29, 40,57,264,301 Thomas, D. W., 214 Thomas, G., 249,294 Thomas, R., 166, 179, 299 Thomas, V. E. M., 446 Thommen, H., 222,307 Thompson, A. C., 8 Thompson, D. J., 144 Thompson, M. J., 484 Thompson, R. M., 327 Thompson, W. E., 4 Thomson, J. A., 284 Thomson, J. B., 212 Thomson, R. H., 6 9 , 2 9 5 ThorCn, S., 39, 113 Thornton, I. M. S., 92, 123, 158 Thornton, M. D., 205 Threlfall, D. R., 249,294, 300,308 Thuillier, A., 53 Thweatt, J. G., 69 Tichy, M., 10 Tieleman, A., 547 Tietze, L.-F., 26 Tikhomirova, 0. B., 444, 460,490,521 Timmons, M. C., 71 Tinelli, E. T., 142, 240 Tirodkar, S. V., 101 Titov, Yu. A., 394, 395, 478 Tkatchenko, I., 360 Toda, M., 9 3 , 1 8 8 , 2 9 2 Todesco, P. E., 357 Tokes, L., 314, 382 Tomorkeny, E., 460,477 Tohma, M., 332,386 Tokane, K., 107 Tolstikov, G. A., 57,328, 332 Tomita, B., 95, 122 Tomita, T., 332 Tomiyama, K., 125 Tomizawa, K., 235 Toneman, L. H., 559 Tonolo, A., 179 Tophan, R. W., 525 Torelli, V., 472 Torgov, I. V., 286, 405, 444, 460, 465, 467, 468, 470, 490, 513, 514,521 Tori, K., 115, 314, 413, 446 Torii, S., 26 Torrado, M. T., 222 Torrance, S., 3 8 Torri, G., 4 0
Torri, J., 3 Tbth, G., 307 Totty, R. N., 553 Toube, T. P., 223 Townend, J., 389 Towns, R. L. R., 540 Townsley, J. D., 398,469, 470,474 Tracey, B. M., 313 Trager, L., 4 5 5 , 4 6 9 Traetteberg, M., 559 Trave, R., 119 Treadgold, C., 22 Trefonas, L. M., 540 Trenkle, R. W., 12 Trivellone, E., 142, 173, 295 Trost, B. M., 8 5 , 3 0 3 Trotter, J., 536 Trueblood, K.N., 531 Trulzsch, D., 287 Trumbull, E. R., 45 Truong, H., 576 Truscott, T. G., 225 Tsai, T. Y. R., 169 Tsatsas, G., 352 Tschesche, R., 172, 286, 299,338 Tsfasman, I. M., 491 Tsizin, Yu. S., 26, 303 Tsong, Y. Y.,462, 485, 500,503,504, 520 Tsou, G., 195, 196 Tsuchikawa, H., 505 Tsuda, K., 176, 179 Tsuneda, K., 338 Tsunetsugu, J., 391 Tsurkova, V. I., 405 Tsuyuki,T.,206,215,216 Tucker, G., 287 Tummler, R., 315 Tbma, J., 405 Tunemoto, D., 86 Turnberg, L. A., 457 Turnbull, J. K., 160 Turner, A. B., 378 Turner, W. B., 77, 92 Tursch, B. M., 193 Turuta, A. M., 313 Tykva, R., 297 Tyler, T. W., 198 Tyminski, I. J., 580 Uata, K., 10 Uchida, T., 390 Uchida, Y., 47 Uchio, Y., 56 Uda, H., 9 6 , 1 0 2 Ueda, S., 24 Uemura, D., 165
Author Index
606 Uh. H.-S., 25, 139 Ulehlova, E.. 218 Uliss, D. B., 73 Ulrich, W., 193, 355 Ulsamer, A. G., 499 Umbreit, J. N.. 246, 296 Umbreit, M. A , , 329 Underwood, B. A., 223 Underwood, R. H., 327 Ungar, F., 283, 394 Unrau, A . M., 277 Uotani, K., 200 Uskokovic, M. R., 26 Uzarewicz, A . , 58 Uzarewicz, I., 58 Vaala, A. R., 222 Vaciago, A.. 179 Vahnier, J. E., 286 Valcavi, U., 352,384,437 Valenta, J. R., 401 VallCn, S., 5 Vanasek, F., 102 Van Belle, H., 5 15 Van Cantfort, J., 286 Van-Catledge, F. A., 580 van der Ende, C. A. M., 535 Van der Gen, A., 136 van dcr Helm, D., 539 Van der Hoeven, T. A., 284 Van der Linde, L. M., 136 Van der Molen, H. J., 284 Van der Sijde, D., 401, 417,421,441,442 Van der Vusse, G. J., 284 Van der Waard, W. F., 398, 401, 408, 413, 417, 441, 442, 446, 452, 511 Van der Weele, C., 5 11 van de Ven, C. F. W., 540 van Dongen, J . P. C. M., 3 Vangedal, S., 285 van Heykoop, E.. 532 van Lier, J. E., 342, 393 van Meerssche, M., 537, 538 van Tamelen, E. E., 183, 300 Van Tongerloo, A., 314 van Wageninger, A.. 245 Varcnn;, H., 72, 74 Varma, K. R., 396 Varma, K. K.. 276 Varner, E. L., 447 Vasileva, V. E., 221 Vaughan, W. R., 44, 45 Vazin, C , 353 Vederas, J. C., 3 1
Velarde, E., 345, 382 Velgova, H., 320 Velluz, L., 472 Vemcera, D., 9 3 Vendt, V. P., 342 Venturella, P., 158 Venzke, B. N., 3 1 , 3 3 Vereshchagin, A. N., 61 Verghese, J., 33, 301 Vernice, G. G., 359 Vestling, C. S., 406, 446 Vetter, W., 223,306,307 Veysoglu, T., 6 VCzina, C., 394,407,416, 419,476,498,499,521 Vialle, J., 52, 53, 60 Viallefont, Ph., 42 Vig, 0. P., 13, 15, 68 Vigevani, A., 369, 457 Vigneron, J.-P., 455 Vilhuber, H. G., 391 Villoutreix, J., 223, 290 Vincent, F., 487 Viswanathan, N., 197, 214, 217 Vitali, R., 327, 339, 345 Vitek, A., 209 Vlasinich, V., 472 Vossing, R., 464 Vogtmann, H., 222 Voigt, D., 315, 535 Voigt, W., 286 Voishvillo, N. E., 458, 460,478,490 Volkova, I. M., 405, 460 von Ardenne, M., 315 von Bahr, C., 286 von Fraunberg, K., 11 von Rudloff, E., 120 von Schantz, M., 12, 264 Vouros, P., 315,326 Vrieze, W. D., 327 Vul’fson, S. G., 61 Vystrcil, A., 208, 209, 218 Wachsman, M. A., 5 5 Wacker, A., 455, 456, 469,487 Wada, H., 125 Wadia, M. S., 102 Waegell, B., 59 Wagner, F., 506 Wahlberg, I., 9, 115, 116, 208,238,302 Waight, E. S., 161, 179 Waisser, K., 209 Waite, M. G., 541 Wall, M. E., 71, 7 3 Wallach, O., 33 Wallen, L. L., 3 9 4
Waller, G. R., 292 Wallwork, J. C., 300 Walters, R. L., 112 Walton, D. C., 142, 240 Wang, A. H. J., 151 Wang, K. C., 462, 485, 500,503,508,520 Wang, P. A., 475 Wang, P. T., 286 Ward, M. G., 489 Warren, C. D., 247 Warren, J. C., 393, 469 Warshel, A., 544 Washuettl, J., 232 Watanabe, A. M., 71 Watanabe, I., 180 Watanabe, M., 164 Watanabe, N., 511, 513 Watanabe, S., 8, 10, 11, 37 Watanatada, C., 5 Watson, J. A., 252, 274 Watson, T. R., 191 Watson, W. H., 537 Watt, A. N., 365 Watt, D. S., 103, 119 Wawrzenczyk, C., 37, 51, 66 Weavers, R. T., 146 Weber, H.-P., 98, 287, 540 Weber, L., 3 18 Weedon, B. C. L., 221, 223,306,307 Weeks, C. M., 535, 537, 539,540,549,576 Weeks, 0. B., 221 Weete, J. D., 299 Wehrberger, K., 528 Wehrli, F. W., 204 Wehrli, H., 387 Weickgenannt, G., 150 Weihing, R. R., 499 Weinert-Orlik, H., 31 Weintraub, H., 487, 488, 489 Weis, E. E., 251 Weisenborn, F. L., 172, 239 Weiss, G., 139, 222, 226 Weiss, J. L., 71 Weiss-Berg, E., 446 Weissenberg, M., 323 Welch, A. D., 487 Welch, S. C., 112 Weldt, E., 196 Wells, D., 16 Welzel, P., 338 Wenkert, E., 108 Wermuth, C.-G., 359 Werstiuk, E. R., 391
607
Author Index
'
Werthermann, L., 86 Wesolowski, M. F., 304 West, C. A., 267, 298, 304 West, P. J., 43 Westcott, N. D., 194 Weston, R. J., 200, 305 Wetter, L. R. 297 Wetzel, M. G., 499 Whalley, W. B., 36, 536 Wharton, P. S., 115 Wheeler, D . M. S., 353 Wheeler, J. W., 9, 143 Whistance, G. R., 308 White, A. F., 268 White, D. N. J., 536 White, I. H., 284, 469 White, J. D.,26, 149,214, 277,530 White, J. G., 55 Whitehead, E. V., 217, 3 06 Whiting, D. A., 20, 75, 228,258 Whitlock, H. W., 300,503 Whittaker, D., 42, 298, 301 Whysner, J. A., 300 W'icha, J., 348, 417 Wickramasinghe, J. A. F., 396 Widin, K.-G., 12, 264 Wiechert, R., 337, 351, 355,405,469,497 Wieglepp, H., 408, 427, 428 Wiehager, A.-C., 93 Wieland, H., 531 Wierenga, W., 183 Wiesner, K., 169, 498 Wigfield, D. C., 328.488, 489 Wikvall, K., 286 Wiley, B. J., 404 Wilke, G., 5 Wilkins, A. L., 219 Wilkomirski, B., 218 Williams, C. N., 286, 461 Williams, J. E., 580 Williams, K. I. H., 314 Williams, R. E. D., 493 Williams, R. G., 387 Williams, S. R., 8 Williams, V. P., 227 Williarns-Ashman, H. G., 576 Williamson, K. L,107 Willoughby, E., 295, 296 Wilson, C. W., tert., 29 Wilson, D. M., 188 Wilson, J. E., 406, 446
Wilson, L. D., 300 Wilson, M. A., 372 Wilton, D. C., 253, 275 Wiltshire, C., 355 Windisch, J., 491 Windreich, S., 225 Winell, B., 220 Wing, R. M., 96, 146, 166, 270 Winter, M., 9 Winter, S. R., 316 Winternitz, F., 75 Wiss, O., 308 Witkiewicz, K., 4 Witschi, E., 284 Witteveen, J. G., 136 Wittstruck, T. A., 314, 338,409 Wix, G., 404, 460, 471 Wojnarowski, W., 45 Wojtowski, R. K., 353 Wolf, G., 234 Wolf, G, C., 326 Wolf, H. R.. 7, 243 Wolff, G., 174 Wolff, M. E., 311, 548, 549,575 Wolinsky, J., 25, 34, 47, 55 Wolkowski, 2. W., 314 Wolter, J., 407 Wong, S., 258 Wong, S.-M., 19, 187, 227,228 Wood, D. L., 12 Wood, R. C., 460 Woodgate, P. D., 398, 446 Woodward, R. B., 51,386 Woolfson, M. M., 532 Wootton, M., 189 Worch, H. H., 535 Worth, G , K., 165 Wrange, O., 285, 484 Wray, V., 332 Wright, P. L., 499 Wu, F., 544 Wu, T., 266 Wuest, H., 39 Wulff, G., 299 Wulfson, N. S., 444,471, 513, 514 Wunderli, A., 344 Wuu, T., 261 Wyatt, R. J., 71 Yagen, B.. 276, 282, 396 Yakhimovich, R. I., 342 Yarnada, A., 408 Yamada, H., 3 , 8 Yamada, K., 9 3 , 1 0 7 , 1 2 5
Yamada, S., 38,206,216, 245, 246, 336 Yamada, Y., 22 Yamaguchi, I., 161 Yamaguchi, M., 230,231 Yamaguchi, R., 249 Yamakazi, K., 386 Yarnamoto, H., 5 Yamamoto, W., 299 Yamamura, S., 119, 139, 188,292 Yarnane, H., 161 Yamanishi, T., 233 Yamashita, K., 20, 22, 24 1 Yamashita, Y., 349 Yamauchi, T., 70 128 Yamazoe, Yan, T. C., 213 Yanagisawa, I., 145 Yanagita, M., 135, 266 Yang, C., 13,244 Yang, P., 13, 244 Yarborough, C., 357 Yaroshenko, Y. F., 342 Yasue, M., 163 Yazawa, H., 93 Yeboah, S. K., 287 Yobiko, Y., 5 Yokoi, T., 212, 216 Yokota, T., 158, 161 Yokoyama, H., 221. 290 Yoo, C. S., 538 Yoshii, E., 340 Yoshikawa, M., 218 Yoshikoshi, A., 96, 102,108 Yoshioka, H., 22 Yoshioko, M., 188 Yosioka, I., 128, 199, 213,218 Yoshizawa, I., 343 Young,T. G., 294 Young, M. R., 1 4 , 1 1 3 Young, R.N., 47 Younglai. E., 442 Yura, Y., 22 Yur'ev, V. P., 57, 328. 332 &'.,
Zabza. A., 37, 41, 51, 66 Zagalak, B., 4 Zahn, W., 506 Zaikin, V G",444 Zaitsev, V. V., 34 Zakharycheva, A. V., 312 Zakrzewski, Z., 476 Zala, A. P.. 371
Author Index
608 Zalkaw, L. H., 123,427 Zaman, A., 160, 181 Zamecnik, J., 132 Zander, J., 457 Zane, A., 164 Zanin, V. A., 460,497, 498 Zank, L. C., 304 Zaretskaya, I. I., 444, 460,490 Zaretskii, V. I., 444, 471, 513, 514, 521
Zbiral, E., 59,334,335 Zdero, C . , 23,127,150 Zedeck, M. S., 487 Zeelen, F. J., 398 Zeevart, J. D., 161 Zelkova, N. T., 468 Zelnik, R., 151, 201 Zhukova, A. I., 497 Ziegler, M. F., 206 Zink, M. P., 7, 243 Zinkel, D . F., 153, 304 Zinsou, C . , 233
Zsindely, J., 344 Zuccarello, F., 559 Zuidweg, M. H. J., 394, 446 zur Nedden, K., 9 Zvyagintseva, D. G., 479 Zvyagintseva, I. S., 405,470,478,479 Zweifel, G., 56 Zyakin, A. M., 479 Zych, L., 421