The Alkaloids: Chemistry and Pharmacology, Volume 49

THE ALKALOIDS Chemistry and Pharmacology VOLUME 49

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THE ALKALOIDS Chemistry and Pharmacology Edited by Geoffrey A. Cordell College of Pharmacy University of Illinois at Chicago Chicago, Illinois

VOLUME 49

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3 2

1

CONTENTS

CONTRIBUTORS. . . . . . . .... . .. . . . . . . . ... . .. . . . .. . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . .. . . . . . . . PREFACE . . . .. . . .. . . . . . ..,. . .. . . ... . .. . . .. . . . ... . . . . . .. . . . . ... . . .. . . . . . . . . . . . .. . . . . . . . . . . .

vii ix

Gelsernium Alkaloids

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

....................................

.

11. Sarpagine-Type Alkaloids. ... . .. . . ... . . ... . .. . . . ... .. ... . ... . . . . . .. .. . . . . .. . , . .

...

. ... . . .. . . . .. . . ... . . . . . ..

IV. Humantenine-Type Alkaloids. . .... . . . . . . . . . . . . . . . . . . . . . .. . . . ... . . V. Gelsedine-Type Alkaloids. . . . . ... . . .. .... . . . . . . . .. . . ... . . . . . . . .. . . .. .. . . . . .. . . .. VI. Gelsemine-Type Alkaloids.. ... .. . . ... ... . . . .. . .. . . . . ... . .. . . . .. . . . . .. . . . . . . . . . . References . . ... .. . . .. .. . . . .. . . .. ... ...................................

1 2 17 21 33 49 75

Alkaloids Containing Quinolinequinone and Quinolinequinoneimine Units TURAN OZTURK

I. Introduction.. . . . . . .. . . .. . . ... . . ... . .... . .. . . . . . . . .. . . .. . . . . . . . . 11. Alkaloids Containing a Quinolinequinone Unit .. . . . . . . . . . . . . . . . .. . . . . . . .. . . . . 111. Alkaloids Containing a IV. Summary.. . . . .. . . . . . . . . . References . .. . . .. . .. . . . .. . . .. . . .. . . . . .. .. . . . . .. . . . . . .. . , . . .. . , . ... . ... . . ... . . . . . .

79 80 143 212 213

Biosynthesis of Terpenoid Indole Alkaloids in Cutharunthus roseus Cells VAN ROBERT VERWORTE, ROBERT

DER

HEIJDEN, AND PAULO R. H. MORENO

I. Introduction.. . .. .. ... . . ... . .... ..... ... . . .. . . ... .. . ... . . . .. . .. . . . . . . . .. . ... . . . . .. 11. Biological Function of Alkaloids .. . ... . . .. . .. ... . .,. . . ... . . . . . . . .. , 111. Pathway Leading to Terpenoid Indole Alkaloids: Intermediates and Enzymes. . .. . ... . . .. . ... . . .. . . . ... . . .... ... . . .. . . .. .. . . ... . . ... . ... . ... . .. . .... ... IV. Genes Encoding Enzymes Involved in Terpenoid Indole Alkaloid Biosynthesis.. . . .. . .... . ... ... .. . ....... . . . .... .... .. ... . ....... . ... . . . V. Metabolic Engineering . .. . .. .. . .... . .... . .... .... . . .... . .... . .... .... . .. . . . ... . . VI. Regulation of Alkaloid Biosynthesis .... . . .. . . .... . ..... .... . ... . ... . . .. . . . ..., VII. Conclusions . . . .... . .... . ... . . .... ..... ...... . ... .. ... . . ... . ..... ... . ... . .... . . . .. References . ... . ..... . ... . .......... . .. . ..... . ... .. . . . . . . . .... . ....,. . . .. ......, .. V

222 225 227 264 270 271 287 288

vi

CONTENTS

Macrocyclic Peptide Alkaloids from Plants HIDEJI ITOKAWA, KOICHITAKEYA, YUKIO HITOTSUYANAGI, AND HIROSHI MORITA

I. Introduction......................................................................

11. Peptide Alkaloids from Higher Plants.......................................... 111. Cyclic Oligopeptides from Higher Plants ......................................

301 303 324 378

CUMULATIVE INDEXOF TITLES.. ....................................................... INDEX ..................................................................................

389 399

References .......................................................................

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

YUKIO HITOTSUYANAGI (301), Department of Pharmacognosy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-03, Japan HIDEJIITOKAWA (301), Department of Pharmacognosy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-03, Japan PAULO R. H. MORENO (221), Division of Pharmacognosy, LeidedArnsterdam Center for Drug Research, University of Leiden, 2300RA Leiden, The Netherlands HIROSHI MORITA (301), Department of Pharmacognosy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-03, Japan

TURAN OZTURK (79), Chemical Laboratory, University of Kent at Canterbury, Canterbury, Kent Cn 7NH, England SHIN-ICHIRO SAKAI (l), Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba, Japan HIROMITSU TAKAYAMA (l), Faculty of Pharmaceutical Sciences,Chiba University, Inage-ku, Chiba, Japan KOICHITAKEYA (301), Department of Pharmacognosy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-03, Japan ROBERT VAN DER HEIJDEN (221), Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, University of Leiden, 2300RA Leiden, The Netherlands ROBERT VERPOORTE (221), Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, University of Leiden, 2300RA Leiden, The Netherlands

vii

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PREFACE

It is only nine years since the last review of the Gelsemium alkaloids appeared in this series. However, two areas that have received substantial attention in the intervening period warrant this timely review by Takayama and Sakai: the isolation of new alkaloids, including several with new skeleta, and the very substantial, and highly successful, efforts at synthesis of the principal alkaloids through both biomimetic and total synthesis. One class of alkaloids, those containing quinolinequinone or quinolinequinoneimine moieties, is unusual because representative members of the series have been isolated from plant, fungal, and marine sources based on their potent biological activities. Ozturk reviews, for the first time in this series, the excitipg growth in the number of structures deduced in the past few years and the intense efforts to synthesize the biologically active members of this series. In addition, advances in the understanding of the biogenesis of these metabolites are discussed. Chapter 3, by Verpoorte, van der Heijden, and Moreno, summarizes the tremendous progress achieved in the past twenty years in the use of cell culture systems to delineate and express the pathway of monterpene indole alkaloid biosynthesis in Catharanthus roseus. The authors review the formation of the precursor units and then examine the enzymatic aspects of secondary metabolism. They conclude with an overview of the influence of exogenous materials on the regulation of alkaloid biosynthesis. The final chapter, by Itokawa, Takeya, Hitotsuyanagi, and Morita, reviews the various macrocyclic peptide alkaloids isolated from plants. In addition to a general overview of the many new peptide and amide alkaloids that have been isolated recently from a diverse range of plant families, this review places particular emphasis on the structure-activity relationships, the conformational analysis, and the antitumor activity of the RA series of cyclic oligopeptides from Rubia spp. Geoffrey A. Cordell University of Illinois at Chicago

ix

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~ H A F T E R 1-

Gelsemiurn ALKALOIDS HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI Faculty of Pharmaceutical Sciences Chiba University Inage-ku, Chiba, Japan

I. Introduction .......................... ...................................................... 11. Sarpagine-Type Alkaloids ....... ...................................................... A. Isolation and Structure Elucidation of New Alkaloids ............................. B. Synthetic Studies .............................................. ....................... 111. Koumine-Type Alkaloids .... A. Isolation and Structure Elucidation of New Alkaloids ............................ B. Synthetic Studies .......... IV. Humantenine-Type Alkaloids ............................. ....................... A. Isolation and Structure El ....................... B. Synthetic Studies .......... ....................... V. Gelsedine-Type Alkaloids ... A. Isolation and Structure Elucidation of New Alkaloids ............................ B. Synthetic Studies ........................................... VI. Gelsemine-Type Alkaloids .................................................................... A. Isolation and Structure cidation of New Alkalo B. Synthetic Studies ....... ............................... References ...........................................

1 2 2

17 17 21 23 33 33 49

I. Introduction The chemistry and biological activity of the Gelsemiurn alkaloids were previously reviewed by Liu and Lu in Volume 33 of this series ( I ) . Since the last review in 1988, the number of Gelsemium alkaloids has increased dramatically, and furthermore, the synthetic aspects of this field have progressed significantly. To bring the record up to date, this chapter focuses on the structure elucidations and synthetic studies, which were mainly published from 1987 to mid-1995. In the tribe Gelsemiue, belonging to the family Loganiaceae, there are two genera: Mostueu and Gelsemiurn, the latter of which comprises three species (Fig. 1). Gelsemium eleguns Benth. (Kou-Wen, or Hu-Man-Teng), which is distributed over southeastern Asia, is known as a toxic plant and THE ALKALOIDS, VOL. 49 0099-9598/97 $25.00

1

Copyright 8 1997 by Academic Press All rights of reproduction in any form reserved.

2

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

Plant Families Containing Indole Alkaloids

I

Apocynaceae

Tribe

Species

Genera

Gelsemieae--(

Loganiaceae -[ Strychneae

Mostuea Gelsemiurn

I

G. sempervirens Ait.

- G.rankinii Small

Rubiaceae

G. elegans Benth.

FIG.1.

has been used in traditional Chinese medicine as a remedy for certain kinds of skin ulcers. Recent clinical experiments with Kou-Wen on malignant tumors such as hepatic cancer have given encouraging results, and furthermore, analgesic activity for the palliation of various acute cancer pains with no addictive side effect has been reported (2). Gelsemium sempervirens Ait. (yellow jasmine) and G. runkinii Small grow in the southeastern United States. Although this plant causes death and abortion in livestock that feed on its leaves, it has been used in the treatment of neuralgia, migraine, and spasmodic disorders such as asthma and whooping cough. All of the species are rich sources of indole alkaloids, and to date 45 indole-related alkaloids have been isolated. The name, molecular formula, sources, and main references of all the Gelsemium alkaloids are listed in Table I. In this review, the Gelsemium alkaloids are classified into five groups, i.e., sarpagine-type, koumine-type, humantenine-type, gelsedine-type, and gelsemine-type, based on their structure. Topics concerning the structure elucidation and synthetic studies are introduced within this structure classification.

II. Sarpagine-Type Alkaloids A. ISOLATIONAND STRUCTURE ELUCIDATION OF NEWALKALOIDS Sarpagine-type indole alkaloids have a structural feature bonding the C-5 and C-16 positions in the Corynanthe-type monoterpenoid indole alkaloids. This type of compound is distributed not only in Gelsemium plants, but also in many genera in the family of Apocynaceae, Rubiaceae, and Loganiaceae. As shown in Table I, six sarpagine-type alkaloids 1-3, 5, 8, and 9 have been isolated from the Gelsemium plants. Koumidine was first obtained from the Chinese G. eleguns Benth. and shown to be a C-16 epimer of normacusine B (3). The presence of an intense NOE between H-19 and H-15, together with the different I3CNMR chemical shifts of C-15 and C-21 from those of gardnerine (28),

1. Gelsemiurn ALKALOIDS

3

TABLE I ALKALOIDS FROM Gelsemiurn SPECIES Alkaloid Koumidine (1) 19(Z)-Akuammidine (Koumicine) (2) Koumicine N-oxide (3) 16-epi-Voacarpine (5) 19(Z)-Anhydrovobasinediol(8) Na-Methoxy-l9(Z)-anhydrovobasinediol(9) Koumine (70) Koumine N-oxide (71) 19(R)-Hydroxy-18,19-dihydrokoumine (72) 19(S)-Hydroxy-18,19-dihydrokoumine(73) 1.2-Dihydrokoumine (74) Humantenine (86) Humantenirine (87) Rankinidine (88) 11-Methoxyhumantenine (89) 11-Hydroxyrankinidine (90) 11-Hydroxyhumantenine (91)

N,-Desmethoxyrankinidine (92) Na-Desmethoxyhumantenine(93) 15-Hydroxyhumantenine (94) 20-Hydroxydihydrorankinidine(95) Gelsemamide (%) 11-Methoxygelsemamide (97) Gelsedine (147) 14-Hydroxygelsedine (148) Gelsemicine (149) 14-Hydroxygelsemicine (150) Gelsenicine (151) 14-Hydroxygelsenicine (152) 19-0x0-gelsenicine (153) Gelsemoxonine (154) Gelselegine (155) 1l-Methoxy-l9(R)-hydroxygelselegine(156) Elegansamine (157) Gelsamydine (158) Gelsemine (118)

Molecular Formula

Source

Ref.

G. elegans G. sempervirens G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. rankinii G. sernpervirens G. elegans G. rankinii G. sempervirens G. rankinii G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. sernpervirens G. elegans G. sernpervirens G. elegans G. sernpervirens G. sernpervirens G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. elegans G. sernpervirens G. elegans

3, 5, 6 4 3, 5-7, 10 9 5, 6, 10 6 13 35-39 6 40, 41 40, 41 42 7, 49-51 52 52 49,51 52 52 52 53 53 53 53 54 54 54 55 55 17, 18 3 67 6 68, 69 70, 71 7, 49 49, 72 6 54 73 73 74 75 89-95 3, 6 (continues)

4

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

TABLE I (continued)

Alkaloid 21-Oxo-gelsemine (233) Gelsevirine (l20)

21-Oxo-gelsevirine (234) Gelsemine N-oxide (235) 19(R)-Hydroxydihydrogelsemine (236)

19(R)-Hydroxydihydrogelsevirine (237) 19(S)-Hydroxydihydrogelsevirine (238) 19(R)-Acetoxydihydrogelsevirine(239) Sempervirine

Molecular Formula

Source G. sernpervirens G. elegans G. sernpervirens G. rankinii G. rankinii G. elegans G. sernpervirens G. rankinii G. elegans G. elegans G. sempervirens G. rankinii G. sempervirens G. elegans

Ref. 96 6, 49 96 97 97 6 98 98 6, 98 98 127, 128 129

indicated that the configuration of C-19 in koumidine (1) should be revised to be a Z configuration instead of the E form of common sarpagine-type alkaloids (4-6). This spectroscopic analysis was confirmed by stereoselective synthesis of this alkaloid (Section 1I.B). The configuration of the ethylidene side chain in koumicine (3,7) was also revised (6). The I3C-NMRchemical shifts of C-15 [5.9 ppm lower field than 19(E) form] and C-21 [3.0 ppm higher field than 19(E) form] of the koumicine compared with those of authentic akuammidine (4), which was obtained from other plant sources (8), can be reasonably interpreted in terms of the y-gauche effect due to C-18 on the double bond having the Z configuration. A differential NOE experiment between H-15 and H-19 (23% enhancement) also gave evidence of the configuration of the side chain. The structure of 2 was confirmed by single-crystal X-ray analysis (6). In the original paper, koumicine was converted into koumicine N-oxide by hydrogen peroxide oxidation (9); therefore, koumicine N-oxide (3) should also have the 19(Z) configuration (Fig. 2). A new sarpagine-type indole alkaloid, 16-epi-voacarpine (9,was isolated from G. eleguns in Thailand (5,6). The stereochemistry at C-16 as well as the presence of a hydroxyl group at C-3 in 5 were demonstrated by the formation of 6 and 7, which were produced by acetylation of 5 with acetic anhydride in pyridine (Scheme 1).The E configuration at C-19 in 5 was determined by the NOE observation at H3-18 (10%)from H-15. The structure was confirmed by single-crystal X-ray analysis (10). Interestingly, 16epi-voacarpine (5) is the one and only compound having a 19(E) ethylidene side chain among the Gelsemiurn alkaloids.

1. Gelsemiurn ALKALOIDS

21

21

19

(1)

Koumidine

5

19

19(Z)-Akuammidine (2) Akuammidine (4) (Koumicine) &-oxide : Koumicine N-oxide (3) FIG.2.

The spectroscopic data for a new alkaloid 8 (6) showed a similarity to those reported for anhydrovobasinediol (taberpsychine) (10) (11,12),but a significant NOE observed between H-19and H-15in the 'H-NMR spectrum of 8 suggested that the configuration of the ethylidene side chain was in the 2 form. The 13C-NMR signal due to C-15 of 8 was observed downfield (7.0 ppm) and, in contrast, that of (2-21 was observed upfield (6.9 ppm) compared to the corresponding signal of 10. The structure of 8 inferred by spectroscopic analysis to be 19(Z)-anhydrovobasinediol was confirmed by chemical synthesis from ajmaline (Section 1I.B). The new alkaloid 9 is the first example of a Gelsemiurn alkaloid having an Na-methoxyindole moiety. Full assignment of the 'H- and I3C-NMR spectra of Na-methoxy-l9(Z)-anhydrovobasinediol(9) was carried out by NMR techniques. The structure was determined by single-crystal X-ray analysis (13) (Fig. 3). This alkaloid may be a biogenetic intermediate to the Na-methoxyoxindole alkaloids.

9-9 9 N. 4

-COCH, Me+

HO"16

'

. ;

NOElO%L

19

*dine

H'

H' \

:

Acd

18

6 16-epi-Voacarpine (6) SCHEME 1.

C C H 3

7

\

6

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

Q q H

\

15

19

Anhydrovobasinediol (10) R=H : 19(Z)-Anhydrovobasinediol( 8 ) R=OMe :Na-Methoxy-l9(Z)-anhydrovobasinediol (9) FIG.3.

B. SYNTHETIC STUDIES 1. Partial Synthesis Based on Biogenetic Speculation

The sarpagine-type alkaloids have the simplest chemical structures compared with other skeletal types of the Gelsemiurn alkaloids. Biogenetically, these compounds would be formed by the intramolecular carbon-carbon bond formation between the C-5 and C-16 positions in the strictosidine derivative l2 (Scheme 2). The olefin migration from the C-18,19 positions to the a,p-unsaturated aldehyde in the hypothetical intermediate 13 would afford two geometric isomers (15 and 16). They would be respectively transformed into 16-epi-voacarpine (5) having the 19(E) configuration, and 19(Z)-akuammidine (2)and koumidine (1) having the 19(Z) form. By C/D ring cleavage and simultaneous ether linkage formation at the C-3 position, one of the biogenetically key intermediates, 19(Z)anhydrovobasinediol(8), would be generated from koumidine (1).Further introduction of an oxygen function on the indole nitrogen would produce a new alkaloid 9 (Scheme 2). The following chemical synthesis of koumidine and 19(Z)-anhydrovobasinediol was achieved by a synthetic route adopting the biogenetic considerations mentioned previously. Ajmaline (14)was chosen as a starting material because its total synthesis and absolute configuration were already established (14-16), and furthermore, the equilibrium isomer 17 from ajmaline chemically corresponds to the hypothetical intermediate 13. Ajmaline (14) was first converted into the hydrazone derivative by treatment with N,N-dimethylhydrazine and a catalytic amount of sulfuric acid. After protection of the resulting secondary amine with methyl carbarnate, the hydrazone was hydrolyzed with copper(I1) chloride in aq. tetrahydrofuran (pH 7) to afford the aldehyde 19 in 75% yield. The hydroxy group in compound 19 was protected with the methoxyethoxymethyl (MEM) ether, and then bromine was selectively

7

1. Gelsemiurn ALKALOIDS

Strictosidine (11)

/

I Intermediate (12)

21

Intermediate (1%

Ajmaline (14)

16-epi-Voacarpine (5)

19(Z)-akuammidine(2)

Na-Methoxy19(Z)-anhydrovobasinediol (8) 19(Z)-anhydrovobasinediol(9)

koumidine (1)

SCHEME2. Tentative biogenetic route of the sarpagine-type Gelsemiurn alkaloids.

19

8

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

introduced to the C-20 position by treatment of the t-butyldimethylsilyl (TBS) enol ether derivative with N-bromosuccinimide (NBS). To create the double bond at the C-19,20 position, the bromide 21 was treated with 1,8-diazabicyclo[5,4,0]undec-7-ene(DBU) to give two a,P-unsaturated aldehydes. The major Z-olefin 22 obtained in 60% yield corresponds to the biogenetically hypothetical intermediate 16.The geometry of the two olefins was confirmed by NOE experiments. The major cr,p-unsaturated aldehyde 22 was reduced with NaBH4 to give the primary alcohol. Deprotection of the carbamate by alkaline hydrolysis gave the amine alcohol, which was then treated with mesyl chloride in pyridine to form the Bring. Removal of the MEM ether afforded the deoxyajmaline derivative 24 By the same sequential treatment, the minor 19(E)-olefin23 gave the Rauwoljia alkaloid tetraphyllicine (25)(17).The transformation of the indoline in 24 into the indole ring was accomplished as follows. The MEM group at the C-17 hydroxy group was substituted with trimethylsilyl (TMS) ether, which was then converted to the indolenine 26 by lead tetraacetate [Pb(OAc)4] oxidation (18) at very low temperature. The unstable indolenine 26 was successively treated with aq. AcOH-THF at 0°C to give 27, and then with sodium cyanoborohydride (NaCNBH3) to afford koumidine (l), which was identical with natural koumidine. Next, koumidine (1)was treated with 2,2,2trichloroethyl chloroformate in aq. THF in the presence of a large excess of magnesium oxide (MgO) to give a carbamate via a fragmentation reaction, which was then reduced with lithium aluminum hydride (LiA1H4) to afford 19(Z)-anhydrovobasinediol (8) [19(Z)-taberpsychine]. Because the absolute configuration of ajmaline has already been established, the structure including the absolute configuration of koumidine (1) and 19(Z)anhydrovobasinediol (8) was determined (Scheme 3) (19,20). Gardnerine (a), one of the major indole alkaloids of Gardneria nutans (21),was converted into koumidine (1) (22).The crucial steps in this transformation are deoxygenation from the indole ring by Pdo-assistedreduction of the aryl triflate derivative (31) and inverting the configuration of the ethylidene side chain in 32 by PdO catalyst (Scheme 4). 2. Total Synthesis of Sarpagine-Type Gelsemiurn Alkaloids a. Magnus Route. The first total synthesis of (+)-koumidine, which is the antipode of the natural sarpagine-type Gelsemiurn alkaloids, as well as (+)-anhydrovobasinediol (taberpsychine), was accomplished starting from (S)-(-)-tryptophan (23,24). This route was further extended to the total synthesis of koumine (Section 1II.B). The synthetic strategy features the construction of the tetracyclic chiral ketone derivative 39 via a Pictet-Spengler condensatiodDieckmann cyclization followed by the formation of the quinuclidine ring system (43,44), which is the

X=$q)-Q v, vi

-code

hie

Ajmaline (14)

Ri' 18: R1=CH=NNMe2,&=H

17

viii-xi -CO2Me

___c

Me

CHI

21

CHO

z-form 22 V. 60%

-

24

19

same (BBctbnconditions

-I-

E-fom 23 y. 12%

as above

1903): Tetraphyllicine (25)

24

19(Z)-Anhydrovobasinediol(8)

Koumidine (1)

Reagents: i. HzN-NMez, cat. HzSO4, MS, EtOH; ii. ClCOOMe, lN-NaOWCH&12,79%; iii. CuClZ, aq. THF' (pH 7),75%; iv. MEMC1, i-PrzNEt, CHzC12,81%; v. TBSOSO2CF3, EtaN, CHzClz, 71%; vi. NBS, THF, 76%; vii. DBU, DMF, viii. NaBH,, MeOH, 88%; ix. NaOH, HOCHzCHzOH,HzO, 8746; x. MsC1, Py., 62% xi. cat.HC1, MeOH, 95%; xii. TMSOTf, Et3N, CH2ClZ;xiii. Pb(OAd4,CHzC12, xiv. AcOH-THF-H20=3:1:1; xv. NaBH3CN;xvi. C1COOCH2CC13,MgO, THF-H20, 58%, wii. 14iAlH4,THF, 86%. SCHEME3.

10

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

...

Gardnerine (28)

I

29 :X=OMe SO :X=OH

81:X=OTf

I

10

Koumidine (1) 19(E):19(E)-Koumidine (33)

32 :X=H

Reagents: i. A&O, pyridine, r.t., 8 h, 97%;TaCl, n-BNNHSO,, SO%KOH-benzene,3 h, 98%; ii. AlCls, EtSH, CH2C12, -18"C, 3 h, 91%;iii. (CFsS02)Z0,EtSN, CHg12, -2OoC, 10 min, 97%;iv. Pd(OAc)z, DPPF, EQN, HCOOH, DMF, 60°C, 2 h, 98%;v. Mg, PdC12, PPhs, MeOH, r.t., 50 h, 48%(l),34%633).

SCHEME4.

basic skeleton of the sarpagine-type alkaloids, using an intramolecular Michael-type addition. The synthesis was initiated with the preparation of ( -)-Na,Nb-dibenzyltryptophanmethyl ester (35). (S)-( -)-tryptophan (34) was converted into its Na-benzylderivative by treatment with sodium amide and benzyl chloride in liquid ammonia. Conversion of the acid into the methyl ester followed by condensation with benzaldehyde and reduction of the resulting imine with sodium borohydride gave 35. According to the method developed by Cook (25), 35 was subjected to Pictet-Spengler condensation with 2-ketoglutaric acid and the resulting acids esterified with MeOH and TMSCl to give a mixture of the diastereomeric methyl esters 36 and 37. These isomers could be separated by fractional crystallization from methanol, from which the major diastereomer was isolated in 58% yield. The minor diastereomer could be isolated in a pure form by chromatography of the mother liquors. Assignment of the stereochemistry was mainly done by comparison of the 13C-NMR chemical shifts with the reported data (26). Typically, N-benzylmethylene carbons for 1,3-cis disubstituted tetrahydro-P-carbolines resonate about 7 ppm downfield of the corresponding peaks in the trans isomers. The major isomer 36 exhibited an a-amino benzylic methylene carbon at 52.63 ppm, while the minor isomer 37 resonated at 61.15 ppm, indicating that the C-3 C02Me and C-1 CH2CH2C02Mein 36 had a trans relationship. Treatment of (-)-37 with sodium hydride (NaH)ltolueneNeOH heated at reflux gave (-)-38 in 5 h, whereas the trans isomer 36 required 15 h to be converted into (+)-38. These experimental results demonstrated that the rate-determining step of the reaction from 36 to 38 is the C-3 epimerization because the trans isomer 36 can only undergo intramolecular Dieckmann cyclization by prior

1. Gelsemiurn ALKALOIDS

11

epimerization at C-3. The antipodal relationship between (+)-38 and (-)38 was demonstrated by rotational data, and the enantiomeric purities were confirmed by an 'H-NMR study. Beginning with a single enantiomer of tryptophan, both enantiomers of the tetracyclic P-ketoesters are available. The major enantiomer (+)-38 was subjected to hydrolysis and decarboxylation under the usual conditions in AcOH/H2S04/H20 under reflux to afford the ketone in 99%yield. Catalytic transfer hydrogenation with 10% Pd/C in 88%formic acid gave the monodebenzylated ketone (+)-(39). The reverse sequence, demonobenzylation followed by decarbomethoxylation, proceeded in 62% overall yield. Starting from (R)-(+)-tryptophan, the (-)39 (natural series) is the major enantiomer produced (Scheme 5). Next, the Nb-alkylation with a linear C-4 unit and the formation of the quinuclidine ring system were examined (Scheme 6). N-Alkylation of the secondary amine (+)-39 with propargyl bromide gave the amine (+)40 in 60% yield. Treatment of (+)-40 with TBS triflatekriethylamine gave the enol ether derivative. Deprotonation of the acetylene moiety with nBuLi followed by quenching the acetylide anion with methyl chloroformate gave the a,P-unsaturated acetylenic ester 41 in 76% yield. Treatment of 41 with a wide range of reagents to deprotect the trialkylsilyl enol ethers for the construction of quinuclidines 43 and 44 gave reaction mixtures that typically consisted of a mixture of the ketone 42 and the required quinuclidine, Z and E olefinic isomers 43 and 44, but always in very poor yield and difficult to purify. Under the conditions used to release the enolate anion from 41, a retro-Dieckmann fragmentation process took place. Ketone 42, which was readily prepared from 41 by treatment with LiBF4,was exposed to a number of conditions to promote intramolecular Michael cyclization to construct the quinuclidines 43 and 44. After several trials it was found that 1.2 eq of lithium diisopropylamide (LDA) at -78 to 0°C gave 43/44 as a separable 1 :1 mixture of E and Z a,S-unsaturated esters in 60% yield. In the scaleup experiments, other reaction conditions, i.e., treatment of 42 with pyrrolidine (0.2 eq) and trifluoroacetic acid (0.2 eq) in dry benzene heated under reflux for 19 h, were employed for the preparation of 43/44. In this case 43 and 44 were obtained in 12 and 68% yield, respectively. Compound 43 equilibrates to a mixture of 43 and 44 on standing in solution, as does 44. The assignment of E and Z isomers was made by comparison of chemical shifts of the olefinic protons in their 'H-NMR spectra. These assignments were confirmed by a single-crystal X-ray analysis of the Z isomer (+)-43. The next task of the synthesis was homologation of the carbonyl group in 43 and 44 to a hydroxymethyl group in a stereospecific manner (Scheme 7). Treatment of 44 with Lombardo's reagent (TiCldZnlCH2Br2) (27) in CH2C12gave a mixture of the desired methylenation product 45 (24%yield)

12

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

vii, viii

vii, viii

(-1-39

(+I439

Reagents; i. NaNHdNH3, PhCH2C1; ii. PhCHO, Na2S04;iii, NaBH4. iv. H02CCOCH2CH2CO2H; v. MeOH. ClSiMe,; vi. NaH, PhMe, MeOH; vii. AcOH, H2SO4, H2O; viii. 104bPd-C, HCOZH. SCHEME 5.

and the unexpected 46 (20%yield). When CH2Br2was replaced by CH21z in Lombardo's method, 45 and 46 were obtained in 1:1 ratio. When the Lombard0 reagent was prepared using a Zn-Cu couple, cyclopropanol47 was the major product. Next, Tebbe's reagent was examined. Treatment of 44 with the Tebbe's reagent (28) (a mixture of titanocene dichloride

1. Gelsemium ALKALOIDS

(+IS9

(+I42

(+)-40

13

(+)-4 1

(+)-43

(+)-44

Reagents:

i. propargyl bromide, EtOH, ii. TBSOTf, EtSN, CHzClz; iii. n-BuLi, ClCOZMe; iv. LiBF,, THF'hIzO; v. pyrrolidine, TFA, benzene or LDA, THF. SCHEME 6.

purified by Soxhlet extraction with CH2C12and trimethylaluminum in toluene), which was prepared using the Grubbs procedure (29), in THF gave the required em-methylene adduct 45 (63% yield) and the methyl ketone 48 (15% yield). Hydroboration of the exo-methylene in compound 45 was attained by treatment with a freshly prepared solution of di-isoamylborane in THF at 0°C to yield the primary alcohol 49 in 69% yield. The stereochemistry of the newly introduced hydroxymethyl group (C-16) was determined by single-crystalX-ray crystallography. Application of the same methylenation/hydroboration sequence to the Z isomer 43 gave the alcohol 51. Reduction of (Z)-51 with diisobutylaluminum hydride (DIBAL) in toluene at room temperature gave the ally1 alcohol 52 in 92% yield, which was then treated with Na/NH3 at -30°C to afford (+)-koumidine. Comparison with the natural sample of (-)-koumidine demonstrated their identity and antipodal relationship and furthermore confirms the spectroscopic reassignment of the stereochemistry of the ethylidene side chain. Reduction of (E)-49 with DIBAL and reductive removal of the N,-benzyl protecting group, as well as the allylic alcohol in 50 with NaMH3 at -3O"C, gave (+)-19(E)-koumidine (33). Treatment of 33 with methyl chloroformate and subsequent reduction of the resulting carbamate with LiA1H4 gave (+)-

14

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

(+)-47

(+)-48

Ii

Me

t

H (+)-49, R=CO,Me, R’=Bn

(+I-60,R=CHzOH, R=Bn R (+)-%I, R=Me, R=H

(+I-Anhydrovobasinediol:(+)-(lo) [(+)-Taberpsychinel

-. . (+)-43

vi, i

ii I$

t-f*4\

iii

c (+)-62. (+Mil, R=CO&e, R=Bn R=CH,OH. R-Bn

c (+)-l,R=Me, k’=H:(+I-Koumidine

Reagents: i. diimamylborane then NaOH/H20z;ii. DIBALH; iii. Na, NH3, -30”C; iv. ClCOaMe, NazC03, aq. THF; v.LiAlH4, THF; vi. Tebbe reagent. SCHEME7.

anhydrovobasinediol (taberpsychine) natural product (Scheme 7).

(lo), which is the

antipode of the

b. Liu Route. A total synthesis of N,-methyl-A18-isokoumidine was reported by Liu and Xu (30). L-Tryptophan methyl ester (53) was treated with succinic half methyl ester acid chloride in pyridine to afford the amide 54 in 81%yield. The dihydro-p-carboline,which was obtained by Bischler-

1. Gebemium ALKALOIDS

15

Napieralski reaction of the amide 54, was subjected to catalytic hydrogenation over PtO, in ethanol to afford the 1,3& disubstituted tetrahydro-pcarboline 55. Nb-Alkylation, respectively with l-bromo-4-acetoxy-2(Z)butene or l-bromo-2(E)-butene in CH3CN in the presence of NaHC03, gave the tertiary amines 56 or 57. After protection of the N , function in 56 and 57 with an acetal group, Dieckmann cyclization was conducted with

iv

68, R:CHZCH=CHCHzOAc (2) 69, R: CH2CH=CHCH3 ( E )

c

55, R: H 56, R: CHZCH~CHCH~OAC (Z)

67, R:CHZC€I=CHCH3 (E)

60, X: H, Y:CH=CH2 61, X, Y: =CHC&OAc (E)

62

H 5

63

II

64

I

Reagents:

65

It

i. Me02CCH2CH2COCl, Py; ii. POCl,; iii, H,, MZ, EtOH; iv.BrCHzCH=CHCHzOAc (2) or BrCH*CH=CHCHs ( E ) , NaHCO3, MeCN; v. CH2=CHOEt, cat. PTS, CH2C12; vi. NaH, cat. MeOH, benzene, reflux or (Me3S02NNa,DME, 100 OC; viii. AcOH, aq. MeOH; ix. KOH, aq. MeOH; vii. M ~ ( O A C ) ~ * HCu(OAc),-H,O; ~O, x. N-hydroxy-2-pyridinethione, DCC, DMAP then t-BUSH, hu; xi. NaH, DMSO then Me3SI, THF;xii. AlCIH,, THF. SCHEME8.

16

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

NaH, cat. MeOH (in benzene, reflux, 6 h) or (Me3Si)JWa (in dimethoxyethane, 100"C, 5.5 h) to give the tetracyclic P-ketoesters 58 and 59. Attempted cyclization of 5 !8 with PdO catalyst to form the sarpagine skeleton was unsuccessful. However, oxidative free-radical cyclization of P-ketoesters with Mn(OAc)3, Cu(0Ac)z (31,32) in AcOH gave the pentacyclic compounds 60 and 61 having a quinuclidine ring system. The N,-protecting group was removed by treatment with aqueous acetic acid. Next, hydrolysis and decarboxylation of the bridgehead carbomethoxy group was carried out using the Barton method (33). Thus, the carboxylic acid 62 obtained by alkaline hydrolysis was converted to the thiol ester, which was then exposed to a 300-W tungsten lamp for several hours to give the ketone 63 in 55% yield. The homologation of the C-16 carbonyl group to the hydroxymethyl group was attained by methylene transfer with dimethylsulfoxonium methylide, followed by reduction of the resulting epoxide with AlHZCl in THF to give Na-methyl-At8-isokoumidine (65) (Scheme 8). The authors claimed that compound 65 should be a useful intermediate for the synthesis of koumine and other sarpaginetype indole alkaloids. c. Bailey Route. L-Tryptophan methyl ester (53) was used as the chiral starting material for the synthesis of the cis-1,3-disubstituted tetrahydroP-carboline 66,which was formed in a kinetically controlled Pictet-Spengler reaction (ratio of 66 to its 3-epimer 67,4 :1). Protection of the two nitrogens in 66 and subsequent cyclization (NaH, cat. MeOH in DMF)/decarboxylation (NaCl, HzO in DMF at 130°C) gave the bridged ketone (-)-68 in

N H

NH2

-

+

methyl 4-oxobutanoate 63

benzene, reflw then TFA. CH&lz

60

1

67

I

N-protection cyclization decarboxylation

(-)-68:R1=Me, R2=C02CH2Ph (-1-89: +Me, &=CH2Ph (-)-59: Ri=CHZPh,R p H SCHEME 9.

1. Gelsemium ALKALOIDS

17

optically pure form. Modifications of the protecting groups in 68 gave (-)69 and (-)-39, which constituted the formal total synthesis of (-)-ajmaline, (-)-koumine, (-)-tabexpsychhe, and (-)-koumidine (Scheme 9) (34).

III. Koumine-Type Alkaloids

Koumine (70) is a principal alkaloid of G. elegans Benth. (35-39). Recent clinical experiments showed the effect of koumine on malignant tumors and its analgesic activity with no additive side effects (2). Because of the novelty of its hexacyclic cage structure, many chemical and synthetic studies have been conducted (I). A. ISOLATIONAND STRUCTURE ELUCIDATION OF NEWALKALOIDS

The presence of koumine Nb-OXide (71)in the leaves of G. eleguns was reported (Fig. 4) (6). Oxidation of koumine (70) with m-chloroperbenzoic acid (m-CPBA) produced two diastereomeric &-oxides, one of which was identical with the natural N-oxide (71).From the ‘H-NMR spectral analysis, the configuration on Nb can be deduced to be (S) in the natural N-oxide. Unnatural N-oxide gave crystals suitable for X-ray analysis, whose result confirmed the structural conclusion obtained from ‘H-NMR analysis. The isolation of a pair of 19-hydroxydihydroderivatives of koumine from G. eleguns was independently reported by two research groups. The Beijing

Koumine (70) lS(R)-Hydroxyl,2-Dihydrokoumine (74) Koumine N-oxide (71) 18,19-dihydrokoumine (72) :Nb-oxide 19(S)-HydrOxy18,19-dihydrokoumine (73) FIG.4.

18

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

group (40) elucidated the structure of the two alkaloids by comparison of the 'H- and I3C-NMRspectra with those of koumine (70) as well as Horeau's method. The Illinois group succeeded in the crystallization of both compounds suitable for X-ray analysis (41). One diastereomer (72) (mp 198200°C) {[(.ID -232.7') has the 19(R) configuration, and the isomer (73) (mp 270-272°C) with DI.[ -184.6"C has the 19(S) configuration. 1,2-Dihydrokoumine (74) was isolated as a natural product (42),although it had been prepared from koumine (70) during its structure elucidation (43). In this report, modified Hofmann degradation of koumine derivative was also described.

B. SYNTHETIC STUDIES 1. Partial Synthesis Based on a Biogenetic Speculation

The biosynthetic route to the cage structure of koumine (70) would diverge from a key intermediate, 19(Z)-anhydrovobasinediol(8). Oxidation of the allylic C-18 position in 8 would give an unnatural (not yet isolated) 18hydroxy-l9(Z)-anhydrovobasinediol (75), and subsequent intramolecular coupling between the C-7 and C-20 positions would produce koumine (70) (Scheme 10). Originally, Lounasmaa and Koskinen proposed the biogenesis

L

18

76

W

19(Z)-Anhydrovobasinediol (8)

'

20

Koumine (70) SCHEME 10.

1. Gelsemium

ALKALOIDS

19

of koumine starting from 18-hydroxy-desoxysarpagine(44). Based on the biogenetic hypothesis, partial synthesis of koumine (70) was attained by two groups independently. As introduced in the last review on the Gelsemium alkaloids, Liu et al. has realized the biogenetic concept using a sarpagine-type alkaloid, vobasine (76) (Scheme 11) (1,45). Vobasine (76)on reduction with LiAlH4 gave vobasinediol, which was dehydrated with aqueous sulfuric acid to afford anhydrovobasinediol (10). Allylic oxidation of 10 with Se02/H202gave koumine (70) in a modest (25%) yield. Using a Gardneria alkaloid, 18-hydroxygardnerine (77), unnatural 11methoxykoumine (78)was initially prepared by Pdo-mediated transannular SN2' cyclization (46). Later, natural koumine was prepared from the same Gardneria alkaloid 77 (Scheme 12) (47). Removing the methoxy group from the indole nucleus in 77 was achieved by reductive deoxygenation of the aryl triflate derivative 79 assisted by a palladium catalyst. The 11demethoxy derivative 80 thus obtained was converted to 18-hydroxyanhydrovobasinediol(81) by C/D ring opening with methyl chloroformate followed by reduction of the carbamate with LiA1H4. Compound 81, which corresponds to a hypothetical precursor to koumine (70), could form two conformational isomers 81s and 81u. Carbon-carbon bond formation between C7 and C20could occur via the r-orbital overlapping in the folded form 81u. Based on calculations by the MNDO method (48), the extended form 81s is more stable than 81u. However, the energetic difference is only 5.5 kcal/mol, so that facile conformational change from 81s to 81u could be expected. Actually, koumine (70) was obtained in 80% yield, when the indole anion prepared from the 18-0-acetate (82)was treated with 0.1 eq of P ~ ( O A Cand ) ~ 0.5 eq of triphenylphosphine at 80-90°C. 2. Total Synthesis

As described in Section II.B.2, Magnus et al. have developed an efficient synthetic route from (S)-( -)-tryptophan to chiral sarpagine-type alkaloids.

Vobasine (76)

Anhydrovobasinediol ( 10) SCHEME 11.

Koumine (70)

20

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

"I*.-

N

0

11-Methoxykoumine(78) 18-Hydroxygardnerine (77)

R=H R=Ac

81 82

X

c--

Koumine (70)

-

81u

81s

'OR

_I

Reagents: i. Ac20, pyridine. r.t., 15 h; TsC1, n-BqNHSO4,5O%KOH-benzene, r.t., 2h, 86%; ii., AlCls, EtSH, CH2C12, -18"C,7 h, 95%iii. (CF$OzhO, Et3N. CH2C12, -18"c,15 min, 82%;iv. aq. 59bKzc03, MeOH, r.t., 10 min, 86%;v. Pd(OAc)p, DPPF, EtaN, HCOzH, DMF, 60°C, 2 h, 9 7 %vi. LiAlG,THF,reflux, 11 h, 96%vii. ClC02Me, MgO, "F-H20, r.t., 2.5 h; viii. L m 4 , THF, r.t., 6.5 h, 41%from (80);ix. Ac20, pyridine, r.t, 1 h, 96%x. NaH, DMF, r.t. 10 min, then Pd(OAc)z, PPh3, 90°C, 1 h, 80%. SCHEME 12.

The synthetic intermediates in this series were further extended to the first total synthesis of antipodal koumine (23,24). Thus, the benzyl protecting group of the indole nitrogen in 49 was removed reductively by treatment with Na/NH3 at -78°C to give 83. Treatment of 83 with methyl chloroformate gave the carbamate derivative, which was then reduced with LiAlH4 to give (+)-18-hydroxy-anhydrovobasinedio1(81).Similarly, the 19(Z) isomer 51 gave 85 via 84. When the 19(Z) isomer 85 was treated with diethyl

1. Gelsemium ALKALOIDS

21

Me

P & 2 i, ii

(+)-49,R=COzMe. R=Bn

(+I-@, R=CH*OH, R=H

iii. iv __t

H Me

(+)-(81)

Me H

(+)-(70)

(+)-Koumine R

(+)-61, R=CO&fe, R=Bn (+)-84, RxCHzOH, R'=H

3 i, ii

iii, iv

(+I4861

__c

Reagents: i. DIBALH; ii. Na, NH,, -78 "C; iii. CICQMe, NazCO3, aq. THF; iv. LiAIb, THF; v. diethyl azodicarboxylate, P h 3 , cat. imidazole, NaH, THF,reflux.

SCHEME13.

azodicarboxylate/Ph3P/imidazole(cata1yst)MaH in dry THF heated under reflux, it was cleanly converted into (+)-koumine (70) in 40% yield (72% based on recovered 85). Conversely, when the 19(E)isomer 81 was exposed to the Mitsunobu conditions just discussed, (+)-koumine was formed in lower yield (34%, no recovered starting material) and at a much reduced rate. The effect of double-bond geometry on the rate and product distribution in the SN2'reaction was discussed in the literature (23) (Scheme 13).

IV. Humantenine-Type Alkaloids A. ISOLATION AND STRUCTURE ELUCIDATION OF NEWALKALOIDS

Humantenine-type alkaloids are the oxindole derivatives of the C/D ringcleaved sarpagine-type indole alkaloids. Only three humantenine-type N,methoxyoxindole alkaloids, i.e., humantenine (86) (7,49-52), humantenirine (87) (49,51,52), and rankinidine (88) (52), had been reported up to

22

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

1988. Recently, nine new humantenine-related alkaloids 89-97 were isolated from G. eleguns Benth. (Fig. 5). 11-Hydroxyrankinidine (W), which was determined by single-crystal Xray analysis (53),and 11-hydroxyhumantenine (91) are the first Gefsemium oxindole alkaloids with a phenol group, although some other oxindole alkaloids have an 11-methoxy group. Two N,-desmethoxy humanteninetype alkaloids 92 (53)and 93 (54) were also found in nature for the first time. 20-Hydroxy-dihydrorankinidine (99, which was determined by

11-Methoxyhumantenine (89) ll-fiydroxyrankinidine (90) Il-HydmXyhumantmine (9 1 Na-Desmethoxyrankinidine (92) Na-Desmethoxyhumantenine (93)

RI=Me, R2=OMe, R3=ORle R p H , R2=OH, R3=OMe RI=Me, R2=0H. R3=OMe

NH dMe

RI=H, R2=H. R3=H R,=Me, R2=H, R3=H

Gelsemamide (96) 11-Methoxygelsemamide (97)

R=H R=OMe

1. Gelsemium ALKALOIDS

23

X-ray analysis (54), and 15-hydroxyhumantenine (94) (54) are the very few members with a hydroxy group at the C-20 or C-15 positions among the Gelsemiurn oxindole alkaloids. Among these alkaloids, 87, 92, 93, and 95 were synthesized from relatively simple indole alkaloids (Section 1V.B). Two novel seco-oxindole alkaloids, gelsemamide (96) and 1l-methoxygelsemamide (97), were isolated from G. elegans (55). The 'H- and 13CNMR spectral assignments were performed in detail using modern NMR techniques, and the structure established unequivocally by single-crystal X-ray analysis. The absolute configuration was inferred by assuming that C-15 is the biogenetically controlled asymmetric center with the same R absolute stereochemistry as the humantenines. This is supported by the observation that 11-methoxygelsemamide(97) has a CD curve identical to those of the humantenines. Later, this new alkaloid was synthesized from gardnerine and the absolute configuration was confirmed (Section 1V.B). The spectral data of gelsemamide (96) closely resembled those of 97, except that % has no aromatic methoxy group at C-11. Therefore, the structure of gelsemamide was determined to be formula 96 (Fig. 5). B. SYNTHETIC STUDIES Biogenetically, the humantenine-type oxindole alkaloids would be generated from the sarpagine-type alkaloids such as 19(Z)-anhydrovobasinediol (8) through rearrangement to the oxindoles and introduction of a methoxy function on the indole nitrogen. Based on this consideration, synthesis of humantenine-type alkaloids was studied as follows. Initially, transformation of sarpagine-type indole alkaloids into the corresponding oxindoles was investigated using the C/D ring-cleaved derivative of gardnerine (28) (Scheme 14). Oxidation of the indole 98 by the conventional method (56) with t-butylhypochlorite (t-BuOC1) in the presence of triethylamine gave the unstable chloroindolenine 99, which was directly treated with aq. acetic acid in methanol to afford two oxindoles 100 and 101 in 9 and 37% yield, respectively. The minor product 100 has the same stereochemistry at C-7 as that of the natural humantenine-type alkaloids, as demonstrated by comparison of the CD spectra. Treatment of 98 with 2.0 eq. of Os04 in pyridine-THF afforded oxindole 103 as the sole product in 77% yield, presumably through the spontaneous pinacol-type rearrangement of the C2-C7 di-a-hydroxy intermediate 102 (57). It is interesting to note that all of the oxindole alkaloids isolated from Gelserniurn spp. (humantenine-, gelsemine-, and gelsedine-type alkaloids) possess the (S)-configuration at the spiro position. Oxidative rearrangement of the indole alkaloids into the oxindole derivatives in Gelsemiurn plants may occur enzymatically via an intermediate similar to that of the osmylation process (Scheme 14).

24

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

Gardnerine (28)

/

(R=McJ

iv, v

iii

10s y. 77%

(ILc€l@Is)

100

y . a

101

y. 57%

Reagents: i. ClCO,R, MgO; ii. rBuOCI, Et3N; iii. AcOH, MeOH, HzO,heat; iv. 2eq. 0804,Py.-THF v. aq. NaHS03 SCHEME 14.

Utilizing this new rearrangement reaction, new minor Gelsemium alkaloids, N,-demethoxyrankinidine (92) and Na-demethoxyhumantenine (93), were synthesized from koumidine (1) (20), which was prepared from ajmaline (14) as described in Section 1I.B.The C/D ring-cleaved compound 104, prepared from koumidine by treatment with 2,2,2-trichloroethyl chloroformate, was exposed to Os04 oxidation to give the oxindole-diol 105 and the diol 106 in 38 and 37% yield, respectively. The indole 106 was again oxidized with OsO4 to give the oxindole-diol 105. The oxindole thus obtained stereoselectively had the natural 7(S) configuration, which was confirmed by comparison of the CD spectrum with that of the humanteninetype alkaloids. The 19(Z)-ethylidene double bond was regenerated by a three-step sequence. Thus, the diollO5 was treated with trimethyl orthoformate in the presence of pyridinium p-toluenesulfonate to give the corresponding 2-methoxy-l,3-dioxolane,which was refluxed in acetic anhydride. The Na-acetyl group was then removed by alkaline hydrolysis to

1. Gelsemiurn ALKALOIDS

25

afford the desired olefinic compound in 74% yield from diollO5. Finally, the protecting group on Nb was removed with Zn in acetic acid to furnish Nademethoxyrankinidine (92)in 78% yield. On &-methylation of 92 with formalin and NaCNBH3 in the presence of AcOH, a new indole alkaloid Na-demethoxyhumantenine (93) was obtained (Scheme 15). The next requirement for the synthesis of humantenine-type alkaloids was the development of a general synthetic procedure to prepare the Na-

Koumidine (1)

H

104

I

i, ii

I iii, iv

105

106

OH OH

6H

\

v-viii

N,-Demethoxyrankinidine (92) R=H N.-Demethoxyhurnantenine (93) R=Me

3 ix

i. Os04, THF-Py, -20 "C,40 min; ii. NaHSO3, rt, 1.5 h, 38%(1061,37% (106); iii. Os04, Py. - THF, -28 "Cto -15 "C, 1.5 h; iv. NaHS03, rt, 2 h, 55%; v. HC(OCH3)3,PPTS, THF, rt. vi. AczO, reflux; vii. NaOH, MeOH, rt, 74% viii. Zn, AcOH, rt, 78%; ix. HCHO, AcOH, NaBH3CN SCHEME15.

26

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAl

methoxyoxindole function, which is one of the characteristics of the Gelsemiurn alkaloids. Biogenetically, two pathways from indole alkaloids to N,-methoxyoxindole derivatives could be considered (Scheme 16).The isolation of a new indole alkaloid, Na-methoxy-19(Z)-anhydrovobasinediol(9), suggests the possibility that an N,-methoxyindole could be a biogenetic precursor of Na-methoxyoxindoles(route A in Scheme 16). Alternatively, Namethoxyoxindolescould be generated by introduction of an oxygen function onto the oxindole nitrogen in Na-demethoxyhumantenine(93)(route B in Scheme 16). Initially, an investigation along with the speculative route A was carried out. By applying Somei’s method (58), indoloquinolizidine 108was reduced with NaBH4 in trifluoroacetic acid to give the indoline derivative 109 in 94% yield. On oxidation with 10 eq of aqueous hydrogen peroxide (H202) in methanol-H20 (10: 1) in the presence of 0.2 eq of sodium tungstate (Na2W04 * 2H20) and successive treatment with ethereal diazomethane (CH2N2),the indoline afforded the N,-methoxyindole 110and Na-methoxyoxindole 111in 49 and 14% yields, respectively. T h e N,-methoxyindole 110 could be converted to the corresponding Na-methoxyoxindoles111and 112 in 30 and 13% yields, respectively, by treating with r-BuOC1 in aqueous THF in the presence of MgO. Using the same procedure, yohimbine could be converted into N,-methoxyoxindole derivatives (59). However, the ste-

rearrangement

*

N,-Demethoxyhumantenine (93)

19Z-Anhydrovobaeinediol(8) Route B

1

&-oxidation

/

&-oxidation

Route A i) Boxidation

ii) rearrangemeni

N,-Methoxy-l9(Z)anhydmvobaeinediol (9)

RZMe: Humantenine (86) EGH: Rankinidhe (88)

SCHEME16. A biogenetic route for the humantenine-type alkaloids.

1. Gelsemiurn ALKALOIDS

27

reochemistry of the spiro center could not be controlled by this procedure (Scheme 17). Next, the transformation of oxindoles into the corresponding Na-methoxyoxindoles was examined (60), along alternative route B in Scheme 16. Murahashi et al. reported the oxidation of 1,2,3,4-tetrahydroquinolines using Na2W04- 2H20 and H202to lead to hydroxamic acids (61,62).This procedure was applied with a slight modification to a relatively simple indoline derivative (Scheme 18). Spiro-indoline 114, which was prepared by LiAlH4 reduction of the oxindole 113, was treated with 5 eq of 31% aqueous H 2 0 2in the presence of 0.1 eq of Na2W04in H20-MeOH (1:10) mixture at room temperature for 8 h to produce the hydroxamic acid 116 in 41% yield. This product could be quantitatively converted to the desired Na-methoxyoxindole 117 by treatment with ethereal CH2N2.The use of the urea-hydrogen peroxide addition complex (H202 - H2NCONH2) in place of aqueous H202in this reaction improved the yield of 116 to 52%. When the oxidation was discontinued at 1 h, the nitrone 115 was obtained as the main product in 52% yield, together with 116 in 12% yield. The

Indoloquinolizidine (108)

/

ii, iii

. t

110

Meb

111 L

Meb

112 J

iv

Reagents: i. NaBH,,, CFsC02H. 94%; ii. Na2W04*2H20,31%H202; iii. CHzN2,49%(110). 14%(111); iv. t-BuOCl, MgO (5 eq), HzO (4 eq), THF, 30%(111). 13%(112).

SCHEME17.

28

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

114

113

iii

= H 116 C RR=Me: 117

Reagents : i. L i A l h , THF; ii. H2Opurea. Na2W04*2H202, aq MeOH; iii. CHzNz, EkO. SCHEME 18.

nitrone 115 could be converted to hydroxamic acid 116 by oxidation with H202,indicating that the nitrone was an intermediate in this oxidation process. This procedure was applied to Gelsemium alkaloid synthesis (60). Gelsevirine (120) is a minor alkaloidal component of Gelsemium species and its structure was determined by chemical correlation with gelsemine (118) through the reductive des-N,-methoxylation of 118. Gelsemine (118) was first reduced with LiAlH4 in quantitative yield, and the resultant indoline 119 was oxidized with 10 eq of the urea-hydrogen peroxide complex and 0.2 eq of Na2W04at 10-15°C for 2 h. The reaction mixture was treated directly with dimethyl sulfide to decompose the excess oxidant and then successively with CH2N2. The crude products were treated with aqueous

ii. iii

6th

H

R-0: Gelsemine (118) R=H2:

119

Ii

Gelsevirine (120)

Reagente: i. LiAlHe, THF; ii. H&*urea, NazWOp2Hz02, aq MeOH then Mez& iii. CH2N2,Et& iv. NaHS03. SCHEME 19.

1. Gelsemiurn ALKALOIDS

29

NaHS03 to reduce the &-oxide function to produce gelsevirine (120) in 53% yield (Scheme 19). By employing the newly developed methods, humantenirine (87),a representative humantenine-type Gelsemiurn alkaloid, was synthesized from a sarpagine-type indole alkaloid (63). The oxindole derivative 103 prepared from gardnerine (28) was used for further transformation (Scheme 20).

Gardnerine (28)

121

122

123

127 Reagents:

i. ClC02CHflCh. MgO, rt; ii. 2eq. 0 ~ 0 4 .Py-THE iii. NaHSO3,78%;iv. S03-pyridine, DMSO.TEA, 7oRD; v. NaBH4, -78°C. 8W vi. 2,2-dimethoxypropane,TsOH, acetone, 98% vii. BHySMe2, THF, reflux;viii. Me3N-0, MeOH, reflux,y. quant; ix. H2&-Urea, Na2W04; x.CH2N2,32%,xi.ag. &OH, reflux, 91%.

SCHEME20.

30

HIROMlTSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

Because humantenines have a 19(Z) configuration, the olefin inversion utilizing the vicinal diol function in 103 was needed. The configuration at C-19 in 103 was inverted by the oxidation-reduction sequence. Thus, compound 103 was oxidized with S03-pyridine complex to afford the CI9keto derivative 121, which was then reduced with N a B 6 in MeOH at -75°C to give the desired C19-(S) alcohol 122, predominantly (122:103 = 16 :1). After protection of the vicinal diol in 122 with 2,2-dimethoxypropane, the lactam residue of the acetonide 123 was reduced with the BH3 - SMe2 complex to yield the secondary amine 124 in quantitative yield. Treatment of the amine 124 with H202 * H2NCONH2 and a catalytic amount of Na2W04 2H20 in aq. MeOH at 10-18°C gave the hydroxamic acid 125, which was methylated with diazomethane to yield the N,-methoxyoxindole 126 in 31% overall yield from 124 (Scheme 20). Next, a vicinal diol function in the humantenine skeleton was converted to the ethylidene double bond as follows. After removal of the acetal group in 126 with aqueous 80% AcOH, the diol 127 was converted to cyclic orthoformate 128 by treatment with trimethyl orthoformate in the presence of pyridinium p toluenesulfonate (PPTS), which was refluxed in acetic anhydride for 5 h to provide the 19(Z)-olefinic compound 129 in 77% overall yield from 127. The Nb-protecting group in 129 was removed with activated zinc in AcOH to furnish humantenirine (87) in 88% yield. A new seco indole alkaloid, 11-methoxygelsemamide (97)(59, might be derived from the humantenine-type oxindole alkaloid, humantenirine (87), by bond cleavage between the N , and C-2 and bond formation between the Nb and C-2 positions. To create the gelsemamide skeleton, humantenirine (87) was treated with NaOMe in dry MeOH to yield the target natural product, 11-methoxygelsemamide (97), in 78% yield. This reaction would proceed via B ring cleavage by the attack of methoxide anion at the C-2 position and subsequent amide formation between the C-2 ester group and the N b function in the resultant flexible molecule. The absolute configuration of the new alkaloid 97 is thereby chemically confirmed (Scheme 21). 20-Hydroxy-dihydrorankinidine( 9 9 , a new humantenine-type alkaloid isolated in 1991 (54), is the only alkaloid that has a hydroxy group at the C-20 position, and is proposed to be an important biogenetic processor of some alkaloids, such as gelselegine, gelsenicine, and gelsedine, via the aziridinium intermediate (Section V.B.1). To prove this proposal by chemical transformation, 95 was prepared from ajmaline (14) in 22 steps utilizing a biogenetically patterned synthesis (64) as follows. Initially, to liberate the masked aldehyde and secondary amine, ajmaline (14)was converted to the carbamate 130.The aldehyde group in 130 was protected as the 13-dioxane 131,which was transformed into the indole 133 by the following four-step sequence: (i) protection of the hydroxy group as a trimethylsilyl ether;

1. Gelsemium ALKALOIDS

127

129

128

Humantenirine (87)

31

1l-Methoxygelsemamide (97)

Reagents: i. HC(OMe)3, PPTS;ii. AczO, reflux, 77%; iii. Zn, AcOH, 88%; iv. NaOMe, MeOH, reflux, 10 h. SCHEME21.

(ii) oxidation of N,-methylindoline with Pb(0Ack leading to indolenine

(132);(iii) deprotection of the hydroxy group, followed by fragmentation under mildly acidic conditions; and (iv) immediate reduction of the resultant aldehyde with NaBH3CN. The primary alcohol in 133 was protected as the benzoyl ester 134 before removal of the aldehyde-protecting group in order to prevent hemiacetal formation between the C1,-OH and the C2,-aldehyde functions. The indole amine in 134 was tosylated under basic conditions, accompanied by some epimerization occurring at C-20, to produce the epimeric mixture 136.The aldehyde function in 136 was converted to a silyl enol ether, and then a hydroxy group was introduced at the C-20 position by treatment with Os04. Separation by medium-pressure liquid chromatography yielded two a-hydroxyaldehydes (137 and 138) and the starting material 136 in 45,22, and 7% yields, respectively. Because the configuration of the epimeric C20 positions in 137 and 138 could not be determined from the spectroscopic analysis at this stage, the major isomer 137 was subjected to ring closure between the C-21 and Nb positions. The aldehyde group in 137 was reduced with NaBH4, and the resulting primary hydroxy group in 139 was selectively mesylated to produce 140. Hydrogenolysis of the carbamate 140 provided the ring-closure compound

OAO

U

140

134

144

146

xix-xxi

xxii

t

___)

20-Hydr0xydihyd.mrankinidine (95)

146

Reagents: i. HaN-NMez, cat.H2SO4,3A-MS, EtOH; ii. CBZ-C1, lN-NaOWCH&; iii. CuCla, aq. THF, iv. HOCH2CH2CHaOH. TsOH, benzene; v. TMSC1, EtsN, CH2C1,; vi. Pb(OAc14, CHzC12; vii. &OH-THF-Ha0 then NaBH3CN; viii. PhCOC1, pyridine; ix. 80%6AcOHx. p-TsC1, n-BuaHSO4, benzene-SO%KOH xi. TBSOTf, EtaN; xii. 0804,THF-pyridine; xiii. NaB&; xvi. MsCl, pyridine; xv. H2,10%PdIC, AcOH, EtOH; xvi. KOH,MeOH, xvii. ClCO~CH2CC13,M a ,aq. THF; nriii.0 8 0 4 , THF-pyridine; xk.BHsoSMe2; a urea-Hfl2, Na2W04, aq. MeOH, xxi. CHzN2, MeOH xxii. Zn, &OH. SCHEME 22.

1. Gelsemium ALKALOIDS

33

141, which was then converted to the sarpagine-type key intermediate 142 by removal of the protecting groups under strong basic conditions. The minor product 138 was also transformed into the epimeric sarpagine-type compound 143 by the same procedure. The stereochemistry at the C-20 position in 142 and 143 was unambiguously determined by a NOE experiment. The major product (142) showed the same S-configuration at the C-20 position as in the natural product 95. Treatment of 142 with 2,2,2-trichloroethylchloroformateand MgO in aq. THF provided the C/D ring-cleaved compound 144. The indole moiety in 144 was stereoselectively converted by oxidation with o S o 4 in THFpyridine to the oxindole 145, which had the desired C-7(S) configuration. The lactam in 145 was chemoselectively reduced with the BH3 * SMe2 complex in THF to produce the indoline, which was then converted to the N,-methoxyoxindole 146 by (i) oxidation with H202 * H2NCONH2and a catalytic amount of Na2W04* 2H20 and (ii) 0-methylation with CH2N2. Finally, removal of the Nb protecting group in 146 with activated zinc in acetic acid provided 20-hydroxy-dihydrorankinidine(95) (Scheme 22).

V. Gelsedine-Type Alkaloids AND STRUCTURE ELUCIDATION OF NEWALKALOIDS A. ISOLATION

The gelsedine-type alkaloids are oxindoles with a novel skeleton similar to that of the humantenine-type oxindole alkaloids, but lacking their C21 carbon. Gelsedine (147) (3,65,66), 14-hydroxygelsedine (148) (6,67), gelsemicine (149) (68,69),14-hydroxygelsemicine(150) (70,71),gelsenicine (151) (7,49),and 14-hydroxygelsenicine(humantenidine) (152) (7,49) were the members of this group known and characterized up to 1988, and since then six new gelsedine-type alkaloids 153-158 have been found (Fig. 6). The isolation of 19-0x0-gelsenicine (153) from the leaves of G. eleguns was reported (6).The structure was established by X-ray analysis and by comparison of the CD spectra with those of gelsenicine (151). A novel gelsenicine-related oxindole alkaloid, named gelsemoxonine (154), was isolated from G. eleguns (54).The COSY spectrum of 154 showed which ) , were two methine doublets (6 3.78, d, H-3 and S 4.99, d, H - ~ ~ c Y coupled only to each other, indicating that C-14 had only one proton and C-15 had no proton. Therefore, both carbons should be oxygenated. The sequential connectivity of the protons, starting from H2-17 to H2-6 via H16 and H-5, such as that in gelsenicine (151), was elucidated by the COSY spectrum. Unambiguous assignments of the protonated carbons were obtained by a HETCOR spectrum. The remaining quaternary carbons were

34

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

&

Rl

19

.,.11-

AMMe

Gelsemoxonine (154)

2

Gelselegine (165) RI=H. Rz=H 11-Methoxy-1NRk Rl=OMe, R2=OH hydmxygelselegine (166)

fl

Gelsamydine ( 168)

Elegansamine (157)

FIG.6.

assigned by the selective INEPT technique. The (2-20 signal ( 8 211.67) of 154 is in the range for a ketonic carbonyl resonance, indicating the existence of a carbonyl and an amine group in 154 instead of the NdC-20 double

1. Gelsemium ALKALOIDS

35

bond in the gelsenicine (151). Selective INEPT irradiation of H-14 with a different 3JcH value (3-7 Hz) more strongly enhanced the C-15 signal than the C-16 signal, indicating an epoxide oxygen bridge between the C-14 and C-15. Gelsemoxonine (154)is the first Gelsemiurnoxindole alkaloid without an NdC-20 bond and might be formed from the corresponding gelsenicine derivative by cleavage of the C =N double bond owing to the strain by the epoxide at the C-14,15 position. The isolation of two members of a new type of oxindole alkaloid having a hydroxymethyl group at the C-20 position in the gelsedine skeleton was reported (73). The new alkaloid 155, named gelselegine, showed a mass 358 spectral fragmentation pattern, i.e., a molecular ion peak at d . 327 [M-CH20H]' and a principal (C20H26N204) with a base peak at d . fragment ion at m/t 296 [M-CH20H-OMe]', corresponding to successive losses of the hydroxymethyl and N,-methoxy groups. The I3C-NMR spectrum indicated the presence of a hydroxymethyl carbon. The HETCOR and selective INEPT spectra of 155 suggested that a rearrangement and ring contraction in the aliphatic portion had occurred to form a quaternary carbon at the C-20 position. The structure was established unequivocally by single-crystal X-ray analysis. The second gelselegine-type new alkaloid, ll-methoxy-l9(R)-hydroxygelselegine(156), has two more oxygen substituents compared with gelselegine itself. Including the new asymmetric center at the C-19, the structure of 156 was determined by X-ray analysis. Biogenetically, gelselegine-type alkaloids might be derived from the corresponding humantenines by rearrangement and ring contraction. According to this biogenetic hypothesis, the gelselegine alkaloids were synthesized from the sarpagine-type alkaloid (Section V.B). A new class of two indole alkaloids, named elegansamine and gelsamydine, were found in G. eleguns Benth. The high-resolution mass spectrum of 157 showed the molecular formula C29H36N206and the base peak at d. 326, corresponding to the molecular weight of gelsenicine (151), C19H22N203,indicating that elegansamine (157) was constructed from gelsenicine (151) or its isomer and a monoterpene unit containing three oxygen atoms. In the 'H-NMR spectrum, in addition to some readily assignable signals due to the gelsenicine moiety, characteristic signals of a doublet for the C-18 protons and a multiplet due to H-19 were observed in place of the ethyl group in 151, suggesting that the monoterpene unit might be connected at the C-19 position. From the 13C-NMR spectrum, the indole alkaloid portion and the monoterpene unit were respectively demonstrated to be gelsenicine and an iridoid skeleton, which possessed a lactone, one C-Me, and a secondary hydroxy group. The structure of elegansamine, including the stereochemistry of the iridoid moiety and the C-19 position, was determined by single-crystal X-ray analysis (74). The CD spectrum of 157closely resembles that of gelsenicine (El),and therefore the gelsenicine

36

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

part and the iridoid residue in 157 have the same absolute configuration as the conventional indole alkaloids and iridoid monoterpenes, respectively. A new oxindole alkaloid gelsamydine (158) (75) isolated from the same species is structurally related to elegansamine (157). The structure was deduced by spectroscopic methods and then established by X-ray analysis. It is also constructed from gelsenicine and a monoterpenoid unit.

B. SYNTHETIC STUDIES 1. Partial Synthesis Based on Biogenetic Speculation

Gelsedine-type alkaloids have a novel oxindole skeleton missing the C-21 carbon of the humantenine alkaloids. A biogenetic route initially speculated for gelsedines involved the release of the C-21 carbon in the early stage of the biogenesis (route A, Scheme 23). Namely, a hypothetical intermediate, D-norsarpagine type alkaloid 159, would be generated by loss of the CZ1-aldehydecarbon from the common intermediate 13. The Dnorsarpagine derivative 159 would provide the C/D ring-opened compound 160 and then be transformed into the gelsedine series via rearrangement to the oxindole derivative (76) (Scheme 23). To execute this proposal (route A) by chemical transformation, synthesis of a hypothetical intermediate 159 and its oxidative rearrangement to the oxindole derivative have been examined. The C-21 aldehyde carbon in ajmaline (14) was removed by C-20,21bond cleavage in the glycol derivative 162, which was prepared from 14 by a six-step operation (Scheme 24).

I

I

Route A

Strictosidine (11) Gelsedine (147)

Q-@

SCHEME 23. A biogenetic route for the gelsedine-type alkaloids.

h e

21.,.DH

i. ii

__L

B-.r"$, kie

kieH 20

iv

-CbZ

___t

H

iii

R,

".,

163: R-TBS. Rl+RpO 164: R=TBS. &=H, %=OH 165: RoTMS, R r H , R.pOAc

161: R-CHO 162: RPCHPH

xiv. xv

xvi

Y

q: 170

159

20

21 R

Ajmaline (14)

1

N-m OH

/

l4

169

""\ 172

173

Reagents: i. HzN-NMel, cat.H$304,3A-MS. EtOH CBZCl, lN-NaOWCH2C12;CuC12. aq. THF(pH 7); ii. TBSOTf, EBN, CH2C12; 0 ~ 0 4 THF-Py., , aq. NaHS03; iii. NaBH.,, MeOH; iv. NaIO.. MeOH v. L-Selectride. 'l". -70°C: vi. AceO-Pv.: vh. n-BurNF. TBF viii. TMF;bTf. CHzCl2; k Pb(OAd4. CH2C12.:70°C; -x. A-dH-THF-HzO then NjaBH4, Me6H; xi. TBSoTf. Et3N. CH2Clz; xii. 5%aq.KOHiMeOH; xiii. MsCl. EtsN, DMAP. CH2C12; xiv. Hq. Pd-C. AcOH. EtOH xv. n-BurNF.THF rvi. BrCN. MeO. benzene:

SCHEME24.

38

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAl

Through conversion of the indoline moiety in 165 into an indole ring by successive operations involving oxidative de-Na-methylation, fragmentation of indolenine to indole, and reduction of the resulting C-17 aldehyde, the norsarpagine derivative 159 having a five-membered D-ring was constructed. On treatment with BrCN and MgO in benzene under reflux conditions, compound 159 afforded the C/D ring-cleaved product 169 in 48% yield (76). Treatment of the N,-BOC derivative 171successively with Os04 and then with AcOH/MeOH/H20gave the oxindole 173 as the sole product in 70% overall yield from 171. However, 173 had the opposite configuration at the spiro C-7 position compared with that of natural gelsedine-type oxindoles. The stereochemical course of this oxidation-rearrangement reaction was speculated to be as follows. The reagent (Os04) might attack from the less hindered @side to generate the diol 172, and subsequent treatment of this intermediate with aqueous acetic acid provided oxindole 173 having the C7(R) configuration via a pinacol-type rearrangement (77) (Scheme 24). This result, i.e., formation of the C7(R)isomer 173 from 169, is in contrast to that obtained in the humantenine-type alkaloid series (Section 1V.B). The appearance of the new Gelsemium alkaloid gelselegine (155) (73) suggested the possibility of an alternative biogenetic pathway for gelsedinetype alkaloids (77) (Route B, Scheme 25). Thus, enzymatic oxidation of sarpagine-type indole alkaloids would first provide the humanteninetype oxindole alkaloids having the C7(S) configuration. An aziridinium intermediate 174 would then be generated from 20-hydroxy-dihydrorankinidine (95) or from rankinidine (88). Ring-opening by the attack of water at the C-21 position in 174 would produce gelselegine (155). Furthermore, gelsenicine (151) and gelsedine (147) would arise from 155 by loss of the (2-21 carbon. By condensation of a monoterpene unit and gelsenicine a new class of alkaloids, elegansamine (157) and gelsamydine (158), would be formed (78) (Scheme 25). Based on this second biogenetic speculation, chemical synthesis of gelsedine-type alkaloids was studied as follows. A sarpagine-type alkaloid, gardnerine (a),was chosen as the starting material and the methoxy group on the indole ring of 28 was initially removed by a six-step sequence as described in Section 1I.B. The resulting 19(E)-koumidine (33) was converted to the C/D ring-opened derivative 175 in 94% yield by treatment with 2,2,2-trichloroethyl chloroformate in the presence of MgO in aqueous THF (Scheme 26) (79).Because of the higher susceptibility to Os04 oxidation of the ethylidene side chain than the indole nucleus, two equivalents of Os04were used to convert the indole ring to an oxindole function. Compound 177 having the S configuration at the C-7 spiro center was produced in 39%yield, accompanied by the indole-

39

1. Gelsemium ALKALOIDS

Route B

r

1

L

Rankinidine (88)

J

2O-H~droxydih~drorankinidine (95)

(oy

174

I

at

&4H

bm,

AM.

Gelsedine (147)

AM.

Gelsenicine (151)

Gelselegine (155)

Iridoids

R=

%

HOi ,

Elegansamine (157) Gelsamydine (158) SCHEME25. A biogenetic route for the gelsedine-type alkaloids.

40

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

-

176

/q R=OMe: Gardnerine (28) R=H: 19(E)-Koumidine(33)

1 R"

&:iii

+

OH

H

176(kTroc)

1

180

'

OH

ii

!

---\I

&iv

cH

0

0

R=Troc: 178 R=H:179

177

RI=H, R p 0 : 1 8 1 RI=H, RpH,: 182 Rl=OMe, &=0:183

3

R=Troc: 184

vj

vii

xii blkle

Gelselegine (166)

bM6l

Gelsenicine (161)

OMe

Gelsedine (147)

Reagents: i. TrocC1, MgO, aqueous THF,rt, 15 h, 94%;ii. 0 6 0 4 , py. THF, -78"C, then aq. NaHSOa; iii. a. CH(OMe)a, PPTS, THF, rt, 2 h; b. Ac20, reflux, 3 h; c. 6% aq. KOH, MeOH, rt, 2 h. 75% overall; iv. TMSCI, NaI, MeCN, rt, 1h, 94%;v. a. Os04,py, THF, rt, 2 h, then aq. NaHSOa, 82%; b. NaBH4, MeOH, rt, 2 h, 97%; vi. BHa-SMe2, THF, reflux, 2 h, then Mefl+O, MeOH, reflux, 2 h, 77%; vii. a. ureamH202, cat. Na2W04, aq. MeOH, rt, 4 h; b. CH2N2, EhO, 61% overall; viii. TMAD, nBuaP, DMF,rt, 4 h, 63%; ix. Zn,AcOH, rt, 4 h; x. standing for 6 days, rt, 60% overall; xi. NaIO4, aq. MeOH, rt, 2 h, 64%; xii. FW2, H2, EtOH, rt, 1h, quant. SCHEME 26.

1. Gelsemium ALKALOIDS

41

dioll76, in 28% yield. The indole 176 was in turn treated with Os04 to give the oxindole 177. The stereochemistry at the C-7 position was confirmed by a comparison of the CD spectrum with that of the humantenine-type alkaloids. The ethylidene side chain was reformed in 75% overall yield from the vicinal diol of 177 by a three-step operation [(i) trimethyl orthoformate, PPTS, T H F (ii) Ac20, reflux; and (iii) 5% KOH aq. MeOH] to give the humantenine-type compound 178. Attempts at the preparation of an aziridine compound, such as 174 from 179, or 20-hydroxy-dihydrorankinidine (95) were unsuccessful. Then, as a clue for the construction of the gelsedine skeleton, double-bond migration from the C-19,20 to the C-20,21 positions was conducted using NaI and TMSCl in MeCN at room temperature to provide the enamine 180. This unusual reaction was considered to occur via the siloxy immonium intermediate and conjugated immonium intermediates (Scheme 27) (77). The enamine 180 was successively treated with Os04 (82% yield) and the NaBH4 (97% yield) to produce the diol 181 stereoselectively. The stereochemistry at C-20 could be deduced from a stereomodel analysis. The reagent (Os04) should approach the double bond of 180 from the less hindered convex side, resulting in the formation of the C-20(S) alcohol (Scheme 27). At this stage, the Na-methoxyoxindole function was introduced. The lactam of 181 was reduced in 77% yield with BH3 - SMez complex and the resultant amine 182 was oxidized with the urea hydrogen peroxide complex, in the presence of a catalytic amount of sodium tungstate, followed by O-methylation with CH2Nz to yield the Namethoxyoxindole 183 in 61%overall yield from 181. To prepare the epoxide from the diol 183, use of conventional reagents, including several MsCl/ base combinations and the Mitsunobu system, was attempted, but gave no

178

-Troc

/ 180 iii

SCHEME27.

42

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

useful results. However, treatment of 183 with N,N,N’,N’-tetramethylazodicarboxamide (TMAD) and n-Bu3P(a modified Mitsunobu reaction) (80,82) in DMF for 4 h at room temperature gave the epoxide 184 in 63% yield. Removal of the Nb-carbamate (Zn, AcOH) gave the primary amine 185, which gradually transformed into the natural product, gelselegine (155), in 50% yield, upon standing for 5 days at room temperature. It appears that the primary amine regioselectively attacked the C-20 position (5-ex0 tetrahedral mode) with complete inversion. The absolute configuration of the new alkaloid 155 is thereby chemically confirmed. In keeping with the foregoing biogenetic speculation, the C-21 carbon of 155 was oxidatively cleaved with NaI04 in aqueous MeOH to yield gelsenicine (151) in 64% yield, which is the most toxic compound among the Gelsemiurn alkaloids [LDS070 pg/kg (iv)]. Furthermore, catalytic reduction of the imine function of 151 furnished gelsedine (147) in quantitative yield (49). Since the total synthesis of (+)-19(E)-koumidine (33) has been accomplished already by Magnus (23,24),the formal total synthesis of these three alkaloids has been completed (79). Gelsemicine (149) was first isolated from G. sempervirens Ait. as a minor constituent (82) and was later found in Mostueu brunonis Didr. var. brunonis forma uugustifolia (83).Using a process similar to the transformation from gardnerine to gelsedine, the alkaloid 149 was synthesized. Gardnerine (28) was first converted to the C/D ring-cleaved derivative 98. The carbamate 98 was regio- and stereoselectively oxidized with 1 eq of Os04 at -70°C to give the oxindole 186. Treatment of compound 186 with TMSCl and NaI afforded the enecarbamate 187. The double bond at the C-20,21 positions was oxidized again with Os04 (1 es) to give a mixture of the dioll89 and the aldehyde 188, which was then reduced with NaBH4 in MeOH to give the diol 190. The glycol in 190 was converted to the epoxide 191 by a conventional method in 93% yield. After removal of Nb-carbamate in compound 191 with Zn/AcOH, the resultant free amine was gradually transformed in 82% yield to the gelselegine-type derivative 192 on standing at room temperature for 5 days. Next, the C-21 carbon in 192 was removed by the oxidative cleavage of the 1,Zamino alcohol system by treatment with NaI04 in MeOH to give the gelsenicine-type derivative 193 in 41% yield. The catalytic hydrogenolysis of the imine 193 (H2, Pt02, EtOH) stereoselectively gave the gelsedine-type compound 194 in 98% yield. The stereochemistry at the C-20 position was unambiguously determined by differential NOE. After protection of the free Nb group as the trichloroethyl carbamate, the lactam in the oxindole 195 was reduced with excess borane-dimethylsulfide complex in THF. The indoline 196 was treated with H202 . H2NCONH2 and Na2W04 2H20 in aq. MeOH at room temperature to give the nitrone 197 and hydroxamic acid 198 in 63 and

1. Gelsemium ALKALOIDS

43

15% yield, respectively. The nitrone 197 was further oxidized with lead tetraacetate (1 eq) in CH2Cl2 at -150°C followed by treatment with potassium carbonate in MeOH to afford the hydroxamic acid derivative 198 in 56% yield. The hydroxamic acid derivative 198 was treated with CH2N2 in MeOH to give the N,-methoxyoxindole 199 in 65% yield. Finally, the protecting group on Nb was removed with Zn/AcOH to yield gelsemicine (149) (Scheme 28) (84). Another member of the gelselegine-type compounds having a 19-hydroxy group was also prepared from gardnerine in a biomimetic manner (85,86). A biogenetic route for ll-methoxy-19(R)-hydroxygelselegine (156) could be assumed as follows (Scheme 29). The double bond at the C-19,20 position in the humantenine-type oxindole alkaloid would be oxidized to form the epoxy derivative 200, and by the subsequent attack of the nitrogen (&) to the C-20 epoxy carbon, an aziridinium intermediate 201would be generated. Furthermore, a new skeletal type alkaloid 156, possessing a hydroxymethyl group at the C-20 position, would arise from 201 by ring opening between the C-21 and Nb positions using water (Scheme 29). To realize the just-mentioned biogenetic hypothesis using chemical was chosen as the means, a sarpagine-type indole alkaloid, gardnerine (a), starting material for the chemical transformations. The C/D-ring cleavage in 28 and stereoselective rearrangement to the oxindole derivative 103 were carried out according to the method previously described in Section 1V.B. Next, the lactam in 103 was chemoselectively reduced with a boranedimethylsulfide complex to give the corresponding indoline derivative in quantitative yield. The secondary amine was then oxidized with H202 * H2NCONH2in the presence of Na2W04. 2H20 followed by treatment of the resulting hydroxamic acid with ethereal CH2N2 to produce the N,methoxyoxindole derivative 202 in 40% overall yield. The diol on the side chain in 202 was converted to the epoxide 203 by a conventional method, i.e., mesylation of the secondary alcohol with mesyl chloride (93% yield) followed by treatment with potassium carbonate in methanol (98% yield). By removal of the & protecting group in 203 with zinc in AcOH, the secondary amine 204 was obtained in 82% yield. This amine-epoxide 204 was then heated in dioxane at 150°Cfor 6 h, producing the aziridine derivative 205 in 61% yield, which corresponded to the biogenetically hypothetical key intermediate. In the 'H NMR spectrum, the protons on the C-21 carbon were observed in the high-field region ( 6 1.62 and 1.58) because of the anisotropic effect of the aziridine ring. The strong NOE (11.6%) between H-16 and H-21 revealed the C-20(S) configuration in 205, indicating that the secondary amine regioselectively attacked the C-20 position with complete inversion of configuration. The stereochemistry at C-19, which was already elucidated by X-ray analysis of the model compound (N,-demethoxy

187

189

im

191

"JlS2

k 193

I xiii

187

-Nc d V

rn-

ISBRH 199ILCH~

Gelserniane (149)

Reagents:

i. ClCO&H&Cl3, MgO, aq. THF; ii. OsO4(l eq.),dry Py.-dry THF;iii.TMSC1, NaI, dry C&CN; iv. 0 6 0 4 , dry -.-dry THF;v. N a B h , MeOH, vi. MsCl, dry Py.dry CH2C12; vii. K&O3, MeOH viii. Zn,AcOH, ix.NaIO4, MeOH x. H2, PtO2, EtOH; xi. ClCOOCHflC13, dry -.-dry CHg12; xii. BH30SMe2, dry THF; Me$J+O*!&O, MeOH; xiii. H$ICONH2*H&, NaaWOp2H20. aq. MeOH xiv. Pb(OAd4, CH2C12; K&O3, MeOH, xv. CHfl2, MeOH; xvi.Zn,AcOH SCHEME 28.

1. Gelsemium ALKALOIDS

19(Z)-Anhydrovobasinediol(8)

L

Humantenirine (87)

/

r

45

1

200

11-Methoxy-19(R)-hydroxygelselegine (156) SCHEME29. A biogenetic route for ll-rnethoxy-19(R)-hydroxygelselegine (156).

derivative of 205), could be considered to be R, like that of the epoxide 204. Finally, the aziridine 205 was refluxed in THF with CF3C02Hfor 0.5 h to furnish ll-methoxy-l9(R)-hydroxygelselegine (156)in 77% yield. Thus, the absolute configuration of the new alkaloid is chemically confirmed (Scheme 30).

2. Synthetic Approach to the Gelsedine- Type Alkaloids a. Kende Route. An approach to the total synthesis of gelsedine was reported by Kende et al. (Scheme 31) (87).The synthetic effort was initiated by the preparation of the requisite pyrrolidine system. The ethyl ester of

46

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

-

RSHKCI,

103:X=H

Gardnerine (28)

iii, iv. v

202: X=OMe

203

204

NOE -1

I .6%

205

11-Methoxy-19(R)hydroxygelselegine (156) Reagents: i. ClCOfiH2CCl3, MgO, aq. THF; ii. Os04 (2 eq.), Py/THF then aq. NaHS03; iii. BHs.Me2S; iv. HzOrurea, Na2WO4; v. CH2N2,40% from 103; vi. MsC1, Em, 9396, vii. K2CO3, MeOH, 984,viii. Zn, AcOH, 824%;ix. dioxane, 120 “C, 61%; x. TFA, THF, 77%. SCHEME 30.

4-tert-butoxycrotonic acid (207) was allowed to react with the ethyl ester of N-carbomethoxyglycine (206) using NaH in benzene under reflux to give the 3-pyrrolidinone ester 208 in 42% yield. Treatment of 208 with 80% aqueous trifluoroacetic acid at room temperature gave the keto lactone

21 1

R=C02Me

R-

xiii

220 222 R=p-phenylbenzoyl 223

( R=H

M902C-N

221 OMe

Reagents: i. NaH, PhH, A; ii.TFA, HzO; iii, (Ph)$'CHCO$Bu, THF; iv. Hz, Pd/C; v. LiBH,, THF; vi. aq. TFA, CH&Iz;vii. Swern conditions; viii. iRzNEt, nBuzBOTf, CHzCIz, -78 OC; ix. CHzCIz, -78 OC; x. Hz, PdK; xi. Li(sBuhBH; xii. T F M F A , CHCI,; xiii. HMDS,Iz. PhMe.

SCHEME31.

48

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

209 in 77% yield, setting the first two ring substituents exclusively in the thermodynamically favored cis configuration. Reaction of 209 with (carbotert-butoxymethy1ene)triphenylphosphorane in THF gave a 10:1 mixture of the Z to E isomers 210 in 72% yield. Catalytic reduction of the mixture 210 with hydrogen over Pd/C gave a quantitative yield of a single crystalline compound 211. The presence of the y-lactone unit in 210 was crucial for

the stereochemistry of this reaction. The catalyst must deliver the hydrogen from the sterically less hindered convex face of the molecule to yield only the all-cis pyrrolidine derivative 211. Selective reduction of the y-lactone unit in the presence of the tert-butyl ester was accomplished using LiBH4to yield the diol2l2, which was directly treated with a catalytic amount of aqueous trifluoroacetic acid in CH2C12 to give the Slactone 213. Treatment of 213 under Swern oxidation conditions gave the moderately sensitive aldehyde 214 Reaction of the Nmethoxyoxindole 215 with di-n-butylboron triflate at -78°C in the presence of diisopropylethylaminegave the vinyloxyborane 216 in nearly quantitative yield. This was allowed to react with the aldehyde 214 to afford a mixture of the E and Z olefins 217 in 42% yield. This mixture was reduced over 5% P d C to give the oxindole 218 as a mixture of C-7 epimers. Chemoselective reduction of the lactone versus lactam ring was carried out using lithium tri-sec-butylborohydridein CH2C12at 0°C. The lactols 219 were obtained as a mixture of C-7 and C-3 epimers. Treatment of the lactols 219 with 1:1 trifluoroacetic acid-trifluoroacetic anhydride mixture in CHC13gave a single pentacyclic product 220 in 53% yield. Selective cleavage of the carbamate protecting group using an in situ preparation of TMSI gave a secondary amine 222. Comparison of the 'H-NMR spectrum of the amine 222 with that of authentic gelsedine showed a major difference. The aromatic proton at C-9 of gelsedine was at a higher field than that of the amine (A 0.77 ppm), indicating that the pentacyclic product 220 had the opposite C-7 configuration to that of the desired compound 221. The structure of the final compound 220 was determined to be 7-epi-20-desethylgelsedine by X-ray crystallographic analysis of the amide derivative 223 (Scheme 31). b. Hamer Route. The tricyclic aza-oxa-undecane ring system present in gelsemicine was synthesized (88).Initially, thermal [3 + 41 cycloaddition of 3,3-dimethoxycyclopropene (225) with 4-methyl-2H-pyran-2-one (224) gave the oxa-bicyclo[3.2.2]nonadienone 226, which was reduced with hydrogen over Rh on alumina to give 227 with high stereoselectivity. The configuration was established by X-ray analysis of the ketone derivative 228. Reduction of the lactone with L i A l b followed by cyclization of the resulting diol gave the required ether 229, which was quantitatively hydrolyzed to 230. Reductive amination of 230 with methylamine/NaBH3CN gave a mix-

1. Gelsemiurn ALKALOIDS

h0+

49

- &%

" O r &OMe i

0

224

225

OW

Qe

'2

X=OMe 227

226

ii c X z = O

P r? 0

'"

X=OMe 229 cX2=0 230

231

232

228

OMe

Gelsemicine (149)

Reagents: i. Hz, Walumina; ii.0.05 M HCYaq. MeCN; iii, LMH4 then TsCl, NaH; iv. HCYaq. MeOH; v. MeNH2, NaBH3CN;vi. NaOCl; vii. hu, TFA then KOH, MeOH. SCHEME 32.

ture (4: 1) of two secondary amines. The NOE experiment clarified that the major isomer is compound 231. N-Chlorination with NaOCl, followed by the Hofmann-Loffler-Freytag reaction (h v/CF3C02Hthen KOHMeOH), gave the desired compound 232 having the tricyclic aza-oxa-undecane ring system present in gelsemicine (149)(Scheme 32).

VI. Gelsemine-Type Alkaloids

The gelsemine-type group involves the oxindole alkaloids having an additional bond between the C-6 and C-20 positions in the humanteninetype alkaloids. Gelsemine (118)(3,6,89-95), one of the principal alkaloidal constituents of the Gelsemiurn species, 21-oxogelsemine (233)(96),gelsevirine (120) (6,49,94,97),and 21-oxogelsevirine (234)(97) were the members of the gelsemine group up to 1988. Later investigation led to the isolation of five new alkaloids (235-239) structurally related to gelsemine. Since the structure elucidation of gelsemine in 1959, a number of studies directed toward total synthesis of this alkaloid have been published.

50

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

A. ISOLATIONAND STRUCTURE ELUCIDATION OF NEWALKALOIDS

In the leaves of G. eleguns Benth., an &-oxide derivative of gelsemine was found (6). The stereochemistry of the quaternary nitrogen in 235 was determined to be the R configuration by 'H-NMR spectral comparison of two diastereomeric N-oxides prepared by m-CPBA oxidation of gelsemine. Both stereoisomers of gelsevirine derivatives (237 and 238) having a secondary hydroxy group at the C-19 position were isolated (6,98). The structure including the 19(R) configuration of one of these two alkaloids was established by single-crystal X-ray analysis (98). The stereochemistry of the first isolated compound initially proposed as the 19(R) configuration became that of S. A new alkaloid 239 having an acetoxy group at the C-19 position in the gelsevirine skeleton was isolated from G. sempervirens and G. runkinii (98). The 'H- and 13C-NMR spectra of 239 were similar to those of 19(R)hydroxy-dihydrogelsevirine(237),except for the presence of an acetyl group in 239, suggesting that 239 had the 19(R) configuration. That was proved by hydrolysis of 239 to produce 237. 19(R)-Hydroxy-dihydrogelsemine (236)was also obtained from G. sempervirens and G. runkinii (98). The stereochemistry at C-19 in 236 was deduced by the similarity of the 'H-and I3C-NMR spectra of 236 and 19(R)-hydroxy-dihydrogelsevirine (237)(Fig. 7).

Gelaemine ( 1 18 ) 21-Oxogeleemine ( 2 3 3 ) Gelsevirine ( 1 2 0 ) 21-Oxogelsevirine (234) Gelsemine N-oxide (235)

RI=H, Rz=Hz RpH, R p O RI=OMe, Rz=Hz RI=OMe. Rz=O R p H , Rz=Hz. Nb-oxide

lS(R)-HydroxyR1=H. R p H , 19(R) dihydrogelsemine (236) lS(R)-HydrOXyRI=OMe, Rz=H, 19(R) dihydrogelsevirine (237) 19(S)-Hyd~yR p O M e , R p H , 19(S) dihydrogaleevirine (238) lS(R)-ACetoxyRI=OMe, R p A c , 19(R) dihydrogelsevirine (238)

FIG.7.

1. Gelsemium ALKALOIDS

51

B. SYNTHETIC STUDIES Gelsemine (118) is one of the principal alkaloids in Gelsemium species, and its unusual and compact hexacyclic skeleton has fascinated many synthetic organic chemists as a challenging target molecule since its structure elucidation. In recent years, many synthetic efforts have been vigorously carried out by distinguished research groups, and more than 20 papers were published from 1988 to 1994. To attain the total synthesis of gelsemine, many synthetic studies to construct the hexacyclic monoterpene unit and to develop new methods for the preparation of the oxindole part have been done. In this section, studies toward the synthesis of gelsemine by six research groups are introduced. 1. Johnson Route

The first synthesis of gelsemine (118) was completed by Johnson and his collaborators (99-101). The successful strategy involved the construction of a caged tetracyclic ketone 240, onto which the spiro-oxindole function was elaborated by photolysis of the alkoxy-substituted-l-alkenylbenzotriazole derivative. The retrosynthetic analysis is based on the application of the Corey rules for the strategic bond recognition in the bridged polycyclic systems in 240. Construction of the ketone 240 by bond formation between C-5 and C-6 (gelsemine numbering) using an intramolecular Mannich reaction of the iminium species 241 was intended (Scheme 33). The first key reaction in this approach involved a photoinduced intramolecular cycloaddition of the triene 245, which was prepared by condensation of 243 and the dimethyl malonate derivative 244. The tricyclic diester 246, which not only possesses 12 of the 13 carbon atoms present in the target ketone 240, but also has the masked vinyl group in the required cis relationship to the hydrogen substituent at the ring fusion, was reduced to the corresponding diol, using a sixfold excess of LiAlH4. The tetrahydropyranyl ring was formed using silver acetate-iodine, which gave the acetoxy alcohol 247. After 247 had been oxidized to the corresponding acid and esterified, the base labile acetoxy group was exchanged for the TBDMS

Gelsemine (118)

24 1

240

SCHEME33.

242

52

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

ether to give the methyl ester 248. To cleave chemoselectively the tetrahydrofuran ring of 248, phenyl trimethylsilylselenide in the presence of catalytic zinc iodide was used to give the tricyclic selenide 249. Oxidative deselenylation of 249 gave the alkene 250. Compound 250 was desilylated to give an unstable cyclobutanol, which was immediately oxidized to the cyclobutane-P-ketoester 251. Cleavage of the cyclobutanone ring of 251 in a retro Claisen reaction was accomplished by treatment with methylamine, which cleanly afforded the amide ester 252 as a single diastereomer, whose stereochemistry was confirmed by NOE experiments. The amide ester 252 was converted into the aldehyde 253 by a two-step process. This involved the chemoselective reduction of the ester function to the alcohol, using lithium methoxyborohydride, and reoxidation using Cr03-pyridine. The unstable aldehyde 253 thus obtained was subjected to further transformation in its crude state immediately after preparation. The aldehyde 253 did not spontaneously cyclize to the hydroxy amide 254. Epimerization of the aldehyde 253 with methanolic potassium carbonate brought the carbonyl group of the aldehyde and the amide nitrogen within bonding distance, and allowed cyclization to occur to give the hydroxy amide 254, which readily dehydrated to the corresponding enamide 255. To prevent the cleavage of the pyran cycle in a vinylogous N-acylaza-acetal system upon treatment with strong acid, enamide 255 was converted to the methoxy bromide 256 by treatment with methanolic bromine in the presence of suspended calcium carbonate buffer. Desilylation and oxidation of 256 gave the key intermediate ketolactam 257, a close relative of the target ketoiminium structure 241, suggested by retrosynthetic analysis. The ketolactam 257 was then converted into its enol silyl ether 258, thus setting the stage for the operation of the key intramolecular Mannich reaction. In practice, the conversion of 258 into bromoketone 259, whose structure was confirmed by X-ray determination, proceeded cleanly in hot trifluoroacetic acid. This cyclization can be viewed as a normally disfavored 5-endo-trig cyclization. Reductive debromination of the ketone 259 with tri-n-butyltin hydride and AIBN in refluxing benzene gave 260 (Scheme 34), which was the 0x0 derivative of the intermediate target derivative 240. The key step remaining involved the conversion of the ketone functionality in 260 to a spiro-oxindole. The conversion of a sterically hindered ketone to an oxindole by applying most of the conventional methods for the creation of a quaternary center at the spiro position was not an easy process. A process involving C-C bond formation by combination of two radical centers generated by photolysis of the alkoxy-substituted-lalkenylbenzotriazole was then investigated (Scheme 35). By applying Wender's procedure (102), the benzotriazole derivative 261 was converted to 262 by metallation-silylation, and then the trimethylsilyl derivative was

1. Gelsemium ALKALOIDS

53

- $T:zk 243

244

-@

OH

iii, iv

A d

V,vi,vii

___)

@Co&le

245

246

viii

TBDMSO

247

q C 0 2 M e

TBDMSO

248

249

xi, xii

ix,X

C O z M x

0

TBDMS

250

OTMS SePh

$

TBDMSO

251

-

e

"'CONHMe

~ ~ V , X V

252

-

-

-

Xvi

xviii

TBDMSO

TBDMSO

254

'Me

255

xxi ___t

TIPSO

256

257

Me

111 258 STlP

xxii

268 R=Br J xxiii 280 R=H Reagent: i. pyridine; ii. hu, MeOH; iii. LiAlH4; iv. AgOAc, 12, AcOH; v. RuC13, NaI04 then

CHzN2; vi. KzCO3, MeOH; vii. TBDMSOTf, 2,6-lutidine; viii. PhSeSiMe3, Zn12; ix. rn-CPBA; x. PrzNH, CCb; xi. K2CO3, MeOH; xii. Swern conditions; xiii. MeNH,; xiv. LiBH(OMe)3; xv. Cr03, pyridine; xvi. K2C03, MeOH; xvii. TsOH; xviii. Br2, MeOH; xix. n-Bu,NF, xx. Swern conditions; xxi.TIPSOTf, EbN; wii. TFA, reflux; xxiii. Bu3SnH, PhH, reflux. SCHEME 34.

54

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

O N : N N

\OM 261

N

i -

N’

ii

Me,a h O M 0 262

264

Me

263

266

Reagents: i. LDA, TMSC1; ii. LDA, adamantanone; iii. hu, MeCN iv. H30*. SCHEME 35.

condensed with adamantanone (which had been used as a model experiment) to give the alkene 263 in a Petersen olefination. Photolysis of 263 gave a 40% yield of the desired cyclized product 264 accompanied by an equal amount of the oxetane 265. Acid hydrolysis of 264 yielded the corresponding spiro-oxindole 266. Given the success in the model series, ketone 260 was subjected to the same reaction sequence. Condensation of the ketone 260 with lithiated 1(methoxytrimethylsilylmethyl)benzotriazole262 gave a mixture of the ( E ) and (2)-methoxymethylene isomers, 267 and 268, in a combined yield of 65%. Next, solutions of the two isomers in acetonitrile were irradiated separately. After chromatographic separation, two cyclized isomers, 269 and 270, were obtained in the ratio 2 :1. The minor isomer 270 displayed spectral data identical to that shown by the imino-ether derived by the action of trimethyloxonium tetrafluoroborate on synthetic 21-0x0-gelsemine (233), which had been prepared by oxidation of natural gelsemine (118). The imino-ether (270) can be converted to gelsemine by hydrolysis to 21-0x0gelsemine (233), followed by selective reduction of the tertiary amide of

1. Gelserniurn ALKALOIDS

55

233 using DIBAL; therefore, the synthesis of 270 constitutes a formal total synthesis of gelsemine (Scheme 36). 2. Hiernstra and Speckamp Route A total synthesis of (?)-gelsemine (118) and (?)-21-oxo-gelsemine (233) was reported by a research group in the Netherlands (103). The synthetic route includes a crucial carbon-carbon bond formation between C-5 and C-16 (gelsemine numbering) by a stereospecific intramolecular ring closure

+ 262

260

+ OMe

268

H V

vi, vii, viii

natural gelsemine (118)

233

Reagents: i. LDA,ii. hu, MeCN, Pyrex; iii. HC1, HzO; iv. Me30BF4; v. DIBALH;vi. DEAD; vii. MeOH; viii. Cr03. SCHEME 36.

56

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

of an N-acyliminium ion with a silyl enol ether to afford a key tricyclic

intermediate 283. The synthesis started with Diels-Alder cycloaddition of (E)-3,5-hexadien-l-01(271)with N-methylmaleimide (272)to give the pure endo adduct 273 as a crystalline solid in 95% yield. Acid-assisted partial reduction of imide 273 with NaBH4 (104), immediately followed by ethanolysis, furnished a complex mixture of products, which mainly contained the desired ethoxy lactam 274 and tricycle 275 (ratio 274275 ca. 70 :30) (105). The crude mixture was subjected to oxidation with Cr03/pyridine in CH2C12. Aldehyde 276 was readily obtained pure from the resulting mixture by column chromatography in 44% overall yield from 273. Treatment of 276 with triisopropylsilyl (TIPS) triflate in the presence of Et3N in CH2C12gave an inseparable mixture of a 70/30 ratio of (E)-277and (2)-277. The next task was the introduction of a vinyl group at the angular position. Lactam 277 was converted into its lithium enolate, which was quenched with 2-(phenylse1eno)ethanalto give a mixture of aldol products. The crude mixture was directly converted into the vinyl compound 278 [ ( E ): ( Z ) , ca. 70 :301 in 70% yield by treatment with MsCl and Et3N in CH2C12.Next, stereochemical aspects of the Lewis-acid-assisted intramolecular reaction of silyl enol ether with N-acyliminium ion were examined using a model compound 278. As a result, BF3 * Et20 was a suitable Lewis acid for this reaction, and the N-acyliminium cyclization appears to be highly stereospecific, i.e., (E)-silyl enol ether 278 gives the aldehyde 281 probably via a stable chair transition-state 280 and (Z)-silyl enol ether 278 gives mainly its C-5 isomer 282. The N-acyliminium cyclization of the E/Z (3/1) mixture of 279 with BF3 - Et20 in CH2C12at 10°C gave a separable 3/1 mixture of epimeric aldehydes, 283 and 284. After chromatography, aldehyde 283 was reduced with sodium borohydride to give the tricyclic alcohol 285 as a crystalline solid. Treatment of 285 with iodine in the presence of Na2C03 furnished the tetracycle 286, whose structure was determined by singlecrystal X-ray analysis (Scheme 37). Construction of the spiro-oxindole moiety at the C-7 position (gelsemine numbering) in 285 was performed using the intramolecular Heck reaction developed by Overman and co-workers (vide infra). Namely, the alcohol in 285 was first protected as a thexyldimethylsilyl (TDS) ether and then subjected to an allylic oxidation with the complex derived from chromium trioxide and 3,5-dimethylpyrazole. This afforded enone 287 in moderate yield along with a minor by-product, the isomeric enone resulting from an allylic rearrangement. Enone 287 was reduced in a 1,4-selective manner with L-selectride, and subsequent in situ trapping of the resultant lithium enolate with N-phenyltrifluoromethanesulfonimidefurnished the enol triflate 288. The triflate 288 was exposed to palladium-catalyzed carbonylation conditions in the presence of 2-bromoaniline to give the anilide 289,

1. Gelsemium ALKALOIDS

57

+ . I

he

he

273

Me

R:CH2CH20H 274 275 R:CHzCHO 276 R: CH=CHOS~(~-PK-)~ 277 (E+z)

iv

c

272

he

(i-Pr),SiO I

R:CH2Ph 281 R: CHzCH2 283

OSi(i-Pr),

280 Me

R I

Ph\

R:CHzPh 278 R: CH=CH2 279 (i-Pr),SiO

OHC

R:CHzPh 282 R CH=CH2 284

283-

vii

viii

0

0

OH H

285

286

Reagents: i. PhMe, reflux; ii. NaBH4, EtOH, cat. H2S04;iii. 6 N H2S04, EtOH; iv. Cr03, pyridine; v. TIPSOTf, Et3N, CH2C12;vi. BF3*Et20,CH2C12; vii, NaBH,, EtOH; viii. 12, Na2C03, MeCN.

SCHEME31.

which was then protected as N-trimethylsilylethoxymethyl(SEM) derivative to give 290. Cyclization of 290 under standard Heck arylation conditions [Pd(OAc)*, PPh3, Et3N, acetonitrile, reflux] gave a single spiro-oxindole product possessing the opposite spiro stereochemistry to that of natural gelsemine

58

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

(118). However, reaction of 290 under modified Heck cyclization conditions newly developed by Overman (vide infra) gave spiro-oxindole 291 in 60% overall yield after removal of the TDS protecting group. In addition, the epimeric spiro-oxindole was obtained in 30% yield. Next, formation of the remaining tetrahydropyran ring was examined. Attempts at iodoetherification or bromoetherification of 291 failed to give the required tetrahydropyran, presumably because of steric crowding of the cyclohexene double bond. Formation of the tetrahydropyran ring was finally achieved by exposure of spiro-oxindole 291 to the complex formed from mercury( 11) triflate and N,N-dimethylaniline. Reduction of the resultant organomercurial compound with alkaline sodium borohydride afforded the SEM-protected 21oxogelsemine (292). Treatment of 292 with tetrabutylammonium fluoride in THF gave 21-0x0-gelsemine (233). Finally, selective reduction of the lactam moiety was achieved by reaction of 233 with aluminum hydride in THF to give racemic gelsemine (118) (Scheme 38). The same authors have developed the synthesis of the enantiomerically pure key intermediate (301) from (R)-malic acid, as follows (206). (S)3-Acetoxysuccinimide (293), readily prepared from (S)-malic acid, was regioselectively reduced with lithium borohydride in THF at -20°C. Isopropanolysis of the resulting aminoacetal function gave 294 as a 1 :4 cishrans mixture. The hydroxy function was acylated to give the trans lactam 295 as the sole product. N-Acetylation of 295 proceeded smoothly, accompanied by elimination of the trichloroacetoxy group, to give the enantiomerically pure dienophile 2%. The Diels-Alder reaction of (R)-2% with the diene 297 proceeded with high endo- and regioselectivity to give the adduct 298. The acetyl function was easily removed from 298 through treatment with dimethylamine. Lactam 299 was then methylated with sodium hydride and methyl iodide, followed by an isopropoxy/ethoxy exchange to give the lactam 300.Finally, the ester group in 300 was reduced with lithium borohydride in the presence of LiBEt3H to give the alcohol 301 in an enantiomerically pure form. The author claimed that the availability of the enantiopure alcohol 301 should allow the synthesis of the natural alkaloids (Scheme 39). 3. Hart Route

The total synthesis of dl-21-0x0-gelseminereported by Hart and collaborators (207)features two free radical cyclizations (C-5-C-16 bond formation in gelsemine) to construct both a tricyclic substructure 314 in the terpene part of gelsemine and the spiro-oxindole moiety. The synthesis was initiated with the Diels-Alder reaction between N-methylmaleimide and the diene 302, followed by treatment of the crude cycloadduct with 2,2-dimethyl-1,3propanediol and a catalytic amount of p-toluenesulfonic acid, to give the perhydro-isoindole 304 in 43% yield. By application of the Grieco dehydra-

1. Gelsemiurn ALKALOIDS

59

iv

i, ii

-0

0 OH

H

H

285

H

287

288

-

vi,vii

0

0

viii, ix

o J j & ~ Me H

H

291 289 R=H 290 R=SEM

3.

H ..

Gelsemine (118)

1.

292

H

21-Oxogelsemine (233)

Reagents : i. TDSCl, imidazole, DMF; ii. Cr03, 3,5-dimethylpyrazole; iii. L-selectride, THF then Tf2NPh; iv. Pd(OA& PPh3, Et3N, CO, 2-bromoaniline, DMF; v. NaH, SEMC1, T H F vi. Pdz(dba)s, Et3N, PhMe, reflux; vii. n-ByNF, T H F viii. HgO, Tf20 N,N-dimethylaniline, MeNO,; ix, NaBH4, NaOH, CH2C12,EtOH; x. n-BudNF, T H F xi. AlH3, THF. SCHEME 38.

tion sequence to the primary alcohol 304, the olefin 305 was obtained in 79% yield, and reduction of the imide with N aBK gave carbinol lactam 306. Because conversion of 306 to 307 with acidic ethanol used in the early studies (108) was capricious as a result of problems associated with ketal hydrolysis, this process was improved using the combination of NaI and ethyl iodide. Alkylation of the lithium enolate of 307 with benzyl chloromethyl ether proceeded smoothly to give 308 in 95% yield. Ozonolysis of 308, in place of the Johnson-Lemieux conditions employed earlier (108), followed by reductive workup with dimethyl sulfide, reproducibly gave crystalline 309 in good yield. Wittig olefination of 309 gave 310, and ethoxythiophenoxy exchange afforded 311 in 65% overall yield. Compound 311

60

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

293

294

296

W

W

2

1

E

t

297 iv

EK)'"

Me (+)-SO1

Rl

vi

C

R1=H, R2-i-Pr 299 R1=Me, &=Et 300

298

Reagents: i. LiBH4, THF then &Sod, i-PrOH; ii. (C13CCO)2,DMAP, EbO; iii. Ac20, pyridine, D W , iv. PhMe, reflux, 3 d; v. Me2NH, DMF; vi. NaH, DMF, Me1 then HaSO4, EtOH; vii, LiBH4, cat. LiEbBH, THF/Et20. SCHEME 39.

was exposed to free-radical cyclization conditions using tri-n-butyltin hydride and AIBN in refluxing benzene to give the gelsemine substructure tricycle 314 in 61% yield (Scheme 40). The next task was introduction of the oxindole moiety at C-4 (C-7in gelsemine numbering). This was also accomplished by free-radical cyclization of appropriate derivatives of vinylogous carbamic acid. From the model experiments (109) using a relatively simple compound, o-bromoN-acryloylanilide derivative, it was found that a variant of the Jones procedure (110,111)was a useful method for the preparation of spiro-oxindoles. Treatment of 314 with phenylmagnesium bromide, alkylation of the resulting tertiary alcohol 315 with iodomethane, and deblocking of ketal 316 using p-toluenesulfonic acid in acetone then gave the ketone 317 in 81% overall yield. Acylation of ketone 317 using NaH, a catalytic amount of potassium hydride, and o-bromophenyl isocyanate gave 318. Free radical cyclizations of several derivatives of 318 were examined, and it was eventually determined that 319 provided the most useful stereochemical results. Thus, treatment of 319 with tri-n-butyltin hydride under photochemical conditions gave oxindole 320 in 40% yield, along with 15%of 321 and 10% of 322. Alcohols derived from structures of the type 321 and 322 were

1. Gelsemium ALKALOIDS

iv i, ii

RCH&H2OH c 304 305 R CH=CH2

vi 306 R1: CH=CH2, &: H.Rg: H vii ' 3 0 , R1: CH=CH2, I(1: H.Rg: Et

viii

302

1

g?q?+

-

'....

xiii

A

c

R1:CH=CH2,I(1: CH20Bn. Rg: Et cc308 309 R1:CHO, &: CH20Bn, Rg: Et

LOTHP

P"

xii

61

J

314 R=C02Et 316 R=C(OH)Ph2 316 R=C(OMe)Ph2

xi

310 L O E t 311 X=SPh)'

Reagents: i. N-methylmaleimide, PhMe, & ii. Dowex-50 (H*h iii. 2,2-dimethylpropane-l,3-diol, p-TsOH; iv. o-N02C,$I&CN, n-Bu3P then H2Oz; v. NaBH4, MeOH; vi. NaH, EtI, THF; vii. IDA, ClCH20Bn; viii. 03,MeOH then Me#; ix. (Ph)3P=CHCOZEt; x.PhSH, pTsOH; xi.n-Bu$%H, AIBN (cat.), PhH, A; xii. PhMgBr, THF; xiii. NaH, MeI, DMF. SCHEME 40.

isomerized to oxindoles having the desired spiro stereochemistry via a retroaldol-aldol sequence. The synthesis of 21-0x0-gelseminefrom 320 was accomplished as follows. Treatment of 320 with p-toluenesulfonic acid in CH2C12followed by the addition of methanol to the reaction mixture gave olefin 323 in 90% yield. Ozonolysis of the double bond gave aldehyde 324 in 65% yield. Treatment of 324 with hydrochloric acid in aqueous DME at 48°C accomplished acetate hydrolysis and isomerization of the aldehyde to afford a mixture of diastereomeric hemiacetals 325 in 64% yield. Reduction of this mixture with triethylsilane-trifluoroacetic acid gave 326, and removal of the benzyl protecting group with BBr3 afforded the alcohol

62

HIROMITSU TAKAYAMA A N D SHIN-ICHIRO SAKAI

327, whose structure was confirmed by X-ray crystallographic analysis. Oxidation of 327 using Dess-Martin periodinane gave aldehyde 328 in 71% yield. Finally, methylenation of 328 using bis(cyclopentadieny1)dimethyltitaniurn afforded dl-21-0x0-gelsemine (233) in 75% yield (Scheme 41). An-

317

iii

<

318 R=H 319 R=Ac

X

viii

ix

C 325 X=OH,R=CH20Bn

c

df-2l-Oxogelsemine (233)

326 X=H,R=CH20Bn 327 X=H,R=CH20H 328 X=H, R=CHO

SCHEME41.

1. Gelsemium ALKALOIDS

63

other model study for the synthesis of gelsemine was also reported by the same group (112). 4. Overman Route

The synthetic approach to the total synthesis of gelsemine by Overman and co-workers involves several original strategies, whose key steps are (i) preparation of cis-hydroisoquinoline by base-catalyzed hydroxy-azaCope rearrangement, (ii) intramolecular Mannich cyclization (C-5-C-6 bond formation in gelsemine) of an N-acyliminium ion to form the azatricycle, and (iii) palladium-catalyzed intramolecular alkene arylation to elaborate the spiro-oxindole. The palladium-catalyzed cyclization was further extended to the development of catalytic asymmetric synthesis of the spiro-oxindole system (vide infra). The synthetic studies have been initiated with the preparation of endo-bicyclo[2.2.2]octenyl amines, which could be expected to transform into the azatricyclo[4.4.0.02.s]decane substructure of gelsemine (113). The A1C13-catalyzedDiels-Alder reaction of l-alkoxy-1,3-cyclohexadienes(329 and 330) with methyl acrylate gave the cycloadduct (331 and 332) in high stereoselectivity (endo:exo = 8-10 : 1). The terminal vinyl substituent, destined for the angular C-20 position of gelsemine, was introduced by Se02 oxidation of 332 followed by Wittig methylenation of the resulting enal to provide 337. In a model series, rearrangement of the formaldiminium ion derived from 335 was initially examined. The intermediate did not undergo [3,3]-sigmatropic rearrangement but underwent Mannich cyclization at the proximal terminus of the alkene double bond to afford ultimately the 10-methylene4-azatricyclo[4.3.1.03~7]decane336. The desired [3,3]-sigmatropicaza-Cope rearrangement was actually accomplished in an electron excessive system by treatment of cyanomethylamine (339 or 340) with potassium hydride at room temperature. In the case of 339, quenching this rearrangement with excess HCN provided the cis-hydroisoquinoline 341 in good yield, whose structure was confirmed by single X-ray crystallographic analysis of the benzamide derivative. Alternatively, imine enolate was acylated by quenching with excess of methyl chloroformate to afford the bicyclic keto enecarbamate 342 (Scheme 42). Next, intramolecular Mannich cyclization using 342 to form the carboncarbon bond between C-5 and C-6 (gelsemine numbering) was examined (114). Simple heating of 342 in refluxing formic acid for 4-6 h cleanly effected the desired transformation and provided a single tricyclic product 344 in 65% yield. To introduce functionality at carbon 16, 342 was treated with one equivalent of Br2 in the presence of 1,2,2,6,6-pentamethylpiperidine to give the P-bromo enecarbamate 343. Cyclization of this bromide with trifluoroacetic acid (reflux, 8-10 h) proceeded regioselectively at C-6 position via the preferential thermodynamic N-acyliminium ion

64

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

OSTlP

methyl acrylate 331 f i C R=Me lzM?

Me

&C02Me 337

329 R=Me 330 R=Si(i-Pr)3

332 R=Si(i-Pr)3

ii, viii, ix

ii, n/

J

iv

(

NHCHzCN

333 R=Me, R'=C02Et 334 R=Si(i-P& R=C02Et 339 R: cH=cH2 335 R=Me, R=Me ",";u 340 R: Me I

338

I xiii

t k 341

342

Reagents: i. AlC13, CH2C12; ii. KOH, aq EtOH; iii. (COC112, CHzCl2 then NaN3, aq acetone; EtOH 4 iv. AlH3, THF; v. Bu,NF, THF then KOH aq MeOH; vi. SeO2, dioxane; vii. Ph3P=CH2, THF; viii. (COClk CHzCl2 then NaN3, aq acetone; PhMe 4 ix. KOH, aq dioxane; x. 1.1eq (CH20)n,0.9 eq RSOsH, MeCN, 100 OC; xi. (CH20)n, HC1-KCN, THF; xii. ClCH,CN, 2 mo1%Bu4NI, i-Pr2NET. THF then Bu4NF, THF; xiii. excess KH, THF, 23 "C; HCN, KCN, H2O; xv. C1CO2Me,pyridine then KOH, aq MeOH

+.

intermediate to afford the tricyclic product 345 in 85% yield. The structure of 345 was confirmed by X-ray analysis of the ethylene ketal derivative. The next stage of the gelsemine synthesis involves the elaboration of the spiro-oxindole at the carbonyl carbon of 345. To achieve this aim, a new synthetic method by intramolecular palladium-catalyzed alkene arylation

1. Gelsemium

65

ALKALOIDS

(Heck reaction) was developed (225). The cyclization substrate, acrylamide 347, was best prepared by conversion to the enol triflate 346 followed by carbonylation in the presence of 2-bromoaniline. After N-methylation of 347, the bromide was exposed to Heck conditions to provide the stereoisomeric pentacyclic products 348 and 349 (1.5-2.0 : 1)in 81% yield. That the major isomer 348 has the desired configuration at the spiro center was established by 'H-NMR NOE experiments (Scheme 43). Overman et al. have further developed a new synthesis of the spirooxindole system utilizing a benzyne intermediate (226). Treatment of 3(silyloxy)acryloyl-2'-(or 3'-)haloanilide with LDA followed by hydrolysis gave 3-acyl-3-alkyloxindoles. The reaction conditions in the Heck-type cyclization used in the spirooxindole construction (347 to 348) was further examined, leading to the observation that from the bromide 350 either oxindole stereoisomer 348

L

342 R=H 343 R=Br

344 R=H 345 R=Br

1

ii

iii __c

Ma&-N' Br

Br

346

341

R

Br'

348

Reagents: i. formic acid, reflux or TFA reflux; ii. Br2, 1,2,2,6,6-pentamethylpiperidine; iii. LDA, PhNTf2, THF iv. 1 mol % Pd(PPh3)4,2-bromoaniline, CO, DMF, 80 "C; v. NaH, RX,THF; vi. 10-20 mol 46 Pd(PPh3)4,MeCN, Et3N,82 "C.

SCHEME43.

66

HIROMITSU TAKAYAMA AND SHIN-ICHIRO S A W 1

or 349 can be formed with high selectivity if the Heck cyclization conditions are chosen properly (117). As shown earlier, the Heck reaction [lo-20% Pd(PPh3)4, Et3N, MeCN, 82"CI of the bromide 350 produced the spiro isomers 348 and 349 in the ratio of 1.5-2.0: 1. Cyclization catalyzed with tris(dibenzy1ideneacetone)dipalladium without added phosphine ligands afforded 348 and 349 in a ratio as high as 9: 1 (80-95% yield) when the intramolecular insertion was carried out in the weakly coordinating solvent, toluene. Remarkably, cyclization of 350 conducted without phosphine ligands in the presence of silver salts such as Ag3P04 occurred with virtually complete selectivity to give the epimeric oxindole 349. The high selectivity in this case is attributed to coordination of the angular vinyl group in 352 during the insertion step (Scheme 44).

350 i /

Br'

\i

i

+

H

351

352

+

[346349=89111

[348349=3:97]

Reagents:

i. 10-20 mol % tris(dibenzy1ideneacetone)dipalladium [Pd2(dba),1, Et3N, PhMe, 110 "C;ii. 10-20 mol % Pd2(dbaI3,Ag3P04,THF, 66 "C.

SCHEME44.

1. Gelsemiurn ALKALOIDS

67

Enantioselective synthesis of spiro-oxindoles using a catalytic asymmetric Heck reaction was investigated by the same group (118). The synthesis of 3,3-spiro-oxindole 354 from the acryloyl 2'-iodoanilide 353 was chosen for initial studies. Enantioselectivities were highest and most reproducible with a catalyst prepared in polar solvents from (R)-BINAP, tris(dibenzylideneacetone)dipalladium,and Ag3P04.Under optimum conditions, (S)-( +)-354 was obtained in 71% enantiomeric excess and a chemical yield of 81%.The absolute configuration of (+)-354 was determined by X-ray crystallographic analysis of the bromide derivative. Remarkably, cyclizations carried out without any added HI scavenger, or in the presence of tertiary amines, proceeded with good enantioselectivity to form the R enantiomer of 354. Reactions conducted in the presence of the basic tertiary amine 1,2,2,6,6-pentamethylpiperidineoccurred slowly at 80°C and afforded (R)-(-)-354 in 77% yield and 66% ee. Thus, the silver- and aminepromoted cyclizations took place with opposite senses of enantioselection. The mechanistic insight of these reactions is discussed in the literature (Scheme 45). In the course of the synthetic study of gelsemine, Overman et al. reported (119) the new chemistry of reactions between an aziridinium ion and Na,Fe(C0)4. To elaborate the cyclopentanone derivative 358 utilizing the aziridinium ion intermediate 356, Na2Fe(COk (120) was chosen for the carbonylation. The nitrogen of the aziridine 355, which was prepared from

(S)-(+)-354 71%ee Me

L

O

(R)-(-)-354 66%ee

Reagents i. 5 % tris(dibenzy1ideneacetone)dipalladiurn [Pd2(dba)31,10%R-(+)-BINAP, 1-2 equiv Ag3P04, MeCONMe2,80 "C; ii. 10 % Pd2(dba)s,20% R-(+)-BINAP, 5 equiv 1,2,2,6,6-pentamethylpiperidine, MeCONMe2,80 "C. SCHEME 45.

68

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

348,was methylated with methyl tritlate; then, the resulting N-methylaziridinium ion 356 was treated with 5 eq of the dioxane complex of NazFe(C0)+ After being purged with CO, the reaction mixture was heated at 50°C under CO atmosphere and finally hydrolyzed with acetic acid to give a single carbonylation product 359 in 54% yield. The structure was first assumed by spectroscopic analysis of the product and its isomer, which was obtained by base treatment of the product via a retro-Michael fragmentation and intramolecular Michael cyclization (359, 360, 361, and 362), and finally determined by single-crystal X-ray analysis of a derivative 363. A plausible mechanism for the formation of 359 from the reaction of aziridinium ion with Na2Fe(CO), was discussed (Scheme 46). 5. Fleming Route

A tetracyclic caged ketone 382, which was a key intermediate for the total synthesis of gelsemine, was prepared by Fleming et al. (121) via several key steps involving a Diels-Alder reaction, a skeletal rearrangement reaction, and an intramolecular reaction between an allylsilane group and an acyliminium ion, by which bond formation between C-20 and C-21 in gelsemine was achieved. The synthetic study of gelsemine began with the Diels-Alder reaction of 1-tetrahydropyranyloxycyclohexa-1,3-diene (364) and P-nitroacrylate (365). By this condensation, a crystalline adduct 366 was obtained in 43% yield accompanied by the formation of 39% of the diastereomer 367. After some functional group modifications, the double bond in 368 was oxidized with p-nitroperbenzoic acid to give a more hindered epoxide 370. When the epoxide 370 was treated with aluminum chloride, the unfunctionalized bridge migrated to give the ketone 372. Using magnesium bromide in place of aluminum chloride, the major product was the bromohydrin 373, which was further converted to ketone 374 by migration of the functionalized bridge antiperiplanar to the bromine atom in 373. Based on this successful information, a tricyclic ketal376 was synthesized from the cycloadduct 366 in 11 steps. These steps include bromination of the ketone 375, protection of the alcohol group, base-catalyzed ether formation, and acid-catalyzed removal of the tetrahydropyranyl group on the secondary alcohol. The structure of the key intermediate 376 was confirmed by single-crystal X-ray analysis of the methylamine derivative. Next, formation of an additional pyrrolidine ring in 376 to construct a tetracyclic compound having a vinyl group at the quaternary center was investigated. The alcohol function in 376 was oxidized to a ketone followed by reaction of vinylmagnesium bromide to give the tertiary allylic alcohol 378. The primary allylic chloride 379 prepared from 378 by treatment with SOC12 was converted into the trimethylvinylsilane derivative 380. The allylsilane

1. Gelsemium ALKALOIDS

69

SEM

SEM

-

-

ii

i

Br'

SEM

355

348

356 iii

-

SEM

/

i SEM

iv

357 SEM

0

362 R=SEM 363 R=H

I

)'

358

i Gelsemine (118)

Reagents:

i. NaCN (100 equiv), Me2S0, 140 "C;ii. MeOTf, CH,Cl,; iii. d i d u r n tetracarbonylferrate-dioxanecomplex, N-methyl-2-pyrrolidone,CO, 50 "C; iv. 1 N NaOH, acetone; v. 6 N HCl. SCHEME46.

380 reacted intramolecularly with the acyliminium ion intermediate 381, which was prepared from 380 by reaction with trioxane in formic acid, to give the desired tetracyclic product 382 in 85% yield (Scheme 47). To accomplish the synthesis of gelsemine from the ketone 382, four new synthetic methods for the construction of a spiro-oxindole from the sterically hindered ketone were developed as follows. Route I (122): The aryl group was attached to the ketones 383 using the o-lithioformanilide salt 384,prepared by halogen-metal exchange between n-butyllithium and o-bromoformanilide, at -105°C. The alcohols 385

+

1

jNo2 i

MeO,

365

364

/ iii, iv

/ii,

366

368 R=COzMe 370 R=COzMe RECHzOAc 371 R=CHzOAc

xi, xii

MazC

J

( 369

-

CQMe

367

372

Et02CHN-

374 R=COZMe 375 R=CHzOAc

373

xiii

EtOZCHN-

EQCHN-

377

EQCHN-

379

378

L

382 I

.

381

380

Reagents: i. benzene, rt; ii. AVHg, MeOH; iii. EtO2CC1, Et3N; iv. HCl, H20; v. LiAlH, then AcCl, Et3N then PPTS, EtOH; vi. p-nitroperbenzoic acid, CHSC12; vii. AlC1,; viii. MgBr2; ix. PhNMe3Br3;x. DHP, H*; xi. K2C03, H2O; xii. PyH'TsO-, EtOH; xiii. PCC, Al2O3; xiv. vinylmagnesium bromide; xv. SOC12; xvi. (Me3Si)2CuLi;xvii. (CH2O)3, HC02H.

1. Gelsemiurn ALKALOIDS

71

reacted with cyanide ion in DMF, without need for acid catalysis, to give the aminoindolenines 388. With adamantanone as the starting material, 386 could be isolated directly by crystallization. Either the aminonitrile 386 or the aminoindolenines 388 gave the corresponding oxindoles in high yield when their hydrochlorides were heated in water. The overall yield from adamantanone was 79%; however, when the ketones were enolizable, the aminoalkenes 387 were major by-products in the key step. Route 2 (222):The epoxides 391, which were prepared in two steps from o-fluorobenzyl bromide and the ketones 383, were subjected to rearrangement in acid. In the cyclohexanone series, aryl migration was the major pathway (3 : l), but, in the norbornyl series, a hydride shift was the major pathway (1.5 :1). The aldehydes 392 were converted into their amides 394, which were then cyclized to the oxindoles 389. The stereochemical implications of the reactions in routes 1 and 2 were discussed in the literature. Route 1 is appropriate for the synthesis of an oxindole such as 389d-1 in which the carbonyl carbon is to be attached to the less hindered face of a diastereotopic ketone group, and route 2 is appropriate when the aryl ring is to be attached to the less hindered face, such as the oxindole (389d-2) (Scheme 48). Route 3 (223):A quaternary carbon atom can be set up by irradiation of the N-methylaniline enamine 395 of adamantane-2-carbaldehyde, giving the indoline 3% in 34% yield. Oxidation of this product gave the spirooxindole 403 in low yield, together with the N-formylindoline 397 (42% yield). Route # (223): A high-yielding, eight-step conversion of adamantanone 383a into the corresponding spiro-oxindole was developed. Conjugate addition of triphenylaluminum to the a,P-unsaturated nitro compound 398 gave the nitroalkane 399 in 91% yield. Dehydration of 399 using successively sodium methoxide and acetyl chloride gave the nitrile oxide 400 in 66% yield. Hydration of the nitrile oxide gave a hydroxamic acid derivative in 56% yield, which was then methylated to afford the methyl hydroxamate 401. By applying Kikugawa’s method (22#,225), the N-methoxyoxindole was prepared from 401. Namely, 401 was first converted to the N-chlorohydroxamate with t-BuOC1, which was then exposed to Lewis-acid-catalyzedcyclization with zinc acetate to give the N-methoxyoxindole 402 in 85%yield. Finally, reduction of the N - 0 bond with sodium amalgam gave the oxindole 403 in quantitative yield. The overall yield from adamantanone was 47% (Scheme 49). 6. Stork Route

The stereoselective construction of a significant portion, namely 411, of the gelsemine structure was reported by Stork and co-workers (226).The

72

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

4cJ

0

Route 1

b: benzophenone c: cyclohexanone d norbornanone

aLi 384

Route

385 a: adamantanone

NLicHo/

h3

0

iii

396

+

1

387

389 a-d

v, vi, vii

393

H

389d-2

3891-1

Reagents: i. NaCN, DMF, 80 "C; ii. HC1 then HzO, 165 "C; iii. mCPBA, iv AlC13 or TFA, v. CrO,; vi. SOCla; vii. NH,; viii. KH, diglyme, reflux. SCHEME 48.

radical cyclization of the bromide 404 with tri-n-butylstannanelAIBN in refluxing benzene gave the bicyclic ester 405 in 95% yield. This was easily converted to the unsaturated ester 406 via selenenylation (LDNPhSeSePh) to a mixture of diastereomers, followed by the usual oxidation-

1. Celsemium ALKALOIDS

73

g;,". 395

I

t

Route 3

40 1

iv

402

\ xiv

Me V

396

397

403

Reagents: i. Me2SO=CH2; ii. BF3*Et20;iii. PhNHMe; iv. hv; v. Mn02; vi. MeN02, ethylenediamine, reflux; vii. Ph3Al. viii. NaOMe; ix. AcCl; x. H2S04; xi. Na2C03, MeI; xii. Bu'OCl; xiii. Z~(OAC)~; xiv. Na/Hg. SCHEME 49.

elimination (m-CPBA; Et,N) reaction sequence. This led to a mixture of tri- and tetrasubstituted isomeric a,@-unsaturated esters (98% yield) in which the desired isomer 406predominated in a ratio of 6 :1.Next, reduction of 406 with DIBALH gave the allylic alcohol, which was then esterified with phenylacetic acid using DCCIDMAP to give 408 in 95%yield. Hydrolysis of 408 to the corresponding hemiacetal (Amberlite-H+ in aq. THF) followed by esterification with trimethylacetyl chloride gave 409 in 85% yield. The alkylation step to 410 could then be carried out by first forming the trimethylsilyl ketene acetal from 409, followed by cyclization using trimethylsilyl triflate to give the somewhat strained lactone 410 in 67% yield as a single isomer. The Claisen rearrangement for the completion of the desired skeleton was easily brought about via the enol silyl ether of lactone 410 (LDA, TMSCI, -78°C to room temperature), which thus gave the desired 411 in 96% yield. The stereochemistry of 411 was confirmed by the formation of a single bromolactone 412 (Scheme 50).

74

HIROMITSU TAKAYAMA AND SHIN-ICHIRO SAKAI

i

ii

OEt M 2 C

OEt

OEt

Br

405

404

Ill

406

R=Et 408 R=COCMe3 409

410

411

412

Reagent:

i. n-BuBSnH,cat. AIBN, benzene, reflux; ii. LDA, PhSeSePh, THF then mCPBA, EtsN, iii. DIBALH, ether; iv. phenylacetic acid, DCC, DMAP; v. Amberlite-H+,aq THF then Me3CCOCl, DMAP, Et3N, CH2C12; vi. LDA, TMSCl then TMSOTf; vii. LDA, TMSCl, -78 "C to rt;viii. Br2, CH2C12,-78 "C. SCHEME 50.

Acknowledgment

The authors are grateful to Dr. Norio Aimi and Dr. Mariko Kitajima,Faculty of Pharmaceutical Sciences, Chiba University, for helpful suggestions and discussions concerning the preparation of the manuscript.

Note Added in Proof

After completing the manuscript, the following relevant papers were published:

1. Gelsemium ALKALOIDS

75

1. Isolation and structure elucidation of a new alkaloid, l9a-hydroxygelsamydine, from Gelsemium eleguns was reported by Cordell and colleagues: L. Z. Lin, S. F. Hu, and G. A. Cordell, Phytochemistry 43,723 (1996). 2. A new and elegant total synthesis of (5)-gelsemine, which features a stereoselective construction of the bicyclo[3.2.1] framework by means of a divinylcyclopropane-cycloheptadiene rearrangement, was accomplished by Fukuyama: T. Fukuyama and G. Liu, J. Am. Chem. SOC. 118,7426 (1996).

References

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70. M. Wichtl, A. Nikiforov, S. Sponer, and K. Jentzsch, Monatsh. Chem. 104,87 (1973). 71. M. Wichtl, A. Nikiforov, G. Schulz, S. Sponer, and K. Jentzsch, Monatsh. Chem. 104, 99 (1W3). 72. J. S . Yan and Y. W. Chen, Acta Pharm. Sinica 19,437 (1984). 73. L. Z. Lin, G. A. Cordell, C. Z. Ni, and J. Clardy, Phytochemistry 29,3013 (1990). 74. D. Ponglux, S. Wongseripipatana, H. Takayama, K. Ogata, N. Aimi, and S. Sakai, Tetrahedron Lett. 29,5395 (1988). 75. L. Z. Lin, G. A. Cordell, C. Z. Ni, and J. Clardy, J. Org. Chem. 54,3199 (1989). 76. H. Takayama, M. Horigome, N. Aimi, and S. Sakai, Tetrahedron Lett. 31,1287 (1990). 77. H. Takayama, H. Odaka, N. Aimi, and S. Sakai, Tetrahedron Lett. 31,5483 (1990). 78. H. Takayama, Y. Morohoshi, M. Kitajima, N. Aimi, S. Wongseripipatana, D. Ponglux, and S. Sakai, Nut. Prod. Lett. 5, 15 (1994). 79. H. Takayama, Y. Tominaga, M. Kitajima, N. Aimi, and S. Sakai, J Org. Chem. 59, 4381 (1994). 80. T. Tsunoda, Y. Yamamiya, and S. Ito, Tetrahedron Lett. 34, 1639 (1993). 81. T. Tsunoda, J. Otsuka, Y. Yamamiya, and S. Ito, Chemistry Lett., 539 (1994). 82. T. Q. Chou, Chin. J. Physiof. 5, 131 (1931). 83. M. Onanga and F. Khuong-Huu, C. R. Acad. Sci. Paris, Ser. C 291,191 (1980). 84. M. Kitajima, H. Takayama, and S . Sakai, J. Chem. Soc., Perkin Trans. I, 1573 (1994). 85. H. Takayama, M. Kitajima, and S. Sakai, J. Org. Chem. 57,4583 (1992). 86. H. Takayama, M. Kitajima, and S. Sakai, Tetrahedron 50, 11813 (1994). 87. A. S. Kende, M. J. Luzzio, and J. S. Mendoza, J. Org. Chem. 55, 918 (1990). 88. N. K. Hamer, J. Chem. Soc., Chem. Commun., 102 (1990). 89. F. M. Lovell, R. Pepinsky, and A. J. C. Wilson, Tetrahedron Lett., 1 (1959). 90. H. Conroy and J. K. Chakrabarti, Tetrahedron Lett., 6, (1959). 91. W. H. Orgell, Lloydia 26,36 (1963). 92. E. Wenkert and J. H. Hansen, Iowa State J. Science 34,163 (1959). 93. E. Wenkert, C. J. Chang, A. 0.Clouse, and D. W. Cochran, J . Chem. SOC.D,961 (1970). 94. E. Wenkert, C. J. Chang, D. W. Cochran, and R. Pellicciari, Experientia 28,377 (1972). 95. Y. Schun and G. A. Cordell, J. Nut. Prod. 48,969 (1985). 96. A. Nikiforov, J. Latzel, K. Varmuza, and M. Wichtl, Monatsh. Chem. 105,1292 (1974). 97. Y. Schun, G. A. Cordell, and M. Garland, J. Nut. Prod. 49,483 (1986). 98. L. Z. Lin, S. Yeh, G. A. Cordell, C. Z. Ni, and J. Clardy, Phyrochemistry 30,679 (1991). 99. Z. Sheikh, R. Steel, A. S. Tasker, and A. P. Johnson, J. Chem. Soc., Chem. Commun., 763 (1994). 100. J. K. Dutton, R. W. Steel, A. S. Tasker, V. Popsavin, and A. P. Johnson, J. Chem. SOC., Chem. Commun., 765 (1994). 101. J. E. Saxton, Nat. Prod. Repts. 9,393 (1992). 102. P. A. Wender and C. B. Cooper, Tetrahedron 42,2985 (1986). 103. N. J. Newcombe, F. Ya, R. J. Vijn, H. Hiemstra, and W. N. Speckamp, J. Chem. Soc., Chem. Commun., 767 (1994). 104. R. J. Vijn, H. Hiemstra, J. J. Kok, M. Knotter, and W. N. Speckamp, Tetrahedron 43, 5019 (1987). 105. H. Heimstra, R. J. Vijn, and W. N. Speckamp, J. Org. Chem. 53,3882 (1988). 106. W. J. Koot, H. Hiemstra, and W. N. Speckamp, J. Org. Chem. 57,1059 (1992). 107. D. Kuzmich, S. C. Wu,D. C. Ha, C. S. Lee, S. Ramesh, S. Atarashi, J. K. Choi, and D. J. Hart, J. Am. Chem. SOC.116,6943 (1994). 108. J. K. Choi, D. C. Ha, D. J. Hart, C. S. Lee, S. Ramesh, and S. Wu, J . Org. Chem 54, 279 (1989). 109. D. J. Hart and S. C. Wu, Tetrahedron Lett. 32,4099 (1991).

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ALKALOIDS CONTAINING QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS TURAN OZTURK* Chemical Laboratory University of Kent at Canterbury Canterbury, Kent, U.K.

I. Introduction

....................................................................................... .........................................

11. Alkaloids Containing a Quinolinequinone Unit

A. Aza-anthraquinone Type Alkaloids . B. Diaza-anthraquinone Type Alkaloids C. Phenanthridine Type Alkaloids ...... D. Quinolinequinone Type Alkaloids ... 111. Alkaloids Containing a Quinolinequinoneimine Unit ................................ A. Pyridoacridine Type Alkaloids ........................................................ B. PyrroloquinolinequinoneimineType Alkaloids .................................... IV. Summary ......................................................................................... References ...............................................................................

79 80

143 143 195 212

I. Introduction Since the first isolation of the alkaloids containing quinolinequinone and quinolinequinoneimine units, phomazarin and calliactine, respectively, more than 30 alkaloids containing these units have been reported. Although the first two alkaloids were isolated in 1940, the remainder were isolated after 1980, except for streptonigrin and isophomazarin, which were isolated in 1959 and 1979, respectively. Alkaloids that possess quinolinequinone and quinolinequinoneimine units (1) have been classified in the literature as members of the azaanthraquinone, diaza-anthraquinone, quinolinequinone, pyridoacridine, and pyrroloquinolinequinoneimine type alkaloids, although they all have common subunits (l),quinolinequinone and quinolinequinoneimine. Most of the alkaloids isolated in recent years with such units fall into the pyrido-

* Present address: TUBITAK-MAM, Marmara Research Center, Department of Chemistry, P. 0. Box 21,41470 Gebze-Kocaeli, Turkey. THE ALKALOIDS, VOL. 49 0099-9598197 $25.00

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Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved.

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R

1

acridine type. Their bioactivity and challenging structures led to the appearance of a series of articles, including reviews (1-4). This chapter deals in depth with the isolation, structure elucidation, synthesis, biological activities, and biosynthesis of these alkaloids.

11. Alkaloids Containing a Quinolinequinone Unit

A. AZA-ANTHRAQUINONE TYPEALKALOIDS 1. Cleistopholine

Cleistopholine (2) was first isolated by Waterman and Muhammad in 1985 from the root bark of Cleistopholis patens (Annonaceae) collected in Ghana (5). A petrol (bp 40-50°C) extract of the bark was partitioned through acid-base (pH lo), and the basic fractions were separated by preparative TLC (silica gel; toluene-EtOAc-AcOH, 94 : 6 : 1) to give cleistopholine as a second fraction (0.006%). Two years later, in 1987, CavC et al. reported the isolation of cleistopholine (2) from the trunk bark of a rain-forest tree Meiogyne virguta (Annonaceae) collected on Mount Kinabalu, in Borneo (Sabah, Malaysia) (6). The basified bark (conc. NH3) was extracted with CH2C12,and the extract was subjected to column chromatography on silica gel using a CH2C12-MeOH gradient to afford cleistopholine, which was further purified by preparative TLC on silica gel (0.0009%). In 1989, Cortes et al. reported that they had isolated cleistopholine from

2

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81

the ripe fruit seeds of Annona cherimolia (Annonaceae) collected at Almunecar (Granada coast, Spain) (7). The finely ground seeds were first extracted with petroleum (50-70°C), and then the extract was continuously percolated with 95% ethanol. The dark brown viscous material from the ethanolic percolation was extracted with chloroform, and then the extract was chromatographed on silica gel eluting with chloroform, which was followed by chloroform-methanol mixtures. The fractions eluted with chloroform-methanol mixtures of 95 : 5 and 90 : 10 were extracted with 5% hydrochloric acid. The acid solution was basified with 5% NH40H and extracted with ether-chloroform (1:1).The extract was subjected to preparative TLC to yield cleistopholine (2) as the fourth fraction. a. Characterization. Although the structure of cleistopholine (2) was established by Waterman and Muhammad ( 5 ) on the basis of conventional spectroscopic methods, CavC et al. (6),with a sufficient amount of material, reported its full 13C, 2D 'H/13C-NMR, COSY, and nOe spectra. NOe of the methoxy protons on the neighboring C-10 carbonyl I3C resulted in a much stronger signal at 181.9 ppm than at 184.7 ppm, which indicated the closeness of the methyl group to C-10. Cleistopholine (2): C14H9N02;yellow glassy solid; mp 185-190°C; EIMS d z 223.06 (M+); UV A 250 (E 44,668), 263sh (E 11,220), 322 (E 6309) nm; IR Y 1695,1685,1600,1300, 980,710 cm-'; 'H-NMR (400 MHz, CDC13) 6 8.95 (lH, d, J = 4.8 Hz,H-2), 7.47 (lH, d, J = 4.8 Hz, H-3), 8.31 (lH, dd, J = 8.5, 2.2 Hz, H-5), 7.79 (2H, m, H-6, -7), 8.21 (lH, dd, J = 8.5, 2.2 Hz, H-8), 2.89 (3H, S, CH3); 13C-NMR (100.6 Hz, CDCl3) S 153.4 (C-2), 131.2 (C-3), 151.6 (C-4), 129.1 (C-4a), 127.4 (C-5), 134.2 (C-6), 134.6 (C-7), 127.2 (C-8), 127.1 (C-8a), 184.7 (C-9), 150.1 (C-9a), 181.9 (C-lo), 132.6 (C-lOa), 22.8 (CH3). b. Synthesis. Synthesis of cleistopholine (2) was achieved by Bracher in 1989 in a one-pot Diels-Alder cycloaddition of bromo-1,4-naphthoquinone (3) to the diene 4 (Scheme 1) (8).

3

4 SCHEME 1. Synthesis of cleistopholine (2) (8).

2

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TURAN OZTURK

In the same year, a rather long synthesis of cleistopholine was reported by Koyama et al. (9)(Scheme 2). a-Tetralone (5) was oxidatively acetylated with lead tetra-acetate to give 6, in which the ring carbonyl group was first protected with ethylene glycol. Oxidative deacetylation with pyridinium chlorochromate in the presence of sodium acetate yielded the ketoketal7. Condensation of 7 with 0-crotylhydroxylamine (8)gave the oxime 9, which, upon thermolysis at 160°C, afforded 4-methylbenzo[g]quinoline (10) in 8% yield, and 2-methylbenzo[g]quinoline (11)in 5% yield, together with

U

0

5

6

n

ii) pytidiniun cHorochromate 50 %

7

75 %

9

0

I

NH, 8

n

10

1

&->

5% 11

8%

Jones Reagent

60%

n

+ 60 % SM

Cleistopholine 2

SCHEME 2. Synthesis of cleistopholine (9).

9

83

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

recovered starting material (60%). Quinoline 10 was oxidized with Jones reagent to give the target compound cleistopholine (2). In 1988, Fillion el al. synthesized four analogs of cliestopholine using Diels-Alder cycloaddition reactions of the diene 4 to the naphthoquinones 12, 14, and 16 to give 13, 15, and 17 and 18 in 1:4 ratio, respectively (Scheme 3) (10). a

12

0

H3CO

+

4

acetonitrile

50 %

14

15

+ cH3coO

0

4

acetoritrile 50%

+$ R 2 0

16 17 R, = H

R2=CH3CO

18 R2=CH3CO R 2 = H 17 : 18 = 1 : 4

SCHEME 3. Synthesis of analogs of cleistopholine (10).

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TURAN OZTURK

Synthesis of the four analogs 460,468,470, and 474 of cleistopholine was reported during the synthesis of 3-methoxysampangine 449, and of the analogs of sampangine 448 by Clark et al. in 1992 (Schemes 53,55, and 56) (12) (see section on sampangines).

c. Biological Activity. Cortes et al. reported in their preliminary screening that cleistopholine (2) inhibited the growth of Staphylococcus aureus and Myobacterium phlei (7). During their studies of inhibitory effects of anthraquinones on Epstein-Barr virus activation, Konoshima and Kozuka reported that cleistopholine did not show significant inhibitory effect (12). Clark et al. also reported their in vitro findings on activities of cleistopholine and its analogs 468 and 474 against the yeasts Candida albicans NIH B311 and Cryprococcus neoformans ATCC 32264, the filamentous fungus Aspergillusfumigatus ATCC 26934, and the atypical mycobacterium M. intracellulare ATCC 23068 (21): d. Biosynthesis. C a d er al. suggested that cleistopholine (2) could be biosynthesized from aza-anthraquinone 20, which is an intermediate on the pathway to eupolauramine (480)suggested by Taylor (13) (Scheme 4). The

19

Cbistopholine 2

-

SCHEME 4. Biosynthetic pathway of cleistopholine (13).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

85

22

catecholic analog 19 of liriodenine may undergo an extradiol cleavage to give 20, in which the side chain could be degraded to afford the aldehyde 21, and reduction of 21 could easily lead to cleistopholine. 2. Dielsiquinone

Dielsiquinone (22) was isolated by Goulart ef al. from trunkwood of Guafferiudielsiana (Annonaceae) collected in the vicinity of Manaus, Amazona State, Brazil, in 1986 (14). The residue from the EtOH extract of the trunkwood was re-extracted with CHCl3 to yield a residue from which the HC1-insoluble fraction was chromatographed over silica gel, eluting with C6H6to afford dielsiquinone as the third fraction. It was further purified by chromatography over alumina (C6H6-EtOAc, 1 : 1) and successive washings with Et,O and EtOAc. a. Characterization. Dielsiquinone (22) (l-aza-3-methoxy-4-methyl-2oxo-l,2-dihydro-9,10-anthracenedione): Cl5H1,NO,; yellow greenish amorphous powder; mp 250-252°C (acetone); EIMS d z 270 (Mt + H), 269 (M+), 268 (M+-H); UV (EtOH) A 247 sh (E 11,748), 274 (E 18,620), 291 (E 17,782), 322sh (E 10,964) nm; UV (EtOH + 2.5 N NaOH) A 251 (E 16,982), 270 (E 16,595), 332 (E 15,488), 363sh (E 10,715) nm; UV (EtOH + NaOAc) A 250 (E 15,135) 273 (E 15,135), 324 (E 14,791), 361sh (E 10,715) nm; IR v

3280,2920,1665,1655,1590,1540,1480,1420,1420,1405,1320,1310,1285,

1270, 1230, 1200, 1120, 1070,1035, 1015, 970,800, 780,725 nm; 'H-NMR (60 MHz, CDCL-CD30D) S 7.5-7.8 (2H, rn) 8.0-8.3 (2H, m), 2.63 (s, CH3), 4.17 (s, OCH3). 3. Phomazarin

Phomazarin (23), an orange pigment, was isolated by Kogl et af. in 1940 along with cynodontin (24) from the mycelium of Phoma terresfris Hansen, the fungus responsible for pinkroot disease of onions and since renamed as Pyrenochaeta ferresfrisHansen (15-1 7). Cynodontin was shown to be an anthraquinone metabolite, and phomazarin was suggested, on the basis of degradation studies, to possess the l-aza-anthraquinone system (25)in which the orientation of the heterocyclic ring was not established. Later, in 1960, an attempt to reisolate phomazarin from a culture of P.

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TURAN OZTURK

23

24

terrestris Hansen, obtained from the Central bureau voor Schimmelcultures (Baarn) and grown by the method of Kogl, failed (28). The presence of one other pigment along with cynodontin was claimed, but no compound resembling phomazarin. In 1961, Birch et al., following the same procedure described by Kogl et al., identified both pigments 23 and 24. Depending on the experiments by which radioactivity from H14C02Na, 14MeC02Na, and MeI4CO2Na was incorporated into phomazarin, followed by analysis of degradation products, it was suggested that the heterocyclic unit had the orientation shown in 26 (29). In 1964, Birch et al. revised the structure and suggested a differently oriented phomazarin (27) depending on chemical and spectroscopic analysis (20). A method to make the aza-anthraquinone skeleton of phomazarin (27) was developed in 1967 (22). The structure and biosynthesis of phomazarin were comprehensively revised in the second half of the 1970s by Simpson et al. Their structure revision was carried out by spectroscopic and chemical analysis, and it was

26

27

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

28

87

29

suggested that phomazarin indeed had the structure 28 (22-24). Their modifications of the medium and method of culture described by Kogl et al. also prevented the failure of the fungus to produce consistent yields of phomazarin, or to produce cynodontin only. Isophomazarin (29)was also isolated as a third product of the culture. Incubation at 25°C in shake culture led to a good growth of thick mycelium, which was extracted with chloroform after treatment of the dried mycelium with acid. Chromatography of the chloroform extract first produced cynodontin (24) on elution with light petroleum. Further elution with chloroform produced a mixture of two compounds, which on precipitation and crystallization with methanol gave an adduct of phomazarin and methanol. Addition of water destroyed the adduct and precipitated phomazarin as second product. Isophomazarin was obtained, contaminated with phomazarin, from the remaining solution. Repeated crystallizations from glacial acetic acid produced deep red needles of isophomazarin (29)(23). a. Characterization. Tri-0-methylphomazarin methyl ester (30) was obtained with silver oxide and methyl iodide treatment in chloroform. Shorter reaction times at room temperature gave mixtures of the partially methylated products 31, 32, and 33. The IR spectrum of 31 showed no carbonyl absorption above 1685 cm-', whereas 32 showed an ester carbonyl at 1748 cm-*, indicating the presence of a hydroxy adjacent to the carboxyl group in phomazarin. Mild hydrolysis of 33 cleaved the ester and the labile pyridinoid methoxyl; re-esterification then gave 32. This established the relationship of 33 to 32,and in particular the presence of the free 8-hydroxy group in both compounds. Hydrolysis and decarboxylation of 30 gave 36. In this reaction a methyl ether was hydrolyzed as well, indicating the presence of a hydroxyl group ortho or para to an aromatic nitrogen. This was also established by replacement of the hydroxyl group by chlorine to give 39 on treatment of 36 with phosphorus oxychloride. The chlorine was then readily displaced by either hydroxide to give back 36 or methoxide to give 37. Hydrogenation of 39 over palladium charcoal gave 40 along with 41. Compound 40 did not react with ortho-phenylenediamine, even under forcing conditions, suggesting the presence of an anthraquinone rather than a phenylanthraquinone skele-

88

TURAN OZTURK

41

ton in phomazarin. The IR spectrum of 40 showed one carbonyl absorption band at 1673 cm-', whereas 41 showed two separate carbonyl bands at 1667 and 1614 cm-'. This suggested the presence of a vinylogous amide system that locates the nitrogen adjacent to the quinonoid ring. The remaining methoxy group in the heteroaromatic ring of 40 showed resistance to acid hydrolysis, and its failure to rearrange to an N-methylpyridone on heating with methyl iodide suggested its position as metu to the ring nitrogen. Thus, the hydroxyl and carboxy groups on the heterocyclic ring must occupy the 2- and 4-positions of phomazarin. The presence of an aromatic proton singlet in the 'H-NMR spectrum of 37 at 8.67 pprn indicated a proton adjacent to the heteroaromatic nitrogen. In "N-enriched 36, this proton was coupled to the ring nitrogen, 2J (15N-'H) 10 Hz. The 'H-NMR spectrum of 40 showed that the proton at 8.67 ppm is metu coupled (J = 3 Hz) to the proton at 7.88 ppm. All of these data suggested that part of phomazarin is 2-carboxy-3,4-dihydroxy-l-aza-anthraquinone (23). Contradictions of the 'H-NMR data obtained by Simpson et ul. (23) to the benzenoid ring suggested by Kogl et ul. (25-17) based on degradation of phomazarin were resolved by repetition of Kogl's chromic acid degradation of phomazarin triacetate (23).The initially obtained 42 was hydrolyzed and decarboxylated to give 43, which was subsequently converted to 44, identical with the synthetic sample prepared as shown in Scheme 5. Bromi-

89

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

nation of ortho-vanillin (45) gave 46,which was methylated to give 47, and then the aldehyde was converted to n-butyl by standard procedures. The lithium derivative of 48, formed with n-butyllithium, reacted with C 0 2 to produce the acid 49, which was subsequently methylated with diazomethane to give 44. The last ambiguity remaining in the structure of phomazarin was the orientation of the heterocyclic ring: that is, whether the heterocyclic nitrogen is on the same side or the n-butyl substituent on the benzenoid ring, or whether they occupy opposite sides. Incorporation studies with I4Cacetate suggested that phomazarin is polyketide in origin, with the carboxyl group originating from the methyl carbon of acetate (19-24). Between the two possible orientations, 23 and 50,only 23 would be consistent with the formation of phomazarin via the normal processes of polyketide biosynthesis.

48 Wuli, C02

I

49 SCHEME 5. Synthetic preparation of 44 (23).

90

TURAN OZTURK

In the I3C-NMRspectrum of 51, the quinone carbonyls appeared at 178.9 and 181.3 ppm. In the fully 'H-coupled I3C-NMR spectrum of 51, the quinone carbonyl resonance at 181.3 ppm appeared as a doublet (J = 4 Hz), which can only be due to a 3-bond coupling to the H-5. In the proton-noise-decoupled spectrum of 15N-enriched51, the other quinone carbonyl resonance at 178.9 ppm appeared as a doublet (J = 8 Hz), which must be due to a 2-bond coupling to 15N (25). These couplings proved the opposite orientation of the heterocyclic nitrogen to the n-butyl substituent on the benzenoid ring of the structure 23. The unusual predominance of the 4-hydroxy tautomer form of the heterocyclic ring was suggested to be due to hydrogen bonding to the quinone carbonyl, as this form predominates even after removal of the electron withdrawing 2-methoxycarbonyl group (24). Phomazarin (20), 6-n-butyl-2-carboxy-3,4,8-trihydroxy-7-methoxy-l-azaanthraquinone: CI9Hl7NO8;mp 196°C; MS d z 387 (M+); UV Y 231 (E 34,673), 277 (E 58,884), 430 (E 8317) nm; IR (CHC13 solution) 1633, 1694 cm-I; 'H-NMR (100 MHz, CF3C02H) S 8.05 (lH, s, H-S), 4.05 (3H, s, 7-OMe), 2.94 (2H, t, 12-CH2), 1.6 (4H, m, 13-, 14-CH2), 1.05 (3H, t, 15-Me), 12.48 (lH, 3-OH). b. Biosynthesis. It was first suggested on the basis of incorporation of radioactivity from HI4CO2Na,I4MeCO2Na,and MeI4CO2into phomazarin that the biosynthesis involved at least eight acetic acid units, and that the nitrogen-containing moiety might be derived from an amino acid, possibly glycine (17). Later, an extensive study of the biosynthetic pathway involving the incorporation of [I3C]acetates and [13C]-and ['4C]malonates by cultures of P. ferrestris revealed that phomazarin contains nine acetate moieties and must be formed from a single nonaketide chain folded, as shown in Scheme 6 (24). 4. lsophomazarin

Isophomazarin (29), a red pigment, was isolated by Birch and Simpson (24) as a minor pigment along with phomazarin (28) from the mycelid of P. ferrestris. Chromatography of the chloroform extract of the acidified mycelia of P. ferrestris gave a red gum, eluted with chloroform, from which phomazarin was separated by crystallization from methanol. The remaining isophomazarin was purified by several crystallizations from acetic acid. a. Characterization. The similarities of the physical properties and spectral data of phomazarin (28) and isophomazarin (29) suggested a close structural relationship. The 'H-NMR spectrum of isophomazarin showed the presence of an uncoupled aromatic proton and of methoxy and n-butyl

9 CH3COSCoA

0

0

0

0

-

Ho

-

OH

"H3l

23

SCHEME6. Biosynthetic pathway of phomazarin (24).

92

TURAN OZTURK

groups, as in phomazarin. Three low-field exchangeable protons indicated the presence of three strongly hydrogen-bonded phenolic groups in isophomazarin. The IR spectrum of isophomazarin methyl ester (52) indicated the presence of an unchelated ester carbonyl at 1730 cm-I, in contrast to phomazarin methyl ester (31), in which the chelated ester and the chelated quinone carbonyls showed IR peaks at 1685 and 1615 cm-', respectively. Di-0-methylisophomazarin methyl ester (53) and tri-0-methylisophomazarin methyl ester (55) were obtained by Purdie methylation of isophomazarin. The IR spectrum of 53 showed the presence of both chelated and nonchelated quinone carbonyls with absorptions at 1625 and 1680 cm-', respectively, whereas in 55 both quinone carbonyls showed absorption at 1680 cm-I. Reduction of 55 over palladium-charcoal gave, after aerial reoxidation of the quinone system, the tetrahydro derivative 56, the IR spectrum of which showed absorptions at 1740,1670, and 1625 cm-'. The last absorption was interpreted as a quinone carbonyl whose absorption frequency was lowered by vinylogous amide conjugation, which locates the nitrogen adjacent to the quinonoid ring, as in phomazarin. The 'H-NMR spectrum of 56 showed that one of the methyl ester functions moved to 3.36 ppm, a shift typical of an aliphatic methoxy, and the aromatic proton resonance in 55 was replaced by a multiplet at ca. 1.6 ppm. The methyl ester resonance moved to 3.70 ppm. Ready hydrolysis of the 4-0-methyl ether of 28 gave 27, which showed a lowered carbonyl absorption frequency at 1620 cm-I, indicating the position of the hydroxyl group para to the heteroaromatic nitrogen and peri to the quinone carbonyl. The I3C spectrum of 52 showed the C-10 and C-9 resonances at 188.3 and 183.6 ppm, respectively. These resonances did not show any longrange coupling in the fully 'H-coupled 13Cspectrum, as was observed for phomazarin derivatives. It was shown that the 13C-NMRspectra of isophomazarin methyl ester 30 and its tri-0-methyl derivative 55 are in full agreement with these structures (26). It was noted that structure 57, with the opposite orientation of the heterocyclic ring, cannot positively be excluded

56

n

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

93

from the data given (26),but it was suggested to be unlikely in view of the co-occurrence of phomazarin and isophomazarin. Again, the isophomazarin structure 29 is fully consistent with a polyketide derivation, whereas 57 is not. Isophomazarin (29) (26), 6-n-butyl-4,5,8-trihydroxy-7-methoxy-l-azaanthraquinone-2-carboxylic acid: C19H17N08;red needles; mp 215-216°C (acetic acid); found C 59.2, H 4.5, N 3.9, requires C 58.9, H 4.4, N 3.6%; 'H-NMR (100 MHz, CF3C02H) S 8.28 (lH, S, H-3), 2.94 (2H, t, 12-CH2), 1.6 (4H, m, 13, 14-CH2), 1.03 (3H, t, 15-CH3), 4.14 (3H, s, OCH3). b. Biosynthesis. Structural similarities between phomazarin (28) and isophomazarin (29) suggested that both are derived from the same nonaketide chain folded as for the phomazarin biosynthetic pathway (Scheme 6). Their initial products are indicated to be different, 58 and 59 (Scheme 7) (26). Both phomazarin and isophomazarin precursors 58 and 59, respectively, undergo a parallel sequence of modifications including C-4 hydroxylation, 0-methylation, oxidative ring cleavage, transamination, and heterocyclic ring closure to give the final compounds.

5. Scorazanone The 1-aza-anthraquinone, scorazanone (60),was isolated by Colegate et al. from the air-dried roots of Goniothalamus scor Techinii (Annonaceae) in Malaysia in 1990 (27). Extraction of the roots in a Soxhlet with petrol yielded a waxy, brown gum, which was chromatographed using a mixture of petrol and EtOAc as eluant. The yellow solid product was further purified by radial chromatography using a mixture of petrol-chloroform as eluant. Recrystallization from E t 2 0 gave yellow needles of scorazanone. a. Characterization. The structure of scorazanone (60) was assigned on the basis of spectroscopic properties and comparison of them with similar compounds such as cleistopholine (2), arborinine (61), 8-0-methylbostrycoidine, and the phomazarin-related compound 36. The ortho-disubstituted benzene fragment was assigned by four aromatic coupled protons. The two aromatic signals at S 8.2 and 8.4 were similar to the H-5 and H-8 signals of cleistopholine at S 8.25 and 8.35, respectively (5), rather than the H-5and H-8 signals of arborinine, S < 7.8 and 8.23, respectively (28). The two absorptions in the IR spectrum at 1690 and 1640 cm-' clearly indicated a free carbonyl and a hydrogen-bonded carbonyl. The presence of a quinolinequinone was explained by a 13C-NMR signal of C-9a at S 142.48, which is consistent with the C-9a signal of the phomazarin-related

94

TURAN OZTURK 9 C&COSCoA

I 0

58

59

I

I

28

29

SCHEME7. Biosynthetic pathway of isophomazarin (26).

compound 21, rather than 8-O-methylbostrycoidin, which gave signals at 6 125 and 143.7, respectively (29). Scorazanone (60): C15HllN03; yellow needles; soluble in aqueous alkaline solution, insoluble in aqueous acid solution; yellow spot on TLC turns pinklpurple on exposure to ammonia vapor; mp 205.5-206.5"C; IR (KBr) v 169.3(m), 1644(s), 1589(m), 1556(m), 1476(m), 1351(s), 1301(s); HRMS d z 285.0638 (M+); EIMS d z 285 (M+); 'H-NMR (300 MHz, CDC13) 6 8.29-8.34 (lH, m, H-5 or H-8), 8.23-8.28 (lH, m, H-8 or H-5), 7.79-7.87 (2H, m, H-6 and H-7), 12.47 (lH, s, OH), 4.22 (3H, s, 2-OMe), 4.05 (3H, s, 3-OMe); 13C-NMR (20.1 MHz, CDC13) 6 161.3 (C-2), 158.9 (C-3 and -4), 142.5 (C-9a), 189.0 (C-9 and -lo), 135.1 (d. C-6 or -7), 134.2 (d, C-7 or

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

95

-6), 133.1 (C-8a or -10a), 132.3 (C-lOa or -8a), 127.8 (d, C-5 or -8), 126.7 (d, C-8 or -5), 60.9 (q, 2-OMe), 54.9 (q, 3-OMe). B. DIAZA-ANTHRAQUINONE TYPE ALKALOIDS

1. Diazaquinomycin

In 1982, Omura et al. reported the isolation of a new diaza-anthraquinone antibiotic called OM-704 (30),which was later characterized as diazaquinomycin A (62) (31),from a fermentation broth of a Streptomyces sp. OM704, a solid isolate. The isolation of diazaquinomycin B (63)was reported during the characterization of diazaquinomycin A as a minor product of the fermentation (31). A reddish orange powder of diazaquinomycin A (62) was extracted with ethyl acetate from a supernatant that was obtained by centrifugation of the culture. The powder was purified by column chromatography on silica gel using an eluant of chloroform-methanol(20 : 1)and subsequent crystallization from chloroform-methanol (9 : 1) to afford red needles (30).

96

TURAN OZTURK

62

63

Diazaquinomycin B (63), the minor component of the culture, was isolated from a slow-moving band of the column chromatography as colorless needles (32). a. Characterization. Characterization of diazaquinomycins was carried out using a range of spectroscopic techniques, including C-H long-range selective proton decoupling (LSPD) and nOe (31). An unsymmetrical derivative of diazaquinomycin A (62) was prepared by methylation of 62 with methyl iodide-silver oxide in N,N-dimethylformamide, which gave two products, the N,N'-dimethyl derivative 64, which is again symmetrical, and an unsymmetrical N,0-dimethyl derivative 65. Methylation of 62 in chloroform gave an 0,O'-dimethyl derivative 66. The 'H-NMR spectra of 64 and 66 showed an N-methyl signal at 6 3.74 and a methoxy signal at S 4.15, respectively. However, the 'H-NMR spectrum of

64

65

66

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

97

65 showed signals for an N-methyl at S 3.91 and a methoxy at S 4.12. The I3C-NMR of the methyl derivatives 64 and 66 possessed a symmetrical structure, whereas 65 did not, which clearly indicated the symmetrical nature of diazaquinomycin A (62). Aerial oxidation of diazaquinomycin B (63) yielded a product that was identical with diazaquinomycin A in every aspect, indicating that 63 is a 9,lO-dihydro derivative of 62. Diazaquinomycin A (62): C20H22N204; deep red needles; mp 291-295°C; EIMS d z 354.157 (M'); UV(Me0H) A 250 (E 11,800), 260 (E 13,600), 278 (E 20,100), 286 ( 6 21,700), 309 (E 9760), 321 (E 8950), 367 (E 4130), 490 (E

1150) nm; IR(KBr) v 2960, 1670,1625 cm-*; 'H-NMR (400 MHz, CDC13CD30D) 82.27 (6H, s, H-3), 3.03 (4H, t, J = 7.8 Hz, H-12), 1.57 (4H, m, H-13), 1.01 (6H, t, J = 7.2 Hz, H-14); I3C-NMR (CDC13 + CD30D) S 162.2 (C-2 and -7), 135.0 (C-3 and -6), 151.2 (C-4 and -5), 117.3 (C-4a and -10a), 136.8 (C-8a and -9a), 173.9 (C-9), 182.9 (C-lo), 12.9 (C-11 and -18), 32.4 (C-12 and -15), 22.9 (C-13 and -16), 14.6 (C-14 and -17). Diazaquinomycin B (63): G0H24N204; colorless needles; mp > 300°C; UV (MeOH) A 277 (E 20,100), 310 (E 14,800), 325 (E 11,750), 356 ( E 6230), 373 (E 6050) nm; 'H-NMR (400 MHz. CDC13-CD30D) S 2.62 (6H, s, H-3), 2.97 (4H, t, J = 8.1 Hz, H-12) 1.72 (4H, m, H-13), 1.13 (6H, t, J = 7.6 Hz, H-14). b. Synthesis. Although analogs of diazaquinomycin A (62) were synthesized for either biological evaluation or characterization of diazaquinomycins by Omura et al. (32) and Alvendano et al. (33,34), the only total synthesis of diazaquinomycin A (62) and B (63) was reported by Kelly et al. in 1988 using double Knorr cyclization successfully for the first time (Scheme 8) (35). The total synthesis of diazaquinomycin B was achieved in three steps starting from 67, and further oxidation of B gave diazaquinomycin A. The product from the reaction between 67 and 68 was deprotected, because phenols are more reactive toward electrophilic aromatic substitution, and hence the phenolic product 69 allowed double Knorr cyclization to proceed successfully. Omura et al. synthesized derivatives of diazaquinomycin A by methylation of the lactam rings to give the imino ethers 70 and 66. Bromination of the methyl groups of 66 and 70 gave their corresponding bromomethyl derivatives. Substituting the bromides 71 and 72 by a series of nucleophiles yielded 73 and 74, which were subsequently hydrolyzed to give the corresponding lactams 75 and 76 (Scheme 9) (32). Avendano et al. synthesized analogs of diazaquinomycin by cycloaddition reactions of l-azadienes 89 to 2,5,8-quinolinetriones 90 to yield the corresponding 1,8-diaza-2,9,10-anthracenetriones91-94 (Scheme 10)

98

TURAN OZTURK

OMOM

hNH* 68

L

YN

67

60

bH

DiazaquinomycinB 83

SCHEME8. Synthesis of diazaquinornycin A and B (35).

(33,34).Aromatization of the pyridone rings of 91 and 94 with phosphorus oxychloride gave 2-chloro-1,8-diaza-9,lO-anthracenediones 95 and 96, respectively. Aromatization of the pyridone ring of 91 was also carried out by methylation, which gave two products 92 and 97.

c. Biological Activity. Omura et al. reported that diazaquinomycin A (62) inhibited the growth of gram-positive bacteria such as S. aureus FDA 209 P, S. aureus ATCC 6538 P, S. aureus FS 1277 (penicillin resistant), S.

aureus KB 199 (erythromycin resistant), S. faeciurn IF03181, and Micrococcus luteus ATCC 9341 at concentrations of 3.13-50 pg/ml, but did not show activity against gram-negative bacteria and fungi. The acute toxicity (LDso) of diazaquinomycin A in mice was reported to be 100 mg/kg intraperitoneally (30).Although both diazaquinomycin A and B are active against grampositive bacteria, their activity was reversed by folate, DHF (dihydrofolate), leucovorin (5-formyltetrahydrofolic acid), and TdR (thymidine) (36,37). The carboxylic acid analogs (78,80, 82, and 84) of diazaquinomycin A were not active against E. faeciurn and HeLa cells, but the corresponding

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

99

70

73 74

71 , 72

Nu : CN, mabnate anion, H20, acetic acid, ethanoland mrpholine H

7 5 , 76

SCHEME9. Synthesis of the analogs of diazaquinomycin A (32).

esters (77, 79, 81, and 83) were active. Both the carboxylic acid and ester analogs were more potent inhibitors of TMP synthase than diazaquinomycin A. The compounds 77 and 81 were active against E. fuecium, though 77 was most cytotoxic. However, 83 exhibited poor activity against E. fuecium. Analogs 85 and 87 were only half as potent as 86 and 88. All Type I analogs

100

TURAN OZTURK

111

I 70

II

CH3

66

111

CH3 CH2CN

111

77

I

CH2CO2Et

70

I

CH2CQH

79

I

CH2CH(C02Et)2

80

I

81

I

CH2CW02W2 CH2CH2C02Et

02

I

CH2CH2C02H

83

I

84

I

c02et c02h

86

I

CH~OAC

111

CH~OAC

86

I

CH~OAC

87

I

CHzOEt

w

I

CH20Et

were more potent inhibitors of TMP synthase than diazaquinomycin A, among which 88 was the most potent inhibitor of TMP synthase. Three analogs 77,86, and 88 exhibited activity against HeLa cells similar to that of diazaquinomycin A, and the analog 84 exhibited significant antitumor activity (TIC 175%) against Meth-A fibrosarcoma in mice (32,38). Omura et al. indicated that changing one or both lactam rings of diazaquinomycin A for pyridine rings substantially reduces the biological activity (32). In contrast to this result, the monolactam analog of diazaquinomycin A showed antitumor activity (34). Diazaquinomycin A was also tested against viral reverse transcriptase, an enzyme essential for human immunodeficiency virus (HIV) replication, which was inhibited to the extent of 76% (39).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

101

U

91

97

SCHEME10. Synthesis of the analogs of diazaquinomycins (33,34).

C. PHENANTHRIDINE TYPEALKALOIDS

2. Phenanthroviridin and Its Aglycone

The first naturally occurring benzo[b]phenanthridine,phenanthroviridin (98), and its aglycone 99 were first isolated from fermentation product of

S. viridiochromgenes DSM3972 in 1987 (40). In 1994, phenanthroviridin aglycone 99 was detected as a minor metabolite of S. murayamaensis (42). The fermentation products were extracted

102

TURAN OZTURK

with EtOAc, from which the nonpolar oils were removed with hexane. The crude material was then chromatographed successively on Silicar CC-4 and then Sephadex LH-20. HPLC analysis of the product revealed a number of components, among which the component eluting at 19.56 min matched an authentic sample of 99. Further confirmation was provided by thermospray (TSP) liquid chromatographylmass spectrometry (LCMS). Both the authentic sample 99 and the metabolite gave the same m/z 305 (41). a. Characterization. Phenanthroviridin trifluoracetate, 1-(2,3,6-tridesoxy-3-met hylamino-a-ribohexopyranosyloxy)-8-hydroxy-3-methylbe~o[b]phenanthridin-7,12-dione-trifluoroacetate: mp 196-197°C; +62” (c = 0.518, MeOH); ‘H-NMR (300 MHz, CDC13) 6 12.21 (lH, bs), 12.05 (lH, bs), 9.49 (lH, bs, H-5), 7.91 (lH, dd, J = 7.6, 1.1Hz, H-11), 7.73 (lH, dd, J = 8.5, 7.6 Hz, H-lo), 7.48 (lH, bs, H-4), 7.40 (lH, dd, J = 8.5, 1.1 Hz, H-9), 7.36 (lH, bd, J = 1.6 Hz,H-2), 2.56 (3H, S, H-13); I3C-NMR (125 MHz, CDCl3) 6 189.4 (C-7), 185.8 (C-12), 162.1 (C-8), 160.0 (C-5), 155.1 (C-1), 145.0 (C-6a), 144.3 (C-3), 137.1 (C-lo), 133.4 (C-4a), 132.4 (Clla), 128.4 (C-l2a), 125.9 (C-9), 124.1 (C-4), 121.6 (C-11), 121.5 (C-2), 120.5 (C-l2b), 114.4 (C-7a), 21.4 (C-13).

b. Synthesis. The total synthesis of phenanthroviridin aglycone was achieved by Gould et al. in 1991 (Scheme 11) (42). The initial step involved coupling of the cyanophthalide 100 and the substituted bromo cinnamate 101, which was prepared in six steps. Aerial oxidation of the product gave the naphthoquinone 102. This compound was reduced and methylated in situ in refluxing acetone to give 103, which was first deprotected with

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

CN

104

\

103

I . bki,-98%

2. DMF

105

108 R = C W 107 R = H

SCHEME11. Total synthesis of phenanthroviridin (99) (42).

103

104

TURAN OZTURK

SCHEME 11. Continued

tetrabutylammonium fluoride and then converted to the amide 104 by treating with Tf20/pyridine followed by ammonia. The formylation of amide 104 using t-BuLi and DMF led to a 1:1 mixture of the desired aldehyde 106 and the debrominated derivative 107, along with the fluorenone 105 as a minor product. The mixture of 106 and 107 was used for the next steps, as they could not be separated by chromatography, but were readily separated from 105. The mixture yielded the corresponding carbamates 108 and 109 by use of the modified Hoffmann rearrangement. Hydrolysis and cyclization of 108 easily led to 110,which was separated from 109 by acidic workup. Deprotection of 110with BBr3and then bubbling O2through the mixture afforded 99, which was identical with an authentic sample of phenanthroviridin aglycone. c. Biosynthesis. During studies on the biosynthetic pathway of the kinamycin antibiotics (43-43, a benzo[b]phenanthridine derivative was suggested as a possible intermediate (Scheme 12) (46). Isolation of phenanthroviridin later on reinforced this suggestion (41). It was established that the kinamycins are of polyketide origin, apparently derived from a benzo[alanthraquinone 112 via a decaketide 111 (Scheme 12). Metabolism of

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

105

111

4

113

112

.1

&

&

Ho

115

114

116

117

SCHEME 12. Possible biosynthetic pathway of 99 (41).

112 to 113 could lead to the benzo[b]phenanthridine nucleus via nitrogen addition and decarboxylation, whereas reduction of 113 to the hydroquinone 114, followed by a biological Friedel-Crafts cyclization, would yield the fluorene system of the kinamycins 115-117 (41). d. Biological Activity. It was reported that both 98 and 99 are active against lung carcinoma MBA9812 implanted in mice (40).

106

TURAN OZTURK

2. Jadomycin and Jadomycin B

In 1991, Ayer et al. reported on the isolation and structure elucidation of a second phenanthridine type alkaloid antibiotic called jadomycin (118), which is a product of the culture of S. venezuelae ISP5230 when the medium is fed with galactose and the temperature is raised to 37°C. When the medium contains glucose-isoleucine at 28"C, the product is chloramphenicol(l20) (47). The filtrate of the culture broth was acidified with 6 M HCl and extracted with EtOAc. After evaporation of the solvent, the product was subjected to a reverse-phase flash column chromatography, which was first washed with H 2 0 and then eluted with a MeOH/H20 step gradient. The fraction eluted with 90% MeOH gave jadomycin, which was further purified by reverse-phase HPLC. A few years later, in 1993, the same group reported the isolation of an antibiotic jadomycin B (119)from the same source, S. venezuelae (48-50). It was observed that the synthesis of jadomycin B in the fermentation medium fed with galactose-isoleucine was triggered by heat shock or ethanol treatment. a. Characterization. The structure of jadomycin (118) was explained on the basis of conventional and modern spectroscopic methods such as DEPT, COSY, HOHAHNMLEV 17, NOESY, and HMBC (47).

&i

110

119

120

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

107

Jadomycin (118),8H-benz[b]oxazolo[3,2-f]phenanthridine: G4HZ1NO6; green crystalline solid; mp 167-168°C; MS m/z 420.14 (MH+);UV (MeOH) A 214 (E 56,234), 239 (E 18,620), 289sh 314 (E 21,379), 429 (E 6025 nrn; IR (thin film from CDC13) Y 1806, 1639, 1607, 1512, 1455, 1424, 1379, 1357, 1296, 1271, 1239, 1226, 1197, 1161, 1104, 1083, 1036 cm-’; ‘H-NMR (500 MHz, CDC13) 65.31 (lH, d, J = 2.7 Hz, H-1), 6.17 (lH, s, H-3a), 6.82 (lH, bs, H-4), 6.91 (lH, bs, H-6), 7.81 (lH, dd, J = 7.5, 1.1 Hz, H-9), 7.67 (lH, dd , J = 8.5,7.5 Hz, H-lo), 7.26 (lH, dd, J = 8.5, 1.1 Hz, H-ll), 1.83 (lH, m, H-l’), 1.83 (2H, m, H-2’), 1.57 m, 1.04 (3H, t, J = 7.2 Hz, H-3’), 0.45 (3H, d, J = 6.6 Hz, H-4’), 2.27 (3H, S , H-5’), 10.2 (lH, S, 7-OH), 11.7 (lH, S , 12-OH); 13C-NMR (125 MHz, CDCl3) S 62.7 (C-l), 170.0 (C-2), 87.2 (C3a), 122.1 (C-3b or C-7b), 114.5 (C-4), 143.4 (C-5), 120.9 (C-6), 154.1 (C7), 111.1 (C-7a), 129.9 (C-7b or C-3b), 183.5 (C-8), 133.1 (C-Sa), 120.9 (C9), 137.7 (C-lo), 124.4 (C-11), 162.0 (C-12), 113.4 (C-l2a), 185.8 (C-13), 142.2 (C-l3a), 40.0 (C-1’), 25.3 (C-2’), 11.9 (C-3‘), 13.8 (C-4’), 21.3 (C-5’). D.

QUINOLINEQUINONE

TYPE ALKALOIDS

1. Lavendamycin

In 1982,Doyle et al. reported isolation of an antibiotic, structurally related to streptonigrin (207),called lavendamycin (121)(51),the structure elucidation of which had been disclosed by the same group in 1981 (52). The nbutanol extract of a culture of S. lavendulae, strain C22030 isolated from a soil sample collected from Woolinton, New York, was treated with excess petroleum ether to precipitate crude lavendamycin, which was purified by silica gel column chromatography eluting with toluene-acetonitriletrifluoroacetic acid (40 :40 :20 and 50 : 30 :20 v/v) solvent systems. Production of lavendamycin from the microorganism, Sfrepfomycessp. 0

10 ’

121

108

TURAN OZTURK

G324 isolated from a soil sample collected in Fujieda City, Shizuoka Prefecture, Japan, was also reported by Abe et al. in 1993 (53,54). a. Characterization. The structure elucidation of lavendamycin (121) was carried out by conventional spectroscopic methods and comparisons with the data for appropriate model compounds (52). Assignment of the position of the -NH2 group (ie., whether it is at the 6- or 7-position) was confirmed after 13C-NMRstudies of the model compounds, 6-aminoquinoLong-range coupling lineJS-quinone and 7-aminoquinoline-5,8-quinone. of the C-8 (6 180.0) to the proton at C-6 was observed in both lavendamycin and 7-aminoquinoline-5,8-quinone. Lavendamycin (121): G2H14N404; dark red solid; mp > 300°C; MS d z 398 (M’); UV (MeOH) A 234 (a = 49.2), 246 (a = 49.8), 391 (a = 21.1) nm; UV (MeOH + dil. acid) h 252 (a = 47.4), 277 (a = 36.0), 385 (a = 19.0) nm; UV (MeOH + dil. base) A 245 (a = 94.1), 309 (a = 42.3), 390 (a = 39.6) nm; IR ~2800-3800,1740,1692,1610,1590;‘H-NMR (DMSO4 ) 6 3.08 (3H, S, C3‘-CH3), 5.91 (IH, S, H-6), 7.37 (lH, dd, J = 8.6, 6.0 Hz,H-ll), 7.40 (2H, bs, NH2), 7.61 (2H, m, H-9’ and H-lo’), 8.28 (lH, d, J = 8.6, H-12’), 8.37 (lH, d , J = 8.0, H-3), 8.93 (lH, d, J = 8.0, H-4), 11.86 (lH, S, NH); 13C-NMR(DMSO-db, 90.5 MHz) 6 157.9 (C-2), 134.4 (C-3), 125.1 (C-4), 129.8 (C-4a or C-4’), 180.7 (C-5), 102.2 (C-6), 150.7 (C-7), 180.0 (C-8), 145.5 (C-8a), 132.7 (C-2’), 128.8 (C-3’), 131.9 (C-4’ or C-4a), 134.6 (C-5’), 136.9 (C-6’), 121.3 (C-7’), 140.3 (C-8’), 112.2 (C-9’), 128.7 (C-lo’), 121.0 (C-ll’), 124.0 (C-12’), 167.3 (COZR), 16.4 (C-3‘-CH3). b. Synthesis. The synthesis of lavendamycin (121)has attracted attention because of its structural similarity to the antitumor antibiotic streptonigrin (207) and its challenging structure. The first of the six total syntheses of lavendamycin methyl ester was reported by Kende et al. in 1984 (Scheme 13) (55,56). Their nine-step synthesis started with the formation of 8-methoxyquinaldic acid (W) by (122) with pyruvic acid condensation of 2-amino-3-methoxybenzaldehyde (123). Nitration of 124 with H2S04/HN03gave 8-methoxy-5-nitroquinaldic acid (125), bromination of which at C-7 was achieved in the presence of AgOCOCF3 to afford 126. Alternatively, because 125 is inert to usual bromination conditions, demethylation to form 8-hydroxy-5-nitroquinaldic acid gave a substrate that underwent bromination at C-7 under usual bromination conditions (56). Amide 128 was synthesized by condensation of the acid 126 with P-methyltryptophan 127 in the presence of the basic carbodiimide. Cyclization of the amide part of 128 to form a pyridine ring using polyphosphoric ester (PPE) afforded the P-carboline ester 129, the nitro group in which was reduced with Na2S204to give the 5-aminoquinoline 130. Oxidation of 130 in a two-phase system gave the quinoline-5S-quinone

109

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

131. Treatment of 131 with sodium azide, to replace the bromine with an azide, followed by reduction to the amino group with Na2S204afforded the desired lavendamycin methyl ester (132).It was reported that conversion of the ester 132 to lavendamycin using conventional methods produced low yields (55). In 1985, Hibino et al. reported the formal synthesis of lavendamycin

L)+: : :

0%

C W

124

122

125 Bq, ASOCOCF3, WAC, 200C. in dark 60 %

Br

* I

HN'

Me2N(CH2)3N=C=NPr 90 %

Br

128 PPE, 31 %

127

R

0

Br

Br

c

~2.~204

70%

129

'=NO2

131

130 R = N k

SCHEME13. Total synthesis of lavendarnycin methyl ester (132) (55,56).

I

110

TURAN OZTURK

0

131

132

SCHEME13. Continued

methyl ester starting from 8-benzyloxyquinolin-2-aldehyde(133)to Kende’s intermediate bromoquinolinequinone (131)(56) in seven steps (Scheme 14) (57). Condensation of the quinoline aldehyde 133 with P-methyltryptophan ethyl ester (134) followed by aromatization with 5% Pd-C in xylene afforded the aromatic pentacyclic 6-carboline 135. Hydrogenation of 135 in the presence of 10% Pd-C cleaved the benzyl group to yield the 8hydroxyquinoline 136,the ethyl ester of which was converted to a methyl ester by hydrolysis with 10% NaOH and then esterification with anhydrous MeOH, BF3 * OEt, to give 137.Dibromination of 137 with 2,2,4,4-tetrabromocyclohexadien-1-one (138)afforded the dibromohydroxyquinoline 139. Oxidation of 139 with ceric ammonium nitrate (CAN) gave the desired quinolinequinone 131 possessing physical and spectroscopic data identical with those of Kende’s intermediate (56). In 1985, Boger et al. reported their total synthesis of lavendamycin (121), completed in 16 steps and using a [4 + 21 cycloaddition reaction to prepare the substituted pyridine ring, and a palladium-mediated P-carboline synthesis (Scheme 15) (58,59). Tetrasubstituted pyridine 142 was prepared by a [4 + 21 cycloaddition reaction of 1,2,4-triazine 140 with the electron-rich olefin 141 (58).Ester hydrolysis of 142 with LiOH was followed by selective Fischer esterification of the accessible carboxylates at 2 and 6 to afford tetrasubstituted pyridine-3-carboxylicacid 143. Application of the Yamada modification of the Curtius rearrangement to 143 gave the 3-aminopyridine 144,which was acetylated using acetyl chloride to obtain the 3-acetamidopyridine 145. Selective hydrolysis of the ester at the 2-position was achieved by base-catalyzed ring closure of 146 to the oxazinone 147, which was opened during aqueous workup to afford 146. Treatment of 146 with dicyclohexylcarbodi-imide(DCC) reformed the oxazinone 147. Introduction of the methyl group to form the 2-acetylpyridine 148 was carried out by the reaction of the lactone 147 with a-lithiomethyl phenyl sulfoxide, which was followed by treatment of the crude product with aluminum amalgam.

111

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

+

1. Benzene 2.5 '/! Pd-C

*ne 75 %

134

9 0

135

10 % Pd-CM2 95 %

139

I

30 % CAN

1.10'ANaOH THF 2. Anhyd. MeoH, BF3.OEt-2

131

c

136 R=Et

137 R = M e

SCHEME14. Formal total synthesis of lavendamycin (57).

The reaction of 148 with 10% HCl-MeOH afforded selective hydrolysis of the amide to form the 3-aminopyridine 149, in which the ring was subsequently closed after treatment with palladium(0) to give the P-carboline 150. Friedlander condensation of 150 with 2-amino-3-benzyloxy-4-bromobenzaldehyde (151), which was synthesized in four steps (58), gave the 2p-carbolinequinoline 152. Hydrogen bromide gas debenzoylation of 152 was followed by oxidation with potassium nitrosodisulfonate (Fremy's salt) to obtain Kende's intermediate 131. In 1986, Rao et al. reported their total synthesis of lavendamycin in 17 steps (Scheme 16) (60).7-Bromo-5,8-dimethoxyquinaldicacid (153),which was synthesized in 10 steps from 8-hydroxyquinoline, was condensed with p-methyltryptophan (127) to furnish the amide 154. In contrast to Kende's cyclization with polyphosphoric ester, which resulted in a 31% yield (55,56),

112

TURAN OZTURK

+ 140

-l i r CHGk

Br&

0

141

142 1. UOH 2.10 % HCI 67 %

I

SCHEME15. Formal total synthesis of lavendamycin (58,59).

Rao et al. carried out the cyclization with P0Cl3 to form the /3-carboline 155 in 86%yield. Demethylation of 155 with 48% HBr afforded 156,which was selectively esterified with methanolic sulfuric acid to obtain 157. Oxidation of 157 with ceric ammonium nitrate (CAN) resulted in Kende's intermediate bromoquinone 131. In 1993,Ciufolini and Bishop reported their formal synthesis of lavendamycin methyl ester (132)starting from quinoline 158(62) to Boger's lavendamycin intermediate 152 (59) in seven steps (Scheme 17) (62).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

113

i

COJ 149

150

151 PtCH2(Me)3NOH, M 52-58%

HBr (gas) o w , 85 %

F I

<

R13~CH2Ph

SCHEME 15. Continued

Condensation of quinoline 158 with 2-azidobenzaldehyde (159)in basic medium gave chalcone 160, reaction of which with a 2 :1 mixture of 2ethoxybut-1-ene and 2-ethoxybut-2-ene in the presence of Yb(fodj3 formed the pyran derivative 161.Treatment of 161 with hydroxylamine hydrochloride converted the pyran ring to a pyridine ring derivative 162. Se02 oxidation of 162 readily afforded the aldehyde 163. Nitrene insertion to obtain the carboline 164 was carried out by refluxing 163 in 1,2-dichlorobenzene. Aldehyde 164 was converted to the acid 165 by oxidation with NaC102. Esterification of 165 with CH2N2provided Boger's intermediate 152. In 1993, Behforouz et al. reported their highly concise, five-step, and overall high-yield (33%) synthesis of lavendamycin methyl ester (l32)

114

TURAN OZTURK

127,EQN CCQCH3 97 %

Br

153

Br HN

156

155

I

MeOH, HpSO4

OH

Br

131

157

SCHEME16. Formal total synthesis of lavendamycin (60).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

115

160

(21, MeCH=C(Me)OEt + CH#(Et).OEt) Yb(fcd)3, 98 %

1

SCHEME17. Formal total synthesis of lavendamycin (62).

(Scheme 18) (63). Diels-Alder condensation of the quinone 168 with the l-azadiene 169, which was prepared by the treatment of o-(ferf-butyldimethylsily1)hydroxylamine (167) with methyl vinyl ketone (166), in refluxing chlorobenzene gave the quinoline-5,8-quinone170. Oxidation of 170 with

116

TURAN OZTURK

171

170

0

SCHEME18. Formal total synthesis of lavendamycin (63).

selenium dioxide produced aldehyde 171. In contrast to Boger el al. (59), the pyridine ring was formed in one step by treatment of the aldehyde 171 with p-methyltryptophan (127) in xylene, which provided the p-carboline in one step. Deacetylation of the amide 172 with H2S04gave lavendamycin methyl ester (132). Because of its biological importance and challenging structure analogs (64,65),partial structures (66,67)and derivatives (68) of lavendamycin have also been reported by various synthetic groups. In 1986, Hibino et al. reported the synthesis of an analog of lavendamycin

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

117

Br

Bromination ___)

Br

I73

174

1

{CAN

CAN

0

0

176

175

SCHEME19. Synthesis of analogs of lavendamycin (64).

176 (Scheme 19) (64) starting from 173, an intermediate similar to that which was used in their total synthesis of lavendamycin (Scheme 14) (57). Bromination of the 8-hydroxyquinoline 173, followed by oxidation of the 5,7-dibromoquinoline 174 with CAN, gave the 6-bromoquinoline-5,8quinone 175. Hydrogenolysis and then oxidation of 175 afforded the analog 176. This analog, 176, was also synthesized directly from the 8-hydroxyquinoline 173 by CAN oxidation, but in poor yield. Hibino et al. reported that the addition of 173 to the liver homogenate obtained from mice, and incubated at 37°C for 30 min, produced 176, which demonstrated that 176 is a metabolic product of 173 (64). QuCquiner's synthesis of the lavendamycin analog 182 involved selective coupling reactions (Scheme 20) (65). First, coupling was carried out between the boronic acid derivative 177 and iodopyridine 178, which was prepared in five steps to afford the biaryl 179. The second coupling between the biaryll79 and 2-trimethylstannylquinoline 180in the presence of Pd(PPh3)4 catalyst gave the polysubstituted triaryll81, treatment of which with boiling pyridinium chloride at 215°C led to the lavendamycin analog 182.

118

TURAN OZTURK

+ 177

178 179 Toluene

182

SCHEME20. Synthesis of analog of lavendamycin (65).

In 1987, Boger et al. synthesized two partial structures of lavendamycin for biological evaluation, using the Friedlander condensation to construct the quinoline ring (Scheme 21) (66,67).Condensation of the 2-aminobenzaldehyde 151 with the 2-acetylpyridine 183, in the presence of N-benzyltrimethylammonium hydroxide, gave quinoline 184. Re-esterification of 184 was carried out in 10%HC1-methanol solution to afford 185. Debenzylation of 185 was achieved with HBr, giving the 8-hydroxyquinoline 186. Conversion of 186 to the quinoline-5,8-quinone was achieved by nitration of 186 at the 5-position, followed by reduction of the nitro group using aluminum amalgam, and finally oxidation with manganese dioxide. 7-Bromoquinoline5,8-quinone derivative 187 was converted to the desired target compound 190 in six steps. The bromine in 187 was replaced by azide reduction with triphenylphosphine, and then hydrolysis of the formed triphenylphosphine imide with a mixture of HOAc-H20-THF afforded the 7-aminoquinoline5,8-quinone 188. Hydrolysis of the methyl ester was achieved best after in situ reduction of 188, which gave the 7-amino-5,8-dihydroxyquinoline (189). Oxidation of hydroquinone 189 to the quinoline-5,8-quinone 190 took place during the workup and exposure to air.

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

HCI-CH3OH(10 %) 85 Yo

I

119

184 R = H 185 R=CH3

HBr (9) 97 %

187

I

1. NaN3,89% 2. Ph3P 3. HOAc-I+OTHF

0

50 %from (2) 1. Na2S2O4

w

2. KOH

90%from (1)

SCHEME21. Synthesis of partial structures of lavendarnycin (66,67)

Synthesis of another partial structure of lavendamycin, by Boger et al., was achieved using the same strategy (Scheme 22) (66).In the Friedlander condensation, methyl 3-acetyl-4-aminobenzoate (191)was used in place of the 2-acetylpyridine 183 to obtain 192, which was converted to the 7-

120

TURAN OZTURK

191

SCHEME22. Synthesis of partial structures of lavendamycin (66)

aminoquinolineJ,8-quinone 193 using the same procedure as described earlier to obtain 190. In 1992, Molina et al. synthesized partial structures of lavendamycin using the aza-Wittig reaction as the main strategy (68) to form a P-carboline at the 2-position of a quinoline (Scheme 23) (68). The reactions of the iminophosphorane 194, prepared from 3-formyl-l-methyl indole by treatment with ethyl azidoacetate and triphenylphosphine, with the 2-formylquinoline derivatives 195 in toluene at 160°C in a sealed tube gave the corresponding quinoline derivatives 1% and 197. Treatment of iminophosphorane 194 with pyruvaldehyde yielded the key P-carboline intermediate 198, which was reacted alternatively with the 2-aminobenzaldehyde 199 and N-(O-formylpheny1)triphenyliminophosphorane 200 to obtain the corresponding partial structures of lavendamycin, 201 and 202, respectively. c. Biosynthesis. Although it has not been established that lavendamycin

(121) is an intermediate in the biosynthesis of streptonigrin (207), the biosynthetic pathway of which has been studied extensively by Gould et al. (69-71), the intermediates in streptonigrin biosynthesis, such as 4-aminoanthranilic acid (203), ~-erythrose-4-phosphate(204), and P-methyltryptophan (206), suggest that lavendamycin either has a similar synthetic pathway, like streptonigrin, or is an intermediate (Scheme 24) (69-71). Gould et al. established that quinoline 205, which is the product of the reaction between 4-aminoanthranilic acid (203), a new metabolite of the shikimate

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

121

196 R = H 8 0 % 197 R = P E k O 75 %

lS8 I

202

201

SCHEME 23. Synthesis of partial structures of lavendamycin (68).

pathway, and ~-erythrose-4-phosphate(204), reacts with P-methyltryptophan (206) to lead to streptonigrin (207). All these intermediates fit nicely into the biosynthetic scheme for lavendamycin, which could be an intermediate in the biosynthesis of streptonigrin. d. Biological Activity. Lavendamycin (121) was found to be slightly active against P-388 leukemia and did not show any inhibitory effect on

TURAN OZTURK

122

a:: k+-[ "rnCW J J" 1 .$ I J

H2N

203

205

204

206

J

1

SCHEME24. Biosynthetic pathway of lavendamycin (69-71).

the standard P-388 or L-1210 leukemias (52). In contrast, Abe et al. reported their findings that lavendamycin shows in vitro cytocidal activity against leukemia cells P388 with an ICso value of 0.06 pglml and colon adenocarcinoma cells W: Dr with an ICso value of 0.09 pg/ml (54). Antimicrobial studies of lavendamycin against various organisms showed that it is less potent than streptonigrin (207),except for the fungi Trichophyton rubrun, T. mentagrophytes, and Microsporum canis, against which lavendamycin was found to be more potent (51). The minimum concentration of lavendamycin (0.003 pg/ml) required to induce bacteriophage production in the lysogenic strain of Escherichiu coli W1709 was found to be comparable to that of streptonigrin (0.008 pgl ml) (52). Hibino et al. subjected lavendamycin analogs (57,64) to the mutation test on Salmonella typhimurium TA 98 and 100 with and without S9 fraction prepared from the liver of rats to use as a metabolism-inducing system (64). The strongest mutagenicity was exhibited by the ethyl ester of 131 to TAlOO with and without S9, but its mutagenicity to TA98 was weak with S9. The analog 176 was the next strongest toward TA 100 with S9. Compound 137,which did not mutate either TA 100 or TA 98 without S9, was lethal to both strains with S9. Ethyl esters of 131,l39,l35,and 136 increased in miitaopnir nrrlpr

f&i\

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

123

2. Streptonigrin, 10'-O-Demethylstreptonigrin, and 10'-Desmethoxystreptonigrin

In 1959, Rao and Cullen reported isolation of a compound with high antitumor activity called streptonigrin (207) from a culture of S. flocculus (72).Its structure was deduced by Rao, Biemann, and Woodward in 1963 (73).A few years later, the same compound was independently isolated by two research groups from a strain of Actinomyces albus var. bruneomycini (7475) and from S. rufochromogenes and S. echinatus (76),being named bruneomycin and rufochromomycin, respectively. Its unusual challenging structure and significant anticancer and antibiotic activity have attracted the attention of many research groups. More than 200 papers, including reviews (77-81), have appeared so far on its synthesis, cytotoxic mechanism of action, and biosynthesis. This highly active compound against experimental lymphomas was subjected to clinical trials, but its severe toxicity to the hematopoietic system prevented its acceptance into human medicine (82). In 1986, Isshiki et al. reported on the isolation and structure elucidation of an analog of streptonigrin called demethylstreptonigrin (208), which was determined to be 10'-O-demethylstreptonigrin,from a strain of S. albus, which also produces streptonigrin (83). In 1992, a third analog of streptonigrin, this time with no substituent at C-lo', was reported by Liu et al. (84). 10'-Desmethoxystreptonigrin (209 was isolated from the fermentation broth of a strain of S. albus, a solid sample collected in Yosemite National Park, California. a. Characterization. The structure of streptonigrin was reported by Rao, Biemann, and Woodward in 1963 on the basis of a combination of intensive chemical degradative methods and spectral measurements (73). Although the structure was determined by this group, its 13C-and 'H-NMR spectra was first reported by Lown and Begleiter in 1974 (85),and by Gould and Weinreb in 1982 (69). During isolation and structure elucidation of demethylstreptonigrin (208) by Isshiki et al., the 13C- and 'H-NMR spectra of streptonigrin were also reported independently in comparison with the spectra of demethylstreptonigrin (83). The elemental composition of streptonigrin could only be determined approximately by elemental analysis, but it was established as C2SH220& by comparing the high-resolution mass spectra of hexamethyldihydrostreptonigrin (210), C31H3608N4 (mol wt 592), and hexadeuteriomethyldihydrostreptonigrin (211), C ~ I H ~ B D ~ (mol ~ O SwtN 610) ~ (73). In 1975, using Xray crystallography, the structure of streptonigrin was verified by Chiu and Lipscomb (86). 13C-1H long-range selective proton decoupling (LSPD) experiments of streptonigrin indicated that irradiating the proton signal at S 6.00 (C7-NH2)

124

TURAN OZTURK

0

Streptonigrin 207 R=OCH3 l(Y-ODemethyIstreptonigrin 208 R = OH 10 ‘-Desmethoxystmptorigrin 209 R = H OR

210 R=CH3 211 R-CDQ

showed a sharp signal at S 181.0 (C-8) and at 6 137.4 (C-6) (83).Assignment of the 13Cchemical shifts at S 137.9 (C-9’) and 154.1 (C-10’) were carried out by irradiation of the proton signals at 6 7.83 (OH-8’), 6.70 (H-ll’), and 6.77 (H-12’). Irradiation of the OCH3 proton signals at S 3.84, 3.88, and 3.96 proved their connectivity to the carbons at 6 137.9 (C-9’), 154.1 (C-10’), and 137.4 (C-6), respectively. After determination of the location of each methoxy group, the missing one on demethylstreptonigrin was easily determined (83). Streptonigrin (207): C25H2208N4; dark brown long rectangular plates; mp 275°C (dec.); slightly soluble in water, lower alcohols, ethyl acetate, or chloroform, and more soluble in dioxane, pyridine, DMF, or THF, monobasic acid with pK, 6.2-6.4 in 1: 1 dioxane-water; MS ndz hexamethyldihydrostreptonigrin (210),592 (M+), hexadeuteriomethyldihydrostreptonigrin (211),610 (M+);UV (MeOH) A 248 (E 38,400), 375-380 (E 17,400) nm; ‘HN M R (400 MHz, dioxane-d8) S 2.34 (3H, s, 3‘-CH3),3.84 (3H, s, 9’-OCH3), 3.88 (3H, s, 10’-OCH3),3.96 (3H, s, 6-OCH3),6.00 (2H, br, 7-NH2),6.70

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

125

(lH, d, J = 8.5 Hz, H-11’), 6.77 (lH, d, J = 8.5 Hz, H-12’), 7.83 (lH, br, 8’-OH), 8.38 (lH, d, J = 9.0 Hz, H-4), 8.93 (lH, d, J = 9.0 Hz, H-3), 11.0 (lH, br, COOH); I3C-NMR (100 MHz, dioxane-d8) S 145.3 (C-2), 126.2 (C-3), 134.2 (C-4), 127.7 (C-4a), 177.2 (C-9,137.4 (C-6), 141.0 (C-7), 181.0 (C-8), 160.8 (C-8a), 133.6 (C-2’), 138.9 (C-3‘), 130.3 (C-4’), 147.5 (C-5’), 135.0 (C-6’), 115.7 (C-7‘), 149.0 (C-8’), 137.9 (C-9’), 154.1 (C-lo‘), 105.2 (C-11’), 125.6 (C-12‘), 165.5 (COOH), 60.7 (9’-OCH3), 60.2 (6-OCH3), 55.9 (10f-OCH3),17.4 (3’-CH3). 10’-O-Demethylstreptonigrin(208): G4H2008N4; amorphous brown powder; mp 177-181°C (dec.); UV (MeOH) A 245 (E 32,800), 385 (E 12,300) nm; *H-NMR(400 MHz, di0Xane-ds) 6 8.95 (lH, d, J = 9.0 Hz, H-3), 8.41 (lH, d, J = 9.0 Hz,H-4), 6.52 (lH, d, J = 8.5 Hz, H-ll’), 6.65 (lH, d, J = 8.5 Hz, H-12’), 2.35 (3H, S, 3’-CH3), 3.83 (9’-OCH3), 3.97 (3H, S, 6OCH3), 5.89 (2H, br, 7-NH2),7.56 (lH, br, 10’-OH),7.85 (lH, br, 8’-OH), 10.9 (lH, br, COOH); 13C-NMR(100 MHz, dioxane-d8) S 181.0 (C-8), 177.2 (C-5), 160.9 (C-8a), 151.4 (C-lo’), 148.9 (C-8’), 147.7 (C-5’), 145.3 (C-2), 141.0 (C-7), 139.2 (C-3’), 137.1 (C-9’), 137.5 (C-6), 134.7 (C-6’), 134.2 (C4), 133.8 (C-2’), 130.4 (C-4‘), 127.7 (C-4a), 126.2 (C-3), 125.9 (C-12’), 114.4 (C-7’), 109.4 (C-11’), 60.6 (C-9‘-OCH3),60.2 (6-OCH3), 17.3 (3’-CH3), 165.5 (COOH). 10’-Desmethoxystreptonigrin (209): C24H2a407; blackish red needles; FABMS m/z 477.14 (MH’); UV (MeOH) A 247 (E 34,800), 379 (E 14,900) nm; UV (acidified methanol) A 247 (E 41,600), 379 (E 17,400) nm; UV (alkaline methanol) A 246 (E 43,600), 380 (E 16,100) nm; IR (KBr) v 3388, 3372,3356,3272,3010,2942,2840,1738,1648,1632,1602,1586,1564,1546, 1480,1442,1400,1372,1344,1276,1234,1214,1184,1166,1096,1074,1038, 1008, 920, 876, 830,808,790,746,712,686,658,582,556,522 cm-I. b. Synthesis. Although the challenging structure of this highly cytotoxic compound, streptonigrin (207),attracted many synthetic groups’ attention (69,77,79-81), its first total synthesis was reported by Weinreb et al. in 1980,20 years after its isolation (87,88).Its second and third total syntheses were achieved in 1981by Kende et al. (89),and in 1983by Boger and Parek (90,91), respectively. In their elegant, but long (30 steps), synthesis of streptonigrin (Scheme 25), Weinreb et al. treated the diene 212,which was prepared in eight steps, with l-(p-chlorophenyl)-4-methoxyhydantoin(213)to obtain the desired Diels-Alder adduct 214 along with an inseparable regioisomer 215, in a ratio of 3 : 1. The mixture was hydrolyzed with Ba(OH)2 to give a mixture of two amino acids, which, on esterification with SOC12/MeOH,gave the methyl ester 216 with some of the isomeric ester 217. Aromatization of the ester mixture with 5% Pd/C afforded the desired pyridine 218 with a trace of its isomer 219. Treatment of 218 with rn-CPBA gave the corresponding

126

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? xylem, ref. 56 Ye, 3 1

+ kH, 212

“I

OYy HN

0% 213

Isc

0

0

w

f

1. K2C03, MeOH

221 X = OCOCH3, 93 %

X=CI

x= 220

SCHEME25. Total synthesis of streptonigrin (207) (87,88).

7N3 CN

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

224

1. Ac20

2. K2CO3, MeOH (dry) 89 %

MllO2,88%( 227 R=CH20H 228 R=CHO 1. Dimethylmethylphosphnate rrBuli, THFMMPT 2. MnO;! 229 R = COCH2PO(OCH3)2 53 %

c

230

231

SCHEME 25. Continued

BdO

127

128

TURAN OZTURK

OH

232

I

Fremy's salt

0

N3,91 % 1.NaN3

c

loo %

233 R = H

R='

1. AIC13 2. KpCQ 63 %

I

207

S c m m 25. Continued

N-oxide 220, which with acetic anhydride afforded the acetate 221. Treatment of 221 first with K2C03/MeOH and then with SOC12 led to the formation of chloride 222. Reaction of 222 with N-cyanomethylpyrrolidine gave the quaternary salt 223, which was, without isolation, converted into aldehyde 224 by treating first with t-BuOK and then with oxalic acid. Oxidation of 224 with pertrifluoroacetic acid and then further oxidation of the N-oxide product with KMn04 gave the carboxylic acid 225. This compound was subjected to the Yamada modification of the Curtius rearrangement, then hydrolysis, to provide the amine 226. Treatment of 226 with

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

129

acetic anhydride and reaction of the crude product with K2C03/MeOH led to alcohol 227, which was oxidized with Mn02 to give aldehyde 228. Ketophosphonate 229 was obtained by treatment of 228 first with dimethylmethylphosphonateln-BuLi and then with Mn02 to oxidize the formed alcohol to the ketone. Condensation of 229 with nitroaldehyde 230 gave nitrochalcone 231, which underwent reductive cyclization with sodium hydrosulfite. Treatment of the product with NaOCH3 removed the sulfonate protecting group leading to the 5-hydroxyquinoline 232. Fremy’s salt oxidation of 232 cleanly afforded quinolinequinone 233. Treatment of 233 with iodine azide led to the introduction of iodine into the 7-position of the quinolinequinone. Iodine was converted to the amine by reacting 234 first with NaN3 to replace iodine with azide, then with sodium hydrosulfite to reduce the azide functionality to the amine. The product 235 was converted to streptonigrin (207) by first debenzylation with AlC13to obtain the methyl ester of streptonigrin and then hydrolysis of the ester group with aq. NH40H. A formal total synthesis of streptonigrin (207) was reported by Kende et al. in 1981 (Scheme 26) (89).This rather short synthesis started with the condensation of the P-keto enamine 236 with methyl acetoacetate, which gave one regiospecificproduct, the acetylpyridone 237. Reduction of ketone 237 with NaBH4 to an alcohol was followed by treatment of the product with PhPOC12 to yield the chloropyridine 238. The chlorine of 238 was replaced by CN on treatment with CuCN to give the cyanopyridine 239; then, reaction with MeMgBr and acid hydrolysis afforded the desired intermediate vinylpyridine 240. Formation of the quinoline ring was achieved by reacting 240 with the aminoimine 241 using t-BuOK. Treatment of the product with TFA for debenzylation led to the formation of the 6hydroxyquinoline 242. Nitration of 242 with HN03/MeN02,and then methylation of the phenolic hydroxyl with Me2S04,gave the 6-methoxy-5-nitroquinoline 243. The olefinic part of 243 was converted to the carboxylic acid 244 by successive treatment with Os0,JNMO (N-methylmorpholine Noxide) and then with NaI04. The methyl group of 244 was first oxidized to an aldehyde with Se02, and then this aldehyde was converted to a carboxylic acid 245 with NaC102. Selective esterification of the less hindered carboxyl group of 245 with AcCVMeOH gave 246. Introduction of an amino group at C-5’ to obtain 247 was achieved by first subjecting 246 to the Yamada modification of the Curtius rearrangement and then reducing with Na2S204.Fremy’s salt oxidation of 247 led to quinone 233, which is an intermediate in Weinreb’s total synthesis of streptonigrin (87,88). A second formal total synthesis of streptonigrin (207) was achieved by Boger and Parek in 1983 (Scheme 27) (90,91).In contrast to the previous two total syntheses, this shortest route started with the modification of commercially available 6-methoxyquinoline (248). Treatment of 248 with

130

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H 1. r-BBuoK 76-82 '70

.1

241

AcCI, 95 %

f

245 R=Rl=CQH 246 R=C@H, R i = C @ C b 247 R = N h , R1= C W H 3

34 '70

F m w s salt 92 %

* 2 3 3

SCHEME26. Formal total synthesis of streptonigrin (89).

QUINOLINEQUINONEAND QUINOLINEQUINONEIMINEUNITS

13 1

p-toluenesulfonyl chloride/potassium cyanide gave 2-cyano-6-methoxyquinoline 249 without isolation of the intermediate Reissert compound. Nitration of 249 with HN03/H2S04provided 5-nitroquinoline 250. The

pTsCI, KCN 81 % R1

248

SCHEME27. Formal total synthesis of streptonigrin (90,91).

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1. PhSeNa ~.~OY~HCVCH~OH

SCHEME 27. Continued

nitrile functional group of 250 was first converted to the thioamide 251 with HzS, and then treatment of 251 with CHJ and NaHC03/H20afforded the desired substituted quinoline 252. Reacting S-methyl thioimidate 252 with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate 253 gave the quinoline 1,2,4-triazine254, which was subsequently subjected to cycloaddition reaction with the pyrrolidine enamine 255. The desired product 256 was separated by chromatography from its isomer 257,2.8 :l, respectively. Because selective de-esterification of the 5'-carbomethoxy group, or both ester groups at 5' and 2', failed, compound 256 was progressed by dealkylative de-esterification using NaSePh, resulting in demethylation of the methoxy group ortho to NOz, along with the methyl ester. Selective methylation of the unhindered ester group with HCl/MeOH gave 258. Conversion of the hindered carboxylic acid to an amino group 259 was carried out with phenylphosphoroazidate, the Yamada modification of the Curtius rearrangement. Methylation of the free phenol provided tetracyclic arnine 247, which is identical to the compound synthesized in the work of Kende et al. (89). c. Biosynthesis. Gould et al. have established through their extensive studies that biosynthesis of streptonigrin (207) involves 4-aminoanthranilic acid (203), ~-erythrose-4-phosphate(204), and P-methyltryptophan (206) (Scheme 28) (70,71,92-95). On the basis of their evidence, they proposed that 7-aminoquinoline-2-carboxylicacid (205) is an intermediate synthesized at an early stage of the biosynthesis (71). They have also suggested

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

204

133

/r 0

206

Streptonigrin 207

SCHEME28. Biosynthetic pathway of streptonigrin (70,71,92-95).

that 4-aminoanthranilic acid (203), which was also isolated as a natural product by Gould and Erickson ( 9 9 , represents a new metabolite of the shikimate pathway (72). d. Biological Activity. Streptonigrin (207) is a highly antitumor (especially against breast, head, and neck cancers, lymphoma, and melanoma (96-98) and antibacterial compound active against both gram-positive and gram-negative bacterias (72,99).Because it is extremely toxic, it has limited clinical application, mainly because of side effects of bone marrow depression in the treated patient (200-103). Besides its antibacterial and antitumor activities, streptonigrin was reported to show potent inhibition of reverse transcriptase (202). Inouye et al. found that it is the most active inhibitor of avian myeloblastosis virus (AMV)reverse transcriptase among ca 150 antibiotics tested in their screening (205). Inouye el al. reported that streptonigrin also inhibits HIV reverse transcriptase, whereas its methyl ester inhibits only A M V reverse transcriptase (106). They also observed that when the carboxyl group on C-2’ was derivatized as an amide, the cytocidal activity of streptonigrin was lost, while no significant change in the inhibitory activity against reverse transcriptase was detected (205). The existence of a quinone pocket on A M V and HIV reverse transcriptase as a specific site of interaction was proposed (205-207). The minimum structural requirement for the cytotoxic activity was sug-

134

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gested as 260 by Rao (98), although this suggestion may need to be modified in light of the fact that the streptonigrin derivative 261 showed significant in vivo activity against mouse mammary tumor (70). Consistent with Rao's findings, isopropylazastreptonigrin did not show any anticancer activity, whereas streptonigrin methyl ester showed weak efficiency due to partial in vivo hydrolysis to streptonigrin (70). It was also reported that at very low concentration, 5 ng/ml, streptonigrin resulted in a 100% inhibition of leukemia virus replication, through inhibition of reverse transcriptase (104). At the same concentration, compound 262 and streptonigrin methyl ester were inactive. Only the methyl ester derivative showed 100% inhibition at 2.5 mg/ml, 262 was not active at any concentration. Although streptonigrin has limited use as an anticancer agent because of its high toxicity, it still receives attention of research groups. This is mainly because of its ability to degrade DNA, like some quinone antibiotics (108-111). Cleavage of a single strand of covalently closed circular DNA (ccc-DNA) in vitro was reported. The reaction was oxygen dependent and required the presence of a reducing agent, NADH (112). Considering the fact that the free-radical scavengers superoxide dismutase and catalase inhibit DNA degradation, Lown et af. proposed that free OH radicals generated by reduced streptonigrin (263),streptonigrin semiquinone radical, and oxygen are the principal species that initiate attack on the DNA. The formation of streptonigrin semiquinone (263) in vivo and in vifro was detected using electron spin resonance (ESR) spectroscopy (114-129). Involvement of divalent metals such as Cu2+,Fe2+,Zn2+,Mn2+,Cd2+, and Pd2+in the ability of streptonigrin to degrade the DNA was reported to accelerate the process (69,120-125). Complex formation of streptonigrin 0

A 261

260

0'

263

262

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

135

U

264

with Au3+was also reported (126).This complex, in the presence of reducing agents such as NADH or reduced glutathione, inhibited glutathione reductase. Degradation of DNA by a streptonigrin-Cu2+-NADPH system was investigated by ‘H-NMR and ESR spectroscopy (103). It was proposed that this system degrades isolated DNA fragments somewhat preferentially at the cytosine bases adjacent to purine base-rich sites. The B ring of the streptonigrin-Cu2+ complex interacts with purine bases, and the tricyclic phenanthridium ring system, including the copper chelate ring, appears to be important for DNA interaction and cleavage by streptonigrin. Fiallo and Garnier-Suillerot showed that streptonigrin forms a 1: 1Pd2+streptonigrin complex which catalyzes the oxidation of NADH by dioxygen (125). The quinone part of streptonigrin in this complex was reduced to a semiquinone by NADH. They found that in the absence of O2 the Pd*+semiquinone complex is very stable. Subsequent addition of dioxygen led to the formation of a superoxo-Pd2+-streptonigrin complex, which in time released the initial Pd2+-streptonigrin complex. Johnson et al. extensively investigated the structure-activity relationships of streptonigrin by designing simple bicyclic analogs (127) in the light of Lown’s results (120) that compound 264 is the most active analog, among the analogs more active than streptonigrin, in their ability to degrade the DNA. It was concluded that the presence of electron-withdrawing groups increases the reduction potential of quinones, which makes their reduction easier. As a result of their investigation, they outlined some conclusions: (i) in contrast to Lown’s claim that NADH could be replaced by NaBH4, NADH is essential; other reducing agents are ineffective; (ii) consistent with Lown’s work, Cu2+has a powerful synergistic effect on the rate of strand scission; DNA was completely protected by Co2+ and other free radical scavengers, such as sodium benzoate or by chelating agents such as EDTA, (iii) optimal time for the DNA degradation by streptonigrin is 8 h under standard experimental conditions; (iv) against a fixed concentration of DNA (200 pM), the optimal concentrations of NADH, Cu2+,and streptonigrin-are 15 mM, 100 pM, and 200 pM, respectively; (v) the optimal pH for the degradation of DNA is 8.5; (vi) the presence of quinoline nitrogen is important for activity, because naphthaquinones had no activity; (vii) isoquinolines and diazanaphthalenes are more active than quinolines, diazanaphthalenes > isoquinolines > quinolines > naphthalenes (265-270);

136

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~~

265 266 267 268 269 270

CH

~

~

N

CH CH N CH

CH CH CH CH

N

CH

CH

N

N N

N CH

~~

CH CH

CH

-0.804 -0.510 -0.418

N

-0.404

CH CH

-0.348

-0.232

0 16 42 51 66 98

(viii) the reduction potential of the compound must be -0.56 (streptonigrin) or less negative to degrade the DNA. Rao and Beach reported their results on the antimicrobial and rootgrowth inhibitory activities of a series of isoquinoline derivatives of streptonigrin (128).They confirmed the necessity of an aminoquinone function in the compound for the activity. Antibacterial activities of the isoquinoline derivatives were found to be less potent than that of streptonigrin. In contrast to the antibacterial activities, most of the isoquinoline derivatives showed activity comparable to that of streptonigrin; in some cases it was higher in the root-growth inhibition assay. The use of streptonigrin in combination with other chemotherapeutic agents such as vincristine, prednisone, and bleomycin was reported to be in clinical trials (70). Although streptonigrin (207) is known to be active against gram-positive and gram-negative bacteria, 10’-O-demethylstreptonigrin(208) was reported to have only weak antibacterial activity (83). The cytotoxic activity of demethylstreptonigrin against P388 leukemia cells in vitro is 0.59 pglml, whereas streptonigrin showed activity of 0.025 pglml. Streptonigrin and demethylstreptonigrin are active on a P388 cell line resistant to adriamycin at 0.01 pglml and 0.41 pg/ml, respectively (83). 10’-Desmethoxystreptonigrin(209) was found to be three-fold more active (IC50 of 2.1 X M) in M ) than streptonigrin (ICs0 of 6.6 X the assay for the farnesylation of rus oncogene p21 proton (84). Interestingly, the more active desmethoxystreptonigrin was reported to be less toxic (approximately five-fold) than streptonigrin, with an LDsDof 8.8 mg/ kg and of 1.8 mglkg, respectively, when injected intraperitoneally in SwissWebster mice. It was also found that desmethoxystreptonigrin is less toxic than streptonigrin to the growth of mouse bone marrow cells. Desmethoxy-

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

137

streptonigrin was reported to show cytotoxicity in vitro to human tumor cell lines such as human colon cells (IC50 of 0.004 pg/ml), human colon cells resistant to etoposide ( IC50of 0.003 pg/ml), human colon cells resistant to teniposide (IC50of 0.001 pg/ml), human ovarian cells (ICso of 0.001 pg/ ml), and human ovarian cells resistant to diamminedichloroplatinum (ICsO of 0.01 puglml). Desmethoxystreptonigrin showed toxicity at doses of 16 and 8 mg/kg in vivo when evaluated in the P388 leukemia model. It did not produce antitumor activity at the nontoxic doses of 4 and 2 mg/kg. Desmethoxystreptonigrin was reported to be a broad-spectrum antibacterial antibiotic, but did not have significant anticandidal activity (84). 3. Streptonigrone

The fifth member of the streptonigrin (207) family of streptomycete metabolites, streptonigrone (271), was isolated as a minor product by Rickards etal. in 1985 along with streptonigrin from an unidentified Streptomyces sp. collected in Fujian Province (129). Five years later, in 1990, Kozlova et al. reported the isolation of streptonigrone as a minor component, along with the major component streptonigrin, in culture broths of S. albus var. bruneomycini (130). The extract from a Streptomyces 114 fermentation was partitioned between CH2C12and cold aq. NaHC03 solution. The bicarbonate portion was acidified and extracted with CH2C12.The residue of the organic phase was chromatographed on Sephadex LH-20 using a MeOH-CHC13 (1 :99) solvent system which afforded streptonigrin as the major component. Chromatography of the CHzClzportion on Sephadex LH-20 using the same solvent system gave a greenish yellow fraction, which, on further purification by preparative TLC on silica gel in toluene-EtOAc (3 : 7), furnished streptonigrone (271) as the minor component (129).

138

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a. Characterization. Streptonigrone (271) was characterized on the basis of conventional spectroscopic methods and comparison with the spectroscopic data of streptonigrin (207) (129). Streptonigrone (271): G4H22N407; mp 268-269°C; CIMS d z 478.148 (M’); UV (MeOH) A 425 (E 9430) shifted to 343 nm upon addition of HC1; ‘H-NMR (200 MHz, CDC13) 6 2.02 (3H, s, 3’-CH3),3.95 (3H, s, OCH3), 3.99 (3H, s, OCH3),4.07 (3H, s, OCH3), 5.06 (2H, s, 5’-NH2),6.65 (lH, d, J = 8.5 Hz, H-11’ or H-12’), 6.83 (lH, d, J = 8.5 Hz, H-11’ or H-12‘), 8.31 (lH, d, J = 8 Hz, H-3), 8.35 (lH, d, J = Hz, H-4).

b. Synthesis. In 1993, Boger et al. reported the total synthesis of streptonigrone (271) in 17 steps starting from Friedlander condensation of 2amino-3-(benzyloxy)-4-bromobenzaldehyde (272) with pyruvic acid (273) to give the quinoline 274 (Scheme 29) (131). Fischer esterification of the carboxylic acid side chain of the quinoline 274 with HCl provided methyl ester 275, which was subjected to low-temperature addition of the lithium enolate of ethyl acetate to yield the P-keto ester 276. Condensation of the ester with 3,4-dimethoxy-2-hydroxybenzaldehyde(277) provided 278. Preparation of the azadiene 280 for Diels-Alder reaction involved two steps, conversion of 278 to oxime 279 with ammonium hydroxide and then formation of the oxime 0-methanesulfinate 280 with methanesulfinyl chloride. Cycloaddition was carried out in benzene between the diene 280 and 1,l-dimethoxypropane281, which led to the formation of the sensitive cycloadduct 282, subsequent aromatization of which, by treating with tBuOK followed by DDQ, provided the pyridine ring of the desired quinoline 283. Hydrolysis of lactone 283 and selective protection of the free phenol in the presence of the free carboxylic acid was effectively achieved in three steps; hydrolysis of lactone 285 with LiOH followed by treatment of the product with methoxymethyl chloride led to the protection of the phenol as well as carboxylic acid esterification.The product was then hydrolyzed with LiOH to afford 284. Replacement of the carboxylic acid functional group of 284 with an amine was achieved by the Yamada modification of the Curtius rearrangement, which provided the quinoline 285. Deprotection of the benzyl and methoxymethyl ethers of 285 was carried out with HBr. Subsequent oxidation with potassium nitrosodisulfonate (Fremy’s salt) gave quinoline-5,8-quinone286 without affecting the 8‘-OH. Methoxide was introduced to the six position of quinone 286 in the presence of Ti(O-i-Prk to afford 287. The bromine of 287 was converted to an amine in two steps, treatment of 287 with sodium azide and then reduction of the azido with sodium borohydride. The corresponding product, 7-amino-6methoxyquinoline-5,8-quinone,was then treated with HBr(g)-CF3CH20H under an H2 atmosphere, which reduced the quinone to the corresponding

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

272

c(

274 R = O H

85 %, Overnll IicLMeoH LiCkCQEt 86%

282

275 R==&

276 R=CH2CQEt

&H,

1.1BlOK I2.DDQ

I Br

SCHEME 29. Total synthesis of streptonigrone (271) (131).

139

140

TURAN OZTURK

283

1. LlOH, 97 % 2.MoMcI,Q6% 3. LIOH, 74 %

*

w

1. NaN3,85 % D 2. NaBHq, 86 % 3. &PdC HBr-CF3CbOH. air

SCHEME29. Continued

271

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

141

hydroquinone and, at the same time, selectively deprotected the 2’-methyl ether. Workup and air oxidation provided the desired streptonigrone (271). In 1992,Balzarini et al. synthesized the analog of streptonigrone, streptonigrone-2’4mine (291)(Scheme 30) (132),from streptonigrin (207).Treatment of streptonigrin with ethyl chloroformate afforded anhydride 288, reaction of which with NaN3 afforded the corresponding azide 289, which was immediately used for further reactions. Decomposition of the azide in

LNlJMI 288

289 I

I

Toluene (dry) 70 %

Af

HJN 291

290

SCHEME 30. Synthesis of analog of streptonigrone (132).

142

TURAN OZTURK

dry toluene gave the dimer 290, which, upon treatment with CF3C02HCH2CClz,afforded streptonigrone-2’4mine (291). Direct conversion of the azide 289 to the imine 291 was achieved by treatment of the azide with CF3CO2H-Hz0. The hydrochloride salt of the imine was obtained after reaction with 0.5 M HCl. Transformation of streptonigrin (207) into streptonigrone (271) over Pd-C was reported by Preobrazhenskaya et al. in 1992 (Scheme 31) (233). Treatment of streptonigrin with diphenyldiazomethane led to the diphenylmethyl ester 292, in which the phenolic hydroxyl was methylated with methyl iodide to give 293. Hydrogenation of either streptonigrin or the streptonigrin derivatives 292 and 293 over Pd-C yielded streptonigrone (271) or its methyl ester 294. The N-methylated derivative of streptonigrin, 295, was obtained along with streptonigrin derivative 296, upon treatment of streptonigrone itself with Me2S04(Scheme 32) (233). c. Biological Activity. Rickards ef al. reported that in contrast to streptonigrin (207), streptonigrone (271) showed no antimicrobial activity in disc assays at 50 pglml against strains of S. aureofaciens, S. fragilis, Bacillus subtilis, E. coli, and Saccharomyces cerevisiae (229). Streptonigrone and derivatives, streptonigrone-2’4mine (291) and its HCl salt, were reported to be less cytotoxic than streptonigrin against murine leukemia (L1210), human T-lymphoblast (MOLT4F), and human To

Pd-C 20 %

N$HPb,

72.7 %

<

207 R1=R2=H 202 R1 =H,&=CHPb

271 R = H 294 U = C b

293 R 1 = C b , & = C H P b

SCHEME 31. Transformation of streptonigrin into streptonigrone (133).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

295

296

26 %

20 %

143

SCHEME32. Synthesis of streptonigrin derivatives (133).

lymphocyte (MT-4) cells (100).They were not found to be effective against HIV-1 or HIV-2 induced cytopathogenicity in MT-4 cells at subtoxic concentrations. Streptonigrone (271) and its derivatives 294,295, and 2% were reported to demonstrate some antimicrobial properties against B. subtilis and B. micoides (133).

111. Alkaloids Containing a Quholinequinoneimine Unit

A. PYRIDOACRIDINE TYPEALKALOIDS 1. Amphimedine

In 1983, Schmitz et al. reported the isolation and structure elucidation of a second example (calliactine 364,isolated in 1940, was the first) of a pyridoacridine type alkaloid called amphimedine (297) (134).It is a fused

297

144

TURAN OZTURK

pentacyclic aromatic alkaloid isolated from a Pacific sponge Amphimedon sp. collected at Guam Island at a depth of 3 m. It was isolated by roomtemperature (CH2C12,CHC13-MeOH, MeOH) and hot Soxhlet extractions followed by successive silica gel and alumina (CHC13-MeOH) chromatography of the extract to obtain pure 297. a. Characterization. The structure of amphimedine (297) was determined by the extensive use of 2D long-range 'H/13C, I3C/l3C(INADEQUATE), and nOe couplings along with conventional spectroscopic techniques (234). Observation of a nOe enhancement between H-4 and H-5 and between H-9 and the N-methyl protons (H-14) indicated the closeness of C-5 and C-4, and the position of N-10, which is bonded to the methyl group (C-14). Amphimedine (297):CI9HllN3O2;yellow solid; mp > 360°C; HRMS d z 313.085 (M'); UV (EtOH) A 210 (e 19,690), 233 (e 39,393), 281 (e 9099), 341 (E 6060) nm; UV after treatment with NaBH4 A 235 (E 12,879),280 (e 9090) nm; IR v 1690,1640cm-'; 'H-NMR (300 MHz, TFA-d/CDC13 (2 : 1) S 8.68 (lH, d, J = 8.5 Hz, H-l), 8.39 (lH, t, J = 8.5 Hz, H-2), 8.22 (lH, t, J = 8.5 Hz, H-3), 8.97 (lH, d, J = 8.5 Hz,H-4), 9.53 (lH, d, J = 7 Hz, H5 ) , 9.29 (lH, d, J = 7 Hz, H-6), 9.20 (lH, S, H-9), 8.52 (lH, S, H-12), 4.10 (3H, S, H-14); 13C-NMR(75 MHz; TFA-dICDC13 (2: 1)) 6 133.1 (C-l), 137.4 (C-2), 132.5 (C-3), 125.8 (C-4), 120.5 (C-4a), 146.2 (C-4b), 125.2 (C5 ) , 139.0 (C-6), 139.8 (C-7a), 175 (C-8), 113.5 (C-8a), 147 (C-9), 165.9 (Cll),115 (C-12), 143.9 (C-l2a), 145.1 (C-l2b), 119.0 (C-l2c), 147.9 (C-l3a), 40.0 (C-14). b. Synthesis. The challenging structure of amphimedine (297)has attracted the attention of many synthetic groups. Four years after its isolation in 1983 (234), the first synthetic study appeared, by Rees et al. (235), and in the following year, the first total syntheses were reported by two separate groups, Stille et al. (136) and Kubo et al. (237). A third total synthesis of amphimedine was achieved by Prager et al. in 1989 (138,239).Other synthetic studies were reported by Thompson and Docter in 1989 (240), Weinreb et al. in 1989 (241), Joule et al. in 1990 (242), and QuCguiner et al. in 1994 (143). Stille et al. completed the total synthesis of amphimedine (297)in eight steps and overall 21-2370 yield (Scheme 33) (236).Their synthesis started with the readily available 4-quinolinone 298 (144), which was reacted with trifluoromethanesulfonic anhydride to give the triflate 299. Coupling of 299 with the organostannane 302 in the presence of Pd(PPh3)4yielded the 4arylquinoline 303,oxidation of which with ceric ammonium nitrate (CAN) or silver(11) oxide failed to produce any of the expected quinone. Thus,

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

145

SCHEME 33. Total synthesis of amphimedine (297) (136).

synthesis of 301 was carried out in two steps: (i) treatment of 303 with trifluoroacetic acid to hydrolyze the carbamate protecting group, and (ii) acylation of the amine with trifluoroacetic anhydride. Direct synthesis of

146

TURAN OZTURK

301 from 299 with organostannane 300 proceeded somewhat sluggishly, so the synthesis from 303 was preferred. Oxidative demethylation of 301 gave the 5,8-quinolinequinone 304, which was subjected to hetero-Diels-Alder reaction with 2-azadiene 305 (145) to give 306. Acid hydrolysis of the amide protecting group of 306 yielded quinolinequinoneimine 307, which was methylated with Me2S04to afford amphimedine (2W). Kubo ef al. achieved the total synthesis of amphimedine (297) in six steps starting from o-nitrobenzoylacetanilide (308) (Scheme 34) (137). Cyclization

308

309

310

1

CAN. 77 %

N4 &o0

1. 305 2. CH3vK2CQ

CI 312

7%

+

\

10 % Pd-C 13 %

Amphimedine 297

SCHEME 34. Total synthesis of amphimedine (297) (137).

311

147

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

of 308 in 80% H2S04 gave quinolone 309,which was aromatized in PC15/ P0Cl3to obtain the 2-chloroquinoline 310. Oxidative demethylation of 310 with CAN gave quinolinequinone 311, which was then subjected to the Diels-Alder reaction with the 2-azadiene 305. This gave a mixture of crude products, which was methylated with methyl iodide/potassium carbonate to give two N-methylisoquinolinequinones312 and 313. Catalytic hydrogenation of 313 with 10%palladium on charcoal gave amphimedine (297).In the same manner, isomer 312 was converted to isoamphimedine (314). Prager's synthesis of amphimedine (297)proceeded in six steps starting from the azafluorenone 315 (Scheme 35) (138,139). Reaction of 315 with

8 bN 1. Me&iCI 2.4-Bromopyidine Bdi

H

87 Yo

NaNPPA. 69 %

-

H

315

317

316

I

PCIyPOc~

90%

1. MeOSQF 2. KOH, K3Fe(CN)6 61 %

319

I""

318

70 %

PPA

297

CN

320

SCHEME35. Total synthesis of amphimedine (297) (138,139).

148

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1. I D A . -7OOC,M F 2. b, -70% THF 62-66W

*

321

322

1 . LDA 2. 4cyanopyridine 3. Pd(PPW4,

82%

J. 318

SCHEME 36. Synthesis of Prager's intermediate (318) (143).

hydrazoic acid resulted in migration of the most electron-rich benzene ring to give the desired diazaphenanthrene 317. The product 317 was converted to the chloropyridine 318 with phosphorus oxychloride/phosphorus trichloride. The nitrogen on the nonfused ring was selectively methylated on treatment with 1eq methyl fluorosulfonate, and alkaline ferricyanide oxidation led to 319. Chloride 319 was converted into the nitrile 320 by reaction with cuprous cyanide. As the last step, amphimedine was synthesized by cyclization of 320 in hot polyphosphoric acid. Prager's crucial intermediate 318 was later synthesized by QuCguiner et al. via a completely different route in two steps in high yield starting from 2-chloropyridine (Scheme 36) (143). Iodination of 2-chloropyridine (321) was carried out by lithiation with LDA followed by iodination to afford 2chloro-3-iodopyridine (322).Conversion of 322 to 318 was achieved by three successive one-pot procedures; (i) lithiation of 322 with LDA, (ii) reaction of the lithiated complex with 4-cyanopyridine, and (iii) Suzuki cross-coupling (146) and cyclization. 2. Ascididemin (= Leptoclinidinone), 11 -Hydroxyascididemin, and 8,9Dihydro-11-Hydroxyascididemin In 1988, Kobayashi et al. reported the isolation and structure elucidation of a pentacyclic aromatic alkaloid called ascididemin (= leptoclinidinone) (323),from the brown-colored tunicate Didemnum sp. collected at Kerama Islands, Okinawa (147,148). The methanol extract of the tunicate was partitioned between ethyl acetate and water. The silica gel chromatography (CHC13/MeOH,95 : 5) of the ethyl acetate soluble material, which exhibited antileukemic activity, was followed by repeated precipitation with chloroform to afford ascididemin (323)(0.006%,wet weight).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

323

149

324

325

Three years later in 1991, Faulkner et al. isolated ascididemin as a major metabolite from the tunicate Eudistoma sp. collected near Ave Maria Rocks on Praslin Island, Seychelles, along with the minor metabolites, the eudistones, 428,429 (249). The CH2C12-MeOH (1 : 10) extract of the tunicate was partitioned between butanol and water. Ascididemin (323) was obtained after the butanol extract was chromatographed on Sephadex LH20 using methanol as the eluant. In 1994, Francisco et al. isolated ascididemin as a major alkaloid from the grey morphs of the ascidian Cystodytes delle chiajei (Polycitor idae) collected near “Punta de la Crev” in the Balearic Isles, Spain (250). The CHClJMeOH extract of the ascidian was first solvent partitioned and then subjected to chromatographic purification, which afforded ascididemin (323) (0.43% dry weight). In 1990,Schmitz et al. reported that they had isolated ll-hydroxyascididemin (324), an analog of ascididemin (323), from an ascidian Leptoclinides sp. collected in Truk Lagoon, Federated States of Micronesia, in the course of reisolating 2-bromoleptoclinidinone (357) (252,252). The ascidian was subsequently extracted with hexane, and then with chloroform. The chloroform extract was subjected to flash chromatography on silica gel using successively hexane, chloroform, and, finally, increasing methanol concentrations in chloroform. The fifth fraction was partitioned between chloroform and 5% HCl. The extraction of the aqueous layer with chloroform, after neutralization with 1N NaOH, afforded 11-hydroxyascididemin (324). The second analog of ascididemin to be isolated, 8,9-dihydro-ll-hydroxyascididemin (325), was obtained by Kobayashi etal. in 1993from the sponge Biemna sp. collected at Unten Habor, Okinawa Island, along with the

150

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alkaloid biemnadin (153).The MeOH extract of the sponge was partitioned between EtOAc and 1 M NaC1. The aqueous layer was further extracted with n-BuOH and the extract was chromatographed on silica gel using CHCl&-BuOH/AcOWH20 (1.5 :6 : 1: 1) as eluant. Further purification by repeated Sephadex LH-20 column chromatographyusing CHC13/MeOH (1 : 1) as eluant gave 11-hydroxyascididemin (324) (0.0001%), and 8,9dihydro-11-hydroxyascididemin(325) (0.0001%). a. Characterization. The structures of ascididemin (323), ll-hydroxyascididemin (324), and 8,9-dihydro-ll-hydroxyascididemin(325) were assigned on the basis of extensive 'H- and I3C-NMR studies including 'H/ I3C correlation by long-range coupling (COLOC), and selective intensive nucleus enhancement by polarization transfer (INAPT). Their spectral data were compared with those of similar alkaloids such as neocalliactine (366), bromoleptoclinidinone (357) (151,161),and amphimedine (297) (134). A nOe enhancement experiment of ascididemin supported the connectivity of C-4a to C-4b on irradiation of H-5, which caused enhancement of H-4 (147). Resemblance of its structure to that of neocalliactine, rather than amphimedine (297), was also proved by I3C resonances at C-7a (6 149.67) and C-12a (6 145.94),which are almost the same as those of neocalliactine (6 149.4 and 145.3), but not of amphimedine (6 145.1 and 139.8) Furthermore, C-7a was coupled with only H-6 in the COLOC experiment. In contrast to the case for meridine (445) (152), no nOe was observed between the exchangeable proton of 11-hydroxyascididemin(324) and any other signals. Besides spectroscopic studies of 8,9-dihydro-ll-hydroxyascididemin (325), treatment of the compound with DDQ afforded ll-hydroxyascididemin (324) (153). Ascididemin (323): C18H9N30;yellow solid; mp > 300°C; EIMS d z 283 (M+);HRFABMS m/z 286.100 (M+ + 2 + H); UV (MeOH) A 220 (E 49,500), 248 (E 48,000), 273 sh (E 27,500), 298 (E 17,000), 308 (E 15,700), 340 sh (E 11,300), 377 (E 13,600) nm; IR (KBr) v 1680, 1600, 1580, 1410, 1260, 860, 740 cm-'; 'H-NMR (500 MHz, CDC13/CD30D,3.5 : 1.5) 6 8.55 (lH, dd, J = 7.7, 1.3 Hz,H-1), 8.05 (lH, ddd, J = 8.1, 7.7, 1.3 Hz, H-2), 7.99 (lH, ddd, J = 8.1, 7.7, 1.3 Hz, H-3), 8.76 (lH, dd, J = 7.7, 1.3 Hz, H4), 5.6 (lH, d, J = 5.6 Hz,H-5), 9.22 (lH, d, J = 5.6 HZ,H-6), 9.14 (lH, dd, J = 4.7, 1.7 Hz, H-9), 7.75 (lH, dd, J = 7.7, 4.7 Hz, H-lo), 8.79 (lH, dd, J = 7.7, 1.7 HZ,H-11); I3C-NMR (125 Hz, CDC13/CD30D (3.5 : 1.5)) 6 132.79 (d, C-l), 132.52 (d, C-2), 131.53 (d, C-3), 123.59 (d. C-4), 123.91 (s, C-4a), 138.51 (s, C-4b), 117.70 (d, C-5), 149.87 (d, C-6), 149.67 (s, C7a), 152. 38 (s, C-7b), 155.67 (d, C-9), 126.30 (d, C-lo), 136.99 (d, C-ll), 129.24 (s, C-lla), 181.99 (s, C-12), 145.94 (s, C-l2a), 118.24 (s, C-l2b), 145.75 (s, C-13a).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

151

11-Hydroxyascididemin (324): CI8H9N302;yellow amorphous solid; mp

> 250°C; LRMS d z 299.0 (M+);HRFABMS m/z 300.076 (M+ + 1); UV

(MeOH) A 203 (E 25,000), 227 (E 38,000), 275 (E 18,000), 285 (E 17,000), 370 (E 11,OOO) nm; IR (film) ~3067,1674cm-'; 'H-NMR (300 MHz, CDC13) S 8.64 (lH, dd, J = 8.1, 1.8 Hz, H-l), 8.06 (lH, dt, J = 8.1, 1.2 Hz, H-2), 8.00 (lH, dt,J = 8.1, 1.8 Hz, H-3), 8.73 (lH, dd, J = 8.1, 1.2 Hz, H-4), 8.58 (lH, d, J = 5.7 Hz, H-5), 9.31 (lH, d, J = 5.7 Hz, H-6), 7.15 (lH, d, J = 5.7 Hz, H-9), 8.89 (lH, d , J = 5.7 Hz, H-lo), 13.06 (lH, s, OH(NH)); 13CNMR (75.4 MHz, CDCl3) S 133.3 (C-l), 132.1 (C-2), 131.5 (C-3), 123.1 (C4), 137.8 (C-4b), 117.2 (C-5), 150.1 (C-6), 149.3 (C-7a), 154.1 (C-7b), 157.7 (C-9), 114.8 (C-lo), 169.5 (C-ll), 115.6 (C-lla), 181.8(C-12), 117.7(C-12b). 8,9-Dihydro-ll-hydroxyascididemin(325): CI8HllN3O2;yellow amorphous powder; mp > 300°C; EIMS d z 301 (M+);HRFABMS m/z 302.092 (M+ + H); UV (MeOH) A 218 (E 17,000), 273 (E 9300), 319 (E 6700), 355 (E 5300) nm; IR (KBr) v 3250, 2900, 1660, 1600, 1020 cm-'; 'H-NMR (CDCl&D3OD (1:l)) S 8.16 (lH, dd, J = 8.0, 1.5 Hz, H-1), 7.81 (lH, ddd, J = 8.0, 7.4, 1.1 Hz, H-2), 7.74 (lH, ddd, J = 8.0, 7.4, 1.5 Hz, H-3), 8.50 lH, dd, J = 8.0, 1.1 Hz, H-4), 8.41 (lH, d, J = 5.7 Hz, H-5), 8.94 (lH, d, J = 5.7 Hz, H-6), 3.82 (2H, t, J = 7.8 Hz, H-9), 2.59 (2H, t, J = 7.8 Hz, H-10); I3C-NMR (CDCl&D30D (1 : 1)) 6 132.0 (d, C-1), 132.5 (d, C-2), 131.5 (d, C-3), 124.5 (d, C-4), 119.0 (d, C-5), 150.5 (d, C-6), 41.0 (t, C-9), 37.0 (6, C-10). b. Synthesis. Synthesis of ascididemin (323)has attracted attention because of its challenging structure. The first synthesis was reported by Bracher in 1989 (Scheme 37) (254). His synthesis, which involved four steps, started with the oxidative amination of quinoline-5,8-quinone 326 with o-aminoacetophenone (327) to give 328, which was cyclized to the tetracyclic quinone 329 on heating with conc. H2SO4 in acetic acid. The last pyridine ring was formed in a one-pot annelation method developed by Bracher (8).The reaction of 329 with dimethylformamide diethyl acetal (DMF - DEA) gave enamine 330, treatment of which with NH4Clwithout purification afforded ascididemin (323). A second total synthesis of ascididemin (323)was achieved by Moody et al. in 1990 in two steps starting from 1,1O-phenanthroline-5,6-quinone (331)(Scheme 38) (155,156). The quinoneimine 333 was first prepared by reaction between the quinone 331 and the sodium salt of diethyl N-(2iodopheny1)-phosphoramidate(332).Photocyclizationof 333 in conc. sulfuric acid gave ascididemin (323). Because of the low yield of especially the first step, 1096, preparation of 333 was carried out via a different route in two steps. The reaction of epoxide 334 with 2-iodoaniline 335 gave the amino alcohol 336,which was readily oxidized with barium manganate to the desired intermediate 333.

152

TURAN OZTURK

U

327

326

328

& N(CH&

330

*

DMF.DEA 120oc

329

I

NHqCI,AcOH 59 %

323

SCHEME 37. Total synthesis of ascididemin (323) (154).

ll-Hydroxyascididemin was synthesized by Kubo et al. (Scheme 39) (157) in 1993 following a strategy like that of Bracher, in his first total synthesis of ascididemin (323)(154). The quinone 338 was synthesized by oxidative demethylation of the quinoline 337 with ceric ammonium nitrate (CAN). Condensation of 338 with 2-aminoacetophenone (327) in the presence of cerium(II1) chloride gave 339. Cyclization of 339 with 10% HZS04in acetic acid yielded the tetracyclic quinone 340,treatment of which with sodium methoxide in methanol substituted the chlorine with a methoxyl group to furnish the desired compound 341. The last pyridine ring was constructed by treatment of 341 with DMF-DEA. Subsequent treatment of the product with ammonium chloride in acetic acid gave the iminoquinone 342. Demethylation of 342 with boron tribromide afforded 11-hydroxyascididemin (324). In 1991, a regioisomer of ascididemin (323), isoascididemin (352),was synthesized by Echavarren et al. (Scheme 40) (258).Their eight-step synthesis started with the bromination of the readily available 343 (159) with Br2 in acetic acid to give 344,which was cyclized in diphenyl ether at 240-250°C to obtain the quinolinequinone 345. Treatment of 345 with trifluorometh-

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

333

153

336

H2SO4,32 %

323

SCHEME38. Total synthesis of ascididemin (323) (155,156).

anesulfonic anhydride gave the quinoline 346. Coupling of 346 with the arylstannane 347 was carried out by heating with Pd(PPh3)4 and LiCl in dioxane at 90°C in the presence of CuBr to afford the 4-arylquinoline 348. Treatment of 348 with a 1: 1 mixture of CF3C02H-(CF3C0)20gave the trifluoroacetamide 349, which was oxidized to the quinolinequinone 350 with CAN. The hetero-Diels-Alder cycloaddition of 350 with acrolein N,Ndimethylhydrazone (351)and then treatment with 6 M HCl-THF at 80°C gave the desired isoascididemin (352).Like ascididemin (323),2-bromoleptoclinidinine (357),and neocalliactine acetate (366), isoascididemin (352) failed to form a red complex with Fe( 11),which is a characteristic of unhindered 1,lO-phenanthrolines. In 1993, Kashman et al. reported the synthesis of 7-phenylascididemin (356)(Scheme 41) (160), which was obtained during their synthetic studies toward the total synthesis of eilatin (413)(160). A double Skraup reaction between 4-nitro-o-phenylenediamine(353)and two moles of 3'-chloropropiophenone (354)gave the diphenylphenanthroline 355,which, upon heating in dodecane under N2 in the presence of (EtO),P, unexpectedly yielded

154

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SCHEME39. Total synthesis of 11-hydroxyascididemin (324) (157).

7-phenylascididemin (356). Its structure was determined by extensive 2DNMR studies (COSY, NOESY, HMQC, and HMBC). c. Biological Activity. Kobayashi et al., who first isolated ascididemin (323),reported that it is cytotoxic against L1210 murine leukemia cells in vitro with an ICSOof 0.39 pglml(147). They also found that it is seven times more potent than caffeine, a well-known Ca2+-releaser,in the Ca2+-releasing activity in sarcoplasmic reticulum. Schmitz et af. later reported that ascididemin and hydroxyascididemin exhibited cytotoxicity to cultures of murine leukemia cells (P388) at 0.3-0.4

155

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

NHBOC

347

Br* 0%

60-64 %

346

2.6 M HCI-MF, 8OOC

&$ 92 %

0 0 0 352

SCHEME 40. Synthesis of isoascididernin (352) (158).

156

TURAN OZTURK

@r+ 15%

2

v

b 353

354

355

SCHEME41. Synthesis of 7-phenylascididemin (356)(160).

mg/ml, and that ascididemin inhibited topoisomerase I1 at 30 and 70 p M concentrations (251). Cytotoxicity of 8,9-dihydro-ll-hydroxyascididemin(325) against the human epidermoid carcinoma KB cell with an IC50 of 0.21 pg/ml and the murine lymphoma cell line L1210 with an IC5, of 0.68 pg/ml in vitro was reported by Kobayashi et al. (253). 3. 2-Bromoleptoclinidinone

In 1987, Schmitz et al. reported the isolation and structure elucidation of a pentacyclic aromatic alkaloid called 2-bromoleptoclinidinone (357) from an ascidian tentatively identified as a Leptoclinides sp. collected in Truk Lagoon, Federated States of Micronesia (252,262). A dichloromethane extract of the ascidian was vacuum flash chromatographed over silica gel using 10 : 90 methanol-acetone eluant, and further preparative thin-layer chromatography, 95 :5 chloroform-methanol elution, or silica gel chromatography, using a dichloromethane-methanol gradient, was followed by methanol addition to a chloroform solution to precipitate pure 2-bromoleptoclinidinone (357).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

157

357

358

a. Characterization. Structure determination of 2-bromoleptoclinidinone (357)was achieved with extensive long-range 'H/13C COSY and nOe experiments along with conventional spectroscopic techniques. After other possible structures were eliminated, structure 358 was first assigned as 2bromoleptoclinidinone on the grounds that treatment of the sample with Fe(11)solution did not form a colored complex,indicative of metal chelation characteristic of the 1,lO-phenanthroline present in structure 357, and no noes were observed upon irradiation of the corresponding H-4 or H-5 (161). In 1989, Schmitz et al. re-examined structure 358 in the light of the newly isolated and fully characterized alkaloid ascididemin (323),which exhibits the same pentacyclic aromatic skeleton (251,152).Kobayashi et al. reported that ascididemin also failed to form a colored complex, which Schmitz et al. used as an argument for assigning the structure 357 to 2-bromoleptoclinidinone. The crucial long-range 'H/13C couplings were re-evaluated using the more sensitive and definitive INAPT experiment with CDCl3 as solvent. On the basis of these results, the structure of 2-bromoleptoclinidinone was reassigned as 357 (162). 2-Bromoleptoclinidinone (357): C18H8BrN30;yellow powder; mp > 300°C; HRMS m/z 362.98 (M+);UV (EtOH) A 371 (E 21,000), 335 (E 18,500), 298 (E 30,600), 278 (E 32,000), 254 sh (E 27,300), 247 (E 27,800), 227 (E 151,600) nm; IR (film from evaporation of a CHC13 solution on a NaCl plate) v 3400 (br), 1680, 1600, 1580,1415, 1270 cm-'; 'H-NMR (CDC13) S 7.68 (lH, dd, J = 7.9, 4.7 Hz,H-lo), 7.98 (lH, dd, J = 8.6, 1.9 Hz, H-3), 8.43 (lH, d, J = 5.7 Hz, H-7), 8.48 (lH, d, J = 8.6 Hz, H-4), 8.71 (lH, d, J = 2.2 Hz, H-1), 8.75 (lH, dd, J = 7.9, 1.9 Hz, H-11), 9.15 (lH, dd, J = 4.7, 1.9 Hz, H-9), 9.23 (lH, d, J = 5.7 Hz, H-6); 13C-NMR(CDC13) S 116.5 (d, C-5), 117.7 (s, C-l2b), 122.1 (s, C-4a), 124.2 (d, C-4), 125.8 (d, C-lo), 126.1 (s, C-2), 128.9 (s, C-lla), 133.9 (d, C-3), 135.1 (d, C-1), 136.6 (d,

158

TURAN OZTURK

C-ll), 137.6 (s, C-4b), 146.2 (s, C-l3a), 146.6 (s, C-l2a), 149.8 (s, C-7a), 150.1 (d, C-6), 152.0 (s, C-7b), 155.6 (d, C-9), 181.2 (s, C-12).

b. Synthesis. 2-Bromoleptoclinidinone (357) was synthesized in four steps from the quinolinequinone 359 by Bracher in 1990 (Scheme 42) (263). The reaction of the quinolinequinone with aniline 360 in the presence of CeC13 yielded 361, which underwent ring closure using conc. H$04 to give 362. Formation of the last ring to afford 2-bromoleptoclinidinone was carried out by reacting 362 with N,N-dimethylformamide diethyl acetal (DMF-DEA) to give 363, and then subjecting 363 to ring closure with ammonium chloride. c. Biological Activity. 2-Bromoleptoclinidone (357)was reported to exhibit cytotoxicity to the cultures of murine leukemia cells (P388) at 0.4 pgI ml (252). Although complexation of 2-~romoleptoclinidinonein a standard colori- 2H20 metric test was not observed, its Fmplex with [~is-RuCl~{(D~)bpy}~]

* 359

DMF-DEA

I

I

361

1

H2so4

c---

Br

363

Br

362

357

SCHEME 42. Total synthesis of bromoleptoclinidinone (357) (163).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

159

was prepared and a study of its interaction with DNA was conducted by Lehn et al. in 1991 (264). 4. Calliactine In 1940, Lederer et al. reported the isolation of a red-violet pigment, calliactine, from the sea anemone Calliactis parasitica (165).A s a result of elemental analysis, the molecular formula C21HZ0N4O5 was assigned. More than 40 years later Barbier, from MS studies, suggested the molecular formula C2'Ht2N405and claimed that calliactine is a paramagnetic compound because it did not give any useful signal in the IR and in the 'Hand I3C-NMR spectra (266). A few years later Cimino et al. carried out extensive 'H- and I3C-NMR studies after establishing a different isolation procedure from the one proposed by Lederer et al. (165). It was suggested that the failure to obtain NMR spectra could be due to the extremely unfavorable solubility properties of calliactine when not freshly prepared (167). Even after these extensive NMR studies, and Schmitz's study on a comparison of the 13C-NMRdata of possible neocalliactine acetate structures (366-369) with ascididemin and meridine, the exact structure of calliactine was not revealed (252,252). Neocalliactine acetate was obtained by first boiling calliactine in 2 N HCl, followed by acetylation of the product to obtain the more soluble and less polar neocalliactine acetate (366-369) for chromatographic purification (267).As a result of these characterization studies, two possible structures, 364 and 365, were suggested (251,152,267),although Cimino et al. (167) and Schmitz et al. (252,252)slightly preferred structure 365 and 364, respectively. The specimens of C. parasitica collected in the Bay of Naples were extracted with EtOH. After removing the solvent, the remaining aqueous residue was first extracted with E t 2 0and then with n-BuOH, which afforded a brown solid. Further purification was carried out by chromatography on cellulose eluting with HzO, MeOH, and MeOH containing 1% AcOH. Fractions were monitored by silica gel TLC using n-BuOH-AcOH-H20 (60 : 15 : 25) as the eluant and spraying the plates with 1 M NaOH. In these conditions calliactine appears as a blue spot (167).

a. Characterization. Calliactine behaves as a pH indicator, changing its color from orange (in acid) to blue (in base), and can be transformed into chlorocalliactine by boiling dilute HC1 and into neocalliactine by boiling water (265). A definitive structure for calliactine has not yet been proposed, although Cimino et al. (267) and Schmitz et al. (252,252) suggested their preferred structures on the basis of extensive NMR studies. Because of the low solubility of calliactine in common solvents, neocalliactine acetate, which

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NH

H

365

384

7N+5

doAC 367

366

\

&OAC \N

N0

/

369

368

370

was prepared in two steps, (i) hydrolysis of imine and aromatization of 4hydroxypiperidine groups in HCl and (ii) subsequent acetylation of hydroxyl group, was the main source of structural information (167). Spectroscopic studies revealed four alternative structures of neocalliactine acetate, 366,367,368,and 369. Cimino et al. favored the structure 367 on the basis of nOe difference spectra. When H-8 was irradiated, besides the enhancement of H-9, the enhancement of H-6 was observed, which is compatible only with structure 367, since in the other cases H-8 and H-6

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

161

are very far away in space. To prove this result, as H-8 and H-6 are also far away in structure 367,the model compound 6-methoxy-l,7-phenanthroline (370)was synthesized. Irradiation of H-10 of 370 resulted in the enhancement of H-2 in the parallel behavior of the favored neocalliactine acetate (367).The failure to form a red complex with iron salts did not support the 1,lO-phenanthroline structures of 366 and 368. However, they also reported some contrasting evidence, including some from the gated I3CNMR spectrum in which C-12 showed a doublet (J = 3.8 Hz). Although this could be the only 4J coupling between C-12 and H-8 in their favored structure 367,Cimino et al. did not rule out the possible structures 366 and 368, which are more likely to give such a 3J coupling. In 1991,Schmitzet al. re-examined the four possible structures of neocalliactine acetates 366, 367, 368, and 369, comparing them with the close structures of ascididemin (323)and meridine (445). Considering the coupling constant of C-12 (J = 3.8 Hz), they preferred structure 366 as neocalliactine acetate and the corresponding structure (364) as calliactine, after ruling out the structure 368 and 369 for which the 13C-NMRsignals of the quaternary carbons adjacent to the carbonyl are not compatible with the meridine model (152,252). Synthesis of neocalliactine acetate (366),which was achieved by Kubo et al. in 1993, proved that the structures of neocalliactine acetate and calliactine are indeed 366 and 364, respectively (157). Calliactine (364):Cl8HI4N4O2; FABMS d z 319 (M+ + 1); EIMS d z 300 (M+-H20);UV (MeOH) A 454 (E 8500), 299 (E 9200); 272 (E 11,700), 267 (E 12,000) nm; UV (MeOH-NaOH) A 580 nm; 'H-NMR (500 MHz, CD30D) 6 3.86 (2H, m), 2.1-2.3 (2H, m), 5.10 (lH, m), 9.02 (1H d, J = 5.8 Hz), 8.61 (lH, d, J = 5.8 Hz), 7.96 (lH, d, J = 2.3 Hz), 7.49 (lH, dd, J = 8.9, 2.3 Hz), 8.18 (lH, d, J = 8.9 Hz); 13C-NMR (67.8 Hz, CD30D/ CD3C02D) 6 38.3 (t), 30.0 (t), 60.2 (d), 103.5 (s), 143.4 (s), 149.1 (d), 120.6 (d), 136.2 (s), 125.8 (s), 108.2 (d), 162.6 (s), 124.0 (d), 134.5 (3), 139.5 (s), 139.8 (s), 156.2 (s), 154.7 (s), 115.0 (s). Neocalliactine acetate (366): mp > 270°C, CZ0H11N303; HREIMS d z 341.079 (M+); EIMS d z 341 (M'); IR (CHC13) v 1765, 1648 cm-I; 'HNMR (270 MHz, CDC13-CD30D) 6 2.39 (3H, S, CH3), 7.66 (lH, dd, J = 7.9,4.6 Hz,H-lo), 7.72 (lH, dd, J = 8.9, 2.3 Hz, H-2), 8.42 (lH, d, J = 2.3 Hz, H-4), 8.47 (lH, d, J = 5.9 Hz, H-5), 8.54 (lH, d, J = 8.9 Hz, H-l), 8.72 (lH, dd, J = 7.9, 1.6 Hz, H-11), 9.08 (lH, dd, J = 4.6, 1.6 Hz, H-9), 9.18 (lH, d, J = 5.9 Hz, H-6); 13C-NMR(67.8 MHz, CDCl3-CD30D) 6 20.80 (CH3), 115.03 (C-4), 117.18 (C-5), 117.72 (C-l2b), 124.42 (C-4a), 125.82 (Clo), 126.71 (C-2), 128.81 (C-lla), 133.99 (C-1), 136.54 (C-11), 137.68 (C-4b), 143.20 (C-l3a), 145.25 (C-l2a), 149.24 (C-7a), 149.31 (C-6), 151.67 (C-7b), 152.24 (C-3), 155.15 (C-9), 169.05 (CH3CO),181.33 (C-12).

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6. Synthesis. Synthesis of the compound 366 was achieved by Kubo et ul. in 1993 in six steps starting from 6-methoxy-5,8-quinolinequinone(371) (Scheme 43) (157).Treatment of 371with 2-amino-5-methoxyacetophenone (372) in ethanol containing cerium(II1) gave 373, which was subjected to ring closure with concentrated sulfuric acid in acetic acid to afford 374. Reaction of 374 with N,N-dimethylformamide diethyl acetal (DMF-DEA) gave 375, treatment of which with ammonium chloride in acetic acid achieved a second ring closure to afford the quinolinequinoneimine 376. Demethylation was carried out in 48% HBr to yield the hydroxy quinolinequinoneimine 377, which was subsequently acetylated with acetic anhydride to afford neocalliactine acetate (366). 5. Cystodamine

Cystodamine (378) was isolated from the ascidian Cystodytes delle chiujei collected near the Bay of Gabes (at 7 m depth), Skhira, Tunisia, in 1992.

lHBl

374 R=CH3 DMF-DEA~375 R = CH=CHN(CH3)2

377

SCHEME43. Total synthesis of neocalliactine acetate (366)(257).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

163

378

CHClJMeOH extraction of the ground animals was followed by extensive chromatographic purification, which yielded cystodamine (6 mg, 0.025% of dry weight) (268). a. Characterization. Cystodamine (378) was characterized on the basis of conventional and modern spectroscopic data (268). Its 'H and I3Cspectra showed close similarities between the phenanthroline subunits of cystodamine, ascididemin (323) (247,248) and meridine (445) (251,252). Further evidence was obtained from the derivatives 378a, 37813, and 378c (Scheme 44). NMR analysis in DMSO-d6revealed the existence of an aminopyridine moiety as the pyridine ring was changed into the pyrrolidine 378a, which was consistent with the behavior of 4-aminopyridine in DMSO where the pyridylamide ion was possible. When the aetamide methyl signals of the

& I

l

378a

l

CHSO,

Ac20, Pyridine

\

+ x-

@ 378b

I

H+, 60%

SCHEME44. Conversion of cystodamine into its derivatives (268).

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monoacetate derivative 378b were irradiated, nOe enhancements of H-2 and H-12 were observed (168). Cystodamine (378):CI8Hl0N40;ESMS m/z 300 (M+ +2); FABMS d z 273; HRFABMS d z 273.08; UV (1 N HCl-CH30H) A 250 (E 4168), 278 (E 2238), 385 (E 1412) nm; IR (CHC13 + THF) v 3025, 1680, 1601, 1462, 1207,1141,845cm-I; 'H-NMR (400 MHz, CDC13 + CF3C02D) S 8.65 (lH, d, J = 8.0 Hz, H-2), 8.28 (lH, t, J = 8.0, 8.0 Hz, H-3), 8.26 (lH, t, J = 8.0, 8.0 Hz, H-4), 8.92 (lH, d, J = 8.0 Hz, H-5), 8.98 (lH, d, J = 5.6 Hz, H-6), 9.35 (lH, d, J = 5.6 Hz,H-7), 8.85 (lH, d, J = 6.8 Hz, H-ll), 7.67 (lH, d, J = 6.8 Hz, H-12); 13C-NMR(CDC13 + CF3C02D)6 145.1 (C-la), 132.4 (C-2), 134.9 (C-3), 134.7 (C-4), 124.6 (C-9,125.2 (C-5a), 139.1 (C-5b), 122.4 (C-6), 150.6 (C-7), 140.5 (C-8a), 175.3 (C-9), 148.3 (C-9a), 146.3 (C-ll), 118.4 (C-12), 158.0 (C-13), 114.6 (C-l3a), 143.2 (C-l3b), 118.0 (C-13c).

b. Biological Activity. It was reported that cystodamine (378)showed activity against CEM human leukemic lymphoblasts (with ICs0 of 1.0 pgl ml) (268). 6. Cystodytins A-J

In 1988, Kobayashi et al. reported the isolation and structure elucidation of three tetracyclic pyridoacridine type alkaloids called cystodytin A (379),

X=

Y=

A 379 R=X,R1 = H

Z=

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

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B (380),and C (381) from the Okinawan tunicate C. dellechiajei, which was collected at Kerama Islands, Okinawa, at a depth of 5 to 10 m (269). The methanol-toluene (3 :1) extract of the brown-colored tunicate was partitioned with toluene and water. The toluene extract, which showed potent cytotoxicity against L1210 murine leukemia cells, was first chromatographed on a silica gel column (CHC13/CH30H,98.5 :1.5). Sephadex LH20 column chromatography (CHC13/CH30H,1:1) then afforded a ca. 3.4 : 1 mixture of cystodytins A (379)and B (380)in 0.022% yield. This mixture could not be separated, because both compounds had the same retention times on HPLC (silica gel or ODS) using several solvent systems. The chloroform extract of the aqueous layer, which exhibited modest cytotoxicity against L1210, was purified by the same procedure to give cystodytin C (381)in 0.0003% yield. In 1991, the same group reported the isolation and structure elucidation of six new cystodytins D (382),E (383),F (384),G (385),H (386),and I (387),along with cystodytins A (379)and B (380),from the same Okinawan marine tunicate C. dellechiajei, which was recollected off the Kerama Islands, Okinawa (270). The methanol extract of the tunicate was successively chromatographed on a Diaion HP-20 using MeOH/H20 system. The fraction eluted with MeOH was subjected to silica gel column chromatography using MeOW CHC13 (from 1.5:98.5 to 3:97). The fraction eluted with MeOH/CHCI3 (1.5 :95.5) was purified by Sephadex LH-20 column chromatography using CHC13/MeOH (1 :1) as eluant to give a mixture of cystodytins F (384)and G (385)(0.001%) and a mixture of cystodytins H (386)and I (387)(0.001%). The fraction eluted with MeOH/CHC13(3 :97) was rechromatographed on Sephadex LH-20 column chromatography using CHC13/MeOH (1 : 1) as eluant to give a mixture of the known cystodytins A (379)and B (380) (0.006%) and a mixture of cystodytins D (382) and E (383) (0.0004%). None of the components of the three mixtures could be separated as the components of each pair had the same retention times on HPLC using several solvent systems. In 1994, Ireland et al. reported the isolation and structure elucidation of a new cystodytin called cystodytin J (388) from a Fijian Cystodytes sp. ascidian (172). a. Charucterization. Although only cystodytins C (381)and J (388)were isolated as pure compounds, and the remainder isolated as mixtures of cystodytins A (379)and B (380),D (382)and E (383),F (384)and G (385), and H (386)and I (387),characterization of each individual compound was achieved successfully using spectroscopic techniques such as 'H-detected heteronuclear multiple-bond 'H-13C correlation (HMBC), COSY, DEPT,

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and 'H-detected heteronuclear multiple quantum coherence (HMQC) (269-1 72). Mixture of cystodytins A (379) and B (380):G2H19N302; yellow crystals; mp 181-183°C; EIMS m / . 359 (M+ +2), 357 (M+);HRFABMS d z 360.17 (M+ + 2 + H); UV (MeOH) h 225 (E 35,000), 272 (E 25,000), 380 (E 11,400) nm; IR (ICJ3r) v 3290,2925,2850,1660, 1640, 1590, 1520, 1330, 1300, 1180,860, 760 cm-'; 'H-NMR for 379 (CDC13/CD30D,2: 1) S 8.07 (lH, dd, J = 8.2, 1.4 Hz, H-l), 7.76 (lH, ddd, J = 8.2, 8.1, 1.3 Hz, H-2), 7.64 (lH, ddd, J = 8.1, 8.1, 1.4 Hz, H-3), 8.30 (lH, dd, J = 8.1, 1.3 Hz, H-4), 8.22 (lH, d, J = 5.5 Hz, H-5), 8.81 (lH, d, J = 5.5 Hz, H-6), 3.08 (2H, t, J = 6.4 Hz, H-12), 3.59 (2H, t, J = 6.4 Hz, H-13), 6.01 (lH, br s, H-14), 5.50 (lH, qq, J = 1.4,1.3 Hz, H-16), 1.65 (3H, d, J = 1.4 Hz, H-18), 1.93 (3H, d, J = 1.3 Hz,H-19); I3C-NMR for 379 (CDC13/CD30D,2: 1) 6 131.6 (C-6), 131.7 (C-2), 129.8 (C-3), 122.8 (C-4), 121.3 (C-4a), 136.9 (C-4b), 119.4 (C-5), 149.0 (C-6), 145.8 (C-7a), 183.2 (C-8), 132.0 (C-9), 152.4 (C-lo), 149.8 (C-lOa), 117.5 (C-lob), 145.0 (C-lla), 31.3 (C-12), 38.4 (C-13), 167.8 (C-15), 118.1 (C-16), 150.8 (C-17), 26.7 (C-18), 19.4 (C-19); 'H-NMR for 380 (CDC13/CD30D,2 : l ) S 8.07 (lH, dd, J = 8.2, 1.7 Hz, H-1) 7.76 (lH, ddd, J = 8.2, 8.1, 1.3 Hz, H-2), 7.64 (lH, ddd, J = 8.1, 8.1, 1.7 Hz, H-3), 8.34 (lH, dd, J = 8.1, 1.3 Hz,H-4), 8.33 (lH, d, J = 5.5 Hz, H-5), 8.89 (lH, d, J = 5.5 Hz, H-6), 6.67 (lH, S, H-9), 3.11 (2H, t, J = 6.4 Hz, H-12), 3.60 (2H, t, J = 6.4 Hz, H-13), 6.19 (lH, qq, J = 6.9, 1.5 Hz, H-17), 1.60 (3H, dq, J = 1.5, 1.2 Hz, H-18), 1.51 (3H, dq, J = 6.9, 1.2 Hz, H-19); 13C-NMR for 380 (CDC13/CD30D,2: 1) S 131.3 (d, C-1), 131.8 (d, C-2), 129.8 (d, C-3), 122.8 (d, C-4), 121.4 (C-4a), 137.0 (C-4b), 119.5 (d, C-5), 149.1 (d, C-6), 145.9 (C-7a), 183.2 (C-8), 132.8 (d, C-9), 152.5 (C-lo), 149.9 (C-lOa), 117.5 (C-lob), 145.0 (C-lla), 30.9 (t, C-12), 39.3 (t, C-13), 170.3 (C-15), 130.3 (C-16), 130.0 (d, C-17), 11.9 (4, C-l8), 13.4 (q, C-19). Cystodytin C (381): C22H21N303; light yellow crystals; mp 257-259°C; EIMS m/z 377 (Mt+2), 375 (Mt); UV (MeOH) 228 (E 29,900), 272 (E 29,100), 380 (E 11,800) nm;IR (KBr) v 3400,2930,2850,2660,1640, 1580, 760 cm-'; 'H-NMR (CDC13/CD30D,2: 1) 6 8.25 (lH, d, J = 8.2 Hz, H-l), 7.90 (lH, dd, J = 8.2, 8.0 Hz, H-2), 7.81 (lH, dd, J = 8.0, 8.0 Hz, H-3), 8.50 (lH, d, J = 8.0 Hz, H-4), 8.44 (lH, d, J = 5.5 Hz, H-5), 9.04 (lH, d, J = 5.5 Hz, H-6), 6.86 (lH, S , H-9), 3.25 (2H, t, J = 6.4 Hz, H-12), 3.75 (2H, t, J = 6.4 Hz, H-13), 2.29 (2H, s, H-16), 1.19 (6H, s, H-18 and -19); I3C-NMR (CDC13/CDOD,2 :1) 6 131.7 (d, C-1 and -2), 129.8 (d, C-3), 122.9 (d, C-4), 121.1 (C-4a), 137.2 (C-4b), 119.5 (d, C-5), 149.9 (d, C-6), 145.3 (C-7a), 183.5 (C-8), 132.4 (d, C-9), 153.2 (C-lo), 149.9 (C-lOa), 117.0 (C-lob), 145.3 (C-lla), 31.4 (t, C-12), 38.6 (t, C-13), 172.7 (C-15), 47.6 (t, C-16), 65.0 (C-17), 28.9 (q, C-18 and -19).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

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brown-colored Mixture of cystodytins D (382)and E (383):C22H19N303; amorphous solid; [aI3OD -160" (c = 0.3, CHCl,); FABMS m/z (M' + 2 + H) 376; HRFABMS m/z 376.166 (M+ + 2 + H); UV (MeOH) A 214 (E 37,000), 274 (E 30,000), 384 (E 11,OOO)nm; IR (KBr) ~ 3 4 0 0(br), 2930,2850, 1650 cm-'; 'H-NMR for 382 (CDCl,) S 8.24 (lH, d, H-l), 7.94 (lH, dd, H-2), 7.64 (lH, dd, H-3), 8.49 (lH, d, H-4), 8.39 (lH, d, H-5), 9.10 (lH, d, H-6), 7.20 (lH, s, H-9), 5.56 (lH, m, H-12), 4.16, 3.96 (2H, m, m, H-13), 6.07 (lH, m, H-14), 5.62 (lH, brs, H-16), 1.64 (3H, q, H-18), 2.16 (3H, brs, H-19); 13C-NMR for 382 (CDCl,) 6 131.7 (d, C-1), 131.9 (d, C-2), 129.9 (d, C-3), 123.0 (d, C-4), 121.8 (C-4a), 137.1 (C-4b), 119.2 (d, C-5), 150.1 (d, C-6), 144.9 (C-7a), 183.5 (C-8), 131.2 (d, C-9), 150.0 (C-lo), 146.7 (C-lOa), 117.4 (C-lob), 144.9 (d, C-lla), 71.2 (d, C-12), 41.6 (t, C-13), 169.3 (C-15), 117.4 (d, C-16), 153.4 (C-17), 17.3 (9, C-18), 20.0 (4,C-19); 'H-NMR for 383 (CDC13) S 6.36 (lH, m, H-14). 6.48 (lH, m, H-17), 1.82 (3H, brs, H-18), 1.73 (3H, dd, J = 6.9, 1.1 Hz, H-19). Mixture of cystodytins F (384)and G (385):C23H21N303: yellow-colored amorphous solids; [Q]"D -133" (C = 0.3, CHCl3); EIMS m/z 389.35 (M' + 2); HREIMS m/z 389.17 (M' + 2); UV (EtOH) A 225 (E 30,000), 272 (E 23,000), 380 (11,000) nm; IR (film) Y 3330,2940,2850,1660,1590,1175, 1110 cm-I; IH-NMR for 384 (CDC13) 6 7.92 (lH, dt, J = 7.3, 1.0 Hz, H-2), 7.82 (lH, dt, J = 8.3, 1.0 Hz,H-3), 8.59 (lH, dd, J = 8.1, 8.1 Hz, H-4), 8.58 (lH, d, J = 5.4 Hz, H-5), 9.25 (lH, d, J = 5.4 Hz, H-6), 7.12 (lH, S, H-9), 5.40 (lH, t, J = 4.9 Hz, H-12), 3.99 (lH, ddd, J = 14.0, 5.4, 3.9 Hz, H-l3a), 3.82 (lH, ddd, J = 14.0, 6.4, 5.4 Hz, H-l3b), 5.86 (lH, m, H-14), 5.57 (lH, brs, H-16), 2.00 (3H, d, J = 1.0 Hz, H-18), 1.81 (3H, d, J = 2.0 Hz, H-19), 3.50 (3H, s, OCH,); ',C-NMR for 384 (CDCl,) S 130.1 (d, C-1), 132.2 (d, C-2), 129.9(d, C-3), 122.0 (d, C-4a), 137.4 (C-4b), 119.5 (d, C-5), 151.2 (d, C-6 or C-lo), 146.8 (C-7a), 183.8 (C-8), 131.7 (d, C-9), 150.1 (d, C-10 or C-6), 149.7 (C-lOa), 118.2 (C-lob), 145.4 (C-lla), 76.72 (d, C-12), 43.25 (t, C-13), 167.0 (C-15), 118.4 (d, C-16), 150.8 (C-17), 27.09 (9, C-18), 19.77 (q, C-19), 57.75 (q, OCH,); 'H-NMR for 385 (CDC13) 6 6.37 (lH, dd, J = 6.8, 1.5 Hz,H-14), 6.24 (lH, t, J = 4.9 Hz, H-17), 1.78 (3H, t, J = 1.2 Hz, H-18), 1.72 (3H, dd, J = 6.8, 1.2 Hz, H-19), 3.52 (3H, S, OCH,); I3C-NMRfor 385 (CDCl,) 6 130.8 (d, C-17), 12.46 (q, C-18), 13.92 (4, C-19), 57.82 (9. OCH3). yellow-colored Mixture of cystodytins H (386)and I (387):C40H51N304; amorphous solid; [(.]"D -29.1" (c = 0.3, CHCl,); FABMS m/z 640.37 (M' + 2 + H); UV (EtOH) A 225 (E 37,000), 273 (E 25,000), 382 (E 10,000) nm; IR (neat) v 3320, 2910, 2840, 1730, 1650, 1590, 1540, 1250, 1190 cm-'; 'H-NMR (CDCl,) S 8.32 (lH, brd, J = 8.3 Hz, H-1), 7.93 (lH, dt, J = 7.8, 1.5 Hz, H-2), 8.58 (lH, dd, J = 8.1, 1.8 Hz, H-4), 7.83 (lH, brt, J = 7.3 Hz, H-4), 8.57 (lH, d, J = 5.4 Hz, H-5), 9.24 (lH, d, J = 5.4 Hz,H-6),

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6.99 (lH, s, H-9), 6.85 (lH, t, J = 3.4 Hz, H-12), 4.14 (lH, m, H-l3a), 3.94 (lH, m, H-l3b), 5.71 (lH, m, H-14), 5.53 (lH, brs, H-16), 1.79 (3H, d , J = 1.5 Hz, H-18), 0.88 (3H, t, J = 6.3 Hz, H-18), 1.97 (3H, d, J = 1.5 Hz, H-19), 5.35 (2H, m, -CH=CH-), 2.83 (2H, m, -CH=CH-CHz-), 2.04 (2H, m, -CH=CH-CHz-), 1.70 (2H, m, CH2), 1.25 (22H, m, CH2). Hydrolysis of H and I gave the mixture of F (384) and G (385). Cystodytin J (388): C19H15N30t;yellow solid; FABMS (3-nitrobenzyl alcohol) d z 318 (M+ + H); FABMS (glycerol) m/z 320 (M+ + 2 + H); 'H-NMR (500 MHz, CDCl3) 6 8.29 (lH, d, J = 8.1 Hz, H-1), 7.94 (lH, ddd, J = 8.1, 7.1, 1.4 Hz, H-2), 7.83 (lH, ddd, J = 8.1, 7.1, 1.4 HZ,H-3), 8.42 (lH, d, J = 8.1 HZ, H-4), 8.18 (lH, d, J = 5.5 Hz, H-5), 8.94 (lH, d, J = 5.5 Hz, H-6), 6.95 (lH, S, H-9), 3.25 (2H, t, J = 6.4 Hz, H-12), 3.79 (2H, dt, J = 6.4, 5.9 Hz, H-13), 6.59 (lH, bs, H-14), 2.02 (3H, S , H-16); "C-NMR (125 MHz, CDCl3) S 131.87 (C-1, -2), 129.83 (C-3), 122.84 (C-4), 121.78 (C-4a), 136.92 (C-4b), 118.99 (C-5), 149.76 (C-6), 146.48 (C-7a), 183.31 (C-8), 132.82 (C-9), 152.17 (C-lo), 150.33 (C-lOa), 117.84 (C-lob), 145.32 (C-lla), 31.72 (C-12), 39.28 (C-13), 170.43 (C-15), 23.30 (C-16). b. Synthesis. The methodology that was developed by Ciufolini et al. led to the synthesis of cystodytins A (379) and B (380) via a modified Knoevenagel-Stobbe pyridine formation and a photochemical nitrene insertion (Scheme 45) (173,274). The synthesis, which was carried out in 18 steps, started with 4ethylhydroxycyclohexanone (389), which was itself prepared in four steps. Condensation of 389 with 2-azidobenzaldehyde yielded the diazide 390, whose hydroxy group was acetylated to give 391. A 1:1 mixture of two diastereomers 392 was obtained when 391 was reacted with ethyl vinyl ether in the presence of a catalyst Yb( 111). Dihydropyran 392 was readily converted into the pyridine 393 by refluxing with hydroxylamine hydrochloride in acetonitrile. Ozonolysis of the 2-azidobenzylidenederivative of 393 gave the key intermediate ketone 394, in which the ketone group was protected with ethylene glycol to afford the ketal 395. Substitution of the acetoxy group of 395 with an amine moiety was accomplished in four conventional steps to obtain 396, which could lead to cystodytin A (379) when reacted with P,P-dimethylacryloylchloride or to cystodytin B (380) when reacted with tigloyl chloride. The reactions of 3% with P,Pdimethylacryloyl chloride and with tigloyl chloride were followed by deprotection of ketone groups to give 3W and 398, respectively. Conversion of 397 and 398 to the natural products 379 and 380, respectively, was successively achieved by photochemical nitrene insertion and then oxidation with DDQ.

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

$8

tE$dA

0

0

393

392

/

/

03,Mew,-78%

then Mes. -78-25OC

AcO

0 394 395

SCHEME45. Synthesis of cystodytins A and B (173,174).

169

170

TURAN OZTURK

1. K2C03,MeOH

2.MscI 3. PotassiwnphVlaYmide 4. Hydradm wmte

396 /ride

1. p,pdi~thylacrYbyl

\

1. llgbyl chkride 2.4NHCI

2.4 N HCI

397

398 2. DDQ 380

379

ScHeuE 45. Continued

c. Biological Activity. The mixture of cystodytins A (379)and B (NO), and pure cystodytin C (381)was found to be potent cytotoxic compounds exhibiting IC50 values of 0.22 and 0.24 &ml against L1210, respectively. Both C (381) and the mixture A (379)and B (380)also showed powerful Ca2+-releasingactivity in sarcoplasmic reticulum, 36 and 13 times more potent than caffeine, respectively (269). The mixtures of cystodytins D (382)and E (383), F (384)and G (385), and H (386)and I (398)were found to be cytotoxic, exhibiting ICs0 values of 1.1 (382 and 383), 0.068 (384 and 389,and 0.080 (386 and 387) pg/ml against murine lymphoma L1210 cells, and values of 1.4 (382and 383),0.078 (384 and 389,and 0.092 (386 and 387) pg/ml against human epidermoid carcinoma KB cells (270).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

171

7. Diplamine

In 1989, Ireland et al. reported the isolation and structure elucidation of diplamine (399), which is a member of the pyridoacridine type alkaloids (275). Diplamine was isolated from a Diplosoma sp. tunicate collected in the Fiji Islands. Flash column chromatography of the extract from the tunicate, using silica gel as support and CHC13/MeOH(90 :10) as the eluting solvent, yielded 0.08% of diplamine. a. Characterization. Diplamine (399) was characterized on the basis of spectroscopic methods, including long-range H-C correlations. The structural assignment was also confirmed by oxidative demethylation of varamine A (400)(276), which gave an identical product to diplamine. burnt orange solid; mp 202-204°C (dec.); Diplamine (399): C20H19N302S; FABMS d z 366 (MH'), 368 (MH+ + 2H); UV A 377 (E 5984), 300 (E 14,722), 263 (E 24,874) nm; IR (neat) v 3323,3066,2984,2923,1651, 1600, 1533 cm-'; 'H-NMR (CDC13) 6 8.46 (lH, dd, J = 8.0, 1.4 Hz, H-1), 7.92 (lH, ddd, J = 8.0, 8.0, 1.2 Hz, H-2), 7.80 (lH, ddd, J = 8.0, 8.0, 1.4 Hz, H3), 8.27 (lH, dd, J = 8.0, 1.2 Hz, H-4), 8.35 (lH, d , J = 5.6 Hz, H-5), 9.06 (lH, d, J = 5.6 Hz, H-6), 3.73 (2H, brs, H-12), 3.73 (2H, brs, H-13), 6.45 (lH, H-14), 1.91 (3H, H-16), 2.62 (3H, S-CH,); 13C-NMR (CDCl3) S 122.76 (C-1), 129.63 (C-2), 131.93 (C-3), 118.94 (C-4), 121.28 (C-4a), 136.59 (C-4b), 131.75 (C-5), 149.71 (C-6), 146.39 (C-7a), 179.43 (C-8), 143.39 (C9), 149.51 (C-lo), 151.70 (C-lOa), 116.90 (C-lob), 145.49 (C-lla), 30.02 (C12), 39.75 (C-13), 170.56 (C-15), 23.32 (C-16), 17.89 (S-CH3). b. Synthesis. . Total synthesis of diplamine (399)was achieved by Heathcock et al. in a rather long synthesis, more than 20 steps, in 1994 (Scheme 46) (277). Their preparation of the quinoline part of diplamine involved the Knorr quinoline synthesis. Cyclization of the P-keto amide 403 prepared by reaction of the p-keto methyl ester 401 and aniline 402, which was synthesized in five steps starting from 4-methoxyphenol, gave quino-

3s9

172

TURAN OZTURK

401

402

SCHEME46. Synthesis of diplamine (145).

403

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

173

A

1. SQ.Py. DMSO 1.sq 4092. H2l 409 2.H2NSQH EtCW y . E

410

\

411

412

I

2.1 MHCl

3. A q O

&H3

1. Zn,50 % AcOH 2. CAN

399

SCHEME46. Continued

lone 404. The pyridone moiety of 404 was converted to the chloropyridine in warm POC13 and then oxidation with conc. nitric acid gave the (2hydroxyethy1)quinone 405. The existence of 405 in the hydroxyethyl form rather than as the hemiacetal form was proved by 13C-NMR,which showed the chemical shifts of two carbonyl groups at 181.48 and 184.54 ppm. Because the activation of the hydroxyl group for displacement by nitrogen nucleophiles generated significant problems, 405 was first treated with P0Cl3 to afford ring closure and the dichlorinated product 406,which was then reoxidized to quinone 407 in nitric acid. Protection of the hydroxy group with acetyl chloride, then reduction with 47%HI, gave hydroquinone 408,which was methylated with diazomethane. The corresponding dimethoxyquinoline was then deacylated with potassium carbonate to afford alcohol 409.Unfortunately, because the displacement of activated alcohol derivatives of 409 with nitrogen nucleophiles did not give the desired yields, an alternative approach, using modified reductive amination, was carried out. Thus, the alcohol side chain of 409 was first oxidized to the aldehyde and

174

TURAN OZTURK

then converted to the nitrile 410 in a one-pot step by condensation with hydroxylamine-0-sulfonic acid, followed by in situ elimination of sulfuric acid. The nitrile side chain of 410 was converted to the primary amine with borane, and after hydrolysis of the intermediate with 1 M HC1, acylation of the resulting amine during the workup gave amide 411. Oxidative demethylation of 411 with ceric ammonium nitrate was followed by treatment of the corresponding quinone with sodium methanethiolate to afford 412. Zinc-acetic acid reduction followed by oxidative workup provided diplamine (399) (177). c. Biological Activity. It was reported that diplamine (399) is among the most biologically active of all the known pyrido[2,3,4-kl]acridinealkaloids (4). It was found to be cytotoxic toward L1210 murine leukemia cells, with an ICso of 0.02 pg/ml, and antimicrobial against E. coli and S. aureus (175).

8. Eilatin In 1988, Kashman et al. reported the isolation and structure elucidation of a novel, highly symmetrical, heptacyclic, aromatic alkaloid, eilatin (413) (ca 0.001% dry wt.), from the marine tunicate Eudistoma sp. collected in the Gulf of Eilat and/or the Straits of the Gulf of Suez, in the Red Sea (178,279). A CHC13/MeOH (8: 2) extract of the tunicate was chromatographed on a silica gel column eluting with CHC13/hexane (7 :3), CHC13, and CHC13 with increasing percentages of methanol, up to 15%, by which eilatin was isolated along with several other alkaloids, including segoline A, segoline B, isosegoline A, norsegoline, and debromoshermilamine (179).Pure eilatin was obtained by crystallization from chloroform-methanol-water (178). a. Characterization. Because of the highly symmetrical nature of the molecule, various 2D-NMR experiments, such as CH correlations and a HETCOR experiment, failed to solve the structure, which was determined

413

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

175

by a single-crystal X-ray diffraction analysis (178). Like neocalliactine acetate (366) (167) and 2-bromoleptoclinidinone (357) (161), eilatin (413) did not form a red complex with iron salts as expected from its 1,lOphenanthroline moiety, but showed UV shifts in the presence of Ni2f cation (178). Eilatin (4W): C24H12N4; bright yellow powder; mp > 310°C; HREIMS m/z 356.1 (Mt); UV (MeOH) A 242 (E 48,200), 286 (E 36,700), 366 (E 11,500), 388 (E 21,000), 408 (E 30,400), 434 (E 27,000) nm; UV in the presence of Ni2+ A 296 (E 38,700), 368 (E 7300), 404 (E 18,300), 426 (E 22,500), 450 (19,900); IR (CHC13) v3000,1240,1200,1120,970 cm-I; 'H-NMR (CDC13) S 8.68 (lH, d, J = 7.2 Hz, H-1), 7.87 (lH, t, J = 7.2 Hz, H-2), 8.00 (lH, t, J = 8.0 Hz, H-3), 8.70 (lH, d, J = 8.0 Hz, H-4), 8.57 (lH, d, J = 5.5 Hz, H-ll), 9.32 (lH, d, J = 5.5 Hz, H-12); I3C-NMR (CDCl3) 6 132.1 (d, C-l), 129.3 (d, C-2), 131.7 (d, C-3), 122.5 (d, C-4), 146.1 (C-4a), 150.2 (C-5a), 118.7 (C-5c), 122.3 (C-lOa), 148.8 (C-lob), 117.1 (d, C-ll), 149.7 (d, C-12), 138.8 (C-13a). b. Synthesis. The synthesis of eilatin (413) was reported almost at the same time in 1993 by two different groups, Kashman et al., who isolated eilatin (160,178,179),and Kubo et al. (180). Starting from two natural products, kynuramine (414) and catechol (415), Kashman et al. achieved the synthesis of eilatin overall in two steps (Scheme 47) (160). The mono-protected trifluoroacetyl kynuramine (414)

414

415

416

SCHEME 47. Synthesis of eilatin (4W) (160).

176

TURAN OZTURK

was reacted with catechol under oxidative conditions to afford the 1,2acridinedione 416, basic treatment of which with NH3-MeOH in the presence of 4-dimethylaminopyridine (DMAP) as catalyst yielded eilatin (413). Kubo et al. started their synthesis with the 2-quinolinone 416, which is an intermediate in their route to amphimedine (297) (133, and completed the synthesis in seven steps (Scheme 48) (180).The pyridone ring of 417 was aromatized with trifluoromethanesulfonic anhydride to give the quinoline triflate 418. Its triflate group was removed by palladium-catalyzed triethylammonium formate reaction to yield 419. Oxidative demethylation of 419 with ceric ammonium nitrate afforded p-quinone 420. Reaction of 420 with 2-aminoacetophenone (421) in the presence of ceric ion gave the desired product 422 regioselectively. Formation of the pyridine ring was carried out with conc. sulfuric acid to afford the tetracyclic quinone 423. The third pyridine ring was constructed on 423 by reacting first with N,Ndimethylformamide diethyl acetal (DMF-DEA) and then with ammonium chloride to afford the iminoquinone 424. The fourth, and last, pyridine ring was finally constructed by catalytic hydrogenation of 424 with 10% Pd/C to afford eilatin (413). Synthesis of eilatin was also achieved from 4-[2-(trifluoroacety1amino)phenyl]quinoline-5,8-dione(425) (136) by the same group, Kubo et al., in a similar fashion (Scheme 49) (180). During the condensation of 425 with 2-aminoacetophenone (421) the second pyridine ring formed as well to yield 426, which was subjected to the third pyridine ring formation using conc. sulfuric acid to give 427; the last pyridine ring was made in the usual way with DMF-DEA to obtain eilatin (413). c. Biological Activity. In 1993, Spector et al. reported their extensive studies on the biological activities of eilatin (413) and related alkaloids (181). They found eilatin to be very potent at inhibiting the proliferation of transformed cells and in the induced differentiation and reverse transformation at concentrations of 0.05-0.2 p M , which did not have any noticeable effects on normal cells. The cells used were a mouse neuroblastoma clone NIE-115, normal hamster fibroblast NIL8 cells, and a derivative of NIL8 cells transformed by hamster sarcoma virus, NIL8-HSV. Eilatin inhibited cell multiplication in these three cell lines, induced differentiation in NIE115 cells and reversed transformation in NIU-HSV cells, and caused a cell flattening and an increase in cell size in all three cell lines. 9. Eudistones A and B

In 1991, Faulkner et al. reported the isolation and structure elucidation of two octacyclic alkaloids, eudistones A (428) and B (429), along with the known alkaloid ascididemin (323) (147,148), from the tunicate Eudistoma sp. collected near Ave Maria Rocks on Praslin Island, Seychelles (182).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

177

& 419

00 0 -

422

423

q l p \

413

424

SCHEME48. Synthesis of eilatin (413) (180).

The tunicate was extracted with CH2C12/CH30H(1: 10) and the extract was partitioned between butanol and water. Chromatographyof the extract from butanol on Sephadex LH-20 using methanol as the eluant gave as the

178

TURAN OZTURK

b 425

426

413

427

SCHEME49. Synthesis of eilatin (4W) (180).

major product ascididemin (0.26%) and a mixture of minor compounds. Further chromatography of the mixture on a Spectral 40s column using 15% aqueous methanol containing 0.01% NaCl as eluant afforded eudistones A (428) (0.0023%)and B (429) (0.0018%). a. Characterization. Structure elucidation of the eudistones was carried out using a variety of NMR techniques, including COSY, HMBC, and nOe, along with conventional spectroscopic methods (282). NOE measurements of eudistone A (428) showed the spatial proximity of H-4 and H-5 and of NH-8 and H-9. The assignment of a cis junction for eudistone A at C-7b and C-13a was made on the basis of a comparison between computer modeling calculations and coupling constant measurements between H-13a and C-7a by a heteronuclear proton-decoupling experiment. Irradiation of the H-6 signal caused the C-7a signal to appear as a doublet with J = 1.5 Hz, which coincided with the calculated cis ring junction, J = 2.6 Hz,rather than the calculated trans ring junction, J = 8.4 Hz. Eudistone B (429) was concluded to be a dehydrogenation product of

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

420

179

429

eudistone A on the basis of spectroscopic measurements. To support this, eudistone A (428) was air oxidized to obtain eudistone B, which confirmed the structure and stereochemistry of B (429). Eudistone A (428): C27H19N50; yellow amorphous powder; HRFABMS d z 430.16 (M+);UV (MeOH) A 210 (E 44,750), 238 (E 48,475), 260 (sh, E 24,670), 323 (E 15,320), 338 (E 15,480), 359 (sh, E 13,060), 395 (E 10,640) nm; CD (MeOH, 25°C) A E 209 (+4.8), 244 (-4.7), 319 (+5.1), 386 (-2.0), 450 (+l.l) nm; IR (neat, AgCl plate) 3360,3220, 1660, 1595,1535, 1200 cm-'; 'H-NMR (DMSO-&/CDC13, 2 :1) S 8.20 (lH, d, J = 8.0 Hz, H-1), 7.93 (lH, br t, J = 8.0 Hz, H-2), 7.79 (lH, br t, J = 7.5 Hz, H-3), 8.72 (lH, br d, J = 8.0 Hz, H-4), 8.52 (lH, d, J = 5.5 Hz, H-5), 8.48 (lH, d, J = 5.5 Hz, H-6), 6.34 (lH, d, J = 8.0 Hz, H-9), 7.04 (lH, br t, J = 7.0 Hz, H-lo), 6.55 (lH, br t,J = 5.5 Hz, H-11), 7.64 (lH, br d, J = 7.5 Hz, H-12), 2.92 (lH, dd, J = 12.0,5.5 Hz, H-l3a), 3.61 (lH, dd, J = 16.5, 12.0 Hz, H-14ax),-3.74 (lH, m, H-14eq),-2.69 (lH, m, H-16ax),-2.92 (lH, m, H-16eq),3.59 (lH, dt, J = 16.0, 6.5 Hz, H-17ax),3.75 (lH, dd, J = 16.0, 5.5 Hz, H-17eq),6.95 (lH, br s, D 2 0 exchangeable, H-8), 10.16 (lH, br, D 2 0 exchangeable, H-18); I3C-NMR (DMSOd6ICD3OD) S 131.0 (C-I), 132.4 (C-2), 130.5 (C-3), 124.3(C-4), 123.6 (C-4a), 137.8(C-4b), 113.7(C-4c), 116.7 (C-5), 148.0 (C-6), 157.9 (C-7a), 55.0 (C-7b), 146.4 (C-8a), 116.0 (C-9), 135.4 (C-lo), 117.9 (C-11), 126.3 (C-12), 117.9 (C-l2a), 191.8 (C-13), 45.7 (C-l3a), 41.4 (C-14), 163.8 (C-l5a), 114.9 (C-l5b), 27.4 (C-16), 38.2 (C-17), 149.9 (C18a), 144.6 (C-Mb), 144.4 (C-19a). Eudistone B (429): G7HI7N5O;white amorphous powder; HRFABMS d. 428.15 (M+); [aID-177.8' (c 0.036, MeOH); UV (MeOH) A 204 (E 45,650), 239 (E 47,365), 259 (E 39,550), 324 (E 14,670) nm; CD (MeOH, 25°C) A E 206 (+35.8), 234 (-40.2), 264 (-22.2), 307 (+34.4), 363 (-17.2) nm; IR (neat AgCl plate) v 3390,1595,1515,1020cm-'; 'H-NMR (DMSOd6ICDC13, 2 : l ) 6 8.35 (lH, br d, J = 8.0 Hz, H-l), 7.89 (lH, br t, J = 7.5 Hz, H-2), 7.74 (lH, br t, J = 7.5 Hz H-3), 8.68 (lH, br d, J = 8.0 Hz, H-4), 8.53 (lH, d, J = 6.0 Hz,H-5), 8.50 (lH, d, J = 6.0 Hz, H-6), 6.35 (lH, d, J = 8.0 Hz, H-9), 7.03 (lH, br t, J = 7.0 Hz, H-lo), 6.56 (lH, br t , J =

180

TURAN OZTURK

5.5 Hz,H-ll), 7.66 (lH, br d, J = 7.5 Hz, H-12), 3.19 (lH, dd, J = 12.0, 6.0 Hz, H-l3a), 3.38 (lH, t, J = 12.0, H-14,), 3.42 (lH, dd, J = 12.0, 6.0 Hz,H-14eq),6.69 (lH, d, J = 6.0 Hz, H-16), 8.32 (lH, d, J = 6.0 Hz, H-17),7.21 (lH, br s, D 2 0 exchangeable, H-8), 7.48 (lH, br, D 2 0exchangeable, H-15); I3C-NMR(DMSO-&/CD30D,2 :1) S 130.7 (C-1), 132.2 (C-2), 129.5 (C-3), 124.4 (C-4), 123.0 (C-4a), 137.9 (C-4b), 113.6 (C-4c), 116.7 (C-5), 147.8 (C-6), 157.7 (C-7a), 55.6 (C-7b), 145.26 (C-8a, -18b, or -19a), 116.2 (C-9), 135.3 (C-lo), 118.17 (C-11), 126.2 (C-12), 118.20 (C-l2a), 191.8 (C-13), 43.9 (C-l3a), 40.1 (C-14), 153.4 (C-l5a), 114.9 (C-l5b), 109.6 (C-16), 146.9 (C-17), 149.3 (C-l8a), 145.28 (C-8a, -18b, or -19a), 145.18 (C-8a, -18b, or -19a).

10. Eupomatidines 1, 2, and 3

In 1978, Taylor el al. isolated a yellow crystalline substance, EL-base-5, from the bark of Eupomatia laurina (Eupomatiaceae) R. Br. collected in North Queensland (183). A more thorough investigation of this material in 1991 revealed that it consisted of two closely related compounds, eupomatidine-2 (431) and eupomatidine-3 (432). Their structure elucidations were reported, along with that of the newly isolated alkaloid eupomatidine-l(430), from the aerial parts of E. bennettii collected in the Mt. Lindsay Forest, New South Wales (184). The bark of E. laurina R. Br. was extracted successively with light petroleum ether and then with methanol; the concentrated methanol extract was further shaken with ethyl acetate/water (1 : 1). The ethyl acetate phase was washed with 2% sodium hydroxide, and the recovered nonacidic material was chromatographed on silica gel. Elution with methanol/ethyl acetate (10: 90) gave EL-base-5 in 0.0011% yield (183). Further chromatography of EL-base-5 on silica gel, eluting with chloroform/ethyl acetate/methanol (80: 15 :5 ) , gave eupomatidine-2 (431) and eupomatidine-3 (432) (184). The methanol extract of the aerial parts of E. bennettii was shaken with ethedwater, the aqueous portion was strongly basified with 2 M ammonium hydroxide, and the solution was further extracted with dichloromethane.

6

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

181

The extracts from the ether and dichloromethane layers were combined and chromatographed on silica gel with a solvent gradient of increasing polarity from light petroleum to ethyl acetate and finally to methanol. Fractions eluted with ethyl acetate/methanol (70 :30) were repeatedly column chromatographed eluting with ethyl acetate/dichloromethane (20 :80), which gave pure eupomatidine-1 (430). a. Characterization. Structures of the eupomatidines 1 (430), 2 (431), and 3 (432) were assigned on the basis of modern NMR spectroscopic methods, including HETCOR, nOe, and insensitive nuclei assignment by polarization transfer (INAPT), along with conventional spectroscopic techniques (284). Eupomatidine-1 (430), 9-methoxynaphtho[l,2,3-ij][2,7]naphthyridin7(7H)-one :Cl6HI0N2o2;yellow prisms; mp 195-197°C (methanol); HREIMS d z 322.095 (M+); UV A 221 (E 14,450), 251 (E 36,300), 294 (E 9600), 305 (E 11,000), 316 (E 14,800), 447 (E 5750) nm; IR v 2928, 2854, 1699,1673,1614,1587,1486,1457,1439,1431,1374,1349,1328,1299,1384, 1263, 1098 cm-'; 'H-NMR (400 MHz, CDC13) 6 9.13 (lH, d, J = 5.6 Hz, H-2), 7.91 (lH, d, J = 5.6 Hz, H-3), 7.65 (lH, d, J = 5.9 Hz, H-4), 8.83 (lH, d, J = 5.9 Hz, H-5), 7.91 (lH, d, J = 2.8 Hz, H-8), 7.34 (lH, dd, J = 8.8, 2.8 Hz, H-lo), 8.74 (lH, d, J = 8.8 Hz, H-11), 4.01 (3H, s, OCH,); I3C-NMR (67.8 MHz, CDCl3) S 55.89 (q), 110.85 (d), 118.19 (d), 119.19, 122.52 (d), 123.45 (d), 127.53 (d), 128.77, 134.05, 138.78, 147.39 (d), 148.25, 148.46 (d), 151.45, 162.46, 181.89. Eupomatidine-2 (431), 4-methoxynaphtho[l,2,3-ij][2,7]naphthyridin7(7H)-one : C16H&02; lemon needles; mp 262-265°C (dec.) (methanol/ dichloromethane); HREIMS m/z 262.074 (M+);UV A 213 sh (E 8200), 245 (E 10,500),260 sh (E 6400),266 (E 5900), 331 (E 1650), 368 sh (E 3000), 392 (E 4550), 409 (E 4450) nm; IR v 1669, 1598, 1573, 1496, 1411, 1380, 1324, 1292, 1280, 1028 cm-'; 'H-NMR (400 MHz, CDC13) S 8.90 (lH, d, J = 6 Hz, H-2), 8.01 (lH, d, J = 6 Hz, H-3), 8.66 (lH, S, H-5), 8.49 (lH, ddd, J = 7.8, 1.2, 0.5 Hz, H-8), 7.69 (lH, ddd, J = 7.8, 7.5, 1.2 Hz, H-9), 7.82 (lH, ddd, J = 8.0, 7.5, 1.5 Hz, H-lo), 8.86 (lH, ddd, J = 8.0, 1.5, 0.5 Hz, H-ll), 4.25 (3H, s, OCH3); I3C-NMR (75 MHz, CDCl,) S 146.5 (d, C-2), 114.1 (d, C-3), 132.6 (C-3a), 152.9 (C-4), 128.7 (d, C-5), 140.5 (C-6a), 180.9 (C-7), 130.2 (C-7a), 128.1 (d, C-8), 131.1 (d, C-9), 134.1 (d, C-lo), 125.2 (d, C-ll), 135.5 (C-lla), 150.2 (C-llb), 119.8 (C-llc), 56.7 (q, OCH3). Eupomatidine-3 (432), 4,9-dimethoxynaphtho[1,2,3-ij][2,7]naphthyridine-7(7H)-one : CI7Hl2N2O3; yellow needles; 245248°C (dec.) (methanol/ dichloromethane); HREIMS d z 292.085 (M'); UV A 220 (E 8710), 230 (E 8320), 248 (E 10,900),269 (E 4900), 285 (E 4790), 324 (E 1860), 337 (E 2190), 391 (E 3390), 414 (E 3470) nm; IR v 1672, 1611, 1602, 1574, 1496, 1464, 1410, 1377, 1340, 1323, 1312, 1306, 1292, 1284, 1095, 1030, 993, 955, 919,

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828 cm-'; 'H-NMR (400 MHz, CDC13) S 8.83 (lH, d, J = 6.0 Hz, H-2), 7.92 (lH, d, J = 6 Hz, H-3), 8.64 (lH, S, H-5), 7.92 (lH, d, J = 3 Hz, H-8), 7.34 (lH, dd, J = 9.0, 3.0 Hz, H-lo), 8.75 (lH, d, J = 9.0 Hz, H-ll), 4.24 (3H, s, R1 = OCH3), 4.00 (3H, s, Rz = OCH,); I3C-NMR (67.8 MHz, CDC13-CF3C02D) (154) 856.87 (q), 58.80 (q), 115.52 (d), 117.07 (d), 118.99, 119.89, 122.91 (d), 129.38 (d), 130.42 (d), 133.69, 134.54, 137.79 (d), 138.49, 149.54, 155.34, 162.17, 177.57.

437a-c

I

43Sa-c

DMFA-DEA

&

N(CW2

NACOH HqCI,

430,431,432

4386c

SCHEME 50. Synthesis of eupomatidines (153).

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b. Synthesis. Total syntheses of eupomatidines 1 (430), 2 (431), and 3 (432) were reported by Kubo et al. in 1992 (Scheme 50) (185). Their fourstep synthesis started with the hetero Diels-Alder cycloaddition of naphthoquinones 433a and 433b with dienes 434a and 434b, which led to the formation of 435a, 435b, 436a, and 436q 435a and 436a as a 1:3 mixture in 48% yield; 436b in 79% yield; and 435b and 436c in 13 and 57% yie'ld, respectively. Oxidation of 436a-c with manganese dioxide gave the azaanthraquinones 437a-c. Condensation of 437a-c with dimethylformamide diethylacetal (DMFA-DEA) afforded 438a-c, which were converted to the corresponding eupomatidine-l(430) in 93% yield from 437a, eupomatidine2 (431) in 66% yield from 436b, and eupomatidine-3 (432) in 42% yield from 436c, respectively. 11. Kuanoniamine A

In 1990, Scheuer et al. reported the isolation and structure elucidation of four thiazolopyridoacridine alkaloids, of which kuanoniamine A (439) possesses a quinolinequinoneimine (1) subunit, from both the lamellariid mollusk Chelynotus semperi and an unidentified purple colonial tunicate collected at the Mante Channel, Pohnpei (186). The tunicates were successively extracted with methanol and then with chloroform/methanol (1 : 1) containing 1%of a 30% ammonium hydroxide solution. The aqueous residue from the combined extracts was acidified with 1 M HCI and partitioned against chloroform. The aqueous layer was basified with 10%ammonium hydroxide and partitioned against chloroform. Evaporation yielded an orange-yellow solid, which was chromatographed initially on silica gel, eluting with chloroform/methanol (80 :20), and then on HPLC, eluting with chloroformlmethanol (98 :2), which resulted in crude kuanoniamine A. A further purification by HPLC on silica eluting with dichloromethane/ethyl acetate (60 : 40) yielded pure kuanoniamine A (439) in 0.09% yield. Kuanoniamine A was also isolated from a single specimen of C. semperi using the same technique just described in 0.22% yield.

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a. Characterization. Kuanoniamine A (439)was characterized by using long-range 1H/'3C coupling (2-3JC-H and 2 - 4 J ~ - ~DEPT, ), and nOe NMR techniques, along with conventional spectroscopic methods (286). Observation of a strong nOe enhancement between H-3 and H-4 suggested their close spatial proximity. Although the regiochemistry of the thiazolo ring of kuanoniamine A was assigned by comparing the 13C-NMR data with that of the structurally similar compounds dercitamine and dercitamide (287),additional information was obtained by a heteronuclear multiple bond 'H/13C correlation experiment (HMBC). Coupling of C-12a (S 157.84) with H-11 gave a doublet (3JC-H = 13.8 Hz), whereas coupling of C-9a (6 135.21) with H-11 gave an unresolved singlet (3JC-H < 5 Hz). The similaritiesof these data with the model compound benzothiazole, in which the H2-C9 cross peak appeared as a doublet (3JC-H = 14.0 Hz), while the H2-C8 cross peak was an unresolved singlet, strongly supported the regiochemistry of the thiazolo ring of kuanoniamine A. This significantly larger coupling was attributed to electron delocalization and conjugation through the C-N bond (288). Kuanoniamine A (439): CI6H7N30S;yellow needles, yellow in neutral and basic solution, purple in acidic solution; mp 255-258°C (dec.) (chloroform); HREIMS d z 289.03 (M'); UV (MeOH) A 214 (E 18,197), 224 (E 19,054), 250 (E 15,849), 258 (E 15,488), 295 (E 6309), 354 (E 4677), 394 (E 4073) nm; UV (MeOH + H+) A 208 (E 16,218), 226 (E 17,782), 244 sh (E 14,791),292 (E 14,791),382 (E 5128) nm; IR (solution in chloroform) ~3020, 1680, 1590, 1290, 1240, 1220, 1050, 800, 750 cm-'; 'H-NMR (300 MHz, DMSO-d6) S 8.69 (lH, d, J = 6.0 Hz,H-2), 8.45 (lH, d, J = 6.0 Hz, H-3), 8.58 (lH, d, J = 8.1 Hz, H-4), 7.60 (lH, dd, J = 8.1,7.6 Hz, H-5), 7.66 (lH, dd, J = 7.8, 7.6 Hz, H-6), 8.01 (lH, d, J = 7.8 Hz, H-7), 9.28 (lH, S, H11); 13C-NMR (75 MHz, DMSO-d6) S 149.04 (C-2), 117.28 (C-3), 137.21 (C-3a), 123.07 (C-3b), 124.08 (C-4), 131.02 (C-5), 132.02 (C-6 and C-7), 144.87 (C-7a), 147.27 (C-8a), 176.17 (C-9), 135.21 (C-9a), 162.72 (C-ll), 157.84 (C-l2a), 147.12 (C-l2b), 116.45 (C-12c).

b. Synthesis. Total synthesis of kuanoniamine A (439)was achieved by Kubo et al. in 1993 starting from 6-methoxybenzothiazole-4,7-dione (440) (189) in three steps (Scheme 51) (257). Treatment of 440 with 2-aminoacetophenone (441)furnished the anilinoquinone 442, which was cyclized with concentrated sulfuric acid in trifluoroacetic acid to give 443. These relatively low yields of two steps, 39 and 36%, respectively, were avoided subsequently by the direct reaction of 440 and 441 in the presence of cerium(II1) chloride in air to give the cyclized product 443 in 73% yield. The quinone 443 was reacted with N,N-dimethylformamide diethyl acetal to form 444,which was treated with ammonium

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

185

SCHEME 51. Synthesis of kuanoniamine A (439) (257).

chloride in acetic acid to give kuanoniamine A (439) in 47% yield from 443 (157).

c. Biological Activity. Kuanoniamine A (439) was reported to be cytotoxic against KB cells exhibiting an ICs0value of 1 pg/ml (186). 12. Meridine

In 1990, Schmitz et al. reported the isolation and structure elucidation of a pentacyclic aromatic alkaloid, meridine (449,and its tautomer, isomeridine (&), from the ascidian Amphicarpa maridiana collected at Stenhouse Bay in South Australia (151,152).

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445

446

The methanol/chloroform (1 : 1) extract of the ascidian was chromatographed using flash vacuum chromatography on silica gel, eluting with increasing concentrations of chloroform in hexane, and then increasing amounts of methanol in chloroform. The impure meridine, which was obtained from the 11th fraction, was further purified either by chromatography on a chromatotron (silica gel plates, 5% MeOH in CHC13 as eluant) or a second flash chromatography on silica gel using CHC13with increasing concentrations of MeOH as eluant, which was followed by preparative layer chromatography (Si02,5% MeOH in CHC13as eluant). Additional meridine was obtained from the other methanolkhloroform extracts by fractionating the extracts with centrifugal countercurrent chromatography (CCCC) using the solvent system of chloroform/methanol/5% aqueous HCl (5 : 5 : 3). The residue from the violet fraction was partitioned between chloroform and 5% aq. HC1. Neutralization of the aqueous layer with 1 N NaOH and re-extraction with chloroform afforded additional meridine (445). Fractions 8 and 9 from the initial flash vacuum chromatography were combined and vacuum flash chromatographed again on a reverse-phase column using methanol. Further purification of the first fraction by preparative layer chromatography on silica using acetonelhexane (1 : 1) afforded isomeridine (446). In 1992, McCarthy et al. reported the isolation of meridine from a different source, the sponge Corticiurn sp. collected at depth of 450 ft at Great Inaqua Island, Bahamas (190). The CH2C12/MeOH(1 : 1) extract of the sponge was chromatographed by planetary coil countercurrent chromatography (PCCC) using MeOH/CH2C12H20(5 :5 :3) as eluant. The fractions that showed antifungal activity were further purified by RPNH2 silica column chromatography using CH2C12/MeOH(9 : 1) saturated with NH3 as eluant to yield meridine (445).

a. Characterization. Although the structure of meridine (445) was determined by long-range 1H/'3Ccorrelation NMRs along with conventional spectroscopic techniques, it was also confirmed by an X-ray analysis (151,152). NOE experiments with meridine showed that irradiation of the

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187

exchangeable proton signal at S 15.26 produced an enhancement at 6 8.23, H-1 signal. Upon being stored in CDCl3 for 1-2 years, isomeridine (446)showed 'HNMR signals indistinguishable from that of meridine, indicating that the two alkaloids are tautomers (152,252). A nOe experiment between the exchangeable proton at 6 12.51 and the proton resonating at 6 7.44, H-1, confirmed the tautomeric nature of isomeridine (446).NOE was also observed between the protons at 6 8.20 (H-1) and 6 7.64 (H-5) of isomeridine. yellow amorphoussolid, mp > 250°C; LRMS Meridine (445): C18H9N302; m/z 299.1 (M'); HRFABMS m/z 300.076 (M+ + H); UV (MeOH) A (E 25,000,227 (e 38,000), 285 (E 17,000), 370 (E 11,000); IR (thin film on KBr) v 3444,3071,1692, 1605 cm-'; 'H-NMR (300 MHz, CDC13) S 8.23 (lH, d, J = 8.1 Hz, H-1), 7.97 (lH, dd, J = 8.1, 7.8 Hz, H-2), 7.86 (lH, dd, J = 8.3, 7.8 Hz, H-3), 8.64 (lH, d, J = 8.3 Hz, H-4), 8.66 (lH, d, J = 5.6 Hz, H-5), 9.38 (lH, d, J = 5.6 Hz, H-6), 8.79 (lH, d, J = 5.5 Hz, H-lo), 7.25 (lH, d, J = 5.5 Hz, H-11), 15.26 (lH, s, OH or NH); I3C-NMR (75.4 MHz, CDCl3) S 132.6 (dd, J = 166.6, 11.2 Hz, C-l), 129.6 (dd, J = 161.0, 7.0 Hz, C-2), 129.4 (dd, J = 161.0, 7.0 Hz, C-3), 123.3 (dd, J = 164.0, 7.0 Hz, C4), 121.7 (dd, J = 14.0, 2.8 Hz, C-4a), 137.9 (t, J = 7 Hz, C-4b), 119.7 (dd, J = 162.4, 8.4 Hz, C-5), 151.5 (d, J = 182.0 Hz, C-6), 147.6 (d, J = 12.6 Hz, C-7a), 180.3 (C-8), 148.9 (d, J = 14.0 Hz, C-8a), 153.7 (d, J = 182.0 Hz, C-lo), 116.8 (dt, J = 166.6, 7.0 Hz, C-ll), 167.3 (t, J = 8.4 Hz, C-12), 116.4 (d, J = 2.8 Hz, C-l2a), 152.1 (C-l2b), 117.9 (d, J = 2.8 Hz, C-l2c), 142.6 (m, J = 7 Hz, C-13a). Isomeridine (446):C18H9N302;'H-NMR (300 MHz, CDC13) S 7.44 (lH, d , J = 8.1 Hz, H-1), 7.63 (lH, t, J = 8.1 Hz, H-2), 7.37 (lH, t, J = 8.1 Hz, H-3), 8.20 (lH, d, J = 8.1 Hz, H-4), 7.64 (lH, d, J = 5.4 Hz, H-5), 8.94 (lH, d , J = 5.4 Hz, H-6), 6.53 (lH, d , J = 8.1 Hz, C-lo), 8.10 (lH, d, J = 8.1 Hz, H-ll), 12.51 (lH, s, OH or NH).

b. Biological Activity. Schmitz et al. reported that meridine (445) exhibited cytotoxicity to cultures of P-388 murine leukemia cells at 0.3-0.4 pgl ml and inhibited topoisomerase I1 at 75 p M concentrations (152). Longley etal. reported that meridine showed selectivecytotoxicitytoward the human colon cancer cell line HT-29 with an IC,, of 0.2 pglml (292). The antifungal activity of meridine was extensively studied by McCarthy et al. (290). Their findings showed that meridine was highly active against the pathogenic yeasts C. albicans with the same minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of 0.2-3.1 pg/ml, and C. neoformans with an MIC of 0.8 pg/ml and MFC at 6.2 pg/ml. Significant activity was also observed against filamentous fungi Trichophyton mentagrophytes, with an MIC of 6.2 pg/ml, and Epidermophy-

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TURAN OZTURK

ton floccosurn, with an MIC of 1.6 pg/ml. It inhibited the growth of the gram-positive bacterium B. subtilis with an MIC of 3.1 pg/ml, but no activity was observed against the gram-negative bacteriae E. coli and Pseudomonus aeruginosa. McCarthy et al., after the incorporation of radiolabeled precursors into macromolecules, suggested that one site of action for meridine was nucleic acid biosynthesis (290). 23. Petrosamine

Petrosamine (447) was isolated from the marine sponge Petrosiu sp. collected in shallow water at Carrie Bow Cay, Belize (292). The methanol extract of the sponge was partitioned between ethyl acetate, n-butanol, and water. Chromatography of the blue-colored extract of n-butanol on Sephadex LH-20 (MeOH) resulted in the separation of a blue band, which showed antimicrobial activity. The blue material was rechromatographed on Sephadex LH-20 to afford petrosamine (447) (0.1%dry wt.). a. Characterization. The structure of petrosamine (447) was determined by X-ray diffraction measurement along with conventional spectroscopic techniques (292). The color of the compound changed with the polarity of the solvent. It gave a purple color (574 nm) in aqueous solution, a blue color (595 nm) in methanolic solution, and a green color (611 nm) in very dilute THF solution. On treatment with base, the aqueous and methanolic solutions turned green. Petrosamine (447): C21H17BrN302;dark green crystals; mp > 330°C; HREIMS d z 422.05; UV (MeOH) A 289 (E 42,600), 346 (E 12,400), 414 (E 6900), 595 (E 5300) nm; UV (H20) A 284 (E 32,000), 345 (E 10,900), 547 (E 4700) nm; UV (THF) A 611 nm; 'H-NMR (CD30D) S 3.93 (6H, s), 4.66 (3H, s), 7,90 (lH, dd, J = 8.2 Hz), 8.46 (lH, d, J = 2 Hz), 9.05 (lH, br d, J = 6 Hz), 9.27 (lH, d, J = 8 Hz),9.52 (lH, d, J = 6 Hz), 9.80 (lH, br s); 'H-NMR (DMSO-d6) S 3.86 (6H, s), 4.62 (3H, s), 7.70 (lH, s), 7.94 (lH, dd, J = 8.2 Hz), 8.34 (lH, d, J = 2 Hz), 9.14 (lH, d, J = 8 Hz), 9.22 (lH, Br

4470

Br

44m

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

189

br d, J = 6 Hz), 9.27 (lH, d, J = 6 Hz), 9.89 (lH, br s); 'H-NMR (DzO) 6 3.70 (6H, s), 4.54 (6H, s), 7.02 (lH, dd, J = 8.2 Hz), 7.12 (lH, d, J = 2 Hz), 8.11 (lH, d , J = 8 Hz), 8.71 (lH, d , J = 6 Hz),8.82 (lH, d, J = 6 Hz), 9.49 (lH, s); '3C-NMR (DMSO-ds) S 187.4, 161.4, 145.6 (d), 142.6 (d), 142.3, 141.7, 140.1, 135.3, 132.1 (d), 131.7, 128.9, 126.4 (d), 123.0, 122.1 (d), 120.4, 114.5, 114.6, 53.4 (2q), 48.6 (q); 13C-NMR(D20) S 187.3, 160.3, 144.6 (d), 142.0 (d), 141.6, 140.9, 138.5, 134.5 (d), 131.1 (d), 127.1, 125.0 (d), 1224. (d), 120.9, 114.9, 54.4 (2q), 48.6 (9). 14. Sampangines

In 1986, Rao et al. reported isolation and structure elucidation of a pentacyclic quinolinequinoneimine alkaloid called sampangine (448)from the stem bark of Cananga odorata (Annonaceae) collected at Visakhapatnam, A.P., India, along with the known alkaloid eupolauridine (450) (193). Four years later, in 1990, 3-methoxysampangine (449) was isolated by Clark et al. from the root bark of Cleistopholis patens (Annonaceae), collected on the University of Ife campus in Ife, Nigeria, along with three known alkaloids eupolauridine (450), liriodenine (451), and eupolauridine N-oxide (452) (194). The stem bark was first extracted with n-hexane in a Soxhlet apparatus, then with chloroform. The chloroform extract was column chromatographed using silica gel and eluting with benzene and benzene-EtOAc (9 : 1) and (4 : 1). Benzene-EtOAc mixtures of 9 : 1 and 4 : 1 gave sampangine (448)and eupolauridine (450), respectively (193).

448 R = H 449 R=OCH3

452

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TURAN OZTURK

The root bark was percolated successively with n-hexane, 95% EtOH, and then hot EtOH. The ethanolic extracts were combined and partitioned between CHC13/H20and then between EtOAc/HzO. The biologically active CHC13 and EtOAc fractions were combined and chromatographed over silica gel using chloroform with increasing percentages of MeOH. The fractions eluted with 5% MeOH-CHC13 showed biological activity. Four compounds were identified as 3-methoxysampangine (449), eupolauridine (450), liriodenine (451), and eupolauridine N-oxide (452). a. Characterization. Although the structure of sampangine (448)was established on the basis of 'H-NMR and other conventional spectroscopic techniques (193), 3-methoxysampangine (449) was characterized by twodimensional 'H/I3C 'J and long-range coupling techniques, along with conventional spectroscopic methods (194). Sampangine (448):Cl5H8N20;bright yellow needles; mp 210°C (dec.) (benzene); MS d z 232 (M+);UV (MeOH) A 220,252, 312, 326, 392 nm; IR (KBr) v 1680, 1620, 1402, 1380, 1320, 1275, 1225, 750 cm-'; 'H-NMR (100 MHz, CDC13) 7.71 (lH, d, J = 5 Hz, H-3 or -4), 7.80 (lH, dd, J = 7.5, 2 Hz, H-lo), 7.86 (lH, dd, J = 7.5, 2 Hz, H-9), 7.94 (lH, d, J = 5 Hz, H-3 or -4), 8.46 (lH, dd, J = 7.5, 2 Hz,H-8), 8.83 (lH, dd, J = 7.5, 2 Hz, H-11), 8.90 (lH, d , J = 5 Hz, H-2 or -5), 9.14 (lH, d, J = 5 Hz, H-2 or -5). 3-Methoxysampangine (449):C16HI0N202; yellow needles; mp 213-215°C (n-hexane-EtOAc); HRMS d z 262.074 (Mt); EIMS d z 262 (M+); UV (MeOH) A 219,253,309,332,429 nm; IR (KBr) v 1673, 1598, 1570,1380, 1300,1238,1021,945,750,720,631cm-'; 'H-NMR (300 MHz) S 8.36 (lH, S, H-2), 8.21 (lH, d, J = 5.4 Hz, H-4), 9.13 (lH, d, J = 5.4 Hz, H-5), 8.43 (lH, dd, J = 7.8, 1.2 HZ, H-8), 7.61 (lH, ddd, J = 7.8, 7.8, 1.2 Hz, H-9), 7.78 (lH, ddd, J = 7.8, 7.8, 1.2 Hz, H-lo), 8.65 (lH, dd, J = 7.8, 1.2 Hz, H-11), 4.18 (3H, S, OCH,); "C-NMR (75 MHZ) 6 126.8 (C-2), 149.9 (C-3), 131.8 (C-3a), 118.8 (C-4), 148.0 (C-5), 147.2 (C-6a), 182.0 (C-7), 131.5 (C7a), 128.5 (C-8), 130.2 (C-9), 134.6 (C-lo), 124.6 (C-11), 135.7 (C-lla), 143.2 (C-llb), 119.7 (C-llc), 56.6 (OCH3). b. Synthesis. A total synthesis of sampangine (448) was achieved by Bracher in 1989 in two steps (Scheme 52) (8). Diels-Alder cycloaddition of 2-bromo-l,4-naphthoquinone (435) to diene 454 yielded the aza-anthraquinone 455, which is the known natural product cleistopholine (2) (5). Reaction of 455 with tripiperidinomethane or dimethylformamide diethyl acetal afforded the enamines 456a or 45613, respectively,which gave sampangine (448)on heating with ammonium chloride in acetic acid without further purification. In 1992, Clark et al. reported synthesis of 3-methoxysampangine (449)

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

453

191

455

454

I

Tripiperidinomethane 62 %

or DMF-DEA 84%

SCHEME52. Total synthesis of sampangine (448)(8).

and analogs of sampangine (11).Total synthesis of 3-methoxysampangine (449) was achieved by modifying the diene 454 used in the synthesis of sampangine (Scheme 52) to 459 (Scheme 53). First, diol457 was monomethylated, and then the second hydroxyl was oxidized to the aldehyde 458 with pyridinium chlorochromate. Treatment of 458 with N,N-dimethylhydrazone provided the desired azadiene 459, which was subjected to Diels-Alder cycloaddition with 2-bromo-1,4-naphthoquinone(453) to give 4’-methoxycleistopholine (460), which is also an analog of cleistopholine. Conversion of 460 to 3-methoxysampangine (449) was carried out in the usual way; reaction with dimethylformamide diethyl acetal and then treatment of the product with ammonium hydrochloride in acetic acid gave synthetic 3methoxysampangine (449). Analogs of sampangine (448)were synthesized by the same group, Clark et al., in 1992 (11).This was achieved either by direct reactions on sampangine itself or by changing the diene 454 for conjugate addition to naphthoquinone (Schemes 54,55, and 56). Bromination of sampangine (448)with pyridinium bromide perbromide

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r

U

460

I

I

459

1. DMF-DEA 2.NH4CI,AcOH

449

SCHEME53. Synthesis of 3-methoxysampangine (449) ( J I ) .

in chloroform gave mainly 4-bromosampangine (461), with a small amount of 462 in which the ethoxy group at position 5 is presumably due to the ethanol present in the chloroform (Scheme 54). Chlorination with N-chlorosuccinimide likewise provided 4-chlorosampangine (464). Substitution of the bromine in 461with sodium azide gave 4-azidosampangine (464), which, after conversion of its azide group to amine, gave a highly fluorescent 4aminosampangine (465). Treatment of 461 with sodium methoxide afforded 4-methoxysampangine (466). On the way to the synthesis of analogs of sampangine (448),another analog of cleistopholine (2 (9,homocleistopholine (468), was obtained from Diels-Alder cycloaddition reaction of 2-bromo-1.4-naphthoquinone (453) with the diene 467. The pyridine ring was constructed in the usual fashion; 468 was first reacted with DMF-DEA, then treatment with ammonium chloride in acetic acid furnished 3-methylsampangine (469) along with 4'-oxohomocleistopholine(470) and the dimer 471 (Scheme 55). The benzo[4,5]sampangine (475) analog was prepared in three steps starting with the conjugate addition of 2'-aminoacetophenone (473) to the

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

193

C5bNHBr3 or NCS, DMF

448

461 Ri=Br,R;!=H 64% 462 Rl=Br,R2=OEt 1 % 463 R l = C I , R z = H 53%

461

"3,

W2CO 80 %

0 NaoMe M e w

464

84%

MeOH 48% l+S, piperidine 95 %

Q 466

465

SCHEME 54. Synthesis of analogs of sarnpanghe (11).

1,4-naphthoquinone472 (Scheme 56). The intermediate from this conjugate addition was cyclized with conc. H2S04in acetic acid to give benzo[2,3]cleistopholine (474). Condensation of 474 with DMF-DEA was followed by treatment with ammonium chloride to afford benzo[4,5]sampangine (475) (12).

194

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453

+

,-.& WW2

0

468

467

I

1. DMF-DEA 2. N&CI, AcOH 6%

+

468 11 Yo

469 6%

+ 470 6%

471 6%

SCHEME 55. Synthesis of 3-methylsampangine (469) (ZZ).

c. Biological Activity. Biological activities of sampangine (448)and its analogs were reported by Clark et al. (11) against the yeasts C. albicans NIH B311 and C. neoformans ATCC 32264, the filamentous fungus A. fimigatus ATCC 26934, and the atypical mycobacterium M . intracellulare

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

472

195

474

473

I

1. DMF-DEA 2. NhCI, AcOH 56 %

0 475

SCHEME56. Synthesis of benzo[4,5]sampangine (475) (11).

ATCC 23068. Antifungal/antimycobacterial activity of the sampangines was enhanced by the presence of a 3-methoxy-, 3-methyl- or 4,5-benzo- group relative to that of sampangine. However, the presence of a 4-halo-, 4-alkoxy-, or 4-amino- substituent reduced the antifungal/antimycobacterial activity relative to that of sampangine. d. Biosynthesis. In 1984,2 years before sampangine (448)was isolated, Taylor suggested its possible presence as an intermediate in the biogenetic pathway for the formation of eupolauramine (480) (Scheme 57) (13). Oxidative cleavage of ring A of the catechol 476 would eventually give the aza-anthraquinone acid 477. Transformation of 477 would lead to 478, in which ring closure to a pyridone 479 would be followed by deoxygenation to form the intermediate sampangine (448).

B. PYRROLOQUINOLINEQUINONEIMINE TYPEALKALOIDS The alkaloids containing the pyrroloquinolinequinoneiminesubunit isolated so far are called discorhabdins (481-486) and prianosins (481, 484, 487,488). They are very similar natural products that are examined together.

196

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(y$:

$ G $

Ho

0

-0 476

+-& J

478

477

479

\

480 SCHEME 57. Biogenetic pathway of sampangine (448)(13).

Some of the compounds that were isolated and named by independent groups were later noticed to be actually the same compounds. Thus, discorhabdin A (481) and discorhabdin D (484)are identical with prianosin A and prianosin D, respectively.

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

197

0

DiscotabdinA (Prianosin A) 481

DiscotabdinB 482

aq 0

I

0

I

H

0

Discorhabdin C

483

Discorhabdin D (Priamsin D)

49 484

&$$ I

0

I

0

I

H

0 DiscotabdinE

485

I

DiscotabdinF

486

Although their isolation, characterization, and biological activities are reviewed individually, synthetic studies toward discorhabdin and prianosin are examined under the same topic because each synthetic study is closely related to both groups of alkaloids. 1. Discorhabdins A-F

In 1986, Munro et al. reported the isolation of discorhabdin C (483)from a red-brown marine sponge of the genus Latrunculia du Bocage (195,196),

198

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0

Ptianosin A (DiscorhabdinA) 481

Ptianosin B 487

which was collected at depths of 20 to 75 meters off the Kaikoura Peninsula, New Zealand. Percolate extraction of the sponge with methanol/toluene (3 : 1) gave a brown paste, which was partitioned by reverse-phase flash chromatography, eluting first with water and then with a methanol/dichloromethane gradient. The cytotoxic fractions were combined and purified by two further stages of RPLC to give discorhabdin C. The isolation of discorhabdin A (481),which was also isolated and named as prianosin A by a different group from an Okinawan sponge, Prianosin melanos (204),and discorhabdin B (482) was reported in 1988 by the same group from a green marine sponge of the genus Latrunculia du Bocage collected at depths of 110 to 145 meters (discorhabdin A) and at depths of 20 to 75 meters (discorhabdin B) off the Kaikoura Peninsula, New Zealand (197-199). The sponges from the depths of 110 to 145 meters were extracted with methanol and dichloromethane to give a green extract, which was partitioned on a reverse-phase column to give a number of cytotoxic fractions containing largely discorhabdin A (481).Pure discorhabdin A was obtained after RPLC. The sponges from depths of 20 to 75 meters were extracted with methanol and toluene to give a green extract, which was separated by reverse-phase column chromatography to give a number of fractions containing largely discorhabdin B (482). Pure discorhabdin B was obtained after two further stages of preparative chromatography followed by semipreparative RPLC. Discorhabdin C (483) was also isolated in about a 3 : 1 B : C ratio. In the same year, 1988, the co-occurrence of discorhabdin A (481)with discorhabdin D (484)was reported (200). The alkaloids were isolated from the sponge Latrunculia brevis, which was collected at depths of about 30 meters off the Sugar Loaf Islands, Tranaki, New Zealand. Isolations of discorhabdin A and discorhabdin D were previously reported under the names of prianosin A and prianosin D, respectively, from a Japanese sponge of the genus Prianos (204,205). The sponge was extracted with methanol

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

199

and methanoYtoluene (3 : 1) to give a green gum, which was partitioned on a reverse-phase column to give a mixture containing largely discorhabdin A. Further preparative RPLC initially gave 484, which was followed by 481. Although discorhabdins E (485) and F (486) have been reported, it appears that no further structural data are available in the literature apart from their biological activities, which are mentioned in the biological activity section. a. Characterization. The structure and absolute configurationof discorhabdin C (483),the first isolated member of the series, was determined by a single-crystal X-ray diffraction analysis, along with other spectroscopic techniques (195,197). The structures of discorhabdins A (481), B (482) (198), and D (484) (200) were determined by spectral comparison with discorhabdin C (483). Assignment of all the protonated carbon signals was carried out using a two-dimensional heteronuclear correlation (HETCOR) experiment. For the assignment of nonprotonated carbon signals, a long-range (2JcHand 3JJH) HETCOR experiment was performed. Further analysis was carried out using COSY, nOe, and HETCOR techniques. The stereochemical configurations of C-8 and C-6 were established by a 4JHH = 1 Hz coupling of H-5 and H-7, as only their "W" conformation could provide such a coupling. 'H and I3C spectra of discorhabdin A (481), B (482),C (483),and D (484)were characterized as their hydrochloride salts. green solid; soluble in MeOH, Discorhabdin A (481): C18HI4BrN3O2S; H20, and DMSO; mp > 360°C; [a],,+ 400" (c 0.05, CH,OH); IR (KBr) v 3700-2400, 1680, 1620, 1585, 1530, 1410, 1385 cm-'; UV (CH30H) A 249 (E 29,500), 351 (E 10,500), 567 (E 900) nm; UV (CH30H/KOH) A 335 (E 14,000),473 (E 1OOO) nm; FABMS m/z 416/418 (MH+),EIMS d z 383/385 (M+-S); 'H-NMR (300 MHz, CD30D, for NH-9, NH-13, and NH-18 in (CD3)zSO) S 7.58 (lH, S, H-l), 2.88 (lH, dd, J = 17, 6.5 Hz, H-4), 3.07 (lH, dd, J = 17, 12 Hz, H-4), 4.53 (lH, dd, J = 12,6.5 Hz,H-5), 2.60 (lH, dt, J = 12.5, 1.0, 1.0 Hz, H-7), 3.0 (lH, dd, J = 12.5, 4.0 Hz, H-7), 5.38 (lH, dd, J = 4.0, 1.0 Hz, H-8), 10.5 (lH, s, NH-9), 13.4 (lH, s, NH-13), 7.19 (lH, t, J = 0.5, 0.5 Hz, H-14), 2.97 (2H, m, H-16), 3.80 (lH, dt, J = 14.5, 10.0, 10.0 Hz, H-17), 3.94 (lH, dt, J = 14.5, 6.0, 6.0 Hz, H-17), 9.8 (lH, S, NH+-18);13C-NMR(75 MHz, (CD3)zSO) 6 147.96 (d, J = 169 Hz, C-1), 125.78 (C-2), 186.67 (C-3), 44.43 (dd, J = 135, 127 Hz, C-4), 53.76 (d, J = 149 Hz, C-5), 49.50 (C-6), 40.4 (C-7), 58.72 (d, C-8), 150.16 (C-10 or -19), 165.83 (C-11), 123.25 (C-12, -15, or -21), 127.28 (d, C-14), 119.76 (C-15, -21, or -12), 18.04 (d, C-16), 43.84 (d, C-17), 153.58 (C-19 or -lo), 103.74 (C-20), 123.13 (C-21, -15, or -12). Discorbabdin B (482): CI8Hl2BrN3O2S; green solid; sparingly soluble in

200

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MeOH and H20, soluble in DMSO; mp > 360°C; [o!]D + 400" (c 0.17, CH30H); IR (KBr) Y 3700-2400, 1740, 1650, 1620, 1520, 1410 cm-'; UV (CH30H) A 248 (E 30,600), 309 (E 10,800), 357 (E 10,600), 567 (E 1100) nm; UV (CH30H/NaOH) A (E 25,500), 306 (E 14,700); 'H-NMR (300 MHz, CD30D, for NH-9, NH-13, and NH-18 in (CD3)2SO) S 7.87 (lH, s, H-l), 6.24 (lH, S, H-4), 2.54 (lH, dd, J = 11.5, 6.0, 3.5 HZ,H-7), 2.81 (lH, d, J = 11.5 Hz, H-7), 5.72 (lH, d, J = 3.5 HZ,H-8), 10.8 (lH, S, NH-9), 13.5 (lH, S, NH-13), 7.21 (lH, S, H-14), 2.92 (2H, dd, J = 9.0, 6.5 Hz, H-16), 3.83 (lH, dt, J = 14.0, 9.0, 9.0 Hz, H-17), 3.94 (lH, dt, J = 14.5, 6.5, 6.5 Hz, H-17), 8.7 (lH, S, NH+ -18); 13C-NMR (75 MHz, (CD3)zSO) S 146.0 (d, C-1). 128.21 (C-2), 174.14 (C-3), 119.32 (d, C-4), 169.99 (C-5), 51.44 (C6), 42.9 (C-7), 61.30 (d, C-8), 150.9 (C-10 or -19), 165.16 (C-11), 123.51 (C12, -15, or -21), 127.42 (d, C-14), 120.53 (C-15, -21, or -12), 18.06 (C-16), 45.15 (C-17), 154.22 (C-19 or -lo), 97.17 (C-20), 123.22 (C-21, -15, or -12). Discorhabdin C (483): CI8Hl4Br2N3O2; red solid; soluble in MeOH, H20, and DMSO; mp > 360°C [.ID 0" (c 0.05, MeOH); IR (KBr) Y 3700-2500, 1675,1585,1540,1325, 1020,695 cm-'; UV (MeOH) A 245 (E 28,500), 351 (E lO,OOO), 545 (E 500) nm; UV (MeOWKOH) A 337 (E 13,000), 481 (E 1500) nm; FABMS (glycerol matrix) m/z 462/464/466 (MH'); 'H-NMR (300 MHz, CD30D, for NH-9, NH-13, and NH-18 in (CD3)2SO) S 7.73 (lH, S, H-l), 7.73 (lH, S, H-5), 2.12 (2H, t, H-7), 3.73 (2H, t, J = 6.0 Hz, H-8), 10.3 (lH, S, NH-9), 13.4 (lH, S, NH-13), 7.22 (lH, S, H-14), 2.90 (2H, t, J = 7 Hz, H-16), 3.79 (2H, t, J = 7 Hz, H-17), 8.3 (lH, s, NH+ -18); 13CNMR (75 MHz, (CD3)2SO) S 151.41 (d, C-1), 122.78 (C-2), 171.52 (C-3), 122.78 (C-4), 151.41 (d, C-5), 44.87 (C-6), 33.83 (d, C-7), 38.46 (d, C-8), 151.98 (C-10 or -19), 165.52 (C-11), 123.35 (C-12), 127.86 (d, C-14), 120.00 (C-15 or -21), 18.22 (d, C-16), 43.87 (d, C-17), 153.39 (C-19 or -lo), 91.89 (C-20), 123.74 (C-21 or -15). Discorhabdin D (484): C18HI4N3O2S;deep green solid; mp > 360°C; [~y]578 -45", [.I546 -160" (C 0.15, CH3OH); v 3700-2300, 1650, 1620, 1550, 1525,1490,1410,1310cm-'; UV (CH30H) A 248 (E 22,387) 281 (E 14,125), 320 (E 8511), 395 (E 8912), 584 (E 692) nm; UV (CH30H/KOH) A 362 (e 30,902), 290 ( E 15,488), 368 ( E 9550) nm; HRFABMS d . 336 (MH+); 'H-NMR ((CD3)2SO) S 3.02 (lH, d, J = 12.8 Hz, H-1), 2.55 (lH, d, J = 12.8 Hz, H-l), 4.47 (lH, S, H-2), 6.22 (lH, S, H-4), 2.80 (lH, d, J = 12.8 Hz, H-7), 2.65 (lH, d, J = 12.8 Hz, H-7), 5.79 (lH, S, H-8), 10.8 (lH, S, NH-9), 13.45 (lH, s, NH-13), 7.37 (lH, s, H-14), 3.15 (2H, m, H-16), 4.13 (lH, m, H-17), 3.92 (lH, m, H-17); 13C-NMR((CD3)2SO)S 30.27 (t, C-1), 62.26 (d, C-2), 183.08(C-3), 112.43 (d, C-4), 173.14 (C-5), 41.19 (C-6), 38.59 (t, C-7), 62.79 (d, C-8), 147.84 (C-10 or -19), 166.47 (C-11), 123.69 (C-12 or -21), 127.0 (d, C-14), 117.71 (C-15), 1949 (t, C-16), 51.24 (t, C-17), 145.90 (C-19 or -lo), 99.64 (C-20), 121.50 (C-21 or -12).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

20 1

b. Biological Activity. Discorhabdins A (481), B (482), and C (483) were reported to be highly active in the in vitro P-388 assays, with EDso values of 0.03,0.05, and 0.1 pglml, respectively. However, in vivo testing in the P-388 leukemia system in mice did not show any antitumor activity for either discorhabdin C or discorhabdin A. Discorhabdin B showed nonsignificant antitumor activity, with a T/C of 117%at a dose of 0.25 mglkg (197,198). Discorhabdins A, B, and C showed antimicrobial activity. In a disk assay, at 30 pgldisk, discorhabdins A and C were active against E. coli, B. subtilis, and C. albicans, but not against Pseudomonas aeruginosa. Discorhabdin B was found to be active against E. coli and B. subtilis, but not against P. aeruginosa or C. albicans ( 197,198). Discorhabdin D (484)was reported to have antimicrobial activity (at 30 pgldisk) against E. coli, B. subtilis, and C. albicans, but not against P. aeruginosa (200).Discorhabdin E (485) was reported to be strongly inhibitory against the gram-negative bacillus E. coli (201). Although all of the discorhabdins have effective in vitro properties, only discorhabdin D showed in vivo antitumor activity (T/C 132%) against the P-388 murine leukemia (201).During a cytotoxicity testing against human colon tumor cell line HCT-116, discorhabdin A was found to be cytotoxic 0.04 pglml) (202,203).Discorhabdin A and C were also reported to be effective against EL-4.1L cell adhesion (191). 2. Prianosins A -D

In 1987, Kobayashi et al. reported on the isolation of a novel alkaloid, prianosin A (481),from the Okinawan marine sponge Prianos melanos (204).The methanol-toluene extract of the green sponge, which was collected at a depth of 2 to 3 meters in the Motobu Peninsula of the Okinawa Island, was partitioned between toluene and water. The water layer was extracted with chloroform and extensive column chromatography afforded prianosin A (481)(0.02%,wet wt.). A year later, the same group reported on the isolation of prianosins B (487), C (488), and D (484) from the same sponge (205). The subsequent silica gel (CHC13/MeOH, 98 : 2 to 80 :20) and Sephadex LH20 (CHClJMeOH, 1 : 1) column chromatographies of the chloroform extract of the sponge gave prianosins C and D, in 0.008 and 0.007% yield of wet weight, respectively, and a mixture of prianosins A and B. The alkaloids were separated by silica gel column chromatography (petroleum ether/CHC13/MeOH, 20 : 5 : 1) in yields of 0.02% for A and 0.001% for B, wet weight. a. Characterization. The structure and absolute configuration of prianosin A (481), which are identical with those of discorhabdin A (1 9 9 ,

202

TURAN OZTURK

were determined by single-crystalX-ray analysis, along with spectroscopic techniques (204). The structure of prianosin B (487) was determined to be the dehydrogenated form of prianosin A at C-16 and C-17. The 'H-NMR data of prianosin B showed only a difference for the vicinal signals at (2-16 and C-17, which showed a typial ortho-coupling pattern of the alpprotons on a pyridine ring (6 7.51 (d, J = 5.9 Hz, H-16) and 8.46 (d, J = 5.9 Hz, C-17)). The absolute stereochemistry of B was assigned by comparison of the CD curve with that of A, whose stereochemistry had been determined unambiguously by X-ray diffraction analysis. The structure elucidation of prianosin C (488)and prianosin D (484) were first carried out with their acetate forms because of their high polarity and instability in solution. Their structures were determined as C (491) and D (492), and the corresponding acetate derivatives as C (489) and D (490) using spectroscopic techniques, including 'H-I3C COSY, DEPT, HOHAHA, and HMBC. Almost at the same time, Blunt et al. (200) reported on the isolation and structure elucidation of discorhabdin D, which corresponds to the dehydro form of the phenol moiety of 492. In 1990, the same group reported 2-

488 R =OH Piiamsin C (2hydroxydiscorhabdlnD) 484 R = H Piiamsin D (Discornabdin D) 0

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

203

hydroxydiscorhabdinD (488),which corresponds to dehydro-491(201). In light of the close structural similarities of these alkaloids, Kobayashi et al. (206) re-examined their results and came to the conclusion that the quinoneimine groups in 488 and 484 were easily reduced to the phenol under the acetylation conditions to give the acetates 489 and 490. The 'H and I3Cdata of C and D were compared with those of 2-hydroxydiscorhabdinD and discorhabdin D as their hydrochloride salts to reveal that the spectra were completely identical. Prianosin A (481): see discorhabdin A. Prianosin B (487): C18H11BrN302S:red crystals; mp 250-251°C (dec.); FABMS (glycerol) d z 414 (M+ + H) and 416 (M+ + 2 + H); [ 0 I 3 O D +360" (c 0.1, CHC13);IR (KBr) v 3350, 1670, 1640, 1600, 1460, 1300, 1210 cm-'; UV (MeOH) A 228 (E 17,800),263 (E 15,000); 410 (sh), 430 (E 11,200) nm; CD (MeOH) A 360 (A E -2.7), 265 (+3.6), 233 (-8.8) nm; 'H-NMR (500 MHz, CDC13KD30D 4 : l ) 87.96 (lH, S, H-1), 2.95 (lH, dd, J = 16.9, 6.7 Hz, H-4), 3.01 (lH, dd, J = 16.9, 12.5 Hz, H-4), 4.79 (lH, dd, J = 12.5, 6.7 Hz, H-5), 2.88 (2H, m, H-7), 5.47 (lH, m, H-8), 7.78 (lH, s, H-14), 7.51 (lH, d, J = 5.9 Hz, H-16), 8.46 (lH, d, J = 5.9 Hz, H-17); 13C-NMR(22.5 MHz, CDCl3/CD30D 4: 1) S 156.8 (d, C-1), 120.2 (C-2), 189.2 (C-3), 45.8 (t, C-4), 56.6 (d, C-5), 51.1 (C-6), 40.3 (t, C-7), 61.8 (d, C-8), 170.5 (C-ll), 125.3 (d, C-14), 113.8 (d, C-16), 142.9 (d, C-17). Prianosin C (488):CI8Hl4N3O3S; green solid; mp > 300°C; HRFABMS m/z 352 (M+),354.09 (M+ +2H); [ ( Y ]+358" ~~~ (c 0.01, MeOH); IR (KBr) v 3400-3100, 2945, 1650, 1630, 1600, 1540, 1500, 1430, 1320, 1220, 1180, 1130, 1090, 1040, 990, 845, 800, 740 cm-'; UV (MeOH) h 231 (E 12,300), 263 (E 3900), 292 (E 2100), 370 (E 1280) nm, CD (MeOH) A 352 (A E +41.1), 308 (-17.3), 258 (-43.3) nm; 'H-NMR (CD30D) S 7.11 (lH, s), 6.07 (lH, s), 5.64 (lH, dd,J = 3.5, 1.3 Hz), 4.40 (lH, m), 3.47 (lH, m), 3.08 (2H, m), 2.85 (lH, d, 12.8 Hz), 2.81 (lH, dd, J = 11.7,3.7 Hz), 2.63 (2H, d, J = 12.8 Hz); 13C-NMR (CD30D) 6 184.6, 175.2, 167.5, 151.3, 148.1, 127.1, 125.8, 123.3, 120.0, 111.8, 101.5, 85.2, 64.3, 45.7, 44.1, 40.7, 39.3, 21.0. Prianosin D (484):see discorhabdin D. b. Biological Activity. It was reported that prianosin A (481) is cytotoxic, with IC50values of 37 and 14 ng/ml against L1210 and L5178Y murine leukemia cells in vitro. It also induced Ca2+release from the sarcoplasmic reticulum and was 10 times more potent than caffeine in this assay. The minimum effective concentrationsof prianosin A and caffeine were 30 and 300 pM,respectively (204). Prianosins B (487), C (488),and D (484) were found to be cytotoxic against murine lymphomas L1210 and L5178Y cells and human epidermoid carcinoma (KB) cells in vitro, with ICs0values of 2.0, 1.8, and B5.0 pg/ml

204

TURAN OZTURK

for 487 (24% inhibition at 5.0 pglml); 0.15, 0.024,and 0.57 pg/ml for 488; and 0.18, 0.048, and 0.46 pg/ml for 484, respectively. Like prianosin A, prianosin D was also found to induce Ca2+release from the sarcoplasmic reticulum, and was 10 times more potent than caffeine in this assay. Such activity was not observed for either prianosin B or prianosin C (205). 3. Synthesis of Discorhabdins and Prianosins

The first total synthesis of discorhabdin C was achieved by Kita et al. using the hypervalent iodine reagent PIFA, phenyliodine( III)bis(trifluoroacetate), in the spirocyclization to form ring B of discorhabdin C (Scheme 58) (207-209). Their synthesis started with the protection of the phenolic hydroxyl group of 2-hydroxy-4-methoxybenzaldehyde(493) with benzyl bromide. Then the aldehyde group was reacted with ethyl azidoacetate to obtain a vinyl azide, which was decomposed on refluxing in xylene to form the 2-(ethoxycarbony1)indole derivative 494 as a result of nitrene insertion. Hydrolysis of the ester group of 494 followed by decarboxylation gave indole 495, which was converted to 3-(cyanomethy1)indole (496) in three steps. Treatment with dimethyl(methy1ene)ammonium iodide gave 3-(dimethylamino)methylindole, in which the amino group was converted to the quaternary salt with methyl iodide, which, on subsequent treatment with NaCN, resulted in the cyanomethylindole 496. The cyano group was reduced to the corresponding amine by catalytic hydrogenation; then the amine group was protected with [(trimethylsilyl)ethoxy]carbonyl (TEOC) to give 497. Debenzylation of 497 followed by oxidation with Fremy’s salt afforded the quinone 498. Because the direct formation of the indoloquinoneimine did not proceed, 498 was first tosylated. Treatment of the product 499 with p-toluenesulfonic acid yielded an unstable indoloquinoneimine 500, which was reacted with 3,5-dibromotyramine hydrobromide without further purification for the substitution reaction of the methoxy group. Subsequent detosylation gave the phenolic aminoindoloquinoneimine 501. Before the oxidative coupling reaction, 501 was converted into the corresponding silyl ether using o-silylated ketene acetal. The coupling reaction with PIFA gave rise to discorhabdin C (483), which was identical with authentic sample. Yamamura et al. developed an efficient route to the synthesis of the A, B, C, D, and E rings of the prianosins and discorhabdins, which led them successfully to synthesize discorhabdin C (Scheme 59) (210-212). Because their route involves the synthesis of the pyrroloquinoline skeleton of the pyrroloquinoline type alkaloids (213-21 7), they have also achieved the synthesis of batzelline C and isobatzelline C (213,214). Their synthesis involved first the construction of the C, D, and E rings as in 505, starting from 502, which was synthesized in three steps (212). Reaction of 502 with

205

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

9"

OH

493

2. Copper chromitd qunoline. 215oC

495

496

500

499

SCHEME 58. Synthesis of discorhabdin C (483)(207-209).

206

TURAN OZTURK

501

I

1. MeCM(OMe)(OSiMe3) 2. PM(OCoCF3)2 (PIFA)

483 SCHEME 58. Continued

503 led to the formation of the indole 504, which was hydrogenated under

acidic conditions to remove both the benzyl and TEOC (2-trimethylsilylethoxycarbonyl) protecting groups. Lactamization using DCC produced 505. To convert 505 to a quinoneimine, the amide-containing ring was first reduced to a pyrimidine and then oxidized with CAN to yield 506. Reaction of 506 with 3,5-dibromotyramine smoothly produced 501, which is also an intermediate in Kita’s synthesis of discorhabdin C (207-209). Anodic oxidation of 501 gave the desired discorhabdin C (483), along with ring expanded product 507. In their total synthesis of mukaluvamine D (519), White el al. synthesized the C, D, and E rings of the discorhabdins and prianosins through a new route to the pyrroloquinoline nucleus common to this type of alkaloids (Scheme 60) (218).Their synthesis started with known hydrazine 508, condensation of which with dihydrofuran produced a 1: 1 mixture of 509 and 510. Fischer indolization of the mixture led to the formation of tryptophol (511)’along with 4-methoxyindole (512) as a by-product, which was separated after sulfonation. Nitration of the desired product 513 with acetyl nitrate gave a 1:1 mixture of the 4-(514) and the 2-nitroindole 515, which were separated by chromatography. The nitro group of 514 was reduced to an amine over Adams’ catalyst; then, because of the product’s airsensitive nature, it was treated immediately with N,N-di-isopropylethylamine to form the ring E, which was converted to quinoneimine 516 with CAN. In contrast to Yamamura (210-212) and Kita (207-209), whose

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

502

207

504

I @

1. HgPdC, 60 % 2. KOH, then DCC, 36 %

0

@.

2. 1.BF3.sw2 CAN, 51 %

wo

H3CO

0

/

H3

506

505

3,5-Dibromotyramine 74 %

OH Br

DiscorhabdinC 483 ,24 YO LiCD4, CCE. 3mA H

+

* Br

501

507 6%

SCHEME59. Synthesis of discorhabdin C (483)(210-212).

pyrroloquinolines 506 and 500, respectively, reacted with tyramine, treatment of 516 with tyramine and a variety of nucleophiles, such as ammonia, primary amines, and azides, to replace the methoxy group unfortunately

208

TURAN OZTURK

517

SCHEME60. Partial synthesis of discorhabdins and prianosins (218).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

NH3, EOH 517

P

209

@J

H2N

Ts

0 518

519

was not successful. In contrast, its tosyl salt 517, which was prepared smoothly with p-toluenesulfonic acid, gave 518 with ammonia, and makaluvamine (519) with tyramine followed by treatment with trifluoroacetic acid. The compound 518 constitutes the C, D, and E rings of the discorhabdins and prianosins. Knolker et al. have developed a one-pot diastereoselective spiroannelation by electrophilic substitution of an aromatic system with an ironcomplexed cation, which led to the construction of the A, B, C, and D rings of the prianosins and discorhabdins. This novel diastereoselective spiroannulation involved the reaction between the tricarbonylironcomplexed cation 520, which was prepared in six steps (219),and an arylamine, the 6-aminoindoline (521), to construct A, B, C, and D rings (512) (Scheme 61) (220).The stereochemistry of the product was determined by X-ray crystallographic analysis; the stereodirecting effect of Fe(C0)3 was indicated to be anti to the aryl ring (219). A synthetic methodology has been under investigation for the synthesis of discorhabdins and prianosins by Confalone et al. using intramolecular

210

TURAN OZTURK

520

+

522

v

SCHEME61. Partial synthesis of discorhabdins and prianosins (219,220).

phenolate alkylation as a key step. This method was initially used to synthesize the naphthalene analog of discorhabdin C in a model study (Scheme 62) (221).3-(2,6-Dibromophenol)naphthoquinone (523) underwent an intramolecular phenolate alkylation to obtain the desired spiro compound 524 in reasonable 34% yield. In contrast, intramolecular phenolate alkylation of the debromo analog 525 gave the aziridine 526 as a major product; 526 was not converted into the spiro derivative 527, showing that 526 is not an intermediate en route to the desired product 527. After this model study, an attempt to synthesize discorhabdin C was carried out with 528, which was synthesized in 15 steps (Scheme 63) (222). The intramolecular phenolate alkylation of 528 gave a similar result, producing aldehyde 529, which with nitromethane gave the nitroethylene 530, in which the alkene functionality was successfully reduced with sodium borohydride, giving 531. As the reduction of the nitro group to the amine

QUINOLINEQUINONE A N D QUINOLINEQUINONEIMINE UNITS

523

21 1

524

525

526

527

SCHEME62. Synthetic studies to the synthesis of discorhabdin C (221).

group seemed problematic in the presence of other potentially reducible groups, an alternative approach, which involves synthesis of indoloquinone 533 from squaric acid (532) (Scheme 63), has been under investigation to synthesize discorhabdin C (222). It was reported that the indoloquinone 533, which was synthesized in 17 steps from squaric acid (532), is currently under investigation to synthesize discorhabdin C (483) (222).

212

TURAN OZTURK

0

529

528

0

530

531 a

532

533

SCHEME63. Synthetic studies to the synthesis of discorhabdin C (222).

IV, summary Alkaloids containing quinolinequinone and quinolinequinoneimine units isolated to date have been reviewed in depth, in terms of their isolation

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

213

from natural sources (e.g., plants and marine sponges), structural elucidation, synthesis, biological activity, and biosynthesis. Widespread effort in gleaning a greater understanding of the properties of these complex and challenging alkaloids has led to the development of synthetic pathways to these compounds and their analogs. These studies have been examined and are presented in this review.

Acknowledgments I thank Dr. J. A. Joule (Univesity of Manchester) for his useful comments on the draft of this manuscript.

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132. V. V. Tolstikov, M. N. Preobrazhenskaya, J. Balzarini, and E. De Clercq, J. Antibiot. 45, 1002 (1992). 133. M. N. Preobrazhenskaya, N. V. Holpne-Kozlova, and E. I. Lazhko, J. Antibioi. 45, 227 (1992). 134. F. J. Schmitz, S. K. Agarwal, and S. P. Gunasekera, J. Am. Chem. SOC. 105, 4835 (1983). 135. C. V. Labarca, A. R.MacKenzie, C. J. Moody, C. W. Rees, and J. J. Vaquero, J. Chem. SOC., Perkin Trans. I, 927 (1987). 136. A. M. Echavarren and J. K. Stille, J . Am. Chem. SOC. 110,4051 (1988). 137. A. Kubo and S. Nakahara, Heterocycles 27,2095 (1988). 138. R. H. Prager and C. Tsopelas, Heterocycles 29,847 (1989). 139. R. H. Prager, C. Tsopelas, and T. Heisler, Ausi. J. Chem. 44,227 (1991). 140. C. M. Thompson and S . Docter, Teirahedron Len. 29,5213 (1988). 141. C. Subramanyam, M. Noguchi, and S. M. Weinreb, J. Org. Chem. 54,5580 (1989). 142. P. Meghani, 0. S. Mills, and J. A. Joule, Heierocycles 30, 1121 (1990). 143. F. Guillier, F. Nivoliers, A. Godard, F. Marsais, and G. Queguiner, Tetrahedron Lei!. 35,6489 (1994). 144. R. Cassis, R. Tapia, and J. A. Valserrama, Synih. Commun. 15, 125 (1985). 145. F. Sainte, B. Serckx-Poncin,A.-M. Hesbain-Frisque, and L. Ghosez, J. Am. Chem. SOC. 104, 1428 (1982). 146. N. Miyavra, T. Yanagi, and A. Suzuki, Synih. Commun. 11,513 (1981). 147. J. Kobayashi, J. Cheng, H. Nakamura, Y. Ohizumi, Y. Hirata, T. Sasaki, T. Ohta, and S. Nozoe, Tetrahedron Lei!. 29, 1177 (1988). 148. J. Kobayashi and Y. Oizumi, Jpn. Kokai Tokkyo Koho, JP 01186885,26 July 1989, Chem. Absir. 112,84161~(1990). 149. H.-Y. He and D. J. Faulkner, J. Org. Chem. 56,5369 (1991). 150. N. Bontemps, I. Bonnard, B. Banaigs, G. Combaut, and C. Francisco, Teiruhedron Len. 35,7023 (1994). 151. F. J. Schmitz, F. S. DeGuzman, Y.-H. Choi, M. B. Hossain, S. K. Pizvi, and D. van der Helm, Pure Appl. Chem. 62, 1393 (1990). 152. F. J. Schmitz, F. S. DeGuzman, M. B. Hossain, and D. van der Helm, J. Org. Chem. 56, 804 (1991). 153. C. Zeng, M. Ishibashi, K. Matsumoto, S. Nakaike, and J. Kobayashi, Teirahedron 37, 8337 (1993). 154. F. Bracher, Heterocycles 29,2093 (1989). 155. C. J. Moody, C. W. Rees. and R. Thomas, Tetrahedron Let!. 31,4375 (1990). 156. C. J. Moody, C. W.Rees, and R. Thomas, Teirahedron Lei!. 48,3589 (1992). 157. Y. Kitahara, S. Nakahara, T. Yorezawa, M. Nagatsu, and A. Kubo, Heierocycles 36, 943 (1993). 158. E. Gomez-Bengoa and A. M. Echavarren, J. Org Chem. 56,3497 (1991). 159. R. Cassis, R. Tapai, and J. A. Valderrama, Synih. Commun. 15, 125 (1985). 160. G.Gellerman, M. Babad, and Y. Kashman, Teirahedron Lei!. 34, 1827 (1993). 161. S . J. Bloor and F. J. Schmitz, J. Am. Chem. SOC. 109,6134 (1987). 162. F. S. de Guzman and F. J. Schmitz, Tetrahedron Lei!. 30,1069 (1989). 163. F. Bracher, Liebigs Ann. Chem., 205 (1990). 164. V. Goulle, J. M. Lehn, B. Schoentjes, and F. J. Schmitz, Helv. Chim. Aciu 74,1471 (1991). 165. E. Lederer, G. Teissier, and C. Huttrer, Bull. SOC. Chim. Fr. 7,608 (1940). 166. M. Barbier, Naiurwissenschafren69,341 (1982). 167. G. Cimino, A. Crispino, S. De Rosa, S. De Stefano, M. Gavagnin, and G. Sodano, Teirahedron 43,4023 (1987).

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TURAN OZTURK

168. N. Bontemps, I. Bonnard, B. Baraigs, G. Combaut, and C. Francisco, Tetrahedron Len. 35,7023 (1994). 169. J. Kobayashi, J. Cheng, M. R. Walchli, N. Nakamura, Y. Hirata, T. Sasaki, and Y. Ohizumi, J. Org. Chem. 53, 1800 (1988). 170. J. Kobayashi, M. Tsuda, A. Tanabe, M. Ishibashi, J. F. Cheng, S. Yamamura, and T. Sasaki, J. Nut. Prod. 54, 1634 (1991). 171. L. A. McDonald, G. S. Eldredge, L. R. Barrows, and C. M. Ireland, J. Med. Chem. 37, 3819 (1994). 172. A. Bax, A. Aszalos, Z. Dinya, and K. Sudo, J. Am. Chem. SOC. 108,8056 (1986). 173. M. A. Ciufolini and N. E. Byrne, Tetrahedron Lett. 30,5559 (1989). 174. M. A, Ciufolini and N. E. Byrne, J. Am. Chem. SOC.113,8016 (1991). 175. G. A. Charyulu, T. C. McKee, and C. M. Ireland, Tetrahedron Lett. 30,4201 (1989). 176. T. F. Molinski and C. M. Ireland, J. Org. Chem. 54,4256 (1989). 177. B. G. Szczepankiewicz and C. H. Heathcock, J. Org. Chem. 59, 3512 (1994). 178. A. Rudi, Y. Benayahu, I. Goldberg, and Y. Kashman, Tetrahedron Lett. 29,6655 (1988). 179. A. Rudi and Y. Kashman, J . Org. Chem. 54,5331 (1989). 180. S. Nakahara, Y. Tanaka, and A. Kubo, Heterocycles 36, 1139 (1993). 181. N. R. Shochet. A. Rudi, Y. Kashman, Y. Hod, M. R. El-Maghrabi, and I. Spector, J. Cell. Physiol. 157,481 (1993). 182. H.-Y. He and D. J. Faulkner, J. Org. Chem. 56,5369 (1991). 183. R. W. Read and W. C. Taylor, Aust. J. Chem. 32,2317 (1979). 184. A. R. Carroll and W. C. Taylor, Aust. J. Chem. 44,1615 (1991). 185. Y. Kitahara and A. Kubo, Heterocycles 34, 1089 (1992). 186. A. R. Carroll and P. J. Scheuer, J. Org. Chem. 55,4426 (1990). 187. G. P. Gunawardana, S. Kohmoto, and N. S . Burres, Tetrahedron Lett. 30,4359 (1989). 188. R. Faure, J.-P. Galy, J. Elguero, and E. Vincent, Can. J. Chem. 56,46 (1978). 189. M. D. Friedman, P. L. St0tter.T. H. Porter, and K. Folkers,J. Med. Chem. 16,1314 (1973). 190. P. J. McCarthy, T. P. Pitts, G. P. Gunawardana, M. Kelly-Borges, and S. A. Pomponi, J. Nat. Prod. 55, 1664 (1992). 191. R. E. Longley, 0.J. McConnell, E. Essich, and D. Harmody,J. Nar. Prod. 56,915 (1993). 192. T. F. Molinski, E. Fahy, D. J. Faulkner, G. D. Van Duyne, and J. Clardy, J. Org. Chem. 53, 1340 (1988). 193. J. U. M. Rao, G. S. Giri, T. Hanumaiah, and K. V. J. Rao, J. Nat. Prod. 49,346 (1986). 194. S. Liu, B. Oguntimein, C. D. Hufford. and A. M. Clark, Antimicrob. Agts, Chemother. 34, 529 (1990). 195. N. B. Perry, J. W. Blunt, J. D. McCombs, and M. H. G. Munro, J. Org. Chem. 51, 5476 (1986). 196. J. W. Blunt, V.L. Calder, G. D. Fenwick, R. J. Lake, J. D. McCombs, M. H. G. Munro, and N. B. Perry, J. Nat. Prod. 50,290 (1987). 197. M. H. G. Munro, N. B. Perry, and J. W. Blunt, PCT Int. Appl., WO 88/00946,11 Feb. 1988, Chem. Abstr. 110, 54771k (1989). 198. N. B. Perry, J. W. Blunt, and M. H. G. Munro, Tetrahedron 44, 1727 (1988). 199. M. H. G. Munro, N. B. Perry, and J. W. Blunt, U.S. Patent 4731366, Mar. 15, 1988. 200. N. B. Perry, J. W. Blunt, and M. H. G. Munro, J. Org. Chem. 53,4127 (1988). 201. J. W. Blunt, M. H. G. Munro, C. N. Battershill, B. R. Copp, J. D. McCombs, N. B. Perry, M. Prinsep, and A. M. Thompson, New J. Chem. 14,761 (1990). 202. D. C. Radisky, E. S. Radisky, L. R. Barrows, B. R. Copp, R. A, Kramer, and C. M. Ireland, J. Am. Chem. Soc. 115,1632 (1993). 203. L. R. Barrows, D. C. Radisky, B. R. Copp, D. S. Swaffar, R. A. Kramer, R. L. Warters, and C. M. Ireland, Anti-Cancer Drug Design 8,333 (1993).

QUINOLINEQUINONE AND QUINOLINEQUINONEIMINE UNITS

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204. J. Kobayashi, J. Cheng, M. Ishibashi, H. Nakamura, Y. Ohizumi, Y. Hirata, T. Sasaki, H. Lu, and J. Clardy, Tetrahedron Lett. 28,4939 (1987). 205. J. Cheng, Y. Ohizumi, M. R. Walchli, H. Nakamura, Y. Hirata, T. Sasaki, and J. Kobayashi, J. Org. Chem. 53,4621 (1988). 206. J. Kobayashi, J. Cheng, S. Yamamura, and M. Ishibashi, Tetrahedron Lett. 32,1227 (1991). 207. Y. Kita, T. Yakura, H. Tohma, K. Kikuchi, and Y. Tamura, Tetrahedron Lett. 30, 1119 (1989). 208. Y. Kita, H. Tohma, M. Inagaki, K. Hatanaka, K. Kikuchi, and T. Yakura, Tetrahedron Lett. 32,2035 (1991). 209. Y. Kita, H. Tohma, M. Inagaki, K. Hatanaka, and T. Yakura, J. Am. Chem. Soc. 114, 2175 (1992). 210. J. F. Cheng, S. Nishiyama. and S. Yamamura, Chem. Lett, 1591 (1990). 211. S. Nishiyama, J. F. Cheng, X. L. Tao, and S. Yamamura, Tetrahedron Lett. 32,4151 (1991). 212. X. L. Tao, J. F. Cheng, S. Nishiyama, and S. Yamamura, Tetrahedron 50,2017 (1994). 213. S. Sakemi, H. H. Sun, C. W. Jefford, and G. Bernardinelli, Tetrahedron Lett. 30, 2517 (1989). 214. H. H. Sun, S. Sakemi, N. Burres, and P. McCarthy, J. Org. Chem. 55,4964 (1990). 215. B. R. Copp, C. M. Ireland, and L. R. Barrows, J. Org. Chem. 56,4596 (1991). 216. D. B. Stierle and D. J. Faulkner, J. Nut. Prod. 54, 1131 (1991). 217. J. R. Carney and P. J. Schever, Tetrahedron 49,8483 (1993). 218. J. D. White, K. M. Yager, and T. Yakura, J. Am. Chem. SOC. 116, 1831 (1994). 219. H.-J. Knolker, R. Boese, and K. Hartmann, Angew. Chem., Int. Ed. Engl. 28,1678 (1989). 220. H.-J. Knolker and K. Hartmann, Synlett, 428 (1991). 221. G. G. Kublak and P. N. Confalone, Tetrahedron Lett. 31,3845 (1990). 222. P. T. Confalone, J. Her. Chem. 27, 31 (1990).

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-CHAPTER 3-

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS IN Catharanthus roseus CELLS ROBERT VERPOORTE, ROBERT VAN DER HEIJDEN, AND PAULOR. H. MORENO Division of Pharmacognosy LeidedAmsterdam Center for Drug Research University of Leiden Leiden, The Netherlands

I. Introduction .................................................................................... 11. Biological Function of Alkaloids ......................................................... 111. Pathway Leading to Terpenoid Indole Alkaloids: Intermediates

IV.

V. VI.

VII.

and Enzymes ................................................................................... A. Biosynthesis of Geraniol ............................................................... B. Biosynthesis of Secologanin ........................................................... C. Biosynthesis of Tryptophan ...... D. Biosynthesis of Tryptarnine ........................................................... E. Biosynthesis of Terpenoid Indole Alkaloids ......... Genes Encoding Enzymes Involved in Terpenoid Indol Alkaloid Biosynthesis ........................ A. HMG-CoA Reductase .................. B. Geraniol 10-Hydroxylase ............... C. Tryptophan Decarboxylase ............................................................ D. Strictosidine Synthase ............................. ..................... E. Desacetoxyvindoline 17-Hydroxylase ............................................... Metabolic Engineering ...................................................................... Regulation of Alkaloid Biosynthesis ..................................................... A. Developmental Regulation ............................................................ B. Compartmentation ....................................................................... C. Light ........................................................................................ D. Plant Growth Regulators ............ .............................. E. Elicitors .................................................................................... F. Jasmonic Acid ................................................... G. Salicylic Acid ..................................................... H. Calcium ............................................................ Conclusions ........................................................... References .................................

THE ALKALOIDS, VOL. 49 00994590t97 $25.00

221

222 225 227 230 235 247

267 268 269 270 271 273 275 279 280 282

Copyright 0 1997 by Academic Press All rights of reproduction in any form resewed.

222

VERPOORTE, VAN DER

HEIJDEN, AND

MORENO

I. Introduction

Carharanthus roseas belongs to the family Apocynaceae, and is particularly known for the presence of terpenoid indole alkaloids. Several of the Carharanthus alkaloids are used as pharmaceutical agents. The bisindole alkaloids vinblastine and vincristine, minor alkaloids from the leaves (1-6) are powerful antitumor drugs. Ajmalicine, the major alkaloid from the roots, is used to improve cerebral circulation (1,2).Because of its economic importance, C. roseus has been studied extensively (Fig. 1). Particularly, the possibilities of producing these alkaloids by means of plant cell cultures have been the subject of numerous publications. Several reviews have been published on the production of the alkaloids in cell cultures (7-11), and the economic feasibility of a plant cell biotechnological production of ajmalicine was assessed (10).Technologically, it is feasible to produce plant secondary metabolites by means of plant cells in bioreactors, but the costs of such a production are high. Although the estimated production costs of ajmalicine are similar to the market price, a considerably improved production is desired to make the process economically interesting. For C. roseus, empirical approaches, such as screening and selection of high-producing cell lines and production media, did result in some increase in the alkaloid production (up to about 200 mg/l, which corresponds to about 0.3% alkaloid on dry weight of cells), but it is still insufficient for commercialization. Therefore, research is now focusing on the regulation of the biosynthesis, aiming at metabolic engineering to improve the production.

I 80

-----patents

I I

1984

1995

-publications

,

1950

1961

1973

Year

FIG.1. Number of publications and patents on Cufhuranfhhusroseus.

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

223

We do not intend to deal here with all of the various approaches to increase alkaloid accumulation in C. roseus plant cell cultures. Rather, we want to evaluate these studies in terms of regulation and signals that affect the alkaloid biosynthesis. Such information may hold clues for the use of metabolic engineering of the alkaloid biosynthetic pathway. Several general reviews have been published on the biosynthesis of terpenoid indole alkaloids (see, e.g., reviews 12-18). The early part of this pathway has been studied in detail by a number of researchers, and several of the genes coding for the enzymes involved have been cloned (for reviews, see 29-21). Some pathways leading to specific products have also been elucidated, particularly for some Ruuvoljiu alkaloids (22,23). Most work has been done on C. roseus; other plants used belong to the genera Tubernuemontuna (Apocynaceae) (24-38), Cinchona (Rubiaceae) (31,39-43, and Cumptothecu (Nyssaceae) (48). As similar pathways may be differently regulated in different species, and as one cannot extrapolate findings for one plant to another plant, we particularly focus on the regulation of the biosynthesis of terpenoid indole alkaloids in C. roseus. Even within one plant the pathway might be differently regulated in different tissues. Findings from related plant species thus are not fully covered; only where necessary is a comparison made of C. roseus with related plant species. Regulation of a pathway encompasses a number of processes, starting with a signal that eventually results in expression of the genes coding for the enzymes catalyzing the various steps of the pathway. Subsequently, transport and processing of the expressed proteins is required to get the active enzyme in the correct cell compartment. Transport of intermediates within the cell, or even from cell to cell; feedback mechanisms on the level of the enzymes; and accumulation of products in cell compartments or specialized cells or tissues are further aspects that involve genes and proteins not directly part of the actual pathway. Regulation is therefore a highly complex system, which is much more than the simple sum of a series of biosynthetic steps. In the present review we divide the pathway leading to the Cutharunthus alkaloids into five parts (Fig. 2). The first two concern the biosynthesis of tryptophan and geraniol diphosphate; they are similar to (or even part of) primary metabolism and occur in all plant species. Whether these pathways in C. roseus are differently regulated, or whether even an additional pathway exists parallel to the normal primary metabolism, is a question not yet answered. The third and the fourth part concern the steps from tryptophan to tryptamine and from geraniol to secologanin, respectively. Both pathways occur also in other plants, including plants that do not produce terpenoid indole alkaloids. The fifth part is the condensation of secologanin and tryptamine to strictosidine and the subsequent conversion into a plethora

TABLE I ALKALOIDS, LISTEDBY BIOGENETIC CLASS,ISOLATED FROM C. roseus CELLAND TISSUE CULTURES' Culture Type Class Alkaloid

vinc08an Strictosidine Strictosidine-lactam Corynsnthean Ajmalicine Ajmalicine, 3-epiAjmalicine, 3-epi-19-epiAjmalicine, 7-hydroxy-indolenine Ajmalicine, pseudo-indoxyl Akuammigine Akuammiline Akuammiline, 10-hydroxy-desacetylAkuammiline, desacetylAlstonine Antirhine Cathindine Cavincidine Cavincine Cyclolochnerine, 21-hydroxyIsositsirikine Isositsirikine, 16R-19,20-EIsositsirikine, 16R-19.20-2Mitraphylline Perivine/perosine Pleiocarpamine Serpentine Sitsirikine Sitsirikine, dihydroTetrahydroalstoqine Yohimbine VPllesischotaman Vallesiachotamine Isovallesiachotamine Strychnan Akuammicine Akuammicine, 12-hydroxyLochneridine

#943-Strychnan-glycoside Aspidospermatan Tubotaiwine Plumeran HBrhamrnericine HBrhamrnerinine Lochnericine Lochnerinine Minovincinine Tabersonine

Suspension Callus

Crown Gall

Root

+ +

+ + t

+ +

+

+

+ + + + + + + + + + + +

+

+

+

+ +

+

+ + + + + + + +

Shoot

+

+

+

+

+ +

+

+ +

+

+ +

+

+

+

+ + + + + + t

+ + t

225

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

TABLE I (continued) Culture Type Class Alkaloid

Suspension

Tabersonine, 19-hydroxy Tabersonine, 19-acetoxy-11-hydroxy Tabersonine, 19-acetoxy-11-methoxy Tabersonine, 19-hydroxy-11-methoxy Vindoline Vindolinine Vindolinine-&oxide Vindolinine, 19-epiVindolinine-Nb-oxide,19-epiIbogan Catharanthine Bisindoles

+ + + +

+ + + + +

Callus

Crown Gall

+

3',4'-Anhydro-~inbIastine Leurosine Catharine Vinblastine a

Root

Shoot

+ t

+

+ + + + +

Data from ref. 7 and references cited therein. Found in callus and in callus with root formation.

of terpenoid indole alkaloids. The formation of strictosidine is found in a number of plant species (mainly in the families Apocynaceae, Loganiaceae, and Rubiaceae) producing terpenoid indole alkaloids, but after strictosidine, the pathways diverge. Even in different parts of C. roseus plants, different products are formed from strictosidine. Consequently, in in vitro cultures of differentiated cells the spectrum of alkaloids produced differs (see Table I). One might thus divide this part of the pathway into a number of separate pathways, which may even occur in parallel in one plant.

II. Biological Function of Alkaloids Regulation of the biosynthesis of secondary metabolites cannot be seen as separate from the role of these products for the plant. However, in most cases this role is not known. In the case of C. roseus an antifeedant activity against Spodoproru larvae has been reported for vinblastine and catharanthine (49). A nematocidal effect has been reported for serpentine (50). Antifeedant activity against Spodopreru caterpillars for C. roseus leaf extracts has been described as well (49,51). Luijendijk et ul. (52,53)found that besides alkaloids, also nonpolar compounds are involved in the antifeedant

226

I

VERPOORTE, VAN DER HEIJDEN, AND MORENO

phoaphoenolpyruvate t o-erythrose4-phoaphate

acetyl-CoA

i

r

uhikimate

HMG-CoA

I

mevalonate

i 3 geraniol

1

I

+

I

10-hydroxygeraniol

i loganin

1 secologanin

I

1

strictmidine

&..

ajmdicine aerpentine

-.-.

v catharanthine

---A &_La___

muemnine

f

vindoline

vinb1astine vincristine I

I

FIG.2. General overview of the pathway leading to the terpenoid indole alkaloids.

activity of leaf extracts against Spodoptera exigua. No antifeedant activity could be detected against S. exigua for strictosidine, the main alkaloid in young leaves, or for the combination of strictosidine with strictosidine

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

227

glucosidase (52,53). In Cinchona seedlings and plants, an antifeedant role for the quinoline and indole alkaloids was found (41-45,54). The formation of bisindole alkaloids increased by treatment of the plants or shoot cultures with near-UV light, suggesting that the oxidation of catharanthine occurs under such conditions (55,56).In C. roseus plants, the levels of the monomers catharanthine and vindoline decrease, from younger to older leaves, whereas the level of the bisindole increases (57,58).The oxidative coupling of the two monomers might play a role in antioxidative processes in the plant. Similarly, the accumulation of serpentine and decrease of ajmalicine, after exposing cell cultures to light (59-61), as well as the induction of the ajmalicine biosynthesis by high levels of oxygen in the medium of cultured cells (62), might be connected to an antioxidant role of ajmalicine (63). Frischknecht et al. (64) reported a strong increase in the alkaloid production (up to 100%)in growing leaf tissue upon wounding, whereas nongrowing tissues did not show any change. The coupling of the monomers is also induced by wounding, as well as the vindoline biosynthesis (65).However, van Dam et al. (66,67) could not find an induction of alkaloid biosynthesis in C. roseus plants upon wounding. Recently, we found that strictosidine in combination with strictosidine glucosidase (SG)strongly enhanced antifungal activity. Minimum inhibitory concentrations were determined of ca 0.33 mM against some Fusarium species and Cladosporium cucumerinum, 1m M against Trichoderma viride, and below 0.008 mM against Phytophthora infestans. Strictosidine itself is also active against P. infestans. Ajmalicine did not show antifungal activity at these levels. Strictosidine levels are particularly high in young leaves. 260 pg/g fresh weight, which is ca 0.5 mM (i.e., sufficient to function as an antifungal defense). Based on the fact that strictosidine is found in the vacuole, whereas the highly specific SG is present at high levels on the tonoplast and in the cytosol, we have postulated that this combination of substrate and glucosidase plays a role in the plant’s defense in the case of wounding (52,53),that is strictosidine is a phytoanticipin (68).

III. Pathway Leading to Terpenoid Indole Alkaloids: Intermediates and Enzymes Here we discuss the intermediates and enzymes involved in the terpenoid indole alkaloid biosynthesis, following the subdivision into the five parts mentioned earlier (Fig. 2). In Table I1 the characteristics of the various

TABLE I1 ENZYMES CHARACTERIZED I N MONOTERPENOID INWLEALKALOID BIOSYNTHESIS IN Curharmhus roseus"

Mw Enzyme Anthranilate

Abbreviation

&factors

lsoforms

As

(ma)

00

-tion

-

cDNA Cloning

Ref.

Optimum

Purification

75-8.3

Apparent homogeneity Apparent homogeneity

i77 178

Tryptophan decarboxylase

TDC

55

037 mM ~Glutamine 67 f l Chorismate 75 f l Tryptophan

Acetoacetyl-CoA

AACT

41

-

-

-

Partial

179 78

-

-

-

Partial

81

-

-

Partial

90

-

-

Partial

b

143

synthase

N

PH

K, value

thiolase HYhxYmethylglutarylCoA synthase Mevalonate base lspentenyl diphosphate isomerase Geraniol 10hydroxylase

(subunit) ca 100

HMGS

MVA base IPP isomerase

Mg2' Mn*'

GlOH

Heme

1

NADPH

1

-

NADPH :cytochrome

ca 35

56

5.5 p M Geraniol 11 pM Nerol

FAD FMN

P-450 reductase S A M :loganic acid

LAW

methyl-transferase Strictmidine synthase

sss

SAM -

-

-

I

38

0.06 mM SAM 12.5 m M Loganic acid 8.2-9.4 u M

cytosol

85

156

Provaculoar membranes

Apparent homegeneity

Rovacuolar membranes

Apparent homogeneity

108 109 111 112 110 113

Partial

122

Apparent homogeneity

204

-

Vacuole

6-7.5

Strinosidine @-glucohidase

3

SG

240 650

Tryptamine 10-18 pM Strictosidine

Tonoplast

6-6.4

Apparent homogeneity

52

930

Peroxidase Tabersonine-llhydroxylase S A M :methoxy-Z.16dihydro-16hydrotabersonineN-methyltransferax Desacetoxyvindoline17-hydroxylax

AcetylCoA : deacetylvindoline 17-0-acetyltransferase

83 uM Geissoxhizine

NADP+

Geissoxhizine dehydrogenase CatheDamine redunase Tetrahydro-alstonine synthase

TllH NMT

NADPH

-

NADPH

1

81

-

60

D17H

2-Oxoglutarate Fe2+ Ascorbate

3

44.7

DAT

Acetyl-CoA

5

54

11 pM Tabersonine 14 pM NADPH -

45 f l 2-Oxoglutarate 45 pM Molecular oxygen 0.03pM Desacetoxyvindoline 6.5 pM AcetylCoA 1.3 pM Vindoline

226

Partial

21 7 222 223 52 182 267 243

Partial

62 pM Iminium cathenamine

15/37

Heme Heme NADPH SAM

Partial

Vacuole Endoplasmatic reticulum Thylakoid membranes

Partial 7.5-8

-

-

Partial

244 245

Cytoplasm

7-8

Partial

241 242 243

Cytoplasm

8-9

Apparent homogeneity

249

* Abbreviations: PP, pyridoxal phosphate; PQQ, pyrroloquinoline quinone; SAM, S-adenosyl-L-methionine A. Ramos ef al., manuscript in preparation.

230

VERPOORTE, VAN

DER

HEIJDEN, AND

MORENO

TABLE I11 RANGEOF ACT~VITIES OF SOME ENZYMES INVOLVED IN THE BIOSYNTHESIS OF TERPENOID INDOLEALKALOIDS IN Carharanthus roseus SUSPENSION-CULTURED CELLS' INDICATIVE

Enzyme

Activity Range (pkathg protein)

Anthranilate synthase Tryptophan decarboxylase Acetoacetyl-CoA thiolase HMG-CoA synthase Mevalonate kinase Geraniol-lO-hydroxylase Strictosidine synthase Strictosidine alucosidase

AS TDC AACT HMGS GlOH

sss SG

1-5 0-50 200-1000 10-100 200-500 0-40 10-400 1000-2000

* Grown under standard (noninduced) conditions in our laboratories.

enzymes are summarized. In Table I11 the activities of some of these enzymes as measured in our laboratories in C. roseus cell cultures are presented. OF GERANIOL A. BIOSYNTHESIS

Terpenoids are a diverse group of plant primary and secondary metabolites, which are involved in many vital processes in the plant and in interactions of the plant with its environment (69). A wide spectrum of chemical structures is connected with these diverse functions. The isoprenoids are derived from mevalonic acid (MVA), which is formed from three molecules of acetyl-CoA (Fig. 3). Two molecules of acetyl-CoA are condensed, yielding acetoacetyl-CoA, Subsequently, this product is coupled with another molecule of acetyl-CoA to yield 33hydroxy-3-methylglutaryl-CoA(HMG-CoA). By reduction of HMG-CoA MVA is obtained. MVA is further converted in some steps to yield the Cs-unit isopentenyl diphosphate (IPP), which is then isomerized to dimethylallyl diphosphate (DMAPP), the starter molecule of the isoprenoid pathway. Coupling of DMAPP with one or more IPP molecules yields the basic structures which form the backbone of terpenoid biosynthesis. A number of reviews on the early steps in the terpenoid biosynthesis have been published (70-77). Quite a few of the enzymes involved in the biosynthesis of HMG-CoA have been studied in C. roseus. Acetoacetyl-CoA thiolase (AACT, acetylCoA: acetyl-CoA C-transferase, EC 2.1.3.9) catalyzing the first step, the Claisen-type condensation of two molecules of acetyl-CoA, was partially purified from a cell suspension culture of C. roseus (78). The enzyme consists

0

0 H3C

SCoA

AACT

0

0 HMGS

H3C

acetoacetylCoA

@ -

0 H,C

3s-hydroxy-3-meth ylglutary l-CoA

COO-

SCoA

acetyl-CoA

1

9 SCoA

COO-

COO-

acetoacetate

acetyl-CoA

COO-

3R-mevalonate MVA kinase

MVAPkinase H

O

)

7

op, co2 A3-isopentenyl &phosphate

COO-

3R-mwalonate-5-diphosphate

COO-

3R-mevalonate-5-phosphate

FIG.3. Early steps of terpenoid biosynthesis: the formation of IPP.

3-methylglutaconyl-CoA

232

VERPOORTE, VAN DER HEIJDEN, AND MORENO

of several identical subunits with a molecular mass of 41 kDa, which is similar to the value reported for the avian enzyme (79). The mammalian and avian thiolases form a family of enzymes, which fulfill a broad range of functions. Their roles in fatty acid degradation and sterol biosynthesis are particularly important. Thiolases are classified based on their subcellular localization and their affinity for short-, medium-, or long-chain CoA-esters. The cytosolic AACT, which is specific for the short-chain-CoA ester acetoacetyl-CoA, is involved in sterol biosynthesis. A mitochondria1AACT is involved in ketogenesis. The subcellular localization and the substrate specificityof the thiolase isolated from C. roseus have not yet been studied. A role in sterol biosynthesis is suggested by the fact that during its chromatographic purification the C. roseus enzyme always coelutes with HMG-CoA synthase, the next enzyme in the MVA biosynthesis (80).From other plants, little is known about AACT activity (81). HMG-CoA synthase (EC 4.1.3.5), catalyzing the formation of HMGCoA and CoASH from acetyl-CoA and acetoacetyl-CoA, and not yet characterized from plants, was partially purified from C. roseus. It is an unstable enzyme that is rapidly inactivated in the presence of a relatively high concentration of salt (>200 mM).Thus far it has not been separated from AACT (81).HMG-CoA synthase activity elutes from gel filtration columns (e.g., Superose 6) with a retention time similar to that of polypeptides with an M, of about 100 kDa. HMG-CoA synthases isolated from other eukaryotic sources are mostly dimeric proteins of which the subunits have M, of 50-55 kDa; for example, HMG-CoA synthase from chicken liver consists of two subunits of 53 kDa (82). When L-659,699, a p-lactone inhibitor of mammalian cytosolicHMG-CoA synthase isolated from, among other sources, Fusuriurn sp. (83),is added to suspension cultured cells of C. roseus, HMG-CoA synthase is, like the mammalian enzyme, inhibited (R. van der Heijden et uL, unpublished results). A modification of the well-established mammalian and microbial HMGCoA pathways was found in plants. In this pathway the formation of HMGCoA from acetyl-CoA may proceed in a single step. In, among others, etiolated seedlings of Ruphunus sutivus (radish), HMG-CoA is formed directly from three molecules of acetyl-CoA, without the release of acetoacetyl-CoA from the enzyme involved (84). In mammalian cells, HMG-CoA is a substrate for several enzymes involved in metabolic processes, including cholesterogenesis, ketogenesis, and leucine metabolism. HMG-CoA lyase (EC 4.1.3.4) catalyzes the formation of the ketone body acetoacetate and acetyl-CoA from HMG-CoA. Acetoacetate is regarded as a transportable form of acetyl-CoA and plays an important role in metabolism during fasting (85).The enzyme is localized in the mitochondria and is also involved in leucine metabolism. Recently, this enzyme has been partially purified from radish (86) and from C. roseus

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

233

(78,87). The C. ruseus enzyme is relatively unstable, and is specific with respect to the 3 (RIS) configuration in HMG-CoA. In C. roseus tissues, the highest HMG-CoA lyase activities were found in stems and roots. 3-Methylglutaryl-CoA hydratase (EC 4.2.1.18) catalyzes the reversible dehydration of HMG-CoA into 3-methylglutaconyl-CoA and is known from mammalian cells to be involved in leucine metabolism. This enzyme was detected in C. ruseus suspension cultured cells and partially purified (87).Further enzymes channeling HMG-CoA away from isoprenoid biosynthesis in C. ruseus cells were detected by a selective HPLC system showing that the CoA-esters may be rapidly dephosphorylated on the 3’-position by the action of 3’-nucleotidases (87). In the presence of NADPH, HMG-CoA is reduced to MVA. The reductase (R-mevalonate: NADP+ oxidoreductase, CoA acylating, EC 1.1.1.34) is one of the few well-characterized plant enzymes, as it is a major site of regulation of terpenoid biosynthesis. The HMG-CoA reductase has been purified from a number of species, and its characteristics and regulation mechanisms have been the subject of extensive reviews (70,72,74).The C. ruseus enzyme(s) has not yet been purified, although a gene encoding HMG-CoA reductase has been cloned (see later discussion) (88,89). MVA is phosphorylated in two steps to the mono- and the diphosphate (MVAP and MVAPP), by the specific ATP-dependent enzymes MVA kinase (ATP-mevalonate-phosphotransferase,EC 2.7.1.36) and MVAP kinase (EC 2.7.4.2), respectively. MVAPP is converted into IPP by a decarboxylase (EC 4.1.1.33). The kinases and the decarboxylase have not yet been given as much attention as HMG-CoA reductase, and have only been characterized in a few plant species. Recently, MVA kinase was partially purified from C. ruseus suspension cultures. The enzyme proved to be unstable and was present only at low activity levels (90). There is not yet a consensus about the subcellular origin of MVA as a precursor for the terpenoid pathways (71-77,80,87). In essence, the two extreme hypotheses are that one cytosolic site of MVA production is a source for all isoprenoid-producing cell compartments, or that each compartment (cytosol, mitochondria, and plastids) has its own complete IPP pathway. Besides the well-documented MVA pathway leading to IPP and DMAPP as precursors for isoprenoid biosynthesis, there is evidence for other pathways leading to IPP and DMAPP as well. Leucine and valine have been shown to be incorporated into squalene and 6-amyrin (91). Based on a study using six different bacteria, it was concluded that IPP could be formed via at least two different pathways (92). The formation of IPP/DMAPP via an alternative, nonmevalonate, pathway has been described (93). In this pathway, a TPP-activated acetaldehyde (generated by pyruvate decarboxylation) is coupled to the C-2 carbonyl

234

VERPOORTE, VAN DER

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group of a dihydroxyacetone derivative. After a transposition reaction, the reduction, isomerization, and elimination reactions may proceed as found in the L-valine biosynthesis; the final product is the C5-unit (DMAPPhPP). Thus far this pathway has been found to lead to, among others, the hopane type of triterpenes (9495) and membrane sterols (96) in certain microorganisms. The first evidence for the occurrence of the nonmevalonate pathway in plants is in the biosynthesis of ginkgolides, a group of diterpenes, in Ginkgo biloba (97). Although no studies have been performed on the level of the enzymes, for the iridoid pathway leading to the terpenoid indole alkaloids in C. roseus the incorporation of MVA, and thus the involvement of the HMG-CoA pathway, seems quite well established (12,13). IPP is the building block of the isoprenoids; however, to start their biosynthesis IPP must be isomerized into dimethylallyl diphosphate (DMAPP), the actual starter molecule for isoprenoid biosynthesis. This isomerization is catalyzed by IPP isomerase (EC 5.3.3.2) (for a review, see ref. 98). The enzyme transforms the relatively unreactive IPP into a highly reactive molecule (DMAPP) (Fig. 4). Compared to other cell cultures belonging to the family Apocynaceae (Tabernaemontana species) and Rubiaceae (Cinchonaspecies), the cultured cells of C. roseus contained relatively low IPP isomerase activity. However, the protein could be clearly observed by immunodetection (Western blotting) using antibodies raised against purified IPP isomerase from Capsicum annuum. By anion-exchange and hydroxyapatite chromatography, IPP isomerase and farnesyl diphosphate IF'P- isomerase

A3-isopentenyldiphosphate

3,3-dimethylallyl&phosphate

Y ropip GPP synthase

phosphatase

geraniol diphosphate

PIP&^= diphosphate estei FIG.4. Pathway from IPP to geraniol.

foH

geraniol

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

235

(FPP) synthase were partially purified from C. roseus suspension-cultured cells (98). IPP isomerase activity was also determined in 5-day-old C. roseus suspension-cultured cells, treated with a Pythium aphanidermatum elicitor preparation. A slight inhibition of the enzyme was observed during the first 120 h after elicitor treatment (99). IPP and DMAPP are coupled in a head-to-tail manner by prenyltransferases. The coupling of one molecule of IPP and DMAPP yields geranyl diphosphate, the precursor for the monoterpenes, including iridoids such as secologanin. The coupling of further C5-unitsyields CIS(sesquiterpenes) and C20(diterpenes) compounds. Coupling of two CISmolecules results in squalene, the precursor for sterols and triterpenes, and that of two C20 molecules leads to the carotenoids. In other words, several pathways with quite different functions use the C5building block. Whether these pathways compete for the same C5 precursor pool or whether separate pathways exist is still a matter of debate, but at least the pathways leading to CIS and C30are believed to be cytosolic, whereas the Cl0,C20,and C4, pathways seem to be plastidial. However, the localization of a geranyl diphosphate synthase in the cytosol has been reported. This enzyme is involved in the biosynthesis of naphthoquinones in Lithospermum erythrorhizon (ZOO). Prenyltransferase activities have been studied in C. roseus both at the enzyme level and at the product level. Biosynthetic capabilities were investigated by incubating [1-'4C]IPP with aliquots of cell-free homogenates prepared from P. aphanidermatum treated and untreated suspension-cultured cells of C. roseus. After elicitation, the total incorporation of IPP into prenyl lipids was decreased, in particular into squalene. But the incorporation of IPP into some (as yet unidentified) compounds was increased (99). The prenyltransferases and subsequent enzyme activities are relatively easily extracted and remain complexed so that the product of one enzyme can be used as a substrate for the next enzyme. With an assay for these enzymes as described in detail in Threlfall and Whitehead (ZOI), about a dozen enzyme activities could be detected in series using cell-free preparations of elicited Tabernaemontana divaricata cells (27). In the elicited C. roseus cells, the activities of IPP isomerase, farnesyl diphosphate synthase, squalene synthase, squalene-2,3-epoxidase (and probably also a squalene-2,3epoxide cyclase) were thus detected. Compared with the control nontreated cells, squalene production seemed to be reduced particularly (99). B. BIOSYNTHESIS OF SECOLOGANIN After the ubiquitous biosynthetic pathway leading to geranyl diphosphate, a series of specific steps, restricted to a few plant species, leads to secologanin (for a general review see ref. 102). The intermediates, which

236

VERPOORTE, VAN

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have been postulated on the basis of tracer studies, are given in Fig. 5. It is noteworthy that geraniol is the substrate for this pathway, as pathways leading to other types of terpenoids such as sesqui- and diterpenes start with the respective diphosphates. Geraniol is hydroxylated on C-10 by the enzyme geraniol-10-hydroxylase (GlOH) (103). This hydroxylation of geraniol occurs exclusively at C-10 with retention of configuration (103105). The enzyme is regarded as a potential site for regulation, as the enzyme catalyzes the first committed step of secologanin biosynthesis. This hypothesis was supported by the observation that GlOH activity was induced in C. roseus cultures transferred to induction medium and by the close relationship of GlOH activity and alkaloid accumulation (106). Another argument for this hypothesis was the noncompetitive inhibition of the enzyme by the alkaloid catharanthine, at a K, of 1 mM. which is similar to the concentration of this product of the terpenoid indole alkaloid pathway in the plant (107). The enzyme is a cytochrome P-450 monooxygenase, which is reflected in its properties. It is membrane-bound, is dependent on NADPH and molecular oxygen, and displays a light-reversible inhibition by carbon monoxide. Cytochrome P-450 enzymes are dependent on a membrane-bound reductase (NADPH :cytochrome P-450 reductase, EC 1.6.2.4), a flavoprotein that is involved in the electron transfer from NADPH to the cytochrome P-450 heme group. Madyastha et al. (108) partially purified GlOH from C. roseus seedlings. The enzyme accepted both geraniol and nerol as substrates, the K , values being 5.5 and 11 p M , respectively. The enzyme was found to be localized in provacuolar membranes (109), unlike many other cytochrome P-450 enzymes that are present in the ER. The reductase was purified to homogeneity (210). The M,of the C. roseus reductase was estimated to be 78 kDa, very similar to the mammalian cytochrome P-450 reductase enzymes. It contained FMN and FAD as cofactors and was strictly dependent on NADPH. Recently, GlOH was purified to apparent homogeneity from C. roseus suspension cultured cells (111,112). After solubilization with cholate, the enzyme was purified in a four-step procedure. In the first step the cytochrome P-450 part was separated from the reductase, necessitating a reconstitution procedure with purified reductase and a lipid fraction from C. roseus cells, for measuring GlOH activity. The M , of the purified enzyme is 56 kDa and has a p l of 8.3. The specific cytochrome P-450 content was 4.7 nmol/mg protein. The enzyme accepted both geraniol and nerol as substrate. The NADPH :cytochrome P-450 reductase was purified by means of affinity chromatography (113). Western blots showed a polypeptide at M ,

geraniol

10-hydroxygeraniol

10-oxogeranial

iridodial

secologanic acid

FIG.5. Pathway from geraniol to secologanh.

iridotrial

238

VERPOORTE, VAN

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74 kDa; in crude membrane fractions, two bands were seen at 82 and 62 kDa. Treatment of this fraction with trypsin resulted in only one band with M , of 74 kDa, similar to the affinity-purifiedprotein. The intact protein is thus believed to have a M , of 82 kDa, a value which is in agreement with the molecular weight calculated from the sequence of the gene (see as follows). The enzyme has a 39% homology with the rat reductase, and 31% with the yeast reductase. The domains involved in cofactor binding are well conserved. Similarly to the rat and yeast reductase, a hydrophobic domain is found close to the N-terminus and probably is responsible for the membrane binding. The cytochrome P450 inducers phenobarbital and beta-naphthoflavone slightly inhibited the growth of C. roseus suspension cultures. Phenobarbital increased serpentine accumulation in cells in a production medium, but not in cells grown in a normal B5-growth medium. The cytochrome P450 inhibitor ketoconazole was found to inhibit serpentine production (114). No measurements of GlOH activities were performed. Soluble oxidoreductases were isolated from C. roseus, which in the presence of NAD' or NADP+ could oxidize 10-hydroxygeraniolinto the dialdehyde 10-0x0-geranial (Fig. 5) (125). By means of extensive feeding experiments with C. roseus (114,117), Lonicera morrowi (113,and L. tatarica plants (223,as well as R. serpenrina cultured cells (217-120), using various possible intermediates, it was found that it is this dialdehyde (10oxogeraniaY10-oxoneral)that is cyclized to iridodial. The enzyme responsible for the cyclization was partially purified from R. serpenrina (127,219,120). The cyclase has a pH optimum of 7.0, and its molecular weight was estimated to be 118 kDa, by means of gel filtration, and, by SDS-PAGE, 28.7 kDa. NADPH, and to a lesser extent NADH, considerably enhanced the activity of the enzyme. These results seem to exclude the possibility that iridotrial is the compound that is cyclized, as was proposed by Balsevich and Kurz (121). The third aldehyde function is thus formed after the cyclization. Of the further steps leading to loganin and secologanin only a few have been studied in more detail. These steps include oxidations, ring closure, glucosylation, and methylation. The carboxylic acid function is methylated by the enzyme S-adenosyl-Lmethionine :loganic acid methyltransferase. The enzyme was partly purified from C. roseus seedlings (122). It catalyzes the transfer of a methyl group from S-adenosyl-L-methionineto loganic acid ( K , 12.5 mM) or secologanic acid. 7-Deoxyloganic acid was not accepted as substrate. The enzyme has a maximum activity just after germination, rapidly converting the loganic acid stored in the seeds (1% of FW). Throughout the germination its activity profile parallels alkaloid accumulation, as only secologanin and not secologanic acid can be used for alkaloid biosynthesis. This makes the methylation of the iridoids a critical step in alkaloid biosynthesis (115,123).

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

239

In the last step, the five-membered ring of loganin is opened to yield secologanin. The nature of this reaction remains unclear. Several mechanisms were proposed, but were subsequently disproven by feeding the putative intermediates. The intermediacy of 10-hydroxy- or 10phosphoryloxyloganin is less likely, because in a series of feeding experiments they were only incorporated in very low ratios into secologanin (for 10-hydroxyloganin less than to 0.0036% compared with 20.3% for loganin) (124,125). Moreover, by dilution experiments only very low levels of 10hydroxyloganin could be detected in the various plants studied. 10Phosphoryloxyloganin is an excellent candidate for the oxidative ring opening (224,225),but it is not clear from the literature whether this compound has been excluded as intermediate based on direct evidence (226),or only on indirect evidence (227). An oxidative cleavage between C-7 and C-8 can be ruled out as the protons at these carbons are retained in secologanin (128-132). The intermediacy of 6-hydroxyloganin was excluded because after different secoiridoid-producing plants were fed with both C-7, 7-2H-labeled stereoisomers, no label could be found in secologanin (226). In feeding experiments with loganin with all protons at C-6, C-7, and C-8 replaced by deuterium, it was found that all labels were retained in the secologanin (232). This also rules out the intermediacy of a 6-thio-derivative. Naudascher et al. (133,134) studied the time course of secologanin and loganin conversion by C. roseus cell cultures. Although alkaloid accumulation increased manyfold (235), the amount of alkaloid produced was less than 1%of the added secologanin. Secologanin fed to the cells disappeared rapidly from the medium; within 4 h only 10%could be detected. Depending on the age of the cells, after 8-10 h no secologanin could be detected in the medium; at that point a maximum was observed in the cells. For 4day-old cells, this was 96% of the added compound; in 10-day-old cells this was 75%. In the younger cells further conversion was much more rapid, reaching a plateau of about 50% of the added compound after about 10 h. For the older cells a plateau of about 20% was reached after about 39 h. Alkaloid levels were slightly increased, although apparently not all of the secologanin was available for alkaloid biosynthesis. Feeding of tryptophan did not further increase alkaloid levels. Evidence was given that secologanin is stored in a reversible form, not recognized as such by the detection methods used in the study. This reversible form is neither loganin nor (seco)loganic acid. Feeding loganin (234)to 4-day-old cells resulted in rapid uptake from the medium. In the cells after 6 h a transient peak was observed of loganin, representing 56% of the fed compound. Two hours later secologanin showed a similar transient peak. After 48 h the levels were less than 1% of the added amount. In 10-day-old cells uptake was much slower, and accumulation in the cells was lower and later (maximum at 15 h). The

240

VERPOORTE. VAN DER

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breakdown is also much slower; after 48 h 25%was still left, and no secologanin could be detected in these cells. As these cells do accumulate alkaloids, the secologanin formed is probably immediately directed into the alkaloid production. In contrast to secologanin feeding, all loganin is converted by the cells. Moreno el al. (136) found that feeding loganin, secologanin, or loganic acid to a C.roseus cell culture resulted in a considerable increase of strictosidine and ajmalicine levels, and a concomitant decrease of tryptamine. Also, after these iridoids were fed to elicited cells a decrease of tryptamine levels was observed, but strictosidine and ajmalicine levels were much lower than in the normal cells to which the iridoids were fed, suggestinga rapid turnover of strictosidine. It was suggested that the enzyme responsible for the conversion of loganin into secologanin might be one of the targets for 2,4-D in its inhibitory effect on alkaloid accumulation in suspension cultures of C. roseus (237). Efforts to detect this enzyme in C. roseus cell-free extracts have not been successful as yet (A. Contin et al., unpublished results).

c. BIOSYNTHESIS OF TRYFTOPHAN The indole moiety of the terpenoid indole alkaloids originates from tryptophan, an aromatic amino acid, which is derived from chorismate via anthranilate. Chorismate is a major branching point in plant primary and secondary metabolism. Here the shikimate pathway (Fig. 6) branches into different pathways (Fig. 7), among others leading to the aromatic amino acids tyrosine, phenylalanine, and tryptophan. The biosynthesis of shikimate, the direct precursor for chorismate, has been reviewed elsewhere (138-141). The shikimate pathway leading to chorismate is located in the plastids. For the two branches from chorismate leading to the aromatic amino acids, it has been postulated that both occur in a plastidial and a cytosolic form (142). The plastidial form is responsible for the aromatic amino acids for primary metabolism, and the cytosolic one for the biosynthesis of the aromatic amino acids used as precursors in secondary metabolism (for a review, see refs. 141,143). The largest flux of carbon atoms from chorismate goes into the phenylalanine/tyrosine pathway, among others leading to lignin and important groups of secondary metabolites such as flavonoids and anthocyanins. The first enzyme in that particular pathway, chorismate mutase (CM, EC 5.4.99.5), catalyzes the conversion of chorismate to prephenate (Fig. 8). Both a cytosolic and a plastidial form have been detected in several plants (e.g., 144147). The plastidial isoform is inhibited by phenylalanine and tyrosine, and activated by tryptophan; the other isoform is not affected by these

OH

Pi 0A : H

-

o +P -

+

OH i

3deoxy-~-arabino-heptulosonate 7-phosphate(DAHP)

H

D-erythrose4phosphate

OH

Pi0

u

COO-

phosphoenolpyruvate(PEP)

3-dehydroquinate

3-dehydroshikimate

shikimate

shikimate-%phosphate

5-enolpyruvylshikimate-3-phosphate

COO-

Pi& phosphate ester

OH

chorisrnate

FIG.6. Shikimate pathway to chorismate.

COO-

OH

coo-

1

chocismate

ICS

coo-

bNb

coo-

anbanilate

N N P

*'

, '.. ,*'

iSOChOliS~te

'\

Coo-

i

COO-

2,3-dihydIo~oate

L-hyptophan

OH Lphenylalanine

L-tyrosine o -succinylbenzoate

betalaines CoUlMIjlls

anthocyanins

phylloquinone antluaquinones

FIG.7. Chorismate as major branching point in secondary metabolism.

indole acetic add fhllmlines indole alkaloids

COOCOO-

' 6H

i

OH

\\

0

COO-

bN&+

PDHY

CO, +-H20

phenylpyruvate

L-phenylalanine

Q

N

e

OH

OH

L-tyrosine

FIG.8. From chorismate to phenylalanine and tyrosine. (PDHY = prephenate dehydratase EC 4.2.1.51;PDH = prephenate dehydrogenase EC 1.3.1.13;TAT = tyrosine aminotransferase EC 2.6.1.5;PREPAT = prephanate aminotransferase; F'TDH = pretyrosine dehydrogenase.)

244

VERPOORTE, VAN DER HEIJDEN, AND MORENO

compounds. From some plants the two isoenzymes have been isolated (148,249).

Also in C. roseus two forms are present (150, R. Bongaerts et al., unpublished results). One is strongly regulated: inhibition by phenylalanine and tyrosine and induction by tryptophan. The other is not influenced by these aromatic amino acids. The apparent molecular mass, determined by gel filtration, of the regulated form CM-1 is 44 kDa. The activity measured for CM is about 100-fold higher than for anthranilate synthase, catalyzing the first committed step in tryptophan biosynthesis (152). L-Tryptophan is formed through a biosynthetic pathway consisting of five enzymatically controlled steps (152). In plants, tryptophan is required for protein synthesis and also provides precursors for secondary metabolites, such as indole alkaloids. The formation of anthranilate and the following four steps in the tryptophan biosynthesis are invariant in all organisms studied to date (153) (Fig. 9). Anthranilate synthase (AS, EC 4.1.3.27) catalyzes the conversion of chorismate to anthranilate, the first step in this pathway. Two forms of anthranilate synthase have been reported in 5methyltryptophan-resistant tobacco cell culures: a plastidial isoenzyme, which is strongly inhibited by tryptophan, and a cytosolic one, which is not affected by tryptophan (154,155). In C. roseus, we have not yet been able to detect a non-tryptophan-regulated form of AS (156). Although two peaks of activity could be detected under certain conditions of protein separation, these peaks were interconvertible. Both were strongly inhibited by tryptophan (Ki of 2-3 p M ) . From C. roseus cells, AS was isolated and purified to apparent homogeneity (156). AS is a tetramer, consisting of two large (ca 67 kDa) and two small (ca 25.5 kDa) subunits. The larger asubunit is responsible for the conversion of chorismate into anthranilate; the smaller p-subunits are responsible for the generation of the substrate NH3 from glutamine. The enzyme shows Michaelis-Menten kinetics for the amino-group donor glutamine, chorismate, and the cofactor Mg2+( K , values respectively 0.37 mM, 67 p M , and 0.26 mM). AS shows positive cooperativity of chorismate binding at higher levels of tryptophan. The tryptophan binding sites showed positive cooperativity at higher concentrations of chorismate. AS is induced by elicitation (151) and the combination of 2,3-dihydroxybenzoic acid (DHBA) and UV light (157). The next enzyme in the tryptophan pathway is anthranilate-5phosphoribosyl transferase (EC 2.4.2.18). In some microbial enzymes it is part of the AS enzyme complex, but this transferase activity could not be measured in the purified C. roseus enzyme. So far, none of the enzymes leading to tryptophan after AS (152,158) has been studied in C. roseus. Only a time course of tryptophan synthase

indole glycerol phosphate

L-tryptophan NH, L-

serine

Pi& phosphate ester

FIG.9. From chorismate to tryptophan.

1-(C-carboxyphenyl~O)-l'deoxyribulosephosphate

246

VERPOORTE, VAN

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(EC 4.2.1.20) activity during the growth of suspension-cultured and immobilized cells of C. ruseus has been determined (259). Immobilized cells presented much lower tryptophan synthase activity than suspension-cultured cells. These findings were consistent with low tryptamine content and a reduced free tryptophan pool in the immobilized cells. There is not enough evidence at the moment to prove or disprove the existence of a dual pathway for tryptophan biosynthesis in C. ruseus. The only higher plant in which tryptophan biosynthesis has been thoroughly studied is Arubidopsis thuliuna. In this plant there is biochemical and genetic evidence that the tryptophan biosynthetic enzymes are localized in the chloroplasts (260,162). A third chorismate utilizing enzyme, isochorismate synthase (ICS, EC 5.4.99.6) is strongly induced after elicitation of C. ruseus cells (257,262,263), with a concomitant formation of 2,3-dihydroxybenzoic acid (DHBA), a product that in microorganisms is known to be formed via isochorismate (Fig. 10).Also, in the plant the induction of ICS was observed after wounding. ICS catalyzes the conversion of chorismate to isochorismate, a precursor of primary metabolites such as menaquinone and enterobactine in bacteria, and phylloquinone in higher plants (143,164). In the secondary metabolism of higher plants, isochorismic acid is also an important precursor for the biosynthesis of anthraquinones (165,166),for example, in Cinchona species also producing terpenoid indole alkaloids (40,267-269). The activity of ICS in the C. roseus cell cultures was 5-10-fold lower than AS activity (262,162). We have purified ICS from elicited C. roseus cell suspension cultures (P. R. H. Moreno er al., unpublished results). This

r-

coo-

chorismate

isochorismate

pyruvate

l

A

8-,

&"" coo-

COO-

DHBADH,

OH

2,3-dihydro-2,3dihydroxybenzoate

2,Mihydroxybenzoate

FIG.10. From chorismate to DHBA.

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

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is the first time that this enzyme has been purified to apparent homogeneity from a plant source. ICS was present in two isoforms that could be separated by anion exchange chromatography. Both isoforms showed a similar molecular weight ( M , 96 kDa) in SDS-PAGE gel electrophoresis, and both are monomers. The enzyme accepted only chorismate as substrate and required Mg2+as cofactor; the K , values for chorismate for both isoforms were 556 and 319 puM, respectively. OF TRYPTAMINE D. BIOSYNTHESIS

For the formation of strictosidine, two precursors are needed: tryptamine and secologanin. Of these, tryptamine can be present at quite high levels in the cultured cells of C. roseus. The enzyme responsible for the tryptamine production is tryptophan decarboxylase (TDC, EC 4.1.1.28) (Fig. 11). TDC has been the subject of extensive studies. It was found to be strongly regulated (170,172).However, from various experiments it is known that in fact the terpenoid pathway is the major limiting factor for alkaloid biosynthesis in cell cultures; for example, feeding of secologanin or loganin results in increased alkaloid levels (133,134,136,159,172-174). Particularly after elicitation, tryptamine is accumulated in cell suspension cultures of C. roseus (136,175,176). Since the first purification of TDC by Noe et al. (277),this protein has been extensively characterized (178-280). TDC is a cytosolic enzyme (35,39,182-183) and consists of two identical subunits (molecular weights of 54 kDa). It showed Michaelis-Menten kinetics ( K , 75 pit4); besides tryptophan, it also accepts 5-hydroxy-, 5-flUOrO-, 4-flUOrO-, and 5methyltryptophan as substrates. D-Tryptophan acts as a noncompetitive inhibitor, tryptamine as a competitive inhibitor ( K , 310 p M ) (177).Pennings et al. (179) reported that TDC contains two molecules of pyrroloquinolinequinone (PQQ) and two molecules of pyridoxal phosphate. Phenylalanine, tyrosine, and L-DOPA are not accepted as substrates in vitro, but in transgenic tobacco plants carrying the Tdc gene, increased tyramine levels were found (284,185). 4

6

7

+ COZ

H1

L-tryptophan

H

hyptamine

FIG.11. Decarboxylation of tryptophan.

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Western blotting of protein extracts of various C. roseus plant materials after SDS-PAGE, with polyclonal antibodies raised against the purified enzyme from elicited C. roseus cells, revealed several bands. During the development of seedlings, first an immunoresponsive 54.8-kDa band, which is devoid of activity, is noted. Subsequently, a transient increase of a 55kDa band is observed, which coincides with the occurrence of TDC activity. With the decrease of the TDC activity some other bands could be detected (40,44,and 67 kDa). In leaves, the 54.8-kDa band could not be detected (278).In a series of in vitro experiments, Fernandez et a/. (286)studied the stability of TDC. The presence of Mn2+,or particularly Mg2+has a stabilizing effect on TDC this might be due to a stabilization of the active dimeric form, the monomer being more susceptible to inactivation by a proteolytic process. ATP increased the rate of inactivation. It was suggested that in this way the TDC activity is regulated by posttranslational controls. In a further study by Fernandez and De Luca (187), it was shown that some protein bands (63 and 68 kDa) detected with TDC antibodies were ubiquinated. The monomer is thus probably irreversibly inactivated through conjugation with ubiquitin, and subsequent proteolysis. Transgenic tobacco plants expressing the Tdc gene were found to contain high levels of tryptamine, over 1 mg/g FW (288-290).Poulsen et al. (292) reported that in such transgenic plants no increase of AS activity could be measured; apparently, the normal tryptophan biosynthetic capacity is capable of producing about 1%on dry weight of tryptamine. This is quite interesting, as most indole alkaloid-producing plants also have about 1% of the total biomass in alkaloid, i.e., probably from normal tryptophan metabolism a plant can make about 1%of tryptophan-derived secondary metabolites. The decarboxylation of the toxic 4-methyltryptophan mentioned earlier has been patented as a basis for a selection procedure of transgenic plants, using the C. roseus Tdc gene as selection marker gene (292). E. BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

Terpenoid indole alkaloid biosynthesis actually starts with the coupling of tryptarnine and secologanin (Fig. 12). In the next step, a glucosidase splits off the sugar moiety and the reactive dialdehyde formed is further converted through different pathways to a cascade of products, including ajmalicine, catharanthine, tabersonine, and vindoline. Strictosidine is the precursor for a broad variety of terpenoid indole alkaloids in a number of plant species, which all share the first part of this pathway. From strictosidine onward, diversification occurs. The basis for this diversity lies in the first place at the dialdehyde, which is formed after

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secologanin

tryptamine

CH,OOC

17

22

strictosidine

FIG.12. Formation of strictosidine.

deglucosylation of strictosidine. In this dialdehyde a number of reactive groups exist: two aldehyde functions, one allylic double bond, and two secondary amine functions. In fact, for all possible combinations of these reactive groups one can find examples among the terpenoid indole alkaloids (Fig. 13). 1. Strictosidine Synthase

The stereospecific condensation of secologanin and tryptamine is catalyzed by the enzyme strictosidine synthase (SSS, EC 4.3.3.2). Smith (193) postulated that strictosidine is the key intermediate for the terpenoid indole alkaloids. Stsckigt and Zenk (194,195) and Scott et al. (196) showed that this intermediate indeed is formed through a specific enzyme, and its presence in a series of plants producing terpenoid indole alkaloids was shown (197). The first partial purification of SSS from C. roseus was reported by Treimer and Zenk (198) and by Mizukami et al. (199). The molecular weight was estimated to be between 34 and 38 kDa. The enzyme is soluble and does not require any cofactors. SSS is highly specific for both substrates (198); for example, tryptophan was not accepted as substrate, nor was any substituted tryptamine derivative. Indole alkaloids such as ajmalicine, vindoline, and catharanthine did

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18

21

-

OH

malindine

C-19+N-4

dialdehyde

CH,

\

15

18 20

16 H3C 1s

-

C-17 + N-1

akagerine

C-21 t N - 1

0

17

OH

kribine

15 CH,OOC

17

C-19 + N-1

decussine

C-21+N-4

17

cathenamine

COOCH,

C-17 + N-4

-

vallesiachotamine

F a . 13. From strictosidine toward the various classes of indole alkaloids.

not inhibit SSS (199).It was reported that a series of isoforms are present in cell cultures (200). Of the seven forms detected, Pfitzner and Zenk characterized four. They differ in p l values (between 4.3 and 4.8) and K , values for tryptamine (from 0.9 to 6.6 mM);all have maximum activity at pH

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

25 1

6.7. Tryptamine inhibits SSS at concentration levels around the Michaelis constant. The molecular mass estimated by gel chromatography is about 31 kDa; by means of SDS electrophoresis it is 41.5 kDa. A similar difference was found for the enzyme isolated from Rauvol~5asp. (201). Also in the leaves different isoforms could be detected. By using polyclonal antibodies raised against one isoform of SSS, Pasquali et al. (202,203) showed that in different parts of the plant different patterns of isoforms are found. Pfitzner and Zenk (200) reported that SSS is glycosylated,which might be the cause of the different isoforms. Antibodies against one of the isoforms not only detected the other isoforms in C. roseus, but also cross reacted with SSS from other Apocynaceaous plants. No cross reactivity with SSS from Rubiaceae was found. The characteristics of four isoforms of SSS isolated from Cinchona robusta cell cultures indeed are quite different from those of C. roseus (38,47). Both enzymes are inhibited by quinine (at 2.2 mM, no activity was left for the Cinchona enzyme; for the C. roseus enzyme 24% of the activity was left. However, the two enzymes differ in substrate specificity (the Cinchona enzyme also accepts 5-methoxytryptophan as substrate) and pZ values (6.5 and 7.5, respectively, for the two pairs of Cinchona isoforms). De Waal et al. (204) reported the characterization of six isoforms of SSS isolated from C. roseus cell cultures. All isoforms were shown to be glycosylated. The pH optima found are broader (6-7.5) than previously reported. For all of the isoforms, very similar kinetics were found; however, they differ considerably from the previously reported data (200). First of all, no tryptamine inhibition (up to 5 mM) could be observed. Measuring the reaction progress curve rather than using a stopped assay, the K,,, determined for all isoforms is around 8.2-9.4 pM (average 8.9 p M ) ; the V,, is 153-312 nkat/mg (average 234 nkatlmg). No substrate inhibition could be found, although strictosidine inhibits the enzyme (Ki 248-442 p M ) . Particularly, the K , values found are much lower than the previously reported values of 0.9-6.6 mM (200). Although earlier reports suggested that SSS occurs in the cytosol (181,205),later studies unequivocally showed that it is located in the vacuole (35,39,52,182,206). The immobilization of SSS on CNBr-activated Sepharose has been described by Pfitzner and Zenk (207); with the immobilized enzyme Iarge amounts of strictosidine can be produced easily (201). The immobilized enzyme is quite stable: After 245 days of storage at 4"C, 50% activity was found. Hallard et al. (208) used SSS isolated from a transgenic tobacco cell line containing the Str gene for an assay of secologanin in plant material. Incubation with the crude enzyme, the plant extract, and tryptamine results

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in the formation of strictosidine, which can be quantified selectively and sensitively by means of HPLC. 2. Strictosidine Glucosidase

From strictosidine, the first step toward the various types of indole alkaloids is deglucosylation by strictosidine glucosidase (SG, EC 3.2.12). The key to the diversification in indole alkaloids must be at the glucosidase or in the steps directly after this enzyme. The glucosidase and the subsequent steps have thus been the subject of quite a few studies. The intermediates leading to ajmalicine have extensively been studied by Zenk, Stackigt, and co-workers (209-211). Cathenamine (210-213) is believed to be a major intermediate in this pathway. Moreover, evidence was presented for the intermediacy of 4,21-dehydrocorynantheinealdehyde (214) and 4,21-dehydrogeissoschizine(215) (Fig. 14). The formation of

FIG.14. From strictosidine to ajmalicine (211).

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253

cathenamine from the latter compound was believed to be catalyzed by cathenamine synthase. Stevens (39)studied the conversion of strictosidine by the partly purified C. roseus glucosidase directly by means of NMR. In these studies no 4,21dehydrogeissoschizine, which previously was pointed out as the central intermediate, could be detected. The reaction was monitored by means of 'H-NMR, using D 2 0 as solvent for the incubation mixture. The formation of free glucose could be observed, but the product of the incubation of the enzyme with strictosidine was not soluble in water, causing a precipitate, which did not show any signals in the NMR spectrum. In three different ways this product was isolated after completion of the reaction: by centrifugation, by extraction with chloroform, and by lyophilization. In the NMR spectrum of the extracted reaction product, cathenamine and 19-epi-cathenamine were the main products; no signals due to 4,21dehydrogeissoschizine could be observed. As the last compound is easily converted into cathenamine in chloroform solution, further experiments were done using methanol as solvent. 4,21-Dehydrogeissoschizineis expected to be stable in that solvent (226).After centrifugation, lyophilization of the residue, and subsequent dissolution in deuteromethanol, the characteristic signal at about 2 ppm of the protons attached to C-18 in 4,21dehydrogeissoschizine could not be observed in the 'H-NMR spectrum of the dissolved product. From its 'H-NMR spectrum, the product was identified as 21-0-methylcathenamine. After removal of the methanol and dissolution in CDC13,the product was spontaneously converted into cathenamine and its 19-epi isomer. Incubation in D 2 0 showed that only one deuterium is introduced at C-18, thus excluding an equilibrium between the dienamines A4-21,19-20 and A20-21.18-19 . B ased on these studies it was postulated that the carbinolamine of cathenamine (21-hydroxyajmalicine)was the intermediate (see Fig. 15) rather than 4,21-dehydrogeissoschizine (225). The formation of the latter compound is probably under enzymatic control and is thus the opening to other biosynthetic pathways. These results are in accordance with those reported by Hemscheidt (227). The formation of cathenamine from strictosidine occurs in the presence of a glucosidase alone; cathenamine synthase is not needed for this conversion. As no major differences were found for the glucolysis products of strictosidine for the C. roseus and the Tubernuernontuna divuricutu enzymes, the key for the formation of the different types of indole alkaloids is probably after the glucosidase, as the two cell cultures do produce quite different alkaloids (39,52,282,228). Scott et ul. (219) reported that some nonspecific glucosidases with a molecular mass of about 55 kDa were responsible for the conversion of strictosidine. Two such enzymes were detected in callus cultures and four

VERPOORTE, VAN

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carbinolamine

end

1

catmolarnine

J

calhenamine

FIG.15. From strictosidine to ajmalicine (39).

in seedlings and plants of C. roseus. The enzymes were believed to be part of a complex that also includes the enzymes responsible for the coupling of tryptamine and secologanin (SSS) and for the formation of ajmalicine out of strictosidine. However, Hemscheidt and Zenk (220) reported the presence two highly specific glucosidases in C. roseus cell cultures. For enzyme I, a molecular weight of about 230 kDa was estimated, and for enzyme 11, one of more than 450 kDa. Also, in some other Cutharunthus species and Apocynaceous species, strictosidine-specific glucosidases were found. For the C. roseus strictosidine glucosidase (SG), no activation by tryptamine could be detected as previously reported by Scott et al. (219); in fact, some inhibition was observed. The K, values of the enzymes I and I1 are respectively 0.1 and 0.2 mM. Enzyme I showed substrate inhibition at 1 mM; no inhibition was observed for enzyme 11. The pH optima are at 6.0-6.4. The two enzymes have different sensitivity for inhibition by the

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

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glucosidase inhibitor Sgluconolactone. Substrates such as secologanin, loganin, strictosidine lactam, and ipecoside are not accepted. Vincoside, the C-3 stereoisomer of strictosidine, is hydrolyzed only at very low rate. Stevens (39) and Luijendijk (52,182)have studied SG extensively. In size exclusion chromatography, a peak with SG activity with a molecular mass above 2000 kDa is observed, as well as major peaks of activity at 650 and 400 kDa. The enzyme apparently occurs in aggregated form. In contrast to the findings mentioned earlier, a broad pH optimum was found above pH 6. At lower pH, activity was lost. Of a series of metal ions tested, only Cu2+inhibits the enzyme (at 1 mM). Sugars do not inhibit SG; tryptamine and secologanin weakly inhibit SG activity (ca 15% at 1 mM); serpentine causes 40% inhibition at 1 mM. Iridoids, such as sweroside, loganin and secologanin, are not converted by the enzyme. Vincoside is only converted at a rate of 10% of that of strictosidine. 10-Methoxystrictosidine is readily converted; its C-3 stereoisomer 10-methoxyvincoside is not. The synthetic used to measure glucosidase activisubstrate p-nitrophenyl-P-D-glucoside, vity, is not accepted as substrate by SG (39,220).The glucosidase inhibitor D( +)-gluconic acid-Slactone weakly inhibits SG (52,182). The enzyme from a C. roscus cell culture was recently purified to homogeneity (52,182). In size exclusion chromatography on Sephacryl s-300 HR, the enzyme is not retained, that is, the molecular weight is above 1500 kDa. However, on Superose 6 one single peak of activity is observed, corresponding to a molecular mass of about 690 kDa. After the latter separation step the enzyme is much less stable. The use of a detergent in the extraction of the enzyme resulted in a 10-fold higher activity, but also in a reduced stability. Apparently, in aggregated form the enzyme is more stable. For the partly purified enzyme on native PAGE gels, using specific SG activity staining (222), a consistent pattern of three equidistant activity bands (at about 930,650 (major band), and 240 kDa) with some variation in molecular weight is observed. SG isolated from the stem of the plant does show other values (720, 450 (major band), and 210 kDa). On SDSPAGE gels only one band of 63 kDa is observed for the denatured enzyme. From this it was concluded that the smallest active form of the enzyme is a tetramer (240 kDa), which may further aggregate to high-molecularweight complexes. Treatment with proteases such as trypsin results in smaller fragments, but with retention of activity. The purified enzyme showed on native PAGE one main activity band at about 330 kDa after treatment with trypsin, with some smaller bands at higher molecular weights. On SDS-PAGE this preparation showed only one band at about 60 kDa. Although the native enzyme is affected, its stability toward proteases is remarkable. This might be important in connection with the possible

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role of SG in the plant's defense (see earlier discussion). The K , for strictosidine estimated by different methods is in the range of 10-18 pM, which is much lower than reported previously (220).The characteristics of the Tabernaemontana SG are similar to those found for C. roseus (39,52,182,218).In Cinchona ledgeriana root cultures, a similar enzyme is also present (52). In a localization study it was found that in isolated vacuoles of C. roseus cultured cells, about 60% of the SG activity was recovered compared with a vacuolar marker enzyme (35,39,52,182).SSS activity was completely recovered from the vacuoles, whereas only very little TDC activity was found in the vacuolar fraction. This means that SG must be associated with the tonoplast, as it is unlikely that SG occurs in the vacuole together with strictosidine. Indeed, a tonoplast preparation obtained from the isolated vacuoles contained most of the SG activity. Also, the pH optimum does not fit a localization in the vacuole. In the plant, the highest SG activities are found in the stems and young leaves; furthermore, activity is found in the flowers and roots. This activity profile more or less parallels that of SSS. These findings fit a model also described for other glucosidases: a substrate stored in the vacuole and an excess of glucosidase outside the vacuole. Damage of the cell results in the rapid conversion of the substrate into a highly reactive compound, that is, the model of a phytoanticipin (see earlier discussion). High levels of strictosidine in young tissues and the strong antimicrobial activity of the combination of strictosidine and SG are in agreement with such a role in plant defense, as discussed earlier. Wounding of leaves does not influence the levels of SG activity. 3. Cathenamine Reductase

The formation of ajmalicine from the carbinolamine or cathenamine requires a reduction. Hemscheidt (217) and Stiickigt et al. (222) described an enzyme cathenamine reductase (CR), which used cathenamine as substrate and NADPH as cofactor, yielding ajmalicine and 19-epi-ajmalicine. Hemscheidt and Zenk (223) reported partial purification of an NADPHdependent tetrahydroalstonine synthase (THAS) from C. roseus cell cultures. This enzyme only yields tetrahydroalstonine, and the substrate was the iminium form of cathenamine. The K,,, for this substrate is 62 pM.The molecular mass of the enzyme was estimated to be 81 kDa. Luijendijk (52,182) could detect both of these activities in cell cultures. The THAS activity being at a constant low level, the CR activity was even lower. THAS is a soluble enzyme. From isolated vacuoles most activity could be recovered, suggesting a vacuolar localization. The CR activity was too low to determine its localization. The role of THAS is not clear, as tetrahydroalstonine has only been detected as a minor compound in C. roseus. As the conversion of strictosidine is believed to occur outside the

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257

vacuole, the localization of the THAS implies that transport of its substrate must occur from the cytosol into the vacuole. It cannot be excluded that this reductase is a more general enzyme which also exhibits THAS activity. 4. Geissoschizine Dehydrogenase

Geissoschizine was found to be incorporated into heteroyohimbinealkaloids, but is not a real intermediate of the pathway (224,225). A dehydrogenase converts this compound into 4,21-dehydrogeissoschizine,which readily forms cathenamine, the precursor for ajmalicine. Geissoschizine dehydrogenase was partially purified from a C. roseus cell culture (226).The reaction is NADP+ dependent; NAD+,FAD, and FMN are not accepted as cofactors. The enzyme was quite stable, having optimum activity at pH 7.6. The K,,, for geissoschizine is 83 pM; other related alkaloids, such as ajmalicine and tetrahydroalstonine, were not accepted as substrate. The reaction is stereoselective and only the hydrogen at the less hindered site (H-21a) is removed.

5. Catharanthine The knowledge of the biosynthesis of catharanthine is very limited; only some feeding experiments with labeled precursors have been described, quite some years ago. Qureshi and Scott (227-229) reported that catharanthine is formed from tabersonine fed to the plant. However, other groups have not been able to confirm these results (230-232). Corynantheine aldehyde (229) and geissoschizine fed to C. roseus plants were reported to be incorporated into catharanthine (233). From these experiments it is believed that the pathway goes from strictosidine via 4,21-dehydrogeissoschizine, stemmadenine, and dehydrosecodine (Fig. 16). Based on the structures, the involvement of tabersonine in the catharanthine pathway is not likely, despite the reports of its incorporation. So far, nothing is known about the enzymes involved in this pathway. 6. Vindoline Pathway

Tabersonine is the precursor for vindoline, as was shown by various incorporation experiments (22J3).Both catharanthine and tabersonine are produced in cultured cells of C. roseus (for reviews see refs. 7-11). Vindoline and the derived bisindole alkaloids accumulate only in the green parts of C. roseus and are not found in the roots or cell suspension cultures (234). The pathway from tabersonine to vindoline can thus only be studied in plants or seedlings. There are, however, some claims of detection of vindoline in newly induced calli and cell suspension cultures from C. roseus plants with high vindoline production (235). Dark-grown seedlings accumulate catharanthine, tabersonine, and 11methoxytabersonine as major compounds and 1l-hydroxytabersonine,

P'

H H

catharanthine

ajmalicine

/

FIG.16. Pathway to catharanthine, tabersonine, and vindoline.

COOCH,

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

259

deacetylvindoline, 17-desacetoxyvindoline, and vindoline as minor compounds. Subjecting these seedlings to light results in the rapid increase of vindoline levels. The levels of the other alkaloids mentioned rapidly decrease. Rather than the three subsequent oxidations as proposed by Fahn et al. (236), Balsevich, De Luca, and co-workers (237-239) proposed the sequence of the biosynthetic pathway shown in Fig. 17: initial formation of 1l-methoxytabersonine, followed by hydroxylation at C-16, N-methylation, hydroxylation at C-17, and finally acetylation of the 17-OH. This sequence is also supported by the substrate selectivity of the various enzymes involved (see later discussion). The enzyme that catalyzes the hydroxylation of tabersonine, tabersoninell-hydroxylase (T1lH), has not yet been purified to homogeneity. However, a first characterization of this enzyme was recently reported (240,241). T l l H activity could be detected in crude protein extracts from young leaves of C. roseus. The hydroxylase activity was shown to be dependent on NADPH and molecular oxygen. It was inhibited by carbon monoxide, clotrimazole, miconazole, and cytochrome C. These characteristics suggested that T l l H is a cytochrome P-450 dependent monooxygenase. In a linear sucrose gradient, the enzyme activity was found in the fraction corresponding to the endoplasmatic reticulum. The enzyme is induced by light. The K , for tabersonine and NADPH are, respectively, 11 and 14 p M . The pH optimum is at 7.5-8.0. An O-methyltransferase was described that transfers a methyl group from S-adenosyl-L-methionine to ll-O-demethyl-17-O-deacetylvindoline, and which has a higher affinity for the deacetyl compound than for vindoline (236).However, later it was found that this enzyme in fact is active much earlier in the pathway, methylating 1l-hydroxytabersonine (237-239). S-Adenosyl-L-methionine:1l-Methoxy-2,16-dihydro-l6-hydroxytabersonine N-methyltransferase (NMT, EC 2.1.1.99) transfers a methyl group from S-adenosyl-L-methionine to N-1 of ll-methoxy-2,16-dihydro-16hydroxytabersonine. The enzyme has a high substrate specificity, the reduced 2,16 double bond in the tabersonine skeleton being essential (242). Absence of the 16-hydroxy group resulted in 60% lower N-methylation rate, compared with the natural substrate. Tabersonine is not accepted as substrate. Also, the 14J5-double bond is an essential requirement for acceptance as substrate. The enzyme is localized in the thylakoids of chloroplasts (181).The partially purified enzyme was further characterized (243). It could be solubilized with CHAPS. By sucrose gradient centrifugation an apparent M , of 60,000 was found. The solubilized enzyme showed some differences in substrate specificity compared with the membrane bound enzyme. The hydroxylation at C-17 of ll-methoxy-2,16-dihydro-16-hydroxy-N( 1)methyltabersonine (desacetoxyvindoline) is catalyzed by the enzyme des-

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acetoxyvindoline 17-hydroxylase (D17H, EC 1.14.11.11) (244-246). The enzyme is a dioxygenase requiring 2-oxoglutarate as cofactor; in the absence of molecular oxygen no reaction occurs. Ascorbate enhances the reaction but is not an absolute requirement. The enzyme has high substrate specificity; it has strict specificity for hydroxylation at the 17-position. No reaction will occur in the presence of a 2,16-double bond, and the presence of the N-1-methyl group is also essential for acceptance as substrate. This substrate specificity supports the position of this step in the vindoline pathway (see Fig. 17). By gel filtration an apparent molecular weight of 45 kDa was determined for D17H. It has an optimum activity at pH 7.5. The enzyme is believed to occur in the cytosol. The enzyme purified to near homogeneity (245) shows a molecular mass of 45 kDa for the native protein and 44.7 kDa for the denatured protein, suggesting that the enzyme is monomeric. Isoelectric focusing under denaturing conditions showed three differently charged isoforms (pZ4.6, 4.7, and 4.8). The active enzyme does not require any divalent cation, but inactive enzyme is activated again by Fe2+. The K, values for 2-oxoglutarate, 02,and desacetoxyvindoline are respectively 45.0, 45.0, and 0.03 p M , and for Fe2+and ascorbate, 8.5 and 200 p M , respectively. Succinate (one of the reaction products) is a competitive inhibitor ( K i 9 mM) for 2-oxoglutarate, but noncompetitive for desacetoxyvindoline. Deacetylvindoline was a noncompetitive inhibitor (Ki 115 p M ) for 2-oxoglutarate, 0 2 . and desacetoxyvindoline. C 0 2causes 50% inhibition at a concentration of 7.5 mM. These binding characteristics are in accordance with an ordered ter ter mechanism, a common feature of 2oxoglutarate-dependent dioxygenases. This means first binding of 2oxoglutarate, followed by binding with O2 and desacetoxyvindoline. The first product released is deacetylvindoline, because of its noncompetitive inhibition with the three substrates. Succinate is the last product released after C02. The enzyme catalyzing the last step in the vindoline route, acetylCoA : 17-0-deacetylvindoline 17-0-acetyl-transferase(DAT, EC 2.3.1.107), has a high selectivity for deacetylvindoline; it also catalyzes the reverse reaction. From the high selectivity it is clear that this enzyme is at the end of the pathway leading to vindoline. It is only found in the vindolinecontaining parts of the plant: mainly in the leaves, less in the stems, not in the roots (247). It is formed after exposing dark-grown seedlings to light (248). DAT is a soluble enzyme occurring in the cytosol (182). After the first characterization of the enzymes in crude enzyme preparations (237,247,248),more detailed studies were made of highly purified DAT. It has a pH optimum of 7.5-9. The enzyme has K, values of 6.5 and 1.3 p M for acetylcoenzyme A and deacetylvindoline, respectively. Tabersonine (45% inhibition at 45 p M ) and coenzyme A (50% inhibition at 37 p M ) inhibit the enzyme, as do K+,Mg2+,and Mn2+(10-100 mM). After SDS-

tabersonine

COOCH, P

11-hydroxytabemnine

11-methoxytakrwnine

D17H

CH30

CH,O CH,

desacetylvindoliie

CH30

COOCH,

desacetoxyvindoline

OCOCH, CH,

COOCH,

vindoline

FIG.17. Pathway from tabersonine to vindoline.

COOCH,

ll-methoxy-2,16dehydrcIbhydroxytabersonine

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PAGE, two major proteins are observed, having molecular masses of 33 and 21 kDa. On native PAGE gels three bands are observed, on isoelectric focusing only one (pl 4.7-5.3) (249). It might thus be a heterodimeric enzyme occurring in differently charged isoforms. Fahn and St6ckigt (250) found five discernible forms of the protein, with isoelectric points between 4.3 and 5.4. They also found two isoforms with similar molecular weights, as mentioned earlier. The Ki for coenzyme A was found to be 8 p M . A nonspecific acylesterase was reported to be present in C. roseus that is capable of hydrolysis of the acetyl group in vindoline. However, this enzyme was thought not to play a role in the vindoline pathway (236). 7. Bisindole Alkaloids

The vinblastine-type bisindole alkaloids are formed by the coupling of catharanthine and vindoline (Fig. 18). This coupling and the subsequent reactions were extensively studied using plant enzymes. 15’,20’-Anhydrovinblastineis converted by C. roseus cultured cells into leurosine, Catharine, and vinblastine (251-256). Later it was shown that the anhydro compound is not a real intermediate, but a reduction product of the true precursor, a highly unstable dihydropyridinium intermediate (see Fig. 18), which is formed in the oxidative coupling of vindoline and catharanthine catalyzed by peroxidases (257-268). Based on increased yields of 15’,20’-anhydrovinblastineand further oxidized bisindole alkaloids in this coupling using crude enzyme extracts of C. roseus cell suspension cultures, FMN and FAD were identified as cofactors of the enzymatic conversion. Moreover, the presence of 1 mM Mn2+increased the yield of bisindole alkaloids (266-268). The coupling leading to the anhydro compound also occurs nonenzymatically in the presence of FMN (or FAD) and Mn2+.Fractionation of the crude enzyme extract resulted in four different active fractions with different p l values (7.8, 8.0, 8.3, 9.0), all having an estimated molecular weight of 37 kDa (267,268). Furthermore, an enzyme with a p l of 10.5 and an apparent molecular weight of 15 kDa was isolated. All these enzymes also had peroxidase activity, and H202could replace FMN as cofactor for the coupling reaction, resulting in an even higher yield (267-269). Horseradish peroxidase could also be used for the coupling reaction. 15’,20‘-Anhydrovinblastine can be oxidized by horseradish peroxidase to yield leurosine and Catharine (251,254).The conversion of vinblastine into vincristine by means of cell cultures of C. roseus has been described by Hamada and Nakazawa (270). 8. Peroxidases

The roots of C. roseus contain ajmalicine and its tetradehydro derivative serpentine as major products. Serpentine is also found in cell cultures;

263

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

COOCH, CH;. COOCH,

Y

catharanthine

vindoline

: COOCH,

CH,O

kOOCH,

OCOCH,

vinblastine

OCOCH,

15'-20'-anhydrovinblastine

FIG.18. Formation of bisindole alkaloids.

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particularly, cells grown in the light produce serpentine. The formation of serpentine was found to parallel the activity of basic peroxidases in the cell cultures (29,60,271). Isolation of these basic peroxidases from vacuoles showed that they readily converted ajmalicine in serpentine. Also, in situ incubation of isolated vacuoles with ajmalicine and H202resulted in increased serpentine levels compared with controls. Peroxidases are also involved in the biosynthesis of the bisindole alkaloids (see earlier discussion). Serpentine, being an anhydronium compound, is unable to pass the tonoplast, and is thus trapped in the vacuole (see later discussion). 9. Catabolism

In cell cultures of C.roseus, ajmalicine is a major product. To determine whether this is an end product or just an intermediate, labeled ajmalicine has been fed to cell cultures of C. roseus (37,272). Extensive turnover of ajmalicine was found, but the products could not be identified. Similar results were found with Tabernaemontana cell cultures, using some "Nlabeled alkaloids specificfor these cultures (32-3436-38). Particularly during the stationary phase, the rate of breakdown equals de novo biosynthesis. The biotransformation of tabersonine, the precursor for vindoline and thus for the bisindole alkaloids vinblastine and vincristine, in C.rozeus cells was studied by Furuya etal. (273).No vindoline was formed; instead, the alkaloid was epoxidized to yield lochnericine and further methoxylated at the 11position (lochnerinine). The rate of the transformation depends on the medium; the highest rate was about 70%conversion after 1 week. Daddona et al. (274) reported on the catabolism of vindoline and catharanthine in the plant. Particularly in apical cuttings, the decrease of levels of labeled alkaloid was much faster than in intact plants. In the intact plants, after pulse or steady-state feeding with 14C02,the amount of labeled alkaloid rapidly reaches a maximum and remains at about that level for at least 30 days. In the apical cuttings the levels started to decrease after having reached a maximum at about 1 to 3 days. In the plant, vindoline seemed somewhat more stable than catharanthine.

IV. Genes Encoding Enzymes Involved in Terpenoid Indole Alkaloid Biosynthesis For several of the enzymes described earlier, the encoding genes have been cloned from plant species other than C. roseus. This especially holds true for enzymes involved in primary metabolism. For example, by genetic

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complementation experiments with yeast mutants, a cDNA encoding AACT was cloned from radish (275), and from Arabidopsis thaliana (276,273,HMG-CoA synthase and mevalonate kinase cDNAs were cloned. A similar procedure is difficult to perform for enzymes involved in secondary metabolism because of the absence of suitable yeast mutants. A. HMG-CoA REDUCTASE Because of its central role in terpenoid biosynthesis, HMG-CoA reductase (HMGR) has been extensively studied, and the gene has been cloned from several plants. Plant HMGRs are encoded by small gene families that display complex developmental and environmental regulation. For example, in Hevea three and in Arabidopsis two distinct genes are found. In maize and potato an even larger gene family is found. Such families of genes may be required by the variety of end products derived from mevalonate and by the diversity of conditions under which those products are made. This topic has extensively been reviewed by various authors (7074,76).A C. roseus Hmg cDNA has been cloned (88).In C. roseus cultures treated with methyl jasmonate, an elicitation signal transducer, Hmg mRNA transiently decreased after 6 h exposure, and then increased in abundance over basal levels at least up to 48 h after exposure. It appeared that the recovery, and later the induction, of Hmg-gene expression is the result of transcription from gene family members distinct from those suppressed by methyl jasmonate. The existence of differentially regulated Hmg isogenes in C. roseus is thus substantiated (89),but the exact number of Hmg genes has still to be determined. B. GERANIOL 10-HYDROXYLASE As most of the enzymes of the terpenoid-iridoid pathway are not well characterized, little work has been done on the level of the genes, with the exception of GlOH. The NADPH :cytochrome P-450 reductase part of this enzyme was obtained pure and antibodies were raised that were used for immunoscreening of a C. roseus cDNA expression library (ZZZJl3). A cDNA clone was isolated and identified by its sequence homology with mammalian and yeast reductase genes. The cDNA expressed in E. coli resulted in a functional protein. The protein consists of 714 amino acids and has an M , of 78,958. Comparison with known sequences of yeast and mammalians showed a high degree of homology for the domains involved in the binding of the cofactors FAD, FMN, and NADPH. The reductase is encoded by a single copy gene; all cytochrome P-450 enzymes in C. roseus are thus probably served by the same reductase. Measuring the expression

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of the gene in different parts of the plant showed that mRNA levels were highest in the flowers. Leaves and stems only had low levels, whereas the levels in the roots were in between these levels. In cell cultures the expression of the reductase mRNA is induced by fungal elicitors and downregulated by auxins, a pattern that was also found for the Tdc and Str gene (see later discussion). Efforts to clone the gene for the P-450 part of GlOH have not been successful so far. Vetter et al. (278) performed a differential screening of cDNA libraries for a normal and an alkaloid-production-induced C. roseus cell culture, using size and the presence of highly conserved areas of P-450 for selection of the clones. Two closely related clones (pCros2 and pCros2) were obtained (98% homology at amino acid level). pCros2 encodes a protein with an M, of 60,557 (524 amino acids). Both genes follow an induction pattern that parallels GlOH activity. But overexpressing pCros2 in yeast did not result in any geraniol or nerol hydroxylating activity. At the amino acid level pCros2 had about 25% homology with some mammalian P450 enzymes. Domains associated with the heme-binding sites were highly conserved. Using the pCros2 cDNA, C. roseus cell cultures were screened. Besides the already known Cros2, a third closely related gene (Cros3) was isolated. This gene was further characterized. It contained conserved areas for P-450 enzymes. As it has only very small divergence in sequence with Cros2, they can be considered as allelic variants of one gene. Cros2 was clearly different, having more than 3% divergence and a completely different 3' noncoding region. Expression of this gene in tobacco and Arubidopsis thaliana resulted in the formation of a protein of the size expected for a cytochrome P-450 enzyme, but no hydroxylase activity could be detected in the protein extracts of these plants (279).The role of these three genes in secondary metabolism remains to be clarified. Meijer et al. purified the GlOH P-450 protein (222,222). The partial sequence determined for the N-terminus of this protein does not fit with the sequence of the pCros2 protein. Immunoscreening of a cDNA expression library of C. roseus roots with antibodies raised against the purified GlOH did not result in the isolation of any cDNA clones having any of the highly conserved domains typical for P-450 genes (222,222). Using PCR, with degenerate primers based on the N-terminal sequence, two cDNA clones could be isolated from a C. roseus root cDNA expression library (222,123). These two cDNA clones showed a high degree of homology (97% at the amino acid level). With other P-450 gene families, despite the presence of certain domains with a high degree of homology, including the highly conserved heme-binding domain, overall less than 40% amino acid homology was found. The genes should thus represent a new gene family. With the aid of Agrobacterium tumefuciens, both genes driven by the CaMV 35s promoter, in a vector also containing the gusA gene and nos terminator,

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were introduced in tobacco and C. roseus. In the transgenic tobacco plants no GlOH activity could be detected; in the C. roseus tumors no increase of GlOH activity could be observed if compared with controls. Further analysis of the regulation of these genes showed that they are involved in a root-specific metabolic pathway that is elicitor inducible. Auxins downregulate both P-450 genes; on the reductase gene the effect is less strong. In a further approach to clone the gene encoding the cytochrome P-450 part of the GlOH enzyme complex, PCR was used with a set of primers consisting of a degenerate primer for the highly conserved heme-binding domain and a nonspecific primer, complementary to the poly(A) tail of the cDNA clones or to a phage vector sequence (280).With this strategy 16 different P-450 sequences were isolated from the cDNA library of C. roseus roots. One of these had 58% homology at the amino acid level with a P-450 gene, of yet unknown function, isolated from avocado (282).This protein sequence is encoded by at least two genes in C. roseus. Further studies will be necessary to determine whether any of the isolated cDNA sequences is part of the gene coding for GlOH. C. TRYPTOPHAN DECARBOXYLASE De Luca et ul. (282) reported the cloning and sequencing of the-full length Tdc cDNA, by screening with TDC-antibodies in an expression library. The sequence has significant homology with dopa decarboxylase of Drosophilu melunoguster (39% at the amino acid level) and other mammalian aromatic amino acid decarboxylase genes. The homology was such that a similar secondary structure for these proteins is expected. With other amino acid decarboxylases sequence similarities are also observed, suggesting an evolutionary link. An open reading frame was found coding for a 500 amino acid protein, corresponding to a 56,142-Da molecular weight. A sequence similar to pyridoxal phosphate binding sites was observed. Goddijn ef ul. (289,290,283)demonstrated that TDC is encoded by a single gene, which does not contain introns. The promoter did not show any tissue specifity when expressed in tobacco in a construct containing a gusA reporter gene. In cell cultures, auxins down-regulate the Tdc gene (202,203),whereas elicitation induces the transcription (202,203,284).The effect of auxins could not be reproduced in transgenic tobacco protoplasts containing a chimaeric Tdc-gus construct. For further studies of the promoter region, transgenic C. roseus is required; however, regeneration of transgenic C. roseus is still hard to achieve (289,290). The expression of the Tdc gene as a function of the cell cycle and the growth of a C. rosem cell suspension culture was extensively studied by Nicoloso (285).Cells grown on the standard medium had a low, but significant, level of Tdc mRNA, after the first cell division a maximum was

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observed. During the stationary phase no expression of the Tdc gene could be detected. Lowering the phosphate concentration of the medium resulted in a strong enhancement of the expression. The combination of phosphate limitation and growth-regulator-free medium resulted in a further increase of the Tdc mRNA levels during the first 5 days of culture. Particularly during the lag phase, a considerable increase of the Tdc mRNA level was observed in the first 12 h after inoculation on the hormone-free medium. This is an accordance with the finding that tryptamine accumulation is strongly enhanced during the lag phase. The switch from primary metabolism to secondary seemed to occur after arrest of cell division: phosphate limitation resulting in low cell division activity is accompanied by high Tdc mRNA levels, and adding phosphate to these cell cultures results in suppression of the expression. Arresting cell division with aphidicolin results in enhanced expression.

D. STRICTOSIDINE SYNTHASE The gene coding for SSS was first cloned from R. serpentina (286). McKnight et al. (287) used part of the sequence of this gene for a probe to screen a C.rosezu cDNA library. The Str gene cloned in this way had an unusual signal peptide without any basic amino acid. It contained 13 hydrophobic amino acids followed by 10 serines. Expression of the cDNA coding for SSS from C.roseus, to which a sequence is added encoding the first nine amino acids of the R. serpentina SSS signal peptide, driven by the CaMV 35s promoter in tobacco, results in plants that have an SSS activity 3-22 times higher than found in C. roseus plants (206).Two different forms are observed in the tobacco plants, one of which migrates on SDSPAGE gels as a protein with a molecular weight similar to SSS from C. roseus, and one with a lower molecular weight. By means of immunolocalization it was shown that SSS is located in the vacuole in C. roseus; in the tobacco plant the enzyme was also targeted to the vacuole. Pasquali et al. (202,203) reported the full mRNA sequence. By screening a cDNA expression library with antibodies raised against SSS, the gene was eventually cloned. Several differences were noted with the sequence reported by McKnight et al. (287); in particular, in the latter the 5' end was missing. C. roseus contains a single copy of the Str gene. The molecular mass calculated from the nucleotide sequence is 39,093 Da for a protein of 352 amino acids. In the plant the highest level of expression is found in the roots. In cell cultures auxins repress transcription, whereas elicitation induces transcription; the Tdc gene behaves similarly (202,203,284). The induction by elicitors is independent of de n o w protein biosynthesis (202,203).

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269

The genomic clone of the Str gene has two introns (202).The nucleotide sequence of the open reading frame of the C. roseus gene has 82%homology to the Rauvolfia gene, whereas the promoter and terminator sequences have only 43 and 41% homology, respectively. On the peptide level, 90% homology exists between the SSS enzymes from these two sources. The promoter of the Str gene was coupled with a gusA reporter gene and the regulation was studied in more detail in transgenic tobacco. Elements could be identified that are connected with the induction by elicitors. The tobacco G-box binding factors (GBF), including the recombinant transcriptional activator TAF-1, bind to a G-box element in the promoter region. However, this element seemed not to play a role in the Str promoter activity. Several other distinct binding activities for nuclear proteins were also detected; the factors GT-1, SSF-1, and 3AF1 bind to some upstream fragments of the promoter region, which are connected with the elicitor-inducible expression. However, evidence for the direct involvement of these nuclear proteins and the corresponding cis-acting elements in the elicitor response is not available. The nuclear protein binding activities may also be connected with developmental or environmental responses. In the studies of the cell cycle and cell culture growth in relation to alkaloid production, Nicoloso (285) found that expression of the Str gene paralleled that of the Tdc gene, with increased levels of mRNA 12 h after inoculation on a growth-regulator-free and phosphate-deficient medium compared with a control cell culture on a normal medium. After this maximum, the Str mRNA transiently decreased. In the second part of the growth cycle it then reached a clear maximum, about twofold higher than in the control cells. The expression-enhancing effect of low phosphate in the medium was much less for the Str mRNA accumulation than for Tdc mRNA. The expression of the Str gene is up-regulated after arrest of cell division, as for the Tdc-gene (see earlier discussion). The Str gene cloned from R. serpentina has been expressed in various organisms, among others E. coli (288) and insect cells (289).In both cases, this resulted in high levels of active enzyme. Two forms were found, a highand low-molecular-mass form; both had activity, the K, values being similar to that of the plant enzyme. It can thus be concluded that the glycosylation is not essential for activity. The C. roseus Str gene was also successfully expressed in E. coli (290).The molecular mass was estimated to be about 34 KDa, which is less than the expected 36,074 Da based on the nucleotide sequence. This might be due to proteolytic processing. E. DESACETOXYVINDOLINE 17-HYDROXYLASE A putative partial cDNA clone coding for D17H was isolated from a cDNA library of C. roseus seedlings. It has 33.3% homology at the amino

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acid level with the protein of hyoscyamine 6-hydroxylase from Hyoscyamus niger and 31.2% with flavanone 3P-hydroxylase from petunia and other plant 2-oxoglutarate dependent dioxygenases (246). The hydroxylase is a single copy gene (292).

V. Metabolic Engineering Presently, one of the main applications of knowledge of the regulation of alkaloid biosynthesis is in metabolic engineering to alter or increase alkaloid accumulation in the plant or plant cell cultures. In fact, the first results have already been reported (for a review, see ref. 292). Also, C. roseus genes have been used to alter metabolic pathways. The Tdc gene driven by the CaMV 35s promoter has been introduced into tobacco (288-292,283). High levels of TDC activity were found, with tryptamine levels up to about 1%of the dry weight. Plants showed normal growth and phenotype. Berlin et al. (293-295) transformed Peganum harmala cells with the Tdc gene under the control of the CaMV 35s promoter. This resulted in a strong increase in TDC activity and an up to 10-fold increase in the levels of serotonin, to which tryptamine apparently was converted directly. However, no increase in the level of harman-type alkaloids, also found in the plant, was observed. In canola (Brussica napus) the introduction of the Tdc gene resulted in a considerably lower level of tryptophan-derived glucosinolates (3% of the control) (296). A comparison made between the expression levels of a construct containing the CaMV 35s promoter and a kanamycin resistance gene in tobacco, potato, and canola showed that in transgenic tobacco plants the highest levels of cDNA are found (297). These plants had a 3- to 10-fold higher level of TDC activity and a 12- to 50-fold higher tryptamine content than the transgenic potato and canola plants. The results of metabolic engineering might thus be species dependent. In the transgenic canola an up to 80% decrease of tryptophan-derived glucosinolates was found. In the high TDC-activity transgenic tobacco, no influence on indole acetic acid levels could be observed. Expression in cells of C. roseus itself resulted in increased TDC activity and tryptamine levels. However, alkaloid levels were little affected, pointing to a possible limitation of secologanin. An antisense Tdc gene almost completely inhibited TDC activity, and alkaloid levels were below the detection limit (290,298). Transgenic tobacco cells containing the Tdc and Str genes produce strictosidine upon feeding of secologanin; this alkaloid is not stored in the

BIOSYNTHESIS OF TERPENOID INDOLE ALKALOIDS

27 1

vacuoles, but is excreted to the medium (364). Introduction of the Str gene into C. roseus cells leads to a considerable increase of SSS activity, but not to increased alkaloid production, as expected because the availability of secologanin is a major limiting factor for alkaloid production (298). The introduction of both the C. roseus Tdc and Str genes into hairy roots of Cinchona ledgeriana resulted in cultures having high TDC and SSS activity and containing high levels of tryptamine and strictosidine. The amounts of quinoline alkaloids found was similar (1-2 mg/g DW) as in an earlier reported hairy root culture (299). Efforts to obtain C. roseus hairy roots containing this gene construct were not successful, as the roots obtained died as soon as they were transferred to liquid medium. From Catharanthus pusillus, hairy root cultures were obtained, but no increased levels of TDC or SSS could be observed in the root cultures transformed with the two encoding genes, if compared with a culture that only contained the empty vector (300).

VI. Regulation of Alkaloid Biosynthesis In the previous section we have separately discussed various steps in the alkaloid biosynthesis on the level of the intermediates, enzymes, and genes. In the past years, numerous studies have been published on factors, such as light, media components, and gas-phase composition, which influence alkaloid biosynthesis in cell cultures. In most cases, the mechanism of action is not known. For reviews on these empirical findings, see van der Heijden ef al. and Moreno et al. (7,lZ). In Tables IV and V, the effects of different signals on alkaloid production and enzyme activities are summarized. Regulation of the metabolic pathways can be achieved at different levels: product, enzyme, mRNA, and DNA (genes). Concerning regulation, two major possibilities can be distinguished: control by endogenous, developmentally controlled signals, or by exogenous signals. In Fig. 19, some of the different signals and their effect on the secondary metabolism in C. roseus are summarized. For regulation through endogenous factors, studies on compartmentation and the role of plant growth hormones are relevant aspects. For the latter type of regulation, studies of the plant-insect and plant-microorganism relationships will be of interest, including the signal molecules and the signal-transduction chains.

TABLE IV EFFECT OF DIFFERENT TREATMENTS ON ALKALOID ACCUMULAITON IN Catharonrhus roseus CELL SUSPENSION CULTURES' Growth Regulators

Light

Elicitation Auxins Cytokinins Heavy Metals Visible UV Total alkaloid production Tryptamine Ajmalicine Serpentine Catharanthine

+I0

+ +I0 +

-

+ +/-

+

+

+I-

+ -

+

+ +

-

Osmotic Addition of Stressb Iridoid Precursors Induction Mediumb

+

+

+

+ + +

+ + +

+ +

+ = induction of alkaloid production; - = inhibition of alkaloid production; 0 = no effect on alkaloid production. See ref. Zl. Further references given in text.

-

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TABLE V EFFECTS OF SEVERAL TREATMENTS ON ENZYME ACTIVITIES, mRNA LEVELS IN Catharanthus roseus CELLSUSPENSION CULTURES' Growth Regulators Heavy Salicylic Induction Elicitation Auxins Cytokinins Metals Acid Mediumb Phenylalanine ammonia lyase Isochorismate synthase Geraniol-10-hydroxylase Anthranilate synthase Tryptophan decarboxylase

E+/EE+ EG + (?) E+ E+

E+ E+ G-

GO

G-

GO

EO

G+

G+

Strictosidine synthase Strictosidine-8-glucosidase

E+/EO G+ EO

G+

E+ E+ G+ EO

EO

E+ = induction of enzyme activity: E- = inhibition of enzyme activity; EO = no effect on enzyme activity; G + = induction of gene transcription: G- = inhibition of gene transcription: GO = no effect on gene transcription. See ref. 11. Further references are given in the text.

A. DEVELOPMENTAL REGULATION The biosynthesis of alkaloids is very much under developmental control, as was shown in several studies using developing seedlings (278,283,237). Plant growth hormones play an important role in these processes, but little work has been done on the direct relationship between growth hormones and alkaloid accumulation in intact plants. Most of this work is in fact done with cell cultures (see later discussion). Light was shown to play a major role; seedlings grown in the dark had tabersonine as a major compound, whereas in the light vindoline was the major alkaloid (237-239). The tabersonine pathway is present in all tissues of the seedling, whereas the vindoline pathway is only present in the aerial parts. S S S is not under strict control; it could be detected in all tissues through the whole period of germination and development of the seedling, with the maximum occurring simultaneous with that of TDC. The activity of TDC is strongly regulated during the development of a seedling (278,283).TDC enzyme activity shows a clear transient maximum at day 5 of the germination, followed after 24 h by some of the enzymes (NMT and DAT) of the vindoline pathway. NMT and DAT are only found in the hypocotyls and cotyledons. Similar results for SSS and TDC were reported for Cinchona seedlings (42). No

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FIG.19. Effect of various signals on secondary metabolism in Curhurunrhus roseus cells.

influence of light was observed in C. roseus on the activity of TDC. The cytosolic DAT was induced by light in etiolated seedlings (10-fold increase); NMT (believed to be chloroplastidic) showed only a 30% increase. Only in light-grown seedlings could vindoline be found; dark-grown seedlings accumulated precursors of vindoline (239). With immunoblots of SDSPAGE gels using anti-TDC antibodies, in mature plants the highest TDC protein levels were detected in the youngest leaves. In seedlings, the levels of a 55-kDa protein coincide with the TDC activity in seedlings, but a 54.8-kDa protein was also detected that had no activity. This protein was not found in the leaves. The highest level of TDC was at day 5 of the development of the seedling, and the other protein peaked just before this. Based on this, it was speculated that the 54.8-kDa protein might be an inactive precursor of TDC. Moreover, some minor bands (40, 44, and 67 kDa) were observed that also reacted with the antibodies. Particularly in older leaves and in seedlings after day 5 these bands were detected (178).

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Treatment of the seedlings with auxins enhanced the TDC activity; the light-induction of DAT (see earlier discussion) was delayed 24 h through such a treatment (302). The finding that heterotrophic cell cultures of C. roseus contain vindoline (235) is in accordance with vindoline biosynthesis being found in the green tissues of the plant. In the plant, the enzymes of the vindoline pathway, DAT (236,247) and NMT (242), are only found in the aerial parts, and in particular the leaves, the site of vindoline biosynthesis. In C. roseus cell cultures these enzymes cannot be detected, either after elicitation or in photoautotrophic or hormone autotrophic cultures (242).However, two enzymes of the early steps of the vindoline pathway, T l l H and 11-O-methyltransferase are also present in cell cultures although at lower levels than in the plant (242). In the plant, the highest T l l H levels are found in young leaves, whereas it is absent in stems and old leaves. B. COMPARTMENTATION Compartmentation of biosynthetic pathways may occur on the cellular and subcellular levels. On the cellular level several studies have been made. Using immunoassays to enable the analysis of small quantities of plant material, it was shown that the highest levels of vindoline and catharanthine are found in the young mature leaves. For the bisindoles, the highest levels are observed in the older leaves (57,58,65). In leaf material, vindoline is mainly located in the parenchymatous intercostal areas. The veins contain much lower concentrations. The base of the leaf also has a lower vindoline content than the middle part (57). Histochemical analysis using alkaloidspecific reagents showed the presence of alkaloids in laticifer cells and in specialized parenchyma cells (302). Mersey and Cutler (303) reported the isolation of idioblast protoplasts from C. roseus leaves. By means of HPLC they showed that these cells are enriched in vindoline and catharanthine, compared with other mesophyll cells. Serpentine and strictosidine lactam did not follow this distribution pattern; the latter alkaloid is even the major component in another cell fraction after centrifugation. Immunolocalization of vindoline confirmed that the mesophyll is the most important site of vindoline accumulation (304). Eilert et al. (305) compared alkaloid- and non-alkaloid-producing cultured cells of C. roseus with leaf cells. They concluded that the producing cells were similar to parenchyma cells. C. roseus cell cultures show both temporal and spatial heterogeneity for the accumulation of anthocyanins; only after cell division has ceased are anthocyanins produced in about 10% of all cells (306).Differences in productivity of these compounds in the cell culture are not determined by the productivity of the individual cell, but by the percentage of producing cells,

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which, however, never exceeds 20% of the total number of cells (307). Similarly detailed studies have not yet been made for alkaloid production. But the microscopic appearance of alkaloid-producing cell cultures clearly shows heterogeneity (F. van Iren, unpublished results). Stafford et al. (308) compared C. roseus cell cultures grown on different alkaloid production media and reported that none of these cultures contained more than 50% alkaloid-accumulating cells. Also at a subcellular level, compartmentation plays an important role, as was described earlier for the terpenoid and tryptophan pathways. Still, some questions remain concerning this aspect of the biosynthesis. In Fig. 20 an effort is made to summarize our present knowledge concerning the localization of the biosynthetic pathway of indole alkaloids. All these reactions do not necessarily occur in the same cell, e.g., ajmalicine and serpentine are typical products of the roots, whereas vindoline and the bisindole alkaloids are typical for the leaves. Using immunofluorescence labeling, Brisson et al. (304) showed that in protoplasts of mesophyll cells, vindoline is located in the vacuoles. With

4

*

ajmalicine proxaase serpentine

f ...*’

c

1.:’

:

w ajmalicine

-

catharanthined b . peroxaase vindoline

alkaloids

a l ,,d

c

f

9

1 w catharanthine + vindoline

-

DAT -

deacetylvindoline

Fro. 20. Compartmentation of secondary metabolism in Carharanthus roseus.

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immunogold labeling, a higher resolution could be obtained. With that technique, vesicles could be observed in chloroplasts and even more in the cytosol, which reacted with the antibodies. These vesicles might be involved in the transport from chloroplast to vacuole, which are, respectively, the site of the last step in vindoline biosynthesis and the storage site of vindoline. For the tryptophan pathway it is a matter of debate whether there is only one pathway that is localized in the plastids, or whether a plastidial and cytosolic pathway occurs in the plant. In the latter hypothesis, the cytosolic pathway is believed to be responsible for the secondary metabolism, whereas the plastidial pathway takes care of the tryptophan production for primary metabolism (for a review, see ref. 243). So far no evidence for such a dual pathway has been found in C. roseus (156; R. Bongaerts et al., unpublished results). Similarly the localization of the biosynthesis of terpenoids is a matter of debate (70-72,74,76).The most likely site for the formation of geraniol is within the plastids, but where the next steps of the pathway leading to secologanin are located is for the most part uncertain. Geraniol 10hydroxylase is localized in provacuolar membranes, and not in the endoplasmatic reticulum as are many other cytochrome P-450enzymes (209). The conversion of tryptophan into tryptamine occurs in the cytosol (see earlier discussion); tryptamine is accumulated in the vacuole, probably by the same ion-trap mechanism as ajmalicine. Because of its stronger basic character it is probably even better retained in the vacuole than ajmalicine. Schripsema et al. (309,310)found different incorporation rates for "N from labeled nitrate and ammonium from the medium into indole alkaloids and tryptamine, in Tubernuernontunacell cultures. This points to the possibility of a difference in cellular compartmentation of parts of the pathway, that is, only certain cells are producing and accumulating tryptamine. In this connection it is interesting to mention the finding of Naudascher et af. (233) that secologanin fed to 10-day-old C. roseus cells was only partly metabolized and remained present in the cells at a constant level of about 50% of the added amount. Apparently, cells also have a storage site for exogenously added secologanin, which is not further used for alkaloid biosynthesis, whereas loganin fed to the cells is metabolized and no secologanin accumulation is observed (134).Cells fed with secologanin and subsequently inoculated on a secologanin-free medium showed an increase of the secologanin level, that is, secologanin is stored in the cells in a reversible form, which is not recognized as such by the analytical methods used. This undetected form of secologanin also occurs in the medium. Strictosidine is formed in the vacuole (35,38,47,52,182,206).The transport of strictosidine has not yet been studied, but the strictosidine-converting enzyme is located on the outside of the tonoplasts and/or in the cytosol (38,52,182), requiring transport of the substrate through the tonoplast.

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Ajmalicine formed as the major end product of this pathway in the cultured cells diffuses through membranes as the neutral base and is accumulated in the vacuole through an ion-trap system (see later discussion) (Fig. 21). In the vacuole, ajmalicine is further converted into serpentine, which remains trapped in the vacuole as it cannot pass the tonoplast (29,60,272). In the vindoline pathway (Fig. 17), the enzyme S-adenosyl-L-methionine : 1l-methoxy-2,16-dihydro-16-hydro~abersonine-N-methyltransferase (NMT) is localized in chloroplasts and is associated with thylakoids. AcetylCoA :17-0-deacetylvindoline 17-0-acetyltransferase (DAT) and desacetoxyvindoline 17-hydroxylase(D17H) are localized in the cytosol(I82). The enzymes catalyzing the two first steps from tabersonine to vindoline, T11H and 11-0-methyl transferase, are also present in the cytosol, the former being localized in the ER, the latter being soluble (242). The fact that the various steps of the biosynthetic pathway of the terpenoid indole alkaloids occur in different cell compartments implies that transport of intermediates and products is involved. Little research has been done on this transport phenomenon, except for the transport to the final storage site of the alkaloids. Deus-Neumann and Zenk (205) postulated an active and selective transport system for Curharunthus alkaloids into the tryptophan

+I. tryptarnine

+

tryptamine

ATP ADP + Pi

H+

PEROXIDASE

T;sloganin VACUOLE ajmalicine + H+ ajmalicine [ H']

strictosidine

t general precursor -

-4

ajrnalicine + H+

many alkaloids

FIG.21. Role vacuole in ajmalicine biosynthesis and storage.

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vacuole. This is, among other reasons, based on the finding that the transport of vindoline and ajmalicine through the tonoplast is ATPase dependent, i.e., requires energy. Moreover, some other types of alkaloids such as morphine, codeine, scopolamine, and nicotine were not taken up by the vacuoles. On the other hand, an ion-trap model for alkaloid uptake was proposed by Renaudin and Guern (312).This hypothesis was further elaborated by Blom et al. (60,271). A mathematical model was presented that fitted the experimental data obtained with the uptake of quinine and cinchonamine in C. roseus and Cinchona ledgeriana cultured cells. The model is based on the free diffusion of the neutral forms of the alkaloids through biomembranes. The model predicts that alkaloids with a high pKa value are more rapidly transported, from a compartment with high pH (e.g., the cytosol) to a compartment with low pH (e.g., the vacuole), than alkaloids with a low pKa. Also, the uptake of ajmalicine in isolated vacuoles was shown to be dependent on the pH gradient (60,271): the larger the gradient, the higher the ajmalicine accumulation. ATP is needed for activation of the proton pumps, resulting in a low pH in the vacuoles (Fig. 21). The pH control of the vacuole thus plays a major regulatory role in alkaloid accumulation. Biphasic alkaloid accumulation kinetics, such as those reported for ajmalicine and vindoline in C. roseus protoplasts (312),might point to the occurrence of both a selective uptake mechanism and an ion-trap mechanism. Because of its highly polar and strongly basic anhydronium system, serpentine cannot pass the tonoplast and will remain trapped in the vacuole. In a cell culture, ajmalicine will move to the compartment with the lowest pH, so an artificial sink outside the cells will result in export from the cells.

C. LIGHT Light is known to influence alkaloid levels in both plants and cell cultures. Under the influence of light, serpentine levels increase in cell cultures, whereas ajmalicine levels decrease (60,62).Hirata et al. (55,56) reported that in C. roseus shoot cultures and plants exposed to near-UV light (370 nm) the production of vinblastine and leurosine is increased, whereas the levels of vindoline and catharanthine, the direct precursors for the bisindoles, decreased. Irradiation with UV light at 254 nm was reported to result in a twofold increase of vindoline levels in the leaves: catharanthine levels decreased to half of the controls. No mention was made of the accumulation of bisindoles (313).The effects of UV light might be due to a stress response of the plant, that is, an antioxidant function of the alkaloids already present. The fact that UV-light irradiation of C. roseus leaves results in increased levels of Tdc and Str mRNAs would fit such a hypothesis

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(365). C. roseus cells grown under UV light (360 nm) did not show any major difference for growth or alkaloid production; only the activity of GlOH was reduced. However, when 2,3-dihydroxybenzoic acid (DHBA, 1 mM) was added to the cultures in combination with growth under UV light, an induction was observed of the activities of AS, TDC, and SSS, whereas phenylalanine ammonia lyase (PAL, EC 4.3.1.5) activity was reduced considerably. Initially, the tryptamine level increases to a maximum at 24 h, but then starts to decrease to slightly below the level of the control. Strictosidine was completely depleted 24 h after the start of the treatment. Treatment with DHBA alone resulted in a small increase of the tryptamine level, and a slight reduction of the strictosidine content of the cells (257). White light also affects the alkaloid biosynthesis. In etiolated seedlings, precursors of vindoline are accumulated, but vindoline is only formed in the light (283,237,239).De Luca and co-workers (183,237,324)found that the last enzyme in the vindoline biosynthesis, the 0-acetyltransferase (DAT), is induced by light in etiolated seedlings of C. roseus. A study, using different treatments with white and red light (activator of phytochrome) and far red light (inactivation), indicated that phytochrome is involved in mediating the induction signal of the light (324).Furthermore, T l l H is also induced by light in seedlings (242).The hydroxylase (D17H) responsible for the introduction of the 17-hydroxy group in desacetoxyvindoline is induced by light in etiolated seedlings, an effect mediated through phytochrome (244). Tyler et al. (325) described a photoautotrophic cell suspension culture of C. roseus. These cultured cells contained chloroplasts, though morphologically somewhat different from those found in mesophyll cells. These cells did not produce vindoline or bisindole alkaloids; only trace amounts of some Aspidosperma-type alkaloids (e.g., vindolinine) could be detected. However, in hkterotrophic cell lines of C. roseus, vindoline has been found (235). Loyola-Vargas et al. (326) developed C. roseus cell lines in which chloroplast biogenesis could be induced: an A. tumefaciens transformed cell line in which induction was possible by growth on benzyladeninecontaining medium, and a nontransformed cell line, in which light induced the formation of chloroplasts. In both cell lines the level of serpentine paralleled the increase in chlorophyll content.

D. PLANTGROWTH REGULATORS The influence of different growth hormones on C.roseus plant cell cultures has been extensively studied (for reviews, see refs. 7,8,11,327). In general, the conclusion is that auxins inhibit the alkaloid production; 2,4-

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D is supposed to be a particularly detrimental factor for alkaloid production. One should be cautious in drawing this conclusion, as most experiments are based on a single step change to a new medium, whereas the real effect of a changed medium might only be observed after a series of subcultures, as was shown by Sierra et af. (328)for the alkaloid production in Tabernaernontana cell cultures. However, an inhibitory effect of auxins on several steps in the biosynthetic pathway of alkaloids is now quite well established on all levels, including the genes (137,189,290,202,203,283).Subculturing cells on an auxin-free medium results in increased Tdc and StrmRNA levels; subsequent addition of auxin results in a rapid decrease of particularly the Tdc mRNA level. Based on feeding experiments, the methylation of loganic acid has been proposed as a possible further target for auxin regulation (237). The stimulating effect of auxins on cell growth and an inhibitory effect on differentiation would thus inhibit alkaloid production. The effect of the auxins also fits with the fact that the alkaloids are particularly formed during the second part of the growth curve, paralleling the differentiation. In C. roseus seedlings, TDC activity was enhanced by auxin treatment (302). Also, in a habituated C. roseus cell line and a cell line normally grown on an auxin-containing medium, it was found that low levels of auxins (0.01-0.1 ppm 2,4-D, IAA, or NAA) result in increased ajmalicine accumulation, whereas higher concentrations resulted in decreased alkaloid levels (329).This shows that extrapolation of results found in cultured cells to an intact plant must be done with great caution. De Gunst (320,322) and Val (322) described mathematical models that fit the cell growth and alkaloid production. These models describe a situation with all cells dividing just after subculturing, and gradually increasing numbers of differentiating cells. This is reflected in the production of secondary metabolites. The models were validated using, among others, C. roseus cell cultures. The studies by Nicoloso et af. (285)showed that alkaloid production only occurred during the linear and stationary growth phases. During the linear phase, yet-unidentified alkaloids with an indole chromophore, not including ajmalicine, were the products. During the stationary phase, ajmalicine and tabersonine were accumulated. Tryptamine was formed as a major product during the lag phase. The expression of both the Tdc and Str genes is clearly enhanced after the arrest of cell division. On phosphate-deficient media and growth-regulator-free media the expression is up-regulated (see earlier discussion). Addition of phosphate to these cells down-regulates these genes. From experiments using oryzalin (arrest in G2 phase) and aphidicolin (arrest in G1 phase) to stop cell division, there is some evidence that cells arrested in the G1 rather than the G2 phase lead to higher levels of Tdc and Str transcripts. Omission of kinetin from the medium of cultured cells has no effect on

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the Tdc and Str mRNA levels (189,202,203).Benzyladenine caused an increase of alkaloid production in 2,4-D depleted cells of C. roseus (see later discussion) (323). Ouelhazi et al. (324) compared the patterns of proteins and poly(A)+ RNAs of cells cultured on hormone-free medium with cells to which, after 3 days on such a medium, 2,4-D, zeatin, or a combination of the two was added. Clear differences could be observed, and some proteins were indicated as having possibly a direct or indirect regulatory role in alkaloid biosynthesis. Abscisic acid (ABA) stimulated the accumulation of catharanthine and vindoline (325). ABA also stimulated Tdc and Str mRNA accumulation, whereas gibberillin and ethylene had no effect (J. Memelink et al., unpublished results). E. ELICITORS

Elicitors are probably the best-studied exogenous signals in plants. They are connected with microbial infections and induce plant defense responses. Among others, the production of certain secondary metabolites (phytoalexins) is induced by biotic or abiotic elicitors (e.g., see reviews in refs. 326328). For C. roseus suspension-cultured cells, elicitation with fungal elicitors results in the induction of TDC activity (99,286,202,203,284,329-332).This is due to the induction of expression of the Tdc gene. Similarly, S S S activity is induced (202,203,284,329,330).The induction by the Pythium aphaniderrnatum or yeast elicitor of the transcription of both genes is not affected by cycloheximide; that is, the induction is independent of de novo protein biosynthesis, and thus follows an already available signal-transduction chain. The response is quite fast, for the enhanced transcription can already be measured 15 min after elicitation (202,203). Also, the NADPH :cytochrome P-450 reductase mRNA level is induced by elicitation with fungal elicitors (223). Moreno et al. (99,152) measured activities of a number of enzymes involved in secondary metabolism in C. roseus before and after elicitation with a P. aphanidermatum preparation. GlOH activity was found to be slightly decreased by elicitation and IPP-isomerase showed similar behavior. The pattern of terpenoids formed by the crude enzyme extracts from elicited and nonelicited cells was different. The total incorporation decreased, that is, the activities of the enzymes of the terpenoid pathway were lower. The relative incorporation decreased particularly for squalene. Chorismate mutase was not induced by elicitation, but PAL activity was strongly reduced at higher concentrations of the elicitor; at lower concentrations an induction was observed. This is contradictory to the results reported by Seitz et al. (331):a very sharp and brief peak of PAL

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activity followed by an increase of the level of phenolics after elicitation. These authors used an assay without applying phenylalanine ammonia transferase (PAT) inhibitors; thus, it can not be excluded that they measured induction of PAT rather than PAL (332). Of the chorisrnate-utilizing enzymes, isochorismate synthase and anthranilate synthase (AS) were induced (99,252).The effect for AS was inhibited by both cycloheximide (inhibition of translation) and actinomycin D (inhibition of transcription); the same results were observed for TDC activity. SSS and SG activities were not significantly affected by the elicitation. The results for alkaloid production after elicitation with a biotic elicitor (e.g., Pythium or Phytophthora preparations) differed in the various studies published. Tryptamine levels were, if determined, found to be increased. The production of ajmalicine, serpentine, and catharanthine was also reported to be increased (329,330,333-337). Nef et al. (334) found that catharanthine and ajmalicine were excreted into the medium, whereas serpentine was mainly found in the cells. The feeding of loganin or secologanin to cells of C. roseus resulted in an increased alkaloid production (ajmalicine and strictosidine) and a decrease of the tryptamine level (236). After elicitation, these cells showed an increase of tryptamine, but the feeding of the iridoid precursors did not result in any further increase of alkaloid accumulation; rather, a decrease was observed. Strictosidine was rapidly depleted from the cells after elicitation. Vazquez-Flota et al. (336) studied the effect of chitinase, macerozyme, and cellulase on hairy root cultures of C. roseus. No major change in catharanthine levels was observed; more ajmalicine was excreted to the medium compared with the control, and the total alkaloid levels were similar. With fungal elicitors no significant changes could be observed in the alkaloid production or excretion of the hairy roots. Only Aspergillus preparations caused some increase of the ajmalicine production. However, methyl jasmonate did effect the production (see later discussion). On the other hand, it was reported that elicitation with Penicillium species enhanced both production and secretion of alkaloids in hairy root cultures of C. roseus (338). Particularly in combination with in situ adsorption on the Amberlite resin XAD-7, a considerable increase of production and release of ajmalicine and catharanthine by the hairy roots was observed, compared with controls. The vindoline pathway is not affected by elicitation (242,244). Besides the induction of some of the steps of indole alkaloid biosynthesis, it was found that the enzyme isochorisrnate synthase was also induced by fungal elicitors (e.g., P. aphanidermatum), at the same time 2,3dihydroxybenzoic acid (DHBA) was produced (251,263,339).5-Chloro-salicylic acid and 2,6-dichloro-isonicotinic acid enhanced the elicitor effect in

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a synergistic way. After a year of subculturing, the cells lost the ability to respond with an increased alkaloid biosynthesis, but the induction of DHBA production was not affected (339). The abiotic elicitor vanadyl sulfate increases alkaloid production (ajmalicine, catharanthine) in C. roseus cell cultures (340,341).The effect is dependent on the age of the cells, the strongest effect being found during the stationary phase. According to Kargi and Potts (342),the treatment results in more excretion of the alkaloids to the medium. For some plant-elicitor systems, the structure of the molecules responsible for the induction have been determined and the receptors identified (327,328). But in the case of C. roseus, so far no elicitor molecules have been identified. Some synthetic oligosaccharides known as elicitors for some other plant species were not active in the C. roseus system (P. R. H. Moreno et al., unpublished results). When a P. megasperma elicitor preparation treated with trypsin was employed, the induction of DHBA and ajmalicine accumulation was not observed in C. roseus cells. This points to the presence of a glycoprotein as an active component (339). Considering the rather diverse reactions occurring after elicitor treatment, it cannot be excluded that several types of molecules are present in the crude elecitor preparations, which each have different effects. The formation of callose as a response to wounding in C. roseus has been extensively studied (356,357),but unfortunately these data cannot be correlated with most of the studies on alkaloid production. However, there is evidence for a role of Ca2+ as second messenger in this process, as well as in the regulation of alkaloid biosynthesis through cytokinins (see later discussion). Lithium, known to reduce the myo-inositol levels, blocks the induction of expression of the Tdc and Str genes by a yeast extract, whereas myoinositol counteracts the effect. The phosphate uptake was not affected by the addition of lithium. This points to the possible involvement of the phosphatidylinositol cycle in the signal-transduction pathway of the induction by elicitation (285). Also, in other cell cultures, evidence has been found for the involvement of the phosphatidylinositol cycle in elicitorinduced phytoalexin production (342,343). For R. canescens cell cultures it was reported that elicitation results in a transient increase of jasmonic acid. The addition of methyl jasmonate to these cultures resulted in an almost 30-fold increase of the accumulation of the indole alkaloid raucaffricine (344).

F. JASMONIC ACID Jasmonate is a well-established endogenous signal compound in the signal-transduction chain in plant development and in the response to

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elicitors (for reviews, see 345-347). During the development of C. roseus seedlings, treatment with methyl jasmonate results in a doubling of the alkaloid accumulation (348).The effect is only found within a small time interval during the development. SSS activity was strongly induced, TDC activity was not affected. The last two enzymes of vindoline biosynthesis, the hydroxylase (D17H) and 0-acetyltransferase activities (DAT), were also strongly enhanced, with a concomitant increase in vindoline levels. The activities of these enzymes are not affected by elicitation (242,244). The catharanthine pathway was less affected by the jasmonate treatment. Treatment of cultured cells of C. roseus with jasmonate resulted in a transient decrease in Hmg mRNA levels of HMGR, after 6 h followed by an increase in Hmg mRNA levels. It was speculated that the biphasic reaction is due to different Hmg genes, which are differently regulated (89). Methyl jasmonate caused both an enhanced accumulation of ajmalicine and catharanthine in the hairy roots (maximum effect at 10 pM methyl jasmonate), as well as an increased excretion of both alkaloids (maximum effect at 100pM methyl jasmonate); the total alkaloid production increased about twofold (336). G. SALICYLIC ACID Salicylicacid was found to play a major role in inducing a plant’s response to microbial infection (for reviews, see 349-351). In the case of C. roseus, a weak inducing effect on Str and Tdc steady-state mRNA levels was observed 8-24 h after addition of 0.1 mM salicylic acid (202,203).Addition to C. roseus cell cultures of DHBA (1 mM), having one more hydroxy group than salicylic acid, induces AS and SSS enzyme activities. TDC activity is only slightly enhanced. In combination with UV light, DHBA causes strong induction of AS, SSS, TDC, and PAL activities, whereas GlOH is inhibited. This results in a large increase of the tryptamine level. The effect might be due to the formation of toxic compounds (radicals?) from DHBA, and not to a signal function of this compound (157). H. CALCIUM

2,4-D depleted Agrobacterium tumefaciens transformed cells and nontransformed cells of C. roseus reacted with an increase of alkaloid production upon inoculation on a benzyladenine-containingmedium. Chlorpromazine, an anticalmodulin agent, and nifedipine, a calcium channel blocker, inhibited the benzyladenine-induced alkaloid production. The cytokinin effect might involve Ca2+ (323). Ca2+ is an important factor as second messenger in signal-transduction chains (352).Calmodulin, involved in the Ca2+second messenger system, was isolated from C. roseus cell cultures

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(353).The authors reported that calmodulin antagonists and Ca2+channel blockers added in the early stationary phase of the C. roseus cultured cells resulted in increased alkaloid levels, Merillon et af. (354) gave further proof of the involvement of the Ca2+-calmodulin system in the alkaloidaccumulation-enhancing effect of cytokinins. Using this system, they found that in the presence of EGTA (Ca2+chelator) the cytokinin effect was less pronounced. A series of calcium entry blockers, including verapamil, nifedipine, Co2', and La3+,all inhibited the cytokinin effect. In all cases a lowered Ca2+ uptake in isolated protoplasts was observed, except for nifedipine, which showed an unexpected increase in the Ca2+uptake. Various calmodulin antagonists, such as chlorpromazine, haloperidol, fluphenazine, tetracaine, and AlC13, representing different mechanisms of action, also counteracted the cytokinin effect, with little effect on growth. As the 1,4-dihydropyridines behaved differently from the other Ca2+ entry blockers, their mode of action was further studied (355).Ca2+uptake was not affected in intact cells. When in nifedipine the dihydropyridine ring is converted into a pyridine ring, Ca2+-modulatingactivity is still found. The dihydropyridines do affect the alkaloid biosynthesis, through an unidentified mode of action, and in any case not by blocking Ca2+ uptake. The cells converted nifedipine through aromatization of the dihydropyridine ring and reduction of the nitro group to an amino group. The increased Ca2+ uptake is also correlated with the induction of callose synthesis (356,357) after treatment with elicitors. The protein phosphatase inhibitor okadaic acid inhibits this reaction, whereas syringomycin, digitonin, and amphotericin B induce callose formation in C. roseus cell cultures. The Ca2+concentration in the medium affects alkaloid production; addition of high levels of Ca2+,as well as Mg2+and Sr2+,to cells grown on low mineral salt concentrations led to higher alkaloid production. In combination with zeatin a synergistic effect was observed when Ca2+was added in the early growth phase (358).GlOH activity was enhanced by adding zeatin to the cells grown for 3 days on a 2,4-D free medium (359). Treatment with zeatin leds to an increase in the ratio ajmalicine :serpentine. Although some increase of alkaloid production was observed with zeatin, the terpenoid pathway remained a limiting factor. Feeding secologanin led to a considerable increase of the alkaloid levels. The zeatin treatment also affected membranes: The ratio of free sterols to phospholipids was lowered, as well as the ratio of 18: 1to 18:2 fatty acids (360).The authors speculated that the lower sterol production could result in more precursors being available for the pathway leading to geraniol, and thus eventually to higher alkaloid production. Considering the localization of the sterol and monoterpenoid biosynthesis in different compartments (see earlier discussion) this needs thorough study, as competition will only happen in the case of a

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single site of MVA production. Besides the enhanced alkaloid production upon subculturing on 2,4-D free medium, an increase of the activity of NADPH-producing enzymes is observed: cytokinins do not further enhance these activities (361).

VII. Conclusions The biosynthesis of the terpenoid indole alkaloids in C. roseus has been studied extensively, but still the pathway has not yet been completely elucidated on the level of the intermediates. Particularly, the secoiridoid pathway, and the different pathways after strictosidine leading to, for example, tabersonine and catharanthine are not yet completely known. On the level of the enzymes, certain steps have now been quite well characterized, but others remain unknown. The conversion of loganin into secologanin is one of the intriguing unresolved problems, although it is not a rate-limiting step. Even possible intermediates and the chemical mechanism behind this conversion are not clear, despite quite extensive studies. Considering the activities of the enzymes TDC, SSS, and SG in cultured C. roseus cells, it is clear that the actual production of alkaloids is considerably lower than that which could have been produced by these enzymes. In other words, there seems to be an overcapacity in the activity of these enzymes. Thus, there is a limiting step in the pathway before these enzymes, or the carbon flux from primary metabolism into the pathway is limited, for example, by competition with other pathways or by transport and compartmentation. A clear limiting factor is the iridoid pathway, in which geraniol-10-hydroxylase is an obvious point of regulation. Catabolism is a further factor which in cell cultures does limit the alkaloid accumulation. For two enzymes in terpenoid indole alkaloid biosynthesis pathway, the encoding genes have been cloned (Tdc and Str genes). The introduction of the Tdc gene into C. roseus, resulting in an increased level of tryptamine, shows that metabolic engineering is feasible. At the same time, these first results showed that, as could be expected, increasing the activity of a single step in the pathway will not necessarily lead to an increase of total alkaloids. But such experiments will be useful to find which steps are limiting factors. Studies on the levels of the Tdc and Str genes showed that they have a similar regulation. Thus, certain parts of the alkaloid biosynthesis pathway might have controlling factors in common. For improving yields by means of metabolic engineering, this opens the exciting perspective of manipulating the gene(s) controlling (parts of) the pathway, provided one will be

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able to identify such genes. However, one has to be careful not to be too optimistic, as rate-limiting steps might also be others than the enzymes directly involved in the alkaloid biosynthetic pathway, e.g., limitations from physical origins such as transport. Because various parts of terpenoid indole alkaloid biosynthesis occur in different cell compartments, further studies on the role of transport and compartmentation in regulation are highly desirable. In this review we have only dealt with alkaloid biosynthesis in C. roseus; the biochemistry of this plant has also been studied in detail for other aspects, such as anthocyanin production, phosphate metabolism, cell growth, and cell division cycle (e.g., ref. 362). Unfortunately, most of the studies concerning the primary metabolism are not linked with those of secondary metabolism. However, one may expect that in the future the studies on secondary metabolism, such as chorismate-derived products (anthocyanins, benzoic acid derivatives, and alkaloids) and terpenoid-derived products such as the alkaloids, will be integrated. This will eventually allow us a much better insight into the overall biochemistry of the plant. All of the available information makes C. roseus an outstanding model system for the study of the regulation of plant metabolism. Studying the regulation of metabolism in a plant is like putting together a four-dimensional jigsaw puzzle, the dimensions being space and time. In doing so, one tries to And a backbone or frame from which one can start to fit together the bits and pieces. In the case of C.roseus, we now have a frame from which we can work further. But, as with a puzzle, though we do have some separated parts ready, they are still far apart, and we cannot see the total picture yet.

References 1. W. A. Creasey, in “Indoles, Part 4 The Monoterpenoid Indole Alkaloids” (J. E. Saxton, ed.), p. 783. Wiley-Interscience,Chichester, 1983. 2. W. A. Creasey, in “Indoles, Part 4, Supplement:The Monoterpenoid Indole Alkaloids” (J. E. Saxton, ed.), p. 715. Wiley-Interscience, Chichester, 1994. 3. H. L. Pearce, in “The Alkaloids” (A. Brossi and M. Suffness, eds.), Vol. 37, p. 145. Academic Press, San Diego, 1990. 4. J. J. McCormack, in “The Alkaloids” (A. Brossi and M. Suffness, eds.), Vol. 37, p. 205. Academic Press, San Diego, 1990. 5. N. Neuss and M. N. Neuss, in “The Alkaloids” (A. Brossi and M. Suffness, eds.), Vol. 37, p. 229. Academic Press, San Diego, 1990. 6. 0. van Tellingen, J. H. M. Sips, J. H. Beijnen, A. Bult, and W. J. Nooijen, Anticunc. Rex 12,1699 (1992).

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-CHAPTER A

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS HIDEJI ITOKAWA, KOICHITAKEYA, YUKIO HITOTSUYANAGI, AND HIROSHI MORITA Department of Pharmacognosy Tokyo University of Pharmacy and Life Science Tokyo, Japan

................................

I. Introduction ...............................

301

11. Peptide Alkaloids from Higher Plant

A. Cyclopeptide Alkaloids from Zizyphus Plants .... B. Cyclopeptide Alkaloids from Other Species ....................................... C. Spermine and Spermidine Alkaloids ................................................. D. Long-Chain Acid Amides .............................................................. E. Miscellaneous ......................................................................... 111. Cyclic Oligopeptides from Higher Plants ...................................... A. RA Alkaloids from Rubia ...................................... B. Astins from Aster tataricus ... .............................................. C. Pseudostellarins from Pseudostellaria heterophylla ............................... D. Yunnanins from Stellaria y ...................................... E. Segetalins from Vaccaria segetalis ..................................................... F. Cyclic Peptides from Stellaria dichotoma L. var. lanceolata Bge. and Stellaria delavayi ........................................................................... G . Cyclic Peptides from Leonurus heterophyllus ................... H. Other Isolations of Cyclic Peptides from Plants . .......................... References .......................................................................................

311 314 317 324 324 355 359 362 364 370 371 378

1. Introduction

To date, many macrocyclic peptides alkaloid have been isolated from plants. Alkaloids are sometimes described as a group of compounds usually containing tertiary or quaternary nitrogen atoms in their molecules and showing alkali and biological activities. Peptides have amide linkages in their molecules, so peptide alkaloids usually have the characteristics of both an alkaloid and a peptide. This chapter review8 macrocyclic peptide alkaloids in dealing with cyclic oligopeptides. Cyclic peptide alkaloids were defined by Schmit and Haslinger as being THE ALKALOIDS, VOL. 49 CUB-9598197 $25.00

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basic compounds embodying an ansa structure, in which a 10- or 12membered peptide-type bridge spans the 1,3- or 1,4-positionsof a benzene ring. Compounds that fit this definition have been isolated from members of the families Rhamnaceae, Sterculiaceae, Pandaceae, and so on. Knowing that cyclic oligopeptides may possess antineoplastic activity, we isolated a number of related compounds from Rubia spp., which showed strong antineoplastic activity against Sarcoma 180A. These compounds usually have two rings in their molecules: One is an 18-membered peptide ring and the other is a 14-membered ring geminal to an 18-membered ring having diary1 ether linkage. These oligopeptides are found in Rubia spp. and Bouvardia spp. of the Rubiaceae. In addition, monocyclic oligopeptides, which comprise a five- to twelveamino acid moiety, have been isolated from many plants, including the families Compositae, Caryophyllaceae, and Linaceae. In this chapter, cyclic peptide alkaloids are discussed in Section I1 and cyclic oligopeptides from higher plants are discussed in Section 111. In some cases, biological activities have been evaluated, especially antitumor activity. Development of novel, clinically useful, anticancer agents is dependent on the screening systems and the sample sources for bioassay. The search for potential anticancer agents from natural sources has mainly been carried out with the guidance of bioassays established by the United States’ National Cancer Institute (NCI). The large number of natural products screened during the NCI program have also been discussed from an overview of the relationship of assessment between experimental analysis and clinical patients for drug development; also, the screening protocols for each tumor system have been well established. It is considered that these are “compound oriented” in vivo screenings that could not lead to the development of new drugs for solid cancers. Recently, NCI has established a “disease-oriented” approach to antitumor activity screening, and a biological response modifiers (BRM) program from the viewpoint of the diversity and specificity of tumors, and the requirements of novel structure types and novel mechanisms of action for anticancer agents. These screening systems led to the isolation of many antineoplastic compounds from plants, microorganisms, marine metabolites, etc. In our laboratory, we have screened higher plants collected in Japan, China, Korea, Southeast Asia, and South America for antineoplastic activity, using as primary screening Sarcoma 180A in mice, P388 lymphocytic leukemia in mice, Chinese hamster lung cells, P388 cells, and human nasopharynx carcinoma (KB) cells (2-4). We reviewed many of the compounds having antineoplastic activity isolated from higher plants in Heterocycles in 1993 (5). This chapter also describes the antitumor and cytotoxic cyclic oligopeptides of the higher plants selected from the screening tests just named.

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11. Peptide Alkaloids from Higher Plants

Peptide alkaloids have mainly been isolated from the families Rhamnaceae, Sterculiaceae, Pandaceae, Rubiaceae, Urticaceae, Hymenocardiaceae, and Celastraceae (6,7). They have been defined as cyclopeptide alkaloids embodying an ansa structure, in which a 13-, 14-, or 15-membered ring is formed either between the 1- and 3-positions or between the 1- and 4-positions of a benzene ring, as shown in Fig. 1. In the continuing expansion

R;

14-memberedring type

13-memberedring type

linear type

spermidine long chain acid amide type

spermine FIG.1. The characteristic types of peptide alkaloids from higher plants.

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of peptide alkaloid isolation, linear peptide alkaloids derived from cyclopeptide alkaloids, the related long-chain acid amides, spermine and spermidine-derived alkaloids, and cyclic peptides forming only peptide linkages have been characterized and are defined as peptide alkaloids. Many reviews on the occurrence, isolation, identification, classification, synthesis, instrumental analysis, biochemistry, and pharmacology of the peptide alkaloids have been published. Greger (8) has reviewed the structural relationship, distribution, and biological activity of alkamides. JoulliC and Nutt (9) have reported on the biology, chemistry, and synthesis of cyclopeptide alkaloids up to 1984. Shah and Panday (10) have described peptide alkaloids that contain a styrylamine unit. Winkelmann et al. (12) have reviewed the structure-activity relationship of siderophores in fungi and studied the structural requirements for their recognition and transport in fungi, using natural and synthetic analogs. Schmidt (12) has reviewed the synthesis of naturally occurring cyclopeptides and cyclopeptolides. Lewis (23) has reported in a series of reviews on peptide alkaloids from natural resources at regular intervals. Schmidt et al. have reviewed peptide alkaloids in a volume of this series (7) published in 1985. They covered the basic peptides that embody an ansa structure in which the 10- or 12-membered peptide-type bridge spans the 1,3- or 1,4-positions of a benzene ring and linear peptide alkaloids. They are defined as those compounds that can be derived formally from cyclic peptide alkaloids by scission of the bridge in an elimination reaction. In this chapter we describe cyclopeptide alkaloids, spermine and spermidine alkaloids, long-chain acid amides, and the cyclic oligopeptides isolated from higher plants since 1985. A. CYCLOPEFTIDE ALKALOIDS FROM Zizyphus PLANTS

From Zizyphus sativa (Rhamnaceae), discarine-related 13-membered cyclopeptide alkaloids, sativanines C (1)(14), D (57), E (2), F (3) (15-17), and G (4) (28) have been isolated. The bark of 2. sativa is used to heal ulcers and wounds. Extensive chromatography of the crude bases furnished a further, previously unknown, 13-membered cyclopeptide alkaloid sativanine H (5) (19). Sativanine K (6) has been obtained from the powdered bark of 2. sativa. This is the first 13-membered cyclopeptide alkaloid to contain an N-formyl group as well as a short side chain (20). A new, minor 13-membered cyclopeptide alkaloid, tscheschamine (W), has been isolated from the bark of 2. sativa, and its structure determined on the basis of spectral studies and hydrolysis (21). (See Figs. 2-6.) From Zizyphus nummularia, in addition to the known alkaloid nummularine B (7), two new peptide alkaloids nummularine M (36)and nummular-

sativanine C (1): RI=Me. R2=CH(Me)CHzMe, R3=CH(Me)z, %=H, R5=COCH(Me)NHMe sativanine E (2): RI=Me. R2=CHzCH(Me)CH2Me, %=R5=Me, R3=

qCH2

sativanine F (3): RI=Me, R2=CHzPh, R3=CH(Me)z, %=H, RS=COCH(NHCHO)CH(Me)z sativanine G (4): RI=Me, R2=CH(Me)CH2Me, R3=CH(Me)CHzMe, %=R5=Me sativanine H (5): RI=Me. Rz=CH2CHzCH(Me)z, R3=CH(Me)z, %=H, R5=COCHzNHMe sativanine K (6): RI=Me, R2=CH(Me)CHzMe. R3=CH(Me)CHzMe. &=H, R5=CHO nununularine B (7): RI=Me, RZ=CHzPh, R3=CH(Me)2, %=H, R5=COCH(NHMe)Me )~, &=R5=Me nummularine C (8): RI=Me, R Z = C H ~ C H ( M ~R3=CHzPh, nummularine N (9): R,=Me, R2=CH2Ph, R3=CH(Me)2. %=H. R5=COCHzNMez nummularine 0 (10): RI=Me, R2=R,=CHzPh, &=H, R5=COCH(CH2Ph)NHMe )~, %=H, R+OCH(Me)NHMe nummularine P (11):RI=Me, R z = C H ~ C H ( M ~R3=CH(Me)z, nummularine R (12): RI=Me, R2=CHzCH(Me)CH2Me, &=R5=Me, R3=

WCH2 ' w

nummularine S (13): RI=Me, Rz=CH2Ph. R3=CHzCH(Me)z, %=Rs=H nummularine T (14): RI=Me. R2=CHzPh, Rj=CH(Me)2, %=H, R5=COCH(NMeCHO)Me tscheschamine (15): RI=Me, Rz=CHzPh, R3=CH(Me)CH2Me. &=R5=H amphibine H (16): RI=Me, Rz=CHzPh, R3=CH(Me)2,&=H, R5=COCH(NMez)Me jubanine A (17): RI=Me, Rz=CH(Me)CHZMe. R3=CH2Ph, %=H, R5=COCH(NMez)CHzPh jubanine B (16): RI=Me, R2=CH2Ph, R3=CHzPh, %=H, R5=COCH(NMe2)CH2Ph lotusine E (19): Rl=H, Rz=CH(Me)CHzMe, R3=CH2Ph. %=H, R5=COCH(NMe2)CH2CHMez lotusine F (20): RI=H. R,=CH(Me)CHzMe, R3=CH2Ph, &=H, R5=Me rugosanine A (21): RI=Me. R2=CH2CHMez, R3=CHMe2, %=H, R+OCH(NMeCHO)Me rugosanine B (22): RI=Me, R,-CHzPh, %=R,=Me, R3=

mCH2 ' w

mucmnine D (23): RI=Me, R2=CH(Me)CHzMe, R3=CH2CHMe2. &=H, R5=COCH(NMe2)CHzPh (24): R,=Me, Rz=CH(Me)CHzMe, R+H2CHMez. %=%=Me (25): RI=H, R2=CH(Me)CH2MeSR3=CH$HMe2, %=H, Rs=COCH(NMe2)CHzPh daechucyclopeptide- I (26): RI=H, R2=CH(Me)CH2Me. R3=CH2Ph, %=R5=Me daechuine-S3 (27): Rl=Me. Rz=CH(Me)CH+le, Rj=CH(Me)CHzMe, %=H, R5=COCH(NMe2)CH(Me)CH2Me daechuine-S6 (28):RI=Me, R2=CH(Me)CHzMe, R3=CH2Ph, &=R5=Me daechuine47 (29): RI=Me. Rz=CH2CHMez, R3=CH2CHMe2,%=R5=Me daechuine-S8-l (30):RI=Me, R2=CH2CHMe2, R3=CH2CHMez, %=H, R5=COCH(NMez)CH2CHMez daechuine-S10 (31): RI=Me, Rz=CH(Me)CHzMe, %=R5=Me. R3=

mCH2 ' w

daechuine-S26 (32): RI=H, Rz=CH(Me)CHzMe, R3=CH2Ph, %=%=Me

FIG.2. Thirteen-membered cyclopeptide alkaloids from Zizyphus plants.

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sativanine A (33): Rl=CH(Me)Z, R2=CH(Me)CH2Me, R3=%=Me, R5=Ph nummularine E (34):RI=CH2CH(Me)2, RZ=CH(OH)Me, Rs=&=Me, Rs=Ph nummularine K (35): RI=CH2CHMe2, R3=&=Me, Rs=CH(Me)Z, Rz=

mCH2 ' N

nummularine M (36):RI=R2=CH(Me)CH2Me, R3=&=Me, R5=Ph integerrinine (37): RI=CHZCH(Me)z, R2=CH(Me)CHzMe, R3=&=Mel Rs=Ph lotusanine A (38): RI=CH(Me)CH2Me,RZ=CHzPh,R3=&=Me, R5=CHMe2 sanjoinine B (39):RI=CH2CHMe2,RNHZPh, R3=H, &=Me, R5=CHMe2 sanjoinine F (40):RI=CH(OH)CHMe2, R&HzF'h, Rs=&=Me, RpCHMez frangufoline (41): RI=CH2CHMe2, RZ=CHzPh, R3=&=Me, R5=CHMe2 frangulanine (42): Rl=CHzCHMcz, Rz=CH(Me)CHzMe, R3=&=Me, Rs=CHMe2 franganine (43): RI=CH$XMe2, R2=CH2CHMe2,%=%=Me, R5=CHMe2 daechuine45 (44): RI=CH2CHMe2,RZ=CHMe2, R3=&=Me, Rs=CHMe2

FIG.3. Fourteen-membered cyclopeptide alkaloids from Zizyphus plants.

ine N (9) have been isolated and their structures elucidated (22).Nummularine M is a 14-membered cyclopeptide alkaloid and belongs to the integerrine type, whereas nummularine N is a 13-membered cyclopeptide alkaloid like nummularine B. Nummularine 0 (10)(23),jubanines A (17), B (18), and mauritine C (50) (24) were obtained from Z. nummularia. The stem bark extract of Z. nummulariu has been re-examined and the 13-membered cyclopeptide alkaloids nummularines P (11)(25), R (12) (26), S (13) (28), and T (14) (29)isolated. The structure was determined by spectroscopic methods and chemical degradation. Z. nummularia seems to be closely related chemotaxonomically to Z. jujuba, Z. sativa, and Z.amphibia. Two new alkaloids obtained from 2. nummulariu, namely nummularines M (36) and N (9), have been studied by high-resolution mass spectrometry (27). Full details of the total synthesis of nummularine F (49), the 14-membered para-ansacyclopeptide alkaloid isolated from Z. nummularia, have now been reported (30).

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lotusine A (45): R1=CH(Me)CHzMe,Rz=CH2Ph, R3=&=Me lotusine B (46): RI=CH(Me)CH2Me,Rz=CH2Ph, R3=H, &=COCH(NMe2)CH2CHMe2 lotusine C (47): R,=CH(Me)CHZMe, R2=CH2Ph, Rj=Me, &=COCH(NHMe)CHMez lotusine D (48): RI=CH(Me)CH2Me,R2=CH2Ph,R3=H, R4=Me nummularine F (49): RI=CH(Me)CHZMe, R2=H, R3=&=Me mauritine C (50): Rl=CH2Ph, R2=CHMe2,R3=H, &=Me mauritine D (51):RI=CH(Me)CHzMe,R2=CH2CHMe2,R3=H, &=COCH(NMez)CH(Me)CHzMe arnphibine B (52): RI=CH(Me)CHzMe,R2=CH(Me)CH2Me,R3=H, &=COCH(NMeZ)CH2Ph mucronine J (53): RI=CH(Me)CHZMe, R2=CH2CHMe2,R3=&=Me mauritine J (54):RI=CH(Me)CHzMe,R3=H, &=COCH(NHMe)CH2CHMe ,R -

2-wcH2 mcH*

amphibine E (55): Rl=CH(Me)CHzMe,R3=H, &=COCH(NMe2)CHzCHMe2, R2=

' w

FIG.4. Fourteen-membered cyclopeptide alkaloids from Zizyphus plants.

From Zizyphus xylopyru, mauritine D (51) and nummularine B (7) have been isolated for the first time (31).From the seeds of Zizyphus spinosus, sedative cyclopeptide alkaloids sanjoinine A (frangufoline 41), sanjoinine B (39), sanjoinine D (61),sanjoinine F (40),sanjoinine GI (62), sanjoinine G2 (63),and sanjoinenine (60) have been isolated (32).Sanjoinine A (41) showed strong prolongation of hexobarbital-induced sleeping time in mice, and its sedative activity was destroyed by heating and the isomer formed enhanced the sedative properties. The methanol extract of zizyphi fructus, that is, the fruits of Zizyphus jujuba, possesses sedative activity. The active principles were identified as nornuciferine and lysicamine. During this investigation a new cyclopeptide alkaloid, daechucyclopeptide-1 (X),was isolated and characterized (33). Sanjoin (seeds of 2. vulguris var. spinosus) and Daechu (fruits of 2.jujubu var. inermis) are traditional Oriental medicines used for the treatment of

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1 sativanine B (56)

>-

nummularine G (58)

sanjoinenine (60)

sativanine D (57)

lotusamine B (59)

sanjoinine D (61)

sanjoinine G, (63)

FIG.5. Cyclopeptide alkaloids from Zizyphus plants.

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309

spinanine A (64):R,=CH2CHMe2 zizyphine G (65):Rl=CH(Me)CH2Me

zizyphine F (66)

FIG.6. Cyclopeptide alkaloids from Zizyphus plants.

insomnia, and other pharmacological properties have also been described for these preparations. From the fruit extract of Daechu five alkaloids were obtained; the one belonging to the peptide group was daechucyclopeptide 1 (%), whereas the root bark extract yielded 12 alkaloids, four being 14membered cyclopeptides such as daechuines-S1 (frangufoline, 41), 4 2 (frangulanine, 42), -S4 (franganine, 43), and -S5 (44, new) and eight 13membered cyclopeptides, daechuinesS3 (27, new), -S6 (28, new), -S7 (29, new), -S8-1 (30, new), -S9 (mucronine D, 23), -S10 (31, new), -S26 (32, new), and -S27 (nummularine B, 7). Traditionally, Sanjoin is produced by heating the plant material, and in this investigation it was found that the effect of heat on sanjoinine A (frangufoline) produced the more sedatively active artifact sanjoininine Ah-1. These compounds were interconvertible,

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and structure determination revealed that Ah-1 was produced by isomerization of the chiral center and not by conformational isomerism of the ring (34).Nine cyclopeptide alkaloids from the seeds of 2. vulgari var. spinosus and the fruits and stem barks of Z. jujuba var. inermis were evaluated for sedative activity by assessment using a hexobarbital-induced sleeping time method in mice (35).The methanol extract of the seeds of Z. jujuba was separated into nine fractions by means of column chromatography using different ratios of chloroform-methanol. Three fractions gave a positive test with Dragendorff's reagent, and the spectral data of the alkaloids isolated from the three fractions indicated the presence of a cyclopeptide alkaloid ring structure (36).Z . jujuba contains many cyclopeptide alkaloids, and in this study two new alkaloids were isolated (37). From the stem bark of Zizyphus rugosa, a new cyclopeptide alkaloid rugosanine A (21)was isolated and its structure determined by spectroscopic methods, coupled with chemical degradation. It is reported to be the third example of an N-formyl cyclopeptide alkaloid containing a 13membered ring (38). Also, from Z. rugosa a new cyclopeptide alkaloid rugosanine B (22)was isolated, together with mummularine F (49) and sativanine H (9, which were identified by chemical degradation and spectroscopic analysis (39). Sanjoin (seeds of Zizyphus vulgaris var. spinosus) is related to Daechu (fruits of Z. jujuba var. inermis) (34).From the seeds of Z. vulgaris, a new cyclic peptide alkaloid sanjoinenine (60) has been obtained together with sanjoinine B (39), sanjoinine D (61),sanjoinine F (40),and sanjoinine G2 (63) (40). The drug Sanjoin is prepared by a heat treatment of the seeds of Z. vulgaris. In an attempt to understand this process, a number of sedative alkaloids, the sanjoinines, have been isolated from untreated seeds and subjected to heating, whereby a number of artifacts have been produced and identified (41). From the Egyptian medicinal plant Zizyphus spina-christi, a new alkaloid has been isolated. It is a 14-membered cyclopeptide spinanine A (64) and is accompanied by zizyphine F (66),jubanine A (17),and amphibine H (16), all of which have 13-membered rings (42). The use of formyl derivatives of cyclopeptide alkaloids in high-resolution MS studies leads to fundamental differences in their fragmentation patterns. A 14-membered cyclopeptide alkaloid, mauritine C (50), was compared to that of the 13-membered sativanine C (1)(43).The cyclopeptide alkaloids franganine (43), mauritine C (50), and sativanine A (33) were isolated from the stem bark of 2. spinachristi. The chloroform and ethanol extracts were active against Staphylococcus aureus and Bacillus subtilis (44). From the root bark of Zizyphus lotus, cyclopeptide alkaloids were isolated. The structures of two new compounds, lotusine A (45) and lotusine

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

31 1

D (48), were elucidated mainly through homo- and heteronuclear NMR techniques (45). Two new 14-membered frangulanine-type cyclopeptide alkaloids, lotusanines A (38) and B (59), have been isolated from the aerial parts of 2. lotus, together with the known alkaloids sanjoinenine (60), sanjoinine F (40),and frangufoline (41) (46). Four new cyclopeptide alkaloids, lotusines B (46),C (47), E (19), and F (20), were isolated from the root bark of 2. lotus. Their structures were elucidated mainly by homoand heteronuclear NMR techniques (47). From the roots of Zizyphus mucronata, in addition to the known alkaloid mucronine D (23), two new cyclopeptide alkaloids 24 and 25 were isolated. The structures were elucidated by 1D- and 2D-NMR techniques (48). A new cyclopeptide alkaloid, named mucronine J (53), was isolated together with known alkaloids abyssenine A and mucronine D (23) from the methylene chloride extract of the root bark of 2. mucronata (49). The structure of mucronine J was elucidated by mass spectrometry and 1D and 2D-NMR techniques, and the solution conformation is proposed on the basis of NOE experiments in combination with MM2 calculations. Leaves of Zizyphus hysodrica were extracted with methanol and the different alkaloidal fractions gave a new compound, hysodricanine B, which is a cyclopeptide alkaloid (50). From the root bark of Zizyphus muuritiana, a new cyclopeptide alkaloid mauritine J (54), together with known alkaloids, was isolated. Its structure was established by homo- and heteronuclear 2D-NMR analysis and compared with that of amphibine E (55) (51). From Zizyphus oenopleu, some cyclopeptide alkaloids were isolated (52-54). B. CYCLOPEP~IDE ALKALOIDS FROM OTHER SPECIES From Discaria febrifuga (Rhamnaceae), four new cyclopeptide alkaloids, discarines F (72) ( 5 9 , G (73) (56), H (74) ( 5 3 , and E (71) (58), have been isolated. These cyclopeptide alkaloids are 14-membered cyclic peptides. A new 14-membered ring peptide alkaloid discarine I (75) and its known relative discarine B (68) have been isolated from D. febrifuga (59). Further studies on the peptide alkaloids produced by D. febrifuga have resulted in the characterization of the tryptophan-containing alkaloid discarine K (76) (60). 'H- and '3C-chemical shift assignments of the cyclopeptide alkaloid discarine B (68) were completed based on 2D-NMR experiments such as 'H-lH and 'H-13C COSY spectra (61). From the methanol extract of the root bark of D. febrifugu, a new peptide alkaloid, discarine L (79), has been isolated and its structure elucidated (62). (See Fig. 7.) From Discaria longispina, a new cyclopeptide alkaloid discarine X (77), together with known cyclopeptide alkaloids adoutine Y' (78), discarine B

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y-4 R3

discarine A (67)

To

R1

NMe2

R2

.

discarine C (69)

RI=CHzCH(CH3)z Rz=CH~CH(CH~)Z

discarine D (70)

R,=CHzph Rz=CHzCH(CH3)2

discarine B (68)

R3=R4=CH3 discarine E (71)

NMe2

discarine F (72)

discarine I(75)

discarine G (73)

Ri=CHzPh Rz=CH(CH~)CH~CH~

discarine H (74)

R,=CH~CH(CH~), RzSHzCH(CH3)z

discarine K (76)

R1=CH(CH3)CHZCH3 Rz= HP

discarine X (77)

adoutine Y'(78)

FIG.7. Cyclopeptide alkaloids from Dbcaria plants.

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313

(68), and discarine E (71),was isolated, and their structures were elucidated by spectroscopic methods and by chemical degradation (63). A chloroform extract of the stem bark of Canthiurn anorldianum (Rubiaceae) contains a pinkish-colored, crystalline substance (anorldianine), which was characterized as a peptide alkaloid (80) by comparing the spectroscopic data with those of other known peptide alkaloids (64). (See Fig. 8.) The methanol extract prepared from the flowers of Sphaeranthus indicus (Compositae) was separated into nine fractions, all of which gave positive tests with Dragendorff's reagent, indicating the presence of alkaloids. Fraction five contained two compounds, both of which are cyclopeptide alkaloids whose structures are tentatively given as 81 and 82 (65). A novel cyclopeptide alkaloid was obtained from the aerial parts of Plectronia odorata (Rubiaceae). Its structure was established as Ndesmethylmyrianthine C (83) (66).

anorldianine (80)

N-desmethylmyrianthineC (83) FIG.8. Cyclopeptide alkaloids from Canthiurn, Sphaeranthus, and Plectronia plants.

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C. SPERMINE AND SPERMIDINE ALKALOIDS 1. Linear Type

Amides derived from spermine and hydroxycinnamic acid have been isolated from the pollen of Alnus glufinosa,Betula verrucosa, and Pterocarya fraxinifolia (67).In Alnus glutinosa, the isolated material contains a mixture of two N,N-di-(E)-p-coumaroyl spermidines. From B. verrucosu, MN'O-di(E)-feruloylspermidine (84) was obtained, and from P. fraxinifolia, the amide was N',N5-di-(E)-coumaroyl spermidine (85). Such conjugates of hydroxycinnamic acids with the aliphatic amines putrescine, spermidine, and spermine have been reported from a large number of families throughout the plant kingdom (68,69). The accumulation was shown to be linked with cell multiplication (70) or the fertility of reproductive organs (71,72). They may also protect plants from viral, bacterial, or fungal infestation (73,74,75) and may be produced as phytoalexins (80). (See Fig. 9.) The new spermidine alkaloid N',A@-dibenzoylsperrnidine (86) is one of the metabolites found in the leaves of Cassiaporibunda (Leguminosae) (76). A hydroxycinnamic acid-spermidine amide has been isolated from the drug "Crataegi flos," which is raw material prepared from the flowers of a Crataegus subspecies and is reported to have cardiotoxic properties. Chemical and spectroscopic methods have indicated that this amide is N',~,N10-tri-4-(E)-coumaroylspermidine (87). Also, the occurrence of this amide in flowers of various members of the Rosaceae has been investigated (77). Of three hydroxycinnamic amides isolated from Iochroma cyaneum (Solanaceae), only one was new, namely N',N'O-di-dihydrocaffeoylspermidine (88) (78).

FIG.9. Linear spermidine alkaloids from higher plants.

MACROCYCLIC PEITIDE ALKALOIDS FROM PLANTS

315

2. Ring Type Using an affinity probe technique it has become possible to identify natural products that bond to DNA (79). Extracts derived from Albizia umara (Leguminosae) were found to demonstrate activity in a recently developed HPLC system designed to detect compounds capable of interacting with DNA. Further investigation led to the procurement of four sets of alkaloid isolates X1-X4 that were found to be macrocyclic pithecolobine alkaloids. Isolate XIfound to inhibit the catalytic activity of DNA polymerase, RNA polymerase, and HIV-1 reverse transcriptase. It has been identified as a mixture of budmunchiamines A (89), B (90), and C (91), in the ratio 4 :1: 1 (80), whose structures were determined by spectroscopic methods (81). In further detailed bioassay-guided isolation, six more spermine macrocyclic alkaloids, budmunchiamines D-I (92-97) were obtained (82).The structures of these substances were confirmed by spectral analysis and comparison with the related alkaloids budmunchiamines A (89)-C (91). (See Fig. 10.) An investigation of the spermine alkaloids present in the roots of several Aphelandru species has indicated that the polyamines putrescine, spermidine, and spermine interconvert, whereas the alkaloid (+)-aphelandrine (98) content varies considerably during the first year. This is presumed to be due to the need for the diamine precursors to be used to transport cations, especially during bud formation (83). New spermidine alkaloids, capparidisine (99) (84), capparisine (100) (85), capparisinine (101) (86), and isocodonocarpine (102) (83,were obtained from the root bark of Capparis decidua (Capparidaceae). A preliminary cardiovascular activity evaluation of capparidisine (99) indicated that it has the ability to depress heart rate and coronary flow in the isolated heart of a rabbit (88). The spermine alkaloid chaenorpine (103) has been isolated from Chaenorhinum minus (Scrophulariaceae) (89). Its structure elucidation was achieved on the basis of chemical degradation and spectroscopic measurements in comparison to the known alkaloid chaenorphine (90).The absolute configuration is depicted as 103. Two macrocyclic spermidine alkaloids have been isolated from whole plants of Clerodendrum myricoides (Verbenaceae). Myricoidine (104) is accompanied by its reduced derivative dihydromyricoidine (105). These two bases are present in C. myricoides in minute amounts (ca. 10 ppm from the dried plant) and were isolated as homogeneous compounds by repeated countercurrent distribution (91). The 13-membered polyamine conjugate alkaloid (-)-N(I)-acetyl-N(1)deoxymayfoline (106)was isolated from the leaves of Maytenus buxifofia (Celastraceae) (92). (+)-lo6 has been synthesized (93).

316

ITOKAWA ET AL.

OH

R2

102

I7 18

I

106

104

105: 17,18-dihydro

H11,.

0 0

107

108

FIG.10. Macrocyclic spermidine and spermine alkaloids from higher plants.

MACROCYCLIC PEIWDE ALKALOIDS FROM PLANTS

317

The medicinal plant Schweinfurthia papilionacea (Scrophulariaceae), which is considered as a tonic to promote diuresis and reduce fever in typhoid, contains five macrocyclic peptide alkaloids of the spermine type. Two of them, ll-epi-ephederadine (107) and schweinine (108),are new alkaloids (94). D. LONGCHAIN ACIDAMIDES An interesting series of polyunsaturated amides (109-113) have been isolated from Achillea ptarmica (Compositae) (95). Because commercial drug preparations involving the roots of Echinacea purpurea (Asteraceae) were frequently adulterated with the roots of Parrhenium integrifolium, a reexamination of this source of insecticidal and immunostimulatory active material has been carried out. Five new alkamides have been isolated, and their structures were determined as undec2E,4Z-dien-8,10-diynoicacid isobutylamide (114),dodeca-2E,4Z-dien-8,10acid diynoic acid isobutylamide (119, dodeca-2E,4E,lOE-trien-8-ynoic isobutylamide (116), dodeca-2E,4E,8Z-trienoicacid isobutylamide (117), and dodeca-2E,4Z-dien-8,1O-diynoic acid 2-methylbutylamide (118)by MS, IR, and NMR spectroscopic techniques (96). (See Fig. 11.) The air-dried fruit hulls of Evodia hupehensis (Rutaceae), collected from the botanic gardens at Miinster Pharmaceutical Institute, contained a new amide (2E,4E)-N-isobutylhexadeca-2,4-dienamide(119)and its known re(97).Alkamide 120 lated derivative (2E,4E)-N-isobutyldeca-2,4-dienamide has been obtained from Canacylus monanthos (98). A new simple amide, violyedoenamide (Ul), with five known compounds (palmitic, p-hydroxybenzoic, trans-p-hydroxycinnamic, and butanedioc acids, and afielin), was isolated from the lipophilic and alcoholic fractions of Viola yedoensis (Violaceae) (99). (See Fig. 12.) The structures of the piperolein acids, guineensine (122) and wisanine (123), isolated from Piper guineense (Piperaceae) have been revised and shown by synthesis to possess the 2,4-transltrans stereochemisty (100). An amide, dehydropipernonaline (124),was isolated as a coronary vasorelaxant from Piper longum (101). Peptide 124,extracted with methanol from the powdered fruit of P. longum, has been found to be a coronary vasodilator and as such has been patented (102). Two P-phenylethylamine-derived amides have been found in the aerial parts of Piper guayranum. Tembamide acetate (125) has been identified as a natural product for the first time, while alatamide (126)was known previously only as a Rutaceous constituent (103). In the search for potent plant insecticides, two new unsaturated amides, brachystamides A and B, have been isolated from the total aboveground parts of Piper brachystachyum. Brachystamide A is N-isobutyl-15-

318

ITOKAWA ET AL.

CH3 (CH& CH=CH-CH=CH-C-NH-CH2Ce E E

119

FIG.11. Acid amides from higher plants.

CH3

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

319

0

OCHa

122 0

129: R X H 3 130: R=H

123

H3CO

131

0

125 132: R=H 133: R=CH3

H3CO

0

126 134:CH3 135:H

0 0

-6

"

id

I"(

127 H3COr

0

136 128

FIG.12. Acid amides from higher plants.

320

ITOKAWA ET AL.

(3',4'-methylenedioxyphenyl)-2E,4E-pentadecadien~de (l27),and brachystamide B is N-isobutyl-15(3',4'-methylenedioxy-phenyl)-2E,4E,14E-pentadecatrienamide (128)(104). The stem bark of Zanrhoxylum rubescens (Rutaceae) has afforded three new amides, belonging to the cinnamoyl series, namely rubescenamide (l29),rubescenamine (130), and zanthosine (131), whose transcinnamoylamide structures were characterized by spectroscopic methods and synthesis (105). In another investigation on the same plant, two new related amides, rubemamide (132)and rubemamine (133),were obtained from the stem bark, and two other novel amides, dioxamide (134) and dioxamine (135)were obtained from the roots (106).Podocarpamide (136), isolated from the bark of Zanthoxylum podocarpum, was prepared in four steps starting from the condensation of piperonal with malonic acid (107). The structures of three minor alkaloids, dinorperipentadenine, peripentamine, and anhydroperipentamine, isolated from the bark of Peripentadenia mearsii (Elaeocarpaceae) have been established as 137,138, and 139 (108). (See Fig. 13.)

137

q CH3

N

-

U

4

O0 H

0

139

140

OH 0 N L N HH N F ( C H 2 ) h

0 141

0ch3 CH3OC CH3

0 142 FIG.13. Acid amides from higher plants.

143

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

32 1

A new cinnamoyl-derived amide regaloside B has been obtained from the bulbs of Lilium regale (Liliaceae). It has been characterized as (?)-A/(4-methylsuccinimido-n-butyl)-p-coumaramide (140) (209). The exudate of Jafropha gossypifolia (Euphorbiaceae) is reported for the first time to be a rich source of alkaloids, two of which are imidazoles. The alkaloids are designated as structures 141 and 142 (220). Thirteen chemical constituents were isolated from the root and stem of Arisfolochia moupinensis (Aristrochiaceae). One of them is a new cinnamoylamide named moupinamide (143), which inhibited rat platelet aggregation and MDA formation in platelets in vifro ( 2 2 2 ) . Five known isobutylamides, namely fagaramide, piperlongumine, 43dihydropiperlongumine, pellitorine, and (2E,4E)-N-isobutylocta-2,4dienamide, were isolated as the molluscidal and insecticidal active principles from Fagara macrophylla (Rutaceae) (212). E. MISCELLANEOUS The pods of Bufea monosperma (Leguminosae), a plant whose constituents are purported to possess important medicinal properties, contain a new imide, palasimide (144), which is identified as N-phenyl imide (213). Four lignanamides, cannabisins A (145) (224), B (146), C (147), and D (148) (225), together with a lignanamide, grossamide (149) (226), and three amides, N-trans-cafferoyltyramine, N-trans-feruloyltyramine, and N-pcoumaroyltyramine, have been isolated from the fruits of Cannabis safiva (Cannabidaceae). Their structures were determined by spectroscopic measurements. (See Fig. 14.) Two carbamates isolated from the bark of Chamaecyparis obfusa (Cupressaceae) have been named obtucarbamate A (150) and its isomer obtucarbamate B (151); their structures were elucidated by spectroscopic and chemical methods (227). Two new peptide alkaloids have been isolated from the flowers of Hibiscus rosasinensis (Malvaceae), and their structures were characterized as 152 and 153 (228). (+)-Iforresthe (154) is a novel heterocyclic oxazole isolated from the unique Australian plant Zsofropis forresfii. This nephrotoxin, whose structure was determined by a single-crystal X-ray diffraction study, is the causative agent for acute renal proximal tubular necrosis associated with animals ingesting the plant (229). Pandamarine (155) obtained from the leaves of Pandanus amaryllifolius (Pandanaceae), is surprisingly a racemic alkaloid, whose structure was determined by X-ray diffraction study (220).(See Fig. 15.) The roots of Sfemonaparvflora contain the alkaloids parvistemonamide (156), parwistemoline (157), and didehydroparvistemonine, whose struc-

322

ITOKAWA ET AL.

0

GfNyJ 0

OH

144

OH

145

OH

HO

149

OH

1 5 2 Rl=H, Rz=CH3 153: RjXH3, R,=H 151

FIG.14. Miscellaneous peptide alkaloids from higher plants.

tures were elucidated by the various spectral methods and chemical conversion (121). Four maytansinoids,trewiasine (IS), treflorine, trenudine, and trenudine diacetate, have been isolated from the seeds of Trewia nudifloru and showed

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

323

155

154

O

-

h

N

H

H

156 Me

0

nFCHMep

0

158

159

FIG.15. Miscellaneous peptide alkaloids from higher plants.

cytotoxic activity against P388 leukemia cells in vitro; trewiasine was the most potent. From the differing activities, it was speculated that the existence of ester side-chain and hydroxyl groups may play an important role in the antitumor activity of the maytansinoids (122). The leaves of Cluusena excuvutu contained clausenlactam (l59),zapoterin, myricetin-3-O-rhamnoside,ferulic acid, 7-hydroxycoumarin, and triethylamine hydroiodide (123).

324

ITOKAWA ET AL.

III. Cyclic Oligopeptides from Higher Plants

A. RA ALKALOIDS FROM Rubia SPP. 1. Structures of RAs

Rubiae Radix is made up of locally common Rubiaceous plants: Rubia akane in Japan, R. cordifolia in China, and R. tinctorum in Europe. The former two showed antineoplastic activity, but the latter did not. Because the extract of Rubiae Radix showed antineoplastic activity against sarcoma 180A, isolation was pursued for the active principles. After repeated fractionation and purification of the extract, oligopeptides were obtained as active principles against P388 leukemia. The extract was partitioned with water and benzene, and water and ethyl acetate. From both fractions, eight components were isolated as crystals, and named as RA-I-VIII (160-167) after Rubia akane (124-128). The physical data for the RA components are shown in Table I. These compounds were assumed to be small peptides from the IR data, showing 3390, 1640 cm-' due to amide bonding. It was determined from the 13CNMR data of RA-VII (166)that there were three C-Me, three CH2-, three N-Me, two O-Me, six CH, 18 aromatic carbons, 11tertiary carbons, seven quaternary carbons (three C-C bond and four C - 0 bond), and six carbonyl carbon groups. By hydrolysis of RA-VII (166), one D-alanine, two molecules of L-alanine, N-methyl-4-methoxy-~-phenylalanine, and N-methyltyrosine dimer having an ether linkage were obtained. The isolate was assumed to be a cyclic hexapeptide consisting of three alanine and three tyrosine molecules. From these findings, the structure of RA-VII was assumed to be a bicyclic hexapeptide having an ether linkage. However, it was difficult to decide the sequence of amino acids and the configuration stereochemically. Finally, X-ray analysis was applied to the p-bromobenzoate of RA-V (164). From various reactions and instrumental analysis, the structural relationships and the structures of RA-I (160) through RA-VIII (167)were determined as illustrated in Fig. 16. RA-VI (165)was elucidated as the configurational isomer of RA-111 (162) at the moiety of D-O-methyl-tyrosine; RA-VIII (167)has L-threonine instead of L-serine in the RA-I11 molecule. It was observed that RA-VII and RA-V were the main components in these oligopeptide mixtures. Subsequently, RA-IX (168)and RA-X (169)were also added to these RA-series (Fig. 17). Their structures were determined by spectroscopic and chemical methods. RA-IX contained a pyroglutamic acid portion instead of Ala-2 in the molecule of RA-VII, and RA-X had glutamic acid instead of Ala-2 in the same compound. RA-XI (170)was a similar compound to

MACROCYCLIC PERIDE ALKALOIDS FROM PLANTS

325

TABLE I RA-SERIES COMPONENTS

PHYSICAL DATA OF

RA-I (160) RA-I1 (161)

RA-I11 (162) RA-IV (163) RA-V (164)

RA-VI (165) RA-VII (166) RA-VIII (167) RA-IX (168)

RA-X (169) RA-XI (170) RA-XI1 (171) RA-XI11 (172)

RA-XIV (173) RA-XV (174) RA-XVI (175)

Colorless powder, mp 284°C (dec. from MeOH) MS m/z: 772 [MI+ (C40H48010N6) [(Y]D-216" (CHCI3) Colorless needles, mp 261°C (dec. from MeOH) MS m/z: 772 [MI+ (c4oH.@9N6) [(Y']D -201°C (CHCl3) Colorless needles, mp > 300°C (from MeOH) O N[M-H20Jt ~), MS m/z: 786 [MI+( C ~ ~ H ~ O O I768 [(.ID -199°C (CHCl3) Colorless powder, mp 247-255°C (from MeOH) MS m/z: 786 [MI+ (C41H50010N6),768 [M-HzO]' [(Y]D-126°C (CHCI3) Colorless powder, mp > 300°C (from MeOH) Colorless needles, mp 219-220°C (from MeOH) MS m/t: 786 [MIt ( C ~ I H S O O I ~ N ~ ) [(Y]D -118.6"C (CHCl3) Colorless needles, mp > 300OC (from MeOH) MS Mz: 770 [MI+( C ~ I H ~ O O ~ N ~ ) [(Y]D -229°C (CHC13) Colorless needles, mp 267-269°C (from MeOH) MS Mz:800 [MI+ ( C ~ ~ H ~ Z O I O N ~ ) [ a ]-159.5"C ~ (CHC13) Colorless needles, mp 242-243°C (from MeOH) MS m/z: 810 [MI' ( C ~ ~ H~O O IO N S) [(Y]D-158.1"C (CHC13) Colorless needles, mp 255-256°C (from MeOH) MS m/z: 828 [MIt (C43H52011N6). [.ID -205.4"C (CHCI3-MeOH 1: 1) Colorless needles, mp 255.5"C (dec. from MeOH) MS Mz:815 [M + 11' ( C ~ ~ H & L I N ~ ) [ a ] -2353°C ~ (MeOH)) Colorless needles, mp 252-255°C (from MeOH) MS m/z: 919 [M + 1]+(C46H59014N6) [.ID -270°C (MeOH) Colorless needles, mp 273-276°C (from MeOH) MS m/z: 999 [M + Na]' (C48Hh0016N6Na) [(Y]D -109.3"C (MeOH) Colorless needles, mp 238-242°C (from MeOH) 959 [M + 11' (C48H59015N6) MS Mz: [(Y]D -173.2"C (MeOH) Colorless needles, mp 218-220°C (from MeOH) MS Mz:961 [M + I]+ (CaH61015N6) [(Y]D -202.4"C (MeOH) Colorless needles, rnp 218-220°C (from MeOH) MS m/z: 977 [M + i j t ( ~ 4 6 ~ ~ ~ 0 ~ ~ ~ ~ ) [ a ] -179.7"C ~ (MeOH)

326

ITOKAWA ET AL.

Rnbia cordiifolh

RA-I(l60) RA-II(161) RA-III(162) RA-IV(163) RA-V(164) RA-VI(165) RA-VII(166) RA-VIII(167)

R1

R2

R3

R4 RS

R6

R7

H Me

Me H

OH H OH H H OH H Me,OH

H H H OH H H H H

H H H H H H H H

H H H H H H H H

H H H H H H H H

H H

W H H H H

Me Me H

Me Me Me

Me Me Me Me Me

Me

B o u v d & temqolia

bouvardin(178) H deoxybouvardin H W - V ) (1 64 )

Me Me

H H

H H

microbial transformation products 0-desmethyl

H

H

H

H

H

M H H

-bouvardin bouvardin -catecho1

H

H

H

H

H

WHOH

FIG.16.

RA-IX (168): R=CH3 RA-XIV (173): R=p-D-glucose

RA-X (169): R1=CHzCOOH, Rz=CH3; R3=H RA-XI (170): RiSHZCOOH; Rz=H; R3=H RA-XII(l71): R I = H Rz=p-D-glucose; R3=H RA-XIII(l72): R,=CHzCOOH Rz=p-D-glucose; R3=H RA-XV (174): R,=H; Rz=~'-Ac-~-D-~Iucos~; R3=H RA-XVI(175): R, = H; Rz = P-D-glucose;R3 = OAc

FIG.17.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

327

RA-X, having a glutamic acid moiety. Recently, RA-XII(l71) through RAXVI (175) have been isolated as glucosides from the same plant. (129-132). As minor constituents, RAI-I11 (176) and RAI-VI (177) were also isolated from the same plants. These compounds had y-turn structures at residues 2, 3, and 4, which were stabilized by a hydrogen bond between Ser-2-OH and D-Ala-l-CO (see, Fig. 18) (133). Conformational issues regarding the RA alkaloids are discussed in the section on conformational analysis (Section III.A.4). Bouvardin (178) was the first compound of this type of cyclic hexapeptide to be isolated from Bouvardia ternifolia (Rubiaceae) by Cole et al. (134).

2. Cytotoxicity and Antineoplastic Activity Cell growth and inhibitory effects of RA derivatives were examined against KB cells, P388 lymphocytic leukemia cells, and MM2 mammary carcinoma cells by using the lead compound RA-V and its n-hexyl ether derivative, which had shown the strongest antitumor activity in the in vivo assay. These results are shown in Fig. 19. The n-hexyl ether showed clear growth-inhibitory effects at concentrations higher than 5 X 10 pg/ml and 5X pg/ml, respectively, in KB cells, and 1 X lo-' pg/ml and 1 X 10 pg/ ml in MM2 cells. Thus, the growth inhibitory effect of the n-hexyl ether derivative was stronger than that of RA-V in each cell line, and showed dose dependency (135,136). Microscopically, mitomycin C-treated KB cells showed enlargement of the nuclei, deformation of the cells, and abnormality of the nuclei, whereas KB cells treated with RA-V and its n-hexyl ether derivatives showed globulization as compared with control cells.

RAI-III(l76) : MI-VI (177) :

LTyr-3 D-Tyr-3

FIG.18. Structures of RAI-111 and VI.

328

ITOKAWA ET AL.

Control 1 x 10-3

1 x 10-2

1 x 10-2

F;: 1 x lo-' 10.0 1.o

1 x 10-1 10.0

1.o

3 1

2

3

4

5

1

2

3

4

5

Time (days)

" h e (days)

1 x 10-3

328 106 105

i3

1 x 10-1

lo4

1.o

1

2 3 4 lEme (days)

5

1

1 x 10-3 Contml 5 x 10-3

106

1 x 10-2

2 3 4 Time (days)

5

106

3

38

10s

Y

5 x 10-2 1 x lo-'

3

1

lo'

3 1

2 3 4 Time (days)

5

1

2 3 4 Time (days)

5

Fro. 19. In vitro antitumor effects of RA-V derivatives. Effects of RA-V and its n-hexyl ether on the growth of P388, MM2, and KB cells. P388 (9.70 x 103, a and b), MM2 (1.24 X lo4,c and d), and KB cells (1.04 X los, e and f ) were treated with various concentrations of drugs, and cell growth was followed daily with a Coulter counter. Drugs: a, c, and e: RA-V. b, d, and f n-Hexyl ether of RA-V.

329

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

RA-IV was considered to have an additional alcoholic hydroxyl group as compared with RA-VII. It was concluded that the hydroxyl group in RA-IV is linked to the p-carbon(p) of Tyr-6 by comparing the 13Cchemical shift values of RA-IV with those of RA-VII; the Cp signal at 35.56(t) ppm due to Tyr-6 of RA-VII was shifted downfield to 73.49(d) ppm in RAIV, whereas other carbon signals in both peptides were similar. Next, to introduce an oxygen functional group into the benzyl position of Tyr-6, (DDQ). RA-V was oxidized with 2,3-dichloro-5,6-dicyano-p-benzoquinone This reaction gave selectively compound E, which was methylated with diazomethane to yield RA-IV. In addition, to confirm the configuration of the hydroxyl group in RA-IV, its epimer could not be acetylated with anhydrous acetic acid pyridine at room temperature. These findings can be reasonably explained by the following stereochemical considerations: The reagent in this series of reactions can approach only from the a-side, because the P-side at the benzyl location of Tyr-6 is strongly blocked by the Nmethyl group of this tyrosine moiety, as noted from the X-ray conformation. Consequently, the hydroxyl group of RA-IV was determined to have an S configuration. (See Chart 1.)

RA-V

OH

'

A

RA-lv

B

Q03

OR'

OCH3 ' C

CHART. 1.

330

ITOKAWA ET AL.

The antineoplastic activity of six native cyclic hexapeptides(RA-I, 11,111, IV, V, and VII) and seven related compounds were also examined against P388 lymphocytic leukemia in mice (Fig. 20). The mice received 10 mg/kg/ day (except for RA-VII and RA-111: 4.0 and 2.0 mg/kg/day) for 5 consecutive days. The results were as follows. The small differences of antitumor activity among these compounds could be explained to some extent by the molecular hydrophobicities, as previously mentioned, but a remarkable decrease in antitumor activity was observed in the RA-IV compounds A, A-diAc, E, E-Me, and E-Ac, whose a-proton at the CP-position of Tyr-6 was replaced with bulky substituent groups. In spite of a similar replacement at Cp, the activity of compounds B and C did not decrease. From these findings, it may be concluded that the introduction of large substituent groups at the a-side of the RA series results in decreased antitumor activity. This seems to play an important role in the mechanism of antitumor activity. The antitumor activity decrease of RA-I1 could be explained from the viewpoint of the molecular hydrophobicity rather than from the a-block hypothesis. To obtain RA analogs with higher pharmacological and lower toxicological activities,several derivatives were synthesized by substituting the phenol moiety of RA-V, and their quantitative structure-activity relationships (QSAR) were investigated from the viewpoint of molecular hydrophobicities. The activity values (log I/ICso) of ether derivatives of RA-V gave Antitumor

R1 R2 R3

R5 activity@

TIC (%) 169.3 RA-IdiAc Ac Me 182.8 RA-I1 Me H 142.2 RA-111 Me Me 179.4b) RA-VI Me Me 149.0 RA-V H Me 187.4 RA-VU Me Me 173.6c) A H Me 126.3 A-diAc Ac Me 98.2 B Me Me 171.9 C Me Me 160.0 E H Me 118.5 E-Me Me Me 132.0 E-Ac Ac Me 116.9 a) P388 : lo6 cellslO.1 ml, i. p., CDFl mice (n = 6). Dosc : 10.0 mgnCg., i. p. day 1-5.

RA-I

OR’

R4

H

Me OH H

H OAC H H H H H OH H H H OH H H H H H H H H OH H H OAc H H =O =O H H OH H OMe H H OMe H H OMe H

b) Dose : 2.0 m@g. c) Dose : 4.0 mgnCg.

FIG.20. Structures and antitumor activities of native cyclic hexapeptides and related compounds.

MACROCYCLIC PEFTIDE ALKALOIDS FROM PLANTS

331

an upward parabolic or bilinear relationship when plotted against log p (p: partition coefficient determined with l-octanoYwater system) as the carbon number of the side chain at the phenol moiety of RA-V was increased. The optimum log p values were in the range of 3.5 to 4.9. (See Table 11.) The ester derivatives showed a similar relationship, the optimum log p values being 6.3-6.7, higher than those of the ether derivatives. The relationships among the ILS (150 and 160%), the minimum lethal dose (MLD), and the hydrophylic coefficient of the ether series of RA-V were analyzed according to both the Hansch-Fujita model and the bilinear model of Kubinyi. When the parabolic model obtained from the HanschFujita equation was applied to the ILS and MLD, significant results could not be obtained. (See Fig. 21.) However, since the optimum log values of ILS 150 and 60% differed from that of MLD, it was considered that the most suitable ether derivatives of RA-V for antitumor activity might be selected from the region away from the optimum log p of MLD and approximating the log 1/D value in the optimum log p of ILS. Thus, RA-VII and the n-hexyl ether of RA-V should be useful compounds on this basis. The therapeutic ratio of RA-VII was 400,compared with 10 of MMC (Table 111). The mechanism of action of RA-VII was also investigated and was assumed to inhibit protein biosynthesis, since 3H-leucine was not taken up. The lethal effect of RA-V on KB cells was clearly different from that of MMC, and RA-VII was concluded to be a “time-dependent drug” like vincristine. Further, RA-VII was effective against Colon 38 (s.c.-i.p., s.c.-i.v.), P388 (i.p.-i.v.), L1210 (i.p.-i.v.), Meth A (s.c.-i.p.), and M5076 (i.p.-i.p.). Inhibition was also found against pulmonary metastasis of B16-BL-6 (s.c.-i.v.). More recently, mechanistic studies using purified elongation factors and ribosomes have identified RA-VII as a peptidyltransferase inhibitor. Thus, similar to the related natural products bouvardin and RA-XII, RA-VII appears to function by binding to eukaryotic ribosomes (137). RA-V is the same as deoxybouvardin (164). Bouvardin (178) has been investigated for development as an antitumor drug at the U.S. NCI. Adriamycin has CH20H in its molecule instead of CH3 as in daunomycin. With such minor chemical differences, adriamycin was revealed to have a much stronger activity and less toxicity than daunomycin. Therefore, it is expected that RA-VII will show different activity from that of bouvardin. RA-VII (RA-700) is now under investigation for Phase I clinical trials at the NCI in Japan. The in vitro phase I1 trial of RA-700 was studied by human tumor clonogenic assay. From the results of a study using the human lung cancer cell line (PC-6), RA-700 appears to possess time-dependent antitumor activity. The chemosensitivity rates of RA-700 were 67, 22, 17, and 10%

TABLE I1 ANTITUMOR ACTIVITIES ON P388 LYMPHOCYTIC LEUKEMIA AND TOXICITIES TO ETHER OF RA-V DERIVATIVES T/C(%)" Comp. No.

0

R

0.05 mglkg

0.5 mgkg

2.0 mgkg

4.0 mgkg

131.1(')

152.5'e)

164.2@'

165.3'e)

Toxicityb Dose (mglkg)

20 30 40 M

on 217 5n 515

A

138.6(''

156.7"'

164.2(4

173.6("'

10 15 20 30

0/3313 313 313

bR

RA-V : R=H RA-W: R=Me

a

1373("

165.4")

162.2

Toxic

138.4")

146.0(')

93.7

Toxic

142.2'"'

175.1('*)

105.4

Toxic

133.0")

144.9'a)

Toxic

Toxic

122.21''

142.7")

165.4tC)

Toxic

110.3'"'

137.3")

153.3")

173.0

111 s ' ) 136.1 112.5'b) 101.0 115.4 93.0 93.0 101.0 126.5 127.6

144.7") 146.8'c) 141.5'")

150.1 162.9@) 150.1(') 132.7") 121.2'"' 105.8 115.4 101.0 164.3'"' 149.2'"

164.0'"' 152.2(") 155.4(b)

120.2("' 108.7 101.0 99.0 98.1 162.2 140.5'8'

5 10 113 313 5 10 113 313 5 10 313 313 5 10 013 313 5 1020 013 013 3/3 10 20 30 013 1 0 313

137.5'") lB.l@) 112.5@) 125.0"' 108.7 Toxic 143.8

Significantly different from control at (a) p < 0.05, (b) p < 0.01, (c) p < 0.001; from RA-V at (d) p

< 0.05, from RA-VII at (e) p < 0.05. Toxicity: number deadnumber tested.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

1.0

333

Q

TIC 150%

0.5

0.0

-0.5 '

TJC 160% Q

-1.0

-1.5

Toxicity

-2.0

FIG.21. Structure-antitumor activity and toxicity relationships of alkyl ethers of RA-V on P-388 leukemia in mice.

for ovarian cancer, non-small-cell lung cancer, breast cancer, and colorectal cancer, respectively. RA-700 showed almost the same chemosensitivity as that of five standard anticancer drugs (adriamycin, mitomycin C, cisplatin, vinbrastine, and 5-FU), but the spectrum of RA-700 activity appeared to be different. Furthermore, the antitumor activity of RA-700 had no relationship with prior chemotherapy. These results indicated that RA-700 is a candidate for Phase I clinical study (138). 3. Metabolites of RA-VII and RA-X

The biotransformation was examined by using rat hepatic microsomes and rabbit bile juice. RA-VII was incubated aerobically with rat liver microsomes in the presence of an NADPH-generating system. In the course of researching the metabolites from the bile juice in rabbits, 11 metabolites 179-187, RA-11, and RA-V were isolated (Fig. 22).

334

ITOKAWA ET AL.

TABLE I11 THERAPEUTIC EFFECTS OF RA-VII ON P388 LEUKEMIA ~~~~

Group

Dose (mg/kg)

Route

Control RA-VII

10 ml 0.005 0.01 0.05 0.5 2.0 4.0 6.0 0.005 0.01 0.1 0.5 1.0 2.0 10 rnl 0.25 1.o 2.5 4.0 6.0 0.1 0.5 1.o 2.0

i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.p. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v. i.v.

MMC

Control RA-VII

MMC

~

~~

~

Survival Time ? S.E.) TIC (%)

(d, mean

101.1 It 0.18 11.0 5 0.26 13.3 t 1.15 15.5 t 1.12 16.7 t 0.33 18.6 2 1.21 23.6 t 2.62O 6.00 t 2.61 10.8 ? 0.31 10.5 ? 0.22 13.7 It 0.67 15.8 t 0.31 18.0 t 0.68 12.7 t 0.33 9.50 ? 0.15 10.0 ? 0.27 11.0 t 0.19 13.4 ? 0.18 14.5 +- 1.25 15.9 t 0.23 10.5 2 0.19 12.5 t 0.19 13.6 2 0.18 12.1 It 0.13

~_____

~~

100.0 109.2 132.3 153.8 165.4 184.3 234.2 62.7 107.5 104.2 135.6 157.1 178.1 125.7 100.0 105.3 115.8 140.8 152.6 167.1 110.5 131.6 143.4 127.6

B. W.(9) +5.0

+3.8 +2.6 +2.1 +1.3 f0.4 -0.6 +4.8 +5.2 +2.8 +0.8 -0.3 -1.8 +4.0 +3.2 +1.9 -0.3 -2.0 -4.7 +4.4 +2.3 +0.9 -3.6

T. R.

=

400

T. R.

=

10

~~~

116 animal survived 60 d. P388 was implanted i.p. (1 X lo6 cells/0.1 ml) in CDFl mice at day 0. Drugs were given daily at indicated doses for 9 consecutive days from day 1 to 9.

RA-V and compound 179 were isolated as the two main metabolites from the chloroform extract of the incubation mixture. The structure of 179was determined to be [N-demethyl-Tyr-3]RA-VII. Compounds 180 and 181 were characterized as [~l-hydroxyl-Tyr-5]RA-VII and [s2-hydroxylTyr-SIRA-VII, respectively. When RA-X was administered in rabbits, the total recovery of RA-XNa was 75%. RA-X-Na did not show antitumor activity against P388 leukemia in mice by i.v. administration. This was also presumed to be attributable to the fast metabolic turnover indicated in this experiment. 4. Conformational Analysis

The therapeutic potential of many biologically active cyclic peptides is intimately related to their conformation. Structural analogs of many of

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

R1

179 180 181 182 183 184 185 186 187 RA-VII(166) RA-II(161) RA-V(164) RA-X(169)

R2

H CH3 CHS CH3 CH, CHJ CH3 CH3 CH3 CH, CH3 H CHS CHI CH3 H CHI H CHj CH3 CHI H CH3 CHI CH3 CH3

R3

R4

RS

H H H OH OH H OH H H

H OH H H OH H H H

::

CHj CH3 CH3 CH, CH3 CH3 H H CH, CH, C?

H H OH H H OH H H OH H

H

CH3

H

H

:: H

R6

H H

H H

335

R7

CH3 CH3 CH3 CH3 CHI CH3 CHg CHs CH3 CHI CH3 CH, CHZCHZCOOH

FIG.22. Structures of RA-VII and its metabolites.

these biologically active RAs have been synthesized for studies of the biological systems with which they interact. The relationship between conformation and biological function of RAs is a topic of great interest and importance. Much work is being done in the field of peptide structural and conformational analysis, using X-ray crystallography, circular dichroism, NMR spectroscopy, and computational methods. These methods provide information about the conformation of RAs in the solid state and in solution. a. Crystal Conformation. Bouvardin (178),originally isolated in 1977 from Bouvardia rernifoliu (Rubiaceae), was the first member of the RA family, and solid-state conformation was reported (242). The molecule consists of five L-amino acids and one D-amino acid joined by peptide linkages (Fig. 23). A characteristic feature of this molecule is that it has,

336

ITOKAWA ET AL.

FIG.23. ORTEP drawing of the crystal structure of bouvardin.

in addition to an 18-membered peptide ring, a 14-membered ring formed by the oxidative coupling of the phenolic oxygen of one tyrosine with a carbon ortho to the phenolic OH group of an adjacent tyrosine. The Nmethyltyrosine residue on the 18-membered peptide ring extends outward. Five of the peptide bonds are in a trans conformation, while the sixth, between Ty? and TyP, is in cis conformation (type VI @turn), which serves to fold the peptide chain to form a cyclic structure. Another turn (type I1 /3-turn) was found between Ala2and Ty9. It is an unusual cyclic hexapeptide because there is an absence of any hydrogen bonding within the 18membered ring. Itokawa et al. reported the X-ray structure of RA-V p-bromobenzoate in 1990 (143) in which the backbone conformation possesses almost the same characteristics as that of bouvardin (Fig. 24). Later, the crystal structures of RA-VI (165)(Fig. 25) (144) and isomerized RA-VII (188) (Fig. 26) (145), neither showing antitumor activity, were reported. The backbone conformation of these molecules differ from those of the 18-membered ring of RA-VII and bouvardin. b. Solution Conformation. The presence of two conformers in chloroform solution was suggested by HPLC analysis (146). However, separation on a preparative scale was not achieved because of fast exchange at room

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

337

FIG.24. Perspective view of the X-ray structure of RA-V p-bromobenzoate. The backbone conformation possesses almost the same characteristics as bouvardin (Fig. 23).

temperature. Conformational analysis of the two states in chloroform solution was performed using NMR and computational methods. Bates et al. indicated that the predominant stereoisomer and conformer in solution for deoxybouvardin and bouvardin is the same as that found in the solid state by X-ray diffraction (247). A minor stereoisomer (ca. 15%)separated by a 20.6 kcal/mol barrier was detected and is believed to contain a rotation about the Tyr5 and/or Tyr3 amide bond; however, its precise conformation could not be deduced. A combination of different homo- and heteronuclear 2D-NMR techniques at 500 MHz have enabled the complete assignment of the 'H and 13Csignals of the two conformers of RA-VII in CDC13 (major conformer: conformer A; minor one: conformer B) (243). The structures of the two conformers (A and B) in CDC13 were elucidated based on temperature effects on NH protons, deuterium exchange rate, vicinal coupling constants, and NOE experiments (Fig. 27). These conformational analyses showed that the structure of these conformers is a result of geometric isomerization and that the predominant conformer A exhibits a typical type I1 P-turn between Ala2 and Tys, similar to the crystal structure as analyzed by Xray diffraction. The minor conformer B exhibits a type VI P-turn between Ala2 and Tyr3, showing a cis amide bond. In the 18-membered ring, the

338

ITOKAWA ET AL.

FIG.25. Perspective view, with each atom numbered, of the crystal structure of RA-VI (165) by PLUTO drawing.

presence of two intramolecular hydrogen bonds between Ala4-NH and DAlal-CO and between D-Alal-NH and Ala4-CO was suggested. This differs between the solid and solution conformations. The reduced biological activity of the N-methyl derivative of RA-VII in comparison with RA-VII may be responsible for the small population of conformer A in solution. Further, the presence of a highly strained 14-membered ring is necessary to maintain the typical type I1 P-turn structure of conformer A. The ring system and structure are considered to play an important role in the antitumor activity (143). The conformation of RA-VII in THF-dg was found to be similar to that observed in CDCl3 (148). The addition of LiCl caused no conformational change and resulted in the adoption of a single dominant solution form (conformer A, 94%) (148).

MACROCYCLIC PEPITDE ALKALOIDS FROM PLANTS

339

FIG.26. Perspective view of the crystal structure of [D-Ty?, ~-Tyr']RA-v11 (188).

FIG.27. NOE enhancements in conformers A and B of RA-VII. Arrows show the NOE relationships confirmed by ID-NOE and NOESY experiments in CDCl3 at 303" K.

340

ITOKAWA ET AL.

Three discernible isomers observed in DMSO-dS were assigned for RAVII (Fig. 28) (249). The dominant form, conformer A, accounting for 64% of the mixture, has a cis configuration only between Tyr' and Tyr6. The second configurational isomer, conformer By32%, has adopted cis configurations between both the TyS and Tyr6 and the Ala2 and Tyr3. The third isomer, conformer C, 4%, has cis configurations for all of the three Nmethyl amide bonds. A molecular design was carried out to lock the type I1 p-turn conformation of RA-VII, by removing the N-methyl group of the Tyr3 residue. Conformational analysis of [N-demethyl-Tyr(OCH3)-3lRA-VII (179), produced by hepatic microsomal biotransformation, was based on 2D-NMR techniques, temperature effects on NH protons, and NOE experiments (250). It showed a restricted conformational state with a typical type I1 pturn between Ala2 and Tyr3 in DMSO-d6. The relationships of the NOE enhancements (all negative NOES) are shown in Fig. 29. This analogue was recently synthesized by Boger et aL, who showed that N-methyl removal does not alter its conformational or biological properties (252). The disfavored cis amide bond between TyP and Tyr6 is the predominant conformation observed with [desmethyl-Tyr6]RA-VII. In contrast, [desmethylTyPlRA-VII in solution possesses a trans amide bond between Tyr' and Tyr6, resulting in a loss of biological activity. Thus, the N-methyl amide group between Ala4 and Tyr' is essential for maintenance of the conformational and biological properties of RA-VII (251).

Conformer A

Conformer B

Conformer C

FIG.28. Molecular structures of three different conformers (A, B, and C) of RA-VII in DMSO-4. Arrows show the NOE relationships confirmed by NOESYPH experiments.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

34 1

FIG.29. NOE relationship of [N-demethyl-Tyr(OCH3)-3]RA-VII. Arrows show the negative NOE relationships by 1D-NOE and NOESYPH in DMSO-d6 at 303" K.

Furthermore, conformational change in the 14-membered ring system of [~l-hydroxyl-Ty~]RA-VII (180) and [~2-hydroxyl-Tyr~]RA-VII (181) was observed in the metabolites of RA-VII and RA-X, produced by using rat hepatic microsomes and rabbit biliary juice (Fig. 30) (252). By conformational analysis using spectroscopicand computational chemical methods, RAI-I11 (176) and VI (177), which were isolated from R.

[ ~-hydroxyl-Ty?]RA-VII l

[~2-hydroxyl-Ty?]RA-VII

FIG.30. NOE relationships of the 14-membered rings of [~l-hydroxyl-Ty~]RA-VII and [e2-hydroxyl-Tyr5]RA-VII.Arrows show the NOE relationships confirmed by NOESYPH experiments in CDC13 at 303" K.

342

ITOKAWA ET AL.

ukune as minor constituents, were shown to have y-turns at residues 2, 3, and 4,which were stabilized by a hydrogen bond between Ser2-OH and D-Alal-CO (Fig. 31) (153). In related work, the glycopeptide RA-XI1 also has been shown to bind to 80s ribosomes (154). In NMR titration experiments, line broadening in the 'H-NMR spectrum for RA-XI1 was observed upon addition of a small quantity of 80s ribosomes to the NMR sample. Selective binding of the major conformer of the peptide was also observed. c. Molecular Modeling. To obtain details of structure and conformation

that agree more closely with the NMR data, calculations of molecular dynamics, starting with the X-ray structure and applying the distance constraints obtained from the NOE experiments, were performed (143). The final structures obtained after several such calculations were examined for their overall energetic favorability, compared with the structure derived from the NMR data, and classified into two conformers, corresponding to conformers A and B deduced by the NMR study (Fig. 32). Conformer A contains Ala2 in the i + 1 and Ty? in the i + 2 positions with stabilization of the transannular H-bridge between Ala4-NH and A1a'-CO. The solution structure of the minor conformer B of RA-VII has not been determined, because no sufficient NOE values are available. This simulation indicated that the type I1 @-turn involving Ala2 and Tyr3 is converted to a type VI @-turn by the NOE enhancements between Ala2-Ha and Tyr3-Ha, suggesting a cis peptide bond.

Q

Ftc. 31. Stereoscopic drawing of the major conformer in RAI-111 (176).

MACROCYCLIC PEITIDE ALKALOIDS FROM PLANTS

343

FIG.32. Perspective view of two stable conformers corresponding to conformers A and B.

A conformational search of RA-VII using Monte Carlo simulation was conducted by Boger et al. (148). Using the OPLSA force field, the three lowest energy conformations corresponded to conformer A, conformer B (AE = 1.3 kcal/mol), and conformer C ( A E = 2.4 kcal/mol) detected by 'H-NMR and paralleled the relative stabilities of the three conformational isomers detected in DMSO-& (149). The conformation of a cyclic bouvardin analog, cycle(-D-Ala-ProMeTyr-Ala-MeTyr-MeTyr-) (189),has been determined by distance geometry cakulation and restrained energy minimization from NMR data (Fig. 33) (155).Calculations done on the major conformer revealed a unique backbone conformation, consisting of two p-turns, a type I1 p-turn at ProMeTyr and a type VI p-turn at MeTyr4-MeTyr'. In this analog, with a proline residue at position 2, the lack of a 14-membered ring formed by the oxidative c mpling of the phenolic oxygen did not affect the backbone conformation I the 18-membered ring. However, an analog with an alanhe residue at position 2 and lacking the 14-membered ring strongly influenced the population n solution (143).

5. Correlation bptween Structures and Biological Activities

A number of analogs of these peptides have been synthesized and biologically evaluated. Y e results suggest some insights into the structure-activity relationships and identification of the pharmacophore for these peptides.

344

ITOKAWA ET AL.

k

P

FIG.33. The conformer of cycle(-D-Ala-Pro-MeTyr-Ala-MeTyr-MeTyr-) (189) with the lowest residual distance violations.

The analogs 190-195 in which the 14-membered cycloisodityrosine moiety was modified were designed and synthesized (156-261). These analogs showed little or no cytotoxicity (Table IV). Since even analog 195, possessing conformational properties similar to the most stable conformation of the natural peptides, showed very weak cytotoxicity,the tetrapeptide moiety (DAla-1-Ala-2-Tyr-3-Ala-4) appeared not to be essential for the activity. TABLE IV CYTOTOXICITY OF RA ANALOGS AGAINST L1210 CELLS' Compound (pg/ml) 166 (RA-VII) 190 191 192 193 194 195 1% 197 198 199 200 a

Refs. 157, 159, 162.

1C.w 0.002

>loo >loo

20

> 10 25 50 0.06 0.03 >lo0 >lo0

>lo0

MACROCYCLIC PEITIDE ALKALOIDS FROM PLANTS

345

Q -9 OH

OH 1so

OH 192

193

196R=H 197 R =Me

198 R' = R2 = H 199 R' = H, R2=Me 200 R' CO&Ie. R' = H

-

Based on these observations and in conjunction with reports regarding cycloisodityrosine analogs 196 and 197 showing fairly potent cytotoxicity, the 14-membered cycloisodityrosine moiety is currently considered to be the pharmacophoric unit for this class of natural peptides (160). However, more simplified analogs 198-200 showed no activity, which defined the C12 amine substituent as also playing an essential role for activity (161). Peptide 166 possesses two methoxy groups on the aromatic ring of the Tyr-3 and Tyr-6 residues. Substitution of the methoxy group at the Tyr-6 residue by a hydrogen atom, as in 204, or a hydroxyl group, as in 164, causes

346

ITOKAWA ET AL.

little effect on the cytotoxicity (Table V) (163). However, when the methoxy group at the Tyr-3 residue is substituted by a hydrogen atom (202 and 203) or a hydroxy group (201 and 205), their cytotoxicity is reduced -100 to 166 R' = R2 = OMe (RA-VII) 201R'=R2=OH 202 R' = R~ m H 203 R' = OH, RZ = H 164 R' = OH, R2 = OMe (RA-V) 204 R' = H, R2 = OMe 208 R' = H, R~ = OH 208 R' =OMe, R 2 = H 207 R' = OMe, R2 = Me 208 R' .IOMe, R2 IEt 209 R' = OMe. R2 = Pr" 210 R' = OMe, R2 =vinyl 211 R' = OMe, R2 = ally1

Me--

\

R'

1000times. Thus, this methoxy group appears to be essential for the activity. However, since the C-alkyl analogs 208-211 showed rather weaker activity than 166, but still significant activity both in vitro (Table V) and in vivo (Table VI), it can be concluded that this appendage moiety is important for the activity, although the ether linkage is not essential (264). CYTOTOXICITV OF

TABLE V RA ANALOGS AGAINST P388 AND KB CELLS'

Compound

P388

166 (RA-VII) 201 202

0.0013 >10 0.37

203

0.031

164 (RA-V) 24M 205 206

0.0027 0.0025

207 208 209 210 211 a

Refs. 163, 164. n.t.: Not tested.

>10

0.22 0.018 0.0072 0.020 0.013 0.0039

KB 0.0023 7.8 0.84 0.36 0.0038

0.0063 >10 0.42 n.teb n.t. n.t. n.t. n.t.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

347

TABLE VI OF RA-VII ANALOGS AGAINST P388 LEUKEMIA ANTITUMOR ACTIVITY IN MICE' TIC (%) #/Doseb:

0.4

0.8

1.6

3.13

6.25

166 (RA-VII) 206 257 208

144 92 105 108 109 100 102

144

152 100 121 120 121 110 130

163

toxic 101 149 151 141 127 130

u)9

210 211 a

Ref. 164. Dose in mg/kg given i.p. on days 1-5.

Since RA-V (164)possesses a reactive phenolic hydroxyl group on the Tyr-6 residue, its derivatization has been extensively studied. A number of alkyl and acyl groups were introduced into this position. Some examples of their antitumor activities are listed in Table VII (165). Interestingly, analogs possessing a rather long alkyl or an acyl group retained potent antitumor activity, which suggests that modification at this position may lead to more biologically promising analogs than for peptide 166. TABLE VII OF ETHER A N D ESTER DERIVATIVES OF RA-V AGAINST P-388 ANTITUMOR ACTIVITY LEUKEMIA IN MICE

T/C (%)

Compound 164 (RA-V) 166 (RA-VII) 212 213 214 215 216 217 218 219 220 221 222 a

Doseb:

0.05

0.5

2.0

4.0

10.0

131.1 138.6 137.3 138.4 110.3 112.5 115.4 133.3 124.1 133.6 126.7 122.0 127.8

152.5 156.7 165.4 146.0 137.3 141.5 108.7 155.6 148.5 143.2 146.7 146.3 185.6

164.2 164.2 162.2 93.7 153.5 150.1 121.2 168.9 162.3 151.6 166.7 151.6 175.6

165.3 173.6 toxic toxic 173.0 155.4 123.1

187.4

Ref. 165. Dose in rng/kg given i.p. on days 1-5.

194.7 189.6 197.2 168.9 150.6 183.3

348

ITOKAWA ET AL.

R 104 H 186 Me 212 Et 213 Pli 214 (CHz)&H3 216 (CHz)&Ha 218 (CHz)ioCH3 217 Ac 218 COCH=CHCHa 219 CO(CH*)&H3 220 CO(CHz)&H3 221 CO(CH&CH3 222 COPh

Various Ala-2 modified analogs of 166 have been synthesized from RAI11 (162) and RA-X methyl ester (223) (166,167). Comparison of their cytotoxicity reveals some tendencies between the side chain structure and the activity (Table VIII). Compounds that possess a polar functionality at TABLE VIII OF RA ANALOGS AGAINST P388 CYTOTOXICITY KB CELLS"

AND

(pdml) Compound

P388

KB

166 (RA-VII)

0.0013

0.0023

162 (RA-111) 223 (RA-X-OMe) 224 225 226 227 228 229 230 231 232 233 234 235 236 237

0.011

0.024 0.030

238 239 a

Ref. 166.

0.034 0.048 0.14 0.18

0.052

0.084

0.14

0.020

0.042

0.031 0.011 0.027 0.99 0.017 0.030 >10 0.0083

0.059 0.019 0.031 1.9 0.040 0.013 >10 0.0060 0.37

0.52

0.29 0.079 0.079

0.31

0.014 0.21

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

349

the side chain showed reduced activity, especially in the case of ornithine (234)and aspartic acid (231)derivatives. In this connection, it is noteworthy that homoserine (224)and 8-hydroxynorleucine (226)derivatives are less toxic than norvaline (229)and methionine (232)derivatives, respectively, having a nonpolar residue with similar length. Another clear relationship exists between the length of the residue and cytotoxicity. In a series of compounds having a hydroxyl group at the end of the side chain, those cytotoxicities are decreased as the carbon chain is lengthened (162 > 224 > 225 > 226). The same tendencies are also observed among other homologs (e.g., 227 > 228; 166 > 229). The observation, however, that

Me-

-

0

OMe

162 CH2OH 223 CH2CH2COzMe 224 (CH2)zOH 226 (CH2)30H 226 (CH2)PH 227 CHsCH2 228 CH&H=CH2 229 CHzCH2CH3 230 CH2CHO 231 CH2CO2H 232 CHzCH2SCH3 2 s (CH2)3N3 234 (CH&NH2 236 CH~SAC 236 CH2S)r 237 CH2SH

the azido intermediate 233, having a rather long residue, shows effective cytotoxicity suggests that a lengthy side chain can be compatible with the activity in the case of a less polar functionarity. As mentioned in the previous section, peptide 166 exists in two or three stable conformational states in solution. However, the proline analog 238

238n=1 238n=2

bMe

350

ITOKAWA ET AL.

showed only one conformation in various solvents, and extensive NMR experiments revealed that this conformation is very similar to the major conformer of peptide 166. Since analog 238 showed potent cytotoxicity, it is concluded that the major conformation of peptide 166 is at least in part responsible for the activity. The pipecolic acid analog 239 showed similar conformational and biological tendencies.

166 R‘ = R2 = R3 .IMe 240 R’ = H, R’ = R3 = Me 241 R‘ = R2 = Me, R3= H 242 R’ R3= Me, R2 .IH 243 R’ -Me, RP = R3 = H 244 R’ = R3 = H, R2 = Me 248 R’ = R2 = H, R3 Me 248 R’ IR2 R 3 = H

Me- -

N-Desmethyl derivatives 240-246 of RA-VII (166)were synthesized and evaluated using L1210 cells (168). Analogs 242, 243, 245, and 246 were found to be biologically inactive (IC~O >10 pg/ml), whereas 240 and 241 were essentially equipotent with 166 (0.0007-0.002 pg/ml). 1D and 2D ‘HNMR studies of these analogs revealed the role of N-methylation in the R

R 166 H 247 Me

262

240 Et 249 (CH&CH3

263

260 (CH2)3CH3 261 (CH*)&H3

264 265 266

268

+

35 1

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

key conformational aspects of the natural agents. The N-methyl group of the Tyr-5 residue is essential for the maintenance of the conformational and biological properties of peptide 166; the N-methyl group of the Tyr-3 residue is not essential, and its removal leads to exclusive population of a single, biologically active conformation. Removal of the N-methyl group of the Tyr-6 residue does not alter the conformational or biological properties of peptide 166. Although a scanty foothold for chemical manipulation of the molecule hampered derivatization of peptide 166, the Ala-2 amide nitrogen proved to be effectively alkylated with reactive alkylating agents under selected conditions. Analogs 247-283 were prepared by direct alkylation or further derivatization (169-171). Simple alkyl analogs 247-258 retained potent cytotoxicity, and some of them (e.g., 256 and 257) showed more promising antitumor activity than peptide 166 in terms of maximum T/C values. (See Tables IX and XI.) Although the cytotoxicity of the amine analogs 259-283, which are believed to improve the water solubility of the peptide, was variable among TABLE IX

CYTOTOXICITY OF RA ANALOGS AGAINST P388 AND KB CELLS" 1 6 0 (pdml)

IC50

Compound

P388

KB

Compound

P388

KB

166 (RA-VII) 247 248 249

0.0013 0.0012 0.035 0.0032 0.010

0.0023 0.0077 0.035 0.0097 0.024

265 266 267 268 269

0.015 0.12 0.071 0.031 0.032

0.034 0.49 0.081 0.060 0.19

0.018 0.015 0.0076 0.010 0.0090

0.063 0.013 0.018 0.022 0.027 0.0064 0.062 0.030 0.19 0.46 0.40

270 271 272 273 274 275 276 277 278 279 280 281 282 283

0.11 0.065 0.74 0.039 0.11 0.036 0.029 0.033 0.025 0.021 0.084 0.30 0.088 1.4

0.19 0.072 0.56 0.16 0.48 0.12 0.073 0.099 0.057 0.051 0.23 0.43 0.21 1.7

250 251 252 253

254 255 256 257

0.0058

258 259 260 261 262 263 264 a

Refs. 169-171.

0.044 0.0094 0.12 0.21 0.25 0.19 0.13 0.19

0.48

0.70 1.1

352

ITOKAWA ET AL.

274

276

u

- N n N e

OMe

278

277

278

279

280

281

-N3N3

282

283

each analog, some of them showed in vivo antitumor activity; the activity, however, was weaker than that of the parent peptide 166. When these peptides were treated with thionating reagents, thioamides 284-292 were obtained (172,173). They retained potent cytotoxicity, and monothioamides at Tyr-3 residue showed 2-4 times more potent activity than the parent analogs (see Table X).NMR studies revealed that these thionated analogs showed very similar conformational properties to the parent peptides in solution. Nickel borohydride reduction of thioamide 284 gave desoxopeptide 293 and its borane complex 294. Analog 293 showed very different conformational properties around the 18-membered ring moiety to peptide 166 both in the crystal and in solution, which may explain

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

353

TABLE X R A THIOAMIDE ANALOGS AND THEIR CYTOTOXICITY AGAINST P-388 CELLS" (172, 173) ~ _ _ _ _

No.

R'

166 284

Me Me Me Me Me Me H H Me Me Me Me Me Me Me Me

285 286 287 288 164

289 223 290

252 291 253 292

293 294'

~

R3

W

H H H H H H H H H H ally1 ally1 crotyl

0

R2

H H H H H H H H CH2C02Me CH2C02Me

H H H H H H

0 0 0 s o

0

0

0

0

0

crotyl

H H

X 0

0 0 0 0 s

Y

Z

ICs0 ( p g h l )

0

0 0 S

0.0013 0.00058 0.0017 0.0026 0.0044 0.0013 0.0027 0.00088 0.034 0.0041 0.015 0.0038 0.0076 0.0032 >10 1.0

s s 0 s s

0

0

0

s

0

0

0

s

0

0

0

0

s

0 0 0 O

0 0 0

0

O

s

s

H2

H

z

0 0 0 0 0 0 0 0 0 0 0 O

~

a

Refs. 172, 173. B Y complex at Ala-4 NH.

Me- -

tw >Me

NH

0

OR'

its loss of activity. Although complex 294 gradually decomposed to compound 293 under the assay conditions, the borane complex 294, possessing a similar conformation to peptide 166, showed only weak activity. These results emphasize the importance of the 18-membered ring structure for the full expression of biological activity. The importance of the 18-membered ring moiety for the activity was also verified by synthesizing the simpified analogs 295 and 296, which

354

ITOKAWA ET AL.

Me-

-

0

OMe

OM0 296

297

showed very weak (L1210, ICso = 2 pg/ml for 295) or no (>lo pg/ml for 2%) cytotoxic activity ( I 74). Although backbone modified analogs that possess very different conformational aspects from the major conformer of peptide 166 generally lose activity, the analog 297, possessing a 19membered ring structure, showed good activity both in vitro (P388, ICs0 = 0.019 puglml) and in vivo (Table XI) (275).

ANTITUMOR Acrivm

#/Doseb:

0.4

OF

TABLE XI RA-VII ANALOGS AGAINST P-388 LEUKEMIA IN MICE"

0.8

1.6

3.13

6.25

12.5

25.0

~~

166 (RA-VII) 253 254 256 257 265 268

276 278 282 297 a

144 112 113 123 119 111 105

144

105 133 Refs. 170, 171, 175. Dose in mg/kg given i.p. on days 1-5.

152 127 125 132 131 117 105 111 119 111

163

toxic

155 155 160 148 128 129 118 137 127 158

174 164 141 143 136 133 130 176

170

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

355

B. ASTINS FROM Aster tataricus 1. Structures of the Astins

The first member of the astin family of cyclic pentapeptides containing a dichlorinated proline residue was isolated in 1993 from the biologically active extracts of the roots of Aster tataricus (Compositae) (176,177).Aster tataricus is known as a Chinese medicine containing several terpenoids and saponins, and is also popular as an ornamental flower (178). The n-butanol extract of this plant showed potent antitumor activity. Chromatographic purification guided by antitumor activity led to the isolation of three new cyclic pentapeptide: astins A-C (298-300) (177). Their structures, containing a novel dichlorinated proline residue, were determined by 2D-NMR and FAB-MS spectroscopy, as well as by degradation, followed by HPLC analysis using Marfey’s method (Fig. 34) (179). Astin A is constructed of L-do-threonine, L-serine (Ser), P-phenylalanine (P-Phe), L-a-aminobutyric acid (Abu), and L-P,y-dichlorinatedproline (Pro(Clz)) residues. ah-Threo nines, though rare in nature, are found as constituents of biologically active peptides (180,181). Astin A was sequenced based on HMBC correlations and MS fragmentation pattern (177,182). Later, cyclic astins D-I (301-306) were isolated as minor constituents and subsequently characterized using degradation and NMR experiments A4”Pro(C1)’

c1

R1 AstinD(301): H E(302): OH H(305): H

Kl R2

H

AbuZ

H

OH

FIG.34. Structures of astins A-I (298-306),and some important HMBC correlations; Pro was provisionally numbered as the first amino acid. Arrows show HMBC correlations.

356

ITOKAWA ET AL.

(183-185). Cyclic astins possess several different types of unique chlorinated proline residues. A @,y-dichlorinatedproline is contained in astins A, B, and C, a A4(5),&chlorinated proline in astins D, E, and H, a @-chlorinated proline in astin F,and a @-hydroxy-y-chlorinatedproline in astin I. All of the chlorine and hydroxy group configurations in these proline residues were elucidated to be &orientated by NOEs, 'H coupling, and HMBC correlations, as shown in Fig. 35. Furthermore, an acyclic astin J (307) has been isolated (186). Cheng et al. reported this kind of acyclic peptide, the asterinins A-C, from the same plant (287). Base-catalyzed cleavage of astins A, B, and C with a @,ydichlorinated proline resulted in acyclic peptides with pyrrole rings, which were considered to be produced by dechlorination and aromatization from Pro(Clz) to pyrrole under basic conditions, following the cleavage of the amide bond in Pro. The acyclic astin B was also produced from the cyclic astin B by hepatic microsomal biotransformation in rats (Fig. 36) (188). Currently, synthetic studies of all of the astins are in progress (189,190), although total synthesis of astins A-C with the cis @,y-dichlorinatedproline has yet to be achieved.

rc 298

301

303

FIG.35. NOE, HMBC,and coupling correlations around Pro residues of 298,301,303, and 306.Arrows show NOEs, dashed arrows show HMBC, and numbers show coupling constants.

MACROCYCLIC PEPITDE ALKALOIDS FROM PLANTS

Astio C (300)

357

Astio J (3W)

FIG.36. Hepatic microsomal biotransformation of astin C (300)to astin J (307).

2. Conformational Analysis

A crystal structure of astin B (299) has been reported (Fig. 37) (282). The solid-state conformation exhibits one cis peptide bond between Abu’ and Pro’. This structural feature is the main difference among the solidstate structures of the astins, cyclochlorotine (292), and islanditoxin (292). The latter two compounds are toxic metabolites of yellow rice mold, Penicillium islandicum spp., whose occurrence on a variety of foodstuffs constitutes a human health hazard. In cyclochlorotine, only the astin Abu’ residue is replaced with serine, yet the molecule adopts a stable type I &turn conformation with a trans proline amide bond and a transannular hydrogen bond (292).

FIG.37. Perspective view of the crystal structure of astin B (299) by PLUTO drawing. Each number refers to the carbon, oxygen, nitrogen, and chlorine atoms of 299.

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ITOKAWA ET AL.

Solution conformational analysis of astin B in DMSO-d6 based on 2DNMR techniques, temperature effects on NH protons, rate of hydrogendeuterium exchange, vicinal NH-CaH coupling constants, and NOE experiments has been reported (Fig. 38) (293). Calculations of molecular mechanics and restrained molecular dynamics were applied to determine the energetic preferences of various conformations of astin B. Distances involving the three intramolecular hydrogen bonds and NOE correlations were used for the refinements using the AMBER program. These results indicated that the conformation in solution was, on the whole, homologous to that observed in the solid state. Astin B, with a cis configuration in a proline amide bond, differed from cyclochlorotine isolated from Penicillium islandicum, which showed an all-trans amide configuration. Furthermore, NMR and molecular dynamics studies suggested that astin B assumed a different backbone conformation from those of astins A and C (294). 3. Biological Activities

Astins A-C, containing chlorines and an ah-Thr and characterized by one cis peptide bond, showed antitumor activity, as determined by the total packed cell volume method using Sarcoma 180 ascites in mice (295).The effectiveness of this activity was evaluated in terms of the tumor growth ratio (GR(%) = (test group packed cell volumekontrol group packed cell volume) X 100). The GR values of astins A, B, and C were 40% (++) at

FIG.38. NOE enhancements of astin B (299). Arrows show the NOE relationships confirmed by a phase-sensitiveNOESY experiment in DMSO-dh at 303" K.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

359

a dose of 0.5 mg/kg/day, 26% (++) at 0.5 mg/kg/day, and 45% (+) at 5 mgkglday, respectively, for 5 consecutive days (277,282).The effective doses of astins A and B were 10-fold stronger than that of astin C. The other astins D-J did not inhibit tumor growth at 10.0 mg/kg/day (288). Various congeners without dichlorinated proline residues, prepared from astins A-C by chemical conversion and hepatic microsomal biotransformation in rats, did not show antitumor activities either, suggesting that the 12-5s dichlorinated proline residues play an important role in the antitumor activity of the astins. Itokawa et al. suggested that astins A and C, with weaker activity than astin B, have different backbone conformations from astin B, and that these backbone conformations affect antitumor activity (294). However, the presence of cis dichlorinated proline residues was concluded to be a more important structural motif for astins to show antitumor activity on S-180A. The backbone conformational difference between astin B and astin C (294)was maintained by backbone modification using Lawesson’s reagents. The produced thionated derivatives, [Ser-3-dCS-NH)-@-Phe-4]astin A (thioastin A), [Ser-3-dCS-NH)-P-Phe-4]astin B (thioastin B) and [Ser3-+(CS-NH)-@-Phe-4]astin C (thioastin C), showed more promising antitumor activity than the corresponding astins (196,197). Although there are few differences between the peptide sequences of the astins and those of cyclochlorotine and islanditoxin, astins exhibited antitumor activity, whereas only hepatotoxicity was shown by cyclochlorotine and islanditoxin (277). The fact that minor structural changes cause such a noticeable change in biological activity in both the astins and the toxins is of interest. C. PSEUDOSTELLARINS FROM Pseudostellaria heterophylla

1. Structures and Biological Activities of Pseudostellarins The series of cyclic peptides known as pseudostellarins was originally isolated from the roots of Pseudostellaria heterophylla (Caryophyllaceae) and showed tyrosinase-inhibitory, melanin-formation-inhibitory, and cellgrowth-inhibitory activities (298-201). The roots of P. heterophylla are a well-known Chinese traditional medicine used as a lung and spleen tonic (202). Tyrosinase inhibitors may control overproduction of the dermal melanin pigment because tyrosinase, which is a bifunctional copper protein widely distributed in animals and plants, plays an important role in the process of melanin biosynthesis (203). The structures of pseudostellarins A (308) through H (315) are shown in Fig. 39. Ning-Hua et al. reported the structure of the cyclic peptides, heterophyllin A [cycEo(-Gly-Ile-Thr-

pseudostellasinA (308)

psndosrellarin E (312)

pseudostellarin B (309)

pseudosrellarin F (313)

psndosrellsrinC (310)

pseudosteUarin G (314)

pseudostellatin H (315)

FIG.39. Structures of cyclic peptides, pseudostellarins A-H (308-315) from Pseudostellaria heterophylla.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

361

Pro-Val-Ile-Phe-)] and heterophyllin B [cycle(-Gly-Gly-Leu-ProPro-Pro-Ile-Phe-)] (204). These peptides are cyclic penta-, hepta-, octa-, and nonapeptides. The structures of pseudostellarins and heterophyllins were determined by chemical degradation, 2D-NMR methods such as 'H-'H COSY, HOHAHA, NOESY, ROESY, HMQC, and HMBC, enzymatic methods, and ESI-MS/MS analysis. The sequencing of pseudostellarins C (310) and D (311),proposed earlier was revised as shown in Fig. 39 by X-ray crystallographic and NMR analysis (205,206).

2. Conformational Analysis Though the number and arrangement of the different amino acids varied among the pseudostellarins, they all displayed the same enzyme-inhibitory activity toward tyrosinase. Cyclic oligopeptides are often used as experimental models for the study of structure-biological activity relationships, because their cyclic structure limits the conformational flexibility of the peptide backbones. To elucidate the mechanisms involved in the action of these cyclic peptides, details of their conformational characteristics are required. To deduce the 3D requirements to show the activity of pseudostellarins A and D, conformational analysis was conducted using high-field NMR studies, computational chemical evidence, and X-ray crystallographic analysis. Pseudostellarin A is characterized by y- and p-turns, fixed by transannular hydrogen bonds between Gly and Leu (207).The crystal form of pseudostellarin D (311) was analyzed by X-ray crystallography (Fig. 40) (205). A single-crystalX-ray analysis showed that pseudostellarin D possesses a type I1 &turn between Leu7 and Gly', and a type I &turn between Pro4 and Leu'. One transannular 4 + 1 hydrogen bond between Ile6-NH and Gly3CO, and two bifurcated hydrogen bonds between Tyr2-NH and Ile6-CO and between Gly3-NH and Ile6-CO were also observed, forming a classical P-bulge unit in the crystal. The dominant conformation in solution, as analyzed by high-field NMR employing vicinal coupling constants, temperature dependence of NH protons, and the ROESY spectrum was, on the whole, homologous to that observed in the solid state. The solution form of pseudostellarin D in DMSO-d6 was confirmed by restrained molecular dynamics calculation in vacuo using an AMBER* force field and Monte Carlo simulation study implemented in MacroModeYBatchmin (206). Conformation may relate closely to mode of action in the pseudostellarins, which probably interact with specific receptor sites, although a suitable pharmacophore model has yet to be proposed. Details as to the precise backbone conformation of other pseudostellarins may be required.

362

ITOKAWA ET AL.

FIG.40. ORTEP perspective view of the crystal structure of pseudostellarin D (311). Only amide and hydrogens (small circles) are included; dashed lines indicate intramolecular hydrogen bonds. The disordered atom (C64’) was omitted.

D. YUNNANINS FROM Stellaria yunnanensis

Stellaria yunnanensis Franch (Caryophyllaceae), which is only distributed in Yunnan Province, China, has been used as a lung tonic. The roots of Stellaria yunnanensis contain several cyclic peptides: yunnanins A-F (316-321)(208-220), whose structures are shown in Fig. 41. The structures of these cyclic hexa-, hepta-, and octapeptides were determined by spectroscopic evidence and chemical degradation (208-210). Both yunnanins B and E contain Shydroxyisoleucine, an unusual amino acid. A few peptides containing a y-hydroxyisoleucine, such as y-amanitin (221,222),have been reported however, none containing a 8-hydroxy isoleucine residue has been detected. To date, several naturally occurring cyclic heptapeptides have been reported, such as ilamycin B1(223),a dolastatin 3 analog (224),cycloheptasarcosine (225), rhizonin A (226), evolidine ( 2 1 3 , hymenamide (228), and phakellistatin (229). The presence of cyclic heptapeptides is very rare in nature. Yunnanins A, C, and D showed cell-growth-inhibitory activity

yunnanin A (316)

yunnanin D (319)

yunnanin B (317)

yUnnanin C (318)

yunnanin E (320)

yunnaOin F (321)

FIG.41. Structures of yunnanins A-F (316-321).

364

ITOKAWA ET AL.

TABLE XI1 STRUCTURESOF STELLARINS D-G Stellarin D: Stellarin E: Stellarin F Stellarin G:

cyclo (-Gly-Tyr-Leu-Phe-Pro-Ile-Pro-) cyclo (-Gly-Ile-Pro-Tyr-I1e-Ala-Ala-) cyclo (-Gly-Ma-Gly-Ser-Pro-Trp-Phe-Pro-) cyclo (-Gly-Ala-Tyr-Leu-Ala-)

yunnanin A: 2.1 pg/ml; yunagainst P388 lymphocytic leukemia cells nanin C: 2.2 pg/ml; yunnanin D: 2.1 pglml). Zhao et al. reported on the structures of several cyclic peptides, stellarins A-G, all from the same plant (220-223). The structures of stellarins A, B, and C correspond to those of yunnanins A, B, and E, whereas the structures of stellarins D, E, F, and G are listed in Table XII.

E. SEGETALINS FROM Vaccaria segetalis 1. Structures and Biological Activities of Segetalins

The seeds of Vaccaria segetalis (Caryophyllaceae) have been used to stimulate blood flow and promote milk secretion, and also to treat amenorrhea and breast infections in China (224). Extraction and purification of chemical constituents showing follicle hormonic activity led to the isolation of the new, bioactive cyclic peptides segetalins A-H (322-329) (225-228). Some segetalins showed potent estrogen-like activity. Although some cyclic peptides such as oxytocin and vasopressin show hormone activities, none show estrogen-like activity. The structures of segetalins A-H (322-329), which are cyclic penta-, hexa-, hepta-, and nonapeptides, are shown in Fig. 42. The molecular formula of segetalin A (322)is C31H43N706. IR and UV absorptions indicated that it is peptidic and contains an indole skeleton. Amino acid analysis of 322 showed the presence of Gly, Ala, Val X 2, Pro, and Trp, all of which were confirmed to be of the L-configuration by Marfey's method. Extensive 2D-NMR analyses, including COSY and HMQC spectra, were used to determine the identity of the six amino acids and to assign the 'H and 13C signals. The sequence of the cyclic peptide was established based on data from an HMBC experiment. As can be seen from Fig. 43, the whole structure was deduced to be cycle(-Gly-Val-ProVal-Trp-Ala-). This was confirmed by ESI-MSIMS (229,230) of an acyclic peptide 330 generated by digestion of 322 with a-chymotrypsin (Fig. 44) (225). The structures of the other segetalins were elucidated based on

MACROCYCLIC PEITIDE ALKALOIDS FROM PLANTS

365

extensive two-dimensional NMR methods, chemical degradation, and ESIMSMS spectra (226-228) as in 322. When the MeOH extract, the EtOAc extract, and segetalins A, B, G, and H were administered to rats for 14 consecutive days, the weight of the uterus increased dose-dependently (231).This estrogenic activity was also confirmed by recording the oxytocin-induced uterine contractions (232). Although isoflavonoids are well known as phytoestrogenic substances (233), they could not be detected in any of the fractions. It has been clarified that the cyclic peptides segetalins A, B, G, and H, which were isolated as active principles from V. segetalis, showed estrogen-like activity in vivo. However, these peptides did not affect follicle-stimulationhormone (FSH), luteinizing hormone (LH), or prolactin activities (231). 2. Conformational Analysis To elucidate the mechanisms of biological action of segetalin A and/or the biologically active conformation and orientation within the hormone binding site, details of its conformational characteristics are required. In addition, on the basis of detailed and exact knowledge of the structure of segetalin A in both crystal and solution under different environmental conditions, the structure-activity relationships may be confidently discussed. The conformation of segetalin A (322), both in the solid state and in solution, was investigated by X-ray analysis, high-field NMR, and computational chemical methods with the aim of developing a pharmacophore model of an estrogen-like cyclic hexapeptide (234). The crystal structure of 322 contains two intramolecular NH.a.0 hydrogen bonds between Gly'-NH and Val4-CO, and between Val4-NH and Glyl-CO [HNl---04 of 2.12 A {Nl---04 3.01(1) A} and HN4---01 of 1.93 A {N4---01 2.85(1) A}] (Fig. 45). These hydrogen bonds make an antiparallel &sheet structure. As a consequence, two &turns are formed by Trp5 and Ala6, and Val2 and Pro3, at the two corners. The TrpS+ Ala6 turn is a type I p-turn formed by the intramolecular hydrogen bond between G1y'-NH and Val4-CO. The other turn, Val2 + Pro3, serves a type VI pturn involving a cis proline amide bond. Results of NMR analysis,such as temperature dependence and deuterium exchange, vicinal coupling, ROE enhancement, and relaxation time, suggested that segetalin A adopts a different backbone conformation in a crystalline environment than in solution. In addition, molecular dynamics simulation indicated that in solution, segetalin A has a type I1 &turn between Trp5 and Ala6, as opposed to the type I /3-turn of the crystal form, and a type VI p-turn between Val2and Pro3,as in the crystal. It is of interest that the conformational energy needed to convert the X-ray backbone into the NMR backbone is relatively small. It is highly probable that 322 in

Tn,

Phe Ala Ala

$HH

"f.

HHyq Val

Pm

0

Val Segetalin A (322)

Gly

His

Segetalin 6 (323)

Segetalin C (324)

914

Pro

Leu Ser

'

Leu

Segetalin D (325)

Trp

Segetalii E (326)

Tyr

sa Ah

sa

v pbc

Segemlin F (327) ebc

sa:

H2

SegetalinH (329)

FIG.42. Structures of segetalins A-H (322-329) from the seeds of Vuccuria segetulis.

368

ITOKAWA ET AL.

A

Trp

Ala

FIG.43. Structure of segetalin A (322). Arrows show selected HMBC correlations.

solution contains two @-turns(type I1 and VI @-turns).These findings rnay relate to the mode of action of segetalin A, which probably interacts with specific receptor sites. The conformations of segetalins B (323), G (328), and H (329), which show estrogen-like activity in solution, were also analyzed by NMR and computational methods (distance geometry calculations) (235).To investigate the 3D requirements necessary for estrogen-like activity, superposition using DISCO was undertaken. Each cyclic peptide was suggested to possess a partial conformation similar to that of segetalin A in the region of TrpAla-Gly-Val (Fig. 46). Sequence and conformation may play an important role in the activity.

322

330

FIG.44. ESI MSMS fragmentation of segetalin A (322 and 330).

FIG.45. ORTEP perspective view of the crystal structure of 322.

FIG.46. Proposed pharmacophore model for estrogen-like activity of segeralins.

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ITOKAWA ET AL.

F. CYCLIC PEPTIDES FROM Stellaria dichotoma L. VAR. lanceolata BGE.AND Stellaria delavayi Some of the medicinal plants belonging to the Caryophyllaceae family contain cyclic peptides. This is rare in higher plants. The roots of Stellaria dichotoma L. var. lanceolata Bge. have been used in folk medicine as an antifebrile agent. A series of dichotomins, cyclic penta- and hexapeptides, have been isolated from the roots of S. dichotoma L. var. lunceolutu, and the structures of dichotomins A-E (331-335), as shown in Fig. 47, were established on the basis of extensive 2D-NMR, chemical degradation, and X-ray crystallographic analysis (236,237).Liu et al. also reported the structure of a cyclic hexapeptide, named stellaria cyclopeptide, cyclo(-Gly-GlyAla-Ala-Val-Tyr-), isolated from the same plant (238). Dichotomins A, B, C, and E showed cell growth inhibitory activities against p388 lymphocytic leukemia cells (I& A: 2.5 pg/ml; B: 3.5 pg/ml; C 5.0 pg/ml; E: 2.0 pg/ml); dichotomin D did not. The amino acid sequences of dichotomins A, B, and C are the same, except at residue 5 (dichotomins A: Val; B: Thr; C: Ala). The cyclic pentapeptide, dichotomin E, contains a sequence (Tyr-Ala-Phe) similar to the Phe-Leu-Tyr sequence present in dichotomins A, B, and C, the central aliphatic amino acid of which is sandwiched between two aromatic amino acids. This inhibitory activity may be due to the sequencing andlor conformation of these dichotomins. In addition, dichotomin D showed moderate cyclooxygenase inhibitory activity (72.6%inhibition at 100 pM),whereas dichotomin A did not show any activity. This may also be due to differences in sequencing andlor conformation. As noted earlier, the conformational features of cyclic peptides are known to be relevant to their biological activities. In the crystal form of dichotomin A, the @-turnsand system of intramolecular hydrogen bonds restrain the 18-membered cyclic frame (Fig. 48). The end of the molecule is constrained by two @-turnsformed by the residues from Val6 to Gly' and from Phe3 to Leu4. The former is denoted as type I1 [Val6 4, $ (-57.9, 130.5); Gly' 4,$ (90, -9.6)], with the intramolecular hydrogen bond between Thr2-NH and TytS-CO, and the other type I [Phe3 4, $ (-76.7, -11.8); Leu4 4, II/ (-114.8, -12.6)] without a transannular intramolecular hydrogen bond. In addition, a side-chain-main-chain interaction has been observed between the backbone NH group of Leu4and the side-chain oxygen of Thr*. The side chain of Th? is directed toward the interior, and significant intramolecular NH.-.O hydrogen bonding exists between Thr2-0 and Leu4-NH. Obviously, because of geometric constraints, an intramolecular hydrogen bond between Thr2-CO and Tyr5-NH cannot occur simultaneously in this system.

MACROCYCLIC PEPTIDE ALKALOIDS FROM PLANTS

37 1

Dichotomins F (336), G (337), H (338), and I (339) were also isolated as minor constituents, and their structures were clarified by 2D-NMR and ESI MSMS analyses (239,240) (see Fig. 47). As a result of the investigation of Stellaria delavayi, which also belongs to the family Caryophyllaceae, six new cyclic peptides, delavayins A-C (340-342) (241) (Fig. 49) and stelladelins A-C (242,243) [stelladelin A: cyclo(-Gly-Pro-Pro-Pro-Leu-Leu-Gly-Pro-Pro-Tyr-Tyr-); stelladelin B: cyclo(-Gly-Ile-Pro-Pro-Ala-Tyr-Asp-Leu-); stelladelin C: cycle(-Val-Pro-Tyr-Pro-Pro-Phe-Tyr-Ser-)] were isolated and their structures determined by spectroscopic methods. G. CYCLIC PERIDESFROM Leonurus heterophyllus The fruits of Leonurus heterophyllus (Labiatae) have been used as a Chinese drug for stimulating blood circulation, regulating menstrual disturbance, and dispelling edema (244,245).Kinoshita reported that the extracts of this drug showed hypotensive and anti-inflammatory effects, as well as increasing blood flow and relaxating the aortic smooth muscle (246). The fruits are alleged to exhibit the same activities. However, except for fatty oils, retinol derivatives, and an uncharacterized basic compound (leonurinine), the constituents have yet to be characterized. Cycloleonurinin (343), a cyclic dodecapeptide, was isolated, sequenced by MS/MS, as well as by protein sequencing of the partial hydrolysates obtained by a-chymotrypsin (247) (Fig. 50) and showed potent immunosuppressive effect on human peripheral blood lymphocytes (286). New proline-rich cyclic nanapeptides, cycloleonuripeptides A-D (344347), showing cell growth inhibitory activity, have been isolated from the fruits of Leonurus heterophyllus (248).Their structures and conformations were elucidated by extensive 2D-NMR (248) and X-ray crystallographic analysis (249),and by distance geometry calculations (250) (Fig. 51).

H. OTHERISOLATIONS OF CYCLIC PEFTIDES FROM PLANTS Cyclic peptides have been isolated from various plant spieces, and these alkaloids are now considered to be a rather popular class of metabolites. Alkaloids reported by other groups are listed as follows:

Annornuncatin A (Annona rnuricata) (252) cyclo(Gly-Pro-Phe-Val-Ser- Ala) Arenarin A (Arenaria junea Bieb) (252)

cyclo(Ser-Ser-Phe-Ile-Pro-Pro-Phe) Citrucin I (Citrus unshiu Marcov.) (253) cyclo(Gly-Thr-Phe-Leu-Ile- Ala-Thr)

OH

AH

dichotomia B (332)

d i c b o t d A (331)

OH

Y N

I

OH dichotomin c (333)

dichotominD (334)

dichotomin E (335)

Vd

Ser'

Leu

Ro2

Leu3

Ro

phc8

dichotomin F (336)

Ro'

Roe

dichotomin G (337)

W 4 W

r 3 '

',\

dichotomin H (338):R=CH3 dichotomin I(339): R=isopropyl

FIG.47. Structures of dichotomins A-I (331-339)from the roots of Stellaria dichotoma L. var. lanceolata Bge.

374

ITOKAWA ET AL.

C

I

C:

c44

FIG.48. Perspective view of the crystal structure of 331;dotted lines indicate two intramolecular H-bonds.

~y~l~konuri@dwA, B,a d C (344- 346)

R

cycloleonuripeptideD (347)

FIG.50. Structures of cycloleonurinin (343) and cycloleonuripeptidesA, B, C, and D (344-347). Alkaloids 345 and 346 are isomers with = 0.

376

ITOKAWA ET AL.

FIG.51. Stereoscopic view of the X-ray structure of cycloleonuripeptide D (347). Hydrogen atoms have been omitted.

Citrucin II (C. sinensis Osbeck., C. nafsudaidai) (253) cyclo( Gly-GI y-Pro-Ala-Pro-Phe-Trp) Citrucin III (C. sinensis) (253) cyclo( Gly-Ser-Pro-Leu-Leu-Pro-Tyr) Citrucin IV (C. sinensis) (253) cyclo( Gly-Glu-Val-Pro-Glu- Ala-Glu-Trp) Cleromyrine I (Clerodendrurn rnyricoides) (254) cyclo( G1y-Pro-Ile-Val-Phe- Ala) Cleromyrine I1 (C. rnyricoides) (255) cyclo( Gly-Tyr-Gly-Pro-Leu-Pro) Curcacycline A (Jufrophu curcus L.) (256) cyclo( Gly-Leu-Leu-Gly-Thr-Val- Leu-Leu) Cycloleonurinin (Leonurus arternisiu S. Y . Hu, L. sibiricus) (257) cycle( Gly-Pro-Thr-Gln-Tyr-Pro-Pro-Tyr-Tyr-Thr-Pro- Ala) Cyclopsychotride A (Psychotriu longipes) (258) cyclo( Gly-Cys-Ser-Cys-Lys-Ser-Lys-Val-Cys-Tyr-Lys-Asn-Ser-Ile-

Pro-Cys-Gly-Glu-Ser-Cys-Val-Phe-Ile-Pro-Cys-Thr-Val-ThrAla-Leu-Leu) Cyclogossine A (Jurrophu gossypifoliu) (259) cyclo( Gly-Val-Leu- Ala-Thr-Trp-Leu) Glabrin A (Annonu glubru (260) cyclo(Gly-Leu-Val-Ile-Tyr-Pro) Heterophyllin A (Pseudostellaria heferophyllu) (262,262) cyclo( Gly-Ile-Thr-Pro-Val-Ile-Phe)

MACROCYCLIC PEPIlDE ALKALOIDS FROM PLANTS

377

349

348

Pyr-

350 351

Y.”&Jq NH

0

Hod 0

Me- -

HO

OH

362

353

Heterophyllin B (P. heterophyllu) (262,262) cyclo( Gly-Gly -Leu-Pro-Pro-Pro-Ile-Phe) Heterophyllin C (P. heterophyllu) (263)

cyclo(Leu-Gly-Pro-Ile-Ile-Pro-Ile) Labaditin (J. rnultifidu L.) (264) cyclo(Gly-Val-Trp-Thr-Val-Trp-Gly-Thr-Ile- Ala) Lyciumin A (348)(265-268), Lyciumin B (349) (265-269, Lyciumin C (350) ( 2 6 3 , Lyciumin D (351) (267) (Lycium chinense Mill) Moroidin (352) (Luporteu moroides) (269,270)

378

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Podacycline A (J. podugricu) (271) cyclo( Gly-Gly -Leu-Leu-Gly- Ala-Val-Trp-Ala) Podacycline B (J. podugricu) (272) cyclo( Gly-Thr-Ile-Phe-Gly -Phe- Ala) RY-III (353) (Rubiu yunnanensis) (272) Stellaria Cyclopeptide (Stellaria dichotoma L. var. lunceoluta Bge.) (273-275) cyclo( Gly-Gly- Ala- Ala-Val-Tyr) Stelladeli A (S. deluvuyi) (276) cyclo( G1y -Pro-Pro-Pro-Leu-Leu-Gly-Pro-Pro-Tyr-Tyr ) Stelladelin B (S. deluvayi) (277) cyclo( Gly-1le-Pro-Pro- Ala-Tyr- Asp-Leu) Stelladelin C (S. delavuyi) (277) cyclo( Val-Pro-Tyr-Pro-Pro-Phe-Tyr-Ser) Stelladelin D (S. deluvuyi) (278) cycle( Gly-Val-Pro-Ser-Pro-Tyr-Phe-Pro- Ala- Ala-Ile) Stellarin A (S. yunnanensis) (279,280) cyclo( Gly-Pro-Phe-Pro-Gly -Tyr-Gly ) Stellarin B (S. yunnunensis) (281,282) cyclo( Gly-Ser-HOIle-Phe-Phe- Ala) Stellarin C (S. yunnunensis) (282,282) cyclo( Gly-Ser-HOIle-Phe-Phe-Ser) Stellarin D (S. yunnanensis) (283) cyclo( Gly-Tyr-Leu-Phe-Pro-Ile-Pro) Stellarin E (S. yunnanensis) (283) cyclo( Ala- Ala-Gly-Ile-Pro-Tyr-Ile) Stellarin F (S. yunnunensis) (284) cyclo( Gly-Ala-Gly-Ser-Pro-Trp-Phe-Pro) Stellarin G (S. yunnanensis) (284) cyclo( Gly- Ala-Tyr-Leu- Ala) Stellarin H (S. yunnanensis (285)

cyclo(Phe-Ser-Leu-Val-Leu-Pro-Pro-Tyr-Ser)

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CUMULATIVE INDEX OF TITLES

Aconitum alkaloids, 4,275 (1954), 7,473 (1960), 34, 95 (1988) CI9diterpenes, l2,2 (1970) Cz0diterpenes, l2, 136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, experimental antitumor activity of acronycine, 21, 1 (1983) N-Acyliminium ions as intermediates in alkaloid synthesis, 32,271 (1988) Ajmaline-Sarpagine alkaloids, 8,789 (1965), 11,41 (1968) enzymes in biosynthesis of, 47, 116 (1995) Alkaloid production, plant biotechnology of, 40, 1 (1991) Alkaloid structures spectral methods, study, 24,287 (1985) unknown structure, 5,301 (1955), 7,509 (1960), 10,545 (1967), 12,455 (1970), 13,397 (1971), 14,507 (1973), 15,263 (1975), 16,511 (1977) X-ray diffraction, 22, 51 (1983) Alkaloids biosynthesis, regulation of, 49, 222 (1997) containing a quinolinequinone unit, 49, 79 (1997) containing a quinolinequinoneimine unit, 49,79 (1997) ecological activity of, 47,227 (1995) forensic chemistry of, 32,l (1988) histochemistry of, 39, 1 (1990) in the plant, 1,15 (1950), 6 , l (1960) Alkaloids from amphibians, 21, 139 (1983), 43, 185 (1993) ants and insects, 31, 193 (1987) Chinese traditional medicinal plants, 32, 241 (1988) mammals, 21,329 (1983), 43,119 (1993) marine organisms, 24,25 (1985), 41,41 (1992) medicinal plants of New Caledonia, 48, 1 (1996) mushrooms, 40,189 (1991) plants of Thailand, 41, 1 (1992) Allelochemical properties or the raison d’Ctre of alkaloids, 43,l (1993) All0 congeners, and tropolonic Colchicum alkaloids, 41, 125 (1992) 389

390

CUMULATIVE INDEX OF TITLES

Alstonia alkaloids, 8,159 (1965), 12,207 (1970), 14,157 (1973) Amaryllidaceae alkaloids, 2, 331 (1952), 6, 289 (1960), 11,307 (1968), 15, 83 (1975), 30,251 (1987) Amphibian alkaloids, 21, 139 (1983), 43, 185 (1983) Analgesic alkaloids, 5, 1 (1955) Anesthetics, local, 5,211 (1955) Anthranilic acid derived alkaloids, 17, 105 (1979), 32,341 (1988), 39, 63 (1990) Antifungal alkaloids, 42, 117 (1992) Antimalarial alkaloids, 5, 141 (1955) Antitumor alkaloids, 25, 1 (1985) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4,119 (1954), 9 , l (1967), 24,153 (1985) Aristolochia alkaloids, 31,29 (1987) Aristotelia alkaloids, 24, 113 (1985), 48, 249 (1996) Aspergillus alkaloids, 29, 185 (1986) Aspidosperma alkaloids, 8,336 (1965), 11,205 (1968), 17, 199 (1979) Azafluoranthene alkaloids, 23,301 (1984)

Bases simple, 3, 313 (1953), 8, 1 (1965) simple indole, 10,491 (1967) simple isoquinoline, 4, 7 (1954), 21, 255 (1983) Benzodiazepine alkaloids, 39,63 (1990) Benzophenanthridine alkaloids, 26,185 (1985) Benzylisoquinoline alkaloids, 4,29 (1954), 10,402 (1967) Betalains, 39, 1 (1990) Biosynthesis in Catharanthus roseus, 49, 222 (1997) isoquinoline alkaloids, 4 , l (1954) pyrrolizidine alkaloids, 46,1 (1995) quinolizidine alkaloids, 46, 1 (1995) tropane alkaloids, 44, 116 (1993) in Rauwolfia serpentina, 47,116 (1995) Bisbenzylisoquinoline alkaloids, 4, 199 (1954), 7, 429 (1960), 9, 133 (1967), 13,303 (1971), 16,249 (1977), 30,l (1987) synthesis, 16,319 (1977) Bisindole alkaloids, 20, 1 (1981) noniridoid, 47, 173 (1995) Bisindole alkaloids of Catharanthus C-20’ position as a functional hot spot in, 37, 133 (1990) isolation, structure elucidation and biosynthesis, 37, 1 (1990) medicinal chemistry of, 37, 145 (1990)

CUMULATIVE INDEX OF TITLES

391

pharmacology of, 37,205 (1990) synthesis of, 37,77 (1990) therapeutic use of, 37,229 (1990) Buxus alkaloids, steroids, 9,305 (1967), 1 4 , l (1973), 32,79 (1988) Cactus alkaloids, 4,23 (1954) Calabar bean alkaloids, 8,27 (1965), 10,383 (1967), 13,213 (1971), 36, 225 (1989) Calabash curare alkaloids, 8, 515 (1965), 11,189 (1968) Calycanthaceae alkaloids, 8,581 (1965) Camptothecine, 21,101 (1983) Cancentrine alkaloids, 14,407 (1973) Cannabis sativa alkaloids, 34,77 (1988) Canthind-one alkaloids, 36, 135 (1989) Capsicum alkaloids, 23,227 (1984) Carbazole alkaloids, 13,273 (1971), 26,l (1985) chemistry and biology of, 44,257 (1993) Carboline alkaloids, 8,47 (1965), 26, 1 (1985) P-Carboline congeners and Ipecac alkaloids, 22, 1 (1983) Cardioactive alkaloids, 5,79 (1955) Catharanthus roseus biosynthesis of terpenoid indole alkaloids in, 49,222 (1997) Celastraceae alkaloids, 16, 215 (1977) Cephalotuxus alkaloids, 23, 157 (1984) Cevane group of Veratrum alkaloids, 41, 177 (1992) Chemotaxonomy of Papaveraceae and Fumaridaceae, 29, 1 (1986) Chinese medicinal plants, alkaloids from, 32, 241 (1988) Chromone alkaloids, 31,67 (1988) Cinchona alkaloids, 3, 1 (1953), 14,181 (1973), 34,332 (1988) Colchicine, 2,261 (1952), 6,247 (1960), 11,407 (1968), 23,l (1984) Colchicum alkaloids and all0 congeners, 41,125 (1992) Configuration and conformation, elucidation by X-ray diffraction, 22,51 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4 249 (1954), 10,463 (1967), 29,287 (1986) Curare-like effects, 5,259 (1955) Cyclic Tautomers of Tryptamine and Tryptophan, 34,1 (1988) Cyclopeptide alkaloids, 15, 165 (1975) Daphniphyllum alkaloids, 15,41 (1975), 29,265 (1986) Delphinium alkaloids, 4 275 (1954), 7, 473 (1960) Clo-diterpenes, 12, 2 (1970) Go-diterpenes, 12,136 (1970) Dibenzazonine alkaloids, 35, 177 (1989)

392

CUMULATIVE INDEX OF TITLES

Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8,336 (1965) Diterpenoid alkaloids Aconitum, 7, 473 (1960), 12,2 (1970), 12,136 (1970), 34,95 (1988) Delphinium, 7,473 (1960), 12,2 (1970), 12,136 (1970) Garrya, 7,473 (1960), 12,2 (1960), 12,136 (1970) chemistry, 18, 99 (1981), 42,151 (1992) general introduction, 12, xv (1970) structure, 17, 1 (1970) synthesis, 17, 1 (1979) Eburnamine-vincamine alkaloids, 8,250 (1965), 11,125 (1968), 20,297 (1981), 42, 1 (1992) Ecological activity of alkaloids, 47,227 (1995) Elaeocarpus alkaloids, 6,325 (1960) Ellipticine and related alkaloids, 39,239 (1990) Enamide cyclizations in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in vitro, 18, 323 (1981) Ephedra alkaloids, 3,339 (1953) Epibatidine, 46, 95 (1995) Ergot alkaloids, 8,726 (1965), 15,l (1975), 38 1 (1990) Erythrina alkaloids, 2,499 (1952), 7, 201 (1960), 9,483 (1967), 18, 1 (1981), 48,249 (1996) Erythrophleum alkaloids, 4,265 (1954), 10,287 (1967) Eupomatia alkaloids, 24, 1 (1985) Forensic chemistry, alkaloids, 12,514 (1970) by chromatographic methods, 32,l (1988) Galbulimima alkaloids, 9,529 (1967), W, 227 (1971) Gardneria alkaloids, 36, 1 (1989) Garrya alkaloids, 7,473 (1960), 12,2 (1970), 12,136 (1970) Geissospermum alkaloids, 8,679 (1965) Gelsemium alkaloids, 8, 93 (1965), 33,84 (1988), 49, 1 (1997) Glycosides, monoterpene alkaloids, 17, 545 (1979) Guatteriu alkaloids, 35, 1 (1989) Haplophyton cimicidum alkaloids, 8, 673 (1965) Hasubanan alkaloids, 16,393 (1977), 33,307 (1988) Histochemistry of alkaloids, 39, 165 (1990) Holarrhena group, steroid alkaloids, 7, 319 (1960) Hunteriu alkaloids, 8,250 (1965)

CUMULATIVE INDEX OF TITLES

393

Zbuga alkaloids, 8, 203 (1965), 11,79 (1968) Imidazole alkaloids, 3, 201 (1953), 22, 281 (1983) Indole alkaloids, 2,369 (1952), 7, 1 (1960), 26, 1 (1985) biosynthesis in Cutharanthus ruseus, 49, 222 (1997) biosynthesis in Rauwul’u serpentinu, 47, 116 (1995) distribution in plants, 11, 1 (1968) simple, 10,491 (1967), 26, 1 (1985) Reissert synthesis of, 31, 1 (1987) Indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) In vitru and microbial enzymatic transformation of alkaloids, 18, 323 (1981) 2,2’-Indolylquinuclidinealkaloids, chemistry, 8,238 (1965), 11,73 (1968) Ipecac alkaloids, 3,363 (1953), 7, 419 (1960), 13, 189 (1971), 22, 1 (1983) Isolation of alkaloids, 1, 1 (1950) Isoquinoline alkaloids, 7,423 (1960) biosynthesis, 4, 1 (1954) I3C-NMR spectra, 18,217 (1981) simple isoquinoline alkaloids, 4,7 (1954), 21,255 (1983) Reissert synthesis of, 31, 1 (1987) Isoquinolinequinones, from Actinomycetes and sponges, 21, 55 (1983) Khat (Cathu edulis) alkaloids, 39, 139 (1990) Kupsiu alkaloids, 8, 336 (1965) Lead tetraacetate oxidation in alkaloid synthesis, 36, 70 (1989) Local anesthetics, 5, 211 (1955) Localization in the plant, 1,15 (1950), 6 , l (1960) Lupine alkaloids, 3, 119 (1953), 7, 253 (1960), 9, 175 (1967), 31, 16 (1987), 47,2 (1995) Lycupudiurn alkaloids, 5,265 (1955), 7,505 (1960), 10,306 (1967), 14, 347 (1973), 26,241 (1985), 45,233 (1994) Lythraceae alkaloids, 18,263 (1981), 35,155 (1989) Macrocyclic peptide alkaloids from plants, 26,299 (1985) 49,301 (1997) Mammalian alkaloids, 21, 329 (1983), 43, 119 (1993) Marine alkaloids, 24,25 (1985), 41,41 (1992) Maytansinoids, 23, 71 (1984) Melanins, 36,254 (1989) Meludinus alkaloids, 11,205 (1968) Mesembrine alkaloids, 9, 467 (1967) Metabolic transformation of alkaloids, 27,323 (1986) Microbial and in vitru enzymatic transformation of alkaloids, 18, 323 (1981) Mitrugynu alkaloids, 8, 59 (1965), 10,521 (1967), 14,123 (1973)

394

CUMULATIVE INDEX OF TITLES

Monoterpene alkaloids, 16,431 (1977) glycosides, 17, 545 (1979) Morphine alkaloids, 2, 1 (part 1, 1952), 2, 161 (part 2, 1952), 6,219 (1960), l3,l (1971), 45,127 (1994) Muscarine alkaloids, 23, 327 (1984) Mushrooms, alkaloids from, 40,190 (1991) Mydriatic alkaloids, 5,243 (1955) a-Naphthophenanthridine alkaloids, 4,253 (1954), 10,485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (1986), 46, 127 (1995) Narcotics, 5, 1 (1955) New Caledonia, alkaloids from the medicinal plants of, 48, 1 (1996) Nuphar alkaloids, 9,441 (1967), 16,181 (1977), 35,215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11,205 (1968) Ourouparia alkaloids, 8, 59 (1965), 10,521 (1967) Oxaporphine alkaloids, 14,225 (1973) Oxazole alkaloids, 35,259 (1989) Oxindole alkaloids, 14, 83 (1973) Papaveraceae alkaloids, 19,467 (1967), 12,333 (1970), 17,385 (1979) pharmacology, 15,207 (1975) toxicology, l5,207 (1975) Pauridiantha alkaloids, 30,223 (1987) Pavine and isopavine alkaloids, 31, 317 (1987) Pentuceras alkaloids, 8, 250 (1965) Peptide alkaloids, 26,299 (1985), 49,301 (1997) Phenanthrene alkaloids, 39,99 (1990) Phenanthroindolizidine alkaloids, 19,193 (1981) Phenanthroquinolizidine alkaloids, 19, 193 (1981) P-Phenethylamines, 3,313 (1953), 35,77 (1989) Phenethylisoquinoline alkaloids, 14,265 (1973), 36, 172 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7,433 (1960), 9, 117 (1967), 24, 253 (1985) Picralima alkaloids, 8, 119 (1965), 10, 501 (1967), 14, 157 (1973) Piperidine alkaloids, 26, 89 (1985) Plant biotechnology, for alkaloid production, 40, 1 (1991) Plant systematics, 16, 1 (1977) Pleiocarpa alkaloids, 8, 336 (1965), 11, 205 (1968) Polyamine alkaloids, 22,85 (1983) Polyamine toxins, 45,l (1994), 46,63 (1995) Pressor alkaloids, 5, 229 (1955) Protoberberine alkaloids, 4,77 (1954), 9,41 (1967), 28,95 (1986) biotransformation of, 46,273 (1995) transformation reactions of, 33, 141 (1988)

CUMULATIVE INDEX OF TITLES

395

Protopine alkaloids, 4,147 (1954), 34,181 (1988) Pseudocinchoma alkaloids, 8, 694 (1965) Purine alkaloids, 38,226 (1990) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960), 11,459 (1968), 26, 89 (1985) Pyrrolidine alkaloids, 1,91 (1950), 6, 31 (1960), 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6,35 (1960), 12,246 (1970), 26,327 (1985) biosynthesis of, 46, 1 (1995) Quinazolidine alkaloids, see Indolizidine alkaloids Quinazoline alkaloids, 3, 101 (1953), 7,247 (1960), 29, 99 (1986) Quinazolinocarbolines, 8, 55 (1965), 21,29 (1983) Quinoline alkaloids related to anthranilic acid, 3, 65 (1953), 7, 229 (1960), 17, 105 (1979), 32,341 (1988) Quinolinequinone alkaloids, 49,79 (1997) Quinolinequinoneimine alkaloids, 49, 79 (1997) Quinolizidine alkaloids, 28, 183 (1985), 47, 1 (995) biosynthesis of, 46,1 (1995) Rauwolfia alkaloids, 8,287 (1965) biosynthesis of, 47,116 (1995) Reissert synthesis of isoquinoline and indole alkaloids, 31, 1 (1987) Reserpine, chemistry, 8,287 (1965) Respiratory stimulants, 5, 109 (1955) Rhoeadine alkaloids, 28, 1 (1986) Salamandra group, steroids, 9,427 (1967) Sarpagine-type alkaloids, 49, 1 (1997) Sceletium alkaloids, 19, 1 (1981) Secoisoquinoline alkaloids, 33,231 (1988) Securinega alkaloids, 14, 425 (1973) Senecio alkaloids, see Pyrrolizidine alkaloids Simple indole alkaloids, 10,491 (1967) Simple indolizidine alkaloids, 28, 183 (1986), 44, 189 (1993) Sinomenine, 2,219 (1952) Solanurn alkaloids chemistry, 3,247 (1953) steroids, 7, 343 (1960), 10, 1 (1967), 19, 81 (1981) Sources of alkaloids, 1, 1 (1950) Spectral methods, alkaloid structures, 24, 287 (1985) Spermidine and related polyamine alkaloids, 22, 85 (1983) Spermine and related polyamine alkaloids, 22, 85 (1983) Spider toxin alkaloids, 45, 1 (1994), 46,63 (1995)

396

CUMULATIVE INDEX OF TITLES

Spirobenzylisoquinolinealkaloids, 13,165 (1971), 38, 157 (1990) Sponges, isoquinolinequinone alkaloids from, 21,55 (1983) Sternonu alkaloids, 9,545 (1967) Steroid alkaloids Apocynaceae, 9,305 (1967), 32,79 (1988) Bums group, 9,305 (1967), 1 4 , l (1973), 32,79 (1988) Holurrhena group, 7,319 (1960) Sulurnundru group, 9,427 (1967) Solunurn group, 7,343 (1960), 10,l (1967), 19,81 (1981) Verutrurn group, 7,363 (1960), 10,193 (1967), 14,l (1973), 41,177 (1992) Stimulants respiratory, 5, 109 (1955) uterine, 5,163 (1955) Structure elucidation, by X-ray diffraction, 22, 51 (1983) Strychnos alkaloids, 1, 375 (part 1, 1950), 2, 513 (part 2, 1952), 6, 179 (1960), 8,515,592 (1965), 11,189 (1968), 34,211 (1988), 36,l (1989), 48,75 (1996) Sulfur-containing alkaloids, 26,53 (1985), 42,249 (1992) Synthesis of alkaloids, Enamide cyclizations for, 22, 189 (1983) Lead tetraacetate oxidation in, 36,70 (1989) Tubernuernontuna alkaloids, 27, 1 (1983) Taxus alkaloids, 10,597 (1967), 39, 195 (1990) Terpenoid indole akaloids, 49,222 (1997) Thailand, alkaloids from the plants of, 41, 1 (1992) Toxicology, Papaveraceae alkaloids, 15,207 (1975) Transformation of alkaloids, enzymatic microbial and in vitro, 18, 323 (1981) Tropane alkaloids biosynthesis of, 44,115 (1993) chemistry, 1,271 (1950), 6,145 (1960), 9,269 (1967), 13,351 (1971), 16,83 (1977), 33,2 (1988), 44,1 (1993) Tropoloisoquinoline alkaloids, 23,301 (1984) Tropolonic Cofchicurnalkaloids, 23, 1 (1984), 41,125 (1992) Tyfophorualkaloids, 9, 517 (1967) Uterine stimulants, 5, 163 (1955) Verutrurn alkaloids cevane group of, 41,177 (1992) chemistry, 3,247 (1952) steroids, 7,363 (1960), 10,193 (1967), 14,l (1973)

CUMULATIVE INDEX OF TITLES

Vincu alkaloids, 8,272 (1965), 11,99 (1968), 20, 297 (1981) Voucungu alkaloids, 8, 203 (1965), 11, 79 (1968) Wasp toxin alkaloids, 45, 1 (1994), 46,63 (1995) X-ray diffraction of alkaloids, 22, 51 (1983) Yohimbe alkaloids, 8, 694 (1965), 11, 145 (1968), 27, 131 (1986)

397

This Page Intentionally Left Blank

INDEX

scorazanone, 93-95 Characterization, 93-95 isolation, 93

19(R)-Acetoxydihydrogelsevirine, 49-50 Achillea ptarmica, long-chain acid amides isolated from, 317 19(Z)-Akuamrnidine, 2-3 Alkaloid biosynthesis, regulation, 271-288 calcium effects, 285-287 compartmentation, 275-279 developmental, 273-275 effects of different signals, 271-273 elicitors, 282-284 jasmonic acid effects, 284-285 light effects, 279-280 salicylic acid effects, 285 Amphimedine, characterization, 143-148

Bisindole alkaloid, biosynthesis, 262 2-Bromoleptoclinidinone, 156-158 biological activity, 158-159 characterization, 157-159 synthesis, 158 Butea monosperma, h i d e isolation from, 321

Calcium, effect on alkaloid biosynthesis regulation, 285-287 Calliactine, 159-162 characterization, 159-161 synthesis, 162 Canacylus monanthos, long-chain acid amides isolated from, 317 Cannabis sativa, amides isolated from, 321 Catharanthus roseus, terpenoid indole alkaloids biological function, 225-227 biosynthesis, 222-288 geraniol, biosynthesis, 230-234 intermediates and enzymes involved in biosynthesis, 227-230 Cathenamine reductase, biosynthesis, 256-257 Chamaecyparisobtusa, carbamates isolated from, 321 Citrucin I, isolation, 371 Citrucins 11-IV, isolation, 376 Cleistopholine, 80-84 biological activity, 84 biosynthesis, 84-85 characterization, 80 synthesis, 81-84 Cleromyrine I, isolation, 376

19(Z)-Anhydrovobasinedio1,2-3 Annomuricatin A, isolation from plants, 371 Arenarin A, isolation, 371 Arisrolochia rnoupinensis, long-chain acid amides isolated from, 321 Aromatic-L-amino-acid decarboxylase gene, terpenoid indole alkaloid biosynthesis, 267-268 Ascididemin, characterization and biological activity, 148-156 Aza-anthraquinone type alkaloids, 80-95 cleistopholine, 80-85 biological activity, 84 biosynthesis, 84-85 characterization, 80 synthesis, 81-84 dielsiquinone, 85 characterization, 85 isolation, 85 isophomazarin, 90-93 biosynthesis, 93 characterization, 90, 92-93 isolation, 90 phomazarin, 85-90 biosynthesis, 90 characterization, 87-90 isolation, 85 399

400 Curcacycline A, isolation, 376 Cyclic oligopeptides, 324-378 astins from Aster tataricus, 355-359 biological activity, 358-359 conformational analysis, 357-358 structure, 355-356 cyclic peptides from Leonurus heterophyllus, 370-371 pseudostellarins from Pseudostellaria heterophylla, 359-361 conformational analysis, 361 structure and biological activity, 359-361 Rubia akane compounds from Rubia species, 324-354 biological activity and structure, correlation, 343-354 conformational analysis, 334-335 crystal conformation, 335-336 cytoxicity and antineoplastic activity, 327-333 metabolites, 333-334 molecular modeling, 342-343 solution conformation, 336-342 structure, 324-327 segetalins from Vaccaria segetalis, 364-369 conformational analysis, 365-369 structure and biological activity, 364-365 Stellaria delavayi, 370-371 Stellaria dichotoma L.var. lanceolata, cyclic Bge. peptides from, 370-371 yunnanins from Stellaria yunnanensis, 362-364 Cyclic peptides. from plants, isolation, 371-378 annomuricatin A, 371 arenarin A, 371 citrucin I, 371 citrucins 11-IV, 376 cleromyrine I, 376 curcacycline A, 376 cyclogossine A, 376 cycloleonurinin,376 cyclopsychotride A, 376 glabrin A, 376 heterophyllin A, 376 heterophyllins B-C, 377 labaditin, 377

INDEX

lyciumins A-C, 377 moroidin, 377 podacyclines A-B, 378 RY-111,378 stelladelins A-D, 378 stellaria cyclopeptide, 378 stellarins A-H, 378 Cyclogossine A, isolation, 376 Cycloleonurinin, isolation, 376 Cyclopeptide alkaloids, 304-313 isolation from species other than Zityphus sativa, 311-313 isolation from Zizyphus sativn plants, 304-31 1 Cyclopsychotride A, isolation, 376 Cystodamine, biological activity, 164 Cystodytins A-J, 164-170 biological activity, 170 structure characterization, 164, 166-168 synthesis, 168 Desacetoxyvindoline 17-hydroxylase gene, terpenoid indole alkaloid biosynthesis, 269 N.-Desmethoxyhumantenine, 22-23 N,-Desmethoxyrankinidine, 22-23 10’-Desmethoxystreptonigrin,123-125 N-Desmethylmyrianthine C, isolated from Plectronia odorata, 311-313 10’-0-Desmethylstreptonigrin, 123-125 Diaza-anthraquinone type alkaloids, 95-1 01 Diazaquinomycins A-B, 96-100 biological activity, 98-100 characterization, 96-97 isolation, 95-96 Dielsiquinone, 85 characterization, 85 isolation, 85 8,9-Dihydro-l1-hydroxyascididemin, 148-150 Diplamine, 171-174 characterization, 171 total synthesis, 171, 173-174 Discaria febrifuga, isolation of discarine cyclopeptide alkaloids, 311-313 Discaria longispina, isolation of discarine X cyclopeptide alkaloid, 311-313 Discarines E-H, 311-313 Discarine X, 311-313

401

INDEX

Discorhabdin A, 197-213 biological activity, 201 characterization, 199-201 isolation, 195-199 total synthesis, 204-213 Discorhabdin B, 197-201,203-213 biological activity, 201 characterization, 199-200 isolation, 197-199 total synthesis, 204-213 Discorhabdin C, 197-201,204-213 biological activity, 201 characterization, 199-201 isolation, 197-199 total synthesis, 204-211 Discorhabdin D, 197-201,204-213 biological activity, 201 characterization, 200 isolation, 197-199 total synthesis, 204-213 Discorhabdin E, 197-199,201.204-213 biological activity, 201 isolation, 197-199 total synthesis, 204-213 Discorhabdin F, 197-199,204-213 isolation, 197-199 total synthesis, 204-213 Echinacea purpurea, long-chain acid amides isolated from,317 Eilatin, 174-176 biological activity, 176 characterization, 174-175 isolation and structure, 174 synthesis, 175-176 Elegansamine, 33,35 Eudistones A-B, synthesis and characterization, 176-180 Eupomatidines 1-3, total synthesis, 180-183 Evodia hupehensis, long-chain acid amides isolated from,317 Fagara rnacrophylla, long-chain acid amides isolated from,321

Geissoschizine dehydrogenase gene, terpenoid indole alkaloid biosynthesis,

257

Gelsamydine, 33,36 Gelsedine-type alkaloids, 33-49 elegansamine, 33,35 gelsamydine, 33,36 gelselegine, 33,35 gelsemoxonine, 33-35 ll-methoxy-19(R-)hydroxygelselegine,

33,35 19-oxogelsenicine,33 partial synthesis, biogenetic speculation basis, 36-45 synthetic approach, 45-49 Hamer route, 48-49 Kende route, 45-48 Gelselegine, 33,35 Gelsemamide, 22-23 Gelsemine N-oxide, 49-50 Gelsemine-type alkaloids, 49-74 isolation and structure, 50 synthetic studies, 51-74 Fleming route, 68-71 Hart route, 58-63,71 Hiemstra and Speckamp route, 55-58 Johnson route, 51-55 Overman route, 63-68,71 Stork route, 71-74 Gelsemiurn alkaloids, structure and synthetic studies, 1-74 gelsedine-type, 33-49 gelsemine-type, 49-74 humantenine-type, 21-33 koumine-type, 17-21 sarpagine-type, 2-7 Gelsemoxonine, 33-35 Geraniol, biosynthesis, 230-234 Geraniol 10-hydroxylase gene, terpenoid indole alkaloid biosynthesis, 265-267 Glabrin A, isolation, 376

Heterophylh A, 376 Heterophyllins B-C, 377 HMG-CoA reductase gene, terpenoid indole alkaloid biosynthesis, 265 Humantenine-type alkaloids, 21-33 N,,-desmethox y humantenine, 22-23

N,-desmethoxyrankinidine,22-23 gelsemamide, 22-23 humantenirine, 22 20-hydroxydihydrorankinidine,22-23

402

INDEX

Humantenine-type alkaloids (continued) 11-hydroxyhumantenine,22 15-hydroxyhumantenine,22-23 11-hydroxyrankinidine,22 11-methoxygelsemamide, 22-23 11-methoxyhumantenine,22 rankinidine, 22 synthetic studies, 23-33 Humantenirine, 22 11-Hydroxyascididemin, 148-150 19(R)-Hydroxydihydrogelsemine,49-50 19(R)-Hydroxydihydrogelsevirine, 49-50 19(S)-Hydroxydihydrogelsevirine, 49-50 19(R)-Hydroxy-18,19-dihydrokoumine,18 19(S)-Hydroxy-l8,19-dihydrokoumine, 18 20-Hydroxydihydrorankinidine,22-23 1I-Hydroxyhumantenine, 22 15-Hydroxyhumantenine,22-23 11-Hydroxyrankinidine,22 Isophomazarin, 90, 92-93 biosynthesis, 93 characterization, 90,92-93 synthesis, 90, 92 Isotropb forrestii, heterocyclic oxazole isolated from,321 Jadomycin, characterization, 106-107 Jadomycin B, characterization, 106-107 Jasmonic acid, effect on alkaloid biosynthesis regulation, 284-285 Jatropha gossypifolia, long-chain acid amides isolated from, 321 Koumicine N-oxide. 2-3 Koumidine, 2-3 Koumine N-oxide, 17 Koumine-type alkaloids, 17-21

19(R)-hydroxy-18,19-dihydrokoumine, 18 19(S)-hydroxy-l8,19-dihydrokoumine,18 koumine N-oxide, 17 partial synthesis, biogenetic speculation basis, 18-19 total synthesis, 19-21 Kuanoniamine A, 183-185 biological activity, 185 characterization, 184 isolation and structure, 183 total synthesis, 184-185

Labaditin, isolation, 377 Lavendamycin, 107-122 biological activity, 121-122 biosynthesis, 120-121 characterization, 108 isolation, 107-108 total synthesis, 108-120 Leukemia P388 cells, cytotoxic effect of Trewia nudiflora seeds, 322-323 Light, effect on alkaloid biosynthesis regulation, 279-280 Lilium regale, long-chain acid amides isolated from,321 Long-chain acid amides, isolation from Achillea ptarmica, 317-321 Lyciumins A-C, isolation, 377 Macrocyclic peptide alkaloids, plant source, 301-378 Meridine, 185-188 biological activity, 187-188 characterization, 186-187 isolation and structure, 185-186 Metabolic engineering, application for increasing alkaloid accumulation in plant, 270-271 N.-Methoxy-l9(Z)-anhydrovobasinediol, 2-3 11-Methoxygelsemamide,22-23 11-Methoxyhumantenine,22 1l-Methoxy-l9(R-)hydroxygelselegine,33,35 Moroidin, isolation, 377

19-Oxogelsenicine,33 Pandanus amaryllifolius, racemic alkaloid, 321 Parthenium integrifolium, long-chain acid amides isolated from, 317 Peptide alkaloids, 303-323 cyclopeptide alkaloids, isolation, 304-313 from species other than Zizyphus sativa, 311-313 from Zizyphus sativa plants, 304-311 isolation from higher plants, 303-323 long-chain acid amides, 317-321 miscellaneous, 321-323 spermidine alkaloids, 314-317 spermine alkaloids, 315-317

INDEX

Peripentadenia mearsii, long-chain acid amides isolated from, 320 Petrosamine, characterization and isolation,

188 Phenanthridine type alkaloids, 101-107 jadomycin and jadomycin B, 106-107 characterization, 106-107 isolation and structure, 106-107 phenanthroviridin, 101-105 biological activity, 105 biosynthesis, 104-105 characterization, 102 total synthesis, 102-104 Phenanthroviridin, total synthesis,

102-104 Phomazarin, 85-90 biosynthesis, 90 characterization, 87-90 isolation, 85-87 Piper brachystachyum, long-chain acid amides isolated from, 317 Piper guineeme, long-chain acid amides isolated from, 317 Piper longum, long-chain acid amides isolated from, 317 Plectronia odorata, N-desmethylmyrianthine C, cyclopeptide alkaloid isolated from,

311-313 Podacycline A, isolation, 378 Prianosins A-D, 201-213 biological activity, 203-204 isolation, 201-203 total synthesis, 204-213 Pyridoacridine type alkaloids, 143-195 amphimedine, 143-148 characterization, 144 isolation and structure, 143-144 total synthesis, 144-148 ascididemin, 148-156 biological activity, 154,156 characterization, 150-151 isolation and structure, 148-150 total synthesis, 151-154 2-bromoleptoclinidinone, 156-159 biological activity, 158-159 isolation and structure, 156-158 synthesis, 158 calliactine, 159-162 characterization, 159-161 isolation and structure, 159 synthesis, 162

403

cystodamine, 162-164 biological activity, 164 characterization, 163-164 isolation, 162-163 cystodytins A-J, 164-170 biological activity, 170 characterization, 165-168 isolation and structure, 164-165 synthesis, 168 8,9-dihydro-ll-hydroxyascididemin, isolation and structure, 148-150 diplamine, 171-174 characterization, 171 isolation and structure, 171 total synthesis, 171-174 eilatin, 174-176 biological activity, 176 characterization, 174-175 isolation and structure, 174 synthesis, 175-176 eudistones A and B, 176-180 characterization, 178-180 isolation and structure, 176,177-179 eupomatidines 1,2,and 3,180-183 characterization, 181-182 isolation, 180-181 total synthesis, 183 11-hydroxyascididemin,isolation and structure, 148-150 kuanoniamine A, 183-185 biological activity, 185 characterization, 184 isolation and structure, 183 total synthesis, 184-185 meridine, 185-188 biological activity, 187-188 characterization, 186-187 isolation and structure, 185-186 petrosamine, 188-189 characterization, 188-189 isolation, 188 sampangine, 189-195 biological activity, 194-195 biosynthesis, 195 characterization, 190 isolation and structure, 189-190 total synthesis, 190-195 Pyrroloquinolinequinoneimine type alkaloids, 195-213 discorhabdins A-F biological activity, 201

404

INDEX

Pyrroloquinolinequinoneiminetype alkaloids, discorhabdins A-F (continued) characterization, 199-201 isolation, 199 total synthesis, 204-213 discorhabdins A-F, 195-213 discorhabdins and prianosins, similarity, 195-1 97 prianosins A-D, 201-213 biological activity, 203-204 characterization, 201-203 isolation, 201-203 total synthesis, 204-213

Quinolinequinone type alkaloids, 107-143 10’-0-demethylstreptonigrin, 123-125 10’-desmethoxystreptonigrin,123-125 lavendamycin, 107-122 biological activity, 121-122 biosynthesis, 120-121 characterization, 108 isolation, 107-108 total synthesis, 108-120 streptonigrin, 123-137 biological activity, 133-137 biosynthesis, 132-133 characterization, 123-125 isolation and structure, 123 total synthesis, 125-132 streptonigrone, 137-143 biological activity, 142-143 characterization, 138 isolation, 137 total synthesis, 138-142 Quinolinequinone unit-containing alkaloids, 80-143 aza-anthraquinone type, 80-95 diaza-anthraquinone type, 95-100 phenanthridine type, 101-107 quinolinequinone type, 107-143 Quinolinequinoneimine unit-containing alkaloids, 143-213 pyridoacridine type, 143-195 pymoloquhobequinoneimine type, 195-213

Rankinidine, 22 RY-111,isolation, 378

Salicylic acid, effect on alkaloid biosynthesis regulation, 285 Sampangine, 189-195 biological activity, 194-195 biosynthesis, 195 characterization, 190 isolation and structure, 189-190 total synthesis, 190-194 Sarpagine-type alkaloids, 2-17 19(Z)-akuammidine, 2-3 19(Z)-anhydrovobasinediol,2-3 koumicine N-oxide, 2-3 koumidine, 2-3 Na-methoxy-19(Z)-anhydrovobasinedioI, 2-3 partial synthesis, biogenetic speculation basis, 6,8 synthetic studies, 6-17 total synthesis, 8, 10-17 Bailey route, 16-17 Liu route, 14-16 Magnus route, 8, 10-14 16-epi-voacarpine,2-4 Scorazanone, 93-95 characterization, 93-95 isolation, 93 Secologanin, 235-240 biosynthesis, 235-240 feeding to C. roseus cell culture, effect, 240 Spermidine alkaloids, 314-317 linear type, 314 ring type, 315-317 Spermine alkaloids, 314-317 linear type, 314 ring type, 315-317 Stelladelins A-D, isolation, 378 Stellaria cyclopeptide, isolation, 378 Stellarins A-H, isolation, 378 Srernona parvijlora, alkaloid structure, 321-322 Streptonigrin, 123-137 biological activity, 133-137 biosynthesis, 132-133 characterization, 123-125 isolation and structure, 123 total synthesis, 125-132 Streptonigrone, 137-143 biological activity, 142-143 characterization, 138 isolation, 137 total synthesis, 138-142

INDEX

Strictosidine glucosidase, biosynthesis, 252-256 Strictosidine synthase, 249-252,268-269 biosynthesis, 249-252 gene, 268-269 Terpenoid indole alkaloids, 227-270 biosynthesis bisindole alkaloids, 262 catharanthine, 257 cathenamine reductase, 256-257 desacetoxyvindoline 17-hydroxylase gene, 269 geissoschizine dehydrogenase, 257 geraniol, pathway, 265-267 geraniol 10-hydroxylasegene, 265-267 HMG-CoA reductase gene, 265 peroxidases, 262-264 secologanin, pathway, 235-240 strictosidine glucosidase, 252-256 strictosidine synthase gene, 249-252, 268-269 tryptamine, pathway, 247-248

405

tryptophan, pathway, 240-247 tryptophan decarboxylase gene, 267-268 vindoline pathway, 257-262 catabolism, 264 Trewiu nudifloru, cytotoxicity to P388 leukemia cells, 321-322 Tryptamine, biosynthesis, 247-248 Tryptophan, biosynthesis, 240-247 Tryptophan decarboxylase, see Aromatic-+ amino-acid decarboxylase

Vindoline pathway, biosynthesis, 257-262 16-epi-Voacarpine,2-4

Zanthoxylum podocurpum, long-chain acid amides isolated from, 320 Zunthoxylum rubescens, long-chain acid amides isolated from, 320 Zizyphus sutivu, cyclopeptide alkaloid isolation from, 304-311

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