Analytical Profiles of Drug Substances and Excipients Volume 16

Analytical Profiles of Drug Substances Volume 16 Edited by Klaus Florey The Squibb Institute for Medical Research New...

Author: Klaus Florey

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Analytical Profiles

of

Drug Substances Volume 16 Edited by

Klaus Florey The Squibb Institute for Medical Research New Brunswick, New Jersey

Contributing Editors

Abdullah A. Al-Badr

Gerald S. Brenner

1987

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers Orlando San Diego New York Ailstin Boston London Sydney Tokyo Toronto

EDITORIAL BOARD

Abdullah A. Al-Badr Steven A. Benezra Gerald S. Brenner Glenn A. Brewer, Jr. Nicholas DeAngelis

Lee T. Grady Eugene Inman Joseph Mollica Milton D. Yudis John Zarembo

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1987 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY B E REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

Orlando, Florida 32887

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road, London NW I 7DX

Library of Congress Cataloging in Publication Data (Revised for volume 16) Analytical profiles o f drug substances. Compiled under the auspices o f the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. Includes bibliographical references and indexes. 1. Drugs-Analysis-Collected works. 2. Chemistry, Pharmaceutical-Collected works. I. Florey, Klaus, 11. Brewer, Glenn A. 111. Academy of Pharmaceutical Sciences. Pharmaceutical Analysis Control Section. [DNLM: 1. Drugs-Analysis-Yearbooks. QV740 A A 1 ASS] RS 189.A58 6 lSI.1 70-187259 ISBN 0-12-260816-X (v. 16 : alk. paper) PRINTED IN THE UNITED STATES OF AMERICA 8 7 888990

9 8 7 6 5 4 3 2 1

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Mohammad A . Abounassif, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Abdul Fattah A . Afify, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Abdullah A . Al-Budr, King Saud University, Riyadh, Saudi Arabia Mohammed A . Al-Yahya, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Salim A . Babhair, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Jos H . Beijnen, Slotervaart Hospital, Amsterdam, The Netherlands Steven A . Benezra, Wellcome Research Laboratories, Research Triangle Park, North Carolina Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania Glenn A . Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick, New Jersey Auke Bult, Faculty of Pharmacy, State University of Utrecht, Utrecht, The Netherlands Nicholas J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania Humeida A. El-Obeid, King Saud University, Riyadh, Saudi Arabia Klaus Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey Dennis K . G . Gorecki, College of Pharmacy, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Lee T . Grady, The United States Pharmacopeia, Rockville, Maryland Mahmoud M . A . Hassan, King Saud University, Riyadh, Saudi Arabia Eugene L. Inman, Lilly Research Laboratories, Indianapolis, Indiana vii

...

Vlll

AFFILIATIONS

Dominic P . I p , Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania Casimir A . Janicki, McNeil Pharmaceutical, Spring House, Pennsylvania Vijay K . Kapoor, Department of Pharmaceutical Sciences, Panjab University, Chandigarh, India Alice E . Loper, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania David J . Mazzo, Travenol Laboratories, Morton Grove, Illinois A . G. Mekkawi, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Joseph A . Mollica, Pharmaceuticals, Du Pont, Wilmington, Delaware Jaber S. Mossa, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Farid J . Muhtadi, King Saud University, Riyadh, Saudi Arabia Davide Pitrc?, Faculty of Pharmacy, University of Milan, Milan, Italy James T. Stewart, College of Pharmacy, University of Georgia, Athens, Georgia Riccardo Stradi, Faculty of Pharmacy, University of Milan, Milan, Italy Abdul Hameed U . Kader Taragan, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Mohammad Tariq, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia WillyJ . M . Underberg, Faculty of Pharmacy, State University of Utrecht, Utrecht, The Netherlands Roger K . Verbeeck, College of Pharmacy, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Milton D . Yudis, Schering-Plough, Kenilworth, New Jersey John E . Zarembo, W. H. Rorer, Inc., Fort Washington, Pennsylvania

PREFACE

Although the official compendia define a drug substance as to identity, purity, strength, and quality, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. Such information is scattered through the scientific literature and the files of pharmaceutical laboratories. I perceived a need to supplement the official compendial standards of drug substances with a comprehensive review of such infoimation, and sixteen years ago the first volume of Anulyticul Profiles of Drug Substances was published under the auspices of the Pharmaceutical Analysis and Control Section of the APhA Academy of Phaimaceutical Sciences. That we were able to publish one volume per year is a tribute to the diligence of the editors to solicit articles and even more so to the enthusiastic response of our authors, an international group associated with pharmaceutical firms, academic institutions, and compendial authorities. I would like to express my sincere gratitude to them for making this venture possible. Over the years, we have had queries concerning our publication policy. Our goal is to cover all divg substances of medical value and, therefore, we have welcomed any articles of interest to an individual contributor. We also have endeavored to solicit profiles of the most useful and used medicines, but many in this category still need to be profiled. Klaus Florey

ix

BROMAZEPAM

Mahmoud M.A. H a h a n , PhO6eAhOh 06 PhahmaceuLLcd C h m h L t y , Depah;tment od PhahmaceuLLcd C h m h L t y , CoMege 06 Phanmacy, King Saud U n L v m L t q , Riyadh, Saudi Axabia. Mohammad A. A b u u m n i d , AnbhXant Phodeddon 04 PhamaceuLic d C h m h i x y , Heud 04 Xhe DepdmenX 06 P h a m a c e d i c d C h m h L t y , College 06 Phahmacy, King Saud U n i v m i , t y , Riyadh, Saudi Arrabia.

1. H i s t o r y and T h e r a p e u t i c Category 2.

Description 2.1

Nomenclature

2.2

Formulae

2.3

Molecular Weight

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

1

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

2

.3.

4,

Physical Properties

3.1

Appearance, Color, Odor and Taste

3.2

Melting Range

3.3

D i s s o c i a t i o n Constants

3.4

Crystal Structure

Spectral Properties

4.1

U l t r a v i o l e t Spectrum

4.2

I n f r a r e d Spectrum

4.3

Nuclear Magnetic Resonance S p e c t r a

4.4

Mass Spectrum

5.

Synthesis

6.

Metabolism

7.

Pharmacokinetic S t u d i e s

8.

Methods of Analysis

8.1

Elemental Analysis

8.2

I d e n t i f i c a t i o n Tests

8.3

Titrimetric

8.4 Spectrophotometric 8.5 Polarographic 8.6 Atomic Absorption

9.

8.7

Radioimunoassays

8.8

Chromatographic

References

BR 0MAZEPAM

3

A n a l y t i c a l P r o f i l e of Bromazepam 1. H i s t o r y and Therapeutic Category The l a s t q u a r t e r c e n t u r y , and i n p a r t i c u l a r t h e decade 1952-1962, has witnessed a break-through i n t h e t r e a t m e n t and outlook of t h e mentally ill. Two groups of drugs were developed namely psychotic and psychotropic a g e n t s . Among t h e l a s t named psychotropic a g e n t s , t h e benzodiazep i n e s . These drugs such as diazepam, chlordiazepoxide and oxazepam have been shown t o e x e r t a n x i o l y t i c , s l e e p inducing, muscle r e l a x a n t and a n t i c o n v u l s a n t e f f e c t s i n g r e a t e r or l e s s e r degree. Search continued f o r making more d e r i v a t i v e s t o produce s u i t a b l e drug f o r s p e c i f i c cases or symptoms. I n t h i s way nitrazepam and more r e c e n t l y flurazepam have been found t o be s p e c i a l l y e f f e c t i v e s l e e p inducers and clonazepam e x e r t s even more p o t e n t l y t h e a n t i c o n w l s a n t a c t i v i t y known a l r e a d y f o r diazepam. Search has continued and i n 1974 bromazepam w a s marketed as a new psychotropic drug of t h e benzodiazepine s e r i e s but belonging t o a new c l a s s of pyridyl-benzodiazepines. It possesses c e r t a i n biochemical f e a t u r e s which d i s t i n guish it from o t h e r benzodiazepines. It has been shown t o e x e r t a more profound a n x i o l y t i c e f f e c t t h a n o t h e r similar substances. 2.

Description 2.1

Nomenclature 2.1.1

Chemical Names

7-Brom0-1,3-dihydro-5-(~2-pyridyl)-2H-l,~benzodiazepine-2-one. 2.1.2

Generic Name Bromazepam.

2.1.3

Trade Names Lectopam; Lexomil; Lexotan; Lexotanil.

4

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

2.2

Formulae 2.2.1

Ehpirical C14H10BrN

2.2.2

0

3

Structural

H

2.2.3

Research Number (1) Ro 5-3350.

2.2.4

Chemical A b s t r a c t s R e g i s t r y Number

[ 1812-30-21 2.3

Molecular Weight 316.16

3.

Physical Properties 3.1

Appearance, Color, Odor and T a s t e A p a l e yellow o d o r l e s s c r y s t a l l i n e s o l i d ( 2 ) .

3.2

Melting Range

BROMAZEPAM

3.3

5

D i s s o c i a t i o n Constants Bromazepam h a s t h r e e pKa v a l u e s o f 2.5, 5.2 and 11.8 corresponding t o p r o t o n a t i o n at t h e azomethine and p y r i d i n e n i t r o g e n atoms, and d e p r o t o n a t i o n a t t h e n i t r o g e n atom i n p o s i t i o n 1, r e s p e c t i v e l y . These pKa v a l u e s were determined by s p e c t r a l and polarographic a n a l y s i s ( 3 ) .

3.4

Crystal Structure

(4)

The c r y s t a l s t r u c t u r e of bromazepam w a s determined u s i n g c r y s t a l s from amyl a c e t a t e - e t h a n o l o b t a i n e d by slow e v a p o r a t i o n . The i n t e n s i t y d a t a w e r e c o l l e c t e d on a n Enraf-Nonius CAD-4 d i f f r a c t o m e t e r , c r y s t a l s i z e 0 . 3 x 0.25 x 0.2 mm, c e l l dimentions from s e t t i n g a n g l e s o f 25 r e f l e c t i o n s and g r a p h i t e monochromated MoKa r a d i a t i o n . Bromazepam i s monoc l i n i c w i t h space groups P 2 l / c (non-centrosymetric) The c e l l dimentions are a = 10.304 ( 4 ) , b = 15.897 ( 5 ) , c = 8.122 ( 3 ) A', 6 = 106.8 (3)', U = 1273.6 AO, Z = 4 , Dx = 1.649 Mg m-3, p(MoKa, A = 0.71069 A') = 3.13 mm-1, F(000) = 6 3 2 , room t e m p e r a t u r e , R = 0.040 f o r 1470 observed r e f l e c t i o n s .

.

I n bromazepam t h e 7-membered r i n g i s i n a b o a t conformat i o n . The a n g l e between t h e benzo-moeity o f t h e 1,4-benzodiazepine system and t h e h e t e r o c y c l i c r i n g system i n t h e ? - p o s i t i o n i s 60.3 ( 6 ) ' . F i n a l atomic parameters f o r bromazepam are l i s t e d i n Table 1; bond l e n g t h s , bond a n g l e s and s e l e c t e d t o r s i o n a n g l e s are l i s t e d i n Table 2. The atomic numbering scheme i s i l l u s t r a t e d i n F i g . 1. Bond l e n g t h s and a n g l e s g e n e r a l l y a g r e e w e l l w i t h v a l u e s found i n analogous molecules ( 5 - 7 ) . The N ( l ) - C ( 2 ) formal s i n g l e bond i s s h o r t e n e d t o 1.351 ( 5 ) A' i n bromazepam and t h e d i s p o s i t i o n o f bonds at N ( 1 ) i s n e a r p l a n a r s o t h a t t h e geometry of t h e bond resembles t h a t of a normal double bond ( c f . t o r s i o n a n g l e s i n Table 2 ) ( 6 ) . The N ( 4 ) - C ( 5 ) l e n g t h i n bromazepam corresponds t o t h a t o f a C = N bond and t h e C ( 5 ) - C ( l ' ) and C ( 5 ) - C ( l l ) l e n g t h s are w i t h i n t h e a c c e p t e d range (1.48-1.50 A') for a C(sp2)-C(sp2) s i n g l e bond. There i s , t h e r e f o r e , no evidence f o r any e l e c t r o n d e l o c a l i z a t i o n between t h e 5-pyridyl r i n g i n bromazepam and t h e 1,4-benzodiazepine system.

a,

c 0

w

c,

F

(ud

BROMAZEPAM

7

The seven-membered r i n g i s i n a c y c l o h e p t a t r i e n e l i k e b o a t conformation, C ( 1 0 ) and C ( 1 1 ) forming t h e ' s t e r n ' and C(3) t h e 'bow'. The bow a n g l e s , 60.3 ( 8O) i n bromazepam and t h e s t e r n a n g l e 30.8 (8' )

.

Table 1.

4

F r a c t i o n a l atomic c o o r d i n a t e s ( x 1 0 ) w i t h e . s . d . ' s i n p a r e n t h e s e s and e q u i v a l e n t i s o t r o p i c t e m p e r a t u r e f a c t o r s ( ~ x2 1 0 3 )

X

-

Y

-

617 (1) -1404 ( 3 ) -528 ( 2 ) -2169 ( 2 ) -1209 ( 2 ) -1298 ( 3 ) -2004 ( 3 ) -1626 ( 5 ) -520 ( 3 ) 237 ( 3 ) 726 ( 3 ) 449 ( 3 ) -324 ( 3 ) -821 ( 2 ) -1841 ( 3 ) -1400 ( 3 ) -2190 ( 3 ) -2820 ( 4 ) -2650 ( 3 )

Z

-

U eq

184 177 94

102

95

110

163 86

101 104

108 119 78 75 79 110

126 125 99

The major conformational d i f f e r e n c e between t h i s compound and o t h e r known 5-phenyl-1,4-benzodiazep i n e s i s i n t h e o r i e n t a t i o n of t h e 5-aryl r i n g . The a n g l e between t h e mean p l a n e o f t h e 5-aryl r i n g and t h e 'benzo' p l a n e i s 60.3 (6') i n bromazepam and 75.5 (9') i n o t h e r benzodiazepines ( 8 ) .

8

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Table 2. Molecular dimensions (b) Bond angles (")

1.351

1.214

1.453 1.278 1.483 1.394 1 371

119 113 127.1 122.1 114.6 123.3 111.3

-

1.376

1.362 1 397 1* 395

(31

(3)

(4) (5) (4) (4)

(4)

117.4 ( 4 ) 126.1 ( 4 )

116.3 (4)

1.402

117.6 (3)

1.892

-

121.3 120.3 119.8 119.8

-

-

(4)

(4) (4) (3)

L

1.378 ( 6)

-

C ( 11) -C( 10)-N( 1) C( 10)-c( 11)-c( 6) c( 1O)-C( ll)-C( 5) C( 6 ) -c( 11) -c( 5 ) C( 5 ) -C( 1' ) -N( 2 ' ) c(5 -C(l')-C(2')

c(5 -c(if)-c(6I)

119.5 121.2 119.5 116.9 123.5 118.1 112.8 119.0 117.9

-

(4)

(4) (4)

(4)

(4) (4)

(4) (4) (4)

120.7 ( 4 )

c(2 )-c( 1'1-c( 6') N ( 2 )-c(it)-c(6') 1 2 2 . 3 (4) C( 2 )-C( 2')-C( 3') 116.8 ( 4 ) C( 1 ) -C( 2')-C( 3') C( 1 ) - C ( 2')-F C( 3 ' ) -C( 2 ' ) -F c( 2 ' )-C(3')-C(4' N( 2')-C( 3' )-C( 4 ' ) 124.4 (5) c( 3' )-c( 4 ' )-c( 5 ' ) 117.8 (5) C( 4 ' )-C( 5')-~(6') 119.2 ( 5 ) C( 5' 1-C( 6 ' 1-C( 1') 1 1 9 . 4 ( 5 )

BROMAZEPAM

(c)

9

Selected torsion angels

C( l O ) - N ( 1 ) - C ( 2)-C( 3) N ( l ) - C ( p ) - C ( 3)-N(4) c(2)-C(3)-N(b)-C( 5 ) C ( 3 ) - N ( 4 ) -C( 5 ) -C( 11) N ( 4 ) - C ( 5 ) -C( ll)-C( 10) C ( 5 ) - C ( 11)-C( 10)-N( 1)

4.

(O);

-1.1

-70.3

72.3

-2.7 -37.6 -1.2

e.s.d.'s

ca

0.6'

C(ll)-C(lO)-N(l)-C(2) 39.8 4 ) - C ( 5 ) -C( 1' )-N( 2 ' ) 141.6 4 ) -C( 5 ) -C( 1' ) -C( 2 ' ) C( 11)-C( 5 ) - C ( 1' ) -N( 2 ' ) -39.8 C( I l ) - C ( 5)-C( 1' )-C( 2 ' ) -

N( Nf

Spectral Properties

4.1

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum o f bromazepam i n methanol i s shown i n F i g . 2. The s p e c t r a of bromazepam i n 1 N NaOH and 1N H C 1 are p r e s e n t e d i n F i g . 3 and Fig. 4 , r e s p e c t i v e l y . The and maximum wavelengths cm are given i n Table 3 ( 9 ) . These v a l u e s a g r e e s w i t h t h e p u b l i s h e d d a t a (10-12). Levillain (13), has s t u d i e d t h e r e l a t i o n s h i p of s t r u c t u r e and t h e W a b s o r p t i o n c h a r a c t e r i s t i c s of a s e r i e s of 1,4-benzod i a z e p i n e s , i n c l u d i n g bromazepam, c o n s i d e r i n g t h e electronic distribution of t h e various substituent s and t h e s t e r e o c h e m i s t r y . The spectrum of bromazepam i s c o n s i s t e n t w i t h t h a t o f o t h e r benzodiazepines w i t h s i m i l a r s t r u c t u r e . Als o bromazepam was i d e n t i f i e d i n s o l i d dosage forms by W a b s o r p t i o n spectrometry

q%

(14).

Table 3. Solvent Methanol

1 N NaOH

1N HC1

UV S p e c t r a l c h a r a c t e r i s t i c s

max ( n m ) 320

258

1%

cm

54.54

233

398.08 1004.78

350 268 238

493.77 794.25

348

270 238

59.33

39 * 23 386.60 552.15

The above v a l u e s a g r e e s w i t h t h a t of bromazepam.

90

80

70

60 M

40

3a

2G

10

11

z z 4

3

.-C N

n

h

P

1cI

0 E

e

a

V

CI

5

0,

3

c

w

m M .r( i*

91

81

7(

6t

5(

4 f

200 Fi:;.

4.

250

The U V s p e c t r u m of bromazeparn

in

IN HCI.

A

4.2

I n f r a r e d Spectrum

An i n f r a r e d a b s o r p t i o n spectrum o f potassium bromide d i s p e r s i o n o f bromazepam (Roche - Reference Standard Lot 0401055) i s shown i n F i g . 5. The s p e c t r a l band assignments (9,16,17) l i s t e d i n Table 4 . Table

4.

I n f r a r e d s p e c t r a l assignments o f bromazepam

Wave number (em-')

Assignment (Vibration m o M

3220,3150 , 3080

N-H

stretch

3000,2940,2860

C-H

stretch

1695

C = 0 stretch

1615

C = N s t r e t c h of both benzodiazepine and pyridine.

1600,1590,1570,14~0

Aromatic C = C s t r e t c h

815

Out o f p l a n e CH-deformat i o n of 1,2,4-tris u b s t i t u t e d aromatic Out o f p l a n e CH deformat i o n of o r t h o - d i s u b s t i t u t e d aromatic 1 , 2 ,h-Trisubst i t u t e d aromatic and o - d i s u b s t i t u t e d aromatic. A l s o 2-substituted pyridine.

Other c h a r a c t e r i s t i c bands a r e 1440, 1385, 1340, 1320, 1230, 1200, 1090, 1040, 1000, 990, 890, and 790 em-1, I n f r a r e d h a s been used f o r i d e n t i f i c a t i o n o f bromazepam w i t h o t h e r psychotropic drugs ( 1 8 ) . Bromazepam was i d e n t i f i e d i n s o l i d dosage forms by I R a b s o r p t i o n spectrometry (14).

15

BROMAZEPAM

4.3

Nuclear Magnetic Resonance S p e c t r a 4.3.1

'H-NMR

Spectrum

A t y p i c a l 'H-NMR spectrum o f bromazepam i s shown i n Fig. 6. The sample w a s d i s s o l v e d i n DMSO d6 and t h e spectrum was recorded on a 400 MHz NMR spectrometer, using TMS as an i n t e r n a l r e f e r e n c e standard. NMR spectrum recorded on a Jeol-FX-100 90 MHz s p e c t r o x e t e r i s a l s o shown i n Fig 6a. The proton assignments are l i s t e d i n Table 5. The spectrum a f t e r a d d i t i o n of D20 i s shown i n F i g . 7.

H Table 5. Proton p o s i t ion

c-3

c-9 C-6 c-5' C-8 c-4'

PMR c h a r a c t e r i s t i c s of bromazepam Group -CH2

-9H -6H -5'H( -8H -4'H( -3'H( -6'H(

(Benzenoid) ( " " ) F'yridine ) (Benzenoid) Fyridine) c-3' Fyridine) c-6' Pyridine) N-1 - l H (Diazepine) (disappeared a f t e r D20 exchange ) s = singlet d = doublet

Chemical s h i f t ppm and split?;irig

4.22 7.23 7.44 7.49 7.41 7.95 8.86 8.57

(s) (d) (s)

(m) (m) (m) (d) (d)

10.66 (s)

rn = m u l t i p l e t

I

Lr

BROMAZEPAM

F i g . 6s.

'H-NYR

Fig. 7 .

17

spectrum of bromazepam i n DMSO d6 and TMS.

'H-NMR

spectrum of bromazepam i n DMSO d6 and

TYS after DzO t=xchsngr.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

18

4.3.2

13C-NMR

Spectra

13C-NMR noise-decoupled and off-resonance s p e c t r a of bromazepam are shown i n F i g . 8, and F i g . 9 , r e s p e c t i v e l y . Both were recorded over 5000 Hz range i n d e u t e r a t e d dimethyl s u l f o x i d e (DMSO d 6 ) , ( c a n e . 100 mg/ 1 m l ) , on J e o l FX-100 90 MHz i n s t r u m e n t . Sample t u b e 1 0 mm and t e t r a m e t h y l s i l a n e as a n i n t e r n a l r e f e r e n c e s t a n d a r d a t 2OoC were used. The carbon chemical s h i f t s a r e a s s i g n e d on t h e b a s i s o f t h e chemical s h i f t t h e o r y and t h e off-resonance s p l i t t i n g p a t t e r n ( T a b l e 6 ) .

0

9

Table 6. Carbon No. c-2

5' Carbon chemical s h i f t s of bromazepam Chemical s h i f t 6 (ppm) 169.72 ( s )

c-5

56.89 ( t ) 167.55 ( s )

C-6 c-7

113.94 ( s f

c-3 C-5a

Carbon No.

127.28 ( s ) 133.50 ( d )

C-8

133.79 ( d )

C-la c-2'

138.49 ( s ) 155.63 ( s )

c-9

c-6' c-5:

c-4

c-3' s = singlet

d = doublet

Chemical s h i f t 6 (ppm) 123.34 ( d )

148.24 ( d ) 122.87 ( a ) 136.96 ( d ) 124.81 ( s )

t = triplet

19

BROMAZEPAM

Fig. 8 .

“C-NMR noise-decoupled Spectrum o f bmmazepam i n OMS0 and TUS.

Pig. 9 .

13C-NMR off-resonance spectrum of bromazepam in DUSO and TUS.

.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

20

4.4

Mass Spectrum

The E l mass spectrum o f bromazepam (Roche Reference Standard, Lot 0401055) was o b t a i n e d on a Finigan 1020 quadrupole mass spectrometer and shown i n Fig. 10. I t s GC t r a c e i s shown i n Fig. 11. The i o n i z i n g e l e c t r o n beam energy w a s at 70 eV. It shows a molecular i o n !d+ a t m/e 316 ( r e l a t i v e i n t e n s i t y 34.87) and M+ + 1 a t m / e 317 ( r e l a t i v e i n t e n s i t y 63.88). Some of t h e most prominent i o n s a r e given i n Table 7 (9). Table 7.

The most prominent fragments of bromaz epam

m/e

Relative i n t e n s i t y

m/e

Relative i n t e n s i t y

317

63.88

236

316

34.87

208

100.00 ( b a s e Peak)

315

60.70

207

30 37

314

26.84

206

23.33

289

33.24

179

30.89

288

59.95

104

21.09

287

35.95

90

35.54

286

52.84

79

19.69

261

19.28

78

31 90

259

19.36

77

24.08

51

25.67

43.56

-

-

F i g . 10.

EI-mass spectrum of bromazeparn.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

22

5.

Synthesis D i f f e r e n t r o u t e s f o r t h e s y n t h e s i s o f bromazepam were r e p o r t e d (19-21). Route I

Br [11

H

CH NH COOC2H5.HC1 2 2 p y r i d i n e /ref lux

I

0

BROMAZEPAM

23

2-(2-Amino-5-bromobenzoylpyridine [ 41 was prepared from 2-phenacylpyridine p-bromophenyl hydrazone [ 11 through cyclization with concentrated hydrochloric acid to give 5-bromo-2-phenyl-3-( 2-pyridy1)indole [ 21. This compound was oxidized with chromium trioxide to give 2-(2-benzamido-5-bromobenzoy1)pyridine [3] ( 2 9 , 2 2 ) which on hydrolysis with concentrated hydrochloric acid afforded the desired compound 2( 2-amino-5-bromobenzoy1)pyridine [4] This compound is considered to be the starting material for the synthesis of 1,4-benzodiazepines. A mixture of [4], glycine ethyl ester hydrochloride and pyridine was refluxed to give bromazepam [ 5 ] .

.

Br2

H I

[5 1

in glacial acetic acid

I

\ /

V

0

[41

Alternatively, the key intermediate compound [4] was prepared by bromination of 2-(2-aminobenzoyl)pyridine [6] with bromine and glacial acetic acid (23,24).

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

24

Route 3

H

O

BrCOCH2Br

H

Br

Heat

-6

[ 51

Bromazepam I n t h i s r o u t e 2-(2-aminobenzoyl)pyridine [ 41 w a s t r e a t e d with bromoacetyl bromide in g l a c i a l a c e t i c a c i d . The crude bromoacetyl d e r i v a t i v e [ 71 w a s converted d i r e c t l y t o bromazepam [ 5 ] by r e a c t i o n with ammonia through t h e intermediate aminoacetamidobenzoyl p y r i d i n e (20).

[a]

BROMAZEPAM

Route

25

4

CH2 0-

\

-/

[41

H-N

H

c o \\

-

QI

IC

Br

Br

'

-

4 - a

L I

CH3COOH

[ 51 An a l t e r n a t i v e method (20)f o r t h e s y n t h e s i s of bromazepam proceeded v i a t h e carbobenzoxyglyclyl d e r i v a t i v e [ 9 ] . T h i s compound i s prepared a c c o r d i n g t o t h e method of Sheehan and Hess d e v i s e d f o r t h e s y n t h e s i s o f p e p t i d e s ( 2 5 , 2 6 ) . A m i x t u r e of t h e 2-(2-amino-5-bromobenzoyl)pyrid i n e and carbobenzoxyglycine i n methylene c h l o r i d e w a s added. The methylene c h l o r i d e s o l u t i o n a f t e r removal of N,N'-dicyclohexylurea w a s c o n c e n t r a t e d i n vacuo a t room t e m p e r a t u r e . The r e s i d u e w a s d i s s o l v e d i n benzene and chromatographed t o g i v e [ g ] . Cleavage o f t h e carbobenzoxy group w i t h hydrogen bromide i n g l a c i a l a c e t i c a c i d (27) gave bromazepam [ 5 ] d i r e c t l y without i s o l a t i o n of t h e i n t e r m e d i a t e , 2-( 2-aminoacetamido-5-bromobenzoy1)pyridine.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

26

Route 5 Clarke e t a1 (1980) (28) have described i n d e t a i l s t h e p r e p a r a t i o n of bromazepam [ 5 ] from t h e corresponding benzophenone by t h e use of hexamine and hydrochloric a c i d as o u t l i n e d below:

H

Br

/ t

-

+ 3MeC6H12N4C1 t

NH4C1

t

3HC02Me

BROMAZEPAM

Three main by-product were i d e n t i f i e d : two i m i d a z o l i d i nones [lo], [ll] and a n aminobenzodiazepine ( 1 2 ) .

27

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

28

6.

Metabolism The f i r s t r e p o r t e d m e t a b o l i c work on bromazepam w a s t h a t published by Sawada i n 1972 ( 2 9 , 3 0 ) . T h i s s t u d y w a s c a r r i e d o u t b o t h i n dogs, r a b b i t s and r a t s . The i s o l a t e d u r i n a r y m e t a b o l i t e was assumed t o be a 3-hydroxylated d e r i v a t i v e o f 2-amino-5-bromobenzoylpyridine [ 1 5 ] . The s t r u c t u r a l e l u c i d a t i o n w a s based on e l e m e n t a l a n a l y s i s , mass spectrometry and n u c l e a r magnetic resonance s t u d i e s .

OH

The b i o t r a n s f o r m a t i o n of bromazepam, was s t u d i e d i n f o u r s p e c i e s namely human, dog, r a t and mouse ( 3 1 ) . Four m e t a b o l i t e s w e r e i d e n t i f i e d and t h e i r e x c r e t i o n w a s q u a n t i t a t i v e l y determined. The major u r i n a r y m e t a b o l i t e s e x c r e t e d by t h r e e humans given s i n g l e 1 2 mg o r a l doses of bromazepam-5- 1 4 C were t h e conjugated forms of 3hydroxy bromazepam [13] accounting f o r 13-30% of t h e dose, and 2-( 2-amino-5-bromo-3-hydroxybenzoyl)pyridine [15] accounting f o r 4-25% of t h e dose. Conjugated [131 w a s a l s o t h e major m e t a b o l i t e i n dogs and mice a d m i n i s t e r e d 14C-bromazepam, but i t s e x c r e t i o n by r a t s was v e r y l i m i t e d . Together w i t h [13], m e t a b o l i t e [ 1 5 ] and 2-( 2amino-5-bromobenzoy1)pyridine [ 4 ] were d e t e c t e d i n t h e e x c r e t a o f t h e human, dog, r a t and mouse. Although t h e p y r i d y l N-oxide [I81 d e r i v a t i v e o f bromazepam w a s not d e t e c t e d as a m e t a b o l i t e i n any s p e i c e s , t h e Nq-oxide [14] was a minor m e t a b o l i t e i n dog u r i n e . T h i s i s t h e f i r s t r e p o r t e d i n s t a n c e o f N4-oxidation o f a 1,4-benzodiazepine-2-one. This s t u d y has shown t h a t t h e metabolism o f bromazepm i n human, dogs but not i n r a t s f i t s t h e same p a t t e r n of o t h e r lY4-benzodiazepine-2-one i n a f f o r d i n g conjugated 3-hydroxylated d e r i v a t i v e (32). The i d e n t i f i c a t i o n and q u a n t i t a t i o n of t h e d i f f e r e n t m e t a b o l i t e s were c a r r i e d o u t by t h i n l a y e r chromatography u s i n g s e v e r a l s o l v e n t systems.

BROMAZEPAM

29

de Silva et al, 1974 (33) have reported a sensitive and specific electron-capture GLC assay for bromazepm and its major metabolites (see under methods of analysis). Table 8 shows the chemical names and physical properties of bromazepam and its major metabolites. Table

8. Chemical names and physical properties of bromazepam and metabolites

No.

5

Chemical name

7-Bromo-1,3-dihydro-5-(2pyridyl)-2H-1,4-benzodiazepin-2-one (bromazepam)

Molecular weight

Melting point

316.16

237438.50 dec

-

13

.

332 2

198-2ooo

14

7-Bromo-1,3-dihydro-5-(2pyridyl)-2H-1,h-benzodiazepin-2-one 4-oxide.

332.2

263O dec.

4

2-Amino-5-bromobenzoylpyridine.

277.12

97* 5-99O

15

2-Amino-5-brom0-3-hydroxybenzoylpyridine.

293.12

190-196O

16

Bromazepam mercapturic acid methylester( 7-bromo-1,3dihydro-5- [ 2 ' - ( 6' -N-ac etylL-cystein-S-y1)-pyridyl] -2H1,4-benzodiazepin-2-one.

228

-

17 18

Met hylthiobromazepam Pyridyl N-oxide of bromazepam

-

-

Taleishi and Shimizu 1976 (34) has recently reported the isolation of a methylthio-containing metabolite which was identified as 7-bromo-l,3-dihydro-5-[2'-( 6I-methylthio)pyridyl]-2H-1,4-benzodiazepin - 2-one (methylthiobromazepam) [17]. Also they have shown that the methionine donor theory (34) is very unlikely in the case of the formation of methylthiobromazepam. Isolation of the mer-

I

0 01

e

Y

Y M

E

BROMAZEPAM

31

capturic acid conjugate of bromazepam from bile of rat given the drug, its identification and subsequent conversion of the mercapturic acid to methylthiobromazepam in rat liver preparation was reported (35). Scheme 1 shows a proposed pathway for the biotransformation of bromazepam to methylthiobromazepam in the rat.

H

H

I

O

II

CH3

[51

' COOH I

H

H

Bromazepam mercapturic acid

I

[MeY

14

C

-SAM]

Br

[I71 : S - @

14CH3

Scheme I. A proposed pathway f o r the biotransformation of bromazepam to methylthiobromazepam in the rat.

14

In this study non-radioactive bromazepam, [5- C] bromazepam and bromazepam-N'-oxide ('j'-bromo-1,3-dihydro-5-[2'(1' -ox0 ) -pyridyl 3 -2H-1,h-benzodiazepin - 2-one were employed. 7-Bromo-l,3-dihydro-5-[ 2 I - ( 6'-methyl ,N-acetylL-cysteinate-5-yl)-pyridyl]-2H-1,4-benzodiazepin-2-one (bromazepam rnercapturate methylester) was synthesised from bromazepam "-oxide and methyl N-acetyl-L-cysteinate in acetic anhydride according to the pyridine-N-oxide

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

32

n u c l e o p h i l i c s u b s t i t u t i o n d e s c r i b e d by Bauer and Dickerhofe ( 3 6 ) . The r e s u l t s i n d i c a t e d t h a t 6'-methylthiobromazepam i s o l a t e d p r e v i o u s l y i n t h e r a t u r i n e i s formed a t l e a s t i n p a r t v i a t h e m e r c a p t u r i c a c i d and t h a t rat l i v e r c o n t a i n s enzyme(s) capable of c a t a l y s i n g t h e conversion from t h e mercapturic a c i d t o methylthiobromazepam. Chemical names and p h y s i c a l p r o p e r t i e s of bromazepam and i t s m e t a b o l i t e s are l i s t e d i n Table 8. Chemical r e a c t i o n s o f bromazepam and i t s known m e t a b o l i t e s are shown i n Scheme 2. H

OH

I

I1

I

""3

COOH

Bromazepam mercapturic acid

0

[I71

Scheme 2. Chemical r e a c t i o n s of bromazepam and i t s known m e t a bolites.

BROMAZEPAM

33

Pharmacokinetic S t u d i e s D i s t r i b u t i o n and e l i m i n a t i o n of bromazepam h a s been s t u d i e d i n 4 human s u b j e c t s f o l l o w i n g s i n g l e o r a l and i n t r a v e n o u s doses o f 6 mg 14C-bromazepam ( 3 7 ) . Absorpt i o n and d i f f u s i o n o f t h e s u b s t a n c e t a k e s p l a c e r a p i d l y , peak plasma l e v e l s b e i n g reached about an hour a f t e r o r a l a d m i n i s t r a t i o n . Bromazepam i s mainly e l i m i n a t e d i n t h e u r i n e , and, u n l i k e most o t h e r benzodiazepines, excret i o n f o l l o w s a r e g u l a r , r e c t i l i n e a r p a t t e r n which i s p r a c t i c a l l y completed a f t e r 120 hours. E x c r e t i o n o f t h e m e t a b o l i t e s c l o s e l y p a r a l l e l s t h a t of t h e unchanged s u b s t a n c e . H a l f - l i f e f o r t h e e l i m i n a t i o n of bromazepam from t h e plasma is 2 0 . 1 hours following i n t r a v e n o u s a d m i n i s t r a t i o n , compared w i t h 22.5 h o u r s f o r t o t a l r a t i o a c t i v i t y (unchanged bromazepam p l u s m e t a b o l i t e s ) . F i g . 1 2 and F i g . 13 f o r i n t r a v e n o u s and o r a l a d m i n i s t r a t i o n a r e p r a c t i c a l l y i d e n t i c a l . Binding o f bromazepam by plasma p r o t e i n s w a s s t u d i e d by e q u i l i b r a t i o n d i a l y s i s , which showed t h a t about 70% of t h e substance p r e s e n t i s bound. T h i s i s c o n s i d e r a b l y l e s s t h a n f o r diazepam and most o t h e r benzodiazepines, and i s a t t r i b u t e d t o t h e i n c r e a s e d p o l a r i t y of t h e molecule due t o t h e p r e s e n c e o f t h e pyridyl radical (38). Another work d e s c r i b e d t h e a p p l i c a t i o n of gas l i q u i d chromatography f o r pharmacokinetic s t u d i e s i n man ( 3 9 ) . The a s s a y can b e used a l s o for r o u t i n e plasma l e v e l monitoring. The important pharmacokinetic parameters have been c a l c u l a t e d from t h e plasma c o n c e n t r a t i o n .

1

-

= e l i m i n a t i o n h a l f - l i f e = 1 8 . 5 hours, C1 = t o t a l T 2 (B) body plasma c l e a r a n c e = 347 m l / m i n and VdR = a p p a r e n t volume of d i s t r i b u t i o n = O.gl/kgm following s i n g l e o r a l However, u s i n g m u l t i p l e dosing w i t h dose o f 6 mg/day. ~

1 ( B ) = 1 9 . 5 hours 3 mg/day, it w a s found t h a t T 2

=

243 ml/min and Vdg = 071/kg. Kaplan e t a l , 1976 (40) have r e p o r t e d plasma c o n c e n t r a t i o n s and t i m e p r o f i l e s which i n agreement w i t h t h e above mentioned work.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

34

Fig. 12

-Bromazepam .....,

Fig. 13

Bromazepam + metabolites

Fig. 12.

14C-Bromazepam and total radioactivity (ug/ml) in plasma after I.V. injection o f 6.0 mg.

Fig. 13.

14C-Bromazepam in plasma (pg/ml) after oral administration of 6.0 mg.

BROMAZEPAM

8.

Methods of Analysis

8.1 Elemental h a l y s i s The elemental composition of bromazepam i s Element

% Theoretical

C

53.18

H

8.2

3-19

Br

25.28

N

13 * 29

0

5.06

I d e n t i f i c a t i o n Tests

8.2.1

Color Reactions Reported c o l o r r e a c t i o n s for bromazepam a r e as follows ( 4 1 ) :

Diazocoupling

React i o n with 1,3dinitrobenzene

In In 1 M N a O H 2 M HC1

I n DMSO

(CH3)bN OH Bromaze-

Pam

Orange red

Red v i o l e t

Yellow

Yellow

Orange brown

i

8.2.2

Thin-Layer Chromatogram (42) Layer

: S i l i c a g e l F254, MERCK pre-

coated p l a t e s 0.25 mm. Mobile phase

- conc. ammonia (100 + 1) ( p r e p a r e f r e s h l y ) without s a t u r a t i o n o f t h e chamber atm0sphel.e.

: Ethyl a c e t a t e

MABMOUD M. A. HASSAN AND MOHAMMADA. ABOUNASSIF

36

Application

: 1 0 p l of each s o l u t i o n

(sample and comparison)

Sample s o l u t i o n : Shake 400 mg of t a b l e t powder w i t h 5 ml of chloroform for approx. 3 min and f i l t e r . Bromazepam comparison solut i o n : Dissolve 60 mg of bromazepam i n 25 m l of chloroform. Front d i s t a n c e : 1 2 cm Detect i o n

: 1. Observe under short-wave

W-light: decrease o f t h e fluorescence through bromazepam. 2. Spray t h e d r i e d p l a t e w i t h a f r e s h l y prepared s o l u t i o n of f e r r o u s s u l f a t e , d r y f o r a s h o r t moment and spray t h e n w i t h an ammonia (1%): Bromazepam appears as a v i o l e t spot.

Rf-value

: Bromazepam approx.

0.6

Ferrous s u l f a t e : Dissolve 1 g of FeS04. 7H20 solution i n 1 0 0 m l of d i s t . water.

8.2.3

Microcrystal T e s t s Bromazepam w a s i d e n t i f i e d i n s o l u t i o n by m i c r o c r y s t a l t e s t s i n microgram q u a n t i t i e s (43) * A micro drop of t h e t e s t s o l u t i o n ( i n 50% e t h a n o l ) w a s placed on t h e g l a s s cover s l i p and a micro drop of t h e reagent was added. The drop being s t i r r e d w i t h a s l i g h t s c r a t c h i n g of t h e g l a s s t o promote t h e formation of c r y s t a l s . The appearance of t h e c r y s t a l s w a s recorded as follows: 1. With potassium cadmium i o d i d e , c r y s t a l s i n t h e form of s m a l l t a b l e t s were obtained a f t e r one hour ( F i g . 14a).

37

BROMAZEPAM

2.

With potassium mercuric i o d i d e , c r y s t a l s i n t h e form of round t r i f e r c a t e b l a c k i s h s t r u c t u r e s , white on black back ground were formed a f t e r one hour ( F i g . 1 4 b ) .

3.

With gold bromide hydrochloride, c r y s t a l s i n t h e form of flowers of d e n d r i t e s were obtained i n s t a n t l y ( F i g . 14c )

.

The s e n s i t i v i t i e s o f t h e t e s t s were 0 . 2 , 0.5 and 0 . 1 microgram r e s p e c t i v e l y . 8.3

Titrimetric 8.3.1

Non-aqueous T i t r a t i o n Among s e v e r a l 1,h-benzodiazepine d e r i v a t i v e s , bromazepam w a s t i t r a t e d with p e r c h l o r i c a c i d i n dioxane or tetrabutylammonium hydroxide i n benzene-methanol ( 4 3 ) .

8.4

Spectrophotometric Unlike o t h e r 1,b-benzodiazepines, t h e e x i s t e n c e of t h e a, a ' - d i p y r i d y l t y p e moiety enables bromazepam t o form complexes w i t h d i v a l e n t metal i o n s . Sabatino -e t a 1 ( 4 5 ) reported t h a t f e r r o u s i o n s form purple complexes with p y r i d y l benzodiazepin-2-ones; compounds having a b a s i c d i p y r i d y l t y p e bond s t r u c t u r e which has e x c e l l e n t metal complexing p r o p e r t i e s . -N

\\c -

HN-

C

The method has been a p p l i e d ( 4 6 ) t o t h e determination o f bromazepam i n b i o l o g i c a l m a t e r i a l s by adding methanol s o l u t i o n of f e r r o u s c h l o r i d e t o t h e bromazepam obtained a f t e r e x t r a c t i o n from blood o r u r i n e and measuring t h e absorbance of t h e s o l u t i o n a t 580 nm. The method proved t o be s p e c i f i c for bromazepam w i t h no i n t e r f e r e n c e from 2-amino-5-bromobenzoylpyridine, one of i t s p r i n c i p a l m e t a b o l i t e s . The procedure w a s a l s o a p p l i e d t o t h e determination o f bromazepam i n r a t blood ( 4 7 ) .

Fig. 14a

Fig. 14b

Fig. 14c

Fig. 14a. Microcrystals of bromazepam with potassium cadmium iodide. Fig. 14b.

Microcrystals of bromazepam with potassium mercuric iodide.

Fig. 14c.

Microcrystals of bromazepam with gold bromide hydrochloride.

BROMAZEPAM

39

Smyth e t a1 (48) s t u d i e d t h e c h e l a t i o n o f bromazepam w i t h F e ( I I ) , C u ( I I ) , and Co(I1) u s i n g s p e c t r o s c o p i c t e c h n i q u e s such as W - v i s . spectrophotometry. C o l o r i m e t r i c d e t e r m i n a t i o n o f bromazepam, as w e l l as some o t h e r benzodiazepines, w a s a l s o performed a f t e r r e a c t i o n w i t h a modified Marquis's r e a g e n t ( 4 9 ) . pKa v a l u e s and mechanism o f h y d r o l y s i s were a l s o r e p o r t e d u s i n g W spectrophotometry ( 3 ) . Another W-spectrophotometric method used by F. Hoffnann - L a Roche & Co Limited i s as f o l l o w s : ( 5 0 ) Assay: The d e t e r m i n a t i o n should b e c a r r i e d o u t w i t h i n one hour under subdued d a y - l i g h t . P u l v e r i z e 20 t a b l e t s and a c c u r e t e l y weigh approximat e l y 1000 mg of t a b l e t powder i n t o a 250 ml-volumetric f l a s k . Add about 200 m l of 0.1 N methanolic s u l f u r i c a c i d and shake f o r 1 5 min o r t r e a t w i t h u l t r a s o n i c s . D i l u t e t o volume w i t h 0 . 1 N methanolic s u l f u r i c a c i d . F i l t e r a part of t h i s solution (discard t h e first 1 5 m l ) and d i l u t e 10.0 m l o f t h e c l e a r f i l t r a t e t o 100.0 m l w i t h 0 . 1 N methanolic s u l f u r i c a c i d (= test solution). Method. o f Analysis: With a spectrophotometer measure t h e e x t i n c t i o n o f t h e t e s t s o l u t i o n a t 285 nm (max.) a g a i n s t 0 . 1 N methanolic s u l f u r i c a c i d as a b l a n k u s i n g 1 em-quartz c e l l s . Calculation: mg of bromazepam t a b l e t . =

E. 25000 300

.B

.A

E = e x t i n c t i o n o f t h e sample s o l u t i o n A = average weight (mg)

B = weight of sample (mg) 300 = E ( 1 % , 1 cm)-value of bromazepam a t 285 nm (maximum) i n 0.1 N methanolic s u l f u r i c a c i d .

Methanolic s u l f u r i c a c i d : D i l u t e 5.0 g o f 98% s u l f u r i c a c i d t o 1000 m l w i t h methanol. P r e p a r e t h i s s o l u t i o n a t l e a s t 24 hours b e f o r e u s e .

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

40

8.5

Polarographic The ease of r e d u c t i o n of t h e azomethine ( > C = N4) group o f bromazepam and i t s 3-hydroxy metabo i t e , and t h e ( > C = 0 ) carbonyl group o f i t s benzophenone m e t a b o l i t e s , promotes t h e i r q u a n t i t a t i o n i n t h e subnanogram range by d i f f e r e n t i a l p u l s e polarography.

7

D e S i l v a et a1 ( 3 3 ) d e s c r i b e d a d i f f e r e n t i a l p u l s e polarographic method f o r t h e d e t e r m i n a t i o n o f bromazepam and i t s m e t a b o l i t e s , 3-hydroxy-bromazepam, 2-amino-3-hydroxy-5-bromobenzoylpyridine and 2-amino5-bromobenzoylpyridine i n u r i n e .

Polarography w a s c a r r i e d o u t u s i n g phosphate b u f f e r as t h e s u p p o r t i n g e l e c t r o l y t e w i t h pH o f 5.5. The peak p o t e n t i a l , Ep, due t o t h e r e d u c t i o n o f t h e azomethine group o f bromazepam and i t s 3-hydroxym e t a b o l i t e occured a t -0.535 and -0.555 V v e r s calomel e l e c t r o d e r e s p e c t i v e l y , whereas t h e P e z due due t o t h e r e d u c t i o n o f t h e c a r b o n y l group o f t h e m e t a b o l i t e s 2-amino-5-bromobenzoylpyridine and 2-amino-3-hydroxy-5-bromobenzoylpyridine occured a t -0.630 and -0.635 V v e r s u s calomel e l e c t rode, r e s p e c t i v e l y ( F i g . 15). S e n s i t i v i t y l i m i t s ranged between 50 and 1 0 0 ng/5 ml u r i n e . Trace l e v e l s of bromazepam i n blood w a s determined by d i f f e r e n t i a l p u l s e polarography ( 5 1 ) . The c u r r e n t r e s u l t i n g from r e d u c t i o n o f t h e 4,5-azomet h i n e bond o f bromazepam w a s measured a t -0.610 V v e r s u s s i l v e r c h l o r i d e e l e c t r o d e . The method has a recovery o f 62.1% 7.8 i n t h e r a n g e of 10-1000 ng bromazepam/l ml o f blood.

*

Polarography w a s a l s o used t o s t u d y acid-base and complexing behaviour o f bromazepam ( 3 ) , and f o r t h e i d e n t i f i c a t i o n o f any one o r more of several 1 , 4 benzodiazepines i n c l u d i n g bromazepam ( 5 2 ) .

8.6 Atomic Absorption Gonzales-Perez e t a1 ( 5 3 ) d e s c r i b e d a method f o r t h e d e t e r m i n a t i o n o f bromazepam by atomic a b s o r p t i o n spectrophotometry. The method i s based on t h e e x t r a c t i o n o f t h e i o n - p a i r formed by t h e bromazepamn i c k e l (11) c a t i o n i c c h e l a t e and p e r c h l o r a t e from aqueous t o m e t h y l i s o b u t y l k e t o n e (MIBK) s o l u t i o n s .

BROMAZEPAM

41

I - 0.350

I

I

-0550 -0.750 POTENrlAL VOLTS VERSUSSATURATED CAZOMELELECTROOE

Cb)

Fig. 15.

Differential pulse polarograms of: (a) I and I 1 (b) I 1 1 and IV in 1.0 M pH 5.5 phosphate buffer as the supporting electrolyte. Key: A, control urine blank; B, authentic standard mixture; and C, authentic recovered from urine.

I.

111.

IV.

Bromazepam 11. 2-Amino-5-bromobenzoylpyridine 3-Hydroxymetabolite of bromazepm 2-Amino-3-hydroxy-5-brombenzoylpyridine

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

42

The absorbance of n i c k e l i n t h e o r g a n i c phase w a s determined a t t h e n i c k e l resonance a 232.0 nm l i n e . The method t h a t showed a d e t e c t i o n l i m i t o f 2 X 10-6M, was a p p l i e d t o t h e d e t e r m i n a t i o n of bromazepam i n drugs.

8.7

Radioimunoassay Robinson e t a1 (54 ) d e s c r i b e d f o u r radioimmunoassays f o r t h e d e t e r m i n a t i o n of bromazepam and o t h e r benzodiazepines. I n t h e most s e n s i t i v e method, t h e antiserum from t h e Finit-tox serum benzodiazepine a a s s a y k i t w a s used w i t h [-H]flunitrazepam and proved t o be s u i t a b l e f o r detecting sub-therapeutic l e v e l s o f a l l benzodiazepines t e s t e d except one. The a s s a y i s p a r t i c u l a r l y a p p l i c a b l e t o blood samples of f o r e n s i c i n t e r e s t which may b e hemolyzed or decomposing, and r e q u i r e o n l y 75 m l o f blood. A r a d i o r e c e p t o r a s s a y f o r benzodiazepines i n c l u d i n g bromazepam i n human blood, plasma, s a l i v a and u r i n e w a s developed ( 5 5 ) . The method i s based upon competit i o n between [ 3H]flunitrzepam and b i o l o g i c a l l y a c t i v e benzodiazepines i n b i o l o g i c a l f l u i d s f o r brain-specific receptors, prepared i n a s t a b l e , dry form and easy t o handle. The method i s s p e c i f i c f o r b i o l o g i c a l l y a c t i v e benzodiazepines. Other method, d e s c r i b e d t h e d e t e r m i n a t i o n o f benzodiazepines i n serum u s i n g t h e enzyme m u l t i p l i e d immuno assay-single t e s t (EMIT-ot) drug d e t e c t i o n system ( 5 6 ) . The method i s u s e f u l t o c l i n i c a l t o x i c o l o g y p r a c t i c e .

8.8

Chromatographic

8.8.1 Paper Chromatography Bromazepam w a s s e p a r a t e d from o t h e r benzodiazepines by u s i n g a t h i n l a y e r system: Merck aluminium o x i d e F254 and CHC13 - PhMe - E t O H (40:60:2) complemented by a Whatman S.G. 81 s i l i c a g e l loaded paper system developed i n CHCl3-EtOH (49:l). The s p o t s were l o c a t e d by s h o r t W wavelength and a c i d i f i e d potassium i o d o p l a t i n a t e ( 57 )

.

43

BROMAZEPAM

8.8.2

Thin-Layer Chromatography Haefelfinger (58) has reported a specific and s e n s i t i v e method for t h e d e t e r m i n a t i o n o f bromazepam i n plasma by q u a n t i t a t i v e t h i n l a y e r chromatography. S i l i c a g e l p l a t e s were used w i t h methylacetate-ammonia 33% (1OO:l) as a s o l v e n t system. The q u a n t i t a t i o n o f t h e s p o t s w a s performed e i t h e r by measuring t h e W r e f l e c t a n c e of t h e p l a t e o r by t h e hydrolys i s o f bromazepam t o 2-amino-5-bromobenzoylp y r i d i n e on t h e p l a t e , d i a z o t i z a t i o n and c o u p l i n g w i t h N-( 1-naphthy1)ethylenediamine t o form an azo-dye, which was t h e n e v a l u a t e d d e n s i t o m e t r i c a l l y . The recovery from plasma was found t o be over 90%. Bromazepam h a s an Rf v a l u e o f 0.6-0.65. Thin-layer chromatographic systems and d e t e c t i o n methods d e s c r i b e d f o r t h e i d e n t i f i c a t i o n and d e t e r m i n a t i o n of bromazepam a r e summarized i n Table 9 .

Table 9 .

Solvent systems and d e t e c t i o n methods f o r br oma z epam Detection

Support

Solvent system

S i l i c a AR 7GF-5 (twc diment ional TLC )

Hept a n e / e t hyl a c e t a t e/ethanol, c o n c e n t r a t e d ammonia ( 50 : 50 :3) and : Heptane/ chloroform/ ethanol/conc e n t r a t e d ammonia (50:50:20:1)

S i l i c a g e l Chloroform/acetone (gO:lO), G

benzene/isopropanol/ammonia ( 85 :15 :10)

S i l i c a g e l Diisopropylether-toluenee t h y l a c e t a t e methanol di6o *254 ethylamine (70:15:10:5:2)

-

W-light

Bratton-Marshall, d r a g e n d o r f f and iodoplati n a te reagents

Ref.

59

60

.

W - l i g h t (254 nm: 50 10% w/v ~ $ 0 4 h e a t a t 120' for 1 0 minutes chamber w i t h n i t r o u s fumes Spray w i t h anapht h y l e t hylmediamine R.

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

44

Thin l a y e r chromatographic d e t e c t i o n and i d e n t i f i c a t i o n of bromazepam among o t h e r lY4-benzodiazepines h a s a l s o been r e p o r t e d

(61-64).

8.8.3

Gas Chromatography

Numerous methods f o r t h e g a s chromatographic a n a l y s i s o f bromazepam i n b i o l o g i c a l f l u i d s have been r e p o r t e d . Most of t h e s e methods d e s c r i b e d t h e d e t e r m i n a t i o n o f t h e compound e i t h e r as t h e i n t a c t 1,b-benzodiazepine-2-0ne o r as t h e hydrolyzed 2-amino-5-bromobenzoylp y r i d i n e (ABBP) (33, 65-69). Bromazepam h a s a l s o been assayed as t h e "-methyl derivative

(70). The presence o f e l e c t r o n e g a t i v e f u n c t i o n a l groups ( t h e halogen i n t h e 7 - p o s i t i o n and t h e 2-carbonyl) r e n d e r s bromazepam amenable t o s e n s i t i v e d e t e r m i n a t i o n by e l e c t r o n - c a p t u r e g a s - l i q u i d chromatography (EC-GLC); t h e r e f o r e a l l ( e x c e p t one) t h e above mentioned methods a p p l i e d (EC-GLC) t o t h e d e t e r m i n a t i o n o f t h e drug. I n p r i n c i p l e , t h e s e methods are c a p a b l e of d e t e c t i n g nanogram amounts o f bromazepam i n b i o l o g i c a l f l u i d s where t h e y compete w i t h radioimmunoassays. Recently, it h a s been coupled w i t h mass spectrometry (71). The experimental c o n d i t i o n s used f o r t h e a n a l y s i s o f t h e drug i n b i o l o g i c a l f l u i d s a r e summarized i n Table ( 1 0 ) .

Table 10. Gas liquid chromatographic conditions f o r the determination of bromazepam and its metabolites

Drug o r metabolite

Column packing

Carrier

2-Amino-5-bromo-benzoylpyridine (ABBP)

2% Carbowax 20M-TPA on silanized Gas ChromP (100-120 mesh)

2-Amino-5-bromo-benzoylpyridine (ABBP)

3% OV 225 (methyl-phenylcyanopropyl. Ar-cH4 silicone) on Gas Chrom Q ( 6 0 / 8 0 Rest (9O:lO)

Bromazepam Bromazepam

Column temp

*

)etector . RT ns -

20

mi

5

2e: -

6-7

EC

65

12.5

EC

66

3% OV-17 on Gas Chrom Q (60/80mesh) Ar-CH4 245 O (90:lO)

6

EC

33

3%

240°

9.6

EC

67

265"

4

FI

68

270°

3-4

EC

70

3-4

EC

69

OV-17

220

N2

on Gas Chrom Q (60/80mesh) Ar-CH4 ( 90-10

1

214O

Bromazepam

10% UCC-W-98 on Chromosorb W AW DMC: (80/100mesh)

N2

N'-Methylbromazepam

0.5% OV-17 Chromosorb G HP (100/200 mesh)

N2

4% SE-52 silica capillary column

He

Programmed 130-260'

20% UCC-W on Chromosorb W AW DMCS (80/100mesh)

He

ProgramMass 71 med spectro100-310° - meter -

Ar = Air

MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

46

8.8.4

High P r e s s u r e Liquid Chromatography A high p r e s s u r e l i q u i d chromatographic procedure f o r t h e d e t e r m i n a t i o n o f bromazepam i n human plasma has been d e s c r i b e d ( 7 2 ) . A f t e r e x t r a c t i o n from plasma, t h e e x t r a c t w a s evaporate& and t h e r e s i d u e w a s suspended i n acetonitrile/tetrabutylammonium hydroxide (60:40,v / v ) b e f o r e i n j e c t i o n i n t o t h e chromatograph. A n a l y s i s w a s performed on a 1-1 Bondpack c18 column w i t h mobile phase made from 20 m l MeOH, 30 m l MeCN, and T O O ml of s o l u t i o n c o n t a i n i n g 20 ml 10% t e t r a b u t y l ammonium hydroxide i n 1 L water. The i n t e r n a l s t a n d a r d w a s carbamazepine and d e t e c t i o n w a s by W spectroscopy. I n o r d e r t o determine t h e lower t h e r a p e u t i c range of drug l e v e l s i n serum (73 ) , bromazepam, among o t h e r b a s i c drug, w a s chromatographed on an ~ ~ Micropack 1 8 MCH 1 0 column u s i n g UV d e t e c t o r and perchlorate/MeCN s o l u t i o n as eluent. Hochmuth et a1 (74) d e s c r i b e d a simple and r a p i d method f o r t h e t o x i c o l o g i c a l a n a l y s i s of some benzodiazepines and a n t i d e p r e s s a n t s i n c l u d i n g bromazepam. A f t e r s o l v e n t e x t r a c t i o n a t pH 9 , t h e a n a l y s i s w a s performed by HPLC on a reverse-phase column w i t h MeCN 0.6% KH PO (1:l) a d j u s t e d t o pH 3. 2 4 Heizmann et a1 ( 7 5 ) r e p o r t e d a HPLC method f o r t h e d e t e r m i n a t i o n o f bromazepam i n plasma and of i t s main m e t a b o l i t e s i n u r i n e . The unchanged drug was e x t r a c t e d from plasma w i t h dichloromethane, and t h e r e s i d u e was subseq u e n t l y analysed by r e v e r s e d phase HPLC w i t h W d e t e c t i o n (230 n m ) . For t h e d e t e r m i n a t i o n of t h e m e t a b o l i t e s , t h e u r i n e samples were incubated t o e f f e c t enzymatic deconjugat i o n b e f o r e e x t r a c t i o n w i t h dichloromethane. The column used w a s a 15 cm S u p e l c o s i l LC18 and t h e mobile phase c o n s i s t e d o f a m i x t u r e o f methanol/phosphate b u f f e r , pH 7 . 5 , a t a flow r a t e of 1 ml/min. The r e t e n t i o n times r a n g i n g from 6 t o 20 minutes.

BROMAZEPAM

9.

41

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Maes, J. Toxicol.

J e n k i n s , F o r e n s i c , S c i . SOC., J.

I

11,1 0

(1978).

58.

P. H a e f e l f i n g e r , Chromatographia,

59.

M.A. Schwartz, W.R. Pool D.L. Hane and E. P o s t n a , Drug Metabolism and D i s p o s i t i o n , 2 , 31 (1974).

60.

H. Schuetz, F r e s e n i u s ' Z. a n a l . Chem.,

61.

L. L a r i n i , P.E.T. Salgado, T.C. Ralpho and M.G. An. Farm. Quim. Sao Paulo, 17,37 (1977).

62.

P. F i c a r r a , M.G. V i g o r i t a and A. Tomanapini, B o l l . Chim. Pharm. , 720 (1977).

63.

D. Dekker and P.M.C. ( 1978 1

64.

A . Hulshoff and J.H. P e r r i n , J . Chromatogr., ( 1976 1.

65.

J.A.

66.

J.P.

294,

135 (1979). Vilela,

116,

.

, 118,465

D e S i l v a and J. Kaplan, J . Pharm. S c i ,

(1966). 1012

P a r e l , Pharm. Weekbl.

Cano, A.M.

(1975).

129,

55,

263

1278

Baile and A . Viala, Arzneim. Forsch,

25

51

BROMAZEPAM

67.

J . A . D e S i l v a , I. Bekersky, V.C. P u g l i s i , M.A. Brooks and R.E. W e i n f i e l d , Anal. Chem. , 48, 1 0 (1976).

68.

T. Kaniewska and W. Wejman, J . Chromatogr., ( 19801

.

69. J.M.F.

Douse, J. Chromatogr.,

182, 81

301, 137 (1984).

222,

501 (1981).

70.

U. K l o t z , J . Chromatogr.,

71.

H. Maurer and K. P f l e g e r , J. Chromatogr.,

72.

H. Hirayama, Y . Kasuya and T. Suga, J . Chromatogr.,

(1981).

222, 409

414 (1983). 73. H. K a e f e r s t e i n 95 (1983). 74. 75.

and G. S t i c h t , B e i t r . G e r i c h t l . Med.,

277

41,

P. Hochmuth, J.L. R o b e r t , H . Schreck and R. Wennig, Anal. Chem. Syrup. S e r . , 20, 143 (1984).

P. Heizmann, R . Geschke and K. Zinapold, J. Chromatogr.,

310, 129 (1984).

Acknowledgement The a u t h o r s would l i k e t o t h a n k M r . Tanvir A. B u t t , College of Pharmacy, King Saud U n i v e r s i t y , for t y p i n g o f t h i s manuscript

.

BUSULPHAN

Muhammad T d q " and AbduReah. A . Al-Ba&**

*U~pumincnX06 P h a c o L o g y and "Vepahtment 06 PhmacclLticd CCZmhfiy, CoReegc 06 P h m a c y , King Saud UniuemLty, Riyadh, Saudi h u b & .

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

Copyright 0 1987 by Academic Press, Inc.

53

A11 rights of reproduction in any form reserved.

MOHAMMADTARIQ AND ABDULLAH A. AL-BADR

54

1.

Foreword

2.

Description 2.1 2.2 2.3

2.4

2.5 3.

Nomenclature Formulae Molecular Weight Elemental Composition Appearance, Color and Odor

Physical Properties 3.1 3.2 3.3 3.4

Melting Point Solubility Storage Spectral Properties

4.

Synthesis

5.

Pharmacokinetics

6.

Toxicology

7.

Methods of Analysis 7.1 7.2 7.3 7.4

Identification Titrimetric Methods Chromatographic Methods Nuclear Magnetic Resonance Methods

References

BUSULPHAN

1.

55

Foreword Busulphanis a chemotherapeutic agent, which has been widely used as antineoplastic drug since its discovery in 1953 (1). This alkylating agent belongs to the series of alkanesulfonic acid esters, possessing significant cytotoxic activity and is the drug of preference in the treatment of chronic myelocytic or granulocytic leukaemia. It has recently been proposed as a drug of choice in polycythaemia and thrombocythemia (2-13). Busulphanhas also been used for the treatment of bronchial carcinoma (18-19) , myasthenia gravis (20) and chronic granulomatous disease in children (21). The drug is also used as reproductive sterilant for insect (22-23) and as a general immunosuppressant for organ transplant in the treatment of autoimmune disease (24-26).

2.

Description 2.1

Nomenclature 2.1.1

Chemical Names 1,4-Butanediol dimethanesulfonate; 1,4-Bis (methanesulf0noxy)butane; 1,4-di(methanesulfonyloxy)butane; 1,4-di(methylsulfonoxy)butane; 1,4-Butanediylbismethane sulphonate;

Methanesulfonic acid tetramethylene ester; Tetramethylene bis (methanesulfonate) (27-29)

2.1.2

Generic Name Busulphan, CB 2041, GT 41, NSC 750, WR 19508 Busulfanum (29).

2.1.3

Trade Names Myleran, Misulban, Mitosan, Myelosan, Myeleukon, Myeloleukon, Sulfabutin, Mielucin (27).

.

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

56

2.2.2

Structural 0

0

0

0

I1 II H C-S-O-CH2-CH2-CH2-CH2-O-S-CH3 3 It II

2.2.3

CAS No. [55-98-17 (27)

2.3

Molecular Weight 246.31

2.4

(31)

Elemental Composition C, 29.26%; H, 5.73%;

2.5

0 , 38.98%;

S,

26.03%

(27).

Appearance, Color and Odor A white almost odorless crystalline powder (29, 32).

3.

Physical Properties 3.1

Melting Point

114-118O, 3.2

119O, 116'.

(27).

Solubility Soluble, at 20°, in 750 parts of water (in which it is slowly hydrolyzed) and in 25 parts of acetone; very slightly soluble in alcohol (27, 32).

3.3

Storage Busulphan should be kept in a well-closed container, protect from light (30).

BUSULPHAN

3.4

Spectral Properties 3.4.1

Ultraviolet Spectrum The ultraviolet spectrum of busulphan in methanol, ethanol and in water was scanned from 200 to 400 nm using Varian DMS 90 Spectrophotometer. No absorbance has been noticed

.

3.4.2

Infrared Spectrum The infrared spectrum of busulphan as KBr disc is shown in Figure 1. It was recorded on a Pye-Unicam SP 1025 infrared spectrophotometer. The assignments of the characteristic bands in the infrared spectrum is shown below: -1

3.4.3

Frequency cm

Assignments

2960-3060

CH stretch

1430-1415

-CH2-0 vibration

1352

S ( = 0 > 2 stretch (Assymm.)

1180

S ( = 0)2 stretch (Symm.)

1000-770

S-0-C

stretch 1

Proton Nuclear Magnetic Resonance ( H NMR) Spectrum The 1H NMR spectrum of Busulphan Figure 2. The drug is dissolved and its spectrum determined on a T60 A NMR spectrometer using TMS internal standard.

is shown in in DMSO-db Varian as the

Assignment of the chemical shifts to the different protons is shown below:

Wavelenqth urn

3500

3ooo 2500 zoo0

1800

1600

1400

1200

Wavenumber Figure 1 : Infrared spectrum of Busulphan, KBr disc.

loo0

800

Jo

625

I

I

"

'

'

500

I

400

'

*

I

'

I

300

6 ! 5 4 3 2 CH3-~-O-CH2-CHj-CH~CH,-0 0

'

.

"

9

1 . "

"

"

"

'

I

0

1

-S- CH3

i;

ch

W

1 -

,

TM:

-

a

.

.

.

a

. . . .

. . . .

8.0 7.0 6.O Figure 2 : Proton nuclear magnetic Resonance spectrum of Busulphan

DMSO -dg

using TMS as reference standard

in

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

60

Chemical shift (6)

Multiplicity

Proton assignment*

1.68-1.90

Multiplet

3 and 4 (Cg2)

3.10

Sing1et

1 and 6 (Cg3)

4.1-4.40

Mu1tiplet

2 and 5 (Cl12)

*Please refer to Figure 2. 3.4.4

Carbon-13 Nuclear Magnetic Resonance Spectra (C-13 NMR) The carbon-13 NMR spectra of busulphan in

DMSO-d6 using TMS as an internal reference are obtained using a Jeol FX 100 MHz spectrometer at an ambient temperature. Figure 3 and 4 represent the proton-decoupled and off-resonance spectra respectively. 0

0

0

0

6 11 5 4 3 2 II CH3-S-0-CH CH CH CH -0-S iI 2 2 2 2 11

The the the are

1 - CH3

carbon chemical shifts are assigned on basis of the chemical shift theory and off-resonance splitting pattern and shown below:

Chemical shift (6)

Multiplicity

Carbon assignments

24.70

Triplet

3 and 4

36.52

Quartet

1 and 6

69.40

Triplet

2 and 5

Figure 3

: Proton - decoupled carbon - I3 nuclear magnetic resonance spectrum of Busulphan in DMSO- dgusing TMS as reference standard.

Ill

Figure 4

I

: Off-

I

resonance carbon-13 nuclear magnetic resonance spectrum of Busulphan in DMSO-dG using TMS as reference standard.

BUSULPHAN

63

3.4.5

Mass Spectrum The electron impact (EI) mass spectrum of busulphan at 70 eV recorded on Varian Mat 311 mass spectrometer and the direct chemical ionization (DCI) mass spectrum obtained with Finnigan 4000 mass spectrometer are shown in figures 5 and 6 respectively. The (EI) spectrum (Figure 5) shows a base peak at m/e 7 1 , the molecularion peak was not detected. Other major fragment are at m/e 175, 122, 111, 109, 97, 79, 55 and 42. A proposed mechanism of fragmentation of the EI fragments is shown in scheme 1. The CI spectrum (Figure 6 ) is simple and shows a base peak at m/e 151 and at molecular ion peak at 247 corresponding to (M + 1) and minor peak at m/e 125.

4. Synthesis By esterifying 1,4-butanediol [HOCH CH CH CH OH] 2 2 2 2 with methanesulfonyl chloride [CH SO2C1] in the 3 presence of pyridine ( 3 1 , 33) as shown below:

0 II

CH3-S-C1 II

+

OH-CH2CH2CH2CH2-OH

0 0

II

0

II

CH3-S-O-CH2CH2CH2CH2-O-E-CH3 11

0

0

Pyridine

c r

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

64

-29568

175

150 200 2 50 300 Electron impact (El) mass spectrum of Busulphan. 100

Figure 5

:

100 150 200 250 300 350 Figure 6:Ckmical imitation(CI1 mass spectrum of Busulphan.

X

+OX

m

5

I

=--a

0

I

'c: 0

m

I

8

t

-

m X V ,m-0

+o- I

c'

0

Y

o=

1

I

0 I

m=o Xm

U

1 m

X

U

O

I

+o=(.=

P

0

-51

0

0

co m I

I +o= m= c

i

0

\

0

re:

..

U

0

w

.rl

CH3

0

,+ ;

/

‘ 0

+

OH

iI

(4*7”J;-m3

0

m/e 246

+

OH

+

0

OH

0

0

m / e 175 Scheme 1:

Continued.

m/e 71

1.

0

0

I m

+O=T=O

E

67

m

T; I II

--m=

51 0 -m=o

P

0

Im T;

m

P

+O

0

.. 4

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

68

5.

Pharmacokinetics Absorption, Metabolism and E x c r e t i o n Busulphan i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and r a p i d l y d i s a p p e a r s from t h e blood. I t i s l a r g e l y e x c r e t e d i n t h e u r i n e as s u l p h u r - c o n t a i n i n g m e t a b o l i t e ( 2 9 ) . Due t o i t s a l k y l a t i n g a c t i v i t y , t h e compound might be bound b o t h r e v e r s i b l y and i r r e v e r s i b l y ( c o v a l e n t l y ) t o blood component (34). The metabolism t a k e s p l a c e mainly i n t h e l i v e r . The c l i n i c a l r e s p o n s e u s u a l l y b e g i n s w i t h i n 1 t o 2 weeks a f t e r i n i t i a t i o n of t h e r a p y . It a p p e a r s t o be p r a c t i c a l l y c o m p l e t e l y e x c r e t e d i n t h e u r i n e as methane-sulphonic a c i d (35) Nadkarni -e t a1 ( 4 ) u s i n g 14C and 35S-labeled busulphan found t h a t , a f t e r i n t r a v e n o u s d o s i n g , t h e r a d i o a c t i v i t y i n t h e blood r a p i d l y d e c r e a s e d , and between 20% and 95% was removed w i t h i n 3 t o 5 minutes. A f t e r o r a l d o s i n g an i n i t i a l l o g phase of about 0.5 t o 2 h o u r s was observed b e f o r e measureable blood l e v e l s were achieved. A f t e r o r a l 3H-labeled busulphan, Vodopick et a l , (36) found a prompt r i s e i n t h e plasma l e v e l of r a d i o a c t i v i t y , which i s peaked w i t h i n t h e f i r s t h o u r , followed by a r a p i d f a l l and t h e n a g r a d u a l r i s e i n r a d i o a c t i v i t y . Busulphan i s e l i m i n a t e d w i t h t$ of about 2.5 h o u r , f o r t h e i n i t i a l p h a s e of r a d i o l a b e l e d d r u g i n plasma. Only about 1% of a dose of busulphan i s e x c r e t e d as unchanged drug w i t h i n 2 4 h o u r s . The major p o r t i o n of busulphan i s probably e l i m i n a t e d by enzymatic a c t i v i t y r a t h e r t h a n pure chemical d e g r a d a t i o n (36). Ehrsson e t a1 (37) have s t u d i e d t h e busulphan k i n e t i c s and r e p o r t e d t h a t k i n e t i c s were followed i n p a t i e n t w i t h c h r o n i c m y e l o c y t i c leukemia a f t e r o r a l dose of 2.4 t o 6 mg. The plasma c o n c e n t r a t i o n t i m e d a t a c o u l d be f i t t e d t o a zero-order a b s o r p t i o n one-component open model. The e l i m i n a t i o n rate c o n s t a n t averaged 0.27 ? 0.05 hr-1 (SD). The plasma AUC w a s l i n e a r l y r e l a t e d t o t h e d o s e . The l o g t i m e f o r t h e s t a r t of a b s o r p t i o n , t h e t i m e absorpt i o n ends, and t h e a b s o r p t i o n rate c o n s t a n t showed

BUSULPHAN

69

some inter-individual variation. About 1% of the drug is excreted unchanged in urine over 24 hours. The fate of the drug in man has been studied after the administration of radiolabelled drug followed by measurement of total radioactivity in plasma and urine (4, 36, 37). The drug is rapidly eleminated from the plasma and is reported to be extensively metabolized; twelve metabolites have been isolated, including methane-sulphonic acid and 3-hydroxytetrahydrothiophene-1,l-dioxide, most of the metabolites have not been identified (38). Wiygul et a1 (39) have studied metabolism of busulphan in the boll weevil. When one-day male boll weevils were force-fed 3H- and Ik-labelled busulphan, 34.34% of total radioactivity was recovered from the feces, about 1.09% as busulphan, at 72 hours after injection. Most metabolism of busulphan took place within 24 hours postingestion. 1,4-Butanediol, 2,3-butanediol, sulfolanes, malic acid, malonic acid, succinic acid, aminoacids, and methanessulphonic acid were identified as principal metabolites. Jones and Campbell (40) have studied the metabolism, reactions and biological activities of some cyclic dimethanesulphonates. Relevance to the mechanism of action of busulphan (Myleran). The results obtained suggest that cycloalkylation of thiol groups by busulphan may represent a normal detoxification route rather than a mechanism of action. Other studies on the metabolism of busulphan in b o l l weevils, are also reported (41, 42, 43).

6. Toxicity Recently Bishop and Wassom (44) have published a detailed review article on the toxicity of busulphan incorporating around 290 references. The most important side effect of busulphan in high doses are thrombocytopenia and damage to haemotological tissues (45,53, 2 9 ) .

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

70

I n t e r s t e t i a l pulmonary f i b r o s i s , known as "Busulphan lung", i s as w e l l r e c o p i s e d s i d e e f f e c t busulphan t h e r a p y (54-64). A w a s t i n g o r Addison - l i k e syndrome has o c c u r r e d i n some p a t i e n t s r e c e i v i n g l o n g term t h e r a p y w i t h Busulphan (59, 65, 6 6 ) . Other a d v e r s e e f f e c t s include endocardia1 f i b r o s i s (67), c a t a r a c t s ( 6 8 ) ) c e l l u l a r d y s p l a s i a of pancreas ( 6 9 ) , a d r e n a l i n s u f f i c i e n c y (70) and bone marrow damage (71-83) Laboratory s t u d i e s i n animals have shown s i g n i f i c a n t immunosuppressive ( 4 8 , 81,) 85, 8 2 , 86) and r e p r o d u c t i v e t o x i c i t y of busulphan (87 - 1 0 0 ) .

.

Busulphan, a d m i n i s t e r e d d u r i n g t h e t h i r d trimester of pregnancy, can a d v e r s e l y a f f e c t f e t a l growth (101). T e r a t o g e n i c e f f e c t of t h i s drug i n animals h a s been r e p o r t e d by s e v e r a l workers (102 - 1 0 6 ) . Busulphan i s p o t e n t i a l l y mutagenic (104 114) and c a r c i n o g e n i c i n man (115 123).

-

7.

-

Methods of A n a l y s i s

7.1

Identification The USP XX (1980) ( 3 2 ) d e s c r i b e s t h e f o l l o w i n g i d e n t i f i c a t i o n t e s t s f o r busulphan. A)

Fuse about 100 mg w i t h about 100 mg of p o t a s s i u m n i t r a t e and a p e l l e t of potassium hydroxide weighing approximately 250 mg. Cool, d i s s o l v e t h e r e s i d u e i n water, a c i d i f y w i t h d i l u t e d h y d r o c h l o r i c a c i d , and add a few d r o n s of barium c h l o r i d e TS; a w h i t e p r e c i p i t a t e i s formed.

B)

To 100 mg add 10 m l of water and 5 m l of 1 N sodium hydroxide. Heat u n t i l a c l e a r s o l u t i o n i s o b t a i n e d ; an odor c h a r a c t e r i s t i c of methanesulfonic acid i s p e r c e p t i b l e .

C)

Cool t h e s o l u t i o n o b t a i n e d i n I d e n t i f i c a t i o n Test B , and d i v i d e i t i n t o two e q u a l p o r t i o n s . To one p o r t i o n add 1 d r o p of potassium permanganate TS; t h e p u r p l e c o l o r changes t o v i o l e t , t h e n t o b l u e , and f i n a l l y t o emeraldgreen. A c i d i f y t h e second p o r t i o n of t h e s o l u t i o n w i t h d i l u t e d s u l f u r i c a c i d , and add 1 drop

BUSULPHAN

71

of potassium permanganate TS: t h e c o l o r of t h e permanganate i s n o t d i s c h a r g e d . B r i t i s h Pharmacopoeia (1980) (30) h a s a l s o r e p o r t e d similar procedures f o r t h e i d e n t i f i c a t i o n of busulphan. 7.2

T i t r i m e t r i c Methods

7.2.1

Aqueous B r i t i s h Pharmacopoeia (1980) (30) d e s c r i b e d an aqueous t i t r a t i o n procedure f o r t h e a s s a y of busulphan a s f o l l o w s : To 0.25 g of t h e drug add 20 m l of w a t e r and b o i l g e n t l y under a r e f l u x condenser f o r t h i r t y minutes. Wash t h e condenser w i t h a small q u a n t i t y of water, c o o l and t i t r a t e w i t h 0.1 M sodium hydroxide VS, u s i n g p h e n o l p h t h a l e i n s o l u t i o n as i n d i c a t o r . Each m l of 0.1 M sodium hydroxide VS i s e q u i v a l e n t t o 0.01232 g of C6H1406S2.

7.2.2

Gravimetric Busulphan t a b l e t s are s u g a r c o a t e d and c o n t a i n o n l y m i l l i g r a m q u a n t i t i e s , hence t h e f l a s k combustion method i s n o t a p p l i c a b l e becuase of t h e l a r g e amount of o r g a n i c matter. It i s n e c e s s a r y t o e x t r a c t t h e busulphan w i t h a c e t o n e and decompose i t i n a s e a l e d t u b e w i t h n i t r i c acid a s follows: T r i t u r a t e an a c c u r a t e l y weighed q u a n t i t y of powdered t a b l e t s e q u i v a l e n t t o about 2 mg of busulphan w i t h 10 m l of h o t a c e t o n e , decant t h e l i q u i d through a f i l t e r i n t o a C a r i u s t u b e and e v a p o r a t e t o s m a l l volume under a j e t of warm a i r . E x t r a c t i n t h e same way w i t h two f u r t h e r 10-ml q u a n t i t i e s of a c e t o n e , d e c a n t i n g and e v a p o r a t i n g a f t e r each e x t r a c t i o n and e v a p o r a t i n g t o d r y n e s s a f t e r t h e t h i r d e x t r a c t i o n . Add 0.5 m l of fuming n i t r i c a c i d , s e a l t h e t u b e and h e a t a t 300° f o r two hours. A f t e r opening t h e

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

12

Carius tube transfer its contents to a small dish with 15 ml of water, evaporate to dryness, dissolve the residue in water and acidify with concentrated hydrochloric acid. Add an excess of 10 per cent barium chloride solution, heat on a water-bath for three hours, filter through a platinum filtering crucible, ignite at 800° for one hour, cool and weigh. Each mg of residue = 0.5276 mg of busulphan (124). 7.3

Chromatographic Methods 7.3.1

Gas Chromatographic Method Hassan and Ehrrson (125) developed a gas chromatographic method for the determination of busulphan in plasma with electron capture detection. The method comprise a new derivatization technique for extraction of busulphan from plasma in a single step combined with high separation efficiency and sensitivity by using capillary column in combination with electron capture detection. The derivatization of busulphan was done directly in plasma by adding sodium iodide in acetone. Plasma sample (1.0 ml) was mixed with 0.1 ml of 1.0 ug/ml of 1,5-bis(methanesulfoxy)pentane in acetone and sodium iodide in water (1 ml acetone and 8 M Na' iodide). After addition of 0.4 ml n-heptane the reaction was carried out at 7OoC for 70 minutes under stirring. The organic phase was separated for analysis on gas chromatography. 1-2 pl of solution was injected into Varian 3700 GC equipped with a constant current 63Ni lectroncapture detector. An OV-I fused silica cappillary column (25 m x 0.3 mm i.d.) with a film thickness of 0.52 um (HewlettPackard) was used. The instrument was operated isothermally with the oven, detector and injector port temperatures at 145OC, 25OoC and 2OO0C respectively. The helium was used as carrier gaswith a flow rate of 2 ml/min,the inlet pressure was

BUSULPHAN

73

0.4 b a r . Nitrogen was used a s make up g a s and added through t h e hydrogen i n l e t a t a rate of 30 ml/min i n t h e d e t e c t o r base. A s p l i t r a t i o of 1 : 10 w a s used. A h i g h s e p a r a t i o n e f f i c i e n c y and s e n s i t i v i t y w a s o b t a i n e d u s i n g t h i s method, t h e r e t e n t i o n t i m e w a s 8 minutes. The s t a n d a r d curve u s i n g plasma w a s l i n e a r w i t h i n t h e range of 5-300 ng/ml. The p r e c i s i o n of t h i s method t 3.9% (c.v.) a t t h e 10 ng/ml l e v e l (n = 5) and ? 2 . 3 % (c.v.) a t t h e 100 ng/ml (n = 5 ) . 7.3.2

Gas Chromatography

-

Mass Spectrometry

(GC-MS) Ehrsson and Hassan (126) developed a chromatographic method f o r t h e determinat i o n of busulphan i n plasma by GC-MS w i t h s e l e c t e d i o n monitoring. Busulphan e x t r a c t e d from plasma with methylene c h l o r i d e and converted t o 1,4-diiodobutane. 1.0 M 1 of plasma was mixed w i t h 0.1 m l of i n t e r n a l s t a n d a r d (1,5-pentanediol d i m e t h a n e s u l f o n a t e 1.0 pg/ml) i n acetone and e x t r a c t e d w i t h 4 m l of methylene c h l o r i d e u s i n g a mechanical shaker (100 s t r o k e s / m i n ) The o r g a n i c phase w a s s e p a r a t e d and evaporated t o d r y n e s s , d e r i v a t i z a t i o n was done by adding 0 . 1 m l of 1 M sodium i o d i d e i n a c e t o n e w i t h a r e a c t i o n t i m e of 20 minutes a t 7OoC. After a d d i t i o n 0.05 m l of n-hexane and 0.1 m l of water t h e m i x t u r e was v i g o r o u s l y shaken. The a l i q u o t (1-2 pg of n-hexane) w a s used f o r t h e a n a l y s i s by GC-MS;LKB 2091 w i t h an i o n i z i n g energy of 70 e V was used. The column(1.5 m x 2 mm i . d . ) w a s packed w i t h 10% SP-2401 on 100-120 Supelcoport and w a s o p e r a t e d a t 140°using a helium flow r a t e of 20 ml/min. The i n j e c t o r and t h e i o n s o u r c e temperature w e r e 230° and 27OoC, respectively.

.

The mininam! d e t e c t a b l e c o n c e n t r a t i o n was 5.7 x 10-16 mole/sec. The s t a n d a r d curve w a s l i n e a r over a range of 10-400 ng/ml. The r e l a t i v e S.D. was 2.6% a t 100 ng/ml and ? 4.3% a t 10 ng/ml (n = 5 ) .

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

74

7.4

NMR S p e c t r o m e t r i c Methods

a)

Truczan and Lau-Cm(l27) d e s c r i b e d a s i m p l e NMR s p e c t r o s c o p i c method f o r t h e a s s a y of busulphan i n t a b l e t s . 20 G r a m of busulphan t a b l e t s are f i n e l y powdered, an a c c u r a t e l y weighed q u a n t i t y of powder e q u i v a l e n t t o 6 mg of busulphan i s t r a n s f e r r e d t o a 15 m l v i a l . To t h i s 1 m l of i n t e r n a l s t a n d a r d s o l u t i o n (11.68 mg of methenamine i n 10 m l of chloroform) was added, t h e v i a l was c l o s e d w i t h a septum and crimper s e a l e d w i t h an aluminium seal. 2 M I of chloroform w a s added u s i n g a s y r i n g e by i n t r o d u c i n g t h e n e e d l e t h r o u g h septum. The c o n t e n t s were shaked v i g o r o u s l y . The i n s o l u b l e matter i s allowed t o s e t t l e and 0.5 t o 1 m l of upper l a y e r i s t r a n s f e r r e d t o NMR t u b e . 1 Drop of r e f e r e n c e s t a n d a r d (50 mg t e t r a m e t h y l s i l a n e (TMS) i n 10 m l chloroform) was added, caped and shaken. The NMR s p e c t r a were r e c o r d e d w i t h a 200 MHz, Wise b o r e , F o u r i e r t r a n s f o r m s p e c t r o m e t e r equipped w i t h 5 mm p r o t o n probe and computer w i t h 24 K memory. The s p e c t r o m e t r y c o n d i t i o n s were: ambient t e m p e r a t u r e = 30OC, frequency 1 h = 200.06 MHz, Observation frequency range = 1600 H2. P u l s e width Pulse r e p e t i t i o n t i m e FTD d a t a p o i n t s Observation t i m e

= =

3

=

9.13 sec. 14 sec. 8 2.2 m i n u t e s .

I n t e g r a t e t h e broad t r i p l e t a t about 4.3 ppm due t o t h e 4-methylene p r o t o n s (-CH2-O-S02-) of busulphan and t h e s i n g l e t s and about 4.7 ppm due t o t h e 12 methylene p r o t o n s of t h e i n t e r n a l s t a n d a r d . The q u a n t i t y of busulphan i n mg i s o b t a i n e d from:(AU/AS) x (EV./ES) x Au

=

As

=

Ev Es

= =

C

=

c,

where

I n t e g r a l v a l u e of signal r e p r e s e n t i n g bulsulphan. I n t e g r a l v a l u e of signal r e p r e s e n t methanamine. Formula weight of busulphan 14, 61.58. Formula wieght of methenamine/l2, 11.68. Weight i n mg of m e t h e n m i n e t a k e n f o r t h e analysis.

BUSULPHAN

b)

15

Feit and Rastrup-Andersen (128) have investigated the hydrolysis of busulphan by means of NMR spectroscopy. The final product was found to be tetrahydrofuran (THF). The rather unstable intermediate, 4-methanesulphonybutanol, was synthesized and proved to undergo cyclization to THF by intramolecular alkylation. The half-life of this first-order reaction in aqueous solution at 3 7 O was determined to be approximately 12 minutes at pH 3 as well as at pH 7.4. From the data obtained, it is concluded that 4-methansulphonyloxybutanol is unlikely to be responsible for the biological action of busulphan.

Acknowledgements The authors would like to thank Mr. Syed Rafatullah, Assistant Researcher, College of Pharmacy, King Saud Univeristy, for his technical assistance and to Mr. Tanvir A. Butt, secretary to the Pharmaceutical Chemistry Dept. for his secretarial services.

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

76

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CHLORAMBUCIL

Mohammad T L q * and AbduReah A . ke-Bad&**

*UepcuLtment 06 P h m a c o R o g y , **UepahGncn;t 06 Phacu~laceuLicaI Chern&in.fhy, College 06 P h m a c y , K i m j Saud LlniLImiXy, P.O. Box 2 4 5 7 , Riyadh-17451, S a u d i RtLabia.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

85

Copyright 01987 by Academic Press, Inc. All rights of reproduction in any form resewed.

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

86

1.

History

2.

Description 2.1 2.2 2.3 2.4 2.5 2.6

3.

Nomenclature Formulae Molecular Weight Elemental Composition Appearance, Color and Odor Crystal Properties

Physical Properties 3.1 3.2 3.3 3.4 3.5 3.6 3.7

Melting Point Extraction Solubility Moisture Content and Hygroscopicity Dissociation Constant Storage Spectral Properties

4. Synthesis 5. Pharmacokinetics 6. Therapeutic Uses

7.

Toxicity

8. Methods of Analysis

8.1 8.2 8.3 8.4

References

Identification Titrimetric Methods Spectrophotometric Methods Chromatographic Methods

87

CHLORAMBUCIL

1. History This aromatic derivative of mechlorethamine was synthesized first at the Chester Beatty Research Institute in England. It was synthesized among other aromatic derivatives of the chloroethylmines by the British chemists, Everett, Roberts and Ross Chlorambucil has shown activity in a variety (1,2). of human malignancies (3). These include chronic lymphocytic leukemia (4,5). Hodgkin's and non-Hodgkin's lymphoma ( 6 ) , choriocarcinoma, ovarian carcinoma, and breast carcinoma (7). It is most often employed in the long-term maintenance management of chronic lymphoet a1 (8) found that a response cytic leukemia. Moore rate of 19% was achieved in 52 advanced breast cancer patients. 2. Description 2.1 Nomenclature

2.1.1 Chemical Names 4-[Bis( 2-chloroethyl)amino]benzenebutanoic acid; 4-[ p- [ Bis ( 2-chloroethyl) amino]phenyl ] butyric acid; N,N-di-2-chloroethyl-y-p-aminophenylbutyric acid; ~-[p-Di(2-chloroethyl)aminophenyl]butyric acid; 4-[ 4-Di-( 2-chloroethyl ) aminophenyl] butyric acid; 4-[4-(Bis(2-chloroethyl)amino)phenyl]butanoic acid; p-Di(2-chloroethyl)aminophenyl butyric acid (9 -12). 2.1.2

Generic Names Chlorambucil, C.B. 1348. NSC 3088 Chlorbuti-

num (10-12).

2.1.3 Trade Names Chlorambucil , Leukeran, Chloroambucil, (11) Amboclorin; Chloraminophene; Linfolysin (10-12)

-

.

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

88

2.1.4

CAS R e g i s t r y No.

( 305-03-3)

.

Wiswesser Line N o t a t i o n

2.1.5

(13)

QV3R DN2G2G

2.2

Formulae 2.2.1

Empirical

(14J 5 )

C14H19C12N02 2.2.2

.

Structural

C 1CH 2CH2

\

/

N

CH CH CH COOH

C1CH2CH2

2.3

Molecular Weight 304.20

2.4

.

(10,15)

Elemental Composition C 55.27%; H 6.30%, 0 10.52% ( 1 0 ) .

2.5

C1 23.31%,

N 4.60%,

Appearance, Color and Odor

A w h i t e or off-white c r y s t a l l i n e or g r a n u l a r powder with a s l i g h t odor (11). 2.6

Crystal Properties F l a t t e n e d n e e d l e s from petroleum e t h e r ( 1 0 ) .

3.

Physical.

3.1

Properties

Melting P o i n t

Melts between

64'

and 69'

(12).

89

CHLORAMBUCIL

3.2

Extract ion Chlorambucil i s e x t r a c t e d by o r g a n i c s o l v e n t s from aqueous a c i d s o l u t i o n s ( 1 2 ) .

3.3

Solubility P r a c t i c a l l y i n s o l u b l e i n water (14). S o l u b l e a t 2OoC i n 1 . 5 p a r t s of a l c o h o l , i n 2.5 p a r t s o f chloroform, and i n 2 p a r t s of a c e t o n e , s o l u b l e i n e t h e r ( 1 0 ) and i n d i l u t e s o l u t i o n of a l k a l i hydroxides (11).

3.4

Moisture Content and Hygroscopicity Not more t h a n O . 5 % , determined by F i s c h e r t i t r a t i o n

(14). 3.5

D i s s o c i a t i o n Constant pKa v a l u e 5.8

3.6

(16).

Storage

It should be s t o r e d i n a c o o l p l a c e i n a i r t i g h t , l i g h t r e s i s t a n t c o n t a i n e r s (11).

3.7

Spectral Properties

3.7.1

U l t r a v i o l e t Spectrum The u l t r a v i o l e t spectrum of chlorambucil i n methanol e x h i b i t s maxima a t 258 nm ( E 1%, 1 cm 650) and 302 nm ( E 1%, 1 cm 8 5 ) ; and minima a t 225 nm and 281 nm ( 1 2 ) . The W spectrum o f chlorambucil i n w a t e r and i n methanol which was scanned from 200 t o 400 nm u s i n g DMS 90 Varian spectrophotometer. It e x h i b i t s a b s o r p t i o n maxima a t 257 nm and a t 302 nm and minima a t 224 and 280 nm. The W s p e c t r a i n w a t e r and i n methanol a r e shown i n Figs [l] and [ 2 ] , respectively.

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR 2

so. 80

90

-

'

80

. 70 .so . 50 .LO

40.

.lo

.20 10.

.10

A 2 00

250

Wavelenplh

2 00

IS0

Wardepth

300

300

150

I50

F i g u r e 2 : U l t r a v i o l e t Spectrum o f Chlorambucil i n Methanol.

91

CHLORAMBUCIL

3.7.2

I n f r a r e d Spectrum The i n f r a r e d spectrum of chlorambucil i s shown i n F i g . [ 3 ] . The spectrum w a s o b t a i n e d a s K B r d i s c and i s recorded i n Pye-Unicam SP 1025 i n f r a r e d spectrophotometer. The f r e q u e n c i e s and t h e i r s t r u c t u r a l assignments are as follows:Frequency (em-')

Assignment

1720

O H s t r e t c h and CH stretch C = 0 acid

1622

C = C aromatic

1450

CH deformation

3100-29'70

730

C-C1

stretch

Other f i n g e r p r i n t bands a r e 1530, 1465 and Clarke ( 1 2 ) r e p o r t e d t h e p r i n c i p a l peak a r e 1695, 1520 and 1229 cm-l.

830 cm-1.

3.7.3

Carbon-13 Nuclear Magnetic Resonance S p e c t r a ( C - 1 3 NMR) The C-13 NMR n o i s e decoupled and o f f resonance s p e c t r a a r e shown i n Figs. [ 4 ] and 151, r e s p e c t i v e l y . Both were recorded i n d e u t e r a t e d chloroform on a Jeol-XL-100 MHz NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e as a r e f e r e n c e s t a n d a r d . The s p e c t r a l assignments are l i s t e d below:-

10 9 H C ~H~

c iC

&@

/ C1CH2CH2

10' 9'

7'

6'

4 3 2 1 CH2-CH2-CH2-COOH

L

92 Wavenumber

Figure 3 : Infrared Spectrum of Chlorambucil as KBr Disc.

93

, 0

W

L

v) .r

Y

E

c

.r

Spectrum o f Chlorambucil in CDC13 (Noise Decoupled). YO

..

d

Figure 4 : "C-NMR

\

a

I

a

a

Figure 5 : 13C-NMR Spectrum o f Chlorambucil i n CDC13 (Off-Resonance).

95

CHLORAMBUCIL

Chemical s h i f t ( PPm 1

Carbon No.

180,3

1

S

2

33.8"

t

3

33.4"

t

4

26.4

t

5

130.2

S

and 6'

129.6

d

7 and 7'

112.1

d

8

144.3

S

9 and 9'

53.5

t

10 and 10'

40.4

t

6

s = singlet

3.7.4

Mult i p l ic ity

d = doublet

t = triplet I

Proton Magnetic Resonance S p e c t r a ( H-NMR)

1 The 60 MHz H-NME3 spectrum of chlorambucil i n d e u t e r a t e d chloroform i s shown i n F i g . [ 61. The spectrum w a s recorded on Varian T ~ O - A NMR spectrometer u s i n g t e t r a m e t h y l s i l a n e (TMS) as t h e i n t e r n a l standard

.

The f o l l o w i n g are t h e s t r u c t u r a l assignments: Chemical s h i f t (ppm)

Assignments

M u l t i p l e t a t 1.8-2.8 -(CH ) -CO p r o t o n s S i n g l e t a t 3.60

23 -N-CH2CH2 p r o t o n s

M u l t i p l e t a t 6.6-7.08

Aromatic p r o t o n s

S i n g l e t a t 11.5

O H ( a c i d i c ) exchangea b l e w i t h D20, Fig. [ T I .

4

500

. - . . .... ,... .. .. "..,".,,',..

l - - - . v

0

100

200

300

400

--,-

TY I

[ . . . . I Qo

7-0

. . . .1

6-0

I

.

.

.

I

5-0

.... .... .... .... .... 6

I

I

I

1

4-0

3-0

2.0

14

Figure 6 : 'H-NMR Spectrum o f Chlorambucil i n Deuterated Chloroform.

0

200

100

L'

Figure 7 : 'H-NMR Spectrum o f Chlorambucil in Deuterated Chloroform + D20.

0

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

98

3.7.5

Mass Spectrum The e l e c t r o n impact ( E I ) mass spectrum o f chlorambucil a t 70 e V , r e c o r d e d on Varian Mat 311 mass s p e c t r o m e t e r u s i n g i o n source p r e s s u r e of 10-6 T o r r , ion s o u r c e temperature of 1 8 0 O c and a n emission c u r r e n t of 300 uA. The spectrum i s shown i n F i g . [ a ] . The spectrum is dominated by m / e 254 i o n ( b a s e peak) r e s u l t i n g from t h e l o s s of CH2C1 and H . A proposed mechanism of f r a g m e n t a t i o n and t h e m / e o f t h e major fragments i s given i n Scheme 1. The chemical i o n i s a t i o n ( C I ) spectrum ( F i g . [ 9 ] ) i s o b t a i n e d on a Finnigan 4000 mass s p e c t r o m e t e r w i t h i o n e l e c t r o n energy of 1 0 0 e V , i o n source p r e s s u r e o f 0.13 Torr, i o n s o u r c e t e m p e r a t u r e o f 1 5 0 O c and emission c u r r e n t o f 300 PA. The spectrum shows a pronounced peak r e s u l t i n g from t h e loss of HC1 (& - 36) c o n s t i t u t e t h e base peak a t m / e 268 and a molecular i o n peak at m / e 304. A proposed f r a g m e n t a t i o n pathway for t h e fragment i n t h e E I spectrum i s shown i n Schemes l a and lb. The mass s p e c t r a l assignments o f t h e prominant i o n s under C I conditions a r e given i n t h e following

m/e

Fragment

306

[ M + 2H]+

305

MH+

304

M+

-

303

[MH

286

[M

-

268

[M

- HCl]'

270

[M + 2 H

254

[MH

232

[M - 2 H C 1 ] +

H20]+

-

-

H20]

+

(H2 + C H 2 C l ] +

1oo.c

50.0

WIE

50

100

1so

ZOO

250

Figure 8 : Mass Spectrum o f Chlorambucil ( E I ) .

300

I

1oo.c

I

3

5O.C

'r MI€

200

220

240

260

'i 2 a

300

320

Figure 9 : Mass Spectrum of Chlorambucil

340

(CI).

I0

310

100

z 0 N

8 6N L W

-N

M

0

a,

M

E

\

4

00

a,

4

E

\

+z

I' Y

z-z u

+z

Ill

db..

W

4

a,

4

E

\

r .

4 4

0

m

0

w

E

rd

+J

.rl

rd

t,

k k

0

w w

a

.d

s8

m

a a, 0 PI 0

k

a

0

m/e 254 H

Scheme l b :

Proposed mechanism of fragmentation of chlorambucil.

103

CHLORAMBUCIL

4.

Synthesis E v e r e t t et g (1) have s y n t h e s i z e d chlorambucil and some o t h e r n i t r o g e n mustard. The s y n t h e s i s have been r e p o r t e d (17,18) as f o l l o w s :

0

N i tr a t ion

CH2CH2CH2COOH

a

02N

-(Ot

02N

CH2CH2CH2COOH

CH2CH2CH2COOCH3

CH2CH2CH2COOCH3

0

HOCH2CH2

4

/

a-

/ C1CH2CH2 ClCH CH

\IN

L

CH2CH2CH2COOCH3

CH2CH2CH2COOH

ClCH ,CH,

L

3

Ethylene oxide CH3COOH

POC 1

CH2CH2CH2COOCH3

HOCH,CH, C~CH~CH~ 2J

Esterification

Reduction Pd/CaCO,

a

HzN

L

-

I

Hydrolysis

P

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

104

5.

Pharmacokinetics

5.1

Absorption, D i s t r i b u t i o n , Metabolism and Excretion

Oral absorption of chlorambucil i s adequate and r e l i a b l e . The drug has a h a l f - l i f e i n plasma of approximately 90 minutes, and it i s almost completely metabolized ( 1 9 ) . Although t h e i n t r a venous r o u t e produced higher c o n c e n t r a t i o n s , absorpt i o n from t h e g a s t r o - i n t e s t i n a l t r a c t w a s consist e n t l y r a p i d and appeared t o be complete; peakplasma concentrations were achieved i n 40 t o 7 0 minutes. A n hour a f t e r a d m i n i s t r a t i o n by e i t h e r r o u t e t h e r a t e of metabolism w a s s i m i l a r and s u f f i c i e n t t o make a c o n t r i b u t i o n t o t h e a c t i v i t y of t h e drug ( 2 0 ) . There appears t o be extensive metabolic degradation of t h e drug and a major m e t a b o l i t e , an aminophenyla c e t i c a c i d d e r i v a t i v e which has a h a l f - l i f e of about 2.5 h r i n man, has been i d e n t i f i e d . The u l t i m a t e m e t a b o l i t e ( s ) of chlorambucil i n man has y e t t o be defined and f u r t h e r work i s r e q u i r e d t o confirm t h e s e i n i t i a l pharmacokinetic observations

(21). McLean e t a1 ( 2 0 ) reported two m e t a b o l i t e s one w a s i d e n t i f i e d as t h e @-oxidation yroduct of chloramb u c i l ; 2[ k-N,N-bis( 2-chloroethyl)aminophenyl] a c e t i c a c i d , a l s o c a l l e d phenylacetic mustard. This m e t a b o l i t e i s known t o have an a l k y l a t i n g a c t i o n i n animals. Dorr and W i l l i a m ( 2 1 ) e x t e n s i v e l y reviewed t h e d i s p o s i t i o n of chlorambucil i n human being and experimental animals. I n r a t s , r a d i o a c t i v e drug appear t o be c l e a r e d from t h e blood r a p i d l y , and a t one h r t h e h i g h e s t t i s s u e c o n c e n t r a t i o n i s found i n t h e l i v e r . However, f a i r l y homogeneous t i s s u e d i s t r i b u t i o n w a s noted. The drug h a s a l s o been shown t o d i s t r i b u t e w e l l i n t o a s c i t i c f l u i d a f t e r subcutaneous administ-ation. S i x t y percent of drug r a d i o a c t i v i t y w a s excreted i n t o t h e u r i n e by 24 h r , with t h e m a j o r i t y of t h e remainder e x i s t i n g as t i s s u e as tissue-bound drug ( 2 2 ) . S i g n i f i c a n t drug binding t o gamma g l o b u l i n p r o t e i n s has been observed. Apparently no drug i s excreted i n t h e f e c e s . Because a p o r t i o n of t h e drug molecule has l i p o p h i l i c

CHLORAMBUCIL

105

p r o p e r t i e s , some f a t ( d e p o t ) drug s t o r a g e may occur. This could e x p l a i n t h e prolonged c l i n i c a l e f f e c t of chlorambucil o c c a s i o n a l l y observed i n man.

6.

Therapeutic Uses C l i n i c a l t r i a l s of c h l o r m b u c i l were i n s t i t u t e d i n 1954. During t h e subsequent 5 y e a r s t h e substance w a s s t u d i e d i n q u i t e some d e t a i l . The t h e r a p e u t i c uses of chlorambucil have been discussed by Larionov ( 2 3 ) . The drug i s given o r a l l y i n t a b l e t form i n doses from 0 . 1 t o 0.4 mg/kg, i . e . 6 t o 25 mg d a i l y . The course of treatment u s u a l l y l a s t s 3-9 weeks. The t o t a l dose on average i s about 400 mg per course but i n i n d i v i d u a l p a t i e n t s it v a r i e s g r e a t l y with t h e form of t h e d i s e a s e , t h e s t a g e , t h e condition of t h e organism, previous t r e a t m e n t , e t c . The t h e r a p e u t i c e f f e c t does not come a t once; but u s u a l l y i n t h e 2nd t o 3rd week a f t e r s t a r t ing t h e drug. A t t h e s e doses s i d e e f f e c t s i n t h e g a s t r o i n t e s t i n a l t r a c t a r e r a r e l y observed. Towards t h e end of t h e course , neutropenia and thrombocytopenia may appear, t h u s n e c e s s i t a t i n g withdrawal of t h e p r e p a r a t i o n . The maintenance dose of t h e compound i s from 0.03 t o 0 . 1 mg/kg, i . e . 2-6 mg d a i l y . Chlorambucil g i v e s t h e b e s t e f f e c t i n chronic lymphoid leumaemia both i n t h e leukaemic and aleukaemic form and, i n p a r t i c u l a r , i n t h a t v a r i a n t known as f o l l i c u l a r lymphoma Galton and T i l l ( 2 4 ) Altman e t a1 ( 2 5 ) , Israels _ e t -a1 (261 Doan e t a1 ( 2 7 ) . I n chronic lymphoid leukaemias, chlorambucil i s given i n doses of 0 . 1 0.2 mg/kg, i . e . 6-12 mg d a i l y f o r 4-8 weeks. The e f f e c t c o n s i s t s remission expressed, i n p a r t i c u l a r , i n r e d u c t i o n of t h e lymph nodes, s p l e e n and l i v e r , i n c r e a s e d haemoglobin, f a l l i n lymphocytosis and sometimes i n c r e a s e i n n e u t r o p h i l s i n t h e blood. The remission l a s t s from 3 months t o 1-3 y e a r s . A f t e r t h e onset of a r e l a p s e , remission can again be obtained i n some p a t i e n t s by t h e same drug ( 2 3 ) .

.

Moore et a1 ( 8 ) used a continuous d a i l y dose of 0 . 1 0.2 mg/kg. The drug may a l s o be administered on an i n t e r m i t t e n t b a s i s ( 0 . 4 mg/kg every four weeks ( 2 8 ) . Both regimens commonly include prednisone. On t h e i n t e r m i t t e n t schedule, monthly pulse doses as high as 1 . 5 2 . 0 mg/kg were w e l l t o l e r a t e d and d i d not produce undue marrow t o x i c i t y . A n e f f e c t i v e biweekly dosing schedule

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

106

w i t h l e s s e n e d hematologic t o x i c i t y i s a l s o r e p o r t e d : 0.4 mg/kg every 2 weeks, i n c r e a s i n g by 0 . 1 mg/kg increments t o t o x i c i t y o r remission ( 5 ) .

7.

Toxicity According t o Dorr and W i l l i a m ( 2 1 ) chlorambucil i s one of t h e b e s t t o l e r a t e d o r a l a l k y l a t i n g a g e n t s . Bone marrow d e p r e s s i o n , i n c l u d i n g n e u t r o p e n i a , thrombocytop e n i a , and lymphopenia, o c c u r s w i t h prolonged u s e . I r r e v e r s i b l e bone marrow damage, however, may o c c u r . Thus myelosuppression i s t h e common d o s e - l i m i t i n g t o x i c i t y with chlorambucil. G a s t r o i n t e s t i n a l d i s t r e s s w i t h l a r g e r doses o c c u r s but i s u s u a l l y not s e r i o u s l Rare h e p a t i t i s and d i s t u r b a n c e s o f l i v e r f u n c t i o n have a l s o been r e p o r t e d . C e n t r a l nervous system s t i m u l a t i o n h a s occurred w i t h chlorambucil but i s uncommon u n l e s s l a r g e doses are used. I n a d v e r t e n t t o x i c i n g e s t i o n s i n c h i l d r e n o f 1.5 5 mg/kg have occurred without f a t a l i t i e s b u t w i t h moderate t o x i c i t y c h a r a c t e r i z e d by vomiting, a g i t a t i o n , i r r i t a b i l i t y , and h y p e r a c t i v i t y followed by l e t h a r g y and e v e n t u a l m i l d pancytopenia ( 2 9 , 3 0 ) . Chromosomal damage i s w e l l documented f o r t h i s agent although t h e e x a c t mechanism i s not w e l l known ( 3 1 ) . Lerner ( 3 2 ) has f u r t h e r a s s o c i a t e d c h r o n i c chlorambucil t h e r a p y w i t h a high i n c i d e n c e o f secondary a c u t e myelogenous leukemia.

-

S i m i l a r t o some o t h e r a l k y l a t i n g a g e n t s , chlorambucil can a l s o cause a l v e o l a r d y s p l a s i a and pulmonary f i b r o s i s w i t h long-term u s e ( 3 3 ) . A good c l i n i c a l response t o drug d i s c o n t i n u a n c e and c o r t i c o s t e r o i d s w a s n o t e d i n t h i s c a s e . Spermatogenesis i s a l s o depressed w h i l e t h e p a t i e n t i s on chlorambucil t h e r a p y ( 3 4 ) . LD 0 i n rats i n a s i n g l e a d m i n i s t r a t i o n i s 21-23 mg/kg ( 3 5 ) .

8.

Methods o f A n a l y s i s

8.1

Identification B r i t i s h Pharmacopoeia 1980 ( 9 ) d e s c r i b e s t h e following i d e n t i f i c a t i o n t e s t s : 1) Mix 0 . 4 g of chlorambucil w i t h 1 0 m l o f 2 M hydrochloric a c i d and a l l o w t o s t a n d f o r t h i r t y minutes, shaking o c c a s i o n a l l y . F i l t e r , wash t h e

CHLORAMBUCIL

107

r e s i d u e with two q u a n t i t i e s , each o f 1 0 m l , o f w a t e r , and r e s e r v e t h e mixed f i l t r a t e s and washi n g s for t e s t f o r i d e n t i f i c a t i o n 2. Melting p o i n t o f t h e r e s i d u e , a f t e r d r y i n g over phosphorous pentoxide a t a p r e s s u r e not exceedi n g 0.7 KPa (about 5 t o r r ) f o r t h r e e h o u r s , about 1 4 6 O . 2)

To 1 0 ml of t h e mixed f i l t r a t e and washings

r e s e r v e d i n t e s t f o r i d e n t i f i c a t i o n , add 0 . 5 m l of potassium m e r c u r i i o d i d e s o l u t i o n ; a b u f f c o l o r e d p r e c i p i t a t e i s produced. To a f u r t h e r 1 0 m l add 0.5 m l of potassium permanganate solution t h e purple color i s discharged.

3 ) Mix 0.6 g w i t h 0.2 m l o f phenylhydrazine, h e a t i n a water b a t h f o r t e n minutes, s t i r r i n g o c c a s i o n a l l y , add 2 m l of a b s o l u t e e t h a n o l , h e a t f o r twenty seconds, f i l t e r immediately, and a l l o w t o stand f o r t h i r t y minutes. C r y s t a l s of phenylhydrazinium c h l o r i d e are formed, which, a f t e r washing with two q u a n t i t i e s , each o f 1 m l , o f a b s o l u t e e t h a n o l and d r y i n g a t 105O, have a m e l t i n g p o i n t of about 245' , w i t h decomposition.

U.S. Pharmacopeia XX ( 1 4 ) d e s c r i b e t h e f o l l o w i n g t e s t s f o r t h e i d e n t i f i c a t i o n o f chlorambucil:

1) The i n f r a r e d a b s o r p t i o n spectrum of a 1 i n 125 s o l u t i o n i n carbon d i s u l f i d e , i n a 1 mm c e l l , e x h i b i t s maxima o n l y a t t h e same wavelength as t h a t o f a s i m i l a r s o l u t i o n o f USP Chlorambucil Reference Standard. 2 ) D i s s o l v e 50 mg i n 5 ml o f a c e t o n e , and d i l u t e w i t h water t o 1 0 m l . Add 1 drop of 2 N s u l f u r i c a c i d , t h e n add 4 drops of s i l v e r n i t r a t e ( t e s t s o l u t i o n ) : no opalescence i s observed immediately (absence of c h l o r i d e i o n ) . Warm t h e s o l u t i o n on a steam b a t h : opalescence develops ( presecne o f i o n i s a b l e c h l o r i n e )

.

8.2

T i t r i m e t r i c Methods U . S . Pharmacopeia XX ( 1 4 ) d e s c r i b e s t h e following method: Dissolve about 200 mg o f c h l o r a m b u c i l , a c c u r a t e l y weighed, i n 1 0 ml o f a c e t o n e , add 1 0 m l o f water,

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

108

and t i t r a t e w i t h 0 . 1 N sodium hydroxide VS, u s i n g p h e n o l p h t h a l e i n TS as t h e i n d i c a t o r . Each m l of 0 . 1 N sodium hydroxide i s e q u i v a l e n t t o 30.42 mg o f C14HlgC12N02. B r i t i s h Pharmacopoeia f o l l o w i n g method:

1980 ( 9 ) d e s c r l b e s t h e

To 0.5 g of chlorambucil, add 20 m l of M sodium hydroxide and b o i l under a r e f l u x condenser f o r one hour. Cool, t r a n s f e r t h e m i x t u r e t o a 100-ml graduated f l a s k w i t h t h e a i d of w a t e r , add 50 m l o f 0 . 1 M s i l v e r n i t r a t e VS and 5 m l o f n i t r i c a c i d , mix, and add s u f f i c i e n t water t o produce 100 m l . F i l t e r and t i t r a t e t h e excess of s i l v e r n i t r a t e i n 50 m l o f t h e f i l t r a t e w i t h 0 . 1 M ammonium t h i o c y a n a t e VS, u s i n g 3 m l o f ammonium i r o n (111) s u l f a t e s o l u t i o n as i n d i c a t o r . Each m l o f 0.1 M s i l v e r n i t r a t e VS i s e q u i v a l e n t t o 0.01521 g of C14H19C12N02*

8.3

Spectrophotometric Methods 8.3.1

C o l o r i m e t r i c Method P e t e r i n g and Van Giessen ( 3 6 ) d e s c r i b e d a c o l o r i m e t r i c procedure f o r d e t e r m i n a t i o n o f n i t r o g e n mustards i n c l u d i n g Chlorambucil , Dopan, U r a c i l Mustard and Mustargen i n plasma. Mix t h e drug (10 t o 70 ug) , d i s s o l v e d i n a 5% s o l u t i o n of dimethylacetamide i n e i t h e r e t h a n o l o r 0.9% N a C l s o l u t i o n , with phthalate buffer s o lu tio n (pH 4 . 0 ) (1m l ) , 5% b ( p - n i t r o b e n z y 1 ) p y r i d i n e s o l u t i o n i n acetone (1 m l ) and 0.9% N a C l s o l u t i o n (1m l ) , and d i l u t e w i t h e t h a n o l t o 4 m l . Heat a t 80' f o r 20 min. , c o o l i n i c e , add N-KOH i n 90% e t h a n o l ( 0 . 1 nil), d i l u t e w i t h e t h a n o l a t 600 mp w i t h i n 2 o r 3 min. Carry o u t a b l a n k determination.

8.3.2

I n f r a r e d S p e c t r o m e t r i c Method Kozlov and Sbezhneva (37) r e p o r t e d an i n f r a r e d s p e c t r o s c o p i c a n a l y s i s of chloramb u c i l i n pharmaceutical p r e p a r a t i o n s . S p e c i a l l y t o determine t h e amount of a c t i v e

109

CHLORAMBUCIL

11

8.3.3

ingredients i n carcinostatic preparations c o n t a i n i n g chlorambucil. 0 . 1 g of sample of t h e p r e p a r a t i o n i s e x t r a c t e d i n 100 m l o f acetone and t h e absorbance o f a l i q u o t i s measured a t 770 cm-1. The e r r o r does not exceed f 1%. U l t r a v i o l e t Spectrometric )Jethod

El-Tarras e t a 1 (38) developed a simple method f o r t h e d e t e r m i n a t i o n of chlorambucil i n pharmaceutical p r e p a r a t i o n s . The powdered t a b l e t s are e x t r a c t e d i n t o e t h a n o l (4 X 1 0 ml). The f i l t e r e d e x t r a c t w a s d i l u t e d t o 50 m l w i t h e t h a n o l . A p o r t i o n of t h i s s o l u t i o n w a s f u r t h e r d i l u t e d as r e q u i r e d w i t h e t h a n o l and t h e absorbance measured a t 250 nm v s e t h a n o l . The c o n c e n t r a t i o n o f t h e drug i s c a l c u l a t e d u s i n g a c a l i b r a t i o n curve. 8.3.4

Mass S p e c t r o m e t r i c Methods

e t -a1 (39) developed a s e n s i t i v e and Chang _ s p e c i f i c method f o r t h e d e t e r m i n a t i o n of chlorambucil and i t s m e t a b o l i t e s i n b i o l o g i c a l samples. The method i s based on s e l e c t e d i o n monitoring d e t e c t i o n f o l l o w i n g simple e x t r a c t i o n of t h e p a r e n t compound, i t s m e t a b o l i t e and i n t e r n a l s t a n d a r d (Chlorambucil-d8) from plasma and u r i n e samples. To determine chlorambucil and b [ b i s - ( 2 chloroethyl)ar,ino]phenyl a c e t i c a c i d i n 0.5 m l plasma or u r i n e add 0.5 m l of 4% HCl04 ( p e r c h l o r i c a c i d ) and [2H8] chlorambucil as i n t e r n a l s t a n d a r d . The mixture i s e x t r a c t e d w i t h e t h y l a c e t a t e and The o r g a n i c l a y e r w a s d r i e d h e x m e (1:l). w i t h p u r i f i e d N2 a t room t e m p e r a t u r e and s e v e r a l drops o f methylene c h l o r i d e were added t o d r y r e s i d u e t o form t r i m e t h y l s i l y l d e r i v a t i v e . T h i s i s submitted t o GLC column ( 5 0 cm X 2 mm) of 3% of OV-17 on gas Chrom Q ( 1 0 0 t o 1 2 0 mesh) a f t e r 40 seconds a t 190°C t h e column t e m p e r a t u r e i s r a p i d l y r a i s e d t o 31OoC; 7 0 eV-m.s detection i s u s e d . T r i m e t h y l s i l y l a t e d chlorambucil i s

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

110

monitored a t m / e 3?6 and 328, t h e d e r i v a t i v e of t h e m e t a b o l i t e of chlorambucil a t m / e 298, and t h a t of t h e i n t e r n a l s t a n d a r d a t m / e 383 and 385. The mean recovery and s t a n d a r d d e v i a t i o n were 94.3 1.3%.

*

et a1 ( 4 0 ) d e s c r i b e d an a n a l y t i c a l Jakhammer procedure f o r simultaneous d e t e r m i n a t i o n of chlorambucil and prednimustine i n plasma. The method i n v o l v e s e x t r a c t i o n and s e p a r a t i o n followed by d e r i v a t i z a t i o n t o chloramb u c i l methyl e s t e r and mass fragmentography. The drugs are e x t r a c t e d i n t o chloroform, hexane ( 3 : 7 ) and s e p a r a t i o n i s done by p a r t i t i o n a f t e r adding 0 . 1 M phosphate 0 . 1 M b o r a t e b u f f e r (pH 9 . 0 ) . Chlorambucil i n t h e aqueous phase add predenimustine i n o r g a n i c phase are each t r a n s e s t e r i f i e d w i t h methanol-3F3 d i e t h y l e t h e r a t e ( 4 : l ) . The compounds are determined u s i n g fragmentography. A Varian MAT mass spectrometer i n combination w i t h Varian Aerograph 1400 g a s chromatograph with a column o f 0.5% o f Carbowax 20 M on Chromosorb W-HP, are used. Analogues, prepared by i n t r o d u c i n g e i g h t 2H atoms i n t o t h e b i s - ( 2-chloroethy1)amino group of b o t h compounds are used as i n t e r n a l s t a n d a r d s and c a r r i e r s . The d e t e c t i o n l i m i t i s about 8 ng/ml f o r b o t h d e t e r m i n a t i o n s and t h e r e l a t i v e s t a n d a r d d e v i a t i o n 3% f o r chlorambucil and 5% prednimustine a t 30-60 ng/rnl l e v e l .

8.4

Chromatographic Methods

8.4.1

Thin Layer Chromatograph (TLC) Norpoth e t a1 ( 4 1 ) d e s c r i b e d a TLC method for t h e d e t e r m i n a t i o n o f a l k y l a t i n g cytos t a t i c a g e n t s on a t h i n l a y e r p l a t e w i t h i s o n i c o t i n a l d e h y d e benzothiazol-2-ylhydrazone. The drug samples are d i s s o l v e d i n methanol. The s p o t i s a p p l i e d on a s h e e t o r precoated p l a t e s . A f t e r developing and drying t h e chromatogram, it i s sprayed w i t h 3% s o l u t i o n of r e a g e n t i n acetophenone dimethylformamide ( 7 : 3 ) c o n t a i n i n g 0 . 0 1 m l of c o n c e n t r a t i o n H C 1 p e r 100 m l . It i s t h e n

111

CHLORAMBUCIL

p l a c e d over a b a t h o f acetophenone and b a t h and p l a t e are h e a t e d a t 2OO0C for 20 minutes; t h e cooled p l a t e i s t h e n sprayed w i t h t r i e t h y l a m i n e . Chlorambucil produces r e d s p o t . For q u a n t i t a t i v e d e t e r m i n a t i o n t h e c o l o r i n t e n s i t y o f t h e spot i s measured u s i n g chromatogram spectrophotometer

.

8.4.2

Gas Liquid Chromatography ( G L C ) Ehrsson _ et _ a1 (42) d e s c r i b e d a GLC t e c h n i q u e f o r d e t e r m i n a t i o n of chlorambucil i n plasma by g a s - l i q u i d chromatography w i t h s e l e c t e d ion-monitoring. 2 m l o f plasma sample c o n t a i n i n g 8 l.~g o f chlorambucil p e r m l w a s mixed w i t h h y d r o c h l o r i c a c i d , phosphoric a c i d , or phosphate b u f f e r ( 0 . 2 m l ) . The mixture w a s e x t r a c t e d with methylene c h l o r i d e ( 2 m l ) f o r 30 minutes. An a l i q u o t o f o r g a n i c phase was s e p a r a t e d and evaporated t o dryness with n i t r o g e n g a s . The r e s i d u e w a s d i s s o l v e d i n methanol-water 0 . 1 M, a c e t i c a c i d (75:15:10) and a n a l y s e d on g a s chromatography. A l t e r n a t i v e l y t h e plasma samples w e r e a d j u s t e d t o pH 3 w i t h 1 M phosphoric a c i d and e x t r a c t e d w i t h e t h y l e n e d i c h l o r i d e f o r 30 minutes. The organic phase w a s s e p a r a t e d and e x t r a c t e d with 0 . 5 m l of 0 . 1 M sod. s u l p h i d e f o r 5 minutes. The aqueous phase w a s s e p a r a t e d and h e a t e d f o r 1 5 minutes a t 80°C t o c o n v e r t chlorambucil i n t o t e t r a h y d r o t h i a z i n e d e r i v a t i v e , then c o n c e n t r a t e d phosphoric a c i d (0.91m l ) was added, and H2S removed w i t h N2 f o r 5 minutes. To t h i s O . 0 5 m l o f 12 M NaOH, 0 . 2 m l of 0.5 M t e t r a b u t y l ammonium, 0.2 ml methylene c h l o r i d e and 0.05 m l a l l y l b r o m i d e were added and t h e mixture w a s shaken f o r 30 min a t 2 5 O C . The o r g a n i c phase (1-2 mcg) w a s i n j e c t e d i n GLC ( a t Z50°C) on a column of 5% of OV-17 on gas-Chrome-Q o p e r a t e d with 70 eV.m. s . d e t e c t i o n . The i n t e r n a l s t a n d a r d used w a s [%8] chlorambucil. The peaks were monitored a t m / e 305 and 313 f o r chlorambucil and

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

112

2

[ H8] chlorambucil r e s p e c t i v e l y .

The c o e f f i c i e n t of v a r i a t i o n f o r 10ng/ml o f chlorambucil w a s 5%. 8.4.3

High-pressure Liquid Chromatography (HPLC) Newel1 et a1 ( 4 3 ) developed a HPLC method f o r e s t i m a t i o n o f c h l o r a m b u c i l , phenyla c e t i c mustard and predenimustine i n human plasma. One m l plasma w a s e x t r a c t e d w i t h 2 ml of e t h y l a c e t a t e , t h e emulsion formed by vigorous a g i t a t i o n on a Whirlimixer was f r o z e n by immersing t h e t u b e s i n a methanol carbondioxide b a t h a t - 6 8 O C . The t u b e s w e r e c e n t r i f u g e d a t 600 pg f o r 10 minutes a t 4 O C u n t i l t h e thawed aqueous phase c o u l d be s e p a r a t e d . The procedure w a s r e p e a t e d with f u r t h e r 2 m l of e th y la c e ta te , t h e pooled e t h y l a c e t a t e e x t r a c t s were d r i e d over anhydrous sodium s u l p h a t e f o r 1 hour a t room t e m p e r a t u r e . 2 m l of d r i e d e t h y l a c e t a t e e x t r a c t was evaporated t o dryness i n a stream o f n i t r o g e n at 4 5 O C . The r e s i d u e w a s d i s s o l v e d i n 100 m l o f e t h y l a c e t a t e f o r chromatographic a n a l y s i s . 50 1.11of t h e e x t r a c t w a s i n j e c t e d on a 1.1 Bondapak c18 column. L i n e a r g r a d i e n t o r e l u t i o n w a s done at 2 m l p e r minute w i t h aqueous 0.175 M - a c e t i c a c i d (from 40 t o 0 % ) i n methanol d u r i n g 1 0 minutes followed by i s o c r a t i c e l u t i o n w i t h methanol. T h i s method gave a good s e p a r a t i o n of chloramb u c i l i n l e s s t h a n 1 2 m i n u t e s . The absorbance o f e l u t e w a s monitored a t 254, and 280 nm simultaneously. D e t e c t o r response w a s r e c t i l i n e a r over a range o f 5 t o 1000 ng o f t h e compound. The recovery o f chlorambucil w a s c o n s t a n t over t h e range o f 0.05 t o 10m.

Leff and Bardsley ( 4 4 ) have r e p o r t e d pharmacokinetic of chlorambucil i n o v a r i a n carcinoma u s i n g a new HPLC assay. The a p p a r a t u s c o n s i s t e d o f a Waters A s s o c i a t e s ~ 6 0 0 0pump, a 10 x 0.46 cm s t e e l column packed w i t h S 5 ODs, a Waters A s s o c i a t e s Model U6K i n j e c t o r , a me-Unicam LC3 v a r i a b l e

113

CHLORAMBUCIL

wavelength W d e t e c t o r o p e r a t i n g at 254 nm. The mobile phase was water: methanol 20:80 ( v / v ) w i t h 0.1% ammonium a c e t a t e ( w / v ) The flow r a t e used w a s 1 ml min-1 ( = 500 p s i ) .

.

Zakaria and Brown ( 4 5 ) r e p o r t e d a r a p i d a s s a y f o r plasma chlorambucil and i t s m e t a b o l i t e ( p h e n y l a c e t i c mustard) u s i n g reversed-phase l i q u i d chromatography. Plasma ( 2 0 o r 30 ~ 1 i)s i n j e c t e d d i r e c t l y i n t h e chromatographic system, which i n c o r p o r a t e s a 5 cm Co: P e l 1 ODS pre-column f o r sample c l e a n up. A p a r t i s i l PXS-10/25 ODS column ( 2 5 cm X 4.6 mm) o r , f o r f a s t e r e l u t i o n , a chromegabond MC-18 column ( 1 5 cm X 4.6 mm) i s used for t h e a n a l y s i s . The mobile phase being methanol - 0.02 M KH2P04 (1:l o r 11:9, r e s p e c t i v e l y ) a t 1 . 5 o r 1 . 0 m l min-l, r e s p e c t i v e l y . C a l i b r a t i o n graphs are r e c t i l i n e a r over t h e range o f i n t e r e s t . Simultaneous d e t e c t i o n a t 280 and 254 nm a l l o w s picomole amounts t o be determined. Chatterji _ et _ a1 ( 4 6 ) have s t u d i e d k i n e t i c s of chlorambucil h y d r o l y s i s u s i n g highp r e s s u r e l i q u i d chromatography. The highp r e s s u r e l i q u i d chromatograph w a s equipped w i t h a fixed-volume l o o p i n j e c t o r and a f i x e d wavelength d e t e c t o r . A 250 X 4.6 mm i . d . reversed-phase column w a s used. S e p a r a t i o n was performed on a reversed-phase column (Zorbax C-8, 6 pm average p a r t i c l e s i z e , Dupont I n s t r u m e n t s , W i l l i n g t o n , D e l . ) and m e t h a n o l - a c e t o n i t r i l e - 0 . 0 1 M a c e t a t e b u f f e r , pH 4.5 ( 6 5 : 5 : 3 0 ) , a t a flow rate of 1 . 6 ml/min was used as t h e mobile phase. A 1 0 p1 full l o o p volume w a s q u a n t i t a t i v e l y i n j e c t e d , and t h e r e c o r d e r w a s set a t 0.32 a u f s (254 nm d e t e c t o r ) . Ekrsson e t a1 ( 4 7 ) d e s c r i b e d a reverse phase HPLC method t o s t u d y d e g r a d a t i o n o f chlorambucil i n aqueous s o l u t i o n . Chloramb u c i l and i t s d e g r a d a t i o n products;4-( 4-( 2-

chloroethyl-2-hydroxyethylamino)-phenyl)

b u t y r i c acid, are determined i n aqueous methanol. The chlorambucil s o l u t i o n i s

114

MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR

prepared by adding 1 0 mg o f compound i n 0.5 m l methanol and t h e volume i s made upto 100 m l with d i s t i l l e d water. A t d i f f e r e n t t i m e i n t e r v a l s e x t r a c t i o n i s done with e t h y l a c e t a t e , t h e solvent i s evaporated t o 0 . 2 m l under a n i t r o g e n atmosphere. Reversed-phase HPLC a n a l y s i s i s done on a column (15 cm X 4 mm) o f Li-Chrosorb RP-18 ( 5 urn). Methanollwater c o n t a i n i n g 0.01 M phosphoric a c i d i s used as mobile phase. A W d e t e c t o r a d j u s t e d a t 253.7 nm w a s used f o r d e t e r m i n a t i o n o f t h e concentration. Ahmed _ et _ a1 (48) d e s c r i b e d a q u a n t i t a t i v e method for d e t e r m i n a t i o n of chlorambucil i n plasma by r e v e r sed-phas e h i gh-pe r f ormanc e l i q u i d chromatography. The plasma samples a r e d e p r o t e i n i s e d by adding 4 volumes o f a c e t o n i t r i l e a t pH 3 . The t u b e s w e r e c e n t r i fuged f o r 2 minutes and t h e s u p e r n a t e n t w a s r a p i d l y f r o z e n i n s o l i d carbondioxide-acetone t o complete d e p r o t e i n i s a t i o n . The t u b e s a r e c e n t r i f u g e d for 2 m i n u t e s . The a l i q u o t s o o b t a i n e d w a s analysed on Water's Radial-PAK c18 ( 1 0 u m ) c a r t r i d g e , w i t h a c e t o n i t r i l e 0.2% a c e t i c a c i d (13:7) as mobile phase. Absorbance was monitored a t 263 nm and recorded on a Data Module. A s t a n d a r d curve was developed u s i n g a c e t o n i t r i l e s o l u t i o n o f chlorambucil s t a n d a r d . Acknowledgement The a u t h o r s would l i k e t o t h a n k Mr. Syed A l i Jawad f o r t h e t e c h n i c a l a s s i s t a n c e and Mr. Tanvir A . B u t t f o r t h e s e c r e t a r i a l a s s i s t a n c e i n t y p i n g t h e manuscript.

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Galton; M. T i l l , E. Wiltshaw,

68,915

(1958).

CH. A. Doan; B.K. Wiseman and B.A. Bouroncle. N.Y. Acad. S c i . , 6, 979 (1958).

&.

117

CHLORAMBUCIL

28. A.

Sawitsky; K.R. R a i ; 0. Glidewell and R.T. Blood, 50, 1049 (1977).

29*

A.A.

Silver,

Green; and J . L . Naiman, Am. J. D i s . C h i l d . ,

190 (1968).

30. S.

Wolfson and M.B.

116,

Olney, JAMA,165, 239

(1957).

4,22 (1975). Treat. Rep. , 62,1135

(1978).

31. B.R. Reeves, Br. Med. J . ,

32.

H.F. L e r n e r , Cancer

Cancer, 33. S.R. Cole; J . J . Myers and A.U. K l a t s k y , _ _ _ -41, 455,

(1978).

34. P. R i c h t e r ; C.J. Calamers and M.C. Morganfeld, Cancer, 25, 1026 (1970). Acad. S c i . , 68, 826

35.

L.A.

E l s o n , Ann. N . Y .

36.

H.G.

P e t e r i n g and G . J .

37

-

Y

~ 5 (1963). 9 N.E.

Kozlov and V.G.

29, 33 (1980).

(1958).

Van G i e s s e n , J . Pharm. S c i . ,

52,

Sbezhneva, Farmatsiya (Moscow),

9

38.

M.F. E l - T a r r a s ; M.M. E l l a i t h y and N.B. Pharma Fenn. , 2, 31 (1983).

39.

S.Y. Chang; B.J. Larcom; D.S. A l b e r t s ; B. Larsen; P.D. Walson and I . G . S i p e s , J . Pharm. S c i . , 80 (1980).

40.

T. Jakhammer; A. Olsson and L. Svensson, Acta Pharma. Suec. , 485 (1977).

41.

K. Norpoth, H.W. Addicks and U. W i t t i n g , A r z n e i m i t t e l Forsch. , 2 3 , 1529 (1973).

42.

H. Ehrsson; S. Eksborg; I . Wallin; Y . Marde and B . Joansson, J . Pharm. S c i . , 6 9 ( 6 ) , 710 (1980).

Tadros,

69,

14,

43. D . R . N e w e l l ; L . I . Heart and K.R. Harrap., J . Chromatogr 164,Biomed. Appl., 6,114 (1979).

44.

P. Leff and W.G.

1289 (1979).

B a r d s l e y , Biochem. Pharmacol.,

28,

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118

45.

46. )

47.

M. Zakaria and P.R. Brown, J. Chromatogr., 230, Biomed. Appl. , 19, 381 (1982). D.C.

Sci

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Chatterji, R.L. Yeager and J.F. Gallelli; J. Pharm. 71, 50 (1982).

H. Ehrsson; S. Eksborg;Inger Wallin and S.G. Nilsson, J. Pharm. Sci., @, 1091 (1980).

48. A.E. Ahmed; M. Moenig; and H.H.J. Farrish, J. Chromatogr. , 233, (1982), Biomed. Appl. , 22, 392 (1982)

.

CHLORZOXAZONE James T. Stewart Department of Medicinal Chemistry and Pharmacognosy College of Pharmacy University of Georgia Athens, Georgia Casimir A. Janicki Director, Analytical Control McNeil Pharmaceutical Spring House, Pennsylvania

Foreword, History, Therapeutic Category Description 2.1 Names, Formula, Molecular Weight 2.2 Appearance, Color, Odor, Taste 3. Synthesis 4 . Physical Properties 4.1 Infrared Spectrum 4.2 Ultraviolet Absorption Spectrum 4.3 Nuclear Magnetic Resonance Spectra 4.4 Mass Spectra 4.5 X-Ray Diffraction Pattern 4.6 Thermal Analysis 4.7 Melting Range 4.8 Solubility 4.9 pKa 5 . Methods of Analysis 5.1 Fluorescence Spectrophotometry 5.2 Ultraviolet Spectrophotometry 5.3 Iodometric Titrimetry 5.4 Gas Chromatography 5.5 Thin-Layer Chromatography 5.6 Paper Chromatography 5.7 High Performance Liquid Chromatography 5.8 Non-Aqueous Titrimetry 5.9 Colorimetry 5.10 Color Test 5.11 Polarizing Microscopy 5.12 Potentiometry 6 . Stability 7. Drug Metabolic Products, Pharmacokinetics, and Bioavailabi1ity 1.

2.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

119

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

JAMES T. STEWART AND CASIMIR A. JANICKI

120

7.1

Drug Metabolic Products 7.11 Urine Metabolism 7.12 In-vitro Metabolism 7.2 Pharmacokinetics and Bioavailability 8. Identification and Determination in Body Fluids and Tissues 8.1 Ultraviolet Spectrophotometry 8.2 Fluorescence Spectrophotometry 8.3 High Performance Liquid Chromatography 8.4 Gas Chromatography 9. Identification and Determination in Pharmaceuticals 9.1 Colorimetry 9.2 Ultraviolet Spectrophotometry 9.3 Fluorescence Spectrophotometry 9.4 Potentiometry 9.5 High Performance Liquid Chromatography 10. References

CHLORZOXAZOIW

1.

121

Foreword, History, Therapeutic Category Chlorzoxazone is a centrally active muscle relaxant for the treatment of painful muscle spasms associated with muscloskeletal disorders, such as fibrositis, bursitis, myositis, spondylitis, sprains, and muscle strains. The skeletal muscle relaxant properties were first discovered by Marsh at McNeil Laboratories In the late 1950s (1). Onset of therapeutic activity is observed within one hour and the duration usually lasts up to 6 hours. The drug was reported to be useful in the treatment of painful spasm of skeletal motor muscles by acting at spinal levels of the central nervous system (2). The drug exhibits minimal adverse effects and almost no gastrointestinal irritation. Chlorzoxazone was ranked among the top 100 most widely prescribed drugs for many years ( 3 ) .

2. Description 2.1 Names, formula, molecular weight Generic name - Chlorzoxazone Trade name Marketed by McNeil Pharmaceutical -__. under the trade name, Paraflex. Chemical names - 5 Chloro-2-(3H)-benzoxazolone, 5-Chloro-2-benzoxazolol, 5-Chloro-2hydroxybenzoxazole, 2-Hydroxy-5-Chlorobenzoxazole, 5-Chlorbenzoxazolin-2-one, 5-Chlorobenzoxazolidone. Chemical Abstracts Registry No. 95-25-0

-

C7H4C1N02

Mol. Wt. 169.58

2.2 Appearance, Color, Odor, Taste - Odorless colorless crystals or a white to creamy white crystalline powder with a bitter taste.

JAMES T. STEWART AND CASIMIR A. JANICKI

122

3. Synthesis Chlorzoxazone is synthesized by either the acid hydrolysis of 2-amino-5-chlorobenzoxazole (4) or by treating 2-amino-4-chlorophenol with phosgene in ethyl acetate (5). It can also be prepared from the reaction of sodium ethoxide with 2-ethoxycarbonylamino4-chlorophenol in tetralin (6).

t 4.

ClCOCl

Physical Properties 4.1 Infrared I SDectrum - An FTIR sDectrum of chlorzoxazone is shown in Figure 1.

-1 cm 3470 3155 3117 3087 3057 2978 2282 1924 1885 1772 1621 1482 1463

%T 72.6 36.3 39.7 37.3 35.2 46.4 69.1 67.4 70.2 3.2 30.5 13.5 34.3

-1

cm % T 1367 43.8

1330 1300 1262 1153 1101 1064 964 923 867 845 805

53.6 27.3 26.2 21.1 58.7 54.8 16.4 33.1 46.1 23.7 19.2

-1

% T cm 732 52.8

713 600 591 552 417 394 382 352 234 216 210

32.2 45.8 41.8 49.0 67.6 70.2 79.1 66.1 8.8

5.0 6.7

11i -

I

rI r I rI

I I I

I I

r

.....

....

I

I

i i

I i

I

i

Fig. 1

FTIR spectrum of chlorzoxazone

JAMES T. STEWART AND CASIMIR A. JANICKI

124

4.2

Ultraviolet Absorption Spectrum - A solution of the drug in absolute methanol shows a maximum at 282 ? 2 nm and a m i n i m u m a i 248 _t 2 nm. There is an inflection at about 228 GII! ( s e e Figure 2).

--

4.3. Nuclear lvlagnetic hpectra - 13C and proton - Resenance nuclear magnetic resonance spectra are shown in Figures 3 and 4.

b

C 13C NMR Carbon 6(ppm) a 39.4 DMSO-dc J b 109.8 C 110.8 d 121.4 e 127.7 f 131.7 g 142.1 h 154.2

C

NH d Proton NMR Proton fi (DDm) r a 2.49 DMSO-d, b 3.36 HDO C 7.10 d 7.13 e 7.28 f 11.8 ~~

~

Y

.r

--I

f

CHLORZOXAZONE

350

220 WAVELENGTH ( n m )

Fig. 2

W spectrum of chlorzoxazone in methanol

I

Fig. 3

13C-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide

127 "-2.1

Fig. 4

- ......7 7 1d.r

I

8.1

..-.--I---* .I

7.1

6.1

PPM

5.0

4.1

3.1

2.1

1.0

'H-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide

JAMES T. STEWART AND CASIMIR A. JANICKI

128

Spectra - Electron impact (EI) and chemical 4.4 EM ionization (CI) mass spectra are shown in Figures 5 and 6. 4.5 X-Ray Diffraction - Figure 7 shows an X-ray diffraction pattern for chlorzoxazone powder.

4.6 Thermal Analysis - A DSC thermogram of chlorzoxazone is shown in Figure 8. 4.7 Melting Range - The melting point range is 189-194°C.

4.8 Solubility - Chlorzoxazone is very slightly soluble in water (0.2 - 0.3 mg/ml), sparingly soluble in ethanol and methanol, soluble in chloroform (1 g in It is freely 250 ml) and ether (1 g in 60 ml). soluble in aqueous solutions of ammonia and alkali hydroxides.

4.9 pKa

-

The pKa of chlorzoxazone in 8.3.

5. Methods of Analysis 5.1 Fluorescence Spectrophotometry - Stewart and Chan have studied the reaction of chlorzoxazone with dansyl chloride, fluorescamine, 2,4-dihydroxybenzaldehyde, and salicylaldehyde to form fluorophores (7). Fluorescamine gave the highest intensity product and was used to analyze chlorzoxazone in the 0.27 - 3.4 pg/ml range. The same authors also reported that chlorzoxazone exhibits native fluorescence in chloroform using excitation and emission wavelengths of 286 and 310 nm, respectively (8). 5.2

Ultraviolet Spectrophotometry - Conney et a1 utilized a back-extraction approach to quantitate chlorzoxazone at 289 nm (9). The drug can also be directly determined at 282 nm in absolute methanol (10)

5.3

*

-

Iodometric Titrimetr Beral et a1 titrated c h l o r z o x a z o d t r e a t m e n t with boiling 10% sulfuric acid, potassium bromide and potassium bromate) with 0.1 N sodium thiosulfate after potassium iodide was added to a cooled solution (11).

5.4

Gas Chromatography -

Kaempe has chromatographed chlorzoxazone on a packed 15% Dexsil 300 on HP

11 9 90 80

-

70 -

I

60 50 -

113

30 -

20

51

63

d

I

d.

I,,,

I

140

.. .

I

.. ,

I;

. . ,[ASS 130 200

40

60

Fig. 5

Electron impact (EI) mass spectrum of chlorzoxazone

30

100

120

160

MH+

'0°1 80 -

60 -

%I

a

!!

50

Fig. 6

90

I30

m/e

I 70

210

250

Chemical ionization (CI) mass spectrum of chlorzoxazone

90 -

80 -

70 -

60 -

50

-

e

W e

1

Fig. 7

X-Ray diffraction pattern of chlorzoxazone

5.00

7

CHLORZOXAZONE. SCAN RATE:

0.00

'

L O T 2933

1.00 mg

WT:

*. MI

5.00 deg/rnxn

380.M

4M.M

42O.M

4 h no

bu). MI

TEMPERATURE

(K)

Fig. 8 DSC thermogram of chlorzoxazone

460.00

5th 01 DSC

CHLORZOXAZONE

133

Chromosorb W 801100 mesh glass column (6 ft x 6.4 m i.d.). The retention time was 0.62 relative to caffeine using a column temperature of 220°C (12). Parker et a1 attempted to chromatograph the drug on SE-30 (0.05% on glass microbeads 60/80 mesh) at 150 and 165°C column temperatures with no success (13). Desiraju et a1 chromatographed chlorzoxazone on a 6 ft x 4 mm i.d. glass column packed with 3% OV-1 on 60/80 mesh Gas Chrom Q (14). Column temperature was 130°C and retention time was 3.25 minutes using a column flow rate of 30 ml/minute. Beckett et a1 - reported that chlorzoxazone can be chromatographed on a 2.5% SE-30 on 80/100 mesh Chromosorb W acid-washed HMDS treated 5 ft x 4 mm i.d. glass column at a column temperature of 225°C (15). Retention time was 0.69 relative to diphenhydramine. Nitrogen was used as carrier gas at 50 ml/min a.nd FID detection was utilized. Ardrey and Moffat also reported that a 2 m x 4 mm i.d. glass column containing 2.5% SE-30 on 80/100 mesh Chromosorb G acid-washed HMOs treated will separate chlorzoxazone using a column temperature of 173°C (16). Retention time is given relative to n-alkanes. Pedroso and Moraes have chromatographed chlorzoxazone on 2.5% SE-30 and 3% OV-17 at 200 and 220°C column temperatures, respectively (17). They used the retention data to perform qualitative analysis of the drug based on Kovat's Retention Indices. 5.5

Thin-Layer Chromatography

Stationary Phase

Mobile Phase

Silica Gel HF254

Chloroform-methanol Chloroform-methanol Chloroform-methanol Chloroform-methanol Chloroform-methanol

Kieselgel 60F254

19:l 9:l 4:l 7:3 3:2

Toluene ethylacetate 85% formic acid (50:45:5)

18 0.32 0.58 0.87 1.00 1.00

0.69

19

134

Kieselgel 60F254

Kieselgel 60F254

Silica Gel 60F254

JAMES T. STEWART AND CASIMIR A. JANICKI

0.6819

-

Toluene Isopropanol concd. ammonium hydroxide (70:29 :1)

0.55

-

19

Toluene-dioxane methanol ammonium hydroxide (20:50:20:10) Toluene Acetone 2N Acetic Acid (30:65:5) -

-

-

0.8320

20

Silica Gel 60F254

Toluene Isopropanol Ethyl Acetate 2N Acetic Acid (10:35:35:20)

0.95

Bonded C18F

Methanol 0.5% Phosphoric Acid 3% Sodium Chloride (60:10:30)

0.3220

Bonded C18F

Isopropanol 0.5% Phosphoric Acid 3% Sodium Chloride ( 4 0 : 10:50)

Bonded C18F

Isopropanol Methanol 0.5% Phosphoric Acid 3% Sodium Chloride (23:23:10:44)

Bonded C18F

Bonded C18F

-

-

-

Tetrahydrofuran Methanol 0.5% Phosphoric Acid 3% Sodium Chloride (28:28:10:34) Methanol 2 N Ammonia 3% Sodium Chloride (60:10:30)

-

20 0.35

0.33

20

20 0.32

20 0.60

135

CHLORZOXAZONE

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

5.6

Isopropanol 2N Ammonia 3% Sodium Chloride (40:10:50)

0.4720

Isopropanol Methanol 2N Ammonia 3% Sodium Chloride (23:23:10:44)

-

0.4320

Tetrahydrofuran Methanol 2N Ammonia 3% Sodium Chloride (28:28:10:34)

0.5520

-

Acetonitrile 3% Sodium Chloride (50:50)

0.4620

Methanol 3 % Sodium Chloride (60 :40)

0.32

Ace tone 3% Sodium Chloride (50:50)

0.25

Tetrahydrofuran 3% Sodium Chloride (43:57)

20 0.18

Isopropanol 3% Sodium Chloride (40:60)

0.35

Methanol Isopropanol Tetrahydrofuran 3 % Sodium Chloride (17:17:17:49)

0.36

20

20

20

20

Paper Chromatography - Clarke reported that chlorzoxazone gave an R of 0.93 using a mobile phase of 87 :13 n-butanol-waEer containing 0.48% citric acid (21)

*

JAMES T. STEWART AND CASIMIR A. JANICKI

136

5.7

5.8

5.9

High Performance Liquid Chromatography - Stewart a1 have chromatographed chlorzoxazone on an octadecylsilane column using varying proportions of absolute methanol-distilled water (22). Chlorzoxazone was effectively separated from acetaminophen in a mixture with 50:50 methanol-water at a 2.0 ml/min flow rate using refractive index detection. The drug has also been chromatographed on an octadecylsilane column using 60:40 watermethanol (23), and a 1:l acetonitrile-water mixture (10). These methods utilized ultraviolet detection at 280 nm. Non-Aqueous Titrimetry - Chlorzoxazone is titrated with 0.1 N sodium methoxide in dimethvlformamide using thymol blue as indicator ( 2 4 ) . The drug can also be titrated in dimethylsulfoxide using O.1N propanolic potassium hydroxide as titrant and metanil yellow as visual indicator (25). Colorimetry - Sanghoi reported a colorimetric procedure for chlorzoxazone based on base hydrolysis of the drug followed by diazotization of the product with nitrous acid formed -in situ (26). The color measured at 405 nm is stable for 60 minutes and obeys Beer's Law in the 4-32 pg/ml range.

5.10

Color Test - The Koppanyi-Zwikker -violet color (27).

5.11

Polarizing Microscopy - Watanabe et al., reported that polarizing microscopy can be used as a qualitative identification tool for chlorzoxazone (28).

5.12

test gives a

-

Potentiometry A potentiometric method for the determination of chlorzoxazone, based on the use of a carbon dioxide gas-sensing electrode, is described by Tagami and Muramoto (29). Upon refluxing the drug with 3N sodium hydroxide, aminophenol and sodium carbonate are formed. Acidification of the reaction mixture yields carbon dioxide which is sensed by the gas-permeable membrane electrode. A line r calibrati n plot was obtained within the 3 x 10-8 to 5 x 10-3 M- range of chlorzoxazone.

6

Stability Chlorzoxazone is stable in the solid state for up to 5 years at room temperature. Storage at temperatures for

CHLORZOXAZONE

137

up to 80°C and in artificial sunlight conditions (1000 foot candles) for up to 4 weeks did not cause significant degradation. Tablets of chlorzoxazone have also been found to be stable for up to 5 years at room temperature. Temperatures for up to 60"C, humidity conditions of 40"C/80% relative humidity and artificial sunlight (1000 foot candles) €or up to 4 weeks have not caused degradation of the product. An aqueous suspension of the drug in distilled water or acidic media is generally stable but in solutions with a pH greater than 7, the solution is generally not stable. Chlorzoxazone yields 2-amino-4-chlorophenol as its major degradation product. 7.

Drug Metabolic Products, Pharmacokinetics, and Bioavailabi1ity 7.1 Drug Metabolic Products 7.11 Urine metabolism - The metabolic product of chlorzoxazone was isolated from urine of human subjects administered an oral dose of the drug. Ether extraction of urine yielded a residue which was identified as 6-hydroxychlorzoxazone (9). Studies showed that the metabolite is eliminated as the glucuronide conjugate to the extent of 74% of the dose. 6-Hydroxychlorzoxazone had little or no muscle relaxant activity when tested in mice and rats. In 1982, Twele and Spiteller identified two additional chlorzoxazone metabolites from human urine, a 5-chloro-2,4-dihydroxyacetanilid produced from 6-hydroxychlorzoxazone by ring cleavage and acetylation of the amino moiety, and 6-hydroxybenzoxazolone, produced by substitution of hydrogen for chlorine and hydroxylation of a neighbor ring-carbon atom (30). 7.12

In-vitro metabolism - Chlorzoxazone was incubated with fortified homogenates of mouse and rat liver. The reaction was stopped by addition of 3N hydrochloric acid. Spectrophotometric assay of a sample preparation identified 6-hydroxy-chlorzoxazone as the major metabolite (31). It has been established that the liver is the principal site of metabolism.

JAMES T. STEWART AND CASIMIR A. JANICKI

138

7.2

Pharmacokinetics and Bioavailability - The following pharmacokinetic data were obtained following a 750 mg oral dose of chlorzoxazone to human subjects ( 1 4 ) : Peak plasma concentration Peak time AUC (0-10 hr) Apparent Volume of Distribution (V/F) Elimination Rate Constant (K) Absorption Rate Constant (ka) Elimination Half Life (t ) Apparent Clearance (KV/F)'

36.3 f 2.3 pg/ml 38 f 3.3 minutes 4084 f 284 pg min/ml 13.70 f 5.07 liters 0.73 2 0.29 2.28 2 2.08 1.12 f 0.48 148.0 f 39.9

hrI: hr hr ml/min

There is rapid absorption and elimination of chlorzoxazone. Chlorzoxazone is widely distributed in plasma and tissues. Less than 1% of the drug is excreted unchanged in urine. The drug concentration in fat is twice the plasma concentration, while liver, muscle, brain and kidney concentrations are one-half or less that found in plasma. The apparent volume of distribution of approximately 14 liters suggests that the drug is not widely distributed and that it would be confined to the circulatory system and possibly the extracellular fluid

.

8.

Identification and Determination in Body Fluids and Tissues 8 . 1 Ultraviolet Spectrophotometry - Conney et a1 reported the extraction and estimation of chlorzoxazone in urine ( 9 ) . Petroleum ether containing 1.5% isoamyl alcohol was shaken with the urine sample for 60 minutes. The organic phase was separated and the drug was back extracted into 0.5 N sodium hydroxide. Absorbance read at 289 nm followed Beers Law. Recovery of drug from urine was 93 f 2%. 8.2

Fluorescence Spectrophotometry - Stewart and Chan reported the quantitation of chlorzoxazone from plasma and/or -urine samples ( 8 ) . Chlorzoxazone was extracted from the biological sample at pH 2.5 with petroleum ether containing 1.5% isoamyl alcohol. The organic phase was re-extracted with strong base, the pH adjusted to pH 6 . 8 with hydrochloric acid, and the drug extracted into chloroform for the measurement step. Excitation and emission wavelengths were 286 and 310 nm, respectively.

CHLORZOXAZONE

139

Drug concentration arid fluorescence were linear from 0.06 - 3.2 pg/ml and 0.13 - 3.0 vg/ml for plasma and urine, respectively. Limits of detection were 6 0 and 130 ng/ml for plasna and urine, respectively. Percent recoveries of chlorzoxazone from plasma and urine were 86.63 2 1.66 and 95.37 2 2.42%, respectively. Stewart and Chan also reported a procedure for the analysis of chlorzoxazone based upon solvent extraction of the drug from human plasma or urine followed by chemical derivatization with fluorescamine (7). Linear detection range was 2-10 pg/ml using excitation and emission wavelengths of 370 and 505 nm, respectively. 8.3

High Performance Liquid Chromatography - Honigberg, Stewart, and Coldren reported an HPLC assay for chlorzoxazone and 6-hydroxy-chlorzoxazone in plasma based on ether extraction of the compounds from acidic plasma (22). The separation was achieved on an octadecylsilane column (30 cm x 3.9 mm, i.d.) at a flow rate of 2 ml/min with UV detection at 280 nm and a mobile phase of 40:60 absolute methanoldistilled water. Retention times for the hydroxy compound and drug were approximately 4 and 8 minutes, respectively. The limit of detection for each compound was calculated to be 8 0 ng at signallnoise = 2. Another HPLC assay for chlorzoxazone in plasma reported by Ng at McNeil Pharmaceutical (32) used at 1:l acetonitrile-distilled water mobile phase, an octadecylsilane column (25 cm x 4..6 mm, i.d.), a 2.0 ml/min flow rate, and UV detection at 280 nm. Retention time for the drug is 2.9 minutes. Ethyl 5-chloro-2-hydroxycarbanilate was used as internal standard (retention time of 4.5 minutes). A plasma sample was made acidic with hydrochloric acid and extracted once with ethyl acetate. The ethyl acetate was evaporated to dryness and the residue dissolved in methanol prior to injection into the HPLC

.

Stewart and Carter reported modifications in the above mentioned HPLC assay by Honigberg, Stewart and Coldren in which an octadecylsilane solid-phase extraction column was used t o separate chlorzoxazone and 6-hydroxychlorzoxazone from human serum (33). Percent recoveries were 90.24 2 4.28 and

JAMEST.STEWART AND CASIMIRA.JANICKI

140

86.06

It: 3.58% for drug and metabolite, respectively. The separation was achieved on an octadecylsilane column using a mobile phase of 5 0 : 5 0 acetonitrile-aqueous 0 . 0 5 M sodium dihydrogen phosphate, pH 4 . 5 with phenacetin as internal standard. The two compounds were detected at the glassy carbon electrode using a cell potential of + 1300 mV. These modifications halved the original HPLC analysis time and increased detection for each compound to 2.5 ng, 30 times the previous limit of detection using 280 nm.

8.4

Gas Chromatography -

Desiraju et a1 reported a packed column method for the quantitation of chlorzoxazone in plasma samples ( 1 4 ) . The drug is extracted from acidified plasma with ethyl acetate containing the internal standard, n-hexadecane. The ethyl acetate is separated andevaporated to dryness. Pyridine and acetic anhydride are added to the residue, the tube is capped, and the reaction allowed to proceed at 42°C for 20 minutes. An aliquot of the mixture is injected into the gas chromatograph.

The GC parameters were flame ionization detection (30 and 300 ml/min flow rates for hydrogen and air, respectively), carrier gas (nitrogen at 30 ml/min), 6 ft x 4 mm i.d. glass column packed with 3% OV-1 on 6 0 / 8 0 mesh Gas Chrom Q, and column temperature of 130°C. The retention times of chlorzoxazone and internal standard were 3.25 and 4 . 5 minutes, respectively. The limit of detection was 0 . 5 ug/ml using 1 ml of plasma. The calibration curve was linear in the 0.5 - 25 ug/ml range. Extraction efficiency for chlorzoxazone was 88 It: 8% (n = 6 ) . 9.

Identification and Determination in Pharmaceuticals Colorimetry - Twenty tablets were weighed and powdered in a dry mortar and pestle. An aliquot of the powder equivalent to 40-60 mg of chlorzoxazone was hydrolyzed with 20% sodium hydroxide solution, cooled, and reacted with nitrous acid to yield the diazonium salt, whose absorbance measured at 405 nm obeyed Beer's Law in the 4-32 ug/ml range ( 2 6 ) . Acetaminophen and indomethacin were shown to interfere with the method. Recovery of chlorzoxazone in bulk powder and tablet samples was in the 9 8 . 3 - 99.9% range utilizing the assay procedure.

9.1

CHLORZOXAZONE

9.2

141

Ultraviolet Spectrophotometry - Kirschner of McNeil Pharmaceutical reported an assay for the drug in tablets as follows (10): Standard Preparation: Dissolve a suitable quantity

of USP Chlorzoxazone KS, accurately weighed, in

methanol, and dilute quantitatively and stepwise with methanol t o obtain a solution having a known concentration of about 20 pg/mL. Assay Preparation: Weigh and finely powder not less than 20 Chlorzoxazone tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of chlorzoxazone, to a 100-ml volumetric flask. Add about 80 ml of warm methanol, and shake by mechanical means for about 15 minutes. Dilute with methanol to volume, and mix. Filter, discarding the first 20 mL of the filtrate, and pipet 2 ml of the filtrate into a 100-ml volumetric flask. Dilute with methanol to volume, and mix. Procedure: Concomitantly determine the absorbances of the Assay and Standard Preparations at 282 nm, with a suitable spectrophotometer, using methanol as the blank. Calculate the quantity, in mg, of chlorzoxazone in the portion of tablets taken by the formula 5C (Au/As), in which C is the concentration, in pg per ml, of USP chlorzoxazone RS in the Standard Preparation and Au and As are the absorbances of the Assay and Standard Preparations, respectively. 9.3

9.4

Fluorescence Spectrophotometry - Stewart and Chan used the intrinsic fluorescence of chlorzoxazone to assay for the drug in commercial tablets containing acetaminophen ( 8 ) . Recovery of drug was in the 98-101% range. These same authors also demonstrated that chlorzoxazone could be successfully analyzed in a tablet dosage form chemical derivatization with fluorescamine to form a fluorophore (7). Potentiometry - Tagami and Muramoto described a method based on the use of a carbon dioxide gas-sensing electrode ( 2 9 ) . Twenty tablets were powdered and a portion equivalent to about 424 mg of drug was weighed and extracted with 20 ml of acetone. After stirring and centrifugation, the supernatant acetone solution was removed and

JAMES T. STEWART AND CASIMIR A. JANICKI

142

diluted with additional acetone. This extraction procedure was performed four times. The collected acetone fractions were evaporated to dryness. The residue was refluxed for 2 hr with 50 ml of 3N sodium hydroxide and after acidification, the carbon dioxide was determined using a gas permeable electrode. Mean recovery of chlorzoxazone was 99.6% with a standard deviation of 0.24. 9.5

High Performance Liquid Chromatography - The USP XXI method for Chlorzoxazone with Acetaminophen tablets is a gradient HPLC method (34). In the method, m-chloroaniline, p-aminophenol (a degradation prozuct of acetaminophen), p-chlorophenol and 2-amino-4-chlorophenol are separated from chlorzoxazone, acetaminophen and the internal standard phenacetin. An example of the separation is as follows: Retention time, minutes Component 2.3 Acetaminophen 6.0 2-Amino-4-chlorophenol p-Aminophenol 7.1 m-Chloroaniline 7.5 Internal Standard (phenacetin) 8.3 9.2 p-Chlorophenol 10.1 Chlorzoxazone

10. References

1. 2. 3. 4. 5. 6. 7. 8. 9.

Current Therapeutic Research (1975) 17,p 123, Literature Services Section, McNeil Laboratories, Inc., Fort Washington, PA. A.P. Roszkowski, J. Pharmacol. Exptl. Therap., 129 (1960) p 75. Pharmacy Times, 48 (1982) 23-32. D. Marsh, U S Patent 2, 895, 877 (1959). J. Sam and J . N . Plampin, J.Pharm. Sci., 53, (1964) 538-544. K. Harsanyi, F. Toffler, KD. Korbonitis and G. Leszkovszky, Hung. Patent 150, 577 (1961); thru Chem. Abstr., 60, 5507b (1964). J.T. Stewart and C.W. Chan, Anal. Letters, Bl1 (9), (1978) 667-680. J.T. Stewart and C.W. Chan, J. Pharm. Sci., 68 (1979) 910-912. A.H. Conney, N. Trousof and J.J. Burns, J. Pharmacol. Exptl. Therap., 128 (1960) 333.

CHLORZOXAZONE

143

10. E.L. Kirschner, McNeil Pharmaceutical, Spring House, PA, (1986), Personal communication. 11. H. Reral, V. Gamentzy and I. Bucur, Rev. Chim. (Bucharest) 16 (1965) 322. 12. B. Kaempe, Arch. Pharm. Chemi., g . Ed., 1. (1974), 145. 13. K.D. Parker, C.R. Fontan and P.L. Kirk, Anal. Chem., 34 (1962) 757. 14. R.K. Desiraju, N.L. Renzi, R.K. Nayak and K. Ng, J, Pharm. Sci. 72 (1983) 991-994. 15. A.H. Beckett,G.T. Tucker and A.C. Moffat, J. Pharm. Pharmac., 19 (1967) 273. 16. "Clarke's Isolation and Identification of Drugs," 2nd Edition, A.C. Moffat, Ed., The Pharmaceutical Press, London, 1986, p 193. 1 7 . R.C. Pedroso and E.D.C.F. Moraes, Rev. Farm. Rioqulm. Univ. S. Paulo, 18 (1982) 183-195. J. 18. I. Ullah, D.E. Cadwallader, and I.L. Honigberg, Chromatogr., 46 (1970) 211-216. 19. R.A. Egli andS. Tanner, Fresenius 2. Anal. Chem. 295 (1979) 398-401. J. Chromatogr., 2 (1984) 20. R.A. Egli and S. Keller, 249-256. 21. E.G.C. Clarke, "Isolation and Identification of Drugs," Volume 1, The Pharmaceutical Press, London (1969), p 34. J. 22 * J.T. Stewart, I.L. Honigberg, and J.W. Coldren, Pharm. Sci., 68, (1979) 32-36. J. 23. I.L. HonigberE J.T. Stewart, and J.W. Coldren, Pharm. Sci., 68, (1979) 253-255. 24. Japan Pharmacopeia, 9th Ed., Society of Japanese Pharmacopeia, Japan, p 157. 25. V.J. Schnekenburger and M. Quade-Henkel, Dtsch. Ap0th.-Ztg., 124 (1984) 1167-1170. 26. N.M. Sanghoi, N.G. Joshi, and K.S. Dubal, Indian Drugs, 18 (1981) 299-302. 27. Tlarke's Isolation and Identification of Drugs," 2nd Edition, A.C. Moffat, Ed., The Pharmaceutical Press, London, 1986, p 465. 28. A . Watanabe. Y. Yamaoka. K. Kuroda, T. Yokoyama and T. Umeda, Yakugaku Zasshi,-l05 (1985) -481-490.29. S. Tagami and Y. MuramotFChem. Pharm. Bull, 32 (1984) 1018-1024. 32 (1982) 30. V.R. Twele and G. Spiteller, Arzneim. Forsch., 759-763. 31. A.H. Conney and J.J. Burns, J. Pharmacol. Exptl. Ther., 128 (1960) 340. Ng, McNeil Pharmaceutical, Spring House, PA (1986), 32. Personal Communication. 33. J.T. Stewart and H.K. Carter, J. Chromatogr., Biomed. Appl., 380 (1986) 177-183.

m.

144

34.

JAMES T. STEWART AND CASIMIR A. JANICKI

Second Supplement, U n i t e d S t a t e s P h a r n a c o p e i a , 2 1st E d i t i o n , U n i t e d S t a t e s Pharmacopeial Convention, B e t h e s d a , MD, (1985), p . 1 8 2 7 .

L i t e r a t u r e surveyed t h r o u g h December, 1986.

CYCLOSPORINE

*Mahmaud M.A. H c u a n and **Mohammed A . AL-Yahya

06 P h c u u n a c W c d Chmbin.thy, Vep&evLt 06 P h m a c e d c d Che.mhin.thy, CoUege 04 P h m a c y , King Saud U n i w m L t y , Riyadh, Saudi Atrabiu.

*Pho6ehboh

PhOdeAhotr 06 Phahmacognob y , Ve-ed 06 Phahmacognon y. VhecZoh 06 ,the MediciMae Akomatic and Pohonoun Plan& Runahch Cenhn, CoLtege 06 Phahmacy, King Saud U n i w m L t y , Riyadh, Saudi k a b i a .

**kshoch~&

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

145

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

146

1. History and Therapeutic Category 2.

Description 2.1 Nomenclature 2.2 Formulae 2.2.3 Degradation and Structural Elucidation 2.2.4 Conformation 2.3 Molecular Weight 2.4 Appearance, Color, Taste, Odor

3. Physical Properties 3.1 3.2 3.3 3.4 3.5

Crystal Properties Melting Range Solubility Optical Rotation Spectral Properties

4. Iso lation 5.

Biosynthesis

6. S y nthesis 6.1 Cyclosporine 6.2 Cyclosporine Analogs 6.3 Structure-Activity Relationship 7.

Metabo1ism

8.

Pharmacokinetics

9. Pharmacological Activity and Clinical Applications 10. Pharmaceutical Forms

11. Methods of Analysis 11.1 11.2 11.3 11.4

Elemental Composition Introduction to Analysis Radioimmunoassay High Performance Liquid Chromatography

12. References 13. Acknowledgement

147

CYCLOSPORINE

Cyclosporine 1.

H i s t o r y and T h e r a p e u t i c Category It w a s from Hardanger Vidda, a b l e a k t r e e l e s s p l a t e a u

i n t h e South o f Norway, t h a t a s o i l sample w a s o b t a i n e d i n t h e e a r l y s e v e n t i e s . The fungus i s o l a t e d from t h i s

sample w a s c a l l e d Tolypocladium i n f l a t m Gams (1), formerly ( 2 ) d e s i g n a t e d as Trichoderma p o l y s p o r m (Link ex P e r s . ) R i f a i or Cylindrocarpon luciderm Booth ( 3 ) . S e v e r a l m e t a b o l i t e s o f t h i s fungus have been i s o l a t e d of which c y c l o s p o r i n s A , C and G t o have so f a r been shown t o p o s s e s s s t r o n g immunosuppressive a c t i v i t y . The main component i n normal f e r m e n t a t i o n b r o t h s w a s named c y c l o s p o r i n A ( A ) , now known as c y c l o s p o r i n e . A s r e p o r t e d by Bore1 e t a1 ( 5 - 9 ) , c y c l o s p o r i n e i s a s e l e c t i v e immunosuppressive drug w i t h a n t i f u n g a l and antiinflammatory p r o p e r t i e s . Today c y c l o s p o r i n e can be regarded as t h e m o s t important r e c e n t i n n o v a t i o n i n t h e f i e l d of organ t r a n s plantation.

2. D e s c r i p t i o n 2.1

Nomenclature 2.1.1

Chemical Names Cyclosporin A; Cyclosporine.

2.1.2

Generic Names Cyclosporine (USAN) and i s adopted f o r t h e b a s i c s t r u c t u r e , meanwhile t h e f o l l o w i n g names h a s been a c c e p t e d i n o t h e r c o u n t r i e s . England, c y c l o s p o r i n (BAN name); F r a n c e , c i c y l o s p o r i n e ; S w i t z e r l a n d and o t h e r s , c i c l o s p o r i n (INN name, WHO).

2.1.3

Trade Names Sandimmune , Sandimmun

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

148

2.2

Formulae 2.2.1

Ehpiricd N O C 6 2 H l l l 11 1 2

2.2.2

Structural

2.2.3

Cyclosporine ( F i g . 1) i s a n e u t r a l , hydrophobic c y c l i c p e p t i d e composed o f 11 amino a c i d r e s i d u e s , a l l having t h e S-configurat i o n o f t h e n a t u r a l L-amino a c i d s , except f o r t h e D-Ala i n p o s i t i o n 8, which has t h e Rc o n f i g u r a t i o n , and S a r i n p o s i t i o n 3. Seven a c i d s a r e N-methylated. Ten are known a c i d s , t h e y are a-Abu i n p o s i t i o n 2 , Sar-in p o s i t i o n 3, N-MeLeu i n p o s i t i o n 4,6,9 and 1 0 , V a l i n p o s i t i o n 5, Ala i n p o s i t i o n 7 , D-Ala i n p o s i t i o n 8 , N - M e V a l i n p o s i t i o n 11 and MeBmt i n p o s i t i o n 1. Degradation and S t r u c t u r a l E l u c i d a t i o n The s t r u c t u r e o f c y c l o s p o r i n e [l] ( F i g . 1) was determined by chemical d e g r a d a t i o n (4) and x-ray c r y s t a l l o g r a p h i c a n a l y s i s (10,ll). Also with an x-ray c r y s t a l o g r a p h i c a n a l y s i s of t h e iodo d e r i v a t i v e [ l a ] . It has t h e molecular formula C62HlllN11012 as deduced by NMR and mass s p e c t r a . Hydrolytic cleavage of c y c l o s p o r i n e f u r n i s h e d 11 amino a c i d s as fragments, among them an a r t i f a c t o f a new C9-amino a c i d . The amino a c i d sequence w a s determined by Edman d e g r a d a t i o n o f i s o c y c l o s p o r i n , a b a s i c rearrangement product formed from c y c l o s p o r i n e by N,Oa c y l m i g r a t i o n . On t h e b a s i s of t h e chemical, s p e c t r o s c o p i c and c r y s t a l l o g r a p h i c e v i d e n c e , t h e s t r u c t u r e of c y c l o s p o r i n e w a s , e l u c i d a t e d as t h e n e u t r a l c y c l i c o l i g o p e p t i d e It i s composed of e l e v e n amino a c i d r e s i d u e s , a l l having t h e L-configuration of t h e n a t u r a l amino a c i d s , except f o r t h e D-alanine i n p o s i t i o n 8 and t h e non-chiral s a r c o s i n e (N-methylglycine) i n p o s i t i o n 3. Seven amino a c i d s i n p o s i t i o n s 1, 3, 4, 6 , 9 , 1 0 and 11- are N-methylated. Ten o f t h e

-

u

A

Fig. I . Struciure of cyclosporine 1 (schematic). McBmt nyl]-4,N-dimcthyl-~-threoninc (see text).

-

(4R)-4-[(€)-2-bute-

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

150

J cyclorporine

not obtoincd

Ib cyclic derivotivool the Nmdhyl-C9-amino ocid

1

cyclosporlnc

1

IS OCYCLOSWRINE

onhydromrthylthio

Ic

hydontoin de rivot ive

Edman d e g r a d a t i o n with CHI-NCS under c a r e l u l l y chosen mild conditions p e p t i d e hydrolysis w i t h c h r o m a tog raphy.

6 N HCI followed by ion erchange

n 'F,.I TlOAcl It

-+

t--Zn I HOAc

cy cloiporlnc

1

n

CYCLOSPORINE

151

eleven ring members are derivatives of known aliphatic amino acids: a-aminobutyric acid (Abu) in position 2, Sarcosine (S.ar) in position 3, N-methylleucine (MeLeu) in positions 4, 6, 9 and 10, Valine (Val) in position 5, Alanine (Ala) in position 7, D-Alanine (D-Ala) in position 8, and N-methylvaline (Me Val) in position 11. The amino acids were readily characterised following acid hydrolysis of cyclosporine. The novel amino acid was found to have the composition ( 2S, 3R, 4R, 6E)-3-hydroxy-bmethyl-2-( methylamino)-6-octenoic acid, and in accord with amino acid nomenclature, is now designated as (4R)-4-[(E )-2-butenyl]4-N-dimethyl-L-threonine and abbreviated Thus the novel amino acid has the MeBmt . polar features of an N-methyl-L-threonine which is substituted at the end of the carbon chain by butenyl and methyl groups. This amino acid was hitherto not known in the free form, since only antifacts and derivatives were obtained from degradation experiments on cyclosporine(4). Hydrolysis of cyclosporine followed by ion exchange chromatography afforded a cyclic derivative [lb]of the MeBmt minoacid. Acidic treatment of cyclosporin [l] in the absence of water effects an N, O-acylmigration of the methylvalyl moiety and furnishes isocyclosporine [lc]. A modified Edman degradation of isocyclosporine [1] using methyl isothiocyanate produced an anhydrothiohydantoin-derivative [l] and thus established MeBmt as the first amino acid in the peptide sequence. The complete sequence was established by repetitive Edman degradation.

2.2.4

Conformation of Cyclosporine The conformation of cyclosporine in the crystal and in solution was reported (11). The

152

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

conformational a n a l y s i s was based on X-ray c r y s t a l l o g r a p h y i n t h e c r y s t a l and on NMR spectroscopy i n s o l u t i o n u s i n g d i f f e r e n t l i p o p h i l i c s o l v e n t s . Cyclosporine molecule i s highly lipophilic, therefore t h e s o l i d s t a t e conformation and t h a t deduced from NMR s t u d i e s i n a p o l a r s o l v e n t s a r e c l o s e l y s i m i l a r . It assumes a r a t h e r r i g i d conformation, b o t h i n t h e c r y s t a l l i n e s t a t e and i n s o l u t i o n ( F i g . 2 ) . The o n l y d i f f e r e n c e s a r e t h e side-chain conformation o f MeBmt ( a major change between t h e c h a i n f o l d e d i n upon t h e molecule i n t h e solid s t a t e , and p o i n t i n g o u t i n t o s o l v e n t i n t h e NMR s t u d y ) , t h e side-chain conformation o f MeLeu-10, and t h e d e t a i l e d backbone conformation i n t h e r e g i o n o f D- A l a - 8 ( F i g . 3 ) . Here t h e X-ray s t r u c t u r e c l e a r l y shows one H-bond from D-Ala-8 ( N H ) t o MeLeu-6 ( C O ) , whereas i n s o l u t i o n a b i f u r c a t e d H-bond t o b o t h t h e c a r b o n y l 0-atom o f MeLeu-6 and itself. t o t h e carbonyl 0-atom of D-&a-8 The conformation change needed t o c o n v e r t t h e X-ray backbone i n t o t h e NMR backbone i s small, and can b e brought about by r e l a t i v e l y s m a l l r o t a t i o n i n t h e backbone t o r s i o n a n g l e s i n t h e r e g i o n MeLeu-6 t o MeLeu-9, p r i n c i p a l l y around t h e 7- and 8residues. The side-chain conformations o f M e V a l and V a l and t h r e e o f t h e f o u r MeLeu-residues (MeLeu -4, -6 and -9) are i d e n t i c a l i n c r y s t a l and i n s o l u t i o n . Only t h a t o f MeLeu-10 i s r o t a t e d by 120°. The side-chain conformation o f MeBmt i n s o l u t i o n can be d e r i v e d from t h e X-ray conformation simply by means o f 120' r o t a t i o n about t h e cl,B-bond. T h i s r o t a t i o n which i s e n e r g e t i c a l l y allowed, could be t h e r e s u l t from t h e formation of an i n t r a m o l e c u l a r H-bond between t h e B-OH and t h e carbonyl 0-atom of MeBmt r e p l a c i n g t h e i n t e r m o l e c u l a r Hbond found i n t h e c r y s t a l . A l a r g e p o r t i o n o f t h e backbone ( r e s i d u e s 1-6) a d o p t s an a n t i p a r a l l e l & p l e a t e d s h e e t conformation which c o n t a i n s t h r e e t r a n s a n n u l a r H-bonds

CYCLOSPORINE

Fig. 2.

153

Stereoview of cyclosporine. a) Solid-state conformation, b) Computer-generated conformation in apolar solvents (in solution the distal atoms of Abu-2 and YeBmt-1 have a high flexibility).

Fig. 3.

Superposition of the solid-state conformation (thin line;) and the computer-generated conformation of cyclosporine in apolar solvent (thick lines).

155

CY CLOSPORINE

and is markedly twisted. The sarcosine in position 3 and N-methylleucine in position 4 participate in a type 11B-turn (12-14). This means that, in the orientations chosen in Figures l and 2, the CC group of Abu-2 and the N-CH? group of MeLeu-4 are up and the CC gEoup of Sar-3 and NH group of Val-5 are down - pro -S proton of the methylene group of Sar 3 is axial, and the side-chain of MeLeu-4 is requatorial. The remaining residues 7-11 form an open loop. This loop contains the only cis- amide linkgage in the molecule between the two adjacent N-methylleucine residues 9 and 10. The remaining H-bond of a y-type and serves to hold the backbone in a folded L-shaped.

As a consequence of this rather rigid conformation of the cyclosporine skeleton, six amino acids have their side-chains directed quasi-perpendicular to the plane of the peptide ring; in Abu-2, Val-5 and MeLeu-6 they are pointing upwards, in MeBmt-1 and ~ e ~ e u -they 6 are pointing downwards. The side-chains of the remaining aminoacids lie more or less in the plane of the peptide ring. The resulting overall molecular shape in apolar solution is that of the previously postulated "butterfly" (10), in which the MeE3mt side-chain, known to be important for immunosuppressive activity, stands out, probiscis - like, in a manner suggestive of special function (Fig. 4).

2.2.5

CAS Registery Number Cyclosporine [79217-60-0] Cyclosporine A [ 59865-13-31

2.2.6 Clinical Code CyAIOL 27-400

156

Fig. 4.

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

Two roughly orthogonal views of a model of native cyclosporine in the conformation suggested as probable. The backbone conformation corresponds to that of iodocyclosporine.

157

CY CLOSPORINE 2.3

Molecular Weight 1202.64

2.4

Appearance, C o l o r , Taste, Odor White p r i s m a t i c c r y s t a l s .

3. P h y s i c a l P r o p e r t i e s 3.1

Crystal Properties

3.1.1

X-ray D i f f r a c t i o n C r y s t a l Data X-ray c r y s t a l s t r u c t u r e s o f c y c l o s p o r i n e and i t s i o d o d e r i v a t i v e [ I a ] were d e t e r m i n e d (10,ll). A summary o f t h e c r y s t a l d a t a and r e f r a c t o m e t r y for c y c l o s p o r i n e i s g i v e n i n Table 1 (11).

T a b l e 1.

C r y s t a l and D i f f r a c t i o n Data

Molecular f o r mula

N O C 6 2 H l l l 11 12

Molecules per c e l l

z = 4

Molecular weight

1202.5

Diffractometer

CAD4 ( E n r a f Nonius)

Crystallisation

from a c e t o n e

Radiation

C a a ( Graphite monochromat o r )

C r y s t a l form

colourless, p r is m a ti c

Intwsity scans

w/2e = 1.0; Ow = 1.0°+0.3 tan 0

Crystal s i z e

ca. 0.2 x 0.2

a ( T ) / I = 0.02

x 0.3 mm

( tmax

= 120s)

Space group

Sphere of ref lexion

sine/X 4 0 . 5 1 ( 36508 r e f l e x i o n r )

Cell dimen-. sions

I n t e n s i t ie: measured Intensities significant

5057 ( u n i q u e )

C r y s t a l dens i t y (calc.)

c = 41.242(33 A; V = 7896A dc = 1 . 0 4 2 g. cm-3

4272(I

2.50(1);

158

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

The c r y s t a l d a t a for iodocyclosporine (10), N 0 + , M = 1327, c o l o r l e s s '62'110 11 1 2 prisms from n-heptene, 2 . 5 : l monoclinic, a = ( 1 0 . 4 7 5 ( 5 ) , b = 1 9 . 6 0 ( 1 ) , c = 21.05(1)A0, f3 = 99.35 (2'1, U = 4264 A o 3 , Dc = 1.03, z = 2, space group ~2~ , ( c,; N O . 4). CUKci r a d i a t i o n , A = 1.54187 A o , g r a p h i t e monochromator. A c r y s t a l o f approximate dimensions 0 . 3 x 0.7 x 0.4 mm w a s used on an Enraf-Nonius CAD4-F automatic d i f f r a c t o meter.

A s t e r e o v i e w o f iodocyclosporine molecule i s shown i n F i g . 5a, and t h a t of c y c l o s p o r i n e i n F i g . 5b. The iodocyclosporine showed t h e a n t i p a r e l l e l & p l e a t e d s h e e t conformation of r e s i d u e s 1-6, t h e open l o o p of r e s i d u e s 7-11, and t h e r a t h e r l a r g e t h e r m a l v i b r a t i o n s of some of t h e side-chain atoms , p a r t i c u l a r l y t h o s e of MeLeu-9 are a p p a r e n t . The a b s o l u t e c o n f i g u r a t i o n w a s n o t determined as p a r t of the structure analysis, since sufficient of t h e h y d r o l y s i s p r o d u c t s could r e l i a b l y be i d e n t i f i e d as L-amino-acids. It became apparent d u r i n g t h e a n a l y s i s , however , t h a t A l a - 8 has t h e D-configuration (15). The conformation of c y c l o s p o r i n e observed i n t h e c r y s t a l ( F i g . 5b) w i t h 50% p r o b a b i l i t y v i b r a t i o n a l e l l i p s o i d s of t h e atomic thermal motion (16) which d i s p l a y t h e r e l a t i v e l y s m a l l , isotopic v ib r a tio n s of t h e backbone atoms - i n d i c a t i n g conformat i o n a l r i g i d i t y - i n c o n t r a s t t o t h e more f l e x i b l e side-chain atoms whose v i b r a t i o n a l amplitudes i n c r e a s e w i t h t h e d i s t a n c e from

(a). Fig. 6a shows t h e backbone of t h e c y c l i c p e p t i d e w i t h t h e 4-, I)-, and w- a n g l e s and some s t r u c t u r a l f e a t u r e s . Three H-bonds b r i d g e t h e two s h o r t @- s t r a n d s adding t o t h e s t a b i l i t y of @-fragment.

/!

Fig. 5a.

A stereoscopic view of the iodocyclosporine molecule showing 50% probability

ellipsoids of thermal vibration. The Cg amino acid is at centre left on the forward side of the molecule and the conventional sequence then extends upwards at the front through a-aminobutyric acid, glycine etc. One of the hydrogen bonds of the 8-pleated sheet is indicated (dotted) in the centre and the interesting hydrogen bond Ala-8 4 Meleu-6 appears at centre right.

Fig. 5b. ORTEP drawing of the crystal structure of cyclosporine. Anisotropic atomic vibrations are represented by 50% - probability ellipsoids. The numbers shown are residue numbers.

loop

Fig. 6 a .

\

bet a Backbone conformation of crystalline cyclosporine with indication of the loop and 6-fragment. The torsion angles 6, $I, and w and in brackets the computermodeled values for the NMR conformation are given. The dashed lines indicate B-bonds, the dotted lines close contacts from CH3N of MeVal to different atoars (0 11:3.04 Ao; C 7:3.36 Ao; 0 7:3.26 AO; C 8:3.41 A O ; N 9 : 3 . 5 4 AO; N 10:3.12 Ao).

F i g . 6b.

Stereoview of a space-filling model of cyclosporine showing the side chain of between MeLeu-6 and-Meleu-4 into the ply of the &fragment

163

CYCLOSPORINE

The s i d e c h a i n s of t h e Abu, V a l , and MeLeu r e s i d u e s are i n t h e s t a g g e r e d conformation, t h e MeLeu s i d e c h a i n s being extended. Rather unexpected i s t h e conformation o f t h e Cg-alkylene s i d e chain of MeBmt-1; it i s n e a t l y f o l d e d i n t o t h e p l y of t h e 6-pleated s h e e t which a l l o w s t h e molecule t o adopt a g l o b u l a r compact shape ( F i g . 6 b ) . 3.2

Melting Range White p r i s m a t i c n e e d l e s from acetone a t - 15', m.p. 148-151' (17). The s y n t h e t i c c y c l o s p o r i n e m.p. 149-150° (18).

3.3

Solubility The compound i s n e u t r a l , r i c h i n hydrophobic amino a c i d s , i n s o l u b l e i n water and n-hexane, b u t v e r y s o l u b l e i n a l l o t h e r o r g a n i c s o l v e n t s . It i s v e r y s o l u b l e i n methanol, e t h a n o l , a c e t o n e , e t h e r and chloroform.

3.4

Optical Rotation

[a]:' 3.5

(17)

-

244' ( C = 0.6 i n c h l o r o f o r m ) .

-

1.89' ( C

= 0.5 i n methanol).

Spectral Properties

3.5.1

U l t r a v i o l e t Spectrum The W spectrum o f c y c l o s p o r i n e i n methanol w a s recorded on a Beck~nann-DK2 spectrophotometer shows only an end a b s o r p t i o n a t 200 nm ( 4 )

3.5.2

I n f r a r e d Spectrum The i n f r a r e d c h a r a c t e r i s t i c s i n methylenec h l o r i d e ( C H 2 C 1 2 ) and i n c a r b o n t e t r a c h l o r i d e ( C C l 4 ) were r e p o r t e d ( 4 , l l ) . The I R spectrum i n methylene c h l o r i d e recorded on a P e r k i n E l m e r 21.IR spectrophotometer i s shown i n Fig. 7. It s h o u l d be n o t e d t h a t s o l u t i o n s t u d i e s , i n d i c a t e d t h e NH groups

Fig. 7.

IR-spectrum of cyclosporine in CH2C12.

165

CYCLOSPORINE

are involved i n H-bonding. Their absorption bonds ( 3330, 3290 cm-l i n C C l 4 s o l u t i o n and 3400-3330 cm-1 i n CH2C12 s o l u t i o n ) are independent o f t h e c o n c e n t r a t i o n i n t h e range o f 4.10-2 t o 4.1Oq4 M i n C C 1 4 s o l u t i o n . The spectrum shows a l s o an i n t e n s i v e amidecarbonyl bond a t 1630-1670 cm-1. 3.5.3

Nuclear Magnetic Resonance S p e c t r a

1

'H-NMR, 13C-NMR and H-NMR-NOE-difference s p e c t r a of cyclosporine i n deuterochloroform and deuterobenzene were r e p o r t e d ( 4 ~ 8 , 11). However, t h e complete assignment of lH-NMR, 13C-NMR and 15N-NMR s p e c t r a i n deuterochloroform and deuterobenzene by a combination of homonuclear and h e t e r o nuclear two d i m e n t i o n a l t e c h n i q u e s were d e s c r i b e d by Kessler e t a1 ( 1 9 ) . 3.5.3.1

'H-NMR

Spectra

1 The H-NMR spectrum of c y c l o s p o r i n e ( F i g . 8 ) i n CDC13 and TMS w a s recorded on Bruker HX-gO-E, 90 MHz instrument ( 4 ) . A p a r t o f 1~-NMR spectrum o f c y c l o s p o r i n e i n ~ 6 ( F~i g .6 9 ) w a s recorded on Bruker HX-360, 360 MHz i n s t r u m e n t . This h a s proved t h e E-configurat i o n o f t h e double bond i n c y c l o s p o r i n e by u s i n g t h e double resonance t e c h n i q u e (JE, 5 = 16 Hz) ( 4 ) . From t h e s e s p e c t r a , i d e n t i f i c a t i o n o f s p i n systems were o b t a i n e d . It i s e v i d e n t from t h e s p e c t r a t h a t one conformation s t r o n g l y dominates. Some minor peaks s p e c i a l l y i n t h e N-methyl r e g i o n (2.5-3.5 ppm) are v i s i b l e i n d i c a t i n g t h e presence o f a n o t h e r conformation i n s l o w exchange. A completely d i f f e r e n t s i t u a t i o n i s observed i n DMSO d6 s o l u t i o n , where a m i x t u r e o f a t l e a s t seven conformations l e a d s t o a spectrum of h i g h complexity. Roughly

8

0

Fig. 8 .

0

T

'H-NMR-spectrum

6

5

I

3

2

*

0 ??a

I

of cyclosporine by 90 !Mz in CDcl3, TMS

--

0 ppm.

167

CYCLOSPORINE

speaking, f i v e more o r l e s s separated s p e c t r a l regions are distinguishable:

NH protons

7

- 8.3 -6

P P ~

a-protons as w e l l as t h e v i n y l i c protons

4.5

N-methyl groups

2.6

C-methyl groups

0.6

A l i p h a t i c f3-, yand 6 protons

1.1 - 2.7 PPRI

pprn

- 3.8 ppm - 1.8 ppm

The proton chemical shifts for cyclosporine i n CDC13 and C6D6 are l i s t e d i n Table 2 .

h C

H-c

c

h

b

0

t

F i g . 9.

1 Part of the H-NMR-spectrum of cyclosporlne by 360 llHz in CsDs. a) Undecoupled spectrum. b) Decoupled s n e c t r m keeping lock on H-C(n) of Cg As c ) Decoupled spectrum keeping lock on H-C(6) of Q As

Table 2.

Residue

1

h- and

chemical shifts of cyclosporine [I] in C D C l

13C-NMR

Amino-acid residue

Group

MeBmt:

CH3N

~~

co H-C(a )

H-C(B) OH

3-52 5.45 3.82 3.87 1.63 2.41 1-73 0.72 5.36

C6D6 3.51

Abu

NH

co

7.93

-

C6D6

CDC13

3.73

-

-

-

5.47

5.7 4.21

5.72 4.20 3.5

2.07 2.65

33.97 169.65 58.75 74.74

-

9

s d d

34.33 U’O * 73 59.96 74.95 -

35.99 d 35.63 t

36.30 36.04

16.76 q 129.68 d 126.32 d

18.30 126.76

17.96

18.69

2.13

0.71

5.35 1.62 2

and C G D ~ ~ )

l3C-NMR

lH-NMR

CDC13

3

1.15 5.64 5-52 1-75

7.96

-

-

q

131.38

8.26

-

-

-

173.04 s

174.29

Table 2.

Residue

No.

(Continued)

Amino-acid

Group CDCl

4

Sar

MeLeu

32K

5-03

5.12

5.12

Val

32K

H-C(

6)

1.6-1.74

1.79

H-C(

Y)

0.87

0.89

3

49.54 26.11

9.93 9 39.40 q

10.73 39.58 171.80 50.10

co

-

3.40 -

H-C( a )

4.76

4.74

4.01

50 37 t.

3.11

2.59

31.32 q

CH N

3

H-C(

~ 1 )

5.34

H1-C(

B)

2.00

-

3.08 -

5.60 2.27 1.58 1.42 0.98 0.91 7.46

C6D6

48.86 d 25.06 t

m3N

co

5

CDCl

C6D6 2K

2K H-C( a)

3

13C-NMR

H-NMR

residue

170.50 s

-

169.35 s 55.51 a

31.39 170.21 56.20

35.99 t

37 * 01

24.90 d 23.49 q 21.18 q

25.79 24.28 22. TOb)

W RW

V

M

d

V

e

4

0

d

c - o r i

A

O

O

W

\

0

0

wlnmrJ&w d

ri

I

W

-

* . . .

?

1 - 3

r

W

I A ( u r l 0

170

?

M

m

(u

0‘

m Ln

d M

0

cu m

L n

cu M

ul

I

a

d

d

I

co

t-

?

co

171

L n d L n L n

? ? ? ?

ti

m t i c o o c u r - J J

L P

m

UJ

cu

t-

co I L n

M

cu

?

0 I L n

cs

cu r-

M

m

co

. .

gt-

cucu

5 9 L n L n

Table 2 .

Residue No.

( Continued)

Amino-acid residue

CDCl

MeLeu

-

H-C(CX)

5.10

6)

H-C(y) CH3(61)

MeVal

13C-NMR

C6D6 2K

6

m3N

-

21.86 9

24.81

29.83 q 169.41 s

30.47 170.98

57.54 d 40.73 t

58.36 42.06

1.79

24.55 d

25.62

1.17

23.85 q

24.34

1.16

23.38 q

22. 6bb)

2.85

5.35 2.42 1.29

2.13 1.24 1.49 0.98 0.98

C6D6

CDC13

32K

0.84 2.70

co

CH3(

32K

0.89

CH 3N

H1-C(

11

3

2K

CH3(62) 10

lH-NMR

Group

2.71

2.42

s

30.96 174.61

5-27

57.93 d

58.84

2.27

29.05 t

2.98

co

-

-

-

H-C(CX)

5-15

5.14

H-C( 6 )

2.17

-

29.81 172.85

q

29 - 99

Table 2.

Group 0

1H-NMR

Pi 3

Amino-acid residue

k 0

Residue

( Continued)

WDV)

19.38

0.66

20.26 q

20.59

0

18.75 q

0

. .

. .

a m

r i m

oco d o

173

e 4 W

0.96

1

At 296 K in pprn from internal TMS. The H-NMR data were obtained from a conventional

w lk k

n

)

Gik

Ld

a

13c-NMR

CDC1,

32K spectrum or from the cross-sections of a IH,$-COSY

with 2K data points in f2 at

300 MHz b,

The signals of MeLeu-4(6 ) and MeLe~-9(6~) as well as those of MeLeu-6(6 ) and MeLeu-lO(6 ) 2 1 2 in C D may be exchanged).

66

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

174

3.5.3.2

13C-NMR

Spectra

I n t h e broadband-decoupled 13C-NMR s p e c t r a i n CDC13 or C6D6 were r e p o r t e d ( 1 9 ) . Almost a l l 62 Cs i g n a l s a r e r e s o l v e d and only few signals overlap, but these s p l i t when t h e s o l v e n t i s changed. A l l carbonyl C-atoms r e s o n a t e i n t h e range between 169 and 175 ppm. P a r t o f t h e l 3 C - m spectrum ( t h e carbonyl r e g i o n ) of c y c l o s p o r i n e i n CDC13 measured on a Bruker-WH-360, 360 MHz instrument i s shown i n Fig. 1 0 (11). The two o l e f i n i c C-atoms of MeBmt are a l s o i n a t y p i c a l range 126-132 ppm, whereas 49 C-signals a r e i n t h e a l i p h a t i c r e g i o n . The number of a t t a c h e d p r o t o n s were o b t a i n e d v i a t h e u s u a l DEPT t e c h n i q u e ( 2 0 , 2 1 ) . A h e t e r o n u c l e a r 2D-J, 6 spectrum (22-26) w a s a l s o performed i n CDC13 The chemical s h i f t f o r a l l carbons and m u t l i p l i c i t i e s i n C X 1 3 and i n C6D6 are given i n Table 2 . 3.5.3.3

15N-NMR

Spectra

AH-decoupled 15N-NMR spectrum shows eleven N-signals ( T a b l e 3 ) . Table 3.

15N-NMR chemical s h i f t s o f 1. i n an e x t e r n a l c a p i l l a r y .

6 ppm from N H 4 l 5 N O 3 ~~

i n C6D6

i n CDC13

-

248.8 A7

- 255.5 Abu

-

256.9 257.3 A8 258.0

258.6 Val

-

255.1 Abu

-

256.7

- 261.8

248.1 A7

256.9 ~8

- 258.3 -

i n CDCl

259.2

-

3

in

C6D6

259.2

-

260.1

- 261.8

260.4 V a l

264.2

-

265.2

- 265.1

263.1 263.9

1

1

m

I73

-6 F i g . 10.

[PpnJ

1

m

170

spectrum of cyclosporlne: CO region. Those CO groups whose signals shifted downfield are marked In black. The CO sign81 of residue 8 (shaded) lowest from those attached to NE groups (residues 1 , 4 , 6 , and 7). ''C-NNFI

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

176

3.6

Mass Spectrum 3.6.1

EI Spectrum The e l e c t r o n impact spectrum o f c y c l o s p o r i n e w a s measured on a CEC-mass spectrometer 21-llOB ( 4 ) . The e l e c t r o n energy w a s 7 0 e V , t h e a c c e l e r a t i o n rate 4.6-8 KV, t h e temperat u r e of t h e source 280-300°C and a p r e s s u r e of 10-5 - 10-6 T o m . The molecules w a s u n s t a b l e g i v i n g a molecular i o n peak a t m/e 1201 which v a n i s h e s q u i c k l y . A s t h e M+ v a n i s h e s a peak a t m / e 1183 i n c r e a s e s i n i n t e n s i t y and t h e spectrum remains s t a b l e . The peak a t m/e 1183 i s formed by eliminat i o n o f a molecule o f water. The e x a c t mass measurement were made at 1183.831 i 0.003 and 1201.842 f 0.003. The molecular f o r m u l a o b t a i n e d by t h i s measurement w a s C62HlllN11012 ( p h y s i c a l molecular mass 1201.841). T h i s w a s i n agreement w i t h t h a t deduced from NMR and o t h e r f i n d i n g s . Other mass s p e c t r a l d a t a w i l l be p r e s e n t e d under metabolism.

3.6.2

FD Spectrum The f i e l d d e s o r p t i o n mass spectrum w a s measured on a MAT 711 mass spectrometer ( 4 ) . The a c c e l e r a t i o n energy r a t e 6 KV, t h e temperature of t h e i o n s o u r c e goo and t h e power f a c t o r f o r m u l t i p l i e r 106. A molec u l a r i o n peak M+ of m / e 1201.8 w a s o b t a i n e d and m / e 1203.6 f o r t h e d i h y d r o c y c l o s p o r i n e . Another FD spectrum w a s r e p o r t e d (18). A molecular i o n peak Mc a t m / e 1202, NH' a t m / e 1203 and ( M + N a + ) a t m / e 1225.

4.

I s o l a t i o n o f Cyclosporine Dreyfuss e t a 1 ( 2 ) and o t h e r s ( 4 , 27-30) have r e p o r t e d t h e i s o l a t i o n of c y c l o s p o r i n e from o t h e r f u n g a l m e t a b o l i t e s of t h e f u n g i Tolypocladium i n f l a t u m Gams. T h i s fungus w a s p r e v i o u s l y known as Trichoderma polysporum (Link ex P r e s . R i f a i ( 2 ) .

CYCLOSPORINE

177

The s t r a i n used i s Tolypocladium i n f l a t u m d e s i g n a t e d No. N RRL 8044. T h i s was grown i n an a e r o b i c submerged culture. A r e p o r t e d method of i s o l a t i o n of c y c l o s p o r i n e from f e r m e n t a t i o n media w a s as f o l l o w s ( 4 ) :

The f e r m e n t a t i o n medium w a s e x t r a c t e d w i t h n-butylacetate. Then a f t e r t h e s e p a r a t i o n of t h e o r g a n i c l a y e r it w a s c o n c e n t r a t e d under vacuum. The crude r e s i d u e o b t a i n e d w a s d e f a t t e d with 90% methanol and petroleum e t h e r . The product o b t a i n e d was d i s s o l v e d i n chloroform and a p p l i e d t o a column chromatography of s i l i c a g e l . The p o l a r i t y of t h e e l u t i n g s o l v e n t w a s g r a d u a l l y i n c r e a s e d by adding methanol t o chloroform. The f r a c t i o n s o b t a i n e d by chloroform-methanol ( 98 :5 :1 . 5 ) c o n t a i n e d c y c l o s p o r i n A . F r a c t i o n s c o n t a i n e d c y c l o s p o r i n -C w a s o b t a i n e d w i t h chloroform-methanol (97:3). The f r a c t i o n s c o n t a i n i n g c y c l o s p o r i n A were s u b j e c t e d t o g e l f i l t r a t i o n with Sephadex LH-20 w i t h methanol. The t o p f r a c t i o n s w e r e d i s s o l v e d i n t o l u e n e and a p p l i e d t o column chromatography o f aluminium oxide u s i n g t o l u e n e - e t h y l a c e t a t e as t h e e l u e n t . The f r a c t i o n s o b t a i n e d were evaporated and d i s s o l v e d i n a l c o h o l , t h e n t r e a t e d w i t h c h a r c o a l . The f i l t r a t e was evaporated t o g i v e c y c l o s p o r i n A as an amorphous powder. Another r e p o r t e d procedure ( 2 7 ) w a s as f o l l o w s : The f e r m e n t a t i o n b r o t h w a s s e p a r a t e d i n t o t h e m y c e l i a l cake and t h e c u l t u r e f i l t r a t e . The former w a s homogen i z e d t h r e e t i m e s w i t h methanol-water (9:l) and t h e combined f i l t r a t e s were c o n c e n t r a t e d under vacuum t o remove t h e methanol. The water s o l u t i o n w a s e x t r a c t e d t h r e e times w i t h l , 2 - d i c h l o r o e t h a n e and t h e o r g a n i c l a y e r s were evaporated t o d r y n e s s . Working-up of t h e c u l t u r e f i l t r a t e by e x t r a c t i o n w i t h 1 , 2 - d i c h l o r o e t h a n e r e s u l t e d i n an a d d i t i o n a l p o r t i o n of crude material. The combined e x t r a c t e d were submitted t o g e l f i l t r a t i o n on Sephadex LH-20 w i t h methanol. Those f r a c t i o n s which c o n t a i n t h e c y c l o s p o r i n e ( a s a m i x t u r e ) were pooled and t h e n s e p a r a t e d i n t o t h e components by s i l i c a g e l column chromatography (Merck , 0.063-0.2 mm) w i t h e t h y l a c e t a t e s a t u r a t e d w i t h water. I n accordance t o t h e i r p o l a r i t y , t h e cyclosporines were e l u t e d i n t h e following o r d e r : D , G, A , B and C . The crude c y c l o s p o r i n e s were f u r t h e r p u r i f i e d by c r y s t a l l i z a t i o n ( c y c l o s p o r i n e G from e t h e r / p e t r o l e u m e t h e r a t room t e m p e r a t u r e ; c y c l o s p o r i n e s A , C and D f r o m a c e t o n e a t - 15OC). The

178

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

r e s u l t i n g pure c y c l o s p o r i n e s A, B, C, D and G w e r e c h a r a c t e r i z e d by a n a l y t i c a l methods ( t h i n l a y e r chromatography, m e l t i n g p o i n t , o p t i c a l r o t a t i o n ) and s p e c t r a l d a t a ( U , I R , 'H- and l3C-NMR, mass s p e c t r u m ) . They were found t o be i d e n t i c a l i n a l l r e s p e c t s w i t h a u t h e n t i c samples i s o l a t e d from f e r m e n t a t i o n i n complex n u t r i e n t media as p u b l i s h e d by Dreyfuss e t a 1 ( 2 ) , Ruegger _ et _ a1 ( 4 ) and Traber e t a1 ( 2 8 , 3 0 ) .

5.

B i o s y n t h e s i s of Cyclosporine Kobel _ e t -a1 ( 3 1 ) and o t h e r s (27,32,33) have r e p o r t e d t h e b i o s y n t h e s i s of c y c l o s p o r i n e and o t h e r f u n g a l metab o l i t e s . I n t h e i n i t i a l b i o s y n t h e t i c s t u d i e s o f Kobel _ et _ a1 (31) , 3H- and 13C-labeled p r e c u r s o r s w e r e f e d t o t h e c u l t u r e and t h e p o s i t i o n o f i n c o r p o r a t i o n o f t h e l a b e l i n c y c l o s p o r i n e w a s determined by NMR s p e c t r o s c o p y . It w a s demonstrated t h a t t h e N-methyl groups of t h e molec u l e and t h e methyl group i n t h e y - p o s i t i o n o f t h e u n s a t u r a t e d amino a c i d MeBmt a r e i n t r o d u c e d as i n t a c t methyl groups from methionine and t h a t t h e remaining carbons of t h e MeBmt moiety a r e d e r i v e d from t h e headt o - t a i l condensation of f o u r a c e t a t e u n i t s . The l3C-NMR spectrum o f t h e e n r i c h e d c y c l o s p o r i n e d e r i v e d from [ l-13C]acetate showed f o u r enhanced s i g n a l s corresponding t o t h e carbon atoms 1, 3, 5 and 7 o f t h e MeBmt u n i t and no '3C i n c o r p o r a t i o n i n t o any o t h e r amino a c i d .

I n e n n i a t i n , a n analogous example of non-ribosomal c y c l i c p e p t i d e s y n t h e s i s by f u n g i , Zocher and Kleinkauf ( 32) demonstrated t h a t a m u l t i f u n c t i o n a l enzyme c a r r i e s o u t t h e N-methylation o f c o n s t i t u e n t amino a c i d s following an a c t i v a t i o n s t e p when t h e a c i d s are bound t o t h e enzyme as t h i o e s t e r s . Supporting evidence f o r a similar mechanism i n c y c l o s p o r i n e b i o s y n t h e s i s w a s provided by t h e o b s e r v a t i o n o f Zocher e t a1 ( 3 3 ) t h a t [14C] s a r c o s i n e w a s n o t ' n c o r p o r a t e d and t h a t t h e r a d i o a c t i v i t y from [Me-16C]methionine i n c o r p o r a t e d i n t o each N-methylated amino a c i d w a s d i r e c t l y proport i o n a l t o t h e number o f t h e corresponding amin a c i d r e s i d u e s i n c y c l o s p o r i n e , t h u s i n d i c a t i n g t h a t Nm e t h y l a t i o n of t h e amino a c i d s o c c u r r e d s i m u l t a n e o u s l y . From t h e r e s u l t s o f Kobel e t a1 ( 3 1 ) t h e s i t e of b i o s y n t h e s i s of t h e Bmt u n i t remains u n c l e a r . However, t h e results o b t a i n e d by t h e Zocher group ( 3 3 ) w i t h short-term f e e d i n g experiments u s i n g l a b e l e d a c e t a t e and methionine, i n which probably o n l y i n c o r p o r a t i o n of

179

CYCLOSPORINE

label in the N-methyl group occurred, suggest that the amino acid Bmt is not biosynthesized on the enzyme matrix but at some other site before incorporation into cyclosporine.

6. Synthesis 6.1 Cyclosporine Winger (18, 34-38) has reported the total synthesis of cyclosporine and analogues. The strategy of the total synthesis of cyclosporine has involved the following steps:-

I. The synthesis of the N-methyl-C-9-amino acid (Scheme 1) (36, 39-44). 11. Building of the tetrapeptide BOC-D-Ala-MeLeuMeLeu-MeVal-benzylester (37). 111. Preparation of the dipeptide BOC-Abu-Sarbenzylester (18,37,46). TV. V. VI

.

VII.

Building of the tetrapeptide BOC-MeLeu-ValMeLeu-Ala-benzylester (18). The synthesis of the hexapeptide BOC-AbuSar-MeLeu-Val-MeLeu-Ala-benzylester (18). Synthesis of the heptapeptide H-Me-C9-AbuSar-MeLeu-Val-MeLeu-Ala-benzylester (18,47)

.

Formation of the undecapeptide BOC-Ala-MeLeuMeLeu-MeVal-Me-Cg-Abu-Sar-MeLeu-Val-MeLeuAla-benzylester (18,48).

VIII. Cyclization of the undecapeptide with a free amino group and free carboxyl group to produce cyclosporine (48,50,51).

180

tio-

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

F:,

7 Steps

b

COOEI

(ZR,3R)-3-methyl-l,2,4,-butanetrio1

6 Steps

(2R,3R,SE)-3-rnethyl-S-heptene1,2-diol

"II_ c y

k

c

y

(2R, 3R, 5E) -2-hydroxy-3-methyl5-heptenal

(2S, 3R, 4R, 6E) -3-hydroxy-4methyl-2-(methylamino)-6-octenoic acid (MeBmt) Scheme 1.

Synthesis of MeBmt

CYCLOSPORINE

VIII.

181

T o t a l Synthesis of Cyclosporine (18, 34, 35,

37 1

S t r a t e g y Used f o r t h e Synthesis o f Cyclosporine For t h e s y n t h e s i s of cyclosporine [l]( F i g . 1) t h e p e p t i d e bond between t h e L-alanine i n p o s i t i o n 7 and t h e D-alanine i n p o s i t i o n 8 w as chosen f o r t h e c y c l i z a t i o n s t e p . There were two main reasons f o r choosing t h i s s t r a t e g y : 1. The intramolecular hydrogen bonds between t h e amide groups of t h i s l i n e a r p e p t i d e could o p e r a t e so as t o s t a b i l i z e t h e open chain i n folded conformations approximating t h e c y c l i c s t r u c t u r e of cyclosporine and t h u s assist c y c l i z a t i o n . 2 . The bond formation between N-methylated amino a c i d s i s more d i f f i c u l t t h a n between non-methylated amino a c i d s (37,45) ; t h e r e f o r e , bond formation between t h e only consecutive p a i r of non-methylated amino a c i d s i n cyclosporine appeared t h e l o g i c a l choice f o r t h e c y c l i z a t i o n s t e p .

For t h e s y n t h e s i s of t h e undecapeptide, a fragment-condensation technique i n t r o d u c i n g t h e amino a c i d MeBmt a t t h e end of t h e s y n t h e s i s w a s used. I n t h i s way, t h e number o f s t e p s a f t e r t h e i n t r o d u c t i o n of t h i s amino a c i d w a s minimized. Scheme 5 shows t h e s t e p sequence used f o r j o i n i n g t h e ( p r o t e c t e d ) p e p t i d e fragments. The carboxy groups were g e n e r a l l y a c t i v a t e d using a v a r i a t i o n of t h e mixed p i v a l i c anhydride method r e p o r t e d by Zaoral ( 4 6 ) and adapted f o r t h e N-methylamino a c i d d e r i v a t i v e s ( 3 7 ) . T h i s s t r a t e g y allowed slow anhydride formation i n chloroform a t - 2OoC with p i v a l o y l c h l o r i d e i n t h e presence of 2 e q u i v a l e n t s of a t e r t i a r y base such as N-methylmorpholine before adding t h e amino a c i d o r p e p t i d e e s t e r s t o be coupled i n t h e form o f t h e i r f r e e bases. The f r e e bases were obtained by removal of t h e Boc-protecting groups with t r i f l u o r o a c e t i c a c i d a t - 2OoC and subsequent

182

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

n e u t r a l i z a t i o n with NaHC03; t h e benzyloxyp r o t e c t i n g groups of t h e p e p t i d e i n t e r mediates were removed h y d r o g e n o l y t i c a l l y with palladium/carbon i n ethanol. The t e t rapept i d e Boc-D-Ala-MeLeu-MeLeuMeVal-OBzl w a s synthesized from t h e l e f t t o t h e r i g h t by forming bonds 1, 2 and 3 (Scheme 2 ) . Bond 4 was formed on synt h e s i s of t h e d i p e p t i d e Boc-Abu-Sar-OBzl. The t e t r a p e p t i d e Boc-MeLeu-Val-MeLeu-AlaOBzl w a s synthesized from t h e r i g h t t o t h e l e f t by forming bonds 5 , 6 and 7 i n t h a t o r d e r . Subsequent coupling (bond 8 ) w i t h t h e p r e v i o u s l y synthesized d i p e p t i d e t h e n furnished t h e hexapeptide Boc-Abu-SarMeLeu-Val-MeLeu-Ala-OBzl. During incorporat i o n of t h e amino a c i d MeBmt, t h e hydroxyand N-methylamino f u n c t i o n s were p r o t e c t e d i n t h e form of a dimethyloxazolidine derivat i v e . This isopropylidene-protecting group was r e a d i l y introduced by r e f l u x i n g t h e amino a c i d i n acetone and has t h e advantage of avoiding epimerization of t h e amino a c i d MeBmt during peptide-bond formation. This means t h a t t h e oxazolidine r i n g r e t a i n s t h e thermodynamically more s t a b l e t r a n s c o n f i g u r a t i o n of s u b s t i t u e n t s during carboxy a c t i v a t i o n and peptide formation. On p r e p a r a t i o n , but before a c t i v a t i o n , t h e p r o t e c t e d MeBmt amino a c i d w a s s t a b i l i z e d by a d d i t i o n of N-methylmorpholine (1 e q u i v . ) . To prepare t h e p r o t e c t e d heptapeptide from t h e p r o t e c t e d MeBmt amino a c i d and t h e hexapeptide, t h e dicyclohexylcarbodiimide ( D C C I ) coupling method w a s used i n presence of N-hydroxybenzotriazole ( 4 7 ) . A f t e r removal of t h e isopropylidene-protecting group (1 equiv. of 1 N HCl/MeOH), t h e f i n a l amide l i n k a g e 1 0 w a s made by coupling Boc-D-Ala-MeLeu-MeLeu-MeVal-OH with t h e heptapeptide i n presence of N-methylmorphol i n e i n CH2C12 at room temperature using t h e reagent (1H-1,2,3-benzotriazol-l-yloxy)tris (dimethylamino)-phosphonium hexafluorophosphate ( B t O P ( NMe2)'3 PFZ) developed by Castro et a1 ( 4 8 ) . A f t e r h y d r o l y s i s of t h e

183

CYCLOSPORINE

step sequence for the peptide bond formation

1

-

4

-

iiiTiiiiii 1

0-Alo

8

HeLeu HeLeu

9

10

HeVol

11

H e 6 m t Abu

Sor

MeLeu

Vol

HeLeu

Alo

1

HeLcu-cHcVol~HeB~t-cAbu~Sar

t

HeLeu

t

0 - A lo --A lo t- H rLeu-Vol --HeLeu

-

Scheme 2.Syniherir olcyclosporinc 1 . Bold arrows: slralrgy lor the synthesis o l the pcptidcs. For delailr see teat. Boc-rrrrbuloxycarbonyl. BzI -bcnzyl. >-OH isopropylidcne.proleacd MeBml.

184

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

e s t e r group (NaOH a t O°C) and removal of t h e Boc group (CF3COOH a t - 2OoC) , t h e unprotected undecapeptide w a s c y c l i z e d a t room temperature t o cyclosporine 1 (0.0002 M CH2C12, 1 equiv. BtOP(NMe); PFZ

( 4 8 ) , N-methylmorpholine). The y i e l d of c r y s t a l l i n e cyclosporine w a s 62%. By using t h e mixed phosphonic anhydride method described by Wissmann and K l e i n e r ( 4 9 ) o r t h e pentafluorophenol-DCCI complex of Kovacs ( 5 0 ) t o e f f e c t c y c l i z a t i o n of t h e unprotected undecapeptide , t h e y i e l d o f cyclosporine could be increased t o 65%. Using t h e fragment-condensation technique described h e r e it i s now p o s s i b l e t o synthesize cyclosporine very e f f i c i e n t l y i n 27.5% y i e l d with r e s p e c t t o t h e amino a c i d MeBmt. Thus, 1 . 6 g of cyclosporine can be produced s t a r t i n g from 1 g of t h e amino a c i d MeBmt

.

7.

Metabolism of Cyclosporine Maurer e t a1 ( 5 2 ) have r e p o r t e d t h e d i s p o s i t i o n of cyclosporine i n man and s e v e r a l animal s p e c i e s and a l s o s t r u c t u r a l e l u c i d a t i o n of i t s m e t a b o l i t e s . The metabol i t e s were i s o l a t e d from dog and human u r i n e and from rat b i l e and f a e c e s . The s t r u c t u r e s of t h e i d e n t i f i e d m e t a b o l i t e s were used t o e s t a b l i s h t h e biotransformation processes. 3H-labeled cyclosporine with s p e c i f i c a c t i v i t i e s ranging from 25 t o 80 uCi/mg w a s prepared b i o s y n t h e t i c a l l y by feeding submerged c u l t u r e s of a s e l e c t e d s t r a i n of t h e fungus inflatum G a m s with [methyl-H3] methionine as l a b e l e d precursor ( 3 1 ) . The radiochemical p u r i t y of t h e l a b e l e d substance w a s b e t t e r t h a n 95%. 3H-NMR a n a l y s i s revealed t h e incorporated tritium t o be l o c a t e d i n t h e seven Nmethyl groups and i n t h e methyl group a t p o s i t i o n 4 (C,) of amino a c i d ( F i g . 11).

r.

The s e p a r a t i o n of m e t a b o l i t e s was performed by HPLC. Nine e t h e r - e x t r a c t a b l e s of cyclosporine were i s o l a t e d from u r i n e of dog and man and from r a t b i l e and f a e c e s ( F i g . 12).

Site and type of biotransformation in cyclosporine

F i g . 11.

c

H

0

r)

..

1)

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

186

Structure of cyclosDorine and i t s m e t a b o l i t e s .

F i g . 12.

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0

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&A8

0

b A I

I

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R ---Ctiz-:ti*.* CkI,-N f

cl'I

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- C ... C H -NI - iII- CH - N - C O - CH 1 I b

I

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

CI.C, I

CI(,/:

'cn,

CII, ,C!l Cn,

CHI

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1202 . C 4

ti

cti,

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CH,

n or.

011

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Cyclosporinc

n 011

1! 9

ho. -

-10 16

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on n

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1214.64

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C H ch-cnZ

z c r . ti

1220.62 1234.64 1214.64 1218.64 A ~ I 1218.64 1188.61

CYCLOSPORINE

187

Despite t h e complex s t r u c t u r e o f c y c l o s p o r i n e , t h e r e a c t i o n s involved i n t h e b i o d e g r a d a t i o n o f t h e molecule are l i m i t e d and produce o n l y a r e l a t i v e l y s m a l l number o f m e t a b o l i t e s . The r e s u l t i n g p r o d u c t s a r e l i p o p h i l i c as i n d i c a t e d by t h e i r occurrence i n t h e e t h e r phase of the liquid-liquid extraction. S i t e and t y p e of b i o t r a n s f o r m a t i o n i n c y c l o s p o r i n e are summarized i n F i g . 11. The c y c l i c o l i g o p e p t i d e s t r u c t u r e of c y c l o s p o r i n e i s p r e s e r v e d i n a l l i d e n t i f i e d metab o l i t e s . Metabolism mainly c o n s i s t s o f o x i d a t i v e p r o c e s s e s ( p h a s e I r e a c t i o n s ) . T y p i c a l phase I1 r e a c t i o n s l i k e c o n j u g a t i o n w i t h s u l f u r i c a c i d or g l u c u r o n i c a c i d could n o t be d e t e c t e d . Only a l i m i t e d number o f t h e c y c l o s p o r i n e s u b u n i t s , namely f o u r of 11 amino a c i d s , undergo r e g i o s p e c i f i c o x i d a t i v e modif i c a t i o n s . Hydroxylation r e a c t i o n s appear t o be r e s t r i c t e d t o t h e rl-position o f amino a c i d 1 (Cg-amino a c i d ) and t h e y - p o s i t i o n o f t h e N-methylleucines 4, 6 , and 9. Oxidative N-demethylation, so f a r , seems t o occur o n l y on t h e N-methylleucine 4. The N-methylleuc i n e s 6 and 9 a r e s u b j e c t o f a s i n g l e r e a c t i o n ( h y d r o x y l a t i o n ) whereas amino a c i d 1 and t h e N-methyll e u c i n e 4 are s u b s t r a t e s f o r two t y p e s of transformat i o n : h y d r o x y l a t i o n and i n t r a m o l e c u l a r e t h e r formation o r h y d r o x y l a t i o n and N-dernethylation. The proposed pathways f o r t h e b i o t r a n s f o r m a t i o n o f c y c l o s p o r i n e ( F i g . 13) are based on t h e s t r u c t u r e o f t h e i s o l a t e d m e t a b o l i t e s . The r e g i o i s o m e r i c monohydroxylated c y c l o s p o r i n e s 1 and 17 and t h e N-demethyl a t e d c y c l o s p o r i n e 2 1 are primary m e t a b o l i t e s . M e t a b o l i t e s 1 and 17 r e s u l t from h y d r o x y l a t i o n o f e i t h e r t h e N-methylleucine 9 a t t h e y - p o s i t i o n or t h e amino a c i d 1 a t t h e q - p o s i t i o n . Metabolite 21 r e p r e s e n t s the product o f N-demethylation on t h e N-methylleucine 4. F u r t h e r o x i d a t i o n o f metab o l i t e l on e i t h e r one o f t h e N-methylleucines 4 and 6 o r on t h e Cg-amino a c i d 1, and o f m e t a b o l i t e 17 on t h e N-methylleucine 9 g e n e r a t e s d i h y d r o x y l a t e d d e r i v a t i v e s of c y c l o s p o r i n e ( m e t a b o l i t e s 8 , 1 0 , and 1 6 ) . The i d e n t i f i c a t i o n o f a d i h y d r o x y l a t e d and N-demethylated d e r i v a t i v e of c y c l o s p o r i n e ( m e t a b o l i t e 9 ) i n d i c a t e s f u r t h e r o x i d a t i o n t o occur on e i t h e r t h e d i h y d r o x y l a t e d m e t a b o l i t e 1 6 by N-demethylation o f t h e N-methylleucine 4 o r t h e primary N-demethylcyclosporine 2 1 by hydroxyl a t i o n o f b o t h N-methylleucines 6 and 9.

8

t

t

21

'En,

I

10

Fig. 13. Proposed pathways for the biotransformation of cyclosporine.

CYCLOSPORINE

189

Metabolite 18, containing a cyclic ether moiety (tetrahydrofuran), may be derived from 17 (monohydroxycyclosporine) by intramolecular addition of the 6-hydroxyl group to the double bond of the C9-amino acid 1. This cyclization is suggested to proceed by a nonenzymatic mechanism. An artifact with a similar cyclic ether structure has previsouly been observed among the acid hydrolysis products of cyclosporine. Three other metabolites possessed the basic cyclic structure of the parent drug. The spectroscopic data of two of them were compatible with hydroxylated and N-demethylated derivatives of cyclosporine, whereas those of the third indicated a dihydroxylated derivative. Their structures remain incompletely determined. It should be noted that metabolites 10 and found in man (53).

16 were not

8. Pharmacokinetics of cyclosporine The absorption, distribution, metabolism and elimination of cyclosporine have been investigated by many authors

-

( 52-57)

The pharmacokinetic data on cyclosporine in man as follows : Absorption Ab solute or a1 bioavailabi1ity

20-50% (mean 34%)

Plasma peak concentration time

1-6 hours

Di stribut ion Binding to plasma proteins

go% (20OC)

Apparent volume of distribution

3.5 1/Kg

Metabolism Extent of metabolism

Ca. 99%

Number of components

10-12(9 of which have been characterised as metabolites).

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

Elimination Terminal e l i m i n a t i o n h a l f - l i f e

14-26 hours

T o t a l body c l e a r a n c e

0.27-0.47

l/h/Kg

because of i t s v e r y low w a t e r - s o l u b i l i t y , t h e o r a l dose form of c y c l o s p o r i n e i n an o l i v e - o i l - b a s e d s o l u t i o n . The a b s o l u t e o r a l b i o a v a i l a b i l i t y from t h i s s o l u t i o n ( i n s t e a d y s t a t e ) i s 20-50% (mean 34%).

I n whole blood, about 50% o f c y c l o s p o r i n e i s t a k e n up by e r y t h r o c y t e s and 10-20% by l e u c o c y t e s . O f t h e t o t a l amount of drug i n t h e plasma, approximately 90% i s protein-bound, mostly t o l i p o p r o t e i n s .

I n animal experiments using3H-cyclosporine, t h e h i g h e s t t i s s u e c o n c e n t r a t i o n s of r a d i o a c t i v i t y were found i n l i v e r , kidney, a d r e n a l s , p a n c r e a s , thymus, t h y r o i d and r e n a l f a t . From blood concentration-time d a t a f o l l o w i n g i n t r a v e n o u s a d m i n i s t r a t i o n i n man, t h e drug was found t o be d i s t r i b u t e d according t o a three-compartment d i s t r i b u t i o n model. The t o t a l a p p a r e n t volume o f d i s t r i b u t i o n was 3.51/Kgm. Cyclosporine i s slowly b u t e x t e n s i v e l y metabolised by l i v e r . I n a d d i t i o n t o t h e p a r e n t d r u g , 10-12 metab o l i t e s have been d e t e c t e d i n human plasma. Nine metab o l i t e s have been i s o l a t e d and i d e n t i f i e d ; a l l c o n t a i n e d t h e i n t a c t c y c l i c o l i g o p e p t i d e s t r u c t u r e of t h e p a r e n t molecule. S t r u c t u r a l m o d i f i c a t i o n s c o n s i s t e d of monoand di-hydroxylation and N-demethylation a t v a r i o u s p o s i t i o n s on t h e c y c l o s p o r i n e . Terminal e l i m i n a t i o n h a l f - l i v e s from blood o r plasma o f 14-26 hours have been r e p o r t e d . The drug and i t s m e t a b o l i t e s are e x c r e t e d mainly v i a t h e l i v e r and o n l y t o a s l i g h t e x t e n t v i a t h e k i d n e y s . I n man t h e t o t a l u r i n a r y e x c r e t i o n o f r a d i o a c t i v i t y i n t h e p e r i o d up t o 96 hours f o l l o w i n g a s i n g l e dose o f 3H-cyclosporine w a s 6% of t h e dose; unchanged cyclos p o r i n e accounted f o r o n l y 0.1% o f t h e o r & dose,

191

CYCLOSPORINE

9 . Pharmacological Activity and Clinical Application As reported by Bore1 et a1 (5-9), cyclosporine is a selective immunosuppressive drug with antifungal and antiinflammatory properties. It has been shown to strongly suppress the production of haemagglutinating antibodies against sheep erythrocytes in mice and to be effective in inhibiting both humoral and cellmediated immune responses. Its effect is reversible, and specific for T-lymphocytes. The hemopoietic tissues in immunosuppressed animals are not affected. Bueding _ et _ a1 (58) demonstrated an antishistosomal activity and Thommen-Scott ( 59) an antimalarial effect of cyclosporine, both antiparasitic activities being independent of immunosuppression. The first transplantation in humans with the aid of cyclosporine were reported in 1978 by Calne et a1 (60) f o r kidney and by F’owles et a1 (61) f o r bone marrow. Today cyclosporine is used successfully to prevent graft rejection following bone marrow and organ transplantations. Other possible applications, such as in the treatment of autoirnune and other diseases, are currently under investigations. 10. Pharmaceutical Forms

Cyclosporine ( Sandimmun) is available as : a)

Drinking Solution: 100 mg/ml cyclosporine dissolved in olive oil and labrafil.

b)

Intravenous Preparation: Ampoules of 5 m l (250 mg cyclosporine) and 1 ml (50 mg cyclosporine).

11.Methods of Analysis 11.1 Elemental Composition

Cyclosporine (C H N 0 ): 62 111 11 12 C

61.90

H

9-30

N

0

12.80

16.00

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

192

11.2

Introduction to Analysis Several methods have been developed for the analysis of cyclosporine in plasma and or blood. The first method reported was the radioimmunoassay (RIA) of Donatsch (62)which is now available as a kit and has been used extensively during the clinical development for cyclosporine. The method was initially developed for plasma, and as shown later, is also applicable to blood samples. The main criticism of the RIA method has been the cross-reactivity of the antisera with some of the circulatinc metabolites of cyclosporine. Other RIA procedures were reported ( 54,55, 63-67). Subsequent to the introduction of the RIA method, Niederberger et a1 (68) developed a high pressure liquid chromatogri:phy ( HPLC) method based on gradient elution. The technical complexity of this method encouraged several investigators, (69-75) to reinvestigate the method. The results of these activities are shown in Table 4.

11.3

Radioimmunoassay ( RIA) Several radioimmunoassay procedures have been developed for both the analysis and immunopharmacological monitoring of cyclosporine ( 54,55,

-

63-67)

One method described recently by Randall et a1 (64) is as follows: Sample Preparation: Venous blood was collected in heparinized tubes from 38 post-transplant patients receiving cyclosporine, just before their next dose and 12 h after their last.

Non-temperature-standardized protocol: About 0.5 to 3 h after collection, an aliquot of each sample f o r whole-blood analysis was set aside and centrifuged the remaining specimen at room temperature to remove cells. The plasma and whole-blood specimens were both then stored at - 20%.

CYCLOSPORINE

193

Temperature-standardized p r o t o c o l : After c o l l e c t i o n , samples were r e f r i g e r a t e d for no l o n g e r t h a n 4 h , r e - e q u i l i b r a t e d t o 37OC by i n c u b a t i o n i n a water b a t h f o r 30 min, t h e n c e n t r i f u g e d without d e l a y (1100 X g , 1 0 min, room t e m p e r a t u r e ) The r e s u l t i n g plasma w a s s t o r e d f r o z e n a t -2OOC u n t i l analysis. Time and t e m p e r a t u r e experiments: A l i q u o t s of whole blood from p a t i e n t s r e c e i v i n g c y c l o s p o r i n e were i n c u b a t e d for 0.25, 0.5, 1, 2, 4 , 6, and 24 h a t 4'12, room t e m p e r a t u r e ( a b o u t 22OC), and 37OC. A t t h e s e s p e c i f i e d t i m e s , samples s t o r e d a t room temperature or 4°C were c e n t r i f u g e d a t room t e m p e r a t u r e ; t h o s e s t o r e d a t 3 7 O C were c e n t r i f u g e d a t 37OC and t h e plasma was decanted and f r o z e n . I n a d d i t i o n , t h e t r e a t e d a l i q u o t s of samples s t o r e d a t room t e m p e r a t u r e and 4 O C f o r t h e above t i m e s b y r e - e q u i l i b r a t i n g t o 3 7 O C by i n c u b a t i n g for 1 5 min i n a 37OC w a t e r b a t h , followed by c e n t r i f u g a t i o n a t 37OC and prompt removal of t h e plasma. E q u i l i b r a t i o n time experiment: A l i q u o t s o f whole blood from p a t i e n t s r e c e i v i n g c y c l o s p o r i n e w e r e maintained a t 4 O C f o r 4 h a f t e r c o l l e c t i o n , t h e n e q u i l i b r a t e d i n a 37OC w a t e r b a t h for 0, 10, 1 5 , 30, 60, 1 2 0 , and 180 min. All specimens were c e n t r i f u g e d a t 3 7 O C , and t h e plasma was immediately decanted and stored a t -2OOC u n t i l a n a l y s i s . Measurement of c y c l o s p o r i n e : Cyclosporine w a s measured by radioimmunoassay, u s i n g t r i t i a t e d c y c l o s p o r i n e as t r a c e r and an a n t i b o d y i n a k i t s u p p l i e d by Sandoz L t d . , B a s l e , S w i t z e r l a n d . D u p l i c a t e 5 pL p a t i e n t ' s samples were mixed with 1 0 0 pL of t r a c e r , TOO 1J.L o f T r i s b u f f e r ( 0 . 5 mol/L, p H 8 . 5 ) , and 100 UL o f sheep a n t i s e r u m t o c y c l o s p o r i n e , t h e n i n c u b a t i n g for 2 h a t room t e m p e r a t u r e . F r e e and bound f r a c t i o n s were s e p a r a t e d by e x t r a c t i o n w i t h c h a r c o a l for min a t 4 O C . The r a d i o a c t i v i t y was measured i n each specimen w i t h a l i q u i d s c i n t i l l a t i o n

194

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

c o u n t e r , u s i n g a quench-corrected c o u n t i n g program. The a n a l y t i c a l recovery o f c y c l o s p o r i n e i n plasma and whole blood supplemented w i t h t h e drug w a s about 75%. C o r r e c t i o n f o r h e m a t o c r i t : Hematocrits of samples f o r c y c l o s p o r i n e a n a l y s i s were measured as p a r t o f t h e r o u t i n e b l o o d count on a Coulter S Plus (Coulter Instruments, Hialeah, F L ) . The plasma c y c l o s p o r i n e r e s u l t s were c o r r e c t e d f o r hematocrit by m u l t i p l y i n g t h e u n c o r r e c t e d plasma c y c l o s p o r i n e r e s u l t s by t h e p a t i e n t ' s hematocrit / O .39, t h e n o m a l v a l u e f o r t h e hematocrit.

1 1 . 4 High Performance Liquid Chromatography S e v e r a l HPLC procedures were r e p o r t e d for t h e e s t i m a t i o n o f c y c l o s p o r i n e i n blood and o r plasma a s w e l l as immunopharmacological monit o r i n g o f c y c l o s p o r i n e ( 5 3 , 5 4 , 67, 69-75). These methods were developed t o overcome t h e drawbacks o f t h e R I A methods p a r t i c u l a r l y t h e overestimation of cyclosporine i n b i o l o g i c a l f l u i d s . This i s because, t h e a n t i b o d i e s c r o s s r e a c t w i t h t h e m e t a b o l i t e s . The HPLC c o n d i t i o n s used f o r t h e a n a l y s i s o f c y c l o s p o r i n e a r e l i s t e d i n Table 5. A r e c e n t HPLC method w a s r e p o r t e d by Kates and L a t i n i ( 7 5 ) and i s as follows : Chemicals and r e a g e n t s : Cyclosporine and t h e i n t e r n a l s t a n d a r d , c y c l o s p o r i n D ( F i g . 14). are o b t a i n e d from Sandoz (Basle, S w i t z e r l a n d ) . Glass-distilled diethyl ether, a c e t o n i t r i l e , q-hexane, and methanol are o b t a i n e d from Burdick and Jackson Labs. (Muskegon, M I , Tris(hydroxymethy1)ainomethane i s U.S.A.). purhcased from A l d r i c h (Milwaukee, W I , U.S.A.) I n s t r u m e n t a t i o n and chromatograghic c o n d i t i o n s : A Waters Instrument ( M i l f o r d , MA, U.S.A.) Model 6 0 0 0 ~HPLC pump, Model 480 v a r i a b l e wavelength UV d e t e c t o r , Model 7lOB sample i n j e c t o r and Model 730 d a t a module a r e employed. The column used i s a Brown-Lee Labs. ( S a n t a Clara, CA, U.S.A.) RP-8 MLPC a n a l y t i c a l c a r t r i d g e

195

CYCLOSPORINE

( 1 0 ern X 4.6 mm I.D.; p a r t i c l e s i z e 1 0 m). A 3-em RP-8 guard c a r t r i d g e i s p o s i t i o n e d a t t h e head o f t h e column. The column i s m a i n t a i n e d a t 7 O o C with an Eldex (Menlo Park, CAY U . S . A . ) column h e a t e r . The h e a t e r i s l e f t on a t a l l t i m e s and a c o n s t a n t flow i s m a i n t a i n e d through t h e column. The flow-rate o f t h e mobile phase i s 0.6 ml/min which produces a precolumn p r e s s u r e o f about 34 bars ( 500 p. s i. ) The e f f l u e n t from t h e column i s monitored a t a wavelength o f 215 nm.

.

Mobile phase: The mobile phase c o n s i s t s o f a simple mixture of a c e t o n i t r i l e - w a t e r ( 72:28). This m i x t u r e i s f i l t e r e d and degassed by vacuum and s o n i c a t i o n . The mobile phase i s c o n t i n u o u s l y r e c y c l s e d and r e p l a c e d about every two weeks. P r e p a r a t i o n o f e x t r a c t i o n columns: The e x t r a c t i o n o f c y c l o s p o r i n e i n v o l v e s t h e use o f a Baker-10 SPE e x t r a c t i o n system ( J . T . Baker, P h i l l i p s b u r g , N J , U.S.A.). The columns used are t h e 3 ml cyano d i s p o s a b l e e x t r a c t i o n columns. These columns a r e p r e p a r e d by washing w i t h 6 ml of methanol and t h e n 6 ml o f water under vacuum. They a r e l e f t approximately one h a l f t o t h r e e q u a r t e r s f u l l of w a t e r u n t i l t h e sample i s added. E x t r a c t i o n procedure: One ml o f whole blood or serum i s added t o PTFE-lined, screw-capped t u b e s . To t h e blood or serum i s added 450 ng o f i n t e r n a l s t a n d a r d , 3 ml o f 0 . 1 M T r i s b u f f e r (pH 9 . 8 ) and 1 0 m l o f d i e t h y l e t h e r . They a r e rocked f o r 20 min on a labquake r o c k e r (Lab. I n d u s t r i e s , Berkeley, CA, U.S.A.) and c e n t r i fuged for 20 min t o s e p a r a t e t h e e t h e r and aqueous l a y e r s . The d i e t h y l e t h e r i s p i p e t t e d i n t o a c l e a n t u b e and t h e blood or serum residue i s d i s c a r d e d . To t h e d i e t h y l e t h e r i s added 200 p1 o f 75% methanol i n w a t e r . The d i e t h y l e t h e r i s t h e n evaporated a t room temperat u r e w i t h a g e n t l e stream of n i t r o g e n . Only t h e d i e t h y l e t h e r i s e v a p o r a t e d , l e a v i n g 150200 p 1 o f t h e methanol-water remaining. To t h i s are added a n o t h e r 100 u1 of methanol. The

196

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

methanol-water i s t h e n t r a n s f e r r e d t o a 3 m l Baker e x t r a c t i o n column, The r e s i d u e i s e l u t e d onto t h e column w i t h water. The column i s t h e n washed w i t h 3 ml of 25% a c e t o n i t r i l e i n w a t e r and 6 m l of n-hexane. The columns a r e t h e n d r i e d by drawing through a i r for 4 min. Cyclosporine and c y c l o s p o r i n D are t h e n d l u t e d off t h e column w i t h t h r e e washings o f 200 p l of methanol. The methanol i s c o l l e c t e d i n t o d i s p o s a b l e t u b e s and evaporated t o dryness w i t h a stream of n i t r o g e n a t 5OoC. The r e s u l t i n g r e s i d u e i s d i s s o l v e d i n 200 p 1 of t h e mobile phase. An a l i q u o t o f 1 0 0 p 1 i s t h e n i n j e c t e d o n t o t h e column ( F i g . 1 4 ) . P r e p a r a t i o n of c a l i b r a t i o n s t a n d a r d s : S t o c k s o l u t i o n s of c y c l o s p o r i n e and c y c l o s p o r i n D are prepared i n methanol and s t o r e d i n amber b o t t l e s a t room t e m p e r a t u r e . A s t o c k s o l u t i o n of c y c l o s p o r i n D i s prepared i n a c o n c e n t r a t i o n of 30 n g / u l . The s t o c k s o l u t i o n of c y c l o s p o r i n e i s prepared i n a c o n c e n t r a t i o n of 10 n g / p l . T h i s s o l u t i o n i s used t o p r e p a r e s t a n d a r d s for t h e c a l i b r a t i o n curve. The c a l i b r a t i o n c u r v e samples are prepared by adding amounts of cyclos p o r i n e (50-800 ng) t o whole blood or serum from a normal v o l u n t e e r . These are t h e n t r e a t e d as d e s c r i b e d i n t h e e x t r a c t i o n procedure. This procedure has a lower l i m i t of s e n s i t i v i t y below 50 ng/ml.

C

d

I n

0

71

I

11

\

L

F i g . 14. Chromatograms of extracted whole blood samples. ( A ) Blood from normal healthy

volunteer, not taking cyclosporine; (B) blood to which has been added 200 ng/rnl of cyclosporine and the internal standard; (C) blood from a patient who was taking cyclosporine ( t h e concentration in this patient sample is 304 nglml). Peaks: I = cyclosporine; I1 = cyclosporin D, internal standard.

Table

4. Comparison o f p u b l i s h e d methods o f a n a l y s i s o f c y c l o s p o r i n e

analysis

Sample t y p e

Sample* preparation

Detect i o n limit pg/litre)

Specificity

S t an dardization Ext

.

-

20

High

Int

.

30

100

-

Ext

Very l o n g

25

High

Int

Short

20

High

Blood/plasma

HPLC-Grad.

Plasma

Long

Serum

Long

.

%

.

Plasma

Long

100

High

. Int . Int .

HPLC-Isocr.

Blood/plasma

Long

100

High

Int

HPLC-Grad.

Serum

Long

30-50

High

Int

HPLC-Isocr.

Plasma

Long

31

High

Int .

ihort s emiaut oma-

8

High

Ext

50

High

Int

HPLC-Isocr. *

1-20

.

Blood/plasma

HPLC-Col Swit Blood/plasma HPLC-Isocr

.

HPLC-Col .Swit 3loodlplasma

HPLC-Isocr "Short:

**RIA:

ted)

.

Long

. .

.

54955, 63-67 68

5 10

69 70

30

71

20

72 63

45 15

.

30

73 74

15

67

.

19

75

p r o t e i n p r e c i p i t a t i o n and i n j e c t i o n ; Long: e x t r a c t i o n & e v a p o r a t i o n ; a d d i t i o n a l sample m a n i p u l a t i o n . Radioimmunoassay. Ex:. =.External; I n t . = Internal; Col-Swit = Column s w i t c h i n g .

Ref.

Time( min)

Metabolite x-react i v i t y

~

RIA**

HPLC-Isocr

Chromat o-

Very Long:

Grad. = G r a d i e n t ; I s o c r . = I s o c r a t i c ;

Table

5.

HPLC c o n d i t i o n s used f o r t h e a n a l y s i s of c y c l o s p o r i n e Flow

Sample

Instrument

rate

Mobile phase

Column

m l /min

I Blood/snrum Waters Model RP-8 YLPC a n a l y t i 6000~

,Blood/

1

Technicon

Acet o n i t r i l e / LC-8 S u p e l c o s i l , 5 urn, s u p e l c o , I n c . , water 55:45 v/v

B e l l e f o n t e , PA., a t 75Oc. A l t e x pumps AX-110. UV d e t e c t o r LCD 725 Uvikon Recorder Tarkan 600 W+W A l t e x m i croproc essor 420.

blood/ plasma

. .

c

~

1

0.6

Acetonitrilel w a t e r 72:28

cal cartridge, 10 cm x 4.6 mm I . D . 3-em RP-8 guard c a r t r i d g e , a t 7OoC.

Column I; A l t e x

p e r i s o r b RP-8 , 40 X 4.6 mm I . D . , 30-40 urn. Column 11; Lichrosorb RP-18, 150 X 4.6 mm I . D . , 5 urn, a t 70Oc. A l t e r n a t i v e column; Beckman U l t r a s p h e r e O D s , 150 X 4.6 mm I . D . , Guard column; A l t e x p e r i s o r b RP-2 40 X 3.2 mm I . D . , 30-40 urn.

I

Tetentior

time

Detect i o n

9.2

UV a t 215 nm

(mins)

3.0

UV a t 202 nm

1-7

W at 210 nm

I

C o n s i s t s of 6 solvent s. 1. A c e t o n i t r i l e 1 methanollwat e r 35 :20 :45 v/v/v 2. Acetonitrile1

water 55:45 v/v 3. A c e t o n i t r i l e l water 72:28 vlv

4. 5-

6.

Tetrahydrofuran Acetonitrile/ water 90110 v/v Methanol

Table

5.

Sample

Serum

Serum

( Continued)

1 Instrument

Varian Vist a HPLC system.

I

Mobile phase

Beckman u l t r a s p h e r e ODs, 25 X 0.46 cm,

S t a r t i n g with t r ifluoroacet i c acidlacetonitrile 5 um. Precolumn Vydac C18, 35:65 and increasing proport ions 4 X 0.4 cm. u n t i l 5:95 a t Both columns a t 1 5 mins. 7OoC.

I Waters syst e m cont r o l l e d with M-660 s o l vent programmer

.

Beckman u l t r a s p h e r e - A c e t o n i t r i l e / o c t y l , 25 cm X 4.6 methanol / w a t er mm, 47 :20 :33 by Vol

5 pm.

Maintained a t 72Oc.

.

Flow Retention time Detect ion 3ef rate (mins ) (ml/min

1

14.1

IUV a t

73

1-5

20.46 and

UV a t 210 nm

74

CYCLOSPORINE

12.

201

References

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2.

M. Dreyf'uss, E. Harri, H. Hofhan, H . Kopel, W. Pache, H. T s c h e r t e r , Eur. J. Appl. Microbiol., 3, 125 (1976).

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R.M.

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H. Kessler, D. Ziessow, Nachr. Chem. T e c h . Lab.,

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R . Wenger, T r a n s p l a n t a t i o n Proceedings 1 5 (4-Suppl.

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R . Wenger, Helv. Chim. Acta, 66, 2308 (1983).

37.

R. Wenger, Helv. Chim. Acta

38.

R . Wenger, Angew. Chem. I n t . Ed. Engl

39.

R.S. Cahn, C . I n g o l d , V. P r e l o g , Angew. Chem. ( 1966 1 ,

40.

D. Seebach, E . Hungerbuhler i n R . S c h e f f o l d , Modern S y n t h e t i c Methods, S a l l e & S a u e r l a n d e r , F r a n k f u r t / Aarau 1980, p. 93.

41. 42.

43.

g,464

(1982).

2230 (1983).

(1985

66,2672

1),

1983).

.

78, 413

M. S c h l o s s e r , K.F. Christman, J u s t u s L i e b i g s Ann. Chem.

708,1

(1967).

D.A. S e e l e y , H . McElwee, J . Org. Chem., ( 1973)

.

R.W.

92,

H e r r , D.M.

3813 (1970).

Wieland, C . R .

3, 1691

Johnson, J . Am. Chem. S O C . ,

44.

K.E.

Pfitzner, J.G.

45.

J.R.

McDermott, N.L.

46.

M. Z a o r a l , C o l l e c t . Czech. Chem. Commun., ( 1962 )

47

W . Konig, R . Geiger, Chem. B e r . ,

48.

B. C a s t r o , J . R . Dormoy, J . G . L e t t . , 1219 (1975).

5670 (1965). (1973)-

M o f f a t t , J . Am. Chem. S o c . , Benoiton, Can. J. Chem.,

.

103,788

3,

51, 2551

3, 1273 (1970).

Evin. C . S e l v e , Tetrahedron

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

204

49. 50

-

H. Wissmann, H . J . K l e i n e r , Angew. Chem., 92, 1 2 9 ( 1 9 8 0 ) ; Angew. Chem. I n t . Ed. E n g l . , 9, 133 ( 1 9 8 0 ) .

a

J . Kovacs "Rcemization and Coupling N - p r o t e c t e d Amino Acid and P e p t i d e Active E s t e r s " , i n t h e ' P e p t i d e s ' eds. E. Gross and J Meinenhofer, Vol. 2 , Academic P r e s s , New York-London, 1980, p. 48s.

51.

S.S. Wang, J . P . Tam, B.S.H. Wang and R.B. M e r r i f i e l d , I n t . J . P e p t . P r o t e i n Res. , 459 (1981).

52 *

G. Maurer, H.R. L o o s l i , E . S c h r e i e r and B. Keller, Drug Metab. D i s p o s i t , 12,120 (1984).

53.

A . J . Wood, G. Maurer, W . Miederberger, and T. Beveridge , T r a n s p l a n t a t i o n Proceedings , Vol. XV , No. Supp-1, 1983, p . 2409.

54.

P.A. Keown, C.R. S t i l l e r , N.R. S i n c l a i r , G . C a r r u t h e r s , W. Howson, M. Stawecki, J . McMichael, J . Koegler, N. McKenzie and W. Wall, T r a n s p l a n t a t i o n Proceedings, Vol. X V , No. 4 , Suppl. 1, (1983) p. 2438.

55 *

J. Newburger and B.D. Kahan, T r a n s p l a n t a t i o n Proceedings, X V , No. 4, Suppl. 1 (1983), p . 2413. Vol. -

56.

F. F o l l a t h , M. Wenk, S . Vozeh, e t a l , C l i n . Pharmacol. Ther. , 638 (1983).

57.

T. Beveridge, A. Gratwohl, F. Michot, e t a l , Cum. Ther. Res., 30, 5 (1981).

58.

18,

.

4,

2,

B.E.

Bueding, J . Hawkins and Y.N.

11, 380 (1981).

Cha, Agents A c t i o n s ,

2, 77 (1981).

59.

K. Thommen-Scott,

60.

R . J . Calne, D . J . White, S . T h i r u , D.B. Evans, P. McMaster, D.C. Dunn, G.N. Craddock, D.B. Pentlow, K . R o l l e s , Lancet i i , 1323 (1978).

61.

R.L. Powles, A . J . Barrett, H . C l i n k , H.E.M. Kay, J . Sloane, T . J . McElwain, Lancet 2 , 1327 (1978).

Agents A c t i o n s ,

205

CYCLOSPORINE

62,

P. Donatsch, E . Abisch, M. Homergen, R . T r a b e r , M. Trapp, R . l'oges, J . Immunoassay, 2(1), 19-32 (1981).

63.

B.D. Kahan, C.T. Buren, S.N. Lin, Y . Ono, G. Agostino, S . J . LeGrue, M. B o i l e a u , W.D. Payne, R . H . Kerman, Transplantation, 36 ( 1 9 8 2 ) .

3,

64.

65.

66.

W.Y. R a n d a l l , D.N. Rush, J . R . J e f f e r y , C l i n . Chem.,

30( 11), 1812 (1984).

P.A. Keown, R.A. Ulan, W . J . Wall, C . R . S t i l l e r , N . R . S i n c l a i r , G. C a r r a t h e r s , W. Howson, The Lancet, March 28 (1981). B.D.

Kahan, M. Ried, J . Newburger, Transp. P r o c e e d . ,

15(1), 446 ( 1 9 8 3 ) .

67.

W.T. Robinson, H.F. Schran, E.P. B a r r y , Transp. P r o c e e d . , 1 5 ( 4 Suppl. 1), 2403 ( 1 9 8 3 ) .

68.

W. Niederberger, P. Schaub, J . Beveridge, J . Chromatogr. Biomed. Appl., 182,454 ( 1 9 8 0 ) -

69.

R. 32

70.

R.J.

71*

K. Nussbaumer, W . Niederberger, H.P. Keller, J. High Resolut Chromatogr Chromatogr. Comun, 2, 424 ( 1 9 8 2 ) .

72.

B . Leyland-Jones, A . C l a r k , W. Kreis, Res. Comun. Chem. P a t h o l Pharmacol., 37, 431 ( 1 9 8 2 ) .

73.

G.C. Yee, D . J .

74.

S.G. C a r r u t h e r s , D . J . Freeman, J . C . Koegler, W. Howson, P.A. Keown, A. Lanpacis, C.R. S t i l l e r , C l i n . Chem., 9, 180 (1983).

75.

R . E . Kntes, R . L a t i n i , J . Chromatogr. Biomed. Appl., 309, 441 (1984).

Lawrence, M.C. Allwood, J . Pharm. Pharmacol., ( S u p p l ) , lOOP ( 1 9 8 0 ) . Sawchuk, L.L.

C a r t i e r , C l i n . Chem.,

(1981).

.

2269 ( 1 9 8 2 ) .

27, 1368

.

Gmur, M.S. Kennedy, C l i n . Chem.,

28,

206

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

13. Acknowledgement The authors would like to express their sincere gratitude to Mr. Abdalla I. Babikir of the Medicinal Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia for his continuous and indispensable assistance throughout the preparation of this profile.

ENALAPRIL MALEATE

Dominic P. Ip and Gerald S . Brenner

1. History and Therapeutic Properties 2. Description 2 - 1 Nomenclature 2.1.1 Chemical Name 2.1.2 Generic Name 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 CAS Registry Number 2.2 Formula and Molecular Weight 2.3 Appearance, Color, Odor 3.

Synthesis

4. Physical Properties 4.1 infrared Spectrum - Nuclear Magnetic Resonance Spectrum 1% - Nuclear Magnetic Resonance Spectrum 4 - 4 Ultraviolet Spectrum 4.5 Mass Spectrum 4.6 Optical Rotation 4 . 7 Thermal Behavior 4.8 Solubility 4.9 Crystal Properties 4.10 Dissociation Constants 4 . 1 1 Partition Behavior 4.12 Confocmational Isomers

::;

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

207

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

DOMINIC P. IP AND GERALD S. BRENNER

208

5. Methods of Analysis 5.1 5.2 5.3 5.4 5.5

Elemental Analysis Colorimetric/Spectrophotometric Chromatographic 5.3.1 Thin-Layer Chromatography 5.3.2 High Performance Liquid Chromatography Identification Tests Titration

6. Stability and Degradation 6.1 Solid State Stability 6.2 Solution Stability

7. Pharmacokinetics and Metabolism 8. Determination in Biological Fluids 9.

References

209

ENALAPRIL MALEATE

1. History and Therapeutic Properties

Enalapril is the ethyl ester of a long-acting angiotensin converting enzyme inhibitor, enalaprilat. Enalapril maleate, discovered and developed by the Merck Sharp and Dohme Research Laboratories (l), is indicated f o r the treatment of essential and renovascular hypertension. Enalapril is a pro-drug; following oral administration, it is bioactivated by hydrolysis of the ethyl ester to enalaprilat, which is the active angiotensin converting enzyme inhibitor. CH3

I

e C H 2 C H 2 y H N H C H -CO

-N

COOH

COOCHPCH3

Enalapril

I

CH3

I

CHZCHzCHNHCH -CO

-N

COOH

COOH

Enalaprilat

Since the oral absorption of enalapril is superior to that of enalaprilat, it is the compound that is used in o r a l dosage forms. Enalaprilat, the deesterified parent diacid of enalapril is the form of the drug that is administered intravenously. Several review articles give a detailed account of the history, design, chernistry and pharmacology of the drug. (2-6). 2. Description 2.1 Nomenclature

2.1.1 Chemical Name ( S) -1-[N-[ 1-( ethoxycarbonyl) -3phenylpropyll-Galany11 ->proline, ixltenedioate(1:l)salt

(2)-2-

DOMINIC P. IP AND GERALD S. BRENNER

210

2.1.2

Generic Name Enalapril Maleate

2.1.3

Laboratory Codes G154,739-001D, MK-0421

2.1.4

Trade Names Vasotec, Renitec, Tnnovace, Pres, Xanef

2.1.5

CAS

Registry Number

76095-16-4 2.2 Formula and Molecular Weight

mol w t 492.53

2.3

Appearance, Color, Odor Enalapril maleate is a white to off-white, crystalline, odorless powder

3. Synthesis

Enalapril maleate has been prepared by the scheme outlined in Figure 1 ( 7 ) . Ethyl 2-0x0-4-phenylbutanoate (1) is condensed with L-alanyl-L-proline (2) to give Schiff base 3, as a mixture of syn and anti isomers. Reduction of the imine bond in 3 results in formation of the diastereomeric mixture 4 (SSS and RSS). The more biologically active diastereomer (SSS) is isolated

o=

t

z

C U

I

z +

0,

I

I

0 ,

a

T

0"

5

l -

A"

a f

3'

5

0"

aJ m

c1

aJ

c

m

rl

I4 .A

I4

m e w 0

w

tn

aJ

c

C h

U

v)

DOMINIC P. IP AND GERALD S. BRENNER

212

by fractional crystallization of the maleate salts to yield enalapril maleate (5) of greater than 99% purity in 32 - 34% overall yield. In addition to this synthetic route, others have also been described in the literature (1, 8, 9). 4. Physical Properties 4.1

Infrared Spectrum The infrared spectrum is shown in Figure 2 (10). The spectrum was obtained as a potassium bromide pellet using a Perkin-Elmer Model 281B spectrophotometer. Assignments for the characteristic bands in the spectrum are listed in Table I. Table I Wavenumber, an-’ 3100-3010

2970 and 2920 2800-2200 (Broad, structured) 1750 1727 1645 1570 1500-1390 1375 1355 1187 750

695 4.2

Assignment Aromatic C-H Stretch Methyl and methylene C-H asymmetric stretch / \NH$ Stretch Acid carbonyl stretch Ester carbonyl stretch Amide carbonyl stretch Monohydrogen maleate carboxyl stretch Mainly methylene bending Methyl symnetric bending Methylene bending Ester C-0 stretch In-phase, out-of-plane hydrogen bending (5adjacent aromatic hydrogens) Phenyl out-of-plane ring bending

’H-Nuclear Magnetic Resonance Spectrum The proton magnetic resonance spectrum is shown in Figure 3 (11). The spectrum was obtained using a Varian Associates Model SC300 spectrometer at a concentration of 0.55% w/v in deuterium oxide (D20). Active protons exchange with D20 in

10

N E

Bb 0

8 0

L2 0 0

s 0

Li 0 0

9 0 0

? 0 0

F 0 0

E 0

5:

N

0 0 0

rr)

0

5: lo

-m

-m

Figure 3 .

The P r o t o n Magnetic Resonance Spectrum of E n a l a p r i l Maleate

ENALAPRIL MALEATE

215

solution and appear in the HOD signal at approximately 4.8-4.9 ppm. Figure 4 shows an expanded scale spectrum which m r e clearly illustrates the signals pattern. For several protons in the molecule, slow rotation about the proline amide C-N bond gives rise to paired signals. The phenomena has been attributed to the existence of rotational conformers as in the case of captopril in aqueous solution ( 1 2 ) . A tabulation of the chemical shifts and assignments for the numbered structure below is shown in Table 11. The notations 'major' and 'minor' denote the relative abundance of the two rotamers relative to each other in the spectrum. H\ m

C II

/COOH

H/'\COOH

o

Table I1 hemical Shift (ppm) Multiplicity

'€I6,

1.30

t

1.54

d

1.59

d

1.75

m

2.02

m

2.02

m

2.33

m

2.33 2.80 3.60 3.95 3.97 4.27 4.09

Assignment CE3CH 0-

Alanyl C% (minor) Alanyl C% (major1 Proline C-4 proton (minor) Proline C-4 proton (major) Proline C-3 protons (minor) Proline C-3 protons (major) C-3 ' protons C-4' protons Proline C-5 protons C-2' proton (minor) C-2' proton (major)

CH3CZ20a ' proton (minor)

--csr I

6.0

4.0

Figure 4 .

3.0

PPM

2.0

1.2

The Proton Magnetic Resonance Expanded Spectrum of Enalapril Maleate

ENALAPRIL MALEATE

217

hemical Shift 'H, 6 (ppm) Multiplicity

4.3

4.33

9

6.33

S

7.37 7.41

m m

Assignment a' proton (major)

-H

@

L'C02H

Aryl protons Aryl protons

-m

-o&p

13C Nuclear Magnetic Resonance Spectrum The l3C magnetic resonance spectrum shown in Figure 5 was run using a Varian Associate Model XL200 spectrometer at a concentration of 8 . 4 % w/v in deuterated methanol (13). The internal standard was tetramethylsilane. The pulse width was 4 ps, acquisition time was 0.9 s. The assignments for the numbered structure below are shown in Table 111. As discussed above, the two rotamers are designated 'major' and 'minor' in order of their relative abundances.

v

160

Figure 5.

PPM

50

The Carbon-13 Magnetic Resonance Spectrum of Enalapril Maleate

219

ENALAPRIL MALEATE

Table 111 Chemical 13c, 6 (ppm) 16.2 17.4 18.1 24 -9 27.7 31.8 33.7 35.0 35.2

Assignment

iE:$2H3 Alanyl ?[I3

(major) (minor)

The signals at 24.9-35.2 p p m arise from the four carbons C-3', C-4' and C-4, C-3 proline including signals corresponding to rotamers. An exact assignment of the signals in this region was not made.

49.8

Proline C5

57.9 58.4 61.7 61.8 62.3 62.4 65.6 65.7

The signals at 57.9-65.7 ppm arise from the four carbons Proline C-2', Alanyl C-a, C-2' and -gH2CH3 including signaIs attributable to rotamers. An exact assignmnt of the signals in this region was not made.

129.3

Phenyl C-4"

131.2 131.4

Phenyl C-2", C-6", and phenyl c-3", c-5"

138.0 142.9

Naleate -HcSHPhenyl C-1"

170.5 171.2 171.6 172.0 172.4 176.5 176.7

The signals at 170.5-176.7 arise from alanyl, proline, ester and maleate carbonyl carbons. However, definitive assignments were not made for the carbonyl carbons.

Carbon-13 NMR may also distinguish enalapril mleate and 'ts RSS diastereomer. Major differences between spectra of the two compounds occur between 31.8 and 34.0 ppm, between 54.7 and 60.8

DOMINIC P. IP AND GERALD S. BRENNER

220

ppm and between 168.6 and 170.7 ppm. However, this technique is not preferred for accurate measurement of small quantities of either compound in the other. 4.4

Ultraviolet Spectrum Enalapril maleate is characterized by inflections or "shoulders" at about 251 and 263 y%and barely discernible maxima at abut 21J nm (A1 cm, about 15) and abut 267 nm (A1 cm, about 8.6). Figure 6 shows the ultraviolet absorption spectra of enalapril maleate in N/10 methanolic hydrochloric acid at concentrations of 0.991 mg/ml and 0.0991 mg/ml. The low intensity and lack of well-defined maxima in the ultraviolet absorption spectrum of enalapril maleate, typical of an unconjugated phenyl moiety, does not make it useful for characterization or quantitation of the compound.

4.5

Mass Spectrum The direct probe electron impact (EI) mass spectrum of enalapril maleate as shown in Figure 7 was obtained on the VG MM7035'mass spectrometer employing 70 eV electron energy and a probe temperature of 16OoC (14). Under the condition of analysis the maleic acid moiety is pumped off before the peptide derivative is vaporized. Molecular ion information for enalapril base is also absent in the spectrum obtained by direct probe EI at 70 eV and 20 eV, respectively. Such information is only possible when a gross sample overload is employed. Under field desorption (FD)/MS however, good molecular ion and pseudomolecular ion [M+H]+ formation (376, 377) is observed (Figure 8 ) . The low resolution EI 70 eV spectrum (Figure 7) shows a base peak at m/e at 91 (benzylic ion), [MH20] atm/e 358 and other major fragment ions. Accurate mass measurements (high resolution, 70 eV, 16OOC) of the diagnostic fragment ions at m/e 358, 313, 285, 257, 254, 234, 208,

c

2

1 al 0 0

c

*u1 a

1

C

\ 220 240 260 280 300 320 340

220 240 260 280 300 320 340

Wavelength ( n m I

F i g u r e 6.

The U l t r a v i o l e t Absorption Spectrum of E n a l a p r i l Maleate i n ( a ) N / 1 0 Methanolic H y d r o c h l o r i c Acid; C o n c e n t r a t i o n : 0.991 mg/ml and (b) N i l 0 Methanolie H y d r o c h l o r i c Acid; C o n c e n t r a t i o n : 0 . ~ 9 9 1 mg/ml

80 c .u)

)r

c 0)

U

60-

x

0

0)

a

50

k

'T' 150

250

350

Mass

Figure 7.

The Low R e s o l u t i o n (EI) Mass Spectrum of Enalapril Maleate

223

Figure 8.

The F i e l d D e s o r p t i o n (FD) Mass Spectrum of E n a l a p r i l Maleate

DOMINIC P. IP AND GERALD S. BRENNER

224

169, 168, 160, 126, 9 1 and 70 facilitated a proposed fragmention pattern as shown in Figure 9. 4.6

Optical Rotation The optical rotation, of enalapril maleate (three asymnetric centers) in methanol is, -42.3' (c=l%).

4.7

Thermal Behavior Enalapril maleate exhibits a single melting endotherm by differential thermal analysis (DTA) under a stream of nitrogen. The peak temperature of the endotherm varies with heating rate, indicating accompanying decompostion during melting of the conpound (- 14OOC at 2OC/min. and 149OC at 2OoC/ min.) A DTA curve for enalapril maleate obtained on a DuPont 1090 equipped with a DTA head is given in Figure 10.

-

- 143-

By capillary melting point determination (USP

Class Ia method) enalapril maleate melts at 144OC (15). 4.8

Solubility The following approximate solubility data (Table IV) have been obtained at ambient temperature. Table IV Solvent Water Methanol Ethanol Isopropanol Acetone Acetonitrile Dimethylformamide Chloroform Diethyl ether Hexane Polyethylene glycol 300

Solubility (mg/ml) 25 200 80

1.4 9.1 3.3 >400 0.6

< 0.1 < 0.1 < 0.1

t 3

\"

I

+5

+*

r

4

Figure 9.

A Proposed Fragmentation Pattern to Explain the Mass Spectrum of Enalapril Maleate

-

O

r

N

r o d - r r ,

(D

0 d-

N

(0

0

" " N

0

N N

0

0

E 0

2 0

5 0

2 0

?

0 (D

0 t

ar

m ar

U

2

4

4

a

k

'rl

m m

4

wG 0

u-i

al

> 3

k

u

fi

m

4 k al

c

b

-4

n

b

0 .-(

ar

3

k

FL(

M 'rl

221

ENALAPRIL MALEATE

The solubility of enalapril maleate increases with pH. A pH versus solubility profile constructed to compare theoretical points to actual data is shown in Figure 11 (16). 4.9

Crystal Properties Enalapril maleate is a white to off-white crystalline corrpound. The compound is polymorphic. TWO nonsolvated polylmrphs have been detected and characterized by high resolution spec oscopic NMR) techniques ( F T I R , Raman, solid state and solution calorimetry ( 1 7 ) . However, these plymrphs, referred to as Form I and Form I1 are very similar in energy, differing only by 0.6 Kcal/ml. The X-ray powder diffraction spectra for Form I and Form I1 of enalapril maleate are shown in Figures 12 and 13, respectively. The spectra were obtained using a Philips Electronics X-ray diffractometer using CuKa irradiation.

%

4.10 Dissociation Constants

Aqueous acidicfiasic potentiometric titration yielded pKa values of 2.97 and 5.35 at 25OC for enalapril (18). 4.11 Partition Behavior At room temperature, enalapril maleate does not partition out of the the aqueous media listed below into mineral oil or n-octanol (19). Solvent Pairs n-Octanol/pH 7 buffer mineral oil/pH 3.5 buffer mineral oil/pH 4.5 buffer mineral oil/pH 5.5 buffer 4.12 Conformational Isomers

Proton NMR and carbon-13 NMR spectra of enalapril maleate are characterized by several paired signals attributed to slow rotation about the proline amide bond, resulting in a slow equilibrium between two rotational conformers (cis and trans). The two rotamers are distinguishable on the NMR

DOMINIC P. IP AND GERALD S. BRENNER

228

1000000

100000

100

10

PH -D-

o Figure 1 1 .

pH

Calculated Pts Experimental Pts

- Solubility Profile of Enalapril Maleate

F i g u r e 12.

Powder X-Ray D i f f r a c t i o n P a t t e r n of E n a l a p r i l Maleate, Form I

In

P

9

R

In (u

m

In

0 O*

f

E

H H

N

0 F a

d

a

N

.d

fd

fd

rl

wc 0

w

E al

PI

d

a:: N

23 1

ENALAPRIL MALEATE

time scale. In addition, HPLC has provided firm evidence for their existence by demonstrating the chromatographic behavior of enalapril maleate at various temperatures (20). Figure 14 shows chromtograms obtained by reverse-phase HPLC at column oven temperatures of 10°C and 80°C. At 10°C, in addition to the early eluting maleic acid peak ( 1.2 minute), the enalapril base peak is resolved into two distinct rotamer peaks. At 8OoC, the rotamer peaks have coalesced to yield a single symmetrical peak. 5.

Methods of Analysis

5.1 Elemental Analysis Analysis of Merck Sharp & Dohme reference l o t , L-154,739-001D22 for carbon, hydrogen and nitrogen was reported as follows: Element C H N

%

Calculated

%

58.53 6.55 5.68

Found 58.53 6.75 5.82

5.2 Colorimetric/Spectrophotometric Due to the lack of strong W absorption maxima, m r e sensitive ways for the analysis of enalapril maleate are needed. One approach has been an autoanalyzer colorimetric assay procedure (21). In this approach enalapril maleate forms a yellow dye complex with bromothymol blue in acidic aqueous, pH 2/CHC13 mixture and is then assayed spectrophotometrically at 415 nm after extraction with chloroform. This method is suitable for content uniformity and dissolution assays of enalapril maleate capsules and tablets. Analysis of enalapril in pharmaceutical dosage forms via flow injection analysis has recently been reported (22). Enalapril is determined by extraction as an ion-pair with bromthyml blue in an unsegmented flow system. The procedure is stability indicating: the degradation products of enalapril and excipients in the dosage forms do not interfere.

DOMINIC P. IP AND GERALD S. BRENNER

232

80"C

10°C

a0

c

r

Time (Minutes 1

Figure 14.

: N

Time (Minutes

HPLC Chromatograms of Enalapril Maleate at 10" and 80°C

233

ENALAPRIL MALEATE

5.3

Chromatographic 5.3.1

Thin-Layer Chromatography Three solvent systems (23) for the thinlayer chromatography of enalapril maleate are given below. The systems separate enalapril base from its salt forming acid (maleic acid) and its hydrolysis product (enalaprilat - see Section 6.1) Solvent System

Rf (Approximate)

A (chloroform/methanol/ 0.6 (enalapril base ) acetic acid 90:10 :1) 0.2 (maleic acid) 0 (enalaprilat) B (n-Butanol/water/ acetic acid 3:l:l)

Solvent System C (n-Butanol/toluene/ water/acetone/acetic acid 1:l:l:l:l)

0.7 (enalapril base and maleic acid) 0.35 (enalaprilat) Rf (Approximate) 0.55 (enalapril

base ) 0.45 (maleic acid and enalaprilat)

These solvent systems use a silica gel adsorbent containing an ultraviolet-fluorescence quench indicator (Analtech GF or Whatman K1F or E Merck Silica Gel G60 type plates). Detection of components is accomplished by visualization of dried plates under short wavelength (254 nm) ultraviolet light. Iodine vapor yields a mre intense spot for the enalapril base but does not visualize the maleic acid. In the systems reported above, the low sensitivity of the method (detection limit 1 to 2 % ) renders it unsuitable for the

DOMINIC P. IP AND GERALD S. BRENNER

234

detection and quantification of low levels of impurities in enalapril maleate bulk substance. 5.3.2

High Performance Liquid Chromatography (HpLC) Several HPLC methods have been developed which are suitable for: (a) quantification of enalapril maleate and process impurities in the bulk substance ( 2 4 ) , (b) analysis of enalapril mleate and degradates in tablets (2 5 ), (c) content uniformity assays of enalapril maleate in tablets (26) and (d) dissolution of enalapril maleate in tablets ( 2 7 ) . Reverse-phase HPLC conditions are employed. Detection is by uv at 210 or 215 nm. Flow rate is normally 1.8 or 2.0 ml/min. A mobile phase of aqueous phosphate buffer and organic modifier (acetonitrile or a mixture of acetonitrile and methanol) and an oven temperature of 7OoC or 80° provide the selectivity and sensitivity required for satisfactory chromatography. The significance of specifying a high column temperature has been discussed in section 4.12. A summary of these HPLC systems is given in Table V.

5.4

Identification Tests Identification of enalapril maleate can be carried out by infrared absorption (section 4.1), HPLC (section 5.3.2) and thin-layer chromatography (section 5.3.1). Supportive evidence for identification can be obtained by differential thermal analysis (section 4 . 7 ) and complexation with bromothyml blue (section 5.2).

5.5

Titration Enalapril maleate can be determined by potentiometric titration with aqueous sodium hydroxide and perchloric acid. With respect to the sodium

&

0

ru

'9

v X

.

cv-l JJ c

u 0

%

X

w

W

In

0

V

m

v

a . .

x

9

0 a3

Y

N

a

z a x

W

X

W

w

0 OD

9 cv X

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obl

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07

0

v

DOMINIC P. IP AND GERALD S. BREWER

236

hydroxide titration, 0.1N sodium hydroxide is the titrant and a combination pH electrode is employed as the electrode system. The perchloric acid titration employs 0.1N perchloric acid in glacial acetic acid as the titrant and an anhydrous electrode system, e.g. Metrohm #EA 441/5 silversilver chloride electrode filled with 0.1N lithium perchlorate in glacial acetic acid (or acetic anhydride), versus a glass electrode is used. 6. Stability and Degradation

6.1 Solid State Stability There are two major potential modes of degradation for enalapril maleate: (a) hydrolysis of the ethyl ester to 11, enalaprilat, the free acid and (b) cyclization to a diketopiperazine derivative 111. The structure of these compounds are shown in Figure 15. Crystalline enalapril maleate is a very stable solid. The compound stored at room temperature (amber glass) for four years shows no evidence of degradation by HPLC analysis. Less than 2% of degradation can be induced by storage at 8OoC for 3 weeks (in screw-cap vials) and < 1% of decomposition at 4OoC and 75% RH (open vial) for 16 weeks. Enalapril maleate tablets are stable when protected from elevated temperatures and high humidity. Degradation can be induced, however, if tablets are stored at 4OoC and 75% RH in open containers, resulting in enalapril maleate loss of about 10% or greater for three months. The major degradation product is 11. The mass balance between loss OF enalapril maleate and formation of I1 and I11 has been demonstrated. 6.2 Solution Stability The stability of enalapril maleate in aqueous buffer solutions has been studied as a function of pH in the range of 2-7 (28). Maximum solution stability occurred around pH 3 . The rate of enalapril loss and the mode of degradation are dependent upon the solution pH. At room temperature, tgO

IN

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V In

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0

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v)

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Ll

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W

238

DOMINIC P. IP AND GERALD S. BRENNER

(time required for 10% loss of starting material) values are 262 days and 114 days €or 0.5 W/ml at pH 2 and 5, respectively. For up to 20% loss at pH 2, I11 was the major degradation product while I1 represented only a minor fraction of the loss. A t pH 3 , I11 was the major degradate but the fraction of I1 was increased. At pH 5 and ahve, I1 accounted for the bulk of enalapril loss with I11 present only in trace quantities. 7. Pharmacokinetics and Metabolism

The pharmacokinetics and metabolism of enalapril maleate follming oral administration have been the subject of a number of investigations and reviews (4, 29-32). Following oral administration of enalapril maleate to man ( 3 3 ) peak serum concentrations of enalapril and enalaprilat occur within 0.5 to 1.5 and 3 to 4 hours respectively. Based on urinary recovery of the total drug (enalapril plus its active metabolite, enalaprilat) absorption is at least 61%. The primary route of excretion of the drug is renal. Approximtely 94% of the dose is recovered in the urine and feces as enalaprilat or enalapril. Enalapril, when administered orally, is rapidly absorbed and bioactivated extensively to enalaprilat. Bioactivation appears to be largely post-absorptive and there is no evidence for metabolism of enalapril beyond bioactivation to enalaprilat in man. The in vitro conversion of enalapril to enalaprilat in human autopsy tissues has been examined (34). Human cadaver liver was the only tissue in which significant conversion was demonstrated. Enalapril absorption is not influenced by the presence of food in the gastrointestinal tract (35). The drug was administered to normal volunteers both in the fasting state and with a normal meal. The area under the serum concentration-time curves for enalaprilat and urinary recoveries for enalaprilat and the total drug did not differ significantly between the fed and fasted conditions. The pharmacokinetics of enalapril maleate was determined in normal volunteers after repeated single daily

ENALAPRIL MALEATE

239

doses for eight days (36). Serum profiles show little accumulation of enalprilat over the course of the study. An average effective half-life for accumulation of approximately 11 hours was calculated from urine data. An average steady state recovery of enalaprilat is achieved by the third or fourth dose. The serum concentration profile of enalaprilat is characterized by a prolonged terminal phase, apparently representing a small fraction of the administered dose that has been bound to angiotensin converting enzyme. The amount bound does not increase with dose indicating a saturable site of binding.

8. Determination in Biological Fluids number of procedures have been developed for the analysis of enalapril and enalaprilat in biological Eluids. Those of primary utility include radiometric, enzyrw inhibition and radioimuno-methods.

A

The physiological disposition and metabolism of enalapril maleate in laboratory animals has been studied with both radiolabeled drug and an enzyme-inhibition assay (37). Following administration of the radioactive drug; urine, feces or plasm samples may be counted by liquid scintillation techniques for total radioactivity. Urinary radioactivity may be separated into enalapril and enalaprilat by thin-layer chromatography * Enalaprilat can also be determined by its in vitro inhibition of angiotensin converting enzyme (37). Plasm, urine and homogenates of feces are placed on xAD.4 columns, washed and then eluted with methanol to yield enalapril and enalaprilat. The enalaprilat component of the mixture is determined by the enzyme assay which uses as a substrate, the tripeptide carbobenzoxycarbonyl-phenylalanyl-histidyl leucine and converting e n z w isolated from porcine plasm. The product, histidyl-leucine is then quantified by fluorescence after reaction with o-phthalaldehydc ( 3 8 ) . For the determination of the total drug (enalapril plus enalaprilat) samples are hydrolyzed at high pH and then reassayed for enalaprilat. Enalapril levels may then be calculated as the difference between the total drug and enalaprilat prior to hydrolysis. An enzyme inhibition assay for enalaprilat has also been developed

DOMINIC P. IP AND GERALD S. BRENNER

240

which uses radiolabeled substrate ( 3 9 ) .thereby eliminating derivatization with o-phthalaldehyde and fluorescence detection of the enzyme roducts. The radiolabeled substrate employed is [flHI hippuryl-kglycylL-glycine. The product, [ 3HI hippuric acid is extracted into a water-immiscible scintillation cocktail and then quantified by counting in a liquid scintillation spectrometer. Enalaprilat has also been determined in serum and urine samples by radioimmunassay procedures. (40,41). In one such procedure ( 4 0 ) antibodies for the radioimunoassay are raised in rabbits using an immunogen prepared by coupling the lysine analog of enalaprilat (lisinopril) to albumin with difluoro-dinitrobenzene. The radiolabeled component is obtained by iodination of the

CH2CH2-c

A

-N

COOH

Lisinopril

- c -c -N

I

I

51

*’

COOH

NH2

amidine derived from the reaction of lisinopril and methyl p-hydroxybenzimidate. The total drug is measured as enalaprilat after sample hydrolysis by rat liver homogenate. Enalapril can then be calculated as the difference between enalaprilat before and after hydrolysis. Acknowledgements The authors wish to thank Mrs. Betty Moyer €or typing the manuscript and Ms. Florence Berg for performing the literature search.

ENALAPRIL MALEATE

24 I

9. References 1. A. A. Patchett, E. Harris, E. W. Tristram, M. J. Wyvratt, M. T. Wu, D. Taub, E. R. Peterson, T. J. Ikeler, J. temroeke, L. G. Payne, D. L. Ondeyka, E. D. Thorsett, W. J. Greenlee, N. S. Lohr, R. D.

Hoffsomer, H. Joshua, W. V. Ruyle, J. W. Rothrock, S. D. Aster, A. L. Maycock, F. M. Robinson, R. Hirschmann, C. S. Sweet, E. H. Ulm, D. M. Gross, T. C. Vassal and C. A. Stone, Nature 288, 280 (1980).

2. A. A. Patchett in "Hypertension and the Angiotensin

System: Therapeutic Approaches", A. E. Doyle and A. G. Bearn Eds., Raven Press, New York, N.Y., 1984.

3. A. A. Patchett, Br. J. Clin. Pharmacol. 18, 201s (1984). 4. P. H. Vlasses, G. E. Larijani, D. P. Conner and R. Ferguson, Clin. Pharrn. 4 , 27 (1985).

K.

-

5. C. S. Sweet, Fed. Proc. 4 2 , 167 (1983).

6. D. M. Gross, C. S. Sweet, E. H. Urn, E. P. Backlund, A. A. Morris, D. Weitz, D. L. Bohn, H. C. Wenger, T. C. Vassil and C. A. Stone, J. Pharmac. Exp. Ther 216, 552 (1981).

7. M. J. Wyvratt, E. W. Tristram, T. J. Ikeler, N. S. Lohr, H. Joshua, J. P. Springer, B. H. Arison and A. A. Patchett, J. Org. Chem. 49, 2816 (1984). 8. A. A. Patchett, E. E. Harris, M. J. Wyvratt and E. W. Tristram (Merck and Co., Inc.) Eur. Pat. Appl. 12,401.

9. E. E. Harris, A. A. Patchett, E. W. Tristram, and J. Wyvratt (Merck and Co., Inc.) US 4,374,829.

M.

10. Merck Sharp and Dohme Research Laboratories, unpublished data.

11. Merck Sharp and Dohme Research Laboratories, unpublished data. 12. D. L. Rabenstein and A. (1982).

A.

Isab, Anal. Chem.,

2, 526

242

DOMINIC P. IP AND GERALD S. BRENNER

13. Merck Sharp and Dohrne Research Laboratories, unpublished data. 14. H. G. Ramjit, Merck Sharp and Dohme Research Laboratories, personal comunication. 15. Nerck Sharp and Dohme Research Laboratories,

unpublished data.

16. R. G. Bergstcorn, Merck Sharp and Dohme Research

Laboratories, personal comunication.

17. D. P. Ip, G. 5. Brenner, J. M. Stevenson? S. Lindenbaum, A. W. Douglas, S. D. Klein and J. McCauley, Int. J. of Pharm., 28, 183 (1986).

A.

18. J. A. McCauley, Merck Sharp and Dohme Research Laboratories, personal comunication. 19. J. P. Draper, Merck Sharp and Dohme Research Laboratories, personal comunication. 20. R. Roman, formerly of Merck Sharp and Dohme Research

Laboratories, unpublished data.

21. J. M. Konieczny, Merck Sharp and Dohme Research

Laboratories, personal comnunication.

22. T. Kato, Anal Chim. Acta,

175,339

(1985).

23. Nerck Sharp and Dohme Research Laboratories,

unpublished data.

24. Merck Sharp and Dohme Research Laboratories,

unpublished data.

25. J. DeMarcO, Merck Sharp and D o h Research

Laboratories, personal comnunication.

26. R. Roman, formerly of Merck Sharp and Dohme Research Laboratories and C. Bell, Merck Sharp and Dohm Research Laboratories, personal comunication. 27. D. J. Mazzo, formerly of Merck Sharp and Dohme

Research Laboratories, unpublished data.

ENALAPRIL MALEATE

243

28. L. L. Ng, Merck Sharp and Dohme Research Laboratories, personal communication. 29. E. H. U l m , Drug. Metab. Rev.,

14 , 99

(1983).

30. P. A. Todd and R. C. Heel, Drugs 31, 198 (1986). 31. R. S. Perry, M e d . A c t u a l . ,

21,

31 (1985).

32. A. F. Lant, R. W. McNabb and F. H. Noormohamed, J. Hypertens., 2, 37 (1984). 33. E. H. U l m , M. Hichens, H. J. Gomez, A. E. T i l l , E. Hand, T. C. V a s s i l , J . Biollaz, H. R. Brunner and J. L. Schnelling, B r . J. Clin. Pharmacol., 14, 357 (1982). 34. I . Larmour, B. Jackson, R. Cubelal and C. I. Johnston, B r . J. Clin. Pharmacol., 19,701 (1985). 35. B. N. Swanson, P. H. Vlasses, R. K. Ferguson, P. A. B e r g q u i s t , A. E. T i l l , J. D. I r v i n and K. Harris, J. Pharm. Sci. 73, 1655 (1984). 36. A. E. T i l l , H. J. Gomez, M. Hichens, J. A. Bolognese, W. R. McNabb, B. A. B r o o k s , F. Noormhamed and A. F. Lant, Biopharm. Drug Dispos., 2, 273 (1984). 37. D. J. TOCCO,F. A. deLuna, A. E. W. Duncan, T. C. Vassil and E. H. U l m , Drug Metab. Dispos. 10, 1 5 (1982). 38. Y. Piquillaud, A. Reinharz and M. Roth, Biochem. Biophys. Acta, 206, 136 (1970). 39. B. N. Swanson, K. L. Stauber, W. C. Alpaugh and S. H. Weinstein, Anal. Biochem. 148, 401 (1985). 40. M. Hirhens, E. L. Hand and W. A. Mulcahy, Ligand Q. 4, 43 (1981). 41. P. J . Worland and B. J a r r o t t , J. Pharm Sci. 75, 512 (1986). The l i t e r a t u r e w a s reviewed t o J u l y , 1986.

HOMATROPINE HYDROBROMIDE

F a i d J. Muktadi, Mahmoud M.A. H u b a n , and AbduR FaMah A . A d i h y

CoUege

F.J. Muktadi M.M.A.

Hanun

A.A. A d i d y

:

:

:

06

Phanmacy, King Saud U n i v m L t y , Riyadh, Saudi A h a b i a .

Uepan;lmeMA: od Phatunacognony. M e d i c i d , AmmaZic a n d P o h a n o u n P&m% R e n u c h Cevtta.

Qep'ahtment 06 Phatunacagnony.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

245

Copylight 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.

FARID J. MUHTADI ETAL.

246

1. Description 1.1 Nomenclature 1.2

Formulae

1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor and Taste

2.

3.

1.6

Dissociation Constant

1.7

pH Range

Physical Properties 2.1

Melting Point

2.2

Solubility

2.3

Crystal Structure

2.4

X-Ray Powder Diffraction

2.5

Spectral Properties 2.5.1

W Spectrum

2.5.2

IR Spectrum

2.5.3

Nuclear Magnetic Resonance Spectra

2.5.4

Mass Spectrum

Preparation of Homatropine Hydrobromide

4. Total Synthesis 4.1 Total Synthesis o f Tropine 4.2 Total Synthesis of Mandelic Acid 4.3 Total Synthesis of Homatropine 5. Therapeutic Uses and Pharmacology 6.

Drug Stability

7. Methods of Analysis

HOMATROPINE HYDROBROMIDE

7.1

Tests of Identification

7.2

Microcrystal Formation

7.3

Titrimetric Methods

7.4

Complexornetry

7.5

Polarographic Methods

7.6

Spectrophotometric Methods

7.7

Chromatographic Methods

Acknowledgement References

247

FARID J. MUHTADI ETAL.

248

1. D e s c r i p t i o n 1.1 Nomenclature 1.1.1 Chemical Names

1.1.2

a- Hydroxybenzene a c e t i c a c i d 8-methyl-8a z a b i c y c l o [ 3 . 2 . 1 ] oct-3-yl e s t e r , hydrobromide. l a H , 5aH-tropane-3a-01 mandelate ( e s t e r ) hydrobromide. Benzeneacetic a c i d , a-hydroxy-, 8-methyl-8a z a b i c y c l o [ 3.2.17 o c t - 3 y l e s t e r hydrobromide , endo-( 2 ) - . Generic Names Homatropine Hydrobromide, DL-Hornatropine Hydrobromide, The hydrobromide of t r o p i n e mandelate , Tropine mandelate Hydrobromide , Mandelyltropine Hydrobromide.

1.1.3

Trade Names Homatrisol, Bufopto, Homatrocel.

1.2

Formulae 1.2.1

Empirical CIGH2103N.

1.2.2

HBr

Structural

7 HO OH II I 0- C-CH

249

HOMATROPINE HYDROBROMIDE

1.2.3

CAS Reg istry No.

[Sl-56-91 C16H2103N. 1.2.4

H Br

Wiswesser Line Notation T56 A ANTJ A -GOVYGR 6 EH

1. 2. 5

(1)

Stereo ch em istry B a m i n a t i o n o f t h e M s p e c t r a of some tro p a n e d e u t e r o h a l i d e s has shown t h a t t h e N-substitue n t i n tro p an es i s predominantly e q u a t o r i a l ( 2 1. X-ray a n a l y s i s o f t r o p i n e hydrobromide has shown t h e presence of c h a i r conformation ( 3 ) . Study of t h e dipole-moment and Kerr-constant measurements o f a number of tro p a n e d e r i v a t i v es h as shown t h a t t h e p i p e r i d i n e r i n g i s i n t h e c h a i r form w i t h t h e N-methyl e q u a t o r i a l ( 4 ) . Another study of t h e dipole-moments and NMR s p e c t r a o f some tr o p a n e d e r i v a t i v e s have confirmed t h a t t h e p i p e r i d i n e r i n g i s i n t h e chair conformation w i t h t h e N-methyl group predominantly e q u a t o r i a l ( 5 ) . I n t r o p i n e , however, t h e predominant conformation i s t h e p i p e r i d i n e r i n g i n a deformed c h a i r form t o gether w i t h a minor amount i n t h e boat form (6

I*

HO Tropine

I n a t r o p i n e , t h e a-3 -s u b s titu e n t i s of greet e r bulk th an t h e hydroxyl, and t h e boat form may w i l l be favored because o f t h e in c re a s e d i n t e r a c t i o n s i n v o l v i n g t h e dimethylene b rid g e i n t h e c h a i r conformation ( 7 ). Hornatropine would be t h e same as a t r o p i n e .

FARID J. MUHTADI ETAL.

250

II

N -CH3

e /

011

0-c-CH

‘‘6HS

A d e t a i l e d review i s a v a i l a b l e f o r t h e boat o r c h a i r conformation i n t r o p i n e s ( 8 ) .

Other PMR s t u d y suggested a p r e f e r e n c e f o r t h e b o a t conformation i n s e v e r a l t r o p a n e d e r i v a t i v e s . T h i s s t u d y showed s t r o n g c r o s s - r i n g i n t r a m o l e c u l a r i n t e r a c t i o n s of t h e t y p e N---C---O and N---H-0were i n d i c a t e d by t h e broadening of t h e p r o t o n s i g n a l due t o t h e c o u p l i n g between 1(5)-H and 2(4)-H p r o t o n s i n t h e boat conformer compared with t h e c h a i r . T h i s broadening a r i s e s a s a consequence of e c l i p s i n g o f t h e s e p r o t o n s i n t h e boat conformer ( 9 ) . 1 A p p l i c a t i o n of H-NMR c o n t a c t s h i f t s u s i n g n i c k e l and c o b a l t d i a c e t y l a c e t o n a t e s , t o t r o p i n e s h a s demonstrated t h a t t h e six-membered r i n g of t r o p i n e s is i n a n e a r l y s e m i p l a n e r form. I t i s s u g g e s t e d t h a t t h e p i p e r i d i n e r i n g of t r o p a n e s is somewhat d i f f e r e n t i n conformation from t h e normal c h a i r form ( 1 0 ) . Carbon-13 magnetic resonance s t u d y has a l s o suggest e d a n o n - c h a i r conformations i n t r o p a n e d e r i.vat i v e s (11).

Me 1.3

Molecular Weight 275.33 356.3

(Homatropine) (Homat r o p i n e HBr)

25 1

HOMATROPINE HYDROBROMIDE

1.4

Elemental Cormosition C , 69.79%; H, 7.69%; N , 5 . 0 9 % ; 0 , 17.43% Il-lomatropine)

.

C , 53.94%; H , 6 . 2 2 % ; B r , 2 2 . 4 4 % ; N , 3.93%; 0 , 13.47%

(Homatropine HBr). 1.5

Appearance, C o l o r , Odor and T a s t e C o l o r l e s s c r y s t a l s o r a w h i t e c r y s t a l l i n e powder, o d o r l e s s , with a b i t t e r t a s t e ( 1 2 ) . Orthorhombic, bipyramidal p r i s m s (from w a t e r ) , odorless (13).

1.6

D i s s o c i a t i o n Constant pKa

1.7

9.9(2O0)

(14)

pH Range -A 2% s o l u t i o n i n water has a pH 5 . 5 t o 7 (12 ) , a 5.67% s o l u t i o n i n water i s i s o - o s m o t i c w i t h serum. pH (1%aqueous s o l u t i o n ) : 5.4 (13).

2.

i'hysical P r o p e r t i.es 2.1

Melting P o i n t (12) 214 - 217O (with decomposition) O t h e r m e l t i n g p o i n t s were a l s o r e p o r t e d : 215 (with decomposition) f 15) 217-218 (with decomposition) (1)

2.2

Solubility One gram d i s s o l v e s i n 6 ml water, 40 m l a l c o h o l , 1 2 m l a l c o h o l a t 60°, 420 m l chlornform. I n s o l u b l e i n e t h e r . The s o l u b i l i t y i n c r e a s e s r a p i d l y as t h e t e E p e r a t u r e rises (12 ) .

2.3

Crystal Structure The c r y s t a l s t r u c t u r e o f b o t h homatropine and homat r o p i n e hydrobromide were r e p o r t e d (16). C r y s t a l s o f homatropine were o b t a i n e d by r e c r y s t a l l i z a t i o n from e t h a n o l , whereas t h e c r y s t a l s of homatropine hydrobromide (C16H21N03. H R r ) were o b t a i n e d by r e c r y s t a l -

FARID J. MUHTADIETAL.

252

l i z a t i o n from water. O s c i l l a t i o n and Weissenberg photographs were used t o determine t h e space group (Table 1 ) . In b o t h homatropine and homatropine hydrobromide t h e n i t r o g e n atom may be o p t i c a l l y a c t i v e , s i n c e t h e s p a c e groups a r e centrosymmetric, t h e u n i t c e l l s must be assumed t o c o n t a i n e q u a l numbers of L - a d D-molecules ( 1 6 ) . T a b l e 1.

The C r y s t a l S t r u c t u r e o f Homatrop i n e and of Homatropine H B r .

a(A")

b(A")

c(A")

Homatropine

14. 5

15.1

6.97

1.21

Homatropine HBr

10.3

16.4

19.25

1.48

Compound

2.4

Density (g.cmR3) observed c a l c u l a t e d

Z

Space group

1.22

4

P211/c

1.46

8

Pcab

X-ray Powder D i f f r a c t i o n

The X-ray d i f f r a c t i o n p a t t e r n o f homatropine hydrobromide was determined with a P h i l i p s X-ray d i f f r a c t i o n s p e c t r o g o n i o m e t e r equipped w i t h PW 1730 generator. X-ray r a d i a t i o n was provided by a copper t a r g e t (Cuanode 2000 W ) . High i n t e n s i t y X-ray t u b e o p e r a t e d a t 40 KV and 20 mA. The monochromator was a s i n g l e curved c r y s t a l ( y = l .5918A0) The u n i t was equipped with P h i l i p s PM 821.0 p r i n t i n g recorded d i g i t a l p r i n t e r . Divergance s l i t and t h e r e c e i v i n g s l i t were 1". The scanning speed o f t h e goniometer used was 0.02 2 e / s e c . , r e c o r d e r f u l l s c a l e was 10,000 c o u n t s a t r e c o r d e r speed o f 4 mm/28. The lower level and t h e upper l e v e l of t h e s i g n a l c o n t r o l waz 35 E 75 r e s pectively. The u n i t was a l i g n e d and examined, u s i n g a S o l i c o n s t a n d a r d , t o a t t a i n maximum o p e r a t i o n c o n d i t i o n s . The X-ray p a t t e r n of homatropine hydrobromide i s p r e s e n t e d i n F i g . 1. I n t e r p l a n n a r d i s t a n c e and r e l a t i v e i n t e n s i t y are t a b u l a t e d i n t a b l e 2 .

253

HOMATROPINE HYDROBROMIDE

Table 2 : X-Ray Powder D i f f r a c t i o n P a t t e r n of Homatropine Hydrobromide

-

~

~~

6.98 6.41 6.00 5.15 5.04 4.84 4.64 4.48 4.35 4.28 4.19 3.96 3.57 3.50 3.43 3.30 3.23 3.17 3.12 3.07 3.00 2.97 ~

17.8% 17.3% 22.1% 15.7% 21.1% 73.5% 12.6% 14.4% 39.0% 85.9% 100.0% 36.3% 48.7% 46.1%

11.2%

38.5% 21.3% 20.2% 22.7% 20.7% 20.8% 17.8%

2.87 7.83 2.72 2.64 2.61 2.58 2.52 2.50 2.47 2.35 2.32 2.29 2.26 2.15 2.07 2.03 1.98 1.93 1.87 1.80 1.77

11,7% 24.1% 19.5% 10.7% 14.2% 20.0% 22.9% 12.1% 22.0% 13.5% 25.9% 13.7% 13.1% 11.9% 11.3% 16.4% 10.7% 13.0% 11.2% 11.0% 13.5%

~~

d = i n t e r p l a n a r distance, I/Io = relat i v e i n t e n s i t y (based on t h e h i g h e s t i n t e n s i t y of 100).

J 1

lY 1

X-RAY DIFFRACTION PATTEW OF HCMATROPINE HYDROIIRO\IIDL.

FARID J. MUHTADI ETAL.

254

2.5

Spectral Properties 2.5.1

U l t r a v i o l e t Spectrum The UV spectrum of homatropine hydrobromide i n methanol ( F i g . 2 ) was scanned from 200 t o 350 nm u s i n g DMS 90 Varian S p e c t r o p h o t o meter. I t e x h i b i t s t h e f o l l o w i n g UV d a t a (Table 3 ) . Table 3 . UV C h a r a c t e r i s t i c s o f Homatropine

Xmax. a t nm 252 258 264.5 273

(A 1%,lcm)

E

224.28 26 7 228.5 78.32

6.3 7.5 6.42 2.2

Other r e p o r t e d UV s p e c t r a l d a t a € o r homatropine i n 0.1 N s u l f u r i c acid ( 1 7 ) . Xmax. a t 252 mu ( E 1%, 1 cm 4 . 8 ) , 258 mu ( E 1%, 1 cm 6.1) and 264 mu ( E 1%, 1 cm 5 ) .

Amax. i n 0 . 1 N h y d r o c h l o r i c a c i d a t 254 nm ( E 1%, 1 cm = 3 ) ; a t 259 nm ( E 1% , 1 cm = 5 . 2 ) and 266 nm. ( 1 8 ) . 2.5.2

I n f r a r e d Spectrum The I R spectrum of homatropine hydrobromide as KBr-disc was r e c o r d e d on a Perkin E l m e r 580 B I n f r a r e d S p e c t r o m e t e r t o which i n f r a r e d d a t a s t a t i o n i s attached (Fig. 3 ) . The s t r u c t u r a l assignment have been c o r r e l a t e d w i t h t h e f o l l o w i n g f r e q u e n c i e s (Table 4 ) .

Table 4. I R C h a r a c t e r i s t i c of Homatropine

Ass i gnment

Frequency cm-’ Base 3350 2940,3050

1735

HBr 3270 2965 2 80 0 - 2.585 1755

OH ( s t r e t c h ) CH ( s t r e t c h )

+

NH

R

-0-C- ( e s t e r )

255

HOMATROPINE HYDROBROMIDE

200

250

300

350

Fig. 2 : ‘THE W SPECTRUM OF HOMATROPINE IIYDROl3KOMIDE IN PE’MANOL

1

I

3500

3000

I

I

I

I

2500

2000

I

I

I

19

1

I

1

1

1

1600

1400

1200

1000

1800

Fig. 3 : ‘IHE IR SPECTRLM OF HOMATRDPINE HYDROBRDMIDE AS KBr DISC.

1

800

1

600

257

HOMATROPINE HY DROBROMIDE

Frequency cm -1 Ease

Assignment

HBr

1600 145 0,1500 1100,1065 755,700

1600 1475 1170,1070 735,700

C=C (aromatic) C=C (aromatic) C-0-C (ether) 5 H (monosubstituted aromatic)

Other characteristic bands are 2750, 2710, 1450, 1420, 1395, 1378, 1328, 1288, 1265, 1195, 1105, 1025, 960, 885, 820 and 615 cm-l. Other IR data for homatropine and the hydrobromide salt have been reported (1,14,17). 2.5.3

Nuclear Magnetic Resonance 2.5.3.1

'H-NMR Spectra The 'H-NMR spectra o f homatropine hydrobromide in DMSO-D6 and in D20 were recorded on a Varian T-60A, 60 MHz NMR spectrometer iising TMS (tet-

8

A H 3

ramethylsilane) as an internal reference. These are shown in F i g . 4 and 5 respectively. The following structural assignment have been made (Table 5).

76& > H $0

'

" OH

Oi4 12

13

0- C-CH 9

10

1 '6 Table 5. The H-NMR Characterjstics of Homatropine Group 5 aromatic protons (12,13,14,15,16 H) 10 H (OH) 10 H

Chemical Shift (pmm) DMSO-d6 D2° 7.33 (s)

7,4 (s)

6.03 (d) 5.13 (d)

5.33 ( s )

J L

. .

1

In

. . . .

I

In

. I. . . ,

.

,

,

1 , l

6 0

so

.

,

.

. . . PPWIII

, l

, .

an

, .

I .

.

.

I

, . 1 0

, .

.

, .

I

. .

.

1 *

.

, I

Y O

Fig. 4 : THE 'H-NMR SPECTRUM HOMATROPINE HYDROBROMIDE IN D I W - L ) ~

. .

L 10

Fig. 5 : THE 'H-NMR SPECTRUM OF HOMATROPINE HYDROBROMIDE IN D20.

FARID J. MUWTADIETAL.

260

Group

Chemical S h i f t (pmm) DMSO- d6 D2°

3H

4.70 ( t )

5.03 ( t )

1 H and 5 H

3.71 ( b s )

3.66 ( b s )

8-N-Me

2.63 (s)

2.67 (s)

2,4,6,7 H

2.4-1.27

(m)

2.4-1.8

(m)

s = s i n g l e t , d = d o u b l e t , t = t r i p l e t , bs=broad s i n g l e t 1 Other H-NMR d a t a f o r homatropine hydrobromide was r e p o r t e d ( 1 ) . Ohashi e t a 1 (10 ) have r e p o r t e d 'H n u c l e a r magnetic resonance c o n t a c t s h i f t s o f some t r o p a n e s i n c l u d i n g homatropine i n CDC13 a t 220 MHz. The c o n t a c t s h i f t s used were n i c k e l and c o b a l t d i a cetylacetonates. 2.5.3.2

13C-NMR

The I3C -NMR complete l y decoupled and o f f resonance s p e c t r a of homat r o p i n e hydrobromide a r e p r e s e n t e d i n Fig.6 and Fig. 7 r e s p e c t i v e l y . Both were r e c o r d e d over 5000 Hz range i n a mixture o f DMSO-D6 and CDC13 (conc. 100 mg/ml), on a J o e l FX-100-90 MHz s p e c t r o m e t e r . A samp l e t u b e ' l 0 mm and TMS a s i n t e r n a l s t a n d a r d a t 20' were used. The carbon chemical s h i f t s are a s s i g n e d on t h e b a s i s o f t h e chemical s h i f t t h e o r y and t h e o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 6 ) .

8

7

12 II

I

0- C-CH 9 10

13

Fig. 6 : 'IHE l 3 C - I W W L E T E L Y DECOUPLED SPECTRUM OF HOMATROPINE HYDROBNNIDE.

Fig.

:

THE

'3C-W

OFF-RESONANCE S P E m M OF Wl'p.OPINE HWWBROFlIDE.

263

HOMATROPINE HYDROBROMIDE

Table 6 . Carbon No. ‘1

Carbon Chemical S h i f t s of Homatropine

Chemical S h i f t [PPml 59.18 (d) 32.11 ( t )

c2

70.74

c3

(d)

32.11 ( t )

‘4

59.18 (d)

c5

21.19 ( t )

‘6

21.19 ( t )

c7

40.21 ( 9 )

‘8

Carbon No. ‘9

5 0 cll 5 2

‘13 14 5 5 ‘16

Chemical S h i f t [PPml 169.38 (s) 62.76 (d) 137.38 (s) 126.34 (d) 124.76 (d) 125.99 (d) 124.76 (d) 126.34 (d)

s = singlet, d = doublet, t = t r i p l e t , q = quartet.

2.5.4

Mass Spectrum The mass s p e c t r u m o f homatropine o b t a i n e d by e l e c t r o n impact i o n i z a t i o n a t 70 eV, was r e c o r d e d on a Finigan-Mat 5100 Mass S p e c t r o meter. The s p e c t r u m ( F i g . 8) shows a molec u l a r i o n peak M+ a t m/e 275. The b a s e peak i s a t m/e 124. The most prominent f r a g m e n t s , t h e i r r e l a t i v e i n t e n s i t i e s and some proposed i o n f r a g m e n t s are shown i n t a b l e 7.

Table 7 .

m/e

Mass Fragements o f Homatropine Relative Intensity %

275

4.4

234

1.2

193

1.2

165

1.3

142

6.4

140

4.6

125

10.5

Ions -

M+

-

+

[IT)]’

I-

Fig. 8 : 'THE MASS SF'ECIRLN OF ~ l R O P I t E

HOMATROPINE HYDROBROMIDE

m/e -

Relative Intensity %

265

Ions 125-H

124

100

122

1.6

107

7.5

106

3.8

105

6.1

96

14

95

9.3

96 -H

94

21.5

95-H

83

20.3

82

29.2

79

14.3

77

19.2

68

5.9

67

19.1

68 -H

55

6.7

[CH2 =N=CH-CH2]

O t h e r mass s p e c t r a l r e p o r t e d (19,20).

-

+

data

of homatropine have been

+

FARID J. MUHTADI ETAL.

266

3.

P r e p a r a t i o n of Homatropine Hydrobromide Atropine i s hydrolyzed w i t h barium hydroxide s o l u t i o n t o g i v e t r o p i n e and t r o p i c a c i d . The t r o p i n e i s h e a t e d w i t h mandelic a c i d i n t h e p r e s e n c e o f hydrogen c h l o r i d e . Ammonia i s added, and t h e l i b e r a t e d homatropine i s e x t r a c t e d with chloroform. The c h l o roform s o l u t i o n i s e v a p o r a t e d t o d r y n e s s . The r e s i d u e i s t r e a t e d with hydrobromic a c i d and t h e homatropine hydrobromide o b t a i n e d is p u r i f i e d by r e c r y s t a l l i z a t i o n (21, 2 2 ) . The p r e p a r a t i o n i s o u t l i n e d i n Scheme I . Scheme I .

H

f) / CH20H 0-C-CH \C6H Hydrolysis

eoi 6 + HOOC -CH-CHZ OH

HOOC\

’

Ho’cH@

1

0

0-E-cii’

OH

‘6HS

Homatropine

HOMATROPINE HYDROBROMIDE

267

An alternative method of hydrolyzing atropine was achieved

microbically by the action of Arthrobacter a t r o p i n i (23).

4. Total Synthesis Since Homatropine is the tropine ester of mandelic acid, schemes for the total synthesis of tropine and of mandelic acid were reported. 4.1

Total Synthesis of Tropine Four schemes for the total synthesis of tropine are known. Scheme I1 was also modified to give a much better yield. Scheme 11: Willstatter's total synthesis of tropine (24) * Suberone (cycloheptanone) [l] is reduced to suberol which is treated with hydrogen iodide to give suberyl iodide [2]. This is treated with potassium hydroxide in ethanol to give cycloheptene [ 3 ] . Cycloheptene is brominated to give 1,Z-dibromocycloheptane [4] which is treated with dimethylamine to yield dimethylaminocyclohept-2-ene [5]. The latter is converted to cyclohepta-1,3-diene [6] by exhaustive methylation. [6] is brnminated at 1,4-positions to give 1,4-dibromocyclohept-2-ene [7]. Elimination of two moles of the hydrogen bromide of [7] is effected by quinaline to give cycloheptatriene [ 8 ] . Substance [8] is treated with hydrogen bromide to give bromocyclohepta-3,5-diene [ 9 ] which is reacted with dimethylamine to give dimethyl aminocyclohepta2,4-diene [ l o ] . The latter is treated with sodium in ethanol followed by bromination to give 1,2-dibromo5-dimethylamino-cycloheptane [ll]. This is warmed in ether when intramolecular alkylation occurs to give 2-bromotropane methobromide [12]. Hydrogen bromide is eliminated from [12] by the action of alkali to yield tropidine methobromide [ 1 3 ] . This is transformed to tropidine methochloride [ 1 4 ] by the action of potassium iodide followed by the action of silver chloride. Sub-rtance [14] is pyrolized to give tropidine [15]. Hydrogen bromide is added to an acetic acid solution of tropidine [15] to yield 3-bromotropane [16! which is hydrolysed with 10% sulfuric acid at 2@0-21@0 to give pseudotropine [ 171. JI-tropine [ 171 is oxidized with chromium trioxide to give tropinone [18]. This ketone is reduced with zinc and hydriodic acid to tropine [ 191.

FARID J. MUHTADI ETAL.

268

Scheme 11 : W i l l s t a t t e r ' s t o t a l s y n t h e s i s o f a t r o p i n e

QBr

exhaust

L methyln

[4

Br

q u i n o l i ne 1500~

*

warm i n Et20 [I21

f

Br

Br

[111

I

Br

269

HOMATROPINE HY DROBROMIDE

Scheme T I l : Robinson's t o t t i l s y n t h e s i s of atropine

CH-OH CH3NH2 121

cond.

G

N -CH 3

CH3

\

/"="

CH3

CH-OH

I31

[GI

+

[41

I51

FARID J. MUHTADI ETAL.

270

Scheme 1 1 1 : Robinson's s y n t h e s i s (25) Succindialdehyde [ 11 i s condensed with methylamine [ 2 ] t o g i v e t h e condensate b i s c a r b i n olamine [3]. This i n t u r n condensed w i t h acet o n e [ 4 ] t o give t r o p i n o n e [ 5 ] ( T h i s mixture i s allowed t o s t a n d i n water a t o r d i n a r y t e m p e r a t u r e f o r h a l f an h o u r ) . Tropinone [ 5 ] i s reduced with z i n c and hydriodic acid t o tropine [6]. The y i e l d can be improved by s u b s t i t u t i o n of t h e more r e a c t i v e acetone d i c a r b o x y l a t e o r i t s e s t e r f o r acetone. Succindialdehyde [l] i s condensed w i t h m t h y l a mine [21 t o g i v e b i s c a r b i n o l m i n e [31. I31 i s condensed w i t h calcium acetonedicarboxyl a t e [4] t o af1'31-d t h e condensate [51. This i s warmed with hydrochloric a c i d t o g i v e t r o pinone [61. Tropinone [61 i s reduced w i t h z i n c and hydriodic a c i d t o t r o p i n e [71. Scheme IV-:

Willstatter I s second s y n t h e s i s ( 2 6 )

S u c c i n y l d i a c e t i c e s t e r [ 11 i s condensed with methylamine [ 2 ] t o g i v e diethyl-N-methylpyrr o l e d i a c e t a t e [ 31. This i s reduced (H2+Pt) t o a f f o r d diethyl-N-methylpyrrolidinediacet a t e [41. The c i s form of [41 is c y c l i z e d i n t h e presence of Na and p-cymene t o g i v e e t h y l tropinone-2-carboxylate [ 51. Hydrolysis of [5] with 10% s u l f u r i c a c i d g i v e s e t h y l t r o p i none-2-carboxylic a c i d [ 61. The l a t t e r i s heated t o y i e l d tropinone [TI which i s reduced w i t h z i n c and hydriodic a c i d t o t r o p i n e [a]. Scheme V: Tropinone can a l s o be s y n t h e s i z e d ( 2 7 ) u s i n g methylamine hydrochloride a c e t o n d i c a r b o x y l i c a c i d and g e n e r a t i n g succindialdehyde i n s i t u by t h e a c t i o n of a c i d on 2,5-dimethoxy t e t r a hydrofuran as follows :

H+

CH3NH2HC.l CO( CH2COOH)2

"0

CHO

HOMATROPINE HYDROBROMIDE

- ';-...

Scheme 111: Robinson's

:li

s y n t h e s i s ( y i e l d improvement) CH-OH

f NHeCH3 121

I11

cond

CH-OIf

[31

Scheme I&

Cli-

CH2-COOC

CH2COOCa

\ c=o

+ /

\4

I

H3CT

1

c=o I

CH -C-CH2-COOC 2

(51

1

H

COOCa

H 2 5

[21

H 2 5 H

.(--------------

CH=

C-

CH2-COOC H

\

' A

CH -C1 2 I III CR -C-

I

2 1 H

2 5

CH

1

I

- c-

i 2 i

II CN

31

CH2-

cI H

-

I

I3]

CH2-COOC

Cli3 CH2-COOC

CH-COOH

I c=o I

&I2

(61

2 5

2 5

[41

H

H

CH,-COOC

I

HI CH2-COOC

1

C-

I

H CH2- C-

[4

CH=

111

I

CH2COOCa

W i l l s t a t t e r ' s second s y n t h e s i s

+ H2NCH3 CH=

27 1

*

II 2 5

H 2 5

FARID J. MUHTADI ETAL.

212

CH2 - C H

I

Me-N

CH2

- CH

I

\

/

-CH2

I

I

C=O

- CH2

Zn HI

L

[71 4.2

CH2

- CH - C H

2

[81

T o t a l S y n t h e s i s of Mandelic Acid S e v e r a l schemes f o r t h e t o t a l s y n t h e s i s a r e known (28-30). Scheme I Benzaldehyde [ l ] i s t r e a t e d w i t h a m i x t u r e of a s a t u r a t e d s o l u t i o n of sodium hydrogen s u l f i t e and aqueous sodium cyanide t o g i v e m a n d e l o n i t r i l e [ 2 ] . [ 2 ] i s hydrolyzed w i t h c o l d c o n c e n t r a t e d hydroc h l o r i c a c i d t o y i e l d mandelic a c i d [3] (28). Scheme I1 Acetophenone [ l ] i s c o n v e r t e d i n t o d i c h l o r o a c e t o phenone [ 2 ] by t h e a c t i o n of c h l o r i n e i n a c e t i c a c i d . [ 2 ] i s t r e a t e d with sodium hydroxide t o a f f o r d t h e sodium s a l t o f mandelic a c i d , which i s t r e a t e d w i t h h y d r o c h l o r i c a c i d t o g i v e mandelic a c i d [ 3 ] ( 2 9 ) . Scheme I11 Amygdalin [ 13 i s t r e a t e d w i t h fuming h y d r o c h l o r i c a c i d t o produce m a n d e l o n i t r i l e [ 2 ] which i s t h e n hydrolyzed w i t h h y d r o c h l o r i c a c i d t o g i v e mandelic a c i d [ 3 ] (30).

4.3

T o t a l S y n t h e s i s of Homatropine Tropine i s t h e n e s t e r i f i e d w i t h (+) -mandelic a c i d i n t h e presence of hydrogen c h l o r i d e ( F i s c h e r S p e i e r e s t e r f i c a t i o n ) t o g i v e ( 2 ) -homatropine.

HOMATROPINE HYDROBROMIDE

213

Total Synthesis of Mandelic Acid Scheme I

NaCN NaHSO

Scheme I1

@Lo

c12 CH3COOH

Scheme I11

HO

HO

b

/

/

CHC 1 2

[21 i) NaOH ii) HCI

FARID J. MUHTADI ETAL.

214

5.

Therapel.itic Uses and Pharmacological E f f e c t s Homatropine i s used a s a m y d r i a t i c and c y c l o p l e g i c d r u g i n ophthalmology. I t i s p r e f e r r e d t o a t r o p i n e f o r d i a g n o s t i c purposes because i t s a c t i o n i s more r a p i d , less prolonged and i s z o r e r e a d i l y c o n t r o l l e d by p h y s o s t i g mine ( 1 2 ) . The e f f e c t of homatropine i s e x e r t e d i n 15 t o 30 minutes and p a s s e s o f f i n 1 2 t o 24 h o u r s . I t j s sornetimes used with c o c a i n e which enhances i t s m y d r i a t i c action (12). Homatropine has a l s o l e s s tendency t h a n a t r o p i n e t o i n c r e a s e t h e i n t r a - o c u l a r p r e s s u r e (18), b u t it produces a l e s s s a t i s f a c t o r y m y d r i a s i s i n c h i l d r e n (18). I t i s seldom used i n t e r n a l l y (12). Homatropine i s an a n t i c h o l i n e r g i c d r u g w i t h e f f e c t s s i m i l a r t o h u t much weaker (about o n e - t e n t h ) t h a n t h o s e o f atropine (12,31l, generally t h e belladonna a l k a l o i d s a r e absorbed r a p i d l y from t h e g a s t r o i n t e s t i n a l t r a c t . They a l s o e n t e r t h e c i r c u l a t i o n when a p p l i e d l o c a l l y t o t h e mucosal s u r f a c e s o f t h e body (31). Some of t h e s e compounds ( a t r o p i n e and homatropine) when a p p l i e d l o c a l l y t o t h e eye can cause mydriasis and c y c l o p l e g i a (31). Homatropine i s used a s a 2% s o l u t i o n i n c a s t o r o i l , b u t more o f t e n i n form of 2 t o 5% hornatropine hydrobromide ophthalmic s o l u t i o n s (12,31). I t has been r e p o r t e d (32) t h a t hornatropine i n doses o f 0 . 5 t o 4.0 mg, induces b r a d y c a r d i a i n man, when it i s a d m i n i s t e r e d subcutaneously

6.

Drug S t a b i l i t y Homatropine hydrobromide i n t h e d r y s t a t e , i n a i r t i g h t c o n t a i n e r a t room temperature and p r o t e c t e d from l i g h t , showed no decomposition a f t e r f i v e y e a r s when examined according t o t h e B.P. ( 1 5 ) . In aqueous s o l u t i o n , homatropine h y d r o l y s e s t o t r o p i n e andmandelic a c i d . Hydrolysis i s c a t a l y z e d by hydrogen i o n s and hydroxide i o n s (18). A t 25", t h e r a t e of hydrol y s i s i s a minimum a t pH 3 . 7 . A s o l u t i o n c o n t a i n i n g homatropine hydrobromide 1%and b o r i c a c i d 1 . 5 5 % , l o s t 2% of i t s homatropine c o n t e n t a f t e r a u t o c l a v i n g f o r 20 minutes a t 120" (12). The pH of t h i s s o l u t i o n f e l l from 4 . 5 t o 3.8 (33,34).

HOMATROPINE HYDROBROMIDE

7.

215

Methods o f A n a l y s i s 7.1

I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e mentioned i n t h e B.P. ( 1 5 ) .

-

Dissolve 10 mg o f homatropine hydrobromide i n 1 m l of water, add a s l i g h t e x c e s s o f 10M ammonia and shake w i t h 5 m l of chloroform. Evaporate t h e c h l o r o form l a y e r t o d r y n e s s on a water b a t h and add 1 . 5 m l of a 2 p e r c e n t w/v s o l u t i o n of mercury (11) c h l o r i d e i n e t h a n o l ( 6 0 % ) ; a yellow c o l o r develops which t u r n t o r e d on warming.

- A 2 p e r c e n t w/v s o l u t i o n y i e l d s t h e r e a c t i o n c h a r a c t e r i s t i c o f a l k a l o i d s and t h e r e a c t i o n s c h a r a c t e r i s t i c o f bromides.

-

The f o l l o w i n g t e s t i s mentioned i n t h e U.S.P. ( 3 5 ) . The i n f r a r e d a b s o r p t i o n spectrum of a potassium bromide d i s p e r s i o n of i t , e x h i b i t s maxima only a t t h e same wavelengths a s t h a t of a s i m i l a r p r e p a r a t i o n of USP Homatropine Hydrobromide Reference Standard

.

-

A s i m p l e , r a p i d and s e n s i t i v e t e s t €or t h e d e t e c t i o n

of homatropine and o t h e r a l k a l o i d s i n p h y s i o l o g i c a l f l u i d s such a s s a l i v a and u r i n e i s r e p o r t e d (36) To t h e s o l u t i o n o f a l k a l o i d s , a methanolic bromophenol b l u e i s added t o g i v e b l u e s t a b l e c o l o r .

- Another c o l o r t e s t was r e p o r t e d a s f o l l o w s ( 3 7 ) :

Homatropine i s e v a p o r a t e d t o d r y n e s s w i t h a mixture of fuming n i t r i c a c i d - a c e t i c anhydride ( 4 : 3 ) and 5% met hano 1i c t e t rame t h y 1ammonium hydroxide so l u t ion i s added t o a s o l u t i o n of t h e r e s i d u e i n a c e t o n e , t h e c o l o r d e v e l o p s and remains c o n s t a n t f o r 6 t o 8 minutes. I t i s p o s s i b l e t o determine t h e a l k a l o i d q u a n t i t a t i v e l y by measuring t h e c o l o r a t 570 ni.1.

- Other i d e n t i f i c a t i o n t e s t s have been a l s o mentioned (38) * 7.2

M i c r o c r v s t a l Tests Homatropine hydrobromide was d i s s o l v e d i n water (10 mg i n 10 m l ) , 1 t o 2 d r o p s of t h i s s o l u t i o n was t r e a t e d w i t h t h e r e a g e n t on a m i c r o s c o p i c a l g l a s s s l i d e . After s p e c i f i c time, t h e c r y s t a l s were m i c r o s c o p i c a l l y examined (39)

.

FARID J. MUHTADI ETAL.

276

7.2.1

Wagner's r e a g e n t was added t o t h e t e s t s o l u t i o n , minute irregular brown b l a d e s were formed a f t e r 2-3 minutes ( F i g . 9 ) .

7.2.2

P i c r i c a c i d was added t o t h e t e s t s o l u t i o n , r a d i a t i n g r o d s and i r r e g u l a r b l a d e s were formed a f t e r 10 minutes ( F i g . 1 0 ) .

7.2.3

D r a g e n d r o f f ' s r e a g e n t was added t o t h e t e s t s o l u t i o n , minute i r r e g u l a r orange c r y s t a l s were formed a f t e r 1 - 3 minutes ( F i g . 1 1 ) . Other m i c r o c r y s t a l t e s t s have a l s o been r e p o r t e d (17,40,41).

7.3

T i t r i m e t r i r Determinations 7.3.1

Aqueous The f o l l o w i n g a s s a y i s mentioned i n U.S.P

(35)

- D i s s o l v e about 400 of Homatropine Hydrohro-

mide, a c c u r a t e l y weighed, i n water t o make 50 m l and mix. T r a n s f e r 10 m l of t h i s s o l u t i o n t o a b e a k e r , add 5 m l of sodium hydroxide TS, and h e a t t h e s o l u t i o n j u s t t o b o i l i n g . Add 10 m l of d i l u t e n i t r i c a c i d ( 1 i n 1 5 ) , add water t o make 50 m l , and c o o l i n an i c e b a t h . Concomitantly add 5 m l o f d i l u t e n i t r i c a c i d (1 i n 15) t o a second 10 m l p o r t i o n o f t h e s o l u t i o n of Homat r o p i n e Hydrobromide, add w a t e r t o make 50 m l , and c o o l i n an i c e b a t h . Add 1 d r o p o f n i t r o p h e n a n t h r o l i n e TS t o each s o l u t i o n and while keeping t h e s o l u t i o n s c o l d , t i t r a t e w i t h O.OSN c e r i c ammonium n i t r a t e u n t i l t h e pink c o l o r i s discharged. Each m l o f t h e d i f f e r e n c e i n volumes of 0.05N c e r i c ammonium n i t r a t e r e q u i r e d i s e q u i v a l e n t t o 8.907 mg of CI6HZ1NO3.HBr.

-

Homatropine was determined by t i t r a t i o n w i t h aqueous 0 . 0 1 M - t e t r a p h e n y l b o r a t e , w i t h t e t r a bromophenolphthalein e t h y l e s t e r as an i n d i c a t o r i n 1,2 - d i c h l o r o e t h a n e (42).

-

A p o t e n t r i o m e t r i c t i t r a t i o n of homatropine hydrobromide i n g a l e n i c a l p r e p a r a t i o n s w i t h sodium t e t r a p h e n y l b o r o n was d e s c r i b e d (43)

.

HOMATROPINE HY DROBROMIDE

277

Fig, 9. Hornatropine HBr with Wagner's reagent

~~

Fig. 10. Hornatropine HBr with picric acid reagent

a-

Fig. 11 Hornatropine HBr with Dragendorff's reagent

FARID J. MUHTADI ETAL.

218

- Homatropine hydrobromide was t i t r a t e d i n

dimethyl sulphoxide medium w i t h 0.1 M AgN03. End-point was determined by conductometric, p o t e n t i o m e t r i c and p o l a r i m e t r i c t e c h n i q u e s , and t h e r e s u l t s were s a t i s f a c t o r y by a l l t h r e e t e c h n i q u e s . Amounts of 0 . 1 mmol (44) could n o t be determined.

7.3.2

Non-aqueous

- The f o l l o w i n g a s s a y i s mentioned i n t h e B.P.

(15) * Dissolve 0 . 3 gm i n 20 m l of anhydrous g l a c i a l a c e t i c a c i d , add 10 m l o f mercury (IT) a c e t a t e s o l u t i o n and c a r r y o u t Method I f o r non-aqueous t i t r a t i o n , Appendix V I I I A, d e t e r m i n i n g t h e end-point p o t e n t i o m e t r i c a l l y . Each m l of 0 . 1 M p e r c h l o r i c a c i d VS i s e q u i v a l e n t t o 0.03563 gm of C16H21N03.HBr.

-

7.4 -

An a l t e r n a t i v e method i n v o l v e s d i r e c t t i t r a -

t i o n of homatropine hydrobromide w i t h p e r c h l o r i c a c i d i n a c e t i c anhydride medium u s i n g naphthalbenzein o r Sudan r e d B a s i n d i c a t o r (45) Other non-aqueous t i t r a t i o n has been a l s o r e p o r t e d (46).

-

Complexometry P r e c i p i t a t i o n of homatropine a s a t h a l i u m complex u s i n g one p a r t 0 . 1 N t h a l l i u m s u l f a t e and 3 p a r t s 0 . 1 N i o d i n e i n 0.15 M potassium i o d i d e s o l u t i o n . The p r e c i p i t a t i o n i n a n e u t r a l medium i s most s e n s i t i v e . The use of t h i s p r e c i p i t a t i o n r e a g e n t f o r an i n d i r e c t d e t e r m i n a t i o n of s m a l l amounts of homatrop i n e i s based on t h e d e t e r m i n a t i o n of t h a l l i u m i n the p r e c i p i t a t e (47).

- Another method u t i l i z i n g merceuric-potassium i o d i d e

r e a g e n t i n 0 . 3 sodium hydroxide s o l u t i o n was r e p o r t e d ( 4 8 ) . The d e t e r m i n a t i o n i s based on t h e e x t r a c t i o n of t h e p r e c i p i t a t e formed with chloroform. The combined e x t r a c t s a r e evaporated and water and 0.1Ms i l v e r n i t r a t e a r e added. Methylthymolblue i s used a s i n d i c a t o r and s u f f i c i e n t 20% h e x a m e t h y l e n e t e t r a mine s o l u t i o n i s added t o g i v e a b l u e c o l o r . The mercury i n t h e s o l u t i o n i s t h e n t i t r a t e d w i t h 0.05M EDTA t o a yellow end p o i n t .

HOMATROPINE HYDROBROMIDE

7.5 -

279

Polarographic The o s c i l l o p o l a r o g r a p h i c behaviour of homatropine and o t h e r a l k a l o i d s was r e p o r t e d ( 4 9 ) . A dropping mercury e l e c t r o d e s e r v e d a s a p o l a r i z a b l e e l e c t r o d e , and a g r a p h i t e one a s a r e f e r e n c e . The d e p o l a r i z i n g p o t e n t i a l s were r e f e r r e d t o t h e p o t e n t i a l s of T1 i o n s . S e m i q u a n t i t a t i v e determinat i o n from t h e depth o f t h e c h a r a c t e r i s t i c C u t s - i n on t h e c u r v e i s p o s s i b l e i n a l k a l i n e s o l u t i o n s .

- Another o s c i l l o p o l a r o g r a p h i c method f o r i d e n t i f i c a t i o n o f homatropine hydrobromide i n a l k a l o i d a l m i x t u r e s was a l s o d e s c r i b e d (50).

7.6

Spectrophotometric methods 7.6.1

Colorimetry

- A c o l o r i m e t r i c method f o r t h e d e t e r m i n a t i o n

of homatropine and o t h e r r e l a t e d a l k a l o i d s was r e p o r t e d ( 5 1 ) . The method i s based on t h e n i t r a t i o n o f t h e drug w i t h a s o l u t i o n of 20% potassium n i t r a t e i n c o n c e n t r a t e d s u l f u r i c a c i d a t 50-60' f o r 30 minutes. The product i s made a l k a l i n e with h o t 20% sodium hydroxide and t h e c o l o r which develops i s measured.

-

A s t a b i l i t y i n d i c a t i n g a s s a y f o r t h e determinat i o n of homatropine hydrobromide and methobromide and t h e i r d e g r a d a t i o n p r o d u c t s i n g a l e n i c a l p r e p a r a t i o n s was r e p o r t e d ( 5 2 ) . The method i s as follows:To 5 ml o f prepared s o l u t i o n ( z = 3 mg o f a l k a l o i d ) i n a 5Q m l f l a s k p l a c e d i n an i c e w a t e r b a t h , add s a t u r a t e d aqueous hydroxylammonium c h l o r i d e s o l u t i o n ( 1 ml) and 1 0 . 5 M potasium hydroxide (1 ml) , mix and s e t a s i d e f o r 1 h r . Add 4 M h y d r o c h l o r i c a c i d ( 2 ml) t o g i v e pH 1 . 2 t o 1 . 4 , m i x , add 0 . 3 7 M F e r r i c chloride solution i n 0.1 M hydrochloric acid (1 ml) and mix a g a i n . Remove t h e f l a s k from t h e b a t h , a l l o w t h e e v o l u t i o n of gas t o s u b s i d e and measure t h e e x t i n c t i o n o f t h e s o l u t i o n a t 540 nm.

- Absorpt i o m e t r i c d e t e r m i n a t i o n o f homatropine hydrobromide i n eye d r o p s ( 5 3 ) ; D i l u t e 2 . 5 m l of sample t o 2 5 m l and t o 1 m l of t h e r e s u l t i n g s o l u t i o n [ 0 . 2 5 t o 2 mg of

FARID J. MUHTADI ETAL.

280

homatropine hydrobromide] add 2.5 m l o f water, 3 m l of 5 % sodium hydroxide s o l u t i o n and a f t e r 5 minutes, 3 . 5 m l of F o l i n r e a g e n t . A f t e r 10 m i n u t e s , measure t h e e x t i n c t i o n of t h e b l u e s o l u t i o n w i t h u s e of a r e d f i l t e r . The s e n s i t i v i t y of t h e method i s 0.25 mg i n t h e f i n a l s o l u t i o n t h e mean e r r o r i s + 3 % .

- Another c o l o r i m e t r i c d e t e r m i n a t i o n o f

homatropine hydrobromide has been a l s o r e p o r t e d (54). The method i s as f o l l o w s : D i s s o l v e 1 mg of sample i n 100 m l of water and t o 1 m l of t h e s o l u t i o n add 3 m l o f mM-bromocresol p u r p l e and 2 m l o f b u f f e r s o l u t i o n (pH 4 ) . E x t r a c t t h e c o l o r e d 1:l p r o d u c t i n t o c h l o r o f o r m (2x5 ml) ; d i l u t e t h e combined e x t r a c t s t o 10 m l w i t h c h l o r o form and measure t h e absorbance a t 405 t o 410 nm. Hydrolysis p r o d u c t s of t h e drug does n o t i n t e r f e r e u n l e s s p r e s e n t i n t h r e e f o l d amounts. The r e l a t i v e e r r o r of t h e method does n o t exceed 2 1%.

-

Other c o l o r i m e t r i c d e t e r m i n a t i o n of homat r o p i n e by c i s - a c o n i t i c anhydride h a s been d e s c r i b e d (55,56). I t i s as f o l l o w s : The sample (0.05 gm) was d i s s o l v e d i n 50 m l t o l u e n e and from t h i s s o l u t i o n , t h e s t a n d a r d s a t 20,50 and 100 y / m l were p r e p a r e d . Each s t a n d a r d ( 2 m l ) was d i l u t e d w i t h 2 m l t o l u e n e and 1 m l r e a g e n t p r e p a r e d by d i s s o l v i n g 0.25 gm c i s - a c o n i t i c a n h y d r i d e i n 100 m l r e d i s t i l l e d a c e t i c anhydride. The s o l u t i o n s were h e a t e d f o r 45 minutes on a steam b a t h and c o o l e d f o r 15 minutes i n t h e d a r k . Then were d i l u t e d t o 10 m l . w i t h 20: 80 a c e t i c a n h y d r i d e - t o l u e n e m i x t u r e and absorbancy measured a t 535 nm. Aqueous s o l u t i o n s o f t h e s a l t was b a s i f i e d w i t h ammonia and e x t r a c t e d w i t h t o l u e n e .

- Homatropine was determined u s i n g t e t r a -

bromophenolphthalein e t h y l e s t e r a t pH 8-10.5 and t h e a d d i t i o n compound formed was e x t r a c t e d w i t h 1 , 2 - d i c h l o r o e t h a n e and e x t i n c t i o n i s measured a t 560 nm (57).

-

C o l o r i m e t r i c d e t e r m i n a t i o n of t h e t h a l i u m complex of homatropine hydrobromide u s i n g c r y s t a l v i o l e t was a l s o r e p o r t e d ( 5 8 ) .

7.7

A 7.7.1

Paper chromatography Homatropine can be d e t e c t e d by paper chromatography. chromatographic c o n d i t i o n s used €or homatropine.

Table 8 i n c l u d e s paper

Table 8 . Paper Chromatography of Hornatropine Conditions Detecting reagent Whatman No. 1 paper impreg- 5% aqueous ammonia s a t u r a t e d w i t h octanol n a t e d w i t h 7 % m e t h a n o l i c o c t a n o l . Descending t e c h n i que. Drying t h e paper i n a i r f o r 5 t o 10 minutes. Solvent system

R -€

value -

Reference (59)

- 4 . 8 gm of c i t r i c a c i d Paper Whatman No.1 dipped

U.V. o r Iodoplatinate

0.3

Clark (17)

- acetate buffer

Location under U . V . Light (254 mu) o r Iodoplatinate spray

0.96

Clark (17)

i n a mixture of 130 water and 870 of n- But an o 1 (pH 4.58)

i n 5% s o l u t i o n of sodium dihydrogen c i t r a t e and d r i e d Whatman No.1 o r No.3 impregnated by d i p p i n g i n 10% s o l u t i o n of T r i b u t y r i n i n a c e t o n e and d r y i n g i n a i r .

- BuOH- AC OH- H 2O

(40 : 10 :50) BUOH - HCO 2H- H2 0 (10: 1 : 10)

Descending t e c h n i q u e

0.2% I o d i n e s o l u t i o n spray

BuOH-C2H5C 02H-H20 (10: 1: 10) Other paper chromatography h a s a l s o been r e p o r t e d (61).

(60)

7.7.2

Thin l a y e r chromatography (TLC) Homatropine can b e d e t e c t e d by t h i n l a y e r chromatography. Table 9 t h i n l a y e r chromatographic c o n d i t i o n s used f o r homatropine. Table 9.

S o l v e n t system

Thin l a y e r chromatography of Eomatropine Conditions

Detecting reagent

S t r o n g ammonia s o l u t i o n : methanol (1.5 : 100)

s i l i c a gel G

acidified iodoplatinate o r under U . V . (254 nm)

Ethanol-pyridine-water : 6 : 4) (1

alumina

iodoplatinate spray

Chloroform-acetone-diethylamine ( 5 : 4 : 1) Acetone - wat e r - 25 % ammonia solution : 6) (90 : 4

K i e s e l g e l GF 254 Kieselgel G

Dragendorf f s p r a y

The upper phase of But ano 1- a c e t i c ac id-wat e r ( 10 : 1 : 5) Other T.L.C.

includes

Ce 1l u l ose

R

f-

value

Reference

0.15

Clark ( 17)

0.87

(65)

Dragendorff e t h y l a c e t a t e reagent

-

(64)

Dragendorff s p r a y

-

(65)

f o r homatropine h a s been a l s o r e p o r t e d ( 6 6 ) .

HOMATROPINE HYDROBROMIDE

7.7.3

283

Electrophoresis

- E l e c t r o p h o r e s i s of homatropine was performed

on Whatman No.1 p a p e r (18x46 cm) a t 8 v./cm f o r 3 h r s . i n t h e p r e s e n c e of u n i v e r s a l buffer The r e l a t i v e d i s p l a c e m e n t s a t pH 2 . 3 , 4 . 3 , 6 . 4 , 8 . 2 , 10.5 and 11.4 were 6 3 , 5 4 , 104,105,100,16 r e s p e c t i v e l y ( 6 7 , 6 8 ) . - Other e l e c t r o p h o r e s i s f o r s e v e r a l a l k a l o i d s have been r e p o r t e d ( 6 9 ) .

.

7.7.4

Column chromatography Chromatographic a s s a y of homatropine hydrobromide and o t h e r r e l a t e d a l k a l o i d s a r e quant i t a t i v e l y e l u t e d w i t h 95% a c e t o n e from a column packed w i t h b a s i c alumina (70). The p u r i t y of homatropine hydrobromide can be determined on alumina column as f o l l o w s (71) ' Homatropine hydrobromide (0.1 gm) was e l u t e d o v e r a column packed w i t h b a s i c alumina (5 gm) by u s i n g aqueous a c e t o n e as s o l v e n t . After e l u t i o n , t h e e l u a t e was d i l u t e d w i t h w a t e r and homatropine hydrobromide i s t i t r a t e d w i t h 0 . 1 N h y d r o c h l o r i c a c i d u s i n g bromophenol b l u e as an i n d i c a t o r .

7.7.5

Gas chromatography (GC)

The gas chromatography f o r homatropine a r e l i s t e d i n t a b l e (lo).

Table 1 0 .

The GC of Homatropine Ref. -

C a r r i e r gas

2.5% SE-30 on 80-100 mesh chromosorb W (5 F t x 4mm i n t e r n a l d i a m e t e r ) . Column t e m p e r a t u r e 225'.

Nitrogen (SO ml/min.)

F . I . D . hydrogen 0.36 r e l a t i v e (50 ml/min.) t o codeine a i r (300 ml/min.)

Clark (17)

3% XE-60 s i l i c o n e n i t r i l e polymer on 100-120 mesh Chromosorb W. Column t e n p e r a t u r e 225".

Nitrogen (50 ml/min.)

F . I . D . hydrogen 0.39 r e l a t i v e (50 ml/min.) t o codeine a i r (300 ml/min.)

Clark (17)

5% SE-30 on 60-80 mesh Chromosorb W AW (5 f t x 1/8 d i a m e t e r ) Column temperature 230'.

Nitrogen (30.7 ml/min.)

F.I.D. hydrogen (22 ml/min.)

0.44 r e l a t i v e t o codeine

Clark (17)

3% J X R on s i l a n i s e d G Chrom P on 100-120 mesh (1.8m x 2mm) Column t e m p e r a t u r e 250".

Nitrogen (50 ml/min.)

F.I.D.

F.I.D.

5% SE-30 on 60-80 mesh a c i d washed Chromosorb W a t 190,210,230, 250-270° 15 % QF-1 on 60-80 mesh, Gas Chrom(R)Q. , (1.5m x 3 . 2 mm) column t e m p e r a t u r e 160".

Detector

Retention _ t i_ me

Column c o n d i t i o n

Nitrogen (50 ml/min)

F.I.D.

Other gas chromatographic a n a l y s i s have been r e p o r t e d (75-78)

-

-

4 . 2 (min)

(73)

HOMATROPINE HY DROBROMIDE

7.7.6

285

High P r e s s u r e Liquid Chromatography (HPLC) Fell _ et _ a l . (73) have r e p o r t e d an a n a l y s i s of homatropine hydrobromide by r e v e r s e d - p h a s e h i g h - p r e s s u r e l i q u i d chromatography. Homatrop i n e hydrobromide was determined on a column of H y p e r s i l ODS ( 5 um) with 5 0 mM. Sodium a c e t a t e i n 10 mM-tetrabutylammonium s u l f a t e (pH 5 . 5 ) - a c e t o n i t r i l e ( 3 : l ) as t h e mobile phase and d e t e c t i o n a t 254 nm. The i n t e r n a l s t a n d a r d was p - t o l u i c a c i d . R e c t i l i n e a r c a l i b r a t i o n graph was o b t a i n e d f o r homatropine hydrobromide ( i n eye d r o p s ) i n t h e range 0 t o 4 mg ml-1.

S t u t z and Sass (80) have d e s c r i b e d a high-speed, high p r e s s u r e l i q u i d chromatography of t r o p a n e a l k a l o i d s i n c l u d i n g homatropine. The compound was s e p a r a t e d on a s t a i n l e s s - s t e e l column (1 meter x 4 . 6 mm) packed with sil-X a b s o r b e n t w i t h 28% aqueous ammonia t e t r a h y d r o f u r a n (1: 100) as t h e s o l v e n t and w i t h a column i n l e t p r e s s u r e o f 500 l b p e r s q . i n c h . A d i f f e r e n t i a l r e f r a c t i v e index d e t e c t o r and a UV d e t e c t o r o p e r a t i n g a t 254 nm were used t o monitor t h e eluate.

ACKNOWLEDGEMENT The a u t h o r s would l i k e t o thank Mr. Uday C. Sharma of Department of Pharmdcognosy, c o l l e g e o f Pharmacy, King Saud U n i v e r s i t y €or h i s s e c r e t a r i a l assistance i n t h e reproduction of t h e manuscript.

FARID J. MUHTADI ETAL.

286

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(Ned.),

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45 (12), 2134, M.H. St.utz, and S. Sass; Analyt. Chem., (1973).

MEBENDAZOLE

A . A . AL-Budrz* und M . T d / t i q * *

*Vepu,ahtmeMA: 06 PhatunuzceuLlcal C h m i ~ f i yand VcpahXment** 0 6 Phahmacology, Coflecje ozj Phahmucy, King Saud UnivehhLty, P.O. Box 2457, Riyudh-11451, Saudi Ahabia.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

29 1

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

A. A. AL-BADR AND M. TARIQ

292

1.

Forward, History

2.

Description 2.1 2.2 2.3 2.4 2.5

3.

Nomenclature Formulae Molecular Weight Elemental Composition Appearance, Color and Taste

Physical properties

4. Spectral Properties 5.

Synthesis

6. Pharmacokinetics 7.

Toxicity

8. Methods of Analysis 8.1 8.2

Identification Titrimetric Methods 8 . 3 Spectrophotometric Methods 8.4 Chromatographic Methods 8.5 Polarographic Method 8.6 Radioimmunoassay Method

References

MEBENDAZOLE

293

Foreword, H i s t o r y Mebendazole i s a broad spectrum a n t h e l m i n t i c a g e n t s y n t h e s i z e d and developed by J a n s s e n Pharmaceutica, Research Laboratody, Beerse, Belgium. A f t e r i t s i n t r o d u c t i o n t h e r e i n 1972, t h e drug became a v a i l a b l e i n numerous c o u n t r i e s around t h e world, i n c l u d i n g t h e United S t a t e s and Canada (1). I t produces high c u r e rates i n i n f e s t a t i o n s by A s c a r i s , threadworms ( E n t e r o b i u s ) , hookworms (Ancylostoma and Necator S p p . ) , and Whipworms ( T r i c h u r i s ) . I t i s a l s o a c t i v e i n some (about 40%) Dwarf tapeworm (Hymenolepis) i n f e s t a t i o n s

(2-5). 2.

Description 2.1

Nomenclature 2.1.1

Chemical Names

(5-Benzoyl-1H-benzimidazol-2-yl)-carbamic

a c i d methyl ester.

5-Benzoyl-2-benzimidazolecarbamic a c i d methyl ester. Methyl 5-benzoyl-2-benzimidozolecarbamate. Methyl-5-benzoylbenzimidazol-2-ylcarbamate. Methyl-(5-benzoyl-1H-benzimidazol-2-yl) carbamat e. Carbamic acid,5-benzoyl-1H-benzimidzol-2-yl, methyl e s t e r . (6) CAS R e g i s t r y Number [31431-29-71 2.1.2

(6)

Generic N a m e Mebendazole; R 17,635

2.1.3

(7).

Trade Names P r o p r e t a r y Names Bendrax, Besantin, E s a d i r a s e , Equivuarm p l u s Forverm, Gendal, Granverm, Lomper, Mebendacin, Mebendozol M K , Mebendazole, Mebenix, Mebutar, Nemasole, Oxiben, Pantelmin, Parelmin, S i r b e n , Telmin, T r o t i l , Vermirax, Vermox, and Zakor (1,6, 8-11).

294

A. A. AL-BADR AND M. TARIQ

2.2

Formulae 2.2.1

Empirical

2.2.2

Structural H

I

2.3

0

Molecular Weight 295.30 and 295 ( 7 E 9 ) ,

2.4

Elemental Composition C , 65.08%, H , 4.44%, N , 14.23%; 0, 16.25%

2.5

Appearance, C o l o r and Taste Off-white to slightly yellow amorphous powder and not unpleasant to taste. (10-12)

3.

Physical Properties 3.1

Melting Point Melts at about 290°, 288.5', decomposition (6-8 6 l o ) .

3.2

above 280' with

Solubility Almost insoluble in water, ethanol, ether, chloroform, and dilute mineral acids; readily soluble in formic acid (9 E 11).

3.3

Hygroscopicity Mebendazole is not hygroscopic and it is stable in air.

MEBENDAZOLE

3.4

295

Extraction Mebendazole i s e x t r a c t e d by chloroform from aqueous a l k a l i n e solutions (11).

4.

Spectral Properties 4.1

U l t r a v i o l e t Spectrum The u l t r a v i o l e t a b s o r p t i o n spectrum o f mebendazole o b t a i n e d from a s o l u t i o n i n n e u t r a l methanol i n t h e r e g i o n o f 200 t o 400 nm u s i n g DMS 90 s p e c t r o p h o t o meter i s shown i n F i g u r e 1. Three a b s o r p t i o n maxima a t about 210, 247 and 310 nm and two minima a t 228 and 273 nm were observed. C l a r k e (11) r e p o r t e d t h e f o l l o w i n g : Mebendazole i n 0.05% f o r m i c a c i d i n isopropanol; maxima a t 248 nm ( E l % , 1 cm 1005) and 313 nm ( E l % , 1 cm 5 1 8 ) , minima a t 230 nm and 275 nm. Mebendazole i n 0 . 0 1 N sodium hydroxide i n i s o p r o p a n o l , maxima a t 270 nm ( E l % , 1 cm 802) and 355 nm ( E l % , 1 cm 653), minima a t 245 nm and 302 nm. Mebendazole i n 0 . 1 N h y d r o c h l o r i c a c i d i n i s o p r o p a n o l , maxima a t 234 nm ( E l % , 1 cm 1000) and 288 nm ( E l % , 1 cm 5 2 4 ) , minima a t 215 nm and 266 nm.

4.2

I n f r a r e d Spectrum The i n f r a r e d spectrum o f mebendazole i s p r e s e n t e d ; i n F i g u r e 2. The spectrum was o b t a i n e d from K B r d i s c u s i n g a Pye Unicam SP 1025 i n f r a r e d s p e c t r o p h o t o m e t e r . The s p e c t r a l a s s i g n m e n t s are presented i n t h e following t a b l e .

A. A. AL-BADR AND M.TARIQ

2%

200

250

300Wavelength 350

400

(nm) FIGURE I : ULTRAVIOLET SPECTRUM OF MEEENDAZOLE IN METHANOL

-E 20.

*20

;"

0

C

0

e

.-

m C

0;

FIGURE 2 . INFRARED S P E C T R U M

OF M E B E N D A Z O L E

( KBr d i s c )

298

A. A. AL-BADR AND M. TARIQ

Frequency cm

-1

Assignment

34 15

NH s t r e t c h

2500-2950

CH s t r e t c h

1720

C = 0 (amide) s t r e t c h

1650

C = 0 stretch

1530 1600

I

141 0-1460 700- 765 878

1

900)

C = C stretch

CH3-0 (C-0) s t r e t c h Monosubstituted benzene

1 , 2 , 4 T r i s u b s t i t u t e d benzene

C l a r k e (11) r e p o r t e d t h e p r i n c i p a l peaks (KBr d i s c ) a r e 705, 1260, 1590, 1635 and 1730 cm-1. 4.3

1 'H Nuclear Magnetic Resonance ( H NMR) Spectrum 1 The H NMR spectrum of mebendazole i s shown i n F i g u r e 3. The drug i s d i s s o l v e d i n T r i f l u o r o a c e t i c a c i d (TFA) and i t s spectrum determined on a Varian T60 A NMR s p e c t r o m e t e r u s i n g TMS ( t e t r a m e t h y l s i l a n e ) as t h e i n t e r n a l s t a n d a r d . Assignment of t h e chemical s h i f t s t o t h e d i f f e r e n t p r o t o n s is shown be low:

Chemical S h i f t PPM (6) 7.5 - 8.3 4.08

Multiplicity

P r o t o n Assignment

mu 1t ip 1et

Aromatic

singlet

methyl

FIGURE 3: PROTON MAGNETIC RESONANCE SPECTRUM OF MEBENDAZOLE IN

TRIFLOUROACE T I C ACID(TFAI

300

A. A. AL-BADR AND M. TARIQ

4.4

I 3 C Nuclear Magnetic Resonance (13C NMR) Spectrum

The I3C NMR s p e c t r a of mebendazole i n formic a c i d u s i n g TMS ( t e t r a m e t h y l s i l a n e ) as an i n t e r n a l s t a n d a r d a r e o b t a i n e d u s i n g a J o e l FX 100 MHz s p e c t r o m e t e r a t an ambient t e m p e r a t u r e . F i g u r e s 4 and 5 r e p r e s e n t t h e 1H-decoupled and o f f - r e s o n a n c e spectra respectively. / 0

.,

The carbon assignments a r e based on chemical s h i f t v a l u e s and by comparison with model compounds and a r e shown below: Chemical S h i f t PPM ( 6 )

Multiplicity

C a r bon Ass ignm en t

57.5

quartet

155.8

singlet

148.4

singlet

139.2

s i n g 1e t

134.4

singlet

118.2

doublet

130.5

singlet

130.9

doublet

115.8

doublet

200.5

singlet

136.7

singlet

132.9

doublet

131.3

doublet

‘13 and ‘15

136.3

doublet

‘14

c1

c2 c3

c4 c5

‘6 c7

c9

clo 5 1 ‘12

and ‘16

MEBENDAZOLE

301

FIGURE L : PROTON OECOUPLEO CARBON-13 NMR SPECTRUM OF MEBENDAZOLE IN FORMIC ACID

FIGURE 5 : OF-RESONANCE CARB3N-I3 NMR SPECTRUM

OF

MEBENDAZOLE IN FORMIC

ACID

A. A. AL-BADR AND M. TARIQ

302

4.5

Mass Spectrum and Fragmentometry F i g u r e 6 shows t h e 70 eV e l e c t r o n impact ( E I ) mass spectrum o b t a i n e d on Varian MAT 311 mass s p e c t r o meter usi ng ion s o u r c e p r e s s u r e of 10-6 T o r r , i o n s o u r ce t e mpe ra ture o f 18OoC and an emission c u r r e n t of 300 PA. The spectrum is dominated by m/e 186 i o n ( ba se peak) r e s u l t i n g from t h e l o s s of CH30H and C6H5 fragments. A proposed mechanism of f r ag m enta ti on and t h e mass/charge r a t i o s of t h e major fragments i s given i n Scheme 1 (a ar,d b ) . Chemical i o n i s a t i o n (CI) spectrum i s p r e s e n t e d i n F i g u r e 7 and i s obt a i ne d on a Finnigan 4000 mass s p e ct r ome t e r wit h ion e l e c t r o n energy of 100 eV, ion s ourc e p r e s s u r e o f 0 . 3 T o r r , ion s o u r c e t e m p e r a ture of 15OoC and emission c u r r e n t of 300 PA. The mass s p e c t r a l assignment of t h e prominent i o n s under C I c o n d i t i o n s i s gi ve n below:

m/e

Species

296

M+ + 1

2 95

M+

218

M+

-

C6H5

MEBENDAZOLE

303

2

lBO(

1

50.C

324.2 J

2180 208 \

I86

,330

50.0

336.2

235.8 251 6 2Ul

32832.

I05

263 218

2 95

M /E FIGURE 7

MASS SPECTRUM OF MEBENDAZOLE ( c l )

b

.+

a

&@+.

H

G

[@!$== 7

m/e 295

N?-NH-c-o

-cH3

'\ 0-C' O+

- co

____)

E

m/e 77

-+0

H

NH

- \\C - OCH3

-CH30H

[-

m/e 105

Ill O 1

IA -co *

C-HNy@ -, i ,

m/e 158

L

m/186 m/e 218

Scheme l a :

Proposed mechanism of fragmentation of mebendazole.

/4

m/e 130

m/e 295

m/e 295

N7rNH-C 0 '& +

OI

s W

-CH30H

t

Figure lb:

m/e 263

= O

-co

4 m/e

Proposed mechanism of f r a g m e n t a t i o n o f mebendazole.

235

A. A. AL-BADR AND M.TARIQ

5.

Synthesis 1- Mebendazole was prepared (13) from 3,4(NH2)2C,H,CoC6H5 and NHz(CH3S)C = N - COOCH3 according to the following scheme.

0

II

NH3

C

0

HN

v

' 3 c

0

II

C-NH-C-0-CH3

.CH3

11

H2N, ,C = N-C-0-CH3

s,

0

CH3 NH -c-0II 0

U

11

0

CH3

-

MEBENDAZOLE

2-

307

Mebendazole was also prepared in 52% yield by decomposition of methyl 7-benzoyl-lH-2,1,4benzothiadiazine-3-y1 carbamate in methanol with 2 N HC1. The latter compound is prepared in two stages from 4 - b e n z o y l - 2 - n i t r o a n i l i n e (14)

.

Two steps

0 II

*

-

CH30H

CH3 2N HC1 H

6.

0

Pharmacokinetics 6.1 Absorption, Distribution, Metbolism and Extraction

Mebendazole is poorly absorbed by gastrointestinal tract after oral administration (10). Only 5-10% of the orally administered frug is found in the blood (1). The poor intestinal absorption of mebendazole is of therapeutic advantage in the treatment of intestinal helminthis. However, for tissue dwelling organism high oral doses are required ( 1 5 ) . The absorption of drug in man is markedly enhanced when the drug is used together with the meals, as concomitent fat ingestion ehances its intestinal absorption. Following oral administration peak plasma level reach in 2-4 hours (16-18). Munst et a1 (19) reported a significant variation in plasma mebendazole level in human subjects following oral ingestion. According to this study the plasma half-life was ranging between 1 . 5 and

308

A. A. AL-BADR AND M. TARIQ

5 . 5 h o u r s . The peak c o n c e n t r a t i o n following o r a l a d m i n i s t r a t i o n were d e t e c t e d a t 30 minutes t o 2 hours of t h e dose. Mebendazole is metabolized p r i m a r i l y i n l i v e r by d e c a r b o x y l a t i o n (20) , t h e major m e t a b o l i t e was 2 -amino-5 (6-benzimidazolyl phenylketone. B r a i t h w a i t e et a1 (21) r e p o r t e d t h a t mebendazole is more l i p o p h i l i c t h a n any of i t s m e t a b o l i t e s . The d i s t r i b u t i o n s t u d y by same a u t h o r showed t h a t 63.3% of mebendazole was d i s t r i b u t e d i n plasma and 36.7% i n c e l l u l a r f r a c t i o n . Most of t h e drug i n plasma (95%) was p r e s e n t i n t h e p r o t e i n bound form. The c o n c e n t r a t i o n of mebendazole i n l i v e r was c o n s i d e r a b l y h i g h e r as compared t o t h e f a t t y t i s s u e . G i l b a l d i and Perrier (22) r e p o r t e d t h a t s t e a d y s t a t e AUC of mebendazole i s independent of a b s o r p t i o n r a t e i n human s u b j e c t s . Approximately 90% o f t h e o r a l l y a d m i n i s t e r e d dose o f mebendazole i s e l i m i n a t e d p r i m a r i l y a s unchanged drug i n faeces. About 5-10% of t h e a d m i n i s t e r e d drug may be recovered i n t h e form o f i t s c o n j u g a t e s , and metabolites i n t h e urine (11). I n r a t s about 85% o f an I . V . dose was e l i m i n a t e d with t h e b i l e and remainder w i t h t h e u r i n e . The m a j o r i t y o f t h e dose was recovered a s conjugated m e t a b o l i t e s (23-24). I n a n o t h e r s t u d y (25), t h r e e m e t a b o l i t e s of mebendazole were i s o l a t e d from t h e b i l e o f r a t s and i d e n t i f i e d as : methyl-5(a-hydroxybenzyl)-2-benzimidazole carbamate; 2amino-5- (a-hydroxybenzyl) benzimidazole and 2-amino5-benzoylbenzimidazole. 7.

Toxicity G a s t r o i n t e s t i n a l symptoms i n c l u d i n g c o n s t i p a t i o n have been r e p o r t e d i n f r e q u e n t l y f o l l o w i n g use o f mebendazole (26). High doses may on o c c a s i o l s b e a s s o c i a t e d w i t h mild colic p a i n o r d i a r r h o e a as r e p o r t e d by C h a v a r r i a ( 2 7 ) u s i n g doses of u p t o 300 mg B I D i n t h e t r e a t m e n t of t a e n i a s i s

.

Acute and c h r o n i c t o x i c i t y s t u d i e s i n animals i n d i c a t e a wide range between t h e r a p e u t i c and t o x i c d o s e s . The LD50 (mg/kg o r a l l y ) was more t h a n 640 mg/kg in r a b b i t s and dogs and more t h a n 1280 mg/kg i n mice and rats (11). S i g n i f i c a n t haematologic, biochemical, o r pathol o g i c a b n o r m a l i t i e s were n o t found i n animals given 40 mg/kg d a i l y f o r 13 weeks b u t were noted i n r a t s

MEBENDAZOLE

309

given a d a i l y dosage o f 130 mg/kg. Mebendazole i s c o n t r a i n d i c a t e d i n p r e g n a n t women because i t h a s shown embryotoxic and t e r a t o g e n i c a c t i v i t y i n s i n g l e p e r o r a l dose a s low as 10 mg/kg (18, 2 8 , 2 9 ) . Rare cases o f leukopenia i n human s u b j e c t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f mebendazole was a l s o reporqed ( 3 0 ) . I n high d o s e s 40-50 mg/kg/day, t h e drug has been a s s o c i a t e d w i t h s e v e r e i r r e v e r s i b l e n e u t r o p e n i a . No s i g n i f i c a n t CNS e f f e c t s i n t h e p a t i e n t s t a k i n g mebendazole t h e r a p y has been r e p o r t e d e x c e p t s l i g h t headache and dizziness (31). 8.

Methods o f A n a l y s i s 8.1

Identification I n f r a r e d Spectrum The i n f r a r e d a b s o r p t i o n spectrum of a potassium bromide d i s p e r s i o n o f i t , p r e v i o u s l y d r i e d , e x h i b i t maxima o n l y a t t h e same wavelength as t h a t o f a s i m i l a r p r e p a r a t i o n of USP mebendazole RS ( 3 2 ) . X-ray D i f f r a c t i o n The X-ray powder d i f f r a c t i o n d a t a f o r i d e n t i f y i n g mebendazole and o t h e r a n t h e l m i n t i c s have been o b t a i n e d by d i f f r a c t o m e t e r and Debye S c h e r r e r camera t e c h n i q u e s ( 3 3 ) . The d a t a were t a b u l a t e d i n terms of t h e l a t t i c e s p a c i n g s and the r e l a t i v e i n t e n s i t i e s of t h e lines. P a t t e r n s u s i n g t h r e e d i f f e r e n t X-ray wavelengths with t h e camera method a r e compared w i t h each o t h e r and w i t h t h e d i f f r a c t o m e t e r p a t t e r n .

8.2

T i t r i m e t r i c Method USP XX (1980) (32) d e s c r i b e d t h e f o l l o w i n g

t i t r i m e t r i c method f o r t h e a s s a y o f mebendazole:

Dissolve about 225 mg of mebendazole, a c c u r a t e l y weighed, i n 30 m l o f g l a c i a l a c e t i c a c i d . T i t r a t e w i t h 0 . 1 N p e r c h l o r i c a c i d VS, d e t e r m i n i n g t h e end-point p o t e n t i o m e t r i c a l l y , u s i n g a calomelg l a s s e l e c t r o d e system. Perform a blank determinat i o n , and make any n e c e s s a r y c o r r e c t i o n . Each ml

A. A. AL-BADR AND M.TARIQ

3 10

o f 0 . 1 N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 29.53 mg Of

C16H13N303'

Wahbi and Onsy, (34) developed t h e two t i t r i m e t r i c methods t o determine t h e mebendazole i n pharmac e u t i c a l p r e p a r a t i o n s . D i r e c t t i t r a t i o n and back t i t r a t i o n methods are mentioned a s f o l l o w s : a)

D i r e c t T i t r a t i o n Method 200 Mg o f mebendazole was t a k e n i n d r y c o n i c a l f l a s k and d i s s o l v e d i n 2 m l o f 99% formic a c i d followed by t h e a d d i t i o n o f 60 m l o f g l a c i a l a c e t i c a c i d . T h i s s o l u t i o n was t i t r a t e d w i t h 0.1 N perchloric acid i n g l a c i a l a c e t i c acid u s i n g c r y s t a l v i o l e t a s i n d i c a t o r , development o f a p e r s i s t e n t green c o l o r was d e s i g n a t e d a s end p o i n t . Blank d e t e r m i n a t i o n was performed and t h e n e c e s s a r y c o r r e c t i o n s were made. The c a l c u l a t i o n was done by u s i n g t h e e q u i v a l e n c e f a c t o r which is 1 m l o f 0 . 1 N p e r c h l o r i c a c i d i s e q u a l t o 29.53 mg o f mebendazole.

b)

B a c k - T i t r a t i o n Method 200 Mg o f mebendazole was t a k e n i n d r y c o n i c a l f l a s k , t h e powder was d i s s o l v e d i n 20 m l o f 1 M p e r c h l o r i c a c i d i n g l a c i a l acetic a c i d . T h i s s o l u t i o n was t i t r a t e d with 0 . 1 N sodium acetate i n glacial acetic acid, crystal violet was used a s i n d i c a t o r , development o f b l u i s h green a s i n d i c a t o r . These methods a r e reproducible with standard deviation of 0.3%.

Mebendazole ( i n amounts u p t o 250 mg) i n anhydrous a c e t i c a c i d medium can be t i t r a t e d w i t h 0 . 1 M p e r c h l o r i c a c i d ( a l s o i n a c e t i c a c i d ) u s i n g 0.5% c r y s t a l v i o l e t solution a s indicator. Results a g r e e with t h o s e o b t a i n e d by spectrophotometry a t 313 nm ( 3 5 ) . 8.3

Spectrophotometric Methods 8.3.1

U l t r a v i o l e t S p e c t r o m e t r i c Methods

--

P a t e 1 e t a1 (36) r e p o r t e d two s p e c t r o p h o t o metric methods f o r t h e e s t i m a t i o n o f

MEBENDAZOLE

311

mebendazole i n raw m a t e r i a l . A s o l u t i o n of t h e sample i n formic a c i d was d i l u t e d w i t h a mixture o f chloroform : methanol (17:3). Thin-layer chromatography was run on s i l i c a g e l i n chloroform : e t h y l a c e t a t e ; a c e t o n e : formic a c i d (70:80:9.5:0.5), developed f o r 15 cm and d r i e d f o r 30 minutes a t 115O. The drug i s t h e n e x t r a c t e d i n t o chloroform and measured a t 313 nm.

In a n o t h e r methods, a s o l u t i o n o f t h e drug i n formic a c i d i s d i l u t e d w i t h i s o p r o p a n o l

and t r e a t e d w i t h MeOH and 30% potassium hydroxide. After 30 min. t h e absorbance was measured a t 420 nm. Wahbi and Osny (34) r e p o r t e d a s p e c t r o p h o t o metric method o f a n a l y s i s of mebendazole i n t a b l e t s with a s t a n d a r d d e v i a t i o n o f 1 . 4 % . Powdered t a b l e t s e q u i v a l e n t t o 50 mg of mebendazole were t a k e n i n t o 50 m l s t a n d a r d f l a s k followed by 10 m l o f 70% p e r c h l o r i c a c i d . The mixture was shaken f o r 10 minutes and f i l t e r e d through a d r y f i l t e r paper. 1 M 1 of f i l t r a t e taken t o a 100 m l f l a s k and d i l u t e d t o volume w i t h d i s t i l l e d w a t e r . The absorbance of t h i s s o l u t i o n was measured i n a 1 cm c e l l a t 288 nm wavelength u s i n g a spectrophotometer. 0 . 2 M 1 o f 70% p e r c h l o r i c a c i d d i l u t e d w i t h 100 m l of d i s t i l l e d w a t e r was used a s b l a n k . C a l c u l a t i o n o f c o n c e n t r a t i o n was done by t a k i n g 566 a s t h e (E 1%, 1 cm) v a l u e a t 288 nm o r by u s i n g a s u i t a b l e c a l i b r a t i o n curve. 8.3.2

Phosphorescence Method Mebendazole h a s been r e p o r t e d t o show good phosphorescence a t room temperature when determined on Whatman 42 f i l t e r paper u s i n g Pb ( I V ) and T 1 ( I ) a s phosphorescence enhancing a g e n t s . In t a b l e t s , t h i s method gave a r e c o v e r y o f 97.6% o f mebendazole (37) *

A. A. AL-BADR AND M. TARIQ

312

8.3.3

F l u o r i m e t r i c Method Abdel F a t t a h et a1 (38) have r e p o r t e d a f l u o r i m e t r i c d e t e r m i n a t i o n method o f mebendazole i n p h a r m a c e u t i c a l dosage forms a f t e r a l k a l i n e h y d r o l y s i s . I n t h i s method 2 t o 5 mg o f mebendazole i s d i s s o l v e d i n 10 m l of 1 M sodium h y d r o x i d e and h e a t e d f o r 1 h r on a b o i l i n g water b a t h . After d i l u t i o n w i t h methanol, 1 M sodium h y d r o x i d e ( 4 9 : 1 ) , t h e s o l u t i o n was s p o t t e d on t o Whatman 42 f i l t e r p a p e r and d r i e d a t 65 f o r 5 t o 10 m i n u t e s . The f l u o r e s c e n c e was measured a t 460 nm ( e x c i t a t i o n a t 365 nm). The l i m i t of d e t e c t i o n was 0 . 1 f o r mebendazole w i t h r e c t i l i n e a r c a l i b r a t i o n graphs u p t o 10 and 50 ppm ( i n t h e f i n a l s o l u t i o n ) . For 8 ng o f mebendazole p e r s p o t , t h e c o e f f i c i e n t o f v a r i a t i o n was 9.5% (n = 1 9 ) . For p h a r m a c e u t i c a l f o r m u l a t i o n s , r e c o v e r y o f mebendazole ranged from 99.04 t o 100.56%, w i t h c o e f f i c i e n t o f v a r i a t i o n of 2.27 t o 2.97%.

8.3.4

C o l o r i m e t r i c Method Rana et& (39) have d e s c r i b e d a method f o r c o l o r i m e t r i c d e t e r m i n a t i o n of mebendazole i n t a b l e t s and s u s p e n s i o n b y adding hydroxyl amine h y d r o c h l o r i d e , dicyclohexylcarbodiimide and f e r r i c c h l o r i d e t o a s o l u t i o n o f t a b l e t s (or a d i l u t i o n o f a s u s p e n s i o n ) i n isopropanol and formic a c i d and measuring t h e absorbance a t 520 nm. The c a l i b r a t i o n graph was l i n e a r f o r 0 . 4 t o 2 . 0 pg/ml of mebendazole .

8.4

Chromatographic Methods 8.4.1

Thin-Layer Chromatography (TLC) C l a r k e (11) h a s d e s c r i b e d t h e f o l l o w i n g TLC system f o r t h e i d e n t i f i c a t i o n o f mebendazole. S o l v e n t system: Chloroform : methanol : formic a c i d (90:5:5). Absorbent:

S i l i c a g e l HF 254 (Merck)

.

MEBENDAZOLE

313

V i s u a l i z i n g a g e n t : I o d i n e vapour; d r a g e n d o r f f s p r a y (Location under u l t r a v i o l e t l i g h t ) . Rf : 0.53. United S t a t e s Pharmacopeia "USPXX 1980 (32) have used TLC t o determine t h e p u r i t y o f mebendazole. The method i s d e s c r i b e d as follows: D i s s o l v e 50 mg i n 1 . 0 m l o f 98 p e r c e n t f o r m i c a c i d i n a 10-ml v o l u m e t r i c f l a s k , add chloroform t o volume, and mix. S i m i l a r l y p r e p a r e a s o l u t i o n of USP Mebendazole RS i n t h e same medium having a c o n c e n t r a t i o n o f 5 mg p e r m l . T r a n s f e r 1 . 0 m l o f t h i s S t a n d a r d s o l u t i o n t o a 200-ml v o l u m e t r i c f l a s k , add a m i x t u r e o f chloroform and 98 p e r c e n t formic a c i d ( 9 : l ) t o volume, and mix ( d i l u t e d S t a n d a r d s o l u t i o n ) . On a s u i t a b l e t h i n - l a y e r chromatographic p l a t e , c o a t e d w i t h a 0.25-mm l a y e r o f chromatog r a p h i c s i l i c a g e l m i x t u r e , s p o t 10-1-11 p o r t i o n s of t h e t e s t s o l u t i o n , t h e S t a n d a r d s o l u t i o n , and t h e d i l u t e d S t a n d a r d s o l u t i o n . Allow t h e s p o t s t o d r y , and develop t h e chromatogram i n a s o l v e n t system c o n s i s t i n g o f chloroform, methanol, and 98 p e r c e n t formic a c i d (90:5:5) u n t i l t h e s o l v e n t f r o n t h a s moved about t h r e e - f o u r t h s o f t h e l e n g t h o f t h e p l a t e . Remove t h e p l a t e from t h e d e v e l o p i n g chamber, mark t h e s o l v e n t f r o n t , a l l o w t h e s o l v e n t t o e v a p o r a t e , and examine t h e p l a t e under s h o r t - w a v e l e n g t h u l t r a v i o l e t l i g h t . The R f v a l u e of t h e main s p o t o b t a i n e d from t h e t e s t s o l u t i o n c o r r e s p o n d s t o t h a t o b t a i n e d from t h e S t a n d a r d s o l u t i o n , and no s p o t , o t h e r t h a n t h e main s p o t , i n t h e chromatogram o f t h e t e s t s o l u t i o n i s l a r g e r o r more i n t e n s e t h a n t h e main s p o t o b t a i n e d from t h e d i l u t e d S t a n d a r d s o l u t i o n . 8.4.2

H i g h - p r e s s u r e Liquid Chromatography (HPLC) A s e n s i t i v e method f o r s e p a r a t i o n and s i m u l t a n e o u s d e t e r m i n a t i o n o f mebendazole and hydroxymebendazole i n human plasma o r c y s t l i q u i d sample u s i n g f l u b e n d a z o l e as

314

A. A. AL-BADR AND M.TARIQ

i n t e r n a l s t a n d a r d by high-performance l i q u i d chromatography w ith e le c tr o c h e m ic a l detector i s described (40). The chromatographic c o n d i t i o n s : The column ( 4 . 6 mm X 5 cm) was packed with S p h er i s o r b 5 ).rm ODS (Phase Sep. Queensferry Clwyd, U.K.). The mobile phase c o n s i s t e d of 20% 2-propanol i n a pH 7 . 0 phosphate b u f f e r . The e l u e n t was d e l i v e r e d by Kipp 9208 h i g h - p r e s s u r e l i q u i d chromatography pump, a t a flow r a t e of 2.5 ml/min. The e l ec t r o ch e m i c a l d e t e c t o r equipped with a r o t a t i n g d i s c working e l e c t r o d e was u se d . The l i m i t of d e t e c t i o n f o r mebendazole and hydroxymebendazole was 5 and 2.5 ng/ml r e s p e c t i v e 1y

_-

.

-

Allan e t a 1 (41) r e p o r t e d two h ig h performance l i q u i d chromatographic methods f o r d et er m i n a t i o n o f mebendazole and i t s m e t a b o l i t e s i n human plasma u sin g a r a p i d Sep Pak C18 e x t r a c t i o n . The methods elimin a t e t h e need f o r s o l v e n t e x t r a c t i o n a s such. One method i n c l u d e s i s o c r a t i c e l u t i o n and o t h e r , g r a d i e n t e l u t i o n . The g r a d i e n t e l u t i o n system p r o v i d es s u p e r i o r r e s o l u t i o n s Plasma samples ( 5 ml) was s p i k e w i t h major m e t a b o l i t e s o f mebendazole, methyl 5fa-hydroxybenzyl) -2-benzimidazole carbamate, 2-amino-5-benzoyl-benzimidazole and 2amino-5- (a-hydroxybenzyl) -benzimidazole and w i t h 5 . 0 pg o f ethyl-5-benzoylbenzimid a z o l e carbamate as i n t e r n a l s t a n d a r d i n t h e range o f 10 ng - 30 pg. Samples were a d j u s t e d t o pH 6 with d i l u t e h y d r o c h l o r i c a c i d o r sodium c a r b o n a te s o l u t i o n and e x t r a c t e d by p a s s i n g through a Sep Pak C18 c a r t r i d g e f i t t e d t o a l u e r lock g l a s s s y r i n g e . A f t e r t h e spikedplasma was p a sse d through t h e c a r t r i d g e , it was washed with 20 m l o f d i s t i l l e d w a t e r , 0 . 5 m l o f 40% methanol i n water and 0.4 m l of methanol. The n e x t 1 . 6 m l of methanol e l u t e d t h e f i v e compounds from c a r t r i d g e , t h i s 1 . 6 m l o f methanol was ev a p o r a t ed t o d r y n e ss i n

MEBENDAZOLE

315

p o i n t e d and t h e r e s i d u e was d i s s o l v e d i n 100 1-11 DMSO. A l i q u o t e s of 20 1~1were i n j e c t e d i n t o HPLC system. The i n s t r u m e n t used was A l t e x Model 322 MP HPLC ( A l t e x S c i e n t i f i c , Berkeley, CA, USA) equipped w i t h f i x e d wavelength d e t e c t o r (254 nm, 8-ul flow c e l l ) , a Rheodyne (Berkeley, CA, USA) Model 7120 i n j e c t o r and Hewlett-Packard (Avondale, PA. USA) Model 3380 A i n t e g r a t o r r e c o r d e r . For mebendazole t h e d e t e r m i n a t i o n limits a r e 20 ng/ml ( i s o c r a t i c system) and 10 ng/ml (gradient system). Chromatographic C o n d i t i o n s : Column

(250 x 4 . 6 mm I.D.) LiChrosorb, RP-8 10-ym reversed-phase packing. A precolumn (35 x 3 . 2 mm I.D.) dry packed with C o r a s i l C18 was a l s o used.

Mobile phase :

Methanol-water (55:45 pump A) and methanol-aqueous ammonium phosphate 0.05 M pH 5 . 5 (55:45 pump B)

.

Flow r a t e Pressure

:

1 . 7 ml/min. 1200-1300 p . s . i .

--

K a r l a g a n i s e t a 1 (42) developed an HPLC method f o r t h e d e t e r m i n a t i o n o f mebendazole i n b i o l o g i c a l samples. 2 M 1 plasma o f t h e p a t i e n t s was a l k a l i n i z e d w i t h 8 ml of sodium c a r b o n a t e b u f f e r (0.05 M, pH 11.3) and was e x t r a c t e d with 10 m l o f chloroform by shaking i n a g l a s s t u b e f o r 15 m i n u t e s . The c o n t e n t s were c e n t r i f u g e d and aqueous phase was a s p i r a t e d and t h e chloroform l a y e r was evaporated under reduced p r e s s u r e . The r e s i d u e was d i s s o l v e d i n 200 1~1mobile phase, 150 1-11 were i n j e c t e d . A n a l y s i s u s i n g Waters Model M6000A High-pressure Liquid Chromatograph equipped w i t h Wisp Model 710 i n j e c t o r and a Perkin-Elmer W d e t e c t o r Model LC55.

A. A. AL-BADR AND M. TARIQ

316

Chromatographic Condi tions: Column

:

25 cm x 3 mm ( i . d . ) LiChrosorb S1 60 5 m .

Mobile phase :

Acetonitrile/water-saturated ch 1o r o f orm/2 5% (weight / volume) ammonia i n water (75/92.5/0.1, by volume) , pH 6 . 5 .

Flow r a t e

:

0 . 8 ml/min

Pressure

:

400 p s i

Column t e mpe ra ture

:

Ambient

De t e c tor wavelength

:

307 nm

Recorder

:

5 mV (0.02 a b s o r p t i o n u n i t s full scale).

C a l i b r a t i o n curve was pr epar ed u s i n g 4 0 , 80, 200 and 400 ng of mebendazole. 1 Mg o f c ic lobe nda z ole was used as a i n t e r n a l s t a n d a r d , d e t e c t i o n r a t e i n t h e range o f 6.117 ng/ml. Alton -e t a1 (43) developed a simple, r a p i d and s p e c i f i c HPLC method €or q u a n t i t a t i v e d e t e r m i n a t i o n of mebendazole i n plasma. 2 M 1 o f plasma was d i l u t e d w i t h 2 n i l o f 0.05 M potassium hydrogen phosphate b u f f e r (pH 7.0) and e x t r a c t e d w i t h 7 m l of e t h y l a c e t a t e . The o r g a n i c l a y e r was removed and d r i e d , t h e r e s i d u e was d i s s o l v e d i n 0.001 N HC1 and r e - e x t r a c t e d w i t h 6 m l o f petroleum e t h e r and c e n t r i f u g e d , s u p e r n a t e n t was d i s c a r d e d t h e aqueous f r a c t i o n was a l k a l i n i z e d w i t h 2 m l 0 . 0 1 N NaOH and e x t r a c t e d t w i c e with e t h y l acetate. The s o l v e n t was d r i e d and d i s s o l v e d i n 60 ~1 of e t h y l acetate and 20 p1 was i n j e c t e d i n chromatograph (Model ALC 202/204 Waters Associates) f o r analysis.

MEBENDAZOLE

317

Chromatographic C o n d i t i o n s : Column

:

u

Bondapak C-18 300 x 3 . 0 mm ( i .d . ) reversed-phase .

Mobile phase :

0.05 M KH2P04-NaOH b u f f e r (pH 6 . 0 ) - a c e t o n i t r i l e ( 7 3 : 2 7 v/v).

Flow r a t e

:

2 . 5 ml/min

Pressure

:

2500 p s i

Column temperature

:

Ambient

Detector wavelength

:

UV (313 nm).

Flubendazole was used as i n t e r n a l s t a n d a r d . i n t h e q u a n t i t y o f 0.15 ug/ml f o r e a c h sample. T h i s method can be used t o measure plasma mebendazole l e v e l as low as 10 ng/ml. A simple and r a p i d method t o d e t e c t t h e presence of d i f f e r e n t benzimidazole compounds i n drug p r e p a r a t i o n s have been r e p o r t e d ( 4 4 ) . The procedure i n v o l v e s two d i f f e r e n t chromatography systems: i-

Reversed-phase g r a d i e n t .

ii- Normal p a r t i t i o n chromatography w i t h isocratic elution. Three d i f f e r e n t m i x t u r e s A , B , and C were used. The benzimidazoles were d i s s o l v e d i n p u r e formic a c i d (5 ml) t h e n i n c o n c e n t r a t e d h y d r o c h l o r i c a c i d (5 ml) and i n 50% (%) aqueous e t h a n o l (90 m l ) . Mixtures A and C c o n t a i n mebendazole. The s o l u t i o n s A and B were s e p a r a t e d by r e v e r s e d - p h a s e g r a d i e n t e l u t i o n with t h e solvent containing aceton i t r i l e - aqueous 1%HzS04 w i t h t h e programme: 10% a c e t o n i t r i l e f o r 2 minutes t h e n from 10% a c e t o n i t r i l e t o 30% a t a r a t e of 5% p e r minute. Mixture C was e l u t e d

A. A. AL-BADR AND M. TARIQ

318

i s o c r a t i c a l l y on a amino phase chemically bonded t o s i l i c a g e l (Type NH2) w i t h t h e s o l v e n t : methylene c h l o r i d e + i s o p r o p y l a l c o h o l (90 + 10) - a c e t o n i t r i l e ( 5 0 : 5 0 ) . I n i t i a l l y f o r d e t e r m i n a t i o n of r e t e n t i o n time each of t h e benzimidazoleswas s e p a r a t e l y chromatographed. Chromatographic Condit ions: Chromatograph :

Varian LC 8500 HPLC w i t h UV d e t e c t o r .

Column

LiChrosorb RP 8 - microp a r t i c u l a t e (15 cm 4 . 7 mm i .d . ) LiChrosorb NH2 (Merck) (15 cm x 4.7 mm i . d . )

Mobile phase : Methylene c h l o r i d e + i s o propyl a l c o h o l (90 + 10) a c e t o n i t r i l e (5O:SO). Flow r a t e

:

80 m l h - l

De te c t or sensitivity

:

0 . 5 AUFS

Column press ure De te c t or wavelength

-

500 p s i :

(UV) 254 nm

This method would be u s e f u l i n t h e q u a l i t y c o n t r o l of raw m a t e r i a l s i n t h e q u a n t i f i c a t i o n o f benzimidazoles i n f o o d s t u f f s of animal o r i g i n , and i n t h e d e t e c t i o n of residues. A simpl e , a c c u r a t e and r e p r o d u c i b l e HPLC

method h a s been d e s c r i b e d (45) f o r deter minat i o n of mebendazole i n t a b l e t s . 20 T a b l e t s were t a k e n and ground i n f i n e powder, e q u i v a l e n t t o about 10 - 20 mg of mebendazole was a c c u r a t e l y measured o u t €or each d e t e r m i n a t i o n . The powder was e x t r a c t e d wi th 5 m l o f formic a c i d . 5 M 1 o f s a l i c y l a mide s o l u t i o n ( 8 . 5 mg/ml) was added t o each

MEBENDAZOLE

319

e x t r a c t as i n t e r n a l s t a n d a r d . The m i x t u r e was c e n t r i f u g e d and t h e s u p e r n a t e n t was used f o r HPLC d e t e r m i n a t i o n . Chromatographic C o n d i t i o n s : Chromatograph:

Waters A s s o c i a t e s Model 440

Column

u

:

Bondapak C-18

Mobile phase :

T e t r a h y d r o f u r a n - 0 . 5 % formic acid (30:60).

Flow r a t e

:

1 . 9 ml/min.

Temperature

:

25-28OC.

Pressure

:

1500 p s i .

Detect o r wavelength

:

(UV) 254 nm

The r e c o v e r y was between 99.58 - 100.15%. An HPLC method f o r t h e e s t i m a t i o n mebendazole,

i t s prodrug, 4-arnin0-3-(3~-methoxycarbonyl 2 -thioureido)benzophenone , and t h e i r known o r expected m e t a b o l i t e s and d e g r a d a t i o n p r o d u c t s i n aqueous media and r a t blood was developed ( 4 6 ) . After o r a l a d m i n i s t r a t i o n o f t h e p r o d r u g , t h e prodrug was r a p i d l y c o n v e r t e d t o mebendazole and t h e area under t h e blood level vs time c u r v e o f mebendazole, i n rats dosed w i t h p r o d r u g , was more t h a n twice t h a t o b t a i n e d a f t e r g i v i n g rats an equimolar amount o f mebendazole. Only t h e p r o d r u g , mebendazole and known m e t a b o l i t e s o f mebendazole were d e t e c t e d i n r a t s g i v e n t h e prodrug.

Behm et a1 (47) have measured t h e c o n c e n t r a t i o n o f mebendazole and i t s major m e t a b o l i t e s by HPLC i n s h e e p plasma a t 12.5, 25, 50 o r 100 mg/kg. A t 1 2 . 5 mg/kg t h e peak plasma c o n c e n t r a t i o n s occured between n i n e and 24 h o u r s f o r a l l dose r a t e s and d e c l i n e r a p i d l y . Two major m e t a b o l i t e s were d e t e c t e d ; t h e i r

A. A. AL-BADR AND M. TARIQ

320

c o n c e n t r a t i o n s exceeded t h a t o f mebendazole a t a l l dose r a t e s . 8.5

P o l a r o g r ap h i c Method P i n z a u t i -e t a1 (48) d e s c r i b e d a d i r e c t - c u r r e n t p o l a r o g r ap h i c r ed u c t i o n method o f mebendazole a t t h e dropping-mercury e l e c t r o d e , and e s t a b l i s h e d t h e optimum c o n d i t i o n s f o r t h e d ete r m in a tio n o f t h i s drug i n dosage form. S i x t a b l e t s o f mebendazole (about 0 . 3 g) were ground. A q u a n t i t y o f t h e powder e q u i v a l e n t t o 50 mg o f mebendazole was t r a n s f e r r e d t o 100 m l v o l u m e t r i c f l a s k . To t h i s powder 8.6 m l o f 70% p e r c h l o r i c a c i d was added and t h e m ix tu r e s t i r r e d f o r 10 minutes and d i l u t e d t o volume w i t h water. The suspension was f i l t e r e d under s u c t i o n through a f i n e p o r s i t y g l a s s c r u c i b l e . 2 M1 p o r t i o n of t h e f i l t r a t e was t r a n s f e r r e d t o a 50 m l volumetric f l a s k , 3 m l o f 1 M p e r c h l o r i c a c i d was added and t h e s o l u t i o n d i l u t e d t o volume w ith t h e McIlvaine b u f f e r pH 2 . 6 (2.18 v o l . 0.2 M sodium phosphate with 17.82 v o l . 0 . 1 M c i t r i c a c i d ) . A 20 m l of t h i s s o l u t i o n (corresponding t o 0 . 4 mg

o f mebendazole) was t r a n s f e r r e d i n t o p o l a r o g r a p h i c c e l l (Metrohm E506 r e c o r d i n g polarograph equipped w i t h polarography s t a n d ) .

Deaeration and polarography were performed on u s i n g Metrohm EA 290 hanging mercury drop system. The number of e l e c t r o n s involved i n t h e r e d u c t i o n was c a l c u l a t e d a t a mebendazole c o n c e n t r a t i o n o f 1 x 10-4 M u s i n g a s o l i d s t a t e c o n t r o l p o t e n t i a l c o u l o m e t r i c a p p a r a t u s . A c a l i b r a t i o n curve was p r e p a r e d u s i n g 5 t o 50 pg/ml mebendazole s o l u t i o n p r e p a r ed i n McIlvaine b u f f e r , u s i n g t h i s method mebendazole a n a l y s i s c o u l d - b e performed with a l i m i t of d e t e c t i o n 100 ng/ml ( 4 8 ) . 8.6

Radioimmunoassay Method M i c h i e l s -e t a 1 (49) developed an e x tr e m e ly h i g h l y s p e c i f i c radioimmunoassay procedure f o r t h e d e t e r m i n at i o n o f mebendazole i n plasma. Mebendazole was converted t o methyl [5-[4-(2-aminoethyl) ben zoy 11-1H-ben zimidazol - 2- y l ] carbamate T h is hapten was coupled t o bovine serum albumin u s i n g a water s o l u b l e carbodiimide as f o llo w s:

.

MEBEND AZOLE

321

~

T

NHH

-

0

0

ICI -0-

C H ~

II

CH3-C-NH-CH2CH2

N

HC1, 1 N

a!a7 H

H 2 NCH2CH2

0

It

NH-C-0-CH3

1. DMF/MeOH

carbodi imide /HC1

2 . Bovine Serum

Albumin (BSA).

H

0 I.

0

II

BSA-C-HN-CH2-CH2

Synthesis of the hapten and the mebendazole-protein conjugate used f o r the immunization of the rabbits (49).

322

A. A. AL-BADR AND M. TARIQ

The r e s u l t i n g co n j cg a t e was used t o immunize r a b b i t s according t o co n v e n t i o n a l p r o c e d u r e s. The a n t i serum e l i c i t e d i n t h i s way was t e s t e d f o r i t s a b i l i t y t o b i n d s p e c i f i c a l l y mebendazole. Blood was c o l l e c t e d on EDTA and plasma was o b ta in e d a f t e r c e n t r i f u g a t i o n of t h e blood a t 2200 g f o r 15 minutes. Unmetabolized mebendazole was measured by d i r e c t radioimmunoassay u s in g a n t i - b o d i e s . Under t h e s t a n d a r d i z e d a s s a y c o n d i t i o n , t h e r a b b i t serum bound s p e c i f i c a l l y n e a r l y 50% o f added 0 . 2 ng H3-Mebendazole a t 1/100 serum d i l u t i o n . Un sp e c if ic a d s o r p t i o n t o plasma c o n s t i t u e n t s d i d n o t exceed 2 % . The method is q u i t e s e n s i t i v e t o a s s a y mebendazole i n plasma a t c o n c e n t r a t i o n as low a s 100 picogram/ml. M et ab o l i t es d i d n o t i n t e r f e r e with t h e binding of parent drug t o antibodies. Acknowledeements The a u t h o r s would l i k e t o Thank M r . Syed R a f a t u l l a h , A s s i s t a n t Researcher, f o r h i s t e c h n i c a l a s s i s t a n c e and Mr. T a n v i r A . Butt f o r h i s s e c r e t a r i a l s e r v i c e s .

MEBENDAZOLE

323

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91, 582 (1979).

2.

W.C. Bowman and M . J . Rand, "Textbook o f Pharmacology", 2nd Ed. Blackwell S c i e n t i f i c P u b l i c a t i o n s , Oxford, page 374 (1980).

3.

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5.

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6.

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8.

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10.

M a r t i n d a l e , The E x t r a Pharmacopoeia, 28th Ed. Pharmaceutical Press, London, page 98 (1982).

11*

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12.

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13.

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16.

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17.

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20.

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21.

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1,

.--

METOCLOPRAMIDE HYDROCHLORIDE

Davide P i t r g and R i c c a r d o S t r a d i F a c u l t y o f Pharmacy, U n i v e r s i t y o f Milan

1.

Description 1.1 Nomenclature 1.2 Chemical names 1.3 G e n e r i c names 1.4 Trade names 1.5 Formula and Molecular Weight 1.6 Appearance, Color, Odor and Taste

2.

Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance S p e c t r a

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

321

Copyright 01987 by Academic Press, Inc. All rights of reproduction in any form reserved.

DAVIDE PITRe AND RICCARDO STRADI

328

2.2.1 2.2.2 2.3 2.4 2.5

2.6 2.7

2.8 2.9 2.10 2.11 2.12 3.

'H-NMR 13

C-NMR Ultraviolet Spectrum Mass Spectrum Fluorescence Spectrum X-Ray, Powder Diffraction Crystal Structure Melting Range Solubility PKa PH Partition Coefficient

Manufacturing Procedures Synthesis

3.1 4.

Stability

5.

Metabolism and Pharmacokinetics 5.1 Metabolism 5.2 Pharmacokinetics 5.3 Acute Toxicity

6.

Methods of Analysis Elementa1 6.1 Identification Tests 6.2 Nonaqueous Titration 6.3 Complexometric Analysis 6.4 Colorimetric Analysis 6.5 Ultraviolet Spectrophotometric Analysis 6.6 Fluorimetric Analysis 6.7 Paper Chromatography 6.8 Thin-Layer Chromatography 6.9 High Performance Thin-Layer Chromatography 6.10 Gas Liquid Chromatography 6.11 High Performance Liquid Chromatography 6.12

7.

Determination in Body Fluids and Tissues

8.

References

METOCLOPRAMIDE HYDROCHLORIDE

1.

Description

1.1

Nomenclature

1.2

Chemical Names

329

Benzamide, 4-amino-5-chloro-N-[2-(diethylamino) ethyl 3-2-methoxy, monohydrochloride, rnonohydrate CAS 54143-57-6 anhydrous CAS 7831-21-5 4-Amino-5-chloro-N-[2(diethylamino)ethyllo-anisasamide

1.3

Generic Names Metoclopramide Hydrochloride USAN-BP 1980-F.U.IX Metoclopramidhydrochloride DAC 1979 Metoclopramidum hydrochloricum 2.AB-DDR

1.4

Trade Names Cerucal (Arzneimittelwerk-Dresden) Elieten (Nippon Kayaku, Tokyo-Japan) Emperal (Neofarma-Helsinki) Maxeran ( Delagrange-Paris) Plasil ( Lepetit-Milan) Peraprin (Taiyo, Gifuken-Japan) Primperan (Delagrange-Paris) Reglan (Robins, Richmond, USA)

DAVIDE PITRh AND RICCARDO STRADI

330

1.5

Formula and Molecular Weight

C0 NH I

- CH-2

CH2-N

OCH3 .HCI * H20

\ c2

CI

NHp C

1.6

H C1N 0 . H C l . H 0 1 4 22 3 2 2

Mol.Wt.

= 354.3

Appearance, C o l o r , Odor, Taste

A w h i t e o r a l m o s t w h i t e c r y s t a l l i n e powder, odorless (1)

33 1

METOCLOPRAMIDE HYDROCHLORIDE

2.

Physical Properties

2.1

I n f r a r e d Spectrum

The i n f r a r e d s p e c t r u m of rnetocloprarnide hydrochlor i d e , i s p r e s e n t e d i n F i g . 1. The spectrum was r e c o r d e d w i t h a s o l i d sample d i s c composed of 1 m g o f compound/200 m g KBr on a P e r k i n E l m e r Model 1310. -1 The f o l l o w i n g bands (cm ) were a s s i g n e d and a r e r e p o r t e d i n Table 1. Table 1

Wave Number

Assignments

,

3200, 3300, 3340, 3400, 3460

VNH

2860, 2950, 2980

VC

H SP3

3030

VC

H SP2

2100, 2500, 2660, 2710

v N+H

1600

v c=o

1540

6NH

1270

v C-0-C

700

v c-c1

VOH

(Amide) (Asymmetric)

Fig. 1

-

I n f r a r e d Spectrum of Metocloprarnide Hydrochloride

333

METOCLOPRAMIDE HYDROCHLORIDE

2.2

Nuclear Magnetic Resonance S p e c t r a

2.2.1

1 H-NMR 1 H-NMR

spectrum o f metoclopramide h y d r o c h l o r i d e , shown i n F i g . 2 , was obtained i n DMSO-d with a Varian spec6 trometer EM-390 o p e r a t i n g a t 90 MHz. The chemical s h i f t s a r e l i s t e d i n t h e Table 2. Table 2

Moltepli city

Number of protons

Assignments

~

1.33

t

6

N-(CH2-CH -3

)

3.0-3.2

m

6

CH -N-(CH -2 -2

-CH

3.72

9

2

NH-CH -2

3.95

S

3

OCH

6.03

broad s

2 ,exch.

NH

6.60

s

1

H~

(arom.)

7.72

S

1

H

(arorn.)

8.44

t

1,exch.

CONH

10.85

broad s

1,exch.

hH

3

2

6

2 ) 3 2

Fig. 2

-

1 H-NMR (90 MHz) Spectrum of Metocloprarnide h y d r o c h l o r i d e i n DMSO-d

6

335

METOCLOPRAMIDE HYDROCHLORIDE 2.2.2

13

C-NMR

13 The C-NMR spectrum o f metoclopramide hydrochlor i d e i s shown i n F i g . 3. The spectrum was r e c o r d e d i n D 0 2 w i t h Varian XL.200 s p e c t r o m e t e r o p e r a t i n g a t 50.391 MHz. The chemical s h i f t s a r e l i s t e d i n Table 3. Table 3

Line

(p.p.m)

Mu1 t i p 1i c i t y

Assignments

N.

N(CH2.CH ) -3 2

1

8.38

q

2

35.15

t

3

48.27

t

N(CH -CH ) -2 3 2

4

51.40

t

NH.CH

5

55.89

q

OCH

6

98.00

d

c3

7

109.18

S

C

8

110.61

S

C

9

131.43

d

10

148.57

S

11

158.07

S

12

167.46

S

2.3

CH -N(C2H512

-2

-2

1

5

3 (aromatic) or C or C

5

1

‘6

C

4

c2

c=o

U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum of metoclopramide hydro-5 c h l o r i d e i n w a t e r ( c = 5.763 10 m/l) was o b t a i n e d (60) with a Beckman Mod. 24 s p e c t r o p h o t o m e t e r and i t is shown i n Fig. 4

i

I

8

-

-

. . '

!

1 Fig. 3

jl I

:

i

i

I h

13 H-NMR

(90 MHz) Spectrum of Metoclopramide hydrochloride in DMSO-d 6

- i'

METOCLOPRAMIDE HYDROCHLORIDE

i

Fig. 4

!

-

,

Ultraviolet Spectrum of Metoclopramide hydrochloride in Water

331

DAVIDE PITRG AND RICCARDO STRADI

338

Mass Spectrum

2.4

The mass spectrum of metoclopramide h y d r o c h l o r i d e was recorded ( F i g . 5 ) on Finnigan 1020 s p e c t r o m e t e r by c o w e n t i o n a l e l e c t r o n impact i o n i s a t i o n a t 70 eV. Prominent fragments and t h e i r r e l a t i v e i n t e n s i t y a r e shown i n Table 4. Table 4

m/e

Intensity

229

0.23

227

0.90

Attribution

~

+

201

7.60

181

6.43

169

0.66

141

1.78

99

27.96

86

100

+

c1QCH3NH2

4fCH3 J$ro

c1

0

II

NH2

c1

-0"'

NH2

c1

CH

+/CZHS

-N

2- \C2H5

METOCLOPRAMIDE HYDROCHLORIDE

339 Ell% N.E:

DulR: STRW16 (72

'108 t 2128

RlCi

n.w.02.cL.

96

367616.

173712.

1W.Q

10.

50.1

99

WE

mss S P E C T M

Wrn.'86 9zs7;oo

t

M n : SIFWI6 172

2128

B P X M'E: P6 PIC: 167616.

WREi Cll.H22.tI3.02.cL. 188.1

se. I

270

i'

272

I M

Fig. 5

281

-

2Qe""'"-

285 ~

"

26

"

29s "

'

Electron impart Mass Spectrum of Monocloprarnide hydrochloride

m

DAVIDE PITRB AND RICCARDO STRADI

340

Fluorescence Spectrum

2.5

A solution of metoclopramide hydrochloride in water exhibits fluorescence when excited with ultraviolet light. When excited at 353 nm, it shows a sharp peak at 353 nm and a second peak at about 273-276 nm. The emission spectrum of the compound consists of a single peak at 355 nm. The spectra obtained in water (5 mcg/ml), on a spectrophotometer Perkin-Elmer MPE 44A,are given in Fig.6. X-Ray Powder Diffraction

2.6

The X-ray powder diffraction data of metoclopramide hydrochloride was determined (2) by a Philips Powder Diffractometer PW 1710 with nichel-filtered copper radiation (1.54051 A) with the following instrumental conditions: TUBE: BF type, CU/Ni, 40 KV, 40 mA; SLITS: lo-0.1 mm-lo; DETE TOR:PW1711 proportional counter + discriminator; SCALE: 2x10 cps; TIME CONSTANT: 1"; SCANNING SPEED: 0.008°xl'1; PAPER SPEED 1 cmxlo; SPECIMEN HOLDER: Niskanen. The X-ray diffraction data are given in Table 5.

2

METOCLOPRAMIDE HYDROCHLORIDE

Fig. 6

-

F l u o r e s c e n c e Spectrum of Metoclopramide hydrochloride i n water

341

DAVIDE PITRB AND RICCARDO STRADI

342

Table 5

N.

d(A)*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

9.23 8.46 7.64 6.98 6.64 6.28 6.02 5.26 5.18 4.87 4.71 4.60 4.48 4.27 4.23 4.03 3.95 3.82 3.78 3.71 362 3.50 3.47 3.41 3.36 3.32

I&** 5 25 20 22 13 65 8 20 10 38 45 30 31 10 20 20 6 47 35 17 10 58 30 75 100 11

I&*+

N. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 50 51 52

3.15 3.07 3.04 2.99 2.95 2.92 2.86 2.82 2.78 2.74 2.69 2.59 2.53 2.51 2.43 2.33 2.31 2.29 2.26 2.16 2.14 2.05 2.01 1.81 1.81 1.71 1.63

12 8 6 11 12 10 20 13 8 21 11 10 9 13 18 10 8 10 11 11 10 8 9 8 8 7 9

PLUS OTHER LINES c5 Interplanar distances = 1.54051/2sin diam.

**

Relative intensities are based on highest intensity of 100

METOCLOPRAMIDE HYDROCHLORIDE

343

Crystal Structure

2.7

The metoclopramide hydrochloride monohydrate is monoclinic, space group P2 /n with a = 12.138(3), b = 1 8.553(2), c = 17.120(4) A and p= 98.06(7) Z = 4 . The structure was solved by direct methods and refined to R = 0.055 (3). The metoclopramide free base ( C A S 364-62-5) is triclinic, space group P1 with a = 13.310 b = 8.728, c=7.477 A , a = 114.20, p = 81.13 and Y = 101.23O, Z = 2. A study of structure-activity relations shows an analogy between metoclopramide and apomorphine(4)

.

Melting Range

2.8

Employing the USP method the metoclopramide monohydrochloride monohydrate melts at 182-185O(5) and with terma1 microscopy the melting begins at 181O and completes at 183O(6). The dihydrochloride monohydrate melts in the range of 148O with decomposition. The melting points f o r metoclopramide free base are about 148O(7) or m.p. 146-148O(8). For dihydrochloride is also reported an eutectic melting point of 160° with phenacetin and of 117O with benzanilide(7). Solubility

2.9

At 25O metoclopramide monohydrochloride is soluble in 0.7 g of water, 3 g of ethanol (96 per cent) and 55 g of chloroform, whereas it is practically insoluble in ether (10). Solubility of the base and of dihydrochloride are reported in Table (6). (11). Table Solubility of metocloprarnide g/100 m l at 25O ~~

Solvent

Base

Water Ethanol 95 Ethanol Benzene Chloroform

0.02 2.90

1.90 0.10 6.60

Dihydrochloride 48 9 6 0.10 0.10

DAVIDE PITRfi AND RICCARDO STRADI

344

2.10

€5

Metoclopramide hydrochloride shows two ionisation costants; pic = 9.71 and pK = 0.42. The determination was 1 carried out spectrometricalfy in aqueous solution and the values are a mean of 16 determinations with a standard deviation of 0.03 and 0.02 (12). 2.11

pH Range

The pH of a 10% water solution must be between 4.6 and 6.5 according to BP 1980. 2.12

Partition Coefficient

The partition coefficient in octanol/water has been determined(l3) by reversed-phase HPLC at 20°. The hydrochloride gives Experimental LogP = 2,667 Calculated from R.A. Deckker (14) LogP = 2.76

METOCLOPRAMIDE HYDROCHLORIDE

3.

Manufacturing Procedures

3.2

Synthesis

345

Among the synthetic methods leading to metoclopramide (I) it seems suitable to report only the ones of greatest interest that are those using p-aminosalicilic acid (II), as starting product, a compound whose properties are economically excellent. The two main processes are listed in Fig 7. In Scheme A, referring to the first invention(l5), the acid (1I)is acetylated to (111) and then treated with methyl sulfate to give the ether-ester (IV). This compound is reacted with (C H ) N-CH CH -NH to the corresponding amide. The 25 2 2 2 subsequent Zesacetylation leads to metoclopramide ( 1). In scheme B the first step in the synthesis is the etherester (VI). The protection of the amino aromatic group was not considered necessary (16). The direct chlorination (18) of (VI) with sodium ipochloride gives (VII) which is transformed in excellent yields into (I). 4.

Stabi1ity Aqueous solution

The maximum stability of metoclopramide, investigated at different pH values, was found at pH = 7.6 while the maximum degradation was found at pH = 2. One of the degradation products was identified as N,N-diethylethylendiamine(l7).

Scheme

A

FOOH

FOOCH3

COOCH3

COOCH3

FOOCH3

COOH

I

60 b

Fig. 7

-

Synthesis of Metoclopramide

METOCLOPRAMIDE HYDROCHLORIDE

341

5.

Metabolism and Pharmacokinetics

5.1

Metabolism

The metabolites identified in the in vitro and in vivo studies are listed in Table 7, in which metoclopramide is indicated as (I). Table 7 Non conjugated metabolites found

CI @ O R 2 NH2 N.

R

R

1

2

-NHCH CH N(C H ) 2 2 252

-CH

-NHCH CH NHC H 2 2 2 5

-C H

-NHCH CH NH 2 2 2

-C Hg

-OH

-CH3

V

-NHCH CH N(C H ) 2 2 2 5 2

-H

VI

-NHCH CH N(C2H512 2 2

-H

-NH 2

-CH

VIII

-NHCH COOH 2

-C H

IX

-NHCH = CH

-CH

-NHCH CH NHCOCH 2 2 3

-CH

I I1 I11 IV

VII

X

3 3

3

2

3 3

3

REFERENCES

348

DAVIDE PITRe AND RICCARDO STRADI

By studies of the in vitro metabolism of (I) using hepatic fractions from several animal species (1) (2) five metabolites (11) (111) (IV) (V) (VI) were detected. Identification studies of the metabolites in urinary excretes of rat and dog (20) after enzimatic hydrolysis evidenced some compounds already known from the in vitro studies and some new ones corresponding to (VII) (VIII) IX) (X). In particular in rat urine, were found metabolites (11) (IV) (V) (VII) (VIII), while in dog urine were present metabolites (V) (VI) (VIII) (IX) (X). In human urine the excretion was found to be 60% in the first 24 hours. After enzimatic hydrolisis was identified metabolite (VIII)(CAS 52702.04.2), but the main metabolite was found to be unchanged metoclopramide originated from the sulfate and glucuronide. Metabolic products were quantified in rabbit urines (21) (Ii) 4 and (IV) but also unchanged (I),as N -glucoronide and N sulfonate were identified. Another product of N-oxidation of 4 (I) was also found. Metoclopramide N -sulfonate (CAS 2726042.0) is the main metabolite in human urine. Pharmacokinetic

5.2

14

The studies of pharmacokinetics of C-metoclopramide in rats, dogs and humans show that the drug is distributed within a few minutes after oral administration and it is eliminated mainly in the urine of the first 24 hrs. (20). Table 8 14 Excretion of C-metoclopramide in rats, dogs, and humans

Dosage p.0. m g k hours

Rats

Dogs

U =

humans

U

F

U

71.9 8.5 1.0

5.7 4.4 1.7

65.3

----

77.8

6.9 1.o

18.3 0.8

8.7 0.8

r

0-24 24-48 48-72

10

20

100

urinary excr. dose

F

=

F

U

F 20

1.0 1.6

fecal excr. % of the administered

METOCLOPRAMIDE HYDROCHLORIDE

349

The pharriiacokinetic of metoclopramide H C 1 was s t u d i e d i n normal male v o l u n t e e r s a f t e r i.v. dosage o f 10 mg. The shape o f t h e plasma l e v e l c u r v e i n t h e s e s u b j e c t s f i t s a two-compartment model w i t h a d i s t r i b u t i o n o f T 1 a = 4.6 p = 165.7 min./2The body min. and a n e l i m i n a t i o n o f T .I/? plasma c l e a r a n c e was 10.9 ml/min g ( 2 2 ) ( 2 3 ) . G. B e r n e r and co-workers ( 2 4 ) r e p o r t e d t h e s t u d y o f b i o a v a i l a b i l i t y of commercial f o r m u l a t i o n s o f metoclopramide i n male h e a l t h y v o l u n t e e r s . The f o l l o w i n g r e s u l t s were o b t a i n e d : f o r t a b l e t s 57.6-80.3%, f o r s o l u t i o n s 49.7-76.7% and f o r c a p s u l e s 54.8%. Acute T o x i c i t y

5.3

(LD ) of t h e metoclopraThe a c u t e t o x i c i t y i . v . 50 mide d i h y d r o c h l o r i d e is 85.7 mg/kg f o r t h e r a t and 71 mg/kg f o r t h e mice ( 2 5 ) . 6.

Methods of A n a l y s i s

6.1

E l e m e n t a l Composition

H , 7.12; C 1 , 20.0; N , 11.9; H 0 , 5.1 f o r t h e 2 monohydrate C , 50.01; H , 6 . 8 9 ; C 1 , 21.08; N, 1 2 . 5 ; 0, 9.50 f o r t h e a n h y d r o u s form C, 5 6 . 9 ; H , 7.40; C 1 , 1 1 . 8 3 ; N , 1 4 . 0 2 ; 0, 10.67 f o r t h e base. C, 47.4;

I d e n t i f i c a t i o n Tests

6.2

a)

I n f r a r e d Spectroscopic Test BP 1980 (10) c i t e s t h e u s e of t h e i n f r a r e d abs o r p t i o n spectrum o f a potassium c h l o r i d e d i s p e r s i o n o f t h e rnetoclopramide h y d r o c h l o r i d e i n a c c o r dance w i t h t h e r e f e r e n c e s p e c t r u m r e p r o d u c e d i n t h e appendix.

DAVIDE PITRk AND RICCARDO STRADI

350

b)

Ultraviolet Spectroscopic Test The light absorption, in the range 230 to 350 nm of a 2-cm layer of a 0.001 per cent w/v solution in 0.01M hydrochloric acid exhibits two maxima, at 273 nm and 303 nm; absorbance at 273 nm about 0.79 and at 309 nm about 0.69. This is another identification test recommended by B.P.1980 (10).

C)

Color Tests With a solution of 4-dimethylaminobenzaldehyde yellow-orange(1); ammonium vanadate test - light brown (sensitivity : 1.0 ug), (7). / Vitali's test: pale-yellow/light brown (sensitivity : 1.0 ug) (7). /

d)

Crystal Tests

-

Gold Cyanide solution needles, sometimes in rosettes (sensitivity: 1 in 1000). Lead iodide solution - feathery rosettes (sensitivity: 1 in 1000) (7).

6.3

Nonaqueous Titration The metocloropramide hydrochloride may be titrated

(10) potentiometrically in glacial acetic acid containing

mercuric acetate with perchloric acid in glacial acetic acid as titrant. 6.4

Complessometric Analysis

The method is ba ed on the use of a column of 5+ form. The released quantity, amberlite IR 120 in the Zn corresponding to the absorbed metoclopramide, is titrated with EDTA using Eriochromo Black T as indicator (26).

METOCLOPRAMIDE HYDROCHLORIDE

6.5

35 1

Colorimetric Analyses

The colorimetric assays have been the most widely used especially to determine metoclopramide in pharmaceutical preparations. They are listed in the following table 9. Table 9 Colorimetric assays of Metoclopramide

N.

max

Bear's law Ref. ,ug/ml

--

1

Citric acid in Ac 0

600

2-14

27

2

NH4SCN t Co (N03)2

620

--

28

3

Z

a , P-dinitrostilbene

390

--

29

4

DiazotizationtN(1-naphthyl)

525

2

0.4-4

30-11

ethylendiamine 5

HNO

6

Diazotization

to form the nitrous 2 derivative

375

10-60

31

+ a-Naphthyl-

510

0.1-11

32

+

500

1-14

33

475

20-120

34

+

495

1-11

35

NH Sulfamate 4 Diazotization +

440

0.8

36

amine 7

Diazotization

8

Dragendorff's

9

Diazotization

10

R-Salt

2,4-dihydroxybenzoic acid

DAVIDE PITRB AND RICCARDO STRADI

352

6.6

Ultraviolet Spectrophotometric Analysis

B.P 1980 (10) has described a spectrophotometric assay for the quantitative determination of metoclopramide tablets. After the extraction of the base in chloroform, the metoclopramide can be determined by measurement of absorbance at 305 nm. E(1%, lcm) = 265. 6.7

Fluorimetric Analysis

Fluorimetric measurements are carried out in pH=2 buffer solution on the basis of intense emission at 360 nm that occurs when the sample is exci ed at 310 nm. The me-1 thod, with detection limit of 3x10 ,ug ml , was successfully applied to analyses of pharmaceutical formulations with a recovery of 100.8-101.8% and a coefficient of variation of 0.9-1.9% (37).

-8

Chromatography 6.8

Paper Chromatography

Several chromatographic assays are summarized in the following table 10. Table 10 Solvent system 4.6 g of citric acid in 130 ml H 0 and 2 870 ml BuOH

Acetate buffer pH = 4.58 Phosphate buffer pH = 7.4 Toluene: Methanol : conc. NH40H

Paper

Detection U.V. Iodoplatinate spray

RfxlOO 45

II

77

II

39

1% of chloranyl in Bz

-

Ref.

METOCLOPRAMIDE HYDROCHLORIDE

353

Paper: A Whatman N 1, s h e e t 11 x 6 cm buffered by dipping i n a 5% s o l u t i o n o f sodium c i t r a t e , b l o t t i n g and drying a t 25O f o r 1 hour. B Whatman N 1 N 3, s h e e t 17x 19 cm impregnated by dipping i n a 10% s o l u t i o n of t r i b u t y r i n i n acetone and drying i n air. C Whatman N 1

-

-

-

6.9

Thin-Layer Chromatographx

The methods f o r s e p a r a t i o n and d e t e c t i o n o f metoclopramide a r e summirized i n t a b l e 11.

Table 11 Solvent system

I I1 III IV V VI VII VIII IX X XI XI1

Plate A,B C C

C C C C F C

C F

F

R f x 100 40 10 46 16 54 29 55

-

98 68 47 13

Reference

DAVIDE PITRfi AND RICCARDO STRADI

354

Solvent Sytem I Strong ammonia solution: methanol (1,5:100) I1 Methanol-chloroform (1:4) I11 1,2-Dichloroethane-ethanol-ammonia solution (sp.gr.0.88) (70: 15:2) IV n-Butanol-acetic acid-water (4:l:l) V Isopropanol-ammonia solution (sp.gr. 0.88) ( 80:4:5) VI VII VIII IX X

Butanol-acetic acid-water (4:l:l) Isopropanol-NH OH-water (80:4:5) 4 Dioxan-NH OH-benzene (8:l:l) 4 Chloroform-methanol-28% ammonia (10:4:1) Chloroform-methanol-dioxane-28% ammonia (90:14:10:3) XI Methanol-ammonia (100:1.5) XI1 Acetone

Detection

-

Acidified iodoplatinate spray - Spray solution: 1% solution sodium nitrite in HC1 N and then with 0.4% of N-(1-naphthy1)-ethylaminediammonium dichloride as coupling agent in methanol. - Spray solution: 0.2% solution of chloranil in acetonitrile followed by heating the plate at 100-105° for 2 min. - Spray solution: 2 g of iodic acid dissolved in 10 m l H20 and made up to volume of 100 m l with 90% (w/v) sulfuric acid (5). - Ultraviolet light (250 nm). Plate

A. B. C. D.8

E. F.

Glassplate, 20 x 20 cm coated with silica gel Pre-coated silica gel G (Merck) Silica1 gel 60 F (Merck) 254 GF Plate. Analtech (6) Kodak Silice Fluorescente K301 Silica dipped in 0.1N KOH and dried

METOCLOPRAMIDEHYDROCHLORIDE

6.10

355

High Performance Thin-Layer Chromatography

HPTLC was performed on metoclopramide base using NH OH HPTLC Fertiplatten Kiegelgel 6OF(Merck)and Me0H:CHCl 3: 4 25% (80:85:0.2) as solvent. The compound may be quantified by means of excitation fluorimetry at 312 nm and emission at 365 nm integrating the peak area with a videointegrator. The results are linear from 160 ug to 20 ug with a standard / / deviation of 2-5% (43). 6.11

Gas Liquid Chromatography

T. Daldrup and co-workers (44) described a GLC assay method on a glass-column (6 ft x 2 inc.) packed with 3% OV-1 on Chromosorb W-HP (100 to 120 mesh) and operated at -1 15Oo-25O0 (lO°C/min), with N as carrier gas (50 ml min ) 2 and a flame ionization detector. Internal standard: 2-amino-5-chlorobenzophenone. Detector and injection temperature were 350° and 250OC. Retention time: 1.87. GLC has been used as the method for the determination of the drug and its 15 metabolites in biological fluids (45) (46) (47) (48)and Y.K. Tamond, J . E . Axelson (49)have been determined the mctoclopramide in picogram quantities in plasma 63 with a Ni-electron capture detector. The procedure involved the extraction of the drug from an alkalinized aqueous solution in benzene and derivatization with heptafluorobutirric anhydride. The determination of the derivative was done using diazepam as internal standard. Working conditions: glass column (1.2 m x 2 inc. packed 3% OV-17, coated with 80-160 mesh, chromosorb W. Injection 2 5 8 O ; oven 250 and detection 350. A mixture of argon-methane (95:5) was employed with a flow -1 rate of 40 ml/min The retention times were 3.76 min for the metoclopramide and 9.1 min for diazepam.

.

356

DAVIDE PITRB AND RICCARDO STRADI

6.12

High-pressure Liquid Chromatography

-?

Teng et al.(l) used a column 15 cm x .5 mm packed with silica gel M 171; flow rate 2.0 ml/min and CH OH3 -CHC1 -NH OH conc. (30:70:0.5) at room temperature. Detect4 ion 280 nm. The retention times of metoclopramide is 2.8 min and 4.5 min for the internal standard (4-amino-5-chloro-ND- ( propylamino ethyl]-Z-me thoxybenz m i de ) W. Block et a1 ( 5 0 ) determined metoclopramide using reverse-phase HPLC with an RP-18 column under isocratic conditi n -4. (CH 0H:H 0:NH OH conc.,75:24.9:0.1). Flow rate 1.2 ml/min 3 2 4 Detection at 308 nm. Elution after 5 min.

.

7.

Determination of metoclopramide in Body Fluids and Tissues

The determination of metoclopramide in biological fluids of man and of various animal species is the object of many communications as reported below: Blood (Plasma, Serum) Colorimetry: (11) : ( 3 ) (41) (40) (52) HPLC: (24) TLC GC : (45) (46) (47) (53) (48) HPLC : (54) (55) (20) (50) (53) (56) (57) GC-Mass Spectrometry: (15) Urine

Colorimetry : (21) (52) TLC : (21) (40) (39) HPCL : (20) (56) GC : (54) (19) GLC-Mass Spectrometry: (58)

Saliva HPLC : ( 59)

METOCLOPRAMIDE HYDROCHLORIDE

Bile Colorimetry : (21)

Feces Radioactivity with 14C-metoclopramide : (20) Microsomal fraction of liver homogenate HPLC : (39)

357

DAVIDE PITRE AND RICCARDO STRADI

REFERENCES F.U. IX G. Liborio, Liiversity of Milan, Ins itute of Mineralogy, Personal Communication. N.M. Blaton, O.M.Peeters, C.J. Be Ranten Cryst. Struct.Comm. 9 , 857 (1980) M. Cesario, C. Pascard, M.E1 Moukhtari, L. Jung Eur.J.Med.Chem.-Chim.Ther. Is, 13 (1981) The Merck Index, IX Ed., 6109 J. Topard, M. Hanocq, Van Damme, L. Molle J.Pharm,Belg. 2, 633 (1973) E.G.L. Clarke, Isolation and Identification of Drugs The Pharmaceutical Press, London 1969 R. Pakula, K. Butkiewics, 2. Trojanowska, J. Ruszczak Arch.Pharm. (Weinheim) 313, 297 (1980) M. Kuhnert-Brandstatter, L. Bosch, G. Eckstein Sci.Pharm. 461, 54 (1978) B.P.1980. Vol. 1, pag. 273 G.P.Pite1, Th.Luce - Ann.Pharm. Fr. 23, 673 (1965) M. Manocq, J. Topart, M. Van Damme, L. Molle J.Pharm.Belg. 28, 649 (1975) N. Verbiese-Genard, M. Hanocq, M. VanDarnme, L. Molle 9, 265 (1981) Int. J. Pharm. R.F. Reckker, H.M. d e Kort 14, 479 Eur. J. Med.Chem.-Chim.Ther. -

-

( 1979)

SOC. d'Etudes Scientifique Industrielle de 1'Ile de France Fr. 1.407.055 M. Murakami, N. Inukai, A. Koda, N. Nakano Chem.Pharm.Bul1 (Tokyo) 19, 1696 (1971) T. Kaniewska, W. Wejman. Farm.Po1. 32, 927 (1976) A.H.Beckett, G. Huing J.Pharm.Pharmaco1. 27, Suppl. 24P (1975) D.A.Cowan, G. Huing, A.H.Beckett Xenobiotica 6, 605 (1976) L. Teng, R.B. Bruce, L.K. Dumming J.Pharm.Sci. 66, 1615 (1977) T. Arita, R. Hori, K. Ito, K. Ichikawa, T. Uesugi Chem.Pharm. Bull. (Tokyo), g , 1663 (1970)

-

METOCLOPRAMIDE HYDROCHLORIDE

359

D.N. Bateman, D.S. Davies, C. Kahn, K. Maskiter Br. J. Clin.Pharmaco1. 4, 6508 (1977) D.N. Bateman, C. Kahn, K. Mashiter, D.S. Davies Br. J. Clin.Pharmaco1. 5 , 401 (1978) G. Beiner, B.Emschermann, E. Glaottke, W. Haase, F. Leuschner, V. Vogtle-Junkert, H.H. Wagener Arzneim. Forsch. 32, 169 (1982) C. Agralo, D. Bulgach, H. Araldi, Jorndas Argent Toxicol. Anal. Actas 1st. 194 (Pub. 1972) 1, 214 - CA 79-73800 J. Jarzebinski, Z. Swajber, G. Klos Pharm. Pol. 34, 237 (1978) J. Emmanuel, T.V. Yogyanaranan East Pharm. 24, 133 (1981) CA 95-1758 8 9 k J. Dobrecky, R.J. Calleja Rev.Farm. (Buenos Aires), 115, 12 (1975) CA 80-100249k P. Dubois, J. Lacroix, P. Levillan, C. Vie1 J.Pharm. Belg. 36, 203 (19811, CA 95-121218~ D.M. Skingbal, K.V. Sawant Indian Drugs 19, 239 (1982) CA 96-223389s D.M. Shingbal, S.D. Naik Indian Durgs 18, 441 (1981) CA 96-24856s O.S.Kamalapurttar, R.S. Priolkar Indian Drugs 20, 108 (1982) CA 98-221907b R.T. Sane, A.Y. Okamantar, U.R. Pandit, A.B. Ambardaker Indian Drugs 20, 73 (1982) CA 98-78232v R.G. Bhatkar, K.V. Nagvenkar East Pharm. 26, 205 (1983) CA 99-164094a O.S. Kamalapurkar, J.J. Chudasama Indian Drugs 3, 298 (1983) CA 99-10952~ J. Emmanuel, S.D. Naik, N. Pradego Indian Drugs 20, 387 (1983) CA 99-181559q W. Balyens, P.De Moerloox Analist 103, 1356 (1978) A.M. Taka, M.A. Abd El-Kader J. Chromatog. 177,405 (1979) G. Huinzing, A. Beckett, J. Segura J. Chromatogr. 172, 227 (1979) O.M. Bakke, J. Segura J.Pharm.Pharmaco1. 28, 32 (1976) D. Schuppan, I. Schmidt, M. Heller Arzneim.Forsch. 9 , 157 (1979)

-

-

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DAVIDE PITRE AND RICCARDO STRADI

360

A.M. Stead, R. Gill, T. Wright, J.P. Gibbs, A.C.Moffat Analyst 107, 1106 (1982) 43 M. Prosek, M.Watre, I. Verbic Comput. suppl. Lab. 1, 223 (1986) T. Daldrup, F. Susanto, P. Michalke 441 Fresenius 2. Anal.Chem. 308, 413 (1981) 45) L.M. Ross-Lee, M.J. Eddie, F. Barier, W.D. Hooper, J.H. Tyrer J. Chromatogr. 183, 173 (1980) K.W. Riggs, J.E. Skelson, D.W. Rurak, D.I. Hasman, 46 B.McErlane, M. Bylsma-Havell, G.H. MacMorland, R.Dugley, J.D.E. Price J.Chromatogr. 276, 319 (1983) Y.K.Tam, J.E. Axelson, R. Ongley J.Pharm.Sci. 68, 1254 (1979) M. Stanley, D. Wazer Neuropsychobiology lo, 108 (1984) Y.K. Tam, J.E. Axelson J.Pharm. Sci. 67, 1073 (1978) W. Block, A . Pingoud, M. Khan, P. Kjeldrap Arzneim.Forsch. 2, 1041 (1981) G. Pitel, Th. Luce Ann.Pharm.Fr. 28, 595 (1970) T. Arita, R. Hori, K.Ito, M. Sckikawa Chem. Pharm. Bull. (Tokyo), 18, 1675 (1970) J.P. Leppard, E. Reid Methodol Surv Biochem. 3, 315 ( 19781, CA 90-66423~ W. Block, B. Pingoud Fresenius Z.Ana1. Chem. 301, 109 (1980) G.B. Bishop-Frendling, H. Vergin J.Chromatogr. 273, 453 (1983) J. Popovic Ther.Drug Monit. 5 , 77 (1984) J.L. Choen, G.M. Hisayasu, E. J. McDermed, S.B. Stram Pharm. Res. (1984) Y.K. Tam, J.E. Axelson J .Pharm.Sci. 67, 1073 (1978) R.P. Kapil, J.E. Axelson, R. Ongley, J.D.E. Price J.Pharm.Sci. 2, 215 (1983) C. Bugatti. Faculty of Pharmacy, University of Milan, Personal Communication 42

-

.

.

-

MITOMYCIN C J o s H. Beijnen Slotervaart HospitalfNetherlands Cancer Institute Amsterdam, The Netherlands

Auke Bult and Willy J.M. Underberg Faculty of Pharmacy, Department of Pharmaceutical Analysis State University of Utrecht Utrecht, The Netherlands 1.

2.

3. 4.

5.

6.

7.

8.

Historv Description 2.1 Name, Formula, Molecular Weight 2.2 Appearance, Colour, Odour Biosynthesis, Chemical Synthesis Physical Properties 4 . 1 Ultraviolet-Visible Spectrum 4 . 2 Infrared Spectrum 4.3 Nuclear Magnetic Resonance (NMR) Spectrum 4.4 Mass Spectrum 4.5 Circular Dichroism (CD) Spectrum 4.6 Optical Rotation 4.7 Melting Point 4.8 Differential Scanning Calorimetry 4 . 9 Solubility 4.10 Partition Coefficient 4.11 Dissociation Constants 4.12 Electrochemistry Methods of Analysis 5 . 1 Elemental Analysis 5.2 Ultraviolet Spectrophotometry 5 . 3 Thin Layer and Paper Chromatography 5 . 4 Gel Filtration Chromatography 5.5 High Performance Liquid Chromatography 5.6 Identification and Purity Tests in Official Compendia Stability, Degradation 6.1 Stability in Aqueous Solutions 6.2 Stability in Pharmaceutical Formulations 6 . 3 Stability in Biological Media Pharmacology 7 . 1 Mechanism of Action 7 . 2 Clinical Antiturnour Activity 7 . 3 Cljnical Toxicity 7 . 4 Pharmacokinetics Determination in Body Fluids References

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

361

Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved.

JOSH. BEIJNEN ETAL.

362

1.

HISTORY

In 1956 Hata and coworkers (1) first reported about the isolation of a new group of antitumour antibiotics which they named mitomycins. These pigmented compounds were obtained by extraction and chromatographic purification of the fermentation broth filtrate of Streptomyces caespitosus. In 1958 another Japanese group, led by Wakaki, succeeded in the isolation of a mitomycin derivative, called mitomycin C, from the same actinomyces strain (2). Several mitomycin derivatives have since been isolated from Streptomyces species (3-8). Extensive chemical degradation studies, X-ray diffraction crystallography, chromatographic and spectroscopic analysis and biosynthesis studies have contributed to the elucidation of the structure and absolute stereochemistry of mitomycin C and related mitomycins (9-23a). Mitomycin C was selected for further development after it had been established that mitomycin C possessed profound antineoplastic activity and that the therapeutic index was more favourable than that of related mitomycin congeners. The drug has activity against an array of solid tumours such as adenocarcinomas of the gastrointestinal tract. Unfortunately, clinical experience with mitomycin C has shown that it causes severe toxicity in particular a delayed, cumulative bone marrow suppression, Mitomycin C is rarely used as a single agent except for the treatment of superficial cancer of the urinary bladder, intravesically administered. This administration mode has been shown to be very effective and devoid of serious haematological toxicities. For more specific information about the clinical usefulness of mitomycin C combinations in lung, breast and stomach cancer the reader is referred to review articles and books ( 2 4 - 3 1 ) . 2.

DESCRIPTION

2.1 Name, Formula, Molecular Weight The generic name is mitomycin C (50-07-7). The drug is marketed under the trade names of MutamycinB, Mitomycin-C The molecular formula for mitomyKyowa@ and Ametycine@ cin C is CI5Hl8N4O5, The molecular weight is 334.13.

.

MITOMYCIN C

363

2.2 Appearance, Colour, Odour Blue-vi.olet crystalline powder which is o d o u r l e s s . 3.

BIOSYNTHESIS, CHEMICAL SYNTHESIS

By using 13C, 14C, 3H and 15N-labeled precursors in feeding experiments, investigators have studied the biosynthetic pathways of mitomycins (20, 21, 32-35). Although considerable progress has been made in elucidating the biosynthesis routes the exact pathway remains to be established. Feeding studies have revealed that D-glucosamine, L-methionine, L-citrulline, L-arginine, pyruvate and D-erythrose are candidates as biosynthetic precursors of the mitomycines (21). The total chemical synthesis of mitomycin C has been accomplished from 2,6-dimethoxytoluene as starting material by Kishi and collaborators (36). The synthesis route is lengthy (47 steps) and complex. 4.

PHYSICAL PROPERTIES

4.1 Ultraviolet-Visible Spectrum The ultraviolet spectrum of mitomycin C ( ~ x ~ O - ~ Min) water is depicted in Figure 1. The spectrum was recorded by using a Shimadzu UV-200 double beam spectrophotometer in 1 cm quartz cell. In the visible region a broad band of low intensity is present. At pH values over 10 the absorption maximum at 360 nm shifts to 295 nm due to keto-enol tautomerization and deprotonation of the 7-amino quinone part of the mitomyzin C molecule (Figure 2)(37). Molar absorptivities (E) and A1 cm values are reported in Table I (9). Table I

360 555

UV-VIS Spectral Data for Mitomycin C in Methanol(9)

689 6.3

23,000 209

364

JOS H.BEIJNEN ETAL.

I

400

MMC

01 absorbance

300 250 wavelength I nrn)

350

200

MMC-

Figure 2. Keto-enol tautomerization and deprotonation of mitomycin C (MMC)

MITOMYCIN C

365

4.2

Infrared Spectrum The infrared spectrum of mitomycin C in a potassium bromide pellet is presented in Figure 3 . The spectrum was recorded with a Jouan-Jasco IRA-grating infrared spectrophotometer. Assignments o f a number of prominent bands is listed in Table 11. Table I1

Infrared Assignments for Mitomycin C -1

wavenumber (cm )

assignment

346OS, 331OS, 3260'

V(NH) and

1735'

carbarnate and 7C-NH2 V(C=O) of carbamate function

16OOs, 1558'

V(C=O)

of quinone

,

,

V

(NH2) of aziridine,

'"1

0

,

,

,

,

,

,

,

1

,

,

Figure 3 . Infrared spectrum of mitomycin C

,

,

,

,

,

,

,

,

,

,

1

1

JOSH. BEIJNEN ETAL.

366

4.3 Nuclear Magnetic Resonance fNMR) Spectrum The proton NMR spectrum was recorded in pyridine-d containing tetramethylsilane as internal reference and wi ?h the use of a Bruker WM-300 spectrometer a t frequency 300.13 MHz (37a). The spectrum (detail) is presented in Figure 4 and refers to the situation after proton exchange. The spectral assignments are presented in Table 111 (in pyridine-d ) and are derived from L o w and Begleiter ( 3 8 ) . Irrigularitges in intensity comparison of the signals make the assignments tentative. Proton exchange with deuterium oxide results in disappearance of NH, NH and H-1 signals, while the H-3, H-9, 2 H-10 and H-10' signals shift 0.10 to 0.20 to lower ppm values (spectrum F i g . 4 ) . Table I11

'H NMR Assignments for Mitomycin C in pyridine-d5 ~

~~~~

Chemical shift 6 (ppm)

Multiplicity

Number of atoms

Assignment

2.00

singlet

3

CH3 (C-6)

2.10

triplet

1

NH (Cl-C2)

2.72

quartet

1

H-2

1

H-1

3.13 3.19

singlet

3

OCH3 ( C - 9 )

3.57

doublet

1

H-3 '

4.02

quartet

1

H-9

4.53

doublet

1

H- 3

5.11

triplet

1

H-10

5.43

quartet

1

H-10'

7.6

broad

4

NH2(C-7)

and CONH2 The natural abundance carbon 13 NMR spectrum was recorded under the same experimental conditions except that the frequency was 75.46 MHz (37a). The proton-noise decoupled spectrum is presented in Figure 5 and the spectral assignments are summarized in Table IV. The assignments of Keller and Hornemann are followed ( 3 9 ) .

MITOMYCIN C

1

,

367

.

.

I

.

5.0

.

,

.

1

.

.

.

.

1

4 .O

. . , .1

.

.

.

.

1

. . . . , . . . . , . . . .

3.O

chemical shift (ppm)

Figure 4 . Proton NMR spectrum of mitomycin C (detail)

2.0

368

JOSH.BEIJNEN ETAL.

180

160

140

120

100

80

chemlcal shift (ppm)

Figure 5.

I3c NMR spectrum of mitomycin

c

60

40

20

0

MITOMYCIN C

369

13C NMR Assignments for Mitomycin C in pyridine-d 5

Table IV

Chemical shift 6(ppm) ~

Ass-ignment

~~

CH3 (C-6)

8.8 32.6

c-2

36.7

c-1

44.2

c-9

49.5

OCH3 (C-9a)

50.6

c-3

62.5

c-10

104.3

C-6

106.8

C-9a

110.7

C-8a

149.6a

c-7

156.1

C-5a

158.1

C-lOa

176.7

C-8 c-5

178.3 ~~~

a

~~

~

obscured by solvent

4.4 Mass Spectrum Electron impact (EI) mass spectroscopy of mitomycins, including mitomycin C, has been investigated in detail by Van Lear (40). His observations were confirmed in later studies (41, 42). For this analytical profile electron impact (EI), chemical ionization (CI) , field desorption (FD) and fast atom bombardment (FAB) in the positive and negative ion mode, spectra of mitomycin C (lot nr. 010783) were recorded. EI mass spectrometry was performed by direct probe analysis on a Kratos MS-80 mass spectrometer. Source conditions were: 200 OC source temperature, 70 eV electron energy and 100 PA ionising current. Direct insertion probe CI mass spectra were obtained by using a Finnigan 3200 quadrupole mass spectrometer combined with a Finnigan 6000 data system. Methane was used as rhe ionising gas. The emission current was 0.20 mA and the multiplier voltage 1800 V. FD mass spectra were obtained with a Varian MAT 711 double focussing mass spectrometer equipped with a MAT 100 data acquisition unit. 10 p m tungsten wire FD emitters containing carbon microneedles

JOSH. BEIJNEN ETAL.

370

Figure 6. EI mass spectrum of mitomycin C

.360

242

292

I

3ab

Figure 7. CI mass spectrum of mitomycin C

x

4

MITOMYCIN C

371

with an average length of 30 p m were used. The mitomycin C sample was dissolved in methanol and then loaded onto the emitters by the dipping technique. An emission current of 12 mA was used to desorb the sample. The ion source temperature was 70 'C. FAB mass spectrometry was carried out using a V.G. micromass ZAB-2F mass spectrometer, an instrument with reverse geometry and fitted with a high field magnet and coupled to a V.G. 11-250 data system. The sample was loaded in thioglycerol solution onto a stainless steel probe and bombarded with Xenon atoms having a 8 keV energy. The recorded spectra are depicted in Figures 6, 7, 8, 9 and 10 and the major ion assignments are given in Table V. A high resolution mass spectrum exhibited the molecular ion at m/z 334.1280 with composition C15H18N405 (calculated mass 334.1277).

4

Figure 8. FD mass spectrum of mitomycin

C

3 72

JOSH. BEIJNEN ETAL.

37

28 18

e

388

L

488.

98. 88 18.

68.

2r

58. 18.

38.

21s

I88

288

388

480

Figure 9/10. FAB(+) mass spectrum (upper spectrum) and FAB(-) mass spectrum (lower spectrum) of mitomycin C

MITOMYCIN C

Table V

373

Mass Spectral Data for Mitomycin C m/z

assignment

FD

334 335

M+* (M+H)+

FAB (+) -

358

(M+H+Na)

357

(M+Na )

336

(M+2 H)+

335 274

(M+H) ( (M+H)-02CNH3)

242

( ( (M+H) -H~COH) -O~CNH~)

+

+

+

+

+

m/z 57, 91 and 215 signals come from the thioglycerol matrix

FAB (-)

334 333

M-'

29 1

(M-CONH) -

(M-H)-

m/z 107, 215 and 321 signals come from the thioglycerol matrix m/z 139 and 265 signals cannot be assigned with certainty CI -

363 335 302 274

EI -

(M-H3COH)+

+

242

(M-02CNH2) ( (M-H3COH) -02CNH2)+

334

M+

302

(M-H~COH)+

291

(M-OCWH)

273 259

(M-0

242

( (M-H3COH) -02CNH2)

+

+

CNH3) ( (M-02CNH2)-CH3)

+

+

JOSH. BEIJNEN ETAL.

374

4.5 Circular Dichroism (CD) Spectrum The chiral centers at C1, C2, C9a and C9 in the mitomycin C molecule are responsible for the CD Cotton effects at 355 nm and 395 nm. The CD spectrum of mitomycin C in a methanolic solution (c=O.OOl%), determined by using a Jobin Yvon Dichrograph 111, is shown in Figure 11.

+A c

F i g u r e 11.

CD spectrum of mitomycin C

MITOMYCIN C

375

4 . 6 Optical Rotation The optical rotation of mitomycin C in methanol (c-0.1%) was determined to be + 2 3 2 " . This analysis was performed with a Thorn NPL Automatic Polarimeter Type 243 at 589 nm. The optical rotatory dispersion (Om)spectrum of mitomycin C has been reported by Hornemann et al. ( 4 3 ) . 4.7

Melting Point In the literature no melting point for mitomycin C is mentioned except for the statement that it should be over 300 " C (11).

4.8

Differential Scanning Calorimetry In nitrogen atmosphere no transitions were recorded up to 330 OC in a closed (air tied) sample holder. In an open sample holder an undefined large exothermic decomposition takes place from 215 "C. The applied apparatus was a Feteram 111, the heating rate was 5 K/min and the sample size was 3 mg ( 4 4 ) . 4.9 Solubility Mitomycin C is readily soluble in methanol, acetonitrile normale saline and water, but is nearly insoluble in hydrocarbon solutions, The following solubility data have been reported ( 4 5 ) , determined by suspending an excess amount of mitomycin C in a solvent, followed by filtration and HPLC analysis.

Table VI

Solubilities of Mitomycin C

solvent

solubility (mM)

water sesame oil isopropylmyristate hexane

2.73

temperature ("C) ~

0.0180

0.0193 0.0234~10-~

25 25 37 25

JOS H.BEIJNEN ETAL.

376

4.10 Partition Coefficient The partition coefficient for mitomycin C has been measured in various organic phaseldistilled water systems. The results have been combined in Table VII. Table V I I

Partition Coefficients for Mitomycin C

solvent chloroform n-octanol ethyl acetate 1-pent an1 chloroform/2-propano1 chloroform/2-propanol chloroform/l-pentanol chloroform/l -pentan01

partition coefficient 0.26; 0.27, 0.41; 0.42

(90+10) (50+50) (90+10) (80+20) dichloromethane/2-propanol (50+50)

a

reference

0.51

2.32 0.50 1.52 1.12 1.85

1.04

the aqueous phase was 0.07M phosphate buffer pH

=

7.4

The organic solvents chloroform (1,2) and ethyl acetate (7, 48) have been used with success €or the isolation of mitomycins from the aqueous fermentation broths of Streptomyces species.

4.11 Dissociation Constants The mitomycin C molecule contains several prototropic functions. Basic groups are the 7-amino group, the N-4 nitrogen and the aziridine nitrogen. Due to rapid degradation the pKa values of the conjugated acid concerning the 7-amino group and N-4 nitrogen can not be deduced from titration experiments with intact mitomycin C. Therefore the acid dissociation constants for these €unctions were derived from titrations with stable analogs (37). Results are listed in Table VIIT. The pKa of the aziridine nitrogen has been determined titrimetrically (11) and kinetically from the pH-rate re1.ationship.s in degradation studies (49-51) although the titrimetrical determination also has been influenced by degradation. Mitomycin C also has acidic properties with an (apparent) pKa 12.44 at room temperature. These acidic properties emerge after keto-enol tautomerization of the 7-amino quinoid moiety which precedes deprotonation in alkaline medium (Figure 2)(37).

MITOMYCIN C

Table V I I I

377

Prototropic Functions of Mitomycin C pKa(25"C)

function

determination method

N-4 7-amino aziridine

-1.2 spectrophotometry spectrophotometry -1.3 potentiometric titration 3.2 via pH-rate profile 2.8 2 via pH-rate profile via pH-rate profile 2.74 via pH-rate profile 2.50(49 spectrophotometry 12.44

7-amino

reference

4 . 1 2 Electrochemistry Due to the presence of the quinone ring of the mitomycin C molecule the compound can be reduced and transformed into its hydroquinone form. This conversion triggers a complicated pattern of chemical consecutive reactions. Although much progress has been made in the elucidation of the processes occurring during and following electrochemical reduction of mitomycin C the exact complicated mechanisms have not yet been unravelled (50). This is due to: rapid degradation of mitomycin C at p H < 3 and p H > 1 2 ; the extremely high reactivity of the hydroquinone form of mitomycin C; the fact that chemical degradation products as well as electrochemically generated degradation products of mitomycin C can be reduced within the region of reduction potentials of mitomycin C itself ( 5 0 ) . Electrochemistry of mitomycin C has been studied in aqueous solutions (50, 52-54) and aprotic solvents ( 5 5 ) , by using polarography ( 5 0 , 52-54) and cyclic voltammetry ( 5 0 , 5 2 , 5 3 , 5 5 ) . Direct current polarographic curves for mitompcin C are shown in Figure 1 2 . Rapid degradation of mltomycin C within the time scale of recording a direct current polarographic curve at p H < 3 and pH > 1 2 strongly influences the shape and heights of these curves ( 5 0 ) .

378

JOS H.BEIJNENETAL.

I

I

I

5

PH = S O

I

0

I

-500

I

-1000

I

-1500

potential (mV)

Figure 12. -4 Direct current polarographic curves of mitomycin C (10 M)

MITOMYCIN C

5.

379

METHODS OF ANALYSIS

5.1 Elemental Analysis The elemental analysis of a purified mitomycin C sample for C, H, N and 0 was reported to be as follows: Table IX

El-emental Analysis of Mltomycin C (11)

element

% theory

% found

C H

53.83 5.43 16.76 23.98

53.55 5.57 16.86 24.13

N

0

5.2 Ultraviolet Spectrophotometry Mitomycin C has been determined by using UV spectrophotometry at 360 nm &n methan-qjl solution. This assay is linear in the range 2x10- to 4x10 M y with a precision of ? 1%. 5.3

Thin Layer and Paper Chromatography

A diverse array of thin layer chromatographic systems

have been described in the literature for analysing mitomycin C and derivatives (55). Some representative systems have been summarized in Table X. The mitomycin C spot can be located by: irradiation with UV light of 254 nm; examination under daylight (mitomycin C has a blue-violet colour) ; spraying with a 1 in 100 solution of ninhydrin in alcohol, followed by heating the plate at 110°C for 15 minutes by which mitomycin C appears as a pink spot (56). Table X

Thin Layer and Paper Chromatography of Mitomycin C

plate

solvent (v/v)

Rf

silicagel

butanol (sat. with water) ethyl acetate (sat. with water) methanol-benzene-water (10:5:5) isopropylalcohol-1ZNH OH (2:l) 4 methano 1

0.57

~

~_________

~

1-octanol-acetone-ligroin

(9O-115°C) (2:5:5) chloroform-methanol (9: 1) cellulose isobutyric acid-0.5M NH40H (10:6) paper butanol-water (84:16) methanol-benzene-water (1:1:2) ethyl acetate (sat. with water)

reference

0.02 0.88 0.80

(12) (12) (12) (57) (57)

0.11 0.12 0.75 0.57 0.02 0.56

(58) (59) (57) (60) (60) (60)

0.56

380

JOS H. BEIJNENETAL.

5 . 4 Cel Filtration Chromatography For the separation of mitomycin C from its degradation products and alkylation adducts Tomasz et a1 (57, 6 1 - 6 4 ) utilized pel filtration chromatography on Sephadex G-25 columns and 0 . 0 2 M NH HCO solution as eluent. By using a 1 . 5 ~ 4 3 4 1 . 5 cm Sephadex G-25 (fine) column the elution volume of mitomycin C is 96 m l ( 5 7 ) . 5.5 High Performance Liquid Chromatography HPLC procedures reported in the literature have been developed mainly for the analysis of mitomycin C and derivatives in decompositinn mixtures ( 5 1 , 5 9 , 6 5 - 7 1 ) in biological matrices (plasma, urine, bile, tissues) ( 4 6 , 7 2 - 8 6 ) and other systems such a s (enzyme containing) culture media ( 6 2 , 6 9 , 87-90). Detection by W absorbance measurements at 365 nm is usually preferred ( 5 5 , 7 5 , 7 6 ) because of the high specificity and sensitivity, but detection at 254 nm, which is less sensitive, is also reported ( 6 6 , 91, 92). The polarographic electrochemical detection mode is an elegant alternative for the detection of mitomycin C ( 5 0 , 75, 93), but demands extensive experimental precautions to be taken, such as removal of oxvgen from the mobile phase and damping of pulsating flow of the solvent delivery system ( 9 3 ) . Important HPLC systems have been enumerated in Table XI,

Table XI

High Performance Liquid Chromatography for Mitomycin C

~~

~~

column

mobile phase

p-Bondapak C 1 8 (10 ,pm) p-Bondapak C18 (10 pm) Lichrosorb RP18 (10 pm) Lichrosorh RP8 (5 p'm) Ultrasphere ODX Hypersil-MOS (5 pm) Hewlett-Packard C8 (10 pm) Radial Pak C18 (10 pm)

Reversed Phase HPLC methanol-water (35: 65) methanol-0.01 M phosphate buffer (pH 6,0)(30:7O;w/w) methanol-water (30:70;w/w) + 0.5% (v/w) phosphate buffer pH 7.0 methanol-water-phosphate buffer pH 7.0 (20:70:10) acetonitrile-0.03M potassium phosphate buffer (12.5:87.5) acetonitrile-0.05M phosphate buffer pH 7 (10:90) acetonitrile-water; gradient from 5% to 41% acetonitrile methanol-0.01M phosphate buffer pH 7; gradient from 0% to 50% methanol

Lichrosorb RP18 (10 pm) Lichrosorb RP18 ( l o p ) Hypersil ODS (5 pm) Hypersil ODS (5 pm) Resolve C18 (5 pm)

Porasil P (10 pm) Porasil (10 pm) Silica Si-60 a

~~

reference (77-80) (46) (67) (82) (91)

(75) (94) (42)

Ion Pair HPLC (65) methanol-water (40:63;w/w) + 0.5X (v/w) glacial acetic acid (~H*=4.3)~ methanol-water (30:70;w/w) + 0.5% (v/w) phosphate buffer pH 7.0 + 0.5% (v/w) tetrabutylammoniumbromide solution (20%, w/w) (59) acetonitrile-water (25:75) + 0.2% sodium laurylsulphate + 0.025M (66) sodium acetate acetonitrile-water (20:80) + 0.2% cetrimide + 0.0125M sodium acetate (66) methanol-1 mM octanesulfonicacid sodium salt solution (30:7O;v/v) (pH* 4.5)a (85)

Normal Phase HPLC tetrahydrofuran-methanol-isooctane-dichloro~t~ane (3:7:15:75)

chloroform-methanol (9:1) ethyl acetate-methanol (95:l) ethyl acetate-methanol-water-dichloromethane (97:2:1:1)

pH values of organic modifier-water mixtures

382

JOS H.BEIJNEN ETAL.

5.6

Identification and Purity Tests in Official Compendia In the United States Pharmacopeia XXI (56) the monographs "Mitomycin" and "Mitomycin for Injection" have been included. In these monographs the identification of mitomycin C is based on IR and UV spectroscopy and chromatography by which the sample should exhibit the same spectroscopic and chromatographic properties as a similar preparation of a USP Mitamycin Reference Standard. The pH of a solution containing 5 mg/ml should be between 6.0 and 8.0. The water content of the raw material may not exceed 5%. Furthermore Mltomycin and Mitomycin for Injection (a dry mixture of mitomycin C and mannitol) must meet the requirements of specific tests described in the USP XXI such as: Safety Tests, Depressor Substances Tests, Pyrogen Tests and Sterility Tests (56). 6.

STABILITY, DEGRADATION

6.1 Stability in Aqueous Solutions Mitomycin C and related mitomycins are not stable when stored as aqueous solutions (11-13, 51, 55, 57, 59, 65-68, 82, 96-108). Degradation is accelerated by acid, alkali, (buffer) ions and high temperatures (108). At p H < 7 mitomycin and at C is converted into l-hydroxy-2,7-diaminomitosenes pH > 7 it is hydrolysed into 7-hydroxymitosane. The overall degradation scheme is given in Figure 13. In the mitosene compounds the C9a methoxy group has been cleaved, a double bond between C 9 and C9a has been introduced and the aziridine moiety has been opened. Aziridine ring opening occurs with configurational retention at C2 and water attachment at C1 with inversion as well. as retention of the C1 stereochemistry yielding 1,2-trans- and 1,2-cis-l-hgdroxy-2,7-diaminomitosenes, respectively. Under drastic acidic conditions progressive degradation reactions lead to hydrolysis of the 7-amino group and the C10 carbamate side chain (9-11, 1 3 ) . The mechanism of the initial degradation step of mitomycin C in acid is shown in Figure 14 (107, 108). In the cascade of reactions an intermediate is proposed, having a positive charge at C1 (49, 107-110). This electrophilic center serves as target for attacking (nucleophilic) water molecules resulting in the formation o f l-hydroxv-2,7-diaminomitosenes. The degree of protonation of the 2-amino function in the proposed intermediate species (pKa:2.8(65)) determines the C1 stereochemistry of the resulting mitosenes. A protonated 2-amino function may direct incoming water molecules to a cis configuration. Whenever the amino function in the intermediate is not protonated the directing electrostatic power is absent and a reacting water molecule may approach the C1 carbonium ion from both

MITOMYCIN C

383

sides with roughly equal chances. This explains the remarkable influence of pH on the C 1 stereochemistry of the resulting mitosenes. At pH=l the cisltrans ratio is about 4 while at pH=6 the ratio approaches 1 (65). When mitomycin C degrades in acidic medium in the presence o f nucleophiles such as acetate, phosphate or ever! DNA parts, l-nucleophile-2,7-diaminomitosenes are generated (57, 103, 111-114). These are important observations which lend credibility to the hypothesis that acid activation may play a role in the in vivo mitomycin C DNA alkylation. In alkaline solution (pH 7 to 13) the 7-amino group of the mitomycin C molecule is substituted by a hydroxy function (59, 100-102). Under prolonged severe alkaline treatment the quinone chromophore is destroyed ( 5 9 ) . In huffered media the degradation kinetics o f mitomycin C can be described adequately by (pseudo) first order kinetics. Some kinetic data are summarized in Table X I I . For further detailed kinetic information is referred to the literature (49, 51, 55, 67, 107, 108).

+ i,i \ cis- mitosene

mitomycin C

\

OH-

7 - hydroxymitosane

Figure 13. Overall degradation scheme of mitomvcin C

trans -mitosene

NH,

384

JOSH. BEIJNEN ETAL.

.."

L 0

1

0

II

c N-H

Figure 14. Acid degradation mechanism of rnitomycin C

+ CH.OH

MITOMYCIN C

Table XI1

385

Degradation Kinetics for Mitomycin C

conditions/method pH 0.87;

perchloric acid solution; 2OoC/ HPLC pH 2.86; 0.001M acetate buffer; 20°C/ HPLC 1.OM phosphate buffer; pH 3.0; 25'C/ HPLC pH 4.64; 0.1M phosphate buffer; 40°C/ spectrophotometry pH 7.4; 0.1M phosphate buffer; 37'C/ HPLC pH 9.0; 0.1M phosphate buffer; 37"C/ spectrophotometry pH 11.1; 0.1M phosphate buffer; 25OC/ HPLC

kobs ( s - l )

1.M

O-~

reference (65)

4.Ox

(65)

1.2~10-~

(114)

1.4~10-~

(107)

4.1~1O-~

(105)

1.8~10-~

(104)

5.3~

(67)

6.2 Stability in Pharmaceutical Formulations Mitomycin C is commercially available in a lyophilized form containing mannitol (Mutamycin@ : 5 mg mitomycin C + 10 mg Mannitol) or sodium chloride (Mitomycin C-Kyowam :10 mg mitomycin C + 240 mg sodium chloride; Ametycine lo@ : 10 mg mitomycin C + 240 mg sodium chloride) as excipients. In this state the drug is stable for at least two years if kept dry and in well closed containers at room temperature in the dark (115). After reconstitution the drug has only limited stability, depending strongly on the pH of the final solution (68). An overview of stability data of mitomycin C in infusion fluids is given in Table XIII. More specific and extended data are described in the references concerned (68, 70, 71, 116-120).

Table XI11

Stability of Mitomycin C in Infusion Fluids at Room Temperature and - 3 O O C

infusion fluid

concentration

stability

reference

5% dextrose

50

pglml

tgO = 3 . 0 h; t50

0.9% sodium chloride

pg/ml mg/ml

93% remained after 5 days

sterile water

50 2

5% dextrose

50

pg/ml

26% remained after 12 hours

0.9% sodium chloride

50

pglml

t50

phosphate buffer pH 7.75

50 pg/ml 0.4 mg/ml

~~

5% dextrose

=

78 h

stable for 7 days = 12

tgO =

tgO =

h

15 days 1 h

0.9% sodium chloride

0 . 4 mg/ml

t

0.9% sodium chloride

0.6 mg/ml

99% remained after 4 weeks storage at -30°C

90

=24h

MITOMYCIN C

387

Further research is required to resolve the problem of conflicting stability data on mitomycin C in 0.9% sodium chloride solution (68, 70, 116, 119, 120). Stability in Biological Media 6.3 Chemical instability of mitomycin C in biological fluids and tissues may be a complicating factor in the bioanalysis of the drug. Ignorance of the chemical stability of the drug during sampling and sample pre-treatment procedures may lead the researcher to misinterpretations. Recently, den Hartigh et al. (86) published an extehsive study concerning the handling of mitomycin C biological samples. The following recommendations and guidelines were given by these authors (86): - whole blood samples should be placed in ice immediately after sampling; separation of plasma and red blood cells should take place within half an hour after collection of the samples; - mitomycin C samples should be protected from long-term exposure to daylight in order to prevent analyte degradation; the pH of urine samples should always be checked and, if necessary, adjusted to neutral values, as a low pH will induce mitomycin C decomposition and the formation of precipitates upon which mitomycin C may be adsorbed: - plasma and urine samples need not necessarily be placed in the refrigerator or freezer immediately after preparation, provided analysis is carried out within a period of 2 h; - plasma and urine samples may be stored in a freezer at -2OOC; however, analysis should take place within 3 weeks as longer storage at this temperature may induce considerable reduction of the mitomycin C concentration; - repeated freezing and thawing of plasma and urine samples does not influence the analytical results of the mitomycin C assay; - extracts of biological samples may be kept for at least 24 h, in the dark, either dry or dissolved in methanol at 4 ° C or 20°C prior to analysis. The in vitro half life of mitomycin C was determined to be > 14 days under conditions of the clonogenic assay ( 21). Mitomycin C is fairly stable (70-802 of the drug remained) when incubated for 5 days at 37°C in Medium E, RPMI, Fischer's or MEM culture media, supplemented with serum (69). In Mark's M-20 culture medium with fetal calf serum, Proctor and Gaulden (90) observed that the amount of mitomycin C was reduced by 2 9 % after 30 minutes incubation.

388

7.

JOS H. BEIJNENETAL.

PHARMACOLOGY

7.1 Mechanism of Action Mitomycin C induced cytotoxicity is believed to be mediated by DNA binding. The drug as such, in its oxidized form, is not cytotoxic although some interaction with nucleic acids may exist (122). Mitomycin C must be activated before it is capable of alkylating DNA (123-128). The alkylating capacity is unmasked by reductive or acidic activation. An outline of the reductive activation pathway is given in Figure 15. Reduction can be mediated by enzyme systems (87-89, 129-132) or by chemical reducing agents (58, 126). The conversion of mitomycin C into its reduced form destabilizes the compound drastically and almost instantaneously the C9a methoxy group is lost (Figure 15). A subsequent rearrangement in species C leads to the formation of the quinone methide D which acts as target for nucleophilic nucleic acid attack, by which a monofu ctional alkylation adduct is formed. An intramolecular 9 SN -displacement of the carbamate moiety leads to the bifunctional cross-linked adduct F (Figure 15) (128). The interaction of mitomycin C with DNA is more than 9OX of the monofunctional type. Chemical structures of monofunctionally linked mitomycin C adducts with DNA, nucleotides and nucleosides have been reported (62-64, 133-136). Reduction of mitomycin C may follow either one or two electron reduction steps. The semiquinone radical is also believed to be capable of producing interstrand cross-links between complimentary strands of DNA (87, 137). Under aerobic conditions, the semiquinone radical formed after a one electron reduction can transfer its electron to oxygen to generate superoxide anion under regeneration of the oxidized quinone (138-142). Reactive oxygen species can cause DNA strand scission and membrane lipid peroxidation, leading to cell damage and can contribute in this manner to mitomycin C induced cytotoxicity. In the absence of any reducing agent or enzymes mitomycin C is also capable of coupling covalently with DNA or nucleic acid parts, but then, activation by acid is a requisite (57, 103, 111-113, 143, 144). Acid catalyzed degradation of mitomycin C (Figure 14) occurs through reaction intermediates possessing an electrophilic C1 center which is the target for nucleophilic nucleic acid attack. More studies are necessary in order to elucidate which of the proposed mechanisms or combination is actually responsible for mitomycin C induced cytotoxicity in vivo.

MITOMYCIN C

389

OH

OH

Figure 15. Reductive activation mechanism of mitomycin C

390

JOSH. BEIJNEN ETAL.

7.2 Clinical Antitumour Activity Single agent activity of mitomycin C has been demonstrated against various solid neoplasms in humans such as breast cancer (29, 145) , stomach cancer (146), pancreatic cancer (147), colorectal cancer (29), non-small cell lung cancer (148) , squamous cell carcinoma of the uterine cervix (28) and head and neck cancer (28). Encouraging results have been obtained on the treatment of superficial transitional cell carcinoma of the bladder when mitomycin C is administered intravesically (28-30, ,l49). Mitomycin C is usually used in combination with other cytostatics (28-30). 7.3 Clinical Toxicity Delayed cumulative myelosuppression is the most important toxicity. Others include nausea, vomiting, anorexia, diarrhea, stomatits, skin rashes and alopecia (24, 28, 150). Extravasation of mitomycin C causes severe cellulitis accompanied with chronic pain and ulceration (151). Interstitial pneumonitis, pulmonary fibrosis, hemolytic-uremic syndrome and renal failure have occasionally also been documented as due to the drug. Following topical intravesical mitomycin C therapy for bladder cancer dermatological toxicity has been encoutered in some cases (154). 7.4 Pharmacokinetics Following intravenous bolus injection of mitomycin C the plasma concentration-time curves show a bi-phasic, exponential decline (72, 155). Mitomycin C pharmacokinetics (at clinically relevant doses) is not dose dependent (72, 155). Maj o r routes of elimination are liver metabolism and urinary excretion. Metabolites have never been identified. Absorption after oral administration is very irregular(l56). After intravesical instillation, limited absorption of mitomycin C into the systemic circulation occurs (30, 157). Pharmacokinetic data after intra-arterial administration are available (73).

MITOMYCIN C

8.

391

DETERMINATION IN BODY FLUIDS

The analysis of mitomycin C in biological samples is hampered by its instability, low concentrations and the polar nature of the mitomycin C molecule (55, 7 6 ) . The following techniques have been used: microbiological assays ( 7 4 , 9 5 , 1 0 4 , 1 5 8 - 1 6 1 , 1 6 6 ) , bioassay by using a repair deficient mutant of Chinese hamster ovary cells ( 1 6 2 ) , immunological assay ( 1 6 3 , 1 6 4 ) , high-performance differential pulse polarography ( 5 4 ) and HPLC assays ( 4 6 , 7 2 - 8 6 , 93-96, 1 5 5 ) . The HPLC assay designed by Den Hartigh et al. ( 4 6 , 7 2 ) has shown to be very suitable for pharmacokinetic studies ( 7 2 - 7 4 ) . The following procedure is followed for plasma samples: 0 . 2 , 0.5 or 1.0 ml samples is mixed with the internal standard porfiromycin (about 500 ng) and with 2 . 0 , 5 . 0 or 10.0 ml, respectively, of chloroform-2-propanol ( l + l , w/w). After shaking for 1 minute and centrifugating for 5 minutes ( 2 5 0 0 g), the clear supernatant is transferred into a conical tube and evaporated to dryness at 30-40°C under nitrogen. The residue is dissolved in 50-100 p l of methanol with the aid of a vortextype mixer. Aliquots of 10-20 PI are injected into the chromatograph. HPLC: column: p Bondapak C18 ( 3 0 cm x 3 . 9 mm i.d., particle size 10 pm); mobile phase: 0.01 M phosphate buffer pH 6.0-methanol (70+30, w/w); flow rate 1.0 ml/min; detection: 365 nm. An overview of published determination methods of mitomycin C in body fluids is given in Table X I V .

Table X I V

Determination Methods for Mitomycin C in Body Fluids

matrix

sample pretreatment

a

b determination limit(ng/ml) - test organism reference microbiological assays ~~~

W tJ

blood, tissues, urine, bile plasma, urine plasma, urine serum, urine

no

2

no no no

10 500 60

E.coli R E.coli B B.subtilis E. coli

bioassay with hamster ovary cells plasma

1

no

(162)

immunological assays serum, urine

no

4

(163)

polarography plasma, urine plasma, urine

lI0

1.s

200 25

(54) (54)

Table XIV (continued) matrix

high performance liquid chromatography sample preinternal chrom to ra hic determination a C g g p treatment standard mode limit(ng/rnl) -

serum, urine, ascites plasma plasma plasma, urine, bile, ascit e s serum, plasma, urine

1.1. 1.1. 1.1.

no no no

1.1. 1.S.

Yes Yes

plasma, urine plasma, urine

1.s. 1.s.

yes no

plasma, urine

1.1.

Yes

plasma, urine serum serum

1.1. (plasma) 1.S. deproteination with methanol 1.S. 1.1.

no no no

w

plasma, urine plasma, urine

Yes Yes

1

RP RP RP

1 5

RP RP

40 (serum)

1

5 0 (urine)

4 (plasma)

2 (plasma) 500 (urine) 1 (plasma) 200 (urine) 1 (plasma)

reference

RP (gradient) RP NP

10 10

RP RP RP

25 10

RP RP

a 1.1.: sample pre-treatment by liquid-liquid extraction; 1.s.: sample pre-treatment by liquid-solid extraction.

b determination limits for 25 p1 ( 8 0 ) , 50 p l ( 8 5 , 1 6 3 ) , 100 p l ( 8 4 ) , 0.2 ml (160), 77, 7 8 , 82, 162), 2.0 ml (54, 7 5 , 9 5 ) and 2.5 ml ( 9 4 ) samples C the internal standard is porfiromycin d RP: reversed phase chromatography; NP: normal phase chromatography.

1.0 ml ( 4 6 , 72-74,

JOSH. BEIJNEN ETAL.

394

Acknowledgements The authors wish t o thank Ms C. de Graaf €or typing the manuscript and Mr P.W.Y. Chan for preparing the figures. REFERENCES 1. T. Hata, Y. Sano, R. Sugawara, A. Matsumae, K. Kanamori, 9, 1 4 1 T. Shima and T. Hoshi, J. Antibiot. Ser. A (1956). 2. S. Wakaki, H. Marumo, K. Tomoika, G. Shimizu, E. Kato,

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natural products, W. Herz, H. Grisebach and G.W. Kirby (Eds.), Springer-Verlag, New York, ( 1 9 7 9 ) p. 1. 36. Y. Kishi, J. Nat. Prod. 4 2 , 5 4 9 ( 1 9 7 9 ) . Underberg and H.-Lingeman, J. Pharm. Sci. 72, 3 7 . W.J.M.

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127,

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107,

156,

MITOMYCIN C

391

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106,

12,

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.

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(1974) 97. W.G. Taylor and W.A. Remers, J. Med. Chem. 18, 307 (1975). 98. J.H. Mowat, J.B. Patrick, R.P. Williams, D.B. Cosulich and J.S. Webb, U.S. Patent (1965) 3,179,671. 99. L. Cheng and W.A. Remers, J. Med. Chem. 20, 767 (1977). 100. E.R. Garrett, J. Med. Chem. 6 , 488 ( 1 9 6 3 r 101. W. Schroeder, U . S . patent (1567) 3,306,821. 102. E.R. Garrett and W. Schroeder, J. Pharm. Sci. 53, 917 (1964). 103. J.W. Lown and G. Weir, Can. J. Biochem. 56, 296 (1978). 104. M. Hashida, Y. Takakura, S . Matsumoto, HTSasaki, A. Kato,

T. Kojima, S . Muranishi and H. Sezaki, Chem. Pharm. Bull. 31, 2055 (1983). 105. H. Saszi, E. Mukai, M. Hashida, T. Kimura and H. Sezaki, Int. J. Pharm. 15, 61 (1983). 106. C.C. Cheng, W.P. Coppola, W.H. Johns, J.B. Bogardus and R.A. Lipper, J. Pharm. Sci. 75, 208 (1986). 107. W.J.M. Underberg and H. Lingzan, J. Pharm. Sci. 72, 549 (1 983)

.

MITOMYCIN C

399

1 0 8 . J.H. Beijnen, Chemical Stability o f Mitomycin and Anthra109. 110. 111. 112. 113. 114. 115. 116. 117.

118.

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3 3 , 767 ( 1 9 7 6 ) . 119. T H . Keller, Am. J. Hosp. Pharm. 43, 5 9 / 6 4 ( 1 9 8 6 ) . 120. E . J . Quebbeman and N.E. Hoffman, Am. J. Hosp. Pharm. 43, 64 (1986). 121. S.U. Hildebrand-Zanki and D.H. Kern, In Vitro Cellular Devel. Biol. 2 2 , 247 ( 1 9 8 6 ) . 122. G. Rodighiero7S.M. Magno, F. Dell'Acqua and D. Veldadi, I1 Farmaco Sci. Ed. 9 , 6 5 1 ( 1 9 7 8 ) . 123. V.N. Iyer and W. Szygalski, Proc. Natl. Acad. Sci., U.S.A. 50, 355 (1963). 124. F N . Iyer and W. Szybalski, Science 1 4 5 , 55 ( 1 9 6 4 ) . 125. V.N. Iyer and W. Szybalski, Microbiology 50, 355 ( 1 9 6 3 ) . 126. W. Szybalski and V.N. Iyer, In: Antibiotics I. Mechanism of action, D. Gottlieb and P.D. Shaw (Eds.), SpringerVerlag, New York ( 1 9 6 7 ) p. 211. 127. H.W. Moore, Science 1 9 7 , 527 ( 1 9 7 7 ) . 1 2 8 . H.W. Moore and R. C z K i a k , Med. Res. Rev. 1, 249 ( 1 9 8 1 ) . 129. J.F. Fisher and R.A. Olsen, Dev. Biochemi2try 21, 240

(1982). 130. S.R. Keyes, P.M. Fracasso, D.C. Heimbrook, S. Rockwell, S.G. Sligar and A . C . Sartorelli, Cancer Res. 44, 5638 (1984). 131. A.C. Sartorelli, Biochem. Pharmacol. 35, 67 ( 1 9 8 6 ) . 132. N.R. Bachur, S.L. Gordon, M.V. Gee a n d H. Kon, Proc. Natl. Acad. Sci. U.S.A. 7 6 , 9 5 4 ( 1 9 7 9 ) . 133. Y. Hashimoto, K. Shudo a z T. Okamoto, Chem. Pharm. Bull. 28, 1961 (1980). 134. Hashimoto, K. Shudo and T. Okamoto, Tetrahedron Lett. 23, 677 ( 1 9 8 2 ) .

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lo,

47,

132,

JG.

MITOMYCIN C

401

161. H. Sasaki, T. Kakutani, M. Hashida, T. Kimura and H. Sezakj, Chem. Pharm. Bull. 33, 2968 (1985). 162. R.S. Marshall, C. Erlichmanand A.M. Rauth. Cancer Res. 4 5 , 5939 (1985). 163. Fujiwara, H. Saikusa, M. Yasuno and T. Kitagawa, Cancer Res. 4 2 , 1487 (1982). 164. K. Fujiwara, H. Saikusa, T. Kitagawa, S. Takahashi and Y. Konishi, Cancer Treat Rep. 1079 (1983). 165. C.P. Schmidbauer, P. Porpaczy, A . Georgopoulos and H. Rameis, J. Urol. 131, 818 (1984). 166. A. Hagiwara, T. Takahashi, R. Lee, T. Ueda, M. Takeda and T. Itoh, Anticancer Res. 5, 1161 (1986).

-

67,

NEOSTIGMINE

A . A . Al-Ba&*

and Ad. lahiq"

*DepahAnent 06 Phanmacaficak? Chemhin.thy and **Vep&men;t Phatrmacotogy, College 06 P h m a c y , King Saud UVLive.&Lty, P.O. Box 2457, Riyadh-77451, (Saudi A m b h l .

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

403

06

Copyright 01987 by Academic Press. Inc. All rights of reproduction in any form reserved.

A. A. AL-BADR AND M.TARIQ

404

1. History 2.

Description 2.1 Nomenclature 2.2

Formulae

2.3

Molecular Weight

2.4 Elemental Composition 2.5 Appearance, Color, Odor and Taste 3.

Physical Properties

4. Synthesis 5. Pharmacokinetics 6.

Therapeutic and Other Uses

7.

Toxicity

8. Methods o f Analysis

8.1 Identification 8.2

Titrimetric Methods

8.3

Biological Method

8.4 Spectrophotometric Methods 8.5 Chromatographic Methods 8.6 Ion-Selective Electrodes Method 8.7

Polarographic Method

References

NEOSTIGMINE

1.

405

Historv As a r e s u l t o f t h e b a s i c r e s e a r c h o f Stedman and

a s s o c i a t e s ( 1 , 2 , 3 ) i n e l u c i d a t i n g t h e chemical b a s i s o f t h e a c t i v i t y of physostigmine, Aeschlimann and R e i n e r t (4) s y s t e m a t i c a l l y i n v e s t i g a t e d a s e r i e s o f s u b s t i t u t e d phenyl esters o f a l l y l c a r b a m i c a c i d s . Neostigmine ( P r o s t i g m i n ) , a most promising member o f t h i s series, was i n t r o d u c e d i n t o t h e r a p e u t i c s i n 1931 f o r i t s s t i m u l a n t a c t i o n on t h e i n t e s t i n a l t r a c t . I t was r e p o r t e d i n d e p e n d e n t l y by Remen (5) and Walker (6) t o be e f f e c t i v e i n t h e symptomatic t h e r a p y of m y a s t h e n i a g r a v i s which is due t o d e f e c t i n s y n a p t i c t r a n s m i s s i o n a t neuromuscular j u n c t i o n . When t h e patient i s given an a p p r o p r i a t e dose o f n e o s t igmine, t h e r e s p o n s e t o t i t a n i c s t i m u l a t i o n i s improved a l o n g w i t h symptomatic improvement i n muscle s t r e n g t h ( 7 ) . Neostigmine was a l s o found u s e f u l i n p a r a l y t i c i l l e u s , a t o n y o f u r i n a r y b l a d d e r and i n glaucoma ( 8 , 9 ) . 2.

Description 2.1

Nomenclature 2.1.1

Chemical Names 3- (Dimethylcarbamoyloxy) -N ,N , N - t r i m e t h y l a n i l i n i u m bromide.

3-(Dimethylcarbamoyloxyphenyl)trimethyl-

ammonium bromide. Benzenaminium, 3- [ [ (dimethylamino)carbonyl] oxy] -N ,N,N-trimethyl bromide. (m-Hydroxypheny1)timethylammonium bromide dimethyl carbamate. 3- [ [ (Dimethylamino)carbonyl]oxy] - N , N , N , trimethylbenzenaminium bromide 3- [ [ (Dimethylamino) carbonyl loxy] -N ,N ,Ntrimethylbenzaminium methyl s u l p h a t e . (m-Hydroxyphenyl) t r i m e t h y l ammonium methyl s u l p h a t e dimethylcarbamate. (3-Dimethylcarbamoxyphenyl)trimethylamonium methyl s u l p h a t e (10-13).

A. A. AL-BADR AND M.TARIQ

2.1.2

Generic Names Neostigmine bromide; Neostigmine DCF; NFN; Neoeserine; P r o s e r i n e ; Synstigmine; Eus t igmine ; P h i 10st igmine ; Neos t igmin i bromidum; N e o s t i g m i n i i bromidum; Neostigmium bromatum (13,14).

2.1.3

Trade Names Neost i pmine bromide P r o s t i g m i n ; J u v a s t i g m i n e ; Konstigmin; Metastigmin; Normastigmin; P r o s t i g m i n ; Leostigmin ( 1 3 , 1 4 ) . Neostigmine methyl s u l p h a t e I n t r a s t i g m i n a ; J u v a s t i g m i n ; Mestastigmin; N e o e s s e r i n ; Normastigmin; P r o s t i g m i n ; Hodostin; S t i g l y n ; Stigmosan (13,14).

2.1.4

CAS R e g i s t r y Number

59-99-4 114-80-7 51-60-5 2.1.5

Neostigmine Neostigmine bromide Neostigmine methyl s u l p h a t e (14)

Wiswesser Line N o t a t i o n 1 N 1 & VOR CK-& E (Neostigmine bromide) (15)

2.2

Formulae 2.2.1

Empirical C

H BrN202 1 2 19

C13H22N206S

Neostigmine bromide Neostigmine methyl s u l p h a t e

401

NEOSTIGMINE

2.2.2

Structural CH ‘I

x0 ’-

X = Br-

II c -N

,CH3

‘CH3

Neostigmine bromide

X = CH3SOi Neostigmine methyl s u l p h a t e

2.3

Molecular Weipht Neostigmine bromide 303.2 Neostigmine methyl s u l p h a t e 334.4

2.4

Elemental Composition C 0 C 0

2.5

47.53%, H 6.32%, N 9.24%, B r 26.36%, 10.55% (bromide). 46.69%, H 6 . 6 3 % ; N 8 . 3 8 % , S 9.59%, 28.71% (methyl s u l p h a t e ) .

Appearance, Color, Odor and Taste Odorless, c o l o r l e s s c r y s t a l o r a white c r y s t a l l i n e , s l i g h t l y hygroscopic powder w i t h a b i t t e r t a s t e (14) *

3.

Physical Properties 3.1

Melting P o i n t Neost igmine bromide C r y s t a l s from a l c o h o l and e t h e r melt a t 167’ w i t h decomposition ( 1 3 ) . Neostigmine methyl s u l p h a t e C r y s t a l s from a l c o h o l melt between 142OC and 145OC (13) *

A. A, AL-BADR AND M. TARIQ

408

3.2

Extraction Neostigmine s a l t s a r e q u a t e r n a r y ammonium compounds and a r e s o l u b l e i n water. The aqueous s o l u t i o n i s a c i d i f i e d w i t h d i l u t e a c e t i c a c i d , evaporated t o dryness and e x t r a c t e d w i t h methanol. The methanol e x t r a c t w i l l c o n t a i n most o f t h e q u a t e r n a r y ammonium compound and may b e p u r i f i e d by paper chromatography ( 1 6 ) .

3.3

Solubility Neostigmine s a l t s a r e f r e e l y s o l u b l e i n w a t e r . Moderately s o l u b l e i n chloroform and e t h a n o l . I n s o l u b l e i n e t h e r (11).

3.4

Acidity Dissolve 0 . 2 0 g i n 20 m l carbon d i o x i d e - f r e e w a t e r and t i t r a t e a t pH 7 . 0 w i t h 0 . 0 2 N sodium hydroxide ( c a r b o n a t e - f r e e ) n o t more than 0 . 2 m l i s r e q u i r e d (17).

3.5

Moisture Content and H y g r o s c o p i c i t y Not more than I%, determined by d r y i n g a t 1 0 5 O C (10)

3.6

-

Storage

I t should b e s t o r e d i n a i r t i g h t c o n t a i n e r s p r o t e c t e d from l i g h t ( 1 0 ) . 3.7

Spectral Properties 3.7.1

U l t r a v i o l e t SDectrum The UV spectrum o f neostigmine bromide i n e t h a n o l i s given i n Figure 1. I t shows two maxima a t 260 nm and 266 nm. The spectrum was recorded on Varian spectrophotometer model DMS 90. Clarke (11) r e p o r t e d t h e following: Neostigmine methyl s u l f a t e i n 1 N H2SO4; maxima a t 260 nm (E 1 % ,1 cm 20) and 266 nm (E 1%,1 cm 1 8 ) .

NEOSTIGMINE

lot

2 00

2SOWavelength 300

(nm)

350

400

Figure I: Ultraviolet spectrum of neostigmine bromide in methanol.

A. A. AL-BADR AND M. TARIQ

410

3.7.2

I n f r a r e d Spectrum - (IR) The IR spectrum of n e o s t i g m i n e bromide i n K B r d i s c i s p r e s e n t e d i n F i g u r e 2 , and was recorded on Pye-Unicam I R s p e c t r o p h o t o meter model SP 1025. The f r e q u e n c i e s and t h e s t r u c t u r a l assignments a s shown below: Frequency cm-l 3020-

1

2900 .) 1730 1610

1

1595) 1490-1450 1030

1

1070) 780 and 895

As s ignment

CH s t r e t c h C = 0 (amide) s t r e t c h

C = C s t r e t c h (aromatic) CH bending v i b r a t i o n s

C - N vibration (aliphatic) CH (aromatic) bending vibrations

Clarke (11) r e p o r t e d t h e f o l l o w i n g : Neostigmine bromide, potassium bromide d i s c t h e p r i n c i p a l peaks a r e 1711, 1215 and 1154 cm-1. 3.7.3

1 Proton Nuclear Magnetic Resonance ( H NMR)

Spectrum 1 The H NMR spectrum o f neostigmine methyl s u l p h a t e i s shown i n F i g u r e 3. The drug was dissolved in deuterium o x i d e (DzO) and i t s spectrum was determined on a Varian - T60 A NMR s p e c t r o m e t e r u s i n g sodium 2,2-dimethyl 2 - s i l a p e n t ane- 5 - s u l p h o n a t e (DSS) a s t h e i n t e r n a l standard.

i

I

--

f I

8.0

7.0

Figure 3: Proton

6.0

S.OPpm(6k.O

3 -0

2 .o

I .o

0

NMR spectrum of neostigmine methyl sulphate in D20 with

TMS as internal reference.

NEOSTIGMINE

413

Assignment o f t h e chemical s h i f t s t o t h e d i f f e r e n t p r o t o n s i s shown below: Chemical shift (ppm)

Multiplicity

0

3.04)

II

singlets

-C-N(CH -3 ) 2

3. 72

singlet

4- ( g 3 )

3.80

s i n g 1e t

CH,- SO4

3.12

1

7.15-7.60 7.66-8.30 3.7.4

Proton assignment

1

m u l t i p l e t s Aromatic p r o t o n s

Carbon-13 Nuclear Magnetic Resonance (C-13 NMR) Spectrum The C-13 NMR s p e c t r a o f neostigmine methyl s u l p h a t e i n deuterium oxide (D2O) u s i n g DSS (sodium 2,2-dimethyl 2 - s i l a p e n t a n e 5-sulphonate) as an i n t e r n a l r e f e r e n c e were o b t a i n e d u s i n g a J e o l FX 100 MHz s p e c t r o m e t e r a t an ambient t e m p e r a t u r e . F i g u r e s 4 and 5 r e p r e s e n t t h e proton-decoupled and o f f resonance s p e c t r a r e s p e c t i v e l y . 12

CH3 I+

13 CH3 - S O i

-C

5

3

0 "

-N

0

1 CH

.CH3 2

The carbon chemical s h i f t s were a s s i g n e d on t h e b a s i s o f t h e chemical s h i f t t h e o r y and t h e o f f - r e s o n a n c e s p l i t t i n g p a t t e r n and a r e shown below:

414

A. A. AL-BADR AND M.TARIQ

Figure 4 : Proton -decoupled carbon-I3 NMR spectrum Of n-stigmine

methyl wlphate

in D20 with DSS as internal reference.

Figure 5 : Off -Resonance carbon -13 NMR spectrum of neostigmine methyl sulphate in 9 0 with DSS a s internal reference.

NEOSTIGMINE

415

Chemical shift (ppm)

3.7.5

Multiplicity

Carbon assignment

38.9

quartet

1

38.8

quartet

2

158.3

singlet

3

149.8

s i n gl e t

4

126.9

doublet

5

134.0

doub 1e t

6

119.8

doublet

7

154.5

singlet

8

117.1

doublet

9

59.6

quartet

10, 11 and 12

57.9

quartet

13

Mass Spectrum The e l e c t r o n impact ( E I ) mass spectrum a t 70 e V was r e c o r d e d on Varian MAT 311 mass s p e c t r o m e t e r i s shown i n F i g u r e 6 . In t h e spectrum an ion a t m/e 428 was observed which most p r o b a b l y a r i s e s from a recombination o f fragments. The spectrum shows a b a s e peak a t m/e 72. The most prominent i o n s a r e shown below:

Figure 6: Electron impld (El)mass spectrum

Of

neostigmine methyl sulphate.

NEOSTIGMINE

417

Fragment 72

208

356

CON ( c H ~+ ) ~

@-

0-CON (cH~$+

428 - CON (CH,),I+

1'

L-. 42 8

OCON ( CH3)

1

CH2

The chemical i o n i s a t i o n ( C I ) mass spectrum was o b t a i n e d on Finnigan 4000 mass s p e c t r o m e t e r and is shown i n F i g u r e 7 . The spectrum shows i o n s at m/e 1 2 7 ( t h e b a s e peak) and a t m/e 303 and both a r e probably a r i s e d from a recombination o f fragments. The most prominent i o n s a r e shown below:

Figure 7 : Chemical ionization ( C I mass Spectrum

Of

neostigmine methyl sulphate.

NEOSTIGMINE

419

m/e 127

Fragment 0 II

CH30-S-O-CH3 II

0

.t

l+

H

209

303

4.

l+

Synthesis Neostigmine bromide can b e p r e p a r e d from 3-hydroxydim e t h y l a n i l i n e , which i s made from 3 - n i t r o a n i l i n e by o r d i n a r y s y n t h e t i c methods. The 3-hydroxydimethyla n i l i n e i s d i s s o l v e d i n an a l c o h o l i c s o l u t i o n o f t h e c a l c u l a t e d amount of potassium hydroxide, and t h e s o l u t i o n o f t h e potassium hydroxide, and t h e s o l u t i o n o f t h e potassium d e r i v a t i v e so formed is t r e a t e d w i t h dimethylcarbamoyl c h l o r i d e , Me2N.COC1 (made from dimethylamine and carbonyl c h l o r i d e ) . T h i s g i v e s a t e r t i a r y b a s e which, by combination w i t h methyl bromide, y i e l d s t h e required quaternary s a l t - i . e . t h e dimethylcarbamic e s t e r o f 3-hydroxy-NNN-trimethylanil i n i u m bromide: (18, 1 9 ) .

A. A. AL-BADR AND M.TARIQ

420

OCON (CH3)

OCON (CH3)

Neostigmine methylsulphate can be made i n the same way, except t h a t i n t h e l a s t s t a g e t h e secondary amine i s combined with methyl sulphate t o form t h e s a l t [ (R-AMe3)MeSOi]

(18).

OK

OCON (CH3)

CH3S0i 0-C-N, ”0 CH3

42 1

NEOSTIGMINE

A modification of t h i s route is t o convert t h e

hydroxydimethylani l i n e i n t o t h e q u a r t e r n a r y s a l t (by combination w i t h methyl bromide o r methyl s u l p h a t e ) , and t h e n t o t r e a t t h e s a l t with dimethylcarbamoyl c h l o r i d e (18).

9

-

+

N(CH3)2

OK ClCON (CH3) P

Neostigmine can be s y n t h e s i z e d e i t h e r d i r e c t l y from 3-hydroxydimethylaniline with phosgene t o g i v e t h e carbonyl c h l o r i d e and t h e n with dimethylamine t o g i v e 3-dimethylaminophenyldimethylurethane o r t h e l a t t e r may be prepared from t h e sodium s a l t of t h e s t a r t i n g m a t e r i a l and dimethylcarbamic c h l o r i d e . In e i t h e r c a s e t h e product i s converted t o t h e m e t h y l s u l p h a t e by t r e a t m e n t with dimethyl s u l p h a t e (20).

Y

OCON (CH31

ON a

COC 1 2 N

OH

2-

.CH3S0i

OCOCl

+

OCON (CH3)

A. A. AL-BADR AND M. TARIQ

422

5.

Pharmacokinetics Neostigmine is absorbed p o o r l y a f t e r o r a l a d m i n i s t r a t i o n , such t h a t much l a r g e r doses a r e needed t h a n by t h e p a r e n t e r a l r o u t e . Whereas t h e e f f e c t i v e p a r e n t e r a l dose o f neostigmine i n man i s 0 . 5 t o 2.0 mg, t h e e q u i v a l e n t o r a l dose may be 30 mg o r more. Large o r a l doses may prove t o x i c i f i n t e s t i n a l a b s o r p t i o n i s enhanced f o r any reason and t h e q u a t e r n a r y a l c o h o l and p a r e n t compound are e x c r e t e d i n t h e u r i n e . P y r i d o s t i g mine and i t s q u a t e r n a r y a l c o h o l are a l s o t h e predominant e n t i t i e s found i n u r i n e a f t e r a d m i n i s t r a t i o n o f t h i s drug t o man (21). Neostigmine was r a p i d l y e l i m i n a t e d from t h e plasma o f 5 p a t i e n t s t o whom 5 mg o f t h e methylsulphate had been given t o a n t a g o n i s e r e s i d u a l neuromuscular b l o c k . The plasma c o n c e n t r a t i o n o f neostigmine d e c l i n e d t o about 8% o f i t s i n i t i a l v a l u e a f t e r 5 minutes with a d i s t r i b u t i o n h a l f - l i f e o f less than one minute. E l i m i n a t i o n h a l f - l i f e ranged from about 15 t o 30 minutes. Trace amounts o f neostigmine could be d e t e c t e d i n t h e plasma a f t e r one hour ( 1 4 ) . O f neostigmine t h a t reaches t h e l i v e r , 98 p e r c e n t is metabolized i n 10 minutes. ( 2 2 ) I t s t r a n s f e r from plasma t o l i v e r c e l l s and then t o b i l e is probably p a s s i v e i n c h a r a c t e r . Since c e l l u l a r membranes permit t h e passage o f plasma p r o t e i n s s y n t h e s i z e d i n l i v e r i n t o t h e blood stream through c a p i l l a r y walls o r lymphatic v e s s e l s , t h e y may n o t p r e s e n t a b a r r i e r t o t h e d i f f u s i o n o f q u a t e r n a r y amines such as n e o s t i g mine. P o s s i b l y t h e r a p i d h e p a t i c metabolism o f neostigmine p r o v i d e s a downhill g r a d i e n t f o r t h e c o n t i n u a l d i f f u s i o n of t h i s compound ( 2 3 ) . A c e r t a i n amount may be hydrolyzed slowly by plasma c h o l i n e s t e r a s e . When neostigmine was given by mouth t o 2 p a t i e n t s with myasthenia g r a v i s less t h a n 5 % was found unchanged i n t h e u r i n e ( 1 4 ) . Following i n t r a m u s c u l a r a d m i n i s t r a t i o n about 65% o f a dose i.s e x c r e t e d i n t h e u r i n e unchanged ( 1 0 ) . 20% of an o r a l dose is e x c r e t e d i n t h e u r i n e and 50% i n t h e faeces; less t h a n 5% o f t h e o r a l dose i s e x c r e t e d i n t h e u r i n e unchanged.

NEOSTIGMINE

6.

423

T h e r a n e u t i c and Ot he r Uses Neostigmine is a q u a t e r n a r y ammonium a n t i c h o l i n e s t e r a s e . I t a c t s a t t h e e s t e r a t i c s i t e o f t h e enzyme t o form t h e i n a c t i v e dimethylcarbamoyl enzyme. Neostigmine h a s widespread a c t i o n s , b u t f o r t u n a t e l y i t s e f f e c t s a r e more prominent on c e r t a i n s t r u c t u r e s t h a n on o t h e r s , b e i n g p a r t i c u l a r l y e f f e c t i v e on t h e bowel, u r i n a r y b l a d d e r , and s k e l e t a l muscle, t h e p u p i l , t h e h e a r t , blood p r e s s u r e , and s e c r e t i o n s b e i n g a f f e c t e d t o a much lesser e x t e n t i n doses t h a t are o r d i n a r i l y e f f e c t i v e on r e l a t e d s t r u c t u r e s (19) . The drug i n h i b i t s c h o l i n e s t e r a s e a c t i v i t y and p r o l o n g s and i n t e n s i f i e s t h e m u s c a r i n i c and n i c o t i n i c e f f e c t s o f a c e t y l c h o l i n e . I t proba bly a l s o has d i r e c t e f f e c t s on s k e l e t a l muscle f i b r e s . The a n t i c h o l i n e s t e r a s e a c t i o n s o f ne ost igmi ne are r e v e r s i b l e . I t is used mainly f o r i t s a c t i o n on s k e l e t a l muscle, and less f r e q u e n t l y t o i n c r e a s e a c t i v i t y o f smooth muscle ( 1 4 ) . Neostigmine me t hylsul pha t e i s used i n t h e t r e a t m e n t o f myasthenia g r a v i s i n u s u a l d o s e s o f 1 t o 2 . 5 mg d a i l y given i n d i v i d e d dose s by subcutaneous, i n t r a m u s c u l a r , o r i n t r a v e n o u s i n j e c t i o n a c c ordi ng t o t h e s e v e r i t y o f t h e c o n d i t i o n (14). Neostigmine i s used i n c o n d i t i o n s o f u r i n a r y b l a d d e r a t o n y due t o p o s t a n e s t h e t i c d e p r e s s i o n o r t o n e u r o l o g i c a l d i s o r d e r s (19). I t is a l s o used i n t h e t r e a t m e n t o f p a r a l y t i c i l e u s postoperative urinary retention, i n d o s es o f 0 . 5 t o 1 mg. For e x p e l l i n g i n t e s t i n a l f l a t u s p r i o r t o r a di ogra phy o f t h e g a l l - b l a d d e r , kidneys , o r u r e t e r s , a s i n g l e dose o f 500 ug h a s sometimes been used ( 1 4 ) . Neostigmine can b e employed as a d i a g n o s t i c t e s t , e s p e c i a l l y a f t e r symptoms have been pur posely a c c e n t u a t e d by t h e a d m i n i s t r a t i o n o f q u i n i n e . Neostigmine is used as d i a g n o s t i c t e s t a gent i n myotonia c o n g e n i t a , i n which c o n d i t i o n ne ostigmine a g g r a v a t e s t h e symptoms. I t may be used as a d i a g n o s t i c agent f o r e a r l y pregnancy o r t o t r e a t delayed menstr uation: given i n t r a m u s c u l a r l y on t h r e e s u c c e s s i v e days i t w i l l i n d u c e m e nst rua ti on w i t h i n 72 hours a f t e r t h e l a s t dose u n l e s s t h e p a t i e n t i s pre gna nt . Neostigmine i s used t o p i c a l l y t o t r e a t primary open-angle glaucoma and i n

424

A. A. AL-BADR AND M.TARlQ

t h e emergency t r ea t m e n t o f primary a c u te - a n g le glaucoma. Miosis lasts 1 2 t o 36 h o u r s . Longer-acting a n t i c h o l i n e s t e r a s e s a r e p r e f e r r e d f o r t h e t r e a t m e n t o f accommod a t i v e e s o t r o p i a (19). 7.

T o x i c i t y and S i d e E f f e c t s Adverse and s i d e e f f e c t s o f neostigmine i n c l u d e s a l i v a t i o n , a n o r e x i a, nausea and vomiting , abdominal cramps and d i a r r h o e a . Symptoms of overdosage i n c l u d e sweating, lachrymation, watery n a s a l d i s c h a r g e , e r u c t a t i o n , i n v o l u n t a r y d e f a e c a t i o n and u r i n a t i o n f l u s h i n g , m i o s i s , c o n j u n c t i v a l co n g e stio n , c i l i a spasm, brow ache, nystagmus, r e s t l e s s n e s s , a g i t i o n , f e a r , e x c e s s i v e dreaming, i n c r e a s e d b r o c h i a l s e c r e t i o n combined wi t h b r o n c h o c o n s t r i c t i o n , b r a d y c a r d ia and hypotension, muscle cramps, s c a t t e r e d f a s i c u l a t i o n s and e v e n t u a l s e v e r e weakness and p a r a l y s i s , c o n v u lsio n s, and coma. I t h as a l s o been s t a t e d t h a t p a r a d o x ic a l e f f e c t could o c c u r due t o i n t e r a c t i o n between n i c o t i n i c and muscarinic a c t i o n s ; a consequence of t h i s could b e some evidence o f an a c c e l e r a t i o n p u l s e - r a t e and e l e v a t i o n o f blood p r e s s u r e . Death may f o llo w due t o cardiac a r r e s t o r central respiratory paralysis and pulmonary oedema. The major symptom o f overdosage i n myasthena g r a v i s i s i n c r e a s e d muscular weakness ( 1 4 ) . I t i s c o n t r a i n d i c a t e d i n a s t h m a t i c p a t i e n t s . I t should n o t be employed al o n g with c h o l i n e esters e x c e p t f o r ophthalmologic u s e. Quinidine i n t e r f e r e s w i t h t h e a c t i o n o f neostigmine ( 1 9 ) .

Neostigmine methyl s u l p h a t e , by i n c r e a s i n g i n t e s t i n a l m o t i l i t y , may cause d i s r u p t i o n o f i n t e s t i n a l s u t u r e l i n e s (10). 8.

Methods o f Analysis 8.1

Identification a)

To 0 . 1 m l o f a 1 p e r c e n t w/v s o l u t i o n , add 0.5 m l of sodium hydroxide s o l u t i o n and e v a p o r a te t o d r y n e s s on a water-bath. Heat q u i c k l y on an o i l - b a t h t o about 250' and m a in ta in a t t h i s temperature f o r about t h i r t y seconds. Cool, d i s s o l v e t h e r e s i d u e i n 1 m l o f water, c o o l i n i c e water, and add 1 m l o f diazoaminobenzene-

425

NEOSTIGMINE

sulphonic acid; a cherry-red colour i s produced ( 1 5 ) . C r y s t a l t e s t s . Micro: l e a d i o d i d e s o l u t i o n b r a n c h i n g n e e d l e s ( s e n s i t i v i t y : 1 i n 400) ; p l a t i n i c i o d i d e s o l u t i o n - bunches o f curved ( s e n s i t i v i t y : 1 i n 400) (11). The i n f r a r e d a b s o r p t i o n spectrum o f a potassium bromide d i s p e r s i o n o f i t , p r e v i o u s l y d r i e d a t 1 0 5 O f o r 3 h o u r s , e x h i b i t s maxima o n l y a t t h e same wavelengths as t h a t of a s i m i l a r p r e p a r a t i o n o f USP Neostigmine Bromide Reference Standard (12).

A s o l u t i o n (1 i n 50) responds t o t h e t e s t o f bromide (neostigmine bromide) (12). 8.2

T i t r i m e t r i c Methods 8.2.1

Aqueous T i t r a t i o n B r i t i s h Pharmacopoeia (1973) (17) r e p o r t e d t h e f o l l o w i n g procedure f o r t h e a s s a y of neostigmine methyl s u l p h a t e : D i s s o l v e 0.15 g i n 20 m l of w a t e r , t r a n s f e r t o a semimicro ammonia d i s t i l l a t i o n a p p a r a t u s , and add 20 m l of a 50 p e r c e n t w/v s o l u t i o n o f sodium hydroxide. Pass a c u r r e n t o f steam through t h e m i x t u r e , c o l l e c t t h e d i s t i l l a t e i n 50 m l o f 0 . 1 N s u l p h u r i c a c i d u n t i l t h e t o t a l volume r e a c h e s about 200 m l , and t i t r a t e t h e e x c e s s o f a c i d w i t h 0.02 N NaOH u s i n g methyl r e d s o l u t i o n as i n d i c a t o r . Repeat t h e o p e r a t i o n w i t h o u t t h e s u b s t a n c e b e i n g examined; t h e d i f f e r e n c e between t h e t i t r a t i o n s represents t h e amount o f a c i d r e q u i r e d t o n e u t r a l i s e t h e dimethylamine formed from t h e n e o s t i g m i n e . Each m l o f 0.02 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 0.006688 g of Cl3HZ2N2O6S. Huang -e t a1 (24) d e s c r i b e d a s i m p l e , r a p i d and accurate method u s i n g t h e a p p l i c a t i o n of a l t e r n a t i n g - c u r r e n t o s c i l l o p o l a r o g r a p h i c t i t r a t i o n i n pharmaceutical a n a l y s i s . In

426

A. A. AL-BADR AND M.TARIQ

t h i s method neostigmine methyl s u l p h a t e i n i n j e c t i o n s was t i t r a t e d w i t h sodium t e t r a p h e n y l b o r a t e . To t h e i n j e c t i o n s o l u t i o n ( 2 0 ml) c o n t a i n i n g 10 mg o f neostigmine methyl s u l p h a t e were added 10 m l of 0 . 0 1 N sodium t e t r a p h e n y l b o r a t e and 10 m l o f a c e t a t e b u f f e r s o l u t i o n (pH 5 . 3 ) . The s o l u t i o n was d i l u t e d t o 50 m l w i t h w a t e r , a f t e r 5 minutes t h e p r e c i p i t a t e was formed, t h e c o n t e n t s were f i l t e r e d . A 25 m l p o r t i o n o f f i l t r a t e was t r e a t e d w i t h 8 drops o f 40% sodium hydroxide s o l u t i o n and t i t r a t e d w i t h 0.01 N TI2S04, t h e d i s appearance o f t h e i n c i s i o n o f sodium t e t r a p h e n y l b o r a t e on t h e curve of d/E/dt vs E b e i n g used t o i n d i c a t e t h e end p o i n t . The r e s u l t s were c o r r e c t e d f o r a blank v a l u e . The r e c o v e r y range from 9 9 . 3 - 100.2% and t h e c o e f f i c i e n t o f v a r i a t i o n was 0 . 4 2 % . Tsubouchi -e t a 1 (25) r e p o r t e d a method o f a n a l y s i s o f neostigmine u s i n g t h e a p p l i c a t i o n o f one-phase end-point change system i n two phase t i t r a t i o n t o amine drug a n a l y s i s . Neostigmine i n aqueous s o l u t i o n ( 5 o r 10 mM) was determined by t i t r a t i o n w i t h aqueous 0 . 0 1 M t e t r a p h e n y l b o r a t e with tetrabromo p h e n o l p h t h a l i e n e t h y l ester a s an i n d i c a t o r i n t h e o r g a n i c phase ( 1 , 2 - d i c h l o r o e t h a n e ) , i n b o r a t e phosphate b u f f e r medium (pH 5 . 5 . - 7 . 5 ) . The end-point was d e t e c t e d by means o f c o l o r change o f t h e i n d i c a t o r i n t h e o r g a n i c phase without t r a n s f e r of i n d i c a t o r between phases. Various common i o n s ( i n c l u d i n g c a r b o n a t e , a c e t a t e , c i t r a t e , and t a n n a t e ) did not i n t e r f e r e but thiamine , papaverine , dodecyle s u l p h a t e and mercury caused interference. Diamandis and C h r i s t o p o u l o s ( 2 6 ) developed a p o t e n t i o m e t r i c t i t r a t i o n o f neostigmine bromide i n pharmaceutical compounds by t i t r a t i n g them w i t h sodium t e t r a p h e n y l b o r a t e . 25 M 1 o f aqueous s o l u t i o n ( 0 . 2 1 mM) o f neostigmine bromide and 5 m l o f t h e a p p r o p r i a t e b u f f e r s o l u t i o n was added.

NEOSTIGMINE

421

The s o l u t i o n was t i t r a t e d p o t e n t i o m e t r i c a l l y a t 0.36 m l p e r minute with 0.01 M sodium t e t r a p h e n y l b o r a t e a t 2 2 O 5 20 w i t h continuous s t i r r i n g . The end-point was d e t e c t e d by a t e t r a p h e n y l b o r a t e s e l e c t i v e e l e c t r o d e . T h i s method was adopted f o r d e t e r m i n a t i o n o f neostigmine i n powders, i n j e c t i o n s , drops and s y r u p s . United S t a t e s Pharmacopeia X I X (1980) (12) d e s c r i b e d t h e f o l l o w i n g procedure f o r t h e a s s a y o f neostigmine methyl s u l p h a t e : Place about 100 mg o f neostigmine methyl s u l p h a t e , a c c u r a t e l y weighed, i n a 500 m l Kjeldahl f l a s k , d i s s o l v e i n 150 m l o f w a t e r , and add 40 m l of sodium hydroxide s o l u t i o n (1 i n 1 0 ) . Connect t h e f l a s k by means o f a d i s t i l l a t i o n t r a p t o a well-cooled condenser t h a t d i p s i n t o 25 m l o f b o r i c a c i d s o l u t i o n ( 1 i n 2 5 ) , d i s t i l about 150 m l o f t h e c o n t e n t s of t h e f l a s k , add methyl p u r p l e TS t o t h e s o l u t i o n i n t h e r e c e i v e r , and t i t r a t e with 0.02 N s u l p h u r i c a c i d . Perform a blank d e t e r m i n a t i o n , and make t h e n e c e s s a r y c o r r e c t i o n . Each m l of 0 . 0 2 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 6.688 mg o f C13H22N206S' B r i t i s h Pharmacopoeia (1973) (17) r e p o r t e d t h e f o l l o w i n g procedure f o r t h e a s s a y of n e o s t igmine t a b l e t s : Weigh and powder 2 t a b l e t s . T r a n s f e r a q u a n t i t y o f t h e powder, e q u i v a l e n t t o 0.15 o f neostigmine bromide, t o a semi-micro ammonia d i s t i l l a t i o n a p p a r a t u s , add 20 m l o f a 50% w/v s o l u t i o n o f sodium hydroxide and 0 . 5 m l o f a 2% s o l u t i o n o f octan-2-01 i n l i q u i d p a r a f f i n and complete t h e a s s a y d e s c r i b e d under neostigmine methyl s u l p h a t e . (above) b e g i n i n g a t t h e words "Pass a c u r r e n t o f steam . . . . I ! . Each m l o f 0.02 N s u l p h u r i c a c i d i s e q u i v a l e n t t o 0.006064 g o f C12H19BrN202.

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8.2.2

T i t r i m e t r i c Methods Non-aqueous T i t r a t i o n United S t a t e Pharmacopeia X I X (1980) (12) d e s c r i b e d t h e f o l l o w i n g procedure f o r t h e a s s a y of neostigmine bromide: Dissolve about 750 mg of neostigmine bromide a c c u r a t e l y weighed, i n a m i x t u r e o f 70 m l of g l a c i a l a c e t i c a c i d and 20 m l o f m e r c u r i c a c e t a t e TS, add 4 d r o p s o f c r y s t a l v i o l e t TS, and t i t r a t e with 0 . 1 N p e r c k l o r i c a c i d t o a b l u e e n d - p o i n t . Perform a blank determinat i o n , and make any n e c e s s a r y c o r r e c t i o n . Each m l of 0 . 1 N p e r c h l o r i c a c i d i s e q u i v a l e n t t o 30.32 mg o f C12H19BrN202 (12). Bayer and Posgay (27) s u g g e s t e d a p e r c h l o r i c a c i d t i t r a t i o n method f o r t h e d e t e r m i n a t i o n o f neostigmine i n pharmaceutical p r e p a r a t i o n s . The neostigmine s o l u t i o n was t i t r a t e d with 0 . 1 N p e r c h l o r i c a c i d which h a s been s t a n d a r d i z e d with anhydrous potassium c a r b o n a t e w i t h 0.1% g e n t i a n v i o l e t i n a c e t i c a c i d c o n t a i n i n g 3% m e r c u r i c a c e t a t e as indicator. Surmann et a 1 (28) r e p o r t e d a t i t r a t i o n method f o r t h e pharmaceutical h y d r o c h l o r i d e s and hydrobromides i n non-aqueous media. T h i s method i n v o l v e d i r e c t t i t r a t i o n o f drug w i t h p e r c h l o r i c a c i d i n a c e t i c anhydride medium, w i t h n a p h t h o l benzein o r Sudan Red B an i n d i c a t o r f o r hydroc h l o r i d e s f o r hydrobromides r e s p e c t i v e l y . Kracmarova and Kracmar (29) r e p o r t e d t h e f o l l o w i n g non-aqueous tit r a t i o n : Evaporate t h e sample o f t h e eye d r o p s (5 ml) on a w a t e r b a t h t o d r y n e s s , d i s s o l v e t h e r e s i d u e i n anhydrous a c e t i c a c i d (2 x 20 m l ) , add c r y s t a l v i o l e t (0.2% i n anhydrous a c e t i c a c i d ) (2 d r o p s ) a s i n d i c a t o r , and t i t r a t e with 0.1 N perchloric acid t o a b l u e e n d - p o i n t , add 5% HgII a c e t a t e s o l u t i o n

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(5 ml), mix and t i t r a t e a g a i n t o a b l u e endp o i n t ; t h e d i f f e r e n c e between t h e t i t r e s corresponds t o neostigmine bromide. 8.2.3

I o d i m e t r i c Methods Koka (30) developed an i o d i m e t r i c determinat i o n o f p r o s e r i n e (neostigmine methyl s u l p h a t e ) i n drug f o r m u l a t i o n s . The drug sample i s d i s s o l v e d i n water and t h e solution is t r e a t e d with 2-3 m l of d i l u t e s u l p h u r i c a c i d , 2-3 m l o f 10% potassium i o d i d e s o l u t i o n and 5 m l of 0 . 1 N i o d i n e , d i l u t e d t o 25 m l with w a t e r and f i l t e r e d ; 10 m l o f f i l t r a t e i s t i t r a t e d with 0 . 1 N sodium t h i o s u l p h a t e . Mitchenko and Kirichenko (31) r e p o r t e d t h e u s e of i o d i m e t r i c method f o r t h e determinat i o n o f neostigmine methyl s u l p h a t e and p i l o c a r p i n e HC1 i n eye-drops. Neostigmine methyl s u l p h a t e and p i l o c a r p i n e HC1 a r e determined by r e a c t i o n with i o d i n e s o l u t i o n i n d a r k , f i l t e r i n g t h e m i x t u r e and t i t r a t i n g u n r e a c t e d i o d i n e w i t h sodium t h i o s u l p h a t e s o l u t i o n . When b o t h a r e p r e s e n t t h e sum o f t h e two i s determined by above method and only p i l o c a r p i n e i s determined a l k a l i m e t r i c a l l y , a r g e n t i m e t r i c a l l y o r mercurim e t r i c a l l y . Neostigmine methyl s u l p h a t e is determined by t h e d i f f e r e n c e . The r e l a t i v e e r r o r i n determining t h e two d r u g s i n eye-drops is 3.56 and 2.85 r e s p e c t i v e l y .

8.3

B i o l o g i c a l Method

Buckles and Bullock (32) r e p o r t e d t h e a p p l i c a t i o n o f enzyme i n h i b i t i o n t o t h e e s t i m a t i o n o f s m a l l q u a n t i t i e s o f drugs p o s s e s s i n g a n t i c h o l i n e s t r a s e a c t i v i t y . The method is based on t h e i n h i b i t i o n of t h e pseudocholinesterase a c t i v i t y of horse serum. 5 M 1 o f sample c o n t a i n i n g 5 ug of neostigmine i s mixed w i t h 1 m l o f h o r s e serum, 1 m l o f 0.2% cresol r e d s o l u t i o n and 37 m l o f w a t e r . The m i x t u r e i s h e a t e d t o 4OoC and pH a d j u s t e d t o 7 . 9 . After 15 minutes add 5 m l o f 3% a c e t y l c h o l i n e p e r c h l o r a t e s o l u t i o n and r e a d j u s t

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t h e pH t o 7 . 9 . Maintain t h e pH between 7 . 8 and 8 . 0 d u r i n g 15 minutes by dropwise a d d i t i o n o f 0.025 N sodium h y d r o x i d e and r e c o r d t h e volume of a l k a l i used. C o r r e c t f o r nonenzymic h y d r o l y s i s by c o n d u c t i n g an experiment w i t h 1 m l b u f f e r i n s t e a d o f h o r s e serum and c a r r y o u t a b l a n k w i t h 5 m l w a t e r i n s t e a d o f sample. The p e r c e n t a g e i n h i b i t i o n i s a l i n e a r f u n c t i o n o f t h e l o g c o n c e n t r a t i o n of n e o s t i g m i n e methyl sulphate. 8.4

S p e c t r o p h o t o m e t r i c Methods 8.4.1

C o l o r i m e t r i c Methods Belal et a l , (33) d e s c r i b e d a s p e c t r o photometric determination o f neostigmine i n t a b l e t s and ampoules. T a b l e t p r e p a r a t i o n s c o n t a i n i n g n e o s t i g m i n e methyl s u l p h a t e were powdered and d i s s o l v e d i n 50 m l o f w a t e r and 1 m l of s o l u t i o n was mixed w i t h 3 m l o f c i t r a t e - p h o s p h a t e b u f f e r s o l u t i o n (pH 7 .8 ) and 2 m l o f a 0.1% s o l u t i o n o f bromothymol b l u e i n 0 . 0 1 N sodium hydroxide. The m i x t u r e was e x t r a c t e d w i t h chloroform b e f o r e measurement o f i t s absorbance a t 416 nm vs a r e a g e n t b l a n k . A l t e r n a t i v e l y 1 0 m l of chloroform e x t r a c t was t r e a t e d w i t h 10 ml of 0 . 0 1 N sodium hydroxide and t h e aqueous l a y e r was s e p a r a t e d and made up t o 25 m l with water, i t s absorbance b e i n g measured a t 615 nm as a r e a g e n t b l a n k . The c a l i b r a t i o n graph were r e c t i l i n e a r f o r 0 . 2 - 1 . 6 mg. The r e c o v e r y by t h i s method was 1 0 0 . 5 % .

Belikov -e t a1 (34) have used sulphonepht h a l e i n dyes as e x t r a c t i o n r e a g e n t s f o r t h e e x t r a c t i o n o f q u a t e r n a r y ammonium compounds. They r e p o r t e d t h e optimum pH v a l u e , Xmax, d i s t r i b u t i o n c o e f f i c i e n t and s e n s i t i v i t y of reactions f o r t h e ion a s s o c i a t i o n complexes o f n e o s t i g m i n e methyl s u l p h a t e . I n a g e n e r a l procedure f o r d e t e r m i n i n g t h e q u a t e r n a r y ammonium compounds, 0 . 5 m l o f 0.01% s o l u t i o n of compound is added t o 5 m l o f a b u f f e r s o l u t i o n o f a p p r o p r i a t e pH, 0 . 5 m l of 0.1% dye

43 I

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s o l u t i o n i s added, t h e complex is e x t r a c t e d i n t o 5 m l o f chloroform and absorbance o f o r g a n i c phase i s measured a t 400-434 nm. Relative e r r o r i s less t h a n 2 % . Tsubouchi (35) r e p o r t e d a s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f o r g a n i c c a t i o n s by s o l v e n t e x t r a c t i o n w i t h tetrabromophenolp h t h a l i e n e t h y l e s t e r . 5 1.11 of t h e sample c o n t a i n i n g less t h a n 0.01 WM of n e o s t i g m i n e was e x t r a c t e d w i t h 2 m l o f 0.07% e t h a n o l i c tetrabromophenolphthalien e t h y l e s t e r (potassium s a l t ) and 5 m l o f b o r a t e phosphate b u f f e r (pH l o ) , d i l u t e t o 25 m l w i t h water and shake f o r 2 minutes w i t h 1 0 m l of 1,2,dichloroethane. S e p a r a t e and f i l t e r t h e o r g a n i c phase and measure i t s absorbance a t 615 nm a g a i n s t r e a g e n t b l a n k . The c o e f f i c i e n t o f v a r i a t i o n was 1.11%. 8.4.2

U l t r a v i o l e t SDectronhotometric Method Rita (36) developed a u l t r a v i o l e t spectrophotometric assay o f neostigmine methyl s u l p h a t e as t h e a l k a l i n e h y d r o l y s i s p r o d u c t , 3- (dimethy1amino)phenol. An a l i q u o t of i n j e c t i o n preparation equivalent t o 5 mg o f n e o s t i g m i n e methyl s u l p h a t e was a c i d i f i e d and any phenol o r p-hydroxy b e n z o a t e p r e s e r v a t i v e p r e s e n t was e x t r a c t e d w i t h chloroform. The r e s i d u a l aqueous s o l u t i o n was made a l k a l i n e and h e a t e d on steam b a t h f o r 30 m i n u t e s and t h e 3- (dimethylamino) phenol formed is determined by UV-spectrophotometry. Kracmarova and Kracmar (29) d e s c r i b e d a s p e c t r o p h o t o m e t r i c method f o r e v a l u a t i o n of n e o s t i g m i n e bromide and n e o s t i g m i n e methyl sulphate with regards t o t h e degradation p r o d u c t s . To 5 m l o f i n j e c t i o n s o l u t i o n , 2 m l o f 0 . 0 5 N H2SO4 was added. The m i x t u r e was d i l u t e d t o 10 m l w i t h water. The absorbance was r e c o r d e d a t 261 nm f o r n e o s t i g m i n e bromide o r a t 260 nm f o r n e o s t i g m i n e methyl s u l p h a t e o r a t 266 nm f o r b o t h o f t h e compounds.

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Mytchenko and Kyrychenko (37) r e p o r t e d a spectrophotometric determination o f p r o s e r i n e ( n e o s t igmine methyl s u l p h a t e ) i n m e d i c i n a l f o r m u l a t i o n s . The t a b l e t s are g r i n d e d i n w a t e r and shaked. 0.5% s o l u t i o n i s f i l t e r e d and t h e absorbance o f f i l t r a t e i s d i r e c t l y r e c o r d e d a t 260 nm. T h i s method can a l s o be used f o r a n a l y s i s o f n e o s t i g m i n e i n eye drops ( c o n t a i n i n g a l s o p i l o c a r p i n e HC1) a f t e r d i l u t i o n . The accuracy o f method i s w i t h i n ? 0 . 6 6 % . 8.4.3

I n f r a r e d S p e c t r o p h o t o m e t r i c Methods - I R Mynka et a1 (38) r e p o r t e d t h e i n f r a r e d s p e c t r o s c o p y o f drugs o f t h e amide class. The I R spectrum of n e o s t i g m i n e methyl s u l p h a t e was determined and i n t e r p r e t e d by measurement a t 1732 cm-l, A n a l y s i s can be done w i t h a maximum e r r o r o f 1 . 6 6 % . Kracmarova and Kracmar (29) r e p o r t e d s p e c t r o p h o t o m e t r i c a n a l y s i s of n e o s t i g m i n e i n i n f r a r e d r e g i o n . Grind t h e sample w i t h l i q u i d p a r a f f i n (12 d r o p s ) and r e c o r d t h e spectrum i n a sodium c h l o r i d e c e l l i n t h e range 3-15 1-1. C h a r a c t e r i s t i c band f o r neostigmine bromide and n e o s t i g m i n e methyl s u l p h a t e a r e observed.

8.4.4

Photometric Method Kracmarova and Kracmar (29) r e p o r t e d a p h o t o m e t r i c method €or t h e e v a l u a t i o n o f n e o s t i g m i n e bromide and n e o s t i g m i n e methyl s u l p h a t e as f o l l o w s : To t h e i n j e c t i o n s o l u t i o n ( 1 . 5 ml) add b u f f e r s o l u t i o n (pH 4 . 6 ) (5 m l ) , bromoc r e s o l p u r p l e s o l u t i o n (2 g i n 15 m l o f water and 3 . 2 m l o f 0 . 1 N - potassium hydroxide d i l u t e d t o 250 m l w i t h w a t e r ) (S ml) and chloroform (25 m l ) . Shake f o r 1 minute i n a s e p a r a t i n g f u n n e l , and f i l t e r t h e chloroform l a y e r ; r e p e a t t h e e x t r a c t i o n w i t h 2 m l of chloroform and d i l u t e t h e combined e x t r a c t s w i t h chloroform t o 50 m l .

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To a 25 m l p o r t i o n add 0 . 1 N potassium hydroxide (20 m l ) , shake f o r 30 seconds, f i l t e r t h e aqueous l a y e r , d i l u t e t o 100 m l w i t h w a t e r and measure t h e e x t i n c t i o n a t 570 nm. 8.5

Chromatographic Methods 8.5.1

Paper Chromatographic Method Giebelmann (39) developed a p a p e r chromatog r a p h i c d e t e c t i o n f o r phenoxy compounds having a phenyl e s t e r f u n c t i o n a l group using t h e following s o l v e n t s - butanol-20% formic a c i d ( 4 : 1 ) , methanol a c e t o n e t r i e t h a n o l a m i n e (100: 100: 3) and b u t a n o l water ( 6 : l ) . As l i t t l e as 2.5-10 1-16 of n e o s t i g m i n e can be d e t e c t e d w i t h M i l l o n ' s reagent. Scott et a l , (40) r e p o r t e d an i n v e s t i g a t i o n on t h e metabolism o f n e o s t i g m i n e i n p a t i e n t s w i t h myasthenia g r a v i s . N e o s t i g m i n e an d m -h y d r oxyphen y 1t r i m e t h y 1ammon iurn bromide are p r e c i p i t a t e d from u r i n e w i t h aqueous bromine. The p r e c i p i t a t e i s e x t r a c t e d with 50% methanol. T h i s e x t r a c t is a p p l i e d t o Amberlite CG-50 r e s i n a t pH 6.86. Ten b a s e s a r e e l u t e d w i t h 0 . 2 N h y d r o c h l o r i c a c i d and s u b m i t t e d t o chromatography on Whatman NO 541 p a p e r w i t h butanol-ethanol-water-acetic a c i d (32: 8 : 1 2 : 1) as s o l v e n t . The Rf v a l u e o f n e o s t i g m i n e was between 0 . 5 - 0 . 3 . Kracmarova and Kracmar ( 2 9 ) d e s c r i b e d procedures f o r t h e determination o f n e o s t i g m i n e bromide and n e o s t igmine methyl sulphate i n various pharmaceutical preparat i o n s , a s w e l l as f o r t h e chromatographic control of t h e degradation products; 3 dimethylaminophenol and (3-hydroxyphenyl) trimethylammonium s a l t s , w i t h t h e u s e o f Whatman No. 1 p a p e r , butanol-conc-aqueous ammonia-water (3: 1:4) as s o l v e n t , and Dragendorff r e a g e n t as t h e d e t e c t i n g a g e n t .

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Clarke (11) d e s c r i b e d t h e f o l l o w i n g : (1) Paper: Whatman No. 1, s h e e t 14 x 6 i n , b u f f e r e d by d i p p i n g i n a 5% s o l u t i o n o f sodium hydrogen c i t r a t e , b l o t t i n g , and d r y i n g a t 250 f o r 1 hour. I t can be s t o r e d immediately.

Sample: 2 . 5 1-11 of a 1%s o l u t i o n ; i n 2 N a c e t i c a c i d i f possible, otherwise i n 2 N h y d r o c h l o r i c a c i d , 2 N sodium hydroxide, o r ethanol. S o l v e n t : 4 . 8 g of c i t r i c a c i d i n a m i x t u r e of 130 m l o f water and 870 m l o f n - b u t a n o l . This may be used f o r s e v e r a l weeks i f water i s added from time t o time t o keep t h e s p e c i f i c g r a v i t y a t 0.843 t o 0.844. Development: Ascending, i n t a n k 8 x 11 x 15% i n ; 4 s h e e t s b e i n g run a t a time. Time of run, 5 h o u r s . Location under u l t r a v i o l e t l i g h t , a b s o r p t i o n ; locat ion reagents : Iodoplati na te spray ; weak r e a c t i o n ; bromocresol green s p r a y , weak r e a c t i o n . Rf 0.20. ( 2 ) Paper: Whatman No. 1 o r No. 3, s h e e t 17 x 19 cm, implegnated by d i p p i n g i n a 10% s o l u t i o n o f t r i b u t y r i n i n acetone and d r y i n g i n a i r .

Sample: 5 p1 o f 1 t o 5% s o l u t i o n s i n e t h a n o l o r chloroform. Solvent:

A c e t a t e b u f f e r (pH 4 . 5 8 ) .

E q u i l i b r a t i o n : The b e a k e r c o n t a i n i n g t h e solvent is equilibrated i n a thermostatically c o n t r o l l e d o v e r a t 95O f o r about 1 5 minutes. Development: Ascending, t h e paper i s f o l d e d i n t o a c y l i n d e r and c l i p p e d , t h e c y l i n d e r is i n s e r t e d i n t h e b e a k e r c o n t a i n i n g t h e s o l v e n t which i s n o t removed from t h e oven. A p l a t e - g l a s s d i s k t h i c k l y smeared with s i l i c o n e g r e a s e may s e r v e as a cover. Time of run, 15 t o 20 minutes.

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Location: Under u l t r a v i o l e t l i g h t , a b s o r p t i o n , Rf 1.00. (3) Paper and sample a s i n 2 above. S o l v e n t : Phosphate b u f f e r (pH 7 . 4 ) . E q u i l i b r a t i o n : The peak c o n t a i n i n g t h e solvent is equilibrated i n a thermostatic a l l y c o n t r o l l e d oven a t 86' f o r about 15 minutes. Development:

as i n 2 above.

Location: Under u l t r a v i o l e t l i g h t , a b s o r p t i o n , Rf 0.66. 8.5.2

Thin-Layer Chromatography (TLC) Anand (41) r e p o r t e d a t h i n - l a y e r chromatog r a p h i c and s p e c t r o p h o t o m e t r i c i n v e s t i g a t i o n on t h e i d e n t i f i c a t i o n o f a p h e n o l i c i m p u r i t y i n n e o s t i g m i n e . The samples o f neostigmine were a p p l i e d on TLC p l a t e s c o a t e d w i t h alumina-D and were developed f o r 30 minutes w i t h chloroform-benzenemethanol (5:4:1) t o g i v e a good s e p a r a t i o n . Neostigmine was d e t e c t e d w i t h i o d i n e vapours o r w i t h D r a g e n d o r f f ' s r e a g e n t . The u l t r a v i o l e t spectrophotometric technique is a l s o used f o r q u a n t i t a t i v e e s t i m a t i o n . Neostigmine h a s a maxima a t 261 nm. P o r s t and Kny (42) d e s c r i b e d a n a l y s i s o f neostigmine eye drops u s i n g TLC system and a b s o r p t i o n s p e c t r o s c o p y . The drug i s d e t e c t e d and determined by TLC on c e l l u l o s e and u l t r a v i o l e t o r v i s i b l e s p e c t r o p h o t o m e t r y is done a f t e r f o r m a t i o n o f c o l o r e d compound w i t h 3,S-dichloro-pbenzoquinonechlorimine o r sodium n i t r i t e o r 4-dimethylaminobenzaldehyde. Clarke (11) d e s c r i b e d t h e f o l l o w i n g : P l a t e : G l a s s p l a t e , 20 x 20 cm, c o a t e d w i t h a s l u r r y c o n s i s t i n g o f 30 g o f s i l i c a g e l G i n 60 m l o f w a t e r t o g i v e - a l a y e r 0.25 mm l i k e and d r i e d a t 110 f o r 1 hour.

A. A. AL-BADR AND M. TARIQ

436

Sample: acid.

1 u1 o f a 1%s o l u t i o n i n 2 N a c e t i c

S o l v e n t : S t r o n g ammonia s o l u t i o n : methanol (1.5 : 100). I t should be changed a f t e r two r u n s . E q u i l i b r a t i o n : The s o l v e n t i s allowed t o stand i n t h e tank f o r 1 hour. Development: 2 1 x 21 x 1 0 covered w i t h evaporation.

Ascending, i n a t a n k , cm, t h e end o f t h e t a n k b e i n g f i l t e r paper t o assist Time o f r u n , 30 minutes.

Location r e a g e n t : A c i d i f i e d i o d o p l a t i n a t e s p r a y , p o s i t i v e r e a c t i o n , R f 0.02, 8.5.3

Gas Liquid Chromatographic Methods e t a1 (43) developed a s e n s i t i v e and Chan -s e l e c t i v e a n a l y t i c a l method f o r t h e measurement o f neostigmine i n human plasma u s i n g gas chromatography. Plasma c o n t a i n i n g neostigmine i s washed w i t h e t h y l e t h e r and b u f f e r e d w i t h potassium iodide-glycine s o l u t i o n , then iodideg l y c i n e - d r u g complexes are e x t r a c t e d i n t o dichloromethane. The e x t r a c t i s e v a p o r a t e d and t h e r e s i d u e is d i s s o l v e d i n methanol. 2 - 5 1-11 o f m e t h a n o l i c s o l u t i o n was i n j e c t e d i n t o gas chromatographic g l a s s column (2 m x 0 . 2 5 i n c h e s O.D.) packed w i t h Diatomite CQ c o a t e d w i t h 3% o f OV-1, 3% o f OV-17 o r 3 . 8 % of SE-30 and s i l a n i s e d ; a n i t r o g e n s e l e c t i v e d e t e c t o r was used. Pyridostigmine bromide was used a s i n t e r n a l s t a n d a r d . The t e m p e r a t u r e was m a i n t a i n e d a t 205OC and t h e flow r a t e o f n i t o g e n was 30 m l p e r minute. The d e t e c t i o n l i m i t was 5 ng/ml and t h e r e was no i n t e r f e r e n c e with o t h e r b a s i c d r u g s . Ward et a 1 (44) d e s c r i b e d a simple GLC a s s a y of neostigmine u s i n g d i e t h y l analog o f neostigmine a s i n t e r n a l s t a n d a r d . Neostigmine and i t s analog were d i s s o l v e d i n 25 1.11 o f chloroform. 2 1.r1o f chloroform s o l u t i o n was i n j e c t e d i n t o a

437

NEOSTIGMINE

gas chromatograph equiped w i t h a flame i o n i s a t i o n d e t e c t o r and 1 . 2 m x 2 mm I . D . g l a s s column packed with 5 % O V - 1 7 on Gas-Chrom Q i s used a t 210' with helium (40 ml/minute) a s a carrier gas and flame i o n i z a t i o n d e t e c t o r . For d e t e r m i n a t i o n o f neostigmine i n b i o l o g i c a l f l u i d s , t h e drug i s e x t r a c t e d i n t o chloroform w i t h added 3-diethylcarbamoyloxy-"N-trimethylanil inium i o d i d e as t h e i n t e r n a l s t a n d a r d . The r e l a t i v e r e t e n t i o n time of t h e drug and t h e s t a n d a r d is 1 t o 1 . 3 5 . De Ruyter et a1 (45) developed a r e v e r s e d phase, i o n - p a i r l i q u i d chromatography o f q u a r t e r n a r y ammonium compounds €or t h e determination of pyridostigmine, n e o s t i g m i n e , and edrophonium i n b i o l o g i c a l f l u i d s . The method i s based on e x t r a c t i o n of neostigmine i n t o w a t e r - s a t u r a t e d dichloromethane, t h e n b a c k - e x t r a c t i o n is done w i t h t e t r a b u t y l ammonium hydrogen s u l p h a t e t o enhance t h e r e c o v e r y t o more t h a n 86%. The e x t r a c t s a r e s u b m i t t e d t o chromatography on a v a r i e t y o f r e v e r s e d phased columns f i t t e d with 214 nm d e t e c t o r s . For b i o l o g i c a l e x t r a c t s a column (15 cm x 4 . 6 mm) o f U l t r a s p h e r e o c t y l ( 5 pm) provided with an RP-2 pre-column was used. For neostigmine e l u t i o n was c a r r i e d o u t w i t h a s o l u t i o n c o n t a i n i n g sodium h e p t a n e s u l p h o n a t e (0.01 M ) , sodium hydrogen phosphate (0.01 M) and t e t r a m e t h y l ammonium c h l o r i d e (2.5 mM) i n aqueous 20% a c e t o n i t r i l e . The mobile phase (2 m l p e r minute) was a d j u s t e d t o pH 3 with s u l p h u r i c a c i d , edrophonium was used a s an i n t e r n a l s t a n d a r d . The c a l i b r a t i o n graphs were r e c t i l i n e a r f o r u p t o 400 ng per m l . The l i m i t of d e t e c t i o n was 5 ng p e r m l . The c o e f f i c i e n t o f v a r i a t i o n was 1 . 5 % . Davison e t a 1 (46) d e s c r i b e d a method f o r simultaneous monitoring of plasma l e v e l of neostigmine and p y r i d o s t i g m i n e i n man. The a s s a y i n v o l v e p r e l i m i n a r y ion p a i r

438

A. A. AL-BADR AND M. TARIQ

e x t r a c t i o n o f t h e drugs and i n t e r n a l s t a n d a r d s (3-diproypl carbamoyloxy) 1-methyl pyridinium bromide) w i t h t h e u s e o f potassium i o d i d e and o f g l y c i n e b u f f e r . The e x t r a c t i s a n a l y s e d by GLC (10% o f OV-17 on Chromosorb W-AW (100-120 mesh) with a n i t r o g e n - s e n s i t i v e d e t e c t o r . The calib r a t i o n graphs a r e r e c t i l i n e a r and reproduced o v e r t h e range 5-100 ng p e r m l o f e i t h e r drugs i n 3 m l samples. Pohlmann and Cohan (47) developed a s i m p l i f i e d d e t e c t i o n o f q u a r t e r n a r y ammonium compounds by gas chromatography. The method h a s been a p p l i e d t o p y r i d o s t i g m i n e , neostigmine and a c e t y l c h o l i n e . The q u a r t e r n a r y s a l t s i n t h e serum o r u r i n e a r e first converted w i t h potassium t r i i o d i d e (K13) i n t o t h e i r i o d i e s which a r e e x t r a c t e d i n t o chloroform. The e x t r a c t i s evaporated i n vacuum and t h e r e s i d u e is d i s s o l v e d i n water o r hexane. The s o l u t i o n is i n j e c t e d on t o a column ( 1 . 8 m x 2 mm) o f Porapak Q (800-100 mesh), Chromosorb 101 (80-100 mesh) o r Chromosorb 105 (80-100 mesh), o p e r a t e d a t 14SoC-17O0C with helium o r n i t r o g e n a s c a r r i e r gas (20 m l p e r ml). On t h e column t h e q u a r t e r n a r y ammonium i o d i d e s a r e t h e r m a l l y decomposed, and t h e methyl i o d i d e r e l e a s e d i s measured w i t h a 63Ni e l e c t r o n - c a p t u r e detector. Extraction y i e l d s a r e a t l e a s t 95% and down t o 1 0 f-mol of q u a r t e r n a r y ammonium compound can be d e t e c t e d w i t h good r e p r o d u c i b i l i t y . Chan and Dehghan (48) d e s c r i b e d a GLC method f o r i s o l a t i o n and d e t e r m i n a t i o n o f neostigmine, p y r i d o s t i g m i n e and t h e i r m e t a b o l i t e s i n human b i o l o g i c a l f l u i d s . The method i n v o l v e s a p r e l i m i n a r y , s e l e c t i v e i o n - p a i r e x t r a c t i o n o f t h e drugs and t h e i r m e t a b o l i t e s i n t o dichloromethane and i n (dichloromethane-acetone) , r e s p e c t i v e l y . The a n a l y s i s o f e x t r a c t was done by GLC on a column o f 3% (drug) o r 10% ( m e t a b o l i t e ) o f OV-17 on Chromosorb W AW,

439

NEOSTIGMINE

with a n i t r o g e n - s e n s i t i v e d e t e c t o r . For t h e d e t e r m i n a t i o n o f 3-hydroxytrimethyl a n i l i n i u m i o n ; a major m e t a b o l i t e o f neostigmine, 3-hydroxy-N-methylpyridinium ion was used a s a i n t e r n a l s t a n d a r d . As l i t t l e as 3 ng/ml o f neostigmine and upto 50 ng/ml o f i t s m e t a b o l i t e can be determined i n b i o l o g i c a l samples. 8.6

I o n - S e l e c t i v e E l e c t r o d e s Method Kina et a1 (49) d e s c r i b e d a method of neostigmine a n a l y s i s based on i o n - s e l e c t i v e e l e c t r o d e s s e n s i t i v e t o some o r g a n i c compounds used a s drugs. Liquid membrane e l e c t r o d e s s e n s i t i v e t o n e o s t i g mine have been made by t h e i o n - a s s o c i a t i o n e x t r a c t i o n method. The exchange components used were c r y s t a l v i o l e t , d i p i c r y l a m i n e , sodium t e t r a p e n t y l b o r a t e , l , l O - p h e n a n t h r o l i n e , 4,7diphenyl- 1,lO-phenanthroline . The s o l v e n t s used f o r t h e s e compounds were 1,Z-dichloroethane and n i t r o b e n z e n e . The c h o i c e of s o l v e n t s a f f e c t e d with s e l e c t i v i t i e s o f t h e membranes b u t t h e c h o i c e o f ion-exchange components d i d n o t . The s e l e c t i v i t i e s r e l a t i v e t o u n i v a l e n t c a t i o n s were mostly b e t t e r than 10-4. The u s e f u l c o n c e n t r a t i o n range o f e l e c t r o d e s was 10 VM t o 0 . 1 M w i t h a response o f 59 mV p e r decade change i n t h e c o n c e n t r a t i o n o f t h e drug. The e l e c t r o d e s could be used i n a n a l y s i s of mixed pharmaceutical preparations. Diamandis and C h r i s t o p o u l o s (26) r e p o r t e d a p o t e n t i o m e t r i c t i t r a t i o n o f neostigmine and o t h e r pharmaceutical compound i n pharmaceutical formul a t i o n with sodium t e t r a h y d r o b o r a t e . (See The end p o i n t was d e t e c t e d by a Section 8.2.1.). tetraphenylborate-selective e l e c t r o d e .

8.7

P o l a r o g r a p h i c Method Novotny (50) devised a p o l a r o g r a p h i c method f o r t h e d e t e r m i n a t i o n o f neostigmine i n pharmaceutical p r e p a r a t i o n s having a c o n c e n t r a t i o n o f 0 . 5 mg/ml o f t h e drug. T h i s method i s based on t h e h y d r o l y s i s of neostigmine and n i t r a t i o n o f r e s u l t i n g phenol, followed by polarography o f

A. A. AL-BADR AND M. TARIQ

r e s u l t i n g d e r i v a t i v e . The neostigmine s o l u t i o n ( 0 . 2 t o 0 . 6 m l of 0 . 1 % ) i s e vapor ated t o dr yness i n a 50 m l be a ke r on a water b a t h . 1 bll o f KOH s o l u t i o n (20%) i s added and warmed on t h e b a t h f o r 20 minutes and a ga i n e va por ated t o dryness. 0 . 5 M 1 of wa te r and 2 m l o f conc. HN03 (65%) i s added and warmed f o r 20 minutes and cooled. 10 M 1 of KOH s o l u t i o n (20%) is added and t h e volume made upto 1 4 . 5 m l w i t h water. The e s t i m a t i o n i s c a r r i e d out by pol a rogra phy, w i t h a galvanometer s e n s i t i v e t o 10-9 amp. To determine t h e c o n c e n t r a t i o n o f neostigmine i n an unknown s o l u t i o n , s o l u t i o n o f t h e unknown c o n c e n t r a t i o n o f neostigmine i s t r e a t e d as d e s c r i b e d above, both with and wit hout a known amount of neostigmine. Acknowledgements The a u t h o r s would l i k e t o thank Mr. Syed A l i Jawad, C o l l e g e of Pharmacy, King Saud U n i v e r s i t y f o r h i s t e c h n i c a l assist a n c e and Mr. Ta nvir A. Butt €or h i s s e c r e t a r i a l a s s i s t a n c e i n t y p i n g t h e manuscript.

441

NEOSTIGMINE

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PIRENZEPINE DIHYDROCHLORIDE

ffumeiida A . ER-Ob&id*, S a L h A . Rubha,&**, and A b d u U a h A . M-Badrr*

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

445

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

HUMEIDA A. EL-OBEID ETAL.

446

Contents 1. Description 1.1 Nomenclature

1.2 Formulae 1 . 3 Molecular Weight 1 . 4 Elemental Composition 1.5 Appearance

2.

Physico-chemical Properties 2.1 2.2 2.3 2.4 2.5

3.

Melting Range Solubility Distribution Ratio Spectral Properties Crystal Structure

Synthesis

4. Stability 5. Methods of Analysis 5.1 Spectrophotometric Methods 5.2 Chromatographic Methods 5 . 3 Radioimmunoassays 6.

Pharmacokinetics 6.1 Absorption 6.2 Distribution 6 . 3 Metabolism 6 . 4 Excretion

7.

Pharmacology Antisecretory Effects Efficacy Adverse React ions Tissue Selectivity 7 . 5 Mechanism of Action

7.1 7.2 7.3 7.4

Acknowledgement Re ferences

PIRENZEPINE DIHYDROCHLORIDE

1.

447

Descrivt ion 1.1 Nomenclature

1.1.1

Chemical Names

5,11-Dihydro-ll-[(4-methyl-l-piperazinyl) a c e t y l ] -6H-pyrido[ 2,3-b] [ 1 , 4 ] b e n z o d i a z e p i n 6-one d i h y d r o c h l o r i d e monohydrate. 11- [ (4-Methyl-1-piperazinlyl) a c e t y l ] - p y r i d o [ 2 , 3-b] [ 1 , 4 ] b e n z o d i a z e p i n - 6 (5 H) -one d i h y d r o c h l o r i d e monohydrate. 1.1.2

G e n e r i c Names L-S 519, P i r e n z e p i n e

1.1.3

Trade Names B i s v a n i l , G a s t e r i l , G a s t r o z e p i n , Leblon, Tabe, Ulcosan.

1.2

Formulae 1.2.1

Empirical (dihydrochl o r i d e )

C 9H2 3C 12N502

C19H21N502 1.2.2

(base)

Structural

I CH2-

.2HC1, H 2 0

NwN CH3

HUMEIDA A. EL-OBEID ETAL.

448

1.2.3 CAS No. [ 29868-97-11 as dihydrochloride salt.

[28797-61-71 as free base. 1.3 Molecular Weight 424.33 as dihydrochloride salt 351.42 as free base. 1.4 Elemental Composition Dihydrochloride salt: C 53.78%, H 5.46%, N 16.50%, 0 7.52%, C1 16.71% (1). Free base: C 64.94%, H 6.02%, N 19.93%, 0 9.11% (2). 1.5

Appearance Pirenzepine dihydrochloride is a white crystalline substance (2).

2. Physico-chemical Properties

2.1 Melting Range Pirenzepine dihydrochloride melts in the range 250-252OC (corrected) with decomposition (I). 2.2 Solubility Pirenzepine dihydrochloride is readily soluble in water, slightly soluble in methanol and practically insoluble in ether (1). 2.3 Distribution Ratio Pirenzepine is a tricyclic compound, but its physioco-chemical properties differ considerably from those of other tricyclic compounds which are used as centrally-acting drugs. Pirenzepine is unusual because of its pronounced hydrophilic properties. Comparison of the distribution coefficient of pirenzepine with those of other

PIRENZEPINE DIHYDROCHLORIDE

449

t r i c y c l i c drugs a t pH 7.4, has been c a r r i e d out (3). The r e s u l t s depicted i n Table 1 show t h a t pirenzepine i s 200-13000 l e s s l i p i d s o l u b l e , Table 1:

D i s t r i b u t i o n c o e f f i c i e n t of pirenzepine and o t h e r t r i c y c l i c drugs.

Drug -

H 7.4

$Ppe---Chlorpromazine 3160

CNS e f f e c t s

+

1600

+

250

+

Dibenzepine

51

+

Pirenzepine

0.23

-

Cyprohept adine Imipramin

Eberlein -e t a1 (3) analysed t h e pirenzepine molecule i n t o f i v e s t r u c t u r a l elements. Each o f t h e f i v e elements can be a l l o c a t e d a hydrophobic s u b s t i t u e n t consant (TX) and compared with t h o s e assigned f o r corresponding s t r u c t u r a l elements i n imipramine molecule (Table 2 ) . A p o s i t i v e T X value i n d i c a t e s contribution t o t h e l i p o p h i l i c c h a r a c t e r of t h e molecule and a negative T X value i n d i c a t e s contribution t o h y d r o p h i l i c i t y of t h e molecules.

HUMEIDA A. EL-OBEID E T A L .

450

Table 2 :

Contribution o f t h e d i f f e r e n t fragments of p i r e n z e p i n e and imipramine m o l e c u l e s t o t h e i r distribution coefficients.

H

CH2CH2CH2

'

CH,

,

CH,

fN I 1

4 -

n

5 -

I m ip r am i n e

Pirenzepine ~~~~

Fragment No.

Fragment structure

TX

-CH2-CH2-

+10.02

- 1 .4 9

+ 1.96

+ O . 65

1.96

+ l .96

+

\ /

N-CH2CH2CH2-

Fragment structure

+ 0.50

\ /

N-COCH2-

nx

- 0 .9 7

n ~

~

LogBase ( c a l c u l a t e d )

+ 5.12

(calculated) LogBase

+O. 010

LogBase(experimental)

+ 4.80

LogBase (experiment a l pH 7 . 4 )

-0.64

PIRENZEPINE DIHYDROCHLORIDE

45 1

I t can b e shown t h a t t h e s t r u c t u r a l elements 1 and 4, i n p a r t i c u l a r , c o n t r i b u t e l a r g e l y t o t h e h y d r o p h i l i c i t y of p i r e n z e p i n e . Fragments 1 and 4 i n p i r e n z e p i n e c o n s i s t o f amide groupings each w i t h two d i p o l e s which can i n t e r a c t s t r o n g l y with water molecules. The p y r i d y l group of fragment 2 i n p i r e n z e p i n e i s a l s o expected t o c o n t r i b u t e t o t h e h y d r o p h i l i c i t y of t h e molecule. Adding up t h e i n d i v i d u a l hydrophobic c o n s t a n t s f o r p i r e n z e p i n e m o l e cu l ar fragments a l o g P of 0.01 i s o b t a i n e d f o r t h e whole molecule, which i n d i c a t e s t h a t p i r e n z e p i n e a s t h e uncharged free b a s e w i l l be d i s t r i b u t e d uniformly between t h e o c t a n o l and wa te r . A t p h y s i o l o g i c a l pH, however, t h e r a t i o s h i f t s even more towards w at er s o l u b i l i t y due i o n i z a t i o n o f t h e molecule. Under p h y s i o l o g i c a l c o n d i t i o n s (pH 7.4) t h e ex p e r i m e n t al l y determined l o g P is - 0.64 (Table 2 ) , i n d i c a t i n g t h a t about 5 times more o f t h e compound i s d i s t r i b u t e d i n t h e aqueous phase t h an i n o c t a n o l . Under t h e same c o n d i t i o n s imipramine accumulates much more s t r o n g l y i n t h e o r g a n i c phase. As w i l l b e shown l a t e r , t h e pronounced h y d r o p h i l i c p r o p e r t i e s o f p i r e n z e p i n e e x p l a i n many o f t h e s p e c i f i c pharmac o k i n e t i c c h a r a c t e r i s t i c s of t h e drug.

2.4

Spectral Properties 2.4.1

U l t r a v i o l e t Suectrum The u l t r a v i o l e t a b s o r p t i o n spectrum o f p i r en z ep i n e i n met h an o 1/HC 1 was ob t a i n ed on a Cary 219 s p e c t r o ph o to m e te r . The spectrum, shown i n F i gu r e 1, i s c h a r a c t e r i z e d by a maximum a t 283 nm and a minimum a t 264 nm. The r e s u l t s a g r e e w i t h t h o s e r e p o r t e d i n t h e l i t e r a t u r e (4, 5 ) . I t is a l s o reported t h a t pirenzepine i n 0 . 1 N sodium hydroxide s o l u t i o n shows a b s o r p t i o n maximum a t 295 nm ( 4 ) .

2.4.2

I n f r a r e d Spectrum The i n f r a r e d a b s o r p t i o n spectrum o f p i r e n z e p i n e o b t a i n ed from a potassium bromide d i s p e r s i o n was r e c o r d e d on a Pye

452

HUMEIDA A. EL-OBEIDETAL.

Figure 1 : ULTRAVIOLET SPECTRUM OF PIRENZEPINE DItiYDRDQiLORIDE IN METHANOLIC HCl.

PIRENZEPINE DIHYDROCHLORIDE

453

Unicam SP 1025 spectrometer and i s shown i n Figure 2 . The c h a r a c t e r i s t i c bands of t h e spectrum with t h e assignments a r e l i s t e d below: Frequency (cm-l)

Assignment

3520, 3463

Amide N-H s t r e t c h

3240

Water molecule Aromatic C-H s t r e t c h

3020, 2990 2700

- 2400

stretch

1 7 1 7 , 1678

Amide C = 0

1605, 1585, 1502,) 1479, 1432 1

Aromatic C - C , C N stretch.

1370

Aryl C

800, 2.4,3

t -Amine s a l t N-H

785,

760

-*

-

N stretch

Aromatic C - H outof-plane bending.

'H Nuclear Magnetic Resonance ('H

NMR)

SDectrum Pirenzepine s o l u t i o n i n D 2 0 was used t o obtain t h e proton magnetic spectrum on a Varian-T60 NMR spectrometer with TMS a s i n t e r n a l standard. The spectrum i s shown i n Figure 3, and assignment of t h e chemical s h i f t s t o t h e d i f f e r e n t protons is summarized below:

Microns

P

VI P

4000

Wavenumber

3000

2500

2000 1800 1600 1400

1000 800

Figure 2 : INFRARED SPECTRUM OF PIRENZEPINE DIHYDROCHLORIDE, KBr DISC.

600 400

200

I

I

400

3

200

100

0

I7

16

Rl u

I

l . . . . l . . . . l . . . . 1 . . . . l . . . . l . . . . 1 . . . . 1 . . . . 7.0 60(ppm)Ei.O ( 6 ) 4-0 3-0 2-0 1.0 0

8.0

Figure 3 : H' SIR SPECIRUM CF PIRENZEPINE DIHYDROCMRIDE IN DZO WITH ?Hs As IkTERUL REFERENCE.

HUMEIDA A. EL-OBEIDETAL.

456

0

I

14

= c 1 2

n N -CH3

1

13

Multiplicity

18

N

CH~-

Chemical s h i f t (6)

15

I

U

6

Proton Assignment

No. of protons

3.05

Sing 1e t

-N-CH -3

3

3.55

Mu I t i p 1et

-CH -(piperazine) -2

8

0 It

4.15

Mult i p l e t

- 3 - C -

2

7.7

Mu 1t i p l e t

Aromat ic-H

6

8.4

Mu 1t i p l e t

Aromatic-H

1

2.4.4

13C Nuclear Magnetic Resonance ( 13C NMR)

Spectrum The 13C NMR s p e c t r a of pirenzepine i n a mixture of D20 and formic acid using dioxane a s i n t e r n a l reference a r e obtained using a J e o l FX 100 90 MHz spectrometer a t ambient temperature. Figure 4 and 5 show t h e IH-decoupled and off-resonance s p e c t r a , r e s p e c t i v e l y . The carbon chemical s h i f t s , assigned on b a s i s of t h e off-resonance s p l i t t i n g p a t t e r n and t h e t h e o r i e s of chemical s h i f t s , are presented below:

PIRENZEPINE DIHYDROCHLORIDE

451

Dioxan 67.4

Figure 4 : 'ti DEaWPLED 13C tW SPEclRuM OF PIRENZEPINE DIHYDROMLORIDE I N DzO/ RIRMIC ACID FlIXlURE W I ' M DIOXANE AS INNWU. REFERNECE.

a'

Figure 5 :

HUMEIDA A. EL-OBEIDETAL.

458

IY

10

121

0-c

I

CH2l3

Chemical S h i f t (61

18

NWN CH3

17

16

Multiplicity

44.83

Quartet

50.72

Triplet

51.87

Triplet

57.77

Triplet

122.41

Doublet

128.22

Doub 1e t

128.88

Doub 1e t

130.57

Doublet

131.57

Singlet

132.15

Doublet

132.74

Doub 1e t

135.24

Singlet

139.43

Doublet

143.61

Singlet

146.77

Singlet

165.21

Singlet

165.31

Sing 1et

Carbon Assignment ‘18 ‘15’

‘16

‘14’

‘17

‘13 clo ‘8

c7 ‘3 ‘6’ c9

c4

c7’ c2

c31 c21

‘12

‘6

PIRENZEPINE DIHYDROCHLORIDE

2.4.5

459

Mass SDectra The 70 eV e l e c t r o n impact mass spectrum of pirenzepine, presented i n Figure 6 , was obtained on Varian MAT 311 mass spectrometer using ion source pressure of Torr, ion source temperature of 18OoC and an emission current o f 300 PA. The molecular ion i s d e t e c t a b l e a t m/e 351 and t h e base peak a t m/e 113. A proposed fragmentation p a t t e r n and t h e mass/charge r a t i o s o f t h e major fragments i s shown i n Scheme 1. The d i r e c t chemical i o n i z a t i o n (DCI) spectrum (Figure 7) was obtained on a Finnigan 4000 mass spectrometer using methane gas a reagent with ion e l e c t r o n energy of 100 eV, ion source pressure o f 0 . 3 Torr, ion source temperature of 150°C and emission current o f 300 PA. The spectrum is dominated by a quasi-molecular ion (M + 1 ) . Two peaks appearing a t m/e 380 and m/e 392 a r e a t t r i b u t e d t o t h e t r a n s f e r o f carbocations from t h e c a r r i e r gas. The mass s p e c t r a l assignments of t h e prominent ions under DCI conditions a r e shown i n Table 3. Table 3.

m/e

Species

392

[M + C ~ H ~ I +

380

[M

.__

2.5

Mass s p e c t r a l assignments o f pirenzepine using DCI with methane a s reagnet gas.

+

352

MH+

35 1

M+

CZH51+

Crystal S t r u c t u r e The c r y s t a l s t r u c t u r e o f pirenzepine f r e e base was s t u d i e d by Ruzic-Toros Prodic (6). Pirenzepine monohydrate monoclinic, P2 a = 13.160 [7], b 1/c

monohydrate and Kojecwas t o be = 12.766 [Sly

460

HUMEIDA A. EL-OBEID ETAL.

Figure 6 : ELECTRON IMPACT W S SPECTHW! OF PSWZEPINE.

Figure 7 : DIRECT WICAL IONIZATION W S SPECIIW OF PIRENZEPINE.

H

@% N

0

-238

=c I

%lac. DN -CH, -N

H,C L

u

dh 0

=c I

____t

+p 'd

-CH -43 3-N = CH2

N

H2C

=

m/e 113 5

m/e 351

-

-42 =N=CHC 2 2 m/e 113

+

w

=CH2

m / e 70

@

N+3 ~cH

1

m/e 71

-

tH 1 ~ A ~ 1N - CHJ 0 = C-CH-N

CH3

- 140

H

m/e 211 Scheme 1.

-cH3-

Proposed f r a p e n t a t i o n of pirenzepine.

m/e 56

HUMEIDA A. EL-OBEID ETAL.

462

c = 12.439 171 A’,

B = 113.2 [4]O, U

Z = 4, Dc = 1 . 2 7 7 Mgm-3, p(Mo Ka)

1920.8 Ao3, -1 = 0.128 mm =

.

F i n a l R = 0.045 f o r 2681 observed r e f l e x i o n s . Bond d i s t a n c e s and a n g l e s (Table 4) i n t h e f u s e d r i n g s , benzene, d i a z e p i n e and p y r i d i n e , a r e a f f e c t e d by c o n j u g a t i o n . Deviation from t h e v a l u e s expected €or t h e given atom type and h y b r i d i z a t i o n could n o t be e x p l a i n e d by t h e thermal v i b r a t i o n s of t h e atoms (Table 5 ) . The s h o r t e n i n g o f C[7] - C [ 8 ] , 1.372[4]Ao, i n t h e benzene r i n g could be due t o i n t e r m o l e c u l a r c o n t a c t s , i n v o l v i n g t h e s e atoms, with N[1][3.352[3]; 3.470[3]Ao. The seven-membered d i a z e p i n e r i n g is f o l d e d a t “51 - C[6] and “111 t o form a b o a t conformations. The p y r i d i n e and benzene r i n g s a r e p l a n a r while t h e p i p e r a z i n e r i n g i n a c h a i r conformation. A v a l u e o f 60.9[41° was o b t a i n e d f o r t h e d i h e d r a l a n g l e between t h e benzene and t h e p y r i d i n e r i n g s . The w a t e r molecule is involved i n hydrogen bonds with t h e amide group o f t h e d i a z e p i n e r i n g , “51 - H[5] .. O[W]I2.800[3])and O(W)-H(W)2 O[ 61 c2.907[ 31 Ao) . N [ 4 ’ ] o f t h e p i p e r a z i n e r i n g is hydrogen-bonded t o t h e water molecule by O(W)- H(W)1 N[4’](2.821[3]A0). Molecular packing i s a l s o i n f l u e n c e d by van d e r Wall i n t e r a c t i o n s . The r e l a t i v e o r i e n t a t i o n o f t h e p y r i d i n e and p i p e r a z i n e r i n g , w i t h a d i h e d r a l a n g l e o f 2 2 . 2 [ 41°, e n a b l e s a s h o r t i n t r a m o l e c u l a r c o n t a c t “11 ... “1’1 o f 3.087[2]Ao. The s t r u c t u r a l formula and atomic numbering o f p i r e n z e p i n e i s given i n F i g u r e 8. The confonnat i o n o f t h e molecule is shown i n F i g u r e 9 and Table 6.

...

. ..

--

Trummlitz e t a l . ( 7 ) , u s i n g X-ray a n a l y s i s , determined t h e c r y s t a l s t r u c t u r e s o f p i r e n z e p i n e d i h y d r o c h l o r i d e and i t s monoprotonated form, p i r e n z e p i n e monohydrochloride (Figure 10) Molecular mechanics (MMPI) and semiempirical quantum chemical (MNDO) c a l c u l a t i o n s showed t h a t t h e c a l c u l a t e d minimum energy conformations o f t h e t r i c y c l e and of t h e e x o c y c l i c amide group are

.

PIRENZEPINE DIHYDROCHLORIDE

T a b l e 4.

463

Bond d i s t a n c e s (A) and a n g l e s (O)

C(lO)-C(15) C(lO)-H(lO) N(l1)-C(12) N(l1)-C(15) N(l1)-C(16) C(12)-C(13)

1 . 3 4 8 (3) 1.315 (3) 1.364 (4) 0 . 9 9 (2) 1 . 3 7 5 (3) 0.97 (2) 1 . 3 9 5 (3) 0 . 9 7 (2) 1.362 (3) 1 . 4 0 3 (3) 0.86 (2) 1 . 2 3 1 (3) 1 . 4 8 4 (4) 1 . 3 9 7 (4) 0.97 (2) 1.372 (4) 1.377 ( 4 ) 1 . 0 0 (3) 1 . 3 8 3 (4) 0.92 ( 2 ) 1 . 3 8 1 (3) 0.94 (2) 1 . 4 3 8 (3) 1 . 4 3 3 (3) 1.362 (2) 1.387 (3)

C(Z)-N(l)-C(12)

c (1) -c (2) -c (3)

N (1) -C ( 2 ) -H (2) C(3)-C(Z)-H(2) c (2) -c (3) -c (4) C (2) -C (3) -H( 3) C (4) -C (3) -H (3) c (3) -c (4) -c (13) C(3)-C (4)-H(4) C ( 1 3) -C (4) -H (4) C(6)-N(S)-C(13) C(6)-N(S)-H(5) C (13) -N (5) -H (5) N (5) -C (6) -0 (6) N (5) -C (6) -C (14) O(6) -C (6) -C (14) C(8) -C (7) -C (14) C (8) -C (7) -H (7)

C (14) -C (15) C(16)-O(16) C (16) -C ( 1 7) C (17) -N (1' ) C(17)-H(17)1 C ( 1 7) -H ( 1 7) 2 N(l')-C(Z') N ( 1 ' ) -C ( 6 ' ) C (2 ' ) -C ( 3 ' ) C (2 ' ) -H(2 ' ) 1 C (2 ' ) -H (2 ' ) 2 C ( 3 ' ) -N (4 ' ) C(3')-H(3')1 C ( 3 ' ) -H ( 3 ' ) 2 N ( 4 ' ) -C ( 5 ' ) N ( 4 ' ) -C ( 7 ' ) C (5 ' ) -C ( 6 ' ) C ( 5 ' ) -1-1 ( 5 ' ) 1 C ( 5 ' ) -H ( 5 ' ) 2 C ( 6 ' ) -H(6 ' ) 1 C (6 ' ) -H ( 6 ' ) 2 C ( 7 ' ) -H ( 7 ' ) C ( 7 ' ) -H( 7 ' ) 2 C (7 I ) -H ( 7 ' ) 3 O(W)-H(W)1 O(W)-H(W)2

116.7 (2) 122.9 (2) 115 (1) 1 2 1 (1) 1 1 9 . 6 (2) 120 ( 1 ) 120 (1) 1 1 8 . 7 (2) 123 (1) 118 (1) 129.8 (2) 114 ( 1 ) 115 (1) 119.3 (2) 1 1 9 . 3 (2) 121.2 (2) 1 2 0 . 5 (2) 121 (2)

1 .3 8 8 (3) 1.229 (3) 1 .5 1 3 (4) 1.452 (3) 1 . 0 1 (2) 1 . 0 5 (2) 1 .4 6 0 (3) 1.454 (3) 1 .5 0 5 (4) 1 .0 4 (2) 1 .0 4 (2) 1 .4 6 5 (3) 1.05 (2) 1 .0 1 (3) 1.462 (4) 1.468 (5) 1.502 (4) 1.03 (2) 1 .0 1 (2) 1 .0 4 (2) 1 . 0 3 (3) 1.09 (3) 0.97 (4) 1 . 0 5 (3) 0 .9 3 (3) 0 . 7 8 (5)

C (12) -N ( 1 1 ) -C (16) C (15) -N (11) -C(16) N ( 1 ) -C ( 12 ) -N ( 1 1 ) N ( l ) -C (12) -C (13) N (11) -C (12) -C(13) C(4) -C (13) -N (5) C (4) -C (13) -C (12) ~ ( s- cj( 1 3 j -c (12) C (6) -C (14) -C (7) C(6) -C (14) -C (15) c (7) -c (14) -c (15) C ( 10) -C (15) -N ( 1 1) C(10) -c (15) -c (14) N (11) -C (15) -C (14) N (11) -C (16) -0 (16) N (11) -C (16) -C (17) 0 (16) -C (16) -C (17) C (16) -C (17) -N ( 1 ' )

1 2 4 .9 120.6 1 1 7 .0 125.0 118.0 119.8 116.8 1 2 3 .1 1 1 8 .1 123.2 1 1 8 .6 1 2 0 .5 1 2 0 .9 1 1 8 .5 120.8 117.9 1 2 1 .3 113.2

HUMEIDA A. EL-OBEIDETAL.

464

Table 4.

Contd .....

C (14) -C (7) -H (7) C (7) -C (8) -C (9) C (7) -C (8) -H (8) C (9) -C (8) -H (8) C (8) -C (9) -C (10) C (8) -C (9) -H(9) C(l0)-C(9) -H(9) C (9)-C (10) -C (15) C(9)-C(lO)-H(lO) C(lS)-C(lO)-H(lO) C( 12) -N(11) -C(15)

Table 5.

118 (2) 120.2 (3) 120 (2) 120 (2) 120.4 (2) 123 (1) 117 (1) 1 1 9 . 4 (2) 1 2 1 (1) 119 (1) 1 1 4 . 3 (1)

C (1 7) -N ( 1 ' ) -C (2 ' ) C(17)-N(lf)-C(6') C (2 ' ) -N ( 1 ' ) -C (6 ' ) N ( 1 ' ) -C (2 ' ) -C ( 3 ' ) C(2')-C(3')-N(4') C(3' ) -N ( 4 ' ) -C(S') C (3 ' ) -N ( 4 ' ) -C ( 7 ' ) C(5 ) -N(4' ) -C ( 7 I ) N (4 ') -C ( 5 ') -C (6' ) N ( 1 ' ) -C(6') -C(S') H(W) l-O(W) -H(W) 2

Final atomic coordinates (x 5

x 10

lo4

111.0 (2) 111.7 (2) 1 1 0 . 6 (2) 109.9 (2) 110.8 (2) 1 0 9 . 3 (2) 1 1 0 . 9 (2) 110.9 (2) 1 1 0 . 1 (2) 1 1 0 . 6 (2) 110 (4)

f o r C , N and 0;

f o r H) and i s o t r o p i c thermal parameters

(x l o 2 ) U

eq U eq

i s derived from t h e a n i s o t r o p i c thermal parameters by 1 = - C . C . U..a* a * . a * . a . . a 3 1 J 1J J 3 1 j or

U

6263 6199 6380 6615 7023 6726 7131 5060 4228 4186 4978 6635 6537 6695 5866 5818 7373 7400 8146 8712

z

Y

X

(1) (1) (1) (1) (1) (1) (1) (1) (2) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

1423 1306 372 -503 -1266 -1540 -2330 -1465 -932 143 698 716 588 -404 -922 162 1361 1426 1997 1375

(1) (1) (2) (1) (1) (1) (1) (1) (2) (2) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

6087 4985 4557 5255 7162 8057 8634 8510 8677 8615 8379 7947 6761 6403 8266 8216 8752 9729 8376 7805

ueq('42) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

(I)

(1) (2) (1)

4.2 4.6 4.7 4.3 4.3 7.2 6.4 4.8 5.1 5.1 4.5 3.7 3.4 3.5 3.9 3.7 4.2 6.2 4.6 3.8

(2) (2) (2) (2) (2) (2) (2) (3) (3) (3) (2) (2) (2) (2) (2) (2) (2) (2) (3) (2)

PIRENZEPINE DIHYDROCHLORIDE

Table 5.

Contd..

465

.. U

Y

X

9214 9766 10604 10082 9536 11162 2165 603 632 676 737 509 366 362 496 868 768 857 978 918 1017 951 1068 1012 917 1175 1152 1057 162 223

(1) (2) (1) (2) (2) (3) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2)

(2) (2) (2) (2) (2) (3) (3) (3) (3) (4)

2042 1377 696 28 688 63 2153 195 32 -118 -175 -222 -133 53 143 237 259 248 255 91 184 -46 -44 112 21 -45 55 -38 167 217

(1) (2) (1) (1) (2) (3) (1) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2)

(2) (2) (2) (2) (2) (3) (3) (3) (3) (4)

7195 6583 7417 8008 8627 6827 8845 451 375 499 696 854 886 871 834 909 780 661 777 594 621 741 857 929 904 747 648 615 843 950

or Ue9(A2)

Z

(2) (2) (1) (2) (2) (3) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (3) (3) (3) (3) (4)

4 . 9 (3) 5 . 8 (3) 5 . 0 (2) 5.4 (3) 4 . 7 (2) 8 .4 ( 4 ) 7.9 (2) 6 . 1 (7) 5.2 ( 6 ) 3.9 ( 6 ) 4 .3 (6) 5 . 7 (7) 7.1 (8) 5.2 (6) 4.2 ( 6 ) 6 . 3 (7) 5.7 (7) 6 .0 (7) 6 . 6 (8) 6 . 6 (9) 8 . 3 (9) 6 . 6 (7) 7 .0 (8) 5 .1 (6) 7.5 (8) 11 (1) 9 (1) 1 0 (1) 1 0 (1) 1 4 (2)

HUMEIDA A. EL-OBEID ETAL.

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Figure

8 : STRUCTURAL FORMULA OF PIRENZEPINE WITH ATOMIC NUMBERING (6).

Figure 9 :

A VIEW OF THE CRYSTAL STRUCTURE SHOWING THE PACKING, HYDROGEN BONDS AND CONFOR-MATION OF THE BENZODIAZEPINE AND PIPERAZINE R I N G S ( 6 ) .

Table 6.

Torsion a n g l e s

C (12) -N (1) -C (2) -C (3) N ( l ) -C(2) -C(3) -C (4) C (2) -C (3) -C (4) -C (13) C(3) -C (4) -C(13)-C (12) C (3) -C (4) -C (13) -N (5) C (4)-C (13) -C(12) -N (1) C(4) -C(13)-C (12) -N(11) C(13)-C (12) -N(1) -C(2) C (7) -C (8) -C (9) -C (10) C(8)-C(S)-C(lO)-C(15) C (9) -C (10) -C (15) -C (14) C (9)-C(lO)-C(15) -N(11) C (10) -C (15) -C (14) -C (7) C(10) -C(15) -C(14) -C(6) C(15) -C (14) -C (7) -C (8) C (14) -C (7) -C(8) -C (9) N(S)-C(6)-C(14)-C(15) C(6) -C (14) -C (15)-N (11)

(O)

1.4 1.5 - 2 .0 - 0 .4 -174.7 3.6 -178.9 -4.1 0.3 - 0 .5 1.1 1 7 8 .7 - 1 .4 1 7 7 .8 1.1 -0.6 -42.4 0 .2

(3) (4) (3) (3) (2) (3) (2) (3) (3) (3) (3) (2) (3) (2) (3) (3) (3) (3)

C (14) -C (15) -N (11) -C (12) N (11 ) -C (12) -C (13) -N (5) C ( 12) -C ( 13) -N (5) -C (6) C ( 1 3 ) -N (5) -C (6) -C (14) C (15) -N (11) -C (12) -C (13) C (13) -N (5) -C (6) - 0 ( 6 ) C(12) -N(11) -C(16) -0(16) C(12) -N(11) -C(16) -C(17) N (11) -C (16) -C (17) -N ( 1 ' ) C (16) -C (17) -N (1' ) -C (2 I ) C (17) -N (I ) -C (2 ' ) -C ( 3 ' ) N ( 1 ' ) -C (2 ' ) -C ( 3 ' ) -N ( 4 ' ) C (2 ) -C ( 3 ) -N (4 ' ) -C ( 5 ) C (2 I ) -C ( 3 ) -N (4 ' ) -C ( 7 ' ) C ( 3 ' ) -N (4 I ) -C ( 5 I ) -C ( 6 ' ) N (4 ' ) -C ( 5 I ) -C (6' ) -N (1I ) C(S')-C(6') -N(1 I ) - C ( 2 ' )

6 9 .5 -4.7 3 8 .3 5.3 -64.5 -178.8 179.8 0.2 -50.0 165.2 -178.4 -57.9 58 .7 -178.7 -58.6 5 8 .6 -57.7

( 2) (3) ( 3) (3) (2) (2) (2) (3) (2) (1) (1) (2) (3) (2) (2) (2) (2)

HUMEIDA A. EL-OBEIDETAL.

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Figure 10: STEREO-PAIRS OF DRAWINGS FOR THE MOLECULAR STRUCTURES OF PIRENZEPINE DIHYDROCHLORIDE(a)AND PI RENfEPlNE MONOHYDROCHLORIDE(b) OBTAINED FROM X-RAY ANALYSIS ( 7 ) .

a-

1.491 (3) 1.503(6)

Figure l l

:

ATOM NUMBERING SCHEME AND CRYSTALLOGRAPHIC BOND ANGLES

(A,

STANDARD DEVIATONS IN PARENTHESES)

FOR PIRENZEFINE DIWDROCHLORIDE ( FIRST VALUE) AND PIRENZEPINE MONOHYDROCHLORIDE (SECOND VALUE I( 7).

PIRENZEPINE DIHYDROCHLORIDE

469

i n agreement w i t h t h e c r y s t a l s t r u c t u r e s . The t r i c y c l i c r i n g system c o n s i s t s of t h e p l a n a r r i n g s A and C and nonplanar r i n g B (Figure 1 1 ) . For b o t h compounds t h e seven-membered r i n g B h a s a d i s t a r t e d boat shape. This r i n g i s f o l d e d a l o n g a f o l d i n g l i n e p a s s i n g through N 1 and t h e middle o f t h e bond N 8 - C9. T h i s s i m i l a r i t y i n t h e geometry o f t h e t r i c y c l i c systems o f b o t h compounds does n o t h o l d f o r t h e s i d e c h a i n s which have t o t a l l y d i f f e r e n t arrangements with r e s p e c t t o t h e t r i c y c l i c system i n both compounds ( F i g u r e s 10 and 1 2 ) . The s t e r i c s i t u a t i o n is q u i t e s i m i l a r along t h e bond N 1 - C16, i n t h a t t h e carbonyl groups are almost i n e c l i p s e d p o s i t i o n s t o N 1 - C15. S i g n i f i c a n t d i f f e r e n c e s are observed, however, a l o n g t h e C16 - C 1 7 bond. For t h e d i h y r o c h l o r i d e N18 i s trans t o N 1 (T N18-Cl7-Cl6-Nl = 171.3') whereas i n t h e monohydrochloride N18 and N 1 a r e i n gauche p o s i t i o n s (T N18-Cl7-Cl6-Nl = 64.1'). In t h i s arrangement t h e p i p e r a z i n e r i n g w i l l be s i t u a t e d below t h e t r i c y c l i c r i n g system i n t h e c r y s t a l s t r u c t u r e o f p i r e n z e p i n e monohydrochloride. The c r y s t a l s t r u c t u r e s o f b o t h s a l t s showed s t r o n g N-H . C 1 - hydrogen bonds f o r t h e p r o t o nated nitrogens of t h e piperazine rings. A r e l a t i v e l y weak 0 C 1 - hydrogen bonds a l s o e x i s t f o r t h e d i s t o r t e d w a t e r molecule i n t h e c r y s t a l l a t t i c e i n t h e dihydrochloride salts. The amide n i t r o g e n N8 o f t h e monohydrochloride s a l t shows a f u r t h e r N-H ... C 1 - hydrogen bond which i s n o t observed i n t h e d i h y d r o c h l o r i d e structure.

..

...

3.

Synthesis P i r e n z e p i n e , t o g e t h e r with o t h e r 1 1 - s u b s t i t u t e d 11Hpyrido[ 2 ,3-b] [ 1 , 5 ] benzodiazepin-5 (6H) -ones , had been prepared by D r . Karl Thomae ( 8 ) . The compounds are p r e p a r e d by t r e a t m e n t of an 11H-pyrido[ 2 ,3-b] [ 1,51 benzodiazepin-5 (6H) -one with a dihalogen compound, XCH2COX' i n which X and X I may be a l i k e o r d i f f e r e n t and d e s i g n a t e an atom o f C 1 , B r and I . The 11h a l o a c e t y l i n t e r m e d i a t e is t h e n t r e a t e d with t h e a p p r o p r i a t e amine. Thus, a b o i l i n g s o l u t i o n o f 2 1 g 11H-pyrido[ 2 ,3-b] [ 1,51 benzodiazepin-5 (6H) -one) i n 300 m l

HUMEIDA A. EL-OBEIDETAL.

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C16 -NI

C 17- C I6 HI71

N 18-C I7 HI8

HI72

Figure 12 : NEWMAN PROJECTIONS DOWN THE BONDS ClbNl, c r C 1 6 &

v8-Ci7

FOR PIRENZEPINE DIHYDROCHLORIDE ( ABOVE ) AND THE MONOHYDROCHLORIDE (BELOW) ( 7 1.

PIRENZEPINE DIHYDROCHLORIDE

47 1

a b s o l u t e dioxane t r e a t e d dropwise sim u lta n e o u sly i n 30 min w i t h 15.8 g ClCH2COC1 i n 40 m l a b s o l u t e dioxane and 1 4 . 4 g E t 3 N i n 30 m l a b s o l u t e dioxane, t h e m ix tu r e r e f l u x e d f o r 8 h r and t h e h o t f i l t r a t e e v a p o r a te d i n vacuo gave ll-chloroacetyl-11H-pyrido[2,3-b][1,5] benzodiazepin-5 (6H) -one (Scheme 2 ) . The c h l o r o a c e t y l d e r i v a t i v e and N-methylpiperazine i n a b s o l u t e a l c o h o l r e f l u x e d f o r 18 h r f i l t e r e d and t h e hot f i l t r a t e e v a p o r a t e d i n vacuo t o g i v e p i r e n z e p i n e .

C 1CH, COC 1 %

N H

0

I =c I

CH2C1

H

H2C -N

0

wNCH3

Scheme 2 .

Synthesis of pirenzepine

HUMEIDA A. EL-OBEID ETAL.

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4.

Stability S t a b i l i t y of p i r e n z e p i n e depends on b o t h m o istu r e c o n t e n t s and pH and t h e e f f e c t o f t h e m o istu r e c o n t e n t s could be minimized by a d j u s t i n g t o optimum pH ( 9 ) . P i r e n z e p i n e d i h y d r o c h l o r i d e i n t a b l e t was shown s t a b l e t o exposure t o l i g h t a t 50 - 5 5 O . No change i n c o l o r , smell, t a s t e o r g e n e r a l appearance o r presence o f d eg r a d a t i o n p r o d u ct s was observed (10).

5.

Methods of A n a l y s i s 5.1

S p e ct r o p h o t o m et r i c Methods The d e t er m i n a t i o n of p i r e n z e p i n e d i h y d r o c h l o r i d e s p e c t r o p h o t o m e t r i c a l l y u s i n g AA method have r e c e n t l y been r e p o r t e d ( 4 ) . The mean p e r c e n ta g e r e c o v er y f o r t a b l e t s , each l a b e l l e d t o c o n t a i n 25 mg,was 100.2 -+ 1 . 1 5 . The drug shows maximum a b s o r p t i o n a t 280 nm i n h y d r o c h l o r i c a c i d and a t 295 nm i n 0 . 1 N sodium hydroxide due t o k e to - e n o l tautomerism. AA a t 300 nm was found t o be l i n e a r l y r e l a t e d t o t h e drug c o n c e n t r a t i o n o v e r a range of 10-80 Ug/ml with a r e l a t i v e s t a n d a r d d e v i a t i o n of 0 . 4 % . A r a p i d and d i r e c t method f o r t h e d e te r m in a tio n

o f p i r e n z e p i n e d i h y d r o c h l o r i d e i n t h e p r e se n c e o f d eg r a d a t i o n p r o d u ct s u s i n g f i r s t d e r i v a t i v e spectrophotometry h as a l s o been r e p o r t e d ( 5 ) . The method was a p p l i e d u s i n g s y n t h e t i c m ix tu r e s o f t h e i n t a c t drug and d e g r a d a tio n p r o d u c ts. The procedure i s s u i t a b l e f o r m o n ito r in g t h e drug s t a b i 1i t y

.

5.2

Chromatographic Eilethods 5.2.1

Thin-Layer Chromatographic (TLC) Methods Thin l a y e r chromatography have been used f o r t h e analysis o f pirenzepine (10). P r e c o a t ed TLC p l a t e s with 0 . 2 mm s i l i c a g e l FZs4 were used.

PIRENZEPINE DIHYDROCHLORIDE

5.2.2

413

High-pressure Liquid Chromatography (HPLC) Daldrup -e t a1 (11) have used reversed-phase l i q u i d chromatography a s a t e s t f o r s c r e e n i n g pharmaceuticals , drugs and insecticides. High performance r e v e r s e - p h a s e l i q u i d chromatography r e t e n t i o n d a t a f o r p i r e n z e p i n e and o t h e r compounds (560 compounds) a r e given. The r e l a t i v e r e t e n t i o n times were c a l c u l a t e d a s t h e r a t i o s of r e t n t i o n times of compound and r e f e r e n c e compound, 5- (p-methylphenyl) -5-phenyl hydantoin. The UV d e t e c t o r wavelength was 220 nm, where most of t h e compounds gave response. Two s o l v e n t programs and a prepacked column C - 1 8 SIL-X-10 were used f o r the analysis. Babhair (12) r e p o r t e d a s i m p l e and s e n s i t i v e method for t h e d e t e r m i n a t i o n o f p i r e n z e p i n e , i n dosage forms and i n b i o l o g i c a l f l u i d s , u s i n g h i g h - p e r formance 1i q u i d chromatography w i t h U . V . d e t e c t o r . A t a b l e t o f 25 mg drug was ground, suspended i n 1 0 m l o f w a t e r , shaken and t h e n f i l t e r e d . A known volume o f t h e f i l t r a t e i s a d j u s t e d t o a p p r o p r i a t e c o n c e n t r a t i o n . Twenty 1.11 o f t h i s s o l u t i o n were i n j e c t e d . Plasma o r u r i n e samples were made a l k a l i n e w i t h ammonia b e f o r e e x t r a c t ion with chloroform which was evaporated and t h e r e s i d u e was d i s s o l v e d i n t h e mobile phase, 20 1-1 o f t h i s s o l u t i o n were i n j e c t e d . The d e t e r m i n a t i o n l i m i t f o r q u a n t i t a t i o n was about 1 pg/ml o f p i r e n z e p i n e . Complete s e p a r a t i o n of t h e drug was achieved i n about 5 . 4 minutes under t h e p r e s e n t chromatographic c o n d i t i o n s . A 30 X 3.9 cm i . d. commercially a v a i l a b l e s t a i n l e s s s t e e l C-18 column was used. Mobile phase c o n s i s t e d o f a c e t o n i t r i l e , methanol and 5% a c e t i c a c i d (70:40:15). High-performance l i q u i d chromatographic determination of pirenzepine dihydrochloride i n i t s pharmaceutical f o r m u l a t i o n have a l s o

HUMEIDA A. EL-OBEIDETAL.

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been r e p o r t e d (13). Normal phase l i q u i d chromatography has been performed on a Micropack Si-10 column u s i n g ammonium hydrox i d e (28-30% NH3) i n methanol (0.75 : 99.25% v/v) mobile phase and a flow r a t e o f 2 ml/min. Clobazam h as been used a s i n t e r n a l s t a n d a r d with r e t e n t i o n times o f 1 . 9 and 2.8 minutes f o r clobazam and p i r e n z e p i n e d i h y d r o c h l o r i d e , r e s p e c t i v e l y a t 254 nm. T a b l e t s each l a b e l l e d t o c o n t ai n 25 mg p i r e n z e p i n e d i h y d r o c h l o r i d e gave mean p er c e n t ag e result o f 99.98 2 0 . 4 . The method have a l s o been r e p o r t e d t o be a s t a b i l i t y i n d i c a t i n g method. 5.3

Radioimmunoassay A radioimmunoassay system developed f o r p i r e n z e p i n e

was used t o s p e c i f y t h e r e q u i re m e n ts f o r u s i n g such system i n pharmacokinetics and t o i l l u s t r a t e t h e general strategy i n establishing s p e c i f i c i t y , sensit i v i t y and r e l i a b i l i t y ( 1 4 ) . The t y p e o f e r r o r i n t h e f i n a l d a t a was c o n s i d er e d . I t is a widespread assumption t h a t weak c r o s s - r e a c t i o n s i n m e t a b o l i t e s r e s u l t i n a r e l a t i v e e r r o r and, t h e r e f o r e , have no r e l e v a n c e t o t h e d e t e c t i o n o f t h e p a r e n t drug. In c o n t r a s t t o t h i s a s s u p t i o n , it was shown t h a t such weak c r o s s - r e a c t i v i t y i n b i o l o g i c a l samples g i v e s r i s e t o an a b s o l u t e e r r o r which i s independent o f t h e c o n c e n t r a t i o n and analogous t o t h a t caused by a b la n k v a l u e i n chemical a n a l y s i s . Tanswell and Zahn (15) a p p l i e d monoclonal a s s a y s f o r s t u d y i n g t h e pharmacokinetics o f p i r e n z e p i n e .

6.

Pharmacokinetics The marked physicochemical p r o p e r t i e s o f p i r e n z e p i n e r e s u l t i n t h e drug b ei n g h i g h l y h y d r o p h i l i c a t p h y s i o l o g i c a l pH. T h i s p r o p e r t y determines t h e f a t e o f p i r e n z e p i n e i n t h e organism due t o t h e l i m i t e d a b i l i t y of t h e drug t o p e n e t r a t e l i p i d membranes. T h i s l i m i t e d p e n e t r a t i o n i s r e f l e c t e d i n i t s a b s o r p t i o n , d i s t r i b u t i o n , b io tr a n sf o r m a t i o n and e x c r e t i o n . 6.1

Absorption O r a l l y a d m i n i s t e r e d p i r e n z e p i n e was almost completely absorbed by r a b b i t s and dogs b u t o n l y about 11%absorbed by r a t s and t h e blood l e v e l maxima reached a f t e r 3 h r (16).

PIRENZEPINE DIHYDROCHLORIDE

475

After o r a l a d m i n i s t r a t i o n o f aqueous s o l u t i o n s o r t a b l e t s 20-30% o f t h e dose is absorbed ( 1 7 ) . In an i n t e r n a t i o n a l pharmacokinetic s t u d i e s 20% o f t h e o r a l dose a d m i n i s t e r e d t o t h e panel o f 87 v o l u n t e e r s was absorbed (18). Uniform plasma l e v e l s were observed i n t h i s l a r g e panel from 10 d i f f e r e n t c o u n t r i e s a f t e r a s i n g l e o r a l dose o f 50 mg, with minimal i n t e r p a t i e n t v a r i a t i o n . Peak serum c o n c e n t r a t i o n were approximately 50 ng/ml o c c u r r i n g w i t h i n 2 h r s . After o r a l dose o f 20 mg/kg, t h e drug appeared i n t h e blood a t a c o n c e n t r a t i o n o f 0 . 1 7 ug/ml o n l y 15 min a f t e r a d m i n i s t r a t i o n . Afterwards, however, i n c r e a s e i n t h e c o n c e n t r a t i o n was shown r e a c h i n g t h e maximum of 0 . 4 2 ug/ml approximately 3 h r a f t e r a d m i n i s t r a t i o n ( 1 9 ) . The r e s u l t s a r e comparable t o t h o s e o f Hammer e t_a1 (16) who r e p o r t e d t h a t t h e maximum blood c o n c e n t r a t i o n was a t t a i n e d 3 h r a f t e r a d m i n i s t r a t i o n a t t h e l e v e l of 0.55 ug/ml. After o r a l a d m i n i s t r a t i o n o f 25 mg o f t h e drug, t h e maximum blood l e v e l occurred i n 2 - 3 h r and was about 24 ng/ml. Computer s i m u l a t i o n was used t o d e r i v e a s i n g l e dosage scheme f o r p i r e n z e p i n e c o n s i s t i n g o f an i n i t i a l dose o f 50 mg and maintenance dose o f 25 mg a t 12 h r - i n t e r v a l s . T h i s schedule maintained a maximum plasma l e v e l o f -> 24.1 ng/ml i n normal s u b j e c t s ( 1 7 ) .

The maximum plasma l e v e l appeared 2 h r a f t e r t h e a d m i n i s t r a t i o n o f 1 2 . 5 - 150 mg p i r e n z e p i n e o r a l l y t o volunteers (20). After multiple administration (25 - 50 mg a day) t h e plasma l e v e l was i n c r e a s e d f o r t h e f i r s t 3 days o f a d m i n i s t r a t i o n and remained constant t h e r e a f t e r . Simulated plasma l e v e l s of p i r e n z e p i n e a f t e r an o r a l t h e r a p e u t i c dosage regimen (50 mg, twice d a i l y ) on t h e b a s i s o f s i n g l e a d m i n i s t r a t i o n d a t a , showed s t e a d y s t a t e w i t h i n 2 days o f t r e a t m e n t (18). P i r e n z e p i n e a d m i n i s t e r e d i . m . was completely absorbed i n r a b b i t s and dogs. Rats t h a t p o o r l y absorb o r a l p i r e n z e p i n e showed complete a b s o r p t i o n o f i . m . dose o f t h e drug. Using monoclonal a s s a y s (15), plasma p i r e n z e p i n e l e v e l s and u r i n a r y e x c r e t i o n were measured over time

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i n 10 h e a l t h y male s u b j e c t s a f t e r 10 mg i . v . or i . m . dose, i n randomized c r o s s o v e r s t u d y . The d a t a were f i t t e d t o a 3-compartment open model. Absorption a f t e r i . m . i n j e c t i o n was r a p i d and complete (mean b i o a v a i l a b i l i t y 1 0 0 . 3 % ) . After i . v . b o l u s i n j e c t i o n h i g h plasma l e v e l peaks are reached which last only f o r a s h o r t time p e r i o d s i n c e due t o t h e d i s t r i b u t i o n o f p i r e n z e p i n e i n t o body t i s s u e s t h e plasma l e v e l d e c l i n e s r a p i d l y with an i n i t i a l h a l f - l i f e o f about 5 min. (17, 2 1 ) . The peak c o n c e n t r a t i o n a f t e r i . v . a d m i n i s t r a t i o n (22) was much h i g h e r than t h e t h e r a p e u t i c l e v e l a f t e r o r a l a d m i n i s t r a t i o n . In t h e f i r s t minutes a f t e r i . v . i n j e c t i o n o f 10 mg p i r e n z e p i n e ( 0 . 1 5 mg/kg) , t h e plasma c o n c e n t r a t i o n s were almost 1 0 - f o l d h i g h e r than t h e s t e a d y s t a t e l e v e l reached a f t e r d a i l y doses o f o r a l 100 mg. I n less t h a n one hour, however, t h e plasma l e v e l o f t h e i . v . dose i s comparable t o t h e s t e a d y s t a t e c o n c e n t r a t i o n s after o r a l a d m i n i s t r a t i o n (18). The a b s o r p t i o n o f S . C . dose o f p i r e n z e p i n e i s very swift and comparable t o t h a t o f i . v . (4) and u n l i k e t h a t o f i . m . r e p o r t e d by Hammer -e t a 1 (16). 6.2

Distribution Informations provided by whole-animal a u t o r a d i o g r a phy of r a t 10 minutes a f t e r i n t r a v e n o u s i n j e c t i o n o f 2 mg/kg o f r a d i o a c t i v e p i r e n z e p i n e (15, 2 1 ) , showed t h a t t h e drug is widely d i s t r i b u t e d i n t h e body. The r a d i o a c t i v e drug i s found i n a l l i n t e r n a l organs and s k e l e t a l muscles. No r a d i o a c t i v i t y was d e t e c t e d i n t h e b r a i n o f t h e r a t o r t h e f e t u s e s o f g e s t a t i n g mouse s u g g e s t i n g t h a t t h e blood-brain b a r r i e r r e p r e s e n t s a genuine b a r r i e r t o p i r e n z e p i n e and t h a t t h e drug does n o t c r o s s t h e p l a c e n t a l b a r r i e r . Continuous i n t r a v e n o u s i n f u s i o n o f p i r e n z e p i n e over a p e r i o d o f 2 days a t a r a t e o f 2 mg/hr/8-9 kg baboon (10 times t h e human i n f u s i o n d o s e ) , showed t h a t t h e h i g h e s t l e v e l s a r e i n t h e organs r e s p o n s i b l e f o r t h e e l i m i n a t i o n of t h e drug i . e . l i v e r and kidneys. The o t h e r i n t e r n a l organs, t h e s k i n and s k e l e t a l muscles, however, showed a h i g h e r p i r e n z e p i n e c o n c e n t r a t i o n p e r g of t i s s u e t h a n t h e plasma.

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Extremely low drug c o n c e n t r a t i o n was found i n t h e brain. The d i s t r i b u t i o n o f p i r e n z e p i n e i n t h e t i s s u e s a f t e r one-time o r a l a d m i n i s t r a t i o n was i n v e s t i g a t e d by Kobayashi -e t a 1 (19) a f t e r i s o l a t i o n o f t i s s u e s and whole-body a u t o r a d i o g r a p h y t e c h n i q u e . After 3 h r , t h e time o f maximum blood c o n c e n t r a t i o n , h i g h c o n c e n t r a t i o n of p i r e n z e p i n e was observed i n t i s s u e s l i k e l i v e r , kidney, p a n c r e a s , lung, p i t u i t a r y g l a n d , s a l i v a r y g l a n d , a d r e n a l gland e t c . These r e s u l t s , t h e r e f o r e , agree w i t h t h o s e o f Hammer and Koss (21) i n t h a t r a d i o a c t i v e p i r e n z e p i n e was found d i s t r i b u t e d i n a l l t i s s u e s e x c e p t t h e c e n t r a l nervous system. The r a d i o a c t i v i t y d i s t r i b u t e d i n t h i s way d e c r e a s e d as time p a s s e d . Twenty-four hours a f t e r a d m i n i s t r a t i o n , t h e c o n c e n t r a t i o n was s l i g h t l y h g i h o n l y i n t h e g a s t r o i n t e s t i n a l t r a c t and l i v e r . R a d i o a c t i v i t y i n t h e t e s t i s showed a tendency t o d e c r e a s e g r a d u a l l y d u r i n g 72 h r . Kaubisch et a1 (23) observed s t r o n g r a d i o a c t i v i t y d i s t r i b u t e d i n t h e g a s t r o i n t e s t i n a l t r a c t , kidney and s a l i v a r y g l a n d s i n a whole-body a u t o r a d i o g r a p h y performed a f t e r i . v. a d m i n i s t r a t i o n of 14C-pirenzepine i n a dose o f 2 mg/kg. The p r e s e n c e o f r a d i o a c t i v i t y i n t h e g a s t r o i n t e s t i n a l t r a c t was c o n s i d e r e d t o r e s u l t from t h e e x c r e t i o n o f t h e r a d i o a c t i v i t y i n t o t h e i n s i d e o f t h e i n t e s t i n a l t r a c t v i a t h e blood v e s s e l s and i n t e s t i n a l w a l l . T h i s i s s u p p o r t e d by t h e f i n d i n g o f Kobayashi e t a 1 (19) who observed c l e a n p i c t u r e s o f r a d i o a c t i v i t y d i s t r i b u t i o n a t t h e wall o r mucus o f t h e stomach and i n t e s t i n e s i n a u t o r a d i o g r a p h y 3 h r and 9 h r a f t e r a d m i n i s t r a t i o n and c o n s i d e r e d t h a t t h e r a d i o a c t i v i t y was s e c r e t e d from t h e g a s t r o i n t e s t i n a l wall o r was d e p o s i t e d t h e r e . L -

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After m u l t i p l e o r a l d o s e , Kobayashi e t a 1 ( 1 9 ) , observed a s l i g h t tendency towards accumulation i n t h e l i v e r , kidney and t e s t i s whereas r a d i o a c t i v i t y d e c r e a s e d s l o w l y i n one-day a d m i n i s t r a t i o n . However, t h e r a d i o a c t i v e c o n c e n t r a t i o n i n each t i s s u e did not increase i n proportion t o t h e a d m i n i s t r a t i o n times b u t d e c r e a s e d as time p a s s e d and l e f t o n l y t r a c e s one week a f t e r t h e t e r m i n a t i o n o f a d m i n i s t r a t i o n . In m u l t i p l e a d m i n i s t r a t i o n

HUMEIDA A. EL-OBEIDETAL.

almost a l l of t h e r a d i o a c t i v i t y was found excreted i n t o u r i n e and feces 24 h r a f t e r each administration. I t i s , t h e r e f o r e , considered t h a t t h e accumulation of t h e drug i n t h e body is small. Jaup and Blomstrand (24) measured serum and cerebrospinal concentrations of pirenzepine i n healthy volunteers a f t e r t h e r a p e u t i c dosage over 2-5 days. The cerebrospinal f l u i d to-serum r a t i o was 0.095 t o 1, i n d i c a t i n g t h a t pirenzepine passed t h e blood-brain b a r r i e r , but only t o a small e x t e n t . Pirenzepine concentrations i n t h e cerebrospinal f l u i d was about 10% those of serum. That t h e penetration of pirenzepine through t h e blood-brain b a r r i e r i s very limited was a l s o demonstrated by o t h e r s t u d i e s (18, 25-27). 6.3

Metabolism Pirenzepine undergoes only s l i g h t metabolism i r r e s p e c t i v e of the route of administration and t h e r e i s no s i g n i f i c a n t f i r s t - p a s s e f f e c t . Only small amounts of metabolites have been detected (16-18, 21), of which t h e desmethyl d e r i v a t i v e accounted f o r s i g n i f i c a n t l y less t h a n 10% of t h e dose. The desmethyl compound has no pharmacological e f f e c t . The t r i c y c l i c moiety, despiperazinly pirenzepine was a l s o detected a s a metabolite i n n e g l i g i b l e amounts ( 1 7 ) . Unlike cimetidine, pirenzepine does not i n h i b i t t h e h e p a t i c mixed function oxidase system (28).

6.4

Excretion In man as well as i n o t h e r animals, pirenzepine i s excreted unchanged i n u r i n e and feces ( 1 7 , 18, 21). Due t o t h e s p e c i a l physicochemical p r o p e r t i e s of pirenzepine only about 10% of t h e dose i s bound t o plasma p r o t e i n s (17, 20) and hence it i s almost completely f i l t e r e d i n t o t h e kidneys. The volume of d i s t r i b u t i o n of pirenzepine i s i d e n t i c a l t o t h e e x t r a c e l l u l a r space which i n d i c a t e s t h a t pirenzepine r a p i d l y e q u i l i b r a t e s between plasma and t h e e x t r a c e l l u l a r f l u i d s of t h e peripheral t i s s u e s . The t o t a l plasma clearance of pirenzepine i s 255 ml/min. Because of i t s limited

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l i p i d s o l u b i l i t y , t h e drug cannot be reabsorbed from t h e renal tubules s o t h a t t h e renal clearance of 129 ml/min corresponds t o the glumerular f i l t r a t i o n r a t e . Hammer -e t a1 (16) reported t h a t approximately equal amounts of pirenzepine were excreted v i a t h e b i l e and u r i n e . The h e p a t i c clearance, mostly b i l i a r y excretion i s about 125 ml/min (17). Following o r a l administration o f a t h e r a p e u t i c dose of labeled drug, 82% of t h e substance excreted i n u r i n e i s unchanged, a s is 92% of t h a t i n t h e feces. The drug i s eliminated from the organism f u l l y and almost equally i n u r i n e and f e c e s , elimination being almost complete a f t e r 4 days (17). Kobayashi -e t a1 (19) reported t h a t i n s i n g l e a s well as i n m u l t i p l e administration, almost a l l of t h e dose was excreted i n u r i n e and feces. In i . v . administration, 44% and 50% of t h e administered dose were excreted i n t o u r i n e and f e c e s , r e s p e c t i v e l y 24 h r a f t e r administration and 46% and 53% r e s p e c t i v e l y , 4 days a f t e r administr a t i o n . Similar r e s u l t s were obtained with subcutaneous administration. Hammer -e t a1 (16) reported t h a t 36% and 56% of t h e administered dose were excreted i n u r i n e and f e c e s , r e s p e c t i v e l y , following i . v . administration. 42% of a 1 0 mg dose was a l s o reported (15) eliminated r e n a l l y with t o t a l clearance o f 253 ml/min and renal clearance of 107 ml/min. The mean t r a n s i t time was 9 . 3 h r . A urinary excretion r a t e of 7-12% was reported by Ohashi -e t a1 (20) within 4 days a f t e r multiple administration of pirenzepine (25-50 mg, 3 times a day f o r 4 days). Hammer e t a1 (16) reported approximately 4% o f the admingt e r e d dose was excreted i n u r i n e a f t e r o r a l administration. According t o Kobayashi -e t a1 (19) about 8% was excreted i n u r i n e a f t e r s i n g l e o r a l administration and about 4.5% i n multiple administration. The d i f f e r e n c e i n t h e value was a t t r i b u t e d t o t h e d i f f e r e n c e i n absorption r a t e s between f a s t e d and nonfasted animals. Elimination h a l f - l i f e of 10 h r was reported (21) following i . v . dose o f 8 mg pirenzepine t o humans. Oral administration o f 25 mg t a b l e t s t o volunteers r e s u l t e d i n elimination h a l f - l i f e of

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11 h r (21). Ohashi e t a1 (20) reported an elimination h a l f - l i f e of 13 h r . The long eliminat i o n h a l f - l i f e was a t t r i b u t e d t o slow r e d i s t r i b u t i o n of pirenzepine from t h e t i s s u e s r a t h e r than t o slow excretion (21).

Intravenous administration o f 14C-pirenzepine t o dams i n a dose of 2 mg/kg caused t h e appearance o f r a d i o a c t i v i t y i n m i l k almost equivalent t o t h a t i n blood (19). Pretreatment of healthy volunteers with t h e r a p e u t i c doses of pirenzepine over 5 days showed no s i g n i f i c a n t e f f e c t on t h e absorption, d i s t r i b u t i o n o r elimination of a n t i p y r i n e (28). 7.

Pharmaco 1ogy 7.1

Antisecretory E f f e c t s Secretory s t u d i e s with pirenzepine c l e a r l y demonstrate t h a t pirenzepine suppresses both basal and stimulated acid and pepsin s e c r e t i o n (30-52). Intravenous pirenzepine reduces b a s a l acid secret i o n by 90% and pentagastrin-stimulated s e c r e t i o n by approximately 50% ( 3 0 ) . F r i t s c h -e t a1 (31) reported t h a t o r a l 50 mg of pirenzepine, i n p a t i e n t s with duodenal u l c e r , i n h i b i t e d t h e basal acid s e c r e t i o n by 39.9% and peptone-induced acid output by 21.3%. The percentage i n h i b i t i o n n e a r l y doubled i f t h e same dose is given a f t e r a treatment with 50 mg pirenzepine twice d a i l y f o r 3 t o 7 days. No acute e f f e c t on serum g a s t r i n l e v e l s can be demonstrated by a s i n g l e dose of pirenzepine. After pretreatment only small increase of basal g a s t r i n l e v e l s i s observed, serum g a s t r i n does not change during stimulation. Oral pirenzepine (25 mg) reduces acid output by 50% and pentagastrin-stimulated s e c r e t i o n by 30-40%. Doubling t h e dose increases t h e degree of i n h i b i t i o n of stimulated s e c r e t i o n by 10% (30). Jaup -e t a1 (32) reported t h a t pirenzepine 50 mg PO b . i . d . reduced b a s a l g a s t r i c a c i d s e c r e t i o n by 54% a t two hours following t h e l a s t dose. Pentagastrin-stimulated s e c r e t i o n o f hydrochloric acid (1 mcg/kg/hr) by continuous i . v . infusion was reduced by 31%. Secretory s t u d i e s by

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J a u p -e t a1 (33) c l e a r l y demonstrated t h a t p i r e n z e p i n e s u p p r e s s e s b a s a l , p e n t a g a s t r i n - s t i m u l a t e d and insulin-stimulated acid secretion in a dose-related manner. In a d o u b l e - b l i n d , placebo c o n t r o l l e d , randomized s t u d i e s on p a t i e n t s w i t h dyspepsia, Bianchi Porro et a1 (34) r e p o r t e d a 48% d e c r e a s e o f g a s t r i c s e c r e t i o n i n t h e first hour and 30% i n t h e second hour a f t e r 50 mg o r a l dose o f p i r e n z e p i n e . P i r e n z e p i n e caused a r e d u c t i o n t o 20% o f t h e concentr a t i o n o f t h e a c i d produced by vagal s t i m u l a t i o n (35). The drug i n h i b i t e d t h e v a g a l l y t r a n s m i t t e d a c i d s e c r e t i o n by about 45% even when t h e v a g a l l y s t i m u l a t e d a c i d s e c r e t i o n amounts t o more t h a n 50% o f t h e p e n t a g a s t r i n - s t i m u l a t e d s e c r e t i o n . These e f f e c t s of pirenzepine a r e simila r t o those of a tro p i n e b u t d i f f e r e n t from t h e e f f e c t s o f c i m e t i d i n e . Eugenides -e t a1 (36) r e p o r t e d t h a t histamines t i m u l a t e d a c i d o u t p u t p e r two hours i n h e a l t h y v o l u n t e e r s was reduced with 25 mg p i r e n z e p i n e b . d . by 12.5% and w i t h 50 mg t . d . s . by 24%. Stockbrugger et a1 (37) s t u d i e d t h e e f f e c t o f t h r e e d i f f e r e n t doses o f p i r e n z e p i n e on a c i d s e c r e t i o n s t i m u l a t e d by modified shamfeeding (MSF) on h e a l t h y v o l u n t e e r s . Oral p i r e n z e p i n e 25 mg b . i . d . , 50 mg b . i . d . o r 50 mg t . i . d . reduced b a s a l o u t p u t (0-30 min) by 48%, 59% and 66% r e s p e c t i v e l y , i n s t i m u l a t e d a c i d o u t p u t (0 120 min) by 45%, 58% and 48% r e s p e c t i v e l y . When t h e a c i d s e c r e t i o n was c a l c u l a t e d a s volume c o r r e c t e d f o r d u o d e n a l - g a s t r i c r e f l u x and p y r o l i c l o s s e s , t h e corresponding f i g u r e s were 33.7%, 3 6 . 3 % and 42.6%. Mignon e t a1 (38) r e p o r t e d a d o s e - r e l a t e d r e d u c t i o n o f meal-stimulated a c i d response w i t h i n c r e a s i n g doses o f i . m . p i r e n z e p i n e . R e s u l t s with 0 . 5 mg/kg o f p i r e n z e p i n e showed 53% r e d u c t i o n i n a c i d secret i o n . P i r e n z e p i n e 10 mg given t o duodenal u l c e r p a t i e n t s 45 min and a g a i n 10 min b e f o r e t h e s t a r t of MSF blocked 48% o f t h e response (39).

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P i r e n z e p i n e (25 pg/kg) a d m i n i s t e r e d i . m . t o dogs 10 min b e f o r e i n f u s i o n o f bombesin, s i g n i f i c a n t l y i n h i b i t e d t h e h y p e r s e c r e t i o n induced by t h e p e p t i d e i n t h e main stomach and i n a dose o f 100 ug/kg p i r e n z e p i n e v e r t u a l l y a b b o l i s h e d t h e a c i d response t o bombesin ( 4 0 ) . I n Heidenhain pouch b o t h doses f u l l y prevented a c i d s e c r e t i o n . Pirenzepine i n j e c t e d during t h e secretory plateau

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induced by bombesin, produced a mean peak i n h i b i t i o n of acid output from t h e main stomach of 76.5% and 80% with doses of 25 and 100 g/kg respectively. Pirenzepine (25 and 100 ug/kg i . v . , 10 min before a meal t e s t ) f u l l y prevented a c i d s e c r e t i o n f o r Hiedenhain pouch. The drug showed no e f f e c t on bombesin - or meal-stimulated g a s t r i n r e l e a s e . Acid and pepsinogen s e c r e t i o n induced by bethanechol i n i s o l a t e d perfused mouse stomach a r e i n h i b i t e d by pirenzepine (41). 7.2

Efficacy Pirenzepine i s e f f e c t i v e i n t h e treatment of g a s t r i c and duodenal u l c e r s . I t i s a l s o e f f e c t i v e i n nonu l c e r dyspepsia and i n preventing u l c e r recurrence. Pirenzepine i n combination therapy with cimetidine can be used s u c c e s s f u l l y f o r t h e treatment o f c i m e t i d i n e - r e s i s t a n t conditions such as duodenal u l c e r and Zollinger-Ellison syndrome. The l i t e r a t u r e r e p o r t s a number of s t u d i e s on t h e e f f i c a c y of pirenzepine i n t h e treatment of g a s t r i c and duodenal u l c e r s (43, 53-130). The published world l i t e r a t u r e (from 1977 t o 1981) on t h e e f f i c a c y of pirenzepine had been t h e s u b j e c t o f a review and commentary by Bianchi Porro and P e t r i l l o (53). The review examined t h e d a t a published i n Europe which deal with pirenzepine i n t h e treatment of g a s t r i c and duodenal u l c e r s , taking i n t o account only those r e s u l t s obtained from c o n t r o l l e d endoscopic t r i a l s . In 12 prospective randomized double-blind placebo-controlled s t u d i e s pirenzepine was administered t o 475 duodenal u l c e r p a t i e n t s with incidence of endoscopically proven healing of 74% when the p a t i e n t s received d a i l y dosage o f 100 o r 150 mg pirenzepine, but only 55% i f t h e dosage of t h e drug was 75 mg o r less. Similar healing rates were observed (72% when d a i l y dose of pirenzepine was 100 o r 150 mg, 55% when t h e dose was 75 mg o r l e s s ) i n 4 randomized doubleb l i n d placebo -controlled s t u d i e s i n which 84 p a t i e n t s with g a s t r i c u l c e r were admitted. In another review, Texter and R e i l l y (83) evaluated the c l i n i c a l evidence concerning t h e healing e f f e c t of pirenzepine i n g a s t r i c and duodenal u l c e r s . The reviewers subjected t h e

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results of t h e previous double-blind, t h e r a p e u t i c s t u d i e s on u l c e r healing t o mathematical treatments. The t h e r a p e u t i c s t u d i e s reviewed included a l a r g e number o f p a t i e n t s i n Europe and Japan. The doseresponse curve f o r duodenal u l c e r incorporating 16 d a t a p o i n t s from 8 t r i a l s i n 530 p a t i e n t s , 343 of whom received pirenzepine, r e s u l t e d i n a c o r r e l a t i o n c o e f f i c i e n t r = 0.887 with P < 0.005. Although t h e a n a l s i s is not s t r i c t l y mathematically c o r r e c t , it suggests t h a t pirenzepine h e a l s duodenal u l c e r compared t o placebo with a p o s i t i v e r e l a t i o n s h i p between dose and e f f e c t . The pred i c t e d healing r a t e a t 150 mg was 79%. The a n a l y s i s a l s o shows t h a t endoscopic healing from g a s t r i c and duodenal u l c e r s i s l i n e a r with respect t o time upto 6 weeks ( r = 0.75, P < 0.001) and suggests t h a t healing r a t e s improve with time on therapy. This observation on time course is strengthened by t h e r e s u l t s of a l a r g e Japanese m u l t i c e n t r e t r i a l on pirenzepine. Therapeutic s t u d i e s on g a s t r i c u l c e r , showed endoscopic healing r a t e o f 66.7%, using 75 mg/day pirenzepine f o r 8 weeks, which increased t o 72% with 150 mg dose f o r 8 weeks. Brunner (94) reported t h a t pirenzepine is equally o r a t least n o t e s s e n t i a l l y l e s s e f f e c t i v e than cimetidine i n t h e treatment o f duodenal u l c e r . The e f f e c t of pirenzepine and cimetidine on healing, symptoms and r e l a p s e r a t e of duodenal u l c e r was s t u d i e d by Eichenberger -e t a1 (95). No s i g n i f i c a n t d i f f e r e n c e s i n u l c e r healing between t h e e f f i c a c y o f t h e drugs when 1 gm/day o f cimetid i n e and 75 mg/day pirenzepine were used. The r e l a p s e r a t e a f t e r treatment with pirenzepine was lower than a f t e r treatment with cimetidine.

Do -e t a1 (96) reported t h e e f f i c a c y of pirenzepine, 100 mg daily, i n t h e treatment of duodenal u l c e r s i n a placebo-controlled study, Pirenzepine produced healing i n 14 out o f 18 p a t i e n t s (78%) following 6 weeks o f treatment as compared t o 7 of 20 p a t i e n t s (35%) receiving placebo. Relief o f daytime and nighttime pain was g r e a t e r i n pirenzepine-treated p a t i e n t s , a s well as lower consumption of antacid.

484

HUMEIDA A. EL-OBEID ETAL.

Cheli -e t a1 (97) reported t h a t pirenzepine i n 100 mg o r a l l y d a i l y f o r 28 days r e s u l t e d i n c l i n i c a l cure i n 34 of 44 duodenal u l c e r p a t i e n t s . Sixteen p a t i e n t s completing treatment, were given pirenzepine 50 mg d a i l y f o r 6 months, r e s u l t e d i n u l c e r r e l a p s e i n 18.7%. In 16 o t h e r p a t i e n t s , who i n t e r r u p t e d treatment completely without pirenzepine, c l i n i c a l recurrence of duodenal u l c e r occured i n 68.7%, with endoscopic recurrence i n 62%, i n t h e following 6 months.

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Pirenzepine was shown by Dal Monte e t a1 (98) t o be e f f e c t i v e i n t h e maintenance treatment of duodenal u l c e r s t o prevent u l c e r recurrence when given i n o r a l doses o f 100 mg d a i l y f o r one year. Pirenzepine has a l s o been e f f e c t i v e i n doses of 50 mg d a i l y f o r one year (100, 104, 107). However, o t h e r i n v e s t i g a t o r s have reported no s i g n i f i c a n t d i f f e r e n c e between pirenzepine 25 mg o r a l l y b . i . d . and placebo i n t h e prevention o f duodenal u l c e r r e l a p s e i n a one-year study (102). Hoffenberg (103) reported pirenzepine i n o r a l dose of 50 mg b . i . d . f o r 4 weeks, i n i t i a l l y , followed by 50 mg b . i . d . f o r 4 weeks, was no more e f f e c t i v e than placebo i n t h e treatment of duodenal u l c e r (48% versus 40% complete healing). These d a t a suggest t h a t longer courses of therapy (6-8 weeks) may be indicated. Pirenzepine i n o r a l d a i l y doses of 100 - 150 mg has been e f f e c t i v e i n producing healing of 74% of p a t i e n t s with duodenal u l c e r s i n c o n t r o l l e d c l i n i c a l t r i a l s (range 52 90%) (53, 55, 57, 60, 74, 76, 104, 105), whereas lower doses of 75 mg d a i l y o r l e s s have produced lower response r a t e s ( z 55%) (53, 54, 106). These results a r e supported by more recent c l i n i c a l s t u d i e s with pirenzepine i n o r a l d a i l y doses of 100 150 mg and t h i s appears t o be t h e optimal dose (107 - 113).

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Available c l i n i c a l t r i a l s on g a s t r i c u l c e r suggest t h a t o r a l doses of 100-150 mg d a i l y can produce healing in approximately 72% of p a t i e n t s with gast r i c u l c e r , s i m i l a r t o duodenal u l c e r , doses of 75 mg d a i l y o r l e s s produce lower healing rates (53, 89, 114, 115).

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Oral 50 mg d a i l y dose of pirenzepine was reported e f f e c t i v e i n t h e treatment o f non-ulcer dyspepsia i n a double-blind comparative t r i a l i n 59 p a t i e n t s f o r a period of 4 weeks (116). A d a i l y dose o f 100 mg was a l s o shown t o be e f f e c t i v e (117). Short-term pirenzepine therapy (100 mg d a i l y f o r 4 weeks) was reported e f f e c t i v e i n improving t h e endoscopic appearance of acute c o n j e s t i v e and e r o s i v e g a s t r i t i s and non-ulcer-associated severe duodenitis (118). The drug was s i m i l a r l y e f f e c t i v e a s cimetidine 1 gm d a i l y f o r 4 weeks. Comparative e f f i c a c y of pirenzepine 100 mg PO d a i l y and cimetidine 1 gm PO d a i l y i n 6-week treatment o f duodenal u l c e r i n o u t p a t i e n t s has been indicated by s e v e r a l c o n t r o l led c l i n i c a l t r i a l s (67, 68, 81, 107, 108). Porro e t a1 (109) have a l s o reported t h a t pirenzepine 150 mg PO d a i l y was comparable t o cimetidine 1 gm d a i l y i n t h e treatment o f a c t i v e duodenal u l c e r i n 90 p a t i e n t s t r e a t e d f o r a period of 4 weeks (72% versus 75% r e s p e c t i v e l y ) . In most c l i n i c a l s t u d i e s , t h e e f f i c a c y of both drugs was approximately 70%. Both drugs have been equally e f f e c t i v e i n preventing duodenal u l c e r recurrence following healing with conventional treatment with e i t h e r drug (99, 101, 120, 121). Combination therapy with cimetidine has been e f f e c t i v e i n Zollinger-Ellison syndrome r e s i s t a n t t o cimetidine alone (121).

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A s i n g l e 400 mg o r a l dose of cimetidine demonstrated

approximately twice t h e a n t i s e c r e t o r y a c t i v i t y of 50 mg pirenzepine s i n g l e o r a l dose i n both basal and stimulated g a s t r i c a c i d s e c r e t i o n following pentagastrin stimulation. With pirenzepine i n doses o f 50 mg, i n h i b i t i o n of p e n t a g a s t r i n stimulated acid s e c r e t i o n was similar t o t h a t o f an o r a l 200 mg cimetidine, t h i s cimetidine dose, however, had a g r e a t e r i n h i b i t o r y e f f e c t on g a s t r i c basal a c i d s e c r e t i o n (122). The authors i n d i c a t e t h a t high doses of pirenzepine should be avoided due t o antimuscarinic e f f e c t s such a s dry mouth and p a l p i t a t i o n s and t h a t cimetidine i s i n d i c a t e d as agent of choice f o r reduction of g a s t r i c a c i d s e c r e t i o n . P a l p i t a t i o n s occurred i n s e v e r a l p a t i e n t s i n t h i s study with doses of 50 mg. In a s i n g l e - b l i n d multicenter study Brunner et a1 (108) evaluated t h e e f f i c a c y of pirenzepine 100 mg d a i l y

486

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(50 mg before b r e a k f a s t and dinner) compared with t h a t of cimetidine 1 gm d a i l y (200 mg t . i . d . with meals and 400 mg h . s . ) i n t h e treatment of endoscopically-proven duodenal u l c e r i n 254 p a t i e n t s . Endoscopy was repeated a f t e r 4 weeks of treatment with both regimens; t h e endoscopist was blinded a s t o treatment regimens. Healing r a t e s of 64% and 73% occurred with pirenzepine and cimetidine, r e s p e c t i v e l y , which was not s t a t i s t i c a l l y s i g n i f i c a n t . Pain r e l i e f was achieved r a p i d l y with both drugs and t h e incidence of s i d e e f f e c t s was s i m i l a r .

Pirenzepine i n an o r a l dose of 50 mg b . i . d . was reported s i m i l a r l y e f f e c t i v e as cimetidine 400 mg b . i . d . o r a l dose (low-dose therapy) i n t h e treatment of duodenal u l c e r s i n multicenter t r i a l (125). Following 4 weeks of therapy, healing occured i n 27 of 37 pirenzepine-treated p a t i e n t s (73%) and 29 of 38 cimetidine-treated p a t i e n t s ( 7 6 % ) . Side e f f e c t s were s i m i l a r i n each group, except f o r more frequently reported antimuscarinic e f f e c t s i n pirenzepine-treated p a t i e n t s . No adverse e f f e c t s on i n t r a g a s t r i c milieu were reported. I n t r a g a s t r i c microbial concentrations were increased s i g n i f i c a n t l y with both drugs but remained within normal limits; n i t r i l e concentrat i o n p r i o r t o o r following treatment did not exceed t h e normal range. Oral 300 mg d a i l y dose r a n i t i d i n e was reported (104) equally e f f e c t i v e as o r a l 100 mg d a i l y dose of pirenzepine i n t h e treatment of duodenal u l c e r i n one c o n t r o l l e d study (response rates of 87% i n each group). Pirenzepine was equally e f f e c t i v e as a t r o p i n e as g a s t r i c a n t i s e c r e t o r y agent, but unlike a t r o p i n e , pirenzepine had no e f f e c t s on CNS, i n c r e a s e i n h e a r t r a t e , mydriasis, urinary bladder c o n t r a c t i o n o r motor function of g a s t r o e n t e r i c t r a c t (124). Londong -e t a1 (125) reported t h a t concomitant pirenzepine and cimetidine therapy r e s u l t i n s i g n i f i c a n t l y g r e a t e r suppression o f stimulated acid s e c r e t i o n than e i t h e r drug alone. I t i s suggested t h a t combination therapy may e x h i b i t s y n e r g i s t i c e f f e c t s on p a r i e t a l c e l l function and could be advantageous i n p a t i e n t s w i t h gastrinoma,

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u l c e r s and h y p e r s e c r e t i o n r e s i s t a n t t o s i n g l e drug t h e r a p y , and i n p r o p h y l a x i s o f stress u l c e r bleeding i n c r i t i c a l l y - i l l p a t i e n t s . Weberg et a1 (126) h a s demonstrated t h a t p i r e n z e p i n e enhances and prolongs t h e e f f e c t s o f a n t a c i d s on i n t r a g a s t r i c a c i d i t y . Addition o f p i r e n z e p i n e t o a n t a c i d t h e r a p y r e s u l t e d i n a more s u s t a i n e d pH e l e v a t i o n t h a n t h a t observed with t w i c e t h e amount o f a n t a c i d a d m i n i s t e r e d a l o n e . Combination t h e r a p y w i t h H2-receptor a n t a g o n i s t s h a s proven more e f f e c t i v e t h a n H2-antagonist t h e r a p y a l o n e (118, 1 2 7 ) . The combination o f p i r e n z e p i n e 50 mg h . s . and r a n i t i d i n e 150 mg b . i . d . given f o r a p e r i o d o f 6-12 weeks was e f f e c t i v e i n a l l of 7 p a t i e n t s with a c t i v e recurrent duodenal u l c e r . Continuous maintenance t h e r a p y with r a n i t i d i n e 150 mg d a i l y and p i r e n z e p i n e 50 mg d a i l y r e s u l t e d i n prevention of u l c e r relapse i n 5 p a t i e n t s who, i n t h e p r e v i o u s y e a r , c o n t i n u a l l y r e l a p s e d while r e c e i v i n g s i n g l e - a g e n t ( c i m e t i d i n e o r r a n i t i d i n e ) t h e r a p y . These r e s u l t s s u g g e s t t h e e f f i c a c y of combined H2-antagonist and p i r e n z e p i n e t h e r a p y i n h i g h l y r e c u r r e n t o r r e s i s t a n t duodenal u l c e r s (127). Mignon e t a1 (121) r e p o r t e d t h e e f f e c t i v e n e s s o f combination t h e r a p y o f p i r e n z e p i n e and c i m e t i d i n e i n 3 of 4 p a t i e n t s w i t h Z o l l i n g e r E l l i s o n syndrome who were unresponsive t o cimet i d i n e a l o n e . - L

Unlike c i m e t i d i n e , inhibitory effects o x i d a s e system and concomitantly w i t h t h i s system (128).

p i r e n z e p i n e appears t o l a c k on t h e h e p a t i c mixed f u n c t i o n it should be safe t o a d m i n i s t e r drugs t h a t a r e metabolized by

The a v a i l a b l e i n f o r m a t i o n s s u g g e s t t h a t p i r e n z e p i n e p o s s e s s e s s e l e c t i v e a n t i m u s c a r i n i c e f f e c t s and minimal CNS effects. S t u d i e s p r e s e n t e d so f a r do n o t show t h a t t h e drug i s more e f f e c t i v e t h a n c i m e t i d i n e o r r a n i t i d i n e and no s i g n i f i c a n t d i f f e r e n c e s i n t o x i c i t y have been observed. P i r e n z e p i n e i n combination t h e r a p y w i t h H2-receptor a n t a g o n i s t s , however, s u g g e s t t h a t p i r e n z e p i n e may be very u s e f u l i n t h e t r e a t m e n t o f r e s i s t a n t duoden a l u l c e r and Z o l l i n g e r - E l l i s o n syndrome.

488

HUMEIDA A. EL-OBEIDETAL.

7.3

Adverse Reactions The major s i d e e f f e c t s of pirenzepine a r e due t o i t s a n t i c h o l i n e r g i c a c t i v i t y which i n d i c a t e s t h a t t h e s e l e c t i v i t y of t h e drug is n o t absolute and t h e a n t i c h o l i n e r g i c e f f e c t s can occur which appear t o be dose-dependents. Dry mouth is t h e most common s i d e e f f e c t of pirenzepine. S a l i v a r y s e c r e t i o n e f f e c t s have been noted i n many t h e r a p e u t i c t r i a l s . In t h e pharmacological s t u d i e s i n man, pirenzepine i n a dose of 50 mg b . i . d . i n h i b i t e d s a l i v a t i o n compared t o placebo (131) but not a s much as R-hyoscyamine i n a dose o f 0.6 mg b . i . d . which was approximately equipotent i n suppression of g a s t r i c a c i d . Ten of 18 p a t i e n t s i n t h e t r i a l s reported dry mouth with pirenzepine and 17 of 18 on R-hyoscyamine. Dry mouth i s reported t o occur i n 13.5% of p a t i e n t s receiving pirenzepine i n doses o f 150 mg d a i l y . The incidence is reduced t o 4% with doses of 100 mg d a i l y and t o 2.5% with doses of 75 mg d a i l y o r l e s s (54). Dry mouth does not appear t o be severe enough t o warrant withdrawal of treatment i n most cases (106). Pirenzepine i n high doses i n h i b i t s t h e c o n t r a c t i l e (132) a c t i v i t y of t h e stomach and colon (132). Bianchi Porro e t a1 (34), however, reported pirenzepine (50 mg o r a l l y ) showed no e f f e c t s on g a s t r i c emptying. Similar r e s u l t s were reported by Stacher -e t a1 (133) when healthy men received e i t h e r 50 mg pirenzepine twice d a i l y o r placebo. Despite t h e considerable e f f e c t s of pirenzepine on a n t r a l m o t i l i t y , its delaying e f f e c t on g a s t r i c emptying was i n s i g n i f i c a n t . Constipation has occurred with pirenzepine i n approximately 2.6% of p a t i e n t s receiving doses of 150 mg o r a l d a i l y dose (54). Diarrhea has a l s o occurred with pirenzepine therapy, however, t h e incidence appears t o be l e s s than t h a t of constipation (23, 106, 134). Jaup (135) measured esophageal p e r i s t a l t i c a c t i v i t y a f t e r pirenzepine, 10 mg i . v . o r 50 mg b . i . d . ( o r a l l y ) i n comparison with a t r o p i n e 0.5 mg i . v . o r R-hyoscyamine 0.6 mg b . i . d . The l a t t e r two agents reduced esophageal p e r i s t a l t i c pressure compared t o placebo while pirenzepine showed I

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marked e f f e c t s only s h o r t l y a f t e r i . v . dosing, when plasma l e v e l s were high. Oral pirenzepine s l i g h t l y decreased (- 15%) p e r i s t a l t i c amplitude compared t o placebo while R-hyoscyamine decreased t h i s parameter over 50%. Other s t u d i e s (136, 137) have shown decreased amplitude of esophageal contractions as well as i n h i b i t i o n of lower esophageal s p i n c t e r pressure with p a r e n t e r a l administration of pirenzepine. Colonic m o t i l i t y is reduced by 0 . 3 mg/kg i . m . (138) and g a s t r i c smooth muscle e f f e c t s a r e s l i g h t a t 0 . 2 mg/kg i . m . (139) o r 50 mg b . i . d . (131). Despite pirenzepine e f f e c t s i n a l t e r i n g esophageal and colonic m o t i l i t y , t h e a v a i l a b l e d a t a does not suggest t h e drug w i l l increase t h e r i s k of p e p t i c g a s t r o esophageal r e f l u x (140). The e f f e c t s of intravenous and o r a l pirenzepine

on various g a s t r o i n t e s t i n a l hormones and metab o l i c parameters a r e s i m i l a r t o those observed f o r antimuscarinic agents except f o r t h e lack of depression of pancreatic bicarbonate s e c r e t i o n (141, 142) and p o s s i b l y lack of g a s t r i n (143-145). Pirenzepine produced no changes i n g a s t r i c i n h i b i t o r y polypeptide, i n s u l i n , glucagon, neurotensin, vasoactive i n t e s t i n a l polypeptide, somatostatin, s e c r e t i n , f r e e f a t t y a c i d s , l a c t a t e , pyruvate, f3-hydroxybutyrate, a c e t o a c e t a t e , p r o l a c t i n and growth hormone. Pancreatic s e c r e t i o n volume, amylase, t r y p s i n , chymotrypsin,. l i p a s e , p a n c r e a t i c polypeptide, enteroglucagon, pepsin and g a s t r i c a c i d s e c r e t i o n s a r e decreased. One study (146) noted a s l i g h t prolongation o f g a s t r i n postp r a n d i a l l y , while o t h e r s (143, 145) observed decreases i n c o n t r a s t t o a t r o p i n e and another t r i a l (144) showed no change. Masala e t a1 (147) reported t h a t pirenzepine i n s i n g l e doses o f 75 mg reduced p r o l a c t i n l e v e l s i n females but produced only minimal e f f e c t s on p r o l a c t i n i n males. S i n g l e doses o f 75 mg pirenzepine were reported by Alagna -e t a1 (148) t o produce no d i r e c t e f f e c t upon t h e adrenal glands. The responsiveness o f t h e adrenal glands t o exogenous ACTH was unchanged, However, a reduction i n basal c o r t i s o l l e v e l s occurred with pirenzepine, suggesting a reduction i n ACTH s e c r e t i o n . The drug has not produced adrenal i n s u f f i c i e n c y .

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HUMEIDA A. EL-OBEIDETAL.

Visual disturbances have been reported i n 42% o f s u b j e c t s (149). Brunner (94) however, reported t h a t only small number of p a t i e n t s t r e a t e d with pirenzepine complained about impairment o f v i s i o n . Blurred vision and double vision have occurred during p i renz ep i n e t r e a t men t i n approximate 1y 6.3% of p a t i e n t s receiving o r a l d a i l y doses of 150 mg (75, 83). Hoffenberg (103) described amblyopia i n 24% of p a t i e n t s receiving dose of 100 mg d a i l y . Urinary r e t e n t i o n has been reported i n 0.3% of p a t i e n t s receiving pirenzepine i n doses o f 100 mg d a i l y ( 8 3 ) . Kuhn -e t a1 (150) reported t h a t pirenzepine (8 mg i.m.) decreased t h e pulse r a t e and s y s t o l i c and d i a s t o l i c blood pressure. Tachycardia has been observed during pirenzepine treatment (103). In most s t u d i e s , however, no e f f e c t s on h e a r t r a t e were observed (83). Pirenzepine i n dose of 50 mg d a i l y caused p a l p i t a t i o n s (122). CNS t o x i c i t y has been minimal i n most s t u d i e s with pirenzepine (83, 151). This can be explained on t h e bases o f pirenzepine s e l e c t i v i t y and a l s o t o hydrophilic c h a r a c t e r i s t i c s . Dizziness, mental confusion, a s t h e n i a and headache have been reported i n a few p a t i e n t s (152, 153). Studies i n healthy volanteers showed t h a t no psychotropic e f f e c t s occurred t h a t a r e commonly encountered with t r i c y c l i c a n t i d e p r e s s a n t s , i n d i c a t i n g t h a t pirenzepine lack appreciable CNS t o x i c i t y (154). Fink and Erwin (149) a l s o reported lack of CNS e f f e c t s by pirenzepine with doses upto 150 mg. 7.4

Tissue S e l e c t i v i t y Pirenzepine blocks t h e acetylcholine receptors of t h e p a r i e t a l c e l l s o f the stomach and i s thereby a marked i n h i b i t o r of g a s t r i c acid s e c r e t i o n both i n animals and men. Pharmacological and c l i n i c a l s t u d i e s have shown t h i s a c t i o n of pirenzepine t o be s e l e c t i v e , s i n c e i n doses which s i g n i f i c a n t l y i n h i b i t g a s t r i c secretion, s i d e e f f e c t s typical of a n t i c h o l i n e r g i c agents do not occur o r minimal (30, 33, 45) and much higher doses a r e needed t o i n h i b i t s a l i v a t i o n (132) and contraction of stomach and colon (138) o r t o induce tachycardia (52, 132).

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Engelhorn (155) has e v a l u a t e d t h e s e l e c t i v i t y o f p i r e n z e p i n e and a t r o p i n e and h i s experiments i n d i c a t e d s u b s t a n t i a l d i f f e r e n c e s between t h e i n f l u e n c e o f e i t h e r a n t i m u s c a r i n i c agent on g a s t r i c u l c e r and g a s t r i c s e c r e t i o n on one hand and on smooth muscle organs on t h e o t h e r . Although t h e a n t i - u l c e r and a n t i s e c r e t o r y e f f e c t s o f p i r e n z e p i n e approach t h o s e o f a t r o p i n e , s i g n i f i c a n t d i f f e r e n c e s a r e demonstrated i n i s o l a t e d muscle preparations a f t e r acetylcholine stimulation as well a s after transmural stimulation. Pirenzepine was 16.5 times weaker than a t r o p i n e i n reducing s a l i v a t i o n i n r a b b i t and is 100 times less e f f e c t i v e than a t r o p i n e i n doubling p a p i l l a r y diameter. Atropine caused a dose-dependent inhibition of t h e gastrointes tin a l tr a n s p o r t w h i l e p i r e n z e p i n e , even a t h i g h e r d o s e s , d i d n o t cause a d e c r e a s e b u t r a t h e r an i n c r e a s e . Atropine caused a dose-dependent i n c r e a s e i n h e a r t r a t e which, however, could only be o b t a i n e d w i t h p i r e n z e p i n e i n doses 10 t o 100 times g r e a t e r t h a n with a t r o p i n e . Jaup et a1 (131, 156) compared t h e effects o f p i r e n z e p i n e and R-hyoscyamine when t a k e n i n e q u i p o t e n t doses i n i n h i b i t i n g g a s t r i c s e c r e t i o n . Both drugs a f f e c t e d t h e s a l i v a r y s e c r e t i o n b u t t h e i n h i b i t i o n by p i r e n z e p i n e was s i g n i f i c a n t l y less t h a n t h a t by R-hyoscyamine. Gastric emptying was s i g n i f i c a n t l y delayed by R-hyoscyamine compared t o p i r e n z e p i n e . Swallowing induced eosphageal p e r i s t a l s i s was s i g n i f i c a n t l y i n h i b i t e d i n 51%. P u p i l s i z e and n e a r p o i n t d i s t a n c e were s i g n i f i c a n t l y a f f e c t e d by R-hyoscyamine b u t n o t by p i r e n z e p i n e . The minimal e f f e c t s o f p i r e n z e p i n e on t h e CNS have been a t t r i b u t e d t o t h e drug s e l e c t i v i t y and h y d r o p h i l i city. 7.5

Mechanism o f Action I t i s g e n e r a l l y accepted t h a t p i r e n z e p i n e i s an a n t i m u s c a r i n i c drug. Among o t h e r examples demonstrating t h i s , is t h e c o m p e t i t i v e i n h i b i t i o n by t h e drug of t h e carbachol e f f e c t on t h e spontaneously b e a t i n g guinea p i g atrium ( 1 5 7 ) , t h e s e l e c t i v e antagonism o f c a r b a c h o l - s t i m u l a t e d a c i d s e c r e t i o n i n t h e p e r f u s e d r a t stomach w h i l e no such antagonism was n o t i c e d when h i s t a m i n e o r

492

HUMEIDA A. EL-OBEID ETAL.

p e n t a g a s t r i n was used as a s t i m u l u s (48), as well a s t h e s e l e c t i v e antagonism, by p i r e n z e p i n e , o f t h e m u s ca r i n i c response t o bethanechol on conscious c a t s with g a s t r i c f i s t u l a (157) and t h e f i n d i n g t h a t p i r e n z e p i n e , i n g r e a t e r amount t h a t a t r o p i n e , blocked o n l y t h e m u s c a r in ic e f f e c t s o f a c e t y l c h o l i n e without a l t e r i n g t h e n i c o t i n i c component (151). S i m i l a r t o o t h e r a n t i m u s c a r i n i c a g e n t s , p i r en z e p i n e a f f e c t s o n ly t h e volume and et a1 a c i d o u t p u t , and n o t g a s t r i c pH (83). B a ld i (158) have demonstrated t h a t p i r e n z e p i n e (0.15 mg/kg, i . m . ) i n h i b i t s t h e motor response i n t h e human colon t o a c h o l i n e r g i c s t i m u l u s w i t h p r o stig m in e . S t u d i e s on r e c e p t o r b i n d i n g , pharmacological i n v e s t i g a t i o n on i s o l a t e d t i s s u e s and s t u d i e s i n man have shown t h a t p i r e n z e p i n e d i s t i n g u i s h e s between d i f f e r e n t s u b c l a s s e s o f m u sc a r in ic r e c e p t o r s (83, 150, 159-170). Using p i r e n z e p i n e a s novel t o o l t h a t r e v e a l s h e t e r o g e n e i t y o f m u sc a r in ic r e c e p t o r s , ev i d en c e s have been p r o v id e d (164-166) o f t h e e x i s t e n c e o f t h r e e d i s t i n c t i v e t y p e s of muscarinic receptors c l a s s i f i e d a s types A, B and C. With p i r e n z e p i n e t h e l o g a f f i n i t y c o n s t a n t f o r t h e A-type is about 7.7, f o r t h e Bt y p e about 6.6 (10-fold weaker) and 4 - f o l d f u r t h e r weaker f o r t h e C-type. In t h e same system t h e l o g a f f i n i t y f o r a t r o p i n e i s about 8.5 t o 9 , i n d i c a t i n g t h a t t h e pharmacological r e sp o n se o f p i r e n z e p i n e as compared t o t h a t o f a t r o p i n e w i l l b e about 1 0 - f o l d weaker a t t h e A-type, 1 0 0 - f o ld weaker a t t h e B-type and 400-fold weaker a t t h e C-type. The pharmacological d a t a a v a i l a b l e on t h e potency of p i r e n z e p i n e as well a s d a t a from b in d in g s t u d i e s s u g g e s t t h a t i n t h e calf sy m p a th e tic g a n g l i a t h e r e seems t o be predominantly Ar e c e p t o r s and i n t h e r a t s y m p a th e tic g a n g l i a more o f B- r e c e p t o r s . The myocardium have predominantly C - r e c e p t o r s , t h e conduction t i s s u e s i n t h e h e a r t c o n t a i n B-type r e c e p t o r s w h i le t h e ileum seems t o have b o t h t y p e B and C r e c e p t o r s and t h e CNS b o t h t y p e A and B r e c e p t o r s . In most of t h e cases t h e r e i s good q u a n t i t a t i v e agreement between t h e b i n d i n g a n a l y s i s and t h e pharmacological r e s u l t s (132, 155). Evidence f o r t h e h e t e r o g e n e i t y o f m u s c ar i n i c r e c e p t o r s i s a l s o p r e s e n t e d (171-173) using t h e s e l e c t i v e ganglionic receptor agonist

PIRENZEPINE DIHYDROCHLORIDE

493

McN-A-343 [ 4- (m-chlorophenylcarbamoyloxy) -2butenyltrimethylammonium c h l o r i d e ] . The compound s e l e c t i v e l y stimulates t h e muscarinic receptors on ganglionic receptors with minimal influence on t h e muscarinic receptors i n t h e h e a r t and smooth muscle. Those receptors stimulated s e l e c t i v e l y by McN-A-343 have been named as M1-receptors and those stimulated by conventional muscarinic drugs are c a l l e d M2-receptors. Further i n v e s t i gations a r e needed on pirenzepine and McN-A-343 t o determine which nomenclature t o follow. I t appears t h a t MI and M2 receptors correspond t o t h e A-type and C-type receptors r e s p e c t i v e l y . Pirenzepine may a l s o have a cytoprotective e f f e c t i n addition t o i t s a n t i s e c r e t o r y e f f e c t s (174, 175). Other mechanism f o r pirenzepine ulcer-heating e f f e c t s were a l s o reported (176). Acknowledgement The authors would l i k e t o thank M r . Tanvir A. B u t t f o r typing t h i s manuscript.

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R. Cheli -et a l , Hephato-Gastroent. Abstr. E23.5, p. 175 (1980).

89.

G . Gasbarrini -e t a l , Scand. J . Gastroent., 14(57), 25 (1979).

90.

M. Giger -e t a l , In: "Die Belhandlung des Ulcus Pepticum m i t Pirenzepin", A . L . B l u m and R . Hammer (Eds.), Demeter-Verlag, Grafelfing, 1979, p. 216.

91.

A. Ishimori, Hephato-Gastroent., 27(51), 175 (1980).

92.

A. Miyoshi -e t a l , Hephato-Gastroent., 27(51), Abstract F1. 14, p. 51 (1980).

93.

G . Sachs -e t a l , In: "Die Behandlung des Ulcus Pepticum m i t Pirenzepin", A.L. Blum and R . Hammer (Eds.), Demeter-Verlag, Grafelfing, 1979, p. 118.

94.

H. Brunner, Scand. J . Gastroent., =(72), (1982).

95.

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96.

D. Do, S. M a r t e l l i and S. D a n i o t t i , Scand. J. Gastroent., E ( 7 2 ) , 2 1 1 (1982).

97.

R. Cheli, A. Giacosa and F. Molinari, Scand. J. Gastroent., 17(72), 2 2 1 (1982).

98.

P.R. Dal Monte, G . Bianchi Porro, M. P e t r i l l o , G . G i u l i a n i - P i c c a r i , N . D'Imperio and S. D a n i o t t i , Scand. J. Gastroent., 17(72), 225 (1982).

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501

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115.

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L

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503

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B.H.

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139.

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140.

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141.

H. Huchzermeyer, In: "Die Behandlung des Ulcus Pepticum rnit Pirenzepin", A . L . Blum and R. Hammer ( E d s . ) , Demeter-Verlag, G r a f e l f i n g , 1979, pp. 136-138.

142.

0. Kuntzen and H. Kaess, I n : "Die Behandlug des Ulcas Pepticum rnit Pirenzepin", A.L. Blum and R . Hammer (Eds.), Demeter-Verlag, G r a f e l f i n g , 1979, pp. 131135.

143.

R. Arnold, L. E b e r t , H. Koop and W. G r e u t z f e l d t , I n : "Die Behandlung des Ulcas Pepticum m i t Pirenzepin", A.L. Blum and R . Hammer ( E d s . ) , Demeter-Verlag, G r a f e l f i n g , 1979, pp. 125-126.

_-

HUMEIDA A. EL-OBEID ETAL.

504

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G. Meller e t a l , I n : "Die Behandlung d e s Ulcas Pepticum m i t P i r e n z e p i n " , A . L . B l u m and R . Hammer ( E d s . ) , Demeter-Verlag, G r a f e l f i n g , 1979, pp. 128-130.

145.

S . J . Kontuner, W. Obtulowicz, N . Kwiecien, M. Dobrazanska, J . Swierczek, B . Kopp and J . Olesky, Scand. J . G a s t o e n t . , 1 5 ( 6 6 ) , 6 3 (1980).

146.

H.N. El-Sabbagh, S.R. Bloom, T . E . Adrian, A. P r i n z , J . H . Baron and R.B. Welbourne, Scand. J . G a s t r o e n t . , 1 5 ( 6 6 ) , 57 (1980).

147.

A. Masala, S . Alagna, L . D e v i l l a e t a l ,-J . Endocrinol . I n v e s t . , 5, 53 (1982).

148.

S. Alagna, A . Masala, A . S a t t a -e t a l , Drug Devel. Res., 3, 385 (1983). - -

149.

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150.

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151.

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152.

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153.

_

L

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Grasso et a l , Curr.

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132, 427 (1982). -

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155.

R. Engelhorn, Scand. J . G a s t r o e n t . , 1 7 ( 7 2 ) , 49 (1982).

156.

B.H. J a u p , R.W. Stockbrugger and G . D o t e v a l l , Scand. J. G a s t r o e n t . , 17(72) , 119 (1982).

157.

K.F. Sewing, Scand. J . G a s t r o e n t . , 1 7 ( 7 2 ) , 265 (1982)

158.

F. B a l d i , F . F e r r a r i n i , M. Cassan et a l , Curr. Ther. Res., 33, 802 (1983). - -

-

PIRENZEPINE DIHYDROCHLORIDE

505

159,

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160.

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161.

R . Hammer, C . P . B e r r i e , N . J . M . B i r d s a l l , A.S.V. Burgen and E.C. Hulme, Nature, - -2 8 3 , 90 (1980).

162.

E.C. Hulme, N . J . M . B i r d s a l l , A.S.V. Burgen and P . Mehta, Molec. Pharmac., 1 4 , 737 (1977).

163.

R. Hammer, Scand. J . G a s t r o e n t . , 1 5 ( 6 6 ) , 5 (1980).

164.

R . Hammer, Scand. J . G a s t r o e n t . , 1 7 ( 7 2 ) , 59 (1982).

165.

J . S t o c k t o n , N . J . M . B i r d s a l l , A.S.V. Burgen and E.C. Hulme, Scand. J . G a s t o e n t . , 17(72), 43 and 268 (1982).

166.

J . S t o c k t o n , N . J . M . B i r d s a l l and R . Hammer, Scand. J. Gastroent., 1 7 ( 7 2 ) , 268 and 270 (1982).

167.

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_ I _ I I _

D.A.

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169.

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170.

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171.

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172.

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Scand. J . G a s t o e n t . , 1 4 ( 5 7 ) ,

, 55, 1119 L

506

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174.

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175.

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176.

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STREPTOMYCIN

J a b e r S . Mossa, Abdul Hameed U . Kader Taragan, and Mahmoud M.A. Hassan,

J a b e r S . Mossa, A s s o c i a t e P r o f e s s o r o f Pharmacognosy, C o l l e g e of Pharmacy, King Saud U n i v e r s i t y , Riyadh, Saudi A r a b i a . Abdul Hameed U . Kader Taragan, L e c t u r e r o f Pharmacognosy, Department o f Pharmacognosy, C o l l e g e o f Pharmacy, King Saud U n i v e r s i t y , Riyadh, S a u d i A r a b i a .

Mahmoud M.A. Hassan, P r o f e s s o r of Medicinal Chemistry, Department of Pharm. Chem., C o l l e g e o f Pharmacy, King Saud U N i v e r s i t y , Riyadh, Saudi Arabia.

ANALYTICAL PROFILES OF DRUG SUBSTANCES

VOLUME 16

507

Copyright 0 1987 by Academic Press, Inc. All rights of reproductionin any form reserved.

JABER S . MOSSA ETAL.

508

1. History 2. Description 2.1 2.2 2.3 2.4 2.5

Nomenclature Formulae Molecular Weight Appearance, Color, Odor and Taste pH Range.

3. Physical Properties 3.1 Melting Range 3.2 Solubility 3.3 Loss on hying 3.4 Optical Rotation 3.5 Crystal Structure 3.6 Spectral Properties

4. Production of Streptomycin 5. Synthesis of Streptomycin

6. Biosynthesis of Streptomycin 7. Pharmacology and Therapeutic Category 8. Pharmacokinetics

8.1 Absorption 8.2 Distribution 8.3 Excretion 8.4 Half -life 9. Toxicity

10. Dosage 11. Pharmaceutical Forms 12. Mechanism of Action 13. Methods of Analysis

STREPTOMYCIN

13.1 13.2 13.3 13.4 13.5 13.6 13.7 13.8 13.9

Elemental Composition Identification Titrimetric Methods Gasometric Electrometric Spectroscopic Methods Chromatographic Methods Bio-Assay Methods. Biochemical Methods.

Acknowledgement References

509

JABER S . MOSSA ETAL.

510

1. HISTORY

Streptomycin is a water soluble organic base biosynthesised by appropriate strain of the actinomycetes,SXkepXornqcU g h i n m . Prior to the development of Penicillin Waksman and his collaborators began to study the production of antibiotic substances in the department of microbiology of New Jersey Agricultural Experiment Station, Rutgers University in 1939 (1). In the succeeding years they examined some thousands of actinomycetes, hundreds of fungi and many bacteria and extracted from their culture media a number of antibacterial substances. Particular attention was devoted to obtaining substances which acted prominently on gram negative bacteria. As a result of this work, the organism producing streptomycin was isolated from heavily manured soil and from a chicken's throat (2,3). The first publication on streptomycin was made by Schatz, Bugie and Waksman (3) in 1944. A complete bibliography of the work on streptomycin from 1944-1952 was recorded by Waksman in "The Literature on Streptomycin" (4). The cultural descriptions and the nutritional requirement of ShepXornqcu g h i n w are also discussed by Waksman ( 5 , 6 ) . The early promising clinical reports of 1944 based on crude Streptomycin prepared in the Merck Research Laboratories showed the importance of the crude drug and started an intensive development program for its large-scale production. The general line of development of the work on streptomycin have closely followed those of the work on penicillin. Rapid progress in the study of streptomycin was greatly helped by the experience gained with penicillin and by the fact that there already existed organizations for large-scale manufacture and exploitation of the latter. 2. DESCRIPTION 2.1 Nomenclature 2 . 1 1 Chemical Names

a) O-2-Deoxy-2-(methylamino)-a-L-glycopyranosyl-

(1 + 2)-0-5-deoxy-3-C-formyl-a-L-lyxofuranosyl-

(1 + 4)-N,N' -bis (aminoiminomethyl)-D-streptamine ( 7 ) .

b) 4-0-(2-0-(2-Deoxy-2-methylamino- a -L-glycopyranosyl)-5-deoxy-3-C-formyl-a-L-lyxofuranosyl)-N

N' -diamidino-D -streptamhe (8).

STREPTOMYCIN

511

c) 2,4-Diguanidino-3,5,6-trihydroxycyclohexyl5-deoxy-2-0-(2-deoxy-2-methylamino-a -L-glucopyranosyl ) -3-formy1-a - L 1yxopentanofuranoside (9). d) N-Methyl-L-glucosamidinostreptosidostreptidine (10). e) 2- (N-methyl-a-L-glucopyranosaminido)- 3-Cforinyl-5-desoxy-L-aldopentofuranose (11) . 2.12 Generic Name Streptomycin A.

(12)

2.13 Trade Names a) b) c) d) e) f) g) h) i) j)

Estreptomicina Streptomycinum Streptomyseen Streptomycini Streptovex Stryzolin Crystamycin Dimycin Oroject NS Streptopen

2.2 Formulae 2.21 Empirical

a) Streptomycin = C21H39N7012 b) Streptomycin sulphate = (C21H39N,012)23H2S04 c) Streptomycin Hydrochloride = C21H39N7012.3HC1. d) Streptomycin calcium chloride = (C21H39N7012. 3HC1) CaC12

JABER S. MOSSA ETAL.

512

2 . 2 2 Structural

MH

NH- C-NH2

0

H3C NH

HO

Dl

Streptomycin i s an o p t i c a l l y a c t i v e , t r i a c i d i c base, C21H39N7012; possessing an aldehydic carbonyl group. The high proportion of oxygen molecule i s c h a r a c t e r i s t i c of a carbohydrate. Streptomycin [ l ] i s made up o f t h r e e components : S t r e p t i d i n e [ Z ] , S t r e p t o s e [ 3 1 , and N-methylL-glucosamine [ 4 ] , linked together by g l y c o s i d i c bonds (16).

STREPTOMYCIN

513

NH

I

Streptomycin

PI

5 14

JABER S. MOSSA ETAL.

2.23 Structural elucidation The difficult task of elucidating the structure of streptomycin was carried out principally by four groups of workers led by K. Folkers, 0. Wintersteiner, H.E. Carter and M.L. Wolfrom. Their work has excellently reviewed f o r several times (11, 17-21). The structural elucidation of streptomycin is based on studies o f its various degradation products. 1) On acid hydrolysis, the weaker glycosidic bond in streptomycin between streptidine and streptose splits to give streptidine [21 and streptobiosamine 151 (16,22-26). 2) Mild methanolysis o f streptomycin [ 11 with methanolic hydrogen chloride gives streptidine [ 21 and methyl streptobiosaminide dimethyl acetal hydrochloride [61. Treating [6] with ethyl mercaptan and hydrogen chloride gives ethyl thiostreptobiosaminide diethyl mercaptal hydrochloride [ 7 1 which is also obtained by the action of ethyl mercaptan and hydrogen chloride on streptomycin. By Raney-nickel and subsequent acid hydrolysis o f [71 , it gives N-methyl-L-glucosamine [4] and bisdeoxy-streptose 181 (27-28). 3) Hydrolysis of streptomycin xith N1 sodium hydroxideofor three minutes at 100 or eighteen hours at 40 yields a weakly acid substance, which has been characterized as maltol [9! (29).

qCH3 OH

0

The degradation reactions of streptomycin is well summarized in Scheme I.

f

t

JABER S . MOSSA ETAL.

516

Streptidine is a meso Form of 1,3Streptidine 121, ~8~18~604, diguanido-2,4,5,6-tetrahydrocyclohexane (24,25, 30, 31). The structure of streptidine [21 was confirmed by synthesis from D-glucosamine (32,33). Streptobioasamine The structure of Nitrogen containing disaccharide, streptobiosamine [51 was determined by a study of hydrolysis products of the methyl ether and the ethyl thio-ether formed by the action of hydrogen chloride in methanol and ethyl mercaptan respectively (27, 29, 34, 35). The hydrolysis of methyl-streptobiosaminide dimethyl acetal gave N-methylglucosamine 141 (27,28). The structure of N-methyl-L-glucosamine has been established by synthesis from L-arabinose (36). Because of its instability, it is very difficult to isolate streptose [ 31 , the second component of streptobiosamine [51. The molecular formula CgH1005 was calculated from the known formulae of streptomycin 111 , Streptidine [ 2 ] and N-methyl-L-glucosamine [41 and its structure was deduced by identifying oxidation and reduction products. It was finally shown to be 3-C-formyl-5-deoxy-L-lyxose, a pentose containing the reactive aldehyde group associated with streptomycin molecule (29,37-40). The structure of streptomycin [I] was finally confirmed by total synthesis (Scheme 11). 2.24 CAS Registry Number 1) Streptomycin 2) Streptomycin sulphate 3) Streptomycin Hydrochloride

(57-92-1) (3810- 74-0) (6160-32-3)

2.25 Wiswesser Line Notation Streptomycin sulphate : T60TJ BlQ CQ -DQ EM1 FO- DT50TJ B CV H CQ EO AL6T J BQ CQ DMYZ UM EQ EMYZU M WSQQ 3.

(41)

STREPTOMYCIN

517

2.26 Stereochemistry and Absolute Configuration The chemistry of the component fragments of streptomycin was thoroughly studied and their structures were established (23-40). The absolute configuration of streptomycin has been elucidated by chemical (applying Reeve's Copper complexing method to various degradation products of streptomycin, dihydrostreptomycin and streptobiosamine) (42,43) and optical correlations (44). The absolute stermch emistry of streptose was established to be (R) at C-4 and (S) at C-2 (37,40,45). The streptidine was shown to possess all mrn configuration (46). Although streptidine is a meso form, the asymmetric attachment of streptobiosamine to it causes each of the ring carbon of streptidine in streptomycin to be asymmetric. The asymmetry of streptidine ring in streptomycin is 1(R), 2(R), 3(S), 4(R), 5(R), 61s). The streptobiosamine moiety of streptomycin has been shown to be attached to position 4 of streptidine in either R or S absolute configuration (46-48). The three units of the streptomycin are attached together by glycosidal bonds which were shown to be formed by condensations between : (i) the C-2 hydroxyl o f streptose and C-1 hydroxyl of N-methyl-L-glucosamine (28,38,39,49-51). (ii) C-1 hydroxyl of streptose and a hydroxl group of streptidine that is adjacent to only one guanidino group (i.e., position 4) (22,38,39,52-54). All glycosidic linkages have an a-S configuration which was confirmed by NMR and crystallographic studies (44, 55-57). By using the configurational assignment, the structure of streptomycin in complete stereochemical details may be written as indicated below :

JABER S. MOSSAETAL.

518

YH2 C=NH I N-H

HOCH2

OH

-I

\,

- 11-

YH2

L-I

II

H NH

HO

2 . 3 Molecular Weight

a) b) c) d)

Streptomycin Streptomycin Streptomycin Streptomycin

=

sulphate = hydrochloride = calcium chloride =

581.6 1457.4 691.0 1492.9 (8)

2 . 4 Appearance, Color, Odor and Taste

Streptomycin occurs as white to slightly pink o r pale brownish powder or granules; odor: odorless o r with a slight c d o r ; taste : slightly bitter. Streptomycin is hygroscopic and may deliquensce on exposure to air but not affected by air or light ( 1 3 ) . 2 . 5 PH Range

per cent w/v solution of streptomycin has a pH between 5 and 7. A 25

519

STREPTOMYCIN

3 . PHYSICAL PROPERTIES

3 . 1 Melting Range I n d e f i n i t e (12). 3 . 2 So 1u b i 1it y 1)

Streptomycin : I t i s very s o l u b l e i n w a t e r ; almost i n s o l u b l e i n a l c o h o l ( 9 5 % ) , chloroform, e t h e r and petroleum e t h e r ( 1 3 ) .

2)

Streptomycin s u l p h a t e : I t i s v e r y s o l u b l e i n water; p r a c t i c a l l y i n s o l u b l e i n a l c o h o l a c e t o n e , chloroform, e t h e r and petroleum e t h e r ( 8 ) .

3)

Streptomycin h y d r o c h l o r i d e : I t i s v e r y s o l u b l e i n water, p r a c t i c a l l y i n s o l u b l e i n a l c o h o l , c h l o r o form and e t h e r ( 8 ) .

4)

Streptomycin calcium c h l o r i d e : I t is v e r y s o l u b l e i n water, p r a c t i c a l l y i n s o l u b l e i n a l c o h o l , c h l o r o form and e t h e r ( 8 ) .

3 . 3 Loss on Drying Strephomycin l o s e s when d r i e d o v e r phosphorus p e n t o x i d e a t 60 C and a t a p r e s s u r e n o t exceeding 5 mm. o f merc u r y , f o r 3 hours, n o t more t h a n 5 p e r c e n t (13). 3.4 O m i c a l R o t a t i o n Streptomycin i s o p t i c a l l y a c t i v e w i t h l e v 0 r o t a t i o n . The f o l l o w i n g o p t i c a l r o t a t i o n s were r e p o r t e d f o r t h e various s a l t s of streptomycin (7,12) : 25

[a1D 1. Streptomycin h y d r o c h l o r i d e . -84: 2 . Streptomycin calcium c h l o r i d e . - 7 6

Solvent Water (C = l . O ) Water (C -1.0)

The s p e c i f i c r o t a t i o n €or s t r e p t o m y c i n s u l p h a t e was determined i n our l a b o r a t o r y on a P e r k i n E l m e r Polar i m e t e r , Model 241 MC and found t o be [a125 - 75' i n D water (C = 1. O ) .

JABER S. MOSSA ETAL.

520

3.5 Crystal Structure Neidle, e t a1 (57) have r e p o r t e d t h e X-ray c r y s t a l l o g r a p h i c a n a l y s i s of s t r e p t o m y c i n oxime s e l e n a t e . S t r e p mycin oxime selenate', C21H40Ng012 [ 1%H2Se04], 4H20 c r y s t a l l i z e s from aqueous methanol as monoclinic n e e d l e s , s a c e groupoC2, a = 17.10, b = 14.36, c = 16.13 B = 108 , D = 1 . 5 4 g . ~ m - ~Dc, = 1 . 5 5 g . cm-3 f o r f o u r formula u n f t s p e r c e l l . The [OOl] p r o j e c t i o n o f one streptomycin oxime i o n i n selenate c r y s t a l i s shown i n F i g . 1.

B,

3.6 Spectral Properties 3 . 6 1 U l t r a v i o l e t spectrum The UV spectrum o f s t r e p t o m y c i n s u l p h a t e i n water was scanned from 200 t o 400 nm on DMS 100 Varian AG Spectrophotometer (Fig. 2 ) . Three maxima were observed a t 241.6, 270 and 328.2 nm. Streptomyc i n i n 0.2N su;phuric a c i d , showed a maxima a t 280 nm w i t h E l 4 = 0.2 ( 9 ) . 1cm 3.62 I n f r a r e d spectrum The i n f r a r e d spectrum of s t r e p t o m y c i n s u l p h a t e a s K B r d i s c recorded on a Pye Unicam i n f r a r e d spectrophotometer i s shown i n F i g . 3. The s t r u c t u r a l assignments have been c o r r e l a t e d with t h e f o l l o w i n g f r e q u e n c i e s (Table 1 ) : Table 1 : I R C h a r a c t e r i s t i c s of Streptomycin Sulphate. Frequency cm

-1

Assignment

3100-3500

NH and OH s t r e t c h (vb).

1630- 1710 1480 1100-1200

-C=O and -C=NH s t r e t c h ( v b ) . (vb) * C-0-C-stretch (vb) *

Y

I

vb = Very broad.

5.

Other r e p o r t e d I R d a t a 41) a r e 3330, 1670, 1470, 1390, 1110 and 1040 cm-

STREPTOMYCIN

F i g . 1 : [OOlI P r o j e c t i o n o f one Streptomycin oxime i o n i n t h e Selenate C r y s t a l .

Fig. 2 : UV Spectrum of Streptomycin Sulphate in Water.

521

80

t 1

2000

1800

1600

I

1400

Fig. 3 : IR S p e c m ~ aof Streptomycin Sdphrte as K k diw.

1

1200

1

1000

800

I

523

STREPTOMYCIN

3 . 6 3 Nuclear Magnetic Resonance Spectra 3.631

1 H-NMR Spectra 1

The H-NMR s p e c t r a of streptomycin i n D 0 ( F i g . 4 ) were recorded on a T60A MHz N M i spectrometer with TMS a s i n t e r n a l reference. The proton chemical s h i f t s a r e shown i n Table 2 . NH

8

I

1

OH

CH

7* 3 Table 2 : PblR C h a r a c t e r i s t i c s o f Stremomycin Sulphate Chemical Group. shift. D20 H-1 N-methylglucosamjne 5.53

H-1

Streptose

3 -ti Formyl s t r e p t o s e

5.30 5.06 3.83 3.56

2 - N Me of N-methyl

glucosamine.

4

- Sec-Me o f S t r e p t o s e .

2.86

1.23

1 1 ,

I

'

.

I . . . .

8.0

,

I .

r.0

I

I

I

I

.

1

I

.

. I . . . , I . . . 1 . . . . 1 . . . , 1 . 6.0 5.0 m (4) 4.0 3.0 Z.0 I

Fig. 4

:

H-NMW Spectrum of Streptomycin Sulphatr iii I)$) and TMS.

I

I

1 . . . . 1 , 1.e

0

STREPTOMYCIN

525

3.632 "C-NMR

Spectra

The n a t u r a l abundance C-13 NMR n o i s e - d e coupled and s i n g l e frequency o f f - r e s o n a n c e decoupled (SFORD) s p e c t r a ( F i g . 5 and F i g . 6 ) i n deuterium o x i d e were o b t a i n e d on a J o e l FX 90 - 90-MHz i n s t r u m e n t . The carbon chemical s h i f t s were a s s i g n e d on t h e b a s i s o f t h e t h e o r y of chemical s h i f t and SFORD s p l i t t i n g p a t t e r n (Table 3 ) . Table 3 : Carbon Chemical S h i f t s o f Streptomycin Sulphate. Carbon No. 1 2 3 4 5 6 7 8 1' 2' 3' 4' 5' 6' 1" 2'7 3" 4" 5" 6" 7''

Chemical S h i f t

6 ppm

57.06 87.13 60.82 108.61 o r 97.11 63.11 61.52 160.33 160.80 108.61 o r 97.11 71.92 84.89 80.67 34.81 186.31 108.61 o r 97.11 64.50 73.33 74.21 80.14 75.97 15.20

Multiplicity d d d d d d S S

d d S

d

4

d d d d d d t

4

Fig. 5 : 13C-NMR Noisc-decoupkd Spectrum of Streptomycin SuIphate in D20 and TMS.

1

hli

Fig. 6 : 13C-NMR Off-Resonance Spectrum of Streptomycin Sulphate in D 2 0 and TMS.

JABER S. MOSSA ETAL.

528

1 6 0 .3 3 (s) 57.06 ( d ) 87.13(d) 160.8 (5)

H2N 60. 82(d)

-

C 0

- HN H 0

63. i 1(d) 108.61 o r 97.11 (d) 9 7 .1 1 (d)

80.14 (d)

0

74.21 (d)

73.33 (d)

64.05 (d)

STREPTOMYCIN

529

4 . PRODUCTION OF STREPTOMYCIN Streptomycin i s produced by t h e micro-organism b;trreptomyca g h h w . I t i s a l s o produced by o t h e r s t r a i n s of organisms l i k e Acfinomyca cj1obhppohu.b ( 5 8 ) , pink v a r i a n t o f bRtreptumqca g h i n e u n (591, bAh.epXumycu g d b u (60). However, f o r l a r g e s c a l e p r o d u c t i o n , a p p r o p r i a t e s t r a i n o f baheptomyca g&mb i s used.

SXtepXomycu g & e u can b i o s y n t h e s i z e s t r e p t o m y c i n i n a medium c o n t a i n i n g g l u c o s e , sodium c i t r a t e and i n o r g a n i c s a l t s , but much h i g h e r y i e l d s are o b t a i n e d by u s i n g t h e media c o n t a i n i n g a l s o an o r g a n i c s o u r c e o f n i t r o g e n such a s meat e x t r a c t , y e a s t a u t o l y s a t e , Soybean f l o u r , c o r n s t e e p l i q u o r o r f e r m e n t a t i o n s o l u b l e s (61-88). One o r more of t h e s e a r e used f o r i n d u s t r i a l b i o s y n t h e s i s . Tremendous amount of work h a s been done on t h e p r o d u c t i o n of s t r e p t o m y c i n (64 , 89-103) . The l a r g e - s c a l e p r o d u c t i o n h a s been accomplished by e i t h e r s u r f a c e c u l t u r e o r deep c u l t u r e p r o c e s s ( 6 4 , 6 5 ) . The deep c u l t u r e p r o c e s s i s more o f t e n used f o r l a r g e - s c a l e p r o d u c t i o n o f s t r e p t o m y c i n . A b r i e f o u t l i n e of t h e p r o d u c t i o n o f s t r e p t o m y c i n i s well summarised below (104): a ) P r o d u c t i o n o f Spore Suspension The s p o r e s o f h%%eptumqcu g h i n ~ .r e~q ~u i r e d f o r i n o c u l a t i o n i s o b t a i n e d from r a p i d l y growing c u l t u r e on s o l i d medium (64, 1 0 5 ) . The s p o r e s are e i t h e r suspended i n s t e r i l e water o r i n skimmed m i l k which i s i n o c u l a t e d w i t h t h e medium i n t h e shake f l a s k s . b) P r e p a r a t i o n o f Medium A l l t h e s o l i d c o n s t i t u e n t s o f t h e c u l t u r e medium a r e p l a c e d i n t h e ferinenter through t h e c h a r g e h o l e and w a t e r i s added t o make up t h e volume. The medium i s s t e r i l i z e d by t h e d i r e c t i n t r o d u c t i o n of steam. S t e r i l i z a t i o n i s c a r r i e d o u t a t 1 5 l b p r e s s u r e f o r 2 5 minutes. A f t e r s h u t t i n g o f f t h e steam, water i s passed through t h e j a c k e t and an a i r p r e s s u r e o f 10 l b i s maintained i n t h e f e r m e n t e r . The s i t r r e r is working d u r i n g s t e r i l i z a t i o n , c o o l i n g and ferment a t i o n .

c ) P r e p a r a t i o n o f t h e inoculum The s t e r i l i z e d medium i n t h e s e e d t a n k (115 L . c a p a c i t y , provided with s t i r r e r and inoculum measuring t a n k ) i s

530

JABER S . MOSSA ETAL.

Flow Sheet 1 : The production of Streptomycin.

STREPTOMYCIN

53 1

i n o c u l a t e d w i t h t h e c o n t e n t o f shake f l a s k . After t h e i n o c u l a t i o n , t h e niycelium i s grown f o r 36 hours w i t h an a e r a t i o n r a t e of 100 L p e r minute. After 36 h o u r s , a heavy growth of mycelium i s o b t a i n e d . About twenty l i t e r s o f t h i s mycelium suspension i s blown i n t o t h e measuring t a n k and f i n a l l y t o t h e f e r m e n t e r s through p i p e - l i n e . d) Fermentation The f e r m e n t e r s a r e made up o f s t a i n l e s s s t e e l , having t h e c a p a c i t y of 15000 g a l l o n s and provided w i t h s i t r r e r , a n t i foam n o z z l e , a i r s p a r g e r and a i r o u t l e t . After t h e inoculum from t h e s e e d tank h a s been added t o t h e ferment e r , a e r a t i o n i s s t a r t e d a t once. Then t h e f e r m e n t a t i o n 0 0 i s c a r r i e d o u t a t an optimum t e m p e r a t u r e of 25 -30 C . Foaming d u r i n g f e r m e n t a t i o n i s c o n t r o l l e d by t h e a d d i t i o n o f a n t i f o a m i n g a g e n t s . The f i n a l c o n c e n t r a t i o n o f s t r e p tomycin (50,000 u n i t s p e r l i t r e ) w i l l be reached i n 72 h o u r s . The p r o d u c t i o n of s t r e p t o m y c i n is given i n flow sheet I .

e) I s o l a t i o n and P u r i f i c a t i o n The s t r e p t o m y c i n from t h e c u l t u r e f l u i d i s s e p a r a t e d by a d s o r p t i o n on t h e c h a r c o a l and by subsequent e l u t i o n w i t h methanolic hydrochloric acid o r other s u i t a b l e solvents (106-117). F u r t h e r p u r i f i c a t i o n i s c a r r i e d o u t by chromatography on alumina (118-122) o r i o n exchange resins (123-164) and by p r e c i p i t a t i o n w i t h v a r i o u s b a s e p r e c i p i t a n t s (165-179). S o l v e n t e x t r a c t i o n techniques (180-188), e l e c t r o d i a l y s i s (189) and e l e c t r o o s m o t i c (190) procedures a r e a l s o employed. 5 . SYNTHESIS OF STREPTOMYCIN

Although s t r e p t o m y c i n was t h e f i r s t aminoglycoside a n t i b i o t i c t o be d i s c o v e r e d , i t was n o t s y n t h e s i s e d u n t i l some 30 y e a r s l a t e r . The t o t a l s y n t h e s i s of s t r e p t o m y c i n was achieved by Sumio Umezawa e t a l , i n 1973 (191,192). The r e a c t i o n s involved a r e i n shown i n Scheme I 1 €j summarized below: Condensation o f blocked d e r i v a t i v e o f d i h y d r o - s t r e p t o s e (benzyl a-L-dihydro-streptose)[lO] with t h e glucosamine d e r i v a t i v e (L-glucopyranosyl b r o m i d e ) [ l l ] i n benzene i n t h e p r e s e n c e of m e r c u r i c c y a n i d e , followed by hydrol y s i s o f t h e S c h i f f b a s e w i t h 50% a c e t i c a c i d and

JABER S . MOSSA ETAL.

532

SCHEME I1

STREPTOMYCIN

533

1111

phcg-J

PhCOO

0

0 1191

PhCOO

JABER S. MOSSA ETAL.

534

phcol&j

PhCOO

0 NH

I1

NHCNH2

1221

tlH

NHCNHz

I

OH

STREPTOMYCIN

535

reaction with methyl chloroformate (Na2Cog, aq. acetone) gave the protected streptobiosaminide[ 121. NMethvlation of [121 gave [131 Deacetylation followed by deisopropylidenation (IN HC1, Aqueous MeOH) gave [ 141 which was identical with benzyl a-S-dihydrostreptobioasminide. Hydrolysis of 1141 with 10% Ba(OH)2 affored benzyl a-L-dihydro-Streptobiosaminide [ 151 . Treatment of [ 151 with 2,2-dimethoxypropane (p-toluenesulfonic acid) gave a diisopropylidene derivative and further blocking by use of p - n i t r o p h en o x y c a r b o n y lc h lo r id e (NaOH, aqueous acetone) gave [ 161. Partial hydrolysis of [161 (25% AcOH in MeOH) gave the diol 1171, This was converted into the dibenzoyl derivative [18]. Partial hydrolysis of [181(75% Acetic acid) removed the isopropylidene group giving a diol which was transformed (p-N02C6H ococ 1, pyridine) into the carbonate [19]: Hydrogenofysis of the glycoside linkages (Palladium Black-dioxane) gave free sugar 1201. The reaction of [201 with thionyl chloride at room temperature offered the a-glycosyl chloride [211. Direct treatment of [ 151 with p-nitrophenoxycarbonylchloride o r phosgene followed by benzolyation also gave [191. But the yield is very poor. Finally condensation of [211 with di-N-acety1-di-N-

b e n z y l o x y c a r b o n y l - 0 - c y c l o h e x y l i d e n e s t r e p t i d i n e gave the 4-0-glycoside [22]. Hydrolysis of 1221 with 0.05 N

Ba(OH2) and dioxane successively removed the carbamate, carbonate, benzoyl and acetyl groups. Removal of the remaining protecting groups with 50% acetic acid followed by hydrogenolysis with Palladium black gave dihydrostreptomycin [23]. Subsequent mild oxidation of [23] afforded streptomycin[l] . 6. BIOSYNTHESIS OF STREPTOMYCIN Biosynthesis of streptomycin has been quite extensively studied. Streptomycin is formed from glucose as a primary precursor (193). Biosynthesis of the components of streptomycin, streptidine, streptose and N-methyl-glucosamine are well summarised by Horner (193) E Snell (194). General indication of the metabolic relationships of glucose L241 to the various components of streptomycin is shown in Scheme 111.

JABER S. MOSSA ETAL.

536

SCHEME I11

Tranunnular

O
Icarranttemcnt.

on

HO Mvoinotitol

-

H,yo3n on

OH

STREPTOMYCIN

537

Experiments w i t h l a b e l e d compounds demonstrated t h a t t h e s p e c i f i c a l l y labeled glucose [24] i s converted i n t o s t r e p tomycin [ l ] i n which each of t h e s u b - u n i t s i s l a b e l e d a t t h e c o r r e s p o n d i n g carbon (Scheme IV). T h i s was e s t a b l i s h e d by s e v e r a l groups (195-203). Some o f t h e i n t e r m e d i a t e s i n t h e b i o s y n t h e t i c pathway of s t r e p t o m y c i n have been i s o l a t e d , b u t some o f t h e key i n t e r m e d i a t e s a r e n o t y e t d e t e c t e d . Recent s t u d i e s e s t a b l i s h e d t h e p r e s e n c e o f s t r e p t i d i n e - 6 phosphate and dihydro-streptose-dTDP, i n t h e s t r e p t o m y c i n b i o s y n t h e t i c pathway (204). Some i n t e r m e d i a t e s between g l u c o s e and d i h y d r o s t r e p t o s e a r e known as a r e s u l t o f t h e work o f Grisebach (205) who demonstrated t h a t [14C-] glucose-dTDP was c o n v e r t e d t o 4-Keto4,6-dideoxyglucose-dTDP which i n t u r n was c o n v e r t e d t o dihydrostreptose-dTDP. Nothing i s known y e t r e g a r d i n g t h e i n t e r m e d i a t e between g l u c o s e and N-methylglucosamine. Recently Kniep and Grisebach (206) e s t a b l i s h e d t h e r o l e o f d i h y d r o s t r e p t o s y l s t r e p t i d i n e 6-phosphate [ 2 5 ] i n t h e b i o s y n t h e s i s o f s t r e p tomycin. They concluded t h a t t h e nucleotide-bound N-methyl4-glucosamine moiety i n S. g h i n w was c a t a l i z e d and t r a n s f e r r e d t o d i h y d r o s t r e p t o s y l s t r e p t i d i n e 6-phosphate [ 2 5 ] which i n t u r n s was c o n v e r t e d t o dihydrostreptomycin 6 phosphate [ 2 6 ] . F u r t h e r o x i d a t i o n of [ 2 6 ] gave s t r e p t o mycin-6-phosphate[27] (207) hid h y d r o l y s i s y i e l d s t r e p t o mycin [ l ] (208). The r e a c t i o n i s summarized i n Scheme V . 7 . PHARMACOLOGY AND THERAPEUTIC CATEGORY

Streptomycin i s an aminoglycoside a n t i b i o t i c which i s a c t i v e a g a i n s t a wide range of b a c t e r i a , both Gram p o s i t i v e and Gram n e g a t i v e (209). I t i s a v a l u a b l e t h e r a p e u t i c a g e n t f o r a l l forms of t u b e r c u l o s i s (210-252). S t r e p t o mycin i s e f f e c t i v e a g a i n s t Tularemia (253,254). I t i s h i g h l y s p e c i f i c and one o f t h e most e f f e c t i v e a g e n t f o r t h e t r e a t m e n t o f a l l forms o f p l a u g e (255). I n combinat i o n w i t h P e n i c i l l i n G , it i s c o n s i d e r e d t o be t h e d r u g o f c h o i c e i n i n f e c t i o n s w i t h SfiepXococcU v h i d a v m and EvLtmococcu ( 2 5 6 ) . I t i s a l s o e f f e c t i v e a g a i n s t h&phyLococcud uLbw (257). Streptomycin i s a d r u g o f second c h o i c e i n i n f e c t i o n s due t o s u s c e p t i b l e s t r a i n s o f a n a e r o b i c bfiCLp,&jCOC&, b a c t e r o i d e s , AmobacXm a e h o g e n a , A . d e c a l h , S h i g e l L a , BhuceRea and Vibh,Lo carnnin and i n combination w i t h t e t r a c y c l i n e , i n P o t l ; t e ~ i n f e c t i o n s and i n b r u c e l l o s i s (256,258-264). Since s t r e p t o m y c i n i s e f f e c t i v e a g a i n s t H. i n d h e n z a e , E. CULL, K&bbLelLa pneurnanhe and S&andl?a, i n combination w i t h

NH

SCHEME I V

II

NH

NHCNH

II

STREPTIDINE

i-koH

HO

-.

0

OH

~241

#q* I

*

0

H3C

OH

STREPTOSE

HQ N-METHYL-

CH20H

#

CH3NH

OH

*

[11

L-GLUCOSAMINE

STREPTOBIOSAMINE

STREPTOMYCIN

539

SCHEME V

nvo

1251

H$NM

OM

OH

Ill

1261

JABER S.MOSSA ETAL.

540

o t h e r a n t i b i o t i c s it i s useful i n t h e treatment of r e s p i r a t o r y t r a c t i n f e c t i o n s , b a c t e r i a l m e n i n g i t i s and u r i n a r y t r a c t i n f e c t i o n s (256,265-273). Streptomycin i s a l s o e f f e c t i v e f o r typhoid f e v e r (274-277) , whooping cough (278) , d i p h t h e r i a (279) and i n murine l e p r o s y (280-283). 8 . PHARMACOKINETICS 8 . 1 Absorption Streptomycin is absorbed p o o r l y and i r r e g u l a r l y from t h e g a s t r o i n t e s t i n a l t r a c t (255,284). T h e r e f o r e , t h e o r a l route i s not s u i t a b l e f o r administration of s t r e p tomycin f o r s y s t e m i c a c t i o n . However, t h e o r a l r o u t e h a s been recommended i n t h e t r e a t m e n t o f i n t e s t i n a l d i s o r d e r s due t o s t r e p t o m y c i n s e n s i t i v e s t r a i n s o f b a c t e r i a o r t o reduce t h e b a c t e r i a l f l o r a of t h e i n t e s t i n e p r i o r t o o p e r a t i o n on i n t e s t i n a l t r a c t . Streptomycin i s r a p i d l y absorbed when a d m i n i s t e r e d by subcutaneous, i n t r a m u s c u l a r o r i n t r a v e n o u s r o u t e s (255,256,285). Streptomycin can a l s o be absorbed through rectum when a d m i n i s t e r e d as cocoa b u t t e r o r carbowax m i x t u r e (286,287). P a r e n t r a l a d m i n i s t r a t i o n must be employed t o e n s u r e t h e r a p e u t i c a l l y a d e q u a t e plasma and t i s s u e l e v e l s of s t r e p t o m y c i n . After an i n t r a m u s c u l a r dose of 1 g o f streptomycinipeak serum c o n c e n t r a t i o n s o f 25 t o 50 mg/ml a r e a t t a i n e d i n 0.52 h o u r s , f a l l i n g t o 5 t o 10 mg/ml a t 12 hours (9, 1 5 ) . The e f f e c t i v e minimum serum c o n c e n t r a t i o n i s about 7 mg/ml ( 1 5 ) . 8.2 Distribution

No m e t a b o l i t e of streptomycin has so f a r been d e t e c t e d . About one t h i r d t h e c i r c u l a t i n g st-reptomycin i s bound t o serum p r o t e i n . Streptomycin i s widely d i s t r i b u t e d t o a l l organs e x c e p t b r a i n . The drug p e n e t r a t e s r a p i d l y i n t o t h e p e r i t o n e a l , p e r i c a r d i a l , p l e u r a l and s y n o v i a l f l u i d s . Very l i t t l e enters i n t o cerebrospinal f l u i d except i n meningitis (256,285,288,289) , 8.3 E x c r e t i o n About 50-80% o f s t r e p t o m y c i n i n j e c t e d i s e x c r e t e d i n u r i n e unchanged i n 24 hours (15,285,290,291). Most of t h e o r a l dose i s e l i m i n a t e d i n t h e f a e c e s (15,256). About 0.5% is e x c r e t e d i n t h e b r e a s t m i l k i n 24 hours and 1%i s e x c r e t e d i n b i l e (15,286).

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8.4 Half-life Serum h a l f - l i f e o f s t r e p t o m y c i n i s about 2 . 5 hours i n young a d u l t s . T h i s w i l l be i n c r e a s e d i n t h e new born and i n a d u l t s above 40 y e a r s . In r e n a l f u n c t i o n impairment, t h e h a l f - l i f e may be i n c r e a s e d t o 2-5 days (15)

*

9 . TOXICITY

A l l e r g i c r e a c t i o n s a r e r e l a t i v e l y v e r y common w i t h t h e a d m i n i s t r a t i o n o f s t r e p t o m y c i n . The u s u a l a l l e r g i c symptoms a r e e o s i n o p h i l i a , f e v e r and s k i n r a s h e s . The d r u g a l s o c a u s e s headache, nausea, a r t h r a l g i a , b l u r r i n g o f v i s i o n , v e r t i g o , r e n a l i r r i t a t i o n and n e u r o t o x i c i t y . The most s e r i o u s t o x i c e f f e c t i s damage t o t h e e i g h t h c r a n i a l n e r v e , c a u s i n g d e a f n e s s which i s permanent. Damage t o r e n a l t u b u l e s , bone marrow and l i v e r r a r e l y o c c u r s a t t h e usual t h e r a p e u t i c t i s s u e l e v e l . Application of s t r e p t o mycin d i r e c t l y t o t h e peritoneum o r i t s i n t r a v e n o u s i n j e c t i o n i n l a r g e doses may cause d e a t h by p a r a l y s i s o f t h e r e s p i r a t i o n (285,286,288, 292-303). 1 0 . DOSAGE

About 0 . 5 t o 1 gram by i n t r a m u s c u l a r i n j e c t i o n d a i l y o r a t long i n t e r v a l s . A s an i n t e r n a l a n t i s e p t i c , 500 m i l l i g r a m s by mouth every e i g h t hours (304). 11. PHARMACEUTICAL FORMS 1) 2) 3) 4)

Streptomycin Streptomycin Streptomycin Streptomycin

s u l p h a t e U.S.P. sulphate i n j e c t i o n B.P. calcium c h l o r i d e B.P. hydrochloride B.P.

(305). (304). (8) * (8)

-

1 2 . MECHANISM OF ACTION

The a n t i b a c t e r i a l a c t i o n of s t r e p t o m y c i n i s a consequence o f i t s combination w i t h t h e 30s ribosomes o f s u s c e p t i b l e organisms, t h e r e b y d i s t o r t i n g t h e t r a n s l a t i o n of t h e g e n e t i c code. As a r e s u l t o f t h i s c o n d i t i o n t h e wrong amino a c i d s a r e i n c o r p o r a t e d i n t o t h e n a s c e n t p o r t i o n o f t h e p r o t e i n molecule, t h e p r o t e i n s y n t h e s i s i s d i s r u p t e d and t h e c e l l d i e s (256, 306, 307).

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13. METHODS OF ANALYSIS 1 3 . 1 Elemental Composition

c = 43.7% H = 6.76%

N = 16.86% 0 = 33.01%

(7).

13.2 I d e n t i f i c a t i o n 13.21 Pharmacopeial T e s t s The f o l l o w i n g t e s t s have been d e s c r i b e d i n B r i t i s h Pharmacopoeia f o r t h e i d e n t i f i c a t i o n of s t r e p t o m y c i n (304). a ) D i s s o l v e about 0 . 2 g o f s t r e p t o m y c i n i n a m i x t u r e o f 2 m l o f methyl a l c o h o l and 0 . 1 m l o f s u l p h u r i c a c i d , f i l t e r i f n e c e s s a r y and allow t o s t a n d a t about 250; c r y s t a l s o f s t r e p t i d i n e sulphate separate i n t h e course of 2 o r 3 d a y s . D i s s o l v e t h e c r y s t a l s i n 10 ml of h o t water c o n t a i n i n g 0.1 g o f t r i n i t r o p h e n o l and c o o l . The m e l t i n g p o i n t o f t h e p r e c i p i t a t e a f t e r r e c r y s t a l l i s a t i o n from hot water i s about 283%. b) Boil a small q u a n t i t y of s t r e p t o m y c i n w i t h 1 N sodium hydroxide f o r a few minutes and add a s l i g h t excess o f h y d r o c h l o r i c a c i d and a few d r o p s o f f e r r i c c h l o r i d e t e s t s o l u t i o n ; a b r i l l i a n t v i o l e t c o l o r i s produced.

13.22 Color T e s t s The f o l l o w i n g c o l o r t e s t s have been d e s c r i b e d f o r t h e i d e n t i f i c a t i o n of streptomycin : a ) Glucosamine Test i ) A 10 mg of s t r e p t o m y c i n i s d i s s o l v e d i n 1 m l o f water, 2 m l o f 1 N h y d r o c h l o r i c a c i d is added and t h e s o l u t i o n is h e a t e d on a steam b a t h f o r 10 minutes. Then 2 m l of 2 N sodium hydroxide and 1 m l o f 20% aqueous a c e t y l a c e t o n e a r e added. The s o l u t i o n i s a g a i n h e a t e d f o r 5 minutes and cooled. About 1 m l o f e t h a n o l and 25 m l o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d

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a r e t h e n added. produced (308).

A cherry red color i s

i i ) To about 2 m l o f an aqueous s o l u t i o n o f s t r e p t o m y c i n , 1 m l of a 2% s o l u t i o n o f a c e t y l a c e t o n e i n w a t e r and 1 m l o f 1 N sodium hydroxide a r e added. The m i x t u r e i s h e a t e d f o r 10 minutes i n a b o i l i n g water b a t h and t h e n cooled. A pink c o l o r i s developed on a d d i t i o n o f 2 m l o f a s o l u t i o n o f E h r l i c h ' s aldehyde (309). b) Guanido Test i ) A 1 0 mg o f s t r e p t o m y c i n i s d i s s o l v e d i n 1 m l o f water and 1 m l of o x i d i z e d n i t r o p n r s s i d e r e a g e n t i s added ( t h e r e a g e n t i s p r e p a r e d by mixing equal volumes of 10% sodium n i t r o p r u s s i d e , 10% potassium f e r r i c y a n i d e and 10% sodium hydroxide, i n t h a t o r d e r ; a f t e r t h e c o l o r becomes b r i g h t green-yellow, 1 m l i s d i l u t e d t o 100 m l w i t h w a t e r ) , a r e d c o l o r devel o p s (310). i i ) Aqueous s o l u t i o n o f s t r e p t o m y c i n when t r e a t e d with d i a c e t y l i n t h e presence o f oxygen g i v e s a pink c o l o r which i s i n t e n s i f i e d by t h e a d d i t i o n o f l-napht h o l (311). i i i ) When s t r e p t o m y c i n i s t r e a t e d with l-napht h o l and sodium hypobromite, a r e d c o l o r i s produced (312,313). i v ) When s t r e p t o m y c i n i s t r e a t e d w i t h sodium hypobromite i n s t r o n g a l k a l i n e s o l u t i o n , a v o l a t i l e bromo amine i s produced which turns acidified starch iodide solutions t o dark b l u e c o l o r (314). c ) Other c o l o r T e s t s i ) An a l c o h o l i c s o l u t i o n o f s t r e p t o m y c i n gives a pink color with sulphuric acid (315).

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ii) An alcoholic solution of streptomycin produces a yellowish-red color with phosphoric acid (315). iii) With Sakaguchi solution (100 mg o f boric acid in 10 ml of concentrated sulphuric acid) streptomycin gives pale-green after 3-5 minutes (315). 13.23 Microbiological Tests a) Test with Streptococcus lactis Streptomycin when treated with milk inoculated with SRhepAococcud ,f!ucLh, prevents the coagulation of the milk in the usual way (316) . b) Triphenyltetrazolium Chloride (TTC) Test When streptomycin is treated with a culture of SR/LepAocaccw t h m o p k i e e u d on a sterile milk medium containing triphenyltetrazolium chloride, prevents the usual color change from leucoform to red (317,318). c) Water-blue Test

A dilute solution of streptomycin prevents

the formation of the blue color when treated with a culture of KLeebtA.eRea pfiewnoniae containing water blue dye (319). 13.3 Titrimetric Methods 13.31 Aqueous Delaby and Stephan (320) have reported an iodimetric method for the determination o f streptomycin. The method is as follows : Dissolve about 0.7 to 1 g of Streptomycin in 15 ml of water, add 15 ml of Nessler's reagent and 1 ml of 10% potassium iodide. Then add 20 ml of water, neutralize with 2N hydrochloric acid and add 1 ml in excess. Add 15 ml of 0.1N iodine solution. Shake and titrate the excess of iodine with 0.05N sodium thiosulphate solution.

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An i o d o m e t r i c method i n v o l v i n g t h e r e a c t i o n o f s t r e p t o m y c i n w i t h sodium hyphobromite has been a l s o r e p o r t e d (321). The method i s a s follows : The sample c o n t a i n i n g s t r e p t o m y c i n s u l p h a t e i s added t o 5-10 m l b a s i c sodium hyphobrom i t e s o l u t i o n and a i r i s swept s u c c e s s i v e l y through t h e sample and 2 U-tubes each cont a i n i n g 5 m l o f 0.1N s u l p h u r i c a c i d and 0.1-0.2 g potassium i o d i d e . A f t e r sweeping a i r (10-15 b u b b l e s / s e c ) through t h e system €or about 15 m i n u t e s , t h e l i b e r a t e d i o d i n e i s t i t r a t e d w i t h 0.005N sodium t h i o s u l p h a t e . A n a l y s i s o f 4-8 mg p o r t i o n s o f 90.2% p u r e s t r e p t o m y c i n s u l p h a t e showed v a l u e s o f 89.2, 88.7 and 8 8 . 4 % . Huttenrauch and Keiner (322) have d e s c r i b e d a t i t r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c i n s u l p h a t e . The method i s a s follows : The sample c o n t a i n i n g about 50 t o 150 mg o f streptomycin sulphate is dissolved i n 25 m l o f w a t e r ; 2 m l o f 1 N ,sodium hydroxide i s added and t h e m i x t u r e i s h e a t e d a t looo f o r 5 minutes. A f t e r c o o l i n g , it i s a c i d i f i e d with 1 N sulphuric a c i d , then d i l u t e d t o 100 m l w i t h water and about 0 . 1 m l o f 2% f e r r i c c h l o r i d e s o l u t i o n is added. The s o l u t i o n i s set a s i d e f o r 7 minutes and t h e n t i t r a t e d w i t h 0.01N c e r i c s u l p h a t e u n t i l t h e c h a r a c t e r i s t i c red colour i s discharged. T h i s method can a l s o b e employed f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n i n c u l t u r e media (323). M o d i f i c a t i o n of Huttenrauch and Keiner method by r e p l a c i n g c e r i c s u l p h a t e s o l u t i o n w i t h p o t assium permanganate s o l u t i o n , was r e p o r t e d by Chandra e t a 1 (324). T h i s modified method g i v e s r e s u l t s w i t h s h a r p e r end p o i n t . 13 32 Non-aaueous Penau e t a1 (325) have d e s c r i b e d a nonaquous method of d e t e r m i n a t i o n o f a n t i b i o t i c s i n c l u d i n g

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s t r e p t o m y c i n . The s p e c i f i e d amount o f S t r e p tomycin s u l p h a t e is d i s s o l v e d i n p e r c h l o r i c a c i d and 2 m l of e t h y l e n e g l y c o l . Then about 0.05M b e n z i d i n e s o l u t i o n i n a c e t i c a c i d i s added. After 10 minutes, 2 d r o p s of i n d i c a t o r (1% thymolphthalein i n e t h a n o l o r 0 . 5 % t h i a z o l e yellow i n e t h a n o l ) is added and t h e e x c e s s o f p e r c h l o r i c a c i d i s t i t r a t e d with a c i d potassium p h t h a l a t e s o l u t i o n . l m l o f 0.1N H C 1 0 4 a c i d i s e q u i v a l e n t t o 581.6/30 mg o f s t r e p t o m y c i n base. G a u t i e r and P e l l e r i n (326) have r e p o r t e d a nonaqueous method o f e s t i m a t i o n o f s u l p h a t e o f t h e o r g a n i c b a s e s l i k e s t r e p t o m y c i n , dihydros t r e p t o m y c i n , and o t h e r s by d i r e c t t i t r a t i o n o f p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d . The procedure i s a s f o l l o w s : To a 0.1N s o l u t i o n of t h e sample i n g l a c i a l a c e t i c a c i d , add s u f f i c i e n t b e n z i d i n e ( a s 0.05M solution i n g l a c i a l acetic acid) t o p r e c i p i t a t e 95 % of t h e s u l p h a t e ; a l l o w t h e p r e c i p i t a t e t o s e t t l e and t i t r a t e t h e s u p e r n a t a n t l i q u i d with 0.1 N p e r c h l o r i c acid i n g l a c i a l a c e t i c a c i d . 13.4 Gasometric Gasometric d e t e r m i n a t i o n o f s t r e p t o m y c i n was r e p o r t e d by Yamagishi and Yokoo ( 3 2 7 ) and V i a l a (328). Pure s t r e p t o m y c i n i n s o l u t i o n o r i n complex p r e p a r a t i o n s o r i n a s s o c i a t i o n w i t h p e n i c i l l i n s , are determined by measuring t h e volume of n i t r o g e n evolved by s t r e p t i d i n e f r a c t i o n c.f tlie molecule under t h e a c t i o n o f sodium h y p o c h l o r i t e o r sodium hypobromite and compari n g it with t h a t f r e e d by r e f e r e n c e s o l u t i o n s o f guanidine hydrochloride o r guanidine a c e t a t e . 13.5 Electrometric 13.51 P o l a r o g r a p h i c Streptomycin i s r e d u c i b l e a t t h e dropping mercury electrode. A polarographic a n a l y s i s has been developed ( 3 2 9 ) . The c o n c e n t r a t i o n d i f f u s i o n current curve i s l i n e a r f o r concent r a t i o n s from 0 . 1 t o 1 . 0 mg/ml. The s o l u t i o n o f s t r e p t o m y c i n i n 3% t e t r a m e t h y l ammonium

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hydroxide is polarographed at 13.6O; the Eh is 1.45 volts vs a mercury electrode. The rate of decomposition o f streptomycin in alkaline solution was polarographically studied by Bricker and Vail ( 3 3 0 ) . Goodey et a1 (331) has described a polarographic method for the assay of streptomycin in fermenter broth and associated recovery stages. A Tinsley Mark 19 pen recording polarograph is used, with mercury capillaries of drop times between 2 and 3 seconds at 50 cm height. Lithium hydroxide is used as base electrolyte. Doan and Riedel (332) have reported a polarographic method for the estimation of streptomycin sulphate.Solutions containing 100-400 y /ml were used for the determination. The pH of the solution was adjusted to 1 2 . 4 by adding 2 ml of 1 N sodium hydroxide. The dissolved oxygen was removed by bubbling nitrogen through the sample f o r 5 minutes after that the surface was flushed with nitrogen. A mercury drop rate of 4 - 5 seconds was maintained. For all concentrations the half wave potential (E%) was 1 . 2 8 1 . 2 9 volts. Plotting the diffusion current against concentration resulted in a straight 1ine. Cunha (333) has published a similar polarographic method f o r determination of streptomycin sulphate. The limit o f identification of streptomycin sulphate was 0 . 5 8 - 7 2 . 8 7 y/ml. Caplis et a1 (334) have reported a method f o r determination o f antibiotics including streptomycin by alternating-current polarography. A survey of the applications of polarographic methods in the production control of antibiotics including streptomycin was described by Weissbuch and Unterman ( 3 3 5 , 3 3 6 ) .

1 3 . 5 2 Amperometric

Streptomycin forms water insoluble salt with various anionic dyes. This can be used €or amperometric estimation of streptomycin ( 3 3 7 ) .

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The method is based on the precipitation of streptomycin with a trivalent dye which is synthesised by coupling diazotized para rosaniline with 1-naphthol-4-sulphonic acid. About 1-3 mg of streptomycin is dissolved in 5 to 10 ml of 0.02M triethylamine citrate buffer, pH 2.8 and is titrated with standard solution of the dye. The potential is maintained at -0.08 volts. Konopik (338) has described the application of metal salt solutions to amperometric determination of organic compounds including streptomycin. 13.53 Potentiometric Machek (339) has carried out a potentiometric titration for the determination of streptomycin in mixed preparations. The method is as follows : Mix the sample with 15 ml o f water and 20 ml of butyl acetate and adjust to pH 2.1 by addition of 2N hydrochloric acid and shake for five minutes. Extract a 1 ml aliquot of the filtered upper layer with 10 ml of phosphate buffer solution (pH 7.2) and determine the penicillin in the extract iodimetrically or microbiologically. Mix the filtered lower layer with 20 ml of butyl acetate, adjust to pH 9 . 5 by addition of 2N sodium hydroxide, add Barium chloride (2ml) and shake for 1 minute; centrifuge, separate and dilute the aqueous phase to 50 ml, adjust the pH of a 20 ml aliquot to 5.8 by addition of 2N hydrochloric acid and titrate the streptomycin potentiometrically with 0.1N NaOH to pH 9.6.

A potentiometric microbiological assay for the

antibiotic substances including streptomycin was proposed (340). The method for the determination of tetracycline hydrochloride with suspension of E b C h d C k i d cofi and a potentiometric C02 sensor (HNC Model 10-22-00 or an Orion Model 95-02) has been extended to the determination of streptomycin. Sample solutions containing 2-4 pg ml-1 of streptomycin

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was used.

The c a l i b r a t i o n graph was r e c t i l i n e a r w i t h range 0.33 t o 16.67 pg m l - 1 .

13.6 S p e c t r o s c o p i c Methods 1 3 . 6 1 C o l o r i m e t r i c Methods 13.611 The m a l t o l Method When s t r e p t o m y c i n is h e a t e d i n d i l u t e a l k a l i n e s o l u t i o n i t forms m a l t o l (29). Maltol r e a c t s w i t h f e r r i c i o n s t o g i v e a p u r p l e c o l o r (304) and w i t h t h e F o l i n - C i o c a l t e u phenol r e a g e n t i t produces a b l u e c o l o r (341). This f a c t may be used f o r t h e a s s a y o f streptomycin. A colorimetric assay involving t h e maltol

formation, f o r t h e estimation of s t r e p tomycin i s d e s c r i b e d i n t h e European Pharmacopoeia (342). The procedure i s as f o l l o w s :

Dissolve 0 . l g o f s t r e p t o m y c i n i n water and d i l u t e t o 100.0 m l with t h e same s o l v e n t . To 5 . 0 m l o f t h i s s o l u t i o n add 5 . 0 m l o f 0.2N sodium hydroxide and h e a t f o r e x a c t l y 10 minutes i n a water b a t h . Cool i n i c e f o r e x a c t l y 5 minutes, add 3 m l of a 1 . 5 p e r c e n t w/v s o l u t i o n o f f e r r i c ammonium s u l p h a t e i n 0.5N s u l p h u r i c a c i d and s u f f i c i e n t water t o produce 25 m l and mix. E x a c t l y 20 minutes a f t e r t h e a d d i t i o n of t h e f e r r i c ammonium s u l p h a t e , measure t h e e x t i n c t i o n o f a 2 cm l a y e r a t about 525 nm, u s i n g a s t h e compensation l i q u i d a s o l u t i o n p r e p a r e d i n t h e same manner, o m i t t i n g t h e s u b s t a n c e t o be examined. The e x t i n c t i o n i s n o t less t h a n 90.0 p e r c e n t o f t h a t o b t a i n e d by c a r r y i n g o u t t h e operations simultaneously, using s t r e p t o m y c i n s u l p h a t e CRS i n s t e a d o f t h e s u b s t a n c e t o be examined. C a l c u l a t e t h e percentage with reference t o t h e subst a n c e t o be examined and t h e streptomyc i n s u l p h a t e CRS, b o t h d r i e d f o r 4 hours

550

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a t 60' o v er phosphorus p e n to x id e a t a p r e s s u r e n o t exceeding 5 T o r r .

Pharmacopoeia of United S t a t e s , 1955 (343) d e s c r i b e d a similar m a l t o l c o l o r i metric a s s a y f o r d e t e r m i n a t i o n o f s t r e p tomycin i n s t r e p t o d u o c i n €or I n j e c t i o n , U.S.P. Boxer e t a1 (344) have r e p o r t e d a s i m p l e and r a p i d c o l o r i m e t r i c a s s a y f o r streptomycin i n c l i n i c a l p r e p a r a t i o n s , u r i n e and b r o t h . The method i s based on t h e formation o f m a l t o l from s t r e p t o mycin, s e p a r a t i o n of t h e m a lto l from t h e i n t e r f e r i n g s u b s t a n c e s by ext r a c t i o n with chloroform and subsequent d e t er m i n a t i o n by e i t h e r phenol r e a g e n t o r a c i d f e r r i c ammonium s u l p h a t e . Eisenman and B r i c k e r (345) have d e s c r i bed a c o l o r i m e t r i c method €or t h e e s t i mation o f s t r ep t om y c in i n which t h e m a l t o l i s s e p a r a t e d from t h e r e a c t i o n m i x t u r e by steam d i s t i l l a t i o n . The method i s s u c c e s s f u l l y a p p l i e d f o r t h e d et er m i n a t i o n of str e p to m y c in i n ferment a t i o n b r o t h s , c o n c e n t r a t e s and c l i n i c a l samples. I f more th a n 500 u n i t s o f streptomycin a r e p r e s e n t t h e colorimet r i c r e a c t i o n w i t h f e r r i c i o n is employed and t h e c o l o r i s read a t 550 nm. I f less t h ea n 500 u n i t s a r e p r e s e n t , t h e phenol r e a g e n t i s used and t h e c o l o r i s read a t 775 nm. The sta n d a r d c u r v e s p r e p a r e d from p u r e str e p to m y c in are linear. Bagdasarian and Michalska (346) have r e p o r t e d the m a lto l method f o r d e te r m in a t i o n of s t r e p t om y c in i n u r i n e without e x t r a c t i n g t h e s tr e p to m y c in p r i o r t o t h e a s s a y , provided t h a t dosage was moder a t e . This method is recommended f o r r o u t i n e u s e , b u t cannot be a p p l i e d t o determining s t r e p to m y c in i n c o n c e n tr a t i o n below 100 y/ml.

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Doery and Mason (347) and Ka r tse v a e t a 1 (348) s e p a r a t e d str e p to m y c in from o t h e r f e r m e n t a t i o n p r o d u c t s by i o n exchange chromatography on c a t i o n exchange r e s i n before convertion i n t o maltol. Final d e t e r m i n a t i o n of str e p to m y c in was done by Maltol method. Bruns e t a 1 ( 3 4 9 ) , have r e p o r t e d a c o lo rimetric method f o r t h e d e t e r m i n a t i o n o f s t r ep t o m y c i n from t h e b r o t h . The a n t i b i o t i c s are c o l l e c t e d from t h e s o l u t i o n a t pH 8 . 3 - 8 . 5 on s i l i c a g e l and extract e d i n s u l p h u r i c a c i d . The c o n t e n t i s determined by m a l t o l method. Desai e t a1 (350) have d e s c r i b e d a d i r e c t method f o r e s t i m a t i o n o f s t r e p tomycin i n f e r m en te r b r o t h sample. The b r o t h sample was d e p r o t e i n i z e d by t h e a d d i t i o n o f 4 m l of t r i c h l o r o a c e t i c a c i d , f i l t e r e d c e n t r i f u g e d and d i l u t e d . After t h e a d d i t i o n o f 2 m l of 2N sodium hydroxide t o 5 m l o f t h e b r o t h , t h e m i x t u r e was heated i n a b o i l i n g water b a t h f o r t h r e e minutes and c o o le d . The absorbance was r e a d a f t e r a d d i t i o n of f e r r i c ammonium s u l p h a t e . A blank was run c o n c u r r e n t l y , c o n s i s t i n g o f t h e same sample without h e a t . F e r r a r i e t a1 (351) have adapted t h e m a l t o l method f o r automated a n a l y s i s o f s t r ep t o m y c i n i n f e r m e n ta tio n media u s i n g d i a l y s i s t o s e p a r a t e m a l t o l from t h e i n t e r f e r i n g s u b s t a n c e s . The m a l t o l t h u s s e p a r a t e d was a u t o m a t i c a l l y t r e a t e d w i t h stream o f f e r r i c c h l o r i d e i n 2.2N h y d r o c h l o r i c a c i d and t h e e x t i n c t i o n o f t h e r e a c t i o n product a t 530 nm was a u to m a t i c a l l y r e c o r d e d . E r r o r s were claimed t o be less t h a n with c o n v e n tio n a l manual methods. F u r t h e r development o f t h e a u t o - a n a l y z e r method f o r e v a l u a t i o n of str e p to m y c in were d e s c r i b e d by Shaw and Fortune ( 3 5 2 ) . They claimed t h a t h ig h p r e c i s i o n could

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be maintained even w i t h c o n s i d e r a b l e s i m p l i f i c a t i o n t o a l l o w f o r h i g h throughp u t o f samples.

Iodimetric-absorptiometric method was adopted by Sauciuc and Ionescu (353) f o r micro-determination o f s t r e p t o m y c i n i n f e r m e n t a t i o n l i q u i d . The method i s a s follows : The sample c o n t a i n i n g 25-150 Ug o f s t r e p t o m y c i n is h e a t e d w i t h 0.1N sodium hydroxide f o r 4 minutes. The s o l u t i o n i s t h e n a d j u s t e d t o pH 7.5 by adding 0.055N phosphoric a c i d t h e n t r e a t e d w i t h f r e s h l y d i l u t e d 0.001N i o d i n e i n potassium i o d i d e s o l u t i o n . After 5 t o 10 minutes 5 m l of r e a g e n t A (300 m l water, 200 m l acetic a c i d , 6 g potassium bromide and l m l h y d r o c h l o r i c a c i d ) is added followed by a s t r o n g j e t o f 1 m l of r e a g e n t B (100 m l water, 0.72g Sulphanilamide 0.02 g N-naphthyl-ethylenediamine d i h y d r o c h l o r i d e and 5g hydroxylammonium c h l o r i d e ) . After 10 minutes t h e e x t i n c t i o n of t h e s o l u t i o n is measured a g a i n s t t h e blank s o l u t i o n which is s i m i l a r l y t r e a t e d . Krystyna (354) h a s r e p o r t e d t h e m a l t o l c o l o r i m e t r i c method €or t h e determinat i o n of streptomycin residues i n milk. A n t i b i o t i c - f o r t i f i e d milk samples were cooled, d e f a t t e d by c e n t r i f u g a t i o n , d e p r o t e i n i z e d with z i n c s u l p h a t e i n t h e p r e s e n c e of barium hydroxide, concent r a t e d and a g a i n d e f a t t e d w i t h l i g h t petroleum. Then t h e sample was t r e a t e d w i t h sodium hydroxide and h e a t e d on t h e water b a t h . The m a l t o l formed was e x t r a c t e d i n t o chloroform from t h e a c i d i c medium and b a c k - e x t r a c t e d i n t o w a t e r a t a l k a l i n e pH. The aqueous e x t r a c t was mixed w i t h F o l i n C i o c a l t e u phenol r e a g e n t , a f t e r 10 minutes t h e absorbance was measured a t 660 nm and r e f e r r e d t o a c a l i b r a t i o n graph.

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The maltol method has been extensively used in studies of stability (355) and in the analysis of streptomycin in pharmaceutical preparations (356), feeds (357) and urine (358). Minor modifications have been suggested by Borowiecka (359), Kartseva and Bruns (360) and Wahbi et a1 (361). 13.6 12 Guanido reaction Method Both streptomycin and dihydrostreptomycin form orange-red color with oxidized nitroprusside reagent (310,362). Based on this principle Valedinskaya (363) has described a colorimetric method for estimation o f streptomycin. The procedure is as follows : 10 ml o f oxidized nitroprusside reagent is added to 10 ml of aqueous sample solution containing approximately 0.1 mg of streptomycin o r dihydrostreptomycin. After 3 minutes the absorbance is determined at 490 nm against a reagent blank prepared by substituting 10 ml of water for the sample aliquot. A working curve is prepared with the appropriate compound following the same procedure. The color is stable for about 4 hour.

Vieira and Pereira (364) have adapted the nitroprusside method for the determination of streptomycin in urine. The urine sample is treated with acetone and centrifuged after 30 minutes. Then the precipitate is stirred with water and filtered o r centrifuged. To an aliquot of the clear solution,water is added to bring to 2.5 ml, then treated with nitroprusside reagent an3 the absorbance is measured. Halliday (311) described the application of the modified Voges-Proskauer reaction to streptomycin, in which a pink color is generated by the reaction of the guanidine moiety with 3iacetyl and

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1-napthol. Quantitative analytical procedure was developed by Szafir and Bennett (365), Ashton et a1 (366) and Banerjee (367). The method is as follows : Transfer 2 ml of the sample solution to a test tube, add 15 ml of water and then 1 ml of 0.4 per cent diacetyl solution, 1 ml of 20% potassium hydroxide solution and 1 ml of 10% solution of 1-naphthol in anhydrous ethanol, in that order. Mix the contents of the tube by inversion after each addition. Forty minutes later determine the extinction at 525 MI in a 1 cm cell against water. Prepare a standard graph from suitable dilutions of a standard streptomycin solution, using the same procedure for color development as described for the sample. Determine the potency of the diluted sample solution from the standard graph. A standard graph should be prepared for each determination to allow for differences in room temperature, reagents, and so on. Schulz and Posada (368) have developed a colorimetric method involving guanidino group reaction for determination of streptomycin. The aqueous sample solution is treated with an ethanolic reagent containing diacetyl and naphthalene-2,7-diol; ethanolic sodium hydroxide is added and the mixture is set aside at room temperature for 45 minutes. The extinction is then measured at 555 nm and the streptomycin content is calculated with reference to a standard solution containing 50 1.18 of streptomycin sulphate per ml. The Sakaguchi reaction (312,313) has been adapted by Buck et a1 (369) to the analysis of streptomycin and dihydrostreptomycin, using spectrophotometric determination of the red color formed by the reaction of 1-naphthol and sodium hypobromite with the antibiotics. Natarajan and Tayal (370) have reported a rapid method of colorimetric estimation of streptomycin, based on the Sakaguchi reaction. The procedure i s as follows :

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Mix a n aqueous s o l u t i o n o f t h e sample w i t h 1 m l o f 10% sodium hydroxide, and 2 m l o f 0.02% e t h a n o l i c 1-naphthol. After 15 m i n u t e s , add 1 m l of sodium hypobramite r e a g e n t and 10 m l o f c a r b o n t e t r a c h l o r i d e . Shake well, and add 5 m l o f a b s o l u t e e t h a n o l , shake a g a i n and measure t h e e x t i n c t i o n o f t h e aqueous p h a s e a t 530 nm i n a 1 cm c u v e t t e with a r e a g e n t b l a n k . The r e s u l t s were comparable w i t h t h o s e from m i c r o b i o l o g i c a l method. The a p p l i c a t i o n of t h e Sakaguchi r e a c t i o n f o r t h e c o l o r i m e t r i c d e t e r m i n a t i o n o f streptomyc i n was a l s o r e p o r t e d by Fontes ( 3 7 1 ) . M o d i f i c a t i o n of t h e above method by u s i n g 8hydroxyquinoline was d e s c r i b e d by T i b a l d i (372). S z i l a g y i and Szabo (373) have r e p o r t e d a simil a r c o l o r i m e t r i c method f o r t h e a n a l y s i s o f S a k a g u c h i - p o s i t i v e a n t i b i o t i c s , u s i n g N-bromosuccinimide. The method i s as f o l l o w s : 5 m l O f t h e s o l u t i o n o f t h e sample i s t r e a t e d w i t h 2 m l o f 1-naphthol r e a g e n t . After b e i n g shaken and set a s i d e f o r 3 minutes, t h e mixt u r e i s r a p i d l y poured i n t o 0.5 m l o f N-bromos u c c i n i m i d e s o l u t i o n , t h e mixture i s shaken v i g o r o u s l y and 1 m l o f 40% u r e a s o l u t i o n i s added i n 1 5 second. A v i v i d r e d c o l o u r devel o p s , t h e e x t i n c t i o n o f which is r e a d i n 5 minutes a g a i n s t b l a n k s , w i t h a b l u e f i l t e r , r e s u l t s being r e f e r r e d t o standard curves. The r e l a t i v e e r r o r o f t h e method i s f 4 % . 13.613 Glucosamine r e a c t i o n Method The glucosamine t e s t f o r t h e i d e n t i f i c a t i o n of s t r e p t o m y c i n u s i n g a c e t y l a c e t o n e and E h r l i c h ' s r e a g e n t , can be used q u a n t i t a t i v e l y (309). Based on t h i s p r i n c i p l e , Kamata e t a1 (374) have d e s c r i b e d a c o l o r i m e t r i c a s s a y f o r s t r e p tomycin. The p r o c e d u r e of t h e method i s as follows :

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To 4 m l of s t r ep t o m y c i n s o l u t i o n , add 1 m l o f 2.5N s u l p h u r i c a c i d . Boil f o r 1 hour, c o o l , n e u t r a l i z e and d i l u t e t o 10 m l . To about 2 m l o f t h i s s o l u t i o n add 5 .5 m l o f r e a g e n t A (2.225 m l a c e t y l a c e t o n e , 113.75 m l 2 N sodium c a r b o n a t e 3 4 . 1 m l 1N h y d r o c h l o r i c a c i d and water) , s t o p p e r , b o i l f o r 20 minutes, c o o l , add 10 m l . o f e t h a n o l , f i n a l l y add r e a g e n t B (0.8 o f Ehrlich'saldehyde, 30 m l o f concent r a t e d h y d r o ch l o r i c a c i d and a b s o l u t e e t h a n o l ) . Measure t h e i n d e n t s i t y o f t h e c o l o r a t 532 nm. The method i s r e l i a b l e o n l y f o r samples o f high p u r i t y . The a c e t y l acetone-Ehrlich's aldehyde method h a s been adapted by Masquelier (375) f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c in i n u r i n e . The u r i n e sample c o n t a i n i n g s t r e p to m y c in i s t r e a t e d w i t h l ea d a c e t a t e , t h e e x c e s s o f l e a d is removed by sodium s u l p h a t e and f i l t e r e d . To about 1 m l o f t h e f i l t r a t e 1 m l o f a c e t y l a c e to n e r e a g e n t and 2 d r o p s o f sodium hydroxide a r e added. The s o l u t i o n i s h ea t ed i n a w a te r h a t h f o r 10 minu t e s and co o l ed . 2 m l o f th e E h r lic h ' s aldehyde r e a g e n t is t h e n added and t h e red c o l o r developed i s compared w i t h a sta n d a r d s o l u t i o n o f s t r ep t o m y c i n and w i t h a blank of u r i n e sample. 13.614 Other Methods The aldehyde group o f str e p to m y c in was expl o i t e d by Marshall e t a1 (358) who developed a q u a n t i t a t i v e p r o ce d u r e based on t h e format i o n o f a co l o r ed semicarbazone with 4-[4(p-chlorophenazo) -1 -naphthyl ] semicarbazide. The ex c e s s of t h e r e a g e n t i s e x t r a c t e d w i t h chloroform and h y d r o c h l o r i c a c i d i s added. The c o l o r developed i s measured a t 580 nm. S a v i t s k a y a and Kartseva (376) used 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e which reacts w i t h s t r e p tomycin t o form a yellow hydrazone. The e x c e s s r e a g e n t is e x t r a c t e d w i t h b u t y l acetate and t h e c o n c e n t r a t i o n o f t h e str e p to m y c in i s determined from c a l i b r a t i o n curve p r e p a r e d with pure streptomycin. Mattick (377) h a s r e p o r t e d a c o l o r i m e t r i c method f o r t h e d e t e r m i n a t i o n o f a n t i b i o t i c s

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i n c l u d i n g s t r ep t o m y c i n i n m i l k which is based on t h e i n h i b i t i o n o f t h e r e d u c t i o n of n i t r a t e t o n i t r o u s o x i d e by Miwiacoccus pqkogcnu. The n i t r o u s o x i d e produced i s determined color i m e t r i c a l l y a f t e r d i a z o t i s a t i o n w i t h su lp h a n i l i c a c i d and c o u p l i n g with l-naphthylamine. B a r i l a r i and K a t z (378) have r e p o r t e d a method u s i n g copper t a r t r a t e and phosphomolybdic a c i d reagents f o r colorimetric determination o f s t r ep t o m y c i n . The f r e s h l y p r e p a r e d s o l u t i o n o f streptomycin s u l p h a t e is heated f o r 8 minu t e s w i t h 2 m l of w a t er and 2 m l of copper t a r t r a t e s o l u t i o n . After c o o l i n g t h e phosphomolybdic a c i d i s added and t h e b l u e c o l o r developed i s r e a d p h o t o c o l o r i m e t r i c a l l y . A p h o t o c o l o r i m e t r i c method f o r t h e determina-

t i o n o f s t r e p t o m y c i n w i t h r e i n e c k a t e s a l t was d e s c r i b e d by B a r i l a r i e t a1 ( 3 7 9 ) . S t r e p t o mycin i s h ea t ed w i t h w ate r a t 40° and e q u a l volume o f ammonium r e i n e c k a t e s o l u t i o n i s added. The p r e c i p i t a t e t h u s formed i s separ a t e d , washed w i t h et h an o l and d i s s o l v e d i n ac e t o n e water m i x t u r e . Then a c e to n e i s evap o r a t e d . The s o l u t i o n i s th e n t r e a t e d w ith sodium hydroxide, d i l u t e d w i t h water and neut r a l i z e d with n i t r i c acid. 5 m l of t h i s solut i o n i s t r e a t e d with 5 m l of f e r r i c n i t r a t e and t h e c o n c e n t r a t i o n o f str e p to m y c in i s d e t e r mined c o l o r i m e t r i c a l l y . The a p p l i c a t i o n o f t h i s method i n t h e pharmaceutical a n a l y s i s was r e p o r t e d by Basu and D utta (380). C o l o r i m e t r i c d e t e r m i n a t i o n o f aldehyde and k e t o n es w i t h o x a l i c a c i d d ih y d r a z id e was adap t e d by Bartos and B u r t i n (381) f o r d e te r m in a t i o n o f s t r ep t o m y c i n . About 1 m l of t h e sample i s t r e a t e d with 2 m l o f p h o s p h a t e - c i t r a t e - b o r a t e b u f f e r and 2 m l of o x a l i c a c i d d i h y d r a z i d e r e a g e n t . The m ix tu r e i s allowed t o s t a n d f o r 4 h o u r s . The b l u e o r v i o l e t c o l o r r e s u l t i n g i s read colorimetrically. Using p i c r i c a c i d r e a g e n t , Agrawal and D u tta (382) have developed a r a p i d c o l o r i m e t r i c a s s a y method f o r s t r ep t om y c in i n m ix tu r e w i t h d i h y d r o s t r e p t o m y c i n . The test sample and

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the blank are treated with 3 ml of 0.1% picric acid,2 ml of 5% sodium hydroxide; heated for 3 minutes, cooled diluted to 10 ml and the extinction is measured at 500 nm. Dihydrostreptomycin does not give this color reaction, but can be determined after being converted into streptomycin. Yavors'kli (383) has reported the application of dinitrobenzene and their derivatives as reagent in colorimetric analysis of organic medicinal substances including streptomycin. A new spectrophotometric method for the assay of streptomycin, by salt formation with bromothymol blue was reported by Nour El-Deen et a1 (384).

Barilari et a1 (385) have reported a new photocolorimetric method for the determination o f streptomycin. Streptomycin solution is treated with potassium iodate solution and sodium arsenite solution. After the disappearance of the yellow color, 0 . 0 3 ml of phenol and 4 ml of sulphuric acid are added, boiled, cooled and the absorbance is read at 490 nm. A spectrophotometric method for the estimation of streptomycin by salt formation with tropaeolin 000, was reported by Nour El-Deen et a1 (384) and Beltagy et a1 (386). The method is as follows :

To about 3 ml of sample in water add about 1 ml o f cold, dilute sulphuric acid, and 3 ml of cold aqueous solution Tropaeolin 000. Mix and cool in ice for one hour, centrifuge, decant and wash the precipitate with 10 n i l of cold dilute sulphuric acid. Dissolve the residue in 2 ml of sodium hydroxide, adjust to 100 ml with water. Dilute a mixture of 5 ml of the solution and 1 ml of dilute sulphuric acid to 50 ml and measure the extinction at 485 nm. A new sensitive colorimetric determination of streptomycin by thiobarbituric acid method was proposed by Erno (387). The method is

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s p e c i f i c and s e n s i t i v e . follows :

The procedure i s a s

The s o l u t i o n c o n t a i n i n g s t r e p t o m y c i n i s mixed w i t h 0.1M potassium i o d a t e i n 0.05 m l o f 0.5M s u l p h u r i c a c i d , a f t e r 15 minutes, a 2% s o l u t i o n o f sodium a r s e n i t e i n 5 m l h y d r o c h l o r i c a c i d i s added. When t h e brown c o l o r i s d i s a p p e a r e d , 1 m l o f t h i o b a r b i t u r a t e r e a g e n t is added. The m i x t u r e i s h e a t e d f o r 10 minutes and t h e n t h e e x t i n c t i o n of t h e s o l u t i o n i s measured a t 415 nm a g a i n s t a b l a n k . The m o d i f i c a t i o n o f t h i o b a r b i t u r i c a c i d method was r e p o r t e d by Erno e t a l . (388) and Fukumoto Hisako (389). Alykov (390) has d e s c r i b e d an a b s o r p t i o m e t r i c method f o r t h e d e t e r m i n a t i o n o f amino g l y c o s i d e a n t i b i o t i c s i n b i o l o g i c a l f l u i d s . Streptomycin and o t h e r amino g l y c o s i d e s form a complex w i t h stilbazochrome P i n a c e t a t e o r c i t r a t e b u f f e r medium. The blood sample is t r e a t e d with 1 m l o f water and 0 . 5 m l o f t r i c h l o r o a c e t i c a c i d s o l u t i o n and c e n t r i f u g e d . To t h e s u p e r n a t a n t l i q u i d 1 m l of s t i l b a z o c h r o m e P r e a g e n t i n c i t r a t e b u f f e r s o l u t i o n , 1 m l o f 1 mM PrC13 and 6 ml o f e t h a n o l added. After 20 minutes t h e mixture i s c e n t r i f u g e d and t h e absorbance o f t h e s u p e r n a t a n t l i q u i d is measured a t 560 nm. A new and s e l e c t i v e s p e c t r o p h o t o m e t r i c d e t e r mination o f s t r e p t o m y c i n u s i n g O-hydroxyhydroq u i n o n e p h t h a l e i n [2',3',6',7'-tetrahydroxyspiro (isobenzofuran; 1( 3 H ) , 9 ' - (9H) xanthen) -3-one) ] and manganese, was r e p o r t e d (391). The method i n v o l v e s f o r m a t i o n o f a t e r n a r y complex i n a medium c o n t a i n i n g 40% of methanol and 1%of p o l y v i n y l p y r r o l i d i n o n e . Measurements a r e made a t 570 nm a t pH 10 t o 1 0 . 3 . A p h o t o m e t r i c method f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n and o t h e r aminoglycoside a n t i b i o t i c s was proposed by Alykov (392). The method i n v o l v e s t h e measurement of t h e d e c r e a s e i n t h e absorbance a t 575 nm, due t o Acid b l a c k S caused by p r e c i p i t a t i o n o f a complex o f C . 1 Acid Black 24 w i t h t h e a n t i b i o t i c s .

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Eriochrome Slack T was used as a precipitant in the photometric determination of streptomycin and other antibiotics in biological fluids (393). Ninhydrin spectrophotometric method has been adapted f o r the determination of aminoglycoside antibiotics in pharmaceutical preparations (394). About 10 l.11portions of the solutions of streptomycin in water were applied to precoated Silica gel plate; the chromatogram was developed in a solution of l g of ninhydrin in 50 ml of 95% ethanol-anhydrous acetic acid (5:l). After drying the colored bands were transferred to test tubes and shaken with 5 ml of propan-2-01 containing 5 drops of ammonium hydroxide and the tubes were centrifuged. The absorbance spectrum o f supernatant solution was recorded in the range 400 to 800 nm against water. Divakar et a1 (395) have reported a spectrophotometric method for the determination of streptomycin, tetracycline and chloramphenicol using brucine and sodium periodate. About 10 ml of streptomycin solution was boiled under reflux f o r 45 minutes with 10 ml o f 2M hydrochloric acid and the excess of the acid was removed in vacuo. The residue was dissolved in 20 ml of warm water and diluted to 50 ml. About 0 . 5 to 3.0 ml of the solution was treated with 3 ml of SmM-brucine, 1.5 ml of 5m.M.sodium metaperiodate and 2 ml of 2.3M.-sulphuric acid. The solution is then diluted, boiled for 15 minutes, cooled and the absorbance was measured at 430-450 nm. 13 62 Ultraviolet Methods When streptomycin is heated with 0.1N sodium hydroxide, it produce maltol (L9), which exhibits an absorption maximum at 322 nm in alkaline solution. Therefore the streptomycin can be assayed by measuring the absorbance at 322 nm before and after hydrolysis in sodium hydroxide solution (396). Katz (397) has reported an ultraviolet spectroscopic method for the determination of

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s t r e p t o m y c i n i n f e e d s . The s t r e p t o m y c i n i n f e e d s was e x t r a c t e d w i t h d i l u t e s u l p h u r i c a c i d , s e p a r a t e d , n e u t r a l i z e d , f u r t h e r s e p a r a t e d , conc e n t r a t e d and p u r i f i e d by i o n exchange. The s t r e p t o s e moiety was c o n v e r t e d i n t o m a l t o l and q u a n t i t a t i v e l y determined by measuring i t s u l t r a v i o l e t absorbance. 1 3 . 6 3 F l u o r i m e t r i c Methods Boxer and J e l i n e k (398) have r e p o r t e d a f l u o r i metric method f o r t h e d e t e r m i n a t i o n o f s t r e p tomycin i n blood and s p i n a l f l u i d . The b a s i s o f t h i s method i s t h e formation o f hydrazone o f s t r e p t o m y c i n w i t h f l u o r e s c e n t 9-hydrazinoa c r i d i n e h y d r o c h l o r i d e . The e x c e s s of t h e r e a g e n t t o g e t h e r w i t h hydrazones of a c i d i c , n e u t r a l and b a s i c compounds are s e p a r a t e d from t h e s t r o n g l y b a s i c s t r e p t o m y c i n hydrazone by e x t r a c t i o n from t h e s o l u t i o n with benzyl a l c o h o l . The aqueous base c o n t a i n i n g s t r e p t o m y c i n hydrazone i s c e n t r i f u g e d and t h e intens i t y of t h e f l u o r e s c e n c e o f t h e clear s o l u t i o n i s measured. The method h a s been extended t o determine s t r e p t o m y c i n i n u r i n e and t i s s u e s (399). Streptomycin and dihydrostreptomycin i n a l k a l i n e s o l u t i o n , produce a s t r o n g f l u o r e s c e n c e w i t h sodium 1,2-naphthaquinone-4-suIphonate (400). Based on t h i s p r i n c i p l e , Faure and Blanquet (401) have developed an a n a l y t i c a l procedure f o r t h e d e t e r m i n a t i o n of s t r e p t o m y c i n . Aqueous s o l u t i o n o f s t r e p t o m y c i n was t r e a t e d w i t h sodium hydroxide and sodium 1,2-naphthaquinone-4-sulphonate s o l u t i o n . Then t h e s o l u t i o n was set a s i d e i n t h e d a r k €or 1 hour a t 20° and t h e n t h e f l u o r e s c e n c e was measured. The d e t a i l e d s t u d i e s o f f l u o r e s c e n c e s p e c t r a were made i n subsequent p u b l i c a t i o n s (402,403). A p p l i c a t i o n o f t h e method f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c i n i n blood serum o r plasma was a l s o e x p l o r e d (404-406). A continuous-flow Auto-Analyzer f l u o r i m e t r i c procedure f o r t h e d e t e r m i n a t i o n of l a c t i c a c i d i n m i l k was a p p l i e d t o t h e d e t e c t i o n o f a n t i b i o t i c s ( 4 0 7 ) . The procedure was based on t h e

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i n h i b i t i o n by a n t i b i o t i c s o f t h e a b i l i t y of s t a r t e r c u l t u r e s t o produce l a c t i c a c i d . Alykov (408) has d e s c r i b e d a new f l u o r i m e t r i c method f o r t h e d et er m i n a tio n o f aminoglycoside a n t i b i o t i c s . The method i n v o l v e s e x t r a c t i o n of f l u o r e s c e i n complexan-Pr3+ aminoglycoside s p e c i e s from an aqueous medium o f pH 5.8 t o 6 . 2 i n t o isoamyl alcohol-benzene (1 :1) r e e x t r a c t i o n o f f l u o r e s c e i n complexan a s s o c i a t e d w i t h t h e aminoglycoside i n t o aqueous 0.1M sodium f l u o r i d e and F l u o r 2 me tr ic d e t e r m i n a t i o n o f f l u o r e s c e i n complexan i n t h i s aqueous s o l u t i o n a t 533 nm. The method i s s u i t a b l e f o r t h e d e t e r m i n a t i o n o f s t r e p t om y c in and t h e r e l a t e d a n t i b i o t i c s i n blood, milk and u r i n e . Prot e i n s a r e removed from t h e blood samples by p r e c i p i t a t i o n with t r i c h l o r o a c e t i c acid. 13. 7 Chromatographic Methods

13.71 Column Chromatography Column chromatographic (CC) method h a s been used e x t e n s i v e l y f o r t h e s e p a r a t i o n and p u r i f i c a t i o n of s t r e p t o m y c i n. S e v e r a l r e p o r t s have been p u b l i s h e d r e g a r d i n g t h e a p p l i c a t i o n o f c h a r c o a l (lQ6-117), a c i d washed alumina (118-122) and i o n exchange r e s i n s (123-164) f o r t h e s e p a r a t i o n and p u r i f i c a t i o n o f s t r e p t o mycin. Okumura e t a1 (409) have used columns o f a magnesium a l u m i n o - s i l i c a t e f o r p u r i f i c a t i o n o f b a s i c a n t i b i o t i c s . R es o lu tio n s such a s s t r e p tomycin and kanamycin from oleandomycin were d e s c r i b e d . The method a p p e a r s t o have a p p l i c a b i l i t y i n t h e i s o l a t i o n o f t h e a n t i b i o t i c s from b i o l o g i c a l f l u i d s such a s c u l t u r e media, blood and u r i n e . S t . John e t a1 (410) used column o f c a t i o n exchange r e s i n . Amberlite IRC-50 t o s e p a r a t e streptomycin and s t r ep t om y c in B from fermentat i o n b r o t h s , a n t i b i o t i c being determined c o l o r i m e t r i c a l l y [maltol method), w ith good a g r e e ment w i t h m i c r o b i o l o g i c a l procedure. Applicat i o n o f t h e above method f o r d e te r m in a tio n of

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s t r e p t o m y c i n i n animal f e e d (397) and i n formul a t i o n s (411) were r e p o r t e d . h b e r l i t e CG 120 i o n exchange r e s i n was u t i l i z e d by Conca and Pazdera (412) f o r s e p a r a t i o n of s t r e p t i d i n e i n s t r ep t o m y c i n . Comparative s t u d y o f c a r b o x y l i c c a t i o n exchangersin r e s p e c t s o f t h e s o r p t i o n and d e s o r p t i o n o f s t r ep t o m y c i n was r e p o r t e d (413). Other r e p o r t s have d e s c r i b e d i n d e t a i l s t h e u s e o f p e r m u t i t (414), phenoxy-acetic a c i d c a t i o n exchanger and p h e n o l i c r e s i n s (415), t h e r e s o l u t i o n o f components of str e p to m y c in complex (416), t h e s e p a r a t i o n o f str e p to m y c in from f e r m e n t a t i o n b r o t h s u s i n g continuous p r o c e s s (417) and e v a l u a t i o n o f v a r i o u s exchangers f o r s e p a r a t i n g c o l o r e d i m p u r i t i e s (418). The chromatographic behaviour of str e p to m y c in on Sephadex G-10 h a s been d e s c r i b e d by S t o r l (419). 13.72 Paper Chromatography Horne and P o l l a r d ( 4 2 0 ) have d e s c r i b e d a method f o r i d e n t i f i c a t i o n o f s tr e p to m y c in by u s i n g p a p e r - s t r i p chromatogram. The p o s i t i o n o f t h e a n t i b i o t i c were d e t e c t e d by means o f t h e Sakaguchi r e a g e n t . 3% ammonium c h l o r i d e was found t o be n e c e s s a r y f o r good s e p a r a t i o n . Wj-nsten and Eigen (421) were s u c c e s s f u l i n r e s o l v i n g m i x t u r es o f s tr e p to m y c in and c l o s e l y r e l a t e d s u b s t a n c e s . B u ta n o l- p ip e r id in e t o l u e n e s u l p h o n i c a c i d m ix tu r e was used as a d e v e l o p i n g a g e n t . The p o s i t i o n o f t h e a n t i b i o t i c s were r e v e a l e d by bioautography on a g a r . Solomons and Regna (422), S t o d o l l a e t a1 (423) Kowezyk (424) have a p p l i e d similar method f o r t h e s e p a r a t i o n o f s t r ep t om y c in . P e t e r s o n and Reineke (425) d e s c r i b e d a s i m i l a r method f o r t h e d e t e r m i n a t i o n o f s t r ep to m y c in i n s a l t - f r e e p r e p a r a t i o n s and a p p l i c a t i o n s o f t h e method t o s t u d i e s o f b i o s y n t h e s i s were p u b lish e d by Hunter e t a1 (199). E f f e c t s o f s a l t s on t h e movement and zone shape of str e p to m y c in i n p ap e r chromatography was d i s c u s s e d by So k o lsk i and Lummis (426).

564

JABER S . MOSSA ETAL. A d e t a i l e d s t u d y by Heding (427,428) produced

a system c a p a b l e of c l e a r l y r e s o l v i n g s t r e p t o mycin, and i t s r e l a t e d compounds w i t h a development time o f 8 hours. The d e v e lo p e r used was p r ep a r e d by mixing e q u a l volume o f amyl a l c o h o l c o n t a i n i n g 1%o f b i s - ( 2 - e t h y l h e x y l ) phosphate and 0.5% sodium c h l o r i d e s o l u t i o n i n b o r a t e b u f f e r . The s p o t s were d e t e c t e d e i t h e r by u s i n g a s p r a y o f 1 - n a p h t h o l - d i a c e t y l o r by bioautogrsphy on a n u t r i e n t a g a r p l a t e seeded w i t h s p o r e s o f B a W bub-. Other r e p o r t s o f r e s o l u t i o n have been p u b l i shed by Makisumi (429), Kondo e t a1 (430) and Pant and Agrawal (431). The former used b u t a n o l , a c e t i c a c i d , p y r i d i n e . water (4: 1:1:2) and b u t a n o l : 6N h y d r o c h l o r i c a c i d ( 7 : 3 ) as developing r e a g e n t and Sakaguchi's r e a g e n t and J a f f e ' s r e a g e n t f o r d e t e c t i o n . The Rf v a l u e s of s t r ep t o m y c i n i n t h e above s o l v e n t systems were 0.17 and 0 . 0 8 r e s p e c t i v e l y . Albu-Budai and Unterman (432) r e p o r t e d a quant i t a t i v e procedure based on paper chromatograp h i c r e s o l u t i o n , e l u t i o n and sp e c tr o p h o to m e tr ic d e t e r m i n a t i o n after c o l o r development w i t h MN 261 paper s t r i p s 1-naphthol-diacetyl. p r e v i o u s l y impregnated with phosphate b u f f e r o f pH 7 were used. The developing s o l v e n t were pentachlorophenol-butanol-sodium hydroxide-water. Numerous s p r a y r e a g e n t s have been used i n t h e d e t e c t i o n o f s t r ep t o m y c in based l a r g e l y on c o lo r i m e t r y . T h i s i n c l u d e s potassium permanganate (433), i o d i n e (434), sodium p e r i o d a t e w i t h p i c r i c a c i d (435), t h e a n i l i n e , l a c t i c a c i d , p i c r i c a c i d m i x t u r e (436) and bromine-starchpotassium i o d i d e , r e a g e n t (9). Couling and Goodey (437) have d e s c r i b e d a new method o f s t r ep t o m y c i n chromatography and i t s u s e i n t h e examination of t h e r e a c t i o n between s t r e p t o m y c i n and ammonia, Paper p r e v i o u s l y impregnated w i t h phosphate b u f f e r a t pH 7 and sodium hydroxide-pentachlorophenol-butanol as developing s o l v e n t were U s e d . To d e t e c t t h e compounds, d i p t h e chromatogram i n p e r i o d a t e ac e t o n e and t h en i n b e n z i d i n e - a c e to n e t o

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g i v e yellow o r white s p o t i n a b l u e background; o r d i p i n a mixture o f d i a c e t y l and 1-naphthol t o g i v e r e d s p o t on a white background. 1 3 . 7 3 Thin-Layer Chromatography

Thin l a y e r chromatographic t e c h n i q u e was ext e n s i v e l y used € o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f s t r e p t o m y c i n and i t s r e l a t e d compounds by Kondo (430) and Borowiecka (438). The former used p l a t e s c o a t e d w i t h a c t i v a t e d carbon whereas t h e latter used p l a t e s c o a t e d e i t h e r w i t h s i l i c a g e l G o r Kieselguhr G. VonSchantz e t a1 (439) s t u d i e d t h e a p p l i c a t i o n of metal p l a t e s f o r t h i n l a y e r d e t e c t i o n o f pharmaceutical compounds. S e v e r a l r e p o r t s have appeared on t h e a p p l i c a t i o n o f t h i n l a y e r chromatography i n i d e n t i f y i n g aminoglycoside a n t i b i o t i c s . The s o l v e n t system and t h e Rf v a l u e s are r e p o r t e d i n t a b l e . 4. Voigt and Bared (443) and Katayama and Ibeda (444) have r e p o r t e d two-dimensional TLC method f o r s e p a r a t i o n and i d e n t i f i c a t i o n of s t r e p t o mycin and i t s r e l a t e d compounds. S a t 0 and Ikeda (445) were a b l e t o r e s o l v e streptomycin and i t s dihydro-and dihydrodeoxy d e r i v a t i v e s by ascending TLC procedure. Simil a r claims were made by Katayama and Ikeda (446). Thin l a y e r - b i o a u t o g r a p h i c method h a s been employed f o r t h e s e p a r a t i o n and i d e n t i f i c a t i o n o f streptomycin (447,448) . The s p o t s o b t a i n e d from streptomycin by TLC procedure have been e l u t e d w i t h phosphate b u f f e r and examined m i c r o b i o l o g i c a l l y w i t h B a W bubLLLi.6 s p o r e s A s i m i l a r procedure h a s been adapted by Zuidweg e t a1 (449) and Hamann e t a 1 (450) t o r e s o l v e streptomycin and o t h e r a n t i b i o t i c s . The former used t h e p l a t e s c o a t e d with sephadex and t h e l a t t e r used t h e c e l l u l o s e l a y e r . A p p l i c a t i o n 4-Dimethylaminobenzaldehyde-antimony t r i c h l o r i d e a s a d e t e c t i n g agent f o r a n t i b i o t i c i n TLC was r e p o r t e d by Mathis and

Table 4 : TLC of Streptomycin

Adsorbent

Solvent System.

Spray Reagent

1. Silica gel G / Aluminium oxide.

PropanolEthyl acetate-waterAmmonium hydroxide (50:10:30:10).

2. Silica gel G

Butanol-watermethanol-tolune -p-sulphonic acid (40:20:10:1)

Sodium Nitroprusside, potassium permanganate and sodium hydroxide mixture.

3 . Silica gel G

1) Acetic Acid : butanol (7 :2) 2) Benzene-acetone acetic acid 2:2:1.

10% aqueous solution Copper sulphate with 2% ammonia.

Ninhydrin.

> J

Rf value

Reference

0.62-07

440

0.4

441

0.32 0.32

442

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Schmitt (451). Paunez (452) described a TLC method utalizing the layer o f ion-exchange resin. More extensive examination of streptomycin group was reported by Heding (453) and More recently Borowiecka ( 4 5 4 ) . Forty seven solvent systems were investigated in deciding on optimum conditions for resolution of streptomycin and related compounds. Visualization was made by a spray reagent of sodium nitroprussidepotassium permanganate. 1 3 . 7 4 High Performance Liquid Chromatography

Bagon (455) has developed an assay of antibiotics in pharmaceutical preparations using reversed-phase high-pressure liquid chromatography. Adapting this method, antibiotics like streptomycin were determined by HPLC on a column packed with Spherisorb SS-ODs. Aqueous methanol at a flow rates of upto 1 ml/min. was used as an eluent. The elutes were monitored by spectrophotometric detection. Whall (456) has reported a HPLC method for the determination of streptomycin and dihydrostreptomycin sulphate. The method is as follows: Samples were dissolved in water and diluted to 50 ml., 3 ml of each solution is then diluted to 50 ml with mobile phase containing 0.02 M sodium hexanesulphonate - 0.025 M sodium phosphate in aqueous acetonitrile, adjusted to pH 6 . A 25 pl portion was subjected to HPLC on a column packed with LI Bondapak C18. The column was fitted with a reversed-phase guard column and elution was carried out at 1 mljmin. at 25O. Solutes being detected at 195 nm. HPLC assay of pyrazinoic acid in human plasma in the presence o f antituberculosis drugs using an automatic sampler was proposed by Ratti et a1 ( 4 5 7 ) . This method could also be used to determine streptomycin. The chromatographic conditions are : Mobile phase : 0.01M ammonium dihydrogen phosphate - acetonitrile ( 3 :7 ) .

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Column condition : Column of 25 cm length 4.6 mm width packed with Lichrosorb-NH2. Flow rate at 2 rnl/min. The elutes are detected at 254 nm. Inchauspe and Samain (458) have described a ion-pair reversed-phase high performance liquid chromatographic method for separating aminoglycoside antibiotics using perfluorinated carboxylic acids. The aminoglycoside antibiotics were separated at ambient temperature on a column (25 cm x 4.6 mm) packed with ultrasphere c18 (5 pm) with methanol - 50 mM-pentafluoropropionic acid (21:29), methanol - 50 mM-heptafluorobutyric acid (3:2), methanol - 50 mM - camphorsulphonic acid (29:21) o r 0.2 M trifluoroacetic acid as mobile phase and refractive index detection. 13.75 Electrophoresis Foster and Ashton (459) described a method for separation of streptomycin and related compounds by paper electrophoresis. The test solutions were applied to the paper which is then wetted with a buffer solution of pH 5. After passage o f current for 16 hours the paper was removed and dried. The spots were revealed by one of following reagents : 1) diacetyle; 2) naphtharesorcinol spray; 3) Elson Morgans reagent. Alternatively, the potency was determined microbiologically by laid down the papers on agar plates seeded with suitable organisms. Takahashi and Amano (460) employed the paper electrophoresis method for the identification o f various antibiotics. Philippe et a1 (461) have reported a similar method of electrophoresis f o r the separation of streptomycin and dihydrostreptomycin. Whatman 3MM paper in borate buffer at pH 9 were used. The electropherogram was run €or 3 hours at 500 V and the spots were developed with Monastero's alkaline nitroprusside-ferric cyanide reagent. Other reports o f resolution of streptomycin and other antibiotics have been published by Paris

569

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and Theallet (462) Lightbrown and de Rossi (463). The application of electrophoresis for separation and identification o f the antibiotics in Pharmaceutical mixtures was reported by Apreotesei and Teodosiu (464). Katayama and Ikeda (465) achieved a similar resolution using electrophoresis layers of silica gel. They also described a two dimensional technique using a combination of electrophoresis and chromatography on silica gel (466). Gorge and Monteoliva (467) used two dimensional techniques on paper (chromatography in one direction and electrophoresis in the second) €or identification of many guanidino compounds including streptomycin. Application of electrphoresis to the identification of several aminoglycoside antibiotics has been reported by Garber and Dobrecky (468) and Ochab (469). Langner et a1 (470) have described a method of determination of antibiotics including streptomycin in mixtures at ultra-micro levels by rapid low-voltage electrophoresis. The antibiotics could be separated on microscopic slides coated with agarose gel. After electrophoresis at 50 V per cm. for 5 to 30 minutes, the antibiotics were located by Bioautography on agar seeded with BaCieeUn c5ub;tieid. 13.76 Counter-Current Distribution Titus and Fried (471) have reported the u s e of counter-current distribution technique f o r the analysis of streptomycin preparation. The Craig technique of counter-current distribution was employed to show the presence of a related substance in crude streptomycin by distribution between butanol and water. Craig countercurrent distribution technique has been extensively employed for the separation of streptomycin from mannosidostreptomycin by Plaut and McCormack (472) and O'Keeffe et a1 (473). The former obtained a better resolution by using the solvent systems of an aqueous phase containing 0.5% sodium bicarbonate and 1% sodium chloride and solvent phase

JABER S. MOSSA ETAL.

570

(Pentasol-amyl a l c o h o l ) c o n t a i n i n g 5% s t e a r i c a c i d and t h e latter employed a system composed o f l a u r i c a c i d i n amyl a l c o h o l and aqueous phospha t e -bora te b u f f e r . Meyer (474) h a s d i s c u s s e d i n d e t a i l s t h e d i f f e r e n t methods o f c o u n t e r - c u r r e n t d i s t r i b u t i o n t e c hnique s employed f o r t h e a n a l y s i s of v a r i o u s compounds i n c l u d i n g s t r e p t o m y c i n . 1 3 .8 Bio-Assay Methods The p r i n c i p l e o f t h e b i o l o g i c a l a s s a y i s based on t h e comparison o f t h e i n h i b i t i o n of t h e growth o f b a c t e r i a produced by known c o n c e n t r a t i o n o f t h e s t a n d a r d p r e p a r a t i o n wi th t h a t produced by measured c o n c e n t r a t i o n o f t h e sample be ing t e s t e d . 13.81 Agar-Diffusion Method on S o l i d Media Many a g a r - d i f f u s i o n methods w i t h t h e s u i t a b l e inoculum o f t h e fol lowing t e s t organisms have been r e p o r t e d : Enchenicki cakX (475-477), BacAYw n u b U (478-483), B a W cd~cf.&ns (484), B a r n ficheni~onmn (485), B a W cetrew v a r . mgcoidu (486-487), SXaphyLocaccw awrew (488490), S.thepRococcub h c ; t i n (491) K L e b n i d h pneumonkw (492) L a c t o b a c i U u b&atLicw (493) a c i d r e s i s t a n t microbacterium V-5(494). The method h a s been e x t e n s i v e l y used i n determin a t i o n o f stre pt omyc i n i n b i o l o g i c a l body f l u i d (495-499), u r i n e (500). food and animal f e e d (501-505) and pha rmaceutical p r e p a r a t i o n s (506).

M o d i f i c a t i o n s of a g a r d i f f u s i o n method have been r e p o r t e d . Paper d i s k method h a s been adapted f o r t h e d e t e r m i n a t i o n o f str eptomycin by Loo e t a1 (507), Koelzer and Giesen ( 5 0 8 ) , R e i l l y and Sobe rs (509), and Kanazawa & Kuramata (510). Resazurin d i s c ha s been employed by Shahani and Badami (511). Waksman and R e i l l y (512) a p p l i e d Agar-str eak method €or a s s a y i n g st reptomycin and o t h e r a n t i The potency o f t h e a n t i b i o t i c substances: b i o t i c s p r e p a r a t i o n s can be determined by

STREPTOMYCIN

571

i n c o r p o r a t i n g graded d i l u t i o n s i n s t a n d a r d n u t r i e n t a g a r media i n p e t r i p l a t e s and s t r e a k i n g t h e agar surface with selected t e s t b a c t e r i a . After o v e r n i g h t i n c u b a t i o n , t h e potency is r e a d and expressed as t h e r e c i p r o c a l o f t h e h i g h e s t d i l u t i o n i n h i b i t i n g t h e d i f f e r e n t t e s t organisms. McGuire e t a 1 (513) h as suggested a new l i n e a r d i f f u s i o n method f o r m i c r o b i o l o g i c a l a ssa y o f s t r e p t o m y c i n . S l i d e - c e l l method (514) and modified d r o p - p l a t e method were a l s o r e p o r t e d . A comparative s t u d y o f cup, p a p e r - d isk , p u lp d i s k and s u p e r p o s i t i o n a s s a y mthods was r e p o r t e d by I s h i d a e t a1 ( 5 1 6 ) . Dewart e t a1 (517) and Light Brown e t a 1 (518) have d e s c r i b e d a d i f f u s i o n a s s a y f o r a n t i b i o t i c s by an automated procedure. The method i s as follows : Disposable p l a s t i c P e t r i d i s h e s loaded with a g a r p r e p a r e d f o r a s s a y are c a r r i e d a u t o m a t i c a l l y t o a d e v i c e t h a t punches h o l e s i n t h e a g a r , t h e n t o a d i s p e n s i n g u n i t t h a t d i s p e n s e s measured volumes ( e . g . , 50 o r 70 1.11) o f t e s t o r s t a n d a r d solution i n t o t h e holes; a f t e r incubation, t h e a r e a o f zones of i n h i b i t i o n a r e measured. Continuous au t o m a t i c m i c r o b i o l o g i c a l a s s a y o f a n t i b i o t i c s was r e p o r t e d by Shaw and Duncombe (519). The method was based on t h e measurement of r e s p i r a t o r y carbon d i o x i d e . Improvements o f Agar d i f f u s i o n method f o r a s s a y i n g s t r e p t o m y c i n were a l s o r e p o r t e d (520,521). B r i t i s h Pharmacopoeia 1958 (304) and Egyptian Pharmacopoeia 1953 (13) d e s c r i b e a m ic r o b io lo g i c a l Assay method f o r d e t e r m i n a t i o n o f s t r ep t o m y c i n . Assay Procedure : F i l l i n uniform t h i c k n e s s p e t r i - d i s h e s , o r r e c t a n g u l a r t r a y s , t o a d e p th o f 3 t o 4 mm. with a g a r c u l t u r e medium which h a s been p r e v i o u s l y inoculated with a s u i t a b l e quantity of a susp en s i o n o f s p o r e s o f a s u i t a b l e s t r a i n o f

BacLUuh h u b U .

572

JABER S. MOSSAETAL.

Allow t h e inoculated p l a t e s t o dry a t room temperature and keep a t 5OC., u n t i l used. Place on the s u r f a c e of the inoculated medium, small s t e r i l e cylinders of uniform s i z e approximately 10 mm high o r having an i n t e r n a l diameter of approximately 5 mm., made of g l a s s , porcelain o r aluminium, and keep a t about 150OC. Five, s i x , e i g h t c y l i n d e r s , o r any o t h e r numbers which can be conveniently adjusted t o t h e s i z e of t h e agar p l a t e , may be placed on each. Bore i n t h e medium, i n place of c y l i n d e r s , holes, 5 t o 8 mm i n diameter, by means of a s t e r i l e borer. Prepare i n s t e r i l e phosphate buffer s o l u t i o n , pH 8 . 0 , s o l u t i o n s of t h e standard preparation o f streptomycin, of known concentrations, as f o r example 0.5 t o 2 u n i t s per m l and s o l u t i o n s of t h e sample t o be t e s t e d presumed t o be o f t h e same order of concentration. F i l l i n t o t h e cylinders o r holes, t h e s o l u t i o n s of t h e s t a n dard preparation and of t h e sample t o be t e s t e d i n a l t e r n a t e order by means of a p i p e t t e which d e l i v e r s a standard amount o f s o l u t i o n s u f f i c i e n t almost t o f i l l t h e holes when they a r e used, Place t h e p l a t e s i n a r e f r i g e r a t o r f o r about 2 hours, and then incubate a t about 30° t o 37OC f o r about 16 hours. Measure, with t h e g r e a t e s t possible accuracy, t h e diameters of t h e i n h i b i t i o n zones produced by t h e varied concentrations of t h e standard preparation of Streptomycin and of t h e sample t o be t e s t e d , and from t h e r e s u l t s t h e potency of t h e l a t t e r is c a l c u l a t e d . L i m i t of Error: The limits of e r r o r (P=O.99) t h a t can be obtained with t h i s method are 79 and 1 2 1 per c e n t . , where 10 cylinders o r holes a r e used; 85Oand l l S O , where 20 cylinders o r holes a r e used; 89.50 and 110.50 where 40 c y l i n d e r s o r holes are used; and 92.50 and 107.50 where 80 cylinders o r holes a r e used f o r t h e standard preparation of streptomycin and f o r t h e sample t o be t e s t e d , r e s p e c t i v e l y .

STREPTOMYCIN

513

13.82 Liquid C u l t u r e Method 13.821 T u r b i d i m e t r i c Method Oswald and Knudsen (522) have r e p o r t e d a t u r b i d i m e t r i c method f o r a s s a y o f s t r e p tomycin. Two series of t u b e s , one w i t h graded amounts o f s t a n d a r d s t r e p t o m y c i n and o t h e r w i t h t h e unknown are i n o c u l a t e d w i t h t h e same amount o f a s t a n d a r d s u s pension o f t h e t e s t organism, i n c u b a t e d a t 37O f o r 4 h o u r s . The p e r c e n t a g e o f transmission read i n a photoelectric c o l o r i m e t e r . Potency of t h e sample under t e s t can be r e a d d i r e c t l y from t h e s t a n dard curve. A p p l i c a t i o n s of t h e t u r b i d i m e t r i c method € o r t h e a n a l y s i s of s t r e p t o m y c i n i n u r i n e , serum and c e r e b r o s p i n a l f l u i d were r e p o r t e d by Gibb (523) and Whitlock e t a l ( 5 2 4 ) . 13.822 Other Methods Bonifas and Chesni (525) have developed t h e d i l u t i o n method f o r t h e d e t e r m i n a t i o n o f s t r e p t o m y c i n : K ~ e b p bn m~o n ~ iae was allowed t o grow a t pH 9-9.5 i n t h e s y n t h e t i c medium of Monod t o which s u g a r and i n d i c a t o r c r e s o l r e d were added. The r e a c t i o n s h i f t e d towards t h e a c i d s i d e upon s p l i t t i n g t h e s u g a r . Decreasing amounts o f s t r e p t o m y c i n were added t o t h e medium. The i n h i b i t i n g amount o f s t r e p tomycin was determined by n o t i n g t h e d i l u t i o n i n t h e t u b e immediately p r e c e d i n g t h a t i n which t h e s e was t h e f i r s t change of color of t h e indicator. Dilution-method was extended t o determine s t r e p t o m y c i n i n u r i n e and body f l u i d s (526). A microbiological assay of a n t i b i o t i c s based on i n h i b i t i o n of ammonia p r o d u c t i o n was r e p o r t e d by Kobos (527). The t e s t s o l u t i o n was i n c u b a t e d f o r 2 hours a t 37O w i t h a s u s p e n s i o n o f E . cofi i n 6% peptone medium., t h e pH o f t h e m i x t u r e was

JABER S. MOSSA ETAL.

514

a d j u s t e d w i t h sodium hydroxide and t h e c o n c e n t r a t i o n of ammonia was determined w i t h u s e of an Orion 95-10 ammonia s e n s o r . 13.9 Biochemical Methods A radioimmuno a s s a y (528,529) was developed f o r d e t e r mination o f s t r e p t o m y c i n i n plasma and u r i n e . The method i n v o l v es enzyme-catalysed i n t r o d u c t i o n o f a l a b e l l e d group i n t o t h e molecule and s e p a r a t i o n o f t h e l a b e l l e d product by paper chromatography b e f o r e counting

.

Schwenzer and Anhalt (530) have r e p o r t e d an automated f l u o r e s c e n c e p o l a r i z a t i o n immunoassay f o r str e p to m y c in i n blood serum. I n t h e a s s a y d e s c r i b e d f l u o r e s c e i n l a b e l l e d streptomycin was used a s t h e tracer and a n t i serum a g a i n s t streptomycin was r a i s e d i n r a b b i t s . On mixing o f t r a c e r , sample and a n tib o d y , t h e p o l a r i z a t i o n o f t r a c e r f l u o r e s c e n c e was measured i n an Abbott TDX f l u o r i m e t e r . Acknwo 1edgement The a u t h o r s s i n c e r e l y thank Mr. A l t a f Hussain Naqvi and M r . Uday C. Sharm €or t h e i r s e c r e t a r i a l h e l p .

STREPTOMYCIN

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Reilly;

J . Bact.

52,

J . Bact;

223 (1946).

51,

STREPTOMYCIN

579

-

Venkataraman;

63.

K.R. Rao,, S . S . Rao and P.R. 23 (1346).

64.

G.A. LePage and E. Campbell; (1946).

65.

G.C. Ainsworth, A.M. 160, 263 (1947). -

66.

A.C.

Thaysen and M. M o r r i s ;

67.

H.B.

Woodruff;

68.

C.V. Hubbard and H.H. (1947); Biol. Abst.,

69.

H.H. T h o r n b e r r y and H.W. Anderson; Arch. Biochem; 389 ( 1 9 4 8 ) ; U.S. 2 , 621, 146 (1952).

70.

J.F.

71.

H.M. Eiser and W.D. McFarlane; (1948).

72.

56, 305 (1948). Eugene L. Dulaney., J. Bact; 271 (1949).

Spilsbury;

N a t u r e 158,

Bown and C. Brownlee;

J . Bact.,

162, 163

J. Biol Chem.,

Nature,

Nature, 1 5 9 , 100 (1947).

54,42

(1947).

T h o r n b e r r y ; Acad. S c . , 22933 (1947).

22,

T r a n s . Brit. Mycol. SOC.,

39, 57

31,

210 (1948).

Canad. J. Res.,

, Brit,

16,

26,

C.A.

164

, p3,

616, 331 (1949);

73.

A y e r s t McKenna and H a r r i s o n Ltd. C.A., 4 3 , 4434 (1949).

74.

Abraham L. Baron, B r i t . (1950).

75.

Paul C. T r u s s e l l ; U.S. 2 , 541, 726 ( 1 9 5 1 ) ; C . A . , 4006 (1951).

76.

L . E . McDaniel and David I-Ie!dlin.; U.S. 2 , 5 3 8 , 943 (1951); C . A . ; 45, 2637 (1951).

77.

Merck and Co, I n c . ; 9814 (1951).

78.

C h r i s t o p h e r J. J a c k s o n , P h i l l i p s D. Coppock and Brendan K . K e l l y . B r i t . j - 660, 203 (1951); C . A . 46. , 4610 (1952).

79.

Merdic E Co. I n c . , Rrit., 305 ( 1 9 5 3 ) , C . A . ,

80.

R . Raghunandana Rao; C u r r e n t S c i . , 4 8 . 4043 ( 1 9 5 4 ) .

,

Brit.,

6 3 9 , 8 6 3 ( 1 9 5 0 ) ; C.A.

44,

650, 087 (1951); C . A . ,

22,

10270

45,

45,

4 8 , 335 (1954).

203 ( 1 9 5 3 ) , C . A . ,

JABER S. MOSSA ETAL.

580

2,

81.

Robert D. Muir and Alden B. Hatch; U.S. (1954), C.A., 48, 11738 (1954).

82.

Kyoichi Kawasaki; Japan; 3750 (56) (1956); C.A. 11666 (1957).

83.

E . I . S u r i k o r a , G.F. Z a v i l e i s k a y a , N.T. Dygern and G.D. P e s t e r o v a ; A n t i b i o t i k i , 4, 12(1959) , C.A. , 53, 22744 (1959).

84.

V.A. S e v e r i n a ; S.V. Gorskaya and I . V . Gracheva., Doklady Akad. Nauk. SS.S.R. 126, 110311959)C.A. 54, 3590 (1960).

85.

Hiromi Nakayama, S h i r o S h i r a t s u c h i and S h i n i c h i r o Esumi; Japan 7969 (1958)., C.A.; 54, 3869 (1960).

86.

H i r o s h i ogawa, Yasuji Sekisawa, Masao ogasawara, S h i n i c h i Kondo and Masako Aoki; J a p a n , 9 , 0 9 9 (1961) C.A., 57, 2681 (1962).

87.

S h i r o S h i r a t o , H i r o s h i Motoyama and Hiroko Ueda; Hakko Kyokaishi, 2, 416 (1961); C . A . , 5 9 , 15919 (1963).

88.

Hideo Kubo, Hiromi Nakayama S h i r o S h i r a t s u c h i H i r o s h i Motoyama and Noriko Yanagita, Japan,11,598 (1960); C.A., 57, 13019 (1962).

89.

C.R. A d d i n a l l ; Drug Cosmetic Ind., C.A., 40, 1279 (1946).

90.

682, 493

,51

,

-

H e r b e r t S i l c o x ; Chem. Eng. N e w s , 252 (1947).

41,

57,

24,

53,

628, 710 (1945);

2762 (1946); C . A . ,

41,

91.

Richard W. P o r t e r ; Chem. Eng. 565 (1947).

92.

P h i l i p D. Coppock; U.S. 2 523, 245 (1950), C . A . , (1951).

93.

Hamao umezawa, Yasusuke o s a t o , Ryozo Utahar, Koki Y a g i s h i t a and Yoshiro okami; J. A n t i b i o t i c s 4 , 2 3 (1951), C.A., 45, 9223 (1951).

94.

Eugene L. Dulaney; U.S. __ 2, 571, 693 (1951); C.A. 694 (1952) *

95.

Hiromi Nakayama, Yonosuke Ikeda and Makoto Kawasaki, Repts. S c i . Research I n s t . , 28, 387 (1952). C.A. , 47, 8833 (1953).

94 (1946); C.A.,

4 5 , 715

,47,

STREPTOMYCIN

581

96.

Merck 4 Co; Inc.; Brit. 698, 113 (1953); C . A . , 4186 (1954).

97.

Idem.,

98.

A. Kramli, A. Kovacs, B. Matkovics, M. Natonek, G. Pulay and P. Turay; Acta Biol. Acad S c i . Hung., 5 , 79 (1954); C . A . , 49, 4938 (1955).

99.

A. Waheed Khan and I . D . Chughtai; P a k i s t a n J. S c i , 7, 15 (1955); C . A . 50, 531 (1956).

I b i d 667, 423 (1952); C.A.,

48 6082 -

48, -

(1954).

100. S.V. Trachuk and S.G. Chekaida, Med. Prom. SSSR 16, l l ( 1 9 6 2 ) ; C . A . , 57, 12639 (1962). 101. S h i r o S h i r a t o and H i r o s h i Motoyama; Hakko Kyokaishi; 19, 549 (1961); C.A., 5 9 , 15919 (1963). 102. Henrik Hedding, Dan. K e m i , 48, 85 (1967), C.A., 10736-.2e (1967).

67,

103. L. A. Voroshilova, E.S. Bylinkina and S.V. Gorskaya; Khim - Farm. Zh., 3, 41 (1969); C . A . , 71, 29266t (1969). 104. H.W. F l o r y , E . Chain, N.G. Heatly, M.A. J e n n i n g s A. G. Sanders; E.P. Abraham and M.E. Florey " A n t i b i o t i c s " Vol. 11, Oxford U n i v e r s i t y Press, London (1949). 105. F. C a r v a j a l ;

Mycologia,

38, 596

(1946).

106. T.J. Woodthrope and D.M. I r e l a n d . , J. Gen. Microbiol., -I , 344 (1948); C.A., 4 2 , 5944 (1948). 107. Merck Fr Co., I n c . , B r i t , 607, 186 (1948); C.A., 1158 (1949). 108. I d e m . ,

Ibid

625, -

820 (1949); C.A.,

44, -

43,

1235 (1950).

109. P h i l i p D. Coppock, L i l y L. Mulligan and Alan G. White; Brit, 622, 682 (1949); C.A. 44, 1656 (1950). 110. HarveyE. Alburn and Eric G. Snyder; U.S., (1950); C . A . , 44, 6087 (1950).

2 , 505, 318

111. Robert D. Babson and Max T i s h l e r ; U.S., 2 , 521, 770 (1950); C.A. , 44, 11041 (1950).

JABER S. MOSSA ETAL.

582

1 1 2 . Robert L. Peck, U.S. 2 , 540, 284 (1957); C.A. (1951).

43, 4894

113. Wm. A. B i t t e n b e n d e r and Robert D. Babson; U.S. 238 (1951); C.A., 45, 4414 (1951).

.

114. Frank J. Wolf; U.S., (195 1)

2, 538, 140 (1951); C.A.,

2,

540,

45, 3131

115. S.A. Waksman, J. H i s t . Med. A l l i e d S c i . , 6 , 318 (1951), C.A. 46, 1220 (1952). 116. David L. Johnson; U.S., 8970 (1953).

2 , 643, 997 (1953), C.A. 47,

117. Arne N. Wick and M i l t o n , J. Vander Brook; U.S. 701, 795 (1955); C . A . , 49, 6550 (1955).

, 2,

118. H.E. Carter; R.K. C l a r k , Jr.; S.R. Dickman, Y.H. Loo, P.S. S k e l l and W.A. S t r o n g ; J. B i o l . Chem; 160, 337 (1945) ; C.A. 40, 3485 (1946). 119. F.A. Kuehl, J r . , R.L. Peck, C.E. H o f f h i n e , Jr., R.P. Graber and K. F o l k e r s ; J. Am. Chem. SOC., 68, 1460 (1946); C.A.; 40, 7211 (1946). 120. M . J . Vander Brook, A.N. Wick, Wm. H. DeVries, R. Harris and G.F. C a r t l a n d ; J. B i o l . Chem., 165,463 (1946); C . A . , 41, 1276 (1947).

121. George P. Mueller; J. Am. Chem. SOC. 69; 195 (1947); C.A., 41, 3927 (1947).

1 2 2 . Herman Sokol and Robert P. Popino; U.S. 2, 653, 151 (1953); C.A., 48, 959 (1954). 123. Roy J. Taylor; U.S. 2 , 528, 188 (1950); C.A. (1951).

45, 2158

124. Eugene E. Howe and I r v i n g P u t t e r ; U.S. 2 ; 541, 420 (1951); C.A. 45; 8726 (1951). 125. C h a r l e s H. McBurney; U.S., 798 (1953). 126. Hideo Kubota;Japan,

2,

613, 200 (1952); C.A.

5248 (1954); C . A . ,

49,

,47,

13605 (1955).

127. Sumio Umezawa and T e t s u o Suami; Japan; 4296 (1954); C.A. 49, 10589 (1955).

583

STREPTOMYCIN

128. Kaneyuki Takagi; Japan; 5649 (1954); C . A . , (1955).

49,

14278

129. J o s e f Hoffman and J i r i n a Capkova; Czech, 84, 779 (1955); C.A. , 50, 10350 (1956). 130. J o s e f Hoffman, J i r i n a Capkova, Miroslav Hermansky and 50, 10351 Zdenek Severa; Czech; 85, 067 (1955); C.A. (1956). 131. O . B . F a r d i g and I . R . Hooper; U.S. 2 , 717, 892 (1955); C . A . , S J , 1272 (1956). 132. O.B. C.A.

Fardig and M.A.

Kaplan; U.S. 2 , 754, 295 (1956);

, S J , 15031 (1956).

2 , 765, 133. C.R. Bartels; B. Berk and W.L. Bryan ; U.S. 302 (1956); C . A . , 51, 3939 (1957). 18, 134. G.V. Samsonov and S.E. Bresler; Kolloid. Zhur., 337 (1956) , C . A . , 51, 799 (1957).

135. C.R. Bartels; G . Kleiman, D . R . I r i s h and J . N . Korzun, 51, 8385 (1957). U.S., 2, 786, 831 (1957), C.A. 136. Zbigniew Kotula, Acta Polon. Pharm. C.A. , 51, 14206 (1957).

, 2, 133

(1957);

137. J o s e f Hoffman. Miroslav Hermansky. Helena Parizkova and Jiri Doskocil; Chem. Tech. , 9 , l k l (1957); C.A. , 51, 17093 (1957). 138. C.R. B a r t e l s , W.L. Bryan and B. Bark; U.S. 779 (1959); C . A . , 53, 6542 (1959). 139. S . I . Kaplan, Med. Prom. S.S.S.R., 53, 11762 (1959).

12, 24

,2,

868,

(1958); C.A.

,

140. S.M. Mamiofe, E.M. S a v i t s k a y a , B.P. Burns, Z.T. S i n i t z y n a and N . N . S h e l l e n b e r g ; Med. Prom. S.S.S.R., 1 2 , 39 (1958); C . A . , 53, 11762 (1959). 141. V.A. Zhelobenko, F.M. Dinulov; V.M. V i s h n e v s k i l , P.S. Romanenko, V.N. Papchinski, G.T. Godunov, R.A. Myrsina, L.V. Fedorovskaya, I . T . Kondokova, I.S. Basenko and I.R. Grekova; U.S.S.R., 114, 385 (1958); C . A . , 53, 11771 (1959).

JABER S . MOSSA ETAL.

584

142. S.E. Bresler, A n t i b i o t i k i , 5 , 33 (1960), C.A., (1960)

.

54; 15840

143. G.V. Samsonov, Yu. B. B o l t a k s , N.P. Kuznetsova, A.P. Bashkovich and R.B. Ponomareva; K o l l o i d , Zhur. 21, 471 (1959). C . A . , 54, 7286 (1960). 144. S.I. Kaplan; Med. Prom. S.S.S.R., 54, 16739 (1960).

13,16

(1959).

C.A.

Samsonov and Yu. B. B o l t a k s ; Trudy Leningrad Khim Farm. I n s t . , 1,35 (1959); C.A., 55, 5085 (1961).

145. G.V.

146. J u s t i n J. Murtaugh and I s i d o r o C a l d a s ; Jr., 138 (1961); C.A. 55, 12782 (1961).

U.S.

-

2 , 970,

147. L.F. Yakhontova;E.M.Savitskaya and B.P. Bruns.; Khromat o g - ee T e o r i y a Primenen; Akad. Nauk. S.S.S.R.; Trudy Vsesoyuz. Scveshchaniya Moscow; 100 (1960). C.A., 55, 19135 (1961). 148. L.F. Yakhontova, B.P. Bruns; Yu. S. Chekulaeva, N.N. S h e l l e n b e r g , N.A. Vakulenko and S.N. Kovardykova., Med. Prom. S.S.S.R., 15,26 (1961); C.A. 55, 27776 (1961). 149. Wolfgang Sonnenkalb and Horst Henkel. Ger. ( E a s t ) ; 18, 098 (1960); C.A. 55, 26380 (1961). 150. L. F.Yakontova, B. P. Bruns, E. S . B y l i n k i n a , A.M.Zisserman,V.V. Matveev, A . B . Pashkov, E.M. S a v i t s k a y a , K.M. S a l d a d z e , M.A. Slobodnik, N.N. S h e l l e n b e r g , Yu. S. Chekulaeva and A.A. Khints, U.S.S.R., 139, 0 5 2 - ( 1 9 6 0 ) ; C.A. 5 6 , 6100 (1962). 151. Shigeo F u j i t a , H i r o s h i Kawabe and Masaya Y a n a g i t a ; Rikagaku Kenkyusho Hokoku; 36, 595 (1960); C.A. 5 6 , 10707 (1962). 152. L.E. Yakhontova, B.P. Bruns, Yu. S. Chekulaeva; N . N . S h e l l e n b e r g , N.A. Vakalenko and S.N. Kovardykova; Ionoobmen S o r b e n t y , v Prom. Akad. Nauk S.S.S.R., I n s t . F i z . Khim. 203 (1963); C . A . , 60, 5281 (1964). 153. S.G. Chekaida, R. Ya. Ladiev-and S.V. Trachuk, 152, 544 (1964); C . A . , 60, 16468 (1964).

U.S.S.R.,

154. All-Union S c i e n t i f i c - R e s e a r c h I n s t i t u t e of a n t i b i o t i c s ; Fr. 1. 402, 160 (1964). C.A., 63, 11263 (1964).

-

STREPTOMYCIN

585

155. Zbigniew Kotula and Teresa Czolowska; P o l . 49, 876 (1965); C.A., 65, 576 (1966). 156. E.S. V a i s b e r g , L . F . Yakhontova and B.P. Bruns, Zh. F i z . Khim., 40, 1884 (1966); C.A. 6 6 , 14337Q (1967). 157. Gheorghe Dragan, Mixcea Rusan and Mircea Toma; F r . 1, 444, 738 (1966). C . A . 66, 27706j (1967). 158. V.A. Ezhov and V . I . D i t y a n t s e v a ; Med. Prom. S.S.S.R., 207, 4.3 (1966). , C.A., 66, 31942a (1967). 3, 313, 693 (1967), C . A . , 159. S t a n l e y W. Ensminger, U.S. 52740u (1967)

.

67,

160. Ionescu, Sabin; J a l u b a M a r g a r e t a ; Danet , Rada; Ionescu L i g i a ; and Dumitrescu, Mihalache; Rom 9- 51, 161 (1968); C.A. 70, 66804s (1969).

-

3,

and D u r s c h F r i e d r i c h , U.S.. 161. Urs F. Nager 992 (1969); C . A . , 71, 59638 h (1969).

-

451,

1, 518, 916 (1968); 162. P f i z e r , Chas., and Go. I n c . F r . C.A., 6525n (1969).

71,

163. N . I . G e l ' p e r i n , L.M. Klyueva and L.L. S t r e m o v s k i i Khim.-Farm. Zh., 4, 23(1970)., L.A. 72, 1 3 6 4 6 3 ~(1970). 164. Kotula Zbigniew, Kunstharz - Ion e n a u s t a u s c h e r P l e n a r - D i s k u s s i o n s v o r t r . Symp. 415(1968); C . A . , 74, 45569 m (1971). 165. P e t e r P. Regna, I s a i a h A. Solomons, I11 and Richard P a s t e r n a c k ; U.S. 2 , 555, 762, 763 (1951), C . A . , 45, 7754 (1951). 166. Idem, i b i d . , U.S. 2 , 555, 760, 761 (1951); C.A. (1951). 167. Idem, I b i d , 2, 560, 890 ( 1 9 5 1 ) , C . A . ,

45,

45,7754

9228(1951).

168. Wm. Braker, Wm. A. Lett and Andrew E.O'keefe; 631, 143 (1953), C . A . , 47, 7742 (1953).

U.S.

, 2,

169. Leon J. Heuser, Morris A. D o l l i v e r and Eric T. S t i l l e r ; J. Am. Chem. SOC. , 75,4013 (1953); C.A., 48, 10624 (1954).

586

JABER S. MOSSA ETAL.

170. Wm. A. Lott, Jack Bernstein and Leon J. Heuser, U.S. 2, 669, 419 (1953), C . A . , 48, 3642 (1954). 171. Robert B. McCormack, Asger F. LagLykke and David Perlman; U.S. 2 , 656, 300 (1953), C.A., 48, 3643 (1954). 172. Hiroshi Ikeda,Hatsuko Ikeda, I t s u o Fujimaki, Kenji Shiroyanagi, Mitsuhiko Katayama and J u n i c h i Sugoyama, J. Sci. Research IASt., 48, 216 (1954); C.A. 4 9 , 4239(1955). 173. Sadajiro Yabuta and Hiroshi Ikeda; Japan 4297 (1954); C.A., 49, 10589 (1955). 174. David A. Johnson; U.S. 2 , 717, 893 (1955), C.A. 12407 (1956).

50,

175. Sadajiro Yabuta and Hiroshi Ikeda; Japan, 98 (1956); C.A. 51, 3938 (1957). 2 , 857, 376 (1958), C . A . 176. F r i t z Ziegler; U.S. (1959).

53,

3611

177. T e i j i r o Yabutta, Hiroshi Ikeda and J o Tanaka; Japan 8295 (1955), C.A. 5 1 , 18494 (1957). 178. V i r g i l V. Bogert; U.S. 2, 872, 442 (1959); C.A. 13518 (1959). 179. Leon J. Heuser; U.S. 2 , 2921, 062 (1960); C.A., 10245 (1960).

53,

-

54,

180. Minoru Tanaka; Hiroshi Yonehara, Takuro Soda and Yusuke Sumiki; J. A n t i b i o t i c s ; 4, 20(1951), C.A.,%, 1720 (1952). 181. Hiroshi Ikeda and Hatsuko Ikeda; J. Sci. Research I n s t . , 45, 161 (1951); C . A . , 46, 4739 (1952).

182. Walter A. Winsten; U.S. 2 , 609, 369 (1952), C . A . , 11670 (1953).

47,

183. E . J . Goett and R . J . Taylor; U.S. 2 , 676, 960 (1954), C.A., 4R, 9023 (1954). 184. Adilia Seixas Antao; Congr. Luso-Espan farm, 277 (1952) ; C . A . , 48, 13784 (1954). 185. Hiroshi Ikeda; Kenji Shiroyanagi, Hatsuko Ikeda, I t s u o Fujimaki, Mitsuhiko Katayama, Saichi Yamagiwa and Teruo Motokawa; J. Sci. Research I n s t . , 48, 2 1 1 (1954); C.A., 49, 4238 (1955).

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587

186. S.I. Kaplan; N.G. Semizhen, E.T. T u f f l i n , N.P. Krivosheev, A . I . Volakhanovich and V.S. Gin'ko, U.S.S.R., 117, 630 (1959), C . A . , 53, 17438 (1959). ~

187. Egon S. Andersen and Henrik Heding; Dan. C.A. , 67,1 4 8 3 3 ~(1967).

, 106,272 (1967);

188. Heding, Henrik, J o e r g e n s e n , J. H a r t v i g and Andersen Egon Steen; J. A n t i b i o t . , 21, 361 (1968); C . A . 70, 27601d [ 1969). 37, 470 189. Henry Fischbach; J. Am. Pharm. Assoc. S c i . Ed. (1948); C . A . , 43, 7195 (1949). 2 , 547, 231 (1951), C.A., 190. Jack D. S a r t a k o f f , U.S. 6352 (1951).

45,

191. Sumio Umezawa, Tsutomu Tsuchiya; T e s t s u r o Yamasaki, H i r o s h i Sano and Yoshikazu Takahashi; J. Am. Chem. SOC. 96, 920 (1974). 192. P.G. Sammes, "Topics i n A n t i b i o t i c Chemistry", Vol. I, E l l i s Horwood Limited, England (1977). 193. W.H. Horner, "Streptomycin", i n " A n t i b i o t i c II-Biosynt h e s i s " Ed. G o t t l i e b , D. and Shaw, P . D . , S p r i n g e r Verlag, N e w York (1967). 194. J . F . S n e l l ; IIBiosynthesis of a n t i b i o t i c s " , Vol. I , Academic Press, New York (1966). 195, D . J . J.,

Candy, N . L . Blumson and J. Baddiley; 31 (1964).

Biochem.

91,

196. D.J. Candy and J. Baddiley; Biochem. J . , 63, 7248 (1965). (1965), C.A. 197, M. Silverman and S.V. Rieder; (1965). 198. J . Burton, W.H. Horner and G.A. L42, 813 (1967). 199. G.D. Hunter and D.J.D. 249 (1955).

J . Biol.

96,

526

Chem. 235, 1251

Russ; J. B i o l . Chem. ,

Hockenhull;

Bio Chem. J . ; 59,

200. R.M. Bruce, H.S. Raghib and H. Weiner; phys. Acta., 158,499 (1968).

Biochem. Bio-

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588

201. W. H. Horner and G . A . 237, 123 (1971). -

Russ;

Biochem. Biophys. Acta;

202. M.H.G. Munro, M. Taniguchi, K . L . R i n e h a r t , J r . , D. G o t t l i e b , T.H. S t o u d t and T.O. Rogers; J. Am. Chem. SOC., 97, 4782 (1975). 203. W.H. Horner; 5777 (lY64).

J . B i o l . Chem.,

239,

578 (1964); C . A . ,

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204. K.L. R i n e h a r t , J r . , and T. Suami; "Aminocycletol A n t i b i o t i c s " , American Chem. SOC., Washington (1980). 2 0 5 . H. G r i s e b a c h ; (19 78)

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Adv. Carbohydr. Chem. Biochem.,

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34, 8 1 -

206. B. Kniep and H. G r i s e b x h ;

J. Antibiot.

207. S. Maier and H. Grisebach; 586, 231 (1979). -

Biochem. Biophys. Acta,

416(1980).

209. S.A. Waksman and H. C h r i s t i n e K e i l l y ; J . i n f e c t i o u s Diseases; 75, 150 (1944) , C.A. , 39, 2089 (1945).

210. W.H. Feldman and H.C. Hinshaw; Proc. S t a f f Meeting Mayo 39, 4677 (1945). C l i n i c , 2, 593 (1944), C . A . , -

211. M.I. Smith, and W . ' l . McClosky, U.S. Pub. H e a l t h R e p o r t s , 60, 1129 (1945); C.A. , 39, 359 (1945). 212. A. Schatz and S.A. Waksman. , Proc. SOC. E x p t l . B i o l . Med., 57, 244 (1944); C.A. , 39, 1193 (1945). 213. Guy P. Youmans and John C. McCarter; Am. Rev. Tuberc., 5 2 , 432 (1945). C.A. 40, 1226 (1946). 214. J . D . C.A;

Wassersug; N e w Engl. J. Med.;

40, 5832 (1946).

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Shaw and K.E. Duncombe; A n a l y s t 8 , 694(1963); 15595 (1963).

59,

520. N.D. I e r u s a l i m s k i i , I . V . Konova and N.M. Neronova; Mikrobiologiya, 28, 433 (1959); Anal. Abst., 8, 1729 (1961), C . A . , 5 3 7 2 2 2 5 2 (1959). 521. M.A. El-Nakeep and R.T. Yousef; Arzneim-Forsch, 20, 103 (1970); C . A . , 72, 829Y7d (1970).

522. E l i z a b e t h J. Oswald and L i l a F. Knudsen; J. Am. Pharm. ASSOC., 39, 61 (1950); C . A . , 44, 4200 (1950). 523. Mary C. Gibb; Am. J. Med. Technol., 17, 14 (1951), C.A., 45,9610 (1951). 524. C.M. Whitlock, J r . , A.D. Hunt, J r . , and S.G. Tashman; J . Lab. C l i n . Med., 37, 155 (1951); C.A., 45, 5755(1951). 525. V. Bonifas and Y. Chesni; E x p e r i e n t i a , 4 , 355(1948); C.A., 4 3 , 1825 (1949). 526. Georg Hennberg and Wolfgang Waterstraat; Z e n t r . Bakt. P a r a s i t e n k I Abt. O r i g . , 258(1950); C . A . , 4 5 , 3448 [ 1951).

-.,

527. D. L. Simpson, R.K. Kobos; Anal. Chin!. Acta, 164, 273 c (1984); Anal. Abst., 47, 9E25 (1985). 528. Arden W. Forrey, Andrew D. Blair, Michelle A. O ’ N e i l and Ralph E - Cutlex;. Methodol. Dev. Biochem., 5, 235 (1976); Anal. Abst., 34, 1D49 (1978).

STREPTOMYCIN

609

529. T. Ya Goncharskaya and V.G. Koroleva; A n t i b i o t i k i , 24, 859 (1979); Anal. Abst., 38, 6 D 68 (1980). 530. K.S. Schwenzer and J . P . Anhalt; Antimicrob Agents Chemother, 23, 683 (1983) ; Anal. Abst. 46, 5D54 (1984).

THIABENDAZOLE

Vijay X. Kapoor 1.

Description 1.1 Name, Formula and Molecular Weight 1.2 Appearance, Color, Odor 1.3 Salts

2.

Physical Properties 2.1 Infrared Spectrum 2.2 Ultraviolet Spectrum 2.3 Optical Rotation 2.4 Melting Range 2.5 Solubility 2.6 Crystal Properties

3.

Synthesis

4.

Stability and Degradation

5.

Metabolism and Phannacokinetics

6.

Methods of Analysis 6.1 Elemental Analysis 6.2 Identification Color Test 6.3 Titrimetric Analysis 6.4 Bioassay 6.5 Polarographic Analysis 6.6 Colorimetric Analysis 6.7 Spectrophotometric Analysis 6.71 Ultraviolet 6.72 Infrared 6.8 Spectrofluorometric Analysis 6.9 Chromatographic Analysis 6.91 Paper Chromatography 6.92 Thin-Layer Chromatography 6.93 Gas Chromatography 6.94 High Performance Liquid Chromatography 6.10 Miscellaneous

7.

Limits of Tolerance

8.

References

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

61 1

Copyright 0 1987 by Academic Press, Inc. AU riehts of reproductionin any form reserved.

VIJAY K.KAPOOR

612

1

Description 1.1 Name, Formula and Molecular Weight Thiabendazole, an anthelmintic and antifungal agent, was developed by scientists of Merck laboratories1 in 1961. Thiabendazole is 2-(4-thiazolyl)-1H_-benzimidazole. The CAS Registry number is 148-79-8. The most common proprietory name is Mintezol

.

Molecular Weight: 201-25 10H7N'3 1.2 Appearance, Color, Odor White to cream-colored, odorless or almost odorless powder. 1.3 Salts Thiabendazole is a weak base. The hydrochloride (sublimed at 265O) and sulfate (melted at 262-6O) have been prepared.2 Watersoluble lactate of thiabendazole has also been prepared along with the citrate, fumarate and salicylate which had limited solubility in water. Physical Properties 2.1 Infrared Spectrum The infrared spectrum4 (KBr disc) of thiabendazole is presented in Figure 1. The principal peaks are at 1408, 906 and 74OCm-1. 2.2 Ultraviolet Spectrum The light absorption, in the range 230 to 350 tun, of a 2-cm layer of a 0.0004 per cent w/v solution in 0.1N hydrochloric acid exhibits two maxima, at 243 ?h~ and at 302 nm; absorbance at 243 nm, about 0.47 and at 302 nm, about 0.98.5 2.

THIABENDAZOLE

613

Wavelength microns 6 7 8 9

10 11 12 131415

90

80 70

60 50 40 30

20 l10 o I 2000

I

1800

I

I

I

1600

1400

1200

I

1000

I I 800 650

Wavenum ber

Figure 1. Infrared Spectrum of Thiabendazole 2.3 Optical Rotation Thiabendazole exhibits no optical activity. 2.4 Melting Range The melting range6 of thiabendazole is between 296O and 303O. 2.5 Solubility The solubility7 of thiabendazole at ordinary room temperature is as follows: Solvent Water Alcohol

Chlorofonn

Ether Acetone

Dilute mineral acids

Solubility, mg/ml practically insoluble 6.66

3.33 0.5 slightly soluble soluble

VIJAY K.KAPOOR

614

2.6

Crystal Properties Crystal structure of thiabendazole is described.* Crystals of thiabendazole are orthorhombic, space group p s, with a = 17.052(7), b = 10.998(4) , and = 10.030?8) A. There are erght formula units per cell; observed -3 and calculated densities are 1.414 and 1.421 g cm Intensity data were collected on an automatic diffractometert the structural parameters were refined by full-matrix least-squares to an index of 0,066 f o r 1805 reflections, The two ring systems are approximately planar, but are twisted by loo with respect to each other. The C-C bond connecting the two ring systems is 1.442(10) %, long. Molecules are linked together by N ( 1 ) H(l)---N(14) hydrogen bonds to form chains parallel to the axis, X-ray powder diffraction data for thiabendazole is given in Table 1.9 The data were obtained by diffractometer and Debye-Scherrer camera techniques, and are tabulated in terms of the lattice spacings and the relative intensities of the line. Patterns using three different X-ray wavelengths w i t h the camera method are given.

.

3.

Synthesis Brown et a1.l from Merck laboratories first reported the synthesis (Scheme I) of thiabendazole by the reaction of 4-thiazolecarboxamide with O-phenylenediamine in polyphos horic acid at 250° for three hours. In a patent18 the synthesis of thiabendazole is reported through another route (Scheme 11). O-Nitroaniline was condensed with lactic acid or-the acid halide to give g-lactoyl2-nitroacetanilide which on oxidation followed by bromination gave ~-(bromopyruvoyl)-2-nitroanilide. The latter on refluxing overnight with thiofomamide hydrate followed by zinc dust reduction gave thiabendazole. Alternate methods of synthesis havs also been described.11812 Synthesis of 14C- or 3 S-labeled thiabendazole i s also described.13

-

Table 1 X-Ray Diffraction Data f o r Thiabendazole Diffractometer CuKa d (ft)

1/11

6.81 5.64 5056

8 4 7 43*

5 007"

-

4r23* 3.72 3 39* 3027

2,814 20695 2,525 2 327

-

-

-14

loo*

43* 2

-10

-33 --

2

Camera

corn

CuKa d (1)

-

6.76 5057 5oO3* 4021~ 4.02

3.71 3 0 38* 3026 3011 2 0803 2.681 20629 2,517 2.419 2.321 2,253 20137 2,055 20022

2,062 2 1 20025 *Most intense lines

1/11 17 I

18 68* loo* 3 26 51* 5 3 19 6 3 12 1

9 4 5 7 4

CrKa.

d
1/11

d (A)

1/11

5.57

-17

6076

-

-1113

6.76

5 04"

4.21* 4001 3071 3 38* 3.26 3010 20806 2 0 685 20630 2.515 20418 2.316 2.252 2.132 2.056 2 0 000

14 75* loof 5 27 5 1*

7 4

5057

5oO3* 4021* (c.

3.70 3 0 38* 3025 I

--

21

2.808

12 17 21 5 4

2.519

7 7

7 5

-

20311

63*

-

loo*

20 43*

7

I

-.14.

8

I

7

VIJAY K.KAPOOR

616

$7

PolYphosphoric acid

NHpCO

N

,

250°/ 3 hr

Scheme I. Synthesis of thiabcndazole

+

UNo2

CHaCHOHCOR

N-H

R =OH, C I

I I H COH I c=o

CH3

aNo2 N -H

I c=o I

c =o

I

0r2/0O0

CHI Br

HC

II

Zn/HCl

NH-C

Scheme

N

n. Alternate

N-H

I

c=o

I I

c=o CHI

'NH2

aNo2 iiya

1

synthetic pathway t o thiabendazole

THIABENDAZOLE

617

4.

Stability and Degradation In general thiabendazole is a stable compound. The USP XXI NF XVI directs that it should be packed and stored in well-closed containers.6 Thiabendazole, however, has been reported14-17 to be slightly uv (sunlight) sensitive. Photolytic degradation products of thiabendazole have been identified as benzimidazole, benzimidazole-2carboxamide, triazole-4-carboxamide, thiazol-4ylamidine and methyl thiazol-4-carboxylate.14.16 A recent study involving photooxidation of thiabendazole in methanol in presence and absence of a photosensitizer, methyleneblue also identified dimethyl oxalate, thiazole-4-(pcarbomethoxy)carboxamlde, methyl benzimidazole-2-carboxylate, benzimidazole-2-carboxamide and benzimidazole as the main products of photolysis.19 Figure 2 gives the structures of the main degradative products,

Benzi midosole

Benzimidozole-2-corboxomide

CH3OOCNHCO

Methyl benrim~dozole- 2 -corboxylote

1hiozote -4

c;sa

- f N-car bomethoxy) - carboxamide

Figure 2. Moin products of photolysis of thiabendazole

A recent review discusses the different breakdown products due to environmental factors of various fungicides including thiabendazole 2o 5, Metabolism and Pharmacokinetics Thiabendazole is metabolized chiefly to 5hydroxythiabendazole which is excreted in the urine conjugated either B S glucuronide or as t h e sulfate (Figure 3) . I 3 Thiabendazole is rapidly absorbed from the gastrointestinal tract and peak plasma levels of 13 to 18 pg/ml are found within an hour of ingestion. The drug and its metabolites are excreted rapidly, 87% in the urine and 5% in

.

VIJAY K.KAPOOR

618

Thiabendazole

5 -Hydroxythiabendazole

Hp

mN Sulfate conjugate

N

Glucuronide conjugate

Figure 3 . Metabolism o f thiabendazole

the faeces within 48 hours. 21 small amounts of both unchanged thiabendazole and free 5-hydroxythiabendazole may be found in both the plasma and urine. Gastrointestinal absorption and secretion of thiabendazole has been studied.22 Wilson & a1.23 have reported that 5-hydroxylation of thiabendazole in rat liver microsomal preparation is dependent on cytochrome P-450 and has a Km of 3.92 x 10-431 and Vmax of 0.484pole of 5hydroxythiabendazole produced/hr/mg of protein. 5-Hydroxythiabendazole gave a type If binding spectrum w i t h cytochrome P-450, Desmethylimipramine and ethoxyquin are reported to inhibit the hepatic microsomal hydroxylation of thiabendazole.24 A single o r a l dose of desmethylimipramine (80 mg/kg) administered to rats inhibited the hydroxylation of thiabendazole by 45% in vitro at 5 hr after dosage but did not decrease cytochrome P-450. A single oral dose of ethoxyquin (200 mg/kg) to rats inhibited the hydroxylation by 65% in vitro at 1 hr after dosage, inhibition was less at 5 hr. Oral ethoxyquin (400 mg/kg) or desmethylimipramine (80 rng/kg) administered 30 minutes before oral thiabendazole (100 and 200 mg/kg, respectively) delayed absorption of thiabendazole and resulted in its decreased plasma concentrations. Bauer

-

THIABENDAZOLE

619

et a1.25

have studied the phamacokinetics of thiabendazole and its metabolites in an anephric patient undergoing hernodialysis and hemoperfusion. The half-life, volume of distribution, and clearance of thiabendazole were 1.17 hr, 2.76 l/kg, and 27.2 ml/rnin/kg, respectively. While thiabendazole and the 5-hydro= metabolite did not accumulate during multiple dosing, the glucuronide and sulfate conjugates accumulated extensively despite hemodialysis and hemoperfusion. Pharmacokinetics of thiabendazole has been studied in cattle,268 27 sheep,28 and avian29 species. The findings are consistent with the literature data that metabolism and excretion of thiabendazole are more extensive in cattle than in sheep. Methods of Analysis 6.1 Elemental Analysis The elemental analysis of thiabendazole is as follows: 11 E 1ement % Calcd. % Found C 59.68 59074 H 3.51 3.62 N 20.88 20.57 S 15.93 15.99 6.2 Identification Color Test The following color reaction can be used to identify a sample of thiabendazole.586 About 5 mg of the sample are dissolved in 5 ml of 0.12 hydrochloric acid and 3 mg of E-phenylenedimine dihydrochloride are added f01 lowed by shaking to dissolve the contents. About 0.1 g of zinc dust is then mixed and the mixture allowed to stand f o r 2 minutes. 5 ml of a solution prepared by dissolving 20 g of ferric ammonium sulphate in 7 5 rnl of water, and 10 ml of 1E sulphuric acid are then added. When diluted with water to 100 ml a blue or blue-violet color is developed6.3 Titrimetric Analysis The titration with perchloric acid is the method of choice to assay thiabendazole.586 6.

620

VIJAY K.KAPOOR

The sample is dissolved i n glsclal acetic acid. After speciEic quantities of acetic snhydride, mercuric aceta%e and two drops of c r y s t a l violet soluticn are added, the titration with 0.1E perchloric acid to a hlue-green end point is carried out. A blank determination is performed and any necessary correction is made. Each r n l of 0.1N perchloric acid is equivalent to 20.13 mg of CIOH7N3S. A titration method utilizing the complex formed by thiabendazole with silver Ions is also described. 30 6.4 Bioassay Several microbiological assay methods using sensitive fungi for the quantitative determination of thiabendazole have been reported. 31-38 These methods have been employed f o r determining the residue of the fungicide on plant materials or in soil. A simple and rapid bioassay €or the direct determination of thiabendazole residue in soil involves placing pellets composed of mixtures of soil (200-500 my> and agar on an agar medium preinoculated with the test organism Penicillium digitatum.37 After cold pre-incubation followed by incubatlon at 2 7 O , the size of the inhibition zone is measured (Figure 4 ) . The lowest de%ectable concentration of thiabendazole in a sand soil was In another method 5 extracts found to be 1.0 ) i g / g . from citrus fruit and bananas were applied as spots to silica gel in P e t r i dishes which were then uniformly inoculated with Penicillium cyclopium spores. Fungicide concentration was detennined from comparison of inhibition zones with those of a standard 48-hr incubation. Detection threshold was 0.5 p g thiabendazole. Other microorganisms employed in the bioassa studies are Penicillium exansum,32 Graphium ulmf3 and Sporobolomyces roseus.38 6.5 Polaroqraphic Analysis A polarographic method for determining thiabendazole is described. 39 The electrochemical behavior of thfabendazole was investigated by sampled doc. polarography and differential pulse polarography. Thiabendazole showed one wave peak in the polarogram when a short drop time (0.4 s ) was used. Detection was most sensitive at pH 8 .

Y

THIABENDAZOLE c

“E 250 c

Y

.-2 .- 200

621

t

C 0

c

.-C

2 4 6 8 10 12 Thiabendazole concentration in soil ( p g / g )

Figure 4. Effect of thiabendazole concentration in-soil on growth inhibition of Penicfllium di-itatum using the soil-agar pellet technique. inhibition is expressed a s the square Grow

+

The current measured was proportional to the concentration. The detection limit was 0.5 ppm. Differential pulse polarography has been used for quantitative determination of thiabendazole In citrus fruit peels. 6.6 Colarimetric Analysis Thiabendazole can be analyzed colorimetrically in feeds.40-43 The method involves: (a) complete extraction of thiabendazole from Eeed w i ’ 3 1 C.2N H C 1 in SO% methanol; (h) separation from other suEstences by ext.raction with chlorof o m from a l k a l i n e solution; (c) further purif ication by reextraction into dil.vte H C l ; ( d ) reduction with

622

VIJAY K. KAPOOR

zinc dust in the presence of pphenylenediamine to form a complex; (el subsequent oxidation with a ferric solution to form a thiazine dye; and (f) extraction of the dye into butanol and measurement at 605 nm against reference standard thiabendazole. The method gives most accurate and reproducible he method described above was automated results. by !~;hite.~~ In the automated system the solution was reduced with 0.3% zinc dust in glycerol, and the products were coupled with 0.17% pphenylenediamine in 2N H2SO4 and 15% Fe NH4 (SOg)2 in 0.1g H2SO4. The glue complex was measured at 610 nm. The relative standard deviation was 1.2%, and the recovery was 95-100% from feed containing 1% thiabendazole. A modified method was used to determine the residues of thiabendazole in citrus fruits-45 A method for estimating thiabendazole in the milligram and submilligram ranges (0.1-2.0 mg/ml) has been descrlbed which involves measuring of absorbance of a copper-thiabendazole-tetramethylguanidine complex at 350-400 nm.46 A modified method in which absorbance of the complex was measured at 311 nm permitted estimation of 2 pg/ml thiabendazole. An improved procedure involves shaking an alkaline aqueous suspension containing thiabendazole with solution of cupric acetate and 1-dimethylamino-2-propanol in chloroform to produce a green color in the chloroform phase.47 The analysis is completed by spectrophotometric measurement. Thiabendazole in concentration range of 0.1-1.0 mg/ml can be estimated by this method. A simple colorimetric method involving the reaction of thiabendazole in ch1.oroform with alkaline buffered bromocresol green is also 6escribed.48 6.7 Spectrophotometric Analysis 6.71 Ultraviolet Thiabendazole is estimated in various fruits as a residual fungicide by ultraviolet spectrophotometry.49-60 The method in general involves extraction with ethyl acetate-ammonium hydroxide, purification by acid-base procedure, and measuring the absorbance of the acid solution at 302-3 nm. In another purification procedure the

THIABENDAZOLE

623

f r u i t p e e l s o r p u l p s are extracted with chloroEorm, t h e e x t r a c t i s a c i d i f i e d and c h l o r o f o m evaporated. The a c i d i c c o n c e n t r a t e i s cleaned up with chloroform, and t h i a b e n d a z o l e e x t r a c t e d a t pH 9.5. Using t h i s method t h i a b e n d a z o l e w a s determined spectrophotom e t r i c a l l y a t 302-3 nm a t c o n c e n t r a t i o n s of 0.1 pprn i n 100 g peel or pulp, o r 0.03 ppm i n whole c i t r u s f r u i t . Aspect of p r o t e i n i n t e r f e r e n c e i n t h i a b e n d a z o l e d e t e r m i n a t i o n i s discussed.61 6.72 Infrared Thiabendazole can a l s o be analyzed by i n f r a r e d spectrophotometry.62 The method i n v o l v e s d i s s o l v i n g 0.12 g of t h i a b e n d a z o l e sample i n 5 m l of l?,~-dimethylformamide and adding 1 g of anhydrous sodium s u l p h a t e . A p o r t i o n of t h e s u p e r n a t a n t i s used for i n f r a r e d spectrophotometry, Absorbance a t scanning between 2.5 and 16.7 )un. 11.09 )xn is used f o r q u a n t i t a t i o n . The s t a t i s t i c a l a n a l y s i s of m u l t i p l e d e t e r m i n a t i o n by t h i s method gave a r e l a t i v e e r r o r o f \ ( 2 % and a c o e f f i c i e n t of v a r i a t i o n 4 1%. 6.8 Spectrofluorometric A n a l y s i s Thiabendazole is a n a l zed by spectroThe f u n g i c i d e f l u o r o m e t r i c method also.54,63-%8 can be detected i n c r o p s by acetone e x t r a c t i o n followed by p a r t i t i o n i n g i n acetone-water and e t h y l a c e t a t e and clean-up on magnesium oxidec e l i t e - a l u n f n i u m oxide column. The cornpound i s t h e n q u a n t i t a t i v e l y measured by d i r e c t fluorometry. The e x c i t a t i o n and emission wavelengths are 305 and 345 nm, r e s p e c t i v e l y f o r thiabendazole. T h e method i s r e p o r t e d t o be s e n s i t i v e t o 0.02 ppm thl sbendazole. 63 An automated f l u o r o m e t r i c method f o r t h e d e t e r m i n a t i o n of t h i a b e n d a z o l e i s a l s o d e s c r i b e d which gives t h e minirnun l e v e l of d e t e c t i o n of t h e f u n g i c i d e asw0.05 pg/ml.6* A s e n s i t i v e f l u o r o m e t r i c method f o r d e t e r m i n a t i o n of t h i a z o l e c o n t a i n i n g compound i s r e c e n t l y described. 68

6.9

described.

Chromatographic A n a l y s i s 6.91 Paper Chromatoqraphy The f o l l o w i n g system h a s been 4

VIJAY K.KAPOOR

624

Paper:

Solvent: Equilibration: Development: Location:

Rft (l:6)

Khatrnan Noel, sheet 1 4 x 6 i n , buffered by dipping i n a 5% s o l u t i o n of sodium d ihydr ogen c i tret e , b l o t t i n g , and drying a t 2 5 O for 1 hour. 4.8 CJ of c i t r i c a c i d in a mixture of 330 m l of water and 870 ml of r p b u t a n o l . None Ascending. Time of run, 5 hours a. Examination under ultraviolet light b. Bromocresol green spray C. Fotassium permanganate spray 0.06

.

A n a l t e r n a t e system employs acetone-water i n ascending mode which g i v e s Rf v a l u e of

Location is done by t h i a b e n d a z o l e as 0.49.69 allowing t h e paper t o react with i o d i n e vapor for 1 minute. 6.92 Thin-Layer Chromatography A v a r i e t y of t h i n - l a y e r chromatog r a p h i c s y s t e m s u s i n g s i l i c a g e l or si"l1ca g e l GF254 p l a t e s f o r i d e n t i f i c a t i o n and e v a l u a t i o n of thiabenda z o l e have been r e p o r t e d . These are t a b u l a t e d below: S o l v e n t System Reference Benzene-acetic acid-acetone-water 70 (10:4: 1:0.4) 71,72 Benzene-acetic acid-acetone-water (70:20: 10: 2) Ethyl a c e t a t e - e t h y l methyl ketone65,67 formic acid-water (50: 30: 10: 10) Ethyl acetate-benzene (50:50) D i e t h y l e t h e r - g l a c i a l acetic acidmethanol (100:5:2)

74

Acetone

74

73

THIABENDAZOLE

625

Solvent. System

Reference

L i g h t petroleum (60-80°)-acetone (30:lO) Chloroform-acetone (80: 20)

74 75

Using aluminium o x i d e F254 n e u t r a l p l a t e 74 d i e t h y l ether-methanol i s t h e s y s tern employed. Cole e t h a s g i v e n t h e Rf v a l u e s of t h i a b e n d a z x e i n d i f f e r e n t s o l v e n t systems. These a r e g i v e n below: R f Value x 100 S o l v e n t System E t h y l acetate

55

E t h y l a c e t a t e s a t u r a t e d wit.h ammonia E t h y l acetate-toluene-anunonia (60:40:2) E t h y l acetate-toluene-ammonia (40:60: 2) E t h y l acetate-toluene-anunonia (20:80:2) Toluene-ethanol-ammonia (80:20:2) Toluene-ethyl a c e t a t e - e t h a n o l ammonia ( 60 :10 :30 :2 1 To 1uene- e t k i y 1 ace t a t e- et h a n o 1 ammonia (70: 15: 15:2) Toluene-ethyl a c e t a t e - e t h a n o l arnmonia (60:10:10:2)

60

-

41 31 21

43 58 52 38

D e t e c t i o n of s p o t of t h i a b e n d a z o l e on t h e p l a t e h a s been c a r r i e d o u t by d i f f e r e n t methods such as v i s u a l i z a t i o n under u l t r a v i o l e t l i g h t , 7 4 8 7 6 # 7 7 by s p r a y i n g with potassium iodobismuthate solution followed by exposure t o bromine vaposs74 o r by exposure t o i o d i n e vapors. 76 A b i o l o g i c a l method f o r d e t e c t i o n and e v a l u a t i o n of t h i a b e n d a z o l e h a s a l s o been d e s c r i b e d O 7 3 I t i s done by s p r a y i n g t h e p l a t e s with spores of any of t h e t h i r t y t w o fungal. species f o l l o w e d b y i n c u b a t i o n . G u a n t i t a t i v e relations were e s t a b l i s h e d b e t w e e n spot s u r f a c e a r e a and t h e amount of f u n g i c i d e . A n enzyme i n h i b i t i o n

VIJAY K.KAPOOR

626

75 technique i s a l s o reported. using t h i n - 1aye r chsornatoy r aphy want i t a t i v e determination of thiabendazole has been c a r r i e d oiit s p e c t r o p h o t o m e t r i c a l l 70,71,72,78,79 or fluorometrically65,67,8~,~1. Otteneder and FIezel65 have r e p o r t e d a method by which thiabenda z o l e can be Getermined d i r e c t l y on t h e chromatogram by r e f l e c t a n c e - a b s o r p t i o n photometry with measurement at 302 nrn o r f l u o r o m e t r i c a l l y a t 355 nm, with e x c i t a t i o n a t 313 nm. Compared with t h e e l u t i o n and subsequent photometry, direct e v a l u a t i o n on t h e chromatogram h a s t h e advantage t h a t it saves t i m e and r e q u i r e s less substance p e r spot. 6.93 G a s Chroniatoqraphy Gas chromatographic methods have been used t o determine r e s i d u e of thiabenda,o1e050,53,67,82-94 An e a r l i e r method t o determine t h e f u n g i c i d e i n whole c i t r u s f r u i t and i n c i t r u s and bansna pulp is d e s c r i b e d by Mestres e t al.50 Samples were made a l k a l i n e with ammonia s o l u t i o n and macerated w i t h e t h y l a c e t a t e and filtered. T h e f i l t r a t e was cleaned up by e x t r a c t i o n i n t o 0.1g HC1, and making t h e aqueous phase a l k a l i n e and r e e x t r a c t i n g back i n t o e t h y l a c e t a t e . The concentrated e x t r a c t was chromatographed on a lo”/, DC-200 s t a t i o n a r y phase using FPD set i n t h e mode f o r determining s u l f u r . The l i m i t o f s 3 d e t e c t i o n was of t h e o r d e r of 0.1 pj. Hey used SE-30 a s s t a t i o n a r y phase with a n i t r o g e n - s e n s i t i v e Tanaka and Fujimoto83 determined detection thiabendazole a s i t s methyl d e r i v a t i v e w i t h flame i o n i z a t i o n d e t e c t i o n and a column of lo”/, Dc-200 on Gas-Chrom Q a t 240O. It p e r m i t s d e t e m l n a t i o n i n c o n c e n t r a t i o n s down t o 0,l ppm, Thiabendazole w a s determined by Nose e t al. 8 4 a s g-pentafluorobenzoyl d e r i v a t i v e by e l e c t r o n c a p t u r e g a s chromatography following r e a c t i o n of thiabendazole with pentafluorobenzoyl c h l o r i d e . The m i n i m u m amount of thiabendazole detectable by t h i s method was 0.01 ppm. P r i n c i p a l o p e r a t i n g c o n d i t i o n s were: 1.5 m x 3 mm I.D. g l a s s column packed with 5% OV-101 on Gas Chrom G (HP) (80-100 mesh), i n j e c t i o n p o r t and d e t e c t o r temperature 270°, column temperature 230O. Nitrogen was used as a c a r r i e r g a s a t a flow-rate of

.

THIABENDAZOLE

627

40 ml/min.

Combined gas chromatography-mass spectrometry was operated with column temperature 2 3 0 ° , separator temperature 260°, ion source temperature 290° and flash heater temperature 270'. The carrier gas was helium at a flow-rate of 30 ml/min. Recently, Bardalaye and Wheeler85 employed this technique to determine thiabendazole in yams with operating conditions as: 1.83 m x 4 mm I . D . glass column packed with 5% OV-17 on 80-100 mesh Gas-Chrom 0, argon-methane carrier gas (95 + 5) at a flow-rate of 60 ml/min. Temperatures of oven, detector and injectors used were 240 300 and 200°, respectively. Tjan and Jansen66 have used pentafluorobenzoyl bromide f o r derivatization of thiabendazole. A combined gas Chromatography mass spectrometry confirniatory assay for thiabendazole and 5-hydroxythiabendazole at 0.1 p m in animal tissue isolates has been developed. 5 On-column methylation converts these compounds to their Emethyl and E,g-dimethyl derivatives, respectively. Identification and quantitation are achjeved by selective ion monitoring of M-1, M and Etl ions from E-methylthiabendazole and the I4 and M-15 i o n s from IJ,g-dimethyl derivative of fj-hydroxy= thiabendazole. 6.94 High Performance Liquid Chromatoqraphy In recent years high performance liquid chromatography (HPLC) has become one of the major analytical tools to determine the residual thiabendazole alone96-100 or simultaneously with other addit.iveslOl-lll in fruits. Isshiki et a l o g 6 have described a simple HPLC method for eetermining thiabendazole in fruits. Thiabendazole is extracted with methanol and the extract is washed with phexane saturated with methanol. The HPLC determination is carried out u s i n g LiChrosorb RP-8 as stationary phase and methanol-2.8"A ammonia wzter (60:40) as mobile phase; 2-methylindole is added as an internal standard. The detection is carried out fluorometrically and the limit of detection is 0.1 pg thiabendazole/ Recoveries are 92%. Collinge and Nairfalisegg have determined thiabendazole in curds (artificial marmalades) and orange and lemon marmalades

-

B

--

.

628

VIJAY K.KAPOOR

by using aqueous solution containing 0,PA of ammonium nitrate and 0.7% of ammonia (buffered solution)-methanol (1:1) as mobile phase for curds, and buffered solution-methanol ( 3 :2) for marmalades. The flow-rate in column was 1.0 ml/min, the column temperature was 22-26O and the detection wavelength was 305 nm. Yamada et &.lo0 have described a detailed procedure for the separation of thiabendazole in fruits in which the uv-absorbing Contaminants are removed by fractional separation with an aqueous-ethyl acetate system. The HPLC conditions for thiabendazole determination reported aret column, Unisil C-18 10 p 4.6 mm x 300 mmt eluant, methanol0.16 M KH2PO4 water ( 5 0 : 5 0 , v/v % ) I flow rate, 1.0 ml/min; pressure, 60 kg/cm2; detector uv (298 nm); range 0.01 AUF; and sample size 5 pl. The detection limit for this method was found to be 0.08 ppm, and it was shown that 1 ppm of thiabendazole in the fruits could be estimated within 10%. An ionggair HPLC has been described by Belinky. A simultaneous HPLC determination of thiabendazole, 0-phenylphenol and diphenyl residues in cityus fruits without prior clean up has been described by Kitada et a1.1°6 A rapid, sensitive and precise HPLC method using fluorescence detection has been developed by Watts et a1,112 for the simultaneous determination of thiabendazole and unconjugated 5-hydroxythiabendazole in human serum. Sample pretreatment consists only of protein precipitation with acetonitrile containing the internal standard, 2-methylindole. Detection limits were found to be 0.1 pg/ml serum for thiabendazole and 0.4 pg/ml serum for 5-hydroxythiabendazole. A microenzymatic method for the conversion of the glucuronide and sulfate esters of 5-hydroxythiabendazole was also developed using P-glucuronidase and sulfatase, respectively. Thus, quantitation of the separate metabolites was possible. A special adaptation of the chromatographic procedure was utilized for the determination of 5-hydroxythiabendazole metabolites in the sera of uremic patients, which can contain large mounts of interfering fluorescent substances. The method should be of particular use for monitoring thiabendazole therapy in

THIABENDAZOLE

629

patients to eliminate the potentially toxic metabolites. HPIC methods for determining thiabendazole in waste waters1 138114 and in textile productsll5-117 are also described. 6.10 Miscellaneous A radioactive indicator method for the determination of thiabendazole residues has been described.118 7.

Limits of Tolerance Tolerances in ppm for the residues of the fungicide thiabendazole in or on various raw agricultural commodities established under the Federal Food, Drug, and Cosmetic Act fixed by the United States Environmental Protection Agency are: 0.02 for sweet otatoes;llg 0.1 for dried beans,l20 eggs,121 meat; 151 and soyabeanso122 0.4 for banana pulp119 and rnilki123 1 for hubbard squashr124 3 for bananas119 and rough rice1125 5 f o r strawberriesol26 6 €or su ar beetsol21 8 for rice hullst127 10 for a les,l 8 carrots,l29 citrus fruits 130 grapes,P93 pears 128 potatoesl31, rice straw,$25 sugar beet tops135 and wheat grains; 131 15 for cantaloupes;126 and 40 for rnushro0m.13~

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D. M. V i c t o r , R. E . H a l l , J. D. Shamis and S. A. Whitlock, J. Chromatogr., 283, 383

G e r i c h t l . Chem.

T. Watts, V. A. R a i s y s and L. J. Chromatogr. 8 230, 79 (1982).

A.

Bauer,

(1984).

114.

R. C. C. Wegman and H. H. Van den Broek, Meded, Fac. Landbouwwet. R i j k s u n i v . G e n t , 47, 361 (1982); C.A., 98, 48523y (1983).

-

Kanetoshi, H. Ogawa, M. A n e t a i , E.

115.

A.

116.

T. Amemiya, M. S a k a i , K. Mori, S. Suzuki and M. Kazama, Kenkyu Nenpo-Tokyo-toritsu E i s e i Kenkyusho, 35, 133 (1984); C.A.8 103, 88997j (198g.

Katsura and H. Kaneshima, E i s e i Kagaku, 31, 245 (1985); C.A., 104, 226211~(1986).

-

,

VIJAY K.KAPOOR

638

117.

T. Amemiya, M. S a k a i , K. Ikeda, K. Mori, S . Siizuki and Y. Watanabe, Kenkyu NenpoTokyo-toritsu Eisel Kenkyusha, 36, 123 (1985); C.A., , & I 1 3 5 3 3 5 ~( 1 9 8 n .

118.

C. P.osenblum anc? H. T , Meriwether, J. Radioanal. Chern., 58 379 (1970); C . A . 8 74, 98714m (1971).

119.

Fed. Regist,., 52304; C.A.,

120.

121. 122. 1230

-

17 Nav 1983, 48 (223), 4922h (1984).

100,

Fed. Regist., 3 A p r 1985, 50 (641, 13195r 1 0 2 8 183870a (1985).

c.A.,

Fed. Regist., 2 2 Jan 1980, 45 (141, C.A.8 92, 145132d (1.980)

3907;

Fed. Regist., 2 8 Jun 19778 42 (1241, 32782; 87, 116494q (1977).

C.A.,

Fed. Regist., 8 Sept 1982, 47 (1’741, 39488; 97, 161179h (1982).

C.A.,

124.

Fed. R e g i s t . , 26 May 1972, 37 (103), 10670; C.A.8 2 0 46820k (1972)

125

Fed. Regist., 9 J u l 1980, 4S (1331, 46073; C.A*, 93, 130751b (1980).

126. 127 1280 129 130. 131,

Fed. Regist.,

C.A.8

19 Mar 1986, 51 (531, 9448;

104, 3 6 7 0 4 4 ~(19$6)

Fed. Regist., 9 July 1980, 45 (1331, 46067; 93, 1 3 0 7 5 2 ~(1980)

C.A.,

Fed, Regist., 7 O c t 1972, 37 (19618 212787 7 8 0 2 8 7 4 ~(1973)

C.A.8

Fed, R e g i s t . , 2 8 NoV 19808 45 (2311, 79068; 94, 63918s (1981).

C.A.,

Fed. R e g i s t . ,

38229; C.A.0

10 A p r 7985, 50 (691, 14106; 2 0 2 7 2 9 ~ (1985)

Fed. Regist.,

C .Am,

102,

30 Oct 1974, 39 (2lO)# 82, 29789t (1975).

THIABENDAZOLE

132.

133.

639

Fed. R e g i s t . , 25 Jun 1973, 38 (121), 16644; 2, 64669~(1073)

C.A.,

Fed. R e g i s t . , 10 hpr 1385, 50 ( 6 9 ) , 14104; 102, 2 0 2 7 2 7 ~ (1985).

C.A.,

TIMOLOL MALEATE

DAVID J. MAZZO* AND ALICE E. LOPER#

*

MANAGER, PARENTERALS RESEARCH AND DEVELOPMENT, TRAVENOL LABORATORIES, MORTON GROVE, ILLINO1 S

#RESEARCH FELLOW, PHARMACEUTICAL RESEARCH AND DEVELOPMENT, MERCK SHARP AND DOHME RESEARCH LABORATORIES, WEST POINT, PENNSYLVANIA

ANALYTICAL PROFILES OF DRUG SUBSTANCES

VOLUME 16

641

Copyright 0 1987 by Academic Press, Inc.

AU rights of reproduction in any form reserved.

DAVID J. MAZZO AND ALICE E. LOPER

642

1.

2.

Introduction 1.1

Therapeutic Category

1.2

History

Description 2.1

Chemical ..me, Formu a, Molecular Weight

2.2

Definition

2.3

Appearance, Color, Odor

3.

Synthesis

4.

Physical Properties 4.1

Infrared Spectrum

4.2

Nuclear Magnetic Resonance Spectrum 4.2.1

Proton NMR

4.2.2

Carbon-13 NMR

4.3

Ultraviolet Spectrum

4.4

Mass Spectrum

4.5

Optical Rotation

4.6

Electroanalytical Behavior

4.7

Thermoanalytical Behavior 4.7.1

Melting Point

4.7.2

Differential Thermal Analysis Behavior

TIMOLOL MALEATE

643

Thermogravimetric Analysis Behavior

4.7.3 4.8

Solubilities

4.9

Crystal Properties

4.10 Hygroscopicity 4.11 Dissociation Constant 5.

Methods of Analysis 5.1

5.1.1

Ultraviolet Spectrophotometry

5.1.2

Infrared Spectroscopy

5.1.3

Elemental Analysis

5.2

Titrimetric Analysis

5.3

Spectrophotometric Analysis

5.4

6.

Identification Tests

5.3.1

Direct Ultraviolet Spectrophotometry

5.3.2

Ultraviolet Spectrophotometry via Flow Injection Analysis

5.3.3

Specific Rotation

Chromatographic Analysis 5.4.1

Thin-layer Chromatography

5.4.2

High Performance Liquid Chromatography

Stability

-

Degradation

6.1

Solid State Stability

6.2

Solution Stability

DAVID J. MAZZO AND ALICE E. LOPER

644

7.

Biopharmaceutics and Metabolism 7.1

Absorption and Bioavailability

7.2

Metabolism

7.3

Pharmacokinetics

8.

Determination in Biological Matrices

9.

Determination in Pharmaceuticals

10.

9.1

Dissolution Testing

9.2

Potency 9.2.1

Assay

9.2.2

Dosage Uniformity

9.2.3

Stability Testing

References

TIMOLOL MALEATE

1.

645

Introduction 1.1

Therapeutic Category (1) Timolol maleate is a beta adrenergic blocker which is non-selective between beta and beta adrenergic receptors. It dhes not ha6e significant intrinsic sympathomimetic, direct myocardial depressant or local anesthetic (membrane-stabilizing)activity. Timolol maleate is effective in lowering intraocular pressure and is widely used in patients with openangle glaucoma and aphakic glaucoma. Timolol maleate is also indicated both for the treatment of hypertension (alone or in combination with other anti-hypertensive agents, especially thiazide-type diuretics) and to reduce cardiovascular mortality and the risk of reinfarction in patients who have survived the acute phase of myocardial infarction and who are clinically stable. Timolol maleate, available for oral dosing as tablets and for injection and ophthalmic dosing as distinct sterile aqueous solutions, is usually well tolerated with most adverse effects being mild and transient. However, a number of adverse effects have been reported with other betaadrenergic blocking agents and should be considered potential adverse effects of timolol maleate. A number of adverse reactions considered to be causally related to timolol maleate have been reported. For example, severe respiratory reactions and cardiac reactions, including death due to bronchospasm in patients with asthma and rarely death in association with cardiac failure, have been reported (1)

DAVID J. MAZZO AND ALICE E. LOPER

646

1.2

History Timolol maleate, belonging to the thiadiazole class of compounds, was first synthesized in the Merck-Frosst Laboratories in Montreal, Canada. The first non-patent literature reference to timolol maleate appeared in 1972 (2). Timolol maleate, since its introduction in pharmaceutical formulations, has gained a wide acceptance as an anti-hypertensive and anti-glaucoma agent. A search of Chemical Abstracts (1966 - 1986) and Pharmaceutical Abstracts (1974 - 1986) produced over 210 unique bibliographic citations for works dealing with timolol maleate.

2.

Description 2.1

Chemical Name, Formula, Molecular Weight The accepted chemical name for timolol maleate (MK-950) is: (S)-l-[(l,l-

dimethylethyl)-aminol-3-[[4-(4morpholinyl)-1,2,5-thiadiazol-3-

ylloxyl-2-propanol, (2)-butenedioate (1:l) salt. The CAS registry number is 26921-17-5. Other names which have been used for timolol maleate include the maleate salts of (s)-(-)-3-(3-tert-butyl-amino2-hydroxypropoxy)-4-morpholino-l,2,5-

thiazide, (-)-3-morpholino-4-(3-tert-

butylamino-2-hydroxypropoxy)-1,2,5-

thiadiazole and (-)-1-(tert-

butylamino)-3-[(4-morpholino-1,2,5thiadiazol-3-yl)oxy]-2-propanol.

TIMOLOL MALEATE

2.2

647

Definition Timolol maleate possesses an asymmetric carbon atom in its chemical structure and is provided as the levoisomer. It has tradenames of BLOCADRENs and TIMOPTICs. Timolol, when referred to, indicates the free base (CAS Registry Number = 26839-75-8).

2.3

Appearance, Color, Odor Timolol maleate is a white, odorless, crystalline powder.

3.

Synthesis Timolol maleate has been prepared through a series of synthetic steps beginning with D-mannitol and acetone (3). The synthetic route is presented in Figure 1. The starting material, I, is reacted with acetone and the product (11) is washed with benzene and crystallized from hexane. This substituted mannitol (I11 is then oxidized in tetrahydrofuran with lead tetraacetate to the substituted glyceraldehyde (1111, hydrogenated with t-butylamine ( I V ) and treated with benzaldehyde to form a substituted phenyloxazolidine (V). In a separate reaction sequence,

C13H24N403S-C4H404 Molecular Weight 432.49 g/mole

H3C..c/0-?H2

HO -CHe I HO-C-H I HO-C-H I H-C-OH

(CH3)zCO

I

H3C'

'0-C-H I Pb(0Ach

HO-C-H I H-C-OH I

H-C-0,

H-C-OH I CHDH

I HeC-O/

C

[""' /o-i:6] H,C/~'O-CH

(CH3)aCNH2

HOCHzCHCHzNHC(CH3)3 I OH

+

6/

m

rn

Y

m

S

\

Y

Ip

H

N,

o&N\C(CH3)3

/CH3 'CH3

II

I

,

CHO

,N

Ix

Figure 1. Synthetic route to timolol maleate.

X

649

TIMOLOL MALEATE

aminoacetonitrile hydrochloride (VI) is reacted in the presence of chlorine with sulfur monochloride to form a chlorinated thiadiazole (VII), which, in turn, is reacted with morpholine to form VIII. Compounds VI and VIII are reacted to timolol free base (1x1 which is then converted to timolol maleate by refluxing with maleic acid in acetone ( X ) .

4.

Physical Properties

4.1

Infrared Spectrum The infrared spectrum of timolol maleate taken in a KBr pellet is shown in Figure 2 (4). A Digilab Model FTS-15C Fourier transform infrared spectrophotometer was used to acquire the spectrum. Frequency assignments for some of the characteristic bands are listed in Table I. Table I Infrared Spectral Assignments for Timolol Maleate

Frequency ( cm’l) 3250 3000 1700 1200 1100

Assiqnment 0-H N-H C=N c-o(c-OH)

c-0-c

stretch stretch stretch stretch stretch

The infrared spectrum of timolol maleate taken in a mineral oil mull is shown in Figure 3 (4). Both of these spectra are consistent with the structure of timolol maleate.

1

1

1

4000 3800

1

1

1

1

1

1

3600 3400

1

1

1

1

3200

1

1

1

1

3000

1

1

1

1

1

1

2800 2600

1

1

1

1

2400

1

1

1

1

2200

1

1

1

1

1

1

1

l

2000 1800

1

l

1

l

1600

l

V l

l

l

1400

l

l

l

l

1200

l

l

l

l

l

l

I

l

i

I

I

I

I

I

I

Wavenumbers Figure 2.

I

1000 900800 700 600500

Infrared spectrum of timolol maleate taken in a KBr pellet.

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

”; 1

1

3700

1

1

3500

1

3300

1

1

3100

2900

2700

2500

2300

2100

1900

1700

1500

1300

1100

900 800700600

Wavenumbers Figure 3.

Infrared spectrum of timolol maleate taken in a mineral oil mull. Mineral oil contributes to the spectrum at ca. 1350-1450, 15001750 and 2875-3000 wavenumbers.

DAVID J. MAZZO AND ALICE E. LOPER

652

4.2

Nuclear Magnetic Resonance Spectrum 4.2.1

Proton NMR Spectrum The proton magnetic resonance spectrum of timolol maleate was obtained using a Nicolet NT 360 spectrometer. The spectrum was acquired from an approximately 0.02 M solution using d DMSO as the solvent. The spgctrum is shown in Figure 4 and the spectral assignments are listed in Table I1 ( 4 , s ) .

-

A 20

1

r

I

ill

LL

/

10

"

'

I

"

7

'

8

Figure 4.

b

4

Proton NMR spectrum of timolol maleate.

DAVID J. MAZZO AND ALICE E. LOPER

654

Table I1

Re lat ive No. Protons

Assignment d - DMSO sglvent effect

- CH- Cg2 -N

2 2*> 3. 3.35

-

3.47

4

CH - C H h / -2 0 -N ‘CH2 - CHf

3.72

4

-N

4.2

1

residual H20/HOD

,CHpE&

- Cgf

‘CH

0

- CHI

0

4.4

2

0-C€12-

5.97

1

-0-g

6.03

2

HC=Cg

8.38

2

I I

H -

I +

-N-C(CH3)3

I

H 20.1

2

I

-C=C-COOH

I -

c02

TIMOLOL MALEATE

4.2.2

655

CI3 NMR Spectrum The CI3 NMR spectrum shown in Figure 5 was obtained on a Varian CFT-20 spectrometer using a 0.5 M timolol maleate fglution in d - DMSO (5). The C spectral assignments are listed in Table 111. Table I11 CI3 NMR Spectral Assignments for

Timolol Maleate 6

(ppm)

Assignment

167.4

C02H/C02-

153.3

c3

149.8

c4 I I HC=CH

135.9 72.0

-0cH2-(Ca)

65.6

O-(CH2-l2 ( a )

65.0

-CHOH-(C@) -

56.5

-NH-C-

43.7

-NHCH2-(Cy)

24.9

C(CH313

I

I

I

I

I

I

I

I

I

I

1

180 170 160 150 145 130 120 110 100 90

I

I

I

I

I

I

I

I

1

80

70

60

50

40

30

20

10

0

(PPm) Figure 5.

CI3 NMR spectrum of timolol maleate.

TIMOLOL MALEATE

657 1

t

1

I

a)

The C and C carbons are equivalent; theregore, onfy a single line is observed for these two carbons.

b)

The C and C carbons are equivalent; there$ore, on?y a single line is observed for these two carbons. Both the proton and CI3 NMR spectra are consistent with the timolol maleate structure.

4.3

Ultraviolet Spectrum The ultraviolet absorption spectrum of timolol maleate in 0.1 N aqueous hydrochloric acid is characterized by maxima at approximately 210 nm and 294

nm.

The absortivity in absorbance units drug solution in a 1 cm is 200 at 294 nm. The W in Figure 6 was obtained using a Hewlett-Packard Model HP8451A diode array spectrophotometer. 4.4

Mass Spectrum A mass spectrum of timolol is shown in

Figure 7. This spectrum was obtained using an LKB model 9000 mass spectrometer operated at 70 eV in the direct probe-electron impact mode. The fragmentation pattern observed is consistent with the chemical structure of timolol. The molecular ion is noted at m/e = 316. A strong M-minus-methyl peak is observed at m/e = 301. The loss of CIHION leads to m/e = 244. Loss of C H produces a fragment at m/e = 259 w h e h lacks the intensity to be seen in a high resolution spectrum. The loss of C H N(H)-CH gives rise to m/e = 230 with4t8e peak it m/e = 86 representing this lost group. Principle fragmentations of the morpholine ring are seen

f

0

658

8

0

w

I 100

80

60

Relative Intensity 40

130

57

20

144 154

0

3

50

100

-

150

187 188 I

200

229 230 l

A 250

A

286

316

l

300

(mle) Figure 7.

Low resolution mass spectrum of timolol maleate taken in the electron impact mode.

DAVID J. MAZZO AND ALICE E. LOPER

at m/e = 286 and m/e = 272. A MacClafferty rearrangement leads to m/e = 187 and two ions at m/e = 130 (see Figure 8). Fragmentation of the heterocyclic nucleus produces ions at m/e = 144 and possible M-144 at m/e = 172. The peak at m/e = 243 is due to the loss of C H 0 from the molecular ion. This fraAe8t also appears as a characteristic doubly charged ion at m/e = 121.5 (due to the same decomposition). The origin of this peak is apparent from the metastable peak at m/e = 196 which corresponds to the transition from m/e = 301 to m/e = 243. 4.5

Optical Rotation Timolol maleate, having one chiral carbon, is optically active. The levorotatory enantiomer is the biologically active form. The optical rotation of a 5% (w/v) solution of timolol maleate in i50 N aqueous HCt5at 25 "C and 405 nm is -12.2" ( A D = -4.2 " ) (6).

r

f7 U N

0 L

mle 187,0407 (187.0415)

(1 30.1232)

I

(major)

/

-(CH,-0-CH-CH,)

154.0616 (154.0625) CH, = N @

H

r

N\s,

f/

0

NH

mle 130.0057 (130,0075) (minor)

Figure 8.

CH,=CH-CH

-H,O

@ CH3

I

= NKC-CH,

I

CH,

mle112

Schematic representation of the mass spectral fragmentation of timolol maleate.

DAVID J. MAZZO AND ALICE E. LOPER

662

4.6

Electroanalytical Behavior Timolol maleate is electrochemically active and it undergoes a single irreversible electro-oxidation. The E of an approximately 40 vg/mL skliition of timolol maleate in 0.01 M aqueous KC1 is +730 mV (vs Ag/AgCl). Figure 9 shows the voltammogram of this solution obtained using a Princeton Applied Research Model 174A Polarographic Analyzer with a glassy carbon working electrode, a silver/silver chloride reference electrode and a platinum auxillary electrode.

4.7

Thermoanalytical Behavior

4.7.1

Melting Point The melting point of timolol maleate varies between 201.5 'C and 2 0 2 . 5 'C as determined by the USP class Ia capillary method (6).

4.7.2

Differential Thermal Analysis Behavior The differential thermal analysis (DSC) behavior of timolol maleate under nitrogen is shown in Figure 10. This curve was obtained using a Perkin-Elmer Series 7 DSC scanning from 40 "C to 2 5 0 "C at 1 0 "C/minute. A primary endotherm corresponding to melting is observed with an actual peak temperature of 2 0 5 . 8 "C. An additional endotherm with a peak temperature of ca. 2 1 5 'C is noted corresponding to compound decomposition.

0

Figure 9.

I

I

I

I

I

I

1

I

I

I

I

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

Potential (Volts vs. AgIAgCI) Scanning voltammogram of timolol maleate in 0.1 M K C 1 (aq).

100.0

90.0 80.0 -

-

n

=3 U

70.0 60.0 -

50.0 -

c

m

40.0 30.0 -

20.0 10.0 0.0

-p

'

I

I

50.0

75.0

I

100.0

I

125.0

1

150.0

I

175.0

I

200.0

225.0

Temperature ("C) Figure 10.

Differential scanning calorimetry behavior of timolol maleate.

TIMOLOL MALEATE

665

Thermogravimetric Analysis Behavior

4.7.3

Thermogravimetric analysis of timolol maleate indicates no weight loss until 205.8 "C. From 2 0 5 . 8 " C to ca. 3 5 0 " C there is an approximately 80% weight loss due to decomposition/vaporization. A typical TGA curve for timolol maleate is shown in Figure 11. The curve was obtained using a PerkinElmer Series 7 TGA scanning from 30 "C to 4 0 0 "C at 1 0 "C/minute. A sample of 3.523 mg of timolol maleate was used. 4.8

Solubilities The solubilities of timolol maleate in a variety of solvents at room temperature ( ~ 2 5"C) is presented in Table IV (6). Note that these solubilities are stated in terms of the current U.S.P. definitions ( 7 ) . Table IV Solubility of Timolol Maleate at

Room Temperature Solvent Water Methanol Ethanol Chloroform Propylene Glycol Ether Cyclohexane Isooctane

Solubility Soluble Soluble Soluble Sparingly Soluble Sparingly Soluble Practically Insoluble Practically Insoluble Practically Insoluble

I

I

I

I

I

I

I

0

8m 8 s: cu

8rr,

0

8 cv

2

8

8 ?

9 m

0

TIMOLOL MALEATE

4.9

667

crystal Properties ( 8 ) Timolol maleate exists as a crystalline powder. No polymorphs of timolol maleate have been reported. Figure 1 2 shows the x-ray powder diffraction pattern of timolol maleate obtained with a Phillips Electronics model APD3720 powder diffractometer using CuKa radiation. Table V lists the interplanar distances and the relative intensities of the major lines in the x-ray powder diffraction pattern of timolol maleate.

Table V X-Ray Powder Diffraction of Timolol Maleate Peak Angle ( 2 0 " ) 7.1 9.9 11.4 14.2 14.8 15.6 16.3 18.1 19.0 19.5 19.8 20.5 20.6 20.9 21.4 22.4 23.0 23.8 24.8 26.0 26.5 27.1 28.3 29.1 29.5 31.6 34.0 38.8 39.3

D Spacinq 12.49 8.95 7.76 6.23 5.96 5.68 5.44 4.90 4.67 4.56 4.47 4.33 4.31 4.24 4.14 3.97 3.86 3.74 3.58 3.42 3.36 3.29 3.16 3.07 3.03 2.82 2.63 2.32 2.29

(I/Imax)(%) 13 13 6 100 27 12 30 43 62 24 18 52 54 67 75 35 9 50 45 7 16 60 32 13 5 26 12 19 14

TIMOLOL MALEATE

669

4.10 Hygroscopicity (9) The maleate salt of timolol is a stable anhydrous compound. No hydrates of timolol maleate have been reported. The free base of timolol, however, exists both as a stable crystalline hemihydrate and anhydrous compound. Anhydrous timolol maleate is nonhygroscopic when stored under ambient temperature conditions (-25 " C ) and relative humidity conditions ranging from 3 3 % to 76%. 4.11 Dissociation Constant ( 9 ) The pKa of timolol base obtained by potentiometric titration in water at The native 25 " C is approximately 9.2. pH of a saturated aqueous solution of timolol maleate is 2 4. 5.

Methods of Analysis 5.1

Identification Tests 5.1.1

Ultraviolet Spectrophotometry (10) Ultraviolet spectrophotometry is used to identify timolol maleate. A solution in 0.1 N HC1 (aq) scanned from 200 nm to 400 nm qualitatively exhibits the same absorbance characteristics at identical wavelengths as does a similarly prepared and concomitantly measured solution of a timolol maleate standard. Quantitatively, equimolar sample and standard solutions will exhibit absorbances at 2 294 nm (Xmax) whi Q differ by no more than 3% (AX&= 2 0 0 ) .

5.1.2

Infrared Spectroscopy Infrared spectroscopy may also

DAVID J. MAZZO AND ALICE E. LOPER

670

be used to identify timolol maleate. The infrared spectrum prepared as a mineral oil dispersion compares qualitatively (with maxima only at the same frequencies) to the spectrum of a similarly prepared timolol maleate standard. 5.1.3

Elemental Analysis Timolol maleate may be identified through the use of elemental analysis. The results of an elemental weight percent determination of carbon, nitrogen, sulfur, hydrogen and oxygen are compared to the respective theoretical values of 49.35%, 17.71%, 10.15%, 7.65% and 15.17%.

5.2

Titrimetric Analysis (10) Timolol maleate may be determined by perchloric acid titration to a visual or potentiometric endpoint. An accurately weighed sample ( = 800 mg) is dissolved in = 90 mL glacial acetic acid and titrated with 0.1 N perchloric acid in dioxane to a green endpoint. Alternatively, the endpoint may be determined potentiometrically as that point at which the greatest change in potential per unit volume change of titrant is noted. For potentiometric titrations, the electrode system should consist of an indicating electrode prepared by emptying a sleeve type calomel electrode, drying it and filling with 0.1 N LiClO in acetic anhydride. A platinum rfng reference electrode is to be used.

5.3

Spectrophotometric Analysis 5.3.1

Direct Ultraviolet Spectrophotometry

TIMOLOL MALEATE

67 1

Timolol maleate exhibits an ultraviolet absorption band near 2 9 4 nm attributed to the thiadiazole core structure. This absorption is the basis for the quantitative determination of the drug. Assay of the compound is performed by comparing the net absorbance at 2 9 4 nm of a sample dissolved in 0.1 N HC1 (as) with the net absorbance of a standard in 0.1 N HC1 (as) of known concentration. The net absorbance is calculated by subtracting the drug free matrix contribution to absorbance at the wavelength of determination from the absorbance of the drug solution at the same wavelength.

where : = Concentration of the sample solution = Concentration of the standard ‘std solution = Absorbance at 2 9 4 nm of the Asam sample solution = Absbrbance at 2 9 4 nm of the standard solution = Absorbance at 2 9 4 nm of the Ab drug free solution matrix 5.3.2

Ultraviolet Spectrophotometry via Flow Injection Analysis Ultraviolet absorbance is also used as the detection mode for timolol maleate determinations by flow injection analysis (11). In the case of the flow injection technique assay, timolol maleate sample and

DAVID J. MAZZO AND ALICE E. LOPER

672

standard solutions are periodically injected into a flowing stream. The resulting changes in UV absorbance at 294 nm of the stream are measured relative to the analyte-free stream. Calculation of the concentration of timolol is made in an identical fashion as direct UV spectrophotometry. 5.3.3

Specific Rotation The specific rotation of timolol maleate is determined in 0.1 N HC1 (aq). Using a solution of = 50 mg/mL, the angle of rotation at 405 nm, 25 "C, in a 10 cm cell is determined with a suitable photoelectric polarimeter. Specific rotation, calculated on the dried basis, is between -11.7" and -12.5'.

5.4

Chromatographic Analysis 5.4.1

Thin Layer Chromatography (10) Normal-phase thin layer chromatography on silica gel has been employed for timolol maleate. A developing solvent system consisting of chloroform/methanol/(28% ammonia water) [80/20/1 (v/v)l is used to develop a spot resulting from 10 pL of an approximately 5 mg/mL solution of the drug in methanol. A reference spot for impurity purposes applied as 10 pL of = 20 pg/mL solution of timolol maleate in methanol is also employed. R for timolol maleate in this &stem is = 0.7. Detection is made under visible light after the developed and air-dried plate has been exposed

613

TIMOLOL MALEATE

to iodine vapor for 2 hours. Sensitivity is estimated to be within an order of magnitude of the amount of timolol maleate in the reference spot solution ( = 200 ng drug). 5.4.2

High Performance Liquid Chromatography (12) High performance liquid chromatography may be employed to analyze timolol maleate. A chromatographic system consisting of a column (300 mm x 4.6 mm i.d.) packing with p-phenyl stationary phase (10 pm particle size) has been used with a mobile phase consisting of 1 part methanol and 2 parts 0.005 M aqueous hexane sulfonic acid (pH adjusted to 1 with acetic acid). A column temperature of 30 'C and a flow rate of 2.00 mL/min are used. Detection is by UV absorbance at 280 nm. Typically, 20 pL injections of a timolol maleate solution are made. Under these conditions, timolol is separated from its known potential process impurities.

6.

Stability 6.1

- Degradation

Solid State Stability ( 1 3 ) Timolol maleate is an extremely stable compound at room temperature in the solid state. In fact, prolonged exposure to extremes of temperature and humidity caused minimal degradation of the solid compound. For example, timolol maleate stored for = 2 months at 105 O C in an air atmosphere (ambient relative humidity) showed no difference in potency by TLC chromatographic

DAVID J. MAZZO AND ALICE E. LOPER

614

profile when compared to a sample of unstressed solid. Only heating timolol maleate to above its melting point and keeping it molten for 10 minutes produced discoloration and an approximate 5% loss in potency. (TLC of this sample did detect small quantities of several unknown decomposition products.) Timolol maleate, when heated at 95 O C for = 3 weeks in an air atmosphere (100% relative humidity), also exhibited a loss in potency of 25% (by W assay). Two degradates were isolated from this sample and were identified as the cis/trans isomer pair of timolol 0-fumarate ester and timolol 0-maleate ester (Figure 13). Timolol maleate exhibited surface discoloration when exposed in an open dish to intense UV light ( z 20 times normal sunlight). This sample did not, however, show any loss of potency by UV assay nor were any decomposition products detected by TLC. 6.2

Solution Stability ( 1 3 ) Timolol maleate is extremely stable in aqueous solutions (protected from light) with no decomposition being detected in injectable formulations stored at room temperature for = 5 years. Timolol maleate can be degraded in solution if exposed to elevated temperatures or intense ultraviolet radiation. The solution decomposition of the drug is pH dependent with maximum stability occurring near pH = 4. Solutions of timolol maleate prepared in each of 0.1 N HC1 (aq) (pH = 1) and phosphate buffer (pH = 7 . 2 ) and then stored in sealed glass vials for = 2 months at 105 OC yielded considerable amounts of degradation products. A solution prepared in distilled water and stored identically

n

OCH,CHCH,NHb-CH,

0

N

\

S

/

N

I

0

I c=o

1

CH3

I

HC = CHCOOH

cis and trans Figure 13.

Potential solid state degradates of timolol maleate exposed to 105 " C / l O O % relative humidity.

DAVID J. MAZZO AND ALICE E. LOPER

676

was found to be virtually the same as unstressed control solution. Studies of timolol aqueous solutions (pH - 5 ) under autoclave conditions indicated that timolol maleate degrades at the rate of approximately l%/hour at 120 OC. Three decomposition products were isolated from solutions which were autoclave degraded. Figure 14 depicts the proposed degradation mechanism and indicates the three modes of thermally induced solution degradation; (1) rearrangement to isotimolol; (2) ether cleavage to form 4-hydroxy-3morpholino-1,2,5-thiadiazole; and (3) oxidation followed by ether cleavage to form 4-hydroxy-3-morpholino-l,2,5thiadiazole 1-oxide. Aqueous solutions of timolol maleate (pH 5 ) have also been shown to be susceptible to degradation when exposed to intense ultraviolet radiation. Exposure for 2 hours to U V light equivalent to = 20 times normal sunlight produced approximately 2% loss in potency. Evidence of the three thermal degradation products mentioned above as well as several unidentified degradation products was found in this solution. Because of the potential for light-induced degradation, it is recommended that aqueous timolol maleate solutions be stored protected from light. 7.

Biopharmaceutics and Metabolism

7.1

Absorption and Bioavailability Timolol maleate is rapidly and completely absorbed after oral administration (14). Maximum blood plasma concentrations ranging from 10 ng/mL to 100 ng/mL are attained within 1 to 2.4 hours after either acute or

,

OH

CH,

I "\_/", n IIOCH,CHCH,NHC-CH, I I N\

/

CH,

N

S

Timolol

n

CH,OH

I

o=N'rrr(opH Y SY

lsotimolol

FH3 CH~NHC-CH,

I

n

oWNllfoH N N

I

' 'S

CH,

I

'OH

0 4-hydroxy-3-morpholino1,2,5-thiadiazole l-oxide

4-hydroxy-3-morpholino1,2,5-thiadiazole

Figure 14.

Proposed aqueous solution degradation mechanism for timolol maleate in solutions exposed to autoclave conditions.

DAVID J. MAZZO AND ALICE E. LOPER

678

chronic administration of 2.5 mg to 20 mg of timolol maleate twice daily (15-16). The bioavailability of oral timolol is reported to be 61% to 75% of a reference intravenous dose (17-18). Bioavailability of less than 100% is attributed to first-pass metabolic extraction by the liver after oral administration rather than to incomplete gastrointestinal absorption (14). The effect of food on the rate and extent of oral absorption of timolol maleate is not significant (19)

Timolol maleate administered directly to the eye as an ophthalmic drop appears to be rapidly absorbed, producing a decrease in intraocular pressure within three hours (20). Systemic absorption of timolol also occurs after ocular administration, producing peak blood plasma levels of no more than 2 to 5 ng/mL at 0.5 hours to 1.5 hours after instillation of 2 drops of 0.5% timolol maleate ophthalmic solution in each eye (21). Some small but statistically significant cardiovascular effects have been reported after ophthalmic administration (20,221. Supporting studies in rabbits indicate that timolol reaches peak levels in most tissues of the eye at ten minutes after ocular administration with blood serum concentrations peaking at 30 to 60 minutes (23). Timolol free base is rapidly absorbed through intact skin (24). 7.2

Metabolism After orf4 administration of a 4 mg C- timolol maleate to man, dose of 72% of the I4C label is excreted within 84 hours with 66% in the urine and 6% in the feces. Only 20% of the timolol

TIMOLOL MALEATE

679

dose is recovered unchanged from the urine. Over 80% of the blood plasma radioactivity represents timolol metabolites (14). Oxidation and hydrolytic cleavage of !be morpholine ring of an oral dose of C- timolol maleate produces two major urinary metabolites in man (25). HO-CH2- CH HOOC-CH<

\

S/

*

0-CH -CH-CH2-NH-C(CH3)3 bH

Metabolite I N-{{4-[3-(l,l-dimethylethyl)aminol-2-hydroxypropoxy}-1,2,5- thiadiazol-3-yl}-N-(2-hydroxyethyl)

glycine.

H O - C H ~ - C H ~ - N H - ~ O--C HC -HC H ~ - N H - C ( C H ~ ) ~ N N bH ’S‘

Metabolite I1 l-(l,l-dimethylethylamino)-3-{[4-(2-hydroxyethylamino)-1,2,5-thiadiazol-3-ylloxy}-2-propanol.

Metabolite I represents approximately 30% of the total urinary radioactivity while Metabolite I1 accounts for 10% (14,251.

Oxidation of the basic oxypropanolamine side chain of orally administered timolol maleate results in two other minor urinary metabolites in man. Metabolites I11 and IV represent 6% and 3 % , respectively, of administered radioactivity (26).

DAVID J. MAZZO AND ALICE E. LOPER

680

..

N\s/

N

OH

Metabolite I11 2-hydroxy-3-[{4-(4-morpholinyl)-l,2,5-thiadiazol-3-yl}oxy]propanoic acid

H-CH2NH-C-CH2-OH O w N -

I

\ /

CH3

S

Metabolite IV l-~[(1,1-dimethyl-2-hydroxy)-ethyllamino}-3-{[4(4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy}-2propanol.

Biological testing of synthetic samples of Metabolites I, I1 and I11 shows only Metabolite I1 to have significant 6-adrenergic blocking activity. However, activity was only one-seventh that of timolol maleate in anesthetized dogs (26). These four metabolites are also produced by other species. Metabolite I11 represents the major metabolic pathway for timolol maleate in the dog. Other metabolites in rodents result from hydroxylation and oxidation of either the morpholino ring or the oxypropanolamine side chain (25). 7.3

Pharmacokinetics Oral administration of timolol maleate in doses ranging from 5 mg to 20 mg

TIMOLOL MALEATE

681

results in peak plasma concentrations of timolol maleate within one to two hours, followed by an exponential decline of plasma concentrations with time ( 1 5 ) . The mean apparent half-life of elimination of unmetabolized drug from plasma is approximately 2 . 5 hours ( 1 5 , 1 7 1 , although plasma half-lives as long as five hours have been reported in normal volunteers ( 2 7 ) . Half-life is independent of the administered dose. Timolol maleate appears to follow simple linear kinetics over the dosage range studied; area under the plasma concentration versus time curve is proportional to the administered dose ( 1 5 ) . The mean renal clearance of unchanged timolol maleate in normal volunteers was reported as 6 9 . 6 mL/minute ( 2 7 ) . Pharmacokinetic parameters do not change upon one week chronic administration of oral timolol maleate ( 1 5 ) . Following intravenous administration, plasma levels decrease biexponentially with time ( 2 8 ) . Reported half-lives for timolol maleate elimination from plasma, 3.3 to 4 . 1 hours, are comparable to the oral half-life. The pharmacodynamics of oral timolol maleate have been well characterized (15,161. Maximal suppression of exercise-induced tachycardia, a measure of @-blockade, occurs when timolol maleate plasma levels reach 27 to 30 ng/mL. Fifty percent of maximum inhibition of exercise-induced tachycardia is produced by timolol maleate plasma concentrations of 3 to 4 ng/mL. 8.

Determination in Biological Matrices Timolol maleate may be determined in blood plasma and urine by isolation and

DAVID J. MAZZO AND ALICE E. LOPER

682

derivatization followed by gas-liquid chromatography with electron capture detection (29). Timolol and the internal standard, desmethyltimolol, are extracted from either 1.0 mL of alkalinized plasma or 0.1 mL of alkalinized urine into 4% isoamyl alcohol in heptane. The compounds are then back-extracted into 0.1 N hydrochloric acid, the acidic phase alkalinized, and the compounds extracted into methylene chloride. After evaporation of the methylene chloride, timolol maleate and the internal standard are derivatized by addition of 0.1 mL of heptaflurobutyrylimidazole-ethyl acetate reagent and immersion in a boiling water bath for one hour. The diheptafluorobutyryl derivatives are recovered in n-heptane, which is evaporated at 60 "C under a stream of nitrogen to concentrate the sample. Typically, 1 to 5 p 1 of sample is chromatographed on a 1.83 m X 0.64 cm column packed with 1% OV-17 on 80-100 mesh Gas Chrom Q. The chromatograph is operated isothermally with injection port, oven and detector temperatures of 250 OC, 185 "C and 300 "C, respectively. Helium, the carrier gas, and 10% methane in argon, the purge gas,63re maintained at 75 mL per minute. The Ni electron-capture detector was operated in the pulse mode of voltage with a pulse interval of 5 0 microseconds. Retention times of derivatized timolol maleate and internal standard are 7.6 and 6.0 minutes, respectively. Limits of detection are 2 ng/mL in plasma and 20 ng/mL in urine. A capillary column gas-liquid

chromatographic-mass spectrometric assay monitoring for timolol with maleate and C-timolol maleate in blood plasma is reported to have a sensitivity of 0.5 ng/mL (30). This method provides the capability to examine administration of drug and its stable isotope-labeled counterpart simultaneously for comparison of two different routes of administration. The

selectefiion

TIMOLOL MALEATE

683

isolation procedure prior to assay consists of passing 9.5 to 1.5 mL of plasma, containing H-timolol maleate as an internal standard, through a cartridge containing an octadecyl-silica bonded reversed-phase packing. The cartridge is flushed and the compounds are then eluted with acetonitrile0.05 N hydrochloric acid, which is subsequently alkalinized. Back extraction from cyclohexane-toluene into an aqueous phase followed by extraction into methylene chloride precedes evaporation and derivatization with bis(trimethylsily1)-trifluoroacetamidepyridine. One to two ~ J Lare injected onto a 16 m X 0.25 mm capillary column coated with SE-30. The splitless injection port temperature is 260 "C and oven temperature is 225 " C . Carrier gas (helium) velocity is 30 cm/second. The mass spectrometer is operated in the electron-impact ionization mode at an ionizing potential of 70 eV, a filament emission current of 0.8 mamp and electron multiplier setting of 1900 V. Selected ion monitoring at m/e 86, 89 and 95, base peaks corresponqing to the side chains f f timolol maleate, C-timolol maleate and H-timolol maleate, respectively, allows construction of a standard curve from the ratios Intensity86/Intensity95 and Intensitygg/ Intensityg5. ~

Other gas-liquid chromatographic methods employing mass fragmentographic detection (31) or nitrogen-selective flame ionization detection (18) have been reported. High performance liquid chromatography with electrochemical detection may also be employed for analysis of timolol maleate in human blood plasma and breast milk (32). A 25 cm X 4.6 m i.d. column packed with PXS 5/25 Partisil ODS 3 (5 pm) is used with a mobile phase of methanol :0.2 M sodium dihydrogen phosphate :88% orthophosphoric acid :water (500:200:3:297) pumped at a flow

DAVID J. MAZZO AND ALICE E. LOPER

684

rate of 1.0 mL per minute. The applied potential for oxidative electrochemical detection is +1.2 V for maximum sensitivity. A glassy carbon working electrode is employed with a silver/silver chloride reference electrode and a platinum auxillary electrode. Timolol maleate and the internal standard, propranolol for plasma or serum, pindolol for breast milk, are extracted from biological matrices with diethyl ether. Compounds are extracted from the organic phase into dilute orthophosphoric acid. For plasma or serum, a hexane wash of the organic phase is sufficient to prepare the acidic aqueous phase for injection of 100 pL into the chromatographic column. For breast milk, the acidic aqueous phase must be alkalinized and the compounds back-extracted into ether. Timolol maleate and internal standard are once again extracted into dilute orthophosphoric acid and the hexane wash and assay are performed as for plasma. Calibration curves are linear from the 2 ng/mL limit of detection up to 100 ng/mL. 9.

Determination in Pharmaceuticals 9.1

Dissolution Testing The dissolution of timolol maleate tablets is evaluated based on methodology and apparatus described in USP XXI(33). The acidic dissolution medium ( 5 0 0 mL of 0.1 N hydrochloric acid) is maintained at 37 5 0.5 "C and basket speed is 100 rpm. After introduction of the tablet, filtered aliquots of the dissolution medium are withdrawn for assay for timolol maleate content against appropriate standards. The official assay is a high performance liquid chromatographic method developed for a system with an ultraviolet detector. The wavelength for detection is 295 nm. A mobile phase consisting of pH 2.8 phosphate

TIMOLOL MALEATE

685

buffer: methanol (3:2) is pumped at a rate of 1.8 mL/minute through a 30 cm X 3.9 mm column packed with octadecylbonded silica. About 15 p 1 of dissolution sample is injected onto the column. Not less than 80% of the labeled amount of timolol maleate is dissolved in 20 minutes. Alternative assay methods, as outlined in the previous sections (direct ultraviolet spectrophotometry, ultraviolet spectrophotometry via flow injection analysis and high performance liquid chromatography), can be used if an official cornpendial method is not required. 9.2

Potency Testing 9.2.1

Assay (33) Official compendial methods are available for both timolol maleate tablets and ophthalmic solution. Tablets are prepared for assay by weighing and finely powdering not less than twenty tablets. An accurately weighed portion of powder is dispersed in 0.05 M monobasic sodium phosphate and sonicated for five minutes. Acetonitrile is added followed by an additional five minutes of sonication. Water is then added and the dispersion is shaken for ten minutes. After dilution to volume with water, the sample may be centrifuged to remove dispersed solids from the timolol maleate solution. The supernatant liquid may be assayed with appropriate standards with the chromatographic system outlined above (Dissolution, Section 9.1) or by an alternative assay if an official cornpendial method is

686

DAVID J. MAZZO AND ALICE E. LOPER

not required. Tablets must contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of timolol maleate. The official compendia1 assay for timolol maleate opthalmic solution requires that an aliquot of the solution be diluted to volume with water to produce a 0.5 mg per mL timolol maleate solution. Five mL each of this solution and reference standard solutions are transferred to separators and made basic with pH 9.7 carbonate buffer. Timolol is extracted into toluene from the alkaline aqueous phase. The aqueous phase in each separator is then transferred to a second set of separators for an additional toluene extraction. The aqueous phase in the second set of separators is then discarded. Additional pH 9.7 buffer is added to the toluene remaining in the first set of separators and, after extraction, the aqueous phase is transferred to the second set of separators. After shaking, the aqueous phase is discarded and the toluene from both sets of separators is combined. Timolol in the combined toluene solutions is extracted into four portions of 0.1 N sulfuric acid. The sulfuric acid extracts are diluted to volume with additional acid solution. The extracts are assayed against a 0.1 N sulfuric acid blank at 2 9 4 nm with an ultraviolet spectrophotometer. The timolol maleate ophthalmic solution must

TIMOLOL MALEATE

687

contain not less than 90.0 percent nor more than 110.0 percent of label claim. 9.2.2

Dosage Uniformity ( 3 3 ) Tablets must also meet official compendia1 standards for uniformity of dosage units. Ten tablets, assayed individually must exhibit 85.0 to 115.0 percent of the label claim and have a relative standard deviation less than or equal to 6.0 percent. If one unit is outside the label claim range or the relative standard deviation is greater than 6.0 percent, or both, an additional twenty tablets must be tested. Of the thirty tested, not more than one may be outside the 85.0 to 115.0 percent range, and that must be within the 7 5 . 0 to 125.0 percent of label claim range ( 3 4 ) . Direct ultraviolet spectrophotometry, ultraviolet spectrophotometry via flow injection analysis and reversedphase high performance liquid chromatography with ultraviolet detection, all as previously described, are acceptable techniques for uniformity determinations. Typically sample preparation involves the disintegration and dissolution of single tablets in solvents identical to those used for assay. A set of extractions and other sample preparation steps identical to those described in the potency assay ( 9 . 2 . 1 ) are then employed.

DAVID J. MAZZO AND ALICE E.LOPER

688

9.2.3

Stability Testing ( 3 3 ) Timolol maleate is tested for stability using the high performance liquid chromatographic method described in the USP XXI. Based upon data generated in accelerated and room temperature stability studies, timolol maleate tablets carry a five year expiration dating.

TIMOLOL MALEATE

689

References

Physicians Desk Reference, 41st edition, Edward R. Barnhart, Publisher, Medical Economics Company, Inc., Oradell, NJ, U.S.A., 1987, pp. 1249-1251, 1340-1342. B. K. Wasson, W. K. Gibson, R. S. Stuart, H. W. R. Williams, C. H. Yates, J. Med. Chem 15(6), 651-655 (1972). L. M. Weinstock, United States of America Patents 3619370, 3655663 and 3657237 (to Merck and Co., Rahway, NJ, U.S.A.), 1971, 1972 and 1972, respectively.

4)

J. A. Ryan, Department of Pharmaceutical Research and Development, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication. S. M. Pitzenberger and J. S. Murphy, Department of Medicinal Chemistry, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication.

6)

The Merck Index, 10th edition, Martha Windholtz, editor, Merck and Company, Inc., Rahway, NJ, U.S.A., 1983, monograph no. 9284, pg. 1353. The United States Pharmacopeia, 21st revision, Mack Publishing Company, Easton, PA, U.S.A., 1985, pg. 7. J. McCauley, Department of Analytical Research, Merck Sharp and Dohme Research Laboratories, Rahway, NJ, U.S.A., personal communication.

9)

E. Oberholtzer, and Joseph V. Bondi; Department of Pharmaceutical Research and Development, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication.

DAVID J. MAZZO AND ALICE E. LOPER

690

10 1

The United States Pharmacopeia, 21st revision, Mack Publishing Company, Easton, PA, U.S.A., 1985, pp. 1063-1064.

111

F. P. Bigley, R. L. Grob, and G. S. Brenner, Anal. Chim. Acta 181, 241-244 (1986).

12 1

P. Tway, Department of Analytical Research, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication.

13

R. Roman, W. Hagerman, Department of Pharmaceutical Research and Development, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication.

14 I

D. J. TOCCO,A. E. W. Duncan, F. A. DeLuna, H.B. Hucker, V. F. Gruber and W. J. A. Vandenheuvel, Druq Metab. Disp. 3, 361 ( 1975 1.

1 51

A. Bobik, G. L. Jennings, P. Ashley and P. I. Korner, Eur. J. Clin, Pharmacol. 16, 243 (1979).

16 1

R. K. Ferguson, P. H. Vlasses, J. R. Koplin, G. I. Holmes, P. Huber, J. Demetriades and W. B. Abrams, Br. J. Clin. Pharmacol. 14, 719 (1982).

17 1

T. W. Wilson, W. B. Firor, G. E. Johnson, G. I. Holmes. M C. Tsianco. P. B. Huber and R. 0. Davies; Clin. Pharmacol. Ther. 32, 676 (1982).

18

0. F. Else, H. Sorensen and I. R. Edwards,

19 1

R. Mantyla, P. Mannisto, S. Nykanen, A. Koponen-and U. Lamminsivu, EuE. J. Clin. Pharmacol. 24, 227 (1983).

20 1

M. B. Affrime, D. T. Lowenthal, J. A. Tobert. J. Shirk, B. Eidelson, T. Cook and G. Onesti, Clin.'Pharmacol. Ther 27, 471

Eur. J. Clin. Pharmacol. 14, 431 (1978).

(19801.

TIMOLOL MALEATE

2 11

G. Alvan, B. Calissendorff. P. Seideman. K. Widmark and G. Widmark, Clin. Pharmacokin. 5, 9 5 ( 1 9 8 0 ) .

22

C. V. Leier, N. D. Baker and P. A. Weber, Ann. Int. Med. 104, 1 9 7 ( 1 9 8 6 ) .

23

C. J. Schmitt, V. J. Lotti and J. C. LeDouarec, Arch. Ophthalmol. 9 8 , 5 4 7 ( 1 9 8 0 ) .

24 1

P. H. Vlasses. L. G. T. Ribeiro, H. H. Rotmensch, J. V. Bondi, A. E. Loper, M. Hichens, R. A. Clementi, W. B. Abrams and R. K. Ferguson, Clin. Pharmacol. Ther. 3 5 , 2 7 9 (1984).

25 1

D. J. TOCCO,A. E. W. Duncan, F. A. DeLuna, J. L. Smith, R. W. Walker and W. J. A. Vandenheuvel, Drug Metab. Disp. 8, 2 3 6 (1980).

26 1

B. K. Wasson, J. Scheigetz, C. S. Rooney, R. A. Hall, N. N. Share, W. J. A. Vandenheuvel, B. H. Arison, 0. D. Hensens, R. L. Ellsworth and D. J. TOCCO, J. Med. Chem. 2 3 , 1 1 7 8 (1980).

27 1

D. T. Lowenthal, J. M. Pitone, M. B. Affrime, J. Shirk, P. Busby, K. E. Kim, J. Nancarrow, C. D. Swartz and G. Onesti, Clin. Pharmacol. Ther. 2 3 , 6 0 6 ( 1 9 7 8 ) .

28 1

J. A. Vedin, J. K. Kristianson and C. E. Wilhelmsson, J. Clin. Pharmacol. 2 3 , 43 ( 1 9 8 2 1.

29 1

D. J. TOCCO, F. A. DeLuna and A. E. W. Duncan, J. Pharm. Sci. 6 4 , 1 8 7 9 ( 1 9 7 5 ) .

30

J. R. Carlin, R. W. Walker, R. 0. Davies, R. T. Ferguson and W. J. A. Vandenheuvel, J. Pharm. Sci. 6 9 , 1111 ( 1 9 8 0 ) .

31

J. B. Fourtillan. M. A. Lefebvre. J. Girault and P. Courtois,'J. Pharm. Sci. 70, 5 7 3 (1981).

69 1

DAVID J. MAZZO AND ALICE E.LOPER

692

32)

M. R. Gregg and D. B. Jack, J . Chromatog.

305,

244 (1984).

33)

The United States Pharmacopeia, 21st revision, Mack Publishing Company, Easton, PA, U.S.A., 1985, pp. 1063-1064.

34)

Ibid, pp. 1277-1278.

TRAZODONE HYDROCHLORIDE

DeMiS K.J. Gorecki and Roger K. Verbeeck College of Pharmacy University of Saskatchewan Saskatoon, Saskatchewan, Canada

1.

Introduction

1.1. History 1.2. Therapeutic Category 2.

Description 2.1.

2.2.

2.3. 2.4. 2.5. 2.6. 3.

Nomenclature 2.1.1. Chemical Name 2.1.2. Generic Name 2.1.3. Proprietary Names Formulae 2.2.1. Empirical 2.2.2. Structural 2.2.3. Registry Numbers Molecular weight Elemental Composition Appearance, Color and Odor Patent Information

Phvsico-Chemical ProDerties 3.1. 3.2. 3.3. 3.4. 3.5. 3.6. 3.7.

Melting Range Solubility Stability and Storage Dissociation Constant Crystal structure Differential Scanning Calorimetry Spectral Properties 3.7.1. Ultraviolet Spectrum 3.7.2. Fluorescence Spectrophotometry 3.7.3. Infrared Spectrum 3.7.4. Nuclear Magnetic Resonance (NMR) Spectra

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

693

Copyright 0 1987 by Academic Press, Inc. All rights of reproductionin any form reserved.

DENNIS K. G. GORECKI AND ROGER K. VERBEECK

694

3.7.4.1. Proton Magnetic Resonance (PMR) Spectra 3.7.4.2. Carbon-13 Nuclear Magnetic Resonance Spectrum (~%-NMEX) 3.7.5. Mass Spectrometry 3.7.5.1. Electron Impact (EI) 3.7.5.2. Chemical Ionization (CI) 4. Synthesis 5.

Pharmacokinetics 5.1. Absorption 5.2. Distribution 5.3. Elimination

6. Methods of Analysis 6.1. Identification 6.2. Chromatographic Analysis 6.2.1. Thin Layer Chromatography (TLC) 6.2.2. Gas-Liquid Chromatography (GLC) 6.2.3. High Performance Liquid Chromatography (HPLC) 7. References

TRAZODONE HYDROCHLORIDE

695

1. Introduction 1.1. History Trazodone hydrochloride was originally synthesized in 1966 by Palazzo and Silvestrini (1) in the chemical laboratories of the firm Francesco Angelini. The molecule consists of a triazolopyridine ring and a phenyl piperazinylalkyl moiety. It is the first triazolopyri -dine derivative to be used clinically, and therefore, represents a new chemical class of antidepressant drugs. Its development was a result of an organized approach rather than fortuitous discovery and was based on the hypothesis that the mechanisms responsible for physical and mental pain are the same (2,3). The history, pharmacology and clinical data on trazbdone hydrochloride is well documented in a number of review articles and monographs ( 3 - 8 ) . In addition, an analytical profile has been described (9). 1.2. Therapeutic Category Trazodone hydrochloride is an antidepressant drug indicated in the symptomatic treatment of moderate to severe depression. Its major advantages include a low incidence of anticholinergic and cardiovascular side effects along with minimal stimulatory effects upon dopamine and norepinephrine receptors and is, therefore, considered particularly useful in the geriatric population (5). 2. Description 2.1. Nomenclature 2.1.1.

Chemical Name

2-{3-[4-(3-chlorophenyl)-l-piperazinyll propyl)-1,2,4-triazolo-[4,3-a]pyridin-3(2H)-one monohydrochloride

696

DENNIS K. G. GORECKI AND ROGER K. VERBEECK

2.1.2. Generic Name Trazodone hydrochloride 2.1.3.

Proprietary Names

Desyrel, Manegan, Molipaxin, Pragmazone, Thombran, Trittico 2.2. Formulae 2.2.1.

Empirical C19H22C1N50 HC1

2.2.2.

Structural

2.2.3.

Registry Numbers

Chemical abstracts; Trazodone:19794-93-5 Trazodone hydrochloride:25332-39-2 2.3. Molecular Weight 408.33 2.4. Elemental Composition C, 55.89%; HI 5.68%; C1 17.36%; N, 17.15%; 0, 3.92%

2.5. Appearance, Color and W o r

White, odorless crystals (plates)with a bitter taste. 2.6. Patent Information U.S. Pat. 3,381,009 (1968 to Angelini Francesco)(l). Brit. Pat. 1,117,068 (11).

TRAZODONE HYDROCHLORIDE

3.

691

Physico-Chemical Properties 3.1. Melting Range The melting point for trazodone free base is 96OC (9). The hydrochboride salt melts with decomposition in the range 222-228 C (grlOr1l). Under vacuum decomposihion does not occur and a melting range of 231-232.5 C is reported (9). 3.2. Solubility Trazodone hydrochloride is soluble in chloroform, sparingly soluble in water, ethanol and methanol (10) and insoluble in common organic solvents (9). 3.3. Stability and Storage Trazodone hydrochloride tablets should be stored at room temperature in tight, light resistant gontainers and protected from temperatures in excess of 40 C (13). 3.4. Dissociation Constant The reported pKa for trazodone, in 50% ethanol, is 6.14 (9). This value was obtained potentiometrically using a glass-calomel electrode. 3.5. Crystal Structure The crystal and molecular structure for trazodone hydrochloride has been determined (14). X-ray diffraction data were obtained using an Oak Ridge diffractometer employing molybdenum Kolradiation with a niobium filter. Trazodone hydrochloride, C H C1N 0-HC1,crystals are monoclinic with space group182?3c a8d with cell dimension8 a=16.866[31, b..1O.66[2lr c=11.230[21, A, =93.04(1) and Z=4 with the final R factor of 0.042 for 3017 reflections. The megsured and calculated densities are 1.344 and 1.345 mg m- respectively for Z=4. Figure 1 depicts the 3-dimensional, X-ray derived, structure of trazodone hydrochloride (58). Bond lengths and angles are tabulated in Tables 1 and 2.

Figure 1.

Three-Dimensional, X-Ray Derived, Structure of Trazodone Hydrochloride.

Table 1.

0

Intramolecular Bond Lengths ( A ) of Trazodone Hydrochloride 0

0

A -

D i s tances

A -

1.222 1.349 1.386 1.308 1.384 1.419 1.344 1.421 1.337 1.384 1.394

N(2)-C(10) C( lO)-C(ll) C(ll)-C(12) C( 12)-N( 13) N( 13)-H(13) N( 13)-C1(1) H( 13)-C1(1) N( 13)-C( 14) C (14)-C ( 15) C (15)-N( 16) N(16)-C(17)

1.451 1.518 1.512 1.498 0.872 3.052 2.180 1.491 1.497 1.455 1.463

Distances C(17)-C(18) C(18)-N(13) N(16)-C(19) C(19)-C(20) C(20)-C(21) C(21)-C(22) C(22)-C(23) C(23)-C(24) C(24)-C(19) C(22)-C1(2)

0

A -

1.506 1.495 1.407 1.394 1.378 1.380 1.363 1.383 1.391 1.747

Table 2.

Bond Angles ( " ) of Trazodone Hydrochloride

Angles 0 (1) -C (1)-N(l) 0 (1)-C( 1)-N(8)

C(l)-N(2)-C(lO) C (l)-N( 2)-N( 3) C ( 1)-N (8) -C ( 9) N(2)-C(l)-N(8) C( 10)-N( 2)-N(3) N( 2)-N( 3)-C(9) N(3)-C(9)-C(4) N (3) -C (9)-N (8) C(9) -C(4) -c(5) C(4)-C(5)-C(6) C (5)-C( 6)-C ( 7 ) C(6)-C(7)-N(8)

("> 130 128 125 114 108 103 121 104 130 112 119 122 121 118

Angles C(7)-N(8)-C(9) N(8)-C(g)-C(4) C(l)-N(8)-C(7) N(2)-C(lO)-C(11) C( lO)-C(ll)-C( 12) C(ll)-C(l2)-N(13) C(12)-N(13)-C(14) C(12)-N(13)-H(13) N (13) -H (13)-C1(1) N (13)-C (14)-C( 15) C ( 14)-C ( 15)-N (16) C(15)-N(16)-C(17) N(16)-C(17)-C(18) C(17)-C(18)-N(13)

(") 123 118 129 113 112 113 109 114 172 112 111 111 111 111

Angles C( 18)-N( 13) -C ( 1 4 ) C(17)-N(16)-C(19) C(15)-N(16)-C(19) N( 16)-C (19)-C(20) N(16)-C(19)-C(24) C(19)-C(20)-C(21) c(20)-c (211-c(221 C(21)-C(22)-C(23) C(22)-C(23)-C(24) C( 23)-c(24)-c( 19) c (24) -c (19) -c (20) Cl(2) -C( 23)-C(22) C1(2)-C(23)-C(24)

108 118 117 121 121 120 122 117 118 120 118 119 118

TRAZODONE HYDROCHLORIDE

70 1

This study revealed a number of interesting features about trazodone which may be useful in proposing structural requirements for neuroleptic activity. The piperazine ring is in the normal chair conformation with “13) extending upward and N(16) down. The bond lengths and angles about N(13) resemble that of a protonated2 mine and those associated with N(16) demonstrate sp character due to the attached chlorophEny1 ring. Torsiogal angles about N(2)-C(10)(93.2 1 C(ll)-C(12) (178.2 ) suggest that the propylene side chain is fully extended. In addition, the triazolopyridine nucleus (including C(10)) and the chlorophenyl moiety are planar groups

.

3.6. Differential Scanning Calorimetry A differential scanning calorimetry (DSC) curve for trazodone hydrochloride has been reported (9). Data obtained using a Perkin Elmer DSC 1B instrument was used as a purity index for trazodone.

3.7. Spectral Properties 3.7.1. Ultraviolet Spectrum The ultraviolet spectrum of trazodone hydrochloride was scanned from 200 to 360 nm with a Gilford Response spectrophotometer at a concentration of 30 and 75!~moles/Lin methanol and 30 Umoles/L water (Figure 2). Each spectrum is characterized by two well defined and two less prominent peaks. The spectra in methanol show maxima at 213, 256, 278 and 312 nm while that in water displays maxima at 211, 246, 275 and 312 nm. The latter are in agreement in previously reported data (9,101. The reported A (l%, 1 cm) and molar absorptivity values for trazodone hydrochloride in water are s m r i z e d in Table 3 (9). Table 3. Ultraviolet Characteristics of Trazodone Hydrochloride max

(m)

211 246 274 312

A( 106,lcm)

1225 287 94 94

E

50,100 11,730 3,040 3,840

A 10

Figure 2 .

Ultraviolet Spectra of Trazodone Hydrochloride (-,

methanol;

---- , water).

TRAZODONE HYDROCHLORIDE

3.7.2.

703

Fluorescence Spectrophotometry

Trazodone hydrochloride, like most 2-alkyl-substituted-l,2,4-triazolo-[4,3-al pyridin-3 ( 2H)-ones , shows an intense fluorescence-when irradiated with ultraviolet light. The excitation and emission wavelengths for trazodone are 317 and 455 nm respectively (9). 3.7.3.

Infrared Spectrum

The infrared spectrum of trazodone hydrochloride is shown in Figure 3. The spectrum was obtained with a Beckman AccuLab 4 infrared spectrophotometer from a compressed KBr disc. Structural assignments of some of the characteristic absorption bands are presented in Table 4. Table 4. Infrared Characteristics of Trazodone Hydrochloride Frequency (cm-' )

Intensity

Assignment

2975-2840 2535,2460 1705 1640 1595

W M S M S

Aliphatic CH +N-H stretching C=O stretching triazoline C=N stretch aromatic C=C stretch

M=Medium; S=Strong; W=Weak

.

Other characteristic bands appe r at 1540, 1491, 1447, 1350, 1273, 943 and 745 cm-'i Principle peaks, wavenumbers and structural assignments are in agreement with previously published results (9,lO). 3.7.4. Nuclear Magnetic Resonance (NMR) Spectra 3.7.4.1.

Proton Magnetic Resonance (PMR) Spectra

The PMR spectra of trazodone base and trazodone hydrochloride were recorded on a Bruker AM 300 NMR spectrometer employing a frequency of 300.13 MHz. Spectral assignments were confirmed by homodecoupling, COSY and heteronuclear correlation experiments.

1

okooo

3

4 I

I

3000

I

MICRONS

5 I

I

I

2000

I

I

1600

I800

WAVENUMBER

Figure 3.

6

7 I

1400

CM

Infrared Spectrum of Trazodone Hydrochloride-KBr Disc.

I

8

9 I

1

1200

10 1

1000

11 I

12 1

800

14

16

10

600

TRAZODONE HYDROCHLORIDE

705

The spectrum of trazodone base obtained in CDCl is presented in Figure 4. Scale expansion $n the region 6.4 to 7.8 ppm is illustrated in Figure 5. The chemical shifts, multiplicities and assignments are compiled in Table 5. These data are consistent with the results reported using a 60 MHz instrument ( 9 ) . Table 5. Proton Magnetic Resonance Assignments for Trazodone Base

3

i

/y\ /-7 9

*\

1

12

10

13

5

6

11

U

Chemical Number Shift of 6 (ppm) Multiplicities Protons Assignment 7.75 7.11 6.84 6.76 6.46 4.09 3.13 2.56 2.49

2.05

d m m m m t t t t m

1

3 1

2

1 2 4 4 2 1

1

314,6 8 517

2

9 13 12 11 10

d=doublet; t=triplet; m=multiplet The PMR spectrum of trazodone hydrochloride in DMSO-d is shown in Figure 6. Scale expansion in the region 6.5 to 8.0 ppm is depicted in Figure 7. Chemical shifts and assignments are given in Table 6. It can be seen that protonation of the piperzine ring nitrogen nearest the propyl chain affects the chemical shift and splitting pattern of adjacent protons in the piperazine ring.

UCJ I " " I " " I ~ " ' " ' 1 " ' " " ' ' " I ' " ' I " " I ' " ' I ' ' ~ ' I " " I ' ' "

8.0

Figure 4 .

7.5

7.0

6.5

6.0

5.5

5.0

PPM

4.5

4.0

3.5

3.0

P r o t o n Magnetic Resonance Spectrum of Trazodone Base i n CDC13.

LL

2.5

2.0

r

1 -

7.8

Figure 5.

'

I

7.6

I

I

7.4

I

7.2

I

7.0

PPM

I

6.6

I

6.6

I

6.4

Proton Magnetic Resonance Spectrum o f Trazodone Base in CDCl with Scale 3 Expansion (6.4-7.8 ppm).

I " " " " ' I

8.0

Figure 6.

7.5

I

1

1

1

1

'

7.0

1

I

I

1

6.5

1

1

"

[

'

I

6.0

1

1

I

l

5.5

"

l

1

'

I

5.0

PPM

1

1

1

4.5

'

1

I

I 1

4.0

1

1

1

~

I

3.5

I

I

I

~I

3.0

1

l

1

1

~

2.5

1

1

1

1

2.0

Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d6.

]

1

1

1

r

I

7.9

Figure 7.

I

7.8

1

7.7

I

7.6

I

7.5

I

7.4

I

7.3

I

PPM

7.2

I

7.1

I

7.0

I

6.9

I

6.6

I

6.7

I

-6.6

Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6 with Scale Expansion (6.5-8.0 ppm).

710

DENNIS K. G. GORECKI AND ROGER K. VERBEECK

Table 6.

Proton Magnetic Resonance Assignments for Trazodone Hydrochloride

Chemical Number Shift of 6 (ppm) Multiplicities Protons Assignment 7.87 7.25 7.04 6.95 6.86 6.64 4.01 3.86 3.53 3.15 2.25

m m m dd dd m t d d m m

1

3 1 1 1 1 2 2 2 6 2

1 31416 8 5 7 2 9 1 2 ,13

12,13

11,14,15 10

d=doublet; dd=doublet,doublet; t-triplet m=multiplet 3.7.4.2.

Carbon-13 Nuclear Magnetic Resonance Spectrum (IjC-NMR)

The 13C-NMR spectrum of trazodone hydrochloride base obtained in DMSO-d6 is given in Figure 8. The spectrum was obtained at ambient temperature on a Bruker AM 300 NMR spectrometer at 75.47 MHz with broad band proton decoupling. Chemical shifts, and assignments are outlined in Table 7. Spectral assignments were confirmed by C-13m-1 heteronuclear correlation experiments and published chemical shift data.

d 1

'

~

160 Figure 8.

'

l

'

~

140

'

l

'

120

,

'

l

'

100

~

'

,

PPM

'

80

,

'

,

'

60

,

'

l

40

'

,

'

"

,

'

l

'

20

Carbon-13 Nuclear Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6'

712

DENNIS K. G. GORECKI AND ROGER K. VERBEECK

Table 7. Carbon-13 Nuclear Mametic _. .~~ Resonance Assignments forTrazodone Hydrochloride

Chemical Shift 6 (ppm) 150.69 147.88 141.06 133.84 130.51 130.47 123.82 119.03 115.12 3.7.5.

Assignment 6 1 7 11

4 , s or 9

4,5 or 9 2 10 12

Chemical Shift 6 (ppm)

114.92 113.99 110.76 52.79 50.23 44.66 42.54 22.78

Assignment 4,s or 9 8 3 13 17 16 15 14

Mass Spectrometry 3.7.5.1.

Electron Impact (EI)

An electron impact mass spectrum of trazodone hydrochloride is presented in Figure 9. This was obtained by direct solid insertion probe at an electron eneljgy of 70 eV and a source temperature of 180 C using a VG Analytical MM 16F single focussing mass spectrometer linked to a VG 2035 data system+ The spectrum shows a molecular ion peak M at a mass/charge (m/z) ratio of 371 with a relative intensity of 13.2% and a base peak at 205. Prominent diagnostic ions are observed at m/z 70, 136, 166, 176, 209, 231 and 278. The fragmentation pattern leading to the various ions is outlined below.

TRAZODONE HYDROCHLORIDE

713

20:91J 231

Mass spectra of trazodone and other related compounds have been reported elsewhere (9,15,16,17,18). 3.7.5.2.

Chemical Ionization (CI)

The chemical ionization spectrum of trazodone hydrochloride is shown in Figure 10. This spectrum was also determined on a VG Analytical MM 16F mass spectrometer, however, using ammonia as a reagent gas. The spectrum shows a pseudo molecular ion at a mass to charge ratio of 372. 4. Synthesis A number of synthetic pathways have been described for the synthesis of trazodone hydrochloride (1,11,19,20). All of the reported routes employ 1,2,4-triazo-[4,3-a]-pyridin3(2H)-one (I) or 2-substituted derivatives as stzrting matFria1. The more important methods are outlined in Scheme 1. The original synthesis reported by Palazzo and Silvestrini (1) consisted of the condensation of l-(3-chloropheny1)-4-(3-~hloropropyl)-piperazine with the sodium salt of I. Other related patented methods were simultaneously developed in the same laboratories (11). More recently, modications of existing pathways were reported (19). One method involves the condensation of the usual triazopyridine (I) with bromochloropropane to yield the 2-(3-~hloropropyl)-derivative (11). Subsequent treatment with N-(3-chlorophenyl)piperazine gives trazodone. Alternatively, I is alkylated with bromochloropropane and treated with diethanolamine to form the di-alcohol (111). This product is then chlorinated with thionyl chloride and subsequently reacted with 3-chloroaniline to yield trazodone.

714

DENNIS K. G.GORECKI AND ROGER K. VERBEECK m

100-

80 > c

-

H

cn

-6 c

60 -

z

-

W

>

0 100

50

1-50

250

200

300

350

MASSKHARGE RAT I0

Figure 9.

Mass Spectrum of Trazodone Hydrochloride Electron Impact.

1s

I

231

I 1 1

Figure 10.

Mass Spectrum of Trazodone HydrochlorideChemical Ionization.

375

Cl-(CH

) -N

2 3

w

CH2 CH20H

c1

I

CH2CH 20H

HC1

I1

.HC1 TRAZODONE c1 HYDROCHLORIDE Synthesis of trazodone hydrochloride

0

Scheme 1.

716

DENNIS K. G . GORECKI AND ROGER K. VERBEECK

The synthesis of 14-C-labelled trazodone hydrochloride has also been published ( 2 0 ) . This procedure also employs the classical condensation reaction described in the original patent.

5. Pharmacokinetics 5.1. Absorption

Following oral administration to man, trazodone is rapidly absorbed. The time to reach m a x i m plasma concentrations typically varies from 0.5 to 4.0 hours in fasting subjects (21-26). Peak plasma concentrations following oral administration of a single 50 mg dose range from 0.5 to 1.0 pg/mL (21,24-26). Following oral ingestion of a single 100 mg trazodone hydrochloride dose average m a x i m plasma concentrations of 1.61 Pg/mt and 1.66 ug/mL were reported for a capsule and liquid formulation respectively ( 2 2 ) . Oral and intramuscular bioavailability of trazodone is almost complete as shown by the virtually identical plasma concentration-time profiles obtained following oral intramuscular and intravenous administration to healthy volunteers ( 2 3 ) . Ingestion of food delays the absorption of trazodone from the gastrointestinal tract but does not seem to affect its bioavailability (23,27). Studies in rats and rabbits have shown that food delays the oral absorption by decreasing the transfer rate of drug from the stomach to the duodenum ( 2 3 ) . 5.2. Distribution

Trazodone is a weak base with a pKa of 6.14 (91, and therefore, exists mostly in the unionized form at physiological pH. The drug distributes in a relatively large volume: Vd values reported in the literature or calculated from published data range approximately from 1-2 L/kg for healthy volunteers (21,22,24,25,28). Trazodone is highly bound to plasma proteins. In vitro protein binding studies with human plasma have shown that trazodone is 96, 98 and 97% bound at concentrations of 1.0, 0.1 and O.Olvg/mL respectively ( 2 9 ) . Studies in laboratory animals show that trazodone readily crosses the blood brain barrier but is minimally transferred across the placenta (30,311. Little information is available in man concerning its distribution in organs and body fluids. Excretion of trazodone in human breast milk has been studied and found to be very small ( 3 2 ) . The average milk/plasma ratio was 0.14, therefore, exposure to babies via breast milk would be minimal.

TRAZODONE HYDROCHLORIDE

717

5.3. Elimination In general, plasma concentrations of trazodone in man decline in a biphasic manner with a mean half-life of approximately 4 hours during the period 3-10 hours following dosing and a terminal elimination half-life of 6-12 hours (21,22,25,28,32). Total body clearance (assuming an oral bioavailability of 100%) ranges from 150 mL/min. to 400 mL/min. (21,24,32). Patients treated with trazodone on a twice daily dosage schedule show significantly higher plasma levels in the morning as compared to the levels at night (33). A possible diurnal variation in clearance may be responsible. Trazodone is extensively metabolized via hydroxylation, N-oxidation and hydrolysis (Scheme 2) (34). Hydroxylation occurs in the benzene ring (4-hydroxyphenyl metabolite, I) and in the triazolopyridine ring (dihydrodiol, 11) while N-oxidation occurs on a piperazine nitrogen (N-oxide, 111). A hydrolytic reaction results in the formation of 3-0~0-1,2,4-triazo10[4,3-a]-pyridin-2-yl propionic acid ( IV) and 1-m-chlorophenylpiperazine ( V ), a pharmacologTcally active metabolite ( 35,361. Small concentrations of 1-m-chlorophenylpiperazine, approximately 1% of 'frazodone plasma concentrations, have been reported in man (37). Whether these small concentrations contribute to the overall effects of trazodone remains unclear. Approximately 70-75% of an oral dose of the drug is excreted in urine within 72 hours of administration, most as metabolites. Only less than 1% of the dose is recovered in urine as unchanged drug (26,34). The remainder of an oral dose is excreted in feces, involving biliary excretion, mainly as trazodone metabolites (23,271. Results from animal studies indicate that trazodone does not induce its own metabolites (38). 6.

Methods of Analysis 6.1. Identification Trazodone hydrochloride reacts with certain reagents to produce characteristic colors which may be useful for its identificahion. When treated with Liebermanns Test reagent at 100 C (sodium nitrite-sulphuric acid) trazodone gives a transient violet color whereas with Mandelin's reagent (ammonium vanadate-sulphuric acid) a grey color develops which later changes to violet (10).

/ QOH

OH

Cl

0

TRAZODONE

\

GLUCURONIDE

IV

0

I

GLUCURONIDE

Scheme 2.

Biotransformation pathways of trazodone in man

TRAZODONE HYDROCHLORIDE

719

An infrared spectroscopic method is described which is useful for the rapid emergency identification of certain psychtropic drugs including trazodone (39). The method employs chloroform extraction of patient's urine at different pH's and utilizes the infrared finger print region and spectra of authentic standards to confirm identity. Infrared spectra are determined as compressed potassium bromide discs from dried chloroform extracts.

6.2. Chromatographic Analysis 6.2.1. Thin-Layer Chromatography ( T L C ) A number of thin-layer chromatographic systems have been developed for the determination of trazodone. These systems have been employed for the identification (9,10,40,41,42)and quantitation (43,44,45) of the drug both as pure drug substance as well as in biological fluids and tissues. A variety of detection methods have been used and most systems use silica gel plates as absorbent. A summary of some of the reported TLC methods is given below.

System I ( 9 )

Absorbent: Merck Kieselgel-0.25 mm Mobile Phase: i) cyclohexane-benzene-diethylamine (5:4:1) ii) chloroform-methanol-isopropanol-ether-NH (60:25:25:2) i ii ) n-butanol :wate? :NH ( 20:10:2 1 iv) abs. ethano1:NH (35:5) Detection: ultra-viol& light (366 MI), ninhydrin, rhodanine, ammonium sulphocyanide and cobaltous chloride or potassium dichromate, sulphuric acid with glacial acetic acid.

System I1 (10)

Absorbent: Silica gel G, 0.25 mm treated with potassium hydroxide in methanol. Mobile Phase: i) methano1:NH (100:1.5) ii) cyclohexane.toluene:diethyl3 amine (75:15:10) Detection: ultra-violet light, Dragendorff's reagent or acidified iodoplatinate solution.

DENNIS K. G . GORECKI AND ROGER K. VERBEECK

720

System 111 (40)

Absorbent: Silica gel, F254, 0.25 mm Mobile Phase: i) methano1:NH (100:1.5) ii) cyclohexane.to1uene:diethyl3 amine (75:15:10) iii) ch1oroform:methanol (9:l) iv) acetone Detection: Dragendorff's reagent

System IV ( 4 2 )

Absorbent: Silica gel 60, F254, 0.25 nun Mobile Phase: i) ethylacetate:methanol:30% NH3 (85:10:5) ii) cyc1ohexane:toluene:diethylamine (65:25:10) iii) ethy1acetate:chloroform (50:50) iv) acetone Detection: 10% sulphuric acid followed by Dragendorff's reagent and acidified iodoplatinate solution

System V ( 4 5 )

Absorbent: Silica gel, 0.25 nun Mobile Phase: i) methano1:ammonia (100:l.S) ii) ethy1acetate:methanol:amonia ( 85:10:5) Detection: 5% sulphuric acid, iodoplatinate solution followed by Dragendorff's reagent

6.2.2. Gas-Liquid Chromatography (GLC) A number of GLC procedures have been published for the determination of trazodone. The technique has been used for routine detection of the drug (9,10,17), biopharmaceutics and pharmacokinetic studies (16,19,28,47), as well as in therapeutic and overdose stiuations.(45,46) Chromatography of trazodone is readily accomplished without derivatization using a variety of stationary phases in packed or capillary columns. Detectors employed include flame ionization (FID), nitrogen-phosphorous (NPD) and mass spectrometry (MS). The conditions for the reported GLC methods are as follows:

TRAZODONE HYDROCHLORIDE

72 1

System I (9)

Type of sample: drug substance Column: 6 ft x 1/4" I . D . glass column packed with 1.5% OV-17 on Chromosorb W HP 80-100 Carrier gas: Nitrogen, &3 lb/inch 2 Temperature: column 268 C, injector 32OoC Retention time: approximately 3.5 min. Detector: not stated, presumably F I D

System I1 (10)

Type of sample: general Column: 2 m x 4 mm I . D . glass packed with 2.5% SE-30 on chromosorb G 80-100 Carrier gas: nitrogen, 45 mt/min. Temperature: column, retention index - 10 Retention index: 3250 Detector: F I D or NPD

System 111 (16)

Type of sample: plasma ( rat) Column: 80 cm x 4 mm I . D . glass column packed with 1% OV-1 on Chromosorb Q 100-120 Carrier gas: nitrogen, 33 mL/min. Tevrature: column, 240 C; injector, 270 C Retention time: 2 min. Detector: F I D and MS

System Iv ( 4 5 )

Type of sample: biological fluids and tissue Column: 4 m x 0.25 mm I.D. DB-1 fused-silica capillary (0.25wm film) Carrier gas: hydrogen, 48 cwsec. Temperature: column, 265 C Retention time: 2.87 min. Detector: F I D (or NPD) In addition: i) Column: 1.8 m column with 3% SP 2250 on Supegcoport 100-120 Temperape: 10 C/min. from 140-300 C, hold 5 min. Retention time: 3.89 min. Detector: NJ?D (MS) ii) Column: 1.8 m column with 3% SP 2100 on Supegcoport 100-120 Temperape: 13 C/min from 160-320 C, hold 7 min. Retention time: 3.10 min. Detector: NPD (MS)

DENNISK. G . GORECKI AND ROGER K. VERBEECK

722

System V (28)

of sample: plasma Column: 1.23 m x 2 mm I.D. glass column packed with 3% OV-101 on Chromosorb W HP 80-100 Carrier gas: helium, 30 p/min Tevrature: column, 260 C, injector, 310 C; detector, 275OC Retention time: 5.1 min. Detector: NPD

System VI (47)

Type of sample: plasma, brain tissue (rat) Column: 1 m x 3 mm I.D. glass column packed with 3% OV-1 on Gas Chrom Q 80-100 Carrier gas: nithogen, 30 ml/min. Temperature: 270 C Retention time: 4.2 min. Detector: NPD (MS)

System V I I (17)

Type of sample: urine (rat) Column: 60 cm x 1 mm I.D. nickel capillary column packed with 20% UCC-W (Hewlett Packard) on Chromosorb W AW DMCS 80-100 Carrier gas: helium, 7 p/min. Temperahure: column, 20 $pin. from 100-300 C; injector, 270 C Detector: MS

System VIII (18)

'rype of sample: plasma

6.2.3.

"ype

Column: 4 m x 0.25 mm I.D. DB-1 fused-silica capillary (0.25 pm film) Carrier gas: helium, 2000cm/sec. Teperature: c o l p , 170 C for 1 gin., 15 C/min. to 255 CA injector, 250 C; separator oven 255 C Retention time: 3 min. Detector: MS

High Performance Liquid Chromatography

3HPLC )

Several HPLC methods have been developed for the determination of trazodone; most of which have been employed in analysis of biological fluids and tissues for unchanged drug and metabolites. The majority of the procedures utilize reverse-phase chromatography with mobile phases comprised of an aqueous buffer with organic solvent modifiers (21,32,43,48,50-52,54). Others include

TRAZODONE HYDROCHLORIDE

723

reversed-phase with ion-pairing agents (49,56,57) and normal-phase systems (53,551. Detector systems include ultra-violet ( w ) at various wavelengths, fluorescence and electrochemical oxidation. A summary of the reported HPLC systems is presented below. System I (48)

Type of sample: serum or plasma

Coluum: 150 x 4.6 mm Supelcosil LC-8 DB Mobile phase: phosphate buffer 0.01M (pH 4.6): methanol: acetonitrile (47:37:16) Temperature: ambient Flow rate: 2.1 mL/min. Retention time: 3.2 min. Detection: UV at 206 nm

System I1 (32)

Type of sample: breast milk Column: 150 x 3.9 mm Nova-Pak C18 (4p m ) Mobile phase: methano1:phosphate buffer 0.082 M (PH 6.5): acetonitrile (39:47:14) Temperature: ambient Flow rate: 1.5 mL/min. Retention time: 5 min. Detection: W at 211 mm

System I11 (49)

Type of sample: plasma and tissue Column: 250 x 4.6 mm trimethyl silyl (5urn) Mobile phase: acetonitri1e:phosphate buffer (pH 3.0) (27:73) with 0.02 M heptane sulfonic acid and 0.04 M triethylamine Temperature: ambient Flow rate: 1.5 mL/min. Retention time: 12.8 min. Detection: W at 214 nm

System Iv (50)

Type of sample: plasma Column: 300 x 4.6 mm Bondapak C18 (10 V M ) Mobile phase: phosphate buffer 0.05 M (pH 4.7): acetonitrhle (72:8) Temperature: 60 C Flow rate: 2.5 mL,/min. Retention time: 8 min. Detection: W at 214 nm

724

DENNIS K. 0 . GORECKI AND ROGER K. VERBEECK

System V (51)

Type of sample: serum Column: 250 x 4 . 6 mm LiChrosorb l o w 8 Mobile phase: acetonitrile: phosphate buffer, 0.6%; pH 3.0 (1:1) Temperature: ambient Flow rate: 2 mL/min. Retention time: 3 . 8 min. Detection: W at 240 nm

System VI ( 5 2 )

Type of sample: biological fluids Column: 250 x 4 . 6 mm Ultrasphere ODs (octadecylsilyl) 5 p m) Mobile phase: i) phosphate buffer, 0.1 M (pH 7.0): acetgnitrile (10:3.8); 3.0 ml/min; 65 C; retention time ( 1 0 : 3 . 8 ) ; 8 min. ii) phosphate buffer 0.1 M (pH 3.0): acgtonitrile (10:3); 1.5 mL/min.; 60 C; retention time, 1.25 min. Detection: W at 242 nm

System VII ( 4 3 )

Type of sample: serum and urine Column: ODs (C18) Mobile phase: methanol: 0.011 M phosphate buffer (pH 7.5): acetonitrile (60:38:2)

Detection: W at 250 nm

System VIII (21)

of sample: plasma Column: 250 x 4.6 rmn Spherisorb S5 ODs Mobile phase: acetonitrile: 0.05 M sulphuric acid ( 1 8 : l ) Temperature: ambient Flow rate: 2 mL/min. Retention tinre: 6 . 8 min. Detection: Uv at 254 nm

System IX (53)

Type of sample: drug substance Column: 125 mm Spherisorb S5W Silica

"ype

Mobile phase: methanolic ammonium perchlorate, 10 mM, pH 6 . 7 Temperature: ambient Flow rate: 2 mL/min. Relative retention time: 0 . 3 1 Detection: W at 254 nm, electrochemical, -I- 1 . 2

v

TRAZODONE HYDROCHLORIDE

125

System X ( 5 4 )

Type of sample: plasma Columns: 250 x 4.6 mm MC-18 with 20% carbon load (5 pm), 300 x 4.6 mm Bondapak C18 (10um) Mobile phase: phosphate buffer, 0.2 M (pH 4.7): tetrahydrofuran: acetonitrile (90:5:5) Temperature: 45'~ Flow rate: 1.5 !nL/min. Retention time: 39 min. Detection: W at 254 nm

System XI (55)

Type of sample: biological fluids Column: 125 x 5 mm Spherisorb S5W Silica ( 5 pm) Mobile phase: methanol with 0.02% perchloric acid Temperature: ambient Flow rate: 2.0 mL/min. Retention time: 3.5 min. Detection: Fluorescence,A ex=200nm

System XI1 (56)

Type of sample: plasma Column: 150 x 4.6 mm Ultrasphere octyl (5urn) Mobile phase: acetonitrile: water (50:50) with 0.05% of each of tetramethylammmonium hydroxide and perchloric acid (70%) Temperature: ambient Flow rate: l.O/min. Retention time: 7.7 min. Detection: Fluorescence, Xex=320 nm; Aem=440 nm

System XI11 (57)

Type of sample: plasma Column: 250 x 4.6 mm LC-1 (trimethysilyl) ( 5 urn) Mobile phase: phosphate buffer (0.05 M I pH 3.0): acetonitrile (9O:lO) with 0.005 M of each of sodium heptane sulfonate and n-nonylamine Temperature: ambient Flow rate: 2.2 mL/min. Retention time: 13 min. Detection: electrochemical, +1.15 V

126

DENNIS K. G . GORECKI AND ROGER K. VERBEECK

7.

References

1.

G. Palazzo and B. Silvestrini, U.S. Patent No. 3,381 - 52146W (1968). 009. CA:69,

2.

B. Silvestrini, V. Cioli, S. Burberi and B. Catanese, Int. J. Neuropharmacol., 1, 587 (1968).

3.

B. Silvestrini. Trazodone. A new approach to the therapy of depression. In Proceedings of "Depression and the Role of Trazodone in Antidepressant Therapy", Eds., G. MOrOZOV, J. Saarma and B. Silvestrini, Edizioni Luigi Pozzi, Roma, 1978, pp. 1-10.

4.

B. Silvestrini, R. Lisciani and M. De Greqorio, in

Pharmacological and Biochemical Properties of Drug Substances, Vol. 3, Ed. M.E. Goldberg, Am. Pharm. Association, Washington, DC, 1981, pp. 94-119.

5.

R.N. Brogden, R.C. Heel, T.M. Speight and G.S. Avery, Drugs, 2, 401 (1981).

6.

M.M. Al-Yassiri, S.I. Ankier and R.K. Bridges, Life Sciences, 28, 2449 (1981).

7.

T.A. Ban and B. Silvestrini, Eds., Trazodone in Modern Problems of Pharmacopsychiatry,Vol 9, S. Karger, 1974.

8.

S. Gershon, K. Rickels and 8. Silvestrini, Eds., Trazodone: A New Broad Spectrum Antidepressant, Amsterdam: Excerpta Medica, 1980.

9.

L. Baiocchi, A. Chiari, A. Frigerio and P. Ridolfi, Arzneim-Forsch., 23, 400 (1973).

10.

A.C. Moffat, Ed., Clarke's Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1986, pp. 1035-1036.

11.

Brit. Patent No. 1,117,068. CA:69, - 67429 q (1968).

12.

The Merck Index, M. Windholz, Ed., 10th edition, Merck and Co., Inc. Rahway, N.J., U.S.A., 1983 p. 1370.

13.

Desyrel (Trazodone hydrochloride) Product Monograph, Mead Johnson and Co., Evansville, Indiana.

14.

J.P. Fillers and S.W. Hawkinson, Acta Cryst., B35, 498 (1979).

.

TRAZODONE HYDROCHLORIDE

727

15.

L. Baiocchi, A. Frigerio, M. GiaMangeli and L. Secchi, Boll. Chim. Farm., 114, 206 (1975).

16.

G. Belvedere, A. Frigerio and C. Pantarotto, J. Chromatogr., 112, 631 (1975).

17.

H. Maurer and K. Pfleger, J. Chromatogr., 305, 309 ( 1984 1.

18.

R.E. Gammans, E.H. Kerns, W.W. Bullen, R.R. Covington 339, 303 (1985). and J.W. Russell, J. Chromatogr., -

19.

L. Baiocchi and M. Giannangeli, Boll. Chim. Farm., 152 (1981).

20.

K. Sugiyama, S. Kono, C. Yamato and T. Fujita, J. Lab. Compd., 9, 723 (1973).

21.

S.I. Ankier, B.K. Martin, M.S. Rogers, P.K. Carpenter and C. Graham, Br. J. Clin. Pharmacol., 11, 505 (1981).

22.

A.W. Harcus, A.E. Ward, S.I. Ankier and G.R. Kimber, J. Clin. Hosp. Pharm., 8, 125 (1983).

23.

R. Javch, Z. Zopitar, A. Prox and A. Zimmer, Arzneim-Forsch., 26, 2084 (1976).

24.

R.E. Gammans, A.V. Mackenthun and J.W. Russell, Br. J. Clin. Pharmacol., 18, 431 (1984).

25.

A.J. Bayer, M.S.J. Pathy and S.I. Ankier, Br. J. Clin. Pharmacol., 16, 371 (1983).

26.

C. Yamato, T. Takahashi and T. Fujita, Xenobiotica., 6,

27.

F.W. Koss and U. Bosch, Pharmacokinetics and metabolism of trazodone in different species. In Proceedings of "Depression and the Role of Trazodone in Antidepressant Therapy", as., G. Morozov, J. Saarma and 8. Silvestrini, Edizioni Luigi Pozzi, Roma, 1978, pp. 11-20.

28.

D.R. Abernethy, D.J. Greenblatt and R.I. Shader, Pharmacology, 28, 42 (1984). R.E. Gammans, Personal communication, 1984.

29. 30,

113,

295 (1976).

F.W. Koss and U. Bosch: Trazodone as an example-drug levels in blood and brain. In "Trazodone--A New Borad-spectrum Antidepressant". Eds. S. Gershon, K. Rickels and B. Silvestrini, Excerpta Medica, Amsterdam, 1980.

728

DENNIS K. G . GORECKI AND ROGER K. VERBEECK

31.

Y. Suzuki: Teratogenicity and placental transfer of trazodone. In "Modern Problems of Pharmacopsychiatry, Vol. 9", Eds. T. Ban and B. Silvestrini, Karger, Basel, 1974.

32.

R.K. Verbeeck, S.G. Ross and E.A. McKenna, Br. J. Clin. Pharmacol., 22, 367 (1986).

33.

L.R. Baxter, J.N. Wilkins and G.B. Smith, J. Clin. 6, 223 (1986). Psychopharmacol., -

34.

L. Baiocchi, A. Frigerio, M. Giannangeli and G. Palazzo, Arzneim.-Forsch., 24, 1699 (1974).

35.

A. Adamus, M. Sansone, M. Melzacka and J. Vetulani, J. Pharm. Pharmacol., 37, 504 (1985).

36.

S. Caccia, M. Ballabio, R. Samanin, M.G. Zanini and S. Garattini, J. Pharm. Pharmacol., 33, 477 (1981).

37.

S. Caccia, M.H. Fong, S. Garattini and M.G. Zanini, J. Pharm. Pharmacol., 34, 605 (1982).

38.

C. Yamato, T. Takahashi and T. Fujita, Xenobiotica, 4, 313 (1974).

39.

F. Pala, G. DeCosmo, A.F. Sabato and A. Bondoli, Resuscitation, 9, 125 (1981).

40.

G. Musumarra, G. Scarlata, G. Romano, S. Clementi and S. Wold, J. Chromatoyr. Sci., 22, 538 (1984).

41.

T. Daldrup, F. Susanto and P. Michalke, Fresenius Z. Anal. Chem., 308, 413 (1981). CA:96, - 29498m (1982).

42.

G. Musumarra. G . Scarlata. G. Cirma, G. Romano, S. Palazzo, S. Clementi and G . Giulietti, J. Chromatogr., 350, 151 (1985).

43.

P. Giovanni, C. DiNunzio, A. Scotto di Tella, P. Schiano di Zenise and P. Ricci, Boll. SOC. Nat. Napoli, 93, 41 (1984). CA: 608, 126695k (1986).

44.

S. Putzolu, J.C. Pecknold, L. Baiocchi, Psychopharmacol. Bull., 12, 40 (1976).

45.

W.H. Anderson and M.M. Archuleta, J. Anal. Toxicol., 8, 217 (1984).

TRAZODONE HYDROCHLORIDE

729

46.

B. Munday, M.J. Kendal, M. Mitchard and T.A. Betts, Br. J. Clin. Pharmacol., 2, 19 (1975).

47.

S. Caccia, M. Ballabio, R. Fanelli, G. Guiso and M.G.

48.

B. Gerson, S. Chan, F. Bell and K.M. Pappalardo, J. 20, 69 (1986). Psychiat. Res., -

49.

R.L. Miller and C.L. Devane, J. Chromatogr., 374, 388 ( 1986 1.

50.

S.H.Y. Wong, S.W. Waugh, M. Draz and N. Jain, Clin. , 30, 230 (1984). Chem. - -

51.

P. Hochmuth, J.L. Robert, H. Schreck and R. Wennig Simultaneous determination of benzodiazepines and antidepressants in emergency toxicology by HPLC. In "Topics in Forensic and Analytical Toxicology", Ed. R.A.A. Mais, Elsevir, Amsterdam, 1984, pp. 143-149.

52.

I. Root and G.B. Ohlson, J. Anal. Toxicol., 8, 91 (1984).

53.

Zanini, J. Chromatogr., 210, 311 (1981).

I. Jane, A. McKiMon and R.J. Flanagan, J. Chromatogr., 191 (1985).

323,

54.

S.H.Y. Wong and N. Marzouk, J. Liquid Chromatogr., 8, 1379 (1985).

55.

R. Caldwell and R.J. Flanagan, J. Chromatogr., 345, 187 (1985).

56.

R.N. Gupta and M. Lew, J. Chromatogr., 342, 442 (1985).

57.

R.F. Suckow, J. Liquid Chromatogr., 6, 2195 (1983).

58.

J.M. Stewart and S.R. Hall. In "The XTAL system of crystallographic programs, Computer Science Centre, University of Maryland, College Park, M.D., 1983.

DENNIS K. G . GORECKI AND ROGER K. VERBEECK

730

8.

Acknowledgements

The authors would like to express sincere appreciation to Mr. Peter Pavlakidis for performing the literature search and providing some spectral data, to Mr. D. Leek for determining and Dr. M. Sardessai, Dr. S . Reid and Dr. J.R. Dimmock for assistance in analyzing the nuclear magnetic resonance spectra, to Dr. G. McKay for determining the mass spectra and to Dr. L. Delbaere, Dr. W. Quail and Mr. G. Loewen for interpretation of X-ray diffraction data and providing the three-dimensional structure of trazodone hydrochloride. Special thanks are extended to Mrs. C. Shuttleworth for typing this manuscript.

YOHIMBINE

A . G . Mehhawi* and A . A . Al-Ua&**

*vep&eitt

06

Phahmacagnany

**'Depcmhen.t 06 Humnaceu;ticcd ~ h m h h y C o l l e g e 0 6 P h m a c y , King Saud UVLivmLty, P.U. Box 2 4 5 7 , Riyadh-] 1451 (Saudi Ahab,i.ul

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

73 1

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form IeSeNed.

A. G . MEKKAWI AND A, A. AL-BADR

732

1.

Description 1.1 1.2 1.3

1.4

Nomenclature Formulae Molecular Weight Elemental Composition

2.

Physical Properties

3.

Spectral Properties

4.

Natural Sources and Isolation

5.

Biosynthesis of Yohimbine

6.

Total Synthesis of Yohimbine

7.

Pharmacology and Medicinal Uses

8.

Methods of Analysis 8.1 8.2

Qualitative Analysis Quantitative Analysis 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5

Acknowledgements References

Gravimetric Methods Titrimetric Methods Spectrophotometric Methods Spectrofluorimetric Methods Chromatographic Methods

YOHIMBINE

133

From t h e h i s t o r i c a l p o i n t of view yohimbine i s t h e most i m p o r t a n t of t h e complex i n d o l e a l k a l o i d t y p e s . I t was known s i n c e a n c i e n t t i m e s , i n t h e c u r d e drug b a r k o r t h e impure form, as an a p h r o d i s i a c by t h e West A f r i c a n n a t i v e s . It o c c u r s i n v a r i o u s t r o p i c a l trees such P a u s i n y s t a l i a yohimbe P i e r r e and r e l a t e d s p e i c e s , Aspidosperma quebrachoblanco S c h l e c h t and as a minor a l k a l o i d i n some Rawaolfia The f i r s t i s o l a t i o n of t h e drug w a s r e p o r t e d by S p i e g e l i n 1896 (1) and Witkop (2) i n 1943 s u g g e s t e d its c o r r e c t constitution.

-.

1.

Description

1.1

Nomenclature

1.1.1

Chemical Name

17a-Hydroxyyohimban-16a-carboxylic

methyl e s t e r ( 3 , 4 ) . 1.1.2

acid

G e n e r i c Names Quebrachine, Corynine, Aphrodine ( 3 , 4 , 5 , 6 ) .

1.1.3

Wiswesser Line N o t a t i o n a)

Yohimbine b a s e : T F6 D5 C666 E M ONbTTTTJ T Q

b)

1.2

.........

(4).

Yohimbine D5 C666 E --h y d r o c h l o r i d e .: MT F6 ON&TTTTJ T Q UVOl &GH . . ( 4 ) .

Formulae

1.2.1

UVOl

Empirical ‘2 l H 26°3N2

A. G . MEKKAWI AND A. A. AL-BADR

134

1.2.2

Structural

10 11

0

1.3

d

I

OH

Molecular Weight 354.45

1.4

Elemental Composition C 71.16%,

2.

H 7.39%,

N 7.90%,

0 13.54%.

Physical Properties 2.1

Color, Odour and Taste

2.2

Colorless needles when crystallized from ethanol, colorless or white in bulk. Taste is bitter, odourless ( 4 , 6 , 7 ) . Melting Point 240'

2.3

-

241'.

Sublimation Under vacuum the base sublimes at 159°/0.01 mm (7).

2.4

Solubility Almost insoluble in water, freely soluble in ethanol, chloroform and sparingly soluble in diethyl ether (4,6).

YOHIMBINE

2.5

735

Salts and Their Melting Point The salts are well crystalline and the hydrochloride is colorless plates m.p. 3oOo - 302OC , the nitrate m.p. 269-270°C, the tarkarate hexahydrate m.p. 213OC, followed by solidification and remelting at 278OC. The thiocyanate is of rectangular crystals (m.p. 233' - 234OC) and the colorless methiodide plates from acetone have the m.p. 249 - 250OC. The melting point for the 0-acetate is 1 3 3 O C and the 0,N-diacetyl derivative melting point is 1 8 3 O C ( 7 , 8 ) .

2.6

Specific Rotations of Base and Salts Various specific rotations have been recorded for the base. [ a ] i O+ EtOH ( 9 ) ;

+ 3.

+

62.2'

107.9'

in pyridine;

+

56 in

in EtOH ( 7 ) ; the hydrochloride

103.3 (H20)

(7).

Spectral Properties 3.1

Ultraviolet Spectrum The U.V. spectra of yohimbine in methanol and in 0.1 N H2SO4 were scanned from 200 to 400 nm using Varian DMS 90 spectrophotometer and the following maxima and (E 1%, 1 cm) values were found: In methanol: 3 maxima at 289, 282 and 225 nm were recorded. The (E 1%, 1 cdfound were : 210 at 289 nm, 247 at 282 nm and 1079 at 225 nm. In the acidic solution a shift to lower wavelengths with some hyperchromic effect was observed (E 1%,1 an) at 280 nm was 225, at 272 nm was 233 and at 221 nm was 1110. Previous reports in 0.1 N H2S04 (6) were as follows: (E 1%, 1 an) at 278 nm 204; at 272 nm 208 and 220 nm 1064. Figure 1 illustrates the UV spectrum behaviour for both the methanolic and the acidic solutions of yohimbine.

736

A. G . MEKKAWI AND A. A. AL-BADR

YOHIMBINE

3.2

737 I n f r a r e d Spectrum

The i n f r a r e d spectrum of yohimbine h y d r o c h l o r i d e i s p r e s e n t e d i n F i g u r e 2. The spectrum i s o b t a i n e d from K B r d i s c on a Perkin-Elmer I n f r a r e d Spectrophotometer model 580 B. I t shows t h e following :

-1 Frequency (cm )

Assignments

3540

OH

- stretch (free)

3190

NH

- stretch

CH

-

2900) 2960) 1730

stretch

C = 0

- stretch

C = C

- aromatic

1460) 1472)) 1448) Other c h a r a c t e r i s t i c a b s o r p t i o n bands are : 1172, 1150, 1025, 1000, 970, 750 and 702 cm-’. I R d a t a of yohimbine h y d r o c h l o r i d e have a l s o been r e p o r t e d (3, 5 , 6 ) .

C l a r k e ( 6 ) r e p o r t e d t h e p r i n c i p l a l peaks a t 1705, 741, 1160 o r 1197 o r 1436 cm-1. 3.3

Nuclear Mangetic Reonance S p e c t r a (NMR) 3.3.1

P r o t o n Nuclear Magnetic Resonance (PMR) Spectra The PMR spectrum of yohimbine h y d r o c h l o r i d e i n DMSO-db and i n DMSO-d6 p l u s D20 were shown i n F i g u r e s 3a and 3b r e s p e c t i v e l y . The s p e c t r a were r e c o r d e d on a Varian T60 A NMR s p e c t r o m e t e r u s i n g t e t r a m e t h y l s i l a n e

Wavelength urn

100.

3

4

7

6

5

8

9

10 11 12 13 1461qm

90 *

80-

*

90

*

80

*

20

70,

Fac

10. r-’ 0, 38003500 3ooo 2500 2000 Wavenumber

-10

1800

1600

1400

1200

Figure 2 : Infrared spectrum of yohimbine, HBr disc.

1000

800

0

625

139

YOHIMBINE

., . . . . , . . 1500

-

I

400

' I . ' . -

I

-

-

1

300

80 7.0 6.0 S.OPpM(r)L.O 30 2.0 1.0 0 Figure 3a: Proton magnetic resonance spectrum of yohimbine hydrochloride in D M s 0 - d ~using TMS as reference standard.

200

. . . I . .

'7 I . . . . , .

. . I .

O

8.0 7.0 60 WPPM(~)L.O 30 2.0 I0 0 Pigurc 3b: Proton magnetic resonance spectrum of yohimbinc hydrochloride in D M s 0 - d ~ PIUS

@ o using

TMS a5 reference standard.

A. G . MEKKAWI AND A. A. AL-BADR

740

(TMS) as reference standard. The structrual assignments as follows:

Chemical shift (PPMJ

Assignments

Multiplets at 0.7-4.60

CH2 and CH protons

Singlet at 3.60

CH3 protons

Multiplet at 6.7-7.4

Aromatic protons

Singlet at 4.05

NH (exchangable with D20).

Singlet at 10.65

OH (exchangable with D 2 0 ) .

Mills et a1 (3) have reported the proton NMR spectrum of yohimbine. 3.4

Carbon-13 Nuclear Magnetic Resonance Spectrum (C-13 NMR) The proton-decoupled and off-resonance carbon-13 NMR spectrum of yohimbine have been determined on Jeol FX 100 MHz spectrometer at an ambient temperature and are presented in Figures 4 and 5 respectively. The sample was dissolved in deuterated chloroform and DMSO-dC; using TMS as reference standard. The carbon assignments are based on chemical shift values and off-resonance spectrum and are listed below:

0

I

OH

(OHIMBINE

741

sil TMS

u

Figure4: Proton- decoupled carbon -13 NMR spectrum of yohimbine in CDCI3 and D M s 0 - d ~using TMS as reference standard.

TMS

0

Figure 5 : Off- resonance carbon-13NMR spectrum of yohimbin inCDCi3 and DMSO-ds using TMS a reference standard.

A. 0.MEKKAWI AND A. A. AL-BADR

142

Chemical s h i f t ( 6 )

Multiplicity

Assignment

135.1

Singlet

c2

70.2

Doublet

c3

52.6

Triplet

‘5

21.7

Triplet

6 ‘

106.9

S i n gl e t

c7

127.0

Singlet

‘8

117.6

Doublet

‘9

118.5

Doublet

clo

120.5

Doublet

c11

111.0

Doublet

c12

136.1

S i n g1e t

‘13

34.7

Triplet

‘14

36.5

Doublet

‘15

52.4

Doublet

‘16

66.8

Doublet

‘17

31.9

Triplet

‘18

23.2

Triplet

‘19

39.8

Doublet

2‘ 0

61.3

Triplet

c2 1

174.8

S i n g 1e t

c22

51.6

Quartet

2 ‘ 3

YOHIMBINE

3.5

143

Mass S p e c t r a The e l e c t r o n impact ( E I ) m a s s spectrum a t 70 e V r e c o r d e d on Varian Mat 311 mass s p e c t r o m e t e r and t h e chemical i o n i s a t i o n (CI) mass spectrum o b t a i n e d w i t h F i n n i g a n 4000 mass s p e c t r o m e t e r are shown i n F i g u r e s 6 and 7 r e s p e c t i v e l y . The E I spectrum (Fig. 6 ) shows a b a s e peak a t m / e 353 and a n o t h e r peak a t m/e 354 (&) of almost e q u a l abundance ( 9 8 % ) , o t h e r major fragments a r e a t m / e 184, m / e 169 and a t m / e 156. The C I spectrum ( F i g . 7) shows a b a s e peak a t m / e 355 (M+ + 1 ) . Other fragments are a t m / e 337 and a t m / e 323. Other m a s s spectrum d a t a h a s a l s o been r e p o r t e d (3) *

4.

N a t u r a l Sources and I s o l a t i o n Yohimbine i s t h e major a l k a l o i d of t h e bark of P a u s i n y s t a l i a yohimba (Syn. Corynanthe yohimbe) and a l s o p r e s e n t i n t h e r e l a t e d s p e c i e s p. m a c r o c e r a s , P. p a n i c u l a t a and 2. t r i l l e s i i ( f . Rubiaceae) ( 7 , 4 , 8 ) . S e v e r a l Rauwolfia s p e c i e s w e r e r e p o r t e d t o c o n t a i n yohimbine ( e . g . &. s e r p e n t i n a ) ( 4 , l O ) . &. c a f f r a ( l l ) , R. t e t r a p h y l l a (12) and &. v o m i t o r i a ( l 3 ) , ( f . Apocynaceae). The i s o l a t i o n of yohimbine from t h e b a r k of s p e c i e s c o n t a i n i n g i t as t h e major a l k a l o i d can be done by t h e method of L e H i r -e t a1 ( 8 ) , where yohimbine i n a m i x t u r e w i t h t h e o t h e r isomers (a-yohimbine) w a s b o i l e d w i t h d - t a r t a r i c a c i d , t h e yohimbine t a r t a r a t e c r y s t a l l i z e d immediately while a-yohimbine t a r t a r a t e remained i n s o l u t i o n and c r y s t a l l i z e d l a t e r . The e x t r a c t i o n of t h e a l k a l o i d s from t h e i r n a t u r a l s o u r c e s i s g e n e r a l l y c a r r i e d o u t by t h e c o n v e n t i o n a l method. The drug powder i s t r e a t e d w i t h Na2C03, d r i e d and e x t r a c t e d w i t h a s u i t a b l e o r g a n i c s o l v e n t e.g. benzene Le H i r -e t a1 (8) a l s o used o r chloroform (14,15). column chromatographic s e p a r a t i o n . Court and h i s s t u d e n t s (16-ZO), working on Rauwolfia s p e c i e s ,used p r e p a r a t i v e t h i n - l a y e r chromatography as a r o u t i n e t e c h n i q u e f o r t h e i s o l a t i o n and c h a r a c t e r i s a t i o n of i n d o l e a l k a l o i d s . Although p r e p a r a t i v e HPLC w a s n o t used i n t h e r o u t i n e i s o l a t i o n of such compounds b u t t h e t e c h n i q u e might be of r o u t i n e u s e i n t h e f u t u r e .

A. G. MEKKAWI AND A. A. AL-BADR

744

-9904.

100.0-

50.0169

467' 100.0-

50.0-

18912.

100.

504

383

337 323 M E 300

320

I

340

360

380

400

420

440

Figure7 : Chemical ionization (CI )mass spectrum of yohimbine

YOHIMBINE

5.

745

Biosynthesis of Yohimbine The major features of the probable biosynthetic pathway to yohimbine were summarized by Atta-ur-Rahman and Basha (21). Scheme 1 illustrates how yohimbine is biosynthesized from tryptamine and secologanin. The condensation of tryptamine [l] with secologanin [2] is catalysed by the enzyme strictosidine synthetase which controls the C-ring closure mechanism, and results in the formation of strictosidine [3]. A second glycosidase enzyme converts strictosidine via a series of reactive intermediates [4]-[6] to 4,21-dehydrocorynantheine aldehyde [7] by D-ring closure. This aldehyde can then isomerize to [8] which cyclizes to yohimbine.

6.

Total Synthesis of Yohimbine The following total synthesis of yohimbine was described by Van Tamelen et a1 (22). p-Benzophenone [l] was condensed with lY4-butadiene to give the adduct 1,4,5,8,9,1O-cis-hexahydronaphthalene1,4-dione [2]. Selective reduction by zinc metal and acetic acid gives the octahydronaphthalenedione [3] which was converted to glycidic acid ester [4], subsequent hydrolysis to the glycidic acid [5] and then final decarboxylation to the ketoaldehyde [ 6 ] . The ketoaldehyde [6] was oxidized to the corresponding ketoacid [7] using silver oxide in the presence of alkali. The keto acid [7] was then converted to its acid chloride [8] and the latter was reacted with tryptamine [9] in the presence of pyridine yielding tryptamide [lo]. The tryptamide [lo] is then converted to the cis-diol [ll] using solution of osmium tetraoxide in tetrahydrofuran added to a solution of amide [lo] in pyridineTHF and cooled at dry-ice-acetone temperature. Hydrogenation of the keto-diol [ll] over Adams' catalyst in ethanol gave a good yield of the desired triol [12]. Intermediate [12] was converted to the dialdehyde [13] by cleavage of the triol amide [12] in aqueous acetone and then was cyclized -in situ using phosphoric acid and heating at 70°, where intermediate [14] was obtained. The lactol [14] was then transformed into the lactollactam methyl ether [15] by means of p-toluenesulfonic acid catalyzed methanolysis. Lithium aluminium hydride reduction of [15] in THF afforded the lactol ether base [16]. Hydrolysis of [16] by means of aqueous hydrochloric

A. G.MEKKAWI AND A. A. AL-BADR

746

,O

c-0-cs3

11

0

+ H20 - glucose

Glu.

[21

r

1

I + + HH 22 00 '

1 I

L

-Glucose

CB

oc

CH3-0-C

3 \\

U

II

OH

CH30-C

II

Scheme 1:

-

Biosynthesis of Yohimbine.

*v , I

OH

YOHIMBINE

-@ 141

Zn-AcOH

Diels-Alder

t --.Condn. --

0

0

111

[21 COOEt

Aqueous NaOH ( h o t ) under nitrogen

Ethyl chloroacetate K t-butoxide/benzene H

O

[41

[31 COOH

(1) Cu powder i n d i e t h y l g l y c o l a t e 155 - 160'

Silver

oxide/alkali H

O

H O [61

[51

Oxalyl c h l o r i d e

0

HZ

. )

i n pyridine 0

Scheme 2:

[91

T o t a l S y n t h e s i s of Yohimbine.

~

A. G . MEKKAWI AND A. A. AL-BADR

748

I

H

HL=O OH

H

OH H

LAH

i n THF

OCH3

OH

[I61 Scheme 2:

[I71 Continued.

YOHIMBINE

749

-

285

[ 181

- 295'

OCOCH3

[ 191

Metaperiodate Cleavage

o = c

'OCHO

H Chromic anhydride at Oo/H2 SO4 Scheme 2 :

Continued.

[211

*

A. G . MEKKAWI AND A. A. AL-BADR

750

i- Chromatography ii- L-camphorsulphonic acid

CH3-O-C

'OH

' H

\\

0

Scheme 2.

[ 23 Continued.

1

-

YOHIMBINE

75 1

a c i d gave t h e l a c t o l b a s e 1171. The l a t t e r w a s t h e n c o n v e r t e d t o t h e l a c t o l acetate [ 1 8 ] ( a s acetate s a l t ) by t r e a t m e n t w i t h a c e t i c a n h y d r i d e - p y r i d i n e . The a c e t a t e s a l t [18] w a s subjected t o pyrolysis c o n d i t i o n s , where a l i m i t e d amount of a c e t a t e s a l t under 0 . 0 1 mm p r e s s u r e i n a s u b l i m e r was exposed t o a metal b a t h p r e v i o u s l y h e a t e d t o 285-295', where t h e e n o l e t h e r [19] have been formed, a s t h e a c e t a t e s a l t . The' o l i f i n i c bond i n t h e c y c l i c e n o l e t h e r system w a s o x i d i z e d u s i n g osmium t e t r a o x i d e - p e r i o d a t e . The ozmylation w a s c a r r i e d i n t h e e n o l e t h e r a t - 7 8 O i n THF-pyridine, where t h e d i o l [ 2 0 ] was o b t a i n e d . M e t a p e r i o d a t e c l e a v a g e of 1,2g l y c o l [ 2 0 ] gave t h e 0-formate of pseudoyohimbaldehyde [ 2 1 ] . The c r u d e p e r i o d a t e was d i s s o l v e d i n methanola c e t o n e and t r e a t e d a t Oo w i t h chromic a n h y d r i d e (2.0-3.5 e q u i v . ) i n s u l f u r i c a c i d f o r 30 m i n u t e s . A f t e r removal of t h e forrnyl group, pseudoyohimbine [ 2 2 ] was o b t a i n e d (low y i e l d ) u s i n g alumina chromatography. The r e s o l u t i o n of t h e s y n t h e t i c b a s e was accomplished by means of l-camphor s u l p h o n i c a c i d where pseudoyohimbine e p i m i e r i z e s t o yohimbine[23]. Scheme 2 i l l u s t r a t e s t h e sequence of s y n t h e s i s d e s c r i b e d above.

7.

Pharmacology and Medicinal Uses Yohimbine produces a c o m p e t i t i v e a - a d r e n e r g i c blockade of l i m i t e d d u r a t i o n and i t i s r e l a t i v e l y s e l e c t i v e f o r t h e presynaptic a2-receptor. A t high doses i t a l s o blocks postsynaptic a-adrenergic receptors. It a l s o b l o c k s 5-HT r e c e p t o r s . It h a s a l i t t l e e f f e c t on smooth muscle and r e a d i l y p e n e t r a t e s t h e c e n t r a l n e r v o u s system and produces a complex p a t t e r n of r e s p o n s e s i n d o s e s lower t h a n t h e r e q u i r e d t o produce p e r i p h e r a l aa d r e n e r g i c blockade. These i n c l u d e v a s o p r e s s i n release and hence m a n i f e s t s an a n t i d i u r e t i c e f f e c t . G e n e r a l CNS e x c i t a t i o n i n c l u d i n g e l e v a t e d blood p r e s s u r e , i n c r e a s e d h e a r t r a t e , i n c r e a s e d motor a c t i v i t y , i r r i t a b i l i t y and t r e m o r s a l s o occured. P a r e n t r a l a d m i n i s t r a t i o n may i n d u c e s w e a t i n g , n a u s e a and vomiting (23 - 3 3 ) . L e c r u b i e r e t a1 (29) i n v e s t i g a t e d t h e p o s s i b l e u s e of yohimbine t o improve t h e c o n d i t i o n of o r t h o s t a t i c hypotension induced by clomipramine. The c o n t r o v e r s i a l a p h r o d i s i a c p r o p e r t y of yohimbine was r e c e n t l y i n v e s t i g a t e d by many a u t h o r s . Morales e t a1 (30) r e p o r t e d t h e a b i l i t y of 6 mg of yohimbine, g i v e n 3 times a day, t o improve p a r a t h e s i a of t h e lower limbs i n 4 p a t i e n t s o u t of 6 d i a b e t i c p a t i e n t s . S t e e r s e t a1 (31)

A. G . MEKKAWI AND A. A. AL-BADR

152

s t u d i e d t h e pharmacological e f f e c t s of yohimbine i n p e n i s and the penis, yohimbine e x h i b i t s a - a d r e n e r g i c b l o c k i n g p r o p e r t i e s and does n o t a f f e c t catecholamine l e v e l i n t h i s t i s s u e . Condra e t a 1 (32) s t u d i e d t h e e f f e c t i v e n e s s of yohimbine i n t h e t r e a t m e n t of impotence and Nakatau e t a1 ( 3 3 ) have s t u d i e d t h e pharmacokinetics of yohimbine i n man. However, t h e p o s s i b l e e f f e c t i v e n e s s of yohimbine as an a p h r o d i s i a c , a t l e a s t i n r a t s , h a s now r e c e i v e d s u p p o r t from a s t u d y c a r r i e d o u t b y Clark e t a1 ( 3 4 ) . The teams of Condra and Nakatau ( 3 2 , 3 3 ) found t h a t i n p a t i e n t s w i t h c l e a r o r g a n i c impotence, t r e a t m e n t w i t h yohimbine h y d r o c h l o r i d e e x h i b i t e d a p o s i t i v e r e s p o n s e r a t e of approximately 30% and p o s t u l a t e d t h a t t h e f a i l u r e t o improve t h e c o n d i t i o n s of t h e o t h e r two t h i r d s of p a t i e n t s may be due t o i n t e r i n d i v i d u a l d i f f e r e n c e s i n t h e pharmacokinetics of t h e drug which a p p e a r s t o be c l e a r e d i n i n d i v i d u a l s by metabolism.

-i n - v i t r o p r e p a r a t i o n s of human and r a b b i t i-n v i v o on r a b b i t s and concluded t h a t , i n

8.

Methods of A n a l y s i s

8.1

Q u a l i t a t i v e Analysis

8.1.1

Color Tests Yohimbine when r e a c t e d upon by ammonium molybdate g i v e s a b l u e c o l o r t h a t changes slowly t o green ( s e n s i t i v i t y 0.025 pg); w i t h ammonium vanadate t e s t i t g i v e s a b l u e changing t o preen and f a d i n g away ( s e n s i t i v i t y i s 0.1 pg). With V i t a l i ' s t e s t t h e c o l o u r sequence i s y e l l o w / y e l l o w / v i o l e t ( 6 ) . Other n o n - s p e c i f i c t e s t s a r e t h e x a n t h y d r o l r e a g e n t and 4-dimethylamino benzaldehyde i n m e t h a n o l i c HC1.

8.1.2

M i c r o c r y s t a l Tests Wachsmuth (35) determined t h e p r e c i p i t a t i n g p r o p e r t i e s of f l a v i n i c a c i d ( i n e t h a n o l d i e t h y l e t h e r s o l u t i o n ) on s e v e r a l a l k a l o i d a l and o t h e r n i t r o g e n c o n t a i n i n g s u b s t a n c e s and r e p o r t e d t h a t abundant p r e c i p i t a t e s were o b t a i n e d w i t h yohimbine ( s e n s i t i v i t y 1:lOO). Habib and Aziz ( 3 6 ) found t h a t yohimbine g i v e s i r r e g u l a r l a r g e r o s e t t e s of curved h a i r s w i t h 0.5% aqueous ammonium r e i n e c k a t e

YOHIMBINE

153

which can be i d e n t i f i e d m i c r o s c o p i c a l l y . With potassium c y a n i d e s o l u t i o n , y o h i m b i n e g i v e s r o s e t t e s of r o d s and w i t h sodium c a r b o n a t e s o l u t i o n i t g i v e s r o s e t t e s of r o d s o r dense r o s e t t e s ( s e n s i t i v i t y i n b o t h r e a g e n t s 1: 1500) ( 4 ) . C h a r a c t e r i s t i c c r y s t a l s with d i f f e r e n t reagents obtained i n o u r l a b o r a t o r y , are i l l u s t r a t e d i n F i g u r e s 8-17. 8.2

Q u a n t i t a t i v e Analysis 8.2.1

G r a v i m e t r i c Methods Raymond (11) reviewed methods used a t h i s t i m e i n gravimetric analysis. He extracted powdered bark of yohimbehe w i t h e t h e r : chloroform ( 4 : l ) m i x t u r e u s i n g 10 m l of s o l v e n t f o r each gram of b a r k m a t e r i a l Feldhoff (15) m o d i f i e d t h e t e c h n i q u e s u s e d b e f o r e him by l i b e r a t i n g t h e b a s e s i n t h e w i t h Na2C03 t r e a t m e n t bark of Yohimbe and e x t r a c t i n g t h e d r u g i n a s o x h l e t w i t h d i - o r - t r i c h l o r o e t h y l e n e mixed i n t h e r e c e i v e r w i t h aqueous o x a l i c a c i d s o l u t i o n . The o r g a n i c s o l v e n t was e v a p o r a t e d , t h e aqueous a c i d s o l u t i o n w a s t r e a t e d w i t h NH40H s o l u t i o n t o l i b e r a t e t h e a l k a l o i d s which were t h e n e x t r a c t e d w i t h e t h e r . The a l k a l o i d s were p r e c i p i t a t e d w i t h an a l c o h o l i c s o l u t i o n of H C 1 i n a b e a k e r and t h e aqueous a l c o h o l i c p a r t was washed twice w i t h e t h e r and methyl a c e t a t e . The secondary a l k a l o i d s remained i n s o l u t i o n a f t e r c r y s t a l l i s a t i o n of yohimbine HC1. A f t e r b e i n g l e f t 24 h o u r s i n a r e f r i g e r a t o r , t h e c r y s t a l l i n e yohimbineH C l was f i l t e r e d on a weighed f i l t e r p a p e r , washed and d r i e d a t 102% t o c o n s t a n t w e i g h t .

8.2.2

T i t r i m e t r i c Methods Precipitation Titration V y t r a s and Riha (37) c a r r i e d o u t p r e c i p i t a t i o n t i t r i m e t r y for 9 alkaloids including yohimbine u s i n g sodium t e t r a p h e n y l b o r a t e r e a g e n t and a C r y t u r valinomycin i o n selective electrode.

A. G . MEKKAWI AND A. A. AL-BADR

754

Figure8: Rosettes of circular plates of yohimbine with picric acide reagent ( a f t e r 3 minutes

1.

Figure91 Radiating pointing plates of phimbine with mercuric chloride reagent ( a f t e r 14minutes).

Figure 10 : Orange coloured radiating relatively wide crystal plates of yohimbine with potassium chromate reagent (after 9 minutes ).

YOHIMBINE

155

Figurell

:

Tagged plates of crystals of yohirnbine with Dragendorf f's reagent ( after 5 minutes ),

Figure12. White crystalline minute rods of yohimbine after reaction w i t h lead iodide reagent (immediately produced )

Figure 13: Crystal of tetragonal or hexagonal structures a f t e r addition of Marme's reagent (after 5 minutes).

A. G . MEKKAWI AND A. A. AL-BADR

156

Figure 14

Radiating rod clusters of yohlrnblne and Naf03 solution (after 10 minutes)

Figure IS

:

Crystal plates, characteristic, to reaction of yohimbine with NH4S CN aqueous solution ( a f t e r 5 - 7 minutes),

Figure

16:immediate sandy crystals w i t h Wagner's reagent.

Figure 17. Fine clusters of rods with KCN solution (after 15minutes)

YOHIMBINE

b)

M i cr 0- he t e r ome t r i c T i t r a t i o n

I n t h e i r t r i a l t o analyze l a r g e organic n i t r o g e n - c o n t a i n i n g compounds, Bobtelsky and B r a z i l y (38) s t u d i e d t h e b e h a v i o u r of micro amounts of yohimbine t o g e t h e r w i t h n i t r o n , quinone , s t r y c h n i n e , p a p a v e r i n e , n i c o t i n e , a t r o p i n e , morphine o r phenanthrol i n e when t i t r a t e d h e t e r o m e t r i c a l l y w i t h t u n g s t o s i l i c i c , tungstophosphoric o r molybdophosphoric a c i d a t pH 1 o r 7. They found t h a t t h e i n f l u e n c e of t h e a c i d i t y of t h e s o l u t i o n was d i f f e r e n t i f t h e o r g a n i c molecule c o n t a i n e d 1 o r 2 h e t e r o c y c l i c N atoms. 8.2.3

S p e c t r o p h o t o m e t r i c Methods a)

Colorimetry

C o l o r i m e t r i c a s s a y s of t h e weakly b a s i c t e r t i a r y a l k a l o i d w e r e f i r s t done on c o l o r r e a c t i o n s c h a r a c t e r i s t i c of t h e i n d o l e group ( 8 , 29). I n p r e s e n c e of o t h e r i n d o l e c o n t a i n i n g compounds, such as t h o s e n a t u r a l l y present i n t h e n a t u r a l source,such a method i s of l i t t l e v a l u e . However, Kolsck (39) used t h e c o l o u r r e a c t i o n of yohimbine i n d o l e n u c l e u s w i t h p-dimethylaminobenzaldehyde and measured t h e c o l o u r produced p h o t o m e t r i c a l l y w i t h f i l t e r S 39 o r S 49. The method i s summarized a s f o l l o w s : Yohimbine-HC1 w a s d i s s o l v e d i n water, i f d e s i r e d w i t h a d d i t i o n of l i t t l e e t h a n o l . I n a 10 m l v o l u m e t r i c f l a s k t o 1 m l of t h e s o l u t i o n c o n t a i n i n g 0.05 - 3 mp, of t h e allcaloid s a l t , 2 m l of p-dimethylylaminobenzaldehyde w e r e added and t h e m i x t u r e w a s l e f t t o c o o l f o r 10 minutes and 0.05 m l of a 5% H202 ( o r 1%NaN02) s o l u t i o n w a s added, shaken, and t h e c o l o r w a s allowed t o develop. To t h i s , d i s t i l l e d water was added t o make 10 m l of m i x t u r e and d e n s i t y was measured p h o t o m e t r i c a l l y u s i n g a f i l t e r S 39 o r S 49. The mechanism of t h i s r e a c t i o n w a s s t u d i e d by P i n d u r (40).

758

A. G . MEKKAWI AND A. A. AL-BADR

Jung assayed reserpine and yohimbine ( 4 1 , 4 2 ) photometrically with xanthydrol reagent in presence of other nitrogen containing compounds. The indole nucleus of the two compounds forms a colored compound with xanthydrol and the extinction is measured at 515 mu. In this method a 50-150 p g of yohimbine were dissolved in 5 ml of a freshly prepared solution o f 0 . 4 % xanthydrol in glacial acetic acid Containing 1% v/v HC1 heated on a boiling water bath for 15 minutes, cooled and the extinction wasmeasured at 515 mu. The color is stable for 5 hr. The procedure can be used for tablets, injections or mixtures after extracting the alkaloid by a suitable method The method was also used by Budavaris a1 ( 4 3 ) . The yohimbine-methyl orange complex color as a quantitative method was utilized by Mohamed and Elsayed ( 4 4 ) for assaying the base in tablet formulations. A solution of the powdered tablets ( e 5 0 mg of yohimbine hydrochloride) in 50 ml of H20 was prepared A measured volume containing about 300 mg of tablet base was diluted to 5 0 ml. A 2 m portion of this solution was transferred to a separating-funnel containing 2 ml of buffer (pH 4 . 0 ) and 1 ml of 0.1% methyl orange solution in 20% ethanol. The yohimbine-methyl orange complex was extracted successively with chloroform, diluted to 25 ml with chloroform and the extinction was measured at 421 nm. For 5 determinations the recovery was 97.6 - 102.5%. 8.2.4

Spectrofluorimetric Methods In their spectrophotofluorimetric study of a vast number of organic compounds of pharmaceutical interest, Underfried et a1 ( 4 5 ) using a Xe arc - source emitting a continuum from 200-800 mu in their spectrofluorometer,reported that yohimbine at pH 7 had its maximum activation at 270 mu and its maximum emission of fluorescence at 360 mu.

YOHIMBINE

759

Chiang and Chen (46) assayed yohimbine after increasing its fluorescence in solutions by subjecting its heated solution in presence of H202 to ultraviolet light. Auterhoff and Schroeder ( 4 7 ) carried a similar assay and evaluated yohimbine in Yohimbe bark. They extracted 1 g of the bark with ethyl ether after alkalinisation with 10% aqueous ammonia. The ethereal extract was re-extracted with 0.1 N H2SO4 solution and the acid solution was basified with NH40H, extracted with chloroform, concentrated and diluted to 10 ml with chloroform. The chloroformic extract was chromatographed on a MN 300 Polygram cellulose plate impregnated with formamide : dimethylformamide : acetone (4:3:12) and developed with heptane : ethylmethyl ketone : 0.25% aqueous ammonia (4:2:1) for 15 cm and the zone of Rf 0.2, correspondin? to yohimbine, was extracted with CHC13 : CH3OH (100 ml). The extract was filtered, the solvent removed under reduced pressure at 35OC and the residue dissolved in 10 ml of 30% acetic acid. To 1 ml of this solution, 1 ml of 3% H202 and 4 ml of 30% acetic acid were added. A similar solution of standard yohimbine was prepared. Both solutions were heated for 2 hr at 80°C, cooled and the volume was adjusted to 10 ml with 30% acetic acid. After excitation at 365 nm the fluorescence was measured at 496 nm. Clarke ( 6 ) reported the quantitative estimation of yohimbine as follows: The drug when in a mixture consisting of reserpine, ajmaline and yohimbine may be separated by thin-layer chromatography and determined by spectrophotometry or spectrofluorometry as described previously for indole alkaloids by some authors (48,49)

A. G . MEKKAWI AND A. A. AL-BADR

760

8.2.5

Chromatographic Methods 8.2.5.1

Paper Chromatography (P.L.C.)

Z a f f a r o n i ' s system (50) w a s t r i e d by Machovicova ( 51 ) on p a p e r chromatography and was found t o be s u i t a b l e f o r t h e s e p a r a t i o n of r e s e r p i n e , r e s e r p i c a c i d and yohimbine. Q u a n t i t a t i o n of yohimbine u s i n g P.L.C. by Auterhoff and Schroeder (47) i s d e s c r i b e d i n s e c t i o n 8.2.4 above C l a r k e (6) r e p o r t e d t h e f o l l o w i n g paper chromatography systems f o r t h e s e p a r a t i o n of yohimbine: a)

Paper: Whatman No. 1 , s h e e t 16 x 4 i n c h , b u f f e r e d by d r i p p i n g i n a 5% s o l u t i o n of sodium dihydrogen c i t r a t e , b l o t t i n g , ( I t can and d r y i n g a t 25' f o r 1 hour. be s t o r e d i n d e f i n i t e l y ) . Sample: 2 . 5 ~1 of a 1X s o l u t i o n ; i n 2 N a c e t i c acid i f possible, otherwise i n 2 N h y d r o c h l o r i c , 2 N sodium h y d r o x i d e , or ethanol. S o l v e n t : 4.8 of c i t r i c a c i d i n a m i x t u r e of 130 m l of w a t e r and 870 m l of n-butanol. T h i s may be used f o r s e v e r a l weeks i f w a t e r added from t i m e t o time t o keep t h e s p e c i f i c g r a v i t y a t 0.843 t o 0.844. Development: Ascending, i n a t a n k 8 x 11 x 15% i n c h , 4 s h e e t s b e i n g run a t a t i m e . Time o f r u n , 5 hour. Location: Under u l t r a v i o l e t l i g h t , p a l e blue fluorescence; location reagent : i o d o p l a t i n a t e s p r a y , week r e a c t i o n ; bromocresol green s p r a y ; weak r e a c t i o n .

b)

Paper: Whatman No. 1 o r No. 3 , s h e e t 1 7 x 19 cm, impregnated by d i p p i n g i n a 10% s o l u t i o n of t r i b u t y r i n i n a c e t o n e and drying i n a i r .

YOHIMBINE

76 1

Sample: 5 1-11of 1 t o 5% s o l u t i o n i s e t h a n o l o r chloroform. Solvent:

Acetate b u f f e r (pH 4.58).

E q u i l i b r a t i o n : The beaker c o n t a i n i n g the solvent i s equilibrated i n a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 95' f o r about 15 m i n u t e s . Development: Ascending. The p a p e r i s f o l d e d i n t o a c y l i n d e r and c l i p p e d , t h e c y l i n d e r i s i n s e r t e d i n t h e beaker c o n t a i n i n g t h e s o l v e n t which i s n o t removed from t h e oven. A p l a t e - g l a s s d i s k t h i c k l y smeared w i t h s i l i c o n e g r e a s e may s e r v e a s a cover. T i m e of r u n , 15 t o 20 m i n u t e s . L o c a t i o n : Under u l t r a v i o l e t l i g h t , blue fluorescence. Rf v a l u e 0 . 6 4 .

c)

Paper and Sample: A s above i n ( b ) Solvent:

.

Phosphate b u f f e r (pH 7 . 4 ) .

E q u i l i b r a t i o n : The beaker c o n t a i n i n g the solvent i s equilibrated i n a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 86' f o r about 15 m i n u t e s . Development: Location: 0.04. 8.2.5.2

A s above i n (b)

A s above i n ( b ) .

Thin-Layer

. Rf v a l u e

Chromatography (T.L.C.)

S i l i c a g e l G. p l a t e s p r e a p r e d w i t h 0 . 1 M NaOH o r 0.1 M KHSO4 developed by 5 s o l v e n t systems were used by Fike (52) i n t h e s t u d y of t h e b e h a v i o u r of 140 b a s i c d r u g s i n c l u d i n g yohimbine and t h e Rf v a l u e s of t h o s e d r u g s w e r e r e c o r d e d and s u c c e s s f u l s e p a r a t i o n w a s achieved b u t none of t h e i n d o l e a l k a l o i d s r e l a t e d t o yohimbine w e r e i n c l u d e d i n t h e s t u d y . However, Court and

162

A. 0.MEKKAWI AND A. A, AL-BADR

and Habib (16) and Court and Timmins ( 1 7 ) , i n t h e i r s t u d i e s on Rauwolfia a l k a l o i d s and t h e i r behaviour on T.L.C. have piven v a l u a b l e i n f o r m a t i o n s on t h e c o l o u r r e a c t i o n s and R f v a l u e s of such r e l a t e d a l k a l o i d s on s i l i c a g e l l a y e r s employing 10 s o l v e n t systems and emphasis can b e c o n c e n t r a t e d on t h e r e l a t e d a l k a l o i d s : Yohimbine, a-yohimbine, s e r p e n t i n e , s a r p a g i n e and a j m a l i c i n e which come i n t h e s t r o n g l y b a s i c f r a c t i o n of Rauwolfia r o o t e x t r a c t . Q u a n t i t a t i o n p h o t o d e n s i t o m e t r i c a l l y on s i l i c a g e l l a y e r s of a l k a l o i d s of p l a n t s o u r c e s w a s c a r r i e d o u t by Massa e t a1 (53) who found t h a t f l u o r s c e n c e r e f l e c t a n c e r e a d i n g s of unsprayed s p o t s of f l u o r e s c e n t a l k a l o i d s were f a v o u r a b l e i n a c t i v a t e d s i l i c a g e l p l a t e s . They used a double-beam p h o t o d e n s i t o m e t e r w i t h r e c o r d e r and i n t e g r a t o r f u n c t i o n i n g i n t h e v i s i b l e and u l t r a v i o l e t l i g h t s . Nine s o l v e n t s systems were used and s u i t a b l e c o n c e n t r a t i o n s of s t a n d a r d s were r u n and t h e s t u d y w a s done on 25 a l k a l o i d s of which 1 4 a l k a l o i d s sprayed w i t h Dragendorff-Schuette r e a g e n t , were d e t e c t e d a t 400 nm and 11 o t h e r s were determined by i n h i b i t i o n of f l u o r e s c e n c e a t 528 nm and 13 a l k a l o i d s e s t i m a t e d by t h e i r n a t u r a l f l u o r e s c e n c e o r induced by a s u i t a b l e r e a g e n t such as KMnO4, I o d i n e o r d i c h l o r o a c e t i c a c i d and h e a t , and e x c i t e d a t 358 nm. However, Court and Habib (16) q u a n t i t a t e d 10 Rauwolfia a l k a l o i d s by combined p r e p a r a t i v e T.L.C. and c o l o r i m e t r y and found t h a t t h e i r method w a s b e s t s u i t a b l e f o r B-carboline a l k a l o i d s . Clarke ( 6 ) d e s c r i b e d t h e f o l l o w i n g TLC system f o r t h e i s o l a t i o n of yohimbine: P l a t e : Glass p l a t e s 20 x 20 c m , c o a t e d w i t h a s l u r r y c o n s i s t i n g of 30 g of s i l i c a p,el G i n 60 m l of w a t e r t o g i v e a l a y e r 0.25 mm t h i c k and d r i e d a t l l O o f o r 1 h o u r .

YOHIMBINE

163

Sample: acid. Solvent:

1 p1 of a 1%s o l u t i o n i n 2 N a c e t i c S t r o n g ammonia s o l u t i o n : methanol I t should be changed a f t e r

(1.5 : 1 0 0 ) .

two r u n s . E q u i l i b r a t i o n : The s o l v e n t i s allowed t o s t a n d i n t h e t a n k f o r 1 hour. Development: Ascending, i n a t a n k 2 1 x 2 1 x 10 cm, t h e end of t h e t a n k b e i n g covered w i t h f i l t e r paper t o a s s i s t e v a p o r a t i o n . Time of run: 30 m i n u t e s . Location reagent: Acidified i d o p l a t i n a t e Rf v a l u e 0.55. spray, p o s i t i v e reaction. 8.2.5.3

(G.L.C.)

Dusci and Hackett (54) d e s c r i b e d a d i r e c t p r o c e d u r e f o r a n a l y s i s of yohimbine and o t h e r n e u t r a l drugs i n t i s s u e s . They used Hewlett-Packard gas chromatograph model 5700A equipped w i t h a f l a m e i o n i z a t i o n d e t e c t o r . The column w a s a 4 f t x 4 mm i . d . g l a s s column pakced w i t h 3% OV-17 on Gas Chrom Q, 80-100 mesh. The i n s t r u m e n t s e t t i n g s were a s f o l l o w s : I n j e c t i o n p o r t t e m p e r a t u r e , 25OoC; d e t e c t o r t e m p e r a t u r e , 30OoC; n i t r o g e n c a r r i e r g a s flow r a t e , 60 ml/min. Oven t e m p e r a t u r e programmed as 15OOC h e l d f o r 2 m i n u t e s , i n c r e a s e d a t 8OC/min t o 29OoC and t h e f i n a l t e m p e r a t u r e was h e l d f o r 4 m i n u t e s . Using c h o l e s t e r o l a s a s t a n d a r d , t h e r e l a t i v e r e t e n t i o n t i m e (RRT) of yohimbine was 0.89. C l a r k e ( 6 ) r e p o r t e d t h e f o l l o w i n g GC system f o r t h e s e p a r a t i o n of yohimbine. 5% SE-30 on 60-80 mesh Chromosorb 5 f t x 118 i n c h i n t e r n a l d i a m e t e r s t a i n l e s s s t e e l column.

Column: W AW,

Column t e m p e r a t u r e : Carrier Gas:

23OoC.

Nitrogen.

A. G . MEKKAWI AND A. A. AL-BADR

764

Gas Flow:

30.7 m l p e r minute.

D e t e c t o r : Flame i o n i z a t i o n d e t e c t o r , hydrogen 22 m l p e r minute. Retention t i m e : 8.2.5.4

0.38 r e l a t i v e t o c o d e i n e .

High-speed L i q u i d Chromatography(HPLC)

The advent of HPLC i n t h e f i e l d o f s e p a r a t i o n and q u a n t i t a t i o n of almost a l l t y p e s of chemical m i x t u r e s made t h e t a s k s of t h e a n a l y t i c a l chemists f a r e a s i e r t h a n two decades ago. HPLC on a l k a l o i d s u s i n g v a r i o u s t y p e s of chromatographic s e p a r a t i o n modes w a s c a r r i e d o u t by v a r i o u s a u t h o r s . Yohimbine s e p a r a t i o n on a d o s r p t i o n HPLC w a s s u c c e s s f u l and V e r p o o r t e and Svendsen ( 5 5 ) were a b l e t o s e p a r a t e i t from r e s e r p i n e on Mercksorb S 1 70 ( 5 pm) column u s i n g 6 systems of e l u e n t and a Packard Model 8200 l i q u i d chromatograph equipped w i t h a 254 nm and 280 nm UV d e t e c t o r w a s used. The column w a s a s t a i n l e s s s t e e l t u b e (30 cm x 2 mm i.d.). The column t e m p e r a t u r e w a s maintained a t 2OoC and p r e s s u r e s of 50-250 kg/cm2 were employed t o c o n t r o l flow rates. However, t h e r e l a t i v e l y high[E 1%,1 cm]of yohimbine i n U.V. l i g h t a t 226 nm makes t h e d e t e c t i o n of minute q u a n t i t i e s p o s s i b l e i f t h e i n s t r u m e n t U.V. wavelength can be v a r i e d t o t h e optimum. Rodgers ( 5 6 ) w a s a b l e t o a n a l y s e s e v e r a l classes of t r a n q u i l i z e r drugs i n c l u d i n g Rauwolfia a l k a l o i d s by t h i s t e c h n i q u e u s i n g a d s o r p t i o n columns of s i l i c a g e l and c a t i o n exchangers. Acknowledgements The a u t h o r would of Pharmacognosy and t o T a n v i r A. Dept. f o r t y p i n g

l i k e t o thank M r . Amir S h e h a t a (B. Pharm.) Dept. f o r performing t h e m i c r o c r y s t a l t e s t s B u t t , S e c r e t a r y t o P h a r m a c e u t i c a l Chemistry t h e manuscript.

YOHIMBINE

765

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100,

742-746

CUMULATIVE INDEX Bold numerals refer to volume numbers

Acetaminophen, 3, I ; 14, 551 Acetohexamide, 1, I ; 2, 573 Allopurinol, 7, I Alpha-tocopheryl acetate, 3, 1 1 1 Amantadine, 12, 1 Amikacin sulfate, 12, 37 Amiloride hydrochloride, 15, I Arninoglutethimide. 15, 35 Aminophylline. 11, I Aminosalicylic acid, 10, I Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 Amphotericin B, 6, 1: 7, 502 Ampicillin, 2, I ; 4, 518 Ascorbic acid, 11, 45 Aspirin, 8, I Atenolol. 13, 1 Atropine, 14, 32 Azathioprine, 10, 29 Bacitracin, 9, 1 Baclofen, 14, 527 Bendroflumethiazide, 5, 1; 6, 597 Benperidol 14, 245 Benzocaine, 12, 73 Benzyl benzoate, 10, 55 Betamethasone dipropionate, 6, 43 Bretylium tosylate, 9, 71

Bromazepam, 16, I

Bromocriptine methanesulfonate, 8, 47

Busulphan, 16, 53 Caffeine, 15, 71 Calcitriol, 8, 83 Camphor, 13, 27 Captopril, 11, 79 Carbamazepine. 9, 87 Cefaclor, 9, 107 Cefamandole nafate, 9, 125; 10, 729 Cefazolin, 4, 1 Cefotaxime, 11, 139 Cefoxitin. sodium, 11, 169 Cephalexin, 4, 21 Cephalothin sodium, 1, 319

Cephradine, 5, 21 Chloral hydrate, 2, 85

Chlorambucil, 16, 85 Chloramphenicol, 4,47, 518; 15, 701 Chlordiazepoxide, I, 15 Chlordiazepoxide hydrochloride, 1, 39; 4, 518 Chloroquine, 13, 95 Chloroquine phosphate, 5,61 Chlorpheniramine maleate, 7, 43 Chlorprothixene, 2, 63 Chlortetracycline hydrochloride. 8, 101 Chlorthalidone, 14, I

Chlorzoxazone, 16, 119

Cholecalciferol. see Vitamin D3 Cimetidine, 13, 127 Cisplatin. 14, 77 : 15, 7% Clidinium bromide, 2, 145 Clindamycin hydrochloride, 10, 75 Clofibrate, 11, 197 Clonazepam, 6,61 Clorazepate dipotassiurn, 4, 91 Clotrimazole, 11, 225 Cloxacillin sodium, 4, 113 Cocaine hydrochloride, 15, 15 I Codeine phosphate, 10,93 Colchicine, 10, 139 Cyanocobalamin, 10, 183 Cyclizine, 6, 83; 7, 502 Cycloserine, 1,53

Cyclosporine, 16, 145 Cyclothiazide.1, 66 Cypropheptadine, 9, 155 Dapsone, 5, 87 Dexamethasone, 2, 163; 4, 519 Diatrizoic acid, 4, 137; 5, 556 Diazepam, 1,79: 4, 518 Dibenzepin hydrochloride, 9, 181 Dibucaine and dibucaine hydrochloride, 12, 105 Diflunisal, 14, 491 Digitoxin, 3, 149 Digoxin, 9, 207 Dihydroergotoxine methanesulfonate, 7, 81

769

770 Dioctyl sodium sulfosuccinate, 2, 199: 12, 713 Diperodon, 6 , 9 9 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disopyramide phosphate, 13, 183 Disulfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Dopamine hydrochloride. 11, 257 Doxorubicine. 9, 245 Droperidol, 7, 171 Echothiophate iodide, 3, 233 Emetine hydrochloride, 10, 289 Enalapril maleate, 16, 207 Ephedrine hydrochloride, 15, 233 Epinephrine, 7, 193 Ergonovine maleate, 11, 273 Ergotamine tartrate, 6, I13 Erythromycin. 8, 159 Erythromycin estolate, 1, 101; 2, 573 Estradiol. 15, 283 Estradiol valerate, 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7, 231 Ethynodiol diacetate, 3, 253 Etomidate. 12, 191 Fenoprofen calcium 6, 161 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 281 Flufenamic acid, 11, 313 Fluorouracil, 2, 221 Fluoxymesterone, 7, 25 I Fluphenazine decanoate. 9, 275; 10, 730 Fluphenazine enanthate, 2, 245; 4, 524 Fluphenazine hydrochloride, 2, 263; 4, 519 Flurazepam hydrochloride, 3, 307 Gentamicin sulfate, 9, 295; 10, 731 Glibenclamide, 10, 337 Gluthethimide, 5, 139 Gramicidin, 8, 179 Griseofulvin, 8, 219; 9, 583 Guanabenz acetate. 15, 319 Halcinonide, 8, 251 Haloperidol, 9, 341 Halothane, 1, 119; 2, 573; 14, 597 Heparin sodium, 12, 215 Heroin, 10, 357 Hexestrol, 11, 347 Hexetidine, 7, 277 Homatropine hydrobromide, 16, 245 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide, 10, 405 Hydrocortisone, 12, 277 Hydroflumethiazide, 7, 297 Hydroxyprogesterone caproate. 4, 209

CUMULATIVE INDEX Hydroxyzine dihydrochloride, 7, 319 Imipramine hydrochloride, 14, 37 Indomethacin, 13, 21 1 loddmide. 15, 337 lodipamide. 2, 333 lopanoic acid, 14, 181 Isocarboxazid. 2, 295 Isoniazide. 6, 183 Isopropamide. 2, 315; 12, 721 Isoproterenol, 14, 391 lsosorbide dinitrate, 4, 225; 5, 556 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Ketoprofen, 10, 443 Ketotifen. 13, 239 Khellin. 9, 371 Leucovorin calcium, 8, 315 Levallorphan tartrate, 2, 339 Levarterenol bitartrate, 1, 49; 2, 573; 11, 555 Levodopa, 5, 189 Levothyroxine sodium, 5 , 225 Lidocaine base and hydrochloride, 14, 207; 15, 761 Lithium carbonate. 15, 367 Lorazepam, 9, 397 Maprotiline hydrochloride, 15, 393 Mebendazole, 16, 291 Mefloquine hydrochloride, 14, 157 Melphalan, 13, 265 Meperidine hydrochloride, 1, 175 Meprobamate, 1, 209; 4, 520; 11, 587 6-Mercaptopurine, 7, 343 Mestranol. 11, 375 Methadone hydrochloride, 3, 365; 4, 520; 9, 601 Methaqualone. 4, 245, 520 Methimazole, 8, 351 Methotrexate, 5 , 283 Methoxsalen. 9, 427 Methyclothiazide, 5 , 307 Methylphenidate hydrochloride, 10,473 Methyprylon, 2, 363 Metocloprarnide hydrochloride, 16, 327 Metoprolol tartrate, 12, 325 Metronidazole, 5, 327 Minocycline. 6, 323 Mitomycin C, 16, 361 Moxalactam disodium, 13, 305 Nabilone, 10, 499 Nadolol, 9, 455; 10, 732 Nalidixic acid, 8, 371 Naloxone hydrochloride, 14, 453 Natamycin, 10, 513 Neornycin, 8, 399 Neostigrnine, 16, 403 Nitrazepam, 9, 487

CUMULATIVE INDEX Nitrofurantoin. 5, 345 Nitroglycerin. 9, 519 Norethindrone, 4, 268 Norgestrel, 4, 294 Nonriptyline hydrochloride. I, 233; 2, 573 Noscapine, 11, 407 Nystatin. 6, 341 Oxazepam. 3,441 Oxyphenbutazone. 13, 333 Oxytocin, 10, 563 Penicillamine. 10, 601 Penicillin-G benzothine, 11, 463 Penicillin C. potassium, IS, 427 Penicillin-V. 1. 249 Pentazocine, 13, 361 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4, 319; 5 , 429 Phenobarbital. 7, 359 Phenoxymethyl penicillin potassium. I , 249 Phenylhutazone, 11,483 Phenylephrine hydrochloride. 3,483 Phenylpropanolamine hydrochloride, 12, 357; 13, 771 Phenytoin, 13, 417 Pilocarpine, 12, 385 Piperazine estrone sulfate. 5 , 375 Pirenzepine dih ydrochloride, 16, 445 Piroxicam. 15, SO9 Primidone, 2,409 Probenecid. 10, 639 Procainamide hydrochloride, 4, 333 Procarbazine hydrochloride, 5, 403 Promethazine hydrochloride, 5 , 429 Proparacaine hydrochloride, 6, 423 Propiomazine hydrochloride, 2, 439 Propoxyphene hydrochloride, I , 301; 4, 520; 6, 598 Propylthiouracil, 6, 457 Pseudoephedrine hydrochloride, 8,489 Pyrazinamide, 12, 433 Pyridoxine hydrochloride, 13,447 Pyrimethamine, 12, 463 Quinidine sulfate, 12, 483 Quinine hydrochloride, 12, 547 Ranitidine. 15, 533 Reserpine, 4, 384; 5 , 557; 13, 737 Rifampin. 5, 467 Rutin. 12, 623 Saccharin, 13, 487 Salbutamol, 10, 665 Salicylamide. 13, 521

77 1 Secobarbital sodium, 1, 343 Silver sulfadiazine, 13, 553 Sodium nitroprusside, 6 , 487; 15, 781 Spironolactone, 4,431 Streptomycin, 16, 507 Strychnine. 15, 563 Succinycholine chloride, 10, 691 Sulfadiazine, 11, 523 Sulfamethazine. 7, 401 Sulfamethoxazole, 2, 467; 4, 521 Sulfasalazine, 5, 515 Sulfisoxazole, 2, 487 Sulindac. 13, 573 Sulphamerazine. 6, 515 Terpin hydrate, 14, 273 Testolactone, 5, 533 Testosterone enanthate. 4, 452 Tetracycline hydrochloride, 13, 597 Theophylline. 4, 466 Thiabendazole, 16, 61 I Thiosrrepton, 7, 423 Timolol maleate, 16, 641 Tolbutamide, 3, 513; 5, 557; 13, 719 Trazodone hydrochloride, 16, 693 Triamcinolone, 1, 367; 2, 571; 4, 521. 524: 11, 593 Triamcinolone acetonide, 1, 397, 416; 2, 571; 4, 521; 7, 501; 11, 615 Triamcinolone diacetate. 1, 423: 11, 651 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2 , 507 Trifluoperazine hydrochloride, 9, 543 Triflupromazine hydrochloride, 2, 523; 4, 521; 5, 557 Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2, 551 Trimethoprim, 7, 445 Trimipramine maleate. 12, 683 Trioxsalen, 10, 705 Tripelennamine hydrochloride. 14, 107 Triprolidine hydrochloride, 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7, 477 Tybamate, 4,494 Valproate sodium and valproic acid, 8, 529 Vidarabine. IS, 647 Vinblastine sulfate, 1, 443 Vincristine sulfate. 1, 463 Vitamin Dlr 13, 655 Warfarin, 14, 243 Xylometazoline hydrochloride, 14, 135 Yohimbine, 16, 731 Zomepirac sodium, 15, 673