Analytical Profiles of Drug Substances Klaus Florey (Eds.) - Academic Press (1976) PDF [PDF]

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Analytical Profiles of Volume 5 Edited b y



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



Contributing Editors



Norman W. Atwater Glenn A. Brewer, Jr. Lester Chafetz



Boen T. Kho Gerald J. Papariello Bernard 2. Senkowski



Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences



Academic Press New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers



1976



EDITORIAL BOARD Norman W. Atwater Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari



Erik H. Jensen Boen T. Kho Arthur F. Michaelis Gerald .J.Papariello Bruce C. Rudy Bernard Z. Senkowski Frederick Tishler



Academic Press Rapid Manuscript Reproduction



COPYRIGHT 0 1976, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE 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. 111 Fifth Avenue, New



York, New' York 10003



United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W I



Library of Congress Cataloging in Publication Data Main entry under title: Analytical profiles of drug substances. Includes bibliographical references. Compiled under the auspices of the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences. 1. Drugs-Analysis-Collected works. 2. Chemistry, I. Florey, Medical and pharmaceutical-Collected works. 11. Brewer, Glenn A. 111. Academy of Klaus, ed. Pharmaceutical Sciences. Pharmaceutical Analysis and 1. Drugs-Analysis-Yearbooks. Control Section. [DNLM: QV740 AA1 A551 RM300.A56 616l.1 70-187259 ISBN 0-12-260805-4 (v. 5)



PRINTED IN THE UNITED STATES OF AMERICA



AFFILIATIONS OF EDITORS AND CONTRIBUTORS H. Y. Aboul-Enein, USV Pharmaceuticals, Tuckahoe, New York, now: Riyadh University, Riyadh, Saudi Arabia



G. D. Anthony, Searle and Company, Chicago, Illinois



N. W.Atwuter, Searle and Company, Chicago, Illinois J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey



G.A. Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey



D. E. Cudwulluder,School of Pharmacy, University of Georgia, Athens, Georgia L. Chufefz,Warner-Lambert Research Institute, Morris Plains, New Jersey A. R. Chamberlin, Stanford Research Institute, Menlo Park, California



2.L. Chung,Searle and Company, Chicago, Illinois A. P. K . Chatng, Stanford Research Institute, Menlo Park, California E. M.Cohen, Merck, Sharp and Dohme, West Point, Pennsylvania J. P. Comer, Eli Ully and Company, Indianapolis, Indiana



R. D. Duley, Ayerst Laboratories, Rouses Point, New York K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey S. A. Fusuri, Parke, Davis and Company, Detroit, Michigan



vii



AFFILIATIONS OF EDITORS A N D CONTRIBUTORS



G. G. Gallo, Gruppo LePetit, Milan, Italy R. Gomez, Hoffmann-LaRoche, Inc., Nutley, New Jersey R. B. Hagel, Hoffmann-LaRoche, Inc., Nutley, New Jersey



D.D.Hong, The Sterling-Winthrop Research Institute, Rensselaer, New York E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan J , H. Johnson, Hoffmann-LaRoche, Inc., Nutley, New Jersey



H. W. Jun, School of Pharmacy, University of Georgia, Athens, Georgia B. T. Kho, Ayerst Laboratories, Rouses Point, New York



P. Lim, Stanford Research Institute, Menlo Park, California J. P. McDonnell, Salsbury Laboratories, Charles City, Iowa



E. A. MacMuZlan, Hoffmann-LaRoche, Inc., Nutley, New Jersey



A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey S. M.Miller, Wyeth Laboratories, Philadelphia, Pennsylvania



N. G.Nash, Ayerst Laboratories, Rouses Point, New York



C E. Onech, Ayerst Laboratories, Rouses Point, New York G. J. Papanello, Wyeth Laboratories, Philadelphia, Pennsylvania A. Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania



P. Radaelli, Gruppo LePetit, Milan, Italy J,



A. Raihle, Abbott Laboratories, North Chicago, Illinois



R, J. Rucki, Hoffmann-LaRoche, Inc., Nutley, New Jersey B. C. Rudy, Schering-Plough,Corp., Bloomfield, New Jersey



viii



AFFILIATIONS OF EDITORS A N D CONTRIBUTORS



F. Russo-Alesi, The Squibb Institute for Medical Research, New Brunswick, New Jersey



B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey



C.M. Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania F. Tishler, Ciba-Geigy, Summit, New Jersey



R J. Warren, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania E. H. Waysek,Hoffmann-LaRoche, Inc., Nutley, New Jersey L. L. Wearley, Searle and Company, Chicago, Illinois



ix



PREFACE Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degredation and metabolism. For drug substances important enough to be accorded monographs in the official compendia such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the fifth. The concept of Analytical Profiles is taking hold not only for cornpendial drugs but, increasingly, in the industrial research laboratories. Analytical Profiles are being prepared and periodically updated to provide physico-chemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not too distant future, the publication of an Analytical Profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors had made this venture possible. All those who have found the profiles useful are earnestly requested to contribute a monograph of their own. The editors stand ready to receive such contributions.



Klaus Florey



xi



BENDROFLUMETHIAZIDE



Klaus FIorey and Frank M.Russo-Alesi



KLAUS FLOREY A N D FRANK M . RUSSO-ALES)



CONTENTS 1.



Description 1.1 History 1.2 Name, Formula, Molecuiar Weight 1.3 Appearance, Color,Odor



2.



Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Thermal Analysis 2.7 Solubility 2.8 Crystal Properties 2.9 pKa



3.



Synthesis



4.



Stability and Degradation



5.



Drug Metabolic Products



6.



Methods of Analysis 6.1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Colorimetric Analysis 6.4 Nonaqueous Titration 6.5 Chromatographic Analysis 6.51 Paper 6.52 Thin-Layer



7.



Identification and Determination in Biological Fluids



8.



Miscellaneous



9.



References



2



- Pharmacokinetics



BENDROFLUMETH IAZIDE



1.



Description 1.1 Histor d l u m e t h i a z i d e belongs to the class of thiazide diuretics. Its synthesis was first reported by Holdrege, Babel and Cheneyl in 1959 and its diuretic activity was first described in 960 almost simultaneously by Lund and Kobing and by Kennedy, Buchanan and Cunningham



s.



1.2



Name, Formula, Molecular Weight Bendroflumethiazide (also bendrofluazide, benydroflumethiazide, and benzylhydroflumethiazide) is 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3,4dihydro-3-(phenyl methyl)-6-trifluoromethyl-l,ldioxide or 3-Benzyl-3,4-dihydro-6-(trifluoromethyl)-2H-1,2,4-benzothiad~az~ne-7-sulfonam~de1,l dioxide [73-48-31, H



>



'



( NH@ SO'



0



0



a



HC-CH2 CF 3



H Mol. Wt. 421.41



1' SH1qF 3N304 2 1.3



Appearance, Color, Odor White or sliqhtly off-white, uniform free flowing crystalline powder i slight floral odor. Physical Properties 2.1 Infrared Spectrum The infrared spectrum f bendroflumethiazide is given in Figure 1 2.



P.



2.2



Nuclear Magnetic Resonance The 60 MHz proton magnetic resonance spectrum in d4-methanol containing tetramethylsilane as an intern93 reference (Figure 2) is assigned as follows :



3



4



P



..



m



rl



h



=w=



cu



(batch #15); KBr, MeOH. cl fd



al



fdk kC\



H H



a



Infrared Spectrum of Bendroflumethiazide Instrument: Perkin-Elmer 621. wrn G G



al



rl



Figure 1.



m



Figure 2.



NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated methanol (Instrument: Perkin Elmer R12B)



KLAUS FLOREY A N D FRANK M. RUSSO-ALES1



H ~1.67(s)



not assigned T2.7



Q



I n d6-DMSO ( F i g u r e 3 1 , t h e NH p r o t o n r e s o n a n c e s were observed near ~ 1 . 7 5( s i n g l e t , superimposed w i t h a r y l p r o t o n ) , ~2 ( b r o a d ) , 2 . 4 5 ( s i n g l e t ) and 2.57 ( s i n g l e t ) ( F i g u r e 3) i n a d d i t i o n t o t h e o t h e r p r o t o n s a s s i g n e d from t h e dq-methanol spectrum. The h i g h e r f i e l d s i n g l e t s a t ~ 2 . 4 5and 2.57 are t e n t a t i v e l y a s s i g n e d t o t h e sulfonamide -NH2 p r o t o n s . N o p r e f e r e n c e of assignment i s made of t h e o t h e r two -NH p r o t o n s . On a d d i t i o n of D 2 0 , t h e -NH p r o t o n s a r e r a p i d l y exchanged f o r d e u t e r i u m , r e s u l t i n g i n t h e d i s a p p e a r a n c e of t h e NH p r o t o n s and sharpening of t h e p r o t o n r e s o n a n c e n e a r ~5 which now a p p e a r s l i k e t h a t of t h e dq-methanol spectrum



.



2.3



U l t r a v i o l e t Spectrum Squibb House S t a n d a r d ( b a t c h #15) i n methanol f x h i b i t e d t h r e e maxima a t 208 mu (El 7 4 5 ) , 1 273 mp (E 565) and 326 mu (Ei 9 6 ) 1 3 . T h i s ag i s w i t h measurements by P i l s b u and JacksonfF and by Kracmar and L a s t o v k o v a f i who d i s c u s s t h e U . V . spectrophotometry of b e n z o t h i a d i a z i n e s and p r e s e n t s p e c t r a . Mass Spectrum The l o w r e s o l u t i o n mass spectrum ( F i g u r e 4 ) shows o n l y a v e r y weak m o l e c u l a r i o n (M+) of m / e 4 2 1 and a b a s e peak o f m / e 319 t h a t could a r i s e by a double p r o t o n r e a r r a n g e m e n t , which i s n o t a common f r a g m e n t a t i o n pathway. The assignment of some of t h e fragment i o n s i s shown below: 2.4



6



0



Figure 3.



L



I



4



c



7



r



S



in



NMR Spectrum of Bendroflumethiazide (batch 15) in deuterated DMSO (Instrument: Perkin-Elmer R12B)



3753 SO15497 BFI#15 12-FEB-76



MR0587



I00



I



I



90.. 80.-



70.60.-



5 0.40-



ie MRSS/CHRRGE



INTENSITY SUM = 1 8 3 3 9



Figure 4.



BRSE PERK Z = 8 . 3 2



Low Resolution Mass Spectrum of Bendroflumethiazide (Instrument: AEI MS9)



BENDROFLUMETHIAZIDE



319



H 2"H2



H



330191



77



m/e 402 6 M+ m/e 4 2 1



-so2NH2



m/e 303 m/e 302 (-HI



m/e 239 \< m/e 255



-so2



m/e 319-



4



m/e 302 -so2NH2, m/e 2 2 2



H



m/e 174 m/e 175 (+HI m/e 176 (+2H)



m/e 158 m/e 159 (+HI



Although t h e s e a p p e a r t o be r e a s o n a b l e a s s i g n m e n t s , t h e y should be c o n s i d e r e d t e n t a t i v e s i n c e t h e a s s i nments a r e n o t confirmed by h i g h - r e s o l u t i o n d a t a 3.



s



2.5



Melting Range The m e l t i n g p o i n t d o e s n o t o c c u r s h a r p l y and depends on t h e r a t e of h e a t i n g . The m e l t i n g p o i n t of t h e Squibb House S t a n d a r d ( b a t c h 15) w a s r e p o r t e d a t 223.20 - 226.20 C. L i t e r a t u r e v a l u e s r a n g e from 220' - 228'.



2.6



D i f f e r e n t i a l Thermal A n a l y s i s Melting endotherm: 2210 C. (17).



2.7



Solubilit d i



n water and c h l o r o f o r m .



9



KLAUS FLOREY AND FRANK M. RUSSO-ALES1



S o l u b l e i n a l k a l i , a c e t o n e and a l c o h o l . S o l u b l e i n one e q u i v a l e n t of 0 . 1 N NaOH. S l i g h t l y s o l u b l e i n e t h e r . I n s o l u b l e i n a c i d , benzene, l i g r o i n and petroleum e t h e r l 7 . S t a b l e c o n c e n t r a t e d s o l u t i o n i n p o l y e t h y l e n e g l y c o l , water and dimethyla c e t a n i l i d e o r N-methyl-2-pyrolidinone have been claimed (18). 2.8



Crystal Properties The powder x-ray d i f f r a c t i o n p a t t e r n i s p r e s e n t e d i n Table I and F i g u r e 517.



TABLE I 0



d (A) *



I/I0**



17.23 13.27 11.63 8.94 8.11 7.64 7.36 6.70 6.34 5.91 5.48 5.31 4.82 4.67 4.53 4.46 4.31 4.19 3.96 3.84 3.75 3.69 3.63 3.54 3.44 3.40 3.19 3.11 2.99



0.528 0.356 0.103 0*246 *d = i n t e r p l a n a r d i s t a n c e 0.148 - 1 0.118 11 n 0.100 Z @ 0.199 0.124 X = 1.539 A 0.341 0*141 Radiation: K a l and 0.194 0.162 Ka2 Copper 0.545 l'ooo elative intensity 0.206 ** R based on h i g h e s t 0.175 i n t e n s i t y of 1 . 0 0 0.516 0.213 0.357 0.185 0.248 0.289 0.205 0.140 0.145 0.166 0.145 0.157



10



I



Figure 5.



Powder X-Ray Diffraction Pattern of Bendroflumethiazide (Instrument: Norelco)



I



KLAUS FLOREY AND FRANK M. RUSSO-ALES1



2.9



pKa



A pKa of 8.5320.05 was determined b using the solubility variation with pH method4




SPACE



MEASURED C E L L COJTI'ENTS



12 cephradine



P 0



4 cephradine Acetonitrile Monohydra t'e Recrystallized from 17.58 M e th an o 1



I 9.4



21.6



90



3568



P212121



1.33



8 cephradine 8 water



Synthesis Cephradine (111, Figure 10) is synthesized by coupling 7-aminodesacetoxycephalosporanic acid (7-ADCA) (11) with a protected derivative of dihydrophenylglycine (I), Figure 10 Synthetic Pathway to Cephradine 3.



0hz CH



-



Intermediate



COOH



+ H2N



I



COOH



0:" -



CO - J < m -J



m2



/



0



CH3



COOH I11



such as the tert.-butoxylcarbonyl derivative which can be converted to a mixed anhydride with ethylchloroformate and reacted with 7-ADCA14. Cephradine can also be made by forming the methyl acetoacetic ester enamine derivative of dihydrophenylglycine-which is converted to a mixed



CEPHRADINE



anhydride w i t h benzoyl c h l o r i d e p r i o r t o coupling Cephradine i s t h e n cr s t a l l i z e d w i t h 7-ADCAI2. It also from a b i p h a s i c MIBK-aqueous s o l u t i o nY2 can be c r y s t a l l i z e d from w a t e r a l o n e , a s w e l l a s from o t h e r s o l v e n t s ( s e e s e c t i o n 2 . 1 1 ) .



.



S t a b i 1i ty-Degrada t i o n 4 . 1 Bulk S t a b i l i t y C e p h r a d i n q w h e n k e p t u n d e r d r y and c o o l s t o r a g e c o n d i t i o n s , h a s shown r e a s o n a b l e b u l k s t a b i l i t y 1 2 . Like o t h e r 1,4 cyclohexadienes such a s 2,5 d i h y d r ~ p h e n y l a l a n i n e ~c~e p, h r a d i n e i s prone t o a slow r a t e of o x i d a t i o n of t h e c y c l o hexadiene r i n g t o t h e benzenoid r i n g . The e x a c t mechanism of t h i s r e a c t i o n i s n o t known, b u t i n a d d i t i o n t o oxygen and w a t e r , t r a c e m e t a l s s u c h a s i r o n seem t o have an a c c e l e r a t i n g e f f e c t . The o x i d a t i o n t o c e p h a l e x i n a s w e l l a s d e g r a d a t i o n (loss of b i o p o t e n c y ) can be p r e v e n t e d o r s i g n i f i c a n t l y r e d u c e d b y s t o r a g e a t low t e m p e r a t u r e and e x c l u s i o n o f oxygen, a s w e l l a s removal of w a t e r . T h i s l a s t method, however, i s i m p r a c t i c a l due t o t h e e x t r e m e l y h y g r o s c o p i c n a t u r e o f anhydrous c e p h r a d i n e 1 2 . On t h e o t h e r hand c e p h r a d i n e d i h y d r a t e 11 b e i n g a t r u e s o l v a t e (see s e c t i o n 2 , l l ) w h e r e water c a n n o t move f r e e l y i n t h e c r y s t a l l a t t i c e e x h i b i t s remarkable r e s i s t e n c e t o c e p h a l e x i n f o r m a t i o n , l o s s o f b i o p o t e n c y and c o l o r d e v e l o p m e n t d u r i n g e x t e n d e d s t o r a g e u n d e r a i r l 2 . The d i f f e r e n c e b e t w e e n c e p h r a d i n e and i t s d i h y d r a t e i s i l l u s t r a t e d i n Table 812. However upon p a r t i a l o r f u l l d e h y d r a t i o n of c e p h r a d i n e d i h y d r a t e u n d e r a v a r i e t y of conditions, drying a t higher temperatures o r c e r t a i n kinds of m i l l i n g t h i s e x c e l l e n t s t a b i l i t y i s not maintainedlz. 4.



45



KLAUS FLOREY



Table 8 Average Bulk S t a b i l i t y Data for Three Batches of Cephradine and f o r Three Batches of Cephradine Dihydrate A f t e r 9 Months of S t o r a g e Under A i r Cephradine ( " a s i s " ) Initial Bioassay, mcg/ml 949 2.7 Cephalexin, % Bioassay, mcg/ml Cepha l e x i n , %



5Oc 947 3.0



RT



942 3.5



4OoC 924 4.6



5OoC 849 7.0



Cephradine Dihydra t e ( " a s i s " ) 870 897 906 870 909 2.3 2.3 2.3 2.2 2.4



Cephradine i s moderately l i g h t s e n s i t i v e . When exposed t o U.V. l i g h t , t h e s o l i d t u r n s yellow on t h s u r f a c e b u t no loss of b i o The s e n s i t i v i t y of a c t i v i t y w a s noted f 2 c e p h a l o s p o r i n C t o l i g h t h a s been p r e v i o u s l y no t e d l 6 . Other than c e p h a l e x i n , no d e g r a d a t i o n p r o d u c t s of s o l i d cephradine have been i d e n t i f i e d TLC examination of degraded (loss of biopotency) samples r e v e a l e d a m u l t i p l i c i t y of U.V. a b s o r b i n g and f l u o r e s c e n t s p o t s 1 7 . 4.2 S t a b i l i t y i n Solution I n aqueous s o l u t i o n c e p h r a d i n e t e n d s t o be q u i t e s t a b l e a t pH 4.0 and below and much l e s s s t a b l e a t h i g h e r pH u n i t s . A p H - s t a b i l i t y p r o f i l e i s p r e s e n t e d i n Table 96 Cephradine was found to be f u l l y p o t e n t for at l e a s t e i g h t h o u r s i n a v a r i e t y of p a r e n t e r a l i n f u s i o n s o l u t i o n s 2 3 . Although cephradine, l i k e o t h e r cephalo s p o r i n s i s much more r e s i s t a n t t o opening of t h e B-lactam r i n g by a l k a l i t h a n p e n i c i l l i n s , t h e r i n g does open up w i t h subsequent f u r t h e r d e g r a d a t i o n , Ring opening a l s o l e a d s t h e loss of U.V. absorbance a t 262 nm.



.



.



46



Table 9 S t a b i l i t y of C e p h r a d i n e i n Phosphate B u f f e r a t Room Temperature



*



p H of S o l u t i o n 4.0 6.0 8.0



10.0



2 days 95.9 73.0 67.1 64.0



P e r c e n t of Remaining B i o a c t i v i t y 4 days 7 days 10 days 105.1 86.9 43.1 41.1



99.3 69.5 24.5 25.2



99.3



30.2 13.4 13. 3



14 days 95.2 18. 5 10.9 11.7



* 5



ph of samples a t s t a r t of s t u d y . The pH o f t h e h i g h pH samples d r i f t e d toward lower pH v a l u e s a s t h e s t u d y p r o g r e s s e d .



One of t h e a l k a l i n e d e g r a d a t i o n p r o d u c t s p r e c i p i t a t e s from t h e s o l u t i o n and h a s been i d e n t i f i e d a s 2-[6-(1,4 cyclohexadien-l-yl)-2, 5-dioxo-3-piperazinyl~-5,6-dihydro-5-methyl-2H-l,3-thiazine-4-carboxylic a c i d , sodium s a l tI8.



KLAUS FLOREY



The B-lactam r i n g o f c e p h r a d i n e i s q u i t e res i s t a n t t o p e n i c i l l i n a s e , b u t opens r e a d i l y w i t h cephalosporinasel9. On t h e a c i d s i d e NMR s t a b i l i t y s t u d i e s w i t h a 2% s o l u t i o n a t pH 1 . 6 , h e l d a t 60°C, d e m o n s t r a t e d t h a t t h e r e i s no 0-lactam r i n g opening. However p o s s i b l e s h i f t i n g o f t h e d o u b l e bond from c a r b o n s 2-3 t o 3-4 was o b s e r v e d b y NMR and c o n f i r m e d by loss of U.V. a b s o r b a n c y a t 262 nm. A f t e r 93 h o u r s NMR i n d i c a t e d a 50% d o u b l e bond i s o m e r i z a t i o n 2 . Very v i g o r o u s treatment with strong a c i d w i l l lead a t l e a s t i n p a r t t o a s p l i t o f t h e amide l i n k a g e t o form 7-ADCA and d i h y d r o p h e n y l g l y c i n e 1 2 . This cleavage can a l s o be a c h i e v e d e n z y m a t i c a l l y w i t h p e n i c i l l i n acylase19. I n a phosphate b u f f e r a t pH 6 v i g o r o u s a e r a t i o n o r e x p o s u r e t o room l i g h t However 10 d i d n o t a c c e l e r a t e degradation2'. h o u r s of e x p o s u r e t o a Hanovia U.V. lamp of an aqueous s o l u t i o n (pH 5 ) of c e p h r a d i n e more t h a n doubled t h e c e p h a l e x i n c o n t e n t w i t h l i t t l e Cephradine was found t o change i n b i o p o t e n c y 1 2 . be s t a b l e f o r a t l e a s t t h i r t y days i n f r o z e n (-S0C) human s e r u m and u r i n e . No significant l o s s was d e t e c t e d by r e p e a t e d thawing and On t h e o t h e r hand when i n c u b a t e d refreezing2'. w i t h s er u m a t 37OC f o r 6 h o u r s t h e r e was a 20% loss of a c t i v i t y . I n c u b a t i o n w i t h whole b l o o d under t h e same c o n d i t i o n s c a u s e d l i t t l e o r no loss of b i o p o t e n c y 2 2 . I t was found5I, t h a t t h e a c t i v i t y of c e p h a l o s p o r i n s c o n t a i n i n g a p h e n y l g l y c i n e m o i e t y ( c e p h a l o g l y c i n and t o a l e s s e r d e g r e e c e p h a l e x i n and c e p h r a d i n e ) i s p r o g r e s s i v e l y 10s t i n t h e p r e s e n c e of c u p r i c i o n . T h i s d e g r a d i n g e f f e c t of c u p r i c i o n can be i n h i b i t e d b y d-penicillamine.



40



CEPHRADINE



Druq Metabolism Cephradine-3H 250 mg d r y f i l l e d c a p s u l e s w e r e a d m i n i s t e r e d t o e i g h t normal male s u b j e c t s a s a s i n g l e o r a l dose i n a b i o a v a i l a b i l i t y study24. Serum and u r i n e s a m p l e s were a s s a y e d i n a d o u b l e b l i n d f a s h i o n f o r b i o l o g i c a l a c t i v i t y and i n a n open f a s h i o n f o r r a d i o c h e m i c a l a c t i v i t y . The mean p e a k c e p h r a d i n e s e r u m c o n c e n t r a t i o n (7.0 2 1 . 0 kgm/ml SE by b i o a s s a y a n d 7 . 8 f 0 . 9 pgm/ml - SE b y r a d i o a s s a y ) o c c u r r e d 55 m i n u t e s a f t e r d o s i n g and d e c r e a s e d w i t h a b i o l o g i c a l h a l f - t i m e o f 40 m i n u t e s . The c u m u l a t i v e a r e a s u n d e r t h e c u r v e s f o r c e p h r a d i n e were 1 6 1 . 5 f 29 and 1 7 7 . 3 f 34.7 min. pgm/ml f o r b i o a s s a y and radioassay curves respectively. C e p h r a d i n e was r a p i d l y e x c r e t e d . A p p r o x i m a t e l y 77% of c e p h r a d i n e was e x c r e t e d w i t h i n t h e t h r e e h o u r p e r i o d f o l l o w i n g d r u g a d m i n i s t r a t i o n . A f t e r 24 h o u r s a p p r o x i m a t e l y 87% o f t h e a d m i n i s t e r e d d o s e of c e p h r a d i n e was r e c o v e r e d i n t h e u r i n e a s m e a s u r e d b y both b i o and r a d i o c h e m i c a l a s s a y25 Four h e a l t h y f e m a l e s u b j e c t s r e c e i v e d a s i n g l e i n t r a m u s c u l a r i n j e c t i o n of 1 gram o f ~ e p h r a d i n e - ~ H ~The ~ . mean p e a k c o n c e n t r a t i o n o f 1 0 f 1 . 7 ug/ml SE i n plasma w a s r e a c h e d a t 2 h o u r s a f t e r a d m i n i s t r a t i o n and t h e n d e c r e a s e d with a b i o l o g i c a l h a l f - l i f e of 2 hours. Binding of c e p h r a d i n e t o plasma p r o t e i n was f o u n d t o be 6% o v e r t h e r a n g e of c o n c e n t r a t i o n s found i n plasma d u r i n g t h e a b s o r p t i v e a n d e x c r e t o r y phases. A b s o r p t i o n , b a s e d on 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 u r i n e o v e r t h e 24 h o u r p e r i o d + 6.6% + SE f o r f o l l o w i n g a d m i n i s t r a t i o n , w a s 92 t h e f o u r s u b j e c t s . A n o t h e r 1%was e x c r e t e d i n f e c e s w i t h i n t h e 72-hr p e r i o d o f t h e e x p e r i m e n t . T h e r e w a s no s i g n i f i c a n t d i f f e r e n c e i n e x c r e t i o n b a s e d on t h e r e c o v e r y o f r a d i o a c t i v i t y o r antimicrobial a c t i v i t y i n urine. E x c r e t i o n was 66 f 6.9% f SE a t t h e e n d o f 6 h r and 8 5 f 6.5% 5.



-



.



-



-



49



-



5 S E a t t h e end of 1 2 hours.



Recovery of antimicrobial a c t i v i t y i n u r i n e and t h e a r e a under t h e c u r v e f o r c o n c e n t r a t i o n o f a n t i b i o t i c i n plasma were i n e x c e l l e n t agreement w i t h t h o s e found when l a b e l l e d c e p h r a d i n e w a s administered o r a l l y . Cephradine a p p e a r s t o be s l o w l y r e l e a s e d from t h e s i t e o f i n j e c t i o n t o g i v e l e v e l s of a n t i b i o t i c i n plasma which, though r e a c h i n g a maximum a t 1/3 t h o s e found a f t e r o r a l a d m i n i s t r a t i o n , p r o v i d e t h e same amount o f b i o a v a i l a b l e a n t i b i o t i c o v e r a l o n g e r p e r i o d of t i m e 2 4 N o c a n i n e o r human drug m e t a b o l i t e s w e r e found so f a r e x c e p t t r a c e amounts of c e p h a l e x i n , a s i d e n t i f i e d by m a s s spectrometry26. The above i n f o r m a t i o n was p u b l i s h e d ( r e f e r e n c e s 27-31). 6.



Methods o f A n a l y s i s 6 . 1 Elemental A n a l y s i s Calc. f o r anhydrous Calc. f o r monohydrate Found f o r c e p h r a d i n e b a t c h NN057NC Calc. f o r d i h y d r a t e Found f o r c e p h r a d i n e d i h y d r a t e b a t c h NNOO5NB



%



C



H



N



55.01 52.30



5.48 5.76



12.03 11.44



51.96 49.85



5.83 6.01



11.16



9.00



10.90



8.31



49.77



6.06



10.95



8.39



S



9.16 8.73



CEPHRADINE



6.2



M i c r o b i o l o g i c a l Assay For b u l k and f o r m u l a t e d p r o d u c t s a t u r b i d i m e t r i c method u s i n g S t r e p t o c o c c u s f a e c a l i s A.T. C. C. 1 0 , 5 4 1 i s c o n v e n i e n t . A l t e r n a t i v e l y a g a r p l a t e methods u s i n g S a r c i n a l u t e a A . T . C. C. 9341, B a c i l l u s pumilus A . T . C. C. 14,884 o r S t a p h y l o c o c c u s a u r e u s , A.T.C. C. 6538P = FDA 209P, a r e a l s o employed. F o r b l o o d and body f l u i d samples an a g a r p l a t e method i s u s e d employing S a r c i n a l u t e a a s t e s t o r anism b e c a u s e of i t s g r e a t s e n s i t i v i t y3 2 , 4 6 , 4 7 , The minimum i n h i b i t o r y c o n c e n t r a t i o n (MIC) of cephradine f o r t h e f o u r t e s t c u l t u r e s i s a s follows: mcq/ml Sarcina lutea 0.04 Staphylococcus aureus 0.40 0.40 Bacillus pumilus Streptococcus f a e c a l i s 50.0 Cephradine i s s l i g h t l y more b i o a c t i v e a g a i n s t S t r e p t o c o c c u s f a e c a l i s and S a r c i n a l u t e a than cephalexin, while the reverse hold t r u e f o r Staphylococcus a ~ r e u s ~ ~ . 6.3 Iodometric Analysis Cephradine can be d e t e r m i n e d by t h e i o d o m e t r i c a s s a y . The B-lactam r i n g i s opened w i t h a l k a l i o r c e p h a l o s p o r i n a s e f o l l o w e d by i o d i n a t i o n a t an a c i d pH (pH 4 . 5 p h o s p h a t e b u f f e r ) . About 4-5 moles o f i o d i n e a r e c o n s u m e d34 , I t i s i n t e r e s t i n g t o n o t e t h a t p e n i c i l l i n s under t h e same c o n d i t i o n consume 8-9 moles o f i o d i n e . The p r e c i s i o n of t h e i o d o m e t r i c a s s a y f o r c e p h r a d i n e i s n o t a s good a s t h a t f o r p e n i c i l l i n s when a l k a l i i s u s e d f o r i n a c t i v a t i o n . The p r e c i s i o n can be c o n s i d e r a b l y improved by u s i n g cephalos o r i n a s e i n s t e a d of a l k a l i f o r r i n g opening A l s o , w i t h t h e l a t t e r method much b e t t e r agreement was o b t a i n e d w i t h t h e m i c r o b i o -



16.



51



logical assay (see Table 10) even with severely degraded bulk samples. It therefore can be considered stability indicating. Table 10 Comparison of the cephradinase-iodometric, alkali-iodometric * and bioassay methods for the determination of cephradine in bulk powders Cephradine potency = mcg/mg Sample NN054ND NNO 5 9ND NN061ND NN054N.D NNO59ND NNO61ND



* Results



Bioassay 812 854 778 578 424 405



Cephalosporinaseiodometric assay 813 043 846 578 466 464



Alkali-iodometric assay 979 991 980



744 643 639



are the means of determinations on two consecutive days. The powders were stored at 5OoC two years in bottles with varying volumes of head space.



CEPHRADINE



The p r e s e n c e of 7-ADCA d o e s n o t i n t e r f e r e w i t h t h e i o d o m e t r i c a s s a y 19 I t was found t h a t when i o d i n a t i o n was c a r r i e d o u t a t an a l k a l i n e r a t h e r t h a n a c i d pH, 1 3 e q u i v a l e n t s o f i o d i n e were consumed 34 6.4 Spectrophotometric Analysis The u l t r a v i o l e t a b s o r b a n c e peak of c e p h r a d i n e a t 262 nm ( s e e s e c t i o n 2 . 4 ) can be u s e d a s an i d e n t i f y a n d homogeneity a s s a y i n formulations35. Opening of t h e B-lactam r i n g w i t h a l k a l i o r preferably, with cephalosporinase a b o l i s h e s t h e U.V. a b s o r b a n c e a t 260nm. This h a s been made t h e p r i n c i p a e o f a q u a n t i t a t i v e a s s a y which a p p e a r s t o be s t a b i l i t y i n d i c a t i n g i n t h e " p r a c t i c a l " r a n g e ( l e s s t h a n 20% loss o f b i o a c t i v i t y ) 19. 6.5 Fluorometric Analysis Cephradine can be a s s a y e d q u a n t i t a t i v e l y i n a l k a l i n e medium b y f l u o r i m e t r y , ( e x c i t a t i o n wave l e n g t h 350 nm, e m i s s i o n wave l e n g t h 495 nm). T h i s method h a s been used f o r b l o o d l e v e l s t u d i e s b u t i s n o t recommended f o r t h e d e t e r m i n a t i o n i n u r i n e because of e r r a t i c r e s u l t s . 6.6 Colorimetric Analysis The w e l l known hydroxylamine method f o r p e n i c i l l i n h a s been a d a p t e d b y FDA t o cephradine a s a batching assay. It i s not s t a b i l i t y indicating. Strongly alkaline reaction c o n d i t i o n s and f e r r i c n i t r a t e w e r e used 5 When f e r r i c ammonium s u l f a t e was u s e d a t pH 7 o n l y 15% o f t h e r e s p o n s e normal f o r p e n i c i l l i n s was o b t a i n e d 3 7. A c o l o r i m e t r i c method u s i n g 5 , 5 ' d i t h i o b i s ( 2 - n i t r o b e n z o i c a c i d ) a t pH 9.2 p r o d u c e s a y e l l o w c o l o r which can be q u a n t i t a t e d b y measuring t h e peak a b s o r b a n c e a t 412 nm38.



.



.



.



-



53



KLAUS FLOREY



6.7



Chromatoqraphic A n a l y s i s 6 . 7 1 Paper Cephalexin (slower moving component) can be s e p a r a t e d from c e p h r a d i n e w i t h a n-butanol-t-amyl alcohol-~ater(7:l:4)system and q u a n t i t a t e d b y b i o a u t o g r a p h y u s i n g S t r e p t o c o c c u s a u r e u s 209P a s t h e a s s a y organisn?? Dihydrophenlyglycine(faster moving component) can be s e p a r a t e d from c e p h r a d i n e w i t h a t e r t . amyl a l c o h o l - s e c . - b u t a n o l - w a t e r (4:4: 1) s y s t e m and q u a n t i t a t e d w i t h a n i n h y d r i n - c o p p e r complex 40



.



6.72



Thin-Layer T h e r e a r e two TLC methods b y which c e p h a l e x i n and o t h e r i m p u r i t i e s can be s e p a r a t e d from c e p h r a d i n e a n d b o t h c e p h a l e x i n a n d c e p h r a d i n e can be q u a n t i t a t e d . Both methods 40 g i v e comparable r e s u l t s . I n t h e f i r s t method , s i l i c a g e l p l a t e s a r e impregnated w i t h s i l i c o n e f l u i d and d e v e l o p e d f o r 2-1/2 h o u r s i n a pH 4 . 1 M c I l v a i n e b u f f e r and a c e t o n e ( 1 0 0 : l . S ) . T h e z o n e s a r e l o c a t e d w i t h U.V. l i g h t , e l u t e d a n d q u a n t i t a t e d s p e c t r o p h o t o m e t r i c a l l y a t 260 nm. The s e p a r a t i o n scheme of t h e known components is a s follows : A t 22OC



compound Dihydrocephradine Cephradine Cephalexin 7-ADCA 7 -ACA Dihydrophenylglycine Phenylglycine



Rf 0.31 0.43 0.51 0.65 0.65 0.74 0.80



Cephradine 0.72 1.00 1.20 1.51 1.51 1.72 1.88



To d e t e r m i n e r e s i d u a l 7-ADCA q u a n t i t a t i v e l y , i t i s a d v a n t a g e o u s t o change t o a s o l v e n t s y s t e m o f pH 6.5 M c I l v a i n e s b u f f e r acetone(50:l). The 7-ADCA zone i s e l u t e d w i t h 0.24 M sodium b i c a r b o n a t e a n d t h e a b s o r b a n c e a t 54



CEPHRADINE



260 nm i s measured41. The s e c o n d method42, a m o d i f i c a t i o n of t h e f i r s t i s p r e f e r r e d because of g r e a t e r e a s e of performance. The p l a t e s a r e i m p r e g n a t e d w i t h t e t r a d e c a n e i n s t e a d of s i l i c o n e and s i n c e t h i s i n t e r f e r e s w i t h t h e u. v. a b s o r b a n c e , q u a n t i t a t i o n is c a r r i e d o u t with ninhydrin. T h i s method can a l s o b e u s e d t o d e t e r m i n e d i h y d r o p h e n l y g l y c i n e and 7-ADCA semiq u a n t i t a t i v e l y . Approximate Rf v a l u e s a r e a s follows: C ep h r a d i n e 0.2 Cephalexin 0.3 7 -ADCA 0.6 Dihydrophenyl- 0.7 glycine 6 . 7 3 Column Chromatoqraphy High p r e s s u r e l i q u i d chromotog r a p h y (HPLC) h a s b e e n u s e d t o q u a n t i t a t e c e p h r a d i n e a n d r e s i d u a l c e p h a l e x i n i n c e p h r a d i n e . The mobile p h a s e i s a pH 4 . 3 g l a c i a l a c e t i c a n h y d r o u s sodium s u l f a t e s y s t e m , a Dupont s t r o n g c a t i o n exchange r e s i n , a t ambient t e m p e r a t u r e and a p r e s s u r e o f 1000 p s i g . Sulfamethazine is u s e d as i n t e r n a l s t a n d a r d and t h e o r d e r of elut i o n i s s u l f a m e t h a z i n e , c e p h a l e x i n and ~ e p h r a d i n e ~ A~ .m o d i f i c a t i o n , u s e s a n a c e t a t e 0.17M sodium s u l f a t e b u f f e r pH 4 . 7 , Dupont Zipax c a t i o n exchange r e s i n , n- (4-methoxy-methyl6 - m e t h y l - 2 - p y r i m i d i n y l s u l f a n i l a m i d e as i n t e r n a l s t a n d a r d and a p r e s s u r e o f 300 p s i g . The o r d e r of e l u t i o n is cephalexin, cephradine, i n t e r n a l s t a n d a r d . 7-ADCA, when p r e s e n t , w i l l a p p e a r i n When a d j u s t i n g t h e p H o f t h e v o i d volume44. t h e mobile p h a s e t o 3.70 a n d r a i s i n g t h e p r e s s u r e t o 1800 p s i g , t h e s y s t e m was a b l e t o s e p a r a t e c e p h r a d i n e ( d - i s o m e r ) from t h e s y n t h e t i c a l l y p r e p a r e d l-isomer which p e a k s between c e p h a l e x i n and c e p h r a d i n e . N o l-isomer was 55



KLAUS FLOREY



A d e t e c t e d i n r e g u l a r c e p h r a d i n e samples4’. r e v e r s e phase system, u s e f u l f o r q u a n t i t a t i o n i n formulation h a s a l s o been d e s c r i b e d 4 9 . A 1 mm x 2.1 mm i . d . column, ODS-Sil-X-I1 packing and 7 % methanol, 93% 0.05M ammonium c a r b o n a t e a s mobile phase were used. P r e s s u r e , 1 0 0 0 p i g ; f l o w 0 . 6 ml/min; d e t e c t o r W (254 nm); s e n s i t i v i t y , 0.08 AUFS.



Determination i n Body F l u i d s and T i s s u e s Cephradine h a s been determined microbiol o g i c a l l y (see s e c t i o n 6.2) i n human serum, i n human u r i n e , human lung t i s s u e , human eye t i s s u e and i n s p i n a l f l u i d . I t has been determined f l u o r o m e t r i c a l l y (see s e c t i o n 6.5) i n dog serum. 7.



Determination i n Pharmaceutical P r e p a r a t i o n I n pharmaceutical p r e p a r a t i o n s ( c a p s u l e s , o r a l suspensions and i n j e c t a b l e s ) i n f r a r e d has been used f o r i d e n t i t y t e s t s , t h e hydroxylamine and iodometric a s s a y f o r b a t c h i n g , t h e microb i o l o g i c a l and cephalosporinase-iodometric a s s a y s f o r s t a b i l i t y and chromatography f o r d e t e c t i o n of impurities. 8.



56



CEPHRADI N E



9.



1. 2.



3. 4. 5. 6. 7.



8. 9.



10.



11. 12.



13. 14.



15. 16. 17.



References B. T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l communi c a t i o n . M. S. P u a r , The S q u i b b I n s t i t u t e , P e r s o n a l communication. P. T. Funke, The S q u i b b I n s t i t u t e , P e r s o n a l communication. J. Dunham, The S q u i b b I n s t i t u t e , P e r s o n a l communication. F. M. R u s s o - A l e s i , The S q u i b b I n s t i t u t e , P e r s o n a l communication. H. J a c o b s o n , The S q u i b b I n s t i t u t e , P e r s o n a l communication. L. P. M a r e l l i , A n a l y t i c a l P r o f i l e s o f Drug S u b s t a n c e s , Vol. 4 , p 21, A c a d e m i c Press, 1974. M. S. A t w a l , The S q u i b b I n s t i t u t e , P e r s o n a l communication. H. J a c o b s o n and I. G i b b s , J. Pharm. S c i . 62, 1543 ( 1 9 7 3 ) . D. E. A u s l a n d e r , The S q u i b b I n s t i t u t e , P e r s o n a l communication, F. D u r s c h , The S q u i b b I n s t i t u t e , U . S . P a t e n t 3,829,620, June 25, 1 9 7 4 . F. D u r s c h , The S q u i b b I n s t i t u t e , P e r s o n a l communication. Q. Ochs, T h e S q u i b b I n s t i t u t e , P e r s o n a l communication. J. E. D o l f i n i , H. E. A p p l e g a t e , G. Bach, H. Basch, J. B e r n s t e i n , J. S c h w a r t z a n d F. L. Weisenborn, J. Med. Chem. 1 4 , 117 (1971) : U . S . P a t e n t 3 , 4 8 5 , 8 1 9 ( 1 9 6 9 ) . M. L. Snow, C. L a u i n g e r and C. R e s s l e r , J. Org. Chem. 3 3 , 1 7 7 4 ( 1 9 6 8 ) . L. Demain, N a t u r e 2 1 0 , 4 2 6 ( 1 9 6 6 ) H. R. Roberts, The S q u i b b I n s t i t u t e , P e r s o n a l communication.



.



57



KLAUS FLOREY



18.



19. 20.



21. 22. 23. 24. 25.



26. 27.



28.



29.



30.



31.



32.



33.



A. I. Cohen, P. T. Funke and M. S. Puar, J. Pharm. S c i , 6 2 , 1559 ( 1 9 7 3 ) . B. M. F r a n t z , The Squibb I n s t i t u t e , P e r s o n a l communication; J. Pharm. S c i . , i n p r e s s , R. V a l e n t i and H. J a c o b s o n , The Squibb



I n s t i t u t e , P e r s o n a l communication. M. A. L e i t z , The S q u i b b I n s t i t u t e , P e r s o n a l commun i c a ti o n , K. J. K r i p a l a n i and A. V. Dean, The S q u i b b I n s t i t u t e , P e r s o n a l communication. D. Adam, Munch. Med. WSchr. 116, 1 9 4 5 ( 1 9 7 4 ) . I. Weliky and R. Vukovich, The S q u i b b I n s t i t u t e , P e r s o n a l communication. A. V a h i d i , R. Vukovich, E. S. N e i s s and E. S c h r e i b e r , The S q u i b b I n s t i t u t e , P e r s o n a l communication. P. T. Funke, The Squibb I n s t i t u t e , P e r s o n a l communication. Weliky, I . , and Z a k i , A . , S e l e c t e d P r o c e e d i n g s from t h e 8 t h I n t e r n a t i o n a l Congress o f Chemotherapy, S e p t . 8-14,1973, A t h e n s , Greece, pp. 1-5. Vukovich, R . , M a r t i n e z , M . , and N e i s s , E . S . , S e l e c t e d P r o c e e d i n g s from t h e 8 t h I n t e r n a t i o n a l Congress o f Chemotherapy, S e p t . 8-14, 1973, A t h e n s , Greece, pp. 30-34. Z a k i , A. , S c h r e i b e r , E . C . , Weliky, I . , K n i l l , J . R . , and Hubsher, J . A . , J. C l i n . Pharmacol, New Drugs 14: 118-126 ( 1 9 7 4 ) . I. Weliky, H. H. Gadebusch, K. K r i p i l a n i , P. Arnow and E. C. S c h r e i b e r , A n t i m i c r o b i a l Agents and Chemotherapy 2, 4 9 ( 1 9 7 4 ) . G. R e n z i n i , G. Ravagnan, B. O l i v a , E. S a l v e t t i , and R. A u r i t i , Q u a d e r n i di A n t i b i o t i c a ; 1972, 17. T. B. P l a t t , The S q u i b b I n s t i t u t e , P e r s o n a l communication. S. Wind, The Squibb I n s t i t u t e , P e r s o n a l c o m u n i c at i o n . 58



CEPHRADINE



34. 35.



36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.



J. Alicino, The Squibb Institute, Personal communication. H. Lerner and S. Willis, The Squibb Institute, Personal communication. A. F. Heald, C. E. Ita and E. Solney, The Squibb Institute, Personal communication. C. Sherman, The Squibb Institute, Personal communication. J. Kirschbaum, J. Pharm. Sci., 63 923 (1974). A. Vahidi and H. R. Roberts, The Squibb Institute, Personal communication. H. R. Roberts, The Squibb Institute, Personal communication. F. P. Targos, The Squibb Institute,Personal communication. I. R. Salmon, The Squibb Institute,Personal communica tion. A. Peterson and D. Guttman, Smith Kline & French Laboratories, Personal communication. J. Kirschbaum, The Squibb Institute, Personal communication. H. H. Pu and R. B. Poet, The Squibb Institute, Personal communication. D. M. Isaacson, The Squibb Institute, Personal communication. J. R. Ster, H. Weisblatt and J. D. Levin, The Squibb Institute, Personal communication. A. N. Niedermayer, The Squibb Institute, Persona1 communication. E. R. White, M. A . Carroll, J.E. Zarembo and A. D. Bender, J. Antibiotics 28, 205 (1975). J. Z. Gougoutas and B. K. Toeplitz, Univ.of Minnesota and Squibb Institute, Personal communication. J. V. Uri, P. Actor, L. Phillips and J. A. Weisbach, Experientia &54(1975). 59



CHMROQUINE PHOSPHATE



Donald D,Hong



DONALD D. HONG



CONTENTS Analytical Profile



-



Chloroquine Phosphate



1.



Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor



2.



Physical Properties 2.1 Ultraviolet Spectrum rescence Spectrum 2.2 2.3 NA ear Magnetic Resonance Spectrum Spectrum 2.4 Ma&% 2.5 ~ p cal t i Rotation morphism and Melting Range 2.6 2.7 Dissociation Constant



2.8 DH 2.9 seezing Point Depression



2.10 Solubility 2.11 Differential Scanning Calorimetry



3.



Synthesis



4.



Drug Metabolic Products



5.



Methods of Analysis 5.1 Phase Solubility Analysis 5.2 Identification by Spot Tests 5.3 Non-Aqueous Titration 5.4 Spectrophotometric Analysis 5.5 Fluorometric Analysis 5.6 Gravimetric Analysis 5.7 Chromatographic Analysis 5.71 Paper 5.72 Thin-Layer 5.73 Gas 5.8 Miscellaneous Methods



6.



Referencea



4.1 Biotransformation Products 4.2 Distribution in Human Tissues



62



CHLOROQUINE PHOSPHATE



1.



Description 1.1



Name, Formula, Molecular Weight



The chemical name of chloroquine phosphate i n Chemical Abstracts is found under t h e heading Quinoline and designated as 7-Chloro- [4- (4-diethylamino-1-methylbutylamino) Iquinoline diphosphate. The hydrochloride and s u l f a t e e a l t e are a l s o a v a i l a b l e .



2H2PO4-



C@&CN3



2H3Po4



Molecular Weight:



515.87



1 . 2 Appearance, Color, Odor Chloroquine phosphate i s a w h i t e , o d o r l e s s , c r y s t a l l i n e powder having a b i t t e r taste: it d i s c o l o r e gradually on exposure t o l i g h t . 2.



Physical P r o p e r t i e s 2 . 1 U l t r a v i o l e t Spectrum



A 10 y/ml s o l u t i o n of chloroquine phosphate i n 0.01 N H C 1 when scanned between 360 and 210 nm e x h i b i t s t h r e e maxima, three minima and s e v e r a l shoulders i n t h e region from 270 t o 225 nm, as shown i n Figure 1. The maxima are located a t 343 nm ( a = 36.1), 328 nm ( a = 32.6) and 222 nm ( a = 59.9). The r a t i o of /%% i s 1.11. Minima



were observed a t 335 nm, 280 nm a342h3 nm. 2.2



Fluorescence Spectrum



Figure 2 shows t h e fluorescence spectrum of chloroquine phosphate obtained on a s o l u t i o n of 0.2 mg/ml pH 7.9 phosphate b u f f e r using an Aminco-Bowman spectrophotofluorometer. Excitation a t e i t h e r 320 nm o r 370 nm produced emission s p e c t r a with a maximum a t 400 nm, t h e 63



.



si



.- . . . . . . . . .



. . . . .



........



...~. . . . . . . . .



...



... ......



.



.



..



1



I



j



.



,. I



1.! .



... . . . . . . . .



'



....



...



.



... . . .



. .



. .



-



64



.



.



I



, i (



+ "I



, . .



i !.



I



3i



i !



,



I



CHLOR OQUlNE PHOSPHATE



Fluorescence Figure2.



70



F luorescence Spectrum of Chloroquine Phosphate i n pH 7.9 Phosphote Buffer (Sterling-Winthrop House Reference Standard Lot N-087- JF)



WAVE L ENGTH-MILL IMICRONS



65



DONALD D. HONG



l a t t e r e x c i t a t i o n wavelength providing a higher emission response.



2.3



Nuclear Magnetic Resonance Spectrum



(NMRl



The spectrum i n Figure 3 was obtained w i t h a Varian A60 NMR Spectrometer using a 20$ s o l u t i o n i n D20 containing TMS as an e x t e r n a l standard. The s p e c t r a l assignments are summarized below (1).



c1 Protons



No. Protons Derived from Integration



CH3 -CH2



6



Cg-



3



CH -CH CH2 CH2-N CH-N



NH



c



4 6 1 exchanged 1 1 1 1



b a C



1



d



2.4



a Chemical Shi



1-68-1-80 1.90-1.9



2.35 3 -55-3 a 9 1 4.38-4.65 5 -39



-



7.15-7 28 7 58-7 -75 7.75 8-33-8.40 8 48-8-55



-



-



;



Multiplic doublet doublet broad s i n g l e t quintet broad s i n g l e t sharp s i n g l e t doublet multiplet singlet doublet doublet



Mass Spectrum



The mass spectrum is shown i n Figure 4 and was obtained using a J o e l JMS-01SC mass spectrometer with an i o n i z i n g energy of 75 eV. The h i g h e s t mass observed a t m/e 319 i s a thermal breakdown product where two phosphoric acid moieties were l o s t from t h e parent compound. The base peak a t m/e 86 i s due t o t h e N,N,N-diethYlmethylene fragment (1).



66



A/-+-



L



L I . . ,



.



I



.



'



.



.



I



.



,



U



Figure 3.



. . , . . . .I . I 71



,



'



. , .



'



I



I



I



1



I



I



00



(0



I



1



Y I 4



I



40



I



I I



an



NMR Spectrum of Chloroquine Phosphate.



I



.



,



.



.



I



I



I



I



10



$8



8



Instrument:



Varian A60



.



.



DONALD D. HONG



Intensity



100



>



t z W v)



80



319



F



5 60



W



L



+ a



40



BACKGROUND



A



W



a 20 0



20



. 40



. 60 ' 80



100 I20 140160 I80



m/e



250



300



Fig.4 Mass Spectrum of Chloroquine Phosphate (Sterling-Winthrop House Reference Standard Lot N-087-JF)



60



CHLOROQU IN E PHOSPHATE



2.5



Optical Rotation



Chloroquine phosphate e x h i b i t s e s s e n t i a l l y no o p t i c a l a c t i v i t y , e x i s t i n g as a racemic mixture. Riegel and Sherwood ( 2 ) have shown t h a t n e i t h e r of t h e o p t i c a l l y a c t i v e enantiomorphs showed any s i g n i f i c a n t d i f f e r e n c e s i n a n t i m a l a r i a l a c t i v i t y i n birds and f o r t o x i c i t y i n dogs. 2.6



Polymorphism and Melting Range



Chloroquine phosphate e x i s t s i n two polymorphic forms giving rise t o two melting ranges. USP xn ( 3 ) r e p o r t s one melting between 193" and 195" and t h e o t h e r between 210" and 215". Mixtures of t h e forms melt between 193" and 215". It i s possible t o obtain one form s e l e c t i v e l y by r e g u l a t i n g t h e rate of c r y s t a l l i z a t i o n ( 4 ) . 2.7



Dissociation Constant



The pKa's f o r chloroquine phosphate by t h e t i t r i m e t r i c method were found t o be 8.10 and 9.94 ( 5 ) . 2.8



@ A 18 aqueous s o l u t i o n has a pH of about 4.2.



2.9



Freezing Point Depression



Cryoscopic measurements were made on (w/v) solutions of t h e drug.



**



Freezing Point Depression Chloroquine phosphate



0.73 "



69



lo$ and 2o$ Calculated Isotonic



7.oe



DONALD 0.HONG



The value f o r "calculated i s o t o n i c " s o l u t i o n was obtained by graphic i n t e r p o l a t i o n t o FPD of 0.550", representing 0 . d sodium chloride s o l u t i o n ( 5 ) . 2.10 S o l u b i l i t y



Chloroquine phosphate i s f r e e l y soluble i n water: p r a c t i c a l l y insoluble i n alcohol, i n chloroform and i n ether (3).



2.11 D i f f e r e n t i a l ScanninR Calorimetry (DSC)



Two polymorphic forms of chloroquine phosphate a r e exhibited by DSC. A mixture of t h e two c r y s t a l forms may be demonstrated a l s o by t h e t r a n s i t i o n temperatures ( 6 ) . The DSC thermogram of a chloroquine phosphate s t a n d a r d shown i n Figure 5 was obtained on a Perkin-Elmer DSC-lB d i f f e r e n t i a l scanning calorimeter a t a heating rate of 10°C per minute under nitrogen. T h i s i s an example of t h e low melting form. Figure 6 shows another sample of chloroquine phosphate containing a mixture of t h e low and high melting forms. Both t h e low and high melting polymorphs may be obtained from t h e same aqueous s o l u t i o n of chloroquine phosphate by s e l e c t i v e c r y s t a l l i z a t i o n . The high melting form usually occurs as small c r y s t a l s while t h e low melting polymorph c r y s t a l l i z e s as s i g n i f i c a n t l y l a r g e r c r y s t a l s . The two forms e x h i b i t s l i g h t d i f f e r e n c e s i n t h e i r I R curves from a KBr matrix.



3.



Synthesis



E l d e r f i e l d (7) and Kenyon i s collaboration w i t h Wiesner and K w a r t l e r (8) and more r e c e n t l y Basu, e t a1 ( 9 ) have summarized t h e American e f f o r t t o develop an a n t i malarial drug necessitated by World W a r 11. T h i s need was compounded when Japan seized c o n t r o l of t h e East Indies, e f f e c t i v e l y c u t t i n g o f f t h e n a t u r a l sources of quinine, which was t h e drug of choice f o r malaria a t t h e time. Chloroquine was one of t h e f r u i t s of t h i s concerted e f f o r t . The synthesis of chloroquine was f i r s t reported by t h e German chemists Andersag, Breitner and Jung (10, 11). Since t h e p a t e n t l i t e r a t u r e lacked t h e d e t a i l information required t o prepare t h e necessary intermediates, a research program w a s s t a r t e d a t Winthrop Chemical Company r e s u l t i n g i n a method o f synthesis for chloroquine by Surrey and 70



CHLOROQUINE PHOSPHATE



Figure 5. OSC Thermogram of Chloroquine Phosphate (Sterling-Winthrop House Reference Standard Lot N - 0 8 7 - J F ) Low Melting Form 189-202.5-207.5 Ic



N



m



t



*t



1



I



I



0



0



t



0



0



0



r-



*



(D



t



0 I



I



TEMPERATURE



71



(Corr.) -4



0



0



OC



I O K



0



0



OD



Q,



t I



t I



DONALD D. HONG



Figure 6 DSC Thermogram of Chloroquine Phosphate (Lot An-K-67) Mixture of Low and High Melting Forms



1-



189-202-1 -207.5 OC (Corrl



215-2232 2 6 O C (Corr) ENDO



t 1



EX0



0 m



O



1



I



e



a



*



P



0 c



Q I



0



0



00



Q,



TEMPERATURE



72



*



d I



I



O K



0 0 In



J



CH LOROQUINE PHOSPHATE



Kammer (12). The two key intermediates required i n t h e synthes is a r e 4 7-d i ch l o r oquinoline and 4-d i e t h y lami no 1 methylbutylamine ( “novol diamine”). The s y n t h e t i c scheme i s shown i n Figure 7.



--



4.



Drug Metabolic Products



4.1



Biotransformation Products



Several metabolites in a d d i t i o n t o t h e unchanged drug have been i s o l a t e d from human t i s s u e s and urine. Titus, e t a1 (13) used counter-current d i s t r i b u t i o n t o i s o l a t e t h e d e s e t h y l compound ( A ) from t h e u r i n e of human volunteers. Kuroda (14) i s o l a t e d four metabolites of chloroquine from human t i s s u e s using a combination of paper chromatography and W spectroscopy. I n a d d i t i o n t o t h e desethyl compound ( A ) of T i t u s , they were t h e b i s d e s e t h y l (B), t h e c a r b i n o l (C) and t h e ~-amino-7-chloroquinoline( D ) d e r i v a t i v e s .



--



Similar observations were seen from u r i n e of human s u b j e c t s b u t no t r a c e of t h e 4-hydroxy-7-chloroquinoline w a s found. The unchanged drug was always found t o be t h e major compound. McChesney, e t a1 (15, 16) using a fluorescence technique confirmed t h e above findings and i n a d d i t i o n found t r a c e s of t h e hl-aldehyde (E) and t h e b’-carboxy (F) d e r i v a t i v e s . They l i s t e d t h e amount of determinable excretory products as TO$ chloroquine, 23s desethylchloroquine, 1-2$ bisdesethylchloroquine and t h e o t h e r s as t r a c e degradation products. I n a d d i t i o n they reported t h a t about one-third of t h e administered chloroquine was unaccounted and remains obscure i n t h i s very complex blotransformation of t h e drug.



73



DONALD D. HONG



Figure 7: Synthesis of Chloroquine Phosphate



m-Chloroaniline



Ethyl oxalacetate



Ethyl(m-chloropheny1imino)succinate



and isomer separation



k



(1) NaOH heat c1



COOH



co0c.$l5



Ethyl-7-chloro-4-hydroxyquinoline-2-carboxylate



a> 1



-.



Cl



Chloroquine diphosphate



CH3CHCH2CH2CH9 (CpHg )2



I ! H 2



N



c1



7-Chloro - 4hydroxyquinoline



I



(2) HC1



7-Chloro-4-hydroxyquinoline-2-carboxylic acid



-COOH



OH



4,7-Dichloroquinoline



-



E \>



-



..



M



0



c



E0 In



Y-



e -..



0



S i l i c a Gel G



..



(0



ul



m



a,



5



Same as 7



a,



62



I



Ceric sulfate: sod i um a r s e n i t e



a,



61



3



Same as 7



0



61



-



P



Ferric chloride: f e r r i cyan i de: arsenious a c i d



-a,



a,



a



Cn



I: -a,



u



0



..



I



E 0 1 ' P 8I



Ch1oroform:methanol : f o r m i c a c i d ( 7 0 : 15: 15, v / v )



I



10.



2I



Ethy1acetate:methanoI:ZN - NH40H (100:40:60, V / V )



Z



9.



V



m



N -



D V a, .._.-



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t c



N + -



0



M



z



(0--0 .3 D n ulna



a,



ul



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a,



uz



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k



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u-3



v



0



Tert-butanol :2N - NH40H:ch l o r o f o r m (376:70:60)



-N



0



n



-0



a



.-L



-0



60



I



!-I



Diazotized s u l f a n i l i c acid, PdC I 2



N i n h y d r i n , PdC12



Same as 5



Cel I u l o s e Powder



8.



58 59



L



ul



Q)



a,



C



m



E 0



c .z



t



L



cn



t W



Lo



*



ul 0



m



N



a,



rub



>x



( I:5)



Formic acid:H20



.-



N i n h y d r i n , PdCI2



Eastman Sheets #6060 ( S i l i c a Gel)



0



-



C



L >



2-0 E t ma, 1 Y ta,L



t - -f



G O -



LL



7.



V



ul



Kieselgel G



(Rf given as reZative Rf of I-, Rf of I- not r e p o r t e d ) Same as 5 Ethano I :methy I e t h y I ketone:ZN - NH40H ( I :4: I )



%W ' F -V



6.



0 t



n



W



w



u



>



0 0



(Rf &ven as r e Z a t i v e Rf of I-, Rf of I- not r e p o r t e d ) Tert-amyl alcoho1:dioxane:iN- NH40H 20% S i I i c a Gel G+ (2:2: I ) 80% C e l l u l o s e MN 300



t



5.



s



Tert-but-nol:tert-amyl alcohol: NH40H :met hy I e t hy I ketone :H20



?!



4.



L



h)



a, + a,



+I



a, .Y



-..



-a, -acn,



0



c



I



I



-V



Z ..



a,



t a,



0 L 0



-V.->



Tert-amy alcohol:acetone:NH40H ( 2 5 : 8 : 7 , v/v) nJ\



3.



9



Ref #



Detect ion



:



L



0



a,



ul



a,



n



ul D



a



m r n



.--



Adsorbent



Mob i l e Phase



ALEX POST AND RICHARD J. WARREN



T a b l e 5 l i s t s t h e R f o f s e v e r a l o f t h e iodoaminoacids i n d i f f e r e n t systems. D e t e c t i o n methods a r e l i s t e d i n T a b l e 6. Several Whatman papers have been used: Whatman I , 3, 4, 3MM b e i n g t h e most p o p u l a r . I n addition, t h e procedure has been c a r r i e d o u t i n t h e ascending, descending and c i r c u l a r modes. I n each case, i t has been t h e e x p e r i e n c e o f t h e a u t h o r s t h a t , under t h e p r o p e r c o n d i t i o n s o f temperature, equi I i b r a t i o n time, and l e n g t h o f run, t h e Rfs o b t a i n e d w i t h any o f t h e s e modes gave e s s e n t i a l l y e q u i v a l e n t r e s o l u t i o n from day t o day and l a b o r a t o r y t o Iaboratory Dei od I n a t I on e f f e c t s d u r i ng paper chromatog r a p hy have been e v a l u a t e d and described I n Section 4.



.



Paper chromatography, u s i n g f o u r d i f f e r e n t s o l v e n t systems on Whatman # I paper, was used t o e s t a b l i s h t h e chromatographic p u r i t y o f t h e L - t h y r o x i n e sodium r e f e r e n c e substance p r i o r t o i t s a d d i t i o n t o t h e B r i t i s h .Pharmacopoeia.54 e f h y r o x i n e v a l u e s a r e r e p o r t e d f o r t h e compounds I i s t e d i n T a b l e 7. 6.32



T h i n Layer Chromatography (TLC)



T h i n l a y e r chromatography o f f e r s some advantages t o paper chromatography i n t h a t b e t t e r s e p a r a t i o n s a r e general l y o b t a i n e d w i t h h i g h e r l o a d i n g s . However, r e p r o d u c i b i l i t y o f R f values i s oftentimes d i f f i c u l t t o o b t a i n because o f t h e b a t c h t o b a t c h d i f f e r e n c e s i n adsorbents, t e m p e r a t u r e v a r i a t i o n s w i t h i n a l a b o r a t o r y , and e q u i l i b r a t i o n t i m e s used by d i f f e r e n t i n v e s t i g a t o r s . Thus, t h e d a t a p r e s e n t e d i n T a b l e 7 s h o u l d be c o n s i d e r e d i n l i g h t o f t h e s e v a r i a b l e s ; and t h e a n a l y s t should be expected t o a l t e r t h e m o b i l e phase, a l t h o u g h s l i g h t l y , t o e f f e c t a s u i t a b l e separation. The r e f e r e n c e s c i t e d 5 5 - 6 2 i n d i c a t e t h e v a r i e t y o f systems a v a i l a b l e f o r t h e s e p a r a t i o n o f t h e s e compounds. Chapters from s e v e r a l t e x t ~ 4 6 , ~ 3 > @c o n t a i n a d d i t i o n a l i nformation. A c r i t i c a l t h i n l a y e r chromatographic a n a l y s i s o f L - t h y r o x i n e sodium was made by a j o i n t committee o f t h e Pharmaceutical S o c i e t y o f G r e a t B r i t a i n and t h e B r i t i s h Pharmacopoeia p r i o r t o e s t a b l i s h i n g t h e sample as a r e f e r e n c e s ~ b s t a n c e . 5 ~F i v e d i f f e r e n t m o b i l e phases on f i v e d i f f e r e n t adsorbants were used t o e s t a b l i s h i t s p u r i t y .



262



SODIUM LEVOTHYAOXINE



Schorn and W i n k l e r S 5 s y s t e m a t i c a l g a t e d more t h a n two dozen s o l v e n t systems on S i I p l a t e s i n t h e s e p a r a t i o n o f L-T4, L-T3, L-T2, LThe r e s u l t s c e a r l y showed t h e v i a b i l i t y o f t h i s t o t h e separa i o n o f t h e iodoaminoacids. 6.33



y investica g e l G I and I-. technicrue



Col umn Chromatography



As column chromatography i s a r a t h e r nons p e c i f i c t e r m t o d e s c r i b e a s e D a r a t i o n orocedure. we have d e l i neated t h i s t e c h n i q u e i n t o ' f o u r s p e c i f i c t y p e s : Ion exchange, g e l f i l t r a t i o n , gas l i q u i d , and h i g h performance I i q u i d chromatography. T h e i r i n d i v i d u a l a p p l i c a t i o n s a r e d e s c r i b e d i n f o l lowing s e c t i o n s . 6.331



I o n Exchange Chromatography ( I E C )



Resin column chromatography has been e v a l u a t e d and employed by many i n v e s t i g a t o r s i n t h e separat i o n and q u a n t i t a i t o n o f iodoamino a c i d s and i o d o t h y r o n i n e s i s o l a t e d from b i o l o g i c a I mater i a I ~ . 6 ~ - 7 8A I though t h e s e procedures a r e amenable t o a s s a y i n g t h y r o x i n e and t h y r o i d powders, t h e new s e p a r a t i o n t e c h n i q u e s d e s c r i b e d i n S e c t i o n s 6.332, 6.333, and 6.334, have, i n g e n e r a l , s u p p l a n t e d i o n exchange chromatographic s e p a r a t i o n s . A n i o n i c r e s i n s , Dowex I-X2 and 50-X4 (Dow Chemical Co., Midland, Mich.), u s i n g ammonium a c e t a t e , sodium a c e t a t e or ammonium formate b u f f e r s a t pH rnages from 3.2-5.6, w i t h o u t o r c o n t a i n i n g up t o 30% e t h a n o l , have been used t o s e p a r a t e s e v e r a l o f t h e i o d o t h y r o n i n e s . Automated procedures f o r d e t e c t i n g t h e components i n e f f l u e n t s have a l s o been reported.68J73J74J77J7&? The c o n v e n t i o n a l c e r i c a r s e n i t e r e a c t i o n d e t e r m i n a t i o n f o r i o d i n e i s t h e most f r e q u e n t l y used d e t e c t i o n method. The a p p l i c a t i o n o f c a t i o n exchange r e s i n s t o s e p a r a t e t h e i o d o t h y r o n i n e s has been r e p o r t e d . 71J 7 7 J 7 8 The c i t e d r e f e r e n c e s deal a l m o s t e x c l u s i v e l y w i t h two iodoaminoacids and two i o d o t h y r o n i n e s , MIT, DIT, T3 and T4, r e s p e c t i v e l y . R e c e n t l y , Sorimachi and U i 7 9 r e p o r t e d t h e s e p a r a t i o n o f e i g h t d i f f e r e n t i o d o t h y r o n i n e s on (1.0 x 15 cm) c a t i o n exchange r e s i n , AG 50W-X4 (30-35 pm), e q u i l i b r a t e d w i t h 0.04 M ammonium a c e t a t e b u f f e r , pH 4.7, c o n t a i n i n g 30% ( v / v ) e t h a n o l a t 5OoC and a g r a d i e n t o f I n c r e a s i n g pH. The



263



ALEX POST AND RICHARD J. WARREN



g r a d i e n t c o n s i s t e d o f s t a r t i n g b u f f e r and 0.65 N NaOH. D e t e c t i o n was by t h e i o d i n e c a t a l y z e d c e r i c - a r s s n l t e r e a c t i o n . T a b l e 8 l i s t s t h e e l u t i o n volumes o f a s e r i e s o f lodoaminoa c i d s and i o d i d e o b t a i n e d by t h i s procedure. Table 8 E l u t i o n Volumes o f lodoam noac ds Com pou nd



Approximate E l u t on Volumes ( m l 1 6 37 50 80 80 I06 91 96 I13 88 96



Iodide MIT DIT TI T2 T3 T4 3'-T I 3,3'-T2 3 , 5 -T2 3,3 ,5' -T3



' ' '



6.332



Gel F i l t r a t i o n Chromatography (GFC)



The appl i c a t i o n o f g e l f i l t r a t i o n s e p a r a t i o n o f t h e i o d o t h y r o n i n e s has been p r i m a r i l y used t o d e t e r m i n e t h e i r i n d i v i d u a l c o n t e n t s I n b i o l o g i c a l samples. The i n f o r m a t i o n p r o v i d e d i n T a b l e 9 i n d i c a t e s t h e chromatog r a p h i c systems used t o s e p a r a t e t h e iodoaminoacids i s o l a t e d from t h e s e samples. Each o f t h e c i t e d r e f e r e n c e s d e s c r i b e s t h e i m p o r t a n t s t e p s r e q u i r e d t o p r e p a r e t h e s e columns (e.g., t h e i r dimensions, mesh s i z e o f t h e g e l p a r t i c l e s , e t c . ) , t h e d e t e c t i o n systems used ( g e n e r a l l y t h e c e r i c - a r s e n i t e r e a c t i o n , u l t r a v i o l e t absorption, l i q u i d s c i n t i l l a t i o n counting o f tagged i s o l a t e s , e t c . ) , and t h e p r e c i s i o n and accuracy o f t h e p a r t i c u I a r v a r i a t i o n employed by t h e r e s p e c t i v e i n v e s i t g a t o r . B l a s i and DeMasiB0 have l i s t e d t h e p a r t i t i o n c o e f f i c i e n t s , Kd, o f s e v e r a l t y r o s i n e s and t h y r o n i n e s as o b t a i n e d from t h e Sephadex G-25 column and t h e i r e l u t i o n conditions. From t h e s e data, r e l a t i o n s h i p s between t h e s t r u c t u r e o f t h e compound and t h e e l u t i o n volume can be e s t a b l i s h e d . The Kd v a l u e s a r e l i s t e d I n T a b l e 10.



264



S O D I UM LEVOTHY ROX INE



Table 9 Gel F i l t r a t i o n Chromatographic S e p a r a t i o n Systems Co I umn Mater i a I



E1uent



Compounds Separated



0.01 ,N NaOH



DIT, T3, T4



81



Sephadex G-25



tert-amyl alcohol saturated w i t h N NH40H 2 -



T3, T4



82



Sephadex G-25



0.02 N NaOH



Ty, MIT, DIT, T, T I , ( b ) T2, T3, T4



80



Sephadex LH-20(a)



MIT, DIT, T3, T4 ethy I acetate: methanol : 2 N NH40H ( l00:25: 10, T/v)



Sephadex G-25



0.1 N NaOH 0.007 N NaCl



I-, T3, T4



84



0.02 N NaOH



T3, T4



85



Sephadex G-25



Sephadex G- I 5



(a )



fa)



fa)Pharmac i a F i ne Chemi ca I s,



I nc.



, P i scataway ,



NJ



I n a d d i t i o n 3 ' ,5'-d i i o d o t h y r o n i ne and fb)3- i o d o t h y r o n i ne. 3,3' ,5'- t r i io d o t h y r o n i ne were a I so separated.



T a b l e 10 Kd Values of l o d o t h y r o n i n e s and R e l a t e d Compounds Comoound



(a )



Kd



TY MIT DIT T 3- l o d o t h y r o n i ne T2 3 , 5 '-d i io d o t h y r o n i ne T3 3,3',5'-tri iodothyron ine T4 (a)l.5



mg i n 0.5 ml 0.02 N NaOH



265



0.32 0.36 0.52 0.52 0.93 1.13 I .95 2.35 4.40 5.20



Ref



# -



83



Table I I Gas L i q u i d Chromatographic S e p a r a t i o n Systems



,



8



Compound Separated



Derivative



Co I umn



T4, T3 MIT, DIT, T



N,O-d p i v a l y l met hy e s t e r



0.5% SE-30 on Gas Chrom Q



T4, T3, T2 MIT, D I T



N,O-b s t r i f I u o r o a c e t y methy I ester



T4, T3, MIT, DIT



Co I umn Temperature



Detector



Amount I nj e c t e d



Program I30-305OC IOo/mi n



FID(~)



ug



66



3.8% SE-30 on D i a t o p o r t S



25OoC



FID



0.01 umol



86



N,O-d p i v a l y I met hy e s t e r



1 % polysulfone on Gas Chrom Q



232OC



ECD f b )



ng



87



T4, T3, MIT, DIT



N, 0-d p i v a l y l methy e s t e r



3% OV-17 on Gas Chrom P



282OC



ECD



P9



87



T4, T3, MIT, DIT



TMS (CJ



3% O V - l on Gas Chrom Q



285OC



ECD



50-150 ng



88



T4, T3, T2, MIT, DIT, T



TMS



0.5% SE-30 o n DMSC-treated f d ) Chromosorb G



75-250°C 8 4.6'/rnin



FIC



20 ng



89



T4, T3, T2



N,O-dipivalyl methyl e s t e r



5% OV-17 on Gas Chrom Q



225-235OC @ 5O/min



FID



< I ug



90



T4, T3, T2, MIT, DIT, T



N,O-dipivalyl methyl e s t e r



5% OV-17 on Gas Chrom Q



285OC



ECD



3 ng



90



Ref



# -



Table I I (continued)



ru



3



Co I umn TemDerature



Detector



Amount Injected



I 50-280'C



FID



50 ng



91



165'C @ 3 m i n FID t o 265' @ 10 m i n



3-15 u g



92



1 % OV-1 on C h romosor b W HP



135-255'C @ 5'/min



FID



'L4 llg



93



T4, T3, T2, T, TMS MIT, DIT, Ty



I % OV- I on Chromosorb WHP



180-225OC



ECD



0.3-1.5



8 25'/min



T4, T3, T2



TMS



3% O V - 1 7 o n D i a t o m i t e CQ



(el



FID



5-20 ng



T4, (f)T3, T2, DIT



N,O-dimethyl methyl e s t e r



3% OV-1 on Gas C h r m Q



25OoC



FID



T4, T3, T2



TFAA(9) methyl e s t e r s



2.3% O V - l on Gas Chrom Q



fe)



ECD



1.4-2.5



T4, T3, T2



N,O-d i p i v a l y I methy I e s t e r s



2.3% OV-1 on Gas Chrom Q



29OoC



ECD



6-8 ng



96



T4, T3,



TMS



1 % OV-l on Gas Chrom Q



165-285'C



FID



4-16



97



Compound Separated



Der i v a t iv e



Co I umn



T4, T3, T2 DIT, Ty



TMS



2% SE-33 on Gas Chrom Q



T4, T3, T DIT, MIT, Ty



TMS



3% OV-17 o n Gas Chrom Q



T4, T3, T2, T, M l T DIT, Ty



TMS



Ref



#--



@ 1o0/min



8 1o0/min



ng



93 94



ng



96



Tab I e I I (continued 1 Compou nd Separated



Der i v a t i ve



Col umn



T4, T3



N,O-dipivalyl methyl e s t e r



3% DEXS 1 L 300 GC on Chromosorb WHP



fa’FID



= Flame I o n i z a t i o n D e t e c t o r



Co I umn Temper a t ure



Detector



Amount Injected



305’C



ECD



0.25-3



ECD = E l e c t r o n Capture Detector



’



OD



fC’Trimethylsl l y l d e r i v a t i v e ‘d’Dimethylchlorosi fe’Variable, ff’As



lane



depending on which compounds a r e t o be separated



t h e sod i um sa I t , h y d r a t e



ng



Ref



# 98



SODIUM LEVOTHYROXINE



6.333



Gas L i q u i d Chromatography (GLC)



S i n c e t h e iodoaminoacids a r e n o t v o l a t i l e and t h u s n o t amenable t o a gas c h r o m a t o g r a p h i c analysis, the preparation o f suitable stable v o l a t i l e derivatives p r i o r t o analysis i s a prerequisite. The f i r s t s u c c e s s f u l gas chromatog r a p h i c s e p a r a t i o n of T4, T3, DIT, MIT, and Ty, a s t h e i r N , O - d i p i v a l y l m e t h y l e s t e r d e r i v a t i v e s , was r e p o r t e d by Stouffer, e t S i n c e t h e n t h i s t e c h n i q u e s has been c r i t i c a l l y e v a l u a t e d because o f i t s i n h e r e n t s e n s i t i v i t y , speed o f a n a l y s i s , and a p p l i c a b i l i t y t o t h e q u a n t i t a t i o n o f t h e s e compounds i n b i o l o g i c a l p r e p a r a t i o n s . As i t i s beyond t h e scope o f t h i s monograph t o p r o v i d e p r e c i s e d e t a i l s o f t h e gas chromatographic methods, a t a b u l a t i o n o f the various r e p o r t s i s l i s t e d i n Table 1 1 . I t i s recommended t h a t t h e c i t e d r e f e r e n c e s be r e f e r r e d t o f o r t h e p r e p a r a t i o n o f t h e v o l a t i l e d e r i v a t i v e s , t h e use o f i n t e r n a l s t a n d a r d s f o r q u a n t i t a t i v e a n a l y s i s , and f o r t h e p r e p a r a t i o n o f t h e column s u b s t r a t e s .



apparent d i p i va I y I former i s they requ prepared



From t h e i n f o r m a t i o n I n T a b l e I 1 i t i s h a t t h e two f a v o r e d d e r i v a t i v e s a r e t h e N,Omethyl e s t e r and t h e TMS. The advantage o f t h e t h a t t h e e s t e r s have g r e a t e r s t a b i l i t y . However, r e a two-step s y n t h e s i s , whereas t h e l a t t e r can be n a s i n g l e step but a r e s e n s i t i v e t o moisture. 6.334



H i g h Performance L i q u i d Chromatography



High performance I i q u i d chromatography (HPLC) has been used by Karger, e t a l . 9 9 t o s e p a r a t e T4, T3 and T2 i n about two m i n u t e s i n a m o b i l e phase o f b u t a n o l and m e t h y l e n e c h l o r i d e on a s i l i c a g e l column c o a t e d w i t h a m i x t u r e o f p e r c h l o r l c a c i d and sodium p e r c h l o r a t e . On a s i m i l a r system T4, MIT and DIT s e p a r a t e d i n l e s s t h a n e i g h t m i n u t e s . DuPont I n s t r u m e n t s l o o r e p o r t e d t h e s e p a r a t i o n o f T4 and T3 on a s t r o n g c a t i o n exchange (SCX) column. Both of t h e s e methods showed e x c e l l e n t s e n s i t i v i t y , i n t h a t nanogram q u a n t i t i e s c o u l d be r e a d i l y d e t e c t e d u s i n g h i g h l y s e n s i t i v e UV d e t e c t o r s a t 254 nm. T h y r o x i n e and t r i i o d o t h y r o n i n e have a l s o been s e p a r a t e d i n l e s s t h a n 12 m i n u t e s on a M i c r o p a k 269



ALEX POST AND RICHARD J. WARREN



C-18 ( r e v e r s e phase) column u s l n g a methanol-ammonium c l t r a t e mobi l e phase.101 Waters Associates102 r e p o r t e d a s l m i l a r s e p a r a t i o n on a C I & o r a s i I column u s i n g a mob i l e phase cons i s t i n g o f a c e t o n i t r i le-n-butanol :0.005 M sodium p e r c h l o r a t e . A c l e a r s e p a r a t i o n was e f f e c t e d i n l e s s t h a n 20 minutes.



6.4



Neutron Act i v a t i on Ana I ys i s



Neutron a c t Iv a t i o n ana I ys 1 s was used by A I soszo3 t o d e t e r m i n e t h e L - t h y r o x i n e sodium c o n t e n t i n t a b l e t s . In this procedure, t h e t a b l e t was i r r a d i a t e d f o r 5 m i n u t e s I n a n e u t r o n f l u x of = 2.5 x 1012n/cm2/sec and q u i c k l y t r a n s f e r r e d t o a c o u n t i n g t u b e f o r measurement o f 1281. Comparison w i t h standards o f potassium i o d i d e I r r a d i a t e d f o r t h e same t i m e y i e l d e d t h e c o n t e n t o f L - t h y r o x i n e sodium. Interfering n u c l e a r r e a c t i o n s were n e g l l g l b l e , and t h e e r r o r was l e s s t h a n 2%. Neutron a c t i v a t i on ana I ys Is was a I so used by G I obe I, e t a I . 104 t o determl ne t h e r e 1 a t i v e amounts o f L - t h y r o x i ne ( T 4 ) and 3 , 5 , 3 ' - t r I i o d o t h y r o n i n e (T3) i n serum. These hormones were removed from t h e p r o t e l ns by pass i ng 20 m I o f serum ( a t pH I I ) t h r o u g h a Dowex IX-2 i o n exchange column i n t h e a c e t a t e form. The e l u a t e was c o n c e n t r a t e d and chromatographed on a c e l l u l o s e t h i n l a y e r p l a t e t o s e p a r a t e t h e T4 and T3 from t h e i n o r g a n i c i o d i d e . The i s o l a t e d T4 and T3 f r a c t i o n s were i r r a d i a t e d I n a t h e r m a l n e u t r o n f l u x o f 5 x 1O2n/cm2/sec u s i n g a T r i g a Mark I r e a c t o r , f o l l o w e d by i d e n t i f i c a t i o n and measurement of induced 1281 a c t i v i t y w i t h a germanium ( L i ) sol i d s t a t e d e t e c t o r . The I i m i t s o f d e t e c t i o n were 5 ng. Schmoelzer and Muel Ier1O5 used a s l m l l a r approach b u t separated t h e T4 and T3 o n a QAE Sephadex A-25 column p r i o r t o activation analysis.



6.5 Po I a r o g r a p h i c Ana I y s i s P o l a r o raphy was employed by Wacholz and P f e i f e r t o assay I h y r o x i n e ~ O 6and t o d e t e r m i n e t h e t h y r o x i n e c o n t e n t o f t h y r o l d powders and t a b I e t s . I n t h e assay o f t h y r o x i n e , 5-50 pg of sample i n I m i o f 2 N n i t r i c a c i d i s heated a t 6OoC f o r I hour. A f t e r c o o l i n g aKd t h e a d d i t i o n o f 5 m l o f 0.06 N sodium h y d r o x i d e , t h e 270



SOD1 U M LEVOTHY R OX INE



s o l u t i o n I s deoxygenated w i t h n i t r o g e n . The t h y r o x i n e c o n t e n t I s determined by comparison o f wave h e i g h t of t h e sample a t E& -0.6 t o -0.7V w i t h t h a t o f standards. The method i s s u f f l c l e n t l y s e n s i t i v e t o determine t h e t h y r o x i n e c o n t e n t o f spots i s o l a t e d by t h i n l a y e r chromatography. I n t h e assay f o r t h y r o x i n e I n t a b l e t s and powders, p r i o r e x t r a c t i o n procedures a r e r e q u i r e d t o remove t h e t h y r o x i n e and o t h e r i o d l n a t e d amino acids. A f t e r s e p a r a t i o n by t h i n l a y e r chromatography, e l u t i o n o f t h e t h r y o x i n e spot, c o n c e n t r a t i o n and n l t r a t i o n , 1 0 6 t h e wave h e i g h t a t t h e p r e v i o u s l y s p e c i f i e d E+ i s obtained. 6.6



K i n e t i c Methods o f A n a l y s i s



The a p p l i c a t i o n of k i n e t i c measurements of t h e i o d i n e c a t a l y z e d c e r i c arseni t e reaction108,109 has been u t i I lzed f o r t h e d e t e r m i n a t i o n o f t h y r o x i n e i o d i n e i n chromatograph I c e l u a t e s . 76~1*0,111 The r e p o r t e d methods a r e rapid, have h i g h p r e c i s i o n and a r e s e n s i t i v e , g e n e r a l l y d e t e c t i n g less than I ng o f T4. 6.7



Double-losotope D i l u t i o n A n a l y s i s



A double-isotope d e r i v a t i v e assay f o r serum iodot h y r o n In e s l l 2 (L-T4 and L-T3) has been mod i f l e d and improved upon by Hagen, e t a1.8 I n t h i s procedure, t h e unknown t h y r o x i n e i s labeled by formation o f an a c e t y l d e r i v a t l v e l l 3 u s i n g t r i t i u m - l a b e l e d a c e t i c anhydride. As t h e s p e c i f i c a c t i v i t y o f t h e t r i t i a t e d d e r i v a t i v e i s known, t h e t h y r o x i n e c o n t e n t of t h e sample can be c a l c u l a t e d . Losses I n t h e complex p u r i f i c a t i o n steps a r e accounted f o r by t h e a d d i t i o n of a h i g h s p e c i f i c a c t i v i t y 1311-labeled t h y r o x i n e . 6.8



Determination o f Stereoisomeric P u r i t y



I n f o r m a t i o n presented by t h e J o i n t Committee of t h e Pharmaceutical S o c i e t y of Great B r i t a i n and t h e B r i t i s h Pharmaceutical Committee54 i n d i c a t e s t h a t a l I D-thyroxine c o n t a i n s t r a c e amounts o f t h e L-isomer. A method f o r d e t e r mining t h e amount o f L-T3, an i n t e r m e d i a t e i n t h e s y n t h e s i s o f D-T4, has been reported.45 An a d a p t a t i o n o f t h i s method has been appl l e d t o D-T4 c o n t a i n i n g less than I % o f t h e Li somer. 114



271



ALEX POST AND RICHARD J. WARREN



6.9



Equi I i b r i u m D i a l y s i s



Several i n v e s t r g a t o r s 115-117 have used equ i I ib r i urn d i a l y s i s t o d e t e r m i n e t h e f r e e t h r y o x i n e i n serum. A c r i t i c a l s t u d y o t h i s p r o c e d u r e was made b y Lee and P i leggT.115 The e f f e c t s of pH, i n c u b a t i o n t i m e and temperature, b u f f e r c o m p o s i t i o n and c o n c e n t r a t i o n , p r o t e i n c o n c e n t r a t i o n , and specimen d l u t i o n were s t u d i e d . U s i n g 1 3 1 1 - L - t h y r o x i n e and a r e u s a b l e p l a s t l c d i a l y s i s c e l I , r e c o v e r i e s of 92-96% were o b t a i n e d when t h e d i a l y s i s was r u n a g a i n s t 0.05 M phosphate pH 7.6 b u f f e r a t 37OC f o r 18 hours. W i t h i n - r u n and betweenr u n p r e c i s i o n s were 10.6% and 14.2%, r e s p e c t i v e l y . Fang and Selenkow,'" using t h e conventional d i a l y s i s bag and 1251-L-thyroxine, determined t h e f r e e t h y r o x i n e cont e n t a f t e r d i a l y s i s a t 4 O C , a g a l n s t pH 7.4 phosphate b u f f e r f o r 18-24 hours. B i r d and A b i o d u n l Z 7 employed e q u i l i b r i u m d i a l y s i s and i o n exchange chromatography t o d e t e r m i n e free t h y r o x i n e . I o n exchange chromatography was used t o s e p a r a t e t h e 1251 i o d i d e from t h e 1251-L-thyroxine p r i o r t o d e t e r m i n i n g t h e f r e e thyroxine content. The l i t e r a t u r e i s r e p l e t e w i t h a s i g n i f i c a n t number of p u b l i c a t i o n s d e s c r i b i n g m o d i f i c a t i o n s o f e q u i l i b r i u m d y a l y s i s o r a c o m b i n a t i o n of t h i s p r o c e d u r e w i t h o t h e r s e p a r a t i o n t e c h n l q u e s . l 1 8 - Z 2 2 The c i t e d papers o f f e r a good b a s i c background t o t h e a p p l i c a t i o n of t h i s method t o t h e s t u d y o f t h e t h r y o x i n e - p r o t e i n b i n d i n g phenomenon.



7.



Methods of A n a l y s i s



-



A Compilation



The f o l l o w i n g t a b l e s (12, 13, 14 and 15) i n c l u d e t h e r e f e r e n c e s t o a n a l y t i c a l procedures f o r t h e a n a l y s i s of chemicals, t a b l e t s , powders, and serum and t i s s u e . S e v e r a l a d d i t i o n a l r e f e r e n c e s which were n o t c i t e d i n t h e s p e c i f i c sections a r e included i n t h e fol lowing compi'lations Two r e v i e w a r t i c l e s , by CahnmannlZ3 and by Ral I , e t a l .i24, a r e a l s o noted.



272



SODIUM LEVOTHYROXINE



Table 12 Analysis o f Thyroxine Chemicals Ana l y s i s :



See Reference # :



I dent I f i c a t ion



3,



T I t r I metry : I odornet r ic N-Bromosuccinimide Coulometric S p e c i f i c Ion E l e c t r o d e



1, 3, 37, 107 38 39, 4 0 43



Cer i rnetry



129



3, 4, 54, 125, 1 2 6



Chromatography:



PC



45, 54, 130, 131, 132, 1 3 3 54, 55, 58, 59, 61, 62, 107, 130, 131, 134 68, 69, 77, 79, 1 3 6 120, 127, 137 86, 87, 89, 90, 91, 92, 93, 94, 97, 98, 138 101, 142, 143



T LC



I EC G FC GLC HP LC



Neutron A c t i v a t i o n



103



Po Ia rog r a p hy



97, 106, 107



Kinetic



76, 78, 111



Spect rophotornet r y



5, 6, 7, 107, 144



Automation



68, 77, 78, 110, 136, 145, 146, 147



Electrophoresis



54



Phase S o l u b i l i t y



54 Table 13



A n a l y s l s o f Thyroxine T a b l e t s Ana I ys i s



See Reference #



Titrimetry: I odometr i c



1, 3, 4



273



ALEX POST AND RICHARD J. WARREN



Table 13 (continued Chromatog rap hy : PC TLC



148 107, 144, 149



Pol arography



107



Spectrop hotometry



107, 144, 148, 149, 150, 151, 152, 153 Table 14



A n a l y s i s o f Thyroxine Powders Ana I ys i s



See Reference #



Cer i metry



61



Chromat o g rap hy PC T LC I EC GLC



154 59, 61, 149, 155, 156 154 94, 97, 157



S pectrophotometry



149, 150, 156



Titrlmetry



1 Table 15



Analysis o f Sera and/or Tissues f o r Thyroxine and Analogs Anal y s 1 s



See Reference #



Cer i met r y



68, 69, 71, 73, 74, 75, 76, 77, 78, 79, 120, 133, 145, 146, 158, 159, 160, 161



Chromatography : I EC GFC G FC GLC



Neutron Act i v a t ion



67, 69, 70, 72, 72, 73, 74, 76, 77, 120, 117, 136, 145, 146, 147, 159, 163, 164 85, 110, 137, 165, 166, 167 87, 90, 98, 138 104, 105 274



SODIUM LEVOTHYROXINE



T a b l e 15 ( c o n t i n u e d ) Kinetic



211



D i a iy s i s



215, 116, 117, 118, 719, 168, 169, 170, 171, 172



Spectrometry



163, 169, 173



Au toma t ion



73, 74, 78, 136, 145, 146, 147, 159, 170



Radloimmunoassay



174, 175, 176



Competitive P r o t e i n Binding



105, 128, 170, 171, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186; 187-



Nephelometry



188



E I e c t r o p hores i s



70, 135, 172



F I uoromet r y



162



8.



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158.



.,



J . M. Ensor and P. Kendal I - T a y l o r , E n d o c r i n o l 46, 527 (1970). P. R. Larsen, Metabolism, 20, 609 (1971). A . J . M a t t y and C. C. Thornburn, J . E n d o c r i n o l . , 46, 417 (1970). T. S o f i a n i d e s , e t a l . , Proc. SOC. Exp. B i o l . Med., 123, 646 (1966). S. Hamada and S . H. Ingbar, J . Chromatogr., 61, 352 (1971 1. R. J . B l o c k and R. H. Mandl, Ann. N.Y. Acad. Sci., 87 (1962). R. H. Osborn and T. H. Simpson, J . Chromatogr., 34, 110 (1968). N. N. N i h e i , e t a l . , A n a l y t . Biochem., 43, 433 (1971). C. H. Bowden, e t a l . , Biochem. J . , 2, 93 (1955). R. Gmelin and A . V i r t a n e n . Acta Chem. Scand.. 13. 1469 (1959). E. Emerson, J . Org. Chem., g, 417 1943). A p p l i c a t i o n High1 i g h t s #19, Waters A s s o c i a t e s , I n c . , Framingham, MA. L i q u i d Chromatography R e p o r t # I 16, DuPont I n s t r u m e n t s , W i lmington, DE. E. Wachholz and S . P f e i e r , Pharma i e , 24, 459 (1969). G. K e s s l e r and V . J . P i eggi, C I i n . Chem., 14, 339 (1968). G. K e s s l e r and V. J . P i eggi, C I i n . Chem., 16, 382 (1970). H. Y . Yee, e t a l . , C I i n Chem., 622 ( 1971 1. R. Lemieux and J. M. Ta mage, J . Pharm. Pharmacol 94 (1966). J . E. Moody, J r . , e t a l , J . Pharm. Sci., 57, 634 ( I968 D. L. Sondack, J. Pharm S c i . , 62, 1342 (1973). I. C o v e l l i , e t a t . , Ana y t . B i o s e m . , 42, 82 (197 1. J . H. Graham, e t a l , J Pharm. S c i . , 63, 763 ( 1 9 4 ) . J. H. Graham. J . Pharm. Sci , 64, 1 3 9 3 7 1 975). S . k b l o g l u , e t a l . , E n d o c r i n o l . , 78, 231 (1966). H. Frey, Scand. J. C I i n . Lab. I n v e s t . , 470 (1964). J o i n t Committee o f t h e Pharmaceutical S o c i e t y and t h e S o c i e t y f o r A n a l y t i c a l Chemistry on Recommended Methods f o r t h e E v a l u a t l o n o f Drugs, A n a l y s t , Lond., 92, 328 (1967). R. B l l o u s , e t a l . , J . Pharm. S c i . , 60, 1270 (1971). E. Makowetz, e t a l . , Mlcrochem. J., 194 (19661.



102,



17,



.



.



16,



10,



280



SODIUM LEVOTHYROXINE



159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186. 287. 188.



2,



K. Horn, e t a t . , Z . K I i n . Chem. u k i n . B ochem., 99 (1972). N. E. K o n t a x i s and D. E. P i c k e r i n c.,I l . J. CI n. E n d o c r i n o l Metab., 18, 774 (1958). J . D. A c E n d , Biochem., g , 177 (1957). E. E. G u s s a k o v s k i i , e t a l . , Mol. B i o l . , 7, 598 (1974 J . A. Hathaway, e t a t . , Am. J . CI i n . Path., 53, 635 (1970). V. V. Row, e t a l . , C I i n . Chim. Acta, 31, 473 (1971). C. H. G. I r v i n e , Proc. A s i a Oceania C z g r . E n d o c r i n o I , 401 (1974). R; Liewendahl, e t a l . , Acta E n d o c r i n o l . , 793 ( 1 9 7 1 ) . P. J . D a v i s and R. I . Gregerman, J. CI i n . E n d o c r i n o l . Metab., 30, 236 (1970). R. R. C a E l i e r i and S. H. Ingbar, Methods Enzyrnol 36, 126 (1975). R. A. Pages, e t a l . , Biochem., 2773 (1973). W. C. G r i f f i t h s , e t a l . , C I i n . Biochem., 5, 13 (1972 N . M. Alexander and J . F. Jenni ngs, CI in.-Chem., 20, 553 ( 1974). S . Harnada, e t a l . , J. C I i n . E n d o c r i n o l . Metab., 2 , 66 ( I 970). I . Posner, J . Lab. C I i n . Med., 57, 314 (1961). A . Bercy, J . BeIge R a d i o l . , 5 7 , 1 8 5 ( 1 9 7 4 ) . M. I . Surks, e t a l . , J. C l i n T l n v e s t . , 52, 805 ( 1 9 7 3 ) . I . J . Chopra, e t a l . , J . C l i n . E n d o c r i n o l . Metab., 36, 31 I ( 1 9 7 3 ) . F. W. S p i e r t o , e t a t . , C l i n . Chim. Acta, 5 , 281 ( 1 9 7 4 ) . B. Murphy and C. Patee, J . C I i n . E n d o c r i n o * ’ 26, 247 (1966) * 24, 187 €3. Murphy and C. Patee, J . C I i n . E n d o c r i n o ( I 964 1 * R. Ekins, C I i n . Chim. Acta, 5, 453 ( 1 9 6 0 ) R. Ekins. e t a l . , C I l n . BiocFem., 2, 253 ( 969) * 46, 345 F. J . L. Crombag, e t I., C l i n . Ch%. Acta ( 1 973). D. E. D a l r y m p l e and R D. U t i g e r , J . Lab. C l i n . Ped., 75, 325 ( 1 9 7 0 ) . S . Barbadoro, Can. J . Med. Technol., 35, 5 ( 1 9 7 3 ) . 38, 199 H. Sel igson and D. Se igson, C I i n . C h x . Acta, (1972) * S. C. Thorson, e t a l . , Acta E n d o c r i n o l . , 64, 630 ( 1 9 7 0 ) . S. Nobel and F. B a r n h a r t , C I i n . Chem., 509 ( 1 9 6 9 ) . G. Hocman and L. Hegedus, E n d o k r i n o l o g i e , 55, 194 (1969).



.



.,



67,



., -



12,



-



.’



-



15,



261



METHOTREXATE



Arthur R. Chamberlin, Andrew P.K. Cheung, and Peter Lim



ARTHUR R . CHAMBERLIN etal.



Contents 1.



Description 1.1 Name, Formulae, Molecular Weight 1.2 Isomeric Forms 1.3 Appearance, Color, Odor



2.



Physical P r o p e r t i e s 2.1 Infrared Spectrum 2.2 Proton Magnetic SpeCtrUm 2.3 Carbon-13 Magnetic Spectrum 2.4 U l t r a v i o l e t spectrum 2 . 5 Mass Spectrum 2.6 Optical Rotation 2.7 D i s s o c i a t i o n Constants 2.8 S o l u b i l i t y



3.



Synthesis



4.



Stability 4.1 Bulk 4.2 S o l u t i o n



5. M e t a bol i s m



6. Methods of A n a l y s i s 6.1 Elemental Analysis 6.2



6.3



6.4 6.5 6.6



6.7



Equivalent Weight Determination 6.21 Nonaqueous t i t r a t ion 6.22 Complex-formation t i t r a t i o n Biological Assay 6.31 Microbiological Assay 6.32 Enzymic Assay Polarographic Assay Spectrophotometric Analysis 6.51 Fluoromet r i c 6.52 U l t r a v i o l e t / v i s i b l e Chromatography 6.61 Paper 6.62 Thin-Layer 6.63 column 6.64 High Speed Liquid Proton Magnetic Resonance



284



METHOTREXATE



7.



Acknowledgment



8.



References



285



ARTHUR R . CHAMBERLIN eta/.



.



1



Description 1.1 Name, S t r u c t u r a l and Empirical Formulae, Molecular



Weight Methotrexate is N-[4-{[(2,4-diamino-6-pteridinyl)met hy 1]met hy 1amine } benz oy1] g l u t ami c a c i d Frequent 1y t h e name i s abbreviated t o MTX. Methotrexate a l s o i s known as ~-amino-l0-methylfolic a c i d and amethopterin and i s i d e n t i f i e d by t h e National Cancer I n s t i t u t e code number NSC-740.



.



'



6'



7



0



Y



,



FooH



COOH CZ0Hz2N8O5



Mol. w t .



454.46



1.2



Isomeric Forms The presence of an asymmetric carbon i n t h e glutamic a c i d moiety provides f o r o p t i c a l isomerism. Unless sp2cif ied, commercially a v a i l a b l e methotrexate i s prepared from L-glutamic a c i d , Recently, L e e and co-workers' prepared methotrexate s t a r t i n g with D-glutamic a c i d . The D-enantioxer of methotrexate was a c t i v e a g a i n s t L-1210 i n t h e mouse and had less t o x i c e f f e c t s t h a n methotrexate i t s e l f , t h e L-enant iomer



.



1.3



Appearance, Color, Odor Methotrexate i s a bright yellow-orange, o d o r l e s s powder. It g e n e r a l l y is hydrated t o t h e e x t e n t of 8 t o lo$ w a t e r . I t a l s o has been prepared a s t h e hydrochloride ( 1:0.3) and hydrate (1:2.5)*. li.



P r i v a t e communication from D r . H.B. Wood, J r . , of t h e National Cancer I n s t i t u t e and M r . D.F. Worth of Parke, Davis and Co.



286



METHOTREXATE



2.



P h y s i c a l Properties 2.1



I n f r a r e d Spectrum ---



Recorded as a s u s p e n s i o n i n m i n e r a l o i l , t h e spectrum i n F i g u r e 1 shows r e l a t i v e l y broad s t r u c t u r e s , i n d i c a t i n g t h e complexity of t h e molecule and s u g g e s t i n g t h e l a c k of c r y s t a l l i n i t y of t h e sample. T a b l e I g i v e s t h e assignments t o t h e major bands. TABLE 1 I n f r a r e d Assignments f o r Methotrexate I R Absorption B a n d b )



I -



Interpretation ---



2.90-3.10



H20,



-W2)



-



-COOH



3 .O0-4.03



9



5.90-6.10 6.20, 6.50-6.60



R B



COOH, -C-N-)



-C-(



Aryl systems



6.50-6.60



Amide I1



11.9



H



ElH



H



H



P r o t o n Magnetic Resonance Spzctrum (pmr) The pmr spectrum i n F i g u r e 2 w a s r e c o r d e d on a Varian A&-A s p e c t r o m e t e r w i t h t h e sample a s a s o l u t i o n i n DMSO-de. The chemical s h i f t s are i n ppm r e l a t i v e t o TMS d e s i g n a t e d a s 0.00. 2.2



Table I1 g i v e s t h e s t r u c t u r a l assignments t o t h e resonances i n F i g u r e 2 .



287



-I 0.10 O



r



w V



z 0.20



N



a, a,



2K



0



0.30 0.40



Q



2



3



4



5



FIGURE 1



6



7 8 9 10 WAVELENGTH - microns



11



12



INFRARED SPECTRUM OF METHOTREXATE



13



14



15



289



0



m



d



w



h



L-



X



w



a L



U



0



f



N Lu



TABLE I1 pnr Assignments for Methotrexate



Assignments



s i c a l Shifts



(ppm)



Multiplicity



J(Hz)



_ _ _ I _



H2N-2



4



H-7 H-9 HSC-11



~ - 2 ~ , 6 ~ ,5' ,3' H-8' H u H-B9y H(COOH, HOH)



7.42 8.64 4.81 3.21 6.81,7.78 8.21



4.43 1.70-2.60 6.21



s( broad) S



s( broad)



----



S



--



d



8.2



d q



7 .o



m



s( broad)



8 .O



---



The values agree reasonable w e l l w i t h those reported by Pastore2 who studied the p m spectra of methotrexate i n solutions a t pH 7.5.



METHOTREXATE



2.3



Carbon-13



magnetic spectrum (cmr)



The "cmr spectrum i n Figure 3 w a s recorded on a Varian XL-100 spectrometer with t h e sample as a s o l u t i o n i n IMSO. The assignments given i n Table 111 f o r Figure 3 are i n general agreement with those reported by Ewers and co-workers,' but minor d i f f e r e n c e s e x i s t . TABLE I11 13C Assignment For Methotrexate (TMS 0 .OO ppm)



Chemical S h i f t ( ppm) 162.67



160.01 148.31 148.96 150.94 rv



Assignment



3



c-2



1



C-6 c-7 c-8a



c-4



C-4a c-9 c-11 c-1



121.66 40 (amid DMSO)



51.96 121.24 128.94 111.12 150.85



~ - 2 1 , C-6' c-31, c-5'



c-4 '



c-7' ca



166.41 54.82 26.17 39.55



173.96 174 .ll



c-B CY U-CoOH Y -COOH



3



Ultraviolet --Figure =the



spectru~ UV spectrum of methotrexate i n 0.1 N NaOH. The longest wavelength maximum appears a t 372 nm and i s a s c r i b a b l e t o t h e diaminopteridine moiety. The i n t e r mediate wavelength maximum occurs a t 303 nm and i s due primarily t o t h e aminobenzoyl group. The s h o r t wavelength maximum is a t 258 nm and is a t t r i b u t e d t o both chromophores. In 0.1 N HC1 methotrexate expsriences a hypsochromic s h i f t r e s u l t i n g i n a UV spzctrum (Figure 5 ) t h a t e x h i b i t s 2.1;



*



UV s p e c t r a were recorded on a Cary Model. 14 and t h e molar absorpt i v i t i e s a r e based on anhydrous methotrexate



291



.



292



U



(3



3



a



W



m



u



(? F



E'



v)



n



W



I0



H 9 a



0



LL



z



L



r



0



I-



a



W



X



a



I-



W



i



3



I



1



I 1



I



293



I I 1



I



0 In



d



0 0 d



5



W



I



P



0



I-



z w



2 2 w



I-



a



X w



a I-



0 I Iw



&I



t; LL



z



a Z



3



3



E



0 > a



J



t;



v)



L



u w



I-



a



3



H



0



z



0



m a



0 0 0



In hl



0



t



w



a



3



12



LL



ARTHUR R. CHAMBERLIN



250



FIGURE



350 NANOMETERS



300



5



294



eta:.



400



METHOTREXATE



maxima a t 307 and 243 nm. The W d a t a are summarized i n T a b l e I V and i n g e n e r a l are i n agreement w i t h t h o s e r e p o r t e d by S e e g e r and co-workers4, TABLE I V A b s o r p t i o n S p a c t r a of M e t h o t r e x a t e Solvent



I



0.1 N HC1 0.1 M



~ ~ 6 T. r 7i s



buffer



0.1 N NaOH 2.5



Mass Spectrum M e t h o t r e x a t e it self d o e s not y i e l d a s a t i s f a c t o r y



.



HoNever, t r e a t m e n t mass spectrum because of non-volat i l i t y of m e t h o t r e x a t e w i t h a TMS r e a g e n t a f f o r d s a m i x t u r e of tri-, tetra-, and psnta-TMS d e r i v a t i v e s t h a t d o e s g i v e u s e f u l mass spzctral d a t a . That s e v e r a l TMS-containing d e r i v a t i v e s are formed i s n o t s u r p r i s i n g , c o n s i d e r i n g t h e number and v a r i e t y of f u n c t i o n a l g r o u p s i n v o l v e d . The mass s p s c t r a l f r a g m e n t a t i o n p a t t e r n o b t a i n e d f o r t h e tri-, tetra-, and psnta-TMS d e r i v a t i v e s is summarized as follows:



I



RZ-NH



c



I



'1



I R1=R,=TMS: Rl,R2=1TMS,



m/e 319 I 1H; m/e 247



I Rl=R2=TMS Rl,R,=lTMS,



COOTMS



m / e 452 1 H m / e 380



I



M1bR1=R2=R3=TMS m / e 814 M-, R1=R2=TMS, R,=H m/e 742 Mf-R1,R2=1TMS, l H , R3=H m/e M-CH, m / e 727 M-HOTMS m/e 652 M-HOTMS-CH, m/e



295



637



670



ARTHUR R . CHAMBERLIN et al.



2.6



Optical Rotation 46



21



= 20.4



2 0.6'



26.9



0.8'



589 546



2.7



=



(c 1, N/10



NaOH)



D i s s o c ia t i on Constants ~ --



Neither t h e a c i d i c nor t h e b a s i c pKa f o r methotrexate has been r e p o r t e d . However, Albert and co-workers5 reported t h a t t h e basic pKa values f o r 2 , b d i a m i n o p t e r i d i n e w e r e < 0.5 and 5.32. Kallen and Jencks' r e p o r t e d t h e a c i d i c pKa values f o r p-aminobenzoylglutamic a c i d a s 4.83 and 3.76. By spectrometry, w e found a pKa of 5.60 2 0.03, which i s a s s i g n a b l e t o t h e diaminopteridinyl moiety.



2.8 ___-Solubility7



Methotrexate is p r a c t i c a l l y i n s o l u b l e i n water, alcohol, chloroform, and e t h e r . I t i s f r e e l y s o l u b l e i n d i l u t e s o l u t i o n s of a l k a l i n e and carbonates; it i s s l i g h t l y s o l u b l e i n d i l u t e hydrochloric acid (1 i n 2 ) .



3. Synthesis The f i r s t reported s y n t h e s i s of methotrexate, t h a t by Seeger and co-workers' is a s follows:



CH2Br



+



I



CHBr



I



+H-N



C-NH-



CHO



COOH



-------+C



----



These s p e c i f i c r o t a t i o n values a r e based on anhydrous methotrexate.



296



METHOTREXATE



T h i s r e a c t i o n o f t e n i s r e f e r r e d t o as t h e Waller r e a c t i o n , because Waller i n i t i a l l y p r e p a r e d p t e r o y l g l u t a m i c a c i d by an analogous method. T h e o r e t i c a l l y , the s u b s t i t u t i o n on t h e p y r a z i n e r i n g c a n be at t h e C-6 o r t h e C-7 p o s i t i o n . Proof of s t r u c t u r e is based on t h e p t e r i n e c a r b o x y l i c a c i d o b t a i n e d from a n a l k a l i n e p3rmanganat.e o x i d a t i o n of m e t h o t r e x a t e . Of t h e two a c i d s p o s s i b l e , o n l y t h e C-6 h a s been found. D i s t i n c t i o n between t h e two p o s s i b l e a c i d s h a s been based on comparative paper chromatography, a l t h o u g h pmr or c m r a l s o c o u l d be u s e d . Because t h e Waller r e a c t i o n g e n e r a l l y y i e l d s impure p r o d u c t s t h a t are v e r y d i f f i c u l t t o p i r i f y, many r e s e a r c h e r s have a t t e m p t e d t o improve t h e s y n t h e s i s of m e t h o t r e x a t e . Among t h e more s u c c e s s f u l a t t e m p t s are t h o s e by Taylor’ The r e s p e c t i v e methods and by P i p 2 r and Montgomery”. are o n t l i n e d as f o l l o w s : Taylor



NC



N



CHZC1



0



0



0



R=N-( g l u t a m y l )



297



I DMF



ARTHUR R . CHAMEERLIN eta/.



Piper and Montgomery NH2



NH2



PMABG = N-( p-methylaminobenzoy1)glutamic acid DMAC = N,N-dimethylacetamide



These improved procedures not only y i e l d products t h a t are easier t o purify, but t h e s u b s t i t u t i o n on t h e pyrazine is unambiguous.



4. ---Stability 4.1



Bulk When stored i n a cappsd, brown b o t t l e a t room temperature, samplings removed over a twelve-month period yielded i n d i s t i n g u i s h a b l e UV and papsr chromatographic d a t a , These r e s u l t s i n d i c a t e t h a t methotrexate is stable f o r a t least one y e a r under these conditions. 4.2



Solution As d i l u t e s o l u t i o n s ( 0 . 5 mg and 0.05 mg/ml) i n pH 7 .O aqueous sodium bicarbonate maintained a t room temperat u r e i n darkness and under laboratory illumination, a l i q u o t s removed over a 24-hour period y i e l d e d i d e n t i c a l papergrams, within experimental e r r o r s . On t h i s basis, these s o l u t i o n s were considered stable under t h e s e conditions.



298



METHOTREXATE



Under s t r o n g l y a c i d i c aqueous conditions, t h e amide i s s u b j e c t t o hydrolysis, y i e l d i n g N10-methyl-4-amino-4deoxypteroic a c i d and glutarnic a c i d . Under highly a l k a l i n e aqueous conditions, e s p x i a l l y a t e l e v a t e d temperatures, t h e p r i n c i p a l decomposition products a r e N1'-methylfolic acid, N1'-methylpteroic acid, and glutamic a c i d - - a l l as t h e carboxylate i o n s . Photodecompwition of c e r t a i n p t e r i n e s is well documented .I1 However, no s p e c i f i c r e p o r t on t h e photochemistry of methotrexate has been found i n t h e literature.



5. Metabolism I n man, a very l a r g e p o r t i o n of t h e methotrexate administered is excreted unchanged, 'la i n d i c a t i n g t h a t l i t t l e metabolism of t h i s drug occurs. Johns and Loo" reported t h a t methotrexate is metabolized r a p i d l y by r a b b i t l i v e r aldehyde oxidase, and t h e metabolite w a s i d e n t i f i e d as 4-amino-k-deoxy-7hydroxy-N1O-methylpteroylglut amic acid (7-hydroxy-MTX)



.



Johns and Valerino13 have observed t h a t , when methotrexate i s incubated with cecal c o n t e n t s from t h e mouse i n v i t r o , ~-amino-~-deoxy-N1O-methylpteroica c i d i s produced. Levy and Goldman'* have demonstrated t h a t t h i s metabolite can be produced from methotrexate by a c t i o n of carboxypiptidases from s t r a i n s of Psudoxona_t.



6. Methods of Analysis --6.1 ---Elemental Analysis



Table I V p r e s e n t s t h e r e s u l t s from a r e p r e s e n t a t i v e elemental a n a l y s i s of methotrexate ( r e f e r e n c e standard)



.



TABLE V



Elemental Analysis of Methotrexate Element



*



C H N



-Theory -52.85



$



9



Found 52.72



4.88



4.85



24.66 Adjusted f o r t h e found water



24.51



299



ARTHUR R . CHAMEERLIN e t a / .



6.2



Equivalent Weight Determinations _ _ l _ l --I_---_



6.21



Nonaqueous T i t r a t i o n



D e Carnevale e t a1.l’ d e s c r i b e d a n e q u i v a l e n t weight d e t e r m i n a t i o n based on a sodium methoxide t i t r a t i o n i n p y r i d i n e t o an a z o - v i o l e t end p o i n t . Because t h e b a s i s of t h e t i t r a t i o n i s a n a c i d l b a s e n e u t r a l i z a t i o n , it l a c k s s p e c i f i c i t y . Consequently, t h e method has not been employed widely as a n a s s a y f o r m e t h o t r e x a t e .



6.22 Complex-Formation ----Titration G u e r e l l o h a s described’’ a second t i t r a t i o n by which e q u i v a l e n t w e i g h t s of m e t h o t r e x a t e samples can be obtain??. The aethod i s basad on t h e complexation between the Ca and t h e glutamyl moiety and t h e r e b y a f f o r d s higher s p e c i f i c i t y than an acidlbase neutralization. Because g l u t a m i c a c i d c o n t a i n i n g i m p u r i t i e s a r e commonly found i n m e t h o t r e x a t e samples, r e s u l t s from t h i s t i t r a t i o n must be i n t e r p r e t e d c a r e f u l l y .



6.3 --------B i o l o g i c a l Assay 6.31 M i c r o b i o l o g i c a l Assay S e v e r a l m i c r o b i o l o g i c a l a s s a y s are d e s c r i b e d i n the 1 i t e r a t ~ r e . l ~ A l l are r e l a t i v e l y n o n s p e c i f i c and very time consuming and t h u s have l i t t l e u s e f u l n e s s f o r routine analyses.



6.32 -Enzymic - - - Assay ~ -



Werkheiser and co-workers” have developed an enzymatic a s s a y f o r m e t h o t r e x a t e u s i n g f o l i c acid reductase M e t h o t r e x a t e b i n d s v e r y t e n a c i o u s l y and s t o i c h i o n e t r i c a l l y t o t h i s enzyme and i s determined c o l o r o m e t r i c a l l y by t i t r a t i o n of t h e drug w i t h t h e enzyme.



.



6.4



P o l a r o g r a p h i c Assay



Asahi” has r e p o r t e d p o l a r o g r a p h i c r e d u c t i o n of m e t h o t r e x a t e . H e a t t r i b J t e s t h e f i r s t wave as being t h e r e d u c t i o n t o dihydromethotrexate, t h e second wave a s being t h e r e d u c t i v e c l e a v a g e of t h e CH2-N bond, and t h e t h i r d wave a s being t h e r e d u c t i o n t o 2,4,-diamino-6-methyl-5,6,7,



8-tetrahydropteridine , 300



METHOTREXATE



6.5



S p e c t r o p h------otometric Analysis



6.51 --Fluorometric Analysis



F l u o r o m e t r i c methods2' have been used w i d e l y i n t h e a n a l y s i s of m e t h o t r e x a t e . The methods a r e based on a n o x i d a t i v e t r a n s f o r m a t i o n of m e t h o t r e x a t e t o the presumed p t e r i n e c a r b o x y l i c a c i d which f l u o r e s c e s i n t e n s e l y . Because many of t h e i m p u r i t i e s commonly found i n m e t h o t r e x a t e a l s o undergo t h e same r e a c t i o n , t h e s e methods l a c k s p c i f i c i t y u n l e s s t h e o x i d a t i o n i s preceded by a s e p a r a t i o n scheme i n which m e t h o t r e x a t e i s i s o l a t e d . 6.52



-Ultraviolet/Visible Analysis



Because commercial m e t h o t r e x a t e s a n p l e s a r e impure, and because t h e o r g a n i c i m p u - i t i e s have W absorpt i o n c h a r a c t e r i s t i c s t h a t a r e similar t o t h o s e of methotrexate, d i r e c t u v / v i s s p s c t r o p h o t o n e t r i c a n a l y s i s i s e n t i r e l y t o o n o n s p e c i f i c t o be of any v a l u e i n t h e q u a n t i t a t i v e a n a l y s i s of m e t h o t r e x a t e . P r a c t i c a l l y a l l t h e contaminants l i k e l y t o be p r e s e n t i n a sample of met h o t r e x a t e- -N1' -me t h y 1p t e r o i c a c i d , and so f o r t h - w m l d i n t e r f e r e w i t h a direct W o r v i s i b l e method.



6.61 -Paper



Nichol and co-workers21 have r e p o r t e d t h e s e p a r a t i o n s of m e t h o t r e x a t e from related compounds on Whatman No. 1 w i t h pH 7.0, 0.1 M sodium phosphate and w i t h pH 5.0, 0.1 M sodium acetate. I n our l a b o r a t o r y , w e have used 0.5% NaHC03 and 0.5% Na2C03 w i t h t h e sane papzr. Balazs and co-workers22 r e p o r t e d a r a p i d a s s a y f o r methotrexate based on ps.per chromatographic s e p a r a t i o n followed by W measurement of t h e i s o l a t e d m e t h o t r e x a t e component. The l i m i t a t i o n s of t h i s a s s a y a r e t h e accuracy of t h e r e f e r e n c e UV v a l u e and t h e r e c o v e r y of m e t h o t r e x a t e from t h e papergram. The molar a b s o r p t i v i t y a t 303 nm f o r m e t h o t r e x a t e i n N / 1 0 NaOH r e p o r t e d by B a l a z s and co-workers i s i n e r r o r ; it should be 24,830. The a s s a y procedure f o r m e t h o t r e x a t e c i t e d i n USP XVIII is s i m i l a r t o t h e a s s a y r e p o r t e d by Balazs et a 1 e x c e p t t h a t t h e r e f e r e n c e s t a n d a r d i s USP m e t h o t r e x a t e



301



ARTHUR R . CHAMEERLIN etal.



which i t s e l f i s impure.



6.62 Thin-layer Copenhaver and O'Brienz3 have used ionexchange thin-layer chromatography t o s e p a r a t e a number of f o l i c a c i d analogs including methotrexate The cationexchange r e s i n was AG50W-X4, and the developing solvent was 154 Na2HF04'12 H20, p H 8.5 b u f f e r containing 0.1 M mercaptoethanol



.



.



6.63 Column Heinrich and c o - ~ o r k e r sreported ~~ the use of Dowex 1-chloride w i t h very d i l u t e HC1 or N@ to separate f o l i c acid and r e l a t e d compounds. Noble described a p u r i f i c a t i o n procedure based on the use of a Dowex 1-acetate w i t h pH 3.2, M a c e t a t e b u f f e r . Oliverio2' cited t h e use of DEAE c e l l u l o s e and pH 8 phosphate b u f f e r gradient t o s e p a r a t e f o l i c acid analogs. G a l l e l l i and YokoyamaZ7 developed a methotrexate assay procedure e n t a i l i n g a DEAE c e l l u l o s e s e p a r a t i o n followed by a spectrophotometric measurement of t h e i s o l a t e d component. The use of DEAE cellulose t o separate f o l i c a c i d analogs is w e l l e s t a b l i s h e d . The p r i n c i p a l l i m i t a t i o n s of t h i s method are t h e t i m e required t o pack t h e column and t h e t i m e needed t o develop t h e chromatogram.



6.64 High



Speed Liquid In our work w i t h other f o l i c a c i d antagonists, cat ion exchange high speed l i q u i d chromatography (HSLC) has been very e f f e c t i v e i n separating c l o s e l y r e l a t e d p t e r i d i n e s . Taking advantage of t h i s s e l e c t i v i t y , we have developed a HSLC method of a n a l y s i s f o r methotrexate that is specific, f a s t , and s e n s i t i v e . W e use it t o assay b u l k and formulated methotrexate. The procedure r e q u i r e s a reference methotrexate sample of known purity, which i s used a s an e x t e r n a l reference o r i n conjunction w i t h a n i n t e r n a l reference that, i n our laboratory, has been 2-amino-bmethylpyridine . The column, l m x 3mm 0 .D. Vydac Cation Exchange Packing, i s held a t 55' during t h e developnent w i t h pH 4.30, 0 . 1 M KH2P04 a t a f l o w rate of 1.0 ml/min. The e f f l u e n t is monitored by a UV-detector set a t 254 nm. With t h e d e t e c t o r s e t a t i t s highest s e n s i t i v i t y , s o l u t i o n s a s d i l u t e d a s 1 pg methotrexate/ml can be i n j e c t e d



302



METHOTREXATE



d i r e c t l y and d e t e c t e d . A second procedure employing a l m x 3mm O.D. column packed with reverse-phase phenyl and a mobile phase of 5% MeOH i n 0.05 M KH2P04, pH 7.0, b u f f e r a l s o has been developed and found u s e f u l , The reverse-phase system is less s e n s i t i v e t o minor pH v a r i a t i o n s r e s u l t i n g from sample i m p u r i t i e s . For t h i s reason, t h i s column produces high p r e c i s i o n more r e a d i l y .



6.7 Proton Magnetic Resonance Assay K t h o t r e x a t e has been assayed by q u a n t i t a t i v e pmr on a Varian A-&A spectrometer. Accurately weighed portions of methotrexate and of an i n t e r n a l standard (2,4-dimethoxy-5-methylpyrimidine) were dissolved i n DMSO, and t h e spectrum was recorded. The psrcentage of anhydrous methotrexate i n t h e sample then was c a l c u l a t e d according t o t h e formula



$



454.4 x - - 1 54.2



MTX = Wr x A s x Ws Ar



P



where W r = weight of i n t e r n a l standard used W s = weight of methotrexate sample A r = i n t e g r a t e d a r e a of i n t e r n a l standard He s i n g l e t (8.02 6 )



As



= i n t e g r a t e d a r e a of methotrexate



H7 proton



(8.64



6)



154.2 = molecular weight of i n t e r n a l standard



454.4 =



molecular weight of anhydrous MTX



P = p u r i t y of t h e i n t e r n a l standard



Although t h e pmr method may lack s e n s i t i v i t y and possibly s p e c i f i c i t y i n complex mixtures, it i s one method of assay t h a t does not r e q u i r e a methotrexate reference sample of known p u r i t y . Thus, it may be u s e f u l i n cases i n which no reference material is a v a i l a b l e .



303



ARTHUR R. CHAMBERLIN etal.



7 , Acknowledgments ---Supported by C o n t r a c t N01-CM-33723 from t h e d i v i s i o n of Cancer Treatment, N a t i o n a l Cancer I n s t i t u t e , N a t i o n a l I n s t i t u t e s of Health, Department of Health, Education, and Welfare. The o p i n i o n s e x p r e s s e d are t h o s e of t h e a u t h o r s and not n e c e s s a r i l y t h o s e of t h e N a t i o n a l Cancer I n s t i t u t e .



The a u t h o r s wish t o acknowledge t h e t e c h n i c a l a s s i s t a n c e r e n d e r e d by Ms. Leslie Pont, M s . Barbara Senuta, Ms. F l o r e n c e Yoshikawa, M r . Martin S t r u b l e and I r John Jee of S t a n f o r d Research I n s t i t u t e .



.



304



METHOTREXATE



8.



Reference? 1.



2.



3.



4. 5. 6.



Lee, A.P. Martinez, and L . Goodman, J . Med. Chem. lJ, 326 (1974) E . J . Pastore, Ann. N.Y. Acad. S c i . -9185 43 (1971). U . Ewers, H. Gunther, and L . Jaenicke, Chem. B e r . W.W.



.



106, 3351 (1973) * D.R. Seeger, D.B. Coslllich, J . M . Smith, and M.E. Hultquist, J . Am. Chein. SOC. 2, 1753 (1943). A . A l b e r t , D.J. Brawn, and G . Chessman, J . Chem. SOC., 4219 (1952). R.G. Kallen and W.P. Jencks, J . B i o l . Chem. 24,



531.~5(1956)



.



I



The United S t a t e s Pharlnacopzia, The XVIII Revision (1970), p. 418. 8. D . R . Seeger, J.M. Smith, and M.E. Hulquist, J . Am. Chem. S O C . 69, 2567 (1947) and Biology of P t e r i d i n e s , 9 . E .C. Taylor,'Chernistry Proceedings of the Fourth I n t e r n a t i o n a l SympDsium on P t e r i d i n e s , Toba, 19s9, I n t e r n a t i o n a l Academic P r i n t i n g Co., Ltd., Tokyo, 1970, p. 79. 10* J . R . Pipzr and J . A . Montgoaery, J . H e t . Chem. 273 (1974) * 11. R.L. B l a k e l y , The Biochemistry of F o l i c Acid and Related P t e r i d i n e s , Nort h-Holland Pub1i s h i n g Compnny, London, 1959, p. 77. l l a . I b i d p.495. 56, 356 12. D.G. Johns and T.L. Loo, J . Pharm. S c i . -



7.



.



11,



(1957) *



13. D.G. Johns and D.M. Valerino, Ann. N.Y. Acad. S c i .



186, 384 (1971).



14. 15. 16. 17.



C.C.



Levy and P. Goldman, J . Biol. Chem. -242,



2933



(1967) * R.C.D. D e Carnevale, J . Dobrecky, and L.O. Guerello, 113, 15 (1971). Rev. F a m . (Buenos Aires) L.O. Guerello, Rev. Asoc. Bioquim. Argent. 34, 33



(1969) * J.H. H.D.



Burchenal, G.B. Waring, R.R. E l l i s o n , and R e i l l y , Proc. SOC. Exp. B i o l . N.Y. i '8, 603 ( 1951) ; A . Z Smolyanskaya and N.N. Agadzhanova, vop. Onkol 13, 58 (1957) Chem. Abst 46, 26036; D.E. Hunt a n r R . F . P i t t i l l o , Cancer R e s . 28, 1095



.



(1958)



.



,



.



305



.,



ARTHUR R . CHAMBERLIN etal.



18.



19. 20.



W.C. Werkheiser, S . F . Zakrzewski, and C.A. Nichol, J . Plannacol Exp. Therap. 162 (19.52) Y, Asahi, Yakugaku Zasshi 1570 (1959), Chea. Abst 54, 10593d. S.G. CcGkrabarti and I . Bernstein, C l i n . Chem. 1157 (1959); S . F . Zakrzewski and C.A. Nichol, J . Biol Chem. 205, -- 364 (1953); M.V. Freeman, J . Pharmacol. E X ~ .Therap. 120, -- 1, (1957) and 121,



m,



.



E,



.



.



9,



154 (1958) *



21.



S . F . Zakrewski, and A.D. Welch, Proc. 272 (1953); S.F. Zakrrewski and C . A . Nichol, J . Biol. Chea. 205, 352 (1953). M.K. Balazs, C.A. Anderson, and K-Lim, J . Pharm. sci. 2002 (1968). J.H. Copmhaver and K.L. O'Brien, Anal. Biochem. C.A.



Nichol,



So. Exp. Biol. Med.



22. 23. 24. 25.



26. 27.



5,



z,



s, 454 (1969) ' M.K. Heinrich, V.C.



Dewey, and G.W. Kidder, J . Chromatog. 2, 296 (1959). E.P. Noble, Biochem. Prep. 8, 20 (1961). V.T. Oliverio, Anal. Chem. 263, (1951). J . F . G a l l e l l i and G. Yokoyama, J . Pharm. S c i .



3,



357 (1957)



306



55,



METHYCLOTHIAZLDE



James A . Raihle



JAMES A. RAIHLE



Contents 1.



Description 1.1 Nomenclature 1.11 Chemical Names 1.12 Generic Name 1.13 Trade Names 1.2 Formulae 1.21 Empirical 1.22 Structural 1.3 Molecular Weight 1.4 Elemental Composition 1.5 General



2.



Physical Properties 2.1 Infrared Spectrum 2.2 Raman Spectrum 2.3 Nuclear Magnetic Resonance Spectrum 2.4 Ultraviolet Spectrum 2.5 Mass Spectrum 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Dissociation Constant 2.9 Solubility 2.10 Crystal Properties



3.



Synthesis



4.



Stability-Degradation



5.



Drug Metabolic Products



6. Methods of Analysis 6.1 Titrimetric Methods 6.11 Argentimetric Titration 6.12 Potentiometric Titration 6.2 Chromatographic Methods 6.21 Column Chromatography 6.22 Paper and Thin-Layer Chromatography 6.3 Spectrophotometric Methods 6.4 Polarographic Method



7. References



308



METHYCLOTH lAZl DE



Methyclothiazide



1.



Description



1.1 Nomenclature 1.11



Chemical Name



Methyclothiazide is 6-chloro-3-(chloromethyl) -3,4-dihydro-2-methy1-2H-1,2,4-benzothiadiazine-7-su1fon-



amide 1,l-dioxide.



(1)



It is also known as 6-chloro-3-



ch1oromethy1-3,4-dihydro-2-methy1-7-su1famoy1-1,2,4-benzothiadiazine lyl-dioxide; 6-chloro-3-chloromethyl-2-methyl7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine 1,l-dioxide (2) and by many slight variations of the particular nomenclature. The GAS Registry No. is [135-07-91.



1.12 Generic Name Methyclothiazide 1.13



Trade Names Enduro@



and Aquatensen@



1.2 Formulae 1.21 Empirical ‘gHl 1C12N304S 2 1.22



Structural



309



JAMES A. RAIHLE



1.3 Molecular Weight 360.23 1.4 0



-



Elemental Composition



-



C 30.00; H 17.77; S 17.80.



-



-



3.08; C1



-



19.68; N



-



11.66;



1.5 General Methyclothiazide occurs as a white to practically white crystalline powder which is principally odorless. 2. Physi'cal Properties 2.1



Infrared Spectrum (IR)



The infrared spectrum of methyclothiazide (NF Reference Standard, Lot No. 69196) is presented in Figure 1. The spectrum of a KBr pellet is taken on a Perkin-Elmer Model 521 Spectrophotometer. The assignments for the characteristic bands in the IR spectrum are listed in Table I. (3) Table I Characteristic Bands in the IR Spectrum of Methyclothiazide Wavelength (em-l)



Characteristic of



3270 and 3360



NH stretching vibration of sulfonamide group



1595 and 1502



C =



1155 and 1330 (doublet)



S = 0 stretching vibrations of sulfonamide groups



C stretch of aromatics



This spectrum is consistent with that published by Fazzari and co-workers. (4)



310



2.5



3



WAVELENGTH 4



(MICRONS) 5



6



FIGURE 1



7



8



9 10



12



15



- INFRARED SPECTRUM 0F METHY CLOTHIAZIDE



JAMES A. RAIHLE



2.2



Raman Spectrum



The raman spectrum of the methyclothiazide reference standard was determined in the solid phase on a Cary Model 83 Spectrophotometer. The assignments of the characteristic bands as shown in Figure 2 are listed in Table 11. (3) Table I1 Characteristic Bands in the Raman Spectrum of Methyclothiazide Wavelength (cm-l)



Characteristic of



3270 and 3363



NH stretching vibration of sulfonamide group



2.3



1600



C = C stretch of aromatics



1160



s = 0 stretching vibrations of sulfonamide groups



Nuclear Magnetic Resonance Spectrum ("El)



The 60 MHz NMR spectrum of methyclothiazide is presented in Figure 3. The spectrum was determined in deuterated acetone (d6) on a Varian T-60 Spectrometer. Spectral assignments are given in Table 111. (5)



312



0



v



0



AlISN31NI



0



0 9



0



0 el



0



0 Qo



313



FIGURE 3



- NUCLEAR MAGNETIC RESONANCE SPECTRUM OF METHYCLOTHIAZIDE



r



d 8.0



7.0



6.0



5.0



4.0 PPM (6)



3.0



2.0



1.o



METHYCLOTH IAZIDE



Table 111 NMR Assignments for Methyclothiazide



Number of Protons



Chemical Shift (ppm)



Aromatic proton at carbon 8



1



8.23



S



Aromatic proton at carbon 5



1



7.22



S



Exchangeable protons: NH, NH2



3



6.87



M(b)



Methyne proton at carbon 3



1



5.53



T



Methylene protons of chloromethyl group at carbon 3



2



4.07



D



Methyl protons of 2-methyl group



3



2.77



S



Assignment



Multiplicity



2.4 Ultraviolet Spectrum (W) The UV spectrum of methyclothiazide prepared as a 1 in 100,000 solution in methanol is shown in Figure 4 . The spectrum exhibits three maxima and two minima characteristic of substituted benzothiadiazines. The maxima are at 226 nm (Em = 39,300), 267 nm (Em = 21,250) and ca 311 nm (Em = 3,300). Minima were observed at 240 nm and 290 nm. The spectrum is consistent with previously published reports by Furman ( 6 ) and Fazzari, et. al. (4) 2.5



Mass Spectrum



The mass spectrum shown in Figure 5 was obtained using an Associated Electrical Industries Model 902 Mass Spectrometer with an ionizing energy of 50 eV and a temperature of 150°C. Methyclothiazide yields a spectrum with a base peak at m/e 359. Subsequent fragments, Table IV, 315



JAMES A. RAIHLE



FIGURE 4



- ULTRAVIOLET SPECTRUM OF METHYCLOTHIAZIDE



Ly



v



2



a m



= 0



VI



m



a



200



250



300



WAVE LENGTH (nm)



316



350



FIGURE 5, MASS 5PEClRUM OF MElHYCLOlHIAZIDE



317



I 250



'



I



'



L



'



I



I



' 300



I,,



.I,



I



.#I,II



I,,



;



I 350



,



JAMES A. RAIHLE



reflect the loss of the chloromethyl and sulfonamide groups as well as the fragmentation of the ring system. (7) Table IV High Resolution Mass Spectrum of Methyclothiazide Mass Found



Relative Intensity



Composition



Error (mu)



-c L!



! 0



s



c135



358.9565



3.06



-0.32



9 1 1



3



4



2



2



309.9711



100.00



-1.21



8



9



3



4



2



1



291.9629



0.56



1.18



8



7



3



3



2



1



229.9914



6.02



-0.26



8



7



2



2



1



1



166.0280



3.52



-1.78



8



7



2



0



0



1



131.0615



5.42



0.57



8



7



2



0



0



0



123.9952



7.89



-0.22



6



3



1



0



0



1



96.9847



5.95



0.16



5



2



0



0



0



1



90.0113



6.77



0.25



3



5



1



0



0



1



42.0345



63.94



0.13



2



4



1



0



0



0



2.6



Melting Range



Methyclothiazide melts with rapid decomposition between 225°C and 227°C. Slight discoloration of the solid may be observed at about 215°C.



2.7



Differential Thermal Analysis



The thermogram depicted in Figure 6 shows a large endothermic melt between 224°C and 231°C. The thermogram shows a subsequent exothermic response confirming the visual observation of rapid decomposition.



318



FIGURE 6



0



20



-



DIFFERENTIAL THERMAL ANALYSIS CURVE OF METHYCLOTHIAZIDE



40



no



140 160 in0 200 1, OC (CORRECTED FOR CHROME1 ALVMEL THERMOCOUPLES) 40



80



100



220



240



JAMES A. RAIHLE



2.8



Dissociation Constant



The pKa of methyclothiazide was determined by the titrimetric method by extrapolation of data from acetonewater mixed solvents to 100% water. The pKa is 9.4 (proton lost). 2.9



Solubility



The following solubilities have been determined for methyclothiazide at room temperature. Solvent Water Chloroform Benzene n-Butanol PEG-600 Ethano1 Acetonitrile Methanol Acetone Pyridine



Solubility 0.06 0.03 < 1 1 1 11 5 > 10 - 200 - 200



-



N



mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml



National Formulary Descriptive Term( 1) Very Slightly Soluble Very Slightly Soluble Very Slightly Soluble



------Slightly Soluble ----



Sparingly Soluble Freely Soluble Freely Soluble



2.10 Crystal Properties The X-ray powder diffraction pattern of methyclothiazide was determined by visual observation of a film obtained with a 143.2 mm Debye-Scherrer Powder Camera. An Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA with nicker filtered copper radiation; ), = 1.5418 was employed. A listing of d-spacings and intensities is presented in Table V. (8)



320



METHYCLOTH IAZIDE



Table V X-Ray Powder Diffraction Pattern d-Spacings and Intensities



dA



-111 1



dA -



I/I1



9.8 7.75 7.5 7.2 6.25 5.75 5.3 5.1 4.85 4.56 4.42 4.3 4.11 4.00 3.90 3.75B 3.6 3.52 3.44 3.32 3.30 3.12 3.07 3.00



5 50 30 5 20 50 5 10 100 80 20 15 20 2 2 20 3 20 5 5 8 5 8 5



2.95 2.90 2.81 2.72 2.68 2.62 2.51 2.42 2.27 2.24 2.15 2.11 1.99 1.88 1.85 1.81 1.77 1.75 1.73 1.69 1.64 1.5 B 1.35B 1.3 B



5 LO 60 3 5 15 10 3 15 10 3 5 8 3 5 8 2 3 3 2 4



3.



4 3 3



Synthesis



Methyclothiazide is synthesized by the reaction sequence shown in Figure 7. Sprague (9) described the reaction of 4-amino-6-chloro-1,3-benzenedisulfonamide with urea to form the 3-keto derivative. Close, et. al. (10) have preferentially alkylated the more acidic cyclic sulfonamide with methyl iodide to form 6-chloro-2-methyl-3oxo-7-sulfamyl-3,4-dihydro-l,2,4-benzothiadiazine 1,ldioxide. This intermediate is readily ring opened by alkaline hydrolysis and then re-cyclized with chloroacetaldehyde to form the desired product. 321



Figure 7 Synthesis of Methyclothiazide



z 0=0 z



I



II



0



0



N



N



+HpNCNHz



+



CH31 N



I



z



N



I



t



0



I 0 I



z



6



6



%



z



I



0



I



0



N



I



2



6



-



*



322



METHYCLOTH IAZIDE



4.



Stability-Degradation



Methyclothiazide is stable in the solid state and under ordinary ambient conditions. It is rapidly decomposed in boiling acidic solutions to 2-methylsulfamyl-4-sulfarnyl-5chloroaniline. In alkaline solution it rapidly loses one chlorine atom to form the 3-hydroxymethyl analog. This product has been shown to degrade further under severe conditions, however none of the alkaline degradation products contain primary aromatic amine centers. Solutions buffered at pH 4.0 show 22% hydrolysis after 7 days at 60"C, 10% after 28 days at 40"C, but only 2% after 28 days at 25°C. Solutions buffered at pH 6.0 gave 1.6% hydrolysis after 7 days at 6OoC, and less than 1% after 28 days at 40°C or 25°C. 5.



Drug Metabolic Products



No report of metabolic products related to methyclothiazide has been published.



6. Methods of Analysis 6.1



Titrimetric Methods 6.11 Argentimetric Titration



The compendia1 procedure for the purity determination of the drug substance is based upon the argentimetric titration of the chloride liberated after reflux in methanolic potassium hydroxide. (1) The equivalent weight is 360.23 since only the chlorine from the 3-chloromethyl group is liberated during reflux. The method is specific since this group is added during the final step of the synthesis and a limit of 0.02% free chloride is imposed on the drug substance. 6.12 Potentiometric Titration Methyclothiazide can be titrated as an acid using either tetra-butylamonium hydroxide in chlorobenzene (11) or potassium hydroxide in isopropanol (12) as the titrant. The solvents are pyridine and acetone, respectively. Methyclothiazide consumes two equivalents of base per 323



JAMES A. RAIHLE



mole of drug substance. The first equivalent is from the neutralization of the free sulfamyl group. The exact location for the reaction of the second equivalent has not been determined, however, it may result from rapid hydrolysis of the 3-chloromethyl function. 6.2 Chromatographic Methods 6.21 Column Chromatography Fazzari (13) has published a collaborative study on a column chromatographic method for the analysis of methyclothiazide from tablets. The drug is eluted from a 0.1 NaHC03-Celite column with chloroform and measured directly at 267 nm by W spectrophotometry. The precision of the method was 99.8 4 1.64% on a commercial preparation of 2.5 mg tablets. 6.22 Paper and Thin-Layer Chromatography Paper chromatography has been applied by Pilsbury and Jackson (14) for the rapid detection and identification of thiazide diuretics in tablets, gastric fluid, and urine. Identification is accomplished by ascending reverse phase chromatography using tributyn treated paper and developing for 20 minutes at 90°C with a phosphate buffer (pH 7.4). The thiazides are located by ultraviolet light (254 nm) and confirmed by the red color given by alkaline sodium 1,2-naphthaquinone-4-sulfonate spray reagent. Paper chromatography can also monitor the extent of manufacturing by-products. Ascending chromatography using butanol saturated with 3% ammonium hydroxide and descending chromatography using butano1:acetic acid:water (50:15:60) have been employed. Visualization is by ultraviolet light. Thin-layer chromatography using the system ch1oroform:methanol:ammonium hydroxide (170:30:2) on 0.25 mm silica gel GF254 plates and ultraviolet detection rapidly isolates and identifies the common manufacturing intermediate s ,



324



METHYCLOTH IAZIDE



6.3



Spectrophotometric Assays



Methyclothiazide can be determined after acid hydrolysis to 2-methylsulfamyl-4-sulfa~l-5-chloroaniline by a modified Bratton-Marshall procedure. This procedure without prior acid hydrolysis also monitors diazotizable substances in the drug substance. (1) Ultraviolet absorption at 267 nm is seldom directly employed since the intermediates and degradation products have similar absorption spectra. The ultraviolet procedure has been utilized after prior separation by chromatography (13) or for non-specific content uniformity measurements. (1)



6.4 Polarographic Method The current compendia1 assay for methyclothiazide tablets is a polarographic assay in an aqueous system containing 6% v/v dimethylformamide and 0 . 1 _M tetra-n-butylammonium chloride as the supporting electrolyte. A mercury anode is used in conjunction with the DME since the halfwave potential of methyclothiazide occurs in the region where potassium ions from classical agar electrodes would interfere. 7.



References 1. The National Formulary, 14th Ed., Mack Publishing G o . , Easton, PA (1975). 2.



The Merck Index, 8th Ed. , Merck and Go. Rahway, NJ (1968).



, Inc.,



3. Washburn, W., Abbott Laboratories, Personal Comunication.



4. Fazzari, F. R., Sharkey, M. F. , Yaciw, C. A. and Brannon, W. L., J. Ass. Offic. Anal. Chem., 2, 1154, (1968).



5.



Egan, R. S., Abbott Laboratories, Personal Communication.



325



JAMES A. RAIHLE



6. Furman, W. B., J . Ass. Offic. Anal. Chem., 1111, (1968).



51,



7. Mueller, S . , Abbott Laboratories, Personal Communication. 8. Quick, J. E. , Abbott Laboratories, Personal Communication.



9. Sprague, J. M., Ann. N. Y. Acad. Sci. , 71, 328, (1958). 10.



Close, W. J., Swett, L. R., Brady, L. E., Short,



J. H. , and Versten, M. , J. Am. Chem. SOC. , 82,



1132 (1960).



11. Luebke, D. 'R., Abbott Laboratories, Personal Communication. 12.



Chang, S . L., Acta. Phann Sinica, 7, (1959). (Anal. Abstr., 1, 1909, (1960)).



13. Fazzari, R. , J. Ass. Offic. Anal. Chem., (1973). 14.



56, 677,



Pilsbury, V. B., and Jackson, J. V., J. Pharm. 713, (1966). Pharmacol., J-8,



326



M ETRONIDAZOLE



Lorraine L. Wearley and Gaylord D. Anthony



LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY



Cantents 1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties



2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9



Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Optical Rotation Melting Range Differential Scanning Calorimetry Thermogravimetric Analysis Solubility



3.



Metabolism



4.



Pharmacokinetics



5. Methods of Analysis 5.1 5.2 5.3 5.4



Titrimetric Analysis Spectrophotometric Analysis Colorimetric Analysis Chromatographic Analysis 5.41 Thin Layer Chromatography 5.42 Gas Chromatography 5.5 Polarographic Analysis



6. Synthesis



7. References 8.



Acknowledgments



328



METRON IDAZOLE



1. Description 1.1 Name, Formula, Molecular Weight Metronidazole is 1-(2-hydroxyethyl) -2-methyl- 5n i troimidazole



CH2 CHZOH



N02a l4olecular Weight:



'SHSN303



1.2



171.16



ADDearance. Color. Odor Metronidazole is a white t o pale yellow, odorless c r y s t a l l i n e powder.



2.



Physical Properties



2.1



Infrared S p e c t m The infrared absorption spectrum of a metronidazole reference standard compressed i n a KBr p e l l e t is shown i n Figure 1. The following assignmenfs have been made f o r absorption bands i n Figure 1. Band (an-1)



Assignment



32 30



OH s t r e t c h



3105



C=CH; C-H s t r e t c h



1538 6 1375



NO2; N-0 s t r e t c h



1078



C-OH; C-0 s t r e t c h



830



C-N02; C-N s t r e t c h



329



W



Figure 1 Infrared Spectrum of Metronidazole



METRON I DAZO LE



2.2 Nuclear Magnetic Resonance Spectrum The M I spectrum of metronidazole in deuterated acetic acid is shown in Figure 2. Below are the assignments of the major signals. Positions of absorption bands are reported as shifts downfield from the signal of the protons in te ramethylsilane which was used as internal standard.



I



u



Assignment



155 singlet 241 (triplet) 274 (triplet) 481 (singlet)



\\



- CH3 -



,C



CH2



$2



a ,C -



--ez- OH



-cFr2-oH H



2 . 3 Ultraviolet Spectrum



Metronidazole exhibits an absorption maxima at about 274 run. using 0.1 N sulfuric acid in methanol as solvent. The molar absorptivity in this solvent is 6333. The ult2aviolet absorption spectrum is shown in Figure 3. 2.4 Mass Spectrum The low resolution mass spectrum of metronidazole is shown h3Figure 4. Structure assignments are as follows: !YE



Assignment



171



M**(molecular ion)



154



M-OH



125



M - NO2



22.1



125



M-NOZ-H



25.7



41



% Relative Intensity



12.5 4.0



M - CH3CN



100



331



. . . . 5.0 . l



'



"



-



I



'



'



~



'



.



..m ( T ). .6.0 , .. .



. . . . . 7.0 . . . . . . . .8.0. . . . . . . .91) . . . . , . . . . ? . . .



'



PD



00



**,



tm



m



tm



o m



0



0 1u



I . . . ko



I 1



1 74



I



.



.



.



.



. . r....l....,



I



. . . II . . .. . , . . . . l



I



.



4.a



.



..



I I



..I



I



. . . . ,



.



Figure 2



Nuclear Magnetic Resonance Spectrum of Metronidazole



I



I . . . . ' .



..



1



.



-



....LA



1



.



..



I



-



Figure 3



u1 traviolet Spectrum of Metronidazole in 0.1 N Sulfuric Acid in Methanol



E 1 J NBMINFIL- MRSSi-



Figure 4 Mass Spectnnn of Metronidazole



METRON IDAZOLE



2.5



Optical Rotation Metronidszole exhibits no optical activity.



2.6 Melting Range Theomelting range given in the USP XIX is 159' 163 C. 2.7



to



Differential Scanning Calorimetry The DSC thermogramoof metronidazole obtained at a heating rate of 20 C/minute is shown in Figuse 5, The endothermic change observed at about 163 C corresponds to the melting of the compound.



2.8



Thermogravimetric Analysis The TGAof metronidazole obtained a t a heatinq r a t e o f 10°C/minute i s shown i n f i g u r e 6. 4



2.9



Solubility Solubilities in various solveps at 2S0C are given in the following table. Solvent



Solubility, mg/ml



Water



10.5



Methanol



32.5



Ethanol



15.4



Chlorofo m



3.8



co.01



Heptane 3. Metabolism



Stambaugh et a1.6 found that the major urinary excretioA products of metronidazole in man and 0 - 1 mice were unchanged metronidazole, 1-(2-hyroxyethyl)-2-hydroxymethyl-5-nitroimidazole and their ether glucuronides



.



335



OaN3 OX3



-



336



Figure 5 DSC Thennogram of Metronidazole



2-



.c-+m



E W E R A T W E OC Figure 6



TGA of Metronidazole



------



LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY



These products represented 70-90% of the total urinary nitro fraction. Minor metabolites were reported to be 1-(2 -hydroxyethyl)- 2 -carboxylic acid-5-nitraimidazole and 1-acetic acid-2-methyl-5-nitroimidazole(see Fig. 7). Oxidation of the methyl group of metronidazole appears to occur more facilely than the hydroxyethyl group. Nitro reduction products have not been found in animal or human urine. The major vaginal products in females given oral doses of drug were found by Manthei et a1 to be unchanged drug, and 1-(2 -hydroxyethyl) - 2 -hydroxymethyl- 5-nitroimidazole. These products were also present in the urine. In addition a fluorescent lipophilic product thought to be a cyclized lactone was found. 4.



Phannacokinetics In a study on healthy females receiving single and multiple doses of 200 mg metronidazole tablets, Welling and Monro reported the biological half-life to be 6.2 hr. Serum concentration data fit a one compartment open model. Steady state serum concentrations of metronidazole on a regimen of 200 mg twice daily averaged 7.07 ug/ml maximum and 2.47 ug/ml m i n h . Ings et al,’ studied the distribution of p4C]jmetronidazole in rats. They found that oral doses were rapidly absorbed from the gastrointestinal tract; and rapidly equilibrated between blood and most tissues. Radioactivity was found to concentrate in the liver, kidney, gastro-intestinal tract and vaginal secretions. The half life of clearance was longest for the gastro-intestinal tract. I.V. doses showed similar distribution.



5.



Methods of Analysis 5.1 Titrimetric Analysis The titration with perchloric acid is the method of choice to assay metronidazole. The sample is dissolved in acetic anhydride, and warmed slightly to effect solution. After cooling, one drop of mlachite green T.S. is added, and the titration with 0.1 N perchloric acid to a yellow-green endpoint is carried out. A blank determination is 338



METRON IDAZOLE



Figure 7: Pathways proposed by Stambaugh et al. for the metabolism of metronidazole in man. (1) metronidazole (2) the corresponding ether glucuronide (R = glucuronide) (3) 1 - (2-hydroxyethy1)2-hydroxymethyl-5-nitroimidazole (4) its corresponding glucuronide (5) 1-acetic acid-2-methyl- S-nitroimidazole (6) 1-(2- hydroxyethyl) - 2-carboxylic acid-5-nitroimidazole



.



339



L O R R A I N E L. WEARLEY A N D G A Y L O R D D. ANTHONY



?ae



performed and any necessary correction is One equivalent of the compound is t i t r a t e d .



*



5.2 Spectrophotometric Analysis



Spectrophotometric analysis of metronidazole m y be carried out using 0 . 1 N sulfuric acid in methanol a s the solvent. The ultraviolet absorption maxima is a t about 274 nm. 5.3 Colorimetric Analysis 5.31 Metronidazole can be analyzed colorimetrically by reducing the n i t r o group t o t h e corresponding m i n e , which is subsequently determined by diazotization and coupling w i t h N- (l-naphthyl) - ethylenediamine dihydrochlor ide (Brat tonMarshall Reagent) .11



5.32 A variation of the above method involves the alkaline hydrolysis of the n i t r o group of metronidazole. The nitrous acid produced diazotizes sulfanilamide i n acidic m e d i u m t o form a diazonium s a l t . After coupling with Bratton-Marshall reagent the concentrat i o n is d e t e n i n e d spectrophotmetrically by canparison t o n i t r i t e standards which have been arried through the colorimetric procedure



15



5.4 Chromatographic Analysis 5.41 Thin Layer Chromatography - Several TLC systems and corre onding Rf values are smnarized below. Pi



340



METRONIDAZOLE



Solvent System



Absorbent Detection



E€



Acetone



Silica Gel



1



0.65



Ch1orofonn:Methanol: Silica Gel Water:Acetic Acid



1



0.76



1



0.36



1, 2



0.66



74:20:4:2



Benzene:methanol: ammonium hydroxide



Silica Gel



79:20:1



Ch1orofonn:methanol: Silica Gel water:acetic acid 70:24 :4: 2 1.



Spray Vith 1% aqueous titanium trichloride; heat at 130 C for 3 minutes. Spray with 1% Dimethylaminobenzaldehyde in 2 N HC1.



2.



Saturate plate with t-butyl hypochlorite vapors. Spray with aqueous 1% starch/l% potassium iodide solution. (This spray has been found to be much more sensitive than #1.) 5.42



Gas-Liquid Chromatography - Metronidazole can be chranatographed as the trimethyl silyl derivative. The silyl derivative is prepared by dissolving metronidazole in a 1:l mixture of dimethylfomide and bis (trimethylsilyl)trifluoroacetamide. The silylation reaction is compete in 30 minutes at room temperature.1 Instrumental Conditions Column: 6 ft. glass column packed with 3% OV-1 on Gas Chrom Q Column Temp.: 160' C Carrier: Nitrogen at 70 ml/min Detector: Hydrogen Flame Ionization Retention Time: 4 . 1 minutes



341



LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY



-0.6 V



Figure 8 Polarograph of Metronidazole, 2% Solution in pH 3.8f 0.2 Buffer



342



METRONIDAZOLE



5.5 Polarographic Analysis A suitable polarographic analysis of metronidazole may be carried out at a concentration of approximately 5 ug/ml in a 2% solution of pH 3 . 8 + 0.2 buffer. The scan shown in figure 8 was ohained on such a solution, using differential pulse mode, 3 electrode system. For less concentrated solutions addition of a maxima suppressant may be necessary. Peak maximuni value is approximately -0.23 volts vs saturated calomel ele~trode.~ 6.0 Synthesis



2-methyl-imidazole (I) is nitrated by reacting with nitric acid in the presence of sulfuric acid catalyst. The resulting 5-nitro product (11) can then be reacted with either chloroethanol or ethylene oxide to produce 1-(2-hydroxyethy1)-2methyl-5-nitroimidazole (111). See figure 9.



NwH3 CH2CH20H



’



CICH2CH20H or H 2 C ~ F H 2 0



m



Figure 9 Synthesis o f Metronidazole



343



LORRAINE L. WEARLEY AND GAYLORD D. ANTHONY



7.



References 1. Damascus, J., Searle Laboratories, personal conmumication. 2. Aranda, E., Searle Laboratories, personal cammicat ion. 3. Hribar, J., Searle Laboratories, personal conmumication. 4. Marshall, S., Searle Laboratories, personal cammication. 5. Aranda, E., Searle Laboratories, personal camnrnicat ion. 6. Stambaugh, J., Feo, L., Manthei, R., J, Phaxm. and Fxp. Therapeutics, Vol. 161, No. 2, 1968. 7. Manthei, R., Feo, L., Stambaugh, J., Wiadmosci Parazytologiczne T. XV, Nr. 3-4, 1969. 8. Welling, P., Monro, A., Arzneim - Forsch (drug Research) 22, Nr. 1 2 (1972). 9. Ings, R., McFadzian J., Onnerod, W., Xenobiotica, 1975, Vol. 5, No. 4, 223-235. 10. USP XIX, p. 327. 11. Bratton, A., Marshall, E., Babbitt, D., Herdrickson, A., J. Biol. Chm., 128, 537, (1939). 12. Lau, E., Yao, C., Lewis, M., Senkwski, B., J. Phaxm. Sci., 58, No. 1, 55, (1969). 13. Quirk, P., Chow,T., Searle Laboratories, personal cammication. 14. Wood, N., Chow, R., Searle Laboratories, personal commmication.



8.



Acknowledgments The authors wish t o express special thanks t o Dr. J. W i t t for the information on synthesis; t o Dr. J. Opperman for his assistance i n preparing the sections on metabolism and pharmacokinetics; to Mr. S. Marshall and Mrs. M. Gerry for generating and interpreting the data on TGA, DSC and Polarography; to Mr, J. Damascus and Dr. J. Hribar for the information on IR, W, and Mass Spectra; and t o Ms. J. Janik for her secretarial assistance i n preparing t h i s manuscript.



344



NITROFURANTOIN



Donald E. Cadwallader and Hung Won Jun



DONALD E. CADWALLADER AND HUNG WON JUN



T a b l e of Contents 1. Description N a m e , F o r m u l a , and Molecular Weight 1. 1 Appearance, C o l o r , Odor and T a s t e 1. 2 2. P h y s i c a l P r o p e r t i e s 2.1 Ultraviolet S p e c t r a 2.2 Infrared Spectrum 2 . 3 N u c l e a r Magnetic Resonance S p e c t r u m 2.4 Dissociation Constant 2.5 Melting Range 2.6 Crystal Properties 2.61 C r y s t a l Shape 2 . 6 2 C r y s t a l Size 2.63 Hydrates 2.7 Salts 2.8 Solubility 2.81 Solubility in Aqueous Media 2.82 Solubility in Organic Solvents 3 . Synthesis



4. Stability 4.1 4.2



Stability to Light and M e t a l Shelf-Life and S t o r a g e Conditions



5 . Metabolism



6 . Methods of Analysis 6.1 Identification 6.2 6. 3 6.4



Color Reaction T e s t E l e m e n t a l Analysis Chromatographic Systems 6.41 Thin L a y e r Chromatography 6.42 P a p e r Chromatography 6 . 4 3 Column Chromatography



346



NITROFURANTOIN



T a b l e of Contents, continued.



6.5



Quantitative Analysis 6.51 A s s a y of Dosage F o r m s 6 . 5 2 Quantitative D e t e r m i n a t i o n in Biological S a m p l e s



7. B i o p h a r m a c e u t i c s and P h a r m a c o k i n e t i c s 7.1 Absorption



7.2 7. 3



7.4 7.5



Distribution Elimination Bioavailability Pharmacokinetics



8. R e f e r e n c e s



347



DONALD E. CADWALLADER AND HUNG WON JUN



I. Description 1. 1 N a m e , F o r m u l a , and M o l e c u l a r Weight Nitrofurantoin is N - ( 5 -Nitro-2-furfurylidene) -1aminohydantoin; I - ( 5- N i ro-2-furfuryliden a r e the hydantoin. F u r a d a n t i n and Macrodantin m o s t commonly u s e d t r a d e m a r k s ; 11 additional a r e listed in the M e r c k Index (1).



smino)



8



I



CH2‘gHgN4O5



,C



/ ‘ 0



Mol. w t . :



238.16



1.2 A p p e a r a n c e , C o l o r , Odor and T a s t e Nitrofurantoin i s d e s c r i b e d as lemon-yellow, o d o r l e s s c r y s t a l s o r fine powder, having a b i t t e r t a s t e (1,2).



2. P h y s i c a l P r o p e r t i e s 2.1 U l t r a v i o l e t S p e c t r a Values of E (170, l c m ) in w a t e r a t 367.5 and 265 n m a r e found to be 760 and 540, r e s p e c t i v e l y (3). T h e m o l a r extinction coefficients, at 367 and 265 n m a r e 13,100 and 17, 300, r e s p e c t i v e l y ( 4 ) .



e



340



NITROFURANTOIN



When t h e U V s p e c t r u m of nitrofurantoin i n 270 d i m e t h y l f o r m a m i d e ( D M F ) w a s s c a n n e d f r o m 260 to 400 n m , t w o maxima o c c u r r e d a t 265 and 367 n m . T h e s p e c t r u m shown in F i g u r e 1 w a s obtained f r o m a solution of 10.00 m g of n i t r o f u r a n t o i n / l i t e r of 270 D M F in w a t e r ( 3 ) . F i g u r e 2 shows the effect of pH on m a x i m a l w a v e length and E (170,I c m ) v a l u e s of n i t r o f u r a n t o i n ( 3 ) . K H 2 P 0 4 - NaOH and b o r i c acid - NaOH buffer s y s t e m s w e r e employed. T h e s p e c t r a l shift w a s n o t due to hydantoin r i n g - opening in the alkaline solution s i n c e the shift w a s r e v e r s i b l e upon reacidification ( 3 ) . 2.2 Infrared Spectrum T h e i n f r a r e d s p e c t r u m of nitrofurantoin (Norwich P h a r m a c a l Reference Standard Purity) a s a m i n e r a l oil m u l l is shown in F i g u r e 3. I n t e r p r e t a t i o n of the s p e c t r u m w a s m a d e by M i c h e l s ( 3 ) using C r o s s , Stevens and Watts ( 5 ) a s a r e f e r e n c e . Table I IR Band A s s i g n m e n t s f o r Nitrofurantoin Wavelength ( m i c r o n s ) 3.05 5.6-5.75 6.6-7.45 10.4 8.05



Assignment NH hydantoin C = 0 d, - n i t r o f u r a n 2,5-disubstituted furan asymmetrical C-0-C symmetrical C-0-C



9. a



349



DONALD E. CADWALLADER AND HUNG WON JUN



1.c



.e



W



0



6



z Q



m



K



0



m



m



a



4



2



0



NANOMETERS F i g u r e 1. U l t r a v i o l e t S p e c t r u m of Nitrofurantoin (Coleman 124) 350



NITROFURANTOIN



390



a z



r



380



-



370



-



360



5



6



7



8



I



I



9



10



F i g u r e 2. U l t r a v i o l e t A b s o r p t i o n of N i t r o f u r a n t o i n as a F u n c t i o n of pH



351



I



4000 3000



2000



1000



1500



900



800



100



h



FR



80



v



Lu



2 k v)



Z



2 fi



60 40



20 0



rcc 5 0



U



.rl



a



m



a cn



k



k



0



E5



k



a,



V



U



k



k



(d



a,



M



5



k



M



Figure 3 . Infrared Spectrum of Nitrofurantoin (Perkin Elmer 467)



700



NITROFURANTOIN



2. 3 N u c l e a r Magnetic R e s o n a n c e S p e c t r u m T h e n u c l e a r magnetic r e s o n a n c e s p e c t r u m f o r nit r ofurantoin ( N o rwic h P h a r m a c a l R e f e r e n c e S t a n d a r d P u r i t y ) in DMSO - d6 containing a t e t r a m e t h y l s i l a n e a s the i n t e r n a l r e f e r e n c e i s shown in F i g u r e 4 ( 3 ) . T h e s p e c t r a l a s s i g n m e n t s a r e presented in T a b l e I1 ( 3 ) . T a b l e I1 NMR S p e c t r a l A s s i g n m e n t s f r Nitr furantoin Band ( p p m , d )



No. of P r o t o n s



1 1 1



hydantoin CH2 f u r a n 3H f u r a n 4H -CH=Nhydantoin NH



-



solvent



2



Singlet 4.40 Doublet 7. 15 ( J = 4 ) Doublet 7 . 6 2 ( J = 4 ) Singlet 7 . 8 3 B r o a d Singlet 1 1 . 4 (exchangeable)



1



2.5



Assignment



2 . 4 Dissociation Constant M i c h e l s ( 3 ) d e t e r m i n e d the pKa of the f r e e a c i d to b e 7 . 0 using t h e method d e s c r i b e d by Stockton and Johnson ( 6 ) . A pKa of 7 . 2 i s a l s o r e p o r t e d f o r n i t r o furantoin ( 1). 2 . 5 Melting Range Nitrofurantoin m e l t s a t 270-272'



353



C.



(1,7).



2.0 I



"



3.0 I



,



'



'



:



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Figure 4. NMR Spectrum of Nitrofurantoin (Varian A60A)



NITROFURANTOIN



2.6 Crystal Properties 2.61 C r y s t a l Shape C r y s t a l l i z a t i o n f r o m diluted a c e t i c a c i d yields needle-like c r y s t a l s (1). 2.62 C r y s t a l S i z e T h e c r y s t a l s i z e of n i t r o f u r a n t o i n h a s been found to affect the d e g r e e of emesis a n d the r a t e s of g a s t r o i n t e s t i n a l a b s o r p t i o n and u r i n a r y excretion following o r a l a d m i n i s t r a t i o n (8). T h e d i f f e r e n t c r y s t a l s i z e s of n i t r o f u r a n t o i n w e r e p r e p a r e d by recrystallization f r o m nitromethane. 2.63 Hydrates It h a s b e e n found that n i t r o f u r a n t o i n c a n e x i s t i n anhydrous and monohydrate f o r m ( 9 ) . Anhydrous n i t r o f u r a n t o i n and p r e v i o u s l y dried n i t r ofurantoin monohydrate b e c o m e h y d r a t e d only a t v e r y high humidity (>92% R . H. ). N i t r o f u r a n toin monohydrate d o e s not l o s e o r gain m o i s t u r e upon s t o r a g e a t v a r i o u s r e l a t i v e humidities i n the r a n g e of 31-9270 R.H. Monohydrate f o r m i s v e r y s t a b l e i n terms of retaining w a t e r a t 5OoC. 2. 7 S a l t s T h e s o d i u m s a l t of nitrofurantoin i s available and is u s e d t o p r e p a r e p a r e n t e r a l f o r m u l a t i o n s ( 1 0 ) . Aqueous solutions of the s a l t a r e v e r y u n s t a b l e .



355



DONALD E. CADWALLADER AND HUNG WON JUN



2 . 8 Solubility 2 . 8 1 Solubility in Aqueous Media Solubilities of nitrofurantoin in aqueous m e d i a have been r e p o r t e d f o r v a r i o u s t e m p e r a t u r e and pH conditions. T h e s e solubility values a r e p r e s e n t e d in T a b l e 111. Table I11 Solubilities of Nitrofurantoin i n Aqueous Media T e m p e r a t u r e , OC



24 24 24 25 30 37 37 37 37 37 45



Solubility ( m g / l ) References.



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76. 3 131.1 79.5 190 113.4 154 125 167.8 174. 1 312. 1 374 251.2



11 11 11 1,12 11 13 14 11 11 11 13 11



2 . 8 2 Solubility in Organic Solvents The solubilities of nitrofurantoin in v a r i o u s organic solvents a r e p r e s e n t e d in T a b l e IV.



356



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T a b l e IV Solubilities of Nitrofurantoin in Organic Solvents:% Solvent



Solubility ( m g / l )



Acetone



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189



70 %



712



9 570



5 10



600



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15,100 1,560



;::The solubility values w e r e r e p o r t e d i n R e f e r e n c e s ( 1 ) and (15). T h e t e m p e r a t u r e w a s not indicated.



357



DONALD E. CADWALLADER AND HUNG WON JUN



3.



Synthesis



Nitrofurantoin c a n b e p r e p a r e d by the r e a c t i o n of 1aminohydantoin a s the sulfate (16) and as the hydrochlor i d e (17) with 5 - n i t r o f u r f u r a l diacetate in isopropylalcohol-water media. Details of the production of n i t r o furantoin w e r e d e s c r i b e d by S a n d e r s -e t at. ( 1 8 ) . T h e r e a c t i o n s c h e m e is shown in F i g u r e 5. Nitrofurantoin w a s a l s o p r e p a r e d by t h e condensation of 1-aminohydantoin with 5-nitro-2-furaldehyde (19). Another method f o r the s y n t h e s i s of nitrofurantoin w a s d e s c r i b e d by J a c k (20). 4.



Stability 4. 1 Stability to Light and M e t a l Nitrofurantoin c r y s t a l s and its solutions a r e d i s colored by alkali and by e x p o s u r e to light, and a r e decomposed upon contact with m e t a l s o t h e r than s t a i n l e s s s t e e l and aluminum ( 2 ) . Since n i t r o f u r a n toin solutions a r e photosensitive, all analytical o p e r a tions m u s t b e conducted under subdued light. Nitrofurantoin solutions a r e a l s o e x t r e m e l y s e n s i t i v e to alkali; t h e r e f o r e , a l l g l a s s w a r e m u s t b e analytically clean and d r y f o r a s s a y p r o c e d u r e s . 4 . 2 Shelf-Life and S t o r a g e Conditions S t o r a g e conditions of nitrofurantoin and o r a l suspensions a r e r e c o m m e n d e d t o be in tight, lightr e s i s t a n t c o n t a i n e r s ( 2 ) . Recommended shelf-life of tablets and suspensions i s f i v e y e a r s when s t o r e d a t r o o m t e m p e r a t u r e and in r e g u l a r g l a s s c o n t a i n e r s (21).



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When the products a r e packaged in l i g h t - r e s i s t a n t c o n t a i n e r s and s t o r e d a t r o o m t e m p e r a t u r e , the d r u g showed negligible l o s s of potency o v e r a f i v e - y e a r period of t i m e . T h e nitrofurantoin products should not be s t o r e d w h e r e t e m p e r a t u r e s a r e expected to exceed 86'F.



5. Metabolism Reckendorf -e t a l . ( 2 2 ) r e p o r t e d that l a r g e amounts of nitrofurantoin (30 -50%) of a n o r a l l y and intravenously a d m i n i s t e r e d d o s e w e r e r e c o v e r e d intact in the u r i n e of r a t , dog and m a n . Beutner _ et -at. ( 2 3 ) a l s o found a s i m i l a r r e covery in urine after o r a l administration. T h e s e studies suggest that nitrofurantoin undergoes metabolic t r a n s f o r mation in t h e body to a significant extent. T h e possible metabolic pathways of nitrofurantoin a r e not completely elucidated i n t h e l i t e r a t u r e . However, nitrofurantoin would follow somewhat s i m i l a r pathways in m e t a b o l i s m to that f o r n i t r o f u r a z o n e which undergoes reduction in the n i t r o g r o u p and hydrolysis in the aaomethane linkage.



6. Methods of Analysis 6. 1 Identification Nitrofurantoin may b e identified by i t s melting point (270-272OC) and by m e a n s of i t s c h a r a c t e r i s t i c infrared spectra (see Figure 3 ) .



6. 2 Color Reaction T e s t A n u m b e r of c o l o r r e a c t i o n t e s t s f o r the identification of nitrofurantoin has b e e n p r e s e n t e d in a publication ( 2 4 ) .



360



NITROFURANTOIN



6. 3 Elemental A n a l y s i s T h e r e s u l t s of a n e l e m e n t a l a n a l y s i s of nitrofurantoin (Norwich P h a r m a c a l C o . , Lot. No. E3769) a r e presented in Table V (25). Table V E l e m e n t a l Analysis of Nitrofurantoin Element



6.4



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Chromatographic S y s t e m s 6.41 Thin Layer Chromatography T h e following T L C s y s t e m s have b e e n found suitable f o r detection of p o s s i b l e postulated impurities (26). Solvent S y s t e m I Acetone: 90



Glacial Acetic Acid: Methanol 5 5 (v/v)



361



DONALD E. CADWALLADER AND HUNG WON JUN



Rf = 0 . 8 1 on B r i n k m a n Silica Gel plate without f l u o r e s c e n t indicator as visualized by s h o r t wave u l t r a v i o l e t light. It w a s found that the l i n e a r dynamic range w a s 0.25-1.10 p g / s p o t . Solvent S y s t e m I1 Acetone: 80



Benzene: Glacial a c e t i c a c i d 20 1 (v/v)



Rf = 0 . 9 5 on Brinkman Silica Gel plate without f l u o r e s c e n t indicator as visualized by s h o r t - w a v e ultraviolet light and H2SO4 c h a r r i n g . Other T L C p r o c e d u r e s f o r the identification and s e p a r a t i o n of nitrofurantoin have been d e s cribed (27).



6.42 P a p e r Chromatography T h e u s e of paper chromatography i n the a n a l y s i s of nitrofurantoin and o t h e r n i t r o f u r a n compounds h a s been r e p o r t e d by B r e i n l i c h ( 2 8 ) , who examined s e v e r a l solvent s y s t e m s and methods f o r identification of the s p o t s .



6.43 Column Chromatography e t al. (29) u s e d column c h r o m a t o Bender -g r a p h i c p r o c e d u r e s to s e p a r a t e nitrofurantoin f r o m o t h e r components of u r i n e s a m p l e s and s u b sequently d e t e r m i n e d the d r u g concentrations by s p e c t r o p h o t o m e t r i c a s say.



362



NITROFURANTOIN



6 . 5 Quantitative Analysis 6.51 Assay of Dosage F o r m s The methods of analysis that a r e used € o r the determination of nitrofurantoin depend on i t s ultraviolet absorption c h a r a c t e r i s t i c s . The U. S. P. XIX ( 3 0 ) d e s c r i b e s a spectrophotometric determination of nitrofurantoin in an acidic 27'0 dimethylformamide -wate r solution. This g e n e r a l method i s applicable to nitrofurantoin in tablet, capsule and suspension dosage f o r m s w h e r e the excipients a r e U V non-absorbing. Under conditions w h e r e separations a r e not made, any drug degradation products a r e reasonably expected to be reflected in the value of the U V ratio; conv e r s e l y , constancy of the U V r a t i o a s compared to the initial formulation value can be taken a s evidence f o r stability (31). A polarographic ( 32) and gravimetric ( 3 3 ) determination of nitrofurantoin in tablets have been described.



6 . 5 2 Quantitative Determination in Biological Samples Paul -et al. (34) utilized the ultraviolet absorption of nitrofurantoin to determine drug concentrations in r a t urine followed by extraction with e t h e r , but the absorption maximum was pH dependent a s indicated by Stoll and c o - w o r k e r s (35).



363



DONALD E. CADWALLADER AND HUNG WON JUN



In 1965 Conklin and Hollifield (36) i n t r o duced the nitromethane-Hyamine p r o c e d u r e f o r the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e , and this method h a s been established a s the p r e f e r r e d a s s a y f o r nitrofurantoin in biological s a m p l e s . T h e original p r o c e d u r e h a s been modified to a s s a y the d r u g in whole blood o r p l a s m a (37). T h i s p r o c e d u r e h a s a sensitivity of 2 r g / m l and i s specific f o r nitrofurantoin. However, this method w a s inadequate f o r quantitating nitrofurantoin blood levels in s u b j e c t s who r e ceived n o r m a l therapeutic d o s e s . Mattock, McGilveray and C h a r e t t e (38) improved the n i t r o methane-Hyamine method f o r the determination of nitrofurantoin in blood s a m p l e s s o that s m a l l e r volumes a r e r e q u i r e d ( 0 . 8 ml) and the sensitivity i s g r e a t e r ( 0 . 2 u g / m l ) . A modified nitromethaneHyamine method w a s d e s c r i b e d by Hollifield and Conklin (39) f o r the a s s a y of nitrofurantoin in u r i n e i n the p r e s e n c e of phenazopyridine and i t s metabolites



.



e t at. ( 4 0 ) developed a c o l o r i m e t r i c Buzard -method f o r the a n a l y s i s of nitrofurantoin in plasma o r s e r u m of the r a t a f t e r v a r i o u s o r a l d o s e s . J o n e s e t at. (41) d e s c r i b e d a polarographic method f o r the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e . T h e sensitivity l i m i t of a s s a y i s l p g / m l . S e v e r a l a u t h o r s (42, 43) have r e p o r t e d polarog r a p h i c p r o c e d u r e s f o r the a s s a y of the drug.



364



NITROFURANTOIN



A microbiological p r o c e d u r e f o r the a s s a y of nitrofurantoin in u r i n e w a s a l s o d e s c r i b e d by J o n e s e t a l . ( 4 1 ) . Gang and Shaikh (44) u s e d a n indicator o r g a n i s m i n a t u r b i d i m e t r i c method f o r the a s s a y of nitrofurantoin and o t h e r n i t r o f u r a n d e r i v a t i v e s in s e r u m and u r i n e s a m p l e s .



--



A column c h r o m a t o g r a p h i c method f o r the d e t e r m i n a t i o n of nitrofurantoin i n u r i n e w a s r e ported by Bender e t a l . ( 2 9 ) . Stone (45) and P u g l i s i (46) have m e a s u r e d a t r a c e of nitrofurantoin i n m i l k by the c o l o r i m e t r i c and s p e c t r o pho t o m e t r i c methods , r e s p e c tive 1y Both p r o c e d u r e s a r e b a s e d on t h e conversion of nitrofurantoin to 5 -nitrofurfuraldehyde phenylhyd r a z o n e and a r e followed by the e x t r a c t i o n and concentration on a c h r o m a t o g r a p h i c column. F i n a l d e t e r m i n a t i o n s depend on the development of a blue c o l o r by the addition of Hyamine base.



.



7. Biopharmaceutics and P h a r m a c o k i n e t i c s 7 . 1 Absorption Nitrofurantoin i s efficiently and rapidly a b s o r b e d a f t e r o r a l a d m i n i s t r a t i o n ( 4 7 ) . Limited d r u g a b s o r p tion o c c u r s when nitrofurantoin is a d m i n i s t e r e d r e c t a l l y (48). T h e f i r s t evidence that a b s o r p t i o n and e x c r e t i o n w e r e affected by d i f f e r e n c e s in p a r t i c l e s i z e of the d r u g w a s published in 1967 ( 8 ) . T h i s paper r e p o r t e d on the r e l a t i o n s h i p of p a r t i c l e s i z e of nitrofurantoin to emesis in dogs and to absorption and u r i n a r y



365



DONALD E. CADWALLADER AND HUNG WON JUN



excretion in man and r a t s . It was found that l a r g e r c r y s t a l s of the drug caused l e s s e m e s i s in dogs and slower absorption in man and r a t s . Thus, i t was concluded that the u s e of l a r g e c r y s t a l s of nitrofurantoin ( M a c r o d a n t i n e could minimize a d v e r s e effects of this drug such a s nausea and vomiting by slowing the r a t e of absorption in the gastrointestinal tract. Nitrofurantoin occasionally causes nausea, vomiting, drowsiness, headache and skin r a s h e s (49). Incidence of these reactions may be reduced to some degree by administration of the drug with food (50,51). Bates and co-workers (52) studied the effect of food on the absorption of nitrofurantoin f r o m c o m m e r cial dosage f o r m s . They found considerably i n c r e a s e d absorption in nonfasting a s compared to fasting subjects.



7 . 2 Distribution After absorption into the blood circulation, n i t r o furantoin is rapidly distributed into most body fluids (53 ) . During n o r m a l o r a l therapeutic regimen, blood o r plasma levels of the drug a r e usually v e r y low, in the neighborhood of 1 p g / m l . However, nitrofurantoin levels about 3-5 t i m e s g r e a t e r than this a r e usually found in these fluids when drug i s administ e r ed intravenous Iy o r intramuscularly. Detailed studies of the absorption and distribution of nitrofurantoin have been c a r r i e d out by Buzard e t al. (54) in small animals.



366



NITROFURANTOIN



7. 3 Elimination The biological half-life of nitrofurantoin in man a p p e a r s to b e 30 min. o r l e s s (55, 56, 57, 22). In clinical studies in human subjects, u r i n a r y drug concentration of 200 to 400 m g / l i t e r have been reported (50, 58). Renal excretion involves glomerular f i l t r a tion and active tubular secretion. S c h i r m e i s t e r -et a t . (59)found that the clearance of nitrofurantoin in human subjects was lower in acid urine than in alkaline urine. During renal impairment, nitrofurantoin u r i n a r y excretion was significantly reduced (60). Conklin and Wagner (61)and Conklin -e t al. (62) reported that a s much a s 20% of the intravenous dose of nitrofurantoin sodium was excreted in hepatic bile in the dog.



7.4 Bioavailability Bioavailability of nitrofurantoin has received a considerable attention in recent y e a r s . Cadwallader (63)presented a monograph on the bioavailability of nitrofurantoin in the special APhA Bioavailability pilot project. The general c h a r a c t e r i s t i c s and experimental c r i t e r i a f o r bioavailability testing of nitrofurantoin w e r e discussed. More recently, Meyer and co-workers (64)evaluated the bioavailabilities of 14 commercially available nitrofurantoin p r e p a r a tions by in vivo and -in vitro procedures. They found that some products that m e t the official Compendia1 requirement, w e r e l e s s bioavailable than other p r o ducts tested. E a r l i e r studies by various authors



367



DONALD E. CADWALLADER AND HUNG WON JUN



(65, 66, 67) a l s o r e p o r t e d bioavailability p r o b l e m s a s s o c i a t e d with the u s e of c o m m e r c i a l n i t r o f u r a n t o i n tablets. An updated monograph on the bioavailability of nitrofurantoin w a s r e c e n t l y p r e s e n t e d by Cadwallader (68). 7. 5 P h a r m a c o k i n e t i c s In m a n , p l a s m a levels of nitrofurantoin a p p e a r t o decline exponentially with a half-life v a l u e of about 20 to 30 m i n u t e s (56, 57, 22). U r i n a r y e x c r e t i o n and b i o t r a n s f o r m a t i o n a p p e a r to b e mainly a n d equally r e s p o n s i b l e f o r the elimination of nitrofurantoin. The o n e - c o m p a r t m e n t m o d e l a p p e a r s to be adequate f o r d e s c r i b i n g the k i n e t i c s involved in nitrofurantoin absorption and elimination.



8. R e f e r e n c e s T h e M e r c k Index, 8 t h e d . , M e r c k and C o . , I n c . , Rahway, N . J . , 1968, p. 738. 2. T h e United S t a t e s P h a r m a c o p e i a , 19th e d . , M a c k Publishing C o . , E a s t o n , Pennsylvania 1975, p. 341. 3. J. G. M i c h e l s , Norwich P h a r m a c a l C o . , P e r s o n a l Communication. 4. V. E g e r t s , J. S t r a d i n s a n d M. S h i m a n s k a , "Analysis of 5 - n i t r o f u r a n D e r i v a t i v e s , t r a n s l a t e d by J. S c h m a r a k , Ann A r b o r S c i e n c e P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 83. 5. A. H. J. C r o s s , S. G. E. S t e v e n s , a n d T . H. E. W a t t s , J . Appl. C h e m . , London, 1,562 (1957). 6. J. Stockton and C. R. Johnson, J. A m , P h a r m . A s s o c . , Sci. E d . , 33, 383 (1944). 7. T h e N i t r o f u r a n s , Vol. 1, "Introduction to the N i t r o f u r a n s , Eaton L a b o r a t o r i e s , Norwich, N. Y. 1958, p. 16. 8 . H. E. P a u l , K . J . H a y e s , M . F. P a u l and A . R. Borgmann, J. P h a r m . S c i . , 882 (1967). 1.



56,



368



NITROFURANTOIN



9. 10. 1 1.



12.



13.



14. 15.



16.



17. 18. 19. 20. 21. 22. 23.



B. G. P a t e l , Norwich P h a r m a c a l C o . , P e r s o n a l Communication. F u r a d a n t i n a Sodium P a r e n t e r a l , Eaton L a b o r a t o r i e s , Norwich, N. Y. L. K. Chen, M.S. T h e s i s , U n i v e r s i t y of G e o r g i a , 1975. H. E. P a u l and M. F. P a u l i n E x p e r i m e n t a l C h e m o t h e r a p y , vol. 11. R. J. S c h n i t z e r and F. Hawking, e d s . , A c a d e m i c P r e s s , New Y o r k , N . Y . , 1964, p. 334. T . R. B a t e s , J . M. Young, C . M. Wu a n d H. A. R o s e n b e r g , J. P h a r m . S c i . , 63, 643 (1974); T . R. B a t e s , SUNY a t Buffalo, P e r s o n a l Communication. M. F. P a u l , R . C. B e n d e r , and E. G. Nohle, A m e r . J . P h y s i o l . , 197,580 (1959). T h e N i t r o f u r a n s , vol. 1, "Introduction to the N i t r o f u r a n s , I ' Eaton L a b o r a t o r i e s , Norwich, N. Y . , 1958, pp. 14-15. A . S w i r s k a , J . L a n g e , and Z . Buczkowski, P r z e m y s l . C h e m . , 11 ( 3 4 ) , 306-308 (1965). Through C. A . , 52, 14079 b (1958). C. J. O'Keefe, Norwich P h a r m a c a l G o . , P e r s o n a 1 C ommunication , H. J. S a n d e r s , R. T . E d m u n d s , and W. B. Stillman, Ind. Enp. C h e m . , 47, 358 (1955). K. H a y e s , U. S. P a t e n t , 2,610, 181 (1952). D. J a c k , J. P h a r m . P h a r m a c o l . , 11,Suppl., 108 T (1959). J. F. S t a r k , Norwich P h a r m a c a l C o . , P e r s o n a l C ommuni ca ti on. H. K. Reckendorf, R . C a s t r i n g i u s , a n d H . Spingler, Med. Welt, 816 (1963). E. H. B e u t n e r , J. J . P e t r o n i o , H. E. Lind, H. M. T r a f t o n , and M. C o r r e i a - B r a n c o , Antibiotics Ann., 1954-1955, 988 (1955).



15,



369



DONALD E. CADWALLADER AND HUNG WON JUN



24.



25. 26. 27.



28. 29. 30.



31. 32.



33. 34.



35. 36.



V. E g e r t s , J. S t r a d i n s , and M. Shimanska, I'Analysis of 5-Nitrofuran D e r i v a t i v e s , I ' t r a n s lated by J. S c h m a r a k , Ann A r b o r Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, p. 18. Atlantic M i c r o l a b , Inc. , Atlanta, Georgia. M. J. Cardone, Norwich P h a r m a c a l Co., P e r s o n a l Communication. V. E g e r t s , J. S t r a d i n s , and M. Shimanska, "Analysis of 5-Nitrofuran D e r i v a t i v e s , t r a n s lated by J . S c h m a r a k , Ann A r b o r Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, pp. 126127. J. Breinlich, Dtsch. Apotheker. Z t g . , 104, 535(1964). R . C . B e n d e r , E . G. Nohle, and M. F . P a u l , Clin. C h e m . , 2, 420 (1956). T h e United S t a t e s P h a r m a c o p e i a , 19th e d . , Mack Publishing Co. , Easton, Pennsylvania, 1975, p. 342. R. A. Boice, Norwich P h a r m a c a l C o . , P e r s o n a l Communication. V. E g e r t s , J. S t r a d i n s , and M. Shimanska, "Analysis of 5-Nitrofuran D e r i v a t i v e s , ' I t r a n s lated by J. S c h m a r a k , Ann A r b o r Science P u b l i s h e r s , Ann A r b o r , Michigan, 1970, pp. 7374. Ibid. , pp. 39-40. H. E. P a u l , F. L. Austin, M. F. P a u l and V. R . E l l s , J. Biol. C h e m . , 180,345 (1949). R . G. Stoll, T. R . B a t e s and J. S w a r b r i c k , J. P h a r m . S c i . , 62, 65 (1973). J . D. Conklin and R . D. Hollifield, Clin. C h e m . , 11, 925 (1965). -



370



NITROFURANTOIN



37.



J . D. Conklin and R. D. Hollifield,



w.,3,



690 (1966). 38. 39.



G. L. Mattock, I. J . McGilveray, and C. Charette, ibid., 820 (1970). R. D. Hollifield and J. D. Conklin, ibid.,



16,



16,



335 (1970). 40. 41. 42. 43. 44.



J. A . B u z a r d , D. M. V r a b l i c , and M. F. P a u l , Antibiotics and Chemotherapy, 6, 702 (1958). B. M. J o n e s , R. J . M. Ratcliffe and S. G. E. S t e v e n s , J. P h a r m . P h a r m a c o l . , 17,525 (1965). T . S a s a k i , P h a r m . Bull. (Tokyo), 2, 104 (1954). H. P. M o o r e and C . R. G u e r t a l , J. A s s o c . Offic. Agr. C h e m i s t s . , 43, 308 (1960). D. M. Gang and K. Z. Shaikh, J. P h a r m . S c i . ,



61,



462 (1972).



45.



L. R. Stone, J. A s s o c . Offic. A g r . C h e m i s t s , 44,



46.



E. P u g l i s i , J. A s s o c . Offic. A g r . C h e m i s t s ,



2 (1961).



44,



30 (1961). 47. 48. 49.



J. 1. J. G.



D. Conklin, R. J. S o b e r s , and D. L. Wagner, P h a r m . S c i . , 58, 1365 (1969). D. Conklin, P h a r m a c o l o g y , S, 178 (1972). C a r r o l l and R . V. Brennan, J. U r o l . , 71,650



( 1954).



50.



51. 52.



P. F. MacLeod, G. S. R o g e r s , and B. R . Anylowar, Intern. R e c o r d Med. and Gen. P r a c t . C l i n . , 169, 5 6 1 (1956). -A. G. S m i t h , J . A m . Med. A S S O C . , 195,1061 (1966). T . R. B a t e s , J. A. S e q u e i r a , and A. V. T e m b o , Clin. P h a r m a c o l . T h e r a p . , 63 (1974).



16,



371



DONALD E. CADWALLADER AND HUNG WON JUN



5 3.



M. F. P a u l , H. E . P a u l , R . C. B e n d e r , F. Kopko, C. M. H a r r i n g t o n , V. R . E l l s , and J. A. B u z a r d , Antibiotics and C h e m o t h e r a p y , 10,287 (1960).



54.



J . A. B u z a r d , J . D . Conklin, E. O'Keefe and M. F. P a u l , J . P h a r m a c o l . Exptl. T h e r a p . 131,



55.



W. M. Bennett, I. S i n g e r , and C. H. Coggins, J. Am. Med. A s s o c . , 214, 1468 (1970). V . D. Kobvletzki. Med. W e l t . .. 19. 2010 (19681. . . , C. M. Kunin, Ann. I n t e r n a l Med. , 1 5 1 (1967). W. A . R i c h a r d , E. R i s s , E. H. K a s s a n d M . Finland, A.M.A. Arch. Internal Med.,



38 (1961).



56. 57. 58.



-



- I



.



I



67, 96,



I



4 3 7 (1955). 59.



60.



61.



J . S c h i r m e i s t e r , F. Stefani, H. Willmann, and W. H a l l o u e r , Antimicrobiol. Agents and C h e m o t h e r a p y , 1965, p. 223. J. S a c h s , T . G e e r , P. Noell, and C. M. Kunin, 1032 (1968). New Engl. J. M e d . , 9 , J . D. Conklin and D. L. Wagner, J. P h a r m a c o l . ,



43, 140 (1971). 62. 63.



64. 65.



J . D. Conklin and R . J. S o b e r s and D . L. W a g n e r , B r . J. P h a r m a c o l o g y , 48, 2 7 3 (1973). D. E. C a d w a l l a d e r , "Nitrofurantoin, I f T h e APhA Bioavailability P i l o t P r o j e c t , A m e r i c a n P h a r m a c e u t i c a l Association, Washington, D. C. J u l y 1973 M. C. M e y e r , G. W . A. Slywka, R. E. Dann and P. L . Whyatt, J . P h a r m . S c i . , 63, 1693 (1974). I. J. M c G i l v e r a y , G. L. Mattok, a n d R . D. H o s s i e , J. P h a r m . P h a r m a c o l . , 23, 246 S ( 1 9 7 1 ) .



372



NITROFUR ANT0 IN



66. G. L. Mattok, R . D. H o s s i e , and I. J . McGilveray 67. 68.



Can. J. P h a r m . S c i . , 1,8 4 (1972). I. J. McGilveray, G. L. Mattok, and R . D. Hossie Rev. Can. Biol. 32, 99 (1973). D. E. Cadwallader, J. Am. P h a r m . A S S O C . , NS15, 413 (1975).



T h e a u t h o r s w i s h to thank D r . John S t a r k and o t h e r p e r s o n n e l at the Norwich P h a r m a c a l Company f o r t h e i r valuable a s s i s t a n c e and support. T h e excellent secretarial support of M r s . Kay W. Oliver i s acknowledged.



373



PIPERAZINE ESTRONE SULFATE



Zui L. Chang



ZUI



L. CHANG



Contents Analytical Profile



-



Piperazine Estrone Sulfate



1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, color, Odor 1.3 Elemental Composition 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Raman Spectrum 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Thermal Analysis 2.9 Solubility 2.10 Crystal Properties 2.11 Dissociation Constant 2.12 Fluorescence 2.13 Hygroscopic Behavior 2.14 Sublimation 3.



Synthesis



4. Stability 5.



-



Degradation



Drug Metabolic Products and Pharmacokinetics



6. Method of Analysis



6.1 Identification 6.2 Chromatographic Analysis 6.21 Thin-Layer Chromatography 6.22 Gas-Liquid Chromatography 6.3 Spectrophotometric Analysis 6.4 Colorimetric Analysis 6.5 Nitrogen Analysis 6.6 Titration



376



PlPERAZlNE ESTRONE SULFATE



7.



Acknowledgements



8.



References



377



ZUI L. CHANG



1. Description 1.1 Name, Formula, Molecular Weight Piperazine estrone sulfate is estra-1,3,5 (10)trien-l7-one, 3-(su1fooxy)-, compound with piperazine (1:1). y 2



0



HO-SO'



II



0



Piperazine Estrone Sulfate Molecular Weight 436.56



c18H2205 s' C4H10N2



1.2 Appearance, Color, Odor Piperazine estrone sulfate is a white to yellowish white, fine crystalline powder. It is odorless. 1.3 Elemental Composition C-60.53; H-7.39; N-6.42; 0-18.32; S-7.34. 2. Physical Properties 2.1 Infrared Spectrum The infrared spectrum of piperazine estrone sulfate is presented in Figure 1. The spectrum was measured in the solid state as a potassium bromide dispersion. The following bands (cm-1) have been assigned for Figure 1.3 a.



3400-2300 cm-l broad complex of bands due mainly to N-H stretching vibrations of the amine salt. 378



FIGURE 1



- INFRARED



S P E C T R U M O F PIPERAZINE E S T R O N E SULFATE WAVELENGTH (MICRONS)



2.5



3



4



5



2 500



2000



6



7



8



9



10



12



15



0 -4 (D



4000



3500



3000



FREQUENCY (CM-1)



1500



1000



700



ZUI L. CHANG



b.



1730 cm-l



characteristic C=O stretching vibration of the 17-keto group.



c.



1600 and 1490 cm-I characteristic skeletal stretching vibrations of the aromatic ring.



d.



1040 cm-l



due to S-0 linkage



2.2 Nuclear Magnetic Resonance Spectrum (NMR) The nuclear magnetic resonance spectrum of piperazine estrone sulfate as shown in Figure 2 was obtained on a Varian HA-100 NMR Spectrometer in deuterated dimethylsulfoxide (d6) containing tetramethylsilane as the internal standard. The spectral peak assignments4 are presented in Table I. 2.3 Ultraviolet Spectrum (W) When the W spectrum of 0.1% solution of piperazine estrone sulfate in 0.04% sodium hydroxide solution was scanned from 400 to 210 nm, two maxima and two minima were observed (Figure 3).l The maxima are at 275 nm ( c = 838) and 268 nm ( c = 851). The minima occur at 272 nm and 239 nm. The spectrum was obtained with a Cary Model 14 Recording Spectrophotometer. 2.4 Mass Spectrum The mass spectrum shown in Figure 4 was obtained using an Associated Electrical Industries Model MS-902 Mass Spectrometer with an ionizing energy of 70 ‘eV. The mass spectrum of piperazine estrone sulfate indicates the presence of estrone, piperazine, and a sulfur-oxygen constituent, but it does not yield a molecular ion f o r the complete chemical entity. This is attributed to the following behavior: (a) Dissociation of the amine salt into the free amine (piperazine) and the free acid (estrone hydrogen sulfate). (b) Possible thermal decomposition of estrone hydrogen sulfate to estrone, S02, OH’, etc.



380



FIGURE 2 - NUCLEAR MAGNETIC RESONANCE SPECTRUM OF PIPERAZINE ESTRONE SULFATE



ZUI L. CHANG



Table I NMR Spectral Assignments for Piperazine



Estrone Sulfate Proton Assignment



Chemical Shift (ppm)



Mu 1 tip 1i c i ty



H



fl



7.17



doublet J=9. 5Hz



6.92



Mu1 t ipl e t



5.5



Broad



2.89



Singlet



0



QIH 0



H



382



PIPERAZINE ESTRONE SULFATE



Table I Cont. Proton Assignment



Chemi c a 1 Shift (ppm)



2



C -CH,



-



0.84



383



3.0



Mu1 tip 1ic i ty



Comp 1ex



Singlet



ZUI L. CHANG



-



0.8



0.7



-



0.6 =



I



FIGURE 3 - ULTRAVIOLET SPECT SPECTRUM RUM OF PIPERAZINE ESTRONE SULFATE SULFATE



\ 0.5



0.4



0.3



0.2



0.1



0



2 00



300



250



WAVELENGTH (nm)



384



350



FIGURE 4 - MASS SPECTRUM OF PIPERAZINE ESTRONE SULFATE



I '



ZUI L. CHANG



The mass spectrum assignments of the prominent ions and subsequent fragments are shown in Table I1 and Figure 5.5 2.5 Raman Spectrum The Raman spectrum of piperazine estrone sulfate a s shown in Figure 6; was obtained in the solid state on a Cary Model 83 Spectrometer. The following bands (cm-l) have been assigned for Figure 6.3 due to the C=O stretching of the 17-keto group.



a.



1733 ane1



b.



1608 cm-l due to skeletal stretching mode of the aromatic ring.



c.



1050 cm-l due to s-0 linkage



2.6 Optical Rotation A 1% solution of piperazine estrone sulfate in 0.4% sodium hydroxide solution exhibited a rotation of [cu]~~ + 87.8" when determined on a Perkin-Elmer Model 141 Po1ar imeter . 6 2.7 Melting Range Piperazine estrone sulfate melts at about 190°C to a light brown, viscous liquid which re-solidifies, on further heatin and finally melts at about 245°C with decomposition.



f3



2.8 Differential Thermal Analysis (DTA) The DTA curve obtained on a Dupont Model 900 Analyzer as shown in Figure 7 confirms the observed melting characteristics described in section 2.7. 2.9 Solubility Approximate solubility data obtained at room temperature are given in the following table:'s8



386



PIPERAZINE ESTRONE SULFATE



Table I1 High Resolution Mass Spectrum of Piperazine Estrone Sulfate Found Mass



Calculated Mass



5 C H N O -



270.1620



270.1620



18



22



0



2



0



213.1287



213.1279



15



17



0



1



0



185.0960



185.0966



13



13



0



1



0



172.0887



172.0888



12



12



0



1



0



146.0725



146.0732



10



LO



0



1



0



86.0843



86.0844



4



10



2



0



0



85.0771



85.0766



4



9



2



0



0



63.9618



63.9619



0



0



0



2



1



47.9669



47.9670



0



0



0



1



1



387



ZUI L. CHANG



FIGURE 5



- FRAGMENTATION PATHWAYS OF PIPERAZINE ESTRONE SULFATE



y '& H



I



HO-S-0 O II



W



II



0



0



II



H



I



HO-S-0



bN>1+*



...I-



--



[



H



O



W



I



m / a 350 not observed



1



m/a 270



L



J



m/a 64



[ so]+'



[..m]+' m/a 172



I



[ H o r n ] " 388



m/a 146



FIGURE 6 2000



- RAMAN SPECTRUM OF PIPERAZINE ESTRONE SULFATE



1800



1600



1400



1200



loo0



800



600



400



200



I



I



I



I



I



I



I



I



I



0



80 -



-



60 -



- 60



80



- 40 - 20 0 2000



I



1



I



1



I



I



I



1



I



1800



1600



1400



1200



lo00



800



600



400



200



RAMAN SHIFT ACM-’



0



0



FIGURE 7



-



DIFFERENTIAL THERMAL ANALYSIS CURVE OF PIPERAZINE ESTRONE SULFATE



0



X



Ly



A -



s



w



(D



0



I-



d '



v -



0 n



t



1



I



I



I



I



I



I



I



I



I



0



50



100



150



200



250



300



3 50



400



450



T " C (CORRECTED FOR CHROME1 ALUMEL THERMOCOUPLES)



PlPERAZlNE ESTRONE SULFATE



Solubility (mg/ml) 8



Solvent Water 95% Ethanol



7.4



Chloroform



1



Ether



Practically insoluble



Acetone



0.2



Benzene



Practically insoluble



Methylene dichloride



1.6



Isopropanol



0.1



Practically insoluble



Sodium hydroxide



57



Propylene glycol



35



Mineral oil



2



Sesame oil



2



2.10 Crystal Properties The X-ray powder diffraction pattern of piperazine estrone sulfate was determined by visual observation of a film obtained with a 1 4 3 . 2 mm Debye-Scherrer Powder Camera (Table 111). An Enraf-Nonius Diffractis 601 Generator; 38 KV and 18 MA with nickel filtered copper radiation; ), = 1 . 5 4 1 8 , were employed.9 2.11 Dissociation Constant The apparent pKa value of the unprotonated piperazine nitrogen (proton gained) was found to be 3 . 6 , by titration in acetonitrile-water ( 8 0 1 2 0 , v/v) with aqueous sodium hydroxide. Attempts to find systems to extrapolate the pKa to 100% water were unsuccessful. The pKa value of the protonated piperazine nitrogen (proton lost) was found to be 9 . 7 by titration in pyridine-water mixtures with methanolic KOH, and extrapolation to 100% water. 8



391



ZUI L. CHANG



Table 111 X-Ray Powder Diffraction Pattern d-Spacings and Intensities



dA



I/I



dA -



16.5



10



2.98



2



7.7



10



2.92



2



7.4



40



2.86



5



6.6



40



2.73



10



6.0



20



2.53



5



5.67



30



2.46



8



5.23



40



2.37



1



4.55B



20



2.32



5



4.38



80



2. 28



5



4.22



60



2.21



1



4.05



5



2.16B



5



3.86



30



2.11



1



3.77



100



2.07B



8



3.61



5



1.95



2



3.54



2



1.89



2



3.4013



20



1.85B



3



3.22



5



1.75B



5



3.05



1



1.70



3



392



if1



__.



PlPERAZlNE ESTRONE SULFATE



2.12 Fluorescence Piperazine estrone sulfate does not exhibit fluorescent properties in either methanol or in an alkaline aqueous solution. However, it does exhibit fluorescence at 488 nmwhen excited at 465 nm in 65% sulfuric acid solution.6 The strong sulfuric acid converts the piperazine estrone sulfate to estrone which reacts with sulfuric acid to yield the fluorescent species. 2.13 Hygroscopic Behavior Piperazine estrone sulfate was not hygroscopic when e posed to a relative humidity of 40-50% for three weeks.3 Piperazine estrone sulfate does not absorb moisture at 79% relative humidity, and is only very slight1y hygroscopic (0.89%) at 100% relative humidity. 7 2.14 Sublimation Piperazine estrone sulfate did not sublime when it was stored at 105'C for one month.8 No evidence of sublimation was noted when it was heated with a hot stage to 204°c.7 3.



Synthesis



Piperazipg estrone sulfate was first prepared by Hasbrouck in 1951. The compound can also be prepared from estrone by a fast, complete conversion reaction using a dimethylformamide/ sulfur troxide complex as the sulfating reactant. Excess dimethylformamide is the solvent. Th reaction is completed by the addition of piperazine.'11 4.



Stability-Degradation Piperazine estrone sulfate was found to be stable when refluxed in water for 3 hours. But it degrades completely after 3 hours in refluxing 1 3 hydrochloric acid to yield estrone and piperazine sulfate. It degrades slightly in refluxing 1 sodium hydroxide after 3 hours to yield less than 10% free estrone.12



393



ZUI L. CHANG



Piperazine estrone sulfate yields about 10% free estrone when heated at 105OC for one month. The rate of hydrolysis of piperazine estrone sulfate to estrone at 90°C was studied over the pH range of 2.5-9.1. The extent of degradation was determined by a spectrophotometric measurement in 0.1 1 sodium hydroxide solution.13 The hydrolysis of piperazine estrone sulfate to estrone follows first order kinetics with respect to the piperazine estrone sulfate concentration remaining. Futh rmore, the degradation is first order with respect to t' 2 hydrogen ion concentration, resulting in a 10-fold rate increase with each pH unit decrease.13 Some rate cons ants at different pH and temperature values are shown in I? oles IV and V. The activation energy of the degradatit n reaction, Ea (obtained from the slope of the plot of 11 K, as a function of 1/T where T is the absolute temp rature), for three pH levels is shown in Table V. 5.



Drug Metabolic Products and Pharmacokinetics The drug substance is hydrolyzed to estrone in acidic media. The known metabolic intercon ersions of the estrone are summarized in Figure 8.1Z Purdy's15 work suggests that estrone, as the sulfate conjugate, is an important transport form of estrone in human plasma. Urinary excretion studies of sodium estrone sulfate have been performed by Twombly and Levitz16, and Brown. 17 Biliary excretion was also studied by Twombly and Levitz.16 A very exhaustive study of urinary and biliary metabolites was described by Jirku and Levitz. 18



Quantitative determination of plasma estrogens by radioimmmunoassay has been developed by Vega. l9



394



PlPERAZlNE ESTRONE S U L F A T E



Table IV First Order Rate Constants of Piperazine Estrone Sulfate as a Function of pH at 90°C



kl -



Initial Concentration mg. /ml.



No. of Samp1e s



2.50



0.3



8



1,830 + - 250



3.03



0.3



8



480 + - 25



3.07



1.5



12



396 + - 16



3.40



1.5



13



3.57



0.3



7



+130 + -



3.88



1.5



15



59.l



4.26



1.5



15



22.4 + - 1.5



4.86



1.5



15



5.16



1.5



15



5.98



1.5



13



6.11



1.5



14



+ 3.63 + 1.85 + 1.12 +



6.48



1.5



15



.68 + - .30



6.88



1.5



14



.72 + - .17



7.50



1.5



13



.42 + - .12



7.90



1.5



15



+ .11 .14 -



8.43



1.5



13



22 + .08



9.10



1.5



12



.18+ - .11



pH



395



95% Conf. Lim. X lo4, hour



201



7.51



-1



14 16 2.9



1.03 .68 .77 .44



ZUI L. CHANG



Table V F i r s t Order Rate C o n s t a n t s and A c t i v a t i o n E n e r g i e s of P i p e r a z i n e E s t r o n e S u l f a t e a t Three pH l e v e l s



pH



kl x



lo4, 90°C. 8OOC. 7OOC.



2.50



pH



3.03



3.57



pH



hr.-’ 1,830 695 262



+



z+



-



250 31 15



480 +_ 25 150 + 4.6 52.2 7 - 2.2



130 44.8 14.6



+ f



16 4.0 2.3



Arrhenius R e l a t i o n s h i p Linear C o r r e l a t i o n Coeff. A c t i v a t i o n Energy, Ea



1.000 24,100



396



.999 27,500



1.000 27,100



Ly



c 4



4



3



Y



v)



Ly



v)



z 0 2! c



Ly



Ly



z



4



-3



A



c 4



-



&



FIGURE 8



METABOLIC PATHWAYS OF PIPERAZINE ESTERONE SULFATE



HO



HO



HO



2 -h yd rox yertrone



\ 178 -errradio1



I 0



OH



397 W



(0



-J



7



HO estriol



Q E



2



L



P)



U



-



X 0



f



Ef



F(



2-methoxyestrone



P HO



M a -h y drox y estrone (also 168-)



HO 15a-hydroxyestradiol



16-epiestriol



ZUI L. CHANG



6. Methods of Analysis 6.1 Identification The presence of piperazine may be identified with or thin-layer chromatography (Section quinone T. S. 6.21). The presence of estrone hydrogen sulfate may be identified by thin-layer chromatography (Section 6.21).



Idied or



The presence of free estrone may be ident with a 2.5% solution of @-naphthol in sulfuric acid thin-layer chromatography (Section 6.21). 6.2 Chromatographic Analysis



6.21 Thin-Layer Chromatography A number of thin-layer chromatographic systems on silica gel have been described for the separation of hydrolysis products of i erazine estrone sulfate from the parent substance. 1 9 6 2f 9 $2 piper azine estrone sulfate chromatographs as the piperazine and estrone sulfate moieties. Various systems,methods of detection and Rf valves are summarized in Table VI. 9



6.22 Gas-Liquid Chromatography Piperazine estrone sulfate can not be directly chromatographed. However, the principal degradation product, estrone, may be silylated with BSA and chromatographed using 3% QF-1 on Gas Chrom Qf2, or it may be silylated with a silylating mixture containing N-trimethylsilylimidazole, BSA and trimethylchorosilane in the ratio 1:30:bf, and chromatographed using 10% SE-30 on Chrom0 sorb W AW .



-



6.3 Spectrophotometric Analysis Direct spectrophotometric analysis of piperazine estrone sulfate is applicable provided significant quantities of interferring contaminents are not present. The drug substance may be examined directly in a methanol solution at 269 nm ( 6 = 860) or in 0.1 N sodium hydroxide solution.6



398



Table VI TLC Systems f o r P i p e r a z i n e E s t r o n e S u l f a t e



(D 0



Piperazine



Rf Estrone Sulfate



A r senomolybda t e spray



Origin



0.64



0.82



6



n-Propanol: Ethanol : Conc. Ammonia (2:1:2)



S h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol



0.42



0.78



0.92



21



Chlorof o m : Methanol (3:1 )



S h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol



0.03



0.35



0.72



21



n-Butanol: Water: Conc. Ammonia (1: 1: 1)



s h o r t wave W o r 10% S u l f u r i c Acid i n Ethanol



Chloroform: Methanol: S t r o n g e r Ammonia TS (85:15: 1 )



I o d i n e Vapor



Rf



Solvent System



Detection



Chloroform: Methanol (5:4)



Rf Estrone



RefePence



(0



ZUI L. CHANG



The estrone content of piperazine estrone sulfate can be determined with a suitable spectrophotometer at 238 nm after the conversion of piperazine estrone sulfate to estrone with the use of hydrochloric acid.l The degradation product, estrone,may be quantitated in the drug substance by a liquid-liquid extraction into chloroform and comparison to an estrone reference standard at 280 nm. Alternately, the chloroform extract may be evaporated and the residue dissolved in 65% suifuric acid solution. The estrone is dehydrated to a species which fluorescence at 488 nm with excitation at 465 nm.6 6.4



Colorimetric Analysis Piperazine estrone sulfate may be determined as dehydrated estrone using a phenol-sulfuric acid mixture as the col r development reagent, The chromophore absorbs at 522 nm.40 Piperazine estrone sulfate may also be determined using 65% sulfuric acid as the reagent. The reaction pro-6 duct with sulfuric acid has a yellow chromophore at 4 5 0 nm. These colorimetric methods may be used for the analysis of piperazine estrone sulfate in tablets or creams. The primary degradation product, estrone, can be removed by prior chloroform extraction. 6.5



Nitrogen Analysis The amount of nitrogen present in piperazine estrone sulfate may be determined by converting the sulfuric nitroqgn to ammonia and titrating with 0.05 acid. 6.6



Titration Piperazine estrone sulfate may be potentiometrically titrated in pyridine-water mixtures using methanolic potassium hydroxide and glass-calomel electrodes.8 7.



Acknowledgements



The author wishes to thank Mr. V. E. Papendick and Mr. J. A. Raihle for their review of the manuscript, and Miss Sandra Hudson for typing and drawing of the manuscript. 400



ZUI



L. CHANG



8. References 1.



The National Formulary, 14th Revision, Mack Publishing Co., Easton, PA (1974).



2.



Chemical Abstracts Service Registry No. 7280-37-7.



3. Washburn, W., Abbott Laboratories, Personal Communication.



4. Cirovic, M., Abbott Laboratories, Personal Comunicat ion.



5.



Mueller, S., Abbott Laboratories, Personal Communication.



6. Chang, 2. L., Abbott Laboratories, unpublished work.



7. Yunker, M., Abbott Laboratories, Personal Communication. 8. Wimer, D. C., Abbott Laboratories, Personal Communicat ion.



9. Quick, J., Abbott Laboratories, Personal Communication. 10. Hasbrouck, R. B., Assignor to Abbott Laboratories, U. S. Patent 2,642,427. 11. Lex, C. G., Assignor to Abbott Laboratories, U. S. Patent 3,525,738. 12. Williamson, D. E., Abbott Laboratories, Personal communication. 13. Borodkin, S., Abbott Laboratories, Personal Communication. 14. Kohn, F. E . , Abbott Laboratories, Personal Communication. 15.



Purdy, R. H., Engel, L. L., and Oncley, J. L., J. Biol. Chem., 236, 1043, (1961). 401



ZUI L. CHANG



16.



Twombly, G H., and Levitz, M., Am. J. Obstet. and Gynec., 80, 889, (1960).



17. Brown, J. B., J. Obstet, and Gynec. Brit. Emp., 66, 795, (1969). 18. Jirku, H. and Levitz, M., J. Clin. Endocr., 615, (1969).



2,



19. Vega., S. M., Abbott Laboratories, Personal Communication. 20. Abbott Laboratories, J. Am. Med. Assoc., 149 (5), 443, (1952). 21. Birch, R. A., Dale, B. J. and Earl, J. M., Abbott Laboratories, Personal Communication. 22. Luebke, D. R., Abbott Laboratories, Personal Communication.



402



PROCARBAZINE HYDROCHLORIDE



Richard J. Rucki



RICHARD J. RUCK1



INDEX Analytical Prof il e



-



Procarbazine H y d r o c h l o r i d e



1.



Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor



2.



Phys i ca 1 Proper t ies 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 U l t r a v i o l e t Spectrum 2.4 F1uorescence Spectrum 2.5 Mass Spectrum 2.6 O p t i c a l R o t a t i o n 2.7 M e l t i n g Range 2.8 D i f f e r e n t i a1 Scanning C a l o r i m e t r y 2.9 Thermogravi met r ic Anal ys i s 2.10 Solubi 1 i t y 2.11 C r y s t a l P r o p e r t i e s 2.12 D i s s o c i a t i o n Constant



3.



Synthesis



4.



S t a b i 1 i t y and Degradation



5.



Drug Metabolic Products



6.



Toxi c i t y



7.



Methods o f A n a l y s i s 7.1 Elemental A n a l y s i s 7.2 Thin-Layer Chromatographic A n a l y s i s 7.3 D i r e c t Spectrophotometric A n a l y s i s 7.4 Coulometric A n a l y s i s 7.5 Pol arog raph ic Ana 1ys i s 7.6 T i t r i r n e t r i c A n a l y s i s



a.



Acknowledgements



9.



References



404



PROCARBAZINE HYDROCHLORIDE



1.



Description



1.1



Name, Formula, Molecular Weight P roca r baz i ne hyd roch 1o r i de i s N- i sop ropy 1-a(2-methylhydrazino)-~-toluamid~h y d r o c h l o r i d e .



Procarbazi ne H y d r o c h l o r i d e C12H19N30-HCl 1.2



Molecular Weight: 257.76



Appearance, Color, Odor White t o p a l e ye1 low c r y s t a l 1 i n e powder w i t h a s l i g h t c h a r a c t e r i s t i c odor.



2.



Phys ic a l P r o p e r t i e s



2.1



I n f r a r e d Spectrum ( 1 R ) The i n f r a r e d spectrum o f procarbazine hydroc h l o r i d e i s presented i n F i g u r e 1 ( 1 ) . The instrument used was a Perkin-Elmer Model 621 G r a t i n g Spectrophotometer. The sample was dispersed i n FluorolubeR t o r e c o r d t h e spectrum i n t h e r e g i o n o f 4000-1340 cm’l and i n m i n e r a l o i l f o r the r e g i o n o f 1340-400 cm-l. The f o l l o w i n g assignments have been made f o r the bands i n F i g u r e 1 ( 1 ) . Band (cm-’)



Assignment



3277 and 3200 3035 2961 and 2853 2760-2300, main band a t 2725



NH s t r e t c h Aromatic CH s t r e t c h A 1 i p h a t i c CH s t r e t c h NH:



405



FIGURE 1 I n f r a r e d Spectrum of Procarbazine Hydrochloride



2.5



3



WAVELENGTH (MICRONS) 6 7 8



5



2500



2000



9



10



12 14



18



22



3550



33NWlllWSNVW %



406



4



4000



3500



3000



1700



1400



FREQUENCY (CM-’1



I loo



800



500



200



PROCARBAZINE HYDROCHLORIDE



1660 and 1636



1556



1299



1361 and 1351



857 2.2



C=O stretch (Amide I ) Amide I t Amide I l l



Aromatic CH out-ofp 1 ane bend i ng



Nuclear Magnetic Resonance Spectrum (NMR) The N M R spectrum shown in Figure 2 was obtained by dissolving 50.4 mg o f procarbazine hydrochloride in 0.5 ml of DMSO-d6 containing tetramethylsilane as internal reference. The spectral assignments are shown in Table 1 (2).



a



Table 1



Protons



Chemi ca 1 Shift 6 (ppm) 1.20 2.72



4.18 4.23



7.55 7.99 8.36 6.33-



10.33



Mu1 tip1 ici ty Doub 1 et Singlet Mu1 ti p let Sing 1 et Doub 1 e t Doublet Doublet Singlet (broad) 407



Coup 1 i ng Constant , J (in Hz)



6.0



--



6.0



--



8.5



8.5 7.0



--



FIGURE 2



N M R Spectrum o f Procarbazine Hydrochloride



408



P



0



0)



1



1



1



1



1



1



6



5



4



3 PPM (8)



2



1



0



PROCARBAZINE HYDROCHLORIDE



2.3



U I t r a v i o l e t Spectrum (UV) The u l t r a v i o l e t spectrum o f procarbazine hydroc h l o r i d e i n the r e g i o n o f 350 t o 200 nm i s shown i n F i g u r e 3. ( 3 ) . The s p e c t r u q e x h i b i t s one maximum a t 232 nm ( E = 1.3 x 10 ) and a m i n i mum a t 213 nm. The s o l u t i o n c o n c e n t r a t i o n was 0.01 mg/ml i n 0 . 1 N h y d r o c h l o r i c a c i d and t h e q u a r t z c e l l width-was 1 cm.



2.4



FI uorescence Spectrum E x c i t a t i o n and emission scans were c a r r i e d o u t f o r procarbazine h y d r o c h l o r i d e i n s o l u t i o n s o f 0 . 1 N h y d r o c h l o r i c a c i d , 0.1N sodium hydroxi d e a n 7 water. No fluorescence, however, was observed under these c o n d i t i o n s (2).



2.5



Mass Spectrum The low r e s o l u t i o n mass spectrum shown i n F i g u r e 4 was o b t a i n e d u s i n g a Varian MAT CH5 mass spectrometer, i n t e r f a c e d w i t h a V a r i a n d a t a system, w i t h an i o n i z i n g energy o f 70 eV ( 4 ) . The d a t a system accepted t h e o u t p u t o f t h e spectrometer, c a l c u l a t e d t h e masses, compared t h e i r i n t e n s i t i e s t o the base peak and p l o t t e d t h i s i n f o r m a t i o n as a s e r i e s o f l i n e s whose h e i g h t s were p r o p o r t i o n a l t o t h e intensities. The molecular i o n o f t h e f r e e base was observed a t m/e 221. The H C l moiety was observed a t m/e 36. The ions a t m/e 191, 177 and 163 c o r respond t o a loss from t h e f r e e base o f NHCH 3’ NHNHCH , and NHCH(CH ) r e s p e c t i v e l y . The ions a ? m/e 149 and 3j$’correspond t o t h e loss o f C H by M c L a f f e r t y rearrangement from m/e I91 an9 177, r e s p e c t i v e l y . The base peak a t m/e 118 r e s u l t s from t h e l o s s o f NHNHCH f r o m m/e



3



163.



A h i g h r e s o l u t i o n scan confirmed t h e r e s u l t s of t h e low r e s o l u t i o n spectrum. Table I I l i s t s t h e elemental compositions f o r t h e i o n s as



409



RICHARD J. RUCK1



FIGURE



3



U l t r a v i o l e t Spectrum o f Procarbazi ne Hydrochlor i de



-



0.5



0.4 -



w



V



z a



8



0.3-



$ m a



1



200



I



I



250 300 NANOMETERS



410



I



35c



FIGURE



4



Mass Spectrum o f Procarbazine Hydrochloride 100



L



90



411



A l l S N 31N I 3A I1 V 138



80



70 60



50 40



30



20 10



0 1 1



I



50



I



I



I



I



I



100



I



I



I



I



Iio m/e



I



I



I



I



I



200



I



I



I



I



2 0



RICHARD J. RUCK1



determined by h i g h r e s o l u t i o n mass spectroscopy (4). Table I I High R e s o l u t i o n Mass Spectrum o f Procarbazine H y d r o c h l o r i d e



-N



Found Mass



Calcd. Mass



C



118.0428 135.0698 149.0753 163.0870 177.1170 191.1288 221.1533



118.0419 135.0684 149.07 1 5 163.0872



8



6



0



8



9



1



9



8



9 11



2 2



177.11 54 191. I 3 1 1



11



15



12



17



1 1



221.1529



12



19



3



2.6



H



Optical Rotation Procarbazine h y d r o c h l o r i d e e x h i b i t s no o p t i c a l a c t i v i t y (3).



2.7



Me1 t i ng Range According t o USP X I X , procarbazine hydroc h l o r i d e m e l t s a t about 223"C, w i t h decornp o s i t i o n (5).



2.8



D i f f e r e n t i a l Scanning C a l o r i m e t r y (DSC)



DSC s p e c t r a f o r procarbazine h y d r o c h l o r i d e a t a scan r a t e o f 10°C/min. e x h i b i t e d an endotherm a t about 23OoC, where m e l t i n g was accompanied by sample decomposition. The endotherm was f o l l o w e d immediately by a slow exotherm (con t inu i n g decompos it ion). The observed temperature o f t h e melting/decomposit i o n endotherm i s dependent upon i n s t r u m e n t a l c o n d i t i o n s and, t h e r e f o r e , i s n o t c h a r a c t e r i s t i c o f t h e compound. 2.9



Thermogravimetric A n a l y s i s (TGA)



A thermogravimetric a n a l y s i s performed on 412



PROCARBAZINE HYDROCHLORIDE



procarbazine h y d r o c h l o r i d e e x h i b i t e d n o l o s s o f w e i g h t from 30-150"C. A t about 150"C, decomposition weight losses began and cont i n u e d t o 500°C (upper l i m i t o f i n s t r u m e n t ) . Solubi 1 i t y



2.10



Approximate s o l u b i l i t y data o b t a i n e d a f t e r 3 hours a t 25°C a r e g i v e n i n Table I l l (6,7). Table I l l S o l u b i l i t y o f Procarbazine H y d r o c h l o r i d e Solubi 1 i t y (mg/ml)



Solvent Water 95% Ethanol Absolute Ethanol Methanol Ace tone Diethyl Ether Petroleum E t h e r (30-60") C h 1o r o f o r m Benzene 3A A l c o h o l l s o p r o p y l Alcohol Cyclohexane Ethyl Acetate D ioxane Acetonitri l e Carbon Tet rach l o r i d e Methylene C h l o r i d e 2.11



200 27



8 59 4.5