{"@context":{"@language":"en","Affiliation":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","AggregatedSourceRepository":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","Campus":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","Creator":"http:\/\/purl.org\/dc\/terms\/creator","DateAvailable":"http:\/\/purl.org\/dc\/terms\/issued","DateIssued":"http:\/\/purl.org\/dc\/terms\/issued","Degree":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","DegreeGrantor":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","Description":"http:\/\/purl.org\/dc\/terms\/description","DigitalResourceOriginalRecord":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","FullText":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","Genre":"http:\/\/www.europeana.eu\/schemas\/edm\/hasType","IsShownAt":"http:\/\/www.europeana.eu\/schemas\/edm\/isShownAt","Language":"http:\/\/purl.org\/dc\/terms\/language","Program":"https:\/\/open.library.ubc.ca\/terms#degreeDiscipline","Provider":"http:\/\/www.europeana.eu\/schemas\/edm\/provider","Publisher":"http:\/\/purl.org\/dc\/terms\/publisher","Rights":"http:\/\/purl.org\/dc\/terms\/rights","ScholarlyLevel":"https:\/\/open.library.ubc.ca\/terms#scholarLevel","Title":"http:\/\/purl.org\/dc\/terms\/title","Type":"http:\/\/purl.org\/dc\/terms\/type","URI":"https:\/\/open.library.ubc.ca\/terms#identifierURI","SortDate":"http:\/\/purl.org\/dc\/terms\/date"},"Affiliation":[{"@value":"Pharmaceutical Sciences, Faculty of","@language":"en"}],"AggregatedSourceRepository":[{"@value":"DSpace","@language":"en"}],"Campus":[{"@value":"UBCV","@language":"en"}],"Creator":[{"@value":"Borel, Anthony Gerard Francis","@language":"en"}],"DateAvailable":[{"@value":"2010-10-04T21:57:50Z","@language":"en"}],"DateIssued":[{"@value":"1990","@language":"en"}],"Degree":[{"@value":"Master of Science - MSc","@language":"en"}],"DegreeGrantor":[{"@value":"University of British Columbia","@language":"en"}],"Description":[{"@value":"Clobazam (CLBZ) is a 1,5-benzodiazepine with potent anticonvulsant activity. The metabolism of this drug was investigated in the rat and in vitro with the aid of stable isotope-labelled analogues and gas chromatography-mass spectroscopy (GCMS).\r\nPentadeuteriophenyl CLBZ [\u00b2H\u2085]CLBZ was synthesized in essentially quantitative isotopic purity, and characterized by \u00b9H-NMR and GCMS. Of the five steps involved in the synthesis of [\u00b2H\u2085]CLBZ, the most susceptible to deuterium exchange was the nucleophilic substitution of 2,4-dichloronitrobenzene by aniline-d\u2087 to form N-(5-chloro-2-nitrophenyl)pentadeuteriophenylamine 18. In this step, the isotopic impurity aniline-2,3,4,5,-d\u2085 introduced protons from nitrogen into the ortho and para positions of the deuteriophenyl ring of 18.\r\nPhenol and catechol metabolites of CLBZ and N-desmethylclobazam (DMC) were synthesized according to the method used for [\u00b2H\u2085]CLBZ using 4-methoxyaniline and 3,4-dimethoxyaniline as starting materials. The methyl ether protecting group was well-suited for the synthetic procedure and was cleanly removed under mild conditions with BBr\u2083 to afford the phenols and catechols in good yield. The 0-methylated catechols of CLBZ and DMC were enzymatically synthesized from the catechol analogues of CLBZ and DMC using rat liver cytosol as a source of catechol O-methyltransferase (COMT) and S-adenosyl-L-methionine as the methyl donor. A meta\/para O-methylation ratio of 2 was obtained from the CLBZ catechol.\r\nCondensation of formaldehyde with DMC catalyzed by K\u2082CO\u2083 produced a compound whose GCMS (EI) spectral properties were consistent with the carbinolamide, however, this compound could not be isolated because of its\r\nmarginal stability. A major product for this reaction was 3-hydroxymethyl DMC which was characterized by \u00b9H-NMR and LCMS. Formaldehyde condensation catalyzed by KOH also afforded 3-hydroxymethyl DMC. Whether formaldehyde addition at the 3-position occurs as a kinetic product or whether it arises as a result of an equilibrium process subsequent to N-hydroxymethylation remains to be resolved.\r\nRats were administered CLBZ:[\u00b2H\u2085]CLBZ as an approximate 50:50 mixture. Isotope shifts detected by GCMS allowed the following CLBZ metabolites to be identified. In bile, decreasing levels of 4'-hydroxy CLBZ, 4'-hydroxy DMC, O-methylated CLBZ catechols, 4'-hydroxy-3'-methoxy DMC and 3',4'-dihydroxy CLBZ appeared as both glucuronide and sulfate conjugates. In urine decreasing levels of the following sulfate conjugates were observed: 4'-hydroxy-3'-methoxy CLBZ, 4'-hydroxy CLBZ, 4'-hydroxy DMC and 3',4'-dihydroxy CLBZ. In bile, the glucuronides were predominant over the sulfates, whereas in urine only sulfate conjugates were detected. There was no detectable evidence of unconjugated CLBZ metabolites in bile or urine. As the biliary glucuronide and urinary sulfate, 3'-hydroxy-4'-methoxy CLBZ constituted \u22642 % of the O-methylated CLBZ catechols, whereas in bile, the sulfate conjugate of this metabolite constituted 30 %. One possible explanation for the higher levels of this metabolite as a biliary sulfate could be the low regiospecific methylation of dihydroxy CLBZ by COMT combined with arylsulfatase activity present in the kidney. Neither the CLBZ carbinolamide nor the N-hydroxy DMC (hydroxamic acid) was present in detectable levels in the conjugated or unconjugated fractions of bile or urine.\r\nIsotope effects in the metabolism of CLBZ and DMC in the rat were marginal for phenyl ring oxidation as measured for phenol metabolites\r\n(kH\/kD =1.103 \u00b1 .090) and for catechols (kH\/kD. = 1.088 \u00b1 .207).\r\nThe metabolism of CLBZ and DMC in vitro was examined using control and phenobarbital-induced rat liver microsomes. CLBZ metabolism resulted in N-demethylation to DMC and phenyl ring hydroxylation at the 4'-position. There was no detectable evidence for the carbinolamide intermediate. Induction appeared to increase N-demethylation compared to phenyl ring hydroxylation. DMC was metabolized to 4'-hydroxy DMC with no detectable production of the hydroxamic acid.\r\nThe isotope effect associated with CLBZ N-demethylation was determined by incubating an approximate 50:50 mixture of pentadeuteriophenyl CLBZ:trideuteriomethyl CLBZ with control and phenobarbital-induced rat liver microsomes. In this way, the source of DMC derived from demethylation ([\u00b2H\u2085]DMC) and dedeuteriomethylation (DMC) could be determined. The values of kH\/kD, determined by protio\/deuterio product ratios, were calculated at 3.65 \u00b1 0.27 (n = 3) and 2.80 \u00b1 0.16 (n = 4) for control and induced microsomes respectively.","@language":"en"}],"DigitalResourceOriginalRecord":[{"@value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/28921?expand=metadata","@language":"en"}],"FullText":[{"@value":"THE METABOLIC PROFILING OF CLOBAZAM IN RATS by ANTHONY GERARD FRANCIS BOREL B.Sc. (Hons.)\/ U n i v e r s i t y of Guelph, 1985 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Fa c u l t y of Pharmaceutical Sciences D i v i s i o n of Pharmaceutical Chemistry We accept t h i s t h e s i s as conforming t o the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1990 (C) ANTHONY BOREL, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Faculty ;P p^ei)tNMiXC( of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada D a t e April 27, 1990 DE-6 (2\/88) i i ABSTRACT Clobazam (CLBZ) i s a 1 , 5 - b e n z o d i a z e p i n e w i t h p o t e n t a n t i c o n v u l s a n t a c t i v i t y . The m e t a b o l i s m o f t h i s d r u g was i n v e s t i g a t e d i n t h e r a t and in vitro w i t h t h e a i d o f s t a b l e i s o t o p e - l a b e l l e d a n a l o g u e s and gas chromatography-mass s p e c t r o s c o p y (GCMS). P e n t a d e u t e r i o p h e n y l CLBZ [ 2Hs]CLBZ was s y n t h e s i z e d i n e s s e n t i a l l y q u a n t i t a t i v e i s o t o p i c p u r i t y , and c h a r a c t e r i z e d by ^H-NMR and GCMS. Of t h e f i v e s t e p s i n v o l v e d i n t h e s y n t h e s i s o f [ 2Hs]CLBZ, t h e most s u s c e p t i b l e t o d e u t e r i u m exchange was t h e n u c l e o p h i l i c s u b s t i t u t i o n o f 2,4-d i c h l o r o n i t r o b e n z e n e by a n i l i n e - d 7 t o form N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l amine 18. In t h i s s t e p , t h e i s o t o p i c i m p u r i t y a n i l i n e - 2 , 3 , 4 , 5 , - d s i n t r o d u c e d p r o t o n s from n i t r o g e n i n t o t h e ortho and para p o s i t i o n s o f t h e d e u t e r i o p h e n y l r i n g o f 18. Phenol and c a t e c h o l m e t a b o l i t e s o f CLBZ and N-de s m e t h y l c l o b a z a m (DMC) were s y n t h e s i z e d a c c o r d i n g t o t h e method used f o r [ 2 Hs]CLBZ u s i n g 4-m e t h o x y a n i l i n e and 3 , 4 - d i m e t h o x y a n i l i n e as s t a r t i n g m a t e r i a l s . The methyl e t h e r p r o t e c t i n g group was w e l l - s u i t e d f o r t h e s y n t h e t i c p r o c e d u r e and was c l e a n l y removed under m i l d c o n d i t i o n s w i t h BBr3 t o a f f o r d t h e p h e n o l s and c a t e c h o l s i n good y i e l d . The O-methylated c a t e c h o l s o f CLBZ and DMC were e n z y m a t i c a l l y s y n t h e s i z e d from t h e c a t e c h o l a n a l o g u e s o f CLBZ and DMC u s i n g r a t l i v e r c y t o s o l as a s o u r c e o f c a t e c h o l 0 - m e t h y l t r a n s f e r a s e (COMT) and S-a d e n o s y l - L - m e t h i o n i n e as t h e methyl donor. A meta\/para 0 - m e t h y l a t i o n r a t i o o f 2 was o b t a i n e d from t h e CLBZ c a t e c h o l . C o n d e n s a t i o n o f fo r m a l d e h y d e w i t h DMC c a t a l y z e d by K2CO3 produced a compound whose GCMS ( E I ) s p e c t r a l p r o p e r t i e s were c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e , however, t h i s compound c o u l d n ot be i s o l a t e d because o f i t s i i i m a r g i n a l s t a b i l i t y . A major p r o d u c t f o r t h i s r e a c t i o n was 3-hydroxymethyl DMC w h i c h was c h a r a c t e r i z e d by ^-NMR and LCMS. Formaldehyde c o n d e n s a t i o n c a t a l y z e d by KOH a l s o a f f o r d e d 3-hydroxymethyl DMC. Whether f o r m a l d e h y d e a d d i t i o n a t t h e 3 - p o s i t i o n o c c u r s as a k i n e t i c p r o d u c t o r whether i t a r i s e s as a r e s u l t o f an e q u i l i b r i u m p r o c e s s subsequent t o N - h y d r o x y m e t h y l a t i o n r e m a i n s t o be r e s o l v e d . R a t s were a d m i n i s t e r e d CLBZ:[ 2H5]CLBZ as an a p p r o x i m a t e 50:50 m i x t u r e . I s o t o p e s h i f t s d e t e c t e d by GCMS a l l o w e d t h e f o l l o w i n g CLBZ m e t a b o l i t e s t o be i d e n t i f i e d . In b i l e , d e c r e a s i n g l e v e l s o f 4'-hydroxy CLBZ, 4'-hydroxy DMC, O-methylated CLBZ c a t e c h o l s , 4'-hydroxy-3'-methoxy DMC and 3 ' , 4 ' - d i h y d r o x y CLBZ appeared as both g l u c u r o n i d e and s u l f a t e c o n j u g a t e s . In u r i n e d e c r e a s i n g l e v e l s o f t h e f o l l o w i n g s u l f a t e c o n j u g a t e s were o b s e r v e d : 4'-hydroxy-3'-methoxy CLBZ, 4'-hydroxy CLBZ, 4'-hydroxy DMC and 3 ' , 4 ' - d i h y d r o x y CLBZ. In b i l e , t h e g l u c u r o n i d e s were predominant o v e r t h e s u l f a t e s , whereas i n u r i n e o n l y s u l f a t e c o n j u g a t e s were d e t e c t e d . There was no d e t e c t a b l e e v i d e n c e o f u n c o n j u g a t e d CLBZ m e t a b o l i t e s i n b i l e o r u r i n e . As t h e b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e , 3'-hydroxy-4'-methoxy CLBZ c o n s t i t u t e d <2 % o f t h e O-methylated CLBZ c a t e c h o l s , whereas i n b i l e , t h e s u l f a t e c o n j u g a t e o f t h i s m e t a b o l i t e c o n s t i t u t e d 30 %. One p o s s i b l e e x p l a n a t i o n f o r t h e h i g h e r l e v e l s o f t h i s m e t a b o l i t e as a b i l i a r y s u l f a t e c o u l d be t h e low r e g i o s p e c i f i c m e t h y l a t i o n o f d i h y d r o x y CLBZ by COMT combined w i t h a r y l s u l f a t a s e a c t i v i t y p r e s e n t i n t h e k i d n e y . N e i t h e r t h e CLBZ c a r b i n o l a m i d e n or t h e N-hydroxy DMC (h y d r o x a m i c a c i d ) was p r e s e n t i n d e t e c t a b l e l e v e l s i n t h e c o n j u g a t e d o r u n c o n j u g a t e d f r a c t i o n s o f b i l e o r u r i n e . I s o t o p e e f f e c t s i n t h e m e t a b o l i s m o f CLBZ and DMC i n t h e r a t were m a r g i n a l f o r phenyl r i n g o x i d a t i o n as measured f o r phenol m e t a b o l i t e s i v ( k H \/ k D =1.103 \u00b1 .090) and f o r c a t e c h o l s (kL|\/kn. = 1.088 \u00b1 .207). The m e t a b o l i s m o f CLBZ and DMC in vitro was examined u s i n g c o n t r o l and p h e n o b a r b i t a l - i n d u c e d r a t l i v e r microsomes. CLBZ m e t a b o l i s m r e s u l t e d i n N - d e m e t h y l a t i o n t o DMC and phenyl r i n g h y d r o x y l a t i o n a t t h e 4 ' - p o s i t i o n . There was no d e t e c t a b l e e v i d e n c e f o r t h e c a r b i n o l a m i d e i n t e r m e d i a t e . I n d u c t i o n appeared t o i n c r e a s e N - d e m e t h y l a t i o n compared t o phenyl r i n g h y d r o x y l a t i o n . DMC was m e t a b o l i z e d t o 4'-hydroxy DMC w i t h no d e t e c t a b l e p r o d u c t i o n o f t h e hydroxamic a c i d . The i s o t o p e e f f e c t a s s o c i a t e d w i t h CLBZ N - d e m e t h y l a t i o n was d e t e r m i n e d by i n c u b a t i n g an a p p r o x i m a t e 50:50 m i x t u r e o f p e n t a d e u t e r i o p h e n y l C L B Z : t r i d e u t e r i o m e t h y l CLBZ w i t h c o n t r o l and p h e n o b a r b i t a l - i n d u c e d r a t l i v e r microsomes. In t h i s way, t h e s o u r c e o f DMC d e r i v e d from d e m e t h y l a t i o n ([ 2H5]DMC) and d e d e u t e r i o m e t h y l a t i o n (DMC) c o u l d be d e t e r m i n e d . The v a l u e s o f ku| \/krj, d e t e r m i n e d by p r o t i o \/ d e u t e r i o p r o d u c t r a t i o s , were c a l c u l a t e d a t 3.65 \u00b1 0.27 (n = 3) and 2.80 \u00b1 0.16 (n = 4) f o r c o n t r o l and i n d u c e d microsomes r e s p e c t i v e l y . V TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF FIGURES v i i i LIST OF SCHEMES x i i i LIST OF TABLES xv LIST OF ABBREVIATIONS x v i ACKNOWLEDGEMENTS x i x DEDICATION xx I INTRODUCTION 1 1. THE MECHANISM OF ACTION OF CLOBAZAM 2 2. THE THERAPEUTIC STATUS OF CLOBAZAM 4 (A) A n t i c o n v u l s a n t E f f i c a c y o f CLBZ 4 (B) R e s t r i c t i o n s t o CLBZ Therapy 5 (C) N-desmethylclobazam: The A c t i v e CLBZ M e t a b o l i t e 6 (D) The P h a r m a c o k i n e t i c s o f CLBZ 7 (E) Drug I n t e r a c t i o n s w i t h CLBZ 8 (F) A n a l y t i c a l T e c h n i q u e s used i n The M o n i t o r i n g o f CLBZ and DMC 8 3. THE METABOLISM OF CLOBAZAM 9 (A) The N - d e m e t h y l a t i o n o f CLBZ 13 (B) The N - o x i d a t i o n o f DMC 17 (C) The A r o m a t i c H y d r o x y l a t i o n o f CLBZ 21 4. THE OBJECTIVES OF THE THESIS 25 I I EXPERIMENTAL 26 1. CHEMICALS AND MATERIALS 26 2. ANIMALS AND SURGICAL EQUIPMENT 28 v i 3. ACCESSORY EQUIPMENT 29 4. INSTRUMENTATION 29 (A) NMR S p e c t r o s c o p y 29 (B) Mass S p e c t r o s c o p y 30 (C) I n f r a r e d S p e c t r o s c o p y 33 (D) M e l t i n g P o i n t 33 (E) U l t r a v i o l e t S p e c t r o s c o p y 33 (F) High P r e s s u r e L i q u i d Chromatography 33 (G) L i q u i d Chromatography-Mass S p e c t r o s c o p y 34 5. METABOLISM EXPERIMENTS 34 (A) Dosage Regimen 34 (B) S u r g e r y and C o l l e c t i o n o f U r i n e and B i l e 35 (C) P r e p a r a t i o n o f B i l e and U r i n e f o r GCMS 36 (D) P r e p a r a t i o n and A n a l y s i s o f L i v e r F r a c t i o n s 36 (E) M i c r o s o m a l I n c u b a t i o n s 38 6. CHEMICAL SYNTHESES 40 (A) Medium p r e s s u r e l i q u i d ( F l a s h ) chromatography 40 (B) S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l c l o b a z a m ( [ 2 H 5 ] C L B Z ) 40 (C) S y n t h e s i s o f T r i d e u t e r i o m e t h y l c l o b a z a m ( [ 2 H 3 ] C L B Z ) 45 (D) S y n t h e s i s o f R i n g - O x i d i z e d M e t a b o l i t e s o f CLBZ 46 (E) S y n t h e s i s o f N-hydroxymethyl CLBZ ( c a r b i n o l a m i d e ) 56 (F) A t t e m p t e d S y n t h e s e s o f N-hydroxy DMC (hy d r o x a m i c a c i d ) 59 I I I RESULTS AND DISCUSSION 62 1. THE SYNTHESIS OF DEUTERATED ANALOGUES OF CLBZ AND CLBZ METABOLITES 62 (A) The S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l CLBZ 62 (B) The S y n t h e s i s o f T r i d e u t e r i o m e t h y l CLBZ 84 (C) The S y n t h e s i s o f Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ 86 (D) GCMS A n a l y s i s o f CLBZ and I t s M e t a b o l i t e s 91 (E) S y n t h e s i s o f N-hydroxymethyl CLBZ ( c a r b i n o l a m i d e ) 104 (F) A t t e m p t e d S y n t h e s e s o f N-hydroxy DMC (hy d r o x a m i c a c i d ) 123 2. THE METABOLISM OF CLBZ 127 v i i (A) Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ i n Rat B i l e and U r i n e 127 (B) M e t a b o l i c F o r m a t i o n o f The P u t a t i v e C a r b i n o l a m i d e 146 (C) M e t a b o l i c F o r m a t i o n o f The P u t a t i v e N-hydroxy DMC (h y d r o x a m i c a c i d ) 148 (D) The I n f l u e n c e o f Cytochrome P-450 I n d u c t i o n on CLBZ M e t a b o l i s m 149 (E) The D e u t e r i u m I s o t o p e E f f e c t a s s o c i a t e d w i t h CLBZ N-d e m e t h y l a t i o n 153 IV SUMMARY AND CONCLUSIONS 154 V REFERENCES 156 VI APPENDIX 174 v i i i LIST OF FIGURES F i g u r e 1. 400 MHz !H-NMR o f [ 2 H 5 ] C L B Z . 66 F i g u r e 2. E l e c t r o n impact mass s p e c t r a o f (A) CLBZ and (B) [ 2 H 5 ] C L B Z . 67 F i g u r e 3. 400 MHz !H-NMR s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a - d e u t e r i o p h e n y l a m i n e 18, and (B) N-(5-c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o - p h e n y l a m i n e 3 7 . 69 F i g u r e 4. E l e c t r o n impact mass s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18, and (B) N-(5-c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o p h e n y l a m i n e 3 7 . 70 F i g u r e 5. 75 MHz broad-band d e c o u p l e d ^C-NMR sp e c t r u m o f N-( 5 - c h l o r o - 2 - n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18. 71 F i g u r e 6. The f o r m a t i o n o f a 6-membered r i n g between t h e n i t r o g roup and amine p r o t o n o f t h e phenyl amine 18 r e s u l t s i n N-H d e s h i e l d i n g and d i m i n i s h e d D2O exchange. 74 F i g u r e 7. Thermal rearrangement o f t h e carbamoyl a c e t a t e 19 t o th e phenylamine 18 r e s u l t s i n i d e n t i c a l r e t e n t i o n t i m e s i n t h e TIC p l o t s o f (A) 19 and (B) 18. 75 F i g u r e 8. Thermal r e a r r a n g e m e n t o f t h e carbamoyl a c e t a t e 19 t o th e phenylamine 18 r e s u l t s i n i d e n t i c a l GC ( E I ) mass s p e c t r a o f (A) 19 and (B) 18. 76 F i g u r e 9. 300 MHz *H-NMR o f t h e carbamoyl a c e t a t e 19. 78 F i g u r e 10. The f o r m a t i o n o f r e s o n a n c e s p e c i e s o f 19 w h i c h r e d u c e t h e a c i d i t y o f t h e methylene p r o t o n s and so i n h i b i t k e t o - e n o l t a u t o m e r i s m . 79 F i g u r e 11. TIC p l o t s o f (A) [ 2H 5]DMC and (B) [ 2H 5]DMC-TMS show t h e improved chromatography o b t a i n e d w i t h TMS-d e r i v a t i z a t i o n . 82 F i g u r e 12. GCMS ( E I ) s p e c t r a o f (A) [ 2H 5]DMC and (B) [ 2H 5]DMC-i x TMS show t h e improvement i n mass s p e c t r o s c o p y o b t a i n e d w i t h T M S - d e r i v a t i z a t i o n . 83 F i g u r e 13. (A) 400 MHz !H-NMR and (B) GCMS ( E I ) s p e c t r a o f [ 2 H 3 ] C L B Z ( 2 2 ) . 85 F i g u r e 14. Mass chromatogram o f CLBZ 0-methyl c a t e c h o l i s o m e r s a t M +-, m\/z 418. 90 F i g u r e 15. TIC p l o t s o f (A) T M S - d e r i v a t i z e d m e t a b o l i t e s t a n d a r d s 26, 28, 32 and 35 a f t e r r e c o v e r y from s p i k e d c o n t r o l b i l e . (B) 1 5 - f o l d d i l u t i o n o f (A) w i t h EtOAc. 94 F i g u r e 16. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-DMC (26) showing t h e p r e s e n c e o f M + -a t m\/z 446. 95 F i g u r e 17. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-CLBZ (28) showing t h e p r e s e n c e o f M +* a t m\/z 388. 95 F i g u r e 18. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3',4'-dihydroxy-DMC (32) showing t h e p r e s e n c e o f M +* a t m\/z 534. 96 F i g u r e 19. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3',4'-d i h y d r o x y CLBZ (35) showing t h e p r e s e n c e o f M +' a t m\/z 476. 96 F i g u r e 20. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy-CLBZ (36a) showing t h e p r e s e n c e o f M+* a t m\/z 418. 97 F i g u r e 21. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3'-hydroxy-4'-methoxy-CLBZ (36b) showing t h e p r e s e n c e o f M +' a t m\/z 418. 97 F i g u r e 22. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) showing t h e p r e s e n c e o f M+-a t m\/z 476. 98 F i g u r e 23. HPLC o f c r u d e p r o d u c t , (A) f r e s h l y p r e p a r e d and X (B) a f t e r o v e r n i g h t s t o r a g e a t 4\u00b0C. 108 F i g u r e 24. (A) LCMS chromatogram o f t h e c r u d e p r o d u c t a f t e r o v e r n i g h t s t o r a g e a t 4 4C shows t h e appearance o f 45, 46 and 47 a t tR 4.68, 6.68 and 7.37 min r e s p e c t i v e l y . (B) LCMS ( C I ) spec t r u m o f 46 shows t h e appearance o f MH + and [M H - H 2 0 ] + a t m\/z 317 and 299 r e s p e c t i v e l y . (C) LCMS ( C I ) spec t r u m o f 47 (DMC) shows t h e appearance o f MH4 + a t m\/z 287 and 304 r e s p e c t i v e l y . 109 F i g u r e 25. (A) GCMS ( C o n d i t i o n , b*) TIC p l o t o f f r e s h l y p r e p a r e d , T M S - d e r i v a t i z e d c r u d e p r o d u c t . P u t a t i v e c a r b i n o l a m i d e - T M S appears a t tR 17.33 min. (B) GCMS ( E I ) mass spec t r u m o f a p p a r e n t c a r b i n o l a m i d e -TMS shows t h e p r e s e n c e o f M+- a t m\/z 388. 110 F i g u r e 26. (A) GCMS ( C o n d i t i o n , b*) TIC p l o t o f p u r i f i e d 46 d e r i v a t i z e d w i t h TMS. (B) GCMS ( E I ) spec t r u m o f 46 shows t h e p r e s e n c e o f M + > a t m\/z 370. 112 F i g u r e 27. 400 MHz ^-NMR spect r u m o f 47. S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e o f DMC. 115 F i g u r e 28. 400 MHz *H-NMR sp e c t r u m o f 46- S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e 3-hydroxymethyl DMC. 116 F i g u r e 29. P a r t i a l 400 MHz *H-NMR s p e c t r a o f (A) compound 40 and (B) compound 46 i n DMS0-d5 r e v e a l t h e o c c u r r e n c e o f m e t h i n e , methylene and h y d r o x y l p r o t o n s as (A) t r i p l e t s i n 40 and as (B) a t r i p l e t , d o u b l e t and broad s i n g l e t r e s p e c t i v e l y i n 46. 119 F i g u r e 30. P a r t i a l 400 MHz ^H-NMR s p e c t r a o f (A) compound 40 and (B) compound 46 i n DMSO-ds w i t h D2O show c h e m i c a l i d e n t i t y f o r t h e 3-hydroxymethyl f u n c t i o n . 120 F i g u r e 31. (A) GCMS ( C o n d i t i o n , d*) TIC p l o t o f p u r i f i e d 40. (B) GCMS ( E I ) spec t r u m o f 40 shows t h e p r e s e n c e o f M+- a t m\/z 370. 121 F i g u r e 32. 400 MHz ^-NMR o f t h e M o V I complex ( 4 2 ) . 125 F i g u r e 33. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h ^ - g l u c u r o n i d a s e . F i g u r e 34. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . F i g u r e 35. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f u r i n e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . F i g u r e 36. P r o f i l e o f c o n j u g a t e d CLBZ m e t a b o l i t e s i n t h e Sprague Dawley r a t d e r i v e d from i n d i v i d u a l l y p o o l e d b i l e and u r i n e samples. F i g u r e 37. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy CLBZ (28) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+- and [ 2H4]M +* a t m\/z 388 and 392 r e s p e c t i v e l y . F i g u r e 38. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy DMC (26) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2H4]M +- a t m\/z 446 and 450 r e s p e c t i v e l y . F i g u r e 39. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy CLBZ (36a) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+* and [ 2H3]M +- a t m\/z 418 and 421 r e s p e c t i v e l y . F i g u r e 40. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) i s o l a t e d f rom b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+* and [ 2 H 3 ] M + - a t m\/z 476 and 479 r e s p e c t i v e l y . F i g u r e 41. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 3 ' , 4 ' - d i h y d r o x y CLBZ (35) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+* and [ 2H3]M +- a t m\/z 476 and 479 r e s p e c t i v e l y . x i i F i g u r e 42. Mass chromatograms (m\/z 418) o f T M S - d e r i v a t i z e d EtOAc e x t r a c t s o f b i l e and u r i n e showing t h e p r e s e n c e o f 0 - m e t h y l a t e d c a t e c h o l s o f CLBZ (36a+b) as (A) a b i l i a r y g l u c u r o n i d e , (B) a b i l i a r y s u l f a t e and (C) a u r i n a r y s u l f a t e . 143 F i g u r e 43. Comparison o f peak a s s i g n m e n t s o f t h e CLBZ 0-m e t h y l a t e d i s o m e r s (36a+b) formed (A) i n v i t r o and (B) as a b i l i a r y g l u c u r o n i d e . 144 F i g u r e 44. Mass chromatograms a t (A) m\/z 418 and (B) m\/z 421 show t h a t t h e peaks o f t h e CLBZ 0 - m e t h y l a t e d c a t e c h o l s (36a+b) o c c u r r i n g as t h e b i l i a r y s u l f a t e s a r e i s o t o p i c a l l y r e l a t e d . 145 F i g u r e 45. (A) SDS-PAGE (Laemmli e t a l . , 1970), and (B) immunoblot (Towbin e t a l . , 1979) o f l i v e r m i c r o s o m a l p r o t e i n o f Sprague Dawley r a t s . F i g u r e 46. SIM p l o t s o f microsomal CLBZ m e t a b o l i t e s . 151 152 LIST OF SCHEMES Scheme I . P u t a t i v e m e t a b o l i c pathways o f clobazam i n man, monkey and r a t . Scheme I I . Mechanisms o f p o t e n t i a l methyl t e r m i n u s t o x i f i c a t i o n o f clobazam. Scheme I I I . Mechanisms o f p o t e n t i a l phenyl t e r m i n u s t o x i f i c a t i o n o f clobazam. Scheme IV. P o s s i b l e mechanism f o r t h e o x i d a t i v e N-d e m e t h y l a t i o n o f N,N-dimethylamine by cytochome P-450. Scheme V. P o s s i b l e mechanisms f o r t h e P-450 c a t a l y z e d o x i d a t i o n o f amides. Scheme V I . M e t a b o l i c pathways i n t h e h e p a t o c a r c i n o g e n i c a c t i v a t i o n o f AAF. Scheme V I I . Pathways i n v o l v e d i n a r o m a t i c o x i d a t i o n . Scheme V I I I . F o r m a t i o n o f r e a c t i v e i n t e r m e d i a t e s from 2-h y d r o x y e s t r o g e n . Scheme IX. The s y n t h e s i s o f [^HsJCLBZ. Scheme X. Mechanism f o r t h e s e l e c t i v e i n t r o d u c t i o n o f p r o t o n s i n t o t h e d e u t e r i o p h e n y l n u c l e u s o f N - ( 5 - c h l o 2 - n i t r o p h e n y l ) - d e u t e r i o p h e n y l a m i n e 37. Scheme X I . Thermal r e a r r a n g e m e n t o f 19 t o form 18 by \/J-p r o t o n a b s t r a c t i o n and e l i m i n a t i o n o f t h e n e u t r a l k e t e n e . Scheme X I I . K e t o - e n o l t a u t o m e r i s m o f 19 showing s t r o n g e q u i l i b r i u m d i s p l a c e m e n t t o t h e k e t o form. Scheme X I I I . S y n t h e s i s o f p h e n o l i c and c a t e c h o l i c m e t a b o l i t e s o f CLBZ . x i v Scheme XIV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n r e t e n t i o n o f b e n z o d i a z e p i n e n i t r o g e n . 99 Scheme XV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n l o s s o f t h e (A) N-phenyl and (B) N-methyl m o i e t i e s . 100 Scheme X V I . Proposed f r a g m e n t a t i o n o f 0-methyl c a t e c h o l s . 101 Scheme X V I I . Proposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds r e s u l t i n g i n t h e l o s s o f a methyl r a d i c a l . 101 Scheme X V I I I . Proposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds 20, 32 and 33 r e s u l t i n g i n l o s s o f t h e N-phenyl r i n g . 102 Scheme XIX. E q u l i b r i u m c o n d e n s a t i o n between an amide and fo r m a l d e h y d e . 104 Scheme XX. The c o n d e n s a t i o n o f form a l d e h y d e w i t h DMC i l l u s t r a t e s a p r o t o t y p e b a s e - c a t a l y z e d f o r m a t i o n o f a c a r b i n o l a m i d e . 104 Scheme X X I . (A) F r a g m e n t a t i o n o f T M S - d e r i v a t i z e d 39 (m\/z 388) shows t h e m i g r a t i o n o f TMS from t h e hyd r o x y m e t h y l t o t h e e n o l oxygen c e n t r e . (B) F r a g m e n t a t i o n o f m\/z 388 r e s u l t i n g i n l o s s o f a methyl r a d i c a l . I l l Scheme X X I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC (50) by o x i d a t i o n o f DMC-TMS. 123 Scheme X X I I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC by r i n g c l o s u r e o f t h e a r y l h y d r o x y l a m i n e . 126 L I S T O F T A B L E S T a b l e 1. GCMS d a t a f o r s y n t h e t i c C L B Z - r e l a t e d compounds. T a b l e 2. GCMS d a t a f o r Phase I CLBZ m e t a b o l i t e s t y p i f i e d by b i l i a r y g l u c u r o n i d e s . M e t a b o l i t e s were e x t r a c t e d from b i l e a f t e r h y d r o l y s i s w i t h ^ - g l u c u r o n i d a s e and d e r i v a t i z e d w i t h TMS. x v i LIST OF ABBREVIATIONS amu a t o m i c mass u n i t s APT a t t a c h e d p r o t o n t e s t asymm asymmetric s t r e t c h ( i n f r a r e d ) BB broad band bp b o i l i n g p o i n t bs broad s i n g l e t BSA b o v i n e serum a l b u m i n \u00b0C d e g r e e s C e l c i u s ca c i r c a ( a p p r o x i m a t e l y ) CBZ carbamazepine CLBZ clob a z a m COMT c a t e c h o l 0 - m e t h y l t r a n s f e r a s e D d e u t e r i u m d d e u t e r i u m ( n o m e n c l a t u r e ) , d o u b l e t (NMR) dd d o u b l e t o f d o u b l e t s DMC N-desmethylclobazam DMF N,N-dimethylformamide DMSO d i m e t h y l s u l f o x i d e EI e l e c t r o n impact e q u i v e q u i v a l e n t e t a l . e t alia EtOH e t h a n o l f o d t r i s ( 6 , 6 , 7 , 7 , 8 , 8 , 8 - h e p t a f l u o r o - 2 , 2 - d i m e t h y l - 3 , 5 -o c a t a n e d i o n a t o ) FT F o u r i e r t r a n s f o r m g gram (mass), g r a v i t a t i o n a l a c c e l e r a t i o n , 9.8 m\/s ( c e n t r i f u g a l a c c e l e r a t i o n ) GC gas chromatography GCMS gas chromatography-mass s p e c t r o s c o p y h hours HPLC h i g h p r e s s u r e l i q u i d chromatography Hz H e r t z i . e . \/tf est ( t h a t i s ) i . p . i n t r a p e r i t o n e a l ( l y ) x v i i i b i d . ibidem ( i n t h e same p l a c e ) IR i n f r a r e d J c o u p l i n g c o n s t a n t i n Hz kg k i l o g r a m LC l i q u i d chromatography m m u l t i p l e t M + m o l e c u l a r i o n m\/z mass t o c h a r g e r a t i o MeOH methanol mg m i l l i g r a m MHz mega H e r t z min m i n u t e s mL m i l l i l i t r e mp m e l t i n g p o i n t MS mass s p e c t r u m MSTFA N - m e t h y l - N - t r i m e t h y l s i l y l - t r i f l u o r o a c e t a m i d e NADH n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ( r e d u c e d ) NADPH n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e phosphate ( r e d u c e d ) NMR n u c l e a r m a g n e t i c r e s o n a n c e P-450 cytochrome P-450 p-TsOH p - t o l u e n e s u f o n a t e PAGE p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s PB p h e n o b a r b i t a l p e t p e t r o l e u m PH p h e n y t o i n ppm p a r t s p e r m i l l i o n p s i pounds p e r square i n c h Rf r e l a t i v e m o b i l i t y by TLC s s i n g l e t SAM S - a d e n o s y l - L - m e t h i o n i n e SDS sodium d o d e c y l s u l f a t e SIM s e l e c t e d i o n m o n i t o r i n g s t r s t r o n g symm symmetric s t r e t c h ( i n f r a r e d ) t t r i p l e t TIC t o t a l i o n c u r r e n t TLC t h i n l a y e r chromatography TMS t e t r a m e t h y l s i l a n e (NMR), t r i m e t h y l s i l y l ( d e r i v a t i v e ) t R r e t e n t i o n t i m e v volume x i x ACKNOWLEDGEMENTS I would l i k e t o than k my s u p e r v i s o r , Dr. Frank A b b o t t f o r h i s k i n d s u p p o r t and g u i d a n c e t h r o u g h o u t t h i s p r o j e c t . I am a l s o g r a t e f u l t o t h e s t a f f o f t h e NMR and Mass S p e c t r o m e t r y F a c i l i t i e s o f The C h e m i s t r y Department, U.B.C. f o r t h e i r a s s i s t a n c e . To Sue P a n e s a r f o r a s s i s t a n c e w i t h m i c r osomal p r e p a r a t i o n s and g e l e l e c t r o p h o r e s i s . To Ron Lee and Abdul M u t l i b f o r t h e i r a s s i s t a n c e i n s u r g e r y . To Rol a n d B u r t o n f o r a s s i s t a n c e w i t h GCMS a n a l y s i s . To my a b l e c o - w o r k e r s Kelem Kassahun, Ron Lee, Abdul M u t l i b , Sue Pan e s a r and J i a o j i a o Zheng f o r t h e i r t o l e r a n c e and generous exchange o f i d e a s . To t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada and B e r l e x L a b o r a t o r i e s I n c . f o r t h e i r f i n a n c i a l s u p p o r t . To Dean M c N e i l l f o r t h e p r o v i s i o n o f a t e a c h i n g a s s i s t a n t s h i p . To Hoechst Canada I n c . f o r t h e s u p p l y o f CLBZ and DMC s t a n d a r d s . And f i n a l l y , t o my f a m i l y ( t h e hard c o r e ) f o r \" b e i n g b e h i n d me a l l t h e way\". XX DEDICATION TO MY FAMILY... THANKS FOR THE SUPPORT. 1 I INTRODUCTION Clobazam (CLBZ) i s a 1 , 5 - b e n z o d i a z e p i n e w h i c h was o r i g i n a l l y s y n t h e s i z e d by R o s s i e t a l . ( 1 9 6 9 ) , and f o u n d t o have p o t e n t a n t i c o n v u l s a n t a c t i v i t y ( G a s t a u t and Low, 1979). A t p r e s e n t CLBZ i s marketed i n 50 c o u n t r i e s as an a n t i e p i l e p t i c , however, i t s c l i n i c a l a p p l i c a t i o n i n Canada i s s t i l l i n v e s t i g a t i o n a l ( P o r t e r , 1989). The f o c u s o f t h i s t h e s i s i s t h e m e t a b o l i s m o f CLBZ, and i n o r d e r t o p r o v i d e a framework w i t h i n w h i c h t h e r e s e a r c h can be a p p r o p r i a t e l y p e r c e i v e d , t h i s i n t r o d u c t i o n s e e ks t o p r o v i d e some background c o n c e r n i n g t h i s d r u g . F i r s t l y , t h e mechanism whereby CLBZ e x e r t s i t s a n t i c o n v u l s a n t a c t i o n w i l l be examined. S e c o n d l y , t h e t h e r a p e u t i c s t a t u s o f CLBZ w i l l be r e v i e w e d w i t h r e g a r d t o (A) a n t i c o n v u l s a n t e f f i c a c y , (B) r e s t r i c t i o n s a s s o c i a t e d w i t h i t s u s e , (C) i t s a c t i v e N-desmethyl m e t a b o l i t e , (D) p h a r m a c o k i n e t i c s , (E) i n t e r a c t i o n w i t h o t h e r a n t i c o n v u l s a n t m e d i c a t i o n and (F) t h e a n a l y t i c a l t e c h n i q u e s used t o m o n i t o r t h e p a r e n t d r u g and i t s N-desmethyl m e t a b o l i t e . T h i r d l y , t h e m e t a b o l i s m o f CLBZ w i l l be r e v i e w e d w i t h r e g a r d t o t h e mechanisms i n v o l v e d i n i t s (A) N - d e m e t h y l a t i o n , (B) p o t e n t i a l N - o x i d a t i o n and (C) a r o m a t i c h y d r o x y l a t i o n . CLOBAZAM 2 1. THE MECHANISM OF ACTION OF CLOBAZAM. The a n t i c o n v u l s a n t a c t i o n o f b e n z o d i a z e p i n e s (BZs) i s e x e r t e d i n r e g i o n s o f t h e b r a i n l i k e t h e s u b s t a n t i a n i g r a and a r e a tempestas ( G a l e , 1989) by t h e p o t e n t i a t i o n o f n e u r o n a l t r a n s m i s s i o n i n v o l v i n g t h e i n h i b i t o r y t r a n s m i t t e r - y - a m i n o b u t y r i c a c i d (GABA) (M a c d o n a l d , 1989; P o r t e r , 1989; DeLorenzo, 1988). The a c t i v i t y o f GABA i s f o r t h e most p a r t , m o d ulated p o s t s y n a p t i c a l l y t h r o u g h an o l i g o m e r i c complex c o n s i s t i n g o f BZ, GABA and b a r b i t u r a t e r e c e p t o r s a d j a c e n t t o a c h l o r i d e i o n o p h o r e ( O l s e n , 1981). The a c t i v i t y o f l i g a n d s a t t h e i r r e s p e c t i v e r e c e p t o r s on t h e complex r e g u l a t e s t h e o p e n i n g o f t h e c h l o r i d e c h a n n e l , t h e l e v e l o f c h l o r i d e i o n i n f l u x , and c o n s e q u e n t l y t h e s t a t e o f n e u r o n a l i n h i b i t i o n . In t h e c a s e o f BZs, r e c e p t o r b i n d i n g i n c r e a s e s t h e f r e q u e n c y o f t h e c h l o r i d e c h a n n e l b e i n g g a t e d i n t h e open s t a t e and i n t h i s way CNS i n h i b i t i o n i s enhanced (Twyman e t a l . , 1989). The a n t i c o n v u l s a n t r e s p o n s e e l i c i t e d by t h e b i n d i n g o f BZs t o t h e i r r e c e p t o r r e p r e s e n t s o n l y a p a r t o f a co n t i n u u m o f a c t i v i t y evoked by BZ r e c e p t o r l i g a n d s ( G a r d n e r , 1988). Indeed, t h e r e i s a s p e c t r u m o f a n t a g o n i s t s t o \" i n v e r s e a g o n i s t s \" t o t h e BZ r e c e p t o r w h i c h e l i c i t from n u l l t o c o n v u l s a n t r e s p o n s e s r e s p e c t i v e l y . A t h r e e - s t a t e model was p r o p o s e d t o a c c o u n t f o r t h i s complex s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p ( P o l e e t a l . , 1982). In t h i s model, t h e BZ r e c e p t o r was vie w e d as a c o u p l i n g u n i t between t h e GABA r e c e p t o r and t h e c h l o r i d e c h a n n e l . Thus, a g o n i s t s , a n t a g o n i s t s and \" i n v e r s e a g o n i s t s \" r e g u l a t e t h e s t a t e o f BZ r e c e p t o r c o u p l i n g which i s r e f l e c t e d i n c h l o r i d e t r a n s p o r t b e i n g enhanced, u n a f f e c t e d o r d i m i n i s h e d r e s p e c t i v e l y . T h i s s c e n a r i o was p e r c e i v e d by E h l e r t e t a l . (1983) as an a l l o s t e r i c r e g u l a t i o n o f t h e GABA r e c e p t o r (and 3 hence c h l o r i d e c h a n n e l - o p e n i n g ) by l i g a n d s t o t h e BZ r e c e p t o r w i t h a g o n i s t s and \" i n v e r s e a g o n i s t s \" a c t i n g as p o s i t i v e and n e g a t i v e m o d i f i e r s r e s p e c t i v e l y . S e v e r a l r e p o r t s s u p p o r t t h e c o n c e p t o f an a l l o s t e r i c model ( S i e g h a r t , 1985; M a r t i n e t a l . , 1983; C h i u and Ro s e n b e r g , 1983), which a c c o u n t s f o r most o f t h e complex a c t i o n s o f l i g a n d s t h a t b i n d t o t h e BZ r e c e p t o r . S t u d i e s on t h e i n f l u e n c e o f s t r u c t u r e on t h e p h a r m a c o l o g i c a l a c t i v i t y o f t h e BZs have f o c u s s e d on t h e 1,4-type o f w h i c h diazepam i s o f t e n c o n s i d e r e d a p r o t o t y p e . CLBZ, on t h e o t h e r hand, i s s t r u c t u r a l l y d i f f e r e n t from diazepam i n two i m p o r t a n t a s p e c t s : ( i ) CLBZ p r e s e n t s a 1 , 5 - d i s p o s i t i o n o f N-atoms i n t h e d i a z e p i n e h e t e r o c y c l e as opposed t o a 1,4- i n d i a z e p a m , and ( i i ) CLBZ p o s s e s s e s a carboxamide f u n c t i o n a t p o s i t i o n 4 as opposed t o t h e more b a s i c i m i n e i n diazepam (Kuch, 1979). CLBZ does p o s s e s s t h e m o l e c u l a r d e t e r m i n a n t s r e q u i r e d f o r BZ a c t i v i t y , namely: ( i ) a benzo group f u s e d t o a 7-membered l a c t a m r i n g i n which t h e c a r b o n y l 0-4 o f CLBZ can be c o n s i d e r e d b i o i s o s t e r i c w i t h t h e i m i n e N-4 o f d i a z e p a m , ( i i ) p h e n y l s u b s t i t u t i o n a t C-5, ( i i i ) m e t hyl s u b s t i t u t i o n a t N - l , and ( i v ) an e l e c t r o n - w i t h d r a w i n g h y d r o p h o b i c s u b s t i t u e n t a t p o s i t i o n 7 ( S t e r n b a c h , 1 9 7 3 ) . In t h i s r e g a r d , CLOBAZAM DIAZEPAM 4 SAR models used t o a c c o u n t f o r t h e a c t i v i t y o f 1,4-BZs s h o u l d by and l a r g e accommodate CLBZ. 2. THE THERAPEUTIC STATUS OF CLOBAZAM. (A) A n t i c o n v u l s a n t E f f i c a c y o f CLBZ. The f i r s t t h e r a p e u t i c a p p l i c a t i o n f o r CLBZ was i n t h e t r e a t m e n t o f a n x i e t y ( R o b e r t s o n , 1986), but t h i s d r u g was s u b s e q u e n t l y found t o be an e f f e c t i v e a n t i c o n v u l s a n t ( G a s t a u t and Low, 1979). In t h e i r t r e a t m e n t o f 103 p a t i e n t s w i t h p r i m a r y g e n e r a l i z e d , s e c o n d a r y g e n e r a l i z e d and p a r t i a l e p i l e p s i e s G a s t a u t and Low (1979) o b s e r v e d an u n p r e c e d e n t e d a n t i c o n v u l s a n t c o n t r o l i n 74 p a t i e n t s (72 % ) , o f whom 29 (28 %) were r e n d e r e d s e i z u r e -f r e e . S e v e r a l o t h e r c l i n i c a l i n v e s t i g a t i o n s a t t e s t t o t h e e f f e c t i v e n e s s o f CLBZ i n a n t i c o n v u l s a n t t h e r a p y ( S c o t t and M o f f e t t , 1988; Munn e t a l . , 1988; C a l l a g h a n and G o g g i n , 1988; K i l p a t r i c k e t a l . , 1 987), e s p e c i a l l y i n t h e t r e a t m e n t o f s e c o n d a r y g e n e r a l i z e d e p i l e p s y ( G a s t a u t , 1981b), complex p a r t i a l s e i z u r e s (Koeppen e t a l . , 1987), and s t a r t l e - i n d u c e d and i n f a n t i l e spasms ( F a r r e l l , 1986). A p a r t from i t s broad a n t i c o n v u l s a n t s p e c t r u m , CLBZ o f f e r s a d d i t i o n a l t h e r a p e u t i c b e n e f i t s i n c l u d i n g a r a p i d o n s e t o f a c t i o n , o r a l e f f e c t i v e n e s s i n c h r o n i c e p i l e p s y and a h i g h t h e r a p e u t i c i n d e x ( G a s t a u t , 1981a). The a n t i c o n v u l s a n t e f f e c t i v e n e s s o f CLBZ has a l s o been d e m o n s t r a t e d i n d i f f e r e n t a nimal s p e c i e s . In t h e c a s e o f a n i m a l s , t h e c o n d i t i o n s o f g r and mal and p e t i t mal g e n e r a l i z e d e p i l e p s y have been mimicked w i t h t h e use o f k i n d l i n g and p e n t y l e n e t e t r a z o l e i n f u s i o n r e s p e c t i v e l y ( G a s t a u t , 1981b). The s u c c e s s f u l use o f CLBZ has been r e p o r t e d i n t h e s e i z u r e 5 c o n t r o l o f k i n d l e d r a t s (Young e t a l . , 1988; V a j d a e t a l . , 1987), and p e n t y l e n e t e t r a z o l e i n f u s e d mice ( F e e l y e t a l . , 1986; Gent e t a l . , 1985; Ha i g h e t a l . , 1984). (B) R e s t r i c t i o n s t o CLBZ Therapy. R e l a t i v e l y m i l d s i d e e f f e c t s o f s e d a t i o n , m u s c l e weakness and a t a x i a were o b s e r v e d by G a s t a u t and Low (1979) i n t h e i r t r e a t m e n t o f p a t i e n t s w i t h CLBZ. A ma j o r drawback, however, was t h e development o f t o l e r a n c e i n 33 % o f t h e i r p a t i e n t p o p u l a t i o n . The development o f t o l e r a n c e t e n d s t o r e p r e s e n t a common d e n o m i n a t o r among c l i n i c a l i n v e s t i g a t i o n s , however, t h e o c c u r r e n c e o f t h i s phenomenon i s v a r i a b l e . Of t h e 22 open s t u d i e s r e v i e w e d by R o b e r t s o n ( 1 9 8 6 ) , t h e i n c i d e n c e o f t o l e r a n c e r a n g e d from 0 t o 86 %, w i t h a mean o c c u r r e n c e o f 36 %. S c o t t and M o f f e t t (1988) r e p o r t e d \" g r e a t b e n e f i t \" f o r 13 (43 %) o f 30 p a t i e n t s w i t h c h r o n i c i n t r a c t a b l e e p i l e p s y f o r up t o 3 y e a r s , F e e l y and G i b s o n (1984) r e p o r t e d c o m p l e t e a v o i d a n c e o f t o l e r a n c e w i t h i n t e r m i t t e n t CLBZ t h e r a p y i n c a t e m e n i a l e p i l e p s y , and F a r r e l l (1986) o b s e r v e d a r e t r i e v a l o f a n t i c o n v u l s a n t c o n t r o l w i t h an i n c r e a s e d dosage i n p e d i a t r i c p a t i e n t s who had d e v e l o p e d p a r t i a l t o l e r a n c e . On t h e o t h e r hand, K i l p a t r i c k e t a l . (1987) o b s e r v e d r a p i d l o s s o f a n t i c o n v u l s a n t p o t e n c y i n 8 o f 11 p a t i e n t s (72 % ) , and Munn e t a l . ( 1 9 8 8 ) , u n l i k e F a r r e l l ( 1 9 8 6 ) , were u n a b l e t o r e s t o r e t h e l o s s o f a n t i c o n v u l s a n t a c t i v i t y by i n c r e a s i n g t h e dosage w i t h i n t h e i r p e d i a t r i c g r o u p . The mechanism by wh i c h t o l e r a n c e t o BZs o c c u r s r e m a i n s u n c l e a r . S e v e r a l w o r k e r s ( F e e l y e t a l . , 1989; H a i g h and F e e l y , 1988; B o a s t and G e r h a r d t , 1987) have p e r c e i v e d t h i s phenomenon t o o c c u r a t a c e l l u l a r l e v e l w h e r e i n a f u l l a g o n i s t t o t h e BZ r e c e p t o r p r e c i p i t a t e s t h e gamut o f 6 r e c e p t o r a s s o c i a t e d a c t i v i t y , namely, t h e a n t i c o n v u l s a n t r e s p o n s e , t o l e r a n c e and a s s o c i a t e d s i d e e f f e c t s , whereas an a p p r o p r i a t e p a r t i a l a g o n i s t t a r g e t s t h e r e c e p t o r more s e l e c t i v e l y f o r b e n e f i c i a l a c t i v i t y . In summary, t h e development o f t o l e r a n c e appears t o be an i d i o s y n c r a t i c phenomenon w i t h i n t h e p a t i e n t p o p u l a t i o n w i t h an u n p r e d i c t a b l e t i m e o f o c c u r r e n c e . (C) N - d e s m e t h y l clobazam: The A c t i v e CLBZ M e t a b o l i t e . The p r i m a r y m e t a b o l i c r o u t e f o r CLBZ i n man and mice i s N-d e m e t h y l a t i o n t o N-desmethylclobazam (DMC) ( G a r a t t i n i , 1985; D a v i e s , 1985) w h i c h a l t h o u g h p h a r m a c o l o g i c a l l y a c t i v e , i s 2.4- t o 7 - f o l d l e s s p o t e n t t h a n t h e p a r e n t d r u g (Koeppen, 1985; F i e l d i n g and Hoffmann, 1979). D e s p i t e i t s l o w e r a n t i c o n v u l s a n t p o t e n c y , DMC o f f e r s s p e c i f i c a dvantages o v e r CLBZ. ( i ) DMC i s more s e l e c t i v e i n i t s a c t i o n t h a n CLBZ, a f f o r d i n g an enhanced d i f f e r e n t i a t i o n between a n t i c o n v u l s a n t a c t i v i t y and s e d a t i v e s i d e - e f f e c t ( F i e l d i n g and Hoffman, 1979). ( i i ) B e t t e r c o r r e l a t i o n e x i s t s f o r DMC between plasma ( H a i g h e t a l . , 1984) and b r a i n ( C a c c i a e t a l . , 1980a + b) l e v e l s i n mice and a c t i v i t y a g a i n s t p e n t y l e n e t e t r a z o l e i n d u c e d s e i z u r e s , w h i c h i s i n a c c o r d a n c e w i t h c l i n i c a l f i n d i n g s w h i c h d e m o n s t r a t e s u p e r i o r a n t i c o n v u l s a n t a c t i v i t y b e i n g d e r i v e d w i t h more r a p i d m e t a b o l i s m o f CLBZ t o DMC (G o g g i n and C a l l a g h a n , 1985). ( i i i ) The development o f t o l e r a n c e i n mice i s much l e s s pronounced w i t h DMC ( H a i g h e t a l . , 1987). In f a c t , i t seems l i k e l y t h a t a c u t e t o l e r a n c e t o CLBZ i s l a r g e l y p r e c i p i t a t e d by t h e p a r e n t d r u g ( F e e l y e t a l . , 1986). ( i v ) The s t e a d y - s t a t e h a l f - l i f e o f DMC i n monotherapy (42 h) ( P u l l a r e t a l . , 1987) i s l o n g e r t h a n t h a t o f CLBZ (34 h) ( G r e e n b l a t t e t a l . , 1981) wh i c h s u g g e s t s t h a t t h e m e t a b o l i t e c o u l d 7 p r o v i d e more e f f e c t i v e a n t i c o n v u l s a n t c o n t r o l i n c h o n i c t h e r a p y . (D) The P h a r m a c o k i n e t i c s o f CLBZ. The p h a r m a c o k i n e t i c s o f CLBZ i s s u b j e c t t o c o n s i d e r a b l e i n t e r - s u b j e c t v a r i a b i l i t y w h i c h c o u l d be i n f l u e n c e d by age, s e x , and c o n c o m i t a n t m e d i c a t i o n (Bun e t a l . , 1985; G r e e n b l a t t e t a l . , 1981; T e d e s c h i e t a l . , 1981). CLBZ i s r a p i d l y a b s o r b e d a f t e r o r a l d o s i n g and peak plasma c o n c e n t r a t i o n s a r e u s u a l l y a c h i e v e d i n 0.5 t o 2.5 h ( G r e e n b l a t t e t a l . , 1981), b u t c o u l d be as l o n g as 4 h (Rupp e t a l . , 1 9 7 9 ) . G r e e n b l a t t e t a l . (1981) s t u d i e d t h e i n f l u e n c e o f age and sex on t h e p h a r m a c o k i n e t i c s o f CLBZ a f t e r a 20 mg o r a l dose. CLBZ was bound t o plasma t o t h e e x t e n t o f 85 % to 91.4 %, and b i n d i n g t o plasma p r o t e i n d e m o n s t r a t e d no c o r r e l a t i o n w i t h age o r s e x . On t h e o t h e r hand, b o t h age and sex i n f l u e n c e d d i s t r i b u t i o n (Vd) wh i c h was h i g h e r i n women and i n c r e a s e d w i t h age i n bo t h s e x e s . T h i s r e l a t i o n s h i p was c o n s i d e r e d a r e f l e c t i o n o f t h e i n c r e a s e d c o n t r i b u t i o n to t o t a l body w e i g h t o f f a t ( i n women and i n t h e e l d e r l y ) i n t o w h i c h the l i p o p h i l i c CLBZ c o u l d be d i s t r i b u t e d t h e r e b y i n c r e a s i n g Vd. Age and sex a l s o i n f l u e n c e d e l i m i n a t i o n and c l e a r a n c e . Whereas i n young s u b j e c t s t h e l a r g e e l i m i n a t i o n h a l f - l i f e i n women (18 - 46 h) compared t o men (11 - 23 h) was a r e f l e c t i o n o f t h e g r e a t e r d i s t r i b u t i o n (Vd) i n women, t h e h a l f -l i f e i n o l d e r males (29 - 77 h) was s i g n i f i c a n t l y l o n g e r t h a n i n t h e i r young c o u n t e r p a r t s (11 - 23 h ) . More e x t e n s i v e m e t a b o l i s m i n t h e younger s u b j e c t s c o u l d p r o b a b l y a c c o u n t f o r t h i s p a t t e r n ( T e d e s c h i e t a l . , 1981). 8 (E) Drug I n t e r a c t i o n s w i t h CLBZ. I n t e r a c t i o n w i t h drugs c o n c o m i t a n t l y used i n a n t i c o n v u l s a n t t h e r a p y a l s o i n f l u e n c e s t h e p h a r m a c o k i n e t i c s o f CLBZ. Thus, Cano e t a l . (1981) o b s e r v e d a d r a m a t i c d e c r e a s e i n CLBZ plasma l e v e l s w i t h a c o r r e s p o n d i n g e l e v a t i o n i n DMC when carbamazepine (CBZ) o r p h e n y t o i n (PH) were c o n c o m i t a n t l y a d m i n i s t e r e d . T h i s phenomenon was c o n s i d e r e d t o be t h e r e s u l t o f i n d u c t i o n o f CLBZ m e t a b o l i s m . However, e l e v a t e d DMC l e v e l s a l s o o c c u r r e d w i t h c o n c o m i t a n t CBZ o r PH a d m i n i s t r a t i o n when DMC was used as t h e p a r e n t d r u g ( P u l l a r e t a l . , 1987). T h e r e f o r e , i t i s a p p a r e n t t h a t t h e i n d u c t i o n o f CLBZ m e t a b o l i s m a c c o u n t s o n l y i n p a r t f o r t h e i n c r e a s e o f DMC plasma l e v e l s , and o t h e r mechanisms c o u l d be o p e r a t i v e . Drug i n t e r a c t i o n a l s o e x i s t s between CLBZ and v a l p r o a t e (VPA) (Cocks e t a l . , 1985). A d d i t i o n o f CLBZ t o t h e t h e r a p y o f e p i l e p t i c p a t i e n t s s t a b i l i z e d on VPA r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n VPA plasma l e v e l s . To a c c o u n t f o r t h i s , i t was p r o p o s e d t h a t VPA m e t a b o l i s m c o u l d be i n h i b i t e d by CLBZ. (F) A n a l y t i c a l T e c h n i q u e s used i n The M o n i t o r i n g o f CLBZ and DMC. A n a l y t i c a l t e c h n i q u e s used i n t h e t h e r a p e u t i c m o n i t o r i n g o f CLBZ and DMC i n c l u d e HPLC ( D u s c i e t a l . , 1987; Tom a s i n i e t a l . , 1985; B r a c h e t -L i e r m a i n e t a l . , 1982), and GC w i t h n i t r o g e n - s p e c i f i c (Pena, 1986), e l e c t r o n - c a p t u r e (EC) (Badcock and Z o a n e t t i , 1987; Douse, 1984; R i v a e t a l . , 1981) and mass s p e c t r o s c o p i c ( D r o u e t - C o a s s o l o e t a l . , 1989) d e t e c t i o n . Each o f t h e r e p o r t e d methods was s u f f i c i e n t l y s e n s i t i v e t o d e t e c t CLBZ and DMC l e v e l s a t t h e i r u s u a l t h e r a p e u t i c serum l e v e l s o f 50 - 300 ng\/mL and 800 - 4000 ng\/mL r e s p e c t i v e l y . However, GC w i t h EC d e t e c t i o n a f f o r d e d t h e 9 h i g h e s t s e n s i t i v i t y , and l e v e l s o f d e t e c t i o n o f 2 ng\/mL and 4 ng\/mL f o r CLBZ and DMC r e s p e c t i v e l y have been a c h i e v e d ( B a d c o c k and Z o a n e t t i , 1987). 3. THE METABOLISM OF CLOBAZAM. There a r e few r e p o r t s i n t h e l i t e r a t u r e c o n c e r n i n g t h e m e t a b o l i s m o f t h e 1,5-BZs, and o f t h e s e , t h e m e t a b o l i c p r o f i l i n g o f CLBZ ( V o l z e t a l . , 1979) (Scheme I ) and t r i f l u b a z a m ( A l t o n e t a l . , 1975a + b) i n man and i n d i f f e r e n t animal s p e c i e s a f f o r d t h e most e l a b o r a t e a c c o u n t s . Because o f t h e c arboxamide f u n c t i o n a t t h e 4 , 5 - p o s i t i o n , CLBZ i s not s u s c e p t i b l e t o 3 - h y d r o x y l a t i o n o r r i n g o p e n i n g l i k e diazepam ( C a l d w e l l , 1985; Kuch 1979). I n s t e a d , m e t a b o l i s m o c c u r s w i t h t h e f o r m a t i o n o f N-d e s m e t h y l c l o b a z a m (DMC) and t h e 4 ' - h y d r o x y , 4'-hydroxy-3'-methoxy and 3 ' , 4 ' - d i h y d r o d i o l m e t a b o l i t e s o f b o t h CLBZ and DMC ( V o l z e t a l . , 1979) (Scheme I ) . A l t h o u g h m e t a b o l i c p r o c e s s e s g e n e r a l l y d e t o x i f y x e n o b i o t i c s , i t i s now w e l l known t h a t m e t a b o l i s m ca n p a r a d o x i c a l l y p o t e n t i a t e t h e t o x i c i t y o f t h e p a r e n t compound. D e s c r i b e d i n Scheme I a r e p o t e n t i a l mechanisms f o r CLBZ t o x i f i c a t i o n o c c u r r i n g e i t h e r a t t h e m e t h y l t e r m i n u s (Scheme I I ) o r TRIFLUBAZAM 10 p h e n y l t e r m i n u s (Scheme I I I ) . In t h i s r e g a r d , t h e f o l l o w i n g b i o t r a n s f o r m a t i o n s w i l l be examined f r o m a t o x i c o l o g i c a l p e r s p e c t i v e : (A) N - d e m e t h y l a t i o n (Scheme I I ) , (B) N - o x i d a t i o n (Scheme I I ) , and (C) A r o m a t i c h y d r o x y l a t i o n (Scheme I I I ) . OH OCH3 OH OCH. Scheme I . P u t a t i v e m e t a b o l i c pathways o f c l o b a z a m i n man, monkey and r a t ( V o l z e t a l . , 1979). 11 Scheme I I . Mechanisms o f p o t e n t i a l methyl t e r m i n u s t o x i f i c a t i o n o f clobazam. A: P-450 o x i d a t i o n t o c a r b i n o l a m i d e ; B: A m i n o m e t h y l a t i o n o f i n t r a c e l l u l a r n u c l e o p h i l e s ; C: R e l e a s e o f t o x i c f o r m a l d e h y d e ; D: P-450 o x i d a t i o n t o hydroxamic a c i d ; E: B i n d i n g t o i n t r a c e l l u l a r n u c l e o p h i l e s e.g. g l u t a t h i o n e (GSH). 12 B i n d i n g t o l i v e r p r o t e i n s OH OH Scheme I I I . Mechanisms o f p o t e n t i a l phenyl t e r m i n u s t o x i f i c a t i o n o f c l o b a z a m . F: P-450 o x i d a t i o n t o arene e p o x i d e ; G: B i n d i n g t o i n t r a c e l l u l a r n u c l e o p h i l e s e.g. g l u t a t h i o n e (GSH); H: O x i d a t i o n t o c a t e c h o l ; I : O x i d a t i o n t o r a d i c a l s e m i q u i n o n e ; J : B i n d i n g t o l i v e r p r o t e i n s ; K: D e t o x i f i c a t i o n by O - m e t h y l a t i o n . 13 Each o f t h e b i o t r a n s f o r m a t i o n p r o c e s s e s i n v o l v i n g N - d e m e t h y l a t i o n , N-o x i d a t i o n and a r o m a t i c h y d r o x y l a t i o n i s c a t a l y z e d by t h e cytochome P-450 enzymes ( W i s l o c k i e t a l . , 1985); however, t h e u n d e r l y i n g mechanism whereby t h e s e a p p a r e n t l y d i f f e r e n t b i o t r a n s f o r m a t i o n s o c c u r can be summarized i n seven s t e p s ( H a l l , 1986; G u e n g e r i c h and L i e b l e r , 1985): ( i ) f o r m a t i o n o f t h e P - 4 5 0 - s u b s t r a t e complex, ( i i ) f i r s t one e l e c t r o n r e d u c t i o n i n v o l v i n g NADPH-P-450 r e d u c t a s e , ( i i i ) b i n d i n g o f m o l e c u l a r O2 , ( i v ) second one e l e c t r o n r e d u c t i o n i n v o l v i n g NADPH-P-450 r e d u c t a s e , (v) a c c e p t a n c e o f 2 H + w i t h c o n c o m i t a n t d i s s o c i a t i o n o f H2O, ( v i ) i n s e r t i o n o f oxygen i n t o t h e s u b s t r a t e and ( v i i ) r e l e a s e o f t h e p r o d u c t from t h e enzyme. A l t h o u g h P-450 c a t a l y s i s can be g e n e r a l l y p e r c e i v e d i n t h i s way, t h e d i v e r s i t y i n r e a c t i o n t y p e s a r i s e s due t o i d e n t i t y o f t h e r e d u c i n g e q u i v a l e n t ( e . g . e\", H*) and r e a r r a n g e m e n t o f t h e p r o d u c t m o l e c u l e . (A) The N - d e m e t h v l a t i o n o f CLBZ. Many o f t h e m e c h a n i s t i c s t u d i e s on t h e P-450 c a t a l y z e d N-d e a l k y l a t i o n s f o c u s on s e c ondary and t e r t i a r y amines ( L i n d e k e and Cho, 1982; W o l f , 1982; W i s l o c k i e t a l . , 1980), however, t h e N - d e a l k y l a t i o n o f amides s h o u l d p r o c e e d i n l i k e f a s h i o n . The N - d e m e t h y l a t i o n o f N,N-d i m e t h y l a m i n e proceeds v i a a-carbon d e p r o t o n a t i o n as shown i n Scheme IV ( H o l l e n b e r g e t a l . , 1985; Miwa e t a l . , 1983). The mechanism i n v o l v e s an i n i t i a l o n e - e l e c t r o n removal from t h e s u b s t r a t e (-e~) t o t h e aminium c a t i o n r a d i c a l , w hich i n t u r n undergoes p r o t o n a b s t r a c t i o n (-H+) t o t h e c a r b o n -c e n t r e d r a d i c a l . Subsequent r e c o m b i n a t i o n w i t h an enzyme-bound h y d r o x y l r a d i c a l (+0H*) g i v e s r i s e t o t h e u n s t a b l e c a r b i n o l a m i n e which e l i m i n a t e s f o r m a l d e h y d e t o a f f o r d t h e d e m e t h y l a t e d p r o d u c t . 14 -e C H 3 R-N-CH3 C H 3 R-N-CH3 OH-H R-N-CH3 ' f C H 2 Q C H 3 R-N-CH2OH Scheme IV. P o s s i b l e mechanism f o r t h e o x i d a t i v e N - d e m e t h y l a t i o n o f N,N-d i m e t h y l a m i n e by cytochome P-450 ( H o l l e n b e r g e t a l . , 1985; Miwa e t a l . , 1983). The N-hydroxymethyl ( c a r b i n o l a m i d e ) i n t e r m e d i a t e s w h i c h o c c u r i n t h e d e m e t h y l a t i o n o f N-methylamides v a r y i n t h e i r s t a b i l i t y . F o r example, N-h y d r o x y m e t h y l - N - m e t h y l b e n z a m i d e ( I ) decomposed r e a d i l y t o t h e N-desmethyl compound, whereas N-hydroxymethylbenzamide (2) was s u f f i c i e n t l y s t a b l e t o be i d e n t i f i e d (Ross e t a l . , 1983). O O 1 2 15 Many a c c o u n t s i n t h e l i t e r a t u r e a t t e s t t o t h e s t a b i l i t y o f t h e c a r b i n o l a m i d e s . F o r example, t h e c a r b i n o l a m i d e s o f bambuterol (3) ( L i n d b e r g e t a l . , 1989), N-methylbenzamide (2) (Ross e t a l . , 1983) and monuron (4) (Ross e t a l . , 1982) were formed in vitro, and ( t h e c a r b i n o l a m i d e s o f ) r e c i p a v r i n t e r t i a r y formamide (5) ( S l a t t e r e t a l . , 1989), Zolpidem (6) ( V a j t a e t a l . , 1 988), monuron (4) (Ross e t a l . , 1982) and d i p h e n a m i d (7) (McMahon and S u l l i v a n , 1965) o c c u r r e d in vivo either as t h e f r e e s p e c i e s o r t r a p p e d as a c o n j u g a t e . Me x Me N Me' V C H 2 O H 6 Two s t r u c t u r a l f e a t u r e s appear t o s t a b i l i z e N-hydroxymethyl i compounds: ( i ) a c a r b o n y l group a d j a c e n t t o t h e N-hydroxymethyl f u n c t i o n (Ross e t a l . , 1982) and ( i i ) t h e l o c a t i o n o f t h e h y d r o x y m e t h y l b e a r i n g n i t r o g e n i n a c o n j u g a t e d system ( G o r r o d and Temple, 1976). McMahon and S u l l i v a n (1965) o b s e r v e d t h a t c a r b i n o l a m i d e s were more s t a b l e t h a n t h e i r 16 c a r b i n o l a m i n e c o u n t e r p a r t s . Ross e t a l . (1983) have s u g g e s t e d t h a t t h e c a r b o n y l group r e d u c e s t h e e l e c t r o n d e n s i t y a t t h e h y d r o x y m e t h y l n i t r o g e n and t h e r e b y m i l i t a t e s a g a i n s t f o r m a l d e h y d e d i s s o c i a t i o n . T h i s r a t i o n a l e was e x t e n d e d t o a c c o u n t f o r t h e enhanced s t a b i l i t y o f N-( h y d r o x y m e t h y l ) b e n z a m i d e (2) o v e r N - ( h y d r o x y m e t h y l ) - N - m e t h y l b e n z a m i d e ( I ) , c l a i m i n g t h a t t h e e l e c t r o n - d o n a t i n g e f f e c t o f t h e methyl m o i e t y a n t a g o n i z e d t h e i n f l u e n c e o f t h e c a r b o n y l group. G o r r o d and Temple (1976) o b s e r v e d t h a t t h e i n c l u s i o n o f n i t r o g e n i n t h e c o n j u g a t e d system o f N-m e t h y l c a r b a z o l e (8) l o w e r e d t h e pKa o f t h e h e t e r o a t o m , and t h e r e b y a c c o u n t e d f o r t h e enhanced s t a b i l i t y c o n f e r r e d on t h e N-hydroxymethyl compound ( 9 ) . 8 9 The c a r b i n o l a m i d e (10) formed from CLBZ p o s s e s s e s a c a r b o n y l and a phenyl group a d j a c e n t t o t h e h y d r o x y m e t h y l b e a r i n g n i t r o g e n , both o f w h i c h c o u l d enhance t h e s t a b i l i t y o f t h i s compound by r e s o n a n c e d e l o c a l i z a t i o n o f n - e l e c t r o n s . On t h i s b a s i s t h e p r o b a b i l i t y o f i s o l a t i n g a c a r b i n o l a m i d e i n t e r m e d i a t e i n t h e f o r m a t i o n o f DMC appeared t o be q u i t e h i g h . 17 10 CLBZ c a r b i n o l a m i d e (10) showing p o s s i b l e r e s o n a n c e f o r m s . The i n v e s t i g a t i o n o f a p o t e n t i a l CLBZ c a r b i n o l a m i d e i s t o x i c o l o g i c a l l y r e l e v a n t f o r two r e a s o n s : ( i ) N-hydroxymethyl i n t e r m e d i a t e s a c t as t r a n s p o r t forms o f t o x i c f o r m a l d e h y d e ( K i n g s t o n , 1965), and ( i i ) t h e y a r e c a p a b l e o f a m i n o m e t h y l a t i o n o f i n t r a c e l l u l a r n u c l e o p h i l e s l i k e g l u t a t h i o n e , p r o t e i n s and n u c l e i c a c i d s (Soloway e t a l . , 1983). (B) The N - o x i d a t i o n o f DMC. T e r t i a r y amides a r e not m e t a b o l i z e d by N - o x i d a t i o n . The l o n e p a i r o f e l e c t r o n s on n i t r o g e n a r e n o t a v a i l a b l e f o r N-0 bond f o r m a t i o n , b u t a r e i n s t e a d d e l o c a l i z e d by t h e c a r b o n y l g r o u p (Damani, 1982). However, subsequent t o t h e N - d e m e t h y l a t i o n o f CLBZ, i t i s c o n c e i v a b l e t h a t t h e se c o n d a r y amide f u n c t i o n o f DMC c o u l d undergo f u r t h e r o x i d a t i o n by P-450 t o N-hydroxy DMC (hy d r o x a m i c a c i d ) ( N e l s o n , 1985) (Scheme V ) . The mechanism whereby amides a r e o x i d i z e d i s u n c e r t a i n , and d e p e n d i n g on t h e P-450 isoenzyme i n v o l v e d one o f s e v e r a l pathways c o u l d be o p e r a t i v e . Some o f t h e s e i n c l u d e : ( i ) s i n g l e t - t y p e oxene r e a c t i o n w i t h t h e i m i n o l t a u t o m e r t o for m an o x a z i r a n e w h i c h e x i s t s i n e q u i l i b r i u m w i t h t h e hyd r o x a m i c a c i d ; ( i i ) s e q u e n t i a l e l e c t r o n and p r o t o n a b s t r a c t i o n s f o l l o w e d by h y d r o x y l 18 r a d i c a l t r a n s f e r t o t h e ami d y l r a d i c a l ; and ( i i i ) h ydrogen atom a b s t r a c t i o n f o l l o w e d by h y d r o x y l r a d i c a l t r a n s f e r . O - F e -HOH + R OH R OH HOO (i) _N=C- -N-C- * -N-C- *- -N-C-HO HO + O HOO . . . . 1 11 -pr 1 11 _u + \u2022 \u00bb OH 1 \" (n _ N -C- -N-C- -H-^-N-C- \u2014 - N - C -H O O HO O ( i i i ) -N-C- -N-C- -N-C-Scheme V. P o s s i b l e mechanisms f o r t h e P-450 c a t a l y z e d o x i d a t i o n o f amides ( N e l s o n , 1985). Cramer e t a l . (1960) were t h e f i r s t t o r e p o r t t h e m e t a b o l i c N-o x i d a t i o n o f amides w i t h t h e d i s c o v e r y o f N - h y d r o x y - N - a c e t y l a m i n o f l u o r e n e (N-OH AAF) (12) as a g l u c u r o n i d a t e d u r i n a r y m e t a b o l i t e o f N-a c e t y l a m i n o f l u o r e n e (AAF) (11) i n r a t s . AAF i s a p o t e n t h e p a t o c a r c i n o g e n , and o x i d a t i o n t o t h e hydroxamic a c i d (N-OH AAF) i s t h o u g h t t o be t h e mechanism o f t o x i c o l o g i c a l a c t i v a t i o n (Scheme V I ) (Hanna and Banks, 1985). However, t h e f o r m a t i o n N-OH AAF r e p r e s e n t s o n l y a p r e r e q u i s i t e s t e p i n t h e b i o a c t i v a t i o n o f AAF, and u l t i m a t e t o x i f i c a t i o n o f N-OH AAF i s mediated by phase I I f o r m a t i o n o f t h e s u l f a t e e s t e r (13), a p o t e n t e l e c t r o p h i l e (Van den Goorbergh e t a l . , 1987), w h i c h r e a c t s n o n - s p e c i f i c a l l y w i t h m e t h i o n y l 19 r e s i d u e s t o form S-adducts w i t h h e p a t i c p r o t e i n s (Hanna and Banks, 1985) 8\/\u2014f\"S\u2014V H 7^\"~\\-^ \/ V N - C O C H J \u20ac 5 4 11 Binding to hepatic proteins a c t i v a t i o n N-hydroxylation \"N-COCHj 13 Scheme V I . M e t a b o l i c pathways i n t h e h e p a t o c a r c i n o g e n i c a c t i v a t i o n o f AAF (Hanna and Banks, 1985). OeBaun e t a l . (1968) o b s e r v e d a c l o s e p a r a l l e l between t h e l e v e l o f s u l f o t r a n s f e r a s e a c t i v i t y i n d i f f e r e n t r o d e n t s p e c i e s and t h e i r s u s c e p t i b i l i t y t o c a r c i n o g e n e s i s w i t h N-OH AAF, and p r o p o s e d t h a t s u l f a t e e s t e r f o r m a t i o n gave r i s e t o t h e u l t i m a t e t o x i f i c a t i o n o f N-OH AAF. Meerman and M u l d e r (1981) s u b s t a n t i a t e d t h i s h y p o t h e s i s when t h e y found t h a t p r e t r e a t m e n t o f r a t s w i t h t h e s u l f o t r a n s f e r a s e i n h i b i t o r p e n t a c h l o r o p h e n o l c o m p l e t e l y a b o l i s h e d t h e c a r c i n o g e n i c e f f e c t s o f N-OH AAF. G l u t a t h i o n e (GSH) p l a y s an i m p o r t a n t r o l e as an i n t r a c e l l u l a r n u c l e o p h i l e by b i n d i n g t o s e v e r a l x e n o b i o t i c e l e c t r o p h i l e s . The n i t r e n i u m c a t i o n g e n e r a t e d by t h e d e c o m p o s i t i o n o f N-OH AAF i s no e x c e p t i o n (van den Goorbergh e t a l . , 1985), and two b i l i a r y GSH c o n j u g a t e s i n r a t s dosed w i t h N-OH AAF were i d e n t i f i e d , namely, 1- and 3 - ( g l u t a t h i o n - S - y l ) AAF (Meerman e t a l . , 1982). D e s p i t e t h e o c c u r r e n c e o f t h e s e c o n j u g a t e s , GSH a f f o r d s 20 l i t t l e p r o t e c t i o n i n t h e d e t o x i f i c a t i o n o f N-OH AAF because o f i t s i n a b i l i t y t o e f f e c t i v e l y compete f o r t h e t r a p p i n g o f t h e n i t r e n i u m i o n (van den Goorbergh e t a l . , 1985). Indeed, when r a t s were p r e t r e a t e d w i t h d i e t h y l m a l e a t e (DEM) t o d e p l e t e h e p a t i c and r e n a l GSH, e x c r e t i o n o f t h e GSH c o n j u g a t e s was d e c r e a s e d by 60 % y e t , no significant increase in the covalent binding of N-OH AAF to hepatic and renal macromolecules was observed (Meerman and T i j d e n s , 1985). I n s t e a d , i t was s u g g e s t e d o t h e r d e t o x i c a n t s l i k e NADH and a s c o r b i c a c i d c o u l d have f a c i l i t a t e d t h e d e t o x i f i c a t i o n o f t h e n i t r e n i u m i o n i n t h e absence o f GSH. U n l i k e t h e p o l y c y c l i c a r o m a t i c s ( e . g . A A F ) , a c e t a n i l i d e s a r e l e s s r e a d i l y p r e d i s p o s e d t o N-hydroxy1 a t i o n (Damani, 1 9 8 2 ) , n e v e r t h e l e s s , t h e i r b i o t r a n s f o r m a t i o n i s t o x i c o l o g i c a l l y r e l e v a n t ( M u l d e r e t a l . , 1977; H i nson and M i t c h e l l , 1976). M u l d e r e t a l . (1977) have s p e c u l a t e d t h a t N-h y d r o x y p h e n a c e t i n (14) c o u l d be r e s p o n s i b l e f o r t h e h e p a t o t o x i c and n e p h o t o x i c a c t i v i t y o f t h e p a r e n t d r u g ( 1 5 ) . The s u l f a t e c o n j u g a t e o f 14, because o f i t s h i g h r e a c t i v i t y , was c o n s i d e r e d t h e t o x i c s p e c i e s i n v o l v e d i n c o v a l e n t b i n d i n g i n t h e l i v e r , whereas t h e g l u c u r o n i d e c o n j u g a t e b e i n g l e s s r e a c t i v e a c t e d as a t r a n s p o r t f o r m o f 14 whose a c t i v i t y was e x e r t e d a f i e l d i n t h e k i d n e y and b l a d d e r where g l u c u r o n i d e s a r e c o n c e n t r a t e d . C l o s e s i m i l a r i t y e x i s t s between t h e s e c o n d a r y amide c h e m i s t r y o f p h e n a c e t i n (15) and DMC w h i c h c o u l d s i g n i f y t h e p o s s i b i l i t y f o r DMC t o be o x i d i z e d , l i k e 15, t o a h ydroxamic a c i d . o 21 (C) The A r o m a t i c H y d r o x y ! a t i o n o f CLBZ. The cytochome P-450 me d i a t e d c a t a l y s i s o f a r o m a t i c h y d r o x y l a t i o n , t o a l a r g e e x t e n t , i n v o l v e s t h e d i r e c t a d d i t i o n o f oxygen a c r o s s t h e C-C a r o m a t i c bond via an o x e n o i d t y p e mechanism t o a f f o r d t h e arene e p o x i d e (Scheme V I I ) ( T r a g e r , 1980). The f o r m a t i o n o f a r e n e e p o x i d e s i n a r o m a t i c o x i d a t i o n i s now w e l l e s t a b l i s h e d , and a l t e r n a t i v e m e t a b o l i c pathways f o r t h i s i n t e r m e d i a t e e x i s t . The e p o x i d e c a n e i t h e r (A) s p o n t a n e o u s l y r e a r r a n g e t o t h e p h e n o l , o r (B) i s b i o t r a n s f o r m e d s e q u e n t i a l l y t o t h e d i h y d r o d i o l and c a t e c h o l ( K a l f , 1987). Scheme V I I . Pathways i n v o l v e d i n a r o m a t i c o x i d a t i o n . The r a t e - d e t e r m i n i n g s t e p i n a r o m a t i c h y d r o x y l a t i o n i s t h e h e t e r o l y t i c c l e a v a g e o f t h e C-0 bond o f t h e a r e n e e p o x i d e t o form t h e c a r b o c a t i o n ( T r a g e r , 1980). The c a r b o c a t i o n i n t e r m e d i a t e c a n , i n t u r n , e i t h e r f o r m t h e phenol d i r e c t l y w i t h t h e l o s s o f X+ o r i s o m e r i z e t o t h e k e t o t a u t o m e r o f t h e phenol w i t h a 1 , 2 - m i g r a t i o n o f X\", known as t h e NIH s h i f t , b e f o r e r e a r r a n g i n g t o t h e f i n a l p r o d u c t . G e n e r a l l y , s u b s t i t u e n t e f f e c t s d e t e r m i n e t h e e x t e n t t o which l a b e l i s r e t a i n e d , w i t h r e t e n t i o n b e i n g f a v o u r e d by e l e c t r o n w i t h d r a w i n g g r o u p s . A p a r t from t h e i r n o n - s p e c i f i c rearrangement t o p h e n o l , arene e p o x i d e s a r e h y d r o l y z e d by e p o x i d e h y d r o l a s e t o t h e trans d i h y d r o d i o l (Oesch, 1972), w h i c h can i n t u r n be c o n v e r t e d by d i h y d r o d i o l dehydrogenase t o c a t e c h o l ( K a l f , 1987; B i l l i n g s , 1985). Arene e p o x i d e s a r e r e a c t i v e e l e c t r o p h i l e s ( J e r i n a and D a l y , 1974) w h i c h have been i m p l i c a t e d i n c o v a l e n t b i n d i n g t o c e l l u l a r n u c l e o p h i l e s ( R i l e y e t a l . , 1989; Gerson e t a l . , 1983). However, r e p o r t s i n t h e l i t e r a t u r e s u p p o r t t h e v i e w t h a t o t h e r r e a c t i v e s p e c i e s d i s t a l f rom t h e e p o x i d e and more i n t i m a t e l y r e l a t e d t o t h e s e c o n d a r y phenol m e t a b o l i t e c o u l d a l s o be i n v o l v e d i n c o v a l e n t b i n d i n g . F o r example, Tunek e t a l . (1978) o b s e r v e d no s i g n i f i c a n t r e a c t i o n between benzene e p o x i d e and microsomal p r o t e i n compared t o t h e p h e n o l , w h i c h prompted them t o c o n s i d e r t h e i n v o l v e m e n t o f a quinone i n t e r m e d i a t e . T h i s h y p o t h e s i s appears t o h o l d t r u e , b e i n g s u b s t a n t i a t e d by t h e c h e m i c a l c h a r a c t e r i z a t i o n o f t h e GSH c o n j u g a t e o f p - h y d r o x y p h e n o l formed m i c r o s o m a l l y from phenol (Tunek e t a l . , 1980), and t h e GSH c o n j u g a t e o f p h e n y l - p - h y d r o x y p h e n o l (Nakagawa and Tayama, 1989) as a b i l i a r y m e t a b o l i t e i n r a t s dosed w i t h o - p h e n y l p h e n o l . A l t h o u g h t h e a c t i o n o f e p o x i d e h y d r o l a s e d e t o x i f i e s arene e p o x i d e s t o t h e d i h y d r o d i o l , subsequent dehydrogenase a c t i v i t y on t h i s i n n ocuous s u b s t r a t e t o form t h e c a t e c h o l c o u l d be p e r c e i v e d as a t o x i f i c a t i o n pathway. The d i r e c t h y d r o x y l a t i o n o f 2-hydroxy e s t r o g e n s (2-HE, 16) t o c a t e c h o l s i s t h o u g h t t o be r e s p o n s i b l e f o r t h e c o v a l e n t b i n d i n g o f t h e l a t t e r t o p r o t e i n s ( P u r b a e t a l . , 1987). The u l t i m a t e s p e c i e s i n v o l v e d i n 23 b i n d i n g , however, i s c o n j e c t u r a l ( I r o n s and Sawahata, 1985) and c o u l d i n v o l v e a r a d i c a l s e miquinone (Marks and He c k e r , 1969) o r an epoxyphenol i n t e r m e d i a t e (Numazawa and Nambara, 1977) (Scheme V I I I ) . Two d e t o x i f i c a t i o n pathways a v a i l a b l e t o 2-HE appear t o be o p e r a t i v e . GSH f u l f i l s i t s r o l e as a d e t o x i c a n t by b i n d i n g t o t h e e l e c t r o p h i l i c i n t e r m e d i a t e s (Maggs e t a l . , 1983); t h u s , 1-, and 4 - g l u t a t h i o n - S - y l 2-h y d r o x y - e s t r a d i o l a d d u c t s o c c u r r e d as b i l i a r y m e t a b o l i t e s i n r a t s dosed w i t h e s t r a d i o l ( E l c e and H a r r i s , 1971) (Scheme V I I I ) . C a t e c h o l 0-m e t h y l t r a n s f e r a s e (COMT), on t h e o t h e r hand, m e t h y l a t e s t h e c a t e c h o l and t h e r e b y a b o l i s h e s f u r t h e r o x i d a t i o n t o e l e c t r o p h i l i c s p e c i e s . Thus, Haaf e t a l . (1987) o b s e r v e d a 70 % r e d u c t i o n o f c o v a l e n t b i n d i n g o f e s t r a d i o l t o hamster l i v e r microsomes when t h e i n c u b a t i o n m i x t u r e was e n r i c h e d w i t h COMT and S - a d e n o s y l - L - m e t h i o n i n e (SAM). The i m p o r t a n c e o f COMT i n t h e d e t o x i f i c a t i o n o f 2-hydroxy e s t r a d i o l ( L i e t a l . , 1989; Haaf e t a l . , 1987; S t r a m e n t i n o l i e t a l . , 1979) c o u l d i n t u r n , s i g n i f y t h e r e l e v a n c e o f 0-m e t h y l a t i o n as a c r u c i a l d e t o x i f i c a t i o n pathway f o r t h e c a t e c h o l m e t a b o l i t e s o f CLBZ. 24 Scheme V I I I . F o r m a t i o n o f r e a c t i v e i n t e r m e d i a t e s f r o m 2-hydroxy e s t r o g e n . Pathway (A) l e a d s t o t h e s e m i q u i n o n e and (B) t o t h e e p o x y p h e n o l . D e t o x i f i c a t i o n mechanisms i n v o l v e (1) GSH b i n d i n g and (2) COMT 0-m e t h y l a t i o n . 25 4. THE OBJECTIVES OF THE THESIS. S e v e r a l m e t a b o l i t e s o f CLBZ have been r e p o r t e d , a l b e i t u n s u b s t a n t i a t e d by p u b l i s h e d s p e c t r a l e v i d e n c e ( V o l z e t a l . , 1979). In a d d i t i o n , CLBZ c o u l d a l s o be b i o t r a n s f o r m e d i n t o r e a c t i v e c a r b i n o l a m i d e o r h y d r o x a m i c a c i d m e t a b o l i t e s . In o r d e r t o c h a r a c t e r i z e t h e m e t a b o l i t e s o f CLBZ w h i c h would i n c l u d e t h e i d e n t i f i c a t i o n and v e r i f i c a t i o n o f t r a c e and p o t e n t i a l l y r e a c t i v e s u b s t a n c e s , t h e f o l l o w i n g o b j e c t i v e s were p r o p o s e d . ( i ) To s y n t h e s i z e p e n t a d e u t e r i o p h e n y l CLBZ f o r t h e a d m i n i s t r a t i o n t o r a t s t o i d e n t i f y CLBZ m e t a b o l i t e s i n b i l e and u r i n e by u s i n g i s o t o p e c l u s t e r a n a l y s i s and GCMS. ( i i ) To p r o v i d e s p e c t r a l e v i d e n c e f o r t h e 4'-hydroxy, 4'-hydroxy-3'-methoxy and 3 ' , 4 ' - d i h y d r o d i o l d e r i v a t i v e s o f b o t h CLBZ and DMC. ( i i i ) To d e t e r m i n e whether N-hydroxymethyl CLBZ and\/or i t s c o n j u g a t e s e x i s t as m e t a b o l i t e s o f CLBZ. ( i v ) To d e t e r m i n e whether N-hydroxy DMC a nd\/or i t s c o n j u g a t e s e x i s t as m e t a b o l i t e s o f CLBZ. 26 I I EXPERIMENTAL 1. CHEMICALS AND MATERIALS. C h e m i c a l s were r e a g e n t g r a d e and p u r c h a s e d f r o m t h e f o l l o w i n g s o u r c e s : A l d r i c h C h e m i cal Co. ( M i l w a u k e e , W i s c o n s i n ) 2 , 4 - d i c h l o r o n i t r o b e n z e n e , 4 - a m i n o v e r a t r o l e ( 3 , 4 - d i m e t h o x y a n i l i n e ) , a c e t i c a n h y d r i d e , boron t r i b r o m i d e , c a l c i u m c h l o r i d e ( a n h y d r o u s ) , CDC!3, DC1 (37 wt. % s o l u t i o n i n D2O, 99 atom % D ) , d e u t e r i u m o x i d e (99.8 atom % D), f o r m a l d e h y d e (37 % s o l u t i o n ) , h y d r o c h l o r i c a c i d , i r o n powder, methyl i o d i d e , n i t r o b e n z e n e - d s (99 atom % D), p-a n i s i d i n e ( 4 - m e t h o x y a n i l i n e ) , p o t a s s i u m h y d r i d e (35 wt. % ) , t r i m e t h y l s i l y l i o d i d e , z i n c d u s t . A l l i e d C h e m i c al (New Y o r k , New Y o r k ) sodium a c e t a t e . Anachemia C h e m i c a l s L t d . ( M o n t r e a l , Quebec) e t h y l a c e t a t e ( d i s t i l l e d i n g l a s s ) . BDH I n c . ( V a n c o u v e r , B.C.) m e t h a n o l , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA), c h l o r o f o r m , benzene, e t h y l e t h e r , p e t r o l e u m e t h e r , magnesium c h l o r i d e ( h e x a h y d r a t e ) , p o t a s s i u m c h l o r i d e , sodium s u l f a t e ( a n h y d r o u s ) , s i l i c a g e l 60 (230 - 400 mesh), p h e n o b a r b i t o n e sodium, p o t a s s i u m h y d r o x i d e , p o t a s s i u m b i c a r b o n a t e , sodium c h l o r i d e , sodium hydrogen o r t h o p h o s p h a t e , sodium d i h y d r o g e n o r t h o p h o s p h a t e . 27 B i o - R a d L a b o r a t o r i e s (Richmond, C a l i f o r n i a ) a c r y l a m i d e N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e ( B I S ) 0 sodium d o d e c y l s u l f a t e (SDS) B o e h r i n g e r Mannheim ( M o n t r e a l , Quebec) ^ - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e , r e d u c e d (NADPH) ^ - n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e , r e d u c e d (NADH) b o v i n e serum al b u m i n (BSA). Cambridge I s o t o p e L a b o r a t o r i e s (Woburn, MA) d i m e t h y l - d ^ s u l f o x i d e . F i s h e r S c i e n t i f i c Co. ( F a i r l a w n , New J e r s e y ) q u i n o l i n e . H o e f e r S c i e n t i f i c I n s t r u m e n t s (San F r a n c i s c o , C a l i f o r n i a ) n i t r o c e l l u l o s e membrane. J.T.B a k e r ( P h i l ! i p s b u r g , New J e r s e y ) s i l i c a g e l (40 n p a r t i c l e d i a m e t e r ) . L i n d e Co. ( U n i o n C a r b i d e , Vancouver, B.C.) n i t r o g e n g a s . M a l l i n c k r o d t Chemical Works L t d . ( M o n t r e a l , Quebec) a c e t i c a c i d ( g l a c i a l ) . Matheson, Coleman and B e l l (Norwood, O h i o ) p o t a s s i u m c a r b o n a t e . 28 MSD I s o t o p e s ( M o n t r e a l , Quebec) methyl-03 i o d i d e . P i e r c e C h e m ical Co. ( R o c k f o r d , I l l i n o i s ) N - m e t h y l - N - t r i m e t h y l s i l y l - t r i f 1 u o r o a c e t a m i d e (MSTFA). Sigma Chemical Co. ( S t . L o u i s , M i s s o u r i ) d i m e t h y l s u l f o x i d e , g l u c u r a s e ( 6 - 4 8 8 2 ) , s u l f a t a s e ( S - 9 7 5 4 ) , S-a d e n o s y l - L - m e t h i o n i n e ( p - t o l u e n e s u f o n a t e s a l t ) , p r o p y l e n e g l y c o l , s u c r o s e , T r i s . H C l , u r e t h a n e . Stanchem L t d . ( W i n n i p e g , M a n i t o b a ) e t h a n o l (95 % ) . S u p e l c o ( O a k v i l l e , O n t a r i o ) 3 % D e x s i l 300 on 100\/120 mesh S u p e l c o p o r t . Whatman L t d . ( M a i d s t o n e , K e n t , England) s i l i c a g e l p l a t e s (AL S I L G\/UV). 2. ANIMALS AND SURGICAL EQUIPMENT. Animal C a r e F a c i l i t y (U.B.C., Vancouver, B.C.) Sprague Dawley male r a t s . F o r l i v e r m icrosomal p r e p a r a t i o n s : 200-225 g F o r in vivo d r u g m e t a b o l i s m : 250-275 g Be c t o n D i c k i n s o n ( R u t h e r f o r d , New J e r s e y ) V a c u t a i n e r tube 6430, Y a l e n e e d l e 19G 1 1\/2\"; 23G 1\", 25G 1\", t u b e r c u l i n s y r i n g e 1 c c . 29 C o e v a l , I n c . ( S t . J o s e p h , I l l i n o i s ) C orn cob g r a n u l e s E t h i c o n , I n c . ( S o m e r v i l l e , New J e r s e y ) 000 S i l k t h r e a d P u r i n a M i l l s I n c . , ( S t . L o u i s , M i s s o u r i ) P u r i n a L a b o r a t o r y Chow 5001 3. ACCESSORY EQUIPMENT. Beckman Model J2-21 c e n t r i f u g e w i t h JA-20 r o t o r Beckman Model L8-60M u l t r a c e n t r i f u g e w i t h 50.2 T i r o t o r Hakke SWB20 w a t e r - b a t h \/ s h a k e r H o e f e r TE 52 T r a n s p h o r u n i t w i t h power l i d P o t t e r - E l v e h j e m t i s s u e g r i n d e r . 4. INSTRUMENTATION. (A) NMR S p e c t r o s c o p y . A l l NMR s p e c t r o s c o p y was pe r f o r m e d i n t h e Department o f C h e m i s t r y , U n i v e r s i t y o f B r i t i s h C o l u m b i a . ^H-NMR s p e c t r o s c o p y was r e c o r d e d on e i t h e r a B r u k e r WH-400 (400 MHz) o r V a r i a n XL-300 (300 MHz) i n s t r u m e n t . 1 3C-NMR s p e c t r o s c o p y was r e c o r d e d on e i t h e r a V a r i a n XL-300 (75 MHz) o r a B r u k e r AC-200 (75 MHz) i n s t r u m e n t w i t h broad-band d e c o u p l i n g on t h e ^H- f r e q u e n c y , and an A t t a c h e d P r o t o n T e s t . 2H-NMR s p e c t r a were t a k e n i n CHCI3 s o l u t i o n on a B r u k e r WH-400 (84.68 MHz) i n s t r u m e n t and c h e m i c a l 30 s h i f t s measured r e l a t i v e t o an e x t e r n a l CDCI3 s t a n d a r d . A l l 1H-NMR and 1 ^ C-NMR s p e c t r a were d e t e r m i n e d i n CDCI3 s o l u t i o n , u n l e s s o t h e r w i s e s t a t e d , and t h e c h e m i c a l s h i f t s a r e i n p a r t s p e r m i l l i o n r e l a t i v e t o t e t r a m e t h y l s i l a n e e i t h e r as an i n t e r n a l o r e x t e r n a l s t a n d a r d . NMR s p e c t r a a r e r e c o r d e d as f o l l o w s : NMR S ( s p l i t t i n g t y p e , c o u p l i n g c o n s t a n t , i n t e g r a l , a s s i g n m e n t ) , e.g. ^ H-NMR 6 6.74 (dd, J = 10 Hz and 2 Hz, 1H, H-4). (B) Mass S p e c t r o s c o p y . ( i ) C a p i l l a r y GCMS. Gen e r a l o p e r a t i n g c o n d i t i o n s a p p l y t o a l l c a p i l l a r y GCMS a n a l y s e s , whereas s p e c i f i c c o n d i t i o n s a r e d e s c r i b e d as ( a ) , ( b ) , ( c ) and ( d ) . G e n e r a l o p e r a t i n g c o n d i t i o n s . C a p i l l a r y GCMS was performed on an HP 5987A GCMS system f i t t e d w i t h an HP-1 25 m x 0.32 mm i . d . x 0.52 \/ i c a p i l l a r y column w i t h a C r o s s l i n k e d M e t h y l S i l i c o n e Gum s t a t i o n a r y phase. The e l e c t r o n impact mode o f i o n i z a t i o n was used w i t h an en e r g y o f 70 eV, e m i s s i o n c u r r e n t o f 300 \/iA and m u l t i p l i e r v o l t a g e o f 2400 eV. Temperature zones were as f o l l o w s . I n j e c t i o n p o r t : 240\u00b0C, i n t e r f a c e oven: 240\u00b0C, GC i n t e r f a c e p r o b e : 250\u00b0C and i o n s o u r c e : 240\u00b0C. The He c a r r i e r gas was s e t a t a f l o w r a t e o f 1 ml\/min w i t h a back p r e s s u r e o f 10 p s i . I n j e c t i o n s were p e r f o r m e d i n t h e s p l i t l e s s mode. 31 S p e c i f i c o p e r a t i n g c o n d i t i o n s . (a) P r e l i m i n a r y c h a r a c t e r i z a t i o n o f b i l i a r y CLBZ m e t a b o l i t e s : Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 30*C\/min from 100\u00b0C t o 200\u00b0C, where i t was h e l d f o r 3 min, t h e n i n c r e a s e d a t 8\u00b0C\/min t o 280\u00b0C, where i t was h e l d f o r 12 min and t h e r e a f t e r a p o s t - r u n t e m p e r a t u r e o f 290\u00b0C h e l d f o r 2 min. T o t a l r un t i m e was 28.33 min. S c a n n i n g i n t h e l i n e a r mode commenced a f t e r 4 min and was o v e r t h e range o f 70 - 500 amu. E l e c t r o n m u l t i p l i e r v o l t a g e was 2,800 eV. GCMS s e n s i t i v i t y was o p t i m a l d u r i n g t h e p r e l i m i n a r y m e t a b o l i t e s t u d i e s . The mass s p e c t r a o b t a i n e d under t h e s e c o n d i t i o n s were o f t h e b e s t q u a l i t y and a r e used i n t h e F i g u r e s 28 t o 32. (b) R o u t i n e c h a r a c t e r i z a t i o n o f CLBZ m e t a b o l i t e s : Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 30\u00b0C\/min from 150\u00b0C t o 200\u00b0C, where i t was h e l d f o r 3 min, t h e n i n c r e a s e d a t 8\u00b0C\/min t o 280\u00b0C, where i t was h e l d f o r 12 min and t h e r e a f t e r a p o s t - r u n t e m p e r a t u r e o f 290\u00b0C h e l d f o r 3 min. T o t a l r un t i m e was 26.67 min. Sc a n n i n g i n t h e l i n e a r mode commenced a f t e r 12 min and was o v e r a range o f 70 - 550 amu. E l e c t r o n m u l t i p l i e r v o l t a g e was 2,400 eV. (c ) S e l e c t e d i o n m o n i t o r i n g f o r CLBZ and i t s m e t a b o l i t e s : Oven c o n d i t i o n s were programmed a c c o r d i n g t o p r o c e d u r e ( b ) . Ions scanned a r e s p e c i f i e d . Dwell t i m e was 100 m s e c \/ i o n . 32 (d) A n a l y s i s o f s y n t h e t i c compounds: Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 30\u00b0C\/min from 100\u00b0C t o 280\u00b0C, where i t was h e l d f o r 10 min and t h e r e a f t e r a p o s t - r u n t e m p e r a t u r e o f 290\u00b0C h e l d f o r 2 min. T o t a l run t i m e was 16 min. S c a n n i n g i n t h e l i n e a r mode commenced a f t e r 2.5 min and was o v e r t h e range o f 70 - 550 amu. E l e c t r o n m u l t i p l i e r v o l t a g e was 2,400 eV. ( i i ) Packed column GCMS. Packed column GCMS was performed on a V a r i a n MAT-111 MS i n t e r f a c e d t o an HP 5700A GC f i t t e d w i t h a 1.8 m x 2 mm i . d . g l a s s column packed w i t h 3 % D e x s i l 300 on 100\/120 mesh S u p e l c o p o r t . Data were r e c o r d e d u s i n g a P a c k a r d B e l l computer (IBM AT c l o n e ) and a program d e v e l o p e d i n o u r l a b o r a t o r y . Mass s p e c t r a were r e c o r d e d from m\/z 14 t o m\/z 750 a t 12 sca n s p e r m i n u t e . T o t a l i o n c u r r e n t (TIC) p l o t s were based on m\/z 50 -500. The e l e c t r o n impact mode o f i o n i z a t i o n was used w i t h an energy o f 70 eV and e m i s s i o n c u r r e n t o f 300 \/iA. I n j e c t i o n p o r t , l i n e and s e p a r a t o r t e m p e r a t u r e s were 250\"C. The He c a r r i e r gas was s e t a t a f l o w r a t e o f 25 mL\/min. I n j e c t i o n s were p e r f o r m e d i n t h e s p l i t l e s s mode. Column t e m p e r a t u r e was programmed t o i n c r e a s e a t 32\u00b0C\/min from 150\u00b0C t o 300\u00b0C, where i t was h e l d f o r 16 min. Mass s p e c t r a were r e c o r d e d as f o l l o w s : GCMS: r e t e n t i o n t i m e , P a r e n t i o n : ( R e l a t i v e i n t e n s i t y , % ) , O t h e r i o n ( s ) : m\/z ( % ) , e t c . e.g. MS: M + - ( 3 5 C 1 ) : m\/z 305 (100 % ) , M +' ( 3 7 C 1 ) : m\/z 307 (34 % ) , [ M - 1 7 ] + * : m\/z 288 (43 % ) , C 6 D 5 + ' : m\/z 82 (48 % ) . 33 (C) I n f r a r e d S p e c t r o s c o p y . IR s p e c t r a were r e c o r d e d on a M i c h e l son BOMEM MB-100 FT-S p e c t r o m e t e r . Samples were a n a l y z e d e i t h e r as a s o l u t i o n i n CCI4 i n NaCl c e l l s o f 0.5 mm p a t h l e n g t h o r as a N u j o l m u l l on NaCl p l a t e s . IR s p e c t r a were r e c o r d e d as f o l l o w s : IR (CCI4 s o l u t i o n \/ N u j o l m u l l ) f r e q u e n c y i n cm\"* ( i n t e n s i t y , a s s i g n m e n t ) , e.g. IR (CCI4 s o l u t i o n ) 1745 ( s , e s t e r c a r b o n y l g r o u p ) , 1693 ( s , amide c a r b o n y l g r o u p ) , 1534 ( s , N O 2 )asymmetric s t r e t c h , 1351 cm\"* ( s , N02)symmetric s t r e t c h . (D) M e l t i n g P o i n t . M e l t i n g p o i n t s were d e t e r m i n e d i n open c a p i l l a r y t u b e s on a Thomas Hoover C a p i l l a r y m e l t i n g p o i n t a p p a r a t u s ( P h i l a d e l p h i a , PA) and a r e u n c o r r e c t e d . (E) U l t r a v i o l e t S p e c t r o s c o p y . UV s p e c t r o s c o p y was pe r f o r m e d on an HP Diode A r r a y 8452A S p e c t r o p h o t o m e t e r i n t e r f a c e d t o an HP V e c t r a Computer. Samples were a n a l y z e d i n q u a r t z c e l l s o f p a t h l e n g t h 1 cm. (F) High P r e s s u r e L i q u i d Chromatography. HPLC a n a l y s i s was done on an HP 1050 S e r i e s System c o n s i s t i n g o f Q u a t e r n a r y Pump and M u l t i p l e Wavelength D e t e c t o r (254 nm) f i t t e d w i t h a H y p e r s i l ODS 5 \/1, 200 cm x 4.6 mm i . d . column. A l l chromatography was 34 p e r f o r m e d i s o c r a t i c a l l y . Flow r a t e s were c o n t r o l l e d a t 1 mL\/min and m o b i l e phase c o m p o s i t i o n s a r e s p e c i f i e d . (G) L i q u i d Chromatography-Mass S p e c t r o s c o p y . LCMS a n a l y s i s was performed on a K r a t o s MS-80 mass s p e c t r o m e t e r c o n s i s t i n g o f a Waters pump and a V e s t e c t h e r m o s p r a y i n t e r f a c e . The system was f i t t e d w i t h an ODS 200 cm x 4.6 mm i . d . column c o n n e c t e d t o a Rheodyne i n j e c t o r . Ammonium a c e t a t e (1 M) was used t o f a c i l i t a t e p o s t -column i o n i z a t i o n . Temperature s e t t i n g s were as f o l l o w s : p r o b e , 114\u00b0C; v a p o r , 172\u00b0C; b l o c k , 219\u00b0C and j e t , 249\u00b0C. The c o m p o s i t i o n o f t h e m o b i l e phase was 50:50 (v \/ v ) Me0H:H20. 5. METABOLISM EXPERIMENTS. (A) Dosage Regimen. CLBZ:[H5]CLBZ was p r e p a r e d as an a p p r o x i m a t e 50:50 (w\/w) m i x t u r e , w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f 53.2:46.8 f o r mass chromatograms a t m\/z 300 and 305 r e s p e c t i v e l y , i . e . CLBZ\/[ 2H5]CLBZ = 1.137. The m i x t u r e was d i s s o l v e d i n p r o p y l e n e g l y c o l (10 mg\/mL) and a d m i n i s t e r e d i . p . a t 10 mg\/kg t o t h r e e r a t s e v e r y 6 h f o r 6 d o s e s . One o t h e r animal was a d m i n i s t e r e d t h e v e h i c l e ( p r o p y l e n e g l y c o l c o n t r o l ) . 35 (B) S u r g e r y and C o l l e c t i o n o f U r i n e and B i l e . A n i m a l s were housed i n s t a i n l e s s s t e e l m e t a b o l i c cages and were a l l o w e d a c c e s s ad libitum t o fo o d and w a t e r . U r i n e c o l l e c t i o n commenced a t t h e f i r s t dose and c o n t i n u e d u n t i l t h e t i m e o f s a c r i f i c e . C o l l e c t e d u r i n e was p e r i o d i c a l l y removed and s t o r e d a t - 2 0 T u n t i l work-up. A f t e r t h e f i f t h dose o f CLBZ:[ 2 Hs]CLBZ, r a t s were a n e s t h e t i z e d w i t h u r e t h a n e (1.2 g\/kg) wh i c h was a d m i n i s t e r e d i . p . as a s o l u t i o n i n d i s t i l l e d w a t e r (0.4 g\/mL). A f t e r a n e s t h e s i a was i n d u c e d ( c a 30 min) an i n c i s i o n o f 3 cm was made on t h e v e n t r a l s u r f a c e o f t h e animal l e f t o f t h e abdominal m i d - l i n e toward t h e diaphragm. The i n t e s t i n e s were d i s p l a c e d , t h e p o r t i o n o f t h e b i l e d u c t a t t h e j u n c t i o n w i t h t h e duodenum, l o c a t e d and s u r r o u n d i n g t i s s u e g e n t l y removed w i t h c o t t o n w o o l . A p u n c t u r e was made i n t o t h e duodenum w i t h a 25G1 n e e d l e t o a c c e s s t h e b i l e d u c t . Through t h i s a p e r t u r e a b e v e l l e d c a n n u l a o f PE-10 t u b i n g was i n s e r t e d 2 cm i n t o t h e b i l e d u c t , l i g a t e d a t t h e duodenal j u n c t i o n w i t h s i l k t h r e a d , and e x t e r i o r i z e d t h r o u g h t h e l o w e r abdominal w a l l g u i d e d by t h e bore o f an e x t e r n a l l y i n s e r t e d 19G1 1\/2 n e e d l e . The d i s p l a c e d i n t e s t i n e s were r e l o c a t e d i n t o t h e abdominal c a v i t y and t h e i n c i s i o n c l o s e d w i t h i n t e r r u p t e d s u t u r e s . B i l e was c o l l e c t e d i n s c i n t i l l a t i o n v i a l s , and u r i n e , a t t h a t t i m e , c o l l e c t e d i n u n s t o p p e r e d V a c u t a i n e r ^ t u b e s s e c u r e d t o t h e u r e t h r a o f t h e animal w i t h t a p e . B i l e and u r i n e were p e r i o d i c a l l y e mptied f o r s t o r a g e a t -20\u00b0C u n t i l work-up. B i l e was c o l l e c t e d f o r 18 h a t which p o i n t t h e a n i m a l , w h i c h was s t i l l u nder a n e s t h e s i a , was s a c r i f i c e d by d e c a p i t a t i o n . 36 (C) P r e p a r a t i o n o f B i l e and U r i n e f o r GCMS. B i l e (2.5 mL) and u r i n e (5 mL) samples were e x t r a c t e d (x 4) w i t h an e q u a l volume o f EtOAc t o remove u n c o n j u g a t e d m e t a b o l i t e s . The o r g a n i c e x t r a c t was s t o r e d a t 4\u00b0C o v e r anhydrous Na2S04 u n t i l f u r t h e r p r e p a r a t i o n . The aqueous f r a c t i o n s o f b i l e (pH 9.0) and u r i n e (pH 7.1) were a d j u s t e d t o pH 5.0 w i t h sodium a c e t a t e b u f f e r , e n r i c h e d w i t h e i t h e r g l u c u r a s e (1,500 U) o r s u l f a t a s e (60 U) and i n c u b a t e d w i t h g e n t l e s h a k i n g i n a w a t e r b a t h a t 37\u00b0C f o r 20 h. The d e c o n j u g a t e d m e t a b o l i t e s were e x t r a c t e d w i t h an equal volume o f EtOAc (x 4) and t h e o r g a n i c e x t r a c t s d r i e d o v e r anhydrous Na2S04 o v e r n i g h t a t 4\u00b0C. The d r i e d o r g a n i c e x t r a c t s o f t h e u n c o n j u g a t e d and d e c o n j u g a t e d m e t a b o l i t e s were c o n c e n t r a t e d t o d r y n e s s i n a w a t e r b a t h a t ca 30\u00b0C under a s t r e a m o f n i t r o g e n . F o r d e r i v a t i z a t i o n , t h e e x t r a c t s were r e c o n s t i t u t e d i n d r y EtOAc (25 \/ i L ) , MSTFA (25 \/xL) added, and t h e m i x t u r e h e a t e d a t 45\u00b0C f o r 90 min. I n j e c t i o n volume f o r GCMS: 3 \/xL. (D) P r e p a r a t i o n and A n a l y s i s o f L i v e r F r a c t i o n s . A n i m a l s were housed i n s t a i n l e s s s t e e l cages w i t h c o r n cob b e d d i n g , and p r o v i d e d a c c e s s ad libitum t o f o o d and w a t e r u n t i l t h e t i m e o f s a c r i f i c e . R e g u l a r 12 h c y c l e s o f l i g h t and d a r k were p r o v i d e d . F o u r a n i m a l s were i n t r a p e r i t o n e a l l y a d m i n i s t e r e d p h e n o b a r b i t o n e sodium (26 mg\/mL) a t 78 mg\/kg-body w e i g h t i n an i s o t o n i c s a l i n e s o l u t i o n once d a i l y f o r 4 d a y s . Four o t h e r a n i m a l s were a d m i n i s t e r e d t h e v e h i c l e ( s a l i n e c o n t r o l ) . A n i m a l s were s a c r i f i c e d by d e c a p i t a t i o n . L i v e r s were i m m e d i a t e l y removed, s h e a r e d w i t h a s c i s s o r s and homogenized i n i c e - c o l d 37 T r i s - H C l (0.05 M)\/KC1 (1.15 %) b u f f e r (pH 7.5) w i t h a P o t t e r - E l v e h j e m t i s s u e homogenizer. The homogenate was c e n t r i f u g e d a t 9,000 g f o r 20 min a t 5\u00b0C and f i l t e r e d t h r o u g h cheese c l o t h . The r e s i d u e was d i s c a r d e d , and t h e s u p e r n a t a n t (S9 f r a c t i o n ) c e n t r i f u g e d a t 105,000 g f o r 60 min a t 5\u00b0C. The c y t o s o l i c f r a c t i o n was r e c o v e r e d as t h e s u p e r n a t a n t and s t o r e d a t -80\u00b0C u n t i l r e q u i r e d , w h i l e t h e microsomal p e l l e t was res u s p e n d e d i n EDTA (10 mM)\/KCl (1.15 %) b u f f e r (pH 7.4). T h i s homogenate was a g a i n c e n t r i f u g e d a t 105,000 g f o r 60 min a t 5\u00b0C. The s u p e r n a t a n t was d i s c a r d e d and t h e microsomal p e l l e t , a f t e r r e s u s p e n s i o n i n an a p p r o x i m a t e l y equal volume o f 0.25 M s u c r o s e s o l u t i o n , s t o r e d a t -80\u00b0C u n t i l r e q u i r e d . Microsomes were d i l u t e d 1:20 i n N a 2 P 0 4 (0.1 M, pH 7 . 4 ) \/ g l y c e r o l (20 %)\/EDTA (0.1 mM) f o r d e t e r m i n a t i o n o f cytochrome P-450 c o n t e n t u s i n g t h e method o f Omura and Sa t o ( 1 9 6 4 ) . Cytochrome P-450 l e v e l s were 18.9 nmol\/mL and 33.9 nmol\/mL f o r c o n t r o l and i n d u c e d microsomes r e s p e c t i v e l y . Cytochrome P-450 a s s a y s were performed a t t h e tim e t h e microsomes were t o be used. P r o t e i n c o n c e n t r a t i o n s o f microsomal and c y t o s o l i c f r a c t i o n s were p e r f o r m e d a c c o r d i n g t o t h e method o f Lowry e t a l . (1951) u s i n g BSA as s t a n d a r d . P r o t e i n c o n c e n t r a t i o n s f o r t h e d i f f e r e n t f r a c t i o n s were as f o l l o w s : c o n t r o l m i c r o s o m a l : 20.8 mg\/mL, c o n t r o l c y t o s o l i c : 16.1 mg\/mL, i n d u c e d m i c r o s o m a l : 18.6 mg\/mL and i n d u c e d c y t o s o l i c : 20.4 mg\/mL. Gel e l e c t r o p h o r e s i s was performed a c c o r d i n g t o t h e method o f Laemmli ( 1 9 7 0 ) , u s i n g 7.5 % sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE). A p p r o x i m a t e l y 10 ug o f microsomal p r o t e i n was l o a d e d on t o each w e l l . The i m m u n o b l o t t i n g p r o c e d u r e o f Towbin e t a l . (1979) was used f o r t h e t r a n s f e r o f s e p a r a t e d microsomal p r o t e i n from t h e g e l t o a n i t r o c e l l u l o s e membrane. Development o f t h e b l o t was a c c o m p l i s h e d w i t h 38 t h e s t e p w i s e use o f r a b b i t a n t i - P - 4 5 0 b\/e p o l y c l o n a l a n t i b o d y as t h e p r i m a r y a n t i b o d y , and p e r o x i d a s e c o n j u g a t e d g o a t a n t i - r a b b i t as t h e s e c o n d a r y a n t i b o d y a t a d i l u t i o n o f 1:25,000. (E) M i c r o s o m a l I n c u b a t i o n s . ( i ) G e n e r a l i n c u b a t i o n p r o c e d u r e . A l l cytochrome P-450 i n c u b a t i o n s i n v o l v e d t h e use o f b o t h c o n t r o l and P B - i n d u c e d microsomes, and were pe r f o r m e d a c c o r d i n g t o t h e method o f R e t t i e e t a l . ( 1 9 8 8 ) . I n t o a c u l t u r e tube were p l a c e d sodium phosphate b u f f e r (0.2M, pH 7.4): 1 mL, M g C l 2 (0.3 M): 50 xxL, NADPH (0.1 M): 50 \/xL, NADH (0.1 M): 50 \/xL, microsomal cytochrome P-450 (10 nmol) and d i s t i l l e d w a t e r t o a f f o r d a f i n a l volume o f 2.1 mL. The enzyme was a l l o w e d t o i n c u b a t e f o r 3 min, a f t e r w h i c h s u b s t r a t e (2 \/xmol) was added i n a 10 % m e t h a n o l i c s u s p e n s i o n . The r e a c t i o n was t e r m i n a t e d by f r e e z i n g t h e m i x t u r e i n an i c e - b a t h . A f t e r 5 min t h e r e a c t i o n m i x t u r e was e x t r a c t e d w i t h an e q u a l volume o f EtOAc (x 4 ) . C e n t r i f u g a t i o n was i n v a r i a b l y r e q u i r e d t o s e p a r a t e t h e e m u l s i o n . The o r g a n i c e x t r a c t was d r i e d o v e r anhydrous Na2S04, c o n c e n t r a t e d t o d r y n e s s under a s t r e a m o f N 2 , r e c o n s t i t u t e d i n 25 \/xL o f EtOAc and d e r i v a t i z e d w i t h 25 \/xL MSTFA a t 45\u00b0C f o r 90 min. I n j e c t i o n volume: 3 \/xL. ( i i ) S p e c i f i c i n c u b a t i o n c o n d i t i o n s . (a) M i c r o s o m a l m e t a b o l i s m o f CLBZ t o i n v e s t i g a t e c a r b i n o l a m i d e f o r m a t i o n . The s u b s t r a t e , CLBZ:[ 2H5]CLBZ, was p r e p a r e d as a m i x t u r e w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f 37.8:62.2 f o r mass chromatograms a t m\/z 300 and 305 r e s p e c t i v e l y , i .e . C L B Z \/ [ 2 r l s ] C L B Z = 0.608. I n c u b a t i o n t i m e s were 15 min and 60 min. (b) M i c r o s o m a l m e t a b o l i s m o f DMC t o i n v e s t i g a t e h ydroxamic a c i d f o r m a t i o n . The s u b s t r a t e , DMC:[ 2H5]DMC, was p r e p a r e d as a m i x t u r e w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f i . e . 45.0:55.0 f o r t h e mass chromatograms o f t h e T M S - d e r i v a t i v e s a t m\/z 358 and 363 r e s p e c t i v e l y , i . e . DMC\/[ 2H5JDMC = 0.818. I n c u b a t i o n t i m e s were p e r f o r m e d o v e r 15 and 60 min. ( c ) D e t e r m i n a t i o n o f N - d e m e t h v l a t i o n i n t e r m o l e c u l a r i s o t o p e e f f e c t . [ 2 H 5 ] C L B Z : [ 2 H 3 ] C L B Z was p r e p a r e d as an a p p r o x i m a t e 50:50 (w\/w) m i x t u r e , w h i c h a f f o r d e d a GCMS peak a r e a r a t i o o f 54.1:45.9 f o r mass chromatograms a t m\/z 305 and 303 r e s p e c t i v e l y , i . e . [ 2H5]CLBZ\/[ 2H3]CLBZ = 1.178. I n c u b a t i o n s were performed o v e r 15, 30, 45 and 60 min. 40 6. CHEMICAL SYNTHESES. (A) Medium p r e s s u r e l i q u i d ( F l a s h ) chromatography. P u r i f i c a t i o n s i n v o l v i n g medium p r e s s u r e column chromatography were p e r f o r m e d a c c o r d i n g t o t h e method o f S t i l l e t a l . (1978) on s i l i c a columns o f d i a m e t e r s 1.5 cm, 2.5 cm o r 5.5 cm, and v a r i a b l e l e n g t h d e p e n d i n g on t h e amount o f m a t e r i a l t o be c h romatographed. M o b i l e phases were p r e p a r e d i n o r d e r t o a f f o r d an Rf o f 0.3 - 0.5 f o r t h e a n a l y t e on TLC. I f improved r e s o l u t i o n was n e c e s s a r y , t h e p o l a r i t y o f t h e m o b i l e phase was i n c r e a s e d i n a g r a d i e n t f a s h i o n d u r i n g e l u t i o n . (B) S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l c l o b a z a m ( T ^ I C L B Z ) . A n i l i n e - d y ( 1 7 ) . I n t o a t h r e e - n e c k e d f l a s k e q u i p p e d w i t h a m e c h a n i c a l s t i r r e r and a d r o p p i n g f u n n e l f i l l e d w i t h DC1 (15 mL, 16.5 mmol) were p l a c e d nitrobenzene - d s (20.2 g, 15.8 mmol), D2O (50 mL) and i r o n powder (24.2 g, 46.9 mmol). The r e a c t i o n f l a s k was f l u s h e d w i t h N 2 and s e a l e d under t h e s l i g h t p o s i t i v e p r e s s u r e o f a N 2 f i l l e d b a l l o o n a t t a c h e d t o a r e f l u x c o n d e n s e r . To t h e s t i r r e d m i x t u r e was added DC1 a t a r a t e t o m a i n t a i n t h e t e m p e r a t u r e below 80\u00b0C. When t h e a d d i t i o n o f DC1 was c o m p l e t e t h e m i x t u r e was s t i r r e d a t 80\u00b0C f o r 12 h. The r e s u l t i n g m i x t u r e was c o o l e d i n an i c e - b a t h and t h e pH a d j u s t e d t o 12 w i t h NaOD w h i l e m a i n t a i n i n g t h e r e a c t i o n m i x t u r e under N 2 . Under a N 2 atmosphere s o l i d s were removed by f i l t r a t i o n and t h e f i l t r a t e appeared as a 41 y e l l o w i s h - g r e e n l i q u i d w i t h a suspended p a l e brown o i l . KC1 ( c a . 10 g) was added t o t h e f i l t r a t e t o \" s a l t o u t \" aqueous a n i l i n e w h i c h was e x t r a c t e d w i t h e t h e r and c o n c e n t r a t e d in vacuuo t o a r u s t c o l o u r e d o i l . F r a c t i o n a l d i s t i l l a t i o n gave 12.7 g (80 %) o f ani l i n e - d z as a s l i g h t l y v i s c o u s c o l o u r l e s s l i q u i d : bp 28 - 30\"C (0.4 t o r r ) . 2H-NMR S 3.47 ( s , 1.80D*, -ND 2), 6.60 ( s , 2D, ortho), 6.78 ( s , ID, para), 7.17 ( s , 2D, meta). (*N,N-D2 i s o t o p i c p u r i t y c a l c u l a t e d a t 90 % from i n t e g r a l ) . N - ( 5 - C h l o r o - 2 - n i t r o p h e n v l ) - p e n t a d e u t e r i o p h e n y l a m i n e ( 1 8 ) . The p e n t a d e u t e r i o p h e n y l a m i n e 18 was s y n t h e s i z e d by u s i n g a p p r o p r i a t e m o d i f i c a t i o n s t o t h e p r o c e d u r e o f Werner and Wagner ( 1 9 7 8 ) . Dry d i c h l o r o n i t r o b e n z e n e (2.23 g, 11.6 mmol) was d i s s o l v e d i n 17 (4.60 g, 3.6 e q u i v ) t o a f f o r d an orange s o l u t i o n . The s t i r r e d s o l u t i o n was heat e d a t 170 - 180\u00b0C under r e f l u x i n a N 2 atmosphere f o r 8.5 h and b e f o r e c o o l i n g , quenched under N 2 w i t h c o n t i n u e d s t i r r i n g by c a u t i o u s a d d i t i o n o f c a . 10 mL o f d r y benzene. The r e s u l t i n g b l a c k s o l u t i o n was a l l o w e d t o r e a c h room t e m p e r a t u r e and f u r t h e r d i l u t e d w i t h an a d d i t i o n a l 30 mL d r y benzene. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (15 cm x 5.5 cm) u s i n g t h e f o l l o w i n g g r a d i e n t e l u t i o n : benzene (100 mL); 2:1 ( v \/ v ) p e t r o l e u m e t h e r : e t h e r (240 mL); 3:2 ( v \/ v ) p e t r o l e u m e t h e r : e t h e r (625 mL). The p r o d u c t - c o n t a i n i n g f r a c t i o n s were p o o l e d and s o l v e n t removed in vacuuo t o a f f o r d 2.69 g (91 %) o f 18 as b r i g h t orange n e e d l e l i k e c r y s t a l s : mp 109 - HO'C. Rf 0.79, m o b i l e phase, 2:1 ( v \/ v ) p e t r o l e u m e t h e r : E t O A c . !H-NMR S 6.74 (dd, J = 10 Hz and 2 Hz, 1H, H-4), 7.16 ( d , J = 2 Hz, 1H, 42 H-6), 7.27 - 7.31 (m, 0.23H*, H-2'\/H-6' and H-4'), 8.18 ( d , J = 10 Hz, 1H, H-3), 9.52 ( b s , 1H, N-H #). ( i n t e g r a t i o n i n d i c a t e d t h a t 8 % p r o t o n a t i o n o f t h e ortho and para p o s i t i o n s had t a k e n p l a c e . ^Does not exchange w i t h D2O) . 1 3C-NMR 8 114.99 ( C - 4 ) , 117.73 ( C - 6 ) , 124.26 ( t , C-2'\/C-6'), 125.77 ( t , C-4'), 127.97 ( C - 3 ) , 129.35 ( t , C-3'\/C-5'), 131.32 ( C - l ' ) , 137.59 ( C - l ) , 142.34 ( C - 5 ) , 143.75 ( C - 2 ) . GCMS ( d ) : t R , 5.35 min, M +'( 3 5C1): m\/z 253 (100 % ) , M + - ( 3 7 C 1 ) : m\/z 255 (37 % ) . IR (CCI4 s o l u t i o n ) 3345 (N-H), 1487 (N0 2)asymm, 1310 cm\" 1 (N0 2)symm. E t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n v l ) - N - p e n t a d e u t e r i o p h e n y l carbamoyl a c e t a t e H D . . To a s t i r r e d s o l u t i o n o f 18 (2.76 g, 10.9 mmol) i n c a . 25 mL d r y benzene was added e t h y l m a l o n y l c h l o r i d e (4.12 g, 27.3 mmol) and t h e r e s u l t i n g s o l u t i o n h e a t e d a t 75 - 80\u00b0C under r e f l u x f o r 20 h. The m i x t u r e was washed w i t h s a t u r a t e d NaHC03, d r i e d , and c o n c e n t r a t e d t o a p a l e brown o i l . The c r u d e p r o d u c t was chromatographed on a s i l i c a column (15. cm x 5.5 cm) u s i n g t h e f o l l o w i n g g r a d i e n t : benzene (100 mL); 2:1 ( v \/ v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (450 mL); 3:2 (v\/ v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (250 mL). P r o d u c t - c o n t a i n i n g f r a c t i o n s were p o o l e d and c o n c e n t r a t e d t o a f f o r d 3.31 g (85 %) o f 19 as p a l e y e l l o w c r y s t a l s : mp 90 - 92\u00b0C. Rf 0.49, m o b i l e phase, 2:1 ( v \/ v ) p e t r o l e u m e t h e r : E t 0 A c . ^-NMR 6 1.26 ( t , 3H, -CH 2CH 3), 3.40 ( s , 2H, -C(=0)CH 2C=0), 4.17 ( q , 2H, -0CH 2CH 3), 7.21 ( d , J = 1 Hz, 1H, H-6), 7.35 ( d d , J = 9 Hz and 1 Hz, 1H, H-4), 7.93 ( d , J = 9 Hz, 1H, H-3). 43 GCMS ( d ) : t R , 5.34 min, [M-114] + - ( 3 5C1): m\/z 253 (100 % ) , [M-114]\" 1\"' ( 3 7 C 1 ) : m\/z 255 (37 % ) . I R (CCI4 s o l u t i o n ) 1745 ( e s t e r c a r b o n y l g r o u p ) , 1693 (amide c a r b o n y l g r o u p ) , 1534 (N02)asymm, 1351 cm\" 1 (N02)symm. P e n t a d e u t e r i o p h e n y l N-desmethvlclobazam ( 2 0 ) . To a s t i r r e d s u s p e n s i o n o f 19 (2.0 g, 5.45 mmol) i n 17 mL e t h a n o l and c o n c e n t r a t e d HCl (10 mL, 110 mmol) was c a u t i o u s l y added Zn d u s t (1.74 g, 26.6 mmol) o v e r 30 min w h i l e m a i n t a i n i n g t h e t e m p e r a t u r e between 25 - 30\u00b0C. The m i x t u r e was s t i r r e d a t 30\u00b0C f o r an a d d i t i o n a l 2 h t o a f f o r d 20 as a w h i t e p r e c i p i t a t e . The p r o d u c t was removed, t h e s u p e r n a t a n t t r e a t e d w i t h a d d i t i o n a l Zn (0.58 g, 8.9 mmol) as p r e v i o u s l y d e s c r i b e d , and s t i r r i n g c o n t i n u e d o v e r n i g h t a t 30\u00b0C t o a f f o r d a second c r o p o f 20. The combined p r o d u c t was washed w i t h c o l d 3:2 ( v \/ v ) ac e t o n e : w a t e r and d r i e d in vacuuo t o a f f o r d 0.56 g (35 %) o f 20 as an amorphous w h i t e s o l i d . ! H - N M R 8 3.55 ( s , 2H, C H 2 ) , 6.93 ( d , J = 3 Hz, 1H, H-6), 7.11 ( d , J = 9 Hz, 1H, H-9), 7.21 (dd, J = 9 Hz and 3 Hz, 1H, H-8), 8.21 ( b s , 1H, N-H). GCMS ( d ) : t R , 8.64 min, M + - ( 3 5 C 1 ) : m\/z 291 (92 % ) , M + - ( 3 7 C 1 ) : m\/z 293 (30 % ) , [M-42] + - ( 3 5 C l ) : m\/z 249 (84 % ) , C 6 D 5 + - : m\/z 100 (41 % ) . TMS d e r i v a t i v e : t R , 7.34 min, M + - ( 3 5 C 1 ) : m\/z 363 (26 % ) , M + - ( 3 7 C 1 ) m\/z 365 (10 % ) , [ M - 1 5 ] + - m\/z 348 (20 % ) , [ M - 1 2 4 ] + - m\/z 239 (46 % ) , [ M - 1 3 8 ] + - m\/z 225 (100 % ) . I R ( N u j o l m u l l ) 3176 (N-H), 1691 and 1673 cm\" 1 (amide c a r b o n y l g r o u p s ) . 44 P e n t a d e u t e r i o p h e n y l clobazam ( 2 1 ) . M e t h y l a t i o n o f 20 was per f o r m e d a c c o r d i n g t o t h e method o f I s e l e and L u t t r i n g h a u s ( 1 9 7 1 ) . To an i c e - c o o l e d (15\u00b0C) s o l u t i o n o f 20 (491 mg, 1.68 mmol) and p u l v e r i z e d KOH (114 mg, 2.05 mmol) i n 6 mL DMSO was added CH3I (193 ZJL, 3.06 mmol) w i t h s t i r r i n g . The r e a c t i o n was m a i n t a i n e d a t t h i s t e m p e r a t u r e f o r 30 min a f t e r w h i c h i t was a l l o w e d t o r e a c h room t e m p e r a t u r e w i t h c o n t i n u e d s t i r r i n g f o r an a d d i t i o n a l 90 min. The r e a c t i o n m i x t u r e was d i l u t e d w i t h 300 mL e t h y l a c e t a t e , washed w i t h a s a t u r a t e d NaCl s o l u t i o n , d r i e d o v e r anhydrous Na2S04, and c o n c e n t r a t e d t o a y e l l o w o i l . The crude p r o d u c t was d i s s o l v e d i n ca 10 mL benzene and chromatographed on a s i l i c a column (15 cm x 2.5 cm) u s i n g 210 mL o f 20:1 ( v \/ v ) CHCl3:Me0H. D e s p i t e s e v e r a l m o d i f i c a t i o n s t o t h e c h r o m a t o g r a p h i c c o n d i t i o n s , u n r e a c t e d 20 c o u l d n ot be s e p a r a t e d from 21 i n i t s l a t t e r f r a c t i o n s . Hence, t h e m i x t u r e was c o n c e n t r a t e d and r e -chromatographed. F r a c t i o n s c o n t a i n i n g o n l y 21 were c o n c e n t r a t e d t o a p a l e y e l l o w o i l and t h e p r o d u c t r e c r y s t a l l i z e d from e t h y l a c e t a t e \/ p e t r o l e u m e t h e r t o a f f o r d 188 mg (37 %) o f 21 i n q u a n t i t a t i v e i s o t o p i c p u r i t y as w h i t e c r y s t a l s : mp 182 - 185\u00b0C. *H-NMR (CDCI3) 6 3.50 ( d , J = 10 Hz, 1H, H-30*), 3.51 ( s , 3H, N-CH3), 3.56 ( d , J = 10 Hz, 1H, H-3a*), 6.93 ( d , J = 3 Hz, H-6), 7.25 (dd, J = 10 Hz and 3 Hz, 1H, H-8), 7.31 ( d , J = 10 Hz, 1H, H-9). (*Assignments c o n s i s t e n t w i t h t h e c o n f o r m a t i o n a l s t r u c t u r e p r o p o s e d by A v e r s a e t a l . ( 1 9 8 0 ) . !H-NMR (C 6D6) 5 2.81 ( d , J = 12 Hz, 1H, H-3\/3), 2.95 ( s , 3H, N-CH 3), 3.46 ( d , J = 12 Hz, 1H, H-3a), 6.38 ( d , J = 9 Hz, 1H, H-9), 6.74 (dd, J = 9 Hz and 3 Hz, 1H, H-8), 6.79 ( d , J = 3 Hz, H-6). 45 MS: M +'( 3 5C1): m\/z 305 (100 % ) , M +- ( 3 7 C 1 ) : m\/z 307 (34 % ) , [ M - 1 7 ] + - : m\/z 288 (43 % ) , CeD5+-: m\/z 82 (48 % ) . IR (CCI4 s o l u t i o n ) 1711 and 1686 cm\"* (amide c a r b o n y l g r o u p s ) . HPLC: The r e c r y s t a l l i z e d 21 was a n a l y z e d by HPLC u s i n g a m o b i l e phase o f 60:40 ( v \/ v ) MeOH:H20 and found t o have a r e t e n t i o n t i m e o f 6.17 min and p u r i t y o f >98 %. (C) S y n t h e s i s o f T r i d e u t e r i o m e t h v l c l o b a z a m ( T ^ I C L B Z ) (22). I2M3lCLBZ (22) was s y n t h e s i z e d from DMC (101 mg, 0.35 mmol) and CD3I u s i n g t h e approach used f o r 21. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 cm x 1.25 cm) u s i n g a m o b i l e phase o f 20:1 ( v \/ v ) Me0H:CHCl3 (60 mL). E a r l y f r a c t i o n s c o n t a i n i n g pure 22 were c o n c e n t r a t e d t o a y e l l o w o i l wh i c h was c r y s t a l l i z e d from EtOAc:pet e t h e r t o a f f o r d 34 mg (32 %) o f w h i t e n e e d l e l i k e c r y s t a l s . The y i e l d on t h i s s y n t h e s i s c o u l d have been improved i f t h e l a t t e r f r a c t i o n s c o n t a i n i n g 22 and c o n t a m i n a t i n g DMC were r e c h r o m a t o g r a p h e d , however, no a t t e m p t was made t o do so. JH-NMR 6 3.52 ( d , J = 12 Hz, 1H, H-30), 3.58 ( d , J = 12 Hz, 1H, H-3a), 6.94 ( d , J = 3 Hz, H-6), 7.20 (dd, J = 8 Hz and 2 Hz, 2H, H-2', H-6'), 7.24 (dd, J = 10 Hz and 2 Hz, 1H, H-8), 7.31 ( d , J = 10 Hz, 1H, H-9), 7.36 (m, 1H, H-4'), 7.43 (m, 2H, H-3', H-5'). MS: M + ' ( 3 5 C 1 ) : m\/z 303 (100 % ) , M+- ( 3 7 C 1 ) : m\/z 305 (34 % ) , [M-17] + *: m\/z 286 (51 % ) , [ M - 6 0 ] + - : m\/z 243 (22 % ) , [ M - 1 1 9 ] + ' : m\/z 184 (23 % ) . 46 (D) S y n t h e s i s o f R i n g - O x i d i z e d M e t a b o l i t e s o f CLBZ. ( i ) C h e m i c al S y n t h e s e s . N - ( 5 - C h 1 o r o - 2 - n i t r o p h e n y l ) - 4 - m e t h o x y p h e n y l a m i n e ( 2 3 ) . 4-Methoxyphenylamine (11.38 g, 92.5 mmol) and 2 , 4 - d i c h l o r o -n i t r o b e n z e n e (4.96 g, 25.8 mmol) were f u s e d , and h e l d under r e f l u x a t 170 - 180\u00b0C f o r 9 h. The r e s u l t i n g s o l u t i o n was d i l u t e d w h i l e h ot w i t h benzene and t h e c r u d e p r o d u c t chromatographed on a s i l i c a column (15 cm x 5.5 cm) u s i n g 1:1 ( v \/ v ) pet e t h e r : e t h e r (800 mL). The p u r i f i e d 23 was c o n c e n t r a t e d in vacuuo t o a f f o r d 6.20 g (86 %) o f r u s t y brown c r y s t a l s . *H-NMR 8 3.85 ( s , 3H, -0CH 3), 6.69 ( d d , J = 8 Hz and 2 Hz, 1H, H-4), 6.96 ( d , J = 2 Hz, 1H, H-6), 7.05 ( d , J = 8 Hz, 2H, H-2', 6 ' ) , 7.20 ( d , J = 8 Hz, 2H, H-3', 5 ' ) , 8.16 ( d , J = 8 Hz, 1H, H-3), 9.43 ( b s , 1H, N-* * H ). ( Does not exchange w i t h D2O). GCMS ( d ) : t R , 7.00 min, M + - ( 3 5 C 1 ) : m\/z 278 (100 % ) , M + ' ( 3 7 C 1 ) : m\/z 280 (31 % ) , [ M - 1 5 ] + - ( 3 5 C 1 ) : m\/z 263 (44 % ) , [ M - 1 5 ] + - ( 3 7 C 1 ) : m\/z 265 (14 % ) . E t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n v 1 ) - N - ( 4 - m e t h o x y p h e n v 1 ) c a r b a m o y l a c e t a t e 1241. The carbamoyl a c e t a t e 24 was s y n t h e s i z e d u s i n g 23 (4.20 g, 14.4 mmol) and e t h y l m a l o n y l c h l o r i d e (4.32 g, 28.6 mmol) f o l l o w i n g t h e method used f o r t h e s y n t h e s i s o f 19. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (20 cm x 5.5 cm) u s i n g t h e f o l l o w i n g g r a d i e n t : 2:1 ( v \/ v ) p e t ether-.EtOAc (900 mL); 3:2 ( v \/ v ) p e t e t h e r : E t 0 A c 47 (550 mL). P u r i f i e d 24 was c o n c e n t r a t e d t o a p a l e y e l l o w o i l w h i c h was c r y s t a l l i z e d f r o m 1:1 ( v \/ v ) p e t e t h e r : e t h e r t o a f f o r d 4.89 g (87 %) o f p a l e y e l l o w c u b e s . !H-NMR 6 1.27 ( t , 3H, -CH 2CH 3), 3.38 ( s , 2H, C(=0)CH 2C(=0), 3.85 ( s , 3H, -0C H 3 ) , 4.17 ( q , 2H, CH 2 C H 3 ) , 6.98 ( d , J = 7 Hz, 2H, H-2', H-6'), 7.21 ( d , J = 1 Hz, 1H, H-6), 7.34 (dd, J = 7 Hz and 1 Hz, 1H, H-4), 7.45 ( d , J = 7 Hz, 2H, H-3', H-5'), 7.92 ( d , J = 7 Hz, 1H, H-3). GCMS ( d ) : t R , 7.00 min, [ M - 1 1 4 ] + - ( 3 5 C 1 ) : m\/z 278 (100 % ) , [M-114] + - ( 3 7 C 1 ) : m\/z 280 (31 % ) , [M-114-15] +- ( 3 5C1): m\/z 263 (44 % ) , [M-1 1 4 - 1 5 ] + ' ( 3 7 C 1 ) : m\/z 265 (14 % ) . 4'-Methoxy N-desmethvlclobazam ( 2 5 ) . The DMC analogue 25 was s y n t h e s i z e d a c c o r d i n g t o t h e method used f o r 20 by u s i n g 24 (2.01 g, 5.12 mmol) as t h e s t a r t i n g m a t e r i a l . The p r o d u c t o c c u r r e d as 620 mg (38 %) o f w h i t e amorphous s o l i d . !H-NMR 6 3.52 ( s , 2H, -CH 2-), 3.84 ( s , 3H, OCH3), 6.96 (dd, J = 9 Hz and 3 Hz, 3H, H-6 and H-2', 6 ' ) , 7.07 ( d , J = 9 Hz, 1H, H-9), 7.12 ( d , J = 9 Hz, 2H, H-3', 5'), 7.19 (dd, = 9 Hz and 3 Hz, 1H, H-8), 7.77 ( b s , 1H, N-H). GCMS ( d ) , TMS d e r i v a t i v e : t R , 8.85 min, M+- ( 3 5C1): m\/z 388 (36 % ) , M+-( 3 7C1) m\/z 390 (14 % ) , [ M - 1 5 ] + - m\/z 373 (15 % ) , [ M - 1 4 9 ] + - m\/z 239 (34 % ) , [ M - 1 6 3 ] + - m\/z 225 (100 % ) . 4'-Hydroxy N-desmethvlclobazam ( 2 6 ) . The s y n t h e s i s o f 26 was c a r r i e d o u t by s u s p e n d i n g 25 i n d r y CHCI3 48 under N 2 i n a septum s e a l e d f l a s k . The s u s p e n s i o n was c o o l e d i n an a c e t o n e \/ d r y i c e bath ( c a . -40\u00b0C) and w h i l e s t i r r i n g BBr3 (252 mg, 1.00 mmol) added by s y r i n g e . S h o r t l y t h e r e a f t e r , t h e s o l v e n t began t o f r e e z e and so t h e f l a s k was t e m p o r a r i l y w i t h d r a w n from t h e b a t h . P e r i o d i c w i t h d r a w a l from t h e ba t h was c o n t i n u e d f o r t h e f o l l o w i n g 5 min t o e n s u r e t h a t t h e s o l v e n t remained l i q u i d . A f t e r t h a t t i m e t h e m i x t u r e was a l l o w e d t o r e a c h room t e m p e r a t u r e and s t i r r i n g c o n t i n u e d f o r 5 h. TLC o f t h e r e a c t i o n i n 10:1 ( v \/ v ) CHCl3:Me0H showed an e s s e n t i a l l y q u a n t i t a t i v e c o n v e r s i o n o f 25 ( R f 0.52) t o a more p o l a r p r o d u c t ( R f , 0.37). The r e a c t i o n was quenched w i t h t h e d r o p w i s e a d d i t i o n o f MeOH, c o n c e n t r a t e d i n vacuuo, r e d i s s o l v e d i n CHCI3 (70 mL), washed w i t h aqueous NaCl s o l u t i o n , and d r i e d o v e r anhydrous Na 2S04. The o r g a n i c s o l u t i o n was c o n c e n t r a t e d i n vacuuo, r e d i s s o l v e d i n c a 2 mL CHCI3 w i t h a few d r o p s o f MeOH and chromatographed on a s i l i c a column (14 cm x 1.25 cm) u s i n g a m o b i l e phase o f 10:1 ( v \/ v ) CHCl3:Me0H. The p u r i f i e d 26 was c o n c e n t r a t e d i n vacuuo t o a f f o r d 10 mg (26 %) o f amorphous w h i t e s o l i d . JH-NMR (DMS0-d 6) 6 3.35 ( b u r i e d i n H 20, - C H 2 - ) , 6.85 ( d , J = 9 Hz, 2H, H-2', H-6'), 7.06 ( d , J = 9 Hz, 2H, H-3', H-5'), 7.25 - 7.40 (m, 3H, H-6, H-8, H-9), 9.77 ( s , 1H, 4'-0H), 10.68 ( s , 1H, N-H). GCMS ( d ) , T M S - d e r i v a t i v e : 12.45 min, M + - ( 3 5 C 1 ) : m\/z 446 (71 % ) , [M-1 5 ] + - : m\/z 431 (27 % ) , [ M - 4 3 ] + - : m\/z 403 (10 % ) , [ M - 1 1 5 ] + - : m\/z 331 (11 % ) , [ M - 2 0 7 ] + - : m\/z 239 (39 % ) , [ M - 2 2 1 ] + - : m\/z 225 (100 % ) . 4'-Methoxy clobazam ( 2 7 ) . The s y n t h e s i s o f 27 from 25 (250.9 mg, 0.79 mmol) and CH3I (90 ul, 1.44 mmol) was a c c o r d i n g t o t h e method used f o r 21. No c h r o m a t o g r a p h i c 4 9 p u r i f i c a t i o n was n e c e s s a r y a f t e r work-up because o f t h e e s s e n t i a l l y q u a n t i t a t i v e f o r m a t i o n o f 27. I n s t e a d , t h e c r u d e p r o d u c t was r e c r y s t a l l i z e d from EtOAc:pet e t h e r t o a f f o r d 180.6 mg (69 %) o f 27 as w h i t e c r y s t a l s . !H-NMR S 3.47 ( d , J = 12 Hz, 1H, H-3\/3), 3.50 ( s , 3H, N-CH 3), 3.55 ( d , J = 12 Hz, 1H, H-3a), 3.83 ( s , 3H, - 0 C H 3 ) , 6.94 ( d , J = 10 Hz, 2H, H-2', H-6'), 7.11 ( d , J = 10 Hz, 2H, H-3', H-5'), 7.23 (dd, J = 8 Hz and 2.5 Hz, 1H, H-8), 7.26 ( d , J = 2.5 Hz, 1H, H-6), 7.28 ( d , J - 8 Hz, 1H, H-9 ) . GCMS ( d ) : t R , 10.11 min, M + - ( 3 5 C 1 ) : m\/z 330 (100 % ) , M+- ( 3 7 C 1 ) : m\/z 332 (32 % ) , [ M - 1 7 ] + - : m\/z 313 (32 % ) , [ M - 5 7 ] + \" : m\/z 273 (50 % ) , [M-149] +\u00ab: m\/z 181 (22 % ) . 4'-Hydroxy clob a z a m ( 2 8 ) . The f o l l o w i n g i s an improved d e m e t h y l a t i o n p r o c e d u r e o v e r t h a t used f o r 26. In d r y CHCI3 27 (51.3 mg, 0.155 mmol) was d i s s o l v e d , t h e f l a s k f l u s h e d w i t h N2, and s e a l e d w i t h a r u b b e r septum. The s o l u t i o n was c o o l e d i n an i c e \/ s a l t b a t h (-5\u00b0C) and w h i l e s t i r r i n g BBr3 (236 mg, 0.94 mmol) was added by s y r i n g e . The r e s u l t i n g s u s p e n s i o n was a l l o w e d t o r e a c h room t e m p e r a t u r e and s t i r r i n g c o n t i n u e d f o r an a d d i t i o n a l 5 h. A t t h a t t i m e TLC a n a l y s i s o f t h e r e a c t i o n m i x t u r e i n 10:1 CHCl3:MeOH showed t h a t t h e r e was an e s s e n t i a l l y q u a n t i t a t i v e c o n v e r s i o n o f 27 ( R f , 0.77) t o a more p o l a r p r o d u c t ( R f , 0.50). The r e a c t i o n was quenched w i t h t h e d r o p w i s e a d d i t i o n o f H2O, s t i r r e d f o r an a d d i t i o n a l hour, t a k e n up i n t o CHCI3 (70 mL), washed w i t h aqueous NaCl s o l u t i o n , and d r i e d o v e r anhydrous Na2S04. The o r g a n i c s o l u t i o n was c o n c e n t r a t e d in vacuuo t o 50 a f f o r d a p u r p l e gum whi c h was d i s s o l v e d i n c a 2 mL EtOAc and t o a v o i d c r y s t a l l i z a t i o n i m m e d i a t e l y chromatographed on a s i l i c a column (14 x 1.5 cm) w i t h t h e f o l l o w i n g m o b i l e phase: EtOAc (2 mL), 10:1 ( v \/ v ) CHCl3:MeOH (45 mL). P u r i f i e d 28, c o n t a i n i n g 27 as a min o r (<5 %) c o n t a m i n a n t , was c o n c e n t r a t e d in vacuuo t o a f f o r d 44.7 mg (91 %) o f w h i t e c r y s t a l s . !H-NMR 6 3.49 ( d , J = 12 Hz, 1H, H-3\/5), 3.50 ( s , 3H, N-CH 3), 3.57 ( d , J = 12 Hz, 1H, H-3a), 6.06 ( b s , 1H, 3'-0H), 6.82 ( d , J = 10 Hz, 2H, H-2', H-6'), 6.97 ( d , J = 2.5 Hz, 1H, H-6), 7.01 ( d , J = 10 Hz, 2H, H-3', H-5' ) , 7.24 (dd, J = 12 Hz and 2.5 Hz, 1H, H-8), 7.30 ( d , J = 12 Hz, 1H, H-9), 7.30 ( d , J = 12 Hz, 1H, H-9). C o n t a m i n a t i n g peaks: 4.14, q, EtOAc; 3.85, s, 4'-methoxy CLBZ (<5 % ) . GCMS ( d ) , T M S - d e r i v a t i v e : t R , 15.65 min, M + - ( 3 5 C 1 ) : m\/z 388 (100 % ) , [M-1 5 ] + - : m\/z 373 (22 % ) , [ M - 1 8 ] + - : m\/z 371 (21 % ) , [ M - 4 2 ] + - : m\/z 346 (19 % ) , [ M - 4 5 ] + - : m\/z 343 (50 % ) , [ M - 5 7 ] + < : m\/z 331 (29 % ) , [M-207J+-: m\/z 181 (17 % ) , [M-235]\"1\"-: m\/z 153 (13 % ) . N - ( 5 - C h l o r o - 2 - n i t r o p h e n y 1 ) - 3 . 4 - d i m e t h o x y p h e n v 1 a m i n e ( 2 9 ) . The s y n t h e s i s o f 29 from 2 , 4 - d i c h l o r o n i t r o b e n z e n e (4.99 g, 26 mmol) and 3 , 4 - d i m e t h o x y a n i l i n e (13.90 g, 91 mmol) was a c c o r d i n g t o t h e p r o c e d u r e used f o r 23. The cr u d e p r o d u c t was t a k e n up i n ca 80 mL benzene, and t h e s o l u t i o n d i v i d e d i n t o 2 equal p o r t i o n s . Each p o r t i o n was chromatographed on a s i l i c a column (19 cm x 5.5 cm) w i t h t h e f o l l o w i n g g r a d i e n t e l u t i o n : 1:2 ( v \/ v ) p e t r o l e u m e t h e r : e t h e r (300 mL); 2:7 ( v \/ v ) p e t r o l e u m e t h e r : e t h e r (450 mL); e t h e r (100 mL). On bo t h o c c a s i o n s t h a t t h e cr u d e 29 was chromatographed some o f t h e p r o d u c t came o u t o f s o l u t i o n and was r e t a i n e d by t h e column; however, no att e m p t was 51 made t o r e c o v e r t h i s p o r t i o n . P u r i f i e d 29 was c o n c e n t r a t e d in vacuuo t o produce f i n e n e e d l e l i k e r u s t c o l o u r e d c r y s t a l s i n 69 % (5.56 g) y i e l d . *H-NMR 6 3.90 ( s , 3H, 4'-0CH 3), 3.94 ( s , 3H, 3'-0CH 3), 6.70 (dd, J = 9 Hz and 2 Hz, 1H, H-6'), 6.79 ( d , J = 2 Hz, 1H, H-2'), 6.87 ( d d , J = 9 Hz and 3 Hz, 1H, H-4), 6.96 ( d , J = 9 Hz, 1H, H-5'), 7.01 ( d , J = 3 Hz, 1H, H-6), 8.16 ( d , J = 9 Hz, 1H, H-3), 9.44 ( b s , 1H, N-H*). (*Does not exchange w i t h D2O). GCMS ( d ) : t R , 7.90 min; M ' + ( 3 5 C 1 ) : m\/z 308 (90 % ) , M' + ( 3 7 C 1 ) : m\/z 310 (31 % ) , [ M - 1 5 ] - + ( 3 5 C 1 ) : m\/z 293 (100 % ) , [ M - 1 5 ] - + ( 3 7 C 1 ) : m\/z 293 (35 % ) . E t h y l N - ( 5 - c h 1 o r o - 2 - n i t r o p h e n y 1 ) - N - ( 3 , 4 - d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e 1301. In t h e f o l l o w i n g p r o c e d u r e , a p p r o p r i a t e m o d i f i c a t i o n s t o t h e method used f o r t h e s y n t h e s i s o f 19 were n e c e s s a r y . E t h y l m a l o n y l c h l o r i d e (3.14 mL, 24.5 mmol) and 29 (3.02 g, 9.79 mmol) were d i s s o l v e d i n d r y benzene (50 mL) and t h e r e s u l t i n g s o l u t i o n r e f l u x e d f o r 22 h a t 75 - 85\u00b0C. The c r u d e p r o d u c t w h i c h o c c u r r e d as a brown s o l u t i o n was e x t r a c t e d w i t h s a t u r a t e d NaHC0 3, d r i e d o v e r Na 2S04 and c o n c e n t r a t e d i n vacuuo t o a brown o i l . The l a t t e r was d i s s o l v e d i n c a 10 mL CHC1 3 and chromatographed on a s i l i c a column (18 cm x 2.5 cm) w i t h t h e f o l l o w i n g g r a d i e n t e l u t i o n : benzene (40 mL), 1:1 ( v \/ v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (80 mL); 2:3 (v\/ v ) p e t r o l e u m e t h e r : e t h y l a c e t a t e (100 mL). P u r i f i e d f r a c t i o n s o f 30 were c o n c e n t r a t e d i n vacuuo t o p a l e y e l l o w c r y s t a l s i n 96 % (3.97 g) y i e l d . ^H-NMR 6 1.27 ( t , 3H, -CH 2CH 3), 3.39 ( s , 2H, -(C=0)CH 2(C=0)-), 3.88 ( s , 3H, 4'-0CH 3), 3.92 ( s , 3H, 3'-0CH 3), 4.16 ( q , 2H, 0 C H 2 C H 3 ) , 6.91 ( d , J = 52 7 Hz, H-5'), 7.07 ( d , J = 2 Hz, 1H, H-2'), 7.08 (dd, J = 7 Hz and 2 Hz, 1H, H-6'), 7.23 ( d , J = 2.5 Hz, 1H, H-6), 7.35 (dd, J = 9 Hz and 2.5 Hz, 1H, H-4), 7.91 ( d , J = 9 Hz, H-3). GCMS ( d ) : tR, 7.83 min; [M-114] * + ( 3 5 C l ) : m\/z 308 (98 % ) , [M-114]- + ( 3 7 C 1 ) : m\/z 310 (33 % ) , [M-114-15]- + ( 3 5 C 1 ) : m\/z 293 (100 % ) , [M-114-1 5 ] ' + ( 3 7 C 1 ) : m\/z 293 (33 % ) . 3',4'-Dimethoxy N - d e s m e t h v l c l o b a z a m ( 3 1 ) . The s y n t h e s i s o f 31 from 30, (2.11 g, 4.99 mmol) was a c c o r d i n g t o t h e method used f o r 20, by t r e a t m e n t w i t h Zn powder (1.6 g, 24.9 mmol) and c o n c e n t r a t e d HCl (10 mL, 110 mmol). TLC a n a l y s i s i n 15:1 ( v \/ v ) CHCl3:MeOH a f t e r 30 h showed t h e p r e s e n c e o f a p r o d u c t a t R f 0.37 w i t h much u n r e a c t e d s t a r t i n g m a t e r i a l a t Rf 0.84. F u r t h e r m o r e , t h e p r o d u c t d i d n ot p r e c i p i t a t e from t h e r e a c t i o n m i x t u r e l i k e 20 and 25 but was s o l u b l e . The r e a c t i o n was t r e a t e d w i t h a d d i t i o n a l HCl (3 mL., 33 mmol) and Zn (0.82 g, 12.7 mmol) and s t i r r i n g c o n t i n u e d f o r a n o t h e r 24 h. TLC a n a l y s i s a f t e r 24 h showed t h a t no f u r t h e r r e a c t i o n had o c c u r r e d and s t i r r i n g was d i s c o n t i n u e d . The r e a c t i o n m i x t u r e was c o n c e n t r a t e d in vacuuo, t a k e n up i n t o CHCI3, backwashed w i t h H2O, and d r i e d o v e r anhydrous Na2S04. The c r u d e p r o d u c t was c o n c e n t r a t e d in vacuuo and chromatographed on a s i l i c a column (20 cm x 2.5 cm) u s i n g a m o b i l e phase o f 12:1 ( v \/ v ) CHCl3:Me0H (130 mL). P u r i f i e d 31 was c o n c e n t r a t e d in vacuuo t o an o i l w h i c h was p r e c i p i t a t e d from CHCl3:pet e t h e r t o a f f o r d 101 mg (6 %) o f an amorphous w h i t e s o l i d . ^H-NMR 6 3.53 ( s , 2H, C H 2 ) , 3.83 ( s , 3H, 4'-0CH 3), 3.90 ( s , 3H, 3'-0 C H 3 ) , 6.69 ( d , J = 2.5 Hz, 1H, H-2'), 6.76 (dd, J = 9 Hz and 2.5 Hz, H-53 6 ' ) , 6.90 (d J = 9 Hz, 1H, H-5'), 6.97 ( d , J = 1.5 Hz, 1H, H-6), 7.07 (m, 2H, H-8, H-9) 9.54 ( b s , 1H, N-H). GCMS ( d ) , TMS d e r i v a t i v e : t R, 9.57 min, M +' ( 3 5 C 1 ) : m\/z 418 (91 % ) , M+-( 3 7 C 1 ) m\/z 420 (35 % ) , [ M - 1 5 ] + - m\/z 403 (28 % ) , [ M - 1 7 9 ] + - m\/z 239 (32 % ) , [ M - 1 9 3 ] + - m\/z 225 (100 % ) . 3 ' , 4 ' - D i h v d r o x y N-desmethylclobazam ( 3 2 ) . The s y n t h e s i s o f 32, l i k e 28, was a c c o m p l i s h e d by t h e t r e a t m e n t o f 31 (51.6 mg, 0.15 mmol) w i t h BBY3 (140 * i L , 1.49 mmol). The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 x 1.5 cm) u s i n g 10:1 (v \/ v ) CHCl3*.MeOH (55 mL). P u r i f i e d 32 was c o n c e n t r a t e d in vacuuo t o a f f o r d 45 mg (96 %) o f an amorphous w h i t e s o l i d . *H-NMR (DMS0-d 6), 8 3.35 ( b u r i e d i n H 20, C H 2 ) , 6.45 ( d d , 1H, J = 8.5 Hz and 2.5 Hz, H-6'), 6.58 ( d , J = 2.5 Hz, 1H, H-2'), 6.78 ( d , J = 8.5 Hz, 1H, H-5'), 6.88 ( d , J = 2.5 Hz, 1H, H-6), 7.23 ( d , J = 8.5 Hz, 1H, H-9), 7.30 (dd, J = 8.5 Hz and 2.5 Hz, 1H, H-8), 9.40 ( s , 2H, 3'-0H, 4'-0H), 10.52 ( s , 1H, N-H). GCMS ( d ) , T M S - d e r i v a t i v e : 13.90 min, M + ' ( 3 5 C 1 ) : m\/z 534 (100 % ) , [M-15]+': m\/z 519 (29 % ) , [ M - 2 9 5 ] + - : m\/z 239 (19 % ) , [ M - 3 0 9 ] + ' : m\/z 225 (50 %). 3'.4'-Dimethoxv clobazam ( 3 4 ) . The s y n t h e s i s o f 34> l i k e 21, was a c c o m p l i s h e d by t r e a t m e n t o f 31 (50.1 mg, 0.14 mmol) w i t h K0H (13.2 mg, 0.24 mmol) i n d r y DMSO. The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 cm x 1.5 cm) 54 u s i n g 25:1 ( v \/ v ) CHCl3:MeOH (52 mL). P u r i f i e d 34 was c o n c e n t r a t e d in vacuuo t o a f f o r d 42 mg (81 %) o f f a i n t l y p u r p l e c r y s t a l s . !H-NMR 6 3.41 ( d , J = 12 Hz, 1H, H-3\/3), 3.44 ( s , 3H, N-CH 3), 3.49 ( d , J = 12 Hz, 1H, H-3a), 3.80 ( s , 3H, 4'-0CH 3), 3.85 ( s , 3H, 4'-0CH 3), 6.64 (dd, J = 10 Hz and 2 Hz, 1H, H-6'), 6.68 ( d , J = 2 Hz, 1H, H-2'), 6.83 ( d , J = 10 Hz, 1H, H-5'), 6.92 ( d , J = 3 Hz, 1H, H-6), 7.18 (dd, J = 10 Hz and 3 Hz, 1H, H-8). GCMS ( d ) : t R , 11.65 min, M + ' ( 3 5 C 1 ) : m\/z 360 (100 % ) , [ M - 1 7 ] + * : m\/z 343 (16 % ) , [ M - 5 7 ] + - : m\/z 303 (26 % ) , [ M - 1 7 9 ] + - : m\/z 181 (24 % ) . 3 ' . 4 ' - D i h v d r o x v clobazam ( 3 5 ) . The s y n t h e s i s o f 35 was a c c o m p l i s h e d by t r e a t m e n t o f 34 (41.2 mg, 0.115 mmol) w i t h B B r 3 (108 \/xL, 1.15 mmol). The c r u d e p r o d u c t was chromatographed on a s i l i c a column (14 cm x 1.5 cm) w i t h 25:1 ( v \/ v ) CHCl 3:MeOH (78 mL). The p u r i f i e d p r o d u c t was c o n c e n t r a t e d in vacuuo t o a f f o r d 33.5 mg (82 %) o f w h i t e c r y s t a l s . !H-NMR 6 3.50 ( d , J = 12 Hz, 1H, H-3\/3), 3.52 ( s , 3H, N-CH 3), 3.57 ( d , J = 12 Hz, 1H, H-3a), 6.28 ( b s , 1H, 4'-0H), 6.44 (dd, J = 8 Hz and 2 Hz, 1H, H-6'), 6.69 ( d , J = 2 Hz, 1H, H-2'), 6.77 ( d , J = 8 Hz, 1H, H-5'), 7.03 ( d \/ b s , J = 3 Hz, 2H, H-6\/3'-0H), 7.25 ( d d , J = 10 Hz and 3 Hz, 1H, H-8), 7.29 ( d , J = 10 Hz, 1H, H-9). GCMS ( d ) , T M S - d e r i v a t i v e : t R , 18.11 min, M + - ( 3 5 C 1 ) : m\/z 476 (100 % ) , [M-1 5 ] + ' : m\/z 461 (24 % ) , [ M - 4 5 ] + - : m\/z 431 (73 % ) , [ M - 5 7 ] + \" . m\/z 419 (52 % ) , [ M - 1 3 3 ] + - : m\/z 343 (14 % ) . 5 5 ( i i ) In vitro s y n t h e s e s . 4'-Hydroxy-3'-methoxy N-desmethvlclobazam ( 3 3 ) . The O-methylated c a t e c h o l o f DMC (33) was s y n t h e s i z e d from 32 u s i n g t h e method o f A l t o n e t a l . ( 1 9 7 5 a ) . Rat l i v e r c y t o s o l (as a s o u r c e o f COMT), phosphate b u f f e r (0.50 M, pH 7.9, 2 mL), M g C l 2 (60 mM, 330 iii) and SAM.p-TsOH (0.50 \/xmol i n 300 ul v\/v\/v H 20:95 7, Et0H:5 % H C l , 2:2:1) were mixed i n a c u l t u r e t u b e , and p r e i n c u b a t e d a t 37\u00b0C w i t h s h a k i n g f o r 3 min i n a w a t e r - b a t h . The s u b s t r a t e 32 (0.52 \/zmol i n 150 tiL MeOH) was t h e n added, and t h e i n c u b a t i o n c o n d u c t e d f o r 80 min. The r e a c t i o n was t e r m i n a t e d by f r e e z i n g t h e m i x t u r e i n an i c e - b a t h . A f t e r 5 min t h e r e a c t i o n m i x t u r e was e x t r a c t e d w i t h an equal volume o f EtOAc (x 4 ) . The o r g a n i c e x t r a c t was d r i e d o v e r anhydrous Na 2S04, c o n c e n t r a t e d t o d r y n e s s under a s t r e a m o f N 2 , r e c o n s t i t u t e d t o 25 IJL i n EtOAc and d e r i v a t i z e d w i t h 25 fil MSTFA a t 45*C f o r 90 min. I n j e c t i o n volume: 3 Hi. GCMS ( b ) , T M S - d e r i v a t i v e : t R , 18.42 min, M + ' ( 3 5 C 1 ) : m\/z 476 (58 % ) , [M-15] + -: m\/z 461 (17 % ) , [ M - 3 0 ] + - : m\/z 446 (8 % ) , [ M - 2 3 7 ] + - : m\/z 239 (18 % ) , [M-251J+-: m\/z 225 (48 % ) . O - methylated c a t e c h o l s o f cloba z a m (36a and 3 6 b ) . The meta- 36a and para- 36b 0-methyl a t e d c a t e c h o l s o f CLBZ were s y n t h e s i z e d from 35 u s i n g t h e p r o c e d u r e d e s c r i b e d f o r 33. GCMS ( b ) , T M S - d e r i v a t i v e : m e t a - i s o m e r , t R , 23.49 min, para-isomer t R , 22.80 min, M + - ( 3 5 C 1 ) : m \/ z 418 (100 % ) , [M-30] + ': m\/z 388 (20 % ) , [M-4 5 ] + - : m\/z 373 (40 % ) , [ M - 7 2 ] + - : m\/z 346 (42 % ) , [ M - 2 3 7 ] + * : m\/z 181 (48 %). (E) S y n t h e s i s o f N-hydroxymethyl CLBZ ( c a r b i n o l a m i d e ) ( 3 9 ) . ( i ) K2CO3 and 37 % f o r m a l d e h y d e . U s i n g t h e approach o f N a i r and F r a n c i s ( 1 9 8 0 ) , DMC (80.4 mg, 0.28 mmol) and K2CO3 (26.2 mg, 0.19 mmol) were s t i r r e d i n 37 % aqueous f o r m a l d e h y d e a t room t e m p e r a t u r e f o r 24 h. A f t e r t h a t t i m e , TLC a n a l y s i s i n 15:1 ( v \/ v ) CHCl3:Me0H r e v e a l e d t h e p r e s e n c e o f t h r e e s p o t s a t Rf 0.20, 0.29 and 0.36 w i t h a p p a r e n t c o r r e s p o n d i n g r e l a t i v e i n t e n s i t i e s -45 %, =45 % and =5 %. A minor amount o f u n r e a c t e d s t a r t i n g m a t e r i a l ( R f 0.41) was a l s o p r e s e n t . The r e a c t i o n was d i l u t e d i n H2O, e x t r a c t e d w i t h EtOAc and d r i e d o v e r anhydrous Na 2S04. A l i q u o t s o f t h e cr u d e p r o d u c t were removed f o r GCMS and HPLC a n a l y s i s . The r e m a i n d e r was c o n c e n t r a t e d in vacuuo t o an o i l w h i c h was chromatographed on a s i l i c a column (14 cm x 1.25 cm) u s i n g a 15:1 ( v \/ v ) Me0H:H20 (50 mL). F l a s h chromatography d i d not s e p a r a t e t h e components o f t h e m i x t u r e and so p r e p a r a t i v e TLC was n e c e s s a r y . The c r u d e p r o d u c t was c o n c e n t r a t e d , and chromatographed on t h r e e s i l i c a p l a t e s (20 cm X 20 cm) u s i n g 15:1 (v\/ v ) Me0H:H 20 as t h e m o b i l e phase. Three major bands were removed from t h e p l a t e s and t h e s i l i c a e x t r a c t e d w i t h EtOAc (5 mL x 2) and 1:1 ( v \/ v ) CHCI3: MeOH (5 mL x 2 ) . The e x t r a c t s were c o n c e n t r a t e d under a stre a m o f N 2 and t h e r e a f t e r in vacuuo. A n a l y t i c a l TLC o f t h e f r a c t i o n s showed pure compounds a t Rf 0.20, 0.30 and 0.43 (DMC). 57 (a) A n a l y s i s o f cr u d e p r o d u c t . HPLC, 50:50 ( v \/ v ) Me0H:H20: ( i ) f r e s h l y p r e p a r e d p r o d u c t , t R 7.85 min (19.5 % ) , 8.57 min (28.4 % ) , 9.61 min (29.4 % ) , 10.68 min (22.6 % ) ; ( i i ) a f t e r o v e r n i g h t s t o r a g e a t 4\u00b0C, tR 7.67 min (19.1 % ) , 8.35 min (58.6 % ) , 10.40 min (22.3 % ) . LCMS, 50:50 ( v \/ v ) MeOH:H20 ( i ) LCMS o f f r e s h l y p r e p a r e d p r o d u c t c o u l d n o t be p e r f o r m e d ; ( i i ) a f t e r o v e r n i g h t s t o r a g e a t 4\u00b0C, tR 4.68 min, scan 94, poor mass s p e c t r u m o b t a i n e d ; 6.68 min, scan 130, MH +: m\/z 317 (100 % ) , [ M H-H 20] +: m\/z 299 (55 % ) ; 7.37 min, scan 145, MH +: m\/z 287 (100 % ) , MNH 4 +: m\/z 304 (40 % ) . GCMS ( b ) , T M S - d e r i v a t i v e : t R , 17.33 min, M + - ( 3 5 C 1 ) : m \/ z 388 (20 % ) , [M-1 5 ] + - : m\/z 373 (24 % ) , [M-30]\"1\"': m\/z 358 (6 % ) , [M-149 ] + ': m\/z 239 (10 % ) , [ M - 1 6 3 ] + - : m\/z 225 (20 % ) , CH 20-TMS +-: m\/z 103 (35 % ) . (b) A n a l y s i s o f t h e p u r i f i e d f r a c t i o n s . Rf 0.20 ( 4 5 ) . 2H-NMR (DMS0-d6), 5 6.64 ( d , J = 2 Hz, 1H, H-6), 7.18 (dd, J = 9 Hz and 1 Hz, 2H, H-2', H-6'), 7.25 ( d , J = 9 Hz, 1H, H-9), 7.30 (d d , J = 9 Hz and 2 Hz, 1H, H-8), 7.42 (m, 1H, H-4'), 7.50 (m, 2H, H-3', H-5'), 10.89 ( s , 1H, N-H). U n a s s i g n e d : 3.75 ( b s , 1H), 3.84 ( b s , 1H), 4.58 ( b s , 1H*). *Exchanges w i t h D 20. Rf 0.30 (46). *H-NMR (DMS0-d6), 8 3.42 ( t , 1H, -CHCH 2), 3.94 ( d , J = 6 Hz, 2H, CHCH 20H), 4.56 ( b s , 1H, -CH 20H), 6.88 ( d , J = 2 Hz, 1H, H-6), 7.20 (dd, J = 9 Hz and 1 Hz, 2H, H-2', H-6'), 7.32 ( d , J = 9 Hz, 1H, H-9), 7.37 58 ( d d , J = 9 Hz and 2 Hz, 1H, H-8), 7.40 (m, 1H, H-4'), 7.48 (m, 2H, H-3', H-5'), 10.80 ( s , 1H, N-H). C o n t a m i n a t i n g peaks: 3.31 ( b s , H 2 0 ) , 2.51 (m, DMSO). GCMS ( b ) , T M S - d e r i v a t i v e : t R , 14.39 min, M + - ( 3 5 C 1 ) : m\/z 370 (12 % ) , [M-1 5 ] + - : m\/z 355 (8 % ) , [ M - 9 7 ] + - : m\/z 301 (10 % ) , [M-119 ] + ' : m\/z 251 (34 % ) , [M-145]\"*\"-: m\/z 225 (46 % ) , TMS +\": m\/z 73 (100 % ) , . Rf 0.43 (DMC) ( 4 7 ) . *H-NMR (DMS0-d6), 6 3.20 ( b s , 0.6H, H-30*), 3.41 ( b s , 0.8H, H-30, H-3a), 3.68 ( b s , 0.6H, H-3a*), 6.82 ( d , J = 3 Hz, 1H, H-6), 7.25 (dd, J = 8 Hz and 1 Hz, 2H, H-2', H-6'), 7.30 ( d , J = 9 Hz, 1H, H-9), 7.36 (dd, J = 9 Hz and 3 Hz, 1H, H-8), 7.42 (m, 1H, H-4'), 7.50 (m, 2H, H-3', H-5'), 10.70 ( s , 1H, N-H). *The c h e m i c a l s h i f t s a t 3.20 and 3.68 ppm d e m o n s t r a t e t h e p a r t i a l l y d i a s t e r e o m e r i c c h a r a c t e r o f H-3. (See R e s u l t s and D i s c u s s i o n ) . ( i i ) KOH and p a r a f o r m a l d e h y d e . The f o l l o w i n g d e s c r i b e s t h e s y n t h e s i s o f 40. I n t o a s t i r r e d s o l u t i o n o f DMC (80.5 mg, 0.28 mmol) and p u l v e r i z e d KOH (24.3 mg, 0.43 mmol), m a i n t a i n e d a t 20\u00b0C, was p l a c e d p a r a f o r m a l d e h y d e (18.9 mg, 0.63 mmol). A f t e r 20 min t h e r e a c t i o n was a l l o w e d t o r e a c h room t e m p e r a t u r e w i t h s t i r r i n g c o n t i n u e d f o r an a d d i t i o n a l 2 h. A t t h a t t i m e TLC a n a l y s i s i n 20:1 ( v \/ v ) CHCl3:Me0H r e v e a l e d t h e p r e s e n c e o f one major p r o d u c t (-95 %) a t Rf 0.17 and u n r e a c t e d s t a r t i n g m a t e r i a l a t Rf 0.34. The r e a c t i o n was d i l u t e d t o c a . 80 mL w i t h EtOAc, washed w i t h aqueous NaCl (2 x 20 mL) and H 20 (2 x 20 mL), d r i e d o v e r anhydrous Na 2S04 and 59 c o n c e n t r a t e d in vacuuo t o a f f o r d 17 mg o f 40, a w h i t e amorphous s o l i d . *H-NMR (DMS0-d 6), 8 3.43 ( t , 1H, -CHCH 2), 3.94 ( t , 2H, CHCH 20H), 4.56 ( t , 1H, -CH 20H), 6.90 ( d , J = 2 Hz, 1H, H-6), 7.20 ( d d , J = 9 Hz and 1 Hz, 2H, H-2', H-6'), 7.32 ( d , J = 9 Hz, 1H, H-9), 7.37 (dd, J = 9 Hz and 2 Hz, 1H, H-8), 7.40 (m, 1H, H-4'), 7.48 (m, 2H, H-3', H-5'), 10.77 ( s , 1H, N-H). C o n t a m i n a t i n g peaks: 4.07 ( q , Et O A c ) , 3.33 ( b s , H 2 0 ) , 2.51 (m, DMSO). *H-NMR (DMS0-d6), D 20 exchange. 8 3.94 ( d , J = 6Hz, 2H, CHCH 20H), 4.56 and 10.77 ppm (e x c h a n g e d ) . Benzo and phenyl c h e m i c a l s h i f t s unchanged. GCMS ( d ) , T M S - d e r i v a t i v e : t R , 7.88 min, M + ' ( 3 5 C 1 ) : m\/z 370 (34 % ) , [M-1 5 ] + - : m\/z 35 (24 % ) , [ M - 9 7 ] + ' : m\/z 301 (24 % ) , [M-119 ] + > : m\/z 251 (76 % ) , [ M - 1 4 5 ] + - : m\/z 225 (100 % ) , TMS+-: m\/z 73 (68 % ) , (F) A t t e m p t e d S y n t h e s e s o f N-hydroxy DMC (hy d r o x a m i c a c i d ) ( 5 0 ) . ( i ) O x i d a t i o n o f DMC-TMS (Mat! i n e t a l . . 1979). (a) S y n t h e s i s o f ( b i s ( N , N - d i m e t h y l f o r m a m i d o ) o x o d i p e r o x o m o l y b d e n u m ( V I ) 1421-M o l y b d i c a c i d (2.00 g, 0.014 mol) was s t i r r e d i n 30 % H 2 0 2 a t 35\u00b0C f o r 15 min t o a f f o r d a y e l l o w s o l u t i o n . The r e a c t i o n m i x t u r e was c o o l e d t o 15\"C, DMF (2.34 mL, 0.30 mol) added, and s t i r r i n g c o n t i n u e d f o r a n o t h e r 20 min. The r e a c t i o n was c o n c e n t r a t e d in vacuuo a t 30\u00b0C and t h e r e s i d u e washed w i t h e t h e r ( 2 x 3 mL), MeOH ( 2 x 3 mL) and e t h e r a g a i n ( 2 x 3 mL). The washed r e s i d u e was d r i e d in vacuuo o v e r phosphorus p e n t a o x i d e t o a f f o r d 1.79 g (45 %) o f an amorphous y e l l o w 60 s o l i d . !H-NMR ( D 2 0 ) S 2.89 ( b s * , 3H, N-CH3), 3.05 ( b s * , 3H, N-CH3), 7.96 ( b s , 1H, N-H). * S o l v e n t - a s s o c i a t e d peak b r o a d e n i n g . C o n t a m i n a t i n g peak: 3.38 ( b s , CH3OH). L i t . 6 2.9 ( s , 3H, N-CH3), 3.0 ( s , 3H, N-CH3), 7.9 ( b s , 1H, N-H). (b) S y n t h e s i s o f DMC-TMS ( 4 8 ) . DMC (10.5 mg, .037 mmol) was suspended i n d r y CHCI3, t h e r e a c t i o n v i a l purged w i t h N 2 and s e a l e d . MSTFA (90 ill, 0.46 mmol) was added by s y r i n g e , and t h e r e a c t i o n s t i r r e d a t 45 \u00b0C f o r 90 min. (c ) A t t e m p t e d s y n t h e s i s o f t h e Molybdenum hydroxamate complex ( 4 9 ) . To t h e c r u d e DMC-TMS, was added 42 (234 mg, 0.69 mmol), t h e v i a l s e a l e d under N 2 and s t i r r i n g c o n t i n u e d a t room t e m p e r a t u r e . A f t e r 48 h, no r e a c t i o n was o b s e r v e d , and so t h e s y n t h e s i s was d i s c o n t i n u e d . ( i i ) R i n g c l o s u r e o f t h e hydroxy!amine. (a) A t t e m p t e d s y n t h e s i s o f t h e hydroxy!amine ( 5 2 ) . A c c o r d i n g t o t h e method o f Ro n d e s t v e d t and Johnson ( 1 9 7 7 ) , e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - p h e n y l c a r b a m o y l a c e t a t e (51) (40.6 mg, 0.112 mmol) and 10 % Pd\/C (3 mg) were suspended i n 50:50 ( v \/ v ) THF:95 % EtOH (3 mL). The m i x t u r e was warmed t o 35\u00b0C and w h i l e s t i r r i n g , h y d r a z i n e monohydrate (190 ill, 1.33 mmol) s l o w l y added i n 50 \/zL 61 t e m p e r a t u r e w i t h c o n t i n u e d s t i r r i n g . TLC a n a l y s i s o f t h e r e a c t i o n i n 3:2 ( v \/ v ) p e t e t h e r : E t O A c a f t e r 40 h r e v e a l e d t h a t no r e a c t i o n had o c c u r r e d , and so s y n t h e s i s was d i s c o n t i n u e d . 62 I I I R E S U L T S AND D I S C U S S I O N T h i s s e c t i o n w i l l be d i v i d e d i n t o t h e a r e a s o f (1) s y n t h e s i s and (2) m e t a b o l i s m . In (1) t h e s y n t h e s i s o f d e u t e r a t e d CLBZ a n a l o g u e s and CLBZ m e t a b o l i t e s w i l l be p r e s e n t e d , and i n (2) t h e m e t a b o l i t e p r o f i l e o f CLBZ i n t h e r a t w i l l be c h a r a c t e r i z e d . 1. THE SYNTHESIS OF DEUTERATED ANALOGUES OF CLBZ AND CLBZ METABOLITES. (A) The S y n t h e s i s o f P e n t a d e u t e r i o p h e n y l CLBZ. The use o f s t a b l e i s o t o p e l a b e l l e d d rugs t o probe t h e mechanisms o f b i o t r a n s f o r m a t i o n p r o c e s s e s i s w e l l - r e c o g n i z e d , and t h e s u b j e c t has been e x t e n s i v e l y r e v i e w e d (Vandenheuvel, 1987; B a i l l i e , 1981). When combined w i t h GCMS, s t a b l e i s o t o p e l a b e l l i n g f a c i l i t a t e s i d e n t i f i c a t i o n o f d r u g -r e l a t e d s p e c i e s i n a m e t a b o l i t e pool t h r o u g h i s o t o p e c l u s t e r s g e n e r a t e d by t h e l a b e l l e d and u n l a b e l l e d a n a l o g u e s . Such an approach was c o n s i d e r e d t o be i d e a l f o r o u r i n v e s t i g a t i o n o f CLBZ m e t a b o l i t e s i n t h e r a t . C u r s o r y a p p r a i s a l o f t h e c h e m i c a l s t r u c t u r e o f CLBZ r e v e a l s s e v e r a l p o s s i b l e s i t e s f o r d e u t e r i u m l a b e l l i n g , namely, t h e N-methyl, C3-methylene and t h e benzo and phenyl r i n g s . The s y n t h e s i s o f t r i d e u t e r i o m e t h y l CLBZ has been r e p o r t e d ( P o u p a e r t e t a l . , 1988); however, s i n c e t h e d e m e t h y l a t i o n o f CLBZ i s a f a c i l e m e t a b o l i c p r o c e s s ( V o l z e t a l . , 1979), such an an a l o g u e would be i n a p p r o p r i a t e f o r t h e i d e n t i f i c a t i o n o f l a b e l l e d m e t a b o l i t e s . The l o c a t i o n o f C-3 i n a b - d i c a r b o n y l system r e n d e r s t h e methylene p r o t o n s a c i d i c ( March, 1985a), and prone t o exchange under b a s i c c o n d i t i o n s . Thus, a 63 m e t h y l e n e - l a b e l l e d a n alogue was an u n s u i t a b l e m e t a b o l i t e p r o b e , and l a b e l l i n g a t t h e a r o m a t i c p o s i t i o n s a f f o r d e d t h e most v i a b l e a l t e r n a t i v e . F a c i l e a pproaches t o t h e de u t e r i u m - e x c h a n g e o f a r o m a t i c systems have been r e p o r t e d , e.g. C6U6 and a l k y l a l u m i n i u m d i c h l o r i d e ( G a r n e t t e t a l . , 1 9 7 2 ) , and BF3 and D2O ( L a r s e n and Chang, 1978). However, n e i t h e r approach was c o n s i d e r e d a p p l i c a b l e t o t h e l a b e l l i n g o f CLBZ f o r t h e f o l l o w i n g r e a s o n s : ( i ) N i t r o g e n - c o n t a i n i n g a r o m a t i c s f a i l t o exchange w i t h C^DQ, and d e a c t i v a t e t h e a l u m i n i u m c a t a l y s t t h r o u g h complex f o r m a t i o n , and ( i i ) t h e s l o w and i n c o m p l e t e exchange t h a t o c c u r s w i t h a r o m a t i c s and BF3.D2O, i s l i k e l y t o r e s u l t i n a m i x t u r e o f d e u t e r a t e d CLBZ a n a l o g u e s . In t h i s r e g a r d , p e n t a d e u t e r i o p h e n y l CLBZ ( [ 2 Hs]CLBZ), appeared t o be t h e most a p p r o p r i a t e c a n d i d a t e f o r a s e l e c t i v e l y d e u t e r a t e d CLBZ a n a l o g u e . D e u t e r i u m i n t h e 5-phenyl r i n g i s n o t r e a d i l y s u s c e p t i b l e t o exchange, p r e s e n t s no e x p e c t e d major i s o t o p e e f f e c t s , and even w i t h t h e f o r m a t i o n o f c a t e c h o l m e t a b o l i t e s a t l e a s t t h r e e 2 H - l a b e l s s h o u l d be r e t a i n e d . F u r t h e r m o r e , t h e p r e s e n c e o f 2 H - l a b e l s a t a l l f i v e p o s i t i o n s o f t h e phenyl r i n g e n s u r e s t h a t a l l u n s u b s t i t u t e d p o s i t i o n s w i l l be l a b e l l e d subsequent t o m e t a b o l i s m d e s p i t e t h e o c c u r r e n c e o f an arene e p o x i d e - a s s o c i a t e d NIH s h i f t ( N e l s o n and Burk e , 1979; N e l s o n and P o w e l l , 1979). The s y n t h e s i s o f [^ HsJCLBZ ( o u t l i n e d i n Scheme IX) was a c c o m p l i s h e d i n e s s e n t i a l l y q u a n t i t a t i v e i s o t o p i c y i e l d , as d e t e r m i n e d by *H-NMR ( F i g u r e 1) and mass s p e c t r a ( F i g u r e 2 ) , w i t h a p p r o p r i a t e m o d i f i c a t i o n s t o t h e p r o c e d u r e s o f Werner and Wagner (1978) and R o s s i e t a l . , ( 1 9 6 9 ) . In t h e f i r s t s t e p , r e d u c t i o n o f nitrobenzene - d s (99 atom % D) a f f o r d e d a n i l i n e - d y 17 i n good y i e l d w i t h an N,N-D2 i s o t o p i c p u r i t y o f 90 % as d e t e r m i n e d by 2H-NMR s p e c t r o s c o p y . GCMS a n a l y s i s was u n s u i t a b l e f o r t h e a c c u r a t e d e t e r m i n a t i o n o f N - d e u t e r a t i o n because t h e l a b i l e d e u t e r i u m atoms on 64 n i t r o g e n were e x t e n s i v e l y exchanged on t h e GC pack e d column p r i o r t o mass s p e c t r a l d e t e c t i o n . Thus, [ M - 2 ] + * a t m\/z 98 ap p e a r e d as t h e most i n t e n s e h i g h mass i o n i n t h e mass s p e c t r u m o f 17 f o l l o w i n g GCMS a n a l y s i s ( A p p e n d i x ) . Scheme I X . The s y n t h e s i s o f pHsJCLBZ. A: DC1, Fe B: 2 , 4 - d i c h l o r o n i t r o b e n z e n e , 170\u00b0C C: e t h y l m a l o n y l c h l o r i d e \/ d r y benzene D: Zn\/HCl E: KOH, DMSO\/CH3I 65 N u c l e o p h i l i c s u b s t i t u t i o n o f 2 , 4 - d i c h l o r o n i t r o b e n z e n e by 17 o c c u r r e d e x c l u s i v e l y ortho t o t h e n i t r o group t o a f f o r d 18 i n h i g h y i e l d . The h i g h r e g i o s p e c i f i c i t y ( f o r ortho s u b s t i t u t i o n ) c l e a r l y d e m o n s t r a t e s t h e r e q u i r e m e n t f o r t h e e l e c t r o n w i t h d r a w i n g i n f l u e n c e o f t h e n i t r o group t o be e x e r t e d t h r o u g h both r e s o n a n c e and f i e l d e f f e c t s (March, 1985c) i n o r d e r t o reduce t h e e l e c t r o n d e n s i t y a t t h e 2 - p o s i t i o n and t h e r e b y f a c i l i t a t e n u c l e o p h i l i c a t t a c k (March, 1985b). The *H-NMR spect r u m o f 18 shown i n F i g u r e 3A r e v e a l e d t h a t r e s o n a n c e a t t h e c h e m i c a l s h i f t o f t h e meta p r o t o n s o f t h e u n s u b s t i t u t e d phenyl r i n g (H-3'\/H-5', 7.48 ppm) was a b s e n t , a l t h o u g h weak s i g n a l s appeared i n t h e r e g i o n c h a r a c t e r i s t i c o f t h e ortho\/para p r o t o n s (H-2'\/6', H-4', 7.27 -7.31). Thus, 2 H - l a b e l s a t t h e meta p o s i t i o n were c o m p l e t e l y r e t a i n e d w i t h m i n o r exchange o c c u r r i n g a t t h e ortho\/para p o s i t i o n s . I n t e g r a t i o n o f t h e ortho\/para p r o t o n s was used t o c a l c u l a t e t h e i s o t o p i c p u r i t y o f 18 a t 92 %. Mass ( F i g u r e 4A) and ^3C-NMR ( F i g u r e 5) s p e c t r a c o n f i r m e d t h e i d e n t i t y o f th e p r o d u c t . 66 F i g u r e 1. 400 MHz *H-NMR o f [ 2 H 5 ] C L B Z . f K N M N u u u a N \u00bb> a A 00 N \u00bb> a a 0 0 a a a a a 0 \u00a3 0 a a a a a a Q s a a a a a a a till I I I. I. I I I I I I I I I t I Relative Intensity (%) H N N N a N a eg a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a o H a a R e l a t i v e i n t e n s i t y (%) 68 A l i k e l y s o u r c e o f t h e p r o t o n s b e i n g i n c o r p o r a t e d i n t o t h e d e u t e r i o p h e n y l r i n g o f 18 c o u l d have been t h e N-H group o f a n i l i n e -2,3,4,5,6-d5 w h i c h o c c u r r e d as an i s o t o p i c i m p u r i t y i n a n i l i n e - d 7 - Indeed, s u b s t a n t i a l i n t r o d u c t i o n o f p r o t o n s a t t h e ortho\/para p o s i t i o n s o f t h e d e u t e r i o p h e n y l r i n g o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o p h e n y l a m i n e 37 o c c u r r e d when aniline-2,3,4,5,6 - d s was used as t h e n u c l e o p h i l e (Scheme X, F i g u r e 3 B ) . In o r d e r t o a s s i g n t h e e x a c t l o c a t i o n o f d e u t e r i u m exchange, t h e ortho\/para s i g n a l s were r e s o l v e d w i t h 0.4 e q u i v a l e n t o f E u ( f o d ) 3 , and b o t h ortho (7.29 ppm) and para (7.26 ppm) p o s i t i o n s were found t o be i n v o l v e d ( F i g u r e 3B, I n s e t ) . The mass s p e c t r u m o f 37 ( F i g u r e 4B) p r e s e n t e d h i g h mass i o n s a t m\/z 253, 252, 251 and 250 w h i c h d e m o n s t r a t e d t h a t i n c o m p l e t e exchange o f t h r e e d e u t e r i o p h e n y l l a b e l s had o c c u r r e d t o a f f o r d a m i x t u r e o f [ ^ 5 ] , [ 2 H 4 ] , [ 2 H 3 ] and [ 2 H 2 l a n a l o g u e s . Harada and T i t a n i (1936) r e p o r t e d t h e t h e r m a l r e a r r a n g e m e n t o f C6H5NH2D\"1\" t o i t s ortho and para r i n g d e u t e r a t e d a n a l o g u e s . O k a z a k i and Okumura (1961) proposed t h a t above 150\u00b0C and below t h e m e l t i n g p o i n t , t h e p r o t o n s o f t h e NH3 + group o f a n i l i n i u m s a l t s were i n t r a m o l e c u l a r l y t r a n s f e r r e d a l o n g t h e w - e l e c t r o n c l o u d t o t h e c a r b o n atoms o f h i g h e r e l e c t r o n d e n s i t y . Thus, t h e ortho and para p o s i t i o n s a r e s e l e c t i v e l y t a r g e t e d f o r p r o t o n exchange. In an a n a l o g o u s f a s h i o n , aniline-2,3,4,5, - d s c o u l d be r e a r r a n g e d t o i t s r i n g p r o t o n a t e d a n a l o g u e s t h r o u g h i t s a n i l i n i u m i o n as shown i n Scheme X. 69 F i g u r e 3. 400 MHz H-NMR s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) p e n t a -d e u t e r i o p h e n y l a m i n e 18, and (B) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o -p h e n y l a m i n e 37 showing s u b s t a n t i a l i n t r o d u c t i o n o f p r o t o n s a t t h e ortho and para p o s i t i o n s o f t h e d e u t e r i o p h e n y l r i n g . R e s o l u t i o n o f t h e ortho\/para p r o t o n s w i t h 0.4 e q u i v a l e n t E u ( f o d ) 3 shows t h a t exchange o c c u r r e d a t both ortho and para p o s i t i o n s ( I n s e t ) . 70 253 S4 30 \u2022ni .ill 172 143 1X34 i i i i i i i i i l iLll i i Jlu .mil, l|, ,.titiiB|. \u201eill 207218 i.. \u2022I..n1. i 23S Ivf + 252 B S3 170 8 1 142 20S 218 235 MAM J l . j M m\/z F i g u r e 4. E l e c t r o n impact mass s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18 showing h i g h i s o t o p i c p u r i t y w i t h M +-, m\/z 253, and (B) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) d e u t e r i o p h e n y l a m i n e 37 showing t h a t i t o c c u r r e d as a m i x t u r e o f [ 2 H 5 ] ( M + * , m\/z 2 5 3 ) , [ 2H4] ( M + > , m\/z 2 5 2 ) , [ 2 H 3 ] (M+-, m\/z 251) and [ 2 H 2 ] ( M + - , m\/z 250) a n a l o g u e s . 71 3 6 PPH F i g u r e 5. 75 MHz broad-band d e c o u p l e d 1 3C-NMR s p e c t r u m o f N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18 showing t h e p r e s e n c e o f d e u t e r i u m a t t h e ortho, para and meta p o s i t i o n s o f t h e p h e n y l n u c l e u s w i t h t h e a p p e a r a n c e o f t r i p l e t s a t 124.26, 125.77 and 129.35 ppm r e s p e c t i v e l y . 72 Scheme X. Mechanism f o r t h e s e l e c t i v e i n t r o d u c t i o n o f p r o t o n s i n t o t h e d e u t e r i o p h e n y l n u c l e u s o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - d e u t e r i o p h e n y l a m i n e 37. (A) N u c l e o p h i l i c a t t a c k , (B) P r o t o n a b s t r a c t i o n and d e p a r t u r e o f C I \" , (C) Thermal rearrangement o f a n i l i n i u m c a t i o n , x = 2, 3, 4, 5, (D) P r o t o n a b s t r a c t i o n t o form r e a r r a n g e d a n i l i n e n u c l e o p h i l e . The s u b s t i t u t i o n o f 2 , 4 - d i c h l o r o n i t r o b e n z e n e by a n i l i n e - 2 , 3 , 4 , 5 , 6 - d 5 p r o c e e d s via a 2- s t e p S^jAr mechanism ( M a r c h , 1985b). In t h e f i r s t s t e p a n i l i n e a c t s as a n u c l e o p h i l e a t t a c k i n g t h e e l e c t r o n d e f i c i e n t 2 - p o s i t i o n o f t h e s u b s t r a t e t o a f f o r d a t e t r a h e d r a l i n t e r m e d i a t e . In t h e second s t e p t h e C I \" l e a v i n g group d e p a r t s and a p r o t o n i s a b s t r a c t e d by a n i l i n e f u n c t i o n i n g as a base. A t t h e r e a c t i o n t e m p e r a t u r e o f 170\u00b0C t h e r e s u l t i n g a n i l i n i u m c a t i o n r e a r r a n g e s w i t h t h e N - p r o t o n s b e i n g i n t r o d u c e d a t t h e 73 ortho and para p o s i t i o n s o f t h e phenyl r i n g . The r e s u l t i n g c o n j u g a t e base o f t h e r e a r r a n g e d a n i l i n i u m c a t i o n i n t u r n , p a r t i c i p a t e s as a n u c l e o p h i l e t o a f f o r d 37. GCMS a n a l y s i s o f t h e r e a c t i o n m i x t u r e a f t e r t h e f o r m a t i o n o f 37 r e v e a l e d t h a t N-D exchanges w i t h N-H (as d i s c u s s e d above) i n t h a t t h e e x c e s s a n i l i n e was no l o n g e r f u l l y d e u t e r a t e d i n t h e phenyl r i n g but i n s t e a d c o n s i s t e d o f a m i x t u r e o f -ds ( M + * , m\/z 98, 45 % ) , -d4 ( M +*, m\/z 97, 1 0 0 % ) , -d3 ( M + - , m\/z 96, 75 % ) , and -d2 ( M + - , m\/z 95, 20 %) a n a l o g u e s . On t h e o t h e r hand, when a n i l i n e - d 7 was used i n t h e s y n t h e s i s o f 18 t h e p e n t a d e u t e r i o p h e n y l n u c l e u s remained e s s e n t i a l l y i n t a c t because o f t h e absence o f a p r o t o n s o u r c e w i t h which exchange c o u l d o c c u r . GCMS: a n i l i n e -d6 ( M + - , m\/z 99, 2 % ) , -d5 (M+-, m\/z 98, 100 % ) , -d4 ( M + - , m\/z 97, 25 % ) , and -d3 ( M + - , m\/z 96, 3 % ) . Because t h e rearrangement o f t h e a n i l i n i u m c a t i o n o c c u r s above l S C C (Scheme X ) , c a r e f u l a t t e n t i o n t o t h e e x c l u s i o n o f p o t e n t i a l p r o t o n s o u r c e s was o n l y r e q u i r e d a t e l e v a t e d t e m p e r a t u r e s . Thus, a f t e r t h e r e a c t i o n was a d j u s t e d t o room t e m p e r a t u r e , t h e work-up o f 18 c o u l d be c o n t i n u e d under ambient c o n d i t i o n s w i t h o u t t h e p o s s i b i l i t y o f d e u t e r i u m exchange. F u r t h e r m o r e , t h e subsequent s t e p s (C -> E, Scheme IX) i n t h e s y n t h e s i s o f [ 2Hs]CLBZ do n o t r e s u l t i n t h e l o s s o f 2 H - l a b e l s . A l t h o u g h t h e in situ d e u t e r i u m r e a r r a n g e m e n t i n t h e f o r m a t i o n o f 18 i s one o f i t s more s a l i e n t f e a t u r e s , t h e r e i s y e t a n o t h e r n o t e w o r t h y s t r u c t u r a l p r o p e r t y , namely t h e pronounced d e s h i e l d i n g and absence o f D 20 e x c h a n g e a b i l i t y a s s o c i a t e d w i t h t h e amine p r o t o n . I n t r a m o l e c u l a r H-bonding w i t h t h e n i t r o group i n a s t r u c t u r e d 6-membered r i n g ( F i g u r e 6) can acc o u n t f o r b o t h phenomena. E l e c t r o s t a t i c i n t e r a c t i o n w i t h t h e n i t r o group r e d u c e s t h e e l e c t r o n d e n s i t y a t N-H and d e s h i e l d s t h e p r o t o n f r o m i t s normal c h e m i c a l s h i f t (=3 - 5 ppm) ( S i l v e r s t e i n e t a l . , 1981) t o 9.52 ppm, and t h e 74 f o r m a t i o n o f a 6-membered r i n g s t a b i l i z e s t h e p r o t o n t h e r e b y m i n i m i z i n g D\u00a30 e x c h a n g e a b i l i t y . F i g u r e 6. The f o r m a t i o n o f a 6-membered r i n g between t h e n i t r o group and amine p r o t o n o f t h e phenylamine 18 r e s u l t s i n N-H d e s h i e l d i n g and d i m i n i s h e d D2O exchange. Treatment o f 18 w i t h e t h y l m a l o n y l c h l o r i d e a f f o r d e d t h e carbamoyl a c e t a t e 19 i n good y i e l d . Two n o t e w o r t h y c h e m i c a l p r o p e r t i e s o f t h e carbamoyl a c e t a t e 19 were o b s e r v e d under GCMS and NMR a n a l y s e s : ( i ) th e r m a l r e a r r a n g e m e n t o f 19 o c c u r s d u r i n g GCMS a n a l y s i s t o a f f o r d 18 (Scheme X I ) , and ( i i ) t h e \/3 - d i c a r b o n y l s y s t e m o f 19 i s n o t r e a d i l y p r e d i s p o s e d t o k e t o - e n o l t a u t o m e r i s m (Scheme X I I ) . TLC w i t h a m o b i l e phase o f p e t e t h e r : E t O A c ( v \/ v ) 2:1 p r o v i d e d an e f f e c t i v e probe i n t o t h e s y n t h e s i s o f 19 ( R f , 0.49) from 18 ( R f , 0.79), because o f t h e d i f f e r e n c e i n p o l a r i t y o f t h e two compounds. However, by GCMS bo t h 19 and 18 appeared t o have i d e n t i c a l r e t e n t i o n t i m e s ( F i g u r e 7) and mass s p e c t r a ( F i g u r e 8 ) . Because o f t h e s u b s t a n t i a l d i f f e r e n c e i n t h e i r p o l a r i t y i t was u n l i k e l y t h a t 19 and 18 would have i d e n t i c a l r e t e n t i o n t i m e s . A p l a u s i b l e e x p l a n a t i o n i s t h a t 19 undergoes t h e r m a l r e a r r a n g e m e n t t o 18 a t t h e i n j e c t i o n p o r t (240\u00b0C) by ^ - p r o t o n a b s t r a c t i o n and e l i m i n a t i o n o f t h e n e u t r a l ketene (Scheme X I ) . o II 7 5 F i l e >RB198 60.6-488.6 amu. d5-PCNN TIC 6888608 5566868 5866668 4566666 4066666 3586068 3088808-2586808 2800608 1500000-1000800-500000 0 S.3S 3.97 \u2022 !\u2022\u2022 t, \u2022\u2022I^Tr r^Tr.-, -s jTT, ,-, |h< p n i l . i i i i n i i i i i i i i i i i i i 3.0 4.0 5.0 6.0 7.0 8.6 9.0 10.0 11.0 12.0 13.0 14.0 15.6 16.0 L01 F i l e >AB 2200000 2000888 1800000-1600000-1400000 1206888 1800008 800000-686008 400000 280000 0 195 60.0-408.0 arou. d5-PCHfl TIC 5.34 B ^BMEg9\u00bb949 11 .99 ,,,,,|,,,,|,,,,,,,,,|,,,,,,,,,, I . \u2022 \u2022 . , 3.8 4.0 5.8 6.0 7.0 8.0 9.8 10.0 11.0 12.0 13.0 14.0 15.0 16.0 F i g u r e 7. Thermal rearrangement o f t h e car b a m o y l a c e t a t e 19 t o t h e phen y l a m i n e 18 r e s u l t s i n i d e n t i c a l r e t e n t i o n t i m e s i n t h e TIC p l o t s o f (A) 19 and (B) 18. 76 F i l e >RB198 Bpk flb 786117 dS-PCNM CLP Scan 241 5.33 min. 800000-700000-600000^ 500000-400000-^ 300000-200000^ 100000-82 \\ 0B195 Bpk fib 394946 d5-PCNfi 400080-369000-320088^ 280000-248888-200000-160000-120000-80000-40080-B 8 2 1 4 3 144 |j 1 7 3 \\ 86 \\ \/ If\/ 1 9 S \/ 189118 jr J | ; \u2022\u20141\u2014\u2022\u20141\u20141\u20141\u20141\u20141\u2014\u2022\u20141\u20141\u20141\u2014\u2022\u20141\u2014\u2022\u2014i\u20141\u20141\u2014\u2022\u20141\u2014<\u20141\u2014\u2022 1 \u2022 1 ' 88 180 128 148 166 180 200 CLP 172 218 287 \/ Scan 235 5.33 min. 236 253 liL ill... -J r l l 0 j-iee |90 r?0 |68 |se J48 r38 J20 j-10 220 248 260 F i g u r e 8. Thermal r e a r r a n g e m e n t o f t h e carbamoyl a c e t a t e 19 t o t h e phen y l a m i n e 18 r e s u l t s i n i d e n t i c a l GC ( E I ) mass s p e c t r a o f (A) 19 and (B) 18. 77 Scheme X I . Thermal rearrangement o f 19 t o f o r m 18 by 0-proton a b s t r a c t i o n and e l i m i n a t i o n o f t h e n e u t r a l k e t e n e . Ample d r i v i n g f o r c e f o r such a r e a r r a n g e m e n t e x i s t s i n t h e a c i d i t y o f t h e meth y l e n e p r o t o n s and t h e l o s s o f t h e s t a b l e c o n j u g a t e d k e t e n e (38) t o form 18. The absence o f an o l e f i n i c p r o t o n (=5 - 6 ppm) and e n o l p r o t o n (-15.0 ppm) ( S i l v e r s t e i n e t a l , 1981) i n t h e lH NMR s p e c t r u m o f 19 ( F i g u r e 9) r e v e a l e d t h a t t h e l a t t e r does not r e a d i l y t a u t o m e r i z e (Scheme X I I ) . Resonance forms w h i c h a r i s e due t o back d o n a t i o n o f t h e l o n e p a i r s o f e l e c t r o n s on t h e amide n i t r o g e n and e s t e r oxygen s e r v e t o reduc e t h e a c i d i t y o f t h e methylene p r o t o n s and hence r e s t r i c t e n o l f o r m a t i o n ( F i g u r e 1 0 ) . H-3 ci 2 ^ N 0 2 0 N' \"OEt 19 -C(=0)CH 2C=0) -0CH 2 CH 3 f ) \\ JL \u2022CH 2 CH 3 LJ 1111111111111111111111111111 1111>111' 111111111111111 i i i [ 1 1 1 1 1 1 1 i 11 11 6 5 4 3 2 Figure 9. 300 MHz *H-NMR of the carbamoyl acetate 19. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 CO 79 CI Scheme X I I . K e t o - e n o l t a u t o m e r i s m o f 19 showing s t r o n g e q u i l i b r i u m d i s p l a c e m e n t t o t h e k e t o form. F i g u r e 10. The f o r m a t i o n o f r e s o n a n c e s p e c i e s o f 19 w h i c h r e d u c e t h e a c i d i t y o f t h e methylene p r o t o n s and so i n h i b i t k e t o - e n o l t a u t o m e r i s m . In t h e t r e a t m e n t o f 19 w i t h Z n \/ H C l , c o n c o m i t a n t r e d u c t i o n o f t h e n i t r o group t o an amine and d e e s t e r i f i c a t i o n t o t h e c a r b o x y l i c a c i d r e s u l t e d i n r i n g c l o s u r e t o [ 2H5]DMC ( 2 0 ) . Because o f i t s r e l a t i v e h i g h p o l a r i t y , 20 chromatographed p o o r l y on t h e m e t h y l s i l i c o n e column. TMS d e r i v a t i z a t i o n o f 20 d r a m a t i c a l l y improved i t s c h r o m a t o g r a p h i c ( F i g u r e 11) and mass s p e c t r a l ( F i g u r e 12) p r o p e r t i e s . A l t h o u g h t h e TMS d e r i v a t i v e i s d i a g r a m a t i c a l l y r e p r e s e n t e d as b e i n g f u n c t i o n a l i z e d a t t h e s e c o n d a r y amide N - p o s i t i o n , t h i s does not p r e c l u d e t h e l i k e l i h o o d o f e n o l i c 0-OEt 80 d e r i v a t i z a t i o n . No d i s t i n c t i o n c o u l d be made between t h e two i s o m e r s , s i m p l y because a l l t h e f r a g m e n t s w h i c h were a s s i g n e d a r e e q u a l l y d i a g n o s t i c o f t h e two for m s . F u r t h e r m o r e , t h e moderate s t a b i l i t y o f t h i s d e r i v a t i v e d i d n o t a l l o w p u r i f i c a t i o n f o r NMR a n a l y s i s . The p o s s i b l e m i g r a t i o n o f t h e TMS group from N- t o O-centres on amides due t o k e t o - e n o l t a u t o m e r i s m has been p r e v i o u s l y d e s c r i b e d f o r h i p p u r i c a c i d (Goodman and Markey, 1981). Lower mass f r a g m e n t s a t m\/z 225 and m\/z 239 w h i c h appeared i n TMS-d e r i v a t i z e d 20 were c h a r a c t e r i s t i c o f a l l T M S - d e r i v a t i z e d D M C - r e l a t e d m e t a b o l i t e s . Proposed pathways w h i c h a c c o u n t f o r t h e s e f ragment i o n s w i l l be d i s c u s s e d i n t h e s e c t i o n on m e t a b o l i t e s y n t h e s i s . The f i n a l s t e p i n t h e s y n t h e s i s o f 21 i n v o l v e d t h e m e t h y l a t i o n o f 20, u s i n g t h e method o f I s e l e and L u t t r i n g h a u s ( 1 9 7 1 ) . S e l e c t i v e m e t h y l a t i o n o f t h e s e c o n d a r y amide o f 20 i n t h e p r e s e n c e o f t h e m e t h y l e n e group, r e q u i r e d c a r e f u l c o n t r o l o f t h e number o f e q u i v a l e n t s o f base. Use o f 1.2 e q u i v a l e n t s o f KOH and e x c e s s CH3I a f f o r d e d [ 2Hs]CLBZ i n 37 % y i e l d and v i r t u a l l y no e v i d e n c e o f t h e d i m e t h y l compound 3-methyl[ 2H5JCLBZ. In t h e *H-NMR spect r u m o f [ 2Hs]CLBZ t h e m e t h y l e n e p r o t o n s appeared as a d o u b l e t o f d o u b l e t s w i t h H-3\/J and H-3a a t 3.50 and 3.56 ppm r e s p e c t i v e l y ( F i g u r e 1 ) . These a s s i g n m e n t s a r e c o n s i s t e n t w i t h 21 e x i s t i n g i n a pseudo-boat c y c l o h e p t a d i e n e - 1 i k e c o n f r o m a t i o n w i t h H-3\/J p s e u d o a x i a l l y d i r e c t e d i n t o t h e s h i e l d i n g r e g i o n o f t h e benzo r i n g ( A v e r s a e t a l . , 1980). The absence o f phenyl r i n g r e s o n a n c e due t o d e u t e r i u m - l a b e l l i n g a l l o w e d t h e c h e m i c a l s h i f t s o f t h e benzo r i n g t o be d i a g n o s t i c a l l y a s s i g n e d w i t h H-6, H-8 and H-9 p r e s e n t a t 6.93, 7.25 and 7.31 ppm r e s p e c t i v e l y . I t i s n o t e w o r t h y t h a t H-6 and H-9 a r e d i a m e t r i c a l l y d i s p o s e d about H-8 w i t h H-6, d e s p i t e i t s p r o x i m i t y t o t h e e l e c t r o n w i t h d r a w i n g CI g r o u p , b e i n g u p f i e l d f r o m H-9. T h i s s u g g e s t s t h a t t h e e l e c t r o n d o n a t i n g i n f l u e n c e o f t h e N-phenyl m o i e t y 81 ortho t o H-6 o u t w e i g h s t h a t o f t h e N-methyl group ortho t o H-9. In t h e GCMS ( E I ) s p e c t r u m o f 21, [M-0H] +- (m\/z 288) and m\/z 181 a r e d i a g n o s t i c ( F i g u r e 2 ) . The d e t a i l s o f t h e s e and o t h e r t y p i c a l C L B Z - r e l a t e d f r a g m e n t a t i o n s w i l l be d i s c u s s e d i n t h e s e c t i o n on m e t a b o l i t e s y n t h e s i s . HPLC a n a l y s i s r e v e a l e d t h a t [ 2Hs]CLBZ was >98 % p u r e , and chromatographed ( t R 6.17 min) s l i g h t l y i n f r o n t o f an a u t h e n t i c s t a n d a r d o f CLBZ ( t R 6.32 m i n ) . The d i f f e r e n c e i n t h e r e t e n t i o n t i m e s between compounds and t h e i r d e u t e r a t e d a n a l o g u e s on r e v e r s e phase HPLC has ample p r e c e d e n c e i n t h e l i t e r a t u r e (Honma e t a l . , 1987). 82 \u2022\u2022\"-le >RB2S3 70.0-5S0.B aau. dS-DMC T I C F i g u r e 11. TIC p l o t s o f (A) [ 2H 5]DMC and (B) [ 2H 5]DMC-TMS show t h e improved chromatography o b t a i n e d w i t h T M S - d e r i v a t i z a t i o n . 83 3 6 6 6 6 -3 2 8 0 0 -2 8 8 6 0 : 2 4 0 8 0 : 2 0 0 0 0 -1 2 8 0 0 ^ 8 0 0 8 -4 0 0 0 -8 2 . . a 1 6 8 1 8 5 1 1 8 1 2 4 N \\ 1 .LILJT^ I\u00ab\u00ab^ H.J(LL>WFIVJLI \u00ab\u2022 \u2022 \u2022 i \u2022 \u2022 \u2022 i 8 8 1 2 6 1 6 8 2 8 0 2 4 0 2 8 0 3 2 0 3 6 0 F i g u r e 12. GCMS ( E I ) s p e c t r a o f (A) [2Hs]DMC and (B) [2Hs]DMC-TMS show t h e i n c r e a s e d mass s p e c t r a l f r a g m e n t a t i o n o b t a i n e d w i t h T M S - d e r i v a t i z a t i o n . 84 (B) The S y n t h e s i s o f T r i d e u t e r i o m e t h y l CLBZ. T r i d e u t e r i o m e t h y l CLBZ ( [ 2 H 3 ] C L B Z , 22) was p r e v i o u s l y s y n t h e s i z e d by P o u p a e r t e t a l . (1988) i n o r d e r t o i n v e s t i g a t e t h e d e u t e r i u m i s o t o p e e f f e c t on t h e p h a r m a c o l o g i c a l a c t i o n o f CLBZ. A l t h o u g h a p r o l o n g e d d u r a t i o n o f a c t i o n was o b s e r v e d , no (k^ \/kg) v a l u e s f o r t h e i s o t o p e e f f e c t were r e p o r t e d . In o r d e r t o d e r i v e t h e kn\/kn, v a l u e f o r t h i s i s o t o p e e f f e c t in vitro, t r i d e u t e r i o m e t h y l CLBZ was s y n t h e s i z e d by t r i d e u t e r i o m e t h y l a t i o n o f DMC w i t h C D 3 I ( I s e l e and L u t t r i n g h a u s , 1971). *H-NMR ( F i g u r e 13A) r e v e a l e d t h e d i a s t e r e o m e r i c n a t u r e o f t h e H-3\/J (3.52 ppm) and H-3a p r o t o n s (3.58 ppm), p r e v i o u s l y d e s c r i b e d f o r [ 2Hs]CLBZ. In c o n t r a s t t o t h e l a t t e r compound, t h e r e s o n a n c e c h a r a c t e r i s t i c o f t h e N-methyl p r o t o n s was ab s e n t i n [ 2H3]CLBZ t h e r e b y d e m o n s t r a t i n g t h e h i g h i s o t o p i c p u r i t y o f t h e N - d e u t e r i o m e t h y l g r o u p . F u r t h e r m o r e , r e s o n a n c e a t 7.20 (H-2', H-6'), 7.36 (H-4') and 7.43 (H-3', H-5') c h a r a c t e r i s t i c o f t h e phenyl p r o t o n s o f [ 2H3]CLBZ a f f o r d e d a c o n v e n i e n t c o m p a r i s o n t o p e n t a d e u t e r i o p h e n y l CLBZ ( i n which t h i s r e s o n a n c e was a b s e n t ) . The mass s p e c t r u m o f [ 2H3]CLBZ s u b s t a n t i a t e d t h e ^-NMR d a t a w i t h M +* a t m\/z 303 and v i r t u a l l y no e v i d e n c e f o r t h e p r o t i o a n a l o g u e . C h a r a c t e r i s t i c f r a g m e n t s a t [M-0H] +* m\/z 286 and m\/z 184 ( t h e d e u t e r i o a n a l o g u e o f m\/z 181 d e s c r i b e d f o r [ 2H5]CLBZ) w i l l be d i s c u s s e d l a t e r . 85 F i l e : Bpk Rl 10000- 95 127 iliiPB294 417.7-418.7 amu.4' 0H-30Me-CLBZ-TMS SMT ADC EIP 23.49 meta ' i I v i i i | i i t i i r*i i i j i i i H s r i i T I i t T-f - i ' f T n ^ T ^ i i f \u2022) i i i v | i i i i | 13.0 14.8 IS. 016.817.0 18.019.8 20.021.0 22.0 23.8 24.0 25.0 26.0 F i g u r e 14. Mass chromatogram o f t h e T M S - d e r i v a t i v e s o f CLBZ 0-methyl c a t e c h o l i s o m e r s a t M + , \u00bb m\/z 418. Meta i s o m e r ( 3 6 a ) : t R 23.49 min, para i s o m e r ( 3 6 b ) : 22.80 min. 91 (D) GCMS A n a l y s i s o f CLBZ and I t s M e t a b o l i t e s . In o r d e r t o d e t e r m i n e t h e r e t e n t i o n t i m e s o f t h e b i o l o g i c a l l y - d e r i v e d m e t a b o l i t e s , c o n t r o l b i l e was s p i k e d w i t h 4'-hydroxy CLBZ ( 2 8 ) , 4'-hydroxy DMC ( 2 6 ) , 3 ' , 4 ' - d i h y d r o x y CLBZ (35) and 3 ' , 4 ' - d i h y d r o x y DMC ( 3 2 ) . The b i l e was e x t r a c t e d and p r e p a r e d f o r GCMS i n a s i m i l a r f a s h i o n t o t h e b i l e o f t h e dosed r a t s . TIC p l o t s f o r t h e T M S - d e r i v a t i z e d s t a n d a r d s a r e p r e s e n t e d i n F i g u r e 15. Because o f t h e l i m i t e d q u a n t i t i e s o f t h e 0-methyl c a t e c h o l a n a l o g u e s o f CLBZ (36a+b) and DMC ( 3 3 ) , t h i s e x p e r i m e n t c o u l d n ot be per f o r m e d w i t h t h e s e samples. Mass s p e c t r a o f t h e T M S - d e r i v a t i z e d s t a n d a r d s a r e p r e s e n t e d i n F i g u r e s 16 - 22, and p o s s i b l e f r a g m e n t a t i o n mechanisms p r o p o s e d i n Schemes XIV - X V I I I . GCMS d a t a a r e summarized i n T a b l e 1. There a r e l i m i t e d r e p o r t s i n t h e l i t e r a t u r e d ocumenting t h e mass s p e c t r o s c o p y o f CLBZ and DMC ( R i v a e t a l . , 1981; C a c c i a e t a l . , 1979) and t h e i r 4'-hydroxy m e t a b o l i t e s ( D r o u e t - C o a s s o l o e t a l . , 1989). V o l z e t a l . (1979) r e p o r t e d s e v e r a l m e t a b o l i t e s o f CLBZ b u t p r o v i d e d no s u b s t a n t i a t i n g s p e c t r a l e v i d e n c e . To o u r knowledge, t h e f o l l o w i n g r e p r e s e n t s t h e f i r s t d e t a i l e d a c c o u n t o f t h e mass s p e c t r o s c o p y o f CLBZ and i t s m e t a b o l i t e s . ( i ) Mass s p e c t r o s c o p y o f CLBZ. The a v a i l a b i l i t y o f CLBZ, and i t s d e u t e r a t e d a n a l o g u e s [2Hs] CLBZ (21) and [ 2h\"3] CLBZ (22) w i t h d e u t e r i u m p r e s e n t i n t h e phenyl and methyl c e n t r e s r e s p e c t i v e l y a l l o w e d s e v e r a l f r a g m e n t s o f CLBZ t o be r e a d i l y e l u c i d a t e d . F r a g m e n t a t i o n s were p e r c e i v e d as a r i s i n g from b o t h k e t o and en o l t a u t o m e r s o f t h e l a c t a m (Scheme X I V ) . When t h e e n o l t a u t o m e r was 9 2 invoked i t was apparent that loss of OH* gave r i se to the highest mass fragments for 21 ([M-0H] +*, m\/z 288) and 22 ([M-0H] +*, m\/z 286). Subsequent loss of CO, charac te r i s t i c of lactams (Budzikiewicz et a l . , 1967) afforded ions m\/z 260 and m\/z 258 for (21) and (22) respect ive ly . It i s noteworthy that th is cascade proceeds with the prel iminary loss of OH*, and not CO as evidenced by the v i r tua l absence of [M-C0]* + at m\/z 277 and 275 in the mass spectra of (21) and (22) respect ive ly . The loss of neutral carbenes i s cha rac te r i s t i c of aromatic amides (Budzikiewicz et a l . , 1967) and could account for the loss of H2=C=0 with concomitant r ing closure to afford [M-42] + *. The loss of isocyanate from semicarbazones has been described (Budzikiewicz et a l . , 1967). In l i k e fashion, th is fragmentation was extrapolated to the lactam of CLBZ to account for the loss of phenylisocyanate and methylisocyanate (Scheme XV). Loss of phenyl isocyanate gave r i se to the ions m\/z 181 and 184 for 2_1 and 22 respect ive ly . Correspondingly, loss of methylisocyanate afforded ions m\/z 248 and 243. ( i i ) Mass spectroscopy of CLBZ metabol i tes. The mass spectral fragmentation cha rac te r i s t i c of CLBZ was extrapolated to CLBZ metabolites with appropriate increments made for -OTMS, di-OTMS and -OTMS-OMe subst i tuted fragments (Scheme XIV). Fragmentation pathways did not allow the TMS-derivatized 0-methyl catechols 36a+b to be d is t ingu ished. TMS-directed loss of the methyl rad ica l from both the M +* and [M-H2CC0]+* ions gave r i se to prominent high mass ions for 28, 35 and 36a+b (Scheme XIV). In add i t ion , loss of C 2 He from M +* and [M-H 2 CC0] + * of 36a+b gave r i se to the major ions m\/z 388 and 346 respect ively 93 (Scheme X V I ) . The l o s s o f Si(013)4 f r o m di-TMS c a t e c h o l s t o form a 5-membered s i l y l o x y r i n g (Goodman and Markey, 1981) i s an a l o g o u s t o t h e l o s s o f C2H6 from 36a+b shown i n Scheme X V I . However, t h e f o r m a t i o n o f t h i s f r a gment w i t h t h e di-TMS c a t e c h o l s o f CLBZ was n o t a p p a r e n t . ( i i i ) Mass s p e c t r o s c o p y o f T M S - d e r i v a t i z e d N-desmethvl m e t a b o l i t e s . Whether T M S - d e r i v a t i z a t i o n o f amides o c c u r s a t t h e k e t o n i t r o g e n o r t h e e n o l h y d r o x y l t h r o u g h t a u t o m e r i s m i s u n c l e a r (Goodman and Markey, 1981). F o r c o n v e n i e n c e , t h e N-TMS d e r i v a t i v e s a r e r e p r e s e n t e d . D i a g n o s t i c f r a g m e n t a t i o n o f t h e N-TMS d e r i v a t i v e s i n v o l v e d l o s s o f th e methyl r a d i c a l (Scheme X V I I ) , and l o s s o f t h e ph e n y l m o i e t y (Scheme X V I I I ) . Loss o f t h e p h e n y l i s o c y a n a t e w h i c h was p r e v i o u s l y d i s c u s s e d g i v e s r i s e t o m\/z 239. The i o n m\/z 225 was a m a j o r f r a g m e n t f o r 20, 26, 32 and 33 and p r o b a b l y a r o s e from t h e l o s s o f a n e u t r a l l a c t a m (Scheme X V I I I ) . The d i a g n o s t i c l o s s o f C2H6, d e s c r i b e d f o r M +* and [M-H 2CC0] +- o f 36a\u00b1b, a l s o o c c u r r e d w i t h 33 t o a f f o r d t h e i o n m\/z 446 (Scheme X V I ) . 94 f i l e >RB313 70.0 17 2000000 1800800 1608000-1400080-1200088^ 1000000-800008-600000 400008-200808 0--550.0 amu. METflB-STB-BILE-SPIKE EIP 98 26, 16 .88 21 .36 ' i \u2022 ' \u2022 \u2022 i ' ' \u2022 ' i \u2022 1 ' ' l \u2022 ' 1 ' l \u2022 \u2022 \u2022 \u2022 i \u2022 \u2022 \u2022 \u2022 i \u2022 \u2022 \u2022 \u2022 i \u2022 \u2022 \u2022 \u2022 i \u2022 \u2022 \u2022 \u2022 i \u2022 \u2022 17.0 18.0 19.8 20.0 21.8 22.0 23.0 24.0 25.0 26.0 f i l e >RG313 70.0-558.0 amu. METfiB-STD-BILE-SPIKE TIC 15 .56 F i g u r e 15. TIC p l o t s o f (A) T M S - d e r i v a t i z e d m e t a b o l i t e s t a n d a r d s 26, 28, 32 and 35 a f t e r r e c o v e r y from s p i k e d c o n t r o l b i l e . (B) 1 5 - f o l d d i l u t i o n o f (A) w i t h EtOAc. 9 5 F i l e >AB313 Bpk Ab 121760 130O0O-120000-110000-100000-98888-80888-70088-60008-50000-40000-38800- 75 20008-1 10000-e-1 METflB-SID-BILE-SPIKE CLP 22S 193 13S 197 88 120 168 208 17 431 331 356 389 446 \/ Sean 83 ,89 M i n . pi 10 i l e a :90 I 8 0 i70 :60 :S0 :40 r30 rZO rl0 \u2022^ 0 240 280 328 360 480 440 F i g u r e 16. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-DMC (26) showing t h e p r e s e n c e o f M +* a t m\/z 446. F i l e >AB313 Bpk Ab 56520 60000-55000-50000-45000-4000O-35008-30008-25000-20000-15000-10000- ^76 500O-0-L A 8Q METAB\u2014STD\u2014BILE-SPIKE CLP 179 153 151 343 76 117 \\ \u201e\u201e\u201e 303 35 B L \u2122 ( I ll 2 1 7 \/ I jl .ijLiuAB313 Bpk Ab 52280 55800-50888-45800-40000 35000-38800-25000-20000-15000-10000 5000 0 METAB-STD-BILE-SPIKE CLP 99 * i i Y j - J i - d l L 225 2 6 7 \" 6 p9 389*?* 4 4 4 4 9 3 I 2 5 0 3 0 0 3 5 8 4 8 0 4 5 8 5 8 8 485 4 108 158 288 Scan 177 19.51 n i n . 534 r l l 0 rl88 |90 :88 r7 0 :68 !se :48 r3 0 |2G 4 3 < [ i i e -0 F i g u r e 18. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 3',4'-dihydroxy-DMC (32) showing t h e p r e s e n c e o f M+* a t m\/z 534. F i l e >AB313 Bpk Ak 47688 52000-1 48000 44888 40000 36800 32888-28000 24000-20808 16800-12000-8000-4080-0-92 1 3 7 1 5 3 _ IIJI.^1 \u2022 ' i t i l lni '^l i METAB-STD-BILE-SPIKE CLP 1 7 9 267 303 L .*c^A . j i\u00ab^-wx^i^ jtLtJt. A. .... Scan 428 23.81 M i n . 476 188 1 5 8 288 258 3 8 8 3 4 3 4 3 1 ' 3 5 8 4 8 8 4 5 ' a ' ' r l l 0 J100 r98 is 8 70 r*8 ise 8 r3 0 :28 8 F i g u r e 19. EI mass s p e c t r u m o f t h e TMS d e r i v a t i v e o f 3 ' , 4 ' - d i h y d r o x y CLBZ (35) showing t h e p r e s e n c e o f M+* a t m\/z 476. F i l e >flG294 Bpk Rb 10895 3' ,4'-0H,0Me-CLBZ CLP S c a n 452 23.70 m i n . 12600-1 11000-10000-9000-8000-7000-6000-5000-4000-3000-2000-1000-0-73 418 O - S i -346 373 166 \/ 153 117 lllill'ii'qi.kllL T\u2014'\u20141\u2014'\u2014I\u2014* 209 303 3 ; 8 245 282 \\ lllltjliL>,,ili,itt\u2014i\u2014\"\u2014r 320 360 400 r-110 ;108 r90 |80 r70 7&0 |50 r46 :30 r20 Ll0 F i g u r e 20. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3' methoxy-CLBZ (36a) showing t h e p r e s e n c e o f M +* a t m\/z 418. F ; i l e >flG294 flb 7087 3' ,4'-0H,0Me-CLBZ CLP 7000H 6000-1 5000-1 40004 3000-1 2000-1 1000-1 73 166 \/ 418 \\ 147 117 \\ 20; \\ 209 \u2022IlLff 303 318 281 { \/ ill u^^tiLyLil.ti 80 120 160 ' 280 240' ' 280' ' 320\" ' 368' ' '40V F i g u r e 21. EI mass spec t r u m o f t h e TMS d e r i v a t i v e o f 3'-hydroxy-4' methoxy-CLBZ (36b) showing t h e p r e s e n c e o f M +* a t m\/z 418. S c a n 414 23.05 m i n . HO j-100 r90 J80 r70 J60 5^0 f40 30 720 :10 98 F i l e >AB291 Bpk Ab 13430 -MeO\u2014DMC\u2014TMS CLP Scan 18 . 42 14000-12000-10000 8000 6000 4000 2000 0-* 73 225 135 ^137 197^ 476 \\ CH3O 239 \/ 266 296 1 \/ S 361 418 446 100 150 200 2S0 300 198 M i n . pi 10 h-00 i90 jso r?0 r60 :S0 ?40 \"-3 0 ^0 ri0 ^0 350 400 450 F i g u r e 22. EI mass spectrum o f t h e TMS d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) showing t h e pr e s e n c e o f M +* a t m\/z 476. 99 Scheme XIV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n r e t e n t i o n o f b e n z o d i a z e p i n e n i t r o g e n . Legend f o r c h e m i c a l s t r u c t u r e s a p p l i e s t o Schemes XIV t o X V I I I . Loss o f t h e methyl r a d i c a l shown f o r 36a i s r e p r e s e n t a t i v e o f 36b. No. R l R 2 R3 R 4 M+- (m\/z) 21 2 H 2 H 2 H CH 3 305 22 H H H CD 3 303 28 H OTMS H CH 3 388 35 OTMS OTMS H CH 3 476 36a 0CH3 OTMS H CH 3 418 36b OTMS OCH3 H CH 3 418 100 B Scheme XV. Proposed f r a g m e n t a t i o n o f C L B Z - r e l a t e d compounds r e s u l t i n g i n l o s s o f t h e (A) N-phenyl and (B) N-methyl m o i e t i e s . Loss o f t h e p h e n y l i s o c y a n a t e i n A gave r i s e t o m\/z 181 f o r 21, 28, 35, 36a and 36b, and m\/z 184 f o r 22. (B) Loss o f methyl i s o c y a n a t e gave r i s e t o t h e c o r r e s p o n d i n g compound and fragment p a i r s , i . e . (Compound No., [M-R 4 N C 0 ] + ' ) . 21, m\/z 248; 22, m\/z 243; 28, m\/z 331; 35, m\/z 419; 36a and 36b. m\/z 361. 101 Scheme X V I . Proposed f r a g m e n t a t i o n o f O-methyl c a t e c h o l s . Ions u n d e r g o i n g t h i s f r a g m e n t a t i o n were M+- o f 36a+b (m\/z, 4 1 8 ) , t h e i r d a u g h t e r i o n s [M-H 2CCO] +- (m\/z 376) and M+- o f 33 (m\/z 4 7 6 ) . L o s s o f C 2 H 6 from t h e s e p r o g e n i t o r i o n s a f f o r d e d t h e c o r r e s p o n d i n g f r a g m e n t s m\/z 388, 346 and 446. Scheme X V I I . Proposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds r e s u l t i n g i n t h e l o s s o f a methyl r a d i c a l . I o n s u n d e r g o i n g t h i s f r a g m e n t a t i o n , 20 (m\/z 2 6 3 ) , 32 (m\/z 534) and 33 (m\/z 476) a f f o r d e d i o n s m\/z 348, 519 and 461 r e s p e c t i v e l y . 102 A B R 2 Scheme X V I I I . P roposed f r a g m e n t a t i o n o f DMC-TMS r e l a t e d compounds 20, 32 and 33 r e s u l t i n g i n l o s s o f t h e N-phenyl r i n g . (A) Loss o f t h e p h e n y l i s o c y n a t e gave r i s e t o t h e i o n m\/z 239 and (B) l o s s o f t h e l a c t a m gave r i s e t o t h e major i o n m\/z 225. Table 1. GCMS data for synthetic CLBZ-related compounds. Compound (No.)a tR (min) M+'(m\/z, %) Diagnostic ions (m\/z, %) [2H53DHC (20) 13.80 363 (26) 225 (100) 239 (46) 82 (42) 348 (21) [2H5]CLBZ (2J.) 15.97 305 (100) 288 (44) 260 (44) 181 (18) 248 (16) [2H3]CLBZ (22) 15.99 303 (100) 258 (94) 261 (76) 286 (52) 184 (22) 4'-OH DMC (26) C 17.98 446 (70) 225 (100) 239 (40) 431 (26) 403 (14) 389 (12) 4'-OH CLBZ (28) C 21.17 388 (100) 343 (75) 181 (33) 331 (42) 153 (32) 3',4'-R2962 70.0-580.0 anu. CfiRBINOLOMIDE-2-TMS 1 TIC -00000-808000-700000 600000-580880 460000-380886 200000 108000 0-- 11.90 14.39 1 16.15 43 i ] i i i \/ | i 1 ! i i | i i i i | i i i i | i i i i | i I'T \u2022\"> f v . .\u2022\u2022 f >-r. \u2022 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.8 20.6 21 .0 22.0 23.0 24.8 25.8 f i l e >R2962 Bpk fib 120392 CfiRBIN0LflMIDE-2-TMS 130000-120000-110000-100000-90000-88008-70000-60000-50000-4W000-30000-20080-10000-0-73 225 251 100 301 327 80 288 X \\ - 1 Z 4 179 \\ i 273 \\ \\ 328 \\ \/ j 155 \/ \\ I ' I \\y 1 I \u2022 \u2022 \u2022 I \u2022 \u2022' I ' ' \u2022 I \u2022 \u2022 \u2022 I ' \u2022 \u2022 I ' \u2022 ' I ' ~ I 1 ' ' I \u2022 \u2022 \u2022 I \u2022 '\" I \u2022 \u2022 J j \u2022 \u2022 \u2022 l \" \u2022 \u2022 I ' \u2022 Scan 219 14.31 min. riie :100 r98 jee :78 6^0 r50 H0 -30 20 18 370 120 168 200 240 280 320 3co F i g u r e 26. (A) GCMS ( C o n d i t i o n , b ) TIC p l o t o f p u r i f i e d 46 d e r i v a t i z e d w i t h TMS. (B) GCMS ( E I ) spect r u m o f 46 shows t h e p r e s e n c e o f M+- a t m\/z 370. *See E x p e r i m e n t a l f o r d e t a i l s . 113 (d) NMR a n a l y s i s . Because t h e T L C - p u r i f i e d components 45, 46 and 47 were p o o r l y s o l u b l e i n CDCI3, DMSO-ds was used as an a l t e r n a t i v e NMR s o l v e n t . Complete d i s s o l u t i o n o f 45, 46 and 47 i n t h i s s o l v e n t a l l o w e d w e l l - d e f i n e d *H-NMR s p e c t r a t o be o b t a i n e d . In t h e *-H-NMR sp e c t r u m o f 47 (DMC) ( F i g u r e 2 7 ) , t h e amide p r o t o n appeared a t 10.70 ppm whi c h was l o w e r d o w n f i e l d t h a n t h e amide p r o t o n o f [2Hs]DMC (20) i n CDCI3 (N-H, 8.21 ppm) ( A p p e n d i x ) . The d e s h i e l d i n g o f t h e amide p r o t o n o f 47 was t h e r e s u l t o f s t r o n g H-bonding w i t h DMSO (Chapman and K i n g , 1964). U p f i e l d r e s o n a n c e d i s p l a y e d by 47 i n DMSO i s a l s o n o t e w o r t h y . Resonance f o r t h e C-3 p r o t o n s o f 47 i n DMSO appeared as broad s i g n a l s a t 3.20 and 3.68 ppm, s y m m e t r i c a l l y d i s p l a c e d about t h e c h e m i c a l s h i f t 3.41 ppm. In CDCI3, t h e C-3 p r o t o n s o f [ 2 H s]DMC (20) appeared as a s i n g l e t (2H, 3.55 ppm) ( F i g u r e 27, I n s e t ) , because r a p i d e q u i l i b r a t i o n between t h e pseudo-boat c o n f o r m e r s ( o f (20)) r e n d e r s t h e (C-3) p r o t o n s e q u i v a l e n t . However, i n DMSO i t a p p ears t h a t t h i s e q u i l i b r a t i o n o c c u r s l e s s r a p i d l y on t h e NMR t i m e s c a l e w i t h t h e a c q u i s i t i o n o f p a r t i a l d i a s t e r e o m e r i c c h a r a c t e r f o r H-3\/J (3.20 ppm) and H-3o: (3.68 ppm). The d i a s t e r o m e r i c d i s p o s i t i o n o f H-3\/5 and H-3a i n t h e b e n z o d i a z e p i n e r i n g was p r e v i o u s l y d i s c u s s e d f o r [ 2Hs]CLBZ ( 2 1 ) . The c h e m i c a l s h i f t s o f 46 f o r t h e amide N-H, benzo and phenyl p r o t o n s ( F i g u r e 28) were s i m i l a r t o 47 (DMC). However, r e s o n a n c e i n t h e u p f i e l d r e g i o n r e v e a l e d t h a t t h e C-3 c e n t r e was c h e m i c a l l y m o d i f i e d by r e a c t i o n w i t h f o r m a l d e h y d e . The p r e s e n c e o f a t r i p l e t , d o u b l e t and broad s i n g l e t ( e x c h a n g e a b l e w i t h D2O) a t 3.42, 3.94 and 4.56 ppm r e s p e c t i v e l y was c o n s i s t e n t w i t h h y d r o x y m e t h y l a t i o n t a k i n g p l a c e a t t h e 3 - p o s i t i o n o f DMC. The -^ H-NMR sp e c t r u m o f 45 (A p p e n d i x ) was s i m i l a r t o t h a t o f 47 f o r 114 t h e a r o m a t i c p r o t o n s and o f f e r e d no d i a g n o s t i c s t r u c t u r a l i n f o r m a t i o n i n t h e u p f i e l d r e g i o n . SHHPLt M : t 2 i ' B.BUCl 082!, 7-5 H-9 r ' \u2022 \u2022 \u2022 \u2022 . \u2022 1 1 I \u2022 ' I ' ' I : \u2022\u2014. \u2014 \u2014 \u2014 ' \u2014 ' r \u2014 ^ \u2014 - r \u2014 \u2022 _ r - r - ^ - -6-\"- 8.8 7.3 7.a 6.5 6.Z 5.5 S .2 t.'s 4*0 3^ 5 P?.1 F i g u r e 27. 400 MHz *H-NMR spectrum o f 47. S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e o f DMC. I n s e t shows t h e e q u i v a l e n c e f o r C -3 p r o t o n s o f [ \u00a3H5]DMC (20) i n CDCI3 . \"\u2022\"Contaminating EtOAc. H-3', H-5' 1 H-2', H-6' , , ^ r - T T - T - r i ; i i . . i i i - p - | i , , , . . i i, , I , . . , , | i , , , . \u2022, , . \u2022 I I . \u2022 - r - P T- -T\u2014r - .\u2014r-r\u2014\u2022-\u2022r\u2014T- -T\u2014T-t\u2014r- *\" T ~ ' !l \u00ab !\u00ab.\u00bb 9.5 9.8 S.v a.B 7.5 I 7 . \u00bb 6 .5 S.Z 5.5 5 . a c. 5 *.\u00ab 3'5 3\u00bb0 I p.=.\u00ab F i g u r e 28. 400 MHz 'H-NMR spectrum o f 46. S p e c t r a l d a t a a r e c o n s i s t e n t w i t h t h e c h e m i c a l s t r u c t u r e 3-hydroxymethyl DMC. H H a* 117 (e) Summary o f a n a l y t i c a l d a t a . * In summary, t h e s e d a t a s u g g e s t t h a t a compound w i t h mass s p e c t r a l p r o p e r t i e s c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e was p r e s e n t i n t h e f r e s h l y p r e p a r e d c r u d e p r o d u c t , but c o u l d n ot be i s o l a t e d because o f i t s m a r g i n a l s t a b i l i t y . The moderate s t a b i l i t y o f s e v e r a l c a r b i n o l a m i d e s has been r e p o r t e d ( V a j t a e t a l . , 1988; Ross e t a l . , 1983; A l t o n e t a l . , 1975b; McMahon and S u l l i v a n , 1965). The compound d e t e c t e d by HPLC which decomposed on s t o r a g e was not c h a r a c t e r i z e d . The s t r u c t u r e o f t h e compound 46 has been e l u c i d a t e d by LCMS and *H-NMR s p e c t r o s c o p y . LCMS r e v e a l e d t h e e x i s t e n c e o f a hydroxym e t h y l a t e d DMC d e r i v a t i v e w i t h MH + a t m\/z 317 and [MH-H20] + a t m\/z 299. The ^ H-NMR sp e c t r u m r e v e a l e d t h e p r e s e n c e o f amide, m e t h i n e , methylene and h y d r o x y l p r o t o n s w h i c h i s c o n s i s t e n t w i t h DMC b e i n g h y d r o x y m e t h y l a t e d a t p o s i t i o n - 3 . A t p r e s e n t , GCMS ( E I ) d a t a does not appear t o s u p p o r t t h e e v i d e n c e o b t a i n e d by LCMS and NMR, because a p a r e n t i o n o f m\/z 370 i s not c o n s i s t e n t w i t h t h e p r e s e n c e o f two d e r i v a t i z a b l e c e n t r e s i n 46, namely t h e amide and h y d r o x y l p r o t o n s . The f u t u r e use o f d i r e c t probe and tandem mass s p e c t r o s c o p i c t e c h n i q u e s , w h i c h were not i m m e d i a t e l y a v a i l a b l e , s h o u l d p r o v i d e some e x p l a n a t i o n f o r t h i s s p e c t r a l phenomenon. ( i i ) S y n t h e s i s o f t h e c a r b i n o l a m i d e u s i n g KOH and p a r a f o r m a l d e h y d e . A n o t h e r approach t o t h e b a s e - c a t a l y z e d c o n d e n s a t i o n o f form a l d e h y d e w i t h DMC i n v o l v e d t h e use o f KOH as base and p a r a f o r m a l d e h y d e , as t h e s o u r c e o f f o r m a l d e h y d e , i n DMSO s o l u t i o n . In t h e ^ H-NMR spect r u m o f t h e p r o d u c t ( 4 0 ) , t h e s i m i l a r i t y o f r e s o n a n c e t o DMC i n t h e a r o m a t i c r e g i o n , 118 and t h e o c c u r r e n c e o f u p f i e l d c h e m i c a l s h i f t s a t 3.43 ( t , 1H, -CHCH2), 3.94 ( t , 2H, CHCH 20H), 4.56 ( t , 1H, -CH 20H) ( F i g u r e 29 A) were c o n s i s t e n t w i t h t h e s t r u c t u r e 3-hydroxymethyl DMC. The s p l i t t i n g o f t h e met h y l e n e and h y d r o x y l p r o t o n s i n t o t r i p l e t s c o u l d be a c c o u n t e d f o r by t h e red u c e d r a t e o f h y d r o x y l p r o t o n exchage i n DMSO due t o hydrogen b o n d i n g w i t h t h e s o l v e n t (Chapman and K i n g , 1964). A l t h o u g h a s i m i l a r s t r u c t u r e was prop o s e d f o r 46, s p l i t t i n g o f t h e h y d r o x y l p r o t o n was not o b s e r v e d ( F i g u r e 29 B ) . One p o s s i b l e e x p l a n a t i o n c o u l d be t h e p r e s e n c e o f t r a c e amounts o f a c i d w h i c h c o u l d c a t a l y z e h y d r o x y l p r o t o n exchange (Chapman and K i n g , 1964). In t h e pr e s e n c e o f D 20, t h e s i m i l a r i t y o f t h e u p f i e l d c h e m i c a l s h i f t s o f 40 and 46 ( F i g u r e 30) l e n d s s u p p o r t t o t h e c h e m i c a l i d e n t i t y o f t h e s e two compounds. T h i s h y p o t h e s i s was s u b s t a n t i a t e d by t h e s i m i l a r i t y o f t h e GCMS ( E I ) spe c t r u m o f 40 ( F i g u r e 31) t o t h a t o f 46 ( F i g u r e 2 6 ) . i 119 F i g u r e 29. P a r t i a l 400 MHz AH-NMR spectra o f (A) compound 40 and (B) compound 46 i n DMSO-ds reveal t h e occurrence o f methine, methylene and hydroxyl protons as (A) triplets i n 40 and as (B) a tr iplet, doublet and b r o a d singlet respectively i n 46. The full NMR spectrum o f 40 i s shown i n t h e A p p e n d i x . Contaminating EtOAc. 120 CHCH 20H J CHCH 20H -CHCH2 -CHCH2 4 . 5 4. a 3.a A. Z F i g u r e 30. P a r t i a l 400 MHz *H-NMR spectra of (A) compound 40 and (B) compound 46 in DMSO-ds with D20 show chemical identity for the 3-hydroxymethyl function. Contaminating EtOAc. 121 F i l e >R225C 260088-240086-.20000-200000-180080-160000-140086-120060-100080-80000-60000-40060-26000-60.0-556.0 amu. N-HYDROXYNETHYL-DMC TIC 7.88 l TMS-DTVE CR-LINK V 9.45 . . l e >R225C N-HYDROXYMETHYL-DMC Bpk Rb 28544 TMS-BTVE CR-LINK MESI Scan 313 7.87 min. 22006-28080-18000-16000-14800-12006-10000-8600-6000-4080-2888-eJJ 73 225 155 188 1 2 4 1 7 9 \\ 1 9 7 251 \\ 273 \\ 3 8 1 \\ 370 \\ 327 316| 80 128 160 280 248 i \u2022 \u2022 ' i 280 r-110 ~-\\00 r90 J60 i-70 f60 rS0 J40 -30 r 2 0 -10 I I \u2022 I I I I I , I 320 368 F i g u r e 3 1 . (A) GCMS ( C o n d i t i o n , d*) TIC p l o t o f p u r i f i e d 40. (B) GCMS ( E I ) s p e c t r u m o f 40 shows t h e p r e s e n c e o f M +- a t m\/z 370. *See E x p e r i m e n t a l f o r d e t a i l s . 122 ( i i i ) C a r b i n o l a m i d e s y n t h e s i s i n p e r s p e c t i v e . I t i s a p p a r e n t t h a t t h e s y n t h e s i s o f t h e CLBZ c a r b i n o l a m i d e o f f e r s a u n i q u e s y n t h e t i c c h a l l e n g e , i n t h a t u n l i k e t h e s y n t h e s e s o f o t h e r c a r b i n o l a m i d e s f r o m t h e i r N-desmethyl d e r i v a t i v e s ( K e d d e r i s e t a l . , 1989; S l a t t e r e t a l . , 1989), t h e r e appears t o be a c o m p e t i t i o n between two a l t e r n a t i v e c e n t r e s on t h e m o l e c u l e f o r f o r m a l d e h y d e a d d i t i o n , namely t h e amide and methylene g r o u p s . I t i s unusual t h a t t h e c o n d e n s a t i o n o f fo r m a l d e h y d e w i t h DMC when c a t a l y z e d by both K2CO3 and KOH s h o u l d r e s u l t i n t h e f o r m a t i o n o f 3-hydroxymethyl DMC as opposed t o N-hydroxymethyl DMC ( c a r b i n o l a m i d e ) . In t h e s y n t h e s i s o f [ 2Hs]CLBZ and [ 2H3]CLBZ t h e s e c o n d a r y amide p r o t o n o f t h e desmethyl compound d i s p l a y e d a h i g h e r a c i d i t y t h a n t h e meth y l e n e p r o t o n s and was s e l e c t i v e l y a l k y l a t e d o v e r t h e methylene group by b a s e - c a t a l y z e d t r e a t m e n t w i t h w i t h CH3I and CD3I r e s p e c t i v e l y . Whether f o r m a l d e h y d e a d d i t i o n a t t h e 3 - p o s i t i o n o c c u r s as a k i n e t i c p r o d u c t o r whether i t a r i s e s as a r e s u l t o f an e q u i l i b r i u m p r o c e s s subsequent t o N-h y d r o x y m e t h y l a t i o n r e m a i n s t o be r e s o l v e d . F u r t h e r m o r e , d e s p i t e t h e p r e s e n c e o f s t r u c t u r e s w h i c h s h o u l d e x e r t a s t a b i l i z i n g i n f l u e n c e on N-hydroxymethyl CLBZ, namely, a phenyl r i n g ( G o r r o d and Temple, 1976) and a c a r b o n y l group (Ross e t a l . , 1983; McMahon and S u l l i v a n , 1965) a d j a c e n t t o t h e hy d r o x y m e t h y l b e a r i n g n i t r o g e n t h e p u t a t i v e c a r b i n o l a m i d e was m a r g i n a l l y s t a b l e . 123 (F) A t t e m p t e d S y n t h e s e s o f N-hvdroxv DMC ( h y d r o x a m i c a c i c ) ( 5 0 ) . Two approaches were used i n an a t t e m p t t o s y n t h e s i z e t h e hydroxamic a c i d ( 5 0 ) : ( i ) M o V I p e r o x i d e o x i d a t i o n o f DMC-TMS (Scheme X X I I ) , and ( i i ) b a s e - c a t a l y z e d r i n g - c l o s u r e o f t h e a r y l h y d r o x y 1 amine carbamoyl a c e t a t e (52) (Scheme X X I I I ) . Scheme X X I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC (50) by o x i d a t i o n o f DMC-TMS. A: MSTFA, 45\u00b0C B: bis(N,N-diDMF)oxodiperoxomolybdenum ( V I ) (42) C: EDTA 124 ( i ) Mo-^ p e r o x i d e o x i d a t i o n o f DMC-TMS. Trea t m e n t o f m o l y b d i c a c i d w i t h H2O2 and N,N-dimethylformamide (DMF) a f f o r d e d bis(N,N-diDMF)oxodiperoxomolybdenum ( V I ) (42) i n 45 % y i e l d . The -^ H-NMR s p e c t r u m o f 42 i n D2O ( F i g u r e 32) was c o n s i s t e n t w i t h i t s s t r u c t u r e . Resonance a t 2.89 (N-CH3) and 3.05 (N-CH3) ppm s u g g e s t e d t h e p r e s e n c e o f two r o t a m e r s o f DMF. *H-NMR a n a l y s i s a l s o i n d i c a t e d t h e p r e s e n c e o f c o n t a m i n a t i n g MeOH, whi c h c o u l d n o t be removed d e s p i t e t h e washi n g s o f 42 w i t h e t h e r , and ext e n d e d d r y i n g in vacuuo o v e r P2O5. The p r o b l e m a t i c r e t e n t i o n o f MeOH by 42 was n o t r e p o r t e d by M a t l i n e t . a l . (1 9 7 9 ) . T r e a t m e n t o f DMC w i t h MSTFA a f f o r d e d t h e T M S - d e r i v a t i v e (48) w h i c h was t r e a t e d w i t h o u t p u r i f i c a t i o n w i t h 42. S t i r r i n g o f t h e m i x t u r e f o r up t o 48 h gave no r e a c t i o n . C o n t a m i n a t i n g MeOH i n 42 c o u l d have h y d r o l y z e d 48 and p r e v e n t e d i t s o x i d a t i o n t o t h e hydroxamate-Mo complex ( 4 9 ) . o 1 M o 1 o ^ r o M e ?x 1 > - C - H M e ' 42 1 N-Me I N-Me O i Mo i Me v n > - C - H Me' F i g u r e 32. 400 MHz XH-NMR o f t h e M o v l complex ( 4 2 ) . 126 ( i i ) R i n g c l o s u r e o f t h e hydroxy!amine carbamoyl a c e t a t e . An a l t e r n a t i v e approach t o t h e hyd r o x a m i c a c i d was sought i n t h e b a s e - c a t a l y z e d h y d r o l y s i s o f h y d r o x y l a m i n e carbamoyl a c e t a t e (52) t o t h e c a r b o x y l i c a c i d w i t h c o n c o m i t a n t r i n g c l o s u r e t o t h e hydroxamate (50) (Scheme X X I I I ) . Treatment o f c h l o r o n i t r o a r e n e s w i t h h y d r a z i n e - P d has been used t o produce t h e h y d r o x y l a m i n e i n l e s s t h a n 3 h r w i t h m i n o r amounts o f c o n t a m i n a t i n g phenylamine ( R o n d e s t v e d t and J o h n s o n , 1977). However, t r e a t m e n t o f 51 under s i m i l a r c o n d i t i o n s f o r 40 h r a f f o r d e d no d e t e c t a b l e r e d u c t i o n . S h o u l d t h e r e d u c t i o n o f 51 t o 52 be a c c o m p l i s h e d , base-c a t a l y z e d r i n g c l o s u r e c o u l d y e t a f f o r d t h e h y d r o x a m i c a c i d ( 5 0 ) . The r e d u c t i o n o f 51 w i t h Zn and NH4CI t o a f f o r d 52 (Kamm, 1941) i s a p o s s i b i l i t y w h i c h c o u l d be examined. Scheme X X I I I . A t t e m p t e d s y n t h e s i s o f N-hydroxy DMC by r i n g c l o s u r e o f t h e a r y l h y d r o x y l a m i ne. A: Pd-C, h y d r a z i n e monohydrate B: sodium m e t h o x i d e 127 2. THE METABOLISM OF CLBZ. In. t h i s s e c t i o n t h e b i l i a r y and u r i n a r y m e t a b o l i t e s o f CLBZ i n Sprague Dawley r a t s w i l l be c h a r a c t e r i z e d . C L B Z - d e r i v e d m e t a b o l i t e s were i d e n t i f i e d by c o m p a r i s o n o f GCMS p r o p e r t i e s t o s y n t h e t i c s t a n d a r d s , and by t h e p r e s e n c e o f i s o t o p e peak s h i f t s o f l a b e l l e d m e t a b o l i t e s from t h e i r u n l a b e l l e d a n a l o g u e s . Mass chromatograms shown i n t h e Ap p e n d i x i d e n t i f y CLBZ m e t a b o l i t e s by t h e p r e s e n c e o f i s o t o p i c a l l y - r e l a t e d p e a k s . (A) Phenol and C a t e c h o l M e t a b o l i t e s o f CLBZ i n Rat B i l e and U r i n e . The m e t a b o l i t e p i c t u r e f o r CLBZ was d e r i v e d from i n d i v i d u a l l y p o o l e d b i l e and u r i n e samples which were o b t a i n e d under t h e f o l l o w i n g c o n d i t i o n s . U r i n e c o l l e c t i o n commenced a f t e r t h e f i r s t dose o f CLBZ:[ 2H5]CLBZ and c o n t i n u e d t h r o u g h o u t t h e d o s i n g regimen ( o f one dose e v e r y 6 h f o r 6 do s e s ) u n t i l t h e t i m e o f s a c r i f i c e . B i l e was c o l l e c t e d s h o r t l y b e f o r e t h e s i x t h d o se, and c o n t i n u e d t h e r e a f t e r u n t i l t h e t i m e o f s a c r i f i c e a p p r o x i m a t e l y 18 h l a t e r . B i l e and u r i n e samples were e x t r a c t e d w i t h EtOAc b e f o r e and a f t e r s e p a r a t e enzyme h y d r o l y s e s w i t h ^ - g l u c u r o n i d a s e and a r y l s u l f a t a s e . E x t r a c t s were T M S - d e r i v a t i z e d w i t h MSTFA, and a n a l y z e d by GCMS. T h i s p r o c e d u r e a l l o w e d CLBZ m e t a b o l i t e s o f t h e u n c o n j u g a t e d f r a c t i o n , as w e l l as g l u c u r o n i d e and s u l f a t e m e t a b o l i t e s o f t h e c o n j u g a t e d f r a c t i o n t o be d i s t i n g u i s h e d . In r e p o r t i n g t h i s d a t a t h e f o l l o w i n g a s s u m p t i o n s were made: ( i ) a s i m i l a r e x t r a c t i o n r a t i o f o r each CLBZ m e t a b o l i t e from b i l e and u r i n e , and ( i i ) a s i m i l a r GCMS r e s p o n s e f o r each T M S - d e r i v a t i z e d m e t a b o l i t e . L e v e l s o f CLBZ m e t a b o l i t e s were s e m i - q u a n t i t a t i v e l y compared u s i n g t h e a r e a under M +* f o r t h e T M S - d e r i v a t i z e d m e t a b o l i t e . 128 There was no d e t e c t a b l e e v i d e n c e o f u n c o n j u g a t e d CLBZ m e t a b o l i t e s i n b i l e o r u r i n e . TIC p l o t s o f c o n j u g a t e d CLBZ m e t a b o l i t e s i n b i l e and u r i n e a r e shown i n F i g u r e s 33 - 35, and r e l a t i v e m e t a b o l i t e l e v e l s a r e compared i n F i g u r e 36. In b i l e d e c r e a s i n g l e v e l s o f t h e f o l l o w i n g m e t a b o l i t e s were d e t e c t e d as g l u c u r o n i d e and s u l f a t e c o n j u g a t e s : 4'-hydroxy CLBZ ( 2 8 ) . 4'-h y d r o x y DMC ( 2 6 ) , 4'-hydroxy-3'-methoxy CLBZ (36a) + 3'-hydroxy-4'-methoxy CLBZ ( 3 6 b ) , 4'-hydroxy-3'-methoxy DMC (33) and 3 ' , 4 ' - d i h y d r o x y CLBZ ( 3 5 ) -In u r i n e t h e o r d e r o f d e c r e a s e f o r s u l f a t e c o n j u g a t e s was 4'-hydroxy-3'-methoxy CLBZ ( 3 6 a ) . 4'-hydroxy CLBZ ( 2 8 ) , 4'-hydroxy DMC ( 2 6 ) , 3',4'-d i h y d r o x y CLBZ ( 3 5 ) . In b i l e , t h e g l u c u r o n i d e s were predominant o v e r t h e s u l f a t e s , whereas i n u r i n e o n l y s u l f a t e c o n j u g a t e s were d e t e c t e d . 129 'le >AB81 60.8-558.0 amu. CLBZ-BILE-GLUC-TMS EIP 788888-4 15.77 688008-500080-480080-308088 288888-188800 8 2%i.%8 \u2022 < t \u00bb i i \u00ab i \u00ab i \u2022 i \u2022 11 \u2022 111111111 i 111 | i 111 | i 1111 11 111 i 1111 \u2022 \u2022 i \u2022 \u2022 ' \u2022 i 15.0 16.8 17.8 18.8 19.8 28.0 21.8 22.8 23.8 24.8 25.8 26.0 F i g u r e 33. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h ^ - g l u c u r o n i d a s e . 26: 4'-hydroxy DMC; 28: 4'-hydroxy CLBZ; 33: 4'-hydroxy-3'-methoxy DMC; 35: 3 ' , 4 ' - d i h y d r o x y CLBZ; 36a: 4'-hydroxy-s' -methoxy CLBZ; 36b: 3'-hydroxy-4'-methoxy CLBZ. 130 f i l e >RB02 68.0-558.8 amu. CLBZ-BILE-SULF-TMS EIP 21 .87 28 21 .56 22.91 22.34 24.12 36. i i i i - J i \u2014 l i i | i i i i |\u2014r\u2014i\u2014i\u2014r -1\u2022 i i i i\u2014j\u2014\u00ab\u2014i\u2014i\u2014i\"i i\u2014i\u2014i\u2014i | i i i \u2014 f | i\u2014i \"11 i | i J. 16.0 17.0 18.0 19.0 20.0 21 .0 22.0 23.0 24.0 25.8 26.0 F i g u r e 34. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f b i l e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . 26: 4'-h y d r o x y DMC; 28: 4'-hydroxy CLBZ; 36a: 4'-hydroxy-3'-methoxy CLBZ; 36b: 3'-hydroxy-4'-methoxy CLBZ. 131 r i l e >fiB84 6 0 . 8 - 5 5 8 . 8 amu. CLBZ-URIN-SULF-TMS EIP 228088 280880-188888^ 16Q000 : 148888-1 1 128888-1J 108888-88888-60000-480O0-28888 : 17.79 .93 18.73 18.49^^ 23 .18 28.feei.56 3 6 a < l 3 ' 4 7 8 \u2022 | \u2014> \u2022\u2014r-'\u2014\u2022\u2014\u2022\u2014>\u2014i\u2014\u2022\u2014<\u2014\u2022 i | \u2014 i \u2014 i \u2014 \u2022 \u2014 i \u2014 | \u2014 i \u2014 i i i | i i\u2014 i \u2014 i \u2014 |\u2014 i\u2014 i \u2014 < \u2014 i | i \u2014 I I i [ i i \u2014 i \u2014 i \u2014 i \u2014 i \u2014 i 17 .8 18 .0 19.8 2 8 . 8 2 1 . 8 2 2 . 8 2 3 . 8 2 4 . 0 25 .8 26 .8 F i g u r e 35. TIC p l o t o f a T M S - d e r i v a t i z e d EtOAc e x t r a c t o f u r i n e a f t e r h y d r o l y s i s w i t h a r y l s u l f a t a s e . 26: 4'-hydroxy DMC; 28: 4'-hydroxy CLBZ; 35: 3 ' , 4 ' - d i h y d r o x y CLBZ; 36a: 4'-hydroxy-3'-methoxy CLBZ. 132 c o c _o > O CD 110 1 0 0 -90 80 + 70 60 + 50 40 3 0 -2 0 -1 0 -0 0 28 26 28: 4'-OH CLBZ 26: 4'-OH DMC 36a: 4'-OH,3'-Olvle CLBZ 36b: 3'-OH,4'-OMe CLBZ 33: 4,-OH,3'-OMe DMC 35: 3',4'-(OH)2 CLBZ \u2022 \u2022 Bile glucuronide VZZ Bile sulfate I I Urine sulfate 1 36a+b 33 Metabolite icf n 35 F i g u r e 36. P r o f i l e o f c o n j u g a t e d CLBZ m e t a b o l i t e s i n t h e Sprague Dawley r a t d e r i v e d from i n d i v i d u a l l y p o o l e d b i l e and u r i n e samples. U r i n e was c o l l e c t e d a f t e r t h e f i r s t dose o f drug t h r o u g h o u t t h e dosage regimen u n t i l t h e t i m e o f s a c r i f i c e . B i l e was c o l l e c t e d a f t e r t h e f i f t h dose u n t i l t he t i m e o f s a c r i f i c e . A f t e r t r e a t m e n t w i t h \/ 3 - g l u c u r o n i d a s e o r a r y l s u l f a t a s e , b i l e and u r i n e samples were e x t r a c t e d w i t h EtOAc, T M S - d e r i v a t i z e d and a n a l y z e d by GCMS. R e l a t i v e amounts o f CLBZ m e t a b o l i t e s a r e based on mass chromatogram peak a r e a s f o r M +* o f t h e TMS d e r i v a t i v e . A s i m i l a r e x t r a c t i o n r a t i o and GCMS r e s p o n s e i s assumed f o r a l l m e t a b o l i t e s . 133 ( i ) G l u c u r o n i d a t i o n vs. S u l f a t i o n . The f o r m a t i o n o f g l u c u r o n i d e and s u l f a t e e s t e r s i s c a t a l y z e d by UDP-g l u c u r o n y l t r a n s f e r a s e ( B u r c h e l l and C o u g h t r i e , 1989) and s u l f o t r a n s f e r a s e ( J a k o b y e t a l . , 1984) r e s p e c t i v e l y , w i t h both h a v i n g o v e r l a p p i n g s u b s t r a t e s p e c i f i c i t i e s . S e v e r a l f a c t o r s g o v e r n t h e b a l a n c e between g l u c u r o n i d a t i o n and s u l f a t i o n p r o c e s s e s in vivo. These i n c l u d e : ( i ) s p e c i e s ( M u l d e r , 1984), ( i i ) dosage ( C a l d w e l l , 1980), ( i i i ) t h e p a r t i t i o n i n g o f t h e s u b s t r a t e i n t h e c e l l u l a r compartments ( M u l d e r , 1984) and ( i v ) t h e s u b s t r a t e s p e c i f i c i t y o f t h e c o n j u g a t i n g enzyme ( M u l d e r and Hagedoorn, 1974). A l t h o u g h t h e r e i s a p r o p e n s i t y t o e i t h e r g l u c u r o n i d a t e o r s u l f a t e among animal s p e c i e s , both c o n j u g a t i o n e v e n t s a r e e f f i c i e n t l y a c c o m p l i s h e d i n t h e r a t ( M u l d e r , 1984). T h i s v i e w g a i n s s u p p o r t w i t h t h e o c c u r r e n c e o f CLBZ m e t a b o l i t e s as both g l u c u r o n i d e and s u l f a t e c o n j u g a t e s . The dosage l e v e l o f a s u b s t r a t e i n f l u e n c e s t h e e x t e n t t o w h i c h each c o n j u g a t i o n pathway i s i n v o l v e d . Thus, a t low doses o f p h e n o l s , s u l f a t i o n t e n d s t o p r e d o m i n a t e , whereas a t h i g h e r d o s e s , g l u c u r o n i d a t i o n i n c r e a s e s ( C a l d w e l l , 1980). Two r e a s o n s c o u l d a c c o u n t f o r t h i s s h i f t i n m e t a b o l i s m ( M u l d e r , 1984). ( i ) S u l f o t r a n s f e r a s e s u s u a l l y have l o w e r Km v a l u e s t h a n U D P - g l u c u r o n y l t r a n s f e r a s e s f o r t h e same s u b s t r a t e s , and ( i i ) t h e c a p a c i t y f o r s u l f o t r a n s f e r a s e t o c o n j u g a t e i s more r e a d i l y compromised by d e p l e t i o n o f i t s c o - s u b s t r a t e . I t i s not known whether t h e dose o f CLBZ a d m i n i s t e r e d was s u f f i c i e n t t o produce l e v e l s o f phase I m e t a b o l i t e s t h a t c o u l d s a t u r a t e t h e s u l f a t i o n pathway i n t h e r a t . I f t h i s were t h e c a s e , a h i g h e r s u l f a t i o n p a t t e r n c o u l d emerge a t l o w e r doses o f t h e d r u g . The n a t u r e o f t h e c o n j u g a t e formed, t o a l a r g e e x t e n t , d e t e r m i n e s t h e 134 e x c r e t o r y pathway i n t o w h i c h i t i s c h a n n e l e d ( C a l d w e l l , 1980). B i l i a r y e x c r e t i o n o f a drug depends on ( i ) an a p p r o p r i a t e h y d r o p h i l i c \/ 1 i p o p h i l i c b a l a n c e , ( i i ) t h e p r e s e n c e o f an a n i o n i c f u n c t i o n , and ( i i i ) a minimum r e q u i r e m e n t f o r m o l e c u l a r w e i g h t which i s dependent on s p e c i e s ( P o w e l l and O l a v e s e n , 1981; P o w e l l , 1981). S t e r o i d c o n j u g a t e s a r e more r e a d i l y e x c r e t e d as g l u c u r o n i d e s i n t h e b i l e ( M a t s u i , 1982; B a i l l i e e t a l . , 1975; Honma and Nambara, 1974), whereas s u l f a t e c o n j u g a t e s a r e g e n e r a l l y e x c r e t e d i n t h e u r i n e ( C a l d w e l l , 1980) by a c t i v e t r a n s p o r t ( M u l d e r , 1984). Phase I m e t a b o l i t e s o f CLBZ a r e r e l a t i v e l y p o l a r and t h e i r m o l e c u l a r w e i g h t s (MW, 302 - 336) a r e i n t e r m e d i a t e i n t h e r e q u i r e m e n t f o r b i l i a r y g l u c u r o n i d e e x c r e t i o n i n t h e r a t (MW > 325 \u00b1 5 0 ) , ( M i l l b u r n e t a l . , 1967). C o n s i s t e n t w i t h t h e s e c h e m i c a l p r o p e r t i e s , h y d r o x y l a t e d CLBZ m e t a b o l i t e s were a b l e t o a c c e s s b o t h b i l i a r y and u r i n a r y e x c r e t o r y pathways. M e t a b o l i t e s o c c u r r e d p r e d o m i n a n t l y i n t h e b i l e as g l u c u r o n i d e s and were d e t e c t e d e x c l u s i v e l y as s u l f a t e s i n t h e u r i n e . T h i s p a t t e r n i s i n a c c o r d a n c e w i t h t h e r e s u l t s o f V o l z e t a l . (1979) who r e p o r t e d t h e predominance o f s u l f a t e s i n t h e u r i n e f o r CLBZ m e t a b o l i t e s . However, i t i s not known how t h e s e w o r k e r s c o u l d have d i s t i n g u i s h e d from which c o n j u g a t e t h e h y d r o l y z e d m e t a b o l i t e s o r i g i n a t e d , because m e t a b o l i t e s were e x t r a c t e d a f t e r t r e a t m e n t w i t h a fi-g l u c u r o n i d a s e \/ a r y l s u l f a t a s e m i x t u r e . ( i i ) Mass S p e c t r a l Data f o r Phase I M e t a b o l i t e s o f CLBZ i s o l a t e d from b i l e and u r i n e . GCMS ( E I ) s p e c t r a f o r d e c o n j u g a t e d CLBZ m e t a b o l i t e s , t y p i f i e d by t h e b i l i a r y g l u c u r o n i d e s , a r e p r e s e n t e d i n F i g u r e s 37 - 41. GCMS d a t a a r e summarized i n T a b l e 2. 135 Fll\u00ab >AB16 Bpk Ab 479939 520000-48QO0Q-44Q0O0-400000-^73 36Q8QQ-320000-28Q0OO-240000-200000-160000-120000-60000-40000-o- - r - l \u2014 r - r - r -CLBZ-BILE-GLUC-THS CLP 153 181 135 388 19 S o n 690 .19 M i n . 388 t.392 343 217 239 27 > 3 '3 I 320 y LJ 371 80 p l l O i i o o r90 :\u00abo r70 ^60 iso :40 i-30 \u00a3 0 rlO 120 160 200 240 280 320 360 400 F i g u r e 37. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d e r i v a t i v e o f 4'-hydr o x y CLBZ (28) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M+- and [ 2 H 4 ] M + * a t m\/z 388 and 392 r e s p e c t i v e l y . ( D e s c r i b e d i n t h e E x p e r i m e n t a l ) . F i l e >AB16 Bpk Ab 934278 CLBZ-BILE-CLUC-THS CLP l e o e o o o -900000 800000 780O0O-600000 s e o o o o 400OOO-388880-208000-l e o o o o o-* 73 22S 103 147 187 eo 1 Scan 53 4 6.42 H I D . . 4 4 6 .450 446 : l l 267 299 331 361 40S 405 I 280 24Q 288 p i l e r t o o r90 :80 r-70 isa r30 SO rlO 320 360 4QO 440 F i g u r e 38. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy DMC (26) i s o l a t e d f r o m b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2 H 4 ] M + * a t m\/z 446 and 450 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . 136 Fil\u00ab > \u00ab B 1 \u00ab ^ B p k i \u00ab 4 4 4 9 z g o a a o -i8oasa-K s e o a -1 2 0 0 0 0 -l a a e a e -s e a a a -\u00ab e a e a : 4 O Q O Q -2 0 0 0 0 -73 CLBZ-B1LE-CLUC-TMS CLP 167 \"AB16 Bpk A I, 24806S 2 6 0 0 0 0 -2 4 0 0 0 0 -2 2 0 0 0 0 -Z O O O O O -i s o o o o -1 6 0 0 0 0 -1 4 Q O O O 1 2 0 0 0 0 I O O Q O O -eaooe-6 0 0 Q 0 -4 0 0 0 0 2 0 0 0 0 -0 - * 73 1 2 9 1 4 7 | *\" If 2 8 1 f *s 4 Z 1 ^ CLBZ-BILC-CLUC-TKS CLP 2 2 S \\ 299 1 9 7 359 361 Scan 593 17.SO M i n . ^ 4 7 6 1.479 l e a 158 2 0 0 2 S O 3SO 111 'JW 476 446 U 4SO r l l O OO r90 i \u00ab e \u202270 rCO i50 a r3 8 O F i g u r e 40. GCMS ( C o n d i t i o n , a ) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 4'-hydroxy-3'-methoxy DMC (33) i s o l a t e d f r o m b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2 H 3 ] M + * a t m\/z 476 and 479 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . 137 Fil\u00ab >AB1C CLBZ-BILE-CLUC-IHS Scan 795 Bpk Ab 2X4337 CLP 21.05 M i n . 100 ISO 200 2SQ 300 3S0 4O0 4SO F i g u r e 41. GCMS ( C o n d i t i o n , a*) ( E I ) s p e c t r u m o f t h e TMS d i d e r i v a t i v e o f 3 ' , 4 ' - d i h y d r o x y CLBZ (35) i s o l a t e d from b i l e a f t e r ^ - g l u c u r o n i d a s e h y d r o l y s i s . The i n s e t shows t h e p r e s e n c e o f M +* and [ 2H3]M +* a t m\/z 476 and 479 r e s p e c t i v e l y ( * D e s c r i b e d i n t h e E x p e r i m e n t a l ) . Table 2. GCMS data for Phase I CLBZ metabolites typified by biliary glucuronides. Metabolites were extracted from bile after hydrolysis with jjj-glucuronidase and derivatized with TMS. Compound (No.)a t. (min) M+*(m\/z, X) Diagnostic ions (m\/z, X) c 4'-OH DMC (26) 17.91 446 (48) 225 (100) 239 (24) 431 (14) 4'-OH [2H4]DMC (26') 17.86 450 (24) 225 (100) 239 (24) 435 (9) 4'-OH CLBZ (28) 21.37 388(100) 343 (56) 181 (35) 331 (30) 153 (28) 4'-OH I2H41CLBZ (28') 21.30 392 (64) 347 (45) 181 (35) 335 (21) 153 (28) 4'-0H,3'-0Me DMC (33) 19.13 476 (16) 225 (20) 239 (8) 461 (5) 446 (5) 4'-0H,3'-0Me 19.11 479 (14) 225 (20) 239 (8) 464 (4) [2H3 DMC(W') 3',4'-(0H)2 CLBZ (35) 23.50 476 (26) 461 (6) 343 (6) 431 (6) 419 (5) 3',4'-(OH)2 23.47 479 (21) 464 (5) 346 (4) 434 (4) 422 (4) [2H3]CLBZ (35') 4'-0H,3'-0Me CLBZ (36a) 23.22 418 (78) 346 (26) 373 (25) 388 (16) 401 (8) 4'-0H,3'-OMe 23.17 421 (72) 349 (18) 376 (14) 391 (14) 404 (9) t2H3]CLBZ(36a') 3'-OH,4'-OMe CLBZ (36b) 22.44 418 (10)d 3'-0H,4'-OMe 22.37 - 421 (7) d t2H3]CLBZ (36b') a: The compound number for the deuterio analogues is indicated by the primed ('). b: Retention times were obtained according to GCMS Condition (b) described in the Experimental. H c: X = Relative intensity of ion for the combined protio\/deuterio spectrum (See Figures 28-31). u CO d: Only M * could be diagnostically assigned in this spectrum. 139 Loss o f one d e u t e r i u m d u r i n g b i o t r a n s f o r m a t i o n a f f o r d e d an i s o t o p e peak s h i f t o f f o u r mass u n i t s i n t h e GCMS ( E I ) s p e c t r a o f t h e TMS-d e r i v a t i z e d b i l e and u r i n e e x t r a c t s , and r e a d i l y a l l o w e d phenol m e t a b o l i t e s t o be i d e n t i f i e d . Comparison o f t h e GCMS p r o p e r t i e s o f t h e l a t t e r t o s y n t h e t i c s t a n d a r d s c o n f i r m e d t h a t m o n o h y d r o x y l a t i o n had o c c u r r e d a t t h e 4 ' - p o s i t i o n o f CLBZ and DMC. I t s h o u l d be no t e d t h a t c y tochrome P-450 can a l s o c a t a l y z e p h e n y l m o n o o x i d a t i o n s a t t h e meta (Korzekwa e t a l . , 1989) and ortho p o s i t i o n s ( J e r i n a e t a l . , 1971). In s p i t e o f t h i s , para h y d r o x y l a t i o n i s l i k e l y t o p r e d o m i n a t e , as borne o u t by t h e v a r i e t y o f s u b s t r a t e s t h a t a r e m o n o h y d r o x y l a t e d i n t h i s f a s h i o n e.g. amphetamine, N-methylamphetamine, diazepam, n o r e p h e d r i n e and p h e n f o r m i n ( T e s t a and J e n n e r , 1976). 3 ' , 4 ' - D i h y d r o x y CLBZ (35) was a minor c o n j u g a t e d m e t a b o l i t e i n b i l e and u r i n e . There was no e v i d e n c e f o r t h e desmethyl c a t e c h o l 3',4'-d i h y d r o x y DMC ( 3 2 ) , p r o b a b l y because t h i s m e t a b o l i t e was p r e s e n t a t l e v e l s below t h e l i m i t o f d e t e c t i o n . 4'-Hydroxy-3'-methoxy CLBZ (36a) was t h e major c o n j u g a t e d u r i n a r y m e t a b o l i t e and was a l s o s i g n i f i c a n t i n t h e c o n j u g a t e d f r a c t i o n o f b i l e . 4'-Hydroxy-3'-methoxy DMC (33) was s i g n i f i c a n t i n t h e c o n j u g a t e d f r a c t i o n o f b i l e and a b s e n t i n u r i i i e . The low l e v e l o f d i h y d r o x y CLBZ (35) and t h e u n d e t e c t a b l e l e v e l o f d i h y d r o x y DMC (32) compared t o t h e i r O - methylated d e r i v a t i v e s speak f o r t h e h i g h h e p a t i c COMT a c t i v i t y p r e s e n t i n t h e r a t ( G u l d b e r g and Marsden, 1975). C o n s i s t e n t w i t h t h e s p e c i f i c i t y o f COMT f o r m e t a - O - m e t h y l a t i o n o f c a t e c h o l a m i n e s in vivo ( G u l d b e r g and Marsden, 1975), t h e meta (36a):para (36b) r a t i o s o f O-methylated CLBZ c a t e c h o l s p r e s e n t as t h e b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e were 98:2 and 100:0 r e s p e c t i v e l y , as 140 d e t e r m i n e d from mass chromatograms o f t h e i r m o l e c u l a r i o n s ( F i g u r e 3 3 ) . In o r d e r t o v e r i f y t h e peak a s s i g n m e n t s f o r t h e O-methylated CLBZ c a t e c h o l s , t h e b i l i a r y g l u c u r o n i d e {meta:para, 98:2) was s p i k e d w i t h t h e m i x t u r e o f CLBZ O-methylated c a t e c h o l s formed in vitro (meta:para, 68:32). The r e t e n t i o n t i m e s f o r t h e O-methylated c a t e c h o l s formed (A) in vitro, (B) as t h e b i l i a r y g l u c u r o n i d e ( u n s p i k e d ) , and (C) as t h e b i l i a r y g l u c u r o n i d e ( s p i k e d ) were c o n s i s t e n t , and t h e l a t t e r a f f o r d e d a metaipara r a t i o o f 74:16 ( F i g u r e 4 3 ) . In t h i s way, t h e peak a s s i g n m e n t s f o r t h e O-methylated c a t e c h o l s f o r t h e b i l i a r y g l u c u r o n i d e , and i n f e r e n t i a l l y t h e b i l i a r y s u l f a t e and u r i n a r y s u l f a t e were c o n f i r m e d . In c o n t r a s t t o t h e e s s e n t i a l l y q u a n t i t a t i v e f o r m a t i o n o f meta-O-m e t h y l a t e d CLBZ c a t e c h o l s as t h e b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e , t h e b i l i a r y s u l f a t e d i s c l o s e d an unus u a l meta (36a) -.para (36b) r a t i o o f 70:30. Repeat i n j e c t i o n s o f t h e b i l e and u r i n e samples v e r i f i e d t h e meta ( 3 6 a ) : p a r a (36b) r a t i o o f t h e b i l i a r y s u l f a t e . In a d d i t i o n , t h e a p p r o x i m a t e l y e q u a l meta (36a) -.para (36b) peak a r e a r a t i o s i n mass chromatograms a t m\/z 418, and f o r t h e d e u t e r a t e d a n a l o g u e s a t m\/z 421 e n s u r e d t h a t t h e peaks were m e t a b o l i t e - r e l a t e d ( F i g u r e 4 4 ) . P a r a - O - m e t h y l a t e d c a t e c h o l s a r e uncommon m e t a b o l i t e s in vivo which u s u a l l y o c c u r as t h e minor i s o m e r compared t o t h e meta-O-methylated d e r i v a t i v e . F o r example t h e p a r a - O - m e t h y l a t e d i s o m e r s o f v a n y l e t h y l amine (O'Gorman e t a l . , 1970) and 3 , 4 - d i h y d r o x y a c e t o p h e n o n e ( D a l y e t a l . , 1960) c o n s t i t u t e d 10 % and 25 % o f t h e O-methylated c a t e c h o l s r e s p e c t i v e l y . The r a t i o o f t h e O-methylated c a t e c h o l s o f CLBZ, however, was u n i q u e i n t h a t a d i f f e r e n t metaipara r a t i o o c c u r r e d w i t h i n t h e same m e t a b o l i t e pool f o r d i f f e r e n t c o n j u g a t e s . In the light of the available data, a h y p o t h e s i s i s pr o p o s e d t o r a t i o n a l i z e t h e e s s e n t i a l l y q u a n t i t a t i v e o c c u r r e n c e o f 4'-141 hydroxy-3'-methoxy CLBZ as a b i l i a r y g l u c u r o n i d e and u r i n a r y s u l f a t e , and th e appearance o f 3'-hydroxy-4'-methoxy CLBZ (36b) as a b i l i a r y s u l f a t e . COMT c a t a l y z e d O - m e t h y l a t i o n o f c a t e c h o l s i s much more r e g i o s p e c i f i c f o r t h e meta i s o m e r in vivo t h a n in vitro ( C r e v e l i n g e t a l . , 1972). Whether t h i s r e g i o s p e c i f i c i t y i s c o n t r o l l e d d u r i n g t h e O - m e t h y l a t i o n p r o c e s s by COMT i n i t s c e l l u l a r e n v i r o n m e n t , o r whether i t a r i s e s from subsequent e n z y m a t i c a c t i v i t y ( A x e l r o d , 1956) i s u n c l e a r . S h o u l d t h e meta ( 3 6 a ) \\ p a r a (36b) r a t i o o b t a i n e d i n t h e COMT c a t a l z y e d O - m e t h y l a t i o n o f t h e d i h y d r o x y s u b s t r a t e (35) in vitro (68:32) be r e f l e c t e d t o some e x t e n t in vivo, t h e n i t i s p o s s i b l e t h a t b o t h meta- (36a) and p a r a - O - m e t h y l a t e d (36b) d e r i v a t i v e s c o u l d s e r v e as s u b s t r a t e s f o r phase I I enzymes. C o n s i s t e n t w i t h such a s c e n a r i o , a h i g h e r s u b s t r a t e \" s p e c i f i c i t y o f UDP-g l u c u r o n y l t r a n s f e r a s e f o r t h e meta i s o m e r (36a) t h a n t h e para i s o m e r ( 3 6 b ) , would a l l o w t h e l a t t e r t o be more r e a d i l y c o n j u g a t e d as a s u l f a t e . The s p e c i f i c i t i e s d e s c r i b e d f o r phase I I c o n j u g a t i o n a r e n o t u n l i k e l y , because t h e f o r m a t i o n o f a s u l f a t e e s t e r a t t h e 3 ' - p o s i t i o n f o r t h e para-O-methyl a t e d i s o m e r (36b) s h o u l d be s t e r i c a l l y more f a v o u r a b l e t h a n t h e f o r m a t i o n o f a g l u c u r o n i d e . S u l f a t e e s t e r s a r e more h y d r o p h i l i c t h a n g l u c u r o n i d e s and as su c h , t h e f o r m e r a r e p r e d o m i n a n t l y d e s t i n e d f o r t h e k i d n e y and subsequent u r i n a r y e x c r e t i o n . Second t o t h e l i v e r , t h e k i d n e y p o s s e s s e s t h e h i g h e s t t i s s u e l e v e l s o f a r y l s u l f a t a s e C i n t h e r a t ( D o l l y e t a l . , 1971). A l t h o u g h e s t r o g e n s u l f a t e s a r e p r o b a b l y t h e n a t u r a l s u b s t r a t e s f o r t h i s enzyme, o t h e r x e n o b i o t i c s u l f a t e s a r e a l s o d e c o n j u g a t e d w i t h v a r y i n g s p e c i f i c i t i e s (Kung e t a l . , 1988; Kawano and Aikaw a , 1987; Zuckerman and Hagerman, 1969). H y d r o l y s i s o f t h e s u l f a t e e s t e r o f t h e p a r a - O - m e t h y l a t e d i s o m e r (36b) by a r y l s u l f a t a s e C c o u l d a f f o r d t h e u n c o n j u g a t e d s p e c i e s . The l a t t e r , b e i n g 142 more l i p o p h i l i c c o u l d be more r e a d i l y r e a b s o r b e d i n t h e k i d n e y t u b u l e s . T h i s would a c c o u n t f o r t h e absence o f t h e p a r a - i s o m e r (36b) i n t h e u r i n e , and i t s p r e s e n c e i n b i l e . The p o s s i b i l i t y o f 3 , 4 - d i h y d r o d i o l CLBZ f o r m a t i o n was i n v e s t i g a t e d i n t h e r a t , inasmuch as d i h y d r o d i o l s a r e p r e c u r s o r s t o c a t e c h o l s ( K a l f , 1987; B i l l i n g s , 1985). Mass chromatograms f o r t h e i s o t o p e c l u s t e r m\/z 478\/483 c o r r e s p o n d i n g t o t h e T M S - d e r i v a t i v e o f 3 , 4 - d i h y d r o d i o l CLBZ r e v e a l e d t h e absence o f d e t e c t a b l e l e v e l s o f t h i s m e t a b o l i t e i n t h e e x t r a c t s o f enzyme-h y d r o l y z e d b i l e and u r i n e . In c o n t r a s t t o o u r o b s e r v a t i o n s , V o l z e t a l . (1979) and A l t o n e t a l . (1975a) r e p o r t e d t h e o c c u r r e n c e o f t h e d i h y d r o d i o l m e t a b o l i t e s o f CLBZ and t r i f l u b a z a m r e s p e c t i v e l y i n t h e r a t , but t h e s e w o r k e r s were u n a b l e t o d e t e c t t h e c o r r e s p o n d i n g c a t e c h o l s . No p l a u s i b l e e x p l a n a t i o n c o u l d be p r o v i d e d f o r t h e d i s c r e p a n c y between o u r r e s u l t s and t h o s e i n t h e l i t e r a t u r e . F u r t h e r m o r e , i t i s u n l i k e l y t h a t i n o u r hands, a c o n j e c t u r a l CLBZ d i h y d r o d i o l would undergo spontaneous d e h y d r o g e n a t i o n t o t h e c a t e c h o l under work-up o r GCMS c o n d i t i o n s . 143 i l e >OB01 4 1 7 . 7 - 4 1 3 . 7 amu.CLBZ-BILE-GLUC-TMS SMT ADC E I P 1600-1 1200-800-400-2 3 . 2 2 >2.4> 36a 12.0 13.0 14.0 15.0 16.0 17 .018.0 19.0 2 0 . 0 2V. 6 2 2 . 0 2 3 .024.0 2 5 . 0 2 6 . 0 36bM F i l e >OB02 4 1 7 . 7 - 4 1 8 . 7 a m u . C L B Z - B I L E - S U L F - T M S SMT ADC E I P 400-3 0 0 2 0 0 : 1 0 8 : 0 2 2 . 8 9 B 22.32 36b. 36a 13.0 14.0 15.0 16.0 17.0 18.8 19.0 2 0 . 0 2 1 . 8 2 2 . 0 2 3 . 0 2 4 . 0 25.0 2 6 . 0 F i l e >OB04 4 1 7 . 7 - 4 1 8 . 7 amu.CLBZ-URIH-SULF-TMS SMT ADC E I P 6Q0-409-~1 23 .12 : c 36a 13.27 I 1 1 '\u2022 1 i ' \u2022 \u2022 i > 1, 12.0 13.0 14.015.0 16.6 17.0 13.0 19.020.0 21 .022.023.024.025.026.0 F i g u r e 42. Mass chromatograms (m\/z 418) o f T M S - d e r i v a t i z e d EtOAc e x t r a c t s o f b i l e and u r i n e showing t h e p r e s e n c e o f O - m e t h y l a t e d c a t e c h o l s o f CLBZ (36a+b) as (A) a b i l i a r y g l u c u r o n i d e , (B) a b i l i a r y s u l f a t e and (C) a u r i n a r y s u l f a t e . 144 F i l e >RB294 417.7-418.7 amu.4'0H-30Me-CLBZ-TMS SMT ADC EIP 23.49 13.0 14.0 1S.0 16.0 17.0 18.0 19.0 20.0 21 .0 22.0 23.0 24.0 25.0 26.0 r i l e >RB0lO 417.7-418.7 amu.ABOl NERT SHT RBC EIP 680 see 400 380 208 106-e B 23.31 36a 18.4 36b22-\u00abRG012 417.7-418.7 an,u .RBOlO'CLBZOH-OHe RB812 (REPEAT) SMT RDC EIP \u2022< 23.53 500 400 300-280 100 8 r2.Sfl 36a 13.814.8 15.8 16.8 17.8 18.0 19.0 20.0 21 .0 22.0 23.0 24.8 25.0 26.0 F i g u r e 43. Comparison o f peak a s s i g n m e n t s o f t h e CLBZ O-methylated isomers (36a+b) formed (A) in vitro and (B) as a b i l i a r y g l u c u r o n i d e . (C) S p i k i n g o f sample-B w i t h sample-A s u g g e s t s t h a t b o t h meta ( 3 6 a , t R , 23.53 min) and para (36b. t R , 22.80 min) i s o m e r s a r e p r e s e n t as a b i l i a r y g l u c u r o n i d e j u d g i n g f r o m t h e i n c r e a s e i n peak a r e a o f t h e min o r m e t a b o l i t e from (B) t o ( C ) . 145 F i l e >AB02 386\"j 288-; 108 : 8-4 1 7 . 7 - 4 1 8 . 7 a m u . C L B Z - B I L E - S U L F - T M S SMT ABC E IP 2 2 . 8 9 22.32 36b L36a 1 T I 'I | lITt | T 1 1 1'J ' T T T 1 J 1 I I 1 [' M \u00bb I] T i l 1 p T Tl \"p'TI I | I 1\"l 1 |T 1 VV^ I I 1 l \" | 1 'VI'l'j'T I'M ] 1 3 . 8 1 4 . 0 15.8 1 6 . 8 17.0 18.0 19 .0 2 0 . 8 2 1 . 8 2 2 . 8 23 .8 24 .0 2 5 . 0 26 .0 F i l e >flB82 386-280-188-4 2 8 . 7 - 4 2 1 . 7 a m u . C L B Z - B I L E - S U L F - T M S SMT ADC E IP B 22 .86 22.21 36a T\"i i i i t11 i | \" n , T T | i r r f T - r , n i \u00ab | t i i T | i T i T | T \\ I I \\ i\" i r r i ' i ' T H T p i ' t r r j i' t\"i ~i \"y i i i Imi | i i i i | 1 3 . 0 1 4 . 8 15.8 16.0 17.8 18.8 19 .0 2 8 . 0 2 1 . 8 2 2 . 8 23 .8 24 .8 25 .8 26 .8 F i g u r e 44. Mass chromatograms a t (A) m\/z 418 and (B) m\/z 421 show t h a t t h e peaks o f t h e CLBZ O-methylated c a t e c h o l s (36a+b) o c c u r r i n g as t h e b i l i a r y s u l f a t e s a r e i s o t o p i c a l l y r e l a t e d . 146 ( i i i ) I s o t o p e E f f e c t s i n Phenyl R i n g O x i d a t i o n . I n t e r m o l e c u l a r i s o t o p e e f f e c t s , c a l c u l a t e d by p r o t i o \/ d e u t e r i o p r o d u c t r a t i o s , were m a r g i n a l f o r phenyl r i n g o x i d a t i o n t o p h e n o l s ( k ^ \/ k p =1.103 \u00b1 .090) and c a t e c h o l s ( k ^ \/ k p = 1.088 \u00b1 .207). These r e s u l t s a r e i n a c c o r d w i t h t h e o b s e r v a t i o n s o f Baba e t a l . (1986) and H o s k i n s and Farmer (1982) who r e p o r t e d modest i s o t o p e e f f e c t s f o r t h e p h e n y l h y d r o x y l a t i o n o f e p h e d r i n e and d i p h e n y l h y d a n t o i n r e s p e c t i v e l y . Our k^\/kg v a l u e s were a l s o c o n s i s t e n t w i t h t h a t r e p o r t e d by Tunek e t a l . (1978) ( k ^ \/ k p = 1.10 \u00b1 .13) f o r t h e in vitro o x i d a t i o n o f benzene t o p h e n o l . The r a t e - d e t e r m i n i n g s t e p i n t h e f o r m a t i o n o f p h e n o l s i s t h e h e t e r o l y t i c c l e a v a g e o f t h e C-0 bond o f t h e arene e p o x i d e i n t e r m e d i a t e t o a c a r b o c a t i o n ( T r a g e r , 1980), and as such phen y l r i n g h y d r o x y l a t i o n i s g e n e r a l l y n o t s u s c e p t i b l e t o an i s o t o p e e f f e c t . C a t e c h o l s a r e a l s o formed from t h e arene e p o x i d e , t h r o u g h t h e s e q u e n t i a l a c t i v i t y o f e p o x i d e h y d r o l a s e (Oesch, 1972) and d i h y d r o d i o l dehydrogenase ( K a l f , 1987; B i l l i n g s , 1 985). Thus, i t i s u n l i k e l y f o r c a t e c h o l f o r m a t i o n t o be a s s o c i a t e d w i t h an i s o t o p e e f f e c t . The m i n o r i s o t o p e e f f e c t a s s o c i a t e d w i t h [ 2H5]CLBZ d e u t e r i o p h e n y l o x i d a t i o n augurs w e l l f o r t h e s u i t a b i l i t y o f t h i s a n a l o g u e f o r m e t a b o l i s m s t u d i e s u s i n g GCMS. No p e r t u r b a t i o n o f CLBZ m e t a b o l i s m i s l i k e l y t o o c c u r as a r e s u l t o f m e t a b o l i c s w i t c h i n g due t o i s o t o p e e f f e c t s ( F o s t e r , 1984). (B) M e t a b o l i c F o r m a t i o n o f The P u t a t i v e C a r b i n o l a m i d e . The o c c u r r e n c e o f c a r b i n o l a m i d e s as s t a b l e N - d e m e t h y l a t i o n i n t e r m e d i a t e s has been r e p o r t e d ( S l a t t e r e t a l . , 1989; V a j t a e t a l . , 1988). 147 In o r d e r t o i n v e s t i g a t e t h e p o s s i b l e m e t a b o l i c f o r m a t i o n o f t h e CLBZ c a r b i n o l a m i d e i n t h e r a t , t h e i s o t o p e c l u s t e r m\/z 388\/393 c o r r e s p o n d i n g t o t h e T M S - d e r i v a t i v e o f t h e CLBZ c a r b i n o l a m i d e was m o n i t o r e d i n t h e c o n j u g a t e d and u n c o n j u g a t e d f r a c t i o n s o f b i l e and u r i n e . There was no d e t e c t a b l e e v i d e n c e f o r t h e CLBZ c a r b i n o l a m i d e . P r e v i o u s w o r k e r s have shown t h a t c o n j u g a t i o n s t a b i l i z e s t h e c a r b i n o l a m i d e s o f monuron (Ross e t a l . , 1982) and d i p h e n a m i d (McMahon and S u l l i v a n , 1965) as d e m o n s t r a t e d by t h e appearance o f t h e N-demethylated s p e c i e s i n t h e c o n j u g a t e d u r i n a r y f r a c t i o n s o f a n i m a l s . S i n c e t h e c a r b i n o l a m i d e o f CLBZ was not d e t e c t e d , we i n v e s t i g a t e d t h e p o s s i b i l i t y o f DMC a r i s i n g from t h e c o n j u g a t e d f r a c t i o n s o f b i l e and u r i n e as i n d i r e c t e v i d e n c e f o r t h e e x i s t e n c e o f t h e c a r b i n o l a m i d e m e t a b o l i t e . Hence, t h e i s o t o p e c l u s t e r m\/z 358\/363 f o r DMC-TMS was m o n i t o r e d , inasmuch as d e f o r m y l a t i o n t o DMC c o u l d have o c c u r r e d c o n c o m i t a n t l y w i t h d e c o n j u g a t i o n . No d e t e c t a b l e e v i d e n c e f o r t h i s i s o t o p e c l u s t e r was fo u n d . The 4'-hydroxy c a r b i n o l a m i d e o f t r i f l u b a z a m was i d e n t i f i e d as a u r i n a r y m e t a b o l i t e i n t h e r a t ( A l t o n e t a l . , 1975b); t h e r e f o r e , m o n i t o r i n g f o r t h e an a l o g o u s CLBZ m e t a b o l i t e a t m\/z 476\/480 was performed f o r t h e c o n j u g a t e d f r a c t i o n s o f u r i n e and b i l e . L i k e t h e c a r b i n o l a m i d e , t h e 4'-hyd r o x y a n a l o g u e o f t h e c a r b i n o l a m i d e c o u l d n o t be d e t e c t e d . R e p o r t s i n t h e l i t e r a t u r e documenting t h e f o r m a t i o n o f c a r b i n o l a m i d e s in vitro ( L i n d b e r g e t a l . , 1989; K e d d e r i s e t a l . , 1989), prompted us t o i n v e s t i g a t e t h e p o s s i b l e f o r m a t i o n o f t h e c a r b i n o l a m i d e by microsomal o x i d a t i o n o f CLBZ. A 50:50 m i x t u r e o f [ 2H5]CLBZ:CLBZ was i n c u b a t e d w i t h c o n t r o l and PB-i n d u c e d microsomes and GCMS a n a l y s i s o f t h e T M S - d e r i v a t i z e d e x t r a c t r e v e a l e d t h a t d e m e t h y l a t i o n t o DMC (m\/z 358\/363) had o c c u r r e d w i t h v i r t u a l l y no e v i d e n c e f o r a c a r b i n o l a m i d e i n t e r m e d i a t e (m\/z 388\/393). 148 I f t h e c a r b i n o l a m i d e i s a s u f f i c i e n t l y s t a b l e i n t e r m e d i a t e t o s e r v e as a s u b s t r a t e f o r c o n j u g a t i o n , i t c o u l d be t r a p p e d in vivo by t h i s p r o c e s s . Indeed, r e p o r t s i n t h e l i t e r a t u r e prompt f u r t h e r i n v e s t i g a t i o n i n t o t h e f o r m a t i o n o f t h e CLBZ c a r b i n o l a m i d e in vivo. F i r s t l y , compared t o t h e mouse ( C a c c i a e t a l . , 1980a+b) and man ( V o l z e t a l . , 1979), N-d e a l k y l a t i o n o f CLBZ does not p r o c e e d as r e a d i l y i n t h e r a t . T h e r e f o r e , i t i s p o s s i b l e t h a t t h e r a t may not be a s u i t a b l e model t o examine t h e f o r m a t i o n o f t h e c a r b i n o l a m i d e o f CLBZ. S e c o n d l y , V o l z e t a l . (1979) o b s e r v e d t h e appearance o f DMC as a s i g n i f i c a n t u r i n a r y m e t a b o l i t e i n man. However, t h e e x p e r i m e n t a l p r o c e d u r e was n o t s u f f i c i e n t l y e x p l i c i t t o d e t e r m i n e whether t h i s m e t a b o l i t e a r o s e b e f o r e o r a f t e r d e c o n j u g a t i o n w i t h a r y l s u l f a t a s e \/ \/ J - g l u c u r o n i d a s e . Were DMC t o a r i s e from t h e c o n j u g a t e d f r a c t i o n , t h i s c o u l d be c o n s i d e r e d i n d i r e c t e v i d e n c e f o r t h e e x i s t e n c e o f t h e c a r b i n o l a m i d e . (CI M e t a b o l i c F o r m a t i o n o f The P u t a t i v e N-hvdroxv DMC ( h v d r o x a m i c a c i d ) . S e l e c t e d i o n m o n i t o r i n g f o r t h e i s o t o p e c l u s t e r m\/z 374\/379 c o r r e s p o n d i n g t o M +* o f t h e TMS d e r i v a t i v e o f t h e h y d r o x a m i c a c i d d i d not p r o v i d e any e v i d e n c e f o r t h i s m e t a b o l i t e i n b i l e and u r i n e . The p o s s i b i l i t y o f N - h y d r o x y l a t i o n o f DMC t o t h e h ydroxamic a c i d was i n v e s t i g a t e d in vitro by i n c u b a t i n g a m i x t u r e o f DMC:[ 2H5]DMC w i t h c o n t r o l and P B - i n d u c e d microsomes. S i m i l a r t o t h e r e s u l t s o b t a i n e d in vivo, no e v i d e n c e f o r o x i d a t i o n a t t h e s e c o n d a r y n i t r o g e n t o form t h e hydroxamic a c i d was o b t a i n e d . A l t h o u g h , N - h y d r o x y l a t i o n o f t h e s e c o n d a r y amide a c e t y l a m i n o f l u o r e n e (AAF) t o t h e h ydroxamic a c i d N-hydroxy AAF i s a s i g n i f i c a n t m e t a b o l i c 149 pathway i n t h e r a t (Cramer e t a l . , 1960) and in vitro ( I b a n e z e t a l . , 1987; As t r o m e t a l . , 1 986), t h i s b i o t r a n s f o r m a t i o n p r o c e e d s l e s s r e a d i l y among t h e a c e t y l a n i l i d e s (Damani, 1982). The absence o f N - h y d r o x y l a t i o n o f 3'-h y d r o x y a c e t a n i l i d e i n t h e mouse and in vitro (Rashed e t a l . , 1989) s u p p o r t s t h i s o b s e r v a t i o n . On t h e o t h e r hand, t h e microsomal N - h y d r o x y l a t i o n o f p h e n a c e t i n ( H i n s o n and M i t c h e l l , 1976) and p - c h l o r o a c e t a n i l i d e ( H i n s o n e t a l . , 1975) has been d e m o n s t r a t e d in vitro w i t h hamster microsomes. I t i s n o t e w o r t h y t h a t t h e hamster i s a more e f f i c i e n t N - h y d r o x y l a t o r o f AAF t h a n t h e r a t ( A s t r o m e t a l . , 1986). Thus, t h e u n d e t e c t a b l e f o r m a t i o n o f N-hy d r o x y DMC i n t h e r a t c o u l d be ( i ) a r e f l e c t i o n o f t h e t r e n d d i s p l a y e d among a c e t y l a n i l i d e s and\/or ( i i ) a r e s u l t o f t h e i n c a p a c i t y o f t h e r a t t o a c c o m p l i s h t h i s b i o t r a n s f o r m a t i o n . (D) The I n f l u e n c e o f Cytochrome P-450 I n d u c t i o n on CLBZ M e t a b o l i s m . The microsomes from u n t r e a t e d r a t s and PB-i n d u c e d r a t s were a n a l y z e d by g e l e l e c t r o p h o r e s i s and i m m u n o b l o t t i n g ( F i g u r e 4 5 ) . PB was r e s p o n s i b l e f o r t h e i n d u c t i o n o f cytochrome P-450b as r e v e a l e d by c o m p a r i s o n t o t h e cyto c h r o m e P-450 s t a n d a r d on t h e g e l , and r e a c t i o n o f t h e t r a n s f e r r e d p r o t e i n when c h a l l e n g e d w i t h a p o l y c l o n a l a n t i b o d y f o r cytochrome P-450b+e. I n c u b a t i o n o f an a p p r o x i m a t e 50:50 m i x t u r e o f CLBZ:[ 2 Hs]CLBZ w i t h c o n t r o l and P B - i n d u c e d microsomes r e v e a l e d t h a t N - d e m e t h y l a t i o n was i n c r e a s e d w i t h PB t r e a t m e n t , whereas phenyl h y d r o x y l a t i o n d i d not appear t o be a f f e c t e d t o t h e same e x t e n t ( F i g u r e 4 6 ) . These r e s u l t s s u g g e s t t h a t cytochrome P-450b i s one o f t h e cytochrome P-450 enzymes w h i c h c a t a l y z e t h e N - d e m e t h y l a t i o n o f CLBZ. In t h i s r e g a r d , i t i s unus u a l t h a t Cano e t a l . (1981) o b s e r v e d no s i g n i f i c a n t i n c r e a s e i n 150 N - d e m e t h y l a t i o n i n p a t i e n t s who were c o n c o m i t a n t l y a d m i n i s t e r e d PB and CLBZ, whereas a pronounced i n c r e a s e ( i n N - d e m e t h y l a t i o n ) o c c u r r e d when ca r b a m a z e p i n e (CBZ) and\/or p h e n y t o i n (PH) were c o - a d m i n i s t e r e d w i t h CLBZ. P a n e s a r and c o - w o r k e r s (1989) have d e m o n s t r a t e d t h a t CBZ i s a t b e s t , as e f f e c t i v e an i n d u c e r o f cytochrome P-450b as PB. Were t h e i n d u c t i o n o f CLBZ d e m e t h y l a t i o n by CBZ r e s p o n s i b l e f o r t h e e l e v a t e d plasma l e v e l o f DMC o b s e r v e d by Cano e t a l . ( 1 9 8 1 ) , t h e n PB c o - a d m i n i s t r a t i o n s h o u l d have a f f o r d e d a comparable degree o f DMC f o r m a t i o n . F u r t h e r m o r e , P u l l a r e t a l . (1987) o b s e r v e d e l e v a t e d DMC plasma l e v e l s w i t h c o n c o m i t a n t CBZ o r PH a d m i n i s t r a t i o n when DMC was used as the parent drug. C o n s i d e r e d a l t o g e t h e r , t h e s e r e s u l t s s u g g e s t , as P u l l a r e t a l . (1987) have p r o p o s e d , t h a t i n d u c t i o n o f CLBZ d e m e t h y l a t i o n i s not s o l e l y r e s p o n s i b l e f o r t h e e l e v a t i o n o f plasma DMC when CBZ and\/or PH a r e c o - a d m i n i s t e r e d w i t h CLBZ. 151 A B est ssi =3: ttSt Ml j O t U B I Y f \u00a7 8 B B 4M\u00bb ^ . ... jatft 1 2 3 4 5 1 2 3 4 5 F i g u r e 45. (A) SDS-PAGE (Laemmli e t a l . , 1970), and (B) immunoblot (Towbin e t a l . , 1979) o f l i v e r m i c r o somal p r o t e i n o f Sprague Dawley r a t s . Lane a s s i g n m e n t s a r e as f o l l o w s . 1: P B - i n d u c e d s t a n d a r d , 2: 3-m e t h y l c h o l a n t h r e n e (3-MC)-induced, 3: 3-M C - c o n t r o l ( c o r n o i l ) , 4: PB-i n d u c e d and 5: P B - c o n t r o l ( s a l i n e ) . 152 F i l * >AF1 24O00Q -220000 -200000 -lflOOQO -1\u00ab0000 -140000 -12QOOO -laaaag -e o o o e -e o o a o -40000-20000-9 9 9 9 . o - o . a 13.014.0 13.a l \u00ab . O 17.016.0X9.020.021.0 22.0 F i l * >AF4 9999.0 - O . O 2O0OO0 ~ l e a o o o 1600Q0 ~ 14O0OQ Z 120000 2_ laofloa -~ \u2022 o o o a -~ t e e e a : 40000 : i 20000: B 19.93 20^03 12 . O 13.014.015.0 16.017 . O 18.019.020.0 21.O 220000 -200000 -l e o e o o -i \u00a3 o a a o -14OOO0 -120000 -l a a o o o -e o o a a -6OOOO-40000-2OOOO-12 . \/ rt\u00ab 7 3 17.03 - A 13.0 1 4 . a i 3 . \u00ab l C . e i 7 . 0 1 S . 0 19.Q20.Q21.0 \u2022 i \u2022 >AFS 220OOO-2O0OOO l a o a o a 140000 -140000 i z a a o a l e o o o a S0O0O c o o o o 4OOO02: 20000 9999 . O-0 . O \u00bb \u00ab \u00bb A\u2014 Ft I* >AFJ) 220000 z a a a a o \u2022 o o o o c o a o o 40000 20000 1 2 . 0 1 3 ' . a i V . o IS'.O o ' i i r ' . o l i e \" . o i i ' . \u00ab 20'.0 21'.o IS. C7 \\ E 11 O i 7 IK 1 39 k s 19.96 70 I -. 1 3 . a 14. a i s . a i \u00ab . 0 1 7 . 0 i a . a 1 9 . 0 2 0 . 0 2 1 . 0 F i g u r e 46. SIM p l o t s o f microsomal CLBZ m e t a b o l i t e s . CLBZ, t R , 15.7 min; DMC-TMS, t R 13.6 min; 4'-hydroxy DMC, t R 17.0 min and 4'-hydroxy CLBZ t R 19.9 min. A: CLBZ, no microsomes, 60 min i n c u b a t i o n ; B: c o n t r o l microsomes 15 min i n c u b a t i o n ; C: c o n t r o l microsomes; 60 min i n c u b a t i o n ; D: PB in d u c e d microsomes, 15 min i n c u b a t i o n ; E: PB i n d u c e d m i c r o s o m e s , 60 min i n c u b a t i o n . 153 (E) The D e u t e r i u m I s o t o p e E f f e c t a s s o c i a t e d w i t h CLBZ N - d e m e t h y l a t i o n . P o u p a e r t e t a l . (1988) o b s e r v e d t h a t when mice were t r e a t e d w i t h t r i d e u t e r i o m e t h y l CLBZ ( [ 2 H 3 ] C L B Z ) , an e x t e n d e d d u r a t i o n o f a n t i c o n v u l s a n t a c t i v i t y was d e r i v e d compared t o CLBZ. They c o n c l u d e d t h a t t h i s phenomenon was a t t r i b u t a b l e t o a p r o l o n g e d a c t i o n o f t h e p a r e n t d r u g w h i c h was caused by an i s o t o p e - a s s o c i a t e d r e d u c t i o n i n N - d e m e t h y l a t i o n . In o r d e r t o i n v e s t i g a t e t h e i s o t o p e e f f e c t a s s o c i a t e d w i t h CLBZ N-d e m e t h y l a t i o n in vitro, an a p p r o x i m a t e 50:50 m i x t u r e o f p e n t a d e u t e r i o p h e n y l C L B Z : t r i d e u t e r i o m e t h y l CLBZ was i n c u b a t e d w i t h c o n t r o l and P B-induced r a t l i v e r microsomes. By t h i s a p p r o a c h , t h e s o u r c e o f DMC d e r i v e d from d e m e t h y l a t i o n ([ 2H5JDMC) and d e d e u t e r i o m e t h y l a t i o n (DMC) c o u l d be d e t e r m i n e d , k \/^kp v a l u e s , d e t e r m i n e d by p r o d u c t r a t i o s , were c a l c u l a t e d a t 3.65 \u00b1 0.27 (n = 3) and 2.80 \u00b1 0.16 (n = 4) f o r c o n t r o l and i n d u c e d microsomes r e s p e c t i v e l y . These v a l u e s , a r e c o n s i s t e n t w i t h t h e d e p r o t o n a t i o n o f an aminium r a d i c a l (k^Ap, < 3.6) (Miwa e t a l . , 1983). Hence, t h e cytochrome P-450 c a t a l y z e d d e m e t h y l a t i o n o f CLBZ s h o u l d p r o c e e d via t h e f o l l o w i n g pathway: e l e c t r o n r e m o v a l , f o l l o w e d by p r o t o n a b s t r a c t i o n , o x y g e n a t i o n t o t h e c a r b i n o l a m i d e , and d e f o r m y l a t i o n t o DMC. 154 IV SUMMARY AND CONCLUSIONS. 1. ['H 5]CLBZ was s y n t h e s i z e d i n e s s e n t i a l l y q u a n t i t a t i v e i s o t o p i c and c h e m i c a l p u r i t y . The most s u s c e p t i b l e s t e p t o d e u t e r i u m exchange i n t h e s y n t h e s i s o f t h i s compound was t h e n u c l e o p h i l i c s u b s t i t u t i o n o f 2,4-d i c h l o r o n i t r o b e n z e n e by a n i l i n e - d 7 t o form N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) p e n t a d e u t e r i o p h e n y l a m i n e 18. In t h i s s t e p c o n t a m i n a t i n g a n i l i n e - 2 , 3 , 4 , 5 , 6 - d 5 i n t r o d u c e d p r o t o n s i n t o t h e ortho and p a r a p o s i t i o n s o f 18. A mechanism a c c o u n t i n g f o r t h i s r e a r r a n g e m e n t has been p r o p o s e d . 2. 4'-Hydroxy CLBZ ( 2 8 ) , 4'-hydroxy DMC ( 2 6 ) , 3 , 4 - d i h y d r o x y CLBZ (35) and 3 , 4 - d i h y d r o x y DMC (32) were c h e m i c a l l y s y n t h e s i z e d as CLBZ m e t a b o l i t e s t a n d a r d s . The 0-methyl e t h e r was a s u i t a b l e p h e n o l i c p r o t e c t i n g group w h i c h was e a s i l y removed i n good y i e l d w i t h BBr3. The 0 - m e t h y l a t e d c a t e c h o l s o f CLBZ (36a+b) and DMC (33) were e n z y m a t i c a l l y s y n t h e s i z e d by u s i n g d i h y d r o x y CLBZ (35) and d i h y d r o x y DMC (32) r e s p e c t i v e l y as s u b s t r a t e s , l i v e r c y t o s o l as a s o u r c e o f c a t e c h o l O - m e t h y l t r a n s f e r a s e and S - a d e n o s y l - L - m e t h i o n i n e as t h e methyl donor. The 0 - m e t h y l a t e d c a t e c h o l s o f CLBZ appeared as a m i x t u r e w i t h a meta (36a):para (36b) r a t i o o f 68:32. 3. T r e a t m e n t o f DMC w i t h formaldehyde i n t h e p r e s e n c e o f K2CO3 p r o d u c e d a compound whose GCMS ( E I ) s p e c t r a l p r o p e r t i e s were c o n s i s t e n t w i t h t h e c a r b i n o l a m i d e ( 3 9 ) . U n f o r t u n a t e l y , t h i s compound c o u l d n o t be p u r i f i e d f o r *H-NMR a n a l y s i s because o f i t s m a r g i n a l s t a b i l i t y . A major p r o d u c t i n t h i s r e a c t i o n was 3-hydroxymethyl DMC (46) which was c h a r a c t e r i z e d by *H-NMR and LCMS. Formaldehyde c o n d e n s a t i o n c a t a l y z e d by KOH a l s o a f f o r d e d 3-hy d r o x y m e t h y l DMC ( 4 0 ) . Whether formaldehyde a d d i t i o n a t t h e 3 - p o s i t i o n o c c u r s as a k i n e t i c p r o d u c t o r whether i t a r i s e s as t h e r e s u l t o f an 155 i s o m e r i z a t i o n subsequent t o N - h y d r o x y m e t h y l a t i o n r e m a i n s t o be r e s o l v e d . 4. GCMS i d e n t i f i c a t i o n o f i s o t o p e c l u s t e r s a l l o w e d t h e f o l l o w i n g c o n j u g a t e d m e t a b o l i t e s o f CLBZ t o be i d e n t i f i e d : 4'-hydroxy CLBZ ( 2 8 ) , 4'-hy d r o x y DMC ( 2 6 ) , 3 ' , 4 ' - d i h y d r o x y CLBZ ( 3 5 ) , 4'-hydroxy-3'-methoxy DMC ( 3 3 ) , 4'-hydroxy-3'-methoxy CLBZ ( 3 6 a ) , and 3'-hydroxy-4'-methoxy CLBZ ( 3 6 b ) . The l a t t e r m e t a b o l i t e was o n l y s i g n i f i c a n t as a b i l i a r y s u l f a t e where i t c o n s t i t u t e d 30 % o f t h e 0-methyl c a t e c h o l a n a l o g u e s o f CLBZ. A mechanism f o r t h i s n o n - u n i f o r m d i s p o s i t i o n o f i s o m e r s has been p r o p o s e d . N e i t h e r t h e c a r b i n o l a m i d e o f CLBZ, n o r t h e h y d r o x a m i c a c i d were d e t e c t a b l e m e t a b o l i t e s o f CLBZ i n t h e r a t . 5. The m e t a b o l i s m o f CLBZ and DMC by c o n t r o l and PB - i n d u c e d microsomes was examined. CLBZ was m e t a b o l i z e d t o DMC, 4'-hydroxy CLBZ and 4'-hydroxy DMC. There was no e v i d e n c e f o r t h e c a r b i n o l a m i d e i n t e r m e d i a t e i n N-d e m e t h y l a t i o n . P B - i n d u c t i o n appeared t o i n c r e a s e N - d e m e t h y l a t i o n o v e r r i n g h y d r o x y l a t i o n . DMC was m e t a b o l i z e d t o 4'-hydroxy DMC. There was no e v i d e n c e f o r N - o x i a t i o n t o t h e hydroxamic a c i d . 6. In t h e r a t , d e u t e r i u m i s o t o p e e f f e c t s were m a r g i n a l f o r phenyl r i n g o x i d a t i o n t o p h e n o l s (ki t \/kg =1.103 \u00b1 .090) and c a t e c h o l s (ku\/kn, = 1.088 \u00b1 .207). 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GCMS ( E I ) spec t r u m o f a n i l i n e - d 7 (17) shows [ M - 2 ] + * as most i n t e n s e h i g h mass i o n because o f e x t e n s i v e d e u t e r i u m exchange d u r i n g GC. 1.4. 84.68 MHz 2H-NMR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a -d e u t e r i o p h e n y l a m i n e (18) shows t h e p r e s e n c e o f d e u t e r i u m a t t h e ortho\/para (7.32 ppm) and meta (7.50 ppm) p o s i t i o n s o f t h e phenyl r i n g . 1.5. FT-IR s p e c t r u m o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a d e u t e r i o p h e n y l a m i n e (CCI4 s o l u t i o n ) ( 1 8 ) . 1.6. FT-IR s p e c t r u m o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - p e n t a d e u t e r i o -phenyl carbamoyl a c e t a t e (CCI4 s o l u t i o n ) ( 1 9 ) . 1.7. 400 MHz ^H-NMR spectrum o f p e n t a d e u t e r i o p h e n y l N-desmethylclobazam ( 2 0 ) . + E t 0 A c . 1.8. FT-IR s p e c t r u m o f p e n t a d e u t e r i o p h e n y l N - d e s m e t h y l c l o b a z a m ( N u j o l m u l l ) ( 2 0 ) . 1.9. FT-IR s p e c t r u m o f p e n t a d e u t e r i o p h e n y l c l o b a z a m (CCI4 s o l u t i o n ) ( 2 1 ) . 1.10. 400 MHz ^H-NMR spect r u m o f p e n t a d e u t e r i o p h e n y l CLBZ (21) i n CQDQ. 1.11. 400 MHz *H-NMR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 4 ' - m e t h o x y -phenylamine ( 2 3 ) . 1.12. 300 MHz !H-NMR spect r u m o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 -m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 2 4 ) . 1.13. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 4 ' - m e t h o x y -p h e n y l a m i n e (23) and (B) e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 -m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 2 4 ) . 1.14. 400 MHz *H-NMR spectrum o f 4'-methoxy-N-desmethylclobazam ( 2 5 ) . 1.15. 400 MHz *H-NMR spectrum o f 4'- h y d r o x y - N - d e s m e t h y l c l o b a z a m (26) i n DMS0-d 6. 1.16. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 4'-methoxy-N-desmethyl clobazam (25) and (B) 4'-methoxyclobazam ( 2 7 ) . 175 1.17. 400 MHz XH-NMR spect r u m o f 4'-methoxyclobazam ( 2 7 ) . 1.18. 400 MHz *H-NMR spect r u m o f 4'- h y d r o x y c l o b a z a m ( 2 8 ) . C o n t a m i n a t i n g p e a k s : 4.14 ( q , E t O A c ) , 3.85 ( s , 4'-methoxy CLBZ (<5 % ) ) . 1.19. 400 MHz *H-NMR spect r u m o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' -d i m e t h o x y p h e n y l a m i n e ( 2 9 ) . 1.20. 400 MHz *H-NMR spect r u m o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 3 , 4 -d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . 1.21. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' -d i m e t h o x y p h e n y l a m i n e (29) and (B) e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) -e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 3 , 4 -d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . 1.22. 400 MHz ^H-NMR spect r u m o f 3',4'-d i m e t h o x y - N - d e s m e t h y l c l o b a z a m ( 3 1 ) . 1.23. 400 MHz *H-NMR spectrum o f 3' , 4 ' - d i h y d r o x y - N - d e s m e t h y l c l o b a z a m (32) i n DMS0-d6-1.24. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 3',4'-dimethoxy-N-desmethyl c l o b a z a m (31) and (B) 3',4 ' - d i m e t h o x y c l o b a z a m ( 3 4 ) . 1.25. 400 MHz ^H-NMR spectrum o f 3',4'-di m e t h o x y c l o b a z a m ( 3 4 ) . 1.26. 400 MHz !H-NMR sp e c t r u m o f 3 ' , 4 ' - d i h y d r o x y c l o b a z a m ( 3 5 ) . 1.27. 400 MHz *H-NMR spect r u m o f compound ( 4 0 ) . 1.28. 400 MHz ^H-NMR spect r u m o f compound ( 4 5 ) . 2. SPECTRA RELATED TO THE METABOLISM OF CLBZ IN THE RAT. 2.1. Mass chromatograms a t m\/z 388 (M +-) and 392 ( [ 2 H 4 ] M + * ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f \/ f - g l u c u r o n i d a s e - h y d r o l y z e d b i l e t y p i f y t h e d e t e c t i o n o f 4'-hydroxy CLBZ (28) a t t R 21.37 and t R 21.30 min r e s p e c t i v e l y . The absence o f t h e i s o t o p i c a l l y r e l a t e d m\/z 388\/393 shows t h a t a c a r b i n o l a m i d e was n o t d e t e c t a b l e . 2.2. Mass chromatograms a t m\/z 446 ( M + > ) and 450 ( [ 2 H 4 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f \/ 3 - g l u c u r o n i d a s e - h y d r o l y z e d b i l e t y p i f y t h e d e t e c t i o n o f 4'-hydroxy DMC (26) a t t R 17.91 and t R 17.86 min r e s p e c t i v e l y . 2.3. Mass chromatograms a t m\/z 476 (M +-) and 479 ( [ 2 H 3 ] M + ' ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f \/ 3 - g l u c u r o n i d a s e - h y d r o l y z e d b i l e t y p i f y 176 t h e d e t e c t i o n o f 4'-hydroxy-3'-methoxy DMC (33) a t tR 19.13 and tR 19.17 min r e s p e c t i v e l y , and d i h y d r o x y CLBZ (35) a t t R 23.50 and tR 23.47 min r e s p e c t i v e l y . Mass chromatograms a t m\/z 418 (M +-) and 421 ([ 2H\"3]M +-) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f \/ J - g l u c u r o n i d a s e - h y d r o l y z e d b i l e show t h e p r e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t tR 23.22 and tR 23.17 min r e s p e c t i v e l y , and 3'-hydroxy-4'-methoxy CLBZ (36b) a t tR 22.44 and t R 22.37 min r e s p e c t i v e l y . Mass chromatograms a t m\/z 418 (M +-) and 421 ( [ 2 H 3 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f a r y l s u l f a t a s e - h y d r o l y z e d u r i n e show t h e p r e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t tR 23.12 and tR 23.08 min r e s p e c t i v e l y . 177 178 S A M P L E N 0 2 3 4 7 A B O R E L S A M P L E I D A B 1 7 6 \/ M T A T \u2014 | \u2014 i \u2014 ; \u2014 i \u2014 i \u2014 j \u2014 i \u2014 I \u2014 I \u2014 I \u2014 | \u2014 ! \u2014 ; \u2014 I \u2014 ; \u2014 j \u2014 : \u2014 i \u2014 I \u2014 i \u2014 [ ~ \u2014 \u2014 i \u2014 i ' j i i i I ] : i i 145 140 135 130 125 120 115 P P M 1.2. 75 MHz 1 3C-NMR sp e c t r u m o f a n i l i n e - a j ( 17) shows t h e p r e s e n c e o f d e u t e r i u m a t t h e C-3, C-4 and C-2 p o s i t i o n s w i t h t h e appearance o f t r i p l e t s . 98 71 28 42 32 33 49 34 37 76 82 88 1.3. GCMS (EI ) spectrum o f a n i l i n e - d y (17) shows [M-2]+* as most i n t e n s e h i g h mass i o n because o f e x t e n s i v e d e u t e r i u m exchange d u r i n g GC. 180 1.4. 84.68 MHz ^H-NMR spect r u m o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a -d e u t e r i o p h e n y l a m i n e (18) shows t h e p r e s e n c e o f d e u t e r i u m a t t h e ortho\/para (7.32 ppm) and meta (7.50 ppm) p o s i t i o n s o f t h e phenyl r i n g . 1.5. FT-IR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - p e n t a d e u t e r i o p h e n y l a m i n e (CCI4 s o l u t i o n ) ( 1 8 ) . 1 . 6 . FT-IR spectrum o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - p e n t a d e u t e r i o - p h e n y l carbamoyl a c e t a t e (CCI4 s o l u t i o n ) ( 1 9 ) . H CO to 183 Hz 5.000 Hz 2,500 4 .000 2 .000 x 8 JL 1.7. 400 MHz *H-NMR spectrum o f p e n t a d e u t e r i o p h e n y l N-desmethylclobazam ( 2 0 ) . + E t O A c . 5 4 3 1.11. 400 MHz *H-NMR spectrum of N-(5-chloro-2-nitrophenyl)-4 '-methoxy-phenylamine (23). J Ju^LJ VARI AN X L - 3 0 0 STANDARO 1H OBSERVE F.XP8 PULSE SEQUENCE STD 1 H DATE 2 1 - 0 6 - 8 9 SOL VENT F I L E CDCL 3 H : 0 l>7 A C Q U I S I T I O N DEC 4 VT TN 1 . 750 DN 1 . 750 SW 4 0 0 0 . 0 DO 350 . 3 A r 3. 752 OW NNN NP J 0 0 1 6 DLP 14 PW 35 0 P i 0 P R O C E S S I N G 01 0 SE 3. 183 02 0 LB 0. 100 TO 300 FN 65536 NT 500 MATH F CT 100 PW90 46. 0 D I S P L A Y SS 0 SP - 6 0 . 1 IL y WP 2 6 9 9 . 5 IN Y VS 300 OP r SC 100 HS NN wc t s RFL RFP TH INS 400 120 5 4 9 . 3 0 20 1 . 000 r \\ 1 1 1 1 1 1 1 1 1 I I I | 1 I I I | I I I I | I I I I [ I M I | I I M | I I I I | I I I I | I I I I | I I I I | I I i I | i I I i | I 7 6 5 '''' 4 3 2 1.12. 300 MHz *H-NMR spectrum o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 -methoxyphenyl)carbamoyl a c e t a t e ( 2 4 ) . 03 03 f i l e >RB141 Bpk fib 20848 4-Otle CNPB Scan 284 7.88 min. 75 92 \/ 123 \/ 111 1,,,,^ 111,1,1 i l l ..i.lnn,, I ill,. . mill, 1 3 9 \/ 182 ^ \\ 188 II, ..III \u2022illlll.l ,.M\\ 216 \\ 217 278 2 6 3 \\ 244 I V 88 128 168 288 240 288 . l i e >RB147 Bpk Rb 2056 2200-2800-1800-1600-1488-1288-1009-888-680-408-0-1' 75 B 92 4-OMe PCMM 123 III I, II 111 \/ ,1 III hlin 154 281 152 182 \\ 216 Scan 285 7.02 min. 278 263 244 \/ \u00bb17 88 120 168 288 248 288 13. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 4 ' - m e t h o x y p h enylamine (23) and (B) e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 4 m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 2 4 ) . 1.15. 400 MHz *H-NMR spectrum o f 4'-hydroxy-N-desmethylclobazam (26) i n DMS0-d 6. 192 f i l e >0B232 Bpk fib 37808 40000-^ 36000-32000-28000-24000-20000^ 16800^ 12000^ 8000^ 75 \/ 4000^ | 1 0-^ L i 80 4*-0Me-DMC-TMS CLP 225 121 \\ 135 167 \\ 197 178 y 273 298 331 347 M I { 120 160 200 240 280 - I -320 Scan 390 8.79 min. r110 400 r90 r80 r70 J60 ^0 388 : \\ |40 r30 r20 jie 360 ...le >RB233 Bpk fib 5504 -MeO-CLBZ Scan 468 10.11 min. 6080-5500-5000-4500-4000-3580-3000-2580-2000-1500-1000-500-J i 92 \\ B 121 108 I LHmllml 153 \\ 111, J l 217 181 V 230 \/ 167 \/ Ji \u2022 III Hill J 0,11 247 80 120 160 200 240 285 273 \/ i i 330 313 280 328 110 j-100 f90 770 r60 rS0 r40 \u202230 E20 10 -^3 1.16. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 4'-methoxy-N-desmethyl c l obazam (25) and (B) 4'-methoxyclobazam ( 2 7 ) . 194 1.18. 400 MHz H-NMR spectrum o f 4 ' - h y d r o x y c l o b a z a m ( 2 8 ) . C o n t a m i n a t i n g peaks: 4.14 ( q , EtOAc), 3.85 ( s , 4'-methoxy CLBZ (<5 % ) ) . 1.19. 400 MHz *H-NMR spectrum o f N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' -dimethoxyphenylamine ( 2 9 ) . \u2014 \u2014 r \u2014 \u2014 , 1 \u2014 i r 5 4 3 2 1 1.20. 400 MHz *H-NMR spectrum o f e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N -( 3 , 4 - d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . 197 f i l e >0B24S Bpk fib 49624 52800-48000-44880-40000-36000-32000-28000-24800-20000-16000-12080-8008-4000-0-3,4-ditleO-CNPR 79 \/ .Inl.j... U l 107 \/ 140 183 \\ 188 07 y I 153 X*\/\" Scan 334 7.84 min. 293 204 231 \/ III. All 274 \\ 246 111. . . . H i . \\\\L 308 \\ 110 100 9^0 6^0 70 ^0 J50 r48 \u202230 20 -10 80 128 160 200 240 280 . l i e >RB248 Bpk Rb 40736 44000-40000-36000-32080-28000-2400frj 20000-16080-12000^ 8009-4080^ 8-3,4-ditleO-PCNM B. 79 \/ \u2022It. . H i . .1 140 183 ' 153 168 \/ J l 1^1... j\\i Jnlll..Jillll,JlJ.illn,,,,, 111 107 y 204 \/ LU Scan 331 7.79 min. 293 308 231 274 \\ 246 I... ..Iii. .ln,L 111- .. 118 100 9^0 i\u00ab0 r70 |60 j\u20ac8 r48 r30 r28 10 80 120 160 200 240 280 1.21. GCMS ( E I ) s p e c t r a o f (A) N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - 3 ' , 4 ' d i m e t h o x y p h e n y l a m i n e (29) and (B) e t h y l N - ( 5 - c h l o r o - 2 -n i t r o p h e n y l ) - e t h y l N - ( 5 - c h l o r o - 2 - n i t r o p h e n y l ) - N - ( 3 , 4 -d i m e t h o x y p h e n y l ) c a r b a m o y l a c e t a t e ( 3 0 ) . SBMPLf N0:6BS6 fl.BORH 1 I 1 1 ' ' I ' ' ' ' I ' 1 ' ' I 1 1 ' 1 r 1 ' 1 ' I 1 ' 1 1 I ' ' ' ' I i i . . I I , , , , , , . . . ! m - ' j i a J.'J -s.'i D.a 7.7i c . ' i F . a s . i b.a ^ . i a . a 3.r> J.B 1.23. 400 MHz H-NMR spectrum o f 3 ' , 4 ' - d i h y d r o x y - N - d e s m e t h y l c l o b a z a m (32) i n DMS0-d 6. 200 . i le >RB250 Bpk Ob 5368 5500-5000-4500-4000-3500-3000-2500-2008- 75 \/ 1508-1000-500- i 0- L,r J 3' ,4'-(0Me)2-DMC-TMS CLP 225 I 124 179 197 \/ 239 X 2 8 5 37 80 120 160 200 240 280 320 360 303 361 375 418 \\ Scan 434 9.52 min. 110 :100 J90 r80 '-79 |60 rse J40 r30 -20 18 400 f i l e >AB288 Bpk Ob 8001 3,4-diMeOCLBZz CLP Scan 555 1 .60 min. 8380-1 7000-^ 6000-1 5800H 4000-^ 3000H 2000-1 79 1800-1 B 151 \/ 117 \\ 138 \\ 4n,.ljulitliiit.iHii. 80 120 315 \\ 1 8 1 \u00bb 8 9 260 24S llli 217 Mill 277 l u l l 360 319 160 200 240 280 320 110 100 r90 r80 \u202270 r60 rS0 r48 \u202230 20 10 CO 360 1.24. GCMS ( E I ) s p e c t r a o f (A) t h e T M S - d e r i v a t i v e o f 3' ,4'-dimethoxy-N-d e s m e t h y l c l o b a z a m (3_i) and (B) 3 ' , 4 ' - d i m e t h o x y c l o b a z a m ( 3 4 ) . 1.25. 400 MHz *H-NMR spectrum o f 3',4'-dimethoxyclobazam ( 3 4 ) . 202 100 so H z 5 . 0 0 0 H i 2 . 5 0 0 4 . 0 0 0 3 . 0 0 0 1.26. 400 MHz *H-NMR spectrum o f 3 ' , 4 ' - d i h y d r o x y c l o b a z a m ( 3 5 ) . 205 F i l e >RB81 387.7-333.7 amu.CLBZ-BILE-GLUC-TMS-SMT ADC EIP 4800-3000-2080-1000-21 .37 15.75 23^0? 2 I - \" 1 \" ! , i f | 12.013.014.015.016.017.818.819.8 20.821.822.823.024.025.026.8 F i l e >RB01 2S00^ 2888 1500: 1880-| See-s' 391 .7-392.7 amu.CLBZ-BILE-GLUC-TMS SMT RDC EIP \u2022 \u2022A. \/A. . . 21 .30 12.8 1 3 . 8 1 4.0 1 5 . 0 1 6.6 1 7 . 8 1 8.0 19* .81 2 0 . 0 2 l'.'eI22.G123:\".'024.0125.0 26.8 F i l e >RB81 888-600-400-200-392 .7-393 .7 amu.CLBZ-BILE-GLUC-TMS SMT RDC EIP 17.87 12.75 i l .38 23.17 22.91 JL 1 2 . 0 1 3 . 8 i V.'o i 5 . 0 i 6 . 0 i 7 . 0 i 8 . 0 i 9.0' 2 0 . 0 2i'.'e 2 2 . 0 2 3 .'024 .'0 25 .'e 26. e 2.1. Mass chromatograms a t m\/z 388 (M +-) and 392 ( [ 2 H 4 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f 0 - g l u c u r o n i d a s e - h y d r o l y z e d b i l e t y p i f y t h e d e t e c t i o n o f 4'-hydroxy CLBZ (28) a t t R 21.37 and t R 21.30 min r e s p e c t i v e l y . The absence o f t h e i s o t o p i c a l l y r e l a t e d m\/z 388\/393 shows t h a t a c a r b i n o l a m i d e was n o t d e t e c t a b l e . 206 J 17-91 1 5 . 9 2 1 5 . 0 9 K-r>f\u00bbB81 1666-i zoo-see-480 41; .7-418.7 amu.CLBZ-BILE-GLUC-TMS SMT ABC EIP 23 .22 12.013.014.015. i i 11 \u2022 * ' i \u2022 I 1 1 ' ' ' ' I * ' ' ' ' ' ' ' ' * ' ' ' ' S16.017.0l8.019.O20.821.02i 623 .024.625.626 .0 F i l e >fiB81 1600-888 608-460 280 426 .7-421 .7 amu.CLBZ-BILE-GLUC-TMS SMT PLC EIP 0-141^ .421 4 17.8419.84 18.41 23.17 48 12.81 3.0 14.0 15.0 16 .8 i 7.018.0 19.020.0 2l '.'o 22 .023 .824.8 25 .8 26.8 2.4. Mass chromatograms a t m\/z 418 (M +-) and 421 ( [ 2 H 3 ] M + - ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f \/ J - g l u c u r o n i d a s e - h y d r o l y z e d b i l e show t h e p r e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t t R 23.22 and tR 23.17 min r e s p e c t i v e l y , and 3'-hydroxy-4'-methoxy CLBZ (36b) a t tR 22.44 and t R 22.37 min r e s p e c t i v e l y . 209 F i l e >flB04 417.7-418.7 amu.CLBZ-URIN-SULF-TMS SMT ADC EIP 800-600-400-200-0 12 13 .27 i I 23.12 0 13.0 14.015.0 16.6 17.6 13.0 19.0 20.021.0 22.0 23.0 24.0 25.0 26.0 F i l e >AB04 420.7-421.7 amu.CLBZ-URIN-SULF-TMS SMT ODC EIP 500-480-300 200-100 23.08 IZ.'e 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.6 2l'.0 22.0 23.0 24.0 25.8 26.0 2.5. Mass chromatograms a t m\/z 418 (M +-) and 421 ( [ 2 H 3 ] M + > ) o f TMS-d e r i v a t i z e d EtOAc e x t r a c t s o f a r y l s u l f a t a s e - h y d r o l y z e d u r i n e show t h e pr e s e n c e o f 4'-hydroxy-3'-methoxy CLBZ (36a) a t t R 23.12 and tR 23.08 min r e s p e c t i v e l y . ","@language":"en"}],"Genre":[{"@value":"Thesis\/Dissertation","@language":"en"}],"IsShownAt":[{"@value":"10.14288\/1.0098100","@language":"en"}],"Language":[{"@value":"eng","@language":"en"}],"Program":[{"@value":"Pharmaceutical Sciences","@language":"en"}],"Provider":[{"@value":"Vancouver : University of British Columbia Library","@language":"en"}],"Publisher":[{"@value":"University of British Columbia","@language":"en"}],"Rights":[{"@value":"For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https:\/\/open.library.ubc.ca\/terms_of_use.","@language":"en"}],"ScholarlyLevel":[{"@value":"Graduate","@language":"en"}],"Title":[{"@value":"The metabolic profiling of clobazam in rats","@language":"en"}],"Type":[{"@value":"Text","@language":"en"}],"URI":[{"@value":"http:\/\/hdl.handle.net\/2429\/28921","@language":"en"}],"SortDate":[{"@value":"1990-12-31 AD","@language":"en"}],"@id":"doi:10.14288\/1.0098100"}