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Pharmacokinetic and metabolism studies of valproic acid using gas chromatography mass spectrometry Acheampong, Andrew Adu 1982

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PHARMACOKINETIC AND METABOLISM STUDIES OF VALPROIC ACID USING GAS CHROMATO-GRAPHY MASS SPECTROMETRY by ANDREW ADU^CHEAMPONG B.Sc. U n i v e r s i t y o f Science and Technology, Ghana, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Pharmaceutical Sciences We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May 1982 (t) Andrew A. Acheampong, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pharmaceutical Science The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date M a y 2 7 > 1 9 8 2 DE-6 (3/81) ABSTRACT i i Di-CS-^H^-propyl ) a c e t i c acid was synthesized and used in pharmaco-k i n e t i c and metabolism studies o f d i p r o p y l a c e t i c a c i d ( v a l p r o i c a c i d ) . 2 K i n e t i c equivalence of v a l p r o i c a c i d and v a l p r o i c a c i d - Hg was demon-st r a t e d i n a s i n g l e dose study i n a human volu n t e e r . An isotope e f f e c t was observed f o r w-oxidation but the d i f f e r e n c e in metabolism of the 2 two i s o t o p i c forms was not s u f f i c i e n t to make v a l p r o i c a c i d - Hg b i o l o g i c a l l y nonequivalent. In a m u l t i p l e dose study, the k i n e t i c s of 2 v a l p r o i c a c i d - Hg were determined i n the presence of steady s t a t e con-c e n t r a t i o n s of v a l p r o i c a c i d in the same volunteer. Concentrations of 2 v a l p r o i c acid and v a l p r o i c a c i d - Hg in serum and s a l i v a were determined by gas chromatography mass spectrometry using s e l e c t e d ion monitoring. S a l i v a drug l e v e l s were measured with good p r e c i s i o n down to 0.1 ug/ml. Compared to s i n g l e dose k i n e t i c data, the t o t a l body clearance o f 2 v a l p r o i c a c i d - Hg increased by 33% at steady s t a t e . This could be explained by an i n c r e a s e i n the serum f r e e drug f r a c t i o n . At steady s t a t e , i n t r i n s i c clearance was found to decrease. Good c o r r e l a t i o n was found between concentrations o f v a l p r o i c a c i d i n s a l i v a and serum. Serum and u r i n a r y metabolites were c h a r a c t e r i z e d as t h e i r methyl, t r i m e t h y l s i l y l or t e r t - b u t y l d i m e t h y l s i l y l d e r i v a t i v e s . The metabolism study was f a c i l i t a t e d by using the s t a b l e isotope t r a c e r technique. A diunsaturated metabolite was i d e n t i f i e d i n serum and u r i n e . The presence of a molecular ion doublet in the mass spectrum reduces the p o s s i b l e s t r u c t u r e s f o r t h i s metabolite. A new m e t abolite, 2-propyl-4-keto-pentanoic a c i d , was detected i n serum and u r i n e and 2 - p r o p y l s u c c i n i c a c i d and 2-propylmalonic a c i d were c h a r a c t e r i z e d as m e t a b o l i t e s . The i d e n t i f i c a t i o n of metabolites was a l s o v e r i f i e d using synthesized reference compounds. i v TABLE OF CONTENTS Page ABSTRACT - i i LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS v i i i I. INTRODUCTION 1 I I . EXPERIMENTAL 17 A. Reagents and m a t e r i a l s 17 B. Methods 19 Synthesis 19 Drug A d m i n i s t r a t i o n 32 Pharmacokinetic Drug Assay 34 Metab o l i t e Assay 38 I I I . RESULTS 43 A. Pharmacokinetic Study 43 B. Metabolism Studies 60 IV. DISCUSSION 83 A. Deuterium-labelling of v a l p r o i c a c i d 83 B. Synthesis of v a l p r o i c a c i d and metabolites 85 C. Pharmacokinetic study 94 D. Metabolism study 109 SUMMARY AND CONCLUSIONS 120 REFERENCES 122 APPENDIX 133 V LIST OF TABLES Table Page I C a l i b r a t i o n curve data o f the t e r t - b u t y l d i m e t h y l s i l y l 49 e s t e r o f v a l p r o i c acid-^Hg i n serum and s a l i v a II C a l i b r a t i o n curve data o f the t e r t - b u t y l d i m e t h y l s i l y l 50 es t e r o f v a l p r o i c a c i d i n serum and s a l i v a III Pharmacokinetic parameters f o r v a l p r o i c a c i d and 58 v a l p r o i c acid-^Hg under s i n g l e dose and m u l t i p l e dose conditions IV R e l a t i o n s h i p of s a l i v a concentrations o f v a l p r o i c a c i d 104 and v a l p r o i c acid-^Hg and serum t o t a l or serum fr e e drug l e v e l s , in the s i n g l e dose and m u l t i p l e dose stud i e s V R e l a t i o n s h i p between t h e o r e t i c a l l y p r e d i c t e d and 107 experimental s a l i v a to serum fr e e v a l p r o i c a c i d concentration r a t i o s in the s i n g l e dose study VI Diagnostic fragment ions and ion doublets i n the mass 112 spectra o f methyl esters of v a l p r o i c acid-C^Hg + ^Hg) and t h e i r metabolites VII Diagnostic ions and ion doublets i n the mass spectra 113 of methyl es t e r s o f unsaturated metabolites o f v a l p r o i c a c i d - ( 2 H 0 + 2 H g ) VIII Diagnostic fragment ions and ion doublets i n the mass 114 spectra o f dimethyl e s t e r s o f d i c a r b o x y l i c a c i d metabolites of v a l p r o i c acid-(2Hg + ^H^) IX Diagnostic fragment ions and ion doublets in the mass 116 spectra o f di-TBDMS d e r i v a t i v e s of v a l p r o i c a c i d metabolites LIST OF FIGURES Proposed metabolic pathways of v a l p r o i c a c i d 2 El-Mass spectra o f v a l p r o i c a c i d and v a l p r o i c a c i d - Hg 2 NMR spectra o f synthesized v a l p r o i c a c i d - Hg and v a l p r o i c a c i d S e l e c t e d ion chromatogram of an extracted s a l i v a sample 2 Semi-log p l o t o f v a l p r o i c a c i d and v a l p r o i c a c i d - Hg serum and s a l i v a concentration-time curves i n the s i n g l e dose study Mass chromatograms o f urin e metabolites i n the s i n g l e dose study 2 Semi-log p l o t o f v a l p r o i c a c i d and v a l p r o i c a c i d - Hg serum concentration-time curves i n the m u l t i p l e dose study Semi-log p l o t o f stimulated and unstimulated v a l p r o i c acid-2H5 s a l i v a concentration-time curves 2 Semi-log p l o t o f serum fr e e v a l p r o i c a c i d - Hg concentration-time curve in the m u l t i p l e c l o s e study Total ion current chromatogram of methylated serum e x t r a c t 2 2 Mass spectrum o f v a l p r o i c a c i d - ( Hg + Hg) methyl esters Total ion current chromatogram o f methylated ur i n e e x t r a c t 2 2 Mass spectrum of 3-heptanone-(. Hg + Hg) i n serum e x t r a c t 2 2 Mass spectrum of 4-keto VPA-(. Hg + Hg) methyl esters 2 ? Mass spectrum o f 3-ene VPA-( Hg + Hg) methyl e s t e r s 2 2 Mass spectrum of 2-ene VPA-( Hg + Hg) methyl esters 2 2 Mass spectrum o f methyl esters o f diene VPA-( Hg + Hg) metabolites Mass chromatogram o f methylated serum metabolites 2 2 Mass spectrum of 4-OH VPA-C Hg + Hg) lactone i n serum e x t r a c t Total ion c u r r e n t chromatogram o f t-BDMS d e r i v a t i z e d u r i n a r y e x t r a c t page 21 Mass spectrum o f di-tBDMS d e r i v a t i v e o f 3-OH VPA-( 2H n + 77 2 H 6 ) ° 22 Mass spectrum of di-tBDMS d e r i v a t i v e o f 5-OH VPA-( 2H n + 77 2H 5) ° 23 Mass spectrum o f methyl e s t e r o f 2 - p r o p y l s u c c i n i c a c i d - 78 ( 2 H 0 + 2 H 3 ) 24 Mass spectrum of di-tBDMS d e r i v a t i v e o f 2-propylmalonic 79 a c i d - ( 2 H ( ) + 2 H 3 ) 25 Mass spectrum o f tri-TMS d e r i v a t i v e of dihydroxy VPA 81 metabolite i n urine e x t r a c t 26 Proposed metabolic pathways f o r new metabolites of 118 v a l p r o i c a c i d LIST OF ABBREVIATIONS VPA V a l p r o i c acid (2-propylpentanoic a c i d ) VPA- 2H 6 V a l p r o i c acid- 2Hg 3- OH VPA 2-Propyl-3-hydroxypentanoic a c i d 4- 0H VPA 2-Propyl-4-hydroxypentanoic a c i d 5- 0H VPA 2-Propyl-5-hydroxypentanoic a c i d 3- keto VPA 2-Propyl-3-ketopentanoic a c i d 4- keto VPA 2-Propyl-4-ketopentanoic a c i d 2- ene VPA 2-Propyl-2-pentenoic a c i d 3- ene VPA 2-Propyl-3-pentenoic a c i d 4- ene VPA 2-Propyl-4-pentenoic a c i d TMS T r i m e t h y l s i l y l t-BDMS Tertiary butyl dimethyl s i l y l MSTFA N-methyl - 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 GCMS Gas chromatography mass spectrometry m m u l t i p l e t t t r i p l e t d doublet s s i n g l e t i . v . intravenous HMPA hexamethylphosphoramide THF tetrahydrofuran RSD r e l a t i v e standard d e v i a t i o n GLC G a s s l i q u i d chromatography FID flame i o n i z a t i o n d e t e c t i o n VPA-D 6 V a l p r o i c a c i d - 2 H 6 VPA-D n v a l p r o i c a c i d ix ACKNOWLEDGEMENT The author wishes to thank Dr. F. S. Abbott f o r his able guidance and s u p e r v i s i o n , and moral support throughout the course o f t h i s study. The author expresses his g r a t i t u d e to Dr. J . Orr f o r gi v i n g h e l p f u l suggestions. The author i s also indebted to Miss S. Ferguson f o r her p a r t i c i p a t i o n i n the prot e i n - b i n d i n g s t u d i e s . The t e c h n i c a l a s s i s t a n c e o f Mr. R. S. Burton i n the mass spectrometric a n a l y s i s i s g r e a t l y appreciated. The f i n a n c i a l support provided by a grant from B.C. Health Care Research Foundation and the f i n a n c i a l support provided by the Faculty of Pharmaceutical Sciences, U n i v e r s i t y of B r i t i s h Columbia are g r a t e f u l l y acknowledged. I. INTRODUCTION Va l p r o i c a c i d ( 2 - p r o p y l v a l e r i c a c i d , dipropyl a c e t i c a c i d ) , I, i s a r e l a t i v e l y new a n t i e p i l e p t i c drug and has a unique chemical CH, CH, CH 9 J L c ^ C H COOH CH^ CH2 CH2 — I s t r u c t u r e among the well-known a n t i e p i l e p t i c drugs ( b a r b i t u r a t e s , deoxybarbiturates, hydantoins, o x a z o l i d i n e d i o n e s , succim'mides). I t is a medium-chain branched f a t t y a c i d . I t contains no benzene r i n g , nor a nitrogen f u n c t i o n . The c l o s e s t resemblance of v a l p r o i c a c i d to some of the a n t i e p i l e p t i c drugs i s the d i s u b s t i t u t i o n a t the alpha carbon of the carbonyl group (dotted enclosure below). \ R X C< 0 / The synt h e s i s o f v a l p r o i c a c i d was f i r s t described in the l i t e r a t u r e by Burton (1) i n 1881. However i t s a n t i c o n v u l s a n t a c t i v i t y was not discovered u n t i l 1963 by Meunier and co-workers (2). Before i t s i n t r o d u c t i o n i n France i n 1967 and i n the United States in 1978, there have been numerous stud i e s o f i t s c l i n i c a l e f f i c a c y as reviewed by Simon and Penry ( 3 ) . V a l p r o i c a c i d i s e f f e c t i v e i n treatment -of absence seizures (petit-mal s e i z u r e s ) , however: primary ge n e r a l i z e d t o n i c - c l o n i c s e i z u r e s a l s o f r e q u e n t l y respond to i t s therapy (3, 4, 5). 2. Mechanism of a c t i o n . In s p i t e o f ext e n s i v e c l i n i c a l and animal experimental work, the mechanism of a c t i o n o f v a l p r o i c a c i d i s s t i l l not c l e a r . Several workers have suggested that v a l p r o i c a c i d exerts i t s a n t i c o n v u l s a n t e f f e c t by i n c r e a s i n g b r a i n l e v e l s o f the i n h i b i t o r y neurotransmitter, gamma-aminobutyric a c i d (GABA) i n animals (6 - 9 ) , po s s i b l y by i n h i b i t i o n of the metabolism of GABA. This increase i n GABA l e v e l s i s thought to be due to competitive i n h i b i t i o n o f GABA transaminase (6, 7). Some i n v e s t i g a t o r s have s t r e s s e d the a b i l i t y o f v a l p r o i c a c i d to i n h i b i t s u c c i n i c semialdehyde dehydrogenase i n lower concentrations (8, 9). Whittle and Turner (10) have reported that v a l p r o i c a c i d i n h i b i t e d the aldehyde reductase a t lower concentrations than required for i n h i b i t i o n o f s u c c i n i c aldehyde dehydrogenase and GABA transaminase. Although the extent o f increase i n GABA l e v e l s c o r r e l a t e s temporally with a n t i c o n v u l s a n t e f f e c t i n some animal model experiments, the c o r r e l a t i o n has not been a c o n s i s t e n t one-to-one correspondence (11). Anzelark and others(12) could not f i n d any s i g n i f i c a n t e f f e c t on brain GABA l e v e l s a f t e r an e f f e c t i v e a n t i c o n v u l s i v e dose of v a l p r o i c a c i d . However regional changes i n GABA l e v e l s have been invoked to ex p l a i n the lack of an apprec i a b l e increase i n t o t a l brain GABA l e v e l s (11, 13). Pharmacokinetics Pharmacokinetic stu d i e s o f v a l p r o i c a c i d were f i r s t reported in the l i t e r a t u r e in 1975 (14, 15). To date, the pharmacokinetics o f v a l p r o i c a c i d have been e x t e n s i v e l y studied i n both p a t i e n t s and volunteers a f t e r s i n g l e and m u l t i p l e dose a d m i n i s t r a t i o n . A summary o f the 3. pharmacokinetic data on v a l p r o i c a c i d has been given i n a number of reviews (4, 16, 17). V a l p r o i c a c i d i s administered as the sodium s a l t or the f r e e a c i d . I t i s r a p i d l y absorbed from the g a s t r o i n t e s t i n a l t r a c t with plasma peak l e v e l s observed i n 1 - 4 hr (16). Its absolute b i o a v a i l a -b i l i t y i s n e a r l y 100% (18, 19). There i s no s i g n i f i c a n t f i r s t - p a s s metabolism in the gut or l i v e r (16). Dosages range from 600 - 3000 mg/ day and the r e l a t i o n s h i p between dose and plasma concentrations i s c u r v i l i n e a r (16, 20). The concentration of v a l p r o i c a c i d in plasma that appears to be a s s o c i a t e d with the t h e r a p e u t i c e f f e c t i s approximately 50 - 100 yg/ml (21). The apparent volume of d i s t r i b u t i o n o f v a l p r o i c a c i d i s r e l a t i v e l y s m a l l , 0.1 - 0.4L/Kg (18, 19, 22) due to i t s high plasma protein binding of 90 - 95% (18, 19, 22). The small volume o f d i s t r i b u t i o n i n d i c a t e s that d i s t r i b u t i o n appears to be r e s t r i c t e d to the c i r c u l a t i o n and e x t r a c e l l u l a r space (18). The plasma pr o t e i n bind-ing of v a l p r o i c a c i d in v i t r o has been shown to decrease in a non-linear manner with i n c r e a s i n g t o t a l plasma co n c e n t r a t i o n s . A s i g n i f i c a n t increase i n the f r e e f r a c t i o n i n plasma i s reported to be observed a t t o t a l plasma concentrations greater than 80 yg/ml (23)., The e l i m i n a t i o n k i n e t i c s of v a l p r o i c a c i d have been described as monoexponential (14, 15, 19) and biexponential (18, 22). The plasma concentration decay curve o f v a l p r o i c a c i d a f t e r an i . v . dose i s b i p h a s i c (18) but the d i s t r i b u t i o n phase a f t e r an o r a l dose i s generally masked by the absorption phase (14, 15, 19). Total plasma clearance o f v a l p r o i c a c i d i s i n the range of 0.3 - 0.6 L/hr (15, 18, 22) and the plasma e l i m i n a t i o n h a l f - l i f e i n the range of 10 - 19 hr (16, 18, 22). The t o t a l plasma clearance i s increased almost two-fold i n p a t i e n t s 4. who a r e r e c e i v i n g o t h e r a n t i e p i l e p t i c drugs ( e . g . p h e n o b a r b i t a l , p h e n y t o i n , c a r b a m a z e p i n e and p r i m i d o n e ) and the h a l f - l i f e i s d e c r e a s e d a l m o s t t w o - f o l d ( 2 4 , 2 5 ) . T h i s i s t h o u g h t to be due to the i n d u c t i o n o f the h e p a t i c d r u g - m e t a b o l i z i n g enzymes by t he se a n t i e p i l e p t i c d r u g s . C o n s i d e r i n g the magn i tude o f the t o t a l p lasma c l e a r a n c e o f v a l p r o i c a c i d and the l i v e r b l o o d f l o w , K l o t z and A n t o n i n (18) f o u n d the l i v e r e x t r a c t i o n r a t i o o f v a l p r o i c a c i d t o be l o w e r than 0.1 ( o r t h e serum f r e e f r a c t i o n i n p l a s m a ) . T h i s i n d i c a t e s t h a t i t i s c l e a r e d i n d e p e n d e n t o f b l o o d f l o w and t h a t o n l y the unbound d rug can undergo m e t a b o l i s m . M e t a b o l i s m M e t a b o l i s m i s t h e ma jo r r o u t e o f e l i m i n a t i o n o f v a l p r o i c a c i d . Renal e x c r e t i o n o f unchanged d rug a c c o u n t s f o r 1 - 4% o f t h e a d m i n i s t e r e d dose ( 2 2 ) . Four ma in m e t a b o l i c pathways have been p roposed based on m e t a b o l i t e s o f v a l p r o i c a c i d found i n human u r i n e (26 - 3 0 ) , r a t u r i n e (30 - 32) and the serum o f s e v e r a l s p e c i e s i n c l u d i n g human ( 2 7 , 33 - 3 6 ) . These may be summar ized ( F i g . 1) as f o l l o w s : a) C o n j u g a t i o n o f v a l p r o i c a c i d w i t h g l u c u r o n i c a c i d . b) B e t a - o x i d a t i o n o f v a l p r o i c a c i d t o 2 - p r o p y l - 3 - k e t o p e n t a n o i c a c i d ( 3 - k e t o VPA) w i t h 2 - p r o p y l - 2 - p e n t e n o i c a c i d ( 2-ene 'VPA ) and 2 - p r o p y l - 3 - h y d r o x y p e n t a n o i c a c i d (3-OH VPA) as i n t e r m e d i a t e p r o d u c t s i n a n a l o g y to b i o c h e m i c a l pathways o f f a t t y a c i d b e t a -o x i d a t i o n . c ) O m e g a - o x i d a t i o n o f v a l p r o i c a c i d l e a d i n g to f o r m a t i o n o f 2 - p r o p y l -g l u t a r i c a c i d ( 2 - P r G l u A ) w i t h 2 - p r o p y l - 4 - p e n t e n o i c a c i d (4-ene 'VPA ) and 2 - p r o p y l - 5 - h y d r o x y p e n t a n o i c a c i d (5-0HVPA) as i n t e r m e d i a t e p r o d u c t s . CH 3 -CH 2 -CH 2 CH 3 -CH 2 -CH 2  VPA glucuronlde ^CHCOOGLu ^ Dlene VPA CH 2-CH-CH 2 C H 3 - C H 2 - C H ^ CH 3 -CH 2 -CH 2 4-ene VPA CHCOOH OH * CH 2 -CH 2 -CH 2 CHCOOH C H 3 " " C H 2 - C H 2 " 5-OH VPA H00C-CH 2-CH 2 \ CH 3 -CH 2 -CH 2 CHCOOH 2-Propy lg lutar ic ac id CHCOOH ->^£: VPA (W-l) -oxtdation CH,-CH-CH. C H j - C ^ - C H ^ 3-ene VPA CHCOOH I OH CH 3-CH-CH 2 CHCOOH CH 3 -CH 2 -CH^ 4-OH VPA CH,-CH,-0< C H 3 - C H 2 - C H 2 / C-COOH 2-ene VPA ',OH CH--CH.-CH \ C H j - C H g - C H ^ 3-OH VPA CH-COOH C H 3 - C H 2 - C X / CH 3 -CH 2 -CH 2 CHCOOH 3-Keto VPA F i g . 1 Proposed metabolic pathways of V a l p r o i c Acid (VPA) in .man, 6. d) (<D - 1) or penultimate carbon o x i d a t i o n o f v a l p r o i c a c i d forming 2-propyl-4-hydroxypentanoic a c i d (4-OH VPA) with 2-propyl-3-pentenoic a c i d as an intermediate product. Although v a l p r o i c a c i d i s a medium chain f a t t y a c i d , i t s metabolism i s quite complex because of i t s branched chain which prevents the removal of successive pairs of carbon atoms o c c u r r i n g as i n the beta-ox i d a t i o n o f f a t t y acids (37). I d e n t i f i c a t i o n of metabolites of v a l p r o i c a c i d i s hindered by the chemical l a b i l i t y o f the metabolites as well as the s t r u c t u r a l s i m i l a r i t y of v a l p r o i c a c i d metabolites to some endogenous f a t t y acids (16). A number of v a l p r o i c a c i d - r e l a t e d compounds have been reported to be present i n b i o l o g i c a l f l u i d s of v a l p r o i c a c i d - t r e a t e d p a t i e n t s or experimental animals (13, 16). I t i s not c e r t a i n whether these are true metabolites o f v a l p r o i c a c i d or endogenous compounds produced i n s i g n i f i c a n t amounts by v a l p r o i c a c i d i n t e r f e r e n c e with normal biochemical degradative pathways i n c l u d i n g f a t t y acid o x i d a t i o n (13). I n t e r e s t i n v a l p r o i c a c i d metabolites .has been st i m u l a t e d by the p o s s i b i l i t y that thevformation of metabolites would explain- the l a t e onset of t h e r a p e u t i c e f f e c t observed i n some c-linical: s i t u a t i o n s (38,39) and animal experiments (40, 41). Metabolites may a l s o e x p l a i n the c a r r y -over e f f e c t s of v a l p r o i c a c i d when plasma t o t a l v a l p r o i c a c i d concentrations are low (40). Among other p o s s i b l e explanations f o r these temporal e f f e c t s i s a delay in formation of a c t i v e metabolites or the slow accumulation of a c t i v e metabolites of longer plasma h a l f -l i v e s . Of f u r t h e r i n t e r e s t i n e l u c i d a t i o n of the mechanism o f v a l p r o i c a c i d a c t i o n i s the e f f e c t of metabolites on brain GABA l e v e l s since prolonged a c t i v i t y o f v a l p r o i c a c i d has been observed a f t e r GABA l e v e l s had d e c l i n e d from peak values (.42). 7. S i d e e f f e c t s , and t o x i c i t y . S i d e e f f e c t s a s s o c i a t e d w i t h the use o f v a l p r o i c a c i d have g e n e r a l l y been mi ld . . ' . N a u s e a , v o m i t i n g , ' a b d o m i n a l cramps and d i a r r h e a a r e the most commonly o b s e r v e d s i d e . e f f e c t s ( 4 , 5 ) . . R e c e n t l y t h e r e have been r e p o r t s o f p a n c r e a t i t i s ( 4 3 ) , t h r o m b o c y t o p e n i a ( 4 4 ) , p l a t e l e t d y s f u n c t i o n ( 4 5 ) , h y p e r g l y c i n e m i a and hyperammonemia (46) a s s o c i a t e d w i t h v a l p r o i c a c i d t h e r a p y i n e p i l e p t i c p a t i e n t s . One s t r i k i n g t o x i c i t y e f f e c t i s the i n c r e a s e i n h e p a t i c enzyme l e v e l s i n serum ( 4 7 ) . T h i s h e p a t i c a b n o r m a l i t y however d i s a p p e a r s when v a l p r o i c a c i d dosage i s r e d u c e d . The more s e r i o u s s i d e e f f e c t o f v a l p r o i c a c i d t h e r a p y i s t h e h e p a t i c t o x i c i t y (48 - 5 1 ) . To da te t h e r e 0 have been o v e r twen t y dea ths due to h e p a t i c f a i l u r e ( 5 2 ) . The o v e r a l l h e p a t o c y t e i n j u r y p roduced by v a l p r o i c a c i d i n p a t i e n t s o r r a t s have been r e p o r t e d t o be s i m i l a r t o a R e y e - l i k e syndrome ( 4 8 , 5 0 ) . p r o d u c e d by t r e a t i n g r a t s w i t h h y p o g l y c i n o r i t s a n a l o g u e , 4 - p e n t e n o i c a c i d , a s h o r t - c h a i n f a t t y a c i d ( 5 3 , 5 4 ) . The i d e n t i f i c a t i o n o f 2-ene VPA, 3 - e n e V P A a n d 4-ene VPA, m e t a b o l i t e s o f v a l p r o i c a c i d w h i c h show t h e c l o s e s t r e s e m b l a n c e t o 4 - p e n t e n o i c a c i d , has l e n t s u p p o r t t o the i d e a t h a t t o x i c i t y s t u d i e s o f v a l p r o i c a c i d m e t a b o l i t e s may h e l p f i n d t h e p r i m a r y cause o f t h e h e p a t i c f a i l u r e . M u l t i p l e dose p h a r m a c o k i n e t i c s t u d i e s The c l i n i c a l e f f e c t i v e n e s s o f v a l p r o i c a c i d i n t r e a t m e n t o f s e i z u r e s i s w e l l e s t a b l i s h e d b u t many q u e s t i o n s r ema in a b o u t t h e c o r r e l a t i o n o f v a l p r o i c a c i d d o s e , serum c o n c e n t r a t i o n s o f v a l p r o i c a c i d and i t s m e t a b o l i t e s , s i d e e f f e c t s a s s o c i a t e d w i t h v a l p r o i c a c i d and m e t a b o l i t e s and management o f s e i z u r e c o n t r o l . Serum c o n c e n t r a t i o n s 8. of v a l p r o i c a c i d are monitored in v a l p r o i c a c i d therapy and knowledge o f i t s d i s p o s i t i o n i s needed f o r i n t e r p r e t a t i o n o f the serum concen-t r a t i o n data. R a t i o n a l i z a t i o n o f the data has not been easy s i n c e other a n t i e p i l e p t i c drugs are o f t e n included i n the dosage regimen. One e f f e c t n o t i c e d i n the i n t e r a c t i o n o f v a l p r o i c a c i d with other a n t i -e p i l e p t i c drugs i s the greater f l u c t u a t i o n of plasma concentrations o f v a l -p r o i c .acid (1.6,24) .• -Most' o f the m u l t i p l e dose k i n e t i c studies o f v a l p r o i c a c i d have been done with patients who r e c e i v e other a n t i e p i l e p t i c drugs. As a consequence l i t t l e i s known about the basic e l i m i n a t i o n k i n e t i c s of v a l p r o i c a c i d i n humans during m u l t i p l e dosing with v a l p r o i c a c i d alone (22, 55, 56). Other a n t i e p i l e p t i c drugs such as carbamazepine, phenobarbital, phenytoin and primidone have been reported to show d i s t i n c t d i f f e r e n c e s between t h e i r steady s t a t e k i n e t i c s and s i n g l e dose k i n e t i c s (57 - 59). This i s thought to be due to the i n d u c t i o n of the mixed fu n c t i o n oxidases by these compounds. Phenytoin also shows a dose-dependent metabolism when high doses are administered (59). In the case o f v a l p r o i c a c i d , terminal h a l f - l i v e s of 15.9 ± 2.6 hr in a s i n g l e dose study and 17.3 ± 3.0 hr i n a m u l t i p l e dose study were reported by Gugler et al (22). They als o reported t h a t steady s t a t e plasma concentrations of v a l p r o i c a c i d in healthy volunteers were about 20% lower than pred i c t e d values from s i n g l e dose s t u d i e s . Recently Bowdle et a l (56) have reported that t o t a l body clearance o f v a l p r o i c a c i d decreased 20% from 8.33 ± 2.44 ml/hr/kg to 6.67 ± 1.25 ml/hr/kg between a s i n g l e dose study and a 500 mg/day m u l t i p l e dose study. However t o t a l body clearance was unchanged between the 500 mg/day dose study and a 1000 mg/day dose study because the e f f e c t of an 9. i n c r e a s e i n mean f r e e f r a c t i o n i n plasma at the higher dose regimen was counteracted by a decrease i n i n t r i n s i c clearance from 114 ±-43~J ml/ hr/kg to 72 .0 ± 20 .8 ml/hr/kg. Between the 1000 mg/day dose study and the 1500 mg/day dose study, they reported that there was no change i n i n t r i n s i c c learance but the t o t a l body clearance increased 21% r e l a t i v e to the 1000 mg/day dose r e s u l t s due mainly to the e f f e c t o f an increase in the fre e f r a c t i o n i n plasma. From the studies reported ( 2 2 , 5 6 ) , i t appears that the e l i m i n a t i o n k i n e t i c s of v a l p r o i c a c i d a f t e r a s i n g l e dose are changed during m u l t i p l e dosing c o n t r a r y to r e s u l t s o f Brum' ejt al_ ( 55 ) who reported no d i f f e r e n c e between the s i n g l e dose and m u l t i p l e dose e l i m i n a t i o n k i n e t i c s . Use o f s t a b l e - i s o t o p e s . The e l i m i n a t i o n k i n e t i c s o f v a l p r o i c a c i d oan be studied in a pa t i e n t without d i s c o n t i n u i n g therapy and r i s k i n g the exacerbation of seizures by using a s t a b l e - i s o t o p e l a b e l l e d analogue of v a l p r o i c a c i d . The use o f the l a b e l l e d analogue allows m u l t i p l e dose a d m i n i s t r a t i o n o f un l a b e l l e d v a l p r o i c a c i d to be c a r r i e d on i n the normal f a s h i o n . The use of a s t a b l e - i s o t o p e l a b e l l e d v a l p r o i c a c i d i n a multiple-dose e l i m i n a -t i o n k i n e t i c study of v a l p r o i c a c i d w i l l e l i m i n a t e i n c l u s i o n o f other a n t i e p i l e p t i c drugs which could i n t e r a c t with v a l p r o i c a c i d . The terminal decay phase of a s i n g l e dose of l a b e l l e d v a l p r o i c a c i d , administered i n the presence of u n l a b e l l e d v a l p r o i c a c i d i n serum, presents the ideal s i t u a t i o n to study the e l i m i n a t i o n k i n e t i c s at steady s t a t e . Normally drug therapy i s discontinued to determine the e l i m i n a t i o n phase in plasma at steady s t a t e (22) because the e l i m i n a t i o n phase i n plasma i s f r e q u e n t l y i l l - d e f i n e d when the phase i s determined between the short dosing i n t e r v a l times of m u l t i p l e dose therapy (44). 10. Use o f a s i n g l e dose o f s t a b l e - i s o t o p e l a b e l l e d drug to study the e l i m i n a t i o n k i n e t i c s of u n l a b e l l e d drugs a t steady s t a t e has been a p p l i e d to methadone (60), propoxyphene (61), phenobarbital (62) and carbamazepine (63). This technique which requires the a n a l y t i c a l p o t e n t i a l o f gas l i q u i d chromatography mass spectrometry (GCMS) has a l s o been used by Von Unruh et al (64) to study the e l i m i n a t i o n k i n e t i c s o f v a l p r o i c a c i d under steady s t a t e c o n d i t i o n s in patients on combined a n t i e p i l e p t i c drug therapy. Di-(2,3- H ^ - p r o p y l ) a c e t i c a c i d was the deuterium-labelled analogue used. The p o s i t i o n f o r the deuterium l a b e l l i n g in v a l p r o i c a c i d i s important s i n c e o x i d a t i o n occurs a l l along the carbon chain of the propyl groups. Von Unruh et al (64) did not r e p o r t the metabolic 2 isotope e f f e c t s observed i n Di-(2,3- H ^ - p r o p y l ) a c e t i c a c i d . In general the deuterium-labelled compound which shows a s i g n i f i c a n t b i o l o g i c a l isotope e f f e c t cannot be used i n a pharmacokinetic study. Isotope e f f e c t s produced by d i f f e r e n c e s i n the absorption and d i s t r i b u t i o n which include binding to macromolecules are g e n e r a l l y small compared to d i f f e r e n c e s in metabolism o f u n l a b e l l e d and deuterium-labelled drug (65). The absorption and d i s t r i b u t i o n processes i n v o l v e p h y s i c a l or r e y e r s i b l e i n t e r a c t i o n s whereas metabolism involves chemical forces i n cleavage of bonds at m e t a b o l i c a l l y a c t i v e s i t e s . The deuterium atoms should also be l o c a t e d at n o n - l a b i l e p o s i t i o n s to reduce l o s s of deuterium i n vivo and during the a n a l y s i s o f deuterated v a l p r o i c a c i d i n b i o l o g i c a l f l u i d s . Owing to the degree o f u n c e r t a i n t y in i d e n t i f i c a t i o n o f v a l p r o i c a c i d m e t a b o l i t e s , confirmation o f metabolites by appropriate s t a b l e - . isotope l a b e l l i n g s t u d i e s i s needed. The use of s t a b l e - i s o t o p e n . l a b e l l e d drugs to f a c i l i t a t e i d e n t i f i c a t i o n o f metabolites i n body f l u i d s has been widely a p p l i e d (66 - 70). Usually an equimolar mixture o f u n l a b e l l e d and s t a b l e - i s o t o p e l a b e l l e d drug i s administered and b i o l o g i c a l f l u i d s are examined by mass spectrometry f o r c h a r a c t e r i s t i c ion doublets i n d i c a t i v e of drug-derived metabolic products. Drug monitoring and s a l i v a drug measurement The therapeutic e f f e c t s o f v a l p r o i c a c i d and other a n t i e p i l e p t i c drugs have been optimized i n patients by monitoring drug l e v e l s i n serum. The value of measuring s a l i v a v a l p r o i c a c i d concentrations has not been determined. S a l i v a sampling o f f e r s a s a f e , convenient, a c c e s s i b l e and non-invasive a l t e r n a t i v e to blood sampling. The usefulness o f s a l i v a r y drug monitoring i n drug treatment has been reviewed by several authors (71 - 73). S a l i v a drug measurement can be useful i f there i s a d i r e c t p r o p o r t i o n a l i t y betwen serum drug l e v e l s and s a l i v a drug l e v e l s . Of p a r t i c u l a r i n t e r e s t i s that s a l i v a drug concentrations seem to r e f l e c t changes i n the pharmacologically a c t i v e , free drug f r a c t i o n in serum. I t has been found that drug l e v e l s i n serum and mixed s a l i v a f o r some commonly used a n t i e p i l e p t i c drugs, ethosuximide (75, 75), phenytoin (74 - 77), phenobarbital (74 - 77), carbamazepine (74, 75), and primidone (77) are h i g h l y c o r r e l a t e d . S a l i v a r y l e v e l s of phenytoin, carbamazepine and ethosuximide have a l s o been reported to r e f l e c t the pharmacologically a c t i v e f r e e drug f r a c t i o n i n plasma (74 - 77). In general serum concentrations can be p r e d i c t e d from s a l i v a r y drug concen-t r a t i o n s without c o r r e c t i n g f o r s a l i v a pH f o r a c i d i c drugs with pKa values greater than 8.5 and f o r basic drugs with pKa-values'1 ess than 5.5 (71). V a l p r o i c a c i d i s a weak a c i d with a pKa of 4.8 ± 0.1 (3) and a high degree of plasma protein b i n d i n g . I t i s l a r g e l y i o n i z e d at blood pH and s a l i v a v a l p r o i c a c i d l e v e l s are expected to be i n f l u e n c e d by s a l i v a pH. The s a l i v a l e v e l s of v a l p r o i c a c i d appear to be much lower than the free f r a c t i o n i n serum (22, 74). S a l i v a v a l p r o i c a c i d l e v e l s are so much lower than serum drug concentrations that most a n a l y t i c a l methods developed f o r assay o f v a l p r o i c a c i d in serum would not be s e n s i t i v e to s a l i v a drug l e v e l s . Thus a more r e l i a b l e and s e n s i t i v e assay i s needed to determine both s a l i v a and serum v a l p r o i c a c i d l e v e l s f o r long periods o f time a f t e r dosing. A n a l y t i c a l methods The p r i n c i p a l methods f o r v a l p r o i c a c i d assay i n serum i n v o l v e g a s - l i q u i d chromatography with f l a m e - i o n i z a t i o n d e t e c t i o n . A n a l y s i s has been c a r r i e d out a f t e r a s i n g l e e x t r a c t i o n step i n the u n d e r i v a t i z e d form o f the a c i d using h i g h l y p o l a r s t a t i o n a r y phases (78 - 80). Other procedures have employed extensive p u r i f i c a t i o n o f the o r i g i n a l b i o l o g i c a l e x t r a c t (81) which increases the time of a n a l y s i s . Increas-ing the s e n s i t i v i t y of the assay by evaporating the e x t r a c t i o n s o l v e n t i s hazardous due to the v o l a t i l i t y o f v a l p r o i c a c i d (16). The gas chromatographic p r o p e r t i e s of v a l p r o i c a c i d have been improved i n gas chromatography assays by forming methyl e s t e r d e r i v a t i v e s (82), t r i -m e t h y l s i l y l (TMS) e s t e r d e r i v a t i v e s (83) and phenylacyl e s t e r derivatives C84). Aside from GLC-FID methods, an enzyme immunoassay f o r v a l p r o i c a c i d has been reported with de t e c t i o n l i m i t s o f 1.5 mg/L (82). A high performance l i q u i d chromatography a n a l y s i s of v a l p r o i c a c i d as i t s phenacyl este r has been developed by Gupta e t al (85). Concentrations o f v a l p r o i c a c i d i n serum have been determined a f t e r forming the phenacyl e s t e r by gas chromatography with e l e c t r o n - c a p t u r e d e t e c t i o n . The l i m i t o f d e t e c t i o n o f the assay was reported to be as low as 100 pg o f v a l p r o i c a c i d (85). A s e n s i t i v e chemical i o n i z a t i o n - mass spectrometric method using the d i r e c t i n s e r t i o n probe has been used to qu a n t i t a t e v a l p r o i c a c i d i n serum (87). The a p p l i c a t i o n o f gas chromatography mass spectrometry f o r v a l p r o i c a c i d a n a l y s i s in serum has been r e c e n t l y used to qu a n t i t a t e 2 2 v a l p r o i c a c i d and v a l p r o i c a c i d - [Di-(2,3- H ^ - p r o p y l ) a c e t i c a c i d ] as t h e i r methyl es t e r s i n the se l e c t e d ion monitoring mode (64). The mass spectra of methyl es t e r s o f v a l p r o i c a c i d and i t s deuterium-labelled analogues are dominated by intense ions a t low masses. The s e n s i t i v i t y and s p e c i f i c i t y of GCMS methods f o r v a l p r o i c a c i d based on s e l e c t e d ion monitoring can be improved by forming t r i a l k y l s i l y l e s t e r s o f v a l p r o i c a c i d . T r i m e t h y l s i l y l d e r i v a t i v e s o f f a t t y acids are the most popular t r i a l k y l s i l y l d e r i v a t i v e s (88). However there has been an in c r e a s i n g use of t e r t i a r y b u t y l d i m e t h y l s i l y l x h l o r i d e as a d e r i v a t i z a t i o n agent to form t e r t i a r y b u t y l d i m e t h y l s i l y l (t-BDMS) d e r i v a t i v e s a f t e r the report (89) that t-BDMS reagent could be used to form t-BDMS ethers to pro t e c t the hydroxy! groups i n pro s t a g l a n d i n s . The t-BDMS d e r i v a t i v e s o f f a t t y acids have been reported to have c h a r a c t e r i s t i c s of good a n a l y t i c a l value (90 - 92). Compared with t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s , t-BDMS d e r i v a t i v e s have much higher h y d r o l y t i c s t a b i l i t y (91, 92). The t-BDMS d e r i v a t i v e s have a strong d i r e c t i n g i n f l u e n c e on the fragmentation o f molecular ions of the f a t t y a c i d d e r i v a t i v e s to provide very intense ions at the high mass regions 14. of the mass s p e c t r a . The electron-impact mass spectra of t-BDMS d e r i v a -t i v e s of f a t t y a c i d s u s u a l l y contain intense [M-57] + ions formed by the l o s s of the r e l a t i v e l y s t a b l e t - b u t y l r a d i c a l . In comparison, the e l e c -tron-impact mass spectra of TMS d e r i v a t i v e s of f a t t y a c i d s contain l e s s intense [M-15] + ions formed by l o s s o f a methyl r a d i c a l (92). Gas chromatograph mass spectrometry a l s o c o n s t i t u t e s one of the most r e l i a b l e methods f o r the a n a l y s i s of metabolites of v a l p r o i c a c i d . The polar nature o f the metabolites d i c t a t e s the formation o f d e r i v a t i v e s to enhance the v o l a t i l i t y and s t a b i l i t y o f the metabolites i n the GCMS assay. A number o f i n v e s t i g a t o r s have reported that some hydroxy-acid metabolites form degradation products i n c l u d i n g unsaturated a c i d s when analyzed u n d e r i v a t i z e d (33, 35, 83). The m e t a b o l i t e , 3-keto VPA, i s de-graded i n t o complex products i n c l u d i n g a decarboxylated product when urine and serum e x t r a c t s are analyzed u n d e r i v a t i z e d on polar columns such as 3% OV-17 or Carbowax 6000 (28, 83). Most of the studies on the a n a l y s i s of v a l p r o i c a c i d metabolites have been c a r r i e d out f o l l o w i n g d e r i v a t i z a t i o n with TMS reagents (16). In one study (26), v a l p r o i c a c i d metabolites were i d e n t i f i e d from methylated u r i n e e x t r a c t s . O b j e c t i v e s . Confirmation o f s p e c i f i c metabolites of v a l p r o i c a c i d ( e s p e c i a l l y isomeric compounds) i n the a c i d i c f r a c t i o n of human serum and u r i n e e x t r a c t s has not been p o s s i b l e i n some studies (33, 34, 93) due to. 7 lack o f r e f e r e n c e compounds. In t h i s study, reported metabolites and new metabolites are to be synthesized. Mass spectra of the synthesized com-pounds and t h e i r d e r i v a t i v e s w i l l be used to e s t a b l i s h s t r u c t u r e - s p e c t r a c o r r e l a t i o n s which should shed some l i g h t on the fragmentation pathways. S t a b i l i t y studies w i l l a l s o be c a r r i e d out with the synthesized compounds In the present study, a d e u t e r i u m - l a b e l l e d analogue o f v a l p r o i c 15. a c i d , D i - ( 3 - 2 H 3 - p r o p y l ) a c e t i c a c i d , I I , i s to he synthesized with the C 2 H 3 CH 2 C H 2 \ ^ ^ C H COOH II deuterium atoms l o c a t e d only at the terminal carbons of the propyl chains o f d i p r o p y l a c e t i c a c i d . The p o s i t i o n f o r the deuterium l a b e l l i n g was thought to be ideal f o r pharmacokinetic and metabolism stu d i e s i n view o f the f a c t that c o-oxidation i s reported to be a minor metabolic pathway. The uniqueness of the present study i s that the deuterium-l a b e l l e d v a l p r o i c a c i d and u n l a b e l l e d v a l p r o i c a c i d are to be given to the same healthy volunteer f i r s t as a s i n g l e o r a l dose and then under m u l t i p l e dose c o n d i t i o n s . The s i n g l e dose study w i l l allow the b i o l o g i c a l isotope e f f e c t s of the deuterated v a l p r o i c a c i d to be examined by determining serum 2 t o t a l and f r e e l e v e l s o f v a l p r o i c a c i d and v a l p r o i c a c i d - Hg. Urinary 2 metabolites of v a l p r o i c a c i d and v a l p r o i c a c i d - Hg in the s i n g l e dose study w i l l be analyzed f o r i n v e s t i g a t i o n o f the metabolic isotope e f f e c t s r e s u l t i n g from the i n c o r p o r a t i o n o f s i x deuterium atoms i n t o the v a l p r o i c a c i d molecule. 2 Serum and s a l i v a l e v e l s o f v a l p r o i c a c i d and v a l p r o i c a c i d - Hg are to be analyzed by GCMS using s e l e c t e d ion monitoring. The assay procedure has been developed by Abbott e t al (94) and invol v e s monitor-ing the intense [M-57j + ion of the t-BDMS d e r i v a t i v e s . The s p e c i f i c i t y , s e n s i t i v i t y and p r e c i s i o n o f the assay w i l l be a p p l i e d to determine 2 the e l i m i n a t i o n phase of v a l p r o i c a c i d - Hfi i n s a l i v a and serum i n the 1 6 . p r e s e n c e o f r e l a t i v e l y h i g h l e v e l s o f t h e u n d e u t e r a t e d v a l p r o i c a c i d i n the m u l t i p l e dose s t u d y . The e f f e c t s o f s t i m u l a t i n g s a l i v a e x c r e t i o n on t h e s a l i v a d rug l e v e l s w i l l be b r i e f l y s t u d i e d . Serum and u r i n e samp les a r e a l s o a n a l y z e d f o r m e t a b o l i t e s o f u n l a b e l l e d and l a b e l l e d v a l p r o i c a c i d by GCMS, a f t e r f o r m i n g e i t h e r m e t h y l d e r i v a t i v e s , t-BDMS d e r i v a t i v e s o r TMS d e r i v a t i v e s o f t h e b i o l o g i -c a l e x t r a c t s . By c o m b i n i n g °the d a t a o b t a i n e d f rom r e c o r d i n g t h e mass s p e c t r a o f t o t a l i o n chromatogram p e a k s , f rom r e c o g n i t i o n o f i o n d o u b l e t s And f rom r e t e n t i o n t imes o f s y n t h e s i z e d r e f e r e n c e compounds, t he m e t a b o l i t e p a t t e r n o f v a l p r o i c a c i d i s to be e x a m i n e d . I I . EXPERIMENTAL 17. A. REAGENTS AND MATERIALS Chemicals were reagent grade and purchased from the f o l l o w i n g sources: 1. ABBOTT LABORATORIES (Montreal, Quebec) V a l p r o i c a c i d (Depakene Syrup, 50 mg/ml) was donated f o r the m u l t i p l e dose study. 2. MERCK SHARP and DOHME CANADA LTD (Montreal , Quebec) 1-Bromopropane 3,3,3-dg, p u r i t y 98 atom % deuterium. Deuterium oxide, p u r i t y 99.8 atom % deuterium. 3. NUTRITIONAL BIOCHEMICALS CORPORATION (Cle v e l a n d , Ohio) Octanoic a c i d 4. CALEDONE (Georgetown, Ontario) Ethyl acetate d i s t i l l e d - i n - glass grade n-Hexane, d i s t i l l e d - i n - glass grade Methanol, d i s t i l l e d - i n - glass grade 5. AMERICAN SCIENTIFIC and CHEMICAL ( S e a t t l e , Washington) Concentrated s u l f u r i c a c i d Concentrated h y d r o c h l o r i c a c i d Phosphorus oxychloride Potassium hydroxide Sodium hydroxide 6. GENERAL CHEMICAL DIVISION (Morristown, New Jersey) Potassium cyanide 7. ALDRICH CHEMICAL COMPANY (Milwaukee, Wisconsin) 1 - Bromopropane 3 - Bromopropanol n - Butyl 1 ithiurn (1.6M inhexane) Calcium hydride Di isopropylamine Ethyl c h loroacetate Ethyl 3-chloropropionate 3- Chloro-l-propene 2-Ethoxyethanol 4- Heptanone Hexamethylphosphorami de N-Isopropylcyclohexylami ne Lithium aluminium hydride N-Methyl-N-nitroso-p-toluenesulfonamide ( D i a z a l d ) Sodium hydride (50% o i l d i s p e r s i o n ) Tetrahydrofuran 8. BRITISH DRUG HOUSE LTD (Poole, U.K.) Diethylmal onate Propionaldehyde Propionyl Chloride V a l e r i c a c i d 9. EASTMAN KODAK COMPANY (Rochester'., New York) 2- Bromovaleric a c i d Ethyl acetoacetate 3- Iodo-l-propene 19. 10. MATHESON COLEMAN and BELL (Norward, Ohio) Phosphorus pentoxide Propylene oxide 11. APPLIED SCIENCE LABORATORIES (State College, Pennsylvania) t-BDMS reagent; 1 mM t e r t - b u t y l d i m e t h y l c h l o r o s i l a n e and 2.5 mM imidazole i n 1 ml dimethylformamide 3% OV-17 on 100/120 Gas Chrom Q 12. PIERCE CHEMICAL COMPANY (Rockford, I l l i n o i s ) TMS reagent: 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 . 13. SUPELCO INC. ( B e l l a f o n t e , Pennsylvania) 37o Dexsil 300 on 100/120 Supelcoport 10% SP-215PS on 100/120 Supelcoport Dimethyldichloros i l ane 14. SIGMA CHEMICAL COMPANY (St. Lou i s , Missouri) C e l l u l o s e d i a l y s i s membranes with molecular weight c u t - o f f o f 10,000 - 12,000. B. METHODS 1 . Synthesis Nuclear Magnetic Resonance (NMR) Spectra were recorded using a Varian XL-100 or -80 spectrometer with deuterated chloroform as s o l v e n t and t e t r a m e t h y l s i l a n e as the i n t e r n a l marker. Infr a r e d (IR) spectra were obtained neat on sodium c h l o r i d e disks using a Perkin-Elmer IR 710B spectrometer. Electron-Impact (EI) mass spectra (80 eV) were recorded with a Varian MAT-111 spectrometer. P r e c i s e isotope 20. composition of deuterated a c i d s was c a l c u l a t e d by GCMS a n a l y s i s of the t-BDMS d e r i v a t i v e s of vdeuterated a c i d s . Melting points (mp) were determined with a Thomas Hoover C a p i l l a r y Melting Point Apparatus and are uncorrected. B o i l i n g points (bp) determined are uncorrected. The I n f r a r e d and Nuclear Magnetic Resonance s p e c t r a of synthesized compounds are shown i n the-Appendix. 2 Di-(3 Hg-propyl)acetic a c i d , II Clean sodium (0.44 g, 0.019 mole), was added to 11.0 ml of absolute . alcohol ( d r i e d with i o d i n e - a c t i v a t e d magnesium) i n a 50 ml round^bottom f l a s k . The mixture was s t i r r e d and r e f l u x e d u n t i l the sodium was completely d i s s o l v e d . Diethylmalonate (2.88 g, 0.018 mole) was then added dropwise. The r e s u l t i n g s o l u t i o n was r e f l u x e d f o r 20 min and then cooled. 1-Bromopropane-3,3,3- H^ (2.5 g, 0.02 mole) was introduced drop-wise to the e s t e r enolate s o l u t i o n and the r e a c t i o n mixture r e f l u x e d f o r 2 hr u n t i l i t was almost neutral to moist l i t m u s . For the second a l k y l a t i o n step, the same amounts, as used p r e v i o u s l y , o f sodium, ethanol , and 1-bromopropane-3,3,3- H^ were added and the r e a c t i o n mixture r e f l u x e d f o r a f u r t h e r 3 hr. Sodium hydroxide s o l u t i o n (5.5 g in 16 ml of water) was added to 2 the r e a c t i o n mixture containing d i e t h y l d i p r o p y l m a l o n a t e - Hg, and the mixture r e f l u x e d f o r 8 hr. A f t e r d i s t i l l i n g o f f the ethanol at atmospheric pressure, the s o l u t i o n was d i l u t e d and cooled in an i c e -water bath. A s o l u t i o n of 9 M s u l f u r i c a c i d was added dropwise. u n t i l the pH o f the r e s u l t i n g s o l u t i o n f e l l to 2.5. A white p r e c i p i t a t e of dipropylmalonic a c i d - Hg was c o l l e c t e d by f i l t r a t i o n under s u c t i o n , and was washed with water and d r i e d under vacuum f o r 6 hr (mp 156° - 158°C). 2 The dipropylmalonic a c i d - Hfi was placed in a d i s t i l l i n g f l a s k and 21. heated at 190 - 200°C using an o i l bath, u n t i l e v o l u t i o n of carbon dioxide had ceased. The residue was d i s t i l l e d under vacuum to give 1.39 g o f v a l p r o i c a c i d - Hg, II (50% y i e l d based on diethylmalonate).. Bp 71 - 72°C at 0.5 mm. L i t e r a t u r e (95) bp 221°C a t 760 mm f o r v a l p r o i c a c i d . NMR: 6 1.1 - 1.8 complex m u l t i p l e t (m), (8H, 2CH 2~CH 2); 2.2 - 2.6, m (IH, CH); , IR: 3600 - 2600 cm"1 ( a c i d 0-H), 2910, 2880, 2845, 2178 (C-D), 1700 (C=0), 1450, 1440, 1410, 1280, 1240, 1195, 1115, 1060, 940, 790. 2-Propy1-3-hydroxypentanoic a c i d (3-OH VPA), I I I . A flame-dried 250 ml three-necked f l a s k equipped with magnetic s t i r r i n g bar, graduated separatory funnel with septum i n l e t , and mercury bubbler, was flushed with nitrogen and immersed in an ice-water bath. N-isopropylcyclohexylamine (0.05 mole) ( d r i e d with calcium hydride) i n 50 ml tetrahydrofuran was placed in the f l a s k and 31.25 ml o f 1.6 M n-bu-t y l l i t h i u m added dropwise from the f u n n e l . j n e mixture was s t i r r e d f o r 20 min and then the f l a s k immersed i n a dry ice-acetone bath. Ethyl v a l e r a t e (0.05 mole) was added dropwise f o r 10 min, followed by drop-wise a d d i t i o n o f propionaldehyde (0.05 mole). A f t e r s t i r r i n g f o r 20 min, the r e a c t i o n was quenched with 15% HC1 and the mixture allowed to a t t a i n room temperature. The product was e x t r a c t e d with ether and the aqueous phase e x t r a c t e d twice with ether. The combined ether e x t r a c t s were washed with s a t u r a t e d sodium bicarbonate followed by washings with d i s t i l l e d water. The ethereal l a y e r was d r i e d with anhydrous sodium s u l f a t e and solvents removed under vacuum using a f l a s h evaporator. F r a c t i o n a l d i s t i l l a t i o n of the residue gave 4.5 g o f ethyl 2-propyl-3-hydroxy-2 2 . p e n t a n o a t e , bp 70 - 72°C a t 0 .2 mm. : [ L i t . (96 ) bp 105°C a t 8 mm]. The e s t e r was h y d r o l y z e d w i t h 5% KOH. A f t e r a c i d i f i c a t i o n w i t h d i l u t e h y d r o c h l o r i c a c i d , t he p r o d u c t was i s o l a t e d by e t h e r e x t r a c t i o n . The e t h e r e a l l a y e r was d r i e d w i t h anhydrous sod ium s u l f a t e and s o l v e n t removed unde r vacuum. D i s t i l l a t i o n o f t h e r e s i d u e gave 3.2 g o f 2 - p r o p y l - 3 - h y d r o x y p e n t a n o i c a c i d (40% y i e l d based on e t h y l v a l e r a t e ) . Bp 115 - 120°C a t 0 . 4 mm. NMR: 6 0 . 8 - 1 . 1 , complex ( 6 H , 2 C H 3 ) ; 1.2 -1 . 9 , m ( 6 H , 3 C H 2 ) ; 2 .4 - 2 . 7 , m ( I H , C H ) ; 3.5 - 3.9 m ( I H , CH-0H) ; 7.1 -7 . 5 , b r o a d s ( I H , OH) ; 11 - 1 3 , b r o a d s ( I H , COOH). I R : 3600 - 2600 cm' ( a c i d 0-H ) , 2 9 4 0 , 2 9 0 0 , 2 8 4 8 , 1700 ( C = 0 ) , 1 4 0 0 , 1 3 7 5 , 1 2 4 0 , 1 1 9 5 , 1 1 0 0 . 2 - P r o p y l - Y - v a l e r o l a c t o n e (4-OH VPA 1 a c t o n e ) , IV C l e a n sod ium ( 0 . 0 5 6 mo le ) was added to 30 ml o f d r y e t h a n o l i n a 100 ml f l a s k . The m i x t u r e was s t i r r e d and r e f l u x e d u n t i l sod ium was c o m p l e t e l y d i s s o l v e d . D i e t h y l p r o p y l m a l o n a t e ( 0 . 0 5 0 mo le ) was then added d r o p w i s e and the s o l u t i o n r e f l u x e d f o r 20 m i n . On c o o l i n g , p r o p y l e n e o x i d e ( 0 . 0 5 6 mo le ) was added d r o p w i s e and the r e a c t i o n m i x t u r e r e f l u x e d f o r 3 h r . S a p o n i f i c a t i o n was c a r r i e d o u t by a d d i n g 20 ml o f a 25% sod ium h y d r o x i d e s o l u t i o n to t h e r e a c t i o n m i x t u r e and h e a t i n g f o r 3 h r under r e f l u x . E t h a n o l was d i s t i l l e d o f f a t a t m o s p h e r i c p r e s s u r e . The r e m a i n i n g s o l u t i o n was a c i d i f i e d w i t h 30 ml d f 8 M s u l f u r i c a c i d and d e c a r b o x y l a t i o n c a r r i e d o u t by r e f l u x i n g t h e a c i d i f i e d s o l u t i o n f o r 8 h r . A f t e r work-up and d i s t i l l a t i o n o f t he c rude p r o d u c t , 4 . 6 g o f 2 - p r o p y l - y - v a l e r o l a c t o n e was o b t a i n e d (65% y i e l d based on 2 - p r o p y l m a l o -n a t e ) . Bp 70° - 73°C a t 0 . 1 5 mm [ L i t . (28 ) bp 110 - 120°C a t 17 mm]. NMR: 6 0 . 8 - 1 . 0 , t ( 3 H , C H 3 ) ; 1.1 - 2 . 0 , complex ( 3 H , CH^-CH-O and 6H , 3 C H 2 ) ; 2 .2 - 2 . 7 , m ( I H , -CH-C=0) ; 4 .2 - 4 . 8 , m ( I H , -CH-0 ) . 2 3 . IR: 2940 c m " 1 , 2 9 0 0 , 2 8 4 5 , 1765 ( O O ) , 1 4 4 5 , 1 3 8 0 , 1 3 4 0 , 1 1 7 5 , 1 1 2 0 . 2 - P r o p y l - 6 - v a l e r o 1 a c t o n e (5-QH'VPA 1 a c t o n e ) , V C l e a n sod ium ( 0 . 0 5 7 mo le ) and 30 ml o f d r y e t h a n o l were s t i r r e d and r e f l u x e d t i l l sod ium was c o m p l e t e l y d i s s o l v e d . D i e t h y l p r o p y l -m a l o n a t e CO.052 mo le ) was added d r o p w i s e and the m i x t u r e r e f l u x e d f o r 20 m i n . A f t e r c o o l i n g , 3 - b r o m o - l - p r o p a n o l ( 0 . 0 5 7 mole ) was added d r o p w i s e and the r e s u l t i n g s o l u t i o n h e a t i n g unde r r e f l u x f o r 3 h r . E t h a n o l was d i s t i l l e d o f f a t a t m o s p h e r i c p r e s s u r e . The p r o d u c t was i s o l a t e d w i t h e t h e r . The e t h e r e a l l a y e r was d r i e d w i t h sod ium s u l f a t e and e t h e r removed under vacuum. D i s t i l l a t i o n o f the r e s i d u e gave 1 2 . 5 g o f d i e t h y l p r o p y l - 3 - h y d r o x y p r o p y l m a l o n a t e (Bp 110°C a t 0.1 mm). Sodium h y d r o x i d e s o l u t i o n ( 7 . 7 g i n 20% e t h a n o l ) was added t o the e s t e r and h y d r o l y s i s e f f e c t e d by r e f l u x i n g the m i x t u r e f o r 8 h r . A f t e r d i s t i l l i n g o f f the e t h a n o l , t he r e s i d u e was h e a t e d a t 40°C f o r 2 h r w i t h 40 ml o f w a t e r and 2 2 . 3 g o f 85% ^ P O ^ . Crude l a c t o n e - a c i d , i s o l a t e d a f t e r e t h e r e x t r a c t i o n , was d i s s o l v e d i n 20 ml t o l u e n e and 3 g p y r i d i n e . D e c a r b o x y l a t i o n was a c c o m p l i s h e d by r e f l u x i n g the s o l u t i o n f o r 6 h r . The r e a c t i o n m i x t u r e was washed w i t h w a t e r . The o r g a n i c phase was d r i e d o v e r anhyd rous sod ium s u l f a t e , t he s o l v e n t was removed by f l a s h e v a p o r a t i o n and t h e r e s i d u e d i s t i l l e d to y i e l d 3 .3 g o f 2 - p r o p y l - 6 - v a l e r o l a c t o n e (45% based on p r o p y l m a l o n i c a c i d d i e t h y l e s t e r ) . Bp 91 - 93°C a t 1.0 mm [ L i t . (28) bp 84°C a t 0 . 4 mm]. NMR: 6 0 . 8 - 1 . 1 , t ( 3 H , C H 3 ) ; 1.1 - 1 . 8 , m ( 8 H , 2 C H 2 - C H 2 ) ; 2 .2 - 2 . 6 , m ( I H , CH-C=0); 3 .8 - 4 . 3 , complex ( 2 H , - C H 2 - 0 ) . IR : 2930 c m - 1 , 2 9 0 0 , 2 8 4 0 , 1710 ( C = 0 ) , 1 4 4 5 , 1 3 8 0 , 1 3 7 5 , 1 3 4 0 , 1 1 6 0 , 1100 . 24. 2-Propy1-3-pentenoic a c i d (3-ene VPA), VI Phosphorus pentoxide (0.09 mole) was added to a mixture of e t h y l -2-propyl-3-hydroxypentanoate (0.06 mole) and 40 ml o f dry benzene. The mixture was s t i r r e d and r e f l u x e d f o r 2 hr. On c o o l i n g , the s o l u t i o n was f i l t e r e d under s u c t i o n . The s o l i d residue was washed with benzene. The f i l t r a t e was d r i e d over anhydrous sodium s u l f a t e and benzene removed by f l a s h evaporation. D i s t i l l a t i o n o f the crude product gave 8.5 g of a mixture of 6 , y-unsaturated e s t e r and a,g-unsaturated e s t e r (bp 77 - 79°C at 0.1 mm). Methanol (6.0 ml) and sodium hydroxide s o l u t i o n (4.4 g i n 20 ml water ) were added to the e s t e r and the mixture heated at 50°C f o r 3 hr. Ethanol and methanol were removed by d i s t i l l a t i o n a t 760 mm. The remaining s o l u t i o n was a c i d i f i e d with d i l u t e hydrochloride a c i d and product extracted with ether. Ether was removed by f l a s h evaporation and the residue f r a c t i o n a l l y d i s t i l l e d . The f r a c t i o n (4.5 g) with bp 89° - 92°C at 0.25 mm was c o l l e c t e d and found to be a mixture of 3-ene VPA and 2-ene VPA i n the r a t i o o f 3 : 1 by GCMS a n a l y s i s of the t-BDMS d e r i v a t i v e s and comparing peak areas. To o b t a i n a higher f r a c t i o n o f 3-eneVPA an a l t e r n a t i v e method was used. A flame-dried 250 ml three-necked f l a s k , graduated separatory funnel with septum i n l e t was flus h e d with nitrogen and immersed i n an ice-water bath. Diisopropylamine (0.018 mole) i n 20 ml tetrahydrofuran was placed i n the f l a s k and 11.5 ml o f 1.6 M n - b u t y l l i t h i u m added dropwise from funnel. The mixture was s t i r r e d f o r 20 min and the f l a s k immersed i n a dry ice-acetone bath. Hexamethylphosphoramide (0.019 mole) was added dropwise and the mixture s t i r r e d f o r 20 min. Ethyl 2-pentenoate, 0.018 mole (synthesized from ethyl 2-bromo-v a l e r a t e ) was added dropwise f o r 10 min followed by dropwise a d d i t i o n of 25. 1- bromopropane (0.02 mole). A f t e r s t i r r i n g f o r 20 min, the r e a c t i o n was quenced with 10% HC1 and the mixture allowed to a t t a i n room temperature. The product was extracted with ether and work-up c a r r i e d out as in 3-OH VPA e s t e r s y n t h e s i s . F r a c t i o n a l d i s t i l l a t i o n of the residue gave 1.5 g of ethyl 2-propyl-3-pentenoate, bp 76 - 78°C at 0.6 mm. Methanol (3 ml) and sodium hydroxide s o l u t i o n (1.1 g in 12 ml water) were added to the e s t e r and the mixture heated at 50°C f o r 2 hr. Ethanol and methanol were d i s t i l l e d o f f at 760 mm. The remaining s o l u t i o n was a c i d i f i e d with d i l u t e h y d r o c h l o r i c a c i d and product extracted with ether. Ether was d i s t i l l e d o f f using an o i l bath and the residue d i s -t i l l e d to give 0.88 g of a f r a c t i o n with bp 86° - 89°C at 1.0 mm. [ L i t . (96) bp 116°C at 8 mm f o r 3-ene VPA]. GCMS a n a l y s i s of the t-BDMS-d e r i v a t i z e d f r a c t i o n showed greater than 10 : 1 r a t i o o f 3-ene VPA to 2- ene VPA. NMR of the f r a c t i o n (see appendix); 6 0.8 - 1.1, d i s t o r t e d t r i p l e t (3H, CH 3_CH 2); 1.1 - 1.6, complex m (4H,,-CH2-CH2-); 1.6 - 2.0, d (3H, CH3-CH=); 2.1 - 2.5, m (CH2-C= from 2-ene VPA); 2.7 - 3.3, m (IH, CH-C=0), 5.2 - 5.9, complex m (2H, CH = CH); 6.8 - 7.1, t (trans CH = C from 2-ene VPA); 9.2 - 10.9, broad S (IH, COOH). 2-Propyl-2-pentenoic a c i d (2-ene VPA), VII. Hydrogen cyanide (8 ml, 0.2 mole), prepared from saturated potassium cyanide and 50% s u l f u r i c a c i d , was added to 4-heptanone (0.12 mole) conta i n i n g a few c r y s t a l s of potassium cyanide. The stoppered f l a s k c o n t a i n i n g the mixture was kept in an ice-water bath. A f t e r s t i r r i n g f o r 30 min, the s o l u t i o n was allowed to a t t a i n room temperature and s t i r r e d f o r a f u r t h e r 3 hr. A few drops of concentrated s u l f u r i c a c i d was added to the r e a c t i o n mixture and excess hydrogen 26. cyanide was removed by d i s t i l l i n g at 760 mm. F r a c t i o n a l d i s t i l l a t i o n o f the residue gave 12.8 g o f 2-propyl-2-hydroxypentanenitrile, bp 80 - 82°C a t 0.6 mm. Phosphorus o x y c h l o r i d e (15 ml) and dry p y r i d i n e Cl6 ml) were added to the cyanohydrin and the r e a c t i o n mixture s t i r r e d and heated a t 90°C f o r 1 hr over a steam bath. The r e a c t i o n f l a s k was then placed i n an ice-water bath. Ice was added to the r e a c t i o n mixture and product extracted with ether. The ethereal l a y e r was washed with d i l u t e h y d r o c h l o r i c a c i d , separated and dri e d over anhydrous sodium s u l f a t e . The ether was removed by f l a s h evaporation and residue d i s t i l l e d to give 9.1 g of 2-propyl-2-pentene-n i t r i l e , bp 52°C a t 5 mm. S u l f u r i c a c i d (60% i n water) was added to the unsaturated n i t r i l e and themixture heated under r e f l u x using an o i l bath f o r 5 hr. [ The intermediate amide was u s u a l l y i s o l a t e d a f t e r h y d r o l y s i s f o r 3 hr (mp 86 - 88°C) and f u r t h e r a c i d h y d r o l y s i s c a r r i e d out f o r 2 h r ] . The unsaturated a c i d was i s o l a t e d by ether e x t r a c t i o n . Ether was removed by f l a s h evaporation and the residue d i s t i l l e d to give 6.0 g of 2-propyl-2-pentenoic a c i d (35% y i e l d based on 4-heptanone), bp 96 - 99°C at 1.3 mm. [ L i t . (97) c i s - a c i d bp 87°C at 1.2 mm, t r a n s -a c i d bp 103°C a t 1.0 mm]. The product was found to be a mixture of c i s - and trans-2-ene VPA in the r a t i o o f 1 : 4. NMR o f the isomeric mixture: 6 0.8 - 1.1, complex m (6H, 2CH 3), 1.2 - 1.6, m (2H, CH 3-CH_ 20CH 2); 2.1 - 2.7, m (4H, CH_2-CH= and -CH_2-C,= ) 5 . 9 - 6.2, t (IH, -CH=C, c i s , weak); 6.8 - 7.1, t (IH, -CH=C, t r a n s , s t r o n g ) . IR: 3600 - 2600 cm"1 ( a c i d 0-H), 3000, 2940, 2900, 2845, 1665 (C=0), 1625 (C=C), 1450, 1410, 1375, 1280, 1245, 1220, 1160, 1110, 940 Ctrans out of plane bending, s t r o n g ) , 750 ( c i s , out o f plane bending, weak). The f r a c t i o n was stored a t -20°C and the trans 2-ene yPA o f high mp i s o l a t e d (NMR spectrum showed t r a c e amounts of cis-2-ene VPA, 27. see Appendix).-.NMR;6 0.8- 1.1, complex m (6H, 2CH 3), 1.2 - 1.7, m (2H, CKg-CHg-CHg); 2.0 - 2.5,. m (4H, CH_2-CH= and -CH 2~C=); 6.8 - 7.1, t OK, -CH=C, t r a n s ) . 2 2 3- H|, 5- H 2-3-heptene-4-carboxy1 i c a c i ' d , J J I J 4- Heptanone (5 gm) was shaken with 2 gm of 2.5 M NaOD (prepared from sodium and deuterium oxide) i n D\,0 .for 1 - 2 days and then the NaOD - D 20 l a y e r was replaced with fresh, NaOD i n D 20. The deuterium-depleted NaOD - D 20 f r a c t i o n was shaken with fresh 4-heptanone to. recover most of the remaining deuterium f o r i n c o r p o r a t i o n into 4-heptanone. The counter-current type o f deuterium e x t r a c t i o n and i n c o r p o r a t i o n i n t o 4-heptanone was c a r r i e d out a f t e r about 15 exchanges.and combined deuterated 4-heptanone f r a c t i o n s examined f o r per cent isotope enrichment. Per cent deuterium was c a l c u l a t e d by GCMS a n a l y s i s o f an ' ethereal s o l u t i o n o f the deuterated 4-heptanone using a simple i t e r a t i v e program, ISO-l and comparing to a n a l y s i s o f an.ethereal s o l u t i o n of, undeuterated 4-heptanone. -Per cent deuterium was 95.3%. 2 Hydrogen cyanide (8 ml, 0.2 mole) was added to 3,3,5,5- H^-4-.. heptanone (0.11 mole) c o n t a i n i n g a few c r y s t a l s o f KCN i n a 50 ml, stoppered f l a s k . The mixture was s t i r r e d at 0 - 4°C f o r 30 min and ., then s t i r r e d a t room temperature f o r 3 hr. A few drops of concentrated ' H 2S0^ was then added and excess HCN removed by d i s t i l l i n g at 760 mm. , The residue was d i s t i l l e d to give 12.7 g of the cyanohydrin, bp 83-,85°C at 0.8 mm. The cyanohydrin was dehydrated with phosphorus o x y c h l o r i d e (15 ml) and dry pyr i d i n e (16 ml), the reactants being heated a t 90°C f o r 1 hr. 2 I s o l a t i o n o f the unsaturated ni trSil e- ;HQ was c a r r i e d out as in the 28. synt h e s i s of 2-ene VPA. The n i t r i l e (8.5 g) was hydrolyzed to the a c i d with s u l f u r i c a c i d (60% i n H 20) f o r 5 hr. Work up as i n 2-ene VPA 2 2 synthesis gave 5.4 g o f 3- Hy 5- H 2-3-heptene-4-carboxy1ic a c i d , bp 88° - 91°C at 0.4 mm. Pure trans-2-ene VPA- 2H 3 was obtained by f r e e z i n g the c i s - t r a n s mixture at -20°C and i s o l a t i n g the s t a b l e , high-melting trans-isomer. NMR (see appendix): 6 0.8 - 1.1, complex m (6H, 2CH 3); 1.2 - 1.7, m (2H, CH 3-CH 2-CD 2); 2.0 - 2.6, quadruplet (2H, CH2>CD=); 10 - 11.8, s (IH, COOH). 2-Propyl-4-pentenoic a c i d (4-ene VPA), IX Clean sodium (0.074 mole) and 35 ml o f dry ethanol were s t i r r e d and r e f l u x e d u n t i l the sodium was completely d i s s o l v e d . To the mixture, d i e t h y l allylmalonate (synthesized from diethylmalonate), 0.070 mole was added dropwise followed by heating under r e f l u x f o r 20 min. 1-Bromo-propane (0.074 mole) was added dropwise and the r e a c t i o n mixture r e f l u x e d f o r 4 hr. A f t e r d i s t i l l i n g o f f the e t h a n o l , work up gave 15.0 g of p r o p y l a l l y l m a l o n i c a c i d d i e t h y l e s t e r (bp 110° - 112°C at 0.6 mm). To the e s t e r , methanol (10 ml) and potassium hydroxide s o l u t i o n (14 g i n 40 ml water) were added and the mixture s t i r r e d and heated under r e f l u x f o r 5 hr. Ethanol and methanol were removed by d i s t i l l a t i o n at 760 mm. The remaining s o l u t i o n was a c i d i f i e d with d i l u t e hydrochloric a c i d and product i s o l a t e d with ether e x t r a c t i o n . Ether was removed by f l a s h evaporation to leave an o i l y r esidue which c r y s t a l l i z e d slowly to give a l l y l p r o p y l m a l o n i c a c i d . The c r y s t a l s were r e c r y s t a l l i z e d from hot benzene to give c o l o u r l e s s needles of a l l y l p r o p y l m a l o n i c a c i d . The a c i d was decarboxylated as described f o r v a l p r o i c a c i d - Hg s y n t h e s i s . D i s t i l l a t i o n o f the residue gave 6.1 g of 2-propy1-4-pentenoic a c i d 2 9 . (60% y i e l d based on d i e t h y l a l l y l m a l o n a t e ) ; bp 85°C a t 1.0 mm. [ L i t . ( 98 ) bp 95 - 100°C a t 5 mm], NMR: 6 0 . 8 - 1 . 1 , t ( 3 H , C H 3 ) ; 1.1 - 1 . 4 , comp lex m, ( 4 H , - C H 2 - C H 2 h 2 .0 - 2 . 7 , complex m ( 3 H , -CJH-CH_2-CH=), 4 . 9 -5 . 3 , m ( 2 H , C H 2 = ) , 5.4 - 6 . 0 , complex m ( I H , -CH=). IR : 3600 - 2600 cm" ( a c i d 0-H ) , 3 0 4 0 , 2 9 3 0 , 2 8 9 0 , 2 8 3 0 , 1695 ( C = 0 ) , 1625 (C=C ) , 1 4 3 0 , 9 9 0 , 9 1 5 . E t h y l 2 - p r o p y l - 3 - k e t o p e n t a n o a t e , X N - i s o p r o p y l c y c l o h e x y l a m i n e ( 1 4 . 2 g , 0.1 m o l e ) , 6 2 . 5 ml o f 1.6 M b u t y l 1 i t h i u r n and 80 ml o f t e t r a h y d r o f u r a n ( d r i e d w i t h l i t h i u m a luminum h y d r i d e ) were s t i r r e d f o r 20 min a t 0 °C under n i t r o g e n . To t h e m i x t u r e c o o l e d t o -78°C w i t h d r y i c e - a c e t o n e b a t h , e thy l v a l e r a t e ( 0 . 0 5 mo le and p r o p i o n y l c h l o r i d e ( 0 . 0 5 mo le ) were added s u c c e s s i v e l y i n d r o p w i s e f a s h i o n . A f t e r s t i r r i n g f o r 20 m i n , t he r e a c t i o n was quenched w i t h 15% HC1. The e s t e r was i s o l a t e d as i n e t h y l 2 - p r o p y l - 3 - h y d r o x y p e n t a -noa te s y n t h e s i s . D i s t i l l a t i o n o f t h e i s o l a t e d c rude p r o d u c t gave 4 . 8 4 g o f e t h y l 2 - p r o p y l - 3 - k e t o p e n t a n o a t e ( y i e l d 49% based on e t h y l -v a l e r a t e ) : bp 70 - 73°C a t 0 . 3 mm. [ L i t . (28 ) bp 64 - 65°C a t 0 . 4 mm]. Mass s p e c t r u m : m/z 57 ( 1 0 0 % ) , 1 0 l ( 5 7 ) , 29 ( 4 8 ) , 73 ( 2 2 ) , 130 ( 2 1 ) , 55 ( .18), 144 ( 1 5 ) , 83 ( 1 0 ) , 115 ( 9 ) , 157 ( 9 ) , 160 ( 7 ) , 111 ( 7 ) , 43 ( 7 ) . 2 - P r o p y l - 4 - k e t o p e n t a n o a t e ( 4 - k e t o VPA),, XI Dry t e t r a h y d r o f u r a n (25 m l ) was p l a c e d i n a 50 ml f l a s k and sod ium h y d r i d e ( 1 . 2 5 g o f 50% o i l d i s p e r s o n ) a d d e d . E t h y l a c e t o a c e t a t e ( 0 . 0 2 5 mo le ) was added d r o p w i s e and t h e m i x t u r e s t i r r e d " f o r . 10. mi,n. E t h y l 2 - b r o m o v a l e r a t e ( 0 . 0 2 8 mo le ) was added d r o p w i s e and the s o l u t i o n r e f l u x e d f o r 4 h r . D i s t i l l e d w a t e r was added and the r e s u l t i n g m i x t u r e 30. f i l t e r e d under s u c t i o n . The product was extracted with ether and the ether removed by f l a s h evaporation. The residue was d i s t i l l e d t o give 3.9 g o f d i e t h y l 2 - p r o p y l - 3 - a c e t y l s u c c i n a t e , bp 98° - 102°C at 0.4 mm. A mixture o f the a c y l s u c c i n a t e , concentrated HC1 (10 ml) and water (2 ml) was heated under r e f l u x f o r 6 hr to e f f e c t h y d r o l y s i s and decar-b o x y l a t i o n . The product was extr a c t e d with ether and the ethereal l a y e r d r i e d over sodium s u l f a t e . Ether was removed by f l a s h evaporation to give a residue c o n t a i n i n g 2-propyl-4-ketopentanoic a c i d (1.35 g ) . D i s t i l l a t i o n o f the residue gave 4-keto VPA contaminated with a p p r e c i a b l e amounts of an unsaturated y - l a c t o n e ; bp 123 - 126°C at 3 mm. [ L i t . (99) bp 136 - 140°C at 3.0 mm]. NMR (see Appendix); 6 0.8 - 1.1, d i s t o r t e d t r i p l e t (6H, CH_3-CH2 from 4-keto VPA, CH_3-CH2 from 8 , y-unsaturated y -l a c t o n e ) ; 1.2 - 2.0, two complex m (11 H, CH 2-CH 2~ from 4-keto VPA, CH 2-CH 2 from y - l a c t o n e , CH 3~C(0) = from B , y-unsaturated y - l a c t o n e ) ; 2.1 - 2.2, s i n g l e t (3 H, CH3-C=0 from 4-keto VPA); 2.2 - 3.1, complex m (4 H, CH-C=0 from 4-keto VPA, CH-C=0 from y - l a c t o n e , CH2-C=0 from 4-keto VPA), 5.0 - 5.2, broad s i n g l e t (1 H, CH= from g , y-unsaturated y - l a c t o n e ) , 7.4 - 7.7 (from CHC1^ in solvent CDC1^)• Mass spectrum o f the unsaturated y - l a c t o n e : m/z 43 (100%), 97 (60), 98 (45), 55 (44), 41 (40), 83 (20), 69 (19), 27 (19), 140 (14), M+. 2 - P r o p y l g l u t a r i c a c i d , XII Clean sodium (0.032 mole) and 25 ml o f dry ethanol were s t i r r e d and r e f l u x e d u n t i l sodium was completely d i s s o l v e d . Diethyl p r o p y l -malonate (0.030 mole) was added and the s o l u t i o n r e f l u x e d f o r 20 min. Ethyl 3-chloropropionate (0.03 mole) was added dropwise and the s o l u t i o n heated under r e f l u x f o r 4 hr. On c o o l i n g , water was added and ethanol d i s t i l l e d o f f at 760 mm. The product was extracted with ether and the ether removed by f l a s h evaporation. The residue was d i s t i l l e d to give 31 . 5.1 g of t r i e t h y l 1 -propylpropane-1,1,3-tri.carhoxylate, hp 123-126°C at 0.4 mm. To the e s t e r , 5.2 g KOH i n 15 ml water and 4 ml ethanol were added and the mixture r e f l u x e d f o r 6 hr. Ethanol was d i s t i l l e d o f f a t 760 mm and the residue a c i d i f i e d with 9 M H^SO^. Following work-up the i s o l a t e d t r i c a r b o x y l i c a c i d was decarboxylated as described in VPA- Hg s y n t h e s i s . D i s t i l l a t i o n o f the residue gave 2.5 g o f 2-propyl-g l u t a r i c a c i d (48% y i e l d based on diethylpropylmalonate) bp 168°C a t 0.65 mm, mp 70°C. [ L i t . (100) mp 66° - 68°C]. NMR: 6 0.8 - 1.1, t (3H, CH 3); 1.2 - 2.1, two complex m (6H, 3CH,2); 2.2 - 2.9., complex m (3H, -CH_2-C00H and -CH-C00H); 9.5 - 11.3, broad s (2H, 2C00H). 2 - P r o p y l s u c c i n i c a c i d , XIII Clean sodium (0.026 mole) and 20 ml o f dry ethanol were s t i r r e d u n t i l sodium was d i s s o l v e d . Diethylpropylmalonate (0.024 mole) was added and the s o l u t i o n r e f l u x e d f o r 20 min. Ethyl chloroacetate (0.027 mole) was added dropwise and the s o l u t i o n r e f l u x e d f o r 4 hr. S a p o n i f i -c a t i o n o f the e s t e r and i s o l a t i o n of the d i c a r b o x y l i c a c i d were c a r r i e d out as i n the synthesis of 2 - p r o p y l g l u t a r i c a c i d . The f i n a l product was r e c r y s t a l l i zed with ether - petroleum ether to give 1.4 g of propyl s u c c i n i c a c i d (40% y i e l d based on diethylpropylmalonate, mp 93° - 96°C, [ L i t . (101) mp 94°C]. NMR: S 0.8 - 1.1, d i s t o r t e d t r i p l e t (3H, CH 3); 1.2 -1.9, complex m (4H, CH 2-CH 2); 2.3 - 2.7, m (2H, CH_2-C00H), 2.7 - 3.0, m (IH, CH_-C00H). 2-Propylmalonic a c i d , XIV Diethylpropylmalonate (0.015 mole) was s a p o n i f i e d with sodium hydroxide s o l u t i o n (2.4 g i n 10 ml water and 3 ml e t h a n o l ) . Ethanol was d i s t i l l e d o f f at 760 mm and the remaining s o l u t i o n a c i d i f i e d with 32. d i l u t e s u l f u r i c a c i d . The s o l u t i o n was extracted with ether and the ether d i s t i l l e d o f f on a water bath. R e c r y s t a l 1 i z a t i o n o f the s o l i d residue with benzene gave 1.8 g of propylmalonic a c i d ; mp 96°C , [ L i t . (102) mp 96°C). 2. Drug A d m i n i s t r a t i o n - Pharmacokinetic and Metabolism s t u d i e s . A healthy male vol u n t e e r , 41 years o f age (weight, 63.64 Kg), who had not taken any drugs during the month p r i o r to the experiment, p a r t i c i p a t e d i n the s i n g l e and m u l t i p l e dose s t u d i e s . a) S i n g l e dose study - A s i n g l e o r a l dose was prepared by d i s s o l v i n g 2 372 mg of v a l p r o i c a c i d and 182 mg of v a l p r o i c a c i d - Hg i n 100 ml o f water and the pH o f the r e s u l t i n g s o l u t i o n was adjusted to pH 7.4 using 1 N NaOH. The or a l dose was taken i n the morning a f t e r an overnight f a s t , and food was not allowed u n t i l 3 hr a f t e r dosing. Blood samples (10 ml) were taken at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 12.0, 24.0 and 48.0 hr using an i n d w e l l i n g catheter (heparin-locked) f o r the f i r s t 4 hr and t h e r e a f t e r by venipuncture. The serum o f each blood sample was sepa'rated immediately a f t e r blood sampling. S a l i v a samples'(unstimulated" 5 ml) were obtained c o i n c i d e n t to blood samples by c o l l e c t i o n v i a expectoration i n t o clean v i a l s . Urine was c o l l e c t e d a t the f o l l o w i n g times: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 - 12.0, 12.0 - 24.0 and 24.0 - 48.0 hr a f t e r dosing. The serum, s a l i v a and urine samples were kept frozen (-20°C) u n t i l they were analyzed. b) M u l t i p l e dose study - A 600 mg oral dose o f v a l p r o i c a c i d (Depakene Syrup 50 mg/ml) was given to the volunteer at 8 pm of day 1. 33. The same dose o f v a l p r o i c a c i d was administered twice a day (.8 am, 8 pm) f o r the next 3 days. On the f i f t h day, 84 hrs a f t e r the i n i t i a l dose, 600 mg o f v a l p r o i c a c i d - H g d i s s o l v e d i n 100 ml o f water (pH 7.4) was s u b s t i t u t e d f o r the v a l p r o i c a c i d dose. Thereafter v a l p r o i c a c i d (Depakene Syrup, 600 mg) was administered every 12 hr f o r a f u r t h e r period of 3 days. During the m u l t i p l e dose study, blood samples ( 5 ml) were drawn p e r i o d i c a l l y ( u s u a l l y at the trough l e v e l s ) p r i o r to the a d m i n i s t r a t i o n of the l a b e l l e d v a l p r o i c a c i d dose. Then f o l l o w i n g the v a l p r o i c a c i d -2 H, dose, blood samples were taken at 0, 0.7, 1.0, 1.6, 2.1, 2.6, 3.1, b 4.0, 5.1, 7.0, 9.0, 10.8, 15.3, 24.8, 26.7, 27.7, 29.8, 32.5, 35.3, 48.5, 59.2 and 72.9 hr. The serum of each sample was .separated and the pH immediately measured using a pH meter. Serum samples were stor e d frozen (-20°C) u n t i l analyzed. S a l i v a samples ( 2 - 5 ml) were obtained unstimulated and by s t i m u l a t i o n o f s a l i v a r y e x c r e t i o n with sucrose. For the c o r r e l a t i o n of s a l i v a and serum drug l e v e l s , unstimulated s a l i v a samples were c o l l e c t e d a t each blood sampling time and a stimulated s a l i v a sample was obtained e i t h e r 5 min before or f o l l o w i n g the blood sample c o l l e c t i o n . S a l i v a samples were als o obtained between blood sampling times. The s a l i v a sample pH was determined immediately f o l l o w i n g c o l l e c t i o n using a pH meter. S a l i v a samples were stored frozen (-20°C)until analyzed. Urine samples were c o l l e c t e d p e r i o d i c a l l y before a d m i n i s t r a t i o n 2 of the v a l p r o i c a c i d - Hg dose. Following the l a b e l l e d v a l p r o i c a c i d dose, urine was c o l l e c t e d at 0 - 4, 4 - 8, 8 - 12, 12 - 16, 16 - 24, 24 - 28, 28 - 32, 32 - 36, 36 - 40, 40 - 48, 48 - 52, 52 - 56, 56 - 60, 60 - 64, 64 - 72 hr. The u r i n e samples were kept frozen (-20°C) u n t i l 34. they were analyzed. 3. Pharmacokinetic Drug Assay a) Serum and S a l i v a Standards Serum standards of l a b e l l e d and u n l a b e l l e d v a l p r o i c a c i d were prepared by the a d d i t i o n o f 0.1 ml o f appropriate stock s b l u t i o n s (500 -6000 yg/ml) in methanol i n t o 5 ml volumetric f l a s k s . Drug-free serum 2 was added to the 5 ml mark to provide v a l p r o i c a c i d - Hg concentrations of 10, 20, 40, 60, 80 and 100 yg/ml and v a l p r o i c a c i d concentrations of 20, 40, 60, 80, 100 and 120 yg/ml. S a l i v a standards were prepared by the a d d i t i o n o f 0.1 ml of appropriate stock s o l u t i o n s (5 - 300 yg/ml) i n methanol i n t o 5 ml volumetric f l a s k s . Drug-free s a l i v a was added to the 5 ml mark to 2 provide v a l p r o i c a c i d - Hg concentrations o f 0.1, 0.25, 0.5, 1.0, 1.5, 3.0, 6.0 yg/ml and v a l p r o i c a c i d concentrations of 0.25, 0.5, 1.0, 1.5, 3.0 and 6.0 yg/ml . b) E x t r a c t i o n and D e r i v a t i z a t i o n Serum standard or sample (50 y l ) was pi p e t t e d i n t o a 1 .0 ml co n i c a l r e a c t i o n v i a l f i t t e d with a PTFE-lined cap. Internal standard (200 y l o f a s o l u t i o n of 10 yg/ml octanoic a c i d i n IN HC1) was added followed by 200 y l of so l v e n t (10% ethyl acetate i n n-hexane) and then the mixture vortex-mixed f o r 60 sec. A f t e r c e n t r i f u g i n g at 2000 rpm (1000 g) f o r 20 min, 100 - 150 y l of the hexane l a y e r was t r a n s f e r r e d to. a second v i a l and 7 y l o f t-BDMS reagent added. The mixture was vortex-mixed f o r 30 sec and then c e n t r i f u g e d f o r 2 - 5 min. An a l i q u o t (5 y l ) of the top organic l a y e r was i n j e c t e d i n t o the GLC-- mass spectrometer. 35. For s a l i v a drug a n a l y s i s , s a l i v a samples (200 y l ) were treated e x a c t l y as serum samples except that the i n t e r n a l standard (octanoic a c i d ) concentration was 6.5 yg/ml in IN HC1. c) GLC - Mass Spectrometry - Selected Ion monitoring L a b e l l e d and u n l a b e l l e d v a l p r o i c a c i d were quantitated i n serum and s a l i v a by s e l e c t e d ion monitoring. The instrument used f o r the assay was a Hewlett Packard 5700A gas chromatograph i n t e r f a c e d to a Varian MAT-111 mass spectrometer v i a a v a r i a b l e s l i t separator. The . GCMS was connected to an o n - l i n e data system Varian 620L computer. The glass column, 1.8 m x 2 mm i . d . was packed with 3% Dexsi.l 300 on 100 -120 mesh Supelcoport. I n j e c t i o n i n t o the GCMS -was done with a Hewlett Packard 761A automatic sampler. Operating c o n d i t i o n s were: i n j e c t i o n port temperature 250°C, column temperature 135°C, helium ( c a r r i e r gas) flow rate of 25 ml/min and i n l e t l i n e temperature 250°C. The mass spectrometer was operated with an e l e c t r o n i o n i z a t i o n energy of 80 eV, trap current of 300 yA, and ion source pressure of 5.0-x 10"^ T o r r . The s e l e c t e d ions were s e t up using a u t h e n t i c samples o f v a l p r o i c a c i d and v a l p r o i c - d g t-BDMS d e r i v a t i v e s . d) C a l i b r a t i o n curves. The intense m/z 201 from the t-BDMS esters o f v a l p r o i c a c i d and octanoic a c i d and the corresponding m/z 207 from the t-BDMS es t e r of 2 v a l p r o i c a c i d - Hg were monitored continuously during assay. The 2 r e t e n t i o n times o f v a l p r o i c a c i d Cor v a l p r o i c a c i d - Hg) and octanoic a c i d d e r i v a t i v e s on the 3% Dexsil 300 column were 2.0 min and 3.6 min 36. r e s p e c t i v e l y . C a l i b r a t i o n curves f o r v a l p r o i c a c i d and v a l p r o i c a c i d -2 Hg were prepared by p l o t t i n g the ion area r a t i o s o f drug and i n t e r n a l standard against the known drug concentrations i n serum or s a l i v a . For v a l p r o i c a c i d curves m/z 201 : m/z 201 area r a t i o s were used whereas 2 m/z 207 : m/z 201 area r a t i o s were used f o r v a l p r o i c a c i d - Hg curves. Li n e a r r e g r e s s i o n a n a l y s i s was used to draw the curves and concentrations 2 o f v a l p r o i c a c i d and v a l p r o i c a c i t i - Hg tn each serum or s a l i v a sample were determined from the l i n e a r equation. e) Plasma P r o t e i n binding studies Free drug l e v e l s i n serum were determined by e q u i l i b r i u m d i a l y s i s using the procedure of Abbott et al ('94'). Two standard curves were prepared f o r the a n a l y s i s . One, a lower con c e n t r a t i o n range curve o f 2 1 - 10 pg/ml of v a l p r o i c a c i d (or 1 - 10 yg/ml of v a l p r o i c a c i d - Hg) was prepared in an i s o t o n i c phosphate b u f f e r (pH 7.4). The other, an upper concentration range curve of 20 - 160 vg/ml v a l p r o i c a c i d (or 10 - 100 2 pg.ml v a l p r o i c a c i d - Hg) was prepared in drug-free serum. The serum and b u f f e r standards were analyzed by GLC - Mass Spectrometry. The e x t r a c t i o n procedure used was the same as f o r serum t o t a l drug described above except that 200 y l o f b u f f e r standard and 200 y l of i n t e r n a l standard (6.5yg/ml of octanoic a c i d in IN HC1) were used. For e q u i l i b r i u m d i a l y s i s , 0.5 ml of serum sample was i n j e c t e d i n t o one s i d e of the d i a l y s i s c e l l and 0.5 ml of i s o t o n i c b u f f e r s o l u t i o n i n j e c t e d i n t o the other side of the c e l l . The d i a l y s i s blocks were placed i n t o a 37°C water bath on a r o t a t i n g device and r o t a t e d twelve times per min. f o r 4 hrs. At the end o f 4 h r s , a 50 y l aliquot from the s:emra chamber and a 200 ul a l i q u o t from the buffer chamber were extracted by 3 7 . t he p r o c e d u r e d e s c r i h e d f o r serum and b u f f e r s t a n d a r d s , f ) R e cove r y S t u d y 2 To d e t e r m i n e the r e l a t i v e r e c o v e r y o f v a l p r o i c a c i d - Hg i n the serum e x t r a c t i o n p r o c e d u r e , serum samp les s p i k e d w i t h known amounts o f 2 v a l p r o i c a c i d - Hg were p r e p a r e d and s u b j e c t e d to t h e same a n a l y t i c a l p r o c e d u r e used f o r serum drug q u a n t i t a t i o n . Then a s e r i e s o f non-e x t r a c t e d r e f e r e n c e s t a n d a r d s o f 1 0 , 2 0 , 4 0 , 6 0 , 8 0 , 100 yg/ml 2 v a l p r o i c a c i d - Hg i n s o l v e n t (10% e t h y l a c e t a t e i n hexane ) were p r e p a r e d , each s t a n d a r d c o n t a i n i n g the same c o n c e n t r a t i o n o f o c t a n o i c a c i d . The 2 c o n c e n t r a t i o n o f v a l p r o i c a c i d - Hg f o u n d i n t h e s p i k e d serum s t a n d a r d s were d e t e r m i n e d u s i n g the n o n - e x t r a c t e d s t a n d a r d c u r v e . S i m i l a r s t u d i e s were done to d e t e r m i n e the r e l a t i v e r e c o v e r y o f 2 v a l p r o i c a c i d - Hg by e x t r a c t i o n f rom s a l i v a . In g e n e r a l serum and s a l i v a s t a n d a r d s were a n a l y z e d i n q u a d r i p l i -c a t e s to d e t e r m i n e t h e p r e c i s i o n o f the a s s a y . S t a n d a r d c u r v e s were p r e p a r e d p r i o r t o e a c h b a t c h o f s a l i v a * a n d serum samp les a n a l y z e d . Serum and s a l i v a samples were a n a l y z e d i n d u p l i c a t e s and 2-3 i n j e c t i o n s o f each s t a n d a r d o r sample i n t o t h e GCMS were .made . G l a s s co lumns were s i l a n i z e d by w a s h i n g w i t h m e t h a n o l , f i l l i n g w i t h 10% d i m e t h y l d i c h l o r o s i l a n e i n t o l u e n e and then a l l o w e d t o s t a n d f o r i h r . The d i m e t h y l d i c h l o r o s i l a n e s o l u t i o n was then removed and the co lumn washed w i t h t o l u e n e , f o l l o w e d by a second w a s h i n g w i t h m e t h a n o l . The column was d r i e d i n an oven a t 100°C and then packed w i t h the co lumn phase . The packed co lumn was c o n d i t i o n e d by h e a t i n g s l o w l y (1 - 4°C per m i n ] up to the maximum t e m p e r a t u r e a t w h i c h the co lumn 38. would be used and using a c a r r i e r gas flow o f 10 ml/min. The column was l e f t a t the maximum temperature f o r at l e a s t 6 hrs-before use. g) C a l c u l a t i o n of Pharmacokinetic Parameters. Serum and s a l i v a concentration-time curves could be described according to a two-compartment model. The e l i m i n a t i o n r a t e constants (K^) of both v a l p r o i c a c i d and v a l p r o i c a c i d - Hg were determined by l e a s t square r e g r e s s i o n a n a l y s i s o f the terminal l o g - l i n e a r part of the decay curves. The e l i m i n a t i o n h a l f - l i f e ( t ^ ) was c a l c u l a t e d from t ^ = 0.693/K^. The area under the curve (AUC) a f t e r o r a l dose a d m i n i s t r a t i o n was c a l c u l a t e d by the t r a p e z o i d a l r u l e up to the l a s t sample (C^) plus the area from time ( t ) to i n f i n i t y . [A U C Q _ „ = A U C O t + C ^ K ] , where C i s the concentration at time t . The area under the curve at one dosing i n t e r v a l during steady s t a t e drug concentration (AUC s s) was c a l c u l a t e d by the t r a p e z o i d a l r u l e summing v a l p r o i c a c i d and v a l p r o i c 2 a c i d - Hg concentrations to obtain steady s t a t e l e v e l s during that i n t e r v a l . The t o t a l body clearance (Cl) was c a l c u l a t e d according to Cl = F >• Dose/AUC assuming complete b i o a v a i l a b i l i ty.i.e.F = 1 (18, 19). The t o t a l body clearance at steady s t a t e (Cl g ) was c a l c u l a t e d according to C l s s = Dose/AUC . I n t r i n s i c clearance (clearance o f unbound v a l p r o i c a c i d in serum) was c a l c u l a t e d according to Cl = Dose/ A U C f r e g . The apparent volume of d i s t r i b u t i o n ( V ( j a r e a ) was c a l c u l a t e d according to V d a r e a = C l / K £ . . 3. I d e n t i f i c a t i o n of metabolites from human serum and urine a) I s o l a t i o n Procedures Serum - Pooled serum, 2.0 ml (obtained by taking 100 - 300 y l 39.: a l i q u o t s o f t h e 0 - 12 h r o r 12 - 24 h r b l o o d samples a f t e r a d m i n i s t r a t i o n t i o n o f v a l p r o i c a c i d - Hg dose i n t h e m u l t i p l e dose s t u d i e s ) was a c i d i f i e d t o pH 1.8 w i t h 4N HC1 , f o l l o w e d by i n c u b a t i o n f o r 2 h r a t 37 °C . Where c o m p l e t e d e c a r b o x y l a t i o n o f b e t a - k e t o a c i d s was no t d e s i r e d i n c u b a t i o n was e x c l u d e d . E x t r a c t i o n was c a r r i e d ou t t w i c e w i t h 2 - 3 ml o f e t h y l a c e t a t e , e a c h - t i m e c e h t r i f u g i n g a t 2 ,000 rpm f o r 30 min t o s e p a r a t e the two p h a s e s . The e t h y l a c e t a t e l a y e r s were s e p a r a t e d a n d c o m b i n e d , d r i e d o v e r anhydrous Na^SO^, and then c o n c e n t r a t e d a t room t e m p e r a t u r e u s i n g a g e n t l e s t r e a m o f d r y n i t r o g e n . The e x t r a c t s were d e r i v a t i z e d and an a l i q u o t (4 - 8 y l ) i n j e c t e d i n t o the GCMS. U r i n e - Enzymic H y d r o l y s i s One ml o f u r i n e (4 h r sample i n the s i n g l e dose s t u d i e s ) was m i x e d w i t h 0 .5 ml c i t r a t e b u f f e r (pH 5 .5 ) and 0 . 5 m l g l u c u r o n i d a s e 5 s o l u t i o n (10 F ishman u n i t s / m l ) and then i n c u b a t e d a t 37°C o v e r n i g h t . The m i x t u r e was a c i d i f i e d w i t h IN HC1 and e x t r a c t e d t w i c e w i t h 0 . 7 ml o f 10% e t h y l a c e t a t e i n h e x a n e , each t i m e c e n t r i f u g i n g a t 2 ,000 rpm f o r 10 m i n . The o r g a n i c phases were c o m b i n e d , d r i e d o v e r anhydrous Na2S0^ and then c o n c e n t r a t e d w i t h n i t r o g e n . The e x t r a c t s were a n a l y z e d i n both u n d e r i v a t i z e d and d e r i v a t i z e d f o r m . U r i n e - A l k a l i n e H y d r o l y s i s To 15 ml o f a u r i n e sample (4 - 8 h r s a m p l e , 8 - 1 2 h r , 12 - 16 h r , 16 -24 h r , 24 - 28 h r sampl e) was added t h e r e q u i r e d vo l ume o f 3N. NaOH to b r i n g t h e pH t o 13 and h y d r o l y s i s o f c o n j u g a t e s - w a s e f f e c t e d by h e a t i n g t h e s o l u t i o n a t 90°C f o r 1 h r ( 1 0 3 ) . A f t e r c o o l i n g , t he s o l u t i o n was a c i d i f i e d t o pH 1.8 w i t h 4N HC1. The a c i d i f i e d u r i n e was e x t r a c t e d t h r e e t imes w i t h 10 ml p o r t i o n s o f e t h y l a c e t a t e . The combined e t h y l a c e t a t e 1 a y e r s were 40. d r i e d over anhydrous NagSO^ and the solvent removed under vacuum. The concentrated e x t r a c t was then d e r i v a t i z e d and an a l i q u o t (. 4 - 8 y l ) i n j e c t e d i n t o the GCMS. Unhydrolyzed urine sample and blank urine sample were subjected to the i s o l a t i o n procedures described above. b) D e r i v a t i z a t i o n Methyl e s t e r d e r i v a t i v e s o f a c i d i c compounds present in the urine and serum concentrated e x t r a c t s were prepared by t r e a t i n g the extr a c t s with diazomethane. Diazomethane was prepared by adding Diazald to an ether-2-ethoxyethanol s o l u t i o n i n a generator glass tube under n i t r o g e n , followed by a d d i t i o n o f 60% potassium hydroxide s o l u t i o n (104). 1. t-BDMS d e r i v a t i v e s were prepared by adding 30 - 50 y l o f t-BDMS reagent to the concentrated e x t r a c t o f urine and serum samples and warming a t 50°C f o r 5 min. The t-BDMS d e r i v a t i v e s were extracted with 60 - 120 y l of sol v e n t (10% e t h y l a c e t a t e i n n-hexane) and phases separated by c e n t r i f u g i n g a t 2000 rpm f o r 2 - 5 min.^ TMS d e r i v a t i v e s were prepared by t r e a t i n g the concentrated e x t r a c t i o n sample with 30 - 50 y l o f N- m e t h y l - N - t r i m e t h y l . s i l y t r i f l u o r o -acetamide a t 50°C f o r 5 min (36). c) Gas Chromatography Mass Spectrometry Separation and i d e n t i f i c a t i o n o f serum and urine 1 metabolites from d e r i v a t i z e d samples were performed by a Hewlett Packard 5700 Gas Chromatograph i n t e r f a c e d to a Varian MAT-111 Mass Spectrometer v i a a 41 . v a r i a b l e s l i t separator. The data was processed by an o n - l i n e Varian 620L computer system. The gas chromatograph was f i t t e d with e i t h e r a 6 m x 2 mm i . d . glass column packed with 3% Dexsil 300 on 100/120 Supelcoport or a 6 m x 2 mm i . d . glass column packed 3% 0V-17 on 100/120 Gas Chrom Q. Operating co n d i t i o n s were: helium ( c a r r i e r gas) flow r a t e of 25 ml/min, i n j e c t i o n port temperature 250°C, separator temperature 250°C, i n l e t l i n e temperature 250°Cand ion source pressure 5.0 x 10 ^ T o r r . The mass spectrometer e l e c t r o n i o n i z a t i o n energy was 80 eV and the emission current 300 yA. Oven temperatures depended on the type o f column and d e r i v a t i z a t i o n agent used. For example, f o r methyl es t e r s using a 3% Dexsil 300 column, c o n d i t i o n s were 80°C (hold 4 min) then programmed at 8°C/min to 250°C. Conditions for methyl esters on a 3% 0V-17 column were 80°C (h o l d 4 min), then programmed at 8°C/min to 280°C. t-BDMS d e r i v a t i v e s were done on a 3% Dexsil 300 or 3% OV-17 from 80°C to 280°C at 8°C/min. TMS d e r i v a t i v e s were done on a 3% 0V-17 column from 80°C to 280°C at 4°C/min. In general the t o t a l ion current was measured from mass 50 to 500. Computerized background s u b t r a c t i o n s were made when anal y z i n g the metabolic ex t r a c t s to p l o t mass spectra and chromatograms. Mass chromatograms were p l o t t e d in the scan mode and s e l e c t e d ion chromato-grams were p l o t t e d i n the s e l e c t e d ion monitoring mode. In one s e t o f experiments i n which methyl d e r i v a t i v e s of the serum e x t r a c t were analyzed on a c a p i l l a r y column, gas chromatography mass spectrometry was performed w i t h a VG 7070 GCMS connected on l i n e with a VG 2035 F/B data system (by courtesy of U n i v e r s i t y of Washington). The gas chromatograph was f i t t e d with a 30 m long, wide bore glass 4 2 . co lumn packed w i t h DB-5. The i o n s o u r c e was m a i n t a i n e d a t 200°C " and h e l i u m was used as t h e c a r r i e r gas a t a head p r e s s u r e o f 15 p s i . Methane was the r e a g e n t gas used and the j ' on s o u r c e p r e s s u r e was 0 .5 T o r r . Column t e m p e r a t u r e was programmed a t 4°C/min f rom 50°C to 280°C. d) S t a b i 1 i t y S t u d i e s To i n v e s t i g a t e the b e h a v i o u r o f s p e c i f i c m e t a b o l i t e s a f t e r d e r i v a t i z a t i o n w i t h d i a z o m e t h a n e , MSTFA and t-BDMCS, s o l u t i o n s o f 3-OH'VPA 4-OH V P A y - l a c t o n e , 5-0H VPA 6 - l a c t o n e , 2-ene VPA, 3-ene VPA, and 4-ene VPA i n e t h y l a c e t a t e - hexane Cl : 9 v/v ) were p r e p a r e d i n s u f f i c i e n t c o n c e n t r a t i o n s to be examined i n the s c a n mode by GCMS. The a p p r o p r i a t e vo lumes o f the s o l u t i o n s were d e r i v a t i z e d and a l i q u o t s i n j e c t e d i n t o the GCMS u s i n g GCMS c o n d i t i o n s used f o r s e p a r a t i o n and i d e n t i f i c a t i o n o f m e t a b o l i t e s . To i n v e s t i g a t e the e f f e c t o f pH on t h e s t a b i l i t y o f serum me tabo -l i t e s d u r i n g e x t r a c t i o n , t h e i s o l a t i o n p r o c e d u r e f o r serum m e t a b o l i t e s was c a r r i e d o u t a f t e r a c i d i f y i n g serum samples to pH v a l u e s r a n g i n g f rom 1.5 to 5 . 0 . The f i n a l c o n c e n t r a t e d e x t r a c t s were then d e r i v a t i z e d and a l i q u o t s i n j e c t e d i n t o the GCMS u s i n g GCMS c o n d i t i o n s d e s c r i b e d above f o r s e p a r a t i o n and i d e n t i f i c a t i o n o f m e t a b o l i t e s ; I I I . RESULTS 4 3 . A P h a r m a c o k i n e t i c S t u d i e s 2 S y n t h e s i s o f v a l p r o i c a c i d - Hg 2 2 D i - ( 3 - H ^ - p r o p y l ) a c e t i c a c i d ( v a l p r o i c a c i d - Hg) was s y n t h e s i z e d i n modera te y i e l d v i a t h e m a l o n i c e s t e r s y n t h e s i s (Scheme 1 ) . The i s o t o p i c d e u t e r i u m c o m p o s i t i o n ,was c l o s e to 97% and was d e t e r m i n e d by m o n i t o r i n g a p r o m i n e n t f r agmen t i o n o f the t-BDMS e s t e r , m/z 207 [ M - 5 7 ] + . F i g u r e 2 shows f o r c o m p a r i s o n the mass s p e c t r a o f t he t-BDMS e s t e r s o f 2 v a l p r o i c a c i d and v a l p r o i c a c i d - Hg . F i g u r e 3 shows the v i r t u a l absence o f the me thy l p r o t o n , peaks . . i n the NMR s p e c t r u m o f v a l p r o i c a c i d - 2 H , . 6 A s s a y The p r o m i n e n t i o n s , m/z 201 and m/z 2 0 7 , [ M - 5 7 ] + o f v a l p r o i c a c i d 2 and v a l p r o i c a c i d - Hg r e s p e c t i v e l y ( F i g 2) were chosen f o r the s e l e c t e d i o n m o n i t o r i n g a s s a y . The i n t e r n a l s t a n d a r d , o c t a n o i c a c i d , a l s o has a p r o m i n e n t m / z 201 [M-57 ] + i n t h e w a s s s p e c t r u m o f i t s t-BDMS e s t e r . The e x t r a c t i o n and d e r i v a t i z a t i o n p r o c e d u r e r e s u l t e d i n c l e a n s e l e c t e d i o n chromatograms as i l l u s t r a t e d i n F i g . 4 w h i c h shows t y p i c a l s e l e c t e d i o n chromatograms f rom an e x t r a c t e d s a l i v a sample c o n t a i n i n g v a l p r o i c a c i d 2 2 and v a l p r o i c a c i d - H g . The e x t r a c t i o n r e c o v e r i e s o f v a l p r o i c a c i d - Hg f rom s p i k e d serum and s a l i v a samples were f o u n d t o be 100% as r e p o r t e d f o r u n l a b e l l e d v a l p r o i c a c i d u s i n g the same a s s a y method ( 9 4 ) . The use o f 10% e t h y l a c e t a t e i n hexane a l s o r e s u l t e d i n the s i l y l e s t e r s b e i n g e x t r a c t e d w h i l e p o l a r m a t e r i a l s , t h a t c o u l d i n t e r f e r e w i t h the s e l e c t e d i o n m o n i t o r i n g , r ema ined i n the d i m e t h y l f o r m a m i d e l a y e r o f the C 2H 3CH 2CH 2Br C 0 0 E t CH 2(C00Et) 2 — > C 2H 3CH CH 2-CH^ NaOEt - EtOH C 0 0 E t A, 2 hr C 2H 3CH 2CH 2Br C 2H 3CH 2CH 2 , COOEt 2 / \ ^ H r.H_ r.H_ / ( NaOEt - EtOH C / H ^ C H ^ COOEt A, 3 hr Ci.) NaOH, A C 2H„CH„CH COOH 6 c c \ y => C. 2 / \ ( i i ) HC1 or H 2S0 4 C^H 3CH 2CH 2 X COOH •C02 C 2H 3CH 2CH 2 A (180 - 200°C) C 2H,CH 0CH CH-COOH 3""2~"2 I I Scheme! - Synthesis o f v a l p r o i c a c i d - H( 45. NXh 207(M-57) 75 co c 1 i CD DC cfHj-CH 2-CH 2^ O C H 3 CHC -0 -S i - c (CH 3 ) 3 cH-CHrCHz CH3 (MW=264) (a) 50 100 "Vz 150 200 100 c . i CO cc (b) 201 (M-57) 75 50 100 ~ 150 200 C H J - C H J - C H ^ jj> f H 3 CHC-O - S i -C fCH j ) , CHj-CHj-CHa C H 3 (MW=258) F i g . 2 El-mass spectra o f t-EDMS deri-vatives o f VPA- 2H g (a) and VPA (b) Fig. 3 NMR Spectra o f VPA ( a l and VPA- 2H g (h): in CDCL 3 47. F i g . 4 Selected ion chromatogram of an extracted s a l i v a sample, m/z 207, VPA- 2H f i; m/z 201, VPA and octanoic a c i d . 4 8 . r e a g e n t . The c a l i b r a t i o n c u r v e s c o n s t r u c t e d f o r v a l p r o i c a c i d and v a l p r o i c 2 a c i d - Hg i n serum and s a l i v a were l i n e a r w i t h i n t h e c o n c e n t r a t i o n ranges. used and gave h i g h c o r r e l a t i o n c o e f f i c i e n t s . T a b l e I shows: t y p i c a l 2 c a l i b r a t i o n c u r v e s f o r v a l p r o i c a c i d - Hg i n serum and s a l i v a w h i l e T a b l e I I shows c u r v e s f o r v a l p r o i c a c i d i n serum and s a l i v a . The p e r c e n t r e l a t i v e s t a n d a r d d e v i a t i o n s , were g e n e r a l l y below. 5% a t t e s t i n g t o t h e r e p r o d u c i b i l i t y o f t h e a s s a y . The l o w e r l i m i t o f s e n s i t i v i t y o f t h e a s s a y was 0.1 yg/ml based on a 200. u l s a l i v a sample o r 50 p i serum s a m p l e . The s e n s i t i v i t y o f t h e a s s a y was l i m i t e d i n p a r t by t h e a p p e a r a n c e o f backg round peaks due t o t h e t-BDMS r e a g e n t . S i n g l e Dose S t u d y 2 The s u i t a b i l i t y o f v a l p r o i c a c i d - Hg f o r t h e p h a r m a c o k i n e t i c s t u d i e s was d e m o n s t r a t e d i n t h e s i n g l e dose s t u d y a f t e r a d m i n i s t r a t i o n o f a m i x -t u r e o f a p p r o x i m a t e l y 2 : 1 w e i g h t r a t i o o f v a l p r o i c a c i d t o v a l p r o i c a c i d -2 2 Hg . The r a t i o o f t o t a l serum l e v e l s o f v a l p r o i c a c i d to v a l p r o i c a c i d - Hg i n each, serum sample was f ound t o be t h e same 2 : 1 as t h a t i n t h e dosage r a t i o ( F i g . 5 ) . The p a r a l l e l i s m between t h e e l i m i n a t i o n phases o f serum 2 v a l p r o i c a c i d and serum v a l p r o i c a c i d - Hg ( F i g . 5) s t r o n g l y d e m o n s t r a t e d t h a t t h e r e was no s i g n i f i c a n t I s o t o p e e f f e c t on t h e e l i m i n a t i o n k i n e t i c s o f v a l p r o i c a c i d a f t e r i n c o r p o r a t i o n o f s i x d e u t e r i u m a t o m s . The r a t i o o f 2 f r e e serum l e v e l s , o f v a l p r o i c a c i d t o v a l p r o i c a c i d - Hg i n each serum sample was f ound t o be 1.8 : 1. The t e r m i n a l phases o f t he s a l i v a drug l e v e l s were no t o b t a i n e d a f t e r s i n g l e dose a d m i n i s t r a t i o n s i n c e t h e l e v e l s were c l o s e t o t h e l i m i t o f d e t e c t i o n . TABLE I C a l i b r a t i o n curve data of the t e r t - b u t y l d i m e t h y l s i l y l e s t e r of v a l p r o i c 2 a c i d - H.K in b i o l o g i c a l samples. SALIVA S a l i v a Concentration (yg/ml) Peak area r a t i o mean* (RSD%) Linear r e g r e s s i o n parameters** 0.1 0.020 (6.67%) 0.25 0.045 (3.77%) a° = -0.008 0.50 0.085 (0.79%) 1.00 0.186 (1.89%) ' a-, = 0.185 1.50 0.238 (0.92%) 3.00 0.546 (1.02%) r 2 = 0.9986 SERUM Total Serum Concentration (yg/ml) Peak area r a t i o mean* (RSD%) Linear regression parameters 10 0.350 (1.75%) 20 0.705 (2.68%) a 0 = -0.0349 40 1.295 (0.77%) 50 2.130 (0.57%) a-j = 0.0358 80 2.880 (0.31%) 100 3.525 (0.44%) r 2 = 0.9981 * n - 4; ** v c i s the c o e f f i c i e n t o f determination, a-j i s the slope and a° i s the i n t e r c e p t . Equation f o r the l i n e i s y = a-jX + a° where y i s the peak area r a t i o mean and x i s the drug conc e n t r a t i o n . Peak area 2 r a t i o i s the r a t i o o f m/z 207 o f v a l p r o i c a c i d - H g e s t e r to m/z 201 o f octanoic a c i d e s t e r • 50. TABLE II C a l i b r a t i o n curve data of the t e r t - b u t y l d i m e t h y l s i l y l e s t e r o f v a l p r o i c a c i d i n b i o l o g i c a l samples SALIVA  S a l i v a Concentration Peak area r a t i o mean* Linear regression Cvig/ml) (RSD/Q parameters** 0.25 0.050 (2.73%) 0.50 0.101 (1.70%) a 0 = 0.005 1.00 0.206 (1.54%) 1.50 0.286 (0.48%) a ] = 0.192 3.00 0.583 (2.38%) 6.00 1.161 (1.86%) r 2 = 0.9998 SERUM Total Serum Peak area r a t i o mean* Linear regression Concentration (yg/ml) (RSD%) parameters** 20 0.631 (1 .91%) 40 1 .305 (3.01%) a° - 0. .014 60 2.001 (0.49%) 80 2.704 (0.77%) a 1 = 0. .0333 100 3.308 (1.04%) 120 3.950 CO.47%) r 2 = 0 .9997 *n.= 4; ** r i s the c o e f f i c i e n t o f determination a-j i s the slope and a° i s the i n t e r c e p t . Equation f o r the l i n e i s y = a-|X = a" where y i s the peak area r a t i o mean and x i s drug c o n c e n t r a t i o n . Peak area r a t i o i s the r a t i o o f m/z 201 o f v a l p r o i c a c i d e s t e r to m/z 201 o f octanoic a c i d e s t e r . F i g . 5 Time course of v a l p r o i c a c i d and v a l p r o i c a c i d - H5 in s a l i v a and serum f o l l o w i n g a ^ s i n g l e oral dose of 3 7 2 mg V P A and 1 8 2 mg V P A - 2 H 6 . r" m/z 1 0 3 v. m/z 1 0 2 • C 3H 7 CH, - CH - CH, - CH - CO 3 L _ o - 1 it - OH - VPA -Y - lactone ^3H7 CH, - CH, - CH, - CH - CO L!—L„i 1 5 - OH - VPA - 6 - lactone m/z 1 2 0 m/z 114 CH - CH - 1 - C H - CH - C H - C H 3 3-heptanone from 3-keto-VPA I . . . . i I • « » • i « • « » I « * • i l < i • • l_j 1 0 2 0 TIME (min.) 3 0 40 F i g . 6 Mass chromatograms of metabolites from urine sample after dose of VPA-H and VPA In single 6 dose study The serum concentration-time curves o f both v a l p r o i c a c i d and 2 v a l p r o i c a c i d - Hg a f t e r the s i n g l e o r a l dose were biphasic ( F i g . 5). 2 The s a l i v a v a l p r o i c a c i d and v a l p r o i c a c i d - Hg l e v e l s showed a s i m i l a r p r o f i l e to t h e i r corresponding serum l e v e l s within an 8 hr i n t e r v a l a f t e r dosing. Peak l e v e l s were observed at 0.5 hr in serum and s a l i v a followed by appearance o f a small r i s e in t o t a l serum drug l e v e l s and s a l i v a l e v e l s at 3 - 4 hr a f t e r dosing. A d d i t i o n a l evidence, for the absence of a major isotope e f f e c t , came from the a n a l y s i s of u r i n a r y m e t a b o l i t e s , where the major metabo-l i t e s , namely v a l p r o i c a c i d glucuronide, 3-keto VPA and 4-OH VPA (measured as the lactone) showed the c o n s i s t e n t 2 : 1 peak height r a t i o o f u n l a b e l l e d to l a b e l l e d metabolites i n the mass chromatograms ( F i g . 6). The 4-OH VPA metabolite and 4-OH VPA- Hg metabolite were present i n the a c i d i f i e d urine e x t r a c t as both d i a s t e r e o i s o m e r i c forms of the lactones which were p a r t i a l l y separated by the GC columns ( F i g . 6). The corresponding diastereoisomer forms of the 4-OH VPA and 4-OH VPA- 2H g lactones a l s o -2 showed a 2 : 1 peak height r a t i o . The minor metabolite, 5-OH VPA- Hg (shown in F i g . 6 as the lactone) showed a metabolic isotope e f f e c t as expected. The peak height f o r the 5-OH VPA lactone i n the m/z 100 mass 2 chromatogram was greater than twice the peak height f o r the 5-OH VPA- Hg in the m/z 102 mass chromatogram f o r the same attenuation value. M u l t i p l e Dose Study 2 Following the pulse dose o f v a l p r o i c a c i d - Hg at steady s t a t e l e v e l s of v a l p r o i c a c i d , absorption of the deuterated analogue was r a p i d with peak serum l e v e l s appearing within 2 - 3 hr ( F i g . 7). 2 Biphasic e l i m i n a t i o n was not evident f o r e i t h e r v a l p r o i c a c i d - Hg as 3 sco-I i g. 7 Time course of serum total (VPA* VPA-2H6) and serum total VPA-2H6 (b) following single dose Of VPA-HglpOOmg) during multiple dosing with VPA '99 5 6 . o b s e r v e d o v e r 72 h r s o r v a l p r o i c a c i d i n the 12 h r d o s i n g i n t e r v a l s . A l l d e c a y c u r v e s appea red t o be m o n o e x p o n e n t i a l i n t h e m u l t i p l e dose s t u d i e s . The u n s t i m u l a t e d and s u c r o s e - s t i m u l a t e d m i x e d s a l i v a v a l p r o i c 2 a c i d - Hg c o n c e n t r a t i o n - t i m e c u r v e s a r e shown i n F i g . 8 . The h i g h u n s t i m u l a t e d s a l i v a l e v e l a t 0 . 9 h r i n F i g . 8 c o u l d be due to r e s i d u a l d rug r e t a i n e d i n the mou th . The appea rance o f a s e c o n d m i n o r peak a t 4 2 h r o c c u r r e d i n b o t h s t i m u l a t e d and u n s t i m u l a t e d s a l i v a v a l p r o i c a c i d - Hg c u r v e s a t s t e a d y s t a t e . The re was g r e a t e r f l u c t u a t i o n s i n s u c r o s e -2 s t i m u l a t e d s a l i v a v a l p r o i c a c i d - Hg c o n c e n t r a t i o n s than i n u n s t i m u l a t e d 2 s a l i v a v a l p r o i c a c i d - Hg c o n c e n t r a t i o n s . S u c r o s e - s t i m u l a t e d s a l i v a samples had c o n s i s t e n t l y l o w e r pH ( 5 . 8 6 ± 0 . 4 8 , mean ± S . D , n = 18) than u n s t i m u l a t e d s a l i v a samples ( 6 . 9 8 ± 0 . 2 9 , n = 18) a t s i m i l a r 2 s a m p l i n g t i m e s . The s a l i v a v a l p r o i c a c i d - Hg decay c u r v e appea red to be m o n o e x p o n e n t i a l and was d e t e r m i n e d up t o 36 h r a f t e r d o s i n g . 2 The t i m e - c o u r s e o f serum f r e e v a l p r o i c a c i d - Hg i n the p r e s e n c e 2 2 o f s t e a d y - s t a t e l e v e l s o f serum f r e e v a l p r o i c a c i d - ( H Q + H £ ) i s 2 shown i n F i g . 9 . Peak l e v e l s o f serum f r e e v a l p r o i c a c i d - Hg were o b s e r v e d a t 2 - 3 h r a f t e r t h e l a b e l l e d v a l p r o i c a c i d d o s e . The decay c u r v e appea red t o be m o n o e x p o n e n t i a l . P h a r m a c o k i n e t i c P a r a m e t e r s . T a b l e III summar izes f o r c o m p a r i s o n t h e p h a r m a c o k i n e t i c d a t a d e r i v e d f rom the s i n g l e dose and f rom the s t e a d y - s t a t e e x p e r i m e n t s . A l t h o u g h t h e - f o c u s o f the m u l t i p l e dose s t u d y was on the pa r ame te r s f o r 2 2 2 v a l p r o i c a c i d - H g , pa ramete r s f o r v a l p r o i c a c i d - C H Q + Hg) a r e i n c l u d e d t o a s s e s s the r e l i a b i l i t y o f t h e d a t a s i n c e o n l y one i n d i v i d u a l 57. 10-1 F i g . 9 C o n c e n t r a t i o n - t i m e c u r v e s o f unbound v a l p r o i c a c i d - H f i and v a l p r o i c a c i d - ( 2 H 0 + 2 H 6 ) i n serum f o l l o w i n g 600 mg o r a l dose o f v a l p r o i c a c i d - 2 H g . 58. TABLE III Pharmacokinetic parameters f o r v a l p r o i c a c i d (VPA) and v a l p r o i c a c i d - H g (VPA- 2H g) under s i n g l e dose and steady s t a t e conditions i n a healthy male volunteer (weight = 63.64 kg). Sing l e Dose Conditions (Mixture o f 372 mg V P A and 182 mg V P A - H R ) Biophase y (hr) K E C h r - l ) c i a ( l i t e r / hr/kq) V D area ( l i t e r / kg) AUC (mg.hr/ l i t e r ) Serum t o t a l V P A - 2 H G 18.7 0.0371 0.0075 0.202 379.1 Serum t o t a l V P A 13.6 0.0372 0.0070 0.188 841 .3 2 Serum free V P A - H G 18.3 0.0378 0.17 4.50 16.83 Serum free V P A 16.8 0.0413 0.20 4.80 29.27 S a l i v a V P A - 2 H G 15.4 0.0451 0.88 19.5 3.25 S a l i v a V P A 17.4 0.0398 1.1 27.6 5.23 Steady State Conditions ( M u l t i p l e Dose V P A , 600 mq V P A - 2 H F I ) 2 b Serum t o t a l V P A - H G 14.3 . 0.0485 0.010 0.206 900 Serum t o t a l V P A + V P A - 2 H F I 1 3.5 0.0513 0.011 0.214 872 2 Serum free V P A - H G 13.2 0.0525 0.12 2.29 79.48 Serum free V P A + V P A - 2 H G 14.8 0.0468 0.13 2.78 72.52 S a l i v a V P A - 2 H G 16.5 0.0420 0.88 20.9 10.66 S a l i v a V P A + V P A - 2 H G 13.1 0.0529 1.1 20.8 8.86 a b c Oral b i o a v a i l a b i l i t y i s assumed to be unity 2 VPA- H G parameters are c a l c u l a t e d from t = o-« 2 For VPA + VPA- H G parameters are c a l c u l a t e d from steady s t a t e VPA and VPA- 2hL concentrations summed up during the dosing i n t e r v a l of 6 o 12 hrs f o l l o w i n g the VPA- H dose. 6 59. was used i n these s t u d i e s . 2 The e l i m i n a t i o n h a l f - l i f e o f t o t a l serum v a l p r o i c a c i d - Hg under m u l t i p l e dose co n d i t i o n s (14.3 hr) was s h o r t e r than the h a l f - l i f e observed in the s i n g l e dose study (18.7 h r ) . The apparent volume o f 2 d i s t r i b u t i o n of v a l p r o i c a c i d - Hg at steady s t a t e was not d i f f e r e n t from the s i n g l e dose value. However the t o t a l body clearance at steady s t a t e (0.010 1 i t e r / h r / k g ) increased by 33% over the s i n g l e dose value. The area-under the curve, AUC K f o r - t o t a l serum v a l -' o-°° proic acid-^Hg at steady s t a t e (900 m g . h r / l i t e r ) was lower than the p r e d i c t e d value from the s i n g l e dose study (379.1 m g . h r / l i t e r f o r 182 mg dose). 2 The h a l f - l i f e o f serum fr e e v a l p r o i c a c i d - Hg under m u l t i p l e dose conditions (13.2 hr) was s h o r t e r than the s i n g l e dose value (18.3 hr) 2 while the clearance of the unbound v a l p r o i c a c i d - Hg decreased under m u l t i p l e dose conditions (0.12 l i t e r / h r / k g ) compared to the s i n g l e dose value (0.17 l i t e r / h r / k g ) . The volume of d i s t r i b u t i o n of unbound v a l p r o i c a c i d - Hg at steady s t a t e (2.29 l i t e r / k g ) was smaller than the s i n g l e dose value (4.50 l i t e r / k g ) . 6 0 . B. M e t a b o l i s m S t u d i e s I d e n t i f i c a t i o n o f m e t a b o l i t e s i n serum and u r i n e A f t e r a d m i n i s t r a t i o n o f v a l p r o i c a c i d as a p u l s e dose a t s t e a d y 2 s t a t e , v a l p r o i c a c i d and v a l p r o i c a c i d - Hg m e t a b o l i t e s were d e t e c t e d i n serum and u r i n e but no t i n a 2 : 1 m o l a r r a t i o as i n t h e s i n g l e dose s t u d y . F i g . 10 shows t h e t o t a l i o n c u r r e n t (T IC) chromatogram o f a m e t h y l a t e d serum e x t r a c t on t h e 6m l o n g 3% OV-17 c o l u m n . The me thy l e s t e r s o f t h e o r g a n i c a c i d s had good c h r o m a t o g r a p h i c p r o p e r t i e s . The most i n t e n s e peak, :No.5 was i d e n t i f i e d as v a l p r o i c a c i d e s t e r . The mass s p e c t r u m i s shown i n F i g . 1 1 . C h a r a c t e r i s t i c i o n d o u b l e t s i n t h e mass s p e c t r u m appea r a t m/z 1 1 6 , 1 1 9 [ M - 4 2 ] + , m/z 1 2 9 , 132 [ M - 2 9 ] + , m/z 9 9 , 105 [ M - 5 9 ] + , m/z 5 7 , 60 [ C 4 H g , C 4 H g 2 H 3 ] + and m/z 4 3 , 46 [ C 3 H ? , 2 + 2 2 ^ 3 H 4 ^ ^ u e t o r e t e n t i o n o f H 3 o r H 5 i n t h e d e u t e r i u m - l a b e l l e d v a l p r o i c a c i d f r agemen t i o n compared t o t h a t o f u n l a b e l l e d v a l p r o i c a c i d . F i g u r e 12 r e p r e s e n t s t h e t o t a l i o n c u r r e n t p l o t o f t h e m e t h y l a t e d u r i n a r y e x t r a c t on t h e 6m l o n g 3% D e x s i l 300 c o l u m n . A l t h o u g h many peaks a p p e a r i n t h e c h r o m a t o g r a m , r e s o l u t i o n o f peaks on t h e l o n g packed co lumn was s u f f i c i e n t t o o b t a i n t h e mass s p e c t r a o f i n d i v i d u a l compounds. The mass s p e c t r a and r e t e n t i o n t i m e s o f s y n t h e s i z e d r e f e r e n c e compounds were a l s o used t o a i d i d e n t i f i c a t i o n o f peaks i n t h e t o t a l i o n c u r r e n t c h r o m a t o g r a m s . K e t o - A c j d M e t a b o l i t e s The 8 - k e t o a c i d m e t a b o l i t e - o f VPA was i d e n t i f i e d i n bo th serum and u r i n e as i t s m e t h y l e s t e r o r as t h e d e c a r b o x y l a t e d p r o d u c t , 3-heptanone (peak 4 i n F i g . 10 and peak 4 i n F i g . 1 2 ) . The mass s p e c t r u m o f 61 F i g . 10 GCMS a n a l y s i s ( t o t a l i o n c u r r e n t p l o t ) o f m e t h y l a t e d human serum e x t r a c t f rom a h e a l t h y v o l u n t e e r on VPA m u l t i p l e dosage a f t e r a d m i n i s -t r a t i o n o f V P A - 2 H 6 (600 m g ) . Column u s e d , 3% 0V-17(6m x 2mm i . d . ) . Temp. P r o g r a m : 80°C (4 m in ) t o 280°C a t 8°C per m i n . Peak numbers c o r r e s p o n d t o : 1 = p r o p i o n i c a c i d , 2 = 2-methyl b u t y r i c a c i d , 3 = unknown, 4 = 3 - h e p t a n o n e , 5 = v a l p r o i c a c i d , ( V P A ) , 6 = 3-ene VPA, 7 = 2 - e t h y l - 4 - k e t o v a l e r i c a c i d 3 , 8 = 2-ene VPA, 9 = 7 - k e t o o c t a n o i c a c i d 3 , 10 = b e n z o i c a c i d , 11 = d i e n e VPA, 12 = 3-ke to VPA, 13 = 4 - k e t o VPA, 14 = p h e n y l a c e t i c a c i d , 1 5 , 16 = l o n g c h a i n f a t t y a c i d s (a = t e n t a t i v e l y i d e n t i f i e d ) . 87 41 27 43 57 2 9 i t 0 0 69-j, 83 ,1 IE .!• CHa—CHt—CH^ CHr^r^H> HC0OCH> TI6(«-42) 119 ("-« +D3) 100 ISO 11 Mass spectrum of peak 5, F i g . 10 con t a i n i n g VPA and VPA- Kg methyl e s t e r s . 6 3 . TIME(sec) 1500 2000 12 GCMS a n a l y s i s ( t o t a l i o n c u r r e n t p l o t ) o f m e t h y l a t e d u r i n a r y e x t r a c t f rom a h e a l t h y v o l u n t e e r on VPA a f t e r a d m i n i s t r a t i o n o f VPA-^Hg (600 m g ) . Column u s e d , 3% D e x s i l 300 (6m x 2mm i . d . ) . Temp, p r o g r a m : 80°C (4 min ) t o 250°C a t 8 °C per m i n . Peak numbers c o r r e s p o n d t o : 1 = unknown, 2 = 2-methyl b u t y r i c a c i d , 3 = 3 - h y d r o x y i s o v a l e r i c a c i d a , 4 = 3-hep t anone , 5 = v a l p r o i c a c i d ( V P A ) , 6 = unknown, 7 = 2-ene VPA , 8 = b e n z o i c a c i d , 9. = g l u t a r i c a c i d , 10 = d i e n e VPA, 11 = 3-OH VPA, 12 = p h e n y l a c e t i c a c i d , 13 = 4 - k e t o VPA , 14 = 2 - p r o p y l s u c c i n i c a c i d , 15 = a d i p i c a c i d , 16 = 4-OH VPA, 17 = 3 - m e t h y l a d i p i c a c i d a , 17b = 5-OH VPA , 18 = 2 - p r o p y l g l u t a r i c a c i d , 19 = a d i c a r b o x y l i c a c i d , 20 = o - m e t h o x y p h e n y l a c e t i c a c i d , 24 = p - m e t h o x y p h e n y l a c e t i c a c i d , 25 = c i t r i c a c i d , 29 = h i p p u r i c a c i d (a = t e n t a t i v e l y i d e n t i f i e d ) . 100 57 CH r - C H * — C H -CH 2—C H ? >=o (Db.L\) 29 \ 41 4 3 85 72 60 5 0 75 88 114(M+ 100 120(M+D6) H—• 1 1 3 Mass s p e c t r u m o f peak 4 , F i g . 1 0 c o n t a i n i n g •3-he.ptanone.--C - H Q + i n serum e x t r a c t . 65. 3-heptanone-[^Hg + ^Hg] i s presented i n F i g . 13. The c h a r a c t e r i s t i c ion doublets i n the mass spectrum appear at m/z 114,120 [ M + ] , m/z 85, 88 (CH 3 -CH 2 -CH 2 -CH 2-C=Q, C 2 H 3 - C H 2 - C H 2 - C H 2-C=0) + ,m/z 72,75 ( C H 3 - C H 2 C H 2 , C 2 H 3 - C H 2-C0H= C H 2 ) + and m/z 57, 60 (CH 3 CH 2C=0,C 2 H 3 CH 2C=0) +. Peak 13 in the TIC chromatogram o f the serum e x t r a c t ( F i g . 10) and ur i n e e x t r a c t ( F i g . 12) was i d e n t i f i e d as 4-keto VPA methyl e s t e r . The metabolite, 4-keto VPA, has not been i d e n t i f i e d before as a product of VPA metabolism. The mass spectrum o f the methyl e s t e r o f 4-keto VPA-2 2 ( Hg + Hg) from peak 13, F i g . 10 i s shown i n F i g . 14a and i s compared with that o f the methyl ester of synthesized 4-keto VPA ( F i g . 14b). The ion doublet at m/z 115,118 ( F i g . 14a) a r i s e s from the l o s s o f CH 3C0CH 2 and C H 3C0CH 2 r a d i c a l s from the molecular ions (Scheme 2). This conforms to p r e f e r e n t i a l cleavage of the carbon-to-carbon CH^O-C-CH CHo-C-CH, " C H 3 C 0 C H 2 ' CH.0-C-CH+ 3 II I 2 || 3 -> 3 ff J 6 C 3 H ? 0 0 C 3 H ? M.W = 172 - C 3 H 6 -CH. CH30-C = CH-CH 2-(j-CH 3 OH 0 m/z 43 (base peak) ( 2H 3, 46) m/z 115 ( 2H 3,118) — C 2H 4 CH,0-C-CH 3 I \ H D + C H 2 m/z 87 m/z 130 ( 2H 3,133) Scheme 2. Mass fragmentation pathways of 4-keto VPA 6 6 . 100, 5CH 43 27 29 U L i i J o ^ \wCOOCH, (t^ Bfe) 87 46 56 59 83 69 Kjn •,,il|fl:,l,ill,...llib m' 100 1$M-57)* t18CM-57*Djt 1 3 0 i i i M " 3 , i l 5 150 2 2 F i g . 14a Mass spectrum of 4-keto VPA-(. H Q + H.g)me.th.yl este r in serum e x t r a c t 1CO, t -z :50 UJ > tr 43 27 29 41 c H ^ H r C H > C O O C H ' 115(M-S7) 87 83 97 101 114 130 4-141 I 146 157(M-Hj l ' • ' ™ , ISO F i g . 14b Mass spectrum o f synthesized 4-keto VPA methyl ester 67. bond beta to the k'eto group (105). The ion doublet m/z 130, 133 a r i s e s from the McLafferty rearrangement i n v o l v i n g the carbonyl group (Scheme 2) . Other ion doublets appear at m/z 43, 46 and m/z 141, 147 (M-31) +. The s e r i e s of ions f o r a l i p h a t i c e sters (105) appear at m/z 59, 73, 87, 101 and 115 ( F i g . 14a). The low i n t e n s i t y of m/z 73 in comparison to the intense m/z 87 or intense m/z 115 i n d i c a t e s that branching occurs at the alpha carbon. Other keto-acids were t e n t a t i v e l y i d e n t i f i e d i n the methylated serum e x t r a c t . Peak 7 i n F i g . 10 was. t e n t a t i v e l y i d e n t i f i e d as 2-ethyl-4-keto pentanoic a c i d methyl e s t e r on the basis o f the mass spectrometric data [m/z 43 (100%), 101(40), 73(25), 130(15), 27(14), 57(13), 86(12), 115(10), 83(8), 99(7), 133(7), 69(6), 60(6), 127(5), 143(4), 46(3)]. The mass spectrum i s s i m i l a r to that o f 3-keto VPA and 4-keto VPA methyl e s t e r s . Ion doublets appear to occur at m/z 130, 133, m/z 57, 60 and m/z 43, 46. Peak 9 i n F i g . 10 was i d e n t i f i e d as 7-ketooctanoate by com-paring the mass spectrum of peak 9 [m/z 43 (100%), 45(62), 41(50), 115(43), 74(38), 57(33), 69(29), 27(17), 39(17), 87(16), 59(15), 83(13), 55(9), 85(9), 100(8), 101(8), 116(6), 125(5)] to that of methyl 7-ketooctanoate i n a r e g i s t r y of mass s p e c t r a l data (106). There was no evidence of ion doublets in the mass spectrum o f peak 9. Unsaturated-acid metabolites Four unsaturated metabolites were i d e n t i f i e d i n serum. Peak 6 in F i g . 10 was i d e n t i f i e d as 3-ene VPA methyl e s t e r by comparing the r e t e n t i o n time and mass spectrum ( F i g . 15a) with that of the methyl este r o f synthesized 3-ene VPA ( F i g . 15b). The ion doublets at 68. 1 5CH 97 2 2 F i g . 15a Mass spectrum of methyl 3-ene valproate-C H Q + H g) in peak 6 of F i g . 10. 113 127 100 CH,-CHrCH, ^H-COOCH, CH,-CH=CH' 97 ,\S. .IL i ,i 113 127 1 F i g . 15b Mass spectrum of methyl est e r o f synthesized 3-ene VPA TOOi 5CH 65 41 27 39 lli -I. ,l. 67 58 59 CHfCHi-CH V C—COOCH. (PoDtJ CHj-CH, CHf 95 127 69 75 81 87 I ,. II. 97 125 113 130 uo 141 - t l — L I 2 2 16a Mass spectrum of peak 8 (methyl 2-ene valproate - H Q + H £ in F i g . 10. KXh 55 41 27 |29 67 59 9 5 81 73 97 127 1 2 5 113 156 141 J _ m/2 too" 150 16b Mass spectrum of methyl est e r of synthesized trans 2-ene 70. 10(h 5 ( H 59 95 41 27 15 4J1 29 LL-]_I 55 43 67 50 73 82 iL M/Z 154(M+) 122 M L 100 111 1 125 139 jLJLtll 150 T 2 2 F i g . 17 Mass spectrum o f methyl ester of diene VPA - H Q + Eg in peak. 11 of F i g . 10. 71. m/z 156, 162 (M +) and ra/z 97, 103 (M-59) + r e f l e c t the presence o f 3-ene 2 VPA and 3-ene VPA- H g i n serum. Peak 8 i n F i g . 10 and peak 7 i n Fig.12 cor-2 2 respond to trans2-ene VPA-[ H Q + H g] methyl e s t e r s . The mass spectrum of peak 8, F i g . 10 i s shown together with the mass spectrum o f the methyl e s t e r of synthesized trans-2-ene VPA ( F i g . 16). The molecular i o n s , 2 m/z 156 and m/z 162 of 2-ene VPA and 2-ene VPA- H g e s t e r s r e s p e c t i v e l y , are of high i n t e n s i t y due to the conjugation o f the double bond to the carbonyl group. Although the mass spectra o f unsaturated p o s i t i o n a l isomers do not show q u a l i t a t i v e d i f f e r e n c e s due to the p o s s i b i l i t y of double bond m i g r a t i o n , some d i f f e r e n c e i n the i n t e n s i t i e s of s p e c i f i c ions are n o t i c e a b l e . Cleavage, of the carbon-to-carbon bond beta to the double bond of 2-ene VPA methyl e s t e r leads to a more intense m/z 127 (Fig.16) while cleavage of a carbon-to-carbon bond beta to the double bond of 3-ene VPA methyl e s t e r leads to a more intense m/z 113 ( F i g . 15). Peak 11 in F i g . 10 and peak 10 in F i g . 12 correspond to the diene VPA o f longer r e t e n t i o n time. The presence o f the molecular ion doublet m/z 154, 160 i n the mass spectrum o f peak 11 ( F i g . 17) proves unequivocably that the double bonds are not located on the terminal p o s i t i o n s of the propyl chain. Two p o s s i b l e s t r u c t u r e s can be postulated from the mass spectrum ( F i g . 17) f o r t h i s diene VPA and are shown below. CH 3-CH=CH X CH 3-CH=CH^ C-COOCH3 a b 72. Of the two s t r u c t u r e s , _b i s more l i k e l y to be the r i g h t s t r u c t u r e . The mass spectra o f a , 6 and 3 , y - u n s a t u r a t e d esters show d i f f e r e n c e s in r e l a t i v e ion i n t e n s i t i e s (107). Apart from the high i n t e n s i t y of the molecular ions o f a , 3 - u n s a t u r a t e s , the ions produced by cleavage of the carbon-to-carbon bond beta to the a,3-double bond or ft.Y-double bond can point to d i f f e r e n c e s between s t r u c t u r e a and s t r u c t u r e b_. Beta cleavage i n s t r u c t u r e b^  would produce m/z 139 (M-15) and a corresponding m/z 142 f o r the diene VPA- 2H g methyl e s t e r ( F i g . 17). Jakobs and Loscher (33) had pointed out that the presence o f fragment m/z 125 i n the mass spectrum o f the diene VPA a c i d i n d i c a t e s the l o s s o f a methyl group from the molecular i o n , m/z 140. The other diene-VPA isomer having the shorter r e t e n t i o n time was detected i n F i g . 10 and F i g . 12 by s e l e c t e d ion monitoring o f ion m/z 154 (M +). Figure 18 shows t y p i c a l mass chromatograms of s e l e c t e d ions corresponding to the TIC p l o t o f F i g . 10. TIC peaks which had c o r r e s -ponding superimposed peaks on the mass chromatograms of the unlabel!ed and l a b e l l e d compounds were considered to be the peaks of metabolites 2 ? o r i g i n a t i n g from VPA- H Q and VPA- Hg. Hydroxy-acid metabolites Although the hydroxy-acid metabolites could not be detected in the TIC p l o t o f the methylated serum e x t r a c t ( F i g . 10) probably because of i n t e r f e r i n g peaks from endogenous compounds, 4-OH VPA gamma lactone and 5-0H VPA d e l t a lactone were i d e n t i f i e d i n the u n d e r i v a t i z e d serum 2 2 e x t r a c t . The mass spectrum o f the 4-OH VPA-( H Q + Hg) lactones detected in serum e x t r a c t i s shown i n F i g . 19. C h a r a c t e r i s t i c ion doublets appeared at tp/z 100, 103 [M-42] + , m/z 113,116 [M-29] + and m/z 127, " ^ 1 6 0 -«-MJUA_WLAJUL»JUJLA. J L 6 i l l ' 7 ' " »t * J M LJL i_JLAiMjlUUL Fi g . 18. Mass chromatograms of methylated serum metabolites of VPA^D^+D )^- Peak numbers correspond to: 6= 3-eneVPA(m/z156),3-eneVPA-D6(m/zl62); 8=2-eneVPA(m/z156), 2-eneVPA-D6 (m/z 162); 10=DieneVPA(m/zl54); 11 =DieneVPA(m/z154), DieneVPA-D6(m/z160); 13= 4-keto\^ (m/z115),4-ketoVR -^D6(m/z118) 74. 10O| 5 C H 41 29 4 3 so 56 69 ,59 100 CH3—OH—CH—CH—C=0 ip3(M-42+03)+ 81 JJJL JJJ ,L 1 0 0 113 127 pp T42 - r ^ 1 2 2 F i g . 19 Mass spectrum of 4-OH VPA -( H Q + H g) lactone in serum ext r a c t . 75. 130 [M-15] + in the mass spectrum. Both metabolites were al s o found i n the methylated u r i n e e x t r a c t ( F i g . 12) and the t-BDMS-derivatized u r i n a r y e x t r a c t ( F i g . 20). The 3-OH VPA metabolite was detected in urine but not i n serum.' Figure 21 shows the mass spectrum of peak 15 in the TIC p l o t of F i g . 20 corresponding to the di-tBDMS d e r i v a t i v e of 3-OH VPA-( 2H Q+ 2H 6). Ion doublets appear at m/z 331, 337 [M-57] +, M/z 1 99, 205 [M-57-132]"1" and m/z 173, 176 (CHgCHgCH-OtBDMSV- C 2H 3CH 2 CH0tBDMS) +. Figure 22 shows the mass spectrum of peak 17 i n TIC p l o t of F i g . 20.corresponding to the di-tBDMS d e r i v a t i v e of 5-OH y.PA ( 2H Q+ 2H 5). C h a r a c t e r i s t i c ion doublets appear at m/z 331, 336 [M-57] + and m/z 199, 204 [M-57-132] +. D i c a r b o x y l i c acid-metabolites Three d i c a r b o x y l i c acids were confirmed as v a l p r o i c a c i d metabo-l i t e s i n the u r i n e e x t r a c t . These were 2 - p r o p y l g l u t a r i c a c i d (peak 18 i n F i g . 12, peak 18 in F i g . 20), 2 - p r o p y l s u c c i n i c a c i d (peak 14 in F i g . 12) and 2-propylmalonic a c i d (peak 14 in F i g . 20). The mass spectrum of peak 14 ( F i g . 12) corresponding to the methyl esters of 2-propyl-? 9-s u c c i n i c a c i d - ( H Q -h H 3) i s shown in Fig.23 t o g e t h e r wi th the'mass-spectrum of the o'd'imethyl e s t e r o f synthesized 2 - p r o p y l s u c c i n i c a c i d . Figure 24 shows the mass spectrum o f peak 14 ( F i g . 20) corresponding to the di-tBDMS 2 2 d e r i v a t i v e of 2-propylmalonic a c i d - ( H Q + H^) and the mass spectrum of thedi-tBDMS d e r i v a t i v e of synthesized 2-propylmalonic a c i d . GCMS a n a l y s i s of the TMS-derivatized e x t r a c t s from u r i n e and serum did not i n d i c a t e any new metabolites other than what had been previous-l y reported in the l i t e r a t u r e f o r a n a l y s i s of TMS-derivatized metabo-l i t e s o f VPA (16), However, one peak f r e q u e n t l y appeared between the 76. 15 I 10 14 J. A 1 1 I 1 1 I I I I I I I I I I I I I 1 I t I I ^ | | 20 1 0 0 0 T I M E (sec.) 2 0 0 0 3000 GCMS a n a l y s i s ( t o t a l ion current p l o t ) o f t-BDMS s i l y l a t e d human urine e x t r a c t from healthy volunteer on VPA m u l t i p l e dosage a f t e r a d m i n i s t r a t i o n of VPA- 2H 6 (600 mg). Column used, 3% OV-17 (6mm x 2mm i . d . ) . Temp, program: 80°C to 280°C at 8°C per min. Peak numbers correspond t o : 1 = b u t y r i c a c i d , 2 = 2-methylbutyric a c i d , 3 = unknown, 4 = v a l p r o i c a c i d (VPA), 5 = cre s o l , 6 = 2-ene VPA, 7 = l a c t i c a c i d 3 , 8 = g l y c o l i c a c i d 3 , 9 = diene VPA, 10 = benzoic a c i d , 11 = 4-keto VPA, 12 = 3-hy d r o x y i s o v a l e r i c a c i d 3 , 13 = 3-hydroxybutyric a c i d , 14 = propylmalonic a c i d , 15 = 3-OH VPA, ^ 16 = 4-OH VPA, 17 = 5-OH VPA, 18 = 2 - p r o p y l g l u t a r i c a c i d , 19 = m-hydroxyphenylacetic a c i d , 20 = p-hydroxyphenylacetic a c i d (a - t e n t a t i v e l y i d e n t i f i e d ) . 10Ch 50H 73 41 f 59 147 97 115 T17 1P3 133 173 H76, F i g . 2 1 Mass spectrum of di-tBDMS d e r i v a t i v e of 3 - O H V P A - C ^ H Q + in urine ex t r a c t 205 „ 247 241. ^ TiL» i- r * 1 i , i i . • i 331(M+-67) ,337 Cfe • M!57 TOCh 504 73 55 147 97 .75 jiiMsl^iii 173 331(M^7) 241, 315 2 2 F i g . 2 2 Mass spectrum o f di-tBDMS d e r i v a t i v e of 5-OH VPA-(.H 0 + E g) in urine e x t r a c t . 78. 5CH m 55 45 41 29 i 59 97 87 157 127 M6 u F i g . 23a Mass spectrum o f peak. 14 In F i g . 20 corresponding to the. dimethyl e s t e r of 2 - p r o p y l s u c c i n i c a c i d - ( 2 H 0 + 2H 3) I i 5CH 55 27 29 59 ^0 TU 97 87 83 157 146 128 "Vi i b o *0 Ik-F i g . 23b. Mass spectrum of dimethyl ester of synthesized 2-propyl s u c c i n i c a c i d . 79. 100i 55CH C M J - C H J C H J - C H ^ XV 317.M-57 289 250 900 3M I (M-57*D3)* M WO ISO M / z200 '250" ' ' W 24a Mass spectrum of peak 14, F i g . 21 corresponding to the di-tBDMS d e r i v a t i v e ; o f 2-propylmalonic acfd-C2'HQ + 2H 3) 55 27 147 75 05 «29| 11«57 **** tn. 317 %>' ' 250 - bfe 1 1 350 2 4 b a c i d S p e C t r U m ° f d l"- t B D M S d e r i v a t i v e o f synthesized 2-propylmal omc 80. di-TMSderivative of 5-OH VPA and di-TMSderivative of p-hydroxyphenyl-a c e t i c acid peaks on the 3% 0V-17 column. Its mass spectrum i s shown in F i g . 25. The peak may be a s c r i b e d to the dihydroxy VPA metabolite suggested by Kochen et al_ (36) who noted a TMS-derivatized compound with m/z 377 and m/z 392 i n the mass spectrum. The molecular weight of a :tri-TMS dihydroxy VPA d e r i v a t i v e w i l l be 392. The m/z 377 [M-15] + i s present i n the mass spectrum ( F i g . 25). The ion m/z 292 i s a rearrangement ion formed from thetri-JMS d e r i v a t i v e of a dihydroxy-c a r b o x y l i c a c i d (92, 108). The ions m/z 147 and m/z 221 are analogues of m/z 73 (see below) and ( C H 3 ) 3 Si S i ( C H 3 ) 3 " l + ( C H 3 ) 3 Si + ° x y ° m/z 73 | H ( C H3>3 H + i s i ( C H 3 ) 3 0 = S i ( C H 3 ) 9 m/z 147 m/z 292 ( C H 3 ) 3 S i x 0-Si(CH.) 9 0 + = S i ( C H 3 ) 2 m/z 221 t h e i r presence i n d i c a t e s a t r i - T M S ; d e r i v a t i v e (108). The intense m/z 117 could be l a r g e l y due to [CH 3CH-0TMS] + in a 4-hydroxy a c i d rather than [C0-0TMS] + while m/z 120 could be due to [C 2H 3-CH-0TMS] +. Although the ions [M-89] + and [M-90] + are absent i n the mass spectrum, the ion m/z 197 could be [M-l5-(2x90)] + and m/z 122 could be [M-(3x90)] + due to 1001 73 5CH 27 58 i55 117 h22 75 100 9* 147 t!35 129 hi W-13=377 f a |269 2923001 fljiJH \\ 1) J . i i H , , , 377 I - i — i — 4 F i g . 25 EI Mass Spectrum of tri-TMS d e r i v a t i v e of di-OH VPA i n TMS d e r i v a t i z e d u r i e x t r a c t . 82. loss o f two or three t r i m e t h y l s H a n o i molecules from the molecular ion. 0 83. IV. DISCUSSION A. Deuterium-labelling of v a l p r o i c a c i d The p o s i t i o n of deuterium l a b e l l i n g i n v a l p r o i c a c i d i s important i f the deuterium-labelled drug i s to be used i n pharmacokinetic and. . metabolism st u d i e s of v a l p r o i c a c i d . Incorporation of deuterium should be at a chemically and m e t a b o l i c a l l y s t a b l e p o s i t i o n so that there i s no s i g n i f i c a n t isotope e f f e c t on the d i s t r i b u t i o n and metabolic k i n e t i c s o f v a l p r o i c a c i d a f t e r i n t r o d u c t i o n of deuterium. The mass fragmentat-ion pattern o f the deuterium-labelled v a l p r o i c a c i d must a l s o be con-s i d e r e d since i t i s imperative to use mass spectrometric d e t e c t i o n f o r q u a n t i t a t i o n andto ;ldcate ion doublets f o r i d e n t i f i c a t i o n of metabol i t e s . From previous studies on the metabol ism of val proic-~acid (16), i t i s known that o x i d a t i o n occurs a l l along the carbon chain o f v a l p r o i c a c i d except at the alpha carbon ( F i g . 1). Deuterium at the alpha carbon Would be in a l a b i l e p o s i t i o n s i n c e hydrogen-deuterium exchange can occur r e a d i l y at that p o s i t i o n . Oxidation at the beta carbon produces the major u r i n a r y m e t a b o l i t e s , 3-keto VPA and 3-OH VPA. Thus the omega and penultimate carbons are probably the most favourable p o s i t i o n s f o r deuterium attachment, Von Unruh et al_ (64) have synthesized d i - ( 2 , 3 - H 2-propyl ) a c e t i c a c i d with deuterium at the omega and penultimate carbons. They used the l a b e l l e d analogue to study the steady-state k i n e t i c s of v a l p r o i c acid in combined a n t i e p i l e p t i c drug therapy. However, they d i d not report the e f f e c t s of d e u t e r i u m - l a b e l l i n g on the formation o f 4-OH VPA-2 H 4 and 5-OH VPA- 2H 4. In the present study i t has been demonstrated that v a l p r o i c a c i d 84. 2 - Hg, synthesized with s i x deuterium atoms introduced at the two methyl groups, does not e x h i b i t a s i g n i f i c a n t isotope e f f e c t with respect to i t s pharmacokinetics. The r a t i o o f u r i n a r y metabolites and serum l e v e l s of 2 v a l p r o i c a c i d to those o f v a l p r o i c a c i d - Hg were determined. A meta-2 b o l i c isotope e f f e c t on the formation o f 5-OHVPA- Hg was c l e a r l y present but d i d not markedly a f f e c t the d i s t r i b u t i o n and e l i m i n a t i o n o f k i n e t i c s of v a l p r o i c a c i d a f t e r deuterium l a b e l l i n g . The formation of 5-OHVPA i s a minor metabolic pathway compared to g l u c u r o n i d a t i o n o f VPA, B - o x i d a t i o n and (w-1)-oxidation (16). 2 V a l p r o i c a c i d - Hg was a l s o useful i n t r a c i n g metabolites of v a l -p r o i c a c i d by the ion-doublet technique. Von Unruh et _al_ (64) reported 2 that the methyl e s t e r o f VPA- H^ synthesized produced a mass spectrum with McLafferty rearrangement ions ra/z 117, 118 (M-42) + i n v o l v i n g 1 2 2 e i t h e r ^ or H-j t r a n s f e r . When methyl esters o f v a l p r o i c a c i d - Hg and i t s metabolites were analyzed using t h e i r mass s p e c t r a , the McLafferty rearrangement ions or other fragmentation pathways did not 1 2 in v o l v e competition between H^  or H^  t r a n s f e r . 2 The number of deuterium atoms i n VPA- Hg reduces the i n t e r f e r e n c e from the natural i s o t o p i c c o n t r i b u t i o n i n u n l a b e l l e d v a l p r o i c a c i d to 2 m/z 207 o f the t-BDMS d e r i v a t i v e o f VPA- H g used in the q u a n t i t a t i o n . 2 The r e t e n t i o n time of v a l p r o i c a c i d and v a l p r o i c a c i d - Hg t-BDMS d e r i v a t i v e s on the 3% Dexsil 300 column were almost i d e n t i c a l . The number of deuterium atoms al s o ensured that under electron-impact mass 2 spectrometric c o n d i t i o n s , some fragment ions of metabolites of VPA- Hg w i l l contain at l e a s t two deuterium atoms to show the ion doublets that are valuable i n i d e n t i f y i n g VPA metabolites. 85. B. Synthesis o f v a l p r o i c a c i d and i t s metabolites V a l p r o i c a c i d synthesis was f i r s t published in the l i t e r a t u r e by Burton (1). The method involved d i a l k y l a t i o n of e t h y l a c e t o a c e t a t e , followed by cleavage of the a c e t y l group by concentrated a l k a l i and then a c i d i f i c a t i o n . The method of synthesis used in t h i s study was the d i a l k y l a t i o n of malonic e s t e r . The malonic e s t e r synthesis o f f e r s one of the most convenient and commonly used procedures f o r the synthesis of medium chain saturated f a t t y a cids (109,110). Sodium ethoxide-ethanol provided an e f f e c t i v e base-solvent system to form the intermediate ester enolate and i t has been used by many workers (109-111). Other base-solvent systems t r i e d were NaH/THF and NaH/ HMPA (95), l i t h i u m N-isopropylcyclohexylamide/THF (112) and the non-solvent system, I^CO^-Crown ether (113). The y i e l d s of v a l p r o i c a c i d using these bases were comparable to the y i e l d of v a l p r o i c a c i d using sodium ethoxide. Sodium ethoxide was chosen because o f the a v a i l a b i l i t y of sodium and ethanol. From the p r e l i m i n a r y experimentation with u n l a b e l l e d propyl bromide, the procedure chosen was the two-step d i a l k y l a t i o n method (Scheme 1, page 44) without i s o l a t i n g the monoalkylated product, diethylpropylmalonate. The one-step d i a l k y l a t i o n method gave comparatively lower y i e l d s of the f i n a l product and an a p p r e c i a b l e amount o f side product, ethylpropylmalonate. 2 In the two-step d i a l k y l a t i o n f o r synthesis o f v a l p r o i c a c i d - Hg, small 2 amounts of the side product, propylmalonic a c i d - H^ (useful f o r r e c y c l i n g ) was recovered in the aqueous phase by e x t r a c t i o n with ether. Propylmalonic a c i d i s more s o l u b l e in water than dipropylmalonic a c i d . The method chosen, although convenient and p r a c t i c a l , n e e d s optimal amounts of reagents, solvent and a l k a l i ( f o r s a p o n i f i c a t i o n ) i f higher y i e l d s are to be gained for production o f the branched chain f a t t y a c i d . 86. 3_-Hydroxy VPA, I I I , was synthesized from propionaldehyde and e t h y l -v a l e r a t e using Rathke'sQ 14) method f o r s y n t h e s i z i n g B-hydroxyesters (Scheme 3). The base used to form the e s t e r enolate was l i t h i u m N-isopropylcyclohexylamide. The r e a c t i o n i s performed at lower temperature and i s l e s s time-consuming compared to the Reformatsky r e a c t i o n between aldehydes and a - h a l o e s t e r s i n the presence o f z i n c . Although 3-hydroxy-acids have a propensity to form B - l a c t o n e s , the NMR and IR spectra of the synthesized 3-OH V i n d i c a t e d the hydroxy-acid. The y i e l d f o r 3-OH VPA synthesis was only 40% probably due to the presence of hexane i n the b u t y l l i t h i u m reagent which decreased the s o l u b i l i t y of the l i t h i u m amide and l i t h i u m e s t e r enolate. 4- Hydroxy VPA l a c t o n e , IV, i s the lactone formed during the synthe-s i s of 4-0H'yPA. It was prepared by f o l l o w i n g the procedure of Schafer and Luhrs (28) using diethylpropylmalonate and propylene oxide (Scheme 4). The y i e l d f o r 4-OH VPAlactone synthesis was i d e n t i c a l to the y % e l d r e p o r t e d by Schafer and Luhrs (28). 5- Hydroxy VPAlactone, V, was synthesized by the method of Schafer and Luhrs (28) using diethylpropylmalonate and 3-bromo-l-propanol (Scheme 5). The y i e l d obtained i n t h i s r e a c t i o n was s l i g h t l y lower than the y i e l d of 57% reported by Schafer and Luhrs (28). The lower y i e l d was thought to be due to the s u s c e p t i b i l i t y o f 5-OH VPA l a c t o n e towards decomposition during d i s t i l l a t i o n at reduced pressure.Delta-lactones have been reported to polymerize more e a s i l y than gamma-1actones (115). The s i d e products formed during d i s t i l l a t i o n probably occurred during depolymerization by heat. 3-Ene VPA, VI, was prepared in two ways. One method involved H / C H 3 0< -N-CH + C 4H 9L,. ^ C H 3 Li CH, -N -CH \ CH, Li CH. -N -CH + C,H 7CH 9C00Et ~'* L > \ 6 1 c THF \ H 3 L i C 3H 7CHC00Et + CH 3CH 2CH0 -78°C THF C 3H 7-CH-C00Et ( i ) KOH / CH 0 / ( i i ) HC1 CH-OH -> C3H7-CH-C00H / CH-OH CH 3 III Scheme 3 - Synthesis o f 3-OH VPA ( I I I ) CH3-CH-^H2 c H C 0 0 E t C 3H 7CH(C00Et) 2 NaOEt-EtOH > CH 3-CH-CH^C=0 A , 3hr 0 1 C ?H 7 ( i ) NaOH, A \ > CHo-CH-CHo-CH-C=0 l o — O I I - I I  ( i i ) H 2S0 4,A(Shr) ' IV Scheme 4 - Synthesis o f 4-OH.VPA lactone (IV) dehydration of 2-propyl-3-hydroxypentanoate using phosphorus pentoxide according to B l a i s e and Bagard (96), The i s o l a t e d f i n a l product con-s i s t e d of a mixture of 3-ene VPA and 2-ene VPA (3:1), Both unsaturated acids c o n s i s t e d of c i s - t r a n s isomers, B l a i s e and Bagard (96) had a l s o reported a mixture o f 3-ene VPA and 2-ene VPA by the dehydration route. 3-Ene VPA and 2-ene VPA could not be e f f i c i e n t l y separated using a 30 cm-fradtionating column. An a l t e r n a t i v e method f o r the preparation of 3,Y-unsaturated esters i s the deconjugative a l k y l a t i o n of the enolate of an a,B-unsaturated e s t e r . 3-Ene VPA was synthesized using the decon-j u g a t i v e a l k y l a t i o n method of Hermann et al_ (116) where l i t h i u m d i i s o -propylamide was the base used (Scheme 6). GCMS a n a l y s i s o f the f i n a l product showed 3-ene VPA to 2-ene VPA r a t i o g r eater than 10:1. NMR of the product showed 3-ene VPA as the major product with t r a c e peaks appearing due to the presence of 2-ene VPA (see appendix). The l i t h i u m amide was found to be an e f f e c t i v e base f o r t h i s method. I t has al s o been used to convert 2-alkenoic acids to 3-alkenoic acids (117), 2-Ene VPA, VII, has been synthesized using two d i f f e r e n t methods, Dehydrobromination of 2 - b r o m o - 2 - p r o p y l v a l e r i c a c i d ethyl ester with N,N-diethylamine, followed by s a p o n i f i c a t i o n and a c i d i f i c a t i o n gave 2-ene VPA (96,118) with small amounts of 3-ene VPA and 4-OH VPA lactone (96). Neuman and Holmes (97) thermally decomposed 2-hydroxy-2-propyl!-pentanoic a c i d to obtain c i s and trans isomers of 2-ene VPA, In the present study, a procedure was required to prepare a deuterated 2-ene VPA from 4-heptanone- H4. Consequently the cyanohydrin of 4-heptanone was prepared but thermal dehydration of 2-hydroxy-2-propylpentanotc a c i d d i d not give q u a n t i t a t i v e y i e l d s of 2-ene VPA, Ac c o r d i n g l y the cyanohydrin of 4-heptanone was dehydrated to 2 - p r o p y l - 2 - p e n t e n e n i t r i l e / C,H7-CH 3 / \ COOEt COOEt HOCH 2CH 2CH 2Br NaOEt-EtOH A,3hr C QH 7 COOEt H0CH 2CH 2CH 2 ^OOEt C,H 7 COOH ( i ) NaOH,A ^  CH7-CH7-CH?-C-C=0 ( i i ) H 3P0 4,A > u ' C 3 H 7 CH2-CH2-CH2-CH-C=0 to l u e n e / p y r i d i n e Scheme 5 - Synthesis o f 5-OH' ;VPA lactone (V) CH, CH, 3\ / 0 0° CH-NH-CH + C,H„Li ^ > CH, Li / C H 3 CH / 4"91-' THF CH, CH, NCH-N-CH CH, CH3CH2CH=CH-C00Et HMPA/THF -78°C Li CH3CH2CH=C-COOEt CH 3CH 2CH 2Br -78*0 CH3CH=CH. CHCOOEt CH 3 -CH 2 -CH 2 ( i ) NaOH ( i i ) HC1 CH 3-CH=CH X CH-COOH VI Scheme 6 - Synthesis o f 3-ene VPA (VI) CH 3~CH 2-CH 2 90. by phosphorus o x y c h l o r i d e (Scheme 7) using the procedure of Bratfde and Wheeler (119) to form a,3-unsaturated n i t r i l e s . H y d r o l y s i s of the n i t r i l e to the a c i d could not be e f f e c t e d with a l k a l i o r concentrated h y d r o c h l o r i c a c i d . H y d r o l y s i s o f the a - p r o p y l -p e n t e n e n i t r i l e with 60% s u l f u r i c a c i d gave the amide e a s i l y a f t e r 3 hr. Further h y d r o l y s i s f o r 2 hr had to be e f f e c t e d to get complete conversion to 2-ene VPA. The f a i l u r e of a l k a l i n e h y d r o l y s i s or h y d r o l y s i s with h y d r o c h l o r i c a c i d could be due to the s t e r i c i n h i b i t i o n posed by the propyl group at the alpha-carbon as observed f o r some branched-chain a l i p h a t i c n i t r i l e s (120). In view of the amide r e s i s t a n c e to hydroly-s i s , the longer h y d r o l y s i s times increased the tendency of 2-ene VPA to l a c t o n i z e to 4-0H: VPA 1 actone. 4-OH VPAy-1actone i s a l s o reported to be formed i n the dehydrobromination of 2-bromo-2-propylpentanoate where a l k a l i i s used to hydrolyze the unsaturated e s t e r (96). In the synthe-t i c method used in t h i s study, 4-OH VPA lactone i s the only s i d e product -compared to formation of two s i d e products, 3-ene VPA and 4-QH VPA lactone i n the dehydrobromination r e a c t i o n (96). NMR. of the d i s t i l l e d product d i d not show the presence of 3-ene VPA (see Appendix) and the 4-OH VPA y-lactone was removed by f r e e z i n g the d i s t i l l e d product. When the product was s t o r e d ' at -20°C,higher - m e l t i n g trans-2-ene VPA cpuld be p a r t i a l l y separated from the cis-2-ene VPA as reported (96). 2 Synthesis of 2-ene VPA- H 3 (VIII) took the same r e a c t i o n route as 2-2 VPA, using heptanone- H 4 of deuterium c o n t e n t g r e a t e r than 95%, The 2 synthesized 2-ene VPA- H 3 had an i s o t o p i c deuterium composition c l o s e to 95%. The NMR'indicated the v i r t u a l absence of peaks at the chemical s h i f t value.( r e l a t i v e to i n t e r n a l marker) 6 of 2,4-2.7 due to -CH9-C= and 6 of 5.9-7.1 due to CH=C (Appendix), 9 1 C=0 + HCN KCN ( c a t a l y s t ) > C H , C H 9 C H 0 / / 3 2 2 C H 3 C H 2 C H 2 X /OH CH 3CH 2CH^ C-CN n n r , . — r - p — ^ C-CN y P 0 C l 3 / p y n d i n e / CH 3CH 2CH 2 CH 3CH 2CH 2 CH 3CH 2CH 6 0 % H 2 S 0 4 \-COOH CH 3CH 2CH 2 VII Scheme 7 Synthesis o f 2-ene VPA (VII) C H 3 C H 2 C H 2 X NaOD7D?0 C H 3 C H 2 C D 2 X ^ C=0 >. C=0 CH 3CH 2CH 2 / / CHgCHgCDg^ 4-Heptanone- 2H 4 CH 3-CH 2-CD^ OCOOH * CH 3~ CH 2 _ CfJ^ VIII 9 2 . 4-Ene VPA, IX ,was p r e p a r e d f o l l o w i n g t h e p r o c e d u r e o f Campos and Amara l C98) by a l k y l a t i n g a l l y ! m a l o n i c e s t e r w i t h 1-bromopropane i n t h e p r e s e n c e o f sod ium e t h o x i d e (Scheme 8 ) . The NMR s p e c t r u m o f t h e f i n a l p r o d u c t con fo rmed t o t h e s t r u c t u r e o f 4-ene VPA. The 4-ene VPA d i d no t fo rm 4-OH V P A l a c t o n e n o r i s o m e r i z e ' t o 3-ene VPA o r 2-ene VPA d u r i n g s y n t h e s i s . 3- Ke to VPA e t h y l e s t e r , X, was s y n t h e s i z e d f rom e t h y l v a l e r a t e and p r o p i o n y l c h l o r i d e u s i n g t h e p r o c e d u r e o f Ra thke and D e i t c h ( 1 2 1 ) . Use o f l i t h i u m N - i s o p o p y l c y c l o h e x y l a m i d e e n a b l e d a c y l a t i o n (Scheme 9) o f t h e e s t e r e n o l a t e t o be pe r f o rmed d u r i n g a s h o r t r e a c t i o n t i m e wh i ch combined w i t h t h e low r e a c t i o n t e m p e r a t u r e m i n i m i s e d t h e r a t e o f d i a c y l a t i o n o f t h e e s t e r and t h e s e l f - c o n d e n s a t i o n o f e t h y l v a l e r a t e . The y i e l d o b t a i n e d was h i g h e r than t h e r e p o r t e d v a l u e ( 2 8 ) . 4- j(eto VPA, X I , has been s y n t h e s i z e d by a l k y l a t i o n o f d i e t h y l -p r o p y l m a l o n a t e w i t h 2 , 3 - d i b r o m o - l - p r o p e n e ( 9 9 ) . Fo r t h e p r e s e n t s t u d y , 4-ketoVPA was s y n t h e s i z e d f rom e t h y l 2 - b r o m o v a l e r a t e and e t h y l a c e t o -a c e t a t e (Scheme 10) due t o t h e a v a i l a b i l i t y o f t h e r e a g e n t s . T h i s s y n t h e t i c pathway was been used f o r t h e s y n t h e s i s o f y - k e t o a c i d s ( 1 2 2 ) . When sod ium e t h o x i d e was used as t h e b a s e , l o w e r y i e l d s o f 4-ketoVPA and s i g n i f i c a n t amounts o f 2 - p r o p y l s u c c i n i c a c i d were f o r m e d . The f o r m a t i o n o f 2 - p r o p y l s u c c i n i c a c i d , i s supposed t o o c c u r d u r i n g a l k y l -a t i o n o f a 3 - k e t o e s t e r i n t h e p r e s e n c e o f sod ium e t h o x i d e by c l e a v a g e o f t h e 3-keto e s t e r a t t h e ke tone f u n c t i o n ( 1 2 3 ) . The 2 - p r o p y l s u c c i n i c a c i d was no t d e t e c t e d when NaH was used as t h e base i n t e t r a h y d r o f u r a n . The f i n a l c r u d e p r o d u c t showed t h e main component t o be 4 - k e t o VPAJby .GCMS.. When d i s t i l l a t i o n o f t h e c r u d e p r o d u c t was c a r r i e d ou t under r educed p r e s s u r e , d e c o m p o s i t i o n o c c u r r e d upon a p p l i c a t i o n o f s u f f i c i e n t / C 0 0 E t CH 3CH 2CH 2Br CH2=CH-CH2-CHx NaOEt-EtOH > COOEt A,3hr C H 3 C H 2 C H 2 \ . C O O " ( i ) K O H CH2=CHCH2 / COOEt H C 1 C H 3 C H 2 C H 2 X ^COOH ^ C H ^ C H ^ yZ\ A d S O ^ O O ^ ) 5 , CHCOOH CH 2=CHCH 2 X COOH CH^CHCH,' IX Scheme 8 Synthesis o f 4-ene VPA (IX) o°c H -N-CH^ + C 4 H g L i T H p CH 3 •N - C H + C , H 7 C H 9 C 0 0 E t "1° S \ 3 7 2 T H F C H 3 Ll 7 P ° r C 3 H 7 C H - C 0 0 E t + C H 3 C H 2 C 0 C 1 ~ T H F > C QH 7-CH-COOEt / C H 2 0 CH, Scheme 9 Synthesis o f 3-keto VPA ethyl e s t e r (X) f 3 H 7 CH 3-C-CH 2C00Et + Br-CH-COOEt ^ ^ a V7 CH,-C-CH-CH-COOEt COOEt Cone HC1 il ?3 H7 CH 3-C-CH 2-CH—COOH XI Scheme 10 Synthesis o f 4-keto VPA (XI) ^ COOEt ClCH 2CH 2COOEt C 3H y-CH NaOEt-EtOH * COOEt A,5hr EtOOC-CH 9CH 0 .COOEt ( 1 ) KOH, A / C \ ( i i ) H 2S0 4 CH 3CH 2CH 2 / COOEt H00C-CH 2CH 2 COOH _ C Q HQOC-CHg-CH^ 2 .CHCOOH / \ —~—> / CH 3CH 2CH 2 COOH A CH 3CH 2CH 2 XII Scheme 1 1 Synthesis o f 2 - p r o p y l g l u t a r i c a c i d (XII) / C 0 0 E t CICHoCOOEt CoH^CH > NaOEt-EtOH A ,3*7 v,n COOEt EtOOC. ^ C H , .COOEt ( i ) KOH, A / C \ ( i i ) H 2 S 0 4 C H 3 C H 2 C H 2 COOEt M00C-CH 2 COOH _ C Q HOOC-CH2 X C ^ 2 CH-COOH / \ > / C H 3 C H 2 C H 2 COOH A C H 3 C H 2 C H 2 XIII Scheme 12 Synthesis o f 2 - p r o p y l s u c c i n i c a c i d ( X I I I ) / C 0 0 E t ( i ) NaOH,A/. / C 0 ° H C,H,CH > C,H 7 CH 0 n 7 u i * O , M 7 O I I COOH XIV Scheme 13 Synthesis o f 2-propylmalonic a c i d (XIV) heat. GCMS o f the d i s t i l l e d product showed the presence of a.ppreciabl e amounts of an unsaturated y-lactone with molecular ion at m / z 140. The dehydration o f y-keto a c i d to unsaturated y-lactones has been reported to occur during d i s t i l l a t i o n and i s catalyzed by the ac i d i t -s e l f or mineral acids at high temperature (124, 125). The dehydration of l e v u l i n i c a c i d i s reported to give 3,y-unsaturated lactone and a,3-unsaturated lactone (124) while a y,6-unsaturated lactone i s al s o reported to be formed i n a d d i t i o n to the two unsaturated lactone (125). The NMR o f the d i s t i l l e d product (see Appendix) i n d i c a t e d the presence of 4-keto VPA contaminated with probably the g;,y-unsaturated lactone. 2 - P r o p y l g l u t a r i c a c i d , XII, was prepared from propylmalonate and 3-chloropropionate (Scheme IT) using the method of Yamakawa (101). The product was obtained i n moderate y i e l d using sodium ethoxide as the base. 2 - P r o p y l s u c c i n i c a c i d , XIII, was synthesized by a l k y l a t i n g d i e t h y l -propylmalonate with chloroacetate (Scheme 12) using the method of Yamakawa (101) to synthesize d i c a r b o x ^ y l i e a c i d s . 2-Propylmalonic a c i d , XIV, was obtained by h y d r o l y s i s of diethylpropylmalonate (Scheme 13). C. Pharmacokinetic Study Assay The assay used in t h i s study was developed by Abbott e_t al_ (94) f o r determination of v a l p r o i c a c i d i n s a l i v a and serum. The i s o l a t i o n procedure involed a s i n g l e e x t r a c t i o n step and the e x t r a c t i o n solvent was also used to e x t r a c t the t-BDMS d e r i v a t i v e s from the dimethyl forma-mide l a y e r (solvent c o n t a i n i n g t e r t - b u t y l d i m e t h y l c h l o r o s i l a n e and imi d a z o l e ) . The high s e n s i t i v i t y and p r e c i s i o n of the s i n g l e ion 98. monitoring assay used f o r v a l p r o i c a c i d (94) was not s u b s t a n t i a l l y decreased when m u l t i p l e ion monitoring was used to determine both v a l p r o i c a c i d and v a l p r o i c a c i d - Hg. The a d d i t i o n a l i o n , m/z 207 monitored was of r e l a -2 t i v e l y high abundance and v a l p r o i c a c i d - H g t-BDMS ester had s i m i l a r r e t e n t i o n time to v a l p r o i c a c i d t-BDMS e s t e r . 2 The high deuterium content of synthesized v a l p r o i c a c i d - Hg and 2 s i x mass d i f f e r e n c e between m/z 201 and m/z 207 allowed v a l p r o i c a c i d - Hg to be quantitated i n serum i n the presence of high concentrations of unlabelled- v a l p r o i c a c i d with minimal i n t e r f e r e n c e from the u n l a b e l l e d drug. The lower l i m i t o f s e n s i t i v i t y was 0.1 ug/ml and s a l i v a l e v e l s 2 of v a l p r o i c a c i d or v a l p r o i c a c i d - Hg were determined with higher p r e c i s i o n than reported GC methods f o r measuring s a l i v a v a l p r o i c a c i d l e v e l s (22, 74). K i n e t i c s - S i n g l e Dose and M u l t i p l e Dose To understand the k i n e t i c s o f v a l p r o i c a c i d a f t e r s i n g l e dose and 2 m u l t i p l e doses, v a l p r o i c a c i d - Hg has been used to detect changes i n the e l i m i n a t i o n k i n e t i c s during m u l t i p l e dosing with u n l a b e l l e d v a l -p r o i c a c i d . The s i n g l e dose e l i m i n a t i o n h a l f - l i f e observed f o r v a l p r o i c a c i d and y a l p r o i c a c i d - 2 H g are p r a c t i c a l l y i d e n t i c a l (Table I I I ) and are com-parable to the 15.9 ± 2.6 hr reported by Gugler et a l (22). The apparent 2 volume of d i s t r i b u t i o n of v a l p r o i c a c i d or v a l p r o i c a c i d - Hg i s a l s o comparable to values o f 0.13 - 0.18 l i t e r / k g obtained by Gugler et al (22) 1n t h e i r s i n g l e dose s t u d i e s . The t o t a l body clearance of v a l p r o i c a d d (0.0070 l i t e r / h r / k g ) and v a l p r o i c acid- 2H 6(0.00.75 l i t e r / h r / k g ) i n the s i n g l e dose studies are s i m i l a r to t o t a l body clearance values of 0.0064 ± 0.0011 l i t e r / h r / k g reported by Gugler et a]_ (22). 99. Under m u l t i p l e dose c o n d i t i o n s , Gugler e_t al_ (22) reported that the terminal plasma h a l f - l i f e o f v a l p r o i c a d d (17.3 ± 3.0 hr) was not s i g n i f i c a n t l y d i f f e r e n t from t h e i r s i n g l e dose values. In the present 2 study the serum t o t a l VPA- Hg e l i m i n a t i o n h a l f - l i f e was shorter at steady state. The s i m i l a r h a l f - l i f e values obtained f o r serum t o t a l VPA- 2Hg (14.3 hr) and serum t o t a l VPA + VPA- 2H g (13.5 hr) was evidence that v a l p r o i c a c i d - 2 H g i s disposed of by the body in k i n e t i c a l l y i d e n t i c a l fashion to the u n l a b e l l e d v a l p r o i c a c i d . I d e n t i c a l k i n e t i c behaviour of 2 v a l p r o i c a c i d and v a l p r o i c a c i d - Hg i s also evident i n the AUC data 2 under m u l t i p l e dose c o n d i t i o n s . The serum t o t a l AUC ^ f o r VPA- Hg (900 m g / h r / l i t e r ) i s e s s e n t i a l l y i d e n t i c a l to the VPA + VPA- 2H g AUC (872 m g / h r / l i t e r ) as i s expected from a p p l i c a t i o n o f the T1" T2 P r i n c i p l e of Sup e r p o s i t i o n where AUC Q_ o oafter f i r s t dose i s equivalent to AUC at steady s t a t e (125). The assumption underlying t h i s p r i n c i p l e T1' T2 i s that the drug does not change in k i n e t i c behaviour ( i . e . l i n e a r ) a f t e r f i r s t dose and during steady s t a t e c o n d i t i o n s . Thus the s i n g l e dose 2 of v a l p r o i c a c i d - Hg, given at steady s t a t e and subjected to p r e d i s -posing f a c t o r s as f o r the u n l a b e l l e d v a l p r o i c a c i d , i s eliminated i n an i d e n t i c a l fashion to the u n l a b e l l e d v a l p r o i c a c i d at that time, In t h i s regard'the hexadeuterated.valproic a c i d can be useful both f o r d e f i n i n g the steady s t a t e parameters and f o r determining possibl e changes i n the k i n e t i c parameters of" VPA -iri-' !patients during drug i n t e r a c t i o n studies without stopping therapy. 2 The observed increase in ithe t o t a l body.clearance o f v a l p r o i c a c i d - Hg (Table 111 X-imay-;best t r e f l e c t the change i n k i n e t i c s from .single dose to mul-t i p l e dose co n d i t i o n s s i nee clearance i s a parameter that i s model-independent (127-129). The change i n t o t a l body clearance i s not l i k e l y due to a TOO, reduction i n the amount of drug absorbed since the oral b i o a v a i l a b i l i t y o f v a l p r o i c a c i d i s n e a r l y 100% (18, 19). There i s the p o s s i b i l i t y . that the 33% increase in t o t a l body clearance i s due to e i t h e r an en-zyme-inducing e f f e c t o f v a l p r o i c a c i d or a change i n binding of v a l p r o i c a c i d to serum pro t e i n s or both. On the enzyme-inducing potency of v a l p r o i c a c i d , several i n v e s t i -gators (130, 131) have reported that the a n t i p y r i n e h a l f - l i f e and u r i n a r y D - g l u c a r i c a c i d e x c r e t i o n in p a t i e n t s treated with sodium v a l -proate were not d i f f e r e n t from those in drug-free c o n t r o l s . There has been one study (132), which reported that v a l p r o i c a c i d increased s i g n i -f i c a n t l y D - g l u c a r i c a c i d u r i n a r y excretion but not gamma-glutamyltrans-peptidase a c t i v i t y i n e p i l e p t i c c h i l d r e n . V a l p r o i c a c i d has a low e x t r a c t i o n r a t i o (18) which means that only the unbound drug can be c l e a r e d from the l i v e r and the e l i m i n a t i o n process i s independent o f blood flow. For drugs that are eliminated almost e n t i r e l y by metabolism without any l i m i t a t i o n s by blood flow to the l i v e r , t o t a l body clearance (CI) i s a f u n c t i o n o f the f r e e f r a c t i o n in serum ( f u ) and the i n t r i n s i c clearance ( C I 1 - n t ) through the expression (133-136) c i = f u c r i n t (i) where fu i s the r a t i o o f f r e e drug in serum to t o t a l drug in serum and C l ' . j n t i s the inherent a b i l i t y of the l i v e r to metabolize unbound drug ( f r e e clearance v a l p r o i c a c i d ) . R e c a p i t u l a t i n g the pharmacokinetic equations and concepts during m u l t i p l e dosing (126, 135, 137) 101 = F.Dose ( 2 ) Ss K E V D t v where C s s i s theaverage steady-state t o t a l serum drug concentrations and F i s assumed to be u n i t y f o r VPA- Since CI = K^ Vp, f o r a one-compartment model r _ Dose/t io) C s s - CT [ ' = f u Dose/x ( 3 a ) u ss CI From equations (1) and (3a) C u = Dose/r ( 4 ) ss , M i n t where C u $ s i s the average steady s t a t e • c o n c e n t r a t i o n s of unbound drug i n se-rum.. For a drug with a low e x t r a c t i o n r a t i o and highly bound to serum proteins the volume of d i s t r i b u t i o n of unbound drug (Vu). i s r e l a t e d to clearance through the expression (133-136) CI = W f (5) where K f i s the r a t e constant of e l i m i n a t i o n of unbound drug. Thus ^ • ft and from equations (5a) and (1) 102. (6) and tv ( f r e e drug) = 0.693.V u (7) C I , Equation (1) p r e d i c t s t h a t an increase i n fr e e f r a c t i o n i n serum (decrease i n serum p r o t e i n binding) should r e s u l t in an increase i n t o t a l body clearance ( C I ) . An increase in t o t a l body clearance would 2 decrease t o t a l serum v a l p r o i c a c i d or v a l p r o i c a c i d - H g steady s t a t e concentrations (Equation 3) with no e f f e c t on steady s t a t e concentrat-ions of unbound v a l p r o i c a c i d or v a l p r o i c - ^ H g in serum (Equation 4 ). In v i t r o s t u d i e s with v a l p r o i c a c i d have shown that p r o t e i n binding of v a l p r o i c a c i d i s decreased at higher v a l p r o i c a c i d concentrations in plasma (23); In v i v o , Gugler et_ a_l_ (22) have observed that steady st a t e plasma concentrations of v a l p r o i c a c i d i n healthy subjects were 20% lower than pre d i c t e d values from s i n g l e dose s t u d i e s . They suggested that the d i f f e r e n c e s may be due to an increase i n the serum f r e e f r a c t i o n of v a l p r o i c a c i d at higher concentrations of t o t a l drug in plasma, under steady s t a t e c o n d i t i o n s . In the present study, t o t a l body clearance increased at steady st a t e compared to s i n g l e dose c o n d i t i o n s (Table III) while the average fre e f r a c t i o n in serum ( f ) increased from 0.05 (VPA.: 0.05 ± 0.01, 2 Mean ± S.D.; VPA- H g:0.05 ± 0.01, F i g . 5) under s i n g l e dose co n d i t i o n s to 0.09 (VPA: 0.08 ± 0.01, Mean ± S.D.; VPA- 2H g: 0.09 ± 0.01, F i g s . 7 and 9) under m u l t i p l e dose c o n d i t i o n s . Unless the l i v e r enzyme a c t i v i t y was a f f e c t e d one would not expect i n t r i n s i c clearance ( C I 1 . ^) to change between s i n g l e dose and steady-s t a t e c o n d i t i o n s . The clearance of the f r e e drug ( C I ' ^ n t ) was observed to decrease (Table I I I , s i n g l e dose: VPA = 0.20 l i t e r / h r / k g , VPA- 2H 6 = 0.17 l i t r e / h r / k g ; steady s t a t e : VPA- 2H 6 = 0.12 1 i t r e / h r / k g ; 2 VPA + VPA- Hg = 0.13 l i t e r / h r / k g ) under steady s t a t e c o n d i t i o n s 2 suggesting an i n h i b i t i o n of v a l p r o i c a c i d or v a l p r o i c a c i d - Hg meta-bolism or s a t u r a t i o n of drug-metabolizing enzymes. Bowdle et al_ (56) have reported s i m i l a r r e s u l t s during m u l t i p l e dosing with v a l p r o i c acid where an increase i n serum f r e e f r a c t i o n o f v a l p r o i c a c i d ( l e a d i n g to an incre a s e i n t o t a l body clearance) co-ex i s t e d with a decrease i n I n t r i n s i c clearance o f v a l p r o i c a c i d . They reported that the dominating e f f e c t depended on the dosage. The i n -t r i n s i c clearance values at dosages of 500 mg/day were higher than the i n t r i n s i c clearance values at dosages of 1000 mg/day whereas the mean serum f r e e f r a c t i o n f o r 500 mg/day dosage (0.070 ± 0.012) were lower than the mean serum f r e e f r a c t i o n f o r 1000 mg/day (0.101 ± 0.015). The net e f f e c t they observed was no change i n t o t a l body clearance f o r the two dosages^ However,•• the r e s u l t s they obtained for" the 1500 mg/day dosages were contrary to pred i c t e d r e s u l t s . The i n t r i n s i c clearance values f o r 1500 mg/day dosage were higher than the values f o r the 1000 mg/day dosage whereas the mean serum f r e e f r a c t i o n f o r the two dosages were s i m i l a r . S a l i v a V a l p r o i c Acid Measurement 2 The concentrations of s a l i v a VPA- Hg and VPA were too low during the e l i m i n a t i o n phase i n the s i n g l e dose studies to obtain a r e l i a b l e estimate of the pharmacokinetic parameters. However, the concentrations of the d e t e c t a b l e s a l i v a l e v e l s were determined to e s t a b l i s h the r e l a t i o n s h i p o f drug l e v e l s i n s a l i v a and serum. Table IV summarizes 104. TABLE IV Re l a t i o n s h i p o f s a l i v a concentrations o f v a l p r o i c a c i d and v a l p r o i c acid- 2Hg and serum t o t a l or serum f r e e drug l e v e l s i n the s i n g l e and m u l t i p l e dose s t u d i e s . S i n g l e Dose Conditions Parameters 3 v a l p r o i c v a l p r o i c a c i d acid- 2H5 Mean Ratio S a l i v a / T o t a l Serum 0.007 0. ,009 Standard Deviation (S.D.) 0.001 0. ,001 C o r r e l a t i o n C o e f f i c i e n t (r) 0.926 0. ,901 Mean Ratio S a l i v a / F r e e Serum 0.196 0. ,200 Standard Deviation 0.027 0. .033 C o r r e l a t i o n C o e f f i c i e n t 0.863 0, ,850 Steady State Conditions Mean Ratio S a l i v a / T o t a l Serum 0.009 0.011 Standard Deviation 0.002 0.002 C o r r e l a t i o n C o e f f i c i e n t 0.953 0.956 Mean Ratio S a l i v a / F r e e Serum 0.112 0.138 Standard Deviation 0.021 0.031 C o r r e l a t i o n C o e f f i c i e n t 0.939 0.916 a Steady s t a t e concentrations were measured during the f i r s t three dosing i n t e r v a l s f o l l o w i n g l a b e l l e d drug a d m i n i s t r a t i o n - (n = 17 samples). S i n g l e dose concentrations cover a period of 24 hrs (n = 10 samples). 105. the c o r r e l a t i o n data obtained from Figs.,. 5, 7, 8 and 9. The mean s a l i v a drug concentration was found to be 0.7 - 0.9% and 0.9 - 1.0% of t o t a l serum drug concentrations in the s i n g l e and m u l t i p l e dose co n d i t i o n s r e s p e c t i v e l y . This i s comparable to values obtained by Gugler et a]_ (22) i n s i x subjects under steady s t a t e c o n d i t i o n s where mean s a l i v a v a l p r o i c a c i d concentrations were 0.7 - 2.4% of plasma t o t a l v a l p r o i c a c i d concentrations. The c o r r e l a t -ion between unstimulated s a l i v a and serum t o t a l drug l e v e l s was f a i r l y high (Table IV) and i s in part due to the p r e c i s i o n of the assay. S i m i l a r c o r r e l a t i o n values have been reported by Abbott et^ al_ (94). The s a l i v a l e v e l s o f v a l p r o i c a c i d were 20% of the serum f r e e v a l p r o i c a c i d concentration under s i n g l e dose c o n d i t i o n s (Table IV). This com-pares to reported values where s a l i v a l e v e l s o f v a l p r o i c a c i d were 10 - 20% of unbound serum l e v e l s in 15 e p i l e p t i c p a t i e n t s (138). Under m u l t i p l e dose c o n d i t i o n s , s a l i v a valproate decreased to approximately 12% of the f r e e serum drug concentrations (Table IV). The observed decrease i n the s a l i v a to fr e e serum va l p r o a t e r a t i o was in co n t r a s t to the r a t i o of s a l i v a to serum t o t a l drug concentrations which were v i r t u a l l y i d e n t i c a l f o r both the s i n g l e dose and m u l t i p l e dose s t u d i e s . The concentration-dependence of the s a l i v a and serum f r e e drug r a t i o suggests that v a l p r o i c a c i d s a l i v a concentration depends on changes i n other f a c t o r s i n c l u d i n g changes i n serum f r e e f r a c t i o n . The pKa of v a l p r o i c a c i d i s 4.8 (3) and the s a l i v a v a l p r o i c a c i d concen-t r a t i o n i s expected to be a f u n c t i o n o f s a l i v a pH, serum pH and the degree of plasma p r o t e i n binding. The t h e o r e t i c a l r e l a t i o n s h i p between the s a l i v a concentration and serum concentration o f an a c i d i c drug can be expressed by the f o l l o w i n g equations derived from the Henderson-Hasselbach equation (139) 1G6. C C P s 1 + 10 1 + 10 (8) C s 1 + 10 ( p H s - P K a ) (8a) o r C u 1 + 10 where C = c o n c e n t r a t i o n o f d rug i n s a l i v a , C = c o n c e n t r a t i o n o f d r u g (pK = 4 .8 f o r v a l p r o i c a c i d ) , C u = c o n c e n t r a t i o n o f unbound d rug i n T a b l e V shows t h e e f f e c t o f s a l i v a and serum pH on v a l p r o i c a c i d l e v e l s i n s a l i v a and t h e t h e o r e t i c a l l y p r e d i c t e d and e x p e r i m e n t a l s a l i v a t o f r e e serum v a l p r o i c a c i d c o n c e n t r a t i o n r a t i o . The d i s c r e p a n c y between a c t u a l e x p e r i m e n t a l r a t i o s and the p r e d i c t e d s a l i v a t o f r e e serum d rug r a t i o shows t h a t t h e r a t i o i s no t r e l a t e d t o s a l i v a and serum pH as p r e d i c t e d . For a c i d i c d r u g s w i t h pK l e s s t h a n 8 . 5 , t h e a s a l i v a t o serum f r e e d rug r a t i o i s e x p e c t e d t o be dependen t on s a l i v a pH ( 7 1 , 7 2 ) . T h i s does not a p p e a r t o be t h e c a s e w i t h v a l p r o i c a c i d . S i m i l a r d i s c r e p a n c i e s between p r e d i c t e d and a c t u a l e x p e r i m e n t a l s a l i v a t o serum d r u g r a t i o s have been r e p o r t e d f o r s a l i c y l i c a c i d (140 ) w i t h a pK o f 3 .0 and f o r p r o c a i n a m i d e (141 ) w i t h a pK o f 9 . 4 . Koup e t a l a a — (141) have s u g g e s t e d t h a t t h e d i s c r e p a n c y c o u l d be due t o t h e d i f f e r e n c e between t h e pH a t t h e s i t e o f s a l i v a e x c r e t i o n and t h e pH o f m ixed s a l i v a . When s a l i v a r y f l o w was s t i m u l a t e d w i t h s u c r o s e , t h e pH o f s t i m u -s P i n s e r u m , p H g = s a l i v a pH , p H p = serum pH, f p = f r a c t i o n o f unbound d rug i n s e r u m , f = f r a c t i o n o f unbound d r u g i n s a l i v a ( f g = 1 , v a l p r o i c a c i d i s assumed t o be o n l y i n t h e unbound form i n s a l i v a ) , p K g = p K g o f d r u g se rum. 107. TABLE V Re l a t i o n s h i p between t h e o r e t i c a l l y predicted and experimental s a l i v a to serum f r e e v a l p r o i c a c i d (VPA) concentration r a t i o s i n the s i n g l e dose study. Time Serum S a l i va VPA S a l i v a VPA Serum Experimental P r e d i c t e d * (hr) PH PH Concentration f r e e Con- S a l i v a / F r e e S a l i v a / F r e e (yg/ml) c e n t r a t i o n (yg/ml) 1.0 7.49 6.73 0.348 1 .703 0.204 0.175 1.5 7.47 6.78 0.341 1.468 0.232 0.206 2.0 7.46 6.92 0.264 1.524 0.173 0.290 2.5 7.48 6.82 0.202 1.425 0.142 0.225 3.0 7.51 6.89 0.215 1.119 0.181 0.241 4.0 7.53 6.73 0.234 1 .041 0.225 0.160 5.0 7.48 6.59 0.183 0.936 0.195 0.131 6.0 7.48 6.69 0.178 0.819 0.217 0.164 7.0 7.53 6.82 0.167 0.794 0.210 0.196 8.0 7.55 6.82 0.162 0.763 0.212 0.188 * Predicted r a t i o s were c a l c u l a t e d using equation (8a) and assuming 108. l a t e d mixed s a l i v a was c o n s i s t e n t l y lower than the pH of unstimulated mixed s a l i v a . This was probably due to the a c i d i t y produced i n the mouth by the a c t i o n o f s a l i v a r y enzymes or b a c t e r i a on sucrose. Instead of stimulated s a l i v a v a l p r o i c a c i d l e v e l s decreasing when s a l i v a pH was lowered as one would p r e d i c t , sucrose-stimulated s a l i v a v a l p r o i c acid l e v e l s during the m u l t i p l e dose s t u d i e s were higher (mean = 0.684 yg/ml) than unstimulated s a l i v a v a l p r o i c a c i d l e v e l s (mean = 0.609 yg/ml) and the d i f f e r e n c e was s i g n i f i c a n t (paired t - t e s t , p = 0.013, n = 22). I n t e r p r e t a t i o n of s a l i v a v a l p r o i c a c i d l e v e l s i s p o s s i b l e i f the mechanism o f e x c r e t i o n o f v a l p r o i c a c i d i n s a l i v a and f a c t o r s that a f f e c t the excretion are understood. The e l e v a t i o n of s a l i v a v a l p r o i c a c i d l e v e l s during the absorption phase could be due to r e t e n t i o n of drug in the mouth. Nevertheless other routes of drug a d m i n i s t r a t i o n such as r e c t a l a d m i n i s t r a t i o n would be more appropriate to confirm the drug r e t e n t i o n i n the mouth. The appearance of a secondary peak i n s a l i v a 4 hr a f t e r dosing suggests that time of sampling w i l l be im-portant s i n c e these l e v e l s do not c o r r e l a t e with serum fr e e or serum t o t a l drug co n c e n t r a t i o n s . It appears that even though a good c o r r e l a t -ion was obtained between s a l i v a and f r e e serum drug l e v e l s during m u l t i p l e dosing, the s a l i v a v a l p r o i c a c i d r e l a t e more to t o t a l serum v a l p r o i c a c i d rather than the f r e e drug concentrations. The s a l i v a to free serum drug r a t i o was found to be concentration-dependent. 109. D. Metabolism Studies Assay The method described f o r the i d e n t i f i c a t i o n o f metabolites i n 2 serum and urine f o l l o w i n g the dose o f v a l p r o i c a c i d - Hg involved GCMS a n a l y s i s o f d e r i v a t i z e d e x t r a c t s . The problems concerning the chemi-cal s t a b i l i t y o f v a l p r o i c a c i d metabolites i n GCMS a n a l y s i s (16) were solved by forming methyl, TMS and t-BDMS d e r i v a t i v e s of serum and u r i n a r y c o n s t i t u e n t s . Each d e r i v a t i z a t i o n method had i t s advantages and disadvantages. Methylation enabled e a r l y peaks in the t o t a l ion chromatograms to be detected. The e a r l y peaks may inc l u d e the degradation products of metabolites formed during e x t r a c t i o n of urine and serum samples. TMS and t-BDMS reagent peaks obscured d e t e c t i o n of smaller molecules such as the 3-heptanone formed from decarboxylation o f 3-keto VPA. On the other hand methylation did not protect hydroxy groups and mul-t i p l e hydroxy compounds would not have good s t a b i l i t y on the polar columns. Apart from p r o t e c t i n g hydroxy groups, TMS and t-BDMS d e r i v a -t i v e s o f f e r e d more d i a g n o s t i c ions in the mass spectra f o r i d e n t i f i c a t -ion o f metabolites.t-BDMS d e r i v a t i v e s provided much more intense ions i n the high mass region. V a l p r o i c a c i d metabolites can only be e f f i c i e n t l y extracted from b i o l o g i c a l f l u i d s a f t e r a c i d i f i c a t i o n of the b i o l o g i c a l f l u i d s . The minimum pH of the e x t r a c t i o n medium should however be 1.5 i f dehydration o f hydroxy-acid metabolites are to be avoided (36). Strong a c i d i c c o n d i t i o n s l e a d i n g to the conversion o f hydroxy-acids to unsaturated acids or unsaturated acids to hydroxy-acid lactones were observed when the no. 2-ene VPA and 3-ene VPA were being synthesized f o r t h i s study. By e x t r a c t i n g serum samples at pH values ranging from 1.5 to 5.0 i t was observed that the four unsaturated metabolites were d e t e c t a b l e w i t h i n the pH range. Thus the unsaturated metabolites i d e n t i f i e d may not be formed by dehydration o f the hydroxy-acids. Nau and Wi t t f o h t (142) have reported that 2-ene VPA and 3-ene VPA are d e t e c t a b l e when serum samples are ext r a c t e d at pH values as high as 5. An a l y s i s o f authentic samples of the hydroxy-acids by GCMS i n the scan mode showed that the d e r i v a t i z e d compounds are s t a b l e under the GCMS c o n d i t i o n s . However, the u n d e r i v a t i z e d 3-OH VPAdegraded i n t o complex products i n c l u d i n g 2-ene VPA when analyzed on e i t h e r the 3% OV-17 or 3% Dexsil 300 columns. The degradation o f 3-OH VPA on polar columns has been reported by several i n v e s t i g a t o r s (35, 83). The new metabolite o f VPA, 4-keto VPA was i d e n t i f i e d i n both serum and u r i n e . I t appeared to be c h e m i c a l l y more s t a b l e than the -3~ke.to VPA metabolite. The TIC peak o f 4-keto VPA was present when serum was extracted with or without incubation i n an a c i d i c medium. I t was als o present when a l k a l i n e h y d r o l y s i s o f the u r i n a r y conjugates was performed and a l s o when u r i n e e x t r a c t s were derivatized with diazomethane or t e r t -b u t y l d i m e t h y l c h l o r o s i l a n e . The 4-keto VPA metabolite was not detected when TMS d e r i v a t i v e s o f u r i n e and serum c o n s t i t u e n t s were prepared. Since most GCMS a n a l y s i s o f v a l p r o i c a c i d metabolites have been conducted with TMS-derivatized e x t r a c t s the metabolites might not have been detected probably because of i n t e r f e r i n g peaks from endogenous compounds. The 4-keto VPA appears i n s i g n i f i c a n t amounts i n serum comparable to those of 3-ene VPA and i n amounts i n u r i n e comparable to those o f 4-OH VPA. Both 2 - p r o p y l s u c c i n i c a c i d and 2-propylmalonic a c i d were c h a r a c t e r -111. 2 ized as d i s t i n c t metabolites of v a l p r o i c a c i d using v a l p r o i c a c i d - Hg as t r a c e r and comparing r e t e n t i o n times and mass spectra of i s o l a t e d compounds with synthesized reference compounds. Both of these d i c a r -b o x y l i c acids have been reported to be present in p a t i e n t u r i n e e x t r a c t s (30). Studies conducted with unhydrolyzed urine samples showed that 4-keto VPA was excreted l a r g e l y i n the unconjugated form. 3-Keto VPA * 4-OHVPAi 3-OH VPAand 5-OH' VPAwere excreted mainly in the unconjugated form as reported (28). A b r i e f study i n v o l v i n g glass c a p i l l a r y GCMS showed an in c r e a s e in separation of the unsaturated metabolites on a 30 m long DB-5 column. Detection o f the 4-ene VPA metabolite, reported by Kochen et al_ (36) proved to be d i f f i c u l t s i nce d e r i v a t i v e s of synthesized 4-ene VPA had the same r e t e n t i o n time as d e r i v a t i v e s o f VPA and could not be separated on the 6 m long packed column or by s e l e c t e d ion monitoring i n the presence of r e l a t i v e l y l a r g e peak of the VPA d e r i v a -t i v e . 4-Ene VPA i s reported to occur in concentrations of 60 - 110 pg/ml in p a t i e n t s ' serum (36). Table's VI, VII, VIII summarize the i n t e n s i t i e s and o r i g i n of c h a r a c t e r i s t i c ions and ion-doublets in the mass spectra of v a l p r o i c 2 a c i d and v a l p r o i c a c i d - Hg metabolites found in e i t h e r the serum or u r i n e methylated e x t r a c t s . In view of the f a c t that u n l a b e l l e d v a l p r o i c a c i d and i t s metabolites were at steady s t a t e l e v e l s i n 2 serum, when v a l p r o i c a c i d - Hg was administered, the i n t e n s i t y r a t i o o f the u n l a b e l l e d ions to t h e i r corresponding ion doublets were not 1:1 but v a r i e d from 2:1 to 8:1. The i n t e n s i t y r a t i o may depend on the peak scan and background scan s e l e c t e d f o r mass spectra data processing TABLE VI D i a g n o s t i c f r agmen t i o n s and i o n - d o u b l e t s i n t h e mass s p e c t r a o f me thy l e s t e r s o f v a l p r o i c a c i d (VPA) and v a l p r o i c a c i d - 2 H 6 (VPA- 2 Hg) and t h e i r m e t a b o l i t e s . C H 3 " ( 2 H 3 ) •CH, •CH 9 / > 2. • f c CH 0 M l c ^ b: \ * R i ai OCH. Compound S u b s t i t u e n t Mass o f f r agmen t i o n ( r e l a t i v e i n t e n s i t y * , %) R l R 2 a b c d e f g VPA methy l e s t e r V P A - 2 H 6 me thy l e s t e r H,H H,H H,H H,H 127 (4 ) 133 (1 ) 99 (5 ) 105 (2 ) 115 (1 ) 43 (26 ) 46 (4 ) 116 (30 ) 119 (4 ) 129 (5 ) 132 (2 ) 29 (22 ) 3 2 ( * * ) 3-ke to VPA me thy l e s t e r 3-ke to V P A - 2 H 6 methy l e s t e r 0 0 H,H H,H 141 (3 ) 113 (3 ) 119 (3 ) 130 (7 ) 1-33(2) 57 (100 ) 60 (25 ) 116 (8 ) 119 (3 ) 143 (3 ) 146 (1 ) 29 (31 ) 3 2 ( * * ) 4 - k e t o VPA methy l e s t e r 4 - k e t o VPA- 2 H6 methy l e s t e r H,H H,H 0 0 141 (3 ) 147(1 ) 113 (1 ) 130 (3 ) 133 (1 ) 57 (1 ) 115 (40 ) 118 (14 ) 129 (1 ) 43 (100 ) 46 (18 ) 3-OH VPA me thy l e s t e r 3-OH V P A - 2 H 6 me thy l e s t e r H,0H H,0H H,H H,H 143 (2 ) 115(1 ) 131 (1 ) 58 (1 ? ) 61(8 ) . 116 (16 ) 119 (3 ) 145 (8 ) 148 (2 ) 29 (25 ) 3 2 ( * * ) * r e l a t i v e i n t e n s i t y i s i o n i n t e n s i t y e x p r e s s e d r e l a t i v e to t h e base peak o f t h e u n l a b e l l e d component i n t h e mass spec t rum o f t h e TIC peak c o n t a i n i n g 2 H Q and 2 H 5 d e r i v a t i v e s . Ions w i t h i n t e n s i t i e s l e s s t h a n 1% a r e not c o n s i d e r e d . * I n t e n s i t y no t g i v e n due t o background e f f e c t o f 0 ? (M + =32) 11-3. TABLE V I I D i a g n o s t i c f r a g m e n t i o n s and i o n - d o u b l e t s i n t h e mass s p e c t r a o f me thy l e s t e r s o f u n s a t u r a t e d m e t a b o l i t e s o f VPA and V P A - 2 H g CH, 'CH, 9 ? \ ( \ ) ^ C H . , 13 \ C -\- OCH, C H 9 \ (^H 3 ) ^ C H -CH, 'CH — C — OCH, CH, •C H 2 A bi a] CH, CH, ^ H 3 ^ 2-ene VPA methy l e s t e r / 2 M \ K 3' 3-ene VPA m e t h y l e s t e r C H 3 -( 2 H 3 ) C H , ( 2 H 3 ) CH-CH, •CH ft J^C — C — OCH, • C H ^ D iene VPA me thy l e s t e r Mass o f i o n ( r e l a t i v e i n t e n s i t y * ) Compound M + a b c d 2-ene VPA me thy l e s t e r 2-ene V P A - 2 H g methy l e s t e r 156 (23 ) 162 (6 ) 125 (10 ) 131 (3 ) 97 (10 ) 103 (2 ) 113 (6 ) 116 (3 ) 127 (43 ) 130 (6 ) 3-ene VPA m e t h y l e s t e r 3-ene V P A - 2 H 6 me thy l e s t e r 156 (4 ) 162 (1 ) 125 (3 ) 131 (1 ) 97 (19 ) 103 (3 ) 113 (13 ) 116 (1 ) 127 (7 ) 130 (2 ) D i ene VPA methy l e s t e r D i ene V P A - 2 H 6 me thy l e s t e r 154 (25 ) 160 (5 ) 123 (20 ) 129 (2 ) 95 (100 ) 101 (7 ) 111 (9 ) 125 (11 ) 128 (4 ) * r e f e r t o T a b ! e V I . 114. TABLE VIII Diagnostic fragment ions and ion doublets in the mass spectra of methyl esters o f d i c a r b o x y l i c a c i d metabolites o f v a l p r o i c a c i d and v a l p r o i c acid-2H 6 C H 3 0 ^ 0 CH 30 0 C \ & 0 2 ^ C H ? |°| 5<CH K -4- PCH-^ C H -4- C -4- OCH^ ^ C H ? V h,1--' J ^ C H 2 ^ 2 ^ ^ * I W {  2 .. 2 ( H 3 } ( H 3 ^ , M.W. = 202 M.W. = 188 Dimethyl 2-propylglutarate Dimethyl 2-propylsuccinate CH 30 ^0 C \ LI ^ C H -4- t -4- 0CH 3 C H 3 - 2 ( H 3 ) M.W. = 174 Dimethyl 2-propylmalonate Mass of fragment ions ( r e l a t i v e i n t e n s i t y * ) Dimethyl ester a b b 1 c d or c+a d' or c+a M-31 M-59 M-60 M-42 M-73 M-74 2-p r o p y l g l u t a r a t e 2 - p r o p y l g l u t a r a t e - 2 H 3 171 (16) 174(4) 143(19) 146(2) :'142(42) 145(2) 160(14) 129(15) 132(2) 128(53) 131(2) 2-propylsuccinate „ 2-propylsuccinate- H 3 157(14) 160(4) 129(13) 132(2) 128(13) 131 (3) 146(10) 115(54) 118(10) 114(100) 117(23) 2-propylmaloriate 2 2-propylmalonate- H 3 143(8) 146(2) 115(7) 114(6) 132(100) 101(46) 100(34) * r e f e r to Table VI. since the undeuterated and deuterated compounds do not have e x a c t l y the same r e t e n t i o n time. The r a t i o may a l s o depend on the c o n t r i b u t i o n of u n l a b e l l e d compound fragmentation to a l a b e l l e d fragment ion or v i c e - v e r s a . Also isotope e f f e c t s in fragmentation may e x i s t . The disadvantage of isotope l a b e l l i n g with three or l e s s deuterium atoms f o r l o c a t i o n o f ion-doublets was seen i n the mass spectra o f the d i c a r b o x y l i c a c i d metabolites. The ions from the deuterated analogues were not divorced from the m/z + 3 ions of the u n l a b e l l e d compounds r e s u l t i n g from c o n t r i b u t i o n s of the n a t u r a l l y o c c u r r i n g isotopes at the high mass region/ It was a l s o observed that exchange or rearrangement 2 of deuterium l a b e l occurred with 4-keto VPA- Hg during fragmentation of the t-BDMS d e r i v a t i v e leading to appearance of ion c l u s t e r s instead of ion doublets. The [M-57] + ion o f 4-keto VPA t-BDMS occurred at m/z 215 while the [M-57] + ion of 4-keto VPA- 2H g t-BDMS, m/z 221, was v i r t u a l l y absent and instead ions m/z 218 and m/z 220 appeared. The presence of a keto group increases the l a b i l i t y of the 3 deuterium atoms in 4-keto VPA. Table IX shows the o r i g i n of c h a r a c t e r i s t i c ions and ion doublets in the mass spectra of dirtBDMS d e r i v a t i v e s of hydroxy"^acid and d i c a r b o -x y l i c a c i d metabolites i s o l a t e d from u r i n e . The [M-57] + ion i s u s u a l l y very intense. The fragmentation pathways f o r t-BDMS d e r i v a t i v e s have been described in d e t a i l by De Jong ejt a ^ (107). The expulsion of the t-butyl r a d i c a l can be followed by l o s s of CO in 2-substituted d i c a r b o -x y l i c a c i d s . Other neutral fragments l o s t from the d e r i v a t i v e s include C0 o, t-BDMSOH and t-BDMSCOOH depending on the s t r u c t u r e of the a c i d . 116. TABLE IX Diagnostic fragment ions and ion doublets from the mass spectra o f di-tBDMS BDMS d e r i v a t i v e s of VPA and VPA-2H5 metabolites i s o l a t e d from u r i n e Mass of ion ( r e l a t i v e i n t e n s i t y * ) D e r i v a t i z e d Compound M.W. M-57 M-57-C0 M-57-C02 M-57-132 M-57-160 3-OH VPA 3-OH VPA-^H 6 388 394 331(22) 337(7) 199(26) 205(6) 171(3) 4-OH VPA p 4-OH VPA- H 6 388 394 331(44) 337(8) 199(30) 205(3) 5-OH VPA 9 5-OH VPA- H 5 388 393 331 (34) 336(3) 199(12) 204(1) 2 - P r o p y l g l u t a r i c a c i d . 2 - P r o p y l g l u t a r i c a c i d - H 3 402 405 345(16) 348(2) 317(2) 301(7) 213(4) 185(8) 2- P r o p y l s u c c i n i c a c i d 2 2 - P r o p y l s u c c i n i c a c i d - H 3 388 391 331(19) 334(3) 303(2) 287(2) 199(2) 171(6) 2-Propylmalonic a c i d 2 2-Propylmalonic a c i d - H 3 374 377 317(20) 320(3) 289(9) 292(2) 273(5) 276(1) 185(8) 157(10) * See Table VI. 1 1 7 . The use o f deuterium-labelled v a l p r o i c a c i d has c e r t a i n l y confirmed some of the fragmentation pathways described f o r methyl, TMS and t-BDMS d e r i v a t i v e s . The presence of the diene VPA- Hg isomer has reduced the p o s i t i o n a l isomers f o r the diene VPA metabolite. Mass s p e c t r a l data f o r the minor diene VPA and i t s deuterated analogue was not s u f f i c i e n t to conclude whether t h i s diene VPA was e i t h e r a p o s i t i o n a l or geometric t s o ~ mer o f the diene VPA having the longer r e t e n t i o n time on the column used. The presence of the diOH VPAmetabolite in u r i n e a l s o requires c o n c l u s i v e evidence. However, on the basis of the mass fragmentation pattern in F i g . 25, t h i s dihydroxy metabolite i s suggested to be one of three p o s s i b l e s t r u c t u r e s ; a 3,4-diOH VPA 3,4'-di'OH'VPAor 2,4-diOH VPA: Metabolic Pathways Figure 26 shows the p o s s i b l e metabolic pathways leading to format-ion of the new metabolites of v a l p r o i c a c i d . Two p o s s i b l e pathways are proposed f o r formation of 4-keto VPA. The f i r s t and most p r e d i c t a b l e i s the o x i d a t i o n of 4-0HVPA si m i l ar to o x i d a t i o n of 3-OH VPA to 3-keto VPA i n 3-oxidation. The other p o s s i b i l i t y i s the mechanism f o r production of carbonyl products from v i c i n a l g l y c o l s c a t a l y z e d by dehydrases (143). The biochemical pathways f o r the formation of 2 - p r o p y l s u c c i n i c a c i d and 2-propylmalonic a c i d would i n v o l v e sequential alpha o x i d a t i o n of 2 - p r o p y l g l u t a r i c a c i d . 2-Propylmalonic a c i d could a l s o be formed by 6-oxidation of 2-propyl gl u t a r i c a c i d . Di.ene VPA could be formed by double dehydrogenation of dihydroxy VPA i n vivo or dehydration of dihydroxy VPA during the a n a l y t i c procedure. 1 1 8 . H O O C — C H 2 — C H ^ H—COOH '3 2 Alpha-oxidation r HOOC—CH L y COOH CH^—~~CH2 C H 2  2-Propylsuccinic acid Al.pha-oxidation HOOC ^ C H — C O O H C H 3 - C H 2 - C H 2 2-Propylmalonic acid C H 3 — C H 2 — C H H—COOH y 2 Valproic acid(VPA) CH 2~~^^2~~^^2 C H 3 — C H = C H C H H 2~~C H 2 (w l)-0xidation :H—COOH ^ 3-ene V P A > S s ,0—COOH C H a - C H C H ^ 3 OH 2 •' ;. 4-OH V P A C H 3 — C H 2 — C H CH ^ — f i -H—COOH 4-Keto V P A H I f H |R2 OH OH OR H,0 C H = C R 2 0—H - > R j — C H 2 J | R 2 0 F i g . 26 Proposed metabolic pathways f o r new. metabol i t e s of v a l p r o i c a c i d 119. R e l a t i v e Importance of V a l p r o i c Acid Metabolites The b i o l o g i c a l s i g n i f i c a n c e o f v a l p r o i c a d d metabolites i s t h e i r p o s s i b l e r o l e in the ther a p e u t i c e f f e c t o f v a l p r o i c a c i d , p o s s i b l e hepatic t o x i c i t y a ssociated with v a l p r o i c a c i d and i n d r u g - i n t e r a c t i o n s . In a recent study (144) of the a n t i c o n v u l s a n t a c t i v i t i e s of 2-ene VPA i n mice, 3-ene VPA, 4-ene VPA, 3-OH VPA, 4-OH VPA, 5-OH VPAand 2-propylglu-t a r i c a c i d , none of the metabolites were as a c t i v e as VPA but 2-ene VPA and 4-ene VPA showed 50 - 90% of the potency o f VPA. 3-Keto VPA has however been reported to be as a c t i v e as VPA (35). The ethyl e s t e r of 3-OH VPAwas reported to give the most p r o t e c t i o n against pentylene-t e t r a z o l e s e i z u r e s i n mice compared to ethyl esters o f VPA and 3-keto VPA (145). The metabolites o f v a l p r o i c acid may be implicated i n the hepatic t o x i c i t y a s s o c i a t e d with the use of VPA. Some long-chain unsaturated f a t t y acids have been reported to depress the a c t i v i t y of drug-meta-b o l i z i n g enzymes of r a t l i v e r microsomes (146). The unsaturated metabolites o f VPA are s t r u c t u r a l l y r e l a t e d to 4-pentenoic a c i d reported to produce a Reye-like syndrome with c h a r a c t e r i s t i c s s i m i l a r to that of VPA (50, 53). It has been reported that 2,4-pentadienoic a c i d , a metabolite o f 4-pentenoic a c i d could be the cause of the Reye-like syn-drome ( 54) . The bismuth s a l t o f 2,5-heptadiene-4-carboxylic a c i d ( a l t e r n a t i v e s t r u c t u r e suggested f o r the diene VPA metabolite) has been reported to cause hepatic t o x i c i t y i n human and the heptadiene-c a r b o x y l i c a c i d i s suggested to be the hepatotoxin (147). SUMMARY AND CONCLUSIONS 1 2 0 . 1 . Ten v a l p r o i c a c i d metabolites were synthesized to serve as reference compounds to i d e n t i f y v a l p r o a t e metabolites i s o l a t e d from human serum and urin e samples. 2 . V a l p r o i c a c i d - Hg was synthesized with the deuterium l a b e l i n the terminal methyl groups o f the propyl chains. 3. From metabolism studies c a r r i e d out a f t e r a s i n g l e dose a d m i n i s t r a t -2 ion o f a mixture o f v a l p r o i c a c i d and v a l p r o i c a c i d - Hg to a human volunteer, an isotope e f f e c t was observed i n the formation o f 2 5-hydroxyval p r o i c a c i d - Hg as expected. 4. A comparison o f the serum e l i m i n a t i o n k i n e t i c s of v a l p r o i c a c i d 2 and v a l p r o i c a c i d - Hg i n the s i n g l e dose study i n d i c a t e d that the two compounds were k i n e t l c a l l y equivalent. 5. Using the hexadeuterated v a l p r o i c a c i d , the e f f e c t s of a s i n g l e dose and 1 2 0 0 mg/day m u l t i p l e dosing on the clearance of v a l p r o i c 2 2 a c i d - Hg were compared. Total body clearance of v a l p r o i c a c i d - Hg increased and i n t r i n s i c clearance decreased at steady s t a t e . The e l i m i n a t i o n k i n e t i c s of v a l p r o i c a c i d appear to be determined by dose-related changes i n the plasma p r o t e i n binding and the a c t i v i t y of drug-metabolizing enzymes. 6. A high l y s e n s i t i v e , s p e c i f i c and p r e c i s e GCMS method was used f o r 2 the simultaneous measurement of v a l p r o i c a c i d and v a l p r o i c a c i d - Hg in serum and s a l i v a . Good c o r r e l a t i o n was found between s a l i v a and t o t a l serum va l p r o a t e concentrations during the post-absorptive phase i n the m u l t i p l e dose study. S a l i v a and serum f r e e v a l p r o a t e concentration r a t i o s were found to depend at l e a s t i n part on the fr e e v a l p r o a t e f r a c t i o n in serum. A new metabolite, 2-propyl-4-ketopentanoic a c i d was found i n serum and u r i n e . 2 - P r o p y l s u c c i n i c a c i d and 2-propylmalonic a c i d were confirmed as v a l p r o i c a c i d metabolites using the stabl e- i/s'otope t r a c e r technique. P o s s i b l e s t r u c t u r e s f o r the major serum diene VPA metabolite have been proposed using the ion-doublet technique. A dihydroxy VPA metabolite i s a l s o suggested to be present i n u r i n e . The hexadeuterated v a l p r o i c a c i d appears to be very s u i t a b l e f o r a p p l i c a t i o n to d r u g - i n t e r a c t i o n studies o f v a l p r o i c a c i d and f o r the determination of r e l i a b l e k i n e t i c constants i n p a t i e n t s at steady state-without discontinuance of t h e i r normal ' >, therapy. 1 2 2 , REFERENCES 1. B u r t o n , R.S . On t h e p r o p y l d e r i v a t i v e s and d e c o m p o s i t i o n p r o d u c t s o f e t h y l a c e t o a c e t a t e . J . Amer . Chem. 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APPENDIX 133. and IR Spectra of synthesized compounds 134. 135. 136. 1 3 7 . 138. 1 3 9 . Nmlr(100MHz) spectrum of 3',5,5-H '- 3 -heptene-4-carboxylic acid in CDCI, ' 140. Nmr(lOOMHz) spectrum of 2-propyl-4-pentenoic acid in CDCI3 141. 1 4 2 . Nmr(lO0MHz) spectrum of 2-propylglutaric acid in CDCI, 143. 144. FREQUENCY^ -1) 146. ifcocf " j ioo " " a j o b " 2400 aooo 1800 * I600 1400 iio» 1000 800 «oo * °< FREQUENCY CpM"1,) 147. 148. FREQUENcr^lVr1) 1 4 9 . 150. ^600 32.00 igoo XfOO 1D0O 800 1600 1400 izoo I O O O " ~ 800 "«00 FReauENcrccM-!) 151, 

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