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The synthesis of 2-((E)-1'-propenyl)-(E)-2-pentenoic acid and its metabolism and pharmacokinetics in… Lee, Ronald Duane 1987

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THE SYNTHESIS OF 2 - ((E)-1'-PROPENYL)-(E)-2-PENTENOIC ACID AND ITS METABOLISM AND PHARMACOKINETICS IN RATS by RONALD DUANE LEE B.Sc. (Pharm.) U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES F a c u l t y of Pharmaceutical Sciences ( D i v i s i o n of Pharmaceutical Chemistry) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1987 , © RONALD D. LEE, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PharmarPirriral S r i o n r - P Q The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date September 1.1987 ABSTRACT The a n t i c o n v u l s a n t drug v a l p r o i c a c i d (VPA) i s e x t e n s i v e l y m e tabolized with 16 m e t a b o l i t e s i d e n t i f i e d i n man. Of i n t e r e s t are the unsaturated m e t a b o l i t e s which appear to be r e s p o n s i b l e f o r the observed secondary a n t i e p i l e p t i c a c t i v i t y and/or i d i o s y n c r a t i c h e p a t o t o x i c i t y . A study by Abbott et al . ( 1986) has shown 2 - ( ( E ) - T -p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d ((E,E)-2,3'-diene VPA) to be a major unsaturated m e t a b o l i t e of VPA. Acheampong (1985) demonstrated that an isomeric mixture of 2,3'-diene VPA prevented p e n t y l e n e t e t r a z o l e - i n d u c e d s e i z u r e s i n mice and had 60% the potency of VPA. Research on (E,E)-2,3'-diene VPA i s l i m i t e d even though the diene appears t o be a c o n t r i b u t o r to the secondary a n t i e p i l e p t i c e f f e c t . A v a i l a b l e s y n t h e s i s of 2,3'-diene VPA r e s u l t i n two or more isomers with very low y i e l d s as shown by Acheampong and Abbott (1985). The o b j e c t of t h i s work was, t h e r e f o r e , to s y n t h e s i z e (E,E)-2,3'-diene VPA i n s u f f i c i e n t q u a n t i t y and isomeric p u r i t y f o r metabolic and pharmacokinetic s t u d i e s i n r a t s . S y n t h e s i s of (E,E)-2,3'-diene VPA was achieved by the a l k y l a t i o n of e t h y l (Z)-2-pentenoate to a f f o r d e t h y l 2 - ( l ' -h y d r o x y p r o p y l ) - ( E ) - 2 - p e n t e n o a t e . Dehydration using methanesulfonyl c h l o r i d e and potassium hydride gave the e t h y l e s t e r of (E,E)-2,3'-diene VPA. H y d r o l y s i s of the e s t e r i n 1 N NaOH a f f o r d e d an 81.2% pure sample of ( E , E ) -i i 2,3'-diene VPA as determined by GCMS and NMR. A second method was used to u n e q u i v o c a l l y s y n t h e s i z e (E,E)-2,3'~ diene VPA whereby an O - t r i m e t h y l s i l y l k e t e n e a c e t a l was o x i d i z e d v i a a hydride a b s t r a c t o r t o y i e l d two isomers of 2,3'-diene VPA plus the s t a r t i n g m a t e r i a l (E)-3-ene VPA. The i d e n t i t y of the products were determined by GCMS with the major isomer being (E,E)-2,3'-diene VPA. In the metabolism s t u d i e s , Wistar r a t s were given 100 mg/kg i . p . of (E,E)-2,3'-diene VPA and b i l e and u r i n e c o l l e c t e d f o r 24 hours. GCMS a n a l y s i s r e v e a l e d three m e t a b o l i t e s present i n both b i l e and u r i n e . These were r e d u c t i o n products of (E,E)-2,3'-diene VPA metabolism and c o n s i s t e d of the monounsatured (E)-3-ene VPA and (E)-2-ene VPA p l u s the f u l l y s a t u r a t e d VPA. These r e s u l t s suggest that t r a c e l e v e l s of (E)-3-ene VPA observed a f t e r VPA dosing may not be a d i r e c t metabolic product of VPA i t s e l f but r a t h e r a reduced m e t a b o l i t e of (E,E)-2,3'-diene VPA. A l l p o l a r m e t a b o l i t e s are yet to be i d e n t i f i e d . For the pharmacokinetic s t u d i e s , two doses, 20 and 100 mg/kg of (E,E)-2,3'-diene VPA were a d m i n i s t e r e d i . v . to Wistar r a t s and the plasma c o n c e n t r a t i o n vs time d e c l i n e curve determined using GCMS a n a l y s i s of the plasma samples. The b i l e duct of these animals was then cannulated and the study repeated. A comparison between the e l i m i n a t i o n r a t e c onstant ( K E ) , c l e a r a n c e (CI), and volume of d i s t r i b u t i o n (V^) between the b i l e duct i n t a c t and cannulated r a t s f o r both dose groups r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s (p>0.1). A comparison of the K E, V^, and CI between dosage groups of both b i l e duct i n t a c t and cannulated r a t s r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e (p>0.08-0.7). T h e r e f o r e , e x t e n s i v e e n t e r o h e p a t i c r e c i r c u l a t i o n was not apparent and the e l i m i n a t i o n of (E,E)-2,3'-diene VPA appeared t o be dose-independent. The diene seems to f o l l o w a simple one-compartment model with a h a l f - l i f e of 35.9+8.9 (S.D.) minutes and a l a r g e volume of d i s t r i b u t i o n of 0.95+0.22 (S.D.) L/kg. In vitro p r o t e i n b i n d i n g s t u d i e s r e v e a l e d that ( E , E ) -2,3'-diene VPA s a t u r a t e s plasma p r o t e i n s between c o n c e n t r a t i o n s of 30 - 600 ug/mL. The percent of (E , E ) -2,3'-diene VPA bound decreases from 92.1% to 28.7% as the c o n c e n t r a t i o n of diene i n c r e a s e s suggesting c o n c e n t r a t i o n -dependent p r o t e i n b i n d i n g . The s y n t h e s i s of (E,E)-2,3'-diene VPA i n s u b s t a n t i a l q u a n t i t i e s has allowed metabolic and pharmacokinetic s t u d i e s to be performed i n r a t s . P r e l i m i n a r y s t u d i e s showed that (,E,E)-2,3' -diene VPA was m e t a b o l i c a l l y reduced to monounsaturated and s a t u r a t e d p r o d u c t s . Pharmacokinetic data appear to i n d i c a t e a p o t e n t i a l f o r the diene to accumulate i n the c e n t r a l nervous system. F u r t h e r s t u d i e s are r e q u i r e d to determine the c o n t r i b u t i o n of (E,E)-2,3'-diene VPA to the secondary a n t i e p i l e p t i c a c t i v i t y of VPA. i v TABLE OF CONTENTS TITLE i ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i LIST OF SCHEMES x i LIST OF ABBREVIATIONS x i i ACKNOWLEDGEMENT xv INTRODUCTION 1 EXPERIMENTAL 21 A. Chemicals and M a t e r i a l s 21 B. Instrumentation 25 C. Chemistry 27 D. M e t a b o l i c and Pharmacokinetic S t u d i e s 44 E. P r o t e i n B i n d i n g Study 54 RESULTS AND DISCUSSION 56 A. Chemistry 56 B. M e t a b o l i c Study 104 C. Pharmacokinetic Study 117 SUMMARY AND CONCLUSIONS 141 REFERENCES 144 APPENDIX 154 v LIST OF TABLES Table Page Summary of the e l i m i n a t i o n r a t e constants 130 (Kg, min 1) of (E,E)-2,3'-diene VPA between b i l e duct i n t a c t and cannul a t e d r a t s given 20 and 100 mg/kg doses i . v . Summary of the c l e a r a n c e s (CI, ml/min/kg) 131 of (E,E)-2,3'-diene VPA between b i l e i n t a c t and cannulated r a t s given 20 and 100 mg/kg doses i . v . Summary of the volume of d i s t r i b u t i o n s 132 (mL/kg) of (E,E)-2,3'-diene VPA between b i l e i n t a c t and cannul a t e d r a t s given 20 and 100 mg/kg doses i . v . Summary of area under the curve (AUC, 133 ug/min/mL), h a l f - l i v e s (t^/^' min), and weight (g) of a l l animals given 20 or 100 mg/kg of (E,E)-2,3'-diene VPA with the b i l e duct i n t a c t and cannulated. Bi n d i n g of (E,E)-2,3'-diene VPA to the 138 u l t r a f i l t r a t i o n apparatus and membrane at 25°C. P r o t e i n b i n d i n g of (E,E)-2,3'-diene VPA 138 to r a t plasma u s i n g u l t r a f i l t r a t i o n at 25°C. v i LIST OF FIGURES Figure Page 1 Metabolic pathways of va lp ro ic ac id in 14 humans (Acheampong, 1985). 2 S i l a s t i c / P E - 5 0 cannula for jugular vein 47 cannulat ion of Wistar r a t s . 3 To ta l ion chromatogram of the two 61 synthesized isomers of 2 ,3 ' -d iene VPA t-BDMS es te r . 4 Mass spectrum of the major synthesized 62 isomer ( E , E ) - 2 , 3 ' - d i e n e VPA t-BDMS es te r . 5 400 MHz proton NMR of e thy l ( E , E ) - 2 , 3 ' - 63 diene VPA in CDCI3 with TMS as an in terna l standard (*=E,Z-isomer) . 6 Tota l ion current chromatogram of e thy l 2- 68 ( (E ) - 1 ' -propenyl ) - (E) -2-pentenoate synthesized using a hydride abstractor (Peaks 1 = e thyl 2 - n - p r o p y l - ( E ) - 3 -pentenoate; Peaks 2 and 3 = Z and E isomers of e thy l 2 - (1 1 -propenyl ) -2 -pentenoate , r e s p e c t i v e l y ) . Mass spectrum of e thy l 2 - n - p r o p y l - ( E ) - 3 -pentenoate corresponding to peak 1 in f igure 6. 69 8 Mass spectrum of the minor isomer of ethyl 70 2- (1 ' -propenyl ) -2-pentenoate corresponding to peak 2 in f igure 6. 9 Mass spectrum of the major isomer of e thy l 71 2- (1 ' -propenyl ) -2-pentenoate corresponding to peak 3 in f igure 6. 10 400 MHz proton NMR of synthet ic 2 -n -propy l - 75 (E)-2-pentenoic ac id (*=Z-isomer). 11 To ta l ion current chromatogram of 2-n- 76 propyl -2-pentenoic ac id methyl ester synthesized by dehalogenation (Peak 1 = Z-isomer; Peak 2 = E- isomer) . 12 Mass spectrum of synthet ic 2 -n -p ropy l - 77 (Z)-2-pentenoic ac id methyl ester v i i c o r r e s p o n d i n g to peak 1 i n f i g u r e 11. Mass spectrum of s y n t h e t i c 2-n-propyl- 78 (E)-2-pentenoic a c i d methyl e s t e r c o r r e s p o n d i n g t o peak 2 i n f i g u r e 11. T o t a l ion c u r r e n t chromatogram of e t h y l 84 2-n-propyl-(E)-2-pentenoate s y n t h e s i z e d u s i n g a hydride a b s t r a c t o r (Peak 1 = e t h y l 2-n-propylpentanoate; Peaks 2 and 3 = Z and E isomers of e t h y l 2-n-propyl-2-pentenoate, r e s p e c t i v e l y ) . Mass spectrum of s y n t h e t i c e t h y l 2-n- 85 propylpentanoate c o r r e s p o n d i n g to peak 1 i n f i g u r e 14. Mass spectrum of s y n t h e t i c e t h y l 2-n- 86 prop y l - ( Z ) - 2 - p e n t e n o a t e c o r r e s p o n d i n g to peak 2 i n f i g u r e 14. Mass spectrum of s y n t h e t i c e t h y l 2-n- 87 pro p y l - ( E ) - 2 - p e n t e n o a t e c o r r e s p o n d i n g to peak 3 i n f i g u r e 14. T o t a l ion c u r r e n t chromatogram of 91 s y n t h e t i c e t h y l 2-n-propyl-2,4-pentadienoate (Peak 1 = Z-isomer; Peak 2 = E-isomer). Mass spectrum of s y n t h e t i c e t h y l 2-n- 92 pr o p y l - ( Z ) - 2 , 4 - p e n t a d i e n o a t e c o r r e s p o n d i n g to peak 1 i n f i g u r e 18. Mass spectrum of s y n t h e t i c e t h y l 2-n- 93 pr o p y l - ( E ) - 2 , 4 - p e n t a d i e n o a t e c o r r e s p o n d i n g to peak 2 i n f i g u r e 18. 400 MHz proton NMR of s y n t h e t i c e t h y l 2-n- 95 pr o p y l - ( E ) - 2 , 4 - p e n t a d i e n o a t e (* = Z-isomer). T o t a l ion c u r r e n t chromatogram of 98 s y n t h e t i c e t h y l 2-n-propyl-3-oxopentanoate (peak 1). Mass spectrum of s y n t h e t i c e t h y l 2-n- 99 propyl-3-oxopentanoate c o r r e s p o n d i n g to peak 1 i n f i g u r e 22. T o t a l ion c u r r e n t chromatogram of 102 s y n t h e t i c e t h y l 2-n-propyl-3-hydroxypentanoate. Mass spectrum of s y n t h e t i c 2-n-propyl-3- 103 hydroxypentanoate from f i g u r e 24. v i i i 26 T o t a l ion c u r r e n t chromatogram of s y n t h e t i c 106 2-n-propylpentanoate methyl e s t e r . 27 Mass spectrum of s y n t h e t i c 2-n- 107 pr o p y l p e n t a n o i c a c i d methyl e s t e r from f i g u r e 26. 28 Mass chromatograms at m/z 199 comparing 108 s y n t h e t i c standards of (E)-2-ene VPA and (E)-3-ene VPA with e x t r a c t e d u r i n e , unconjugated f r a c t i o n from b i l e and conjugated f r a c t i o n from b i l e a f t e r a l k a l i n e h y d r o l y s i s from a r a t dosed with (E,E)-2,3'-diene VPA. 29 Mass s p e c t r a comparing (A) s y n t h e t i c 110 (E)-3-ene VPA, (B) u r i n e , (C) unconjugated, and (D) conjugated b i l e f r a c t i o n s from a r a t dosed with (E,E)-2,3'-diene VPA. 30 Mass s p e c t r a comparing (A) s y n t h e t i c 111 (E)-2-ene VPA, (B) u r i n e , (C) unconjugated, and (D) conjugated b i l e f r a c t i o n s from a r a t dosed with (E,E)-2,3'-diene VPA. 31 Mass chromatograms at m/z 201 comparing 112 s y n t h e t i c VPA and e x t r a c t e d u r i n e , unconjugated f r a c t i o n from b i l e , and conjugated f r a c t i o n from b i l e a f t e r a l k a l i n e h y d r o l y s i s from a r a t dosed with ( E , E ) -2,3'-diene VPA. 32 Mass s p e c t r a comparing (A) s y n t h e t i c VPA, 113 (B) u r i n e , (C) unconjugated, and (D) conjugated b i l e f r a c t i o n s from a r a t dosed w i t h (E,E)-2,3'-diene VPA. 33 P o s s i b l e metabolic pathways f o r (E , E ) - 116 2,3'-diene VPA i n humans and animals. 34 A summary of the e x t r a c t i o n and 118 d e r i v a t i z a t i o n procedures f o r the assay of (E,E)-2,3'-diene VPA i n r a t plasma. 35 Mass chromatogram at m/z 207 of the 119 t-BDMS e s t e r of 2H 6~VPA e x t r a c t e d from r a t plasma. 36 Mass chromatogram at m/z 197 of the 120 t-BDMS e s t e r of (E,E)-2,3'-diene VPA e x t r a c t e d from r a t plasma. 37 C a l i b r a t i o n curve f o r (E,E)-2,3'-diene 122 VPA from r a t plasma with a c o n c e n t r a t i o n ix range between 0 - 240 ug/mL. 38 C a l i b r a t i o n curve f o r (E,E)-2,3'-diene 123 VPA from r a t plasma with a c o n c e n t r a t i o n range between 0 - 600 ug/mL. 39 Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e 125 duct i n t a c t r a t s dosed 20 mg/kg i . v . with (E,E)-2,3*-diene VPA (S.D. shown). 40 Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e 126 duct cannulated r a t s dosed 20 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). 41 Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e 127 duct i n t a c t r a t s dosed 100 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). 42 Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e 128 duct cannulated r a t s dosed 100 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). x LIST OF SCHEMES Scheme Page 1 S y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - 57 pentenoic a c i d by deh y d r a t i n g a /3-h y d r o x y a l k e n y l e s t e r . 2 S y n t h e s i s of e t h y l 2 - ( ( E ) - 1 ' - p r o p e n y l ) - 65 (E)-2-pentenoate using a hy d r i d e a b s t r a c t o r . 2a The a l l y l i c hydride a b s t r a c t i o n of a d i a l k y l 67 O - e t h y l - 0 - t r i m e t h y l s i l y l k e t e n e a c e t a l by t r i t y l t e t r a f l u o r o b o r a t e (TTFB). 3 S y n t h e s i s of 2-n-propyl-(E)-2-pentenoic 73 a c i d by dehalogenation of 2-bromo-2-n-pr o p y l p e n t a n o i c a c i d 4 S y n t h e s i s of e t h y l 2-n-propyl-(E)-2- 79 pentenoate by deh y d r a t i n g a j3-hydroxyalkyl e s t e r . 5 S y n t h e s i s of e t h y l 2-n-propyl-(E)-2- 82 pentenoate u s i n g a hy d r i d e a b s t r a c t o r . 6 S y n t h e s i s of e t h y l 2-n-propyl-(E)-2,4- 89 pentadienoate by d e h y d r a t i n g a /3-hyd r o x y a l k e n y l e s t e r 7 S y n t h e s i s of e t h y l 2-n-propyl-3- 97 oxopentanoate. 8 S y n t h e s i s of e t h y l 2-n-propyl-3- 101 hydroxypentanoate. 9 S y n t h e s i s of 2-n-propylpentanoic a c i d . 105 x i LIST OF ABBREVIATIONS 2,3'-diene VPA 2,4-diene VPA 3,3'-diene VPA 4,4'-diene VPA 2- ene VPA 3- ene VPA 4- ene VPA 4-ene-3-keto VPA 3- keto VPA 4- keto VPA 3- OH VPA 4- OH VPA 5- OH VPA AUC bp CBZ CI cm CNS d dd DDQ DPH dt E 2-(1'-propenyl)-2-pentenoic a c i d 2-n-propy1-2,4-pentadienoic a c i d 2-(1'-propenyl)-3-pentenoic a c i d 2-(2'-propenyl)-4-pentenoic a c i d 2-n-propyl-2-pentenoic a c i d 2-n-propyl-3-pentenoic a c i d 2-n-propyl-4-pentenoic a c i d 2-n-propyl-3-oxo-4-pentenoic a c i d 2-n-propyl-3-oxopentanoic a c i d 2-n-propyl-4-oxopentanoic a c i d 2-n-propy1-3-hydroxypentanoic a c i d 2-n-propyl-4-hydroxypentanoic ac i d 2-n-propyl-5-hydroxypentanoic ac i d area under the curve b o i l i n g p o i n t carbamazepine c l e a r a n c e centimeter c e n t r a l nervous system doublet doublet of d o u b l e t s 2,3-dichloro-5,6-dicyano-1,4-benzoquinone phenytoin doublet of t r i p l e t s t r a n s XI1 eV e l e c t r o n v o l t G gauge GABA gamma aminobutyric a c i d GABA-t gamma aminobutyric acid-transaminase GAD g l u t a r i c a c i d decarboxylase GCMS gas chromatography mass spectrometry i .d. i n t e r n a l diameter i .p. i n t r a p e r i t o n e a l IR i n f r a r e d i . v . i n t ravenous J c o u p l i n g constant K E e l i m i n a t i o n r a t e constant kg k i l o g r a m L l i t r e l i t . l i t e r a t u r e l o g l o g a r i t h m m m e t e r / m u l t i p l e t M m o l a r i t y M + molecular ion mg m i l l i g r a m MHz megahertz min. minute mL m i l l i l i t r e mm m i l l i m e t e r mmoles m i l l i m o l e s MW molecular weight m/z mass/charge x i i i N n o r m a l i t y NMR nuc l e a r magnetic spectrometry o.d. outer diameter PB p h e n o b a r b i t a l PE p o l y e t h y l e n e ppm p a r t s per m i l l i o n q q u a r t e t t t r i p l e t ty/2 h a l f l i f e t-BDMS t e r t i a r y - b u t y l d i m e t h y l s i l a n e THF t e t r a h y d r o f u r a n TIC t o t a l ion chromatogram TTFB t r i t y l t e t r a f l u o r o b o r a t e U u n i t s uA microampere ug microgram uL m i c r o l i t r e USP U n i t e d S t a t e s Pharmacopeia volume of d i s t r i b u t i o n VPA v a l p r o i c a c i d Z c i s x i v ACKNOWLEDGEMENT The author s i n c e r e l y thank Dr. Frank S. Abbott f o r h i s support and e x c e l l e n t guidance throughout t h i s program. The author i s g r a t e f u l to the committee members Dr. James Orr, Dr. John S i n c l a i r , Dr. K e i t h McErlane, Dr. Marc Levi n e , and Dr. Robin Ensom f o r t h e i r e f f o r t and h e l p f u l s u g g e s t i o n s . Thanks a l s o go to Mr. Roland Burton f o r h i s t e c h n i c a l a s s i s t a n c e ; Dr. S h e i l a Innes f o r her. guidance i n animal surgery; Dr. James Axelson f o r h i s h e l p f u l h i n t s i n pharmacokinetics and animal surgery; my c o l l e a g u e s Greg S l a t t e r f o r h i s a s s i s t a n c e i n NMR and animal surgery, Kelem Kassahun, Sue Panesar, and Dr. Andrew Acheampong f o r t h e i r h e l p f u l a d v i c e and Matthew Wright f o r h i s a s s i s t a n c e i n animal surgery. The f i n a n c i a l support p r o v i d e d by the F a c u l t y of Pharmaceutical Sciences and Berlex L a b o r a t o r i e s Inc. are g r a t e f u l l y acknowledged. xv DEDICATION To mom and dad. xvi INTRODUCTION V a l p r o i c a c i d (2-n-propylpentanoic a c i d , 2-p r o p y l v a l e r i c a c i d , n - d i p r o p y l a c e t a t e , VPA, Depakene 1*), i s a shor t branched-chain f a t t y a c i d with the f o l l o w i n g s t r u c t u r a l formula: C H o - C H 2 _ C H 2 ^ CH-COOH CH 3-CH 2-CH 2^ Meunier et al . (1963) s e r e n d i p i t o u s l y d i s c o v e r e d that VPA p r o t e c t s mice and r a b b i t s from p e n t y l e n e t e t r a z o l e - i n d u c e d s e i z u r e s . Today, VPA i s widely used f o r the treatment of primary g e n e r a l i z e d , myoclonic, absence or p e t i t e mal, and p a r t i a l s e i z u r e s . Pharmacokinetics of VPA In humans, VPA i s r a p i d l y and completely absorbed a f t e r o r a l a d m i n i s t r a t i o n with peak plasma c o n c e n t r a t i o n s reached w i t h i n 1 to 3 hours ( S c h r e i b e r , 1981). VPA i s 90% bound to plasma albumin with f r e e f r a c t i o n i n c r e a s i n g s t e a d i l y at c o n c e n t r a t i o n s over 100 ug/mL (Johannesson and Henriksen, 1980; M o r s e l l i and F r a n c o - M o r s e l l i , 1980; Schobben et al., 1980; von Unruh et a l . , 1980). Steady-s t a t e plasma c o n c e n t r a t i o n i s achieved w i t h i n 48 hours, however, maximum pharmacological response i s not observed f o r s e v e r a l weeks (Schobben et al ., 1980). T h e r a p e u t i c plasma c o n c e n t r a t i o n s i n a d u l t s range from 60 - 120 ug/mL 1 with a r e p o r t e d h a l f - l i f e of 8 - 16 hours (Johannessen and Henriksen, 1980; Schobben et al., 1980; von Unruh et al., 1980) and a mean c l e a r a n c e of 0.0064 L/kg/h (Gugler et al., 1977). F a r r e l l et al . (1986) have r e p o r t e d t h e r a p e u t i c plasma c o n c e n t r a t i o n s f o r c h i l d r e n (9 months to 18 years) to range from 30 - 80 ug/mL with one p a t i e n t being c o n t r o l l e d on as l i t t l e as 20 ug/mL. The h a l f - l i v e s r e p o r t e d i n 52 e p i l e p t i c c h i l d r e n ranged from 2.5 to 15.2 hours which i s s h o r t e r compared t o a d u l t s ( H a l l et al., 1985). VPA i s a r e s t r i c t i v e l y c l e a r e d drug with an e x t r a c t i o n r a t i o of 0.02 which i s s m a l l e r than the f r e e f r a c t i o n ( K l o t z and Antonin, 1977; Levy and L a i , 1982). The volume of d i s t r i b u t i o n of VPA i s r e l a t i v e l y s m a l l , 0.15 -0.4 L/kg; hence, 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/or r a p i d l y exchangeable e x t r a c e l l u l a r water ( K l o t z and Antonin, 1977; Johannessen and Henriksen, 1980; von Unruh et al., 1980). The l o g VPA c o n c e n t r a t i o n vs time d e c l i n e curve f o r humans has been r e p o r t e d to be monophasic (Schobben et al., 1975); however, other s t u d i e s have shown VPA to d e c l i n e b i p h a s i c a l l y ( K l o t z and Antonin, 1977; Bruni et al ., 1978; Perucca, 1978; B a i l e r et al., 1985). The b i e x p o n e n t i a l d e c l i n e has been a t t r i b u t e d to the e n t e r o h e p a t i c c y c l e s i n c e VPA i s e x t e n s i v e l y conjugated to g l u c u r o n i c a c i d ( L o i s e a u et al ., 1975; B a i l e r el al ., 1985). The pharmacokinetics of VPA i n r a t s have been s t u d i e d but not t o the same extent as i n humans. The l o g VPA 2 c o n c e n t r a t i o n vs time curve f o r r a t s a l s o e x h i b i t s a b i p h a s i c d e c l i n e and when the b i l e duct was l i g a t e d , the e l i m i n a t i o n curve became monophasic ( D i c k i n s o n et al . , 1979; Ogiso et al ., 1986). The b i e x p o n e n t i a l d e c l i n e i s t h e r e f o r e due to e n t e r o h e p a t i c r e c i r c u l a t i o n of VPA. The h a l f - l i f e was r e p o r t e d to be 4.6 hours i n r a t s given 200 mg/kg i . v . of VPA (Loscher, 1978). In a study by D i c k i n s o n et al. (1979), r a t s dosed i . v . with 15, 150, and 600 mg/kg of VPA r e s u l t e d i n h a l f - l i v e s of 11.7, 41, and 125 minutes, r e s p e c t i v e l y . T h i s d i s c r e p a n c y i n h a l f - l i v e s between the two s t u d i e s i s probably due to the methodology used by Loscher (1978). S e v e r a l r a t s were dosed with VPA and the blood samples from the s a c r i f i c e d animals were pooled r a t h e r than s e r i a l l y sampling blood from one r a t as performed by D i c k i n s o n et al. (1979). VPA i n r a t s i s 63.4% bound to serum albumin with no apparent change i n b i n d i n g at c o n c e n t r a t i o n s below 190 ug/mL (Loscher, 1978). The volume of d i s t r i b u t i o n of VPA i n r a t s i s 0.44 - 0.66 L/kg ( D i c k i n s o n , 1979; Nau and Loscher, 1984) which i s r e l a t i v e l y l a r g e , i . e . g r e a t e r than 0.4 L/kg (MacKichan, 1984). The f a c t t h a t VPA i s not h i g h l y bound t o plasma p r o t e i n s p a r t i a l l y e x p l a i n s the l a r g e volume of d i s t r i b u t i o n r e p o r t e d i n r a t s . The p r o t e i n b i n d i n g study of Loscher (1978) only determined p r o t e i n b i n d i n g at two c o n c e n t r a t i o n s , 105 and 190 ug/mL. VPA plasma c o n c e n t r a t i o n s f o r r a t s given 600 mg/kg were r e p o r t e d to be as h i g h as 1700 ug/mL (Dickinson 3 et al., 1979). Although VPA appears to e x h i b i t dose-independent b i n d i n g at c o n c e n t r a t i o n s below 190 ug/mL, s a t u r a t i o n of the plasma p r o t e i n b i n d i n g s i t e s i s p o s s i b l e at the higher plasma c o n c e n t r a t i o n s . T h e r e f o r e , Loscher (1978) should have i n c l u d e d a broader range of VPA plasma c o n c e n t r a t i o n s when determining p r o t e i n b i n d i n g . In summary, a comparison between r a t and human data i n d i c a t e t h at r a t s have a g r e a t e r t o t a l body c l e a r a n c e and thus a s h o r t e r h a l f - l i f e f o r VPA. VPA i s l e s s p r o t e i n bound in r a t s which may account f o r the l a r g e r volume of d i s t r i b u t i o n . The e n t e r o h e p a t i c c y c l e i s r e s p o n s i b l e f o r the b i p h a s i c e l i m i n a t i o n of VPA i n r a t s and humans. Mechanism Of A c t i o n 1. Role of Gamma Aminobutyric A c i d (GABA) The mechanism of s e i z u r e a c t i v i t y and the p h y s i o l o g y of the c e n t r a l nervous system (CNS) i s not w e l l understood. T h i s e x p l a i n s why mechanisms of a c t i o n of a n t i c o n v u l s a n t s are u n c l e a r . Before the mechanism of VPA and other a n t i e p i l e t i c drugs i s understood, the p h y s i o l o g y and pathology of e p i l e p s y i t s e l f must be determined. Although VPA has been the center of a t t e n t i o n among i n v e s t i g a t o r s f o r s e v e r a l y e a r s , the mechanism by which VPA prevents s e i z u r e s i s s t i l l u n c l e a r . The most commonly accepted mechanism of a c t i o n i s v i a gamma aminobutyric a c i d (GABA), an i n h i b i t o r y n e u r o t r a n s m i t t e r . VPA i s thought to decrease GABA-transaminase (GABA-t) a c t i v i t y , a GABA 4 m e t a b o l i z i n g enzyme (Loscher and Schmidt, 1980; K u r u v i l l a and Uretsky, 1981; Hammond et al., 1982), or i n c r e a s e g l u t a r i c a c i d decarboxylase (GAD) a c t i v i t y , a GABA s y n t h e s i z i n g enzyme (Loscher and Nau, 1982; Nau and Loscher, 1982). The mechanism of a n t i e p i l e p t i c a c t i v i t y may not be s o l e l y dependent on GABA. Rats g i v e n gamma-vinyl GABA, a GABA-t i n h i b i t o r , e x h i b i t an i n c r e a s e i n b r a i n GABA c o n c e n t r a t i o n however, a n t i c o n v u l s a n t a c t i v i t y was low (Gale and I a d a r o l a , 1980). P r o t e c t i o n a g a i n s t e l e c t r o s h o c k -induced s e i z u r e s i n r a t s was observed w i t h i n 2 minutes of VPA dosing whereas b r a i n GABA l e v e l s remained s t a t i o n a r y (Kerwin et al., 1980). Hence, the involvement of GABA with VPA a n t i c o n v u l s a n t a c t i v i t y appears to onl y p a r t i a l l y e x p l a i n the mechanism and a non-GABA system i s probably i n v o l v e d . 2. Secondary A n t i e p i l e t i c A c t i v i t y An i n t e r e s t i n g f e a t u r e of VPA i s the presence of a secondary a n t i e p i l e p t i c or c a r r y - o v e r e f f e c t observed i n humans and animals upon c e s s a t i o n of VPA therapy (Nau and Loscher, 1982). A l i k e l y e x p l a n a t i o n would be the accumulation of VPA and/or i t s m e t a b o l i t e ( s ) i n s p e c i f i c areas of the b r a i n r e s p o n s i b l e f o r the c o n t r o l of s e i z u r e a c t i v i t y . In a study by Loscher and Nau (1983), VPA and 8 m e t a b o l i t e s were measured i n the CNS of dogs f o l l o w i n g an acute dose of VPA, and i n r a t s a f t e r continuous dosing with 5 VPA f o r 2 weeks. Both VPA and 2-n-propyl-2-pentenoic a c i d (2-ene VPA) were found i n v a r i o u s regions of the b r a i n i n dogs and r a t s . F o l l o w i n g prolonged treatment with VPA i n r a t s , 2-ene VPA was found to p r o g r e s s i v e l y accumulate i n the b r a i n . In an e a r l i e r study where mice were given VPA c o n t i n u o u s l y f o r 12 days, the presence of 2-ene VPA, the only VPA m e t a b o l i t e , i n the b r a i n was r e p o r t e d (Loscher and Nau, 1982). Upon the d i s c o n t i n u a t i o n of VPA, a n t i e p i l e p t i c a c t i v i t y was s t i l l e vident and while 2-ene VPA was s t i l l p r e s e nt, VPA c o u l d not be d e t e c t e d i n the b r a i n . The c o n c l u s i o n d e r i v e d from these s t u d i e s i s that 2-ene VPA may be r e s p o n s i b l e f o r the secondary a n t i c o n v u l s a n t e f f e c t . F u r t h e r s t u d i e s by Loscher et al. (1984) have shown 2-ene VPA not to be embryotoxic i n rodents i n c o n t r a s t t o VPA. Since 2-ene VPA and VPA are app a r e n t l y equipotent a g a i n s t t o n i c - c l o n i c s e i z u r e s i n mice (Loscher et a l . , 1984) and 2-ene VPA i s ap p a r e n t l y not t e r a t o g e n i c i n mice, t h i s m e t a b o l i t e has p o t e n t i a l as an a l t e r n a t e a n t i c o n v u l s a n t . Acheampong (1985) found that an isomeric mixture of 2-(1'-propenyl)-2-pentenoic a c i d (2,3'-diene VPA) d i s p l a y e d a n t i c o n v u l s a n t a c t i v i t y a g a i n s t p e n t y l e n e t e t r a z o l e - i n d u c e d s e i z u r e s i n mice. Although the c o n c e n t r a t i o n s of 2,3'-diene VPA i n the b r a i n were not measured, the diene appears t o be another p o s s i b l e c o n t r i b u t o r to the c a r r y - o v e r e f f e c t . 6 Drug I n t e r a c t i o n s The p o t e n t i a l f o r drug i n t e r a c t i o n s with VPA are numerous s i n c e VPA can d i s p l a c e or be d i s p l a c e d from plasma p r o t e i n s . The metabolism of VPA can be induced by s e v e r a l agents, and VPA can i n h i b i t the metabolism of c e r t a i n drugs. Drug i n t e r a c t i o n s between VPA and other a n t i c o n v u l s a n t s are l i k e l y to occur s i n c e VPA i s commonly used with other a n t i e p i l e p t i c agents. 1) VPA I n t e r a c t i o n s With Other Drugs P a t i e n t s on p h e n o b a r b i t a l (PB) therapy show an in c r e a s e i n PB l e v e l s of up to 100% upon the a d d i t i o n of VPA ( P a t e l et al ., 1 980; Suganuma et al., 1981). The use of PB i n c o n j u n c t i o n with VPA i s not uncommon. Hence, an adjustment of PB d o s i n g i s r e q u i r e d to prevent PB t o x i c i t y . The mechanism by which VPA e l e v a t e s plasma PB l e v e l s i s unclear although displacement of PB from plasma p r o t e i n s by VPA i s a p o s s i b i l i t y . PB i s not e x t e n s i v e l y bound to plasma p r o t e i n s (50%) consequently, displacement of PB by VPA would not r e s u l t in such a s i g n i f i c a n t i n c r e a s e i n PB l e v e l . Kapetanovic et al. (1981) suggest VPA c o m p e t i t i v e l y i n h i b i t s the metabolism of PB thereby i n c r e a s i n g plasma PB c o n c e n t r a t i o n s . The i n t e r a c t i o n between VPA and phenytoin (DPH) i s more complex. Upon c o a d m i n i s t r a t i o n with VPA, t o t a l DPH plasma l e v e l s have been known to decrease to the p o i n t of s e i z u r e a c t i v i t y as w e l l as i n c r e a s e c a u s i n g DPH t o x i c i t y (Rodin et 7 al., 1981). DPH i s e x t e n s i v e l y bound to plasma p r o t e i n s and shares the same b i n d i n g s i t e with VPA (Kober et al., 1980). F r i e l et al. (1979) r e p o r t e d an i n i t i a l i n c r e a s e i n f r e e plasma DPH l e v e l s due to the displacement of DPH by VPA. An i n c r e a s e i n DPH e l i m i n a t i o n f o l l o w s r e s u l t i n g i n a decrease i n t o t a l DPH l e v e l s with f r e e DPH l e v e l s r e t u r n i n g to t h e i r i n i t i a l v a l u e . A l v a r e z (1985) a l s o reported i n c r e a s e s and decreases of t o t a l DPH plasma c o n c e n t r a t i o n s with f r e e DPH l e v e l s i n c r e a s e d i n most s u b j e c t s . C o n s i s t e n t r e s u l t s r e g a r d i n g the i n t e r a c t i o n between VPA and DPH have yet to be r e p o r t e d . These s t u d i e s have shown DPH plasma c o n c e n t r a t i o n s to i n c r e a s e or decrease upon VPA c o a d m i n i s t r a t i o n c o n f i r m i n g the complexity of t h i s i n t e r a c t i o n . Carbamazepine (CBZ) l e v e l s were found to i n c r e a s e , decrease, and remain unchanged when VPA was added to therapy (Wilder et al., 1978). Since VPA binds to plasma p r o t e i n s t o a gr e a t e r extent, the displacement of CBZ would decrease t o t a l CBZ l e v e l s by i n c r e a s i n g the a v a i l a b i l i t y of f r e e CBZ f o r metabolism (Mattson et al ., 1980). P a t i e n t s on combined VPA and CBZ therapy have i n c r e a s e d serum l e v e l s of CBZ-10,11-epoxide, a CBZ m e t a b o l i t e , which decreased when VPA therapy was d i s c o n t i n u e d ( M e i j e r et al., 1984). I f VPA i n h i b i t s CBZ metabolism, an i n c r e a s e i n t o t a l CBZ l e v e l s would occur however, there i s no evidence as yet f o r t h i s i n t e r a c t i o n . 8 2) Drug i n t e r a c t i o n s of other drugs with VPA P a t i e n t s on VPA therapy at s t e a d y - s t a t e given DPH or PB showed a decrease i n mean serum VPA and h a l f - l i f e (Kutt, 1984). T h i s e f f e c t can be a t t r i b u t e d to the displacement of VPA from plasma p r o t e i n s by DPH or PB r e s u l t i n g i n an i n c r e a s e d c l e a r a n c e of VPA. Since f r e e VPA l e v e l s remain constant and only t o t a l VPA l e v e l s decrease, the l i k e l i h o o d of t h i s i n t e r a c t i o n being c l i n i c a l l y s i g n i f i c a n t i s minimal. The metabolism of VPA can be induced by PB c a u s i n g a decrease i n t o t a l VPA l e v e l s with f r e e l e v e l s unchanged. Although the c l i n i c a l i m p l i c a t i o n s are minimal t h i s i n t e r a c t i o n cannot be completely d i s c o u n t e d . I f the i n d u c t i o n of VPA metabolism i s e x t e n s i v e , a decrease i n t o t a l plasma VPA to sub t h e r a p e u t i c l e v e l s c o u l d p r e c i p i t a t e s e i z u r e a c t i v i t y . The i n t e r a c t i o n between VPA and a c e t y l s a l i c y l i c a c i d (ASA) has been s t u d i e d by Orr et al . (1982) and F a r r e l l et a l . (1982). An i n c r e a s e i n f r e e and t o t a l VPA l e v e l s were r e p o r t e d i n humans given VPA and ASA co n c o m i t a n t l y (Orr et al., 1982). S a l i c y l a t e s have been r e p o r t e d to d i s p l a c e VPA from serum albumin in vitro (Fleitman et al., 1980). In a study i n v o l v i n g an a d u l t s u b j e c t and 7 p e d i a t r i c p a t i e n t s on VPA at s t e a d y - s t a t e , the i n t r o d u c t i o n of ASA decreased 2-ene VPA, 2-n-propyl-3-hydroxypentanoic a c i d (3-OH VPA), and 2-n-propyl-3-oxopentanoic a c i d (3-keto VPA) e x c r e t i o n i n the u r i n e (Abbott et al., 1986). T h e r e f o r e , the i n c r e a s e 9 i n f r e e and t o t a l VPA may be due to an i n h i b i t i i o n of 0-o x i d a t i o n of VPA by ASA. T o x i c i t y The e f f e c t i v e use of VPA i s not without some minor adverse e f f e c t s (nausea, v o m i t i n g , a n o r e x i a , changes i n s l e e p i n g h a b i t s , and/or e n u r e s i s (Browne, 1980)) or major c o m p l i c a t i o n s (most noted h e p a t i c t o x i c i t y ) . T h i s h e p a t o t o x i c i t y i s a r a r e but f a t a l i d i o s y n c r a t i c r e a c t i o n o c c u r r i n g p r i m a r i l y i n young p a t i e n t s on m u l t i - d r u g therapy and appears w i t h i n the f i r s t s i x months of VPA therapy (Gram and Bentsen, 1983; Green, 1984). The f a t a l r e a c t i o n appears to resemble s e v e r a l other c o n d i t i o n s such as Reye's syndrome, Jamaican v o m i t i n g s i c k n e s s , a f l a t o x i n p o i s o n i n g , and t e t r a c y c l i n e h e p a t o t o x i c i t y (Itoh et al., 1982; Zimmerman and Ishak, 1982). Hypoglycin and 4-pentenoic a c i d were found to be h i g h l y h e p a t o t o x i c producing s i m i l a r h e p a t i c l e s i o n s . H y p o g l y c i n , a compound found i n an u n r i p e t r o p i c a l f r u i t , i s metabolized to methylene c y c l o p r o p y l a c e t i c a c i d which i s r e s p o n s i b l e f o r causing Jamaican v o m i t i n g s i c k n e s s ( B r e s s l e r et al., 1969). Whereas a R e y e ' s - l i k e syndrome can be produced i n r a t s t r e a t e d with 4-pentenoic a c i d (Glasgow and Chase, 1975). The cause of VPA h e p a t o t o x i c i t y i s thought to o r i g i n a t e from VPA m e t a b o l i t e s i n which 2-n-propyl-4-pentenoic a c i d (4-ene VPA) appears to be a major c o n t r i b u t o r (Zimmerman 10 and Ishak, 1982). The 3 compounds, methylene c y c l o p r o p y l a c e t i c a c i d , 4-pentenoic a c i d , and 4-ene VPA, are s t r u c t u r a l l y s i m i l a r which may e x p l a i n t h e i r p o t e n t i a l to produce h e p a t o t o x i c i t y . CH 2=C-,CH-CH2-COOH methylene c y c l o p r o p y l a c e t i c CH 2 a c i d ( m e t a b o l i t e of hypog l y c i n ) CH 2=CH-CH2-CH 2-COOH 4-pentenoic a c i d C H 3—CH 2 — C H 2 ^CH-COOH 4-ene VPA CH 2=CH-CH 2' Rettenmeier et al . ( 1985)' p o s t u l a t e that 4-ene VPA may be i n v o l v e d i n a " s u i c i d e s u b s t r a t e " mechanism. The m e t a b o l i t e , 4-ene VPA, may be metabolized to a r e a c t i v e e l e c t r o p h i l l i c i n t e r m ediate which i n h i b i t s f a t t y a c i d metabolism by a l k y l a t i n g key enzymes. 0-oxidation of 4-ene VPA y i e l d s 2-n-propyl-3-oxo-4-pentenoic a c i d (4-ene-3-keto VPA) which i s a r e a c t i v e s p e c i e s due t o i t s a b i l i t y to form a r e a c t i v e resonance s t r u c t u r e : CH2=CH-C^ CH2-CH=C ^CH-COOH — ^CH-COOH H 7 C 3 H 7 C 3 However, attempts by Rettenmeier et al. (1985,1986b) to i s o l a t e 4-ene-3-keto VPA i n r a t s have f a i l e d , p o s s i b l y due to the r e a c t i v e nature of t h i s compound. 11 H i s t o l o g i c a l examination of the l i v e r s of VPA h e p a t o t o x i c v i c t i m s r e v e a l e d the presence of m i c r o v a c u o l a r s t e a t o s i s with n e c r o s i s o c c u r r i n g r e g i o n a l l y (Lewis et a l . , 1982; Zimmerman and Ishak, 1982; Kesterson et a l . , 1984). The p r o d u c t i o n of s t e a t o s i s i n r a t s was accomplished with a dose of VPA g r e a t e r than t h a t used c l i n i c a l l y i n man (Granneman et al., 1984b). U r i n a r y a n a l y s i s of p a t i e n t s t h a t succumbed to VPA h e p a t o t o x i c i t y r e v e a l e d s e v e r a l i n t e r e s t i n g f e a t u r e s . Most remarkable was the i n h i b i t i o n of 0 - o x i d a t i o n noted by the absence of 3-keto VPA (Kochen et al., 1983; Kuhara et a l . , 1985; T u r n b u l l et al., 1986). Kuhara et al. (1985) noted an i n c r e a s e i n 2 - p r o p y l g l u t a r i c a c i d , an end product of c o -o x i d a t i o n whereas Kochen et al. (1983) found low c o n c e n t r a t i o n s of 2 - p r o p y l g l u t a r i c a c i d . The c o n t r a s t i n r e s u l t s c o u l d be due to the p a t i e n t ' s pathology. The f i r s t p a t i e n t had Reye's syndrome and was p l a c e d on VPA therapy (Kuhara et al., 1985) whereas the l a t t e r p a t i e n t developed a R e y e ' s - l i k e syndrome while on VPA therapy (Kochen et a l . , 1983). However, a mechanism was proposed by J e z e q u e l et al . (1984) r e g a r d i n g the i n h i b i t o n of 0 - o x i d a t i o n . An abnormal m e t a b o l i t e of VPA would bi n d acetyl-CoA and compete with endogenous s u b s t r a t e s thereby i n h i b i t i n g /3-oxidation and p o s s i b l y damaging the m i t o c h o n d r i a . T h i s process may be m agnified i n s e v e r i t y by the presence of an enzymatic d e f e c t , an in-born e r r o r i n metabolism, or an a s s o c i a t e d pathology. 12 Of i n t e r e s t was the presence of s e v e r a l d i u n s a t u r a t e d m e t a b o l i t e s found i n u n u s u a l l y h i g h c o n c e n t r a t i o n s i n the plasma and u r i n e of a VPA-hepatotoxic v i c t i m . Kochen et a l . (1983), f o r the f i r s t time, i d e n t i f i e d these d i u n s a t u r a t e s to be 2- n - p r o p y l - ( E ) - 2 , 4 - p e n t a d i e n o i c a c i d ((E)-2,4-diene VPA), 2-(2'-propenyl)-4-pentenoic a c i d (4,4'-diene VPA), and two s t r u c t u r a l l y unknown diene m e t a b o l i t e s . The unknown dienes were thought to be 2,3'-diene VPA and 2~(1'-propenyl)-3-pentenoic a c i d (3,3'-diene VPA)(Kochen et a l . , 1983) . The h y p o t h e s i s by Jez e q u e l et al. (1984) mentions an abnormal m e t a b o l i t e of VPA being r e s p o n s i b l e f o r • the i n h i b i t i o n of 0 - o x i d a t i o n . Since e l e v a t i o n of dienes was observed i n the u r i n e and plasma of he p a t o t o x i c v i c t i m s , and 4,4'-diene VPA was not present i n normal p a t i e n t s on VPA, the i n h i b i t i o n of /3-oxidation and p o s s i b l y the cause of the hepatoxic r e a c t i o n may be due to one or more of the d i u n s a t u r a t e d m e t a b o l i t e s . Metabolism VPA i s mainly metabolized by the l i v e r v i a a h i g h l y complex scheme with 16 m e t a b o l i t e s i d e n t i f i e d i n humans and animals. Since VPA i s a f a t t y a c i d , s e v e r a l routes of metabolism are p o s s i b l e : g l u c u r o n i d a t i o n , 0 - o x i d a t i o n , CJ o x i d a t i o n , and to-1 o x i d a t i o n (Loscher, 1981b; Abbott et al., 1986). F i g u r e 1 i l l u s t r a t e s the v a r i o u s metabolic pathways f o r VPA and i t s m e t a b o l i t e s i n humans. Of these, 13 C H , - C H , - C M -C H , - C H j - C H | /  *H> «liKurontd» CHCOOGlu ON C H j - C M j - C M ^ CHCOOH C H J - C H J - C H J S-OM tf* WOC-CHj -CM ? ,CHCOOH CH,-CHj-CH? f-rro»»1fl1utirlt »dd WIWOIC ACID CHj— CH^-CH 2 ;CHCOOH CHj -CH j -CH j OH • C M j - C K - C H ? C N , - C H 2 - C H { CHCOOH <-OH <P« 0 C H j - C M 2 - t H ? CHCOOH CH?—CH-CM? CHCOOH C H j - C H 2 - C H 2 4-ene VP* CH.-CH—CH CHj=CH-CH { C H y = C H - C H ^ M ' - d l t w t »P« CHCOOH C H , - C H ? - C H ^ CHCOOH COOH C H , - C H , - C H ^ C H j - C H , - C H ^ ?-tnt « H l ,OM C H j - C H ^ - C H ^ C-COOH CH-COOH C H j - C H ; - C H ? 3-OH m C H — CHj-CH CH,-CH, -CH. -CH: .COOH V COOH HOOC-CH, C H j - C H ? - C H ? CHCOOH Z-Propyltucclntc «cld C H j - C M ^ C ^ C H , - C H ? - C H ^ CHCOOH 3-mo »P» Figure 1: Metabolic pathways of v a l p r o i c a c i d i n humans (Acheampong, 1985). 0 - o x i d a t i o n and g l u c u r o n i d a t i o n are the most prominent m e t a b o l i c pathways whereas w and CJ-1 o x i d a t i o n occurs to a l e s s e r extent (Granneman et al ., 1984). In humans, 20 - 70% of VPA i s e x c r e t e d i n the u r i n e as VPA-glucuronide while 1 - 3% i s e l i m i n a t e d as the f r e e a c i d (Bruni and Wilder, 1979; Gugler and von Unruh, 1980; B a i l e r et al., 1985). The m e t a b o l i t e 2-ene VPA i s the r e s u l t of 0-o x i d a t i o n which occurs i n the mitochondria (Lazarow, 1978) and p o s s i b l y i n peroxisomes (Bjorkhem et al., 1984; Van den Branden and Roels, 1985). Approximately 12% of VPA i s e x c r e t e d as 2-ene VPA whereas the remaining f r a c t i o n of 2-ene VPA i s f u r t h e r metabolized to 3-OH VPA and 3-keto VPA (Granneman et al ., 1984; Abbott et al., 1986). The minor routes of metabolism, CJ and CJ-1 o x i d a t i o n , occur r e a d i l y v i a the l i v e r microsomal enzyme system and are cytochrome P-450 dependent ( P r i c k e t t and B a i l l i e , 1984 and r e f e r e n c e s t h e r e i n ) . The o - o x i d a t i o n pathway m e t a b o l i z e s VPA to 2-n-propyl-5-hydroxypentanoic a c i d (5-OH VPA), 2 - p r o p y l g l u t a r i c a c i d , and 2-propylmalonic a c i d while CJ-1 o x i d a t i o n produces 2-n-propyl-4-hydroxypentanoic a c i d (4-OH VPA), 2-n-propyl-4-oxopentanoic a c i d (4-keto VPA), and 2 - p r o p y l s u c c i n i c a c i d (Acheampong et al . , 1983; Granneman et al ., 1984). Since CJ and OJ-1 o x i d a t i o n are known to be cytochrome P-450 dependent, enzyme indu c e r s and i n h i b i t o r s may a f f e c t m e t a b o l i t e c o n c e n t r a t i o n s . In f a c t , Heinemeyer et a l . (1985) have shown i n r a t s t hat enzyme in d u c e r s ( p h e n o b a r b i t a l , 3-methylcholanthrene, 0-15 naphthoflavone, and c l o f i b r a t e ) s i g n i f i c a n t l y i n c r e a s e the presence of h y d r o x y l a t e d m e t a b o l i t e s of VPA. An in vitro study by P r i c k e t t and B a i l l i e (1984) u s i n g r a t l i v e r microsomes showed that h y d r o x y l a t i o n of VPA was suppressed by CO, SKF-525A, and metyrapone (enzyme i n h i b i t o r s ) . Hence, the p o t e n t i a l f o r s i g n i f i c a n t l y a l t e r i n g the metabolic p r o f i l e of VPA i n p a t i e n t s on m u l t i - d r u g therapy ( e s p e c i a l l y enzyme inducers and i n h i b i t o r s ) i s p o s s i b l e . The u n s a t u r a t e d and d i u n s a t u r a t e d m e t a b o l i t e s of VPA are produced by l e s s e r known pathways, except f o r 2-ene VPA. Granneman et al. (1984) suggest that the unsaturated m e t a b o l i t e s 4-ene VPA and 2-n-propyl-3-pentenoic a c i d (3-ene VPA) a r i s e from 7 and A dehydrogenation pathways, r e s p e c t i v e l y . The m e t a b o l i t e 4-ene VPA can undergo f u r t h e r metabolism v i a 0 - o x i d a t i o n to y i e l d 2,4-diene VPA and even f u r t h e r t o a suspected hepatogen, 4-ene-3-keto VPA (Granneman et al., 1984; Rettenmeier et al ., 1984, 1986b). In humans 4-ene VPA and 2,4-diene VPA comprise 1.4 and 0.4% of VPA i n serum, r e s p e c t i v e l y (Abbott et al., 1986). The m e t a b o l i t e 3-ene VPA i s f u r t h e r m e tabolized to 2-ene VPA and 3-keto VPA i n r a t s which suggest the involvement of enoyl CoA isomerase (Granneman et a/., 1984). Mean serum c o n c e n t r a t i o n of 3-ene VPA i n humans i s 0.94 ug/mL which i s 2% of VPA i n serum (Abbott et al., 1986) Two other d i u n s a t u r a t e d m e t a b o l i t e s , (E,E)-2,3'-diene and 4,4'-diene VPA, have been r e p o r t e d i n humans and are termed secondary m e t a b o l i t e s a r i s i n g from monounsaturates. 16 Abbott et al. (1986) and Panesar (1987) have q u a n t i t a t e d (E,E)-2,3'-diene VPA i n humans and found the diene to be a major unsaturated m e t a b o l i t e of VPA (3 - 6% of VPA i n serum). Singh (unpublished) has shown (E,E)-2,3'-diene VPA to a r i s e from (E)-2-ene VPA i n r a t s probably v i a a 7 -dehydrogenation pathway. (E,E)-2,3'-diene VPA c o u l d a l s o a r i s e from (E)-3-ene VPA v i a 0 - o x i d a t i o n . The m e t a b o l i t e 4,4'-diene VPA was t h e o r i z e d to o r i g i n a t e from 4-ene VPA and was i d e n t i f i e d i n a p a t i e n t with VPA h e p a t o t o x i c i t y (Kochen and Sprunck, 1984). Since 4-ene VPA has been a s s o c i a t e d with VPA h e p a t o t o x i c i t y (Browne, 1980) and 4,4'-diene VPA was only present d u r i n g VPA h e p a t o t o x i c i t y , 4,4'-diene VPA may have a r i s e n v i a the metabolism of 4-ene VPA. The complete metabolic p r o f i l e of VPA has not been e n t i r e l y e l u c i d a t e d i n man or animals. The c o n t r i b u t i o n of the m e t a b o l i t e s to a n t i c o n v u l s a n t a c t i v i t y and h e p a t o t o x i c i t y warrants the need to completely determine the metabolism of VPA. A r e l a t i v e l y non-toxic or i n a c t i v e VPA m e t a b o l i t e c o u l d be converted to a t o x i c or a c t i v e compound. Th e r e f o r e , f u r t h e r s t u d i e s are r e q u i r e d to thoroughly understand the d i s p o s i t i o n and p h y s i o l o g i c a l i m p l i c a t i o n s of VPA and i t s m e t a b o l i t e s . S i g n i f i c a n c e Of The Unsaturated VPA M e t a b o l i t e s The unsaturated m e t a b o l i t e s of VPA appear to be i n v o l v e d i n the secondary a n t i e p i l e t i c e f f e c t and the f a t a l 17 i d i o s y n c r a t i c h e p a t o t o x i c i t y . Animal s t u d i e s by Loscher and Nau (1982, 1983) have found 2-ene VPA to accumulate upon c h r o n i c a d m i n i s t r a t i o n of VPA and that 2-ene VPA l e v e l s p a r a l l e l a n t i c o n v u l s a n t a c t i v i t y . The unsaturated m e t a b o l i t e s , 4-ene and 2,4-diene VPA, have been shown to induce p a t h o l o g i c a l changes, i n c l u d i n g m i c r o v e s i c u l a r s t e a t o s i s , i n r a t l i v e r (Kesterson et al., 1984). A p a t i e n t d i s p l a y i n g VPA h e p a t o t o x i c i t y had abnormally h i g h l e v e l s of 4-ene and 2,4-diene VPA and i s i n keeping with the h e p a t o t o x i c i t y r e s u l t s seen i n the animal experiments. A d i u n s a t u r a t e d m e t a b o l i t e which has not caught the a t t e n t i o n of i n v e s t i g a t o r s i s (E,E)-2,3'-diene VPA. Although (E,E)-2,3'-diene VPA has been termed the major d i u n s a t u r a t e d m e t a b o l i t e of VPA by Abbott et al. (1986) and Panesar (1987), l i m i t e d i n f o r m a t i o n i s known about t h i s m e t a b o l i t e . S e v e r a l s t u d i e s have termed (E,E)-2,3'-diene VPA as diene-VPA because of the i n v e s t i g a t o r s i n a b i l i t y to i d e n t i f y t h i s m e t a b o l i t e (Kochen and S c h e f f n e r , 1980; Acheampong et al., 1983; Kochen et al., 1983; Granneman et al . , 1984). The i d e n t i f i c a t i o n and study of (E,E)-2,3'-diene VPA r e q u i r e s the presence of a s y n t h e t i c r e f e r e n c e sample which i s not r e a d i l y a v a i l a b l e i n s i g n i f i c a n t amounts. The d i f f i c u l t y i n s y n t h e s i z i n g (E,E)-2,3'-diene VPA i s the l a c k of a s t e r e o s p e c i f i c procedure. The method of Acheampong and Abbott (1985) f o r the s y n t h e s i s of (E,E)-2,3'-diene VPA a f f o r d e d a mixture of isomers with the (E,E)-isomer being 18 the major product. Although t h i s product i s not i s o m e r i c a l l y pure, the s y n t h e s i s appears to be the most s u c c e s s f u l . With the a v a i l a b i l i t y of s y n t h e t i c 2,3'-diene VPA, Acheampong (1985) demonstrated the diene's a b i l i t y to p r o t e c t a d u l t male Swiss mice a g a i n s t p e n t y l e n e t e t r a z o l e -induced s e i z u r e s . In f a c t , 2,3'-diene VPA was as potent as 2-ene VPA a g a i n s t p e n t y l e n e t e t r a z o l e - i n d u c e d s e i z u r e s . These r e s u l t s appear to i n d i c a t e 2,3'-diene VPA may c o n t r i b u t e to the secondary a n t i c o n v u l s a n t a c t i v i t y observed in animals and humans. Since 2,3'-diene VPA, the major d i u n s a t u r a t e d m e t a b o l i t e of VPA, has been shown to possess a n t i c o n v u l s a n t a c t i v i t y , i n f o r m a t i o n r e g a r d i n g i t s t o x i c i t y , p harmacokinetics, metabolism, and t i s s u e d i s t r i b u t i o n should be known. T h i s i n f o r m a t i o n can a i d i n the understanding of VPA's mechanism of a c t i o n and t o x i c i t y which can only r e s u l t i n b e t t e r p a t i e n t care and s a f e t y . Object i v e s 1. Using the procedures of Acheampong and Abbott (1985) f o r the s y n t h e s i s of (E,E)-2,3'-diene VPA, attempts were to be made to improve the y i e l d as w e l l as the isomeric p u r i t y by modifying these procedures. Another method f o r the s y n t h e s i s of (E,E)-2,3'-diene VPA was to be i n v e s t i g a t e d with the i n t e n t of improving both the y i e l d and isomeric 19 p u r i t y . T h i s method i n v o l v e s the o x i d a t i o n of a t r i a l k y l s i l y l k e t e n e a c e t a l u s i n g a hydride a b s t r a c t o r . 2. VPA and other VPA m e t a b o l i t e s (2-ene VPA, 3-ene VPA, 2,4-diene VPA, 3-OH VPA, and 3-keto VPA) were t o be s y n t h e s i z e d i n order t o have r e f e r e n c e compounds and s t a r t i n g m a t e r i a l f o r f u r t h e r s y n t h e t i c methods. 3. The metabolism of (E,E)-2,3'-diene VPA was to be i n v e s t i g a t e d i n male Wistar r a t s . U r i ne and b i l e samples were to be c o l l e c t e d and analyzed by GCMS f o r t h e i r m etabolic c o n t e n t . 4. The pharmacokinetic p r o f i l e of (E,E)-2,3'-diene VPA was to be determined i n Wistar r a t s with and without t h e i r b i l e duct i n t a c t . In vitro plasma p r o t e i n b i n d i n g of (E,E)-2,3'-diene VPA was to be determined using u l t r a f i l t r a t i o n at v a r i o u s c o n c e n t r a t i o n s . 5. Plasma, u r i n e , and b i l e samples of (E,E)-2,3'-diene VPA and i t s m e t a b o l i t e s were to be assayed by GCMS us i n g s e l e c t e d ion m o n i t o r i n g . 20 EXPERIMENTAL A. Chemicals and M a t e r i a l s 1 . S y n t h e s i s Chemicals were reagent grade and o b t a i n e d from the f o l l o w i n g s ources. a. A l d r i c h Chemical Co.(Milwaukee, Wisconsin) a c r o l e i n t - b u t y l d i m e t h y l s i l y l c h l o r i d e n - b u t y l l i t h i u m (1.6 M i n hexane) 18-crown-6 de u t e r o c h l o r o f o r m (g o l d l a b e l ) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone d i isopropylamine 4-dimethylaminopyridine l i t h i u m aluminum hydride methanesulfonyl c h l o r i d e pentanoic a c i d 2-pentanone potassium h y d r i d e (35% o i l d i s p e r s i o n ) p r o p i o n y l c h l o r i d e p r o p y l bromide p r o p y l i o d i d e t e t r a h y d r o f u r a n t r i e t h y l a m i n e t r i p h e n y l c a r b e n i u m t e t r a f l u o r o b o r a t e 21 b. BDH Chemicals (Toronto, O n t a r i o ) benzene c h l o r o f o r m ether (anhydrous) h y d r o c h l o r i c a c i d magnesium s u l f a t e (anhydrous) sodium hydroxide sodium s u l f a t e (anhydrous) s u l f u r i c a c i d c. B r i t i s h Drug House (Poole, U.K.) iodoethane p y r i d i n e d. Caledon L a b o r a t o r i e s L t d . (Georgetown, O n t a r i o ) dichloromethane e t h a n o l e t h y l a c e t a t e e. Eastman Kodak Co. (Rochester, New York) propionaldehyde f. F i s h e r S c i e n t i f i c Co. ( F a i r l a w n , New J e r s e y ) bromine t - b u t a n o l hexamethylphosphoramide ( D o r c o l R ) 22 hydrogen bromide (48%) q u i n o l i n e g. M a l l i n k r o d t Chemicals ( S t . L o u i s , M i s s o u r i ) potassium carbonate (anhydrous) sodium bicarbonate h. Matheson Coleman and B e l l Co. (Norward, Ohio) c h l o r o t r i m e t h y l s i l a n e phosphorus t r i b r o m i d e 2 , 4 , 6 - t r i m e t h y l p y r i d i n e 2. Animal Experiments a. A l l e n and Hanburys (Toronto, O n t a r i o ) h e p a r i n sodium i n j e c t i o n USP b. Animal Care F a c i l i t y (U.B.C., Vancouver, B.C.) Wistar male r a t s (200 - 300g) c. Becton D i c k i n s o n (Rutherford, New J e r s e y ) Yale needle 22G, 1 1/2" t u b e r c u l l i n s y r i n g e 1cc d. Clay Adams (Parsippany, New J e r s e y ) p o l y e t h y l e n e t u b i n g PE 10, PE 50 e. Dow Corning (Midland, Michigan) 23 S i l a s t i c 1 * medical grade t u b i n g - 0.02" i . d . x 0.037" o.d. - 0.058" i . d . x 0.077" o.d. f . E t h i c o n L t d . (Peterborough, O n t a r i o ) 4-0 s i l k g. F i s h e r S c i e n t i f i c Co. (Toronto, O n t a r i o ) Caraway R micro blood c o l l e c t i n g tubes (red, h e p a r i n i z e d ) h. Permabond I n t e r n a t i o n a l D i v . (Englewood, New J e r s e y ) 910 R Adhesive i . Sherwood Me d i c a l Ind. (St. L o u i s , M i s s o u r i ) C r i t o c a p s R ( r e g u l a r ) j . T r a v e n o l Canada Inc. ( M i s s i s s a u g a , O n t a r i o ) 0.9% sodium c h l o r i d e i n j e c t i o n USP 3. P r o t e i n B i n d i n g Experiment a. Amicon Corp. (Danvers, MA) m i c r o p a r t i t i o n system, MPS-1 u l t r a f i l t r a t i o n membrane, type YMT 24 B. Instrumentation 1. I n f r a r e d Spectrometry IR s p e c t r a were obtained, using sodium c h l o r i d e d i s k s , as neat l i q u i d f i l m s on a Unicam SP1000 spectrometer. 2. Nuclear Magnetic Resonance Spectrometry Proton NMR s p e c t r a were performed on a Bruker WP-80, a Nicole t - O x f o r d - 2 7 0 , and a Bruker WH-400 spectrometer at the NMR l a b o r a t o r y i n the Department of Chemistry, U n i v e r s i t y of B r i t i s h Columbia. The s o l v e n t and i n t e r n a l standard used were deuterated c h l o r o f o r m (CDC1 3) and t e t r a m e t h y l s i l a n e , r e s p e c t i v e l y . 3. Gas Chromatography - Mass Spectrometry a. Packed column GCMS a n a l y s i s was performed on a Hewlett Packard 5700A gas chromatograph i n t e r f a c e d to a V a r i a n MAT-111 mass spectrometer using a v a r i a b l e s l i t s e p a r a t o r . E l e c t r o n impact data were obtained with the source p r e s s u r e at 5 X 10"*^ T o r r and an emission c u r r e n t of 300 uA at 70 eV. The instrument recorded data i n scan mode with a range of 15 - 750 mass u n i t s every 5 seconds. The data was processed using an o n - l i n e V a r i a n 620L computer. The f o l l o w i n g c o n d i t i o n s were used: a column (1.8 m x 2 mm i.d.) packed with 3% D e x s i l 300 on 100 - 120 mesh Supelcoport (Supelco, B e l l e f o n t e , Penn.); oven temperature 25 50°C to 270°C at 8°C/min; i n j e c t i o n p o r t temperature 250°C; s e p a r t o r temperature 250°C; c a r r i e r gas (helium) flow r a t e 25 mL/min. b. C a p i l l a r y Column C a p i l l a r y GCMS a n a l y s i s was performed on a Hewlett Packard 5987A instrument i n t e r f a c e d t o a HP-1000 o n - l i n e computer. The gas chromatograph i s coupled to the mass spectrometer v i a an o p e n - s p l i t i n t e r f a c e . E l e c t r o n impact data were recorded with the source pressure at 1.8 x 10"^ Tor r and an emission c u r r e n t of 300 uA at 70 eV. S e l e c t e d ion m o nitoring f o r ions m/z 197 (2,3'-diene VPA) and m/z 207 ( 2Hg-VPA, i n t e r n a l standard) were used f o r the pharmacokinetic study. For the m e t a b o l i t e study, the instrument was switched to l i n e a r mode with a mass range of 10 to 400 u n i t s . Analyses of t - 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 of v a l p r o i c a c i d analogs were performed using the f o l l o w i n g parameters: OV-1701 f u s e d - s i l i c a column (25 m x 0.32 mm i.d.)(Quadrex Corp., New Haven, Conn.); oven temperature, 50°C to 100°C at 30°C/min, 100°C to 230°C at 8°C/min; i n j e c t i o n p o r t temperature 240°C; t r a n s f e r l i n e temperature 250°C; source temperature 240°C; s p l i t l e s s mode; c a r r i e r gas (helium) flow r a t e 1 mL/min. c. D e r i v a t i z a t i o n 26 The f r e e a c i d s of VPA and i t s m e t a b o l i t e s are not w e l l r e s o l v e d by GCMS and t h e r e f o r e r e q u i r e d e r i v a t i z a t i o n with t-BDMS reagent t o i n c r e a s e r e s o l u t i o n . The reagent c o n s i s t s of 50 mg of dim e t h y l a m i n o p y r i d i n e , 1 mL of anhydrous p y r i d i n e , and 1 g of 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 dimethylaminopyridine was d i s s o l v e d f i r s t i n anhydrous p y r i d i n e and then added to a v i a l c o n t a i n i n g 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 . Upon mixing, the p r e c i p i t a t e was allowed t o s e t t l e and the supernatant was used f o r d e r i v a t i z i n g . A 60 uL a l i q u o t of t-BDMS reagent was added to an e x t r a c t e d u r i n e or plasma sample i n a 1 mL R e a c t i - v i a 1 R. The v i a l was t i g h t l y capped and heated f o r 4 hours at 60°C to ensure complete d e r i v a t i z a t i o n . A 1 uL a l i q u o t of d e r i v a t i z e d sample was i n j e c t e d i n t o the GCMS f o r a n a l y s i s . 4. Beckman C e n t r i f u g e Model J2-21 The p r o t e i n b i n d i n g study used the Amicon R m i c r o p a r t i o n system which was c e n t r i f u g e d f o r 20 minutes u s i n g a 45° r o t o r at 3500 rpm or I650g. The temperature of the c e n t r i f u g e was e q u i l i b r a t e d to 25°C before s p i n n i n g . C. Chemistry 1. S y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d 1.1 S y n t h e s i s v i a deh y d r a t i o n of a 0-hydroxyunsaturated e s t e r a. S y n t h e s i s of 1,3-dibromo-2-pentanone 27 A mixture of 48% HBr (100 mL/mole ketone) and 2-pentanone (86.1 g, 1 mole) i n a 1 l i t r e f l a s k equipped with a mechanical s t i r r e r , dropping f u n n e l , r e f l u x condenser, and HBr t r a p was c o o l e d i n an i c e bath to 0°C. Once c o o l , bromine (102.4 mL, 2 moles) was added dropwise over 4 t o 6 hours at a ra t e such that the temperature of the mixture never exceeded 10°C. The mixture was quenched with water (200 mL/mole B ^ ) , the heavier o r g a n i c l a y e r separated, and then f r a c t i o n a l l y d i s t i l l e d under reduced p r e s s u r e . The l a r g e r f r a c t i o n , bp 78°C/10 mm ( l i t . Rappe and Adestrom (1965) 84 - 89°C/10 mm), co n t a i n e d the dibromo compound as the major component (65% y i e l d ) . Mass spectrum (MW=244) m/z(%): 41(100), 121/123(50/50), 149/151(42/42), 70(23), 55(15), 93/95(12/12), 163/165 (10/10), 214/216/218(5/9/5). b. S y n t h e s i s of (Z)-2-pentenoic a c i d To a 1 l i t r e s o l u t i o n of 1 M potassium b i c a r b o n a t e i n a 2 l i t r e f l a s k equipped with a dropping funnel and mechanical s t i r r e r , 1,3-dibromo-2-pentanone (48.8 g, 0.2 moles) was added dropwise. The mixture was s t i r r e d f o r 48 hours, e x t r a c t e d with 2 x 100 mL of ether ( d i s c a r d ) , and ad j u s t e d to pH 1 - 2 with d i l u t e HC1. The aqueous mixture was e x t r a c t e d with 6 X 100 mL of ether and the e t h e r e a l l a y e r d r i e d over anhydrous MgSO^. Upon removal of the ether by f l a s h e v a p o r a t i o n , f r a c t i o n a l d i s t i l l a t i o n gave 17.6 g 28 (88% y i e l d ) of (Z)-2-pentenoic a c i d , bp 55 - 56°C/0.2 mm ( l i t . Rappe and Adestrom (1969) 39 - 41°C/0.4 mm). Mass spectrum m/z(%): 55(100), l00(M +,67), 20(40), 39(34), 45(27), 82(29), 73(10), 60(9). 80 MHz proton NMR ( C D C 1 3 ) : ppm 1.1(t,3H,CH 3), 2.7(q,2H,CH 2), 5.75(d,1H,CH-COOH,J=12Hz), 6.2-6.5(m,1H,CH2~ CH,J=12Hz). IR ( f i l m ) : 330 - 2500 cm - 1 (O-H), 1730 cm"1 (C=0), 740 cm - 1 (=CH,(Z)). c. S y n t h e s i s of e t h y l (Z)-2-pentenoate In a 300 mL f l a s k equipped with a dropping f u n n e l , d r y i n g tube, mechanical s t i r r e r , and r e f l u x condenser, c i s -2-pentenoic a c i d (23.3 g, 0.23 moles), e t h y l i o d i d e (7l.76g, 0.46 moles), l8-crown-6 (3 g, 0.05 M), and potassium carbonate (23.84 g, 0.17 moles) i n 230 mL of THF were r e f l u x e d f o r 6 hours. The mixture was f i l t e r e d by s u c t i o n and s e p a r a t i o n by f r a c t i o n a l d i s t i l l a t i o n a f f o r d e d 17.1 g (58% y i e l d ) of e t h y l (Z)-2-pentenoate, bp 43 -45°C/10 mm ( l i t . Acheampong (1985) 50 - 52°C/12 mm). Mass spectrum m/z(%): 100(100), 83(95), 55(77), 29(65), l28(M +,30). 80 MHz proton NMR ( C D C I 3 ) : ppm 1.1(t,3H,CH 3), 1.3(t,3H,CH 3), 2.65(q,2H,CH 2), 4.2(q,2H,OCH 2), 5.6 5.8(d,1H,=CH,J=10 Hz), 6.2 - 6.5(dt,1H,HC=,J=10 Hz). d. S y n t h e s i s of e t h y l 2-(1'-hydroxypropyl)-(E)-3-pentenoate 29 In a 500 mL f l a s k equipped with a dropping f u n n e l , mechanical s t i r r e r , and r e f l u x condenser, d i i s o p r o p y l a m i n e (11.13 g, 0.11 mole) i n anhydrous THF (90 mL) over a n i t r o g e n atmosphere was c o o l e d t o 0°C using an i c e bath, n-B u t y l l i t h i u m (69 mL of 1.6 M i n hexane, 0.11 mole) was added dropwise over 15 minutes and mixed f o r 20 minutes at 0°C. The mixture was c o o l e d to -78°C using a dry ice/ a c e t o n e bath and hexamethylphosphoramide (19.57 g, 0.11 mole) added dropwise i n t o the f l a s k with s t i r r i n g f o r 15 minutes. E t h y l (Z)-2-pentenoate (12.8 g. 0.1 mole) i n 10 mL of THF was added dropwise over 10 minutes. A f t e r 30 minutes propionaldehyde (5.8 g, 0.1 mole) i n 10 mL of THF was in t r o d u c e d i n t o the mixture dropwise over 15 minutes and s t i r r e d a f u r t h e r 30 minutes. The mixture was quenched with 15% HC1 to y i e l d a pH of 1 f o l l o w e d by the e x t r a c t i o n of the aqueous phase with 3 x 100 mL of et h e r . The e t h e r e a l l a y e r was c o n s e c u t i v e l y washed with a s a t u r a t e d s o l u t i o n of NaHCC>3 and water and f i n a l l y d r i e d over anhydrous Na 2S0 4. The 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 f r a c t i o n a l d i s t i l l a t i o n of the r e s i d u e gave 11.2 g (60.2% y i e l d ) of e t h y l 2-(1'-hydroxypropyl)-(E)-3-pentenoate, bp 78°C/0.22 mm ( l i t . Acheampong (1985) 95 - 100°C/1 mm). Mass spectrum (MW=186) m/z(%): 29(100), 100(50), 55(49), 82(44), 128(40), 113(5), 141(2), 157(2). 80 MHz proton NMR (CDCI3): ppm 0.9(t,3H,CH 3), 1.25(t,3H,OCH 2CH 3), 1.55(m,2H,CH 2), 1.7(d,3H,CH 3-CH=), 2.75 30 (broad s, 1H,0H), 3.4(m,1H,CH-C=0), 3.8(m,1H,CH-0), 4.2(q, 2H,0CH ?-CH ?), 5.3 - 5.9(m,2H,CH=CH). e. S y n t h e s i s of e t h y l 2-((E)-1'-propenyl)-(E)-2-pentenoate E t h y l 2-(1'-hydroxypropyl)-(E)-3-pentenoate (9.3 g, 0.05 mole), t r i e t h y l a m i n e (8.1 g, 0.08 mole), and dichloromethane (40 mL), i n a 250 mL f l a s k equipped with a dropping funnel and r e f l u x condenser were c o o l e d i n an i c e bath to 0°C. Methanesulfonyl c h l o r i d e (6.87 g, 0.06 moles), c o o l e d to 0°C, was slowly i n t r o d u c e d i n t o the mixture and s t i r r e d f o r 60 minutes at 25°C. The s o l u t i o n was f i l t e r e d and the s o l v e n t removed by f l a s h e v a p o r a t i o n . The r e s i d u e was r e c o n s t i t u t e d i n 150 mL of anhydrous THF ( d i s t i l l e d over l i t h i u m aluminum h y d r i d e ) , c o o l e d to 0°C with an i c e bath, and potassium h y d r i d e (4.01 g, 0.1 mole) c a u t i o u s l y added. Care should be taken to ensure that no moisture e n t e r s i n t o the r e a c t i o n v e s s e l . The mixture was s t i r r e d at 25°C f o r 12 hours and the excess potassium hydride was decomposed using t - b u t a n o l and water (1:2). The aqueous f r a c t i o n was e x t r a c t e d with 3 x 100 mL of ether and the combined organic f r a c t i o n d r i e d over anhydrous Na2SC>4 The 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 f r a c t i o n a l d i s t i l l a t i o n of the r e s i d u e a f f o r d e d 2 isomeric d i e n o a t e s , bp 70°C/0.7 mm ( l i t . Acheampong (1985) 65 - 70°C/0.1 mm), 3.9 g (46.3% y i e l d ) . GCMS a n a l y s i s r e v e a l e d that the major d e s i r e d isomer, e t h y l 2-((E)-1'-propenyl)-(E)-2-pentenoate, c o n t r i b u t e d t o 81.2% of the t o t a l mixture. 31 Mass spectrum (MW=168) m/z(%): 95(100), 168(M +,90), 79(64), 67(49), 123(46), 140(37), 111(30), 153(7). 400 MHz proton NMR (CDCI3): ppm 1.0 - 1.1(t,3H,CH 3-CH 2), 1.32(t,3H,OCH 2-CH 3, 1.84(dd,J=7Hz,3H,CH3~CH=), 2.3 (m,2H,CH2-CH=), 4.2(q,2H,OCH 2-CH 3), 5.75(m,1H,CH=CH,(Z)), 6.0 7(m,1H,CH=CH,(E)), 6.13(d,J=18Hz,1H,CH=CH,(E)), 6.55(t, J=7Hz,1H,CH 2-CH=,(E)). f. S y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d E t h y l 2-((E)-1'-propenyl)-(E)-2-pentenoate (2.7 g, 0.016 mole) was added t o 25 mL of 2.2 M NaOH and s t i r r e d f o r 48 hours at 60°C. The mixture was e x t r a c t e d with 25 mL hexane ( d i s c a r d ) and a d j u s t e d t o a pH of 1 with d i l u t e HC1. The mixture was e x t r a c t e d with 3 x 50 mL of ether and the combined e t h e r e a l p o r t i o n d r i e d over anhydrous Na2SC>4. T n e ether was removed by f l a s h e v a p o r a t i o n and f r a c t i o n a l d i s t i l l a t i o n a f f o r d e d 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d (1.2 g, 52.1% y i e l d , bp 84°C/0.05 mm). An a l i q u o t was r e c o n s t i t u t e d i n 2 mL of e t h y l a c e t a t e and 100 uL of the s o l u t i o n was d e r i v a t i z e d with 100 uL of t-BDMS reagent. The mixture was heated to 60°C f o r 4 hours and analyzed by GCMS. Mass spectrum (MW=282) m/z(%): 197(100), 74(40), 95(25), 67(12), 123(12), 254(3), 139(2), 153(2), 167(2). 1.2. S y n t h e s i s v i a the o x i d a t i o n of a t r i a l k y l s i l y l k e t e n e a c e t a l u s i n g a hydride a b s t r a c t o r a. S y n t h e s i s of e t h y l 2-n-propyl-(E)-3-pentenoate 32 In a dry 100 mL f l a s k equipped with a r e f l u x condenser, mechanical s t i r r e r , and a dropping f u n n e l , n - b u t y l l i t h i u m (26.88 mL of 1.6M i n hexane, 0.043 moles) was added dropwise t o a s o l u t i o n of d i i s o p r o p y l a m i n e (4.35g, 0.043 moles) i n 40 mL of THF at 0°C. A f t e r 15 minutes, the mixture was c o o l e d t o -78°C u s i n g a dry ice/acetone bath and hexamethylphosphoramide ( 7 . 7 l g , 0.043 moles) added dropwise i n t o the f l a s k and s t i r r e d f o r 15 minutes. E t h y l (Z)-2-pentenoate (5g, 0.04 moles) i n 10 mL of THF was added dropwise and a f t e r 20 minutes, p r o p y l bromide (7.38g, 0.06 moles) i n 10 mL of THF was added dropwise. The mixture was s t i r r e d f o r 30 minutes and quenched with 15% HC1 u n t i l a pH of 1 was a t t a i n e d . The s o l u t i o n was e x t r a c t e d with 3 X 100 mL of ether and the combined o r g a n i c f r a c t i o n was washed with a s a t u r a t e d NaHCC^ s o l u t i o n f o l l o w e d by water and then d r i e d over anhydrous Na 2S0 4. The 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 the re s i d u e f r a c t i o n a l l y d i s t i l l e d to a f f o r d 1.2 g (17.7% y i e l d ) of e t h y l 2 - n - p r o p y l - ( E ) - 3 -pentenoate, bp 42°C/0.5mm ( l i t . Acheampong (1982) 76-78°C/0.6mm). Mass spectrum m/z(%): 55(100), 97(29), 127(11), 141(5), 113(2), 170(M +,2). 300 MHz Proton NMR (CDCI3): 0.9 (t,3H,CH 3-CH 2), 1.3(t,3H,OCH 2-CH 3), 1.32(m,2H,CH 2-CH 2), 1.7(d,J=8Hz,5H,CH3-CH=,CH 2-CH 2), 2.95(dt,1H,CH,(E)), 3.3(dt,1H,CH,(Z)), 33 4.15(q,2H,OCH 2), 5.31-5.42(dd,J=8Hz,1H,=CH,(Z)), 5.38-5.47(dd,J=8Hz,1H, =CH,(E)), 5.48-5.62(m,J=8Hz,1H,CH=,(E)). b. S y n t h e s i s of e t h y l 2-((E)-1'-propenyl)-(E)-2-pentenoate In a dry 100 mL f l a s k equipped with a dropping f u n n e l , mechanical s t i r r e r , and r e f l u x condenser, n - b u t y l l i t h i u m (4.55 mL of 1.6M i n hexane, 7.06 mmole) was added dropwise to d i i s o p r o p y l a m i n e (0.7l4g, 7.06 mmole) in 7 mL of THF at 0°C. A f t e r 10 minutes the mixture was c o o l e d to -78°C, u s i n g a dry ice/acetone bath, f o l l o w e d by the dropwise a d d i t i o n of e t h y l 2-n-propyl-(E)-3-pentenoate (1g, 5.88 mmoles) i n 2 mL of THF. Once the mixture was s t i r r e d f o r 60 minutes, c h l o r o t r i m e t h y l s i l a n e ( l . 0 9 g , 10 mmoles) i n 2 mL of THF was added dropwise. The temperature of the r e a c t i o n was i n c r e a s e d to 25°C and the mixture s t i r r e d another 60 minutes. THF was removed by vacuum using an o i l pump equipped with a l i q u i d n i t r o g e n t r a p . The r e s i d u e was r e c o n s t i t u t e d i n 5 mL dichloromethane and analyzed by GCMS to c o n f i r m the presence of the s i l y l e s t e r . Mass spectrum m/z(%): 73(100), 95(80), 124(50), 169/170/171(11/7/2), 213/214/215(11/3/2), 242/243/244 (M +,11/3/2), 141(10). The s i l y l e s t e r was added dropwise to a s o l u t i o n of c o l l i d i n e (0.86g, 7.06 mmoles) and t r i p h e n y l c a r b e n i u m -t e t r a f l u o r o b o r a t e (2.9g, 8.82 mmoles) i n 20 mL of dichloromethane. A f t e r 30 minutes of s t i r r i n g the mixture was quenched with 50 mL of water and e x t r a c t e d with 3 X 100 34 mL of e t h e r . The combined o r g a n i c f r a c t i o n was washed with water and d r i e d over anhydrous Na2SC>4. GCMS a n a l y s i s r e v e a l e d two isomers of e t h y l 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 -pentenoate and the i n i t i a l compound, e t h y l 2 - n - p r o p y l - ( E ) -3-pentenoate. S e p a r a t i o n of the products by f r a c t i o n a l d i s t i l l a t i o n was i m p o s s i b l e . Other means of s e p a r a t i o n w i l l be i n v e s t i g a t e d i n the f u t u r e . Mass spectrum (apparent E,E-isomer) m/z(%): 29(100), 67(79), 95(70), 55(42), 111(40), 140(35), 79(30), 123(30), 168(M +,29). 2. S y n t h e s i s of 2-n-propyl-(E)-2-pentenoic a c i d 2.1. S y n t h e s i s by dehalogenation a. S y n t h e s i s of 2-bromo-2-n-propylpentanoic a c i d In a 250 mL f l a s k equipped with a r e f l u x condenser, gas t r a p , and s t i r bar, v a l p r o i c a c i d (21.5g, 0.15 moles), bromine (25.6g, 0.16 moles), and phosphorus t r i b r o m i d e (2g, 7.39 mmoles) were combined. Using an o i l bath, the mixture was heated to 70°C f o r 30 minutes and then t o 100°C f o r 4 to 6 hours u n t i l a l l the bromine had r e a c t e d . Excess HBr was removed using a water pump and the mixture was f r a c t i o n a l l y d i s t i l l e d t o a f f o r d 2-bromo-2-n-p r o p y l p e n t a n o i c a c i d 09.6g, 55% y i e l d , 80-90°C/0.03mm). Mass spectrum (MW=237) m/z(%): 55(100), 97(60), 41(42), 73(42), 157(40), 125(10), 165/167(10), 194/196(10). b. S y n t h e s i s of e t h y l 2-bromo-2-n-propylpentanoate 35 In a 250 mL f l a s k equipped with a Dean-Stark apparatus, 2-bromovalproic a c i d (I9.6g, 0.08 moles), benzene (100 mL), ethanol (11.04g, 0.24 moles), and 1 mL of c o n c e n t r a t e d s u l f u r i c a c i d were r e f l u x e d f o r 12 hours. The o r g a n i c s o l u t i o n was c o n s e c u t i v e l y washed with s a t u r a t e d NaHCC>3 and water u n t i l a pH of 6 - 7 was a t t a i n e d . The mixture was d r i e d over anhydrous MgS0 4 and the so l v e n t removed by f l a s h e v a p o r a t i o n . F r a c t i o n a l d i s t i l l a t i o n a f f o r d e d e t h y l 2-bromo-2-n-propylpentanoate (I0.8g, 54% y i e l d , 1lO°C/6mm). Mass spectrum (MW=251) m/z(%): 55(100), 97(41), 143(22), 171(19), 208/210(10), 99/101(7), 151/153(3). c. S y n t h e s i s of 2-n-propyl-(E)-2-pentenoic a c i d In a 25 mL f l a s k equipped with a d i s t i l l a t i o n column, e t h y l 2-bromovalproate (7.8g, 0.031 moles) and q u i n o l i n e (12.2g, 0.094 moles) were heated to 160°C with s t i r r i n g f o r 15 minutes. The temperature was then r a p i d l y i n c r e a s e d to f r a c t i o n a t e the mixture to y i e l d e t h y l 2 - n-propyl-(E)-2-pentenoate. The product was washed with d i l u t e HC1 and GCMS a n a l y s i s r e v e a l e d two isomers (E and Z) of e t h y l 2-n-propyl-2-pentenoate with E being the major isomer. Mass spectrum (E-isomer) m/z(%): 55(100), 113(80), 95(57), 125(30), 170(M +,29), 141(27). The e t h y l e s t e r was hy d r o l y z e d by s t i r r i n g i n 20 mL of 3N NaOH f o r 6 days f o l l o w e d by the a d d i t i o n of d i l u t e HCL u n t i l a pH of 1 - 2 was a t t a i n e d . The aqueous mixture was 36 e x t r a c t e d with 3 X 100 mL of ether and d r i e d over anhydrous N a 2 S 0 4 . The 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 the r e s i d u e r e c o n s t i t u e d i n 10 mL of c h l o r o f o r m and s t o r e d at -15°C f o r 3 days. C r y s t a l s of 2-n-propyl-(E)-2-pentenoic a c i d were removed and d i s s o l v e d i n 10 mL of f r e s h c h l o r o f o r m and s t o r e d at -15°C f o r a f u r t h e r 3 days. T h i s was repeated as o f t e n as necessary u n t i l pure 2-n-propyl-(E)-2-pentenoic a c i d (2g, 45.4% y i e l d ) was o b t a i n e d . 400MHz proton NMR ( C D C I 3 ) : 0.92(t,3H,CH 3-CH 2~CH 2), 1.06 (t,3H,CH 3-CH 2-CH=), 1.4-1.5(m,2H,CH 3-CH 2-CH 2), 2.2-2.3 (m,2H,CH2-C= andCH 2-CH=), 6.9(t,1H,J=7Hz,CH=). Mass spectrum m/z(%): 55(100), 95(55), 127(50), 67(35), 156(M +,25), 59(20), 81(10), 113(7), 141(3). 2.2. S y n t h e s i s by the dehydration of a /3-hydroxysaturated e s t e r In a 250 mL f l a s k equipped with a r e f l u x condenser, dropping f u n n e l , and mechanical s t i r r e r , e t h y l 3-hydroxy-2-n-propylpentanoate (5.6g, 0.03 moles), t r i e t h y l a m i n e (5.1g, 0.05 moles), and 75 mL of dichloromethane were combined and c o o l e d to 0°C. Methanesulfonyl c h l o r i d e (3.7g, 0.032 moles) at 0°C was added dropwise to the mixture and allowed to r e a c t f o r 30 minutes. Approximately 2 to 3 mL of ether was added to f a c i l i t a t e the p r e c i p i t a t i o n of the amine h y d r o c h l o r i d e s a l t . The suspension was f i l t e r e d by s u c t i o n , the s o l v e n t removed by f l a s h e v a p o r a t i o n , and the r e s i d u e r e c o n s t i t u t e d i n 100 mL of THF. 37 The s o l u t i o n was c o o l e d to 0°C, potassium hydride (2.4g, 0.06 moles) added, and the mixture allowed to react f o r 12 hours. The r e a c t i o n was quenched with water slowly at -78°C and the aqueous f r a c t i o n e x t r a c t e d with 3 X 100 mL of e t h e r . The combined o r g a n i c phase was d r i e d over anhydrous Na2S04 and the s o l v e n t removed by f l a s h e v a p o r a t i o n . The crude r e s i d u e was analyzed by GCMS r e v e a l i n g that e t h y l 2-n-propyl-(E)-2-pentenoate was absent. The r e a c t i o n was repeated s e v e r a l times with the same r e s u l t s . 2.3. S y n t h e s i s by the o x i d a t i o n of a t r i a l k y l s i l y l k e t e n e a c e t a l u s ing a hy d r i d e a b s t r a c t o r In a dry 100 mL f l a s k equipped with a dropping f u n n e l , condenser, and mechanical s t i r r e r , d i i s o p r o p y l a m i n e (2.12g, 0.021 moles) and n - b u t y l l i t h i u m (13.1 mL, 1.6 M i n hexane) were combined with 25 mL of THF at 0°C. A f t e r 10 minutes the mixture was c o o l e d to -78°C and e t h y l 2-n-propylpentanoate (3.44g, 0.02 moles) i n 5 mL of THF was added dropwise and allowed to r e a c t f o r 60 minutes. Then, c h l o r o t r i m e t h y l s i l a n e (3.69g, 0.034 moles) i n 5 mL of THF was added dropwise while the mixture was brought to 25°C and a l l o w e d to react f o r another 60 minutes. THF was removed by d i s t i l l a t i o n and the residue r e c o n s t i t u t e d i n 10 mL of benzene. In 30 mL of benzene, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2.08g, 9.18 mmoles) was d i s s o l v e d f o l l o w e d by c o l l i d i n e ( 1 . l 8 g , 9.76 mmoles) dropwise. A f t e r 10 minutes the s i l a n i z e d e s t e r i n 38 benzene was added dropwise to the mixture and s t i r r e d f o r 2 hours. The r e a c t i o n was quenched w i t h 25 mL of 1 N NaOH and the aqueous phase e x t r a c t e d with 3 X 50 mL of e t h e r . The combined o r g a n i c f r a c t i o n was washed c o n s e c u t i v e l y with d i l u t e HC1 and water and d r i e d over anhydrous Na2S0 4. The so 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 the r e s i d u e f r a c t i o n a t e d by d i s t i l l a t i o n to a f f o r d a product c o n t a i n i n g 3 compounds, e t h y l 2-n-propylpentanoate and (Z) and (E) e t h y l 2-n-propyl-2-pentenoate. Mass spectrum ( e t h y l 2-n-propylpentanoate, MW=172) m/z(%): 101(100), 57(85), 130(40), 73(35), 115(7), 143(4). Mass spectrum ( e t h y l 2-n-propyl-(E)-2-pentenoate) m/z(%): 55(100), 113(35), 67(25), 95(25), 125(20), 141(17), 170(M +,15). 3. S y n t h e s i s of e t h y l 2-n-propyl-(E)-2,4-pentadienoate a. S y n t h e s i s of e t h y l v a l e r a t e In a 1 l i t r e f l a s k equipped with a Dean-Stark apparatus, d r y i n g tube, and a r e f l u x condenser, v a l e r i c a c i d (163.6g, 1.6 moles), ethanol (220.8g, 4.8 moles), 2 mL of c o n c e n t r a t e d s u l f u r i c a c i d , and 500 mL of benzene were r e f l u x e d f o r 48 hours. The mixture was c o n s e c u t i v e l y washed with s a t u r a t e d NaHC0 3 and water u n t i l the wash was n e u t r a l . The e t h e r e a l s o l u t i o n was then d r i e d over anhydrous Na2S0 4 and f r a c t i o n a t i o n by d i s t i l l a t i o n gave 129.7g (62.3% y i e l d ) of e t h y l v a l e r a t e (bp 137°C/760mm). 39 Mass spectrum m/z(%): 88(100), 85(85), 29(85), 57(74), 60(55), 101(29), 115(5), 130(M +,2). b. S y n t h e s i s of e t h y l 2-n-propyl-3-hydroxy-4-pentenoate In a 500 mL f l a s k equipped with a dropping f u n n e l , r e f l u x condenser, d r y i n g tube, and mechanical s t i r r e r , d i i s o p r o p y l a m i n e (20.24g, 0.2 moles) i n 150 mL of THF was c o o l e d to 0°C with an i c e bath. n - B u t y l l i t h i u m i n hexane (130 mL of 1.6M, 0.2 moles) was added dropwise over 15 minutes and the mixture s t i r r e d f o r 15 minutes. The mixture was c o o l e d t o -7B°C and e t h y l v a l e r a t e (26g, 0.2 moles) i n 10 mL of THF added dropwise over 15 minutes and s t i r r e d f o r 60 minutes. A c r o l e i n (11.2g, 0.02 moles) i n 10 mL of THF was added dropwise over 10 minutes and the mixture s t i r r e d a f u r t h e r 60 minutes at -78°C. A f t e r quenching with 15% HC1 to a pH of 1, the aqueous phase was e x t r a c t e d with 3 X 100 mL of ether and the combined e t h e r e a l l a y e r c o n s e c u t i v e l y washed wi t h s a t u r a t e d NaHC0 3 and water. A f t e r d r y i n g the e t h e r e a l l a y e r over anhydrous MgSO^ and removing the s o l v e n t by f l a s h e v a p o r a t i o n , f r a c t i o n a l d i s t i l l a t i o n gave 23.6g (63.4% y i e l d ) of e t h y l 2-n-propyl-3-hydroxy-4-pentenoate (bp 74°C/0.1mm). Mass spectrum (MW = l86) m/z(%): 101(100), 73(80), 55(78), 130(32), 84(27), 141(7), 159(2), 169(2). 80MHz proton NMR (CDCI3): 0.9(t,3H,CH 3-), 1.2(t,3H, -CH 3), 1.3-1.7(m,4H,CH 2-CH 2), 2.3-2.7(m,1H,CH), 2.4(s, 40 broad,1H,OH), 4-4.4(q,2H,0-CH 2), 4.2-4.5(t,1H,CH-0), 5.1-5.4(m,2H,CH 2=), 5.65-6.1(m,1H,=CH). c. S y n t h e s i s of e t h y l 2-n-propyl-(E)-2,4-pentadienoate In a 500 mL f l a s k equipped with a dropping f u n n e l , mechanical s t i r r e r , d r y i n g tube, and condenser, e t h y l 2-n-propyl-3-hydroxy-4-pentenoate (7.4g, 0.04 moles) and t r i e t h y l a m i n e (4g, 0.04 moles) i n 40 mL of dichloromethane were c o o l e d to 0°C. Methanesulfonyl c h l o r i d e (4.89g, 0.04 moles) c o o l e d to 0°C was added dropwise to the mixture and allowed t o r e a c t f o r 60 minutes. The mixture was f i l t e r e d by s u c t i o n , the s o l v e n t removed by f l a s h e v a p o r a t i o n , and the r e s i d u e r e c o n s t i t u t e d i n 100 mL of THF. The mixture was c o o l e d to 0°C with an i c e bath and potassium h y d r i d e ( 4 . 8 l g , 0.12 moles) added and s t i r r e d f o r 12 hours. Excess potassium hydride was decomposed with t - b u t a n o l and water (1:2) and the aqueous f r a c t i o n e x t r a c t e d with 3 X 100 mL of e t h e r . The e t h e r e a l p o r t i o n was d r i e d over anhydrous Na 2S0 4, the ether removed by f l a s h e v a p o r a t i o n , and the r e s i d u e f r a c t i o n a t e d by d i s t i l l a t i o n . Upon GCMS a n a l y s i s , two isomers (bp 70°C/lmm) were present i n 22% and 78% (Z and E, r e s p e c t i v e l y ) . Mass spectrum (E-isomer) m/z(%): 9 5 ( 1 0 0 ) , 168(M +,97), 67(80), 111(74), 123(74), 140(65), 79(40), 153(5). 400 MHz proton NMR (CDCI3): 0.92(t,3H,CH3-), 1.31(t,3H,-CH 3), 1.4-1.52(m,2H,-CH 2-CH 2), 2.4(t,2H,J=7Hz, CH 2-CH 2), 4.22(q,2H,0-CH 2), 5.28-5.33(d,1H,J=10Hz,CH 2=, 41 ( Z ) ) f 5.34-5.41(d,1H,J=16Hz,CH 2=,(Z)), 5.44(d,1H,J=1OHz, CH 2=,(E)), 5.57(d,1H,J=17Hz,CH 2=,(E)), 6.62-6.73(m,1H,=CH), 7.17(d,1H, J=12Hz,CH=). 4. S y n t h e s i s of e t h y l 2-n-propyl-3-oxopentanoate In a 500 mL f l a s k equipped with a r e f l u x condenser, d r y i n g tube, and dropping f u n n e l , n - b u t y l l i t h i u m i n hexane (103 mL of 1.6 M, 0.16 moles) was added dropwise over 15 minutes to a s o l u t i o n of d i i s o p r o p y l a m i n e ( I 6 . l 9 g , 0.16 moles) i n 80 mL of THF at 0°C. The mixture was r e a c t e d f o r 20 minutes and was then c o o l e d to -78°C with a dry ice/ a c e t o n e bath. E t h y l v a l e r a t e (I0.4g, 0.08 moles) i n 10 mL of THF was added dropwise over 10 minutes and allowed to s t i r f o r 20 minutes. The dropwise a d d i t i o n of p r o p i o n y l c h l o r i d e (7.24g, 0.08 moles) over 10 minutes i n 10 mL of THF f o l l o w e d and the mixture was allowed to r e a c t a f u r t h e r 30 minutes. A f t e r quenching the mixture with 15% HC1 to a pH of 1, the aqueous p o r t i o n was e x t r a c t e d with 3 X 100 mL of ether and the e t h e r e a l p o r t i o n c o n s e c u t i v e l y washed with s a t u r a t e d NaHCC>3 and water. Upon d r y i n g over anhydrous Na2SC>4 and removing ether by f l a s h e v a p o r a t i o n , the residue was f r a c t i o n a t e d by d i s t i l l a t i o n to y i e l d e t h y l 2-n-propyl-3-oxopentanoate, bp 85 - 92°C/4mm ( l i t . Acheampong (1982) 70 - 73°C/0.3mm). Mass spectrum (MW = 188) m/z(%): 57(100), 101(50), 29(40), 73(20), 130(15), 144(12), 57(5). 5. S y n t h e s i s of e t h y l 2-n-propyl-3-hydroxypentandate 42 In a 500 mL f l a s k equipped with a dropping f u n n e l , r e f l u x condenser, d r y i n g tube, and mechanical s t i r r e r , n-b u t y l l i t h i u m i n hexane (156 mL of 1.55 M, 0.25 moles) was added to d i i s o p r o p y l a m i n e (25.3g, 0.25 moles) i n 200 mL of THF at 0°C. The mixture was s t i r r e d f o r 30 minutes and c o o l e d to -78°C u s i n g a dry ice/acetone bath. E t h y l v a l e r a t e (32.5g, 0.25 moles) i n 10 mL of THF was added dropwise over 10 minutes to the mixture and s t i r r e d a f u r t h e r 60 minutes. A f t e r quenching with 15% HC1 to a pH of 1, the aqueous l a y e r was e x t r a c t e d with 3 X 100 mL of ether and the e t h e r e a l p o r t i o n washed c o n s e c u t i v e l y with s a t u r a t e d NaHCC^ and water. The e t h e r e a l s o l u t i o n was d r i e d over anhydrous Na2SO^ and the ether removed by f l a s h e v a p o r a t i o n . F r a c t i o n a t i o n by d i s t i l l a t i o n of the r e s i d u e gave 22.8g (48% y i e l d ) of the d e s i r e d compound, bp 94 -96°C/4.5mm ( l i t . Acheampong (1982) 70 - 72°C/0.2mm). Mass spectrum (MW = 190) m/z(%): 101(100), 73(67), 55(50), 113(50), 130(27), 84(20), 159(20), 143(15). 6. S y n t h e s i s of 2-n-propylpentanoic a c i d In a 1 l i t r e f l a s k equipped with a d r y i n g tube, dropping f u n n e l , mechanical s t i r r e r , and r e f l u x condenser, n - b u t y l l i t h i u m i n hexane (231.3 mL of 1.6 M, 0.37 moles) was added to a s o l u t i o n of d i i s o p r o p y l a m i n e (37.44g, 0.37 moles) i n 250 mL of THF at 0°C and s t i r r e d f o r 30 minutes. V a l e r i c a c i d (0.17 moles, I7.36g) i n 10 mL of THF was added 43 dropwise so that the temperature of the r e a c t i o n never exceeded 5°C. Once s t i r r e d f o r 30 minutes, p r o p y l i o d i d e (32.3g, 0.19 moles) i n 10 mL of THF was added dropwise over 10 minutes at 0°C. A f t e r s t i r r i n g the r e a c t i o n f o r 2 hours, the mixture was quenched with 25% HC1 to a pH of 1. The aqueous p o r t i o n was e x t r a c t e d with 3 X 100 mL of ether and the e t h e r e a l mixture c o n s e c u t i v e l y washed with 10% HCl and water. The e t h e r e a l s o l u t i o n was d r i e d over anhydrous Na2S0 4, the ether removed by f l a s h e v a p o r a t i o n , and the re s i d u e f r a c t i o n a l l y d i s t i l l e d . The r e s u l t was 223g (91% y i e l d ) of 2-n-propylpentanoic a c i d , bp 84°C/0.2mm ( l i t . Acheampong (1982) 71 - 72°C/0.5mm). A small p o r t i o n (100 uL) of the sample was d e r i v a t i z e d with diazomethane ( L e v i t t , 1973) f o r GCMS a n a l y s i s . Mass spectrum (MW = 172) m/z(%): 87(100), 116(40), 141(20) , 99(5), 129(5). D. M e t a b o l i c and Pharmacokinetic Study 1. Animals A d u l t male Wistar r a t s weighing between 200 - 300g were used i n a l l the experiments. The animals were housed i n p l a s t i c cages with wood shavings f o r bedding and kept i n the animal q u a r t e r s i n the F a c u l t y of Pharmaceutical S c i e n c e s f o r 24 hours before use. The animals were allowed access to food (Purina Rat Chow) and water ad libitum except d u r i n g the experiments. The photo-period was c o n t r o l l e d to provide l i g h t between 6:00 am to 8:00 pm. 44 2. S u r g i c a l Procedures < a. B i l e Duct Cannulation The r a t was a n e s t h e t i z e d with ether and the h a i r removed from the abdominal area and at the back of the neck. The shaved area was c l e a n s e d with d i l u t e S a v l o n R h o s p i t a l c o n c e n t r a t e and a 2 cm t r a n s v e r s e i n c i s i o n was made 1 cm below the r i b cage on the animal's r i g h t s i d e . Another s i m i l a r i n c i s i o n was made through both muscle l a y e r s u n t i l the i n t e s t i n e s were exposed. Using two c o t t o n swabs soaked i n s a l i n e , the stomach was i s o l a t e d and kept moist using a gauze pad dipped i n s a l i n e . The common b i l e duct was i d e n t i f i e d and a g l a s s rod p l a c e d under the duct to i s o l a t e i t from the i n t e s t i n e s . The b i l e duct was l i g a t e d at the lower end near the duodenum using 4-0 s u r g i c a l s i l k causing the duct to s w e l l with b i l e . The duct was punctured with a 23 gauge needle and a 30 cm p i e c e of PE-10 t u b i n g , with a 45° b e v e l , i n s e r t e d i n t o the duct. The tube was advanced slowly i n t o the duct u n t i l i t stopped. The cannula was then withdrawn 0.5 cm and secured t o the b i l e duct with 4-0 s u r g i c a l s i l k . Two drops of 91 0 R adhesive were p l a c e d on the knots to anchor the cannula to the duct. The cannula was e x t e r n a l i z e d through the muscle w a l l near the base of the abdomen and e x i t e d through the s k i n v i a a 0.5 cm i n c i s i o n at the back of the neck. The muscle l a y e r s and s k i n were j o i n e d s e p a r a t e l y with 4-0 45 s u r g i c a l s i l k u s i n g d i s c o n t i n u o u s s u t u r e s . The p r o t r u d i n g cannula at the back of the neck was secured with 4-0 s u r g i c a l s i l k . b. J u g u l a r V e i n C a n n u l a t i o n Using the m o d i f i e d procedure of Venkataramanan (1978), the animal was a n e s t h e t i z e d with ether and the area under the jaw c l e a n s e d with d i l u t e S a v l o n R . A 1 cm t r a n s v e r s e i n c i s i o n was made j u s t above the shoulder t o expose the u n d e r l y i n g t i s s u e . A blunt d i s e c t i o n was performed to separate the t i s s u e l a y e r s and expose the j u g u l a r v e i n . The v e i n was i s o l a t e d and the upper s e c t i o n l i g a t e d with s i l k s u t u r e . Another p i e c e of s i l k suture was p l a c e d 1 cm below the f i r s t s uture but not t i e d . Using a p a i r of tenotomy s c i s s o r s , a small nick was made i n the v e i n . A cannula f i l l e d with h e p a r i n i z e d s a l i n e (20 U/mL) with a 45° bevel t i p was i n s e r t e d i n t o the v e i n . The tube was advanced i n t o the v e i n up to the s i l a s t i c - p o l y e t h y l e n e connection ( p r e p a r a t i o n of the P E - 5 0 / s i l a s t i c c o n n e c t i o n seen i n f i g u r e 2). The cannula was secured using the s i l k suture p r e v i o u s l y prepared. The area behind the neck near the shoulder blade was c l e a n s e d with S a v l o n R and a small i n c i s i o n (0.5 cm) was made to e x i t the cannula. At the p o i n t where the s i l a s t i c tube e x i t s the v e i n , two drops of 910 R adhesive were p l a c e d to secure the cannula. 46 F i g u r e 2: S i l a s t i c / P E - 5 0 cannula f o r j u g u l a r v e i n c a n n u l a t i o n of Wistar r a t s . 47 The i n c i s i o n s were c l o s e d u s i n g 4-0 s u r g i c a l s i l k . The cannula was shortened so that approximately 4 cm was exposed and a p i n was used to s e a l the cannula. To ensure that the cannula stayed patent, h e p a r i n i z e d s a l i n e (20 U/mL) was f l u s h e d through the cannula every other day. The animal was allowed to recover f o r two days before beginning the experiments. 3. M e t a b o l i c Study a. Study Design The metabolic study of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 3 -pentenoic a c i d ((E,E)-2,3'-diene VPA) was performed i n animals with t h e i r b i l e duct cannulated to allow f o r the c o l l e c t i o n of b i l e . The animals were kept i n a r e s t r a i n t cage equipped with a f e c a l c o l l e c t o r , f u n n e l , and a g l a s s c o n t a i n e r to c o l l e c t the u r i n e . b. Metabolism of (E,E)-2,3'-diene VPA An i . p . i n j e c t i o n of I00mg/kg of (E,E)-2,3'-diene VPA was a d m i n i s t e r e d once the animal had recovered from the a n e s t h e t i c . The m e t a b o l i t e s of (E,E)-2,3'-diene VPA were recovered i n pooled b i l e and u r i n e samples over 24 hours. The b i l e was c o l l e c t e d i n a l i g h t p r o t e c t e d c o n t a i n e r and the samples immediately f r o z e n . U r i n e samples were a l s o f r o z e n u n t i l a n a l y z e d . c. Assay 48 i . U r i n e Samples The assay was a m o d i f i e d procedure of Abbott et al. (1986). To a 1 mL u r i n e sample i n a 10 mL screw cap t e s t tube, 120 uL of 3N NaOH was added t o i n c r e a s e the pH to 12. The mixture was heated f o r 1 hour at 60°C to hy d r o l y z e any con j u g a t e s . Once.cool, 100 uL of 4N HC1 was added to reduce the pH t o 1. The sample was e x t r a c t e d with 3 mL of e t h y l a c e t a t e by r o t a t i o n f o r 20 minutes and the organic f r a c t i o n d r i e d over anhydrous Na 2S0 4. The volume of organic s o l v e n t was reduced to 200 uL under n i t r o g e n , the so l v e n t t r a n s f e r r e d i n t o a 1 mL R e a c t i - v i a l R , and 60 uL of t-BDMS reagent was added and heated f o r 4 hours at 60°C. A 1 - 2 uL a l i q u o t was i n j e c t e d i n t o the GCMS i n scan mode f o r a n a l y s i s . i i . B i l e Samples Two mL of b i l e i n a 10 mL screw capped tube were c e n t r i f u g e d and the supernatant e x t r a c t e d with 3 mL of e t h y l a c e t a t e and d i s c a r d e d . The aqueous p o r t i o n was made a c i d i c (pH 2) with d i l u t e HCl and e x t r a c t e d with 3 mL of e t h y l a c e t a t e . The o r g a n i c f r a c t i o n was d r i e d over anhydrous Na 2S04 and the volume of org a n i c s o l v e n t reduced t o 200 uL under n i t r o g e n . The sample was t r a n s f e r r e d to a 1 mL R e a c t i -v i a l R , d e r i v a t i z e d with 60 uL of t-BDMS reagent, and heated f o r 4 hours at 60°C. A 1 - 2 uL sample was analyzed by GCMS i n scan mode. 49 The aqueous p o r t i o n was a d j u s t e d to a pH of 4.5 - 5, l y o p h i l y z e d , and r e c o n s t i t u t e d with 2 mL of 0.1M NaAc b u f f e r at pH 5. 0-glucuronidase (200 uL) was added to the sample and allowed to incubate at 38°C f o r 12 hours. The samples were a c i d i f i e d with d i l u t e HC1 to a pH of 1, c e n t r i f u g e d , and the supernatant e x t r a c t e d with 3 mL of e t h y l a c e t a t e . The org a n i c f r a c t i o n was d r i e d over anhydrous N a 2 S 0 4 and the volume reduced to 200 uL under n i t r o g e n . To d e r i v a t i z e the e x t r a c t , 60 uL of t-BDMS reagent was added to the sample and heated at 60°C f o r 4 hours. An a l i q u o t of 1 - 2 uL was i n j e c t e d i n t o the GCMS us i n g the scan mode f o r a n a l y s i s . 4. Pharmacokinetic Study a. Sample P r e p a r a t i o n Two s o l u t i o n s of (E,E)-2,3'-diene VPA were prepared by the a d d i t i o n of an equimolar amount of NaOH and the pH ad j u s t e d to 7.4 with d i l u t e HC1. The c o n c e n t r a t i o n of each s o l u t i o n was 40 and 200 mg/mL and when approximately 0.1 mL was i n j e c t e d , doses of 20 and 100 mg/kg were a d m i n i s t e r e d , r e s p e c t i v e l y . b. Study Design Two groups of 5 r a t s were given 20 and 100 mg/kg of (E,E)-2,3'-diene VPA v i a the j u g u l a r v e i n cannula. Once the drug was admi n i s t e r e d the cannula was f l u s h e d with 1 mL of 50 s t e r i l e s a l i n e to ensure no drug remained i n the cannula. Blood samples of 0.1 mL were withdrawn at v a r i o u s i n t e r v a l s . For the 20 mg/kg dose, the i n t e r v a l s were -15, 1.5, 3, 6, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 minutes and f o r the 100 mg/kg dose -15, 1.5, 3, 6, 10, 15, 20, 40, 60, 90, 120, 180, 300, and 480 minutes. The animals were kept i n a r e s t r a i n t cage, equipped with a f e c a l c o l l e c t o r and a g l a s s f u n n e l , when the samples were c o l l e c t e d d u r i n g the experiment. The l o s s of blood due to sampling was r e p l a c e d immediately with s t e r i l e s a l i n e . The samples were withdrawn us i n g 1cc t u b e r c u l i n s y r i n g e s with 22 gauge needles and the samples t r a n s f e r r e d t o 370 uL heparin z e d Caraway R tubes. The tubes were c e n t r i f u g e d at 2000 rpm f o r 30 minutes and the plasma immediately separated and s t o r e d at -15°C u n t i l a n a l y z e d . Cummulative u r i n e samples were c o l l e c t e d at times 0 -12 and 1 2 - 2 4 hours. The r a t s were gi v e n a 1 week drug wash out p e r i o d before the procedure was repeated with the b i l e duct c a n n u l a t e d . Cummulative b i l e samples were c o l l e c t e d at times 0 - 12 and 1 2 - 2 4 hours v i a the b i l e duct cannula. Plasma and u r i n e samples were a l s o c o l l e c t e d u s i n g the i d e n t i c a l times i n the p r e v i o u s experiment. c. Pharmacokinetic Drug Assay i . C a l i b r a t i o n Curves of (E,E)-2,3'-diene VPA f o r plasma 51 Two c a l i b r a t i o n curves were r e q u i r e d due to v a r i a t i o n s i n plasma c o n c e n t r a t i o n observed f o r the two doses. Rats given 20 mg/kg i . v . of (E,E)-2,3'-diene VPA r e q u i r e d a standard curve with plasma c o n c e n t r a t i o n s ranging from 0 -240 ug/mL whereas r a t s given 100 mg/kg i . v . r e q u i r e d c o n c e n t r a t i o n s between 0 - 600 ug/mL. P r e p a r a t i o n of the c a l i b r a t i o n curves f o r plasma were from a stock s o l u t i o n of 3 mg/mL of (E,E)-2,3'-diene VPA i n water. To prepare a s o l u t i o n of 300 ug/mL, 100 uL of stock s o l u t i o n were d i l u t e d to 1 mL with c o n t r o l r a t plasma. To prepare a 12 ug/mL s o l u t i o n , 40 uL of the 300 ug/mL plasma s o l u t i o n were d i l u t e d to 1 mL with c o n t r o l r a t plasma. Using the f o l l o w i n g d i l u t i o n s , the c a l i b r a t i o n standards of 0 - 240 ug/mL were prepared: Amount of stock Amount of c o n t r o l F i n a l p l a s m a _ s o l u t i o n rat_plasma c o n c e n t r a t i o n 0 u l of l2ug/mL 50 uL 0 ug/nL 10 40 2.4 20 30 4.8 40 10 9.6 50 0 12 10 u l of 300ug/mL 40 60 20 30 120 40 10 240 To prepare a s o l u t i o n of 600 ug/mL, 200 uL of stock s o l u t i o n were d i l u t e d to 1 mL with c o n t r o l r a t plasma. Using the f o l l o w i n g d i l u t i o n s , c a l i b r a t i o n standards of 0 -600 ug/mL were prepared: 52 Amount of stock Amount of c o n t r o l F i n a l glasma_solut ion rat_rjlasrna c o n c e n t r a t i o n 0 uL of 12ug/mL 50 u l 0 ug/mL 10 40 2.4 20 30 4.8 40 10 9.6 50 0 12 10 u l of 600ug/mL 40 120 30 20 360 50 0 600 i i . Plasma Sample A n a l y s i s The procedures of Abbott et al . (1986) were m o d i f i e d to assay the s m a l l volumes of r a t plasma. To a 50 uL plasma sample i n a 3.5 mL screw cap septum v i a l , 100 uL of 2Hg-VPA (10 ug/mL i n water) were added as an i n t e r n a l standard. The sample was a d j u s t e d to a pH of 12 with 25 uL of 1N NaOH and heated at 60°C f o r 1 hour to h y d r o l y z e any conjugates. The sample was c o o l e d t o room temperature and 35 uL of 1N HC1 were added u n t i l a pH of 1 was a t t a i n e d . A f t e r 10 minutes, the sample was e x t r a c t e d twice f o r 20 minutes with 500 uL of e t h y l a c e t a t e . The combined s o l v e n t l a y e r s weres d r i e d over anhydrous Na 2S0 4, the s o l v e n t t r a n s f e r r e d i n t o a 1 mL R e a c t i - v i a l R , and the volume reduced to 200 uL under n i t r o g e n . To d e r i v a t i z e the samples, 60 uL of t-BDMS reagent were added and the mixture heated at 60°C f o r 4 hours. A 1 uL a l i q u o t was i n j e c t e d i n t o the GCMS f o r a n a l y s i s . 53 E. P r o t e i n B i n d i n g Study 1. In vitro p r o t e i n b i n d i n g Pooled Wistar r a t plasma was c o l l e c t e d and spiked with (E,E)-2,3'-diene VPA. The c o n c e n t r a t i o n s used were 30, 100, 300, and 600 ug/mL. To prepare the samples 30, 100, 300, and 600 uL of a 3 mg/mL ( i n water) stock s o l u t i o n of (E, E) -2,3'-diene VPA were made to 3000 uL with r a t plasma to obt a i n the r e q u i r e d c o n c e n t r a t i o n s . The four s o l u t i o n s were incubated i n a 37°C water bath f o r 2 hours. To determine the amount of f r e e drug present i n each s o l u t i o n , 4 a l i q u o t s of 500 uL f o r each plasma sample were p l a c e d i n a separate Amicon R m i c r o p a r t i t i o n apparatus. The samples were c e n t r i f u g e d at 25°C f o r 20 minutes at 1650 g to separate the f r e e drug from the p r o t e i n . A 50 uL a l i q u o t of the u l t r a f i l t r a t e s and a 50 uL a l i q u o t of the o r i g i n a l s piked plasma samples were e x t r a c t e d u s i n g the procedures o u t l i n e d i n the pharmacokinetic study. The e x t r a c t s were d e r i v a t i z e d to y i e l d t-BDMS e s t e r s and 1 uL i n j e c t e d i n t o the GCMS f o r a n a l y s i s . The r e s u l t s were e x t r a p o l a t e d from two standard curves. T o t a l drug i n plasma was determined u s i n g the c a l i b r a t i o n curve i n the k i n e t i c study (0 - 600 ug/mL) and the f r e e drug r e s u l t s were c a l c u l a t e d u s i n g the same c a l i b r a t i o n curve except that the drug was spi k e d i n water. 2. B i n d i n g Of The Drug To The YMT Membrane 54 To determine the amount of (E,E)-2 , 3'-diene VPA th a t binds to the u l t r a f i l t r a t i o n membrane, d u p l i c a t e samples (500 uL a l i q u o t s ) of drug i n water a t c o n c e n t r a t i o n s of 30, 100, and 600 ug/mL were c e n t r i f u g e d u s i n g the Amicon R m i c r o p a r t i t i o n apparatus. The procedures used f o r f i l t e r i n g were i d e n t i c a l to that used f o r the plasma samples. A f i f t y uL a l i q u o t from the u l t r a f i l t r a t e s was e x t r a c t e d and d e r i v a t i z e d u s i n g t-BDMS reagent. The t o t a l drug f o r each s o l u t i o n was determined by a s s a y i n g 50 uL a l i q u o t s of (E,E)-2 , 3'-diene VPA i n water f o r each c o n c e n t r a t i o n used. The standard curve was i d e n t i c a l to t h a t used f o r determining the f r e e drug i n plasma. 55 RESULTS AND DISCUSSION A. Chemistry 1. S y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d ((E,E)-2,3'-diene VPA) a. S y n t h e s i s v i a the dehydration of a /3-hydroxyalkyl e s t e r The s y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) ~ 2 - p e n t e n o i c a c i d was f i r s t performed by Acheampong and Abbott (1985) but the y i e l d was low and the presence of other unsaturated isomers was h i g h . M o d i f i c a t i o n s t o these procedures to i n c r e a s e the y i e l d and isomeric p u r i t y were attempted. Scheme 1 o u t l i n e s the method which e v e n t u a l l y produced s u f f i c i e n t q u a n t i t i e s of 2 - ( ( E ) - p r o p e n y l ) - 1 ' - ( E ) - 2 -pentenoic a c i d to perform the metabolic and pharmacokinetic s t u d i e s i n animals. The s y n t h e s i s of (Z)-a,/3-unsaturated a c i d s has been rep o r t e d i n the l i t e r a t u r e by Rappe and Adestrom (1965) and Rappe (1979) and these methods were a p p l i e d to the s y n t h e s i s of (Z )-2-pentenoic a c i d . The y i e l d obtained was s l i g h t l y g r e a t e r than the r e p o r t e d y i e l d of Rappe and Adestrom (1965). E s t e r i f i c a t i o n of the (Z )-2-pentenoic a c i d by the a c i d c a t a l y z e d method was too vig o r o u s f o r t h i s compound due to the p o s s i b i l i t y of i s o m e r i z a t i o n . However the method of Acheampong and Abbott (1985) whereby potassium carbonate, e t h y l i o d i d e , and (Z )-2-pentenoic a c i d are r e f l u x e d i n anhydrous THF i s a m i l d e r method of e s t e r i f i c a t i o n . Although the r e a c t i o n occurs i n a b a s i c 56 0 II C H ^ - C H p C H p C — C H 3 2 B r ? I? C H j - C H p C H B r — C — C H ^ B r KHCOj HC1 CH 5 - C H j - C H = C H - C -E t l K 2 C 0 3 OH 1 8 - c r o w n - 6 THF C H 3 - C H p C H = C H — [ ' - O C H ^ - C H 3 p r o p i o n a l d e h y d e LDA HMPA THF C H T - C H = C H 0 3 \ C H J - C H ^ - C H 2 - p e n t a n o n e 1 , 3 - d 1 b r o m o - ? - p e n t e n o n e ( Z ) - 2 - p e n t e n o i c a c i d e t h y l ( 7 ) - ? - p e n t * n o a t e — { ! — O C H ^ - C H j e t h y l 2 - ( 1 ' - h y d r o x y p r o p y l ) - ( E ) - 3 - p e n t e n o a t e \>H M s C l E t 3 N . C H ? C 1 2 C H F C H = C H ^ 0 m e s y , e s t e r o f C H — t — O C H = - C H / e t h y l 2 - ( r - h y d r o x y p r o p y l ) - ( E ) - 3 - p e n t e n o a t r C H ^ C H ^ - C H V O S O ? C H 3 KH THF C H T - C H = C H 0 J v II CHJ-CHJ-CH \ I! - C — C — O C H ^ - C H 2 3 e t h y l 2 - ( ( E ) - r - p r o p e n y l ) - ( E ) . ? . p e n t e n o a t e NaOH HC1 C H 3 - C H = C H Q \ — \ — OH CHJ-CHJ-CH 2 - ( ( C ) - l ' - p r o p e n y l ) . ( F ) . ? . p e n t e n o 1 c a c i d Scheme 1: S y n t h e s i s of 2-((E)-1'-propenyl)-(E)-2-pentenoic a c i d by de h y d r a t i n g a j 3 - h y d r o x y a l k e n y l e s t e r . medium which i s l e s s l i k e l y to isomerize the a c i d , the r e a c t i o n f a i l e d to produce the e s t e r i n a p p r e c i a b l e amounts. The e s t e r i f i c a t i o n of (Z)-2-pentenoic a c i d appeared to be the most i n e f f i c i e n t step i n the s y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . The use of a c a t a l y s t , a crown e t h e r , made a s i g n i f i c a n t i n c r e a s e i n y i e l d to a f f o r d e t h y l (Z)-2-pentenoate. The y i e l d was 58% using l8-crown-6 as a c a t a l y s t (Cook et al., 1974; Durst, 1974; Grushka et al., 1975). The crown ether s o l v a t e d the c a t i o n (K +) i n a non-polar environment by complexing with the a l k a l i metal and forming an ion p a i r with the anion (pentenoic a c i d ) at the l i q u i d - s o l i d i n t e r f a c e (Gokel and Durst, 1976). The ion p a i r then complexes with e t h y l i o d i d e to y i e l d the e t h y l e s t e r (Fedorynski et al., 1978). The formation of e t h y l 2 - ( 1 ' - h y d r o x y p r o p y l ) - ( E ) - 3 -pentenoate was performed by a l k y l a t i n g the (Z)-pentenoate e s t e r e n o l a t e with propionaldehyde. The a d d i t i o n of aldehydes to a,^-unsaturated e n o l a t e s has been r e p o r t e d by s e v e r a l i n v e s t i g a t o r s ( P f e f f e r and S i l b e r t , 1971; Rathke and L i n d e r t , 1971; Rathke and S u l l i v a n , 1972; P f e f f e r et al., 1973; Kende and Toder, 1982) but was r e c e n t l y a p p l i e d by Acheampong and Abbott (1985) f o r the s y n t h e s i s of e t h y l 2-(1'-hydroxy)-(E)-2-pentenoate. The method used to a l k y l a t e the enolate was i d e n t i c a l t o the method of Acheampong and Abbott (1985) except the s t a r t i n g m a t e r i a l s were d r i e d and/or p u r i f i e d by d i s t i l l a t i o n . M o i s t u r e can g r e a t l y reduce the y i e l d . The e q u i l i b r i u m between the e n o l 58 and e n o l a t e can be a f f e c t e d i n the presence of water to favor the e n o l . A l k y l a t i o n of e t h y l (Z)-2-pentenoate r e q u i r e s the formation of the en o l a t e t h e r e f o r e , c o n d i t i o n s that d i s f a v o r the presence of the enolate w i l l reduce the y i e l d of e t h y l 2-(1'-hydroxypropyl)-(E)-2-pentenoate. A y i e l d of 60.2% was obt a i n e d u s i n g dry p u r i f i e d r e a c t a n t s compared to a 50% y i e l d o b t ained by Acheampong and Abbott (1985). Acheampong and Abbott (1985) i n v e s t i g a t e d the use of s e v e r a l d e hydrating agents f o r the con v e r s i o n of a /3-hydroxy unsaturated e s t e r to a d i u n s a t u r a t e d e s t e r . The use of methanesulfonyl c h l o r i d e and potassium hydride was found to produce the l e a s t number of isomeric d i e n o l a t e s . A p p l y i n g t h i s method on l y two isomers of e t h y l 2-(1'-propenyl)-2-pentenoate were obtained from the potassium hydride c a t a l y z e d mesyl e s t e r e l i m i n a t i o n . The r e s u l t s shown by GCMS a n a l y s i s were c o n s i s t e n t with t h a t of.Kende and Toder (1982) who found mesyl e s t e r e l i m i n a t i o n by treatment w i t h potassium h y d r i d e t o be a s t e r e o s p e c i f i c process f o r producing diene e s t e r s . H y d r o l y s i s of e t h y l 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 -pentenoate to y i e l d the f r e e a c i d was obtained by r e f l u x i n g the e s t e r i n 1 N NaOH f o r 24 hours., reducing the pH of the mixture to 1 - 2, and e x t r a c t i n g the product with e t h e r . Upon i s o l a t i n g and d e r i v a t i z i n g the f r e e a c i d with t-BDMS reagent, the i d e n t i f i c a t i o n of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d was determined by GCMS with the TIC ( f i g u r e 59 3) r e v e a l i n g two peaks. The mass spectrum ( f i g u r e 4) of the major peak at 9.5 minutes c o n t a i n s ions at m/z 197, 167, 139, 123, 95, and 67 cor r e s p o n d i n g to the t-BDMS e s t e r of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . S t r u c t u r e s f o r the fragment ions correspond to M-57 +, CH 3CH 2CHC(CH 3CHCH)COOSi +, CH 3CH 2CHC(CH 3CHCH)COO +, CH 3CH 2CH-C(CH 3CHCH)CO +, CH 3CH 2CHC(CH 3CHCH) +, and CH 3CH 2CHC(CH) +, r e s p e c t i v e l y . The major peak r e p r e s e n t s 81.2% of the t o t a l p roduct. NMR data of the e t h y l e s t e r i n f i g u r e 5 agrees with the GCMS data r e g a r d i n g the i d e n t i t y and p u r i t y of the product. Peaks with an a s t e r i s k are those of the (E,Z)-isomer (16% based on i n t e g r a t i o n ) with the peaks from the major (E,E)-isomer r e p r e s e n t i n g 84% of the mixture. The s i g n a l c o r r e s p o n d i n g to CH2~CH= i s s p l i t by two methylene protons producing a t r i p l e t at 6.55 ppm with a c o u p l i n g constant of 7 Hz. The ( E ) - v i n y l i c proton CH=CH i s s p l i t by one adjacent proton to y i e l d a doublet at 6.13 ppm with a c o u p l i n g constant of 18 Hz. The other v i n y l i c proton, CH=CH, i s s p l i t by the adjacent methyl protons and by a non-equivalent v i n y l i c proton t o g i v e a m u l t i p l e t at 6.07 ppm. The corres p o n d i n g proton, CH=CH, for the (Z)-isomer occurs s l i g h t l y u p f i e l d at 5.75 ppm due to the g r e a t e r s h i e l d i n g e f f e c t from the c a r b o n y l group. The methylene protons OCH 2 and CH2-CH= at 4.2 and 2.3 ppm are r e p o r t e d as a q u a r t e t and a m u l t i p l e t , r e s p e c t i v e l y . The methyl adjacent t o the v i n y l i c proton at 1.84 ppm i s s l i g h t l y u p f i e l d compared to the other two methyl protons at 1 - 1.1 60 (E .E) (E.Z) i —« 12 —r -10 i — 11 TIME (MINUTES) F i g u r e 3: T o t a l ion chromatogram of the two s y n t h e s i z e d isomers of 2,3'-diene VPA t-BDMS e s t e r . 61 Z9 c - 2 P] Q) - 01 PJ 01 I Ul ro Xi - (Tj 00 o - rr 1 -1 a c ro 3 O <T> i-n < r i -T J rr > ro rr 3 1 0) a o 2 n 01 ro •< oi 3 rr ri-a> rr N re a a o -f=-a o ^ u i M O o o 2 RELATIVE INTENSITY cn CO ui ro U) a) CD a t CO cn o o 3: 3: W| co| i o o 3i 3: || 1 o M o 3: / | 1 OO o o a: oo oo o 3: C O l \3 CO 1 0) O 3 ro cn o F i g u r e 5: 400 MHz p r o t o n NMR of e t h y l ( E , E ) - 2 , 3 ' - d i i n CDC1 3 w i t h TMS as an i n t e r n a l s t a n d a r d (*= E,Z - i s o m e r ) . and 1.32 ppm due to g r e a t e r s h i e l d i n g of the f i r s t methyl protons from the v i n y l i c and c a r b o n y l group. The y i e l d and p u r i t y of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d obtained u s i n g the above s y n t h e s i s surpassed that of Acheampong and Abbott (1985). b. S y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d by the o x i d a t i o n of an 0 - t r i a l k y l s i l y l k e t e n e a c e t a l using a hydride a b s t r a c t o r The s y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d from a ^-hydroxy unsaturated e s t e r gave a s a t i s f a c t o r y y i e l d but the s y n t h e s i s c o u l d be more s t e r e o s p e c i f i c . The goal was to o b t a i n pure 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d f o r metabolic and pharmacokinetic s t u d i e s i n animals without the i n t e r f e r e n c e of the other isomer. Using a hydride a b s t r a c t o r to o x i d i z e an 0 - t r i a l k y l s i l y l k e t e n e a c e t a l to produce 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d was • an attempt to i n c r e a s e the isomeric p u r i t y . The s y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d as o u t l i n e d i n scheme 2 r e q u i r e d an unsaturated e s t e r as the s t a r t i n g product. The e s t e r was s y n t h e s i z e d by the a l k y l a t i o n of an a,^-unsaturated e s t e r enolate with p r o p y l bromide to a f f o r d e t h y l 2-n-propyl-(E)-3-pentenoate. T h i s s y n t h e s i s was s i m i l a r to the p r e v i o u s method (scheme 1) where an aldehyde was used to a l k y l a t e an enolate e s t e r . As p r e v i o u s l y mentioned, a c c o r d i n g t o Kende and Toder (1982), a l k y l a t i o n of e t h y l (Z)-2-alkenoate y i e l d s s t e r e o s p e c i f i c a l l y ( E)-3-alkenoate. The presence of e t h y l 64 CHj-CHpCH=sCH—8—0—CH^-CH3 CH3CH2CH2Br LDA HMPA THF 2 3 CHr-CH=CH 0 3 \ II ^ C H — C — 0—CH-s-CH, C H 2~ C H C H 2 LDA THF TMSC1 e t h y l ( Z)-2 - p e n t e n o a t e e t h y l 2 - n - p r o p y l - ( E ) -3-pentenoate CH^-CH=CH 0—TMS c = c C H — C H ^ C H ^ 0—CH^-CHg d i a l k y l 0 - e t h y l - O - T M S k e t e n e a c e t a l T r i t y l t e t r a f l u o r o b o r a t e D i c h l o r o m e t h a n e C H ^ - C H = C H 0 3 \ it X — C — 0 — C H T T - C H , # 2 3 C H ^ - C H ^ C H ' e t h y l 2 - ( ( E ) - l ' - p r o p e n y l ) • ( E)-2 - p e n t e n o a t e Scheme 2: S y n t h e s i s of e t h y l 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 -pentenoate u s i n g a h y d r i d e a b s t r a c t o r . 65 2-n-propyl-(E)-3-pentenoate was i d e n t i f i e d by GCMS with ions a t m/z 170, 141, 97, 55, and 41 that correspond t o M +, M-CH 2CH 3 +, CH 3CH=CHCH(C 3H 7) +, CH 3CH 2CH=CH +, and CH 3CH=CH +, r e s p e c t i v e l y . The 300 MHz proton NMR (appendix) confirms the presence of e t h y l - 2 - n - p r o p y l - ( E ) - 3 - p e n t e n o a t e . The ( E ) - v i n y l i c proton adjacent to a methyl and another v i n y l i c proton at 5.48 - 5.62 ppm should be a doublet of a q u a r t e t however, i t a c t u a l l y occurs as a m u l t i p l e t with a c o u p l i n g constant of 8 Hz. The other v i n y l i c proton at 5.38 - 5.47 ppm i s s p l i t by two adjacent non-equivalent protons with a c o u p l i n g constant of 8 Hz. The methyl group adjacent to the v i n y l i c proton i s represented as a doublet at 1.7 ppm with a c o u p l i n g constant of 8 Hz. The e s t e r methylene and methyl protons are at 4.15 and 1.3 ppm, r e s p e c t i v e l y where as the methyl protons adjacent to the methylene group occur at 0.9 ppm. The methylene proton adjacent to the methyl group and the remaining methylene proton are at 1.32 and 1.7 ppm, r e s p e c t i v e l y . The methinyl proton at 2.95 ppm i s r e p o r t e d as a doublet of a t r i p l e t . The unsaturated e s t e r was converted to a d i a l k y l 0-e t h y l - O - t r i m e t h y l s i l y l k e t e n e a c e t a l v i a an eno l e s t e r i n t e r m e d i a t e using t r i m e t h y l s i l y l c h l o r i d e (Chan et al., 1979; Rathke and S u l l i v a n , 1973). The d i a l k y l O-ethyl-O-t r i m e t h y l s i l y l k e t e n e a c e t a l was re a c t e d with t r i t y l t e t r a f l u o r o b o r a t e which removes hydride to y i e l d a d i u n s a t u r a t e d e s t e r (Jung and Pan, 1977; Fleming and 66 P a t t e r s o n , 1979; Ryu et al ., 1978). The sequence of events, shown i n scheme 2a, i n v o l v e s an a l l y l i c h ydride a b s t r a c t i o n to an oxygenated a l l y l i c c a t i o n and upon workup y i e l d s a o,^-unsaturated e s t e r with the ( E ) - c o n f i g u r a t i o n . Scheme 2a: The a l l y l i c h y d ride a b s t r a c t i o n of a d i a l k y l 0-e t h y l - O - t r i m e t h y l s i l y l k e t e n e a c e t a l by t r i t y l t e t r a f l u o r o b o r a t e (TTFB). R 1-CH 2^ ^OTMS -CrU + ^OTMS R,~CH ,0 C=C — C~C — V - c ' ' Rj ^0R 3 T T F B R 2 XOR 3 H 2 0 R 2 V 0 R 3 GCMS a n a l y s i s of the product r e v e a l e d the presence of s e v e r a l compounds as shown i n the TIC i n f i g u r e 6. The mass spectrum of the major peak ( f i g u r e 7) corresponds to the s t a r t i n g m a t e r i a l 2-n-propyl-(E)-3-pentenoate. Ions m/z 170, 141, 97, 55, and 41 are c h a r a c t e r i s t i c of fragments M +, M-CH 2CH 3 +, CH 3CH=CHCH(C 3H 7) +, CH 3CH 2CHCH +, and CH 3CHCH +. The remaining two peaks are isomers of e t h y l 2-(1'-propenyl)-2-pentenoate. The mass s p e c t r a of the two isomers ( f i g u r e s 8 and 9) c o n t a i n ions m/z 168, 139, 123, 95, and 55 which correspond to fragments M +, M-CH 2CH 3 +, M-OCH 2CH 3 +, M-COOCH 2CH 3 +, and CH 3CH 2CHCH +. Attempts to separate the products by f r a c t i o n a l d i s t i l l a t i o n were u n s u c c e s s f u l due to the presence of i n s o l u b l e t r i t y l s a l t s ; any f u t u r e attempts t o p u r i f y the e s t e r s w i l l r e q u i r e the i n i t i a l removal of the s a l t s before f r a c t i o n a l d i s t i l l a t i o n . Column chromatography techniques should be 67 4 . 6 8 TIME (Minutes) 10 12 14 F i g u r e 6: T o t a l ion c u r r e n t chromatogram of e t h y l 2 - ( ( E ) -1'-propenyl)-(E)-2-pentenoate s y n t h e s i z e d using a hy d r i d e a b s t r a c t o r (Peaks 1 = e t h y l 2-n-propyl-(E)-3-pentenoate; Peaks 2 and 3 = Z and E isomers of e t h y l 2-(1'-propenyl)-2-pentenoate, r e s p e c t i v e l y ) . 68 5 5 CHy-CH^- C H = C H [ + 2 9 4 1 97" C H T - C H ^ - C H , '3 w , ,2 2 N CH — CH = CH \ J 41—A 0 CH-f-C — 0 + ' C H p C H 3 141" 9 7 6 9 J lilili I Hill iHl> Ii 1 2 7 1 1 3 JiU L 1 f 1 4 1 1 7 0 (M +) 5 0 1 0 0 M/Z 1 5 0 2 0 0 Figure 7: Mass spectrum of e t h y l 2 - n - p r o p y l - ( E ) - 3 -pentenoate corresponding to peak 1 i n f i g u r e 6. OL % RELATIVE INTENSITY C 1 n> oo ">TJ 2 i-.. i-i ro vo o n C TJ CO >-t fD (D 3 Ul •< TJ CTi t-1 0) . — O I rr ro n I C TJ 3 0) 3 O rr i-ti fD 3 rr O =r Q) fD rr fD 3 3 o o o •-i i-t >-t fD cn cn TJ o 3 Cu »-i >-•• 3 O iQ rn rT fD O rr 3* TJ •< fD H-1 ?r ro i cn o r v j o C/1 o CO ro CD cn cn — o 3: col CO CD o 3: col CO cn o 3: ro O ool o 3: I II O 3: ro CO r->=: o ro CO CO CO cn CO o — h o 3: ro, o 3: CO ro o o 100 _ 29 >-i— •—i c n LU CE _ l LU CH 67 95 C H y - C H - p C H = C ^ + 41 55 lil illlllll J_±J 79 JliliLl 123—, C H ~ - C H = C H 0 J \ I' C H F C H 2 ~ C H 111 95-C-r-0 + C H ^ - C H 3 139—' 50 100 M/Z 123 140 168 (M ) 150 200 Figure 9: Mass spectrum of the major isomer of e t h y l 2-(1'-propenyl)-2-pentenoate c o r r e s p o n d i n g to peak 3 i n f i g u r e 6. i n v e s t i g a t e d . A l a r g e q u a n t i t y of product was comprised of s t a r t i n g m a t e r i a l . In n e a r l y a l l cases o x i d a t i o n was incomplete with some s t a r t i n g m a t e r i a l present upon aqueous workup (Jung and Pan, 1977). Since s e p a r a t i o n of the two isomers was not p o s s i b l e , NMR data c o u l d not be o b t a i n e d . The c e r t a i n t y of the products being the (E,Z) and (E,E)-isomers c o u l d not be confirmed however, the peak with the longer r e t e n t i o n time i s u s u a l l y the (E)-isomer. Although the s y n t h e s i s f o r 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 -pentenoic a c i d by t h i s method d i d not y i e l d product i n any a p p r e c i a b l e amount, the s y n t h e s i s does appear to be e a s i e r and a p p a r e n t l y more s t e r e o s p e c i f i c than the potassium hydride c a t a l y z e d mesyl e s t e r e l i m i n a t i o n r o u t e . T h e r e f o r e , methods to improve the y i e l d of the hydride e x t r a c t i o n process, and to separate the products and reduce the amount of unreacted s t a r t i n g m a t e r i a l i s warranted. 2. S y n t h e s i s of 2-n-propyl-(E)-2-pentenoic a c i d a. S y n t h e s i s by dehalogenation The VPA m e t a b o l i t e 2-n-propyl-(E)-2-pentenoic a c i d was r e q u i r e d as a r e f e r e n c e compound to compare with 2 - ( ( E ) - 1 ' -p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d with respect to mass s p e c t r a , NMR s p e c t r a , t o t a l ion chromatogram, and pharmacokinetics. The method of Acheampong (1985) was used to s y n t h e s i z e 2-n-propyl-(E)-2-pentenoic a c i d as o u t l i n e d i n scheme 3. The 2-n-propylpentanoic a c i d was f i r s t brominated to y i e l d 2-bromo-2-n-propylpentanoic a c i d . The 72 CHr-CHr-CH-CHj-CHpCH^ CH—C—OH Br 2 PBr, 2-n-propylpentanoic acid CHr-CHr-CH, C—OH 3 2 2 / A C H J - C H J - C H ^ Br H 2S0 4 EtOH Benzene 0 I C H - C H r C H , C - O — C H r - C H . 3 2 2 ^ / 2 3 CHj -CH^-CH^ \ r 2 -bromo-2 -n-propylpentanoic acid ethyl 2-bromo-2-n-propylpentanoate Qui no! i ne Heat, 160°C CHr-CHr-CH 0 3 2 <^  II C—C—0-CHr-CH, / 2 3 CH — CH^CH,, ethyl 2 -n -propy l - (E)-2-pentenoate NaOH HC1 CHr-CHr-CH 0 3 2 ^, ,| C—C—OH CHj-CH—CH 2 -n -propy l - (E ) -penteno ic ac id Scheme 3: S y n t h e s i s of 2 - n - p r o p y l - ( E ) - 2 - p e n t e n o i c a c i d by d e h a l o g e n a t i o n of 2-bromo -2-n-p r o p y l p e n t a n o i c a c i d 73 a c i d was then e s t e r i f i e d , u s ing the a c i d c a t a l y z e d method and Dean-Stark apparatus. Debromination of the e t h y l e s t e r with q u i n o l i n e a f f o r d e d the (Z) and (E) isomer of 2-n-prop y l - 2 - p e n t e n o i c a c i d . S e p a r a t i o n of the isomers was achieved by d i s s o l v i n g the isomeric mixture i n c h l o r o f o r m and s t o r i n g at -15°C f o r 3 days. Pure c r y s t a l s of 2-n-pr o p y l - ( E ) - 2 - p e n t e n o i c a c i d were removed by decanting and r e c r y s t a l l i z e d from f r e s h c h l o r o f o r m to f u r t h e r p u r i f y the (E)-isomer. The p u r i t y of 2-n-propyl-(E)-2-pentenoic a c i d obtained was 94% based on NMR a n a l y s i s ( f i g u r e 10). The a s t e r i s k on peaks i n the spectrum represent 2 - n - p r o p y l - ( Z ) -2-pentenoic a c i d a small amount of which was c a r r i e d over d u r i n g the r e c r y s t a l l i z a t i o n procedure. The TIC of the methyl e s t e r d e r i v a t i v e of 2-n-propyl-(E)-2-pentenoic a c i d ( f i g u r e 11) confirms the p u r i t y of the product with the major peak being the (E)-isomer and the minor peak the ( Z ) -isomer. The mass s p e c t r a of both the (Z)-isomer ( f i g u r e 12) and (E)-isomer ( f i g u r e 13) of 2-n-propyl-2-pentenoic a c i d c o n t a i n the common ions m/z 156, 141, 127, 113, 97, and 55 that correspond to M +, M-CH 3 +, CH 3CH 2CHC(CH 2)COOCH 3 +, CH 3CH 2CHCCOOCH 3 +, CH 3CH 2CHCC 3H 7 +, and CH 3CH 2CHCH +. b. Attempted s y n t h e s i s by the deh y d r a t i o n of a 0-h y d r o x y a l k y l e s t e r T h i s procedure to s y n t h e s i z e 2-n-propyl-(E)-2-pentenoic a c i d as o u t l i n e d i n scheme 4 was i d e n t i c a l to that used t o s y n t h e s i z e 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d from e t h y l 2-(1'-hydroxypropyl)-(E)-2-pentenoate. The s y n t h e s i s 74 F i g u r e 10: 400 MHz p r o t o n NMR of s y n t h e t i c 2 - n - p r o p y l - ( E ) - 2 -p e n t e n o i c a c i d (*=Z-isomer). 2 VJ /VJ 4 6 8 TIME (Minutes) 10 12 F i g u r e 11: T o t a l ion c u r r e n t chromatogram of 2-n-propyl-2-pentenoic a c i d methyl e s t e r s y n t h e s i z e d by dehalogenation (Peak 1 = Z-isomer; Peak 2 = E-isomer). 76 100 _ >-28 C H r - C H r - C H „ 0 3 2 2 \ II C — C — 0 — C H 3 C H ^ C H p C H ' -j CO LU CX _ J LU CH 41 55 67 50 95 127 113 100 M/Z 156 (M ) 150 200 F i g u r e 12: Mass spectrum of s y n t h e t i c 2 - n - p r o p y l - ( Z ) - 2 -pentenoic a c i d methyl e s t e r c o r r e s p o n d i n g to peak 1 in f i g u r e 11. 100 _ 55 >-i— i—i c n C H r - C H ^ - C H , 0 3 2 2 N || C — C — 0 - f C H C H ^ - C H T - C H ' 3 2 1 4 1 . cr _i UJ cr 28 41 67 . i l l i |J lBl l i i l l 50 95 127 113 } j L 156 (M ) 141 100 M/Z 150 200 Figure 13: Mass spectrum of s y n t h e t i c 2 - n - p r o p y l - ( E ) - 2 -pentenoic a c i d methyl e s t e r c o r r e s p o n d i n g to peak 2 in f i g u r e 11. CH^CHpCh^ \ CHr-CHr-CH 3 2 \ CH—C—0—CHpCH^ OH ethyl 2 - n - p r o p y l - 3 -hydroxypentanoate MsCl E t 3 N , CH 2C1 2 C H = - C H T - C H _ 0 3 2 \ II CH—C—0—CH^-CH, / 2 3 CHx-CH^-CH '3 w " 2 \ 0 S 0 2 C H 3 KH THF mesyl ester o f ethyl 2-n-propyl -3 -hydroxypentanoate CH^ CHjj— CH2 0 CH—CH—CH C — C — 0 — C H r - C H . # 2 3 ethyl 2 - n - p r o p y l - ( E ) -2-pentenoate Scheme 4: S y n t h e s i s of e t h y l 2-n-propyl-(E)-2-pentenoate by d e h y d r a t i n g a j3 _hydroxyalky 1 e s t e r . 79 of 2-n-propyl-(E)-2-pentenoic a c i d v i a the dehydration method was an attempt to develop a new and simpler s y n t h e t i c method f o r t h i s compound. The dehydration of e t h y l 3-hydroxypropylpentanoate r e q u i r e d methanesulfonyl c h l o r i d e and potassium h y d r i d e . However, i n c o n t r a s t to the s y n t h e s i s of e t h y l 2 - ( ( E)-1'-propenyl)-(E)-2-pentenoate, the s y n t h e s i s of e t h y l 2-n-propyl-(E)-2-pentenoate by t h i s method d i d not r e s u l t i n product. Repeated attempts using the same procedures were u n s u c c e s s f u l . An e x p l a n a t i o n f o r the f a i l u r e of t h i s s y n t h e s i s may be r e l a t e d to the s t a b i l i t y of the e n o l a t e i n t e r m e d i a t e . The above method was s u c c e s s f u l f o r producing 2 - ( ( E ) - 1 ' -p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d , where the a d d i t i o n a l double bond appears to s t a b i l i z e the i n t e r m e d i a t e a l l o w i n g the r e a c t i o n to proceed to product. In the attempt to s y n t h e s i z e 2-n-propyl-(E)-2-pentenoic a c i d , the i n t e r m e d i a t e , i f p r e s e n t , l a c k s the double bond necessary f o r s t a b i l i z i n g the complex. A l s o the r e a c t i o n was allowed to proceed f o r 12 hours at room temperature. The longer r e a c t i o n times at ambient temperature may have been too v i g o r o u s f o r the s u c c e s s f u l completion of t h i s r e a c t i o n . Future attempts using t h i s r e a c t i o n sequence to produce 2-n-propyl-(E)-2-pentenoic a c i d w i l l r e q u i r e some m o d i f i c a t i o n s to the procedure. F i r s t l y , a s h o r t e r r e a c t i o n p e r i o d c o u l d be t r i e d . A l i q u o t s of the r e a c t i o n mixture o b t a i n e d every hour would determine i f the product was p r e s e n t , hence, reducing the r i s k of d e g r a d a t i o n . Secondly, 80 the temperature of the r e a c t i o n mixture c o u l d be reduced to 0°C or as low as -78°C. Shorter r e a c t i o n times and/or lower temperatures may be r e q u i r e d t o k i n e t i c a l l y f a v o r the formation of 2-n-propyl-(E)-2-pentenoic a c i d . c. S y n t h e s i s by the o x i d a t i o n of an O - t r i a l k y l s i l y l k e t e n e a c e t a l u s ing a hydride a b s t r a c t o r The method employing a h y d r i d e a b s t r a c t o r to produce 2-( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 ^ p e n t e n o i c a c i d c o u l d a l s o be used to s y n t h e s i z e 2-n-propyl-(E)-2-pentenoic a c i d as o u t l i n e d i n scheme 5. The use of t h i s procedure was an attempt to develop a simpler s t e r e o s p e c i f i c s y n t h e s i s f o r 2-n-propyl-(E)-2-pentenoic a c i d . Using the method of Fleming and Paterson (1979), e t h y l 2-n-propylpentanoate was converted to the d i a l k y l 0 - e t h y l - 0 - t r i m e t h y l s i l y l k e t e n e a c e t a l by s u b j e c t i n g the e s t e r e n o l a t e of 2-n-propylpentanoic a c i d t o t r i m e t h y l s i l y l c h l o r i d e . The h y d r i d e a b s t r a c t o r 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) o x i d i z e s the a c e t a l complex to y i e l d e t h y l 2-n-propyl-2-pentenoate and 2-n-propylpentanoate as shown by Jung and Pan (1977). The h y d r i d e a b s t r a c t o r used was DDQ which d i f f e r s from the a b s t r a c t o r ( t r i t y l t e t r a f l u o r o b o r a t e ) used f o r the s y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . The mechanism of a l l y l i c h y d r i d e a b s t r a c t i o n by DDQ i s s i m i l a r to t r i t y l t e t r a f l u o r o b o r a t e seen i n scheme 2a. The s y n t h e s i s of 2-n-propyl-(E)-pentenoic a c i d was performed i n i t i a l l y u s i ng DDQ. Upon s e a r c h i n g the l i t e r a t u r e f o r a more e f f i c i e n t method, Jung and Pan (1975) r e p o r t e d that 81 CHr-CHr-CH. 0 6 1 < \ II CH—C—0—CH^—CH^ CHj-CH^—CH^ ethyl 2-n-propylpentanoate LDA THF TMSC1 CH rCHr-CH 0 0—TMS 3 2 2 S / C = C • \ CHyCH^—CH,, O-CH^CH d i a l k y l O-ethyl-O-TMS ketene aceta l DDQ Benzene CHr-CH^-CH- 0 3 2 2 \ II CH^CH^CH C—C—0—CH^-CH. S 2 3 ethyl 2 - n - p r o p y l - ( E ) - 2 -pentenoate Scheme 5: S y n t h e s i s of e t h y l 2-n-propyl-(E)-2-pentenoate u s i n g a h y d r i d e a b s t r a c t o r . 82 t r i t y l t e t r a f l u o r o b o r a t e a f f o r d e d much higher y i e l d s when compared to DDQ. Thus, t r i t y l t e t r a f l u r o b o r a t e was u l t i m a t e l y used f o r the s y n t h e s i s of 2 - ( ( E ) - 1 ' - p r o p e n y l ) -(E)-2-pentenoic a c i d i n s t e a d of DDQ. Upon GCMS a n a l y s i s , the TIC ( f i g u r e 14) c o n t a i n s 3 peaks. The mass spectrum ( f i g u r e 15) of the f i r s t peak (7.2 minutes) i n d i c a t e d the presence of e t h y l 2-n-propylpentenoate, the s t a r t i n g m a t e r i a l . Ions at m/z 143, 127, 99, and 57 correspond to the fragments M-CH 2CH3 +, M-OCH 2CH 3 +, M-COOCH 2CH 3 +, and CH 3CH 2CH 2CH 2 +. The remaining two peaks were the Z and E isomers of e t h y l 2-propyl-2-pentenoate. The isomers were i d e n t i f i e d a c c o r d i n g to t h e i r r e l a t i v e r e t e n t i o n times and mass s p e c t r a . The peak at 8.2 minutes r e p r e s e n t s the (Z)-isomer with i t s corresponding mass spectrum shown i n f i g u r e 16. The major peak at 8.8 minutes r e p r e s e n t s the (E)-isomer with i t s corresponding mass spectrum shown i n f i g u r e 17. Both the mass s p e c t r a f o r the Z and E isomer c o n t a i n the c h a r a c t e r i s t i c ions at m/z 170, 141, 125, and 97 corresponding to M+, M-CH 2CH 3 +, M-OCH 2CH 3 +, and M-COOCH 2CH 3 +. Separation of the product from the s t a r t i n g m a t e r i a l by f r a c t i o n a l d i s t i l l a t i o n was impo s s i b l e due to the s i m i l a r i t i e s i n b o i l i n g p o i n t . From the TIC shown i n f i g u r e 14, t h i s s y n t h e s i s appears to be h i g h l y s t e r e o s e l e c t i v e f o r the (E)-isomer. Future work w i l l be necessary to e l i m i n a t e the unreacted s t a r t i n g m a t e r i a l , e t h y l 2-n-propylpentanoate, s i n c e i t i n t e r f e r e s with the 83 • i > 1 i i i 0 2 4 6 8 10 12 TIME (Minutes) F i g u r e 14: T o t a l ion c u r r e n t chromatogram of e t h y l 2-n-p r o p y l - (E)-2-pentenoate s y n t h e s i z e d u s i n g a hydride a b s t r a c t o r (Peak 1 = e t h y l 2-n-p r o p y l p e n t a n o a t e ; Peaks 2 and.3 = Z and E isomers of e t h y l 2-n-propyl-2-pentenoate, r e s p e c t i v e l y ) . 84 CD 100 _ >-I — I — I cn LU LU CH 57 I CH—CH^-CH^-CH 101 29 41 J lilWl hll i Hi 73 127—j CHr-CH^CH- 0 3 2 K II | ^ C H —C-fO-[-CH^CH 3 C H r C H r C H 2 130 115 143 "I 1 50 100 M/Z 150 200 F i g u r e 15: Mass spectrum of s y n t h e t i c e t h y l 2-n-propylpentanoate corresponding to peak 1 i n f i g u r e 14. 125-C H y - C H ^ - C H ^ C H - y C H y - C H \ l c — c -141-• O - r - C H j - C H 3 113 95 55 67 81 125 141 170 (M ) "T 50 100 M/Z 150 200 F i g u r e 16: Mass spectrum of s y n t h e t i c e t h y l 2-n-propy1-(Z)-2-pentenoate corresponding to peak 2 i n f i g u r e 14. 100 _ 55 CD ^— i—i cn LU > I I I— cr _i LU CH 29 41 125-C H r C H r C H 2 C H ^ - C H ^ - C H c — c -141-O-hCHj-CH3 113 67 95 81 il i I III I I I iiy u 125 141 170 (M 50 100 M/Z 150 200 F i g u r e 17: Mass spectrum of s y n t h e t i c e t h y l 2 - n - p r o p y l - ( E ) -2-pentenoate corresponding to peak 3 i n f i g u r e 14. s e p a r a t i o n of the end product. Other techniques of s e p a r a t i o n other than d i s t i l l a t i o n may be necessary. 3. S y n t h e s i s of e t h y l 2-n-propyl-(E)-2,4-pentadienoate The VPA m e t a b o l i t e 2-n-propyl-(E)-2,4-pentadienoic a c i d i s the second most abundant d i u n s a t u r a t e d m e t a b o l i t e and appears to be i n v o l v e d i n the hepatotoxic r e a c t i o n i n animals and humans. A s y n t h e t i c sample of 2 - n - p r o p y l - ( E ) -2,4-pentadienoic a c i d would prove u s e f u l f o r h e p a t o t o x i c s t u d i e s and to compare i t s chromatographic c h a r a c t e r i s t i c s and mass spectrum with 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d . Since 2-n-propyl-2,4-pentadienoic a c i d has i d e n t i c a l molecular weight to 2-((E)-1 '-propenyl.)- (E)-2-pentenoic a c i d , a r e f e r e n c e sample of 2-n-propyl-(E)-2,4-pentadienoic a c i d was r e q u i r e d . The s y n t h e s i s of 2-n-propyl-(E)-2,4-p e n t a d i e n o i c a c i d was f i r s t r e p o r t e d by Rettenmeier et al. (1985) where aluminum t r i c h l o r i d e was u t i l i z e d t o dehydrate e t h y l 3-hydroxy-2-n-propyl-4-pentenoate. The method of Rettenmeier et al. (1985) a f f o r d e d 2 isomers, E and Z, i n the r a t i o of approximately 9:1, r e s p e c t i v e l y . Attempts were made to s y n t h e s i z e 2-n-propyl-2,4-pentadienoic a c i d u s ing methanesulfonyl c h l o r i d e and potassium h y d r i d e as the d e h y d r a t i n g agent i n order to i n c r e a s e y i e l d s and maintain the s t e r e o s e l e c t i v i t y . The s y n t h e s i s of e t h y l 2-n-propyl-(E)-2,4-pentadienoate by t h i s route i s summarized i n scheme 6. V a l e r i c a c i d was e s t e r i f i e d with ethanol and s u l f u r i c a c i d i n benzene to y i e l d e t h y l v a l e r a t e . The e s t e r was 88 C H y C H ^ C H ^ C H ^ - ! — O H H 2 S 0 4 EtOH Benzene 0 C H y C H y C H p C H y C — 0 — C H y C ^ acrolein LDA THF CH^-CH2-—CH2 CH==CH—CH T \ JLW—C—0— CH^-CHg OH valeric acid ethyl valerate ethyl 2-n-propyl-3-hydroxy-4-pentenoate CH 2C1 2 MsCl Et 3N CH^-CH5 -CH 0 0 3 2 K II C H — C — 0 — C H 5 - C H -/ 2 3 CHS=CH—CH T \ 0 S 0 2 C H 3 KH THF mesyl ester of ethyl 2-n-propyl -3-hydroxy-4-pentenoate CH^-CH«-CH 0 0 3 2 \ II X—C—0—CH^-CH, CH^CH—CH ethyl 2-n-propyl-(E)-2,4-pentadienoa±e Scheme 6: S y n t h e s i s of e t h y l 2 -n-propyl-(E)-2,4-pentadienoate by d e h y d r a t i n g a 0-hydroxyalkenyl e s t e r 89 c o n v e r t e d to an e s t e r e n o l a t e v i a LDA and combined with a c r o l e i n u s i n g the procedures f o r the s y n t h e s i s of 2 - ( ( E ) -1'-propenyl)-(E)-2-pentenoic a c i d to gi v e e t h y l 3-hydroxy-2-n-propyl-4-pentenoate. A n a l y s i s by GCMS r e s u l t e d i n ions at m/z 169, 159, 141, 130, and 101 which correspond to M-OH +, M-CH 2=CH +, M-OCH 2CH 3 +, CH 3CH 2CH 2CH(CHOH)COO +, and CH 3CH 2CH 2CH 2COO +, r e s p e c t i v e l y . The 80 MHz NMR data (appendix) i n d i c a t e the presence of e t h y l 2-n-propyl-3-hydroxy-4-pentenoate. Absorption s i g n a l s at 5.65 - 6.1 and 5.1 - 5.4 ppm correspond to =CH and CH 2=, r e s p e c t i v e l y . The protons CH-0 and CH occurs at 4.2 - 4.5 and 2.3 - 2.7 ppm, r e s p e c t i v e l y . The hydroxy proton occurs at 2.4 ppm as a broad s i g n a l with the methinyl proton present w i t h i n t h i s s i g n a l . The methylene protons occur together at 1.3 - 1.7 ppm while the methyl protons CH 3 and OCH 2CH 3 occur at 0.9 and 1.2 ppm, r e s p e c t i v e l y . The methylene protons of the e s t e r i s present at 4 - 4.4 ppm. The unsa t u r a t e d hydroxy e s t e r was dehydrated with methanesulfonyl c h l o r i d e and potassium hydride to a f f o r d e t h y l 2-n-propyl-(E)-2,4-pentadienoate. The TIC ( f i g u r e 18) r e v e a l s the presence of 2 isomers i n the p r o p o r t i o n s of 21.1% and 72.4% with the major peak at 8.3 minutes being the (E)-isomer. F i g u r e s 19 and 20 show the mass s p e c t r a of the Z and E isomers, r e s p e c t i v e l y . The c h a r a c t e r i s t i c ions f o r both isomers of e t h y l 2-n-propyl-2,4-pentadienoate are m/z 168, 139, 123, 95, and 55 which correspond to M +, M-CH 2CH 3 +, M-OCH 2CH 3 +, CH 2CHCHCC 3H 7 +, and CH 3CH 2CHCH +, 90 1 I 2 4 6 i TIME (Minutes) — ? — 10 —i— 12 F i g u r e 18: T o t a l ion c u r r e n t chromatogram of s y n t h e t i c e t h y l 2-n-propyl-2,4-pentadienoate (Peak 1 = Z-isomer; Peak 2 = E-isomer). 91 26 % RELRTIVE INTENSITY TJ C fD C fD . , OJ in I cn TJ fD Ul 3 TJ fD CO 0) • a »-»• fD 3 O ro CO o 0 3 M rr 3* fD fD 01 rr TJ O 3 a. rD o 3 3-L Q r r O ro I TJ 3 fD I 0) TJ O - T J << M- f—' 3 I tsJ I o r v j o cn' o ro o ' o ro CO a i CD CO CO cn ro CO CO CO o o ro| I "| CD GO s \ / I | _ ro co co vo I r-o 3: rol o CO 29 C H C H^ -— C H 2 CHjj=CH—CH 123— 0 N n C — C - 4 - O - f C H ^ - C H . 95-1 139— 67 95 41 55 123 140 79 111 lillllllil 50 1.11 Ml ll 100 M/Z 168 (M ) 150 200 F i g u r e 20: Mass spectrum of s y n t h e t i c e t h y l 2 - n - p r o p y l - ( E ) -2,4-pentadienoate corresponding to peak 2 i n f i g u r e 18. r e s p e c t i v e l y . F i g u r e 21 r e p r e s e n t s the NMR spectrum f o r the dienes with the (E)-isomer c o n t r i b u t i n g 74% of the t o t a l mixture. The methyl protons at 0.92 ppm and the e s t e r methyl protons at 1.31 ppm appear as t r i p l e t s . The s i g n a l f o r the methylene protons adjacent to the methyl group appear at 1.41 - 1.52 ppm as a m u l t i p l e t whereas the methylene protons adjacent to the double bond occur as a t r i p l e t at 2.4 ppm with a c o u p l i n g constant of 7 Hz. The e s t e r methylene protons are s p l i t by the adjacent methyl group producing a q u a r t e t at 4.22 ppm. The v i n y l i c s t r u c t u r e r e p r e s e n t s an ABX system with the CH2= protons, from the (E)-isomer, being non-equivalent at 5.44 and 5.57 ppm with each proton s p l i t by each other and by the adjacent v i n y l i c proton (J B X=10 Hz, J ^ x " 1 6 H z ' r e s p e c t i v e l y and J A B = 2 H Z ) . For the (Z)-isomer, the CH 2= protons are s t r o n g l y s h i e l d e d by the c a r b o n y l oxygen with the s i g n a l o c c u r r i n g s l i g h t l y u p f i e l d at 5.28 - 5.33 and 5.34 - 5.41 ppm. Although t h i s s y n t h e s i s r e s u l t e d i n good y i e l d s the isomeric p u r i t y d i d not exceed the r e s u l t s of Rettenmeier et al. (1985). As shown f o r the s y n t h e s i s of 2 - ( ( E ) - 1 ' -p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d , dehydration u s i n g methanesulfonyl c h l o r i d e and potassium hydride r e s u l t e d i n mainly the (E)-isomer at the C-2 p o s i t i o n . An e x p l a n a t i o n f o r the decrease i n s t e r e o s e l e c t i v i t y f o r the s y n t h e s i s of 2-n-propyl-2,4-pentadienoic a c i d u s ing the same deh y d r a t i n g agent may be due to the c o n j u g a t i o n of the double bonds 94 F i g u r e 21: 400 MHz proton NMR of s y n t h e t i c e t h y l 2-n-propyl-(E)-2,4-pentadienoate (* = Z-isomer). with the c a r b o n y l group of the mesylate e s t e r i n t e r m e d i a t e . The a b i l i t y t o form long range c o n j u g a t i o n may s t a b i l i z e the mesylate e s t e r d e c r e a s i n g the ease of i t s removal by potassium h y d r i d e thereby d e c r e a s i n g s t e r e o s p e c f i c i t y . Attempts to i s o l a t e the f r e e a c i d from the e t h y l e s t e r r e s u l t e d i n the degradation of the a c i d . Using base h y d r o l y s i s (1 N NaOH), the s o l u t i o n proceeded to a yellow mixture over a p e r i o d of 24 hours. Q u a n t i t a t i o n of the f r e e a c i d p r e s e n t s a problem i f the a c i d i s c o n s t a n t l y being degraded over time. There have been no attempts to i d e n t i f y the breakdown p r o d u c t ( s ) . Knowledge of the s t r u c t u r e of these breakdown p r o d u c t ( s ) c o u l d prove u s e f u l i n minimizing d e g r a d a t i o n . 4. S y n t h e s i s of e t h y l 2-n-propyl-3-oxopentanoate E t h y l 2-n-propyl-3-oxopentanoate was r e q u i r e d as a s y n t h e t i c r e f e r e n c e compound to i d e n t i f y i t as a VPA m e t a b o l i t e i n b i o l o g i c a l samples. The s y n t h e s i s of e t h y l 2-n-propyl-3-oxopentanoate o u t l i n e d i n scheme 7 i s based on the method of Acheampong (1982). The e s t e r e n o l a t e of v a l e r i c a c i d was a l k y l a t e d with p r o p i o n y l c h l o r i d e to y i e l d e t h y l 2-n-propyl-3-oxopentanoate. The TIC ( f i g u r e 22) i n d i c a t e s the presence of two peaks with the major peak at 10.5 minutes being e t h y l 2-n-propyl-3-oxopentanoate. The minor peak at 9 minutes i s an amide formed by the condensation of p r o p i o n y l c h l o r i d e and LDA. F i g u r e 23 r e p r e s e n t s the mass spectrum of e t h y l 2-n-propyl-3-96 0 il C H j - C H y C H y C H y C — 0 — CHsr-CHg ethyl v a l e r a t e propionyl c h l o r i d e LDA THF C H r C H r c \ i C H r C H r C ^ 0 CH- -0—CHyCH-j ethyl 2 - n - p r o p y l - 3 -oxopentanoate Scheme 7: S y n t h e s i s of e t h y l 2-n-propyl-3-oxopentanoate. 97 1 8 10 • \ 12 — r -14 TIME ( M i n u t e s ) F i g u r e 22: T o t a l i on c u r r e n t chromatogram of s y n t h e t i c e t h y l 2-n-propyl-3-oxopentanoate (peak 1). 98 vo VO 100 _ >-I— I — I CO -ZL LxJ r— LU > » \ \— CE _ l LU or 29 57 0 C H r C H - ( V \ ^ C H — C — 0 -C — C H'2"—C H 2 1 5 7 — : H 2 ~ C H 3 101 73 UlLl U J IL C H y C H j - C H j - C H—C — 0 130 115 II I I 144 157 h 50 100 M/Z 150 200 F i g u r e 23: Mass spectrum of s y n t h e t i c e t h y l 2-n-propy1-3-oxopentanoate corresponding to peak 1 i n f i g u r e 22. oxopentanoate with ions m/z 141, 115, and 57 corresponding to fragments CH 3CH 2COCHC 3H 7CO +, CH 3CH 2COHCHCOO +, and CH 3CH 2CO +, r e s p e c t i v e l y . S e p a r a t i o n of these two peaks was not p o s s i b l e , t h e r e f o r e , e l i m i n a t i o n of the amide r e q u i r e d the use of N-isopropylhexylamine i n s t e a d of d i i s o p r o p y l a m i n e . The condensation product formed with N-isopropylhexylamine and p r o p i o n y l c h l o r i d e has a lower b o i l i n g p o i n t and hence, can be e a s i l y separated by d i s t i l l a t i o n . 5. S y n t h e s i s of e t h y l 2-n-propyl-3-hydroxypentanoate The compound e t h y l 2-n-propyl-3-hydroxypentanoate was r e q u i r e d as s t a r t i n g m a t e r i a l f o r the attempted s y n t h e s i s of e t h y l 2-n-propyl-(E)-2-pentenoate. The procedures of Acheampong (1982) were m o d i f i e d to s y n t h e s i z e e t h y l 2-n-propyl-3-hydroxypentanoate (scheme 8 ) . T h i s method was s i m i l a r t o the s y n t h e s i s of e t h y l 2-n-propyl-3-oxopentanoate e:;cept f o r the a l k y l a t i n g agent. Propionaldehyde was combined with the e s t e r e n o l a t e of v a l e r i c a c i d and the crude mixture was f r a c t i o n a t e d by d i s t i l l a t i o n to y i e l d pure e t h y l 2-n-propyl-3-hydroxypentanoate. F i g u r e 24 d e p i c t s the TIC of e t h y l 2-n-propyl-3-hydroxypentanoate with i t s mass spectrum seen i n f i g u r e 25. C h a r a c t e r i s t i c i o n s , m/z 159, 143, 101, and 73, are r e p r e s e n t e d by the fragments CH 3CH 2CHOHCHC 3H 7COO +, CH 3CH 2CHOHCHC 3H 7CO +, CH 3CH 2CHOHCHCOH +, and CH 3CH 2CHOHCH 2 +, r e s p e c t i v e l y . 100 0 CHyCHyCHyCHyS-O-CHyC^ ethyl va le ra te propionaldehyde LDA THF CH^-CH^-CH 0 0 3 2 K 11 ^CH— C—0—CHyCH-j C H ^ - C H T T - C H '3 1,1'2 ethyl 2-n-propyl-3-hydroxypentanoate 0 H Scheme 8: S y n t h e s i s of e t h y l 2-n-propyl-3-hydroxypentanoate. 101 1 I 1 % 1 1 I 1— 0 2 4 6 8 10 12 14 TIME (Minutes) F i g u r e 24: T o t a l ion c u r r e n t chromatogram of s y n t h e t i c e t h y l 2-n-propy1-3-hydroxypentanoate. 102 e o i X RELATIVE INTENSITY iD. c rt fD tO cn =r 3: k< 0, Cu co rt CO o X cn <^ TJ TJ r» fD O 3 r f r f rt Q) C 3 3 O 0) O r f m fD CO r-h>< rt 3 O r f 3 3" fD rt» r f vO O C 1-1 ro fD l 3 rO I * » T J • r| O TJ r-« I CO I o r v j o cn o ro o ' o ro CD 4^  co cn cn CD ^1 —1 CO 0 "I o n: I m CO CD CO CO O O "I o ro CO to o * i o o cn CD 0 = 0 C O cn O - 4 -o CO 6. S y n t h e s i s of 2-n-propylpentanoic a c i d The s y n t h e s i s of 2-n-propylpentanoic a c i d was r e q u i r e d as s t a r t i n g m a t e r i a l f o r the s y n t h e s i s of 2 - n - p r o p y l - ( E ) -pentenoic a c i d by dehalogenation. Using the procedure of Acheampong (1985) o u t l i n e d i n scheme 9, pure 2-n-p r o p y l p e n t a n o i c a c i d was s y n t h e s i z e d by a l k y l a t i n g the d i a n i o n of v a l e r i c a c i d with p r o p y l i o d i d e . The crude product was separated by f r a c t i o n a l d i s t i l l a t i o n (91% y i e l d ) and upon GCMS a n a l y s i s of the methyl e s t e r d e r i v a t i v e , the TIC ( f i g u r e 26) i n d i c a t e d the presence of a s i n g l e peak o c c u r r i n g a t 6 minutes. The mass spectrum of the methyl e s t e r of 2-n-propylpentanoic a c i d ( f i g u r e 27) gave c h a r a c t e r i s t i c ions at m/z 127, 99, 57, and 43 corresponding to (CH 3CH 2CH 2) 2CHCO +, (CH 3CH 2CH 2) 2CH+', CH 3CH 2CH 2CH 2 +, and CH 3CH 2CH 2 +, r e s p e c t i v e l y . B. M e t a b o l i c Study S t u d i e s with Wistar r a t s dosed i . p . with 100 mg/kg of 2 - ( ( E ) - 1 ' - p r o p e n y l ) - ( E ) - 2 - p e n t e n o i c a c i d ((E,E)-2,3'-diene VPA) r e v e a l e d the presence of s e v e r a l m e t a b o l i t e s i n u r i n e and b i l e . Reduced products of (E,E)-2,3'-diene VPA that were i d e n t i f i e d were 2-n-propyl-3-pentenoic a c i d ((E)-3-ene VPA), 2-n-propyl-2-pentenoic a c i d ((E)-2-ene VPA), and 2-n-p r o p y l p e n t a n o i c a c i d (VPA). F i g u r e 28 compares the mass chromatograms at m/z 199 of the m e t a b o l i t e s i n u r i n e and b i l e (conjugated and unconjugated) to known s y n t h e t i c standards of (E)-2-ene and (E)-3-ene VPA. The mass s p e c t r a 104 0 C H y C H y C H y C H y C — O H propyl iodide LDA THF CH^-CH^-CH, 0 3 2 \ 11 ^ C H — C — C H y CH^y CH 2 OH valeric acid 2-n-propylpentanoic acid Scheme 9: S y n t h e s i s of 2-n-propylpentanoic a c i d . 105 0 2 4 6 8 TIME (Minutes) 10 12 F i g u r e 26: T o t a l ion c u r r e n t chromatogram of s y n t h e t i c 2-n-pro p y l p e n t a n o a t e methyl e s t e r . 106 87 29 41 CHg CH—CH,j 127—1 0 II CH-fC+O—CH. C H y CH^ -CH^ 99_J 57 116 (M-42 ) 69 50 _Lil_JJ U_ 99 100 M/Z 129 150 200 F i g u r e 27: Mass spectrum of s y n t h e t i c 2-n-propylpentanoic a c i d methyl es t e r from f i g u r e 26. V J A J 1 A 1 1 1 Synthesi zed (E)-2-ene VPA Synthesi zed (E)-3-ene VPA Urine Unconjugated Bile 8 Conjugated Bile TIME (Minutes) F i g u r e 28 Mass chromatograms at m/z 199 comparing s y n t h e t i c s t a n d a r d s of (E)-2-ene VPA and (E)-3-ene VPA with e x t r a c t e d u r i n e , unconjugated f r a c t i o n from b i l e and conjugated f r a c t i o n from b i l e a f t e r a l k a l i n e h y d r o l y s i s from a r a t dosed with (E,E)-2,3'-diene VPA. 108 comparing s y n t h e t i c (E)-3-ene and (E)-2-ene VPA to peaks found i n u r i n e and b i l e f r a c t i o n s are shown i n f i g u r e s 29 and 30, r e s p e c t i v e l y . The mass chromatogram a t m/z 201 comparing the u r i n e and b i l e (conjugated and unconjugated) f r a c t i o n to s y n t h e t i c VPA i s seen i n f i g u r e 31. The cor r e s p o n d i n g mass s p e c t r a ( f i g u r e 32) confirms the presence of VPA. The r e d u c t i o n of an unsaturated compound to a s a t u r a t e d product i s not unique. T e s t o s t e r o n e i s reduced to 5-a-d i h y d r o t e s t o s e r o n e by the enzyme 5-a-reductase i n r a t l i v e r microsomes (Breuer et al., 1968). S t i g m a s t e r o l i s reduced to c h o l e s t e r o l v i a the metabolic processes i n the nematode C a e n o r h a b d i t i s elegans (Lozano et al., 1985). The f a t t y a c i d 1 5 -keto-prostaglandin i s reduced to 13,14-dihydro-15-k e t o - p r o s t a g l a n d i n by 13,14-prostaglandin reductase i n guinea p i g and swine ( H a l l and Behrman, 1982). Granneman et al. (1984) demonstrated that when r a t s were given 2-ene VPA or 3-ene VPA, 2% of the dose was recovered as VPA. The enzyme that reduces (E,E)-2,3'-diene VPA to (E)-2-ene, ( E ) -3-ene VPA, and VPA i s l i k e l y to be s i m i l a r to the reductase that reduces 2-ene and 3-ene VPA to VPA. In a study by Acheampong (1985) where mice were given 280 mg/kg of (E,E)-2,3'-diene VPA i . p . f o l l o w e d by 85 mg/kg of p e n t y l e n e t e t r a z o l e s . c , 60% of the p o p u l a t i o n were p r o t e c t e d a g a i n s t t o n i c - c l o n i c s e i z u r e s . The r e d u c t i o n of (E,E)-2,3'-diene VPA to (E)-3-ene, (E)-2-ene VPA, and VPA in r a t s may c o n t r i b u t e to the a n t i c o n v u l s a n t a c t i v i t y i n 109 A) mo 50 l'J<J(H-57 ) CHJ-CHJ-CH j 100 llikii, lL_ull ,JJ.. rL i— ISO M/Z 2-11 —1—. B) 55 ILil 99 9^  115 ~I— 150 M/Z c) D) . J _ J 50 155 150 M/Z 150 M/Z 250 F i g u r e 29: Mass s p e c t r a comparing (A) s y n t h e t i c (E)-3-ene VPA, (B) u r i n e , (C) unconjugated, and (D) conjugated b i l e f r a c t i o n s from a r a t dosed with (E,E)-2,3'-diene VPA. I'J'j (n-w C l l j - C l l j - C H ? 0 C— C - 0 — S i C l C H ^ ) j ( C H 3 ) ? 125 I I "3 IflLjM. I lik I I . i L. — 1 ' — 15(1 M/Z B) 155 - I 150 M/Z 1 251) 100 -liiljli 125 150 M/Z D) 1 15 1 150 M/Z _U-, 1—jl— —I 250 Figure 30: Mass s p e c t r a comparing (A) s y n t h e t i c (E)-2-ene VPA, (B) u r i n e , (C) unconjugated, and (D) conjugated b i l e f r a c t i o n s from a r a t dosed with (E,E)-2,3'-diene VPA. Synthesized VPA Urine Unconjugated B i l e Conjugated B i l e ~ i 1 6 7 TIME (Minutes) F i g u r e 31: Mass chromatograms a t m/z 201 comparing s y n t h e t i c VPA and e x t r a c t e d u r i n e , u n c o n j u g a t e d = f r a c t i o n from b i l e , and c o n j u g a t e d f r a c t i o n from b i l e a f t e r a l k a l i n e h y d r o l y s i s from a r a t dosed with (E,E)-2,3'-diene VPA. 112 e i i o 3 01 O 3 Di C re OO r o rt rj Q, 0) ^ - 01 0 3 0) rt- f0 - 01 TJ ro n o •—• rr ri C 0) 3 o o o o 3 g c-i.TJ ri C Q» OIlO T rr fo rr 3 Qi fD iQ 0 Cu 01 -» - ~ ro > Qj Q) • -3 « Cu 01 r t ^ 3 r t —- rr ro n n i t - o m 3 o ^—'i i. I c < to iQ 13 - 0) > Co rr -- ro i a — a a fD 3 t— fD fD I RELATIVE INTENSITY ' 1 I L t RELATIVE INTENSITY —i 1 1 i _ I RELATIVE INTENSITT I RELATIVE INTENSITY s J i u mice. In other words, (E,E)-2,3'-diene VPA i t s e l f may not possess a n t i c o n v u l s a n t a c t i v i t y , i . e . the p r o t e c t i o n a g a i n s t p e n t y l e n e t e t r a z o l e s e i z u r e i n mice may be due to VPA and/or 2-ene VPA. Rats dosed 100 mg/kg i . p . with (E,E)-2,3'-diene VPA have r e l a t i v e l y low c o n c e n t r a t i o n s of VPA and 2-ene VPA present i n the u r i n e and b i l e compared to (E,E)-2,3'-diene VPA. Since the metabolism study was only q u a l i t a t i v e , the c o n c e n t r a t i o n s of the m e t a b o l i t e s were not determined. A dose of 100 mg/kg of VPA was r e q u i r e d t o prevent p e n t y l e n e t e t r a z o l e - i n d u c e d c o n v u l s i o n s i n E1 mice whereas a 50 mg/kg dose of VPA only p r o t e c t e d 40% of the p o p u l a t i o n (Sugaya et al., 1986). T h e r e f o r e , to p r o t e c t a g a i n s t p e n t y l e n e t e t r a z o l e - i n d u c e d s e i z u r e s , there must be a s u b s t a n t i a l q u a n t i t y of VPA i n the body. The amount of VPA formed by the r e d u c t i o n of (E,E)-2,3'-diene VPA does not appear to be e x t e n s i v e enough to be t h e r a p e u t i c a l l y e f f e c t i v e . Thus (E,E)-2,3'-diene VPA appears t o l a r g e l y possess a n t i c o n v u l s a n t a c t i v i t y i n mice as the parent substance. As p r e v i o u s l y mentioned, VPA i s metabolized e x t e n s i v e l y by the l i v e r t o 16 known m e t a b o l i t e s . The metabolic pathways of VPA are q u i t e complex and are f u r t h e r c o m p l i c a t e d by r e d u c t i o n of the mono and d i u n s a t u r a t e d m e t a b o l i t e s back to VPA. The o x i d i z e d VPA m e t a b o l i t e s 5-OH VPA and 4-OH VPA occur v i a co and co-1 o x i d a t i o n , r e s p e c t i v e l y , by the l i v e r microsomes while 3-OH VPA and 3-11 4 keto VPA occur v i a 0-oxidation i n the mitochondria (Schafer and Luhrs, 1978). The metabolism of (E,E)-2,3'-diene VPA may p o s s i b l y p a r a l l e l some of the metabolic pathways of VPA. F i g u r e 33 o u t l i n e s the p o s s i b l e metabolic routes f o r (E,E)-2,3'-diene VPA where (*) denotes confirmed m e t a b o l i t e s and (+) denotes m e t a b o l i t e s d e t e c t e d but not confirmed. One new m e t a b o l i t e of VPA i d e n t i f i e d i n human u r i n e by Kassahun (unpublished) which was not confirmed as a m e t a b o l i t e of (E,E)-2,3'-diene VPA i n r a t s i s 2-(2'-oxopropyl)-2-pentenoic a c i d (4'-keto-2-ene VPA). Attempts to i d e n t i f y t h i s m e t a b o l i t e i n r a t u r i n e f o l l o w i n g (E,E)-2,3'-diene VPA dosing f a i l e d . T h i s can probably be a t t r i b u t e d to the assay technique s i n c e the u r i n e sample was not concentrated s u f f i c i e n t l y to observe t r a c e m e t a b o l i t e s . An i n t e r e s t i n g o b s e r v a t i o n from the metabolism study of (E,E)-2,3'-diene VPA was the presence of (E)-3-ene VPA. Since (E)-3-ene VPA i n serum has been r e p o r t e d to e x i s t at r e l a t i v e l y low c o n c e n t r a t i o n s (Abbott et a l . , 1986), t h i s m e t a b o l i t e may not be a d i r e c t m e t a b o l i t e of VPA but i n s t e a d , a reduced product of (E,E)-2,3'-diene VPA. In f u t u r e s t u d i e s , c o n c e n t r a t e d u r i n e samples from r a t s dosed with (E,E)-2,3'-diene VPA w i l l be used to i d e n t i f y other m e t a b o l i t e s . S t u d i e s r e g a r d i n g the metabolism of (E,E)-2,3'-diene VPA w i l l center around the cytochrome P-450 enzyme system which i s r e s p o n s i b l e f o r to and w-1 o x i d a t i o n of x e n o b i o t i c s . 115 CH=-CH=CH. 0 3 \ II * confirmed metabol i tes + metabol i tes detected a f te r VPA dosing in humans 2 ,3 ' -d iene VPA glucuronide-C - C - O H C H ^ C H — C H ^ 2 , 3 ' , 4 - t r i e n e VPA CHtr-CHrrCH 0 3 \ II C—C—OH CKr-CH=CH 0 3 \ II CH-C-OH / CHr-CHr-CK 3 2 \ OK 3'-0H-3-ene VPA CH^-CH=CH 0 3 \ „ « CHj-CH^-CH 2 ,3 ' -d iene VPA OH I CHr-CH-CH, 0 3 2 N II CHj-CK^-CH 4'-0H-2-ene VPA C—C —OH CH-C-OH C H r - C - C H , 0 CH^CH^C 3 2 - ^ 0 3 ' -oxo-3-ene VPA Z — C — O H // CHj-CHjpCH 4 ' -oxo-2-ene VPA CHs-CH=CH 0 •> \ II CH—C —OH CHj-CH^-CH 2 3-ene VPA C H T - C H T - C H . 0 3 2 2 N ii C — C — O H C H ^ - C H ^ - C H ^ 2-ene VPA C H ^ - C H T T - C H , 0 3 2 2 V II CH— C — OH CHj-CH^-CW^ VPA F i g u r e 33: P o s s i b l e m e t a b o l i c pathways f o r (E ,.E)-2 , 3' -diene VPA i n humans and animals. 116 C. Pharmacokinetic Study 1. Animal Model Male Wistar r a t s weighing 200 - 300 grams with a blood volume of 15 mL were used i n a l l the s t u d i e s (Hurwitz, 1971). The pharmacokinetic study r e q u i r e d 1.5 mL of blood to be c o l l e c t e d over a 4 hour p e r i o d . The sample volume was kept to a minimum to prevent any s i g n i f i c a n t changes in hemodynamics s i n c e the experiment was to be repeated a week l a t e r . 2. Assay The procedures of Abbott et al. (1986) to measure VPA and i t s m e t a b o l i t e s were m o d i f i e d to q u a n t i t a t e (E,E)-2,3'~ diene VPA i n r a t plasma by GCMS using s e l e c t e d ion mo n i t o r i n g . The small volumes of r a t plasma r e q u i r e d the procedures to be s c a l e d down s i n c e the method of Abbott et al. (1986) a p p l i e d to human samples where l a r g e r volumes of serum were a v a i l a b l e . A summary of 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 procedures f o r r a t plasma can be seen i n f i g u r e 34. The ions chosen f o r s e l e c t e d ion monitoring were m/z 207 and m/z 199 which correspond to the base peak, [M-57] +, f o r 2H 6-VPA ( i n t e r n a l standard) and (E,E)-2,3*-diene VPA, r e s p e c t i v e l y . 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 procedures f o r r a t plasma r e s u l t e d i n c l e a n ion chromatograms f o r ions at m/z 207 and 199 ( f i g u r e s 35 and 36, r e s p e c t i v e l y ) . The 117 50 uL PLASMA, 100 UL 2H 6-VPA (INT STD), 25 uL 1 N NaOH HEAT AT 60°C FOR 1 HOUR ACIDIFY WITH 35 UL 1 N HC1 EXTRACT WITH 2 X 500 uL OF ETHYL ACETATE, DRY WITH ANHYDROUS Na 2S0 4 REDUCE VOLUME (200 uL) WITH NITROGEN ADD 60 uL t-BDMS REAGENT, HEAT AT 60°C FOR 4 HOURS INJECT 1 uL F i g u r e 34: A summary of 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 procedures f o r the assay of (E,E)-2,3'-diene VPA i n r a t plasma. 118 6.0 6.2 6.4 6.6 TIME (Minutes) 6.8 7.0 F i g u r e 35: Mass chromatogram at m/z 207 of the t-BDMS e s t e r of ^Hg-VPA e x t r a c t e d from r a t plasma. 119 ( E . E ) I ( E , Z ) _ A i : 1 1— 6.0 6.5 7.0 TIME (Minutes) F i g u r e 36: Mass chromatogram at m/z 197 of the t-BDMS of (E,E)-2,3'-diene VPA e x t r a c t e d from r a t plasma. 120 e x t r a c t i o n recovery of (E,E)-2,3'-diene VPA from r a t plasma was 98% when compared t o the recovery from water. The c a l i b r a t i o n curves f o r r a t s given 20 mg/kg and 100 mg/kg ( f i g u r e s 37 and 38, r e s p e c t i v e l y ) were l i n e a r w i t h i n the c o n c e n t r a t i o n ranges used and r e s u l t e d i n a c o e f f i c i e n t of d e t e r m i n a t i o n , r 2 , exceeding 0.99. The c a l i b r a t i o n curve c o n c e n t r a t i o n ranges used f o r r a t s given 20 mg/kg and 100 mg/kg were 0 - 240 ug/mL and 0 - 600 ug/mL, r e s p e c t i v e l y . The lower l i m i t of d e t e c t i o n was 800 pg of (E,E)-2,3'-diene VPA and the s e n s i t i v i t y of the assay was l i m i t e d by the presence of background peaks c o n t r i b u t e d by the t-BDMS reagent and plasma. 3. S i n g l e Dose Study Wistar r a t s given 20 mg/kg i . v . of (E,E)-2,3'-diene VPA d i d not d i s p l a y any v i s i b l e changes i n behavior. A 100 mg/kg i . v . dose e l i c i t e d tremors and an i n c r e a s e i n r e s p i r a t i o n minutes a f t e r the' i n j e c t i o n . Sedation then followed suggesting that the diene c r o s s e s the b l o o d - b r a i n -b a r r i e r r e a d i l y . A s e d a t i v e e f f e c t f o r (E)-2-ene VPA was a l s o r e p o r t e d by Loscher et al. (1984) i n r a t s . VPA has been noted to cause s e d a t i o n but the i n c i d e n c e was minimal (Loscher et a l . , 1984). The amount of (E,E)-2,3'-diene VPA recovered i n u r i n e was only 0.1 - 1% i n r a t s (Granneman et al . , 1984) and 3 - 6% in man (Abbott et al., unpublished) a f t e r VPA do s i n g . T h e r e f o r e , s i n c e the dose of (E,E)-2,3'-diene VPA ad m i n i s t e r e d to r a t s was s e v e r a l times g r e a t e r 121 15-1 (E ,E)-2 ,3 ' -DIENE VPA CONCENTRATION (ug /mL) F i g u r e 37: C a l i b r a t i o n curve f o r (E,E)-2,3'-diene VPA from ra t plasma with a c o n c e n t r a t i o n range between 0 -240 ug/mL. F i g u r e 38: C a l i b r a t i o n curve f o r (E, E) -2 , 3'-diene VPA from rat plasma with a c o n c e n t r a t i o n range between 0 -600 ug/mL. than normally seen i n plasma a f t e r VPA dosing may e x p l a i n the observed CNS e f f e c t s seen with (E,E)-2,3'-diene VPA. Fo l l o w i n g a s i n g l e 20 mg/kg i . v . dose of (E,E)-2,3'-diene VPA i n b i l e duct i n t a c t r a t s , the plasma c o n c e n t r a t i o n vs time curve d i s p l a y e d a monophasic d e c l i n e as seen i n f i g u r e 39. A monophasic d e c l i n e was a l s o observed ( f i g u r e 40) i n the same r a t s with t h e i r b i l e ducts cannulated. A comparison of the e l i m i n a t i o n r a t e constants (Kg), volume of d i s t r i b u t i o n ( V ^ ) , and t o t a l body c l e a r a n c e s (CI) between the b i l e duct i n t a c t and cannulated animals r e v e a l e d no s i g n i f i c a n t d i f f e r e n c e s (p>0.1) using the p a i r e d t - t e s t . The e l i m i n a t i o n r a t e constants were obtained from the slope of the plasma c o n c e n t r a t i o n vs time p l o t u s ing the computer program, "AUTOAN" (Sedman and Wagner, 1976). T o t a l body c l e a r a n c e and volume of d i s t r i b u t i o n were obtained from equation 1 where area under the curve (AUC) was c a l c u l a t e d u s i n g the method of t r a p e z o i d s from zero to i n f i n i t y . The l a s t plasma c o n c e n t r a t i o n was d i v i d e d by the e l i m i n a t i o n r a t e constant to o b t a i n the area under the curve from the l a s t c o n c e n t r a t i o n to i n f i n i t y . dose CI = Kg x V J = equation 1 AUC A 100 mg/kg i . v . dose r e s u l t e d i n a monophasic plasma c o n c e n t r a t i o n vs time curve f o r the b i l e duct i n t a c t ( f i g u r e 41) and cannulated ( f i g u r e 42) animals. There were 124 F i g u r e 39: Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e duct i n t a c t r a t s dosed 20 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). F i g u r e 40: Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e duct cannulated r a t s dosed 20 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). F i g u r e 41 : Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e duct i n t a c t r a t s dosed 100 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). F i g u r e 42: Plasma c o n c e n t r a t i o n - t i m e p l o t f o r b i l e duct cannulated r a t s dosed 100 mg/kg i . v . with (E,E)-2,3'-diene VPA (S.D. shown). no s i g n i f i c a n t d i f f e r e n c e s i n K E, V^, and CI between the b i l e duct i n t a c t and cannulated r a t s (p>0.1) u s i n g the p a i r e d t - t e s t . The e l i m i n a t i o n process of (E,E)-2,3'-diene VPA can be d e s c r i b e d by a simple one-compartment f i r s t order k i n e t i c model. T h i s d i f f e r s from VPA (Dickinson et al., 1979; Ogiso et al., 1980) and (E)-2-ene VPA (Singh et al., unpublished) where the e l i m i n a t i o n k i n e t i c s i n b i l e duct i n t a c t r a t s was r e p o r t e d to be b i p h a i s c due to ex t e n s i v e e n t e r o h e p a t i c r e c y c l i n g . E x t e r i o r i z i n g the common b i l e duct r e s u l t e d i n a monophasic e l i m i n a t i o n process f o r both VPA and 2-ene VPA. In c o n t r a s t , the e l i m i n a t i o n k i n e t i c s of (E,E)-2,3'-diene VPA were s i m i l a r whether or not the b i l e duct was i n t a c t . T h e r e f o r e , the amount of (E,E)-2,3'-diene VPA e x c r e t e d i n t o the b i l e was r e l a t i v e l y small compared to VPA and r e c y c l i n g of the b i l e does not g r e a t l y a f f e c t (E,E)-2,3'-diene VPA e l i m i n a t i o n . The r e s u l t s f o r R E, CI, and V^ are summarized i n t a b l e s 1, 2, and 3, r e s p e c t i v e l y , f o r the 20 and 100 mg/kg dosed animals with t h e i r b i l e duct i n t a c t and cannulated. The AUC, h a l f - l i f e ( t ^ / 2 ) , and weight of each animal f o r the 20 and 100 mg/kg dose with t h e i r b i l e duct i n t a c t and cannulated are r e p o r t e d i n t a b l e 4. A comparison of K E, CI, and Vjj between the 20 and 100 mg/kg dose f o r b i l e duct i n t a c t animals showed no s i g n i f i c a n t d i f f e r e n c e s (p>0.08, p>0.4, and p>0.2, r e s p e c t i v e l y ) u s i n g the Student's t - t e s t . There were a l s o no s i g n i f i c a n t d i f f e r e n c e s (p>0.3, p>0.2, 129 Table 1: Summary of the e l i m i n a t i o n r a t e c o n s t a n t s (K E, m i n - 1 ) of (E,E)-2,3'-diene VPA between b i l e duct i n t a c t and cannulated r a t s given 20 and 100 mg/kg doses i . v . Dose- 20 mg/kg ?AT BILE DUCT INTACT BILE_DUCT_CANNULATED 2 0.0248 0.0222 3 0.0248 0.0221 4 0.0343 0.0187 5 0.0247 0.0229 6 0.0146 0.0171 mean 0.0246+0.007 (S.D.) 0.0206+0.003 No s i g n i f i c a n t d i f f e r e n c e between the two procedures using the p a i r e d t - t e s t (p>0.1, t = 1.32, 0 = 0.1). Rat 1 d i e d d u r i n g the experiment. Dose- 100 mg/kg RAT BILE DUCT INTACT BILE_DUCT_CANNULATED 7 0.0149 0.0151 8 0.0134 0.0160 9 0.0137 0.0213 10 0.0175 0.0214 1 1 0.0258 0.0200 0.0171+0.005 0.0188+0.003 No s i g n i f i c a n t d i f f e r e n c e between the two procedures u s i n g the p a i r e d t - t e s t (p>0.1, t = 0.76, 0 = 0.1). No s i g n i f i c a n t d i f f e r e n c e between doses f o r b i l e duct i n t a c t (p>0.8, t = 1.96, 0 = 0.45) and b i l e duct cannulated (p>0.3, t = 1.05, 0 = 0.45) animals using the Student's t - t e s t . 130 Table 2: Summary of the c l e a r a n c e s (mL/min/kg) of (E , E ) -2,3'-diene VPA between b i l e i n t a c t and cannulated r a t s given 20 and 100 mg/kg doses i . v . Dose- 20 mg/kg RAT BILE DUCT INTACT §1LE_DUCT_CANNULATED 2 23.36 20.10 3 12.35 20.73 4 26.48 17.65 5 30.69 21.74 6 15.61 17.57 mean 21.70+7.6 (S.D.) 19.56+1.9 No s i g n i f i c a n t d i f f e r e n c e between the two procedures u s i n g the p a i r e d t - t e s t (p>0.1, t = 0.6459, 0 = 0.1). Rat 1 d i e d d u r i n g the experiment. Dose- 100 mg/kg RAT BILE DUCT INTACT BILE_DUCT_CANNULATED 7 13.16 9.64 8 15.78 21.28 9 18.85 19.53 10 20.42 18.38 1 1 24.49 15.92 18.54+4.3 16.95+4.5 No s i g n i f i c a n t d i f f e r e n c e between the two procedures u s i n g the p a i r e d t - t e s t (p>0.1, t = 0.684, 0 = 0.1). No s i g n i f i c a n t d i f f e r e n c e between doses f o r b i l e duct i n t a c t (p>0.4, t = 0.81, 0 = 0.45) and b i l e duct cannulated (p>0.2, t = 1.19, 0 = 0.45) animals using the Student's t - t e s t . 131 Table 3: Summary of the volume of d i s t r i b u t i o n s (mL/kg) of (E,E)-2,3'-diene VPA between b i l e i n t a c t and cannulated r a t s given 20 and 100 mg/kg doses i . v . Dose- 20mg/kg RAT BILE_DUCT_INTACT §1LE_DUCT_CANNULATED 2 942.40 904.50 3 498.55 938.25 4 771.00 946.49 5 1241.42 950.92 6 1071.31 1027.00 mean 857.66+280 (S.D.) 953.43+45 No s i g n i f i c a n t d i f f e r e n c e between the two procedures using the p a i r e d t - t e s t (p>0.1, t = -0.4, /3 = 0.1). Rat 1 d i e d d u r i n g the experiment. Dose- I00mg/kg RAT BILE_DUCT_INTACT BILE_DUCT_CANNULATED 7 883.81 640.53 8 1178.57 1328.21 9 1375.77 917.76 10 1169.47 858.34 1 1 947.72 795. 1 0 1111.66+198 907.99+257 No s i g n i f i c a n t d i f f e r e n c e between the two procedures u s i n g the p a i r e d t - t e s t (p>0.05, t = 2.0, 0 = 0.1). No s i g n i f i c a n t d i f f e r e n c e between doses f o r b i l e duct i n t a c t (p>0.2, t = 1.33, 0 = 0.35) and b i l e duct c a n n u l a t e d (p>0.7, t = 0.39, 0 = 0.35) animals using the Student's t - t e s t . 132 Table 4: Summary of area under the curve (AUC, ug min/mL), h a l f - l i v e s ( t j / i , min), and weight (g) of a l l animals given 20 or 100 mg/kg of (E,E)-2,3'-diene VPA with the b i l e duct i n t a c t and cannulated. RATS* AUC -ill WEIGHT 1 + 1277.99 27.63 262 2 856.11 27.95 271 3 1618.92 27.97 206 4 755.34 20. 18 212 5 651.76 28.03 218 6 1281.23 47.56 233 mean 1032.67+406 (S.D.) 30.34+10.2 228+26 7 7598.11 46.54 247 8 6336.67 51.76 269 9 5305.64 50.58 257 10 4897.51 39.69 277 11 4083.26 26.82 225 5644.24+1361 43.08+10.2 255+20 2B 995.12 31.19 325 3B 964.89 31 .37 307 4B 1 132.94 37. 16 227 5B 919.97 30.31 237 6B 1138.11 40.50 257 1030.21+100 34.11+4.5 270+43 7B 10373.81 46.05 277 8B 4699.71 43.26 280 9B 5120.38 32.57 275 1 OB 5441.73 32.37 299 1 1B 6282.03 34.62 257 6383.53+2305 37.77+6.4 278+15 * Rats 1 - 6 = 20 mg/kg b i l e duct i n t a c t 7 - 11 = 100 mg/kg b i l e duct i n t a c t 2B - 6B = 20 mg/kg b i l e duct cannulated 7B - 11B = 100 mg/kg b i l e duct cannulated + Rat 1 d i e d , excluded from the mean c a l c u l a t i o n 133 and p>0.7) using the Student's t - t e s t i n K E, CI, and V d, r e s p e c t i v e l y , between the 20 and 100 mg/kg dose f o r b i l e duct cannulated animals. The data suggest the e l i m i n a t i o n of (E,E)-2,3'-diene VPA i s dose-independent; however, c l a i m s r e g a r d i n g dose dependency or independency cannot be a c c u r a t e l y made based on the study d e s i g n . Since d i f f e r e n t animals were used between doses, the r a t i o n a l e f o r comparing t h e i r pharmacokinetic parameters i s l i m i t e d . A cr o s s - o v e r study of 5 animals u s i n g 3 to 4 d i f f e r e n t dosages should d e t e c t trends i n d i s t r i b u t i o n and e l i m i n a t i o n ; however, the e f f e c t of c o n c e n t r a t i o n on drug e l i m i n a t i o n was not the emphasis of t h i s study. T h e r e f o r e , the e f f e c t of dose on K E, CI, and V^ app a r e n t l y cannot be r a t i o n a l i z e d e a s i l y . Changes i n dose and hence f r e e f r a c t i o n (see p r o t e i n b i n d i n g study) should a f f e c t at l e a s t one or a l l of the re p o r t e d parameters (K E, CI, and V ^ ) . Although there i s a tr e n d f o r K E and CI to decrease and V^ to i n c r e a s e , t h i s change i s not s i g n i f i c a n t p o s s i b l y due to the study design or the small number of animals s t u d i e d . The c o n t r i b u t i o n of (E,E)-2,3'-diene VPA to the c a r r y -over e f f e c t can be r a t i o n a l i z e d by the immediate CNS e f f e c t s observed upon i . v . d o s i n g . A comparison between VPA and (E,E)-2,3'-diene VPA, with r e s p e c t t o the b l o o d - b r a i n -b a r r i e r , may e x p l a i n the CNS e f f e c t s observed with the diene i n r a t s . Hammond et al. (1982) r e p o r t e d that VPA i n the c a t b r a i n was r a p i d l y c l e a r e d with a small volume of 134 d i s t r i b u t i o n and that the brain:plasma r a t i o was low. The brain:plasma r a t i o f o r VPA was found to be lower when compared to other a n t i e p i l e p t i c agents l i k e phenytoin and p h e n o b a r b i t a l (Vajda et al ., 1974). C o r n f o r d et al. (1985) suggested an a c t i v e t r a n s p o r t mechanism was present to remove VPA from the b r a i n which accounted f o r the lower l e v e l s observed. Permeation of molecules a c r o s s the b l o o d - b r a i n - b a r r i e r can occur by two methods: p a s s i v e d i f f u s i o n and a c t i v e t r a n s p o r t . P a s sive d i f f u s i o n depends mainly on l i p o p h i l i c i t y , the degree of i o n i z a t i o n , and the extent of p r o t e i n b i n d i n g . The l i p o p h i l i c i t y of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA have been determined by Acheampong (1985) by HPLC. VPA was the most l i p o p h i l i c and (E,E)-2,3'~ diene VPA the l e a s t l i p o p h i l i c but by a small margin. Thus, i t may be assumed, based on l i p o p h i l i c i t y a l o n e , that a l l three compounds penetrate the b l o o d - b r a i n - b a r r i e r to the same e x t e n t . The pKa of the molecule i s a l s o a c o n t r i b u t i n g f a c t o r . The pKa of VPA i s approximately 4.6 and the pH i n plasma i s 7.4 i . e . , the m a j o r i t y of VPA w i l l be i o n i z e d ( D i C a r l o et al., 1986). Although the pKa's of (E)-2-ene VPA and (E,E)-2,3'-diene VPA are not known, based on the s t r u c t u r a l s i m i l a r i t i e s of these compounds to VPA, the pKa's are probably s i m i l a r . T h e r e f o r e , with s i m i l a r pKa v a l u e s , p e n e t r a t i o n i n t o the b l o o d - b r a i n - b a r r i e r should be equal based on pKa. P r o t e i n b i n d i n g of VPA i n r a t s has been r e p o r t e d to be 63.5% at a c o n c e n t r a t i o n of 75 ug/mL using 135 e q u i l i b r i u m d i a l y s i s (Loscher, 1978). The p r o t e i n b i n d i n g of (E)-2-ene VPA has been r e p o r t e d to be 98% i n humans (Nau et al., 1984). B i n d i n g of (E,E)-2,3'-diene VPA i n r a t s i s g r e a t e r than VPA ( r e p o r t e d l a t e r ) . Since only f r e e drug i s a v a i l a b l e f o r e n t r y i n t o the b l o o d - b r a i n - b a r r i e r , VPA in r a t s should achieve g r e a t e r l e v e l s i n the b r a i n . The f a c t that VPA l e v e l s i n the plasma do not r e f l e c t the l e v e l s i n the r a t b r a i n (Loscher and Nau, 1982) j u s t i f i e s the p o s s i b i l i t y of an a c t i v e t r a n s p o r t mechanism present to remove VPA from the CNS. Since r a t s given (E,E)-2,3'-diene VPA d i s p l a y e d a h i g h degree of s e d a t i o n soon a f t e r d o s i n g — t h i s i s absent upon VPA d o s i n g — the diene must e i t h e r be a c t i v e l y t r a n s p o r t e d i n t o or perhaps not a c t i v e l y t r a n s p o r t e d out of the CNS to the same extent as VPA. S i n c e Acheampong (1985) has shown 2,3'-diene VPA isomers to possess a n t i c o n v u l s a n t a c t i v i t y i n mice, i t i s apparent that the diene i s present i n the CNS. The same t e s t f o r a n t i c o n v u l s a n t a c t i v i t y using p e n t y l e n e t e t r a z o l e -induced s e i z u r e s should be performed i n r a t s u s i n g (E,E)-2,3'-diene VPA. As p r e v i o u s l y s t a t e d , b r a i n l e v e l s of (E,E)-2,3'-diene VPA a f t e r i . v . dosing of the diene should be determined. These f u r t h e r experiments would e x p l a i n the observed CNS e f f e c t s and the pharmacokinetic r e s u l t s a s s o c i a t e d with (E,E)-2,3'-diene VPA. 136 4. In Vitro Plasma P r o t e i n B i n d i n g The plasma samples f o r the p r o t e i n b i n d i n g study were prepared using a s i n g l e stock s o l u t i o n of (E,E)-2,3'-diene VPA i n water. T h i s r e s u l t e d i n the d i l u t i o n of p r o t e i n s as the c o n c e n t r a t i o n of (E,E)-2,3'-diene VPA i n c r e a s e d . The magnitude of d i l u t i o n was 1, 3.3, 10, and 20% which correspond to c o n c e n t r a t i o n s of 30, 100, 300, and 600 ug/mL, r e s p e c t i v e l y . The d i l u t i o n e f f e c t can be c o r r e c t e d by assuming the diene only binds to albumin and that the albumin c o n c e n t r a t i o n present i n r a t s i s 3.7 g/dL (Canadian C o u n c i l on Animal Care, 1980). A 3 mL plasma sample w i l l c o n t a i n 111 mg of albumin a v a i l a b l e f o r b i n d i n g (E,E)-2,3'-diene VPA. T h e r e f o r e , by knowing the observed amount of (E,E)-2,3'-diene VPA bound and the a c t u a l amount of albumin present f o r each c o n c e n t r a t i o n , a c o r r e c t e d value f o r the bound diene can be determined f o r 111 mg of albumin. The b i n d i n g of diene to the u l t r a f i l t r a t i o n apparatus and the membrane was f i r s t examined and was found to be 5.18% ( t a b l e 5). The c o n c e n t r a t i o n of f r e e and percent bound of (E,E)-2,3'-diene VPA were determined at plasma c o n c e n t r a t i o n s 30, 100, 300, and 600 ug/mL and the r e s u l t s are r e p o r t e d i n t a b l e 6 with and without the c o r r e c t i o n f o r p r o t e i n d i l u t i o n . The in vitro data suggest that as the (E,E)-2,3'-diene VPA c o n c e n t r a t i o n i n c r e a s e s , the amount of f r e e drug present i n plasma i n c r e a s e s . The diene appears to be h i g h l y 137 Table 5: Bi n d i n g of (E,E)-2,3'-diene VPA to the u l t r a f i l t r a t i o n apparatus and membrane at 25°C CONCENTRATION IN_WATER *FILTRATE_(ug/mL) *TOTAL__(ug/mL) 30 ug/mL 100 ug/mL 600 ug/mL * mean (S.D.) 27.13+1.76 87.09+4.79 561.97+19.88 29.75+1.08 92.04+3.80 569.62+2.31 8.81 5.38 1 .34 5.18 + 3.74 Table 6: P r o t e i n b i n d i n g of (E,E)-2,3'-diene VPA to r a t plasma u s i n g u l t r a f i l t r a t i o n a t 25°C. CONCENTRATION 30 ug/mL 100 ug/mL 300 ug/mL 600 ug/mL f r e e (ug/mL) t o t a l (ug/mL) bound (%) f r e e (ug/mL) t o t a l (ug/mL) bound (%) f r e e (ug/mL) t o t a l (ug/mL) bound (%) f r e e (ug/mL) t o t a l (ug/mL) bound (%) OBSERVED ACTUAL+ *2.42(2.301+0.39) 27.362 91.16 92.1 *23.34(22.19+1.42) 90.221 74.13 76.7 * 173. 16( 164.63 + 5.44) 264.051 34.42 38.3 *433.67(412.31+21 . 1 1 ) 562.784 22.96 28.7 * c o r r e c t e d f o r drug b i n d i n g to the membrane/apparatus mean (S.D.) + c o r r e c t e d f o r the d i l u t i o n of p r o t e i n 138 bound at 30 ug/mL (92.1%) so that i n c r e a s i n g the c o n c e n t r a t i o n of (E,E)-2,3'-diene VPA s a t u r a t e s the b i n d i n g s i t e s on the p r o t e i n s a l l o w i n g f o r more drug to remain unbound i n plasma. P r o t e i n b i n d i n g of VPA has a l s o been r e p o r t e d t o be dose-dependent i n p a t i e n t s on c h r o n i c VPA therapy (Riva et al., 1982). Loscher (1978) has r e p o r t e d the same t r e n d f o r VPA i n dogs. Rats, however, d i d not e x h i b i t dose-dependency at 105 and 190 ug/mL (60.6 and 57.3% bound, r e s p e c t i v e l y ) ( L o s c h e r , 1978). In vitro p r o t e i n b i n d i n g of (E,E)-2,3'-diene VPA appears to be g r e a t e r than VPA and may be a c o n t r i b u t i n g f a c t o r i n d e l a y i n g the diene e l i m i n a t i o n i n the r a t . However, the longer h a l f - l i f e of (E,E)-2,3'-diene VPA compared to VPA may be due to the l a r g e r volume of d i s t r i b u t i o n of the d i e n e . Because of the i n i t i a l d i l u t i n g e f f e c t of the plasma p r o t e i n s , the b i n d i n g r e s u l t s may not be e n t i r e l y a c curate s i n c e two assumptions were made, that (E,E)-2,3'-diene VPA o n l y binds albumin and b i n d i n g to albumin at each c o n c e n t r a t i o n i s l i n e a r . Although the trend f o r f r e e f r a c t i o n t o i n c r e a s e with an i n c r e a s e i n c o n c e n t r a t i o n i s a c c u r a t e the a c t u a l values may not be. T h e r e f o r e , the study should be repeated with the d i l u t i o n e f f e c t avoided. F u r t h e r s t u d i e s w i l l i n c l u d e comparisons between VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA with r e s p e c t to p r o t e i n b i n d i n g , e l i m i n a t i o n , and CNS d i s t r i b u t i o n . I f l e v e l s of (E,E)-2,3'-diene VPA i n the b r a i n prove to be 139 higher than f o r VPA, t h i s c o u l d p a r t i a l l y e x p l a i n the a p p a r e n t l y g r e a t e r h a l f - l i f e found f o r the diene. 140 SUMMARY AND CONCLUSIONS 1. The VPA m e t a b o l i t e (E,E)-2,3'-diene VPA was s y n t h e s i z e d i n r e l a t i v e l y pure amounts to be used as a r e f e r e n c e f o r i t s i d e n t i f i c a t i o n i n u r i n e , b i l e , and plasma samples and fo r metabolic and pharmacokinetic s t u d i e s i n r a t s . Two methods were used to s y n t h e s i z e (E,E)-2,3'-diene VPA with the m o d i f i e d method of Acheampong and Abbott (1985) a f f o r d i n g the gr e a t e r y i e l d . The second method, u s i n g a hydride a b s t r a c t o r , appeared to r e s u l t i n g r e a t e r s t e r e o s p e c i f i c i t y but i s o l a t i n g (E,E)-2,3'-diene VPA was not p o s s i b l e . Future work w i l l focus on the second method of s y n t h e s i s with the i n t e n t i o n of improving the y i e l d of the f i n a l product. 2. F i v e other VPA m e t a b o l i t e s were s y n t h e s i z e d to be used as r e f e r e n c e compounds f o r t h e i r i d e n t i f i c a t i o n i n u r i n e , plasma, and b i l e and as s t a r t i n g m a t e r i a l f o r s y n t h e s i z i n g other VPA m e t a b o l i t e s . The s y n t h e s i s of the m e t a b o l i t e ( E ) -2-ene VPA was attempted by three methods with only two of the procedures l e a d i n g to product. The two s u c c e s s f u l methods were that of Acheampong (1985) and a new method us i n g a l l y l i c hydride a b s t r a c t i o n . The l a t t e r method w i l l r e q u i r e f u r t h e r work t o i s o l a t e the product from the s t a r t i n g m a t e r i a l . 141 3. The metabolism of (E,E)-2,3'-diene VPA was s t u d i e d i n male Wistar r a t s . U r i ne and b i l e samples were c o l l e c t e d over 24 hours a f t e r a s i n g l e i . p . dose of 100 mg/kg of (E,E)-2,3'-diene VPA was a d m i n i s t e r e d . 4. GCMS a n a l y s i s of the u r i n e and b i l e samples r e v e a l e d the presence of s e v e r a l m e t a b o l i t e s . The confirmed m e t a b o l i t e s u s i n g s y n t h e t i c r e f e r e n c e samples were (E)-2-ene VPA, (E)-3-ene VPA, and VPA a l l a r i s i n g from metabolic r e d u c t i o n of the ad m i n i s t e r e d (E,E)-2,3'-diene VPA. The metabolic r e d u c t i o n of (E,E)-2,3'-diene VPA co m p l i c a t e s an a l r e a d y complex metabolic scheme f o r VPA. The search t o i d e n t i f y the o x i d i z e d m e t a b o l i t e s of (E,E)-2,3'-diene VPA w i l l c o n t i n u e . 5. The pharmacokinetics of (E,E)-2,3'-diene VPA were performed u s i n g two dose l e v e l s , 20 and 100 mg/kg i . v . i n r a t s , with and without the b i l e duct i n t a c t . The pharmacokinetic r e s u l t s r e v e a l e d that (E,E)-2,3'-diene VPA was not e x t e n s i v e l y r e c i r c u l a t e d v i a the e n t e r o h e p a t i c c y c l e . The l o g (E,E)-2,3'-diene VPA c o n c e n t r a t i o n vs time p l o t appeared to be s i m i l a r f o r the b i l e duct i n t a c t and cannulated animal i n both dosage groups. The e l i m i n a t i o n r a t e c o n s t a n t , c l e a r a n c e , and volume of d i s t r i b u t i o n d i d not d i f f e r s i g n i f i c a n t l y between r a t s with and without the b i l e duct i n t a c t or between dosages. T h e r e f o r e , (E,E)-2,3'-diene VPA appears to d i s p l a y dose-independent e l i m i n a t i o n 142 i n r a t s however, more e x t e n s i v e pharmacokinetic s t u d i e s are r e q u i r e d to c o n f i r m t h i s r e s u l t . 6. In vitro plasma p r o t e i n b i n d i n g of (E,E)-2,3'-diene VPA was performed at four d i f f e r e n t c o n c e n t r a t i o n s using u l t r a f i l t r a t i o n . In vitro plasma p r o t e i n b i n d i n g f o r (E,E)-2,3'-diene VPA was dose-dependent between 30 and 600 ug/mL in r a t s . 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V a l p r o i c a c i d : i n t e r a c t i o n with other a n t i c o n v u l s a n t drugs. N e u r o l . , 28, 892 (1978). Zimmerman, H.J. and Ishak, K.G., V a l p r o a t e induced h e p a t i c i n j u r y : Analyses of 23 f a t a l c a s es. H e p a t o l . , 2, 591 (1982). 153 APPENDIX IR AND NMR SPECTRA OF SYNTHESIZED COMPOUNDS 154 il: • I .. .U . i : . l - i H.J.hl'liL:: - . . i l X u : ! . • 0 L . , i . .ML. • ' is.. . M l * . ; : i l "! .1:1 . IL '.[[, ' ! J Li I 11 i l l 1 j : • ' I! JIJ l i i i n l . ! : . i ; . . ! : . . . ! . J.tUO 3U.IU aOOO 2SUO 2000 2uoO tttOO HiOO 14UU i^uO IOOO SOU 0 2 * W.p>«nun>ii«l I n f r a r e d spectrum of (Z)-2-pentenoic a c i d (neat f i l m on sodium c h l o r i d e d i s k s ) MHz proton NMR of (Z)-2-pentenoic a c i d i n CDC1 80 MHz proton NMR of e t h y l (Z)-2-pentenoate i n C D C I 3 IS 10 s '"T~ 35 10 5 7J 30 IS Kill, m 0 IHlAm • proton NMR of e t h y l 2-n-propyl-3-hydroxy-4-pentenoate i n CDCI3 MHz p r o t o n NMR of e t h y l 2 - ( 1 ' - h y d r o x y p r o p y l ) - ( E ) - 3 - p e n t e n o a t e i n CDC1 300 MHz proton NMR of e t h y l 2-n-propy1-(E)-3-pentenoate i n CDC1 

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