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Quantitative structure-anticonvulsant activity studies of valproic acid analogues Acheampong, Andrew Adu 1985

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QUANTITATIVE STRUCTURE-ANTICONVULSANT ACTIVITY STUDIES OF VALPROIC ACID ANALOGUES By ANDREW ADU ACHEAMPONG B . S c , The U n i v e r s i t y of Science and Tech., Ghana, 1978 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED AS PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty 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 required standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1985 ©  Andrew Adu Acheampong, 1985  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  f k c ^ ^ q . cg_vAJ>  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  Q cfcMaJtv"  CU  \ \ Q  f  i ^ J ^ n i_g_J>  ABSTRACT  Valproic a c i d (2-propylpentanoic  a c i d ) i s an a n t i e p i l e p t i c drug  w i d e l y used f o r t r e a t m e n t of absence s e i z u r e s .  V a l p r o i c a c i d has a  unique c h e m i c a l s t r u c t u r e which does not c o n t a i n the i m i d e s t r u c t u r e found i n most conventional a n t i e p i l e p t i c drugs. antagonism of p e n t y l e n e t e t r a z o l - i n d u c e d  An i n vivo study of the  c l o n i c s e i z u r e s by  alkyl-  substituted c a r b o x y l i c acids and t e t r a z o l e s was of i n t e r e s t owing to the known b i o i s o s t e r i s m between the c a r b o x y l i c and the t e t r a z o l y l moiety. The main o b j e c t i v e of t h i s study was  to i n v e s t i g a t e the r o l e played  by  the l i p o p h i l i c i t y , the e l e c t r o n i c properties and the s t e r i c influence of compounds on t h e i r anticonvulsant potency. Quantitative s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of the a l i p h a t i c and a l i c y c l i c substituted c a r b o x y l i c acids and t e t r a z o l e s have been performed using the Hansch l i n e a r free-energy  r e l a t i o n s h i p s model.  The  study  proceeded by s y n t h e s i s of compounds u s i n g known p r o c e d u r e s . The d i u n s a t u r a t e d d e r i v a t i v e s of v a l p r o i c a c i d , 2 - [ ( E ) - l ' - p r o p e n y l ] - ( E ) - 2 pentenoic  a c i d and  2-[(Z)-l'-propenyl]-(E)-2-pentenoic  prepared v i a a s t e r e o s e l e c t i v e s y n t h e t i c r o u t e .  acid  were  The s y n t h e s i z e d d i -  unsaturated acids were used i n i d e n t i f i c a t i o n of the major diunsaturated metabolite of v a l p r o i c a c i d as 2-[(E)-l'-propenyl]-(E)-2-pentenoic  acid.  The anticonvulsant potency of t e s t compounds was determined i n mice (CD1 s t r a i n , 20-32g) by the standard subcutaneous p e n t y l e n e t e t r a z o l e seizure threshold t e s t .  The pentylenetetrazole c l o n i c seizure t e s t  was  found to be more s e n s i t i v e to s t r u c t u r a l e f f e c t s than the  pentylene-  t e t r a z o l e m o r t a l i t y assay. The l i p o p h i l i c i t y ( o c t a n o l - w a t e r  partition  c o e f f i c i e n t ) of compounds was  determined i n d i r e c t l y by reversed phase  l i q u i d chromatography employing an octadecylsilane column (Hypersil ODS) ii  and mobile phase as 70% methanol : 30% phosphate buffer  (pH 3.5).  The  e l e c t r o n i c character of the compounds was monitored by the apparent acid i o n i z a t i o n constant raethanol-water The  obtained  from p o t e n t i o m e t r i c t i t r a t i o n  system.  ED5Q of 0.70 mmol/kg found f o r v a l p r o i c a c i d was  literature  i n 10%  values.  5-Heptyltetrazole was  similar  to  found to be the most potent  compound i n the s e r i e s of analogues s t u d i e d .  The  t e t r a z o l e group gave a low c o r r e l a t i o n ( r = 0.63)  c a r b o x y l i c plus  between the anticon-  vulsant potency and a l i n e a r combination of l i p o p h i l i c i t y and apparent i o n i z a t i o n constant. the anticonvulsant  However, i n the s e r i e s of a c t i v e c a r b o x y l i c a c i d s , activity  was  noted to be  significantly  correlated  with l i p o p h i l i c i t y and apparent i o n i z a t i o n constant (r = 0.91). The  usefulness of the e l e c t r o n i c parameters, acid i o n i z a t i o n con-  stant and  dipole moment, were explored  substituted Addition  anticonvulsant  of the  significantly  compounds with  different  polar  dipole moment term to the l i p o p h i l i c i t y  alkyl-  moieties.  term led to  better c o r r e l a t i o n s (r = 0.81) as compared to that with an  added pKa term. dipole  i n an extensive set of  The negative dependency of anticonvulsant a c t i v i t y  moment supported  previous  findings i n studies  of  on  1,4-benzo-  diazepines and phenyl-substituted anticonvulsant compounds. There were some exceptions activity succinamic  on l i p o p h i l i c i t y  and  to the  dependence of  anticonvulsant  d i p o l e moment or pKa.  N,N-dibutyl-  acid showed convulsant  lack of a c t i v i t y  properties at sublethal doses.  of c y c l o h e x y l a c e t i c acid and  5-cyclohexylmethyltetra-  z o l e , i n comparison to the a c t i v e 1-methylcyclohexanecarboxylic has  some pharmacological  molecular analogues.  specificity The  significance.  i n the  acid,  I t shows a c e r t a i n degree of  anticonvulsant  cyclohexylmethyl  The  conformation  a c t i o n of was  valproic acid  suggested, from  a  proposed model, to be l e s s e f f e c t i v e i n hydrophobic binding due to a s t e r i c e f f e c t at a s t e r e o s e l e c t i v e p o s i t i o n the GABA receptor complex.  on the hydrophobic s i t e of  Thus i t can be concluded that while l i p o -  p h i l i c i t y governed access to s i t e s of a c t i o n , the dependence of a c t i v i t y on  the polar  convulsants  character  provided  binding s i t e are met.  may  explain the diverse  that the s t e r i c  structures of  requirements of the hydrophobic  S t e r i c e f f e c t s may  lead to i n a c t i v i t y or even  convulsant properties of a l k y l - s u b s t i t u t e d compounds.  iv  anti-  TABLE OF CONTENTS  Pa  Abstract  R  e  i i v  Table of Contents L i s t of Tables  xi  L i s t of Figures  xiv  L i s t of Schemes  xvi  Symbols and Abbreviations  xvii  Acknowledgements  xix  INTRODUCTION  1  S p e c i f i c Objectives  6  LITERATURE SURVEY 1.  11  C l i n i c a l Use and Anticonvulsant Properties of V a l p r o i c Acid (VPA)  2.  Pharmacological  11 Testing  11  a. b.  Experimental Models of Epilepsy Mechanism of Action of Pentylenetetrazole  11 13  c.  Pharmacokinetics of Pentylenetetrazole  15  3.  Chemistry and Physicochemical  4.  Mechanism of Action of V a l p r o i c Acid  16  5.  Studies on Anticonvulsant A c t i v i t y of V a l p r o i c Acid Analogues General S t r u c t u r e - A c t i v i t y Relationships of A n t i e p i l e p t i c  19  Drugs  23  7.  S t r u c t u r a l S p e c i f i c i t y of Anticonvulsants  27  8.  Pharmacokinetics of V a l p r o i c Acid  33  a. b. c.  33 34  6.  Properties of V a l p r o i c Acid  Human Rodents K i n e t i c s of V a l p r o i c Acid i n the Central Nervous System v  16  34  TABLE OF CONTENTS (Contd) Page 9.  Metabolism of Valproic Acid  35  10.  T o x i c i t y of Valproic Acid  37  11.  A Quantitative S t r u c t u r e - A c t i v i t y Model  37  12.  Physicochemical Parameters used i n Quantitative StructureA c t i v i t y Relationships  40  Hydrophobic Parameters  41  13.  a. b. c.  Determination of L i p o p h i l i c i t y by the Shake-Flask Procedure  41  High-Performance Liquid Chromatographic Determination of L i p o p h i l i c i t y  42  Determination of L i p o p h i l i c i t y using Substituent Constants  45  EXPERIMENTAL  47  A.  Chemicals and M a t e r i a l s  47  1.  Synthesis  47  2.  Thin Layer Chromatrography  48  3.  High-Performance Liquid Chromatography  48  4.  Potentiometric T i t r i m e t r y  49  5.  Gas Chromatography-Mass Spectrometry  49  6.  Pharmacological Testing  50  B.  Instrumentation  50  1.  Nuclear Magnetic Resonance Spectrometry  50  2.  I n f r a Red Spectrometry  50  3.  U l t r a v i o l e t Spectrometry  51  4.  Gas Chromatography Mass Spectrometry  51  a. b.  51 51  Packed Column C a p i l l a r y Column  vi  TABLE OF CONTENTS (Contd) Page C.  Synthesis of A l k y l Carboxylic A c i d s , Tetrazoles and Succinaraic Acids  52  1.  Synthesis of Alpha-Substituted A l i p h a t i c Acids  52  a. b. c.  Synthesis of 2-Butylhexanoic Acid Synthesis of V a l p r o i c Acid Synthesis of 2-Propyl-(E)-2-Pentenoic  52 53 53  d.  Synthesis of 2-Propyl-4-0xopentanoic Acid  2.  3.  4.  5.  6.  Acid  55  Synthesis of Alpha, Alpha-Disubstituted A l i p h a t i c Acids  56  a. Synthesis of 2,2-Dimethylbutyric Acid b. Synthesis of 2,2-Dimethylvaleric Acid Synthesis of Beta-Substituted A l i p h a t i c Acids a. Synthesis of 3-Ethylpentanoic Acid b. Synthesis of Cyclohexylacetic Acid c. Synthesis of 3-Methylvaleric Acid d. Synthesis of 3-Methylhexanoic Acid  56 57 57 57 59 59 60  Synthesis of 5 - A l k y l t e t r a z o l e s  60  a. b. c.  60 61 62  Synthesis of 5-Isoamyltetrazole Synthesis of 5-Cyclohexylmethyltetrazole Synthesis of 5-Heptyltetrazole  Synthesis of Succinamic Acids  63  a.  Synthesis of N,N-Diethylsuccinamic  Acid  63  b.  Synthesis of N,N-Dibutylsuccinamic  Acid  63  Synthesis of Diunsaturated Analogues of V a l p r o i c Acid la. lb. II  Synthesis of E t h y l 2-(l'-Hydroxypropyl) -3-Pentenoate from E t h y l (E)-2-Pentenoate Synthesis of E t h y l 2-(l'-Hydroxypropyl) -3-Pentenoate from E t h y l (Z)-2-Pentenoate Dehydration of E t h y l -3-Pentenoate a. b. c.  III IV  64 65  2-(l'-Hydroxypropyl)  Phosphorus Pentoxide Toluenesulfonyl C h l o r i d e - P y r i d i n e Methanesulfonyl Chloride-Potassium Hydride  Semi-Preparative Argentation Thin Layer Chromatography In Vivo Metabolism-Isolation Procedure vii  64  66 66 67 67 69 70  TABLE OF CONTENTS (Contd) Page.  D.  V  D e r i v a t i z a t i o n of Acids  70  VI  Photochemical Isomerization  71  VII  C a p i l l a r y Gas Chromatography Mass Spectroraetric Resolution of Isomeric 2-(l'-Propenyl)-2-Pentenoic Acid and 2-Propyl-2,4-Pentadienoic Acid  71  Subcutaneous Pentylenetetrazole Seizure Threshold Test  72  1.  Animals  72  2.  Drugs  72  3.  Drug Solutions  73  4.  Experimental Procedure  73  a.  E.  F. G.  C h a r a c t e r i z a t i o n of Pentylenetetrazole Seizures (85 mg/kg dose)  73  b.  Antagonism of Pentylenetetrazole Clonic Seizures  74  c.  M o r t a l i t y Test  75  d.  Toxic E f f e c t s of Drugs  75  e. Convulsant A c t i v i t y Test High Performance L i q u i d Chromatographic Method f o r Determination of Octanol-Water P a r t i t i o n C o e f f i c i e n t  75  1.  Instrumentation  76  2.  Column  76  3.  Eluents  76  4.  Compounds  76  5.  Sample Preparation  77  6.  Retention Time Measurements  77  7.  Column Void Time  78  76  Determination of Octanol-Water P a r t i t i o n C o e f f i c i e n t s by the Shake-Flask Procedure Determination of Apparent I o n i z a t i o n Constants (pKa) Potentiometric T i t r a t i o n  viii  78 by 80  TABLE OF CONTENTS (Contd) Page RESULTS AND DISCUSSION  82  A.  Chemistry  82  1.  Alpha-Alkyl Substituted A l i p h a t i c Acids  82  2.  Alpha-Alpha D i a l k y l Substituted A l i p h a t i c Acids  82  3.  Alpha-Alkyl Substituted A l i p h a t i c Acids with F u n c t i o n a l i t y i n the Carbon Chain  82  4.  Beta-Substituted Carboxylic Acids  86  5.  N,N-Dialkylsuccinamic  86  6.  5-Alkyltetrazoles  90  7.  Diunsaturated Derivatives of V a l p r o i c Acid Synthesis and Metabolism Study  92  8.  a.  Attempted Synthesis of 2-(l'-Propenyl) -3-Pentenoic Acid  94  A l d o l Condensation Reactions Toward Synthesis of 2-(l'Propenyl)-2-Pentenoic Acid  96  c.  Dehydration of B-Hydroxyunsaturated Esters  99  d.  Photochemical Isomerization  103  e.  GC E l u t i o n Order i n Gas Chromatography Mass Spectrometric Analysis  104  f.  Argentation Thin Layer Chromatography  104  g.  I d e n t i f i c a t i o n of the Major and Minor Diunsaturated Metabolites of V a l p r o i c Acid  109  b.  B.  Acids  High-Performance L i q u i d Chromatographic Determination of Lipophilicity  113  1.  Assay Method  113  2.  Void Time and Retention Mechanism i n Reversed-Phase L i q u i d Chromatography  122  3.  Eluent E f f e c t s on Capacity Factor  125  4.  High-Performance L i q u i d Chromatographic Log P (OctanolWater P a r t i t i o n C o e f f i c i e n t ) Values of V a l p r o i c Acid and Analogues  129  ix  TABLE OF CONTENTS (Contd) Page 5.  Comparison of L i p o p h i l i c i t y from Reversed Phase L i q u i d Chromatography and Other Methods  133  Intramolecular Bonding E f f e c t s of Amic Acids  143  6. C.  E l e c t r o n i c S t r u c t u r a l Effects-Determination of Apparent I o n i z a t i o n Constants  147  1.  A n a l y t i c a l Method  147  2.  E f f e c t of S t r u c t u r a l C o n s t i t u t i o n on I o n i z a t i o n Constants  D.  E.  Pharmacological  156 Studies  159  1.  Evaluation of Anticonvulsant A c t i v i t y  159  2.  T o x i c i t y of Compounds  168  S t r u c t u r e - A c t i v i t y Relationships  170  1. 2.  170  Quantitative S t r u c t u r e - A c t i v i t y Relationships S t r u c t u r a l Features that Enhance or Diminish Anticonvulsant A c t i v i t y  183  a.  A l i p h a t i c Substituents  183  b.  A l i c y c l i c Substituents  184  c.  E f f e c t s of a Polar F u n c t i o n a l i t y i n the A l k y l Chain  187  d.  Model to Show S e l e c t i v e E f f e c t s of A l i p h a t i c and A l i c y c l i c Substituents at the Hydrophobic Binding Site  189  SUMMARY AND CONCLUSIONS  197  REFERENCES  203  APPENDIX  219  x  LIST OF TABLES  Composition and chromatographic data of a mixture of synthesized isomeric dienoates NMR (400 MHz) data f o r diene VPA e t h y l esters Retention times of reference compounds using unbuffered mobile phase (CH3CN/H20) E f f e c t of a d d i t i o n of phosphate buffer (pH 3.5), i n mobile phase (Me0H/H20), on the retention times of reference compounds Retention times of seven reference compounds at d i f f e r e n t percentages of methanol i n the mobile phase (MeOH/O.OlM NaH 2 P0 4 ) Retention times of seven reference compounds a t d i f f e r e n t flow rates a) i n 70% MeOH : 30% 0.01M NaH 2 P0 4 mobile phase b) i n 60% MeOH : 40% 0.01M NaH 2 P0 4 mobile phase Retention times of seven reference compounds at d i f f e r e n t percentages of a c e t o n i t r i l e i n the mobile phase Comparison of void times ( t ) determined from a) i n j e c t i o n of methanol ana b) dead time i t e r a t i o n of the r e t e n t i o n times of the homologous s e r i e s from C3H7COOH to CyH-^COOH C o r r e l a t i o n of l o g k' and l o g P Q / W f o r seven reference compounds at various compositions of the mobile phase (MeOH/O.OlM NaH 2 P0 4 ) C o r r e l a t i o n of l o g k' and l o g P Q / W f o r seven reference compounds at various compositions of the mobile phase (CH3CN/O.OIM NaH 2 P0 4 ) Summary of l i n e a r regression parameters f o r l o g P versus k' HPLC method f o r determining the l i p o p h i l i c i t i e s of the a c i d i c compounds using 70% MeOH : 30% 0.01M NaH 2 P0 4 as mobile phase HPLC method f o r determining the l i p o p h i l i c i t i e s of the a c i d i c compounds using 50% CH3CN : 50% 0.01M NaH 2 P0 4 as mobile phase C a l i b r a t i o n curve data of t r i m e t h y l a c e t i c acid i n 0.1N HC1 C a l i b r a t i o n curve data of N,N-dibutylsuccinamic acid i n 0.1N HC1 xi  LIST OF TABLES (Contd) Page 16  C a l i b r a t i o n curve data of 5-isoamyltetrazole i n 0.1N HC1  136  17  C a l i b r a t i o n curve data of 5-cyclohexylmethyltetrazole i n 0.1N HC1  137  18  Octanol-water p a r t i t i o n c o e f f i c i e n t s of selected compounds determined by the shake-flask procedure  138  19  Hansch u-values used i n c a l c u l a t i n g log P Q / W  139  Rekker's fragmental values ( f ) used i n c a l c u l a t i n g log o/w  140  20 21  P  L i p o p h i l i c i t i e s (log P Q / W ) by d i f f e r e n t methods  of the a c i d i c compounds obtained 141  22  Determination of the i o n i z a t i o n constant of a monobasic a c i d , v a l p r o i c a c i d , i n 10% MeOH  148  23  Determination of the i o n i z a t i o n constant of 5-isoamylt e t r a z o l e i n 10% MeOH  149  24  Determination of the i o n i z a t i o n constant of t r i m e t h y l a c e t i c acid i n 10% MeOH  150  25  Determination of the i o n i z a t i o n constant of d i b u t y l a c e t i c acid i n 50% MeOH  151  26  Determination of the i o n i z a t i o n constant of N,N-diethylsuccinamic acid i n 50% MeOH  152  27  Determination of the i o n i z a t i o n constant of 5-cyclohexylmethyltetrazole i n 50% MeOH  153  28  Determination of the i o n i z a t i o n constant of N,N-dibutylsuccinamic acid i n 50% MeOH  154  29  pka values of v a l p r o i c acid and analogues  155  30  Polar e f f e c t of s u b s t i t u t i o n i n an a l i p h a t i c s e r i e s  158  31  Protection against PTZ-induced seizures i n mice by v a l p r o i c acid and i t s analogues  161  32  Anticonvulsant potency of v a l p r o i c acid and i t s analogues against the c l o n i c phase of PTZ-induced seizures i n mice  164  33  Anticonvulsant potency of v a l p r o i c acid and i t s analogues against the c l o n i c phase of PTZ-induced seizures i n mice. Dose range of a c i d s , 0.2-2.0 mmol/kg  165  xii  LIST OF TABLES (Contd) Page 34  Anticonvulsant a c t i v i t y of VPA and i t s analogues on the threshold of PTZ-induced seizures determined by protection against c l o n i c seizures and by percent m o r t a l i t y i n mice  167  35  Observed t o x i c e f f e c t s of t e s t compounds i n mice  169  36  B i o l o g i c a l data and physicochemical properties of compounds  171  37  Equations obtained c o r r e l a t i n g the anti-PTZ e f f e c t of v a l p r o i c acid and analogues with t h e i r physicochemical parameters  174  38  Equations obtained c o r r e l a t i n g the anti-PTZ e f f e c t s of VPA and analogues (excluding 5-heptyltetrazole) with t h e i r physicochemical parameters  175  39  Anticonvulsant a c t i v i t y of various drugs against c l o n i c seizures induced by PTZ ( s . c . 85 mg/kg) i n mice and t h e i r physicochemical constants  178  40  Equations c o r r e l a t i n g anti-PTZ a c t i v i t y and physicochemical properties of a l k y l - s u b s t i t u t e d anticonvulsants  179  xiii  LIST OF FIGURES Page 1  Chemical structures of v a l p r o i c acid and analogues  2  Chemical structures of t r a d i t i o n a l a n t i e p i l e p t i c drugs  24  3  S t r u c t u r a l l y - r e l a t e d convulsant and anticonvulsant barbiturates  28  Model of GABA-Benzodiazepine receptor-chloride complex .  32  4  7  ionophore  5  Metabolic pathways of v a l p r o i c acid  36  6  NMR spectra of N,N-diethylsuccinamic acid and N,N-dibutylsuccinamic acid  91  7  Stereoisomers i n a l k y l a t i o n and a l d o l reactions of ester enolates  100  8  C a p i l l a r y GCMS separation of t-BDMS esters of seven isomeric dienoic acid mixture derived from dehydration reaction with phosphorus pentoxide a) Before UV i r r a d i a t i o n b) After 6hr UV i r r a d i a t i o n  101  9  a.  Mass chromatograms of t-BDMS esters of four isomeric dienoic acid mixture derived from dehydration with p-toluenesulfonyl chloride  102  b.  Diene-VPA metabolites i n urine  extract  10  GCMS analysis of dienoates eluted from TLC p l a t e s , using a 3% Dexsil 300 packed column  107  11  Chemical structures of diunsaturated d e r i v a t i v e s of v a l p r o i c acid investigated as p o t e n t i a l metabolites of v a l p r o i c acid  110  12  C a p i l l a r y GCMS separation  112  of the TMS d e r i v a t i v e s of  a.  A mixture of 2-propyl-2,4-pentadienoic acid and 2-(1'-propenyl)-2-pentenoic acid  b.  Diene-VPA metabolites i n urine  extract  13  UV absorption spectra of four a c i d i c compounds  114  14  Superimposed HPLC chromatograms of a c i d i c compounds using an unbuffered mobile phase (20% CH3CN : 80% H 2 0)  117  xiv  LIST OF FIGURES (Contd) Page 15  Superimposed HPLC chromatograms of 23 a c i d i c compounds using 70% MeOH : 30% 0.01M NaH 2 P0 4 as mobile phase  132  16  Dose-response curves of v a l p r o i c acid and analogues using the subcutaneous pentylenetetrazole seizure threshold t e s t i n mice  163  17  Active and i n a c t i v e a l i c y c l i c and a l i c y c l i c a l k y l - s u b s t i t u t e d 186 compounds  18  Conformations of (a) GABA, (b) v a l p r o i c acid amide and (c) 3-ethylpentanoic acid amide  190  19  Model of pharmacophoric s t r u c t u r a l features i n carboxylic acids and t e t r a z o l e s  193  xv  LIST OF SCHEMES Page 1  Synthetic pathway for alpha-substituted a l i p h a t i c acids  83  2  Synthetic route for alpha, alpha-disubstituted a l i p h a t i c acids  84  3  Outline for synthesis of 2-propyl-(E)-2-pentenoic  85  4  Synthetic sequence for preparation of 2-propyl-4-oxopentanoic acid (4-Keto VPA)  87  5  Pathways for synthesis of the beta-substituted carbo x y l i c acids  88  6  Synthetic route for the succinamic acids  89  7  Synthetic pathway for 5 - a l k y l t e t r a z o l e s  93  8  Outline for synthesis of dienol ether used i n attempted preparation of 2-(1'-propenyl)-3-pentenoic acid  95  9  Stereoselective synthetic routes for preparation of 2-(1'-propenyl)-2-pentenoic acid  97  10  K i n e t i c model proposed by some i n v e s t i g a t o r s (183,184) f o r the h y d r o l y s i s of maleamic acids  xvi  acid  145  SYMBOLS AND ABBREVIATIONS P  octanol-water p a r t i t i o n c o e f f i c i e n t  pKa  negative logarithm of the apparent acid i o n i z a t i o n constant e f f e c t i v e dose i n 50% of mice  TD50  t o x i c dose i n 50% of mice  k'  capacity factor retention time of retained solute  fc  o  i.p.  e l u t i o n time of unretained solute intraperitoneal  s.c.  subcutaneous  r  correlation coefficient  5  standard error of estimate  y  dipole moment  Eg  Taft s t e r i c f a c t o r  O*  polar substituent constant  E  trans  Z  cis  m/z  mass to charge r a t i o  f  fragmental constant  s  singlet  t  triplet  d  doublet  dd  doublet of doublets  q  quadruplet  m  multiplet  6  chemical s h i f t  J  coupling constant xvii  SYMBOLS AND ABBREVIATIONS (Contd) o/w  octanol-water  i.d.  i n t e r n a l diameter  ACN  acetonitrile  BDZ  benzodiazepine  C-18  octadecylsilane  CNS  c e n t r a l nervous system  DHP  dihydropicrotoxinin  diene  diunsaturated  GABA  gamma-aminobutyric acid  GABA-T  Gamma-aminobutyrate transaminase  GAD  glutamic acid decarboxylase  GCMS  gas chromatography mass spectrometry  HMPA  hexamethylphosphoramide  HPLC  high-performance l i q u i d chromatography  IR  i n f r a red  LDA  l i t h i u m diisopropylamide  lit.  literature  MES  maximal electroshock seizure test  NMR  nuclear magnetic resonance  ODS  octadecylsilane  PTZ  pentylenetetrazole  QSAR  quantitative structure-activity relationships  RP  reversed phase  SAR  structure-activity relationships  s.c. PTZ  subcutaneous pentylenetetrazole  SSA  Succinic semialdehyde  xviii  seizure threshold  test  SYMBOLS AND ABBREVIATIONS (Contd) t-BDMS  tertiarybutyldimethylsilyl  TBPS  t-butylbicyclophosphothionate  THF  tetrahydrofuran  TLC  t h i n layer chromatography  TMS  trimethylsilyl  2,3'-diene VPA 2-(l'-propenyl)-2-pentanoic acid 2,4-diene VPA 2-propyl-2,4-pentadienoic  acid  2E-3'E Diene  2-[(E)-l'-propenyl]-(E)-2-pentenoate  2E-3'Z Diene  2-[(Z)-l'-propenyl]-(E)-2-pentenoate  2Z-3'E Diene  2-[(E)-l'propenyl]-(Z)-2-pentenoate  2Z-3'Z Diene  2-[(Z)-l'-propenyl]-(Z)-2-pentenoate  3Z-3'Z Diene  2-[(Z)-l'-propenyl]-(Z)-3-pentenoate  3Z-3'E Diene  2-[(E)-l'-propenyl]-(Z)-3-pentenoate  3E-3'E Diene  2-[(E)-l'-propenyl]-(E)-3-pentenoate  3,3'-Diene  2-(l'-propenyl)-3-pentenoate  VPA  v a l p r o i c acid  2- ene VPA  2-propyl-2-pentenoic  acid  4-ene VPA  2-propyl-4-pentenoic  acid  3- ene VPA  2-propyl-3-pentenoic  acid  4- OH VPA  2-propyl-4-hydroxypentanoic  acid  3- OH VPA  2-propyl-3-hydroxypentanoic  acid  4- Keto VPA  2-propyl-4-oxopentanoic  xix  acid  ACKNOWLEDGEMENTS  I would l i k e t o express my sincere thanks to Dr. Frank Abbott f o r h i s e x c e l l e n t supervision throughout the course of my graduate s t u d i e s . I  greatly appreciate h i s h e l p f u l advice and e n t h u s i a s t i c response t o  academic and personal a f f a i r s . The  helpful  discussions with members of my graduate  committee,  Dr. Terence Brown, Dr. David Godin, Dr. S i d Katz and Dr. Jim O r r , a r e gratefully  acknowledged.  I wish  t o e x p r e s s my g r a t i t u d e t o  Dr. R.A. Wall and Dr. B. D. Roufogalis f o r t h e i r h e l p f u l suggestions. Special  thanks  t o Mr. Roland  Burton  for h i s valuable technical  assistance i n the gas chromatography - mass spectrometric a n a l y s i s . I appreciate the various assistance by my l a b mates and f e l l o w graduate  students, Greg  Slater,  Jeanine Kassam, Sukhbinder  Panesar,  Kuldeep Singh, Ron Lee and David Kwok. T h i s work would not have been p o s s i b l e w i t h o u t the f i n a n c i a l support  p r o v i d e d by a grant  Foundation  and t h e k i n d  from  support  the B.C. H e a l t h Care provided  Research  by t h e F a c u l t y o f  Pharmaceutical Sciences. F i n a l l y , I wish to thank my parents, brothers and s i s t e r s f o r t h e i r unflagging support.  xx  INTRODUCTION  The  medium branched-chain f a t t y a c i d , v a l p r o i c a c i d ,  I,(2-propyl-  p e n t a n o i c a c i d ) i s a r e l a t i v e l y new a n t i e p i l e p t i c drug, i n t r o d u c e d i n North America i n 1978.  I t i s used i n treatment of absence seizures and  i n combination therapy f o r generalized t o n i c - c l o n i c seizures. covery of the anticonvulsant properties a l . (1) has d i r e c t e d  The d i s -  of v a l p r o i c a c i d by Meunier e t  some a t t e n t i o n toward s i m i l a r p o t e n t i a l a n t i -  c o n v u l s a n t compounds w i t h o u t t h e i m i d e s t r u c t u r e  as found i n most  conventional a n t i e p i l e p t i c drugs. Valproic acid presents a good lead compound f o r s t r u c t u r e - a c t i v i t y studies because i t appears to have a novel mechanism of a c t i o n i n v o l v i n g augmentation of GABAergic a c t i v i t y .  I t has been r e p o r t e d t o i n c r e a s e  brain GABA l e v e l s i n vivo and i n h i b i t enzymes involved a t i o n pathways (2).  i n GABA degrad-  E l u c i d a t i o n o f the m o l e c u l a r a c t i o n s of v a l p r o i c  a c i d t h a t a r e d i r e c t l y r e l a t e d t o i t s a n t i c o n v u l s a n t e f f e c t would be f a c i l i t a t e d by the development of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (SAR) w i t h i n a s e r i e s of c l o s e l y - r e l a t e d analogues of v a l p r o i c a c i d .  Invest-  i g a t i o n o f the m o l e c u l a r s p e c i f i c i t y of t h e a n t i c o n v u l s a n t a c t i o n o f v a l p r o i c acid analogues i s also of i n t e r e s t i n d e t e r m i n i n g s t r u c t u r a l requirements at the s i t e of a c t i o n . E a r l i e r studies on the r e l a t i o n s h i p s between structure and a c t i v i t y explored the p o s s i b i l i t y of modifying the carboxylic f u n c t i o n a l group to enhance a n t i c o n v u l s a n t a c t i v i t y .  Most of t h e compounds t e s t e d f o r  anticonvulsant a c t i v i t y were d i p r o p y l a c e t i c a c i d d e r i v a t i v e s such as amides (3-5), ureides (6), esters (3,5) i n c l u d i n g an oxazepam d e r i v a t i v e (7) and a h y d a n t o i n d e r i v a t i v e o f v a l p r o i c a c i d (6).  1  Ureas ( 8 ) , a l c o -  hols (5,9), carbamates (9) and ketones (9) with the 1-propylbutyl c h a i n , as i n v a l p r o i c a c i d , were a l s o examined f o r anticonvulsant a c t i v i t y . A number of a l i p h a t i c and have been evaluated  a l i c y c l i c - s u b s t i t u t e d c a r b o x y l i c acids  f o r anticonvulsant a c t i v i t y but only at a s i n g l e  dose (5,10,11) or b i o l o g i c a l response was determined using a l e s s specific  t e s t , i . e . protection against  m o r t a l i t y (10). the  pentylenetetrazole (PTZ)  -induced  However, there have been recent studies which reported  dose-dependency of the anti-PTZ  c l o n i c seizure a c t i v i t y of homo-  logous s t r a i g h t - c h a i n and alpha-branched f a t t y acids (12,13,14). Anticonvulsant drugs have diverse chemical s t r u c t u r e s which suggest they may  have d i f f e r e n t mechanisms of a c t i o n or that they may  at a s i m i l a r s i t e of a c t i o n by v i r t u e of having c o p h o r e groups. pharmacophoric drugs.  interact  some s i m i l a r pharma-  There have been s e v e r a l attempts t o uncover the  structural  features  of  the  conventional  antiepileptic  Andrews (15) reported that there was no r e l a t i o n s h i p between the  anticonvulsant a c t i v i t y and the e f f e c t i v e atomic charges at the quaternary carbon common to the anticonvulsant drugs with the imide or ureide structure.  Some i n v e s t i g a t o r s have looked at the v a r i e t y of f u n c t i o n a l  groups that w i l l confer anticonvulsant a c t i v i t y on compounds with a l k y l or  phenyl substituents (16).  P a t r i c k and  Bresee (17)  reported  that  hydrogen-bonding strengths of the major a n t i e p i l e p t i c drugs, measured by the  hydrogen-bonding  enthalpies  with  phenol, were the  compounds studied and thus unrelated to a c t i v i t y .  same f o r  the  Camerman and Camerman  (18) examined the x-ray s t r u c t u r e s of some conventional  antiepileptic  drugs i n c l u d i n g phenytoin, diazepam and phenylacylurea and proposed that the s p a t i a l configurations of these compounds allow superposition of the phenyl groups and  a l s o the carbonyl groups or an equivalent e l e c t r o n -  donor group.  2  Valproic acid has the basic s t r u c t u r a l features, a polar moiety with an electron-donor  group or hydrogen-bonding  group and hydrophobic  s u b s t i t u e n t s , i n common with conventional anticonvulsant drugs.  I t also  possesses a c a r b o x y l i c group and a l k y l chain as i n the structure of gamma-aminobutyric acid (GABA).  However, v a l p r o i c a c i d does not have a  nitrogen function as found i n the structure of GABA.  Different a l i -  phatic and a l i c y c l i c groups may enhance or have an adverse e f f e c t on the i n t e r a c t i o n of the c a r b o x y l i c acids at the s i t e of a c t i o n . been shown i n some barbiturates  This has  where the presence of an i s o a l k y l ,  i s o a l k e n y l and 6-cyclohexylidene-ethyl  chain at the quaternary carbon  r e s u l t s i n convulsant a c t i v i t y ( 1 9 ) . From studies on a homologous alpha-branched a l i p h a t i c c a r b o x y l i c acid s e r i e s , Keane et a l . (13) and Meldrum et a l . (12) reported there was a s i g n i f i c a n t c o r r e l a t i o n between the anticonvulsant and the length of s i d e - c h a i n . the  increase  that  potency  They a l s o found good c o r r e l a t i o n s between  i n GABA brain l e v e l s and anticonvulsant  potency.  In a  d i f f e r e n t study, Perlman and Goldstein (14) used a fluorescent probe t o show that the a b i l i t y of these homologous c a r b o x y l i c acids to disorder synaptosomal plasma membranes c o r r e l a t e d w e l l with t h e i r potency.  anticonvulsant  From the fluorescent p o l a r i z a t i o n s t u d i e s , they suggested that  the anticonvulsant  e f f e c t of v a l p r o i c a c i d i s mediated by nonspecific  mechanisms s i m i l a r to those of general  anesthetics.  I t seems that a  wide v a r i e t y of s t r u c t u r e s are required to i n v e s t i g a t e the s t r u c t u r a l s p e c i f i c i t y of v a l p r o i c a c i d analogues. The present i n v e s t i g a t i o n i s concerned with the e f f e c t of diverse substituents on anticonvulsant a c t i v i t y of v a l p r o i c a c i d analogues. The physicochemical p r o p e r t i e s , namely l i p o p h i l i c i t y , e l e c t r o n i c properties  3  and s t e r i c f a c t o r s have been determined to f i n d whether a c t i v i t y i n vivo i s determined by a nonspecific property such as l i p o p h i l i c i t y or whether there i s a s t e r e o e l e c t r o n i c f a c t o r determining anticonvulsant a c t i v i t y . Multiparametric  r e l a t i o n s h i p s increase  the l i k e l i h o o d  of s t r u c t u r a l  s p e c i f i c i t y i n a c l a s s of s t r u c t u r a l l y - r e l a t e d compounds. This approach has been used by various i n v e s t i g a t o r s t o r e c o n c i l e the high s t r u c t u r a l s p e c i f i c i t y of drug-receptor i n t e r a c t i o n s and the common physicochemical p r o p e r t i e s of d i s p a r a t e s t r u c t u r e s w i t h a common b i o l o g i c a l  act-  i v i t y (20). Different  tests  have  been  used to evaluate  potency of compounds f o r development of SAR. experimental purposes.  models of epilepsy  have  the anticonvulsant  Currently two i n vivo  been widely  employed f o r such  These are the maximal electroshock seizure t e s t (MES) and the  subcutaneous pentylenetetrazole seizure threshold t e s t . the  anticonvulsant  potency of a l k y l - s u b s t i t u t e d  In t h i s study,  c a r b o x y l i c acids and  t e t r a z o l e s have been determined by the subcutaneous pentylenetetrazole seizure  threshold  test.  Several  i n v e s t i g a t o r s have pointed  out the  b i o i s o s t e r i s m between t h e c a r b o x y l i c group and t h e t e t r a z o l e nucleus (21-23).  Comparative studies on s u b s t i t u t e d c a r b o x y l i c acid and  t e t r a z o l e s have revealed s i m i l a r , greater or i n f e r i o r b i o l o g i c a l a c t i v ity acid  of the t e t r a z o l e analogues ( 2 3 ) . and i t s corresponding  Kraus (24) found both v a l p r o i c  t e t r a z o l e , 4-tetrazolylheptane,  inhibited  succinic-semialdehyde  dehydrogenase i n the GABA metabolic  inhibitory  (K^) of 0.7 mM and 0.75 mM r e s p e c t i v e l y .  constants  shunt with  compounds have a polar a c i d i c group and a l k y l s u b s t i t u e n t s . pentylenetetrazole i s a convulsant  Both  However,  and has no a c i d i c p r o p e r t i e s .  The  e f f e c t of a c i d i c properties of compounds on anticonvulsant potency has made i t necessary that the e l e c t r o n i c e f f e c t of the polar moiety, which 4  is  influenced  by the a l k y l  substituents,  be quantified  by physical  methods. By expressing physicochemical properties of s t r u c t u r a l features and the anticonvulsant a c t i v i t y i n q u a n t i t a t i v e terms, m u l t i p l e  regression  a n a l y s i s i s used to obtain q u a n t i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (QSAR).  This l i n e a r free-energy r e l a t i o n s h i p s model was pioneered by  Hansch and co-workers (29). regression of SAR.  The use of s t a t i s t i c a l a n a l y s i s i n the  equation appears to allow much more objective establishment Several researchers have used the Hansch approach to i d e n t i f y  molecular properties that account f o r the anticonvulsant a c t i v i t y of the s t r u c t u r a l l y diverse  a n t i e p i l e p t i c drugs (15,19,26,27).  Lien and co-  workers (26,28) found that 1,4-benzodiazepines, which are known to have s p e c i f i c binding  s i t e s , could not be included with other a n t i e p i l e p t i c  drugs to develop a s i g n i f i c a n t QSAR.  There have been suggestions from  QSAR studies that CNS-acting drugs have d i f f e r i n g l i p o p h i l i c i t y requirements which determine t h e i r d i s t r i b u t i o n a l l o c a l i z a t i o n and hydrophobic binding  at the a c t i v e s i t e (20).  Hansch and co-workers (29) developed  q u a n t i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s f o r a s e r i e s of hypnotic barbiturates  and  suggested  an o p t i m a l  octanol-water  partition  c o e f f i c i e n t of about 100 (log P = 2.0). This QSAR approach has not been applied to e i t h e r a l k y l substituted carboxylic  acids  or t e t r a z o l e s which may s p e c i f i c a l l y antagonize PTZ-  induced c l o n i c seizures because of the i n s i m i l a r i t y i n structure and the known b i o i s o s t e r i s m between c a r b o x y l i c and t e t r a z o l e groups. that  the semi-rigid  structures action  cycloalkyl-substituted  to i n v e s t i g a t e  for valproic  compounds  I t appears  are p o t e n t i a l  the s t r u c t u r a l requirements at the s i t e of acid  analogues.  5  Apparently  5-alkyl-  t e t r a z o l e s (21,23) and  diunsaturated d e r i v a t i v e s of v a l p r o i c acid  (2)  have not been evaluated for t h e i r anticonvulsant a c t i v i t y . U n i d e n t i f i e d diunsaturated metabolites of v a l p r o i c acid have been reported to be present i n the serum and acid therapy ( 2 ) . help  The a v a i l a b i l i t y of synthetic reference material w i l l  characterize  diunsaturated  urine of patients on v a l p r o i c  the  stereochemical  metabolites.  Their  configuration  synthesis  requires  these  stereoselective  methods owing to the number of p o s i t i o n a l isomers and the stereoisomers.  of  multiplicity  of  Compounds with polar groups i n the a l k y l chain, i n  a d d i t i o n to the terminal carboxylic f u n c t i o n , have also been included i n the study. S p e c i f i c Objectives 1.  Valproic acid has been reported to e x h i b i t s e l e c t i v e actions may  mediate i t s anticonvulsant e f f e c t s .  that  A wide v a r i e t y of s t r u c t -  u r a l analogues of v a l p r o i c acid were to be used to i n v e s t i g a t e the degree of The  structural specificity  of  the  anticonvulsant  actions.  s e r i e s of v a l p r o i c acid analogues (Figure 1) include compounds  with y - a l k y l substituents; stituents;  a-alkyl  a , a - d i a l k y l substituents;  substituent,  alicyclic  substituents;  groups i n a l k y l chain; unsaturated a l k y l groups. the study was  B - a l k y l subpolar  The f i r s t part of  to apply synthetic methods to prepare the  substituted  c a r b o x y l i c acids and t e t r a z o l e s . 2.  In the course of the study, i t was  of i n t e r e s t to employ a stereo-  s e l e c t i v e synthetic method to prepare the diunsaturated analogues of  valproic  acid,  2-[(E)-l'-propenyl]-(E)-2-pentenoic  2-[(Z)-l'-propenyl]-(E)-2-pentenoic anticonvulsant  activity  and 6  acid,  for  identification  evaluation of  the  acid  and  of  the  major d i -  CH3-CH2-CH2  CH3-CH2-CH2-CH2  ^CHCOOH  CH3 \HCOOH  CH3-CH2-CH£  CH3-CH2-CH2-CH£  I  II  CH3  CH  III  CH3  CH3-C-COOH  ^CHCOOH  3  CH3-CH2 N  CH 3 -CH 2 -C-COOH  CH3  CH CH33  IV  CH3-CH2  V  ChL-CH9-CH9 /  VI  CHQ-CH=CH  C-C00H  CH3-CH2-CH '  LM  ^C-COOH  0 r, •CH2 3 2v L LH  ^ CHCOOH CH^-CH^  CHg-CHg-CI?  VII  CH-CH2COOH  VIII  IX  H  H  / N  CH^-CH9-CH9-CH7CH?-CH?-CH9-Cf 3 222 222  Vn*  <  CHJ 2  2  CH-CH 9 -CH 9 -C^  v  >  N N-N  X  XI H CH  3  -C00H XII  XIII  CH3-CH2  XIV  CH3-CH2-CH2-CH2 N  Vc-CH 9 CH 9 C00H  /  CH3-CH2  II  2  /  2  0  CH3-CH2-CH2-CH2  XV Figure 1.  N-C-CH9-CH9C00H  II  2  2  0  XVI Chemical Structures of Valproic Acid and analogues  7  unsaturated metabolites of v a l p r o i c a c i d . 3.  Aside from a p p l i c a t i o n of reverse-phase HPLC as an a n a l y t i c a l t o o l , one can r e l a t e the observed chromatographic r e t e n t i o n parameters of compounds  to  physicochemical  Octanol-water  partition  properties  coefficients  such  as  indirectly  lipophilicity. determined  by  reverse phase (RP) - HPLC have been shown to be i n close agreement with  values  obtained  by  the  traditional  Since chromatographic methods are  shake-flask  generally  more rapid  procedure. than  s t a t i c method owing to the higher rate of e q u i l i b r a t i o n of between the  phases, t h i s study u t i l i z e s a RP-HPLC  the  solute  procedure to  determine the octanol-water p a r t i t i o n c o e f f i c i e n t s of v a l p r o i c a c i d analogues. diverse  The e f f e c t s of d i f f e r e n t mobile phases and compounds of  chemical structures on the accuracy of the RP-HPLC method  were examined. 4.  V a r i a t i o n of the s u b s t i t u t i o n pattern  i n the carboxylic acids  and  t e t r a z o l e s i s manifested by the r e s u l t a n t e l e c t r o n i c e f f e c t s at the a c i d i c groups of the polar moiety. constants (pKa) in  the  of the compounds may  literature,  the  conditions  values usually d i f f e r from one the i n t e r e s t of accuracy and  While the apparent i o n i z a t i o n have been v a r i o u s l y reported used  laboratory  for  measurement of  to the other.  I t was  pKa in  consistency to employ an appropriate  method to determine the pKa of v a l p r o i c acid and analogues. 5.  Valproic acid and a l k y l - s u b s t i t u t e d compounds have been reported to be more e f f e c t i v e against PTZ-induced c l o n i c seizures compared to other chemical-induced seizures  or e l e c t r i c a l l y induced  seizures.  Since there have been r e l a t i v e l y few studies on the dose-dependent anti-PTZ a c t i v i t y of v a l p r o i c acid analogues, the aim of the study 8  was to determine the anti-PTZ potency of not only the homologous abranched acids but also compounds with other s u b s t i t u t i o n a l characteristics. Since the QSAR approach has not been applied to a l k y l - s u b s t i t u t e d carboxylic acids and t e t r a z o l e s , i t was of i n t e r e s t to evaluate the r o l e played by the e l e c t r o n i c properties of the polar group and the hydrophobicity of the a l k y l group on the anticonvulsant potencies of test compounds.  A d d i t i o n a l s t r u c t u r a l properties considered i n  the present study were the conformational or s t e r i c e f f e c t s and the molecular dipole moments.  (5) HC-  NH  (1)  N  (2)  II (4)  N N (3)  Tetrazole  (8) (7) CH2  CH2  (9)  (6) CH2  CH2  (10)  (5) C  11 (1)  II (4) N  N (2) N (3)  Pentylenetetrazole  (PTZ)  Although t h i s study p e r t a i n s s p e c i f i c a l l y t o a l k y l s u b s t i t u t e d carboxylic acids and t e t r a z o l e s , comparative studies with other a l k y l substituted h e t e r o c y c l i c compounds were made possible by the a v a i l a b l e l i t e r a t u r e data on t h e i r anti-PTZ p o t e n c i e s and p h y s i c o c h e m i c a l properties.  The b a s i s of such comparison i s t h e p o s s i b i l i t y of a common  molecular action of the a l k y l substituted compounds as suggested from biochemical,  pharmacological and neurophysiological  researchers (30-34).  10  studies by several  LITERATURE SURVEY 1.  C l i n i c a l Use and Anticonvulsant Properties of V a l p r o i c Acid (VPA) The  therapeutic e f f i c a c y of VPA (Depakene®) has been demonstrated  i n several c l i n i c a l studies (35,36).  VPA i s now widely used i n primary  generalized e p i l e p s i e s , p a r t i c u l a r l y those of the absence seizure type. V a l p r o i c a c i d i s considered  to be at l e a s t as e f f e c t i v e as ethosuximide  i n the treatment of absence seizure (37).  I t s broad spectrum of a n t i -  e p i l e p t i c e f f e c t s has, however, proven valuable i n combination therapy for myoclonic epilepsy and generalized t o n i c - c l o n i c seizures (35,36). VPA shows s e l e c t i v e a c t i v i t y against several types of chemically or e l e c t r i c a l l y - i n d u c e d seizures i n a v a r i e t y of species. VPA has a weak a c t i v i t y against maximal electroshock  seizures i n mice compared to the  a c t i v i t y of phenobarbital and phenytoin (38). prevention  of c l o n i c  p i c r o t o x i n (38-40).  or t o n i c  seizures  induced  by PTZ and  High doses are reported  to block  tonic-clonic,  b i c u c u l l i n e and strychnine-induced  seizures (38-40).  2.  Pharmacological Testing  a.  Experimental Models of Epilepsy Quantitative  VPA i s more e f f e c t i v e i n  e f f e c t s of s t r u c t u r a l variants on the pharma-  c o l o g i c a l a c t i v i t y of the a n t i e p i l e p t i c drugs have been obtained by the  use of numerous t e s t s i n experimental animals.  experimental techniques f o r inducing electroshock PTZ.  seizure  and systemic administration  The maximal electroshock  The common  i n rodents  include  of convulsants such as  seizure t e s t (MES) and the sub-  cutaneous pentylenetetrazole seizure threshold t e s t ( s . c . PTZ) are 11  widely recognized models f o r determining the anticonvulsant a c t i v i t y of compounds.  A c t i v i t y i n the MES  test has been c o r r e l a t e d  with a compound's a b i l i t y to modify maximal seizures or i n h i b i t the seizure spread through the b r a i n .  In contrast the s.c. PTZ  test  measures the a b i l i t y of a compound to elevate the degree of seizure threshold (41). clonic  The MES  t e s t i s thus a model of generalized t o n i c -  s e i z u r e w h i l e the s . c . PTZ  seizures.  t e s t i s a model of absence  Other models have been developed to simulate  partial  seizures. Extensive i n v e s t i g a t i o n s have been c a r r i e d out to standardize the MES  t e s t (41-43) and the s.c. PTZ  t e s t (41,44,45).  These two  methods have been preferred over other t e s t s since they are reported  to be r a p i d , s i m p l e , e a s i l y c o n t r o l l e d and  non-erratic i n  producing the c l o n i c or t o n i c seizure component (38).  The value of  these t e s t s has been shown by good c o r r e l a t i o n between t e s t r e s u l t s and e f f i c a c y i n c l i n i c a l epilepsy (38,46).  In the MES  t e s t , max-  imal seizures are induced by passing high current ( f i v e to seven times  threshold  electrodes  value,  f o r 0.2 sec  i . e . 50  mA,  i n mice (46).  60  Hz)  through  In the MES  corneal  test, active  compounds protect against the t o n i c extension of hind l i m b s . is  administered  i n mice or r a t s at doses r a n g i n g  convulsant to nearly l e t h a l doses. clonic  seizures  are  produced  as  absence of c l o n i c  frank  In the s.c. PTZ t e s t , threshold when  PTZ  subcutaneously i n a dose of 85 mg/kg i n mice. CDoj dose i n mice (46).  from  PTZ  is  administered  This i s the reported  P r o t e c t i o n i n the s.c. PTZ t e s t i s defined spasms of d u r a t i o n g r e a t e r  than 5 s e c .  Another PTZ seizure threshold t e s t , the timed i . v . i n f u s i o n method,  12  has  also  been  used  to  determine  anticonvulsant  potency  in  r a t s (45). Whereas there neuronal discharge,  i s a need f o r the use  of i n v i t r o models of  e s p e c i a l l y i n i d e n t i f y i n g the s e l e c t i v e mol-  ecular actions of a n t i e p i l e p t i c drugs (31), the e m p i r i c a l i n vivo models have a l s o been d i s c r i m i n a t i v e i n showing  differential  actions of a n t i e p i l e p t i c drugs.  trimethadione,  Ethosuximide and  both a l k y l - s u b s t i t u t e d compounds, are e f f e c t i v e only f o r absence s e i z u r e s and  i n the s . c . PTZ  test (44,47).  e f f e c t i v e f o r absence seizures and  Phenytoin i s i n -  i n the s.c. PTZ  prevents generalized  tonic-clonic seizures, p a r t i a l  maximal electroshock  seizures (42-44).  suppress the spread of s e i z u r e s . the t o n i c seizure component i n MES  Phenytoin  t e s t , but i t seizures  and  i s thought to  1,4-Benzodiazepines, which block t e s t and  the c l o n i c spasms i n  s.c. PTZ t e s t at approximately the same dose, may  act by e l e v a t i o n  of seizure threshold (48).  Barbiturates are e f f e c t i v e i n the  MES  test  separated  the  and  s.c. PTZ  t e s t at  doses suggesting  that  mechanism of a c t i o n involves both e l e v a t i o n of seizure threshold and  i n h i b i t i o n of seizure spread (41,48).  The  e f f e c t of v a l p r o i c  acid i s s i m i l a r to b a r b i t u r a t e s , except that i t i s more e f f e c t i v e i n e l e v a t i o n of seizure threshold (39,40). b.  Mechanism of Action of  PTZ  PTZ i s known to act on the whole CNS but there are c o n f l i c t i n g reports  as  to whether the  PTZ-induced convulsions  s p e c i f i c areas such as the c e r e b r a l cortex (49). PTZ  convulsant actions i s s t i l l not defined.  originate i n  The mechanism of  However, the d i r e c t  e f f e c t on c e l l membrane e x c i t a b i l i t y , together with i t s a b i l i t y to 13  s e l e c t i v e l y block the e f f e c t of GABA on c h l o r i d e conductance may account for the convulsant properties of PTZ ( 4 9 , 5 0 ) . A number of workers have reported that PTZ enhances the e x c i t a t o r y system through d i r e c t e f f e c t on membrane p r o p e r t i e s t o increase  spontaneous  ance ( 5 1 , 5 2 ) . and  discharge  by a l t e r i n g  ionic  conduct-  Using cultured mouse s p i n a l cord neurons, Macdonald  Barker ( 5 3 ) noted that PTZ r e v e r s i b l y antagonizes i n a dose-  dependent manner the conductance produced by iontophoresed GABA, without an e f f e c t on the r e s t i n g membrane p r o p e r t i e s . was  The response  s e l e c t i v e for GABA since PTZ did not a f f e c t the response due t o  g l y c i n e , 8-alanine and glutamic a c i d .  The synaptic a c t i o n of PTZ  i s reported to block the increase i n c h l o r i d e conductance induced by iontophoresed GABA i n neurons of Aplysia C a l i f o r n i c u s ( 5 4 ) . In the  same study ( 5 4 ) , PTZ was observed to show minimal e f f e c t on  e x c i t a t o r y response of inward sodium current and i n h i b i t o r y e f f e c t of  outward  potassium c u r r e n t .  Unlike valproic  a c i d , PTZ i s  reported to have no s i g n i f i c a n t e f f e c t on brain GABA l e v e l s ( 5 5 ) . PTZ  i n h i b i t e d the a c t i v i t y of glutamic acid decarboxylase i n v i t r o  at concentrations r e l a t e d PTZ  to p h y s i o l o g i c a l l e v e l s ( 5 5 ) .  In v i v o ,  i n h i b i t e d the a c t i v i t y of glutamic acid decarboxylase at twice  the CDgj dose i n mice ( 5 5 ) . I t has been observed that PTZ seizures  are s i m i l a r to those  induced by p i c r o t o x i n but u n l i k e those of strychnine ( 5 0 ) .  Ticku  and  Olsen ( 3 3 ) reported that PTZ shows a s i g n i f i c a n t a f f i n i t y f o r  the  picrotoxin  binding s i t e (IC^Q = 30 yM) hence i t i s l i k e l y to  act at the benzodiazepine-GABA-chloride ionophore receptor complex. Recently Squires et a l . (56) examined a s e r i e s of convulsant t e t r a -  14  zoles  for their  potencies  i n displacing  S-t-butylbicyclo  phosphonothionate (TBPS) from the p i c r o t o x i n s i t e on the BDZ-GABACl~  ionophore r e c e p t o r  relative  complex.  A good c o r r e l a t i o n between  JJ  affinities  f o r S-TBPS b i n d i n g s i t e and c o n v u l s a n t  potencies (IC5Q of PTZ = 0.54 mM) was found. Tetrazole d e r i v a t i v e s vulsant a c t i v i t y .  have been investigated  f o r t h e i r con-  Potent convulsant e f f e c t s were obtained when  1,5-positions are bridged by a penta to heptamethylene chain (57). Tetrazole d e r i v a t i v e s  possessing a methyl or e t h y l group at pos-  i t i o n - 5 and a substituent atoms  (aliphatic  at p o s i t i o n 1(N) with four to s i x carbon  or a l i c y c l i c )  are a c t i v e  as convulsants (58).  Depressant e f f e c t s of some 1,5-disubstituted t e t r a z o l e s have been obtained with s u b s t i t u t i o n of aminophenyl groups at p o s i t i o n 1 or 5 and a l i p h a t i c groups at the unsubstituted p o s i t i o n (59). Shunkla (60) investigated  5-acetamido-substituted  Ahmad and  t e t r a z o l e s and  reported anticonvulsant a c t i v i t y of 5-(N-phenylacetamido)-tetrazole with an i . p . dose of 100 mg/kg.  c.  Pharmacokinetics of PTZ The  pharmacokinetic properties  of PTZ are a t t r i b u t e d to i t s  ready s o l u b i l i t y i n both l i p i d s and water. from a l l s i t e s of administration  PTZ i s r a p i d l y absorbed  i n mice and r a t s and i s r a p i d l y  d i s t r i b u t e d i n t o t a l body water (61,62).  Brain ^C-PTZ uptake i s  rapid w i t h i n 10 min a f t e r s.c. administration  i n r a t s and the h a l f -  l i f e of PTZ i n brain ranged from 16-21 min (191). l i f e of r a d i o l a b e l e d  The serum h a l f -  PTZ i n r a t s was found to be 2-4 hr (61,62).  Metabolism i s the major route of PTZ e l i m i n a t i o n .  A low percentage  of unchanged drug i s excreted i n urine (61,64).  The major metab-  15  o l i t e s i n r a t s are 6-hydroxy PTZ and 8-hydroxy PTZ (23,61). ing  Bind-  to t i s s u e constituents i s almost n e g l i g i b l e and approximately  9% of the drug i s bound t o plasma proteins (65). 3.  Chemistry and Physicochemical Properties of V a l p r o i c Acid Valproic  Burton (66) acetate.  acid  (2-propylpentanoic  i n 1882 by a l k a l i n e  water.  decomposition  The p h y s i c a l and chemical  been reviewed by Chang (67). The c r i t i c a l  acid) was f i r s t of  synthesized by  2,2-dipropylaceto-  properties of v a l p r o i c a c i d have  VPA has a s o l u b i l i t y of 1.27 mg/ml i n  m i c e l l a r c o n c e n t r a t i o n of i t s s t r a i g h t  isomer, octanoic a c i d i s reported to be 8.0 mmol/L (68).  chain  The p a r t i t i o n  c o e f f i c i e n t of VPA between various organic solvents and phosphate buffer (pH 7.4) has been reported to be 0.013 f o r heptane, 0.064 f o r benzene and 0.21 f o r chloroform (69).  V a l p r o i c a c i d showed a pKa value of 4.56  when determined by potentiometric  titration  of the a c i d i n aqueous  medium (70). 4.  Mechanism of Action of V a l p r o i c Acid The basic mechanism by which v a l p r o i c a c i d exerts i t anticonvulsant  effect  i s not yet c l e a r .  Several biochemical  and neurophysiological  studies have shed some l i g h t on i t s mode of a c t i o n (2,71,72). logical  specificity  anticonvulsants,  of v a l p r o i c  i s broad.  At h i g h  acid,  like  that  The b i o of  other  d o s e s , v a l p r o i c a c i d shows  n o n s p e c i f i c a c t i o n s such as i n h i b i t i o n of m i t o c h o n d r i a l o x i d a t i v e phosphorylation (73), actions shared by non-anticonvulsants  (73).  Some  i n v e s t i g a t o r s have used the s e l e c t i v e actions of anticonvulsants, such as v a l p r o i c a c i d , a t lower drug c o n c e n t r a t i o n s  16  to pinpoint  their  mechanism  of a c t i o n  (31,74).  The p o s s i b i l i t y  of a  nonspecific  anticonvulsant a c t i o n of v a l p r o i c a c i d has received some a t t e n t i o n (14) although more d i r e c t e f f e c t s have been observed f o r v a l p r o i c a c i d . Four possible  modes of a c t i o n  have been postulated from  e f f e c t s observed f o r v a l p r o i c a c i d .  direct  These are e l e v a t i o n of brain GABA  l e v e l s , enhancement of GABAergic i n h i b i t i o n at the post-synaptic GABA r e c e p t o r complex, r e d u c t i o n of e x c i t a t o r y  a c t i o n o f a s p a r t a t e and  increase i n membrane conductance t o potassium i o n s . Valproic acid has been repeatedly shown to r a i s e l e v e l s of GABA i n the brain (75,76). The increase i n GABA l e v e l s induced by v a l p r o i c acid appears to take place i n nerve terminals ( 7 7 ) . In v i t r o studies showed that v a l p r o i c acid l e v e l s up to 1 mM had no e f f e c t on the release of preloaded  radiolabelled  GABA  from  r a t brain  synaptosomal  preparations (78). GABA i s synthesized by GAD-catalyzed decarboxylation of  glutamate and i s m e t a b o l i z e d t o s u c c i n i c  r e v e r s i b l e GABA-T catalyzed metabolized  either  reaction.  to s u c c i n i c  acid  Succinic  semialdehyde  by the  semialdehyde (SSA) i s  by SSA-dehydrogenase  catalyzed  oxidation or metabolized by aldehyde reductase-catalyzed reduction to 4hydroxybutyric a c i d .  The increase i n GABA brain  l e v e l s produced by  v a l p r o i c acid was a t t r i b u t e d from early studies t o i n h i b i t i o n of GABAT (75). (K i f  I t appears that VPA more potently  i n h i b i t s SSA-dehydrogenase  0.5 mM) compared to i n h i b i t i o n of GABA-T, K± >20 mM (79,80). Thus  an increase i n s u c c i n i c semialdehyde l e v e l s might elevate GABA l e v e l s by indirectly  inhibiting  increase the a c t i v i t y  GABA-T.  Valproic  acid  has also  of the GABA biosynthetic  been shown to  enzyme, glutamic a c i d  decarboxylase, i n whole brain and i n brain synaptosomes i n mice ( 7 7 ) . Recent studies suggest that VPA may rather act at the post-synaptic  17  membrane of the GABAergic synapse.  The administration of v a l p r o i c a c i d  by iontophoresis potentiates the i n h i b i t i o n e f f e c t of GABA on neurons i n the  brain (81,82), i n mouse s p i n a l cord neuronal c u l t u r e (74) and i n  cuneate f i b r e  preparations (83).  Iontophoresed v a l p r o i c a c i d had no  s i g n i f i c a n t e f f e c t on responses from e i t h e r iontophoresed glycine or glutamate ( 7 4 ) .  The c o n c e n t r a t i o n  of v a l p r o i c a c i d a c t i n g on t h e  neuronal membrane during iontophoretic a p p l i c a t i o n of valproate was not ascertained i n some of the s t u d i e s . Harrison and Simmonds (83) reported that v a l p r o i c a c i d enhances GABA e f f e c t s a t 3.0 mM i n v i t r o . Benzodiazepines and barbiturates are a l s o reported the postsynaptic i n h i b i t o r y e f f e c t s of GABA (31).  to potentiate  This a c t i o n of a n t i -  convulsant barbiturates and v a l p r o i c a c i d i s suggested to occur a t the p i c r o t o x i n binding s i t e , i . e . the c h l o r i d e ionophore (30,33,34).  The  GABA receptor appears to be part of a p r o t e i n complex containing receptor  s i t e s f o r GABA, BDZ and p i c r o t o x i n as w e l l as the c h l o r i d e iono-  phore (30).  Loscher (84)  has reported  i n h i b i t nor enhance the binding of concentrations up to 1 mM.  that  v a l p r o i c a c i d does not  H-GABA t o r a t b r a i n membranes at  V a l p r o i c a c i d also does not bind to benzo-  diazepine receptors at concentrations up to 1 mM (34). the site  The component of  GABA-receptor-chloride ionophore, the p i c r o t o x i n "receptor" at which several convulsants  isa  ( i n c l u d i n g pentylenetetrazole) and  anticonvulsants appear to a c t (30,33,34).  Ticku and Davis (34) found  that v a l p r o i c a c i d i n h i b i t s the binding of H-dihydropicrotoxinin to r a t brain membrane (IC^Q = 0.5 mM).  V a l p r o i c a c i d i s reported to have no  affinity  of  f o r the b i n d i n g  site  H-phenytoin  t o r a t b r a i n mem-  branes ( 8 5 ) . In an i n t r a c e l l u l a r  study of the e f f e c t s of valproatec  18  on non-  mammalian species, Johnson (71) found that concentrations of VPA (530 mM), 15-50 times the serum l e v e l of patients on VPA therapy, caused an increase i n potassium membrane conductance to produce  hyperpolariz-  a t i o n of the r e s t i n g membrane. Valproic aspartate  a c i d i s reported by several  i n vivo (12,75,86).  The time course f o r the decrease i n  aspartate concentrations correlated induced  protection  against  researchers to reduce brain  with the period of v a l p r o i c  audiogenic  seizure  acid-  i n mice and with the  increase i n brain GABA l e v e l s (86). So f a r i t i s not c e r t a i n whether v a l p r o i c acid exerts i t s anticonvulsant e f f e c t s e x c l u s i v e l y through one of the four possible action.  Potentiation  of GABAergic post-synaptic  inhibition  modes of may act  together with elevated GABA l e v e l s r e s u l t i n g from i n h i b i t i o n of SSAdehydrogenase.  Moreover t h e r e appear t o be i n c o n s i s t e n c i e s  as t o  whether the suggested molecular actions of valproate occur at therapeuti c a l l y r e l e v a n t v a l p r o i c a c i d b r a i n and serum l e v e l s i n mammalian species.  In patients  receiving  therapeutic doses of v a l p r o i c  serum l e v e l s of 0.3-0.8 mM have been reported to be e f f e c t i v e ( 2 ) . present study, by valproic  acid, The  i n v e s t i g a t i n g the SAR of c l o s e l y - r e l a t e d analogues of  a c i d , may be useful  i n determining which of the molecular  actions of v a l p r o i c acid can be correlated with anticonvulsant a c t i v i t y . 5.  Studies on Anticonvulsant A c t i v i t y of Valproic Acid Analogues Valproic  acid  i s not the only a c t i v e  among i t s congeners.  Carraz (10) reported  a l i p h a t i c carboxylic protection  acid  of PTZ-induced  m o r t a l i t y by some alpha-branched f a t t y acids of the general form, X V I I . These studies were conducted at a s i n g l e dose, 200 mg/kg i . p . i n mice.  19  R2-CH-COOH | Rj  Rj . R2 == CH Rj = R2 ^2 5 Rj = R2 = C^Hy n  XVII Activity  rose w i t h i n c r e a s e s i n c h a i n l e n g t h up t o v a l p r o i c a c i d .  Dibutylacetic  a c i d was reported  to be i n a c t i v e .  Recent studies by  Chapman et a l . (12) and Keane et a l . (13) showed that the a c t i v i t y of alpha-branched f a t t y acids continued side-chain.  to increase with elongation of the  However, they found s t r a i g h t - c h a i n f a t t y acids (C^-C^) to  be i n a c t i v e up to 4.0 mmol/kg doses. Anticonvulsant a c t i v i t i e s were also noted with t r i s u b s t i t u t e d f a t t y acids (10,87) of the s t r u c t u r a l form, X V I I I . investigated  the anti-PTZ a c t i v i t y  R3 j R2~C—C00H R  =  = R  T a i l l a n d i e r e t a l . (5)  of a s e r i e s of 3-substituted and  =  Rj= ^2 = 3 ^^3 R^ R3 = CH 3 , R2 =— ^2^5 = R 3 CH 3 , R 2 ^3^7  l  XVIII a,3-disubstituted acids of the type XIX.  R2  ^CH-CH-COOH / I  R^  R^  R2 R2 R2 R i\2 2 R2 R2  = = = = =  =  R3 R3 R3 R3 R3 R3  = CH3, RL 1 = 2 5 0 U ' D = C 3 H 7 , Rj — ^C23 H 77*, Rj ^1 ^2 5 : H = C H , R, 3 ? C fl — R l = 3 n  =  n  A  xix  20  9  As i n most of these s t r u c t u r e - a c t i v i t y s t u d i e s , the PTZ t e s t was p e r formed a t a s i n g l e  dose i n m i c e ,  0.9 mmol/kg i n t h i s  instance.  According to the i n v e s t i g a t o r s (5), isopentanoic a c i d , 3-ethylpentanoic a c i d , 2-ethyl-3-propylhexanoic a c i d and 2 , 3 - d i p r o p y l h e x a n o i c a c i d were i n a c t i v e while 3-propylhexanoic  and 2-butyl-3-propylhexanoic a c i d were  as a c t i v e as v a l p r o i c a c i d . C y c l i c analogues of v a l p r o i c a c i d have also been studied.  Cyclo-  heptane c a r b o x y l i c a c i d i s i n a c t i v e compared t o v a l p r o i c a c i d when a d m i n i s t e r e d a t a dose of 200 mg/kg i . p . i n mice (11). Both a - c y c l o pentylpentanoic acid  and a - c y c l o h e x y l p e n t a n o i c a c i d a r e weaker  anticonvulsants than v a l p r o i c acid (5). Two recent studies explored the p o s s i b i l i t y of r i g i d a l i c y c l i c c a r b o x y l i c compounds revealing the nature of the a c t i v e c o n f i g u r a t i o n o f the a l k y l groups t h a t i n t e r a c t at the s i t e of a c t i o n o f v a l p r o i c a c i d .  Brana and c o - r e s e a r c h e r s (5) found  compounds, XX and X X I , t o be as a c t i v e as v a l p r o i c a c i d i n the s.c. PTZ  XXI  XX  t e s t when compounds were a d m i n i s t e r e d i . p . a t a dose of 200 mg/kg i n mice. S c o t t e t a l . (88) looked a t the a n t i - P T Z a c t i v i t y o f supposedly metabolically  s t a b l e s p i r o c a r b o x y l i c a c i d s , i n c l u d i n g XXII and XXIII.  Compound XXIII, S p i r o [ 4 , 6 ] u n d e c a n e - 2 - c a r b o x y l i c a c i d , the most a c t i v e  21  COOH  OCT  OOH  XXIII  XXII  compound  i n the s e r i e s , was s l i g h t l y more potent than v a l p r o i c  acid  (ED50 of 0.42 mmol/kg compared to 0.9 mmol/kg f o r sodium v a l p r o a t e ) . Early published studies on anticonvulsant a c t i v i t y of v a l p r o i c acid analogues focused on various d e r i v a t i v e s of the carboxylic a c i d such as esters,  amides and ureas (3-9).  Most of the compounds  tested  were  b a r e l y a c t i v e except the primary amide of v a l p r o i c a c i d which was equally to  potent.  be a c t i v e  However, d i a l k y l u r e i d e s have been previously reported  compounds ( 8 9 ) .  The secondary  and t e r t i a r y  v a l p r o i c acid were found to be mostly convulsants (3,4). the carboxylic acids to the primary alcohols  amides of  Conversion of  i n a d i a l k y l a l k a n o i c acid  s e r i e s did not reduce d r a s t i c a l l y t h e i r anticonvulsant a c t i v i t i e s while esterification The  of the r e s u l t i n g alcohol  anticonvulsant  alcohols  properties  destroyed t h e i r a c t i v i t y ( 5 ) .  of the primary  were p a r t l y a t t r i b u t e d  amide and the primary  to the a c t i v e carboxylic  a c i d metab-  olites (5). Some workers  have evaluated analogues  with b i f u n c t i o n a l  groups.  1,3-Propanediols and dicarbamates such as 2,2-dipropyl-l,3-propanediol and  meprobamate are known anticonvulsants (38,90).  d e r i v a t i v e of the s p i r o c a r b o x y l i c slightly  less active  The malonic  acid  compound, X X I I I , was observed to be  than v a l p r o i c  acid (88).  Schwartz  and co-  workers (91) reported that 2-ethyl-2-propylcyanoacetamide and 2-ethyl-222  workers (91) reported that 2-ethyl-2-propylcyanoacetamide  and 2-ethyl-2-  b u t y l m a l o n d i a m i d e possessed a n t i c o n v u l s a n t a c t i v i t y  with  sedative properties at a dose of 400 mg/kg i n the s.c. PTZ A number of v a l p r o i c a c i d possessed  metabolites  anticonvulsant activity  minimal  test.  with greater  polarity  weaker than t h a t of  valproic  acid (92). Among the known m e t a b o l i t e s of v a l p r o i c a c i d , the urated metabolites, namely 4-ene VPA, most p o t e n t .  unsat-  3-ene VPA and 2-ene VPA were the  However, these m e t a b o l i t e s occur i n serum and u r i n e of  human and rodents i n r e l a t i v e l y s m a l l q u a n t i t i e s (<10%) when compared to l e v e l s of v a l p r o i c a c i d (92). 2-propylglutaric acid had  3-Keto VPA,  the hydroxy metabolites  s l i g h t anticonvulsant p r o p e r t i e s .  and  There are  no data on the a n t i c o n v u l s a n t p r o p e r t i e s of the r e c e n t l y d e s c r i b e d m e t a b o l i t e s of v a l p r o i c a c i d , 4-Keto VPA and the d i u n s a t u r a t e d compounds (93). W h i l e no  r a t i o n a l b a s i s f o r the p r e d i c t i o n of a n t i c o n v u l s a n t  a c t i v i t y of a l k y l s u b s t i t u t e d c a r b o x y l i c a c i d d e r i v a t i v e s has been e s t a b l i s h e d , the a n t i c o n v u l s a n t data suggest t h a t the nature of the a l k y l group i s of b a s i c i m p o r t a n c e to p h a r m a c o l o g i c a l  action.  The  s i g n i f i c a n c e of d e t e r m i n i n g the QSAR of v a l p r o i c a c i d analogues i s highlighted by the experimental o b s e r v a t i o n s t h a t v a l p r o i c a c i d , v a l proamide, d i p r o p y l u r e i d e and 5 , 5 - d i p r o p y l b a r b i t u r i c a c i d a r e potent anticonvulsants (4,6) despite d i f f e r e n c e s i n l i p i d 6.  solubility.  General SAR of A n t i e p i l e p t i c Drugs Although v a l p r o i c acid d i f f e r s from the t r a d i t i o n a l a n t i e p i l e p t i c s  (Figure 2) which c o n t a i n a n i t r o g e n f u n c t i o n a l group, t h e r e are s i m i l a r i t i e s i n s t r u c t u r e . Common s t r u c t u r a l features of the a n t i e p i l e p t i c  23  II o (i) C  H  C2H5  3 7, \_  CH' 3  (ii)  CH—COOH  CH3-Y  7  Valproic acid (Carboxylic acid)  '  c=o  J ,  Ethosuximide (Succinimide)  Tn'methadione (Oxazolidinedione)  c=o  Phenobarbital (Barbiturate)  Primidone (Deoxybarbiturate)  Figure 2.  Di phenylhydantoin (Hydantoin)  Clonazepam (1,4-Benzodiazepine)  Phenacemide (acetyl urea)  Carbamazepine (Dibenzoazepine-5-Carboxamide)  Chemical structures of t r a d i t i o n a l a n t i e p i l e t i c agents.  24  drugs are embodied i n the polar carboxyl or imide f u n c t i o n a l group and the d i s u b s t i t u t e d quaternary or t e r t i a r y carbon, i and i i i n Figure 2. Anticonvulsant from the  activities  literature  and  convulsant drugs (94).  of classes of compounds have been compiled summarized to determine SAR  among the  anti-  S i m i l a r s t r u c t u r a l requirements appear to e x i s t  i n the conventional anticonvulsant drug groups such as the b a r b i t u r a t e s , hydantoins, oxazolidinediones, succinimides and a c y c l i c u r e i d e s . Certain d i s t i n g u i s h i n g features i n the SAR drugs have been recognized.  The i n t r o d u c t i o n of at l e a s t one phenyl or  s i m i l a r aromatic group at Rj or R2  (Figure 2) i s required f o r optimal  a c t i v i t y against MES-induced s e i z u r e s . at  Rj  or  R2  confer  high  of these a n t i e p i l e p t i c  activity  In c o n t r a s t , a l k y l substituents  against  s.c. PTZ-induced s e i z u r e s .  Increasing the length of the a l k y l chain usually maximizes the activity.  Most of the c l a s s i c a l a n t i e p i l e p t i c drugs produce maximal  a c t i v i t y i n the s.c. PTZ  t e s t when the sum of carbon atoms i n the sub-  s t i t u e n t s i s between Cg and C-^Q. MES  t e s t (16).  Diphenylacetic acid i s a c t i v e i n the  5,5-Diphenyloxazolidinedione,  2,2-diphenylsuccinimide ity  5,5-diphenylhydantoin,  and 5,5-diphenylbarbiturate  (or even i n a c t i v i t y )  i n the s . c . PTZ  butylsuccinimide i s i n a c t i v e i n the MES The nitrogen atom may  have minimal a c t i v -  test (94).  anticonvulsant  2-Methyl-2-  t e s t (94).  be s u b s t i t u t e d preferably by a methyl group,  lower a l k y l groups and a l k o x y a l k y l groups without reducing the  hypnotic  activity,  e.g.  metharbital,  significantly  N-methyldipropyl-  acetamide (5,8), mephenytoin, methsuximide and trimethadione  (5,8).  Rigid analogues of a l k y l substituents have been tested f o r anticonvulsant a c t i v i t y .  These compounds can a l t e r the s t e r i c properties and  metabolism of the f l e x i b l e a l k y l groups.  25  Thus cyclo C^-C^  spiro  com-  pounds  of hydantoin  (95),  oxazolidinediones (95),  succinimides (95),  c a r b o x y l i c acids (88) and barbiturates (94) have been s t u d i e d . have  been  no g e n e r a l i z a t i o n s as to whether  certain  There  spirocycloalkyl  groups confer d e s i r a b l e pharmacological p r o p e r t i e s . The tures  1,4-benzodiazepines and dibenzazepines  with  different  substitution  structure-activity  i s considered  diazepines (96).  have more r i g i d s t r u c -  requirements.  5-Phenyl  to be necessary f o r a l l a c t i v e 1,4-benzo-  Diazepam and carbamazepine (Figure 2) both have l i p o -  p h i l i c groups and the t y p i c a l amido s t r u c t u r e containing the e l e c t r o n donor group or hydrogen-bonding group as i n other a n t i e p i l e p t i c drugs. According  to Camerman and Camerman (18), the phenyl groups i n phenytoin  overlap with those of diazepam when c r y s t a l structures are examined by x-ray c r y s t a l l o g r a p h y .  Other c l a s s e s of compounds with anticonvulsant  a c t i v i t y a r e the y - b u t y r o l a c t o n e s  (XXIV) and g l u t a r i m i d e s .  Klunk  a,a-disubstituted y-butyrolactones  to be  XXIV  et  a l . (47) recently  found  potent anticonvulsants when examined i n the s.c. PTZ t e s t .  Somers (98)  has described the anticonvulsant properties of B-methyl-B-butyl g l u t a r i mide  and  convulsant  (Bemegride®).  Klunk  butyrolactones  such  properties  of  B-methyl-B-ethylglutarimide  and co-workers (97) have found as  B-ethyl-B-methyl-y-butyrolactone  26  B , B - d i a l k y l yt o be con-  vulsants. Anticonvulsant  or convulsant properties may be obtained by minor  s t r u c t u r a l changes i n the side chains.  There are s t r u c t u r a l features  that  i n the SAR of  may  confer  drugs (94). urates (94), chain  convulsant Certain  branched  properties  benzylethylbarbiturates (94), chain  anticonvulsant dibenzylbarbit-  a l k y l e t h y l b a r b i t u r a t e s (17,99),  a l k e n y l a l k y l b a r b i t u r a t e s (17,99), N-alkylated  branched  and di-N-alkylated  compounds, e.g. secondary and t e r t i a r y amides of v a l p r o i c a c i d (3,4), 1,3,5,5-tetraalkylbarbiturates  (94).  2,2,3,3-tetraalkyl  imides (97) and 5-methyl-l,4-benzodiazepine (30) are known  succin-  convulsants.  According to studies by Andrews et a l . (19) and Downes et a l . (100), the terminal  isopropyl  and isopropenyl  groups i n 5 , 5 - d i a l k y l b a r b i t u r a t e s  (Figure 3) appear to be necessary f o r convulsant a c t i o n . 7.  S t r u c t u r a l S p e c i f i c i t y of Anticonvulsants A n t i e p i l e p t i c drugs appear to show low s t r u c t u r a l and b i o l o g i c a l  specificity.  This may be due to the few pharmacophoric groups i n these  compounds and t h e i r a b i l i t y drugs show absolute  to modulate GABA e f f e c t s .  biological  s p e c i f i c i t y , most drugs act by i n t e r -  a c t i o n with s p e c i f i c bioreceptors.  S p e c i f i c i t y of b i o l o g i c a l a c t i o n i s  determined by the combination and stereochemical groups and t h e i r  Although few  arrangement of chemical  p h y s i c a l i n t e r a c t i o n with s p e c i f i c binding  s i t e s or  receptors. By v i r t u e of t h e i r nonspecific perturbation of c e l l membranes and lack of s t r u c t u r a l s p e c i f i c i t y , general anesthetics have been c l a s s i f i e d as s t r u c t u r a l l y nonspecific drugs (101).  General anesthetics have been  regarded as non-polar, i n e r t and nonionizable  27  compounds which do not  ANTICONVULSANTS  CONVULSANTS  DIFFERENCES double bond (isopropenyl)  methyl group (isopropyl)  methyl group (isopropenyl)  double bond (cyclohexylidene)  methylene group (benzyl)  Figure 3. S t r u c t u r a l l y - r e l a t e d convulsant and anticonvulsant barbiturates.  28  have h i g h l y r e a c t i v e p o l a r groups t o i n t e r a c t w i t h s p e c i f i c receptors but p a r t i t i o n i n t o c e l l membranes to secondarily disrupt i o n i c channels i n the membrane s t r u c t u r e (101). Most a n t i e p i l e p t i c d r u g s , however, show s e d a t i v e e f f e c t s .  It i s  g e n e r a l l y assumed t h a t s e d a t i v e , h y p n o t i c and a n e s t h e t i c a c t i o n s of anticonvulsants occur w i t h increasing the dose of the compound. In some compounds, s e d a t i v e  and  anticonvulsant  effects  overlap  although  phenytoin i s reported to exert i t s anticonvulsant e f f e c t at non-sedative doses (42).  I t i s known that anticonvulsants are not the only c l a s s of  compounds with sedative a c t i o n s .  Most CNS-active agents show sedative  properties probably because they have s u f f i c i e n t l i p o p h i l i c i t y to penet r a t e the blood b r a i n b a r r i e r t o i n t e r a c t n o n s p e c i f i c a l l y w i t h b r a i n membranes. Even c e r t a i n convulsants have been reported to show sedative properties (59).  I t may  be premature to conclude that v a l p r o i c a c i d and  analogues a c t by n o n s p e c i f i c mechanisms as suggested by P e r l m a n and Goldstein (14).  These workers i n d i c a t e d t h a t the a b i l i t y of e t h a n o l ,  v a l p r o i c a c i d and  homologous congeners to d i s o r d e r c e l l membranes  correlated w e l l w i t h t h e i r sedative and anticonvulsant potencies.  Some  i n v e s t i g a t o r s studied other d i r e c t p h y s i o l o g i c a l and biochemical e f f e c t s of v a l p r o i c a c i d analogues and a l s o r e p o r t e d good c o r r e l a t i o n w i t h anticonvulsant potencies (12,13,20,81,82). A n t i e p i l e p t i c drugs can be described as more s t r u c t u r a l l y s p e c i f i c than anesthetics. Barbiturates are known to show marked s p e c i f i c i t y . Small changes i n the a l k y l side chain of c e r t a i n anticonvulsant b a r b i t u r a t e s can produce c o n v u l s a n t  p r o p e r t i e s ( F i g u r e 3).  The  selective  nature of a n t i e p i l e p t i c drug therapy w i t h anticonvulsants such as phenyt o i n , p h e n o b a r b i t a l , v a l p r o i c a c i d and e t h o s u x i m i d e a t t e s t t o some degree of s t r u c t u r a l s p e c i f i c i t y .  These f i n d i n g s suggest that anticon29  v u l s a n t s may show d i s c r e t e or s e l e c t i v e e f f e c t s on n e u r o n a l c e l l structures. Recent advances i n neurophysiological and biochemical have allowed  extensive  i n v e s t i g a t i o n s of the s e l e c t i v e  techniques synaptic or  c e l l u l a r actions of some of the a n t i e p i l e p t i c drugs (74,102).  Further-  more, r a d i o l i g a n d b i n d i n g s t u d i e s (30) have added support t o t h e suggestion that d i f f e r e n t subgroups of anticonvulsants e x i s t , each with a d i f f e r e n t binding s i t e but i n close a s s o c i a t i o n and probably each with its  own s t r u c t u r e - a c t i v i t y  affinity,  relationships.  The observation  of high  saturable and i n some cases s t e r e o s p e c i f i c binding of 1,4-  benzodiazepines (103), GABA and analogues (30), d i h y d r o p i c r o t o x i n i n (33) and phenytoin (85) have added weight to proposals of s p e c i f i c  molecular  mechanisms of a n t i e p i l e p t i c and convulsant a c t i o n s . 1,4-Benzodiazepines have been documented to enhance GABA binding and also vice-versa (30).  Barbiturates are known to enhance the binding  of benzodiazepines (EC^Q of pentobarbital  100 uM) and GABA(30).  Picro-  t o x i n at concentrations of 1 uM i s reported to i n h i b i t the enhancement of benzodiazepine binding by barbiturates (30).  Barbiturates and v a l -  proic a c i d are suggested to act on the same s i t e s as convulsants such as p i c r o t o x i n , 1,5-alkyl-substituted t e t r a z o l e s and t e r t - b u t y l b i c y c l o p h o s p h o n o t h i o n a t e (30,33,34,56).  The c o n v u l s a n t  suggested  a t the s i t e  chloride (DHP)  to exert ions,  i t s effects  controlling  the c h l o r i d e ionophore (30).  has been  the f l u x of  H-Dihydropicrotoxinin  and "^S-tert-butylbicyclophosphothionate (TBPS) have been used t o  assay p i c r o t o x i n binding with  picrotoxin  s i t e s (30,33,56,104).  Barbiturates competed  H-DHP binding with r e l a t i v e potencies that c o r r e l a t e d with t h e i r  ability  to enhance  H-GABA  binding (33,104).  30  P e n t o b a r b i t a l , pheno-  b a r b i t a l and v a l p r o i c acid are reported to i n h i b i t the binding of H-DHP with I C 5 0  of 50 uM, 400 yM and 500 yM r e s p e c t i v e l y (33,34).  Valproic  a c i d , i n comparison to b a r b i t u r a t e s , did not i n h i b i t the binding of HGABA (87) or H-diazepam (34) at concentrations up to 1 mM.  Studies on 35  other anticonvulsants have shown that ethosuximide i n h i b i t e d binding by 30% at 1 mM while a-ethyl-a-methylbutylrolactone 35  S-TBPS inhibited  S-TBPS binding by 23% at 0.5 mM ( 3 2 ) . 3  Phenytoin  i n h i b i t e d H-DHP binding with an I C 5 Q  of 100 yM (30).  The binding of phenytoin to i t s r e c o g n i t i o n s i t e i s reported to i n t e r a c t with  the GABA-BDZ-receptor C l ~ ionophore complex ( 8 5 ) .  Using  spinal  cord neuronal c u l t u r e , Macdonald and McLean (102) i n d i c a t e d that phenytoin  augmented  (>8yM) than  postsynaptic  inhibition  GABA  response  at higher  concentrations  of post-tetanic p o t e n t i a t i o n (high  frequency  r e p e t i t i v e f i r i n g of neurons) which occurred a t therapeutic free serum concentrations  (4-8 yM).  Phenytoin  and carbamazepine, which show  s i g n i f i c a n t e f f e c t on p o s t - t e t a n i c p o t e n t i a t i o n compared t o other a n t i e p i l e p t i c s , are reported to decrease calcium i n f l u x across synaptosomes and  affect  significantly  phosphorylation  calcium-calmodulin  dependent  protein  (105).  Figure 4 depicts  a model  of the GABA  receptor-benzodiazepine-  c h l o r i d e ionophore complex developed by various i n v e s t i g a t o r s f o r s i t e s of  a c t i o n of some anticonvulsants  and convulsants.  The f u n c t i o n a l  coupling between the GABA receptor and the 1,4-benzodiazepine receptor and p i c r o t o x i n i n s i t e s have given suggestive evidence of a common mechanism  of a c t i o n i n v o l v i n g  modulatory  effects  on the i n h i b i t o r y  neurotransmitter, GABA. The model a l s o shows the wide v a r i e t y of s t r u c tures which i n t e r a c t on each r e c o g n i t i o n s i t e . indicated m u l t i p l e r e c o g n i t i o n s i t e s 31  Detailed studies have  f o r the GABA and  benzodiazepine  GABA Receptor -GABA -Muscimol -Bicuculline  Chloride Ionophore  Benzodiazepine Receptor  -Picrotoxin  -1,4-Benzodiazepines  -t-Butylbicyclophosphonothionate  -Purines  -Anticonvulsant b a r b i t u r a t e s  -Nicotinamide  -Convulsant b a r b i t u r a t e s  -B-Carbolines  - V a l p r o i c acid -Ethosuximide -Convulsant t e t r a z o l e s -Purines  Figure 4.  Model of GABA-benzodiazepine-receptor complex  32  c h l o r i d e ionophore  r e c e p t o r complex ( 3 0 , 1 0 3 ) .  A r e c e n t study (107)  i n v e s t i g a t e d the  35 k i n e t i c s of i n h i b i t i o n  of  S-TBPS by barbiturates f o r evidence that  there i s one c l a s s of binding s i t e s f o r p i c r o t o x i n and b a r b i t u r a t e s . I t was  suggested  linked  that  picrotoxin/TBPS  to b a r b i t u r a t e s i t e s .  binding  sites  are a l l o s t e r i c a l l y  There are also reports of d i f f e r e n c e s  between a l i p h a t i c - s u b s t i t u t e d  b a r b i t u r a t e s and p h e n y l - s u b s t i t u t e d  barbiturates i n t h e i r a c t i o n t o augment GABA responses (31,108). 8.  Pharmacokinetics  a.  Human Valproic  of V a l p r o i c Acid  a c i d i s r a p i d l y and n e a r l y c o m p l e t e l y  absorbed  f o l l o w i n g o r a l c l i n i c a l doses of 15-60 mg/kg with peak blood l e v e l s occurring w i t h i n 1-4 hr (109).  The e l i m i n a t i o n h a l f - l i f e i s i n the  range of 6-18 h r , with shorter h a l f - l i v e s obtained i n the presence of  antiepileptic  acid (109).  drugs which induce the metabolism of v a l p r o i c  T o t a l plasma clearance i s 0.07-0.14 ml/min/kg with an  apparent volume of d i s t r i b u t i o n of 0.1-0.4L/kg (109-111). protein binding of the drug at therapeutic plasma (50-100 mg/L) ranges  from 80-95% (111).  Plasma  concentrations  P r o t e i n binding  sites  become saturated at concentrations greater than 80 mg/L (111,112). Plasma clearance of VPA i s dependent on free f r a c t i o n and i n t r i n s i c clearance. These  pharmacokinetic  parameters  i n d i c a t e that  VPA  d i s t r i b u t i o n i s l i m i t e d mostly to e x t r a c e l l u l a r f l u i d s with only minor  tissue  uptake.  Such  distributional  c h a r a c t e r i s t i c s are  a t t r i b u t e d to the low pKa of VPA and the high plasma p r o t e i n bind-  33  ing.  Studies on s i n g l e dose and m u l t i p l e dose k i n e t i c s of VPA by  Acheampong et a l . (113) and Bowdle et a l . (114)  indicate  increased  plasma clearance at higher doses due t o an increase i n free f r a c tion.  There i s a tendency f o r a decrease i n i n t r i n s i c clearance at  higher doses probably as a r e s u l t of saturation of metabolism or a u t o i n h i b i t i o n of metabolism (113,114). Rodents A f t e r o r a l administration  of 200 mg/kg i n mice, valproate i s  reported to be r a p i d l y absorbed with maximum serum concentrations reached w i t h i n 5-15 min (70).  There are q u a n t i t a t i v e  i n pharmacokinetic properties  differences  of the drug i n rodents compared to  human, apparently due to differences i n protein b i n d i n g . life  of v a l p r o i c  r a t s (70,115). and  acid  The h a l f -  i s 0.8 h r i n mouse and 1-4 h r i n  Volume of d i s t r i b u t i o n i s about 0.66 L/kg i n r a t  0.33 L/kg i n mice with t o t a l body clearance of 4.17 ml/min/kg  i n r a t s and 4.33 ml/min/kg i n mice (110).  At plasma concentrations  of 50-80 ug/mL, the plasma protein-binding of the drug i s about 90% i n human, 63% i n rat and 12% i n mice (110). K i n e t i c s of Valproic Acid i n the CNS Valproic acid has been shown t o enter the CNS r a p i d l y with maximal  brain  concentrations  reached  at 5-10 min i n mice (70).  Several studies have demonstrated that regional d i s t r i b u t i o n of the drug  i n the brain  genous (116,117). showed bulbs  of mice Analysis  and r a t s  i s relatively  of d i s c r e t e  gradual  accumulation  (116,117).  Valproic 34  of acid  brain  the drug does  areas,  homo-  however,  i n olfactory  not bind  to brain  t i s s u e s (118).  Studies on the s u b c e l l u l a r d i s t r i b u t i o n of the drug  by A l y and A b d e l - L a t i f (119) i n d i c a t e d that i t i s mainly associated with the soluble and mitochondrial f r a c t i o n s .  I t has a l s o been  documented that the drug i s r a p i d l y cleared from the b r a i n (120). The rapid b r a i n uptake of v a l p r o i c acid i s suggested to be due to an a c t i v e - t r a n s p o r t mechanism (120) or r a p i d  d i s s o c i a t i o n of  protein-bound drug w i t h i n the brain c a p i l l a r i e s (121).  Human b r a i n  v a l p r o i c l e v e l s were found t o be 7-28% of l e v e l s i n plasma (122). In mice, b r a i n drug concentrations (5-60 mg/L) were found to be about 15-20% of those  i n serum (20-250 mg/L) a f t e r  o r a l admin-  i s t r a t i o n of 200 mg/kg of sodium valproate (70,117). 9.  Metabolism of V a l p r o i c Acid Metabolism i s the major route of v a l p r o i c acid e l i m i n a t i o n i n human  and rodents (123).  Several metabolites have been i d e n t i f i e d i n a number  of metabolism studies [reviewed i n (123)]. main m e t a b o l i c  pathways, namely g l u c u r o n i d a t i o n , B - o x i d a t i o n , w-  oxidation and ( w - l ) - o x i d a t i o n . Figure 5.  These studies i n d i c a t e four  The metabolic pathways are summarized i n  Glucuronidation and B-oxidation are the major pathways. The  major metabolites i n human are the glucuronide and 3-keto VPA (123).  3-  Keto VPA and 2-ene VPA are major plasma metabolites i n human, r a t s and mice (115).  Other metabolites that occur i n s i g n i f i c a n t q u a n t i t i e s i n  serum of p a t i e n t s are 2,3'-diene VPA, 4-0H VPA and 5-0H VPA. Acheampong et a l . (93) have i d e n t i f i e d a new metabolite, 4-keto VPA i n human serum and u r i n e . malonic  The urinary compounds, 2-propylsuccinic a c i d and 2-propyl-  acids were confirmed  as v a l p r o i c acid metabolites by s t a b l e -  isotope and GCMS techniques (93).  35  CH2-=CH-CH2  CH,—CH,—CH  CH2~CH-CH? CHCOOH  / CH3-CH2-CH2  CHCOOH CHpCH-CH^ 4,4'-d1ene VPA  4-eneVPA  CHpXH-CH  CH,-CH-CH  C-COOH  CHCOOH  OH  CHj-CH^CH^  CH2-CH2-CH2  (E) 2,4-dl ene VPA 3-ene VPA  CH3-CH2-CH2  CH,—CH,—CH v 3 2 ^ C—COOH  5-OH VPA  CH;J-CH2-CH2/  ;CHCOOH CHj-CH2-CH2  CH3—C—CH2  I  2-Propvlglutarlc a d d  1 1  COOH CHj™ CH^**"" CH^"™ C H ^ COOH 2-Propylmalonlc a d d  Figure 5.  i  /  CHCOOH  CH 3 -CH 2 —CH 2  /  0  2(E).3'(E)-d1ene VPA  H  CH3-CH2-CHX CH-COOH CH3—CH2—CH2  4-Keto VPA  1  HOOC-CH,  CHCOOH CH3—CH2—CH2 2-Propylsucdnic acid  Metabolic pathways of valproic acid (VPA).  COOH CH—CH 2 — CH  2-ene VPA  HOOC-CH2—CH2  CH,—CH=CH  3-OH VPA  C„_CH2-C  /  CH3-CH2-CH2  3-Kcto VPA  CHCOOH  There have been more recent studies which sought to f u r t h e r d e l i n eate the metabolic pathways of the drug, with emphasis on the i d e n t i t y of  diunsaturated  u r i n e (124,125). pentenoic  acid  metabolites Only two  and  elusive,  diunsaturated  human  metabolites,  serum  and  2-[2'-propenyl]-4acid  have  been  I d e n t i f i c a t i o n of the major diene metabolite  primarily  acid  diene  in  2-propyl-(E)-2,4-pentadenoic  i d e n t i f i e d (128,129). been  detected  due  isomers and  to m u l t i p l i c i t y a l s o the  of  the  unavailability  has  possible  of s y n t h e t i c  reference m a t e r i a l . 10.  T o x i c i t y of V a l p r o i c Acid Nausea, v o m i t i n g , abdominal cramps and  commonly reported especially  at high  ammonemia and  side e f f e c t s (35). doses and  generally been mild ( 3 5 ) .  Drowsiness and  with combination  hyperglycinemia  v a l p r o i c acid therapy (127).  d i a r r h e a are the most sedation  therapy ( 3 5 ) .  Hyper-  have been observed to occur  during  Side e f f e c t s associated with the drug have However, i n more recent years more serious  side e f f e c t s have been observed.  These include thrombocytopenia (128),  p a n c r e a t i t i s (126) and hepatic damage (128-131). t o x i c i t y i s of major concern.  The p o t e n t i a l hepatic  In most serious cases, l e t h a l e f f e c t s of  v a l p r o i c a c i d due to hepatic f a i l u r e have been v a r i o u s l y reported 130).  occur,  (128-  The proposed mechanism f o r the hepatic t o x i c i t y and/or the Reye-  l i k e syndrome (131) induced by v a l p r o i c a c i d include d i r e c t t o x i c e f f e c t of a metabolite, possibly 4-ene VPA  which i s a s t r u c t u r a l analogue of  the hepatotoxin, 4-pentenoic acid (130,132).  11.  A Quantitative S t r u c t u r e - A c t i v i t y Model One appropriate method of examining the r e l a t i o n s h i p between s t r u c -  tures of c l o s e l y - r e l a t e d congeners and 37  anticonvulsant a c t i v i t y was  to  determine the q u a n t i t a t i v e r e l a t i o n s h i p between s t r u c t u r a l properties or s u b s t r u c t u r a l d e s c r i p t o r s and p h a r m a c o l o g i c a l a c t i v i t y .  Hansch and  c o l l a b o r a t o r s (25,29) have a p p l i e d the l i n e a r f r e e energy r e l a t i o n s h i p approach to i n v i t r o and i n vivo conditions where physicochemical i n t e r a c t i o n s between drugs and  b i o m a c r o r a o l e c u l a r s i t e s of a c t i o n can  be  q u a n t i t a t i v e l y expressed i n terms of r e g r e s s i o n e q u a t i o n s i n v o l v i n g hydrophobic (TT), e l e c t r o n i c (o), s t e r i c (Eg) and other s t r u c t u r a l parameters. The  Hansch method models the dynamic s i t u a t i o n i n a b i o l o g i c a l  system where a drug i s applied at a dose C and proceeds through several biophases to reach the t a r g e t s i t e w i t h p r o b a b i l i t y of a c c e s s , A and e f f e c t s response reaction with a rate or e q u i l i b r i u m constant, K x «  Thus  the r a t e of b i o l o g i c a l response at the r e a c t i o n s i t e can be expressed mathematically as  d(response)/dt = ACKX  (i)  For most drugs the transport process or p r o b a b i l i t y of reaching the s i t e of a c t i o n i s d e t e r m i n e d l a r g e l y by l i p i d s o l u b i l i t y or p a r t i t i o n  co-  e f f i c i e n t (TT or l o g P ) , i . e .  A = f Or)  (ii)  On the b a s i s of an e m p i r i c a l p a r a b o l i c r e l a t i o n s h i p between a drug's b i o l o g i c a l a c t i v i t y and p a r t i t i o n c o e f f i c i e n t , Hansch (25) expresses the function i n the form of a normal Gaussian d i s t r i b u t i o n .  38  2  _ L _ e -(TT-TT 0 ) /2S2  f = S/2TT  .(iii)  From equations ( i ) , ( i i ) ,  d(response)/dt  =  (iii)  2 -(TT-7r ) /b. C .K x AE 0  (iv)  1 where a = s/2-n, b = 2s , s = standard d e v i a t i o n parameter  At a f i x e d time i n t e r v a l a f t e r a d m i n i s t r a t i o n of e q u i e f f e c t i v e doses C (e.g. EDIJQ), a constant b i o l o g i c a l response i s determined under steady s t a t e c o n d i t i o n s , i.e.  d (response) = constant = ae"^ dt  71-7  2 ^) /°.KXC  Taking logarithms, 2 D TI n log (constant) = l o g [ae-( '-' 'o) / « C . K X ] and  log 1/C = -n lTr 2 + n2Tr.Tr0 -  n3TT0  2  + l o g K x + 114  (v)  The Hansch method r e l i e s on the physicochemical i n t e r a c t i o n phenomenon a t the c r i t i c a l  site  of a c t i o n  which  i s determined  by  the  hydrophobic (IT), e l e c t r o n i c (s) and s t e r i c (Eg) f a c t o r s . log K x = kTT + k30 + k4E  .....(vi)  s  Thus 39  log  1/C = k-j^-rr + k27T + k^O + k^Eg + ks  Equations  ( v i ) and ( v i i ) are l i n e a r  (vii) free  energy  relationships  showing the a d d i t i v e combination of the free-energy related parameters. 2 The f i r s t two terms i n TT describe the transport process. The TT v a r i able i n equation ( v i i ) i s generally agreed to be e s s e n t i a l i n complex b i o l o g i c a l systems, f o r instance i n whole animal s t u d i e s . In simpler 9 b i o l o g i c a l systems TT may not be required and TT describes hydrophobic effects (25).  On the basis of the a d d i t i v e - c o n s t i t u t i v e properties of  p a r t i t i o n c o e f f i c i e n t (P) revealed by Hansch (133), equation ( v i i ) can also be expressed as log  1/C = k x l o g P + k 2 ( l o g P )  2  + k3a + k4Es + k5  (viii)  where log P =  ZTT  or l o g P  ( R - X ) = TT(R)  + TT(X)  L i p o p h i l i c i t y can be considered as the r e l a t i v e a f f i n i t y of a drug f o r the  l i p i d biophase and i s q u a n t i t a t i v e l y defined i n terms of p a r t i t i o n  coefficient  (P) which c h a r a c t e r i z e s t h e e q u i l i b r a t i o n between t h e  aqueous and l i p i d phases. 12.  Physicochemical Parameters Used i n  QSAR  S t e r i c and e l e c t r o n i c e f f e c t s i n drug structures are not as w e l l defined as l i p o p h i l i c i t y .  Examination of the structures of v a l p r o i c  a c i d analogues i n Figure 2 shows that the l i p o p h i l i c parameter describes the  hydrophobic character of the a l k y l groups, the e l e c t r o n i c parameter  evaluates the e l e c t r o n i c properties of the polar moiety and the sub-  40  stitution  pattern  or s t e r i c  effects  characterized by the s t e r i c parameter.  o f the a l k y l  chain  can be  Since the regression  equation  ( v i i i ) i s a l i n e a r free energy r e l a t i o n s h i p , other free-energy r e l a t e d parameters can be used.  The e l e c t r o n i c parameters used frequently i n  QSAR are the Hammett's s u b s t i t u t i o n constant ( a * ) , pKa, ApKa and d i p o l e moment ( u ) . function  I o n i z a t i o n constant  of p o l a r e f f e c t s  (pKa) i s generally considered  as a  d e s c r i b i n g the e l e c t r o n - w i t h d r a w i n g  or  e l e c t r o n - r e l e a s i n g e f f e c t s (134). There are various methods f o r the determination acidic  compounds.  These  include  potentiometric  of pKa values of titration  spectrophotometry methods, and c o n d u c t i v i t y methods. such as valproate analogues, s o l u b i l i t y  the  For many drugs  and UV absorption  i s t i c s appear to determine the methods of choice.  methods,  character-  In one study (69),  pKa of v a l p r o i c acid was determined by potentiometric  titration  using acetone-water media. Dipole moments of a number of anticonvulsant drugs have a l s o been c a l c u l a t e d by measurement of the d i e l e c t r i c constant and molar r e f r a c t i o n  of the drug s o l u t i o n s and solvents (135).  L i p o p h i l i c i t y of compounds i s e i t h e r determined experimentally or c a l culated using substituent constants. 13.  Hydrophobic Parameters  a.  Determination of l i p o p h i l i c i t y by the shake-flask procedure Determination  of the l i p o p h i l i c i t y of a drug u s u a l l y requires  measurement of the e q u i l i b r i u m constant  or p a r t i t i o n  between the non-polar and aqueous phases using cedures.  coefficient  shake-flask  pro-  The choice of a model f o r the l i p i d phase i n biomembranes  has been a r b i t r a r y , ranging from highly nonpolar solvents t o mod41  e r a t e l y nonpolar solvents (133). used nonpolar s o l v e n t .  Octan-l-ol i s the most frequently  Apart from preferred advantages of octanol  i n terms of a v a i l a b i l i t y , p u r i t y and b i f u n c t i o n a l properties (133), octanol i s probably as appropriate as other nonpolar solvents f o r modelling biomembranes. values can be converted has  Collander (136) has showed that p a r t i t i o n between organic solvent-water systems.  It  been found, however, that f o r a successful conversion, con-  sideration  ought to be  given  properties between octanol and  to d i f f e r e n c e s i n hydrogen-bonding other organic solvents as w e l l as  hydrogen-donor or acceptor properties of the solutes (133,136). b.  HPLC determination of l i p o p h i l i c i t y The  t r a d i t i o n a l shake-flask procedure of determining octanol-  water p a r t i t i o n c o e f f i c i e n t (log P) has been observed to have many a n a l y t i c a l problems (137-141).  I t i s tedious and  time-consuming.  S o l u b i l i t y d i f f i c u l t i e s i n aqueous media, i n s t a b i l i t y of compounds i n l i q u i d phases and l o s s of m a t e r i a l by adsorption or other mechanisms have been a major problem.  A l t e r n a t i v e methods, e s p e c i a l l y  chromatography methods (TLC, HPLC) that provide a rapid and  accur-  ate estimate of l i p o p h i l i c i t y have been sought.  years,  reverse-phase silica  (RP)-HPLC, with a hydrophobic column c o n s i s t i n g of  particles  c h a i n , has  In recent  coated with covalent-bonded C-18  and  C-8  alkyl  been used to study hydrophobic e f f e c t s (137-143) and  s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (144). The reverse-phase HPLC method i s based on the determination of r e t e n t i o n parameters which are then c o r r e l a t e d with log P values. I t i s a l s o required that the r e t e n t i o n parameter be  42  reproducible  and  accounted  f o r by  a mechanism  to r e f l e c t  behaviour of solute i n l i q u i d phases.  only  The l i n e a r  partitioning relationships  reported between l o g P and capacity f a c t o r of compounds found by numerous i n v e s t i g a t o r s (137-150) appear to be a close p a r a l l e l to the  l i n e a r regression equation found by Collander (136) to e x i s t  between p a r t i t i o n c o e f f i c i e n t s determined i n two d i f f e r e n t nonpolar solvent-aqueous p a r t i t i o n i n g systems. The  unique features of the monomeric bonded-phase chromato-  graphic  column  i s the  presence  of  a nonpolar C-18  hydrocarbon chain chemically bonded to the s i l i c a various methods f o r attaching porous  or p e l l i c u l a r  surface s i l a n o l (-Si-N-)  and  silica  or  C-8  support.  The  a covalently-bonded phase to the support r e l y  on  the conversion of  groups to s i l i c a t e esters (-Si-OR), aminosilanes  siloxanes  (-Si-O-Si-R^).  The  most w i d e l y  used  chemically bonded stationary phases are those based on siloxanes These relatively 8 (151).  stable  phases  against h y d r o l y s i s w i t h i n  are  known  to  be  the pH range of 2-  Siloxane bonded phase packings are commercially a v a i l a b l e  with m i c r o p a r t i c u l a r supports and a monomeric C-18  organic layer  such as H y p e r s i l ODS used i n t h i s study. Different P0/w  reverse phase HPLC procedures of determining l o g  have emerged over the years of i t s a p p l i c a t i o n .  treated (138,140,141) and s i l y l a t e d (140) octadecylsilane phase columns have been used as the stationary phase. ent  Both unreverse  In a d i f f e r -  procedure, M i r r l e e s et a l . (139) coated s i l y l a t e d s i l i c a with a  l a y e r of n-octanol and used n-octanol-saturated water as the mobile phase. water  This was suggested to be c l o s e l y analogous to the octanolpartitioning  system. 43  Unger and  co-workers (137)  used  octanol-coated  C-18  bonded  silica  as  the  s t a t i o n a r y phase  and  octanol-saturated phosphate buffer as the e l u e n t . HPLC methods have been used to demonstrate good c o r r e l a t i o n s between  capacity  alkylcarboxylic  f a c t o r s and  log P Q / W  acids (145,146).  In a  values study  a l i p h a t i c a c i d s (C7-C20), d'Amboise and  of s t r a i g h t - c h a i n of  long  Hanai (145)  chain  n-  reported  excellent c o r r e l a t i o n s with u n a c i d i f i e d 50% a c e t o n i t r i l e - w a t e r as the mobile phase among d i f f e r e n t compositions n i t r i l e and tetrahydrofuran.  of methanol, aceto-  Tanaka and Thorton (153) studied the  hydrophobic e f f e c t s of various c a r b o x y l i c acids using a u-Bondapak C^g-coluran  and methanol-O.OlM sodium phosphate buffer (pH 3.0-3.5)  of d i f f e r e n t compositions.  To  suppress i o n i z a t i o n  minimize adsorption (NH^+) of a s e r i e s of aromatic  (low pH)  and  acids i n one  study (147), 50% methanol-ammonium phosphate (pH 2.15) and H y p e r s i l ODS  was used to determine the capacity f a c t o r s .  In t h i s study the  HPLC method i s used to determine the c a p a c i t y f a c t o r s of compounds as w e l l as the log P Q / W  values.  the  This requires use of  reference compounds and o p t i m i z a t i o n of the a n a l y t i c a l procedure to be s e n s i t i v e to s t r u c t u r a l hydrophobic e f f e c t s of varied c a r b o x y l i c acids and t e t r a z o l e s .  The log P values obtained i n the HPLC method  are evaluated i n terms of accuracy  by comparison to r e s u l t s from  the shake-flask procedure. Although the nature of the s t a t i o n a r y phase during the chromatographic  separations  chemically-bonded  and  the  phases i s s t i l l  mechanism not  of  retention i n  completely  understood, a  p a r t i t i o n - a d s o r p t i o n mechanism has been postulated by various i n v e s t i g a t o r s (148-150) f o r the r e t e n t i o n of s o l u t e s .  44  According  to  the  proposed mechanism, the stationary phase i s a combination of  C-18 hydrocarbon l a y e r , r e s i d u a l s i l a n o l present on the s i l i c a surface and associated solvent molecules from the mobile phase. solute  molecules are suggested  to adsorb  on a modified  The  silica  surface with an adsorbed layer of mobile phase components or partition  into a l i q u i d  phase formed by the s i l i c a  bonded-C^g layer  with associated solvent molecules from the bulk e l u e n t . The  application  of l i q u i d  chromatography  to measurement of  physicochemical properties such as l o g P has been based on chromatographic theory.  A t h e o r e t i c a l treatment of a pure p a r t i t i o n  process i n r e l a t i o n to the r e t e n t i o n parameter by Snyder  and  Kirkland (151) showed, i n the e q u i l i b r i u m s i t u a t i o n , the r e l a t i o n ship between the observed chromatographic r e t e n t i o n parameter and the d i s t r i b u t i o n c o e f f i c i e n t k» = K_Vs V  m  where k' i s the capacity f a c t o r , V m i s the t o t a l volume of solvent w i t h i n the column, V g i s the t o t a l volume of the stationary phase and K i s the d i s t r i b u t i o n c o e f f i c i e n t which defines the e q u i l i b r i u m d i s t r i b u t i o n of solute between the s t a t i o n a r y phase and the mobile phase. c.  Determination of l i p o p h i l i c i t y using substituent constants Other methods of estimating l o g P values are the Hansch TT method (133,152) and Rekker fragmental method (153). are  Both methods  based on the a d d i t i v e - c o n s t i t u t i v e properties of p a r t i t i o n co-  e f f i c i e n t of compounds, but the approaches are d i f f e r e n t . 45  Hansch TT  values are c a l c u l a t e d from the r e l a t i o n , TTx = l o g P (R-X) - l o g P (R-H). The  Hansch method  coefficient  usually  requires  of the parent compound  the octanol-water p a r t i t i o n (R-H).  Rekker adopted an  e m p i r i c a l approach based on the r e l a t i o n log P (R-X) = Z a n f n or l o g P (R-X) = f(R) + f ( X ) Thus f  i s the fragment constant f o r the molecular fragment, n and  a n i s the numerical f a c t o r i n d i c a t i n g  the number of times a given  fragment n appears i n the s t r u c t u r e .  The fragment constants are  obtained from s t a t i s t i c a l data reduction procedures (153) i n which contributions from the molecular fragment, n, i n various molecular structures are averaged.  Thus, the l i n e a r regression method has a n  as the independent parameter, l o g P as the dependent v a r i a b l e and the  f  n  values are determined as the regression c o e f f i c i e n t .  One  r e s u l t of the Rekker estimation method i s the p r e d i c t i o n that the hydrogen atom i n a molecular s t r u c t u r e contributes s i g n i f i c a n t l y to the  partition coefficient.  Thus compared to Hansch Tr-values, f -  values d i f f e r f o r C, CH, CH 2 and CH 3 .  46  EXPERIMENTAL  A.  Chemicals and M a t e r i a l s  1.  Synthesis Chemicals  were reagent  grade and procured from t h e f o l l o w i n g  sources. a.  A l d r i c h Chemical Co. (Milwaukee, Wisconsin) Aluminium  chloride  (anhydrous),  n-Butyl  bromide, n-Butyl-  l i t h i u m (1.6M i n hexane), Calcium hydride, Cyclohexylmethylbromide, Deuterochloroform acid,  (gold  2-Ethylhexanoic  label), acid,  Hexamethylphosphoramide, hydride,  Magnesium  Diisopropylamine, 2-Ethylbutyric  2-Ethoxyethanol,  Isobutyric  sulfate  acid,  (anhydrous),  n-Heptyl Lithium  bromide, aluminium  N-Methyl-N-nitroso-p-  toluenesulfonamide (Diazald®), 1-Methylcyclohexanecarboxylic a c i d , Pentan-2-one,  (E)-2-Pentenoic  acid,  Potassium  hydride  (35%  oil  d i s p e r s i o n ) , n-Propyl bromide, n-Propyl i o d i d e , Sodium hydride (50% o i l d i s p e r s i o n ) , Tetrahydrofuran, Triethylamine. b.  B r i t i s h Drug House (Poole, U.K.) D i e t h y l malonate, Diethylamine, P y r i d i n e , Sodium cyanide.  c.  Eastman Kodak Co. (Rochester, New York) Sec-Butyl Ethyl  alcohol,  acetoacetate,  l-Bromo-3-methylbutane,  Methanesulfonyl  Sodium a z i d e , Succinic anhydride.  47  chloride,  Di-n-Butylamine, Propionaldehyde,  d.  Fisher S c i e n t i f i c Co. ( F a i r l a w n , New Jersey) Bromine, t-Butanol, Hydrogen bromide (48%), Potassium cyanide, Quinoline.  e.  M a l l i n k r o d t Chemicals ( S t . L o u i s , Missouri) E t h y l bromide, E t h y l i o d i d e , Potassium carbonate (anhydrous), Sodium bicarbonate, p-Toluenesulfonyl c h l o r i d e .  f.  Matheson Coleman and B e l l Co. (Norward, Ohio) Dimethylsulfoxide  (anhydrous),  Phosphorus  pentoxide,  Phos-  phorus t r i b r o m i d e . 2.  Thin-layer Chromatography Glass plates (20 x 20 cm) - CAMAG, B e r l i n , Germany. Ether solvent - USP grade Petroleum ether, 30°-60°C - USP grade Benzene - USP grade S i l i c a g e l G - E. Merck, Darmstadt, FR Germany S u l f u r i c a c i d (96%) - B r i t i s h Drug House S i l v e r n i t r a t e - Nichols Chemical Co., Vancouver, B.C. TLC Streaking Apparatus - Applied Science Lab L t d . , State C o l l e g e , Pennsylvania Spreader - Desaga, Heidelberg, FR Germany  3.  High-Performance L i q u i d Chromatography Norganic  c a r t r i d g e s , membrane  48  filters  ( f o r preparation  of HPLC  grade water  from deionized d i s t i l l e d  water) - M i l l i p o r e Corporation,  Bedford, Massachusetts. Sodium dihydrogen orthophosphate, monohydrate - B r i t i s h Drug House (Canada) L t d . , Toronto, O n t a r i o . 2-Ethylbutyric a c i d , 2-Ethylhexanoic a c i d , Trimethylacetic a c i d , nV a l e r i c acid - A l d r i c h Chemical Co. n-Heptanoic a c i d , n-Hexanoic a c i d - Eastman Kodak Co. n-Butyric  acid,  Octanoic  acid  - Nutritional  Biochemicals  Corp-  o r a t i o n , Cleveland, Ohio. 4.  Potentiometric T i t r i m e t r y Potassium hydrogen phthalate - B r i t i s h Drug House. Potassium  h y d r o x i d e - American  Scientific  and  Chemical  Co.,  S e a t t l e , Washington. Phosphate  pH  7.0  buffer  - VWR  Scientific  I n c . , San  Francisco,  California. Potassium dihydrogen orthophosphate - M a l l i n k r o d t Chemicals. 5.  Gas Chromatography - Mass Spectrometry 3% D e x s i l 300 on 100/120 Supelcoport - Supelco I n c . , B e l l e f o n t e ,  Pennsylvania. Fused s i l i c a  capillary  column  SE-54 - Hewlett Packard.  49  (25 m x 0.3 mm  i.d.) coated  with  Fused s i l i c a c a p i l l a r y column (25 m x 0.3 mm i.d.) with bonded 0V1701 - Quadrex Co. t-Butyldimethylsilyl  chloride  - A p p l i e d S c i e n c e Lab.,  State  C o l l e g e , Pennsylvania. N-Methyl-N-trimethylsilyltrifluoroacetamide,  trimethylanilinium  hydroxide (0.2M i n methanol) - P i e r c e Chemical Co., Rockford, I l l i n o i s . 2-Propyl-(E)-2,4-pentadienoic acid was a g i f t from Dr.T.A. B a i l l i e , School of Pharmacy, U n i v e r s i t y of Washington, S e a t t l e , Washington. 6.  Pharmacological Testing Pentylenetetrazole - A l d r i c h Chemical Co. Sodium c h l o r i d e - B r i t i s h Drug House. Hydrochloric acid - American S c i e n t i f i c and Chemical Co.  B.  Instrumentation  1.  Nuclear Magnetic Resonance Spectrometry Proton NMR spectra were recorded on a Bruker WP-80, Varian XL-100,  Nicolet-Oxford-270 or Bruker WH-400 spectrometer at the NMR f a c i l i t y i n the Department of Chemistry, U.B.C.  Spectra were taken with CDClg as  solvent and tetramethylsilane as an i n t e r n a l standard. 2.  I n f r a Red Spectrometry IR  spectra were obtained  with  sodium c h l o r i d e  d i s k s e i t h e r as  l i q u i d f i l m s or nujol mulls on a Unicam SP1000 spectrometer.  50  3.  U l t r a v i o l e t Spectrometry UV spectra were recorded i n 50% methanol on a Beckman Model 24 UV-  v i s i b l e spectrophotometer. 4.  Gas Chromatography Mass Spectrometry (GCMS)  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 Varian MAT-111 mass spectrometer v i a a  variable  slit  separator.  Electron  impact  mass spectra were  recorded at 70eV, i o n source pressure of 5.0 x 10 sion current of 300yA.  Computerized background  made to plot mass s p e c t r a .  Torr and emis-  subtractions were  The scan range was 5 to 500 daltons  with one scan taken every 5 s e c . T o t a l i o n chromatographic p l o t s were based on m/z 50 to 500.  Mass chromatograms were p l o t t e d i n  scan mode. The data were processed by an on-line Varian 620L computer system. GCMS a n a l y s i s was c a r r i e d out under the f o l l o w i n g c o n d i t i o n s : 3% D e x s i l 300 column (1.8rax 2 mm i . d . ) ; Oven temperature, 50°C t o 280°C at 8°C/min; Helium ( c a r r i e r gas) flow r a t e , 25 mL; I n j e c t i o n port temperature, 250°C; Separator temperature, 250°C; I n l e t l i n e temperature, 250°C. b.  F u s e d - s i l i c a C a p i l l a r y Columns C a p i l l a r y GCMS a n a l y s i s was done on a Hewlett Packard 5987A instrument equipped with an HP gas chromatograph, mass spectrometer and on-line data system.  E l e c t r o n impact mass spectra were record-  ed a t 70eV, i o n source pressure of 2.0 x 10~^ Torr and emission 51  current of 300 uA. The gas chromatograph was interfaced to the mass spectrometer via an open-split interface. GCMS analysis of t-BDMS derivatives was performed under the following conditions:  Dimethylsilicone column (12.5 m x 0.2 mm  i.d.), 0V-1701 column (25 m x 0.3 mm i.d.); Oven temperature, 50°C to 100°C at 30°C/min, 100OC to 260OC at 8QC/min; Helium flow rate, 1 mL/min; Splitless mode of injection; Source temperature, 200°C; transfer line temperature, 240°C; injection port temperature, 240°C. C.  Synthesis of Alkyl Carboxylic Acids, Tetrazoles and Succinamic Acids  1.  Synthesis of Alpha-Substituted Aliphatic Acids  a.  Synthesis of 2-Butylhexanoic Acid (II) A flame-dried 500 mL three-necked f l a s k , equipped with a graduated separatory funnel with septum inlet and reflux condenser connected to a mercury bubbler, was immersed in an ice-water bath. The reaction vessel was flushed with nitrogen and maintained under a nitrogen atmosphere throughout the reaction. Diisopropylamine (0.13 mol) in 100 mL anhydrous THF was placed in the flask.  After  cooling the mixture to 0°C, n-butyllithium i n hexane (81 mL of 1.6 M, 0.13 mol) was added dropwise and with stirring. The mixture was stirred for 20 min and then n-hexanoic acid (0.055 mol) added at a slow rate to maintain the reaction temperature below 0°C A milky white precipitate formed and then HMPA (0.13 mol) in THF was added. The resulting homogenous mixture was stirred at 0°C for an additional 20 min.  n-Butylbromide (0.055 mol) was added rapidly to 52  the dianion of hexanoic a c i d , during which time the r e a c t i o n temperature approached room temperature.  The r e a c t i o n was allowed to  proceed f o r a f u r t h e r 90 min b e f o r e being quenched w i t h 25% (80 mL).  HC1  The o r g a n i c l a y e r was separated and the aqueous l a y e r  extracted twice with petroleum ether, 30°-60°C (100 mL p o r t i o n s ) . The  combined o r g a n i c l a y e r s were washed i n t u r n w i t h 10%  s a t u r a t e d NaHC0 3 and s a t u r a t e d NaCl s o l u t i o n .  HC1,  The o r g a n i c l a y e r  was dried over anhydrous Na2S04» f i l t e r e d and the solvents removed with a rotary evaporator.  The residue was  then subjected to f r a c -  t i o n a l d i s t i l l a t i o n under reduced p r e s s u r e to o b t a i n 2 - b u t y l hexanoic acid 89°C/0.1 NMR  (70%  yield),  bp  96O-98OC/0.35 mm  [ L i t (154),  mm]. Spectrum:  6 0.7-1.0 ( t , 6H,  2CH 3 ), 1.1-1.8 (m,  12H,  6CH2>» 2.1-2.5 (m, 1H, CH), 8.3-10 (broad s, 1H, COOH). Synthesis of V a l p r o i c Acid ( I ) V a l p r o i c a c i d was  prepared,  i n a s i m i l a r f a s h i o n to t h a t  described above f o r 2-butylhexanoic a c i d , by r e a c t i o n of n - v a l e r i c acid (0.084 mol) w i t h n-propylbromide. a c i d , was  separated  distillation  [bp  from unreacted of  valproic  erature (69) bp 221°C at 760 NMR  Spectrum:  The f i n a l product, v a l p r o i c v a l e r i c a c i d by  acid  fractional  110°-ll2°C/1.4 mm;  lit-  mm].  60.8-1.1 ( t , 6H, 2CH 3 ), 1.1-1.8 (m, 8H, 4CH 2 ),  2.2-2.5 (m, 1H, CH), 8.0-10 (broad s, 1H, COOH). Synthesis of 2-Propyl-(E)-2-Pentenoic  Acid (VII)  V a l p r o i c acid (0.15 mol), synthesized as described above,  was  placed i n a 250 mL f l a s k equipped w i t h a r e f l u x condenser the top 53  of which was connected t o a gas a b s o r p t i o n (0.16 mol)  device.  Bromine  was added i n t o the f l a s k followed by 0.7 mL of phos-  phorus tribromide.  The mixture was s t i r r e d and heated with an o i l  bath at 70°C f o r 30 min and then at 100°C f o r 4 hours u n t i l a l l the bromine had reacted. reduced pressure bromide.  The r e a c t i o n mixture was then d i s t i l l e d under  using a water pump to remove r e s i d u a l hydrogen  The d i s t i l l a t i o n  assembly was then connected to an o i l  pump and the f r a c t i o n containing 2-bromovalproic a c i d and 2-propyl2-pentenoic a c i d c o l l e c t e d at 70°-80°C/0.01 mm. The  acids were converted  to the e t h y l ester d e r i v a t i v e s by  r e f l u x i n g a mixture of the acids f o r 12 hours i n an excess of e t h y l alcohol i n benzene and catalyzed by a small amount of concentrated sulfuric acid. water.  A Dean-Stark apparatus was used to separate out the  The i s o l a t e d  e t h y l ester d e r i v a t i v e s and quinoline were  placed i n a f l a s k connected to a Vigreux column s e t f o r downward distillation. ring  during  The r e a c t i o n mixture was heated to 160°C with s t i r a 15 min p e r i o d , a f t e r which heating  was increased  r a p i d l y t o d i s t i l l the r e s u l t i n g 2-propyl-2-pentenoate.  The f r a c -  t i o n with bp 188°-196°C was c o l l e c t e d and p u r i f i e d by washing with dilute sulfuric acid. unsaturated acid  The unsaturated ester was hydrolyzed and the  a c i d obtained  was extracted with  after diethyl  acidification. ether.  The unsaturated  The ether  e x t r a c t was  washed w i t h w a t e r , d r i e d w i t h anhydrous Na2S0^, f i l t e r e d and solvent  removed w i t h  a rotary evaporator.  r e c r y s t a l l i z e d from a chloroform at  -20°C f o r a p r o l o n g e d  recrystallization  steps,  pure  54  The product was  s o l u t i o n by keeping the s o l u t i o n  period.  A f t e r two  2-propyl-(E)-2-pentenoic  successive a c i d was  obtained, mp  32°C [ l i t . (155) mp  35°C].  These p u r i f i c a t i o n  steps  were necessary to remove small amounts of the low-melting Z-isomer. NMR CH 2 ),  Spectrum: 60.8-1.1 (m, 6H, 2CH 3 ), 1.2-1.7 (m, 2H, CH^-CH?-  2.1-2.5 (ra, 4H, CH^-CH = and CH2-C=), 6.8-7.1 ( t , 1H, - CH =  C, t r a n s ) . d.  Synthesis of 2-Propyl-4-oxopentanoic a c i d (IX) 2-Bromopentanoic a c i d was synthesized, i n a s i m i l a r fashion to that  described  above f o r synthesis  of  2-bromovalproic a c i d , by  reaction of pentanoic a c i d with bromine i n the presence of a c a t a l ytic  amount of phosphorus t r i b r o m i d e .  distilled  to  give  Mass spectrum: m/z  2-bromopentanoic 55 (100%), 138  The  crude product  a c i d , bp  (30%), 140  was  102°-105°C/2.5  mm.  (28%), 27 (20%), 41  (18%), 29 ( 1 5 % ) , 94 (12%), 43 (10%). The  e t h y l e s t e r of 2-bromopentanoic a c i d was  prepared  r e f l u x i n g a mixture of 2-bromopentanoic a c i d (0.43 mol), (1.0 mol), benzene (100 mL) and concentrated  by  ethanol  s u l f u r i c a c i d (1.5  mL)  f o r 12 hour using a Dean-Stark water separation u n i t .  Pure e t h y l  2-bromopentanoate  the  work-up.  was  obtained  Bp 60°-62°C/3.0 mm.  by  distillation  Mass spectrum:  after m/z  usual  29 (100%),  166  synthesis of 2-propyl-4-oxopentanoic a c i d , anhydrous  THF  (26%), 168 (24%), 101 (22%), 140 (12%), 138 (10%). For (100 mL)  was  (0.084 mol)  placed  added.  in a  250 mL  flask  E t h y l acetoacetate  and  sodium  (0.08 mol) was  hydride  added drop-  wise over 30 min and the mixture s t i r r e d f o r an a d d i t i o n a l 10 E t h y l 2-bromopentanoate (0.08 mol) was s o l u t i o n refluxed f o r 5 hours. and  the  resulting  55  added drop by drop and  D i s t i l l e d water (30 mL)  mixture f i l t e r e d  min.  under s u c t i o n .  was  The  the  added organic  layer  was  ether. and  separated  and  the  aqueous phase extracted twice with  The combined organic l a y e r was dried over Na2S0^, f i l t e r e d  ether removed by a rotary evaporator.  tilled  to  115°C/0.2  HC1  give  ethyl  The  2-propyl-3-acetylsuccinate  (75%  dis-  yield)  bp  mm.  A mixture  of the acylsuccinate (0.018 mol)  (0.18 mol)  was  h y d r o l y s i s and times  residue was  heated under r e f l u x  decarboxylation.  with ether  and  the ether  The  and  concentrated  f o r 10 hours to  product was  effect  extracted three  e x t r a c t dried over ^2^0^.  The  residue l e f t a f t e r evaporation of the solvent was d i s t i l l e d to give pure 2-propyl-4-oxopentanoic a c i d , bp bp 165°C/20 NMR 2.15  135°-140°C/9 mm  [lit.  (156)  mm].  Spectrum: 60.8-1.1 ( t , 3H, CH 3 ),  1.2-1.7 (m, 4H,  ( s , 3H, CH3-C=0), 2.5-3.1 (complex m, 3H, CH2  2CH 2 ),  - C = 0, CH - C  = 0). Synthesis of Alpha, Alpha-Disubstituted A l i p h a t i c Acids Synthesis of 2,2-Dimethylbutyric Acid (V) Anhydrous  THF  (80 mL)  and  diisopropylamine  (0.1 mol)  added to a dry, nitrogen-flushed f l a s k immersed i n an bath.  ice-water  n - B u t y l l i t h i u m i n hexane (62 mL of 1.6 M, 0.1 mol) was added  drop by  drop to the w e l l - s t i r r e d  (0.045 mol) ution.  were  was  then  solution.  added to the l i t h i u m  Isobutyric acid  diisopropylamide  sol-  The r e s u l t i n g milky white s o l u t i o n was turned i n t o a c l e a r  homogenous s o l u t i o n by a d d i t i o n of tetrahydrofuran. continued  f o r 60 minutes at room t e m p e r a t u r e .  Stirring  Ethyl iodide  (0.048 mol) was then added at once i n t o the r e a c t i o n f l a s k . 56  was  After  2 hours of a d d i t i o n a l s t i r r i n g mixture was  at room temperature, the r e a c t i o n  quenched by a d d i t i o n of 15% HC1  organic l a y e r was  (65 mL)  at 0°C.  The  separated and the aqueous l a y e r extracted twice  with d i e t h y l e t h e r .  The combined organic l a y e r s were washed with  10% HC1, and saturated NaCl s o l u t i o n .  The organic l a y e r was  then  dried with anhydrous Na 2 S0 4 , f i l t e r e d and solvent removed using an oil acid  bath. The (50%  81°C/11 NMR  residue was  yield),  bp  distilled  to give  183°-185°C/760 mm  2,2-dimethylbutyric  [ l i t (157)  bp 7 9 ° -  mm]. Spectrum:  60.8-1.0 ( t , 3H, CH 3 ),  1.2  ( s , 6H, 2CH 3 ),  1.4-  1.8 (m, 2H, CH 2 ), 10.5-11.0 (broad s, 1H, COOH). b.  Synthesis of 2,2-Dimethylvaleric Acid This compound was  prepared using a procedure s i m i l a r to that  described above f o r 2,2-dimethylbutyric a c i d , by r e a c t i o n of i s o b u t y r i c acid (0.05 mol) and n-Propyl i o d i d e (0.055 mol).  The  2,2-  dimethylvaleric acid thus obtained was p u r i f i e d by d i s t i l l a t i o n , bp 203°-204°C/760 mm. NMR 1.7  Spectrum:  [ L i t . (158) bp 110°C/20 60.8-1.0 (m, 3H, CH 3 ),  mm]. 1.2  ( s , 6H, 2CH 3 ),  1.3-  (m, 4H, 2CH 2 ), 10.8 (broad s, 1H, COOH).  3.  Synthesis of Beta-Substituted A l i p h a t i c Acids  a.  Synthesis of 3-Ethylpentanoic Acid (VI) Lithium aluminium hydride (700 mL)  (0.48 mol)  and  ether  were introduced i n t o a 2 l i t r e f l a s k equipped with mech-  a n i c a l s t i r r e r , dropping and r e f l u x condenser. mixture  sodium-dried  After s t i r r i n g  the  for 15 min, e t h y l 2-ethylbutyrate (0.70 mol) i n anhydrous  57  ether was  added such that the ether refluxed g e n t l y .  of the e s t e r , the mixture was reaction.  Excess hydride  Following  filtration  s o l u t i o n was  b u t a n - l - o l (80% intense i o n s ) : m/z cold  destroyed  separation  of  by  the  a d d i t i o n of phases, the  yield),  bp  146°-149°C.  water. ethereal  the ether removed by  D i s t i l l a t i o n of the residue afforded  2-ethyl-  Mass spectrum ( e i g h t  43, 70, 71 (M-31), 55, 41, 56, 29, 84 [M-18].  2-ethylbutan-l-ol  (0.30 mol)  was  10°C.  s o l u t i o n was  The  was  f o r 20 min to complete the  dried over MgSO^, f i l t e r e d and  f l a s h evaporation.  To  and  stirred  On a d d i t i o n  (0.54 mol),  phosphorus  tribromide  added dropwise and with s t i r r i n g over 2 hours at then s t i r r e d  at ambient temperature f o r 2  hours before being heated f o r 20 min with a steam bath.  Water was  added to the cooled r e a c t i o n mixture and the product extracted with hexane.  The  removed.  Distillation  (50% y i e l d ) ,  dried over ?2®5 and  hexane e x t r a c t was  of the residue  bp 145°-148°C/760 mm.  tense i o n , r e l a t i v e i n t e n s i t y ) : m/z  Mass spectrum (eight most i n 43 (100%), 85 (51%), 55  l-Bromo-2-ethylbutane (0.18 mol)  was  refluxed f o r 24 hours.  product was organic  extracted  three  solvent  gave l-bromo-2-ethylbutane  71 (27%), 29 (23%), 116 (15%), 135 (12%), 137  aqueous-methanol s o l u t i o n of KCN  the  (10%).  i n methanol was  (0.26 mol).  (48%),  added to an  The r e a c t i o n mixture  A f t e r d i s t i l l i n g of the a l c o h o l , the times with  e x t r a c t s washed s u c c e s s i v e l y  hexane and w i t h 30%  the  HC1,  combined saturated  NaHCO-j, water and dried over anhydrous MgSO^. D i s t i l l a t i o n a f f o r d ed 2 - e t h y l p e n t a n e n i t r i l e (50% y i e l d ) , bp 100-102°C/ca 20 mm. spectrum: m/z  Mass  43 (100%), 28 (40%), 71 (35%), 41 (30%), 55 (28%), 29  (20%), 54 (16%), 82 ( 8 % ) .  58  2-Ethylpentanenitrile (0.05 mol) was hydrolyzed to the corresponding acid by hours.  r e f l u x i n g with  50%  sulfuric  acid (25 mL)  for 9  On e x t r a c t i o n of the a c i d with ether, the ether e x t r a c t was  dried with tillation  anhydrous Na^O^ of the  residue  y i e l d ) , bp 80°-82°C/4 mm Mass Spectrum:  m/z  and  Spectrum:  ether  distilled  off.  produced 3 - e t h y l p e n t a n o i c  [ l i t . (5) bp 87°C/1  Dis-  acid  (80%  mm].  60 (100%), 43 (64%), 70 (61%), 55  61 (23%), 71 (18%), 83 (12%), 101 NMR  the  (58%),  (10%).  60.8-1.0 (m, 6H, 2CH 3 ), 1.2-1.5 (m, 4H, 2CH 2 ),  1.6-1.9 (m, 1H, CH), 2.2-2.4 (d, 2H, CH2-C00H), 9.5-10.5 (broad s, 1H, C00H). Synthesis of Cyclohexylacetic Acid ( X I I I ) Cyclohexylmethylcyanide (bp 76°-80°C/7 mm)  was  prepared from  cyclohexylraethyl bromide and hydrolyzed to c y c l o h e x y l a c e t i c a c i d i n a s i m i l a r procedure to that described ethylpentanoic duced  acid ( a ) .  pressure  [ l i t . ( 1 5 9 ) bp  above f o r synthesis of  3-  D i s t i l l a t i o n of crude product under r e -  gave c y c l o h e x y l a c e t i c a c i d , bp 137°C/0.2 mm]  136°C/0.15  mm  which s o l i d i f i e d under ambient temp-  erature. Mass Spectrum:  m/z  60 (100%), 82 (75%), 55 (65%), 83  (52%),  61 (51%), 67 (43%), 41 (39%), 27 (17%). NMR  Spectrum:  60.8-1.4 (m;  5H;  C2,  C5 protons on r i n g  CH), 1.5-2.0 (m, 6H, C 3 -C 5 protons on a l i c y c l i c r i n g ) , 2.2 CH2-C00H), 10.2  and  ( d , 2H,  (broad s, 1H, C00H).  Synthesis of 3-Methylvaleric Acid Diethyl  1-methylpropylmalonate was 59  synthesized  from  diethyl  malonate  and  s e c - b u t y l bromide.  E t h a n o l and sodium e t h o x i d e  (prepared i n s i t u ) were used as the solvent and base, r e s p e c t i v e l y . Saponification acidification  of  diethyl  1-methylpropylmalonate,  followed  by  gave upon decarboxylation 3-methylvaleric a c i d , bp  190°-196°C/760 mm [ l i t . (160) bp 187°-200°C/760mm]. NMR Spectrum:  60.8-1.1 (m, 6H, 2CH 3 ), 1.2-1.5 (m, 2H,  CH 2 ),  1.6- 2.0 (m, 1H, CH), 2.1-2.5 (m, 2H, CH2C00H), 10.2 (broad s, 1H, C00H). Synthesis of 3-Methylhexanoic Acid Diethyl  1-methylbutylmalonate  was  synthesized  malonate and 2-bromopentane (from pentan-2-ol). ethoxide  were  used  as  the  solvent  and  from  diethyl  Ethanol and sodium  base  respectively.  S a p o n i f i c a t i o n of the monoalkylmalonate, followed by a c i d i f i c a t i o n gave upon decarboxylation 3-methylhexanoic acid [ l i t . (5) bp 60°-70°C/0.5  bp 91°-94°C/5 mm  mm].  NMR Spectrum: 60.8-1.1 (m, 6H, 2CH 3 ), 1.2-1.5 (m, 4H, 2CH 2 ), 1.7- 2.0 (m, 1H, CH), 2.1-2.5 (m, 2H, CH2C00H), 10.1 (broad s, 1H, C00H). Synthesis of 2-propyl-4-pentenoic a c i d (4-ene VPA) and 2-propyl-3hydroxypentanoic a c i d (3-OH VPA) was accomplished as described i n a previous work ( 9 3 ) .  60  4.  Synthesis of 5 - A l k y l t e t r a z o l e s  a.  Synthesis of 5-Isoamyltetrazole 5-Methylpentanenitrile that  described  (XI)  was  prepared  f o r synthesis  of  i n a s i m i l a r manner to  3-ethylpentanoic  a c i d , from  1-  bromo-3-methylbutane and an aqueous-methanol s o l u t i o n of potassium cyanide.  F r a c t i o n a l d i s t i l l a t i o n gave 5-methylpentanenitrile,  150°-155°C/760  bp  mm.  Mass Spectrum:  m/z  55 (100%), 41 (51%), 43 (47%), 27  (35%),  29 (30%), 54 (21%), 39 (15%), 57 (13%), 82 (M-CH3, 11%). 5-Methylpentanenitrile i n anhydrous THF  (0.06 mol)  and sodium azide (0.18  mol)  (60 mL) were treated at room temperature with an-  hydrous aluminium c h l o r i d e (0.063 mol) dissolved i n cold anhydrous THF.  The mixture was refluxed f o r 24 hours and with s t i r r i n g . The  solvent was then d i s t i l l e d o f f under reduced pressure using a steam bath. Water (80 mL) was added to the residue and the s o l u t i o n a c i d i f i e d to pH 2 with concentrated again The  under reduced pressure  s o l u t i o n was  HC1.  Evaporation  c a r r i e d out  to remove r e s i d u a l hydrazoic  acid.  f u r t h e r d i l u t e d with water to d i s s o l v e the alum-  inium c h l o r i d e and sodium c h l o r i d e formed. was  was  The o i l y organic l a y e r  separated and the aqueous phase extracted with e t h y l a c e t a t e .  The organic layers were l e f t i n the r e f r i g e r a t o r u n t i l the 5 - a l k y l t e t r a z o l e c r y s t a l l i z e d out.  The c r y s t a l l i n e s o l i d was f i l t e r e d  and  r e c r y s t a l l i z e d from petroleum ether-diethylether solvent mixture to give 5-isoamyltetrazole, mp 95°-96°C [ l i t . (161) mp 95°-96°C]. NMR  Spectrum:  60.95 ( d , 6H, 2CH 3 ), 1.5-2.0 (m, 3H, CH-CH2),  3.0-3.3 ( t , 2H, CH2-C=), 11.4  ( s , 1H, 61  NH).  Synthesis of 5-Cyclohexylmethyltetrazole Cyclohexylethanenitrile  was  (XII)  prepared, as  synthesis of c y c l o h e x y l a c e t i c a c i d , from The  n i t r i l e was  similar  fashion  tetrazole.  converted to  that  aluminium c h l o r i d e ) .  described  110°C [ l i t . (161) mp  109°C].  r i n g and CH),  cyclohexylmethylbromide.  f o r synthesis  of  5-isoamyl-  of c y c l o h e x y l e t h a n e n i t r i l e and  The i s o l a t e d crude product was to give  Spectrum:  the  i n s i t u from sodium azide and anhydrous  from e t h y l acetate  NMR  in  i n t o 5-cyclohexylmethyltetrazole, i n a  T h i s r e q u i r e d use  aluminium azide (prepared  described  recrystallized  5-cyclohexylmethyltetrazole, mp  60.9-1.4 (m;  5H;  C2,  CQ  109-  protons on a l i c y c l i c  1.5-2.0 (m, 6H, C3-C5 protons on r i n g ) , 2.9-3.2 ( d ,  2H, CH2C=), 10.5 (broad s, 1H,  NH).  Synthesis of 5-Heptyltetrazole (X) 1-Bromoheptane (0.32 mol) stirred  was  added slowly to a warm, w e l l -  s o l u t i o n of sodium cyanide (0.41 mol) i n dimethylsulfoxide  (130 mL).  Stirring  was  continued  for 1 hour u n t i l  the  solution  temperature cooled from 130°C to ambient temperature. The r e a c t i o n mixture was poured i n t o water and the product extracted three times with ether.  The  combined ether extract was washed with saturated  NaCl and dried over anhydrous MgSO^. The l i q u i d l e f t upon evapora t i o n of ether was d i s t i l l e d under reduced pressure to give octanen i t r i l e , bp 85°-88°C/10 O c t a n e n i t r i l e was THF,  mm. reacted with aluminium azide i n r e f l u x i n g  employing the procedure f o r synthesis of 5-isoamyltetrazole.  62  The  crude  nitrile  solid  product  i s o l a t e d was  recrystallized  to give 5 - h e p t y l t e t r a z o l e , mp  41°-42°C  from aceto-  [ l i t . (161)  mp  41.5°C]. NMR Spectrum:  60.7-1.0 ( t , 3H, CH 3 ), 1.1-1.5 (m, 8H, 4CH 2 ),  1.6-2.0 (m, 2H, CH2 CH2-C=), 2.9-3.3 ( t , 2H, CH2-C=), 10-11  (broad  s, 1H, NH). 5.  Synthesis of Succinamic Acids  a.  Synthesis of N,N-Diethylsuccinamic Acid (XV) Succinic  anhydride  (0.1 mol)  was  added w i t h s t i r r i n g  diethylamine (0.1 mol) i n cold absolute ethanol (25 mL).  to  Stirring  was continued u n t i l the r e a c t i o n mixture attained room temperature. N,N-Diethylsuccinamic a c i d c r y s t a l l i z e d  out of the homogenous s o l -  ution on cooling and was f i l t e r e d under suction before  recrystal-  l i z a t i o n from benzene, mp 83°-85°C [ l i t . (162) mp 82-84°C]. IR Spectrum ( m u l l ) : 1690-1720 cm  -1  (C = 0 i n COOH), broad),  1630 (C = 0 i n amide). NMR Spectrum: 61.1-1.5 ( d i s t o r t e d t , 6H 2CH 3 ), 2.6 CH2CH2-C=0), 2.8-3.3 ( q , 4H,  CH2-N-CH2), 5.0-6.0 (broad  ( s , 0=Cs,  2H,  COOH). b.  Synthesis of N,N-Dibutylsuccinamic Acid (XVI) Succinic anhydride (0.15 mol) was added to anhydrous benzene (120 mL)  and  hydride.  the  solution  warmed to p a r t i a l l y  Dibutylamine (0.15 mol)  i n benzene (30 mL) was  added to the mixture at a c o n t r o l l e d generated.  The  r e a c t i o n m i x t u r e was  63  d i s s o l v e the  an-  rapidly  rate to contain the then r e f l u x e d u n t i l  heat the  reaction mixture turned homogenous. The r e s u l t i n g r e a c t i o n mixture was  allowed  refrigerator  t o c o o l to ambient temperature until  Dibutylsuccinamic  the s o l i d acid  and  left  i n the  product c r y s t a l l i z e d o u t .  was  filtered  under  suction  N,Nand  r e c r y s t a l l i z e d with benzene, mp 78°-80°C. IR Spectrum ( m u l l ) :  1720 cm  -1  (C = 0 i n C00H), 1615 (C = 0 i n  amide). NMR  Spectrum:  60.8-1.1 ( t , 6H, 2CH 3 ), 1.2-1.9 (m, 8H, 2CH2-  CH 2 ), 2.6 ( s , 4H, 0 = C-CH2 CH2 C = 0 ) , 2.8-3.1 ( t , 4H, CH2-N-CH2), 8.2-8.9 (broad s, 2H, C00H). 6.  Synthesis of Diunsaturated Analogues of V a l p r o i c Acid The  diunsaturated d e r i v a t i v e s of v a l p r o i c acid  were prepared  by  dehydration of the 8-hydroxy unsaturated esters followed by h y d r o l y s i s of  the  diunsaturated e s t e r s .  The  3-hydroxy unsaturated esters were  prepared by deconjugative a l d o l condensation of the l i t h i u m dienolates of e t h y l 2-pentenoate and propionaldehyde.  The dienolate of e t h y l ( E ) -  2-pentenoate was expected to give mainly the whereas 8-hydroxy-(E)-3-pentenoate  B-hydroxy-(Z)-3-pentenoate  was expected from r e a c t i o n with e t h y l  (Z)-2-pentenoate. Ia.  Synthesis of E t h y l 2-(l'-hydroxypropyl)-3-Pentenoate from E t h y l (E) -2-Pentenoate E s t e r i f i c a t i o n of ( E ) - 2 - p e n t e n o i c a c i d ( E t I , K 2 C 0 3 , r e f l u x , 6 hr) gave ethyl-(E)-2-pentenoate, bp 36-38°C/0.07 NMR  Spectrum:  2H, CH2-C=), 4.2  61.0  ( t , 3H, CH 3 ),  1.3  THF,  mm.  ( t , 3H, CH 3 ), 2.2  (q,  ( q , 2H, 0CH 2 ), 5.6-6.0 ( d , 1H, = CH, J = 16Hz),  6.8-7.3 ( d t , 1H, HC ==, J = 16 Hz).  64  n - B u t y l l i t h i u m i n hexane (69 mL of 1.6M,  0.11 mol) was added  dropwise with s t i r r i n g to a s o l u t i o n of diisopropylamine (0.11 i n anhydrous THF  (120 mL) at 0°C.  min  then  at  0°C and  cooled  mol)  The s o l u t i o n was s t i r r e d f o r 20  to -78°C.  Hexamethylphosphoramide  (0.11 mol) was added to the s o l u t i o n followed by dropwise a d d i t i o n of  ethyl-(E)-2-pentenoate  (0.10 mol).  hyde (0.10 mol) was added.  A f t e r 30 min, propionalde-  The r e a c t i o n mixture was s t i r r e d f o r a  further 30 min at -78°C before the r e a c t i o n was quenched with 15% HC1.  The  product was  extracted with ether and the ether e x t r a c t  washed successively with 10% HC1, NaCl.  The  moved with  organic layer was a  saturated NaHC03 and  saturated  dried over Na 2 S0 4 , and solvents r e -  rotary evaporator.  The  residue a f t e r  fractional  d i s t i l l a t i o n under reduced pressure gave the major product, e t h y l 2-(l'-hydroxypropyl)-3-pentenoate, bp 85-90°C/0.25 mm (48% y i e l d ) . IR Spectrum: (C=C), 1175 cm 745  -1  3400 cm  -1  (0-H),  (C-0), 985 cm  -1  1735 cm  -1  (C=0), 1635  (medium i n t e n s i t y , =CH,  cm  -1  trans),  -1  cm . NMR  Spectrum:  (m, 2H, CH 2 ),  1.75  60.95 ( t , 3H, CH 3 ), 1.25 ( d , 3H, CH3-CH=), 2.55  (m, 1H, CH-C=0), 3.8  (m, 1H, CH-0), 4.2  ( t , 3H, O C ^ C H j ) , 1.55 (broad s, 1H, OH),  3.40  ( q , 2H, 0CH 2 CH 3 ), 5.3-5.9  (m, 2H, CH = CH). f  Synthesis of E t h y l 2-(l -hyrodxypropyl)-3-pentenoate -2-Pentenoate Ethyl  (Z)-2-Pentenoate  was  prepared  from Ethyl-(Z)  i n three stages, ( i ) A  mixture of 2-pentanone (0.5 mol) and precooled 48% HBr (50 mL) c h i l l e d to 0°C and  bromine (1.0 mol) added dropwise.  was  A f t e r add-  i t i o n of bromine, water (200 mL) was added and the heavier organic  65  layer d i s t i l l e d  to give a f r a c t i o n with bp 84-89°C/10 mm.  analysis  1,3-dibromo-2-pentanone to be the main product,  (ii)  showed  GCMS  1,3-dibromo-2-pentanone (0.3 mol) was added to a s o l u t i o n of  KHCOg (2.1 mol) i n 1200 mL water. f o r 24 hours. carded  after  The r e a c t i o n mixture was s t i r r e d  Nonacidic by-products i n basic s o l u t i o n were d i s extraction  with  ether.  The b a s i c s o l u t i o n  was  a c i d i f i e d to pH 2 with d i l u t e HC1 and the product extracted with ether.  The ether phase was i n turn dried over MgSO^, f i l t e r e d and  concentrated with a rotary evaporator. D i s t i l l a t i o n of the residue gave  (Z)-2-pentenoic a c i d ,  f i c a t i o n of the acid  bp  39°-41°C/0.4 mm.  (iii)  Esteri-  ( E t I , K 2 C 0 3 , THF, r e f l u x , 6 hr) gave e t h y l  (Z)-2-pentenoate, bp 50°-52°C/12  mm.  NMR Spectrum; 61.0 ( t , 3H, CH 3 ), 1.3 ( t , 3H, ( X ^ C H j ) , 2.65 ( q , 2H, CH2-C=), 4.2 ( q , 2H, 0CH 2 -), 5.6-5.9 ( d , 1H, CH=, J = 10 Hz), 6.0-6.4 ( d t , 1H, CH=, J = 10 H z ) . . The l i t h i u m dienolate of e t h y l (Z)-2-pentenoate (0.05 mol) was prepared 60 mL  at -78°C using l i t h i u m  THF  aldehyde  and  diisopropylamide (0.055 mol) i n  hexamethylphosphoramide (0.055 mol).  (0.05 mol) was  added dropwise  Proprion-  to the s o l u t i o n and the  r e a c t i o n s t i r r e d f o r 30 min and f i n a l l y quenched with 10% HC1. major product, e t h y l  2-(l'-hydroxypropyl)-3-pentenoate, was  The iso-  l a t e d , bp 95-100°C/l mm (50% y i e l d ) . IR Spectrum: (C=C), 985 cm  -1  3400  cm  -1  (0-H), 1735 cm  -1  (C=0), 1640 cm  (strong i n t e n s i t y , = CH, t r a n s ) , 745  66  -1  cm .  -1  II.  Dehydration of e t h y l 2-(l'-hydroxypropyl)-3-pentenoate  a.  Phosphorus Pentoxide Phosphorus  pentoxide (36 mmol) was added  to the B-hydroxy  unsaturated ester (39 mmol, from e t h y l (E)-2-pentenoate) i n 60 mL of anhydrous benzene and the mixture refluxed f o r 4 hours.  The  diunsaturated esters i s o l a t e d were saponified with d i l u t e NaOH and subsequently a c i d i f i e d with d i l u t e H 2 S0 4 to give a mixture of seven isomeric dienoic a c i d s , bp 105°-110°C/2.5 mm. b.  Toluenesulfonyl c h l o r i d e - p y r i d i n e Dehydration of the S-hydroxyunsaturated ester (33 mmol, from e t h y l ( E ) - 2 - p e n t e n o a t e ) i n 20 mL of p y r i d i n e w i t h  p-toluene-  s u l f o n y l c h l o r i d e (45 mmol) was c a r r i e d out by f i r s t s t i r r i n g the r e a c t i o n mixture at room temperature f o r two days, d i l u t i n g with ice-water CHCI3.  and e x t r a c t i n g  the p-toluenesulfonate  derivative  with  The i s o l a t e d p-toluenesulfonate was refluxed with d i l u t e  NaOH and a c i d i f i e d  t o give a mixture of four  isomeric  dienoic  a c i d s , bp 120°-128°C/6 mm. NMR Spectrum (270 MHz) of mixture:  60.95-1.1 ( t , CH3), 1.55  (dd, CH3-C=, 2E-3'Z), 1.64 (dd, CH3-O, 3Z-3'Z), 1.70 (dd, CH3-C=, 2Z-3'E), 1.85 ( d , CH3-C=, 2E-3'E), 2.16 (m, CH2-C=, 2E-3'Z), 2.35 (m, CH2-C=, 2E-3'E), 2.55 (ra, CH2-C=, 2Z-3'E), 4.05 ( t , CH, 3Z3'Z), 5.65 (m, CH=CH, 3Z-3'Z), 5.73 (m, CH=CH, 2E-3'Z), 5.85 (m, CH=CH, 2Z-3'E), 5.95 ( t , CH=, 2Z-3'E), 6.03 (m, CH=, 2E-3'E), 6.14 ( d , CH=, 2E-3'E), 6.83 ( t , CH=, 2E-3'E), 7.00 ( t , CH=, 2E-3'Z). IR Spectrum of mixture: 1690 cm 965 cm  -1  -1  -1  -1  (=CH, t r a n s ) , 935 cm , 735 cm . 67  (C=0), 1633 cm  -1  (C=C),  NMR assay  of the four dienoic a c i d isomeric mixture, using  appropriate integrated areas, gave the r a t i o of 2-(l'-propenyl)-2pentenoic acid and 2-(l'-propenyl)-3-pentenoic acid isomers 3Z-3'Z, 2Z-3'E, 2E-3'Z, 2E-3'E as approximately 12 : 14 : 56 : 18. Methanesulfonyl c h l o r i d e - Potassium hydride Dehydration  of the 6-hydroxy  unsaturated  ester  (0.05 mol),  derived from e t h y l (E)-2-pentenoate, was c a r r i e d out by forming the sulfonate with methanesulfonyl c h l o r i d e (0.06 mol) i n the presence of triethylamine (0.08 mol) and methylene c h l o r i d e (40 mL) at 0°C. KH  (0.10 mol) was added a t 0°C t o t h e i s o l a t e d s u l f o n a t e and  r e a c t i o n mixture s t i r r e d f o r 12 hours a t room temperature. hydride was decomposed by a d d i t i o n of t-butanol and water. ing  Excess Follow-  the u s u a l work-up, the i s o l a t e d product m i x t u r e , bp 75-  80°C/2 mm, contained three isomeric dienoates. IR Spectrum of mixture:  1716 cm  -  -1  (C=0), 1636 cm  -1  (C=C),  -  968 cm"'' (=CH, t r a n s ) , 756 cnT^, 690 cm''" (=CH, c i s more intense than =CH, t r a n s ) . NMR assay of the e t h y l e s t e r s of the three dienoic acid mixt u r e , using appropriate integrated areas, gave the r a t i o of isomers 2Z-3'E: 2E-3'Z: 2E-3'E as 7:71:22. NMR Spectrum of isomeric mixture (270 MHz): 61.0-1.1 (m, CH3), 1.3 (m, CH 3 ), 1.55 (dd, CH3-C=, 2E-3'Z), 1.7 (dd, CH3-C=, 2Z-3'E), 1.83 ( d , CH3-C=, 2E-3'E), 2.12 (ra, CH2-C=, 2E-3'Z), 2.3 (m, CH2-C=, 2E-3'E), 2.52 (m, CH2-C=, 2Z-3'E), 4.2 ( q , CH 2 0), 5.75 (m, CH =, 2E-3'Z), 5.98 ( d , CH=, 2E-3'Z), 6.04 (m, CH=, 2E-3'E), 6.13 ( d , CH=, 2E-3'E), 6.55 ( t , CH=, 2E-3'E), 6.8 ( t , CH=, 2E-3'Z).  68  The isomeric dienoates mixture was saponified with d i l u t e NaOH and subsequently a c i d i f i e d with d i l u t e H 2 S0^ t o give the i s o l a t e d product mixture of the corresponding acids of two major isomers 2E3'Z, 2E-3'E and trace amounts of the isomer 2Z-3'E. NMR Spectrum (400 MHz) of the mixture:  61.0-1.1  (m, CH3),  1.56 (dd, CH3-C=, 2E-3'Z), 1.73 (dd, CH3-C=, 2Z-3'E), 1.84 ( d , CH3~ C=, 2E-3'E), 2.15 (m, CH2-C=, 2E-3'Z), 2.23 (m, CH2-C=, 2E-3'E), 2.50 (m, CH2-C=, t r a c e , 2Z-3'E), 5.8 (m, CH=, 2E-3'Z), 5.96 ( d , CH=, 2E-3'Z), 6.07 (m, CH=, 2E-3'E), 6.13 ( d , CH=, 2E-3*E), 6.78 ( t , CH=, 2E-3'E), 6.97 ( t , CH=, 2E-3'Z). (ii)  Dehydration of the 8-hydroxyunsaturated e t h y l  ester  (0.02 mol), derived from e t h y l (Z)-2-pentenoate, was c a r r i e d out by treatment  of the mesylate d e r i v a t i v e  with  KH (0.04 mol).  The  i s o l a t e d product mixture, bp 65-70°/0.1 mm contained three isomeric dienoates, 3Z-3'Z, 2E-3'Z, 2E-3'E i n the r a t i o  of approximately  44:8:48 (determined by NMR a n a l y s i s ) . IR Spectrum: (C=C), 985-965 cm  1733 cm -1  -1  (C=0), 1716 cm  -1  1  (C=0), 1655-1640 cm"  -1  (CH=, t r a n s ) , 745 cm .  NMR Spectrum of the isomeric mixture (270 MHZ):  61.0-1.1 (m,  CH 3 ), 1.2-1.25 (m, CH 3 ), 1.53 ( d , CH3-C=, 2E-3'Z), 1.64 (dd, CH3~ C=, 3Z-3'Z), 1.68 (dd, CH3-C=, 3Z-3'Z), 1.82 ( d , CH3-C=, 2E-3'E), 2.1 (m, CH2-C=, 2E-3'Z), 2.35 (m, CH2~C=, 2E-3'E), 3.5 (m, CH, 3Z3'Z), 5.5-5.7 (m, CH=CH, 3Z-3'Z), 6.04 (m, CH=, 2E-3'E), 6.13 ( d , CH=, 2E-3E), 6.57 ( t , CH=, 2E-3'E), 6.77 ( t , CH=, 2E-3'Z). I I I . Semi-Preparative Argentation TLC Silica  g e l G was impregnated with AgN03 (20% w/w) and TLC 69  p l a t e s prepared w i t h 0.5 dienoates (20-40 mg) 20 cm)  mm  thickness.  i n CHCI3 was  i n a narrow band.  A m i x t u r e of  isomeric  applied across the plates (20 x  Benzene: p e t r o l e u m e t h e r ,  30°-60°:  d i e t h y l e t h e r (80 : 20 : 5) was s e l e c t e d as the m o b i l e phase a f t e r experimenting with d i f f e r e n t solvent systems. developed i n a r e c t a n g u l a r  glass tank.  The  The p l a t e s were  solvent front  allowed to run to 15 cm on the p l a t e s , from the o r i g i n .  The  was  plates  were then sprayed w i t h 50% H2SO4 and heated at 200°C f o r 10 values of three charred of p l a t e s was  bands were determined.  Another s e r i e s  developed under the same c o n d i t i o n s .  k n o w l e d g e of  band p o s i t i o n s , s t r i p s  of  min.  With  support  pre-  material  c o r r e s p o n d i n g to the t h r e e bands were scraped o f f the unheated plates i n t o centrifuge tubes. The e t h y l esters were extracted w i t h methanol.  Following  c e n t r i f u g a t i o n and  filtration  of  the  m e t h a n o l i c s o l u t i o n s , the t h r e e f r a c t i o n s were c o n c e n t r a t e d and reconstituted i n CDCI3 f o r GCMS and NMR IV. a.  analysis.  In vivo metabolism - i s o l a t i o n procedure Serum.  A serum sample (2.0 mL) from a p a t i e n t on VPA,  a c i d i f i e d to pH 2.0 with 4N HC1.  E x t r a c t i o n was  c a r r i e d out  was twice  with 2.0 mL of e t h y l acetate, each t i m e c e n t r i f u g i n g at 2000  rpm  f o r 20 min to s e p a r a t e the phases. The e t h y l a c e t a t e l a y e r s were combined, dried over anhydrous Na2S04 and then concentrated at room t e m p e r a t u r e u s i n g a g e n t l e stream of dry n i t r o g e n .  The  concen-  trated e x t r a c t s were d e r i v a t i z e d and a l i q u o t s i n j e c t e d i n t o the  gas  chromatograph mass spectrometer. b.  Urine.  To 5 mL  of a u r i n e sample was added 3N NaOH to b r i n g 70  the pH to 13.0 and h y d r o l y s i s of conjugates was effected by heating the  s o l u t i o n a t 60°C f o r 1 h r .  a c i d i f i e d to pH 2.0 with AN HC1. twice with e t h y l a c e t a t e .  A f t e r c o o l i n g , the s o l u t i o n was The a c i d i f i e d urine was extracted  The combined e t h y l acetate l a y e r s were  d r i e d , concentrated and d e r i v a t i z a t i o n c a r r i e d o u t . V.  D e r i v a t i z a t i o n of Acids t-BDMS d e r i v a t i v e s were prepared by adding 30-50 uL of t-BDMS reagent to synthesized sample (3-5mg) or urine e x t r a c t i o n samples (see above) and warming at 60°C for 5 min.  The t-BDMS d e r i v a t i v e s  were extracted  ( e t h y l acetate-hexane)  with  100-150 u l of solvent  and a l i q u o t s i n j e c t e d i n t o the GCMS. TMS-derivatives were formed by t r e a t i n g the concentrated urine extraction  samples  or synthesized  samples with  methyl-N-trimethylsilyl-trifluoroacetamide  30-50 u l of N-  at 50°C f o r 5 min.  E t h y l esters of acids were formed using appropriate volumes of t r i r a e t h y l a n i l i n i u m hydroxide s o l u t i o n and e t h y l i o d i d e .  The mix-  ture was warmed at 50°C f o r 5 min. Methyl esters of acids were prepared by reaction of the acid sample  with  diazoraethane  generated  from  Diazald®  (dissolved i n  ether/2-ethoxyethanol) and 60% K0H. VI.  Photochemical  Isomerization  A mixture of seven isomeric dienoic acids ( l g ) , derived from dehydration of e t h y l 2-(l'-hydroxypropyl)-3-pentenoate with was  dissolved  P2^5»  i n 150 mL of hexane and added to a pyrex w e l l .  Propyl-(E)-2-pentenoic acid  2-  (2-ene VPA) was added as an i n t e r n a l  71  standard.  A quartz immersion w e l l with a 450W u n f i l t e r e d Hanovia  lamp was set i n t o place and i r r a d i a t i o n c a r r i e d out f o r 6 hours. A l i q u o t s of the r e a c t i o n mixture were removed p e r i o d i c a l l y and the t-BDMS esters of photolyzed acids analyzed by c a p i l l a r y GCMS.  V I I . C a p i l l a r y GCMS r e s o l u t i o n of 2,3'-diene VPA and 2,4-diene VPA Samples  of synthetic  diene  a c i d s , 2-propyl-(E)-2,4-penta-  dienoic acid and 2-[(Z)-l'-propenyl]-(E)-2-pentenoic a c i d , or the urine extract from one patient on VPA therapy, were made with e t h y l acetate and d e r i v a t i v e s formed.  Urine extracts were also spiked  with synthetic diene acids p r i o r to TMS or t-BDMS d e r i v a t i z a t i o n . C a p i l l a r y GCMS a n a l y s i s of the TMS d e r i v a t i v e s was performed using a 25 m x 0.3 mm i . d . SE 54 column a t oven temperatures from 50°C t o 90°C at 30°/min and then held at 90°C f o r 10 min.  C a p i l l a r y GCMS  a n a l y s i s of the t-BDMS d e r i v a t i v e s was performed using e i t h e r the SE-54 column or 0V-1701 column.  Oven temperatures were programmed  from 50°C to 100°C at 30°/min and then 100°C t o 200°C at 8°/min. D.  Subcutaneous Pentylenetetrazole (PTZ) Seizure Threshold Test  1.  Animals Adult  male Swiss mice  (CD1 s t r a i n ,  20-32g, Charles R i v e r ,  Quebec, Canada) were used as experimental animals.  A l l animals  were allowed free access t o food and water except during the time of t e s t .  Mice were housed a f t e r a r r i v a l f o r a t l e a s t 24 hours  before use.  72  Drugs Isobutyric a c i d , t r i m e t h y l a c e t i c a c i d s , carboxylic  acid  and  1-Methylcyclohexane-l-  pentylenetetrazole (PTZ)  A l d r i c h Chemical Company, Milwaukee, USA.  were obtained  from  Valproic a c i d was  pur-  chased from Abbott L a b o r a t o r i e s , I l l i n o i s , USA.  2-Butylhexanoic  a c i d , 2,2-Dimethylbutyric a c i d , 3-Ethylpentanoic a c i d , Cyclohexyla c e t i c a c i d , 2-Propyl-A-oxopentanoic  acid (4-keto VPA),  2-Propyl-  (E)-2-pentenoic a c i d (2-ene VPA), 2-(l'-Propenyl)-2-pentenoic acid (2,3'-diene VPA), as a mixture of 2E-3'Z and 2E-3'E i n a r a t i o of 7 :  3,  N,N-Diethylsuccinamic  Cyclohexylraethyltetrazole, were synthesized as p u r i t y and  acid,  N,N-Dibutylsuccinamic  acid,  5-  5-Isoamyltetrazole and 5-Heptyltetrazole  described i n the experimental  section.  i d e n t i t y of these compounds were v e r i f i e d by IR,  The NMR  spectroscopy and GCMS a n a l y s i s . Drug Solutions Pentylenetetrazole was make a concentration of soluble  dissolved  1.7%.  The  i n water, were dissolved  solutions  of the f a t t y a c i d s and  i n 0.9%  sodium c h l o r i d e  succinamic  i n 0.9%  to  a c i d s , which are  sodium c h l o r i d e .  t e t r a z o l e s were prepared  The by  n e u t r a l i z i n g a known amount of the drug with IN NaOH and the pH of the r e s u l t i n g s o l u t i o n adjusted to 7.4 with 1.0N  HC1.  A l l drugs  were administered to mice i n concentrations which permitted optimal accuracy i n dosage.  Drugs were administered i . p . i n a volume not  more than 7 mL/kg body weight i n mice and PTZ i n j e c t e d s.c. i n a volume of 5 mL/kg.  73  4.  Experimental Procedure  a.  C h a r a c t e r i z a t i o n of PTZ Seizures (85 mg/kg dose) Mice received a subcutaneous i n j e c t i o n of PTZ, 85 mg/kg, i n a loose f o l d of s k i n , on the back of the neck.  This i s the CDgy dose  of PTZ i n mice (46). Control mice, i n a group of 12, were i n j e c t e d s.c. w i t h PTZ, 85 mg/kg. A f t e r PTZ a d m i n i s t r a t i o n , a n i m a l s were placed i n i n d i v i d u a l cages f o r observation. 30 min f o l l o w i n g the s.c. PTZ  Observation  time  was  injection.  The r e a c t i o n of each mouse to PTZ (85 mg/kg) f o l l o w e d a defi n i t e p a t t e r n , c h a r a c t e r i z e d by a b r i e f episode of body t w i t c h e s which passed i n t o a s e r i e s of c l o n i c j e r k i n g s usually accompanied by audible squeaks and Straub t a i l phenomenon. The c l o n i c spasms occurred between r e s t i n g phases.  The  convulsive syndrome usually  ended w i t h the swimming phase, characterized by coordinated f l e x o r e x t e n s o r movement of the l e g s .  The  post s e i z u r e phase u s u a l l y  ended f a t a l l y w i t h tonic extension of hind limbs or was  character-  ized by a stupor phase a f t e r which mice recovered. During the 30 minute observation period, sustained synchronous c l o n i c j e r k i n g s o c c u r r e d i n 100% of c o n t r o l m i c e . The onset t o c l o n i c e p i s o d e s ( e p i s o d e s of 5 second d u r a t i o n or l o n g e r )  was  between 3-9 m i n u t e s . S e v e n t y - f i v e p e r c e n t of c o n t r o l mice d i e d w i t h i n the 30 min observation period. b.  Antagonism of PTZ c l o n i c seizures Mice i n groups of eight per dose of drug, received i.p. i n j e c t i o n s of t e s t drug i n a maximum of four doses between 0.2 mmol/kg and 2.0 mmol/kg. For each d r u g , a f i x e d t i m e i n t e r v a l of 15 74  min  a f t e r i . p . administration was chosen before mice received a s.c. 85 mg/kg PTZ i n j e c t i o n . Each mouse was i n d i v i d u a l l y caged f o r observation a f t e r the s.c. PTZ i n j e c t i o n .  Each mouse was observed f o r a maximum time of  30 min a f t e r PTZ administration but c l o n i c seizures r a r e l y occurred a f t e r times longer than 20 min.  During the period of observation,  the incidence and timing of c l o n i c convulsions was recorded  with  absence of sustained c l o n i c spasms (5 sec duration or longer) being defined as p r o t e c t i o n . Eight mice were used f o r each point on the dose-effect curves. At l e a s t three points were established between the l i m i t s of complete protection and no p r o t e c t i o n .  The probit of the percentage  protected was plotted against the l o g dose.  The data were analyzed  by the s t a t i s t i c a l method of L i t c h f i e l d and Wilcoxon (192). ED^Q  values, slope of the curves, and t h e i r 95% confidence  The limits  were then recorded for each anticonvulsant drug. M o r t a l i t y Test The  percentage of mice which survived w i t h i n 30 minutes a f t e r  the s.c. 85 mg/kg PTZ i n j e c t i o n was determined at the 1.0 mmol/kg i . p . dose l e v e l of t e s t compound.  Test drugs were  administered  i . p . 15 minutes before PTZ a d m i n i s t r a t i o n . Toxic e f f e c t s of drugs S p e c i f i c t o x i c i t y t e s t s were not conducted.  However, observ-  ations of t o x i c signs were made during the 15 minute time i n t e r v a l before a d m i n i s t r a t i o n of PTZ. Neurotoxic e f f e c t s were i n d i c a t e d by  75  sedation, abnormal g a i t or a t a x i a , h y p e r a c t i v i t y , activity,  rapid-circling  abnormal body posture, abnormal spread of the  limbs,  p r o s t r a t i o n (immobility or r e s t i n g on b e l l y ) , f a s c i c u l a t i o n (movement of muscles on the back), tremors, and convulsions. e.  Convulsant A c t i v i t y Test Compounds which induced tremors, h y p e r e x c i t a b i l i t y and convulsions  i n mice  administration  were  tested  of sublethal  for convulsant a c t i v i t y doses and  by i . p .  the mice observed  f o r 30  minutes without administration of PTZ. E.  HPLC Method Coefficients  1.  Instrumentation  f o r Determination  Analyses were performed HPLC equipped  with an HP  of  Octanol-Water  Partition  with a Hewlett-Packard Model 1082B  79850B LC t e r m i n a l .  Detection of the  a c i d i c compounds was at 210 nm with a H i t a c h i Model 155-10 v a r i a b l e wave length spectrophotometer. (AUFS) was set at 0.05 2.  The  Absorbance Units F u l l Scale  AU.  Column The  analyses were c a r r i e d  out using a 20 cm  x 4.6mm i . d .  column packed w i t h 5 um, H y p e r s i l 0DS (Hewlett-Packard). 3.  Eluents HPLC-grade a c e t o n i t r i l e and methanol were used i n the prepa r a t i o n of the mobile phases.  D i s t i l l e d and d e i o n i z e d water  ( M i l l i - Q system, M i l l i p o r e Corp., Bedford, MA) was used to prepare 76  0.01M  NaH 2 P0 4 b u f f e r , pH 3.5.  buffer  containing  nitrile.  different  The eluents  percentages  were 0.01M  of methanol or  NaH2POA aceto-  The solvents were mixed and degassed with s t i r r i n g f o r 5  min under a water-aspirator vacuum. Eluent flow rates were between 1.0-1.5 mL/rain r e q u i r i n g a pressure of 900-1500 p s i . 4.  Compounds Seven a l i p h a t i c efficients  p a r t i t i o n co-  had been determined i n the same laboratory served as  reference compounds. to 3.20.  a c i d s whose o c t a n o l - w a t e r  The l o g P Q / W of these acids ranged from 0.98  S t r a i g h t - c h a i n f a t t y acids obtained  commercially, pro-  vided a homologous s e r i e s f o r estimation of void time. acid,  trimethylacetic acid  and  Isobutyric  1-methylcyclohexane-l-carboxylic  acid were purchased from A l d r i c h Chemical Company, Milwaukee, USA. The other a c i d i c compounds used were synthesized as described i n the experimental s e c t i o n . 5.  Sample Preparation Standards of a c i d i c compounds were prepared i n e i t h e r methanol or a c e t o n i t r i l e to give concentrations of saturated f a t t y acids of 0.2-0.6 Ug/uL, unsaturated  fatty  acids  of 0.01-0.05 ug/yL, N,N-  dibutylsuccinamic a c i d of 0.1-0.3 Ug/uL, N,N-diethylsuccinaraic a c i d of 0.8-1.2 ug/uL and t e t r a z o l e s of 0.02-0.07 ug/uL. 6.  Retention Time Measurements The a c i d i c compounds were studied i n d i v i d u a l l y . t i o n volumes were 20 uL.  Sample i n j e c -  The r e t e n t i o n times were expressed i n  77  terras of log (capacity f a c t o r s ) or l o g k'.  Log k' = l o g ( t R - t Q ) fc o  where t-g represents the r e t e n t i o n time of the compound and t Q , the e l u t i o n time of  unretained  peak generated by methanol or  n i t r i l e . The recorder chart speed was of the  eluent was  aceto-  1.0 cm/min and the flow r a t e  between 1.0 mL/min and  1.5 mL/min.  Retention  times were measured by the i n t e g r a t o r and the average of two r e p l i cate i n j e c t i o n s was used. 7.  Column Void Time While the dead volume (v ) i s independent of flow r a t e , the column void (or dead) time i s dependent on the eluent and  flow  rate.  I t was  therefore determined by  composition  injection  of  substance that i s expected to be unretained, i . e . methanol. void time was iteration  The  compared to the void time determined by dead time  of the r e t e n t i o n times of homologous n - a l k y l c a r b o x y l i c  acids (181). (a-1).  a  By  The  equation  plotting  tj^+1  i s expressed by tp  t  = atg N ~  ( r e t e n t i o n time of the N+l  o  = carbon  homologue) versus tp ^ ( r e t e n t i o n time of the N-carbon homologue), t  can  be  c a l c u l a t e d from the  slope  of the regression l i n e ,  ( r e l a t i v e retention) and the i n t e r c e p t .  a  Values of t Q and t ^ were  not corrected f o r the small time l a g between column and d e t e c t o r . F.  Determination of Octanol-Water P a r t i t i o n C o e f f i c i e n t s by the ShakeFlask Procedure The acid,  p a r t i t i o n c o e f f i c i e n t of four compounds, t r i m e t h y l a c e t i c 5-isoamyltetrazole,  5-cyclohexylmethyltetrazole  and  N,N-  dibutylsuccinaraic a c i d , were determined i n l-octanol-0.IN HC1.  For  the  and  partitioning  s t u d i e s , 1-octanol saturated  78  with 0.1N  HC1  0.1N HC1 saturated with 1-octanol were used. The amount of compound and volume of the aqueous phase were chosen such that the i n i t i a l concentrations of compounds i n 0.1N -3  HC1 saturated with 1-octanol were i n the range, 1.0 x 10 M t o 5.0 x 10 M.  Trimethylacetic acid and N,N-dibutylsuccinaraic were s o l -  uble i n the volume of aqueous phase used.  The weighed amount of  the t e t r a z o l e s were rendered soluble i n the aqueous phase by f i r s t d i s s o l v i n g i n a small volume of MeOH ( 2 % , v/v) and the c a l c u l a t e d volume of 0.1N HC1 added. The r a t i o s of the volumes of the aqueous and octanol phase were 2:1 f o r t r i m e t h y l a c e t i c a c i d and 10:1 f o r the other compounds.  The r a t i o s were chosen so that a n a l y t i c a l  errors can be decreased. The separatory  funnel (50-120 mL capacity) was shaken gently  and inverted several times f o r 15 min.  The aqueous phase was  c o l l e c t e d i n t o centrifuge tubes and centrifuged a t 2000 rpm f o r 20 min.  The concentration of the compounds i n the aqueous phase a f t e r  p a r t i t i o n i n g was determined by HPLC a n a l y s i s . for  concentrations  i n the range (0.01 mg/mL to 0.6 mg/mL) were  obtained f o r each compound s t u d i e d . for  C a l i b r a t i o n curves  Duplicate i n j e c t i o n s were made  standard and sample s o l u t i o n s . The concentration of compounds  i n the aqueous phase was deduced from the c a l i b r a t i o n curves. HPLC analyses  were conducted with a Hewlett Packard Model  1082B, HPLC. Detection of the a c i d i c compounds was accomplished a t 210 nm with a H i t a c h i Model v a r i a b l e wave length spectrophotometer. The spectrophotometer was s e t a t 0.05 absorbance u n i t s f u l l s c a l e . A 20 cm x 4.6 mm i . d . column packed w i t h  5 ym H y p e r s i l 0DS  (Hewlett-Packard) was used i n the a n a l y s i s .  Mobile phase of 70%  MeOH : 30% 0.01M NaH 2 P0 A was used f o r t r i m e t h y l a c e t i c a c i d , 579  isoamyltetrazole and  N,N-dibutylsuccinamic  phase of 50% CH3CN : 50% 0.01M methyltetrazole a n a l y s i s .  measurements.  Mobile  NaH 2 P0 4 was used f o r 5-cyclohexylEluent  volumes of 20 uL were i n j e c t e d . peak area  a c i d analyses.  f l o w r a t e was  1 mL/min  and  Quantitation was by peak height or  HPLC analyses  were conducted at room  temperature. The  p a r t i t i o n experiment f o r each compound was  ruplicate.  The  partition  coefficient  done i n quad-  (p) of a compound  was  calculated from the r e l a t i o n s h i p .  J  =  v  where X^"  and  Xgq  are  the respective amount of compound i n the  aqueous phase before and a f t e r p a r t i t i o n i n g and V Q q  and V Q are the  volumes of the aqueous and organic phases r e s p e c t i v e l y . G.  Determination of apparent i o n i z a t i o n constants (pKa) by potentiometric t i t r a t i o n The pKa values of the a c i d i c compounds were determined by the method  of  titration titration  A. was  A l b e r t and carried  instrument  recorder (Beckman).  E.  out  Serjeant with  an  (185).  Potentiometric  automatic  potentiometric  containing a motor-driven microburet and  The electrode system was a glass electrode i n  combination with a saturated calomel e l e c t r o d e . t i t r a t i o n the instrument phthalate  a  Before  each  was c a l i b r a t e d against potassium hydrogen  (0.05M, pH 4.00)  and  phosphate buffer (pH 7.00).  vents were deionized water and HPLC-grade methanol.  80  Sol-  The apparent i o n i z a t i o n constants were determined i n aqueous methanol (10% and 50% MeOH, v/v) by potentiometric t i t r a t i o n with standard  KOH  (standardized  with  d e l i v e r e d from a microburet.  potassium  hydrogen  Samples of compounds were weighed out  i n t o beakers and dissolved i n the solvent mixture that  the mean  concentration  of acids  during  (47.5 mL) such  the t i t r a t i o n (or  concentration at h a l f - n e u t r a l i z a t i o n ) was approximately low  molecular  0.002M f o r  weight acids and 0.001M f o r high molecular  (>130 Daltons) points  phthalate),  i n each  weight  a c i d s . Values of pKa were c a l c u l a t e d at several titration  by applying  equation and a mean pKa determined. used.  81  the Henderson-Hasselbalch  An average of 6 pKa values was  RESULTS AND DISCUSSION  A.  Chemistry  1.  Alpha-alkylsubstituted a l i p h a t i c acids Valproic a c i d , I , and 2-butylhexanoic a c i d , I I , were prepared according to the method of P f e f f e r and co-workers (154). t h e t i c sequence i s shown i n Scheme 1.  The syn-  I t i s a one-pot synthetic  method i n v o l v i n g treatment of a l k y l c a r b o x y l i c acids with the strong base, l i t h i u m diisopropylamide Alkylation  of the dianions  (LDA) to generate  with  the appropriate  the alkyl  dianions. bromide  occurred r e g i o s p e c i f i c a l l y at the a-anionic s i t e to produce the asubstituted a l i p h a t i c acids i n good y i e l d s . 2.  a.q-Dialkylsubstituted a l i p h a t i c acids The synthetic procedure of Creger (163) was used to make the a,a-disubstituted a c i d s , 2,2-dimethylvaleric methylbutyric acid usually  (V) (Scheme 2 ) . The r e a c t i v e c e n t r e s a r e  l e s s accessible  multiple  reaction  with  other  steps i n other  c l a s s i c a l malonic ester 3.  acid (XXV) and 2,2-di-  bases (e.g. NaH) or require  synthetic  methods such as the  synthesis.  a - A l k y l s u b s t i t u t e d a l i p h a t i c acids with f u n c t i o n a l i t y i n the carbon chain 2-Propyl-(E)-2-pentenoic acid was prepared employing standard procedures (Scheme 3 ) .  The f i r s t step involves bromination of v a l -  proic a c i d , I , i n the a - p o s i t i o n .  The bromo-acid was converted to  the  carried  ester  and dehydrobromination 82  out s u c c e s s f u l l y  with  2LDA a)  C3H7-CH^COOH  C3H7-CHCOO  THF/HMPA/0°C  (i) (ii)  C  2Li  C 3 H 7 Br H  +  H  3 7. CHCOOH  C3H7  2LDA b)  (^Hg-Ch^COOH THF/HMPA/0°C  C 4 H G -CHC00  2Li  ( i ) C 4 H g Br (ii) H  C  +  4H9.  >  HCOOH  C  H  4 9  II  Scheme 1.  Synthetic pathway f o r alpha-substituted a l i p h a t i c acids.  83  CH. 2LDA :HCOOH  e  e  ( C H 3 ) 2 C-COO  2Li  THF/0°C CH-  (i) (ii)  (i)  C2H5I  (11)  H+  C3H?I CH,  I  H+  3  CH,-CH9-C-COOH 3 2 , CH.  CH 0  CH 3 CH 2 CH 2 -C-COOH CH,  XXV  Scheme 2.  Synthetic route f o r alpha,alpha-disubstituted a l i p h a t i c acids  84  C  C  H  3 7 \  C  Br 2 9  H  3 7  CHCOOH  . CBr — COOH  H  3  3 7  C  H  3 7  +  EtOH/H  _  -  CH3 CH2 CH2 \  (i)Qui no!ine, A  C3H.  C-COOH  :Br - COOEt ( i i ) OH"  C  H  C  2 5~ |'  H  (iii) H  C3H?  VII  Scheme 3.  Outline f o r synthesis of 2-propyl-(E)-2-pentenoic acid (VII)  85  quinoline.  The  synthetic product which occurs as a mixture of  geometric isomers (~5:1) was  E:Z  p u r i f i e d by f r a c t i o n a l c r y s t a l l i z a t i o n  to give the thermodynamically stable E isomer. The  synthesis of 4-keto VPA  where (93)  involved  (Scheme 4 ) , IX, as reported e l s e -  a p p l i c a t i o n of the c l a s s i c a l acetoacetic  acid  synthesis. B-substituted carboxylic The  malonic  ester  synthesis was  methylvaleric  acid and  ways of  synthesis  the  acids employed to  3-methylhexanoic a c i d . involved  synthesize  The  3-  f a m i l i a r path-  a l k y l a t i o n of  malonate  with  secondary h a l i d e s , followed by base and acid hydrolysis to convert the d i e s t e r to a monoacid through d e e s t e r i f i c a t i o n and  decarboxyl-  ation . 3-Ethylpentanoic acid V I , and cyclohexylacetic  acid X I I I , were  synthesized using a one-carbon homologation method as outlined i n Scheme 5, s t a r t i n g from a v a i l a b l e chemical reagents. N,N-Dialkylsuccinamic acids Formation of N,N-diethylsuccinamic acid XV, succinamic  acid  anhydride and  the  XVI,  proceeded r e a d i l y  isolation  reaction  secondary bases (Scheme 6 ) .  ether (162), ethanol (164) reactions.  by  and  and  N,N-dibutylof  succinic  Various  solvents,  benzene have been used for  these  In t h i s study, d i s s o l u t i o n of s u c c i n i c anhydride of  c r y s t a l l i n e product proceeded more r e a d i l y  and with  ethanol than benzene. The  IR spectroscopic data of the t e r t i a r y succinamic acids  86  are  0 II CH3-C-CH2-C00Et  (i)  Na OEt  ( i i ) C,H,CHCOOEt 7 Br  0 II -•CH3-C-CH-C00Et  /  C3H7CH-C00Et  Cone. HC1/A 0 II CH3-C-CH2-CHC00H C3H?  IX  Scheme 4.  Synthetic sequence f o r preparation of 2-propyl-4-oxopentanoic acid (IX)  87  XIII  Scheme 5.  Pathways for Synthesis of the beta-substituted Carboxylic acids  88  C  2H5  a) C  CH 2 -CO  \  NH  +  V  0 -  CH2-C0  2H5  C  X  2H5.  -  0 ^N-C-CH2CH2C00H  E T 0 H  XV  CH 2 -CO  \NH  b) C4HG  +  \  C  4 H 9 ,\  \  0  M N-C-CH 2 CH 2 COOH  Benzene  CH 2 -C0'  C4HG  XVI  Scheme 6.  Synthetic route f o r preparation of the succinamic acids  89  i n agreement with l i t e r a t u r e data (165).  The IR spectra of the  succinamic acids show two c h a r a c t e r i s t i c bands a t 1690-1725 cm and  1610-1640 cm  -1  corresponding  amide carbonyl r e s p e c t i v e l y . succinamic acid  acids  t o the carboxylic and t e r t i a r y  The NMR spectra (Figure 6) of the  show differences  (XV) and N,N-dibutylsuccinamic  have two groups.  units  -1  between acid  N,N-diethylsuccinamic  (XVI).  Both compounds  between the amide and carboxylic  functional  While N,N-dibutylsuccinamic acid gives a s i n g l e t at 2.66  corresponding  to four equivalent hydrogens of the two CH^ u n i t s  (Figure 6b), N,N-diethylsuccinamic acid spectrum shows a s i n g l e t a t 2.66  equivalent to l e s s than 4 protons.  N,N-dibutylsuccinamic  acid  The ^H-NMR spectrum of  shows a broad  peak at 8.56,  f o r the  hydrogen-bonded carboxylic proton, equivalent t o two protons. I n c o n t r a s t , the •'•H-NMR of N,N-diethylsuccinamic a c i d shows a broad peak at 5.56 f o r the hydrogen-bonded carboxylic proton, equivalent to two protons.  I t appears l i k e l y that the amide and carboxylic  group i n t e r a c t i n t r a r a o l e c u l a r l y , e s p e c i a l l y i n N,N-diethylsuccinamic a c i d .  In a study of molecular i n t e r a c t i o n s of N,N-diethyl-  succinamic acid and N,N-diisobutylsuccinamic acid by IR spectroscopy,  Antonenko (165)  reported  that,  i n dilute  s o l u t i o n s , the  amides probably e x i s t i n a c y c l i c form through i n t e r a c t i o n of the acidic also  function with the amide group. suggested  Other workers (162) have  the existence of a c y c l i c  structure i n N - a l k y l -  succinamic acid and N,N-dialkylsuccinamic a c i d s .  6.  5-Alkyltetrazoles The synthesis of 5-isoamyltetrazole (XI) 5-heptyltetrazole (X) and  5-cyclohexylmethyltetrazole (XII) has been 90  reported  in  the  (a)  6(ppm) Figure 6.  NMR spectra of (a) N,N-diethyl succinamic dibutylsuccinamic a c i d .  91  acid and (t>)  N,N-  l i t e r a t u r e (161) and involved r e a c t i o n of alkylcyanide with hydrazoic acid (from NaN3 and a c e t i c acid) i n benzene at 150°C for 96120 hrs  in a  sealed  tube.  The  most frequently employed open-  reaction method requires r e a c t i o n of n i t r i l e s with ammonium azides i n dimethylformamide (166). s y n t h e s i s of the  Attempts to apply t h i s method f o r the  5 - a l k y l t e t r a z o l e s proved u n s u c c e s s f u l .  The  reaction products were apparently not a c i d i c and possessed d i f f e r ent melting points from expected values (161). data did not conform to expectations. releasing  alkyl  groups  decreases the r e a c t i v i t y  attached  In a d d i t i o n , •'•H-NMR  I t i s known that e l e c t r o n -  to  the  cyano  of the a l k y l n i t r i l e  functionality  towards  hydrazoic  acid. An a l t e r n a t i v e procedure (167), r e q u i r i n g r e a c t i o n of a l k y l nitrile  with  aluminium  azide  (prepared  in situ  from  anhydrous  aluminium c h l o r i d e and sodium azide) i n tetrahydrofuran (Scheme 7) was  successful i n producing  desired products.  The  disadvantages  associated with t h i s method include the excess of sodium azide used and the presence of inorganic aluminium s a l t i n the crude product. As  a  result  i s o l a t i o n and  of  the  known d i f f i c u l t i e s (161,166) encountered i n  p u r i f i c a t i o n of the 5 - a l k y l t e t r a z o l e , the y i e l d s of  products were not determined. 7.  Diunsaturated d e r i v a t i v e s of v a l p r o i c acid-synthesis and metabolism study The synthesis of selected analogues of v a l p r o i c acid have been reported  i n the  literature  propenyl)-2-pentenoic acid (XXVI).  acid  except (VI)  and  for the  synthesis of  2-(l'-  2-(l'-propenyl)-3-pentenoic  These two dienoic acids have been previously proposed  as possible s t r u c t u r e s f o r 92  the major diunsaturated metabolite of  CH. a)  >  NaN3/AlC13  ,CHCH2CH2CN  THF, A  CH.  H N  CH. 3 CHCH,CH9-C 2  / CH.  2  \\.  XII  c)  C77H11(.-CN 5  NaN,7AlCl, THF, A  (  \  C7H15-C  H N  ^ II  N— N  X  Scheme 7.  Synthetic  pathway f o r 5-alkyltetrazoles  93  // •N  CH3-CH=CH  CH3-CH=CH C-COOH  CH-COOH  —  CH3-CH=CH  CH3—CH2 CH VI  XXVI  v a l p r o i c a c i d (93).  There are four geometric isomers of VI and  three  of XXVI.  stereoisomers  stereoisomers,  Due t o the m u l t i p l i c i t y  of the  s t e r e o s e l e c t i v e synthetic methods are necessary to  characterize these dienoic a c i d isomers. a.  Attempted synthesis of 2-(1'-propenyl)-3-pentenoic acid (XXVI) In attempts to synthesize the  procedure  2-(1'-propenyl)-3-pentenoic a c i d ,  of Normant (168) was followed.  E t h y l a-methoxy-  acetate was treated with two equivalents of the Grignard  reagent,  propenylmagnesium bromide to a f f o r d the d i - o l e f i n t e r t i a r y a l c o h o l , 1  XXVII (Scheme 8)- whose structure was established by MS, H-NMR and IR spectroscopic  data.  Mass spectrum of dienol ether, XXVII:  m/z  111  (M-CH2OCH3,  100%), 43 (35%), 55 (33%), 41 (27%), 77 (22%), 91 (20%), 45 (17%), 123 (14%), 138 (M-HOH, 9 % ) . NMR spectrum of dienol ether, XXVII:  61.8 (m, 6H, 2CH3-C=),  2.45 (broad peak, 1H, OH), 3.43 (m, 5H, CH 2 0CH 3 ), 5.4-5.8 (m, 4H, 2CH=CH). However,  dehydration  94  of the d i e n o l  ether  followed  by  CH30CH2C00C2H5 + 2 CH3-CH = CHMgBr • THF/ether  (CH3 - CH = C H ) 2 > C-CH20CH3  HCOOH or (COOH),  OH  XXVII  (CH3- CH = CH) 2 =C=CH0CH3  CH3-CH  CH3-CH  CH  \CHCOOH  /  AgNO,  (CH3-CH=CH)2==C = CHOH  CH3-CH = CH  NaOH  CH  CH3-CH = CH  \ /  H-CHO  XXVI  Scheme 8.  Outline f o r synthesis of dienol ether i n an attempt to prepare 2-(l-propenyl)-3-pentenoic a c i d .  95  hydrolysis with e i t h e r anhydrous o x a l i c acid or formic acid yielded a dark viscous polymeric product. duct  was  The polymolecular pro-  presumed to r e s u l t from p o l y m e r i z a t i o n of the  aldehydes,  although  this  problem was  diene  not r e p o r t e d i n s i m i l a r  conditions f o r synthesis of monounsaturated aldehydes b.  (168)  (168).  A l d o l Condensation reactions towards synthesis of 2 - ( l ' - p r o p e n y l ) 2-pentenoic a c i d , VI A recent study by Kochen et a l . (124) attempted synthesis of 2-(l'-propenyl)-2-pentenoic a c i d v i a n u c l e o p h i l i c a c y l a t i o n of  1-  bromopropane with "umpolung" or carbonyl carbanion equivalent of crotonaldehyde, e v e r , t h a t the  followed by s a p o n i f i c a t i o n . synthetic  products  They reported, how-  c o n s i s t e d of a m i x t u r e  of  diunsaturated acid isomers of unknown stereochemistry. The synthetic s t r a t e g i c sequence used f o r the synthesis of 2(1'-propenyl)-2-pentenoic acid proceeded v i a an a l d o l condensation of e t h y l followed  2-pentenoate by  propionaldehyde,  1  dehydration of the 6-hydroxy-8 .y'-unsaturated  with  dehydration  NMR  and  IR  (E or Z isomer) w i t h  agents of varied  spectral  kinetically-controlled regioselectively  analysis reaction  stereoselectivity demonstrated  procedure,  at the a - p o s i t i o n  of  that  (Scheme 9 ) . under  unsaturated  adds  the a,6-unsaturated  ester  A d d i t i o n of aldehydes  (XXVIII)  to enolates of a , 8-  esters i s reported to occur at e i t h e r the y- or a-  carbon of the esters (169-173). condensation  the  propionaldehyde  enolates to a f f o r d e t h y l 2-(l'-hydroxypropyl)-3-pentenoate as the major product.  ester  The geometric aspects of the a l d o l  r e a c t i o n have been s u b j e c t e d t o s e v e r a l i n v e s t -  i g a t i o n s (170-173).  96  /  <  O  O  E  «  OH  (ll)CH CH CHO 3  (jj)KH,THF  2  f = I S 2Z-3'E (-7%)  Z.mainr 2,major  (E)  ;ooEt CH  3  CH3  C H^sC CH=C  2E-3'Z (-71%)  ^CHCOOEt  ^  ^  ^ OOEt  3Z-3'E+3Z-3'Z+3E-3'E  +  3Z-3'Z (~12%) COOEt +  + CH-j  CHj—Cf  CH  ™XOOEt CHSSCH^  3  2E-3'E (-22%)  COOEt  2Z-3'Z+2Z-3,E+2E-3'Z+2E-3'E  2Z-3'E (-14%) +  y ^ ^ —  C O O E t  +  ^"V-COOEt  2E-3'Z (-56%)  2E-3'E(-18%)  OOEt  OH  (i) LDA,HMPA.THF,-78^ (II) C H33C CH H22C CH HO O  \ ^ £ - C O O E t ' E,major  (Z)  (XXVIII)  Ai)MsCI,Et3N,CH2CI2 («)KH,THF  r X ^ ^ C O O E t  +  3Z-3'Z (-44%)  ^ ^ - C O O E t 2E-3'E (-48*)  •  ^ - C O O E t 2E-3'Z ("8X)  Scheme 9. Stereoselective synthetic routes f o r preparation of 1 2-(1 -propenyl)-2-pentenoic a c i d .  97  CH3 - CH - CH = CH - COOEt  CH3 - CH = CH - CH - COOEt  X-anion  a-anion  In the present study, NMR  and IR a n a l y s i s of the a l d o l con-  densation products indicated that e t h y l 2-(l'-hydroxypropyl)-(Z)-3pentenoate  predominated  e t h y l (E)-2-pentenoate  over was  the other hand, e t h y l  the  corresponding  (E)-isomer  the s t a r t i n g m a t e r i a l (Scheme 9 ) .  2-(l'-hydroxypropyl)-(E)-3-pentenoate  dominated over the (Z)-isomer when e t h y l (Z)-2-pentenoate was s t a r t i n g reagent. intensity 690 cm  -1  IR  On prethe  In p a r t i c u l a r , the (Z)-isomer showed a medium  absorption band at 985 cm  -1  and  a strong band at  corresponding to the Z-configuration at the 6,y-ethylene  group i n XXVIII. at 985 cm ethylenic  when  -1  The E-isomer showed a strong IR absorption band  characteristic  group  of an E - c o n f i g u r a t i o n at the  (see Experimental  5-Ib).  8,y-  These r e s u l t s are i n  accord with s i m i l a r f i n d i n g s reported f o r a l d o l condensation  and  a l k y l a t i o n reactions (170,172,173). The mechanism of the i n v e r s i o n of the geometrical ( o l e f i n i c ) configuration  from  ( Z ) - p r e c u r s o r to  (E)-product  i s probably  analogous to that proposed by Kende and Toder (170) as shown i n Figure 6. on  product  The stereochemical course of the r e a c t i o n has been based stereoselectively  and  to f a c t o r s  stabilizing  the  i n c i p i e n t intermediates. Kende and Toder (170) have asserted that the  base-induced  deprotonation  of  the  2Z-precursor  occurs  preferably from the conformation XXIXa to a f f o r d the more stable carbanion, XXXIa and  subsequently  98  the 3E-product.  The  carbanion  intermediate XXXIIa was allylic  steric  considered to be l e s s stable due to  repulsion  geometry of the product  between R and  1,3-  COOEt (Figure 7 ) .  from the 2E-precursor was  The  considered to  a r i s e from the greater s t a b i l i t y of the carbanion XXXIb although 1,3-allylic While  non-bonded i n t e r a c t i o n s are not  i t seems l e s s ob  stable  than  the  apparent  in  XXXIIb.  vious that the carbanion XXXIb i s more  carbanion  XXXIIb  as  put  forward  by  the  authors (170), the p o s s i b i l i t y of s l i g h t differences i n s t a b i l i t y between XXXIb and XXXIIb could explain reports (172,173) that the reaction with 2Z-precursor i s more s t e r e o s e l e c t i v e than that with 2E-precursor. product  Moreover, t h i s  together  pentenoate  as  with  the  starting  study  major  showed the presence  of  3Z-product using ethyl  material  (see  IR  spectral  3E-  (E)-2-  data  in  Experimental 6 - I a ) . c  Dehydration of B-hydroxyunsaturated  e s t e r s (XXVIII)  Var ious dehydration agents were used f o r dehydration of the 8— hydroxyunsaturated reaction.  ester  formed  from  the a l d o l  condensation  Phosphorus pentoxide dehydration of e t h y l 2-(l'-hydroxy-  propyl)-3-pentenoate, derived from e t h y l (E)-2-pentenoate, gave the seven possible isomeric acids of 2,3'-diene VPA (VI) and 3,3'-diene VPA, XXVI (Scheme 9 ) .  Figure 8a shows the t o t a l i o n chromatogram  of the t-BDMS e s t e r s of the dienoic acid isomeric mixture  after  conversion of the e t h y l e s t e r s to the dienoic a c i d s . Dehydration  of e t h y l 2-(1'-hydroxypropyl)-3-pentenoate,  der-  ived from e t h y l (E)-2-pentenoate, with p-toluenesulfonyl c h l o r i d e afforded four diunsaturated isomeric e s t e r s (Scheme 9 ) .  Figure 9a  +  shows the mass chromatogram of the [M-57] ion of the t-BDMS esters of the four diene VPA isomers i n the synthetic product mixture. 99  Conformation  Ester  (E)  Favored  Resulting Carbanion  Disfavored  XXIXb  XXXb  Favored  XXXIb  Disfavored  Product  XXXIIb  (Z)  R = CH ;R=CHCH CHj| 3  R=CH  2  3  R=CH (CH ) 3  Figure 7.  2  n  Stereoisomers in a l k y l a t i o n and aldol reactions of ester enolates.  100  5"  s-  ' *  5  «  CH • COW  3  /  CHj - CH2 - CH  3,3-diene VPA  23-<JieneVPA ,  (2Z^(fe: 22-3*2; 2E-3TZ; 2E-3"E)  [ 3 Z - * ; 3Z-3TZ ; 3E-3**c)  52  1  i  60  C - COOH  3^  CH, - CH - CH  — i  / r  CHj - CH • CHV  CH, - CH - CH  i  i  i  6.8  i  76  —  84  TIME{nfa)  Figure 8„ Capillary GCMS separation of t-BDMS esters of isomeric diene-VPA. a) before UV i r r a d i a t i o n , b) after 6 hr UV i r r a d i a t i o n . Peak numbers correspond to 1: ( Z,E)-3,3'-diene; 2: (Z,E)-3,3'-diene; 3: (Z,Z)-3,3'-diene; 4: ( E,E)-3,3'-diene; 5: (Z,E)-2,3'-diene; , 6: (E)-2-ene VPA; 7: (E,Z)-2,3'-diene; 8: (Z,Z)-2,3 -diene; 9: (E,E)2,3'-diene.  101  2  I  6.0  -»  1  i  7.0  u —  8.0  m/z 197  %  9.0  i  10.0  11.0  TIME (min)  B.  r  I  m/z 197 6.0  70  8.0  ' . 9>0  10.0  11.0  TIME (min) Figure 9. Mass chromatograms of t-BDMS esters of a) four diene-VPA isomeric mixture prepared using p-TsCl, b) diene-VPA metabolites in urine extract. Peak 1: (Z,Z)-3,3'-diene; 2: (Z,E)-2,3'-diene; 3: ( E,Z)-2,3'-diene; 4: (E,E)-2,3'-diene. 102  The  combination  of  methanesulfonyl  c h l o r i d e and  hydride i n the dehydration of the B-hydroxyunsaturated  potassium esters gave  the minimum number of isomeric dienoic acids (Scheme 9 ) .  These  r e s u l t s , along with the studies of Kende and Toder (170), demonstrate  the  highly s t e r e o s e l e c t i v e nature  of CH2SO2CI-KH i n  the  dehydration r e a c t i o n s . d.  Photochemical Isomerization Photochemical isomerization of the unsaturated acids provided an i n d i r e c t method of determining the p o s i t i o n a l isomers and  their  c a p i l l a r y GC r e t e n t i o n times.  acids  C i s and trans a,B-unsaturated  have been reported to isomerize photochemically 8,y-unsaturated  acid  esters  usually possess a strong absorption band i n the 220-250 nm  region  compared  to  unsaturated  acids (174,175).  to c i s and/or trans  less  strong  compounds.  a,8-Unsaturated  absorption  of  their  isomeric  B,y-  UV i r r a d i a t i o n of the seven isomeric a c i d  mixture (from dehydration of B-hydroxyunsaturated  esters with P2O5)  resulted i n a s t r i k i n g build-up of four peaks (peaks 1, 2, 3, 4 i n F i g . 7) which were assigned to B,y-B',y'-diunsaturated acids ( V I I ) . Peaks 1 and 2 can be described as diastereoisomers of 3Z-3'E-diene VPA  or one of the peaks may  double bond.  be due to a diene VPA with a terminal  The peaks whose height decreased with UV  (peaks 7, 9 i n F i g . 8) were described as trans acids.  irradiation  a,B-diunsaturated  The peaks whose height did not show any s i g n i f i c a n t change  (peaks 5, 8) were described as c i s a,B-diunsaturated esters since under the conditions of photoisomerization they can be formed from the corresponding B,y-isomers  trans a,B-isomers and  (174,175). 103  i n turn isomerize to the  e.  GC E l u t i o n Order i n GCMS A n a l y s i s A d d i t i o n a l support f o r a t e n t a t i v e assignment of s t e r e o chemical configuration of the dienoic acid peaks i n Figure 8 obtained from the GC r e t e n t i o n data.  was  Shorter retention times f o r  the cis-isomer of a given p o s i t i o n compared to the trans-isomer on non-polar columns have been frequently observed (176,177).  The  peaks b e f o r e peak 1 i n F i g u r e 8b, which were confirmed  two  using  authentic samples to be 3-ene VPA and c i s 2-ene VPA, were produced by photochemical i s o m e r i z a t i o n of trans 2-ene VPA (peak 6) added as an i n t e r n a l standard. With a t e n t a t i v e d e s c r i p t i o n of the GC e l u t i o n order (12.5m d i m e t h y l s i l i c o n e column) of the seven isomeric a c i d s , peaks 3 and 4 i n Figure 9a were due to trans a,8-8,y'-diunsaturated a c i d s . was supported by NMR  spectra of the four diene VPA isomeric mixture  obtained from dehydration of the 8-hydroxyunsaturated toluenesulfonyl c h l o r i d e . 66.83 and  67.0  This  ester with p-  The i n t e n s i t y of the •''H-NMR t r i p l e t s at  (see Experimental  l i b ) due  to the trans B - v i n y l  proton indicated the high proportion of 2E-3'Z and 2E-3'E isomers i n the mixture (Scheme 9 ) . f.  Argentation TLC The  synthesized isomeric mixtures of dienoates obtained from  dehydration of the major products, e t h y l 2-(l'-hydroxypropyl)-(Z)3-pentenoate and CH3SO2CI-KH  e t h y l 2-(l'-hydroxypropyl)-(E)-3-pentenoate  were  argentation thin  subjected  to  purification  l a y e r chromatography. 104  The  by  with  preparative  o b j e c t i v e was  to  i s o l a t e the isomers from the bands on the TLC p l a t e s , c h a r a c t e r i z e the NMR  s p e c t r a l data for each isomer and confirm stereochemical  designation of the isomeric dienoates and dienoic a c i d s . Table  1 shows the i d e n t i t y , proportion and Rf values of the  isomers i n the three bands, f o l l o w i n g argentation TLC. ortion  of isomers  argentation TLC was  i n the mixture  of dienoates  determined by NMR  analysis.  The  prop-  before and  after  Figure 10 shows  the p r o f i l e of components i n the three bands f o l l o w i n g argentation TLC  and GCMS a n a l y s i s of the e t h y l esters on a D e x s i l 300 packed  column.  The order of e l u t i o n of isomeric dienoates on the D e x s i l  300 column and the m o b i l i t y of the isomers on the TLC p l a t e (Table 1) agree with the chromatographic properties of the stereoisomers. Trans-trans  diunsaturated  esters are  reported  to be  less  polar  (high Rf) than t h e i r corresponding c i s - c i s , t r a n s - c i s or c i s - t r a n s isomers (177-178). Moreover conjugated dienoates have been reported to have higher Rf values than t h e i r non-conjugated congeners using s i m i l a r eluents (181,182). Mass spectra of the four dienoic acid e t h y l esters show the +  intense M , (Table  1)  m/z  168 of the 2,3'-dienes compared to the 3,3'-dienes  indicating  the  effect  of conjugation  Diene e t h y l e s t e r s eluted from the TLC e i t h e r as a s i n g l e Purified  i n 2,3'-dienes.  bands could be  isomer or a mixture of isomers  (Figure 10).  synthesized products were characterized by NMR  scopy and Table 2 summarizes the NMR  isolated  spectro-  data f o r the dienoate isomers.  The chemical s h i f t s assigned the o l e f i n i c protons, 8 - v i n y l proton, methylene and characteristic  methyl of  the  protons  adjacent  stereochemical  105  to the features  double bands are of  the  dienoate  Table 1 Composition of chromatographic data of a mixture of synthesized isomeric dienoates  Diene VPA Ethyl Ester  Product-ratio with r e a c t a n t , ethyl 2-pentenoate E  3Z-3»Z  a  0.07  2E-3'Z  0.71  2E-3'E  0.22  D  C  tR (min)  Mass spectrum (70ev) m/z ( r e l . i n t e n s i t y )  9.37  95 (100%), 168 ( 7 % ) , 139 ( 2 % ) , 122(1%)  Z 0.44  2Z-3»E  TLC R f  0.42-0.49 (Band l a ) 0.53-0.62 (Band I l l b )  10.0  95 (100%), 168 (50%), 140 (41%), 122 (24%)  0.08  0.45-0.53 (Band l i b )  10.5  95 (100%), 168 (57%), 140 (41%), 122 (28%), 153 (4%)  0.48  0.53-0.62 (Band I l i a , b)  11.1  95 (100%), 168 (54%), 140 (35%), 122 (32%), 153 (4%)  r a t i o of i s o m e r s i n s y n t h e t i c product m i x t u r e before TLC u s i n g e i t h e r e t h y l (Z) or (E)-2-pentenoate as reactant. band i n which isomer i s concentrated. GCMS r e t e n t i o n time of isomeric peaks analyzed with 3% D e x s i l 300 column (1.8m x 2 mm i.d.) H e l i u m , 25 ml/min. Column temp of 50°C to 280°C at rate of 8°C/min.  106  Ia 1  I  Ilia  3 u  nb  11  1Mb  m/z168 0  TO"  —1%  TIME (min) Figure 10. GCMS analysis of dienoates eluted from TLC p l a t e s . Total ion chromatograms I, I I , III correspond to ethyl esters i n bands I, I I , III respectively, a and b refer to esters synthesized from(Z) and (E)-2-pentenoate respectively. Peak 1: (Z,Z)-3,3'diene; 2: (Z,E)-2,3'-diene; 3: (E,Z)-2,3'-diene; 4: (E,E)-2,3'diene 0 107  Table 2 NMR (400 MHz) data for diene VPA ethyl esters  Dienote  CH3  2E-3'E  1.04(t)  2E-3»Z  1.0A(t) 1.54(dd) 2.11(m)  2Z-3'E  1.04(t)  3Z-3'Z  a  CH3-C= 1.84(d)  1.70(d)  CH2 2.31(m)  2.44(m)  1.66(dd) 1.69(d)  CH  H(3')  a  H(4')  a  H(3)  a  6.17(d) J=16Hz  6.08(dq) J=16Hz  6.59(t)  6.01(d) J=11.4Hz  5.79(dq) J=11.4Hz  6.79(t)  b  b  5.92(t)  3.5(t) 5.5-5.6 (m)  5.6-5.7 (dq)  Position of hydrogen in the branched-carboxylic acid ester (Scheme 9).  ^Resonance peaks were very weak due to small amounts of isomer obtained.  108  isomers  and agree with l i t e r a t u r e  structures 9.  precedence (170,179,180).  The  of the 2,3'-dienes and 3,3'-dienes are shown i n Scheme  The stereochemistry of the isomers i s c l a r i f i e d by the observ-  ation  that  the 2E-3'Z  deshielded isomers  methyl have  Similarly  and the 3Z-3'Z  isomers  have  (CH3~C=) doublets while the 2E-3'E  the more  deshielded  methyl  the  and  (CH3-C=)  less  2Z-3'E  doublets.  the 2E-3'Z has the l e a s t deshielded methylene  (CH2-C=)  m u l t i p l e t s i n comparison with those of 2E-3'E and 2Z-3'E.  The 2E-  3'E and 2E-3'Z have the more deshielded 8 - v i n y l proton occurring as a t r i p l e t at 66.59 and 66.79 r e s p e c t i v e l y . also  2E-3'E and 2E-3'Z can  be d i f f e r e n t i a t e d by the coupling constants obtained f o r the  coupling of the o l e f i n i c protons, H(3') and H(4') i n Table 2. g.  I d e n t i f i c a t i o n of the Major and Minor Diene VPA Metabolites The has  i d e n t i f i c a t i o n of one of the minor diene VPA  been  reported  acid (124,125). diene  VPA  retention  and  respectively  be  diene VPA were  found  with the minor and major  i n human  urine  (Figures  a c q u i s i t i o n of a synthetic sample of acid,  2-propyl-(E)-2,4-pentadienoic  In t h i s study, the t-BDMS d e r i v a t i v e s of 2E-3'Z2E-3'E  times  to  metabolites  to have  diene 8  and  VPA 9).  identical metabolites With  the  2-propyl-(E)-2,4-pentadienoic  i t was included i n the GCMS a n a l y s i s of  synthetic  dienoic  acid d e r i v a t i v e s and urine metabolites. Separation of the t-BDMS and TMS d e r i v a t i v e s was using  a 25m long SE-54 and 0V-1701 columns.  structures were  accomplished  Figure 11 shows the  of the diunsaturated d e r i v a t i v e s of v a l p r o i c a c i d which  considered i n the i d e n t i f i c a t i o n of the diunsaturated  olites.  metab-  The r e t e n t i o n times of the t-BDMS d e r i v a t i v e s of 2-propyl109  5'  4'  3'  5'  CH  CH  CH 3 - CH = C  4'  3'  V  5  4  CH  •/  2  - COOH  CH  3,3'-DIENE VPA  4  CH:  CH 2 - CH  2,3'-DIENE VPA  •3'E ; 3Z-3'Z ; 3E-3'E)  (2Z-3'E ; 2Z-37 ; 2E-37 ; 2E-3'E)  CH 3 - CH 2 - CH 2 \  t - COOH 3 /  5  CH 2 = CH - CH - COOH  CH - COOH  CH 2 = CH - D T  CH 2 = CH - CH 2  2,4-EIENE VPA (2E j 2Z)  M'-DIENE VPA  Figure 1 1 . Chemical structures of diunsaturated derivatives of valproic acid investigated as the potential metabolites of valproic acid.  110  (E)-2,4-pentadienoic acid To  acid and  2-[(Z)-l'-propenyl]-(E)-2-pentenoic  as w e l l as the minor diene VPA metabolite were a l l i d e n t i c a l . resolve  the problem of which of the two dienoic acids  ascribed to the minor metabolite, c a p i l l a r y GCMS. acids two  was  can  be  TMS d e r i v a t i v e s were analyzed by  Separation of the TMS d e r i v a t i v e s of the  dienoic  accomplished (Figure 12) and i n d i c a t e d that one of  minor diene VPA metabolites and  the  2-propyl-(E)-2,4-pentadienoic  acid had i d e n t i c a l r e t e n t i o n times which was d i f f e r e n t from that of 2-[(Z)-l'-propenyl]-(E)-2-pentenoic  acid (peak 3 i n  Figure  12a).  The major diene VPA metabolite (peak 5 i n Figure 12b) had the  same  retention  time as 2-[(E)-l'-propenyl]-(E)-2-pentenoic a c i d .  This  confirmed  e a r l i e r r e s u l t s obtained with the t-BDMS d e r i v a t i v e s of  dienoic  acids i n a synthesized product sample and i n a  t r a c t (Figures 8, 9 ) . retention  time  pentenoic  acid  urine  The small peak i n Figure 12b had a  similar (peak  to  4  that  of  i n Figure  ex-  relative  2-[(E)-l-propenyl]-(Z)-2-  12a),  a  by-product  synthesis of 2-[(Z)-l'-propenyl]-(E)-2-pentenoic a c i d .  in  This  the diene  has been t e n t a t i v e l y suggested to be another diene VPA metabolite. On  examination  of  the stereochemical outcome of  the  aldol  condensation and subsequent dehydration r e a c t i o n s , i t i s c l e a r that there i s an i n v e r s i o n of the geometric c o n f i g u r a t i o n i n ing ester  the  a8-unsaturated  while  dehydration However, VPA  the  6'y'-unsaturated  reactions with the more s e l e c t i v e dehydration  the presence of s i g n i f i c a n t amounts of  is  B-hydroxy  the 2E-isomer i s favoured over the 2Z-isomer  i n the dehydration  (XXVIII)  ester to  transform-  in  of  the  agents.  3Z-3'Z-diene  2-[-l'-hydroxypropyl]-(E)-3-pentenoate  contrast to the  111  the  in  established  direction  of  the  (a)  m/z197  TIME (min)  I • I •» • i • 4.0 4B  i -1 • i i I•I 5.6 64 1  TIME (min) Figure 12. Capillary GCMS separation of TMS derivatives of (a) synthesized 2,3'-diene VPA and 2,4-diene VPA; (b) diene VPA metabolites in urine extract. Peak 1: (Z)-2,4-diene VPA; 2: ( 2,4-diene VPA; 3: (E,Z)-2,3'-diene VPA; 4: (Z,E)-2,3'-diene VPA; 5: (E,E)-2,3'-diene VPA.  112  dehydration This  reaction  unexpected  of  2-[-l'-hydroxypropyl]-(Z)-3-pentenoate.  by-product  could  arise  from  deconjugation  and  i n v e r s i o n of geometric c o n f i g u r a t i o n during e l i m i n a t i o n of mesylate with  KH. I t i s evident from the "''H-NMR spectra of synthesized  that the chemical  s h i f t s are  consistent with the  assignment of the 2,3'-diene VPA with the 3,3'-diene VPA  isomers.  isomers.  The  expected GC  elution  stereochemical not the case  geometry about the double  bond could not be determined from the NMR combined with  This was  dienoates  pattern alone except when  order  (Figures 8 and  9)  and  photoisomerization r e s u l t s (Figure 8 ) . HPLC determination of l i p o p h i l i c i t y Assay method The cratic  RP-HPLC procedure was  chromatographic a n a l y s i s of r e f e r e n c e  different conditions. by UV absorption. based  developed s y s t e m a t i c a l l y by i s o -  on  the  compounds under  Detection of the compounds was accomplished  The s e l e c t i o n of the wave length of 210 nm  absorption  maximum a b s o r p t i o n  spectra  i n the  range  of  was  those compounds which show of  205-215 nm  (Figure  13).  Methanol and a c e t o n i t r i l e were chosen as the organic co-solvents, i n a n t i c i p a t i o n of s e l e c t i v i t y d i f f e r e n c e s between these two vents and  t h e i r i n d i v i d u a l e f f e c t s on the octanol-water  sol-  partition  c o e f f i c i e n t values as determined by RP-HPLC. RP-HPLC i s generally considered to be a combination of partition  (dynamic  e q u i l i b r i u m c o n d i t i o n s ) and  e q u i l i b r i u m conditions) processes.  adsorptive  (non-  Minimization of the adsorption 113  UV spectrum of tiN-Obutytouccinwnic add UV spectrum of 2,3'-Dtone VPA  300 Wavetength,nm UV spectrum of SHsoamyltetrazole Uttraviotet spectrum of Valproic acid  Wsvslertgttvwn  200  -i  1  r-  WavalengtMm  Figure 13.  UV absorption spectra of four a c i d i c compounds.  114  300  of compounds on r e s i d u a l s i l a n o l s i t e s ensures hydrophobic  inter-  actions as the sole process i n retention of compounds evidenced by the c o r r e l a t i o n between r e t e n t i o n f a c t o r s and  l o g P.  The  seven  reference compounds selected f o r the c o r r e l a t i o n of r e t e n t i o n f a c t ors and log P are v a l p r o i c a c i d and several analogues. values f o r these compounds have been determined  The l o g P  from the  shake-  f l a s k procedure by Keane et a l . ( 1 3 ) . d'Amboise and study hydrophobic  Hanai (145)  used unbuffered mobile  e f f e c t s of medium chain and  a l i p h a t i c acids i n RP-HPLC.  phases to  long chain normal  In t h i s study, the retention  times  (tg) of the polar and i o n i z a b l e v a l p r o i c acid analogues were very close using a low  percentage  mobile phase (Table 3 ) .  of organic modifier i n unbuffered  Figure 14 shows asymmetric peak shapes or  even double peaks f o r the i o n i z a b l e compounds using the H y p e r s i l ODS  column  and  the  unbuffered  acetonitrile-water  mobile  phase.  Reducation of solute adsorption with a higher percentage of organic co-solvent  i n unbuffered water  (higher e l u t i n g  strength) caused  them to elute c l o s e l y to each other and to the unretained solvent (Table 4 ) . Sodium phosphate buffer (pH 3.5) was used to suppress ation  of  stationary  ionized phase.  forms which By  interacted  selecting  strongly  form-  with the  ODS  the appropriate flow rates  and  varying the composition of the methanol-buffer and  acetonitrile-  buffer mobile phases, the r e t e n t i o n times of the reference compounds were determined  (Tables 5-7).  Longer r e t e n t i o n times and  broad, t a i l i n g peaks were observed f o r the highly l i p o p h i l i c compound 2-butylhexanoic acid  i n methanol-buffer  115  compositions below  Table 3 Retention times of reference compounds using unbuffered mobile phase (CH3CN/H20)  Retention Time (min) % CH3CN (v/v)  Compounds  20%  22%  25%  1.68  -  2.23  1. B u t y r i c acid  1.66  -  2.03  2. V a l e r i c acid  1.76  1.72  2.09  3.  2-Ethylbutyric a c i d  1.87  1.79  2.16  4.  Hexanoic a c i d  1.99*  1.94  2.38  5.  Valproic acid  2.87*  2.35  6.  2-Ethylhexanoic a c i d  7.  2-Butylhexanoic a c i d  MeOH  3.55*  *Broad peak  116  2.42 —  —  6  2  4  §  §  10  Time(min)  Figure 14. Superimposed HPLC chromatograms of a c i d i c compounds using unbuffered mobile phase. Column; Hypersil ODS reverse phase column (20cm x 4.6mm). Mobile phase; 20% a c e t o n i t r i l e in water ( v / v ) . Flow r a t e , 1.0 mL/min. a) solvent, b) valproic acid in solvent, c) heptanoic acid in solvent, d) v a l e r i c acid in sol vent.  117  Table 4 E f f e c t of a d d i t i o n of phosphate buffer (pH 3.5), i n mobile phase (MeOH/r^O), on the r e t e n t i o n times of reference compounds. Flow rate i s 1.0 mL/min  Retention Time (min) Compounds  Presence of Buffer  Absence of Buffer  60% MeOH  70% MeOH  2.40  2.39  2.28  2.39  1. B u t y r i c a c i d  3.23  2.94  1.92  2.09  2. V a l e r i c a c i d  4.36  3.37  2.19  2.27  5.50  3.91  2.29  2.34  4. Hexanoic a c i d  6.28  4.18  2.30  2.39  5. V a l p r o i c acid  13.85  6.53  3.24*  2.65*  6. 2-Ethylhexanoic a c i d  13.96  6.58  Methanol  3. 2-Ethylbutyric  acid  7. 2-Butylhexanoic a c i d  >30.0  13.70  *Broad and t a i l i n g peaks.  118  60% MeOH  -  70% MeOH  2.95* 2.89*  Table 5 Retention times of seven reference compounds a t d i f f e r e n t percentages of MeOH i n the mobile phase (MeOH/O.OlM NaH 2 P0 4 ). Flow rate of mobile phase i s 1.0 mL/min.  Retention Time (min) % MeOH (v/v) 50% 60% 70%  Compound  1.54  2.40  2.39  1. Butyric a c i d  2.49  3.23  2.94  2. V a l e r i c acid  3.80  4.36  3.37  3. 2-Ethylbutyric a c i d  5.54  5.50  3.91  4. Hexanoic acid  6.85  6.28  4.18  5. Valproic a c i d  20.45'  13.85  6.53  6. 2-Ethylhexanoic a c i d  19.45  13.96  6.58  Methanol  7. 2-Butylhexanoic a c i d  >50.0  119  >30.0  13.70  Table 6 Retention times of seven reference substances a t d i f f e r e n t flow r a t e s (a)  I n 70% MeOH: 30% 0.01M NaH 2 P0 A mobile phase  Compound  Retention Time (min) Flow Rates 1.5mL/min 1.OmL/min 1.2mL/min 2.39  2.03  1.54  1. B u t y r i c a c i d  2.94  2.45  1.90  2. V a l e r i c a c i d  3.37  2.83  2.21  3. 2-Ethylbutyric a c i d  3.91  3.27  2.54  4. Hexanoic a c i d  4.18  3.50  2.73  5. Valproic a c i d  6.53  5.49  4.37  6. 2-Ethylhexanoic a c i d  6.58  5.49  4.40  7. 2-Butylhexanoic a c i d  13.70  11.46  9.50  Methanol  (b)  In 60% MeOH: 40% 0.01M NaH 2 P0 A mobile phase Retention Time (min) Flow Rates 1.5mL/min 1.OmL/min  Compound  2.40  1.63  1. B u t y r i c a c i d  3.23  2.19  2. V a l e r i c a c i d  4.36  2.94  3. 2-Ethylbutyric a c i d  5.50  3.71  4. Hexanoic a c i d  6.28  4.25  5. V a l p r o i c a c i d  13.85  9.26  6. 2-Ethylhexanoic a c i d  13.96  9.32  Methanol  >30.0  7. 2-Butylhexanoic a c i d  120  >23.0  Table 7 Retention times of seven reference compounds at d i f f e r e n t percentages of a c e t o n i t r i l e i n the mobile phase  Retention Time (min) Compounds  (Flow Rate=1.0mL/min) (Flow Rate=1.5mL/min) % CH3CN (v/v) % CH3CN (v/v) 50%  55%  2.00  2.00  2.00  1. B u t y r i c acid  2.72  2.78  2. V a l e r i c a c i d  3.17  3. 2-Ethylbutyric a c i d  45%  50%  1.47  1.42  1.37  2.70  2.06  1.93  1.85  3.16  3.00  2.67  2.33  2.18  3.65  3.52  3.25  3.24  2.72  2.47  4. Hexanoic acid  3.96  3.73  3.40  3.73  3.02  2.69  5. Valproic a c i d  6.32  5.51  4.63  7.71  5.31  4.30  6. 2-Ethylhexanoic a c i d  6.34  5.50  4.63  7.77  5.35  4.33  7.88  23.23  Methanol  7. 2-Butylhexanoic a c i d  13.51  10.25  121  60%  40%  13.39  9.40  60% and a c e t o n i t r i l e - b u f f e r compositions below 40%. 2.  Void time and retention mechanism i n RP-HPLC The unretained e l u t i o n time or void time ( t Q ) i n the RP-HPLC method was determined  by the e l u t i o n  component, methanol or a c e t o n i t r i l e . tQ  time of the mobile  phase  Table 8 shows the values of  obtained by organic co-solvent and l i n e a r regression of homo-  logous s t r a i g h t - c h a i n c a r b o x y l i c acids (^HyCOOH-CyH-j^COOH) according to the method of Berendsen et a l . (181).  For the same flow  r a t e , the void times i n the two methods appear to be s i m i l a r and stay reasonably constant with increases i n the volume percentage of a c e t o n i t r i l e i n the buffered mobile phase.  For the same flow r a t e ,  anomalous values are obtained f o r the methanol-buffer mobile phase. The  void  appear  times determined  by e i t h e r of the two methods do not  to decrease or stay constant with increases i n the prop-  o r t i o n of methanol i n the mobile phase.  S i m i l a r f i n d i n g s of void  times i n methanol-water mixtures, e s p e c i a l l y i n the region of 6070%  methanol,  have  searchers (149,150,181).  been  reported  by  several  re-  However, f o r the same mobile phase, the  void time decreases with an increase i n the flow r a t e . Berendsen et a l . (181), i n a study of r e t e n t i o n mechanisms i n RP-HPLC, have proposed that the normal e f f e c t of decrease i n void time with increase i n volume percentage of methanol or a c e t o n i t r i l e could be explained by solvophobic e f f e c t s .  Thus, with a decrease  i n volume percentage of organic m o d i f i e r , the s o l v a t i o n of the C-18 monomeric layer by the organic modifier decreases.  Solvation was  suggested to be minimal f o r pure water, hence i n t e r a c t i o n between  122  the hydrocarbon chain and water eluent i s minimal.  According  to  the authors, minimal s o l v a t i o n implied a maximal i n t e r n a l porosity of the m i c r o p a r t i c u l a t e supports  and  hence a maximal hold-up or  void time. In comparison of methanol-water and a c e t o n i t r i l e - w a t e r e f f e c t s on  void  time,  Yonker  et  a l . (149,150) explained  the  anomalous  values of void time i n methanol-water mixtures as due to a s t a t i o n ary phase phenomenon. They also proposed a model of the s t a t i o n a r y phase composed of the C-18  monomeric l a y e r , s i l i c a  s o l v a t i o n layer from mobile phase components.  support and  a  With increases i n  the volume of percentage of methanol up to 70%,  they postulated  that the mobile phase components, methanol or water, hydrogen-bond to  the exposed s i l i c a  support between the C-18  phenomenon predominates over that of s o l v a t i o n . plateau  with approximately  70%  chains  and  this  They explained the  methanol-water  ( i n s t e a d of  a  decrease i n void time) to be due to the dominating e f f e c t of s o l v a t i o n layer on C-18  hydrocarbon c h a i n .  Void times of the mobile  phase component, methanol i s then held up by i n t e r a c t i o n with the s o l v a t i o n layer on the C-18  chain.  On  the other hand, the void  time determined i n a c e t o n i t r i l e - w a t e r mixtures  decreases s l i g h t l y  or stays nearly constant with an increase i n volume percentage of a c e t o n i t r i l e (Table 8 ) .  The e f f e c t s of a c e t o n i t r i l e - w a t e r mobile  phase i n t h i s study could be explained accordingly by the lower hydrogen-bonding properties of a c e t o n i t r i l e with the e f f e c t s of the solvation  layer i n opposition to the higher e l u t i n g strength of  acetonitrile. In  view of  the  differing  s t a t i o n a r y phase c o n d i t i o n s , void  times were determined experimentally f o r each mobile phase compos123  Table 8 Comparison of void times ( t Q ) determined from (a) i n j e c t i o n of methanol, and (b) dead time i t e r a t i o n of the retention times of the homologous s e r i e s from C3H7COOH to C7H15COOH Mobile Phase using 0.01M NaH^PO/ as co-solvent (v/v)  Flow Rate (mL/min)  t Q (Me0H) (min)  tQ(iteration)a (min)  50% MeOH  1.5  1.54  1.38  60% MeOH  1.0  2.40  2.13  70% MeOH  1.0  2.39  2.33  70% MeOH  1.2  2.03  1.86  70% MeOH  1.5  1.54  1.51  60% MeOH  1.5  1.63  1.33  50% CH3CN  1.0  2.00  2.06  50% CH3CN  1.5  1.37  1.43  55% CH3CN  1.0  2.00  2.02  60% CH3CN  1.0  2.00  1.80  40% CH3CN  1.5  1.47  1.41  45% CH3CN  1.5  1.42  1.38  = at determined using equation 1L R,N+1 R,N- - t 0 ( a - l )  124  ition.  Capacity  factor  (k') for each reference  d i f f e r e n t mobile phase was then 3.  compound i n a  calculated.  Eluent e f f e c t s on capacity f a c t o r Mobile  phases containing various percentages of organic  co-  s o l v e n t were used to determine which mobile phase p r o v i d e s  a  s i g n i f i c a n t high c o r r e l a t i o n f o r the l i n e a r regression equation log P = a + b log k'. correlation  Tables  9-11  parameters obtained  show the log k' values and for the  various  the  percentages  of  organic modifier i n buffered mobile phases. Several i n t e r e s t i n g  points emerge from the regression data.  For the same organic co-solvent and  flow r a t e , the slope of the  plot of log P versus log k' decreased with a decrease i n the percentage  of  organic  co-solvent  i n the  mobile  phase.  Thus  s e n s i t i v i t y to changes i n the l i p o p h i l i c character increased with the  more aqueous mobile phase, suggesting  that the  hydrophobic  e f f e c t i s a function of the p o l a r i t y d i f f e r e n c e between the mobile and s t a t i o n a r y phases.  On the other hand, there was v i r t u a l l y no  change i n the slope with the same mobile phase but d i f f e r i n g flow rates between 1.0 ml/min and 1.5 ml/min. The regression equations with methanol-buffer 50-70% methanol and phases  gave  mixture between  40-60% a c e t o n i t r i l e - b u f f e r mixture  s i g n i f i c a n t l y high  correlation  as mobile  coefficients.  The  l i n e a r model explained greater than 97% of the v a r i a t i o n s observed. The  correlation  coefficient  increased  at  lower  organic modifier  volume percentage due  to the  l i p o p h i l i c character.  However, lower percentages could not e l u t e  125  greater s e n s i t i v i t y to changes i n  Table 9 C o r r e l a t i o n of l o g k' and l o g P Q / W f o r seven reference compounds at various compositions of the mobile phase (MeOH/O.OlM N a H 2 P 0 4 ) . log P Q / W = a + b l o g k'  Compound  5 0 % MeOH  6 0 % MeOH  70% MeOH  70% MeOH  70% MeOH  (FR*=1.0)  (FR=1.0)  (FR=1.0)  (FR=1.2)  (FR=1.5)  log k'  log k'  log k'  log k'  log k'  ** o/w  P  8  1. B u t y r i c acid  -0.2098  -0.4611  -0.6380  -0.6842  -0.6312  0.98  2 . V a l e r i c acid  0.1666  -0.0879  -0.3872  -0.4044  -0.3614  1.51  3 . 2 - E t h y l b u t y r i c acid  0.4145  0.1111  -0.1965  -0.2141  -0.1875  1.68  4 . Hexanoic acid  0.5376  0.2086  -0.1255  -0.1402  -0.1120  1.93  5 . V a l p r o i c acid  1.089  0.6786  0.2386  0.2316  0.2643  2.75  6 . 2-Ethylhexanoic a c i d  1.065  0.6827  0.2438  0.2316  0.2688  2.64  7 . 2-Butylhexanoic a c i d  >1.498  0.6751  0.6670  0.7134  3.20  2.1955 1.9461 0.9930 0.090 281  2.2223 1.8822 0.9928 0.091 273  2.1576 1.9179 0.9944 0.080 357  2.1463 1.7618 0.990 0.122 246  2.1755 1.7222 0.991 0.117 270  2.1098 1.726 0.990 0.120 255  a n = 6 b (excluding compound # 7 ) r s F  n = 7  1.2336 1.3348 0.9957 0.070 467  >1.061 1.6279 1.5218 0.9944 0.080 355  a b r s F  *  L O  FR i s F l o w Rate (mL/min) o f e l u e n t , s i s s t a n d a r d e r r o r of e s t i m a t e , FQ Q J = 16.0, r i s c o r r e l a t i o n c o e f f i c i e n t , F s t a t i s t i c from a n a l y s i s of variance  ** Values obtained from P.E. Keane et a l . ( 1 3 ) .  Table 1 0 C o r r e l a t i o n of l o g k' and l o g P Q / W f o r seven reference compounds at various compositions of the mobileP phase (CH3CN/O.OIM N a H 2 P 0 4 ) . L O 8 o/w = a + b l o g k* 50% (FR*=1.0)  Compound  log k'  log k'  60% (FR=1.0)  log k'  40% (FR=1.5) f  45% (FR=1.5)  50% (FR=1.5)  log k»  log k'  log k  L O  ** o/w  P  8  1. B u t y r i c a c i d  -0.4437  -0.4089  -0.4559  -0.3965  -0.4447  -0.4555  0.98  2. Valeric acid  -0.2328  -0.2366  -0.3010  -0.0881  -0.1932  -0.2282  1.51  3. 2-Ethylbutyric acid  -0.0835  -0.2744  -0.2041  0.0806  0.03834  -0.09533  1.68  4 . Hexanoic a c i d  -0.00877  -0.063  -0.1549  0.1870  0.05183  -0.01615  1.93  5. Valproic acid  0.3345  0.2443  0.1189  0.6279  0.4377  0.3301  2.75  6 . 2-Ethylhexanoic acid  0.3365  0.2430  0.1189  0.6320  0.4421  0.3346  2.64  7 . 2-Butylhexanoic a c i d  0.7600  0.6154  0.4683  1.1703  0.9258  0.7680  3.20  a b r s F  1.9178 1.9104 0.9880 0.133 205  2.0623 2.1190 0.9798 0.173 120  2.2436 2.4780 0.9834 0.157 146  1.6309 1.4793 0.9895 0.125 235  1.7941 1.6945 0.9836 0.156 148  1.9266 1.8879 0.9877 0.135 200  a b n=6 r (excluding compound #7) s F  1.9506 2.1840 0.9958 0.069 478  2.1153 2.4244 0.9822 0.143 109  2.3472 2.9534 0.9968 0.061 616  1.6236 1.6765 0.9967 0.062 607  1.8003 1.9276 0.9875 0.120 157  1.9623 2.1740 0.9968 0.061 613  n=7  *  55% (FR=1.0)  FR i s Flow Rate (mL/min) o f e l u e n t , s i s s t a n d a r d e r r o r of e s t i m a t e , FQ Q J = 1 6 . 0 , r i s c o r r e l a t i o n c o e f f i c i e n t , F s t a t i s t i c from a n a l y s i s of variance  ** Values obtained from P.E. Keane e t ' a l . ( 1 3 ) .  Table 11 Summary of l i n e a r regression parameters f o r l o g P versus l o g k Log P = a + b l o g k'  1  n* = 6 Mobile Phase (Aqueous b u f f e r organic solvent)  Flow Rate (mL/min)  a  b  r  s  50% MeOH  1.0  1.2336  1.3348  0.9957  0.070  60% MeOH  1.0  1.6279  1.5218  0.9944  0.080  70% MeOH  1.0  2.1955  1.9461  0.9930  0.090 -  70% MeOH  1.2  2.2223  1.8822  0.9928  0.091  70% MeOH  1.5  2.2389  2.0530  0.9944  0.080  50% AcN  1.0  1.9506  2.1840  0.9958  0.069  55% AcN  1.0  2.1153  2.4244  0.9822  0.143  60% AcN  1.0  2.3472  2.9534  0.9968  0.961  40% AcN  1.5  1.6236  1.6765  0.9967  0.062  45% AcN  1.5  1.8327  1.9334  0.9965  0.120  50% AcN  1.5  1.9623  2.1740  0.9968  0.961  n* = 7 70% MeOH  1.0  2.1463  1.7618  0.990  0.122  70% MeOH  1.2  2.1755  1.7222  0.991  0.117  70% MeOH  1.5  2.1561  1.7278  0.990  0.120  50% AcN  1.0  1.9178  1.9104  0.9880  0.133  55% AcN  1.0  2.0623  2.1190  0.9798  0.173  60% AcN  1.0  2.2436  2.4780  0.9834  0.157  40% AcN  1.5  1.6309  1.4793  0.9895  0.125  45% AcN  1.5  1.8144  1.6842  0.9879  0.156  50% AcN  1.5  1.9266  1.8879  0.9877  0.135  •number of reference compounds (solutes) used; r i s c o r r e l a t i o n c o e f f i c i e n t , s i s standard error of estimate.  128  the highly l i p o p h i l i c 2-butylhexanoic acid without peak broadening. Consequently when t h i s compound was included, the c o r r e l a t i o n coe f f i c i e n t decreased. Thus reduction of the extent of adsorption i n the p a r t i t i o n process using optimum flow rate and e l u t i n g strength produced high c o r r e l a t i o n c o e f f i c i e n t s f o r the set of compounds.  A decrease i n  the adsorption phenomenon then ensures optimum rate of exchange and mass t r a n s f e r of solute between the l i q u i d phases. 4.  HPLC l o g P values of v a l p r o i c acid and analogues Mobile phases selected f o r the determination of l o g P values were  70% methanol-buffer and 50% a c e t o n i t r i l e - b u f f e r .  The two  mobile phases gave high c o r r e l a t i o n c o e f f i c i e n t s i n the regression equation and were able to e l u t e the highly  lipophilic  hexanoic acid without excessive peak broadening.  2-butyl-  Having selected  the mobile phases and experimental conditions the r e t e n t i o n times of  reference compounds  (Table 12, 13).  reference  were  determined  f o r comparison, using 70% methanol-buffer  Twenty-three  compounds.  sharp peaks.  compounds  Figure 15 shows the HPLC chromatograms of the  compounds, superimposed mobile phase.  and remaining  compounds were analyzed i n c l u d i n g the  A l l the compounds  showed  symmetrical and  Values of l o g P were c a l c u l a t e d from the respective  regression equation below and are shown i n Tables 12, 13. log P = 2.1463 + 1.7618 l o g k' n = 7, r = 0.990, s = 0.122, F = 246 (Mobile phase: 70% MeOH-30% NaH 2 P0 4 , 0.01M) log P = 1.9506 + 2.1840 l o g k  1  n = 7, r = 0.9958, s = 0.069, F = 478 (Mobile phase: 50% CH3CN - 50% NaH 2 P0 A , 0.01M) 129  Table 12 HPLC method f o r determining the l i p o p h i l i c i t i e s of the a c i d i c3 compounds using 70% MeOH: 30% 0.01M NaH 2 P0 4 as mobile phase  Compounds  log k'  lo  P  S HPLC  lo  8  pb  o/w  1.  Isobutyric acid  3.00  -0.5931  1.10  2.  Butyric acid  2.94  -0.6380  1.02  3.  Trimethylacetic acid  3.47  -0.3450  1.54  4.  Valeric acid  3.37  -0.3872  1.46  1.51  5.  2-Ethylbutyric a c i d  3.91  -0.1965  1.80  1.68  6.  2,2-Dimethylbutyric acid Hexanoic acid  4.12  -0.1403  1.90  4.18  -0.1255  1.93  5.49  0.1130  2.35  3-Ethylpentanoic acid  4.93  0.02644  2.19  10. Cyclohexylacetic acid  5.35  0.09289  2.31  11. 1-Methylcyclohexane-lcarboxylic a c i d 12. Valproic acid (VPA)  5.65  0.1348  2.38  6.53  0.2386  2.57  13. 4-Keto VPA  3.19  -0.4753  1.31  14. 2-Ene VPA  5.89  0.1658  2.44  15. 2,3'-Diene VPA  5.21  0.07185  2.27  16. 2-Ethylhexanoic acid  6.58  0.2438  2.58  17. Octanoic acid  7.66  0.3434  2.75  13.70  0.6751  3.34  19. 5-Isoamyltetrazole  3.17  -0.4863  1.29  20. 5-Heptyltetrazole  4.39  -0.07737  2.01  21. 5-Cyclohexylmethyltetrazole 22. N,N-Diethylsuccinamic acid 23. N,N-Dibutylsuccinamic acid  3.54  -0.3177  1.59  7.  8. Heptanoic acid 9.  18. 2-Butylhexanoic acid  a  t R (min)  5.20  0.07031  2.27  5.31  0.08699  2.30  0.98  1.93  2.75  2.64 3.20  Flow rate i s 1.0 mL/min, t Q (Me0H) i s 2.39 min.  'Used i n l i n e a r c o r r e l a t i o n of l o g P Q / W v e r s u s l o g k'. V a l u e s taken from P.E. Keane e t a l . (13).  130  • Table 13 HPLC method f o r determining the l i p o p h i l i c i t i e s of the a c i d i c3 compounds using 50% CH3CN: 50% 0.01M NaH 2 P0 A as mobile phase  Compounds  log k'  lo  P  § HPLC  !°8  pb  o/w  1.  Isobutyric a c i d  2.72  -0.4437  1.07  2.  Butyric a c i d  2.72  -0.4437  1.07  3.  Trimethylacetic a c i d  3.24  -0.2076  1.52  4.  Valeric acid  3.17  -0.2328  1.47  1.51  5.  2-Ethylbutyric acid  3.65  -0.0835  1.76  1.68  6.  2,2-Dimethylbutyric acid  3.82  -0.04096  1.84  7. Hexanoic a c i d  3.96  -0.00877  1.90  8. Heptanoic acid  5.17  0.2000  2.30  3-Ethylpentanoic acid  4.61  0.1156  2.14  10. Cyclohexylacetic acid  4.97  0.1717  2.25  11. 1-Methylcyclohexane-lcarboxylic acid  5.24  0.2095  2.32  12. Valproic acid (VPA)  6.32  0.3345  2.56  13. 4-Keto VPA  3.03  -0.2882  1.37  14. 2-Ene VPA  6.08  0.3096  2.51  15. 2,3'-Diene VPA  4.99  0.1746  2.25  16. 2-Ethylhexanoic acid  6.34  0.3365  2.56  17. Octanoic a c i d  7.31  0.4241  2.72  13.51  0.7600  3.37  19. 5-Isoamyltetrazole  3.00  -0.2967  1.35  20. 5-Heptyltetrazole  4.09  21. 5-Cyclohexylmethyltetrazole  3.52  -0.1192  1.69  22. N,N-Diethylsuccinamic acid  4.92  0.1651  2.23  23. N,N-Dibutylsuccinamic acid  5.35  0.2240  2.35  9.  18. 2-Butylhexanoic acid  a  t R (min)  0.01912  0.98  1.93  2.75  2.64 3.20  1.95  Flow rate i s 1.0 mL/min, t Q (Me0H) i s 2.0 min.  Taken from P.E. Keane e t a l . (13). Values used i n l i n e a r c o r r e l a t i o n o f l o g P Q / W v e r s u s l o g k'. 131  6  4  §  10  12  14  16~  Time(min) Superposed HPLC chromatograms of a c i d i c compounds. Column; h y p e r s i l reverse phase column (20cm x 4.6mm). 0. 01M  NaH 2 P0 4 (pH3.5). 2.  Flow r a t e , 1.Oml/min.  1.  Butyric acid  4.  4-Keto VPA  7.  5-Cyclohexylmethyltetrazole  9.  2,2-Dimethylbutyric a c i d  12.  3-Ethylpentanoic a c i d  5.  14. 2.3-Diene VPA  22. Octanoic a c i d  Isobutyric  Valeric acid  acid 6.  3.  20.  5-Isoamyltetrazole  Trimethylacetic  8. 2 - E t h y l b u t y r i c 10. Hexanoic a c i d  acid acid  11.  5-Heptyltetrazole  13. N.N-Diethylsuccinamic a c i d 16.  IB. 1-Methylcyclohexane c a r b o x y l i c Valproic a d d 23.  Methanol:30%  S,solvent;  15. N.N-Dlbutylsuccinamic a c i d  17. Heptanoic a c i d 19. 2-Ene VPA  Mobile phase; 70%  ODS  (VPA)  21.  2-Butylhexanoic a c i d  132  Cyclohexylacetic acid  2-Ethylhexanoic a c i d  acid  5.  Comparison of l i p o p h i l i c i t y from RP-HPLC and other methods Retention of the t e s t compounds on the RP-HPLC column appeared to  be based on the hydrocarbon  s t r u c t u r e , the presence of polar  f u n c t i o n a l groups, the type of a l k y l s u b s t i t u t i o n , the number of double bonds, the presence of a r i n g structure and molecular s i z e (Tables 12, 13). values determined  As shown i n Tables 12 and 13, the HPLC l o g P from  both mobile phases are i n good agreement  with an average difference of ± 0.06 l o g u n i t s . The shake-flask method was used to determine the octanol-water p a r t i t i o n c o e f f i c i e n t of four compounds of diverse structures i n order to v e r i f y the accuracy of the HPLC l o g P v a l u e s . Tables 1417  show the c a l i b r a t i o n  using HPLC a n a l y s i s .  data f o r the four compounds  determined  High c o r r e l a t i o n c o e f f i c i e n t s were obtained  for the c a l i b r a t i o n curves. The l o g P values of the four compounds determined log  by the shake-flask method are shown i n Table 18.  The  P values are f o r the unionized form of the a c i d i c compounds  since 0.01N HC1 was used as the aqueous phase. the l o g P values was high with average  The p r e c i s i o n of  standard deviations  less  than 0.05 l o g u n i t s , except f o r N,N-dibutylsuccinamic a c i d with a deviation of 0.08 l o g u n i t s .  Probable sources of error include low  s e n s i t i v i t y f o r detection of these compounds by HPLC and propensity of  N,N-dibutylsuccinamic a c i d to be unstable i n an a c i d i c aqueous  phase.  In a d d i t i o n , d i s s o l u t i o n of t e t r a z o l e s i n the 0.01N HC1  phase was d i f f i c u l t and had to be effected using small volumes of methanol.  133  Table 14 C a l i b r a t i o n curve data of t r i m e t h y l a c e t i c a c i d i n 0.1N HC1  Concentration mg/mL  Mean Peak Height  0.05  193  0.1  392  Linear Regression 3 Parameters  a  0.2  767  a  0.4  1465  r  0.6  2210  r  1  0  1  = 22.81 = 3639 = 0.9999  2  = 0.9998  i s t h e 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 , a-- i s t h e s l o 0p e and a ° i s the i n t e r c e p t . E q u a t i o n f o r t h e l i n e i s y = a*x + a where y i s the peak height and x i s concentration of compound.  134  Table 15 C a l i b r a t i o n curve data of N,N-Dibutylsuccinamic acid i n 0.1N HC1  Concentration mg/mL  Mean Peak Height  Linear Regression Parameters  0.1  128  a ° = 3.14  0.2  260  a  0.4  530  r = 0.99994  0.6  787  r  1  2  3  = 1321  = 0.99988  r i s the c o e f f i c i e n t of determination, a i s the slope and a i s the i n t e r c e p t . Equation f o r the l i n e i s y = a x + a where y i s the peak height and x i s concentration of compound.  135  Table 16 C a l i b r a t i o n curve data of 5-Isoamyltetrazole i n 0.1N HC1  Concentration mg/mL  Mean Peak Height  Linear Regression 3 Parameters  0.01  1099  a ° = 235.9  0.02  2153  a  0.05  4889  r  0.1  9496  r  1  = 92742 = 0.9999  2  =  0.9998  r2 i s t h e 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 , a l i s the s l o p e and a ° i s the i n t e r c e p t . E q u a t i o n f o r the l i n e i s y = a^x + a ° where y i s the peak height and x i s concentration of compound.  136  Table 17 C a l i b r a t i o n curve data of 5-cyclohexylmethyltetrazole i n 0.1N HC1  Concentration mg/mL  Mean Peak Height  Linear Regression 3 Parameters  0.01  455  a ° = -72.67  0.02  974  a  0.05  2581  r  0.1  5200  r  1  = 52782 = 0.99999  2  = 0.99998  r 2 i s t h e 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 , a l i s the s l o p e and a ° i s the i n t e r c e p t . E q u a t i o n f o r t h e l i n e i s y = a^x + a ° where y i s the peak height and x i s concentration of compound.  137  Table 18 Octanol-water p a r t i t i o n c o e f f i c i e n t s of selected a c i d i c compounds determined by the shake-flask procedure  Compound  log  b  (+SD )  PQ/W  1.54  +  0.01  2.27  +  0.08  3. 5-Isoamyltetrazole  1.38  +  0.03  4. 5-Cyclohexylmethyltetrazole  1.61  +  0.04  1. Trimethylacetic a c i d 2. N,N-Dibutylsuccinamic  acid  a  Shake-flask method used with 1-octanol as organic phase and HC1 as aqueous phase.  k  Standard d e v i a t i o n i n l o g u n i t s , from four separate measurements.  138  0.01N  experimental  Table 19 3 Hansch-ir - v a l u e s used i n c a l c u l a t i n g l o g P Q / W  A l i p h a t i c Group  a  1.  CH3  0.5  2.  CH2  0.5  3.  -OH  -1.16  4.  -0-  -0.98  5.  C00H  -0.65  6.  C=0  -1.21  7.  C0NH2  -1.71  8.  NH2  -1.19  9.  -CON-  -2.27  10. N  -1.32  11. Tetrazole  -1.04  12. NMe2  -0.32  13. Double bond  -0.30 (ATT)  14. Chain branch ( s i n g l e )  -0.20 (ATT)  15. Branching i n r i n g closure  -0.09 (Air)  16. Ring closure (per bond)  -0.09 (ATT)  17. Intramolecular H-bonding  -0.65 (ATT)  Taken from A. Leo et a l . (133).  \ ( t e t r a z o l e ) obtained from C. Hansch and A. Leo (152).  139  b  Table 20 a  Rekker's fragmental values ( f ) used i n c a l c u l a t i n g l o g P Q / W  Fragment  a  f(aliphatic)  1. CH 3  0.702  2. CH 2  0.527  3. CH  0.236  4. C  0.14  5. H  0.175  6. CH 2 = CH  0.93  7. COOH  -1.003  8. C = 0  -1.69  9. NH2  -1.38  10. NH  -1.864  11. -N-  -2.133  12. CON  -2.894  13. C0NH2  -1.99  14. Tetrazole  -2.93  15. NH ( h e t e r o c y c l i c )  -0.70  16. N ( h e t e r o c y c l i c )  -1.06  17. C ( h e t e r o c y c l i c )  0.157  18. H ( h e t e r o c y c l i c )  0.199  19. CH ( h e t e r o c y c l i c )  0.35  20. Single conjugated pattern  0.314 ( A f )  21. Proximity e f f e c t f o r 1 C separation  0.80  22. Proximity e f f e c t of electronegative group f o r 2C separation 23. -N = C-NH  0.46 -0.79  24. -N = N  -2.14  Taken from R. F. Rekker (153).  140  Table 21 L i p o p h i l i c i t i e s (log P Q / W ) of the a c i d i c compounds obtained by d i f f e r e n t methods lo  Compounds  3  HPLC (MeOH)  b 0  ^  b  HPLC Hansch (CH3CN)  0  Rekker  d  ShakeFlask  1. Isobutyric a c i d  1.10  1.07  0.65  0.64  2. B u t y r i c acid  1.02  1.07  0.85  0.76  0.98  1.54  1.52  0.95  1.24  1.54  1.46  1.47  1.35  1.28  1.51  acid  1.80  1.76  1.65  1.69  1.68  2,2-Dimethylbutyric acid 7. Hexanoic acid  1.90  1.84  1.45  1.45  1.93  1.90  1.85  1.81  8. Heptanoic acid  2.35  2.30  2.35  2.33  9.  3-Ethylpentanoic acid 10. Cyclohexylacetic acid 11. 1-Methylcyclohexane1-carboxylic acid 12. Valproic acid (VPA)  2.19  2.14  2.15  2.22  2.31  2.25  2.22  2.39  2.38  2.32  2.13  2.47  2.57  2.56  2.65  2.75  13. 4-keto VPA  1.31  1.37  1.14  0.99  14. 2-Ene VPA  2.44  2.51  2.35  2.56  15. 2,3-Diene VPA  2.27  2.25  2.05  2.26  16. 2-Ethylhexanoic acid 17. Octanoic acid  2.58  2.56  2.65  2.75  2.75  2.73  2.85  2.86  18. 2-Butylhexanoic acid  3.34  3.37  3.65  3.80  3.20  19. 5-Isoamyltetrazole  1.29  1.35  1.26  -1.03  1.38  20. 5-Heptyltetrazole  2.01  1.95  2.46  0.83  21. 5-Cyclohexylmethyltetrazole 22. N,N-Diethylsuccinamic acid 23. N,N-Dibutylsuccinamic acid  1.56  1.69  1.85  0.50  2.27  2.23  0.08  0.08  2.30  2.35  2.08  2.18  3.  Trimethylacetic  4.  Valeric acid  5.  2-Ethylbutyric  acid  6.  3  P  8 o/w  determined using 70% MeOH: 30% 0.01M NaHoPO, determined using 50%CHoCH: 50% 0.01M NaH 2 P0 4 calculated using Hansch ir-approach c a l c u l a t e d using Rekker's fragment constant experimentally determined by P. E. Keane et a l . (13) octanol-water p a r t i t i o n c o e f f i c i e n t determined i n t h i s study 141  e f  e  1.93  2.75  e  e  e  2.64  1.61  e  e f  f  2.27  f  Hansch-rr a nd Rekker-f values (Tables 19, 20) have been used to predict l o g P values of the compounds studied (Table 21).  Com-  parison of the HPLC log P values with Hansch and Rekker values i n Table  21 shows that there i s good agreement f o r the homologous  straight-chain CyH-j^COOH.  and alpha-branched a l i p h a t i c  a c i d s , C^HgCOOH-  The discrepancies i n l o g P values f o r the HPLC and  c a l c u l a t e d methods a r e , however, f o r highly s u b s t i t u t e d compounds (e.g. t r i m e t h y l a c e t i c a c i d ) , s u b s t i t u t e d a l i c y c l i c compounds (e.g. 1-methylcyclohexanecarboxylic  acid),  tetrazoles,  bonded compounds e.g. N,N-diethylsuccinamic VPA  intramolecular  a c i d (165) and 4-keto  (162). The  shake-flask l o g P values of the h i g h l y - s u b s t i t u t e d t r i -  methylacetic  acid,  highly  lipophilic  2-butylhexanoic  acid,  t e t r a z o l e s and l e s s l i p o p h i l i c b u t y r i c a c i d agree better with HPLC log P values than the c a l c u l a t e d log P values (Table 21).  This  i n d i c a t e s that the Hansch and Rekker l o g P values may lead to l e s s accurate  values  hydrophobicity. not  hold  f o r compounds  showing  constitutive  e f f e c t s on  Obviously, a d d i t i v i t y of group c o n t r i b u t i o n does  f o r the t e t r a z o l e s  and t h e l o g P v a l u e of t h e un-  substituted t e t r a z o l e would have to be used, as noted i n Table 19. There i s close agreement among the various methods with the shakeflask  value  f o r N,N-dibutylsuccinamic  acid  (XVI).  Lower  values  from  additivity  methods were obtained  f o r N,N-diethylsuccinamic  acid  (XV) which may show s i g n i f i c a n t  intramolecular e f f e c t s to  increase the log P value.  142  6.  Intramolecular bonding e f f e c t s of amic acids The HPLC log P value f o r N,N-diethylsuccinamic a c i d (XV) was considerably explained  higher  by  a  than  predicted  (Table 21).  greater intramolecular  bonding  This could  f o r N,N-diethyl-  succinamic a c i d than f o r N,N-dibutylsuccinamic a c i d . the  NMR  be  In a d d i t i o n ,  spectra of N,N-diethylsuccinamic appear to i n d i c a t e  an  i n t e r a c t i o n between the c a r b o x y l i c group and the amido group compared  to t h a t of N , N - d i b u t y l s u c c i n a m i c  study (165)  of  NMR  acid  (Figure  6).  spectra of N,N-dialkylsuccinamic acids  A also  showed evidence of an i n t e r a c t i o n between the amido and carboxyl f u n c t i o n a l groups i n N,N-diethylsuccinamic acid  through  hydrogen  bonding i n d i l u t e s o l u t i o n s . Another a s p e c t of amic a c i d s i n v o l v e s t h e i r s t a b i l i t y i n a c i d i c media.  Both N,N-dibutylsuccinamic and N,N-diethylsuccinamic  are soluble i n water. stable  i n 0.1N  HC1  N,N-dibutylsuccinaraic acid was and  0.01M  NaH 2 P0 4  as  shown by  relatively the  high  c o r r e l a t i o n c o e f f i c i e n t of i t s l i n e a r c a l i b r a t i o n curves used to determine  the concentration i n the aqueous phase of the octanol-  0.1N HC1 p a r t i t i o n system. succinaraic acid was diethylsuccinamic  was  The long-term s t a b i l i t y of N,N-dibutyl-  not i n v e s t i g a t e d . not  stable  On  i n 0.1N  the other hand, HC1  and  N,N-  i t s partition  c o e f f i c i e n t i n the octanol/O.lN HC1 system could not be determined. In  order  to observe  chromatographic  peaks,  amounts of 20ug or  greater of N,N-diethylsuccinamic acid i n methanol or a c e t o n i t r i l e had to be i n j e c t e d . There i s l i t e r a t u r e precedence (183,184) f o r the of  some amic acids i n a c i d i c media. 143  instability  I t has been documented that  the presence of a carboxylic group adjacent to an amide w i t h i n the molecular structure usually leads to h y d r o l y s i s of the amic a c i d . The degree of h y d r o l y s i s appears to depend on the type of amic a c i d and  N-alkyl  reported  to  where the  substituents be  even  (183,184).  greater  Thus the  hydrolysis  i n N-alkylmaleamic  acids (182,183)  c i s - c o n f i g u r a t i o n favours intramolecular  In a k i n e t i c study of N-alkylmalearaic  is  interactions.  a c i d h y d r o l y s i s , Kluger  and  Chin (184) proposed a mechanism where f o r compounds with more basic leaving groups, the amine group e l i m i n a t i o n XXXIV-XXXV i s the r a t e determining  step  (Scheme 10).  in  formation  of  the  Aldersley et a l . (183)  internal  anhydride, XXXV  proposed a s i m i l a r mechanism  to account f o r the rapid h y d r o l y s i s of the N-alkylmaleamic a c i d s . However, the rate-determining step was the C-N  bond, XXXIII-XXXV.  jection  of  The  HPLC chromatograms, f o l l o w i n g i n -  N,N-diethylsuccinamic  expected solvent peak.  suggested to be cleavage of  This was  acid,  indicated  a  bigger  probably due to c o - e l u t i o n of the  degraded products, s u c c i n i c a c i d , diethylamine and methanol. amide group has  than  The  also been reported to give underestimated log P  values, c a l c u l a t e d by the Hansch method, i n compounds with m u l t i p l e f u n c t i o n a l groups (182). In comparative s t u d i e s , both HPLC and shake-flask methods have advantages and  limitations.  The  shake-flask  method i s l a b o r i o u s ,  time-consuming, subject to e r r o r s from i n s o l u b i l i t y and of compounds i n the octanol-O.lN HC1  instability  p a r t i t i o n system and  requires  s e n s i t i v e a n a l y t i c a l techniques to determine the concentration solute i n the aqueous phase. flask  procedure include  of  Advantages associated with the shake-  high  144  sensitivity  to hydrophobic e f f e c t s  Scheme 10.  Kinetic model proposed by some investigators the hydrolysis of maleamic acids.  145  (182, 183) f o r  since aqueous s o l u t i o n s were used instead of mixed solvents.  The  shake-flask method can a l s o be used f o r compounds of widely d i f f e r ent chemical s t r u c t u r e s . rapid technique.  The HPLC method, on the other hand, i s a  I t o f f e r s h i g h r e p r o d u c i b i l i t y of l o g P v a l u e s  s i n c e the o n l y parameter, r e t e n t i o n t i m e , can be accurately. pounds.  determined  I t does not r e q u i r e q u a n t i t a t i v e a n a l y s i s of com-  I t may  be appropriate f o r compounds prone to degradation  i n octanol-water systems or f o r compounds i n l e s s pure samples. The RP-HPLC method, however, requires reference compounds w i t h l o g P v a l u e s determined by the s t a n d a r d s h a k e - f l a s k method.  The  range of l i p o p h i l i c i t y i s such that d i f f e r e n t chromatographic  con-  d i t i o n s have t o be e m p l o y e d f o r compounds o f h i g h or  low  lipophilicity. In t h i s study the HPLC method under i s o c r a t i c conditions was l i m i t e d to compounds w i t h l o g P of about 0.8-3.2. shortcoming  One  notable  of the HPLC method observed i n t h i s study i s that the  order of r e t e n t i o n t i m e between s t r u c t u r a l i s o m e r s , f o r example b u t y r i c a c i d and i s o b u t y r i c a c i d or v a l p r o i c a c i d and  2-ethy-  hexanoic a c i d , can change depending on the composition and type of mobile phase.  Some workers (141,147) have tackled t h i s problem by  e x t r a p o l a t i o n of the plot of log k' versus mobile phase composition to o b t a i n l o g k' f o r water.  A p p a r e n t l y t h i s was not p o s s i b l e f o r  the compounds studied since the mobile phase composition needed to elute a l l the t e s t compounds while maintaining dynamic e q u i l i b r i u m conditions was l i m i t e d to a range of 40-60% a c e t o n i t r i l e - b u f f e r and 60-70% methanol-buffer.  Nevertheless, the accuracy of HPLC l o g P  v a l u e s was h i g h , and v a l u e s were comparable t o s h a k e - f l a s k l o g P values.  The HPLC method c o u l d a l s o be a p p l i e d t o a v a r i e t y of  146  chemical structures unlike  the a d d i t i v i t y  methods of Hansch and  Rekker. C.  E l e c t r o n i c S t r u c t u r a l E f f e c t s - Determination of Apparent I o n i z a t i o n Constants  1.  A n a l y t i c a l method The apparent i o n i z a t i o n constants (pKa) of t e s t compounds were determined  by potentiometric t i t r a t i o n  due to the l i m i t e d s o l u b i l i t y Potentiometric  and  UV  determine  of most of the compounds i n water.  spectrophotometric  frequently used to determine compounds (185,187).  i n aqueous-methanol media  the i o n i z a t i o n  The conductivity  the i o n i z a t i o n  methods  have been  constants of a c i d i c  method has been used to  c o n s t a n t s of a l i p h a t i c a c i d s ( 1 8 8 ) .  Spectrophotometric methods were not appropriate f o r the compounds studied since there i s no change i n the UV spectra upon i o n i z a t i o n . In  the potentiometric procedure, s o l u t i o n s of the compounds  were progressively n e u t r a l i z e d with q u a n t i t i e s of standard KOH and the pH recorded.  Tables 22-28 show t y p i c a l r e s u l t s obtained i n the  potentiometric t i t r a t i o n Henderson-Hasselbalch  procedure.  The c o r r e c t i o n f a c t o r i n the  equation, which i s reported (185) to be more  s i g n i f i c a n t at pH values lower than 5 and higher than 9, was u n i formly applied at a l l pH v a l u e s .  As shown i n Tables 23, 26, 28,  v a l u e s of pKa a t the b e g i n n i n g of the t i t r a t i o n  occasionally  deviated from expected values. Table 29 shows the pKa values of 23 compounds determined water.  i n e i t h e r 10% methanol-water or 50% methanol-  The i o n i z a t i o n constants were determined at 23°C + 1.0°C.  Values of pKa were determined  to w i t h i n a p r e c i s i o n of _+ 0.05 pKa  147  Table 22 Determination of the i o n i z a t i o n constant of a monobasic a c i d , v a l p r o i c acid i n 10% MeOH Temperature - 24°C Titrant 0.0105M KOH (mL)  [HA] x 10 pH  3  [A~] x 10  1  (mol. L  (mol. L" )  -1  3  )  THAI - fH+] [A"] + {H-M  pKa  0.5  4.24  0.9218  0.105  5.325  4.97  1.0  4.47  0.8178  0.210  2.930  4.94  1.5  A.6A  0.7128  0.315  2.042  4.95  2.0  4.82  0.6078  0.420  1.362  4.95  2.5  4.99  0.5028  0.525  0.9204  4.95  3.0  5.18  0.3978  0.630  0.6147  4.97  148  Table 23 Determination of the i o n i z a t i o n constant of 5-Isoamyltetrazole i n 10% MeOH Temperature - 23°C  Titrant 0.0105M KOH (mL)  [HA] x 10 pH (mol. L  -1  3  -  [A ] x 10  )  (mol. L  -1  3  )  THA] - {H+} [A"] + {IF}  pKa  0.5  4.36  0.888  0.105  5.7043  5.12  1.0  4.69  0.783  0.210  3.3099  5.21  1.5  4.91  0.678  0.315  2.0339  5.22  2.0  5.12  0.543  0.420  1.3223  5.24  2.5  5.28  0.468  0.525  0.8729  5.22  3.0  5.46  0.363  0.630  0.5672  5.21  3.5  5.65  0.258  0.735  0.3470  5.19  149  Table 24 Determination of the i o n i z a t i o n constant of t r i m e t h y l a c e t i c acid i n 10% MeOH Temperature - 23°C  Titrant 0.01997M KOH (mL)  [HA] x 10 pH  (mol. L  -1  3  [A~] x 10 (raol. L  )  _1  3  )  [HA] - {H+} 1 [A"] + {H""}  pKa  0.5  4.27  1.8983  0.1997  7.2794  5.10  1.0  4.52  1.6986  0.3994  3.8836  5.10  1.5  4.77  1.4989  0.5991  2.4053  5.15  2.0  4.91  1.2992  0.7988  1.5866  5.11  2.5  5.08  1.0995  0.9985  1.0838  5.11  3.0  5.22  0.8998  1.1982  0.7422  5.09  3.5  5.38  0.7001  1.3979  0.4963  5.08  150  Table 25 Determination of the i o n i z a t i o n constant of d i b u t y l a c e t i c a c i d i n 50% MeOH Temperature - 23°C  Titrant 0.0105M KOH (mL)  [HA] x 10 pH  (mol. L  -1  3  [A~] x 10  )  (mol. L  -1  3  )  [HA] - {H+} + [A"] + {H }  pKa  0.5  5.11  0.8776  0.105  7.7110  6.00  1.0  5.38  0.7726  0.210  3.5873  5.93  1.5  5.58  0.6676  0.315  2.0938  5.90  2.0  5.74  0.5626  0.420  1.3295  5.86  2.5  5.98  0.4576  0.525  0.8681  5.92  3.0  6.29  0.3526  0.630  0.5584  6.04  3.5  6.53  0.2476  0.735  0.3363  6.06  151  Table 26 Determination of the i o n i z a t i o n constant of N,N-diethylsuccinamic a c i d i n 50% MeOH Temperature - 2A°C  Titrant 0.0098M KOH (mL)  [HA] x 10 pH (mol. L  _1  -  3  [A ] x 10 (mol. L  )  -1  3  )  [HA] - {H+} + [A"] + {H }  pKa  0.5  6.29  0.896  0.098  9.030A  7.2A  1.0  6.39  0.792  0.196  A.0305  7.00  1.5  6.50  0.69A  0.29A  2.3571  6.87  2.0  6.61  0.596  0.392  1.5191  6.79  2.5  6.76  0.A98  0.A90  0.0155  6.77  3.0  6.91  0.A00  0.588  0.6800  6.7A  3.5  7.12  0.302  0.686  O.AAOA*  6.76  4.0  7.A3  0.20A  0.78A  0.2607*  6.85  • c a l c u l a t e d using the c o r r e c t i o n f a c t o r  152  [HA] + {0H~} [A~] - {OH }  Table 27 Determination of the i o n i z a t i o n constant of 5-cyclohexylmethyltetrazole i n 50% MeOH Temperature - 23°C  Titrant 0.0105M KOH (mL)  [HA] x 10 pH  3  [A~] x 10  1  1  (mol. L" )  3  (mol. L" )  THAI - {H+} [A~] + {H"n  pKa  0.5  4.75  0.9552  0.105  7.658  5.63  1.0  5.05  0.8502  0.210  3.843  5.63  1.5  5.27  0.7452  0.315  2.309  5.63  2.0  5.45  0.6402  0.420  1.503  5.62  2.5  5.62  0.5352  0.525  1.010  5.62  3.0  5.79  0.4302  0.630  0.6786  5.61  3.5  5.97  0.3252  0.735  0.4403  5.57  153  Table 28 Determination of the i o n i z a t i o n constant of N,N-dibutylsuccinamic a c i d i n 50% MeOH Temperature - 24°C  Titrant 0.0098M KOH (mL)  [HA] x 10 pH (mol. L  -1  3  [A~] x 10 (mol. L  )  _1  3  )  [HA] - {H+} [A"] + {H+}  pKa  0.5  6.21  0.9060  0.0980  9.1825  7.17  1.0  6.33  0.8080  0.1960  4.1094  6.94  1.5  6.42  0.7100  0.2940  2.4103  6.80  2.0  6.56  0.6120  0.3920  1.5593  6.75  2.5  6.69  0.5140  0.4900  1.0481  6.71  3.0  6.82  0.4160  0.5880  0.7069  6.67  3.5  7.01  0.3180  0.6860  0.4635  6.68  4.0  7.23  0.2200  0.7840  0.2809*  6.68  4.5  7.58  0.1220  0.8820  0.1388*  6.72  5.0  8.29  0.0240  0.9800  0.0265*  6.71  -  * c a l c u l a t e d using the c o r r e c t i o n f a c t o r  154  [HA]- + {OH-} [ A ] - {OH }  Table 29 pKa Values of v a l p r o i c a i d and analogues pK;3t +_ s.d.(n=6) Compound ( i n 10% MeOH)  ( i n 50% MeOH)  1. Isobutyric acid  4.96  0.02  5.85 + 0.06  2.  Trimethylacetic acid  5.11 + 0.02  5.94 + 0.06  3.  3-Methylpentanoic acid  4.85 + 0.03  5.76 + 0.05  4.  D i e t h y l a c e t i c acid  4.83 + 0.03  6.02  5.  3-Methylhexanoic acid  5.02 + 0.01  5.72 + 0.05  6.  2,2-Dimethylbutyric  5.26 + 0.03  6.52 + 0.02  7.  2,2-Dimethylpentanoic acid  5.26 + 0.05  6.36  8.  Valproic a c i d (VPA)  4.95 + 0.01  6.03 + 0.01  9.  3-OH VPA  4.39 + 0.02  5.56 + 0.04  10. 4-keto VPA  4.58 + 0.01  5.28 + 0.06  11. 4-Ene VPA  4.63 + 0.01  5.51 + 0.06  5.05 + 0.03  6.14 + 0.13  13. D i b u t y l a c e t i c a c i d  5.05 + 0.06  5.95 + 0.08  14. 1-Methylcyclohexane-l-carboxylic acid  5.17 + 0.05  6.58 + 0.01  15. 3-Ethylpentanoic  acid  4.78 + 0.08  5.99 + 0.02  16. Cyclohexylacetic acid  4.78 + 0.02  5.77 + 0.04  12. 2-Ethylhexanoic  acid  acid  ±  ±  0.02  0.01  17. N,N-Diethylsuccinamic  acid  5.58 + 0.05  6.80 + 0.05  18. N,N-Dibutylsuccinamic  acid  5.58 + 0.05  6.72 + 0.03  19. 5-Isoamyltetrazole  5.22 + 0.02  5.60 + 0.01  20. 5-Cyclohexylmethyltetrazole  5.37 + 0.03  5.62 + 0.01  21. 5-Heptyltetrazole  5.31 + 0.05  5.60 + 0.01  22. 2,3'-Diene VPA  4.02 + 0.02  5.47 + 0.06  23. 2-Ene VPA  4.36 + 0.05  5.51 + 0.06  155  units.  Standard  deviations of the pKa values were i n a few cases  higher than +0.06 pKa. The  pKa  of v a l p r o i c a c i d , 4.95,  determined i n 10%-methanol-  water i s higher than the reported values of 4.82  i n 5% methanol-  water (189) and 4.6 as the aqueous value from extrapolated acetonewater mixtures (67). methanol-water  than  The pKa values i n Table 29 are higher i n 50% i n 10%  methanol-water  d i e l e c t r i c constant of 50% methanol-water.  due  to  the  lower  However, the order of  i o n i z a t i o n strength i n 50% methanol-water d i d not r e f l e c t the same order i n 10% methanol-water. between pKa reported  and  S i m i l a r f i n d i n g s on the n o n - l i n e a r i t y  the c o m p o s i t i o n  of o r g a n i c s o l v e n t have been  f o r some compounds ( 1 8 5 - 1 8 7 ) .  There have a l s o been  reports that the conventional approach of e x t r a p o l a t i n g values of pKa to aqueous values may be u s e f u l when lower solvent compositions are  used (185,186).  The  pKa  values i n 10% methanol-water were  considered to be more r e l i a b l e than values i n 50% methanol-water. Compared to pKa values of a l i p h a t i c acids reported by Dippy (188) i n c o n d u c t i v i t y methods (pKa of t r i m e t h y l a c e t i c a c i d = 5.05, pKa of d i e t h y l a c e t i c acid = 4.75, pKa of i s o b u t y r i c acid = 4.86), the pKa values  i n 10% methanol-water are 0.1-0.2 pKa  u n i t s above the  aqueous values. 2.  E f f e c t s of s t r u c t u r a l c o n s t i t u t i o n on i o n i z a t i o n constants To the extent that the pKa likely  values i n 10% methanol are most  to be d i f f e r e n t from the aqueous v a l u e s by a  constant  amount, the e l e c t r o n i c e f f e c t s can be determined by the pKa v a l u e s . This i s supported  by the observation that the pKa values i n Table  29 are consistent with the i n d u c t i v e e f f e c t s of substituent groups 156  expressed  i n Table  Electron-withdrawing  30  by  the  polar  substituent  substituents have a p o s i t i v e polar substituent  constant while e l e c t r o n - r e p e l l i n g groups have negative The  constants.  values.  i o n i z a t i o n constants of the t e t r a z o l e s (Table 29) conform  to the g e n e r a l i z a t i o n (21) that they are about a tenth to one-half as large as t h e i r corresponding  c a r b o x y l i c a c i d values.  The  pKa  values of the t e t r a z o l e s are s i m i l a r to r e s u l t s obtained by Mihina and Herbst (161) i n 25% by weight methanol-water. The influence of s u b s t i t u t i o n p a t t e r n , polar groups, a l i c y c l i c groups, unsaturation the pKa values. by  and  intramolecular bonding are r e f l e c t e d i n  B e t a - s u b s t i t u t i o n i n the a l k y l c h a i n , exemplified  3-ethylpentanoic  a c i d and  c y c l o h e x y l a c e t i c a c i d , leads  to  r e l a t i v e l y low pKa values compared to alpha-substituted compounds. D i e t h y l a c e t i c and  v a l p r o i c a c i d , both alpha-branched a c i d s , have  comparable or s l i g h t l y higher i o n i z a t i o n strength than i s o b u t y r i c a c i d , i n agreement with r e s u l t s of Dippy (188). a,a-dimethylvaleric  Compounds such as  a c i d , t r i m e t h y l a c e t i c a c i d and  hexane c a r b o x y l i c a c i d  have r e l a t i v e l y  high  pKa  combined i n d u c t i v e e f f e c t s of the methyl groups. pKa  of the  unsaturated  1-methylcyclovalues The  compounds compared to t h e i r  analogues i s known to be due  due  to  decrease i n saturated  to p o l a r i z a t i o n of the double bond.  Values of the polar substituent constants i n d i c a t e that c y c l o h e x y l methyl and  cyclohexyl s u b s t i t u t i o n diminishes  the  ionization  strength of the unsubstituted c a r b o x y l i c acids and the t e t r a z o l e s (Table 30). The chain  presence of the keto group or hydroxy group i n the a l k y l  increases  the  ionization  157  strength  i n accordance with  the  Table 30 Polar e f f e c t of s u b s t i t u t i o n i n an a l i p h a t i c s e r i e s , R-Y where Y i s the f u n c t i o n a l group.  a * 3 (Polar Substituent Constant)  R C1 3 C  2.65  CH3CO  1.65  CH3C0CH2  0.60  H0CH2  0.55  H  0.49  CH3-CH=CH  0.36  CH3—CH=CH—CH2  0.13  CH 3  0  C2H5  -0.1  i - C4H9  -0.125  n - C4H9  -0.13  n - C3Hy  -0.115  i - C3H7  -0.19  Cyclo - C 6 H n C H 2  -0.06  Cyclo - C 6 H n  -0.15  s - C4H9  -0.21  ((^2^5) 2  -0.225  CH  -0.30  t - C4H9  a  t a k e n from R. W. Taft (134).  158  polar substituent constants.  The presence of an amido group close  to a carboxyl group u s u a l l y leads to a decrease i n pKa r e l a t i v e to an a l k y l substituent because of the i n d u c t i v e e f f e c t .  The opposite  results  acids.  are obtained  f o r the N,N-dialkylsuccinamic  The  r e l a t i v e l y high pKa values could be explained by hydrogen-bonding between the c a r b o x y l i c proton and the amido group.  Intramolecular  bonding i s even stronger i n N-propylmaleamide, pKa of 10.53 (184) where  the  c i s c o n f i g u r a t i o n favours such  an  interaction.  The pKa values f o r most of the compounds studied are a v a i l a b l e in  the l i t e r a t u r e  but  the values  are u s u a l l y determined under  d i f f e r e n t conditions and by d i f f e r e n t workers.  This consideration  prompted the determination of the pKa values of t e s t compounds by the potentiometric t i t r a t i o n method using the same solvent-mixture for a l l the compounds. Pharmacological  Studies  Evaluation of anticonvulsant a c t i v i t y The  anticonvulsant  activity  of each  test  compound  was  determined using the i n vivo s.c. PTZ seizure threshold t e s t .  The  t e s t appears to be s u i t a b l e f o r v a l p r o i c acid and analogues.  Val-  proic acid and a l k y l - s u b s t i t u t e d a n t i e p i l e p t i c drugs are known to be more e f f e c t i v e i n the s.c. PTZ threshold t e s t than i n maximal electroshock seizure t e s t s . appear  to e x e r t  s i t e (53,54,81).  In a d d i t i o n , v a l p r o i c a c i d and  opposing a c t i o n s a t the GABA Recent  PTZ  post-synaptic  studies (30,31,33,34,57) have suggested  that v a l p r o i c a c i d and PTZ may act at the p i c r o t o x i n s i t e of the  159  GABA-Benzodiazepine-receptor-chloride ionophore complex. The speci f i t y of t h e s.c. PTZ t e s t has been r e c e n t l y r e - i n v e s t i g a t e d and suggested  t o be r e l a t e d t o t h e dosage l e v e l of PTZ used (190).  C l o n i c t h r e s h o l d s e i z u r e s were found t o be induced r e p r o d u c i b l y w i t h 85 mg/kg s.c. PTZ (CDg7) i n mice (190). The c o n t r o l t e s t i n t h i s study showed t h a t 100% of t h e mice responded to the s.c. i n j e c t i o n of 85 mg/kg PTZ w i t h well-defined c l o n i c spasms. The time of onset of c l o n i c seizures was between 39 minutes which i s consistent w i t h previous studies on the onset of c l o n u s a t d i f f e r e n t PTZ doses (191).  PTZ was i n j e c t e d s.c. 15  minutes a f t e r i.p. i n j e c t i o n o f each t e s t compound. The s e l e c t e d time i n t e r v a l was based on l i t e r a t u r e reports where 2 min (14), 15 min (5,12,13) and 30 min (10,11,88) have been chosen as s u i t a b l e e l a p s e d t i m e s b e f o r e s.c. i n j e c t i o n of PTZ.  The i . p . dose of t e s t  compounds ranged from 0.2 mraol/kg to 2.0 mmol/kg. The observation t i m e of 30 min a f t e r s.c. PTZ i n j e c t i o n was chosen s i n c e c l o n i c seizures r a r e l y occurred a f t e r times longer than 20 min w i t h i.p. doses of compounds that prevent the i n i t i a l s e r i e s of c l o n i c a c t ivity. The dose-response Table 31.  data f o r t e s t compounds a r e presented i n  Nine compounds prevented the c l o n i c seizures induced by  the threshold dose of PTZ i n a dose-dependent fashion.  Isobutyric  a c i d , 4-keto VPA, c y c l o h e x y l a c e t i c a c i d , 5 - c y c l o h e x y l m e t h y l t e t r a z o l e and 5 - i s o a m y l t e t r a z o l e were i n a c t i v e i n the dose range studied.  N,N-Diethylsuccinamic a c i d was i n a c t i v e a t doses between  0.2-2.0 mmol/kg but induced h y p e r a c t i v i t y ( r a p i d - c i r c l i n g a c t i v i t y ) when i n j e c t e d alone i n mice at doses of 3.0 mmol/kg.  On the other  hand, N , N - d i b u t y l s u c c i n a m i c acid exhibited convulsant a c t i v i t y a t  160  Table 31 3  Protection against PTZ-induced s e i z u r e s i n mice by v a l p r o i c a c i d and i t s analogues  % Protected against c l o n i c Dose, mmol/kg  seizures  Compounds  1.  Valproic a c i d (VPA)  2.  2-Butylhexanoic acid  3.  1-Methylcyclohexane-lcarboxylic a c i d  4.  2,2-Dimethylbutyric acid  5.  3-Ethylpentanoic acid  6.  Trimethylacetic  7.  5-Cyclohexylmethyltetrazole  8.  Cyclohexylacetic  9.  Isobutyric a c i d  0.3  0.5  1.0  -  -  25  87.5  12.5  37.5  87.5  100  -  12.5  37.5  75  87.5  0  50  37.5  62.5  -  12.5  25  62.5  0  25  50  37.5  12.5  12.5  0  12.5  12.5  0  -  12.5  50  37.5  62.5  37.5  50  62.5  -  acid  -  acid  -  -  10. 5-Isoamyltetrazole 11. 2,3'-Diene VPA 12. 2-Ene VPA  -  13. 4-keto VPA  -  14. 5-Heptyltetrazole  25  37.5  -  -  15. N,N-Diethylsuccinamic a c i d 16. N,N-Dibutylsuccinamic a c i d  -  -  0 12.5  12.5  87.5 100  -  12.5  Convil l s a n t  1.5  2.0  0.2  87.5  -  -  100  -  0  0  0  -  Anticonvulsant a c t i v i t y of compounds was evaluated 15 min a f t e r i.p. administration. Mice were observed f o r c l o n i c spasms (>5 sec duration) w i t h i n 30 min a f t e r 85 mg/kg s.c. PTZ a d m i n i s t r a t i o n . Adult male Swiss mice (CD1 s t r a i n , 20-32g) were used. Eight mice were used per dose of t e s t compounds.  161  s u b - l e t h a l doses of 1.0 mmol/kg or l e s s .  The dose-response curves  of a c t i v e compounds are depicted i n Figure 1 6 . curves  of the most potent  The  drugs, v a l p r o i c a c i d ,  a c i d , 5 - h e p t y l t e t r a z o l e and  dose-response 2-butylhexanoic  1-methylcyclohexane-l-carboxylic a c i d  are r e l a t i v e l y steep compared with the moderately a c t i v e drugs. Calculation Litchfield  and  of  ED^g  values  Wilcoxon ( 1 9 2 ) .  was  based  on  the  method  Goodness-of-fit of the  of  estimated  curve to the observed dose-response data was tested using the C h i Square t e s t .  Tables 3 2 and  3 3 show the ED^Q  values, slopes of  dose-response curves and chemical s t r u c t u r e s of a c t i v e compounds. The slope values i n Tables 3 2 and 3 3 were determined by the method of L i t c h f i e l d and Wilcoxon ( 1 9 2 ) and are the a n t i l o g a r i t h m of the inverse of the slope constant i n the dose-response curves.  The  dose-response curves did not deviate s i g n i f i c a n t l y from p a r a l l e l ism.  The estimated ED^Q  of v a l p r o i c a c i d , 0 . 7 0 mmol/kg i s i n the  range of values of 0.57 mmol/kg ( 1 4 ) and 0 . 9 0 mmol/kg ( 4 0 ) reported i n previous studies using the s.c. PTZ activity  of  five  aliphatic  acids  test.  The anticonvulsant  studied have previously been  examined i n a single-dose study ( 1 0 ) where the endpoint the p r o t e c t i o n of m o r t a l i t y .  The  used  r e s u l t s of the m o r t a l i t y  was  study  are summarized i n Table 3 4 along with the r e s u l t s of t h i s study at 1 mmol/kg i . p . dose. does not  As shown i n Table 3 4 , the m o r t a l i t y endpoint  reveal the q u a n t i t a t i v e d i f f e r e n c e s i n a c t i v i t y of  the  f i v e a l i p h a t i c a c i d s compared to the d i s c r i m i n a t i v e c l o n i c s e i z u r e test.  Moreover, d i b u t y l a c e t i c a c i d was reported to be i n a c t i v e at  1.39 mmol/kg, which  i s contrary  162  to  the  results  of  the  present  8.2-Oim«ttiytX«yrie »cld TrtnaltiylacaMc acid  10  Do—(mmota/ko)  10  DoMftranoto/kg)  150  I  tt 2 3-Ettiyk»ntanole acid 1-M«ttiylcyelohexan«cartooxy»c acid  10  10  Doaa<imio4a/kg)  Do*a{mmola/kg)  96 BO  <80  SO  |  30  I  cl  » Valproic acid  i 160 6-Heptyltatraiole  ho *  2 8-Butytwxanofc: add  10  10  Poaaftwiufci/fcg)  DoaaOimoai/ka)  03  10 Doaeinmola/kQ)  Figure 16. Dose-response curves of valproic acid and analogues using the subcutaneous pentylenetetrazole seizure threshold test i n mice. 163  |  130 *  Table 32 Anticonvulsant potency of v a l p r o i c a c i d and i t s analogues against the c l o n i c phase of PTZ-induced seizures i n mice EDCQ, mmol/kg, i . p .  3  (95X confidence b limits)  Compounds  1.  5-Heptyltetrazole  0.31 (0.23-0.42)  1.56 (1.22-2.00)  2.  2-Butylhexanoic acid  0.57 (0.33-0.97)  1.72 (1.02-2.91)  3.  Valproic a c i d (VPA)  0.70 (0.50-0.98)  1.63 (1.14-2.33)  4.  1-Methylcyclohexane-lcarboxylic a c i d  1.08 (0.71-1.64)  1.83 (1.06-3.16)  5.  2,2-Dimethylbutyric a c i d  1.43 (0.56-2.58)  2.84 (0.72-11.2)  6.  2,3'-Diene VPA  1.45 (0.82-2.57)  2.76 (0.29-25.9)  7.  2-Ene VPA  1.46 (0.78-2.74)  3.06 (0.24-38.8)  8.  3-Ethylpentanoic a c i d  1.91 (1.43-2.56)  1.69 (0.81-3.52)  9.  Trimethylacetic  2.02 (1.11-3.68)  2.88 (0.77-10.8)  acid  10. Isobutyric a c i d 11. Cyclohexylacetic  a  b  c  Slope (95% confidence b limits)  -  inactive acid  inactive  12.5-Cyclohexylmethyltetrazole  inactive  13. 5-Isoamyltetrazole  inactive  14. 4-keto VPA  inactive  0  15. N,N-Diethylsuccinamic  acid  inactive  16. N,N-Dibutylsuccinamic  acid  convulsant  Drugs administered 15 min before s.c. 85 mg/kg PTZ i n j e c t i o n . Dose range of 0.2-2.0 mmol/kg. Data analyzed by the method of L i t c h f i e l d and Wilcoxon (192). R a p i d - c i r c l i n g a c t i v i t y of t e s t drug at 3.0 mmol/kg.  164  -  -  Table 33 Anticonvulsant potency of v a l p r o i c a c i d and i t s analogues against the c l o n i c phase of PTZ-induced seizures i n mice. Dose range of a c i d s , 0.2-2.0 mmol/kg ED^Q, mmol/kg, i . p . Slope (95% confidence (95% confidence limits) limits)  Compounds H CH3CH2CH2CH2CH2CH2CH2C*N_N  0.31 (0.23-0.42)  1.56 (1.22-2.00)  C 0 0 H  0.57 (0.33-0.97)  1.72 (1.02-2.91)  0.70 (0.50-0.98)  1.63 (1.14-2.33)  1.08 (0.71-1.64)  1.83 (1.06-3.16)  1.43 (0.56-2.58)  2.84 (0.72-11.2)  CHo - CH = CH ^ 0 0 0 0 1 1 CH3 - CH 2 - CH** "  1.45 (0.82-2.57)  2.76 (0.29-25.9)  CHo - CH 2 - CHo. ^tt o CH3 - CH 2 - CH^C - COOH  1.46 (0.78-2.74)  3.06 (0.24-38.8)  1.91 (1.43-2.56)  1.69 (0.81-3.52)  2.02 (1.11-3.68)  2.88 (0.77-10.8)  c $ p ™  ~  § H ^ C H - COOH C H 3  / X  V /^COOH CHo  1  CH 3 CH2 - C - COOH CH3  °  2H 5  ) C H - CH 2 - COOH CH3  CH 3 - C - COOH CH 3 C H ^ C H - COOH  inactive  165  Table 33 (Cont'd)  Compounds  <^  CH2_COOH  Slope ED5Q. mmol/kg, i . p . (95% confidence (95% confidence limits) limits) inactive  -  inactive  -  H / \  N_  \>_CH J} C / / 2 - ^N-N  C H  3K H ^CH - CHo - CHo - C —N-N CH 3 N—-N  CH 3 - ^ - CHo  inactive  ^CH - COOH CH 3 - CH 2 - C H ^  inactive  -  ^ N-C - CH2CH2 COOH C2H5^ 0  inactive  -  convulsant  -  CAH9  ^ N-C - CH2_CH2-C00H C4H9^ 0  166  Table 34 Anticonvulsant a c t i v i t y of VPA and i t s analogues on the threshold f o r PTZ-induced seizures determined by protection against c l o n i c seizures and by percent m o r t a l i t y i n mice % Protected Against (n = 8/dose) Clonic 3 Seizures  Compounds  3  Mortality  b  Mortality  1. Valproic a c i d (VPA)  87.5  100  100  2.  2-Butylhexanoic a c i d  87.5  100  0  3.  2,2-Dimethylbutyric a c i d  50  100  80  4.  Trimethylacetic  25  100  80  5.  Isobutyric a c i d  0  75  20  6.  3-Ethylpentanoic a c i d  12.5  100  7.  1-Methylcyclohexane-lcarboxylic acid  37.5  100  8.  Cyclohexylacetic  12.5  100  9.  2-Ene VPA  acid  acid  37.5  0  62.5 100  10. 2,3'-Diene VPA  50  11. 4-keto VPA  12.5  25  12. 5-Isoamyltetrazole  12.5  62.5  13. 5-Cyclohexylmethyltetrazole  12.5  75  14. 5-Heptyltetrazole  100  100  15. N,N-Diethylsuccinamic acid  12.5  62.5  16. N,N-Dibutylsuccinamic a c i d  0  0  A c t i v i t y at 1.0 mmol/g i . p . of t e s t compounds administered before 85 mg/kg s.c. PTZ i n j e c t i o n .  15 min  ^Conditions same as i n (a) above with m o r t a l i t y determined w i t h i n 30 min a f t e r PTZ a d m i n i s t r a t i o n . c  Taken from G. Carraz (4) where 1.39 mmol/kg i . p . before 80 mg/kg s.c. PTZ a d m i n i s t r a t i o n . 167  administered  30 min  study.  Table 34 a l s o shows that compounds i n a c t i v e against c l o n i c  seizures would have been described as a c t i v e by the m o r t a l i t y t e s t . 2.  T o x i c i t y of compounds The acute t o x i c e f f e c t s o f the t e s t compounds were noted d u r i n g t h e 15 min i n t e r v a l b e f o r e i n j e c t i o n of PTZ (Table 3 5 ) . Sedation was a common side e f f e c t of the anticonvulsant compounds. V a l p r o i c a c i d showed remarkable sedation at 2 mmol/kg doses.  The  TD^Q of v a l p r o i c a c i d i s reported to be 2.56 mmol/kg i n mice ( 4 0 ) . The unsaturated  analogues of v a l p r o i c a c i d and d i b u t y l a c e t i c a c i d  exhibited marked sedation even at 1 mmol/kg doses while the most potent anticonvulsant 5 - h e p t y l t e t r a z o l e showed s e d a t i o n a t much lower doses.  D i b u t y l a c e t i c a c i d appeared to have the lowest  pro-  t e c t i v e index w i t h i t s l e t h a l e f f e c t s o c c u r r i n g a t 2.0 mmol/kg. Compared to the c a r b o x y l i c a c i d s , the t e t r a z o l e s possessed greater t o x i c properties with a t a x i a occurring a t low doses.  N,N-Dibutyl-  s u c c i n a m i c a c i d had c o n v u l s a n t p r o p e r t i e s a t s u b l e t h a l doses o f 0.5-1.0 mmol/kg. Among the compounds s t u d i e d , v a l p r o i c a c i d appeared t o have the most d e s i r a b l e pharmacological  p r o p e r t i e s s i n c e i t possessed  h i g h a n t i c o n v u l s a n t a c t i v i t y w i t h marked s e d a t i o n appearing a t doses g r e a t e r than 2.0 mmol/kg.  I t i s a p p a r e n t l y not y e t c l e a r  whether anticonvulsant and sedative e f f e c t s occur at the same s i t e i n the CNS (14,30).  I t has been suggested that the d i s t r i b u t i o n a l  l o c a l i z a t i o n of anticonvulsant drugs may be the predominating f a c t or to determine the sedative side e f f e c t s (20,29).  The unsaturated  analogues of v a l p r o i c a c i d showed more sedative e f f e c t s and l e s s anticonvulsant a c t i v i t y than v a l p r o i c a c i d (Table 3 5 ) .  168  Table 35 Observed t o x i c e f f e c t s of t e s t compounds i n mice Observed Toxic E f f e c t s  Compounds A.  Isobutyric acid 4-keto VPA Trimethylacetic a c i d 2,2-Diraethylbutyric acid 3-Ethylpentanoic acid Cyclohexylacetic a c i d  Minimal sedative e f f e c t s a t doses not greater than 2.0 mmol/kg.  B.  Valproic acid (VPA) 1-Methylcyclohexane-lcarboxylic a c i d  Sedative e f f e c t s a t 2.0 mmol/kg dose.  C.  2,3'-Diene VPA 2-Ene VPA  Sedative e f f e c t s a t doses greater than 1.0 mmol/kg.  D.  2-Butylhexanoic a c i d  Sedation and a t a x i a a t doses greater than 1.0 mmol/kg. Lethal dose a t 2.0 mmol/kg.  E.  5-Cyclohexylmethyltetrazole 5-Isoamyltetrazole  Ataxia a t doses greater than 1.0 mmol/kg. P r o s t r a t i o n and abnormal spread of hind limbs a t 2.0 mmol/kg dose.  F.  5-Heptyltetrazole  Sedative e f f e c t s and a t a x i a at doses greater than 0.2 mmol/kg. Abnormal body posture. Occasiona l f a s c i c u l a t i o n a t 1.0 mmol/kg dose.  G.  N,N-Diethylsuccinamic acid  Abnormal spread of hind limbs a t doses greater than 0.5 mmol/kg. Hyperactivity ( r a p i d - c i r c l i n g a c t i v i t y ) a t doses of 3.0 mmol/kg  H.  N,N-Dibutylsuccinamic acid  Tremors, jumping or hopping movements and convulsions a t doses tested (0.5-1.0 mmol/kg). Tonicc l o n i c spasms. Lethal dose at 2.0 mmol/kg.  169  E.  Structure-Activity Relationships  1.  Q u a n t i t a t i v e S t r u c t u r e - A c t i v i t y Relationships The  investigation  of  the molecular  specifity  of the  anti-  convulsant a c t i o n of v a l p r o i c a c i d analogues was one of the major objectives of t h i s study. free-energy  model  was  Towards t h i s end, the Hansch  applied  to  study  s t r u c t u r a l l y - d i v e r s e valproate analogues. of Meyer and  Overton,  i t has  the  SAR  linear  among  the  Since the c l a s s i c a l work  become evident that  lipophilicity  plays a predominant r o l e i n determining the i n vivo a c t i v i t y many CNS-active ability  drugs.  According to the Hansch model, the  t h a t CNS-active  determined  drugs r e a c h t h e i r  by l i p o p h i l i c i t y ( l o g P ) .  site  prob-  of a c t i o n i s  In a d d i t i o n , pharmacokinetic  f a c t o r s such as p r o t e i n - b i n d i n g , d i s t r i b u t i o n , metabolism and c r e t i o n which play a major r o l e  of  i n determining  ex-  the e f f i c a c y  of  these drugs are at l e a s t governed by l i p o p h i l i c i t y (193,194).  On  the other hand, s t e r i c and e l e c t r o n i c f a c t o r s appear to be c r i t i c a l for the pharmacodynamic a c t i o n of drugs that a r i s e by i n t e r a c t i o n with s p e c i f i c r e c e p t o r s . In t h i s study, the l i p o p h i l i c i t y of compounds was described by the octanol-water  partition coefficient  ( l o g P) values, obtained  from HPLC studies using 70% methanol-phosphate buffer as the mobile phase.  The e l e c t r o n i c e f f e c t s were represented by the pKa values  of the compounds as determined  i n 10% methanol-water.  The  pKa  values express the r e l a t i v e i o n i z a t i o n strength of the compounds. The b i o l o g i c a l a c t i v i t y evaluated was the antagonism of PTZ-induced clonic  seizures i n mice.  Table  36  170  shows the s t r u c t u r e ,  anti-  Table 36 B i o l o g i c a l data and physicochemical properties of compounds tested  ED5  ?/w mmol/kg i.p.  Compounds  ES(R) *  log 1 ED 5 F J  l o g P pKa ES(R) (HPLC) (10% * * MeOH)  H CH3CH2CH2CH2CH2CH2CH2C^v.j^_j^  0.31  -0.35  0.509  2.01  5.31  -1.54  C ^ > C H - COOH  0.57  -2.23  0.243  3.34  5.05  -3.47  r l ^cn  0.70  -2.11  0.155  2.57  4.95  -3.35  1.08  -2.03 -0.0334  2.38  5.17  -3.27  1.43  -1.60 -0.156  1.90  5.26  -3.41  1.45  -0.161  2.27  4.02  -  1.46  -0.164  2.44  4.36  -  1.91  -2.00 -0.281  2.19  4.78  -2.17  2.02  -1.54 -0.305  1.54  5.11  -2.78  1.10  4.96  -1.71  3 7  - COOH C  H  / - V 3 V X^-COOH CH  3  CHo-CH9 - C - COOH 3 ! CH 3 C  CH3 - CH 2 - C H *  C _  C 0 0 H  cl : S*: Ssc - < w » 3  3  CoHc ^CH - CH 2 - COOH C2H5/ CHo  1  3  CHo3 - C - COOH  1  CH 3  CH ^ 3 ^ C H - COOH  i n a c t i v e -0.47  171  Table 36 (Cont'd)  ED5  9/w  Compounds  ^  mmol/kg i.p.  yCH 2 -COOH  *  log 1 ED 5 Q  log P pKa (HPLC) (10% ** MeOH)  i n a c t i v e -0.98  2.31  4.78  -3.22  i n a c t i v e -0.98  1.59  5.37  -3.22  •^CH - CHo - CHo - C ^ ~ i n a c t i v e -0.35 2 2 CHf \\ II N —N  1.29  5.22  -1.59  inactive  1.31  4.58  inactive  2.27  5.58  convulsant  2.30  5.58  H (  /"  CH  _ C  2  -N-N N  N  9 CHo —  C — CHo  ^CH - COOH CH^ — CH 2 — CH 2 2  5  ^ N-C - CH0CH0-COOH C H ' " C4H9 ^ C  H  4 9  N-C - CH0-CH0-COOH 0  *  Taken from  R. W. Taft (134).  **  Taken from C. Hansch and A. Leo (152).  172  convulsant potency and physicochemical properties of v a l p r o i c a c i d and the other analogues t e s t e d .  The l o g P values range from 1.0 t o  3.A and pKa values from A.O t o 5.6.  The s t e r i c bulk of the a l k y l  substituents was described by the Taft s t e r i c  constant, E g * or  Hansch s t e r i c constant, E * * . In accordance with the l i n e a r free-energy r e l a t i o n s h i p model, log I/ED5Q was c o r r e l a t e d with l o g P, pKa and E' using m u l t i p l e r e gression MIDAS).  analysis  (Michigan  Interactive  Data  Analysis  System,  In Table 37 are l i s t e d the regression equations along with  the m u l t i p l e c o r r e l a t i o n c o e f f i c i e n t , r (from r e s i d u a l and p r e d i c t ed  v a l u e s ) , the standard  error  of the r e g r e s s i o n , (S) and the  attained s i g n i f i c a n c e l e v e l , p (from a n a l y s i s of variance f o r the regression). served  From the c o r r e l a t i o n matrix i n Table 35, i t i s ob-  that a l l interdependence  among the v a r i a b l e s of the nine  a c t i v e compounds are i n s i g n i f i c a n t except between l o g P and E g ( r = -0.5A).  This i s expected from the increase i n l o g P with molecular  s i z e which i n turn c o r r e l a t e s with E g (195). For the nine a c t i v e compounds, i t can be seen from Table 37 that the regression equations 1-A are not s i g n i f i c a n t .  The a n t i -  PTZ potency could not be described by e i t h e r l o g P (equation 1) or pKa (equation 2) alone. where both accounts  The c o r r e l a t i o n improved i n equation 3  l o g P and pKa a r e i n c l u d e d .  f o r only  However, e q u a t i o n 3  A0% ( r ) of the v a r i a t i o n s  i n anticonvulsant  potency. Examination of the graphic p l o t of l o g l/ED^g versus l o g P or pKa revealed that 5 - h e p t y l t e t r a z o l e was a c t u a l l y more a c t i v e than anticipated.  When 5 - h e p t y l t e t r a z o l e was deleted from the QSAR, the  potency was better c o r r e l a t e d with l o g P (Table 38, equation 5 ) . 173  Table 37 Equations obtained c o r r e l a t i n g the anti-PTZ e f f e c t of v a l p r o i c a c i d and analogues with t h e i r physicochemical parameters  Equation  a  n  1. - l o g EDCQ = 0.223 le o g P - 0.534 (+0.186) (+0.435)  a c  r  b  S  c  F  P  d  9  0.413  0.264  1.442  0.269  2. - l o g ED 5 0 = 0.254 pKa - 1.263 (+0.215) (+1.05)  9  0.408  0.265  1.398  0.276  3. - l o g EDr n = 0.263 l o g P + (+0.173) 0.300 pKa - 2.09 (+0.199) (+1.11)  9  0.631  0.243  1.986  0.218  4. - l o g ED 5 Q = -0.265 E g - 0.517  7 -0.291  nuraber of compounds s t u d i e d , ^correlation coefficient, standard error of estimate, l e v e l of s i g n i f i c a n c e of F-value, number i n parentheses gives standard error of c o e f f i c i e n t .  e  C o r r e l a t i o n matrix f o r the physicochemical parameters with nine observations log P log P  pKa  Es  1  pKa  -0.153  E_  -0.540  174  1 0.298  1  Table 38 Equations obtained c o r r e l a t i n g the anti-PTZ e f f e c t s of VPA and analogues (excluding 5-heptyltetrazole) with t h e i r physicochemical parameters  Equation  n  5. - l o g ED 5 0 = 0.322 l o g P - 0.837 (+0.079) (+0.188) 6. - l o g ED 5 0 = 0.102 pKa - 0.582 (+0.180) (+0.876)  r  S  F  p  8  0.856  0.110 16.48  0.0067  8  0.225  0.207  0.59  7. - l o g ED 5 0 = 0.331 l o g P + 0.136 pKa 8 . (+0.171) (+0.086)  0.907  0.098  0.321  11.56  0.013  -1.516 (+0.461) 8. - l o g ED s n = 0.065 l o g P (+0.622) 2 +0.052 ( l o g P ) - 0.536 (+0.126) (+0.751)  0.861  0.118  7.190  0.034  9. - l o g EDc J Un = 0.478 l o g P (+0.647) -0.0297 ( l o g ?) z + 0.147 pKa (+0.130) (+0.107) -1.743 (+1.12)  0.908  0.109  6.263  0.054  For meaning of n, r , s , F, P: Correlation log P  pKa  Matrix (log P )  1  log P (log P )  r e f e r to Table 35.  :  0.991  1  -0.0841  175  -0.0206  2  pKa  The a d d i t i o n of a pKa term to equation 5, gave a s i g n i f i c a n t and better c o r r e l a t i o n (equation 7 ) .  The p o s i t i v e c o e f f i c i e n t f o r l o g  P and pKa i n equation 7 i n d i c a t e d that an increase i n l o g P and pKa enhanced  the anticonvulsant a c t i v i t y .  apparent i n equation 3 f o r Table 36. equation  7 agrees with e a r l i e r  The same conclusion was The r e l a t i o n s h i p expressed by  studies (196) which showed that  l i p i d s o l u b i l i t y and i o n i z a t i o n constants of a c i d i c and basic drugs are of greater importance i n drug entry i n t o the CNS. Since a n t i convulsant a c t i v i t y of the c a r b o x y l i c acids increases with high l o g P values, r e a l i z a t i o n of an optimum log P value could be obtained from the p a r a b o l i c r e l a t i o n s h i p between i n vivo b i o l o g i c a l a c t i v i t y and log P suggested by Hansch and co-workers (19,199).  However, as  shown i n equation 9 (Table 38), the parabolic r e l a t i o n s h i p i n log P was  not s i g n i f i c a n t , probably  because the l o g P values of the  a c t i v e compounds are l e s s than the optimum l o g P value but f a l l i n t o the l i n e a r portion of the curve. The  f a i l u r e of l o g P and pKa t o e x p l a i n t h e v a r i a t i o n i n  antagonism  of PTZ by the tested compounds i n c l u d i n g  5-heptyl-  t e t r a z o l e could be due to the absence of a physicochemical parameter to c h a r a c t e r i z e the i n t r i n s i c a c t i v i t y of compounds a r i s i n g from i n t e r a c t i o n with the r e c o g n i t i o n s i t e .  The Hansch model has  been extended t o replace the Hammet constant, a , with other e l e c t r o n i c parameters such as pKa, dipole moment and net atomic charges (205).  However, the e l e c t r o n i c e f f e c t s expressed by the pKa values  have been noted to have a dual f u n c t i o n (197). proportion barrier.  of unionized  drug  which  The pKa a f f e c t s the  penetrates  the blood  brain  I t a l s o describes the e l e c t r o n i c e f f e c t s of the a l k y l  176  substituent on the charge density of the c a r b o x y l i c group or the other polar moiety.  Dipole moments have been shown t o be u s e f u l i n  c h a r a c t e r i z i n g the polar moiety which may i n t e r a c t  electrostatical-  l y a t the recognition s i t e (26-28,182). Due  to the p o s s i b i l i t y that a l k y l - s u b s t i t u t e d anticonvulsant  compounds a c t v i a i n t e r a c t i o n a t the p i c r o t o x i n s i t e of the GABA receptor  complex, the physicochemical  anti-PTZ a c t i v i t y were i n v e s t i g a t e d .  properties that determine  The anti-PTZ potency f o l l o w -  ing standard procedures and physicochemical properties of the other alkyl-substituted  compounds, i n c l u d i n g  the m o l e c u l a r  dipole  moments, were obtained from l i t e r a t u r e sources and are summarized i n Table 39. It  The r e s u l t s of the QSAR are presented i n Table 40.  can be seen  insignificant. (M)  that  the r e g r e s s i o n equations  1-7 a r e  Equations 1-3 show that l o g P, pKa or dipole moment  alone, do not account f o r the v a r i a t i o n s i n anticonvulsant  potency. slight  A d d i t i o n of a term i n ( l o g P)  to equation 1 gives a  improvement i n the c o r r e l a t i o n (equation 4 ) .  The combin-  a t i o n of l o g P and pKa (equation 5) or l o g P and u (equation 6) a l s o s l i g h t l y improves the c o r r e l a t i o n i n equation 1. gave the best c o r r e l a t i o n . strates  Equation 8  Comparison of equations 7 and 8, demon-  t h a t e q u a t i o n 8 i s s u p e r i o r i n terms of s t a t i s t i c a l  s i g n i f i c a n c e (p = 0.01) and reduction of the standard error of the estimate.  Thus the regression equation  moment term accounts activity  than  with the added d i p o l e  better f o r the v a r i a t i o n  that with the added pKa term.  i n anticonvulsant The optimum l o g P  value i n equation 8 below was 1.45. The negative c o e f f i c i e n t of  177  Table 39 Anticonvulsant a c t i v i t y of various drugs against c l o n i c seizures induced by PTZ ( s . c . 85 mg/kg) i n mice and t h e i r physicochemical constants Compound  0.70  2.  2-Butylhexanoic acid  0.57  3.  Ethosuximide  0.922  5.  0.057  Barbital  8. 9.  Butobarbital 5-Heptyltetrazole  10. Dimethadione 11. Trimethadione 12. Paramethadione  d  0.293  0.243  3.34  0.035  0.016  0.533  h  0.085 c  5.75  2.083 0.399 e  3.51  14. a,a-Ethylmethy1Y-butyrolactone  1.17  e  d  4.95°  1.15  b  9.1  c  e  1.15 1.47 1.47  2.198  7.938  1.13  0.688  7.758  1.13  b  1  1.70§  0.399  a.b y» ' (debyes)  9.1  1.070  -0.319  pKa  5.05  f  -0.49  1.21  -0.760 d  c  2.973  0.509  b  c  2.57  1.244  h  0.051  0.31  h  P  8 o/w  0.155  -0.623  13. a,a-Dimethyl-ybutyrolactone  Taken from Taken from °Taken from d Taken from ^Taken from f Calculated bBTaken from xTaken from Taken from b  d  e  4.2  7. Metharbital  a  c  4. a,a-Dimethylsuccinimide  6.  ED 5 0  c  1. V a l p r o i c a c i d  Pentobarbital  lo  ED 5 0 mmol/kg  2.01° -0.93 -0.37 0.13  -0.545  1.01  -0.157  1.51  b  b  b  f  f  8.45  7.818 5.31  c  6.13  1  1.13 1.13 2.65 1.74  -  4.13  -  4.13  1.74 1.69  A. L . McClellan (198). E. J . Lien et a l . ( 2 8 ) . t h i s study. R. L. K r a l l et a l . ( 3 8 ) . W. E. Klunk et a l . ( 4 7 ) . using Hansch 7T-parameters, or fragraental constants (152). S. Toon and M. Rowland (194). A. Raines et a l . (200). D. M. Woodbury e t a l . ( 4 6 ) .  178  Table 40 Equations correlating anti-PTZ activity and physicochemical properties of anticonvulsants in Table 39 n  Equations  s  b  r  c  d  F  P  1. -log ED5Q = 0.2961 log P + 0.0327  14  0.925  0.387  2.12  0.17  2. -log ED50 = 0.150 pKa - 0.537  10  1.10  0.232  0.454  0.52  3. -log ED5Q = -0.37u  14  0.917  -0.406  2.37  0.15  14  0.859  0.573  2.70  0.11  0.900  0.668  2.81  0.13  14  0.874  0.552  2.41  0.14  10  0.830  0.772  2.95  0.12  14  0.643  0.811  6.40  0.01  + 1.03 2  4. -log ED5Q = -0.873 log P - 0.262 (log P) + 0.103  10  5. -log ED50 = 0.586 log P + 0.431 pKa - 3.263 6. -log ED5Q = 0.286 log P - 0.360u + 0.710 2  7. -log ED50 = 1.11 log P - 0.302 (log P) + 0.205 pKa - 1.31 2  8. -log ED5Q = 1.14 log P - 0.392 (log P) -0.559u + 1.19  c  a  number of compounds used, correlation coefficient,  ^standard error of estimate, level of significant of F-value and degrees of freedom. Correlation matrix log P  log P (log P) pKa U  1  (log P) 0.8723 1  pKa  V  -0.5691 -0.0336 -0.7342 -0.1971 1 -0.3076  2  log 1/ED50 = 1.14 l o g P - 0.392 ( l o g P ) - 0.559 y + 1.19 (n = 14, r = 0.811, s = 0.643, l o g P Q = 1.45)  the d i p o l e moment v a r i a b l e i n equation 8 implied that an increase i n d i p o l e moment without the necessary l o g P value would the anticonvulsant potency.  decrease  S i m i l a r f i n d i n g s , i n d i c a t i n g the r o l e  of dipole moment i n anticonvulsant a c t i v i t y , have been reported i n the l i t e r a t u r e .  L i e n et a l . (26) reported that the best equation  for phenyl- and a l k y l - s u b s t i t u t e d h e t e r o c y c l i c a n t i e p i l e p t i c drugs was  2  log 1/ED50 = 0.852 l o g P - 0.301 ( l o g P ) - 0.629u + 4.139 (n = 12, r = 0.915, s = 0.227, l o g P Q = 1.42)  As i n t h i s study, highly a c t i v e compounds i n d i f f e r e n t classes of a n t i e p i l e p t i c drugs were used i n the regression a n a l y s i s . and  Webb (27) i n v e s t i g a t e d  activity  and physicochemical  diazepines.  Blair  t h e r e l a t i o n s h i p between a n t i - P T Z properties i n a s e t of 1,4-benzo-  The best equation was indicated to be  log 1/ED5Q = -0.50y + 3.26 (n = 52, r = 0.626, s = 0.866)  The equation showed that i n a s e r i e s of 1,4-benzodiazepines with s i m i l a r l o g P values or with almost equal access  to the CNS s i t e s  of a c t i o n , d i p o l e moment played the major r o l e i n determining the anticonvulsant a c t i v i t y . 180  The dependence of a c t i v i t y on d i p o l e moment may explain the low  structural  specificity  of anticonvulsant  s t e r i c e f f e c t s are not apparent. trum  of s t r u c t u r a l l y  diverse  compounds i n which  Thus there i s a continuous speccompounds with  varying  molecular  dipole moments. The physical meaning of the negative dependence of anticonvulsant  activity  on dipole moment i s not c l e a r .  compounds with a low molecular  Those  dipole moment are not n e c e s s a r i l y  the most potent anticonvulsant compounds.  The 1,4-benzodiazepines  are reported to have higher dipole moments and l o g P values than other t r a d i t i o n a l a n t i e p i l e p t i c drugs (26).  The dipole moment of  diazepam was found to be 2.65 debyes ( 2 6 ) . However, the benzodiazepines  show  very  high  activity  against  PTZ-induced  clonic  seizures.  The e f f e c t i v e n e s s of these compounds may be due to  favourable conformations f o r hydrophobic binding and e l e c t r o s t a t i c i n t e r a c t i o n at target s i t e s .  Since the benzodiazepines could not  be included i n a s e r i e s of anticonvulsants to develop QSAR (26), i t appears  that  different  c h a r a c t e r i s t i c molecular trostatic  interaction  c l a s s e s of a n t i c o n v u l s a n t  drugs have  dipole moments that determine t h e i r e l e c -  at s p e c i f i c  binding  sites.  I t has been  suggested that i n whole animal s t u d i e s , the s i g n i f i c a n c e of dipole moment i n QSAR of anticonvulsant drugs may be i n d i c a t i v e of d i p o l e dipole or charge-dipole i n t e r a c t i o n not only at s i t e s of a c t i o n but also at s i t e s of nonspecific binding ( 2 0 ) . Pentylenetetrazole and 5-heptyltetrazole show opposing pharmac o l o g i c a l e f f e c t s , and have d i f f e r e n t physicochemical  properties.  5 - A l k y l t e t r a z o l e s have an average dipole moment of 2.65 whereas 1 , 5 - d i a l k y l t e t r a z o l e s have an average value of 5.30 (198).  181  Both  compounds are l i p i d - s o l u b l e while PTZ alone i s r e a d i l y soluble i n water. in  PTZ, however, lacks a c i d i c p r o p e r t i e s . I t i s obvious that  such comparisons, d i f f e r e n c e s e x i s t i n h y d r o p h i l i c i t y , d i p o l e  moment values and a c i d i c properties (pKa).  Also s t e r i c d i f f e r e n c e s  cannot be ruled o u t . The  dominating influence of dipole moment i n the preceding  regression equation does not r u l e out the r o l e of pKa i n determining  anticonvulsant  activity.  I t would  be expected that among  s t r u c t u r a l congeners such as the a l k y l c a r b o x y l i c acids that there w i l l be high c o l l i n e a r i t y between pKa and u as reported f o r homologous a l k y l - s u b s t i t u t e d  compounds (134).  However, as shown i n  Table 40, f o r compounds with diverse polar groups the c o r r e l a t i o n between pKa and u i s low ( r = 0.3076). In t h i s study, approximately 66% ( r ) of the variance i n a n t i convulsant  a c t i v i t y of the s t r u c t u r a l l y diverse a l k y l - s u b s t i t u t e d  anticonvulsants could be accounted f o r by l i p o p h i l i c i t y and dipole moment (equation 8, Table 38). perfect activity  correlation  could  be obtained  and p h y s i c o c h e m i c a l  d i f f e r e n t sources.  I t appears u n l i k e l y that a near-  be  In a d d i t i o n , the c o r r e l a t i o n i n the QSAR study  variously such  reported  could  effects  and e x c r e t i o n .  on absorption, metabolism,  These p r o c e s s e s  have a l s o been  t o be governed by physicochemical  as l i p o p h i l i c i t y  factors  of the b i o l o g i c a l t e s t system,  The v a r i a b i l i t y i n the anticonvulsant potency could  influenced by s t r u c t u r a l  distribution  the anticonvulsant  p r o p e r t i e s were determined from  i s l i m i t e d by the complex nature whole animal.  since  and pKa (193,194).  e x p l a i n the r e s i d u a l  regression equations. 182  parameters  Furthermore,  variance unaccounted  steric f o r by  Structural activity  features  that  enhance or d i m i n i s h  anticonvulsant  A l i p h a t i c substituents The lack of a c t i v i t y of i s o b u t y r i c acid and reported  inactiv-  i t y of non-branched C^HyCOOH to C ^ j C O O H (12,13,20) may be due to t h e i r low l i p o p h i l i c i t y . as e f f e c t i v e  I t appears that a or 3-branching may  as s t r a i g h t - c h a i n a l k y l  anticonvulsant a c t i v i t y .  be  substitution i n conferring  5-Heptyltetrazole, with a s t r a i g h t - c h a i n  a l k y l substituent possessed potent anticonvulsant a c t i v i t y .  From  QSAR studies i t i s known that a l i p h a t i c substituents increase the lipid  solubility  anticonvulsant dimethylbutyric multiple  of compounds f o r e n t r y activity  acid  groups  recognition s i t e .  on  oxidative  f o r binding Increased  the CNS.  trimethylacetic acid  probably r e f l e c t  a-branching  hydrophobic  of  into  and  The 2,2-  the s t a b i l i z i n g  e f f e c t of  metabolism  increased  or  to a hydrophobic portion of the  hydrophobic binding by the Cy-chain  may also account f o r the high a c t i v i t y of 5 - h e p t y l t e t r a z o l e . V a l p r o i c acid and  i t s analogues probably bind to a p r o t e i n  complex i n the GABA-metabolizing enzyme system or the reported (30) protein complex. other  complex  of  the  GABA-benzodiazepine  picrotoxin  receptor  In v i t r o studies have shown that v a l p r o i c a c i d (112) and  medium-chain f a t t y acids (199) bind with high a f f i n i t y  bovine or human serum albumin.  to  Bovine or human serum albumin has  been used as a convenient model to study the molecular basis of specific-ligand receptors  protein  i n t e r a c t i o n s (112,199),  since  have r e c o g n i t i o n s i t e s on protein subunits.  many  drug  Valproic  a c i d and other medium chain f a t t y acids have been suggested, from i n v i t r o protein-binding s t u d i e s , to bind with high a f f i n i t y to a 183  site  on  human  albumin  indole/benzodiazepine  identical  s i t e (112,199).  or  close  to  the  In a recent study, Brown et  a l . (199) i n v e s t i g a t e d the a l b u m i n b i n d i n g of normal a l i p h a t i c a c i d s ( p e n t a n o i c a c i d up to nonanoic a c i d ) u s i n g u l t r a f i l t r a t i o n techniques.  A s i n g l e h i g h a f f i n i t y s i t e was  observed f o r each  f a t t y acid w i t h an increase i n number of secondary s i t e s w i t h chain elongation.  From the b i n d i n g a f f i n i t i e s and competitive binding  data, the authors suggested that there are d i s t i n c t albumin binding s i t e s f o r the short-chain « C ? ) and the medium chain (C8-C9) f a t t y acids.  On the o t h e r hand, the h i g h a f f i n i t y s i t e s of l o n g c h a i n  f a t t y acids are reported to be d i f f e r e n t from that of medium-chain f a t t y acids (112,199). 5-Isoamyltetrazole was analogue, isohexanoic  inactive.  a c i d has  The c a r b o x y l i c b i o i s o s t e r i c  a l s o been r e p o r t e d  to be i n -  a c t i v e ( 5 ) . T h i s c o u l d be due to the low l i p o p h i l i c i t y of these compounds. Thus the e f f e c t of the i s o p r o p y l terminus could not be investigated i n t h i s s e r i e s .  The convulsant properties of b a r b i t -  urates w i t h the i s o p r o p y l and isopropenyl terminus (Figure 3) have been a t t r i b u t e d to e i t h e r a s t e r i c i n f l u e n c e from conformation  of  these groups a t the r e c o g n i t i o n s i t e or t o s t e r i c i n f l u e n c e on o x i d a t i v e metabolism at the (co-l) p o s i t i o n (19,99,100). b.  Alicyclic-substituents C y c l o h e x y l a c e t i c a c i d and i t s t e t r a z o l e analogue, 5 - c y c l o hexylmethyltetrazole were found to be i n a c t i v e i n the dose range s t u d i e d (Table 29).  However, 1-methylcyclohexanecarboxylic  acid  was r e l a t i v e l y a c t i v e even though the combined e f f e c t of log P and  184  pKa  values i s s i m i l a r to that of the cyclohexylmethyl-substituted  compounds.  Owing to the enormous d i f f e r e n c e i n a c t i v i t y between  the cyclohexyl and cyclohexylmethyl-substituted compounds, i t i s apparent t h a t s u b t l e s t e r i c e f f e c t s were r e s p o n s i b l e f o r the difference i n a c t i v i t y .  The Taft s t e r i c parameter, E_, has been  frequently found to be inadequate that  influence biological  i n describing s t e r i c properties  a c t i v i t y (195).  L i t e r a t u r e E_  values  showed covariance with l o g P values (Table 3 7 ) . The uniqueness of the cyclohexylmethyl group i n reducing a n t i convulsant a c t i v i t y was evident i n other classes of anticonvulsants although i t appears to have been overlooked.  5-Cyclohexylmethyl  b a r b i t u r a t e , XXXVI, has been reported to have l i m i t e d activity  compared  to b a r b i t a l (201), while  biological  5-ethyl-5-benzylbarb-  i t u r a t e , XXXVII, has convulsant properties (201).  The urea d e r i v -  a t i v e , XXXVIII ( 8 ) , a c y l u r e a d e r i v a t i v e , XL ( 8 9 ) , and  spiro-  c a r b o x y l i c acid d e r i v a t i v e , XLI (98), have been reported to have l i m i t e d a c t i v i t y against PTZ-induced s e i z u r e s . On the other hand, 1-methylcyclohexylurea,  XXXIX (205),  and  the  spiro  carboxylic  d e r i v a t i v e , XXIII (88), have been found to be a c t i v e against PTZinduced seizures at comparative doses. The lack of a c t i v i t y of cyclohexylmethyl-substituted compounds may be due to t h e i r i n a b i l i t y to assume a required conformation to i n t e r a c t with binding s i t e s .  I t seems possible that t h i s may be  due to i n vivo metabolic e f f e c t s . how  metabolic  effects  However, i t i s d i f f i c u l t to see  can uniformly apply  to a l l the d i f f e r e n t  classes and a l s o not to those with cyclohexyl groups.  The e f f e c t s  of s t r u c t u r a l features such as the cyclohexylmethyl group, benzyl g r o u p , i s o p r o p y l o r i s o p r o p e n y l g r o u p may 185  due t o  steric  Figure 17. compounds.  Active and inactive a l i c y c l i c and  186  alicyclicalkyl-substituted  i n t e r a c t i o n s at the s i t e of a c t i o n .  The molecular conformation of  the  an i s o p r o p y l or i s o p r o p e n y l  convulsant barbiturates with  terminus i n the b u t y l side chain (Figure 3) has been i n v e s t i g a t e d using  NMR  spectroscopy  c a l c u l a t i o n s (99).  ( 1 0 0 ) and  molecular  orbital  I t was suggested from these studies that the  convulsant properties may be due t o the s t e r i c influence of the isopropyl  or isopropenyl  terminus.  Confirmation  of the s t e r i c  influence of these s t r u c t u r a l features w i l l have to await r e s u l t s of further i n v i t r o and i n vivo s t r u c t u r e - a c t i v i t y s t u d i e s . Recent studies on s p i r o analogues of v a l p r o i c a c i d (88) and r i g i d c y c l o a l k y l compounds (11) have been aimed at r e v e a l i n g configurations  which  analogues.  I n analogy to structures of the GABA a g o n i s t s , i s o -  guvacine  would  and THIP  confer  high  activity  of v a l p r o i c  acid  (4,5,6,7-tetrahydro-isoxazolopyridin-3-ol),  methylcyclohexanecarboxylic a c i d d e r i v a t i v e s w i l l  1-  be of i n t e r e s t  for future s t u d i e s . c.  E f f e c t s of a polar f u n c t i o n a l i t y i n the a l k y l chain N,N-dibutylsuccinamic properties  while  acid  was found  N,N,-diethylsuccinamic  acid  h y p e r a c t i v i t y a t the dose range t e s t e d . r e l a t i v e l y high  convulsant  appeared to induce Both compounds have  pKa and l o g P values when compared to the other  compounds s t u d i e d . amides (3,4,50),  to possess  Secondary and t e r t i a r y amides of a l i p h a t i c a c i d succinic  acid-bis-dialkylamides,  tetra  alkyl-  ureas (49), and polymethylene lactams (26) have been reported t o have  stimulant  succinamic  acids  and/or  convulsant  properties.  The N,N-dialkyl-  and these compounds share a common f u n c t i o n a l  187  group, the s u b s t i t u t e d amido group.  According to L i e n et a l . (26)  the convulsant a c t i v i t y of some a l k y l - s u b s t i t u t e d ureas and lactams increased w i t h higher molecular d i p o l e moments. The two succinamic acids studied appear to show some s t r u c t u r a l s i m i l a r i t i e s to the putative e x c i t a t o r y t r a n s m i t t e r s , a s p a r t i c and glutamic a c i d s .  I t has been proposed that g l u t a m i c - l i k e a c t -  i v i t y required a c a t i o n i c and two anionic centres i n the molecular structure (204).  Due t o amide resonance i n t h e s u c c i n a m i c a c i d s  there could be two centres of negative charge and a p o s i t i v e centre but with charge separation d i f f e r e n t from that of glutamic acid or aspartic acid.  0  _ II 0-C-CH2-CH2-CH-C00~  glutamic acid  NH 3 + 0  _ II 0-C-CH2-CH-C00~  a s p a r t i c acid  NH 3 + 0  _ II  0  0-C-CH2-CH2-C-06-(jj-06-  — —  _ II  0-C-CH2-CH2-C=C -C=0 0-C-CH2-CH 2  N,N-dialkylsuccinamic acid  L i k e PTZ, the two s u c c i n a m i c a c i d s a r e w a t e r - s o l u b l e and lipid-soluble.  I t has been pointed out that an optimal balance of  water and l i p i d s o l u b i l i t y may a l s o be important i n the development 188  of convulsant a c t i v i t y due to d i s t r i b u t i o n a l l o c a l i z a t i o n of compounds i n the CNS (49).  Succinamic  acids and glutaramic a c i d are  the open-chain forms of the anticonvulsant succinimides and g l u t arimides, r e s p e c t i v e l y .  They are reported t o be formed i n minor  amounts from i n vivo transformation of the succinimides and g l u t arimides (49,98).  Published studies have indicated  the lack of  anticonvulsant a c t i v i t y of a s e r i e s of N-alkyl-2-phenylsuccinamic acids (203)  and s l i g h t  convulsant  activity  of  3,3-ethylmethyl-  g l u t a r a m i c a c i d , the open-chain form o f t h e c o n v u l s a n t bemegride d.  R  (49).  Model showing s e l e c t i v e e f f e c t s of a l i p h a t i c substituents at the hydrophobic binding s i t e  and  alicyclic  The r e s u l t s of x-ray or t h e o r e t i c a l a n a l y s i s on GABA s t r u c t u r e have indicated  the presence of two p r e f e r e n t i a l conformers, one  planar extended conformation, the other a non-planar folded conformation (Figure 18a).  Ferrandes et a l . (205) a l s o determined the  conformation of c r y s t a l l i n e amides of v a l p r o i c acid and pointed out that the molecular conformation of v a l p r o i c acid may overlap with the GABA conformers. adopt a planar  The d i p r o p y l branched-chain  extended conformation  was shown to  which i s symmetrical  respect to the amido group (Figure 18b).  with  Cohen-Addad (206) deter-  mined the molecular conformation of c r y s t a l l i n e amides of 3 - e t h y l pentanoic  acid and indicated that the 6,8-diethyl branched chain  was not oriented symmetrically with respect to the amido group as in  valproic  acid  (Figure 18c).  However, the conformation  of a  segment of 3-ethylpentanoic a c i d could be compared to that observed for valproic a c i d .  As shown i n Figure 18, the geometry of the 189  1*4-1  4.22A  -617A -5-58 A  GABA extended planar conformation  GABA folded conformation  (a)  Figure 18. Conformation of (a) GABA, (b) Valproic acid amide, and (c) 3-ethylpentanoic amide.  190  atoms [0^02,03,C"pC"2] of 3-ethylpentoic acid was shown to be equivalent to that of [ C p C 2 , C ^ . C ^ . C ' g ] from v a l p r o i c a c i d , and a l s o the folded conformation of GABA. However, the e t h y l groups i n 3-ethylpentanoic are not stereochemically i d e n t i c a l .  I n a previous  s i n g l e dose study ( 5 ) , 3-ethylpentanoic acid was found t o be i n active.  This r e s u l t i s contrary t o the present study which has  shown i t s e f f e c t i v e n e s s i n antagonizing PTZ-induced seizures (Table 31). From the conformations  shown i n Figure 18, i t i s l i k e l y that  the more s t a b l e conformations this  study  will  indistinguishable GABA.  of the anticonvulsant compounds i n  have s e g m e n t s o f t h e a l k y l from  the folded or extended  chain  almost  conformations  of  The f l e x i b l e normal a l i p h a t i c s i d e c h a i n i n 5 - h e p t y l -  tetrazole  would  superimposes  likely  with  adopt  an e q u i l i b r i u m  the extended  conformation  GABA conformation.  that  Convulsant  t e t r a z o l e s have been reported t o i n t e r a c t at the p i c r o t o x i n s i t e of the GABA receptor complex l i k e v a l p r o i c acid ( 7 ) .  Kraus (24) has  also reported that v a l p r o i c a c i d and i t s t e t r a z o l e analogue, 4tetrazolylheptane i n h i b i t e d  succinic  an enzyme i n the GABA metabolism  semi-aldehyde dehydrogenase,  shunt, with i n h i b i t o r y constants  of 0.7 mM and 0.75 mM r e s p e c t i v e l y .  The conformation of the amide  of the highly a c t i v e d i b u t y l a c e t i c acid was a l s o reported t o be similar oriented  to that of v a l p r o i c  with  the d i b u t y l a l k y l  symmetrically to the amide group (206).  conformations  of t h e a l k y l  compounds may overlap conformation. and  acid  with  chain either  chain  S i m i l a r l y the  i n the unsaturated  active  the extended or folded GABA  The a l k y l c o n f i g u r a t i o n s i n 2,2-dimethylbutyric a c i d  trimethylacetic  acid  would  191  likely  overlap  with  e i t h e r the  extended or the folded GABA conformation. In the a c t i v e a l i c y c l i c - s u b s t i t u t e d compound, 1-methylcyclohexanecarboxylic  a c i d , the s e m i - r i g i d cyclohexyl chain conformation  (Figure 19) would  likely  conform t o the GABA extended or folded  conformation with overlap of the atoms [C^.C^.C^] i n Figure 18a. The cyclohexylmethyl group i n the i n a c t i v e compounds, 5-cyclohexylmethyltetrazole and c y c l o h e x y l a c e t i c a c i d , appears to have segments which overlap  with  atoms [C2, C3, C^] of the GABA extended or  folded conformation (Figure 18a). methylcyclohexanecarboxylic  However, i n comparison with 1-  a c i d , the presence of the methylene  group between the polar f u n c t i o n a l group and the a l i c y c l i c r i n g may cause the terminal portion of the cyclohexyl r i n g t o appear i n the p o s i t i o n occupied by the nitrogen group of GABA. Thus, u n l i k e the 3,8-diethyl  chain  i n 3-ethylpentanoic  methylene groups connecting C2" and unexpected i n a c t i v i t y  a c i d , the presence of two (Figure 18c) may explain the  of c y c l o h e x y l a c e t i c a c i d and 5-cyclohexyl-  raethyltetrazole. The  structural  model (Figure  19) proposed  f o r the pharma-  c o p h o r e features i n v a l p r o i c analogues i s based on the i n a c t i v i t y and anticonvulsant a c t i v i t y of the c a r b o x y l i c acids and t e t r a z o l e s studied.  Both a l k y l s u b s t i t u t e d c a r b o x y l i c acids and t e t r a z o l e s  were included possible zoles  i n the pharmacophoric model because of reports of  similar  binding  sites  of v a l p r o i c a c i d  and t e t r a -  (33,34,55,57,104). Although  t h e e v i d e n c e t h a t v a l p r o i c a c i d and c o n v u l s a n t  t e t r a z o l e s enhance or diminish GABA-mediated postsynaptic  192  inhib-  \  Position of N in GABA conformations C  Q  C"  \  C  "' ~ - C — / C \  Q electron-donor r0U s 9 P  \ OH hydrogenbonding groups  /  w  I  electron-donor groups  H  hydrogenbonding groups Zone f o r alpha alkyl groups  Steric Effect  C ,  IK  h. H  Cyclohexylacetic acid  5-Cyclohexylmethyltetrazole  CH. 1-Methylcyclohexanecarboxylic acid  Pentylenetetrazole (PTZ)  Figure 19. Model of pharmacophoric structural features in carboxylic acids and t e t r a z o l e s .  193  ition  may  n o t be s t r o n g  or complete  (compared  t o 1,4-  benzodiazepines and b a r b i t u r a t e s ) , there has been a growing body of evidence implying i n h i b i t i o n of GABA metabolism by v a l p r o i c acid a t synaptic s i t e s ( 2 ) .  Valproic acid may be a p o t e n t i a l substrate of  GABA-transaminase or s u c c i n i c semialdehyde dehydrogenase. V a l p r o i c acid and i t s t e t r a z o l e analogue, 4-tetrazolylheptane were shown t o i n h i b i t s u c c i n i c semialdehyde dehydrogenase (24). In the s t r u c t u r a l model (Figure 19), the a l k y l chain probably binds to a hydrophobic s i t e on the GABA recognition s i t e .  This  hydrophobic s i t e i s probably between binding s i t e s f o r the nitrogen and c a r b o x y l i c groups i n GABA. The p o s s i b i l i t y that GABA e x i s t s i n e i t h e r the extended or folded conformation has been considered i n the proposed s t r u c t u r a l model. The lack of anticonvulsant a c t i v i t y of  cyclohexylacetic acid  suggested  and 5 - c y c l o h e x y l r a e t h y l t e t r a z o l e i s  to be due to s t e r i c  e f f e c t s at the hydrophobic s i t e ,  close to the GABA nitrogen binding s i t e on the GABA-receptor complex.  The zone of a l k y l  groups a t t h e a - p o s i t i o n i n f l u e n c e s  anticonvulsant a c t i v i t y as shown by the a c t i v i t y of a-cyclohexylpentanoic acid which was, however, reported to be l e s s than that of VPA  (5).  Thus i f  metabolism e f f e c t s on the cyclohexylmethyl-  substituted and cyclohexylsubstituted compounds are s i m i l a r ,  then  the difference i n a c t i v i t y i s suggested to be due to conformational e f f e c t s at the s i t e of a c t i o n .  The  polar moiety plays a s i g n i f i c a n t  anticonvulsant  activity  role' i n determining the  of compounds as shown by the presence o f  the dipole moment term i n the QSAR.  I t i s considered that favour-  able o r i e n t a t i o n of the electron-donor 194  or hydrogen-bonding groups  i n the c a r b o x y l i c and t e t r a z o l e m o i e t i e s , i n a d d i t i o n t o hydrophobic binding of a l k y l chain, i n f l u e n c e a n t i c o n v u l s a n t  activity.  Valproic  protein,  acid  albumin (112).  i s strongly  bound  t o t h e serum  A t h e o r e t i c a l evaluation of the i n t e r a c t i o n between  v a l p r o i c a c i d and human serum albumin has been made by Andrews et a l . (207).  V a l p r o i c a c i d was i n d i c a t e d t o meet the g e o m e t r i c  requirements of the s p e c i f i c binding s i t e s and the c a r b o x y l i c group was i m p l i c a t e d i n the high strength of the non-covalent bonding. The presence o f p o l a r f u n c t i o n s i n the a l k y l c h a i n o f the succinamic acids may imply d i f f e r e n t d i p o l e - d i p o l e or dipole-charge i n t e r a c t i o n s compared with that of v a l p r o i c a c i d . I t i s l i k e l y that the c o n v u l s a n t or s t i m u l a t o r y s u c c i n a m i c a c i d s i n t e r a c t a t a d i f f e r e n t s i t e from that of a l k y l s u b s t i t u t e d c a r b o x y l i c acids and tetrazoles.  The i n vivo antagonism between v a l p r o i c a c i d and N,N-  dibutylsuccinamic a c i d was, however, not i n v e s t i g a t e d . The s t r u c t u r e - a c t i v i t y c o r r e l a t i o n s i n t h i s study i m p l i c a t e both the c a r b o x y l i c and t e t r a z o l e analogues of v a l p r o i c a c i d i n the s t r u c t u r a l pharmacophore model shown i n Figure 19.  X-ray a n a l y s i s  of c r y s t a l s of p e n t y l e n e t e t r a z o l e - i o d i n e monochloride complex by Baenziger et a l . (208) i n d i c a t e d that the t e t r a z o l e r i n g i s planar with the C^-Cy-Cg-Cg-Cio a l i c y c l i c r i n g i n the c h a i r conformation (Figure 19). The electron-donor  group, bound to the iodine-mono-  c h l o r i d e was found t o be the n i t r o g e n atom at p o s i t i o n - 4 of t h e tetrazole r i n g . I t has been r e p o r t e d t h a t s u b s t i t u t i o n of a m e t h y l or i s o propyl group at C8 °f pentylenetetrazole increased the s t i m u l a t o r y a c t i v i t y while s u b s t i t u t i o n of s i m i l a r a l k y l groups at Cg and C^Q led to a decrease i n s t i m u l a t o r y a c t i v i t y (58,59). 195  I t i s conceiv-  able that s u b s t i t u t i o n of a l k y l groups i n the pentylene chain of PTZ  exerts stereochemical e f f e c t s s i m i l a r to that proposed for the  carboxylic  and  t e t r a z o l e analogues of v a l p r o i c acid (Figure  19).  However, these p o s s i b i l i t i e s can only be evaluated adequately when the x-ray structures of these compounds have been determined. i s also worth i n v e s t i g a t i n g i f the  It  p r e f e r e n t i a l conformations i n  s o l u t i o n phase correspond to those i n the s o l i d s t a t e . It  appears  that  further  structure-activity correlations  analogues of GABA, v a l p r o i c a c i d , p i c r o t o x i n and may  provide supporting evidence f o r the  structural  basis  This could lead  for  interactions  at  pentylenetetrazole  a c t i v e conformations  the  of  and  GABA-receptor complex.  to compounds with greater s p e c i f i c i t y i n t h e i r  anticonvulsant a c t i o n s .  196  SUMMARY AND CONCLUSIONS  A wide range of s t r u c t u r a l l y - r e l a t e d analogues of v a l p r o i c a c i d have been synthesized  and examined f o r anticonvulsant a c t i v i t y .  The  s e r i e s o f analogues i n c l u d e d compounds w i t h a - a l k y l s u b s t i t u e n t s , 6,6-dialkyl  substituents, B-alkyl substituents, Y-alkyl substituents,  a l i c y c l i c and a l i c y c l i c a l k y l groups, an unsaturated a l k y l chain and the t e t r a z o l e f u n c t i o n a l group as a b i o i s o s t e r i c function of the c a r b o x y l i c group. A  The compounds were synthesized using known procedures. s t e r e o s e l e c t i v e method  was used  to prepare  the diunsaturated  analogues of v a l p r o i c a c i d , 2-[(E)-l'-propenyl]-(E)-2-pentenoic a c i d and 2-[(Z)-l'-propenyl]-(E)-2-pentenoic the  deconjugative  acid.  The stereochemical course of  a l d o l - t y p e r e a c t i o n of e t h y l 2-pentenoate w i t h  propionaldehyde was i n v e s t i g a t e d using the (E) or (Z)-isomer of e t h y l 2pentenoate.  The a d d i t i o n r e a c t i o n was shown t o occur with high r e g i o -  s e l e c t i v i t y and deconjugation occurred with high s t e r e o s e l e c t i v i t y . The 6'-hydroxy-B',y'-unsaturated products were dehydrated with various dehydration agents.  The combination of methanesulfonyl c h l o r i d e and KH  resulted i n the minimum number of diunsaturated VPA e t h y l ester isomers. NMR s p e c t r o s c o p i c data f o r each d i e n o a t e  isomer was c h a r a c t e r i z e d  f o l l o w i n g p u r i f i c a t i o n of the product mixture by argentation TLC. diunsaturated  Using  synthesized  reference  a c i d s , the u n i d e n t i f i e d major d i -  unsaturated  VPA metabolite has been assigned the chemical s t r u c t u r e , 2-  [(E)-l'-propenyl]-(E)-2-pentenoic a c i d . L i p o p h i l i c and e l e c t r o n i c properties of t e s t compounds have been determined experimentally t o i d e n t i f y the molecular properties relevant to t h e i r anticonvulsant a c t i v i t y .  L i p o p h i l i c i t y was described by the  197  octanol-water octanol-water  partition  coefficient.  The  possibility  of  estimating  p a r t i t i o n c o e f f i c i e n t s (P) of compounds of diverse s t r u c -  tures by RP-HPLC was D i f f e r e n t compositions nitrile-phosphate  explored.  A 5ym  H y p e r s i l ODS  column was  of methanol-phosphate buffer (pH 3.5)  buffer were i n v e s t i g a t e d f o r t h e i r  used.  or aceto-  suitability  as  mobile phase to determine hydrophobic e f f e c t s . Analysis of the l i n e a r regressions of known log P values of r e f e r ence substances mobile  and  their  phases culminated  log  [capacity f a c t o r s ] f o r the  i n the  selection  of 70%  different  methanol-phosphate  buffer and 50% acetonitrile-phosphate buffer as the mobile phases to be used i n determining the HPLC log P values.  The two mobile phases showed  high c o r r e l a t i o n c o e f f i c i e n t s , good s e n s i t i v i t y to hydrophobic e f f e c t s and  eluted highly l i p o p h i l i c  compounds w i t h m i n i m i z a t i o n of  e q u i l i b r i u m adsorption phenomena.  non-  HPLC log P values, determined from  the regression equations, were found to be better than Hansch or Rekker predicted log P values i n determining accurate log P values f o r h i g h l y substituted a l i p h a t i c and a l i c y c l i c c a r b o x y l i c a c i d s , h e t e r o c y c l i c t e t razoles and intramolecularly-bonded compounds. The accuracy of the HPLC log P values was v e r i f i e d by determining the shake-flask log P values of four compounds of diverse s t r u c t u r e .  An HPLC method was described f o r  measuring compounds i n aqueous s o l u t i o n s .  Owing to i t s speed, high  r e p r o d u c i b i l i t y , and a b i l i t y to determine the log P values of unstable compounds, the RP-HPLC method complements and provides an a l t e r n a t i v e to the conventional shake-flask procedure. The  e l e c t r o n i c properties of the t e s t compounds were described by  the apparent i o n i z a t i o n constants.  The  i o n i z a t i o n strength was  mined with good p r e c i s i o n by a potentiometric t i t r a t i o n method. aqueous-methanol  solvents  were  used 198  because of  the  limited  deterMixed aqueous  solubility  of the compounds.  pKa Values i n 10% methanol-water were  found to be more meaningful than values i n 50% methanol-water.  The  i s d s t e r i s m between carboxylic acid and t e t r a z o l e was indicated by the pKa  v a l u e s of t e t r a z o l e s which were j u s t s l i g h t l y h i g h e r than t h e  corresponding carboxylic a c i d s . The standardized s.c. PTZ seizure threshold was used to e s t a b l i s h the anticonvulsant potency of t e s t compounds i n mice. Anti-PTZ a c t i v i t y of each compound was determined a t i . p . doses between 0.2 and 2.0 mmol/kg.  The r e l i a b i l i t y of the pharmacological t e s t i n g was shown by  the close agreement of the measured ED^Q of v a l p r o i c acid t o l i t e r a t u r e values.  The s.c. PTZ seizure t e s t was noted to be more s e l e c t i v e i n  d i s c r i m i n a t i o n of the anticonvulsant potency than the PTZ m o r t a l i t y t e s t used i n previous studies of some a l i p h a t i c a c i d s . the most potent compound t e s t e d . desirable  5-Heptyltetrazole was  However, v a l p r o i c acid had the most  pharmacological properties  i n terms of high anticonvulsant  a c t i v i t y and minimum q u a l i t a t i v e t o x i c e f f e c t s . QSAR studies  showed that the dependency of anticonvulsant potency  on a combination of l o g P and pKa was s t a t i s t i c a l l y s i g n i f i c a n t when 5h e p t y l t e t r a z o l e was deleted from the s e r i e s of a c t i v e compounds. The stated r e l a t i o n s h i p suggested that the i n vivo a c t i v i t y was governed by the proportion  and l i p o p h i l i c i t y of the unionized form f o r entry i n t o  the CNS. The f a i l u r e of l o g P and pKa to account f o r the v a r i a t i o n i n potency of the nine a c t i v e  compounds i n c l u d i n g  5-heptyltetrazole  was  a t t r i b u t e d t o lack of an e f f e c t i v e parameter to measure the e l e c t r o n i c properties acts  of the polar f u n c t i o n a l group which e l e c t r o s t a t i c a l l y i n t e r -  with the recognition  sitei  A comparison between pKa and dipole moment i n describing the e l e c -  199  tron  properties  was undertaken using highly  active  alkyl-substituted  compounds from d i f f e r e n t classes of anticonvulsant drugs. substituted  compounds have been suggested by various workers t o have a  common mechanism of a c t i o n . ED^Q  These a l k y l  The physicochemical properties and anti-PTZ  values were obtained from l i t e r a t u r e sources.  Regression a n a l y s i s  of the QSAR showed that for the d i f f e r e n t classes of a l k y l - s u b s t i t u t e d compounds, the dependence of a c t i v i t y on l o g P and u terms was s t a t i s t i c a l l y s i g n i f i c a n t compared to dependence of a c t i v i t y on l o g P and pKa.  D i p o l e moment v a l u e s showed low c o v a r i a n c e w i t h pKa v a l u e s .  S i m i l a r f i n d i n g s of negative dependence of anticonvulsant a c t i v i t y on dipole  moment have  been  groups of anticonvulsants.  reported  f o r 1,4-benzodiazepines and other  Further studies may be required f o r e l u c i d -  a t i o n of the negative dependence of a c t i v i t y on dipole moment. A major f i n d i n g of the s t r u c t u r e - a c t i v i t y studies was the unique structural  property of the cyclohexylmethyl configuration  reducing the anticonvulsant a c t i v i t y cyclohexylmethyltetrazole. s i t e of a c t i o n activity  of cyclohexylacetic  i n greatly acid  and 5-  A model of the s t r u c t u r a l requirement at the  has been proposed to account f o r the a c t i v i t y and i n -  of v a l p r o i c  acid analogues.  T h i s model i s based on t h e  reported X-ray structure of GABA, c r y s t a l l i n e amides of v a l p r o i c a c i d , 3-ethylpentanoic a c i d , d i b u t y l a c e t i c a c i d , pentylenetetrazole and the h y p o t h e s i z e d mechanism o f a c t i o n tetrazole. alicyclic  of v a l p r o i c acid  The modulatory influence substituents  and p e n t y l e n e -  of the d i f f e r e n t a l i p h a t i c and  probably r e s u l t s from binding t o a hydrophobic  s i t e on t h e GABA-receptor complex or t h e GABA m e t a b o l i z i n g enzyme system.  The cyclohexylmethyl conformation was suggested t o be l e s s  e f f e c t i v e i n hydrophobic bonding due to i t s s t e r i c e f f e c t at a stereos e l e c t i v e p o s i t i o n on the hydrophobic recognition 200  site.  The  s i g n i f i c a n c e of t h i s research l i e s i n the d e l i n e a t i o n of the  dependence of anticonvulsant a c t i v i t y on the physicochemical p r o p e r t i e s : lipophilicity, steric  apparent acid  factors.  ionization  constant,  dipole moment and  While the proportion and l i p o p h i l i c i t y of unionized  species governed t h e i r access to CNS s i t e s of a c t i o n , the dependence of a c t i v i t y on dipole moment may e x p l a i n the diverse s t r u c t u r e s of a n t i convulsants.  There appears to be a p r o v i s i o n that s t e r i c requirements  of the hydrophobic binding s i t e are accommodated i n a c t i v e compounds. Steric  effects,  substituted  as suggested  t o be present  compounds, may lead to i n a c t i v i t y  i n cyclohexylmethylor probably  convulsant  properties as i n b a r b i t u r a t e s with an i s o p r o p y l terminus or benzyl substituents. A survey of the a v a i l a b l e l i t e r a t u r e data i n d i c a t e d that there has been no report of the high potency of a 5 - a l k y l t e t r a z o l e (e.g. 5-heptylt e t r a z o l e ) i n antagonizing the convulsant e f f e c t s of a c l o s e l y - r e l a t e d analogue,  pentylenetetrazole.  These  f i n d i n g s have  pharmacological  s i g n i f i c a n c e i n e l u c i d a t i n g the nature of the a c t i v e s i t e of v a l p r o i c acid and i t s analogues.  I t can be postulated from the r e s u l t s of t h i s  s t r u c t u r e - a c t i v i t y study that the a c t i v i t y of v a l p r o i c a c i d , 5-heptylt e t r a z o l e , c y c l o h e x y l a c e t i c a c i d , 5-cyclohexylmethyltetrazole, p e n t y l e n e t e t r a z o l e and the other  t e t r a z o l e and c a r b o x y l i c a c i d s may be  governed by common s t r u c t u r a l requirements at a s i m i l a r s i t e of a c t i o n . This  study  a l s o reports the stereochemical  course of a stereo-  s e l e c t i v e synthesis of 2-[(Z)-l'-propenyl]-(E)-2-pentenoic a c i d and 2[(E)-l'-propenyl]-(E)-2-pentenoic metabolite properties  acid.  The major  of v a l p r o i c a i d was i d e n t i f i e d . of the diunsaturated  diunsaturated  Studies of the t o x i c  analogues of v a l p r o i c a c i d could be 201  undertaken with due regard to t h e i r importance as metabolites of v a l proic a c i d . The  i n vivo  limitations  of  s t r u c t u r e - a c t i v i t y studies  whole  animal  studies,  are u s e f u l , despite the  for establishing  correlations  between i n vivo and i n v i t r o actions of v a l p r o i c a c i d analogues.  202  REFERENCES 1.  H. M e u n i e r , G. C a r r a z , Y. M e u n i e r , P. Eymard and M. A i m a r d . P r o p r i e t e s p h a r m a c o d y n a m i q u e de l ' a c i d e d i p r o p y l a c e t i q u e . T h e r a p i e , 18, 435 (1963).  2.  A. Chapman, P.E. K e a n e , B.S. M e l d r u m , J . S i m i a n d and J.C. Vernieres. Mechanism of a n t i c o n v u l s a n t a c t i o n of v a l p r o a t e . P r o g . N e u r o b i o l . , 19, 315 (1982).  3.  J . L. Benoit-Guyod, A. B o u c h e r l e , M. Benoit-Guyod, R. Rupp, P. Eymard, G. C a r r a z , M. B o i t a r d , S. L e b r e t o n , H. B e r i e l and H. M e u n i e r . Derives de l ' a c i d e d i p r o p y l a c e t i q u e . I I I . Nouveaux amides e t e s t e r s . Eur. J . Med. Chem., _3» 336 (1968).  4.  G. C a r r a z , A. B o u c h e r l e . S. L e b r e t o n , J.L. Benoit-Guyod and M. B o i t a r d . Le n e u r o t r o p i s m e de l a s t r u c t u r e d i p r o p y l a c e t i q u e . Therapie 19, 917 (1964).  5.  G. T a i l l a n d i e r , J.L. Benoit-Guyod, A. B o u c h e r l e , M. B r o i l and P. Eymard. Recherches dans l a s e r i e d i p r o p y l a c e t i q u e X I I . A c i d e t a l c o o l a l i p h a t i q u e s r a m i f i e s anticonvulsants. Eur. J . Med. Chem., jLO, 453 (1975).  6.  G. C a r r a z and N. Emin. A c t i o n a n t i c o n v u l s a n t e du monooureide de l ' a c i d e d i p r o p y l a c e t i q u e e t du d e r i v e h y d a n o i n i q u e de c e t a c i d e . T h e r a p i e , 22, 641 (1967).  7.  F. De M a r c h i and M. V. T o r r i e l l i . Synthese e t p r o p r i e t e s a n t i convulsantes de l a chloro-7 dihydro-1,3 d i p r o p y l a c e t o x y - 3 phenyl5(H) benzodiazepine -1,4 one-2. Eur. J . Med. Chem., 3.. 430 (1968).  8.  M. B e n o l i t - G u y o d , J.L. Benoit-Guyod, A. B o u c h e r l e , M. B r o i l and P. Eymard. Recherches dans l a s e r i e d i p r o p y l a c e t i q u e . V I I I . Structures homologues: amides et urfes de l a propyl-2 pentylamine. Eur. J . Med. Chem., 7, 393 (1972).  9.  M. Benoit-Guyod, J.L. Benoit-Guyod, M. B r o i l , A. B o u c h e r l i e , J.P. Werbenec and P. Eymard. Recherches dans l a s e r i e d i p r o p y l a c e t i q u e . X. Cetones et a l c o o l s e c o n d a r i e s a c h a i n e p r o p y l - 1 b u t y l e . A c t i o n s u r l e systeme nerveux c e n t r a l . E u r . J . Med. Chem., 8, 419 (1973).  10.  G. Carraz. Approaches pour une theorie sur l ' a c t i v i t e de l a s t r u c ture dipropylacetique. Agressologie, j5, 13 (1967).  11.  M. F. B r a n a , M. M a r t i n e z , J . G a r r i d o and C M . R o l d a n , S i n t e s i s y a c t i v i d a d f a r m a c o l o g i c a de homogos c i c l i c o s d e l a c i d o d i p r o p i l a c e t i c o . An. Quim., 79, 47 (1983).  12.  A.G. Chapman, B. S. Meldrum and E. Mendes. Acute a n t i c o n v u l s a n t a c t i v i t y of s t r u c t u r a l analogues o f v a l p r o i c a c i d and changes i n brain GABA and aspartate content. L i f e S c i . , 32, 2023 (1983). 203  13.  P.E. Keane, J . S i m i a n d , E. Mendes, V. S a n t u c c i and M. M o r r e . The e f f e c t of analogues of v a l p r o i c acid on pentylenetetrazole-induced s e i z u r e s and b r a i n GABA c o n t e n t i n m i c e . Neuropharmacology, 22, 875 (1983).  14.  B.J. Perlman and D.B. Goldstein. Membrane disordering potency and anticonvulsant a c t i o n of v a l p r o i c acid and other short-chain f a t t y a c i d s . M o l . Pharmacol., 26, 83 (1984).  15.  P.R. Andrews. M o l e c u l a r o r b i t a l c a l c u l a t i o n s on a n t i c o n v u l s a n t drugs. J . Med. Chem., 12, 761 (1969).  16.  T.J. Putnam and H.H. M e r r i t t . C h e m i s t r y of a n t i c o n v u l s a n t drugs. A r c h . N e u r o l . P s y c h i a t . , 43, 505 ( 1940).  17.  T.B. P a t r i c k and R.R. Bresee. E n t h a l p i e s of hydrogen bonding i n p s c y h o t r o p i c drugs. J . Pharm. S c i . , 65, 1066 (1976).  18.  A. Camerman and N. Camerman i n " A n t i e p i l e p t i c Drugs: Mechanism of A c t i o n " , G.H. G l a s e r , J.K. Penry and D.M. Woodbury, Eds., Raven P r e s s , New York, p.223 (1980).  19.  P.R. Andrews, G.P. Jones and D. Lodge. C o n v u l s a n t , a n t i c o n,v u l s a n t and a n e s t h e t i c b a r b i t u r a t e s . 5 - E t h y l - 5 - ( 3 ' - m e t h y l b u t - 2 - e n y l ) b a r b i t u r i c a c i d and r e l a t e d compounds. Eur. J . Pharmacol., 55, 115 (1979) .  20.  G.L. Jones and D.M. Woodbury. A n t i c o n v u l s a n t S t r u c t u r e - A c t i v i t y R e l a t i o n s h i p s . H i s t o r i c a l Development and P r o b a b l e Causes of F a i l u r e . Drug. Dev. Res., 2, 333 (1982).  21.  R.M. H e r b s t . T e t r a z o l e s as c a r b o x y l i c a c i d a n a l o g s . Biochemistry". J . Wiley, New York, p.141 (1956).  22.  P.F. Juby, T.W. Hudyma and M. Brown. P r e p a r a t i o n and a n t i inflammatory properties of some 5-(2-Anilinophenyl)tetrazoles. J . Med. Chem., 1_1, 111 (1968).  23.  H. S i n g h , A.S. Chawla, V.K. Kapoor, D. P a u l and R.K. Mahhotru. M e d i c i n a l c h e m i s t r y of t e t r a z o l e s . P r o g r . i n Med. Chem., 17, 151 (1980) .  24.  J.L. Kraus. Isosterism and molecular m o d i f i c a t i o n i n drug design. New dipropylacetate analogs as i n h i b i t o r s of s u c c i n i c semialdehyde dehydrogenase. Pharmacol. Res. Commun., 15, 119 (1983).  25.  C. Hansch and T. F u j i t a . A method f o r the c o r r e l a t i o n of b i o l o g i c a l a c t i v i t y and c h e m i c a l s t r u c t u r e . J . Amer. Chem. S o c , 86, 1616 (1964).  26.  E.J. L i e n , R.C.H. L i a o and H.G. Shinouda. Q u a n t i t a t i v e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s and d i p o l e moments of a n t i c o n v u l s a n t s and CNS d e p r e s s a n t s . J . Pharm. S c i . , 68, 463 (1979).  204  "Essays i n  27.  T. B l a i r and G.A. Webb. E l e c t r o n i c f a c t o r s i n the s t r u c t u r e a c t i v i t y r e l a t i o n s h i p o f some l,4-benzodiazepine-2-ones. J . Med. Chem., 20, 1206 (1977).  28.  E.J. L i e n , G.L. Tong, J.T. Chou and L.L. L i e n . S t r u c t u r e r e q u i r e ments f o r c e n t r a l l y acting drugs. J . Pharm. S c i . , 62, 246 (1973).  29.  C. Hansch and J.M. C l a y t o n . L i p o p h i l i c c h a r a c t e r and b i o l o g i c a l a c t i v i t y o f drugs. The p a r a b o l i c c a s e . J . Pharm. S c i . , 62, 1 (1973).  30.  R.W. Olsen. GABA-Benzodiazepine-Barbiturate receptor i n t e r a c t i o n s . J . Neurochem., 37, 1 (1981).  31.  R.L. Macdonald and J.L. Barker. Enhancement of GABA-mediated postsynaptic i n h i b i t i o n i n cultured s p i n a l cord neurons. A common mode of anticonvulsant a c t i o n . B r a i n Res., 167, 323 (1979).  32.  B.A. Weisraan, T.R. B u r k e r , K.C. R i c e and 3P. S k o l n i c k . A l k y l - s u b s t i t u t e d gamma-butyrolactones i n h i b i t [ ^S]-TBPS binding t o GABA l i n k e d c h l o r i d e ionophore. Eur. J . Pharmacol., 105, 195 (1984).  33.  M.K T i c k u and R.W. O l s e n . I n t e r a c t i o n o f b a r b i t u r a t e s w i t h d i h y d r o p i c r o t o x i n i n b i n d i n g s i t e s r e l a t e d t o t h e GABA-receptor ionophore system. L i f e Sci., 22, 1643 (1978).  34.  M.K T i c k u and W.C. D a v i s . E f f e c t o f v a l p r o i c a c i d on ^H-diazepam and 3 H -d i h y d r o p i c r o t o x i n i n binding s i t e s a t the benzodiazepine-GABA receptor-ionophore complex. B r a i n Res., 223, 218 (1981).  35.  D. Simon and J.K. Penry. Sodium d i p r o p y l a c e t a t e i n the t r e a t m e n t of epilepsy. A review. E p i l e p s i a , 16, 549 (1975).  36.  R.M. P i n d e r , R.N. Brogden, T.M. S p e i g h t and G.S. Avery. Sodium valproate: A review of i t s p h a r m a c o l o g i c a l p r o p e r t i e s and t h e r a peutic e f f i c a c y i n epilepsy. Drugs, 13, 81 (1977).  37.  S. S a t o , B.G. W h i t e , J.K. Penry, F.E. D r e i f u s s , J.C. S a c k e l l a r e s and H.J. K u p f e r b e r g . V a l p r o i c a c i d v e r s u s e t h o s u x i m i d e i n t h e treatment of absence seizures. Neurology, 32, 157 (1983).  38.  R.L. K r a l l , J.K. P e n r y , B.G. W h i t e , H.J. K u p f e r b e r g and E.A. Swinyard. A n t i e p i l e p t i c drug development. I I . Anticonvulsant drug screening. E p i l e p s i a , 19, 409 (1978).  39.  H.H. Frey and W. L o s c h e r . D i p r o p y l a c e t i c a c i d - p r o f i l e o f a n t i convulsant a c t i v i t y i n mice. Arzneim. Forsch., 26, 299 (1976).  40.  A.K. Shenoy, J.T. M i y a h a r a , E.A. Swinyard and H.J. K u p f e r b e r g . Comparative anticonvulsant a c t i v i t y and n e u r o t o x i c i t y of clobazam, diazepam, phenobarbital and valproate i n mice and r a t s . E p i l e p s i a , 23, 399 (1982).  205  41.  E.A. S w i n y a r d , W.C. Brown and L.S. Goodman. Comparative assays of a n t i e p i l e p t i c drugs i n mice and r a t s . J . P h a r m a c o l . Exp. Ther., 106, 319 (1952).  42.  H.H. M e r r i t t and T.J. Putnam. A new s e r i e s of anticonvulsant drugs t e s t e d by e x p e r i m e n t s on a n i m a l s . A r c h . N e u r o l . P s y c h i a t . , 39, 1003 (1938).  43.  J.E.P. Toman, E.A. S w i n y a r d and L.S. Goodman. P r o p e r t i e s of maxi m a l s e i z u r e s and t h e i r a l t e r a t i o n by a n t i c o n v u l s a n t drugs and o t h e r a g e n t s . J . N e u r o p h y s i o l . , 9, 231 (1946).  44.  G.M. Everett and R.K. Richards. Comparative a n t i c o n v u l s i v e a c t i o n of 3 , 5 , 5 - t r i m e t h y l o x a z o l i d i n e - 2 , 4 - d i o n e ( T r i d i o n e ) , d i l a n t i n and p h e n o b a r b i t a l . J . P h a r m a c o l . Exp. Ther., 81, 402 (1944).  45.  M.J. O r l o f f , H.L. W i l l i a m s and C.C. P f e i f f e r . Timed i n t r a v e n o u s i n f u s i o n of m e t r a z o l and s t r y c h n i n e f o r t e s t i n g a n t i c o n v u l s a n t drugs. P r o c . Soc. Exp. B i o l . Med., 70, 254 (1949).  46.  E.A. S w i n y a r d and J.H. Woodhead i n " A n t i e p i l e p t i c D r u g s " , D.M. Woodbury, J.K. Penry and C.E. P i p p e n g e r , Eds., Raven P r e s s , New Y o r k , p . l l l (1982).  47.  W.E. K l u n k , D.F. Corey and J.A. F e r r e n d e l l i . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p of a l k y l substituted gamma-butyrolactones andsuccinimides. Mol. Pharmacol., 22, 444 (1982).  48.  R. Testa, L. G r a z i a n i and G. G r a z i a n i . Do d i f f e r e n t anticonvulsant t e s t s provide the same i n f o r m a t i o n concerning the p r o f i l e of a n t i e p i l e p t i c a c t i v i t y ? Pharmacol. Res. Commun., 15, 765 (1983).  49.  F. Hahn. A n a l e p t i c s . Pharmacol. Rev., 12, 447  50.  D.M. Woodbury i n " A n t i e p i l e p t i c Drugs: Mechanism of A c t i o n " . G.H. G l a s e r , J.K. Penry and D.M. Woodbury, Eds., Raven P r e s s , New Y o r k , p.249 (1980).  51.  R.J. D a v i d , W.A. W i l s o n and A.V. E s c u e t a . V o l t a g e clamp a n a l y s i s of pentylenetetrazole e f f e c t s on A p l y s i a neurons. Brain Res., 67, 549 (1974).  52.  M. K l e e n , D. Faber and W. H e i s s . S t r y c h n i n e and 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 changes of e x c i t a b i l i t y i n A p l y s i a Neurons. Science, 179, 1133 (1973).  53.  R.LJ^acdonald and J.L. B a r k e r . P e n i c i l l i n and p e n t y l e n e t e t r a z o l e s e l e c t i v e l y antagonize GABA-mediated p o s t s y n a p t i c i n h i b i t i o n of cultured mammalian neurones. Neurology, 27, 337 (1979).  54.  T.C. P e l l m a n and W.A. W i l s o n . S y n a p t i c mechanism of p e n t y l e n e t e t r a z o l e : S e l e c t i v i t y f o r c h l o r i d e conductance. S c i e n c e , 197, 912 (1977).  206  (1960).  55.  W. Loscher and H.H. Frey. E f f e c t of convulsant and anticonvulsant agents on l e v e l and metabolism of gamma-aminobutyric a c i d i n mouse b r a i n . N.S. A r c h . Pharmacol., 196, 263 (1977).  56.  R.F. S q u i r e s , E. Saederup, S.N. C r a w l e y , P. S k o l n i c k and S.M. P a u l . Convulsant p o t e n c i e s o f t e t r a z o l e s are h i g h l y c o r r e l a t e d w i t h a c t i o n on G A B A / B e n z o d i a z e p i n e / P i c r o t o x i n r e c e p t o r complexes i n b r a i n . L i f e S c i . , 35, 1439 (1984).  57.  W.E. Stone. Convulsant a c t i o n of t e t r a z o l e d e r i v a t i v e s . c o l o g y , 3, 367 (1970).  58.  E.G. Gross and R.M. F e a t h e r s t o n e . S t u d i e s w i t h t e t r a z o l e d e r i v a t i v e s . I . Some pharmacologic properties of a l i p h a t i c substituted pentaraethylene t e t r a z o l e d e r i v a t i v e s . J . P h a r m a c o l . Exp. Ther., 87, 291 (1946).  59.  E.G. Gross and R.M. Featherstone. V. Some pharmacologic properties of aminophenyl t e t r a z o l e s . J . Pharmacol. Exp. Ther., 92, 330 (1948).  60.  I . Ahmad and J.S. S h u n k l a . S t u d i e s on the n e u r o p h a r m a c o l o g i c a l a c t i o n of some newer substituted t e t r a z o l e s and quinazolones. Ind. J . P h y s i o l . P h a r m a c o l . 26, 289 (1982).  61.  H.W. Vohland and W. Koransky. E f f e c t of a-hexachlorocyclohexane on m e t a b o l i s m and e x c r e t i o n of p e n t y l e n e t e t r a z o l e i n the r a t . N.S. A r c h . Pharmacol., 273, 99 (1972).  62.  D.W. E s p l i n and D.M. Woodbury. The f a t e and e x c r e t i o n of C ^ labeled pentylenetetrazole i n the r a t , w i t h comments on a n a l y t i c a l methods f o r pentylenetetrazole. J . Pharmacol. Exp. Ther. 118, 129 (1956).  63.  J . J . B r i n k , H. C a r i g l i a , D.G. S t e i n and L.A. G a l i p e a u . Uptake of [l^C]pentylenetetrazole by d e v e l o p i n g r a t b r a i n . B r a i n Res., 19, 445 (1970).  64.  S.G. R o w l e s , G.S. B o r n , H.T. R u s s e l l , W.V. K e s l l e r and J.E. C h r i s t i a n . B i o l o g i c a l d i s p o s i t i o n of p e n t y l e n e t e t r a z o l e - 1 0 C i n r a t s and humans. J . Pharm. S c i . , 60, 725 (1971).  65.  J.T. Stewart and J.L. Story. In v i t r o binding of pentylenetetrazol to plasma p r o t e i n s . J . Pharm. S c i . , 61, 652 (1972).  66.  R.S. Burton. On the propyl d e r i v a t i v e s and decomposition products of. e t h y l a c e t o a c e t a t e . Am. Chem. J . , 3, 383 (1982).  67.  Z.L. Chang. Sodium v a l p r o a t e and v a l p r o i c a c i d . In " A n a l y t i c a l p r o f i l e s of drug s u b s t a n c e s " , V o l . 8, K. F l o r e y , ed., Academic P r e s s , New Y o r k , p.529 (1979).  68.  M.A. T a y l o r and L.H. P r i n c e n . F a t t y a c i d s i n s o l u t i o n . In " F a t t y acids". American O i l Chemists Society, I l l i n o i s , p.195 (1979).  207  Pharma-  69.  W. Loscher and H.H. Frey. K i n e t i c s of penetration of common a n t i e p i l e p t i c drugs i n t o c e r e b r o s p i n a l f l u i d . E p i l e p s i a , 25, 346 (1984).  70.  W. Loscher and H. Esenwein. i n dog and mouse. A r z n e i m .  71.  D. J o h n s t o n . V a l p r o i c a c i d : Update on i t s mechanism o f a c t i o n . E p i l e p s i a , 25 ( S u p p l . l ) , SI (1984).  72.  R.W. Kerwin and P.V. Toberner Minireview: The mechanism of a c t i o n of sodium valproate. Gen. Pharmacol., 12,71 (1981).  73.  R. Haas, D.A. Stumpf, J.K. Parks and L. Eguren. I n h i b i t o r y e f f e c t s of sodium valproate on o x i d a t i v e phosphorylation. Neurology, 31, 1473 (1981).  74.  R.L. Macdonald and G.K. Bergey. V a l p r o i c a c i d augments GABAmediated postsynaptic i n h i b i t i o n i n c u l t u r e d mammalian neurons. B r a i n Res., 170, 558 (1979).  75.  Y. G o d i n , L. H e i n e r , J . Mark and P. Mandel. E f f e c t s o f d i p r o p y l a c e t a t e , an a n t i c o n v u l s a n t compound, on GABA m e t a b o l i s m . J . Neurochem., 16, 869 (1969).  76.  S. S i m l e r , L. G i e s i e l s k i , M. M a i t r e , H. R a n d r i a n a r i s o a and P. Mandel. E f f e c t s of sodium d i p r o p y l a c e t a t e on a u d i o g e n i c s e i z u r e s and b r a i n y - a m i n o b u t y r i c a c i d l e v e l s . Biochem. Pharmacol., 22, 1701 (1973).  77.  W. L o s c h e r . V a l p r o a t e , i n d u c e d changes i n GABA m e t a b o l i s m a t t h e s u b c e l l u l a r l e v e l . Biochem. Pharmacol., 30, 1364 (1981).  78.  A.S. A b d u l - G h a n i , J . C o n t i n h o - N e t t o , D. Druce and H.F. B r a d f o r d . E f f e c t s o f a n t i c o n v u l s a n t s on the i n v i v o and i n v i t r o r e l e a s e o f GABA. Biochem. Pharmacol., 30, 363 (1981).  79.  S.R. W h i t t l e and A.J. Turner. E f f e c t s of the anticonvulsant sodium valproate on aminobutyrate and aldehyde metabolism i n ox b r a i n . J . Neurochem., 31, 1453 (1978).  80.  J.W. Van Der Laan, T. De Boer and J . B r u i n v e l s . D i p r o p y l a c e t a t e and GABA degradation. P r e f e r e n t i a l i n h i b i t i o n o f s u c c i n i c s e m i aldehyde dehydrogenase and i n d i r e c t i n h i b i t i o n of GABA-transminase. J . Neurochem., 32, 1769 (1979).  81.  J.P. Gent and N.I. P h i l i p s . Sodium d i p r o p y l a c e t a t e ( v a l p r o a t e ) p o t e n t i a t e s responses t o GABA and muscimol on s i n g l e c e n t r a l neurons. B r a i n Res., 197 (1980).  82.  R.W. K e r w i n , H.R. Olpe and M. S c h u l t z . The e f f e c t o f sodium d i p r o p y l a c e t a t e on gamma-aminobutyric a c i d dependent i n h i b i t i o n i n the r a t cortex and substantia n i g r a i n r e l a t i o n t o i t s a n t i c o n v u l s ant a c t i v i t y . B r . J . Pharmacol., 71, 545 (1980).  Pharmacokinetics of sodium valproate F o r s c h „ 28, 782 (1978).  208  83.  N.L. Harrison and M.A. Simmonds. Sodium valproate enhances responses to GABA receptor a c t i v a t i o n only at high concentrations. Brain Res., 250, 201 (1982).  84.  W. Loscher. E f f e c t of i n h i b i t o r s of GABA transmission on the s y n t h e s i s , b i n d i n g , uptake and metabolism of GABA. J . Neurochem., 34, 1603 (1980).  85.  W.M. Burnhanu L. Spero, M.M. Okasaki and B.K. Madras. Saturable binding of H-phenytoin to r a t b r a i n membrane f r a c t i o n . Can. J . P h y s i o l . Pharmacol., 59, 402 (1981).  86.  P . J . Schecter, Y. Tranier and J . Grove. E f f e c t of dipropylacetate on amino a c i d concentrations i n mouse b r a i n : c o r r e l a t i o n s with anticonvulsant a c t i v i t y . J . Neurochem., 31, 1325 (1978).  87.  A. Lespagnol, J . Mercier, F. Erb-Deruyne and S. Dessaigne. Sur l e s proprietes neurosedatives et a n t i e p i l e p t i q u e s d'acides organiques a chaines r a m i f i e e s . Ann. Pharm. F r . , 30, 193 (1972).  88.  K.R. S c o t t , J.A. Moore, I.B. Lalusky, J.M. Nicholson, A.M. Lee and C.N. Hinko. Spiro [4,5] and Spiro [4,6] c a r b o x y l i c a c i d s : c y c l i c analogues of v a l p r o i c a c i d . Synthesis and anticonvulsant e v a l u a t i o n . J . Med. Chem., 28, 413 (1985).  89.  M.A. Spielman, A.0. G e i s z l e r and W.J. C l o s e . Anticonvulsant drugs. Some Acylureas. J . Amer. Chem. S o c , 70, 4189 (1948).  90.  F.M. Berger and B.J. Ludwig. The anticonvulsant a c t i o n of 2,2d i e t h y l 1,3-propanediol and some of i t s homologues and e s t e r s . J . Pharmacol., Exp. Ther., 99,27 (1951).  91.  H.F. Schwartz, L.F. W o r r e l l and J.N. Delgado. Synthesis of some d i s u b s t i t u t e d cyanoacetamides as p o t e n t i a l a n t i c o n v u l s a n t s . J. Pharm. S c i . , 56, 80 (1967).  92.  W. Loscher. Anticonvulsant a c t i v i t y of metabolites of a c i d . Arch. I n t . Pharmacodyn., 249, 58 (1981).  93.  A. Acheampong, F. Abbott and R. Burton. I d e n t i f i c a t i o n of v a l p r o i c acid metabolites i n human serum and urine using hexadeuterated v a l p r o i c acid and GCMS a n a l y s i s . Biomed. Mass Spectrom., 10, 586 (1983).  94.  J.A. Vida and E.H. Gerry i n "Anticonvulsants", Academic Press, York, p.151 (1977).  95.  M.A. Davis, S.0. Winthrop, R.A. Thomas, F. Herr, M. Charest and R.0. Gandry. Anticonvulsants. 11. Spiro compounds Dibenzo [a,d] cycloheptadiene-5,5'-hydantoins,-5,5'-oxazolidinediones and -5,2'succinimides. J . Med. Chem., 7, 439 (1964).  96.  L.H. Sternbach i n "The Benzodiazepines", S. G a n a t t i n i , E. Mussini and L.O. R a n d a l l , eds., Raven P r e s s , New York, p . l (1973). 209  valproic  New  97.  W.E. K l u n k , D.F. Corey and J.A. F e r r e n d e l l i . Comparison of epileptogenic properties of unsubstituted and B - a l k y l substituted gamma-butyrolactones. Mol. Pharmacol., 22, 431 (1982).  98.  T.C. Somers. New a n a l e p t i c s and hypnotics r e l a t e d to the b a r b i t urate antagonist, 'Bemigride'. Nature, 178, 996 (1956).  99.  P.R. Andrews and G.P. Jones. Convulsant and anticonvulsant b a r b i t u r a t e s . Molecular o r b i t a l c a l c u l a t i o n s . Eur. J . Med. Chem., 16, 139 (1981).  100. G.D. Davies, R.B. Belshee and W.R. Anderson. S o l u t i o n conformations of ethyl-1'-methylbutylbarbituric a c i d . Implications f o r drug receptor s i t e i n t e r a c t i o n s . Mol. Pharmacol., 11, 470 (1975). 101. P. Seeman. The membrane actions of anesthetics and Pharmacol. Rev., 24, 583 (1972).  tranquilizers.  102. R.L. Macdonald and M.J. McLean. C e l l u l a r basis of barbiturates and phenytoin anticonvulsant drug a c t i o n . E p i l e p s i a , 23 (Suppl. 1 ) , S7 (1983). 103. R.F. Squires and C. Braestrup. b r a i n . Nature, 266, 732 (1977).  Benzodiazepine receptors i n r a t 3  104. R.F. Squires, J.E. Casida, M. Richardson and E. Saederup. [ - % ] - t butylbicyclophosphorothionate binds with high a f f i n i t y to b r a i n s p e c i f i c s i t e s coupled to y-aminobutyric acid-A and i o n recognition s i t e s . Mol. Pharmacol., 23, 326 (1983). 105. J.A. F e r r e n d e l l i and S. Daniels-McQueen. Comparative actions of phenytoin and other anticonvulsant drugs on potassium and verat r i d i n e stimulated calcium uptake i n synaptosomes. J . Pharmacol. Exp. Ther., 220, 29 (1982). 106. R.J. DeLorenzo. Phenytoin: calcium- and calmodulin-dependent prot e i n p h o s p h o r y l a t i o n and n e u r o t r a n s m i t t e r r e l e a s e . In " A n t i e p i l e p t i c Drugs. Mechanism of A c t i o n " . G. H. G l a s e r , J.K. Penry and D.M. Woodbury, Raven P r e s s , New York, p.399 (1980). 107. R. T r i f u l e t t i , A.M. Snowman and S.H. Snyder. Barbiturate recogn i t i o n s i t e on the GABA/Benzodiazepine receptor complex i s d i s t i n c t from the picrotoxin/TBPS recognition s i t e . Eur. J . Pharmacol., 106, 44 (1984). 108. M.J. I a d a r o l a , R.F. F a n e l l i , J.0. McNamara and W.A. Wilson. Comparison of the e f f e c t s of d i p h e n y l b a r b i t u r i c a c i d , phenobarbital, pentobarbital and s e c o b a r b i t a l on GABA-mediated i n h i b i t i o n and benzodiazepine b i n d i n g . J . Pharmacol. Exp. Ther., 232, 127 (1984). 109. U. Kotz and R.H. Antonin. Pharmacokinetics and b i o a v a i l a b i l i t y of sodium v a l p r o a t e . C l i n . Pharmacol. Ther., 21, 736 (1977).  210  110. W. Loscher. Serum protein binding and pharmacokinetics of v a l proate i n man, dog, r a t and mouse. J . Pharmacol. Exp. Ther., 204, 255 (1978). 111. R. Gugler and G. M u e l l e r . Plasma protein binding of v a l p r o i c a c i d i n healthy subjects and i n patients with renal d i s e a s e . B r . J . C l i n . Pharmacol., 5, 441 (1978). 112. A. Kober, Y. Olsen and T. Sjoholm. Binding of drugs t o human serum albumin: XIV. The t h e o r e t i c a l basis f o r the i n t e r a c t i o n between phenytoin and v a l p r o a t e . Mol. Pharmacol., 18, 237 (1980). 113. A. Acheampong, F . S . A b b o t t , J . M . O r r , S.M. F e r g u s o n and R.W. Burton. Use of hexadeuterated v a l p r o i c a c i d and GCMS to determine the pharmacokinetics of v a l p r o i c a c i d . J . Pharm. S c i . , 73, 489 (1984). 114. T.A. Bowdle, I.H. P a t e l , R.H. Levy and H.J. Wilensky. Valproic acid dosage and plasma protein binding and clearance. C l i n . Pharma c o l . Ther., 28, 486 (1980). 115. H. Nau and W. Loscher. V a l p r o i c acid and metabolites: Pharmac o l o g i c a l and t o x i c o l o g i c a l s t u d i e s . E p i l e p s i a , 25 (Suppl. 1 ) , S14 (1984). 116. F. Schobben, E. Van der K l e i j n and T.B. Vree. Therapeutic monitoring of v a l p r o i c a c i d . Ther. Drug Monit., 2, 61 (1980). 117. E. Mesdjian, L . C i e s i e l s k i , M. V a l l i , B. B r u g u e r o l l e , G. Jadot, P. Bouyard and P. Mandel. Sodium valproate: k i n e t i c p r o f i l e and e f f e c t s on GABA l e v e l s i n various b r a i n areas of the r a t . Prog. Neuro-Psychopharmacol. B i o l . P s y c h i a t . , 6, 223 (1982). 118. M.A. Goldberg and T. Todoroff. Brain binding of anticonvulsants: carbamazepine and v a l p r o i c a c i d . Neurology, 30, 826 (1980). 119. M.L. A l y and A.A. A b d e l - L a t i f . Studies on d i s t r i b u t i o n and metabolism of valproate i n r a t b r a i n , l i v e r and kidney. Neurochem. Res., 5, 1231 (1980). 120.  H.H. Frey and W. Loscher. D i s t r i b u t i o n of valproate across the i n t e r f a c e between blood and cerebrospinal f l u i d . Neuropharmacol., 17, 637 (1978).  121. E.M Cornford. Blood-brain b a r r i e r permeability to anticonvulsant drugs. In "Metabolism o f A n t i e p i l e p t i c Drugs", R.H. L e v y , W.H. P i t l i c k , M. Eichelbaum and J . M e i j e r , eds., Raven P r e s s , New York, p.129 (1984). 122. R.J.E. Vadja, G.A. Donnan, J . P h i l l i p s and P.F. B l a d i n . Human b r a i n , plasma and cerebrospinal f l u i d concentrations of sodium v a l proate a f t e r 72 hours of therapy. Neurology, 31, 486 (1981). 123. R. Gugler and G.E. Von Unruh. C l i n i c a l pharmacokinetics of v a l proic a c i d . C l i n . Pharmacok., 4, 433 (1980). 211  124. W. Kochen, H.P. Sprunck, B. Tauscher and M. Klemens. Five doubly unsaturated metabolites of v a l p r o i c a c i d i n urine and plasma of patients on v a l p r o i c acid therapy. J . C l i n . Chem. C l i n . Biochem., 22, 309 (1984). 125. G.R. Granneraan, S . I . Wang, J.M. Machinist and J.W. Kesterson. Aspects of the metabolism of v a l p r o i c a c i d . X e n o b i o t i c a , 14, 375 (1984). 126. D.L. Coulter and R.J. A l l e n . P a n c r e a t i t i s associated with v a l p r o i c a c i d therapy f o r e p i l e p s y . Ann. Neurol., 7, 92 (1980). 127. J . Jaekens, P. Casear and L . C o r b e l l . Hyperammonemia and hyperglycinemia during sodium valproate therapy. Lancet 2, 260 (1980). 128. D.A. W i n f i e l d , P.B. Benton and M.L.E. E s p i r . Sodium Valproate and thrombocytopenia. B r . Med. J . , 2, 981 (1976). 129. L . J . Wilmore, B . J . Wilder, J . Bruni and H.J. V i l l a r e a l . E f f e c t of v a l p r o i c a c i d on hepatic f u n c t i o n . Neurology, 28, 961 (1978). 130. H.J. Zimmerman and K.G. Ishak. Valproate-induced hepatic i n j u r y : a n a l y s i s of 23 f a t a l cases. Hepatology, 2, 591 (1982). 131. N. Gerber, R.G. Dickinson, R.C. Harland, R.K. Lynn, D. Houghton, J . I . Antonias and J.C. Schimschock. Reye-like syndrome associated with v a l p r o i c acid therapy. J . P e d i a t r . , 95, 142 (1979). 132. A.W. R e t t e n m e i e r , K.S. P r i c k e t t , W.R. Gordon, S.M. B j o r g e , S.L. Chang, R.H. Levy and T.A. B a i l l i e . Studies on the biotransformation i n the perfused r a t l i v e r of 2-propyl-4-pentenoic a c i d , a metabolite of the a n t i e p i l e p t i c drug, v a l p r o i c a c i d . Evidence f o r the formation of chemically r e a c t i v e intermediates. Drug Metab. Disp., 13, 81 (1985). 133. A. Leo, C. Hansch and D. E l k i n s . P a r t i t i o n C o e f f i c i e n t s and t h e i r uses. Chem. Rev., 71, 525 (1971). 134. R.W. Taft i n " S t e r i c E f f e c t s i n Organic Chemistry", M.S. Newman, ed., Wiley, London, p.559 (1956). 135. C M . Lee and W.D. Kumler. The dipole moment and structure of the imide group. I . F i v e - and six-raembered c y c l i c imides, lactams and the carbamate group. J . Amer. Chem. S o c , 83, 4586 (1961). 136. R. C o l l a n d e r . The p a r t i t i o n of organic compounds between higher alcohols and water. A c t a . Chem. Scand., 5, 774 (1951). 137. S.H. linger, J.R. Cook and J.S. Hollenberg. Simple procedure f o r determining octanol-aqueous p a r t i t i o n , d i s t r i b u t i o n and i o n i z a t i o n c o e f f i c i e n t by reversed-phase HPLC. J . Pharm. S c i . , 67, 1364 (1978).  212  138. R.M. C a r l s o n , R.E. Carlson and H.L. Kopperman. Determination of p a r t i t i o n c o e f f i c i e n t by l i q u i d chromatography. J . Chromatogr., 107, 210 (1975). 139. M.S. M i r r l e e s , S.J. Moulton, C.T. Murphy and P . J . T a y l o r . D i r e c t measurement of octanol-water p a r t i t i o n c o e f f i c i e n t by HPLC. J . Med. Chem., 19, 615 (1976). 140. J.M. M c C a l l . L i q u i d - l i q u i d p a r t i t i o n c o e f f i c i e n t s by high-pressure l i q u i d chromatography. J . Med. Chem., 18, 549 (1975). 141. M. Harnisch, H.S. Mockel and G. S c h u l t z e . Relationship between l o g P shake-flask values and capacity f a c t o r derived from reversed phase HPLC f o r n-alkylbenzenes and some OECD reference substances. J . Chromatogr., 282, 315 (1983). 142. R.E. Koopmans and R.F. Rekker. HPLC of alkylbenzenes as determined from octanol-water p a r t i t i o n c o e f f i c i e n t or c a l c u l a t e d from hydrophobic fragmental data and c o n n e c t i v i t y i n d i c e s . J . Chromatogr., 285, 267 (1984). 143. S. Toon, T. Mayer and R.M. Rowland. LC determination of l i p o p h i l i c i t y with a p p l i c a t i o n to a homologous s e r i e s of b a r b i t u r a t e s . J . Pharm. S c i . , 73, 625 (1984). 144. R. K a l i s z a n . Chromatography i n studies of q u a n t i t a t i v e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s . J . Chromatogr., 220, 71 (1981). 145. M. d'Amboise and T. Hanai. Hydrophobicity and r e t e n t i o n i n reversed phase l i q u i d chromatography. J . L i q . Chromatogr., 5, 229 (1982). 146. N. Tanaka and E.R. Thornton. S t r u c t u r a l and i s o t o p i c e f f e c t s i n hydrophobic binding measured by HPLC. A stable and highly precise method f o r hydrophobic i n t e r a c t i o n i n biomembranes. J . Amer. Chem. S o c , 99, 7300 (1977). 147. T.L. H a f k e n s c h e i d and E. T o r a l i n s o n . C o r r e l a t i o n between alkane/water and octanol/water d i s t r i b u t i o n c o e f f i c i e n t . I s o c r a t i c reversed phase LC capacity f a c t o r s of a c i d s , bases and n e u t r a l s . I n t . J . Pharmaceutics, 16, 225 (1983). 148. C. Horvath, W. Melander and I . Molnar. Solvophobic i n t e r a c t i o n s i n l i q u i d chromatography with nonpolar stationary phases. J . Chromat o g r . , 125, 129 (1976). 149. C.R. Yonker, T.A. Zwier and M.F. Burke. Comparison of s t a t i o n a r y phase formation i n reverse phase f o r methanol-water systems. J . Chromatogr., 241, 257 (1982). 150. C.R. Yonker, T.A. Zwier and M.F. Burke. I n v e s t i g a t i o n of s t a t i o n ary phase formation f o r RP-18 using various organic m o d i f i e r s . J . Chromatogr., 241, 269 (1982).  213  151. L.R. Snyder and J . J . K i r k l a n d . "Introduction to Modern Chromatography". Wiley, New York, p.22 (1979). 152. C. Hansch and A . J . Leo. "Substituent Constants f o r C o r r e l a t i o n Analysis i n Chemistry and B i o l o g y " . J . Wiley, New York (1979). 153. R.F. Rekker. "The Hydrophobic Amsterdam (1977).  Fragmental  Constant".  Elsevier,  154. P.E. P f e f f e r , L.S. S i l b e r t and J.M. C h i r i n k o . Alpha-anions of c a r b o x y l i c acids I I . The formation and a l k y l a t i o n of alpha-metalated a l i p h a t i c a c i d s . J . Org. Chem., 37, 451 (1972). 155. G. T a i l l a n d i e r , J . L . Benoit-Guyod, C. L a r u e l l e and A. Boucherle. I n v e s t i g a t i o n i n the d i p r o p y l a c e t i c acid s e r i e s , C8 and C9 branched chain ethylenic acids and amides. Arch. Pharm. (Weinheim), 310, 394 (1977). 156. N. Thoai. Isomerization of 2,5-dimethyl-3-propyl-2-pentyl-2,3dihydrofuran. B u l l . S o c Chim., 2, 225 (1964). 157. T. R e i c h s t e i n , H.R Rosenberg and R. E b e r h a d t . Eine einfache methode zur gewinning g e s a t t i g t e r f e r t i a r e r carbonsauren. HelvChim. Acta., 18, 721 (1935). 158. S. Pawlenko.  Carboxylic a c i d s .  Chem. A b s t r a c t s , 68, 2055 (1968).  159. K. B o t t . Carboxylic acid synthesis with 1,1-dichloroethylene. Preparation of sec. and t e r t . a l k y l a c e t i c a c i d . Chem. Ber., 100, 978 (1967). 160. K.B. Wiberg and T.W. Hutton. The stereochemistry of the Wolff rearrangement. J . Amer. Chem. S o c , 78, 1640 (1956). 161. J.S. Mihina and R.M. Herbst. The r e a c t i o n of n i t r i l e s with hydrazoic a c i d . Synthesis of monosubstituted t e t r a z o l e s . J . Org. Chem., 15, 1082 (1950). 162. D. Preissman, J.H. Bryder and L . P a u l i n g . The reactions of a n t i serum homologous to the p-Azosuccinilate i o n group. J . Amer. Chem. S o c , 70, 1352 (1948). 163. P.L. Creger. Metalated c a r b o x y l i c acids I . A l k y l a t i o n . Chem. S o c , 89, 2500 (1967).  J . Amer.  164. A.W.D. Avison. The a p p l i c a t i o n of l i t h i u m aluminium hydride to the preparation of some amino-alkanols. J . A p p l . Chem., 1, 469 (1951). 165. N.S. Antonenko. Study of the molecular i n t e r a c t i o n of amic acids by IR spectroscopy. Zhur. Obskich. Khim., 35, 425 (1965). 166. W.G. Finnegan, R.A. Henry and R. L o f q u i s t . An improved synthesis of 5-substituted t e t r a z o l e s . J . Amer. Chem. S o c , 80, 3908 (1958).  214  167. H. Behringer and K. K o h l . aminoathyl-tetrazoles und Ber., 89, 2648 (1956).  Uber t e t r a z o l e d i e synthese des 5-6einiger tetrazolylmethyl derivative.  168. H. Normant. Recherches sur l e s magnesien v i n y l i q u e s V I I . Synthese d'aldehyde a',B-ethyleniques. Comptes Rendus, 240, 1435 (1955). 169. A. Kajikawa, M. Morasaki and N. Ikekawa. Exclusive y-coupling i n the a l d o l r e a c t i o n of a8-unsaturated e s t e r s . T e t t . L e t t . , 4135 (1975). 170. A.S. Kende and B.H. Toder. Stereochemistry of deconjugative a l k y l a t i o n of ester d i e n o l a t e s . S t e r e o s p e c i f i c t o t a l synthesis of the l i t s e n o l i d s . J . Org. Chem., 47, 163 (1982). 171. M.W. Rathke and D. S u l l i v a n . The preparation and reaction of enolate anions derived from aB-unsaturated e s t e r s . T e t t . L e t t . , 4249 (1972). 172. P.E. P f e f f e r , L.S. S i l b e r t and E. K i n s e l . A re-examination of unsaturated carboxylate dianion r e a c t i o n s . Evidence f o r a- and y s u b s t i t u t i o n i n alkenoic a c i d s . T e t t . L e t t . , 1163 (1973). 173. P.E. P f e f f e r and L.S. S i l b e r t . y-Anions. I V . P o s i t i o n and stereochemical isomerization of 2- and 3- unsaturated c a r b o x y l i c acid d i a n i o n s . J . Org. Chem., 36, 3290 (1971). 174. R.R. Rando and W. von E. Doering. 6,y-unsaturated acid and esters by photochemical isomerization of Y,B-congeners. J . Org. Chem., 33,1671 (1968). 175. R.M. Duhaime, D.A. Lombardo, I.A. Skinner and A.C. Weedon. Conversion of a,6-unsaturated esters to t h e i r 8,y-unsaturated isomers by photochemical deconjugation. J . Org. Chem., 50, 873 (1985). 176. J.A. Barve, F.D. Gunstone, F.R. Jacobsberg and P. Winlow. Fatty Acids. B e h a v i o u r o f a l l t h e m e t h y l o c t a d e c e n o a t e s and octadecynoates i n a r g e n t a t i o n chromatography and gas l i q u i d chromatography. Chem. Phys. L i p i d s , 18, 117 (1972). 177. H.B.S. Conacher. Chromatographic determination of c i s - t r a n s monoe t h y l e n i c unsaturation i n f a t s and o i l s - A review. J . Chromatogr. S c i . , 14, 405 (1976). 178. M.S.F. L i e Ken J i e and C H . Lam. The t h i n - l a y e r chromatographic behaviour of a l l the c i s , c i s and t r a n s , t r a n s - dimethylene i n t e r rupted methyl octadecadienoates and methyl octadecadiynoates. J . Chromatogr., 124, 147 (1976). 179. K.C. Chan, R.A. J e w e l l , W.H. Nutting and H. Rapoport. Synthesis and stereochemical assignment of c i s and trans 2-methyl-2-pentenoic a c i d and the corresponding e s t e r s , aldehydes and a l c o h o l s . J . Org. Chem., 33, 3382 (1968).  215  180. M. Anteneunis, A. De Bruyn, H. De Proter and G. Verhegge. C i s and trans-isomers of e t h y l heptenoates. « B u l l . Soc. Chim., 11, 371 (1968). 181. G.E. Berendsen, P . J . Schoenmakers, L. De Galan, G. Bigh, Z. VargaPuchony and J . Inczedy. On the determination of the hold-up time i n reversed phase l i q u i d chromatography. J . L i q . Chromatogr., 3, 1669 (1980). 182. M.S. Tute. P r i n c i p l e s and p r a c t i c e of Hansch a n a l y s i s : A guide t o s t r u c t u r e - a c t i v i t y c o r r e l a t i o n f o r the medicinal chemist. Adv. Drug Res., 6, 1 (1971). 183. M.F. A l d e r s l e y , A . J . K i r b y , P.W. Lancaster, R.S. McDonald and C.R. Smith. Intramolecular c a t a l y s i s of amide h y d r o l y s i s by the carboxyl group. Rate-determining proton t r a n s f e r from external general acids i n the h y d r o l y s i s of substituted maleamic a c i d s . J . Chem. S o c , Perkin I I , 1487 (1974). 184. R. Kluger and J . C h i n . Carboxylic acid p a r t i c i p a t i o n i n amide h y d r o l y s i s . Evidence that separation of a non-bonded complex can be rate determining. J . Amer. Chem. S o c , 104, 2891 (1982). 185. A. Albert and E.P. S e r j e a n t . "The determination of i o n i z a t i o n constants". Chapman and H a l l , London (1971). 186. R.F. Cookson. The determination of a c i d i t y constants. Chem. Rev., 74,5 (1974). 187. L.Z. Benet and J.E. Goyan. Potentiometric determination of d i s s o c i a t i o n constants. J . Pharm. S c i . , 56, 665 (1967). 188. J . F . J . Dippy. Chemical c o n s t i t u t i o n and the d i s s o c i a t i o n constants of monocarboxylic a c i d s . Part X. Saturated a l i p h a t i c a c i d s . J . Chem. S o c , 1222 (1938). 189. A.E. Hershey, J.R. Patton and K.H. Dudley. Gas chromatographic method f o r the determination of v a l p r o i c acid i n human plasma. Ther. Drug Monit., 1, 217 (1979). 190. S.G. P i r e d d a , J.H. Woodhead and E.A. Swinyard. E f f e c t of stimulus i n t e n s i t y on the p r o f i l e of anticonvulsant a c t i o n of phenytoin, ethosuximide and v a l p r o a t e . J . Pharmacol. Exp. Ther., 232, 741 (1985). 191. W.D. Yonekawa, H.J. Kupferberg and D.M Woodbury. Relationship between pentylenetetrazole-induced seizures and b r a i n pentylenet e t r a z o l e l e v e l s i n m i c e . J . P h a r m a c o l . E x p . T h e r . , 214, 59 (1980). 192. J.T. L i t c h f i e l d and F . Wilcoxon. A s i m p l i f i e d method of evaluating dose-effect experiments. J . Pharmacol., Exp. Ther., 96, 99 (1949). 193. J.K. Seydel and K.J. Schaper. Structure-pharmacokinetic r e l a t i o n ship and drug design. Pharmacol. Ther., 15, 131 (1982). 216  194. S. Toon and M. Rowland. Structure-pharmacokinetic r e l a t i o n s h i p s among the barbiturates i n the r a t . J . Pharmacol. Exp. Ther., 225, 752 (1983). 195. P.N. C r a i g . Interdependence between p h y s i c a l properties and s e l e c t i o n of substituent groups f o r c o r r e l a t i o n s t u d i e s . J . Med. Chera., 14, 680 (1971). 196. B.B. Brodie, H. Kurtz and L.S. Schanker. The importance of d i s s o c i a t i o n constant and l i p i d - s o l u b i l i t y i n i n f l u e n c i n g the passage of drugs i n t o the CSF. J . Pharmacol. Exp. Ther., 130, 120 (1960). 197. Y.C. M a r t i n i n " P h y s i c a l C h e m i c a l P r o p e r t i e s o f D r u g s " , 5. H. Yalkowsky, A.A. Sinkula and S.C. V a l v a n i , eds., Marcel Dekker, p.49 (1980). 198. A.L. M c C l e l l a n . "Tables of Experimental Dipole Moments:, V o l . 1. Freeman and Co., San Francisco (1963). 199. N.A. Brown, A.G.E. Wilson and J.W. B r i d g e s . Chain-length dependency of f a t t y acid and carbamate binding to serum albumin. Biochem. Pharmacol., 31, 4019 (1982). 200. A. Raines, G.J. Blake, B. Richardson and M.B. G i l b e r t . D i f f e r e n t i a l s e l e c t i v i t y of several barbiturates on experimental seizures and n e u r o t o x i c i t y i n the mouse. E p i l e p s i a , 20, 105 (1979). 201. F . F . B l i c k e and M.F. Z i e n t r y . A c i d amides as h y p n o t i c s . I V . B a r b i t u r i c a c i d s . J . Amer. Chem. S o c , 63, 2891 (1984). 202. E.S. Echague and R.K.S. L i m . Anticonvulsant a c t i v i t y of some c a r b i n y l u r e a s . J . Pharmacol. Exp. Ther., 138, 224 (1962). 203. C.A. M i l l e r and L.M. Long. A n t i c o n v u l s a n t s . An i n v e s t i g a t i o n of N-R-a-RT-a-Phenylsuccinimides. J . Amer. Chem. S o c , 73, 4895 1 (1951). 204. D.R. C u r t i s and J.C. Watkins. The e x c i t a t i o n and depression of s p i n a l neurons by s t r u c t u r a l l y r e l a t e d amino a c i d s . J . Neurochem., 6, 117 (1960). 205. B. Ferrandes, C. Cohen-Addad, J . L . Benoit-Guyod and P. Eymard. Etude des r e l a t i o n entre l a structure c r i s t a l l i n e et l ' a c t i v i t e biologique de molecules de s e r i e s d i - et t r i - a l c o y l acetiques. Biochem. Pharmacol., 23, 3363 (1974). 206. C. Cohen-Addad, G. d'Assenza, G. T a i l l a n d i e r and J . L . Benoit-Guyod. Structures c r i s t a l l i n e s de derives des acides dipropylace"tiques et t r i p r o p y l a c e t i q u e . 111. Diethylpropionamide and dipropylpropionamide. Acta C r y s t . , 31, 835 (1975). 207. P.R. Andrews, D.J. Craik and J . L . M a r t i n . Function group c o n t r i b u t i o n s t o d r u g - r e c e p t o r i n t e r a c t i o n s . J . Med. Chem., 27,1648 (1984). 217  208. N.C. B a e n z i g e r , A.D. N e l s o n , A. T u l i n s k y , J.H. B l o o r and A . I . Popov. Two independent d e t e r m i n a t i o n s of the c r y s t a l and m o l e c u l a r s t r u c t u r e s of the i o d i n e m o n o c h l o r i d e complex of pentylenetetrazole. J . Amer. Chem. S o c , 89, 6463 (1967).  218  APPENDIX NMR spectra of some of the synthesized compounds.  219  H-NMR ( 400 MHz) spectrum of isomeric mixture of ethyl 2-(Z-l'-propenyl)-E-2 pentenoate and ethyl 2-(E-l'-propenyl)-E-Z-pentenoate.  H-NMR (400 MHz) magnified s pectrum of isomeric mixture of ethyl 2-(Z-l'-propenyl)E-2-pentenoate and ethyl 2-( E-l'-propenyl)-E-2-pentenoate.  5  4  3  2 6(ppm)  H-NMR (400 MHz) spectrum of ethyl 2-(Z-l'-propenyl)-Z-3-pentenoate with trace amount of ethyl 2-(Z-l'-propenyl)-E-2-pentenoate.  1  0  r S3 4>  1 i  -A.  6(ppm)  *H-NMR ( 400 MHz) spectrum of 2-(Z-l'-propenyl )-E-2-pentenoic acid with trace amount of 2-(E-l'-propenyl)-Z-2-pentenoic acid.  H-NMR ( 400 MHz) spectrum of isomeric mixture of 2-(Z-l'-propenyl)-E-2-pentenoic acid, 2-(E-l'-propenyl)-E-2-pentenoic acid, 2-(E-l'-propenyl)-Z-2-pentenoic acid, and 2-(Z-l 1 -propenyl)-Z-3-pentenoic acid.  227  H-NMR( 80 MHz) spectrum of N,N-diethylsuccinamic shown in Figure 6a.  acid from the same synthesized product  

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