DESIGN AND SYNTHESIS OF SOME ARYLOXYARYL SEMICARBAZONES AND RELATED COMPOUNDS AS NOVEL

A Thesis Submitted to the College of Graduate Studies and Research in Partial fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmacy

BY Puthucode Ramanan Narayan Spring 1996

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DEGREE OF DOCTOR OF PHILOSOPEW by Puthucode Ramanan Narayan College of Pharmacy and Nutrition University of Saskatchewan Spring 1996

Examining Committee: Dr. R.T. Card Dean's Designate, Chair College of Gradauate Studies & Research Dr. E. M. Hawes Chair of Advisory Committee College of Pharmacy and Nutrition Dr. J. R Dimmock Supervisor, College of Pharmacy and Nutrition Dr. J. W. Hubbard CoHege of Pharmacy and Nutrition Dr. J. D. Wood Department of Biochemistry Dr. J. M. Tuchek Department of Pharmacology

External Examiner: Dr. C. R Clark School of Pharmacy Auburn University Alabama 36849-550 1 USA DESIGN AND SYNTHESIS OF SOME ARYLOXYARYL SEMICARBAZONES AND RELATED COMPOUNDS AS NOVEL ANICONWLSANTS

Epilepsy is one of the most common neurological disorders affecting approximately 2% of the world's population. Unfortunately, currently available drugs are effenive in only 65% of the patients and their use may be associated with sigdicant side effects causing disability with considerable socioeconomic implications. Thus. there is an urgent need for new antiepileptic drugs with greater efficacy, specificity and lower toxicity. The development of new antiepileptic drugs remains a challenging problem. since both the prirnw pathologies of epilepsy and the precise mechanisms by which available anticonvulsants act are not well understood- Therefore. the search for new antiepileptic drugs continues to be an active area of investigation in medicinal chemistry.

Previous studies revealed that a number of aryl sernicarbazones on oral administration to rats possessed signilicant activity (ED5, figures in the 20-25 mgkg range) in the MES screen. In addition some of these compounds displayed good protection indices (PI viz. TDr&Dr, of appro-uirnately 25). Lf the aryl sernicarbazones displaying activity in the MES screen interact at a specific binding site. it is likely that the semicarbazono group and the aryl ring align at complementq areas on a macromolecular complex in vivo which have been referred to as the hydrogen bonding area and the aql binding site respectively (Fig. 1).

Auxilianr Bindina Area

Awl Bindina Site

Hvdmsen bond in^ Area

Fig. 1 Proposed binding site of aryl semicarbazoncs

The principal aim of the study was to investigate the area around the postulated aryl binding site. which is shown in Fig. I as the ausiliaxy binding area with the main view of improving the potency. Ideally an ED5()of 1-3 mg/kg when administered orally to rats will be achieved. The biodata generated on these compounds may afford a clearer picture of the nature of the postulated binding site. A number of axyloxyaqd semicarbazones and related compounds WUE synthesized and evaluated for anticonvulsant activities. After intraperitoneal injection to mice, the Semicart,azones were examined in the MES , scPTZ and neurotoxicity screens. The resuits indicated that greater protection was obtained in the MES test than the scPrZ screen Quantitation of approximately one-third of the compounds revealed an average protection index of approximately 9. After oral administration to rats, a number of compounds displayed significant potencies in the MES screen (EDMof 1-5 mgkg) accompanied by very high protection indices. In fact over half the compounds had PI figures of greater than 100 and four were in excess of 200 and two compounds displayad protection &dices greater than 300.

These compounds are ranked very high in terms of pure activity. A number of compounds displayed greater potencies and PI figures in the mouse intraperitoneal and rat oral screens than three clinically used drugs viz , cubamazepine and . Patent protection for these novel semicarbazones were made at the US Patent Oifice on June 7, 1995. The data generated fiom these studies supported a binding site hypothesis. Quantitative structure-activity relationships indicated a number of physicochemicaf parameters which contributed to activity in the MES screen X-ray crystallography and moiecular modeling of five compounds suggested the importance of certain interatomic distances and bond angles for activity in the mouse and rat MES screens. In presenting this thesis in partial fulfdment of the requirements for a postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor who supervised my thesis work or, in his absence, by the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis.

Request for permission to copy or to make other use of material in this thesis in whole or part should be addressed to :

Dean of the College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, CANADA S7N SC9 TO PARENTS, MEMORY OF MOTHER-lN-LAW,

WIFE SHREEDEVI AND DAUGEmR SHALINI ACKNOWLEDGMENTS

There are many people to whom I owe a debt of gratitude for the help I received throughout the course of this project. First and foremost, I wish to sincerely thank Dr. J. R Dirnmock for his personal interest, guidance, and encouragement during the course of the research work and in the preparation of this thesis. I acknowledge the appropriate suggestions provided to me by the members of my advisory committee: Dr. E. M. Hawes, Dr. J. W. Hubbard, Dr. J. M. Tuchek and Dr. J. D. Wood. I would especially like to express my appreciation to Dr. S. N. Rao, Dr. J. M. Tuchek and Dr. V. Gopal for the time they devoted towards my academic and research endeavors.

The rich facilities and the Eendly atmosphere that were bestowed on me by the College of Pharmacy and Nutrition, University of Saskatchewan are also acknowledged. I would like to thank Nordic Merrell Dow Research, Laval, PQ, Canada, for financial suppon of this project and Mr. J. P. Stables (NDlJ for generating most of the biological data. Appreciation is recorded to Dr. J. W. Quail, Dr. L. Prasad and Mrs. U. Pugazhenthi for assistance in the interpretation of the X-ray clystdographic data.

My thanks are also due to C. Savithi, M. A. Saeed, (Department of Chemistry), A Lo, L M. Smith, C. Thomson (summer students), and M. Hetherington for their contributions to my research project. The timely help offered by K. G. Tischler, N. M. Kandepu, H. V. Raghuram, H. Jyothsna, V. Iowkin and V. Khanna are greatly appreciated. I also extend my thanks to all my colleagues for their cooperation throughout this work.

Finally I would like to thank all my family members especially my wife Shreedevi and daughter Shalini for their love, patience and understanding during the long months of intense labor needed to complete this task. They provided the comfort and support necessary to sustain the human psyche that science never can grant. ABSTRACT

Epilepsy is one of the most common neurological disorders affecting approximately 2% of the world's population. Unfortunately, currently available drugs aneffective in only 65% of the patients and their use may be associated with significant side effects causing disability with considerable socioeconomic implications. In addition, between 10 and 25 % of epileptic patients have chronic intractable seizures that are inadequately controlled with the currently available antiepileptic drugs. Thus, there is an urgent need for new antiepileptic drugs with greater efficacy, specificity and lower toxicity.

The development of new antiepileptic drugs remains a challenging problem, since both the primary pathologies of epilepsy and the precise mechanisms by which available anticonvulsants act are not well understood. Therefore, the search for new antiepileptic drugs continues to be an active area of investigation in medicinal chemistry.

Two major pharmacological screening tests used to evaluate compounds for anticonvulsant activities are the maximal electroshock screen (MES) and the subcutaneous pentylenetetrazol (scPTZ) screen. These tests are claimed to detect compounds possessing activity against generalized tonic-cIonic (grand mal) and generalized absence (petit mal) seizures. The structural requirements for activity in the MES screen have been stat& t; be the presence of a Iarge hydrophobic group which is in close proximity to at least two electron donor atoms. For activity in the scPTZ screen, a smaller, less hydrophobic group than is required for activity in the MES screen should be present near to a minimum of two electron donor atoms.

Many currently available antiepileptic drugs possess a dicarboximide function (-CONHCO-)which may be associated with toxic side effects. Therefore, a number of compounds (aryl semicarbazones and related analogs) were designed to be bereft of this group yet still fulfill the structuml requirements for activity in the maximal electroshock screen (MES) and/or subcutnneous pentylenetetrazol (scPTZ) screen.

Previous studies revealed that a number of aryl semicarbazones on oral administration to rats possessed significant anticonvuisant activity (EDw figures in the 20-25 mg/kg range) in the MES screen. In addition, some of these compounds displayed good protection indices (PI viz TD&DSO of approximately 25). If the aryl semicarbazones displaying activity in the MES screen interact at a specific binding site, it is likely that the semieubazono group and the aryl ring align at complementary areas on a macromoIecular complex in vivo which have been referred to as the hydrogen bonding area and the aryl binding site respectively (Fig. 1).

Auxiliarv Bindina Area

Arvl Bindins Site

Hvdrouen Bondinn Area

Fie. 1 Proposed binding site of aryl semicarbazones The principal aim of the study was to investigate the area around the postulated aryl binding site, which is shown in Fig. I as the auxiliary binding area with the main view of improving the potency. Ideally an EDSDof 1-3 mg/kg when administered ordy to rats will be achieved. The biodata generated on these compounds may afford a dearer picture of the nature of the postulated binding site.

A number of aryloxyaryl semicarbazones and related compounds were synthesized and evaluated for anticonvulsant activities. After intraperitoneal injection to mice, the semicarbazones were esamined in the MES , scPTZ and neurotoxicity screens. The results indicated that greater protection was obtained in the MES test than the scPTZ screen. Quantitation of approximately one-third of the compounds revealed an average protection index of approximately 9. After oral administration to rats, a number of compounds displayed significant potencies in the MES screen (ED- of 1-5 mglkg) accompanied by very high protection indices. In fact over half the compounds had PI figures of greater than 100 and four were in excess of 200 and two compounds displayed protection indices greater than 300.

These compounds are ranked very high in terms of pure activity. A number of compounds displayed greater potencies and PI figures in the mouse intraperitoneal and rat oral screens than three clinically used drugs viz. phenytoin, and valproate. Patent protection for these novel semicarbazones were made at the US Patent Oflice on June 7,1995.

A semicarbazone designated ADD 222036 (NC 1285 / N4) is the subject of a NIH "red book'' and detailed pharmacological evaluations have been undertaken. This compound displayed high potency in electrically induced seizure models (MES, corneal and hippocampal kindled rats) and genetic epilepsy models (Frings audiogenic mice and epileptic fowl). This compound did not display any proconvulsant activities and its effect on hepatic microsomal protein content and

vii drug metabolizing enzymes were minimal. ADD 222036 has the potential to limit focal firing and hence may be dinidly useful in the treatment of complex partial seizures in addition to its use in generalized tonic-clonic seizures: This compound appears to act by one or more mechanisms which are dimerent from established anticonvulsant drugs.

The data generated from these studies supported a binding site hypothesis. Quantitative structure-activity relationships indicated a number of physicochemical parameters which contributed to activity in the MES screen. X- ray crystallography and molecular modeling of five compounds suggested the importance of certain interatomic distances and bond angles for activity in the mouse and rat MES screens. TABLE OF CONTENTS

DEDICATION ACKNOWLEDGMENTS ABSTRACT TABLE OF CONTENTS LIST OF FIGURES xiv LIST OF TABLES xv LIST OF SCHEMES xviii LIST OF ABBREVIATIONS xix

1.0 INTRODUCTION 1.1.0.0 A brief history of epilepsy and antiepileptic drug development 1-2.0.0 Classification of epilepsies 1.3.0.0 Antiepileptic therapy 1 40.0 The search for new antiepileptic dmgs 1.5-0.0 Strategies for the development of new antiepileptic dmgs 1.6.0.0 Animalmodelsofepilepsy

1 -6.1.O Electrically-induced seizures 1.6.2.0 Chemicall y-induced seizures 1.6.3.0 Genetic animal models of epilepsy

1.7.0.0 Biochemistry of seizure generation and propagation 1.8 -0.0 Targets for antiepileptic drug development

1.8.1.0 Antiepileptic drugs that enhance central inhibition 1.8.2.0 Antiepileptic drugs that diminish excitatory amino acid transmission 1.8.3-0 Antiepileptic dmgs which modulate ionic channel activity

1 -9.O. 0 Conformational. stereochemical features and structure-activity relationships of potential anticonvulsant agents 2.0 RATIONALE OF 'llU3 PRESENT INVESTIGATION 2.1 -0.0 Drug design and development 2.2.0.0 Development of novel audconvuisants 2.3 -0-0 Previous work undertaken in these Iaboratories 2.4.0.0 Objectives of the present investigation 2.5.0.0 Rationale for selecting series I- XVI

3.0 DESCRIPTION OF THE EXPERIMENTAL WORK 3.1.0.0 Chemistry

General method of the preparation of 4-bromo-

benzaldehyde acy Ihydrazones ( 11 -5 ) Synthesis of 4-bromobenzaldehyde guanyhydrazone ( h) General method for the preparation of some 4-bromo-

benzaldehyde thiosexnicarbazones ( 17-9 ) General method for the preparation of aryloxyaryl or arylthioaryl aldehydes General method for the preparation of the aryl or aryIoxyary1 or arylthioaryl benzaldehyde semicarbazones (H-VZ v.4XIandJW Synthesis of 4-benzyoyloxybeddehyde semicarbazone and 4+-chlorobenzoyloxy)benzaldehyde semicarbazone mu) Synthesis of 4-phenylsuffonylbeazaldehyde semicarbazone om) Synthesis of 4-@henylsulfonyloxy)benzaldehyde semicar- bazone and 4-(4'-methylphenylsuIfony1o~)benzaidehyde semicarbazone ( v&6) Synthesis of 4-(4'-fluorophenoxy)benzaldehyde thiosemi- carbarone and 4-[(4'-fluorop henyI)tho] benzaldehyde thiosernicarbamne ( IX ) Synthesis of 444'-fluorophenoxy)benzaldehyde guanyl- hydrazone and 4-[(4'-fluoropheny1)thio J benzaldehyde gu=y~yd==e ( E3.4 Synthesis of q4'-fluorophenoxy)benzaldehyde formyl- hydrazone. 4-[(4'-fluorophenyf)thio]benzaldehyde formy l- hydrazone and 4-(4'-fluorop henoxy)benzaldehyde acety 1- hydrazone ( ) Synthesis of 4-(4'-fluorophenoxy)benzaldehyde carbo- hydrazone and 4x4'-fluorophenoxy)benzaldehyde oxamic- hydrazone ( x4.5 Synthesis of 3-aqlproped semicarbazone and related =m~ounds (J(IIL1-3) Synthesis of 4,4'-dichlorobenzophenone semicarbazone

( m2) General synthesis of some 1,3-diaryl-2-propen- 1-one semicarbazones ( XV14) Synthesis of I,j-dipheny1-2,4-pentadien-3~ne semicar- bazone ( XVI ) Attempted synthesis of 444'-hydroxyphenoxy)- benzaldehyde semicarbazone Attempted synthesis of 344-phenoxypheny1)- I -pheny l-2- propen- 1-one semicarbazone Attempted synthesis of benzophenone semicarbarone analogs Attempted synthesis of 4-(2',3',4',5',6'-pentafhoro- p henoxy), 4-(2',3',5',6'-tetranuorophenoxy), 4-(4'-amino- phenoxy), 444'-trifluoromethylphenoxy), 4-(4'-carboxy- phenolcy)-benzaldehyde semicarbazones Attempted synthesis of 344'-£luorophenoxy)benzaldehy de semicarbazone Partition coefficient measurements Stability studies Statistical analyses and physicochernid constants X-ray crystallographic study 3.1.26.0 Evaluation of the binding site hypothesis using X-ray 100 crystallography and mo1ecuIar modeling 3.2.0.0 Pharmacology

3.2.1 -0 Materid and methods 3.2.2.0 Determination of median effective ( EDSo) or toxic dose

( -l-50 3.2.3 -0 Determination of acute toxicity 3.2.4.0 Anticonvulsant and differentiation tests 3.2.5.0 Maximal electroshock seizure (MES)and corneal kindled rat test 3.2.6.0 Hippocampd krndled rat test 3 -2.7.0 Amygdala kindled rat test 3.2.8.0 Subcutaneous pentylenetetrazol (Metrazol) seizure threshold test (scPTZ/scMET), subcutaneous bicuculline (scBic), picrotoxin (scPic), and strychnine (scStr) tests 3 -2.9.0 Timed intravenous pentylenetetrazol test

3 2 1 10 Overt tolerance and liver enzyme studies 3 -2.12.0 Epileptic fowl genetic model 3 -2.13 -0 Frings audiogenic seizure genetic model

4.0 RESULTS AND DISCUSSION 4.1.0.0 Introduction 4.2.0.0 General introduction to the formation of semicarbazones and related derivatives

4.2.1 -0 Synthesis of 4-(4'-chlorophenoxy)benzaldehyde semicar- bazone ( N lj ) 4.2.2.0 Reaction mechanism for the formation of aryloxyaryl aldehydes and related compounds

4 -3.0-0 Anticonwlsant identification

4.3.1.0 Intrapentoned injection in mice (Phase la) 4.3.2.0 Threshold tonic extension (TTE) test 4.3-3 -0 Oral administration to rats (Phase I b)

xii 4.4.0.0 Anticonwlsant quantification

4.4.1.0 Intraperitoned injection in mice (Phase 2a) 4.4.2.0 Oral administration to rats (Phase Zc)

4.5 -0-0 Anticonvulsant drug differentiation

4.5.1.0 Subcutaneous bicuculline (scBic), picroto.uin (scPic). and strychnine (scS tr) tests (Phase 3a) 4.5.2.0 Timed intravenous pentylenetetrazol test (Phase 3c) 4.5.3 -0 Corneal kindled rat test 4.5 A.0 fippocampal kindled rat test 4.5.5 -0 Amygdala kindled rat test 4.5-6.0 Measurement of intracetlular calcium transients

4.6.0.0 Overt tolerance and liver enzyme studies (Phase 5) 4.7.0.0 Genetic models

47.1.0 Epileptic fowl 4.7.2.0 Frings audiogenic seizure susceptible mice

4.8.0.0 Anticonvulsant activity profile of [V, 49.0.0 Statistical analyses and quantitative st~~chlre-acti~i~relationships studies (QSAR) 4.10.0.0 Determination of log P values 4.1 1-0.0 Stability studies 4.12.0.0 Evaluation of the binding site hypothesis using X-ray crystallography and molecular modeling 4.1 3.0.0 Structure-activity relationships (SAR)

5.0 SUMMARY

REFERENCES LIST OF FIGURES

Fig. 1.1 Possible sites of interaction of antiepileptic drugs on GABA-mediated transmission

Fig. 1.2 Possible sites of interaction of antiepileptic drugs on glutamate-mediated transmission

Fig. 2.1 Proposed binding site of aryl semicarbazones

Fig. 2.2 Proposed binding site of aryloqary l semicarbazones

Fig. 2.3 General binding sites for anticonvulsants

Fig. 4.1 ORTEP diagram of Illl

Fig. 4.2 ORTEP diagram of Nl

Fig. 4.3 ORTEP diagram of lVJ

Fig. 4.4 ORTEP diagram of IVr,

Fig. 4.5 ORTEP diagram of VIII,

Fig. 4.6 Orientation of the proximal and distal aryl rings of 1111 (colorless), IVI (red), IV4 (green). IVn (orange). VIII,, (blue) and Mnls (yellow) when the N3. C 14. (02 or 0) and N2 atoms of each molecule are superimposed

Fig. 4.7 -A Distances and 8 angles behveen the C 14 atom and centres of both the pro?cimal (CAr,) and distal (CArd) rings in IV1

-B Displacement and y angles of the centres of the proximal (CAr,) and distal (CArd) rings from the N3 -C 14-(02)-N2 plane in IV,

Fig. 4.8 Orientation of the proximal and distal aryl rings of 111,. IVI. IVL IV= and WIl,

Fig. 4.9 Orientation of the proximal and distal aryl rings of IV4 with phenytoin

xiv LIST OF TABLES

Table 1.1 Chemical structures of antiepileptic drugs

Table 1.2 hernational classification of epileptic seizures

Table 1.3 Suggested first- and second- line antiepileptic drugs

Table 1.4 Drawbacks of current antiepileptic therapy

Table 1.5 Minimal target characteristics of a new antiepileptic dnrg

Table 1.6 Experimental models of epilepsy

Table 1.7 Anticonvulsant drug development

Table 1.8 Antiepileptic that enhance central inhibition

Table 1.9 Antiepileptic drugs that diminish excitatory amino acid transmission

Table 1.10 Action of various antiepileptic drugs

Table 3.1 Physical data of 4-bromobenzddehyde acylhydrazones

Table 3.2 Physical data of substituted benzaldehyde and semicarbazones

Table 3.3 Physical data of substituted 3-phenoxybenzaldehyde sernicarbazones

Table 3.4 Physical data of substituted 4-aryloqbenzaldehyde semicarbazones

Table 3.5 Physical data of substituted 4-aryloxyacetophenone and 4-aryloxy- propiophenone semicarbazones

Table 3.6 Physical data of substituted 2-aryloxybentaIdehyde semicarbazones

Table 3.7 Physical data of 4-benzoy loxybenzaldehyde. 4-pheny Isul fony loxy- benzaldehyde and 4-phenylsulfonylbe~Idehyde semicarbazones and related compounds Table 3.8 Physical data of 4-phenylthiobeddehyde, J-phenylthioacetophenone and 4-phenylthiopropiophenonesemicarbazones and related compounds

Table 3.9 Physical data of 4-(4'-fluoropheno?cy)- and 4-(4'-fluorophenythio)- benzaldehyde thiosemicarbazones and guanylhydrazones

Table 3.10 Physical data of 4-(4'-fluorophenoxy)- and 4-(Ct-fluorophenythio)- benzaldehyde acy hydrazones

Table 3.1 1 Physical data of 4-substituted benzaldehyde semicarbazones

Table 3.12 Physical data of 1,4-bis(4'-fomylphenoxy) bissemicarbazone

Table 3.13 Physical data of 3-arylpropenal semicarbazone and related compounds

Table 3.14 Physical data of some bisarylketone semicarbazones

Table 3.15 Physical data of some 1,3-diaryl-2-propen-1-onesemicarbazones

Table 3.16 Physical &ta of 1,5-dipheny i-2,4-pentadien-3-one sernicarbazone

Table 4.1 Anticonvulsant evaluation after innaperitoneal injection into mice and oral administration to rats of the compounds in Series 1 - XVI

Table 4.2 Evaluation of selected compounds in the MES, scPTZ and neurotoxicity screens after intraperitoneal injection in mice

Table 4.3 Evaluation of selected compounds in the MES and neurotoxicity tests after oral administration to rats

Table 4.4 Evaluation of selected compounds for protection against seizures induced by subcutaneous injection of bicuculline7 picrotoxin and strychnine in mice

Table 4.5 Evaluation of selected compounds on the threshold for minimal seizures induced by the timed intravenous inttsion of pentylentetrazol in mice

Table 4.6 Evaluation of selected compounds against stage V seizures in the corned-kindled rats

Table 4.7 Evaluation of selected compounds against stage V seizures in the hippocampal-kindled rats

Table 4.8 Mean baseline and vehicle values (SEM) for behavioral and electrical seizure parameters using the amygdala kindling rat model

Table 4.9 Changes in mean behavioral seizure stage and percent reduction in seizure stage for in amygdala-kindled rats Table 4.10 Effects of N4on seizure parameters in amygdala-kindled rats

Table 4.1 1 Effect of N4on [ca2'-ji transients of mouse cerebellar granule cells (6-9 days in vitro)

Table 4.12 Effect of 7-day subchronic oral administration of N4and some related antiepileptic drugs (1OOmgkg) on the liver drug metabolizing eve system of rats

TabIe 4.13 Evaluation of selected compounds in the adult epileptic chicken model and comparison of the results with other epiieptic screens

Table 4.14 Pharmacological activity profile of IV4 and clinically used antiepileptic bgs

Table 4.15 Distances between the C 14 atoms and the centres of the proximal and distal rings in I&, IVI,N5, IV, and Vml and the angles 0, and ed

Table 4.16 Distances and angles of displacement of the centres of the aryl rings f?om the N3-C 14402 or 0)-N2plane in IIII, lVI,IVS, Nn_ and VIIIl

Table 4.17 Distances between the C14 atoms and the centres of the proximal and distal rings in I&, NI, WI, Nz and V'IIIl and the angles 8, and ed calculated using the Hyperchem II programme LIST OF SCHEMES

Scheme 4.1 Formation of an aminohydrin 114 Scheme 4.2 Elimination phases of imine formation 115 Scheme 4.3 General mechanisms for formation of irnine derivatives 1 16 Scheme 4.4 Formation of I& L 17

Scheme 4.5 Reaction mechanism for the formation of aryloxyaryi aldehydes and 1 I8 related compounds LIST OF ABBREVIATIONS

ADD program antiepileptic drug development program ADD afterdischarge duration AD afterdischarge threshold AEDs antiepiIeptic drugs ASP anticonvulant screening project ASTM american society for testing and materials BSS behaviorai seizure score [ca*']i calcium ion influx CBZ carbamazepine CD97 convulsive dose c.1. confidence intend CNS central nervous system EDSO median effective dose EAA excitatory amino acid EEG electroencephalogram GABA y-aminobutyric acid GAB A-T GmAa-ketoglutarate transaminase ILAE international league against epilepsy i.p. intraperitoneal 1.v. intravenous MR molar refractivity NM national institutes of health NNCDS national institute of neurological and communicative disorders and stroke NMDA N-methy I-D-aspartate NMR nuclear magnetic resonance NT neurotoxicity MES madelectroshock screen P.I. protection indices (PI viz. TD50/ EDSO) p.0. per oral QSAR quantitative structure-activity relationships SA surface area SAR structure-activity relationships S.C. subcutaneous scBic subcutaneous bicuculline scMET subcutaneous rnetrazol scPTZ subcutaneous pentylenetetrazol scPic subcutaneous picrotoxin TDso median tosic dose TLC thin layer chromatography TPE time of peak effect TTE threshold tonic extension VSCC voltage-sensitive calcium channels

xix INTRODUCTION

1.1.0.0 A brief histow of e~ilepsvand antie~ilepticdrup develo~ment

Epilepsy is one of the oldest of the common human ailments and is mentioned in ancient writings such as the Babylonian Code of Hammurabi 2084 BC and in the Hebrew scriptures. Epilepsy has been known since the beginning of history and over the millennia it has been referred to as "the dread disease", "the sacred disease" and "the falling sickness" (Temkin, 1971 : O'Leary and Golding 1976). According to the Indian system of medicine, Ayurveda, epilepsy has been defined as apsmara; apa meaning "negation or loss of" and smara meaning "recoUection or consciousness".

The history of epilepsy abounds with records of association with evil spirits and curses, hence it is a litany of human suffering and medical ignorance. The first known monograph on epilepsy "On the sacred disease" was written by Hippocrates about 400 B.C. (Temkin, 1971). Although Hippocrates recognized epilepsy as an organic process of the brain, many ancient writers considered it to be a product of supernatural forces that were usually believed to be evil; thus, the term epilepsy comes £?om the Greek word, epilambanien meaning "to be seized by forces from without". During the middle ages, with the advent of Christianity, epileptics were considered to be possessed by evil spirits which tormented victims by the onset of seizures. Consequently, the treatment of epilepsy emerged from ignorance, religious beliefs and superstition. Until the 19th century, patients with epilepsy were subjected to remedies of antiquity which inevitably resulted in the patient's death, as illustrated by the record of treatment given to King Charles I1 at the time of his death (Swinyard, 1980). "In 1685, the king [Charles II] fell backward and had a violent convulsion. Treatment was begun immediately by a dozen physicians. He was bled to the extent of 1 pint fiom his right arm. Next, his shoulder was incised and cupped, depriving him of another 8 oz. of blood. After an emetic and two purgatives, he was given an enema containing antimony, bitters, rock salt, mallow leaves, violets, beet root, camomile flowers, fennel seed, linseed, cinnamon, cardamom seed, s&on and aloes. The enema was repeated in two hours and another purgative was given. The king's head was shaved and a blister raised on his scalp. A sneezing powder of hellebore root and one of cowslip flowers were administered to strengthen the king's brain. Soothing drinks of barley water, licorice and sweet almond were given, as well as extracts of mint, thistle leaves, rue, and angelica. For external treatment, a plaster of Burgundy pitch and pigeon dung was applied to the king's feet. After continued bleeding and purging, to which were added melon seeds, manna, slippery elm, black cheny water, and dissolved pearls, the king's condition did not improve and, as an emergency measure, 40 drops of human skull extract were given to allay convulsions. As his condition grew increasingly worse, pearl julep and ammonia water were forced down the dying king' s throat. ' '

The advent of the renaissance era brought about the renewed influence of physicians and the written word. Early attempts at chemical treatments were made by the iatrochernists of the time. Unfortunately the 1800's saw a return to superstitious beliefs. such as occult forces or sexual activity being the instigators of seizures. In fact, to the end of the 19th century the theory that masturbation was involved in the etiology of epilepsy was accepted to the point that castration was an accepted medical treatment of severe cases (Solomon et al., 1983). Such superstitious beliefs were gradually discarded with the advancement in medical sciences.

The first actual treatment of epilepsy was recorded in 1857, when Sir Charles Locock introduced potassium for the treatment of catarnenial seizures (Locock. 1857). Although it was used regularly for the next 50 years, it was found to cause severe skin eruption and psychosis, prompting a search for less toxic drugs. Further advances occurred during the late 1800's, due to the work of John Hughlins Jackson, who postulated that the fbnction of the brain was based on electrical activity (Jackson, 193 1) and he believed a convulsion occurred due to alterations in discharges of nerve tissues which caused a "storing up" of electrical activity until it reached a certain degree when it could be spontaneously discharged causing abnormal physiological discharges. The first synthetic - anticonvulsant drug, was introduced in 1912 by Hauptmann. This drug proved to be more effective than and soon become the drug of choice in controlling seizures although it was associated with significant side effects (Hauptmann, 1912). In the absence of experimental models of seizures which could be used to test anticonvulsant activity, the discovery of the antiepiletic effect of bromide and phenobarbital was serendipitous.

The development of the electroencephalogram (EEG) by Hans Berger in 1929 (Gloor, 1974) and the correlation of EEG patterns to different seizure types led to important developments in the classification and treatment of epilepsy (Gibbs q aJ.. 1938). The year 1937 marked the beginning of the experimental evaluation of promising anticonvulsant chemicals prior to clinical use. Using a seizure model based on a new electroshock technique for producing convulsions in animals (Spiegel. 1 937), Menitt and Putnam ( t938a and 1938b) screened a group of compounds supplied by Parke-Davis and discovered the anticonvulsant properties of diphenylhydantoin (phenytoin). The discovery was very important because it was the result of efficacy testing in animal models of epilepsy, and unlike phenobarbital, phenytoin was not a sedative and had a wider spectrum of activity. Phenytoin is still the most widely used anticonwlsant despite its serious side effects. In 1944, Richards and Everett introduced the first anti-absence drug, and they also showed that these seizures were prevented by phenobarbital, but not phenytoin (Richards and Everett, 1944). In 1948 Goodman and his coworker standardized the maximal electroshock seizure (MES) test and later introduced the subcutaneous pentylenetetrazol (scPTZ), I Metrazol (scMET) seizure threshold test. The impetus generated by the discovery of phenytoin and later trimethadione resulted in the laboratory screening, using standardized animal models of epilepsy, of thousands of candidate drugs. Between 1945 and 1950, several investigators conducted tests with a variety of seizure models, but failed to tind one in which all drugs were active. In 195 1, Chen et al., investigated the anticonvulsant activity of approximately 65 phenylsuccinirnides and found that among the most potent antipentylenetetrruol compounds were phensuximde and methswimide (Chen gt d., 1951), and a third , ethoswcirnide was introduced in 1960 for the same purpose. Interestingly, all antiepileptic drug developments from 1912 to 1960 were based on a simple heterocyclic ring structure ( Table 1.1 ). During this period, genuinely novel structures were ignored in the development of antiepileptic drugs; instead, attention centered on the , , , succinirnides and acetylureas. There followed a period of marked decline in the development of antiepileptic drugs, from 196 1 to 1973, as it was realized that most of the drugs in use contained a modified ureide moiety embedded within their chemical framework (Gallery, 1982). This structural theme had been extensively developed, prompting some researchers to suggest that hrther elaboration would not lead to an improved drug. Moreover, the search for new antiepileptic agents had failed to produce significant numbers of new lead compounds possessing novel structures.

In the late 1960s. research in antiepileptic drug development was stimulated by the creation of the Epilepsy Branch and the Epilepsy Advisory Committee within the United States National Institute of Neurological and Communicative Disorders and Stroke (MNCDS). These programs were established to collate and review the neuroscience literature pertinent to epilepsy and encouraged to screen large numbers of potentially new anticonvulsants. From this point, the development of new drugs concentrated on different molecular structures, resulting in the marketing of carbamazepine ( 1974). ( 1975) and valproate ( 1978). Most recently, a number of new antiepileptic drugs have been approved or are at the moment awaiting approval in a number of countries and these drugs include (l989), (l99 l), (l993), , and zonisarnide. In addition, a number of active compounds with novel structures have been identified and are at various stages of preclinical and clinical development and these drugs include , eterobarb, remacemide, , flunaridne, and .

1.2.0.0 Classification of epilepsies

Epilepsy is one of the most common neurological disorders characterized by recurrent, usually transient, seizures having a sudden onset and a spontaneous resolution (Parker, 1984). Epilepsies are due to the activation or inactivation of cerebral neurons which exhibit an abnormal and sudden degree of electrical discharge. This synchronous electrical discharge results in an area of aberrant tissue called the focus or focal lesion.

The differential diagnosis and classification of epilepsy are vital to the treatment of epilepsy. The majority of epilepsies are primary or idiopathic in which no identifiable cause can be determined. A second smaller group is comprised of the secondary or organic epilepsies in which case seizures exist in conjunction with an identifiable precipitating factor. Many precipitating factors can trigger abnormal electrical discharges in individuals and subsequently cause seizures. Precipitants of seizures include metabolic disorders, head injury, central nervous system infections, degenerative diseases such as Alzheimer's disease and multiple sclerosis, drug overdose and abrupt drug withdrawal. Table 1.1 Chemical structures of antiepileptic drugs

Nature of X Class of Druq Re~resentative Substitution drugs fiom ( RL,R2 and R3) each class

COW Barbiturates Phenobarbital RI = C&, R2 = C2H5,Rj = H NH Hydantoins Phenytoin RI and R2 = C&, R3= H

0 Oxazolidinediones Trimethadione RI,Rz and R3 = CH3 cH2 Ethoslucimide RI = C~HS,R2 = CH3, R3 = H

m2 Acetylureas * RI = C&. R2 = H, R3 = H * Structure represented below

Phenacemide

Carbamazepine Valproic acid . Epileptic seizures are classified on the basis of the aSected cerebral area and the subsequent clinical symtomatology (Porter a 4.. 1984 ; Dreiffus. 1990). The classification of epileptic seizures have become more complex as neurological abnormalities underlying seizure activity have been more completely understood. The accepted scheme for clinical and electroencephalographic classifications of epileptic seizures was first developed by the International League Against Epilepsy (ILAE) in 198 1 and subsequently it was modified in 1985 and 1989 respectively (Commission on Classification and Terminology of the ILAE, 198 1, 1985, 1989). A simplified version of the international classification of epileptic seizures is given in Table 1.2, distinguishing the two main types of seizures, namely partial and generalized seizures (Commission on Classification and Terminology of the EAE, 198 1, Dreifbs, 1990).

Partial seizures begin locally, and can be divided into three subtypes : simple partial seizures in which consciousness is not impaired, complex partial seizures in which there is some impairment of consciousness. and partial seizures that evolve secondarily into generalized seizures IDreifks, 1990).

Simple partial seizures are "focal" in that they begin in a limited portion of the brain. These seizures do not disrupt consciousness, and their clinical manifestations reflect the portion of the brain involved, e.g. a twitching movement or tingling sensation on one side of the body.

Complex oartial seizures are characterized by a loss of /disturbed consciousness. Complex partial seizures may occur without other symptoms, with symptoms like those seen with simple partial seizures, and/or with development of automatism. The patient stops what he is doing and unconsciously starts exhibiting inappropriate behavior such as lip smacking, picking of clothes, staring and sometime the patient may also experience visual, olfactory and auditory hallucinations. Many complex partial seizures begin with a simple partial seizure, often termed an aura or warning. Any of the seizures described above may generalize or spread to other portions of the brain leading to partial seizures secondarilv generalized . Once a seizure spreads, its symptoms are indistinguishable &om those of a generalized seizure.

Generalized or non-focal seizures involve large portions of the brain from the outset and are invariably associated with a loss of consciousness. A variety of generalized seizures have been identified and the two most important seizures are tonic- clonic seizures and absence seizures (Bastone, 1986).

Tonic-clonic seizures are the most fiequently encountered of the generalized seizures, which were formerly referred to as "grand mal" seizures. They are associated with an abrupt loss of consciousness and motor control. The patient falls to the ground and suffers tonic-clonic movements, periods of increased muscle tone (the tonic phase), the patient becomes very rigid and begins to secrete large amounts of saliva. This rigid phase then changes to a clonic phase which involves severe rhythmic body contractions and relaxation, urinary incontinence sometimes also ensues (the clonic phase). The clonic phase usually lasts for 2-5 minutes.

Absence seizures were formerly referred to as "petit mal" seizures and are predominantly of childhood onset. Consciousness is partially blunted during these seizures, but motor activity is usually fairly mild. Like tonic-clonic seizures. the onset of absence seizures is sudden and associated with an interruption of ongoing activity. Absence seizures may last from a few seconds to a few minutes, and their disappearance is as sudden as their onset. Absence seizures are rare in adults. Table 1.2 Internationai classification of epileptic seizures'

I Partial seizures (seizures beginning locally)

A. Simple partial seizures (consciousness not impaired)

I. With motor symptoms 2. With somatosensory or special sensory symptoms 3. With autonomic symptoms 4. With psychic symptoms

B. Complex partial seizures (with impairment of consciousness)

1. Beginning as simple partial seizures and progressing to impairment of consciousnes~ a. With no other features b. With features as in partial seizures c. With automatism

2. With impairment of consciousness at onset a. With other features b. With features as in partial seizures c. With automatism

C. Partial seizures secondarily generalized

XI Generalized seizures (bilaterally symmetrical and withou .t locd onset)

A. Absence seizures 1. Classical 2. Atypical B. Myoclonic seizures C. Clonic seizures D. Tonic seizures E. Tonic-clonic seizures F. Atonic seizures

III Unilateral seizures (or predominantly)

N Unclassified epileptic seizures (due to incomplete data)

* Reproduced with permission of the copyright owner @reiffus, 1990) The degree of success obtained with antiepileptic drugs is largely dependent on the type of seizure and the extent of associated neurological abnormalities. The major rationale for initiating therapy should be the prediction that a patient's seizure is of a type that is expected to respond to antiepileptic drugs (Solomon a, 1983). It is unlikely that a wide variety of epileptic seizures could be managed successfidly with just one drug since more than one mechanism may be responsible for the various seizures and drugs usehl for one seizure type may actually aggravate other types. Drugs usehl for the treatment of the different seizure types are suggested in Table 1.3 (Patsalos and Sander, 1994). , phenytoin, lorazeparn and phenobarbital are the drugs most widely accepted for treating status epilepticus which is generally recognized as a clinical emergency (Shaner et al., 1988). The most difficult seizures to control are of the complex partial type and combination therapy is often required. The majority of patients respond to a single drug, and polytherapy should be avoided if possible. Only when all drugs appropriate for use as monotherapy have failed should combination therapy be tried. The traditional antiepileptic drug decreases the incidence or severity of spontaneous seizures. However. drastic procedures such as brain surgery have become an increasingly accepted therapeutic alternative when seizures fail to respond to drugs (Andermann, 1987).

1.4.0.0 The search for new antiepileptic drugs

There has been a resurgence of interest in the development of new antiepileptic drugs with novel structures and mechanistic properties. The magnitude of the problem of epilepsy as a manifestation of a medical or neurological disease can hardly be overstated. Epilepsy is second only to stroke as the most common neurological disorder, and since epilepsy affects people at the peaks of their productive lives, its socioeconomic importance is disproportionate to its prevalence (Engel, 1989 ; Niedermeyer, 1990). Approximately 1.5% of the general population experience recurrent seizures and 7-8% of the population experience at least one seizure during life. Regrettably, less than 50% of these people achieve seizure control with currently available anticonvulsants (Schmidt and Morselli, 1986). Approximately 10% of patients with epilepsy have at least one seizure a month despite optimal doses of antiepileptic drugs (Kupferberg, 1990) and only 70-80% experience partial seizure control (Jones, 1982). Medical therapy, which depends on the seizure type, often fails to produce complete control of the seizures and some of the major problems associated with current therapy are presented in Table 1.4 (Shin and McNamura, 1994 ; Pellock, 1994). Moreover, the use of these drugs is associated with significant untoward effects, ranging from unpleasant cosmetic side- effects to serious hematological or hepatic toxicity, including sedation, teratogenicity, cognitive dulling and blood dyscrasia, (Davies, 1978 ; Eadie and Tyrer, 1989). Despite considerable advances in our understanding of this neurologic disorder and the modes of drug action, development of new therapies is hampered by a lack of hndamental knowledge of drug mechanisms and of epilepsy itself (Levy et al., 1989). Nevertheless. the different selectivities of clinically useful substances suggest multiple mechanisms of action that may involve regulation of several neuronal properties (Ames et aL, 1992).

Since both the primary pathology of epilepsy and the precise mechanisms by which the available anticonvulsants act are not well understood, the development of new agents remains a challenging problem. The vast majority of uncontrolled patients. therefore, are at the mercy of the medications available to them and their sole hope is the eventual development of more effective medications. Thus there is a need to design new anticonwlsant drugs which are more effective than existing drugs in intractable seizures,

d such as those with complex partial seizures and Lennox-Gastaut syndrome; in particular, there is a need to apply drug design techniques to the development of receptor site specific drugs with greater pharmacological efficiency and decreased toxicity (Meldrum and Porter, 1986). Therefore, the search for new anticonvulsant drugs continues to be an active area of investigation in medicinal chemistry (Moreau et al., 1994). Table 1.3 Suggested first- and second- line antiepileptic drugs*

Seizure Type Drug of choicet

Generalized seizures

Tonic-clonic (grand mal) First choice carbamazepine, phenytoin or valproic acid

Second choice acetazolarnide, , clonazepam lamotrighe, phenobarbital, or vigabat~ Absence (petit mal) Fist choice ethoswdmide or valproic acid Second choice clonazepam Myoclonic seizures First choice piracetarn or valproic acid Second choice clobazam, clonazepam, ethosludmide or nitrozeparn Tonic, atonic seizures First choice any of the above

Partial seizures

Simple and complex First choice carbamazepine, phenytoin or valproic acid Second choice acetazolarnide, clobazam, clonazepam, gabapentin, larnotrigine, phenobarbital, primidone or vigabatrin Secondarily generalized First choice carbamazepine, phenytoin or valproic acid Second choice acetazolarnide, clo bazam, gabapentin, lamotrigin, phenobarbital, prirnidone or vigabatrin

* Reproduced with permission of the copyright owner (Patsalos and Sander, 1994) Drugs are listed alphabetically under each subheading 1.5.0.0 Strategies for the development of new antie~ile~ticdmes

The ideal new antiepileptic drug is one that which will be effective in all seizure disorders in all patients and have no adverse effects. It must be easy to monitor, allowing infi-equent clinic visits by the patients, can be administered once or twice daily and is inexpensive. In addition, water solubility, a long half-life and negligible protein binding properties are preferable. However, because of the heterogeneity of epilepsy and interpatient variability in metabolic handling and receptor responses to drugs, the possibility of achieving such a drug is at present negligible (leppik, 1994). Thus, current drug development is based on the target characteristics listed in Table 1.5 and focuses primarily on efficacy and tolerability (Patsalos and Sander, 1994).

There are at least three strategies which are currently used for the development of new antiepileptic drugs : (1) random screening of chemicals of diverse structural categories for anticonwlsant activity, (2) structural variation of known antiepileptic drugs, and (3) rational drug design (or rational drug development). All three strategies have generated clinically usehl antiepiletic drugs (Loscher and Schmidt, 1994).

Historically all standard antiepileptic drugs have been found or developed by serendipity, screening or structural variations of known drugs. The anticonvulsant properties of the sleeping medication phenobarbital were discovered accidentally in 19 12 when Hauptmann a junior physician working on an epilepsy ward, administered it to epileptic patients in the hope that they would sleep through the night (Hauptmann, 19 12). Likewise, the anticonvulsant drug valproic acid was discovered serendipitously in 1962 by Eymard when it was accidentally used as a solvent to dissolve a khellin derivative under evaluation as a potential antiepileptic agent (Kaufhan, 1982 ; Loscher and Schmidt, 1994).

This time-honored "drug discovery technique" of serendipity is unpredictable and inefficient. The important need for new anticonvulsant drugs must be addressed through "rational drug design". The technique of rational drug designing is an evolving technology. Most traditional antiepileptic drugs available today were developed empirically, whereas development of the newer agents has been based on modem knowledge of putative pathophysiological epileptogenic mechanisms. A rational approach to the development of antiepileptic drugs (AEDs) must be based on (a) the knowledge of the molecular and cellular events responsible for epilepsy and @) the choice of an appropriate animal model that reflects all of the pathophysiological processes and symptomatologies of the epilepsies.

The animal models of epilepsy and overview of molecular and cellular events responsible for epilepsy are discussed in sections 1.6 and 1.7 respectively. Some of the drugs developed recently using the rational approach include vigabatrin, tigabine. flunarizine, ralitoline, rnilacemide, and taltrimide. Drugs developed by random screening or structural variation of known compounds include clobazam, eterobarb, felbamate, , gabapentin, lamotrigine, , losigamone, nafimidone, oxcarbazepine, remacemide, stiripentol, topiramate and . An alternative strategy to the traditional structural variation of known drugs is the development of transport forms of known neuroactive compounds to facilitate drug distribution into the brain using a chemical system for delivery, and these are referred to as chemical delivery systems (Pop and Bodor, 1992).

It can be seen that most of the AEDs have been developed by screening or structural variation of known drugs and not by rational strategies based on knowledge of - epilepsy mechanisms. Most conventional antiepileptic drugs exert their activities by more than one mechanism, while severd of the novel agents (designed by rational approaches) are often selective for one type of mechanism. The fact that several of the novel drugs that emerged &om screening projects were subsequently found to act by one or several mechanisms proposed to form novel strategies for drug development (rational approach) only demonstrates that serendipity is still an important factor in drug discovery (Loscher and Schmidt, 1994). Table 1.4 Drawbacks of current antiepileptic therapy*

Available compounds do not treat the disease, but rather control only the symptoms

The majority of antiepileptic drugs fail, not because of efficacy but because of unforeseen toxicity

Current therapy fails to adequately control 50% of patients with severe complex partial seizures

Few current drugs are available for pediatric use

Therapy is lacking to prevent epileptogenesis. Thus, a percentage of patients with head injury or infants with febrile seizures will subsequently develop the epileptic syndrome

Choice of drug is highly limited with respect to treatment of absence seizures

Control of life-threatening status epilepticus with current therapy is to some extent inadequate

Some highly effective drugs produce tolerance and others cause fetal malfonnations

Side effects often results in poor patient compliance

* Adapted €kom Palmer and Miller, 1996

Table 1.5 Minimal target characteristics of a new antiepilpetic drug

As effective as existing therapy

Improved therapeutic ratio ( i.e. less toxic in proporfion to its observed benefit)

Simple pharmoacokinetics with a half-life of 12-24 hours so that once or twice daily administration would be possible

Should not induce or inhibit Liver enzymes and consequently have the potential to cause drug interactions

Should not cause tolerance or withdrawal problems 1.6.0.0 Animal models of e~ilepsv

A very important step in antiepileptic drug discovery is the choice of an appropriate animal model for the initial screening as well as for the more complex procedures that elucidate mechanisms of actions. The ideal animal model would be one that can identi@ all drugs active in all forms of epilepj: independent of the mechanism of action. Unfortunately, there is no single animal model that reflects all of the pathophysiological processes and syrnptomatologies of the epilepsies (Kupferberg, 1992). However, there are several more recent so-called genetic animal models of epilepsy. which resemble idiopathic epilepsy in humans more closely than any other experimental model. The innumerable animal models that are used in epilepsy research have been the subject of one volume (Purpura et al., 1972) and several reviews (Loscher and Schmidt. 1988). However, for practical reasons, the most commonly used animal models are not models of chronic epilepsy but models of single epileptic seizures, in which seizures are induced in small laboratory animals (rats, mice) by simple chemical or electrical means (Loscher and Schrnidt,1988). The most popular and widely used of these models are the maximal electroshock seizure (MES) test and the subcutaneous pentylenetetrazol (scPT.2) seizure test (metrazol I scMET seizure test). The MES test is thought to predict drugs effective against generalized seizures of the tonic-clonic (grand mal) type, whereas the scPTZ test is used to find drugs effective against the generalized seizures of the petit ma1 (absence) type (Woodbury, 1972). The most important epilepsy models that are currently used are summarized in Table 1.6.

The epilepsy branch of the National Institute of Neurological and Communicative Disorders and Stroke (MNCDS) established the Antiepileptic Drug Development (ADD) program in 1975. The three components of the ADD program include the Anticonvulsant Screening Project (ASP), the Toxicology Project, and the support of controlled clinical trials of potential new antiepileptic drugs. Chemists from academia and the pharmaceutical industry submit compounds to the ASP and the anticonvulsant evaluations are carried out as per the guidelines of ADD (Table 1.7). Table 1.6 Experimental models of epilepsy*

- - Models of epilepsy 1 Electricallv induced seizures

a) Threshold models (minimal and ~~~a.vimalelectroshock seizure threshold ) b) Maximat electroshock seizure (MES)test (generalized tonic seizure) c) KindIed (focal and secondarily generalized) seizures induced by repeated stimulation of various brain regions, the amygdala being the most sensitive structure

2 Chemicallv induced seizures

a) Chemoconvulsants inducing generaiized seizures after systemic administration Pentylenetetrazol (generalized clonic and (in higher doses) tonic seizures) Other GABA antagonists (e-g., bicuculline, picrotoxin. 'cage convulsants'. convuIsant barbiturates. penicillin, hdane) Inhibitors of GABA synthesis Inverse receptor agonists Glycine antagonists (e.g. strychnine) Cholinomimetic drugs (e.g. pilocarpine) Excitatory amino acid receptor agonists (e-g., NMDA and kainic acid) MiscelIaneous convdsants (e.g., y - hydroxybutyric acid DDT (myoclonus. petit ma1 seizures), rnethionine sulfoximine, flurothyl ) b) Chemoconvulsants used to induce fdseizures (after centrai administration), cg.. penicillia kainic acid, quinolinic acid, pentylenetetrazoi, ouabin

3 Genetic animd models

a) With spontaneous recurrent seizures Epileptic dogs (focal seinues, generalized tonic-clonic seizures), rats with petit mal epilepsy, tottering mice (focal seizures, petit ma1 seizures), AE mice (tonic-clonic seizures). quaking mice and BIO 86.93 mutant hamsters (spontaneous and reflex myoclonic and generalized tonic seizures), C57BUlOBg mice (generalized seizures)

b) With reflex seizures Baboons with photomyoclonic seizures, photosensitive fowl (generalized toniccIonic seizures), audiogenic seizure susceptible mice and rats ( running fits, generalized tonic- clonic seizures), gerbils (facial myoclonic and generalized myodonic and tonicclonic - seizures in response to handling, change in environment or air blast), EI mice (limbic and generalized seizures in response to vestibular stimulation)

4 Focal seizures induced by (topical convulsant) metals applied to cortical areas

5 Neuro~hvsio~o~cdseizure models using recordings &om single neurons in intact animals, isolated tissue preparations or tissue cultures

* This table summarizes the most important models that are currently used. For additional models see Purpura et al., 1972 and Loscher and Schmidt, 1988 Table 1.7 Anticonvulsant drug development'

Phase 1 ~nticonvukantidentification

a mice1.P. Dose range: 30, 100, and 300 mgkg Tesu : MES, scMET(scPTZ),rotorod. and general behavior Time of test : 112 and 4 hours b rats P.O. Dose : 50 mgkg Tests : MES or scPTZ and minimal neurotoxicity Time of test : 1/4, 1/2, 1.2, and 4 hours

Phase 2 Anticonvulsant quantification

a mice I.P. TPE :MES, scPTZ, rotorod EDSo: MES, SCPTZ TDsO: rotorod b mice P.O. TPE : MES. scPTZ. rotorod EDSo: MES, SCPTZ TDSo: rotorod c rats P.O. TPE : MES, scPTZ, minimal neurotoxicity EDs0 : MES, SCPTZ TDSo: minimal neurotoxicity

Phase 3 Anticonvulsant drug differentiation

a mice LP. EDSo: scBic EDso: =Pic EDSo: scStr b mouse whole brain ( in vitro ) benzodiazepine receptor binding GABA receptor binding adenosine uptake c mice LP. timed i.v. infusion of MetrazoI (Pentylenetetrazol) d rats P.O. TPE : EST (kindled) EDs0: EST (khdled)

Phase 4 Toxicity profile, mice I.P.

behavior induced by 1 TDSo,2 TDSos, and 4 TDSOS TPE : loss of righting reflex HDSO: loss of righting reflex

Table 1.7 Contd. ... Phase 5 Subchronic administration and overt tolerance studies

a Subchronic administration : in vivo tolerance EDM for 5 days MES or scPTZ sleep time b Subchronic administration : Iiver parameters

1 EDw or TDJ or 100 mgkg for 7 days weight rnicrosomal protein yield qtochrome P-450 concentration p-nitroanisole Odemethylase NADPH cytochrome c reductase UDP-glucuronosyltransferase @-nitrophenol) qtosol glutathione S-transferase (l

significant differences in I, then all or some additional isozyme selected qtochrome P-450 oxidations ethoxyresorufin deethylase activity pentoxyresoruh dealbylase activity norbenzphetamine MI complex formation p-nitrophenol hydroxylase activity erythromycin demethylase activity troleandomycin MI complex formation UDP-glucuronosyltranferase of 1-naphthol estrone flllfotransferase (cytosol) p-nitro

3 significant induction in I or 2 a) phenytoin MES b) phenytoin hydroqlase in vitro

4 signifmnt inhibition in I, acute dose (EDl6) on a) phenytoin (EDts) MES b) phenytoin hydroxylase in vitro c) hexobarbitai sleep time

Phase 6 Pharmacodynamic interactions (with prototypic drugs), mice LP.

TPE : MES, SCPTZ, rotorod EDs : MES, scPTZ TD, : rotorod * Table reproduced with permission of the copyright owner (Levy et al., 1989) The anticonvulsant evaluation of compounds described in this project was carried out by the ADD program using their reported protocols (Table 1.7). Active anticonvulsant compounds flow through a series of tests designed to give information on their pharmacodynamic and pharmacokinetic profiles (Levy et al., 1989).

1.6.1.0 Electricallv-induced seizures

Electrically induced seizures are the most frequently used animal model for the identification of anticonvulsant activity. Using either mice or rats (although other species have been used) an electrode is either placed directly on the cornea or ear or implanted into the cortex or hippocampus. Depending upon the intensity, wave form and frequency of the current delivered, three major types of electrical seizure models can be differentiated (Swinyard, 1972). (1) Threshold models, in which the current (or voltage) necessary to elicit a minimal (clonic) or maximal (tonic extension) seizure is quantitated, (2) the MES test with supramaximal stimulation; and (3) focal electrical stimulation. as performed in the kindling model, in which repeated stimulation of a brain region with initially subconvulsive electrical stimulation leads to the development of focal and secondarily generalized seizures.

Due to the difference in the severity of convulsions, the MES test is used to identify compounds that prevent seizure spread and represents a good grand mal seizure model. The minimal electroshock induced seizure was considered initially as a model for petit mal, but later it was not validated as a good model for petit ma1 seizure. True models of partial seizures, such as the kindling test are used to test anticonvulsant efficacy against partial epileptic activity (Loscher and Schmidt, 1988). 1.6.2.0 Chemicallv-induced seizures

Innumerable chemicals induce seizures at toxic doses and hence chemicals that induce seizures can be used as a primary screen of anticonvulsant activity. They produce both clonic and tonic seizures when administered parenterally. The most commonly used model involving a systemic administration of a chemoconvulsant is the scPTZ model

(pentylenetetrazol, metrazol, leptazol). Pentylenetetrazol induces generalized clo NC and, in higher doses, tonic seizures using different routes of administration. By means of i.v. infusion, the threshold doses to the different components of PTZ seizures can be quantitated. Convulsant doses of PTZ after i-p. administration are similar to those after

S.C. injection, but latency to the seizure is somewhat shorter. A PTZ-induced tonic(maximal) seizure can be blocked by those antiepileptic drugs which are also effective in the MES test, whereas PTZ-induced clonic seizures are widely used as a model that predicts drugs effective against generalized seizures of the petit ma1 (absence) type (Woodbury, 1972). The in vivo tests used to elucidate possible mechanisms of drug action include seizures in mice induced by bicuculline (GABA receptors), picrotoxin (chloride channels), and strychnine (glycine receptors).

Thus for the routine identification, quantification, and evaluation of anticonvulsant activity, the following five tests are used; MES test, subcutaneous pentylenetetrazol seizure threshold test, S.C. bicuclline, S.C. picrotoxin, and S.C. strychnine seizure pattern tests.

1.6.3.0 Genetic animal models of e~ile~sy

Despite the specific potential of genetic animal models for the development of new antiepileptic drugs. they are only rarely used in preclinical drug testing. As shown in Table 1.6, genetic animal models can be subdivided into animals with spontaneously occumng recurrent seizures, and models in which seizures are induced by specific sensory stimulation in genetically susceptible animals. Animals with chronically recurring, spontaneous seizures represent ideal models for human epilepsy; however, the disadvantage of such models for drug evaluation is that in most of the animals the naturally occurring seizures cannot be elicited at will by an investigator, which makes drug efficacy studies time-consuming, especially when the seizure frequency is low. The advantages and drawbacks of these models have been reviewed recently (Loscher, 1984. Loscher and Schmidt 1988).

1.7.0.0 Biochemistrv of seizure eeneration and ~ro~agation

The normal flow of information throughout the CNS is maintained by a relatively delicate balance between excitatory and inhibitory activity within the neurond circuits. Any event that disturbs the delicate balance between excitation and inhibition can produce a seizure or an epileptic discharge. A seizure is a clinical event associated with aberrant electrical activity within the central nervous system. The human brain contains more than 10 billion neurons and predisposition to aberrant electrical activity arises from hyperexcitability of neuronaf tissue manifesting as increased random firing of individual neurons. When a neuron is electrically at rest, a transmembrane charge asymmetry results in a membrane potential of 65 millivolts, with the intracellular compartment being negative with respect to the extracellular compartment. When the neuron becomes electrically excited, voltage regulated ion channels open. This event permits sodium ions to enter the neuron producing a local depolarization in the transmembrane potential. This voltage change then induces conformational changes in the adjacent sodium ion channels and a wave of depolarization subsequently travels along the neuron. This phenomenon is the " action potential" and is the hndamental physiological unit of electrical transmission within the central nervous system. Thus action potential begins with a rapid increase in the sodium ion permeability through voltage sensitive sodium channels. When the action potential reaches the terminus of the neuron, another voltage regulated ion channel, the calcium channel, opens. Coupled to the opening of this channel is the release of a chemical messenger ("neurotransmitter") which diffuses across a synaptic space to an adjacent neuron and interacts with postsynaptic receptors. Excitatory neurotransmitters, such as glutamate or aspartate, dock with the postsynaptic receptor regulated ion channels causing these channels to open. This in turn permits actions to enter the postsynaptic neuron and reestablishes the process of electrical excitation.

The action potential is terminated by voltage sensitive inactivation of sodium channels and activation of voltage sensitive potassium channels mediating the outward movement of potassium ions leading to repolarization of the cell. Chloride ion movement also plays an important role as a carrier of negative current in the regulation of cellular excitability. Thus voltage regulated ion channel proteins and receptor regulated ion channel proteins are of crucial importance to the initiation and propagation of aberrant electrical activity within the brain (Dichter, 1994 ; Faingold, 1992).

1.8.0.0 Tamets for antie~ilepticdru~ develoament

The search for new antiepileptic drugs has not reached the point of focusing entirely on compounds that modify precise neuronal transmission. but it is still based on compounds active in various animal models of epilepsy (Heinemann et al., 1994). Much current research is based on the premise that a greater understanding of mechanisms of epilepsy will facilitate the development of new AEDs or other intervention strategies that better alleviate epilepsy.

Three main problems with present model-specific strategies of AED development are (1) animal models do not reveal anything specific about epilepsy mechanisms (2) none of the seizure studies in current animal models are truly epileptic; rather they are acute seizures induced chemically or electrically and (3) most acute seizure models do not consider different brain regions (Dichter. 1994). Thus. the scientifWmedical community is moving fiom a "model-specific" strategy for AED development to a "mechanism-specific" strategy. How antiepileptic drugs work is inextricably related to how epilepsy itself occurs.

Mechanism-specific strategies to develop AEDs are aimed at the following sitesAeve1 involved with epilepsy and include : (1) drugs that act at an ionic level on synapses and membranes, (2) drugs with actions at the molecular level, with primary effects on receptors, neurotransmitters, and peptides, (3) drugs that act at the cellular level, (4) drugs that act at the level of rnulticelluiar neuronal synchrony and (5) drugs where the evidence for action is only at the organism level (Porter, 1989).

Many of the present AED developments are concept-oriented and are based on the argument that the development of seizures depends on a disturbed balance between excitation and inhibition. This balance is organized on a cellular and on a synaptic level. On the cellular level, depolarizing inward currents such as sodium and calcium ion currents are balanced by repolarking outward currents such as the different varieties of voltage and calcium-dependent potassium ion currents. On the synaptic level. synaptic transmission. which is mostly due to glutanate and other excitatory amino acids and cholinergic neurotransmissions, is balanced by inhibitory neurotransmission predominantly mediated by GABA and glycine. Thus drugs that augment inhibitory neurotransrnissions or reduce excitatory neurotransrnissions are effective in protecting against many seizure models. Presently available AEDs share three basic mechanisms of action nameIy:

( 1) Enhancing central inhibition (2) Diminishing excitatory amino acid transmission and (3) Modulation of ionic channel activity 1.8.1.0 Antieoile~ticdmps that enhance central inhibition

Gamma arninobutyric acid (GABA) is one of the two amino acids (the other being glycine) that hnction as major inhibitory neurotransmitters in the mammalian brain. Two subtypes of GABA receptors have been described and are referred to as GABA, and GAB& subtypes. The GABAA receptor appears to exist in a macromolecular complex (ligand-gated ion channel) which consists of the GABA recognition site, the chloride channel, the benzodiazepine and the -binding site. There are allosteric modulatory sites on the GABAA-receptor complex, several of which are sites of action of commonly prescribed AEDs. GABA is synthesized from glutamic acid by the enzyme glutamic acid decarboxylase and is metabolized by the enzyme GABA a-ketoglutarate nansaminase (GABA-T). GABA-mediated inhibition plays a critical role in the epileptic process (Browning, 1992). Strategies for enhancing GAB A-mediated inhibitions are presented in Table 1.8, and the possible sites of interaction of AEDs on GABA-mediated transmission are portrayed in Fig 1.1.

1.8.2.0 Antieuile~ticdrum that diminish excitatory amino acid transmission

The second major neurotransmitter system believed to be extensively involved in the development of epileptic activity is that which normally mediates CNS excitatory effects and utilizes glutamate and, possibly aspartate, as neurotransmitters. This system is responsible for most information transfer within the CNS and is thought to become excessively active during epileptic events (Mayer and Westbrook, 1987). The five distinct excitatory amino acid FAA) receptors which have been identified include the NMDA receptor, the kainate receptor, the quisqualate (AMPA), L-AP4 and the ACPD receptors. Except for the ACPD receptor all the EAA receptors are believed to be ligand-gated ion channels. The NMDA receptors are believed to be important in learning and memory (Browning, 1992). The NMDA receptor is known to contain multiple regulatory sites (Fig 12) and has become a target for AED development. The receptor is activated by glutamate or aspartate in conjunction with another amino acid, glycine, which acts as an obligatory "co-transmitter" at most synapses. Blockade of the NMDA receptor, either by direct interference with the amino acid binding site or by action at any of the modulatory sites, has antiepileptic effects in a variety of epilepsy models (Rogawski, 1992 ; Meldrum, 1992b). Strategies to diminish the excitatory amino acid transmission are presented in Table 1.9.

1.8.3.0 Antiepile~ticdrugs which modulate ionic channel activitv

The ionic environment is vital to the excitability of neurons and the importance of different ions in seizure spread and propagation have been explained in section 1.7.0.0. The action of anticonvuisant drugs on the passage of ions through cell membranes has been shown to be an important general mechanism by which many of these agents exert their seizure-suppressing effects (Browning, 1992). The importance of membrane stabilization and ionic transport is due to the fact that alteration in neuronal membrane ion conductance can (i) reduce the burst-firing tendency of neurons, (ii) change the properties of the membrane and neurons by rnodifjing their passive and electrical features, and (iii) alter the active transport of ions across the membrane leading to changes in resting and action potentials. Thus drugs having direct actions on neuronal membrane ion conductance can act directly on abnormal epileptic neurons in focus and thus prevent the spread of seizure activity (Saxena and Saxena, 1995). AEDs that reduce repetitive firing due to interference with sodium currents include phenytoin. carbamazepine, valproate, and probably lamotrigine.

Drugs usehi in the treatment of generalized tonic-clonic and partial seizures. with the notable exception of valproate, are ineffective against absence seizures. indicating that the pathophysiological mechanisms underlying absence seizures are distinct from those mediating the other seizure types. Recently, it has been proposed that the antiabsence activity of drugs such as ethoswcimide and dirnethadione (Coulter et al.. 1989)- and possibly valproate as well (Kelly et al., 1990)- is a consequence of their ability to inhibit T-type calcium ion channels in thalmic neurons. The T-type calcium channels have been shown to mediate the burst firing of thalmic neurons (Heinemann et a1 ., 1994 ; Rogawski et al., 1989).

An alternative approach to diminishing excitability is to stimulate the opening of the potassium ion channel. Activation of this channel would be expected either to hyperpolarize neurons and thus inhibit them or to Limit action potential firing by increasing the opposing influence that potassium currents normally have on depoiarizing sodium currents (Porter and Rogawski, 1992). The mechanisms of action of prototypic antiepileptic drugs are presented in Table 1.10.

With the recent development of new molecular biology techniques for the study of CNS functions and the cloning of cDNAs for specific neurotransmitter receptors and ion channels that are targets of AEDs, it may be possible to study more closely the interaction of AEDs with their target receptors or channels. This will assist in elucidating channel and receptor structures and it may also provide insights into the interactions of MDs with receptors and channels. Insights gained From these studies may assist in the design of improved AEDs, which may act on the same receptors or channels as standard AEDs but may have more specific or selective actions. Table 1.8 Antiepileptic drugs that enhance central inhibition'

Mechanism for enhancing central inhibition Antiepileptic drugs

GAB4 GABA ago& GABA prodrugs - muscimol,

Enhanced GABA synthesis and / or synaptic release - benzodiazipines , vigabatrin

GAB A-transaminase inhibition - vigabatrin, L-cycloserine, y-acety1enicGA.B A GABA uptake inhibition - nipecotic acid, tiagabine

Action at GAB AJbenzodiazepine allosteric site -, p carbolines

Action at chloride ionophore site -barbiturates

Non GAB Aergic mechanism -milacemide, taltrimide

* Table reproduced with permission of the copyright owner (Meldrum, 1992a) terminal

benzodiazepines

neurone Cl-

Possible sites of interaction of antiepileptic drugs on GABA-mediated transmission

Reproduced with permission of the copyright owner (Upton, 1994) Table 1.9 Antiepileptic drugs that diminish excitatory amino acid transmission'

Approaches to decrease glutamatergic transmission Antiepileptic drugs

1 Inhibition of glutarninase - azaserine 2 Presynaptic effects on release

a) adenosine (A1 ) receptors b) GABAs agonist -

C) benzodiazepines d) , carbamazepine, lamo trigine e) glutamatd2-amino-4-phosphonobutyrate

3 Postsynaptic antagonists

a) Non-seIective b) NMDAantagonists i) competitive - APH, CPP, D-CPf ene ii) noncompetitive - MK 801, PCP iii) glycine antagonist (HA 966) - 7-chlorokynurenic acid 5,7-dichlorokynurenic acid iv) polyarnine site antagonist - ifenprodil c) Non-NMDA (kainate, quisqualate) antagonist - DNQX,CNQX, NBQX

4 Enhancing glutamate reuptake 5 Long term down regulation of receptors

* Table reproduced with permission of the copyright owner (Meldrum, 1992a) carbamazepine phenytoin

stimulus val~roate* lamotrigine gabapentin? @ felbarnate? excitatory nerve terminal voltage-dependent

glutamate ( felbarnate?

postsynaptic neurone -.- .. - .

NMDA I receptor K+

Fig. 1.2 Possible sites of interaction of antiepileptic drugs on glutamate- mediated transmission

Reproduced with permission of the copyright owner ( Upton, 1994) Table 1.10 Action of various antiepileptic drugs*

Antiepileptic drugs sodium GABAA T-type calcium NMDA receptor channels receptor channels Carbamazepine ++ - -

Phenytoin ++ - - -

Primidone + - ? -

Valproate ++ ?/+ ?/+ -

Barbiturates + + - -

B enzodiazepines i- ++ - - - - + + - Fetbamate + - ? +

Gabapenth + - - ?

Lamotrigine ++ ? ?

* Adapted from Macdonald and Kelly, 1993 1.7,0,0 Conforrnational, stereochemical features and structure-activitv relationshi~sof ~otentialanticonvulsant apents

Structure-activity relationships of anticonvulsant drugs have been extensively investigated in the past few years (Vida and Gerry, 1977 ; Camerman and Carnerman ; 1980 ; Klopman and Contreras, 1984). Several major factors can be cited that have impeded progress in anticonvulsant structure-activity relationships (SAR) research : (a) the chemical diversity of molecules known to possess anticonvulsant activity, (b) the anatomic, physiological and biochemical complexity of the central nervous system and (c) the lack of entirely suitable models of epilepsy. Among the chemically and structurally diverse anticonvulsants agents (Table 1.1). certain structural requirements have been found to be necessary for molecules to be effective against generalized tonic-clonic seizures and absence seizures (Jones and Woodbury 1982). At least two electron donor atoms (usually oxygen or ) in some proximity to a hydrophobic site are required for activity against both MES- and scPTZ-induced seizures. For activity against MES- induced seizures, the hydrophobic group should be a bum phenyl or other aryl group. Smaller alkyl groups confer activity against scPTZ-induced seizures. The incorporation of both aryl and alkyl substituents in the presence of two electron donor atoms has produced agents effective against both petit ma1 and grand mal epilepsies (Jones and Woodbury, 1982).

A stereochemical model for anticonvulsant activity was developed by Cameman and Cameman (1980, 1981) initially on the basis of their study of the similarities in shape between diphenyhydantoin and diazepam. With several structural analyses. Camerman and Carnerman identified a specific geometrical arrangement between two hydrophobic groups and two electron-donating groups that are separated by 2.4-4.6 A and demonstrated that the possession of certain hydrophobic areas and electrophilic functions which occupy relatively similar positions in space, regardless of the gross confonnational features of the molecules, was responsible for anticonvulsant activity. This work had a major impact on studies of seizure-control drug mechanisms. yet the model does not differentiate antiabsence drugs from those effective against generalized tonic-clonic seizures. However, in a review, Jones and Woodbury ( 1982) opposed this point of view and showed that it did not work when it was applied to several other cases involving known anticonvulsant chemicals and the proposed stereochemical model seems to apply only to anticonvulsants effective against generalized tonic-clonic seizures. In addition, Jones and Woodbury (1982) stated that molecular structure descriptors seems to provide only a very Limited amount of information and that they are frequently unsuitable for quantitative correlation and hence it is more desirable to use physicochemical parameters for quantitative structure-activity relationship studies in anticonvulsant chemistry.

When pharmacological activities of the major anticonvulsant compounds were compared with a variety of independent variables, correlations between anticonvulsant activity and partition coefficients, molecular weights and dipole moments have been observed (Jones and Woodbury, 1982 ; Lien et al., 1979). In general, anticonvulsants are of low molecular weight, possess relatively high lipid solubility, are weak acids or bases and are poorly water soluble. Hydrogen bonding has been shown by theoretical (Andrews, 1969) and experimental studies (Wong d., 1986) to be necessary for activity, but there does not appear to be any correlation between the strength of hydrogen bonding and the type or extent of anticonwlsant activity ( Wong et al., 1986). Molecular orbital calculations on a range of anticonvulsants did not reveal any significant correlation between the electronic indices and activity (Andrews and Defina, 1980).

The preponderance of anticonwlsant SAR research during the past several decades has involved the application of qualitative methods. However, it is apparent that quantitative techniques have now been developed to the level of sophistication necessary to extend our knowledge of anticonwlsant mechanisms to the molecular-electronic level. RATIONALE OF THE PRESENT INVESTIGATION

2.1.0.0 Drug desi~nand development

In general, clinically used drugs are not discovered. What is more likely discovered is known as a lead compound (Silverman, 1992). The lead is a prototypic compound that has the desired biological or pharmacological activity, but it may have many undesirable characteristics. The structure of the lead compound is then modified by synthesis to amplify the desired activity and to minimize or eliminate the unwanted properties. Most anticonwlsant agents have been investigated and developed based on results obtained £%om the screening of potential drugs. There are a variety of approaches used to identie a lead compound and these include random screening, nonrandom screening, drug metabolism studies, clinical observations and rational approaches.

The following chemical approaches can be utilized in the structural modifications of a lead compound in order to improve the desired pharmacological properties (Korolkovas and Burckhalter ; lW6, Silverman, 1992).

(I) The preparation of a series of homologous compounds or modifications of compounds by chain branching or by ring-chain transformations which causes changes in tipophilicity and structural features. (2) The preparation of isosteres which involves the insertion of substituents with similar steric, electronic or other physical or chemical properties. Bioisosterism is considered a very good approach to lead modification. (3) Resolution of isomeric mixtures. (4) The concept of disjunction or molecular simplification which involves the synthesis and evaluation of simpler and simpler analogues of the prototypic compound. (5) Preparation of topological analogs i.e. retention of general topological relationships while making alterations in the reference or lead compound.

Structural modifications of the lead compound are designed to achieve specific goals such as the following improvements over the prototypic molecule. (1) The development of more potent analogs. (2) To eliminate or minimize toxic effects. (3) To discover the pharmacophoric moiety and to identify and separate the molecular features responsible for the desired activity and the undesirable or toxic effects. (4) Modification of the pharmacokinetic properties of the compound.

The process of rational drug design has three hndamentai steps: (a) identification and molecular-level understanding of a specific etiologidpathogenic mechanism to be exploited in the drug discovery, @) identification of a clw of molecules (such as heterocycles or peptides) to be exploited as the molecular template, or prototype, for the new drug, and (c) identification of appropriate techniques for determining the properties (such as molecular shape and geometry) of the prototypic drug and related analogues.

The strategies used for the development of novel anticonwlsants (section 2.2.0.0) include application of some of these chemical approaches to structural modification of the lead compound linked with X-ray crystallographic and molecular modeling studies.

2.2.0.0 Develo~mentof novet anticonvulsants

The development of semicarbarones as potential anticonvulsant agents evolved from the modifications and integration of fhctional groups present on compounds already known to possess anticonvulsant activity. Various semicarbazones. thiosemicarbazones and related compounds have been found to exhibit a wide range of biological activities including anticonvulsant activity (Pandeya and Dimmock, 1993). Numerous sernicarbazones, thiosemicarbazones, semicarbazide, thiosemicarbazide, hydrazones, hydrazides and amides have been found to possess anticonvulsant activity (Craig, 1967 ; Popp, 1977 ; Kornet, 1980 ; Clark and Davenport, 1987). Many anticonvulsant agents contain an amide, hide or urea subunit and the Fragment I , which is chemically similar to the sernicarbazones and thiosemicarbazone moieties, has been proposed as being necessary for anticonvulsant activity (Murray and Kier, 1977).

Many currently available anticonwlsant drugs possessing the general formula 2 possess a dicarboximide fbnction (-CONHCO-)which may be associated with toxic side effects (Kadaba, 1984 ; Andrews, 1969). Hence, compounds were designed to be bereft of this group (dicarboximide) yet still fblfill the structural requirements for activity in the maximal electroshock (MES) and/or subcutaneous pentylenetetrazol (scPTZ) screens.

The structural requirements for activity in the MES screen have been stated to be the presence of a large hydrophobic group which is in close proximity to at least two electron donor atoms and for activity in the scPTZ screen, a smaller, less hydrophobic group than is required for activity in the MES screen should be present near to a minimum of two electron donor atoms (Jones and Woodbury, 1982). Nature of X Class of Drug CONH Barbiturates NH Hydantoins 0 Oxazolidinediones CH2 S uccinimides

m2 Acetylureas

In the light of these general requirements for activity, coupled with the knowledge that various hydrazones which contain the hydrazo function (-NHNH-), as well as various amides (-Corn-), have displayed anticonvuisant propenies, the incorporation of the ureido, hydrazo and amidic group into a single hnctional entity namely a sernicarbazido group (-NHNHCOMI2) or the related semicarbazono (=NNHCONI&) groups was considered to be viable plan.

2.3.0.0 Previous work undertaken in these laboratories

Previous studies by Dimmock et al. have produced synthetic anticonvulsants which incorporated these molecular features and yet were structurally dissimilar from many common monocyclic anticonvulsants. In the first study. a series of thiosernicarbazones and semicarbazones of arylidene methyl ketones were prepared and evaluated in the MES, scPTZ and neurotoxicity screens (Dimmock g d., 1986). Seventeen of the 22 compounds examined were active in the MES and/or scPTZ screens when given by the intraperitoneal route in mice (Phase la, NTH screening). Both neurotodcity and lethality in mice were higher in the thiosernicarbazones than the semicarbazono analogs. Since the most active compound 2 was a thiosernicarbazone (scPTZ, mice i-p. EDSO: 6.96rngkg P.I. :10.37), development of this series of derivatives rather than the corresponding semicarbazones was considered more likely to produce potent anticonvulsants.

A second study (Dimmock et al., 1990) involved principally variation of the aikyl groups of some thiosemicarbazones of arylidene ketones and aldehydes. Activity was found in eight of the 14 compounds in the Phase la screen. In addition. removal of the olefinic double bond led to the formation of aryl alkyl ketone thiosernicarbazones and compound 4 displayed activity in the MES test not only by the intraperitoneal route in mice (EDs0 : 18 -97 mg/kg, P. I. : 2.32) but also when given orally to rats (EDSO: 1 6.89 mg/kg, P.I.:4.33).

A third study (Dimmock et a[., 199 1) revealed that 25 of 28 various aryl alkyl ketone thiosemicarbazones and related compounds were active in the Phase la screen. Many of these derivatives demonstrated activity in the MES but not in the scPTZ screens when given orally to rats. No firther improvement in activity was noted with the thiosemicarbazone analogs and hrthermore neurotoxicity and death were observed with the thiosernicarbazone analogs in contrast to the semicarbazone derivatives. Hence the decision was made to prepare sernicarbazone analogs.

Subsequent studies (Dimmock d., 1993, 1995% 1995b) involved the preparation of a number of semicarbazones of various aryl aldehyde and ketones and related compounds (frozen and flexible analogs) for evaluation in the MES and scPTZ screens. Most of the compounds displayed anticonvulsant activity in the MES and scPTZ screens accompanied by neurotoxicity when given to mice by the i-p. route. Quantitative data revealed protection indices of less than 4 in general. However on oral administration to rats, two interesting features were observed. First, marked activity in the MES screen was noted whereby some of the compounds had EDlo figures in the 20-25 m@g range while activity in the scPTZ test was virtually abolished. Second, neurotoxicity was diminished and P.I. figures of approximately 25 were detected in some of the compounds. Most of the semicarbazones had a rapid onset of action and the data generated suggested that a common mode of action may be interaction with chloride channels. Correlations were noted between the o and d values of the aryl substituents, the interplanar angles made by the aryl ring with the adjacent carbimino groups and the shapes of certain semicarbazones determined by X-ray crystallography with the activities in the rat oral MES screen. Molecular modeling studies revealed a number of statistically significant descriptors which contributed to anticonvulsant activity. These studies resulted in the identification of the first lead compound 5 , designated NC 1 13 2 (ADD 199002). The lead compound 5 was generally bereft of neurotoxicity at the maximum dose administered orally to rats (500 mgkg) and yet had significant potency when administered orally to these animals in the MES screen. Compound 5 had an EDs fiwre of 22.3 mdke and P.I. of 44 (rat. D.o.. MES screen). This safety margin compared favorably with established drugs such as phenobarbital and with P.I. indices of 6.7 and 4.7 respectively. This compound was the subject of a NM "red book and its detailed pharmacological evaluation has been described (Dimmock and Baker, 1994).

2.4.0.0 Obiectives of the Present Investigation

1. Svnthetic Chemistrv Obiective : This objective involves the molecular modification of the lead compound 5 (ADD 199002) with a view to improving potency; ideally an EDIo of 1-3 mgkg when administered orally to rats will be achieved.

2. Molecular Modeling Ob-iectives : This aspect of the work involves the use of empirical and semiempirical conformational calculations and X-ray crystallographic studies to evaluate the viability of a binding site hypothesis.

3. Structure-Activity Relationships : Structure-activity relationships (from both qualitative and quantitative viewpoints) of this new group of anticonwlsants would be sought using various physicochemical and computational techniques. including data fiom X-ray crystallography . 4. Pharmacoloeical Mechanism Ob-iectives : An evaluation of anticonvulsant activities in chemical, electrical and genetic epilepsy models was planned as well as probing the mechanisrn(s) of anticonvulsant activity.

5. Physicochemical Studies : This aspect of the work includes determination of log P values, stability studies, QSAR and elucidating the structures of various compounds.

Aryl semicarbazones displaying activity in the MES screen are believed to interact at a specific binding site, and it is likely that the sernicarbazono group and the aryl ring align at complementary areas on a macromolecular complex in vim which has been referred to as the hydrogen bonding area and the aryl binding site respectively (Dimmock et al., 1995b ; 1996). These possible interactions are represented in Fie. 2.1.

Auxiliaw Bindina Area

Arvl Binding Site

Hvdroaen Bondina Area

Fie. 2.1 Proposed binding site of aryl semicarbazones The principal aim of the present study was to investigate the area around the postulated aryl binding site, which is shown in Fig. 2.1 as the auxiliary binding area. The biodata generated on these compounds may atFord a clearer picture of the nature of the postulated binding site. In addition, the primary amino group and methine proton of the semicarbazone fbnction may be replaced by different atoms or groups. The biodata generated will provide evidence regarding the hydrogen bonding properties of the terminal group which the hydrogen bonding area can accommodate.

2.5.0.0 Rationale for selecting series I -XIV

The following series (I -MV) were prepared for evaluation as anticonvulsants with a view to refining the binding site hypothesis and also to delineate structure-activity relationships.

Series I

A previous study by Dimrnock et ai., indicated that the atomic charge on the terminal nitrogen atom was a significant descriptor at the 85% confidence interval (Dimrnock a a., 1993). In order to determine the importance of the terminal arnido amino group of 5 , [\-5.&9 were prepared.

The semicarbazone analogs are virtually insoluble in water but solutions of representative semicarbazones in deuterated DMSO were shown by 'H NMR spectroscopy to be stable and isomerically pure up to and including the time of peak effects in the MES screen. In order to study the stability and effect on bioactivity due to changes in solubility, water soluble derivatives of 5 were prepared using Girard reagents (I)16.~ were prepared in order to study how isosterism may affect bioactivity. The biodata generated on these compounds may afford some appreciation of the size, electronic nature and hydrogen bonding properties of the terminal group which the hydrogen bonding area can accommodate.

Series I

Cornpourid R X

N"2

N"2 NHCH,

NHC,H,

Series U

This series was designed mainly to investigate the area around the postulated aryl binding site ( Fig. 2.1 ) i.e. to delineate the structural requirements for high activity by optimal interaction at the binding site. Additional groups placed in the aryl ring could strengthen attachment at the binding site and increase the potency or alternatively due to aeric interactions the potency may be decreased.

Initially the attachment of a second aryl group, designated the distal ring, to the proximal aromatic ring located nearest to the semicarbazono group was considered (It5). This compound could increase the van der Wads bonding on a receptor and enhance bioactivity. However I& displayed no bioactivity in the MES, scPTZ and neurotoxicity screens when doses up to and including 300 rng/kg were given intraperitoneally to mice (Dimmock et al., 1995a). Nevertheless oral administration of 115 to rats indicated no neurotoxicity at a dose of 500 m@g and results in the MES screen were ambiguous i.e. 0/8, 2/8, 3/8 and 0/16 animals were protected using doses of 20. 40. 80 and 160 mgkg respectively. No oral activity in rats was noted in the scPTZ screen at a dose of 250 mag.

Thus one can conclude that neurotoxicity is not exhibited by a distal aryl ring and the MES activity, while weak, was retained when the compound was administered orally. Subsequently the distal aryl ring of II5 was replaced by I-piperidinyl and 1- morpholinyl groups leading to the formation of n6.7 respectively. The mouse i.p. MES test revealed that & afforded protection after 0.5 and 4 h using a dose of 300 rng/kg, while Ib was inactive under these conditions. No activity in the scPTZ or neurotoxicity tests was observed. The sernicarbazone 117 was inactive in the MES screen when given orally to rats at a dose of 30 mg/kg over the time period 0.25-4hr.

Thus the biodata of 115-7 suggested that the use of an aryl group rather than heterocyclic rings to interact at the auxiliary binding site should be incorporated into the general structure of II. In addition. the realignment of the distal aryl ring at a greater distance from the proximal ring than is found in U5was considered i.e. the placement of spacer groups between the two aryl rings. - Compound RI R2 Rf k

a : I-Piperidinyl group, b : 4-Morpholinyl group, c : Benzyloxy group

Series M and IV

Based on the results from series 11. the following three structural isomers of phenoxybenzaldehyde semicarbazone (IIIl. IV, and VII) were synthesized. These isomers differ in the location of the phenoxy group to the proximal aryl ring. The ortho (MI) and the meta (III,) isomers were either inactive or demonstrated weak anticonvulsant properties in the mouse and rat screen and the para isomer (IV,) afforded good protection in both tests (Table 4.1). The structure of aryloxyaryl semicarbazones may be conceptually divided into four regions viz. the distal aromatic ring, spacer group, proximal aromatic ring and the semicarbazono group. Molecular modification of IVI were carried out systematically in the above four regions in order to gain fiuther insight into the nature of the putative binding site. It is conceivable that while the phenoxy group can aLgn at different places on the auxiliary binding site, the presence of a distal ring in the "para" location to the proximal aromatic ring is preferable. In other words, favorable interactions of the distal ring and an area designated the distal binding site may occur (Fig. 2.2).

Auxiliarv Bindincr Area

Distal Bindim Site

Distal Awl Ring Awl Bindincr Site

Proximal Arvl Ring

Hvdrorren Bondina Area

Fie. 2.2 Proposed Binding Site of Aryloxyaryl ~emicarbazokes In order to discern the magnitude of the distal binding site, groups of varying sizes were placed at the aryl ring. Since IV4 and Nn showed good anticonvulsant activity, the aryl substitution pattern in series IV was developed using principally halogens and in particular fluorine (N2.19)as wen as alkyl groups (Nuuo).

Series III

Compound R

a : 3 -(3'-~rifluorometh~l~henox~)group Series LV

Compound R R~ Compound RI R' Series V

Replacement of the methine proton by small alkyi groups leading to series V was considered for the following reasons. First, if the compounds in both series IV and V initially align at the hydrogen bonding area in an identical way then the presence of small alkyl groups in series V may cause changes in the positioning of the aryl rings in V compared to IV. This possibility may be evaluated by X-ray crystallography and molecular modeling. The biodata generated could afford valuable information as to the size and location (relative to the hydrogen bonding area) of the aryl and distal binding sites. Second, the alkyl groups could interact at a hydrophobic pocket by forming van der Wads bonds thereby assisting alignment at the receptor site leading to an increase in anticonvulsant activity. Alternatively unfavorable steric interactions between the alkyl groups in V and a place on the receptor could occur resulting in a lowering of potency.

Series V

*, ,lu-C-N-N=C H pi 4

Compound R' R' Series VI

Since series V displayed good anticonvulsant properties , the decision was made to prepare the structural isomers VI. The sernicarbazone VII was inactive in the mouse intraperitoned screen and afforded only weak protection when given orally to rats in the MES test (Table 4.1). Compounds VIzs were proposed since reduction or elimination of anticonvulsant properties in series VI would confirm the limitations in the size of the sites on the receptor where interactions with the distal aryl rings occurs.

Series VI

Compound R' R~ Wl H H Series VII

This series was designed which incorporated different spacer groups which could affect not only the distances behveen the two aryl rings but also their orientation in relation to each other. In other words, a lack of coplanarity between the proximal and distal rings may vary among the compounds in series W and hence the biodata generated would afford some insight into the area on the receptor with which the distal aryl ring aligns.

Series VII

Compound RI R2 WI 0CH2 H

m2 OCO H

m3 OCO CI

m4 so2 H WS OSOz H

m6 osoz (333

Series Vlll

Since the conversion of some biologically active compounds into the corresponding partial bioisosteres has led to retention of activity and increased potency (Taylor and Ke~ewell,1981). series Vm was contemplated. In this series, isosteric replacement of the oxygen atom of representative compounds in IV and V by sulfbr was planned. Series VIII

Series IX and X

In order to gain an improved understanding of the steric and electronic requirements of the hydrogen bonding area, the decision was made to prepare compounds in series IX and X whose anticonvulsant activity could be compared to certain 0x0 analogs in the semicarbazones IV and IX. In order to discern the importance of the semicarbazono amino group, the decision was made to prepare series X in which the amino finetion was replaced by substituents with different hydrogen bonding capabilities. Thus bioevaluation of series IX and X may shed some light on the size of the hydrogen bonding area on the binding site. Series IX

Series X

Compound RI R2 The importance of the distal aromatic ring was evaluated by the replacement of the phenyl ring of IVl by a heterocyclic(4-pyridyl), P napthyl and 3- dimethylaminopropoxy groups, which may lead to compounds with differing alignments at a binding site and also induce an alteration in the spatial arrangements between the distal and proximal aromatic rings.

-a : p Napthyl group ; b : Pyridyl group; g : 3-Dimethylaminopropoxy group

Series ?UI

If the aryloxyaryl sernicarbazono group contributes sigruficantly to bioactivity, then the presence of two such moieties 0may lead to compounds with marked anticonvulsant properties, since two rather than one parts of the molecule may align at the binding site. On the other hand, if W displayed little or no anticonwlsant activity, the result may indicate steric impedance at the binding site i.e. the molecule has too large a group at the 4' position of a phenoxyaryl group to be accommodated. Series XIll

Previous studies by Dimrnock et al. have produced active aryl semicarbazono analogs possessing two carbon atom spacer groups between the aryl ring and the azornethine carbon atom (Dimmock g d., 1993). When the two carbon spacer is unsaturated , the potency was considerably increased. This series is designed mainly to improve the potency of semicarbazone analogs.

Series XIII

Compound R1 R2

=I Br H

m2 C6&FO ' H

=3 H Br

a : 4-Fluorophenoxy group Series XIV

The aryi semicarbazones may be considered to consist of a hydrophobic component as well as a group with hydrogen bonding capacity. If the binding area which interacts with the hydrophobic groups is capable of accommodating hydrophobic groups larger than the presently used functions, then anticonvulsant activity may be increased with additional aryl rings and similar moieties. Secondly, even if only one aryl ring is responsible for the critical drug-receptor (binding) interactions, the alignment may be easier in compounds possessing two aryl rings since either of two groups may position at the appropriate site. Thus some bis-aryl derivatives were also suggested. The biodata generated may give some insight about the hydrophobic portion of candidate anticonvulsants. Series XIV

Compound RI R2 Compound RI R2

XIV I H H XW 7 CI H

xn/ 2 CI CI 8 F H

xw 3 F F xw 9 Br H XIV Br Br XIV to OH H

XIV OH OH XIV 11 OCHj H

XIV 6 OCHj 0CH3 XIV 12 C6H5 H Series XV and XVI

The chalcone semicarbazones (XV and a related analogue XVI) were planned based on previous studies undertaken by Dimmock et aL(1993). Currently, a theory that may explain the anticonvulsant activities is that there are two general binding sites (hydrogen bonding and hydrophobic bonding areas) which act as concertina i.e. they can move to accommodate a wide variety of structures (Fig. 2.3). One of the objectives of preparing this series was to determine the size of the aryl binding site which is probably larger than the diagram in Fig. 2.3 suggested since it was deduced from aryl semicarbazones. Two pieces of evidence suppon this contention. First, replacement of the proton on the carbirnino group of beddehyde sernicarbazone by a phenyl group led to retention of activity in the rat oral MES screen. Second, the insertion of a carbon chain between the aryl ring and the carbirnino carbon atom enabled activity to be retained and in the case of cinnarnaldehyde semicarbazone. potency was increased (Dimmock g

--a1 3 1993). Hvdrocren Bondincl Area /

\ Hvdro~hobicBonding Area

Fie. 2.3 General binding sites for anticonvulsants Series XV

Compound Rl R2

-a : Phenoxy group ; b : 4-Fluorophenoxy group Series XVI

In summary, the principal aim of this study was to prepare a number of aryloxyaryl and related sernicarbazones for evaluation as candidate anticonvulsants with a view to understand the chemical features which contribute to interactions at a binding site. DESCRIPTION OF THE EXPERIMENTAL WORK

3.1.0.0 CHEMISTRY

Drying and purification of solvents and reagents were carried out according to literature procedures (Vogel, 1989a). Weighings were canied out on an analytical Mettler AE 100 balance. Thin layer chromatography (TLC) was performed on Eastman chromatogram sheets, type 13 18 I (silica gel with fluorescent indicator). Compounds were observed under short-wave ultraviolet light or in an iodine chamber. Column chromatography was carried out using Merck's silica gel 60, 70-230 mesh size (ASTM). All compounds reported were homogeneous by TLC unless otherwise stated in the text. Melting points were observed visually on a Gallenkamp MF-370 instrument and are uncorrected. Elemental analyses were performed by Mr. K. Thoms. Department of Chemistry, University of Saskatchewan using a Perkin-Elmer CHN elemental analyzer. All samples were dried in an Abderhdden drying pistol before being analyzed.

'H Nuclear magnetic resonance (NMR) spectra were obtained using a Varian T-60 spectrometer. High resolution NMR spectra were determined on a Bruker AM400 FT NMR spectrometer. Tetramethylsilane (TMS)was used as an internal standard for spectra recorded in -d and dimethylsulfoxide-ds. Inf?ared spectra (IR) were recorded on a Beckrnan Acculab TM 4 spectrophotometer. Ultraviolet (UV) and visible (WS) spectra were recorded on a Gilford UV-VIS spectrophotometer. 3.1.1.0 General method for the ore~aration of 4-bromo benzaldehvde

The preparation of Ilj was achieved by the condensation of 4- bromobenzaldehyde (0.0 1 1 mol) with the appropriate acid hydrazide (0.0 12 mol). For the synthesis of I,, the reaction mixture in (20mL) was stirred at room temperature for 2 h and for the synthesis of I2 the reaction mixture in methanol (30mL)was heated at

60'~for 0.5 h. For the synthesis of 13-s , the Girard reagents D, T and P respectively were dissolved in alcoholic acetic acid (LO%, 30rnL) and the mixture was heated under reflux for 3 h. In alI cases, the precipitates were collected, dried and recrystallized from

95% ethanol (11.3. s), methanol (Iz) or isopropanol a). The purity of the compounds were confirmed by TLC using a solvent system of chloroform : methanol (9: 1) and elemental analyses. The physical data for these compounds are presented in Table 3.1.

3.1.2.0 Svnthesis of 4- bromobenzaldehvde euanvIhvdrazone ( 4 1

A mixture of arninoguanidine bicarbonate (0.OI moi) and 4- bromobenzaldehyde (0.01 rnol) in ethanol : 50% aqueous hydrochloric acid (20mL : 2mL) was heated under reflux for 3h. The reaction mixture was monitored by TLC on silica gel using a solvent mixture of benzene : methanol : acetic acid (9: 1:0.5). On cooling, the solution was basified and extracted with ether. After addition of an ethanol- water mixture (7:3) to the ethereal extract, the reaction mixture was cooled in the refrigerator. The resultant colorless compound which precipitated was purified by recrystallization from methanol to give the title compound. The physical data of the compound are presented in Table 3.1. 3.1.3.0 General method for the preparation of some 4-bromobenzaldehvde

thiosemicarbazones ( 17-91

These compounds were prepared according to a literature procedure @immock et al., 1996). A solution of the appropriate thiosernicarbadde (0.01 mol) in ethanol (95%, 50mL) was added to a solution of 4-bromobenzaldehyde (0.01 moi) in ethanol (95%, 20mL) containing hydrochloric acid (37%, 2m.L). The reaction mixture was heated under reflux with stirring for 3.5 h. On cooling, the precipitate was collected, dried and recrystallized from ethanol (95%) to give the desired product. The physical data for these compounds are presented in Table 3.1.

3.1.4.0 General method for the ~re~arationof arvlsxvarvl or arvlthioarvl aldehvdes

3-Phenoxybenzaldehyde and 3-(3'-trifluoromethylphenoxy)benzaldehyde required in the synthesis of were obtained fiom the Aldrich Chemical Company, Milwaukee, WI. The intermediate aryloxyaryl and arylthioaryl aldehydes required in the synthesis of compounds in Series IV - VI, Vm, XI and XII were prepared using a literature procedure with a slight modification ( Yeager and Schissel, 1991 ; Trust et al., 1979).

To a solution containing the substituted phenol or thiophenol (0.15 mol) and either Cfluorobenzaldehyde, 4-fluoroacetophenone or Cfluoropropiophenone (0.14 mol) in dimethylacetamide (100rnL) was added anhydrous potassium carbonate (0.12 mol). For Series W, Cfluorobenzaldehyde (0.28 mol) was used. The reaction mixture was heated under reflux at lS°C under nitrogen and the progress of the reaction was monitored by TLC using a solvent system of benzene : methanol (9: 1). After = 5- 10 h, the mixture was cooled to room temperature and diluted with water (100rnL). The reaction mixture was extracted with chloroform (2xlOOmL) and the combined organic extracts were washed with aqueous sodium hydroxide solution (4% w/v) and water. After drying over anhydrous magnesium sulfate, the solvent was removed in vacuo and the resultant oil was distilled under reduced pressure to give the appropriate aryloxyaryl or arylthioaryl aldehyde in 70 - 85% yield. The purity of the distillate was checked by TLC and NMR spectroscopy.

The 'H NMR spectrum (300MHz) of representative intermediate (4-phenoxybenzaldehyde) was as follows: 6 (CDCC) : 9.94 (s, LH, CHO), 7.82-7.88 (2t, 2H, ortho H of proximal aryl ring), 7.38-7.46 (m, 2H, meta H of proximal aryl ring), 7.20-7.27 (m, lH, para H of distal aryl ring), 7.03-7.12 (m,4H, ortho and meta H of distal aryl ring).

3.1.5.0 General method for the ~re~arationof awl or aryloxaryl or arvlthioarvI benzaldehvde semicarbazones UI- VI, Vm. XI and XI1 1

A mixture of semicarbazide hydrochloride (0.0 1 moi) and water ( 1OmL) was added slowly to a stirring solution of the aryi, aryloxyaryl or arylthioaryl aldehyde (0.01 mol) in ethanol ( 95%, 30mL). The reaction mixture was stirred at room temperature for 1-28 the precipitate was collected, washed with ether and dried. Most of the compounds were recrystallized from ethanol(95%) except for [VU (methanol). The physical data for these compounds are presented in Tables 3.2 - 3.6 (Series I1 - W), and Tables 3.8, 3.1 1 and 3.12 (Series Vm, XI and XU).

The 'H NMR spectrum (300MHz) of a representative compound 4-(4'-fluorophenoxy)benzaldehyde semicarbazone was as follows: 6 (DMSO-d6) : 9.40 (s, lH, NH), 6.94 (s, lH, CH), 6.82-6.88(2t, 2qmeta H of distal aryl ring), 6.33-6.42(m, 2H, onho H of distal aryl ring), 6.05-6.30 (m, 4Y ortho and meta H of proximal aryl ring), 5.60 (s.2H. NH2). 3.1.6.0 Svnthesis of 4-beazovlowbenzaldehyde semicarbazone and

The intermediate aldehydes required for the synthesis of ViIu were prepared according to literature procedure (McGookin, 1949 ; Kenneth and Walter, 1940). Benzoyl chloride or 4-chlorobenzoyl chloride (0.05 mol) was added to a solution of 4- hydroxybenzddehyde (0.04 mol) in pyridine (100rnL). AAer standing overnight at room temperature, the reaction mixture was poured onto acetic acid (2N, 100rnL). The solution was extracted with ether and washed thoroughly with dilute hydrochloric acid to remove the pyridine and subsequently with dilute aqueous sodium bicarbonate solution (LO%w/v) to remove excess of the aroyl chloride. The ether was removed in vacuo and the precipitate was washed with water and recrystallized from a water-methanol (7:3 ) mixture to give 4-benzoyloxybenzaldehyde m-p. 92-94OC,lit. m.p. 92-94OC (McGookin* 1949) in 80% yield and 4-(4'-chlorobenzoyloxy)benzaldehyde rn-p. 1 17- 1 8OC, lit. m.p. 1 17- 18°C (Raiford and Milbery, 1934) in 76% yield. The intermediate aldehydes were converted to the corresponding semicarbazones as per the procedure mentioned under section 3.1.5.0. The physical data for the compounds are presented in Table 3.7.

3.1.7.0 Synthesis of 4-~henvlsutfonvlbenzaldehvdesemicarbazone (ViQ

The intermediate 4-phenylsulfonylbemaldehyde required for the synthesis of V& was synthesized according to a literature procedure (Ulrnan and Urankar, 1989). A mixture of sodium benzenesulfinate (0.1 1 mol) and Cfluorobenzaldehyde (0.1 mol) in dry dimethylsulfoxide (75mL)was stirred at 100°C for 18 h under nitrogen and the reaction mixture was poured onto ice (-2009). The precipitate was collected. washed with water and recrystallized from ethanol (95% v/v) to give 4-phenylsulfonylbenzaldehyde in 85% yield. m.p. 130-3 1°C (lit. rn-p. 124-25"C, Arnold and Vogeslag, 1956). The purity of the compound was confirmed by elemental analyses and NMR spectroscopy. The 'H NMR (60MHZ) spectrum for 4-phenylsulfonylbenzaldehy de was : 6 (CDCla) : 9.86 (s,lH, CHO),7.98 (t, 4Y aryl H), 7.90 (m, 2H, aryl H), 7.50 (m,3K

ary1 HI-

The intermediate aldehyde was converted to the corresponding sernicarbazone Wq as per the procedure mentioned under section 3.1.5.0. The physical data for this compound are presented in Table 3.7.

3.1.8.0 Svnthesis of 4-~~henvlsulfonvlolrv)benzaIdehvdesemicarbazone and

The intermediate aldehydes required for the synthesis of VIGS6 were synthesized according to a literature procedure (Battersby et d., 1980 ; Crossland and S ervis, 1970). Benzenesulfonyl chloride or 4-methylbenzenesulfonyl chloride (0.20 mol) was added dropwise to a stirred solution of 4hydroxybenzaldehyde (0.16 rnol) in dichlorornethane (90mL) and triethylarnine (3-5mL) at 0- IO°C over a period of 10 min. Mer a fbrther IS min, the reaction mixture was diluted with and successively extracted with water, hydrochloric acid (10% wlv), saturated sodium bicarbonate solution and saturated sodium chloride solution. After drying the organic extract, the solvent was removed affording the products required in the synthesis of V11s.6. The purity of the compounds were confirmed by elemental analyses and NMR spectroscopy .

The 'H NMR (60 MHz) spectrum for 4-(phenylsulfonyloxy)benzaldehyde was as follows : 5 (CDCI3) : 9.98 (s, l El, CHO) and 8.5-8.0(m, 9H,ArH).

The intermediate aldehydes were converted into the corresponding semicarbazones (VIIS6)as per the procedure mentioned under section 3.1.5.0.Physical data for these compounds are presented in Table 3 -7. 3.1.9.0 Svnthesis of 444'- fluoroohenow)benzaldehvde thiosemicarbazone and

The intermediate aldehydes required for the synthesis of the title compounds QX13were prepared as per the procedure mentioned under section 3.1.4.0.. and the corresponding thiosemicarbazone were prepared by a Literature procedure with slight modifications (Dimmock et al., 1996). A solution of thiosemicarbazide (0.0 1rnol) in ethanol (9596, SOmL) was added to a solution of 4-(4'-fluorophenoxy)-benzaldehyde or 4-[(4'-fluorophenyl)thio]benzaldehyde (0.01 mot) in ethanol (95%, 20rnL) containing hydrochloric acid (37%, 2mL). The reaction mixture was heated under reflux with stirring for 5-7 h. On cooling the precipitate was collected, dried and recrystallized fiorn ethanol (95%) to give the title compound. The purity of the compounds was confirmed by elemental analyses and NMR spectroscopy . The physical data for these compounds are presented in Table 3.9.

The 'H NMR (60MHz) spectrum for 4-(4'-fluorophenoxy)benzaldehyde thiosernicarbazone was as follows : 6 (DMSO-&) : 9.68 (s, lH, NH), 6.84(s, lH, CH), 6.06-6.74 (m, 8H, ArH), 5.42 (s, 2H, m).

3.1.10.0 Synthesis of 4-(4'-fluorophenoxv)benzaldehvde ~uanvlhvdrazone and 444'-fluoroahenvl~thiol benzaldehvde rmanvlhvdm

The title compounds were prepared fiom the corresponding 4-(4'- fluorophenoxy)benzaldehyde or 4-[(4'-fluorophenyl)thio]benzaldehyde as per the procedure mentioned under section 3.1.2.0. A mixture of aminoguanidine bicarbonate (0.Olmol) and 4-(4'-fluorophenoxy)beddehyde or 4-[(4'-fluorophenyl)thio]- benzaldehyde (0.0 1 mol) in a mixture of ethanol : SO% aqueous hydrochloric acid (20mL : 2mL) was heated under reflux for 8 h. The reaction mixture was monitored by TL,C on silica gel using a solvent of benzene : methanol : acetic acid (9: 1: 0.5). On cooling, the soIution was basified and extracted with ether. After addition of an ethanol-water mixture to the ethereal extract, the reaction mixture refrigerated. The precipitate was collected, washed with water and the compounds were purified by recrystallization from ethanol (95%) to give the title compound. Physical data for these compounds are presented in Table 3 -9.

The 'H NMR (300 MHz) spectrum for 4-(4'-fIuorophenoxy)benza~dehyde guanylhydrazone was as follows : 6 (DMSO-d6) : 7.83 (s, IH, CH), 7.20-7.65 (m, 4H, ortho and meta H of distal aryl ring), 6.90-7.10 (m, 4H, ortho and meta H of proximal aryi ring), 5.80-5.90 (s, 262NH), 5.45 (s, 2H, w).

3.1.11.0 Svnthesis of 4-(4'-fIuoro~heooxv)benzaIdehvde formvlhvdrazone, 4114'- fluoro~henvlYhiolbenzaldehydeformvlhvdrazone and 4-14'fluorophenoxv)-

benzaldehvde acetvlhvdrazone (XI.3)

The title compounds were prepared by condensing 4-(4'-fluorophenoxy)- benzaldehyde or 4-[(4'-fluorophenyl)t~benzaldehyde (0.0 1 1 mol) with formic acid hydrazide or acetic acid hydrazide (0.0 12 rnol) in methanol (30mL). The reaction mixture was stirred for 24 h at room temperature for Xl,z and for Xj the reaction mixture was heated at 60°C for 0Sh. The compounds were recrystallized from ethanol (95%. v/v). The physical data for these compounds are presented in Table 3.10.

The 'H NMR (300 MHz) spectrum for 4-(4'-fluorophenoxy)benzaldehyde formylhydrazone was as follows : 6 (DMSO-d6) : 8.60 (s, IH, NH). 7.95 (s,lH, CHO), 6.90-7.65 (m, 8H, ArH ), 6.95 (s, IH, CH). 3.1.12.0 Svnthesis of 4-(4'-fluoro~henoxv)benzaldehvde carbohvdrazone and

The title compounds were prepared by condensing 4-(4'-fluorophenoxy)- benzaldehyde(0.0 1 1 mol) with carbohydrazide or oxamichydraride (0.0 12 mol) in methanol (30m.L). The reaction mixture was stirred at room temperature for 24 h. The precipitate was collected, washed with water and dried. The compounds were recrystallized Eom absolute ethanol. The purity of the compounds was confirmed by elemental analyses and NMR spectroscopy. The physical data for these compounds are presented in Table 3.10.

The 'H NMR (300 MHz) spectrum for 4-(4'-fluorophenoxy)benzaldehyde oxamichydrazone was as follows : 6 (DMSO-d6) : 8.48 (s, lH, NH), 7.90-8.25 (d, 2H. NH2 ),7.25-7.65 (m,4H., ortho and meta H of distal aryl ring), 6.98-7.23 (m, 4H, ortho and meta H of proximal aryl ring), 7.00 (s, lH, CH).

3.1.13.0 Synthesis of 3-arvtpropenal semicarbazone and related compounds

cxm 1-3)

a-Bromocimamaldehyde required for the synthesis of XI& was obtained from the Aldrich Chemical Company, Milwaukee, WI. The other substituted cinnamaldehydes were prepared f?om the corresponding aryl aldehyde according to a literature procedure (Billman and Tonnis, I97 1).

A mixture of the aryl aldehyde (0.084 mol), triethylamine orthoformate (0.092 mol) and phosphoric acid (894, 0.2mL) was stirred at 2S°C under a nitrogen atmosphere for 24h. The resulting diethyl acetal was cooled to O°C , and a zinc chloride- ethylacetate (10%) solution was added. Freshly distilled ethylvinyl ether (0.17 mol) was then added dropwise at a rate to maintain the temperature below 0°C. After complete addition, the solution was stirred at room temperature for 12 h. To the reaction mixture, a solution of glacial acetic acid (83rnL), sodium acetate(8.7g) and water (7.7rn.L) was added and the solution was heated at 95°C for 2 h. The solution was then poured into cold water (700 rnL) and the precipitate was collected and dried. The intermediate arylpropenals were converted to the corresponding semicarbazones by the procedure mentioned under section 3.1 S.O. The purity of the compounds were established by TLC. elemental analyses and NMR spectroscopy. The physical data for these compounds are presented in Table 3.13.

The 'H NMR (300 MHz) spectrum for 4-(4'-fluorophenoxy)ci~amaldehyde sernicarbazone was as follows : 6 @MSO-d6) : 7.80 (s, lH, NH), 6.70-7.20 (m, 1 lH, olefinic and aryl hydrogen), 6.2 (s, 26N& ).

A solution of 4,4'-dichlor~benzo~henone(0.0 1 mol) in ethanol (95%, 30mL) was added to a solution of sernicarbazide hydrochloride (0.01 mol) and sodium acetate (0.01 mol) in water (IOrnL). The reaction mixture was heated under reflux for 96 h and the progress of the reaction was monitored by TLC using a solvent system of benzene:rnethanol(7:3). The reaction mixture stood at room temperature and the solvent was removed in vacuo. The precipitate was fbrther purified by column chromatography using ethyl acetate as the solvent. The yield of the title compound was 20 %. The purity of the compound was checked by TLC and elemental analyses. Compound XIVl was synthesized as per a literature procedure (Dimmock et al., 1993). The physical data for the compounds are presented are Table 3.14. 3.1.15.0 General svntbesis of some 1.3-diarv!-2-oro~en-Lonesemicarbazones (XVd

The substituted chalcones were synthesized by the Claisen-Schmidt reaction according to a literature procedure (Vogel, 1989~). To a cold solution of sodium hydroxide (4.4g), water (40mL) and ethanol (95%, 40mL) was added the appropriate aryl aldehydes and ketones (0.086 mot). The reaction mixture was stirred vigorously for 2-3 h. The reaction mixture was lefi overnight in a refrigerator. The precipitate was filtered, washed with cold water untiI the washings were neutral to litmus, and then with ice cold absolute ethanol (1 0m.L). The crude chalcone was recrystallized f?om absolute ethanol.

The intermediate chalcone were converted to the corresponding semicarbazone described in section 3.1.5.0. The physical data for these compounds are presented in Table 3.15.

3.1.16.0 Svnthesis of 1.5-di~henvl-2.4-~entaddee-3-onesemicarbazone (XVQ

The title compound was prepared as per a literature procedure (Vogel, 1989~). To a cold solution of sodium hydroxide(l2Sg), water (125mL) and ethanol (95%. 1OOmL), was added one half of a previously prepared mixture of benzaldehyde (0.125 mol) and (0.0625 mol). The reaction mixture was stirred vigorously. After 15 min, the remainder of the benzaldehyde-acetone mixture was added. The stirring was continued for lh and the precipitate was filtered, washed with cold water to eliminate alkali and dried. The crude precipitate was recrystallized fiom absolute ethanol and converted to the corresponding semicarbazone (XVI) as per the procedure mentioned under section 3.1 S.O. The physical data for the compound are presented in Table 3.16. Table 3.1 Physicd data of 4-hromobenzaldehyde acylhydrazones

Compound R X Yield M.B. Molecular Elemental Analysis (%) (?"4 ("c) Formula Calculated Wound C I H I N I C I H 1 N I I H 0 40 200 C8H7BrN20 42.31 3.10 12.34 42.46 3.06 12.12

I 1

16 NH2 NH 40 209-21Oa CaHsBrNr 39.85 3.76 23.23 39.75 3.77 23.23 17 NH2 S 60 226h CeHsBrN3S 37.22 3.12 16.27 37.23 3.01 16.13 1 H NHCH, S 59 208-210 CUHloBrN3S 39.86 3.35 15,50 39.90 3.67 15-57 19 NHC,H, S 72 196-198 C1JH12BrN3S 50.3 1 3.62 12.57 50.16 3.67 12.38 a : Lit. 1n.p. 209-2 1 1 "C, (Soman et al., 1986) b : Lit. m.p 24 1°C (Bayer, 1953)

Table 3.4 Physical dr tr of substituted 4-aryloxybenzaldehyde semicarbazones

Compound R' it2 Yield M.P. Molecular Elemental Analysis (%) (%) ("c) Formula Calculated Found C I H I N I C I R I N IV 1 H H 60 224-225 CIJHI3N3O2 65.87 513 16.46 65.68 5.05 16.17 Table 3.4 Physical data of substituted 4-nryloxybenzddehyde semicarbazones

Compound R' R~ Yield M.P. Molecular Elemental Analysis (%) (%I ("C) Formula Calculated Found

76 Titblc 3.4 contd. .,. Table 3.4 Physical data of substituted 4-aryloxybenzaldehyde semicarbazones

------pp - Compound R' RI Yield M.P. Molecular Elemental ~nal~sis(%) (%) ("c) Formula Calculated Found C I W I N I C I H I N Table 3.4 Physical data of substituted 4-aryloxybenzrldehyde semicarbazones

Compound R' 3 Yield M.P. Molecular Elemental Analysis (?AD)

Calculated Found Table 3.5 Physical data of substituted 4-aryloxyacetophenone and 4-aryloxypropiophenone semicarbazones

Compound R' R' Yield M.P. Molecular Elemental Analysis (%) (%) ("c) Formula Calculated Found C I H I N I C 1 H I N

UOOOOC n~oovw~ Table 3.8 Physical data of 4-phenylthiobenz~ldehyde, 4-phenylthioacetophenone and 4-phenylthiopropiophenone semicarbrzones and related compounds

Compound R' R* Yield M.P. Molecular Elemental Analysis (%) (%) ("c) Formula Calculated Found C 1 H I N I C I H 1 N VIII 1 H H 40 226-227 CIjHI3N30S 61.97 4.83 15.48 62.11 4.65 15.48 VlIlZ H CH3 60 208-210 CI~HIjN30S 63.13 5.30 14.73 63.06 5.07 14.53 Vlll3 H C2H5 16 131-133 C16HI7N30S 64.19 5.73 14.04 64.1 1 5.53 13.90 VIII., F H 52 230-23 1 CIjH12FN30S 58.12 4.18 14.53 58.04 4.06 14.62 VIII 5 F CHI 9 1 204-207 C I SH jFN30S 59.39 4.65 13.85 59.35 4.82 13,93 VIII 6 F C2H5 18 1 SO-152 CI6Hl6FN3OS 60.55 5,08 13.24 60.13 5.44 12.87 VII1.l CI H 40 216 CIJH12CIN30S 54.99 3.95 13.74 54.96 4.04 13.66 Vill Br H 30 2 12-2 13 CI4HI2BrN30S 48.14 3.47 12.04 48.40 3.16 11.68 Vlll t, Br CH3 46 2 14-2 16 C15H14BrN30S 49.46 3.87 11.54 50.12 3.52 11.23 Vllllo Ch H 32 225-227 CI5HI5N30S 63.13 5.30 14.73 63.39 4.86 14.65 VIII 11 (343 CH3 60 222-224 ClgH17N30S 64.19 5.72 14.04 64.33 5.59 13.82 'hble 3.9 Physical data of 4-(4'-fluurophenoxy)- and 4-(4'4uoropheny t hio)- benzaldehyde t hiosemicarbazones md guanylhydrazones

Compound R' R~ Yield M.P. Molecular Elemental Analysis (YO) (%) ("c) Formula Calculated Found

Table 3.1 1 Physical data of 4-substituted benzaldehyde semicarbazones

- .------. Compound R Yield M.P. Molecular Elemental Analysis (YO)

Calculated Found I

a : p Napthyl group b : Pyridyl group c : 3-Dinietliylami~iopropoxygroup Table 3.12 Physical data of 1,4-bis(4'-formy1phenoxy)benzene bissemicarbazone

Compound Yield M.P. Molecular Elemental Analysis (%) ("/.I ("c) Formula Calculated Found C IHI N I C IHI N

Tiible 3.14 Physicrl data of some bisrrylketone semicarbazones

Compound R' R* Yield M.P. Molecular Elemental Analysis (%) (%) ("c) Forniula Calculated Found C I H I N 1 C I H I N XIVia H H 44 166- 167 CI4Hl3N30 70.28 5.48 17.56 70.56 5.74 17.33

XIV 3 C1 Cl 20 196~ C IAHI r C12N3O 54S6 3.60 13.64 54.48 3.58 13.73 a : Data reproduced with pern~issionof the copyright owner (Dimmock et al., 1993) b : Lit. map.192- 193°C ( Speroni, 1952 ; Pearson elal, 1953) Compound R' R~ Yield M.P. Molecular Elemental Analysis (%) (%) ("c) Formula Calculated Found C I H I N I C I H 1 N xv I ff H 60 168a CI~HI~N~~72.43 5.70 15.84 72.17 5.72 15.56 XVZ F H 49 202-204 CI6HIJN3FO 67.83 4.98 14.83 67.90 5.22 14.87 XV3 H F 40 2 12-214 CI6HI4N3FO 67.83 4.98 14.83 47.76 5.14 14,91 XV 4 F F 20 2 18-220 CI6H13N3FZ0 63.78 4,35 13-95 63.68 4.52 13.99 a : Lit, 111.p. 168°C (Vogel, l98Od)

3.1.17.0 Attemoted svnthesis of 4-~4~hvdro~ohenoxv)benzaldehvdesemicarbazone

The preparation of this compound was attempted as per the procedure mentioned under section 3.1.4.0. To a solution of Chydroxyphenol (0.15 mol), 4- fluorobenzaldehyde (0.14 rnol) in dimethylacetamide (100 mL) was added anhydrous potassium carbonate (0.12 mol). The reaction mixture was heated at 156" C for 24-48h under nitrogen and the progress of the reaction was monitored by TLC using a solvent system of benzene : methanol (94. TLC suggested numerous compounds had been formed and the work up of the reaction mixture did not yield the desired compound. The reaction was attempted at both a lower temperature (100" C) and a higher temperature (175" C) but were unsuccessfid in forming the desired product. Hence the same reaction was repeated by protecting the phenolic hydroxy group of hydroquinone.

Synthesis of 4-hvdroxyphenoxy tetrahvdro~vranvlether

The phenolic hydroxy group of hydroquinone was protected as per the literature procedure (Lam et al., 1982). To a solution of hydroquinone (0.05 mol) and p- toluenesulfonic acid (Srng) in anhydrous ether (200m.L)was added dropwise a solution of dihydropyran (0.055 mol) over a period of 2 h at room temperature. The reaction mixture stood at room temperature for 24 h and was neutralized with solid sodium bicarbonate to pH 6. The excess sodium bicarbonate was removed by filtration and the filtrate was evaporated in vacuo . The crude product was fbrther purified by column chromatography using petroleum ether, b.p. 38.4-52S°C - ether (4:l v/v containing 0.025% acetic acid) mixture as the eluting solvent. The yield of the product was 80% and the m.p. was 88°C (lit. m.p. 88-8g°C, Lam et ai., 1982). Reaction of 4-hydroxyphenoxy tetrahydropyranyl ether with 4-fluorophenol as per the procedure outlined under section 3.1.4.0 resulted in the formation of an oily residue. TLC indicated that a number of compounds were present and the mixture was not investigated further.

3.1.18.0 Attem~ted svnthesis of 3-i4-phenolrv~henv[)-1-~henvl-2-aro~en-I-one semicasbazone

The preparation of this compound was attempted as per the procedure outlined under section 3.1.15.0. To a cold solution of sodium hydroxide (I. lgm), water ( 1OrnL) and ethanol (95%, l OrL) was added 4-phenoxybenzaldehyde (0.01 1 4 mol) and acetophenone (0.01 14 mol). The reaction mixture was stirred vigorously for 2-3 h and the mixture refrigerated for 48 h. The precipitate was filtered, washed with cold water until the washings were neutral to litmus, and then with cold absolute (IOrnL). The purity of the product was checked by TLC and NMR spectroscopy. The intermediate 4-phenoxyphenyl styryl ketone was treated with semicarbazide as per the procedure mentioned under section 3.1.5.0 with slight modifications. The reaction mixture was stirred for 4-5 days and the reaction was monitored by TLC. The work up of the reaction mixture yielded a product the TLC of which showed two distinct spots of equal intensity different from the starting material. These two compounds (A and B) were separated by column chromatography using ethyl acetate as the eluting solvent. Compound A had a m.p. of 180°C and B had a m.p. of 176OC. These compounds may represent isomers which are sometimes observed with phenylstyryl ketone semicarbazones (Heilbron and Wilson, 1912). Elemental analysis of A and B indicated traces of impurity. Further purification of the compounds A and B was not possible due to lack of sufficient quantities of materials.

Elemental analvsis:

Calculated for CnH19N302 C : 73.93 H : 5.36 N : I 1-76 Found Compound A C : 72.40 H : 4.90 N : 1 1.03 Found Compound B C : 64.00 H : 5.40 N : 12.40 Found Compound A and B C : 73. LO H : 4.90 N : 1 1.05

3.1.19.0 Attempted synthesis of benzo~henonesemicarbazone analo~s

R R = Br, F, OH, OCH, The preparation of the above substituted benzophenone sernicarbazone analogs were attempted as per the procedure outlined under section 3.1.15.0. The appropriate benzophenone (0.01 mol) in alcohol (30rnL) was added to a solution of semicarbazide hydrochloride (0.0 1 mol) and sodium acetate (0.0 1 mol) in water (1OmL). The reaction mixture was heated under reflux for 5-10 days and the progress of the reaction was monitored by TLC using a solvent system of benzene : methanol (9: 1). The reaction was not complete even after prolonged heating and TLC indicated the formation of a product along with unreacted materials. The compound was soluble in hot water and had a m.p. of 270°C and was identified as a byproduct of semicarbazide formed due to prolonged heating of sernicarbazide. NMR spectroscopy (60MHz) and a literature search revealed the compound as hydrazido dicarbamide (MI2CONH-NHCOMI,, lit. m.p. 247-50°C. Vogel, 1989b).

The 'H NMR (60MHZ) spectrum for hydrazido dicarbarnide was as follows : 6 (CDC1;) : 8.86 (s.~H,NH), 7.73(s94H,NH2).

Changing the reaction conditions by taking different proportions of semicarbazide and benzophenone (2: 1. 3: 1, 5: 1) and stirring the reaction mixture at room temperature for 10 -I5 days did not yield the desired product. TLC indicated the presence of more unreacted materials and traces of components which were not identified. Attemoted svnthesis of 4-~2'.3',4'.5'.6'-~en~uoro~henow). 4-(2',3'.5',6'- tetrafluoroohenorvb 4-(4'-aminoohenoxvb 4-14'-trifluoromethvl~henoxv), 4-14'-carbow~henorv)-benzaldehvdesernicarbazones

The preparation of the substituted aryloxyaryl aldehydes was attempted as per the procedure outlined under section 3.1.4.0. The reaction mixture was heated at I 50°C for 24 to 48 h and the progress of the reaction was monitored by TLC. The reaction did not proceed when the substituents were pentafluoro [R = 2,3,4,5,6 (F)s] , tetrafluoro [R

= 2,3,5,6 (F)4] or carboxy (R = 4COOH) group. The work up of the reaction mixtures for the 4-amino and Ctrifluoromethyl analogs resulted in oily residues which showed a streak of components by TLC which were not purified.

3.1.2 1.0 Attem~tedsynthesis of 3-(4'-fluoro~henow)benzaldehvdesernicarbazone The preparation of this compound was attempted as per the procedure outlined under section 3.1.4.0. To a solution containing 4-fluorophenol (0.15 mol), 3-fluoro- benzaldehyde (0.14 mol) in dimethylacetamide (100 mL) was added anhydrous potassium carbonate (0.12 mol). The reaction mixture was heated at 156" C for 24-48h under nitrogen and the progress of the reaction was monitored by TLC using a solvent system of benzene : methanol (93). The reaction did not proceed even after 48 h, and prolonged heating (72 h-90 h) resulted in resinous masses with streaks of components. No attempt was made to purify the resinous mass.

3.1.22-0 Partition coefficient measurements

Partition coefficients were determined by Mr. M. Hetherington using a Gilford WMS spectrophotometer. The log P figures were determined by a previously reported procedure @immock et al., 1989) except that solutions were made using 1-octanol to which buffer was added. The X- and E values of the compounds were obtained in 1- oaanol and not in phosphate buffered saline (PBS), pH 7.4 due to the low aqueous solubilities of the compounds.

Accurately prepared standard solutions of the compounds (I&, NI,9. 43. 4s,

Vj, VI&? X3.4 ) were made in 1-octanol and aliquots of these solutions were then shaken with phosphate buffered saline in a horizontal shaker for lh at 37°C. The separated 1- octanol layer was then withdrawn and analyzed. The partition coefficients were from 1- - octanol to PBS so the inverse of these coefficients are then the standard partition coefficient (i.e. &om PBS to 1-octanol). All measurements were performed in triplicate. The values obtained for replicate samples were well within the accepted instrumental error of + lnm. Error limits for the E values are quoted on the basis of 0.2% error in the concentration of solutions. 3.1.23.0 Stability Studies

Compounds (tL V3.4) were dissolved in DMSO-d6 to give 4OmM solutions. Immediateiy after dissolution, a 300 MHz NMR spectrum (to) was taken for each compound at 3 7°C. Following this, the solutions were incubated at 3 7.3"C + 0.0 1OC in a circulation water bath and another NMR spectrum (t3) of each compound was recorded after 3 h. Stability studies for compounds Tf-Jy W2and W5was carried by dissolving the compounds in phosphate buffer pH 7.4 and incubating the solution at 37°C for 24 h and studying the W absorption and & for each compound at the end of 24 h.

3.1.24.0 Statistical analvses and P hvsicochemical constants

The physicochemical constants sigma (o), hydrophobic (x) and molar refractivity (MR) constants were taken from the literature (Hansch and Leo, 1979) and the Tafk a' values were obtained from a reference source (Taft, 1956). The solvent accessible surface areas calculated with a probe radius of 1.4 18 were obtained using a MacroModel Version 4.5 program (MacroModel Version 4.5, 1994 ; Moharnadi et al., 1990) and a silicon graphics indigo Extreme workstation. A correlation coefficient of 0.8 was chosen arbitrarily as indicating a relationship between the physicochemical constants and bioactivity and when observed, the specific r values were determined.

The X-ray crystallographic study was carried out under the supervision of Dr. J. W. Quail, Department of Chemistry in the laboratory of Dr. L. T. J Delbaere, Department of Biochemistry, University of Saskatchewan. The compounds were crystallized from ethanol-dimethylsulfoxide (I&), ethyl acetate (IVI), acetone-methanol

(IVp),and hexane-methanol (Wl)by vapor diffusion while (I&) was crystallized from hot ethanol. An End-Nonius CAD4 diffiactometer with a d20 scan was used for data collection and the structure was solved by direct methods using NRCVAX (Gabe g -al., 1989). Atomic scattering factors were taken fiom the literature ( International table for X-ray crystallography, 1974). All non-hydrogen atoms were found on the E map and refined anisotropically. Hydrogen atoms were calculated and not refined. Crystallographic R factor [R (F)]is considered good , when its value is less than or equal to 5% and poor with values more than 9% which were rejected. Merging of the R factor (to check the validity of the data set) was considered good with values less than or equal to 3% and poor with values more than or equal to 6%. Merging was based on intensities of the least square refinement.

The data for III I were as follows : C 14H13N302, M- = 255.27, colorless plates.

0.3 x 0.22 x 0.15 mm, a = 11.809(3), b = 7.1862(20), = 15.4816(20)& fi = 109.220(20)0, Y = 1240.6(5) Bi?, Z = 4, space group = P21/& monoclinic,Q. = 1.367 gcm3, b(MoKa) = 0.7093 & = 0.88 cm", E(000) = 536, 1 =123K, (~inO)/hmJ.~=

0.6 180 8;', -145 h 5 13, 0s k 5 8, OS I 5 18. Merging R is based on intensities 0.0 12 for

480 replicate reflections, lX@) = 0.046, Rw- = 0.049, S = 3 -82. A total of 2652 reflections were measured of which 2 172 were independent. The refinement of the structure used

1728 observed reflections [I> 20( (! )], parameters refined = 172,Iw = k2(l?)];final

= 0.000. & in the final difference map within +OX and -0.240e A?

The data for IV1were as follows : ClsH13N30a,M, = 255.27, colorless blocks,

0.55 x 0.5 x 0.1Smm. 3 = 12.4637(15), = 7.6840(22), c = 12.9457(15)& @ =

9 1.32 1( = 123 9.S(4) A', Z = 4, space group = P2 I/c, monoclinic,QK = 1 -368 gcm- j, h(Mo&a) = 0.7093 & = 0.90 ern-', M000) = 536, I =287K, = 0.6 1 80 k',- 145 h 14, 05 k 9, 0< I r 15. Merging R is based on intensities 0.007 for 3 16 replicate reflections, RE) = 0.039, RE = 0.044, S = 2.33. A total of 2489 reflections were measured of which 2 173 were independent. The refinement of the structure used 1670 observed reflections @52u( 1)], parameters refined = l72,[w = l/d(F)+ 0.000 1 1; final (Ah),, = 0.000. & in the final difference map within H.24 and -0.260e A?

The data for NJwere as follows : CLJI~ZN~O~F,= 273 -27, colorless plates,

0.55 x 0.40 x 0.05 rnm, 4 = 12.7066(19), b = 7.7389(15), c = 13.2838(23)& B =

103.965(15)", = 1267.7(4) 2,Z_ = 4, space group = P2,/a, monoclinic,QK= 1.432 gcm3, X(MoEa) = 0.7093 4 = 1.0 cm-', E(000) = 568, 1 =123K, (sin@)/A-..=

0.6180 kl,-1% h 5 14, 05 k 5 9, 05 l 5 15. Merging R is based on intensities 0.017 for

301 replicate reflections, IZQ = 0.038, RE = 0.05 1, S = 2.15. A total of 2508 reflections were measured of which 2207 were independent. The refinement of the structure used

1750 observed reflections CI> 2a( 1)], parameters refined = 18 1,[w = l/a2(~)+ 0.0002]; final (Nr), = 0.000. & in the final difference map within i0.17 and -0.260e it3.

The data for IV* were as follows : CLSHI~N~O~, M- = 269.30, colorless plates.

0.45 x 0.20 x 0.10 mm, g = 13.0692(6), h = 7.8606(4), c = 13.5724(6)& fi =

101.857(4)", = l364.57(11) ti3, Z = 4, space group = P2,/a, monoclinic,& = 1.3 1 1 gcm-3, h(MoKa) = 0.7093 = 0.80 cm-', E(O00) = 608, 1 =287K, (sin9)/kLK=

0.5958 kL,-155 h < 15,W k 5 9, 0s I 5 16. Merging R is based on intensities 0.006 for 1 19 replicate reflections, BE) = 0.04 1, RE = 0.056, S = 2.5 1. A total of 25 13 reflections were measured of which 2394 were independent. The refinement of the structure used

1858 observed reflections [b 2Sa( )I, parameters refined = 18 1,[w = I/&(F) + 0.00021; final (Ak), = 0.000. & in the final difference map within +0.22 and -0.ZSOe P.

The data for WIII were as follows : CLSHI~N~OS, M- = 271.32, colorless plates, 0.40 x 0.20 x 0.10 mm, 3 = 12.9365(15), = 5.3 1 1 1(1 I), c = 19-06l(4)& = 98.46(3)', = 1295.3(4) p, Z = 4, space group = P21/a, rnonoclinic,QK= 1.391 gcrn". L(MoKa) = 0.7093 & = 2.3 ern-', E(000) = 568, T_ 423K. (sinO)/hM, = 0.61 80 kt. -15s h < 15, 05 k 5 6, 0s l r 22. Merging R is based on intensities 0.012 for 1 18 replicate reflections, RE) = 0.062, Rw- = 0.078, S = 2.95. A total of 2394 reflections were measured of which 2276 were independent. The refinement of the structure used

15 19 observed reflections [b2.b( I )], parameters refined = 23 5.[w = l/a2(~)];final (Ah)- = 0.036. & in the final difference map within +0.3 1 and -0.3 10e 8;'.

3.1.26.0 Evaluation of the bindinr site hvoothesis using X-rav crvstallograohv and molecular modeling

In order to compare the shapes of four active analogs (N!,N,, IV2 and VIII,) and one inactive analog I&, four atoms in the semicarbazono group namely the N3-C 14-(02 or 0)-N2 atoms were superimposed and the overlapping of the X-ray diagrams were undertaken using MacroModel Version 4.5. The determination of the precise positions of the aryl rings from the X-ray crystallographic data of all five compounds was undertaken. The distance between the C 14 atom and the centers of both aryl rings were measured. In addition, the angle 9 between the centers of the aromatic rings and the C14-N2 bonds were obtained. Furthermore the displacements of the aryl rings above or below the N3-C14-(02 or 0)-N2 plane were calculated for each compounds in terms of both distances and angles ly.

The data generated from X-ray crystallographic study were compared with the molecular modeling data obtained using a HyperChem program (~~~er~hern"'Release 2 for Windows). Compounds were minimized (geometry optimization) by using a MM' molecular mechanics force field in vacuo, applying the Polak-Ribiere algorithm (conjugate gradient), and the calculations finished when the RMS gradient was ~.~lkca~(hol)or 500 cycles had elapsed. The minimized molecules were used for comparison with the X-ray crystallographic data. The evaluation of the compounds for anticonvulsant activities (Phase 1 to 5, Table 1.7) was undertaken by the Division of Convulsive, Developmental and Neuromuscular Disorders, The National Institute of Neurological Disorders and Stroke. National Institutes of Health 0,using their reported procedures ( Porter et al., 1984). In addition, the effect of the lead compound (IVJ) on [ca2']i transients of mouse cerebellar granule ceUs and whole-cell peak currents of mouse cortical neurons and the effect of N4on Frings audigenic mice (White @ d., 1992 ; Frings and Frings, 1952 ; Collins, 1972) and hippocampal kindled rat test was undertaken by the NIH. The effect of 1V4 in amygdala kindled rats was undertaken by the Department of Pharmacology, The University of Toledo, Ohio, USA. The neuroprotective effect of IV4 on PC 12 gene expression and an in vivo ischaernia model ( Li et al., 1992 ) was undertaken at the Neuropsychiatry Research Unit, Department of Psychiatry, University of Saskatchewan. Selected compounds were further evaluated using the photosensitive fowl screen (Johnson and Tuchek, 1987 ; Fisher gt d., 1985). in the Department of Pharmacology, University of Saskatchewan.

3-2.1.0 Material and Methods

Male albino CF No. I mice (1 2-2Sg, Charles River, Wilmington. MA) and male albino Sprague-Dawley rats (100-150g, Simonsen, Gilroy, CA) were used as experimental animals for the anticonwlsant evaluations (Phase I to 5, Tablel.7. Page 18). Male Sprague-Dawley rats (150- 175 g, Harlan Sprague-Dawley, Indianapolis, M) were used in the amygdala kindling test. Male and female epileptic and carrier chicks were obtained from Dr. Hank Classen of the Department of Animal and Poultry Sciences, University of Saskatchewan. The breeding of a heterozygote (carrier) with an epileptic (homozygous recessive) animal results in 50% of the offspring bearing carriers and 50% epileptics. Male and female broiler chickens (normal) were obtained f?om a local hatchery (Miller Hatcheries, Saskatoon). Male and female Frings audiogenic seizure- susceptible mice (18-25 g) were obtained from an in-house colony at the University of Utah. Salt Lake City, UT. All animals were allowed fiee access to both food and water. Compounds and standard antiepileptic drugs (phenytoin and carbarnazepine) were administered in 0.5% w/v methylcellulose in water, whereas valproate was administered in 0.9% sodium chloride solution. These drugs were administered either orally ( p.0) or intraperitoneally (i.p) in a volume of 0-Oldgbody weight in mice and 0.04 mUlOg body weight in rats. The chemical convulsants were administered subcutaneously. For &z -vitro studies (measurement of intracellular calcium) the compounds were dissolved in DMSO (10-20 mM stock solution) and for epileptic fowls, the compounds were dissolved in DMSO and administered by the i-v. route.

All quantitative studies were conducted at the previously determined time of peak effect (TPE). Groups of at least eight mice or rats were tested with various doses of the candidate drug until at least two points were established between the limits of 100% protection or minimal impairment (toxicity) and 0% protection or toxicity. The dose of drug required to produce the desired endpoint in 50% of the animals (ED50 or TDSo)in each test, the 95% confidence interval, the slope of the regression line, and the S.E. of the slope were then calculated by a computer program (Finney, 1971). TPE indicates how rapidly the drug is absorbed, while EDSOand TD5. data disclose how adequately the drug is absorbed. 3.2.3.0 Determination of acute toxicitv

Abnormal neurological status disclosed by the rotorod test (Dunham and Miya, 1957) and loss of righting reflux are commonly taken as the end points for minimal neurotoxicity and the hypnotic state, respectively in mice. The inability of a mouse to maintain its equilibrium for one minute in each of three trials on a rotating rod was used as an indication of such impairment. Abnormal neurological status disclosed by the positional sense test, or the gait and stance test, is taken as the end point for minimal neurotoxicity in rats. The inability of rats to perform normally in at least two of these tests indicated that the animal has some neurological deficit.

3.2.4.0 Anticonvulsant and Differentiation Tests

Anticonvulsant activity was established by electrical, chemical (Swinyard et al., 1989) and genetic seizure or reflex models (Meldrum 3 &., 1975). The electrical tests used were the maximal electroshock seizure (MES) test and the kindled rat test (corneal, amygdala and hippocampus). The chemical tests included the subcutaneous pentylenetetrazol (scPTZ), bicuculline (scBic), and picrotoxin (scPic) seizure threshold tests. The genetic seizure model includes the photosensitive epileptic fowl and Frings audiogenic mice model.

Selected substances are hrther characterized in special studies to differentiate possible mechanisms of action. The timed intravenous ifision of pentylenetetrazol is used to measure an increase or decrease in seizure threshold. Studies in kindled rats are used to identify substances that may be usehl in complex partial seizures (Loscher and Schmidt, 1988 ; McNamara, 1989). 3.2.5.0 Maximal electroshock seizure (MES) and coneal kindled rat test

At the previously determined time of peak effect (TPE)of the test substance, a drop of anesthetidelectrolyte solution (0.5% butacaine hemisulfate in 0.9% saline) was applied to the eyes of each animal prior to placement of the corneal electrode and the electrical stimulus (for MES test , SOW60- for mice and iSOmA, 60Hq for rats) was delivered for 0.2 sec by an apparatus similar to that originally described by Woodbury (Woodbury and Davenport, 1952). Abolition of the hindleg tonic extensor component was used as the endpoint. The absence of this response indicates that the test substance has the ability to prevent seizure spread.

The kindled rat test was undertaken by reported procedures ( Racine, 1972 ; Skeen et al., 1990). For the kindled rat test, the animals were electrically stimulated with 8M60E for 4 sec daily to a criterion of I0 consecutive stage 5 seizures. Five seizure scores are recorded: (1) abnormal mouth and facial movement, (2) as in I plus head nodding, (3) as in 2 plus forelimb clonus, (4) as in 3 plus rearing and (5) as in 4 plus falling (Racine. 1972). Compounds were administered orally and the animals were challenged with electric$ stimuli 2h later. Abolition of the generalized seizure characterized by clonus and the rearing and falling observed in Stages 4 and 5 seizures was taken as the endpoint for this test. The EDa is the dose required to reduce seizures from stage 5 to stage 3 or less.

3.2.6.0 Hippocam~alkindled rat test

The rapid hippocampal kindling model of Lothman et al. appears to offer some distinct advantages for the routine screening and evaluation of new anticonwlsants (Lothman et al.. 1958). A bipolar stimulating electrode was stereotactically implanted in the ventral hippocampus of adult male Sprague-Dawley rats (250-300 gms) under ketamine- anesthesia. Three anchor screws were attached to the skull and the electrode assembly anchored to the skull with dental acrylic cement. After the incision is closed with sutures, the animals receives a single dose of Bicillin (60,000 Units, i-m.) and are returned in their home cages in the animal quarters. Animals were kindled according to the reported procedure (Lothman and Wiliamson, 1994). Briefly , after one week, animals were stimulated with suprathreshold trains of 2OOpamps for 10 sec, 50Hz, every 30 min for 6 h on alternate days until they were fully kindled. One week later the effect of a single dose of test substance (50 mg/kg imp.)on the behavioral seizure score (BSS) and afterdischarge duration (ADD) was assessed in a single group of kindled rats (n = 6- 8) at 15, 45, 75, 105, 135, 165, and 195 minutes after drug administration. The results obtained at various time points were compared with the last control stimulus delivered 15 minutes prior to the drug administration. Thus each animal serves as its own control. Seizures are scored and recorded to stage 5 (Racine, 1972) as already mentioned under section 3.2.5.0.

When a drug treatment is observed to sigmficantly lower seizure score and decrease afterdischarge, a dose-response study is initiated. For this study the ability of a candidate substance to block afterdischarge and reduce seizure severity is quantitated by varying the dose between 0 and 100% effect. Drugs that are active against focal seizures would be expected to reduce the BSS to 2 or less and significantly lower the ADD.

3.2.7.0 Amv~dalakindled rat test

Five rats were anesthetized with ketamine (60 mgkg, i.m) and sodium (65 mgkg, i-p). AU animals received atropine (0.05 rng/kg, i.m) 10 min prior to sodium pentobarbital dosing. Bipolar, twisted nichrome wire electrodes were surgically implanted in the right basolateral amygdala nucleus (Tietz et al., 1989). After 10-20 days, afterdischarge threshold(AD) is determined with the delivery of 1 Opamp stimulation for 1 second to the amygdala and the current increased in lopamp increments at 3 min interval until an AD was recorded that lasted a minimum of 2 seconds (Tietz gt -al., 1989). The rats were further stimulated once a day using a suprathreshold stimulus of 200pamp for 1 second until the criterion of 10 consecutive Stage 5 seizures were recorded (Racine, 1972).

Compound N4was administered orally 2h prior to the electrical stimulus as an 0.5% methylcellulose suspension at six different doses (5, l5,30,45,65 and 100 mg/kg). The electrical stimulus was administered 2h post drug administration and this pretreatment time was chosen based on the data collected by the ADD program of the NIH and their calculated TPE of 2 h used in the MES and corneal kindling test. The initial dose of 5mgkg was chosen based on the calculated EDso for corneal kindled rats of 3 -93rnglkg. Abolition of the generalized seizure characterized by clonus and the rearing and falling observed in Stage 4 and 5 seizures was taken as the endpoint for this test.

Seizure parameters were recorded and can be divided into two types. behavioral and EEG. Behavioral parameters include: behavioral seizure latency, behavioral seizure duration, forelimb clonus latency, and forelimb clonus duration. Behavioral seizure latency was the time span between the application of stimulus and the appearance of the first sign of motor seizure. Behavioral seizure duration was the length of time that the motor seizure was observed. Forelimb clonus latency was measured from the application of stimulus to the onset of unilateral or bislaterai forelimb clonus. Forelimb clonus duration was the length of time from onset to disappearance of forelimb clonus. The EEG parameter recorded was amygdala AD duration, defined as the time from application of stimulus to the end of the last arnygdda AD spike. No statistically significant reductions in behavioral seizure latency were observed at any dose tested. Forelimb clonus latency was significantly delayed at doses of 30 mgkg, 45 mg/kg and 65 mgkg. Lengthening of behavioral seizure duration by the 5 mgkg, 30 mg/kg and 45 mg/kg doses was significant. Prolongation of forelimb clonus duration at doses of 5 rngfkg, 15 mgkg 45 mg/kg and 65 mgkg was significant and arnygdala AD duration was significantly prolonged at doses of 5 mg/kg, 30 rnglkg, 45 m@g and 65 mg/kg (Table 4.10). All animals were subjected to motor function tests to determine any presence of motor impairment immediately prior to electrical stimulation. The motor tests have been previously described (section 3.2.3.0) and include assessment of gross motor impairment. [n addition, an inclined screen test, and a test for loss of hindlimb or abdominal muscle tone are also included (Tietz a &, 1989). Upon conclusion of the study, histological verification of electrode placement was performed on each rat and only rats with histological verification of amygdda electrode placement were used in data analysis (Schwark and Loscher, 1985). AU statistical analysis were performed on version 5 of the statgraphic computer program (199 1).

3.2.8.0 Subcutaneous ~entvlenetetrazol IMetrazol) seizure threshold test JscMet/scPTZ), subcutaneous bicuculline (scBic), picrotoxin (scPicL and strvchnine (scStr) tesl

The convulsive dose (CD97) of Metrazol (85 rnglkg) , bicuculline (2.70 mg/kg), picrotoxin (3.15 mg/kg) or strychnine (1.2 mg/kg) was injected subcutaneously into each of the requisite number of mice at the previously determined TPE for the test substances. In rats, a dose of 70 mgkg of Metrazol was used. The animals were observed for 30 min for the presence or absence of an episode of clonic spasm persisting for at least five sec. Picrotoxin-treated animals are observed for 45 min. The absence of a clonic seizure in metrazol- , bicuculline- and picrotoxin-treated animals indicated that the substance had the ability to elevate the respective seizure threshold. In strychnine-treated animals. abolition of the hindleg tonic-extensor component is taken as the end point and indicates that the test substance has the ability to prevent seizure spread. AII subcutaneously injected convulsants were dissolved in 0.9% saline and injected in a volume of O.OlmL/g body weight in mice. Metrazol was administered in a volume of 0.02 mL/lOg body weight in rats. 3.2.9.0 Timed intravenous ~entvlenetetrazoltest

This test measured the minimal seizure threshold of each animd and identified the potential proconvulsant effect of a test compound (Orlof et al., 1949). Compounds in methylcellulose solution(O.S%) were injected intraperitonedy into mice. The two doses used were the approximate EDSO values in the MES test and the TDso figures. After 1h, the convulsant solution (0.5% pentylenetetrazol in 0.9% sodium chloride containing 10 U.S.P. units/mL of heparin sodium) was infiised into the tail vein at a constant rate of 0.37 mUmin. The time in seconds From the start of the infusion to the appearance of the first twitch and onset of clonus is recorded for each experimental and control animal. These values were converted to mgkg of pentylenetetrazol and the mean doses and standard errors (first twitch and clonus) for each group were calculated.

3.t.LO.O Measurement of intracellular calcium transients

To gain some insight into the mechanism of action of candidate anticonvulsant substances, studies are conducted which determines their ability to limit ca2+influx into primary cultures of mouse cerebellar granule cells via both voltage-sensitive ~a" channels and tetradotoxin-sensitive ~a**channels. Granule cells were derived from the cerebellum of 8-day-old mice and grown on 25 rnm glass cover slips according to the method of Parks g d.(199 1). Measurement of intraceilular-free ca2+([ca2-]i) was determined according to the methods of Peeters et aL(1987) and Ikenouchi d. (199 1). Cells were excited at 360 nm by means of a high pressure mercury-arc lamp (OSRAM. HBO 100 W, Bulbman, Reno, NV). The emitted fluorescent light was collected by a 2OxFluor objective lens (Nikon) and divided with a dichroic beam splitter to permit simultaneous measurement of both 410 and 480 nm wavelengths by use of two photomultiplier tubes. The ratio of 4 1OM80 run emitted fluorescence. obtained electronically with an analog divider circuit, is an indirect indicator of [ca2-]i. After obtaining several seconds of baseline recording (K' = 4.2 mM), cells were exposed to either elevated K- (55 mM) or veratridine (10 pM) for 60 seconds. Following a four-minute washout with normal buffer, the candidate substance was bath applied for I minute. After this time, the cells were simultaneously challenged with a solution containing either 55mM K' and the candidate substance (10- 100 pMJ or 10 pM veratridine and the candidate substance (NJfor 60 seconds. Cells were then re-exposed to the candidate substance for an additional 5 minute, after which they were challenged a second time with either KC1 or veratridine plus candidate substance. When a drug effect was recorded, the perfusate was switched to control buffer for a 5-minute washout. Cells were then rechallenged with agonist alone. This procedure attempts to establish the reversibility if the drug effect; in this respect, the cells on each cover slip serve as their own predrug and postdrug control.

The results obtained from these studies were expressed as a percent of the control [ca2+]itransient induced by KC1 or veratridine. In order to gain an appreciation for the overall effect of a candidate substance on the [ca2']i transient, results were expressed as a percent of the peak height, the area under the curve, and the height of the [ca2-]i transient at 60 seconds.

3.2.11.0 Overt tolerance and liver enzyme studies

Subchronic Administration : Overt Tolerance

To determine the effect of 5-day subchronic treatment on anticonwlsant activity (MES or scMet tests), three groups of eight animals were treated as follows. One group was given the MES EDso of the test drug (IV4) for 5 days; the second group was given the requisite volume of vehicle for 4 days and a single dose (EDSO)of the test drug on day 5 at the time of peak effect of the candidate substance and the third group received only the requisite vehicle for 5 days. All groups were subjected to the MES test and the number protected was recorded. A1 three groups of rats were subjected to the hexobarbital sleep time test (100 mgkg of hexobarbital LP. and sleep time measured to the nearest minute).

Subchronic Administration : Liver Studies

Four animals f?om each chronically treated and vehicle control group subjected to the hexobarbital sleep time test were continued on their respective treatment regimens two more days (days 6 and 7) and 24 h later subjected to a short screen that determined changes in some hepatic parameters. In addition to these two groups, four rats were given either the MES (ED9,), the TD3 or 100 mg/kg for 7 days. The livers were removed and homogenized and subcellular fractions were prepared by differential centrifigation and the following biochemical determinations were made : measurement of cytochrome P-450 concentration and activities of p-nitroanisole 0-demethylase, NADPH cytochrome c reductase and p-nitrophenol UDP-glucuronosyltransferase activities (Franklin and Estabrook, 197 I ; Franklin, 1974 ; Papac and Franklin, 1988).

3.Ll2.O Epileptic fowl ~eneticmodel

Homozygous recessive birds display primarily generalized grand ma1 type seizures that occur spontaneously throughout the bird's life span or that can be induced reproducibly with intermittent light stimulation (ILS) at a stimulation rate of 14 flashes per second (lee, 1973). Seizures induced by ILS consist of three well defined phases. Phase I occurs within 10 to 20 s after the onset of ILS and consists of opisthotonoid arching of the neck lasting approximately 5 s. This progresses to Phase I1 where the bird staggers back on its haunches extending its wings outward and downward. The duration of this phase is approximately 5 s and progresses to Phase 111 where the bird loses its balance and violently tumbles about flapping its wings and displaying tonic/clonic leg movements. Phase lII usually lasts from 25 to 35 sec (Fagnou and Tuchek, 1996). The ability of the compounds to block IPS-induced seizures (Stage 3) is taken as the end point.

3.2.13.0 Frings audioeenic seizure penetic model

Frings mice possess a life-long susceptibility to sound induced seizures. Groups of eight mice each are treated with varying doses of the test substance. At the time of peak effects as determined in the MES test, individual mice are placed into a round plexiglass jar (diameter, 15 cm; height, 18 cm) and exposed to a sound stimulus of 110 decibels (11 KHz) delivered for 20 sec. Mice are observed for 25 sec for the presence or absence of hindlimb tonic extension. Mice not displaying hindlimb tonic extension are considered protected. RESULTS AND DISCUSSION

4.1.0.0 Introduction

The synthesis of compounds in series I to XVI was accomplished successfblly. Elemental anaiysis (C, H, N) for all the compounds were within 0.4% of the calculated values except for IV3 (found: H : 3.94, calculated for C&&N~O~,H : 4-42}, IV, (found: N : 13.96, calculated for C1&IIIFZN3O2,N : 14-42), IVio (found: C : 58.36. calculated for ClJIlIF2N302,C : 57-73}, IVzo (found: N : 14.95, calculated for C15H15N302,N : 15-60),VI, (found: N: 15.99, calculated for CIJH13N302,N : 16-46), V12 (found: C : 66.35, calculated for C~SK~~N~O~, C : 66-90), VIIs (found: N: 12.63, calculated for CI+H13N0& N : 13-16), Vl& (found: C : 50.12, calculated for

CISHIJBrNaS, C : 49.46) and MI (found: N : 18.88, calculated for czzHzoN6Or, N : 19.43). The testing phases mentioned under the discussion are described in Table 1.7 (pages 18 and 19). All of the compounds were initially examined in the maximal electroshock seizure (MES), subcutaneous pentylenetetrazol seizure threshold test (scPTZ) and neurotoxicity (M) screens after intraperitoneai injection into mice. Virtually all of the compounds were administered orally to rats and these data are presented in Table 4.1.

Compounds found to possess significant anticonvulsant activity in the primary screens proceed through a multiphase evaluation to establish the pharmacological profile. Quantification of selected compounds in mice (imp.)and rats (oral) are presented in Tables 4.1 and 4.3. Selected compounds based on the quantification results were hrther characterized to differentiate the possible mechanism(s) of action and these results are presented in Table 4.4 to Table 4.1 1. Results of overt tolerance and liver enzyme studies for promising anticonwlsants are presented in Table 4.12. Evaluation of selected compounds in seizure specific genetic models are presented in Table 4.13. The overall anticonwlsant activity profile of N4 are presented in Table 4.14. Statistical analyses, determination of log P values and stability studies for selected compounds are explained in sections 4.9.0.0 - 4.1 1-0 -0 respectively.

X-ray crystallography and molecular modeling data for selected compounds are presented in section 4.12.0.0 in order to delineate structure-activity relationships and to evaluate the viability of the binding site hypothesis pig 2.2). The overall structure- activity relationships are explained in section 4.13.0.0.

4.2.0.0 General introduction to the formation of semicarbazones and related derivatives

Sernicarbadde and other hydrazine derivatives add to the carbonyl group of ketones resulting in the formation of the corresponding semicarbazone or hydrazone. The general reaction showing the formation of imine derivatives involves addition of the amino compound to form an aminohydrin followed by elimination to form the imine. Addition involves the attack of an amino compound on the carbonyl group producing a high-energy, unstable intermediate which is converted to the aminohydrin by two proton transfers. Strong nucleophiles such as hydroxylamine will attack an unactivated carbonyl carbon but weaker nucleophiles such as sernicarbazide or phenylhydrazine require acid catalysis to activate the carbonyl group (Jencks, 1969), The formation of an aminohydrin is depicted in Scheme 4.1 . Free base Salt (nucleophilic) (not nucleophilic)

Scheme 4.1 :Formation of an aminohydrin

Protonation of the carbonyl oxygen makes the carbonyl carbon more susceptible to nucleophilic attack and the addition will be favored by high acidity. However the hydrazine, NH2-R, can also undergo protonation to form the ion, MI& which lacks unshared electrons and is no longer nucleophilic. As the concentration of the free base falls with increasing acidity, the mine becomes progressively more protonated and the reaction is retarded or prevented. Considering the arnine, the addition is favored by low acidity. The solution therefore must be acidic enough for an appreciable fraction of the carbonyl compound to be protonated but not so acidic that the concentration of the free hydrazine is too low, as indicated in scheme 4.1.

The tetrahedral intermediates formed by addition of certain nucleophilic species are unstable and break down to form a new double bond. The elimination or dehydration step may proceed with acid (A) or base catalysis (B) depending on the base strength. Strong base arninohydrins may dissociate without catalytic assistance (C) or with acid catalysis whereas less strongly basic arninohydrins may require basic catalysis, as represented in Scheme 4.2. H I H I I /' - R-N-C-OH C) *. R-NL~+ OH

Scheme 4.2 : Elimination phases of imine formation

The reaction rates for imine formation vary with pH in a characteristic manner. which is a consequence of a change in the rate limiting step. At neutral and alkaline pH the dehydration step is generally slow and rate limiting. As the solution is made more acidic, the rate increases as a result of the acid catalysis of the dehydration until the maximum rate is attained, generally at a pH between 2 and 5 (Jencks, 1959 . 1969). A decrease of rate occurs on fbrther acidity since although the dehydration is facilitated, the addition step is inhibited because only unprotonated amine is reactive. The entire mechanism is represented in Scheme 4.3. fast above pH 4 -,, + \ /OH + HA - ,c=N-R + A- + H,O /'\NHR slow below pH 4 I H

fast \ ,C=N, \ R

Scheme 4.3 :General mechanisms for formation of irnine derivatives

4.2.1.0 Svnthesis of 4-(4'-chloro~~henorv~benzaldehvdesemicarbazone W1d

The synthesis of the aryloxyaryl sernicarbazone Nl3was initially carried out by reacting 4-chlorophenol (0.0202 mol) with 4fluorobenzaldehyde semicarbazone (0.0 144 mol) in the presence of potassium carbonate in dimethylacetamide at 155°C for several hours. The work up of the reaction mixture was carried out as per the procedure outlined in section 3.1.4.0. However, the product isolated was found to be different fiom the expected title compound I'I~.This product (IVx) was identified by mass spectrometry to have a mass of 460.08 and based on the elemental analysis results and L H NMR (300 MHz) spectroscopy, the structure of IVx was proposed (Scheme 4.4). Scheme 4.4 : Formation of lVx

The possible way of formation of IVs may be synchronous hydrolyses of the -C=N- and -C(O)N- bonds of IVia to give the aldehyde and hydrazone respectively, which in turn would react to give the product Nx(Scheme 4.4). The formation of IVs was hrther confirmed by treating 4-chlorophenoxybenzaldehyde (2 mole) with hydrazine hydrate (1 mot) in methanol and comparing the I.R. spectra of this product with the I.R. spectra of IVx . Funher conformation was done by elemental analyses. The m.p. of IVx was 205-207OC and the m.wt was 46 1.3503 respectively.

Compound IVs was also submitted to MH for anticonvulsant evaluation , and it was found however to be inactive compared to IVI1. Compound IV13 was synthesized by reacting 4-chlorophenol(0.15 mol) with Cfluorobenzaldehyde (0.14) rather than using 4- fluorobenzaldehyde semicarbazone as per the procedure outlined under sections 3.1.4.0 and Ll.5.O. 4.2.2.0 Reaction mechanism for the formation of arylollcvawl aldehvdes and related

The reaction mechanism for the formation of substituted aryloxyaryl aldehydes is depicted in scheme 4.5. Reaction of the appropriate phenol with 4-fluorobenzaldehyde in the presence of potassium carbonate in diethylacetarnide is by aromatic nucleophilic substitution. Step 1

___,fast

Scheme 4.5 : Reaction mechanism for the formation of aryloxyaryl aldehydes and related compounds

The reaction is activated by electron withdrawing groups para or ortho to the leaving group. Fluoro is the best leaving group among the halogens in most aromatic nucleophilic substitutions. The electron-withdrawing formyl group (CHO) activates the reaction with strong nucleophdes (alkoxide ion) by withdrawing the electron density in the aryl ring and stabilizing the intermediate and transition states. The first step in the aromatic nucleophilic substitution (SNAR) mechanism is usually the rate determining step and it is promoted by groups with strong -I effects. The increase in eiectronegativity caused by the fluorine atom causes a decrease in electron density at the site of attack resulting in a faster attack by a nucleophile. The nucleophilicity of the alkoxide ion in the presence of a polar aprotic solvent (dimethylacetamide) is enhanced by solvation of the cation (potassium) which leaves the anion free to attack. If polar protic solvents are used, then carbon alkylation competes with oxygen allqlation i.e. solvation of the anion oxygen occurs which decreases the nucleophilic power to the extent that carbanion alkylation can compete favorably (March, 1992 ; Sykes, 1986 ; Solomons, 1988).

Ifthe electron-withdrawing group is present in the meta position to the leaving group, the reaction may not be easily activated and this may be one of the reasons for the difticulties encountered during the attempted synthesis of 3-(4'-fluoro- phenoxy)benzaldehyde semicarbazone (section 3.1.2 1.0).

4.3.0.0 Anticonvulsant Identification

4.3.1.0 Intrapentoned iniection in mice (Phase la)

The initial anticonvulsant evaluations were undertaken by administering the compounds by the i.p. route to mice. Two tests are used in Phase la for the detection of anticonvulsant activities in candidate substances. The MES screen detects agents that prevent seizure spread and the scPTZ threshold test indicates agents that elevate minimal seizure threshold. In addition, the rotorod test is used to identlfy minimal neurotoxicity. Protection and/or neurotoxicity was noted 0.5 and 4 h after administrating doses of 30, 100 and 300 magof each semicarbazone to the animals. These results are presented in Table 4.1. The following observation may be made based on the data presented in Table 4.1.

First, in general, the compounds demonstrated a selective protection in the MES screen rather than the scPTZ test which may be noted by comparing not only the percentage of compound which were active in both screens but the fact that lower doses were required to afford protection. Thus the percentage of compounds which were active at minimum doses of 30, 100 and 300 mgkg or were inactive were 43, 39, 26 and 18 respectively in the MES screens whereas in the scPTZ test, the comparable figures were 13, 17, 29 and 47 respectively.

Second, the percentage of compounds causing neurological deficit at the minimum doses of 30, 100 and 300 rngkg or were not neurotoxic at the maximum dose utilized were 5, 28, 54 and 34 respectively. Thus in the 30-100 mgkg dose range, while 67% of the compounds did not display neurotoxicity, 82% demonstrated activity in the MES screen which suggested that favorable P.I. values may be displayed in this group of compounds.

Third, a comparison of the bioactivity of each series was undertaken in order to discern the general effects of structural modifications. Both series I and I1 displayed weak activity andor neurotoxicity compared to the first lead compound 5 (see page 40). These results (Table 4.1) reveal that the primary amino group of & and presumably related compounds such as other members of series II, is not essential for anticonvulsant properties. The lack of activity of Lrc may be due to the greater size of the R group in these compounds compared to Ilj or alternatively their complete ionization may impede transportation via cell membranes to a site of action.

The three structural isomers IlI 1, NIand VI I (rneta, para and ortho) of phenoxybenzaldehyde semicarbazone displayed low potency, significant activity and inactivity in the MES screen respectively. Comparison of the bioactivity of series N with the series having four or more compounds i.e. I, II, V-X, XIV, XV was made. The percentage of compounds in series N which displayed activity in the MES, scPTZ and NT screens was 96, 58 and 75 respectively. A similar trend was observed for series V, VIII, X and XV respectively. Series I, II and VI displayed weak activity in the MES and /or scPTZ test compared to series N.

Thus in general, activity was retained by molecular modification of N except VI suggesting that the placement of the aryloxy group in the para position of the proximal ring is preferable than the ortho position. The results of Iv35 are noteworthy, since it displayed MES activity but no neurotoxicity at the end of 0.5 h at a dose of 300 mgkg and also at the end of 0.4 h at a low dose of 3 mg/kg. Thus IVS5may display high P.I. values in the MES screen at the end of 4h.

In order to evaluate the effect of replacing the methine hydrogen atom in IV by methyl and ethyl groups, series V was prepared. In general, the anticonvulsant activity was retained with series V in the MES screen but most of the compounds in V displayed

neurotoxicity.

Replacement of the oxygen atom between the two aryl rings by different spacer groups led to the formation of W . The data in Table 4.1 reveal that VIIS has potency similar to IVJ in the MES screen, but it demonstrated neurotoxicity. The compounds with the smallest and largest distances between the two aryl rings namely IVI and W5have comparable activity. It is conceivable that the distal aryl ring of MIr is at the periphery of the distal binding site (Fig. 2.2), since the presence of an additional methyl group in V& inhibits alignment at the binding site leading to the abolition of anticonvulsant activity. The lower activity of W14 compared to N4may be due to differences in the orientation of the distal aryl ring caused by the carbonyloxy, methyleneoxy and sulfonyl groups respectively.

Replacement of the ether oxygen atom of Nl.r.13.1~.u by sulfbr led to VIIII.4Zs.~orespectively and the anticonvulsant activity of V14 was compared with the related sulfur isosters VIIIU.5.6. With the exception of IVI and W12, all the compounds displayed activity at the lowest dose used. Thus the increase in size of sulfbr over oxygen did not significantly affect alignment at the binding site and hence activity was retained.

With the exception of 1x1and X3, the compounds in IX and X displayed weak anticonvulsant activity and all the compounds displayed neurotoxicity. Replacement of the distal aromatic ring by heterocyclic or other aliphatic groups resulted in reduction in activity (XI). Series W and XVI displayed neither activity nor neurotoxicity, while the remaining analogs (XIII-XV) displayed weak activity.

Side effects were noted in the scPTZ screen for the following compounds at various doses and time intervals. Continuous seizure activity (CSA) was noted in the case of N1o.1213.1r17.1p~12r_~34~839.40.41.47,VZ-9, WS and m7. Death following CSA was caused by the following compounds namely Iz, NZ1,V8 and VT&. Tonic extension was observed during the evaluation of IV32. The mice died during the test without having seizure in the case of 4 VI, WS,IX3 and K.Stretching and rolling were noted for mice which was caused by Vmt and tremors resulted fiom the administration of I&.

Death occurred after injection of iX3 and &. In the NT screen, mice were unable to grasp the rotorod after administration of the following compounds IL

~8.1~13.l5.17.18.2124.2931343839.5~lVl-8- & WI, m3-6, 1x1-3and X3. A loss of righting reflex was noted with K.

4.3.2.0 Threshold tonic extension (TTE)test

This new test is believed to be more sensitive than the MES screen. This test is useful to uncover agents which may be working by a different mechanism of action. Compounds which are inactive in the initial phase la screen are again administered to the mice by the i-p. route and a subthreshold level of electrical stimulation is given to mice and they are screened at LOO mgkg over time and observed for signs of protection. If effective, these compounds are rescreened in the standard MES test (Phase 1a). Among the compounds tested in the TTE test, only VI5 was found to be active and this compound was further evaluated and the EDso value in the MES screen (mice, i-p.) was 106.45 mgkg respectively.

Table 4.1 Asticonvulsant evaluation after intraperitoneal injection into mice and oral administration to rats of the co~upoundsin Series I - XVl

Compd. Intraperitoneal injection in Micea Oral Administration to ~ats~ No. ------MES Screen ScPTZ Screen Toxicity Screen MES Screen 0.5h 4h 0.5h 4h 0.5h 4h Dose 0.25h 0.5h lh 2h 4h

~VI 100 300 ------50 -- 3 4 4 4 1v2 100 300 300 ------50 2 4 4 4 4 Iv3 30 300 100 -- 300 300 50 4 4 4 4 4 IVJ 30 100 ------"- 50 2 4 4 4 4 Ivs 100 100 300 ------12.5 1% 30 30 1 00 ------so 3 4 4 4 4 lv7 100 300 100 -- 3 00 300 12.5 - - 1 1 4 1 M 30 30 300 300 300 300 12.5 -- 2 4 4 4 IVs 100 30 30 300 -- - - 50 2 4 4 4 4 ~VIO 30 30 -- -- 300 3 00 12.5 1 3 4 4 4 ~VII 30 30 1 00 300 300 - - 50 3 4 4 4 4 Iv12 30 100 30 300 300 100 50 - - 4 4 4 4 Iv13 30 30 30 - - 300 30 50 4 4 4 4 4 lh 3 00 30 ------300 50 - - 2 4 4 4 IVI5 30 30 -- - - 300 30 50 1 4 4 4 4 Iv\6 30 30 100 300 300 100 50 3 4 4 4 4 Iv17 30 30 - - - - 100 30 12.5 2 4 4 4 4 ~VJS 30 30 - - -- I00 300 50 4 4 4 4 4 1 V 10 100 I00 3 00 - - 300 300 50 4 4 4 4 4 124 Table 4.1 cuntd. ...

loOO 00 1000100 mmm --m

0 000 0 Iolc. : Iooo' mmm m 4.3.3.0 OraI administration to rats (Phase lb)

Phase lb provides information relative to whether or not the test substance is active or toxic after oral administration in rats over a 0.254 hour time period. With few exceptions, the compounds in series LXVI were administered ordy to rats and examined in the MES screen. These data are presented in Table 4.1. Initially a dose of 50 mgkg was employed. However under these circumstances, virtually all of the compounds afforded complete protection and hence the dose was reduced principally fourfold in order to detect candidate anticonvulsants with marked potencies.

The data in Table 4.1 reveal that in most cases complete protection was displayed by the rats which received 12.5 mgkg of the compounds. Using principally the doses Listed in Table 4.1, twenty one compounds were examined in the scPTZ screen of which half were inactive and in general the remaining analogs had marginal potencies whereby 25% of the rats were protected against seizures. Compounds rzh N3,69.1W31, Ws and were devoid of activity in the scPTZ screen. On the other hand, the following compounds had activity viz. N11.1610142633, V1, V&, & and XIIS. With the exception of N18,which caused neurological deficit in one of four rats, using the doses listed in Table 4.1 all of the compounds in Table 4.1 which were examined in the MES screen were bereft of neurotoxicity.

Most of the compounds were active in the MES screen and the results support several of the general conclusions drawn fiom the mouse i.p data. In addition, this qualitative examination revealed that complete protection was demonstrated in either all or the majority of the compounds in series IV, V, VIII and XI suggesting that these molecules are accommodated well at the binding site. On the other hand, the diminished activity of the compounds in III and VI reinforces the belief that alignment at the binding site is reduced when the aryloxy group is placed in the meta or ortho positions of the distal aryl ring. Other structural alterations whereby the size of the spacer group was greater than oxygen and suffir atoms (VII) or replacements of the oxygen and primary amino group of the sernicarbazono function (IX,X) appeared to be detrimental.

4.4.0.0 Anticonvulsant auantification

4.4.1.0 Intra~eritonedinjection in mice (Phase 2al

Phase 2a anticonvulsant quantification results (Table 4.2) reveal for each compound the time of peak effect (TPE), the ED50 in the MES and scPTZ tests, the TDIo by the rotorod test, the 95% confidence interval, slope of the regression line and protective index. The TPE data provide fbrther insight into the time of onset and the duration of anticonvulsant and neurotoxic activities. Also, the median effective dose information and protective indices reveal reliable information as to possible clinical usefblness.

Quantitative evaluations of the anticonvulsant efficacy and neurotoxicity of approximately one-third of the compounds in series I-XVI were undertaken and the results are presented in Table 4.2. The following observations may be made based on the data presented in Table 4.2.

First, the selective efficacy of these anticonwlsants in reducing seizures in the MES rather than the scPTZ screen was confirmed. A comparison of the P.I. values in twelve cases where EDIo figures were available in both the MES and scPTZ screens revealed that higher figures were always obtained from MES screening. The average PI value of the MES screen was 5.2 times the EDIo figures generated in the scPTZ test. All the compounds displayed activity in the MES tests but ED50 figures were obtained in only 38% of these analogs for the scPTZ screen. In the remaining cases, the doses were elevated substantially above the EDSOfigures in the MES screen. Subsequent discussion will be focused mainly around the MES screening.

Second, a comparison was made with the three reference drugs phenytoin, carbarnazepine and valproate in terms of potencies and PI values. The EDSofigures in the mouse MES screen for compounds m1724 ; VJ9 and Vmg were lower than phenytoin and carbamazepine; in addition those of N34 ; V1,5.8 ; VIII, were less than that of carbamazepine. Furthermore, the percentage of compounds which had greater PI values than phenytoin and carbamazepine were 63 and 80 respectively. All of the compounds listed in Table 4.2 were considerably more potent and had higher PI values than valproate. Hence a number of these aryloxyaryl semicarbazones and related compounds compare favorably with these three reference drugs.

Third, with the exception of the data for W5and XI&, the activity of the compounds listed in Table 4.2 is found in three series of compounds namely IV, V and VIII. Since the substitution in the aryl ring were not constant in each of these three series, a strict comparison between the potencies and PI values cannot be made. However the following general observations (A-D) may be made.

(A) In series IV, all para substituents of the distal ring had ED5(]figures in the 3-15 mg/kg range. In contrast, the potencies of compounds containing ortho (IV2) or meta (IV12) substitution were 21 and 28 mg/kg. With the exception of IV8.1a , the structural isomers having para substitution displayed higher potency compared to the isomer lacking para substitution (IVsg> IVs ; IVu >IV2s). A similar trend was also noted with IV17and IV2 (IV17> I&). These results reinforce the hypothesis that the distal binding site contains a hydrophobic region which interacts with a small substituent in the para position of the aryl ring. Substitution at other positions while tolerated. reduced anticonwlsant activity in the MES screen. (B) It is of interest to note that IV17 is four times more active than the structural isomer N18.Thus it is conceivable that in addition to steric effects, the hydrophobic and electronic properties of the aryl substituents contribute to activity in the MES screen.

(a The PI values of IV3I and Vmlo which are approximately 22 and 21 respectively are particularly noteworthy. Increasing the size of the spacer function in MIl by a ~ffonyloxygroup gave rise to W5.However this molecular modikation resulted in reduction in potency and neurotoxicity compared to Vml.

@) The average ED50 figures in the MES screen for the compounds in series IV, V and VIU were 14.96, 8.90 and 13 -64mg/kg respectively i.e. the highest potency was found in series V. It is conceivable therefore that replacement of the methine proton by allcyl groups may indicate a hydrophobic bonding area on the binding site. However the average PI values for the compounds in series IV, V and VIII were 8.8, 7.8 and 9.8 respectively indicating that neurotoxicity was lower in series Vm. The data recorded in Table 4.2 do not conflict with the hypothesis that these compounds align at the binding site represented in Fig. 2.2.

Table 4.2 Evuluation of selected compounds in the MES, wPTZ and neurotoxicity screens after intraperitoneal injection in mice

MES Screen scPTZ Screen Neurotoxicity Screen P.I." Compd. t(h) EDSO(mg/kg) Slope t(h) EDSO(mg/kg) Slope t(h) TDso (mg/kg) Slope MES scPTZ No. (95% CI) (SE) (95% CI) (SE) (95% CI) (SE)

(9.27- 12.65) (3.12) (44.17- 139.19) (0.65) (1 72.03-304.50) (2.04)

135 Table 3.2 conttl. ,.,

4.4.2.0 Oral administration to rats mhase tcl

Phase 2c screening, which consists of the quantification of the anticonvulsant effect in rats after oral administration, defines the TPE, the experimental anticonwlsant activity and neurotoxicity in another rodent species, and develops dose information which is a prerequisite to subsequent chronic toxicity studies. More importantly, the phase 2c data obtained in rats must be carehlly reviewed and compared with similar data in mice before it can be determined if the accumulated results are sufficiently promising to warrant moving the candidate anticonvulsant into costly pharmacokinetic and chronic toxicity studies. Under clinical conditions, the oral route of administration is the preferred route for the long term administration of an anticonvulsant and significant activity with a high PI value are mandatory if candidate drugs are to emerge from this study. In addition, from the biodata generated (Table 4.3) , observations could be made whether the structural correlation obtained fiom the mouse data, especially those leading to the formulation of the postulated binding site (Fig. 2.2) could withstand hnher scrutiny using another route of administration and a different rodent.

Quantitative evaluation of one third of the compounds, principally from series IV, V and WII were undertaken in the oral MES screen and the data are summarized in Table 4.3. Almost 60% of the compounds tested in Phase 2a (Table 4.2) were selected for phase 2c testing. The remaining compounds from phase 2a are not included in the phase 2c as they all displayed neurotoxicity and or side effects (e-g. continuous seizure activity) in the scPTZ test. Compound IV6 was not selected in phase 2c due to an apparent discrepancy in the phase la and phase 2a results.

All the compounds tested demonstrated MES-selectivity and further discussion will refer to their evaluation in the MES screen only. Neurotoxicity was absent in virtually all compounds at the highest doses employed. The data in Table 4.3 reveal the very high potencies of many of these compounds in the rat oral MES screen. In fact the desired goal of an EDIo figure of 2-3 mgkg or less was achieved with ten compounds (~49~171131343) ; V5,6.7) and EDm figures of 3- 10 magwere achieved by fifteen compounds reported in Table 4.3. With the exception of I2 , IVJ4 and V2, all the remaining compounds in Table 4.3 had higher potencies than the first lead compound 5 (ADD199002). Thus one of the main objectives of improving the potency of 3 was achieved. The extremely high PI values displayed by most of these compounds suggests a wide margin of safety. Comparison with three established standard anticonvulsant drugs revealed that almost 91% of the compounds in Table 4.3 had greater potencies (lower ED50 figures) and 58 % had greater PI values than phenytoin; the analogous figures for carbarnazepine were 39 and 39 % respectively. All of the compounds were more potent than valproic acid and had higher PI values.

The marked increase in potencies of IVJ and V3 compared to X3 and V14 respectively indicates the importance of both the primary amino group and also the position of the aryloxy fbnction in the proximal aryl ring in conferring anticonvulsant activity.

The activities of IVa , and V7 are remarkable in terms of pure potency and PI. These compounds possess potencies of approximately i 5. 2 and 255 times that of phenytoin, carbarnazepine and valproate respectively. In addition, the lack of neurotoxicity at the highest dose examined reveals exceptional safety margins. All the three compounds (IV4 . N~sand V,) have similar activity and the EDso figures are in the range of 1 -52- 1-59 mgkg and the PI in the range of >3 15-328 respectively. Since one of the earliest compounds to be evaluated in the rat oral MES screen was IV4, this compound was selected for extensive bioevaluation and is the subject of a NM "red book".

Evaluation in the scPTZ screen was undertaken with 55% of the semicarbazones and related compounds which are listed in Table 4.3. At the maximum doses administered, the majority of the compounds were inactive while few demonstrated very marginal anticonvulsant properties. The only exception was IVzo which afforded protection in 50% of the rats using a dose of 50 mgkg. Table 4.3 Evaluation of selected compoi~ldsin the MES and neurotoxicity tests after oral administration to rats

Compd . MES Screen Neurotoxicity Screen P.1." No. t (11) EDa 95% CI Slope SE t (h) TDso 95% CI Slope SE (MES)

4.5.0.0 Anticonvulsant drun differentiation

Once promising lead molecules have been identified using the primary screens, more advanced mechanistic and seizure type models are needed to refine the choice of a lead compound. Antiepileptic drug differentiation delineates antiepileptic profile and possible mechanisms of action of the candidate substance using in vivo and /or in vitro models. The $ vivo tests include antagonism of seizures in mice induced by bicuculline, picrotoxin and strychnine (Phase 3a). The @ vitro tests include receptor- binding studies which are designed to characterize the effect of the candidate substance to displace radiolabeled [3~flunitrazepamand L~H]GABAfrom crude synaptic membranes (mouse whole brain) and to inhibit [3~adenosineuptake into synaptosomes (Phase 3b ). Phase 3c and 3d studies are used to evaluate more definitively the effect of the candidate substance on seizure threshold in mice (Phase 3c) and rats (Phase 3d). In addition, the effect of IVJ to limit calcium idux into primary cultures of mouse cerebellar granule cells are undertaken to gain some insight into the mechanism(s) of action of the potential drug.

4.5.1.0 Subcutaneous bicuculline (scSicb oicrotoxin (scPic), and strvchnine (scStr) tests (Phase 3a)

Seizures in mice induced by bicuculline, picrotoxin and strychnine (Phase 3a) act via different neurotransmitter systems and the resulting EDsos may reflect the activity profile of the test compound, which can be compared with those of clinically effective drugs. For example, bicuculline blocks GABA receptors and thus interferes with GABA- mediated inhibitory transmission; picrotoxin interferes with chloride channels regulated by GABA receptors; and strychnine blocks postsynaptic inhibition mediated by glycine. Thus anticonvulsants which block seizures induced by bicuculline. picrotoxin and strychnine do so by acting on y-arninobutyric acid (GABA) receptors, chloride channels and glycine receptors respectively (Kupferberg, 1989). If a compound blocks seizures induced by bicuculline, picrotoxin or strychnine, it always demonstrates pharmacological activity in either the MES or scPTZ tests (Kupferberg, 1989). The choice of seizure endpoint is critical as these chemical convulsants produce almost identical seizure patterns. Those compounds which block the hindlimb tonic extension induced by the maximal electrical stimulation will also inhibit the tonic extension produced by the chemical convulsants. A differential pattern of pharmacological activity can be attained by using clonic seizures as the end point. The absence of a clonic seizure in rnetrazole- , bicuculline- and picrotoxin-treated animals indicates that the substance has the ability to elevate the respective seizure threshold. In strychnine-treated animals, abolition of the hindleg tonic-extensor component is taken as the end point and indicates that the test substance has the ability to prevent seizure spread.

The testing results for the selected compounds (IV4$, 31 ; VIIIl) in phase 3a are presented in Table 4.4. The data in Table 4.4 reveal that only Vml interferes with the binding of bicucuhe to the GABA receptors while N3 interferes with the binding of picrotoxin to the chloride channels. Both N3and VIIIl had higher potencies and PI values compared to the standard drugs and compound 5 in preventing the respective seizure spread. No sigruficant protection was noted by any of the compounds listed in Table 4.3 against seizures induced by strychnine and although some activity was exhibited by other compounds in these tests, the doses were at or near the toxicity levels.

Thus the data suggest that GABA receptors and chloride channels may be the sites of action of these anticonvulsants. This results are similar to the results obtained for some of the aryl semicarbazones reported earlier (Dimmock et al., 1993). Table 4.4 Evaluation of selected compounds for protection against seizures induced by subcutaneous injection or bicuculline, picrotoxiu and strychnine in mice

ED50 (mglkg), (95% C.I.), Time of [slope] and PI Compound Testa Tho (mdkt.9, sc bicuculline sc picrotoxin sc strychnine (h) (95% C.I), [slope] - (ADD 199002) 1,1 427 >700 76.8, (50.60- 102.00), >500

[9.30], PI : 1.1 [3.92], PI : 1.6 [14.7], PI : 1.4 n: First number. TDI,,; secoud nurubcr ED$,,'s b: Data rcproduccd with pcrlnission of tlrc copyright owner (Sw'iayiird aJ., 1992) 4.5.2.0 Timed intravenous aentvlenetetrazol test (Phase 3c)

This screen is designed to provide information on the ability of a compound to lower seizure threshold. It is not uncommon that certain substances while effective in blocking the spread of seizures can lower the threshold at the same time. Several compounds that exhibit anticonvulsant activity appear to be proconvulsant when administered in high doses. These compounds prevent seizure spread in both mice and rats but are ineffective in blocking seizures induced by chemical convulsants. This is a negative characteristic and would essentially cause hrther development of such substances to cease.

The timed intravenous pentylenetetrazol test (Phase 3c) is used to evaluate whether a compound is simultaneously both proconvulsant (lowers seizure threshold) and anticonvulsant (prevents seizure spread). Pentylenetetrazol is ifised into the tail veins of mice. The time to the appearance of the first focal seizure (first twitch) and clonic seizures is measured. A significant increase from control in the time to the first twitch or clonus indicates the test substance increases seizure threshold. Proconvulsants require less pentylenetetrazol to produce these end points.

Selected compounds were tested in Phase 3c at two doses namely the approximate EDXI in the MES test and the TDlo figures (Table 4.5). As shown in Table 4.5 , none of the compounds lowered seizure threshold at the TDlo dose and thus were not proconvulsants. At the MES EDSOdose IV34 lowered the time to first twitch and the remaining compounds increased the time to first twitch or clonus. Table 4.5. Evaluation of selected compounds on the threshold for minimal seizures induced by the timed intravenous infusion of pentylentetrazol in mice

i-p. Approximate Time of Pentylenetetrazol (m@g 2 S. E.) Dose Equivalent Test Substance (mgkg) (h) First Twitch Clonus -5 (ADD 199002f 0 32-30? 1.2 38.7Ok1.5 MES EDso 220 TD3 34.70k1.2 53.30&5.0* -*-.-.f*f*.-l.----.---.~----*----~ -*------.-* -.------...... ** m4 0 30.40+1. I 35.20kI.3 13 MES EDso I 32.30kI -4 37.60k1.5 108 TDso 32.60-3 42. IOk1.4

rv9 0 33.335t1.03 44.38f2.47 15 MES EDSo 1 32.9421-38 4 1 -2Ofl.60 95 mso 34.56k1.54 44.3 8S.47

0 30.99k1.07 35.4421.41 15 MES EDSo 1 30.58k1 -54 36.37fl-58 204 'no 29.4321.62 48.45k5.76

0 26.88k1 -31 3 1.81k1.53 11 MES EDso 1 29.57k1.00 32.88k1.08 243 SO 30.20&1.56 38.3 7e.27

0 4 33.34k1 -06 38.52k1. 1 1 9 MES EDso 30.41f1.92 37. I8k1.7 1 106 TDso 35.92f1 -86 5 1.43k6.99

VIII 1 0 30.70k0.8 33.20+,0.9 16 MES EDSo 1 30.70kO.9 36.3Ok1.5 ...... 181 TDso _.___._.-.--__.-.11.-...-.~~~..--~~~~....~~.~...... -.~~~~...~--~~.-~.....___...__.32-70? 1.3 37.20+1 .3 Phenytoin 0 33.60kO.6 42.60t1.5 6.5 MES EDso 35.50k1.6 42.5052.1 ...... 43 T@o 28.20?1.9* ...... 50.10+3.1° Carbamazepine 0 35 -2040.7 46.20k I.5 11 MES EDSO 36.1Of 1.3 48.60k4.2

.---..-.__..----.._____..--._._.__-...... f._____._...__1...... ___.___..__._-...f.__.__..___....._.~....~~~...48 TDso --...-.--...--..-.-.---~.-~...-~---....------.-..--...~+-.-~~.~...... ~...-.~.~.~~~~.~.~~.~....~~.-~~.~~~~~~---.36.50+1. 1 54.50fl.8 Valproate 0 34.80&1.8 43.40k1.8 60 37.1Ok 1.1 45.00k2.3 120 sc Met ED3 40.40k2.0 55.20kI -9'

*Significantly diaerent from solvent control. a : Swinyard q 4..1992 4.5.3.0 Corneal kindled rat test

The advanced evaluation of novel compounds includes models which are thought to be specific for a seizure type. In recent years, the kindling models (corneal. hippocampal and arnygdala kindled rat tests) have been an usehl adjunct to the more traditional anticonvulsant tests for identifying the potential utility of a test substance for treating complex partial seizures. The relatively slow development of seizure generalization within the brain during kindling compared to that of maximal electroshock and pentylentetrazol models of epilepsy, offers the opportunity to study intermediate stages of epileptogenesis prior to the development of Mly kindled, generalized seizures (Stark, 1992). The convulsions produced in the kindled rat test (Phase 3d) are believed to constitute a suitable model of complex partial seizures evolving into generalized motor seizures in humans (Loscher and Schmidt, 1988 ; McNarnara, 1989). Kindling can be produced both electrically and chemically. Seizures evoked in corneally kindled rats provide a suitable model consistent with human complex partial seizures secondarily generalized. Electrical kindling of rats via corneal electrodes produces a progression of behavioral seizures identical to that seen with stimulation via the amygdala. The ability of a candidate substance to prevent the expression of stage 5 seizures in this model may be predictive of its effectiveness against complex partial seizures in man. The EDSo is the dose required to reduce seizures from stage 5 to stage 3 or less.

Evaluation of IV, and WIl in the corneal kindled rat test are presented in i Table 4.6. As shown in Table 4.6, compound displayed excellent activity and lowered the seizure score from stage 5 to 0 and the ED50 was 3.93 mg/kg. Compound IV4 displayed very good potency compared to the standard anticonwlsants. Compound VIII, lowered seizure score to one at 50 mgkg and the EDSO was approximately 24 mgkg and the potency was comparable to carbamazepine but higher than other standard anticonvulsants. These results suggests that IVA and VIIIl have the potential to limit focal firing and hence may be usefbl in the treatment of complex partial seizures. Table 4.6. Evaluation of selected compounds against stage V seizures in the corneal-kindled rats

Test Substance Route Time of EDso 95% Test (h) (mg/kg) Confidence Interval -5 (ADD 199002)' p.o 2 I 150 mgkg --

P henytoin P-0 1/2 WOO --

P henyt oin i. p 112 48.25 24.57-78.36

Valproate P-0 112 117.41 67.98- 189.02

Felbamate P-0 4 83 -9 28.70- 192.10

a : Data taken with permission of copyright owner ( Dirnmock and Baker, 1994) 4.5.4.0 Hippocamoaf kindled rat test

Of the various kindling paradigms described in the literature, the rapid hippocarnpal kindling model of Lothrnan a d(1988) appears to offer some distinct advantages for the routine screening and evaluation of a new anticonvulsant substance. Kindled seizures not only provide an experlnental model of focal seizures, but also provide a means of studying complex brain networks that may contribute to seizure spread and generalization fi-om a focus (Lothman et al., 1988).

One potentially important advantage of the rapidly recurring hippocampal seizure model is its ability to provide a framework for assessing drug efficacy in a focal seizure model in a temporal fashion. When a drug treatment is observed to significantly lower seizure score and decrease afterdischarge, a dose-response study is initiated. For this study the ability of a candidate substance to block afterdischarge and reduce seizure severity is quantitated by varying the dose between 0 and 100% effects. Drugs that are active against focal seizures would be expected to reduce the behavioral seizure score (BSS) to 2 or less and significantly lower the afterdischarge duration (ADD).

The potential of NJ to limit focal firing was challenged by another sensitive hippocarnpal kindling model and the testing results are presented in Table 4.7.

Compound N4was quite active (dose Smg/kg) in lowering the seizure scores peaking at 45 rnin. The decrease in afterdischarge duration also correlated well at this time period. Future studies include looking at acquisition and dose responses in the kindled animal. Compound N4has the potential to be useful in the clinic against focal seizures that become secondarily generalized. Compound V4 was also active in this model and had an

EDsofigure 3 6.48 mg/kg. Compounds IV, I and V15 did not display any significant activity at the dose tested. Since IV~Idisplayed promising activity in both mice i.p and rat oral screens against MES induced seizures (Tables 4.2 and 4.3). the lack of significant activity by IV;I (dose Srngkg) in this screen suggests that IV;, may find limited use in controlling focal seizure. Table 4.7. Evaluation of selected compounds against stage V seizures in the hippocampal-kindled rats

Test Substance Time Dose Seizure Score AAerdisc huge Course (W/kg) 2 S.E.M. Duration @in) (sec) -t S.E.M. w Control 5 5.00 +_ 0.00 84.71 + 10.84

Control

Control 36.4gb

Control

* Significantly different from control a : Compound was tested from +I 5 to +255 min and was found to be inactive (--) b : 95% C.I. 29.53-43.06; slope 7.60 k 2.30 4.5.5.0 Amvpdala kindled rat test

The most common way to produce kindling is stimulation of a specific area of the amygdala via implanted electrodes. A fked current is applied until an afterdischarge is produced at the site of stimulation. Repeated daily stimulation results in behavioral changes that are classified or graded. These seizures progress to where the animal rears with loss of balance, appearing to have a generalized clonic seizure.

Since the pre-drug base line and vehicle values (Table 4.8) were significantly different for three of the five parameters measured, the effects of the drug treatment were analyzed against vehicle treatments given for that drug and not baseline values. The data in Table 4.9 suggested that IV4 has no signs of gross motor impairment. Compound IV, was effective at three doses (5, 15 or 30 mgkg) in lowering seizure stage, however. none of these doses reduced seizure stage by more than 14% (Table 4.9). Even a high dose of 100 rn@g had minimal effect and the resultant average seizure severity was only 4.8. The data in Table 4.10 display the effect of I& on the various behavioral and electrical seizure parameters. No statistically sigruficant reductions in behavioral and electrical seizure parameters were observed. Thus. there appears to be slight effect of IV, on amygdala kindled seizures but the effect is more than an order of magnitude less than what was observed in corneal kindled rats as mentioned under section 4.5.3.0.

Phenytoin is the drug of choice for the treatment of complex seizures in man and a dose-response study of this drug in arnygdaloid. fkuy kindled rats revealed slight and insignificant changes in the expression of the convulsions, with alterations of afterdischarge occurring only at doses which produced ataxia in all animals tested (Albertson a &., 1980). Though this model is a very sensitive model it has some limitations as amygdala is not a homogenous neuroanatornic structure and further analysis of relevant networks will require even more detail about these fUnctionally interactive groups of brain structures (McNarnara et al.. 1986 ; McNamara, 1988). Table 4.8. Mean baseline and vehicle values (SEM) for behavioral and electrical seizure parameters using the amygdala kindling rat model

Drug Behavioral Behavioral Forelimb Forelimb Amygdala seizure seizure clonus clonus AD duration latency duration latency duration (sec) (sec) (sec) (set) m4

baseline 2.6 k 0.6 85.0 + 2.4 10.3 k 3.5 65.2 + 3.5 82.8 k 2.5

* Statistically diirent from corresponding baseline value for the same drug (p < 0.05) a : n = 40 measures (8 per rat)

Table 4.9. Changes in mean behavioral seizure stage and percent reduction in seizure stage for N4in amygdda-kindled rats

Dose (mgflcg) Mean seizure stage (SEM) Percent reduction in seizure stage* (%) Vehiclea 5.0 t 0.00 -

* Values represent percent reduction from vehicle (n = 5 rats) a : n = 40 measures (8 per rat) b : n = 10 measures ( 2 per rat) Table 4.10. Effects of IVI on seizure parameters in amygdala-kindled rats

Dose Behavioral Behavioral Forelimb Forelimb Am ygdal a (mglkg) seizure seizure clonus clonus duration latency duration latency duration Time Time Time Time Time (sec + SEM)' (sec k SEM)' (sec f SEW (sec + SEWa (sec + SEM)'

* Significantly different fiom vehicle @<0.05) a : Values are means + SEM for all animals exhibiting the parameter 4.5.6.0 Measurement of intracellular calcium transients

Calcium channels play a major role in neurond function, in part, because of the important second messenger actions exerted by free intracellular calcium ions. The passage of calcium ions into the cell can be mediated by the ligand- and voltage-gated NMDA receptors, which may be very important in epilepsy-induced cell damage (excitotoxicity). Voltage-gated calcium ions channels have been categorized into four main types (T, N, L and P). The L, P and N currents are high threshold currents. The T-type (transient or type I) calcium channel can open with minor cellular depolarization, resulting in a small rise in calcium ion current (Tsien a aJ., L988). Phenytoin, carbamazepine, benzodiazepines and barbiturates have been reported to reduce calcium ions influx into synaptic terminals and to reduce the presynaptic release of neurotransmitters, although in some cases the concentrations required were in excess of therapeutic levels (Macdonald and Meldrum, 1989).

The results of the effect of N4 on [ca27i transients of mouse cerebellar granule cells are presented in Table 4.1 1. The biodata generated may give some insight of the ability of to limit calcium ions influx into granule cells via both voltage-sensitive calcium ions channek and tetrodotoxin-sensitive sodium ion channels. The results are expressed as a percent of the control [ca2']i transient induced by potassium chloride or veratridine. In order to gain an appreciation for the overall effect of a candidate substance on the [ca27i transient, the results are expressed as a percent of the peak height, the area under the curve and the height of [ca2']i transient at 60 sec.

The results from Table 4.1 1 suggest that [ca2+]itransients evoked by 55mM potassium chloride and 10 pM veratridine are reduced by 10 pM of IV4 in a reversible manner. A pretreatment enhanced the effect in both assays. The effects of tV4 on veratridine-evoked transients was most evident on the peak height which was reduced 3040% in the presence of 10 pM of IV4 and the corresponding reduction in the area under the curve was around 20%. In contrast, IV4 limited potassium chloride-induced calcium ions influx by 14 % reduction in the area under the curve and this effect is similar to valproate. The effects on peak height and area under the curve were concentration- dependent over the range of concentrations tested. It is not clear why there is an enhancement of veratridine-evoked[~a~~]itransients in the presence of O.lpM of IV4. The effect of I& on whole-cell peak currents of mouse conical neurons ( 13- 16 days in vitro) was also undertaken, and the results suggest that 100 pM of IV4 does not interact acutely with GABA-or glutamate-sensitive ion channels (control : LOO f 3, + drug : 105 r 14, evoked by 1pM GABA : control 100 + 2, + drug 107 f 1 1, evoked by 10 pM glutamate and 1 pM glycine).

Anticonwisants like phenytoin and carbamazepine have known effects at the voltage-sensitive calcium channels (VSCC) and voltage-dependent sodium channels attenuating both potassium chloride-and veratridine-induced calcium ion influx (Table 4-11). On the contrary, ethosuxirnide and valproate, which do not affect voltage- dependent sodium ion channels but do decrease conductance through the t-type VSCC, do not affect veratridine-induced calcium ions influx. In contrast, valproate at the concentration tested (500pM) does limit potassium chloride-induced calcium ions influx.

These results suggest that IVJ has the ability to interact with the voltage- dependent sodium ion and/or calcium ions channels. However. before any firm conclusion can be made, additional studies would be necessary to understand the mechanism(s) of action of IVJ.

4.6.0.0 Overt tolerance and liver enzvme studies Whase 5)

An understanding of the pharmacokinetic and phmacodynamic drug-drug interactions is essential for the rational use of antiepiletic drugs. Patients with intractable epilepsy usually receive more than one antiepileptic drug. Examples of enzyme induction, inhibition and changes in protein binding with polytherapy are wen documented (Perucca and Richens, 1985). It is therefore necessary to obtain preliminary information on these interactions during the preclinical development of any new antiepileptic drug. Changes in the efficacy of anticonvulsant activity upon repeated administration of an experimental drug may also indicate the development of tolerance to its anticonvulsant effect.

Since most antiepileptic medication is taken for prolonged periods and often in combination with other drugs, and because most anticonvulsant drugs undergo significant hepatic metabolism, it becomes important to assess the effect of subchronic therapy and combination therapy on anticonwlsant efficacy and on liver metabolizing enzymes. In addition, the absence of any significant changes in the liver weights and microsomal protein contents would be si@cant positive attributes.

The effects of chronic oral administration of N4 and related prototypic agents on the liver microsomal system of rats are presented in Table 4.12 (Phase 5b). The results of the hepatic microsomal studies revealed that neither liver weight, hepatic microsomal protein content nor the activities of five drug metabolizing enzymes were affected by chronic administration of IVJ to rats. Subchronic administration of IVA significantly increased ethoxyresorufin 0-deethylase activity by 100%. Its effect on other hepatic metabolizing enzymes were small when contrasted with the magnitude of the increase observed for phenytoin and carbamazepine (Table 4.12). Since the short screen (Phase 5b-I) did not show significant differences in liver parameters, hnher liver studies were not undertaken (i.e. Phase 5b-2,3 and 4). Table 4.12. Effect of 74ay subchronic oral administration of N4 and some related antiepileptic drugs (100mg/kg) on the liver drug metabolizing enzyme system of rats I Percentage chGgea I Parameter Control N4 w4 -5b phenytoinb CBZ~.' Liver weight 7.05 k 0.38 i 6.83 f 0.27 f -13% / - - -

-7 f ('------..I. microsomd 1 32.3 t 4.2 i 32.3 + 4.1 1 no - - - protein yield f change f (mgikg of liver) 1_____.-. cytochrome P-450 f 0.87 f 0.05 1 0.85 + 0.05 1 -12% f &5% ?52% ?45% (nmoledmg) -----.--.---- ! 4- pa.--- p-nitroanisole 1 0.77 t0.03 1 0.83 k0.05 [ ?7.8% 1 -127% ! ?220% f ?210% 0-demethylase I (nrn~les/rng/min)~/ --l----i----lp --I___--.--- ! NADPH - -- - % ?37% / f46% cytochrome reductase

a : Percentage changes are with respect to control for each group respectively b : Data reproduced with permission of the copyright owner (Dimmock and Baker. 1994) c : Carbamazepine (CBZ) The lines - indicate that the compound was not tested and the designation - indicates data are not avaihble d : Enzyme activity nrnoledmg of proteinfmin. 4.7.0.0 Genetic models

4-7.1.0 E~ile~ticFowl

The development o f the epileptic chicken as a valid model o f human epilepsy was based on the premise that in order to provide a suitable model, the common anticonvulsants used in the treatment of human epilepsy should not only protect against seizures induced by intermittent photic stimulation (IPS) but also do so at appropriate plasma concentrations found in clinical conditions (Johnson and Tuchek, 1987). AII common anticonvulsants used in the therapy of human grand ma1 epilepsy (phenytoin, phenobarbitd carbamazepine, primidone and valproate) were found to prevent IPS- evoked seizures at plasma concentrations similar to those reported in humans (Johnson q -al., 1977 ; 1978 ; Davis et al., 1978 ; Johnson and Tuchek, 1987). An additional benefit of this model is the availability of non-epileptic (carrier) hatchmates of a uniform genetic background. These carriers provide a usefbl paradigm for the pharmacological and biochemical studies as they serve as excellent controls. Selected compounds were injected by the intravenous route and are challenged with a strobe light one hour after administration of the compound.

Two series of compounds were examined in this model with the aim of observing whether oxygen or sulfbr is a preferable spacer atom between the two aryl rings and also to compare the ED5o figures with those obtained in the rat oral and mouse intraperitoneal screens. The results of the evaluation of selected compounds in epileptic chickens are presented in Table 4.13. The following aspects are noteworthy.

First, all the compounds tested exhibited potency range (1-2.5 mgkg) comparable with diazepam and were more active than phenytoin, carbamazepine and phenobarbital. These results are similar to the mouse i.p. test (phase 2a) and the rat oral MES test (phase 2c).

Second, the ED50 results for I&, VIIII.~are similar to the rat oral MES screen (Phase 2c), and are in the range of 1-5 rngfkg. However. VIIIl is better tolerated in rats than chickens. The EDso figures in the mouse i.p test (Phase 2a) for N4,Vml,4,7.t are approximately in the range of 12-25 mgkg.

Third, the thio compounds are marginally more potent than the 0x0 analogues and in general are more f?ee from side effects. The potencies however are unaffected by whether oxygen or sulfur are used as the spacer group.

Fourth, the most interesting compound seemed to be ViII, which gave protection after 6 hours and may have been active at the end of 24 hours. In both series, substitution in the aryl ring by a halogen increased potency compared to the unsubstituted derivative. The lowest EDSOfigures were displayed by compounds containing a chloro substituent. Hence the results from the epileptic chicken model are comparable with the quantitative data provided in the rat oral screen.

4.7.2.0 Frinns audioeenic seizure susce~tiblemice

Frings audiogenic-susceptible mice are genetically susceptible to sound- induced seizures. Beginning at about 21 days of age, they display prominent seizure activity in response to a high-intensity sound stimulus. Frings mice possess a life-long susceptibility to sound seizures. In contrast, DBN2 mice are only susceptible to sound- induced seizures during a narrow period of development (18-2 1 days of age). Their seizures respond to a wide range of CNS-active drugs, and in this respect they are a highly usehl screening model for the early identification of potentially usefid anticonvulsant drugs.

All of the commonly used anticonvulsant drugs protect against sound-induced seizures in mice with the same order of rank as that seen with clinical use (Chapman q -at., 1984). This model provides a method for the acute screening of potential anticonvulsants and allows a simple determination of ED50 values for comparing potencies between agents. Thus this model is a highly usefbl genetic model for identifying and characterizing the underlying cause(s) of inherited epilepsy and also for acute and chronic pharmacological investigations.

The lead compound N4was administered by the i.p route to Frings mice and after one hour the animals were exposed to a sound stimulus of 110 decibels. Mice not displaying hindlimb tonic extension were considered protected. Compound IV4 was active in this model and had a EDIo figure of 17.74 mgkg [(95% C I : 10.38-36.66), (slope: 2.20 t- 0.64)]. The EDSa (mgkg, i.p) figure for the standard antiepileptic drugs in this model using DBN2 mice are as follows namely phenytoin 2.3-14, valproate 55-300, phenobarbital 2.7 and diazepam 0.04-0.12 mag respectively. The corresponding figures for the standard AEDs in this model using Frings mice are not available. Table 4.13. Evaluation of selected compounds in the adult epileptic chicken model and comparison of the results with other epileptic screens

Diazepam 0.52~

Dose of 3 .S mgkg was lethal himaIs exhibited sedation as side effect Animals exhibited light ataxia and vocalization Animals exhibited light ataxia After 24 h, a 2mg/kg dose gave complete protection but no protection was noted after 96 hours; in addition animals also exhibited Light vocalization Dose of 4 mgkg was lethal After 6h, a 3 mgkg dose gave complete protection ( long acting) Data reproduced with permission of the copyright owner (Fisher et al.. 1985) The designation -- indicates that compounds were not tested, and the line - indicates that data not available The overall pharmacological activity profile for is presented in Table 4.14.

[n summary, I& is an effective anticonvulsant with high potency and a very favorable protective index. Its profile of anticonvulsant efficacy diEers from established drugs. The activity against MES-induced seizures ranks it very high in terms of pure potency. Compound I& was not effective against chemically induced seizures. Doses equivalent to the TD5o have no significant effect in the i.v. pentylenetetrazol threshold screen and thus it is not a proconvulsant.

A seven-day subchronic administration of 100 mgkg of IV4 revealed that neither liver weight, hepatic microsomal protein content nor the activities of five out of eight drug metabolizing enzymes were affected. These characteristics are looked upon as positive aspects of the compound.

Compound IV4 was also effective in the rat kindling models(corneal and hippocampal) and displayed high potency. However, it displayed minimal effect in an

amygdala kindling model. Thus N4 has the potential to limit focal firing and hence may be clinically usehl in the treatment of complex partial seizures. Compound IV., was also active in both the genetic model (epileptic fowl and Frings audiogenic mice). Compound IV4 has the ability to interact with the voltage-dependent ~al-and/or ~a"channels. This compound appears to act by one or more mechanisms which are different from established anticonvulsant drugs.

Compound IV4 also stimulated gene expression in PC 12 cells and had activity comparable with (-) deprenyl which is used as a neuronal rescue agent. However, hnher studies in the in vivo ischaemia model (Li et al., 1992) showed negative results and thus IV., does not seem to have significant neuronal protective action. Compound IV4 is the subject of a NIH "red book" and detailed pharmacological evaluations have been undertaken. Since antiepileptic drugs are generally administered orally, it is important to know how well the candidate drug is absorbed from the gastrointestinal tract. The absorption ratio was calculated as follows :

oral dose MES rat fitraperitoneal dose MES rat = 1.59/2.37 = 0.670.

It is generally agreed that the absorption ratio should be equal to or less than 4. Therefore, Na is well absorbed after oral administration (Swinyard, 1989).

4.9.0.0 Statistical analyses and quantitative structure-activity relationships studies (OSAR)

In order to evaluate the effects of different substituents in the distal aryl ring on anticonvulsant activity and neurotoxicity, various linear and semilogarithmic plots were made. The relatively few EDSOfigures in the scPTZ test precluded their consideration; hence comments will be confined to activities in the MES screen. The physicochernical constants chosen reflected the electronic (qo'), hydrophobic (z), steric (MR i.e. molar refractivity) and topological (S A i.e. surface area) characteristics of the aryl substituents. A correlation coefficient of 0.8 was chosen arbitrarily as indicating a relationship between the physicochemical constants and bioactivity and when observed, the specific r values are indicated as r r. which refers to linear and semilogarithmic correlation coefficients. QSAR studies were undertaken for only compounds in series IV and the correlation coefficients were determined for both linear and multiple linear regression plots.

Statistical analyses are divided into three sections. The first and second section deal with correlations of MES EDso (mice i.p, Table 4.2), and MES EDIo (rat p.0, Table 4.3) data with different physicochemical parameters. The third section deals with correlations of the MES EDso (mice i-p., Table 42), and MES ED50 (rat p.0.. Table 4.3) results with partition coefficients. Table 4.14. Pharmacological activity profde of IV4 and clinically used antiepileptic drugs

EDSompjkg , (PI) or percentage changes P henytoin CBZ" Valproate Testing models

Etectricaltv induced seizures MES (mice, i.p) 6.32 9.85 287 (6.52) (4.85) ( 1.68) MES (rat, pa) 23 -2 3 -57 395 (>2 1.6) (101) (2.17) MES (rat, i. p) - - -

Corneal kindled rats (p.0) >I00 28.90 117.41 Hippocampal kindled rats 34b 4.6b 1lob Amygdala kindled rats 3 0-60" 1 0-3 od 1god

Chemicallv induced seizures sc pentylenetetrazol >50 >so 209 (2.3 1) sc bicuculline >60 >60 43 7 (1-1) sc picrotoxin >60 28.9 311 (1-7) ( 1 -6) sc strychnine >60 >60 345 ( 1-41

Genetic epile~svmodel Epileptic fowl - - - Frings audiogenic mice 2- 14' - 55-3 00'

Liver enzvme studies J% changes) Cytochrome P 450 ? 52% ?- 45% - p-Nitroanisole 0-demethylase T 220 % T 210% - NADPH cytochrome t37% ? 46% - reductase UDP-glucuronosyltransferase t 15% T 12% - a : Carbamazepine b : Suppression of kindled motor seizures (Lothman gt a.. 1988) c : Compound IV, had minimal effect and reduced seizure severity by 14 % at a dose of 45 mgkg d : Data reproduced with permission of cop_vrighrowner ( Kupferberg, 1989) e : The results represents the EDso dose in DBN2 mice (Kupferberg, 1989) Six groups of compounds k-fJ were analyzed in this section. Plots were made between the o, d , x, MR and SA constants of the aryl substituents in the distal aryl ring against MES EDIo figures of (aJ all members of series IV listed in Table 4.2

(I?)V1.V3. Vs. V7. and V9 V4, V6 and V8 (dl Vml. VIIL. W7.Vm 8 and Vmlo In addition, in order to obtain some insight into the importance of the size and shape of the group attached to the azomethine carbon atom and also at the para position of the distal aryl ring, the ED50 figures of @ EV4, V3 and Vq and (f) IV4. IVn W29,N3 and N were plotted against the MR and SA values.

A positive correlation between the MES activity of the compounds in (g) i.e.

I&,V3 and V4 was noted with both the MR (r, = 0.955, r,l = 0.93 1) and SA (r, = 0.919, r,l= 0.899) constants i-e. activity increased as the size of these physicochernical constants rose. This result strengthens the hypothesis that the alkyl groups of V3 and VJ interact with the hydrophobic area which may be present on the binding site. In the case of (0, the r values for the linear plots of the MES EDIo figures against the MR and SA constants were 0.693 and 0.709 which shows a general trend whereby activity increased as the MR and SA constants were elevated. In the remaining cases, no correlations were noted. QSAR studies correlating log (1/EDlo) for compounds in series N with o,r, MR and SA indicated a poor correlations (r

Section 2

Six groups of compounds &- f) were analyzed in this section. Plots were made between the a, d , R, MR and SA constants of the aryl substituents in the distal aryl ring against the MES EDSOfigures of (4) ail members of series IV listed in Table 4.3

@) V,, V3, VI and V7 @ V2,VJ. Vs and V8 and (d) Vml, Vl& and Wrlo In addition, an evaluation of the importance of the size and shape of the groups attached to the azomethine carbon atom and the para position of the distal aryl ring were found by plotting the MES ED Jo figures against the MR and SA constants found in (g) 1%. V3 and v4 and (B m4. w22,N29. w31.N33. rV34 and NU-

The compounds in (b3 correlated positively with the o (rl = 0.808, rsl= 0.825). n (rl = 0.786, r,, = 0.85 l), MR(rl = 0.692, r,l= 0.819) and SA (rl = 0.899, rsl = 0.926) constants. Similarly, the compounds in @) correlated positively with the o (q = 0.998,r,,

=1.000) , n (r, = 0.847, rsl = 0.865), MR(r, = 0.956, r,, = 0.966) and SA (r] = 0.778, r,l= 0.799) constants. except a negative correlation was noted between the a values and the MES EDla. The potencies of the semicarbazones listed in (s)correlated positively with the a (ri = 0.765, r,~=O.8 17) and SA (rl = 0.834, r,l= 0.86 1) constants. Plots of the MR and SA constants of I&. V3 and V4 (g) against the MES EDro figures in Table 4.3 revealed a negative correlation. No correlations were observed in the remaining cases.

Thus relationships were noted with various compounds in series V, VIII but not IV. Though sigruficant relationships of individual parameters with the compounds in series N was not observed, QSAR studies revealed that the biological activity displayed by compounds in series IV is dependent on all the four parameters viz. o, n, MR and SA (r =0.87). In general, increases in the o, K, MR and SA constants in V and VtII caused increases in activity and this observation can be utilized in subsequent drug design. However the exception to this general trend is the group of compounds IV4, Vj and V, (g) whereby activity was diminished with increasing size and shape of the substituents.

Hence fkture work should be directed to placing various groups on the azomethine carbon atom in order to evaluate further the structural requirements for interaction at the binding site. Section Ill

Linear and semilogarithmic plots between the partition coefficients and the mouse intraperitoneal ED5o figures of N4, N9,V3 and VIII did not show any correlation (r<0.8). Similarly no relationship was established between the partition coefficients of IVd, IVg, V3, Wll J and X3 and the ED50rat oraI data.

4.10.0.0 Determination of log P values

The biological activity of a compound is influenced by steric and electronic parameters as well as the partition coefficients of the molecule. Previous studies by Dimmock et d(1993) suggested that the activity of semicarbazone anticonvulsants was dependent on both pharmacokinetic and pharrnacodynamic properties. The theoretical log P values reflect the ability of drugs to cross biological barriers and membranes before effecting anticonvulsant bioactivity. The binding site for the interaction of semicarbazones have been explained in a previous section (Figs. 2.1 and 2.2).

The hydrophobicity of the molecule (log P) will be expected to affect the ratdextent of transportation to a site of action and may align with the liphophilic pocket of the binding site. The separation and orientation of the lipophilic pocket and hydrogen bonding surface influence activity.

Since bioactivity is considered to be influenced by the rate and extent of the passage of a drug to its site of action (Silverman, 1992), the partition coefficients between 1-octanol and buffer (pH 7.4) of ten representative compounds and carbarnazepine were determined. The log P values of these compounds and error are indicated in parentheses viz. HIl (2.07,0.08), IV, (2.66, 0.1 I), IV4 (1 -91, 0.08), IV9

(2.76, 0.1 I), W.4;(1.29, 0.05), IVja (1.92,0.05), Vj (2.03,0.08), VIII 4 (2.80, 0.1 I ), X3(1 -96,0.08), X4(2.06, 0.08)and carbamazepine (2.20,0.09). The data support the concept that the compounds align at a specific binding site and are not structurally nonspecific (Albert, 1985). First, the log P values of compounds which were either very weakly active or inactive in the mouse irrtraperitoneal and rat oral MES screen (Tables 4.2 and 4.3) had similar partition coefficients to compounds as N4 V3, X3 and XJwhich demonstrated marked anticonvulsant activity.

Second, linear and semilogarithmic plots between the partition coefficients and mouse intraperitoneal EDso figures of IVJ, ,V3 and VIII 4 did not show any correlation (60.8). Similarly no relationship was established between the partition coefficients of IV4 IVg ,V3, Vm 4 and X3 and the EDso rat oral data (rC0.8).

Third, comparisons of the mouse and rat data for X and JC, (Table 4.1) indicate the greater anticonvulsant activity of X3 yet both have the same partition coefficients.

Fourth, in the MES screens, the isosteres IV4 and VI& had the same activity in the mouse intraperitoneal test and both compounds are highly potent when given orally to rats yet their partition coefficients were markedly different. Of interest is the fact that the average log P value for all of the active compounds were 2.15 which is similar to the figure for carbamazepine.

4.1 1.0.0 Stabilitv studies

In solution sernicarbazones are capable of displaying E/Z isomerization pertaining to the carbirnino double bond. The ratio of isomers could be influenced by the sizes of the groups on the carbimino carbon atom. Previous studies by Dimmock et al.. ( 1990) revealed that the percentage of E isomers diminished considerably as the size of the group was increased. Hence, three representative compounds namely IV1, Vj and V4 were selected for stability studies. Compounds N4,V3 and Vq were dissolved in deuterated dWiethylsuKoxide (10mM)and incubated at 37°C until the time of peak effect (TPE) of each compound was reached. The data in Table 4.2 and 4.3 reveal that the TPE for N4V3 and V4 in the mouse and rat MES screens were one and two hours respectively. Hence 'H NMR spectra were recorded at dissolution and three hours after incubation of the solution at 37°C. The spectra at both time intervals were identical and indicated the presence of one isomer. Since the stereochemistry of the carbirnino group in different semicarbazones was shown by X-ray crystallography to have the E configuration vide inf?;5 the compounds were considered to retain this stereochemistry vivo and, it is likely that the anticonvulsant activity is due to the molecules per se displaying the E configuration.

Since the water soluble analogs (I4 displayed no anticonvulsant activity (Table 4. l), the stability of these compounds were determined by dissolving the compounds in phosphate buffer pH 7.4, and incubating the solution at 37OC for 24 hours. The UV absorption and L- for each of the compounds were determined and the percentage breakdown was calculated. Degradation was observed for L and 1s and the percentage breakdown was approximately 10 and 30% respectively. Compound G was stable and did not undergo degradation.

A wide variety of nonspecific esterases are found in plasma, liver, kidney and intestine and there are some esterases which catalyze the hydrolysis of aromatic esters (Silverman, 1992). In order to determine the stability of representative esters, compounds W2and Ws were selected and the compounds were dissolved in phosphate - buffer pH 7.4 and the solution incubated at 37OC for 4 hours. Both compounds were stable for 4 hours and the weak anticonvulsant activities exhibited by these esters (Table 4.1) is unlikely to be due to their undergoing hydrolysis. 4.12.0.0 Evaluation of the binding site hv~othesisus in^ X-rav crvstalloera~hvand molecular modeling

X-ray crystallography involves bombarding a compound in the c~stalform by X-rays. From the resultant dfiaction patterns, the structure and shape of a molecule in the crystal form may be found. The X-ray crystallographic data of bioactive ( and on occasions related inactive) compounds can be used to compare the results from molecular modeling data, to compare physicochernical data (bond angles. interatomic distances etc.), to construct postulated binding sites and in addition, overlapping of molecules having similar or different boactivities may give some insight about the shapes of the molecules in confemng bioactivity (Dimmock 1995b. 19952).

The binding site which was postulated (Fig. 2.2) was evaluated using X-ray crystallographic data obtained for four compounds which displayed activity in the MES mouse intraperitoneal and rat oral screens (TV1.J2LwI1) and one semicarbazone which was very weakly active or inactive at the doses employed in both test (1111). The ORTEP diagrams of 1V1.4.~and Vm, are portrayed in Fig. 4.1-4.5. In the case of VIIII, the C5. C4, S. C8. C9. C11 and C12 atoms occupy two different positions each with an occupancy of 0.5. These two moIecules are referred to as VIIIl.4 and VIIIIB. The carbirnino group had the E configuration in all five compounds.

In order to compare the shapes of the five compounds. the following assumptions were made that the alignment of compounds at a binding site involved the terminal four non-hydrogen atoms and the two aryl rings (Fig. 2.2). Thus the molecules were aligned so that the four atoms N3-C14-(02 or 0)-N2 of the semicarbazono groups were superimposed (i-e. occupied the same location). The aryl rings then will occupy different positions (Fig. 4.6). Fie. 4.1 ORTEP diagram of I& Fie. 4.2 ORTEP diagram of NI Fk. 4.3 ORTEP diagram of N4 Fig. 4.4 ORTEP diagram of IV22 Fie. 4.5 ORTEP diagram of Vml Fie. 4.6 Orientation of the proximal and distal aryl rings of 1111 (colorless), WI (red), w4 (green), WU (orange), (blue) and WIIB(yellow) when the N3, C14, (02 or 0) and N2 atoms of each molecule are superimposed The results show that in the crystal state while the proximal ring occupies a similar position in all five compounds, the distal aryl rings were found in three distinct locations which may be designated as location A (IVl and Wl), location B (IVa and ZVn) and location C (I&). The determination of the precise positions of the aryl rings from the X-ray crystallographic data of all five compounds was undertaken in order to obtain further information pertaining to the nature of the binding site. The distances between the C14 atom and the centres of both aryl rings were measured. In addition, the €Iangles between the centres of the aromatic rings and the C14-N2 bonds were obtained. Furthermore the displacements of the aryl rings above or below the N3-C 14-(02 or 0)-

N2 plane were calculated for each compound in terms of both distances and angles t~. The determination for a representative compound NI is illustrated in Fig. 4.7(A and B) and the data are summarized in Tables 4.15 and 4.16.

The results indicated in Table 4.15 reved that the distances between the C 14 atom and the centres of the proximal aryl rings (d,) are similar for all compounds and are in the range of 6.0-6.LA. On the other hand, the dd figures for the compounds in location A-C are 10.9-1 1.1, 10.1- 10.2 and 8. SA respectively. The low/weak activity of III may be due to the spatial arrangements of the compound with the binding site and the dd figures may be one of the factors which affects anticonvulsant activity.

Since compounds having the distal aryl rings in location B had better anticonvulsant activity than compounds having the distal aryl ring in location A, future design of aryloxyaryl sernicarbazones should confine the distal aryl ring to location B in order to increase the potencies.

The 8, angles were between 29" and 34". The synthesis and anticonvuisant evaluation of analogs in which this angle is altered, may reveal the importance of this angle in confemng anticonvulsant activity. The 8d angles of IVJ and IVzz in location B are approximately twice that for IVI and Vml in location A. However a hrther increase in the edangle from approximately 5 1' to that found in Inl is detrimental to activity. The data in Table 4.16 indicate the displacement of the proximal and distal aryl rings from the N3-C14-(02 or 0)-N2 plane. The proximal rings are nearly coplanar with the ureido group as revealed by the dlp and W, figures. With the exception of NI,there was a greater displacement of the distal than the proximal rings from the ureido group in m1, N4, NU and m1-

The distal aryl ring in IV4, which had the lowest EDro figures in both the quantitative MES screens, was located above the N3-C 14-(02)-N2 plane while the marginally less active compounds IVzz and Vml had distal rings below this plane. The distal aryl ring of the inactive analog (I&) was displaced to a greater extent compared to other analogs. This observation may indicate that there is pocket at location B and the distal rings of IV4 could interact with the top of this cavity. The X-ray data thus provided some insight into the putative binding site of these novel anticonvulsants.

Molecular modeling

To support some of the observations obtained From X-ray crystallography studies, compounds I&, IV1, IV+ IVz2 and WII were minimized using the Hyperchem II program, and the minimized molecules were then used for comparison with the X-ray crystallographic data. The distances between the C14 atoms and the centers of the proximal and distal aryl rings (d, and dd) and the angles 8, and €Id were calculated and are presented in Table 4.17.

The values obtained are only approximate as the molecules were not minimized sufficiently to represent the conformation near or at the global minimum energy level.

With the exception for the 86 values, the data obtained using molecular modeling are comparable with the data found from X-ray crystallography. All the five molecules were aligned so that the terminal four non-hydrogen atoms were superimposed (Fig. 4.8). The distal aryl rings occupied different positions similar to what was observed in the X-ray crystallography studies (Fig. 4.6). However, compared to Fig. 4.6, the regions A and B in Fig 4.8 are too close to be distinguished separately but the inactive molecule (IDI) occupies region C as observed in Fig. 4.6.

A comparison of the shape of the prototypic anticonvulsant phenytoin with the lead molecule lV4 is portrayed in Fig. 4.9. From Fig. 4.9, it appears that phenytoin is able to occupy the proposed aryloxyaryl semicarbazone binding site and both aryl rings of phenytoin are close to the proximal aromatic ring of TVJ- Thus, one can assume, that any compound which can occupy the proposed binding site (Fig. 2.2) would display promising anticonvulsant activity. Fig. 4.7 A Distances and 0 angles between the C14 atom and centres of both the proximal (CAr,) and distal (CArd)rings in IVI -B Displacement and yr angles of the centres of the proximal (CAr,) and distal (CArd)rings from the N3-C14-(02)-N2 plane in IVI Table 4.15. Distances between the C14 atoms and the centres of the proximal and distal rings in Illl, IVl, NS,Na and Vml and the angles 8, and Od

a : The letters d, and 4 refer to the distances between the C 14 atom and the centres of the proximal and distal rings respectively b : The designation 8, and ea refer to the angles made between the centres of the aryl ring (CAr)-C14-N2 planes in respect of the proximal and distai rings respectively Table 4.16. Distances and angles of displacement of the centres of the aryl rings from the N3-C14-(02 or 0)-N2 plane in I&, IV,, Ws,IVU and Vml

a : The symbols di and dd refer to the distances of displacement of the proximal and distal aryl rings respectively either above (+) or below (-) the N3-C 14-(02 or 0)-N2 plane b : The designation W, ,.d yd refer to the angles of displacement of the proximal and distal rings respectively either above (+) or below (-) the N3-C 14-(02 or 0)-N2 plane Table 4.17. Distances between the C14 atoms and the centres of the proximal and distal rings in I&, IVl, Ns,ZVt2 and WIIl and the angles 0, and Od calculated using the Hyperchem II programme

a : The letters d, and dd refer to the distances between the C 14 atom and the centres of the proximal and distal rings respectively b : The designations 8, and Bd refer to the angles made between the centres of the aryl ring (CAr)-C 14-N2 planes in respect of the proximal and distal rings respectively Fig. 4.8 Orientation of the proximal and distal aryl rings of III,, IV,, IV,. IVz2 and VIlll Phenytoin F

Fi9 Orientation of the proximal and distal aryl rings of IV, with phenytoin The structure of aryloxyaryi semicarbazones may be conceptually divided into four regions viz. the distal aromatic ring , spacer group, proximd aromatic ring and the semicarbazono group. Molecular modifications were carried out systematically in the above four regions in order to gain a further insight into the nature of the putative binding site. The aryloxyaryl semicarbazones displayed selective efficacy in reducing seizures in the MES test rather than the scPTZ screen. The possible interactions of the aryloxyaryl semicarbazones at a putative binding site is postulated in Fig. 2.2. Based on the pharmacological testing results, X-ray crystallography and molecular modeling studies, the structural requirements for binding of the aryloxyaryl semicarbazones with the proposed binding site are indicated below.

Distal Aromatic Ring

The presence of the distal ring in the 'para' location to the proximal aromatic ring favors alignment at the binding site . In other words , favorable interaction of the distal ring and an area designated the distal binding site may occur. The distal binding site contains a hydrophobic region which interacts with substituents in the para position of the aryl ring. Substitutions at other positions of the distal aryl ring while tolerated, reduced anticonvulsant activity in the MES screen. An increase in the size of the para substituent reduced anticonvulsant activity (optimum activity with the t-pentyl group). Replacement of the distal aryl ring by a heterocyclic or other aliphatic groups caused a change in its alignment at a receptor site resulting in loss of activity.

Spacer Group

The two aromatic rings are perpendicular to each other and separated by a spacer atom (0,S) which holds the rings at an angle of about 120" (data fiom molecular modeling and X-ray crystallography). The spacer group could affect not only the distance between the two aryI rings but also their orientation in relation to each other. An increase in the size of the sulfur over oxygen atoms did not significantly affect the alignment at the receptor site and hence activity was retained. The carbonyloxy, methyleneoxy and sulfonyl spacer groups caused a difference in the orientation of the distal ring and activity was lowered.

Proximal Aromatic Rine and Semicarbazono Grow

Since the two aromatic rings of the aryloxyaryl sernicarbazones are perpendicular to each other at an angle ranging from 120"-13Y, any substitution in the proximal aromatic ring, or replacement of the methine hydrogen atom of the azomethine carbon by methyl and ethyl groups may cause a change in the alignment of the molecule with the binding site. Replacement of the methine hydrogen atom attached to the azomethine carbon atom by methyl and ethyl groups led to retention of activity although neurotoxicity increased in some cases. The retention of activity may indicate interactions of the alkyl group with a hydrophobic pocket by van der Wads bonds.

Future molecular modifications should preferably retain a proton on the carbimino carbon atom. The presence of a two carbon atom spacer group between the proximal aryl ring and the azomethine carbon did not improve anticonwlsant activity. Isosteric replacement of the carbonyl oxygen of the terminal aminocarbonyl group by sulphur and imino substituents resulted in the retention of activity but increased neurotoxicity. Retention of activity strengthens the concept of the presence of a hydrogen bonding area on the receptor. The terminal amino carbonyl group can be replaced by other functions which are capable of hydrogen bonding at a receptor with retention of activity. Activity was retained when the amino group was replaced by a methyl group but not a hydrazino fbnction or a proton. These results reveal that the primary amino group is not essential for anticonwlsant properties. The X-ray crystallographic data supported with molecular modeling studies suggested that the distal aryl ring occupies different positions at the distal binding site and that certain interatomic distances and bond angles affected potency. QSAR indicated a number of physicochemical parameters which contributed to activity in the MES screen and in general the hydrophobicity of the molecules did not influence anticonvulsmt activity. Antiepileptic dnrg discovery has evolved from serendipity through random screening to a scientific era where drugs are designed rationally according to modem principles of neuroscience and the art of medicinal chemistry.

The present study revealed that many aryloxyaryl semicarbazones and related compounds have marked potencies in the rat oral MES screen and did not demonstrate neurotoxicity at the highest doses administered and thus displayed very high protection indices. Most of the objectives outlined in the present investigation were achieved. The deduction from the screening and X-ray crystallographic results suggest that the putative binding site at which MES-active compounds interact is a valid hypothesis which will be scrutinized carefblly as additional data are generated. From the QSAR data, the size and shape of various groups in these molecules correlated positively with activity in the MES screen while the use of n, as well as partition coefficient determinations, indicates that in general the hydrophobicity of the molecules did not influence anticonvulsant activity. The X-ray crystallographic data and molecular modeling studies supported the viability of the binding site hypothesis. These novel semicarbazones appears to act by one or more mechanisms which are different from established anticonvulsant drugs.

The application of modem approaches to drug discovery provides some optimism that effective new compounds will be marketed in the coming decade. with the promise of diminished suffering by persons with uncontrolled epilepsy. Albert A*,Selective Toxicity. 7th. Ed., 1985, Chapman and Hall, London, p.6 1 1.

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