'':-g
`‘'I_-.I
`
`'
`
`.»
`
`2-Substituted-2—acetamido-N-benzylacetamides. Synthesis, Spectroscopic
`and Anticonvulsant Properties
`
`A Thesis
`
`Presented to
`
`the Faculty of
`
`the Department of Chemistry
`
`University of Houston-University Park
`
`In Partial Fulfillment
`
`of the Requirements for
`
`the Degree
`
` 4/ //
`
`EXHIBIT NO. g
`
`
`
`P. Antone, ORR
`
`DEF_O00O077
`MYLAN - EXHIBIT 1104.03
`
`
`
`Master of Science
`
`‘:4
`
`{;‘'.-£'
`
`By
`
`Philippe Le Gall
`
`December. 1987
`
`MYLAN - EXHIBIT 1104.03
`
`

`
`
`
`2-substztutéd-2-acetamido-151-bénzylaoetamides.
`Synthesis, Spectroscopic and Anticonvulsant Properties
`
`
`
`APPROVED:
`
`«A 5-%..._._
`Dr. Harold L. Kohn
`
`Dr.
`
`Joseph
`
`.
`
`treat
`
`
`
`
`Dean, Coll
`
`
`
`
`a 91 Natural Sciences and Mathematics
`
`
`
`DEF_0O0OO78
`
`
`
`ii
`
`
`
`
`
`‘'-‘."";\1'.':.'_.;,=-'-;--';',-;~:“-v'--_---a:.—.;e-
`
`
`
`
`
`
`
`
`-_,-..-.-:~..-...____'.viii-«la...-fz4:$:.i.:‘e.$L;5a';::::-=.ap;.;g..,.'&?§“;q'-Li.‘-‘-_‘__-'1Yfl'g:m'f-"'\!‘_._._.-_._:-_-._-_:-._-._-__5__
`
`

`
`
`
`ACKNOWLEDGEMENT
`
`I would like
`to
`thank Dr. Kchn, whose
`knowledge were always on my side when needed.
`
`enthusiasm and
`I also want
`to '
`
`thank all
`
`the past
`
`and present members of this
`
`research group.
`
`i
`
`am indebted to
`
`their
`
`friendship and assistance.
`
`l would like to acknowledge all
`
`those who have contributed
`
`to the completion of
`
`this work.
`
`In
`
`particular.
`
`I would like to thank
`
`Dr. Gary E. Martin and the N.M.R.
`
`laboratory at
`
`the University cl
`
`Houston, Dr.
`John Chinn at
`the University of Texas at Austin, and
`Dr. J. David Leander
`and Dr. David Robertson
`at
`the Eli
`Lilly
`
`Research Center,
`
`Indianapolis,
`
`Indiana.
`
`for
`
`their kind cooperation.
`
`finally,
`
`i would like to thank Dr. Kurt L. Loening. Director of
`
`Nomenclature. Chemical Abstracts Services._ Columbus, Ohio.
`
`for his
`
`help in naming the synthesized compounds.
`
`DEF_0O00079
`
`

`
`
`
`2-Substituted-2-acetamido-Ll-benzylacetamides. Synthesis. Spectroscopic
`and Anticonvuisant Properties
`
`
`
`An Abstract of . a Thesis
`
`Presented to
`
`the Faculty of
`
`the Department of Chemistry
`
`University of Houston-University Park
`
`In Partial Futfiliment
`
`of the Requirements for
`
`the Degree
`
`Master of Science
`
`
`
`BY
`
`Philippe Le Gail
`
`December. 1987
`
`iv
`
`DEF_0OO0080
`
`

`
`W
`
`2%
`Q
`
`
`
`
`
`b="-r.1'..'.E-.7.-=_'_._;...,-_-.'_;._'_'__,-_,;.__.__":._-_
`
`
`
`
`
`it
`
`ABSTRACT
`
`Select
`lunctlonalizedt amino acid derivatives of the potent anti-
`convulsant agent or-acetamido-N;benzylphenyiecetamide
`(5.611)
`and
`
`2-acetamido-N,-benzylpropionamide
`
`(5.83)
`
`have been
`
`prepared and
`
`evaluated.
`
`Attention has
`
`been focused on the replacement of
`
`the
`
`ct-phenyl and u-methyl groups in ma and 551; by five-membered ring
`
`hetereoaromatic moieties. benzo-fused
`
`heteroaromatic groups. and
`
`simple polar substituents.
`
`The synthetic and pharmacological
`
`studies revealed
`
`several
`
`notable
`
`findings.
`
`First.
`
`the use of
`
`amidoalkylation procedures using
`
`boron tritluoride etherate provided
`
`a
`
`straightforward
`
`and
`
`reliable
`
`method to" introduce
`
`an electron-rich
`
`heteroaromatic
`
`substituent
`
`at
`
`the
`
`or-carbon
`
`in
`
`the
`
`amino acid derivatives.
`
`This
`
`technology
`
`permitted
`
`the
`
`incorporation
`
`of
`
`acid sensitive,
`
`unsubstituted
`
`heteroaromatic
`
`compounds
`
`(i.e..
`
`pyrrole
`
`(L4),
`
`indole
`
`(Z2)
`
`and
`
`benzoturan (25)) within the molecule. Second.
`
`all
`
`the five-rnembered
`
`ring heteroaromatic analogues of
`
`on-acetamido-Ltbenzylphenylaoeta—
`
`mide
`
`proved highly active in the MES seizure test.
`
`In particular,
`
`on-acetamido-M-benzyl-2-furanacetamide (593) and oz-aceta-
`
`mido-N,-benzyI-2-pyrroleacetamide
`
`(sen)
`
`exhibited activities similar to
`
`phenytoin
`
`and diazepam.
`
`Third.
`
`the or-alkoxy derivatives,
`
`2-'aceta-
`
`rnido-b1-benzyl-2-methoxyacetamide (£53) and 2-acetamido-h1-benzyl-2-
`
`ethoxyacetamide. exhibited sinificant activity in the MES seizure test.
`
`V
`
`DEF_00O0081
`
`

`
`Fourth.
`
`neither
`
`the benzo-fused
`
`heteroaromatic derivatives nor
`
`t:'ompounds
`
`bearing
`
`an
`
`electron-withdrawing
`
`substituent
`
`at
`
`the
`
`the
`
`Fifth,
`the composite pharmacolog-
`activity.
`oz-carbon had signifimnt
`substituents are
`small
`icat data suggested that
`required
`at
`the
`at-carbon
`of
`the
`2-aoetamido-Isl-bennzylglycine
`derivative
`for maximal
`
`activity in
`
`the MES test and that
`
`this
`
`activity is enhanced by the
`
`presence of electron-donating groups at
`
`this site.
`
`v i
`
`
`
`DEF__00O0082
`
`

`
`TABLE OF CONTENTS.
`
`Acknowledgements
`List of Tables
`List of Schemes
`
`List of Figures
`Introduction.
`
`iii
`viii
`xiii
`
`xiv
`1
`
`_
`
`I.
`II.
`
`Blogenesls of the Epileptic Seizure
`Symptornatology and Classification of
`Epileptic Seizures.
`13
`III. Evaluation of Anticonvulsant Agents.
`17
`Iv.
`Antiepileptic Drugs.
`25
`v.
`Clinical Applications
`30
`VI. Mechanism of Drug Action.
`32
`VII.
`Structure-activity Relationships.
`VIII. Anticonvulsant Amino Acids and Amino 35
`Acid Derivatives
`
`2
`5
`
`_
`
`Chapter I. Synthesis. Spectroscopic and AnticonvulsantProper-
`ties of Structural Analogues 53 of 2-Aoetamido-N-hem
`zylphenylacetamide Lefilz) and 2-Acetamido-N-benzyl
`2-alkoxyacetamides (56).
`
`44
`
`I .
`II.
`
`Introduction
`Results and Discussion
`
`1. Synthesis.
`2. Spectral Evaluation.
`a. Infrared Spectra.
`b. Mass Spectral Data.
`c. ‘H NMR Spectral Data.
`cl. 130 NMR Spectral Data.
`3. Pharmacological Evaluation.
`III. Experimental Section.
`Chapter II . Synthesis, Spectroscopic and Anticonvulsant Proper-
`'
`ties of Polar Analogues 1.01 of 2-Acetamido-N-hem
`zylpropionamide (fiaa)
`I .
`Introduction.
`II.
`Results and Discussion
`1. Synthesis.
`2. Spectral Evaluation.
`a.
`infrared Spectra.
`
`44
`47
`
`47
`74
`74
`79
`so
`93
`102
`109
`132
`
`132
`132
`132
`139
`139
`
`vii
`
`9;?’
`
`
`
`DEF_000OO83
`
`

`
`b. Mass Spectral Data.
`c. ‘H NMR Spectral Data.
`d. 130 NMR spectra! Data.
`3. Pharmacological Evaluation.
`III. Experimantai Section
`'
`General Conclusions.
`References.
`
`.
`
`144
`144
`147
`153'
`155
`164
`166
`
`if
`
` u‘.‘:?v‘.
`
`
`
`é
`
`
`
`viii
`
`DEF_0000084
`
`

`
`
`
`
`
`
`
`1
`
`
`
`
`
`
`
`7:_‘;:_::;W.“:'’’-—:.‘’./:‘;V“‘_~l.__N‘-2Em."VI..g.........
`
` 37
`
`Sequential Test Phases Utilized for the Anticonvulsant
`Screening Project of
`the Antiepileptic Drug Develop-
`men! Program.
`
`Antiepileptic Drugs Marketed in the United States.
`
`Monocyclic Heteroaromatic Analogues 53 of
`2-Acetamido-,u—benzyIphenyiacetamide (flan).
`
`_
`
`Benzotused Heteroaromatic Analogues 53 of
`2-Acetamido-Ll-benzylphenylacetamide (5512).
`
`Examples of Amino Acids with an on-Heterocyclic
`Substituent.-
`-
`
`Synthesis of Amino Acid Derivatives by the Amido-
`alkylation Technique.
`
`Amidoalkylation Reactions
`2-Methylfuran (1012).
`
`Involving Furan(1Qa) and
`
`53
`
`Amidoalkyiation Reactions Involving Pyrrole (11) and
`Substituted Pyrroles.
`
`8
`
`9
`
`18
`
`29
`
`45
`
`46
`
`48
`
`50
`
`55
`
`56
`
`57
`
`LIST OF TABLES.‘
`
`Iabie.
`
`.
`
`Ban:
`
`1.
`
`The lntemational Classification of Epileptic Seizures.
`
`2..
`
`The Revised Classification of Epilpetic Seizures._
`
`3.
`
`4._
`
`5a.
`
`5b.
`
`.9’
`
`7.
`
`8.
`
`9.
`
`10.,
`
`Amidoalkylation Reactions Involving Indole
`
`(Z2).
`
`11.
`
`Amidoalkylation Reactions Involving Thiophene (11,).
`
`12.
`
`Amidoalkylation Reactions Involving Other Heterocycles.
`
`58
`
`ix
`
`DEF_0O00085
`
`

`
`Selected Physical and Spectral Data for Alkyl 2-Aceta-
`_ mido-2-alkoxyacetate (59).
`
`e1 _
`
`Selected ‘Physical and Spectral Data for u-Substitu-
`ted Alkyl 2-Acetamidoacetates (81).
`
`62
`
`64
`
`s7
`
`'_ 15.
`
`Selected Physical and Spectral Data for a-Substitu-
`ted 2-Acetamidoacetic Adds (B2).
`
`% Selected Physical and -Spectral Data for u-Substitu-
`ted 2-Acetamido-N-benzylacetamides (as).
`
`13.
`
`14.
`
`
`.'f':%v.-.."~51vwu.
`..-.«.-,
`
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21_.
`
`22.
`
`23.
`
`24.
`
`selected Physical and Spectral Data for 2-Ace_tamido- 69
`51-benzyl-2-alkoxyacetamides (B5).
`'
`
`Comparison of Several Amidoalkylation Reactions tnvoI- 73
`ving F_uran (ma). Pyrrole (E), Thiophene (Z1),
`lndole
`(Z2). Benzofuran (Z5) and Benzo[b]thiophene (25) Be-
`ginning with Either B51. 523 and 12 or _8_5_tz.
`
`Selected infrared spectra! Data for Alkyl 2-Acetamido-2- 75
`alkoxyacetates gag).
`
`Selected Infrared Spectra! Data for Alkyl 2-Substituted
`-2-acetamidoacetates
`(£1).
`
`76
`
`Selected Intrared Spectral Data for 2-Substituted-2-ace 77
`tamidoacetic Acids (£2).
`
`Selected Intrared Spectral Data for 2~Substitutedv2-ace- 78
`tamido-N,-benzylacetarnides (62.85).
`
`1H NMR Spectral Properties for Alkyl 2-Acetamido-2-
`alkoxyacetates (B9).
`
`‘H NMR Spectral Properties for Alky: 2-Substituted-2
`Acetamidoacetates (51).
`
`as
`
`87
`
` -
`
`- -,--
`
`.-.
`
`-.—.e~ —. -- =----—.-.-'r..-~.r---7=‘3-».'-.~.~.
`
`DEF_00O0O86
`
`

`
`25.
`
`26.
`
`27.
`
`28.
`
`29.
`
`30.
`
`32.
`
`33.
`
`34.
`
`35.
`
`36.
`
`‘H NMR Spectral Properties lor 2-Subslituted—2-ace1a-
`midoacetic Acids (H2).
`
`‘H NMR Spectral Properties for 2-Substituted-2-aoeta
`mido-u-benzyiaceiamides (fig. 55).
`
`13c NMR Spectral Properties for Alkyl 2-Acetamido-
`2-alkoxyacetates (89,).
`
`13c NMR Spectral Properties lor Alkyl 2-Acetamido-
`2-acetamidoacetates (81).
`
`13C NMR Spectral Properties for Alkyl 2-Substiiuted-
`2-acetamidoacelic Acids (52).
`
`89
`
`9o
`
`94
`
`95
`
`96
`
`130 NMR Spectral Properties for 2-Substituted-2-aceta ea
`mido-bl-benzylacetamides (£2. 5&1.
`
`Pharmacological Evaluation of 2-Substituted-2-aoetamh
`do-M-benzylacetamides (59) Containing a Monocyciic He-
`terocyclic Moiety.
`
`104
`
`Pharmacological Evaluation of 2-Substituted-2-acetami
`do-N-benzylacelamides (59,) Containing a Benzo-fused
`Heterocyciic Moiety.
`
`Pharmacological Evaluation oi 2-Alkyl-2-acetamido-by
`benzylacetamides (£5).
`
`Pharmacological Activity 01 Some Proven Anticonvul-
`sants.
`
`2-Acetamido-1}benzylpropionamide Analogues _1_Q_Z.
`
`the Po-
`Selected Physical and Spectral Properties for
`lar Analogues oi 2-Acetamido-u-benzylpropionamide
`lfifial.
`
`105
`
`106
`
`107
`
`133
`
`137
`
`
`
`DEF_0O00087
`
`

`
`37.
`
`38.
`
`39.
`
`40.
`
`selected Physical and Spectral ‘Data for Oxazola De-
`rivatives 111 and _1_1A_.
`
`the Polar Analo-
`Selected Intrared. spectral Data for
`gues _1_QZa;e of 2-Acetamido-H-benzylpropionamide
`(.683)-
`
`Selected Physical and Spectra! Data for Oxazole De-
`rivatives _]_u and 115,
`
`the Polar Analogues
`‘H NMFI Spectral Properties for
`.1QZa-.9. of 2-Aoetamido-M-benzylpropionamide (ma).
`
`41..
`
`‘H NMR Spectral Properties for the Oxazole Deriva-
`tives
`;L1_]_ and J15.
`
`141
`
`142
`
`143
`
`145
`
`148
`
`149
`
`42.
`
`43.
`
`44.
`
`the Polar Analo-
`130 NMR Spectral Properties for
`gues .1.Q.Za-.2
`of 2-Acetamido-N-benzylpropionamide
`(.6.8.a)-
`
`13C NMR Spectral Properties
`tives JJ_1_ and 115.
`
`for Oxazole Deriva-
`
`151
`
`the Volar Analogues
`Pharmacological Evaluation 0!
`of
`2-Acetamido-fl~benzyIpropionamide (§_8_a).
`
`154
`
`xii
`
`
`
`DEF_0000088
`
`

`
`LIST OF SCHEMES
`
`Page
`
`Synthesis of 2-Substituted-2-acetamido-fl-benzy|-
`aoetamldes (§.9_a.b,,Ln) by Method A.
`
`Preparation of an-Acetamido-2-benzo[b]thiophenacetic
`Acid mag).
`
`Synthesis of 2-Substituted-2-acetamido-M-benzylaceta
`mides (fi9_a,m;n) by Method B.
`
`Acid catalysed Trimerization ot Indole (25).
`
`Selected Mass Spectral Patterns Observed tor AIkyi-2-
`substituted-2-acetarnidoacetates (51).
`
`Selected Mass Spectral Cleavage Patterns Observed
`for 2~SubstItuted-2-acetamido-bl-benzyiacetamides
`(£2. 35)-
`
`Selected Mass Spectral Cieavage Patterns Observed
`tor Alkyi 2-Acetamido-2-alkoxyacetates (39).
`
`Selected Mass Spectral Patterns Observed for 2-
`Substituted-2-acetamidbacetic Acids Q2).
`
`the Polar Analogues _1_QZa-_e of 2-
`Preparation of
`Acetamido—fl-benzylpropionamide (fifla).
`
`10.
`
`11.
`
`12.
`
`Preparation ot 5—Ethoxy-2-methyloxazole-4-carboxyiic
`Acid N-Banzylamide (_1_]_1).
`
`Preparation oi 5-Amino-2-methyloxazole-4-carboxylit:
`Add H-Benzylamide.
`
`the Conversion of Com-
`Proposed Mechanism tor
`pound 191 to the Oxazole Derivative _‘LJ_4_.
`
`xiii
`
`59
`
`65
`
`68
`
`72
`
`81
`
`82
`
`83
`
`84
`
`134
`
`138
`
`138
`
`140
`
`DEF_00O0089
`
`

`
`LIST OF FIGURES.
`
`Page
`
`34
`
`37
`
`38
`
`40
`
`the Three-dimensional
`Prespective ‘Drawing of
`conformations of Phenyltoin (Flight) and Diaze-
`pam (Left).
`
`Amino Acids and Amino Acid Dertvatives
`acting with Excitatory Neurotransmission.
`
`Inter-
`
`Amino Acids and Amino Acid Derivatives
`
`Inter-
`
`acting with the GABAergic System: GABA Prodrugs.
`
`Amino Acids and Amino Acid Derivatives
`
`Inter-
`
`acting with the GABAergic System: Compounds
`Limiting GABA Uptake.
`
`Amino Acids and Amino Acid Derivatives
`Inter-
`acting with the GABAergic System: GABA Re-
`ceptors Agonists.
`
`xiv
`
`
`
`DEF_0000O9O
`
`

`
`lNTFIODUCTlON '
`
`Epilepsy is a major neurological disorder.
`
`It effects at least 0.5
`
`percent of
`
`the world population‘-2 and its symptoms generally appear
`
`early in life.‘-3
`
`If
`
`the origin of
`
`the disease is unknown,
`
`it
`
`is termed
`
`idiopathic. Correspondingly, when the epilepsy is of a known origin, the
`
`disease is referred to as
`
`symptomatic.3 In the latter situation. prenatal
`
`and postnatal ‘trauma. brain tumor. and vascular disorders are the major
`
`etiologies associated with the disease.1~3
`
`The occurrence of seizures
`
`is the manisfestation of epilepsy. Seizures may be
`
`of different
`
`types
`
`and may have sensory, motor and autonomic components.
`
`Historically. epilepsy has
`
`been
`
`referred to as
`
`the
`
`‘dread
`
`disease‘.
`
`the “Sacred Disease‘,
`
`the ‘falling sickness‘ or
`
`‘St. John's
`
`disease".
`
`It was mentioned in
`
`the babylonian civil code of
`
`Hammurabi
`
`(2080 B.C.)
`
`and
`
`early
`
`akkadian
`
`and
`
`hebrew texts.
`
`Hippocrates (ca. 400 B.C.) wrote the tirst monograph on the attliction and
`
`first anticipated its brain origin.3v4
`
`Despite the early recognition of this disease. significant activity
`
`- directed towards the treatment and the understanding of epilepsies
`
`began in earnest
`
`In the mid-nineteenth century.1v3-5
`
`in
`
`1870, H..J.
`
`Jackson described epilepsy as ‘an occasional and abnormally intense
`
`disorderly discharge of nervous tissue‘ of which the seizures are the
`
`symptom.3
`
`This statement
`
`is still accepted today. Our‘ knowledge of
`
`the brain malfunction has not permitted a more detailed definition.
`
`It
`
`is
`
`generally accepted that
`
`epilepsy is
`
`reserved for
`
`these diseases
`
`
`
`DEF_0O0O091
`
`

`
`2
`
`rnanistesting themselves
`
`by
`
`a chronic occurrence
`
`of
`
`seizures.
`
`Accordingly. seizures elicited by punctual
`
`tactors. such as alcohol and
`
`drug intoxication or withdrawal are not considered to be a form at
`
`this
`
`disease.
`
`I.
`
`Blogenesls oi
`
`the Epileptic Seizures.
`
`Although the neurophysiology of
`
`the central nervous system
`
`and the biochemical processes associated with neuronal activity are
`
`now fairly well established5'5.
`
`the key
`
`events leading to epileptic
`
`activity have not been determined.
`
`it was early recognized that epilepsy
`
`may affect only a part of the brain at
`
`the onset.
`
`Subsequently,
`
`the
`
`existence oi epileptic loci were revealed.7
`
`in 1975. Ebersole and
`
`Levine9 demonstrated that Enhanced Physiological Response (EPR) oi
`
`neurons submitted to an epileptogenic application of penicillin (1) could
`
`lead to Paroxysmal Depolarization Shifts(PDS).
`
`The PDSs are charac-
`
`terized by abnormally large. prolonged and repetitive excilatory post-
`
`synaptic potential and are believed to participate in the spread of
`
`electrical discharge during the seizure.1°-11
`
`The origins of EPRS are
`
`considered the key step in the epileptogenesls.‘°
`
`These observations have led to a six stage model
`
`tor
`
`the gen-
`
`eration oi epileptic seizures: (:3)
`
`the Initiation ol
`
`the EPR; (b) the gen-
`
`eration of PDSs; (c)
`
`the recruitment of nonnal neurons;
`
`(d)
`
`the inter-
`
`ventlon oi control mechanisms that
`
`limit
`
`the spread oi
`
`the epileptic
`
`discharges or promote selective routes of
`
`spread; (e) the establishment
`
`DEF_0O00092
`
`

`
`3
`
`ol satellite and independent epileptic loci; and (f)
`
`the engagement of
`
`the brain stem and of
`
`the spinal motor neuron pooIs.1°
`
`‘J
`
`cu.
`
`COOH
`
`1.
`
`Both
`
`the
`
`generation ot
`
`the EPR and the
`
`recruitment oi
`
`normal neurons
`
`in
`
`the
`
`epileptic
`
`seizure
`
`have
`
`been
`
`extensively
`
`examined.
`
`The EPR has sometimes been related to local deteriora-
`
`tion
`
`of
`
`neurons
`
`by extraneuronal
`
`lactors.
`
`in
`
`1880. Sommer
`
`observed an extensive loss oi neural mass
`
`in
`
`the hippocampal area
`
`of one
`
`third of
`
`the
`
`epileptic patients
`
`examined.
`
`Shortly
`
`after,
`
`Alzheimer
`
`and Chaslin independently reported an associated abnor-
`
`mal
`
`proliferation
`
`of
`
`glial cells
`
`in
`
`the same area?
`
`Gliosis has
`
`been related to seizures
`
`in was ways.
`
`First.
`
`this process may be
`
`a biological
`
`response to a decrease in neural matter.7 Second. ab-
`
`normal
`
`proliferation oi
`
`glial cells may mechanically
`
`irritate
`
`the
`
`neurons
`
`and
`
`initiate
`
`the EPFl—mediated prooesses7.
`
`Third. glial
`
`cells are known to participate in the regulation ot
`
`the potassium ion
`
`concentration
`
`in
`
`the
`
`brain
`
`and
`
`in
`
`the metabolism of 1-amino
`
`DEF_OO0O093
`
`

`
`4
`
`butyric acid
`
`(2).
`
`These
`
`substances may influence
`
`the
`
`seizure
`
`processes.”
`
`l-‘lnally. De Moor and Westrum noticed dendritic
`
`alterations
`
`adjacent
`
`to
`
`epileptic
`
`foci
`
`in
`
`human
`
`cortices
`
`and
`
`experimemally generated epileptic centers,
`
`resper:tively.7-12
`
`H!"/\/\n,OH
`O
`
`E
`
`Other factors which may be responsible for
`
`the generation oi
`
`the
`
`EPR are associated with the blood—brain
`
`barrier.
`
`Scheibel7
`
`observed
`
`that
`
`neurons
`
`in epileptic
`
`loci were
`
`often
`
`altered
`
`in
`
`regions nearby damaged capillaries.
`
`He attributed the
`
`generation
`
`of
`
`epileptic neurons
`
`to
`
`exogenic
`
`lactors
`
`such as vimses
`
`and
`
`toxins.
`
`Despite
`
`these important
`
`initiation
`
`processes,
`
`the majority ol
`
`researchers
`
`consider metabolic disorders
`
`existent
`
`in
`
`the
`
`epileptic
`
`patient
`
`to be the primary cause ol
`
`the disease.-3-‘O Most sub-
`
`stances
`
`involved in the
`
`normal neuronal
`
`activity as well as
`
`their
`
`associated metabolic cycles have been claimed to participate in
`
`the
`
`EPR
`
`generation.”-13
`
`Extensive
`
`experimental
`
`evidence
`
`exists
`
`which demonstrate
`
`the involvement both of potassium ion8-1°-“H5
`
`and
`
`the neuronal
`
`sodum-potassium pump“)
`
`in the first step oi
`
`the
`
`DEF_OOOOO94
`
`

`
`S
`
`seizure.
`
`Other ions
`
`(i.e.. Cl’. Ca“. NH4*) may also be involved
`
`in
`
`ebnonnal membrane depolarization processes.3
`
`Disorders
`
`in
`
`the metabolic
`
`cycles
`
`of
`
`all
`
`the substances regulating
`
`the concen-
`
`tration
`
`and the transport
`
`of
`
`these ions across
`
`the membrane or
`
`the neuron, as well as structural
`
`disorders
`
`in the neuron mem-
`
`brane can ultimately be responsible tor
`
`the EPR generation.
`
`Several explanations have been olfered tor
`
`the mechanism
`
`involved
`
`in
`
`the
`
`recruitment or
`
`normal neurons
`
`in
`
`the
`
`seizure.
`
`Variations
`
`in
`
`the protelnic constitution ot
`
`the synapses have been
`
`invoked to explain
`
`the propagation of
`
`the epileptic discharge by
`
`selective
`
`neuronal
`
`circuits.
`
`The
`
`same
`
`explanation
`
`has been
`
`suggested tor
`
`the creation of
`
`secondary epileptic ioci.‘°
`
`Exten-
`
`sive documentation does exist
`
`in support
`
`at various
`
`other
`
`phenom-
`
`ena.
`
`These include
`
`the enhancement
`
`of
`
`the excitalory neuro-
`
`transmissions
`
`induced by
`
`substances
`
`such as
`
`acetylcholine (3).
`
`aspartic
`
`acid (5), glutamic
`
`acid
`
`(5)
`
`and some
`
`neuropeptides as
`
`well
`
`as
`
`the
`
`decrease
`
`in
`
`inhibitory
`
`regulation
`
`promoted
`
`by
`
`substances
`
`such as norepinephrine lfi),
`
`dopamine (1).
`
`serotonin (a)
`
`and
`
`7-aminobutyric acid
`
`(GABA)
`
`(z).3- "40
`
`Finally.
`
`transient
`
`modifications oi
`
`the membrane at
`
`the normal neuron and anomalies
`
`in
`
`the
`
`dendritic
`
`and
`
`axon
`
`structures
`
`are
`
`also
`
`believed
`
`to
`
`participate
`
`in the epileptic
`
`discharge propagetion.‘°~‘9-2‘
`
`11. Symptomatology and Clesslllcatlon
`
`oi Epileptic Seizures.
`
`The part of
`
`the brain affected by the epileptic discharges has
`
`DEF_0000095
`
`

`
`HO
`
`N”!
`
`0H
`
`HO
`
`OH
`
`OH
`
`NH,
`
`DEF_O0O0O96
`
`

`
`7
`
`drect bearing
`
`on the clinical manislestation
`
`oi
`
`the disease.
`
`in
`
`some cases. careful monitoring of
`
`the patient can permit
`
`the physi-
`
`cian to follow the spread of
`
`the abnormal
`
`brain electrical activity.
`
`The observed clinical
`
`symptoms serve as a basis
`
`for
`
`the currently
`
`employed classifications of the various
`seizure states.
`The first widely accepted ‘classification of
`the epileptic seizures
`
`(Table
`
`1) was
`
`established
`
`by the
`
`international League Against
`
`Epilepsy in 1969.132
`
`This classification
`
`attempted
`
`to correlate
`
`the symptoms
`
`and
`
`electroencephalographic
`
`(EEG)
`
`data
`
`to the
`
`anatomic substrate.
`
`the etiology. and the age of
`
`the patient.
`
`This
`
`system cfitierentiated
`seizures
`which
`evolved
`into
`generalized
`seizures.
`generalized from the
`beginning.‘
`
`and
`onset
`a
`focal
`had
`are
`which
`those
`from
`A revised classification (Table
`
`I
`
`This new ciassiticaiion was
`2) appeared in 1981.
`development of
`better methods
`to
`analyze the
`
`by the
`lostered
`seizures.
`(i.e.,
`
`telemetered EEG. videotaping) The major changes- incorporated
`
`in
`
`the
`
`new system were
`
`the exclusive use
`
`of
`
`the
`
`clinical
`
`and
`
`electroencephalographic data
`
`to determine the seizure
`
`type.
`
`and
`
`the
`
`restructuring ot
`
`the
`
`partial
`
`seizures
`
`into
`
`simple
`
`partial
`
`seizures and complex partial
`
`seizures depending
`
`on whether
`
`or
`
`not
`
`consciousness
`
`is
`
`altered
`
`at
`
`the onset or
`
`in
`
`the
`
`further
`
`evolution
`
`of
`
`the seizure.‘
`
`Although.
`
`a clinical
`
`discussion
`
`of
`
`the various types
`
`of
`
`seizures
`
`is beyond the scope oi
`
`this
`
`thesis e briel
`
`review 01 the
`
`major
`
`terms of
`
`the disease tollows.
`
`A
`‘I
`
`‘V
`
`DEF-'_O0OOO97
`
`

`
`8
`
`IaD.i£_1.. The
`
`lntemationai Classification oi Epileptic Seizures.‘
`
`1.
`
`(seizures beginning locally)
`seizures
`Partial
`A. Partial
`seizures with elementary symptomatology (generally
`without
`impairment of consciousness)
`.
`1. With motor symptoms (includes Jacksonlan seizures)
`2. With special sensory or somatosensory symptoms
`3. With automatic symptoms
`4.
`Compound lorms
`B. Panial seizures with complex symptomatology (generally
`with impairment oi consciousness) (temporal
`lobe or psycho-
`motor seizures)
`1.
`with impairment oi consdousness only
`2.
`with cognitive symptomatology
`3. With altective symptomatology
`4. With 'psychosensory" symptomatoiogy
`5. With 'psychomotor' symplomatology (automatisms)
`6.
`Compound forms
`C. Partial
`seizures secondarily generalized
`
`II.
`
`Generalized seizures (bilaterally symmetrical and without
`onset)
`A. Absence (pelit mai)
`8. Bilateral massive epileptic myoclonus
`C.
`lnlantile spasms
`D. Clonic teatures
`E. Tonic features
`
`local
`
`F. Tonic-clonic seizures (grand mai)
`G. Atonic seizures
`H. Akinetic seizures
`
`III. Unilateral seizures
`
`(or predominately)
`
`IV.
`
`Unclassified epileptic seizures
`
`(due to incomplete data)
`
`DEF_0000098
`
`

`
`
`
`1.
`
`II.
`
`III.
`
`.
`2
`,;
`.
`
`_
`
`is
`
`Igp_|_g__2. The Revised Classification of Epileptic Seizures.‘
`
`9
`
`seizures (consciousness not
`Simple partial
`A. With motor signs
`1.
`Focal motor without march
`
`impaired)
`
`2.
`3.
`4.
`
`Focal motor with march (Jacksonian)
`Verslve
`Postural
`
`(simple
`
`Phonatory (vocalization or arrest of speech)
`5.
`B. With somatosensory or
`special-sensory symptoms
`hallucinations. e.g.. tingling. light flashes. buzzing)
`Somatosensory
`Visual
`
`Auditory
`Oltactory
`Gustatory
`Verliglnous
`.
`with automatic symptoms or signs
`Vlfith psychic symptoms (disturbance of higher cortical function)
`Dysphasic
`Dysnesmic (e.g., deja vu)
`Cognitive (e.g.. forced thinking)
`Affective (e.g.. fear. anger)
`Illusions (e.g.. macropsia)
`Structured hallucinations (e.g.. music. scenes)
`Complex partial seizures (generally with
`impairment ot conscious-
`ness; may sometimes begin with simple symptomatology)
`A. Simple partial onset followed by impairment of consciousness
`1. With
`simple
`partial
`features
`(AD)
`and
`impaired
`consciousness
`2. With automatisms
`
`
`
`9.0wwswwrmwewwr
`
`B. Vlfith impairment of consciousness at onset
`1. With impairment ol consciousness only
`2. With automatlsms
`Partial
`seizures
`evolving to generalized
`seizures (GTC with partial or focal onset)
`A. Simple partial seizures (1) evolving to GTC
`B. Complex partial seizures (II) evolving to GTC
`0. Simple partial seizures evolving to complex
`partial seizures
`evolving to GTC
`
`tonic-clonic
`
`(GTC)
`
`_
`
`DEF_O00OO99
`
`

`
`Ianie_2 000'!-
`
`IO
`
`IV.
`
`Generalized seizures (bilaterally symmetrical
`onset)
`Absence (pelit mal)
`. Bilateral massive epileptic myocionus
`Infantile spasms
`Clonic features
`Tonic features
`
`Tonic-clonic seizures (grand mal)
`. Atonic seizures
`
`C-7.'“!“!3.°°’.>
`
`and without
`
`local
`
`v.
`v1.
`
`Unilateral seizures (or predominately)
`Unclassified epileptic seizures (due to incomplete data)
`
`Y.
`
`i‘.;
`
`
`
`:'FI-.r~*-...u'Ir.
`
`DEF_00O0100
`
`

`
`11
`
`Simple partial
`
`seizures atiect any part or
`
`the body depend-
`
`ing on the origin
`
`or
`
`the discharge in
`
`the motor
`
`strip.
`
`The clini-
`
`cal observations
`
`are characterized by tonic contraction of
`
`the activa-
`
`ted muscles
`
`iollowed by a clonus.
`
`Generally. one muscle or a
`
`group
`
`of muscles are
`
`involved in
`
`the seizure.
`
`resulting in
`
`invol-
`
`untary movements.
`
`involuntary actions (i.e.. vocalization) or posture
`
`change.
`
`The
`
`step by step spread of
`
`the electrical disorder
`
`from
`
`strictly
`
`focal
`
`to adjacent
`
`areas produce sequential
`
`involvement
`
`of
`
`body
`
`parts
`
`and
`
`is
`
`reterred to
`
`as
`
`the
`
`epileptic march
`
`or
`
`These muscular disorders tind their origin
`seizure.‘-5
`Jacksonian
`in the part of
`the cortex responsible
`for motor
`activity.
`Ukewise.
`
`epileptic discharges
`
`originating or
`
`spreading in
`
`the part
`
`oi
`
`the
`
`cortex
`
`responsible
`
`for automatic functions
`
`result
`
`in manisfestations
`
`such as
`
`vomiting,
`
`incontinence.
`
`pupil
`
`dilatation
`
`and
`
`palior.‘
`
`involvement
`
`of
`
`the
`
`cortex area
`
`subversing
`
`somatosensory and
`
`psychic
`tunctions
`lead to
`additional
`seizure maniiestations within
`this general
`class oi
`seizures
`(Table 2).
`in
`all
`instances. partial
`
`seizures are not associated with consciousness
`
`Impairment.
`
`partial
`by
`characterized
`are
`seizures
`partial
`Complex
`symptoms with accompanying
`impairment
`oi consciousness.
`seizure
`This often
`results
`in automatisms
`(i.e., verbal. ambulatory. gestural.
`
`masticatory).
`
`and
`
`sometimes
`
`results
`
`in
`
`confusion.‘-23
`
`This
`
`disease
`
`state
`
`is
`
`always
`
`associated with
`
`various degrees
`
`of
`
`amnesia.5_
`
`Generalized seizures which are non-convulsive
`
`are reierred
`
`_
`
`_
`iv
`5
`
`..
`
`'-
`
`DEF_0000 101
`
`

`
`12
`
`to as absence
`
`(petit mal)
`
`seizures.
`
`Absence
`
`seizures
`
`atlect
`
`‘children
`
`before
`
`puberty and are characterized
`
`by trequent
`
`(5-100
`
`per day)
`
`attacks
`
`of
`
`impaired
`
`consciousness
`
`lasting usualy tess
`
`than 20 seconds.
`
`Interruption
`
`of
`
`ongoing
`
`activities.
`
`staring. occa-
`
`sional
`
`eyes movements,
`
`arm jerks
`
`and modification
`
`at
`
`the
`
`postural
`
`tone
`
`are manistestations
`
`of
`
`this type ot
`
`seizure.
`
`The
`
`associated EEG is characterized
`
`by a regular
`
`three per second
`
`spike
`
`and wave pattern.
`
`The
`
`classification
`
`ot
`
`generalized seizures,
`
`involving
`
`con-
`
`vulsions
`
`has
`
`been subdivided
`
`according to
`
`the characteristics of
`
`convulsive episodes.
`
`Bilateral massive
`
`epileptic myoclonus
`
`3.
`V
`
`
`
`V".:'.:L-J.'-J‘.
`
`.‘:_‘_
`
`the
`
`and intantile
`
`spasms (West's
`
`syndrome) are manlslested by sudden
`
`of major muscle groups.
`The latter seizure type occur
`contractions
`age of
`tour
`and
`Involves
`legs,
`arms
`and
`trunk
`betore
`the
`is often
`related
`to phenylketonuria,
`iipidosis and
`the
`It
`muscles.
`metabolism of
`piridoxine.
`The EEG
`shows
`high voltage
`slow
`
`waves and spikes.
`
`Both 01
`
`these seizure types
`
`involve the right
`
`and left
`
`brains.1v5
`
`Clonic
`
`seizures occur especially
`
`in
`
`childhood
`
`and are manistested by a
`
`succession
`
`of
`
`shock-like
`
`spasms affect-
`
`ing one or both sides ot
`
`the body and
`
`are
`
`associated with con-
`
`sciousness
`
`impainnent.
`
`Tonic seizures
`
`are characterized
`
`by tonic
`
`contractions
`
`of
`
`long duration
`
`(up to one minute) of
`
`the trunkal
`
`and
`
`limbs musculatures,
`
`and
`
`are
`
`associated with
`
`autonomic activ-
`
`ity.
`
`The EEG shows high vottage
`
`activity.
`
`Tonic-clonic seizures
`
`(grand mal)
`
`are
`
`a succession
`
`of
`
`tonic
`
`and clonic episodes with
`
`DEF_OO0Ot02
`
`

`
`I3
`
`loss of consciousness
`
`and autonomic
`
`hyperactivity.
`
`This
`
`form of
`
`epilepsy
`
`can occur
`
`In both children
`
`and adults.
`
`Grand mat
`
`is
`
`the most common type ol epilepsy
`
`and
`
`the most disrupting
`
`in
`
`the
`
`lite oi
`
`the patient.
`
`The
`
`chronlcity
`
`of
`
`the
`
`seizures varies
`
`trom one
`
`or more
`
`a day to
`
`intervals
`
`of momhs between the
`
`seizure
`
`episodes.‘ -5
`
`:11. Evaluation cl Anticonvulsant Agents.
`
`Many procedures have been designed and
`
`evaluated for the
`
`screening of potential anticonvulsant drugs?‘ Current
`
`protocols can
`
`be
`
`divided into four general categories:
`
`(a)
`
`the employment
`
`of a
`
`chemical convulsant
`
`to induce the artiiical
`
`seizures in
`
`animals:
`
`(b)
`
`the use of electrically induced seizures
`
`in
`
`animals;
`
`(c)
`
`the em-
`
`ployment ol genetically predisposed
`
`animal
`
`strains;
`
`(d)
`
`the
`
`use
`
`in in mm experiments."
`
`,5
`
`;4."“;.‘u.:-r..:.’V-X
`
`_
`
`’.-:x..'g...
`
`of nervous tissues
`
`Many chemical
`
`convulsants
`
`have been utilized to generate
`
`artiliclal seizures."-24-25
`
`The most widely used chemical convul-
`
`sants are:
`
`strychnine (2),
`
`picroloxin L19),
`
`bicuculline u_1_) and pen-
`
`lylenetetrazol (Metrazol)
`
`(12).
`
`All
`
`these subtances
`
`are subcutan-
`
`eously injected
`
`in the test animals after the administration of
`
`the
`
`compound
`
`to be
`
`evaluated
`
`for arrliconvulsant properlies.25
`
`The
`
`augmentation
`
`of
`
`the
`
`seizure
`
`threshold
`
`reveals
`
`the
`
`anticonvul-
`
`sarri
`
`activity of
`
`the
`
`dmg.
`
`The
`
`pentylenetetrazol
`
`(scMet)
`
`seizure
`
`threshold test27'29
`
`is
`
`ol
`
`particular
`
`interest
`
`in
`
`the evaluation
`
`of
`
`new compounds.
`
`it
`
`is widely
`
`recognized that
`
`substances
`
`active
`
`DEF_O00O103
`
`

`
`3
`1:R‘=Ph, R2=Ph,R =H
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`
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`DEF__00O0104
`
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`
`F..5
`~ : .
`
`'-1‘
`it
`‘Li
`
`2%».1
`
`15
`
`In
`
`this
`
`test
`
`are
`
`likely to
`
`be effective
`
`in the treatment
`
`at petit
`
`mal
`
`seizures provided threshold doses oi pentylenetetrazol are em-
`
`ployed to produce clonic seizures.
`
`The use
`
`of
`
`electrically
`
`induced seizures was introduced in
`
`the mid-18005
`
`after
`
`the
`
`pioneering studies
`
`of Fritsch. Hitzig and
`
`Alberloni.25
`
`The systematic use of
`
`electrically
`
`induced seizures in
`
`cats
`eventually
`anticonvulsant
`Dilantin) use).
`
`led
`Putnam and Merritt3°
`to discover
`the
`properties
`of
`5,5-diphenylhydantoln
`(phenytoin.
`This medicinal agent
`is
`the most extensively used
`
`drug today for
`
`the treatment of grand mal epilepsy.
`
`Subsequently,
`
`Goodman
`
`and his
`
`colleagues
`
`standardized the
`
`technique which
`
`is
`
`now named the Maximal Eleclroshock Seizure (MES)
`
`test.27’29-3‘
`
`This
`
`test
`
`is believed
`
`to identify substances that will be useful
`
`in
`
`the treatment
`
`oi partial and generalized tonic-clonic seizures.3
`
`In
`
`this
`
`technique a
`
`60-cycle
`
`alternating
`
`current
`
`is
`
`applied for
`
`0.1
`
`electrodes.
`
`A..‘‘A'v...a'‘‘T.-
`~35-Lemur:‘
`
`'-e
`
`
`
`'~..s;"’.‘
`
`2-'
`
`,;:»:.o.i'm'-‘?~‘i..
`
`"-__‘"'.~L
`
`or 0.3
`
`seconds to mice and
`
`rats
`
`by means of corneal
`
`Many
`
`other
`
`useful
`
`electrically induced
`
`seizure
`
`tests have
`
`also.
`
`been described.24v27‘29«31v32
`
`More
`
`recently, kindling procedures
`
`have
`
`been
`
`introduced
`
`in
`
`the hopes
`
`oi
`
`identifying
`
`new drugs
`
`specific
`
`for partial
`
`seizures.”
`
`The kindling
`
`process
`
`refers
`
`to
`
`an experimental
`
`protocol where subthreshold
`
`stimuli
`
`are
`
`repeat-
`
`edly
`
`appiied
`
`to
`
`the animal
`
`until
`
`they become
`
`convulsant stimuli.
`
`This method
`
`requires the implantation
`
`of electrodes
`
`in the brain
`
`area where the epileptic
`
`discharge is
`
`to be generated.’
`
`Genetically
`
`predisposed animal
`
`strains have been employed
`
`DEF_0O00105
`
`

`
`and
`
`guinea-pigs
`
`as
`
`well
`
`as
`
`neuronal
`
`cultures
`
`have
`
`employed
`
`in the experimental
`
`protocols.
`
`In
`
`the first case,
`
`burst
`
`tiring
`
`are
`
`generated
`
`by
`
`altering
`
`the
`
`extracellular
`
`ionic
`
`concentration
`
`or by the application
`
`of
`
`convulsant
`
`agents such as
`
`bicuculline (3) or
`
`penicillin (§).
`
`In the second case.
`
`intracellular
`
`electrodes
`
`are used to deliver a current
`
`pulse
`
`that generates the
`
`epileptic
`
`activity.
`
`In
`
`these tests,
`
`the anticonvulsant properties of
`
`the substances are
`
`determined by their
`
`ability to limit
`
`the
`
`spread
`
`ol
`
`the epileptic
`
`electrical
`
`phenomena
`
`to
`
`non-stimulated areas.”
`
`None ol
`
`the currently
`
`available
`
`test procedures indepen-
`
`dently provides a
`
`definitive method tor
`
`the
`
`recognition
`
`of anti-
`
`convulsant
`
`dmgs.
`
`Each
`
`procedure
`
`can give
`
`talse-positive
`
`and
`
`DEF_OOOO106
`
`'
`
`
`
`
`
`
`
`
`
`7‘~'-ii#~":¢5'.'§¥§-gr-£‘:.'s-:n.<*~J:-.1-fat-g-£?‘n..‘i‘.I:.—_'~;:1_--mm-;=2>_-a-'mv.\_-«..-:.‘.."d‘#.’4*i“r«=6.'l=.-.'}i'§27"
`
`‘I6
`
`in
`
`the anticonvulsant
`
`testing of new substances.
`
`Among
`
`these.
`
`three species
`
`have found
`
`extensive
`
`use.
`
`They are
`
`the DBA/2
`
`(Dilute Brown Agouti)
`
`strain of
`
`house mouse,
`
`in which
`
`seizures
`
`are elicited by audiogenic
`
`stimun
`
`such as
`
`a door bell,33
`
`the
`
`Wlstar
`
`strain ol
`
`rat.
`
`in which
`
`seizures
`
`appear to
`
`be similar to
`
`petlt mal:34
`
`and
`
`the
`
`baboon _Eapi_o_ _p_apig_,
`
`in which responses to
`
`anticonvulsant
`
`dmgs are similar
`
`to that observed in human.“-3535
`
`In
`
`this last
`
`strain
`
`of
`
`animal.
`
`the seizure
`
`is
`
`induced
`
`by flicker-
`
`ing light.
`
`Various
`
`in mm test systems
`
`have been developed for
`
`the
`
`evaluation
`
`of
`
`anticonvulsant
`
`properties
`
`of drug
`
`candidates.”
`
`These
`
`procedures
`
`were
`
`originally
`
`developed to
`
`study
`
`the
`
`biogenesis
`
`of
`
`the
`
`epiIepsies.24
`
`Hippocampal
`
`slices
`
`of
`
`rats
`
`been
`
`
`
`.,--.5‘.7._35,-‘‘£5,‘-‘L.5,1-"-*,5..>__,'-
`~5..-V_.L\."V“'\._
`
`
`

`
`l7
`
`false-negative
`
`respons’es.‘7v25
`
`Meldmm has
`
`urged
`
`the use
`
`oi
`
`as many
`
`tests as
`
`poss