`
`
`United States Patent
`Gobert et al.
`
`[11]Patent Number:
`4,943,639
`[45]Date of Patent:*
`Jul. 24, 1990
`
`[54](S)-ALPHA-ETHYL-2-0X0-1-PYR-
`[52]U.S. CI ..................................................... 548/550
`
`ROLIDINEACETAMIDE
`
`
`
`[58]Field of Search ................. 548/546, 550; 514/424
`
`ABSTRACT
`
`[21]Appl. No.: 311,631
`
`[22]Filed:Feb. 16, 1989
`
`
`
`
`
`Related U.S. Application Data
`
`[75]Inventors:
`Jean Gobert, Brussels; Jean-Pierre
`
`B. Springer
`Primary Examiner-David
`
`
`Geerts, Leglise; Guy Bodson,
`
`Lind & Ponack Attorney, Agent, or Firm-Wenderoth,
`
`
`
`Bellefontaine, all of Belgium
`[57]
`[73]Assignee:
`
`
`U C B Societe Anonyme, Brussels,
`(S)-alpha-ethyl-2-oxo-1-pyrrolidineacetamide, its prepa
`
`Belgium
`
`
`
`
`ration and pharmaceutical compositions containing the
`[ *] Notice:The portion
`of the term of this patent
`
`
`
`same . .It can be prepared either by reacting (S)-alpha
`
`subsequent to Jun. 6, 2006 has been
`
`
`ethyl-2-oxo-1-pyrrolidineacetic acid successively with
`disclaimed.
`
`
`an alkyl haloformate and with ammonia, or, by cycliz
`
`ing an (S)-2-amino-butanamide of the formula
`
`
`X-CH2CH2-Y-NHCH(C2Bs)CONH2 wherein Y is
`
`
`a -CH2- radical when X represents a ZOOC-radi
`
`
`cal and Y is a -CO-radical when X represents a
`
`
`HalCH2-radical, Z being a C1-C4 alkyl radical and
`Mar. 12, 1987, Pat. No.[62]Division of Ser. No. 25,277,
`
`
`
`Hal a halogen atom.
`
`
`
`4,837,223, which is a division of Ser. No. 733,790, May
`This laevorotatory enantiomer has been found to have
`
`
`24, 1985, Pat. No. 4,696,943.
`
`
`
`
`
`significantly higher protective activity against hypoxia
`
`
`and ischemia than the corresponding racemate.
`
`May 15, 1984 [GB] United Kingdom ................. 8412357
`
`
`
`
`
`[30]Foreign Application Priority Data
`
`
`
`
`
`
`
`[51]Int. CI.5
`
`•.••.•.•••.•••••••••••••••••.•.•••.••••.••
`
`C07D 207/277
`
`2 Claims, No Drawings
`
`ARGENTUM Exhibit 1095
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
`
`Page 00001
`
`
`
`1
`
`(S)-ALPHA-ETHYL-2-OXO-1-PYR
`ROLIDINEACETAMIDE
`
`5
`
`4,943,639
`2
`and with (2) ammonia. The alkyl haloformate is prefera
`bly ethyl chloroformate.
`This reaction is generally carried out in dichloro
`methane at a temperature between — 10° and —60° C.
`The (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid,
`used in this reaction, can be obtained from the racemic
`(i)-alpha-ethyl-2-oxo-l-pyrrolidineacetic
`acid
`by
`chemical resolution in accordance with methods known
`per se, for example by forming a salt of this acid with an
`optically active base and isolating the salt formed with
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid by suc
`cessive crystallizations an appropriate solvent (for ex
`ample benzene).
`By way of examples of optically active bases which
`. can be used for this resolution there may be mentioned
`alkaloids such as brucine, quinine strychnine, quinidine
`and cinchonidine and amines such as alpha-methylben
`zylamine and dehydroabietylamine (cf. S. H. WILEN et
`al., Tetrahedron, 33,(l977),2725-2736). Particularly
`favourable results are obtained by using alpha-methyl
`benzylamine and dehydroabietylamine.
`The racemic (i)-alpha-ethyl-2-oxo-l-pyrrolidinea
`cetic acid used as the starting material can be obtained
`by saponifying the corresponding alkyl esters, the syn
`thesis of which has been described in British Pat. No.
`1,309,692.
`(b) cyclizing an (S)-2-amino-butanamide of the for
`mula
`
`This application is a division of application Ser. No.
`025,277, ?led Mar. 12, 1987, now U.S. Pat. No.
`4,837,223, which application is, in turn, a division of
`application Ser. No. 733,790, ?led May 24, 1985, now
`US. Pat. No. 4,696,943.
`The present invention relates to the novel compound
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide, as well
`as to processes for the preparation thereof. It also re
`lates to pharmaceutical compositions containing the
`said compound.
`British Pat. No. 1,309,692 describes the compound
`alpha-ethyl-2-oxo-l-pyrrolidineacetamide
`(melting
`point 122° C.) and states that the compounds of this type
`can be used for therapeutic purposes, for example for
`' the treatment of motion sickness, hyperkinesia, hyperto
`nia and epilepsy.
`Moreover, it also mentions that these compounds can
`be applied in ?eld of memory disorders in normal or
`pathological conditions.
`I
`It is‘ also known that alpha-ethyl-Z-oxo-l-pyr
`rolidineacetamide possesses a protective activity against
`25
`aggressions of the central nervous system caused by
`hypoxias,
`cerebral
`ischemia,
`etc.
`(Phar
`mazie,37/ l l,( 1982), 753-765).
`Continuing research work in this ?eld, we have pre
`pared and isolated the laevorotatory enantiomer of
`30
`alpha-ethyl-Z-oxo-l-pyrrolidineacetamide and have
`found that this compound differs in a completely unpre
`dictable manner from the known racemic form, by
`(1) having a 10 times higher protective activity against
`hypoxia (antihypoxia) and
`(2) having a 4 times higher protective activity against
`ischemia (antiischemia).
`As a result of this unexpected combination of proper
`ties the laevorotatory enantiomer of alpha-ethyl-Z-oxo
`l-pyrrolidineacetamide is more suitable for the treat
`ment and prevention of hypoxic and ischemic type
`aggressions of the central nervous system. The impor
`tant contribution of the hypoxic phenomenon in certain
`pathological conditions of the central nervous system
`suggests that this compound has a therapeutic effect in
`the treatment of the consequences of cerebral vascular
`accidents and of cranial traumas, of the sequelae of the
`ageing process or of circulatory insuf?ciencies or the
`central nervous system resulting from cerebral-ischemic
`or hypoxic accidents occurring for example during
`birth. The compound may also be used in hypoxic-type
`diseases of other organs or tissues, such as the heart and
`kidneys.
`Accordingly, the present invention relates to the
`laevorotatory enantiomer of alpha-ethyl-Z-oxo-l-pyr
`rolidineacetamide which has the S absolute con?gura
`tion, the said compound being substantially free from
`the dextrorotatory enantiomer which has the R absolute
`con?guration.
`ac
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide
`cording to the present invention cannot be obtained
`directly from the racemic form by separating the two
`enantiomers. It can be prepared by one or other of the
`following processes:
`(a) reacting (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetic
`acid successively with (1) an alkyl haloformate of the
`formula HalCOOZ in which Hal represents a halogen
`atom and Z an alkyl radical having 1 to 4 carbon atoms
`
`35
`
`40
`
`45
`
`55
`
`65
`
`in which
`X represents a ZOOC- or HalCH2— radical, Z being
`an alkyl radical having 1 to 4 carbon atoms, and Hal
`a halogen atom, preferably chlorine or bromine, and
`Y represents a --CH2— or —CO— radical,
`with the proviso that Y is a —CHz- radical when X
`represents a ZOOC—- radical and Y is a —CO—- radical
`when X represents a HalCH2-- radical. The cyclization
`of the (S)-2-amino-butanamide of formula A is carried
`out in an inert solvent, such as toluene or dichlorometh
`ane, at a temperature of from 0° C. to the boiling point
`of the solvent. This cyclization is advantageously car
`ried out in the presence of a basic substance as a cata
`lyst. This catalyst is preferably 2-hydroxypyridine
`when the compound of formula A is an ester
`(X=ZOOC--) and tetrabutylammonium bromide
`when the compound of formula A is a halide (X: HalC
`H2—-—).
`When X represents a ZOOC- radical and Y is a
`—CH;— radical the compound of formula A is an alkyl,
`(S)-4-[[l-(aminocarbonyl)propyl]amino]butyrate of the
`formula ZOOCCHZCHZCHZNHCHKIZHQCONHZ, in
`which Z has the meaning given above. The latter can be
`prepared by condensing .(S)-2-amino-butanamide with
`an
`alkyl
`4-halobutyrate
`of
`the
`formula
`ZOOCCH2CH2CH2Hal, in which Z has the meaning
`given above and Hal is a halogen atom.
`When X represents a HalCH2— radical and Y is thus
`a —CO— radical, the compound of formula A is (S)-N
`[l-(aminocarbonyl)propyl]—4-halobutanamide of the
`formula HalCH2CH2CH2CONHCH(C2H5)CONH2, in
`which Hal has the meaning given above. This latter
`compound can be prepared by condensing (S)-2-amino
`butanamide with a 4~halobutyryl halide of the formula
`HaICHZCHZCHZCOHaI, in which Hal is a halogen
`atom.
`
`Page 00002
`
`
`
`4,943,639
`3
`4
`The reaction between the (S)-2-amino-butanamide on
`is distilled and replaced at the distillation rate, by 14
`the one hand and the alkyl 4'halobutyrate or 4-halobu
`liters of toluene from which the product crystallizes.
`tyryl halide on the other hand, is generally carried out
`The mixture is cooled to ambient temperature and the
`in an inert solvent, such as benzene, toluene, dichloro
`crystals are ?ltered off to obtain 2.78 kg of (S)-alpha
`methane or acetonitrile, at a temperature of from -— 5' to
`ethyl-Z-oxo-l-pyrrolidineacetic acid.
`+100° C. and in the presence of an acid acceptor such
`Melting point: 125.9‘ C. [alpha]p2°= —26.4° (c=l,
`as a tertiary organic base (for example triethylamine) or
`acetone). Yield: 94.5%.
`an inorganic base (for example potassium carbonate or
`(0) Preparation of (S)-alpha-ethyl-2-oxo-l-pyr
`hydroxide or sodium carbonate or hydroxide).
`rolidineacetamide
`When X represents a HalCH2-- radical and Y a
`34.2 g (0.2 mole) of (S)-alpha-ethyl-2-oxo-l-pyr
`—CO-- radical, it is not absolutely necessary to isolate
`rolidineacetic acid are suspended in 225 ml of dichloro
`the compound of formula A obtained from the starting
`methane cooled to —30' C. 24.3 g (0.24 mole) of trieth
`materials mentioned above. In fact, the compound of
`ylamine are added dropwise over 15 minutes. The reac
`formula A, obtained in situ, can be cyclized directly to
`tion mixture is then cooled to —40' C. and 24.3 g (0.224
`the (S)-alphaethyl-2-oxo-l-pyrrolidineacetamide ac
`mole) of ethyl chloroformate are added over 12 min
`cording to the present invention (see Example 4 below).
`utes. Thereafter, a stream of ammonia is passed through
`The (S)-2-amino-butanamide used as starting material
`the mixture for 4! hours. The reaction mixture is then
`can be obtained from (S)-2~amino~butyric acid by am
`allowed to return to ambient temperature and the am
`monolysis of the corresponding methyl ester in accor
`monium salts formed are removed by ?ltration and
`dance with the method described by K. FOLKERS et
`washed with dichloromethane. The solvent is distilled
`in J.Med.Chem.14,(6),(l971),484-487.
`The following examples are given for the purpose of
`off under reduced pressure. The solid residue thus ob
`illustration only
`tained is dispersed in 55 ml toluene and the dispersion is
`In these examples, the optical purity of the com
`stirred for 30 minutes and then ?ltered. The product is
`pounds obtained was veri?ed by calorimetric determi
`recrystallized from 280 ml of ethyl acetate in the pres
`nation of the differential enthalpies (C. FOUQUEY and
`ence of 9 g of 0,4 nm molecular sieve in powder form.
`J. JACQUES, Tetrahedron,23,(1967),4009—19).
`24.6 g of (S)-alpha-ethyl-2-oxo-l-pyrrolidineaceta
`mide are obtained.
`EXAMPLE 1
`Melting point: 115‘-l18' C. [alpha]p25=-89.7‘
`(a) Preparation of the (R)-alpha-methyl-benzylamine
`(c= 1, acetone). Yield: 72.3%.
`salt of (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid
`Analysis for C3H14N2O1 in % calculated: C 56.45. H
`8.7 kg (50.8 moles) of racemic (i)-alpha-ethyl~2-oxo
`8.29. N 16.46. found: 56.71. 8.22. 16.48.
`The racemic (i)-alpha-ethyl-2-oxo-l-pyrrolidinea
`l-pyrrolidineacetic acid are suspended in 21.5 liters of
`cetic acid used in this synthesis has been prepared in the
`anhydrous benzene in a 50 liter reactor. To this suspen
`sion is added gradually a solution containing 3.08 kg
`manner described below.
`(25.45 moles) of (R)-(+)-alpha-methyl-benzylamine and
`A solution containing 788 g (19.7 moles) of sodium
`2.575 kg (25.49 moles) of triethylamine in 2.4 liters of
`hydroxide in 4.35 liters of water is introduced over 2
`anhydrous benzene. This mixture is then heated to re
`hours into a 20 liter ?ask containing 3.65 kg (18.34
`?ux temperature until complete dissolution It is then
`moles) of ethyl (i)-alpha-ethyl-2-oxo-l-pyrrolidineace
`cooled and allowed to crystallize for a few hours. 5.73
`tate at a temperature not exceeding 60' C. When this
`kg of the (R)-alpha—methyl-benzylamine salt of (S)
`addition is complete, the temperature of the mixture is
`alpha-ethyl-Z-oxo-l-pyrrolidineacetic acid are thus ob
`raised to 80' C. and the alcohol formed is distilled off
`tained.
`until the temperature of the reaction mixture reaches
`Melting point: l48'-151" C. Yield: 77.1%.
`100' C.
`This salt may be puri?ed by heating under reflux in
`The reaction mixture is then cooled to 0° C. and 1.66
`48.3 liters of benzene for 4 hours. The mixture is cooled
`liter (19.8 moles) of 12N hydrochloric acid is added
`and ?ltered to obtain 5.040 kg of the desired salt. Melt
`over two and a half hours. The precipitate formed is
`ing point: 152‘-153.5’ C. Yield: 67.85%.
`?ltered off, washed with 2 liters of toluene and recrys
`tallized from isopropyl alcohol. 2.447 kg of racemic
`(b) Preparation of
`(i)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid, melting
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid
`at 155'~l56' C., are thus obtained. Yield: 78%.
`5.04 kg of the salt obtained in (a) above are dissolved
`Analysis for C3H13NO3, in % calculated: C 56.12. H
`in 9 liters of water. 710 g of a 30% sodium hydroxide
`7.65. N 8.18. found: 55.82. 8.10. 7.97.
`solution are added slowly so that the pH of the solution
`reaches 12.6 and the temperature does not exceed 25‘ C.
`The solution is stirred for a further 20 minutes and the
`alpha-methylbenzylamine liberated is extracted repeat
`edly with a total volume of 18 liters of benzene.
`The aqueous phase is then acidi?ed to a pH of 1.1 by
`adding 3.2 liters of 6N hydrochloric acid. The precipi
`tate formed is ?ltered off, washed with water and dried.
`The ?ltrate is extracted repeatedly with a total vol
`ume of 50 liters of dichloromethane. The organic phase
`is dried over sodium sulfate and ?ltered and evaporated
`to dryness under reduced pressure.
`The residue obtained after the evaporation and the
`precipitate isolate previously, are dissolved together in
`14 liters of hot dichloromethane. The dichloromethane
`
`EXAMPLE 2
`(a) Preparation of ethyl
`(S)-4-[[l-(arninocarbonyl)propyl]amino]butyrate
`143.6 ml (1.035 mole) of triethylamine are added to a
`suspension of 47.75 g (0.345 mole) of (S)-2-amino
`butanamide hydrochloride ([alpha]D25: +26.1'; c=l,
`methanol) in 400 ml of toluene. The mixture is heated to
`80' and 67.2 g (0.345 mole) of ethyl 4-bromobutyrate
`are introduced dropwise.
`The reaction mixture is maintained at 80' C. for 10
`hours and then ?ltered hot to remove the triethylamine
`salts. The ?ltrate is then evaporated under reduced
`pressure and 59 g of an oily residue consisting essen
`
`55
`
`60
`
`65
`
`20
`
`25
`
`35
`
`40
`
`45
`
`Page 00003
`
`
`
`(b) Preparation of
`4
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide
`54 g of the crude product obtained in a) above are
`dissolved in 125 ml of toluene in the presence of 2 g of
`Z-hydroxypyridine. The mixture is heated at 110° C. for
`12 hours.
`The insoluble matter is ?ltered off hot and the ?ltrate
`is then evaporated under reduced pressure.
`The residue is puri?ed by chromatography on a col
`umn of 1.1 kg of silica (column diameter: 5 cm; eluent:
`a mixture of ethyl acetate, methanok and concentrated
`ammonia solution in a proportion by volume of 85: 12:3).
`The product isolated is recrystallized from 50 ml of
`ethyl acetate to obtain 17.5 g of (S)-alpha-ethyl-2-oxo-I
`pyrrolidineacetamide.
`Melting point: 117° C. [alpha]D25: —90.0° (c=1, ace
`tone). Yield: 41%.
`
`15
`
`25
`
`4,943,639
`5
`6
`tially of the monoalkylation product but containing also
`latter is removed by hot ?ltration to give 3.10 g of (5)»
`alphaethyl-Z-oxo-l-pyrrolidineacetamide.
`a small amount of dialkylated derivative are obtained.
`Melting point: 1l6.7° C. [alpha]D25: —90.1° (c= 1,
`The product obtained in the crude state has been used
`as such, without additional puri?cation, in the prepara
`acetone). Yield: 60.7%.
`tion of (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide by
`cyclization.
`EXAMPLE 4
`Preparation of
`(S)-alpha-ethyl-2-oxo- l-pyrrolidineacetamide
`This example illustrates a variant of the process of
`Example 3, in which the intermediate 4-chlorobutana
`mide obtained in situ is not isolated. 84 g of anhydrous
`sodium sulfate are added to a suspension of 69.25 g (0.5
`mole) of (S)-2-amino-butanamide hydrochloride in 600
`ml of dichloromethane at ambient temperature. The
`mixture is cooled to 0° C. and 115 g of ground potas
`sium hydroxide are added, followed by 8.1 g (0.025
`mole) of tetrabutylammonium bromide dissolved in 100
`ml of dichloromethane. A solution of 77.5 g of 4
`chlorobutyryl chloride in 100 ml of dichlorometha is
`added dropwise at 0° C., wih vigorous stirring. After 5
`hours’ reaction, a further29 g of ground potassium
`hydroxide are added. Two hours later, the reaction
`mixture is ?ltered over Hy?o-cel and the ?ltrate evapo
`rated under reduced pressure. The residue (93.5 g) is
`dispersed in 130 ml of hot toluene for 45 minutes. The
`resultant mixture is ?ltered and the ?ltrate evaporated ’
`under reduced pressure. The residue (71.3 g) is dis
`solved hot in 380 ml of ethyl acetate to which 23 g of 0,4
`nm molecular sieve in powder form are added. This
`mixture is heated to re?ux temperature and filtered hot.
`After cooling the ?ltrate, the desired product crystal
`lizes to give 63 g of (S)-alpha-ethyl-2—oxo-l-pyr
`rolidineacetamide.
`Melting point: 117'’ C. [alpha]D25: -9l.3° (0: l, ace
`tone). Yield: 74.1%.
`Pharmcological tests
`Racemic alpha-ethyl-Z-oxo-l-pyrrolidineacetamide
`(compound A) and (S)-alpha-ethyl-2-oxo-l-pyr
`rolidineacetamide (compound B) of the present inven
`tion were subjected to pharmcological tests.
`1. Protection against hypoxia (mouse)
`a. Principle (C. GIURGEA and F. MOURAVIEFF
`LESUISSE; Proc.Xth Intern. Congr. of the C011. In
`tern. Neuro-psych.- Pergamon Press, Oxford and New
`York, 1978, p.1623-163l).
`The principle of this test lies in measuring the possi
`bilities of survival of the organism subjected to an atmo
`sphere in which the oxygen level is progressively de
`creased. Due to the particular sensitivity of the nervous
`system to this type of aggression, the results obtained in
`this test can be interpreted as a measure of the resistance
`of the central nervous system. Compounds which in
`crease the resistance of the animals to this stress are
`suitable for the treatment and prevention of hypoxic
`type aggressions of the central nervous system.
`b. Method
`The apparatus consists of an airtight transparent cage '
`37 cm high, 39 cm deep and 97 cm wide. This 140 liter
`cage is provided with 60 transparent compartments
`each 6X10><10 cm, making it possible to separately
`accomodate 60 mice.
`_ A fan ensures circulation of the atmosphere between
`the compartments through a grid ?oor. The cage is
`equipped with a device for introducing nitrogen at a
`constant ?ow rate, and with an ori?ce communicating
`
`EXAMPLE 3
`(a) Preparation of
`(S)-N-[1(aminocarbonyl)propyl]-4-chlorobutanarnide
`345.6 g (2.5 moles) of ground potassium carbonate are
`mixed with 138.5 g (1 mole) of (S)-2-amino-butanamide
`hydrochloride in 2.5 liters of acetonitrile. The reaction
`mixture is cooled to 0° C. and a solution of 129.2 g (1.2
`mole) of 4-chlorobutyryl chloride in 500 ml of acetoni
`trile is introduced dropwise. After the addition, the
`reaction mixture is allowed to return to ambient temper
`ature; the insoluble matter is ?ltered off and the ?ltrate
`evaporated under reduced pressure. The crude residue
`obtained is stirred in 1.2 liter of anhydrous ether for 30
`minutes at a temperature between 5° and 10° C. The
`precipitate is ?ltered off, washed twice with 225 ml of
`ether and dried in vacuo to obtain 162.7 g of (S)-N-[1
`(aminocarbonyl)propy]-4-chlorobutanamide.
`Melting point: ll8°—l23° C. [alpha]D25: —l8° (c= l,
`methanol). Yield: 78.7%.
`The crude product thus obtained is very suitable for
`the cyclization stage which follows. It can however be
`puri?ed by stirring for one hour in anhydrous ethyl
`acetate.
`Melting point: l20°—122° C. [alpha]D25: -22.2° (c= l,
`methanol).
`
`(b) Preparation of .
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide
`6.2 g (0.03 mole) of (S)-N-[1(aminocarbonyl)propyl]
`4-chlorobutamine and 0.484 g (0.0015 mole) of tet
`rabutylammonium bromide are mixed in 45 ml of di
`chloromethane at 0° C. under a nitrogen atmosphere.
`2.02 g (0.036 mole) of potassium hydroxide powder are
`added over 30 minutes, at such a rate that the tempera
`ture of the reaction mixture does not exceed +2° C.
`The mixture is then stirred for one hour, after which a
`further 0.1 g (0.0018 mole) of ground potassium hydrox
`ide is added and stirring continued for 30 minutes at 0°
`C. The mixture is allowed to return to ambient tempera
`ture. The insoluble matter is ?ltered off and the ?ltrate
`is concentrated under reduced pressure. The residue
`obtained is recrystallized from 40 ml of ethyl acetate in
`the presence of 1.9 g of 0,4 nm molecular sieve. The
`
`60
`
`65
`
`Page 00004
`
`
`
`4,943,639
`7
`.
`with the ambient atmosphere. Male mice (NMRI strain)
`weighing 20 to 22 g, are kept fasting as from the day
`before the test. The experiment is effected on the fol
`lowing day, simultaneously on 3 groups of 20 mice; a
`control group is given water (25 ml/ltg) orally, and the
`other two groups are each given orally a compound to
`be tested.
`25 minutes after the administration, the animals are
`distributed at random amongst the compartments so
`that none of the three groups is concentrated in a pre
`ferred area of the cage.
`30 minutes after administration, the cage is closed and
`nitrogen is admitted into it at a constant ?ow rate (7.75
`liters of technical grade nitrogen per minute) for about
`37 minutes, at which stage the atmosphere contains
`3.7% oxygen.
`The cage is left closed until the critical moment
`where no more than 3 survivors are observed among
`the 20 control animals. At that moment, the cage is
`opened and atmospheric air admitted into it. A few
`moments later the survivors in each group of animals
`are counted.
`>
`For each dose of compound to be tested, the experi
`ments are repeated once or twice, and the results pooled
`25
`to obtain a minimum of 40 (or 60 animals treated per
`dose and 40 (or 60) corresponding control animals. For
`each dose of compound tested, the number of surviving
`animals among those treated with the compound is
`compared with the number of surviving animals among
`30
`the control animals. The difference between these num
`bers expresses the protective activity of the compound
`against hypoxia caused by oxygen deprivation. The
`statistical signi?cance (P) of this difference is evaluated
`by the Fischer-Yates test.
`c. Results
`Table I below gives the results obtained for increas
`ing doses of compounds A and B.
`TABLE I
`Number of surviving
`animals
`control
`treated
`
`15
`
`20
`
`35
`
`Oral dose
`Compound tested in mmol/kg
`
`P
`
`B
`
`A
`
`0.032
`0.1
`0.16
`0.32
`0.016
`0.032
`0.1
`0.16
`0.32
`NS = statistically non-significant.
`
`12/60
`8/60
`12/60
`10/60
`5/40
`8/40
`6/40
`6/40
`5/40
`
`16/60
`7/60
`12/60
`30/60
`11/40
`17/40
`19/40
`19/40
`17/40
`
`NS
`NS
`NS
`<0.001
`NS
`<0.6
`(0.005
`<0.005
`<0.01
`
`d. Conclusions
`In this test, the laevorotatory enantiomer of the in
`vention (compound B) increases the survival of the
`animals deprived of oxygen when administered at doses
`from 0.032 mmol/ltg upwards. The racemate (com
`pound A) exerts a similar activity only from 0.32
`mmol/kg upwards (1st effective dose). Thus, the la
`evorotatory enantiomer of the present invention is 10
`times more active than the corresponding racemate.
`
`II. Protection against cerebral ischemia (rats)
`a. Principle (C. GIURGEA and F. MOURAVIEFF
`~ LESUISSE; see above under Ia.) Electroencephalo
`graphic controls have shown that the ligature of the 2
`common carotids in the rat causes a true cerebral isch
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`emia: the electroencephalogram trace ?attens and even
`becomes isoelectric (electric silence).
`b. Method
`-
`Male Wistar rats weighing between 250 and 350 g are
`anesthetized with pentobarbital administered intraperi
`toneally at a dose of 50 mg/kg (0.5 ml/ 100 g).
`Immediately after the anesthesia, the animals are
`administered intraperitoneally with an amount of 0.5
`ml/ 100 g, either the compound to be tested dissolved in
`an isotonic sodium chloride solution (treated animals),
`or only an isotonic sodium chloide solution or placebo
`(control animals). About 20 minutes later, the 2 com
`mon carotids are exposed and about 10 minutes later
`ligatured simultaneously. This operation is effected
`simultaneously on the control animals and the treated
`animals.
`An hour after administration of the compound to be
`tested or of the placebo, there is again administered
`intraperitoneally the same dose of either the compound
`to be tested (to the treated animals) or the place (to the
`control animals).
`5 hours after the ?rst administration, there is adminis
`tered for the third time the same dose of either the
`compound to be tested (to the surviving treated ani
`mals) or the placebo (to the surviving control animals).
`24 hours after the ?rst administration the efficacy of
`the ligature is veri?ed in all animals, under pentobarbi
`tal anesthesia, by section or the carotids downstream of
`the ligature. The number of surviving animals is re
`corded among both the treated animals and the control
`animals. For each dose of compound tested, the number
`of surviving animals among those treated with the com
`pound is compared with the number of surviving ani
`mals among the control animals. The difference ex
`presses the protective activity of the compound against
`the lethality induced by the simultaneous ligature of the
`2 carotids. The statistical signi?cance (P) of this differ
`ence is evaluated by the Brandt-Snedecor test.
`c. Results
`Table II below gives the results obtained for increas
`ing doses of compounds A and B.
`TABLE II
`Number of surviving
`animals
`control
`treated
`
`Compound
`tested
`
`lntraperitoneal
`dose in mmol/kg
`
`P
`
`A
`
`B
`
`0.32
`0.64
`0.1
`0.16
`0.32
`
`6/29
`11/30
`9/29
`6/29
`8/30
`
`8/29
`21/30
`14/29
`14/30
`19/29
`
`NS
`0.01
`NS
`0.05
`0.01
`
`NS = non-signi?cant difference.
`
`d. Conclusions
`Table II shows that the racemate (compound A) is
`only active from a dose of 0.64 mmol/kg upwards. In
`contrast, the laevorotatory enantiomer of the invention
`(compound B) protects the animals, from 0.16 mmol/kg
`upwards, against the lethality induced by the simulta
`neous ligature of the two carotids and thus proves to be
`4 times more active than the racemate.
`III. Toxicity
`Table III below gives, for compounds A and B, the
`LD50, in mg/kg, determined on the male mouse and the
`male rat after intravenous administration:
`
`Page 00005
`
`
`
`9
`TABLE III
`LD50 in mg/kg
`mouse
`rat
`
`Compound tested
`
`A
`B
`
`1790
`1081
`
`1500
`1038
`
`4,943,639
`10
`calcium carbonate, lactose, sucrose and magnesium
`stearate.
`The percentage of active product in the pharmaceuti
`cal compositions can vary within very wide limits de
`pending upon the mode of administration and the condi
`tion of the patient. The human posology can vary be
`tween 250 mg and 3 g per day.
`There is given below a non-limiting example of a
`composition containing the compound of the invention
`i.e. a 100 mg gelatine capsule for oral administration:
`
`10
`
`As can be seen from this table the laevorotatory enan
`tiomer of the invention (compound B) has, like the
`racemate (compound A), very low toxicity and the
`toxic dose is well above the active dose.
`The compound of the present invention can be ad
`ministered either orally in the form of solid or liquid
`compositions for example, in the form of tablets, pills,
`degrees, gelatine capsules, solutions or syrups, or paren
`terally in the form of injectable solutions or suspensions.
`Pharmaceutical forms such as solutions or tablets are
`prepared according to conventional pharmaceutical
`20
`' methods. The compound of the invention may be mixed
`with a solid or liquid non-toxic pharmaceutically ac
`ceptable carrier and optionally with a dispersant, a sta
`bilizer and where necessary, colorants and sweeteners.
`Similarly the solid or liquid pharmaceutical carriers
`used in these compositions are well known.
`Solid pharmaceutical excipients for the preparation
`of tablets or capsules include, for example, starch, talc,
`
`compound B
`avicel (microcrystalline cellulose)
`Mg stearate
`
`100 mg
`217 mg
`5 mg
`
`We claim:
`1. (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide sub
`stantially
`free
`of
`(R)-alpha-ethyl-2-oxo-l-pyr
`rolidineacetamide.
`2. (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide sub
`stantially
`free
`of
`(R)-alpha-ethyl-2-oxo-l-pyr
`rolidineacetamide, prepared by the process which com
`prises reacting (S)-alpha-ethyl-2-oxo-l-pyrrolidinea
`cetic acid successively with (1) an alkyl haloformate of
`the formula HalCOOZ in which Hal represents a halo
`gen atom and Z represents an alkyl radical having 1 to
`4 carbon atoms, and with (2) ammonia.
`# i i i i
`
`15
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`Page 00006
`
`
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`(12)
`
`CERTIFICATE EXTENDING PATENT TERM
`UNDER 35 USC. § 156
`
`(68) PATENT NO,
`
`(45) ISSUED
`
`(75) INVENTOR
`
`(73) PATENT OWNER
`
`(95) PRODUCT
`
`;
`
`1
`
`:
`
`:
`
`:
`
`4,943,639
`
`July 24, 1990
`
`Jean GObBl't, et a1,
`
`UCB SOCIETE ANONYME
`
`KEPPRA® (levetiracetam)
`
`This is to certify that an application under 35 USC. § 156 has been ?led in the United
`States Patent and Trademark Of?ce, requesting extension of the term of US. Patent No.
`4,943,639 based upon the regulatory review of the product KEPPRA (levetiracetam) by
`the Food and Drug Administration, Since it appears that the requirements of the law have
`been met, this certificate extends the term of the patent for the period of
`
`(94)
`
`1,157 days
`
`from June 6, 2006, the original expiration date of the patent, subject to the payment of
`maintenance fees as provided by law, with all rights pertaining thereto as provided by
`35 USC § 156(b).
`
`Page 00007
`
`
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`
`PATENT NO. : 4,943,639
`DATED
`: July 24, 1990
`INVENTOR(S) : Jean Gobert et al.
`
`Page 1 of 1
`
`It is certified that error appears in the above-identi?ed patent and that said Letters Patent is
`hereby corrected as shown below:
`
`Title page,
`Item [*] Notice, “The portion of this patent subsequent to June 6, 2006 has been
`disclaimed.” should read -- This patent is subject to a terminal disclaimer.
`Item [86], 35 USC § 156, “from June 6, 2006,” should read -- from May 14, 2005,
`
`Signed and Sealed this
`
`Fourteenth Day of June, 2005
`
`m Wan,”
`
`JON W. DUDAS
`Director ofthe United States Patent and Trademark O?‘ice
`
`Page 00008
`
`
`
`Disclaimer
`
`4,943,639—Jean Gobert, Brussels; Jean-Pierre Geens, Leglise; Guy Bodson, Bellefontaine, all of
`Belgium.(S)-ALPHA-ETHYL-2-OXO-l-PYR- ROLIDINEACETAMIDE. Patent dated Jul. 24, 1990. Dis
`claimer ?led Mar. 22, 2005, by the Assignee, U C B Societe Anonyme.
`The term of this patent which would extend beyond the expiration date of Pat. Nos. 4,837,223 and
`4,696,943.
`
`(Official Gazette March 18, 2008)
`
`Page 00009