[191
`
`
`United States Patent
`Gobert et al.
`
`4,837,223
`
`[U]Patent Number:
`[ 45] Date of Patent:*
`Jun.6, 1989
`
`References Cited
`
`[54] (S)-ALPHA-ETHYL-2-0X0-1-PYR·
`
`[56]
`
`ROLIDINEACETAMIDE COMPOSITIONS
`FOREIGN PATENT DOCUMENTS
`Jean-Pierre
`Brussels;
`Jean Gobert,
`[75]Inventors:
`2081508 12/1971 France.
`
`
`Geerts,
`Leglise;
`Guy Bodson,
`
`2368275 5/1978 France.
`
`Bellefontaine, all of Belgium
`B. Springer
`Primary Examiner-David
`
`
`UCB Societe Anonyme, Brussels,
`[73]Assignee:
`
`Lind & Ponack Attorney, Agent, or Firm-Wenderoth,
`
`
`Belgium
`
`ABSTRACT
`
`[21]Appl. No.: 25,277
`
`[22]Filed:Mar. 12, 1987
`
`[57]
`[ *] Notice: The portion of the term of this patent
`
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide, its prepa­
`
`
`subsequent to Jun. 6, 2006 has been
`
`
`
`
`ration and pharmaceutical compositions containing the
`disclaimed.
`
`
`
`same. It can be prepared either by reacting (S)-alpha­
`
`
`
`ethyl-2-oxo-l-pyrrolidineacetic acid successively with
`
`
`an alkyl haloformate and with ammonia, or, by cycliz­
`
`ing an (S)-2-amino-butanamide of the formula
`
`
`
`X-CH2CH2-NHCH (C2Hs)CONH2 wherein Y is a
`
`
`-CH2-radical when X represents a ZOOC-radical
`
`
`and Y is a -CO-radical when X represents a HalC­
`
`
`H2-radical, Z being a C1-C4 alkyl radical and Hal a
`
`halogen atom.
`This laevorotatory enantiomer has been found to have
`
`
`
`May 15, 1984 [GB] United Kingdom ............... 84/12357
`
`
`
`
`
`significantly higher protective activity against hypoxia
`
`
`and ischemia than the corresponding racemate.
`
`
`[51]Int. Cl.4 .................. C07D 207/277; A61K 31/40
`
`[52]U.S. Cl, ..................................... 514/424; 548/543
`
`
`
`
`[58]Field of Search ......................... 548/543; 514/424
`
`2 Claims, No Drawings
`
`
`
`
`
`Related U.S. Application Data
`
`[62]Division
`
`of Ser. No. 733,790, May 14, 1985, Pat. No.
`4,696,943.
`[30] Foreign Application
`
`Priority Data
`
`
`
`
`
`ARGENTUM Exhibit 1094
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
`
`Page 00001
`
`

`
`(S)-ALPHA-ETHYL-Z-OXO-l-PYR
`ROLIDINEACETAMIDE COMPOSITIONS
`
`4,837,223
`2
`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 in an appropriate solvent (for
`example 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-methyl-ben~
`zylamine and dehydroabietylamine (cf. 8. H. WILEN et
`al., Tetrahedron, 33, (1977), 2725-2736). Particularly
`favourable results are obtained by using alpha-methyl
`benzylamine and dehydroabietylamine. The racemic
`(i)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid used as
`the starting material can be obtained by saponifying the
`corresponding alkyl esters, the synthesis of which has
`been described in British Pat. No. 1,309,692.
`(b) cyclizing an (S)-2-amino-butanamide of the for
`mula
`
`15
`
`20
`
`25
`
`This application is a division of application Ser. No.
`733,790 ?led May 14, 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-Z-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 the ?eld of memory disorders in normal or
`pathological conditions.
`It is also known that alpha-ethyl-Z-oxo-l-pyr
`rolidineacetamide possesses a protective activity against
`aggressions of the central nervous system caused by
`hypoxias, cerebral ischemia, etc. (Pharmazie, 37/11,
`(1982), 753-765).
`Continuing research work in this ?eld, we have pre
`pared and isolated the levorotatory enantiomer of al
`pha-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 sequels of the
`ageing process or of circulatory insuf?ciencies of 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
`and with (2) ammonia. The alkyl haloformate is prefera
`bly ethyl chloroformate.
`
`X-CI-IgCI-h-Y-NHCPKCZH5)CONH2
`
`(A)
`
`in which
`X represents a ZOOC—- or HalCHg-— 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 ——CH;— or ——CO—— radical,
`with the proviso that Y is a ——CH;;— radical when X
`represents a ZOOC- radical and Y is a —CO— radical
`when X represents a HalCHg- 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
`—_—CH2—- radical the compound of formula A is an alkyl
`(S)~4~[[l-(aminocarbonyl)propyl]amino]butyrate of the
`formula ZOOCCHZCHZCHZNHCH(C2H5)CONH2, 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
`ZOOCCHZCHZCHZHaI, 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
`[1'(aminocarbonyl)propyl]-4-halobutanamide of the
`formula HalCH2CH1CH2CONI-ICH(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
`HalCHgCHgCHgCOHal, in which Hal is a halogen
`atom.
`The reaction between the (S)-2~amino-butanamide on
`the one hand and the alkyl 4-halobutyrate or 4-halobu
`tyryl halide on the other hand, is generally carried out
`
`35
`
`45
`
`60
`
`65
`
`Page 00002
`
`

`
`3
`in an inert solvent, such as benzene, toluene, dichloro
`methane or acetonitrile, at a temperature of from — 5° to
`+100° C. and in the presence of an acid acceptor such
`as a tertiary organic base (for example triethylamine) or
`an inorganic base (for example potassium carbonate or 5
`hydroxide or sodium carbonate or hydroxide).
`When X represents a HalCH2— radical and Y a
`—CO— radical, it is not absolutely necessary to isolate
`the compound of formula A obtained from the starting
`materials mentioned above. In fact, the compound of 10
`formula A, obtained in situ, can be cyclized ‘directly to
`the (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide ac
`cording to the present invention (see Example 4 below).
`The (S)-2-amino-butanamide used as starting material
`can be obtained from (S)-2-amino-butyric acid by am
`monolysis of the corresponding methyl ester in accor
`dance with the method described by K. FOLKERS et
`al in J. Med. Chem. 14, (6), (1971), 484-487.
`The following examples are given for the purpose of
`20
`illustration only,
`In these examples, the optical purity of the com
`pounds obtained was veri?ed by calorimetric determi
`nation of the differential enthalpies (C. FOUQUEY and
`J. JACQUES, Tetrahedron, 23, (1967), 4009-19).
`EXAMPLE 1
`(a) Preparation of the (R)-alpha-methyl—benzylamine
`salt of (S)-alpha-ethyl-2-oxo-1—pyrrolidineacetic acid
`8.7 kg (50.8 moles) of racemic (i)-alpha-ethyl-2-oxo- 30
`l-pyrrolidineacetic acid are suspended in 21.5 liters of
`anhydrous benzene in a 50 liter reactor. To this suspen~
`sion is added gradually a solution containing 3.08 kg
`(25.45 moles) of (R)-(+)-alpha-methyl~benzylamine and
`2.575 kg (25.49 moles) of triethylamine in 2.4 liters of 35
`anhydrous benzene. This mixture is then heated to re
`flux temperature until complete dissolution It is then
`cooled and allowed to crystallize for a few hours. 5.73
`kg of the (R)—alpha-methyl-benzylamine salt of (S)
`alpha-ethyl-Z-oxo-l-pyrrolidineacetic acid are thus ob
`tained.
`Melting point: 148°~151° C. Yield: 77.1%.
`This salt may be puri?ed by heating under re?ux in
`48.3 liters of benzene for 4 hours. The mixture is cooled
`and ?ltered to obtain 5.040 kg of the desired salt.
`Melting point: 152°—153.5° C.
`Yield: 67.85%.
`
`25
`
`45
`
`50
`
`(b) Preparation of
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid
`5.04 kg of the salt obtained in (a) above are dissolved
`in 9 liters of water. 710 g of a 30% sodium hydroxide
`solution are added slowly so that the pH of the solution
`reaches 12.6 and the temperature does not exceed 25“ C. 55
`The solution is stirred for a further 20 minutes and the
`alpha-methyl-benzylamine 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- 6O
`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
`
`65
`
`4,837,223
`
`4
`is distilled and replaced at the distillation rate, by 14
`liters of toluene from which the product crystallizes.
`The mixture is cooled to ambient temperature and the
`crystals are ?ltered off to obtain 2.78 kg of (S)-alpha
`ethyl-2-oxo-l-pyrrolidineacetic acid.
`Melting point: 125.9° C.
`[alpha] D20: —26.4° (c=1, acetone).
`Yield: 94.5%.
`
`(0) Preparation of
`(S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide
`34.2 g (0.2 mole) of (S)-alpha-ethyl-2-oxo-l-pyr
`rolidineacetic acid are suspended in 225 ml of dichloro
`methane cooled to —30“ C. 24.3 g (0.24 mole) of trieth
`ylamine are added dropwise over 15 minutes. The reac
`tion mixture is then cooled to ~40“ C. and 24.3 g (0.224
`mole) of ethyl chloroformate are added over 12 min
`utes. Thereafter, a stream of ammonia is passed through
`the mixture for 4% hours. The reaction mixture is then
`allowed to return to ambient temperature and the am
`monium salts formed are removed by ?ltration and
`washed with dichloromethane. The solvent is distilled
`off under reduced pressure. The solid residue thus ob
`tained is dispersed in 55 ml toluene and the dispersion is
`stirred for 30 minutes and then ?ltered The product is
`recrystallized from 280 ml of ethyl acetate in the pres
`ence of 9 g of 0,4 nm molecular sieve in powder form.
`24.6 g of (S)-alpha-ethyl-2-oxo-l-pyrrolidineaceta
`mide are obtained.
`Melting point: 115°—118° C.
`[alpha] D25: — 89.7° (c: 1, acetone).
`Yield: 72.3%.
`‘
`Analysis for CgH14N202 in %: calculated: C ‘56.45; H
`8.29; N 16.46; found: 56.71; 8.22; 16.48;
`The racemic (i)-alpha~ethyl-2-oxo-l-pyrrolidinea
`cetic acid used in this synthesis has been prepared in the
`manner described below.
`A solution containing 788 g (19.7 moles) of sodium
`hydroxide in 4.35 liters of water is introduced over 2
`hours into a 20 liter ?ask containing 3.65 kg (18.34
`moles) of ethyl (i-)-alpha-ethyl-2-oxo-l-pyrrolidineace
`tate at a temperature not exceeding 60° C. When this
`addition is complete, the temperature of the mixture is
`raised to 80° C. and the alcohol formed is distilled off
`until the temperature of the reaction mixture reaches
`100° C.
`The reaction mixture is then cooled to 0° C. and 1.66
`liter (19.8 moles) of 12N hydrochloric acid is added
`over two and a half hours. The precipitate formed is
`?ltered off, washed with 2 liters of toluene and recrys
`tallized from isopropyl alcohol. 2.447 kg of racemic
`(i)-alpha-ethyl-2-oxo-l-pyrrolidineacetic acid, melting
`at 155°-156° C., are thus obtained.
`Yield: 78%.
`Analysis for C3H13N03, in %: calculated: C 56.12; H
`7.65; N 8.18; found: 55.82; 8.10; 7.97;
`EXAMPLE 2
`(a) Preparation of ethyl
`(S)-4-[[1-(aminocarbonyl)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: 1,
`methanol) in 400 ml of toluene. The mixture is heated to
`80° C. and 67.2 g (0.345 mole) of ethyl 4-bromobutyrate
`are introduced dropwise.
`
`Page 00003
`
`

`
`4,837,223
`6
`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
`latter is removed by hot ?ltration to give 3.10 g of (S)
`alpha-ethyl-2-oxo-l-pyrrolidineacetamide.
`Melting
`point: 116.7” C.
`[alpha]D25: —90.1° (C: l, acetone).
`Yield: 60.7%.
`
`5
`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
`tially of the monoalkylation product but containing also
`a small amount of dialkylated derivative are obtained.
`The product obtained in the crude state has been used
`as such, without additional puri?cation, in the prepara
`tion of (S)-alpha-ethyl-2-oxo-l-pyrrolidineacetamide by
`cyclization.
`
`15
`
`(b) Preparation of
`(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
`2-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, methanol and concentrated
`ammonia solution in a proportion by volume of 85zl2z3).
`The product isolated is recrystallized from 50 ml of
`ethyl acetate to obtain 17.5 g of (S)-alpha-ethyl-2~oxo~l~
`25
`pyrrolidineacetamide.
`Melting point: 117° C.
`[alpha]D25: —90.0° (c=l, acetone).
`Yield: 41%.
`
`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 dichloromethane is
`added dropwise at 0° C., with vigorous stirring. After 5
`hours’ reaction, a further 29 g of ground potassium
`hydroxide are added. Two hours later, the reaction
`mixture is ?ltered over Hyflo-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 ?ltered 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° (c: l, acetone).
`Yield: 74.1%.
`
`PHARMACOLOGICAL 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 pharmacological tests.
`I. Protection against hypoxia (mouse)
`a. Principle (C. GIURGEA and F. MOURAVIEFF
`LESUISSE; Proc. Xth Intern. Congr. of the C011.
`Intern. Neuro-psych.-Pergamon Press, Oxford and
`New York, 1978, p. 1623-1631).
`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.
`
`35
`
`40
`
`50
`
`EXAMPLE 3
`(a) Preparation of
`(S)-N-[1-(aminocarbonyl)propyl]-4-chloro-butanamide
`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
`45
`ether and dried in vacuo to obtain 162.7 g of (S)-N-[l
`(aminocarbonyl)propyl]-4-chlorobutanamide.
`Melting point: 1l8°~123° C.
`[alpha]D25: - 18° (0: 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-[l-(aminocarbonyl)propyl]
`4-chlorobutanami 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
`
`55
`
`60
`
`65
`
`Page 00004
`
`

`
`4,837,223
`7
`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 6><l0>< 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 flow rate, and with an ori?ce communicating
`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/kg) 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 flow rate (7.75
`liters of technical grade nitrogen per minute) for about
`37 minutes, at which stage the atmosphere contains
`25
`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
`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 com
`pound is compared with the number of surviving ani
`mals among the control animals. The difference be
`tween these numbers expresses the protective activity
`of the compound against hypoxia caused by oxygen
`deprivation. The statistical signi?cance (P) of this dif
`ference is evaluated by the Fischer-Yates test.
`0. 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
`
`35
`
`45
`
`Compound tested
`
`Oral dose
`in mmol/kg
`
`50
`
`P
`
`B
`
`A
`
`0.032
`0.1
`0.16
`0.32
`0.016
`0.032
`0.1
`0.16
`0.32
`NS = statistically non-signi?cant.
`
`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/kg upwards. The racemate (com
`pound A) exerts a similar activity only from 0.32
`mmol/kg upwards (1st effective dose). Thus, the la
`
`55
`
`60
`
`65
`
`8
`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.
`Electroencephalographic controls have shown that
`the ligature of the 2 common carotids in the rat causes
`a true cerebral ischemia: 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 chloride 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 placebo (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 pentobarbital
`anesthesia, by section of the carotids downstream of the
`ligature. The number of surviving animals is recorded
`among both the treated animals and the control animals.
`For each dose of compound tested, the number of sur
`viving animals among those treated with the compound
`is compared with the number of surviving animals
`among the control animals. The difference expresses the
`protective activity of the compound against the lethal
`ity induced by the simultaneous ligature of the 2 ca
`rotids. The statistical signi?cance (P) of this difference
`is evaluated by the Brandt-Snedecor test.
`0. 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
`
`Intraperitoneal
`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
`
`Page 00005
`
`

`
`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
`LD5Q, in mg/ kg, determined on the male mouse and the
`male rat after intravenous administration:
`TABLE III
`LDQQ in mg/kg
`mouse
`
`Compound tested
`
`rat
`
`A
`B
`
`1790
`1081
`
`1500
`1038
`
`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,
`dragees, 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
`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 capsule include, for example, starch, talc,
`calcium carbonate, lactose, sucrose and magnesium
`stearate.
`
`4,837,223
`10
`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:
`compound B: 100 mg
`avicel (microcrystalline cellulose): 217 mg
`Mg stearate: 5 mg
`We claim:
`1. A pharmaceutical composition comprising a thera
`peutically effective amount of (S)-alpha-ethyl-2-oxo-l
`pyrrolidineacetamide and a pharmaceutically accept
`able solid or liquid diluent or carrier therefor, said com
`position being substantially free of (R)~alpha-ethyl-2
`oxo-l-pyrrolidineacetamide.
`2. (S)-a1pha-ethyl-2-oxo-l-pyrrolidineacetamide sub
`stantially
`free
`of
`(R)-alpha-ethyl—2~oxo-l-pyr
`rolidineacetamide, prepared by the process which com
`prises cyclizing, in an inert solvent and in the presence
`of a basic substance, an (S)-2-amino-butanamide of the
`formula
`
`5
`
`X—CH-_)CHZ—Y—NHCl-I(C2l-l5)CONHz
`
`in which
`X represents ZOOC— or HalCl-l2-—, wherein Z is
`alkyl of l to 4 carbon atoms and Hal a halogen
`atom, and
`Y represents —CH1-— or —CO—,
`with the proviso that Y is --—CH;—- when X represents
`ZOOC—, and Y is ——CO—- when X represents HalC
`H2——.
`
`* * * * *
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Page 00006
`
`

`
`Disclaimer
`
`4,837,223 — Jean Goberl, Brussels, Belgium; Jean-Pierre Geens. Leglise, Belgium; Guy Bodson,
`Bellefonlaine, all of Belgium. (S)-ALPHA~ETHYL-2-OXO-l-PYRROLIDINEACETAMIDE COMPOSI
`TIONS. Patent dated June 6, 1989. Disclaimer ?led March 22. 2005, by the assignee. UCB Sociele Annnyme.
`The term of this patent, subsequent 10 the term of patent number 4.696943. has been diselaimed.
`
`(Official Gazette Ma)‘ 17, 2005)
`
`Page 00007