US005439930A United States Patent 19 11 Patent Number: 5,439,930 Seredenin et al. 45. Date of Patent: Aug. 8, 1995

54 BIOLOGICALLY ACTIVE al., pp. 293-301, "Variations in the Turn-Forming N-ACYLPROLYDIPEPTDES HAVING Characteristics of N-Acyl Proline Units’. ANTIAMNESTIC, ANTIHYPOXIC AND European Biophysical Journal, vol. 14, No. 1, (1986) pp. ANOREXIGENCEFFECTS 43-51, M. McHarfi et al., "Backbone Side-Chain Inter 75) Inventors: Sergei B. Seredenin; Tatiana A. actions in Peptides'. Voronina; Tatiana A. Gudasheva; Rita Inorganic Chemistry, vol. 27, No. 22, (Nov. 2, 1988) pp. U. Ostrovskaya; Grigori G. 4046-4052, Y. N. Belokon, “Copper(II) Ion Promoted Rozantsev; Alexander P. Skoldinov; Direct Hydrolysis of 2-Cyanopyridine to Picolinic Sergei S. Trophimov, all of Moscow, Acid, Intramolecular Catalysis by the Coordinated Russian Federation; James A. N-beta-Hydroxyethyl Group'. Halikas, North Oaks, Minn.; Taisija Peptides 1990, Proceedings of the 21. Peptide Sympo L. Garibova, Moscow, Russian sium, Sep. 2-8, (1990), E. Giralt and D. Andreu, 1991 Federation ESCOM Publishers, Leiden, NL, pp. 462-464, G. Pietr zynski et al., "Conformational Propensities of Moel 73) Assignee: Russian-American Institute for New Peptides with Alpha, Beta-Dehydroamino Acids'. Drug Development, Bloomington, Journal of the Chemical Society, Perkin Transaction 1, Minn. (1981) pp. 1639-1646, J. S. Davies and R. J. Thomas, 21 Appl. No.: 960,905 "Studies on the Diastereoisomeric and Conformational Aspects of Benzoyl Dipeptide Esters, as a Means of 22 Filed: Oct. 14, 1992 Assessing Racemisation using Neuclear Magnetic Reso nance Spectroscopy'. Related U.S. Application Data 63 Continuation-in-part of Ser. No. 868,000, Apr. 14, (List continued on next page.) 1992, abandoned. Primary Examiner-Joseph Paul Brust 51) Int. Cl...... A61K 31/40; C07D 207/12 Attorney, Agent, or Firm-Merchant, Gould, Smith, 52 U.S.C...... 514/423: 548/533; Edell, Welter & Schmidt 54.8/537 58) Field of Search ...... 548/533, 537; 514/423 57 ABSTRACT A novel class of substances of N-acyl-prolyldipeptides, 56) References Cited which possess psychotropic activity and particularly U.S. PATENT DOCUMENTS facilitate learning and memory are described. The N 4,093,713 6/1978 Sestanjet al...... 424/177 acyl-prolyldipeptides of the invention have the formula: 4,743,616 5/1988 Tanaka et al...... 514/423 (1) OTHER PUBLICATIONS O R2 O Journal of the Chemical Society, Perkin Transactions 1, No. 11, (Nov. 1990) pp. 3103-3108; B. C. Challis et al. N C-NHCHCCH2)C “Synthesis and Characterisation of Some New N-Ni o=-R R3 trosodipeptides'. wherein R=(C4-Cs) alkyl, cycloalkyl, aralkyl, or aryl; Biopolymers, vol. 28, No. 1, (Jan. 1989) pp. 109-122, A. R2=H (C1-C4) alkyl, carbamidoalkyl, or carbalkoxy Aubry and M. Marraud, "Pseudopeptides and beta alkyl; R3 = NH2, NH(alkyl), N(alkyl)2, OH, or alkoxy; Folding: X-Ray Structures Compared with Structures and n=0-3, preferably 0–2. in Solution'. Biopolymers, vol. 32, No. 3, (Mar. 1992) G. B. Liang et 25 Claims, No Drawings 5,439,930 Page 2

OTHER PUBLICATIONS T. A. Gudasheva et al., Chem. Pharmac. J., 1988, N 3, pp. 271-275. Chemical Abstracts, vol. 111, No. 19, (Nov. 6, 1989), G. B. Liang et al., Chem. Abs., 116, Abstract No. Columbus, Ohio, U.S.; abstract No. 16676, T. A. Guda 116:214897k (1992). sheva et al., "Topological Proline-based Analogs of V. Y. Podlipskii et al., Chem. Abs., 112, 780, Abstract Piracetam and their Nootropic Activity'. No. 112:158881d (1990). L. Amaducci et al., Pharmacopsychiatry, 23, 171-175 V. Y. Podlipskii et al., Chem. Abs., 112, 786, Abstract (1990). No. 112:158955f (1990). A. Aubry et al., Chem. Abs., 87, 614, Abstract No. R. C. Thompson et al., Chem. Abs., 78, 184, Abstract 87:168404e (1977). No.78:107639c (1973). G. Boussard et al., Chem. Abs., 81, 458, Abstract No. T. A. Voronina et al., Ann. Ist. Super. Sanita, 24, N 3, 81:136515b (1974). pp. 461–466 (1988). G. Boussard et al., Chem. Abs., 91, Abstract No. T. A. Voronina et al., Ann. Ist. Super. Sanita, 26, N 1, 91:141207p (1979). pp. 55-60 (1990). T. A. Gudasheva et al., Chem. Pharmac. J., 1985, N 11, T. A. Voronina et al., Drug Development Research, 14, pp. 1322-1329. 353-358 (1988). 5,439,930 1.

BIOLOGICALLY ACTIVE (l) N-ACYTLPROLYDIPEPTOES HAVING O ANTIAMNESTIC, ANTIHYPOXIC AND ( y i. ANOREXIGENCEFFECTS N c-NHCHCH) o=l-R R3 CROSS REFERENCE TO RELATED APPLICATIONS wherein: R=(C4-Cs) alkyl, cycloalkyl, aralkyl, or aryl; The present application is a Continuation-In-Part 10 R2=H1 (C1-C4) alkyl, carbamidoalkyl, or carbalk application of U.S. application Ser. No. 07/868,000, oxyalkyl; filed Apr. 14, 1992, now abandoned. R3=NH2, NH(alkyl), N(alkyl)2, OH, or alkoxy; and BACKGROUND OF THE INVENTION n=0-3, preferably 0-2. In the compound represented by Formula 1, R is Nootropic agents (cognitive enhancers) constitute a 15 preferably selected from the group consisting of isobu promising group of medicines. Known nootropic agents tyl, pentyl, 1-adamantyl, phenyl, phenylmethyl, and include piracetam (N-carbamido-methyl-pyrrolidone phenylpropyl, and more preferably selected from the 2), which was introduced into wide medical practice in the early 1980s. Other N-substituted-2-pyrrollidones group consisting of phenylmethyl and phenyl. R3 is 20 preferably selected from the group consisting of amino (e.g. etiracetan, oxiracetam, aniracetam, pramiracetan, (NH2), methylamino (NHCH3), dimethylamino rolziracetam, etc.) have been synthesized. (N(CH3)2), hydroxy (OH), and ethoxy (OC2H5), and U.S. Pat. No. 4,743,616 to Tanaka et al. describes more preferably selected from the group consisting of N-acyl pyrrolidine compounds having endopeptidase amino and ethoxy. inhibitory activity and which are reported to exhibit 25 Compounds of Formula 1 differ from known noo antiamnestic effects. Unlike the piracetam derivatives, tropic agents in that the compounds of Formula 1 con the compounds described by Tanaka et al. contain a tain in their structure a residue of the natural amino proline group. acid, L-proline (instead of pyrrolidone), together with a Biologically active N-terminal pyroglutamic acid residue of a second natural amino acid. These N-acyl-L- compounds having the formula: 30 proline derived dipeptide compounds have an ex tremely low toxicity and are highly active. Following are compounds illustrative of the scope of this invention: OA N \,C-NHCHCONH2 . 35 H I. N-phenacetyl-L-prolylglycine ethyl ester II. N-phenacetyl-L-prolylglycine amide have been reported by T. Gudasheva and R. Ostrov III. N-phenacetyl-L-prolyl-6-alanine ethyl ester skaya (Chem. Pharmac. J., 1985, N 11, pp. 1322-1329). IV. N-phenylacetyl-L-prolyl-3-alanine amide Another reported (T. A. Gudasheva et al., Chem. Phar V. N-phenylacetyl-L-prolyl-L-aspartic acid diethyl mac. J., 1988, N 3, pp. 271-275) biologically active ester compound is N-acylproline having the formula: VI. N-phenylacetyl-L-prolyl-L-asparagine amide VII. N-benzoyl-L-prolylglycine ethyl ester VIII. N-isovaleryl-L-prolylglycine ethyl ester 45 IX. N-phenylacetyl-L-prolyl-L-valine ethyl ester X. N-benzoyl-L-prolyl-L-valine ethyl ester XI. N-benzoyl-L-prolyl-g-alanine ethyl ester XII. N-benzoyl-L-prolyl-g-alanine amide XIII. N-benzoyl-L-prolylglycine amide 50 XIV. N-phenylacetyl-L-prolylglycine N-methylamide XV. N-phenylacetyl-L-prolylglycine dimethylamide A substantial need exists for highly active and non XVI. N-phenylacetyl-L-prolyl-L-glutamic acid diethyl toxic nootropic agents which can be used for treatment ester of mental decline, caused by different damaging factors. XVII. N-phenylacetyl-L-prolyl-L-leucine amide The present invention is directed to addressing this 55 XVIII. N-phenylacetyl-L-prolylglycine need. XIX. N-phenylacetyl-L-prolyl-GABA methylester XX. N-phenylacetyl-L-prolyl-L-alanine ethyl ester SUMMARY OF THE INVENTION XXI. N-caproyl-L-prolylglycine ethyl ester The present invention relates to a novel group of XXII. N-(1-adamantoyl)-L-prolylglycine ethyl ester compounds, which possess different types of psycho 60 XXIII. N-phenylbutyl-L-prolyl-glycine ethylester tropic activity, particularly antiamnestic, antihypoxic and anorexigenic effects. The invention also relates to a The present invention is also directed to a pharma method for chemical synthesis of such novel con ceutical compositions and methods of medical treat pounds as well as their use as drugs for treatment, espe 65 ment that include as an active substance a pharmaceuti cially of different forms of memory disturbances. cally effective amount of an N-acylprolyldipeptide of The present invention provides N-acyl-prolyldipep Formula 1, as defined above, preferably a compound of tides of the formula: the formula: 5,439,930 3 4 by converting it into a phosphite-amide or by resorting to the “phosphoraso' method. R--L-pro-Gly-R The most common techniques for the above conden O sation reactions are: the carbodiimide method; the azide 5 method; the method of mixed anhydrides; and the wherein R is preferably selected from the group con method of activated esters. These methods are de sisting of iso-butyl, pentyl, 1-adamantyl, phenyl, phe scribed in “The Peptides', Vol. 1, 1965 (Academic nylmethyl, and phenylpropyl; and R3 is preferably se Press), E. Schröder and K. Libke, or in “The Pep lected from the group consisting of NH2, NHCH3, tides', Vol. 1, 1979 (Academic Press), E. Gross and L. N(CH3)2, OH, and OC2H5. More preferably, the phar 10 Meinhofen. maceutical compositions and methods of treatment of The preferable condensation methods of obtaining the present invention include an effective dose of a Formula 1 peptides are the method of mixed anhydrides compound of the formula: or the carbodiimide method. The condensation reaction performed by the method of mixed anhydrides is prefer 15 ably conducted under "Andersonian' conditions G. ch:CH-i-L-pro-Gly-och, W. Anderson et al., J. Am. Chem. Soc., 89, 5012-5017 O (1967)). The carboxyl component (i.e., for the formation of a mixed anhydride of N-acylproline) is preferably Herein, a “pharmaceutically effective amount’ or “ef activated by isobutyl chloroformate. Ethyl chlorofor fective amount' is an amount capable of effectively mate and methyl chloroformate may also be used. treating the syndrome from which the patient suffers. Preferred solvents are a mixture of ethyl acetate and The compounds of the invention possess psycho dimethylformamide, pure dimethylformamide, and tropic activity, including antiamnestic activity, and chloroform. Preferred tertiary bases are N-methylmor improvement of cognitive function damaged by brain pholine, N-ethylmorpholine, and triethylamine. trauma, intoxication, aging, and hypoxia. These com 25 The condensation reaction performed by the carbodi pounds also display anorexigenic effect, antialcoholic imide method is preferably conducted in the presence of activity, and cause diminished mental decline in prena oxybenzotriazole W. Konig and R. Geiger, Chem. Ber, tally alcoholized offsprings. 103,788-798 (1979)). Condensation by the carbodiimide The present invention is also directed to various method may also be conducted in the presence of other methods of treating human suffering by administrating 30 additives, such as para-nitrophenol, pentachlorphenol, an effective dose of the compounds of the invention. or N-oxysuccinimide. Such methods include treating human suffering from The reactive groups that are not supposed to be in mental decline caused by brain trauma, aging, senile volved in the condensation reaction can be protected by dementia, and mentally retarded children; obesity; groups that can be easily removed through hydrolysis chemical toxicity CNS effects, preferably caused by 35 or reduction, for example. Thus, a carboxyl group may lead poisoning; sickle cell anemia; benzodiazepine with be protected by esterification with ethanol, methanol, tertiary butanol, benzyl alcohol. The groups that usu drawal syndrome, which is manifested by aggression, ally effectively protect an amino group are acid groups, anxiety, and seizures; and alcohol withdrawal. for example: acid group from aliphatic, aromatic, heter DETAILED DESCRIPTION OF THE ocyclic carboxylic acid, such as acetyl, benzoyl, pyri INVENTION dine carboxyl; the acid group from carbonic acid, such Compounds according to Formula 1 above were as ethoxycarbonyl, benzyloxycarbonyl, tetra-butylox obtained by the well-known methods of peptide synthe ycarbonyl group; or the acid group derived from sulfo sis. The ordinary process for producing the compounds acid, such as the para-toluenesulfonyl acid group. 45 Functional peptide derivatives of Formula 1 mean: under consideration consists of combining the required (1) N-acyl derivatives from aliphatic or aromatic acids; amino acids by condensation, as a rule, in the homoge (2) esters derived from low-alkyl alcohols; and (3) am nous phase. ides or monoalkyl- or dialkyl-substituted amides, in Condensation in the homogenous phase may be per which the alkyl groups have one or two C-atoms. formed in the following way: 50 During the synthesis of the claimed prolyl dipeptides, a) condensation of an amino acid having a free car N-acyl derivatives were preferably obtained through boxyl group and another protected reactive group, the use of proline, preliminarily acylated by a suitable with an amino acid having a free amino group and acyl group. This acyl group also functioned as a protec other protected reactive groups in the presence of tive group in the course of further synthesis. It is also a condensing agent, such as carbodiimide; 55 possible to insert the desired amine group after the pep b) condensation of an amino acid having an activated tide synthesis by acylating the dipeptide by routine carboxyl group and another protected reactive methods. The preferred N-acyl groups are N-phenyl group, with an amino acid that has a free amino acetyl and N-benzoyl. group and other protected reactive groups; Peptide esters according to Formula 1 are preferably c) condensation of an amino acid having a free car obtained by the use of an amino acid in the form of the boxyl group and another protected reactive group, desired ester. They may also be obtained by suitable with an amino acid having an activated amino esterification of the obtained peptide. Preferably, the group and other protected reactive groups. esters are derived from methanol or ethanol. Activation of the carboxyl group may be performed Amides of the peptides according to Formula 1 are by converting it into a chloranhydride, azide, anhydride obtained by ammonolysis (i.e., reaction with NH3) of group or activated ester, such as N-oxysuccinimide, the alkyl ester of a corresponding dipeptide, or by using N-oxybenztriazole, penthachlorophenylicorpara in the reaction an amino acid in the form of the desired nitrophenylic ester. The amino group may be activated amide. Amides of dipeptides may also be obtained by 5,439,930 5 6 inserting an amide group into the corresponding dipep tide by some suitable method, for example, treatment EXAMPLES with an amine in the presence of a condensing agent. The invention is further illustrated by the following The preferable amides are nonsubstituted amides, specific examples. monomethyl-amides, and dimethyl-amides. The following abbreviations are used: The compounds of the present invention were as sessed for their ability to prevent memory impairment Pro-prolyl induced by maximal electroshock, or scopolamine ad Asp-aspartyl ministration. Unlike N-acylpyrrolidine compounds of ASn-asparaginyl the type described by Tanaka et al., supra, the com 10 Val-valy pounds of the present invention are not believed to Ala-alanyl inhibit prolyl endopeptidase activity. As is described in y-Ala-y-alanyl the Examples, the tested compounds exhibit antiamnes Leu-leucyl tic activity (Example 12). A preferred compound hav Gly-glycyl ing Formula I (see Table 1) was demonstrated to be able 15 Glu-glutamyl to facilitate different phases of memory formation: ac EtOH-ethanol quisition of the information, retention and retrieval in MeOH-methanol various procedures of passive and active avoidance DMF-dimethylformamide (Examples 12, 13). This compound was shown to be DCC-dicyclohexylcarbodimide able to increase the degree of acute habituation (Exam 20 DCU-dicyclohexylurea ple 14). The Compound I prevents mental decline, in EtAC-ethyl acetate duced by frontal lobectomy (Example 15), prenatal BZ-benzoyl alcoholization or prenatal hypoxia (Example 16) and Ad-1-adamantyl aging (Example 17). This substance exhibited an OHBt-1-oxibenztriazole tihypoxic activity (Example 18). 25 Et3N-triethylamine While piracetam was observed to exert some of the Phac-phenylacetyl above-described activities, the level of active dose for TLC-thin layer chromatography piracetam is 200-800 mg/kg. In contrast to this, sub stances in accordance with the present invention exert In examples the following apparatus were used: melt their effects in a dose between about 0.1-0.5 mg/kg. 30 ing points were determined on a sulfur acid apparatus in Compound I described herein has an additional useful the open capillars and were not corrected. Specific property: anorexigenic effect (Example 19). In contrast optical rotations were recorded on automatic polarine to known anorexigenic drugs, Compound I fails to ter Perkin-Elmer-241. Nuclear magnetic resonance evoke the adrenergic stimulation (excitation, increase of spectra were obtained on AC-250 Bruker spectrometer. blood pressure, etc.). Compound I is also nontoxic (Ex 35 ample 20), and active when administered perorally (Ex Chemical shifts are expressed ppm downfield from ample 12). Compound I was shown to be able to dimin Me4Si. For the resonance signals the following abbrevi ish the syndrome of benzodiazepine withdrawal dimin ations are used: s-singlet, d-doublet, t-triplet, q-quartet, ishing the anxiety, aggressiveness and pentyllentetrazole m-multiplet. Spin-spin coupling constants are given in induced kindling (Example 21). Hz. Thin layer chromatography (TLC) was performed Substances according to the present invention can be on Merk silica gel 60F254, plates and spots were devel used in any form which is suitable for oral administra oped in an iodine chamber or in UF light. tion, such as pills, tablets and dragee. The presently In vivo experiments employed randomly bred male described substances can be administered parenterally, albino rats of approximately 180-220 g and/or ran by injection or infusion in the form of pharmaceutical 45 domly bred male albino mice of about 18-22 g. In vivo preparations composing the active ingredient in combi alcohol tests employed offspring of alcoholized mothers nation with a pharmaceutically accepted carrier. The of similar type. Wister strain male albino rats at 24-26 substances of the present invention can be used for the months were used in the experiments with aging. treatment of human mental decline, induced by brain EXAMPLE 1. trauma, intoxications, age-related processes, Korsakoff 50 Synthesis of Ethyl Ester of syndrome, Alzheimers disease, organic brain syndrome, N-phenylacetyl-L-prolyl-glycine alcoholism including prenatal alcohol damage, hypoxia, N-Phac-L-Pro-Gly-OEt (I) mental retardation of children, obesity arteriosclerotic cerebrovascular disease, brain damage due to congeni a) N-Phenylacetyl-L-proline, N-Phac-L-Pro-OH tal malformations or genetic abnormalities, chemical 55 To 5.75 g (0.05 mole) L-proline in 25 ml 2N NaOH toxicity CNS effects including lead poisoning, drug were added dropwise under stirring at a temperature abuse treatment including withdrawal and abstinence below 10° C., 12.5 mi 4NNaOH and 6.6 ml (0.05 mole) maintenance, and certain hematologic disorders includ N-phenylacetyl chloride (b.p. 89-90 C/10 Torr). ing sickle cell anemia. Preferably, the substances can be Reaction mixture was stirred 15min, extracted by EtAc used in doses of 0.5-5.0 mg per day. to remove chloride then it was acidified by 2N HCl to The invention has been described with reference to pH-3, extracted by chloroform, dried over sodium various specific and preferred embodiments and will be sulfate and evaporated. 6.2 g N-Phac-L-Pro-OH were further described by reference to the following detailed obtained as white crystals, m.p. 150-152 C., examples. It is understood, however, that there are (a) D20-60.5 (c. 0.4, DMF), Rf 0.66 (Kiesel-gel, diox many extensions, variations, and modifications on the 65 ane-water 9:1). basic theme of the present invention beyond that shown NMR-spectrum in CDCl3 8 (ppm): 1.77-2.29 (m, in the examples and detailed description, which are CBH2-CyH2 Pro, 4H); 3.40-3.63 (m, C6H2, Pro 2H); within the spirit and scope of the present invention. 3.63 and 3.73 (each s, CH2-C6H5, 2H); 4.56 and 4.38 5,439,930 7 8 (each dd, CaH Pro, 1H); 7.18-7.39 (m, CH2C6H5, 5H); and the solvent evaporated on a rotary evaporator. The 11.38 (broad s, COOH, 1H). Analysis Calcd. for remainder was dissolved in CHCl3 and washed with 5% C131sNO3: C, 66.93; H, 6.49; N, 6.00. Found: C, 66.65; solution of NaHCO3, water, 1N HCl, water, and then H, 6.40; N, 5.86. b) Ethyl ester of N-phenylacetyl-L-prolyl-glycine. dried over sodium sulfate and evaporated. The yield To solution of 2.33 g (0.01 mole) N-Phac-L-Pro-OH was 0.58 g or (98%) N-Phac-L-Pro-g-Ala-OEt, which (m.p. 150-151 C) in 50 ml DMF were added under was obtained as a transparent oil. Rf 0.52 (Kiesel-gel, stirring at -10° C., 1.39 ml (0.01 mole) Et3N and then CHCl3-MeOH, 9:1); Rf 0.53 (Kiesel-gel, dioxane 1.34 ml (0.01 mole) isobutylchloroforniate. In 2 minutes water, 9:1); ap2092.25 (c. 0.3, CHCl3). to reaction mixture were added dropwise for 25 min 10 H-NMR-spectrum in (CD3)2SO, 6 (ppm): 1.17 (t, utes, 1.4 g (0.01 mole) hydrochloride of glycine ethyl CH3CH2-O, 68% 3H); 1.13 (t, CH3CH2O, 32% 3H); ester (m.p. 140°-142 C.) and solution 1.39 ml (0.01 1.7-2.2 (m, C6H2-CyH2, Pro, 4H); 2.42 (m, Co-H2(3- mole) Et3N in 25 ml DMF to avoid temperature rise. Ala, 2H); 3.2-3.3 (m, C6H2, Pro, 2H); ~3.40 (s, Then stirring was prolongated to 30 min. at cooling and CH2C6Hs, 32% 2H) under HDO-Signal; 3.66 (m, 1.5 hours at room temperature. The precipitate was 15 CH2C6Hs, 68% 2H); 4.01 (q, CH3-CH2O, 32% 2H); filtered and the filtrate evaporated in vacuo, the residue 4.04 (q, CH3-CH2O, 68% 2H); 4.41 (m, C3H2E-Ala, was dissolved in CHCl3, solution was washed by 5% 2H); 4.21 (dd, CH, Pro, 1H); 7.1-7.36 (m, CH2-C6H5, NaHCO3, water, 1N HCl and again water, dried over 5H); 7.93 (t, NH, 1H). Analysis Calcd for C18H24N2O4: sodium sulfate and evaporated. A resulting 1.66 g (54% C, 65.03; H, 7.29; N, 8.42. Found: C, 65.37; H, 7.77; N, ) of oil was blended with ether into white crystals, m.p. 8.38. 96-97° C.; alp20-120° C. (c. 0.4, CHCl3). Rf 0.80 20 (Kiesel-gel, dioxane-water, 9:1). H-NMR spectrum in EXAMPLE 4 (CD3)2SO, 6 (ppm): 1.18 (t, CH3CH2O, 55% 5H); 1.17 Synthesis of amide of (t, CH3CH2O, 45% 3H); 165-2.35 (m, C8H2-CH2 N-phenylacethyl-L-prolyl-g-alanine Pro, 4H); 3.2-3.4 (m, C8H2, Pro, 2H); 3.40 (s, 25 N-Phac-L-Pro-g-Ala-NH2 (IV) CH2-C6H5, 45% H); 3.67 (s, CH2-C6Hs, 55% 2H); 3.80 (d, CaH2 Gly, J =5.9, 55% 2H); 3.86 (d, J = 5.9, Gaseous ammonia was bubbled to saturation through CaH2Oly, 45% 2H); 4.08 (q, CH3-CH2-O, 55% 2H); an ethanol solution (15 ml ) of 0.36 g ethyl ester of 4.09 (q, CH3-CH2-O, 45% 2H); 4.32 (dd, CoH, Pro, N-phenylacetyl-L-prolyl-g-alanine, obtained as in Ex 55%. 1H); 4.48 (dd, CoH, Pro, 45% 1H); 7.1-7.6 (m, ample 3. After standing overnight the solvent was evap CH2C6Hs, 5H); 8.29 (t, J=5.9, NH Gly, 55% 1H); 8.63 orated and the residue was purified by column chroma (t, J=5.9, NHGly, 45% 1H). Analysis Calcd. for tography (silica gel), using CHCl3 as eluent. The amide C17H22N2O4: C, 64.15; H, 6.92; N, 8.80. Found: C, IV was obtained as an oil, yield 0.22 g (61%). Rf 0.28 63.93; H, 6.81, N, 9.07. (Kiesel-gel, CHCl3-methanol, 9:1); alp20-22.8 (c 0.33; CHCl3). EXAMPLE 2 35 H-NMR (Me2SO-dg), 6 (ppm): 1.69-2.2 (m, Synthesis of N-phenylacethyl-L-prolyl-glycin-amide C6H2-CyH2 Pro, 4H); 2.16-2.31 (m, CaH26-Ala, 2H); Phac-L-Pro-Gly-NH2 (II) 3.1-3.3 (m, CPH2, 6-Ala., 2H); 3.3-3.45 (m, C6H2, Pro, N-Phac-L-Pro-Gly-OEt (0.53 g, m.p. 96-97 C., 2H); 3.66 (s, CH2-C6H5, 2H); 4.22 and 4.41 (every dd, a)D20-122.0 in CHCl3, see Example 1b) was dissolved CoH Pro, 1H ); 6.84, 7.36 and 6.86, 7.38 (each br. s, in 25 ml of saturated NH3 ethanol. The reaction mixture NH2, 2H); 7.12-7.35 (m, CH2C6Hs, 5H); 7.89 and 8.22 was maintained for 12-16 hours at room temperature. (each t, NH 6-Ala, 1H). Analysis Calcd. for Ethanol was then evaporated and the residue was C16H21N3O3: C, 63.34; H, 6.99; N, 13.85. Found: C, washed with ether. The yield of amide II was 0.5 g, 63.81; H, 7.03; N, 14.01. which was obtained as an oil. Rf 0.36 (Kiesel-gel, 45 CHCl3-MeOH, 9:1). ap2058.5 (c, 0.2, CHCl3). H EXAMPLE 5 NMR spectrum in CDCl3, 6 (ppm): 1.8-2.3 (m, Synthesis of diethyl ester of C6H2-CyH2, Pro, 4H); 3.3-3.5 (m, C8H2, Pro, 2H); N-phenylacetyl-L-prolyl-L-asparaginic acid, 3.55-3.75 (m, AB part ABX system, CH2 Gly, 2H); N-Phac-L-Pro-L-Asp (OEt)2 (V) 3.66 (s, CH2-C6H5, 2H); 4.07 (dd, CoH, Pro, 1H); 4.37 50 (t, NHGly, 1H); 5.63 and 7.86 (every s, NH2); 7.2-7.4 To a well-stirred solution of N-phenylacetyl-L-pro (m, C6H5, 5H). Analysis Calcd for C15H19N3O3: C, line (1 g; 4.3 mmol) in absolute EtAc (40 ml), N-methyl 62.53; H, 6.48; N, 14.61. Found: C, 62.28; H, 6.54; N, morpholine (0.48 ml; 4.3 mmol) was added at -10° C. 14.53. Following this addition, iso-BuOC(O)Cl (0.57 ml; 4.3 55 mmol) was added, and after 2-3 minutes a mixture of EXAMPLE 3 L-Asp (OEt)2.HCl (0.91 g; 4.3 mmol), N-methylmor Synthesis of ethylester of N-phenyl-L-prolyl-g-alanine pholine (0.48 ml; 4.3 mmol), and EtAc (15 ml) were N-Phac-L-Pro-g-Ala-OEt (III) added. Stirring was continued for 1 hour at -10° C. To solution of 0.61 g (2.6 mmol) N-phenylacetyl-L- After the mixture was allowed to stand for 1 hour, the proline (m.p. 151 C., ap20-60.5 in DMF) in 25 ml 60 precipitate was separated by filtration, the solvent was CHCl3 at -10° C., 0.29 ml (2.6 mmol) N-methylmor evaporated, and the residue was dissolved in a mixture pholine was added followed by 0.35 ml (2.6 mmol) of ethanol and ether. The resulting precipitate was sepa isobutylchloroformate. At 2 minutes, 0.4 g (2.6 mmol) rated, mother liquor was evaporated, and the residue hydrochloride of 6-alanine ethyl ester (m.p. 69-71 C.) (1.65 g) was purified by column chromatography (Silica and 0.29 ml N-methylmorpholine in 4 ml CHCl3 were 65 gel), using CHCl3 and the mixture CHCl3-EtOH, as added to the reaction mixture solution. Stirring was eluents. The diethyl ester of N-phenylacetyl-L-prolyl prolonged under cooling for 40 minutes and then 2 L-asparaginic acid was obtained as an oil; Rf 0.87 (Kie hours at room temperature. The residue was filtered sel-gel, CHCl3-EtOH, 9:3) ap20-38.0 (C 2.2; CHCl3). 5,439,930 9 10 1H-NMR (Me2SO-d6), 8 (ppm): 1.23 (t, J =7.16 Hz, cooling and for 1.5 hours at room temperature the sol CH3CH2O, 90%3H); 1.24 (t, J=7.16, CH3CH2O, 10% vent was removed in vacuo. The residue was dissolved 3H ); 4.10 (q, CH3CH2-O, 90% 2H); 4.12 (q, in chloroform, washed with 5% aq. NaHCO3, 1N aq. CH3CH-O, 10% 2H); 1.25 (t, J=7.14, CH3CH2O, 90 HCl, H2O and then dried with Na2SO4. After filtration %3H); 1.26 (t, J=7.14, CH3CH2O, 10%3H); 4.19 (q, the solvent was evaporated. To the residue ether was CH3CH2O, 90%. 2H); 4.21 (q, CH3CH2O, 10% 2H); added, and the resulting crystals of ester VII were sepa 1.75–2.40 (m, C6H2-CH2, Pro, 4H); 3.45-3.65 (m, rated. Yield 1.67 g (76%), m.p. 63-65 C., ap20148.0 C6C2 Pro, 2H); 4.58 (dd, J-8.00; J-2.59; CaH Pro, (c 04; CHCl3); Rf 0.71 (Kiesel-gel, CHCl3-CH3OH, 1H); 2.78; 2.95 (dd, AB-part of ABX-system, 9:1). JAB = 17.04; JAY=4.88; JBx=4.88; C3H2 Asp, 90% 2H); O 2.80; 3.00 (dd, C6H2 Asp, 10% 2H); 4.81 (dt, J = Co-H 1H-NMR (Me2SO-d6) (ppm): 1.18 (t, J =7.04, NH 8.50; Ca H Asp 1H); 7.5 (d, J = 8.50; NH Asp, 90% CH3CH2O, 75%3H); 1.09 (t, J=704, CH3CH2O, 25% 1H) 7.03 (d, J = 8.40; NHAsp, 10% 1H); 3.70 (s, 3H); 1.73-2.28 (m, CRH2-CH2 Pro, 4H); 3.3-3.4 (m, CH2C6H5, 2H); 7.20-7.36 (m, C6Hs, 5H). Analysis CyH2 Pro, 2H); 3.60 and 3.74 (dd, J = 5.87 and J = 8.22 Calcd. for C2H28N2O6: C, 62.35; H, 6.99; N, 6.92. 15 CaH2 Gly, 25% 2H); 3.85 (d, J =5.87, CaH2Oly, 75% Found: C, 62.63; H, 7.01; N, 6.74. 2H); 4.10 (q, CH3CH2O), 75% 2H); 4.13 (q, CH3CH2O, 25% 2FH); 4.47 (dd, CaH Pro, 25% 1H); 4.48 (dd, Cah EXAMPLE 6 Pro, 75% 1H); 7.33-7.62 (m, C6Hs 5H); 8.36 (t, J = 5.87; Synthesis of amide of NH, 75% 1H); 8.40 (t, J = 5.87; NH, 25% 1H). Analysis N-phenylacetyl-L-prolyl-L-asparagine, 20 Calcd. for C16H2ON2O4: C, 63.14; H, 6.61; N, 9.20. N-Phac-L-Pro-L-Asn-NH2 (VI) Found: C, 63.56; H, 6.87; N, 9.48. Gaseous ammonia was bubbled to saturation through EXAMPLE 8 a methanol solution (25 ml) of 0.5 g N-Phac-L-Pro-L- Asp (OEt)2, obtained as in Example 5, at 0° C. After Synthesis of ethyl ester of standing overnight the solvent was evaporated, the 25 N-iso-valeryl-L-prolylglycine, residue was dissolved in the warm mixture of ethanol iso-C4H9C(O)-Pro-Gly-OEt (VIII) and chloroform, and pentane was added to precipita a) N-iso-valeryl-L-proline tion. The resulting precipitate was collected and dried To a well stirred solution of L-proline (1.15 g; 0.01 to obtain the amide of N-phenylacetyl-L-prolyl-L- mole) in 2N aq. NaOH (5 ml), 4N aq. NaOH (2.5 ml) asparagine (0.45 g): m.p. 170-172. C., Rf 0.24 (Kiesel 30 and iso-valeryl chloride (1.4 ml; 0.012 mole) were added gel, CHCl3-EtOH, 9:3), ad20-55.7 (C14; DMSO). simultaneously dropwise maintaining the temperature 1H-NMR (Me2SO-d6) (ppm): 1.60-2.30 (m, near 0-4 C. After 15 minutes, the mixture was acidi C3H2-CyH2 Pro, 4H); 2.35-2.50 (m, C6H2 Asn, 2H) fied (pH 2-3) with 1N HCl and extracted with CHCL3. under signal of solvent; 3.63 (s, CH2-C6H5, 2H); 4.36 The combined organic extract was dried using MgSO4, (m, CoH Pro, 1H); 44-46 (m, CoHAsn, 1H); 6.80-7.60 35 and the solvent was removed. N-iso-valeryl-L-proline (m, C6H5, 5H); 6.8-7.1 (s, NH2, 4H); 8.15; 8.25; 8.35 was obtained as an oil. Yield 0.85g, Rfs=0.66 (Silica gel, (each d, NH. Asn., 1H). Analysis Calcd. for n-C4H90H-AcOH-H2O, 5:1:2); ap20-129.3 (c. 0.6; C17H22N4O4: C, 58.94; H, 6.41; N, 16.16. Found: C, CHCl3). 59.23; H, 6.66; N, 16.04. H-NMR CDCl3, 6 (ppm): 0.99 (d, -CH(CH3)2, 6H); EXAMPLE 7 2.25 (m, CHCCH3)2, 1H); 2.3 (m, CH2CHCCH3)2, 2H); Synthesis of ethyl ester of N-benzoyl-L-prolylglycine, 1.9-2.65 (m, CaH2-CyH2,4H); 3.45-3.70 (m, C8H2Pro, N-BZ-L-Pro-Gly-OEt (VII) 2H); 8.85 (br. s, COOH, 1H). Analysis Calcd. for C10H17NO3: C, 60.27; H, 8.62; N, 7.02. Found: C, 60.34; a) N-benzoyl-L-proline H, 8.74; N, 7.23. To a well-stirred solution of L-proline (5.75 g, 0.05 45 b) Ethyl ester of N-iso-Valeryl-L-prolylglycine mmole) in 2N NaOH (25 ml), 4N NaOH (12.5 ml) and To a well stirred solution of N-iso-valeryl-L-proline benzoyl chloride (5.8 ml, 0.05 mole) were added drop wise from different drop funnels, maintaining the tem (0.74 g; 0.0037 mole) in abs. EtAc (15 ml), N-ethylmor perature near 0-4°C. The mixture after 15 minutes was pholine (0.47 ml; 0.0037 mole) and iso-BuOC(O)Cl (0.5 acidified with 1N HCl. The resulting oil was extracted 50 ml; 0.0037 mole ) were added. After 2-3 minutes, a with CHCl3, the combined organic extract was dried mixture of NH2CH2COOEt.HCl (0.52 g; 0.0037 mole), (MgSO4), and the solvent was removed. Ether was N-ethylmorpholine (0.47 ml; 0.0037 mole), and DMF added to the residue and the mixture was allowed to (10 ml) was added dropwise, while maintaining the stand overnight at 0° C. The crystals of N-benzoyl-L- temperature at -10° C. to -5° C. Stirring was contin proline were separated. Yield 3.42 g (60%), m.p. 55 ued for 30 minutes under cooling and 1.5 hours at room 152-154 C., ap20-68.5' (c O.4; CHCl3); R0.13 (Kie temperature. The precipitate was separated by filtra sel-gel, CHCl3-CH3OH, 9:1). Analysis Calcd. for tion, and the mother liquor was evaporated. The residue C12H13NO3: C, 65.73; H, 5.99; N, 6.39. Found: C, 65.64; was dissolved in chloroform, washed with 5% aq. NaH H, 6.03; N, 6.54. CO3, H2O, 1N aq. HCl, H2O and then dried with b) Ethyl ester of N-benzoyl-L-prolylglycine 60 MgSO4. After filtration the solvent was evaporated and To a well stirred solution of N-benzoyl-L-proline the residue (0.6 g) was purified by column chromatog (2.19 g, 0.01 mole) in the mixture of absolute EtAc (50 raphy (Silica gel), using CHCl3 as eluent. Yield VIII as ml) and DMF (10 ml), N-methylmorpholine (1.12 ml; viscous colorless liquid was 0.49 g (49%); RO.55 (Silica 0.01 mole) and iso-BuOC(O)Cl. (1.34 ml; 0.01 mole) gel, CHCl3-CH3OH, 9:1), ald20-113.4 (c 0 3; were added dropwise at -10° C. After 2 minutes a 65 CHCl3). mixture of NH2CH2COOEt.HCl (1.4 g; 0.01 mole), 1H-NMR (CDCl3), 6 (ppm): 0.99 (d, J =5.97, N-methylmorpholine (1.12 ml; 0.01 mole), and DMF CH(CH3)2, 6H); 2.22 (m, CHMe2, 1H); 1.27 (t, J =7.16; (20 ml) was added. After stirring for 30 minutes under OCH2CH3, 3H); 4.18 (q, OCH2CH3, 2H); 1.75-2.60 (m,

5,439,930 15 16 TABLE 1. Antiamnestic activity of drugs (dose of 0.1 mg/kg) checked in passive avoidance paradigm with maximal electroshock Substance Latency (sec.) Amnesia -- A.A. No. Rl R2 R3 Control Amnesia subst. 9% I. CH2C6H5 H OC2H5 O 45 4 25 36: II. CH2C6H5 H NH2 O 153 19 56 27s III. CH2C6Hs H OC2H5 13 25 56 29* IV. CH2C6H5 H NH2 98 26 64 52 V. CH2C6H5 CH2COOC2H5 OC2H5 O 173 41 80 29.5-k VI. CH2C6H5 CH2CONH2 NH2 O 133 28 55 26 W. C6H5 H OC2Hs O 95 3 37 37+ VIII. iso-C4H9 H OC2H5 O 38 78 95 28 IX. CH2C6H5 CHCCH3)2 OC2H5 O 133 39.6 39.7 0.0001 X. C6H5 CH(CH3)2 OC2H5 O 133 39.6 75.4 38 XI. C6H5 H OC2H5 102.7 51.6 62.8 2.9 XI. C6H5 H NH2 102.7 51.6 59.4 15.2 XII. C6H5 H NH2 O 102.7 51.6 42.6 - 17.6 XIV. CH2C6H5 H NHCH3 O 102.7 51.6 47.7 -7.6 XV. CH2C6H5 H N(CH3)2 O 1.0 19 4.0 23.0 XVI. CH2C6H5 (CH2)2COOC2H5 OC2H5 O 11.0 19 72.8 58.3% XVII. CH2C6H5 CH2CH(CH3)2 NH O 38.9 60 66.3 7.9 XVIII CH2C6Hs H OH O 102.7 56 56.6 9.8 XX. CH2C6Hs H OCH3 2 138.9 60 95.8 45.4 XX. CH2C6H5 CH3 OC2H5 O 138.9 60 68.6 0.8 XXI. (CH2)4CH3 H OC2H5 O 33.9 60 76.1 20.2 XII. C10H15(Ad) H OC2H5 O 1.0 19 31.6 3.1 XXIII. (CH2)3CHs H OC2H5 O 1.0 19 31.0 12.6 Piracetan, 26.8 12 47.7 30.5% 200 mg/kg AA - antiamnestic activity estimated according to the Butler's modified formula. *P < 0.05; "'P ( 0.01 in comparison with control.

TABLE 2 min. before trial, immediately after it or 15 min. before Influence of substances on the amnestic effect the retrieval test (Table 4). of scopolamine (1 mg/kg subcutaneously 30 min. TABLE 4 before the trial) in passive avoidance test. Comparative activity. of Compound I and piracetam Latency (sec.) in passive avoidance with different 0.9% Scopolamine -- 35 regimes of administration. Substances Saline Scopolamine substance Aa: Mnestic activity (Ma, 96%) I 86.6 36 64.1 55.5 Administration Administration Administration II 1614 55.2 45.6 -9.0 Substance 15 in. before immediately 15 min. before III 163.9 52.1 78.6 23.7 (mg/kg) trial after trial retrieval IV 138.9 47.1 94.9 52.1 V 158.9 58.3 48. -10 Compound I 23. 140 32 VI 148 50.5 46.3 - 4.3 0.5, i.p. Piracetan 118 28.9 64.2 39.3 Piracetam 20.0 1.0 - 15% (200 mg/kg) 200, i.p. Substances were administered in the dose of 0.1 mg/kg 15 nin. before the learning *P < 0.05; "Aa is calculated according to the formula: 'P < 0.01 in comparison with control animals. Latency subst. - Latency Scopolanine Ma was calculated according to the formula: x 100% 45 Latency drug. - Latency control x 100% Latency saline - Latency Scopolamine 180 seconds - Latency control Taking into consideration excellent antiamnestic ac Compound I may facilitate all main phases of mem tivity of Compound I in both kinds of amnesia, this ory formation: input of information, consolidation and compound was chosen for further study. retrieval. Piracetam did not facilitate the retrieval in It was shown that effectiveness of this substance is 50 these experiments. preserved after its peroral administration; converted U-shape dose-response relationship was demonstrated EXAMPLE 13 in the experiments with maximal electroshock amnesia The influence of Compound I on the active avoid (Table 3). ance learning was studied in shuttle-box test (Ugo TABLE 3 Dose-response relationship for Compound I revealed in passive avoidance test with MES amnesia. Dose (mg/kg) per 0.1 0.3 0.4 0.5 0.7 0.9 10 1.2 1.5 os compound I) Antiamnestic 19 1983: 23° 43 52: 36. 10s 16 10 activity (Aa) P < 0.05; P C 0.01 in comparison with control animals. Aa - antiamnestic activity in %. The positive amnestic effect of Compound I was 65 revealed also in the situation of undertraining of the Basile, Italy). The conditioned reflex of active avoid passive avoidance reflex. The facilitating effect of com ance had been developed for 5 days with 50 daily trials pound I was shown in the case of its administration 15 for each rat. The learning task consisted in the rats' 5,439,930 17 18 avoiding of the electric shock applied through the cage days. In the control group (false operation) the passive floor directly after sound. Rats could avoid the shock and active avoidance learning did not change. The fron by moving into another compartment of the cage, but tal lobectomy has as a consequence the decrease of the only while the sound was present (3 sec.). Compound I latency of the entering of the dark compartment; Com (0.1 mg/kg daily i.p.), administered 14 days before the 5 pound I restored the passive avoidance reaction (Table learning and every day during the learning, was demon 8). strated to be able to facilitate the learning ability (Table Lobectomized rats showed the whole absence of the 5). active avoidance reflex at the 4th day and weak ten dency to its restoration at 9th day. Compound I in TABLE 5 10 The influence of Compound I on the learning ability creased the percentage of the animals performing the estimated in shuttle-box test. active avoidance reaction and the coefficient of the Percentage of the rats reached preserving (Table 8). Group the stable avoidance TABLE 8 Days of learning 1 - 2 3 4. 5 Control O 0 0 29 7 15 The influence of Compound I on mnestic Substance I O 15 43 57+ 67 functions in the lobectomized rats P < 0.05 in comparison to control. FRONTAL LOBECTOMY 4th day 9th day In other experiments, stable active avoidance reac False Con- Comp Con- Comp. tion was damaged by simultaneously switching of sound 20 Groups Operation trol I trol I and electrical footshock for 5 trials. Compound I pre Passive avoidance 80 51.9 1412 vented the impairment of avoidance (Table 6). test (latency, sec) TABLE 6 Active avoidance 7.8 O 28.1% 9 38.4 test - coeff. of The influence of Compound I on the conditional active 25 preserving, 7%%** avoidance reflex after damage. %% of avoiding rats 100 O 42.8 16.6 57. Percentage of avoidances during 5 trials "P < 0.05 compared with control; Before 1-5th After damage "this coefficient was calculated according to the formula: Group damage trials 6-10th trials 11-15th trials Control 85.0 - 6.3 60.0 - 82.5 - 10.3 85.0 - 7.3 N - N4 N - N9 11.3 Coeff= - N'- x 100% or N x 100% Compound I 88.9 - 4.8 86.7 - 88.9 it 4.8 97.8 - 2.2 where "N" means the amount of the trials, sufficient for the stable reflex before the 3.3tt operation (Ni), 4th (N), and 9th (N3) days, respectively. "P < 0.01 in comparison with the amount of the avoidances before damage "P < 0.01 in comparison with control 35 EXAMPLE 6 EXAMPLE 1.4 The cognitive impairment in the rat's offspring was The effect of Compound I on acute habituation of locomotor activity was estimated according to diminu provoked by prenatal alcoholization or prenatal hyp tion of locomotions during 30 minutes. Experiments oxia. Prenatal alcoholization was achieved by peroral were performed on outbred albino male mice (18-22 g) administration of 5 g/kg/day (25% solution) ethanol to placed in groups of 10 animals in registration cage (Op pregnant female rats during whole pregnancy. To tovarimex, Colomb., USA). achieve prenatal hypoxia the rats on the 15th day of Compound I, administered 15 minutes before the pregnancy were placed into hypobaric barometric beginning of registration in the dose range 0.05-5.0 45 chamber (the "elevating" up 8500 m altitude; exposition mg/kg was demonstrated to increase the degree of for 2 hours). habituation ("negative learning') without changing of The treatment of the offspring was performed from initial locomotor activity (Table 7). 8th day to 20th day. Compound I was administered in TABLE 7 the dose of 0.1 mg/kg/day subcutaneously. The testing Effect of Compound I on habituation (extinction 50 was performed on two-month old rats. of exploratory behavior reaction) It has been established that both kinds of damage Habituation index (alcoholization, hypoxia) caused learning disorders in Dose, mg/kg 0.05 0.1 0.5 2.5 5.0 active avoidance test. Early postnatal administration of Compound I 30 On 15ss 25& 27t Compound I restored learning activity (Table 9). Control 55 53 67 89 63 55 TABLE 9 (saline) ^ Habituation index - ratio of motor activity during last 5 minutes of recording to Influence of Compound I on rat's offspring learning, motor activity in the first 5 minutes of recording, in %%. damaged by prenatal alcoholization or tP < 0.05 prenatal hypoxia •P < 0.01 Rats with reflex of active avoidance in %% 60 Days of EXAMPLE 15 - Days of learning - retention The impairment of learning and memory was Group 2 3 4 5 1 2 Control 0. 9 50 73 86 68 79 achieved also by frontal lobectomy, which was per Prenata O O 33 50 25 M 65 formed after passive avoidance or active avoidance 65 alcoholization learning. Compound I (0.1 mg/kg) was administered Prenata O 0 55 82 78t 89 beginning from the first post-operational day and lasting alcoholization -- for 9 days. The testing was performed at 4th and 9th Compound I Prenata O O O 3 54 hypoxia Prenatal - O 0 25 688 88ss hypoxia 5,439,930 19 20 TABLE 9-continued EXAMPLE 1.8 Influence of Compound I on rat's offspring learning, damaged by prenatal alcoholization or The protective effect of Compound I in hypoxia was prenatal hypoxia assessed using the barometric chamber by "elevating' Rats with reflex of active avoidance in %% of the outbred albino mice (18-22 g) to the altitude of Days of 11,000 m at a speed of 1000 m/min. After 1 hour exposi Days of learning retention tion the percentage of surviving mice after 0.9% saline Group 1 2 3 4 5 1 2 pretreatment was 15%. Compound I (0.5 mg/kg) 15 Compound I po 10 minutes before hypoxia increased this value to 43.7%. tP < 0.05 *P < 0.01 in comparison with damaged group EXAMPLE 19 * P (0.05 in comparison with control The influence of Compound I on body weight and food consumption was studied in the experiments on the Prenatal alcoholization was shown to diminish the outbred rats. The substance, administered during 3 degree of the habituation in the open field. Chronic 15 weeks in dose of 0.1 mg/kg/day intraperitoneally postnatal administration of Compound I restored the slowed the dynamics of weight increase in comparison normal course of the habituation (Table 10). to control: the increase of the weight (in %% to the TABLE 10 initial one) was 12% in the treated group and 24% in the Influence of compound I on locomotor activity control group. of offspring after prenatal alcoholization 20 The amount of the consumed chow checked between 19th and 21st days was less for treated animals (39.5 Number of horizontal movements g/day/rat) than for control ones (68.6 g/day/rat). The Days of observation Coefficient of obtained data shows an anorexigenic effect of com Groups 5 Habituation pound I. Control 7.6 3.1 0.4 25 Prenatal 10.0 8.1 - 0.81 alcoholization EXAMPLE 2.0 Prenatal 8.2 3.7s 0.45 Compound I was checked for gross behavior and alcoholization - acute toxicity in outbred male mice (18-22 g). Com Compound I pound I in the dose range 1-10 mg/kg i.p. neither stimu PC 0.05 in comparison with alcoholized group 30 lated nor depressed the spontaneous motor activity and P < 0.05 in comparison with control did not enhance the stimulating effect of amphetamine on it. In this dose range Compound I also did not EXAMPLE 17 change rectal temperature. In the dose of 25 mg/kg compound I diminished the stimulating effect of am The influence of Compound I on age-related mental 35 phetamine. decline was studied in experiments on 24-month Wistar Up to the dose of 500 mg/kg it did not change motor rats. The substance was administered chronically in the coordination (rota rod test) and did not disturb the dose of 0.1 mg/kg/day during 24 days, intraperitoneally general condition (gross behavior) of the animals. before the testing and 14 days during testing. In the dose of 500-3000 mg/kg i.p. it did not cause Registration of the locomotor activity in the Op 40 lethality at 24 hours. In the dose of 1000 mg/kg com tovarimex multichannel analyzer has shown that Com pound I caused excitement in 50% of animals. In the pound I increased the initial horizontal activity being dose of 5000 mg/kg i.p. compound I caused death in administered chronically. The testing of the activity 50% of animals. during 5 minutes revealed the decrease of the motility, due to acute habituation (“negative learning'). This test 45 EXAMPLE 21 is suitable for the study of nootropics. In the control The influence of Compound I on withdrawal syn group the coefficient of the habituation was 0.29. Com drome caused by the interruption of chronic administra pound I pretreated animals showed more pronounced tion of benzodiazepine tranquilizer phenazepam (T. A. habituation with coefficient 0.03 (Table 11). Voronina et al., In Fenazepam; A. V. Bogatsky, Ed.; 50 Naukova dumka Publishers: Kiev, 1982; pp. 67-169) TABLE 11 was studied in experiments on male albino rats weighing The influence of Compound I on the age-related 190-200 g at the beginning and 300-350 g at the end of mental decline the experiment. - Locomotor activity (counts) Time of 55 Phenazepam (2 mg/kg intraperitoneally) or saline regis- Coefficient (control group of rats) were given once daily for 57 tration of habituation Passive avoidance days. The withdrawal syndrome occurred 24-72 hours 1st 5th (ratio - test (latency, see) after the last injection. The withdrawal syndrome in Groups min. min. 5th min/1st min) 24 hours 14 days rats consisted in "anxiogenic-like' state aggressiveness Control 244 72 0.29 t O7 60 and accelerated development of kindled seizures caused Compound I 422° 13' 0.03% 128 132 by pentyllentetrazol. Compound I was administered to the withdrawn rats in doses of 0.5 mg/kg intraperitone *P C0.05 as compared to the control. ally 15 minutes prior to testing. The conflict situation test was used to reveal the Chronic administration of Compound I facilitated the "anxiety' of rats (J. R. Vogel et al., Psychopharmacolo retention tested 24 hours and especially 14 days after 65 gia, 21, 1-7 (1971)). This experiment involved previous learning in passive avoidance test (Table 11). It is evi training of water deprivated rats to drink water from a dent from these experiments that Compound I is able to trough. Next day electrical stimulation was given by a improve amnestic function in the old rats. 0.5 mA electric current through the trough. A conflict 5,439,930 21 22 situation was created by clashing of the two different TABLE 14 reflexes (drinking and self-defense). The Effect of Compound I on Seizures Produced by Rats withdrawn from phenazepam 24 hours after Pentylenetetrazol (Kindling Test) During Phenazepam stopping a chronic exposure showed a reduction in the Withdrawal, 24, 48 and 72 hours after incidence of drinking compared with the control group. the Last Chronic Injection This behavior in the conflict situation test can be con Seizure Reactions in Points (M = m) sidered as "anxiogenic-like' one. Compound I antago Groups 24 h. 48 h. 72h nized the "anxiogenic-like' response to benzodiazepine Control 0.75 - 0.32 10 : 0.35 1.37 - 0.33 withdrawal and increased incidence of drinking in the 10 Phenazepam 1.0 - 0.41 1.86 - 0.25 2.37 - 0.45 conflict situation (Table 12). withdrawai -- Saline TABLE 12 Phenazepam 0.25 - 0.2 0.87 - 0.1 0.5 - 0.24 withdrawal -- The Effect of Compound I on the "Anxiogenic-Like" Compound I State Evoked by Phenazepam Withdrawal 15 The amount of the "P < 0.01 in comparison with 48 h phenazepam withdrawal group (u-test). Groups Punishment Response It is obvious from these experiments that Compound Control 31.75 6.44 Phenazepam withdrawal - I is able to diminish the syndrome of benzodiazepines Saline 12.08 - 83 20 withdrawal: it decreases the anxiety, aggressiveness and Phenazepam withdrawal -- pentylenetetrazol induced kindling. Compound I 48.40 - 12.39 All patents, patent documents, and publications cited "P < 0.05 in comparison with the control group (Student t-test) herein are incorporated by reference. The foregoing 'P < 0.05 in comparison with the phenazepam withdrawal. detailed description and examples have been given for 25 clarity of understanding only. No unnecessary limita The threshold of the aggressive behavior was deter tions are to be understood therefrom. The invention is mined in the experiment on the rats (R. Tedeshi et al., J. not limited to the exact details shown and described, for Pharmacol. Exp. Ther, 125, 28-34 (1959)). The paired variations obvious to one skilled in the art will be in rats were placed on the electrified floor; the strength of cluded within the invention defined by the claims. the current, which provokes the fighting, was regis 30 We claim: tered. 1. A compound of the formula: Phenazepam withdrawn rats showed the decrease of this threshold. Compound I, being administered to the O 24 hours withdrawn rats, was demonstrated to be able 35 R-C-L-Pro-NH-CH-(CH2)-C to increase the threshold of aggressive response (Table O R2 R3 13). wherein: TABLE 13 (a) R1 is CH2C6Hs; The Effect of Compound I on the Aggressiveness (b) R2 is selected from the group consisting of H, Evoked by Phenazepam Withdrawal Groups Aggression Threshold (mA) Control 0.71 - 0.02 Phenazepamf 2.16 0.2 (c) R3 is selected from the group consisting of OH, Phenazepam withdrawal -- 0.48 0.03 45 OCH3, OC2H5, NH2, NHCH3, and N(CH3)2; and Saline## (d) n=0-3. Phenazepam withdrawal -- 0.92 0.1 2. The compound of claim 1 wherein R2=H and Compoundiff n=0, said compound having the formula: 'P < 0.05 in comparison with phenazepam withdrawal group (U- criterium). fimmediately after the last administration of phenazepam. “24 hours after the last administration of phenazepam. 50 R!---Pro-Gly-r The seizure reactions of rats were studied using O chemical kindling (C.R. Mason et al., Epilepsia, 13, wherein R3 is selected from the group consisting of OH, 663-674 (1972); R. M. Post et al., Handbook of Biological 55 OC2H5, NH2, NHCH3, and N(CH3)2. Psychiatry, Part IV, No. 7, Marcel-Dekker (1981) pp. 3. The compound of claim 2 wherein Ri=CH2C6Hs 609-651) produced by small doses of pentylene-tetrazol and R3 = OC2H5. (35 mg/kg, intraperitoneally) administrated 24, 48 and 4. The compound of claim 1 wherein R2=H and 72 h after phenazepam withdrawal. The degree of sei n=1, said compound having the formula: zures was evaluated by 4 points: tremor (1 point); jerk (2 point); clonic seizures (3 points); tonic seizures and death (4 points). R!---Pop Al-R Our experiments pointed out that the pentylenetetra O zol seizures were more pronounced in phenazepam 65 wherein: withdrawn rats than in the control one. Compound I (a) R is CH2C6H5 and diminished the degree of kindling provoked by penty (b) R3 is selected from the group consisting of NH2 lenetetrazol (Table 14). and OC2H5. 5,439,930 23 24 5. The compound of claim 1 wherein R2=CH(CH3)2 (b) R2 is selected from the group consisting of H, and n=0, said compound having the formula: CH3, CH(CH3)2, CH2CH(CH3)2, CH2C(O)OC2H5, (CH2)2C(O)OC2H5, and R-C-L-Pro-L-Val-OCHs CH2C(O)NH2; I 5 (c) R3 is selected from the group consisting of OH, O OCH3, OC2H5, NH2, NHCH3, and N(CH3)2; and (d) n=0-3. wherein R1 is CH2C6H5. 13. The pharmaceutical composition of claim 12 6. The compound of claim 1 wherein Ri=CH2C6H5, wherein R2 = H and n=0, said compound having the R2=CH2C(O)NH2, R3=NH2, and n=0, said com- 10 formula: pound having the formula: R!--L-pro-Gly-R O O 15 wherein R3 is selected from the group consisting of 7. The compound of claim 1 wherein R1 = CH2C6H5, OC2H5 and NH2. R2=CH2C(O)OC2H5, R3 = OC2H5, and n=0, said 14. The pharmaceutical composition of claim 13 compound having the formula: wherein R1 = CH2C6Hs and R3 = OC2H5. 20 15. A method of treating obesity, said method con prising administrating an effective dose of a compound catch--L-pro-i-Asp-och9. of the formula: O 8. The compound of claim 1 wherein R1 = CH2C6H5, 25 R!--L-pro-Gly-r R2=CH2CH2C(O)OC2H5, R3 = OC2H5, and n=0, said compound having the formula: O wherein: (a) R is CH2C6H5; and ch:CH-i- L-Pro-L-Glu-(OC2H5)2. 30 (b) R3 is selected from the group consisting of OC2H5 O and NH2. 16. The method of claim 15 wherein the compound is 9. The compound of claim 1 wherein R1 = CH2C6H5, of the formula C6H5CH2-C(O)-L-Pro-Gly-OC2H5. R2=CH2CH(CH3)2, R3=NH2, and n=0, said com 17. A method of treating sickle cell anemia, said pound having the formula: 35 method comprising administering an effective dose of a compound of the formula: CHCH-i-L-Pro-L-leu-NH. O R!--L-pro-Gly-r 10. The compound of claim 1 wherein 40 O R=CH2C6H5, R2s CH3, R3 = OC2H5, and n=0, said wherein: compound having the formula: (a) R1 is CH2C6H5; and (b) R3 is selected from the group consisting of OC2H5 C6H5CH2-C-L-Pro-L-Ala-OCHs. 45 and NH2. 18. The method of claim 17 wherein the compound is O of the formula C6H5CH2-C(O)-L-Pro-Gly-OC2H5. 19. A method of diminishing mental decline in prena 11. The compound of claim 1 wherein tally alcoholized offsprings, said method comprising R=CH2C6H5, R2 = H, R3 = OCH3, and n=2, said 50 administering an effective dose of a compound of the compound having the formula: formula: R!--L-pro-Gly-R 55 O O OCH3 wherein: 12. A pharmaceutical composition containing as an (a) R is CH2C6H5; and active substance a pharmaceutically effective amount of (b) Ris selected from the group consisting of OC2H5 an N-acylprolyldipeptide having the formula: 60 and NH2. 20. A method of diminishing benzodiazepine with drawal syndrome, said method comprising administer R!--L-pro-NH-H-CH2 ing an effective dose of a compound of the formula: O R2 R3 6S R!---Pro-Gly-r wherein: O (a) R1 is CH2C6Hs; 5,439,930 25 26 wherein: (a) R1 is CH2C6Hs; and R--L-pro-Gly-R (b) R is selected from the group consisting of OC2H5 O 5 and NH2. wherein (a) R1 is CH2C6H5; and 21. A method of treating alcohol withdrawal, said (b) R3 is selected from the group consisting of OC2H5 method comprising administering an effective dose of a and NH2. compound of the formula: 10 23. A method of treating chemical dependency and toxicity, said method comprising administering an effec tive dose of a compound of the formula: R--L-pro-Gly-R O 15 R!--L-pro-Gly-R O wherein wherein: (a) R is CH2C6H5; and (a) R1 is CH2C6H5; and (b) Ris selected from the group consisting of OC2Hs 20 (b) R3 is selected from the group consisting of OC2H5 and NH2. and NH2. 24. The method of claim 23 wherein the compound is 22. A method of improving CNS functions, said of the formula C6H5CH2-C(O)-L-Pro-Gly-OC2H5. method comprising administering an effective dose of a 25. The method of claim 23 wherein said chemical 25 toxicity CNS effects are caused by lead poisoning. compound of the formula: k

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