Peptide derivatives and processes for their preparation

Abstract: Peptide ester and amide derivatives of the general for­ mula I in which X is H or acyl, a C₂ to C₅ alkanecarbonyl, a C₃ to C₆ car­ boxylalkanecarbonyl or a p-toluenesulfonyl, A is a glycine, alanine, leucine, phenylalanine, tyrosine, lysine or arginine residue, B is a glutamine, proline, arginine or lysine residue or a direct chemical bond, n is an integer of from 1 to3, R¹ is H or a C₁ to C₃ alkyl and R² is a C₁ to C₃ alkoxyl or an amino group when C is a peptidic­ ally bound glycine, alanine, leucine, phenylalanine, glutam­ ine or serine residue or R² is a peptidically bound glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide residue when C is a direct chemical bond, and pharmaceutically acceptable acid addition salts thereof are converted under physiological conditions, by enzymic hydrolysis in pathologically altered tissues and subsequent spontaneous cyclization, into pharmacodynamically active spirocyclic peptide derivatives of the general formula III, in which R³ and R⁴ are H atoms, optionally substituted alkyl groups of jointly an aliphatic chain forming preferably a 2,5-­ piperazinedione ring, and hence can act as pro-farmaca (drug precursors) of prolonged biological effect.

Description: The invention relates to peptide derivatives of the general formula I in which X is H, a C₂ to C₅ alkanecarbonyl, a C₃ to C₆ carboxyalkanecarbonyl or a p-toluenesulfonyl, A is a glycine, alanine, leucine, phenylalanine, tyrosine, lysine or arginine residue, B is a glutamine, proline, arginine or lysine residue or a direct chemical bond, n is an integer of from 1 to 3, R¹ is H or a C₁ to C₃ alkyl and R² is a C₁ to C₃ alkoxyl or an amino group when C is a peptidically bound glycine, alanine, leucine, phenylalanine, glutamine or serine residue or R² is a peptidically bound glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide residue when C is a direct chemical bond. The subject peptides are of possible interest since under the action of proteolytic enzymes present in pathological tissues they decompose to give C-terminal peptide esters or amides of the general formula II in which n and R¹ are the same as in formula I and R² is as defined above when respectively C is an aforementioned amino acid residue or alternatively a hydrogen atom. These peptide derivatives cyclize spontaneously and so afford spirocyclic peptides of the general formula III in which n and R¹ are the same as in formula I, R³ is H, a methyl, an isobutyl, a 2-aminoethyl, a 2-hydroxyethyl or a benzyl and R⁴ is a hydrogen atom, or R³ jointly with R⁴ form a di-, tri- or tetramethylene chain. Spirocyclic peptides of formula III are partly known, partly new. They are pharmacodynamically active substances that influence different biological systems. Thus, cyclo (L-alanyl-1-amino-1-cyclopentanecarbonyl) shows remarkable effects on the environmental behaviour of experimental animals (I Krejci et al, Activitas neurosa superior, in print) and in certain other pharmacological tests (Czechoslovak author certificate No. 231 227). Said spirocyclic peptides are comparatively less soluble, which is disadvantageous in preparing medicinal dosage forms. A possible route to overcoming the above obstacle is based on preparing a sufficiently soluble, more hydrophilic precursor compound comprising an additional optionally modified amino acid or lower peptide moiety and affording under pathophysiological conditions, by enzymic cleavage in the affected (eg, infected or inflamed tissue, the respective pharmacodynamically active substance, which is thus immediately liberated in or safely conveyed to the target organ in an amount substantially corresponding to the actual requirement of the organism without untoward effects of possible overdosage and, due to its gradual liberation, elicits the desired effect over a prolonged time period. According to the present invention, the object of safe, efficient and economical medication with said spirocyclic compounds of formula III is attained with the use of the subject peptide derivatives of formula I as drug precursors (pro-drugs or intermediates). The intact, non-cleaved peptide derivative of formula I is physiologically inert and under non-pathological conditions is metabolized and eliminated from the organism without liberating biologically active substances or leaving undesirable residua in healthy tissues. Said pathophysiological cleavage of the subject peptide derivatives of formula I is effected at bodily temperature and pH either under the action of certain pure proteolytic enzymes (proteases), eg, trypsin, kallikrein, chymotrypsin, cathepsin G, thrombin, plasmin or "factor X", as well as aminopeptidases, eg, leucinaminopeptidase, or aminodipeptidyl-peptidases, eg, the DAP IV peptidase, or also under the action of native, endogenous enzymic systems of brain, liver, kidney, pancreas, lungs, heart or intestine tissues or of blood serum. The spontaneous cyclization of the so liberated intermediary compounds of formula II to give said pharmacodynamically active spirocyclic peptide 2,5-piperazinedione derivatives of formula III proceeds at similar mild conditions in the pathologically affected tissue during a period of about from 4 to 12 hours. The above cleavage mechanism was demonstrated in vitro by thin-layer chromatography in n-butanol - acetic acid - water 4 : 1 : 1 for cleaving ethyl leucyl-alanyl-1-amino-1-cyclopentanecarboxylate (R f 0.44) with leucinaminopeptidase (LAP) in "tris" (abbreviated name for tris(hydroxymethyl)aminomethane) buffer of pH 7.4 at 37°C. The formed ethyl alanyl-1-amino-1-cyclopentanecarboxylate (R f 0.16) cyclizes spontaneously to afford cyclo(alanyl-1-amino-1-cyclopentanecarbonyl) (R f 0.64). This two-stage conversion of the starting peptide derivative into the spirocyclic product proceeds markedly even after two hours of incubation. A similar evidence was also obtained for other peptides and other enzymic systems. Thus, ethyl N-acetyltyrosyl-alanyl-1-amino-1-cyclopentanecarboxylate, ethyl 3-carboxypropionylphenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate and N-acetyltyrosyl-1-amino-1-cyclopentanecarbonylserine methyl ester were cleaved with chymotrypsin, and ethyl glycyl-prolyl-alanyl-1-amino-1-cyclopentanecarboxylate was similarly cleaved with DAP IV. Comparable results were obtained with the use of native tissue extracts with enzymic activity in place of the above mentioned pure enzymes. The structural design of the subject peptide derivatives of formula I was primarily focused on desired negligible toxic properties and physiological aspects of administration. Therefore, the corresponding esters with Et (an ethyl) as the C-terminal protective group were preferred, and conversion of the products into appropriate acid addition salts, predominantly citrates, was found advantageous. The preparative strategy was chosen so as to facilitate the cyclization of the intermediary compounds into the biologically active spirocyclic peptide derivatives. The subject compounds of the general formula I are available by conventional methods of the preparative chemistry of peptides. A suitable route for preparing the subject compounds consists principally in reacting a peptide derivative of the general formula IV Y-A-B-W IV in which A and B are the same as in formula I, Y is a C₂ to C₅ alkanecarbonyl or a protective group such as a benzyloxycarbonyl or a tert-butyloxycarbonyl and W is a carboxyl activating moiety such as a reactive halogen atom or an anhydride or reactive ester function, with a peptide derivative of the general formula II, in which n, R¹, R² and C are as defined hereinbefore, subsequent deprotecting of the so obtained peptide derivative and isolation of the liberated product, whereupon if required the product is converted into an acid addition salt or acylated. A convenient alternative route to the subject compounds of formula I consists in reacting a peptide derivative of the general formula V in which A, B, n and R¹ are the same as in formula I and Y and W are the same as in formula IV, with glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide and subsequent deprotection, isolation and optional salt formation or acylation. Said acylation step is effected with the use of a reactive derivative of a carboxylic acid of the general formula X - COOH in which X is the same as in formula I, preferably with an acid anhydride or halide thereof. Starting materials required for the preparation are known substances available by methods described in the pertinent literature. Further particulars of the procedure are illustrated by the following non-limitative examples. Melting points were determined on Kofler apparatus and are not corrected. Analytical samples were dried at 70 Pa. Optical rotations: Perkin-Elmer 141 polarimeter. Chromatography on silica gel ("Kieselgel" Merck) thin layer in 1-BuOH -AcOH - water 4:1:1 (S₁) and 1-BuOH - AcOH - pyridine - water 15:3:10:6 (S₂) systems. Evaporation of solutions was done on a rotary vacuum evaporator. Example 1 Ethyl benzyloxycarbonylleucyl-alanyl-1-amino-1-cyclopentanecarboxylate To a solution of benzyloxycarbonylleucyl-alanine (6.72 g, 20 mmoles) in dimethylformamide (70 ml) was added N-hydroxysuccinimide (2.3 g, 20 mmoles) and on cooling to - 5°C N,N'-dicyclohexylcarbodiimide (4.4 g) was admixed. After 1 hour of stirring and cooling (-5°C) there was added a dimethylformamide (40 ml) solution of ethyl 1-amino-1-cyclopentanecarboxylate liberated from its hydrochloride (3.86 g, 20 mmoles) with N-ethylpiperidine (2.8 ml) and the reaction mixture was stirred for a further 2 hours at room temperature. After a subsequent 12 hours of standing the N,N'-dicyclohexylurea precipitate was filtered off, washed with dimethylformamide and the filtrate was evaporated. The residue was dissolved in an ethyl acetate - water mixture and the organic phase was shaken successively with 1M hydrochloric acid, water, 5% sodium hydrogen-carbonate and water, dried over anhydrous sodium sulfate and evaporated. The residue was crystallized from ethyl acetate (25 ml) and petroleum ether (200 ml) to give 7.6 g (80%) of the title compound, m.p. 102 to 104°C, after similar recrystallization 104 to 106°C. [α] 20 / D -35.9° (c 0.2, methanol). Ethyl leucyl-alanyl-1-amino-1-cyclopentanecarboxylate citrate To a solution of the preceding ester (7.5 g, 15.8 mmoles) in methanol (100 ml) was added a suspension of 5% Pd/C catalyst (0.8 g) in toluene (30 ml). Hydrogenolysis was conducted at room temperature in an autoclave at an initial hydrogen pressure of 2 MPa and stirrer speed of 2000 rpm. After 10 minutes the autoclave was degassed, the catalyst was filtered off and the filtrate was evaporated. The residue was dissolved in methanol (30 ml) and a solution of citric acid (3.3 g) in methanol (20 ml) was added. The resulting solution was mixed gradually with 300 ml of ether and after 2 hours of standing the citrate precipitate was separated, washed with ether and dried in a desiccator over phosphorus pentoxide to give 4.7 g of the title product, R f 0.44/S₁, 0.58/S₂. [α] 20 / D -10.9° (c 0.2, MeOH). Similar procedures afforded the following compounds: Ethyl benzyloxycarbonylalanyl-alanyl-1-amino-1-cyclopentanecarboxylate, yield 76%, m.p. 134 to 136°C (ethyl acetate - petroleum ether). [α] 20 / D -39.2° (c 0.2, methanol). Ethyl alanyl-alanyl-1-amino-1-cyclopentanecarboxylate citrate, m.p. 99 to 102°C, R f 0.40/S₁, 0.56/S₂.[α] 20 / D -14.8° (c 0.2, MeOH). Methyl benzyloxycarbonylalanyl-alanyl-1-amino-1-cyclobutanecarboxylate, m.p. 101 to 103°C, [α] 20 / D -43.0°. Methyl alanyl-alanyl-1-amino-1-cyclobutanecarboxylate citrate, R f 0.27/S₁, 0.52/S₂. [α] 20 / D -12.9°. Ethyl benzyloxycarbonylalanyl-alanyl-1-amino-1-cyclohexanecarboxylate, m.p. 110 to 112°C. [α] 20 / D -42.2°. Ethyl alanyl-alanyl-1-amino-1-cyclohexanecarboxylate citrate, R f 0.37/S₁, 0.68/S₂. [α] 20 / D -42.2°. Ethyl benzyloxycarbonylphenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate, m.p. 93 to 96°C (toluene - petroleum ether). [α] 20 / D -22.5°. Ethyl phenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate citrate, R f 0.64/S₁, 0.78/S₂. [α] 20 / D -4.7°. Ethyl tert-butyloxycarbonyl-N g -nitroarginyl-alanyl-1-amino-1-cyclopentanecarboxylate (N g represents the guanidine N atom in the arginine residue), by the carbodiimide technique using N-hydroxysuccinimide from N alpha -tert-butyloxycarbonyl-N g -nitro-arginine and ethyl alanyl-1-amino-1-cyclopentanecarboxylate hydrobromide, m.p. 112 to 115°C (ethyl acetate), [α] 20 / D -20.4° (c 0.2, MeOH). Ethyl acetylleucyl-N g -nitroarginyl-alanyl-1-amino-1-cyclopentanecarboxylate, similarly from N-acetylleucine and ethyl N g -nitroarginyl-alanyl-1-amino-1-cyclopentanecarboxylate hydrochloride (yield 69%), m.p. 223 to 225°C (aqueous ethanol), R f 0.33/S₁, 0.69/S₂, [α] 20 / D -11.8° (c 0.2, dimethylformamide). Methyl acetylleucyl-N epsilon -benzyloxycarbonyllysyl-alanyl-1-amino-1-cyclopentanecarboxylate, similarly from N-acetylleucine and methyl N epsilon -benzyloxycarbonyllysyl-alanyl-1-amino-1-cyclopentanecarboxylate hydrochloride (yield 72%), m.p. 202 to 204°C, R f 0.52/S₁, 0.63/S₂, [α] 20 / D -41.6° (c 0.2, MeOH). Ethyl acetylleucyl-arginyl-alanyl-1-amino-1-cyclopentanecarboxylate acetate, by pressure hydrogenolysis (6 MPa) deprotection from the corresponding N g -nitroarginyl analog (yield 78%), m.p. 203 to 205°C (methanol - ethyl acetate) [α] 20 / D -32.1° (c 0.2, MeOH). Methyl acetylleucyl-lysyl-alanyl-1-amino-1-cyclopentanecarboxylate acetate, similarly by hydrogenolysis (2 MPa) of the corresponding benzyloxycarbonyl derivative (yield 85%), m.p. 210 to 212°C (methanol - ethyl acetate), [α] 20 / D -47.1° (c 0.2, MeOH). Example 2 Benzyloxycarbonylglycyl-prolyl-alanine methyl ester To a solution of benzyloxycarbonylglycyl-proline pentachlorophenyl ester (5.55 g, 10 mmoles) in dimethylformamide (30 ml) was added alanine methyl ester liberated from its hydrochloride (1.4 g, 10 mmoles) with N-ethylpiperidine (1.4 ml). After 5 hours of stirring and 12 hours of standing at room temperature the solution was evaporated, the residue was taken into ethyl acetate and the organic phase was washed successively with 1M hydrochloric acid, water, 5% sodium hydrogencarbonate and water, dried over anhydrous sodium sulfate and evaporated. The residue was crystallized from ethyl acetate - petroleum ether to give 1.95 g (50%) of the title product, m.p. 103 to 106°C. An analytical sample was recrystallized similarly, m.p. 105 to 108°C [α] 20 / D -100.1° (c 0.2, MeOH). Benzyloxycarbonylglycyl-prolyl-alanine, by alkaline hydrolysis of the preceding ester, yield 79%, m.p. 144 to 145°C (methylene chloride - petroleum ether). [α] 20 / D -95.6°. Ethyl benzyloxycarbonylglycyl-prolyl-alanyl-1-amino-1-cyclopentanecarboxylate, by the procedure of Example 1 from the preceding compound and ethyl 1-amino-1-cyclopentanecarboxylate, yield 78%, [α] 20 / D -61.5°. Ethyl glycyl-prolyl-alanyl-1-amino-1-cyclopentanecarboxylate citrate, by hydrogenolytic deprotection of the preceding, m.p. 115 to 118°C. R f 0.22/S₁, 0.58/S₂. [α] 20 / D -62.7°. Example 3 Ethyl 3-carboxypropionylphenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate A solution of ethyl benzyloxycarbonylphenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate (590 mg, 1 mmole) in methanol was hydrogenated by the procedure of Example 1. The methanolic solution was evaporated, dissolved in tetrahydrofuran, evaporated and acylated with glutaric anhydride (175 mg) in dimethylformamide for 2 hours at 80°C. The reaction mixture was evaporated and the product was precipitated with water to give the title compound, m.p. 193 to 196°C (dimethylformamide - water). Example 4 N,O-Diacetyltyrosyl-alanine methyl ester To a solution of N,O-diacetyltyrosine (13.3 g, 50 mmoles) and N-ethylpiperidine (7 ml) in tetrahydrofuran (100 ml) precooled to -15°C was added ethyl chloroformate (5 ml). After 15 minutes of stirring and cooling (-15°C) alanine methyl ester liberated from its hydrochloride (7.0 g, 50 mmoles) with N-ethylpiperidine (7 ml) was admixed. After 2 hours of stirring at 0°C and 12 hours of standing at room temperature the solution was evaporated, the residue was taken into ethyl acetate and the organic phase was successively washed with 1M hydrochloric acid, water, 5% sodium hydrogencarbonate and water, dried over anhydrous sodium sulfate and evaporated. Crystallization from 2-propanol - petroleum ether yielded 10.6 g (60%) of the title product. An analytical sample was recrystallized similarly, m.p. 173 to 176°C. [α] 20 / D -7.5°. N-Acetyltyrosyl-alanine The preceding protected ester (7 g, 20 mmoles) dissolved in methanol (100 ml) was hydrolyzed for 1 hour with 2M sodium hydroxide (25 ml). The reaction mixture was processed in the known manner to give an amorphous product. R f 0.59/S₁, 0.67/S₂. Ethyl N-acetyltyrosyl-alanyl-1-amino-1-cyclopentanecarboxylate, by the procedure of Example 1 from the preceding compound and ethyl-1-amino-1-cyclopentanecarboxylate, yield 88%, m.p. 168 to 171°C, [α] 20 / D -3.1° (c 0.2, MeOH). Similar procedures afforded the following compounds: Ethyl tert-butyloxycarbonylglutaminyl-1-amino-1-cyclopentanecarboxylate, from tert-butyloxycarbonylglutamine and ethyl 1-amino-1-cyclopentanecarboxylate, yield 62%, m.p. 131 to 132°C. Ethyl glutaminyl-1-amino-1-cyclopentanecarboxylate hydrochloride, by deprotection of the preceding compound with hydrogen chloride in glacial acetic acid. R f 0.38/S₁, 0.44/S₂. Ethyl N alpha -tert-butyloxycarbonyl-N omega -benzyloxycarbonyllysyl-glutaminyl-1-amino-1-cyclopentanecarboxylate, by the anhydride technique from the respective protected lysine compound and the preceding dipeptide ethyl ester, yield 93%, m.p. 127 to 130°C. Ethyl N omega -benzyloxycarbonyllysyl-glutaminyl-1-amino-1-cyclopentanecarboxylate hydrochloride, by analogous deprotection of the preceding compound. R f 0.28/S₁, 0.63/S₂. Ethyl acetylleucyl-N omega -benzyloxycarbonyllysyl-glutaminyl-1-amino-1-cyclopentanecarboxylate, by the carbodiimide technique in the presence of N-hydroxysuccinimide from N-acetylleucine and the corresponding tripeptide ethyl ester in dimethylformamide, m.p. 231 to 232°C (aqueous dimethylformamide). Ethyl acetylleucyl-lysyl-glutaminyl-1-amino-1-cyclopentanecarboxylate acetate, by hydrogenolytic deprotection of the preceding derivative, after crystallization from ethanol - ether R f 0.24/S₁, 0.51/S₂. Ethyl N alpha -tert-butyloxycarbonyl-N omega -benzyloxycarbonyllysyl-glycyl-1-amino-1-cyclopentanecarboxylate, by the anhydride technique from the respective protected lysine and ethyl glycyl-1-amino-1-cyclopentanecarboxylate hydrobromide, R f 0.41/S₁, 0.72/S₂. Ethyl N omega -benzyloxycarbonyllysyl-glycyl-1-amino-1-cyclopentanecarboxylate hydrochloride, by analogous deprotection of the preceding compound as above. R f 0.28/S₁, 0.63/S₂. Ethyl acetylleucyl-N omega -benzyloxycarbonyllysyl-glycyl-1-amino-1-cyclopentanecarboxylate, from N-acetylleucine and the preceding tripeptide ethyl ester in dimethylformamide, m.p. 179 to 182°C (aqueous dimethylformamide). Ethyl acetylleucyl-1-lysyl-glycyl-1-amino-1-cyclopentanecarboxylate acetate, by hydrogenolytic deprotection of the preceding compound, after crystallization from methanol - ether R f 0.25/S₁, 0.54/S₂. Methyl N,O-diacetyltyrosyl-1-amino-1-cyclopentanecarboxylate, by the anhydride technique from N,O-diacetyltyrosine and methyl 1-amino-1-cyclopentanecarboxylate, yield 43%, m.p. 210 to 212°C (ethanol). N-Acetyltyrosyl-1-amino-1-cyclopentanecarboxylic acid, by alkaline hydrolysis of the preceding ester; m.p. 252 to 254°C (aqueous ethanol). N-Acetyltyrosyl-1-amino-21-cyclopentanecarbonylserine methyl ester, similarly from the preceding acid and serine methyl ester, m.p. 128 to 132°C (2-propanol - petroleum ether), [α] 20 / D +8.6°. Example 5 Ethyl benzyloxycarbonylalanyl-glycyl-1-amino-1-cyclopentanecarboxylate To a dimethylformamide (25 ml) solution of ethyl 1-amino-1-cyclopentanecarboxylate liberated from its hydrochloride (1.0 g, 5 mmoles) with N-ethylpiperidine (0.7 ml) there was portionwise added benzyloxycarbonylalanylglycine p-nitrophenyl ester (2.1 g, 5 mmoles). After 3 hours of stirring and 12 hours of standing at room temperature the solution was evaporated, the residue was taken into ethyl acetate and extracted successively with 1% ammonia and water, dried over anhydrous sodium sulfate and evaporated. The residue was crystallized from ethyl acetate - petroleum ether to give 1.4 g (67%) of the title product, m.p. 119 to 121 °C. An analytical sample was crystallized similarly, m.p. 137 to 139 °C. [α] 20 / D -3.4°. Hydrogenolytic deprotection of this compound similarly as described in Example 1 yielded ethyl alanyl-glycyl-1-amino-1-cyclopentane-carboxylate, citrate R f 0.28/S₁, 0.71/S₂. [α] 20 / D +7.3°. Recent pharmacological report on the effect of a typical compound of the invention, Leu-Ala-Acp-OEt.Cit (hereinafter Compound X in which Acp represents 1-amino-1-cyclopentanecarboxylic acid residues Cit - citrate salt) in an in vivo behavioural test in rats. The effect of compound X on learning and memory processes was verified using the standard "conditioned taste aversion" (CTA) test generally accepted as conclusive in this respect. Compound X was found to inhibit the partial amnesia of experimental CTA induced by hypoxia in male rats. This is considered to evidence a significant beneficial effect of the substance under test on memory and learning in mammals. Method Male Wistar rats of 280 to 320 g were placed on a water deprivation schedule. Throughout the experiment they received water of 10% sucrose solution for 15 min in individual drinking boxes (from a calibrated 25 pipette) and for 60 min in their home cages. After 4 days of training their daily water intake stabilized. On the subsequent conditioning day 5 the animals were allowed to drink sucrose solution in the boxes for 15 min as before, and after 60 min they were injected i.p. (intraperitoneally) with saline or C.075 M LiCl, 2 ml/100 g. Twenty-four hours thereafter, on day 6, they received s.c. (subcutaneously) 55 mm/kg of sodium nitrite dissolved in 1 ml/kg of water. On day 7 the animals were allowed to drink water according to the previous schedule and the water intake values were recorded; these posterior values did not differ substantially from the intakes measured prior to conditioning. On the subsequent 8th (test) day the degree of taste aversion was determined by offering the animals sucrose solution for 15 min the same way as before. Compound X was administered subcutaneously at two dosage levels, in a single dose of 1.0 or 10.0 mg/kg, over 5 days starting with the last training day. The administration time preceded by 45 min the sucrose drinking on the conditioning day, nitrite administration and sucrose drinking on the test day. Piracetam (PIR, 50 mg/kg sc) was administered analogously using the same time schedule. Results The intake volumes of sucrose solution on the test day are summarized in the following Table 1. The hypoxia induced by sodium nitrite resulted in a partial amnesia of the acquired CTA. This experimental amnesia was significantly inhibited by either dose of compound X of the present application. Antiamnestic effect of PIR used as reference compound for comparison, at the above dosage level, was not significant. Group (N=10) Treatment on the Sucrose intake on the test day mean in ml ± S E Drug mg/kg condit day 6th day 1 saline NaNO₂ 13.5 ± 1.7 saline 2 LiCl saline 6.0 1.8 saline 3 LiCl NaNO₂ 8.1 2.2 saline 4 LiCl NaNO₂ 5.4 1.7 x 1 5 LiCl NaNO₂ 6.3 1.9 x 10 6 LiCl NaNO₂ 7.0 3.5 PIR 50 Significant difference (p 0.05) between the group 1 and all other groups 2 and 3 3 and 4 4 and 5

Claims: Claims for the following Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE Peptide derivatives of the general formula I in which X is H, a C₂ to C₅ alkanecarbonyl, a C₃ to C₆ carboxyalkanecarbonyl or a p-toluenesulfonyl, A is a glycine, alanine, leucine, phenylalanine, tyrosine, lysine or arginine residue, B is a glutamine, proline, arginine or lysine residue or a direct chemical bond, n is an integer of from 1 to 3, R¹ is H or a C₁ to C₃ alkyl and R² is a C₁ to C₃ alkoxyl or an amino group when C is a peptidically bound glycine, alanine, leucine, phenylalanine, glutamine or serine residue or R² is a peptidically bound glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide residue when C is a direct chemical bond, and pharmaceutically acceptable acid addition salts thereof. Ethyl leucyl-alanyl-1-amino-1-cyclopentanecarboxylate and its citrate. Ethyl alanyl-alanyl-amino-1-cyclopentanecarboxylate and its citrate. Methyl alanyl-alanyl-1-amino-1-cyclobutanecarboxylate and its citrate. Ethyl alanyl-alanyl-1-amino-1-cyclohexanecarboxylate and its citrate. Ethyl phenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate and its citrate. Ethyl glycyl-prolyl-alanyl-1-amino-1-cyclopentanecarboxylate and its citrate. Ethyl 3-carboxypropionylphenylalanyl-phenylalanyl-1-amino-1-cyclopentanecarboxylate. Ethyl N-acetyltyrosyl-alanyl-1-amino-1-cyclopentanecarboxylate. Ethyl acetylleucyl-1-lysyl-glutaminyl-1-amino-1-cyclopentanecarboxylate and its acetate. Ethyl acetylleucyl-1-lysyl-glycyl-1-amino-1-cyclopentanecarboxylate and its acetate. N-Acetyltyrosyl-1-amino-1-cyclopentanecarbonylserine methyl ester. Ethyl glutaminyl-1-amino-1-cyclopentanecarboxylate and its hydrochloride. Ethyl alanyl-glycyl-1-amino-1-cyclopentanecarboxylate and its citrate. Ethyl acetylleucyl-arginyl-alanyl-1-amino-1-cyclopentanecarboxylate and its acetate. Methyl acetylleucyl-lysyl-alanyl-1-amino-1-cyclopentanecarboxylate and its acetate. Claims for the following Contracting State : AT A process for the preparation of peptide derivatives of the general formula I in which X is H, a C₂ to C₅ alkanecarbonyl; a C₃ to C₆ carboxyalkanecarbonyl or a p-toluenesulfonyl, A is a glycine, alanine, leucine, phenylalanine, tyrosine, lysine or arginine residue, B is a glutamine, proline, arginine or lysine residue or a direct chemical bond, n is an integer of from 1 to 3, R¹ is H or a C₁ to C₃ alkyl and R² is a C₁ to C₃ alkoxyl or an amino group when C is a peptidically bound glycine, alanine, leucine, phenylalanine, glutamine or serine residue or R² is a peptidically bound glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide residue when C is a direct chemical bond, and pharmaceutically acceptable acid addition salts thereof, comprising reacting a peptide derivative of the general formula IV Y-A-B-W IV in which A and B are the same as in formula I, Y is a C₂ to C₅ alkanecarbonyl or a protective group and W is a carboxyl activating moiety with a peptide derivative of the general formula II, in which n and R¹ are the same as in formula I and R² is a C₁ to C₃ alkoxyl or an amino group when C is a peptidically bound glycine, alanine, leucine, phenylalanine, glutamine or serine residue or R² is a peptidically bound glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide residue when C is a hydrogen atom, subsequent deprotecting of the so obtained peptide derivative and isolation of the liberated product, whereupon if required the product is converted into an acid addition salt or acylated. A process for the preparation of peptide derivatives of formula I of claim 1 comprising reacting a peptide derivative of the general formula in which A, B, n and R¹ are the same as in formula I and Y and W are the same as in formula IV, with glycine, alanine, leucine, phenylalanine or serine methyl ester, ethyl ester or amide, subsequent deprotecting of the so obtained peptide derivative and isolation of the liberated product, whereupon if required the product is converted into an acid addition salt or acylated. A process of claim 1 or 2 comprising acylating the product with a reactive derivative of a carboxylic acid of the general formula X - COOH in which X is the same as in formula I. A process according to claim 3 wherein the reactive derivative of a carboxylic acid is an acid anhydride or halide thereof. A process according to claim 1 wherein Y is a benzyloxycarbonyl or tert-butyloxycarbonyl protective group. A process according to claim 1 wherein W is a reactive halogen atom or an anhydride or reactive ester group. A peptide derivative of the general formula I, in which X, A, B, C, n, R¹ and R² are the same as in claim 1, whenever prepared by a procedure substantially in accordance with any of the preceding claims or by an obvious equivalent thereof.

Inventors: BARTIK MICHAL, KASAFIREK EVZEN, KREJCI IVAN, KREPELA EVZEN, ROUBALOVA ALENA, RYBAK MIROSLAV, STURC ANTONIN, VANZURA JIRI, BARTIK, MICHAL, KASAFIREK, EVZEN, KREJCI, IVAN, KREPELA, EVZEN, ROUBALOVA, ALENA, RYBAK, MIROSLAV, STURC, ANTONIN, VANZURA, JIRI
Applicants: VYZK USTAV FARM BIOCHEM SP, VYZKUMNY USTAV PRO FARMACII A BIOCHEMII
Application year: 1987
Main classification: C07K5
IPC codes: C12P21, C07K5, C07D241, A61K38, C07K7