Tetrapeptide derivative

Abstract: A tetrapeptide derivative represented by the following formula or a salt thereof has a higher cytostatic activity than dolastatin 10, and is useful as an antitumor agent: wherein

Description: Technical Field This invention relates to a novel tetrapeptide derivative having an antitumor activity, and relates more detailedly to a tetrapeptide derivative represented by the following formula or a salt thereof: wherein (a) R 4 = CH 3 - and R 5 =PhCH 2 - (b) R 4 = CH 3 - and R 5 = PhCH 2 -; (c) R 4 = H- and R 5 = PhCH 2 -; (d) R 4 = CH 3 - and R 5 = PhCH 2 -; (e) R 3 = CH 3 CH 2 CH 2 -R 4 = CH 3 - and R 5 = PhCH 2 -; (f) R 4 = CH 3 - and R 5 = PhCH 2 -; (g) R 2 = PhCH 2 -, R 4 = CH 3 - and R 5 = PhCH 2 -; (h) R 4 = CH 3 - and R 5 = PhCH 2 -. Background Art Peptides having a cytostatic activity and/or an antineoplasm activity have been isolated from marine molluscs, sea hare Dolabella auricularia and these peptides are called dolastatins 1 to 15. Among them, dolastatin 10 is a pentapeptide extracted from Dolabella auricularia from the Indian Ocean in 1987 by G. R. Pettit, et al. and having the following structural formula, and is said to be the strongest cytostatic substance presently known (see G. R. Pettit, et al., J. Am. Chem. Soc., 109 , 6883 (1987) and Japanese Laid-Open Patent Publication No. 167278/1992). Further, recently, publication was made on the total synthesis of dolastatin 10 itself (see, US Patent No. 4,978,744), but its derivatives have not so far been known at all. The present inventors had intensely studied about derivatives of dolastatin 10, and as a result they found that certain dolastatin analogs represented by the above formula (I) have a higher cytostatic activity than dolastatin 10. They further found that many of these compounds have a larger therapeutic ratio (maximum effective dose/dose at 30% prolongation of life) and a lower toxicity than dolastatin 10, and are thus excellent as an antitumor agent. Namely, they found that amino acid analogs of dolastatin 10 exhibit a higher activity than the native dolastatin 10. Disclosure of Invention The embodiments of this invention completed based on these findings are amino acid-substituted compounds of dolastatin 10 and synthesis thereof. The synthesis of compounds according to the present invention is described in the later-shown Flow sheets 1, 2 and 3. In the present description, the term "lower" means that the number of the carbon atoms of a group or compound to which this term is attached is 6 or less, preferably 4 or less. In the present description, a "lower alkyl group" may be either of straight-chain and branched-chain, and there can, for example, be mentioned a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl'or isohexyl group, or the like and "aralkyl group" means an aryl-lower alkyl group, and there can, for example, be mentioned a benzyl or phenethyl group, or the like. Further, "lower alkoxy group" means a lower alkyl-0-group wherein the lower alkyl part has the above meaning, an there can, for example, be mentioned a methoxy, ethoxy, n-propoxy, isopropoxy or tert-butoxy group, or the like, and "aralkyloxy group" means an aralkyl-O-group wherein the aralkyl part has the above meaning, and there can, for example, be mentioned a benzyloxy or phenethyloxy group, or the like. Further, a "lower alkylene group" may be either of straight-chain and branched-chain, and there can, for example, be mentioned a methylene, ethylene, trimethylene, tetramethylene, methylmethylene, propylene, ethylethylene or 1,2-dimethylethylene group, or the like, and as a "cycloalkyl group", there can be mentioned a cycloalkyl group having 3 to 7 carbon atoms such as, for example, a cyclopropyl, cyclopentyl, cyclohexyl or cycloheptyl group, and as "aryl group", there can, for example, be mentioned a phenyl or naphthyl group, or the like. In the compounds of the aforesaid formula (I) of this invention, the carbon atoms to which the substituents R 1 , R 2 , R 3 , R 4 , R 5 and methoxy groups are bonded are asymmetric carbon atoms, and can take arbitrary R or S configurations, and all these are included in the scope of this invention, but in view of the point of pharmacological activities, compounds which have the same configuration as dolastatin 10 are preferable. The compounds of the above formula (I) provided by this invention, are listed in the following table: Compound R 1 R 2 R 3 R 4 R 5 (a) i-Pr i-Pr i-Bu Me Bzl (b) i-Pr i-Pr i-Pr Me Bzl (c) i-Pr i-Pr s-Bu H Bzl (d) i-Pr s-Bu s-Bu, Me Bzl (e) i-Pr i-Pr n-Pr Me Bzl (f) H i-Pr s-Bu Me Bzl (g) i-Pr Bzl s-Bu Me Bzl (h) Me i-Pr s-Bu Me Bzl In the above groups, Me, Pr, Bu, and Bzl mean a methyl group, a propyl group, a butyl group, and a benzyl group, respectively. The tetrapeptide derivatives of the above formula (I) can exist as a salt, and examples of such salts, there can be mentioned hydrochlorides, hydrobromides, trifuloroacetates, p-toluenesulfonates and acetates. According to this invention, a tetrapeptide derivative of the above formula (I) can be prepared by condensing amino acid or peptide fragments, for example according to the liquid phase synthesis method (see, E. Schröder and K. Lübke, "The Peptides", volume 1, pages 76-136, 1965, published by Academic Press) well known in the field of peptide chemistry. However, it is most preferable to synthesize these tetrapeptide derivatives by condensing a tripeptide fragment of the following formula (II) wherein R 1 , R 2 and R 3 have the same meanings as defined above, with a fragment of the following formula (III) wherein R 4 and R 5 have the same meaning as defined above, because each fragment of the formulae (II) and (III) is easy to synthesize and is free from racemization at their condensation step. The reaction can generally be carried out by treating the above fragments (II) and (III) with a condensing agent, e.g. dicyclohexylcarbodiimide (DCC), diphenyl phosphoryl azide (DPPA) or diethyl phosphorocyanidate (DEPC), a so-called BOP reagent, or the like in an inert solvent such as, for example, chloroform, ethyl acetate, tetrahydrofuran (THF), dimethylformamide (DMF) or acetonitrile, if necessary in the presence of an organic base such as, for example, triethylamine, N-methylmorpholine or diisopropylethylamine (DIEA). The reaction temperature is usually -10°C to room temperature, preferably around 0°C, and as for use rates of the compound of the formula (III), the organic base and the condensing agent based on the compound of the formula (II), it is advantageous to use the compound of the formula (III) of at least one mole, preferably of the order of 1.0 to 1.1 moles, the organic base of the order of 2 moles, and the condensing agent of the equimolar order, respectively per mole of the compound of the formula (II). Thus, the desired tetrapeptide derivatives of the formula (I) are formed, and the isolation and purification thereof from the reaction mixtures can be carried out by recrystallization, ion exchange chromatography, gel filtration, high performance liquid chromatography, etc. The compounds of the above formulae (II) and (III) used as starting materials in the above reaction are novel compounds not disclosed in prior literatures, and can easily be prepared by condensing amino acids, which are constituents thereof, according to a liquid phase synthesis method. The tetrapeptide derivatives of the formula (I) of this invention have a higher cytostatic activity than dolastatin 10, and are useful for treatment of acute myelocytic leukemia, acute lymphocytic leukemia, chronic melanoma, pulmonary adenocarcinoma, neuroblastoma, pulmonary small cell carcinoma, breast cancer, colon cancer, ovary cancer, bladder cancer, etc. The screening of cytostatic activity was made using lymphocytic leukemia P388 cells. The results are shown in the following Table. Compound ED 50 (µg/ml) (a) 3.1 x 10 -6 (b) 1.7 x 10 -6 (c) 3.1 x 10 -5 Dolastatin 10 7.0 x 10 -4 The compounds of this invention, when used as a drug, can be used by formulating them into any dosage form of solid forms (e.g., tablets, hard capsules, soft capsules, granules, powders, fine granules, pills, troches, etc.), semi-solid forms (e.g., suppositories, ointments, etc.) and liquid forms (e.g., injections, emulsions, suspensions, lotions, sprays, etc.). As nontoxic additives usable in the above formulations, there can, for example, be mentioned starches, gelatin, glucose, lactose, fructose, maltose, magnesium carbonate, talc, magnesium stearate, methylcellulose, carboxymethylcellulose or salts thereof, gum arabic, polyethylene glycol, p-hydroxybenzoic acid alkyl esters, syrups, ethanol, propylene glycol, vaseline, carbowax, glycerol, sodium chloride, sodium sulfite, sodium phosphate, citric acid, etc. The drug can also contain another therapeutically effective drug. The content of the compound of this invention in the drug varies depending on the dosage form, but it is generally preferable that the drug contains the compound at a concentration of 0.1 to 50 wt% in the case of solid and semi-solid forms, and at a concentration of 0.05 to 10 wt% in the case of liquid form. The dose of the compound of this invention can widely be varied depending on the kind of warm-blooded animals including human beings and a subject, administration routes, the seriousness of symptoms, the diagnoses of doctors, etc., but can generally be on the order of 0.01 to 50 mg/kg per day. However, it is of course be possible to administer the compound in an amount smaller than the lower limit of the above range or in an amount larger than the upper limit thereof in accordance with the seriousness of the symptom of the patient and the diagnosis of the doctor as mentioned above. The above dose can be administered once a day or in divided several portions per day. This invention is further described below according to Referential Examples and Examples. As for the structure of compounds corresponding to compound numbers used in Referential Examples and Examples, please refer to the following Flow sheets 1 to 3. Therein, Z, Me, Bu t , Boc and Bzl represent a benzyloxycarbonyl group, a methyl group, a tert-butyl group, a tert-butoxycarbonyl group and a benzyl group, respectively, and R 1 , R 2 , R 3 , R 4 and R 5 have the aforesaid meanings. Referential Example 1-A Preparation of compound 1-A (Compound 1 wherein R 3 =CH 3 ) 11.15 g (50 mmoles) of Z-alanine is dissolved in 140 ml of tetrahydrofuran, 9.72 g (60 mmoles) of carbonyldiimidazole is added, and the mixture is stirred at room temperature for 4 to 5 hours. On the other hand, 17.16 g (110 mmoles) of malonic acid monomethyl ester potassium salt and 7.60 g (80 mmoles) of anhydrous magnesium chloride are suspended in 150 ml of tetrahydrofuran and is stirred for 6 hours with heating on a water bath of 55°. The reaction mixture is cooled with ice, the above reaction solution is poured thereinto all at once, the cooling bath is immediately removed, and the mixture is stirred at room temperature for 24 to 48 hours. A small amount of water is added to the reaction solution, and a clear supernatant is decanted from the deposited waxy precipitate, and concentrated under reduced pressure to obtain an oily matter. Ethyl acetate and ice-cooled 4N hydrochloric acid are added to each of 'the above waxy residue and this oily matter, each of the mixtures is shaken to dissolve, the two solutions are combined, the layers are separated, and the aqueous layer is extracted again with ethyl acetate. The ethyl acetate layer is washed with ice-cooled 2N hydrochloric acid and saturated aqueous sodium bicarbonate and dried, and the solvent is distilled off to obtain 13.50 g of a pale yellow oily matter. This is purified by silica gel column chromatography (eluent : ethyl acetate-n-hexane (1 : 1)) to obtain the desired compound 1-A as a color-less - pale yellow oily matter. 12.96 g (92.9%). [α] D 26 -17.7° (c=1.01, MeOH) 1 H-NMR (CDCl 3 , δ) 1.38(3H, d, J=7.1 Hz), 3.55(2H, s), 3.72(3 H, s), 4.45(1H, m, J=7.1 Hz), 5.11(2H, s), 5.25∼5.55(1H, m), 7.34(5H, s) Referential Examples 1-B, 1-C, 1-D and 1-E were carried out in all the same manner as in Referential Example 1-A to obtain compounds 1-B, 1-C, 1-D and 1-E as oily matters, respectively. Referential Example 2-A Preparation of compound 2-A (Compound 2 wherein R 3 =CH 3 ) 12.96 g (46.45 mmoles) of compound 1-A obtained in Referential Example 1-A is dissolved in 380 ml of methanol, and 3.56 g (93.67 mmoles) of sodium borohydride is added thereinto all at once under stirring at -78°. Cooling and stirring are continued for 6 hours, ice-cooled 1N hydrochloric acid is added gradually, and when it is confirmed that the mixture becomes acidic, the mixture is concentrated under reduced pressure, and the deposited oily matter is extracted with ethyl acetate. The ethyl acetate layer is washed with saturated aqueous sodium bicarbonate and dried, and the solvent is distilled off to obtain 12.93 g of crystals. The crystals are recrystallized from isopropyl ether to obtain the desired compound 2-A as colorless needles of a melting point of 78°. 11.09g (85.0%). [α] D 28 -4.4° (c=1.00, MeOH) As C 14 H 19 NO 5 Calculated C=59.77% H=6.81% N=4.98% Found C=59.83% H=6.92% N=5.07% 1 H-NMR (CDCl 3 , δ) 1.15(3H, d, J=6.8 Hz), 2.35∼2.55(2H m), 3.70(3H, s), 3.85∼4.15(1H, m), 5.09(2H, s), 7.34(5H, s) Referential Examples 2-B, 2-C, 2-D and 2-E were carried out in all the same manner as in Referential Example 2-A to obtain compounds 2-B, 2-C, 2-D and 2-E, respectively. Referential Example 3-A Preparation of compound 3-A (Compound 3 wherein R 3 =CH 3 ) 9.78. g (34.80 mmoles) of the compound obtained in Referential Example 2-A is dissolved in 100 ml of dimethylformamide, 40.0 g (172.41 mmoles) of silver oxide and 50 ml of methyl iodide are added, and the mixture is stirred for 5 hours in a water bath of 35°. The mixture is filtered, silver oxide is washed with dimethylformamide, and the filtrate and washings are combined and concentrated under reduced pressure at 50° or below. The residue is thoroughly extracted with ethyl acetate, the ethyl acetate layer is washed with 5% sodium thiosulfate and saturated aqueous sodium bicarbonate and dried, and the solvent is distilled off to obtain 10.43 g of a yellow oily matter. The oily matter is purified by silica gel column chromatography (eluent : benzene-ethyl acetate (5 : 1)) to obtain the desired Compound 3-A as a pale yellow oily matter. 7.63 g (71.0%). [α] D 25 -39.8° (c=1.03, MeOH) 1 H-NMR (CDCl 3 , δ) 1.21(3H, d, J=6.8 Hz), 2.47(2H, d, J=6.2 Hz), 2.80(3H, s), 3.38(3H, s), 3.64(3H, s), 5.13(2H, s), 7.34(5H, s) Referential Examples 3-B, 3-C, 3-D and 3-E were carried out in all the same manner as in Referential Example 3-A to obtain compounds 3-B, 3-C, 3-D and 3-E as oily matters, respectively. Referential Example 4-A Preparation of compound 4-A (Compound 4 wherein R 3 =CH 3 ) 6.60 g (21.36 mmoles) of the compound 3-A obtained in Referential Example 3-A is dissolved in 100 ml of dioxane, 23.5 ml (23.5 mmoles) of 1N sodium hydroxide is added, and the mixture is strirred at room temperature for 2 to 3 hours. 20% citric acid is added to the reaction solution to make the pH 4.0, the mixture is concentrated under reduced pressure, and the deposited oily matter is extracted with ethyl acetate. The ethyl acetate layer is washed with saturated saline and dried, and the solvent is distilled off to give a colorless - pale yellow oily matter. This dissolved in 60 ml of dichloromethane, 0.8 ml of concentrated sulfuric acid is added, and the mixture is shaken with 25 ml of isobutene in a pressure bottle at room temperature for 48 to 96 hours. The reaction solution is poured into saturated aqueous sodium bicarbonate, nitrogen gas is blown thereinto remove isobutene and the greater part of dichloromethane, the deposited oily matter is extracted with ethyl acetate, and the ethyl acetate layer is washed with saturated aqueous sodium bicarbonate and dried. The solvent is distilled off, and the remaining yellow oily matter (7.32 g) is purified by silica gel column chromatography (eluent : benzene-ethyl acetate (10 : 1)) to obtain 6.31 g (84.1%) of the desired compound 4-A as a colorless - pale yellow oily matter. [α] D 27 -33.0° (c=1.02, MeOH) 1 H-NMR (CDCl 3 , δ) 1.21(3H, d, J=6.8 Hz), 1.44(9H, s), 2.38(2 H, d, J=6.2 Hz), 2.82(3H, s), 3.38(3H, s), 3.5∼3.85(1H, m), 3.85∼4.4(1H, m), 5.13(2 H, s), 7.34(5H, s) Referential Examples 4-B, 4-C, 4-D and 4-E were carried out in all the same manner as in Referential Example 4-A to obtain compounds 4-B, 4-C, 4-D and 4-E as oily matters, respectively. Referential Example 5-A Preparation of compound 5-A (Compound 5 wherein 0.70 g (2.00 mmoles) of compound 4-A obtained in Referential Example 4-A is dissolved in 20 ml of t-butanol-water (9 : 1), 0.1 g of 5% palladium carbon is added, and the mixture is stirred under a hydrogen stream for 2 hours. After the reaction, the catalyst is filtered and washed, and the filtrate and the washings are concentrated under reduced pressure. The remaining oily matter is dissolved in 30 ml of benzene, the solution is concentrated again under reduced pressure, and the operation is repeated once more. The resultant oily matter is dissolved together with 0.56 g (2.23 mmoles) of Z-valine in 10 ml of acetonitrile, and 0.43 g (2.09 mmoles) of DCC is added under ice cooling and stirring. Soon, crystals appear. Stirring is continued at 0° for at least 3 hours, and then overnight during which time the temperature is allowed to rise as the ice melts, the reaction mixture is diluted with ethyl acetate, and the crystals are filtered and washed with ethyl acetate. The filtrate and the washings are concentrated under reduced pressure, the syrupy residue is dissolved in ethyl acetate, the insoluble matters, if any, are removed by filtration, the ethyl acetate solution is washed with ice-cooled 2N hydrochloric acid and saturated aqueous sodium bicarbonate and dried, and the solvent is distilled off to obtain 1.01 g of a colorless oily matter. This is purified by silica gel column chromatography (eluent : benzene-ethyl acetate (5 : 1)) to obtain 0.67 g (74.4%) of the desired compound 5-A as a colorless oily matter.. [α] D 28 -31.4° (c=1.02, MeOH) 1 H-NMR (CDCl 3 , δ) 0.8∼1.1(6H, m), 1.17(3H, d, J=6.8 Hz), 1.45(9H, s), 2.25∼2.45(2H, m), 3.00(3H, s), 3.37(3H, s), 3.68(1H, dd, J=12.1 Hz, 6.2 Hz), 4.35∼4.75(2H, m), 5.09(2H, s), 5.56 (1H, br, d), 7.33(5H, s) The following compounds were obtained in all the same manner as in Referential Example 5-A. Referential Example 6-A Preparation of compound 6-A (Compound 6 wherein 0.65 g (1.44 mmoles) of compound 5-A obtained in Referential Example 5-A is dissolved in 15 ml of t-butanol-water (9 : 1), 50 mg of 5% palladium carbon is added, and the mixture is stirred under a hydrogen stream for 2 hours. After the reaction, the catalyst is removed by filtration and washed, and the filtrate and the washings are concentrated under reduced pressure. The oily residue is dissolved in 30 ml of benzene and the solution is concentrated again under reduced pressure, and this operation is further repeated once more. The resultant oily matter is dissolved in 6 ml of dimethylformamide, 0.25 g (1.72 mmoles) of N,N-dimethylvaline and 0.29 g (1.78 mmoles) of DEPC are added, the mixture is stirred at room temperature until clear solution is obtained, then the solution is cooled with ice, and the solution of' 0.17 g (1.68 mmoles) of triethylamine in 1 ml of dimethylformamide is added dropwise over a period of 4 minutes. Stirring is continued at 0° for at least 4 hours and then overnight during which time the temperature is allowed to rise as the ice melts, the resultant clear reaction mixture is diluted with ethyl acetate, and the ethyl acetate solution is thoroughly washed with saturated aqueous sodium bicarbonate and then dried. The solvent is distilled off, and 0.66 g of the remaining pale brown oily matter is purified by silica gel column chromatography (eluent : ethyl acetate-hexane (1 : 1)) to obtain 0.46 g (71.9%) of the desired compound 6-A as a colorless oily matter. [α] D 27 -56.5° (c=1.00, MeOH) 1 H-NMR (CDCl 3 , δ) 0.8∼1.1(12H, m), 1.15(3H, d, J=7.0 Hz), 1.45(9H, s), 2.27(6H, s), 3.05(3H, s), 3.38(3H, s), 3.55∼3.85(1H, m), 4.35∼ 4.65(1H, m), 4.65∼4.95(1H, m), 0.88(1H, br, d) The following compounds were obtained in all the same manner as in Referential Example 6-A. Referential Example 7 Preparation of compound 7-A (Compound 7 wherein R 4 =H) 7 ml (15.5 mmoles) of a 23.8% LDA tetrahydrofuran : hexane (1 : 1) solution is added dropwise to 10 ml of anhydrous tetrahydrofuran at -20°C under a nitrogen atmosphere and stirring. Then the mixture is cooled to -78°C in a dry ice-acetone bath. 2.3 g (15 mmoles) of benzyl acetate is added dropwise over a period of 30 minutes, the mixture is stirred at -78°C for 5 minutes, and a solution of 2.0 g (10 mmoles) of Boc-L-prolinal in 10 ml of tetrahydrofuran is added dropwise over a period of 1 hour. The mixture is stirred at -78°C for 10 minutes, 30 ml, of ice-cooled 1N hydrochloric acid is added, and the temperature is elevated up to room temperature. The mixture is extracted with ethyl acetate, the extract is washed with water and dried, the solvent is distilled off under reduced pressure, and the residue is purified by silica gel flash chromatography using hexane : ethyl acetate (5 : 1) as an eluent to obtain 1.12 g (32.0%) of the desired compound 7-A as an oily matter. [α] D 25 -23.7° (c=1.26, CHCl 3 ) MS 331, 276 1 H-NMR (CDCl 3 , δ) 1.46(9H, s), 2.47(2H, d, J=6.8 Hz), 3.7∼ 4.3(2H, m), 5.15(2H, s), 7.2∼1.4(5H, m) Referential Example 8 Preparation of compound 8-A (Compound 8 wherein R 4 =H) 560 mg (1.6 mmoles) of compound 7-A obtained in Referential Example 7 is dissolved in 27 ml of dichloromethane, 202 µl (1.6 mmoles) of BF 3 .Et 2 O is added thereto under cooling in ice-salt bath, and an ether solution of diazomethane (32 mmoles) is added dropwise over a period of 30 minutes. After stirring for further two hours under cooling in ice-salt bath, 2 ml of saturated aqueous sodium bicarbonate is added. The insoluble matter is removed by filtration, the filtrate is extracted with ethyl acetate, and the extract is washed with water and dried. The solvent is distilled off under reduced pressure, and the residue is purified by silica gel flash chromatography using hexane : ethyl acetate (5 : 1) as an eluent to obtain 378 mg (65.0%) of the desired compound 8-A as an oily matter. [α] D 25 -58.7° (c=0.52, CHCl 3 ) MS 241, 218 1 H-NMR (CDCl 3 , δ) 1.46(9H, s), 2.47(2H, d, J=7.5 Hz), 3.34(3 H, s),3.6∼4.3(2H, m), 5.14(2H, s), 7.2∼ 7.4(5H, m) Referential Example 9 Preparation of compound 9-A (Compound 9 wherein R 5 =CH 3 ) 6.82 g (60 mmoles) of cysteamine hydrochloride is dissolved in 50 ml of dimethylformamide, and the solution is neutralized with 8.4 ml of triethylamine. The solution is mixed with a solution of crude Boc-L-alaninal obtained by oxidizing 8.75 g (50 mmoles) of Boc-alaninol in 50 ml of dimethylformamide, and mixture is stirred overnight at room temperature. The solvent is distilled off under reduced pressure, the formed crystals are dissolved in ethyl acetate, the solution is washed with 10% citric acid and saturated aqueous sodium bicarbonate, and the ethyl acetate layer is dried. The solvent is distilled off under reduced pressure to obtain 4.93 g (42.6%) of the desired compound 9-A as white crystals. Melting point 81.0-84.1°C MS 232 (M + ), 159 1 H-NMR (CDCl 3 , δ) 1.24(3H, d, J=6.6 Hz), 1.45(9H, s), 2.1(1 H, br), 4.42(1H, d, J=8.1 Hz) Referential Example 10 Preparation of compound 10-A (Compound 10 wherein R 3 =CH 3 ) 1.34 g (5.77 mmoles) of the compound 9-A obtained in Referential Example 9 and 12.5 g of manganese dioxide are stirred in 58 ml of benzene at 55°C for 1.5 hours. The suspension is filtered, the solvent is distilled off under reduced pressure, and the residue is purified by silica gel flash chromatography using hexane : ethyl acetate (5 : 1) as an eluent to obtain 118 mg (9.0%) of the desired compound 10-A as an oil matter. [α] D 22 -36.0° (c=1.29, CH 2 Cl 2 ) MS 228(M + ) 172 1 H-NMR (CD 2 Cl 2 , δ) 1.43(9H, s), 1.56(3H, d, J=7.2 Hz), 4.9 ∼5.2(2H, m), 7.26(1H, d, J=3.3 Hz), 7.67 (1H, d, J=3.3 Hz) Referential Example 11-A Preparation of compound 11-A (Compound 11 wherein R 4 =H and R 5 =PhCH 2 ) 220 mg (0.604 mmole) of compound 8-A obtained in Referential Example 8 is dissolved in 9 ml of t-butanol : water (9 : 1), 50 mg of palladium carbon is added, and the mixture is stirred under a hydrogen stream. After completion of the reaction, the reaction solution is filtered, and the solvent is distilled off under reduced pressure to obtain 165 mg (0.604 mmole) of a solid matter. This is dissolved in 3 ml of acetonitrile, 267 mg (0.604 mmole) of the BOP reagent and 192 mg (0.604 mmole) of a trifluoroacetate salt obtained from known compound 10-B (compound 10 wherein R 5 =CH 2 Ph) are added, and then 195 mg (1.51 mmoles) of diisopropylethylamine is added dropwise under ice cooling. The mixture is stirred overnight at room temperature, the solvent is distilled off under reduced pressure, the residue is dissolved in dichloromethane, and the solution is washed successively with 10% citric acid, saturated aqueous sodium bicarbonate and saturated saline and dried. The solvent is distilled off under reduced pressure, and the resultant crude product is purified by silica gel column chromatography using dichloromethane-methanol (50 : 1)) as an eluent to obtain 262 mg (94.4%) of the desired compound 11-A as amorphous powder. [α] D 27 -86.4° (c=0.43, MeOH) MS 428, 368 1 H-NMR (CDCl 3 , δ) 1.47(9H, s), 2.29(2H, d, J=6.2 Hz), 3.27(3 H, s),5.63(1H, m), 7.1∼7.3(6H, m), 7.40(1 H, d, J=3.3Hz) Referential Example 11-B Preparation of compound 11-B (Compound 11 wherein R 4 =CH 3 and R 5 =CH 3 ) Compound 8-B (compound 8 wherein R 4 =CH 3 ), known compound, and compound 10-A obtained in Referential Example 10 are treated in all the same manner as in Referential Example 11-A to obtain the desired compound 11-B. Yield 62.0%, an oily matter. [α] D 26 -100° (c=1.3, MeOH) MS 365, 324, 309 1 H-NMR (CDCl 3 , δ) 1.26(3H, d, J=6.7 Hz), 1.48(9H, s), 1.63(3 H, d, J=6.7 Hz), 3.43(3H, s), 5.2∼5.6(1H, m), 7.23(1H, d, J=3.3 Hz), 7.69(1H, s, J= 3.3 Hz) Example 1 Preparation of compound (a) (Compound of formula (I) wherein R 4 =CH 3 and R 5 =PhCH 2 ) 1 ml of concentrated hydrochloric acid is added with ice cooling to 108 mg (0.222 mmole) of compound 6-G obtained in Referential Example 6-G, and the mixture is stirred for 1 hour. The mixture is evaporated to dryness under reduced pressure, the residue is dissolved in 2 ml of dimethylformamide, and 0.15 ml of triethylamine is added under ice cooling. Although triethylamine hydrochloride is deposited, the.mixture is evaporated to dryness under reduced pressure and dried. On the other hand, 105 mg (0.222 mmole) of known compound 11-C (compound 11 wherein R 4 =CH 3 and R 5 =PhCH 2 ) is dissolved in 0.4 ml of ethyl acetate, and 3.3 ml of 2N hydrogen chloride/ethyl acetate is added under ice cooling. The mixture is stirred at room temperature for 1 hour, the solvent is distilled off under reduced pressure, and the residue is dried. The resultant hygroscopic crystals are dissolved in 1.6 ml of dimethylformamide and is added to the above tripeptide carboxylic acid, and 40 mg (0.222 mmole) of 90% DEPC and 62 µl (0.444 mmole) of triethylamine are added under ice cooling. The mixture is stirred for 1 hour under ice cooling and then overnight at room temperature. The solvent is distilled off under reduced pressure, the residue is dissolved in dichloromethane, and the solution is washed with saturated aqueous sodium bicarbonate and saturated saline and dried. The solvent is distilled off, the residue is purified by silica gel flash chromatography using dichloromethane-methanol (20 : 1) as an eluent, and the fractions containing the desired compound is further purified by Sephadex LH-20 chromatography using hexane : dichloromethane : methanol (2 : 7.5 : 2.5) as an eluent to obtain 137 mg (78.4%) of the desired compound (a) as amorphous powder. [α] D 27 -89.0° (c=0.60, MeOH) MS 741, 693 1 H-NMR (CDCl 3 , δ) 2.32(6H, s), 2.96(3H, s), 3.32(3H, s), 3.40(3H, s), 5.56(1H, m), 6. 8∼7. 3(9H, m), 7.73(1H, d, J=3.3 Hz) Examples 2-8 The following compounds were obtained by carrying out Examples 2-8 in all the same manner as in Example 1.

Claims: Claims for the following Contracting States : AT, BE, CH, LI, DE, DK, FR, GB, IE, IT, LU, MC, NL, SE A tetrapeptide derivative represented by the following formula or a salt thereof wherein (a) R 4 =CH 3 - and R 5 =PhCH 2 - (b) R 4 = CH 3 - and R 5 = PhCH 2 -; (c) R 4 = H- and R 5 = PhCH 2 - ; (d) R 4 = CH 3 - and R 5 = PhCH 2 -; (e) R 3 = CH 3 CH 2 CH 2 - , R 4 = CH 3 - and R 5 = PhCH 2 -; (f) R 4 = CH 3 - and R 5 = PhCH 2 -; (g) R 2 = PhCH 2 -, R 4 = CH 3 - and R 5 = PhCH 2 -; (h) R 4 = CH 3 - and R 5 = PhCH 2 -. An antitumor agent containing a tetrapeptide derivative or a salt thereof according to claim 1 as an effective ingredient. Claims for the following Contracting States : ES, GR A tetrapeptide derivative represented by the following formula or a salt thereof wherein (a) R 4 = CH 3 - and R 5 =PhCH 2 - (b) R 4 = CH 3 - and R 5 = PhCH 2 -; (c) R 4 = H- and R 5 = PhCH 2 -; (d) R 4 = CH 3 - and R 5 = PhCH 2 -; (e) R 4 = CH 3 - and R 5 = PhCH 2 -; (f) R 4 = CH 3 - and R 5 = PhCH 2 -; (g) R 2 = PhCH 2 -, R 4 = CH 3 - and R 5 = PhCH 2 - ; (h) R 4 = CH 3 - and R 5 = PhCH 2 -. An antitumor agent containing a tetrapeptide derivative or a salt thereof according to claim 1 as an effective ingredient. Use of a tetrapeptide derivative or a salt thereof as defined in claim 1 for the preparation of an antitumor agent. A process for the preparation of a tetrapeptide derivative represented by the following formula or a salt thereof wherein (a) R 4 = CH 3 - and R 5 =PhCH 2 - (b) R 4 = CH 3 - and R 5 = PhCH 2 -; (c) R 4 = H- and R 5 = PhCH 2 -; (d) R 4 = CH 3 - and R 5 = PhCH 2 -; (e) R 3 = CH 3 CH 2 CH 2 - , R 4 = CH 3 - and R 5 = PhCH 2 - ; (f) R 4 = CH 3 - and R 5 = PhCH 2 - ; (g) R 2 = PhCH 2 -, R 4 = CH 3 - and R 5 = PhCH 2 -; (h) R 4 = CH 3 - and R 5 = PhCH 2 -, which comprises condensing a tripeptide fragment of the following formula (II) wherein R 1 , R 2 and R 3 have the same meanings as defined above, with a fragment of the following formula (III) wherein R 4 and R 5 have the same meaning as defined above, followed by isolation and purification of the desired tetrapeptide derivative and optionally converting said tetrapeptide derivative into its salt. Process as claimed in claim 4, wherein a condensing agent selected from dicyclohexylcarbodiimide, diphenylphosphorylazide or diethylphosphorocyanidate in an inert solvent optionally in the presence of an organic base is used. Process as claimed in claims 4 or 5, wherein the reaction temperature is from -10°C to room temperature. Process as claimed in claim 6, wherein the reaction temperature is 0°C. Process as claimed in claims 5 to 7, wherein the compound of the formula (III) is used in an amount of at least 1 mol, preferably 1.0 to 1.1 mols per mol of the compound of the formula (II) and wherein the organic base is used in an amount of 2 mols per mol of the compound of the formula (II) and wherein the condensing agent is used in an amount equimolar to the compound of the formula (II).

Inventors: GONDO MASAAKI, MIYAZAKI KOICHI, SAKAKIBARA KYOICHI, GONDO, MASAAKI, MIYAZAKI, KOICHI, SAKAKIBARA, KYOICHI, GONDO, Masaaki, 5-310, Sunstage-Ryokuentoshi
Applicants: TEIKOKU HORMONE MFG CO LTD, TEIKOKU HORMONE MFG. CO., LTD.
Application year: 1992
Main classification: C07K5
IPC codes: A61K38, C07K5, C07K7