WATER-SOLUBLE BIS-DIOXOPIPERAZINE DERIVATIVES WITH ANTITUMORAL PROPERTIES

Abstract: An acid addition salt of a bis-dioxopiperazine derivative represented by general formula (I), wherein R¹ represents C₁ to C₆ alkyl; R² represents hydrogen or (a); R³ represents hydrogen, C₁ to C₆ alkyl or -(CH₂) n -A, wherein A represents carboxyl, benzyloxycarbonyl, mercapto, amino, benzyloxycarbonylamino or (b), wherein X represents hydrogen, trifluoromethyl, hydroxy, acetoxy or benzyloxy, and n represents an integer of 1 to 4; and R⁴ represents hydrogen or alternatively R⁴ and R³ are combined together to represent a 5-membered cyclic group optionally containing sulfur.

Description: TECHNICAL FIELD Compounds of the present invention are acid addition salts of bis-dioxopiperazine derivatives represented by the formula (I) wherein R¹ represents a C₁₋₆ alkyl group, R² represents a hydrogen atom or a group of and R3 and R4 together form a -(CH₂)₃-group, wherein a -CH₂-group can be replaced by a sulfur atom. Synthetic intermediates of the compounds of the present invention are water soluble bis-dioxopiperazine derivatives having antitumor activity represented by the formula (II) wherein R¹ is as defined above, R⁵ represents a hydrogen atom or a group of R6 and R7 together form a -(CH₂)₃-group, wherein a -CH₂-group can be replaced by a sulfur atom, and R⁸ represents a protecting group for amino group. BACKGROUND ART Several kinds of bis-dioxopiperazine derivatives have been already reported. Among them, especially known as compounds having antitumor activity are 1,2-bis(4-morpholinomethyl-3,5-dioxopiperazin-1-yl)ethane (see Abstract, 8th International Congress of Pharmacology p441. 1981), dl-1,2-bis(4-morpholinomethyl-3,5-dioxopiperazin-1-yl)propane (see EP-A-0 125 475, corresponding to Japanese Patent Publication (Kokai) No. 190976/1984) and 1,2-bis(4-isobutoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane (see Abstract, 14th International Congress of Chemotherapy p324, 1985 and Japanese Patent Publication (Kokai) No. 97963/1985). Bis-dioxopiperazin derivatives are also described in EP-A-0 256 137, corresponding to the Japanese Patent Publication (Kokai) No. 281870/1987, and similar compounds also in Japanese Patent Publication (Kokai) No. 152660/1986, 89668/1987 and 279875/1989. Though these compounds have excellent antitumor activity, water soluble salts thereof with retained activity cannot be formed so that route of administration and pharmaceutical form of the compounds are much limitative. Therefore, there has been a demand for a water soluble bis-dioxopiperazine derivative which may be expected to have wider clinical applicabilities. Under these circumstances, we, the inventors carried out studies on novel bis-dioxopiperazine derivatives to find out that acid addition salts of the compounds represented by the formula (I) exhibit remarkably excellent antitumor activity, thus completing the present invention. Subject matter of the present invention are pharmaceutically acceptable acid addition salts of a bis-dioxopiperazine derivatives represented by the formula (I) according to Claim 1. Preferred embodiments thereof are subject matter of Claims 2 to 6. The terms used for definition of variables in the above-mentioned formulas (I) and (II) by which the compound of the present invention and synthetic intermediates thereof are respectively represented are defined and exemplified in the following. The "C₁₋₆" refers to a group having 1 to 6 carbon atoms unless otherwise indicated. The "C₁₋₆ alkyl group" refers to a straight- or branched-chain alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl. Preferably, methyl is used for R¹. The "protecting group for amino group" is preferably tert-butoxycarbonyl, or benzyloxycarbonyl. The compounds of the present invention are for example as follows: 2,3-bis(4-pyrrolidinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)butane 2,3-bis(4-thiazolydinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)butane 2-(4-pyrrolidinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)-3-(3,5-dioxopiperazin-1-yl)butane 2-(4-thiazolidinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)-3-(3,5-dioxopiperazin-1-yl)butane. The compound (I) of the present invention has asymmetric carbon atoms in its molecule. It is to be understood that isomers due to such asymmetric carbon atom or combination of any of the isomers are included in the category of the compound (I). Especially, meso- or erythro-form is preferred. Preferably, salts of the compounds of the present invention may be pharmaceutically acceptable salts such as hydrochloride, hydrobromide or trifluoroacetate. A further subject matter of the present invention is a synthetic intermediate of a bis-dioxopiperazine derivative, represented by the formula (II) according to Claim 7, Preferred embodiments thereof are subject matter of Claims 8 to 12. A synthetic intermediate (II) of the present invention is a compound wherein R⁸ being a protecting group for the amino group which bonds R⁴ of compound (I) and is for example as follows: 2,3-bis(4-N-tert-butoxycarbonylpyrrolidine carbonyloxymethyl-3,5-dioxopiperazin-1-yl)butane 2,3-bis(4-N-tert-butoxycarbonylthiazolidinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)butane 2-(4-N-tert-butoxycarbonylpyrrolidinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)-3-(3,5-dioxopiperazin-1-yl)butane 2-(4-N-tert-butoxycarbonylthiazolidinecarbonyloxymethyl-3,5-dioxopiperazin-1-yl)-3-(3,5-dioxopiperazin-1-yl)butane The compounds (I) of the present invention may be prepared by the reaction of compound represented by the formula (III): wherein R¹ is as defined above, with a compound represented by the forumula (IV): wherein R⁶, R⁷ and R⁸ are as defined above, (A) in the presence of a condensing agent to thereby readily afford the synthetic intermediate (II), followed by (B) removal of the protecting group R⁸ of amino group by acidic hydrolysis or by catalytic reduction in the presence of an acid to thereby prepare the compound of the formula (I) or acid addition salt thereof. In the reaction (A), the condensing agent may be, for example, 1-methyl-2-chloropyridinium iodide, dicyclohexylcarbodiimide or N,N'-carbonyldiimidazole. Preferably, methyl iodide is used as reaction accelerator in the case of N,N'-carbonyldiimidazole being a condensing agent used; and N,N-dimethylaminopyridine, in the case of the other condensing agents. The reaction temperature may range from 0 to 30°C, and the reaction time may range from 16 to 48 hours depending on reaction temperature. In the above-mentioned reaction, 1.2 to 2.4 molar amount of the compound of the formula (IV) is used for one molar amount of the compound of the formula (III). As for the reaction solvent, an aprotic polar solvent such as N,N-dimethylformamide (DMF), pyridine, dichloromethane, chloroform, acetonitrile or their mixture may be used. In the above-mentioned reaction, the, synthetic intermediates of the formula (II) with R⁵ being hydrogen atom and the group of are concurrently generated, and are separated and purified according to ordinary method using silica gel column chromatography or the like. Moreover, when the synthetic intermediates (II) contain a protective group in addition to the protective group R⁸, such protective group or groups can be removed together with the protective group R⁸ by the reaction (B). When the acidic hydrolysis is used in the reaction (B), the acid may be for example hydrogen chloride, hydrogen bromide or trifluoroacetic acid. The amount of the acid between 20 and 30 moles is used for one molar amount of the synthetic intermediate (II). The reaction temperature may range from 0 to 70°C and the reaction time may range from 2 to 4 hours depending on reaction temperature. As for the reaction solvent, dioxane, acetic acid or the like may be used. In the catalytic reduction, 10% palladium on carbon, 5% palladium on barium sulfate, platinum oxide or the like may be used as a catalyst; 10% palladium on carbon or platinum oxide is preferable. The acid used may be, for example, acetic or hydrochloric acid; and the reaction solvent may be methanol, ethanol, acetic acid, dioxane or the like. The reaction temperature may range from 0 to 70°C and the reaction time may range from 1 to 24 hours depending on reaction temperature. The thus obtained compound of the present invention or acid addition salt thereof may be isolated by usual manners such as extraction, concentration, filtration or recrystallization. The compound of the formula (III) which is the starting material in the above-mentioned preparation can be readily obtained from the reaction of bis(3,5-dioxopiperazin-1-yl)alkane, which disclosed in British Patent No. 1234935, with formaldehyde. The antitumor activity of the compounds of the formula (I) according to the present invention and prepared by the above-mentioned preparation process was verified by the below-mentioned tests. Test samples in these tests were as follows: Sample 1: Meso-2,3-bis[4-(2-pyrrolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]butane·hydrochloride Sample 2: Meso-2,3-bis[4-(4-thiazolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]butane·hydrochloride Comparative Compounds Sample A: 1,2-bis(4-isobutoxycarbonyloxymethyl-3,5-dioxopiperazin-1-yl)ethane Sample B: 1,2-bis[4-((R)-2-amino-2-phenylacetoxymethyl)-3,5-dioxopiperazin-1-yl]ethane·trifluoroacetate (Typical water soluble compound disclosed in Japanese Patent Publication (Kokai) No. 152660/1986) I) Growth Inhibition of Tumor Cells of P388 Lymphocytic Leukemia in vitro: Tumor cells were collected aseptically with capillary tube from ascites in DBA/2 male mice transplanted intraperitoneally with 1 x 10⁶ cells of P388 lymphocytic leukemia 5 days before. Cell suspension was prepared at 5 x 10⁴ cells/0.5 ml in a RPMI1640 medium supplemented with 10% fetal calf serum, kanamycin (0.1 mg/ml) and 2-hydroxyethyldisulfide (0.01 mM). Each test sample was dissolved or suspended in the medium at a concentration of from 2 x 10⁻⁴ to 2 x 10⁻¹⁰ M. A test tube involving 0.5 ml each of the cell suspension and the sample solution was kept for 48 hours at 37°C for cultivation in the atmosphere containing 5% carbon dioxide. Afther the cultivation, the cells were counted by using of Coulter Counter and the inhibition of cell growth was calculated by the following formula: Growth Inhibition (%)=(1 - T C ) x 100 T: number of cells in the culture containing test sample C: number of cells in the culture of control 50% Inhibitory concentration of cell growth (IC₅₀) was calculated based on the inhibition in various concentrations of test compound and is shown in Table 1. Sample IC₅₀(M) 1 1.8 x 10⁻⁸ 2 6.5 x 10⁻⁹ A 4.8 x 10⁻⁵ B 3.0 x 10⁻⁵ II) Increase in Life Span on L1210 Lymphoid Leukemia Tumor-transplanted Mouse: The treated group to which the test sample was administered consisted of seven mice, while the control group consisted of ten mice. Six weeks old male mice (CDF₁, 25±2 g of body weight) were employed as host animals. Tumor cells (1.0 x 10⁶) of L1210 lymphoid leukemia were transplanted subcutaneously into each mouse. The treatment was effected for 5 days from one day after the transplantation by administering prescribed dose of each test sample dissolved in physiological saline solution intravenously to the mice. Antitumor activity of the test sample was evaluated by the rate of increase in life span (ILS) which was calculated with the following formula. ILS (%)=( T' C' - 1) x 100 T': median survival time of treated mice C': median survival time of control mice The results obtained are shown in Table 2. Sample Daily Dose (mg/kg) ILS(%) 1 17 113 2 18 108 B 20 20 The above results of the tests revealed that the compounds of the present invention exhibit remarkably strong antitumor activity in comparison with the known water soluble bis-dioxopiperazine derivative (comparative compound B). The following descriptions are given for the administration routes, pharmaceutical forms and doses when bis-dioxopiperazine derivatives of the present invention are applied to human. The compounds of the present invention may be administered orally in forms such as tablets, coated tablets, powders, granules, capsules, microcapsules, syrups and so on. They may be also administered parenterally in forms such as injections which may include dissolvable freeze-dryed form, suppositories and so on. In the preparation of these forms, pharmaceutically acceptable diluents, binders, disintegrators, lubricants, suspensions, emulsifiers, antiseptics, stabilizers and dispersing agents, for example, lactose, sucrose, starch, dextrin, crystalline cellulose, kaolin, calcium carbonate, talc, magnesium stearate, distilled water and physiological saline solution may be used. Although the daily doses of these compounds may be varied according to the conditions, ages and weights of the subjects to be treated, the daily doses to adult humans may normally fall within the range of 1 to 600 mg, preferably 5 to 100 mg, and may be divided into two or three portions. BEST MODE FOR CARRYING OUT THE INVENTION [Preparation and Examples] The present invention is illustrated by the following preparation and examples. Production Meso-2,3-bis(4-hydroxymethyl-3,5-dioxopiperazin-1-yl)butane A mixture of meso-2,3-bis(3,5-dioxopiperazin-1-yl)butane (2.7 g, 10 mmol) and DMF (30 ml) was heated at 110°C for 10 minutes. To the mixture, 37% aqueous formaldehyde solution (2.2 ml) was added, and then the mixture was heated at 140°C with stirring for 1.5 hours. The reaction mixture was condensed under reduced pressure and the residue was treated with absolute ethanol. The resulting precipitates were collected by filtration and dried over calcium chloride to give 2.8 g (yield: 86%) of the titled compounds. Melting Point: 299 - 301°C IR Spectrum (KBr)cm⁻¹: 1670, 1730 (C=O) NMR Spectrum (DMSO-d₆) δ ppm: 0.89(6H,d,J=4Hz) 2.77(2H,m) 3.37 and 3.47(8H,AB,J=17Hz) 5.01(4H,d,J=7Hz) 6.19(2H,t,J=7Hz) Example 1 To a mixture of a corresponding compound of formula (IV) as defined above (0.96 mmol) and anhydrous dichloromethane (10 ml), N,N'-carbonyldiimidazole (156 mg, 0.96 mmol) and methyl iodide (0.14 ml, 2.2 mmol) was added successively with stirring at room temperature. Then, the mixture was further stirred at room temperature for 1.5 hours. Meso-2,3-bis(4-hydroxymethyl-3,5-dioxopiperazin-1-ly)butane (274 mg, 0.8 mmol) was added to the mixture. After stirred for 16 hours at room temperature, the reaction mixture was condensed under reduced pressure and the residue was extracted with dichloromethane. The extract was washed with 1N-hydrochloric acid and water, successively, and dried over magnesium sulfate. The extract was condensed under reduced pressure and the resulting residue was purified by column Meso-2,3-bis[4-(N-tert-butoxycarbonyl-2-pyrrolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]butane Yield: 58% Melting Point: 79 - 82°C (dec.) IR Spectrum (KBr)cm⁻¹: 1700, 1750 (C=O) chromatography on silica gel, using ethyl acetate and n-hexane (6 : 4) as an eluant, to give the following compounds : Meso-2,3-bis[4-(N-tert-butoxycarbonyl-4-thiazolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]butane Yield: 22% Melting Point: 94 - 97°C (dec.) IR Spectrum (KBr)cm⁻¹: 1700, 1750 (C=O) Erythro-2-[4-(N-tert-butoxycarbonyl-4-thiazolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]-3-(3,5-dioxopiperazin-1-yl)butane Yield: 48% Melting Point: 157 - 160°C (dec.) IR Spectrum (KBr)cm⁻¹: 1700, 1750 (C=O) Example 2 0.06 mmol of a corresponding compound of formula (II) as defined above, wherein R8 is tert.-butoxycarbonyl, were added to 5 ml of dioxane dissolved with hydrogen chloride (4N) at 0°C and stirred at 0°C for 1 hour and was further stirred at room temperature for 1 hour. The reaction mixture was condensed under reduced pressure, treated with anhydrous ether and stirred at room temperature for 1 hour. The resulting precipitates were collected by filtration and dried over potassium hydroxide under reduced pressure to give the following compounds: Meso-2,3-bis[4-(2-pyrrolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]butane·hydrochloride Melting Point: 110 - 115°C (dec.) IR Spectrum (KBr)cm⁻¹: 1700, 1750 (C=O) NMR Spectrum (D₂O) δ ppm: 1.21(6H,d,J=5Hz) 2.0 - 2.2(6H,m) 2.3 - 2.5(2H,m) 3.1 - 3.2(2H,m) 3.4 - 3.5(4H,m) 3.86 and 3.94(8H,AB,J=17Hz) 4.4 - 4.5(2H,m) 5.91 and 5.93(4H,AB,J=5Hz) Meso-2,3-bis[4-(4-thiazolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]butane·hydrochloride Melting Point: 161 - 166°C (dec.) IR Spectrum (KBr)cm⁻¹: 1700, 1750 (C=O) NMR Spectrum (D₂O) δ ppm: 1.17(6H,d,J=5Hz) 3.0 - 3.1(2H,m) 3.4 - 3.6 (4H,m) 3.79 and 3.89(8H,AB,J=17Hz) 4.44 and 4.50(4H,AB,J=10Hz) 4.9 - 5.0(2H,m) 5.92 and 5.98(4H,AB,J=10Hz) Erythro-2-[4-(4-thiazolidinecarbonyloxymethyl)-3,5-dioxopiperazin-1-yl]-3-(3,5-dioxopiperazin-1-yl)butane·hydrochloride Melting point: 140 - 145°C (dec.) IR Spectrum (KBr)cm⁻¹: 1700, 1750 (C=O) NMR Spectrum (D₂O) δ ppm: 1.18(3H,d,J=7Hz) 1.30(3H,d,J=7Hz) 3.1 - 3.2(1H,m) 3.3 - 3.4(1H,m) 3.4 - 3.5(2H,m) 3.76 and 3.84(4H,AB,J=15Hz) 3.88 and 3.98(4H,AB,J=17Hz) 4.44 and 4.50(2H,AB.J=10Hz) 4.9 - 5.0(1H,m) 5.92 and 5.97(2H,AB,J=10Hz) CAPABILITY OF EXPLOITATION IN INDUSTRY As described above, the water soluble bis-dioxopiperazine derivatives according to the present invention are water soluble salts having excellent antitumor activity and may have expanded pharmaceutical forms of administration which enables wider clinical applicability.

Claims: A pharmaceutically acceptable acid addition salt of a bis-dioxopiperazine derivative represented by the formula (I) wherein R1 represents a C1-C6 alkyl group, R2 represents a hydrogen atom or a group of and R3 and R4 together form a -(CH₂)₃-group, wherein a -CH₂-group can be replaced by a sulfur atom. The compound according to Claim 1 wherein R1 is methyl and R2 is a group of and R3 and R4 are coupled to form pyrrolidinyl or thiazolidinyl. The compound according to Claim 2 which is in a meso form. The compound according to Claim 1 wherein R1 is methyl, R2 is hydrogen atom and R3 and R4 are coupled to form pyrrolidinyl or thiazolidinyl. The compound according to Claim 4 which is in an erythro form. The compound according to any one of Claim 1 to 5 wherein the acid addition salt is a hydrochloride, hydrobromide or trifluoracetate. A synthetic intermediate of a bis-dioxopiperazine derivative, represented by the formula (II): wherein R1 represents a C1-C6 alkyl group, R5 represents a hydrogen atom or a group of R6 and R7 together form a -(CH₂)₃-group, wherein a -CH₂-group can be replaced by a sulfur atom, and R8 represents a protecting group for the amino group. The compound according to Claim 7 wherein R8 is tert-butoxycarbonyl or benzyloxycarbonyl. The compound according to Claim 7 or 8 wherein R1 is methyl, R5 is a group of and R6 and R7 are coupled to form pyrrolidinyl or thiazolidinyl. The compound according to Claim 9 which is in a meso form. The compound according to Claim 7 or 8 wherein R1 is methyl, and R5 is hydrogen atom and R6 and R7 are coupled to form pyrrolidinyl or thiazolidinyl. The compound according to Claim 11 which is in an erythro form.

Inventors: KOMATSU TOSHIHIKO - UWADO-SHIN, NARITA TOSHIHARU - AOBACHO -CH, TAKASE MUNEAKI - OIZUMI-MACHI, KOMATSU, TOSHIHIKO, 2-1, UWADO-SHINMACHI, NARITA, TOSHIHARU, 10-9, AOBACHO 3-CHOME, TAKASE, MUNEAKI, 24-13, OIZUMI-MACHI 1-CHOME
Applicants: ZENYAKU KOGYO KK, ZENYAKU KOGYO KABUSHIKI KAISHA
Application year: 1991
Main classification: C07D241
IPC codes: A61K31, C07D403, C07D241, C07D417, A61P35