2'-Methylidenepyrimidine Nucleoside Compounds, their use and method for production thereof

Abstract: 2'-Methylidenepyrimidine nucleoside compounds of the general formula : wherein R¹ stands for amino or hydroxy group; R² stands for a halogen or a lower alkyl when R¹ is amino or R² stands for an alkyl having 2 to 4 carbon atoms, an alkynyl having 2 to 4 carbon atoms or a haloalkyl when R¹ is hydroxy group; and R³ stands for hydrogen or a phosphoric acid residue, or salts thereof, anticancer compositions containing one or more of these compounds and methods for production of these compounds. Said compounds and salts thereof exhibit noticeable antitumor activities and are useful as anticancers.

Description: This invention relates to 2'-deoxy-2'-methylidene-5-fluorocytidine possessing an excellent antitumor action and its pharmaceutical use. Under the circumstances in which death due to cancer has increased in number, chemotherapy and immunotherapy in addition to surgical therapy have been widely conducted. In this connection, in chemotherapy, cytosine arabinoside and 5-fluorouracil as antimethabolites, which are considered effective against acute leukemia, have been clinically used. However, the hitherto-known anticancer agents leave much to be desired in respect to therapeutic effect and involve various problems such as side effects. Thus, the development of excellent anticancer agents has been desired from various fields. Under such situations, the present inventors created 2'-deoxy 2'-deoxy-2'-methylidenecytidine and its pharmaceutically acceptable salts. However, since 2'-deoxy-2'-methylidenecytidine is inferior in solubility and its acid addition salts are unstable, they are unsatisfactory in the aspect of pharmaceutical production. Further, EP-A-0 310 673 (relevant under Art. 54(3) EPC) generically discloses 2'Alkylidenepyrimidine nucleoside derivatives represented by formula: wherein R 1 is an amino or hydroxy group, R 2 is a hydrogen or halogen atom or a lower alkyl group, R 3 is a hydrogen atom or a lower alkyl group, and R 4 is a hydrogen atom or a phosphate residue, or salts thereof. However, 2'-deoxy-2'-methylidene-5-fluorocytidine is not expressly mentioned. SUMMARY OF THE INVENTION The object of the present invention is to provide novel compounds possessing excellent antitumor actions and their pharmaceutical use. The present inventors had conducted extensive studies to develop novel compounds useful as an anticancer agent and having no problems in the aspect of pharmaceutical production and found that 2'-deoxy-2'-methylidene-5-fluorocytidine possessed excellent antitumor actions and no problems in the aspect of pharmaceutical production, which culminated in the completion of the present invention. This invention relates to a 2'-deoxy-2'-methylidene-5-fluorocytidine compound of the general formula : wherein R 3 stands for hydrogen or a phosphoric acid residue, or a salt thereof. Further, the present invention relates to an anticancer composition characterized by containing, as the active ingredient, a compound of the general formula (I) or a salt thereof. As the salts of the compounds of the general formula (I), in case where R 3 is hydrogen, mention is made of acid addition salts such as hydrochlorides, sulfates, hydrobromides, phosphates, maleates, fumarates, tartrates, succinates, citrates and p-toluenesulfonates, and in case where R 3 is a phosphoric acid residue, mention is made of pharmaceutically acceptable salts exemplified by metal salts such as sodium salt, potassium salt, lithium salt, and calcium salt, or ammonium salt. The present invention encompasses hydrates (1 hydrate, 1/2 hydrate, 1/4 hydrate, 3/2 hydrate) or other solvates of the compounds of the general formula (I) or their salts. As the compound of the general formula (I), there can be mentioned, for example, 2'-deoxy-2'-methylidene-5-fluorocytidine and 2'-deoxy-2'-methylidene-5-fluorocytidine-5'-phosphate. The compound of the general formula (I) of the present invention can be produced by, for example, the following method: After the hydroxy groups at the 3'- and 5'-positions of the sugar moiety of a compound of the general formula : are protected, the hydroxy group at the 2'-position of the sugar moiety is oxidized, followed by reaction with a Wittig reagent to obtain the compound having methylidene at the 2'-position of the sugar moiety, followed by converting the hydroxy group at the 4-position of the base into an amino group, for example, by reacting the obtained compound with 1,2,4-triazol in the presence of phosphorus oxychloride and triethylamine to obtain the 4-(1,2,4-triazol-1-yl) compound and converting the obtained compound to the 4-amino compound by passing ammonia gas thereto, and thereafter removing the protective groups of the hydroxy groups at the 3'- and 5'-positions of the obtained compound, whereafter, if desired, the 5'position of the sugar moiety is subjected to phosphorylation. In the above-mentioned reactions, as the protective groups for the hydroxy groups at the 3'- and 5'-positions, any protective groups that are conventionally used as the protective groups for hydroxy groups can be applied. As such protective groups, for example, mention can be made of acyl groups such as acetyl, propionyl, butyryl, benzoyl and naphthoyl; acetal- or ketal-type protective groups such as ethylidene, propylidene, isopropylidene, benzylidene, cyclohexylidene, cyclopentylidene, methoxymethylidene, ethoxymethylidene and dimethoxyethylidene; aralkyl groups such as benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, diphenylmethyl, triphenylmethyl, α- or β-naphthylmethyl and α-naphthyldiphenylmethyl; and silyl groups such as trimethylsilyl, t-butyldimethylsilyl, melthyldiisopropylsilyl, triisopropylsilyl and tetraisopropyldisiloxyl (TIPDS). As the oxidation method for the hydroxy group at the 2'-position, there can be adopted chromic acid oxidation (A method) which comprises using a complex of chromic acid-pyridine-acetic anhydride and the like or activated dimethylsulfoxide oxidation (B method) which comprises using activated dimethylsulfoxide produced from oxalyl chloridedimethylsulfoxide. The oxidation reaction can be carried out in the presence of 1-10 times mole of an oxidizing agent relative to 1 mole of the compound to be oxidized at a temperature ranging from -10°C to room temperature in the case of A method or at a temperature ranging from -10°C to -80°C in the case of B method for 1-10 hours. As the Wittig reagent to be used for the methylidenation reaction, specifically, triphenylphosphinemethylene can be mentioned, and preferably used is a compound which is prepared, immediately before use, from a triphenylphosphonium compound (methyltriphenylphosphonium bromide, methyltriphenylphosphonium iodide) and a strong alkali (potassium hydride, sodium hydride, n-butyl lithium, sodium amide) in accordance with the conventional methods. The Wittig reagent can be used suitably in an amount ranging from 1 to 3 times mole relative to 1 mole of the compound. The methylidenation with the use of a Wittig reagent can be carried out by reacting in a solvent (tetrahydrofuran, dioxane, ether, benzene, dimethylsulfoxide, singly or in mixture) at -30 to 30°C for 0.5 to 20 hours. The reaction with 1,2,4-triazole proceeds under the flow of inert gas such as argon gas in a solvent such as acetonitrile at a temperature ranging from 0°C to about room temperature for 30 minutes to 5 hours. The ammonia gas is preferably in advance passed through sodium hydroxide. Preferably, the amination reaction with ammonia gas is usually conducted for 1-5 hours. The deprotection reaction for the hydroxy groups of the sugar moiety can be conducted by suitably selecting conventional treatment such as acidic hydrolysis, alkaline hydrolysis, ammonium fluoride treatment or catalytic reduction in accordance with the protective group used. For example, when a silyl group is used as the protective group for the hydroxy group, the silyl group can be removed by ammonium fluoride treatment or acidic or alkaline hydrolysis. The compounds wherein R 3 is a phosphoric acid residue can be obtained by reacting with a phosphorylating agent (phosphorus oxychloride, tetrachloropyrophosphoric acid, etc.) which is conventionally used for selective phosphorylation at the 5'-position of nucleosides. The objective compounds of the present invention can be isolated and purified by conventional purification and isolation methods (e.g. various chromatography method, ion exchange and adsorption chromatography, recrystallization) suitably in combination. Specifically, for example, in the case of the nucleoside compounds, after the solvent is distilled off, they are, if necessary, subjected to column chromatography and crystallized from a suitable organic solvent. In the case of nucleotide compounds (wherein R 3 is a phosphoric acid residue), they are purified by ion exchange chromatography with ion exchange resin adsorption column chromatography with activated charcoal and lyophilization or crystallization to obtain the objective or intermediate compounds in the free acid form, or, if desired, as the salts. The thus-obtained compounds of the general formula (I) can be converted into the above-mentioned acid addition salts, metal salts or ammonium salts. Since the compounds of the present invention exhibit remarkable multiplication-suppressive actions against tumor cells and have low toxicity, the pharmaceuticals of the present invention containing them as the active ingredient are useful as anticancer agents. The anticancer compositions of the present invention can be in forms such as powders, granules, tablets, sugar-coated tablets, capsules, syrups, suppositories, medicines for external use, injections and medicines for instillation in a mixture of an effective amount of the compound (I) and pharmaceutically acceptable carriers, excipients and diluents. While 2'-deoxy-2'-methylidenecytidine is not soluble in saline at the concentration of 25 mg/ml at room temperature, the compound of Example 1 is easily soluble under the same conditions. The anticancer compositions of the present invention can be orally or non-orally administered to mammals including human. Any dosage is preferable so long as the compounds can inhibit the objective cancers effectively at the dosage. While the dosage varies depending on the animal to be treated, the kind of the cancer, the administration route and the composition form the daily dosage is generally 10 - 400 mg/kg body weight, preferably 50 - 200 mg/kg body weight, in the case of medicine for oral administration and 1 - 20 mg/kg body weight, preferably 1 - 5 mg/kg body weight, in the case of injections. The administration frequency can be suitably selected in the range from once to 4 times a day. The anticancer compositions of the present invention can be used in combination with the other anticancer agents, immunostimulant or other acceptable medicaments. PREFERED EMBODIMENTS: The invention will be hereinbelow described more specifically by way of examples. Example 1 (1) 5-Fluorouridine, 2.62 g, was dissolved in 30 ml of pyridine, 3.3 ml of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane was added at 0°C, and the mixture was stirred at 0°C for 2 hours and then at room temperature for 1.5 hours. By adding 5 ml of water, the solvent was distilled off under reduced pressure. After 100 ml of ethyl acetate and 100 ml of water were added to partition the solution, the organic layer was washed with water twice and then, dried over sodium sulphate and the solvent was distilled off under reduced pressure. The residue was packed into a silica gel column (diameter, 2.4x23 cm) and eluted with 25% ethyl acetate/ hexane to afford 3',5'-0-tetraisopropyldisiloxanyl-5-flurouridine as a syrupy substance. Yield: 4.24 g, 83.9%; Mass (m/z): 504(M + ), 461(M + -isopropyl) (2) The compound obtained in (1) above, 5 g, was dissolved in 10 ml of methylene chloride and mixed with 4 equivalents of chromic acid complex (4 g of chromium oxide, 6.67 ml of pyridine, and 4 ml of acetic anhydride in 100 ml of methylene chloride). The mixture was stirred at room temperature for 1 hour and added dropwise to 500 ml of ethyl acetate and the solution was filtered through a silica gel filter (diameter, 6.0x1.5 cm). The filtrate was distilled off under reduced pressure and the residue was partitioned by 200 ml of ethyl acetate and 200 ml of water. The organic layer was washed with water, dried over sodium sulphate, and the solvent was distilled off under reduced pressure. The residue was placed into a silica gel column (diameter, 2.4x28 cm), eluted with 20% ethyl acetate/hexane, and crystallized from hexane to give 3',5'-0-tetraisopropyldisiloxanyl-2'-oxo-5-fluorouridine. Yield: 4.3 g, 86%; M.p,183-186°C; Mass(m/z): 502(M + ) Elementary analysis : for C 21 H 35 N 2 O 7 FSi 2 : Calculated C, 50.17; H, 7.02; N, 5.57 Found C, 50.29; H, 7.33; N, 5.58 (3) Methyltriphenylphosphonium bromide, 8.57 g, was suspended in 80 ml of tetrahydrofuran under argon gas stream, 15.1 ml of n-butyllithium (1.58 mol/µℓ hexane solution) was added dropwise at 0°C, and the mixture was stirred at room temperature for 1 hour. A solution of 3.02 g of the compound obtained in (2) above in 25 ml of tetrahydrofuran was added dropwise under argon gas stream at 0°C. After 20 minutes, the temperature was restored to room temperature, at which stirring was continued for 3 hours. Neutralization was conducted with 1 N ammonium bromide and the solution was concentrated. The solution was partitioned with 100 ml of ethyl acetate and 100 ml of water, and the organic layer was washed with water, dried over sodium sulphate and distilled off under diminished pressure. The residue was placed into a silica gel column (diameter, 3.5x18 cm) and eluted with 20% ethyl acetate/hexane to afford a syrupy product of 3',5'-0-tetraisopropyldisiloxanyl-2'-deoxy-2'-methylidene-5-fluorouridine. Yield: 2.43 g, 81%; Mass (m/z): 500(M + ) (4) Triethylamine, 1.4 ml, and 684 mg of 1,2,4-triazole were dissolved in 10 ml of acetonitrile and the solution was cooled under argon gas stream to 0°C. Phosphorus oxychloride, 280 µℓ, was added dropwise, and the mixture was stirred at room temperature for 1 hour. The precipitate was filtered off, and to the filtrate was added dropwise a solution of 500 mg of the compound obtained in (3) above in 5 ml of acetonitrile. After 3 hours at room temperature, the solution was stirred at 50°C for 1 hour and ammonia gas which had been passed over sodium hydroxide was passed through for 4 hours. The solution was partitioned with 100 ml of chloroform and 100 ml of water, and the aqueous layer was washed with chloroform twice. The collected chloroform layer was dried over sodium sulphate and distilled off under diminished pressure. The residue was placed on a column of silica gel (diameter, 3.5x14 cm) and eluted with 5% ethanol/chloroform to give 3',5'-0-tetraisopropyldisiloxanyl-2'-deoxy-2'-methylidene-5-fluorocytidine as a syrupy substance. Yield: 435 mg, 87%; Mass(m/z): 499(M + ) (5) The compound obtained in (4) above, 400 mg, was dissolved in 10 ml of tetrahydrofuran, 1.8 ml of tetra-n-butylammonium floride was added, and the mixture was stirred at room temperature for 30 minutes. After neutralization with acetic acid, the solvent was distilled off under reduced pressure. The residue was packed into a silica gel column (diameter, 2.4x18 cm), eluted with ethanol/chloroform, and crystallized from ethanol/ethyl acetate to give 2'-deoxy-2'-methylidene-5-fluorocytidine. Yield: 177 mg, 86.2%; M.p. 185-187°C; Mass(m/z): 257(M + ) Elementary analysis: for C 10 H 12 N 3 O 4 F: Calculated: C, 46.70; H, 4.70; N, 16.34 Found : C, 46.06; H, 4.67; N, 16.00 Example 2 2'-Deoxy-2'-methylidene-5-fluorocytidine and phosphorus oxychloride are made to react and treated in a usual manner to give 2'-deoxy-2'-methylidene-5-fluorocytidine-5'-phosphate. Pharmaceutical Preparation Example 1 Tablets comprising: Compound of Example 1 50.0 mg Finely divided cellulose 25.0 mg Lactose 49.5 mg Starch 40.0 mg Talc 5.0 mg Magnesium stearate 0.5 mg The tablets may be subjected to sugar coating or film coating treatment, if desired, thereby to obtain sugar-coated tablets or film-coated tablets. Pharmaceutical Preparation Example 2 Capsules encapsulated with the ingredients: Compound of Example 1 50.0 mg Lactose 50.0 mg Starch 15.0 mg Talc 5.0 mg Pharmaceutical Preparation Example 3 Fine powder comprising: Compound of Example 1 10% Lactose 80% Starch 10% Pharmaceutical Preparation Example 4 Granules comprising: Pharmaceutical Preparation Example 5 Compound of Example 1 50.0 mg Glucose 100.0 mg The above ingredients are dissolved in purified water to prepare 2 ml of a injection solution. Pharmaceutical Preparation Example 6 Suppository comprising: Compound of Example 1 100 mg Witepsole H 15 950 mg Witepsole E 75 950 mg Pharmaceutical Preparation Example 7 Compound of Example 1 2 g Ethyl para-hydroxybenzoate 0.025 g Propyl para-hydroxybenzoate 0.015 g Sodium laurylsulphonate 1.5 g Propylene glycol 12.0 g Stearyl alcohol 22.0 g White vaseline 25.0 g The above ingredients are dissolved in purified water to prepare 100.0 g of hydrophilic ointment. Pharmacological Experiments: Effect of the compounds on in vitro cell growth of various tumor cell lines: The assay of cytotoxicity is dependent on the cellular reduction of MTT [3'-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by the mitochondrial dehydrogenase of viable cells to a blue formazan product which can be measured spectrophotometrically. Cell suspension, 100 µl, containing 10 4 tumor cells with or without test sample was incubated for 72 h in 5% CO 2 -95% air at 37°C in 96-well microculture plates (Falcol 3072). The 10 µl of MTT(5 mg/ml) in saline was added to each culture well and incubated for 4 hr at 37°C. The formazan crystals were solubilized by the addition of 100 µl of 10% SDS/0.01 N HC1 to each well, and incubated for 24 hr at 37°C. The plates were then read at 570 nm on the plate reader. The percentage of cytotoxicity was calculated from the formula: 2'-Deoxy-2'-methylidene-5-fluorocytidine was dissolved in distilled water, and diluted in RPM1 1640 medium. Tumor cell lines were maintained in RPM1 1640 medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan). Table 1 below shows effect of 2'-deoxy-2'-methylidene-5-fluorocytidine on the growth of various tumor cell lines. IC 50 values gave the concentrations showing the 50% cytotoxity. Effect of 2'-deoxy-2'-methylidene-5-fluorocytidine on the growth of various tumor cell lines Tumor cell line and its origin IC 50 (µg/ml) L1210/c; mouse leukemia 0.33 B16; mouse melanoma 6.9 CCRF-CEM; human T cell leukemia 0.14 MOLT4; human T cell leukemia 0.036 U937; human histiocytic leukemia 0.44 HL60; human promyelocytic leukemia 0.040 PC13; human lung large cell carcinoma 1.0

Claims: A 2'-deoxy-2'-methylidene-5-fluorocytidine of the general formula: wherein R 3 stands for hydrogen or a phosphoric acid residue or a salt thereof. An anticancer composition which is characterized by containing 2'-deoxy-2'-methylidene-5-fluorocytidine of the general formula: wherein R 3 stands for hydrogen or a phosphoric acid residue, or a salt thereof as the active ingredient.

Inventors: FUJII AKIHIRO, MATSUDA AKIRA, SASAKI TAKUMA, UEDA TOHRU, YAMAGAMI KEIJI, FUJII, AKIHIRO, MATSUDA, AKIRA, SASAKI, TAKUMA, UEDA, TOHRU, YAMAGAMI, KEIJI, MATSUDA, AKIRA 12-1-7-501, KITA 24-JO NISHI
Applicants: YAMASA SHOYU KK, YOSHITOMI PHARMACEUTICAL, YAMASA SHOYU CO., LTD., YOSHITOMI PHARMACEUTICAL INDUSTRIES, LTD.
Application year: 1993
Main classification: C07H19
IPC codes: C07H19