AGENTS PROMOTING THE ACTIVITY OF SOME ANTITUMOR AGENTS AND METHODS FOR THEIR PRODUCTION

Abstract: This invention relates to new and useful 2,6-dimethyl-4-­ (pyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-di­ carboxylate derivatives of the following formula; and pharmaceutically acceptable salts thereof having the potent promoting activity of some antitumor agents against various kinds of tumor cells including multiple drug resistant tumor cells.

Description: This invention relates to new and useful 1,4-dihydropyridine derivatives promoting the activity of some antitumor agents against various kinds of tumor cells including multiple drug resistant cells and also to their use, and further, to processes for the preparation of the novel derivatives. In more detail, this invention relates to new 2,6-dimethyl-4-(pyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate derivatives of the formula (I) and their hydrates and salts, wherein R₁ is a lower alkyl group, R₂ is a hydrogen atom, halogen atom, lower alkoxy, or a nitro group, and R and R₃ are each independently alkyl, lower alkoxyalkyl, aralkyl group or (A is straight or branched alkylene group, and R₄ and R₅ are each independently a hydrogen atom, lower alkyl, aralkyl group or connected with each other to make five- or six-membered saturated heterocycles which may contain other hetero atoms, to processes for their preparation and to their use in cancer chemotherapy. The term"alkyl group" as it is employed herein includes a straight or branched chain containing 1 to 10 carbon atoms, e.g. a methyl, isopropyl,or octyl group. The term "lower alkyl group" includes a straight or branched chain containing between 1 and 6 carbon atoms, e.g. a methyl, ethyl or isopropyl group. The term "aralkyl group" includes a lower alkyl group which is substituted by an aryl group, e.g. benzyl or phenethyl. The term "lower alkoxyalkyl group" includes an alkyl group which is substituted by a straight or branched chain containing 1 to 6 carbon atoms, e.g. a methoxymethyl or methoxyethyl group. The term "straight or branched alkylene group" includes, e.g. an ethylene or 2-methylpropylene group. The term "five- or six-membered saturated heterocycle" which may contain other hetero atoms includes, e.g. piperazine, pyrrolidine,piperidine, morpholine or thiomorpholine. The term "halogen atom" includes e.g. fluorine, chlorine and bromine. It is going on with new trials which stamp out cancerous growth by using various novel antitumor agents. However, an appearance of resistance to antitumor drugs is a serious problem in the clinical treatment of various tumor and therefore, the circumvention of such resistance is an important subject in the cancer chemotherapy. Further, there are some intricate factors which make these problems more difficult to solve. In other words, the tumor cells resistant to one of the antitumor agents show cross-resistance to the other antitumor agents (Multiple Drug Resistance : MDR), and overcoming this type of resistance is more difficult in the clinical cancer chemotherapy. Tsuruo et al . (CANCER RES., 44 , 4303, 1984; 43 , 2267, 1983) reported that the drugs used clinically as calcium channel-blocker, calmodulin inhibitor and antiarrhythmic agent promoted the activity of antitumor agents against resistant tumor cells. Practically, it was found that verapamil, caroverine, clomipramine, trifluoperazine, prenylamine, No. 233 and quinidine enhanced the activity of antitumor agents in vitro against the resistant sub-lines of mouse leukemia cells (P388/VCR, P388/ADR) and also verapamil enhanced the antitumor activity in vivo . Tsuruo et al . (CANCER RES., 43 , 2905, 1983) also reported that the diltiazem and nicardipine as calcium influx antagonist and quinidine as antiarrhythmic agent promoted the activity of antitumor agents against mouse P388 cells resistant to VCR or ADR (P388/VCR, P388/ADR) in vitro and in vivo . Promoting agents of antitumor activity reported so far mainly belong to calcium antagonists or blockers. Intracellular calcium ions are the chemical mediator which carries on the physiological function in some particular tissues, that is, plays an important role in the excitation-contraction coupling of heart muscle and also in the excitation-contraction coupling of vascular smooth muscle. Intracellular distribution and transport of calcium ion play a key role in their physiological function. The pharmacological action of various calcium antagonists is induced as a consequence of inhibition of calcium influx or function. However, with respect to therapeutic efficacy of the calcium blockers as a promotor of antitumor agents, the striking action of these calcium blockers becomes a factor restricting their usefulness due to serious side-effects. Therefore, their clinical application is limited. This invention as a result of analyzing these various factors described above, provides novel compounds with following characteristic properties, a) They show low toxicity, negligible Ca-antagonizing and hypotensive activities, and therefore, it may be easy to bring in clinical application different from a calcium blocker. b) They are able to completely reverse very high resistance to anthracyclines or vinca-alkaloids in vitro at as low concentration as 0.5-3.0 µg/ml. c) This combined treatment with both the antitumor agents and these derivatives clearly induces synergistic antitumor effects in therapeutic experiments. d) This combination of relatively low doses of antitumor agents and the compound of this invention in an as high dose as possible is an optimal treatment regimen. Therefore, it may be useful for preventing the serious side-effects of antitumor agents in clinical chemotherapy, because of reducing the dose of the antitumor agents. e) This combination treatment exhibits not only clear synergistic effects, but also curable effect with long survivor against non-resistant tumor cells, indicating that these novel derivatives are able to overcome the heterogeneity as to sensitivity to antitumor agents in such a cell propulation. In conclusion, this type of combination treatment may have therapeutic efficacy not only at the late stage when tumor acquired resistance, but also at the initial stage of chemotherapy. In the following the processes for the preparation of the compounds of the invention are explained. wherein R₁ is lower alkyl group, R₂ is a hydrogen atom, halogen atom, lower alkyl, lower alkoxy, or nitro group, and R and R₃ are each independently alkyl, lower alkoxyalkyl, aralkyl group, or (A is straight or branched alkylene group, and R₄ and R₅ are each independently a hydrogen atom, lower alkyl, aralkyl group, or connected with each other to make five- or six-membered heterocycles which may contain other hetero atom). (1) Initially, the starting compounds represented by the formula (II) are formylated in the presence of formylation agents such as phosphorus oxychloride-dimethylformamide (DMF), and so on, to give the compounds represented by the formula (III). (2) The compounds represented by the formula (III) are condensed with the compounds represented by the formula (IV) in the presence of condensing agents to give the compounds represented by the formula (V). The suitable condensing agents of this reaction are organic and inorganic acids as acid catalyst, for example, hydrochloric acid or acetic acid and amines as base catalyst, for example, piperidine or triethylamine, and benzene, toluene, chloroform, dimethylformamide, tetrahydrofuran, alcohol, or the like are used as solvent. The reaction temperature is selected appropriately in a range of 50-150 °C and the reaction time is selected appropriately in a range of 0.5-15 hours. In more detail, it is preferable to react compounds represented by the formula (III) with 0.8 to 1.5 times mole of compounds represented by the formula (IV). (3) The compounds represented by the formula (V) are condensed with the compounds represented by the formula (VI) to give the compounds represented by the formula (I) in the presence or absence of the solvent by heating. The suitable solvent of this reaction is benzene, toluene, chloroform, dimethylformamide, tetrahydrofuran, alcohols such as isopropylalcohol, or the like. In more detail, it is preferable to react compounds represented by the formula (V) with 0.8 to 1.2 times mole of compounds represented by the formula (VI) for 3 to 20 hours at 50-150 °C. Furthermore, the compounds of the formula (I) can be converted, if desired, to pharmaceutically acceptable acid salts by the treatment with acid wherein either of R and R₃ are a group. The acid may be an organic or inorganic acid such as, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, methanesulfonic acid, oxalic acid, fumaric acid or lactic acid. The compound of the formula (I), hydrates and salts thereof may be used as medicine in the conventional form of pharmaceutical preparations, which may be, for example, tablets, capsules, powder, ointment, suppositories, syrup, liquor, suspension, spray or injection, suitable for peroral, parenteral, enteral or local administration. The following examples will further illustrate the present invention without, however, limiting it thereto. Example 1 Dimethyl 2,6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]-pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate a) Methyl 2-acetyl-3-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-acrylate To a solution of 4 g of 3-formyl-2-isopropylpyrazolo[1,5-a]-pyridine, 2.7 g of methyl acetoacetate and 256 mg of glacial acetic acid in 11 ml of benzene was added dropwise a solution of 0.36 g of piperidine in 2.2 ml of benzene by a fifth at 20 minutes intervals under stirring at room temperature. After the addition was completed, the mixture was refluxed for 4 hours while resulting azeotropic water was separated by isobaric distillation. Then 40 ml of benzene was added, and washed with 20 ml of saturated sodium hydrogencarbonate, 20 ml of water, and 20 ml of salt water. The benzene solution was dried over anhydrous barium sulfate, and then concentrated to give the title compound in a yield of 5.9 g as a crude dark reddish liquid. Silica gel thin layer chromatography of the title compound gave Rf=0.36 value using benzene as an eluent. NMR (δ in CDCl₃), 7.66 (1H, s), 6.45-8.33 (4H, m). 3.89 (3H, s), 3.34 (1H, m), 2.46 (3H, s), 1.34 (6H, d, J=6.59 Hz) b) A mixture of 5.9 g of methyl 2-acetyl-3-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)acrylate and 2.5 g of methyl 3-aminocrotonate was stirred for 4.5 hours at 60-65 °C, and then for 8.7 hours at 100-105 °C. The reaction mixture was purified with silica gel column chromatography using benzene-ethyl acetate mixtures as an eluent. The eluate was concentrated and recrystallized from hexane-benzene mixtures to give the title compound in a yield of 2.6 g (31 %) as yellow needles, mp 244-246 °C. Analysis (%) for C₂₁H₂₅N₃O₄, Calcd. (Found): C, 65.78 (65.91); H, 6.57 (6.59); 10.96 (10.83). Using the procedure described in Example 1, following new compounds have been obtained. Example 2 Methyl ethyl 2,6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate Yield 22 %, mp 219-221.5 °C. Analysis (%) for C₂₂H₂₇N₃O₄, Calcd. (Found): C, 66.48 (66.73); H, 6.85 (6.87); 10.57 (10.58). Example 3 Butyl methyl 2,6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3-5-dicarboxylate Yield 21 %, mp 160-161 °C. Analysis (%) for C₂₄H₃₁N₃O₄, Calcd. (Found): C, 67.74 (66.79); H, 7.34 (7.46); 9.87 (9.91). Example 4 tert-Butyl methyl 2-6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1-4-dihydropyridine-3,5-dicarboxylate Yield 8 %, mp 205.5-206 °C. Analysis (%) for C₂₄H₃₁N₃O₄, Calcd. (Found): C, 67.74 (66.75); H, 7.34 (7.41); 9.87 (9.91). Example 5 Methyl octyl 2,6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate Yield 20 %, mp 154-156 °C. Analysis (%) for C₂₈H₃₉N₃O₄, Calcd. (Found): C, 69.83 (69.81); H, 8.16 (8.24); 8.72 (8.79). Example 6 N-Benzyl-N-methyl-2-aminoethyl methyl 2,6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate Yield 14 %, mp 172-175 °C. Analysis (%) for C₃₀H₃₆N₄O₄·0.1 C₆H₆, Calcd. (Found): C, 70.08 (69.90); H, 7.03 (7.07); 10.68 (10.47). Example 7 Methyl 2-dimethylaminoethyl 2,6-dimethyl-4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate Yield 6 %, mp 136-138 °C. Analysis (%) for C₂₄H₃₂N₄O₄, Calcd. (Found): C, 65.43 (65.44); H, 7.32 (7.48); 12.72 (12.98). Example 8 Methyl 2-(4-methyl-1-piperazinyl)ethyl 2,6-dimethyl4-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridne-3,5-dicarboxylate Yield 6 %, mp 102-104 °C. Analysis (%) for C₂₇H₃₇N₅O₄·0.3 C₆H₆, Calcd. (Found): C, 66.64 (66.78); H, 7.53 (7.54); 13.49 (13.25). Example 9 Methyl 2-(N-benzyl-N-methylamino)ethyl 2,6-dimethyl-4-(2-methylpyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate a) 3-Formyl-2-methylpyrazolo[1,5-a]pyridine To 116.6 ml of dimethylformamide was added 105 ml of phosphorus oxychloride, followed by 99 g of 2-methylpyrazolo[1,5-a]pyridine under stirring at keeping the temperature below 20 °C. The reaction mixture was stirred at 50-60 °C for 1 hours, then poured into ice water. Acidic aqueous solution was neutralized with potassium carbonate, then extracted with dichloromethane, washed with salt water, dried over anhydrous sodium sulfate and then concentrated. The resulting residue was recrystallized from methanol to give the title compound in a yield of 54.8 g (46 %) as colorless crystals, mp 77-79 °C. b) Methyl 2-acetyl-3-(2-methylpyrazolo[1,5-a]pyridine-3-yl)acrylate A solution of 54 g of 3-formyl-2-methylpyrazolo[1,5-a]pyridine, 47.1 g of methyl acetoacetate, 20.6 g of benzoic acid and 2.9 g of piperidine in 200 ml of toluene was refluxed for 9 hours while resulting azeotropic water was separated by isobaric distillation, by adding 2.9 g of piperidine at 3 hour intervals. The reaction mixture was washed with saturated sodium hydrogenecarbonate, then salt water, dried over anhydrous sodium sulfate and then concentrated. After cooling, resulting crystals were removed by filtration to give the title compound in a yield of 40.7 g as a crude dark reddish liquid. c) A solution of 24.9 g of methyl 2-acetyl-3-(2-methylpyrazolo[1,5-a]pyridin-3-yl)acrylate and 28.8 g of 2-(N-benzyl-N-methylamino)ethyl 3-aminocrotonate in 125 ml of isopropylalcohol was refluxed for 8.5 hours, then concentrated. The resulting residue was dissolved in 100 ml of ethyl acetate, washed with 1 N-hydrochloric acid, and then extracted with 2 N-hydrochloric acid three times. The resulting hydrochloric acid layer was alkalized with 5 N-aqueous sodium hydroxide at pH 9, and then extracted with ethyl acetate. The ethyl acetate layer was washed with salt water, dried over anhydrous sodium sulfate and then concentrated. Hexane was added to the resulting residue, and insoluble layer was collected, then recrystallized from diethyl ether-hexane mixtures. The crystals were separated from the solution by filtration, washed with diethyl ether, and then recrystallized from benzenehexane mixed solvent to give the title compound in a yield of 1.6 g (3 %) as yellow ocher crystals, mp 118-120 °C. Analysis (%) for C₂₈H₃₂N₄O₄, Calcd. (Found): C, 68.83 (68.85); H, 6.60 (6.72); N, 11.47 (11.35). The utility of this invention Experiment 1 It was observed that the derivatives of this invention promoted the activity of antitumor agents, and the potency was equal to those of verapamil and nifedipine in vitro culture of P388/VCR and P388/ADR cells. The derivatives showed low values (pA₁₀ 5.6-6.1) of calcium antagonizing activity as compared with values of verapamil (pA₁₀: 7.5) and nifedipine (pA₁₀: 8.8) by assessing the dose-dependent relaxing effect using the Magnus's method with isolated preparation of guinea-pig caecum strip. From the results of examination on the blood pressure of SHR rats, verapamil and nifedipine as positive control demonstrated the typical effects of calcium antagonist, that is, observed the depression of 41 and 25 mmHg, respectively. However, the derivatives of this invention did not show hypotensive activity in the same experiment (Table 1). Experiment 2 One million P388/VCR cells were inoculated intraperitoneally into female CDF₁ mice. VCR (0.1 mg/kg) or Example 6 or verapamil (at doses indicated in Table 2) dissolved in sterile physiological saline were administered twice a day (in the morning and evening) intraperitoneally for 5 consecutive days. Compound of this invention demonstrated significantly better life-prolongation effect in the combined treatment with VCR than that of verapamil. The resistance to VCR of these resistant tumor cells was almost entirely reversed by the compound of this invention (Table 2). Experiment 3 The therapeutic effect of the compound of this invention was examined against P388 parent line (sensitive to VCR) in the same condition described in Experiment 2. A dose of VCR (0.1 mg/kg) alone produced about twice life-prolongation effect as compared with the control group, but no cured mouse was observed. On the other hand, combination treatment with VCR and the compound of this invention resulted in not only the highest life-prolongation effect but also cured mice (one-sixth mouse at dose of 50 × 2 mg/kg and three-sixth mice at dose of 75 × 2 mg/kg, respectively).

Claims: Claims for the following Contracting States : BE, CH, DE, FR, GB, IT, LI, NL, SE 2,6-dimethyl-4-(pyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate derivatives of formula (I) wherein R and R₃ are each independently a straight or branched alkyl chain containing 1 to 10 carbon atoms, a straight or branched alkyl chain containing 1 to 10 carbon atoms which is substituted by a straight or branched alkoxy chain containing 1 to 6 carbon atoms, a straight or branched alkyl chain containing 1 to 6 carbon atoms which is substituted by an aryl group, or (A is straight or branched alkylene group, and R₄ and R₅ are each independently a hydrogen atom, a straight or branched alkyl chain containing 1 to 6 carbon atoms, a straight or branched alkyl chain containing 1 to 6 carbon atoms which is substituted by an aryl group or connected with each other to make five- or six-membered saturated heterocycles which may contain other hetero atom), R₂ is a hydrogen atom, halogen atom, a straight or branched alkyl chain containing 1 to 6 carbon atoms, a straight or branched alkoxy chain containing 1 to 6 carbon atoms or a nitro group, and R₁ is a straight or branched alkyl chain containing 1 to 6 carbon atoms, and the pharmaceutically acceptable salts thereof. A process for the preparation of novel 2,6-dimethyl-4-(pyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate derivatives of formula (I) wherein R, R₁, R₂, and R₃ have the meaning specified in claim 1, which comprises reacting by heating 2-acetyl-3-(pyrazolo[1,5-a]pyridin-3-yl)acrylate derivatives of formula (V) wherein R, R₁, and R₂ have the meaning specified in claim 1, with 3-aminocrotonate derivatives of formula (VI) wherein R₃ has the meaning specified in claim 1. A process for the preparation of the compound of formula (I) which comprises reacting a 3-formylpyrazolo[1,5-a]pyridine derivative of formula (III) wherein R₁ and R₂ have the meaning specified in claim 1, with an acetoacetate derivative of formula (IV) wherein R has the meaning specified in claim 1, and a 3-aminocrotonate derivative of formula (VI) wherein R₃ has the meaning specified in claim 1. The compound of claim 1 for use as a pharmaceutically active compound. The compound of claim 1 for use as a pharmaceutically active compound in the treatment of tumors including multiple drug resistant tumors. A pharmaceutical composition containing at least one compound of claim 1 and optionally an inert pharmaceutically acceptable carrier. A pharmaceutical composition for promoting the activity of antitumor agents against tumors including multiple drug resistant tumors in tumor patients containing at least one compound of claim 1 and optionally an inert pharmaceutically acceptable carrier. A pharmaceutical composition for the treatment of tumors including multiple drug resistant tumors containing at least one compound of claim 1 and at least one antitumor agent the activity of which is promoted by said compound(s) and optionally an inert pharmaceutically acceptable carrier. Use of a compound of claim 1 for the production of a pharmaceutical composition according to anyone of claims 6 - 8. A potent effective composition which possesses the activity of some antitumor agents against various kinds of tumor cells including multiple drug resistant tumor cells, containing the compound according to claim 1 and an inert pharmaceutically acceptable carrier. Claims for the following Contracting State : ES A process for the preparation of 2,6-dimethyl-4-(pyrazolo[1,5-a]pyridin-3-yl)-1,4-dihydropyridine-3,5-dicarboxylate derivatives of formula (I) wherein R and R₃ are each independently a straight or branched alkyl chain containing 1 to 10 carbon atoms, a straight or branched alkyl chain containing 1 to 10 carbon atoms which is substituted by a straight or branched alkoxy chain containing 1 to 6 carbon atoms, a straight or branched alkyl chain containing 1 to 6 carbon atoms which is substituted by an aryl group, or (A is straight or branched alkylene group, and R₄ and R₅ are each independently a hydrogen atom, a straight or branched alkyl chain containing 1 to 6 carbon atoms, a straight or branched alkyl chain containing 1 to 6 carbon atoms which is substituted by an aryl group or connected with each other to make five- or six-membered saturated heterocycles which may contain other hetero atom), R₂ is a hydrogen atom, halogen atom, a straight or branched alkyl chain containing 1 to 6 carbon atoms, a straight or branched alkoxy chain containing 1 to 6 carbon atoms or a nitro group, and R₁ is a straight or branched alkyl chain containing 1 to 6 carbon atoms, or of the pharmaceutically acceptable salts thereof, which comprises reacting by heating 2-acetyl-3-(pyrazolo[1,5-a]pyridin-3-yl)acrylate derivatives or formula (V) wherein R, R₁, and R₂ have the meaning specified above with 3-aminocrotonate derivatives of formula (VI) wherein R₃ has the meaning specified above. A process for the preparation of the compound of formula (I) as defined in claim 1 which comprises reacting a 3-formylpyrazolo[1,5-a]pyridine derivative of formula (III) wherein R₁ and R₂ have the meaning specified in claim 1, with an acetoacetate derivative of formula (IV) wherein R has the meaning specified in claim 1, and a 3-aminocrotonate derivative of formula (VI) wherein R₃ has the meaning specified in claim 1. A process for the preparation of a pharmaceutical composition which comprises combining at least one compound of claim 1 and an inert pharmaceutically acceptable carrier. Process according to claim 3 whereby the pharmaceutical composition is for promoting the activity of antitumor agents against tumors including multiple drug resistant tumors in tumor patients. A process for the preparation of a pharmaceutical composition for the treatment of tumors including multiple drug resistant tumors which comprises combining at least one compound of claim 1 and at least one antitumor agent the activity of which is promoted by said compound(s).

Inventors: IINUMA, FUJIO, INABA, MAKOTO / C/O JAP. FOUND. CANCER RESEARCH, SHINODA, HIROTAKA
Applicants: JAPANESE FOUNDATION FOR CANCER RESEARCH, KYORIN SEIYAKU KABUSHIKI KAISHA
Application year: 1987
Main classification: C07D471
IPC codes: A61K31, A61P35, C07D471