JPH0692401B2 - 1,4-dihydropyridine derivative - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel 1,4-dihydropyridine derivative having excellent pharmacological activity, and more specifically, a 1,4-dihydropyridine derivative effective as a therapeutic agent for tumors and the like. Regarding

[Conventional technology]

Many compounds are already known as 1,4-dihydropyridine derivatives. Many of these known 1,4-dihydropyridine derivatives are known to have pharmacological activity, but most of them have pharmacological activity on the circulatory system. Regarding the pharmacological activity, only a few substances having anti-inflammatory action, hepatoprotective action, etc. have been reported.

On the other hand, as for the 1,4-dihydropyridine derivative having some pharmacological activity against tumor, there is a 1,4-dihydropyridine compound having no substituent at the 4-position in Japanese Patent Publication No. 55-500577. It has been described that it has a metastasis-inhibiting effect on tumors of Further, JP-A-60-6613 describes an antitumor and antitumor metastasis agent containing 1,4-dihydropyridine such as nifedipine and nimodipine as an active ingredient. Further, JP-A-62-87516 describes a method for treating a malignant tumor in which a platinum coordination compound and a compound such as nifedipine or nimodipine are used in combination.

[Problems to be Solved by the Present Invention]

However, the compounds described in the above publications are
A 1,4-dihydropyridine compound having no substituent at the 4-position or having a substituted phenyl group such as a nitrophenyl group bonded at the 4-position, and a 1,4-dihydropyridine compound having a special heterocycle such as dithiene bonded at the 4-position -No mention is made of dihydropyridine derivatives.

For the 1,4-dihydropyridine derivative, the present inventors
As a result of extensive screening for a combination effect with an antitumor drug, surprisingly, it was found that a certain compound has an action of significantly increasing the sensitivity of tumor cells, particularly tumor cells that have acquired resistance, to an antitumor drug. The heading has arrived at the present invention.

[Means for solving problems]

According to the invention, the formula (I) (In the formula, R ₁ represents a hydrogen atom or a C ₁ -C ₃ alkyl group,
R ₂ is a C _{1 to} C ₆ alkyl group, preferably a C _{1 to} C ₅ alkyl group,
More preferably, it represents a C ₃ -C ₅ alkyl group, and n is 2-4.
R ₃ represents a hydrogen atom or a C ₁ -C ₃ alkyl group, R ₁ represents a hydrogen atom, R ₂ represents a methyl group, n represents an integer 2, and R ₃ represents a hydrogen atom. A 1,4-dihydropyridine derivative represented by

In the present invention, among the 1,4-dihydropyridine derivatives represented by the above formula (I), those having a particularly remarkable drug sensitivity enhancing action are compounds in which R ₂ is a C ₃ -C ₅ alkyl group.

As will be described in detail in Test Examples below, the compounds having these specific substituents have a more remarkable drug-sensitivity-enhancing action as compared with other compounds, and are particularly useful compounds as pharmaceuticals.

Each of the 1,4-dihydropyridine derivatives represented by the above formula (I) can be produced by utilizing a well-known reaction conventionally used for producing 1,4-dihydropyridines. For example, a compound of the formula (I) in which R ₃ is a hydrogen atom is represented by the formula (II) (In the formula, R ₁ represents a hydrogen atom or a C _{1 to} C ₃ alkyl group)
Is reacted with the β-aminocrotonic acid ester and acetoacetic acid ester by heating or heating under reflux in the presence or absence of an organic solvent (method A), or represented by the formula (II) It can be produced by reacting an aldehyde with acetoacetic ester and aqueous ammonia in the presence or absence of an organic solvent by heating, preferably by heating under reflux (method B). Further, in the compound of formula (I), wherein R ₃ is a C ₁ -C ₃ alkyl group, the target compound obtained by the above method A or method B is further added with sodium hydride in an organic solvent. It can be produced by reacting with a C ₁ -C ₃ alkyl chloride in the presence of (method C). These reactions are shown in reaction formulas as follows.

The reaction used in these production methods is conventionally 1,4-
Known reactions used in the production of dihydropyridine compounds (for example, reactions used in the methods described in JP-B-46-40625, JP-A-56-37225 and JP-A-60-214786). ) Is basically the same. Therefore, the 1,4-dihydropyridine derivative of the present invention can be produced by appropriately applying other reactions described in these known documents in addition to the above method.

The raw material compounds used in the above production method are all known compounds except for some aldehydes, and can be easily obtained or produced by those skilled in the art as needed. That is, both acetoacetic acid ester and β-aminocrotonic acid ester are compounds that are commonly used as raw materials for producing 1,4-dihydropyridine compounds,
If necessary, a commercial product can be obtained at any time and can be easily synthesized. The aldehyde represented by the formula (II) is 1,4-dithiene or 1,4-dithiene substituted with a lower alkyl group at the 2-position as a raw material, which is reacted with dimethylformamide and phosphorus oxychloride. It can be produced by hydrolyzing the obtained product.

According to the present invention, the reaction product produced by the above method,
That is, the 1,4-dihydropyridine derivative represented by the formula (I) can be separated from the reaction mixture and purified by a conventional method, for example, extraction with a solvent, chromatography, crystallization and the like.

〔Example〕

Next, reference examples and examples are shown, and 1,4-
An example of the synthesis of a dihydropyridine derivative and the results of a pharmacological test conducted to confirm its usefulness will be described.
It goes without saying that the scope of the present invention is not limited to these examples.

Reference Example 1 Synthesis of 2-formyl-3-methyl-p-dithiene Phosphorus oxychloride 53 was added to 131 ml (1.70 mol) of dimethylformamide under ice-cooling stirring while maintaining the temperature of the solution at 0-5 ° C.
ml (0.565 mol) was added dropwise. Next, 62.2 g (0.471 mol) of 2-methyl-p-dithiene was added dropwise to the obtained reaction solution while maintaining the temperature of the solution at 10 ° C or lower, then gradually returned to room temperature, and further heated in an oil bath at 75 ° C. The reaction was completed by stirring with heating for 1 hour. After the reaction, the obtained reaction solution was poured into ice water, hydrolyzed with sodium hydrogen carbonate, and extracted with dichloromethane. The dichloromethane layer was dried over sodium sulfate, the solvent was distilled off, and the obtained crystals were recrystallized with a mixed solvent of n-hexane and dichloromethane to obtain 45.5 g (yield: 60.3%) of yellow target substance crystals. The analytical values of this substance are as follows.

Melting point: 79.5-81.0 ° C IR: ν ^KBr _max cm ^-1 1640 (C = O), 1200 (CO) NMR (CDCl ₃ , TMS, PPM) 2.45 (3H, s, CH ₃ ) 2.88-3.42 (4H , m, dithien ring SCH ₂ CH ₂ S) 9.91 (1H, s, CHO) Example 1 4- [2- (3-Methyl-5,6-dihydro-1,4-dithiynyl)]-2,6- Dimethyl-1,4-dihydropyridine-3,
Synthesis of 5-dicarboxylic acid bis (2-methoxyethyl) ester 2-formyl-3-methyl-1,4-dithiene 12.0 g (0.07
5 mol), acetoacetic acid-2-methoxyethyl ester 37.
5 g (0.234 mol) and 28% ammonia water 42 ml were dissolved in isopropyl alcohol 60 ml, and the mixture was heated under reflux for 5 days.
After cooling, the reaction solution was concentrated, the residue was dissolved in ethyl acetate, and impurities were extracted and removed with a small amount of water. The ethyl acetate layer was dried over sodium sulfate, the solvent was distilled off, and the residue was diluted with ethyl acetate.
Silica gel column chromatography was performed with a mixed solvent of -hexane to obtain crystals, which were then recrystallized from isopropyl alcohol to give 10.2 g of the target substance as pale yellow crystals (yield 3
0.7%) was obtained. The analytical values of this substance are as follows.

Melting point: 99.0 to 101.0 ° C IR: ν ^KBr _max cm ^-1 3300 (NH), 1680 (C = O), 1190, 1260 (CO) NMR (CDCl ₃ , TMS, PPM) 2.20 (3H, s, dithien Ring CH ₃ ) 2.28 (6H, s, 2,6 position CH ₃ ) 2.82-3.17 (4H, m, SCH ₂ CH ₂ S) 3.37 (6H, s, 2 × OCH ₃ ) 3.58 (4H, t, 2 × COOCH ₂ CH ₂ O) 4.26 (4H, t, 2 × COOCH ₂ CH ₂ ) 5.25 (1H, s, 4th H) 5.51 (1H, b, NH) Example 2 4- [2- (5,6- Dihydro-1,4-dithiynyl)]-2,
Synthesis of 6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid bis (3-methoxypropyl) ester 2-formyl-1,4-dithiene 7.30 g (0.050 mol), acetoacetic acid-3-methoxypropyl ester 26.1 g (0.15
Mol) and 7.5 ml of 28% aqueous ammonia were dissolved in 50 ml of isopropyl alcohol, and a sealed tube reaction was carried out at 130 ° C. for 20 hours. After cooling, the reaction solution was concentrated, the residue was dissolved in ethyl acetate, and impurities were extracted and removed with a small amount of water. The ethyl acetate layer was dried over sodium sulfate, the solvent was distilled off, and the residue was diluted with ethyl acetate.
Silica gel column chromatography was performed with a mixed solvent of -hexane to obtain crystals. Then recrystallize with a mixed solvent of ethanol and ether to obtain the target substance as pale yellow crystals.6.
53 g (yield 28.6%) was obtained. The analytical values of this substance are as follows.

Melting point: 104.5 to 105.5 ° C IR: ν ^KBr _max cm ^-1 3300 (NH), 1690 (C = O), 1200,1280 (CO) NMR (CDCl ₃ , TMS, PPM) 1.80-2.07 (4H, m , 2 × COOCH ₂ CH ₂ CH ₂ O) 2.31 (6H, s, 2,6th CH ₃ ) 3.04 (4H, s, SCH ₂ CH ₂ S) 3.35 (6H, s, 2 × OCH ₃ ) 3.50 (4H , t, 2 × COOCH ₂ CH ₂ CH ₂ O) 4.07−4.36 (4H, m, 2 × COOCH ₂ CH ₂ CH ₂ ) 4.72 (1H, s, 4th H) 5.98 (1H, s, vinyl H) 6.07 (1H, b, NH) Example 3 4- [2- (3-Methyl-5,6-dihydro-1,4-dithinyl)]-2,6-dimethyl-1,4-dihydropyridine-3,
Synthesis of 5-dicarboxylic acid bis (3-methoxypropyl) ester 2-formyl-3-methyl-1,4-dithien 5.00 g (0.03
1 mol), acetoacetic acid-3-methoxypropyl ester 1
6.9 g (0.097 mol) and 17% of 28% aqueous ammonia were dissolved in 30 ml of isopropyl alcohol and heated under reflux for 3 days. After cooling, the reaction solution was concentrated, the residue was dissolved in ethyl acetate, and impurities were extracted and removed with a small amount of water. After drying the ethyl acetate layer with sodium sulfate, the solvent was distilled off and the residue was subjected to silica gel column chromatography with a mixed solvent of ethyl acetate and n-hexane to obtain crystals, and then recrystallized with a mixed solvent of n-hexane and dichloromethane. Thus, 3.70 g (yield 25.7%) of the target substance was obtained as pale yellow crystals. The analytical values of this substance are as follows.

Melting point: 102.0 to 105.0 ° C IR: ν ^KBr _max cm ^-1 3320 (NH), 1690 (C = O), 1110,1200 (CO) NMR (CDCl ₃ , TMS, PPM) 1.73-2.10 (4H, m , 2 × COOCH ₂ CH ₂ CH ₂ O) 2.16 (3H, s, dithiene ring CH ₃ ) 2.27 (6H, s, 2,6 position CH ₃ ) 2.80-3.18 (4H, m, SCH ₂ CH ₂ S) 3.32 (6H, s, 2 × OCH ₃ ) 3.44 (4H, t, 2 × COOCH ₂ CH ₂ CH ₂ O) 4.19 (4H, t, 2 × COOCH ₂ CH ₂ CH ₂ O) 5.21 (1H, s, 4th place H) 5.72 (1H, b, NH) Example 4 1-Ethyl-4- [2- (5,6-dihydro-1,4-dithinyl)]-2,6-dimethyl-1,4-dihydropyridine-3 ,
Synthesis of 5-dicarboxylic acid bis (3-methoxypropyl) ester 4- [2- (5,6-dihydro-1,4-dithynyl)]-2,6-dimethyl-1,4-obtained in Example 3 Dihydropyridine
6.58 g (0.014 mol) of 3,5-dicarboxylic acid bis (3-methoxypropyl) ester was dissolved in 15 ml of dimethylformamide, and sodium hydride (60% in oil) 1.20 was added while stirring with ice cooling.
g (0.030 mol) and stir for 30 minutes, then ethyl iodide
12.0 g (0.078 mol) was added dropwise, and the mixture was reacted with stirring at room temperature for 1 hour. Ethyl acetate was added to the reaction solution, and impurities were extracted and removed with water. The ethyl acetate layer was dried over sodium sulfate, the solvent was distilled off, and the residue was subjected to silica gel column chromatography with a mixed solvent of ethyl acetate and n-hexane to obtain crystals, and then n
Recrystallization was performed with a mixed solvent of hexane and ether to obtain 3.19 g (yield 45.7%) of the target substance as pale yellow crystals. The analytical values of this substance are as follows.

Melting point: 45.0 to 47.0 ° C IR: ν ^KBr _max cm ^-1 1690 (C = O), 1670 (C = C), 1160,1200 (C-O) NMR (CDCl ₃ , TMS, PPM) 1.18 (3H, t , N−CH ₂ CH ₃ ) 1.80−2.10 (4H, m, 2 × COOCH ₂ CH ₂ CH ₂ O) 2.43 (6H, s, 2,6th CH ₃ ) 3.01 (4H, s, SCH ₂ CH ₂ S ) 3.33 (6H, s, 2 × OCH ₃ ) 3.48 (4H, t, 2 × COOCH ₂ CH ₂ CH ₂ O) 3.67 (2H, q, N−CH ₂ CH ₃ ) 4.10−4.30 (4H, m, 2 × COOCH ₂ CH ₂ CH ₂ O) 4.75 (1H, s, 4-position H) 5.85 (1H, s, vinyl H) Example 5 1-Ethyl-4- [2- (3-methyl-5,6-dihydro) Synthesis of bis (2-methoxyethyl) ester of 1,4-dithiynyl)]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid 4- [2- (3 -Methyl-5,6-dihydro-1,4-dithynyl)]-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid bis (2-methoxyethyl) ester 4.00 g (0.009 mol) 80 ml of dimethoxyethane After melting, stirring with ice cooling, 1.00 g (0.025 mol) of sodium hydride (60% in oil) was added and stirred for 30 minutes, then 3.2 g (0.021 mol) of ethyl iodide was added dropwise, and the mixture was reacted at room temperature for 3 hours with stirring. . Ethyl acetate was added to the reaction solution, and impurities were extracted and removed with water. The ethyl acetate layer was dried over sodium sulfate, the solvent was distilled off, and the residue was subjected to silica gel column chromatography with a mixed solvent of ethyl acetate and n-hexane to obtain crystals.
Then, it was recrystallized from n-hexane to obtain 3.10 g (yield 72.9%) of the target substance as pale yellow crystals. The analytical values of this substance are as follows.

Melting point: 69.0 to 70.5 ° C IR: ν ^KBr _max cm ^-1 1680 (C = O), 1200, 1270 (C-O) NMR (CDCl ₃ , TMS, PPM) 1.30 (3H, t, N-CH ₂ CH ₃ ) 2.15 (3H, s, dithiene ring CH ₃ ) 2.39 (6H, s, 2,6 position CH ₃ ) 2.80-3.16 (4H, m, SCH ₂ CH ₂ S) 3.35 (6H, s, 2 × OCH ₃ ) 3.58 (4H, t, 2 × COOCH ₂ CH ₂ O) 3.65 (2H, q, N−CH ₂ CH ₃ ) 4.13−4.35 (4H, m, 2 × COOCH ₂ CH ₂ O) 5.27 (1H, s, 4 Position H) Example 6 4- [2- (3-Methyl-5,6-dihydro-1,4-dithynyl)]-2,6-dimethyl-1,4-dihydropyridine-3,
Synthesis of 5-dicarboxylic acid bis (2-n-butoxyethyl) ester 2-formyl-3-methyl-1,4-dithiene 3.17 g (0.02
(0 mol), 8.2 g (0.041 mol) of acetoacetic acid-2-n-butoxyethyl ester and 11 ml of 28% aqueous ammonia were dissolved in 50 ml of isopropyl alcohol, and the mixture was heated under reflux for 4 days. After cooling, the reaction solution was concentrated, the residue was dissolved in ethyl acetate, and impurities were extracted and removed with a small amount of water. After drying with sodium sulfate in the ethyl acetate layer, the solvent was distilled off, and the residue was subjected to silica gel column chromatography with a mixed solvent of ethyl acetate and benzene to obtain crystals, and then recrystallized with a mixed solvent of n-hexane and ether to give a crystal. Target substance 1.29 as yellow crystals
g (yield 12.0%) was obtained. The analytical values of this substance are as follows.

Melting point: 63.0-65.0 ° C IR: ν ^KBr _max cm ^-1 3310 (NH), 1685 (C = O), 1205 (CO) NMR (CDCl ₃ , TMS, PPM) 0.8-1.05 (6H, m, 2 × −CH ₂ CH ₃ ) 1.1−1.75 (8H, m, 2 × OCH ₂ CH ₂ CH ₂ CH ₃ ) 2.20 (3H, s, dithiene ring CH ₃ ) 2.28 (6H, s, 2, 6th CH ₃ ) 2.75-3.25 (4H, m, SCH ₂ CH ₂ S) 3.42 (4H, t, 2 × OCH ₂ CH ₂ CH ₂ CH ₃ ) 3.60 (4H, t, 2 × COOCH ₂ CH ₂ O) 4.24 (4H, t , 2 × COOCH ₂ CH ₂ O) 5.22 (1H, s, 4th position H) 5.51 (1H, b, NH) Example 7 (Test Example) Test of tumor cell growth inhibitory effect in combination with antitumor drug Human the KB-Ch ^R -24 cells is KB-3-1 cells and its MDR clones from nasopharyngeal carcinoma were used as test cells.
The culture solution is 10% calf serum (Flow Laboratories (Flow
Laboratories), 1 mg / ml Bactopeptone (Difco), 0.292 mg / ml L-glutamine, 100
An Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing a unit / ml penicillin G was used. The test for the resistance overcoming effect by the combined use of the antitumor drug vincristine and the test compound was carried out as follows.

Spread each test cell in the culture medium and adjust the cell density to 75 cells / ml. Dispense 4 ml of this cell suspension into a Petri dish and place it in a carbon dioxide gas incubator (5% CO ₂ , 95% air).
Incubate at ℃ for 24 hours. After 24 hours of culture, 20 μl each of a dimethylsulfoxide (DMSO) solution of a predetermined concentration of vincristine and a DMSO solution of a test compound of a predetermined concentration are added, and the culture is further continued for 10 days. After the culture, Giemsa staining was performed, the number of colonies in each dish was counted, and a dose-response curve was prepared. Thus, the vincristine concentration (D ₁₀ value) at 10% cell viability was calculated, and the resistance overcoming effect was determined.

The results are shown in Table 1. In the table, the control is vincristine (VC
R) alone group, a is VCR and the compound of Example 1 (100 μg / m
l) a combination group, b is VCR and the compound of Example 5 (100 μg / m
l) Shows the combination group.

Example 8 (Test Example) Test of Growth Inhibitory Effect on Tumor Cell by Combination with Antitumor Drug Antitumor Drug Sensitive Cell of Mouse P388 Leukemia Cell (P388 / S)
The antitumor drug vincristine resistant clone (P388 / VCR) was used as a test cell. The culture solution is 10% fetal bovine serum (GIBCO), 10 μM 2-hydroxyethyl disulfide (Aldrich), and 10 μg.
RPMI-1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing / ml kanamycin was used. The test for the resistance overcoming effect by the combined use of the antitumor drug vincristine and the test compound was carried out as follows.

The test cells are seeded in a culture medium and the cell density is adjusted to 1 × 10 ⁵ cells / ml. Dispense 2 ml of this cell suspension into a 24-well microplate, and then add 20 μl each of a dimethylsulfoxide (DMSO) solution of vincristine at a predetermined concentration and a DMSO solution of a test compound at a predetermined concentration, and add a carbon dioxide incubator (5% CO 2 ₂ , 95% air) at 37 ℃ 72
Incubate for hours. After the culture was completed, the number of each cell was counted and a dose-response curve was prepared. From this, the 50% growth inhibitory concentration (IC ₅₀ ) by vincristine was calculated, and the resistance overcoming effect was determined.

The results are shown in Tables 2 and 3. In Tables 2 and 3,
Controls, a, and b have the same meaning as in Table 1,
c shows a group in which VCR and the compound of Example 2 (10 μg / ml) were used in combination.

Example 9 (Test example) Vincristine-resistant mouse leukemia Tumor-carrying effect in cancer-bearing mice 10 ⁶ vincristine (VCR) in ⁶ CDF1 mice per group
Transplanting resistant mouse leukemia (P388 / VCR) cells intraperitoneally,
The compound of the present invention and VCR were intraperitoneally administered for 5 days and then observed, the number of survival days of each was determined, and the survival rate (T / C) relative to the control was determined. The enhancing effect (T / V) of the anticancer agent was calculated by the following formula. The results are shown in Table 4. Compound 1 in the table
Is the compound of Example 1 and the compound 5 is the compound of Example 5.

[Effects of the Invention] The 1,4-dihydropyridine derivative according to the present invention enhances its action when used in combination with an antitumor drug. The effect is particularly pronounced on clones that have acquired resistance to antitumor drugs. For example, as is clear from Table 1 above, KB-Ch ^R- 24 cells, which are multidrug-resistant clones of KB-3-1 cells derived from human nasopharynx, were compared to cells that have not acquired resistance. The same effect (10% cell viability) cannot be obtained without using a double concentration of the antitumor drug, whereas the combination of the compound of the present invention is a (compound of Example 1).
100 μg / ml combined), the same effect can be obtained at 0.59 times the concentration.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Haruaki Inada 2-1344-1 Owada-cho, Omiya City, Saitama Prefecture Matsukaze Mansion 201 (72) Inventor Akira Kigami 2977-2-834 (72) Iwatsuki, Iwatsuki City, Saitama Prefecture ) Inventor Tetsuro Sano 2-161, Sunomachi, Omiya-shi, Saitama Prefecture (72) Inventor Elgis Albidwich Viseniex Soviet Union, Riga, Ulyza Tarabas Gatobe, 11, Kuvartilla 13 (72) Inventor Gnar Janowitch Dubourg Soviet Union, Riga, Uritsa Erik, 43, Kvartilla 2

Claims (5)

[Claims] 1. A formula (I) (In the formula, R ₁ represents a hydrogen atom or a C ₁ -C ₃ alkyl group,
R ₂ represents a C _{1 to} C ₆ alkyl group, n represents an integer of 2 to 4, R ₃ represents a hydrogen atom or a C _{1 to} C ₃ alkyl group,
R ₁ represents a hydrogen atom, R ₂ represents a methyl group, and n is 2
Of the 1,4-dihydropyridine derivative represented by the formula (1), and R ₃ represents a hydrogen atom. 2. R ₁ is a hydrogen atom or a methyl group, and R ₂ is C _1.
~C is ₅ alkyl group, R ₃ is a hydrogen atom, a methyl group or an ethyl group, 1,4-dihydropyridine derivatives of the range preceding claim in which n is an integer of 2 or 3 claims. 3. The 1,4-dihydropyridine derivative according to claim 1 or 2, wherein R ₂ is a C ₃ -C ₅ alkyl group. 4. The 1,4-dihydropyridine derivative according to claim 1 or 2, wherein R ₂ is a methyl group. 5. The 1,4-dihydropyridine derivative according to claim 1 or 2, wherein R ₃ is a methyl group or an ethyl group.