WO1994004701A1

WO1994004701A1 - Process for producing optically active 1,4-dihydropyridine compound

Info

Publication number: WO1994004701A1
Application number: PCT/JP1992/001074
Authority: WO
Inventors: Takashi Adachi; Mayuma Ikeda; Takako Hadachi; Kazunori Hanada
Original assignee: Taisho Pharmaceutical Co., Ltd.
Priority date: 1991-03-01
Filing date: 1992-08-26
Publication date: 1994-03-03
Also published as: AU2486392A; JPH0584089A

Abstract

A process for producing an optically active 3-(2-nicotinoylaminoethyl)ester of (-)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid by asymmetrically hydrolyzing bis(2-nicotinoylaminoethyl) 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate or a salt thereof with an enzyme which can asymmetrically hydrolyze the carboxylate groups bonded to the 3- and 5-positions of a 1,4-dihydropyridine ring. The invention provides a process for producing a very important intermediate for producing only that optical isomer which is suitable as a medicine among the isomers of 3-(2-nicotinoylaminoethyl) 5-(3-nitroxypropyl) ester of 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid.

Description

Description Method for producing optically active 1,4-dihydropyridine compounds

The present invention relates to a method for producing an intermediate for producing an optically active 1,4-dihydropyridine compound, and more particularly, to an important optically active 1,4 when producing an optically active 1,4 dihydropyridine compound which is extremely useful as a pharmaceutical. The present invention relates to a method for producing a dihydropyridine intermediate. Background art

If the two carboxylic acid esters bonded to the 3- and 5-positions of the dihydropyridine ring of the 1,4-dihydropyridine compound are different from each other, they have an asymmetric carbon at the 4-position and there are two optical isomers I do. Recently, as a result of a detailed study of the biological properties of this class of compounds, it has been reported that there are differences in pharmacological activity, pharmacokinetics, safety, etc. among the optical isomers. When a compound having such an asymmetric carbon is used as a drug, it is generally considered that only one isomer that is preferable as a drug is given to a living body from the viewpoint that no extra load is applied to the living body. It is becoming more and more.

Conventionally, methods for producing various optically active 1,4-dihydropyridine compounds have been reported, but these are roughly classified into the following two methods. That is, a method of forming a salt with an optically active compound and then performing optical resolution by fractional crystallization, and a method of combining a compound having an asymmetric carbon to induce a diastereomer and separating it. However, optical resolution by fractional crystallization requires the use of expensive resolving agents such as cinchonidine and cinchonine, and requires crystallization several times. [Japanese Unexamined Patent Publication No. 63-185960, Japanese Unexamined Patent Publication No. 64-52757, Japanese Unexamined Patent Publication No. Hei 1-1254661, Chem.Phar rm.Bull 1., 28, 2809 (1980), etc.]. In addition, the method of separation by guiding to diastereomers is expensive optical optics. ₀

-2-

It has drawbacks such as using active compounds as raw materials, and the reaction process is long and complicated.

[Japanese Unexamined Patent Publication No. Sho 61-431,872, Japanese Unexamined Patent Publication No. Hei 2-11592, etc.]

Whichever method is used, the desired optical isomer can be obtained only with a maximum yield of 50% through complicated operations.

2,6-Dimethyl-41- (3-ditrophenyl) 1-1,4-dihydropyridine-3.5-dicarboxylic acid 3- (2-nicotinylaminoethyl) ester 5- (3-Ditrooxypropyl) ester (Hereinafter, it may be referred to as compound A.) and a salt thereof are disclosed in Japanese Patent Application Laid-Open No. 223580/1990, a process for producing its racemate and its pharmacological action as a calcium antagonist. . Compound A is expected to be a prophylactic and therapeutic agent for ischemic heart disease, hypertension, etc., because it has a selective coronary vasodilator effect, excellent sustained drug efficacy, and a c-GMP increasing effect Has been done. Further, it is expected that Compound A has an optical isomer in its structure, and only one of the isomers is preferable as a pharmaceutical.

It is an object of the present invention to provide a method for producing a very important intermediate for producing only the optical isomer of compound A which is preferable as a pharmaceutical. Disclosure of the invention

The present inventors have diligently studied a method using an enzyme in producing optically active compound A. As a result, in the course of this research, hydrolyzing enzymes produced by microorganisms belonging to the genera Aspergillus, Benicillium, Streptomyces and Bacillus, and hydrolysis prepared from animal organs using compounds that can be easily produced as raw materials The present inventors have found that the use of an enzyme and a hydrolase prepared from a plant gives a production intermediate of compound A exhibiting a preferred optical rotation in high yield and high optical purity, and completed the present invention. .

That is, the present invention relates to 2,6-dimethyl-14- (3-nitrophenyl) 1-1,4-dihydropyridine-13,5-dicarboxylic acid bis (2-nicotinylaminoethyl) ester or a salt thereof. 6-dimethyl-1- (3-nitrophenyl) 1-1,4-dihydroxypyridine-1,3,5-dicarboxylic acid bis (2-nicotinoylaminoethyl) ester or a salt thereof bonded to the 3- and 5-position of the carboxylic acid ester Asymmetric hydrolysis by the action of an enzyme having the ability to asymmetrically hydrolyze ter, and (1) 1,2,6-dimethyl-4- (3-ditrophenyl) 1-1,4-dihydroviridine-13,5 —Dicarboxylic acid 3 -— (2-nicotinoylaminoethyl) ester is recovered, and its optical activity is characterized by (1) 1,2,6-dimethyl-41- (3-ditrophenyl) 1-1,4-dihydropyridine This is a method for producing 3,5-dicarboxylic acid 3- (2-nicotinylaminoethyl) ester.

In the present invention, the salt is a hydrochloride, a sulfate or a nitrate. The enzymes used in the present invention include enzymes produced by microorganisms belonging to Aspergillus, Benicillium, Streptomyces, and Bacillus, enzymes prepared from animal organs, and enzymes prepared from plants. However, any object can be used as long as the object of the present invention can be achieved, and there is no particular limitation.

Some of these microbial enzymes are commercially available and are readily available. Specific examples of commercially available enzymes include, for example, enzymes derived from the genus Aspergillus (Aspergillus oryzae); protease A “Amano”, protease M “Amano”, enzymes derived from the genus Aspergillus (Aspergillus melleus); Mano ", Seabrose" Amano ", an enzyme derived from the genus Aspergillus (Aspergillus sp.); Cellulase A" Amano ", Acylase" Amano "1500, Piozyme A (registered trademark)" Amano ", Deamisym ( (Registered trademark) "Amano", an enzyme derived from the genus Penicillium sp .; nuclease "Amano" (above, manufactured by Amano Pharmaceutical Co., Ltd.), an enzyme derived from the genus Aspergillus sojae; Protease type XI X; Sigma), an enzyme derived from Streptomyces griseus; Actinase E (Kaken Pharmaceutical) Proteinase type XIV (Sigma), an enzyme derived from Streptomyces caespitosus; Proteinase type IV (Sigma), an enzyme derived from Bacillus licheniformis; Subtilisin A (Novo ), Proteinase (manufactured by Huriki Co., Ltd.), proteinase type VIII (manufactured by Sigma), enzymes derived from Bacillus subtilis; alkaline protease (manufactured by Nagase Seikagaku), proteinase (manufactured by Nagase Seikagaku) Full Rikisha).

Some of the enzymes prepared from animal organs are commercially available, _t

One four one

Can be obtained. Specific examples of commercially available enzymes include, for example, pig liver esterase (manufactured by Sigma) and kidney lipase (manufactured by Tokyo Chemical Industry). Some enzymes prepared from plants are commercially available and are also readily available. Specific examples of commercially available enzymes include, for example, bean nut phospholipase (Phospholipase D) (manufactured by Lucerna Chem), and pine-atre proteases (Bromelain) (manufactured by Sigma).

Further, a protease manufactured by Tokyo Chemical Industry Co., Ltd. may be mentioned.

In addition, various additives can be used in the enzyme reaction, and addition of 1,10-0-phenanthroline is particularly effective. By using this additive, the reaction rate can be improved and the amount of required enzyme can be reduced.

Next, the enzymatic reaction of the present invention will be described. Basically, each is set according to the optimum conditions of the enzyme to be used. Here, the general reaction conditions will be described. A culture solution obtained by culturing the microorganism, a cell isolated from the culture, a culture filtrate containing the enzyme, or a crude enzyme separated from the cell or culture filtrate by various enzyme separation methods; a purified enzyme further purified; 6-Dimethyl-4- (32-nitrophen, nil) -11,4-dihydropyridine-13,5-dicarboxylic acid bis (2-nicotinolaminoethyl) ester or a salt thereof is stirred or stirred in an aqueous solution. This is done by shaking.

As the reaction solution, it is preferable to use a buffer such as a phosphate buffer or a Tris-HCl buffer in order to keep the pH of the reaction solution constant. The concentration of the buffer varies depending on the type of the buffer, but is preferably 2 M or less. In addition, the reaction can be carried out without using a buffer. In this case, it is preferable to control the pH of the reaction solution by a pH stat using an aqueous solution of sodium hydroxide, potassium hydroxide or the like.

The reaction conditions may be set according to the enzyme used. For example, various conditions when using protease P are such that the pH is 6.0 to 8.0, and most preferably, the pH is 7.0 to 7.5. The reaction temperature is from 25 ° C to 50 ° C, most preferably from 30 ° C to 35 ° C. The concentration of S substance in the reaction solution is 0.05 to 25% by weight based on the reaction solution. The amount of enzyme used depends on the enzyme titer and the amount of substrate. _c

One five one

It may be determined appropriately. The reaction time may be set while confirming the progress of the reaction by TLC analysis or HPLC analysis.

Further, in the present enzyme reaction, various organic solvents can be used as a substrate dissolution aid. Examples of the organic solvent that can be added include acetone, methyl ethyl ketone, dimethyl sulfoxide, dioxane, N, dimethylformamide, and acetonitrile. These may be used alone or in combination of two or more. When adding an organic solvent to this enzyme reaction, it is necessary to add it within a range that does not reduce the enzyme activity. For example, when acetone is added, the power that can be added at a concentration of 1 to 15% to the reaction solution is most preferably 4 to 6%. The organic solvent may be added either at the beginning of the reaction or during the reaction. In addition, surfactants such as sodium lauryl sulfate, triton 100, and Tween 80, and emulsifying agents such as polyethylene glycol # 400, gum arabic, and lecithin can also be used.

Furthermore, if the substrate is added all at once in the early stage of the reaction when performing this enzyme reaction, the substrate precipitates and aggregates, and the progress of the reaction is inhibited. As a solution to this problem, a method of dissolving a substrate (hydrochloride, sulfate, or nitrate) in water and adding the solution to the enzyme solution little by little continuously showed a great effect. When the continuous addition of the substrate and the addition of the organic solvent are performed at the same time, it is preferable to add the organic solvent in the middle of the reaction rather than adding the organic solvent at the beginning of the reaction in which the substrate concentration is low.

The use of the substrate solubilizing agent and the continuous addition of the substrate can greatly increase the final substrate charge concentration.

In addition, by using various additives in the present enzyme reaction, the reaction rate can be increased and the amount of the enzyme used can be reduced. When the enzyme used was protease II, various organic reagents and metal ions were added and examined. As a result, 1.10-0-funanthroline showed a particularly excellent effect. Before the start of the reaction, 1,10—ο-phenanthroline can be dissolved in an organic solvent such as dimethylsulfoxide or acetone and added to the enzyme solution. It is effective to add at a concentration of O mM, and the optimal concentration is 1 mM to 4 mM.

After completion of the reaction, adjust the reaction solution to pH 5.0 by adding 1 N phosphoric acid, and add ethyl acetate. .

One six one

The desired product can be isolated by extraction with an organic solvent such as, for example, and, if necessary, purification by column chromatography or the like. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described with reference to Examples and Reference Examples, but the present invention is not limited only to Examples.

The reaction products were confirmed by the TLC method and the HPLC method shown below.

TLC; TLC Plate RP-18 (Merck)

Developing solvent: Me OHZH ₂₀ = 8Z2

HPLC; Column ODS Cie (4.60X15 Omm)

Eluent Me OHZH ₂ 0 = lZl

i 1 m 1 / m i n

Temperature 50 C

Detection UV 240 nm

Optical purity was determined by optical rotation measurement or HPLC analysis using a chiral column. HPLC analysis was performed under the following conditions.

Analysis conditions; Column ULTRON E S-OVM

(4.60X15 Omm) (Shinwa Kako)

Eluent i-Pr 0HZ2 OmM phosphate buffer (pH 3.0)

= 1/9

'Ο ^, τΒ 1 m 1 Zm i n

Temperature 25

Detection UV 240 nm

Example 1

Dissolve 20 g of Protease P and 2,6-dimethyl-41- (3-ditrophenyl) -1.4 dihydroxypyridine-13,5-dicarboxylate bis (2-nicotinylaminoaminoethyl) ester.1 hydrochloride in 1 L of water. This solution was adjusted to pH 7.5 by adding 1N sodium hydroxide solution, and 30. The mixture was stirred at C. pH After the mixture was stirred for 24 hours while maintaining the pH at 7.5, 1 N phosphoric acid was added to adjust the pH to 5.0, and the mixture was extracted twice with 1 L of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated, and the resulting residue was subjected to reverse phase chromatography [Column: LiChroprep RP-18 (40-63 ^ m) (Merck), eluent: Me OH / H20 = 6/4], and (1) 1,2,6-dimethyl-4-1 (3-nitrophenyl) 1-1,4-dihydropyridine-1,3,5-dicarboxylic acid 3- (2-nicotinol 574 mg (yield: 85%) of aminoethyl) ester was obtained as a pale yellow foam.

m.p. 116-: L19. C

[a] D ³⁰ — 77.9 ° (c = ll 3, MeOH)

The optical yield measured by the HPLC method was 100%.

_{NMR (DMSO- d 6, 20 ΟΜζ} ) δ (ppm);

2.28 (6H, s), 3.51 (2H, q, J = 6Hz),

4.13 (2H, t, J = 6Hz), 4.99 (1H, s),

7.42 (1 H, t, J = 8H z),

7.50 (1 H, d d, J = 5 Hz, 8 Hz),

7.60 (1 H, d, J = 8H z),

7.93 (1 H, d, J = 8H z), 7.98 (1 H, s),

8.13 (1 H, d, J = 8 Hz),

8.71 (1H, d, J = 5Hz), 8.57 to 8.78 (1H, m), 8.94 (1H, s), 8.96 (1H, s),

11.83 (1 H, b r s)

Examples 2 to 14

20 mg of the enzyme shown in Table 1 was dissolved in 2 ml of phosphate buffer (20 mM, pH 7.5), and 2,6-dimethyl-4-1 (3-ditrophenyl) 1-1,4 dissolved in 201 of water. 1 mg of bis (2-nicotinoylaminoethyl) dihydropyridine-1,3-dicarboxylate, dihydrochloride was added. This reaction solution was shaken at 30 ° C. for 24 hours, and the obtained reaction mixture was adjusted to pH 5.0 with 1N phosphoric acid and extracted with 2 ml of ethyl acetate. The residue obtained by concentrating the organic layer was analyzed by HP LC, The yield was calculated. Further, the portion corresponding to the mono-functional ruponic acid of the target substance was separated by HPLC, and the optical yield was calculated by an HPLC method using an optically active column.

The results are shown in Table 1.

Example Enzyme Z origin Chemical yield (%) Optical yield (%)

2 Brothese P "Amano" 86.7 100

/ Aspergillus me Ileus

3 Seabrose "Amano" 97.6 100

/ Aspergillus me Ileus

4 Acylase Amano 15000 10.2 100

/ ^ Aspergillus sp.

5 Deamizam `` Amano '' 34.3 100

. Aspergillus sp.

6 Actinase E (Bronase E) 23.1 100

ZStreptomyces griseus

7 Protease type X I V 11.7 100

ZStreptomyces griseus

8 Subtilisin A 25.7 100

// Bacillus licheniformis

9 Proteinase 34.1 100

/ "Bacillus licheniformis

0 Protease type V I I I 40.4 100

// Bacillus licheniformis

1 Alkaline protease 36.2 100

ZBasillus subtilis

2 Proteinase 12.6 100

ZBasillus subtilis

3 Lipase 21.8 100

/ Pancreas

4 Protease 17.3 100

Example 15

Dissolve 6.0 g of proteases P in 500 ml of water and add 10 ml of One three —

0.3 g of 1,10-0-phenanthroline dissolved in tylsulfoxide was added. This solution was adjusted to pH 7.5 by adding a 0.5 N aqueous sodium hydroxide solution. While stirring the above SL enzyme solution at 30 ^e C, 2 This was dissolved in water 5 Om 1, 6- dimethyl Chiru 4 i (3 two Torofueniru) one 1, 4 Jihi Dorobirijin one 3, 5-dicarboxylic Bis (2-nicotinoylaminoethyl) ester ′ dihydrochloride (6.0 g) was added little by little continuously so that the substrate did not precipitate. Although the pH of the reaction solution decreased with the addition of the substrate, the pH was maintained at 7.5 by adding a 0.5 N aqueous sodium hydroxide solution according to the pH status. After 48 hours, when the addition of the substrate was completed, the progress of the reaction was confirmed by HPLC, and the substrate was almost completely eliminated. The reaction mixture was adjusted to pH 5.0 by adding 1N phosphoric acid, and extracted twice with 300 ml of ethyl acetate. After the ethyl acetate layer was concentrated to 200 ml, it was extracted twice with 200 ml of a 1N aqueous sodium hydroxide solution. The obtained sodium hydroxide solution was washed once with 10 Om 1 of ethyl acetate, adjusted to pH 5.0 again by adding 1 N phosphoric acid, and extracted twice with 100 m 1 of ethyl acetate. The combined ethyl acetate layers were washed with saturated saline, dried over anhydrous magnesium sulfate, filtered, and concentrated to give (1,) 1,2-dimethyl-4-1 (3-ditrophenyl) -11,4-diethyl 3.74 g (yield: 92%) of dropyridine-3,5-dicarbonic acid 3- (2-nicotinoylaminoethyl) ester was obtained. ·

Reference example 1

(I) 1,2,6-dimethyl-4-1 (3-nitrophenyl) 1,1,4-dihydroxypyridine 1,3,5-dicarboxylic acid 3_ (2-nicotinylaminoethyl) ester 25 Omg of dimethylformamide To 1 Om 1 solution, 12 Omg of 3-nitropropyl oxypropyl 11-bromide and 9 Omg of potassium carbonate were added, and the mixture was stirred at room temperature for 10 hours. The reaction solution was poured into water and extracted with ethyl acetate. The organic layer was washed with water and an aqueous solution of sodium hydrogen carbonate, dried, and the solvent was distilled off. After dissolving the residue in 10 ml of ethyl acetate, 0.2 ml of 4N hydrochloric acid ethyl acetate solution was added, and the mixture was stirred for 1 hour. The resulting insoluble matter is filtered and dried under reduced pressure to obtain the desired (1) 1,2,6-dimethyl-4,1- (3-ditrophenyl) -11,4-dihydropyridine-13,5-dicarboxylic acid 3- ( 2-nicotinoylaminoethyl) ester 5- (3-nitro Xypropyl) ester hydrochloride 24 Omg was obtained.

m.P. 126-128 ° C

[a] _D ²⁵ one 25.2 ° (c = l .12, MeOH)

Reference example 2

(I) 1,2,6-dimethyl-14- (3-ditrophenyl) -1,4-dihydroxypyridine-1,3,5-dicarboxylic acid 3- (2-nicotinoylaminoethyl) ester 25 Omg of methylene chloride 160 mg of acetic anhydride and Molecular Sieves 3 A25 Omg were added to the 10 ml solution, and the mixture was stirred for 1 hour. After filtering off the insoluble matter, 10 Omg of 3-nitroxypropyl alcohol and 1 drop of acetyl chloride were added to the filtrate, and the mixture was stirred for 3 hours. The reaction solution was poured into water, extracted with methylene chloride, and the organic layer was washed with an aqueous sodium hydrogen carbonate solution and dried. After distilling off the solvent, the residue was treated in the same manner as in Reference Example 1 to obtain the desired ((1) -1,6-dimethyl-4- (3-ditrophenyl) -1-1.4-dihydropyridine-1,3,5-dicarboxylic acid There was obtained 23 Omg of the acid tri- (2-nicotinoylaminoethyl) ester 5- (3-nitrooxypropyl) ester hydrochloride. Industrial applicability

According to the present invention, it is possible to provide an intermediate for the production of a compound A which is simple, has a high yield, and is optically active (levorotatory) with high optical purity. In particular, in the method of the present invention, the raw material (prochiral compound) used and the product have a 1: 1 balance relationship, and the yield of the desired optical isomer is at most 50% as in the conventional optical resolution method. The biggest feature is that it has no shortcomings. Furthermore, the method of the present invention has the following advantages: the reaction proceeds under mild conditions, so that no special reactor is required, an expensive resolving agent is not required, and the waste liquid after the reaction is easily treated. It has very advantageous advantages over the method.

By using this optically active production intermediate, an optically active compound A can be produced extremely easily and in good yield. That is, the compound can be reacted with 3-nitrooxypropyl-111-bromide or 3-nitrooxypropyl alcohol to obtain an optically active compound A.

The optically active compound A obtained here is a preferred isomer (Levorotary) as a pharmaceutical U

It is. Levorotatory compound A is significantly more effective than racemic and dextrorotatory compound A in tests of calcium channel binding ability, coronary blood flow increasing action, heart rate inhibitory action and hypotensive action. Therefore, the compound A having an optical activity of levorotation produced by the intermediate of the present invention is extremely useful as a drug for preventing and treating angina pectoris, hypertension and the like.

Claims

Scope of request

1. 2,6-Dimethyl 4- (3-ditrophenyl) -1,4-dihydroxypyridine-1,3,5-dicarboxylic acid bis (2-nicotinoylaminoethyl) ester or its salt, 2,6- Dimethyl 4- (3-ditrophenyl) 1-1,4-dihydropyridine-13,5-dicarboxylic acid bis (2-nicotinoylaminoethyl) ester or a salt thereof bound to the carboxylic acid ester bonded to the 3- and 5-positions Asymmetric hydrolysis Asymmetric hydrolysis is caused by the action of an enzyme capable of hydrolyzing. (1) 1,2-Dimethyl-4- (3-ditrophenyl) -1,4-dihydroviridine-3,5-dicarboxylic acid Optical activity characterized by recovering 3- (2-nicotinoylaminoethyl) ester (I) 1, 2, 6-dimethyl 4- (3-ditrophenyl) 1, 1, 4-dihydroviridine 1, 3, 5 —Dicarboxylic acid 3— (2-nico Noiruamino Echiru) process for the preparation of esters.

2. The salt of 2.6-dimethyl-41- (3-ditrophenyl) 1-1,4-dihydropyridine-13,5-dicarboxylate bis (2-nicotinoylaminoethyl) ester is hydrochloride, sulfate or nitrate The production method according to claim 1, wherein:

3. The process according to claim 1, wherein the enzyme is an enzyme obtained from a microorganism selected from the group consisting of Aspergillus, Penicillium, Streptomyces, and Bacillus.

4. The process according to claim 1, wherein the enzyme is an enzyme obtained from Aspergillus oryzae, Aspergillus meleus, Aspergillus sodiae, Streptomyces griseus, Streptomyces casbitosas, Bacillus licheniformis or Bacillus subtilis. .

5. The production method according to claim 1, wherein the enzyme is an enzyme prepared from animal organs.

6. The method according to claim 1, wherein the enzyme is an enzyme prepared from a plant.

7. The process according to claim 1, wherein 1,10-o-fenanthroline is added.