JP3388803B2 - Method for producing optically active 7-amidinonaphthalene derivative - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to an optically active 2- [4-[[(3S) -1-acetimidoyl-3-, which is useful as a blood coagulation inhibitor or a prophylactic / therapeutic agent for thrombus or embolus.
It relates to a method for producing pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionic acid or a salt thereof.

[0002]

2. Description of the Related Art Conventionally, the problems to be solved by the invention
Although an antithrombin agent has been developed as a thrombus formation inhibitor, it is known that this antithrombin agent has a risk of causing a bleeding tendency because it suppresses the platelet aggregation effect by thrombin together with the anticoagulant effect. Therefore, it was not possible to easily control the coagulation ability.
Therefore, the development of an anticoagulant based on a mechanism of action other than the thrombin inhibitory effect has been carried out, and the present applicant has proposed that activated blood coagulation
An aromatic amidine derivative or a salt thereof that reversibly inhibits a factor (hereinafter abbreviated as FXa) and exhibits a strong anticoagulant action has good water solubility and is strong in FXa specificity and reversibly even in oral administration. It was found to be useful as a prophylactic and therapeutic drug for various diseases caused by thrombosis and embolism, and it applied for a patent (Patent Application No. 4-
292892).

Of these aromatic amidine derivatives or salts thereof, optically active 2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl]-
3- [7-amidino-2-naphthyl] propionic acid or its salt is ethyl (2RS) -2- [4-[[(3S)
-1-acetamidoyl-3-pyrrolidinyl] oxy]
Ethyl (2RS) which is a precursor of phenyl] -3- [7-amidino-2-naphthyl] propionate or a salt thereof.
-2- [4-[[((3S) -1-tert-butoxycarbonyl-3-pyrrolidinyl] oxy] phenyl] -3
-[7-Cyano-2-naphthyl]] propionate was resolved by fractional crystallization and these were separated from ethyl 2- [4-
By derivatizing [[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate or a salt thereof, ester hydrolysis with hydrochloric acid is performed. Manufactured.

However, these methods have a problem that the operation of fractional crystallization is complicated and the target compound cannot be obtained industrially advantageously. Therefore, optically active 2- [4-[[(3S) -1-acetimidoyl-
A method for easily obtaining 3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionic acid or a salt thereof under mild conditions has been desired.

[0005]

Under these circumstances, the inventors of the present invention have earnestly studied and found that alkyl-2- [4
-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-
Naphthyl] propionate or a salt thereof is treated with one or more selected from the group consisting of a microorganism, a culture solution thereof and a fraction thereof, and ester hydrolyzed to give an optically active 2- [4-[[[ (3S) -1-acetimidoyl-
The present invention was completed by finding that 3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionic acid or a salt thereof can be easily obtained under mild conditions.

That is, the present invention is based on the general formula (2)

[0007]

[Chemical 7]

(Wherein R represents a lower alkyl group having 1 to 6 carbon atoms) alkyl-2- [4-[[(3
S) -1-Acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl]
General formula (1) characterized in that propionate or a salt thereof is treated with one or more selected from the group consisting of microorganisms, culture solutions thereof and fractions thereof.

[0009]

[Chemical 8]

Optical activity represented by 2- [4-[[(3
S) -1-Acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl]
The present invention provides a method for producing propionic acid or a salt thereof.

In the present invention, alkyl-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3 used as a starting material compound
-[7-amidino-2-naphthyl] propionate or a salt thereof is represented by the general formula (2),
For example, it can be manufactured by the method described in Japanese Patent Application No. 4-292892. In the general formula (2), examples of the alkyl group having 1 to 6 carbon atoms represented by R include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a pentyl group, a hexyl group, and the like. Groups are preferred.

These starting compounds are preferably used in a concentration of 0.1 to 10% by weight, particularly 0.1 to 1.0%.

The microorganisms used in the present invention, their culture solutions and fractions thereof include yeasts of the genus Debyromyces, and the bacteria of genus Brevibacterium, genus Sitophaga, sphingobacterium, and filamentous fungus Aspergillus. , Microorganisms belonging to the genus Silacinera, the genus Cuavararia, the genus Mortierella, culture solutions thereof, fractions thereof, and the like are used. Among these microorganisms, Debyromyces species are preferred as Debyromyces spp .; Brevibacterium linens is preferred as Brevibacterium spp. Aspergillus humigetas, Aspergillus unilateraris, Aspergillus nigia, Aspergillus usami; as preferred species of Aspergillus spp. Mention may be made of Mortierella Isabella.

Examples of the culture solution of the microorganism include those obtained by culturing the microorganism in an appropriate medium. Also,
As the fractionated product, microbial cells or a culture solution of a microorganism are extracted with water or an appropriate buffer solution; when the culture is carried out in a liquid medium, the culture filtrate, ammonium sulfate or alcohol is added to the extract or the filtrate. Precipitate obtained by adding; the extract or filtrate concerned was fractionated by using gel filtration, ultrafiltration, ion exchange chromatography, etc .; crushed cells; and the crushed product as described above And the like. The medium contains a carbon source, a nitrogen source, inorganic salts and trace amounts of nutrients that can be assimilated by the microorganism used. That is, carbon sources include carbohydrates such as glucose, maltose, and dextrose; nitrogen sources include natural organic nitrogen sources such as yeast extract, corn steep liquor, peptone, meat extract, and mixed amino acid powder; inorganic salts include magnesium and iron. Salts of copper, manganese, potassium, sodium and the like are appropriately used. Further, if necessary, various organic substances, inorganic substances, or commonly used defoaming agents necessary for the growth of microorganisms can be added.

The amounts of these microorganisms, cultures, and fractions used are not particularly limited, but may be 1 to the starting compound.
It is preferably 30 times by weight.

When a filamentous fungus is used as the microorganism, the (+)-form of the racemic raw material is selectively hydrolyzed to give (+)-2- [4-[[(3S)- 1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl]-
3- [7-Amidino-2-naphthyl] propionic acid is advantageously obtained. On the other hand, when yeast or bacteria is used as the microorganism, the (-)-form is selectively hydrolyzed,
-2- [4-[[(3S) -1-acetimidoyl-3
Pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionic acid is advantageously obtained.
Therefore, the (+)-form or the (-)-form can be produced separately by selecting the microorganism.

The reaction is carried out at 10 to 50 ° C., preferably 20 to 40 ° C., by mixing the raw material compound solution with the microbial cells.
It is carried out by stirring at pH 4 to 8, preferably 5 to 7, for 3 to 48 hours, preferably for about 72 hours. After completion of the reaction, the reaction solution is subjected to known means such as filtration, concentration under reduced pressure, ultrafiltration, column chromatography, etc.
A highly pure target compound can be isolated.

The target compound thus obtained can be further converted into a pharmaceutically acceptable salt by a conventional method. Examples of such a salt include mineral salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate and sulfate; methanesulfonate, 2-hydroxyethanesulfonate, p-toluene. Organic sulfonates such as sulfonates; as well as acetate, propionate, oxalate, malonate, succinate, glutarate, adipate, tartrate, maleate, malate, mandel Organic carboxylic acid salts such as acid salts may be mentioned.

[0019]

INDUSTRIAL APPLICABILITY According to the present invention, an optically active 2- [4-[[(3S) -1-acetimidoyl-3-, which is useful as a blood coagulation inhibitor or a prophylactic / therapeutic agent for thrombus or embolus.
Pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionic acid or a salt thereof can be easily obtained in high purity by a simple method under mild conditions, and is industrially advantageous. is there.

[0020]

EXAMPLES Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Reference Example 1 850 ml of glucose peptone liquid medium was inoculated with a filamentous fungus (Aspergillus fumigatus IFO-8866; purchased from Fermentation Research Institute) and shake-cultured at 30 ° C. for 3 days. The filamentous fungus body of this culture and the culture solution were used for the reaction.

Example 1 (+)-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7
-Preparation of amidino-2-naphthyl] propionate hydrochloride: ethyl (2RS) -2- [4-[[(3S) -1-
Acetoimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate hydrochloride (850 mg) was dissolved in the culture of Reference Example 1 and shake-cultured at 30 ° C for 72 hours. did. After the reaction,
When the reaction solution was analyzed by high performance liquid chromatography, it was found that (+)-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3-
The production rate of [7-amidino-2-naphthyl] propionic acid was 49%. The obtained reaction solution was filtered, the filtrate was concentrated under reduced pressure, purified by an adsorption resin, and the fraction containing the target compound was concentrated under reduced pressure. 1N-HCl was added to the obtained residue.
After adding 50 ml and hydrochloric acid salification, desalting with an adsorption resin, concentration under reduced pressure, crystallization from water-containing ethanol, and the desired compound (+)-2- [4-[[(3S) -1-acetimidoyl] -3-Pyrrolidinyl] oxy] phenyl]-
519 mg of 3- [7-amidino-2-naphthyl] propionate hydrochloride was obtained. The instrumental analysis data of the obtained target compound were as follows. When this product was analyzed by optical isomer separation chromatography, the optical purity of the compound was 99% ee.

[Α] _D ²⁴ = + 57.4 ° (c = 1.00
0, H ₂ O) (dissolved at 40 ° C., measured after heating at the same temperature for 30 minutes) ¹ H-NMR (DMSO-d ₆ ) δ: 2.20-2.35
(2H, m), 2.29 (1.5H, s), 2.32
(1.5H, s), 2.80-2.95 (1H, m),
3.30-4.00 (6H, m), 5.16 (0.5
H, br), 5.22 (0.5H, br), 6.90-
7.00 (2H, m), 7.45-7.51 (2H,
m), 7.57 (1H, d, J = 8.3 Hz), 7.6
6 (1H, d, J = 8.3 Hz), 7.93 (1H,
d, J = 8.3 Hz), 7.97 (1H, d, J = 8.
3Hz), 8.11 (1H, s), 8.68 (1H, b
r), 8.70-9.30 (br), 11.50-1
2.20 (br) X-ray analysis showed that this compound was (+)-2- [4-[(3S)
-1-acetamidoyl-3-pyrrolidinyl] oxy]
It was determined to be phenyl] -3- [7-amidino-2-naphthyl] propionic acid.

Example 2 (+)-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7
-Amidino-2-naphthyl] propionate hydrochloride production: The filamentous fungi shown in Table 1 and the culture solution thereof were obtained by the same operation as in Reference Example 1 and reacted in the same manner as in Example 1. Table 1 shows the results of analysis of the reaction completed liquid by high performance liquid chromatography.

[0025]

[Table 1]

Reference Example 2 850 ml of glucose peptone liquid medium was inoculated with yeast (Debyromyces species HUT-7060; purchased from Department of Fermentation Engineering, Faculty of Engineering, Hiroshima University) and incubated at 30 ° C. for 3 days.
Culture was carried out with shaking for one day. The yeast of this culture and the culture solution were used for the reaction.

Example 3 (-)-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7
-Production of amidino-2-naphthyl] propionate hydrochloride: Ethyl (2RS) -2- [4-[[(3S)-was added to the culture obtained in Reference Example 2 by the same procedure as in Example 1.
850 mg of 1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate hydrochloride were reacted. When the reaction-completed liquid was analyzed by high performance liquid chromatography, the production rate of hydrolyzed products was 46.6%. The reaction-terminated liquid was used in Example 1.
Purify in the same manner as in (-)-2- [4-[[(3S) -1
494 mg of -acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate hydrochloride were obtained. The instrumental analysis data of the obtained target compound were as follows. When this product was analyzed by optical isomer separation chromatography, the optical purity of the compound was 98% ee.

¹ H-NMR (DMSO-d ₆ ) δ: 2.0
5-2.4 (2H, m), 2.28 (1.5H, s),
2.31 (1.5H, s), 3.10-3.30 (1
H, m), 3.40-4.10 (6H, m), 5.13.
(0.5H, br), 5.20 (0.5H, br),
6.90-7.00 (2H, m), 7.35-7.40
(2H, m), 7.60 (1H, d, J = 8.3H
z), 7.81 (1H, d, J = 8.3 Hz), 7.8
4 (1H, s), 7.94 (1H, d, J = 8.3H
z), 8.06 (1H, d, J = 8.3 Hz), 8.4
2 (1H, s), 8.55 (0.5H, br), 8.6
4 (0.5H, br), 9.30-9.70 (5H) H
PLC column: Ligand exchange type column having D-penicillamine as an optically active site (SUMICHIRAL OA-
5000 4.6φ x 150 mm Sumika Chemical Analysis Service Center) Solvent: 2 mM Copper sulfate aqueous solution: Acetonitrile = 85:
15 (v / v) Flow rate: 1 ml / min Column temperature: 60 ° C Retention time: 38.14 minutes

Reference Example 3 Bacteria (Brevibacterium linens JCM-1327; purchased from RIKEN) were inoculated into 850 ml of a normal broth liquid medium and cultured at 30 ° C. for 3 days with shaking. The bacterial cells of this culture and the culture solution were used for the reaction.

Example 4 (-)-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7
-Production of amidino-2-naphthyl] propionate hydrochloride: Ethyl (2RS) -2- [4-[[(3S)-was added to the culture obtained in Reference Example 3 by the same procedure as in Example 1.
850 mg of 1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate hydrochloride were reacted. When the reaction completed liquid was analyzed by high performance liquid chromatography, the production rate of hydrolyzed product was 41.2%. The reaction-terminated liquid was used in Example 1.
Purify in the same manner as in (-)-2- [4-[[(3S) -1
436 mg of -acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propione hydrochloride was obtained. The instrumental analysis data of the obtained target compound completely agreed with those of Example 3. In addition, when this product was analyzed by optical isomer separation chromatography under the same analysis conditions as in Example 3,
The optical purity of the compound was 94% ee.

Example 5 (-)-2- [4-[[(3S) -1-acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7
-Amidino-2-naphthyl] propionate hydrochloride production: The bacterial cells and the culture solution shown in Table 2 were obtained by the same operation as in Reference Example 3, and the reaction was carried out in the same manner as in Example 3. Table 2 shows the results of analysis of the reaction completed liquid by high performance liquid chromatography.

[0032]

[Table 2]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 645) C12R 1: 685 (C12P 17/10 1:01 C12R 1: 685) C12R 1:13 (C12P 17/10 C12R (1:01) (C12P 17/10 C12R 1:13) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 17/00-17/18 C07D 207/12 CA / REGISTRY (STN)

Claims (5)

(57) [Claims] 1. A compound represented by the general formula (2): (In the formula, R represents a lower alkyl group having 1 to 6 carbon atoms) Alkyl-2- [4-[[(3S) -1-
Acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate or a salt thereof was added to Aspergillus spp, Silascinera spp,
1 selected from the group consisting of filamentous fungi belonging to the genus Cuavariaria or the genus Mortierella, a culture solution thereof and fractions thereof
General formula (3) characterized by treating with one or more species Optical activity represented by (+)-2- [4-[[(3S)
-1-acetamidoyl-3-pyrrolidinyl] oxy]
A method for producing phenyl] -3- [7-amidino-2-naphthyl] propionic acid or a salt thereof. 2. The filamentous fungus is Aspergillus fumigatus, Aspergillus unilateraris, Aspergillus
Nigia, Aspergillus Usami, Syracinella mucoloides, Cuavariaria trifoli or Mortierella
The production method according to claim 1, which is Isaverina. 3. A compound represented by the general formula (2): (In the formula, R represents a lower alkyl group having 1 to 6 carbon atoms) Alkyl-2- [4-[[(3S) -1-
Acetimidoyl-3-pyrrolidinyl] oxy] phenyl] -3- [7-amidino-2-naphthyl] propionate or a salt thereof, yeast belonging to the genus Debyromyces,
Bacteria belonging to the genus Brevibacterium, the genus Sitophaga or the genus Sphingobacteria, a culture solution thereof, and a treatment with one or more species selected from the group consisting of fractions thereof (4) ) [Chemical 4] Optical activity represented by (-)-2- [4-[[(3S)
-1-acetamidoyl-3-pyrrolidinyl] oxy]
A method for producing phenyl] -3- [7-amidino-2-naphthyl] propionic acid or a salt thereof. 4. The production method according to claim 3, wherein the yeast or bacterium is Debyromyces species, Brevibacterium linens, Sitophaga avensicora or Sphingobacterium spiritibolum. 5. The method according to claim 1, wherein R in the general formula (2) is an ethyl group.