JP2000247950A - Polymorphic crystal of amidinoaniline derivative sulfate dihydrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide 2-[N-(3-amidinophenyl)-N-(3-carboxymethyloxybenzyl) amino]-N-[4-[1-(1-iminoethyl)piperidin-4-yl]phenyl]acetamide sulfate dihydrate polymorphic crystal which has a strong and selective activated blood coagulation factor X-inhibiting activity and is useful as an agent for preventing or treating thromboembolism. SOLUTION: This polymorphic crystal exhibits strong diffraction peaks at 5.0, 16.1,18.8,19.1,20.7,21.1 and 23.9 degree at a diffraction angle (2θ±0.1 in a powder X-ray diffraction figure. The polymorphic crystal is produced by adding and thermally dissolving 2-[N-(3-amidinophenyl)-N-(3-carboxymethyloxybenzyl) amino]-N-[4-[1-(1-iminoethyl)piperidin-4-yl]phenyl]acetamide or its salt in an aqueous sulfuric acid solution containing the equivalent mole or more of sulfuric acid, removing insolubles, when the insolubles are contained, gradually cooling or standing to cool the filtrate, and then filtering the cooled product to obtain the deposited crystals.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel polymorph of amidinoaniline derivative sulfate dihydrate useful as a pharmaceutical.

More specifically, the present invention provides a compound of formula (I) having potent and selective activated blood coagulation factor X inhibitory activity, which is useful as an agent for preventing or treating thromboembolic diseases.

[0003]

Embedded image

The amidinoaniline derivative (chemical name: 2- [N- (3-amidinophenyl) -N- (3-
Carboxymethyloxybenzyl) amino] -N- [4
-[1- (1-Iminoethyl) piperidin-4-yl]
[Phenyl] acetamide).

[0005]

2. Description of the Related Art 2- [N- (3-amidinophenyl)-
N- (3-carboxymethyloxybenzyl) amino]
-N- [4- [1- (1-iminoethyl) piperidine-
4-yl] phenyl] acetamide and a salt thereof are novel compounds not described in the literature, and their physical properties and pharmacological activities are not known at all.

[0006]

DISCLOSURE OF THE INVENTION The present inventors have found that 2-
[N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4- [1-
A study on (1-iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate hydrate revealed that there were several types of polymorphs in the compound. And the existence ratio fluctuated, and it was found that a product of a certain quality could not be obtained.

[0007] Usually, compounds having crystal polymorphs have different properties depending on the crystal polymorphs, so that even the same compound may exhibit completely different effects. In particular, it is known that there is a difference in solubility, dissolution rate, stability, etc. in pharmaceuticals, and even when the same compound is used, the intended effect cannot be obtained due to a difference in crystal polymorphism. Or produce a different effect than expected,
This can lead to unexpected situations. Furthermore, it is conceivable that the formulation itself becomes difficult or the properties of the manufactured formulation change due to the difference in physical properties between the crystalline polymorphs. Therefore, there is a need to provide compounds of the same quality that can always be expected to have certain properties and effects. Therefore, when a compound having a crystalline polymorph is used as a drug, it is necessary to stably provide a compound having a certain crystallinity in order to ensure uniform quality and certain effects required as a drug. In addition, a stable polymorph that can maintain the same quality in storage is desired.

2- [N- (3-amidinophenyl) -N
-(3-carboxymethyloxybenzyl) amino]-
N- [4- [1- (1-Iminoethyl) piperidine-4
[-Yl] phenyl] acetamidosulfate hydrate is a compound having several types of polymorphs, and a plurality of polymorphs may be mixed depending on the production method. In addition, some polymorphs vary depending on the external environment during storage, and may not have a certain quality. Therefore, in order to ensure a certain effect and quality, which are indispensable requirements for pharmaceuticals, we have found a stable crystalline polymorph of the compound and established a manufacturing method to always obtain the crystalline polymorph constantly. I was eager to do it.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have proposed 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino useful as an agent for preventing or treating thromboembolic diseases. ] -N- [4- [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate hydrate. As a result of intensive studies, it was found that the polymorph of the present invention was constant according to the following method. That the polymorph of the present invention is excellent in moisture resistance and the like, and is found to be extremely useful as a pharmaceutical,
The present invention has been made.

The present invention provides a powder X-ray diffraction pattern with a diffraction angle (2θ ± 0.1 degrees) of 5.0, 16.1, 1
8.8, 19.1, 20.7, 21.1 and 23.9
Novel 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4- [1- (1-iminoethyl) piperidine-4 showing a very strong diffraction peak -Yl] phenyl] acetamidosulfate dihydrate.

The polymorph of the present invention is 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4- [1- (1-iminoethyl) piperidine. -4-yl] phenyl] acetamide or a salt thereof is added to an aqueous solution of sulfuric acid containing equimolar or more, preferably 5 to 10-fold mol of sulfuric acid, preferably 5 to 10% aqueous sulfuric acid, and dissolved by heating. Can be produced by slowly cooling or allowing to cool after removing insolubles, and collecting precipitated crystals by filtration. The dissolution temperature can be adjusted appropriately depending on the type and amount of the raw material used, the concentration and amount of the aqueous sulfuric acid solution, and the like. Slow cooling or cooling may be performed with stirring.

The thus obtained polymorph of the present invention has excellent moisture resistance and is very desirable for storage.

The contents of the present invention will be described in detail with reference to the following Reference Examples, Examples and Test Examples. The melting points of the various polymorphs were determined by Rigaku Co., Ltd.
/ DTA) Measurement was performed at a heating rate of 10 ° C./min using Thermo plus TG8120, and the result was indicated by the extrapolation melting onset temperature. In addition, powder X-ray diffraction data of various polymorphs was obtained by X-ray diffractometer RINT1400 of Rigaku Corporation.
The measurement was performed using a uKα ray (1.541 °).

Reference Example 1 4-phenylpiperidine hydrochloride 125 g of 1,2,5,6-tetrahydro-4-phenylpyridine hydrochloride and 12.0 g of 10% palladium carbon were suspended in 1 L of methanol, and the suspension was kept at room temperature under a hydrogen atmosphere. Stirred under pressure for 5 hours. The insoluble material was removed by filtration with Celite, and the filtrate was concentrated under reduced pressure. The obtained residue was suspended in ethyl acetate and a small amount of ethanol, and the insoluble material was collected by filtration to obtain 60.3 g of 4-phenylpiperidine hydrochloride. Was.

¹ H-NMR (DMSO-d ₆ ) δ pp
m: 1.79-2.01 (4H, m), 2.73-3.08 (3H, m), 3.20-3.4
5 (2H, m), 7.10-7.40 (5H, m), 9.18 (2H, br-s)

Reference Example 2 10.0 g of 4-phenyl-1-trifluoroacetylpiperidine hydrochloride and 25 ml of triethylamine were added to 50 ml of methylene chloride, and 8.5 ml of trifluoroacetic anhydride was added dropwise under ice cooling. After reacting at room temperature for 2 hours, the reaction mixture was concentrated under reduced pressure, and the residue was suspended in diethyl ether. The insolubles were removed by filtration through Celite, and the filtrate was washed successively with water, dilute hydrochloric acid, a saturated aqueous solution of sodium hydrogen carbonate and a saturated aqueous solution of sodium chloride. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 12.45 g of 4-phenyl-1-trifluoroacetylpiperidine.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.63-1.80 (2H, m), 1.90-2.07 (2H, m), 2.74-2.93 (2
H, m), 3.17-3.32 (1H, m), 4.06-4.21 (1H, m), 4.62-
4.78 (1H, m), 7.11-7.40 (5H, m)

Reference Example 3 4- (4-nitrophenyl) -1-trifluoroacetylpiperidine 4-phenyl-1-trifluoroacetylpiperidine 1
23.7 g was dissolved in 600 ml of trifluoroacetic acid, and 82.0 g of sodium nitrate was added under ice cooling. The reaction was allowed to continue at room temperature for 14 hours, the insolubles were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. Ethyl acetate was added to the residue, washed sequentially with water, a saturated aqueous solution of sodium hydrogen carbonate and a saturated aqueous solution of sodium chloride, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and diethyl ether was added to suspend the mixture.
90.4 g of-(4-nitrophenyl) -1-trifluoroacetylpiperidine was obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.66-1.82 (2H, m), 1.95-2.10 (2H, m), 2.81-3.04 (2
H, m), 3.20-3.34 (1H, m), 4.12-4.25 (1H, m), 4.68-
4.80 (1H, m), 7.38 (2H, d, J = 8.7Hz), 8.20 (2H, d, J
= 8.7Hz)

Reference Example 4 4- (4-aminophenyl) -1-trifluoroacetylpiperidine 70.5 g of 4- (4-nitrophenyl) -1-trifluoroacetylpiperidine, 10.0 g of 10% palladium carbon and 2 g
N-hydrochloric acid (100 ml) was suspended in methanol (50 ml) and reacted at room temperature and normal pressure for 8 hours in a hydrogen atmosphere. The insolubles were removed by filtration through Celite, and the filtrate was concentrated under reduced pressure. The obtained residue was dissolved in water, made sufficiently alkaline with a saturated aqueous solution of sodium hydrogen carbonate, and the precipitate was collected by filtration. The residue was dissolved in methylene chloride, washed with a saturated saline solution, the organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to give 4- (4
-Aminophenyl) -1-trifluoroacetylpiperidine (60.7 g) was obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.50-1.75 (2H, m), 1.87-2.00 (2H, m), 2.62-2.76 (1
H, m), 2.77-2.91 (1H, m), 3.15-3.28 (1H, m), 3.61
(2H, br-s), 4.05-4.18 (1H, m), 4.61-4.74 (1H, m),
6.65 (2H, d, J = 8.4Hz), 6.98 (2H, d, J = 8.4Hz)

Reference Example 5 Methyl 2- (3-formylphenyloxy) acetate 10.0 g of 3-hydroxybenzaldehyde was dissolved in 50 ml of dimethyl sulfoxide, 13.0 g of potassium carbonate was added, and 8.14 ml of methyl bromoacetate was added dropwise at room temperature. Stir for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with diethyl ether. The organic layer was washed successively with water, a saturated aqueous solution of sodium bicarbonate and brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. 16.3 g of methyl (formylphenyloxy) acetate were obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
3.82 (3H, s), 4.71 (2H, s), 7.20-7.41 (1H, m), 7.3
5-7.41 (1H, m), 7.44-7.56 (2H, m), 9.97 (1H, s)

Reference Example 6 Methyl 2- (3-hydroxymethylphenyloxy) acetate 10.0 g of methyl 2- (3-formylphenyloxy) acetate
Was dissolved in 50 ml of tetrahydrofuran, 1.01 g of sodium borohydride was added under ice-cooling, 10 ml of methanol was added dropwise, and the mixture was stirred at room temperature under an argon atmosphere for 2 hours. Water and 30 ml of 2N hydrochloric acid were added to the reaction mixture, and the mixture was extracted with diethyl ether. The organic layer was washed with water and a saturated saline solution, and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the residue was subjected to silica gel column chromatography (eluent:
Purification with ethyl acetate-hexane) gave 4.37 g of methyl 2- (3-hydroxymethylphenyloxy) acetate.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.69 (1H, t, J = 6.1Hz), 3.81 (3H, s), 4.61-4.72 (4
H, m), 6.83 (1H, dd, J = 8.3Hz, 2.6Hz), 6.92-7.03 (2
H, m), 7.23-7.32 (1H, m)

Reference Example 7 Methyl 2- (3-bromomethylphenyloxy) acetate 4.20 g of methyl 2- (3-hydroxymethylphenyloxy) acetate and 6.18 g of triphenylphosphine were dissolved in 30 ml of methylene chloride. 7.81 g of carbon bromide were added in several portions. After reacting for 15 minutes at room temperature under an argon atmosphere, the reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (elution solvent: ethyl acetate-hexane) to give 2- (3-bromo 5.5 g of methyl (methylphenyloxy) acetate were obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
3.81 (3H, s), 4.45 (2H, s), 4.65 (2H, s), 6.81-6.8
8 (1H, m), 6.93-6.98 (1H, m), 7.00-7.06 (1H, m), 7.
23-7.30 (1H, m)

Reference Example 8 Ethyl 2- (3-cyanophenylamino) acetate 200.6 g of 3-aminobenzonitrile and 353.9 ml of N-ethyldiisopropylamine were added to 500 ml of N, N-dimethylformamide, and 206.5 ml of methyl bromoacetate was added dropwise. I
Stirred at 60 ° C. for 4 hours. 1000 ml of water is added to the reaction mixture,
The precipitate was collected by filtration, and the obtained solid was washed successively with 1000 ml of 1N hydrochloric acid and 1000 ml of ethanol to obtain 298.6 g of ethyl 2- (3-cyanophenylamino) acetate.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.32 (3H, t, J = 7.1Hz), 3.90 (2H, d, J = 5.0Hz), 4.27
(2H, q, J = 7.1Hz), 4.48-4.62 (1H, m), 6.75-6.85 (2
H, m), 6.97-7.05 (1H, m), 7.20-7.30 (1H, m)

REFERENCE EXAMPLE 9 2- (3-cyanophenylamino) acetic acid To 80.0 g of ethyl 2- (3-cyanophenylamino) acetate was added 100 ml of methanol and 500 aqueous 1N sodium hydroxide solution.
ml was added and stirred at room temperature for 2 hours. The reaction mixture was acidified by adding dilute hydrochloric acid, and extracted with diethyl ether. The organic layer was washed with brine and dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain 64.3 g of 2- (3-cyanophenylamino) acetic acid.

¹ H-NMR (CDCl ₃ ) δ ppm:
3.99 (2H, s), 6.78-6.87 (2H, m), 7.00-7.10 (1H,
m), 7.20-7.30 (1H, m)

Reference Example 10 2- (3-cyanophenylamino) -N- [4- (1-
Trifluoroacetylpiperidin-4-yl) phenyl] acetamide 2- (3-cyanophenylamino) ethyl acetate 5.2 g,
8.0 g of 4- (4-aminophenyl) -1-trifluoroacetylpiperidine and 5.0 g of 1-hydroxybenzotriazole hydrate are added to 300 ml of acetonitrile, and 1-ethyl-3- (3-dimethylaminopropyl) is stirred under ice cooling. )
6.25 g of carbodiimide hydrochloride were added in several portions. After stirring at room temperature for 6 hours, dilute hydrochloric acid was added to the reaction mixture to make it sufficiently acidic, and the mixture was extracted with methylene chloride. The organic layer was washed sequentially with an aqueous sodium hydrogen carbonate solution and brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (elution solvent:
(Ethyl acetate-hexane) to give 2-
8.2 g of (3-cyanophenylamino) -N- [4- (1-trifluoroacetylpiperidin-4-yl) phenyl] acetamide was obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.55-1.75 (2H, m), 1.88-2.01 (2H, m), 2.72-2.91 (2
H, m), 3.16-3.28 (1H, m), 3.94 (2H, d, J = 5.4Hz), 4.
07-4.19 (1H, m), 4.62-4.77 (2H, m), 6.85-6.94 (2H,
m), 7.06-7.20 (3H, m), 7.25-7.36 (1H, m), 7.47 (2
(H, d, J = 8.5Hz), 8.24 (1H, s)

Reference Example 11 2- [N- (3-cyanophenyl) -N- (3-ethoxycarbonylmethyloxybenzyl) amino] -N-
[4- (1-trifluoroacetylpiperidin-4-yl) phenyl] acetamide 2- (3-cyanophenylamino) -N- [4- (1-
Trifluoroacetylpiperidin-4-yl) phenyl] acetamide 6.14 g, methyl 2- (3-bromomethylphenyloxy) acetate 5.5 g and sodium iodide 2.
57 g was suspended in ethanol (100 ml), N-ethyldiisopropylamine (3.73 ml) was added, and the mixture was heated under reflux for 16 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate.
After washing with dilute hydrochloric acid and a saturated saline solution sequentially and drying over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue is purified by silica gel column chromatography (elution solvent: tetrahydrofuran-methylene chloride-hexane) to give 2- [N- (3-cyanophenyl) -N- (3-ethoxycarbonylmethyloxybenzyl) amino]-. N
-[4- (1-trifluoroacetylpiperidine-4-
4.69 g of yl) phenyl] acetamide were obtained.

¹ H-NMR (CDCl ₃ ) δ ppm:
1.28 (3H, t, J = 7.1Hz), 1.60-1.73 (2H, m), 1.89-2.0
1 (2H, m), 2.72-2.91 (2H, m), 3.17-3.28 (1H, m), 4.
05-4.19 (3H, m), 4.24 (2H, q, J = 7.1Hz), 4.59 (2H,
s), 4.63-4.74 (3H, m), 6.77-6.90 (3H, m), 6.97-7.0
6 (2H, m), 7.10-7.19 (3H, m), 7.23-7.42 (4H, m), 7.
83 (1H, br-s)

Reference Example 12 2- [N- (3-amidinophenyl) -N- (3-methoxycarbonylmethyloxybenzyl) amino] -N-
[4- (4-Piperidinyl) phenyl] acetamide dihydrochloride 2- [N- (3-cyanophenyl) -N- (3-ethoxycarbonylmethyloxybenzyl) amino] -N-
[4- (1-Trifluoroacetylpiperidin-4-yl) phenyl] acetamide (3.0 g) was added with a saturated hydrogen chloride-methanol solution (100 ml), sealed, and allowed to react at room temperature for 5 hours. The reaction mixture was concentrated under reduced pressure. And got an imidate. Ethyl acetate and methylene chloride were added to 1.0 g of the imidate, and the mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 20 ml of methanol, a solution of 172 mg of ammonium chloride in 0.69 ml of water was added, and the mixture was refluxed for 6 hours. After concentrating the reaction mixture under reduced pressure,
Purification was performed by S column chromatography (elution solvent: water-methanol), and the obtained target fraction was acidified by adding diluted hydrochloric acid, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in 5 ml of methanol, 10 ml of water, 5 ml of acetonitrile and 1.0 g of potassium carbonate were added, and the mixture was reacted at room temperature for 15 hours. After dilute hydrochloric acid was added to the reaction mixture to make it acidic, the mixture was concentrated under reduced pressure, methanol was added to the residue, and insolubles were removed by filtration through celite. The solvent was distilled off under reduced pressure, the obtained residue was dissolved in methanol (10 ml), a small amount of a saturated hydrogen chloride-methanol solution was added, and the mixture was reacted at room temperature for 8 hours. After the reaction mixture was concentrated under reduced pressure, the residue was purified by ODS column chromatography (elution solvent: water-methanol), and the obtained target fraction was acidified by adding dilute hydrochloric acid, and the solvent was distilled off under reduced pressure. N- (3-amidinophenyl) -N- (3-
Methoxycarbonylmethyloxybenzyl) amino]-
418 mg of N- [4- (4-piperidinyl) phenyl] acetamide dihydrochloride were obtained.

¹ H-NMR (DMSO-d ₆ ) δ pp
m: 1.73-2.00 (4H, m), 2.72-2.85 (1H, m), 2.90-3.0
4 (2H, m), 3.66 (3H, s), 4.37 (2H, s), 4.77-4.88 (4
H, m), 6.77-7.40 (10H, m), 7.52-7.70 (2H, m), 8.60-
9.55 (6H, m), 10.30 (1H, s)

Reference Example 13 2- [N- (3-amidinophenyl) -N- (3-methoxycarbonylmethyloxybenzyl) amino] -N-
[4- [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamide dihydrochloride 2- [N- (3-amidinophenyl) -N- (3-methoxycarbonylmethyloxybenzyl) amino] -N −
300 mg of [4- (4-piperidinyl) phenyl] acetamide dihydrochloride and 74 mg of ethyl acetylimidate hydrochloride were added to 3 ml of methanol, and 0.167 ml of triethylamine was added dropwise. After stirring at room temperature for 4.5 hours, the reaction mixture was concentrated under reduced pressure, the residue was purified by ODS column chromatography (elution solvent: water-methanol), diluted hydrochloric acid was added to the desired fraction, and the mixture was concentrated under reduced pressure. The obtained residue was suspended by adding diethyl ether and a small amount of methanol, the supernatant was removed, and the residue was dried under reduced pressure to give 278 mg of 2- [N
-(3-amidinophenyl) -N- (3-methoxycarbonylmethyloxybenzyl) amino] -N- [4-
[1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamide dihydrochloride was obtained.

¹ H-NMR (DMSO-d ₆ ) δ pp
m: 1.54-1.94 (4H, m), 2.30 (3H, s), 2.79-2.92 (1
H, m), 3.09-3.35 (2H, m), 3.66 (3H, s), 3.95-4.06
(1H, m), 4.17-4.28 (1H, m), 4.37 (2H, s), 4.67-4.7
9 (4H, m), 6.75-7.40 (10H, m), 7.56 (2H, d, J = 8.5H
z), 8.66 (1H, s), 8.96 (2H, s), 9.21 (1H, s), 9.28
(2H, s), 10.27 (1H, s)

Reference Example 14 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
[1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamide dihydrochloride 2- [N- (3-amidinophenyl) -N- (3-methoxycarbonylmethyloxybenzyl) amino] -N-
1 ml of 1N hydrochloric acid and 1 ml of acetonitrile were added to 71 mg of [4- [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamide dihydrochloride, and the mixture was reacted at 60 ° C. for 4 hours. The reaction mixture was concentrated under reduced pressure, the residue was purified by ODS column chromatography (elution solvent: water-acetonitrile), the resulting target fraction was acidified by adding dilute hydrochloric acid, and the solvent was distilled off under reduced pressure. 58 mg of N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4- [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamide dihydrochloride were obtained. Was.

¹ H-NMR (DMSO-d ₆ ) δ pp
m: 1.52-1.93 (4H, m), 2.31 (3H, s), 2.77-2.92 (1
H, m), 3.05-3.35 (2H, m), 3.94-4.07 (1H, m), 4.20-
4.35 (1H, m), 4.41 (2H, s), 4.63 (2H, s), 4.73 (2H,
s), 6.72-7.40 (10H, m), 7.59 (2H, d, J = 8.6Hz), 8.
80 (1H, s), 9.14 (2H, s), 9.25-9.50 (3H, m), 10.48
(1H, s)

Reference Example 15 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
[1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamide hydrochloride 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
16.69 g of [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamide dihydrochloride is purified by ODS column chromatography (elution solvent: water-acetonitrile), and the desired fraction is concentrated by distillation. [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
11.32 g of [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamide hydrochloride was obtained.

¹ H-NMR (DMSO-d ₆ ) δ pp
m: 1.40-1.95 (4H, m), 2.30 (3H, s), 2.83 (1H, t,
J = 11.6Hz), 3.79-4.49 (6H, m), 4.69 (2H, br-s), 6.56
-6.85 (3H, m), 6.87-7.40 (7H, m), 7.56 (2H, d, J = 8.
4Hz), 8.40-11.35 (6H, m)

Reference Example 16 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
[1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
4.4 g of [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamide hydrochloride is added at 50 ° C. with 1N sulfuric acid.
Dissolved in 50 ml. After stirring at room temperature for 6 hours, the precipitate was collected by filtration and washed sequentially with water and acetone to give 2-
[N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4- [1-
3.9 g of (1-iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate was obtained.

¹ H-NMR (DCOOD) δ ppm:
1.69-1.99 (2H, m), 2.00-2.22 (2H, m), 2.48 (3H, m
s), 2.90-3.12 (1H, m), 3.28-3.60 (2H, m), 4.10-4.35
(2H, m), 4.79 (2H, s), 4.82 (2H, s), 5.00 (2H, s),
6.87-7.08 (3H, m), 7.31 (2H, d, J = 8.5Hz), 7.32-7.
43 (1H, m), 7.46 (2H, d, J = 8.5Hz), 7.50-7.85 (4H,
m)

Example 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
Crystal polymorph of [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino ] -N- [4-
253 g of [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate was added to 1730 g of
After dissolving in a 7.4% sulfuric acid aqueous solution at 65 ° C to remove insolubles, the mixture was cooled to 42 ° C, stirred for 1 hour, and then cooled to room temperature. The resulting precipitate was collected by filtration and washed with water to give 214 g of 2- [N- (3-amidinophenyl) -N-
(3-carboxymethyloxybenzyl) amino] -N
-[4- [1- (1-Iminoethyl) piperidine-4-
A crystalline polymorph of [yl] phenyl] acetamidosulfate dihydrate was obtained.

Melting point: 230.7 ° C. 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
The powder X-ray diffraction pattern of the polymorph of [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate is as shown in FIG. 1 below.

[0048]

FIG.

Test Example 1 Measurement of Inhibitory Activity of Activated Blood Coagulation Factor X 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
5 μl of [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate in dimethyl sulfoxide, pH 8.4, Tris-HCl buffer 3
75 μl and 100 μl of an aqueous solution of 1 mM S-2222 (manufactured by Daiichi Kagaku) are mixed, and 20 μl of a 0.6 unit / ml gelatin-glycine buffer solution of human activated blood coagulation factor X (manufactured by Calbiochem) is added. And incubated at 37 ° C. for 10 minutes. The reaction was stopped by adding 100 μl of 60% acetic acid, and the absorbance (405 n
m) was measured.

The group without the test compound was used as a control,
A group without human activated blood coagulation factor X was used as a blank. The concentration (IC ₅₀ ) of the test compound at which the inhibition was 50% relative to the control was determined and used as an index of the activated blood coagulation factor X inhibitory activity. Table 1 shows the results.

Test Example 2 Measurement of thrombin inhibitory activity 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
5 μl of [1- (1-Iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate in dimethyl sulfoxide, pH 8.4, Tris-HCl buffer 3
75 μl and 100 μl of an aqueous solution of 1 mM S-2238 (manufactured by Daiichi Kagaku) were mixed with 2.0 units / ml of human-thrombin (manufactured by Sigma) gelatin.
20 μl of glycine buffer solution was added and incubated at 37 ° C. for 10 minutes. The reaction was stopped by adding 100 μl of 60% acetic acid, and the absorbance (405 nm) was measured.

A group without the test compound was used as a control.
The group without human-thrombin was used as a blank. Control
Of test compound at 50% inhibition against
(IC ₅₀) Was determined as an index of the thrombin inhibitory activity.
Table 1 shows the results.

Test Example 3 Measurement of anticoagulant action (plasma prothrombin time) 2- [N- (3-amidinophenyl) -N- (3-carboxymethyloxybenzyl) amino] -N- [4-
A dedicated cuvette containing 2 μl of a solution of [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate in dimethyl sulfoxide was heated to 37 ° C., and human normal plasma (manufactured by George King Co., Ltd.) ) 50 μl
One minute later, 100 μl of a plasma prothrombin time reagent (Neoplastin Plus (registered trademark), manufactured by Boehringer Meinheim KK) kept at 37 ° C. is added, and coagulation time is set using ST4 (manufactured by Boehringer Mannheim KK). Was measured.

A group without the test compound was used as a control.
The concentration (CT ₂ ) of the test compound that prolonged the control coagulation time by a factor of ₂ was determined and used as an index of the anticoagulant effect.
Table 1 shows the results.

[0055]

[Table 1]

[Brief description of the drawings]

FIG. 1 shows 2- [N- (3-amidinophenyl) of the present invention.
Powder X-ray of crystalline polymorph of -N- (3-carboxymethyloxybenzyl) amino] -N- [4- [1- (1-iminoethyl) piperidin-4-yl] phenyl] acetamidosulfate dihydrate Diffraction data (using a monochromator). The vertical axis indicates the intensity of X-rays (kcps), and the horizontal axis indicates the diffraction angle (2θ).

Claims (1)

[Claims] 1. A diffraction angle (2θ ± 2) in a powder X-ray diffraction pattern.
0.1 degrees), 5.0, 16.1, 18.8, 1
2- [N- (3-amidinophenyl)-showing strong diffraction peaks at 9.1, 20.7, 21.1 and 23.9 degrees
N- (3-carboxymethyloxybenzyl) amino]
-N- [4- [1- (1-iminoethyl) piperidine-
4-yl] phenyl] acetamidosulfate dihydrate crystal polymorph.