JP2606643B2 - Benzoic acid amidinophenyl ester derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel benzoic acid amidinophenyl ester derivative and an addition salt thereof, and more particularly to a novel benzoic acid amidinophenyl ester derivative having an inhibitory activity at an extremely low concentration against serine proteases such as trypsin, thrombin and factor Xa. The present invention relates to a novel benzoic acid amidinophenyl ester derivative and an addition salt thereof, and a protease inhibitor containing them as an active ingredient.

[0002]

2. Description of the Related Art It is well known that various proteolytic enzymes exist in a living body. For example, trypsin-like enzymes such as plasmin, trypsin, kallikrein, thrombin, urokinase, chymotrypsin-like enzymes, and pepsin-like enzymes are known. Enzymes and the like are known. It is known that these proteases cause various diseases when abnormally activated for some reason. Therefore, substances showing an inhibitory activity against these proteases are useful as some clinical therapeutic agents. For example, antiplasmin agents are useful as hemostatic agents, anti-inflammatory agents, antiallergic agents, antithrombin agents are useful for treating thrombosis, antitrypsin agents are useful for treating pancreatitis, antikallikrein agents are useful for treating inflammation, It is useful as a therapeutic agent for ulcers, and an anti-urokinase agent is useful for suppressing bleeding symptoms during thrombolytic therapy with urokinase.

[0003] Hitherto, development of protease inhibitors having such an action has been promoted, but their protease inhibitory activity is low, and they are not sufficient for practical use as pharmaceuticals. In addition, a protease inhibitor having sufficient inhibitory activity against a plurality of proteases has not yet been developed. For example, tripeptide derivatives containing certain argininals are widely known as protease inhibitors. That is, acetyl-L-leucyl-L-leucyl-argininal (leupeptin)
Is one of the tripeptide derivatives containing argininal produced by certain microorganisms (eg, J. Anti).
biotics (Tokyo) 1969, Vol. 22, 283
Page, but its inhibitory activity is low (eg, metabolism 197).
(2007, Vol. 14, No. 6, page 1087). D-phenylalanyl-L-prolyl-L-argininal is known as a thrombin inhibitor (for example, Symposia).
Biologica Hungarica 1984
25, p. 277), but its ability to inhibit other similar trypsin-like enzymes is weak. Have synthesized a large number of leupeptin derivatives, all of which have a small inhibitory activity on trypsin-like enzymes (J. Antibiotic).
s (Tokyo), 1988, Vol. 41, No. 2, page 220).

[0004]

SUMMARY OF THE INVENTION The present invention solves the problems of the prior art and has a practically sufficient inhibitory activity.
Moreover, it is an object of the present invention to develop a compound having a sufficient inhibitory activity against a plurality of proteases and a protease inhibitor containing the compound as an active ingredient.

[0005]

Means for Solving the Problems The present inventors have made intensive efforts to search for compounds having a stronger and broader inhibitory activity than conventional protease inhibitors. That is, the present inventor has synthesized various benzoic acid amidinophenyl ester derivatives,
Upon examining its pharmacological effects, it was found that certain benzoic acid amidinophenyl ester derivatives had an inhibitory effect on various proteolytic enzymes, such as trypsin, thrombin, factor Xa, and the like at extremely low concentrations. Reached.

Accordingly, the present invention provides the following formula (I)

Embedded image (Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen atom,
It represents a hydroxyl group, an amino group, a halogen atom, a carboxyl group or a 4-amidinophenyl-1-oxycarbonyl group. However, R ₁ and R ₂ are not simultaneously a hydrogen atom or a 4-amidinophenyl-1-oxycarbonyl group, and R ₁ and R ₂ may be protected. The present invention provides a benzoic acid amidinophenyl ester derivative represented by the formula (1) and an acid addition salt thereof.

In the formula (I), when R ₁ and / or R ₂ represent a halogen atom, examples of the halogen atom include halogen atoms such as chlorine, fluorine, bromine and iodine. In the formula (I), when R ₁ and / or R ₂ represent a hydroxyl group, an amino group, a carboxyl group or a 4-amidinophenyl-1-oxycarbonyl group, these functional groups may be protected. The protected hydroxyl group, for example, C ₁ -C ₅ alkyl carboxy group or a benzyloxy group, such as methyl carboxyethyl, ethyl carboxy. The protected amino group, such as acetylamino, propionylamino, C ₂ -C ₆ acylamino group such as hexanoylamino or the like benzyloxycarbonylamino group, and the like. Examples of the protected carboxyl group include a C ₁ -C ₆ alkyloxycarbonyl group such as methoxycarbonyl and ethoxycarbonyl. Examples of the protected 4-amidinophenyl-1-oxycarbonyl group include an N-benzyloxycarbonyl-4-amidinophenyl-1-oxycarbonyl group. Among the protected functional groups exemplified above, particularly R ₁ and / or
Or R ₂ is a C ₁ -C ₅ alkylcarboxy group, C ₂ -C
_{When it is a 6} acylamino group or a C ₁ -C ₆ alkyloxycarbonyl group, the compound of formula (I) becomes a so-called prodrug which, when administered as a drug, is gradually hydrolyzed in the body to be converted into an active compound. What you get.

The compound of formula (I) may be an acid addition salt, such as an inorganic acid salt such as hydrochloride, hydrobromide or sulfate; or an acetate, Oxalate, succinate, malate, citrate, lactate,
Organic acid salts such as benzenesulfonate and methanesulfonate are exemplified.

The compound of the formula (I) of the present invention can be produced as follows. (A) R ₁ and R ₂ represent a hydrogen atom, a hydroxyl group, an amino group,
The compound of the formula (I) in the case of a halogen atom or a carboxyl group or a protected functional group thereof can be produced by the following reaction formula.

Embedded image R ₁ ′ and R ₂ ′ in the general formula (II) represent a hydrogen atom or a halogen atom, or a protected hydroxyl, amino or carboxyl group, and R _{3 in the} general formula (III) represents a protected amidino group . These protected functional groups include the same as those described above.

The reaction between the benzoic acid derivative represented by the general formula (II) and the protected amidinophenol (III) is carried out by a usual esterification reaction known per se, for example, the benzoic acid derivative is protected in the presence of a condensing agent. The reaction is carried out by reacting with amidinophenol. Deprotection can also be carried out by a conventional method known per se. (B) A compound of the formula (I) wherein either R ₁ or R ₂ is a 4-amidinophenyl-1-oxycarbonyl group or a protected functional group thereof can be produced as follows. .

Embedded image The reaction between the isophthalic acid derivative of formula (IV) and the protected amidinophenol (III) can be carried out in the same manner as described above, and the deprotection can be carried out in the same manner.
The compound of the formula (I) thus obtained may be converted to an acid addition salt, and such a conversion reaction can be carried out by a method known per se.

The compound of formula (I) and the acid addition salt thereof obtained according to the present invention are novel compounds not described in the literature,
It has an inhibitory effect on proteolytic enzymes such as trypsin, thrombin, factor Xa and the like, and these inhibitory effects were observed at extremely low concentrations. The compound of the present invention has good solubility and is suitable for administration as a drug in an aqueous solution, physiological saline or other solutions as a drug. When the compound of the present invention is used as a medicament or a veterinary therapeutic agent, there is no particular limitation on the method of administration, and a suitable formulation (eg, tablet, injection, etc.) may be prepared by a commonly used pharmaceutical method, and may be injected intravenously, intramuscularly. It is used by injection, intravenous drip, oral administration, etc. The dose is 1 day per day based on the case where the weight of the individual to be administered is 60 kg.
1 to 1000 mg per individual is appropriate. However, it goes without saying that it can be increased or decreased as needed.

[0012]

EXAMPLES The present invention will be described in more detail with reference to the following Examples and Test Examples, but the present invention is not limited thereto. The abbreviations used in the examples have the following meanings. DMAP: 4-dimethylaminopyridine DMF: N, N-dimethylformamide WSC · HCl: 1-ethyl-3- (3-dimethylaminopropyl) -carbonylimide / hydrochloride Chf: chloroform MeOH: methanol EtOH: ethanol Et ₂ O : Diethyl ether AcOEt: ethyl acetate n-BuOH: n-butanol AcOH: acetic acid Z-Cl: benzyloxycarbonyl chloride ZS: benzyl S-4,6-dimethylpyrimidine-2
- thiocarbonate Ac-Cl: Acetyl chloride IPA: isopropyl alcohol n-Hex: n-hexane Pd-Black: palladium black MgSO _4: magnesium sulfate TLC: Thin layer chromatography _{Rf A = n-BuOH: AcOH} : H 2 O (4: 1:
2) Rf _B = Chf: MeOH (20: 1) Rf _C = Chf: MeOH (50: 1) Rf _D = Chf: MeOH (75: 1) Rf _E = Chf: MeOH (100: 1) Rf _F = Chf : MeOH: formic acid (7: 3: 0.2) Rf _G = Chf: MeOH: AcOH: H ₂ O (80:
20: 2.5: 5)

Embodiment 14'-amidinophenyl-
3,5-dihydroxybenzoate · HCl (Compound N
o. 1) 3,5-dibenzyloxybenzoic acid [Koichi Nakazawa: Organic
Compound synthesis method, Gihodo, Vol. 16, 20 (196
8)] 16.7 g, N- (benzyloxycarboni
Ru) -p-amidinophenol [Yuichi Kanaoka et al .: Che
m. Pharm. Bull. 28, (6) 1899-1
905 (1980)] with 13.5 g and 4-dimethyla
1.2 g of minopyridine in N, N-dimethylformamide
Dissolve in 200 ml and cool to 0-5 ° C with ice, WSC ・ HCl
 9.6 g was added as a powder and reacted overnight at room temperature with stirring. Anti
After the reaction, the solvent was distilled off under reduced pressure.
After adding and dissolving, wash with 150 ml of 5% hydrochloric acid,
After washing with 150 ml of a saturated saline solution, the organic phase was dried over anhydrous magnesium sulfate.
After drying with nesium, the solvent was distilled off under reduced pressure to obtain an oily substance (19.8).
g was obtained. Silica gel chromatography (chloroform
Target fraction in methanol: methanol 100: 1)
Was collected and the solvent was distilled off under reduced pressure to obtain 14.3 g (49%) of crystals.
%)Obtained. Melting point: 176-183 ° C (decomposition) Dissolve the crystals in 300 ml of EtOH and add 0.1N-H
Palladium black with addition of 25 ml of Cl / MeOH
3 g is added and catalytic reduction is carried out for 5 hours under a stream of hydrogen. Catalyst
After filtration, the solvent is distilled off under reduced pressure to obtain a crystalline residue. this
To MeOH / Et_TwoRecrystallized with O to give 4'-amidinophenyl
4. 3,5-dihydroxybenzoate.HCl
5 g (74%) were obtained. Melting point: 209-225 ° C TLC Rf value Rf_A 0.82 Rf_F 0.50 Elemental analysis C₁₄H₁₃N_TwoO_FourCl 3/5 H_TwoOCHN Analysis (%) 52.54 4.36 8.50 Theory (%) 52.63 4.48 8.77

Example 2 4'-Amidinophenyl-3
-Hydroxybenzoate.HCl (Compound No. 2) Example 1 using 15.9 g of 3-benzyloxybenzoic acid
Was reacted with N- (benzyloxycarbonyl) -p-amidinophenol to obtain 37.6 g of crude N- (benzyloxycarbonyl) -4'-amidinophenyl-3-benzyloxybenzoate. The desired fractions were collected by silica gel chromatography (chloroform: methanol 100: 1), and the solvent was distilled off under reduced pressure to obtain 13.7 g (41%) of crystals. Melting point: 108-136 ° C This was subjected to catalytic reduction according to Example 1 to give 4'-amidinophenyl-3-hydroxybenzoate.HCl.
0 g was obtained. Melting point: 202-219 ° C. TLC Rf value Rf _A 0.80 Elemental analysis C ₁₄ H ₁₃ N ₂ O ₃ Cl. 1/10 H ₂ O CHN Analysis value (%) 57.09 4.60 9.34 Theory Value (%) 57.09 4.52 9.51

Embodiment 3Di (4'-amidinopheni
) -5-Hydroxyisophthalate.2HCl (compound
Object No. 3) 72.5 g of 5-aminoisophthalic acid was added to 6N-sulfuric acid 230
suspended in 0 ml, cooled to 0-5 ° C, and stirred, 70.4 ml of water
Slowly drop 27.6 g of sodium nitrite dissolved in water
I do. After reacting for 1 hour, add 40 ml of boiling concentrated sulfuric acid.
The above reaction solution was added to 400 ml of water, and the mixture was boiled for 5 minutes.
After cooling, the precipitated crystals were collected by filtration, washed well with water, and dried under reduced pressure.
64.8 g (89%) of dried 5-hydroxyisophthalic acid
I got This was treated according to Example 1 with 5-benzyloxyi
41.8 g (65%) of sophthalic acid were obtained. This 5-b
21.7 g of benzyloxyisophthalic acid and N- (benzyl
Oxycarbonyl) -p-amidinophenol 43.2
g. Further, 3.9 g of 4-dimethylaminopyridine was added to WSC
-According to Example 1 in DMF using 30.6 g of HCl
The reaction was performed to obtain 73.4 g of an oily substance. This oil is
Kagel chromatography (chloroform: methanol
 100: 1 → 50: 1 → 20: 1)
The desired fractions were collected, the solvent was distilled off, and methanol
And crystallize until it becomes slightly cloudy, and leave it to cool overnight.
The precipitated crystals are collected by filtration, washed with methanol and dried. 2
Obtain 1.5 g (35%). Melting point: 95 ° C-decomposition TLC Rf value Rf_C 0.51 This di [N- (benzyloxycarbonyl) -p-a
Midinophenyl] -5-benzyloxyisophthalate
16.4 g of ethanol 300 ml and 1N-HCl / M
Catalytic reduction as in Example 1 in 42.2 ml of OH solution
MeOH / Et_TwoRecrystallized from O, 9.7 g
(94%). Melting point: 255-260 ° C Decomposition TLC Rf value Rf_A 0.51 Elemental analysis C_{twenty two}H₂₀N_FourO_FiveCl_Two CH N analysis (%) 53.50 4.01 11.28 Theoretical (%) 53.78 4.10 11.40

Example 4 4'-Amidinophenyl-3
-Aminobenzoate · 2HCl (Compound No. 4) 2.73 N NaOH in 27.4 g of m-aminobenzoic acid
Add 110 ml, dissolve with stirring, and then add
A solution of S60.3 g in 1,4-dioxane 110 ml was added with stirring and reacted overnight. Then, add 300 m
and wash twice with 400 ml of ethyl acetate. When the aqueous phase is adjusted to pH 2 with cold 5N HCl, the desired product, 3-benzyloxycarbonylaminobenzoic acid, crystallizes. This is collected by filtration, washed with water and dried. Recrystallization from MeOH / IPA gave 33.4 g (61%). This crystal 17.
6 g was reacted with 17.5 g of N- (benzyloxycarbonyl) -p-amidinophenol according to Example 1 to obtain 36.4 g of crude N- (benzyloxycarbonyl) -4'-amidinophenyl-3-benzyloxycarbonylaminobenzoate. I got it. This is subjected to silica gel chromatography (chloroform: methanol 100: 1).
The objective fraction was collected, the solvent was distilled off under reduced pressure, and the crystal residue was washed with EtOH and dried, and 12.6 g was obtained.
(37%). Melting point: 181 to 183 ° C. TLC Rf value Rf _C 0.51 The obtained N- (benzyloxycarbonyl) -4′-
Amidinophenyl-3-benzyloxycarbonylaminobenzoate (12.3 g) was subjected to catalytic reduction according to Example 1, the solvent was distilled off under reduced pressure, and the residue was recrystallized from MeOH / Et ₂ O to obtain 7.0 g (91%). Was. Melting point: 222-233 ° C Decomposition TLC Rf value Rf _A 0.67 Elemental analysis C ₁₄ H ₁₅ N ₃ O ₂ Cl ₂ CH N Analysis value (%) 51.20 4.63 12.74 Theoretical value (%) 51 .24 4.61 12.80

Embodiment 54'-amidinophenyl-
3,5-diaminobenzoate-3HCl (compound N
o. 5) 30.4 g of 3,5-diaminobenzoic acid was added to 2N-NaOH
 After dissolving in 300 ml with stirring, Z-C at 0-5 ° C under ice cooling.
61.5 ml is slowly added dropwise. 0-5 during the reaction
C. and maintained at pH 9-10 using 2N NaOH.
React under stirring for 3 to 4 hours while adjusting. Then the reaction solution
To Et_TwoO, washed twice with 500 ml, and the aqueous phase was washed with 5N-HC.
to pH 2 with l and once with 500 ml of ethyl acetate,
Extract twice more with 300 ml. Combine the extracts and add 5% H
And then washed twice with 50 ml of saturated saline.
After washing twice, anhydrous MgSO_FourDry with. Solvent is distilled under reduced pressure
After removal, recrystallized from AcOEt / n-Hex, 55.2 g
(66%). 25.2 g of this crystal and N- (benzyl
(Oxycarbonyl) -p-amidinophenol 16.2
g, DMAP1.5g, WSC ・ HCl 11.5g
The reaction was carried out in DMF using Example 1 to obtain crude N-
(Benzyloxycarbonyl) -4'-amidinopheni
-3,5-dibenzyloxycarbonylaminobenzo
39.6 g (98%) of the ate were obtained. This is silica gel
Chromatography (chloroform: methanol 10
0: 1), collect the desired fraction and reduce the solvent.
The residue was dried under reduced pressure to give 21.8 g (54%). TLC Rf value Rf_C 0.31 13.4 g of this was mixed with 200 ml of EtOH and 1N HCl /
The catalytic reduction was carried out in the same manner as in Example 1 in 60 ml of MeOH.
The solvent is distilled off under reduced pressure, the residue is washed with EtOH and dried.
Then, 6.3 g (83%) of the desired product was obtained. Melting point: 201-247 ° C Decomposition TLC Rf value Rf_A 0.51 Elemental analysis C₁₄H₁₇N_FourO_TwoCl_Three CH N analysis (%) 44.65 4.71 14.74 Theoretical (%) 44.29 4.51 14.76

Example 6 4'-Amidinophenyl-3
-Acetylaminobenzoate (Compound No. 6) 6.9 g of m-aminobenzoic acid and 3.9 ml of Ac-Cl
Was reacted in the same manner as in Example 5 to obtain 2.1 g of crude crystals of 3-acetylaminobenzoic acid. This was washed with AcOEt to give 1.5 g (17%). 1.3 g of this crystal and N
2.0 g of-(benzyloxycarbonyl) -p-amidinophenol and 1.8 g of DMAP were added to WSC · HCl.
Using 1.7 g, the reaction was carried out in DMF according to Example 1, and M
Recrystallization from OH / AcOEt gave 2.3 g (73%) of N- (benzyloxycarbonyl) -4'-amidinophenyl-3-acetylaminobenzoate. TLC Rf value Rf _G 0.61 1.0 g of these crystals was added to 130 ml of ethanol and 0.45 N
Carried out in the same manner as in the catalytic reduction as in Example 1 in -HCl / MeOH5.2ml, the solvent to dissolve the residue was evaporated under reduced pressure to MeOH, collected by filtration and reprecipitation after the precipitate in Et ₂ O, dried purposes 0.7 g (88%) of the product was obtained. Melting point: 244 to 249 ° C. Decomposition TLC Rf value Rf _F 0.65 Elemental analysis C ₁₆ H ₁₆ N ₃ O ₃ Cl. 1/10 H ₂ O CHN Analysis value (%) 57.16 4.85 12.32. Theoretical value (%) 57.27 4.87 12.52

Example 7 4'-Amidinophenyl-3
-Amino-4-chlorobenzoate · 2HCl (compound
No. 7) 2N-N in 13.7 g of 3-amino-4-chlorobenzoic acid
Add 80 ml of aOH and dissolve with stirring. This is 0-5
12.3 ml of Z-Cl is slowly added dropwise while stirring at ice-cold temperature. Thereafter, the same procedure as in Example 5 was carried out, and the aqueous phase was changed to 5N-HC.
When acidified with 1, crystals of 3-benzyloxycarbonylamino-4-chlorobenzoic acid are precipitated. The crystals are collected by filtration, washed sufficiently with water, further washed with AcOEt, and dried, and 13.1 g is obtained.
(53%). 27.2 g of these crystals and N- (benzyloxycarbonyl) -p-amidinophenol.
0 g and 2.2 g of DMAP were further added to 18.7 of WSC / HCl.
Using g, the reaction was carried out in DMF according to Example 1, and the crude product was purified by silica gel chromatography (chloroform: methanol 100: 1), and the desired fraction was collected to obtain 32.5 g (65%). Mp: 157-159 ° C. TLC Rf value Rf _D 0.46 EtOH 400 ml and 1N-H The crystals 32.3g
The catalytic reduction was carried out in the same manner as in Example 1 with 115.7 ml of Cl / MeOH, the solvent was distilled off under reduced pressure, and the desired fraction was collected by column chromatography (Sephadex LH-20 MeOH). Me
Recrystallization from OH / Et ₂ O gave the desired product in 9.7 g (46%). Melting point: 226-233 ° C. TLC Rf value Rf _F 0.58 Elemental analysis C ₁₄ H ₁₄ N ₃ O ₂ Cl ₃ .1 / 2 H ₂ O CHN Analysis value (%) 45.17 4.11 11.28 Theoretical value (%) 45.25 4.07 11.31

Example 8 4'-Amidinophenyl-5
-Amino-2-chlorobenzoate · 2HCl (compound
No. 8) According to Example 7, 5-benzyloxycarbonylamino-
2-Chlorobenzoic acid was synthesized, and 38.8 g (6
4%). This was recrystallized from AcOEt / n-Hex to obtain 30.4 g (50%). 21.4 g of this crystal
And N- (benzyloxycarbonyl) -p-amidinophenol (18.9 g) and DMAP (1.7 g) were added to WSC.H
Reaction was carried out according to Example 1 in DMF using 14.8 g of Cl and the crude obtained was chromatographed on silica gel (chloroform: methanol 100: 1 → 50: 1).
And the solvent was distilled off under reduced pressure to obtain 16.8 g (43%) of the desired product. TLC Rf value Rf _E 0.43 This N- (benzyloxycarbonyl) -4′-amidinophenyl-5-benzyloxycarbonylamino-
13.4 g of 2-chlorobenzoate in 300 m of EtOH
1 and 48.0 ml of 1N HCl / MeOH in the same manner as in Example 1, and the solvent was distilled off under reduced pressure to obtain Me.
Recrystallization from OH / Et ₂ O gave the desired product in 8.1 g (93%). Melting point: 217-228 ° C Decomposition TLC Rf value Rf _F 0.54 Rf _A 0.64 Elemental analysis C ₁₄ H ₁₄ N ₃ O ₂ Cl ₃ .2 / 3 H ₂ O CHN Analysis value (%) 42.24 4.11 11.31 Theoretical value (%) 42.21 4.13 11.22

Embodiment 9Di (4'-amidinopheni
) -5-aminoisophthalate.3HCl (compound N
o. 9) 23.6 g of 5-aminoisophthalic acid was added to 1N HCl 2
Dissolved in 60 ml and cooled to 0-5 ° C. under stirring and Z-Cl 2
0 ml is slowly added dropwise. Then, as in Example 5,
The obtained crude crystals yielded 38.8 g (95%). This is
After washing with PA and drying, 31.9 g (78%) were obtained.
This 5-benzyloxycarbonylaminoisophthal
22.1 g of acid and N- (benzyloxycarbonyl) -p
Amidinophenol 37.8 g, and DMAP 3.4 g
In DMF using 26.8 g of WSC.HCl
The reaction was carried out according to 1, and the solvent was distilled off under reduced pressure.
Chromatography (chloroform: methanol 50:
Perform 1) to collect the desired fraction and evaporate the solvent under reduced pressure
After washing with EtOH and drying, 12.7 g of the desired product was obtained.
(22%). Melting point: 124 ° C-decomposition TLC Rf value Rf_B 0.49 12.6 g of these crystals were mixed with 300 ml of EtOH and 1N-HC.
Contact as in Example 1 in 46.2 ml of 1 / MeOH
After reduction, the solvent is distilled off under reduced pressure, and the residue is washed with MeOH, Et.
OH / Et_TwoRecrystallized with O to obtain 8.0 g (99%) of the target compound
I got Melting point: 217 ° C-decomposition TLC Rf value Rf_A 0.50 Elemental analysis C_{twenty two}H_{twenty two}N_FiveO_FourCl_Three・ 6/5 H_TwoOCHN Analysis (%) 48.18 4.29 12.67 Theoretical (%) 48.18 4.48 12.77

Test Example The test for the inhibitory effect of the compound (I) thus obtained on the protease was carried out as follows. The synthetic substrate used to test the action of inhibiting the enzyme from decomposing the substrate was PS-994 [H-DL] for plasmin.
ys (Tos) -Phe-Lys-CHA.2HCl,
Nitto Boseki CHA: 3-carboxy-4-hydroxyanilide, hereinafter referred to as CHA], thrombin and trypsin are PS-915 [HD-Phe-Pro.
-Arg-CHA.2HCl, manufactured by Nitto Boseki Co., Ltd.), and for Factor Xa, PS-2000 [ZD-Lys
(HCO) -Gly-Arg-CHA.2HCl, manufactured by Nitto Boseki Co., Ltd.)
-D-Pro-Phe-Arg-pNA, mold p
NA: p-nitroanilide, hereinafter referred to as pNA], and urokinase

Embedded image Was used. Human plasmin was 0.3 CU / ml of Green Cross, bovine thrombin was 1.2 NIH / ml of Park Davis, and bovine trypsin was 2 μg / m2 of code 3703 manufactured by Worthington.
1, human factor Xa was 0.25 U / ml from Boehringer Mannheim, human plasma kallikrein was 0.12 U / ml from Mold, and human urokinase was urokinase 60,000 (60,000 U / ml) from Mochida Pharmaceutical. V
ial) at 1000 U / ml.

(1)Plasmin inhibitory action 0.4 ml buffer (100 mM Tris, 150 mM
NaCl, pH 7.8) in various concentrations of inhibitors
(0.1 ml) and warm for 5 minutes. Plasmin
Solution (0.2 ml), and reacted for 5 minutes.
Add S-994 solution (0.1 ml) and react for 5 minutes
You. After the reaction is completed, 2.0 ml of pentacyanoamine
Add a color stop solution after stopping roating and leave for 10 minutes, then 700 nm
Measure the absorbance at, and measure the absorbance at half of that without inhibitor.
The concentration of the inhibitor in the reaction system showing ₅₀Asking.

(2) Thrombin inhibitory action 0.4 ml of buffer (150 mM Tris, 150 mM
NaCl, pH 8.5) and aqueous solutions (0.1 ml) of the inhibitors at various concentrations are added and heated for 5 minutes. A thrombin solution (0.2 ml) was added and reacted for 5 minutes.
Add S-915 solution (0.1 ml) and react for 5 minutes. After completion of the reaction, 2.0 ml of a pentacyanoamine ferroate-stopped coloring solution was added, and the mixture was allowed to stand for 10 minutes and then 700 nm.
The absorbance is measured by using the above method, and the IC ₅₀ is determined by the above method.

(3) Trypsin inhibitory action 0.4 ml of buffer (150 mM Tris, 150 mM
NaCl, pH 8.0) and aqueous solutions (0.1 ml) of various concentrations of the inhibitor are added and heated for 5 minutes. A trypsin solution (0.1 ml) was added, and the mixture was reacted for 5 minutes.
Add S-915 solution (0.1 ml) and react for 5 minutes. After the completion of the reaction, 2.0 ml of pentacyanoamine ferroate-stopped color solution [containing 2.5 mg / ml soybean trypsin inhibitor (Sigma, T-9003)]
After 10 minutes, absorbance is measured at 700 nm,
The IC ₅₀ is determined by the above method.

(4) Factor Xa inhibitory action 0.4 ml of buffer (50 mM Tris, 150 mM N
aCl, pH 8.5) and various concentrations of aqueous solutions of the inhibitor (0.1 ml) are added and heated for 5 minutes. Factor X
Solution a (0.2 ml) was added and reacted for 5 minutes.
1 of PS-2000 solution (10 mM) is added and reacted for 5 minutes. After the completion of the reaction, 2.0 ml of a pentacyanoamine ferroate-stopped coloring solution was added, and the mixture was allowed to stand for 10 minutes.
The absorbance is measured at 0 nm, and the IC ₅₀ is determined by the method described above.

(5) Kallikrein inhibitory action 0.4 ml of buffer (50 mM Tris, 150 mM N
aCl, pH 8.0) and aqueous solutions (0.1 ml) of inhibitors at various concentrations are added and heated for 5 minutes. Kallikrein solution (0.5% bovine serum albumin, Sigma No. A
-8022), and reacted for 5 minutes.
Of S-2302 aqueous solution (10 mM) was added and reacted for 5 minutes. After completion of the reaction, 5.0 ml of a 20% aqueous acetic acid solution was added to stop the reaction, and the absorbance was measured at 405 nm, and the IC ₅₀ was determined by the above method.

(6) Urokinase inhibitory action 0.3 ml of buffer (50 mM Tris, 150 mM N
aCl, pH 8.2) and aqueous solutions (0.1 ml) of inhibitors at various concentrations are added and heated for 5 minutes. Urokinase solution (50 mM Tris, 150 mM NaCl, pH
8.2 containing 0.1% bovine serum albumin)
After reacting for 0.1 minute, 0.1 ml of MUK-34 aqueous solution (10 m
M) and react for 5 minutes. After the reaction, 2.0m
The reaction was stopped by adding 1 l of a 10% acetic acid aqueous solution.
The absorbance is measured in m, and the IC ₅₀ is determined by the above method.

The concentration of the compound represented by the general formula (I), which suppresses the action of each protease to hydrolyze each substrate by 50%, is as shown in Table 1. For control, PPACK: D-phenylalanyl-L-prolyl-L-arginine chloromethylketone · 2HCl, FOY: [ethyl 4- (6
-Guanidino hexanoyloxy) benzoate methanesulfonate, FOY-305: N, N-dimethylcarbamoylmethyl p- (p-guanidinobenzoyloxy) phenylacetate methanesulfonate,
FUTHAN: 6-amidino-2-naphthyl-p-guanidinobenzoate dimethanesulfonate was used.

[0030]

[Table 1]

The test compound numbers and structural formulas are as shown in Table 2 below.

[Table 2]

[Table 3]

As is evident from the results shown in Table 1, the compounds of the present invention have an excellent inhibitory activity on proteolytic enzymes.
It has stronger inhibitory activity than FOY-305, FUTHAN and PPACK. The mechanism of blood coagulation includes an intrinsic system based on blood and an extrinsic system based on tissue factor. In both cases, factor Xa activates factor Xa, activates prothrombin to thrombin by factor Xa, and thrombin acts on fibrinogen to form fibrin in common. Strong inhibition of factor Xa and thrombin in this coagulation mechanism makes this compound useful as a therapeutic drug for diseases such as thrombosis and disseminated intravascular coagulation (DIC).

[0033]

As described in detail above, the novel amidinophenyl benzoate derivative and the acid addition salt thereof provided by the present invention are useful for various proteases, particularly trypsin, thrombin, factor Xa and the like. It has an extremely strong inhibitory effect and is therefore extremely useful as a therapeutic agent for diseases such as thrombosis and disseminated intravascular coagulation (DIC).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location A61K 31/24 ACJ A61K 31/24 ACJ ACL ACL AED AED C12N 9/99 C12N 9/99 (56) References JP-A-60-174724 (JP, A) JP-A-58-41855 (JP, A) CHEM. PHARM. BULL. 32 [11] P. 4466-4477 (1984)

Claims (3)

(57) [Claims] 1. A compound represented by the following formula (I): (Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen atom,
It represents a hydroxyl group, an amino group, a halogen atom, a carboxyl group or a 4-amidinophenyl-1-oxycarbonyl group. However, R ₁ and R ₂ are not simultaneously a hydrogen atom or a 4-amidinophenyl-1-oxycarbonyl group, R ₁ and R ₂ may be protected, and _one of R ₁ and R ₂ is hydrogen. When the other atom is an amino group, the amino group is substituted at the 2, 3, 5 or 6 position. Amidinophenyl benzoate derivatives and acid addition salts thereof. 2. 4'-amidinophenyl-3,5-dihydroxybenzoate, 4'-amidinophenyl-3-hydroxybenzoate, di (4'-amidinophenyl)
-5-hydroxyisophthalate, 4'-amidinophenyl-3-aminobenzoate, 4'-amidinophenyl-3,5-diaminobenzoate, 4'-amidinophenyl-3-acetylaminobenzoate, 4'-amidinophenyl-3 -Amino-4-chlorobenzoate,
The amidinophenyl benzoate derivative and its acid addition according to claim 1, wherein the derivative is selected from the group consisting of 4'-amidinophenyl-5-amino-2-chlorobenzoate and di (4'-amidinophenyl) -5-aminoisophthalate. salt. 3. A compound represented by the following formula (I): (Wherein R ₁ and R ₂ are the same or different and each represents a hydrogen atom,
It represents a hydroxyl group, an amino group, a halogen atom, a carboxyl group or a 4-amidinophenyl-1-oxycarbonyl group. However, R ₁ and R ₂ are not simultaneously a hydrogen atom or a 4-amidinophenyl-1-oxycarbonyl group, R ₁ and R ₂ may be protected, and _one of R ₁ and R ₂ is hydrogen. When the other atom is an amino group, the amino group is substituted at the 2, 3, 5 or 6 position. A benzoic acid amidinophenyl ester derivative represented by the following formula) or an acid addition salt thereof: