JPH01139561A - Guanidine derivative and enzyme inhibitor - Google Patents

Abstract

NEW MATERIAL:A guanidine derivative shown by formula I [n is 3 or 4; R<1> and R<2> are H (with the proviso that R<1> and R<2> are not simultaneously H), 1-4C alkyl, phenyl (may be replaced with hydroxycarbonyl or alkoxycarbonyl) or R<1> and R<2> are bonded to form piperidyl (may be replaced with alkyl, benzyl or alkoxycarbonyl)] or a salt thereof. EXAMPLE:N<2>-(trans-4-Aminomethylcyclohexylcarbonyl)-L-arginine 4- methylpiperidinoamide. USE:A protease inhibitor. PREPARATION:For example, a compound shown by formula (7) is reacted with a compound shown by formula (8) to give a compound shown by formula (9), which is then reacted with a compound shown by the formula HN(R<1>)R<2> and hydrobromic acid to give a compound shown by formula (10). This compound is further reacted with a compound shown by formula (13) to give a compound shown by formula (11), which is then treated with a solution of trifluoromethanesulfonic acid-thioanisole-trifluoroacetic acid to give a compound shown by formula (12).

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to novel guanidine derivatives, and more particularly to guanidine derivatives having protease inhibitory activity or pharmaceutically acceptable salts thereof and their use as active ingredients. The present invention relates to a protease inhibitor.

[Prior art and problems to be solved by the invention] It is well known that various proteolytic enzymes exist in living organisms, such as plasmin, trilcine, kallikrein, and urokinase. ing. It is known that these proteolytic enzymes cause various diseases if they are abnormally activated for some reason. For example, when a large amount of abnormally activated plasmin is present in the blood, bleeding disorders occur. Plasmin is also involved in inflammation and is thought to cause inflammatory diseases. Therefore, substances that exhibit inhibitory activity against these proteolytic enzymes are useful as some kind of clinical therapeutic agent, and various studies have been conducted to develop them. For example, anti-plasmin agents are useful as hemostatic agents, anti-inflammatory agents, and anti-allergic agents, anti-trigsin agents are useful in the treatment of pancreatitis, anti-kallikrein agents are useful as agents for treating inflammation, etc., and anti-urokinase agents are useful as urokinase agents. It is useful for controlling bleeding symptoms during thrombolytic therapy. The development of protease inhibitors with such effects has been progressing for some time, and agrotinin, a protease inhibitor known as a drug, is an extract from bovine lung and is a high-molecular-weight protein. However, there are problems with stability, side effects, etc. The present invention solves the problems of the prior art and provides a compound that has sufficient inhibitory activity for practical use and also has sufficient inhibitory activity against some proteolytic enzymes, and a proteolytic compound containing the compound as an active ingredient. #The aim is to develop an elementary inhibitor.

[Means for solving the above problems]

According to the present invention, the general formula (1) in which n is 3. and 4, and R1 and R are each independently a hydrogen atom (R and R are never hydrogen atoms at the same time).C-C4 alkyl group phenyl group (hydroxycardanyl group, C4-C6 noalkoxycar? or a pyridyl group (C1-C4 alkyl group, benzyl group, or C1-C4 alkoxycargenyl group), or R and R may be substituted with a C1-C4 alkyl group, benzyl group, or A guanidine derivative or a pharmaceutically acceptable salt thereof is provided.

Such salts include, for example, hydrochloride, hydrobromide,
Inorganic acid salts such as hydroiodide, sulfate, nitrate, phosphate, oxalate, succinate, glycolate, malate,
Citrate, maleate, lactate, benzenesulfonic acid4. ) Examples include organic acid salts such as luenesulfonate, methanesulfonate, and acetate.

According to the present invention, there is further provided a protease inhibitor containing the guanidine derivative of general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Detailed description of the invention]

Typical examples of the compounds represented by the general formula (I) of the present invention are shown in Table 1.

Although the compounds in the table are numbered, in the following explanation, for convenience, individual compounds will be indicated by the compound numbers.

Furthermore, all of the asymmetric carbon atoms in the structural formula of the compound are five atoms. IR in physical properties mK means infrared absorption spectrum,
Measured as potassium bromide tablets unless otherwise specified.9. Note that the numbers indicate wave numbers, and the unit is 3-1. Also, the absorption peaks only show the main ones.

(Hereinafter in the margin) The compound of the present invention can be synthesized by a combination of various methods called so-called pegtide synthesis.

1) Mixed acid anhydride method [Ann, Cham, +572
+2) Acid chloride method [Bioehsmlmtry, 4
+ 22193) Phosphazo method (Ch@n, B@r
,,93,23874) Cal? Diimide method (J,
Am, Ch@m, 8oc,.

77.1067 (1955)) 5) Activated ester method [for example, method using N-hydroxysuccinimide] [J, Am.

Ch@m, Soc, 85, 3039 (1963
)) However, not all of the compounds of the present invention can be synthesized by any of the methods described herein. A combination of nine synthetic methods suitable for each compound is required.

Among these methods, general reaction routes for typical examples are shown below.

Route (1) Route (3) To synthesize from ■ to ■, dissolve HNR2 (0.5 to 3 equivalents to
/3 to 3 parts) and then add ■. The reaction time is 0.5 to 24 hours, preferably 6 to 12 hours. The reaction temperature at this time is -30°C to 100°C, preferably -20°C.
~60°C. After the reaction, the solvent is distilled off, extracted with ethyl acetate, and recrystallized with a suitable solvent such as chloroform, methylene chloride, ethyl acetate, etc., or purified with a silica gel column (eluent: chloroform, methanol, etc.).

To synthesize ① from ①, ② is added to a hydrobromic acid/acetic acid solution and stirred. Reaction time is 0.1 to 2 hours, preferably 0.4 to 2 hours.
It will be over in 1 hour. The reaction temperature is -10°C to 30°C. After the reaction, a suitable solvent such as hexane, ether, etc. is added to crystallize, and the mixture is washed several times with ether.

To synthesize ■ from ■, dissolve ■ in dimethylformamide and add triethylamine (0.8 to 5 equivalents to ■).
Furthermore, a solution prepared by dissolving ■ (0.8 to 2 equivalents relative to ■) in chloroform and adding thionyl chloride (1 to 3 equivalents relative to ■) is added. The reaction time is 1 to 24 hours, preferably 6 hours.
~12 hours.

The reaction temperature is θ°C to 30°C. After distilling off the solvent, extract with ethyl acetate, chloroform, etc., and use an appropriate solvent such as chloroform,
Recrystallize with alcohol, ethyl acetate, etc., or purify with a silica gel column (eluent: chloroform, methanol).

The reaction from ■ to ■ involves dissolving ■ in a mixed solvent of water and acetic acid and performing catalytic reduction under a palladium catalyst.The reaction time is from 1 hour to 2 hours.
4 hours, preferably 6 to 10 hours. The reaction temperature is 10°C to 50°C.

After the reaction, the catalyst is removed, the solvent is distilled off, and the residue is dissolved in water and freeze-dried.

For the reaction from ■ to ■, ■ is 1 to 5 N hydroxylated) IJum solution (2 to 3 equivalents to ■) - Acetone mixed solution is suspended ■ (0.8 to 3 equivalents to ■) acetone solution of
- Drop in 5 to 60 minutes. Reaction time is 1 hour to 1
The reaction time is 2 hours, preferably 2 to 4 hours, and the reaction temperature is -10°C to 30°C. After the reaction, the mixture is neutralized and the solvent is distilled off. The residue is extracted with ethyl acetate, chloroform, etc. Evaporate the solvent and dissolve in methanol to dissolve cyclohexylamine (
(1 to 5 equivalents relative to (1)) to obtain the cyclohexylammonium salt (2).

The reaction from ■ to ■ is the same as the method from ■ to ■.

The reaction from ■ to @ is to convert ■ to trifluoromethanesulfonic acid -
Add to thioanisole-trifluoroacetic acid (3:2:5) solution. The reaction time is 0.5 to 3 hours, and the reaction temperature is -10°C to 30°C. After the reaction, ether is added to precipitate the product, which is then purified using an ion exchange resin and freeze-dried.

The reaction from (1) to (2) involves dissolving (1) in a suitable solvent such as tetrahydrofuran, dimethylformamide, diethyl ether, dioxane, dichloromethane, etc., and adding a suitable base such as triethylamine, pyridine, etc.
Add 5 equivalents, preferably 2 to 3 equivalents. To this reaction solution, ethyl chlorocarbonate (0.7 to 1.2 equivalents relative to ■) is added as it is, or dissolved in the solvent used as the reaction solvent, all at once or in several portions. The reaction temperature is -10℃~
The temperature is 30°C. Reaction time is 1 to 50 hours, preferably 5 to 50 hours.
It is 20 hours. 0.5 to 2 equivalents of ■N after normal post-treatment
Add R2. The reaction temperature is 0°C to 30°C.

Reaction time is 5-20 hours.

After normal post-treatment, it is recrystallized from ethyl acetate, chloroform, alcohol, etc.

For reactions from (1) to (2), 0 is added to a 4-8 N hydrochloric acid/dioxane solution (1-10 equivalents 1L for (1)) and stirred. The reaction temperature is -10°C to 30°C.

Reaction time is 5-60 minutes. After the reaction, ether is added to precipitate and washed several times with ether to obtain (2).

Example 1 4-methylpiperidine (1,0II) was converted into pyridine (25
m/) and cooled to -20°C.
Add 1d) and stir for 15 minutes. N-Cal? Benzoxy N2-=-Draw L,-Fluinine (3,5N) was added and stirred at 60°C for 2 hours and at room temperature for 10 hours. After evaporating the solvent, the residue was extracted with ethyl acetate and diluted with IN-hydrochloric acid water and 5t
(s-carbonic acid) Wash with IJum water. After drying the ethyl acetate solution over anhydrous sodium sulfate, the solvent was distilled off. The residue was dissolved in chloroform (3i/), adsorbed on a silica gel column, and developed with a mixed solvent of chloroform and 1% methanol-chloroform to give pale yellow N-lumepenzoxy N7.
-Nitro L-arginine 4-methylpiperidinoamide (1) (1,5 g) was isolated and 7'j, IR and N
After confirmation by MR, 25% hydrobromic acid/acetic acid solution (2i/) was added to the compound (1) (0.87#) under ice cooling, and after another 10 minutes, acetic acid (2i/) was added at room temperature. Stir for 40 minutes. Ether was added to precipitate, and the precipitated solid was washed several times with ether. After drying this solid under reduced pressure, it was dissolved in dimethylformamide (10 m) and triethylamine (0.56 m) was added under water cooling.

On the other hand, 4-calgepenzoxyaminomethylcyclohexylcarmenic acid (o,5sy) was dissolved in chloroform (lQm/), thionyl chloride (1,8i/) was added, and the mixture was stirred at room temperature for 3 hours. Add to the dimethylformamide solution and stir overnight at room temperature. After evaporation of the solvent, the residue was extracted with ethyl acetate, subjected to the same post-treatment as described above, and isolated using a silica gel column in the same manner to obtain white N-(trans-4
-cargopenzoacyaminomethylcyclohexylcargenyl) + PJ7-nitro-L-arginine 4-,
'Tilpiperidinoamide (2) (0,6s i ) obtained. After confirmation by IR and NMR, the compound (2) (0,
Dissolve 41 N) in 50 liters of acetic acid (1211 Ll) and perform catalytic reduction with palladium black (100 ~) for 9 hours.

The catalyst was removed and the solvent was distilled off, and the t0 residue was dissolved in water (5d) and freeze-dried to give a pale yellow solid N2- () lance-
4-aminomethylcyclohexylcarbonyl)-L-arginine 4-methylpiperidinoamide diacetate (0
, 28, F) was obtained.

Example 2 Xyanilide diacetate N2-Calgbenzoxy L-alpnin (20 g) was suspended in a mixed solvent of 4N sodium hydroxide solution (60 ml) and acetone (600 In/) under cooling with water to form mesitylene-2-sulfonyl chloride. (An acetone solution (60 ml) of 30,6,1 was added dropwise over 30 minutes, stirred for 2 hours, and after the reaction, neutralized with IN-hydrochloric acid water and distilled off the solvent. The residue was reduced to 5%
- Extracted with sodium bicarbonate solution and washed with ether. The water-cooled sewage layer was acidified with hydrochloric acid and extracted with ethyl acetate.
．． Wash with 1N hydrochloric acid water. After drying the ethyl acetate solution with anhydrous sulfuric acid, the solvent was distilled off. The residue was dissolved in methanol (200 m/) and cyclohexylamine (6
i/ ) and precipitated with ether to obtain white N2-carmebenzoxyN7-mesitylene-2-sulfonyl-arginine cyclohexylammonium salt (1)
(25N) was obtained. Said compound (1) (4,8,9
) was suspended in ethyl acetate (120 liters), and under water cooling, IN-hydrochloric acid (8 liters) was added and stirred for 20 minutes. After washing this with water, the ethyl acetate solution was dried with anhydrous sodium sulfate. After distilling off the solvent, pale yellow N2-calgepenzoxy N-menthylene-2-sulfonyl-L-arginine was obtained, which was dissolved in pyridine (i Qm/). -10,000-4-
Methyl aminobenzoate (1,2F) was converted into pyridi7 (40
rnt) and cooled to -20°C to dissolve phosphorus trichloride (0
, 36 m/) and stirred for 15 minutes. The above-mentioned pyridine solution (lQm/) was added thereto, and the mixture was stirred at 60°C for 2 hours and at room temperature for 10 hours. After evaporation of the solvent, the residue was extracted with ethyl acetate and washed with IN-hydrochloric acid water and 5-chloride sodium carbonate.

After drying the ethyl acetate solution over anhydrous sodium sulfate, the solvent was distilled off. The residue was recrystallized from a mixed solvent of ethyl acetate and petroleum ether to obtain white crystals of N2-cargopenxoxyN'-mesitylene-2-sulfonyl-L-arginine 4-methoxycargenylanilide (2) (3,9N). Get it. The compound (2) (1,2,9) was dissolved in methanol (100
IN-hydrochloric acid (2 i/ml) was added to the solution, and catalytic reduction was carried out for 1.5 hours using a radium black catalyst (150~). The catalyst was removed and the solvent was distilled off, followed by drying under reduced pressure. This was dissolved in dimethylformamide (20 ml) and triethylamine (1,2 i/h) was added under water cooling. Separately, trans-4-carmebenzoxyaminomethylcyclohexyl carmine acid (1.1 g) was dissolved in chloroform (4Q tnl
) and added thionyl chloride (2.4 ml), stirred at room temperature for 2 hours, and after eating, added to the dimethylformamide solution and stirred overnight at room temperature. After evaporation of the solvent, the same post-treatment as in Example 2 was carried out to give N2- () lance-4-calR benzoxyaminomethylcyclohexylcal I-Nyl)-N7-
, mesitylene-2-sulfonyl-L-arginine 4-
Methoxycargenyl anilide (3) (0,81) was obtained. The above compound (3) (o, a s tt ) was dissolved in methanol (40 m/), IN-sodium hydroxide (1,5 i/) was added under water cooling, stirred at room temperature for 2 hours, and then IN-sodium hydroxide (3 i/) was added. ) was added and stirred for 6 hours. Under cooling with water, neutralize with acetic acid and distill off the solvent. The residue was extracted with ethyl acetate and washed with 3% acetic acid and saturated brine. After drying the ethyl acetate solution with anhydrous sulfuric acid) and distilling off the solvent, a pale yellow solid of N2- () lance-4-car♂benzoxyaminomethylcyclohexylcarbonyl) was obtained.
-N'-mesitylene-2-sulfonyl-L-arginine 4-carxyanilide (4) (0,33,9)
Get a friend. The above compound (4) (0,26,F) was added to t M − in the presence of 3-cresol (038 ml) under water cooling.
) Trifluoromethanesulfonic acid-thioanisole trifluorovinegar #R (0.58ml-0.43ml-2r
nl) was added and stirred for 1 hour. Ether was added to the reaction solution to precipitate a solid, which was further washed with ether and diluted with water (lQm
J) was added and further washed with ether. The aqueous layer was prepared using Amberlite IRA-45 and freeze-dried. Dissolve this solid in 5% vinegar # (:31/) and use Sephadex G-2.
5 and freeze-dried to obtain white powder N2- (trans-4-7minomethylcyclohexylcargenyl)-L-
Arginino 4-cal? Xyanily diacetate (0,
1611) f! :Obtained.

Example 3 L-homoarginino hydrochloride (5g) in concentrated sulfuric acid (lQmJ)
) was added little by little while stirring, the generated hydrochloric acid gas was removed under reduced pressure, and stirring was continued for 30 minutes. Ammonium nitrate (2,5,9) was added little by little to this reaction solution, and the mixture was stirred for 3 hours under reduced pressure. Add the reaction solution to ice water and adjust the pH to 6 with aqueous ammonia.
, 8 and left overnight at 4°C. The precipitated solid was collected and washed with cold water to obtain a pale yellow powder oN''-nitro L-homoalinine (1) (4 g).

The above compound (1) (4, Sj) was suspended in water (59 ml) and mixed with triethylamine (2.8 ml) and 2-t under water cooling.
-Ptoxycargenyloxyimino-2-phenylacetonitrile (4,71) in a solution of 150ml
) and stirred overnight. After evaporation of the solvent, the residue was extracted with t-5 sodium bicarbonate solution and washed with ether.

Citric acid was added to the aqueous layer to adjust the pH to 3, and the mixture was extracted with ethyl acetate. After drying the ethyl acetate layer over sodium sulfate, the solvent was distilled off to give a white powder of N2-t-ptoxycargenyl-N8-.
2) Obtain day-L-homoarginine (2) (4,2N) t0 Said compound (2) (1 g') was added to tetrahydrofuran (
50 mJ) and cooled with water, triethylamine C0,42
m1), further added ethyl chlorocarbonate (0,294), and stirred for 5 minutes. A solution of 4-benzylpiperidine (0,531) in tetrahydrofuran (50ml) was added to this reaction solution.
/) was added and stirred for 6 hours. After evaporating the solvent, the residue was extracted with ethyl acetate and washed with 10 citric acid water. After drying the ethyl acetate layer with sulfuric acid, the solvent was distilled off. The residue was recrystallized with a mixed solvent of ethyl acetate and petroleum ether to form a white solid, N2-t-phthoxycargenyl-N8-nitro-
L-*more/L/4'nin 4-benzylpiperidinoamide (3) (0,75N) was obtained.

The above compound (3) (013N) was heated at 7.6N under water cooling.
Add hydrogen chloride/dioxane solution (o, asmj) and add 10
After a few minutes, dioxane (lat) was added and the mixture was stirred at room temperature for 50 minutes. Add ether to precipitate a solid, wash with ether, dry under reduced pressure, and dimethylformamide (5i/)
It was dissolved in Add triethylamine (Q,
94 mA was added.

Separately, thionyl chloride CO (0,9ml) was dissolved in trans-4-carmepenzoxyaminomethylcyclohexylcargenic acid (0,12I) t'dichloroform (2d) at room temperature, stirred for 3 hours, and then added to the dimethylformamide solution overnight. Stirred. After evaporation of the solvent, the residue was extracted with ethyl acetate, diluted with aqueous hydrochloric acid and 5% carbonic acid, and washed with an IJ solution and water. After drying the ethyl acetate layer with sulfuric acid, the solvent was distilled off. Petroleum ether was added to the residue to obtain a white powder of N2-(trans-4-carbopendixyaminomethylcyclohexyl-N8-2)H-I.
, -Homoarginine 4-benzylpiperidinoamide (
4) We get (0,11!) and nine.

The compound (4) (0,08lI) was dissolved in 50% acetic acid (F
3 #It) and 4+ radium black (0,05F
) was added and catalytic reduction was carried out for 6 hours. Remove the palladium black and evaporate the solvent. Water (3M) was added to the residue and lyophilized to give a white powder w2-(trans-4-aminemethylcyclohexylcar♂yl)-L-homoarginine 4-benzylpiperidinoamide diacetate (0,
03II) was obtained at t0 [Effect of the Invention] As is clear from the following experimental results, the guanison derivative of the general formula (1) or its salt, which is the active ingredient of the proteolytic enzyme inhibitor of the present invention, is grasmino, kallikrein, and trigsin. It has very strong inhibitory activity against and urokinase. The action of the compound of the present invention is different from that of conventionally known drugs, such as tranexamic acid and ε-7 minocagulonic acid, which selectively inhibit only Grasmino among proteolytic enzymes. For example, some of the active ingredients of the protease inhibitors according to the present invention exhibit inhibitory activity against urokinase, but since urokinase is a grasminordan activating enzyme as is well known, it is preferable to inhibit this as a hemostatic agent. Becomes a drug. Some of the protease inhibitors according to the present invention exhibit anti-kallikrein and anti-trigsin effects, but exhibiting these effects together with the above-mentioned anti-grasmin effects makes them effective as more powerful anti-inflammatory agents. It means something. However, anti-trinosine action is effective in treating pancreatitis. Nineth, the compound of the present invention has a lower molecular weight and is easier to supply with constant quality than agrotinin, which is a protease inhibitor known as a pharmaceutical.

The anti-grasmin activity, anti-kallikrein activity, anti-thrombin activity, anti-thrombin activity and anti-thrombin activity of the compounds of the present invention will be specifically explained below by showing typical test examples.

The measurement method used in the following test examples is as follows. The test results for the compounds of the present invention are shown in Table 2 using the compound numbers in Table 1 above.

l) Anti! Measurement of rasmin activity (1) Measurement method of inhibition of fibrin degradation Inhibitor was added to 0.18M boric acid saline buffer (-7,4)
Dissolve the whole in 600μ! Then, in a thermostat at 37°C, 0.2% of the fibrinodan from the cow was dissolved in the same buffer solution.
200 μl of 1 human Grasmino 0.3 casein unit/ml solution, 100 μl of bovine thrombin 50
Unit/WLl solution 1Mk 100μ1m, then measure the dissolution time of the formed fibrin clot, and adjust the concentration of the inhibitor to double the dissolution time (approximately 5 minutes under the present experimental conditions) when no inhibitor is added. .

(50% inhibitor concentration, unit μmot) Find f.

(ii) Assay method for inhibition of S-2251 decomposition Dissolve the inhibitor in 0.05M Tris weir acid buffer (PH7.4),
Make the total volume 400μl and add 3mM of S-2251 here.
Add 50 μl of the bath solution, incubate for 5 minutes in a constant temperature bath at 37°C, add 0.2 zein units/mg'tso μl of human plasmin, incubate for 4 minutes at 37°C, and then incubate with 50% vinegar. Add 50 μl of M to stop the reaction.

The absorbance of paranitroaniline produced in the system is 405
The concentration of inhibitor (μmot) is determined to give an absorbance of 1/2 of that without inhibitor when measured in nm. Close and ask for it.

2) Measurement of antithrombin activity (Measurement method of inhibition of rifibrin degradation)
), make a total of 500 μl, and place in a constant water bath at 37°C.
Add to this the bovine fibrinodan 0.2 dissolved in the same buffer.
400 μm of lysate, 4 units/ml of bovine thrombin
100 μ of solution! In addition, the clotting time was measured, and the concentration of inhibitor (μ
mat) 'r: A friend sought as Iso.

(ii) Assay method for inhibition of S-2238 degradation Dissolve the inhibitor in 0.05M) Lis-HCl buffer (pH 8.3) to a total volume of 400μ, and add 50μ of 0.2mM IL solution of 8-2238. ! Incubate for 5 minutes in a constant temperature bath at 37°C, and collect 0.2 unit/ffi of bovine thrombin! 50 μ of solution! Addition, measure the absorbance of paranitroaniline produced per minute at 405 nm using the initial rate method at 37°C, and find the inhibitor concentration (μmot) that shows an absorbance of 172 when no inhibitor is added! ,
. Closed and looking for a friend.

3) Measurement of antitrypsin activity Measurement of S-2238 degradation inhibition Inhibitor was added to 0.05M) lisimidazole buffer (pH 8)
, 1), a 1 mM bath solution of S-2238 12
5μ! Then, the whole was heated to 1.20 d and incubated for 5 minutes in a constant temperature bath at 37°C. Add 0.05 au of bovine trizocine to this, and the absorbance of 9-9 ranitroaniline generated per minute at 37°C by the initial velocity method was 40.
The inhibitor concentration (μmob), which was measured at 5 am and showed an absorbance of 172 when no inhibitor was added, was determined by dividing 5°.

4) Measurement of anti-plasma kallikrein activity Assay method for inhibition of S-2302 degradation Dissolve the inhibitor in 0.05M) Lis-HCl buffer (pH 7, 8), make a total volume of 400 μl, and add 50 μl of a 2 mM solution of S-2302 to the solution. Add 50 μl of human plasma kallikrein 0.12 units/ml solution and incubate for 5 minutes in a 37°C constant temperature bath. After the addition and incubation at 37°C for 5 minutes, 50μ of 50% acetic acid was added to stop the reaction.

The absorbance of nitroaniline produced in the system is 405
am was measured, and the inhibitor concentration (μmot) showing 1/2 the absorbance when no inhibitor was added was determined as Iso.

5) Measurement of anti-urokinase utilization S-2444 degradation inhibition measurement method Dissolve the inhibitor in 0.05M) Lis-HCl buffer (PH8,8), make a total volume of 400μ, and add 1mM bath solution of 8-2444 thereto. 50μ! Add human urokinase (500 units) and incubate for 5 minutes in a 37°C constant temperature bath.
/d bath solution 50μ! After addition and incubation at 37°C for 5 minutes, 50 μl of 50% acetic acid was added to stop the reaction.

Generated within the system and ftノ! The absorbance of ranitroaniline was set to 40
The inhibitor concentration (μmot) measured at 5 nm gives an absorbance of 172 without inhibitor. Looking for a friend.

When the compound of the present invention is used as a medicine, there are no restrictions on the method of administration, and suitable formulations may be used such as intravenous injection, intramuscular injection, intravenous drip, etc.
It is used by oral administration, respiratory tract inhalation, eye drops, nasal drops, external skin application, etc. Also, the dose is 1 to 10 per person per day.
00IIg9 is correct. However, it goes without saying that the number can be increased or decreased as necessary.

Claims (1)

[Claims] 1) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) In the formula, n represents 3 and 4, and R^1 and R^2 each independently represent a hydrogen atom ( However, R^1 and R^2 cannot be hydrogen atoms at the same time) C_1 to C_4 alkyl group phenyl group (hydroxycarbonyl group, C_1 to C_6
may be substituted with an alkoxycarbonyl group of C_1 to C_4 or an alkylcarbonyl group of C_1 to C_4), or R^1 and R^2 may be substituted with a piperidyl group (an alkyl group of C_1 to C_4, a benzyl group, or an alkyl group of C_1 to C_4) 2) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) In the formula, n is 3, and 4 and R^1 and R^2 are each independently a hydrogen atom (however, R^1 and R^2 cannot be hydrogen atoms at the same time) C_1 to C_4 alkyl group phenyl group (hydroxycarbonyl group, C_1 to C_6
may be substituted with an alkoxycarbonyl group of C_1 to C_4 or an alkylcarbonyl group of C_1 to C_4), or R^1 and R^2 may be substituted with a piperidyl group (an alkyl group of C_1 to C_4, a benzyl group, or an alkyl group of C_1 to C_4) A protease inhibitor containing a guanidine derivative (which may be substituted with a carbonyl group) or a pharmaceutically acceptable salt thereof as an active ingredient.