JPS6272656A - Lysine derivative and proteolytic enzyme inhibitor - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [X is formula II or III; Y is formula IV (R1 and R2 are H, phenyl, etc.) or 4-benzylpiperidino; Z is formula V, VI, etc.]. EXAMPLE:N<2>-( trans-4-Aminomethylcyclohexylcarbonyl )-N<6>-(benzyloxycarbonyl)- L-lysine 4-benzoylanilide. USE:A proteolytic enzyme inhibitor capable of exhibiting inhibitory activity against plasmin, kallikrein, trypsin and urokinase. PREPARATION:A 6N-hydrochloric acid/dioxane solution is added to N<2>-(trans-4-tert-butyloxycarbonylaminomethylcyclohexylcarbonyl)-N<6> -(benzyloxycarbonyl)-L- lysine 4-benzoylanilide and dioxane is further added thereto. The resultant mixture is stirred at room temperature for 30min to afford the aimed compound expressed by formula I.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to novel L-lysine derivatives, and more particularly to L-lysine derivatives having protease inhibitory activity or pharmaceutically acceptable salts thereof and their pharmaceutically acceptable salts. This invention relates to a protease inhibitor containing as an active ingredient.

[Prior art and problems to be solved by the invention] It is well known that various proteolytic enzymes exist in living organisms, such as tridicin-like enzymes such as plasmin, trypsin, kallikrein, and urokinase. , chymotrigsin-like enzymes, and pepsin-like enzymes are known. It is known that when these proteolytic enzymes are abnormally activated for some reason, they cause various diseases.

For example, if a large amount of abnormally activated plasmin exists in the blood, it can cause bleeding disorders, and plasmin also plays a role in inflammation, increasing blood vessel permeability, causing edema, etc., and causing inflammation. Since these proteolytic enzymes can cause diseases, substances that exhibit inhibitory activity against these proteolytic enzymes are useful as some kind of clinical therapeutic agent, and various studies have been made 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 and ulcers, and anti-urokinase agents are useful as agents for treating inflammation and ulcers. It is useful in controlling bleeding symptoms during thrombolytic therapy with urokinase. Therefore, although efforts have been made to develop protease inhibitors with such effects, their protease inhibitory activity is low and is not sufficient for practical use as pharmaceuticals. Compounds with sufficient inhibitory activity have not yet been developed. 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 enzyme inhibitors.

[Means for solving problems]

According to the present invention, general formula (1), an elementary atom (however, it must not be a hydrogen atom at the same time)
A phenyl group (which may be substituted with an alkylcarnyl group, a phenylcarnyl group, an alkoxycarbunyl group, an alkyl group, or an alkylcarnylalkyl group)].

Further, Y represents a 4-benzylpiperidino group.

2 is show.

The L-IJ gin derivative represented by the general formula (1) or a pharmaceutically acceptable salt thereof is provided. Such salts include, for example, inorganic acid salts such as hydrochlorides, bromates, sulfates, nitrates, phosphates, oxalates, succinates, phosphates, citrates, lactates, benzenesulfonic acids, etc. Salts, organic acid salts such as toluenesulfonate, methanesulfonate, etc. can be mentioned.

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

Typical examples of the compounds represented by the above general formula of the present invention are shown in Table 1. Although the compounds in the table are numbered, in the following explanation, individual compounds will be indicated by the compound numbers for convenience. Note that Lys in the compound represents L-lysine. NMR in the physical properties column means nuclear magnetic resonance spectral, and the number is usually a δ (delta) value used to indicate a chemical shift, and the unit is ppm. solvent CDct,
(C deuterium chloroform), (CD,)2So(d'-dimethylsulfoxide), and CD30D (deuterium methanol) were used alone or in combination. TMS (tetramethylsilane) was used as an internal standard. In addition, δ
The number in parentheses next to the value is the number of hydrogen atoms, and the numbers that follow are S for a single line, d for a double line, and t for a triple line.

q means a quartet, 9m means a multiplet, and broad means a wide absorption. Note that absorption derived from the solvent has been omitted.

IR means infrared spectral and was measured as potassium bromide tablets unless otherwise specified. Note that the numbers indicate wave numbers, and the unit is Confucian-1. Also, only the main absorption peaks are shown.

MS means mass spectrum, and the number indicates M/e, which is the mass of the cation fragment divided by the charge.

Note that only the main peaks are shown.

The compounds of the present invention can be synthesized by a combination of various methods, so-called peptide synthesis.

1] Mixed acid anhydride method (Ann, Ch@m, 572
190 (1951)>2) Acid chloride method (Bioch@m
1stry, 4 2219 (1965)) 3) Phosphazo method (Cham, Ber, 932387C196
0) ] 4) Synchronohexylcardidiimide method (J,
Am, Chem, Soc, 771067 (1955
):) 5) Activated ester method (e.g. method using N-hydroxysuccinimide) [J, Am, Chem, Soc, 853039 (1
963)) However, not all of the compounds of the present invention were synthesized by all of the methods described herein. A combination of synthetic methods suitable for each compound is required.

Hereinafter, a typical method for producing a compound will be explained with specific examples.

Example 1 Synthesis of N2- (t-butyloxycarvinyl) -N6-(
henzyloxycarbunyl) -L -IJ syn (1
), 4.78.9 in dry tetrahydrofuran, 30rn
Triethylamine was dissolved in
g was added thereto, and further 1.57.9 g of ethyl chlorocarbonate was added and stirred for 20 minutes. Here, 4-benzoylaniline 2
．． 5.0 ml of a dry tetrahydrofuran solution of 48.9 was added thereto, and the mixture was stirred under cooling for about 2 hours, and then left overnight at room temperature. After normal post-treatment, 5N-(t-butyloxycarbinyl)-N6-(pennoloxycarbonyl)-I, -
IJ resin 4-benzoylanilide (II), 4°55
I got g.

3.0 ml of 6N hydrogen chloride/dioxane solution was added to 1.63 g of the compound (n), and after 10 minutes, 3.0 ml of 6N hydrogen chloride/dioxane solution was added.
．． 0 ml was added and stirred at room temperature for 30 minutes. Add 20 ml of ethyl ether to this.
- The hydrochloride salt of benzoylanilide (1) is precipitated. Remove ethyl ether by decantation. After repeating this operation several times, it was dried in a desiccator.

On the other hand, trans-4-t-butyloxycar? Nylaminomethylcyclohexyl caldenic acid 890μ and triethylamine 440rn9 were dried in tetrahydrofuran 15
m7! of ethyl chlorocarbonate while cooling with ice-salt.
After adding 62■ and stirring for 20 minutes, the hydrochloride of (1) 1.
42.9 was added, stirred under cooling for about 1 hour, and then left at room temperature overnight. By normal work-up, N-(trans-4-t-butyloxycardenylaminomethylcyclohexylcargenyl)-N-(pencyloxycarbunyl)-L-lysine 4-benzoylanilide (R/
) 1.6.9 was obtained.

1.4 d of 6N hydrogen chloride/dioxane solution was added to 1.0 g of compound (IV), and 1.4 d of dioxane was added after 10 minutes.
m/ was added and stirred at room temperature for 30 minutes. This solution was subjected to a conventional work-up treatment, and N-(trans-4-aminemethylcyclohexylcarbunyl)-N'-(pencyloxycarbunyl)-L-lysine 4-benzoylanilide (
V) Obtained 680 ■.

Example 2 N2- () lance-4-t-butyloxycarboxylic acid synthesized by a conventional method. Nylaminomethylcyclohexylcar♂
nyl)-N-(penzyloxycarbunyl)-L-
Dissolve 1.6II of IJ lysine 4-benzoylanilide (1) in 20d of ethanol, ・Mother of cysts black so o
rn9 and cyclohexene 3 were added, and the reaction was carried out for 5 hours with stirring under heating and reflux. After removing insoluble matter, it was distilled off under reduced pressure, and the resulting crude oil was dissolved in 10 m of dry tetrahydrofuran.
To this solution, 688 da of hetodiethylamine was added, and further 534 η of acetyl chloride was added, and the mixture was stirred at room temperature for 10 hours.

By normal work-up, N-(trans-4-t-butyloxycal?nylaminomethylcyclohexylcardenyl)+N6-acetyl-L-17dine 4-benzoylanilide (II) 250In9 was obtained.

1.5 ml of a 6N hydrogen chloride/dioxane solution was added to 250 ml of the compound (II), and after 10 minutes, 1.5 ml of dioxane was added.
5 were added and stirred at room temperature for 1 hour. When ethyl ether 201d is added thereto, the hydrochloride of N-(trans-4-aminomethylcyclohexylcalzenyl)-N6-acetyl-L-lysine 4-benzoylanilide (III) is precipitated. After removing ethyl ether by decantation and repeating this operation several times, the mixture was dried in a desiccator to obtain 125 da of the hydrochloride of (1).

Example 3 N -(4-cyanobenzoyl)- synthesized by a conventional method
N4- (pencyloxycarbunyl) -L -IJ
Dissolve 210 m9 of lysine 4-acetylanilide C1) in 2.0 g of pyridine, add 0.5 ml of triethylamine, and blow hydrogen sulfide gas for 3 hours under water cooling.
It was left for 8 hours. 50 g of dichloromethane was added to the reaction mixture, and the organic layer was mixed with dilute NaQH, water, and dilute hydrochloric acid.

After washing with water, it is dried over anhydrous potassium carbonate and evaporated under reduced pressure. The obtained crude oil was powdered with ethyl ether,
N-(4-aminothiocal?nylbensoyl)-N'
-(pencyloxycarbunyl)-L-lysine 4-acetylanili)'(If)213rn9 is obtained.

190 ml of the above compound ([[)] was dissolved in 15 acetate, 0.51 trrl of methyl iodide was added, and the mixture was stirred at room temperature for 5 hours. After concentrating under reduced pressure, powder it with ethyl ether,
After stirring, 5 rnl of an ethanol solution containing 23 parts ammonium acetate was added. This solution was heated at 60-70°C for 6 hours. After cooling, ethyl ether was added and the precipitated crystals were taken out and dried to give N2-(4-amidinophenylcarbunyl)-N-(penzyloxycarbunyl).
100 Da of hydroiodide of -L,-lysine 4-acetylanilide (IV) was obtained.

〔Effect of the invention〕

As is clear from the following experimental results, the L-lysine derivative of the general formula (I) or its salt, which is the active ingredient of the protease inhibitor of the present invention, is a plasmino.

It is a low-molecular compound that has inhibitory activity against kallikrein, trypsin, and urokinase, but shows almost no inhibitory activity against thrombin, and has a unique enzyme inhibitory activity.
This is a compound that exhibits 4 turns.

The fact that the plasmino-inhibiting effect is different from that of conventional anti-plasmin agents is due to the fact that conventionally known drugs, such as tranexamic acid and ε-amitucasolonic acid, selectively inhibit only plasmino in the liE autolytic enzyme. are in contrast. The pharmacological effects of tranexamic acid and e-amitsukagulonic acid are thought to be expressed primarily as a result of binding to the so-called lysine binding site (LBS) of glasminordan and plasmino, and preventing 74 purine from binding to plasminodan and plasmino. (for example, C
h@m, Rev, 81431 (1981), B1
och@m, J., 163389 (1977), E
ur, J. Bloehem, 84573 (1978
), etc.]. On the other hand, these drugs hardly inhibit the degradation of synthetic substrates (for example, S-2251 by Kabi) and fibrinodan by plasmino. This means that there are various substrates for plasmino in the body other than fibrin [for example, fibrinodan, etc.], but the above-mentioned drugs have no effect on inhibiting their degradation.

The compound of the present invention is expected to have a remarkable effect of inhibiting the degradation of fibrin by plasmino, as well as of synthetic substrates and in-vivo proteins such as fibrinodan. Medicinal effect,
For example, it has the effect of inhibiting the decomposition of fibrinodan and is a novel anti-plasmin agent. Note that these known anti-plasmin agents selectively inhibit only plasmin in proteolytic enzymes.

Some of the compounds of the present invention exhibit inhibitory activity against urokinase, but urokinase is known to inhibit plasminogenesis.
It is a dan-activating enzyme, so inhibiting it is meant to give favorable results as a hemostatic agent. Furthermore, some of the compounds of the present invention exhibit anti-kallikrein and anti-trypsin effects, which means that, in combination with the above-mentioned anti-grasmin effect, they may become more powerful anti-inflammatory agents.

The following is a list of the compounds of the present invention. Typical test examples for rasmin activity, anti-kallikrein activity, anti-torinosine activity, and anti-urokinase activity will be shown and specifically explained.

The measurement method used in the following test examples is as follows. In addition, the test injection results for the compounds of the present invention are shown in Table 4 using the compound numbers in Table 1 above, and for the commercially available anti-plasmin agent as a comparative example, the structure of the compound is shown in Table 2, and the test results are shown in Table 3. It was shown to.

The inhibitor was dissolved in 0.18M borate saline buffer (pH 7.4).
), the total volume was 600 μL, and the mixture was placed in a 37C thermostat.
This was supplemented with 0.0% of bovine physolinodan dissolved in the same buffer.
200 µt of 1 human plasmin 0.3 100 µt of casein unit solution, 100 µt of bovine thrombin 505
-2. Measure the dissolution time of the fibrin clot formed after adding 100μ of the t/d solution, and measure the dissolution time when no inhibitor is added (approximately 5 minutes under the present experimental conditions) t? Determine the concentration of the inhibitor that causes a doubling of the length of time, I5o' (50% inhibitor concentration).

(Measurement method for inhibition of iD s -22s 1 degradation) Dissolve the inhibitor in 0.05M lithium-hydrochloric acid buffer (pH 7.4),
The whole is 400μt, and 3mM of S-2251 is added here.
Add 50 µt of bath solution and incubate for 5 minutes in a constant temperature bath at 37°C. Add 0.2 casein units/ml of human plasmin at 50 µAm and incubate at 37°C for 4 minutes, then add 50 µt of 5° acetic acid to stop the reaction.

The absorbance of /# ranitroaniline produced in the system is 405
Measured in nm, the concentration of inhibitor that shows 1/2 the absorbance of the case without inhibitor! It was calculated as 50.

The inhibitor was dissolved in 0.18M borate saline buffer (pH 7.4).
), the total volume was 500 uL, and 0.0.
Add 400 μL of 21 solution and 100 μL of bovine thrombin 4 units/co solution, measure the clotting time, and find the concentration of inhibitor that doubles the clotting time when no inhibitor is added!
It was calculated by dividing the angle by 5°.

(ii), 5-223g Decomposition inhibition measurement method Dissolve the inhibitor in 0.05M) Lis-HCl buffer (pH 8,3) to make a total of 400μt, and add 50μ of 0.2rmVf solution of 8-2238 to this. 5 in a 37℃ constant temperature bath
50μ of a 0.2 unit/d solution of bovine thrombin was added and heated at 37°C for 1 minute using the initial velocity method. The absorbance of nitroaniline produced in 9 was measured at 405 nm, and 1 in the case of no inhibitor was measured.
The inhibitor concentration exhibiting an absorbance of /2 was determined as I5°.

inhibitor (0.05M) in lisimidazole buffer (pH 8)
, 1), add 125μ of 1mM solution of S-2238.
was added to make the total 1.20a/, and heated in a thermostat at 37°C for 50 minutes.
Incubate for a minute. 0.051 d of bovine trypsin was added to this, and the absorbance of the nitroxypropylene produced in 1 minute at 37°C using the initial velocity method was 405.
Measured in nm, 1/l of without inhibitor! The inhibitor concentration exhibiting the absorbance was determined as Iso.

Inhibitor (0.05M) in Lis-HCl buffer (pi(7,8)
Dissolve it in the solution to make the total 400μt, and add S-2302 here.
Add 50μ of a 2mM solution of 50 μg of human plasma kallikrein and ink for 5 minutes in a constant temperature bath at 37°C. After that, 50 μt of 5° acetic acid was added to stop the reaction.

The absorbance of nine-halanitroaniline produced in the system is 405.
The concentration of the inhibitor was measured by nm, and the concentration of the inhibitor showing 1/2 the absorbance of the case without the inhibitor was determined as Iso.

Dissolve the inhibitor in 0.05M) Lis-HCl buffer (PH 1,8) to make a total of 400μt, add 1mM solution of 8-2444 & 50μ and incubate for 5 minutes in a 37°C thermostat. Add 50 μL of human urokinase 5003-nit/p solution, incubate at 37° C. for 5 minutes, and then add 50 μt of 504 acetic acid to stop the reaction.

The absorbance of the mother nitroaniline produced in the system is 405
Inhibitor concentration that shows 1/2 absorbance when measured in nm and without inhibitor! ,. I asked.

When the compound of the present invention is used as a medicine, there are no restrictions on the method of administration, and it can be formulated into an appropriate formulation using pharmaceutically customary formulation methods, and can be administered by intravenous injection, intramuscular injection, intravenous injection, etc.
It is used by methods such as oral administration. The appropriate dose is 100 to 1000 my per person per day. However, it goes without saying that the number can be increased or decreased as necessary.

Claims (1)

[Claims] 1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (L-form) [In the formula, etc. Indicates ▼. Y is a formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [R_1, R
Either of _2 is a hydrogen atom (however, it cannot be a hydrogen atom at the same time), a phenyl group (alkylcarbonyl group,
(optionally substituted with a phenylcarbonyl group, an alkoxycarbonyl group, an alkyl group, or an alkylcarbonylalkyl group). ] Or represents a 4-benzylpiperidino group. Z has ▲mathematical formulas, chemical formulas, tables, etc.▼, ▲mathematical formulas, chemical formulas,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼
, ▲There are mathematical formulas, chemical formulas, tables, etc.▼,▲Mathematical formulas, chemical formulas,
There are tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Indicates. ] An L-lysine derivative compound represented by this general formula. 2) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (L form) [In the formula, X indicates ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (trans) or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ . Y is a formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [R_1, R
Either of _2 is a hydrogen atom (however, it cannot be a hydrogen atom at the same time), a phenyl group (alkylcarbonyl group,
(optionally substituted with a phenylcarbonyl group, an alkoxycarbonyl group, an alkyl group, or an alkylcarbonylalkyl group). ] Or represents a 4-benzylpiperidino group. Z has ▲mathematical formulas, chemical formulas, tables, etc.▼, ▲mathematical formulas, chemical formulas,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼
, ▲There are mathematical formulas, chemical formulas, tables, etc.▼,▲Mathematical formulas, chemical formulas,
There are tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Indicates. ] A protease inhibitor containing an L-lysine derivative represented by this general formula or a pharmaceutically acceptable salt thereof as an active ingredient.