JP2515119B2 - Pharmaceutical composition containing a piperazine derivative, which has an active oxygen production suppressing effect and an active oxygen removing effect - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a pharmaceutical composition containing a piperazine derivative, which has an activity of suppressing active oxygen production and an activity of removing active oxygen.

Oxygen in the stable state (triplet oxygen) obtains one electron due to the involvement of xanthine oxidase or NADPH oxidase to generate superoxide anion, and further, hydroxyl radical, hydrogen peroxide, anion of hypochlorous acid,
Active oxygen such as singlet oxygen is generated.

These free radicals of active oxygen are involved in bactericidal action of granulocytes, inflammatory action, fibrogenesis, alloxan diabetes, lipid peroxidation, histamine release, DNA damage and the like.

Therefore, substances that suppress abnormal production of active oxygen are anti-inflammatory agents, anti-rheumatic agents, therapeutic agents for digestive tract diseases, anti-cataract agents, anti-arteriosclerotic agents, anti-cerebral infarction agents, therapeutic agents for liver diseases, autoimmune disease treatment It is useful as an agent.

Therefore, the present inventors have conducted earnest research to find a compound that suppresses active oxygen production. As a result, they have found that the compound represented by the following general formula (I) has excellent active oxygen production suppression and active oxygen removal effects, and completed the present invention.

Therefore, an object of the present invention is to provide a pharmaceutical composition having a useful active oxygen production suppressing and active oxygen removing action.

Specifically, the following general formula (I) (In the formula, R represents a hydrogen atom or a lower alkyl group, and n
Represents an integer of 0 to 3), and a pharmaceutical composition containing a non-toxic salt of a piperazine derivative as an active ingredient, which has an activity of suppressing active oxygen production and an activity of removing active oxygen.

It is known by the present inventors that the compound represented by the general formula (I) has already been effective in an experimental model of myocardial infarction by coronary artery ligation and is useful as a preventive and therapeutic agent for myocardial infarction. (JP 57-169478, JP Sho
58-126879) Examples of the case where R in the general formula (I) is a lower alkyl group include a methyl group, an ethyl group, a propyl group, an isobutyl group, an n-butyl group and a sec-butyl group.

The non-toxic salts include sodium, potassium, calcium, magnesium, trialkylamine, dibenzylamine, N-lower alkylpiperidine,
Non-toxic salts such as α-phenethylamine, 1- (1-naphthyl) ethylamine and N-benzyl-β-phenethylamine, or non-toxic with hydrochloric acid, hydrobromic acid, formic acid, sulfuric acid, fumaric acid, maleic acid, tartaric acid, etc. Examples include salt.

The compound represented by the general formula (I) can be obtained, for example, by the following method.

(In the formula, R and n have the same meanings as described above.) Specific examples of the compound represented by the general formula (I) include
The following can be mentioned.

(2R, 3R) -3-[(s) -1- {4- (4-methoxyphenylmethyl) piperazin-1-ylcarbonyl}-
3-Methylbutylcarbamoyl] oxirane-2-carboxylic acid, (2R, 3R) -3-[(s) -1- (4- (3,4-dimethoxyphenylmethyl) piperazin-1-ylcarbonyl}
-3-Methylbutylcarbamoyl] oxirane-2-carboxylic acid, (2R, 3R) -3-[(s) -3-methyl-1- {4-
(2,3,4-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid, (2R, 3R) -3-[(s) -3-methyl-1- {4-
(3,4,5-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid, (2R, 3R) -3-[(s) -1- (4-benzylpiperazin-1-ylcarbonyl) -3-methylbutylcarbamoyl] oxirane- 2-carboxylic acid, (2S, 3S) -3-[(s) -1- {4-methoxyphenylmethyl) piperazin-1-ylcarbonyl} -3-methylbutylcarbamoyl] oxirane-2-carboxylic acid, (2S , 3S) -3-[(s) -1- (4- (3,4-dimethoxyphenylmethyl) piperazin-1-ylcarbonyl}
-3-Methylbutylcarbamoyl] oxirane-2-carboxylic acid, (2S, 3S) -3-[(s) -3-methyl-1- {4-
(2,3,4-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid, (2S, 3S) -3-[(s) -3-methyl-1- {4-
(3,4,5-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid, (2S, 3S) -3-[(s) -1- (4-benzylpiperazin-1-ylcarbonyl) -3-methylbutylcarbamoyl] oxirane- 2-carboxylic acid.

Esters or non-toxic salts of these compounds are also the active ingredients of the present invention.

The usefulness of the compound represented by the general formula (I) and the non-toxic salt thereof in the present invention as a pharmaceutical composition having an active oxygen production inhibitory effect means that in vitro and in vivo production of active oxygen from rabbit granulocytes. It became clear by observing the influence of the drug on it. That is,
As the test drug, (2R, 3R) -3-[(s) -3-methyl-1- {4- (2,3,4-trimethoxyphenylmethyl) piperazin-1-ylcarbonyl} butylcarbamoyl] oxirane- Formyl-methionyl-leucyl-pheny of rabbit granulocytes using ethyl 2-carboxylate 1/2 sulfate
As shown in Table 1 in the experiment observing the effect on the luminol-dependent chemiluminescence generated by stimulation with lalanine (FMLP), the test drug suppressed the production of active oxygen by 90% at 30 μM as compared with the control.

Also, in vivo experiments, that is, 20m of test drug in rabbits
Experiments using granulocytes obtained after intravenous injection of g / kg also showed significant suppression as compared with the control (Table 2).

Further, the fact that the compound represented by the general formula (I) in the present invention also has a scavenging effect, that is, an effect of removing generated active oxygen, is that the influence of the drug on the active oxygen generated in the hypoxanthine-xanthine oxidase system. Experiment (Experiment 3) and H ₂ O ₂ −
It was clarified by an experiment for examining the effect of the drug in the NaOCl system (Experiment 4). As shown in Table 4, in the H ₂ O ₂ —NaOCl system, the drug inhibited chemiluminescence by 60% at 30 μM.

Further, the compound represented by the general formula (I), which is the active ingredient of the present invention, is found to be a highly safe substance for the living body by the acute toxicity test in mice.

The dose of the compound of the general formula (I) and the non-toxic salt thereof according to the present invention varies depending on the kind of the compound and the degree of symptom of the patient, but usually about 10 mg to 1 g per day may be administered to the patient.

When the compound represented by the general formula (I) or a salt thereof is used as a pharmaceutical composition having active oxygen production inhibitory activity and active oxygen removing activity, it is usually in the form of a pharmaceutical composition together with a pharmaceutical carrier. As the carrier, diluents or excipients such as a bulking agent, a binder, a disintegrating agent, a lubricant and the like which are usually used for preparing a drug depending on the usage form are used.

The dosage form may be any of injections, powders, capsules, granules, tablets and the like.

When used in the form of tablets, examples of carriers include lactose, sucrose, sodium chloride, glucose, starch,
Excipients such as calcium carbonate, crystalline cellulose, silicic acid,
Water, ethanol, propanol, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, methylcellulose, binders such as potassium phosphate, dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, stearic acid monoglyceride, starch, Disintegrators such as lactose, stearates, boric acid powders, lubricants such as solid polyethylene glycol, and the like widely used in this field can be used.
Further, if necessary, sugar-coated tablets, gelatin-coated tablets, film-coated tablets and the like can be used.

When prepared as an injection, examples of the diluent include water, ethyl alcohol, propylene glycol, polyoxyethylene sorbit, sorbitan ester and the like. At this time, salt, glucose or glycerin may be contained in an amount sufficient to prepare an isotonic solution, and a usual solubilizing agent, buffer, soothing agent, preservative, etc. are required. You may make it contain according to it.

As described above, the piperazine derivative represented by the general formula (I) or a non-toxic salt thereof has an excellent active oxygen production inhibitory and active oxygen scavenging effect, and has anti-inflammatory agent, anti-rheumatic agent, digestive tract. It is useful as a disease therapeutic agent, anti-cataract agent, anti-arteriosclerotic agent, anti-cerebral infarction agent, liver disease therapeutic agent, autoimmune disease therapeutic agent.

Next, in the present invention, it is shown that the compound of the general formula (I) and its non-toxic salt used as the active ingredient are useful as a pharmaceutical composition having an active oxygen production suppressing action and an active oxygen removing action and are highly safe. The present invention will be specifically described with reference to Test Examples and Examples showing formulation examples of the compound of the general formula (I).

Example 1 Method Reagents Formyl-methionyl-leucyl-phenylalanine (abbreviated as FMLP), luminol, xanthine oxidase (abbreviated as XOD) (0.6 U / mg protein), catalase (26,000 U /)
(mg protein) used was manufactured by Sigma. Hypoxanthine, hydrogen peroxide (30%), and hypochlorous acid (5% solution) manufactured by Wako Pure Chemical Industries, Ltd. were used.

FMLP and Luminor are HEPES-Saline (5mM N-2-
Hydroxyethylpiperazine-N'-2-ethanesulfonic acid was dissolved in dimethylsulfoxide (DMSO) before dilution with saline buffered at pH 7.4). The final concentration of DMSO in the reaction mixture was 10 μM and this concentration had no effect on the reaction.

Preparation of granulocytes Blood (70-80 ml) supplemented with heparin was added to a male white rabbit (2.
2 to 3.0 kg) and mixed with 2% dextran (manufactured by Hanai Chemical Co., Ltd.) in physiological saline. Erythrocytes were allowed to settle for 30 minutes at room temperature. Leukocyte-rich plasma sodium metrizoate Ficoll solution (manufactured by Nyegaard)
Then, it was centrifuged at 4 ° C. for 40 minutes at 800 G. A 155 mM ammonium chloride solution was added to the pellet to lyse the red blood cells. The remaining granulocytes were washed twice with cold HEPES-Saline and resuspended in the same solvent.

Measurement of chemiluminescence (abbreviated as CL) CL was measured with a Lumiphotometer (model TD4000; manufactured by LabScience) and recorded in millivolts. Unless stated otherwise the reaction mixture is cell suspension was suspended in HEPES-Saline containing KCl of CaCl ₂ and 5mM of 2.5 mM (1 × 1
( ⁷ cells / ml) 0.2 ml, luminol (final concentration 30 μM) and drug, and the reaction was carried out in a 0.5 ml polystyrene cuvette. The drug was also tested for its ability to suppress luminol-dependent CL produced in the H ₂ O ₂ + NaOCl and hypoxanthine-XOD systems.

The same amount (0.1 ml) of 10 μM H ₂ O ₂ and 30 μM luminol and the drug or buffer were premixed and placed in a counting room of a Lumiphotometer controlled at 37 ° C.
It was activated by injecting 1 (0.1 ml).

XOD-catalyzed production of superoxide anion was continuously monitored by a similar method. 20μ
XOD (4 U / ml) was added to cuvettes containing hypoxanthine (1 mM), luminol (30 μM) and various concentrations of drug or buffer. The amount of luminescence was evaluated by the area under the CL curve, and the inhibition rate was calculated by comparing with the CL of the control.

As the test drug, (2R, 3R) -3-[(s) -3-
Methyl-1- {4- (2,3,4-trimethoxyphenylmethyl) piperazin-1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid ethyl 1/2 sulfate was used.

Experiment 1 Effect of drugs on luminol-dependent chemiluminescence generation (in vitro) Granulocytes (1 × 10 ⁸ cells / ml) were treated with various concentrations of drugs at 37 ° C.
After incubating at 30 ° C. for 30 minutes, it was centrifuged and washed twice with HEPES-saline (4 ° C.) to remove the drug. Then 1
After incubating for 0 min (37 ° C.), CL produced by FMLP stimulation was measured. Table 1 shows the results.

Experiment 2 Effect of Drug on Luminol-Dependent Chemiluminescence Generation (In Vivo) Twelve male white rabbits (2.0 to 2.3 kg) were randomly assigned to drug treatment or control group. 5 ml of drug (20 mg / kg)
The above solution was dissolved in physiological saline solution and the solution was intravenously injected over 5 minutes. Similarly, saline was administered to the control.

 Heparinized blood was obtained 5 minutes after injection.

Granulocytes were obtained by the method described in the preparation of granulocytes. The results are shown in Table 2.

Experiment 3 Effect of drug on luminol-dependent chemiluminescence of hypoxanthine-xanthine oxidase system (removal effect) 20μ XOD (4U / ml) was added to hypoxanthine (1mM),
Luminol (50 μM), added to cuvettes containing various drugs. CL production was evaluated by the area under the CL curve and compared with a control in which the CL reaction was set to 100%. Table 3 shows the results.

Experiment 4 Effect of drug on luminol-dependent chemiluminescence of H ₂ O ₂ + NaOCl system (removal effect) The compounds represented by the general formula (I), which is the active ingredient of the present invention, were revealed from Experiments 1 and 2 to have an active oxygen production inhibitory action, and also had the effect of removing active oxygen generated in Experiments 3 and 4. It became clear to have.

Example 2 Acute toxicity test Male ddN mice having a body weight of 20 to 28 g were used. The drug was administered through the tail vein. As a result, as shown in Table 5, it was confirmed that the drug of the present invention has high safety.

However, Compound 1 (2R, 3R) -3-[(s) -3-methyl-1- {4- (2,3,4-trimethoxyphenylmethyl) piperazin-1-ylcarbonyl} butylcarbamoyl] oxirane- Ethyl 2-carboxylate 1/2 sulfate Compound 2 (2R, 3R) -3-[(s) -3-methyl-1
-{4- (2,3,4-Trimethoxyphenylmethyl) piperazin-1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid sodium compound 3 (2S, 3S) -3-[(s) -3- Methyl-1
-{4- (2,3,4-Trimethoxyphenylmethyl) piperazin-1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid ethyl 1/2 sulfate Compound 4 (2S, 3S) -3-[( s) -3-methyl-1
-{4- (2,3,4-trimethoxyphenylmethyl) piperazin-1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylate sodium Example 3 Formulation example (tablet) 1 tablet (220 mg) The film-coated tablet contained.

(2R, 3R) -3-[(s) -3-methyl-1- {4-
(2,3,4-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butyl carbamoyl] oxirane-2-carboxylate 50 mg Lactose 100 mg Crystalline cellulose 50 mg Magnesium stearate 1 mg Hydroxypropyl methylcellulose 15 mg Hydroxypropyl cellulose 4 mg Other compounds used as active ingredients in the present invention are also filmed by the same formulation. It can be a coated tablet.

Example 4 Formulation Example (Granule) The following components are contained in 1 g of granules.

(2S, 3S) -3-[(s) -3-methyl-1- {4-
(2,3,4-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid ethyl 1/2 sulfate 200 mg lactose 500 mg corn starch 300 mg Other compounds used as active ingredients in the present invention can be granulated by the same formulation. .

Example 5 Formulation Example (Injection) 1 ampoule contains the following components.

(2R, 3R) -3-[(s) -3-methyl-1- {4-
(2,3,4-Trimethoxyphenylmethyl) piperazine-
1-ylcarbonyl} butylcarbamoyl] oxirane-2-carboxylic acid ethyl 1/2 sulfate 20 mg Aseptic distilled water is added to the above ingredients to a volume of 10 ml.

Other compounds used as active ingredients in the present invention can be made into injections by the similar formulation.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 31/495 ABN A61K 31/495 ABN ABX ABX ACJ ACJ ACS ACS C07D 303/48 C07D 303/48

Claims (1)

(57) [Claims] 1. A general formula (In the formula, R represents a hydrogen atom or a lower alkyl group, and n
Is an integer of 0 to 3), and a pharmaceutical composition having an active oxygen production inhibitory action and an active oxygen removing action, which comprises a piperazine derivative or a nontoxic salt thereof as an active ingredient.