JPS63264525A - Medicine composition containing piperazine derivative and having active oxygen production inhibiting and active oxygen removing action - Google Patents

Abstract

PURPOSE:To obtain a medicine composition containing piperazine derivative as an ingredient, having active oxygen production inhibiting action and active oxygen removing action, useful as antiphlogistics, antirheumatics, remedy for digestive tract disease, anticataract agent, etc., and having high safeness. CONSTITUTION:The aimed composition is obtained by using a piperazine deriva tive expressed by the formula (R is H or lower alkyl; n is 0-3) or nontoxic salt thereof as an active ingredient, blending the derivative (salt) with a conven tional carrier (e.g. extender, bonding agent, disintegrator, lubricant or diluting agent) and formulating the blend according to ordinary method. The composition can be prepared into a form of injection, inhalant, capsule, granule, tablet, etc. and administered in an amount of about 10mg-1g daily. The compound expressed by the formula includes e.g. (2R,3R)-3-{(S)-4-[4-(4-methoxyphenylmethyl) piperazine-1-ylcarbonyl]-3-methylbutylcarbamoyl}oxirane-2-carboxylic acid.

Description

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

Stable oxygen (triplet oxygen) acquires one electron through the involvement of xanthine oxidase and NADP11 oxidase, producing superoxide anion, which further generates hydroxyl radical, hydrogen peroxide, hypochlorous acid anion, and singlet oxygen. Active oxygen such as is generated.

These active oxygen free radicals are involved in granulocytes.

Therefore, it is a substance that suppresses the abnormal production of active oxygen.

Therefore, the present inventors have conducted intensive research to find a compound that suppresses active oxygen production.

The present invention was completed based on this discovery.

Therefore, an object of the present invention is to provide a pharmaceutical composition that has useful effects of inhibiting active oxygen production and removing active oxygen.

In detail, piperazine mA represented by the following general formula (1) where n is an integer of 0 to 3)
The present invention relates to a pharmaceutical composition containing active oxygen or a non-toxic salt thereof as an active ingredient and having an active oxygen production inhibiting and active oxygen removing action.

The compound represented by the above general formula (1) has already been shown to be effective in an experimental myocardial infarction model using coronary artery ligation by the present inventors, and is known to be useful as a preventive and therapeutic agent for myocardial infarction.

(Japanese Patent Publication No. 57-169478, Japanese Patent Application Publication No. 58-1268
79) 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, a 5ec-butyl group, and the like.

In addition, non-toxic salts include sodium, potassium, calcium, magnesium, trialkylamine, dibenzylamine, N-lower alkylpiperidine, α
- Non-toxic salts of phenethylamine, 1-(1-naphthyl)ethylamine, N-benzyl-β-phenethylamine, or non-toxic salts with hydrochloric acid, hydrobromic acid, formic acid, sulfuric acid, fumaric acid, maleic acid, tartaric acid, etc. can be mentioned.

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

C113Cl5--→ (1) (b) (In the formula, R and n have the same meanings as above, next year = C--
-i Specific examples of the compound represented by formula (1) include the following.

(2R,3R) -3-((S) -1-(4-(4-
methoxyphenylmethyl)piperazin-1-ylcarbonyl)-3-methylbutylcarbamoyl]oxirane
2-carboxylic acid, (2R,3R) -3-(TS)-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-)dimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylic acid, (2R,3R) -3-((5)-3-methyl-1-
(4-(3,4,5-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]
oxirane-2-carboxylic acid, (21?, 31?) -3-(TS)-1-(4-benzylpiperazin-1-ylcarbonyl)-3-methylbutylcarbamoyl]oxirane-2-carboxylic acid, (2
S, 3S) -'3-((S) -1-(4-(4-methoxyphenylmethyl)piperazine-neeylcarbonyl)-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-((Sl-3-methyl-1-
(4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]
Oxirane-2-carboxylic acid, (2S,3S)-3-((S)-3-methyl-1-(
4-(3,4,5-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylic acid, (28,35) -3-((S)-1-(4-benzyl) Piperazin-1-ylcarbonyl-3-methylbutylcarbamoyl]oxirane-2-carboxylic acid.

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

The fact that the compound represented by general formula (I) and its non-toxic salt in the present invention is useful as a pharmaceutical composition having an active oxygen production inhibiting effect has been demonstrated in 1nvjtro and in vitro studies.
This was revealed by observing the effects of drugs on active oxygen production from rabbit granulocytes in vivo.

That is, as a test drug, (2R,3R) -3-(
(Sl -3-methyl-1-(4-(2,3,4-1-
rimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylic acid ethyl% sulfate was used to formyl granulocytes from rabbits.
-methiony+-1eucyl-phenyla
In an experiment to observe the effect on luminol-dependent chemiluminescence generated by lanine (FMLP) stimulation, as shown in Table 1, the test drug inhibited the production of active oxygen by 90% at 30 μm compared to the control.

In addition, an in vivo experiment using granulocytes obtained after intravenously injecting the test drug into rabbits at 20 mg/kg also showed significant inhibition compared to the control (Table 2
).

Furthermore, the fact that the compound represented by the general formula (1) in the present invention also has a scavenging effect, that is, an effect of removing generated active oxygen, indicates that the drug has no effect on active oxygen generated in the hypoxanthine-xanthine oxidase system. Experiment examining the tongue (Experiment 3) and HtOz-NaOC/! This was revealed through an experiment to investigate the effect of drugs on the system (Experiment 4). As shown in Table 4, H20□-NaOCjl! In the system, the drug is
Chemiluminescence was suppressed by 60% at μH.

Further, the compound represented by the general formula (1), which is the active ingredient of the present invention, has been found to be a highly safe substance for living organisms through an acute toxicity test in mice.

The dosage of the compound of general formula (1) and its nontoxic salt in the present invention varies depending on the type of compound and the severity of the patient's symptoms, but it is usually sufficient to administer about 10 rrg to 1 g per day to the patient.

When the compound represented by general formula (1) and its salts are used as a pharmaceutical composition having active oxygen production inhibiting and active oxygen removing effects, they are usually combined with a pharmaceutical carrier and in the form of a pharmaceutical composition. be done.

As the carrier, diluents or excipients such as fillers, binders, disintegrants, lubricants, etc., which are commonly used to prepare drugs depending on the usage form, are used.

The dosage form may be any form such as injection, powder, capsule, granule, or tablet.

When used in tablet form, carriers include, for example, lactose, sucrose, sodium chloride, glucose, starch,
Excipients such as calcium carbonate, crystalline cellulose, and silicic acid;
Water, ethanol, propatool, grape starch solution,
Gelatin solution, carboxymethylcellulose, methylcellulose, binders such as potassium phosphate, dried starch,
Disintegrants such as sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, stearic acid monoglyceride, starch, lactose, stearate, boric acid powder,
Lubricants such as solid polyethylene glycol and other lubricants widely used in this field can be used. Furthermore, it can be made into sugar-coated tablets, gelatin-encapsulated tablets, film-coated tablets, etc., if necessary.

When prepared as an injection, examples of diluents include water, ethyl alcohol, propylene glycol, polyoxyethylene sorbisoto, and sorbitan ester. At this time, sufficient common salt, glucose, or glycerin may be included to prepare an isotonic solution, and conventional solubilizing agents, buffering agents, soothing agents, preservatives, etc. may be necessary. It may be included depending on the situation.

As described above, the piperazine derivative represented by the general formula (I) or its nontoxic salt has excellent effects of inhibiting active oxygen production and removing active oxygen.

Next, the present invention will be specifically described with reference to test examples showing that it is used as an active ingredient, useful as a pharmaceutical composition, and highly safe, and examples showing formulation examples of the compound of general formula (1). Explain in detail.

Example 1 - Hirokou formyl-methionyl-leucyl-phenylalanine (
Luminol, xanthine oxidase (abbreviated as XOD) (0.61/mg protein), and catalase (26.0 OOU/■ protein) manufactured by Sigma were used.

Hypoxanthine, hydrogen peroxide (30%), coffee.

Hypochlorous acid (5% solution) manufactured by Wako Hyakuyaku Co., Ltd. was used.

FMLP and Luminol are IIEPBs-5aline
(5mFI N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid was dissolved in dimethyl sulfoxide (DMSO) before dilution with normal gold saline buffered pH 7.4. The final concentration of DMSO in the reaction mixture was 10 μH; this concentration did not affect the reaction. Heparinized blood (70-80 mj2) was obtained from a male white rabbit (2,2-3.0 kg) and diluted in saline for 2 h. % dextran (manufactured by Hanui Kagaku ■).

Red blood cells were allowed to settle for 30 minutes at room temperature. Plasma rich in white blood cells is added to sodium metrizoate ficoll solution.
(manufactured by Nyegaard), and then heated at 4°C.
Centrifuged at 8004 for 40 minutes.

A 155 mM ammonium chloride solution was added to the pellet to lyse the red blood cells. The remaining granulocytes were placed in cold HEPES-Sa.
The cells were washed twice with 100% chlorine and resuspended in the same solvent.

Higaku Hikari (CL) measurement CL is measured using a Lumiphotometer (n+odel TD400
0; manufactured by Labo Science) and recorded in millivolts. Unless otherwise stated, reaction mixtures were HEPHS containing 2.5 m+ CaC12 and 5mM KCl.
Cell suspension suspended in -3aline (I x 10
) cells/ml) 0.2111 luminol (final concentration 3
(0 μM) and drug, and the reaction was carried out in a 0.5 mj2 polystyrene cuchette. The drug is H20
The ability to suppress luminol-dependent CL produced by the □+Na0Cl and hypoxanthine-XOD systems was also tested.

Same ff1 (0,1nj ri no 10 #M 8202 and 30
．． crM luminol and drug or buffer were premixed and placed in the counting chamber of a lumiphotometer controlled at 37° C., and activated by injecting 10 μH of Na0C1 (0.1 ml).

The production of superoxide anion catalyzed by XOD was continuously monitored by a similar method. 20μl
=;1.Φ.$61416 (4tJ/mj?) was added to cuvettes containing hypoxanthine (1mM), luminol (30μM) and various concentrations of drug or buffer. The luminescence amount was evaluated from the CL curved surface area, and the suppression rate was determined by comparing it with the control CL.

The test drug was (2R,3R)-3-((31-
Ethyl 3-methyl-1-(4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbutyl)butylcarbamoyl]oxirane-2-carboxylate 2
A sulfur salt was used.

Experiment 1 Effect of drugs on luminol-dependent chemiluminescence production (in vitro) Granulocytes (7 ml of IXIO" cells) were incubated with various concentrations of drugs for 30 minutes at 37°C, then centrifuged and incubated twice with HEPES to remove the drugs. -saline (4°C
). Then, incubate for 10 minutes (37
℃), and then the CL generated by FMLP stimulation was measured. The results are shown in Table 1.

Table 1 Experiment 2 Effect of drugs on luminol-dependent chemiluminescence production (in vivo) Twelve male white rabbits (2.0-2.3 kg) were randomly assigned to drug treatment or control groups. Drugs (20r
rw/' kg) was dissolved in 5 ml of physiological saline,
It took 5 minutes for intravenous injection. Physiological saline was also administered to controls in the same manner.

Heparinized blood was obtained 5 minutes after injection.

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

Table 2 * p < 0.05 (Mann-Whitney
U test) Experiment 3 Effect of drugs on luminol-dependent chemiluminescence of hypoxanthine-xanthine oxidase system (removal effect) 20 μl of XOD (4 U/ml) containing hypoxanthine (1 mM), luminol (50 μM), and various drugs Added to cuvette. CL production was evaluated by the area under the CL curve and compared with a control in which the CL response was set at 100%. The results are shown in Table 3.

Table 3 Experiment 4 1 (Effect of drugs on luminol-dependent chemiluminescence of zo□+Na0C12 system) It has become clear that it has the effect of inhibiting the production of active oxygen, and from Experiments 3 and 4, it has also become clear that it has the effect of removing the active oxygen generated.

Example 2 2 Hōsendan Hirokei ddN male mice weighing 20 to 28 g were used.

- The drug was administered through the tail vein. As a result, as shown in Table φ, it was confirmed that the drug of the present invention is highly safe. However, compound 1 (2[?, 3R) -3-((S
) -3-Methyl-1-(4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylic acid ethyl disulfate Compound 2 (2R, 31?) ) -3-((51-3
-Methyl-1-(4-(2,3,4-1-rimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylic acid sodium compound 3 (25,3S) -3-( (31-3-
Ethyl methyl-1-(4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylate% sulfate Compound 4 (2S, 3S) -3-( (Sl-3
-Sodium methyl-1-(4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylate Example 3 Formulation example (tablet) 1 tablet (220 mg) Film-coated tablets containing the following ingredients.

(2R,3R) -3-((31-3-methyl-I-(
Sodium 4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylate 50■ Milk $J!
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 can also be made into film-coated tablets using the same formulation.

Example 4 Formulation example (granules) 1 g of granules contains the following ingredients.

(2S,3S) -3-((31-3-methyl-1-(
Ethyl 4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylate 'A g acid salt 00mg Lactose 500
mg Corn starch 300rrg Other compounds used as active ingredients in the present invention can also be made into granules using a similar formulation.

Example 5 Formulation example (injection) One ampoule contains the following ingredients.

(2R,3R) -3-((S) -3-methyl-1-
(4-(2,3,4-trimethoxyphenylmethyl)piperazin-1-ylcarbonyl)butylcarbamoyl]oxirane-2-carboxylic acid ethyl disulfate 20 mg Add sterile distilled water to the above ingredients to a volume of 10 ml.

Other compounds used as active ingredients in the present invention can also be made into injections using the same formulation.

Patent applicant: Nippon Chemifa Co., Ltd. Representative Keizo Ushiyama

Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R represents a hydrogen atom or a lower alkyl group, and n
is an integer of 0 to 3) or a non-toxic salt thereof as an active ingredient, the pharmaceutical composition has active oxygen production inhibiting and active oxygen removing effects.