JPH0535128B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention relates to lipid peroxide production inhibitors, and more particularly, to lipid peroxide production inhibitors useful as preventive and therapeutic agents for various ischemic diseases and accompanying various brain diseases, heart diseases, and peripheral circulation disorders. This invention relates to lipogenesis inhibitors. [Prior art and its problems] In circulatory disorders in the brain, heart, or periphery, unsaturated fatty acids such as arachidonic acid released from cell membranes due to ischemia (a state in which blood is not supplied to tissues) are Active oxygen (OH, radical superoxide, etc.) generated in the surrounding tissues acts to generate lipid peroxide. These changes occur not only during ischemia, but also at an accelerated pace due to reoxygenation through the blood after ischemic recanalization, leading to damage to biomembrane cells rich in unsaturated fatty acids, destruction of surrounding tissues, and vascular endothelium. It is known that this causes destruction of blood vessels, vasospasm, or edema, etc., and that the disease condition progresses due to a vicious cycle of these reactions. (“Cerebral Ischemia and Cellular Damage” edited by Takao Asano, Niyuronsha,
1980; “Cerebral Ischemia and Free Radicals” edited by Takao Asano, Niyuronsha, 1983). Therefore, by suppressing lipid peroxide production caused by active oxygen, it is possible to prevent tissue destruction, vascular endothelium destruction, vasospasm, edema, etc., and it is possible to prevent conventional drugs that improve circulation by increasing blood flow. It is a new type of circulatory disorder prevention and treatment agent that acts on the cause of the disease. Particularly in recent years, the effectiveness of increasing blood flow in the infarcted area has been questioned, and it has even been said to have the opposite effect in acute cerebrovascular disorders, and such drugs have become even more important. As a drug that suppresses lipid peroxidation by active oxygen, vitamin E, the following formula: Idebenone (Biochemical and Biophysical Research Communications)
Research Communications) 125, 1046
(1984); Takeda Institute Bulletin 44 , 30 (1985)) and the following formula: Nizophenone (Journal of Neurochemistry)
chemistry) 37 , 934 (1981)). However, vitamin E has insufficient action, idebenone and nizophenone have a long synthesis route, and idebenone is difficult to solubilize in water, so there may be problems in making an injection formulation, and nizophenone has a depressant effect on the central nervous system. is strong (Pharmaceutical Research 16 , 1
(1985)). Various pyrazolone derivatives are known. JP-A-51-13766 describes the following formula (A): The pyrazolin-5-one derivative represented by: The application is; JP-A-59-175469 describes the following formula: (wherein, X represents a group -CH ₂ CH ₂ O-, -CH ₂ CH ₂ S-, etc., and R represents an aryl group) and its use as a lipoxygenase inhibitor. The application is; JP-A No. 59-512 describes the following formula: (In the formula, R ₁ represents a hydrogen atom or an amino group,
R ₂ represents an aryl group, and X represents a group -CH ₂ CH ₂ -
etc. ) and its use as a diuretic, antihypertensive, and antithrombotic; West German Patent Application No. 2836891 describes the following formula: (In the formula, R ₁ and R ₂ represent a hydrogen atom or a substituent.) A pyrazolin-5-one derivative represented by the formula and its use as an anti-inflammatory agent has been described, but lipid peroxidation by active oxygen There is no description regarding the effect of suppressing . In addition, the compound represented by formula (A) is said to be effective in ischemic heart disease models using rats, rabbits, and dogs, but is ineffective in a model using pigs that approximates the hemodynamics of the human heart. This result is in good agreement with reports that it is ineffective against ischemic heart disease in humans (European Journal of Pharmacology).
Journal of Pharmacology) 114 , 189 (1985)).
Furthermore, there is no description of specific effects regarding protection after cerebral ischemia recanalization. Therefore, the present inventors conducted extensive research with the aim of providing a drug that has the effect of suppressing lipid peroxidation caused by active oxygen, and as a result, the following formula (): (In the formula, R ₁ represents a hydrogen atom, an aryl group, an alkyl group having 1 to 5 carbon atoms, or an alkoxycarbonyl alkyl group having a total of 3 to 6 carbon atoms; R ₂ represents a hydrogen atom, an aryloxy group, an arylmercapto group; group, an alkyl group having 1 to 5 carbon atoms, or an alkyl group having 1 to 3 carbon atoms
represents a hydroxyalkyl group; or
R ₁ and R ₂ jointly represent an alkylene group having 3 to 5 carbon atoms; R ₃ is a hydrogen atom, and R 3 is a hydrogen atom;
5 alkyl group, cycloalkyl group having 5 to 7 carbon atoms, hydroxyalkyl group having 1 to 3 carbon atoms, benzyl group, naphthyl group, or unsubstituted, or unsubstituted, or cycloalkyl group having 1 to 3 carbon atoms
-5 alkyl group, alkoxy group having 1 to 5 carbon atoms, hydroxyalkyl group having 1 to 3 carbon atoms, alkoxycarbonyl group having 2 to 5 total carbon atoms, 1 carbon number
-3 alkylmercapto group, alkylamino group having 1 to 4 carbon atoms, dialkylamino group having 2 to 8 total carbon atoms, halogen atom, trifluoromethyl group,
carboxyl group, cyano group, hydroxyl group, nitro group,
It represents a phenyl group substituted with 1 to 3 same or different substituents selected from the group consisting of an amino group and an acetamido group. ) It was discovered that the pyrazolone derivative represented by the following formula has a strong lipid peroxidation inhibitory effect, and that it has protective effects such as recovery of brain waves in an animal model of cerebral ischemia recanalization, which is similar to the actual pathological condition. It was completed. [Structure of the Invention] The lipid peroxide production inhibitor of the present invention is characterized in that it contains a pyrazolone derivative represented by the above formula () or a pharmaceutically acceptable salt thereof as an active ingredient. The compound () used in the present invention has the following formula (')
Or (″):

【formula】

It can also take the structure shown by [Formula]. Therefore, compounds having the structure of the above formula (') or ('') are also included in the active ingredients of the present invention. In the above formula (), the aryl group in the definition of R ₁ includes a phenyl group, a methyl group,
Examples thereof include a butyl group, a methoxy group, a butoxy group, a phenyl group substituted with a substituent such as a chlorine atom, and a hydroxyl group. Examples of the alkyl group having 1 to 5 carbon atoms in the definitions of R ₁ , R ₂ and R ₃ include methyl group, ethyl group, propyl group, isopropyl group, butyl group,
isobutyl group, sec-butyl group, tert-butyl group,
Examples include pentyl group. Examples of the alkoxycarbonylalkyl group having a total of 3 to 6 carbon atoms in the definition of R ₁ include a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group, a propoxycarbonylmethyl group, a methoxycarbonylethyl group, a methoxycarbonylpropyl group, and the like. Examples of the aryloxy group in the definition of R ₂ include phenoxy group, p-methylphenoxy group, p-methoxyphenoxy group, p-chlorophenoxy group, p-hydroxyphenoxy group, etc., and examples of the arylmercapto group include is a phenylmercapto group, p
-methylphenylmercapto group, p-methoxyphenylmercapto group, p-chlorophenylmercapto group, p-hydroxyphenylmercapto group, and the like. Examples of the hydroxyalkyl group having 1 to 3 carbon atoms in the definition of R ₂ and R ₃ include a hydroxymethyl group, a 2-hydroxyethyl group, a 3-hydroxypropyl group, and the like. Examples of the cycloalkyl group having 5 to 7 carbon atoms in the definition of R ₃ include a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and the like. In the definition of _R3 , the alkoxy group having 1 to 5 carbon atoms in the substituent of the phenyl group includes methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, pentyloxy group, etc., and the total number of carbon atoms is 2
Examples of the alkoxycarbonyl group of ~5 include methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, butoxycarbonyl group, etc., and examples of the alkylmercapto group having 1 to 3 carbon atoms include methylmercapto group, ethylmercapto group, propyl Examples of the alkylamino group having 1 to 4 carbon atoms include methylamino group, ethylamino group, propylamino group, butylamino group, and dialkylamino groups having 2 to 8 total carbon atoms. Examples include dimethylamino group, diethylamino group, dipropylamino group, and dibutylamino group. Specific examples of the compound () used in the present invention include the compounds shown below. Γ3-methyl-1-phenyl-2-pyrazoline-
5-one Γ3-methyl-1-(2-methylphenyl)-2-
Pyrazolin-5-one Γ3-methyl-1-(3-methylphenyl)-2-
Pyrazolin-5-one Γ3-methyl-1-(4-methylphenyl)-2-
Pyrazolin-5-one Γ3-methyl-1-(3,4-dimethylphenyl)
-2-pyrazolin-5-one Γ1-(4-ethylphenyl)-3-methyl-2-
Pyrazolin-5-one Γ3-methyl-1-(4-propylphenyl)-2
-pyrazolin-5-one Γ1-(4-butylphenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(3-trifluoromethylphenyl)-3-
Methyl-2-pyrazolin-5-one Γ1-(4-trifluoromethylphenyl)-3-
Methyl-2-pyrazolin-5-one Γ1-(2-methoxyphenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(3-methoxyphenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(4-methoxyphenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(3,4-dimethoxyphenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-ethoxyphenyl)-3-methyl-2
-Pyrazolin-5-one Γ3-methyl-1-(4-propoxyphenyl)-
2-pyrazolin-5-one Γ1-(4-butoxyphenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(2-chlorophenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(3-chlorophenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(4-chlorophenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(3,4-dichlorophenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-bromophenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(4-fluorophenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(3-chloro-4-methylphenyl)-3-
Methyl-2-pyrazolin-5-one Γ1-(3-methylmercaptophenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-methylmercaptophenyl)-3-methyl-2-pyrazoline- 5-one Γ4-(3-methyl-5-oxo-2-pyrazolin-1-yl)benzoic acid Γ1-(4-ethoxycarbonylphenyl)-3-
Methyl-2-pyrazolin-5-one Γ1-(4-nitrophenyl)-3-methyl-2-
Pyrazolin-5-one Γ3-ethyl-1-phenyl-2-pyrazoline-
5-one Γ1-phenyl-3-propyl-2-pyrazolin-5-one Γ1,3-diphenyl-2-pyrazolin-5-one Γ3-phenyl-1-(p-tolyl)-2-pyrazolin-5-one Γ1-(4-methoxyphenyl)-3-phenyl-
2-pyrazolin-5-one Γ1-(4-chlorophenyl)-3-phenyl-2
-pyrazolin-5-one Γ3,4-dimethyl-1-phenyl-2-pyrazolin-5-one Γ4-isobutyl-3-methyl-1-phenyl-
2-pyrazolin-5-one Γ4-(2-hydroxyethyl)-3-methyl-1
-Phenyl-2-pyrazolin-5-one Γ3-methyl-4-phenoxy-1-phenyl-
2-pyrazolin-5-one Γ3-methyl-4-phenylmercapto-1-phenyl-2-pyrazolin-5-one Γ3,3',4,5,6,7-hexahydro-2-
Phenyl-2H-indazol-3-one Γ3-(ethoxykicarbonylmethyl)-1-phenyl-2-pyrazolin-5-one Γ1-phenyl-2-pyrazolin-5-one Γ3-methyl-2-pyrazoline-5- Γ1,3-dimethyl-2-pyrazolin-5-one Γ1-ethyl-3-methyl-2-pyrazoline-5
-one Γ1-butyl-3-methyl-2-pyrazoline-5
-one Γ1-(2-hydroxyethyl)-3-methyl-2-
Pyrazolin-5-one Γ1-cyclohexyl-3-methyl-2-pyrazolin-5-one Γ1-benzyl-3-methyl-2-pyrazoline-
5-one Γ1-(α-naphthyl)-3-methyl-2-pyrazolin-5-one Γ1-methyl-3-phenyl-2-pyrazoline-
5-one Γ3-methyl-1-(4-methylphenyl)-2-
Pyrazolin-5-one Γ1-(4-butylphenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(4-methoxyphenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(4-butoxyphenyl)-3-methyl-2
-pyrazolin-5-one Γ1-(4-chlorophenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(4-hydroxyphenyl)-3-methyl-
2-pyrazolin-5-one Γ1-(3,4-dihydroxyphenyl)-3-methyl-2-pyrazolin-5-one Γ1-(2-hydroxyphenyl)-3-methyl-
2-pyrazolin-5-one Γ1-(3-hydroxyphenyl)-3-methyl-
2-pyrazolin-5-one Γ1-(4-hydroxyphenyl)-3-methyl-
2-pyrazolin-5-one Γ1-(3,4-hydroxyphenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-hydroxyphenyl)-3-phenyl-2-pyrazoline-5- One Γ1-(4-hydroxymethylphenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-aminophenyl)-3-methyl-2-
Pyrazolin-5-one Γ1-(4-methylaminophenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-ethylaminophenyl)-3-methyl-2-pyrazolin-5-one Γ1 -(4-Butylaminophenyl)-3-methyl-2-pyrazolin-5-one Γ1-(4-dimethylaminophenyl)-3-methyl-2-pyrazolin-5-one Γ1-(acetamidophenyl) -3-methyl-2
-pyrazolin-5-one Γ1-(4-cyanophenyl)-3-methyl-2-
Pyrazolin-5-one Some of the compounds used in the present invention are known compounds used as intermediate raw materials for dyes and the like, but their use as medicines is not known. Among the salts of the compound () used in the present invention, pharmaceutically acceptable salts include salts with mineral acids such as hydrochloric acid, sulfuric acid, hydrobromide, and phosphoric acid; methanesulfonic acid, p-toluenesulfonic acid , salts with organic acids such as benzenesulfonic acid, acetic acid, glycolic acid, glucuronic acid, maleic acid, fumaric acid, oxalic acid, ascorbic acid, citric acid, salicylic acid, nicotinic acid, tartaric acid; with alkali metals such as sodium and potassium. salts with alkaline earth metals such as magnesium and calcium; ammonia, tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine, 2-amino-2-
Methyl-1-propanol, ethanolamine,
Examples include salts with amines such as N-methylglucamine and L-glucamine. The compound used in the present invention can be synthesized by any purposeful method, but one of the preferred methods is
For example: (In the formula, R ₁ , R ₂ and R ₃ have the same meanings as above,
R' represents an alkyl group having 1 to 5 carbon atoms. ) That is, a β-keto acid derivative represented by the formula () and a hydrazine derivative represented by the formula () are mixed in the presence or absence of a solvent such as an alcohol such as methanol or ethanol, or an aromatic solvent such as benzene or toluene. Solvents, if necessary, potassium carbonate, sodium ethoxide, sodium hydroxide,
In the presence of a catalyst such as a base such as potassium hydroxide or sodium acetate, a mineral acid such as hydrochloric acid, sulfuric acid, or hydrobromic acid, or an organic acid such as acetic acid or para-toluenesulfonic acid,
Compound () can be obtained by reacting at a temperature of ~200°C. Further, depending on the substituent of the aryl group of R ₃ , the desired product can be synthesized as shown below. (In the formula, R ₁ , R ₂ and R ₃ have the same meanings as above,
R'' represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or an alkoxycarbonyl group having 2 to 6 total carbon atoms. Alternatively, it can be obtained by decomposing a carboxylic acid or a derivative thereof with a suitable reducing agent such as lithium aluminum hydride, sodium borohydride, diborane. For example, a target product in which the substituent is an amino group can be obtained by reducing a nitro group under appropriate conditions, such as hydrogen-Pd/
C, can be obtained by reduction with hydrochloric acid-stannic chloride. When applying compound (2) orally, it is preferable to administer 1 to 100 mg of compound (2) 1 to 3 times a day to adults, and when administering intravenously to adults. In contrast, it is preferable to administer 0.01 to 10 mg of the compound () 2 to 5 times a day, or to administer these doses by continuous drip infusion.
In the case of intrarectal administration, it is preferable to administer 1 to 100 mg of the compound (2) 1 to 3 times a day. Further, it is more preferable that the above dosage is adjusted as appropriate depending on the age, pathological condition, and symptoms. In addition, in the case of oral or rectal administration, it may be used as a sustained release preparation. When formulating a formulation, one or more of the compound () or its pharmaceutically acceptable salt is usually used as a composition containing commonly used pharmaceutical carriers, excipients, and other additives. . Pharmaceutical carriers can be solid or liquid; examples of solid carriers include lactose, kaolin, sucrose, crystalline cellulose, cornstarch, talc, agar, pectin, acacia, stearic acid, magnesium stearate, lecithin, and sodium chloride. etc. Examples of liquid carriers include syrup, glycerin, peanut oil, polyvinylpyrrolidone, olive water, ethanol, benzyl alcohol, propylene glycol, water, and the like. It can take a variety of dosage forms, including tablets, powders, granules, hard gelatin capsules, suppositories, or troches when solid carriers are used. The amount of solid carrier can vary widely but is preferably from about 1 mg to about 1 g. When using a liquid carrier, syrup, emulsion,
It can be a sterile injectable solution, such as a soft gelatin capsule, or an ampoule, or an aqueous or non-aqueous suspension. Further, the compound () can also be used by inserting it into a cyclodextrin clathrate or liposome. [Effects of the Invention] The lipid peroxide production inhibitor of the present invention has excellent effects and is effective against various ischemic diseases and various diseases based thereon, including cerebrovascular disorders such as cerebral infarction and cerebral apoplexy;
or brain function decline, vascular dementia,
It is useful as a preventive/therapeutic agent for various brain diseases such as age-related cerebrovascular tissue lesions, various heart diseases caused by myocardial ischemia such as myocardial infarction and heart failure, and various peripheral circulation disorders. [Examples of the Invention] Hereinafter, the present invention will be explained in more detail based on Synthesis Examples and Examples, but these are not intended to limit the scope of the present invention in any way. Synthesis Example 1 13.0 g of ethyl acetoacetate and 10.8 g of phenylhydrazine were added to 50 ml of ethanol, and the mixture was stirred under reflux for 3 hours. After cooling, the precipitated crystals were collected and recrystallized from ethanol to give 3-methyl-1-phenyl-2-pyrazolin-5-one (Compound No. 1) 11.3
g was obtained as colorless crystals. Yield: 65% Melting point: 127.5-128.5°C Synthesis Examples 2-43 Compounds shown as Compounds Nos. 2-43 in Table 1 were synthesized in the same manner as in Synthesis Example 1. Synthesis example 44 1-(2-methoxyphenyl)-3-methyl-2
-1.80g of pyrazolin-5-one in 47% hydrobromic acid
It was added to a mixture of 18 ml and acetic acid, and the mixture was stirred under reflux for 6.5 hours. After evaporating the solvent, aqueous NaHCO ₃ solution was added to adjust the pH to 4, followed by extraction with ethyl acetate. After drying the organic layer and concentrating, the residue was recrystallized from ethanol to give 1-(2-hydroxyphenyl)-3-methyl-2-pyrazolin-5-one (Compound No. 44).
1.19 g was obtained as colorless crystals. Yield: 67% Melting point: 212.5-214°C Synthesis Examples 45-48 Compounds shown as Compounds Nos. 45-48 in Table 1 were synthesized in the same manner as Synthesis Example 44. Synthesis Example 49 4-(3-methyl-
5.0 g of 5-oxo-2-pyrazolin-1-yl)benzoic acid and 25 ml of triethylamine were added, and then 12.5 ml of ethyl chlorocarbonate was added dropwise under ice cooling. After distilling off the solvent, the residue was dissolved in 200 ml of THF, and after removing insoluble matter, a solution of 2.08 g of NaBH ₄ dissolved in 60 ml of water was added dropwise to the solution, and the mixture was stirred at room temperature for 2 hours. After evaporating the solvent, water was added to the residue, and the pH was adjusted to 4 to 5 with dilute hydrochloric acid, followed by extraction with chloroform. The organic layer was dried, concentrated, purified on silica gel column chromatography using chloroform-ethanol (100:1) as a developing solvent, and recrystallized from chloroform-ethyl ether to give 1-(4-hydroxymethylphenyl). -3-methyl-2-pyrazoline-
1.16 g of 5-one (Compound No. 49) was obtained as colorless crystals. Yield 35% Melting point 139-140℃ Synthesis example 50 500 mg of 3-methyl-1-(4-nitrophenyl)-2-pyrazolin-5-one in 310 ml of methanol
was dissolved, 50 mg of 5% Pd/C and 0.6 ml of concentrated hydrochloric acid were added, and the mixture was stirred under a hydrogen atmosphere to consume the calculated amount of hydrogen, then the catalyst was removed and the liquid was concentrated. The residue was recrystallized from methanol-ethyl ether to give 1-
409 mg of (4-aminophenyl)-3-methyl-2-pyrazolin-5-one dihydrochloride (Compound No. 50) was obtained as light brown crystals. Yield: 68% Melting point: 196-200°C Synthesis Example 51 In the same manner as Synthesis Example 1, a compound shown as Compound No. 51 in Table 1 was synthesized. Example 1 (1) Lipid peroxidation inhibitory effect (a) Preparation of brain homogenate Brain homogenate was prepared using male Wistar rats according to the following operating procedure. Thoracotomy was performed under anesthesia with intraperitoneal administration of 45 mg/Kg of sodium pentobarbital.
A polyethylene tube was inserted into the aorta from the left ventricle and fixed. Next, the brain was perfused with ice-cold 50 mM phosphate buffered saline (PH7.4) (hereinafter referred to as "PBS") through this tube. The whole brain was removed. After removing the cerebellum,
Measure the wet weight of the cerebrum and add 9 times the amount of PBS.
was added and homogenized by crushing with a Teflon homogenizer in ice water. This brain honestenate was centrifuged at 2200 rpm for 10 minutes at 4°C, and 0.3 ml of the supernatant was aliquoted into a light-shielding test tube with a stopper, and used as brain honestenate for drug evaluation. (b) Evaluation of test drug Add PBS0.6 to the brain homogenate prepared in (a).
ml and 10μ of ethanol solution of test drug (final concentration 500μM or concentration at common ratio 3 of 0.3-100μM)
was added and heated in a 37°C hot bath for 30 minutes.
Next, after adding 200μ of a 35% aqueous perchloric acid solution, centrifugation was performed at 2600 rpm for 10 minutes at 4°C to obtain a supernatant. In addition, for blank measurement, 10μ of ethanol was added instead of the 10μ of the ethanol solution of the test drug (blank), and the same operation was performed. (c) Quantification of lipid peroxide To 0.1 ml of the supernatant obtained in (b), add 0.2 ml of 8.1% sodium dodecyl sulfate aqueous solution, 1.5 ml of 20% acetate buffer (PH3.5), and 0.67% 2-thiobarbituric acid. 1.5 ml of the aqueous solution and 0.7 ml of distilled water were added and mixed. This mixture was then heated in a boiling water bath for 60 minutes, rapidly cooled with ice water, and diluted with distilled water 1.0
ml and pyridine-butanol mixture (1:15)
Add 5.0ml and shake for about 30 seconds, then at 3000 rpm.
The mixture was centrifuged for 10 minutes, and the supernatant was used as a sample for lipid peroxide measurement. In addition, Lipoperoxide-test (Wako Pure Chemical Industries, Ltd.)
Co., Ltd.; containing 5 nmol/ml of 1,1,3,3-tetraethoxypropane) was added in place of the brain homogenate obtained in (b) to serve as a standard solution. Lipid peroxide was measured using a fluorescence spectrophotometer (Model 204, manufactured by Hitachi, Ltd.) at an excitation wavelength of 515 nm and a fluorescence wavelength of 550 nm, and the amount of lipid peroxide (TBA value) was determined according to the following formula. TAB value = 0.5 × f / F × 1.1 / 0.3 × 10 (nmol/ml) F: Fluorescence intensity of standard solution f: Fluorescence intensity of test drug Next, suppress the concentration of each test drug against the blank TAB value in (b) The IC ₅₀ value was calculated according to the least squares method. The results are shown in Table 1. (2) Protective effect in cerebral ischemia recanalization model Male Wistar rats weighing approximately 400 g were immobilized by intramuscular administration of 0.6 mg of d-tubocurarine, and after a tracheal cannula device, the head was placed under artificial respiration. The patient was held in a stereotaxic apparatus. After incising the scalp and drilling a hole in the skull, an electrode for deriving electroencephalograms was placed on the surface of the frontal lobe of the left cerebral cortex under the dura mater.
After fixing the electrode to the skull using dental cement, the animal was held in a dorsal position. Next, a cannula for measuring systemic pressure was placed in the left femoral artery, and a cannula for additional administration of d-tubocurarine was placed in the left femoral vein. Heart rate was measured and recorded using a heart rate meter driven by arterial waves. After stabilizing various parameters such as blood pressure, heart rate, and electroencephalogram, administer 1 ml/Kg of 1% gum tragacanth solution.
Active ingredient 10 of the present invention prepared in suspension so that
mg/Kg was administered directly into the intestine 30 minutes before cerebral ischemic challenge. The same volume of 1% gum tragacanth solution alone was similarly administered to the control group. Versatile monitoring and recording device (manufactured by Nihon Kohden Co., Ltd., RM-
85 type), the following procedure was performed to reduce cerebral ischemic stress. First, the rib was released at the end of the left costal cartilage, and the chest was opened. Next, the left common carotid artery and left vertebral artery exposed at the aortic root were simultaneously exposed, followed by the innominate artery.
Cephalic blood flow was blocked for 10 minutes by occlusion using an arterial clip 30 minutes after drug administration. Recanalization of blood flow to the head was performed by simultaneously releasing the two arterial clips attached to each site. The protective effect of drugs against damage after recanalization of cerebral ischemic stress was examined based on the presence or absence of recovery of electroencephalograms. During the experiment, the animal's rectal temperature was maintained at 37 to 38°C using a heating mat. In addition, rectal temperature was continuously recorded on a recorder along with electroencephalogram, femoral artery pressure, and heart rate. When cerebral ischemia was applied for 10 minutes, the voltage of the brain waves decreased immediately after the ischemia, and after about 15 seconds, the brain waves disappeared and became flat. Such flattening of the electroencephalogram during ischemic stress was observed in both the control group and the group administered with the active ingredient of the present invention. Even after 10 minutes of cerebral ischemia was released and recanalization was performed, no electroencephalograms were observed in all cases in the control group, and they remained flat as during ischemic stress. Due to the persistence of these flat EEGs, the animals died an average of 75 minutes after recanalization. However, in the group administered with the active ingredient of the present invention, the brain waves recovered during recanalization, and the so-called recovery of brain function and the function of the cardiovascular system were activated and normalized. As a result of these, the survival time of the animals was clearly extended. Table 2 shows the results.
Shown below. Example 2 Myocardial ischemic protective effect Coronary blood flow that nourishes the myocardium is interrupted for some reason (blood flow disorder) or recanalized (recanalization disorder)
When this occurs, the myocardium is injured (myocardial ischemic injury). The degree of such myocardial damage progresses as the ischemic time increases, causing various ischemic heart diseases such as so-called myocardial infarction and heart failure. Therefore, the ischemic myocardial protective effect of Compound () as a representative compound was investigated as follows. Male Wistar rats weighing 300-400 g were used in the experiments. Pentobarbital sodium 50
Under anesthesia with mg/Kg intraperitoneal injection, the heart was exposed to 95% O ₂
- Krebs-Henseleit oxygenated with 5% _CO2
The aortic cannula was immediately inserted into the ascending aorta and fixed in bicarbonate (KH) solution.
Perfusion by Langendorff method (75 cmH ₂ O) was started. Subsequently, the left atrial cannula was inserted and fixed into the left atrium. After this, perfusion using the Langendorff maneuver was continued for another 10 minutes, and then the left atrial cannula was opened.
Working heart method (preload 10cmH ₂ O, afterload 80cm
perfusion ( _H2O ) (American Journal of
Physiology, 212 , 804 (1967)) and perfused for 15 minutes, followed by the method of Ichihara et al. (Journal of
Cardiovascular Pharmacology, 5 , 745
(1983)), the afterload on the aortic valve was removed and ischemia was induced for 20 minutes. The perfusion solution containing the test drug was switched to the perfusion solution containing the test drug 5 minutes before ischemia, and the perfusion solution containing the test drug was used during ischemia. Reperfusion afterload 80cmH ₂ O
This was then performed for 30 minutes with a perfusate solution that did not contain the study drug.
Cardiac function includes coronary perfusion (CF), cardiac output (CO),
Heart rate (HR), aortic pressure (AP) and rate pressure products
product) (HR×AP) was measured. CF was measured using a 10ml graduated cylinder. CO was measured using an electromagnetic blood flow meter (Nihon Kohden MFV-2100) and a bioelectrical amplifier (Nihon Kohden AB-621G) using an invasive blood flow measurement probe (Nihon Kohden FF-030γ 3φ) placed just before the left atrial cannula. It was measured using AP
was measured using a pressure transducer (Nihon Kohden MPU-0.5) connected to the aortic cannula and a strain pressure amplifier (Nihon Kohden AP-601G). HR was measured using an instantaneous heart rate monitor (Nihon Kohden AT-601G). HR×AP was calculated by multiplying HR by systolic aortic pressure. The perfusate was KH oxygenated with 95% _O2 - 5% _CO2 .
The solution was heated to 37°C before use. The composition of the KH solution (unit: mM) is as follows. NaCl118,
KCl4.7, _CaCl2 2.5, _MgSO4 1.2, _{KH2PO4 1.2} ,
_NaHCO3 25, glucose 11. The test drug compound () is 1 equivalent of 1N-
After dissolving in NaOH aqueous solution, it was diluted with KH solution and used. Changes in cardiac function after treatment with the study drug and recovery of heart function during reperfusion were expressed as percentages of the values before treatment with the study drug. All experimental results are expressed as mean value ± standard error. After applying myocardial ischemia for 20 minutes and perfusing with KH solution again
When calculating the degree of cardiac function recovery in 30 minutes using cardiac output (CO) as an index, compound () averaged 24%.
showed a restorative effect. However, in the negative control group in which the same dose of physiological saline was administered instead of compound (2), the effect was slight, averaging 3% (Table 3).

[Table] Table 4 also shows cases where the cardiac output (CO) reached 50% or more after 30 minutes of ischemic stress reperfusion as cases of cardiac function recovery.

[Table] From the above results, it can be said that compound () has myocardial ischemia protective effect. Example 3 Formulation of lipid peroxide production inhibitor of the present invention (1) Tablet The following ingredients were mixed according to a conventional method and tableted using a conventional device. Active ingredients of the present invention 10 mg Crystalline cellulose 21 mg Cornstarch 33 mg Lactose 65 mg Magnesium stearate 1.3 mg (2) Soft capsules The following ingredients were mixed according to a conventional method and filled into soft capsules. Active ingredients of the present invention 10 mg Olive oil 105 mg Lecithin 6.5 mg (3) Injection preparation The following ingredients were mixed according to a conventional method to prepare a 1 ml ampoule. Active ingredients of the present invention 0.7mg Sodium chloride 3.5mg Distilled water for injection 1.0mg

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Claims (1)

[Claims] Primary formula: (In the formula, R ₁ represents a hydrogen atom, an aryl group, an alkyl group having 1 to 5 carbon atoms, or an alkoxycarbonyl alkyl group having a total of 3 to 6 carbon atoms; R ₂ represents a hydrogen atom, an aryloxy group, an arylmercapto group; group, an alkyl group having 1 to 5 carbon atoms, or an alkyl group having 1 to 3 carbon atoms
represents a hydroxyalkyl group; or
R ₁ and R ₂ jointly represent an alkylene group having 3 to 5 carbon atoms; R 3 is a hydrogen atom, and R ₃ is a hydrogen atom and 1 to 5 carbon atoms;
alkyl group, cycloalkyl group having 5 to 7 carbon atoms, hydroxyalkyl group having 1 to 3 carbon atoms, benzyl group, naphthyl group, or unsubstituted, or cycloalkyl group having 1 to 3 carbon atoms;
-5 alkyl group, alkoxy group having 1 to 5 carbon atoms, hydroxyalkyl group having 1 to 3 carbon atoms, alkoxycarbonyl group having 2 to 5 total carbon atoms, 1 carbon number
-3 alkylmercapto group, alkylamino group having 1 to 4 carbon atoms, dialkylamino group having 2 to 8 total carbon atoms, halogen atom, trifluoromethyl group,
carboxyl group, cyano group, hydroxyl group, nitro group,
It represents a phenyl group substituted with 1 to 3 same or different substituents selected from the group consisting of an amino group and an acetamido group. ) A lipid peroxide production inhibitor characterized by containing a pyrazolone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.