JPH07258098A - End serine acceptor bond inhibitor - Google Patents

Abstract

PURPOSE: To obtain the subject inhibitor containing a ursonic acid expressed by a specific formula as active ingredients, having a marked inhibiting action of endothelin receptor binding and useful as a treating and preventing agent for hypertension, angina pectoris, myocardosis, arteriosclerosis, myocardial infarct, etc. CONSTITUTION: This inhibitor for endothelin receptor binding consists of a ursonic acid expressed by formula I (R1 , R2 are each H or OH, provided that at least one of the R1 and R2 or H, or R1 and R2 combined to form =O, =NOH, =N-NH2 , formula II or formula III; R3 and R4 are each H or a halogen, or R3 and R4 combined to form a double bond between carbons bonded therewith) or a pharmaceutically acceptable salt thereof as active ingredients. Further, the ursonic acid is e.g. obtained by extracting Chinese medicinal plant Xylanche himalaica, separating and purifying.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an endothelin receptor binding inhibitor useful for treating hypertension, cardio-cerebral circulatory disease, renal disease, asthma and the like.

[0002]

Endothelin (ET) is a vasoconstrictive peptide consisting of 21 amino acids, which was isolated from the culture supernatant of porcine arterial endothelial cells by Yanagisawa et al. In 1988 and whose structure was determined (Yanagisawa et al., Nature 332). 411-4
Item 12). Then, the study of the gene encoding endothelin revealed the existence of peptides with a structure similar to endothelin, and endothelin 1 (E
T1), endothelin-2 (ET2), and endothelin-3 (ET3). The structures of these endothelins are as follows (the constituent amino acids in ET1, 2, 3 are all L-forms) (Inoue et al., Proc.
Natl Acad. Sci. USA Volume 86, 286
3 to 2867).

[0003]

[Chemical 3]

Since the above endothelins are present in the body and have a strong vasoconstrictor action and pressor action, they are expected to be endogenous factors involved in the regulation of the circulatory system, such as hypertension, heart / brain. A relationship with circulatory disease (eg myocardial infarction), renal disease (eg acute renal failure) and vasospasm has been estimated (J. Med. Chem., 35 , 1493 (1).
992)). It also has a bronchial smooth muscle contractile action, and its relationship with asthma has been estimated.

Various peptides having an inhibitory action on endothelin receptor binding have been reported so far, but as non-peptidic compounds, anthraquinone derivatives (Japanese Patent Laid-Open No. 3-47163) and pheophorbide derivatives (Japanese Patent Laid-Open No. 5-163163). -331063) has only been reported.
On the other hand, ursonic acid has been synthesized since the oxidation of ursolic acid in 1963 (Bull. Calcutta S.
school Troop. Med. , 11 , 20 (196
3)), various studies have been conducted, but no report has been made on the inhibitory action on endothelin receptor binding.

[0006]

If a compound that inhibits the receptor binding of the above endothelins is obtained, it will be useful not only for elucidating the mechanism of action of the endothelins but also as an effective therapeutic drug for the above diseases. It is considered highly likely.
The present invention provides ursonic acids as a non-peptidic low molecule having an endothelin receptor binding inhibitory action.

[0007]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have proceeded with the extraction, separation and purification of Chinese medicinal plants using the action of suppressing the strong vascular smooth muscle contractile activity of endothelins as an index, and They found that they have a remarkable inhibitory action on endothelin receptor binding, and completed the present invention. That is, the present invention provides the following formula (I):

[0008]

[Chemical 4] (In the formula, R ₁ and R ₂ each independently represent H or OH, provided that at least one of R ₁ and R ₂ is H, or R ₁ and R ₂ are taken together and are represented by the following formula: :

[Chemical 5] The present invention provides an endothelin receptor binding inhibitor containing the ursonic acid represented by or a pharmacologically acceptable salt thereof as an active ingredient. Ursonic acid (I ′), which is one of the compounds represented by the general formula (I):

[0009]

[Chemical 6] Is a triterpene compound, which can be obtained by separating and purifying from Chinese medicinal plants by a known method. Further, known methods (Khim. Farm. Zh., 20 , 568 (198
6) etc. ), Ursolic acid (R ₁ : OH,
R ₂ : H, Aldrich 30, 172-8) can be produced by oxidation. Also, R ₁ and R ₂ together form the following formula:

[0010]

[Chemical 7] The compound represented by can be prepared by reacting a compound represented by ═O for R ₁ and R ₂ with hydroxylamine, hydrazine, ethylene glycol or ethanedithiol. Also, R ₃ and / or R ₄
The compound in which R represents halogen can be produced by reacting a compound in which R ₃ and R ₄ combine to form a double bond with halogen or hydrogen halide, and R ₃ and R ₄ both represent H. The compound can be produced by hydrogenation.

In the present invention, the pharmacologically acceptable salt of ursonic acids means a salt with an inorganic base, a salt with an organic base,
Examples thereof include salts with amino acids. As an inorganic base,
Alkali metals such as lithium, sodium, potassium and the like and alkaline earths such as magnesium, calcium and the like. However, other metals such as aluminum, zinc, iron, etc. are also included in the invention. Examples of the organic base include amines such as ammonia, primary amine, secondary amine, tertiary amine, and nitrogen-containing heterocycle (including aliphatic and aromatic).

More specifically, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, isopropylamine, diisopropylamine,
Butylamine, dibutylamine, isobutylamine, t
-Butylamine, monoethanolamine, diethanolamine, triethanolamine, pyrrolidine, piperidine, morpholine, pyrrole, pyridine, and the like. Examples of amino acids include lysine, arginine, histidine and the like.

The present invention relates to an endothelin receptor binding inhibitor containing ursonic acids and salts thereof as an active ingredient. The endothelin receptor binding inhibitor is specifically a mammal (eg, human, dog, cat, rabbit, Rat, mouse, etc.), various diseases caused by endothelin, such as hypertension, angina, cardiomyopathy, arteriosclerosis, myocardial infarction, Raynaud's disease, cerebral artery spasm, cerebral ischemia, subarachnoid hemorrhage It can be used as a vasodilator for the treatment and prevention of aortic spasm, asthma, acute renal failure and the like.

The endothelin receptor binding inhibitor of the present invention can be administered orally or parenterally. Dosage forms include tablets, pellets, capsules, granules, emulsifiers, suspensions, injections and the like. These dosage forms are produced according to a standard method using appropriate carriers, excipients, and other additives as necessary.

The daily dose of the ursonic acid represented by the formula (I) varies depending on the condition and weight of the patient, the kind of the derivative, the administration route, etc., but, for example, parenterally,
About 0.01 to 20 subcutaneously, intravenously, intramuscularly, or intrarectally
mg / kg / day, preferably about 0.1-10 mg / kg / day. As an oral agent, about 0.1-100 mg / kg / day,
It is preferable to administer about 1 to 10 mg / kg / day. The ursonic acids of the present invention, which are non-peptidic low molecular weight compounds, can be administered orally without being decomposed compared with various peptides which have already been reported to have an inhibitory action on endothelin receptor binding. It has the advantage that it can be easily obtained from things.

[0016]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. The thin-layer chromatography plate used in the examples is a Merck product (Silica Gel 60).
F 254) was used.

Example . Isolation of ursonic acid [Xylanche] collected from Sichuan, China
himalica (Hook.f.et Thom
s) G. Beck (Orobanchaceae)] was dried and crushed, extracted with 95% ethanol at room temperature, and the obtained extract was concentrated under reduced pressure at 60 ° C. or lower. 30 g of this extract was separated into a soluble component and an insoluble component of ethyl acetate, and the soluble component (7.93 g) was subjected to column chromatography using 1 kg of silica gel to eluate 96-to-4 hexane-isopropanol. separated. The yield of active fraction is 3.44 g, and Rf =
It was 0.42 (hexane-isopropanol = 10: 1).

This fraction was further added with 180 g of silica gel, eluent hexane-isopropanol 97: 3 to 9
Separation by chromatography with a 0 to 10 step gradient. The yield of the active fraction was 3.19 g,
f = 0.38 (hexane-isopropanol = 90: 1)
It was 0). Further, this fraction was separated by chromatography using an eluent of 80:20 to 0: 100 hexane-ethyl acetate and 150 g of silica gel.
The active fraction yield was 257 mg, and Rf = 0.49 (hexane-ethyl acetate = 75: 25).

This fraction was diluted with dichloromethane-methanol 9
Separation by column chromatography using 25 grams of silica gel with a 9: 1 eluent. The active fraction yield was 101 mg, and Rf = 0.69 (dichloromethane-methanol = 95: 5). Finally HPLC (S
himadzu LC-6A system, Merc
k Hibar RT250-25 LiChroso
rb Si60 7 μm, 254 nm, 9.9 ml / min,
The active fraction was purified at 20 ° C. and hexane-isopropanol = 95: 5) to isolate ursonic acid as an endothelin receptor binding inhibitor. The yield was 16.7 mg, Rf = 0.69 (dichloromethane-methanol = 9
It was 5: 5).

Next, the pharmacological action of the ursonic acid of the present invention will be described. Receptor binding test (1) Endothelin receptor preparation Rat fetal thoracic aorta-derived cell line A10 (Bmax:
20.3 PM, ATCC CRL 1476) was used as a receptor cell membrane preparation, and a measurement kit (manufactured by DuPont, USA) was used. According to the measurement manual, dilute the solution (CaCl ₂ , BSA, Bacitr) into 5 ml of A10 cell suspension.
38 ml of 50 mM Tris buffer (pH 7.4) containing acin was added to prepare a diluted receptor preparation.

(2) ¹²⁵ I-endothelin binding experiment ¹²⁵ I-endothelin-1 (8) as a radioligand
1.4 TBq / mmol, DuPont, USA) 8.5 KBq
175 μl of receptor dilution standard and 25 μl of sample were added. The amount of non-specific binding is 1 × 10 ^-6 M
Endothelin-1 was added and measured. The reaction is 250
It was performed at room temperature for 2 hours in μl of Tris buffer.

After the reaction, rapid filtration was performed on a glass filter (GF / B, Whatman, USA) using a cell harvester (M-30, Brandel, USA), and 5
After washing 3 times with 0 mM Tris buffer (pH 7.4), the radioactivity remaining on the filter was measured with a gamma counter. As a result, the binding of endothelin-1 to the receptor was 5
The concentration at which 0% inhibition (IC ₅₀ ) was about 5 μg / ml.

[0023]

INDUSTRIAL APPLICABILITY The present invention can provide an endothelin receptor binding inhibitor containing ursonic acid (I) or a pharmacologically acceptable salt thereof as an active ingredient. The receptor binding inhibitor is useful as a therapeutic and prophylactic agent for hypertension, angina, cardiomyopathy, arteriosclerosis, myocardial infarction, vasospasm, asthma, acute renal failure and the like.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 31/705 ACV AED C07J 63/00 9051-4C // C07C 62/24 9450-4H 251/44 251/84 (72) Inventor Din Li Shen, People's Republic of China, Suitsang Sen, Cheng Dous, Renmin Nanru 4-9 (72) Inventor Norio Kawabe 1111 Tehiro, Kamakura City, Kanagawa Prefecture Toray Industries Co., Ltd. Basic Research Laboratory (72) Invention Toshi Togami, 1111 Tehiro, Kamakura City, Kanagawa Prefecture, Toray Co., Ltd.

Claims (1)

[Claims] 1. The following formula (I): (In the formula, R ₁ and R ₂ each independently represent H or OH, provided that at least one of R ₁ and R ₂ is H, or R ₁ and R ₂ are taken together and are represented by the following formula: : [Chemical 2] R ₃ and R ₄ independently of each other represent H or halogen, or R ₃ and R ₄ combine to form a double bond between the carbon atoms to which they are attached). An endothelin receptor binding inhibitor comprising an ursonic acid or a pharmacologically acceptable salt thereof as an active ingredient.