JP5376418B2 - Arteriosclerosis preventive drugs containing brassinosteroid derivatives - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new arteriosclerosis-preventing agent. <P>SOLUTION: This arteriosclerosis-preventing agent comprises a compound expressed by general formula (1) [wherein, R1 is a hydrogen atom, a methyl group, an ethyl group, a propyl group or the like; R2 to R5 are each independently selected from the group consisting of a hydrogen atom, a lower alkyl group and an acyl group; X is selected from the group consisting of -(CH)2-, -(CH)3, -CO-CH2-, -CO-(CH2)2-, -CH2-CO-CH2, -O-CO-CH2 and the like; and Y is a hydrogen atom or a lower alkyl group]. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a prophylactic agent for arteriosclerosis comprising a brassinosteroid derivative.

  In many countries including Japan and Europe and America, diseases caused by arteriosclerosis such as cerebral infarction and myocardial infarction account for most of the causes of death. Among arteriosclerosis, atherosclerosis (atherosclerosis), in which foam cells derived from macrophages form plaques in the intima, has a high onset frequency and is regarded as a particular problem. In the onset of atherosclerosis, oxidized LDL (ox-LDL) commonly called “bad cholesterol” is deeply involved. When LDL that has entered the vascular intima undergoes oxidative modification to become ox-LDL, macrophages take up oxidized LDL into cells via scavenger receptors in order to eliminate this. Eventually, the macrophages become foam cells containing a large amount of lipid droplets, leading to the deposition of lipids in the intravascular subcutaneous space, resulting in arteriosclerosis.

  Plant-derived components are known which are effective in preventing arteriosclerosis mainly by antioxidant action. Among them, isoflavones, which are contained in a large amount in soybeans, are widely known to be effective in promoting health including prevention of arteriosclerosis because of their antioxidant action. However, isoflavones, on the other hand, are thought to bind to the estrogen receptor and mimic its action due to its three-dimensional structural similarity to the female hormone estrogen (estrus hormone). Therefore, it is pointed out that there is a possibility of miscarriage if pregnant women and so on ingest a large amount. Estrogen is also known as a causative agent of breast cancer and uterine cancer, and it is necessary to recognize that it is dangerous to take it indiscriminately.

  Brassinosteroids are physiologically active substances unique to plants that play important roles in plant development, chloroplast control, seed formation, and the like. Although the basic structure having a steroid skeleton is similar to that of animal steroid hormones, it is considered to exhibit different physical properties from steroid hormones due to the structural features rich in hydroxyl groups. In fact, brassinosteroids and steroid hormones differ greatly in the molecular structure and information transmission mechanism of the receptors. Animal steroid hormones are mainly produced in endocrine organs such as the adrenal glands and gonads, and act on remote target organs through the bloodstream to act on reproduction, sex differentiation, sugar metabolism, electrolyte metabolism, and so on. The steroid hormone receptor is a transcription factor type receptor present in the cytoplasm of the target cell. When the hormone binds, it moves into the nucleus and directly acts on the transcription of the target gene. On the other hand, brassinosteroid receptors are membrane-bound receptors on the cell membrane, and their information is transferred into the cell by Ser / Thr kinase in the cytoplasmic domain phosphorylating transcription factors. To do. Therefore, brassinosteroids have been thought to have no physiological effect on animal cells.

  An object of the present invention is to provide a novel preventive agent for arteriosclerosis.

  In order to investigate the possibility that brassinosteroids known as plant sexually active substances suppress the onset of atherosclerosis, brassinosteroids against macrophage proliferation induced by ox-LDL The effect of was examined. As a result, it was shown that brassinosteroid and its derivatives can suppress the growth of macrophages induced by ox-LDL and suppress the onset of atherosclerosis. Unlike dexamethasone, a synthetic glucocorticoid known as a potent immunosuppressant, brassinosteroids have been shown not to inhibit the production of NO associated with macrophage activation and cytotoxic activity against cancer cells. From the above results, it was shown that brassinosteroid has an effect of suppressing the onset of arteriosclerosis without affecting the cellular immune mechanism of macrophages. The present invention has been completed based on these findings.

According to the present invention, a prophylactic agent for arteriosclerosis comprising a compound represented by the following formula (1) is provided.
Wherein R ¹ is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a 2-methylpropyl group, a 1,2-dimethylpropyl group, and 1-ethyl-2-methyl. R ^{2 to} R ⁵ are independently selected from the group consisting of hydrogen, a lower alkyl group, and an acyl group, X is — (CH) ₂ —, — (CH ) ₃ -,-(CH) ₄ , -CO-CH _2- , -CO- (CH ₂ ) _2- , -CO- (CH ₂ ) _3- , -CH ₂ -CO-CH _2- , -CO- O-CH _2- , -CO-O- (CH ₂ ) _2- , -O-CO-CH _2- , -O-CO- (CH ₂ ) _2- , -CH ₂ -O-CH ₂ -,- _{CH 2 -O- (CH 2) 2} -, - O-CH 2 -, - O- (CH 2) 2 -, - O- (CH 2) 3 -, - NHY-CO-CH 2 -, - NHY Selected from the group consisting of —CO— (CH ₂ ) ₂ —, —CO—NHY—CH ₂ —, and —CO—NHY— (CH ₂ ) ₂ —, wherein Y represents hydrogen or a lower alkyl group.

Furthermore, according to this invention, the macrophage growth inhibitor containing the compound shown by following formula (1) is provided.
Wherein R ¹ is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a 2-methylpropyl group, a 1,2-dimethylpropyl group, and 1-ethyl-2-methyl. R ^{2 to} R ⁵ are independently selected from the group consisting of hydrogen, a lower alkyl group, and an acyl group, X is — (CH) ₂ —, — (CH ) ₃ -,-(CH) ₄ , -CO-CH _2- , -CO- (CH ₂ ) _2- , -CO- (CH ₂ ) _3- , -CH ₂ -CO-CH _2- , -CO- O-CH _2- , -CO-O- (CH ₂ ) _2- , -O-CO-CH _2- , -O-CO- (CH ₂ ) _2- , -CH ₂ -O-CH ₂ -,- _{CH 2 -O- (CH 2) 2} -, - O-CH 2 -, - O- (CH 2) 2 -, - O- (CH 2) 3 -, - NHY-CO-CH 2 -, - NHY Selected from the group consisting of —CO— (CH ₂ ) ₂ —, —CO—NHY—CH ₂ —, and —CO—NHY— (CH ₂ ) ₂ —, wherein Y represents hydrogen or a lower alkyl group.

Preferably R ¹ is a 1,2-dimethylpropyl group.
Preferably, R ^{2 to} R ⁵ are hydrogen atoms.
Preferably, X is -CO-O-CH _2-, -CO-CH _2- , -CO- (CH ₂ ) _2- , -CO- (CH ₂ ) _3- , -O-CO-CH _2- , Or —CH ₂ —CO—CH ₂ —.

Preferably, the compound represented by the formula (1) is any one of the following compounds.

Preferably, the medicament of the present invention can be used as a health food or food additive.
According to another aspect of the present invention, there is provided a health food or food additive containing the above-mentioned preventive agent for arteriosclerosis.

  It has been shown that the brassinosteroids used in the present invention do not activate animal steroid hormone receptors. Therefore, brassinosteroids used in the present invention do not have to worry about side effects, unlike existing steroidal agents and steroid mimetics such as isoflavones (which can be called environmental hormones), and are ideal health foods and treatments. It can be a medicine. Also, brassinosteroids do not inhibit the production of anticancer substances by macrophages and do not inhibit the cytotoxic activity, so unlike the case of using dexamethasone, the cells do not suppress the host's cellular immunity mechanism and enter macrophage foam cells. It is expected to have an effect of preventing the development of arteriosclerosis.

Hereinafter, embodiments of the present invention will be described in more detail.
Explanation of terms:
Arteriosclerosis refers to lesions including atherosclerosis or atherosclerosis and the like, which are mainly characterized by a decrease in vascular function associated with arterial thickening and lipid deposition. Atherosclerosis causes acute coronary syndromes such as angina and myocardial infarction.
Low density lipoprotein (LDL) is a low density lipoprotein that increases subcutaneously in the blood vessels with hyperlipidemia.
Oxidized LDL is bad cholesterol.
Macrophages (macrophages) are white blood cells that play an important role in the innate immune mechanism that prey on bacteria and viruses that have entered the body.
Foam cells are macrophages that have taken up oxidized LDL and foamed, and appear in early arteriosclerotic lesions.

As described in the Examples described below, the present inventors administered 3% TGC to BALB / c mice and adjusted macrophages from the abdominal cavity three days later. 1 × 10 ⁵ / ml and 0.1 ml / well were cultured in 96-well-plate for 3 hours, and the cells attached to the plate were used for the experiment. The cells were cultured for 6 days in the presence of ox-LDL (50 μg / ml) and Brassinosteroids (0.2 to 20 μM), and the effect of Brassinosteroids on macrophage proliferation was examined by MTT assay. As a result, it was shown that Brassinosteroids and derivatives thereof suppress the proliferation of macrophages induced by ox-LDL and suppress the onset of atherosclerosis. Brassinosteroids, unlike Dexamethasone, a kind of synthetic glucocorticoid known as a potent immunosuppressant, has been shown not to inhibit the production of NO associated with macrophage activation and cytotoxic activity against cancer cells. From the above results, it was demonstrated that Brassinosteroids has an effect of suppressing the onset of arteriosclerosis without affecting the cellular immune system that macrophages play. For suppression of macrophage proliferation induced by ox-LDL, Takeshi Biwa et al, The Journal of Biological Chemistry, Vol. 275, No. 8, pp5810-5816, 2000, and Masakazu Sakai et al., The Journal of Biological Chemistry, Vol.271, No.44, pp27346-27352, 1996, and the like.

In the arteriosclerosis preventive agent and macrophage growth inhibitor of the present invention (in the present application, these are collectively referred to as the drug of the present invention), a compound represented by the following formula (1) is used as an active ingredient.
Wherein R ¹ is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a 2-methylpropyl group, a 1,2-dimethylpropyl group, and 1-ethyl-2-methyl. R ^{2 to} R ⁵ are independently selected from the group consisting of hydrogen, a lower alkyl group, and an acyl group, X is — (CH) ₂ —, — (CH ) ₃ -,-(CH) ₄ , -CO-CH _2- , -CO- (CH ₂ ) _2- , -CO- (CH ₂ ) _3- , -CH ₂ -CO-CH _2- , -CO- O-CH _2- , -CO-O- (CH ₂ ) _2- , -O-CO-CH _2- , -O-CO- (CH ₂ ) _2- , -CH ₂ -O-CH ₂ -,- _{CH 2 -O- (CH 2) 2} -, - O-CH 2 -, - O- (CH 2) 2 -, - O- (CH 2) 3 -, - NHY-CO-CH 2 -, - NHY Selected from the group consisting of —CO— (CH ₂ ) ₂ —, —CO—NHY—CH ₂ —, and —CO—NHY— (CH ₂ ) ₂ —, wherein Y represents hydrogen or a lower alkyl group.

  The lower alkyl group referred to in the present invention is preferably an alkyl group having 1 to 6 carbon atoms, more preferably an alkyl group having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, a propyl group, or a butyl group. These may be linear or branched.

  The acyl group referred to in the present invention is preferably an acyl group having 1 to 6 carbon atoms, more preferably an acyl group having 1 to 4 carbon atoms, and examples thereof include a formyl group, an acetyl group, a propionyl group, and a butyryl group. .

  The compound represented by the formula (1) can be synthesized according to a known method described in the literature or a method analogous thereto as described below.

(A) R ¹ is a 1,2-dimethylpropyl group, R ^{2 to} R ⁵ are hydrogen atoms, and X is —CO— (CH ₂ ) ₂ —, —CO— (CH ₂ ) ₃ —, or The compound of formula (1), which is —CH ₂ —CO—CH ₂ —, is Preparation, conformational analysis and biological evaluation of 6a-carbabrassinolide and related compounds: H. Seto, S. Hiranuma, S. Fujioka, H. Koshino, It can be synthesized according to the method described in T. Suenaga and S. Yoshida, Tetrahedron, 58, 9741-9749 (2002).

(B) R ¹ is a hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, tert-butyl group, 2-methylpropyl group, 1,2-dimethylpropyl group, and 1-ethyl-2-methylpropyl group R ^{2 to} R ⁵ are hydrogen atoms, and X is —CO— (CH ₂ ) ₂ or —CO—O— (CH ₂ ) ₂ , —O—CO— (CH) ₂ — The compound of the formula (1) selected from the group consisting of can be synthesized according to the method described in the following literature.
(i) Synthesis of (24R) -28-homobrassinolide analogues and structure-activity relationships of brassinosteroids in the rice-lamina inclination test: S. Takatsuto, N. Yazawa, N. Ikekawa, T. Morishita and H. Abe, Phytochemistry, 22, 1393-1397 (1983).
(ii) Synthesis and biological activity of brassinolide analogues, 26,27-bisnorbrassinolide and its 6-oxo analogue: S. Takatsuto, N. Yazawa and N. Ikekawa, Phyrochemistry, 23, 525-528 (1984).
(iii) Synthesis of brassinosteroids and relationship of structure to plant growth-promoting effects: MJ Thompson, WJ Meudt, NB Manava, SR Dutky, WR Lusby and DW Spaulding, Steroids, 39, 89-105 (1982).

(C) X is — (CH) ₂ —, — (CH) ₃ —, — (CH) ₄ , —CO—CH ₂ —, —CO— (CH ₂ ) ₂ —, —CO— (CH ₂ ) ₃ -, -CH ₂ -CO-CH _2- , -CO-O-CH _2- , -CO-O- (CH ₂ ) _2- , -O-CO-CH _2- , -O-CO- (CH _{2 ) 2 -, - CH 2 -O} -CH 2 -, - CH 2 -O- (CH 2) 2 -, - O-CH 2 -, - O- (CH 2) 2 -, - O- (CH 2 ) _3- , -NHY-CO-CH _2- , -NHY-CO- (CH ₂ ) _2- , -CO-NHY-CH _2- , and -CO-NHY- (CH ₂ ) _2- The compound of the formula (1), which is selected and Y represents hydrogen or a lower alkyl group, can be synthesized according to the method described in the following literature.
(i) structure activity studies of brassinolide B-ring analogues: DL Baron, W. Luo, L. Janzen, RP Pharis and TG Back, Phyrochemistry, 49, 1849-1858 (1998).
(ii) Brassinosteroid activity of brassinolide B ring structural change: Sayoko Hiranuma, Hideharu Seto, Shozo Fujioka, Makoto Kobayashi, Shigeo Yoshida, Proceedings of the 36th Annual Meeting of the Society of Plant Chemistry Regulation, pp. 13- 14, 2001.

(D) The compound of the formula (1) in which R ^{2 to} R ⁵ are independently selected from the group consisting of a hydrogen atom, an alkyl group, and an acyl group can be synthesized according to the method described in the following document. it can.
(i) Bioactivity of brassinolide methyl esters: W. Luo, L. Janzen, RP Pharis and TG Back, Phyrochemistry, 49, 637-642 (1998).
(ii) Synthesis and bioactivity of C-2 and C-3 methyl ester derivatives of brassinolide: TG Back, L. Janzen, RP Pharis, Z. Yan, Phyrochemistry, 59, 627-634 (2002).

  Specific examples of arteriosclerosis to be prevented in the present invention include, but are not limited to, atherosclerosis or atherosclerosis. Preferred is atherosclerosis.

  The dosage when the drug of the present invention is administered to a patient can be appropriately set according to the age, weight, symptom, etc. of the patient. 1000 mg / kg body weight, particularly 1 to 100 mg / kg body weight can be administered in 1 to several divided doses.

  The administration route of the drug of the present invention is not particularly limited, and for example, it can be administered by oral administration or parenteral administration such as injection. In the case of administration by injection, intravenous injection, arterial injection, subcutaneous injection, intradermal injection, intraperitoneal injection, intramuscular administration, and the like can be performed.

  The drug of the present invention can contain additives usually used in pharmaceutical compositions in addition to the compound represented by the formula (1) contained as an active ingredient. Such optional additives include excipients, binders, disintegrants, emulsifiers, solubilizers, dispersants, lubricants, coating agents, colorants, stabilizers, isotonic agents, and the like. However, it is not limited to these. As an acupuncture agent, saccharides such as lactose, sucrose, glucose etc., inorganic substances such as starch, calcium carbonate, calcium sulfate, crystalline cellulose, distilled water, purified water, sesame oil, soybean oil, corn oil, olive oil, cottonseed oil, etc. What is being done can be illustrated. The drug of the present invention can be formulated by a conventional method using these additives. Moreover, the chemical | medical agent of this invention can be mixed and used together with other pharmaceuticals (for example, other arteriosclerosis preventive drugs etc.), and can also be used together.

  The compound represented by the formula (1) of the present invention can also be used by adding to foods and the like. For example, the compound represented by the formula (1) may be directly added to the food to make a health food, or the compound represented by the formula (1) may be added to the fat used as a raw material of the food, It is also possible to produce food.

  Specific examples of health food include beverages such as confectionery such as cookies and yogurt, frozen confectionery such as ice cream, tea, soft drinks (including juice, coffee, cocoa, etc.), nutritional drinks, and beauty drinks. , Any food and drink such as bread, ham, soup, jam, spaghetti and frozen food. It can be expected that the effect of preventing arteriosclerosis will be exhibited by ingesting the health food of the present invention.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

(1) Preparation and culture of mouse intraperitoneal macrophages
3% thioglycolate medium (Difco) was intraperitoneally administered to BALB / c mice. After 3 days, intraperitoneal cells were collected, 10% fetal bovine serum and penicillin (20 U / ml), streptomycin (100 μg / ml) in an RPMI-1640 medium at 37 ° C. in the presence of 5% CO 2. After 3 hours of culture, non-adherent cells were removed, and the adherent cells were subjected to experiments as macrophages.

(2) Cell proliferation test The influence of brassinosteroid on the proliferation of macrophages by ox-LDL stimulation was examined using MTT assay. Macrophages brassinolide (see Figure 1), castasterone (see Figure 1), or various brassinosteroids in the presence of a steroid derivative (see A in FIG. 3) 96-well-plate ( 1 × 10 4 / well). After 5 days of culture, 1 mg / ml of MTT was added. After 4 hours, the medium was removed, and 04N hydrochloric acid-added isopropanol was added to dissolve formazan, and OD570 nm was measured.

FIG. 1 shows the results of an experiment conducted using brassinolide and castasterone. FIG. 1A shows the chemical structures of brassinolide and castasterone, which are plant steroid hormones. FIG. 1B shows the effect of brassinosteroids on macrophage proliferation induced by ox-LDL. TGC-induced intraperitoneal macrophages (1 × 10 ⁴ cells) were isolated from ox-LDL (in the presence of brassinolide (Bra) or castasterone (Cas) at the indicated concentrations (0.2, 2 and 20 μM). (50 μg / ml) and cultured for 6 days, and cell proliferation was measured by MTT assay. Data are shown as mean OD575nm + SD. **, p <0.001

The results of experiments conducted using various brassinosteroid derivatives are shown in FIG. FIG. 3A shows the chemical structure of the brassinosteroid derivative. FIG. 3B shows the effect of brassinosteroid derivatives on macrophage proliferation induced by ox-LDL. TGC-induced intraperitoneal macrophages (1 × 10 ⁴ cells) were combined with ox-LDL (50 μg / ml) in the presence of brassinosteroid derivatives at the indicated concentrations (0.2, 2 and 20 μM). Cultured for days, cell proliferation was measured by MTT assay. Data are shown as mean OD575nm + SD. * And ** indicate p <0.01 and p <0.001, respectively.

(3) Action of brassinosteroid on cellular immune activity of macrophages To investigate the effect of brassinosteroid on NO production by macrophages, LPS stimulation (100 ng / ml) was performed in the presence of 20 μM brassinosteroid or dexamethasone. Then, the NO2 concentration in the supernatant for 24 hours of culture was analyzed by “Griess Reagent System” manufactured by Promega. In addition, in order to examine the effect of brassinosteroid on the cytotoxic activity of macrophages, macrophages (2 × 10 ⁶ / ml) were treated with IFN-γ (100 U / ml) in the presence of brassinosteroids or dexamethasone (20 μM). Stimulated with. After 24 hours, the cells were washed, and HeLa cells (5 × 10 ⁵ ) were added and cultured for 24 hours. The cytotoxic activity was measured using the “Cytotoxicity detection kit” manufactured by Roche. It was measured.

The results are shown in FIG. FIG. 2 (A) shows the results of comparing the effect of brassinosteroid on NO production in mouse intraperitoneal macrophages with dexamethasone. TGC-induced intraperitoneal macrophages (1 × 10 ⁶ cells) were combined with LPS (100 ng / ml) in the presence of 20 μM brassinolide (Bra), castasterone (Cas), or dexamethasone (Dex). Cultured for 48 hours. NO ²⁻ in the culture supernatant was used as an index for NO production and measured using Griess reagent. FIG. 2 (B) shows the effect of brassinosteroids and dexamethasone on the cytotoxic activity of macrophages. Macrophages were cultured for 48 hours with IFN-γ (100 U / ml) and brassinosteroids or dexamethasone (20 μM). The cells were washed 3 times with medium and co-cultured with HeLa cells (5 × 10 ⁵ cells) for 24 hours. The cytotoxic activity of macrophages was measured by measuring LDH activity in the culture supernatant. Results are expressed as mean + SD. **, p <0.001.

FIG. 1 shows the antiproliferative effect of brassinosteroids on mouse peritoneal macrophage proliferation induced by ox-LDL. FIG. 2 shows the non-inhibitory effect of brassinosteroids on macrophage tumoricidal activity. FIG. 3 shows the antiproliferative effect of brassinosteroid derivatives on the proliferation of mouse intraperitoneal macrophages induced by ox-LDL.

Claims (5)

A macrophage growth inhibitor comprising a compound represented by the following formula (1).
Wherein R ¹ is a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a 2-methylpropyl group, a 1,2-dimethylpropyl group, and 1-ethyl-2-methyl. R ^{2 to} R ⁵ are independently selected from the group consisting of hydrogen, a lower alkyl group, and an acyl group, X is — (CH) ₂ —, — (CH _{) 3 -, - (CH)} 4 -, - CO - CH 2 -, - CO - (CH 2) 2 -, - CO - (CH 2) 3 -, - CH 2 -CO-CH 2 -, -CO--O-CH _2- , -CO--O- (CH ₂ ) _2- , -O-CO-CH _2- , -O-CO- (CH ₂ ) _2- , -CH _{2- -O-CH 2 -, - CH} 2 -O- (CH 2) 2 -, - O-CH 2 -, - O- (CH 2) 2 -, - O- (CH 2) 3 -, - NHY- Selected from the group consisting of CO—CH ₂ —, —NHY—CO— (CH ₂ ) ₂ —, —CO—NHY—CH ₂ —, and —CO—NHY— (CH ₂ ) ₂ —, where Y is hydrogen Alternatively, it represents a lower alkyl group.) The macrophage growth inhibitor according to claim 1, wherein R ¹ is a 1,2-dimethylpropyl group. The macrophage growth inhibitor according to claim 1 or 2, wherein R ^{2 to} R ⁵ are hydrogen atoms. X is -CO-O-CH _2-, -CO-CH _2- , -CO-(CH ₂ ) _2- , -CO-(CH ₂ ) _3- , -O-CO-CH _2- , or The macrophage growth inhibitor according to any one of claims 1 to 3, which is -CH ₂ -CO-CH _2- . The macrophage growth inhibitor according to any one of claims 1 to 4, wherein the compound represented by the formula (1) is any one of the following compounds.