JPH08310949A - Acyl Coenzyme A: Cholesterol Acyltransferase Inhibitor - Google Patents

Abstract

(57) [Summary] [Structure] An acylcoenzyme A: cholesterol acyltransferase inhibitor, a blood cholesterol lowering agent, and a prophylactic / therapeutic agent for arteriosclerosis, which comprises the piperidine derivative of the formula (1) as an active ingredient. Embedded image [Effect] Since it has a strong ACAT inhibitory activity and high safety, it lowers blood cholesterol and suppresses the accumulation of cholesterol, and thus is useful as a prophylactic and therapeutic agent for human arteriosclerosis.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an acylcoenzyme A: cholesterol acyltransferase (hereinafter referred to as ACAT) inhibitor and a blood cholesterol lowering agent which are useful for preventing arteriosclerosis.

[0002]

2. Description of the Related Art With the westernization of eating habits, the frequency of ischemic heart disease in Japan has remarkably increased. Many epidemiological studies have clarified the relationship between blood cholesterol levels and arteriosclerosis that are elevated by Western diets. That is, there is a significant correlation between the serum cholesterol level and the occurrence frequency of ischemic heart disease, and it is said that treating hypercholesterolemia prevents arteriosclerosis and eventually ischemic heart disease. Arteriosclerosis is a lesion characterized by thickening of the intima due to lipid accumulation in blood vessels. Recently, macrophage-derived cells store foam of cholesterol ester as lipid droplets in the lesion of atherosclerosis. It is estimated that cells are observed and are deeply involved in the development of lesions.

On the other hand, ACAT is a fatty acid esterifying enzyme of cholesterol, and an inhibitor of this enzyme is expected as a therapeutic drug for arteriosclerosis (Drago R. et.
al. , Tips 194, (12), 1991). That is, it has been reported that ACAT activity is high in the blood vessel wall at the site of arteriosclerotic lesion, and cholesterol ester is accumulated in the blood vessel wall. Therefore, if a substance having an activity of suppressing ACAT activity is used, it is possible to inhibit esterification of cholesterol, and free cholesterol present in cells is converted to high density lipoprotein (HDL) by
Since it is removed from the deposition site, transported to the liver and metabolized, the accumulation of cholesterol ester at the lesion site is suppressed and a direct anti-atherogenic effect can be obtained.

Cholesterol contained in the diet is esterified by ACAT in small intestinal mucosal cells and then released into the bloodstream as a chylomicron component. Therefore, by using the ACAT inhibitor, blood cholesterol can be lowered by inhibiting the absorption of dietary cholesterol in the intestinal epithelial cells, and as a result, arteriosclerosis can be suppressed.

Cholesterol ester carried to the liver by chylomicrons is decomposed into free cholesterol by cholesterol esterase, and then converted into cholesterol ester by ACAT together with free cholesterol synthesized in the liver, and ultra-low-density lipoprotein ( It is incorporated into VLDL) and released into the blood.
Therefore, if the esterification of cholesterol is blocked by such an ACAT inhibitor, the amount of free cholesterol in the liver is increased, the cholesterol synthesis system is suppressed, and 7α, which is a rate-limiting enzyme in the process of converting cholesterol into bile acid, is involved. -Hydroxylase activity is enhanced and the catabolic excretion of cholesterol to bile acids is enhanced.

[0006] Thus, if an ACAT inhibitor is used,
By inhibiting the absorption of cholesterol in the diet from the small intestine, it also suppresses the re-esterification of cholesterol in the liver, and can further reduce blood cholesterol by the action of promoting catabolism and excretion of cholesterol, thereby improving arteriosclerosis. It can be prevented or treated.

From such a background, search for ACAT inhibitors has been conducted, and some ACAT inhibitors produced by microorganisms are known, but these substances are still satisfactory in view of both action and safety. It wasn't possible.

[0008]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a pharmaceutical composition which has a high ACAT inhibitory activity, has no safety problems, and is useful as a blood cholesterol lowering agent and an agent for preventing and treating arteriosclerosis. is there.

[0009]

Under such circumstances, the present inventors have continued to search for an active ingredient having an ACAT activity inhibitory action in the plant ingredients, and as a result, pepper (Piper nigrum L. ) Unripe fruit of dried black pepper and matured fruit with outer pericarp removed to dry white pepper, or unripe fruit of lyophilized green pepper and pepper pepper plant (Piper longu)
m L. It was found that the alcohol extract of (1) has an ACAT inhibitory action and a cholesterol ester accumulation inhibiting action and is useful as a medicine. As a result of further isolating and purifying a substance having an ACAT inhibitory activity from the extract, it was found that the active substance was a piperidine derivative represented by the following formula (1), and the present invention was completed.

That is, the present invention uses the following equation (1)

[0011]

[Chemical 4]

The present invention provides an acylcoenzyme A: cholesterol acyltransferase inhibitor containing a piperidine derivative represented by the following as an active ingredient.

The present invention also provides a blood cholesterol lowering agent containing the piperidine derivative (1) as an active ingredient.

Furthermore, the present invention provides a preventive / therapeutic agent for arteriosclerosis containing the above-mentioned piperidine derivative (1) as an active ingredient.

The piperidine derivative (1) has four stereoisomers based on two ethenylene groups, and any stereoisomer or a mixture thereof can be used in the present invention. You can Of these isomers, the trans-trans type compound is known as piperine, and the cis-cis type compound is known as xavicin. Of these, piperine is particularly preferable.

The piperidine derivative (1) used in the present invention can be chemically synthesized by a known method, or can be extracted from a plant belonging to the pepper family.

The plant used for the extraction of the piperidine derivative (1) is not particularly limited as long as it belongs to the pepper family, but pepper (Piperis nigri fru)
ctus) and hihatsu (Piperis longi)
fructus) is particularly preferred.

The pepper is a creeping evergreen woody plant native to Southeast Asia and is cultivated in various places such as Southeast Asia and South America. The stem is about 10m long, roots are laid out at each node and wrapped around other things, and the leaves are oval, dark green, and smooth leathery. The flower season is from April to October. The inflorescences are spike-shaped and the flowers are yellow-green. The fruits are spherical and dioecious.
Green pepper is freeze-dried on the immature fruit, black pepper is sun-dried, and white pepper is on the shade after removing the pericarp of the mature fruit. Pepper is generally used as a folk medicine against dyspepsia, diarrhea, etc. by stimulating the gastrointestinal and activating peristaltic movement, promoting sweating, and is also used mainly as a spice in Western dishes and is also effective for healthy stomach. But AC
Nothing is known about AT inhibitory action.

From these plants, piperidine derivatives (1)
In order to collect the product, for example, first, a polar organic solvent such as alcohol represented by methanol is extracted, and the obtained extract is concentrated under reduced pressure. Then, the concentrate was layered on a silica gel column (internal diameter 5 cm, length 30 cm) and eluted with a mixed solvent of chloroform and methanol to obtain ACA.
It is preferable to fractionate the T-inhibiting active ingredient. Further, the active fraction can be dried under reduced pressure to obtain a crude active substance. Further, the active fraction can be purified by preparative thin layer chromatography.

In the present invention, as long as it contains the piperidine derivative (1), the alcohol-extracted fraction of the above pepperaceous plant may be used as it is, or a more purified fraction may be used.

The piperidine derivative has an excellent ACAT inhibitory action and intracellular cholesterol ester accumulation inhibitory action as shown in the Examples below, and is extremely safe since it is derived from foods. It is useful as a prophylactic / therapeutic agent for arteriosclerosis.

The drug of the present invention is a piperidine derivative (1).
Administered alone or together with other medicinal components (for example, anti-arteriosclerotic drugs having other mechanisms of action), and optionally orally or parenterally such as injection with a pharmaceutically acceptable carrier. However, it is preferably administered orally.

Examples of oral preparations include tablets, capsules, granules, powders, troches and syrups. In solid compositions such as tablets, capsules, granules, powders, troches, etc., excipients such as starch, lactose, carboxymethylcellulose, precipitated calcium carbonate; gum arabic, tragacanth, gelatin, methylcellulose, carboxymethylcellulose, etc. A binder such as alginic acid and corn starch; a lubricant such as magnesium stearate, hydrous silicon dioxide and light anhydrous silicic acid; a sweetener such as saccharose; and a flavoring agent such as menthol. In liquid compositions such as syrups, sorbitol, gelatin, methyl cellulose, vegetable oil,
In addition to an emulsifier, a sweetening agent, a flavoring agent, a coloring agent and the like can be contained. 1 to 95% by weight in these formulations
It is desirable to contain the piperidine derivative (1).

The dose of the drug of the present invention varies depending on the symptoms, body weight, administration route, etc., but in the case of oral administration, it is 10 to 1,5 per adult per day as the piperidine derivative (1).
It is preferable to administer 00 mg in 1 to several divided doses.

[0025]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Production Example 1 (1) 500 g of green pepper was cold-extracted with 10 L of methanol for 5 days at room temperature. The extract was filtered, concentrated under reduced pressure and dried to give 77.1 g of crude extract. This crude extract was then passed through a silica gel column (inner diameter 5 cm, length 90 c).
m) was layered and all the components eluted with chloroform were eluted, and then extracted with a solvent of chloroform: methanol (98: 2) to fractionate the ACAT inhibitory active component.

The eluted liquid was collected in 150 ml portions, and the active fraction was dried under reduced pressure to obtain 12.8 g of crude active substance. Next, silica gel column chromatography (5 cmφ x 6
(0 cm) with the whole crude extract, 600 ml of chloroform was eluted, and then 95% chloroform: methanol, 90
% Chloroform: methanol and 80% chloroform:
The ACAT inhibitory activity fraction was collected by sequentially eluting with 600 ml of methanol each. The amount of crude active substance obtained was 9.8 g.

A component showing an ACAT inhibitory activity in 1000 mg of this crude active substance was further analyzed by preparative thin layer chromatography (made by Merck: Silica gel 60F ₂₅₄ Art.5) using chloroform: methanol (98: 2) as a developing solvent.
Purification was carried out according to 717). After confirming the active ingredient by UV absorption, the fraction was scraped off, eluted from the silica gel with chloroform and then concentrated to obtain 480 mg of crude crystals.

Next, the total amount of this crude crystal substance was analyzed by high performance liquid chromatography (System 600 manufactured by Waters Co., Ltd.).
E) was used for elution with a reversed phase C ₁₈ column and a solvent system of acetonitrile: water (50:50) to obtain 288 mg of crystals. Finally, the crystals were recrystallized in ethyl acetate to obtain 170 mg of young greenish yellow crystals.
Its purity was 9 by analysis by high performance liquid chromatography.
It was 5% or more.

The physicochemical properties of the obtained crystal were as follows.

[Table 1] (1) Molecular formula: C ₁₇ H ₁₉ O ₃ N (2) Molecular weight: 285 (m / z 2 by mass spectrum)
85 (M ⁺ ) was observed. (3) Ultraviolet absorption spectrum (in ethanol): As shown in FIG. (4) Infrared absorption spectrum (in KBr): As shown in FIG. (5) Solubility in solvent: methanol, ethanol,
Soluble in acetonitrile, ethyl acetate, chloroform, benzene, insoluble in water. (6) Distinction between basic, acidic and neutral: neutral (7) Color and shape of substance: pale yellowish green, powder (8) Proton nuclear magnetic resonance spectrum (in deuterated dimethyl sulfoxide): As shown in FIG. . ¹ H-NMR (DMSO-d ₆ , 400MHz, 37c) δ: 1.46 (4H, m), 1.57 (2
H, q), 3.50 (4H, m), 6.02 (2H, s), 6.65 (1H, d), 6.84 (1H,
d), 6.88 (1H, d), 6.94 (1H, q), 6.97 (1H, q), 7.15 (1H, d),
7.20 (1H, q)

Based on these physicochemical properties, this compound was identified as piperine having the following chemical structure.

[0032]

Embedded image

Example 1 (Inhibitory effect on rat-derived ACAT) The effect on ACAT activity was obtained by using a crude enzyme prepared from a rat liver microsomal protein fraction and using ( ¹⁴ C) -oleoyl CoA.
Was used as a substrate to produce ( ¹⁴ C) -cholesteryl oleate from endogenous cholesterol. That is, when the culture supernatant was used as a sample, the reaction solution composition was 20 μl of microsomal protein (1.83 mg / ml), 20 μl of ( ¹⁴ C) -oleoyl CoA (5 μCi / ml, BSA: 96 nmol / ml), 90
1 μl of 100 mM phosphate buffer (containing 5 mM dithiothreitol, pH 7.4), 20 μl of culture supernatant, total 1
It consists of 50 μl. On the other hand, the composition of the reaction solution when the fraction was used as a sample was 20 μl of microsomal protein (1.83 mg /
ml), 20 μl of ( ¹⁴ C) -oleoyl CoA (5 μC
i / ml, BSA: 96 nmol / ml), 106 μl of 100
mM phosphate buffer (containing 5 mM dithiothreitol, pH
7.4) Samples dissolved in 4 μl of methanol or dimethylsulfoxide, 150 μl in total.

The reaction was carried out at 37 ° C., and after completion of a predetermined reaction time (usually 18 minutes), 5.65 ml of chloroform: methanol (2: 1) was added to stop the reaction, and then 0.04 N was added. 0.98 ml of an aqueous hydrochloric acid solution and cholesteryl oleate as a carrier were added, and the mixture was extracted with a shaker for 20 minutes and then left overnight in a cool dark place. The next day, the whole amount of the separated lower chloroform layer was extracted, dried under a nitrogen stream, and the remaining upper layer was a distribution solution of Holch, lower layer 5.6.
5 ml was added and extraction was performed again. This was left overnight in a dark place, then the whole amount of the lower layer was extracted and combined with the previously dried chloroform lower layer fraction to dryness.

Next, this was dissolved in 200 μl of chloroform, and the entire amount was applied to a silica gel thin layer chromatography G60 plate (aluminum sheet). 10 with diethyl ether: petroleum ether (10: 190)
After being developed in cm, autoradiography was performed to confirm the produced ( ¹⁴ C) -cholesteryl oleate. Next, this portion was cut off, and the radioactivity was measured for 30 minutes in toluene by liquid scintillation counting for 5 minutes or by betascope (manufactured by Beta Gen). Concentration (I
The IC ₅₀ value of piperine was 18 μM as a result of measurement of C ₅₀ ).
Met.

Example 2 (Cholesterol Ester Accumulation Inhibiting Action on Macrophages) The cholesterol ester accumulation inhibiting action on macrophages was carried out using the J774.1 strain, which is a mouse macrophage-established cell.

The metabolism of LDL in endothelial cells and smooth muscle cells is
Although down-regulation of the L receptor is involved, uptake of denatured LDL in macrophages is via the scavenger receptor. Since this pathway is unregulated, cholesterol esters accumulate indefinitely.

Therefore, liposomes consisting of cholesterol and phosphatidylcholine labeled with ¹⁴ C are incorporated into macrophage cells, and ACA present in the rough endoplasmic reticulum.
By acting on T, it was investigated whether or not the piperine suppresses intracellular cholesterol ester accumulation.

Macrophage cells were treated with 10% fetal bovine serum, penicillin 50 U / ml, streptomycin 50 μg.
RPMI 1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.)
Incubation was carried out for 2 days under the condition of 5% CO ₂ . Then, trypsin treatment is performed to remove the cells attached to the bottom of the well to obtain a cell suspension, and the cells are washed with the above-mentioned fresh medium, and then the 24-well multiple well plate (Corning 25820-24). Each well was inoculated with 750 μl of a cell solution of 2 × 10 ⁶ cells / ml. After culturing at 37 ° C. under 5% CO ₂ for 37 hours, the medium was exchanged with the fresh medium and further added with 80 μg of cholesterol (containing 0.4 μCi of ( ³ H) -cholesterol) and 160 μg of phosphatidylcholine.
40 μl of a 3 M glucose solution and 10 μl of a methanol solution in which a test sample was dissolved were added, and the mixture was cultured for 12 hours. These operations were performed aseptically.

After completion of culture, serum-free RPMI 1
The cells attached to the bottom surface were washed 5 times with a Komagome pipette using 640 medium, and then 1 ml of isopropanol was added to extract intracellular cholesterol and produced cholesterol ester. This extract was subjected to silica gel thin layer chromatography (Merck, Inc., Art. 5553).
Hexane: ether: formic acid (80: 2)
After development at 0: 2), autoradiography was performed. Next, the cholesterol ester and the cholesterol moiety labeled with ³ H were cut out, and the radioactivity was measured with a liquid scintillation counter to obtain the cholesterol ester accumulation inhibition rate as compared with the control. As a result, the 50% cholesterol ester accumulation inhibitory concentration (IC ₅₀ value) of piperine was 7 μM.

[0041]

[Table 2] Formulation Example 1 (tablets) Piperine 40 g Lactose 360 g Talc stearate 40 g Starch 60 g

The above ingredients are mixed in a mixer and tabletted to give 0.5 g.
1000 tablets were obtained.

[0043]

Since the active ingredient of the present invention has a strong ACAT inhibitory activity and high safety, it lowers blood cholesterol and suppresses the accumulation of cholesterol, so that it is useful as a prophylactic and therapeutic agent for human arteriosclerosis. Is.

[Brief description of drawings]

FIG. 1 is a diagram showing an ultraviolet absorption spectrum of a substance obtained in Production Example 1.

FIG. 2 is a diagram showing an infrared absorption spectrum of a substance obtained in Production Example 1.

FIG. 3 is a diagram showing a proton nuclear magnetic resonance spectrum of the substance obtained in Production Example 1.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takako Chatani 1-1-19 Higashishimbashi, Minato-ku, Tokyo 1-1-19 Yakult Honsha Co., Ltd. (72) Inventor Chie Mori 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Tsunekazu Watanabe 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Teruo Yokokura 1-1-19 Higashishimbashi, Minato-ku, Tokyo Shares Company Yakult Head Office

Claims (3)

[Claims] 1. Formula (1): An acylcoenzyme A: cholesterol acyltransferase inhibitor comprising a piperidine derivative represented by 2. Formula (1): A blood cholesterol lowering agent comprising a piperidine derivative represented by 3. Formula (1): An agent for preventing and treating arteriosclerosis, which comprises a piperidine derivative represented by