HU182069B - Process for preparing monacoline k, a compound with antihypercholesteremic activity - Google Patents

Abstract

The invention relates to a process for the preparation of monacolin K for use as a medicament for hypercholesterolemia. The aim of the invention is to provide a compound having an improved action for lowering the cholesterol content in the blood. According to the invention, a Monacolin K compound of the following formula is prepared by culturing microorganisms of the culture strain Monascus ruber, strain 1005, in a suitable culture medium at a temperature of from 7 to 40 ° C, especially from 20 to 35 ° C.

Description

The present invention relates to a novel antihypercholesterolemic compound of the formula (I), termed Monacolin K. Monacolin K can be prepared by culturing the Monascus ruber 1005 strain of the genus Monascus in an appropriate medium.

It has been previously recognized that one of the main causes of heart disease, such as myocardial infarction or multiple sclerosis, is high blood cholesterol levels. As a result of this discovery, extensive research has been carried out to produce physiologically acceptable compounds that inhibit cholesterol biosynthesis and thereby lower blood cholesterol levels. One such compound known as ML-256 by researchers is disclosed in U.S. Patent No. 1,455,425. British Patent Specification No. 4,684,198. ML-256 is produced by culturing microorganisms of the genus Penicilliuai.

Examining fungi of the genus Monascus, it was found that the strain Monascus ruber 1005 produces an antihypercholesterolemic agent with a much higher activity than ML-2J6. This new compound of formula (1) was named Monacolin K.

Monascus ruber 1005 is a newly isolated microorganism with the following microbiological properties. The strain was isolated from food produced in Thailand and deposited on 16 February 1979 under the accession number FERM 4822 in Japan (Fermentation Research Institute, Ministry of International Trade and Industry), and December 1979, Jl-int. No. NRRL 12075 to U.S. Agricultural Research Service, Northern Region Research Laboratory.

The microorganism can be well grown on potato glucose agar medium at 25 ° C. The diameter of the rapidly growing colonies reaches 6-6.5 cm on the 10th day after inoculation. The surface of the battery develops smooth and has a relatively thin hyphae base. Airborne hyphae are poorly developed; these hyphae are white and mostly woolly. Many clelstothecia are formed on the hyphae basal layer which, upon maturation, turn reddish brown. The surface and bottom of the colony are brown to reddish brown.

The strain grows very rapidly on Sabouraud's agar medium at 25 ° C and the colony diameter after inoculation

On day 10, it reaches 6-6.5 cm. The colony surface is very smooth and the surface hyphae and legumes develop better than in potato-glucose agar medium. Very few cleistothecia develop '. The surface of the colony is reddish-yellow and the lower surface is red-brown-dark brown.

The strain grows slowly on oatmeal agar at 25 ° C and colonies are 1.5 to 2 cm in diameter on day 10 after inoculation. The battery is smooth; air hyphae and cleistothecia are both very poorly developed. The surface and bottom of the battery are both dark red and reddish brown.

The microorganism develops very slowly on Czapek agar at 25 ° C; the diameter of the colony on the 10th day after inoculation was only 1.6-1.8 cm.

In each of these media, the growth rate at 57 ° C is practically the same as the growth rate at 25 ° C.

As for the morphological characteristics of the strain, the cleis

-2182.069 tothecium spherical in shape and 30-60 mm in diameter * have thin and membranous walls, partitions at the handle and each one

3.5-4.5 / u. diameter and 15-80 / u length consists of hyphae. The ascus consists of 8 spores, almost spherical and easily disintegrates. Ascospores are colorless, oval or ellipsoidal, 4-5 x 4-7 / u in size, with smooth surfaces. Conidia are colorless, spherical or pear-shaped, with a 6-9 x 6-11 / 1 · base, truncated, relatively thin and smooth. The conidia are petal-like to the base, a kind of arthorspora of the meristem. Conidiospores are similar to vegetative hyphae, branched or unbranched; conidia are formed on the upper part. Mycelia are colorless, branched and have partitions, most of them 3-5 µm in diameter.

Based on the above characteristics, the microorganism was found to belong to a strain of Monascus ruber van Tieghem.

For example, the microbiological properties of Monascus ruber are described in Takada, Transactions of the Micrological Society of Japan, 125-130 (1969), 'Materials for the fungus flora of Japan (7); van Tiegham, Bull. Soc. Botan. Francé * 31 »227/1884 /. The ascospore generation of the strain has been described by Colé et al., Canadian Journal of Botany, 46, 987 (1968), at Conidium Ontogeny in Hypomycetes. The iniperfect state os Monascus ruber and its mer arthrospores. ”

Although the use of Monascus ruber strain 1005 is illustrated herein, examples of mutants of said strain can be used to carry out the process of the present invention.

Monacolin K can be prepared by aerobically culturing the selected microorganism in a suitable medium; the cultivation is carried out by methods well known in the art for culturing fungi and microorganisms. For example, a microorganism producing Monacolin K may first be cultured in a suitable medium, and then the microorganisms developed therein may be harvested and transferred to another medium on which it produces the desired Monacolin K. The medium used for the growth of the microorganism and for the production of Monacolin K may be the same or different.

For culture, any medium known in the art for growing mushrooms is suitable provided that it contains the appropriate nutrients, in particular well-assimilable sources of carbon and nitrogen. Suitable, well assimilable carbon sources include, for example, glucose, maltose, dextrin, starch, lactose * sucrose, and glycerol. Of these, the use of glucose, glycerol and starch is preferred for the preparation of Monacolin K. Suitable, well assimilable nitrogen sources include, for example, peptone, huse extract, yeast, yeast extract, soybean meal, peanut flour, corn jam, rice bran and inorganic carbon sources. Of these, the use of peptone is particularly preferred. In preparing Monacolin K, an inorganic salt and / or metal salt may be added to the medium * if necessary. In addition, small amounts of heavy metals may be added if necessary.

Preferably, the microorganism is cultured under aerobic conditions using techniques well known in the art,

-3182.069 for example solid or shaken or aerated and mixed culture. The microorganism can be cultivated over a wide range of temperatures, for example between 7 and 40 ° C, but it is most preferred to produce between 20 and 35 ° C for the production of Monacolin K.

During cultivation of the microorganism, the formation of Monacolin K can be observed by sampling the medium and measuring the physiological activity of Monacolin K in the sample as described below. The culture can be continued until a significant amount of Monacolin K accumulates in the medium, and then Monacolin K can be isolated from the medium by separation techniques selected according to its physical and chemical properties. For example, any of the following separation techniques may be employed: extraction of the fermentation broth with a hydrophilic solvent such as diethyl ether, ethyl acetate, chloroform or benzene, extraction of the microorganisms with a hydrophilic solvent such as acetone or alcohol, concentration, a more polar solvent, e.g. norgestrel acetone or alcohol / to the removal of impurities with a less polar solvent / e.g. petroleum ether or hexane / using · /, gel filtration with a suitable material such as a packed column Sephadexszel, absorption chromatography ^- nude IVSZ ene or silica. By properly combining these techniques, Monacolin K can be recovered from the culture in pure form.

Monacolin K has the following properties:

Colorless crystals melting at 157-159 ° C with decomposition. Elemental analysis of the formula: Tin ^ H ^ gOt-: C = 71.56%, II = 8.85% θθ 0 = 19.59%. It has a mass spectrometry molecular weight of 404. The ultraviolet absorption spectrum of methanol is shown in Figure 1; maxima can be observed at 232, 238 and 246 m / u. The infrared absorption spectrum of potassium bromide in a paste is shown in Figure 2; spectrum of a deuterochloroform using tetramethylsilane internal standard / proton 60 MHz / a is shown in Figure 3. Figure 4 shows the MR spectrum of deuteromethanol (13c). Monacolin K is soluble in lower alcohols (e.g. methanol, ethanol and propanol), and in acetone, chloroform, ethyl acetate and benzene. Insoluble in light petroleum and hexane. Optical rotation: λmax _{= +} 307.6 (c = 1, in methanol). TLC Rf = 0.47 · spot occurs; run by Merek et. Co. Inc. Ltd., No. 5715 Kiesel gel 6OF254 on silica gel, eluting with methylene chloride: acetone = 4: 1. Detection is based on the ultraviolet absorbing property of the spot or by spraying the plate with 50% (v / v) sulfur, acid or iodine. When sprayed with sulfuric acid, the spot becomes light red to reddish-brown when heated. Monacolin K is an insoluble substance and is insoluble in a neutral or aqueous acidic medium. When treated with an alkali, it is converted to an acidic substance which is soluble in water · This acidic compound can be extracted with acidic pH η ethyl acetate or chloroform and converted to Monacolin K by evaporation of the solvent. The physiological activity of Monacolin K can also be quantified using the following in vivo assay.

Carrier test in rabbits

-4182.039

This test measures how well the Monacolin K arrows lower the blood cholesterol level. The experimental animals weighed 2.5-3.0 kg. Immediately before the experiment, blood was drawn from the vein of each rabbit ear and blood serum cholesterol levels were measured in a known manner. Thereafter, a predetermined amount of Monacolln K is administered to the rabbits orally continuously for 1 to 5 days, and the serum cholesterol is measured after administration. The efficacy of Monacolln K and Monacolln K containing media can be quantified by measuring cholesterol levels before and after treatment.

The blood and liver cholesterol lowering effect of Monacolih K was determined in various in vivo tests.

Reduction of blood cholesterol levels in rats

About 300 g of testulyu Wistar Imamlchi rats are divided into groups of 5 to 5 members. Each animal is given 400 mg / kg Triton TO-1139 intravenously (this increases blood cholesterol levels), and at the same time 10 mg / kg Monacolln K is administered intraperitoneally. 14 hours after intraperitoneal administration of Monaoolin K, the animals are bled * blood is collected and cholesterol is measured in a known manner. It was found that the blood cholesterol level in Monacolln K treated animals was reduced by 23.9% compared to the control group, which received only Triton WR-1339.

Rabbit blood cholesterol lowering

The experimental animals are 2.7 to 2.9 kg body-weight rabbits administered 1 mg / kg Monacolln K orally twice daily in the morning and evening for five days. Blood is drawn from the rabbit ear vein and blood serum cholesterol is measured before and 3 days after and 5 days after administration of Monacolin K. We found that after 3 and 5 days after administration of Monacolin K, cholesterol levels were 15% and 29%, respectively; these values are lower than those before administration of Monacolin K.

In addition to its significant inhibitory effect on cholesterol biosynthesis, the toxicity of Monacolin K is very low. Acute oral toxicity in mice / LD ^ q / 1 g / kg body weight.

Monacolin K may be administered orally or parenterally in the form of capsules, tablets, injections or any other pharmaceutical preparation; oral administration is generally preferred.

The dosage used will vary with the age and weight of the patient and the severity of the disease, but usually an adult can be given 0.5 to 50 mg daily in two, three or three divided doses. Due to the very low toxicity of Monacolin K, higher doses may be used if necessary.

The process of the present invention is illustrated by the following example, but is not intended to be limiting.

Example of a medium containing mixed% glucose, 2.5% peptone, 0.5% corn jam and 0.5% ammonium chlorine time was inoculated with Monascus ruber 1005. The breeding is aerobic. under tin conditions at 28 ° C for 10 days. The filtrate (5 L) of the culture filtrate was adjusted to pH 3 ~ 6 with 6N hydrochloric acid and the filtrate was extracted with an equivalent volume of ethyl acetate.

-5182.069

The solvent was evaporated under reduced pressure and the residue was dissolved in 100 ml of benzene; insoluble matter is filtered off.

The filtrate was washed twice with 100 ml of 5% aqueous sodium bicarbonate solution, 100 ml of 0.2 N aqueous sodium hydroxide solution was added and the mixture was stirred at room temperature. Thin layer chromatography is used to verify that Monacolln K is completely removed from the benzene phase, and the aqueous phase is separated and adjusted to pH 3 with 6N hydrochloric acid. The solution was extracted with ethyl acetate (2 x 100 mL) and the extract was concentrated to dryness under reduced pressure to give 260 mg of an oil. The resulting oil was dissolved in benzene and allowed to crystallize and recrystallized from aqueous acetone to give 87 mg of Monacolin K as colorless needles; the identification data are the same as those described in this section.

Claims (3)

Patent claims CLAIMS 1. A process for the preparation of a compound of formula (I), Monacolin K, wherein the microorganism or mutant of Monascus ruber strain 1005 is cultured on a suitable medium. The method of claim 1, wherein the culture is carried out at a temperature of 7 to 40 ° C. The method of claim 2, wherein the culture is carried out at a temperature of 20-35 ° C.