FO-2546 SUBSTANCE, ITS PRODUCTION AND ITS USE AS INHIBITOR OF ACYL-COA CHOLESTEROL ACYLTRANSFERASE

Abstract: FO-2546 substance of formula (I) comprising FO-2546A substance, FO-2546B substance, FO-2546C substance and FO-2546D substance, FO-2546I substance, FO-2546J substance and FO-2546K substance, which has inhibitory activity for acylcoenzyme A cholesterol acyltransferase is produced by culturing microorganism strain FO-2546 belonging to Coelomycetes. The substance is useful for treatment and prevention of a disease attributed by accumulation of cholesterol.

Description: FO-2546 SUBSTANCE ITS PRODUCTION AND ITS USE AS INHIBITOR, OF ACYL-COA CHOLESTEROL ACYLTRANSFERASE The present invention relates to a novel FO-2546 substance having an inhibitory activity for acylcoenzyme A cholesterol acyltransferase (hereinafter designated as acyl CoA cholesterol acyltransferase). Heretofore some pharmaceutical products for treatment of hyperlipemia have been reported. Among them, a drug having an action on (1) cholesterol synthesis inhibition, (2) cholesterol absorption inhibition, (3) stimulation of cholesterol catabolism and (4) activation of lipoprotein lipase (suppression of lipoprotein synthesis), has been known. In recent years, the symptoms attributable to accumulation of cholesterol in vivo such as hyperlipemia and arteriosclerosis in adult are becoming world-wide problems. Hyperlipemia is known to be a factor for stimulating a progress of arteriosclerosis, and hence ischemic cardiopathy can be decreased by lowering blood cholesterol level. The crisis of yocardial infraction caused by hyperlipemia is observed at a high rate. Accordingly more effective and safe medicament for treatment of hyperlipemia, in particular hypercholesterolemia, is desirable. Cholesterol is converted to cholesterol ester by an action of acyltransfer from acyl CoA which is accumulated in the cells and in blood lipoprotein. An enzyme which catalyses acyltransfer reaction is acyl CoA cholesterol acyltransferase, and is specifically related to adsorption of cholesterol from the intestinal tract and a formation of the foam cells in the coronary artery. Thererfore acyl CoA cholesterol acyltransferase inhibitor is estimated to be effective for treatment of the hypercholesterolemia. To provide acyl CoA cholesterol acyltransferase inhibitor is expected to be useful for treatment of the diseases of adult people such as hyperlipemia or arteriosclerosis attributed therefrom. We have found according to continued studies on the metabolic products produced by microorganisms that substances having an inhibitory activity for acyl CoA cholesterol acyltr ¬ ansferase are produced in a fermentation broth of microorganism Strain FO-2546 isolated from soil sample. Further we have isolated the substances having inhibitory activity for acyl CoA cholesterol acyltransferase which have physico-chemical properties described hereinbelow. These substances are novel compounds and were designated as F0-2546A substance, F0-2546B substance, F0-2546C substance, F0-2546D substance, F0-2546I substance, F0-2546J substance and F0-2546K substance. In the present invention, these substances were given a general name FO-2546 substance. An object of the present invention is to provide FO-2546 substance of the formula wherein R . is hydrogen or hydroxyl, and W is no bonding or oxygen, X is, together with Y, a group 0 in which R 2 is -CH= C (CH 3 ) 2 or -CH - C (CH 3 ) and R 3 is hydrogen or hydroxyl. Another object of the present invention is to provide F0-2546A substance, F0-2546B substance, F0-2546C substance, FO- 2546D substance, F0-2546I substance, F0-2546J substance and FO- 2546K substance. Further object of the present invention is to provide a process for production of FO-2546 substance which comprises culturing the FO-2546 substance producing microorganism belonging to Coelomycetes in a nutrient medium, accumulating the FO-2546 substance therein, and isolating the FO-2546 substance therefrom. More further object of the present invention is to provide a microorganism strain belonging to Coelomycetes having an ability to produce FO-2546 substance. More further object of the present invention is to provide are pharmaceutical composition for treatment and prevention of a disease attributed of cholesterol, and also provide of a method of treating hypercholesterolemia in mammals. The microorganism having an ability to produce FO-2546 substance (herein designated as FO-2546 substance producing microorganism) belongs to Coelomycetes. For example a microorganism strain FO-2546 belonging to Coelomycetes isolated by the inventors of the present invention is one of the most preferable strain used in the process of the present invention. Taxonomical properties of the strain FO-2546 are shown in the followings. Taxonomical properties: 1. Observation on various media: (1) Cultural observations of the strain are illustrated in Table 1. Observations were taken macroscopically after cultivation at 25°C for 14 days. Table 1 (To be continued Table 1) 2. Physiological properties: (1) Growth temperature 8.5 - 33°C (2) Optimum growth temperature 18.5 - 28°C (3) Growth pH 2 - 10 (4) Optimum growth pH 3 - 6 (5) Nature aerobic In comparison with the above properties of the Strain FO- 2546 based upon the morphological properties, various properties on culture and physiological properties, and the taxonomical properties of known microorganism species, the present Strain FO-2546 is defined as the strain belonging to Coelomycetes, however the strain is not detailed identified in its taxonomical position. Therefore, the present strain is given a temporary name Strain FO-2546. The strain has been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1-chome Tsukuba-shi Ibaraki-ken Japan with the deposition No. FERM BP-4406. The deposition was made on July 17th, 1992. Although a description of the Strain FO-2546 was given hereinabove, taxonomical properties of the microorganism strain are in general very mutable and are not stable. Spontaneous mutation and artificial mutation with ultraviolet radiation or treatment with mutagen such as N-methyl-N-nitro-N-nitrosoguanidine or ethyl methanesulfonate or microorganisms can occur. Therefore artificial and spontaneous mutants of Strain FO-2546 which belong to Coelomycetes and have FO-2546 substance producing ability are usable in the present invention. Further, the mutated strain obtained by cell technology method such as cell fusion and gene manipulation technology which produce FO-2546 substance are included within the scope of the FO-2546 substance producing microorganism of the present invention. In the present invention, a microorganism FO-2546 substance producing strain belonging to Coelomycetes is cultured in a nutrient medium. A culturing method generally used for fungi can be used on the present invention. A nutrient medium containing assimilable carbon source, assimilable nitrogen source and, if required together with inorganic salt can be used. Examples of assimilable carbon sources are glucose, sucrose, molasses, dextrin or cellulose etc. and combinations thereof. Examples of assimilable nitrogen sources are organic nitrogen source such as peptone, meat extract, yeast extract, dry yeast, soybean powder, corn steep liquor, cotton seed cake, casein, soybean protein hydrolysate, amino acid or urea, and inorganic nitrogen source such as ammonium salt, and combinations thereof. If required, inorganic salt such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate salt, and heavy metallic salt can be added to the medium. Further, trace nutrient element, growth stimulant or precusor can be added in the medium, if desired. Cultivation should be performed under aerobic condition, e. g., by shaking or agitation. Submerged aeration agitation cultivation ispreferable for industrial production. Cultivation can be preferably performed at neutral pH. Cultivation temperature is usually at 20- 37 β C. preferably at 24 - 30°C. FO-2546 substance is accumulated in the cultured mass by culturing for 2 - 3 days, and when maximum production of the FO-2546 substance is obtained, cultivation can be terminated. These culturing condition such as medium composition, pH of the medium, culturing temperature, agitation time and aeration rate can be varied depending upon the microorganism strain used and the atmospheric conditions for obtaining the preferable result. Anti-foaming agent such as silicon oil, vegetable oil or surface active agent can also be used if necessary. The FO- 2546 substance is accumulated in mycelia and in the cultured broth. The FO-2546 substance can preferably be isolated from supernatant and mycelia separated by centrifugation of cultured broth. The FO-2546 substance can be extracted from supernatant with water-immiscible organic solvent such as ethyl acetate, butyl acetate or benzene, and crude FO-2546 substance can be obtained by concentrating the extract in vacuo. The crude substance is subjected to a known purification process for 1ipophilie substance, for example, by column chromatography using a carrier such as silica gel or alumina. Each component of the FO-2546 substance such as F0-2546A, -B, -C, -D, -I, -J and -K substance can be isolated. The FO-2546 substance can also be isolated from mycelial cells by extracting the mycelia with water- iscible organic solvent such as aqueous acetone or aqueous methanol. The extract is concentrated under reduced pressure and the concentrate is subjected to further extraction with water-immiscible organic solvent such as ethyl acetate, butyl acetate or benzene. The extract which is optionally combined with the extract obtained from cultured filtrate hereinabove explained, can be purified, or it can be purified and separated by the same procedure as of the above described to obtained each component of the FO-2546 substance. Physico-chemical properties: Physico-chemical properies of F0-2546A substance, F0-2546B substance, F0-2546C substance. F0-2546D substance, F0-2546I substance, F0-2546J substance and F0-2546K substance are illustrated in Table 2 and Table 3. Some properties of the F0-2546A substance, F0-2546B substance, F0-2546C substance, F0-2546D substance, F0-2546I substance, F0-2546J substance and F0-2546K substance are shown as follows. Solubility in solvent : soluble in methanol, ethanol, aceto- nitrile, ethyl acetate and benzene; insoluble in water Nature of substance : neutral Appearance : yellowish powder Table 2 (To be continued Table 2) Table 3 (To be continued Table 3) Chemical structure F0-2546A substance : HJ C CH, F0-2546B substance H 3 C CH 3 F0-2546D substance H 3 C CH 3 F0-2546I substance H3 C CH3 F0-2546J substance H 3 C CH 3 F0-2546K substance Biological properties: (1) Inhibitory action on rat acyl CoA cholesterol acyltransferase: An inhibitory action of the FO-2546 substance for acyl CoA cholesterol acyltransferase is tested according to the method of Kyoda et al. (J. Antibiotics. 44: 136, 1991). A crude enzyme prepared from rat liver microsome fraction is added in the 100 mM phosphate buffer solution (pH 7.4) 200 #M containing 300 li M bovine serum albumin, 30 /zM Cl- 1 C 3 oleoyl-CoA (0.02 zCi) and 30 #M cholesterol (solubilized with 1/30 w/w Triton WR-1339) and incubated at 37°C for 30 minutes. Total lipid is extracted with a mixture of chloroform-methanol (2 : 1), then each lipid is separated by thin layer chromatography (Kiesel gel GF 2 64, developer : petroleum ether-diethyl ether-acetic acid = 90 : 10: 1). Radioactivity incorporated in the cholesterol ester fraction is analysed by RI scanner (Ambis Corp.) to assay an activity of acyl CoA cholesterol acyltransferase. The 50 % inhibition concentration on the enzyme is shown in Table 4. Table 4 Effect of the invention: As illustrated hereinabove, FO-2546 substance and the individual components thereof, of the present invention show » strong inhibitory action on acyl CoA cholesterol acyltransferase. Accordingly the substances are useful in the treatment of high serum cholesterol in mammals, including humans. As used herein, treatment is meant to include both the prevention and alleviation of high serum cholesterol. The substances may be administered to a subject in need of treatment by a variety of conventional routes of administration, including oral and parenteral. In general, these substances will be administered orally or parenterally at dosages between about 0.1 and about 10 mg/kg body weight of the subject to be treated per day, preferably from about 0.5 to 5 mg/kg. However, some variation in dosage will necessarily occur depending on the condition of the subject being treated and the activity of the compound being employed. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. A substance of the invention may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutically carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. The resulting pharmaceutical compositions are then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch, alginic acid and certain complex silicates, together with binidng agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesiu stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type may also be employed as fillers in sort and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents coloring matter or dyes and. if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof. For parenteral administration, solutions of substance of the invention in seasa e or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Such solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitioneal administration. Brief explanation of the drawings; Figure 1 UV spectrum of F0-2546A substance, Figure 2 IR spectrum of F0-2546A substance, Figure 3 'H-NMR spectrum of F0-2546A substance, Figure 4 13 C-NMR spectrum of F0-2546A substance, Figure 5 UV spectrum of F0-2546B substance, Figure 6 IR spectrum of F0-2546B substance, Figure 7 'H-NMR spectrum of F0-2546B substance. Figure 8 13 C-NMR spectrum of F0-2546B substance. Figure 9 : UV spectrum of F0-2546C substance, Figure 10 : IR spectrum of F0-2546C substance, Figure 11 : 'H-NMR spectrum of 2546C substance. Figure 12 : 13 C-NMR spectrum of F0-2546C substance, Figure 13 : UV spectrum of F0-2546D substance. Figure 14 : IR spectrum of F0-2546D substance, Figure 15 : 'H-NMR spectrum of 2546D substance, Figure 16 : 13 C-NMR spectrum of F0-2546D substance. Figure 17 : UV spectrum of F0-2546I substance, Figure 18 : IR spectrum of F0-2546I substance, Figure 19 : 'H-NMR spectrum of F0-2546I substance, Figure 20 : 13 C-NMR spectrum of F0-2546I substance. Figure 21 : UV spectrum of F0-2546J substance, Figure 22 : IR spectrum of F0-2546J substance, Figure 23 : 'H-NMR spectrum of 2546J substance, Figure 24 : 13 C-NMR spectrum of F0-2546J substance Figure 25 : UV spectrum of F0-2546K substance, Figure 26 : IR spectrum of F0-2546K substance, Figure 27 : 'H-NMR spectrum of 2546K substance and Figure 28 : 13 C-NMR spectrum of F0-2546K substance. Following example illustrates the present invention but is not construed as limiting. Example 1. A medium 100 ml (pH adjudted to 6.0) comprising glucose 21 yeast extract 0.21 MgS0 4 • 7H 2 0 0.051 polypeptone 0.51 potasium dihydrogen phosphate 0.1% and agar 0.1 % in 500 ml Erlenmeyer flask with cotton seal was steam sterilized. One loopful microorganism strain F0-2546 FERM BP-4406 grown on a nutrient agar medium was inoculated aseptically into the medium and shake cultured at 27°C for 48 hours to prepare seed culture liquid. A medium comprising soluble starch 3. 0 %, glycerol 1.0 1 soybean powder 2.01 dry yeast 0.31 potasium chloride 0.31 calcium carbonate 0.21 MgS0 4 • 7H 2 0 0.05 % and potasium dihydrogen phosphate 0.05 % (pH adjusted to 6.5) in 30 lit. jar- fer enter was sterilized and cooled. The seed culture 200 ml was transferred aseptically thereto and the inoculated medium was cultured aerobically at agitation 250 rp , aeration 10 lit./min. at 27°C for 72 hours. Cultured liquid 20 lit. including mycelia was extracted with ethyl acetate 18 lit. The extract was concentrated under reduced pressure to obtain crude substance 19g. The crude substance was charged on a column of silica gel (400g, Merk, Art 9385) and eluted with a mixture of chloroform- methanol (99 : 1). Active fractions containing FO-2546 substance comprising each 100 ml fraction were collected and concentrated in vacuo to obtain the crude substance 1.0 g. The crude substance was purified separately divided with 10 times by high performance liquid chromatography (Trirota V, Nihon Bunko Co.. column YMC-Pack A-343, ODS resin. Yama ura Kagaku Kenkyusho) with linear gradient elution solvent sysytem of 75 - 90 % aqueous acetonitrile for 20 min., detection UV 225 nm, flow rate 8 ml/min. The F0-2546A substance 13 mg. F0-2546B substance 4.6 mg. F0-2546C substance 13 mg, F0-2546D substance 335 mg, F0-2546I substance 41 mg, F0-2546J substance 134 mg and F0-2546K substance 11 mg were obtained.

Claims: CLA IMS (1) A FO-2546 substance of the formula wherein Ri is hydrogen or hydroxyl, and W is no bonding or oxygen, X is, together with Y, a group O in which R 2 is -CH=C (CH 3 ) 2 or -CH-C (CH 3 ) _ and R 3 is hydrogen or hydroxyl. (2) A process for production of FO-2546 substance which comprises culturing FO-2546 substance producing microorganism belonging to Coelomycetes in a nurtrient medium, accumulating the FO-2546 substance in the cultured mass, and isolating the FO-2546 substance therefrom. (3) A process according to claim 2, wherein the FO-2546 substance producing microorganism is belonging to Coelomycetes is Strain FO-2546 FERM BP-4406. (4) A microorganism strain belonging to Coelomycetes which has ability to produce FO-2546 substance. (5) A microorganism strain according to claim 4, which is Strain FO-2546 FERM BP-4406. (6) A F0-2546A substance of the formula (7) A F0-2546B subs tance of the f ormu la H 3 C CH3 (8) A F0-2546D substance of the formula H 3 C CH 3 (9) A F0-2546I subs tance of the formu la H 3 C CH 3 (10) A F0-2546J substance of the formula H 3 C CH 3 (11) A F0-2546K substance of the formula H 3 C CH 3 (12) A pharmaceutical composition for the treatment of a disease attributed by accumulation of cholesterol in a mammalian subject which comprises a therapeutically effective amount of a compound of the formula wherein Ri is hydrogen or hydroxyl, and W is no bonding or oxygen. X is, together with Y, a group Ha C CHa 0 V in which R 2 is -CH= C (CH 3 ) 2 or -C ' H-t (CH 3 ) 2 and R 3 is hydrogen or hydroxyl, and a pharmaceutical acceptable carrier. (13) A method for the treatment of a disease attributed by accumulation of cholesterol in a mammalian subject, which comprises administering to said subject a therapeutically effective amount of a compound of the formula, wherein Ki is hydrogen or hydroxyl, and W is no bonding or oxygen, X is, together with Y ι a group in which R 2 is -CH = C (CH 3 ) 2 (CH 3 ) _ and R 3 is hydrogen or hydroxyl.

Inventors: MASUMA ROKURO, OMURA SATOSHI, TOMODA HIROSHI, MASUMA, ROKURO, OMURA, SATOSHI, TOMODA, HIROSHI
Applicants: KITASATO INST, MASUMA ROKURO, OMURA SATOSHI, TOMODA HIROSHI, THE KITASATO INSTITUTE
Application year: 1993
Main classification: C07D491
IPC codes: C07D491, C12P17