JPH08245691A - Compound tan-2177, its production and use - Google Patents

Abstract

PURPOSE: To obtain a new compound, TAN-2177, having a squalene-synthesizing enzyme-inhibiting action and useful as a hyperlipidemia-preventing and treating medicine, etc., by culturing a microorganism belonging to the genus Talaromyces and having capability of producing the compound, TAN-2177. CONSTITUTION: A new compound, TAN-2177, represented by the formula (R is H, methyl), having a squalene-synthesizing enzyme-inhibiting action and a blood cholesterol-reducing action, excellent in oral absorbability, and useful for preventing and treating ischemic heart diseases, cerebrovascular diseases, renal diseases, etc. The compound is obtained by inoculating a microorganism having an ability to produce the compound, TAN-2177, [e.g. Talaromyces flavus FL-55755 (FERM BP-4967)] on a medium, subjecting the inoculated microorganism to a shaking culture at 28 deg.C for 2 days to accumulate the product in the culture product, collecting the product, and subsequently purifying the collected product by silica chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel compound TAN-2177 having a squalene synthase inhibitory action and useful as a therapeutic agent for hyperlipidemia.

[0002]

BACKGROUND ART Hyperlipidemia is known as the three major risk factors for ischemic heart disease as well as hypertension and smoking. Proper control of blood cholesterol level is an ischemic heart disease caused by hyperlipidemia. It is extremely important for the prevention or treatment of diseases, cerebrovascular disorders, renal diseases and the like. As a natural product-derived compound that inhibits squalene synthase and has a blood cholesterol lowering action, zaragodic acid (US Pat.
No.), squalestatin 1 [Journal of Biological Chem
mistry), Vol. 267, No. 17, pp. 11705-11708].

[0003]

However, these compounds are not sufficient in terms of blood cholesterol lowering action, oral absorbability and the like.

[0004]

[Means for Solving the Problems] In view of such circumstances,
As a result of repeated studies of the present inventors for a squalene synthase inhibitor having a strong activity among microbial metabolites,
It was found that a compound having a strong squalene synthase inhibitory activity was contained in the culture solution of a filamentous fungus belonging to the genus Talaromyces, and the active compound was successfully isolated. These compounds are called TAN-2177A and B. I decided. Detailed examination of the physicochemical and biological properties of these compounds confirmed that they were novel compounds and completed the present invention.

That is, the present invention is based on the equation (1)

Embedded image [Wherein, R represents a methyl group or a hydrogen atom] TAN-2177 or a salt thereof, (2) belongs to the genus Talalomyces, and compound TAN-2177 according to (1)
A method for producing the compound TAN-2177 or a salt thereof according to (1), characterized in that the microorganism having the ability to produce is cultured in a medium, the compound is produced and accumulated in the culture, and the product is collected. (3) Compound TAN- described in (1)
A microbial organism, Talalomyces having the ability to produce 2177.
Flavas, the compound TAN-2177 described in (4) (1)
The microorganism TA having the ability to produce Tararomysces flavus strain FL-55755, the compound TA according to (5) (1)
A drug comprising N-2177 or a salt thereof, (6)
(1) A squalene synthase inhibitor comprising the compound TAN-2177 or a salt thereof described in (1), and (7)
(1) An antihyperlipidemic agent containing the compound TAN-2177 described above or a salt thereof.

In the formula [I], -Arg-Gly-Gly-Leu- is an amino acid sequence consisting of amino acid residues, Arg is an arginine residue, Gly is a glycine residue and Leu is a leucine residue.
These amino acid residues are included in the present invention in both the D-form and L-form, but the arginine residue is preferably a D-arginine residue, and the leucine residue is preferably a D-leucine residue. . In the compound of formula [I], wherein R represents methyl (referred to as TAN-2177A), the amino acid sequence C
The amino acid residue on the terminal side represents a 4-methyl-proline residue. In the formula [I], compounds in which R represents a hydrogen atom (TAN
(Referred to as −2177B), the amino acid residue on the C-terminal side of the amino acid sequence represents a proline residue. The proline residue is preferably L-proline residue. In addition, the compound of the present invention is in solution, depending on the pH of the solution.

Embedded image But

[Chemical 4] There may be compounds converted to the present invention, but those converted compounds or mixtures thereof are also included in the present invention. T
Since AN-2177 has a double bond (> C = C <), geometric isomers (cis and trans forms) may exist.
Further, since it contains an asymmetric carbon, optical isomers (D-form, L-form)
Isomers such as body) may exist, but each isomer thereof and a mixture thereof are also included in the present invention. TAN-21
77 can exist as a mixture of conformers,
Also included in the invention are those conformers and mixtures thereof.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Any microorganism can be used in the present invention as long as it belongs to the genus Talaromyces and produces the compounds TAN-2177A and / or B. For example, the filamentous fungus Tararomysces flavus FL-55 isolated from Indian soil
The 755 (Talaromyces flavus FL-55755) strain can be used as an example.

The FL-55755 strain exhibits the following properties. (A) Morphological characteristics Conidia peduncle: The surface is smooth. It branches from the basal hyphae.
80-100 × 2-2.5 μm. Penisiri: Bicycle living body. Metre: 4-6 clusters. 8-10 x 1.5-
2.5 μm. Fear Ride: Nib type. 4 to 6 form a living body. 6
~ 9 x 1.5-2 μm. Conidia: oval. Both ends are often sharp. The surface is smooth. Form a long chain. 0.75 to 1 x 1.5 to 2μ
m. Ascomyate: spherical. A yellow hyphal net develops and becomes a wall. Diameter 200-300 μm. Ascus: Oval to spherical. 6-8 cells. 7-9 μm. Ascospore: Oval shape. 4-5 × 2.5-3.5 μm.
The surface is spiny.

(B) Properties on agar medium 1) Malt extract agar medium Growth is good and colony diameter is 70 after 2 weeks at 24 ° C.
mm. The surface consists of a slightly raised wool-like mycelium, the central part is slightly raised, and a mass of yellow mycelia is observed, and the outer edge is regularly framed. Development of aerial hyphae and conidia formation are good. The color of the surface of the colony is yellow to yellowish brown in the central part and yellowish green to dull green in the peripheral part. The back surface is pale yellow to pale yellowish brown. No formation of soluble pigment is observed. After 3 weeks, the formation of a large number of ascospores of yellow to reddish brown is observed. 2) Potato-glucose agar medium The growth was good, and the diameter of the colonies after 2 weeks at 24 ° C was 80.
mm. The surface consists of a slightly raised wool-like mycelium, a reddish brown water drop is observed in the center, and the outer edge is regularly framed. Development of aerial hyphae and conidia formation are good. The color tone of the colony surface is pale yellow to pale reddish brown in the central part, and pale yellow gray to greenish yellow gray in the peripheral part. From the center part of the back surface to the peripheral part, it is yellowish brown to light yellowish brown. No formation of soluble pigment is observed. Three
After a week, the formation of ascidian to orange-yellow ascidian fruits is observed. 3) Czapek agar medium Medium growth, 24 ° C., colony diameter after 2 weeks was 3
It was 7 mm. The surface is flat and consists of wool-like mycelium, and the outer edges are regularly framed. The color tone of the colony surface is pale yellow to pale yellow gray. The back surface has an elephant bud color. No formation of soluble pigment is observed. 4) Oatmeal agar medium The growth was good and the diameter of the colony after 2 weeks was 75.
mm. The surface consisted of a slightly raised wool-like mycelium, and a yellow mycelium mass and water droplets were observed in the center,
The outer edge is regularly bordered. Development of aerial hyphae and conidia formation are good. The color tone of the surface of the colony is pale yellow to yellowish brown in the central part, and pale yellowish green to greenish yellow from the peripheral part to the outer peripheral part. The central part of the back surface is pale yellow to pale yellow-brown, and the peripheral part is pale yellow-orange to orange-yellow. No formation of soluble pigment is observed. After three weeks,
The formation of a large number of ascospores of pale yellow-orange to reddish brown is observed.

(C) Physiological properties When the growth conditions of this strain were examined on a potato-glucose agar medium, the growth was good in any of PH3 to PH10, and the growth temperature range was 10 ° C to 35 ° C, which was optimal. The growth temperature is 24 ° C to 28 ° C. Does not grow at 37 ° C.

The above-mentioned various properties are described by D. Mall
och), Translated by Shunichi Udagawa "Mold Separation, Culture and Identification"
(Showa 58, Ito Denshaku Publishing Co., Ltd.) When compared with the identification search table on page 51, this strain was identified as Talaromyces (Talaromyces).
myces), it is clear that the strain belongs to the genus Talaromyces flavus by referring to "Fungus Encyclopedia (1)" by Shunichi Udagawa and Keisuke Tsubaki (1978, Kodansha Scientific). It is in good agreement with the property described in Therefore, the bacterium is Talaromyces flavus FL-55 FL-55755.
755).

This strain was deposited with the Fermentation Research Institute (IFO) on Dec. 12, 1994 by deposit number IFO-32670.
Also, on January 11, 1995, the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology (NIBH), the contract number F
Deposited as ERM BP-4967, respectively.

The compound TAN-2177 of the present invention, specifically TAN-2177A and / or B, or a salt thereof is not limited to these strains, but may be a method known per se including genetic engineering techniques (eg, X Ray, gamma ray, irradiation with radiation such as ultraviolet rays, drug treatment, culture on a drug-containing medium, etc.), and the mutant strain having the ability to produce the present compound derived from these strains It can be produced by culturing a microorganism in a medium, allowing the compound of the present invention to accumulate in the medium, and collecting the compound.

The medium used for culturing the bacterium producing the compound of the present invention may be liquid or solid as long as it contains a nutrient source that can be utilized by the bacterium, but it is suitable to use a liquid medium when treating a large amount. Is. A carbon source, a nitrogen source, an inorganic substance, and a trace nutrient source that can be assimilated by the compound-producing bacterium are appropriately added to the medium. As the carbon source, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, oils and fats (eg, cottonseed oil, soybean oil, lard oil, chicken oil, etc.), n-paraffin and the like are used. . As the nitrogen source, for example, meat extract, yeast extract, dry yeast, soybean flour, corn steep liquor, peptone, raw soybean flour, cottonseed flour, tomato paste, peanut meal, molasses, urea, ammonia salts (example ,
Ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) are used. Further, salts containing sodium, potassium, calcium, magnesium, etc., metal salts such as iron, manganese, zinc, cobalt, nickel etc., salts such as phosphoric acid, boric acid, etc., salts of organic acids such as acetic acid, propionic acid etc. are appropriately used. May be. Other amino acids (eg, glycine, leucine, arginine, proline, glutamic acid, aspartic acid, etc.), peptides (eg, dipeptide, tripeptide, etc.), vitamins (eg, vitamin B ₁ , vitamin B ₂ , nicotinic acid, vitamin B) ₁₂ , vitamin C, etc.), nucleic acids (eg, purines, pyrimidines, derivatives thereof, etc.) and the like may be contained. For the purpose of adjusting the pH in the medium, inorganic or organic acids or alkalis, buffers, etc. are added,
Alternatively, an appropriate amount of oils and fats, surfactants, etc. may be added for the purpose of defoaming. In liquid culture, the pH of the medium is preferably around neutral, preferably pH 6-8. The culturing temperature is preferably about 14 to 30 ° C., and the culturing time is preferably about 1 to 14 days. The culturing means may be according to a method known per se such as static culturing, shaking culturing or aeration and stirring culturing. For large-scale processing,
The aeration stirring culture method is preferred. Usually, the maximum production of TAN-2177A and / or B is reached in 5 to 10 days of culture.

Target compound TAN-21 from culture
A method for collecting 77A and / or B will be described below. The separation means usually used for collecting the metabolite produced by the microorganism from the culture of the microorganism is appropriately used.
For example, TAN-2177A and B are contained in the culture filtrate and the bacterial cells, so that acetone or methanol or the like is first added to the culture to extract the active substance, and the obtained extract is concentrated, and then the adsorbent resin, for example, Diaion HP-2
0 or SP-207 (manufactured by Mitsubishi Kasei), Amberlite XAD-I or II (manufactured by Rohm and Haas),
The method of subjecting to chromatography using US etc. is advantageously used. In order to elute the target active substance from the column, an organic solvent (eg, methanol, ethanol, acetone, acetonitrile, etc.) that is miscible with water and an aqueous solution [eg, water, alkali (eg, sodium hydroxide, hydroxide Aqueous solution containing potassium, sodium hydrogen carbonate, etc.), acid (eg, hydrochloric acid, acetic acid, formic acid, phosphoric acid, etc.), salt-containing aqueous solution (eg, saline, acetate buffer, phosphate buffer, etc.)] A mixed solvent of an appropriate ratio is used. Also, TAN-2177A and B can be extracted with an organic solvent immiscible with water (eg, isobutanol, methyl isobutyl ketone, etc.). The eluate or extract thus obtained is concentrated under reduced pressure to give TAN-217.
A crude material containing 7A and / or B is obtained.

The crude material was further purified to give pure TAN-2.
Various well known chromatographic methods are advantageously used to obtain 177A or B. As carriers, those that utilize the difference in the adsorptivity of compounds such as activated carbon, silica gel, microcrystalline cellulose, adsorptive resins, or those that utilize the differences in the functional groups of compounds such as ion exchange resins, ion exchange cellulose, and ion exchange Sephadex. Alternatively, a resin that utilizes the difference in the molecular weight of the compound, such as a molecular sieving resin, is advantageously used. In order to elute the target compound from these carriers, the combination varies depending on the type and properties of the carrier, but suitable organic solvents (eg, halogenated hydrocarbons such as dichloromethane and chloroform, aromatic hydrocarbons such as toluene) , Esters such as ethyl acetate, ketones such as acetone, alcohols such as methanol, ethanol, isobutanol, nitriles such as acetonitrile), organic acids (eg, acetic acid, formic acid, etc.), aqueous solutions [eg,
Water, aqueous solution containing alkali (eg, sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, etc.), aqueous solution containing acid (eg, hydrochloric acid, acetic acid, formic acid, phosphoric acid, etc.), salt-containing aqueous solution (eg, saline solution, acetate buffer) Liquid, phosphate buffer, etc.)]
Or a mixed solvent of an appropriate ratio is used.

More specifically, as a carrier, activated carbon for chromatography (manufactured by Takeda Pharmaceutical Co., Ltd.), Kieselgel 60 (manufactured by Merck, Germany), microcrystalline cellulose [eg, Avicel (manufactured by Asahi Kasei), Funacell (manufactured by Funakoshi Co., Ltd.) Etc.], adsorptive resin [eg, DIAION HP-20 or SP-20]
7 (manufactured by Mitsubishi Kasei), Amberlite XAD-I or
II (Rohm and Haas Company, USA), etc., cation exchange resin [eg, Amberlite IR-120, IRC
-50 or CG-50 (Rohm and Haas Company, USA), Dowex 50W (Dow Chemical Company, USA), Diaion SK1A (Mitsubishi Chemical Corporation)
Etc.], anion exchange resin [eg, Amberlite IRA-
402 or IRA-68 (Rohm and Haas, USA), Dowex 1 (Dow Chemical, USA), Diaion SA10B, PA-404 or W
A-30 (manufactured by Mitsubishi Kasei Co., Ltd.), ion exchange Sephadex [eg, QAE or CM-Sephadex (Pharmacia, Sweden), etc.], molecular sieving resin [eg, Sephadex LH-20 (Pharmacia) Made,
Sweden) etc.] are advantageously used.

Further, when the compound is purified, a preparative high performance liquid chromatography (HPLC) method is also advantageously used. When this method is applied, octadecylsilane (ODS) type, polymer type and silica gel type carriers are advantageously used. For example, in the case of ODS, YMC gel (manufactured by Yamamura Chemical Laboratory) or TSK gel (manufactured by Toyo Soda Kogyo Co., Ltd.) is used. In the case of a polymer type, octadecyl group-introduced ODP (manufactured by Asahi Kasei Corporation) or polyamine Introduced NH2P
(Manufactured by Asahi Kasei Co., Ltd.) or the like is used, and as the mobile phase, a mixed solution of methanol or acetonitrile and water or a salt-containing aqueous solution is advantageously used.

Since TAN-2177A and B are basic substances, they can be obtained as an acid addition salt, particularly a pharmacologically acceptable acid addition salt, by a method known per se. For example, an inorganic acid (eg, hydrochloric acid, Sulfuric acid, phosphoric acid) or organic acids (eg acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, methanesulfonic acid, octanesulfonic acid)
And the like.

The compounds TAN-2177A and B of the present invention or salts thereof have low toxicity and can be used safely. The compounds TAN-2177A and B or salts thereof have a squalene synthase inhibitory action and exhibit an effect of suppressing cholesterol synthesis. Compound TAN-
2177A and B, or salts thereof, are used as a squalene synthase inhibitor as a prophylactic or therapeutic agent for hyperlipidemia in humans and mammals (eg, monkey, horse, cow, pig, cat, dog, rabbit, etc.). To be More specifically,
Ischemic heart disease caused by hyperlipidemia (eg, angina, myocardial infarction, etc.), cerebrovascular disorder (eg, transient cerebral ischemia, cerebral infarction, cerebral thrombosis, etc.), renal disease (eg, renal sclerosis) Disease, renal failure, renal hypertension, etc.), abdominal aortic aneurysm, peripheral arteriosclerosis associated with intermittent claudication, etc.
The compounds TAN-2177A and B, or their salts, are used in yeasts such as Candida albicans.
bicans) and filamentous fungi, such as Aspergillus niger, exhibit excellent antibacterial activity and can be used, for example, in the treatment of infectious diseases caused by these microorganisms in humans and mammals. The compound is provided as a pharmaceutically suitable parenteral or oral preparation by mixing with a pharmaceutically acceptable carrier. Parenteral agents include, for example, injections, drip infusions, external preparations (eg, nasal preparations, transdermal preparations, etc.), suppositories (eg, rectal suppositories, vaginal suppositories, etc.), etc. Capsules,
Examples thereof include tablets, syrups, powders and granules.

These formulations can be produced by a method generally used in the formulation process. For example, the compound TAN-2177A or B, or a salt thereof is a dispersant (eg, Tween 80 (manufactured by Atlas Powder Co., USA), HCO 60 (manufactured by Nikko Chemicals), polyethylene glycol, carboxymethyl cellulose, sodium alginate, etc. ), Preservatives (eg, methylparaben, benzyl alcohol, chlorobutanol, etc.), isotonicity agents (eg, sodium chloride, glycerin, sorbitol, glucose, etc.) with aqueous injections, or olive oil, sesame oil, peanut oil, cottonseed oil It may be dissolved in, suspended in, or emulsified with vegetable oil such as corn oil, propylene glycol, etc., and molded into an oily injection to give an injection.

To prepare a preparation for oral administration, the compound TAN-2177A or B or a salt thereof can be prepared according to a method known per se, for example, by an excipient (eg, lactose, sucrose, starch etc.), a disintegrating agent (eg, starch). , Calcium carbonate, etc.),
Binder (eg, starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose, etc.) or lubricant (eg, talc, magnesium stearate, polyethylene glycol 6000)
Etc.) etc. are added and compression molding is carried out. Tablets, granules, and fine granules may be coated by a method known per se for the purpose of taste masking, enteric property, and sustainability. Examples of the coating agent include general film-forming coating agents [eg, hydroxypropylmethylcellulose, TC-5 (Shin-Etsu Chemical Co., Ltd.), ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, etc.], aqueous coating Agents [eg, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate,
Hydroxypropyl methylcellose acetate succinate, etc.], methyl methacrylate / methacrylic acid copolymer, Euragid-L, S (Rohm, Germany), methyl acrylate / methacrylic acid copolymer, dye (eg, talc, titanium oxide, Bengal, etc.] etc. are used.In the case of performing enteric coating, one or more intermediate layers of the above film-forming coating agent are formed between the central core containing the active ingredient and the enteric coating according to a method known per se. It is also effective to provide.

As the external preparation, the compound TAN-2177A or B or a salt thereof can be made into a solid, semi-solid or liquid external preparation according to a method known per se. For example, as the above solid, the compound TAN
-2177A or B, or a salt thereof as it is, an excipient (eg, glycol, mannitol, starch, microcrystalline cellulose, etc.), a thickener (eg, natural rubber, cellulose derivative, acrylic acid polymer, etc.), etc. Is added and mixed to obtain a powdery composition. The above-mentioned liquid form is almost the same as the case of the injection, and is an oily or aqueous suspension. In the case of semi-solid form, an aqueous or oily gel or ointment form is preferable. In addition, all of these are pH adjusting agents (eg, carbonic acid, phosphoric acid, citric acid,
Hydrochloric acid, sodium hydroxide, etc.), preservatives (eg, paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride, etc.) and the like may be added.

To make a suppository, according to a method known per se,
The compound TAN-2177A or B, or a salt thereof, can be made into an oily or aqueous solid, semisolid or liquid suppository. The oily base used in the above composition,
For example, glycerides of higher fatty acids [eg, cocoa butter, witepsols (manufactured by Dynamite Nobel Co.), etc.], vegetable oils (eg, sesame oil, soybean oil, cottonseed oil, etc.) and the like can be mentioned. Examples of the aqueous base include polyethylene glycols and propylene glycol, and examples of the aqueous gel base include natural rubbers, cellulose derivatives, vinyl polymers, acrylic acid polymers and the like.

When the compound TAN-2177 is used in humans, the dose may vary depending on the type and degree of the target disease, the age of the patient, etc., but usually the content of the compound TAN-2177A or B or its salt is 1 Adults (weight 50k
g) About 1 mg to about 500 mg, particularly preferably about 2 mg to 100 mg per person is used for the prevention and treatment of diseases. These formulations may be taken once daily or from 2 to 4
It can be administered in divided doses. Compound TAN-21
When 77A or B or a salt thereof is parenterally administered subcutaneously, intravenously or intramuscularly as an injection, the dose is about 0.5 to about 200 mg / day, preferably about 1 to
It is about 50 mg / day.

[0025]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto. The percentages (%) in the examples and test examples are weight / volume percentages unless otherwise specified. Further, the mixing ratio of the solvent indicates a volume ratio unless otherwise specified. ^{The 13} C NMR spectrum is measured by Bruker AC-300 type spectrum meter (manufactured by Bruker Co.,
Germany), tetramethylsilane was used as an internal standard, and all δ values are shown in ppm. The symbols in this specification have the following meanings. Q: Quaternary carbon

[0026]

[Example 1] Talaromyces flavus FL-557 grown on a slant medium made of Bacto potato dextrose agar medium (manufactured by Difco, USA).
Spore suspension of 55 strains (in 10% glycerol), glucose 2.0%, maltose 3.0%, raw soybean flour 1.5
%, Corn steep liquor 1.0%, polypeptone 0.5%, yeast extract 0.3%, salt 0.3%, pH
Disinfection sterilization of 500 ml of 6.0 seed culture medium 2
A liter Sakaguchi flask was inoculated and shake-cultured at 28 ° C. for 2 days. 1 liter of this culture solution was added with glucose 1.0%, mannitol 4.0%, raw soybean powder 0.5%,
Tomato paste 0.5%, peanut meal 0.5%,
Polypeptone 0.5%, glycine 0.1%, yeast extract 0.1%, cottonseed oil 0.5%, dipotassium hydrogen phosphate.
05%, iron sulfate heptahydrate 0.05%, magnesium sulfate heptahydrate 0.05%, manganese sulfate tetrahydrate 0.05
%, Calcium carbonate 0.5%, Actol 0.05
%, Silicon 0.02%, pH 7.0, 120 liters of the main culture medium was transferred to a steam-sterilized 200-liter fermentor. This main fermentation was cultivated at 28 ° C., aeration 120 liter / min, stirring 150 rpm, and internal pressure 1.0 kg / cm ^{2 for} 162 hours to produce and accumulate TAN-2177A and B.

[0027]

Example 2 Methanol (110 liters) was added to the culture solution (110 liters) obtained in Example 1, stirred for 30 minutes, and then filtered using a filter aid (Radiolite 600, Showa Chemical Industry Co., Ltd.). did. Water (60 liters) was added to the filtrate and subjected to column chromatography with Amberlite XAD-II (10 liters), 50% (v / v)
After washing with methanol water (30 liters), 80% (v / v)
It was eluted with methanol water (50 liters). After the eluate was concentrated to remove methanol, water was added to make 15 liters. After adjusting the pH of the aqueous solution to 3.0, it was extracted twice with isobutanol (5 liters) and washed with water (3 liters). After the organic layer was concentrated to dryness, the residue was treated with hexane to obtain a crude powder I (17.8 g). The culture solution (225 liters) obtained in the same manner as in Example 1 was treated in the same manner as above to obtain crude powder II (24.2 g). Obtained coarse powder I
And II are combined and silica gel (Kieselgel 60, 5,
(00 ml) column chromatography, and eluted with an eluent of sequentially increasing amounts of methanol added to a chloroform-formic acid mixture, and chloroform / methanol / formic acid (12:
A powder (1.63 g) was obtained from the 8: 1) elution fraction. The powder was subjected to column chromatography on Sephadex LH-20 (1 liter) in two batches, and fractionated by elution with methanol. The active fractions were collected and concentrated to dryness to give a powder (926 mg).

The obtained powder was divided into 4 portions and preparative HPLC
[Column; Asahipak ODP-90, diameter 21.
5 mm x length 300 mm (Asahi Kasei), mobile phase: 25%
(v / v) acetonitrile / 0.05M phosphate buffer (pH
2.5), flow rate; 10 ml / min], and HPL for analysis
At C, it was divided into two fractions giving a single peak of TAN-2177A or B. After adjusting the pH of the fraction containing TAN-2177A to 7.0, acetonitrile was distilled off under reduced pressure, and Amberlite IRA-402 (Cl type, 400 m
It was passed through a column packed with l) and washed with water (400 ml). The passing solution and the water washing solution were combined and adjusted to pH 6.5, and then subjected to Amberlite XAD-II (50 ml) column chromatography. After washing with water (150 ml), eluting with 80% (v / v) methanol water (100 ml), concentrating the eluate, and freeze-drying it to TAN-2177A.
-The monohydrochloride salt (256 mg) was obtained as a white powder.
The same operation was performed on the fraction containing TAN-2177B to obtain TAN-2177B.monohydrochloride (54 mg) as a white powder.

The physicochemical properties of the obtained TAN-2177A · monohydrochloride and TAN-2177B · monohydrochloride are shown below. TAN-2177A monohydrochloride (1) Appearance: white powder (2) Specific rotation: -32 ° (c 0.50, H ₂ O, 22 ° C) (3) Molecular weight: SI-mass spectrum; m / z 877
(M + H) ⁺ high resolution FAB-mass spectrum; m / z measured value; 877.5281 calculated value; 877.5259 (4) Elemental analysis value: (%) (calculated as 4 mol of water content) measured value; C, 48.69; H, 7.82; N, 17.36; Cl, 4.05 Calculated value; C, 48.75; H, 7.87; N, 17.05; Cl, 3.60 (5) Molecular formula: C ₄₀ H ₆₈ N ₁₂ O ₁₀ HCl (6) UV spectrum: methano- (7) IR spectrum: major absorption in KBr tablets (wavenumber, cm ^-1 ). (Fig. 1) 3340, 2960, 1660, 1550, 1460, 1390, 1250, 1100, 10
30, 610 (8) ¹³ C NMR spectrum: 75 Mz in deuterated methanol,
δppm (Fig. 2) (TAN-2177A monohydrochloride in deuterated methanol, 2
It exists as a mixture of species conformers and thus shows the signal of the main conformer) 180.1 (Q), 175.0 (Q), 174.1 (Q), 173.9 (Q), 172.5
(Q), 172.2 (Q), 171.8 (Q), 171.0 (Q), 161.2 (Q), 15
8.7 (Q), 134.6 (Q), 121.8 (CH), 76.2 (CH), 64.5 (C
H), 55.5 (CH ₂ ), 54.9 (CH), 54.3 (CH), 52.1 (CH), 5
0.5 (CH), 49.4 (CH ₂ ), 44.4 (CH ₂ ), 43.9 (CH ₂ ), 43.7
(CH ₂ ), 42.3 (CH ₂ ), 42.1 (CH ₂ ), 39.1 (CH ₂ ), 38.4 (CH
₂ ), 37.4 (CH ₂ ), 35.7 (CH), 34.8 (CH), 29.2 (CH ₂ ),
26.3 (CH ₂ ), 25.6 (CH), 23.9 (CH ₃ ), 22.0 (CH ₃ ), 21.
3 (CH ₃ ), 17.3 (CH ₃ ), 15.5 (CH ₃ ), 15.2 (CH ₃ ), 13.6
(CH ₃ ) (9) Amino acid analysis: Reaction in 6N hydrochloric acid at 110 ° C. for 15 hours, then analysis. Glycine (2 mol), leucine (1 mol), arginine (1 mol) (10) Color reaction: positive; Sakaguchi, phosphomolybdic acid reaction negative; Dragendorff, Ehrlich reaction (11) High performance liquid chromatography: column; Asahipak ODP-50, Diameter 6.0 m
m × length 150 mm (manufactured by Asahi Kasei) mobile phase; 28% (v / v) acetonitrile / 0.05 M phosphate buffer (pH 2.5) flow rate; 1.0 ml / min detection method; 214 nm retention time; 9 .8 minutes (12) Thin layer chromatography: carrier; silica gel 60F ₂₅₄ (Merck, Germany) developing solvent; chloroform: methanol: water: formic acid (6:
4: 1: 0.1) Rf value: 0.49

TAN-2177B / monohydrochloride 1) Appearance: white powder 2) Specific rotation: -31 ° (c 0.51, H ₂ O, 22 ° C) 3) Molecular weight: SI-mass spectrum; m / z 863 ( M + H)
⁺ High-resolution FAB-mass spectrum; m / z measured value; 863.5110 calculated value; 863.5103 4) Molecular formula: C ₃₉ H ₆₆ N ₁₂ O ₁₀ · HCl 5) UV spectrum: end absorption in methanol 6) IR spectrum: KBr Main absorption in tablets (wavenumber, cm ^-1 ). (Fig. 3) 3350, 2960, 1660, 1550, 1450, 1390, 1250, 1100, 10
30, 610 7) ¹³ C NMR spectrum: 75 Mz, in deuterated methanol, δp
pm (Fig. 4) (TAN-2177B monohydrochloride in deuterated methanol, 2
It exists as a mixture of species conformers and thus shows the signal of the main conformer) 179.9 (Q), 175.0 (Q), 174.1 (Q), 173.9 (Q), 173.3
(Q), 172.5 (Q), 172.1 (Q), 171.0 (Q), 161.1 (Q), 15
8.6 (Q), 134.5 (Q), 121.7 (CH), 76.1 (CH), 63.7 (C
H), 54.8 (CH), 54.3 (CH), 52.0 (CH), 50.5 (CH), 4
9.1 (CH ₂ ), 48.5 (CH ₂ ), 44.5 (CH ₂ ), 43.8 (CH ₂ ), 43.6
(CH ₂ ), 42.5 (CH ₂ ), 42.0 (CH ₂ ), 38.4 (CH ₂ ), 37.0 (C
H ₂ ), 35.7 (CH), 31.0 (CH ₂ ), 29.0 (CH ₂ ), 26.2 (C
H ₂ ), 25.7 (CH ₂ ), 25.6 (CH), 23.9 (CH ₃ ), 22.0 (C
H ₃ ), 21.3 (CH ₃ ), 15.5 (CH ₃ ), 15.2 (CH ₃ ), 13.6 (C
H ₃ ) 8) Amino acid analysis: Reaction in 6N hydrochloric acid at 110 ° C. for 15 hours, then analysis. Glycine (2 mol), leucine (1 mol), arginine (1 mol), proline (1 mol) 9) Color reaction: positive; Sakaguchi, phosphomolybdic acid reaction negative; Dragendorf, Ehrlich reaction 10) High performance liquid chromatography Graphography: Column; Asahipak ODP-50, diameter 6.0 m
m × length 150 mm (manufactured by Asahi Kasei) mobile phase; 28% (v / v) acetonitrile / 0.05M phosphate buffer (pH 2.5) flow rate; 1.0 ml / min detection method; 214 nm retention time; 6.9 minutes 11) Thin layer chromatography: carrier; silica gel 60F254 (Merck, Germany) developing solvent; chloroform: methanol: water: formic acid (6:
4: 1: 0.1) Rf value; 0.46

From the detailed examination of the above-mentioned physicochemical data and NMR spectrum, it was determined that TAN-2177A · monohydrochloride and B · monohydrochloride had the following structures.

Embedded image

[0032]

[Test Example 1] Squalene synthase inhibition test [Method] 1. Preparation of human cell-derived enzyme Dulbecco's modified Eagle medium (Gibco, USA) containing 10% (v / v) fetal bovine serum (Gibco, USA) ) Human hepatoma cells HepG2 (about 1 × 10 ⁹ cells)
s) in ice-cold buffer (100 mM potassium phosphate buffer (pH 7.
4), 30 mM nicotinamide, 2.5 mM MgCl ₂ ] 10 ml, and the cells were disrupted by ultrasonication (30 seconds, twice). The obtained sonicate was centrifuged at 10,000 × g for 20 minutes at 4 ° C., and the obtained supernatant was further centrifuged at 4 ° C. for 10 minutes.
Centrifuged at 5,000 xg for 90 minutes. The obtained precipitate was suspended in ice-cold 10 mM potassium phosphate buffer (pH 7.4) and then centrifuged again at 45,000 at 105,000 xg for 90 minutes. The precipitate (microsome fraction) thus obtained was ice-cooled 100 mM potassium phosphate buffer (pH 7.4).
Was suspended (protein concentration 4 mg / ml) to prepare an enzyme solution.

2. Measurement of squalene synthase inhibitory activity 5 μM [1- ³ H] farnesyl pyrophosphate (25 μCi / μmol
e, New England Nuclear Company, USA)
1 mM NADPH, 5 mM MgCl ₂ , 6 mM glutathione, 100 mM potassium phosphate buffer (pH 7.
4), and a solution containing TAN-2177A monohydrochloride,
Human cell-derived enzyme solution (protein amount 0.8 μg) was added to bring the total amount to 50
After making it to μl, it was reacted at 37 ° C. for 45 minutes. After adding 150 μl of chloroform / methanol mixture (1: 2) to the reaction solution to stop the reaction, 50 μl of chloroform and 50 μl of 3N sodium hydroxide solution were added to extract the reaction product into the chloroform layer (lower layer). The squalene synthase inhibitory activity was calculated by comparing the radioactivity incorporated in the chloroform layer with a control in which water was added instead of the test compound. 50% of squalene synthase of test compound
The inhibitory concentration is shown in [Table 1]. 〔result〕

TABLE 1 squalene synthase inhibitory activity Compounds _{IC 50 (nM) TAN-2177A} · monosalt salt 37

[0034]

[Test Example 2] Cholesterol biosynthesis inhibition test in rat hepatocytes [Method] Method of Seglen (Seglen PO)
In-cell biology, vol. 13, p. 29, 1976), hepatocytes were isolated from 6-week-old male SD rats by the collagenase perfusion method and seeded on a 24-well multiplate (10 ⁵
Cells / well) and then 5% (v / v) fetal bovine serum and 1n
M insulin, 1 nM dexamethasone, 100 uni
It was cultured overnight in Williams E medium (Dainippon Pharmaceutical Co., Ltd.) containing t / ml penicillin and 100 μg / ml streptomycin. The primary hepatocytes obtained in this manner were treated with TA
250 μl of Dulbecco's modified Eagle medium (manufactured by Gibco, USA) supplemented with 10% (v / v) lipoprotein-deficient human serum containing N-2177A monohydrochloride (manufactured by Sigma, USA) was added and incubated for 1 hour. , 25 mM [ ¹⁴ C]
10 μl of acetic acid (2.8 μCi / μmole) was added, and the mixture was further incubated for 4 hours. After washing twice with Dulbecco's phosphate buffered saline, 100 μl of 15% potassium hydroxide was added to lyse the cells at 37 ° C. 15% potassium hydroxide / 8
Add 400 μl of 0% (v / v) ethanol and add 1 at 75 ° C.
After saponification for an hour, 300 μl of distilled water and hexane 8
00 μl was added to extract the unsaponifiable lipid. Hexane layer
After 400 μl was evaporated to dryness under reduced pressure, it was dissolved in 200 μl of 0.1% cholesterol solution (in acetone: ethanol = 1: 1), and 400 μl of 0.5% digitonin solution (in 50% (v / v) ethanol) was added, and overnight. It was left at room temperature. The obtained precipitate was filtered with a glass filter (Advantech Toyo, GC-5
0) collected above and washed with 50% (v / v) acetone. The cholesterol biosynthesis inhibitory activity was calculated by comparing the radioactivity incorporated into the digitonin precipitate with a control in which water was added instead of the test compound. The 50% inhibitory concentration of cholesterol biosynthesis of the test compound is shown in [Table 2]. 〔result〕

[Table 2] Compound _{50 for} inhibiting cholesterol biosynthesis in rat hepatocytes IC ₅₀ (nM) TAN-2177A / monohydrochloride 140

[0035]

[Test Example 3] Mouse toxicity test TAN-2177A / monohydrochloride was added to two mice at 200 m.
No death was observed even after intraperitoneal administration of g / kg.

As shown in the above test examples, the compound of the present invention or a salt thereof has low toxicity, inhibits the activity of squalene synthase derived from human cells at a very low concentration, and also produces cholesterol in rat hepatocytes. It strongly inhibited the synthesis.

[0037]

[Formulation Example 1] Using the compound TAN-2177A monohydrochloride, all ingredients of the formulation shown below were mixed and filled in a gelatin capsule, and 30 mg of the compound TAN-2177A monohydrochloride was added per capsule. The containing capsules were manufactured. Compound TAN-2177A / monohydrochloride 30 mg Lactose 100 mg Corn starch 40 mg Magnesium stearate 10 mg Total 180 mg

[0038]

[Formulation Example 2] Compound TAN-2177A monohydrochloride, lactose, corn starch (half of the amount shown below) and hydroxypropyl cellulose were mixed, and water was added to the mixture to knead and granulate. Then, after vacuum drying, this was mixed with a mixture of magnesium stearate and corn starch (half of the amount shown below). The obtained mixture was compression-molded to produce tablets having the formulations shown below. Compound TAN-2177A monohydrochloride 60 mg Lactose 68.4 mg Corn starch 65 mg Hydroxypropyl cellulose 6 mg Magnesium stearate 0.6 mg Total 200.0 mg

[0039]

[Formulation Example 3] Compound TAN-2177A / monohydrochloride
A 0.05% (w / v) solution of the compound TAN-2177A monohydrochloride was prepared by dissolving it in a physiological saline solution containing 0% (w / v) polyethylene glycol 400, sterilized by filtration, and then put into a vial. 30 ml each was dispensed. Per vial,
An IV formulation was prepared containing 15 mg of compound TAN-2177A monohydrochloride.

[0040]

[Formulation Example 4] Using the compound TAN-2177B monohydrochloride, all the ingredients of the formulation shown below were mixed and filled into a gelatin capsule, and 30 mg of the compound TAN-2177B monohydrochloride was added per capsule. The containing capsules were manufactured. Compound TAN-2177B / monohydrochloride 30 mg Lactose 110 mg Corn starch 30 mg Magnesium stearate 10 mg Total 180 mg

[0041]

[Formulation Example 5] Compound TAN-2177B / monohydrochloride, lactose, corn starch (half of the amount shown below) and hydroxypropyl cellulose were mixed, and water was added to the mixture to knead / granulate. Then, after vacuum drying, this was mixed with a mixture of magnesium stearate and corn starch (half of the amount shown below). The obtained mixture was compression-molded to produce tablets having the formulations shown below. Compound TAN-2177B / monohydrochloride 60 mg Lactose 68.4 mg Corn starch 65 mg Hydroxypropylcellulose 6 mg Magnesium stearate 0.6 mg Total 200.0 mg

[0042]

[Formulation Example 6] Compound TAN-2177B / monohydrochloride
A 0.05% (w / v) solution of the compound TAN-2177B monohydrochloride was prepared by dissolving it in a physiological saline solution containing 0% (w / v) polyethylene glycol 400, sterilized by filtration, and then put into a vial. 30 ml each was dispensed. Per vial,
An intravenous solution was prepared containing 15 mg of compound TAN-2177B monohydrochloride.

[0043]

INDUSTRIAL APPLICABILITY The compounds TAN-2177A and B or salts thereof have low toxicity and excellent squalene synthase inhibitory activity, and are useful as prophylactic and therapeutic agents for hyperlipidemia. Further, the compound is yeast, for example, Candida albicans, and filamentous fungi,
For example Aspergillus niger
Excellent antibacterial activity against bacteria such as

[0044]

[Brief description of drawings]

FIG. 1 Infrared absorption spectrum of compound TAN-2177A (in KBr tablet).

FIG. 2 ¹³ C nuclear magnetic resonance (NMR) spectrum of compound TAN-2177A (tetramethylsilane added as an internal standard in deuterated methanol).

FIG. 3: Infrared absorption spectrum of compound TAN-2177B (in KBr tablet)

FIG. 4 ¹³ C Nuclear Magnetic Resonance (NMR) spectrum of compound TAN-2177B (in deuterated methanol, tetramethylsilane was added as an internal standard).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 1/14 C12R 1: 645) (C12P 21/00 C12R 1: 645) (72) Inventor Eiji Sunahara 2-5-3 Umezono, Tsukuba City, Ibaraki Prefecture Umezono Square B Building No. 307

Claims (7)

[Claims] (1) Formula (1) [In the formula, R represents a methyl group or a hydrogen atom] A compound TAN-2177 or a salt thereof. 2. A microorganism belonging to the genus Talaromyces and having the ability to produce the compound TAN-2177 according to claim 1, is cultured in a medium, and the compound is produced and accumulated in the culture,
The TAN according to claim 1, wherein the product is collected.
A method for producing a compound according to -2177 or a salt thereof. 3. A microorganism Tararomys flava having the ability to produce the compound of TAN-2177 according to claim 1. 4. A microorganism, Talaromyces flavus, having the ability to produce the compound TAN-2177 according to claim 1.
FL-55755 strain. 5. A medicine comprising the compound TAN-2177 according to claim 1 or a salt thereof. 6. A squalene synthase inhibitor comprising the compound TAN-2177 according to claim 1 or a salt thereof. 7. An antihyperlipidemic agent containing the compound TAN-2177 according to claim 1 or a salt thereof.