JPH09124655A - Novel squalene synthase inhibitory compounds - Google Patents

Abstract

(57) Abstract: The present invention provides a compound having squalene synthase inhibitory activity, a salt thereof or a compound having a protected carboxyl group. SOLUTION: General formula (I) (Wherein R represents a hydrogen atom or a methyl group, X and Y each represent a hydrogen atom, or together form a single bond), a salt thereof or a protected carboxyl group thereof. Compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a compound having squalene synthase inhibitory activity, a salt thereof or a compound having a protected carboxyl group.

[0002]

2. Description of the Related Art Hyperlipidemia is one of the three major risk factors for ischemic heart disease such as arteriosclerosis. It recognized. Mevalotin (registered trademark; MEVALOTIN, general name; pravastatin sodium) currently on the market is one of the powerful therapeutic agents for hyperlipidemia, and its action is HMG-CoA reductase, which is a rate-limiting enzyme in the cholesterol biosynthesis system. Is the inhibition of. Squalene synthase is an enzyme involved in the first participating reaction of the de novo cholesterol biosynthesis pathway, and is located in the cholesterol synthesis system several steps downstream of HMG-CoA reductase. After this stage, the cholesterol synthesis system is biosynthesized by a route different from that of other isoprene-derived compounds. That is, inhibiting squalene synthase without blocking the biosynthesis of ubiquinone and dolichol,
It is possible to suppress the biosynthesis of cholesterol.

Isoprenoid (phosphinylmethyl) phosphonates have been reported as squalene synthase inhibitors in US Pat. No. 4,871,721. As a fermentation product, a compound having a basic skeleton of a dioxabicyclooctane ring (zaragozic aci)
ds) in U.S. Pat. No. 5,102,907, TAN-
1607A is described in EP-568946A.

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a result of many years of earnest research on a compound having a squalene synthase inhibitory activity, the present inventors have found that Mollisia sp.
The present invention was completed by finding that a compound having squalene synthase inhibitory activity is present in the culture of p.) SANK10294 strain.

[0005]

The present invention provides (1) the general formula (I)

[0006]

[Chemical 6]

(Wherein R represents a hydrogen atom or a methyl group, and X and Y each represent a hydrogen atom, or together form a single bond), a salt thereof or a carboxyl group thereof. A protected compound of.

More specifically, the present invention relates to (2) the formula (I
I)

[0009]

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Compound A having the formula (1), a salt thereof or a compound having a protected carboxyl group, (3) Formula (III)

[0011]

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A compound B having, a salt thereof or a compound having a protected carboxyl group, (4) Formula (IV)

[0013]

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A compound C, a salt thereof or a compound protected with a carboxyl group thereof, (5) Formula (V)

[0015]

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A compound D having, a salt thereof or a compound having a protected carboxyl group.

The present invention also includes (6) culturing a bacterium that produces a compound having the formula (I) belonging to the genus Moricia and collecting the compound having the formula (I) from the culture. The manufacturing method of the compound which has these.

More specifically, the present invention relates to (7) a bacterium producing a compound having the formula (I) is Moricia sp.
The production method according to (6), which is SANK10294 strain.

The present invention also relates to (8) a compound having the formula (I), a pharmacologically acceptable salt thereof or a compound having a protected carboxyl group thereof as an active ingredient.

More specifically, the present invention provides (9) a squalene synthase inhibitory active compound comprising as an active ingredient a compound having the formula (I), a pharmacologically acceptable salt thereof or a compound having a protected carboxyl group thereof: (10) A therapeutic and / or prophylactic agent for hyperlipidemia, comprising a compound having the formula (I), a pharmacologically acceptable salt thereof or a compound having a protected carboxyl group thereof as an active ingredient.

Physics of compounds A, B, C and D of the present invention
The chemical properties are as follows. 1. Compound A 1) Properties of substance: Acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 766 3) Molecular formula: C₄₁H₅₀O₁₄ 4) UV absorption spectrum (λ_max nm): The ultraviolet absorption spectrum measured in ethanol is as shown below. 205.0, 251.5, 283.9, 293.1.

5) Infrared absorption spectrum (ν _max cm ^-1 ): An infrared absorption spectrum measured by the potassium bromide (KBr) tablet method is as shown below. 2967, 2923, 2611, 1710, 1694, 1409, 1264, 905.

6) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured with tetramethylsilane as an internal standard in deuterated methanol is as follows. 174.7 (s), 174.4 (s), 173.6 (s), 172.1 (s), 170.4 (s), 139.8 (s), 139.6 (s), 133.3 (d), 132.2 (d), 131.7 (d), 130.1 (d), 130.0 (d), 130.0 (d), 130.0 (d), 130.0 (d), 128.4 (d), 128.3 (d), 127.5 (d), 127.5 (d), 127.4 (d), 127.4 (d), 107.1 (s), 92.0 (s), 83.2 (d), 82.1 (d), 78.6 (d), 77.7 (d), 76.5 (s), 38.48 (d), 38.3 (t), 37.1 (t), 35.6 (t), 34.5 (t), 33.2 (t), 30.8 (t), 30.5 (t), 30.4 (t), 26.3 (t), 21.7 (q), 20.6 (t), 15.1 (q).

7) High Performance Liquid Chromatography: Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65: 35 Flow rate: 1.2 ml / min Detection: UV 210 nm retention time: 4.2 minutes.

2. Compound B 1) Properties of substance: Acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 780 3) Molecular formula: C ₄₂ H ₅₂ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm (ε)): in methanol The ultraviolet absorption spectrum measured in 1. is as shown below. 205.0 (4.59), 251.5 (4.49), 283.9 (3.38), 293.1 (3.18).

5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the solution method (chloroform) is as shown below. 3450, 3000, 2925, 2850, 1725, 1595, 1490, 1435, 1370, 1250, 1140, 1015, 965, 695.

6) Optical rotation: [α] ^D ₂₅ = + 14.8 (c 0.869, methanol solution).

7) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 173.7 (s), 173.1 (s), 172.5 (s), 169.1 (s), 168.5 (s), 139.3 (s), 139.1 (s), 132.9 (d), 131.7 (d), 131.2 (d), 129.6 (d), 129.5 (d), 129.5 (d), 129.5 (d), 129.5 (d), 128.0 (d), 127.8 (d), 127.0 (d), 127.0 (d), 126.9 (d), 126.9 (d), 107.2 (s), 91.0 (s), 82.2 (d), 81.0 (d), 78.0 (d), 77.0 (d), 75.8 (s), 52.5 (q), 38.0 (d), 38.0 (t), 36.3 (t), 35.0 (t), 34.0 (t), 32.7 (t), 30.3 (t), 30.0 (t), 29.9 (t), 25.8 (t), 21.1 (q), 20.1 (t), 14.6 (q).

8) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65: 35 Flow rate: 1.2 ml / min Output: UV 210 nm, retention time: 5.4 minutes.

[0030] 3. Compound C 1) Properties of substance: Acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 782 3) Molecular formula: C ₄₂ H ₅₄ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm (ε)): in methanol The ultraviolet absorption spectrum measured in 1. is as shown below. 205.6 (4.44), 251.9 (4.25), 283.3 (3.15), 293.2 (2.94).

5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the solution method (chloroform) is as shown below. 3350, 3000, 2925, 2850, 1725, 1595, 1490, 1370, 1250, 1175, 1140, 1015, 965, 690.

6) Optical rotation: [α] ^D ₂₅ = + 14.2 (c 0.874, methanol solution).

7) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 174.6 (s), 173.9 (s), 173.4 (s), 169.9 (s), 169.4 (s), 144.6 (s), 139.5 (s), 133.4 (d), 132.9 (d), 130.8 (d), 130.1 (d), 130.3 (d), 130.0 (d), 128.5 (d), 128.5 (d), 128.4 (d), 127.5 (d), 127.5 (d), 127.2 (d), 107.2 (s), 91.6 (s), 82.7 (d), 81.6 (d), 78.6 (d), 77.6 (d), 76.4 (s), 53.2 (q), 38.4 (d), 38.4 (t), 37.4 (t), 36.8 (t), 34.8 (t), 33.2 (t), 33.1 (t), 30.9 (t), 30.7 (t), 30.7 (t), 30.6 (t), 26.3 (t), 20.5 (q), 20.5 (t), 15.0 (q).

8) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65: 35 Flow rate: 1.2 ml / min Output: UV 210 nm, retention time: 6.8 minutes.

[0035] 4. Compound D 1) Property of substance: Acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 768 3) Molecular formula: C ₄₁ H ₅₂ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm): Measured in methanol The ultraviolet absorption spectrum is as shown below. 205, 251, 283.

5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the solution method (chloroform) is as shown below. 3350, 3000, 2925, 2850, 1725, 1595, 1490, 1370, 1250, 1175, 1140, 1015, 965.

6) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 173.7 (s), 173.1 (s), 172.5 (s), 170.2 (s), 168.5 (s), 144.0 (s), 139.1 (s), 132.9 (d), 129.6 (d), 129.5 (d), 129.5 (d), 129.4 (d), 129.4 (d), 129.2 (d), 129.2 (d), 128.0 (d), 127.0 (d), 127.0 (d), 126.6 (d), 107.2 (s), 91.0 (s), 82.2 (d), 81.0 (d), 78.0 (d), 76.6 (d), 75.6 (s), 38.0 (d), 38.0 (t), 36.9 (t), 36.3 (t), 35.0 (t), 32.7 (t), 32.7 (t), 30.4 (t), 30.3 (t), 30.3 (t), 30.1 (t), 25.8 (t), 21.1 (q), 20.1 (t), 14.6 (q).

7) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65: 35 Flow rate: 1.2 ml / min Output: UV 210 nm, retention time: 4.7 minutes.

The compound of the present invention represented by the general formula (I) can be converted into a salt according to a conventional method. Such salts are preferably alkali metal salts such as sodium salts, potassium salts, lithium salts; alkaline earth metal salts such as calcium salts, magnesium salts; aluminum salts, iron salts, zinc salts, Metal salts such as copper salts, nickel salts and cobalt salts; ammonium salts; t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine. Salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris (hydroxy) Methyl) amino Amine salts such as organic salts such as tan salts; hydrohalides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide; nitrates, perchlorates, sulfates , Inorganic salts such as phosphates; lower alkane sulfonates such as methane sulfonate, trifluoromethane sulfonate, ethane sulfonate; benzene sulfonate;
aryl sulfonates such as p-toluene sulfonate; organic acid salts such as acetic acid, malic acid, fumarate, succinate, citrate, oxalate, maleate;
Glycine salt, lysine salt, arginine salt, ornithine salt,
Amino acid salts such as glutamate and aspartate; Preferably, it is a pharmacologically acceptable salt.

The carboxyl-protecting group means a protecting group in the reaction (when used as a so-called intermediate) and a protecting group for forming a prodrug when administered to a living body, and examples thereof include methyl, ethyl, propyl, Isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl,
"Lower alkyl group" such as hexyl: 2,2,2-trichloroethyl, 2-bromoethyl, 2-chloroethyl, 2-fluoroethyl, 2,2-
“Halogeno lower alkyl group” such as dibromoethyl: benzyl, phenethyl, 3-phenylpropyl, α
A lower alkyl group substituted with 1 to 3 aryl groups such as naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, α-naphthyldiphenylmethyl, 9-anthrylmethyl; 4-methylbenzyl 2,4,6-trimethylbenzyl, 3,4,5-trimethylbenzyl, 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, 2-nitrobenzyl, 4
-Nitrobenzyl, 4-chlorobenzyl, 4-bromobenzyl, 4-cyanobenzyl, 4-cyanobenzyldiphenylmethyl, bis (2-nitrophenyl) methyl, lower alkyl such as piperonyl, lower alkoxy, nitro, halogen, cyano An “aralkyl group” such as a lower alkyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with: methoxymethyl, 1,1-dimethyl-1-methoxymethyl, ethoxymethyl, Lower alkoxymethyl groups such as propoxymethyl, isopropoxymethyl, butoxymethyl, t-butoxymethyl; lower alkoxylated lower alkoxymethyl groups such as 2-methoxyethoxymethyl; 2,2,2-trichloroethoxymethyl, bis ( Two
-Alkoxymethyl group "such as halogenated lower alkoxymethyl group such as -chloroethoxy) methyl; lower alkoxylation such as 1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1- (isopropoxy) ethyl "Substituted ethyl group" such as ethyl group; halogenated ethyl group such as 2,2,2-trichloroethyl; aryl-selenylated ethyl group such as 2- (phenylzelenyl) ethyl; acetoxymethyl, dimethylaminoacetoxymethyl , An acyloxyalkyl group such as propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, 1-acetoxyethyl; methoxycarbonyloxymethyl, ethoxycarbonyloxymethyl, propoxycarbonyloxymethyl, isopropoxycarbonyloxymethyl, bromo. Alkoxycarbonyloxy methyl,
Isobutoxycarbonyloxymethyl, cyclohexyloxycarbonyloxymethyl, cyclohexyloxycarbonyloxy (cyclohexyl) methyl, 1- (methoxycarbonyloxy) ethyl, 1- (ethoxycarbonyloxy) ethyl, 1- (propoxycarbonyloxy) ethyl, 1- (Isopropoxycarbonyloxy) ethyl, 1- (butoxycarbonyloxy) ethyl, 1- (isobutoxycarbonyloxy) ethyl, 1
-(T-Butoxycarbonyloxy) ethyl, 1- (cyclohexyloxycarbonyloxy) ethyl, 1-
Alkoxycarbonyloxyalkyl groups such as (ethoxycarbonyloxy) propyl; oxodioxolenyl such as 4-methyl-oxodioxolenylmethyl, 4-phenyloxodioxolenylmethyl, oxodioxolenylmethyl Examples thereof include "carbonyloxyalkyl groups" such as alkyl groups; "phthalidyl groups" such as phthalidyl, dimethylphthalidyl, and dimethoxyphthalidyl: "aryl groups" such as phenyl and indanyl.

The compound of the present invention represented by the general formula (I) has various isomers. In the compounds of the present invention, these isomers and mixtures of these isomers are all represented by the general formula (I). Therefore, the present invention also includes all of these isomers and mixtures of these isomers.

The compound of the present invention represented by the general formula (I) may absorb water by adsorbing water or become a hydrate by being left in the air or recrystallized. Yes, such hydrates are also included in the present invention.

Next, Morishia SP SANK10
The 294 strain will be described. SANK10294 strain is 1
It is a disc fungus isolated from fruiting bodies on dead trees, which was collected in April 992 at Mount Tsukuba, Ibaraki Prefecture.

The mycological properties are as follows. Ascosal is flat and disc-shaped, dark gray and slightly whitish on the edges. When dry, the edges fold inward and have a maximum diameter of 1 mm. The outer skin layer has a round fungal tissue-like appearance, is brown, and consists of thin-film cells. The pulp layer presents a dense entangled bacterial tissue. The asci are valveless, cylindrical, contain 8 spores, and are 58-66 x 5-6.5 μm in size.
The tip of the ascium turns blue due to the Meltzer reagent. The side thread is a thin cylinder, and its size is 74.5-100 x 3.5.
-4 μm. Ascospores are rod-shaped to spindle-shaped, single-celled, colorless, and have a size of 18-26.5 × 2.5-
5 μm.

The culture properties of this bacterium are as follows. When this strain is cultured on PDA at 23 ° C. for 3 weeks, the diameter of the colony reaches 3.5 to 4.0 cm, and the surface becomes fluffy and partially powdery. It may be slightly raised and have radial wrinkles. The reverse side is greenish gray with concentric and radial wrinkles sometimes visible. No soluble pigment is secreted. Conidia are not observed.

As a result of searching the strains corresponding to this bacterium based on the above-mentioned mycological properties, it was identified as a bacterium of the genus Morrisia. Considering the characteristics of this bacterium, as species similar to this bacterium, M. lignii and M. cinerea (M. cine)
rea) and so on. However, these species show very large mutations and lack of definitive characteristics, so that this bacterium cannot be identified as either. In addition, more than 300 species of the genus Morrisia have been published, and there is no suitable monograph that covers all of them. Therefore, identifying species levels here can be rather confusing. Therefore, the identification of the species level of this bacterium is withheld, and this bacterium is identified as Moriscia sp. SA.
It was identified as strain NK10294.

It should be noted that this strain is Moricia sp. SA.
NK10294 strain deposited on September 19, 1995 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry,
Deposit number FERM BP-5234 is attached.

As is well known, discophytes are used in the natural world or artificially (for example, ultraviolet irradiation, irradiation,
Mutation is likely to occur due to chemical treatment, etc.), and the SANK10294 strain of the present invention is the same in that respect. The SANK10294 strain referred to in the present invention includes all mutants thereof. In addition, among these mutants, genetic methods,
For example, those obtained by recombination, transduction, transformation, etc. are also included. That is, compounds A, B, C and / or
The SANK10294 strain producing D or D, mutants thereof, and strains not clearly distinguished from S are all S strains.
Included in the ANK10294 strain.

[0049]

BEST MODE FOR CARRYING OUT THE INVENTION The compound having the above general formula (I) is obtained by culturing this microorganism in a liquid nutrient medium containing assimilable carbon and nitrogen sources, preferably under aerobic conditions. Can be done. Further, the nutrient medium may contain mineral salts and an antifoaming agent.

The preferred carbon sources in the nutrient medium are glucose, starch, dextrins, glycerin and other carbohydrates. Other carbon sources that may be included are fructose, maltose, lactose, sucrose, mannitol and the like. Furthermore, complex nutrient sources such as oat flour, rye flour, corn starch, potato, corn flour, soybean flour and malt extract can also be used as carbon sources.

The exact amount of carbon source used in the medium will depend, in part, on the other components in the medium, but is usually 0.5 to 5.
It is in the weight percent range. These carbon sources may be used alone in the medium, or some carbon sources may be used in combination in the medium.

Preferred nitrogen sources are glutamine, cystine, alanine, glycine, proline, threonine and other amino acids, as well as bran, peanut flour, casein hydrolyzate, fish meal, malt extract, soy flour, soy casein, casamino acid. , Pharmamedia, cottonseed flour, wheat germ, meat extract, peptone, corn steep liquor, self-digesting yeast, dried yeast, yeast extract and so on. Ammonium salts (eg ammonium sulphate,
Inorganic nitrogen sources such as ammonium nitrate, ammonium chloride, ammonium phosphate) can also be used.

The various nitrogen sources can be used alone or in combination in amounts ranging from 0.1 to 10 weight percent in the medium. The carbon and nitrogen sources are generally used in combination, but need not be in pure form. Less pure ones containing trace amounts of growth factors, vitamins and mineral nutrition may be used. In addition, usual salts capable of obtaining ions such as sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride, and carbonate may be added to the medium. In addition, when a sulfur compound that can be assimilated by bacteria is added to the medium, the production amount of the target substance may increase. For example, zinc sulfate, copper sulfate, ferrous sulfate, sulfates such as ammonium sulfate, thiosulfates such as ammonium thiosulfate, inorganic sulfur compounds such as sulfites such as ammonium sulfite; cystine, cystine, L-thiazolidine- Sulfur-containing amino acids such as 4-carboxylic acid, organic sulfur compounds such as hypotaurine and sulfur-containing peptides such as glutathione, ferrous sulfate, heavy metals such as copper sulfate, vitamin B ₁ , vitamins such as biotin, thiamine If necessary, a cell growth promoting substance such as the above may be added. It also contains manganese, iron, molybdenum, zinc and other trace metals. If necessary, an antifoaming agent such as silicone oil, polyalkylene glycol ether, vegetable oil, animal oil, or a surfactant may be added to the medium, particularly if the nutrient medium is considerably foamed (especially during liquid culture). Is preferred).

A preferred method of producing the compounds of the present invention comprises inoculating the spores or mycelia of the producing microorganism into a suitable medium and then culturing under aerobic conditions. Generally, in the fermentation process, a culture medium that has been stored is first inoculated into a nutrient seed culture medium, and a microorganism serving as an inoculum for the production of an active compound is sometimes grown through three stages. After inoculation, shake the flask at 15-30 ° C, preferably 22-25 ° C,
Incubate in the temperature range of. Shaking speed is 400
Range up to rpm, preferably 190 to 220 rp
m. The seed flask is incubated for 2 to 10 days, preferably 3 to 7 days. Once grown well, the culture is used to inoculate the production medium flask. The second stage seed culture may be performed, especially when transferred to a larger vessel. In that case, part of the culture growth is inoculated into the second seed flask and cultured in the same manner,
Shorten the time.

The culturing method is not particularly limited and may be a culturing method generally used for microorganisms, such as a stirring culturing method,
A shaking culture method, an aeration culture method and the like can be used. Suitably, the stirring culture method which is an aerobic liquid culture method,
The shaking culture method or the aeration culture method is more preferable, and the shaking culture method is more preferable. The aeration and agitation culture method is industrially preferable.

After inoculating the seed flask, the fermentation production medium is shaken for 3 to 20 days, preferably 4 to 6 days (depending on whether the fermentation medium is liquid or solid), or without shaking. Incubate. Fermentation is carried out aerobically in the temperature range of 20 to 30 ° C. For shaking, speeds of 180 to 220 rpm are preferred.

For optimum results, the temperature should be 22
To 28 ° C, preferably 23 to 26 ° C. The pH of the nutrient medium suitable for producing the active compounds is in the range 3.5 to 8.5, preferably 5.0 to 7.0.

The active compound is isolated after the formation of the desired compound for a suitable time.

The production amount of the product after the culture can be measured by measuring the squalene synthase inhibitory activity.

The product after the culture is adjusted to pH 1 to 9 (suitably 2 to 5), preferably mixed with a solvent miscible with water, and the mycelium is filtered. afterwards,
The active compound is isolated from the aqueous filtrate by several methods, including the following.

That is, (1) preferably after adjusting the pH of the aqueous filtrate to between 1.5 and 2.5, in a solvent immiscible with water, such as methyl ethyl ketone, ethyl acetate, dichloromethane, butyl acetate, butanol. To liquid phase-liquid phase extraction.

(2) A method commonly used for separating and purifying organic compounds, for example, Amberlite XAD-11 (manufactured by Rohm and Haas), Diaion HP-20
Solid phase-liquid phase extraction of the aqueous filtrate onto an organic matrix such as (Mitsubishi Kasei Co., Ltd.) and methanol-water (90: 1).
0) or with an organic solvent such as acetone-water (90:10).

[0063] (3) Dowex 1 (Cl ^-), etc. to adsorb the active compound ion-exchange aqueous filtrate onto the resin, methanol - by high ionic strength organic / aqueous solvent such as aqueous 30% ammonium chloride (90:10) Elute.

Next, desalting can be carried out by using any one of the above methods 1 and 2.

Each of the three methods may also be used to further purify the active compound.

The fractions obtained by the above method can then be dried under reduced pressure to give the crude active compound. The crude compound is then generally subjected to several separation steps such as adsorption and partition chromatography. At each separation step, fractions are collected and combined based on the results of the assay and / or HPLC analysis.

Separation by chromatography can be carried out using conventional column chromatography using ionic and nonionic adsorbents. When silica gel is the adsorbent, ethyl acetate, methanol, chloroform, acetic acid, water and the like are suitable as the eluent. The preferred adsorbent for reverse phase chromatography is C8 or C18 stationary phase silica gel. A suitable eluent for reverse phase chromatography is a mixed solvent of water and acetonitrile buffered to low pH with sulfuric acid or 0.3% triethylamine and phosphoric acid.

The active compounds can also be precipitated as ammonium salts from polar solvents such as methanol.

The compounds of the general formula (I) have at least 2, and optionally 3, carboxyl groups. The reaction for protecting these carboxyl groups is performed by the following method.

1) When the protecting group for the carboxyl group is a "lower alkyl group", a "halogeno lower alkyl group" or an "aralkyl group", 1-1) the reaction is carried out in a solvent containing the carboxyl group and the corresponding alcohol. (Preferably, the alcohol itself corresponding to the ester residue portion is used as a solvent.), And it is achieved by reacting with an acid catalyst and performing solvolysis.
The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but preferably, aliphatic hydrocarbons such as hexane and heptane;
Aromatic hydrocarbons such as benzene, toluene, xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, halogenated hydrocarbons such as dichlorobenzene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane ,
Ethers such as dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-
Examples thereof include amides such as dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide. As the acid catalyst used,
It is not particularly limited as long as it is used as an acid catalyst in a usual reaction, but preferably, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, perchloric acid and phosphoric acid, acetic acid, formic acid, oxalic acid and methane. Bronsted acids such as organic acids such as sulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid; Lewis acids such as boron trichloride, boron trifluoride, boron tribromide; or acidic ion exchange resins The organic acid is preferable, and the strong organic acid is more preferable. The reaction temperature is 0 ° C. to the boiling point of the solvent used, and preferably 50 ° C. to the boiling point of the solvent used. The reaction time varies depending mainly on the reaction temperature, the starting compound, the reaction reagent or the type of solvent used, but is usually 10 minutes to 6 days, and preferably 30 minutes to 3
Days.

1-2) Alternatively, the reaction is carried out by reacting a carboxyl group with (i) a halogenating agent (for example, phosphorus pentachloride, thionyl chloride, oxalyl chloride, etc.) at room temperature for about 30 minutes to 5 minutes.
Treatment with a corresponding acid halide or (ii) a chloroformate such as methyl chloroformate and ethyl chloroformate in the presence of an organic base such as (ii) triethylamine to convert to a corresponding acid anhydride. After that, in an inert solvent (not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, preferably benzene,
Aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as methylene chloride and chloroform; esters such as ethyl acetate and propyl acetate; ethers such as ether, tetrahydrofuran, dioxane and dimethoxyethane. -Ters or nitriles such as acetonitrile), bases (eg triethylamine etc.)
In the presence of a corresponding alcohol (potassium t-butoxide is preferable when producing t-butyl ester) and −10 ° C. to 150 ° C., preferably around room temperature,
10 minutes to 15 hours, preferably 30 minutes to 10 hours,
It can also be achieved by a reaction method.

1-3) Alternatively, the reaction is carried out by reacting the carboxyl group with a diazoalkane such as diazomethane or diazoethane (usually an ether solution of diazoalkane) at around room temperature (depending on the type of reaction system, if necessary, under heating). It may be carried out.), Also achieved by the method of contacting.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, when an acidic ion exchange resin is used as the acid catalyst, it is obtained by filtering and distilling the solvent from the filtrate, and when using another acid catalyst, the reaction mixture is appropriately neutralized. Then
If insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, the organic layer containing the target compound is separated and dried over anhydrous magnesium sulfate or the like. , Obtained by distilling off the solvent. If necessary, the obtained target compound can be obtained by a conventional method,
For example, recrystallization, reprecipitation, or a method commonly used for separation and purification of organic compounds, such as Sephadex L.
H-20 (Pharmacia), Amberlite XAD
-11 (manufactured by Rohm and Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.), a method using a synthetic adsorbent such as partition column chromatography, or silica gel or It can be separated and purified by appropriately combining normal-phase / reverse-phase column chromatography methods (preferably high-performance liquid chromatography) using alkylated silica gel and eluting with a suitable eluent.

2) When the protective group for the carboxy group is "alkoxymethyl group", "substituted ethyl group", "carbonyloxyalkyl group", "phthalidyl group" or "aryl group", 2-1) the reaction is carboxyl This is achieved by reacting ^a group in which the hydrogen atom of the group or the carboxyl group is substituted with a metal such as sodium or potassium with a compound having the general formula ^Ra- Xa.

In the above formula, R ^a represents the above-mentioned “alkoxymethyl group”, “substituted ethyl group”, “carbonyloxyalkyl group”, “phthalidyl group” or “aryl group”.

X ^a is, for example, a halogen atom such as chlorine, bromine or iodine; a lower alkanesulfonyloxy group such as methanesulfonyloxy or ethanesulfonyloxy; a halogeno such as trifluoromethanesulfonyloxy or pentafluoroethanesulfonyloxy. Lower alkanesulfonyloxy group; benzenesulfonyloxy,
p-toluenesulfonyloxy, arylsulfonyloxy groups such as p-nitrobenzenesulfonyloxy, etc.,
A group that leaves as a nucleophilic residue is shown.

Examples of such compounds having R ^a -X ^a include acetoxymethyl chloride, pivaloyloxymethyl bromide, acyloxyalkyl halides such as pivaloyloxymethyl chloride, ethoxycarbonyloxymethyl chloride and iso Alkoxycarbonyloxyalkyl halides such as propoxycarbonyloxymethyl chloride, 1- (ethoxycarbonyloxy) ethyl chloride, 1- (ethoxycarbonyloxy) ethyl iodide, phthalidyl halides or (5-methyl-2-oxo- 5-Methyl-1,3-dioxolen-4-yl) methyl halides can be mentioned.

The reaction is carried out in a solvent in the presence of a base. The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, and is preferably an aliphatic hydrocarbon such as hexane or heptane;
Aromatic hydrocarbons such as benzene, toluene, xylene; methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, halogenated hydrocarbons such as dichlorobenzene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane ,
Ethers such as dimethoxyethane and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone and cyclohexanone; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-
Examples thereof include amides such as dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide.

The base used is not particularly limited as long as it is used as a base in a usual reaction, and preferably alkali metal carbonates such as sodium carbonate, potassium carbonate and lithium carbonate; carbonic acid. Alkali metal hydrogen carbonates such as sodium hydrogen, potassium hydrogen carbonate, lithium hydrogen carbonate; alkali metal hydrides such as lithium hydride, sodium hydride, potassium hydride; sodium hydroxide, potassium hydroxide, barium hydroxide , Alkali metal hydroxides such as lithium hydroxide;
Inorganic bases such as alkali metal fluorides such as sodium fluoride and potassium fluoride; alkali metal such as sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium t-butoxide, lithium methoxide Alkoxides; mercaptan alkali metals such as sodium methyl mercaptan and sodium ethyl mercaptan; N-methylmorpholine, triethylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4 -(N, N-dimethylamino) pyridine,
2,6-di (t-butyl) -4-methylpyridine, quinoline, N, N-dimethylaniline, N, N-diethylaniline, 1,5-diazabicyclo [4.3.0] nona
5-ene, 1,4-diazabicyclo [2.2.2] octane (DABCO), 1,8-diazabicyclo [5.
4.0] organic bases such as undec-7-ene (DBU) or organic metal bases such as butyllithium, lithium diisopropylamide, lithium bis (trimethylsilyl) amide. Generally, the reaction is at -20 ° C to 120 ° C, preferably 0-80 ° C.
It is achieved by reacting for 0.5 to 10 hours.

2-2) Alternatively, in the reaction, a carboxyl group or a group in which a hydrogen atom of the carboxyl group is substituted with a metal such as sodium or potassium is reacted with a compound represented by the general formula ^Ra- OH.
It is achieved by reacting a compound having the formula (I) with a condensing agent and a catalytic amount of a base.

(In the above formula, R ^a has the same meaning as described above.) The reaction is preferably carried out in the presence of a solvent. The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, and is preferably aliphatic hydrocarbons such as hexane and heptane; benzene, toluene and xylene. Aromatic hydrocarbons; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate Ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethyl Acetamide, N- methyl-2-pyrrolidone, N-
Amides such as methylpyrrolidinone and hexamethylphosphorotriamide can be mentioned. Examples of the condensing agent used include N-hydroxysuccinimide,
1-hydroxybenzotriazole, N-hydroxy-
N-hydroxy derivatives such as 5-norbornene-2,3-dicarboximide; disulfide compounds such as 2,2′-dipyridyldisulfide; N, N′-
Succinic acid compounds such as disuccinimidyl carbonate; phosphinic chloride compounds such as N, N'-bis (2-oxo-3-oxazolidinyl) phosphinic chloride; N, N'- Disuccinimidyl oxalate (DSO), N, N'-diphthalimido oxalate (DPO), N, N'-bis (norbornenyl succinimidyl) oxalate (BNO), 1,
1'-bis (benzotriazolyl) oxalate (BB
TO), 1,1'-bis (6-chlorobenzotriazolyl) oxalate (BCTO), 1,1'-bis (6-
Oxalate derivatives such as trifluoromethylbenzotriazolyl) oxalate (BTBO); triarylphosphines such as triphenylphosphine, diethyl lower azodicarboxylates such as diethyl azodicarboxylate-triphenylphosphine -Triarylphosphines such as triarylphosphines; N-ethyl-
N-lower alkyl-5-arylisoxazolium-3'-sulphonates such as 5-phenylisoxazolium-3'-sulphonate; N ', N'-dicyclohexylcarbodiimide (DCC) Like N ', N'
-Dicycloalkylcarbodiimides, 1-ethyl-3
-(3-dimethylaminopropyl) carbodiimide (E
Carbodiimide derivatives such as DAPC); diheteroaryl diselenides such as di-2-pyridyl diselenide;
Arylsulfonyltriazolides such as p-nitrobenzenesulfonyltriazolide; 2-halo-1-lower alkylpyridinium halides such as 2-chloro-1-methylpyridinium iodide; such as diphenylphosphoryl azide (DPPA) Diarylphosphoryl azides; 1,1′-oxalyldiimidazole,
Imidazole derivatives such as N, N'-carbonyldiimidazole; 1-hydroxybenzotriazole (HO
Benzotriazole derivatives such as BT); dicarboximide derivatives such as N-hydroxy-5-norbornene-2,3-dicarboximide (HONB); preferably diarylphosphoryl azides. Is. The base used is not particularly limited as long as it is used as a base in a usual reaction, but preferably N-methylmorpholine, triethylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methyl are used. Piperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4-
Examples of organic bases such as (N, N-dimethylamino) pyridine, 2,6-di (t-butyl) -4-methylpyridine, quinoline, N, N-dimethylaniline, N, N-diethylaniline. You can In addition, 4- (N, N-dimethylamino) pyridine and 4-pyrrolidinopyridine are
Catalytic amounts can also be used in combination with other bases,
In addition, in order to effectively carry out the reaction, quaternary ammonium salts such as benzyltriethylammonium chloride and tetrabutylammonium chloride, dibenzo-
Crown ethers such as 18-crown-6 can also be added. Reaction temperature is -20 ° C to 80 ° C
The temperature is preferably 0 ° C. to room temperature. The reaction time varies depending mainly on the reaction temperature, the starting compound, the reaction reagent or the type of solvent used, but is usually 10 minutes to 3 days, and preferably 30 minutes to 1 day.

2-3) Alternatively, in the reaction, a carboxyl group or a group in which the hydrogen atom of the carboxyl group is substituted with a metal such as sodium or potassium is prepared by the general formula ^Ra- OH.
It is achieved by reacting a compound having ## STR6 ## with a halogenated phosphoric acid diester such as diethyl chlorophosphate and a base in a solvent.

[In the above formula, R ^a has the same meaning as described above. The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, preferably aliphatic hydrocarbons such as hexane and heptane; benzene, toluene and xylene. Such aromatic hydrocarbons;
Halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate; diethyl ether, diisopropyl ether Ethers such as ter, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N- Methyl-2-
Mention may be made of amides such as pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide. As the base to be used, the same base as described in 2-1) above can be used. The reaction temperature is 0 ° C to the reflux temperature of the solvent used, and preferably room temperature to 50 ° C. The reaction time is
The reaction time is usually 10 minutes to 3 days, preferably 30 minutes to 1 day, varying mainly depending on the reaction temperature, the raw material compound, the reaction reagent or the type of solvent used.

In the case of lower alkylation, it can also be carried out by reacting it with a dialkylsulfate such as dimethylsulfate or diethylsulfate according to a conventional method.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, the organic layer containing the target compound is separated. It is obtained by distilling off the solvent after drying over anhydrous magnesium sulfate and the like. If necessary, the obtained target compound is subjected to a conventional method such as recrystallization, reprecipitation, or a method commonly used for separation and purification of organic compounds, such as silica gel, alumina, magnesium-silica gel florisil, etc. Adsorption column chromatography using a carrier; Sephadex LH-20 (Pharmacia), Amberlite XAD-11 (Rohm and
Haas Co., Ltd.), Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.), a method using a synthetic adsorbent such as partition column chromatography, or normal phase / reverse using silica gel or alkylated silica gel. It can be separated and purified by appropriately combining a phase column chromatography method (preferably high performance liquid chromatography) and eluting with a suitable eluent.

The intrinsic squalene synthase inhibitory activity of the compound of the present invention represented by the general formula (I), a salt thereof or a compound having a protected carboxyl group thereof is shown in, for example, US Pat. No. 5,102,907, and RMTait, Anal.Bi
It can be measured by the standard in vitro protocol described below according to the method described in Ochem., 203, page 310 (1992).

For example, under anaerobic conditions, rat liver microsome suspension and [ ¹⁴ C] -farnesyl pyrophosphate (FPP) were used.
Is incubated at 37 ° C for 20 minutes, the radioactivity uptake into squalene is measured, and the inhibitory activity is quantitatively measured.

The reaction is carried out, for example, as follows.

It is carried out in a 16 × 110 mm test tube. The composition of the reaction solution is shown below. Reaction solution 1 assay 100 μl
50 mM potassium dihydrogen phosphate / dipotassium hydrogen phosphate (pH 7.5), 10 mM sodium fluoride, 10 mM magnesium chloride, 2 mM dithiothreitol (DTT), 50 mM ascorbate oxidase, 1 mM reduced nicotinamide adenine dinucleotide Phosphate (NADPH), 1
0μM [4- ¹⁴ C] -FPP, including rat liver microsomes suspension, an inhibitor solution.

To measure the inhibitory activity, the rat liver microsome suspension is added last to start the reaction. The reaction was carried out in a constant temperature bath at 37 ° C, incubated for 20 minutes, and then 100
μl of 40% potassium hydroxide and 95% ethanol 1:
Stop the reaction by adding 1 mixture. 65 more
After heating at ℃ for 30 minutes, cool, add 2 ml of hexane to extract unsaponifiable matter, and collect 1 ml of hexane layer.
Radioactivity is measured with a liquid scintillation counter by mixing with 10 ml of scintillator.

The compound of the present invention having the general formula (I), a salt thereof or a compound having a protected carboxyl group has squalene synthase inhibitory activity and is useful as a therapeutic and / or prophylactic agent for hyperlipidemia. is there. When the compound having the general formula (I), a salt thereof or a compound having a protected carboxyl group is used as, for example, a therapeutic and / or prophylactic agent for hyperlipidemia, it can be administered in various forms.

Examples of the dosage form include tablets, pills,
Examples thereof include oral administration by capsules, powders, granules, syrups, solutions, suspensions, emulsions, etc., rectal administration by suppositories, and injections (intravenous, intramuscular, subcutaneous) and the like. These various preparations are prepared by using known auxiliary agents such as an excipient, a binder, a disintegrating agent, a lubricant, a solubilizer, a flavoring agent, a coating agent, etc., which are commonly used in the field of known pharmaceutical preparations, as the main ingredient according to a conventional method. It can be formulated.

In the case of molding into tablets, those conventionally known in this field can be widely used as carriers, for example, lactose, sucrose, sodium chloride, glucose, urea, starch,
Binding of excipients such as calcium carbonate, kaolin, crystalline cellulose, silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone sugar Agent, dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, disintegrants such as lactose, white sugar, stearin, cacao Decay inhibitors such as butter and hydrogenated oil, quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid. , Ltd. talc, stearates,
Lubricants such as boric acid powder and polyethylene glycol can be exemplified. Furthermore, tablets are tablets that have been given a normal coating as necessary,
For example, a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, or a multi-layer tablet can be used.

In the case of molding in the form of pills, those conventionally known in this field can be widely used as carriers, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, kaolin and talc, and Arabic. Examples thereof include rubber powder, tragacanth powder, a binder such as gelatin and ethanol, a disintegrating agent such as laminaranthantene, and the like. In the case of molding in the form of suppositories, various carriers conventionally known in this field can be widely used, and examples thereof include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semisynthetic glycerides. it can.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood, and when formed into a solution, emulsion or suspension, they are diluted. All the agents conventionally used in this field can be used as the agent, for example, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol,
Examples thereof include polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. may be added. You may add.

If necessary, coloring agents, preservatives, fragrances,
Flavors, sweeteners, etc. and other pharmaceuticals may be included.

The amount of the active ingredient compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and may be appropriately selected within a wide range, but it is usually 1 to 70% by weight, preferably 1 to 30% by weight in the whole composition. Is appropriate.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and it is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered alone or mixed with an ordinary replenisher such as glucose or amino acid, and then intravenously administered. Further, if necessary, it may be administered intramuscularly, intracutaneously, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally.

The amount used varies depending on symptoms, age, weight, administration method, dosage form, etc., but is usually 1 for adults.
Day, 1,000 mg as an upper limit (preferably 100 m
g, more preferably 10 mg), and as the lower limit
0.001 mg (preferably 0.01 mg, more preferably 0.1 mg) can be administered.

[0100]

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

Example 1 Preparation of compounds A, B, C and D
Construction 1) Culture MBG-3-with the following composition for both seed culture and production medium
8 media were used.

[0102]

[Table 1] Medium composition (MBG-3-8 medium) Glycerin 70 g Glucose 30 g Soybean flour 10 g Peptone (Kyokuto Pharmaceutical Co., Ltd.) 10 g Corn steep liquor 10 g Magnesium sulfate 1 g Sodium nitrate 0.5 g ────────────────────────── Tap water 1,000 ml pH 6.5 (before sterilization).

100 ml of the medium having the above composition was placed in a 500 ml Erlenmeyer flask with a baffle, and the medium was kept at 121 ° C. for 30 minutes.
Sterilized for a minute. As a seed culture, one platinum loop was inoculated from a slant of SANK10294 strain into an Erlenmeyer flask containing this medium, and a rotary shaker (200 rp) was used at 23 ° C. for 6 days.
m, 5 cm throw). afterwards,
As the second stage seed culture, 500 ml of the same medium was placed in a 2000 ml Erlenmeyer flask, and 3% of the above culture was inoculated into the Erlenmeyer flask containing the medium that had been sterilized at 121 ° C. for 30 minutes.
m, 5 cm throw).

This second stage seed culture was added to 30 liters of a medium of the same composition and sterilized at 121 ° C. for 30 minutes.
3% was added to a 60 liter tank. A third-stage seed culture was prepared by performing aeration and stirring culture at 23 ° C. for 2 days with an aeration rate of 1.0 vvm. This third stage seed culture solution was added to a 600 liter tank in which 300 liters of a medium having the same composition was placed and sterilized at 121 ° C. for 35 minutes, and 3% was added. Main culture was carried out at 23 ° C., an air flow rate of 1.0 vvm, and a dissolved oxygen concentration (DO) of 5 ppm for 7 days.

B) Isolation To 900 liters of the culture broth, 1000 liters of acetone was added, and the mixture was stirred at room temperature for 2 hours. Then, the mixture was filtered through Celite to obtain 1790 liters of an acetone extract. The extract was adjusted to pH 3 with hydrochloric acid and then extracted with 2000 liters of ethyl acetate to obtain 2760 liters of ethyl acetate extract as the upper layer. This was washed with 500 liters of saturated saline and then concentrated to 58 liters. After adding 10 liters of water and adjusting the pH to 9.5 with sodium hydroxide, the mixture was stirred and transferred to the water layer to obtain 10 liters of water layer. 20
1.5 liters of a% saline solution was added thereto for salting out. The resulting precipitate was filtered through Celite, and the precipitate was collected and dissolved in 10 liters of water. After adjusting the pH of this solution to 3 with hydrochloric acid, 1
Extraction was performed with 5 liters of ethyl acetate to obtain 15 liters of ethyl acetate extract as the upper layer. Saturated saline solution 500
The extract was washed with liter, dried over anhydrous sodium sulfate, concentrated and dried to obtain 97 g of an oily substance. 50 oil
It was dissolved in 0 ml of methanol and methanol-water (40:
It was applied to a 6-liter column (Cosmo Seal 140C18-OPN, manufactured by Nacalai Tesque, Inc.) equilibrated with 60). 20 liters of methanol-water (40:60),
Methanol-water (60:40) was eluted with 30 liters. The respective eluates were combined and concentrated to dryness to obtain 64 g of an oily substance.

The obtained oily substance was purified by HPLC fractionation under the fractionation conditions shown below. Column: YMC Packed column ODS-1050-20-SR (10 cm x 50 cm) (manufactured by YMC Co., Ltd.) Mobile phase: acetonitrile-0.01% sulfuric acid aqueous solution (65:35) Flow rate: 200 ml / min Detection: UV The oily substance dissolved in the 254 nm mobile phase was applied to a column equivalent to 6 g at a time, and the peak reacted with the UV detector was fractionated into 5 fractions. This was repeated 10 times.

The obtained five fractions were analyzed by HPLC under the following conditions to contain fraction 2 containing compound A showing a substantially single peak at a retention time of 3.3 minutes and compound B showing a peak at 4.4 minutes. Fraction 4 was obtained.

The analysis conditions are as follows. Column: Radial pack cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = (65:35) Flow rate: 1.5 ml / min Detection: UV 254 nm fractionation Acetonitrile was distilled off separately from fraction 2 and fraction 4, and each was extracted with ethyl acetate.
The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and dried to give 26.4 g of crude compound A and compound B 1.
1.85 g was obtained. Fractions 1, 3 and 5 were combined and similarly concentrated to dryness to obtain 10.5 g of the other fractions. In order to further purify Compound A and Compound B, the above-mentioned HPL
C Same method as for preparative conditions, but mobile phase only 60:40
Each was re-sorted. Acetonitrile was distilled off and extracted with ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and dried to give pure compound A (15.5 g) and compound B (2.2 g). The other fractions were similarly subjected to HPLC fractionation, and pure compound D showing a single peak at 6.8 minutes under the above-mentioned analysis conditions showed a single peak at 7.7 g and 4.7 minutes. 0.7 g of the pure compound C shown was obtained.

Example 2 Preparation of ammonium salt of Compound A A 1 mmol sample of the free acid of Compound A is dissolved in 100 ml of ethyl acetate. By saturating the resulting solution with gaseous ammonia, the ammonium salt precipitates from the solution.

Example 3 Preparation of Compound A Potassium Salt A 1 mmol sample of the free acid of Compound A is dissolved in 100 ml of ethyl acetate. An aqueous solution containing 3 mmol of potassium hydroxide or a methanol solution was added to the obtained solution. Evaporation of the solvent produces the tripotassium salt. 1mm
The addition of 3 mmol of potassium hydroxide from ol likewise gives a mixture of monopotassium, dipotassium and tripotassium salts, the composition of which varies depending on the amount of potassium hydroxide added.

Similarly, sodium salt and lithium salt can be formed.

Example 4 Preparation of Compound A Calcium Salt A 1 mmol sample of the free acid of Compound A is dissolved in 100 ml of methanol / water (60:40). Treatment with 1 mmol of aqueous calcium hydroxide solution and removal of the solvent gives the corresponding calcium salt.

Example 5 Preparation of Arginine Salt of Compound A A 1 mmol sample of the free acid of Compound A is dissolved in 100 ml of methanol / water (60:40). Treated with an aqueous solution of 1 mmol to 0.3 mmol of L-arginine,
Removal of the solvent gives the corresponding arginine salt.

The composition is determined by the molar ratio of amino acid to free acid of compound A.

Example 6 Preparation of Compound A Ester 10 mg of the free acid of Compound A was dissolved in 10 ml of methanol and reacted with a slight excess of ethereal diazomethane. After 20 minutes, excess diazomethane and solvent were removed to give the trimethyl ester of Compound A.

The obtained compound had a molecular weight of 808 according to the FAB-MS method and had a composition of C ₄₄ H ₅₆ O ₁₄ according to high resolution measurement.
It was decided.

Further, the δ value (multiplicity) of the ¹³ C-nuclear magnetic resonance spectrum (90 MHz) measured by using tetramethylsilane as an internal standard in deuterated methanol was as follows.

173.4 (s), 173.0 (s), 172.2 (s), 168.7 (s), 167.4 (s), 139.2 (s), 139.1 (s), 132.9 (d), 131.6 (d), 131.3 ( d), 129.6 (d), 129.5 (d), 129.5 (d), 129.5 (d), 129.5 (d), 128.0 (d), 127.8 (d), 127.0 (d), 127.0 (d), 126.9 ( d), 126.9 (d), 107.5 (s), 91.1 (s), 81.8 (d), 81.0 (d), 78.0 (d), 77.9 (d), 76.2 (s), 53.6 (q), 53.0 ( q), 52.8 (q), 38.0 (d), 38.0 (t), 36.0 (t), 34.9 (t), 33.9 (t), 32.6 (t), 30.3 (t), 29.9 (t), 29.8 ( t), 25.8 (t), 21.1 (q), 20.1 (t), 14.7 (q).

[0119]

[Test Examples] Next, the effects of the present invention will be described with reference to test examples.

Test Example 1 Squalene synthase inhibitory activity Squalene synthase inhibitory activity was assayed by the following method. That is, under anaerobic conditions, rat liver microsome suspension and [ ¹⁴ C] -farnesyl pyrophosphate (FPP) 37
Incubation at 20 ° C for 20 minutes, the uptake of radioactivity into squalene was measured, and the inhibitory activity was quantitatively measured (US Pat. No. 5,102,907, etc., and RMTait, Anal.Bio.
chem., 203, 310 (1992)).

That is, the reaction was carried out in a 16 × 110 mm test tube. The composition of the reaction solution is shown below.

[Reaction Solution Composition] Reaction Solution 1 Assay 100
In μL, 50 mM potassium dihydrogen phosphate / dipotassium hydrogen phosphate (pH 7.5), 10 mM sodium fluoride, 10 mM magnesium chloride, 2 mM DT
T, 50 mM ascorbate oxidase, 1 mM
^{NADPH, 10μM [4- 14 C]} -FPP, including rat liver microsomes suspension, an inhibitor solution.

[Measurement of Inhibitory Activity] To measure the inhibitory activity, the rat liver microsome suspension was added last to start the reaction. The reaction was carried out in a constant temperature bath at 37 ° C, and after incubation for 20 minutes, 100 µl of 40% potassium hydroxide and 9
The reaction was stopped by adding a 1: 1 mixture of 5% ethanol. This was further heated at 65 ° C. for 30 minutes and then cooled, 2 ml of hexane was added to extract the unsaponifiable matter, 1 ml of the hexane layer was collected and mixed with 10 ml of scintillator, and the radioactivity was measured by a liquid scintillation counter.

The results are shown below.

The 50% inhibitory concentration (IC ₅₀ ) in the above assay was as follows.

[0126]

[0127]

[Formulation Example] Next, a formulation example will be given. Formulation Example 1 Oral capsule

[0128]

Table 3 Formulation Compound A 30 mg Lactose 170 mg Corn starch 150 mg Magnesium stearate 2 mg ─────────────────────────── 352 mg The powders of the above formulation were mixed, passed through a 30 mesh sieve, and 352 mg of this powder was placed in a gelatin capsule.
Use as a capsule.

[0129]

As described above, the physiologically active substance of the present invention is
Since it not only inhibits squalene synthase but also has high safety, it is useful as, for example, a therapeutic and / or prophylactic agent for hyperlipidemia.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication (C12N 1/20 C12R 1:01) (C12P 17/18 C12R 1:01) (72) Inventor Takayuki Yasuyuki 389-4 Oigata, Shimokawa, Izumi-cho, Iwaki, Fukushima Prefecture Sankyo Co., Ltd. (72) Inventor Takeshi Kinoshita 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Go Hosoya Ibaraki 33 Miyukigaoka, Tsukuba City, Japan Sankyo Co., Ltd.

Claims (14)

[Claims] 1. A compound of the general formula (I) (Wherein R represents a hydrogen atom or a methyl group, X and Y each represent a hydrogen atom, or together form a single bond), a salt thereof or a protected carboxyl group thereof. Compound. 2. The following formula (II): A having the formula (1), a salt thereof or a compound having a protected carboxyl group. 3. The following formula (III): A compound B having the formula (1), a salt thereof or a compound having a protected carboxyl group. 4. The following formula (IV): C, a salt thereof or a compound having a protected carboxyl group. 5. The following formula (V): A compound D having the formula (1), a salt thereof or a compound having a protected carboxyl group. 6. A compound A having the following physicochemical properties:
A compound in which the salt or the carboxyl group is protected. 1) Properties of substance: Acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 766 3) Molecular formula: C ₄₁ H ₅₀ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm): Ultraviolet absorption measured in ethanol The spectrum is as shown below. 205.0, 251.5, 283.9, 293.1. 5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the potassium bromide (KBr) tablet method is as shown below. 2967, 2923, 2611, 1710, 1694, 1409, 1264, 905. 6) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 174.7 (s), 174.4 (s), 173.6 (s), 172.1 (s), 170.4 (s), 139.8 (s), 139.6 (s), 133.3 (d), 132.2 (d), 131.7 (d), 130.1 (d), 130.0 (d), 130.0 (d), 130.0 (d), 130.0 (d), 128.4 (d), 128.3 (d), 127.5 (d), 127.5 (d), 127.4 (d), 127.4 (d), 107.1 (s), 92.0 (s), 83.2 (d), 82.1 (d), 78.6 (d), 77.7 (d), 76.5 (s), 38.48 (d), 38.3 (t), 37.1 (t), 35.6 (t), 34.5 (t), 33.2 (t), 30.8 (t), 30.5 (t), 30.4 (t), 26.3 (t), 21.7 (q), 20.6 (t), 15.1 (q). 7) High-performance liquid chromatography: Column: Radial pack cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65:35 Flow rate: 1.2 ml / min Detection: UV 210nm retention time: 4.2 minutes. 7. A compound B having the following physicochemical properties,
A compound in which the salt or the carboxyl group is protected. 1) Properties of substance: Acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 780 3) Molecular formula: C ₄₂ H ₅₂ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm (ε)): Measured in methanol The ultraviolet absorption spectrum is shown below. 205.0 (4.59), 251.5 (4.49), 283.9 (3.38), 293.1 (3.18). 5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the solution method (chloroform) is as shown below. 3450, 3000, 2925, 2850, 1725, 1595, 1490, 1435, 1370, 1250, 1140, 1015, 965, 695. 6) Optical rotation: [α] ^D ₂₅ = + 14.8 (c 0.869, methanol solution). 7) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 173.7 (s), 173.1 (s), 172.5 (s), 169.1 (s), 168.5 (s), 139.3 (s), 139.1 (s), 132.9 (d), 131.7 (d), 131.2 (d), 129.6 (d), 129.5 (d), 129.5 (d), 129.5 (d), 129.5 (d), 128.0 (d), 127.8 (d), 127.0 (d), 127.0 (d), 126.9 (d), 126.9 (d), 107.2 (s), 91.0 (s), 82.2 (d), 81.0 (d), 78.0 (d), 77.0 (d), 75.8 (s), 52.5 (q), 38.0 (d), 38.0 (t), 36.3 (t), 35.0 (t), 34.0 (t), 32.7 (t), 30.3 (t), 30.0 (t), 29.9 (t), 25.8 (t), 21.1 (q), 20.1 (t), 14.6 (q). 8) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65:35 Flow rate: 1.2 ml / min Detection: UV 210 nm retention time: 5.4 minutes. 8. A compound C having the following physicochemical properties,
A compound in which the salt or the carboxyl group is protected. 1) Property of substance: acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 782 3) Molecular formula: C ₄₂ H ₅₄ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm (ε)): Measured in methanol The ultraviolet absorption spectrum is shown below. 205.6 (4.44), 251.9 (4.25), 283.3 (3.15), 293.2 (2.94). 5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the solution method (chloroform) is as shown below. 3350, 3000, 2925, 2850, 1725, 1595, 1490, 1370, 1250, 1175, 1140, 1015, 965, 690. 6) Optical rotation: [α] ^D ₂₅ = + 14.2 (c 0.874, methanol solution). 7) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 174.6 (s), 173.9 (s), 173.4 (s), 169.9 (s), 169.4 (s), 144.6 (s), 139.5 (s), 133.4 (d), 132.9 (d), 130.8 (d), 130.1 (d), 130.3 (d), 130.0 (d), 128.5 (d), 128.5 (d), 128.4 (d), 127.5 (d), 127.5 (d), 127.2 (d), 107.2 (s), 91.6 (s), 82.7 (d), 81.6 (d), 78.6 (d), 77.6 (d), 76.4 (s), 53.2 (q), 38.4 (d), 38.4 (t), 37.4 (t), 36.8 (t), 34.8 (t), 33.2 (t), 33.1 (t), 30.9 (t), 30.7 (t), 30.7 (t), 30.6 (t), 26.3 (t), 20.5 (q), 20.5 (t), 15.0 (q). 8) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65:35 Flow rate: 1.2 ml / min Detection: UV 210 nm retention time: 6.8 minutes. 9. A compound D having the following physicochemical properties,
A compound in which the salt or the carboxyl group is protected. 1) Properties of substance: acidic fat-soluble white powder 2) Molecular weight (FAB-MS method): 768 3) Molecular formula: C ₄₁ H ₅₂ O ₁₄ 4) Ultraviolet absorption spectrum (λ _max nm): Ultraviolet absorption measured in methanol The spectrum is as shown below. 205, 251, 283. 5) Infrared absorption spectrum (ν _max cm ^-1 ): The infrared absorption spectrum measured by the solution method (chloroform) is as shown below. 3350, 3000, 2925, 2850, 1725, 1595, 1490, 1370, 1250, 1175, 1140, 1015, 965. 6) ¹³ C-nuclear magnetic resonance spectrum (δ ppm): The ¹³ C-nuclear magnetic resonance spectrum (90 MH) measured in deuterated methanol with tetramethylsilane as an internal standard is as follows. 173.7 (s), 173.1 (s), 172.5 (s), 170.2 (s), 168.5 (s), 144.0 (s), 139.1 (s), 132.9 (d), 129.6 (d), 129.5 (d), 129.5 (d), 129.4 (d), 129.4 (d), 129.2 (d), 129.2 (d), 128.0 (d), 127.0 (d), 127.0 (d), 126.6 (d), 107.2 (s), 91.0 (s), 82.2 (d), 81.0 (d), 78.0 (d), 76.6 (d), 75.6 (s), 38.0 (d), 38.0 (t), 36.9 (t), 36.3 (t), 35.0 (t), 32.7 (t), 32.7 (t), 30.4 (t), 30.3 (t), 30.3 (t), 30.1 (t), 25.8 (t), 21.1 (q), 20.1 (t), 14.6 (q). 7) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184, manufactured by Waters) Mobile phase: Acetonitrile: Triethylamine-phosphate buffer (pH 3.2) = 65:35 Flow rate: 1.2 ml / min Detection: UV 210 nm retention time: 4.7 minutes. 10. A compound of formula (I), which comprises culturing a bacterium producing a compound of formula (I) belonging to the genus Morrisia and collecting the compound of formula (I) from the culture. Manufacturing method. 11. A bacterium producing a compound having the formula (I) is Moricia sp. SANK10294 strain.
The production method according to 0. 12. A medicament comprising a compound having the formula (I) as an active ingredient. 13. A squalene synthase inhibitory active compound comprising a compound having the formula (I) as an active ingredient. 14. A therapeutic and / or prophylactic agent for hyperlipidemia, which comprises a compound having the formula (I) as an active ingredient.