JP2003012607A - Novel mevastatin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel mevastatin derivative expected to express excellent inhibiting action to 3-hydroxy-3-methylglutaryl.coenzyme A reductase. SOLUTION: This novel mevastatin derivative is expressed by general formula (I) (wherein R1 and R2 express each H or OH, but the case when R1 and R2 express H at the same time is excepted). The derivative is produced by adding mevastatin to a microorganism belonging to the genus Streptomyces and having activity to hydroxylate 3" and 4" positions of mevastatin, e.g. Streptomyces sp. TM-6 strain, a culture solution obtained by culturing the TM-6 strain or a solution containing the cells separated from the culture solution and reacting them.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel derivative of mevastatin that inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is a rate-limiting enzyme in a cholesterol biosynthesis system.

[0002]

BACKGROUND ART Mevastatin represented by the following formula is separated from metabolites of Penicillium citrinum, a kind of blue mold, and 3-hydroxy-3-methylglutaryl coenzyme A, which is a rate-limiting enzyme in the cholesterol biosynthesis system. Known to inhibit reductase (JP-A-5
0-155690, J. Antibiotics, 29, 1346 to 1348 (197
6)).

[0003]

[Chemical 4]

[0004] Pravastatin, which was found during the administration of mevastatin to animals, is a compound in which the 6-position of the lactone ring-opened form of mevastatin is hydroxylated, and more strongly 3-hydroxy-3-methylgluta. Inhibition of Ryl-Coenzyme A reductase was found (Japanese Patent Laid-Open No. 57-2240), and it is marketed as a therapeutic agent for hyperlipidemia all over the world.

[0005]

As a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor that can be used as a therapeutic agent for hyperlipidemia, many compounds have already been proposed, and as described above, one of them has been proposed. The department is widely used clinically. However, there is still a need for better therapeutic agents for hyperlipidemia.

[0006]

[Means for Solving the Problems] In order to develop a new production method of pravastatin by a bioconversion method using mevastatin as a starting material, the present inventors have identified the 6-position hydrogen atom of mevastatin from a wide range of microbial groups. As a result of an attempt to screen a microorganism that can be converted into a hydroxyl group, a strain belonging to the genus Streptomyces was found to have the conversion ability, and a patent application was previously filed (Japanese Patent Application No. 2001-1664).
No. 12). As a result of further research, a metabolite of mevastatin, which is different from pravastatin, was found in the conversion reaction solution of the strain, and the present invention was completed.

That is, the present invention has the following general formula (I)

[Chemical 5] (In the formula, R ₁ and R ₂ represent a hydrogen atom or a hydroxyl group, except when R ₁ and R ₂ are both hydrogen atoms), and a mevastatin derivative and a salt thereof. is there.

Specifically, a mevastatin derivative represented by the following formula (II) in which the 4 "-position of mevastatin is hydroxylated and a mevastatin derivative represented by the following formula (III) in which the 3" -position of mevastatin is hydroxylated And a salt thereof.

[Chemical 6]

[Chemical 7]

The derivative of the present invention is a hydroxide of mevastatin, which has an inhibitory action on 3-hydroxy-3-methylglutaryl coenzyme A reductase, and is an excellent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Expected to have action.

The mevastatin derivative of the present invention includes a microorganism that belongs to the genus Streptomyces and has the activity of hydroxylating the 3 "-position or 4" -position of mevastatin, and the culture solution or a cell separated from the culture solution. It can be produced by adding mevastatin to the solution and reacting it to produce and accumulate these derivatives in the reaction solution and collect them.

The microorganism used in the present invention may be any microorganism belonging to the genus Streptomyces and having the ability to hydroxylate the 3 "or 4" position of mevastatin. For example, Streptomyces sp. sp.) TM-6 strain and Streptomyces sp.
Examples thereof include TM-7 strains, but not limited to these strains, any strains capable of hydroxylating mevastatin to the mevastatin derivative of the present invention, including mutants thereof, can be used in the present invention . The above TM-6 strain and
TM-7 strains have been deposited with the National Institute of Advanced Industrial Science and Technology as FERM P-18311 and FERM P-18312, respectively.

Any medium can be used in the present invention as long as the microorganism used can grow well and can express the enzyme activity of hydroxylating the 3 "or 4" position of mevastatin. However, any of various synthetic media, semi-synthetic media, natural media and the like can be used. The nutrient sources of these media are glucose, fructose, glycerol, sorbitol, alcohols,
Acetic acid, starch and the like can be used alone or in combination, and the concentration used is not particularly limited and is appropriately 1 to 10%. As a nitrogen source, ammonia, urea, ammonium sulfate, ammonium nitrate,
One or more compounds such as ammonium vinegar can be used. As the inorganic salt, salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, manganese sulfate, and ferrous sulfate can be used. Furthermore, as an organic nutrient source having a growth promoting effect on the bacteria used, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, etc. are used, and a small amount of vitamins and nucleic acids can be contained in the medium.

The culturing conditions can be appropriately selected within a range in which the microorganism used can grow well.
Cultivate under aerobic conditions at ~ 9, 28-30 ° C for 2-8 days. The various culture conditions described above can be appropriately changed depending on the type and characteristics of the microorganism used, external conditions and the like. The substrate, mevastatin, may be added to the culture medium either before the start of the culture or after the culture has started for a certain period of time, and the cells are recovered from the culture solution and the cells are converted into an appropriate aqueous solution. You may add in the suspended liquid. The added mevastatin can be used at various concentrations,
It is desirable to use it at a concentration of 0.1 g / l to 2 g / l, and this may be added all at once or in portions. After the addition of the substrate, it is operated at 27 to 31 ° C. for 3 to 10 days, preferably 6 to 8 days, such as shaking or aeration and stirring, and the reaction is allowed to proceed under aerobic conditions. Can be converted to the desired mevastatin derivative.

In order to separate and purify the desired mevastatin derivative thus produced from the culture solution or cell suspension,
Various known methods can be selected and combined. For example, solvent extraction using ethyl acetate, n-butanol, etc., polystyrene adsorption resins such as Amberlite XAD (Rohm and Haas Co.), Diaion HP-20 (Mitsubishi Chemical Co.), silica gel, alumina, activated carbon A method by column chromatography using a carrier such as can be used. The method of eluting the target substance from these carriers varies depending on the type and properties of the carrier, but for example, in the case of a polystyrene-based adsorption resin, hydrous alcohol, hydrous acetone or the like can be used as the elution solvent. In addition, gel filtration with Sephadex LH-20 (Pharmacia), Bio-gel P-2 (Bio-Rad), thin-layer chromatography with silica gel, alumina, etc. High performance liquid chromatography (preparative HPLC) and the like can be used, and these methods can be separated or purified by using these methods alone or in appropriate combination, and in some cases repeatedly used. The mevastatin derivative thus obtained can be precipitated in the form of a salt in the presence of an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide or an amine such as triethylamine in an organic solvent.

Among the mevastatin derivatives thus obtained, the triethylamine salt of the 4 "-position hydroxide of mevastatin represented by the formula (II) has the following physicochemical properties. (1) Appearance: white powder (2) Molecular formula: C ₂₃ H ₃₆ O ₇ (3) Mass spectrum (FAB-MS): Positive: 447 [(M + Na) ⁺ ] Negative: 423 [(MH) ^- ]

(4) ¹ H-NMR spectrum (400 MHz, CD ₃ OD):
As shown in Table 1.

[Table 1]

(5) HPLC retention time: HPLC under the following conditions
The retention time is 10.6 minutes. HPLC analysis conditions: Column: YMC-Pack Pro C18 AS307 (YMC) Internal diameter 4.6 mm x 75 mm Mobile phase: Methanol / water / acetic acid / triethylamine = 55: 4
5: 0.1: 0.1 Flow rate: 1.5 ml / min Detection wavelength: 238 nm

Further, 3 "of mevastatin represented by the formula (III)
The position hydroxide has the following physicochemical properties. 3 "
The position hydroxide has two types of optical isomers, and the triethylamine salts thereof have exactly the same physicochemical properties from the following (1) to (4), but the property of (5) is different. (1) Appearance: White powder (2) Molecular formula: C ₂₃ H ₃₆ O ₇ (3) Mass spectrum (FAB-MS): Positive: 447 [(M + Na) ⁺ ] Negative: 423 [(MH) ^- ]

(4) ¹ H-NMR spectrum (400 MHz, CD ₃ OD):
It is as shown in Table 2.

[Table 2]

(5) HPLC retention time: HPLC under the following conditions
The retention time when performing is 11.1 minutes and 11.9 minutes. HPLC analysis conditions: Column YMC-Pack Pro C18 AS307 (YMC) Inner diameter
4.6mm × 75mm Mobile phase: Methanol / water / acetic acid / triethylamine = 55: 4
5: 0.1: 0.1 Flow rate: 1.5 ml / min Detection wavelength: 238 nm

[0021]

The present invention will be described in detail below with reference to examples. Example 1 Preparation of conversion culture solution In a 250 ml Erlenmeyer flask containing 3 glass beads, trypsin soybean medium (Difco: casein digest 1.7
%, Soybean powder papain digest 0.3%, glucose 0.25%,
Sodium chloride 0.5%, dipotassium phosphate 0.25%, pH 7.
3) Add 50 ml and streptomyces sp.
Frozen seed of omyces sp.) TM-7 strain (FERM P-18312) was inoculated at 2% and cultured at 28 ° C. and 220 rpm for 24 hours to prepare a seed culture. Then, 2% of the seed culture solution was inoculated into 2 of 3 l mini jars containing 1.5 l of double concentration trypsin soybean medium containing 500 mg / l of mevastatin and 100 mg / l of ferrous sulfate heptahydrate. Aeration and agitation culture was performed at 28 ° C., 400 rpm, 0.5 vvm. 26 hours after starting the culture, the mevastatin solution
(25 g / l) was continuously added, and the culture was continued until 160 hours later. About 3 liters of the culture broth was obtained by combining the two mini jars. The total addition amount of mevastatin was 18.1 g per one mini jar.

Example 2 Purification and isolation of the desired product A column of an adsorption resin Diaion HP-20 (Mitsubishi Chemical Co., Ltd.) in which the filtrate obtained by filtering 3 l of the culture broth obtained in Example 1 was previously filled with water. The solution was passed through 0.5 l. Acetone and water ratios changed to 1: 9, 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3 were passed through 1 liter of mixed solution, and the adsorbed purpose The product was eluted. Analyze under the following HPLC conditions to obtain the desired product (retention time: about 5.8
Fractions containing) were collected.

HPLC conditions: Column YMC-Pack Pro C18 AS307 (YMC) Internal diameter
4.6 mm × 75 mm Mobile phase: A) Water / triethylamine / acetic acid = 100: 0.1: 0.1 Mobile phase: B) Methanol / triethylamine / acetic acid = 100:
0.1: 0.1 hours (min) Mobile phase (B) 0.0-5.0 40% -90% 5.0-6.0 90% 6.0-6.1 90% -40% 6.1-10 40% Flow rate: 1.5 ml / min Detection wavelength: 238 nm

The fractions thus collected were concentrated under reduced pressure and extracted with 500 ml of ethyl acetate. When the obtained ethyl acetate layer was concentrated to dryness, 1 g of residue was obtained. 20 ml ethyl acetate residue
And was adsorbed on the upper part of a column of 80 g of silica gel (Wako gel C200, Wako Pure Chemical Industries, Ltd.) packed with ethyl acetate / hexane (1: 1). Ethyl acetate / hexane (1: 1) 0.5l, ethyl acetate / hexane (5: 1) 0.5l, chloroform / methanol (1: 1) 0.5l, methanol 0.5l were sequentially eluted to obtain a fraction containing the target substance. collected. This is concentrated to dryness, and the residue is 780 m.
g was obtained and dissolved in a small amount of methanol. Preparative HPLC
Then, three kinds of compounds were sequentially eluted. The conditions of preparative HPLC are as follows.

Preparative HPLC conditions: Column: Inertsil ODS-3 (GL Science) Internal diameter 10 mm
× 250mm Mobile phase: 50% methanol (0% acetic acid and trimethylamine.
Flow rate: 30ml / min Detection wavelength: 238nm

Each of the obtained fractions was analyzed under the following HPLC conditions, and the fractions containing only the compound having a retention time of 10.6 were collected, concentrated under reduced pressure, and lyophilized to hydroxylate mevastatin at the 4 "position. In the same manner, 15 mg of the triethylamine salt of the compound was obtained. Similarly, the fractions containing only the compound having a retention time of 11.1 minutes or 11.9 minutes were collected, concentrated under reduced pressure, and lyophilized to obtain each of the optical isomers of the 3 "-position hydroxide 23 mg and 10 mg of triethylamine salt were obtained.

HPLC analysis conditions: Column: YMC-Pack Pro C18 AS307 (YMC) Inner diameter 4.6 mm × 75 mm Mobile phase: Methanol / water / acetic acid / triethylamine = 55: 4
5: 0.1: 0.1 Flow rate: 1.5 ml / min Detection wavelength: 238 nm

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 7/62 C12P 7/62 // (C12P 7/62 C12R 1: 465 C12R 1: 465) F term ( Reference) 4B064 AD65 BA05 CA04 CB02 CB12 CD08 CE10 DA01 4C206 AA03 DB02 DB56 NA14 ZC20 ZC33 4H006 AA01 AB20 AB23 BJ30 BN10 BS10

Claims (3)

[Claims] 1. A compound represented by the general formula (I): (In the formula, R ₁ and R ₂ represent a hydrogen atom or a hydroxyl group, except when R ₁ and R ₂ are both hydrogen atoms.), And a salt thereof. 2. Formula (II): The mevastatin derivative and its salt represented by. 3. Formula (III): The mevastatin derivative and its salt represented by.