JP2931971B1 - How to make pravastatin - Google Patents

Abstract

SOLUTION: When cytochrome P-450 3A is allowed to act on a compactin salt or a lactone form thereof and a lactone form is used as a substrate, the lactone ring is hydrolyzed as necessary to cleave or hydrolyze the lactone ring. Pravastatin, a salt thereof or a lactone thereof. [Effect] Pravastatin, a salt thereof or a lactone thereof useful as a therapeutic agent for hyperlipidemia can be efficiently produced.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing pravastatin sodium salt or a lactone thereof useful as a therapeutic agent for hyperlipidemia. In this specification, compound names are used as follows.

The compactin lactone compound represents a compound having the following structure.

[0003]

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[0004] Compactin sodium salt means a compound having the following structure.

[0005]

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[0006] The pravastatin lactone is a compound having the following structure.

[0007]

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[0008] Pravastatin sodium salt represents a compound having the following structure.

[0009]

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[0010]

BACKGROUND OF THE INVENTION Pravastatin sodium is a compound represented by the above formula, which inhibits the biosynthesis of cholesterol specifically and antagonistically by its rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase. It has a potent serum cholesterol lowering effect and is widely used as a therapeutic agent for hyperlipidemia.

The pravastatin or a salt thereof is a lactone of compactin (ML-236) isolated from a metabolite of Penicillium titrinum, a kind of blue mold.
It is known that a free compound of pravastatin can be obtained from urine of a dog to which B) has been administered or by allowing a liver homogenate of a rabbit or a dog to act on the ML-236B lactone (Japanese Patent Publication No. 61-13699). ). ML-23 can also be obtained using cytochrome P-450 derived from actinomycetes such as Streptomyces carbophilus.
It is known that pravastatin sodium salt is produced from 6B sodium salt (Japanese Patent No. 2603677).

[0012]

However, all of these conventional methods for producing pravastatin have drawbacks, such as an insufficient yield and a risk of contamination of trace components derived from microorganisms.

[0013]

The present inventors have conducted various studies on an efficient method for producing pravastatin. As a result, among many cytochromes P-450, cytochrome P-4 has been described.
The present inventors have found that pravastatin can be obtained efficiently and easily by allowing 503A to act on compactins, thereby completing the present invention.

That is, in the present invention, when a cytochrome P-450 3A is allowed to act on a compactin salt or a lactone thereof, and when the lactone of compactin is used as a substrate, hydrolysis is carried out to cleave the lactone ring or hydrolyze the lactone ring if desired. It is intended to provide a method for producing pravastatin, a salt thereof or a lactone derivative, which is decomposed to form a salt.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The salt of compactin or its lactone used in the present invention is penicillium.
It can be isolated from a culture of Titrinum (JP-A-50-155690).

On the other hand, cytochrome P-45 used in the present invention
As for 03A, those belonging to many cytochrome P-450 3A can be used, but are preferably derived from human or rat, more preferably human-derived cytochrome P-450 3A4, cytochrome P-45.
03A5 or cytochrome P-4503 from rat
A2.

These cytochromes P-450 3A are:
Although enzymes may be used separately, it is preferable to use the microsomal fraction of cells producing these enzymes. Such a microsomal fraction can be easily obtained by homogenizing and disrupting cells that produce these enzymes.

Further, these cytochromes P-450 3
The A-producing cells may be human or rat hepatocytes in which the cytochrome P-450 3A has been induced by, for example, administration of dexamethasone.
50 3A gene and NADPH cytochrome P-45
It is more preferable to use cells transformed with a vector incorporating the 0 reductase gene. Host cells used for this transformation include human B lymphoblastoid cells, insect cells, yeast, E. coli and the like. Commercially available microsomes of these hepatocytes and microsomes of genetically modified cells can be used.

In the present invention, the cytochrome P-450 3A and the NADPH cytochrome P-450
In addition to the reductase, more preferably it reacted with the addition of cytochrome b _5. Means for coexisting such cytochrome b ₅ include the aforementioned cytochrome P-450 3
A gene is a method using a microsomal fraction of NADPH cytochrome P-450 reductase gene and cytochrome b ₅ cells transformed gene in vectors incorporating. Commercially available microsomes of these transgenic cells may be used.
The microsomal fraction of the recombinant yeast described in -289252 may be used.

The reaction of a compactin salt or a lactone form thereof with the cytochrome P-450 3A is carried out, for example, by
10 minutes to 8 hours at 30 to 40 ° C. in a buffer having a pH of 7 to 8,
Preferably, it may be performed for 30 minutes to 4 hours.

When the lactone form of compactin is used as a raw material, the lactone form of pravastatin is formed by the above reaction. Is obtained. The salt of pravastatin is preferably a sodium salt.

The target compound is isolated from the reaction mixture by a conventional method, for example, extraction with an organic solvent, purification via an ester derivative,
It is performed by appropriately combining washing, chromatography and the like.

[0023]

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

Example 1 (1) Microsomal human cytochrome P-450 3A4 used and NADPH P-
Microsomes of human B lymphoblastoid cells AHH-1 TK +/− (10 mg protein / ml, 0.56 μM P-45) simultaneously expressing 450 reductase cDNA
0, hereinafter 3A4 lymph cell line); human cytochrome P-4
Microsomes of insect cells BTI-TN-5B1-4 infected with baculovirus incorporating 503A4 and cDNA of NADPH P-450 reductase (4.
7 mg protein / ml, 2 μM P-450, hereinafter 3A4 insect cell line); human cytochrome P-450 3A4, NA
DPH P-450 reductase and cytochrome b ₅
BTI-TN-5B1-4 microsomes (7.1 mg pr
otein / ml, 1 μM P-450, hereinafter 3A4 + b ₅ insect cell line); human cytochrome P-450 3A5 and NA
BTI-TN-5B1 transfected with baculovirus incorporating DHP P-450 reductase cDNA
-4 microsomes (3.6 mg protein / ml, 2 μM
Four types of microsomes of P-450 (hereinafter referred to as 3A5 insect cell line) were liver microsomes (37 mg protein / ml, 41 mg) of Sprague-Dawley rats (male) induced cytochrome P-450 3A with dexamethasone by Daiichi Kagaku. .
4 μM P-450, hereinafter referred to as 3A-induced rat liver) was purchased from Charles River Japan and used in experiments.

(2) Production of pravastatin from compactin using cytochrome P-450 3A In a 0.1 M potassium phosphate buffer (pH 7.4),
3.3 mM MgCl ₂ , 3.3 mM glucose-6-phosphate, 1.3 mM NADP ⁺ , 0.4 U / ml glucose-6-phosphate dehydrogenase and 0.1 mM glucose-6-phosphate dehydrogenase.
1 mM compactin sodium or 0.185 mM
A 1% solution of compactin lactone in acetone (all final concentrations) was added and pre-incubated for 5 minutes at 37 ° C. The aforementioned cytochrome P-450 3A microsome was added with 40 nM P-450 (final concentration), and the reaction was started at 37 ° C. (final volume 250 μl). Cyanomethane 12
The reaction was stopped by adding 5 μl. When compactin sodium is the substrate, the supernatant obtained by centrifugation at 10,000 × g for 3 minutes is added. When compactin lactone is the substrate, 250 μl of ethyl acetate is added.
The upper layer after centrifugation for 3 minutes was dissolved in methanol after distilling off the solvent with nitrogen gas, and after adding an internal standard, quantification was performed by HPLC.

(3) The obtained results are shown in Tables 1 and 2. In the table, the reaction rate is the consumption rate of compactin,
The conversion indicates the conversion of compactin to pravastatin.

[0027]

[Table 1]

[0028]

[Table 2]

According to Tables 1 and 2, 3A4
A comparison of the activities of 3A5 and 3A5 indicated that 3A4 had greater activity. Further, in the presence cytochrome b ₅ it has been shown to be greater 10 times several times in the absence. Also,
The obtained pravastatin lactone was easily converted to pravastatin sodium by hydrolysis using an aqueous sodium hydroxide solution.

[0030]

According to the present invention, pravastatin, a salt thereof, or a lactone thereof useful as a therapeutic agent for hyperlipidemia can be efficiently produced.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 17/06 C12N 5/00 B // (C12P 7/40 C12R 1:91) (C12P 17/06 C12R 1:91) C07M 7:00 (56) References JP-A-9-206085 (JP, A) JP-A-7-289252 (JP, A) International Publication 96/40863 (WO, A1) Shinji Serizawa, “The 31st Plant Chemistry Summary of Symposium Lectures ", December 5, 1994, p. 11-19 Jacqueline, L .; Et al., "Ga steroidology", Vol. 106 (2), February 1994, p. 464-472 Tomas A. Et al., "Durg Metabolism and Disposition," Vol. 24 (11), November 1996, p. 1197-1204 C.I. Transon et al., "European Journal of Clinical Pharmacology", 50 (3), 1996, p. 209-215 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 C07C 69/03 C07D 309/30 C12P 7/40 C12P 17/06 C12N 5/10 BIOSIS (DIALOG ) WPI (DIALOG) JICST file (JOIS)

Claims (1)

(57) [Claims] To 1. A salt of compactin or its lactone form, cytochrome P-450 3A gene, the NADPH cytochrome P-450 reductase gene and cytochrome P-450 3A-producing cells transformed with vectors incorporating cytochrome b ₅ gene The microsomal fraction is allowed to act, and when a lactone form of compactin is used as the substrate, pravastatin, a salt thereof or a pravastatin characterized by being hydrolyzed to cleave the lactone ring or to form a salt by hydrolysis, if desired. Production method of lactone body.