JPS6328595B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention produces ML-236B lactone, ML-236B carboxylic acid, its lower alkyl ester, or its alkali metal salt (hereinafter referred to as ML-236B carboxylic acid) through the action of microorganisms.
Collectively called 236B compounds. ) to convert the formula The present invention relates to a method for producing the ML-236B derivative or its ring-closed lactone. In the above formula, R ₁ represents a hydrogen atom; a lower alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl; or an alkali metal such as sodium or potassium. R ₂ is the group

【formula】

【formula】 or

[Formula] is shown. The ML-236B derivative having the general formula () or its ring-closed lactone has the effect of lowering blood lipids by inhibiting the synthesis of cholesterol, and is used, for example, as a therapeutic agent for hyperlipidemia and as a preventive agent for arteriosclerosis. It can be used as a medicine. Among the substances represented by the above formula (), the formula The substance represented by is abbreviated as M-3 and has the formula The substance represented by is abbreviated as M-4 and has the formula The substance represented by is abbreviated as isoM-4'. M-3 is a chemical conversion product of ML-236B (Japanese Patent Application Laid-open No. 56-150037), and M-4 is a chemical conversion product for animals.
It had already been isolated in the applicant's laboratory as a metabolite of ML-236B lactone during an administration experiment (Japanese Patent Application Laid-Open No. 57-2240). The ML-236B derivative represented by the above formula () or its ring-closed lactone form has an inhibitory effect on cholesterol synthesis, but the inhibitory effect on cholesterol synthesis of M-4 is particularly remarkable, and ML-236B
Shows 10 times more inhibitory action than lactone. However, M-4 was obtained as a metabolite of animals administered ML-236B lactone, and it was difficult to mass-produce it. Therefore, when we were considering an economical method for producing this substance, we found that it According to M.L.
The present invention was completed by discovering that the -236B compound can be converted into an ML-236B derivative having the formula () or a closed ring lactone thereof. ML-236B lactone and ML-236B carboxylic acid themselves are known substances, separated and purified from the metabolites of Penicillium titrinum, a type of blue mold, and have the chemical structure shown in the following formula. It inhibits cholesterol biosynthesis in isolated enzyme systems and cultured cell systems by competing with its rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and is a powerful serum cholesterol inhibitor even at the individual animal level. (Japanese Patent Application Laid-Open No. 155690/1976, Journal of Antibiotics Vol. 29, pp. 1346-1348, 1976).

[expression] and

[Formula] Similarly, ML-236B carboxylic acid lower alkyl ester is expressed by the following formula: (In the formula, R ₃ represents a lower alkyl group.) (Japanese Patent Laid-Open No. 53-84954), and the alkali metal salt of ML-236B carboxylic acid is a metal salt (Japanese Patent Laid-Open No. 53-56314).
These are also known to exhibit cholesterol inhibitory effects. The ML-236B compound is represented by the above formula ()
Microorganisms that can be converted into ML-236B derivatives or closed ring lactones include Mucor genus and Rhizopus, which belong to Zygomycetes.
The genera Zygorhynchus, Circinella, Actinomucor, Gongronella, Phycomyces and Mortierella and the non-zygomycete Pycnoporus (formerly known as Trametes
genus) and Rhizoctonia. Among the microorganisms belonging to these, Mucor hiemalis f.hiemalis (Mucor hiemalis f.hiemalis) 242.35 CBS 110.19 CBS 201.65 Mucor Basiliformis
bacilliformis) NRRL 2346 Mucor circinelloides f.
circinelloides) IFO 4554 IFO 5775 Mucor hiemalis f.corticolus SANK 34572
(Feikoken Bibori No. 5913) Mucor dimorphosporus (Mucor
dimorphosporus) IFO 4556 Mucor fragilis (Mucor fragilis) CBS
236.35 Mucor genevensis
IFO 4585 Mucor globosus
SANK 35472 (Fiber Science and Technology Research Institute No. 5915) Mucor circinelloides f. griseo-
cyanus) IFO 4563 Mucor heterosporus (Mucor
NRRL 3154 Mucor spinescens
IAM 6071 Rhizopus chinensis
IFO 4772 Rhizopus circinans
circinans) ATCC 1225 Rhizopus arrhizus
ATCC 11145 Zygorhynchus
IFO 4833 Circinella muscae
IFO 4457 Circinella rigida
NRRL 2341 Circinella umbellata
umbellata) NRRL 1713 IFO 4452 IFO 5842 Actinomucor elegans
elegans) ATCC 6476 Phycomyces blakesleeanus (Phycomyces blakesleeanus) SANK 45172
(Feikoken Bacteria No. 5914) Mortierella Isabelina (Mortierella
isabellina) IFO 6739 Gongronella butleri
IFO 8080 Pycnoporus coccineus
coccineus) SANK 11280 (Feikoken Bacillus No. 5916
No.) Rhizoctonia solani
SANK 22972 (Feikoken Bibori No. 5917) is suitable. Among these microorganisms, especially Mucor hiemalis f.
hiemalis) has the ability to convert the ML-236B compound to the compound of formula () with a conversion rate of more than 90%. All microorganisms suitably used in the present invention have been deposited with the Institute of Fine Technology, or have been deposited with public storage institutions (IFO, CBS, IAM, ATCC, etc.).
Available from NRRL). The ML-236B compound is represented by the above formula ()
In order to convert the ML-236B derivative or closed ring lactone, a microorganism (live or dormant system) is cultured in a medium containing the ML-236B compound, or
In some cases, this can also be achieved by bringing enzyme extracts (cell-free extracts) of these microorganisms into contact with the ML-236B compound. In this case, in the culture of microorganisms (living bacteria), it is produced as a carboxylic acid type, lactone type, alkali metal salt, or lower alkyl ester type depending on the culture conditions. In addition, it is obtained as an alkali metal salt in a resting bacterial system and an enzyme extract (cell-free extract). Alternatively, the carboxylic acid form obtained from the ML-236B compound by microbial conversion can be used as is or after being converted into the lactone form, if desired, by treatment with a conventional chemical method, such as a diazoalkane or a salt-forming substance, These can also be obtained as lower alkyl esters or alkali metal salts. Similarly, for example, an alkali metal salt can be converted into a lower alkyl ester type by treating it with a conventional chemical method, if desired. A method for quantifying M-4 produced by treating the cultured cells of the above strain with the ML-236B compound is shown below. Method for quantifying M-4 lactone The method for quantifying M-4 lactone was carried out by high performance liquid chromatography. That is, Microbonder Pack C ₁₈ (manufactured by Waters) was used as a carrier, and a 62% methanol aqueous solution was used as a solvent.
Liquid volume 1ml/min, detection method is ultraviolet absorption
When detected at 237 nm, M-4 lactone showed a peak at a retention time of 10 minutes, and was quantified based on this peak. Next, examples will be shown. Example 1 Twenty 500 ml Erlenmeyer flasks containing 100 ml of the medium with the following composition were inoculated with Mucor hyimalis forma hyimalis IFO 5834, cultured with shaking at 26°C and 220 rpm, and after 4 days, ML-236B lactone was finally added. Added to a concentration of 0.05% for another 6 days26
Incubate at 220 rpm at °C. Medium composition Glucose 1.0% Peptone 0.2 Meat extract 0.1 Yeast extract 0.1 Corn steep liquor 0.3 Remaining tap water (PH not corrected) After completion of the culture, the conversion reaction solution was filtered and the pH of the solution was adjusted to 3 with trifluoroacetic acid. It is then extracted three times with ethyl acetate of 1 to obtain the fraction containing M-4. M-4 is thin layer chromatography (TLC) (plate; silica gel manufactured by Merck & Co.)
Art5715 Solvent; Benzene: Acetone: Acetic acid = 50:
50:3) indicates an R _f value of 0.45. Conversion rate 90%. The above extract was washed with a saturated sodium chloride solution, an ether solution of diazomethane was added, and after standing for 30 minutes, it was dried under reduced pressure. The residue was transferred to a Rover column (Merck Si60,
Size A), benzene: ethyl acetate = 1:
When purified using system 1, M-4 methyl ester is approximately
600 mg was obtained. Note that if a suitable diazoalkane is used in place of diazomethane in this operation, the corresponding alkyl ester of M-4 can be obtained. M-4 methyl ester has the following properties. (1) NMR spectrum Measured at 200MHz in deuterated chloroform using TMS as an internal standard. (CDCl ₃ ) δppm: 0.88 (3H, t, J = 7.3Hz) 0.89 (3H, d, J = 6.5Hz) 1.12 (3H, d, J = 6.8Hz) 1.1~1.7 (10H, m) 2.34 (1H , sex, J=7Hz) 2.3-2.5 (2H, m) 2.49 (2H, d, J=6.4Hz) 2.58 (1H, m) 3.72 (3H, s) 3.78 (1H, m) 4.25 (1H, quin, J = 7Hz) 4.4 (1H, m) 5.42 (1H, m) 5.56 (1H, m) 5.56 (1H, m) 5.90 (1H, d, d, J = 9.8, 5.6Hz) 5.99 (1H, d, J =9.8Hz) (2) Mass spectrum After silylation with N,O-bis(trimethylsilyl)trifluoroacetamide, measurement was performed using JEOL Model D-300. M/e: 654 (M ⁺ ), 552, 462, 372, 290, 272,
233, 231 (3) Ultraviolet absorption spectrum (ethanol solution) λ _nax (nm): 230.1, 237.3, 246.4 (4) Infrared absorption spectrum (thin film method) cm ^-1 :
3400, 2950, 1730 (5) TLC TLC plate; Merck silica gel
Art5715 Solvent; Benzene: Acetone (1:1) R _f value 0.88 Example 2 Mucor hyimaris Forma hyimaris
Using IFO 5834, operate in the same manner as in Example 1,
ML-236B lactone conversion reaction solution 1.9 was obtained (conversion rate 90%). The conversion reaction solution was filtered, and the pH of the solution was adjusted to 3 with trifluoroacetic acid. It is then extracted three times with ethyl acetate of 1 to obtain the fraction containing M-4. The above extract was washed with saturated saline, then dehydrated with sodium sulfate, and then lactonized by adding a catalytic amount of trifluoroacetic acid. Next, the above extract was washed with a 5% aqueous sodium bicarbonate solution, dehydrated with sodium sulfate, and concentrated to dryness to obtain a lactone. This was crystallized from ethyl acetate and approx.
560 mg of M-4 lactone was obtained. Yield 56%. M-4 lactone has the following physical properties. (1) NMR spectrum Figure 1 shows the NMR spectrum measured at 60MHz in deuterated chloroform using TMS as an internal standard. (2) Ultraviolet absorption spectrum (methanol solution) λ _nax (nm): 230, 236.7, 244.6 (3) Infrared absorption spectrum (thin film method) cm ^-1 :
3400, 2950, 1725 (4) TLC TLC plate: Merck Silica Gel Art 5715 Solvent; Benzene: Acetone: Acetic acid = 50:50:3 R _f value 0.62 Example 3 Mucor hyimaris Forma hyimaris
Using IFO 5834, operate in the same manner as in Example 1,
ML-236B lactone conversion reaction solution 1.9 was obtained (conversion rate 90%). The conversion reaction solution was filtered, and the pH of the solution was adjusted to 3 with trifluoroacetic acid. It is then extracted three times with ethyl acetate of 1 to obtain the fraction containing M-4. Immediately obtain the M-4 Na salt fraction by dissolving in 5% NaHCO ₃ water. then 2N
-Adjust the pH to 7.0 with HCl and adjust the M-4 Na salt classification.
After adsorption on HP-20 resin (manufactured by Mitsubishi Chemical Industries, Ltd.) and washing with water, the fraction containing M-4 Na salt was eluted with 50% acetone to obtain 570 mg of a lyophilized product.
Yield 52%. M-4 sodium salt has the following properties. (1) NMR spectrum Measured at 200MHz in heavy methanol using TMS as an internal standard. (CD ₃ OD) δppm: 0.91 (3H, t, J = 7.5Hz) 0.92 (3H, d, J = 7Hz) 1.12 (3H, d, J = 7Hz) 1.1~1.8 (10H, m) 2.25 (1H, d, d, J=15, 7.6Hz) 2.34 (1H, d, d, J=15, 5.5Hz) 2.2-2.4 (3H, m) 2.48 (1H, m) 3.68 (1H, m) 4.07 (1H, m) 4.28 (1H, m) 5.36 (1H, m) 5.48 (1H, d, d, J = 3.2Hz) 5.88 (1H, d, d, J = 9.6, 5.3Hz) 5.98 (1H, d, J = (9.8Hz) (2) Ultraviolet absorption spectrum (methanol solution) λ _nax (nm): 230.0, 237.2, 245.0 (3) Infrared absorption spectrum (KBr method) cm ^-1 :
3400, 2900, 1725, 1580 (4) TLC TLC plate: Merck Silica Gel Art 5715 Solvent: Benzene: Acetone: Acetic acid (50:50:
3) R _f value 0.45 Example 4 According to the method of Example 2, each M-4 lactone was obtained using the microorganisms shown in Table 1.

【table】

[Table] Example 5 Circinella
Inoculated with Muscae IFO 4457, 26℃, 220r.pm
After 4 days, ML-236B lactone was added at a final concentration of 0.05%, and the mixture was further cultured for 6 days at 26° C. and 220 rpm. After the cultivation was completed, the conversion reaction solution was filtered, and the pH of the solution was adjusted to 3.0 with trifluoroacetic acid. This is then extracted three times with 1 of ethyl acetate to obtain the fraction containing isoM-4'. IsoM-4' is thin layer chromatography (TLC)
(Plate: silica gel Art5715 manufactured by Merck, solvent: benzene:acetone:acetic acid=50:50:3) shows an R _f value of 0.45. The above extract was washed with saturated saline, then dehydrated with sodium sulfate, and then lactonized by adding a catalytic amount of trifluoroacetic acid. Next, the above extract was washed with a 5% aqueous sodium bicarbonate solution, dehydrated with sodium sulfate, and concentrated to dryness to obtain a lactone. The residue was purified using a Rover column (Si60 size A manufactured by Merck & Co.) and an ethyl acetate system to obtain 12 mg of isoM-4' lactone. IsoM-4' lactone has the following properties. (1) NMR spectrum Figure 2 shows the NMR spectrum measured at 100MHz in deuterated chloroform using TMS as an internal standard. (2) Ultraviolet absorption spectrum (methanol solution) λ _nax (nm): 229, 234.8, 244.5 (3) Infrared absorption spectrum (thin film method): Shown in Figure 3. Example 6 IsoM-4' lactones were obtained in the same manner as in Example 5 using the microorganisms shown in Table 2.

[Table] Example 7 Mucor hiimaris IFO 8565 was inoculated into 100 500 ml Erlenmeyer flasks containing 100 ml of a medium with the same composition as that shown in Example 1, and the mixture was incubated at 26°C and 220 rpm.
After 4 days, ML-236B lactone was added to a final concentration of 0.05%, and cultured for 6 more days.
Incubate at 26°C and 220 rpm for 1 day. After culturing,
Filter the conversion reaction solution and PH the solution with trifluoroacetic acid.
Adjusted to 3. Then, by extracting three times with 10 parts of ethyl acetate, a fraction containing M-4 and M-3 is obtained. Among these, M-3 is thin layer chromatography (TLC) (plate; silica gel manufactured by Merck & Co.)
Art5715, solvent; benzene: acetone: acetic acid =
50:50:3) indicates an R _f value of 0.2. The above extract was washed with saturated brine, dehydrated with sodium sulfate, and then lactonized by adding a catalytic amount of trifluoroacetic acid. Next, the above extract was washed with a 5% aqueous sodium bicarbonate solution, dehydrated with sodium sulfate, and then dried under reduced pressure. The residue was collected using a Rover column (manufactured by Merck & Co., RP-8 size A).
Elution was performed with 25% acetonitrile to obtain 5 mg of M-3 as a lactone form. Characteristic values of M-3 lactone (1) Melting point: 63-67℃ (2) Mass spectrometry value (M ⁺ ): 424 (C ₂₃ H ₃₆ O ₇ ) (3) Ultraviolet absorption spectrum (methanol): Only terminal absorption (4) Infrared absorption spectrum (KBr) ν _nax cm ^-1 :
3300, 1720 (5) NMR spectrum (CDCl ₃ ) δppm: 5.9 (1H, double doublet, J=1.5, 5Hz) 5.5 (1H, multiplet) 4.3 (2H, multiplet) 3.9 (1H, doublet Double line, J = 3, 5 Hz) Example 8 M-3 lactones were obtained in the same manner as shown in Example 7 using the microorganisms shown in Table 3.

【table】

[Table] Cholesterol synthesis inhibitory effect The compound represented by the above formula () is 3-hydroxy-3-methylglutaryl coenzyme A, which is known as the rate-limiting enzyme in the cholesterol synthesis pathway.
Reductase (3-hydroxy-3-methyl-
glutaryl-CoA reductase). Table 4 shows the cholesterol synthesis inhibitory effects of these compounds [measured by the method described in Journal of Biological Chemistry (J. Biol. Chem.) Vol. 234, p. 2835 (1959)].

【table】

【table】 [Brief explanation of the drawing]

Figure 1 shows the NMR spectrum of M-4 lactone. Figure 2 shows the NMR spectrum of isoM-4' lactone, and Figure 3 shows the infrared absorption spectrum of the same substance.

Claims (1)

[Scope of Claims] 1. ML-236B lactone, ML-236B carboxylic acid, lower alkyl ester thereof, or alkali metal salt thereof (hereinafter collectively referred to as ML-236B compound) having the formula (In the formula, R ₁ represents a hydrogen atom, a lower alkyl group, or an alkali metal, and R ₂ represents a group [Formula] [Formula] or [Formula].) microorganisms belonging to the genus Mucor, Rhizopus, Tigorhynchus, Circinella, Actinomucor, Gongronella, Huicomyces, Mortierella, Pycnoporus or Rhizoctonia that can be cultured in a medium containing the ML-236B compound; Alternatively, by bringing the enzyme extract of these microorganisms into contact with the ML-236B compound,
ML-236B compound has the above formula ()
ML-236B derivative having the above formula () or the ring-closed lactone form thereof is obtained by converting the ML-236B derivative or the ring-closed lactone form thereof from a system containing a conversion reaction product.
A method for producing an ML-236B derivative or a closed ring lactone thereof.