KR840001055B1 - Process for preparing ml-236 b decivatives - Google Patents

Abstract

Cholesterol formation inhibitors were produced from ML 236B (I) by fermn. with fungi or bacteria. Thus, spores of Absidia coerulea IFO 4423 were inoculated into a pH 7 medium contg. glucose 2, K2HPO4 0.15, MgSO47H2O 0.15, NH4NO3 0.1, 7H2O 0.001% at 26≰C for 2 days with shaking. Then, 0.05% I Na salt was added and incubation continued for 5 days. The broth filtrate was pH 3 with the addn. of TCA and was extd. with EtOAc. The ext. was chromatographed on silica gel to sep. M-4 (II). M-4 was lactonized with a catalytic amt. of TCA to form 50.1 mg of M-4 lactone (III).

Description

Method for preparing ML-236B derivative

1 shows the NMR spectrum of M-4 lactone,

2 shows the NMR spectrum of IsoM-4'-methyl ester,

3 shows the infrared absorption spectrum of IsoM-4 'methyl ester,

4 shows the NMR spectrum of IsoM-4 'lactone,

5 shows infrared absorption spectra of IsoM-4 ′ lactones, respectively.

The present invention relates to a series of novel thermostats of known compounds ML-236B, methods for their preparation and pharmaceutical compositions comprising them.

ML-236B having the above compound structure is described in US Pat. No. 3,983,140. This substance has been isolated and purified from the genus Penicillium, particularly the metabolite of the penicillium citrinum, a type of blue fungus. This substance exhibits cholesterol biosynthesis-inhibiting activity by cultured cells isolated from enzymes or experimental animals by competing with the rate-limiting enzyme, ie 3-hydroxy-3-methylglutaryl-coenzyme A reducing agent, in cholesterol biosynthesis. As a result, it plays an important role in reducing serum cholesterol levels in animals. Journal of Antibiotics, 29, 1346 (1976). Many compounds chemically related to ML-236B have been found and found to have the ability to inhibit cholesterol biosynthesis.

The inventors have discovered a series of novel compounds or derivatives thereof that can be prepared by enzymatic hydroxylation of ML-236B, which in some cases substantially inhibits cholesterol biosynthesis at least comparable to its own capabilities. Has

Compounds of the present invention are oxycarboxylic acids of formula (I) and ring closure lactones, salts and esters thereof:

(Wherein R represents a group (A) or (B) having the structure

The present invention also provides a process for preparing a compound of formula (I) or a closed ring lactone, salt or ester thereof by enzymatic hydroxylation of ML-236B, or a process for preparing ML-236B carboxylic acid, salt or ester thereof. To provide.

ML-236B carboxylic acid has the following structural formula.

One class of compounds according to the invention is a compound of formula (II), a pharmaceutically toxic salt of an acid of said compound (II) wherein R ₁ represents a hydrogen atom, Lactone corresponding to compound (II).

Wherein R ¹ represents a hydrogen atom or an alkyl group of C ₁ to C ₅ . In view of the number of sub-carbons in these compounds, various geometric isomers are possible. Among the most important isomers are lactones corresponding to the compound of formula (IV), a pharmaceutically toxic salt of an acid in which R ₁ represents a hydrogen atom, and a compound (IV) having structure (V). And a compound of formula (VI), a pharmaceutically toxic salt of an acid in which R ₁ represents a hydrogen atom, and lactone corresponding to formula (VI) having formula (VII).

In the formula, R ¹ represents a hydrogen atom or an alkyl group of C ₁ to C ₅ .

The oxycarboxylic acid of formula (IV) wherein R ¹ represents a hydrogen atom is referred to herein as M-4, and derivatives of this acid, in particular salts and esters, are referred to as M-4 derivatives and correspond to formula (IV). The lactone having (V) is called M-4 lactone.

Similarly, lactones having the formula (V) corresponding to formula (VI) in which R ¹ represents a hydrogen cloud are called M-4 lactones.

Another preferred class of compounds according to the invention are the compounds of formula (VII), the weakly toxic salts of acids of the compound (VII) in which R ¹ represents a hydrogen atom, and the formula (VII) Lactone corresponding to compound (iii) having

Wherein R ¹ is as defined above.

These compounds are also possible that many of the geometric isomers, the major isomer is the compound is combined with, R ₁ of the compound of the following structural formula (Ⅹ) with a salt thereof and the structural formula (ⅩI) of the acid of the compound) Ⅹ) represents a hydrogen atom Lactone corresponding to (iii); and a compound of formula (VII), a salt of an acid of compound (X) in which R ₁ represents a hydrogen atom, and a lactone of (VII) having structural formula (VII), etc. to be.

R ¹ in the above formulas is as defined above. The compound of formula (VII) is referred to herein as IsoM-4, and its derivatives such as salts and esters are called derivatives of IsoM-4, and the lactones corresponding to formula (XI) having formula (XI) are IsoM-4. It is called lactone. The acid of structural formula (XII) wherein R ¹ represents a hydrogen atom is called IsoM-4 ', its derivative is called derivative of IsoM-4', has the structural formula (XIII), and the lactone corresponding to formula (XII) is IsoM- 4 'Lactone.

Among the esters of the oxycarboxylic acids of formula (I), C ₁ to C ₅ alkyl esters are preferred. These alkyl groups are linear or branched groups to which methyl, ethyl, propyl, isopropyl, butyl and isobutyl groups and the like belong, among which methyl group is particularly preferred.

Oxy carboxylic acids also form salts with various cynos, especially metal ions, with alkali metals such as sodium or calum being preferred, in particular sodium being most preferred.

Among the compounds according to the invention, the most preferred compounds are M-4 lactone, M-4 sodium salt, M-4 methyl ester, IsoM-4 'lactone, IsoM-4' sodium salt and IsoM-4 'methyl ester, and the like. -4 sodium salt is preferred. The compounds of the present invention can be prepared by enzymatic hydroxylation of ML-236B or its derivatives, in particular ML-236B carboxylic acid or its salts or esters.

This enzymatic hydroxylation reaction is part of the mammalian metabolism of ML-236B or its derivatives, for example by administering ML-236B to an appropriate animal to collect its metabolites (eg urine) and then The desired compound or the compound of the present invention is separated from the compound. Alternatively, enzymes containing livers or extracts from livers can be used instead of live animals. However, metabolism of animals or methods using animal products are relatively low in productivity and difficult to reproduce. Therefore, the inventors have used enzymes containing extracts from microorganisms or microorganisms.

Thus, the process according to the invention can be preferably achieved using microorganisms capable of converting ML-236B or its derivatives into the compounds of the invention, or by using enzymes containing extracts of such microorganisms. Particularly preferred microorganisms belong to the following genus: Muco, Rhizopis, Zygorynchus, Sircinella, Circimella, Actinumucor, Gongronella, Pico Phycomyces, Martierella, Pycnoporus, Rhizictonia, Absidia, Cunninghamella, Sync ephalastrum, and Streptomyces ). In particular, the following Species are preferred.

Apsidia koerulea, Kunningella quinulata, syncephalus racemicium, streptomyces roseochromomogenus, muco hyemalis f. Hiemalis, Muco Basiliformis, Muco Sircinellodes f. Sircinelloides, Muco Hiemalis f. Corticolus, muco dimorphosporus, muco pragillis, muco genenevensis, muco glogus, muco sircinelloides f. Griseo-Cyanus, Muco Heterosporus, Muco Spinesense, Rizopus Kinesis, Rizopus Sircinus, Rizopus Arhijus, Zygorincus Moeleri, Sircinella Muscae, Sircinella Lidida, Sircinella Umbellata, Actinomuco Elegance, Picomises Blakesreanus, Martierella Isabella, Gogronella Butleri, Pycnphorus Coxineus, Lizatonia Solani, Cyncephalast Cerium nigricans, apsidia glaca var, paradox,

Among the strains of this species, the following are particularly preferred.

Apsidia Koerurea IFO-4423, Koningamella Echinulata, IFO-4445, Kuningamella Echinulata IFO-4444 Kuningamella Echinulata ATCC-9244, Cinsepalastrum racemium IFO-4814, Syncephalus racemosium IFO-4828, Streptomyces roseochromomogenus IFO-3411, muco hyemalis f. IFO-5834, Muco Hiemalis f. Muco Hiemalis IFO-5303, Muco Hiemalis f. IFO-8567, Muco Hiemalis f. IFO-4889, Muco Hiemalis f. Hiemalis CBS-117.08, Muco Hiemalis f. CBS-109.19, Muco Hiemalis f. CBS-200.28, Muco Hiemalis f. CBS-242.35, Muco Hiemalis f. CBS-110.19, Muco Hiemalis f. Hiemalis f. CBS-201.35, Muco Hiemalis f. Hiemalis CBS-110.19, Muco Hiemalis f. CBS-201.65, Muco Basiliformis NRRL-2346, Muco Sirnelloides f. Sircineloides iFO-4554, Muco dimorphosporus IFO-4556 Muco Fragilis CBS-236.35, Muco Genebensis IFO-4585, Muco Globounce NRRL-3154, Muco Spinesense IAM-3071, Rizopus Kinensis IFO-4772, Resorpus Sircinus ATCC-1225, Resorpus Arhijus ATCC-11145, Zygorincus Moeleri IFO-8433, Resorpus Sircinus ATCC-1225, Resorpus Arhijuss ATCC- 11145, Jigorinkus Moeleri IFO-8433, Sircinella Muska IFO-4457, Sircinella Lida NRRL-2341, Sircinella Umbellata NRRL-1713, Sircinella Umbellata IFO-4452 , Sirinella Umbellata IFO-5842, Picomises Blaques Reeanus NRRL-12475, Martiella Isabella IFO-3739, Gogronella Burleri IFO-3080, Pycnopurus Coxineus NRRL-12476, Lizatonia Solani NRRL-12477, Cincepalstrium nigricans NRRL-12478, Cincepalstrium nigricans NRRL-1 2479, syncephalum nigricans NRRL-12480, apsidia glaca var. Paradoxone IFO-4431, Actinomuco Elegance ATCC-4676

The microorganisms listed above are available from international strain collections, by means of the symbols attached to their accession numbers, which have the following meanings.

IFO = Institute for Fermentation, Osaka, Japan

NRRL = Agricultural Reserarch Culture Collection, Illinois, USA

CBS = Centraal Bureau voor Schimmelcultures, Netherlands

IAM = Institute of Applied Microbiology, Tolyo, Japn

ATCC = Americal Type Culture Collection, Maryland, USA.

Among the aforementioned species, the following species are particularly preferred.

Apsidia coerleaa, kunningamella echinulata, cinsepalastrum racemosium, muco hyemalis f. Hiemalis, Muco Basiliformis, Muco Sircinellodes f. Sircinelloides, Muco Hiemalis f. Corticus Muco dimorphosporus, Muco Fragilis, Muco Genenvensis, Muco Glogus, Muco Sircineloides f. Griseo-Cyanus, Muco Heterosporus, Muco Spinesense, Pycnophus Coxineus, Lizatonia Solani, Cincepalstrium Nigricans

And the following are particularly preferred strains of species:

Apsidia Koerurea IFO-4423, Kunningamella Echinulata IFP-4445, Kunningamella Echinulata IFO-4444, Kunningamella Echinulata ATCC-9244, Shincephalastrium racemium IFO-4814, Syncephallium racemosium IFO-4828, muco hyemalis f. Hiemalis IFO-5834, Muco Hiemalis IFP-8567, Muco Hiemalis f. Hiemalis IFO-8449, Muco Hiemalis IFO-8448, Muco Hiemalis f. Hiemalis IFO-8565, Muco Hiemalis f. Hiemalis CBS-117.08, Muco Hiemalis f. Hiemalis CBS-109.19, Muco Hiemalis f. Hiemalis CBS-200.28, Muco Hiemalis f. Hiemalis CBS-242.35, Muco Hiemalis f. Hiemalis CBS-110.19, Muco Hiemalis f. Hiemalis CBS-201.65, Muco Basiliformis NRRL-2346, Muco Sircinelloides f. Sircinelloides IFO-4554, Muco Sircinideus f. Sircinelloides IFO-4556, Muco Fragilis CBS-236.35, Muco Genebensis IFO-4585, Muco Globounce NRRL-12474, Muco Sircinidedes f. Griseo-Cyanus IFO-4563, Muco heterosporus NRRL-3154, Muco Spinesense IAM-3071, Pycnphorus coccinus NRRL-12476 Lizatonia Solani NRRL-12477, Cincepalastium nigricans NRRL-12478 , Cefepalastrum nigricans NRRL-12480.

In order to prepare the compounds of the formulas (IV) and (V) and salts thereof, the following species are preferred.

Muco Hiemalis f. Hiemalis, Muco Sircinelloides f. Sircineloides, Muco Fragilis, Muco Genebensis, Muco Sircinellodes f. Griseo-Cyanus, Pycnphorus Coxineus, Lizatononiasolani

In order to prepare the compounds of the formulas (VI) and (iii) and salts thereof, species of Syncephalastrum nigricans and Syncephalastr um recemisum are preferred.

Preferred are the metabolic Absidia coerulea species and the Cunninghamella echinulata species that produce the compounds of formulas (iii) and (iii) and salts thereof.

Among all the species listed above, muco hyemalis f. Especially preferred is Hiemalis (Mucor hiemalis f. Hiemalis) because ML-236B and its derivatives can be converted to the desired compounds of formula (I) at 90% or even higher conversions.

ML-236B or derivatives thereof can be converted to the compound of formula (I) by contacting a complete cellular microorganism or, in some cases, cell-free extracts from the microorganism with ML-236B or a derivative thereof.

The type of compound produced will depend on the culture conditions and the type of microorganism used. Thus, for example, if a complete cellular microorganism is cultured in the presence of ML-236B or a derivative thereof, the product may be a carboxylic acid, lactone or alkali metal salt, depending on the culture conditions, in particular pH. On the other hand, when ML-236B or its derivatives are simply contacted with resting cellular or cell-free extracts, the compounds of the present invention are obtained in the form of alkali metal salts.

The progress of the conversion reaction can be measured by analyzing a sample of the reaction mixture to determine the degree of conversion during the reaction. For example, by means of liquid chromatography using Microvanda Pect C ₁₈ (MICRD BON DAPAK) (manufactured by Water Co., USA) as a carrier and using 62% V / V aqueous methanol at a rate of 1 ml / minute as solvent. The presence of M-4 lactones can be analyzed. When it detects its ultraviolet absorption at 237 nm, M-4 peaks, which can be used for analysis. Similar techniques are used to analyze other compounds of the invention.

In order to produce the compounds of the present invention, the culture conditions and culture medium to be used to cultivate the microorganisms in the presence of ML-236B or derivatives thereof may be carefully selected for the specific microorganism to be cultured. Since the species of microorganism to be used in the method of the present invention are well known, the culture conditions and culture medium to be used for the microorganism are also well known.

The compounds of the present invention are subjected to conventional methods, for example, by filtration of microbial cells and discarded (if necessary), the residue mixture of which is optionally combined with thin layer chromatography, column chromatography or high performance liquid chromatography. May be separated into the reaction mixture. The various compounds of the invention are prepared together with stones or more, which may be separated from one another during the purification step by one or more chromatography.

In some cases, in addition to the compounds of the present invention, the inventors have named M-3, and 3 ', 5'-dihydroxy (dihydro-) in the pending specification entitled 계 hydronaphthalene derivatives, their preparation and use. Compounds already known under the name ML-236B) may also be formulated. This too can be separated in the same way.

The inventors have found that the compounds according to the invention show a 50% cholesterol biosynthesis effect at a much lower concentration in some cases comparable to the concentrations required for ML-236B and any other similar known compounds. The inhibitory effect of the compounds according to the invention is as follows in terms of the concentration (μg / ml) required to inhibit cholesterol biosynthesis by 50%.

M-4 Methyl Ester 0.001 IsoM-4 'Lactone 0.013

M-4 Sodium Salt 0.008 M-4 0.019

M-4 Lactone 0.016 M-4 'Sodium Salt 0.00049

IsoM-4 'Methyl Ester 0.007 ML-236B 0.01

The invention will be explained in more detail by the following examples.

Example 1

Preparation of M-4 Lactones

Twenty 500 ml Sakaguc hi flasks each containing 100 ml of the medium having the following composition were inoculated with a hole seed of Absidia coerulea IFO 4423.

The flask was vibrated for 2 days at 26 ° C and 120 s.p.m. At the end, the sodium salt of ML-236B is added to each flask until the final concentration is 0.05W / V. Incubation is continued for 5 days at 26 ° C and 120s.p.m.

Composition of medium (% is W / V):

Glucose 2.0% Corn Bile Solution 0.2%

K ₂ HPO ₄ 0.15% Yeast Extract 0.1%

_{MgSO 7H 2 O 0.15% ZnSO 4} · 7H 2 O 0.001%

NH ₄ NO ₃ 0.1% tap water balance

0.1% peptone (pH is adjusted to 7.0)

After the incubation is completed, the reaction solution is filtered and the filtrate is adjusted to pH 3 using trifluoroacetic acid. The reaction mixture is extracted three times with 1 L of ethyl acetate every time to give an extract containing M-4. This compound exhibits an Rf value of 0.45 by thin layer chromatography (TLC) (plate: Merk silica gel Art 5715; volume ratio of solvent: benzine, acetone, acetic acid 50: 50: 3). The combined extracts were washed with a saturated aqueous solution of sodium chloride, and then trifluoroacetic acid was added in an ultra-light amount to lactone. The resulting mixture was washed with 1% W / V aqueous sodium bicarbonate solution, dried over sodium sulphate and evaporated under reduced pressure to dryness. This residue is liquid chromatographed with a System 500 using Prep PAK-500 / C18 Caratridge (manufactured by Waters Associcaets, Prep PAK is a trademark). Purification by 55% V / V aqueous methanol system yields 50.1 mg of M-4 lactone.

M-4 lactones have the following physical properties.

1) NMR spectrum: NMR spectrum measured at 60 MHz in deuterochloroform using tetramethylsilane as an internal standard is shown in FIG.

: 230; 236. 7; 244.62) UV absorption spectrum) Methanol solution

: 230; 236. 7; 244.6

: 3400. 2950., 17253) infrared absorption spectrum (liquid film)

3400. 2950., 1725

4) thin layer chromatography: TLC plate: Merk silica gel Art 5715; Solvent: benzene, acetone, acetic acid (50: 50: 3, volume ratio) Rf value: 0.62

Example 2

48 mg of M-4 lactone is prepared by using Kunninghamella echimulata IFO 4445 and performing the same procedure as in Example 1.

Example 3

30 mg of cotton M-4 lactone was prepared using Streptomyces Roseeochromogenus NRRL 1233 and the remaining procedure was performed in the same manner as in Example 1.

Example 4

When using Cefepalastrum racemosium IFO 4828 and the rest of the procedure is carried out in the same manner as in Example 1, 5 mg of M-4 lactone is obtained.

Example 5

6 mg of M-4 lactone is prepared when using Cefepalastrum racemosium IFO 4828 and the rest of the procedure is performed in the same manner as in Example 1.

Example 6

Preparation of IsoM-4 'Methyl Ester

Twenty 500-ml sacchaguchi flasks, each containing 100 ml of the medium of the following composition, were inoculated with apsidia coerurea IFO 4423. The flask is vibrated for 2 days at 26 ° C., 120 s.p.m. At the end, Sodium salt of ML-236B has a final concentration of 0.5 W / V):

Composition of medium (% is W / V):

Glucose 20.% Corn Dipped 0.2%

K ₂ HPO ₄ 0.15% Yeast Extract 0.1%

MgSO ₄ 7H ₂ O 0.15% ZnSO7H ₂ O 0.001%

NH ₄ NO ₃ 0.1% tap water balance

0.1% peptone (pH is adjusted to 7.0)

After the incubation is completed, the reaction solution is filtered and the filtrate is adjusted to pH 3 using trifluoro acetic acid. To obtain an extract containing IsoM-4 ', it is extracted three times with 1 L of ethyl acetate each time. The compound was subjected to thin layer chromatography (plate: Merk silica gel Axt 5715; mixture of solvent: benzene, acetone, and acetic acid in a volume ratio of 50: 50: 3) to obtain an Rf value of 0.45. The extract is washed with a saturated aqueous solution of sodium chloride and then an ethereal solution of diazomethane is added. The mixture is left for 30 minutes and then evaporated under reduced pressure to dryness. This residue is placed on a Lobar column (Merk Si 60, Size A) and purified as a solvent using a mixture of benzene and ethyl acetate in a volume ratio of 1: 1. 200 mg of IsoM-4 'methyl ester fraction is obtained. This fraction was purified further by Lobar column (Merk RP-8, Size A) using 35% V / V aqueous acetonitrile as eluent. Obtaining 78 mg of IsoM-4 'methyl ester, having the following characteristics:

1) NMR spectrum

An NMR spectrum measured at 100 MHz in duterochloroform using tetramethylsilane as an internal standard is shown in FIG.

2) Mass Spectrum:

Measurement was carried out [after silylation with N, o-bis (trimethylsilyl) trifluoroacetamide] using a D-300 mass spectrometer manufactured by Nippon Electromics.

M / e: 654 (M < + >), 552, 462, 372, 272, 233, 231.

: 229;234.9;244.5.3) UV absorption spectrum (methanol solution)

: 229; 234.9; 244.5.

4) Infrared Absorption Spectrum (Liquid Film): Shown in Attached Drawing 3.

5) Thin layer chromatography:

TLC plate: Merk silica gel Art 5715; Solvent: benzine, acetone (volume ratio 1: 1); Rf value: 0.88.

By operating in the same manner as described above using other suitable diazoalkanes instead of diazomethane, Other esters of IsoM-4 'can be produced.

Example 7

Preparation of IsoM-4 'Lactone.

Repeat the procedure described in Example 6 completely, Extract with ethyl estate to obtain an extract containing IsoM-4 '. The combined extracts are washed with a saturated aqueous solution of sodium chloride and then evaporated to dryness to obtain the lactone product. The resulting residue was placed on a Merk column (Merk Si 60 Size A) and purified using a mixture of benzine and ethyl acetate as a solvent (volume ratio 1: 1). Obtain 198 mg of IsoM-4 'lactone. The product was further purified on a Lobar column (Merk RF-8 Size A) eluting with 35% V / V aqueous acetonitrile. 82 mg of IsoM-4 'lactone is obtained, having the following characteristics.

1) NMR spectrum: The NMR spectrum measured at 100 MHz in tetrachlorosilane using tetramethylsilane as an internal standard is shown in FIG.

: 229;234.9;244.5.2) UV absorption spectrum (methanol solution)

: 229; 234.9; 244.5.

3) Infrared absorption spectrum (liquid film): shown in attached drawing 5.

Example 8

If Kunningamella Echinulata IFO 4445 is used and the rest of the procedure is carried out in the same manner as in Example 7 63 mg of IsoM-4 ′ lactone is prepared.

Example 9

If Cyncephallium racemosium IFO 4814 is used and the rest of the procedure is carried out in the same manner as in Example 7, 24 mg of IsoM-4 'lactone is prepared.

Example 10

Using Cefepalastrum racemosium IFO 4828 and the rest of the procedure was performed in the same manner as in Example 7, 35 mg of IsoM-4 'lactone is prepared.

Example 11

If Streptomyces Roseeochromogenus NRRL 1233 is used and the rest of the procedure is performed in the same manner as in Example 7, 12 mg of IsoM-4 'lactone is prepared.

Example 12

Preparation of IsoM-4 'Sodium Salt.

In a small amount of acetone After dissolving 10 mg of IsoM-4 'lactone, an equal amount of sodium hydroxide is added to the solution, and the mixture is left for 1 hour. The pH of this mixture is adjusted to 8.0 with 0.1 N hydrochloric acid. Acetone is then distilled off and the residue is placed on an XAD-20 column (about 20 ml). The column is washed with distilled water and then eluted with 50 ml of 50% V / V aqueous acetone. Acetone is distilled off again, and the residue is freeze-dried to have the following characteristics: 6 mg of IsoM-4 'sodium salt are obtained:

: 229(쇼을더);225;245(쇼울더).1) UV absorption spectrum (methanol solution)

229 (Shoulder); 225; 245 (Shoulder).

: 3400, 2850, 1710, 1580.2) infrared absorption spectrum (KBr)

: 3400, 2850, 1710, 1580.

3) Thin layer chromatography:

TLC plate: Merk silica gel Art 5715; Solvent: benzene, acetone, acetic acid (volume ratio; 50: 50: 3); Rf value: 0.45.

Example 13

Preparation of M-4 Methyl Ester.

Twenty 500-ml sagagucci flasks, each containing 100 ml of the medium having the same composition as shown in Example 1, were inoculated with Holcidia coerurea IFO 4423. The flask was vibrated for 2 days at 26 ° C and 120s.p.m. To each flask add the sodium salt of ML-236B until the final concentration is 0.05% W / V. Incubation is continued for 5 days at 26 ° C and 120s.p.m.

After incubation is complete, the reaction solution is filtered and the filtrate is adjusted to pH 3 with trifluoroacetic acid.

The reaction mixture is extracted three times with 1 L of ethyl acetate each time to obtain an extract containing M-3, M-4 and IsoM-4 '. Both M-4 and IsoM-4 'have an Rf value of 0.45 by thin layer chromatography (plate: Merk silica gel Art 5715 solvent: mixture of benzene, acetone and acetic acid with a volume ratio of 50: 50: 3). The compound extract is washed with a saturated aqueous solution of sodium chloride and then an ethereal solution of diazomethane is added. The mixture is left for 30 minutes and then dried under reduced pressure until dryness. This residue was placed on a Lobar column (Merk si 60, Size A) and purified using a mixture of benzene and ethyl acetate with a volume ratio of 1: 1 and fractions containing IsoM-4 'methyl ester and M Fractions containing -4 methyl ester are separated. 185.3 mg of the latter fractionated fraction was obtained, which was eluted with 35% V / V aqueous acetonitrile using a Lobar column (Merk RP-8, Size A) to give a colorless oily pure M-4 methyl ester 10MG. Get

M-4 methyl ester has the following characteristics.

1) NMR Spectrum:

It was measured at 200 MHz in deuterochloroform using tetraethylsilane as internal standard.

δ ppm: 0.88 (3H, triplet, J = 7.3 Hz); 0.89 (3H, doublet, J = 6.5 Hz); 1.12 (3H, doublet, J = 6.8 Hz); 1.1-1.7 (10H, multiplet); 2.34 (1H, doublet, J = 7 Hz); 2.3-2.5 (2H, multiplet); 2.49 (2H, doublet, J = 6.4 Hz); 2.58 (1 H, multiplet); 3.72 (3H, singlet); 3.78 (1 H, multiplet); 4.25 (1H, quartet, J = 7 Hz); 4.4 (1H, multiplet); 5.42 (1 H, multiplet); 5.56 (1 H, multiplet); 5.90 (1H, doublets of overlap, J = 9.8 and 5.6 Hz); 5.99 (1H, doublet, J = 9.8 Hz);

2) Mass Spectrum:

Measured using a Nippon D-300 mass spectrometer (after silylation with N, O-bis (trimethylsiline) trifluoroacetamide). M / e: 654 (M < + >), 552, 462, 372, 290, 272, 233, 231.

:230.1;237.3;246.4.3) Ultraviolet absorption spectrum (ethane jade solution)

: 230.1; 237.3; 246.4.

:3400, 2950, 1730.4) Infrared absorption spectrum (liquid film)

: 3400, 2950, 1730.

5) Thin layer chromatography

TLC plate: Marck silica gel Art 5715; Solvent: benzine and acetone (volume ratio 1: 1); Rf value: 0.88.

Other esters of M-4 can be prepared by operating in the same manner as described above using other suitable diazoalkanes instead of diazomethane.

Example 14

Preparation of Sodium Salts of M-4 and IsoM-4 '.

The procedure of Example 1 was completely repeated except that NaHPO 4 was used instead of K 2 HPO 4, and the reaction solution was filtered. The filtrate is absorbed on an HP-20 column (manufactured by Mitsubishi Chemical Industries). The column is washed with water and then eluted with 50% V / V aqueous acetone to give a fraction containing M-4 sodium salt, IsoM-4 'sodium salt and M-3 sodium salt. The fraction was lyophilized to yield 830 mg of lyophilized product, which was purified by repeated high performance liquid chromatography (column: MICRO BONDAPAK C18, 40% V / V aqueous methanol 1 ml'min), followed by M-4 sodium 32 mg of salt and 280 mg of IsoM-4 ′ sodium salt are obtained.

The properties of the IsoM-4 'sodium salt are the same as those of the product of Example 12, and the properties of the M-4 sodium salt are as follows.

1) NMR Spectrum:

It measured at 200 MHz in deuterochloroform using tetramethylsilane as an internal standard. δ ppm: 091 (3H, triplet, J = 7.5 MHz); 0.92 (3H, doublet, J = 7 Hz); 1.12 (3H, doublet, J = 7 Hz); 1.1-1.8 (10H, multiplet); 2.25 (1H, doublets of overlap, J = 15 and 7.6 Hz); 2.23 (1H, doublets of overlap, J = 15 and 5.5 Hz); 2.2-2.4 (3H, multiplet); 2.48 (1H, multiplet); 3.68 (1 H, multiplet); 4.07 (1H, multiplet); 4.28 (1H, multiplet); 5.36 (1H, multiplet); 5.48 (1H, doublets of overlap, J = 3 and 2 Hz); 5.88 (1H, doublets of overlap, J = 9.6 and 5.3 Hz); 5.98 (1H, doublet, J = 9.8 Hz).

: 230.0;237.2;245.0.2) UV absorption spectrum (methanol solution

: 230.0; 237.2; 245.0.

: 3400, 2900, 1725, 15803) infrared absorption spectrum (KBr)

: 3400, 2900, 1725, 1580

4) Thin layer chromatography

TLC plate: Merck silica gel Art 5715; Solvent: benzine, acetone and acetic acid (volume ratio 50: 50: 3): Rf value: 0.45

Example 15

18 mg of M-4 methyl ester is prepared by using Kuningamella equinulata IFO 4445, the remainder following the same procedure as in Example 13.

Example 16

33 mg of M-4 methylester is prepared by using Streptomyces roseochromogenus NRRL 1233 and performing the same procedure as in Example 13.

Example 17

Cinsepallastrum racemodium IFO 4814 is used and the remaining procedure is the same as in Example 13, 12 mg M-4 methyl ester is prepared.

Example 18

16 mg of M-4 ethyl ester is prepared by using Cyncephallium racemosium IFO 4828 and the remaining procedure is the same as in Example 13.

Example 19

Preparation of M-4 Methyl Ester

-Bondapak C₁₈)에 의해 정제한다. 네번째 피크를 나타내는 부분을 분리하고, 용매를 증류시켜 버리면 실시예 13에 표시한 성질을 갖는 무색유상의 M-4메틸 에스테르 4mg이 수득된다.Five beagles (males, males, average weight 10 kg) are given 200 mg / kg / day of ML-236B and their urine is collected for three days. Three liters of urine collected are collected in 500 ml of 50% V / V aqueous acetone. Pass the eluting 500 ml, XAD-2 column. After distilling acetone under reduced pressure, the residue is adjusted to 3 by adding trifluoroacetic acid. This mixture is extracted three times with 1 L of ethyl acetate each time to give M-4. This compound had a thin layer chromatography (TLC plate: Silck & Co., Znc. Silica gel Art 5715; Rf value 0.45 by a mixture of benzene, acetone and acetic acid with a solvent: volume ratio of 50: 50: 3). Wash with a saturated aqueous solution of saturated sodium chloride and add an ethereal solution of diazomethane and leave for 30 minutes, evaporate to dryness under reduced pressure, and dissolve the residue in 10 ml of a 55% aqueous V / V methanol solution. Pass through column chromatography (Merck & CO., Inc .; RP-8, SiZe B.) 200 ml of a 55% V / V methanol aqueous solution and then elute with 60% aqueous V / V methanol solution. Discard the first 240 ml and collect the last 120 ml. The fraction obtained by evaporation to dryness is dissolved in 2.5 ml of 65% V / V methanol aqueous solution, and high performance liquid chromatography (JASCO Triritar, column:

Purified by Bendapak C ₁₈ ). When the part showing the fourth peak is separated and the solvent is distilled off, 4 mg of a colorless oily M-4 methyl ester having the properties shown in Example 13 is obtained.

Example 20

M-4 recipe

In this M-4 example, homogenized rabbit liver was used to obtain M-4 from ML-236B.

(a) enzyme solution

Three volumes of 1.15% W / V potassium chloride-10 mM phosphate (pH 7.4) buffer are added to 1 volume of rabbit liver and the mixture is homogenized. The homogenized mixture is centrifuged at 9000 G for 20 minutes and the upper fraction is taken as enzyme solution.

(b) cofactor solution

β-type nicotinamide adenine

Reduced nucleotide phosphate (MADPH) 3 mg

0.1 ml MgCl2 solution (508 mg / 10 ml)

1.15% W / V KCl-0.3ml

0.2M phosphate buffer solution (pH7.4)

The above materials are mixed at a total volume of 1 ml to make a cofactor solution.

(c) reaction solution

80 µl of the enzyme solution, 20 µl of the cofactor solution, and 2 µl of the methanol solution of ML-236B are mixed to bring the final concentration of ML-236B to 1 mM. The resulting solution is vibrated at 37 ° C. for 30 minutes. M-4 is produced in the reaction mixture, which is confirmed by TLC (same conditions as in Example 19).

Example 21

2 mg of M-4 methyl ester is dissolved in 1 ml of 0.1 N aqueous sodium chloride solution and hydrolyzed at 30 ° C. for 1 hour. The reaction mixture is washed with 1 ml of chloroform, the resulting aqueous phase is adjusted to pH 8 with 0.1 N hydrochloric acid and then passed through an XAD-2 column (about 5 ml). The column is washed with 10 distilled water and the desired compound is eluted with 15 ml of 50% V / V acetone aqueous solution. Acetone is distilled off from the eluate. Confirmation of the residue by high performance liquid chromatography yields a single peak (eluted with 40% V / V methanol solution at 1 ml / min). The residue was then lyophilized to yield 0.8 mg of sodium salt of M-4 having the same properties as the product of Example 14.

Example 22

Preparation of M-4 Methyl Ester

Muco Hiemalis for 20 ml of 500 ml of Erlenmeyer flasks each containing 100 ml of a medium having the composition listed below. Inoculate with spores of Hiemalis IFO-5834. The seed is vibrated at 26 ° C 220s.p.m. After 4 days, add ML-236B until the final concentration is 0.05% W / V, and then continue incubating for 6 more days at 26 ° C, 220s.p.m. Composition of medium (% is W / V):

After the incubation was completed, the filtrate was adjusted to pH 3 using trifluoroacetic acid, and then the mixture was extracted three times with 100 ml of ethyl acetate each time. A fraction containing M-4 is obtained. M-4 has an Rf value of 0.45 by thin layer chromatography (plate: Merck silica cell Art 5715; mixture of benzene, acetone and acetic acid having a solvent: volume ratio of 50: 50: 3). The conversion rate is 90%. The extract is washed with a saturated aqueous solution of sodium chloride and then an ethereal solution of diazomethane is added. The resulting mixture is left for 30 minutes and then concentrated under reduced pressure until dry. The residue was placed on a Lobar column (Merck Si 60, Size A) and purified with a mixture of benzene and ethyl acetate (volume ratio 1: 1) to afford M-4 methyl ester having the same properties as the product of Example 13. 600 mg are obtained.

Example 23

Preparation of M-4 Lactones

The method described in Example 22 was completely repeated and the extract extracted three times with ethyl acetate was washed with a saturated aqueous solution of sodium chloride. The resulting solution is evaporated to dryness to yield the lactone product. Recrystallization of this product with ethyl acetate yielded about 560 mg (56%) of M-4 lactone, having the same resolution as the product of Example 1.

Example 24

Preparation of M-4 Sodium.

The method described in Example 22 was repeated to obtain 1.9 liters of filtrate from the conversion reaction. To obtain a fraction containing M-4, this filtrate is extracted three times with 1 L of ethyl acetate each time. When this is immediately placed in an aqueous 5% W / V sodium carbonate solution, a fraction containing M-4 sodium salt is obtained. This M-4 sodium fraction is adjusted to pH 7.0 with 2N hydrochloric acid and absorbed onto an HP-20 column (from Mitsubishi Chemical Industries). Washing with water and eluting with 50% aqueous V / V acetone yields a fraction containing M-4 sodium salt, which gives 170 mg (52%) of lyophilized product. This material has the properties described in Example 14.

Example 25

Manufacture of M-4

The method described in Example 22 is repeated except that the following microorganisms are used, and the conversion to M-4 is as indicated by the relevant symbols.

Codes indicating a switch to M-4 have the following meanings.

Trace amount = 0.5% or less

+ 1 = 0.5-5% + 2 = 5.10-0% + 3 = 10.0-30.% + 4 = 70.0-90.0%

Example 26

Preparation of IsoM-4'lactone

Twenty 500-ml sagagucci flasks, each containing 100 ml of the medium having the composition described in Example 22, were inoculated in Sircinella musca with IFO-4457. The seed is vibrated at 26 ° C. and 120 s.p.m. After 4 days, add ML-236B until the final concentration is 0.05% W / V and continue incubating for 6 days at 120s.p.m at 26 ° C.

After incubation is complete, the conversion reaction mixture is filtered and the filtrate is adjusted to pH 3.0 with trifluoroacetic acid. Then, the mixture is extracted three times with 1 liter of ethyl acetate each time to obtain a fraction containing IsoM-4 '. The extract is washed with a saturated aqueous solution of sodium chloride and evaporated to dryness. Lactone product is obtained. Absorption of this residue on a Lobar column (Merck Si 60, Size A) and purification with ethyl acetate yielded 12 mg of IsoM-4 'lactone with the sincerity described in Example 7.

Example 27

Preparation of M-4'lactone

Twenty 500 ml Erlenmeyer flasks containing 100 ml of each medium having the following composition were inoculated with C. cepenastrium nigricans NRRL-12478. The seed is vibrated at 220 ° C. at 220 s.p.m.

After 3 days, ML-236B is added until the final concentration is 0.05% W / V, and the culture is continued at 26 ° C and 220s.p.m.

The composition of the medium is as follows (% is W / V):

Glucose 1% Yeast Extract 0.1%

Peptone 0.2% Corn Dipped 0.3%

Meat Extract 0.1% (pH not controlled)

After the incubation is completed, the conversion reaction mixture is filtered and the filtrate is adjusted to pH 3 using trifluoroacetic acid. Then, the mixture was extracted three times with 1 L of ethyl acetate each time to obtain a fraction containing M-4 ', which was thin layer chromatography (plate: Merck silica gel Art 5715; benzene with volume ratio 50: 50: 3, Rf value of a mixture of acetone and acetic acid) is 0.46. The extract is washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and then a catalytic amount of trifluoroacetic acid is added to lactone. The resulting mixture is washed with 5% W / V aqueous sodium hydrogen carbonate solution, dried over anhydrous sodium sulfate and evaporated to dryness. Crystallization of the residue with ethyl acetate gave about 180 mg of M-4'lactone having the following physical properties.

1) NMR spectrum: measured at 100 MHz in duterchloroform using tetramethylsilane as internal standard.

δppm:

6.01 (1H, doublet); 5.50 (1H, multiplet);

5.90 (1 H, quartet); 4.60 (1 H, multiplet);

5.75 (1 H, multiplet); 4.25 (1 H, multiplet);

maxnm; 230,237,245.2) UV absorption spectrum (methanol)

maxnm; 230,237,245.

3) Infrared absorption spectrum (KBr) ν cm -1: 3500, 1720.

4) Mass spectrum: M / e: 406 (M-), 304, 286.

+310.9°(c=0.66,메탄올).5) Optical rotation:

+ 310.9 ° (c = 0.66, methanol).

6) Melting Point: 141 ~ 143 ℃

7) Elemental Analysis:

Calculated Value: C, 67.95% H, 8.43%

Found: C.68.05%, H, 8.37%

8) Thin layer chromatography:

TLC version: Merck silica gel Art 5715.

Solvent: benzine-acetone (volume ratio 1: 1)

Rf value: 0.64

Example 28

Preparation of M-4'Sodium Salt

Substantially, the conversion reaction mixture is obtained according to the same culture method as in Example 27.

After incubation is complete, the conversion reaction mixture is filtered and the filtrate is adjusted to pH 3 with trifluoroacetic acid. Three times each time, 1 L of ethyl acetate can be used to obtain a fraction containing M-4 '. After washing with a saturated aqueous solution of sodium chloride, immediately put into 5% W / V sodium hydrogen carbonate solution and add M-4' sodium. Fractions containing salts are obtained. The aqueous layer thus obtained was adjusted to pH 8.0 using 0.1 N hydrochloric acid and then absorbed onto a Dision HP 20 resin column (manufactured by Mitsubishi Chemical Industmies). Elution with 50% V / V acetone aqueous solution, acetone is distilled off and the residue is lyophilized to give 1.41 g of M-4'sodium salt having the following physical properties:

1) NMR spectrum: Measured at 600MHz in duterochloroform using tetramethylsilane as internal standard.

δppm:

6.00 (1 H, doublet); 5.70 (1H, broad singlet);

5.95 (1 H, quartet); 5.50 (1H, broad singlet);

maxnm : 230,238,246.2) UV absorption spectrum) Methanol

maxnm: 230,238,246.

3) Infrared absorption spectrum (KBr) ν cm ^-1 ; 3400,2900,1680

Example 29

Preparation of M-4'Methyl Ester

A conversion reaction mixture is obtained substantially following the same culture method as in Example 27.

After incubation is complete, the conversion reaction mixture is filtered and the filtrate is adjusted to pH 3 with trifluoroacetic acid. Extract three times with 1 L of ethyl acetate each time. The combined extracts are washed with a saturated aqueous solution of sodium chloride, and then an ethereal solution of diazomethane is added thereto. The resulting mixture is held for 30 minutes and then concentrated under reduced pressure until dry. The residue is purified using Lobar column (Merck RP-8, Size A) and a mixture of benzene and acetone (volume ratio 1: 1) as developing solvent. 150 mg of aster is obtained in a colorless oily substance M-4'methyl having the following properties:

1) NMR spectrum: Measured at 60 MHz in deuterochloroform using tetramethylsilane as internal standard.

δppm

6.01 (1H, doublet); 5.50 (1H, broad singlet);

5.90 (1 H, quartet); 3.70 (3H, singlet);

5.75 (1H, wide single line)

maxnm : 230,238,246.2) UV absorption spectrum) Methanol

maxnm: 230,238,246.

3) Infrared absorption spectrum (liquid film) v cm ^-1 ; 3400, 1730.

4) Mass spectrometry: After silylation with N, O-bis (trimethylsilyl) trifluoroacetamide, it was measured using a D-300 mass spectrometer manufactured by Nippon Electronics.

Claims (1)

Process for the preparation of a compound of formula (I) or a cyclized lactone, salts or esters thereof, characterized by enzymatically hydroxylating a substrate selected from ML-236B, or ML-236Bcarboxylic acid or salts or esters thereof .

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