JPH05123184A - Production of (+)monodesmethoxyschizandrin - Google Patents

Abstract

PURPOSE:To selectively obtain the subject compound useful as an optically active intermediate for the production of gomisin A known as a treating agent for hepatitis by treating dl-schizandrin with a specific microorganism. CONSTITUTION:The objective compound of formula II is produced by treating dl-schizandrin of formula I with a microbial strain belonging to genus Mortierella [e.g. Mortierella sp.-TM-11104 (FERM P-10967)] to convert exclusively the (+)schizandrin to (+)monodesmethoxyschizandrin of formula II and removing the unmetabolized (-)schizandrin from the metabolic product of the microorganism by partition process with acid and alkali. The treatment of the compound of formula I with the microorganism of genus Mortierella is preferably carried out by culturing the microorganism in the presence of di- schizandrin at 28-29 deg.C and pH5-6 under aerobic condition for 8-12 days under stirring.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a synthetic intermediate of the following formula (I) for producing gomisin A which is useful as a therapeutic agent for liver diseases.

[Chemical 1] And an advantageous method for producing (+) monodesmethoxyschizandrin.

[0002]

PRIOR ART The following formula (II)

[Chemical 2] Gomisin A represented by is a compound having an action of improving liver function, and is currently under development as a therapeutic agent for hepatitis.

This compound is obtained mainly by extraction and separation from Schisandra chinensis, but its yield is low, which has been a problem in industrial production. In addition, in the method of using a natural product as a raw material, in general, the content of the target product may vary depending on the place of production of the natural product, the production time, the climate at the time of production, and the production amount itself also varies. There was a problem that a stable supply could not be guaranteed.

[0004]

In developing and providing Gomisin A as a medicine, it is necessary to produce it as economically and stably as possible, and for that purpose, There has been a demand for the development of not only a method for efficiently obtaining gomisin A from natural products but also a method for producing it by chemical synthesis.

The present inventors have conducted various studies on the method for producing gomisin A, and previously prepared the following formula (III), which is a synthetic intermediate for gomisin A.

[Chemical 3] The present inventors have found a chemical synthesis method capable of advantageously producing schizandrin represented by:

However, the schizandrin obtained by this synthetic method is a dl-form, but is a pharmaceutically effective gomisin A.
Is a (+)-form, and in order to finally obtain Gomisin A by the chemical synthesis method, it is essential to develop a method for selectively utilizing only the (+)-form from the chemically synthesized dl-schizandrin. there were.

[0007]

As a result of further studies, the present inventor has found that when a specific microorganism is allowed to act on dl-schizandrin, only (+) schizandrin is selectively converted to (+) monodesmethoxyschizandrin. The present inventors have completed the present invention by finding that the converted product can be easily separated from (−) schizandrin and that gomisin A can be advantageously obtained from the converted (+) monodesmethoxy schizandrin.

That is, the present invention is characterized in that a microorganism belonging to the genus Mortierella is allowed to act on dl-schizandrin, and then (-) schizandrin is separated from this metabolite, which is a method for producing (+) monodesmethoxyschizandrin. Is.

The method of the present invention is represented, for example, by the following formula.

[Chemical 4]

The starting material for the process of the present invention, dl-schizandrin, can be prepared according to the following formula.

[Chemical 5]

That is, dl-schizandrin (III)
Is, for example, a tetracyclic compound represented by the formula (IV) as a starting material, which is reduced to a dihydroxy compound represented by the formula (V), which is then subjected to an oxidation reaction and then a methanesulfonylation reaction. Thus, dl− represented by the formula (VI)
It can be obtained by obtaining schizandrine mesylate and finally removing the methanesulfonyl group.

Specifically, the reduction reaction of the 4-ring compound (IV) is carried out by using diisobutylaluminum hydride, lithium aluminum hydride or the like as a reducing agent in an inert gas such as argon or nitrogen, and using this as tetrahydrofuran or ether. ,
It is carried out by reacting in a general organic solvent such as toluene, benzene, or dioxane under a temperature condition of about 0 ° C. to 100 ° C. for 10 minutes or more.

Further, the oxidation of the dihydroxy compound (V) obtained as described above is specifically carried out by using osmium tetroxide as an oxidizing agent in a solvent such as pyridine, water or acetone.
The reaction is carried out by stirring at 0 ° C. to 50 ° C. for 10 minutes or more, and the methanesulfonylation is performed using methanesulfonyl chloride or anhydrous methanesulfonyl,
In the presence of a tertiary amine such as pyridine or triethylamine, in a general organic solvent such as ether, benzene, tetrahydrofuran, dichloromethane, chloroform,
It is carried out by reacting for 5 minutes to 1 day under a temperature condition of about 30 ° C. to room temperature.

The final demethanesulfonylation reaction is carried out by a method similar to the above-mentioned reduction method.

Of course, in the present invention, not only the above method but also dl-schizandrin synthesized by another method can be used.

On the other hand, examples of the Mortierella microorganism used in the method of the present invention include:
The present inventors separated Mortierella sp. TM-I0501 and sp. TM- from the soil of Inashiki-gun, Ibaraki prefecture.
I0702, the same sp. TM-I0703, the same sp. T
M-I104, sp. TM-I1105 and the like can be mentioned, and the following are the mycological properties of Mortierella sp. TM-I1104, which is a typical one of them.

(1) Morphology Mycelium: Pure culture can be easily carried out in an ordinary medium. The young hypha lacks the septum. Sporangia: The sporangiophores are mainly formed on aerial hyphae. The sporangium is subspherical and has a diameter of 17 to 28 x 12 to 2
0 μ. The sporangium is smooth and melts to release spores. Spores: Colorless and smooth surface. The shape is often kidney-shaped to magatama-shaped, and irregular spores such as an isosceles triangle are sometimes observed. The size is 5-12 x 1-5
μm.

(2) Growth state in each medium potato agar medium (PDA): vegetative mycelia develop well,
It forms an aerial mycelium and the colonies show a clear tongue-like pattern. Oatmeal agar medium (OMA): The amount of aerial mycelium is smaller than that of PDA, but the tongue-like pattern of colonies is observed. Tuapec Agar: PDA and OMA for aerial mycelia
Extremely less than. Also, the tongue-like pattern of the colony is only slightly recognized.

(3) Physiological properties Growth temperature 23 to 30 ° C. (optimum temperature 28 to 29 ° C.) Growth pH 4.0 to 9.0 (optimal pH 5.0 to 6.0)

From the above properties, the microorganism TM-I1104 used in the present invention was judged to be a strain belonging to Mortierella. Therefore, this strain was deposited on August 18, 1989 at the Institute for Microbial Engineering, Ministry of International Trade and Industry, Ministry of International Trade and Industry (Ministry of Industrial Science, No. 10967).

In order to allow the microorganisms belonging to Mortierella to act on dl-schizandrin, first, TM- in a liquid medium is used.
A microorganism of the genus Mortierella such as I1104 may be seed-cultured, and the obtained seed may be cultured together with dl-schizandrin in a medium containing a carbon source, a nitrogen source and the like.

The addition of dl-schizandrin is preferably carried out before the first half of the logarithmic growth phase, particularly in the first half of the logarithmic growth phase. Culturing is performed at a temperature of 23 to 30 ° C., particularly 28 to 29 ° C. for about 5 to 15 days, preferably 8 to 12 days. The optimum pH of the medium is 5 to 6, and it is preferable to culture under aerobic conditions with stirring.

When the concentration of dl-schizandrin added to the medium is too high, the growth of microorganisms is inhibited. Therefore, the concentration of dl-schizandrin is generally about 1 g / l or less, particularly about 0.5 g / l or less. Is preferred.

As the medium used for the culture, soluble starch as a carbon source, fructose, maltose,
Galactose, glucose and the like containing peptone as a nitrogen source, yeast extract, casein, kasiton and the like and vitamins, minerals and the like are preferable, and commercially available potato-dextrose medium (DIFCO), Tuapeck agar medium (DIFCO). A medium in which a nitrogen source is added can be used.

In order to separate unmetabolized (-) schizandrin from the culture obtained as described above and obtain (+) monodesmethoxy schizandrin, it is present in (+) monodesmethoxy schizandrin. The property of the phenolic hydroxyl group may be used. That is, it is possible to separate (+) monodesmethoxyschizandrin from the culture by using an acid-alkali partitioning method or an ion exchange resin.

Of course, the culture solution is extracted with an organic solvent, and this extract is subjected to silica gel column chromatography and / or ODS column chromatography,
It is possible to obtain (+) monodesmethoxyschizandrin, or to obtain it in combination with the above method.

As a specific method for purifying the culture, the culture solution is sterilized by autoclaving, etc., and the filtered filtrate is adjusted to a basic liquid by using ammonia, sodium hydroxide, potassium hydroxide or the like. , N-hexane, petroleum ether, toluene, benzene, ether, chloroform,
After partitioning with a water-insoluble organic solvent such as acetic acid ethyl ester or n-butanol, the water tank is acidified with acetic acid, hydrochloric acid, sulfuric acid or the like, and n-hexane, petroleum ether, toluene, benzene, ether, chloroform is prepared. , A water-insoluble organic solvent such as ethyl acetate, n-butanol and the like.

The filtered culture broth was directly applied to an anion exchange resin, washed with water, and then eluted with an acidic aqueous solution of acetic acid, hydrochloric acid, sulfuric acid or the like, and the eluate was n-hexane, petroleum ether, toluene, Benzene, ether, chloroform,
It may be washed with a water-insoluble organic solvent such as acetic acid ethyl ester or n-butanol, or may be adsorbed to a resin such as Diaion or Sepa beads and washed with water, and then eluted with a water-soluble organic solvent such as acetone, ethanol or methanol .. In addition, when washing with water or eluting metabolites when using an anion exchange resin, the amount of elution is 3 minutes by adding a water-soluble organic solvent such as acetone, acetonitrile, ethanol, or methanol to the eluate. Can be set to 1 or less.

The extract of relatively high purity (90% or more) obtained by these methods is further subjected to column chromatography or preparative thin layer chromatography using a carrier such as silica gel, alumina, celite, polyamide or ODS. By applying it to the graph once or more,
(+) Monodesmethoxy schizandrin can be separated and obtained with high purity. At this time, as an elution solvent, n-
It is possible to use a single solvent or a mixed solvent such as hexane, petroleum ether, toluene, benzene, ether, chloroform, ethyl acetate, acetone, tetrahydrofuran, acetonitrile, ethanol, methanol and water.

[0030]

INDUSTRIAL APPLICABILITY The (+) monodesmethoxy schizandrin thus obtained is obtained by selectively demethylating only the 13-position alkoxy group according to the formula shown below.
Can be didesmethoxy schizandrin, and
12 and 1 of this (+) didesmethoxy schizandrin
The hydroxyl group at the 3-position can be converted to gomisin A by methylenedioxylation by a known organic synthesis method.

[Chemical 6] Selective demethylation of (+) monodesmethoxyschizandrin is carried out by reacting a Lewis acid in the presence of a base. More specifically, it is carried out by sufficiently dissolving (+) monodesizandrin in a solvent such as anhydrous dichloromethane, adding a base and stirring for about 10 minutes, and then adding a Lewis acid.

The reaction is carried out at temperatures between 0 ° C. and room temperature and takes about 24 to 120 hours to complete. Examples of the base used in the selective demethylation reaction include triethylamine, N, N-diisopropylamine, dicyclohexylamine, 1,8-bis (N, N-dimethylamino) naphthalene, pyridine, 2,6-dimethylpyridine, and 2 , 6-
Di-tert-butyl-4-methylpyridine, 2,2,6,6
-Tetramethylpiperidine, 1,1,1,3,3,3-hexamethyldisilazane, etc. are exemplified, and Lewis acids include boron trichloride, boron tribromide, aluminum trichloride, diethylaluminum chloride, Examples are dimethylboron bromide and the like.

The (+) didesmethoxy schizandrin produced by demethylation is extracted with a solvent such as ethyl acetate, if necessary, and subjected to silica gel column chromatography using a mixed solvent of ethyl acetate-hexane as an elution solvent. Can be purified.

The methylene dioxygenation reaction of (+) didesmethoxy schizandrin is carried out according to a conventional method using methylene iodide,
50 to 7 using methylene bromide, methylene chloride bromide, etc.
It is carried out at a temperature of about 0 ° C. for about 1.5 hours.

After completion of the methylenedioxylation, for example, gomisin A can be obtained by purification by silica gel column chromatography using a mixed solution of ethyl acetate-hexane as an elution solvent.

As described above, according to the method of the present invention, an optically active intermediate for the production of gomisin A, which is useful as a therapeutic agent for liver diseases, can be produced from dl-schizandrin obtained by the chemical synthesis method. , Which is extremely useful industrially.

[0036]

The present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited to these Examples.

Reference Example 1 (3aRS, SRbiar) -5,6-dihydro-6,7
-Bis (hydroxymethyl) -1,2,3,10,11,1
Synthesis of 2-hexamethoxydibenzo [a, c] cyclooctene: Tetrahedor (Tetrahedor) in Argon Stream
on), Vol. 47, pp. 3787 to 3804 (199)
(3aRS, SRbiar) -3 prepared according to 1)
a, 4-Dihydro-6,7,8,9,10,11-hexamethoxydibenzo [4,5: 6,7] cycloocta [1,2-
c] Furan-1 (3H) -one (137.7 mg, 0.3
THF solution of 1 mmol) was cooled to 0 ° C., and a toluene solution of hydrogenated diisobutylaluminum (1.50 mol /
1, 0.6 ml, 0.9 mmol) was added and stirred for 30 minutes. Acetone (20 ml) was added, and ethyl acetate (20 ml) was added.
ml), 2N HCl (50 ml), water (5 ml)
0 ml), saturated aqueous sodium hydrogen carbonate (50 ml), saturated saline (50 ml)
ml). After drying over anhydrous magnesium sulfate,
The solvent was evaporated under reduced pressure to give a colorless oil (155 mg). Purify by column chromatography (silica gel [Merck # 9385] 10 g, ethyl acetate),
(3aRS, SRbiar) -5,6-dihydro-6,7
-Bis (hydroxymethyl) -1,2,3,10,11,1
2-Hexamethoxydibenzo [a, c] cyclooctene (GO-45) (139 mg, 100%) was obtained as a colorless foam.

Melting point: 161.5-162.5 ° C. (colorless prism crystal, ethyl acetate-hexane) ¹ HNMR (CDCl ₃ ): 1.57 (2H, br, OH),
2.51 (1H, dd, J = 13,18Hz, C5-H), 2.
97-3.06 (2H, m, C5, 6-H), 3.52 (3H,
_{s, C1-OCH 3),} 3.68 (3H, s, C12-OC
_{H 3), 3.85 (6H,} s, C2,3-OCH 3), 3.87
(3H, s, C10-OCH 3), 3.91 (3H, s, C11-
_{OCH 3), 3.79-4.00 (2H,} m, C6-CH
₂ O), 4.09 (1H, d, J = 122Hz, C7-CH
₂ O), 4.16 (1H, d, J = 12Hz, C7-CH
₂ O), 6.43 (1H, s, C4-H), 6.48 (1H, s,
C9-H), 6.53 (1H, s, C8-H). IR (KBr, cm- ¹ ): 3524 (OH), 148
8 (Aromatech) MS: 446 (M ⁺ , base) Elemental analysis (as C ₂₄ H ₃₀ O ₈ (M ⁺ )) Calculated value; C, 64.56; H, 6.77. Actual value; C, 64.34; H, 6.87.

Reference Example 2 Synthesis of dl-schizandrin mesylate and dl-schizandrin: (1) (3aRS, SRbiar) -5,6-dihydro-
6,7-bis (hydroxymethyl) -1,2,3,10,1
1,12-Hexamethoxydibenzo [a, c] cyclooctene (102 mg, 0.23 mmol) in pyridine (2
osmium tetroxide (62 mg, 0.24 m)
mol) was added, and the mixture was stirred at room temperature for 45 minutes. Further, saturated aqueous sodium hydrogen sulfite (2 ml) was added, and at room temperature,
Stir for 3.5 hours. Add ethyl acetate (25 ml),
It was washed with water (10 ml), 6N HCl (10 ml), saturated aqueous sodium hydrogen carbonate (10 ml) and saturated saline (10 ml), dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure to give an anhydrous oil (66 mg). Was given. This oily substance was dissolved in pyridine (1 ml), methanesulfonyl chloride (0.05 ml) was added, and the mixture was stirred at room temperature for 0.5 hr.
Dissolve in ethyl acetate (20 ml) and add 2N HCl (20
ml), water (20 ml), saturated aqueous sodium hydrogen carbonate (20 ml) and saturated saline (20 ml), and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to give a pale yellow oil (9
Dl-schizandrin mesylate was obtained as 9.5 mg).

(2) TH of lithium aluminum hydride
F solution (0.25 mol / l, 2 ml, 0.5 mmol)
Was heated to reflux under an argon atmosphere, to which was added a solution of the mesylate (28 mg) obtained above in THF (1 ml), and the mixture was further heated to reflux for 30 minutes and cooled with ice. Ethyl acetate (50 ml) was added, and the mixture was washed with 2N HCl (50 ml), water (50 ml), saturated aqueous sodium hydrogen carbonate (50 ml) and saturated brine (50 ml). After drying over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure to give a colorless oil (21 mg). Column chromatography (silica gel [Merck # 9385] 20 g, ethyl acetate-hexane)
Purification at 1: 3) gave dl-schizandrin as a colorless oil (8 mg, 29%). Dl thus obtained
-The NMR spectrum of Schizandrin was in agreement with that of natural Schizandrin.

Example (1) Glucose 40 g / l, peptone 10 g / l,
0.7 g / l potassium dihydrogen phosphate, magnesium sulfate
Heptahydrate 0.5 g / l, medium 10 adjusted to pH 5.5
ml was put into an Erlenmeyer flask and sterilized by using an autoclave at 121 ° C. for 15 minutes. This was inoculated with Mortierella TM-I1104 and seed culture was carried out for 3 days. 1 part of the cells that had been seed-cultured were inoculated into the same medium as described above, shake-cultured at 25 ° C. and 120 rpm, and after 48 hours, 5 mg of racemic schizandrin was added,
Cultivation was continued for 12 days.

(2) After culturing, the culture solution was autoclaved and sterilized, and the filtrate obtained by filtration was adjusted to pH 13 with 1N-sodium hydroxide and then extracted with ether.
The obtained ether extract was subjected to ODS column chromatography, and colorless (-) schizandrin was obtained as an unmetabolite from a fraction eluted with a water / methanol / tetrahydrofuran / acetonitrile mixed solution (100: 35: 8: 35). It was

(3) Furthermore, the aqueous layer (p
H13) was adjusted to pH 4 with acetic acid, extracted with ether again, and the obtained ether extract was subjected to ODS column chromatography to give a mixed solution of water / methanol / tetrahydrofuran / acetonitrile (100: 35: 8: 3).
Colorless (+) monodesmethoxyschizandrin was recovered as a metabolite from the fraction eluted in 5).

(4) The CD spectra of these separated and purified metabolites and unmetabolites were measured.
The spectra showed the same positive cotton effect as the native schizandrin, while the CD spectrum of the unmetabolite showed the negative cotton effect opposite to the natural schizandrin. This indicates that the metabolism of racemic schizandrin by TM-I1104 is preferential to (+)-schizandrin. Furthermore, the instrumental data of the compound obtained by methylating this metabolite with methyl iodide and potassium carbonate in pyridine are as follows, and the instrumental data other than the specific rotation were in agreement with the instrumental data of natural schizandrin. .. The optical purity obtained by comparing the specific optical rotation of 82.0 ° with the specific optical rotation of natural schizandrin of 88.1 ° was 93% or more, and most of the metabolites were (+) monodesmethoxyschizandrin. Was confirmed. As is clear from these results, by allowing Mortierella microorganisms to act on dl-schizandrin, it is possible to separate the naturally occurring optically active substance from dl-schizandrin with high purity.

[Α] _D : + 82.0 ° (C = 0.755,
CHCl ₃ ) M S (m / z (%)): 432 (M ⁺ ). ¹ HNMR (δppm, in CDCl ₃ ); 0.82 (3H,
d, J = 7.3), 1.25 (3H, s), 1.85 (1H,
m), 2.34 (1H, dd, J = 14.2, 7.8Hz),
2.37 (1H, d, J = 13.2Hz), 2.60 (1H,
dd, J = 14.2,1.5Hz), 3.55 (6H, s),
3.90 (3H, s), 3.91 (3H, s), 3.92 (3
H, s), 5.72 (1H, s), 6.62 (2H, s). ¹³ CNMR (δppm, in CDCl ₃ ); 15.8 (C 1
7), 29.8 (C18), 34.0 (C9), 40.9 (C6),
41.9 (C8), 56.0 (C1-OMe), 60.2 (C1
4-OMe), 60.6 (C3-OMe), 61.0 (C13-)
OMe), 61.0 (C2-OMe), 72.0 (C7), 1
10.2 (C4), 113.1 (C11), 122.0 (C1
5), 124.1 (C16), 132.1 (C5), 134.7
(C10), 137.8 (C13), 140.9 (C2), 14
7.8 (C12) 150.4 (C14), 152.1 (C3), 1
52.4 (C1). And above

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yukinobu Iketani 3586 Yoshiwara, Ami-cho, Inashiki-gun, Ibaraki Tsumura Co., Ltd.

Claims (1)

[Claims] 1. A method characterized in that a microorganism belonging to the genus Mortierella is allowed to act on dl-schizandrin, and then (-) schizandrin is removed from this metabolite (+).
Method for producing monodesmethoxy schizandrin.