JPH0742280B2 - γ-Lactone derivative and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a γ-lactone derivative useful as a substance or a raw material for inducing the formation of a production system of an antibiotic (for example, staphyromycin) at the start of production. Regarding the manufacturing method.

2. Description of the Related Art Recently, several kinds of hormone-like substances that regulate the production, resistance, morphological differentiation, etc. of antibiotics of actinomycetes have been isolated.

For example, Streptomyces
A-factor produced by griseus) is a substance that controls the production and resistance of streptomycin, and morphological differentiation such as spore settlement. [Doklady Akademii Nauk SSSR] 177 ,
232-235 (1967)] In addition, Streptomyces viridumogenes (Streptomyces viridumogenes) is an inducer that restores the production of anthracycline-producing block mutants of Streptomyces griseus.
ochromogenes), Streptomyces bikinensis (St
reptomyces bikinensis) and Streptomyces cyaneofuscatus (Streptomyces cyaneofuscatus) and the like. [Journal of Antibiotics 35 , 1722-1723 (1982);
Biotechnological Let
ter) 5, 591-596 (1983) Reference invention although you are trying to problems peptide antibiotics resolution widely used as fattening effect agent livestock culture titer of these antibiotics other When compared to those of antibiotics, many are much lower and there is a problem with compound productivity. Various basic studies for improving the culture titer have been carried out by making full use of conventional fermentation technology, but the technology as shown in this patent is still unknown in many fields, and it is overcome and put into practical use. If the technology is established, it will contribute to the productivity improvement of the above antibiotics.

Means for Solving the Problems As a result of studies in view of the above circumstances, the present inventors have found that certain microorganisms produce a novel γ-lactone derivative that induces the production of the antibiotic stafilomycin. Further, knowing that the γ-lactone derivative can be produced by a synthetic method, etc., the compound (II) described later among this γ-lactone derivative is referred to as virginie butanolide,
In the compound (II) described later, a compound in which R is methyl is named as Virginie-butanolide B, and a compound in which R is hydrogen is named as Virginie-butanolide C.

The present inventors have completed the present invention as a result of further research based on these findings.

That is, the present invention provides (1) the general formula [Wherein R is hydrogen or methyl, and A is And X represents a hydroxyl group which may be protected. ] The γ-lactone derivative (compound (I)) represented by the formula (2) belongs to the genus Streptomyces, and has the general formula [In the formula, R has the same meaning as described above. ] The virginie-butanolide (compound (II)) represented by the following formula is cultured in a medium, the virginie-butanolide is produced and accumulated in the culture, and this is collected. Method for producing butanolide and (3) general formula [In the formula, R and X have the same meanings as described above. ] The compound (III) represented by these is reduced, and the protecting group is eliminated as needed, It relates to the manufacturing method of the compound (II).

Examples of the protecting group for the optionally protected hydroxyl group represented by X above include trimethylsilyl, benzyloxycarbonyl, acetyl, etc. Among them, trimethylsilyl is preferable.

The bacterium producing compound (II) used in the method of the present invention may be any bacterium belonging to the genus Streptomyces and having the ability to produce compound (II).
An example thereof is Streptomyces virginiae. A specific example is Streptomyces
The virginie ACTT 13161 is mentioned.

The properties of the Streptomyces bacterium used in the method of the present invention are generally liable to change, and can be easily mutated by an artificial mutagenesis means using, for example, ultraviolet rays, X-rays, chemicals (eg, nitrosoguanidine, ethylmethanesulfonic acid) and the like. However, any mutant strain having the ability to produce the compound (II) of the present invention can be used in the method of the present invention.

In culturing the bacterium producing compound (II), examples of carbon sources include glucose, maltose, lactose, waste sugar concentrate,
Fats and oils (eg, glycerin, soybean oil, olive oil, etc.),
Grouped acids (eg citric acid, succinic acid, gluconic acid, etc.)
Those that can be assimilated by bacteria are appropriately used. As the nitrogen source, for example, a casein enzymatic degradation product [eg, Bact Kasiton (manufactured by Difco, USA)] Soybean flour, cotton seed flour, corn steep liquor, dry yeast, yeast extract, meat extract, peptone, urea, Organic or inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, ammonium chloride, and ammonium phosphate can be used. As the inorganic salt, for example, inorganic salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, dipotassium phosphate, and disodium phosphate, which are necessary for culturing ordinary bacteria, may be used alone or as appropriate. Used in combination.

Further, a defoaming agent such as silicone oil or polyalkylene glycol ether or a surfactant may be added to the medium. In addition, an organic substance or an inorganic substance that assists the growth of bacteria and promotes the production of compound (II) may be added as appropriate.

The culture method may be solid culture or liquid culture. In the case of liquid culture, any of static culture, stirring culture, shaking culture, aeration culture and the like may be carried out, but aeration and stirring culture is particularly preferable. The culture temperature is preferably in the range of about 20 ° C to 37 ° C, more preferably about 24 ° C to 28 ° C, and the pH of the medium is in the range of about 5 to 8, more preferably in the range of about 6 to 8, The culture is performed for about 18 to 66 hours, preferably about 18 to 48 hours.

In order to collect the target compound (II) from the culture, a separation means usually used for collecting the metabolite produced by the microorganism from the culture of the microorganism is appropriately used. For example, since compound (II) is mainly contained in the culture filtrate,
First, a filter aid is added to the culture solution, and the cells are removed by filtration or centrifugation. The obtained culture filtrate is extracted with an active ingredient of an appropriate organic solvent (eg, ethyl acetate, chloroform, etc.). Next, a means for bringing the extraction section into contact with an appropriate carrier to adsorb the active ingredient, desorbing the active ingredient with an appropriate solvent, and separating and collecting the active ingredient is advantageously used. The extract may be treated with an alkaline aqueous solution to remove impurities in the extract. As a carrier for chromatography, use the difference in the adsorptivity of compounds such as alumina, silica gel, powdered cellulose, and adsorbent resins, or the difference in the functional groups of compounds such as non-aqueous anion exchange resins, or compounds such as non-aqueous Sephadex Those that utilize the difference in the molecular weight of are advantageously used. In order to elute the target compound from these carriers, the combination varies depending on the type and properties of the carrier, but organic solvents such as alcohol (eg, methanol, iso-propanol etc.), chloroform, ethyl acetate, n-hexane etc. or those Are appropriately combined and used.

It is also possible to obtain a purified product by subjecting the crude substance containing the active ingredient obtained by these chromatography to preparative high performance liquid chromatography.

More specifically, the extract extracted with ethyl acetate or the like from the culture filtrate is separated with an n-hexane-methanol-water system to remove waxes, and potassium hydroxide is added to the methanol-water layer for heating. Reflux to hydrolyze impurities,
The solution is acidified and extracted with ethyl acetate. When the extract is subjected to chromatography using a non-aqueous anion exchange resin such as Amberlyst A-21 (manufactured by Organo, USA) or an adsorbent carrier such as alumina (manufactured by Merck, USA) as a carrier, the active ingredient Is adsorbed, methanol,
It is eluted with ethyl acetate or a combination thereof. The fractionated elution fraction is purified as an oily substance by concentrating, dissolving it in doubling solution, and the like. When the oily substance thus obtained has poor purity, high performance liquid chromatography (HPLC) is advantageously used for further purification. Examples of the carrier used include silica gel (Prep
PAK ^TM -500, manufactured by Waters, USA) and octadecyl silica gel (ODS, manufactured by Hanai Chemical Co., Ltd.)
As the moving bed, n-hexane-iso-propanol system and methanol-water system are used, respectively.

Vergnier butanolide B obtained in Example 1 described later
The physicochemical properties of C and C are as follows.

[1] Vergnier Butanolide B 1) Appearance: colorless oily substance 2) Circular dichroism (CD) spectrum: 214-2 in methanol
Shows + cotton effect around 18 nm.

3) Mass spectrum (EI-MS): m / z 230 (M ⁺ ) 4) Molecular formula: C ₁₂ H ₂₂ O ₄ 5) Ultraviolet absorption (UV) spectrum: End absorption in methanol 6) ¹ H nuclear magnetism Resonance (NMR) spectrum: in CDCl ₃ , 270MHz,
δppm 4.40 (1H, dd), 4.10 (2H, dd and m), 3.73 (1H, m),
2.85 (1H, m), 2.52 (1H, dd), 1.61-1.11 (4H, m), 0.89
(1H, d), 0.88 (1H, t) (however, dd: double doublet,
m) multiplet, d: doublet, t: triplet) 7) ¹³ C NMR spectrum: in CDCl ₃ , 68MHz, δppm 178.46,71.22,69.42,63.27,48.06,38.13,34.39,32.68,3
2.45,29.32,19.23,11.32 [2] Vergnier Butanolide C 1) Appearance: colorless oily substance 2) CD spectrum: + in the vicinity of 214 to 218 nm in methanol
Shows the cotton effect of.

3) Mass spectrum (EI-MS): m / z 216 (M ⁺ ) 4) Molecular formula: C ₁₁ H ₂₀ O ₄ 5) UV spectrum: end absorption in methanol 6) ¹ H NMR spectrum: in CDCl ₃ 270MHz, δppm 4.42 (1H, dd), 4.14 (1H, m), 4.10 (1H, dd), 3.74 (1H,
m), 2.87 (1H, m), 2.57 (1H, dd), 1.64-1.32 (3H, m),
1.32 (1H, m), 0.90 (1H, t) 7) ¹³ C NMR spectrum: 68 MHz, in CDCl ₃ , δppm 178.39,70.89,69.33,63.41,48.07,38.16,34.83,31.60,2
5.49,22.57,13.98 Next, a method for producing the compound (I) of the present invention will be described.

A of compound (I) And a compound in which X is a protected hydroxyl group (hereinafter referred to as compound (IV)) has the general formula (V) [In the formula, X represents a protected hydroxyl group. ] The compound (V) represented by the general formula (VI) [In the formula, R has the same meaning as described above. ] It is obtained by subjecting the compound (VI) represented by the following to a condensation reaction with lithium diisopropylamide (LDA). The reaction temperature is room temperature to −100 ° C., preferably 0 ° C. to −80 ° C., and the reaction solvent used is tetrahydrofuran (THF), diethyl ether, dimethoxyethane or the like.

Here, the compound in which X is trimethylsilyloxymethyl in the compound (V) is a known compound listed in Tetrahedron Letters 22 , No. 35, 3431-3342, and X is a protected compound other than trimethylsilyloxymethyl. The compound as the group can be similarly obtained by a method known per se. For the synthesis of the compound (VI), a general method of converting a carboxylic acid group halide of a lower fatty acid is used.

Compound (V) can also be produced by the following method. That is, the general formula [In the formula, Y represents lower alkyl. ] The compound represented by the formula is reacted in the presence of sodium borohydride (NaBH ₄ ) It is obtained by obtaining the compound represented by and then protecting the compound.

In the above formula, the lower alkyl represented by Y is preferably one having 1 to 2 carbon atoms, and examples thereof include methyl and ethyl.

Examples of the protecting group include the same protecting groups as described above.

The reduction reaction with NaBH ₄ is carried out in a solvent such as methanol, ethanol or THF, and the reaction temperature is 0 ° C to 50 ° C.
The temperature is preferably 10 to 40 ° C., and the reaction time is 30 minutes to 6 hours, preferably 1 to 3 hours. The amount of NaBH ₄ added is preferably about 0.25 mol with respect to the raw material compound.

A method known per se for introducing and removing a protective group for a hydroxyl group [TW Greene, Protective groups in organic synthesis]
rganic synthesis) p.10, John Wiley & Sons, 1981].

Furthermore, in compound (I), A is And X is a hydroxyl group, that is, compound (II)
Can be obtained by subjecting a compound (III) in which X is a hydroxyl group (hereinafter referred to as compound (VII)) to a reduction reaction. It is desirable to use alcohols such as methanol and ethanol as a reaction solvent, the reaction temperature is -10 ° C to 50 ° C, preferably 10 ° C to 35 ° C, and the reaction time is 15 minutes to 4 hours, preferably 30 minutes to 2 hours. NaBH
The amount of ₄ is preferably about 1.5 mol based on the starting compound.

The target compound (I) thus obtained can be isolated and purified by a means known per se, for example, concentration, phase transfer, solvent extraction, fractional distillation, chromatography and the like.

Among the compounds (I) thus obtained, the compound (II) is useful as a substance that induces the production system of antibiotics (for example, staphyromycin) at the initiation stage, and the compound (IV) And (VII) are compounds (I
It is useful as a raw material for I).

Examples The present invention is described in more detail below with reference to Reference Examples and Examples, but the present invention is not limited thereto. The percentage (%) means weight / volume percentage unless otherwise specified.

Reference Example 1 Production of 3-hydroxymethylbutanolide (compound (6)) Into a 500 ml eggplant flask, take 49.61 g of diethylformyl succinate (5) and add 200 ml of ethanol. 9.28 g NaBH ₄ are added in small portions with stirring in an ice bath. After the addition, a condenser was attached and the mixture was stirred at room temperature for 2 hours. A suitable amount of 4N HCl is added to the obtained reaction solution, and the precipitate formed in an acidic state is removed by suction filtration, the obtained filtrate is concentrated under source pressure, extracted with ethyl acetate, and the extracted portion is dried over anhydrous sodium sulfate. After concentration, 30.83 g of oil was obtained. To this oil, 120 ml of methanol and 40 ml of water were added, and 15 g of potassium carbonate was added little by little while stirring the solution to dissolve it. 1 this solution
After heating and refluxing in an oil bath at 10 ℃ for 2 hours, 4NHC again
The appropriate amount of l was added to acidify, the reaction mixture was concentrated, the residue was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated to 20.79 g.
Oil was obtained. Add this to a 200 g silica gel column.
Adsorbed in _{CH 2 Cl 2, CH 2 Cl} 2 -MeOH: eluting with (9 1, V / V) , to give an oil of 14.46 g. This oil is distilled under reduced pressure,
6.24 g of 3-hydroxymethylbutanolide (6) was obtained.

Physical properties of compound (6) ¹ H-NMR spectrum: 60 MHz, δ ppm, in CDCl ₃ The following signals are observed.

4.6 to 4.0 (2H, m) 3.61 (2H, d) 3.0 to 2.3 (3H, m) IR spectrum (liquid film method) Wavenumber showing major absorption (cm ^-1 ): 3400,1770 Reference Example 2 3-Trimethylsilyl Production of oxymethylbutanolide (compound (1)) 6.2 g of the compound (6) obtained in Reference Example 1 was dissolved in 6 ml of pyridine, and hexamethyldisilazane [((CH ₃ ) ₃
6 ml of Si) ₂ NH] was added, and then 6 ml of trimethylsilyl chloride ((CH ₃ ) ₃ SiCl) was added dropwise. After dropping, seal tightly 2
After stirring for an hour, benzene-n-hexane (1: 1) was added to the reaction solution.
The resulting precipitate was filtered off, and the filtrate was concentrated and dried to give a trimethylsilyl ether compound (1) (8.2 g) of compound (6).

Physical properties of compound (1) ¹ H-NMR spectrum: 60 MHz, δ ppm, in CDCl ₃ The following signals are observed.

4.5 to 4.1 (2H, m) 3.6 (2H, d) 3.0 to 2.3 (3H, m) 0.14 (9H, s) IR spectrum (liquid film method) Main absorption wavelength (cm ^-1 ): 1780 Example 1 ISP-2 Agar (manufactured by Difco, USA) Streptomyces virginie ATCC 13161 grown on a slope was inoculated into a 2-volume Sakaguchi flask containing 500 ml of the following composition (a), and shaken reciprocally at 28 ° C for 24 hours. Cultured.

Medium composition (a) Bacto-Casiton (Difco, USA) 7.5g Yeast extract (Difco, USA) 7.5g Glycerin 15.0g Sodium chloride 2.5g Distilled water 1 pH 6.5 The total amount of this culture solution is the following composition (b). Volume containing 30 media
50 tanks were inoculated and cultured for 24 hours under the conditions of 28 ° C., aeration 1 vvm, stirring 280 rpm, and internal pressure 1 kg / cm ² .

Medium composition (b) Polypeptone (manufactured by Daigo Nutrition Chemicals Co., Ltd.) 7.5g Powdered yeast extract (manufactured by Daigo Nutrition Chemistry Co., Ltd.) 7.5g Glycerin 15.0g Sodium chloride 2.5g Distilled water 1 pH 6.5 This culture solution The total volume 2000 including the following composition (b) 1200
Inoculate the tank of 28 ℃, aeration 1vvm, stirring 140rpm, internal pressure
The cells were cultured for 24 hours under the condition of 1 kg / cm ² .

The culture broth 1150 obtained above was adjusted to pH 5.0 to 6.0 with 2N sulfuric acid, and then a filter aid was added, followed by filtration and washing with water to obtain a filtrate 1280 (pH
5.7) was obtained. The filtrate was adjusted to pH 3.0 and then extracted with 1/3 volume of ethyl acetate of the filtrate to obtain an ethyl acetate layer 320. The ethyl acetate layer 315 obtained by washing this with 1/3 volume of water was concentrated under reduced pressure to obtain 318.8 g of an extract.

The ethyl acetate extract obtained above was treated with methanol-water (9:
It was dissolved in 1) and extracted with n-hexane 2 to remove the wax component in the n-hexane layer, and then methanol-
40 g of potassium hydroxide was added to 3 liters of the aqueous layer, and the mixture was allowed to stand overnight and then heated under reflux at 50 ° C. for 1 hour. It was acidified with hydrochloric acid, extracted with ethyl acetate, dried over sodium sulfate, and concentrated to obtain 166.6 g of extract.

The obtained extract was subjected to column chromatography using a non-aqueous anion exchange resin Amberlyst A-21 (3 kg, manufactured by Organo). The eluate with methanol was fractionated and the active fraction was concentrated to obtain 35.90 g of oil.

Next, alumina (700 g, manufactured by Merck & Co., Inc., USA) was packed in a column and column chromatography was performed. The eluate from ethyl acetate-methanol (8: 2) was fractionated, and the active fraction was concentrated to give 21.87 g of oil. This was dissolved in n-hexane-iso-propanol (8: 2) to remove insoluble matter, and 16.6 g of oil was obtained in the soluble portion. Next, preparative high performance liquid chromatography was performed using a silica gel column (Pr
ep PAK ^™ -500 / SILICA, manufactured by Waters, USA). The conditions are as follows.

Instrument: System Waters mobile phase: n-hexane: iso-propanol (8: 2) Flow rate: 100 ml / min Column pressure: 20 atm Detector: RI Since active fraction was present, concentrated 2.01 g of oil. Obtained.

Next, purification was performed again using the silica gel column under the same conditions as above. There was an activity peak, so concentrate
461 mg of oil was obtained.

Further, it was purified by HPLC under the following conditions using an ODS column.

Equipment: Tri-Rotor V, Ubidec-100-V, RID-300,
RC-250 (manufactured by JASCO Corporation) Column: Cosmosil 5C ₁₈ (manufactured by Hanai Chemical Co., Inc.) Mobile phase: Methanol: Water (1: 1) Detector: RI and UV210nm HPLC elution pattern is shown in FIG. When the eluate was fractionated, three active fractions were present, and Vergnier butanolides C and B were obtained in the fractions that eluted quickly.
The B and C fractions were concentrated together, and the HPLC purification was repeated to obtain virginier butanolide B 1.271 mg, C
1.210 mg was obtained.

Example 2 Production of 2-hexanoyl-3-trimethylsilyloxymethylbutanolide (Compound (2)) Lithium diisopropylamide (LDA) was prepared by reacting 3.61 ml of diisopropylamine and 25.70 ml of n-butyllithium (1.05M, n-hexane solution) at −78 ° C. in 80 ml of tetrahydrofuran (THF). The compound (1) 2.20 g THF was dissolved in this solution and added dropwise. 1 hour after dropping 15
Min, stirred at -78 ° C. Next, hexanoyl chloride 1.
While keeping the temperature at -78 ° C, 64 ml was dropped for 1 hour, then at -78
After reacting at 0 ° C, cooling was stopped and the temperature was raised to 0 ° C. The obtained reaction solution was poured into 15 ml of acetic acid and 100 ml of ice water, and extracted with CH ₂ Cl ₂ . The CH ₂ Cl ₂ layer was collected and concentrated, 200 ml of CH ₂ Cl ₂ was added to the residue again, extracted with saturated aqueous sodium hydrogen carbonate, the CH ₂ Cl ₂ layer was washed with a small amount of water, dried over sodium sulfate and concentrated. Thus, 3.05 g of an oily compound (2) was obtained.

Example 3 Production of 2-hexanoyl-3-hydroxymethylbutanolide (Compound (3)) 3.05 g of the compound (2) obtained in Example 2 was added to 40 m of ethanol.
The mixture was heated to reflux for 30 minutes in a mixed solution of 10 ml of water and 10 ml of water. 2.51 g of the crude reaction product obtained by concentrating the reaction solution was developed on a silica gel column (20 g) using an n-hexane-ether type solvent to obtain a fraction of n-hexane-ether (1: 1). 0.67 g of compound (3) was obtained.

Physical properties of compound (3) ¹ H-NMR spectrum: 60 MHz, δ ppm, in CDCl ₃ The following signals are observed.

4.6 to 3.9 (2H, m) 3.5 to 3.8 (2H, dd) 3.4 to 2.9 (2H, m) 2.6 to 2.9 (2H, dt) 2.0 to 1 (6H, m) 0.9 (3H, t) IR spectrum (liquid Membrane method) Wave number (cm ⁻¹ ) showing main absorption: 3450,1770,1720 Example 4 Production of 3-hydroxymethyl-2- (1′-hydroxyhexyl) butanolide (Compound (4)) 200 mg (0.93 mmol) of the compound (3) obtained in Example 3 was added to 2
Dissolve in 0 ml of methanol under ice cooling (-5
℃), sodium borohydride (NaBH ₄ ) 13.4mg (1.5 equivalents) was added and reacted at room temperature for 1 hour. 4N in this reaction solution
A suitable amount of HCl was added to acidify, the resulting precipitate was removed by suction filtration, the filtrate was concentrated, extracted with ethyl acetate, dried over sodium sulfate and the solvent was distilled off to obtain 175.28 mg of compound (4). Was obtained.

Physical properties of compound (4) ¹ H-NMR spectrum: 60 MHz, δ ppm, in CDCl ₃ The following signals are observed.

4.6-3.9 (3H, m) 3.8-3.6 (2H, dd) 3.0-2.3 (2H, m) 2.0-1.1 (8H, m) 0.9 (3H, t) Effect of the Invention The γ-lactone derivative of the present invention is It is useful as a substance or a raw material for inducing the production system of antibiotics (eg, staphyromycin) at the start of production.

[Brief description of drawings]

1 and 2 show the ¹ H-NMR spectra of virginier butanolides B and C obtained in Example 1, and FIG. 3 shows the elution pattern of HPLC in Example 1 using an ODS column. B and C in FIG. 3 show elution fractions of the virginie butanolide active ingredient.

Claims (3)

[Claims] 1. A general formula [Wherein R is hydrogen or methyl, and A is And X represents a hydroxyl group which may be protected. ] The γ-lactone derivative represented by 2. A general formula belonging to the genus Streptomyces [In the formula, R represents hydrogen or methyl. ] The method for producing virginie butanolide, which comprises culturing a microorganism having the ability to produce virginier butanolide represented by the formula [3] in a medium, allowing virginier butanolide to be produced and accumulated in the culture, and collecting the virginie butanolide. 3. General formula [In the formula, R represents hydrogen or methyl, and X represents an optionally protected hydroxyl group. ] The general formula characterized by reducing the compound represented by and removing the protective group as needed. [In the formula, R has the same meaning as described above. ] The manufacturing method of the compound represented by these.