JPH08149986A - Production of 12-hydroxy-6-o-alkylerythromycin and its analog - Google Patents

Abstract

PURPOSE: To provide a microbial process for easily producing the subject com pound by adding a 6-O-alkylerythromycin and its analog to a solution containing actinomycotic cell or its enzyme capable of hydroxylating the erythromycin compound and its analog and reacting the compound with the enzyme. CONSTITUTION: A 6-O-alkylerythromycin having hydrogen atom at 12-site and its analog are added to a solution containing the cells of actinomycete capable of hydroxylating the 6-O-alkylerythromycin and its analog (e.g. Micromonospora megalomycea subsp. nigra IFO14114) or an enzyme produced by the cell and the cells are cultured under agitation and shaking at 28 deg.C for 96 hr to convert the 12-hydrogen atom into hydroxyl group and obtain the objective 12-hydroxy-6- O-alkylerythromycmn of formula I [R<1> is a 1-4C alkyl; R<2> is H or methyl; R<3> is H or OH; R<4> is H or group of formula II (R<5> is H or methyl)] and its analog.

Description

Detailed Description of the Invention

[0001]

The present invention relates to 12-hydroxy-6-
The present invention relates to a method for producing O -methylerythromycin and its analogs using a microorganism.

[0002]

2. Description of the Related Art It is extremely difficult to directly convert a hydrogen atom at the 12-position of 6- O- alkylerythromycin and its analogs to a hydroxyl group by an organic chemical method, and an example thereof has not been reported yet. In addition, a method for directly converting the hydrogen atom at the 12-position of 6- O- alkylerythromycin and its analogs into a hydroxyl group by an enzymatic chemical method using a microorganism has not been reported so far.

[0003]

The object of the present invention is to provide a method for obtaining 12-hydroxy-6- O -methylerythromycin and its analogs by an easier means.

[0004]

The present inventors have found that the hydrogen atom at the 12-position of 6- O- alkylerythromycin and its analogs can be directly converted into a hydroxyl group by utilizing a specific microorganism, and the present invention Was completed. The present invention, 6- O - alkyl erythromycin and actinomycetes cells hydroxylating analogue thereof or a solution thereof bacterial cells containing an enzyme produced, 6- O having a hydrogen atom at position 12
-Adding alkyl erythromycin and its analogs to convert the hydrogen atom into a hydroxyl group 12-
A method for producing hydroxy-6- O- alkylerythromycin and its analogs.

The production method of the present invention is a method of directly hydroxylating the 12-position of 6- O- alkylerythromycin and its analogs having a hydrogen atom at the 12-position in one step.
It may have any substituent other than the 2-position. Therefore, 6- O- having a hydrogen atom at the 12-position in the present invention.
Alkylerythromycin (hereinafter sometimes simply referred to as a substrate) means, for example, 6- O -methylerythromycin B, 6- O -ethylerythromycin B, 6- O-.
Examples include propylerythromycin B, 6- O -butylerythromycin B, 6- O -methylerythromycin D, and the like. Also, 6- O having a hydrogen atom at position 12 - analogues of alkyl erythromycin (hereinafter, simply referred to as substrate) and is, for example, 6- O - methylerythromycin B oxime, Dekurajinoshiru-6-O - methylerythromycin B, 14-hydroxy-6- O -methylerythromycin B, 6,11-di- O -methylerythromycin B, 6,4 "-di- O -methylerythromycin B, 6,11,4" -tri- O- Methylerythromycin B, 6- O -methylerythronolide B and the like.

The actinomycetes used in the present invention are 6- O-
It is a bacterium having the ability to convert the hydrogen atom at the 12-position of methylerythromycin and its analogs to a hydroxyl group. They are, for example, Bergey's Manual of Systemic Bacteriology.
tematic Bacteriology), Vol. 4, a actinomycetes 1989., typically Akuchinomajura (Actinomadura) genus Amikorata (Amycolata) genus Nocardia (Nocardia) genus Micromonospora (Micromon
genus ospora ), Saccharopolyspor
a ) genus, Streptomyces ( Streptomyces ) genus and the like can be mentioned, preferably Micromonospora, Streptomyces, Saccharopolyspora actinomycetes, most preferably Micromonospora megalomicea subspecies Nigra (Micromonospora megalomicea subsp. ni
gra ) IFO14114, Streptomyces felleus JCM4031, Saccharopolyspora eryt
hraea ) IFO 13426.

Micromonospora megalomicea subsp. Nigra IFO14114 ("Agricultural Research Cult
Strains of the same origin as the preserved strain NRRL3275 of the "ure Collection"; Reference: US Pat. No. 3,632,750
No.) and Saccharopoli spora erythrea IFO13
426 ["Agricultural Research Culture Collectio
Strain that has the same origin as the "n" preserved strain NRRL2338; Reference: International Journal of Systematic Bacteriology (Int. J. Syst. Bac
teriol.), 37, 458, 1987, and International Journal of Systematic Bacteriology (Int. J. Syst. Bacteriol.), 37, 19-22, 1987]. Conserved strains of the Fermentation Research Institute, each of which is called “FERM B
P-5194 "and" FERM BP-5195 "have been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, and the mycological properties of these strains have been clarified by the above literature. Streptomyces ferreus JCM4031
(A strain having the same origin as the conserved strain NRRL2251 of "Agricultural Research Culture Collection";
U.S. Pat. No. 2,693,433) is a preserved strain of the microbial strain preservation facility of the Institute of Physical and Chemical Research (FERM BP-5).
193 ”has been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology, and the mycological properties of this strain have been clarified by the above-mentioned literature.

Further, the cells or spores of actinomycetes are treated with, for example, ultraviolet rays or NTG (N-methyl-N'-nitro-N-nitrosog).
It is also possible to mutate it by subjecting it to uanidine) treatment or the like to obtain and use a more preferable strain.

In order to obtain the cells of actinomycetes required for the reaction in the present invention, the strain is cultured in a medium under aerobic conditions. As the medium, a liquid medium is mainly used, and glucose, maltose, dextrose, starch, oatmeal, arabinose, xylose, and sucrose are used alone or as a mixture as a carbon source. As a nitrogen source, peptone,
Casein, polypeptone, soybean peptone, yeast extract,
Meat extract, corn steep liquor, malt extract, cottonseed meal, gluten meal, soybean flour and the like are used alone or in combination. In addition, organic substances and inorganic salts that help the growth of this strain and promote the production of 12-hydroxy-6- O- alkylerythromycin and its analogs can be added if necessary. As for the culture method, culture under aerobic conditions such as shaking culture and aeration stirring culture is suitable, and the pH is 6 to 8, 26.
Incubate at -30 ° C for 2-7 days.

The actinomycete cells obtained by this culture or a solution containing the enzyme produced by the cells is used in the reaction for producing 12-hydroxy-6- O- alkylerythromycin and its analogs. That is, the substrate is added to the culture solution containing the microbial cells in culture, or the substrate is added to a suspension of the microbial cells separated by centrifugation or filtration after the completion of the culture to start the reaction. After the completion of the culture, the bacterial cells separated by centrifugation or filtration may be crushed to extract the enzyme produced, and a substrate may be added to this to react. In addition, α-cyclodextrin, β-
Cyclodextrins such as cyclodextrin and γ-cyclodextrin, organic substances such as adsorbents such as Amberlite XAD-7 [trade name; manufactured by Organo Corporation], surfactants such as Tween 80 and magnesium chloride Inorganic salts can be added for the purpose of promoting the reaction. Furthermore, actinomycetes and enzymes produced by the cells are photocrosslinkable resin prepolymers such as ENT340.
0 [trade name; manufactured by Kansai Paint Co., Ltd.], urethane prepolymers such as PU-9 [trade name; manufactured by Toyo Tire & Rubber Co., Ltd.], and after being immobilized on polysaccharides such as κ-carrageenan It may be added to the solution. Also, freeze-dried cells of the pre-stage cells can be used in the same manner as above.

The reaction solution used in the present invention is a culture solution obtained by culturing using the above-mentioned medium, or tris-acetic acid, tris-hydrochloric acid, sodium succinate-succinic acid,
Potassium succinate-succinic acid, sodium citrate-citric acid, phosphates such as potassium and sodium, buffer solutions such as sodium cacodylate-hydrochloric acid, imidazole-hydrochloric acid, boric acid-borax, etc., alone or mixed. . Other solutions include the desired 12-hydroxy-6- O-
If necessary, an organic substance, a surfactant and an inorganic salt that accelerate the production of alkylerythromycin and its analogs can be added.

In the present invention, it is suitable to carry out a shaking operation, an aeration stirring operation, etc. in a solution containing the above-mentioned bacterial cells under aerobic conditions, and the pH is 5 to 9 and 18 to 37 ° C.
Shake for 10 days. Further, the reaction can be performed under an oxygen stream. An appropriate amount of substrate is added at the start of stirring and shaking.
When using a culture solution containing cells in culture, the substrate is added, and then the reaction is performed under the same conditions as above. In order to isolate 12-hydroxy-6- O- alkylerythromycin and its analogs produced by these reactions, a general method for collecting macrolide antibiotics such as erythromycin A from a culture solution can be performed according to a general method. Good. For example, after completion of the reaction, the reaction solution is extracted with an organic solvent such as ethyl acetate or methylene chloride. After separating and concentrating the organic solvent layer, a normal phase silica gel column (Kieselgel 60F2
54, manufactured by Merck & Co., Inc. and a reversed-phase silica gel column (row bar column, lycroprep C18, manufactured by Merck & Co., Inc.) for purification of desired 12-hydroxy-6- O.
-Methylerythromycin and its analogs can be isolated. Also, partition chromatography using Sephadex LH-20 (manufactured by Pharmacia), thin layer chromatography on silica gel, and Diaion HP.
-20 (trade name; manufactured by Mitsubishi Kasei Co., Ltd.)
-Hydroxy-6- O- alkylerythromycin and its analogs can be isolated.

[0013]

By the method of the present invention, 6- O having a hydrogen atom at position 12 - alkyl erythromycin and 12 of its analogs directly hydroxylating 12-hydroxy-6-O
It has become possible to produce alkyl erythromycin and its analogs.

[0014]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 Sterile liquid medium of pH 7.0 consisting of starch 2.4%, dextrose 0.1%, peptone 0.5%, meat extract 0.3%, yeast extract 0.5%, calcium carbonate 0.2%. (Hereinafter abbreviated as GM medium) 200 ml containing 40 ml
Inoculate an Erlenmeyer flask with branches with 1 platinum loop of Micromonospore megalomicea subsp. Nigra IFO14114 strain,
Culture was carried out at 28 ° C. for 96 hours with shaking. After the completion of the culture, the culture solution was centrifuged to collect the cells, and the cells were mixed with 15 mM Tris-hydrochloric acid, 25 mM sodium succinate, and 2 mM magnesium chloride at a pH of 7.4 (hereinafter referred to as buffer solution A).
It is suspended in 40 ml, and the suspension is centrifuged again to collect the cells. The cells were sufficiently stirred and suspended in 40 ml of buffer solution A, and placed in a 200 ml Erlenmeyer flask with a branch. In this Erlenmeyer flask, 4 mg of 6- O -methylerythromycin B dissolved in 400 μl of ethanol was added.
[Reference: The Journal of Antibiotics, Vol. 43, No. 5,
Page 544, 1990] was added, and the reaction was carried out by stirring and shaking at 28 ° C. for 4 days. P of the reaction solution after completion of the reaction
After adjusting H to 10 with sodium hydrogen carbonate, the methylene chloride layer extracted with 40 ml of methylene chloride was concentrated under reduced pressure, and then subjected to thin layer chromatography on normal phase silica gel (thin layer plate: Kieselgel 1.05715, manufactured by Merck & Co., Inc.). , Developing solvent: methylene chloride: methanol: 25% aqueous ammonia = 100: 10: 1). After development, this was subjected to iodine color development, and the silica gel in the portion corresponding to the clarithromycin standard product (manufactured by Taisho Pharmaceutical Co., Ltd., reference: US Pat. No. 4,331,803 and US Pat. No. 4,990,602, the same in the following examples) was scraped off, and ethyl acetate It was extracted with. The ethyl acetate extract was concentrated under reduced pressure, and then subjected to thin layer chromatography on reverse-phase silica gel (thin layer plate: Kieselgel Art.5747, manufactured by Merck & Co., a developing solvent; methanol: water: ammonium acetate 15% solution = 5: 2:
It is attached to 1). After the development, this is colored with iodine, and the silica gel part that matches the clarithromycin standard is scraped off.
It was extracted with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to dryness to obtain 1.2 mg of a product. This is a clarithromycin standard and liquid chromatography (TSK-gel
ODS-120A: manufactured by Tosoh, developing solvent: acetonitrile: methanol: 50 mM sodium phosphate pH
6.8 = 6: 2: 3, flow rate 1 ml / min, column temperature 50 ° C., retention time of photodiode array detector (manufactured by Nippon Millipore)), ultraviolet absorption spectrum, mass spectrum cleavage pattern completely matched. . Retention time in liquid chromatography: 8.7 minutes UV: λ _max = 288 nm (acetonitrile) FAB-MS (m / z): 748 ([M + H] ⁺ ), 5
90 ([M + H] ⁺ -cladinose), 158.

Example 2 Micromonospore megalomicea subsp. Nigra IFO1411, which was previously cultivated in GM medium in each of three jars having a volume of 1.8 L containing 1 L of sterilized GM medium
40 ml of the culture solution of 4 strains was inoculated, and the cells were cultured at 28 ° C. for 72 hours with aeration and stirring. The culture solution was collected by centrifugation, suspended in buffer A2L, and then collected again by centrifugation. A suspension prepared by suspending the cells in buffer A3L was added to each of the three jars having the internal volume of 1.8 L used in the above culture, 1 L each. Substrate 6
- O - methylerythromycin B jar per group 2
00 mg was added, and the reaction was carried out at 28 ° C. with aeration and stirring for 96 hours. After completion of the reaction, the reaction solutions were collected, adjusted to pH 10 with sodium hydrogen carbonate, and extracted with 1.5 L of ethyl acetate. After concentrating this under reduced pressure, column chromatography on normal phase silica gel (Bond Elute: trade name, manufactured by Uniflex, size 20 ml, elution solvent: methylene chloride-methanol-25% aqueous ammonia =
10: 1: 0.1). 140 ml of the eluting solvent was concentrated to obtain a reddish brown oily concentrate. This is 12 at 5 ℃
After standing for a period of time, the obtained crude crystals were dissolved in 5 ml of acetonitrile-methanol-methylene chloride = 10: 2: 1,
Reversed-phase silica gel column chromatography (Rover column, size B, Licroprep C18; trade name, manufactured by Merck, 25 mm ID × 310 mm, elution solvent: acetonitrile-methanol-50 mM sodium phosphate buffer pH 6.8 = 6: 2: 3). The clarithromycin elution fraction was subjected to thin layer chromatography on reversed-phase silica gel (thin layer plate: Kieselgel Art.57.
47, manufactured by Merck & Co., Inc., a developing solvent; methanol: water: 15% ammonium acetate solution = 5: 2: 1), and the elution fractions were collected and concentrated to dryness. This was recrystallized from ethanol to obtain 46.4 mg of clarithromycin. This was in perfect agreement with the clarithromycin standard in melting point, ultraviolet absorption spectrum, mass spectrum cleavage pattern, IR absorption spectrum and ¹³ C-NMR.

M. p. : 225-227 degreeC UV: (lambda) _max = 288nm (acetonitrile) FAB-MS (m / z): 748 ([M + H] ^< +>), 5.
90 ([M + H] ⁺ -cladinose), 158 IR (KBr) ν _max = 3474,1733,1693
cm ^{-1 13} C-NMR (100.4 MHz, CDCl ₃ ) δ
(Ppm): 221.0, 175.8, 102.7, 9
6.1, 80.9, 78.4, 78.3, 77.9, 7
6.6, 74.2, 72.7, 70.9, 69.0, 6
8.6, 65.8, 65.6, 50.6, 49.5, 4
5.2, 45.0, 40.3, 39.3, 39.1, 3
7.2, 34.9, 29.0, 21.5, 21.4, 2
1.0, 19.7, 18.7, 18.0, 15.9, 1
2.3, 10.6, 9.1.

Example 3 The strain used in the procedure of Example 1 was micromonospores.
Saccharopolispora erythraea IFO134 in place of the megalomicea subsp. Nigra IFO14114 strain
26 strains and Streptomyces ferreus JCM40
31 was used. For strains of the genera Saccharopolispora and Streptomyces, oatmeal 2.0
%, Glucose 2.0%, meat extract 0.3%, sodium chloride 0.3%, ferric sulfate 0.04%, manganese chloride 0.04%, calcium carbonate 0.3%, pH 7.0 Sterile liquid medium was used for the culture. As a result, the production of clarithromycin was confirmed in each strain. The clarithromycin production in each strain is shown in Table 1 below.

[0018]

[Table 1]

Example 4 In the procedure of Example 1, Saccharopolyspora erythrea IFO 13426 was used as the strain to be used, and the substrate 6-
When O -methylerythromycin B was added and β-cyclodextrin (manufactured by Mercian) was added at a final concentration of 0.2%, the result was 0.4 mg of clarithromycin. Example 5 As a result of carrying out the reaction in Example 2 with a reaction time of 48 hours, 191 mg of clarithromycin was obtained.

Example 6 Micromonospore megalomicea subsp. Nigra IFO1
4114 strain and Saccharopolyspora erythrea IF
The O13426 strain was used as a GM medium and a J3 medium (glucose 2%, Bactosoyton (trade name: manufactured by Defico), respectively.
1.5%, corn steep liquor 0.5%, salt 0.4
%, Dibasic hydrogen phosphate 0.04%, manganese chloride 4
Hydrate 0.04%, yeast extract 0.2%, calcium carbonate 0.2%, pH 7.0) 28 ° C. using 80 ml of each
After culturing with aeration and stirring for 3 days, buffer solution C (Tris-hydrochloric acid 8
It was washed by centrifugation with 0 mM, pH 7.5, dithiothreitol 2 mM, glycerol 15%). Obtained Micromonospore megalomicea subsp. Nigra IFO14114 strain and Saccharopolyspora erythrea IFO1342
The washed bacterial cells of the 6 strains were suspended in buffer C, sonicated and disrupted, and the bacterial cells were removed by centrifugation to obtain 40 ml of a crude enzyme solution as a supernatant fraction. In the enzyme reaction, NADPH 0.26 mM, ferredoxin NADP reductase 0.4 unit, ferredoxin 1.6 m was added to a part of each crude enzyme solution obtained.
g, glucose 6-phosphate 14 mM, glucose 6-phosphate dehydrogenase 1 unit, nicotinamide 10 m
It was carried out by aeration stirring at 30 ° C. for 12 hours in 2 ml of a reaction solution containing 0.4 mg of M, 6- O -methylerythromycin B. The amount of clarithromycin produced by extracting each reaction solution with ethyl acetate was measured by liquid chromatography (TSK-gel ODS-120A: manufactured by Tosoh Corporation, developing solvent: acetonitrile: methanol: 50).
mM sodium phosphate pH 6.8 = 6: 2: 3, flow rate 1 ml / min, column temperature 50 ° C., detection with photodiode array detector (manufactured by Nippon Millipore), quantitative analysis). The results are shown in Table 2 below.

[0021]

[Table 2]

Example 7 Preparation of 6,4 "-di- O -methylerythromycin A:
Reference [The Journal of Antibiotics (Th
e Journal of Antibiotics), Vol. 43, No. 5, No. 5
P. 44, 1990] 6,4 "-Ji-
According to the same method as in Example 1 using 4.0 mg of O -methylerythromycin B as a raw material, 6,4 ″ -di- O—
Methylerythromycin A (melting point: 208-210 ° C,
¹³ C-NMR: FIG. 1) (1.0 mg) was obtained.

Example 8 6,11,4 "-tri- O -methylerythromycin A
Production: 6,11,4 "described in the literature cited in Example 7
-Tri- O -methylerythromycin B 4.0 mg was used as a starting material according to the same method as in Example 1,6,11,
4 "-tri- O -methylerythromycin A [Reference: The Journal of Antibiotics]
l of Antibiotics), Vol. 43, No. 3, p. 286,
1990].

Example 9 Preparation of decladinosyl-6- O -methylerythromycin A: 6- O -methylerythromycin B is acid-treated [Reference: Experientia Vol. 27, p. 362, 1971]. The decladinosyl-6- O -methylerythromycin B (melting point: 162-164 ° C, ¹³ C-NMR: Fig. 2) thus obtained was synthesized, and 4.0 mg thereof was used as a starting material according to the same method as in Example 1. Decladinosyl-6- O -methylerythromycin A [Reference: Chemotherapy, Volume 36 (S-3), 264, 1988] 0.6 mg
I got

[0025] Example 10 (14 R) -14- hydroxy-6-O - producing (14 R) methyl erythromycin A-14-hydroxy-6-O - methylerythromycin B [Reference: The Journal of Anti Biotics (The Journal of Antibiotics) Vol. 41,
No. 7, pp. 908-915, 1988] 4.0 mg.
In the same manner as in Example 1 using
R ) -14-Hydroxy-6- O -methylerythromycin A (literature: same as above) (1.0 mg) was obtained.

[Brief description of drawings]

FIG. 1: 6, measured in deuterated chloroform at 75 MHz
¹³ C-NM of 4 "-di- O -methylerythromycin A
The R spectrum is shown.

FIG. 2: ¹³ C of decladinosyl-6- O -methylerythromycin B measured at 75 MHz in deuterated chloroform.
-Shows an NMR spectrum.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shigeo Morimoto 3-24-1 Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd.

Claims (3)

[Claims] 1. A 6- O -alkyl having a hydrogen atom at the 12-position in a solution containing a 6- O- alkylerythromycin and an actinomycete that hydroxylates actinomycetes or an enzyme produced by the fungus. A method for producing a 12-hydroxy-6- O -alkylerythromycin and its analogs, which comprises adding erythromycin and its analogs to convert the hydrogen atom into a hydroxyl group. 2. The production method according to claim 1, wherein the 6- O- alkylerythromycin having a hydrogen atom at the 12-position and its analogs are compounds represented by the following formula. Embedded image [In the formula, R ¹ represents an alkyl group having 1 to 4 carbon atoms, R ² represents a hydrogen atom or a methyl group, R ³ represents a hydrogen atom or a hydroxyl group, R ⁴ represents a hydrogen atom or a formula 2] (In the formula, R ⁵ represents a hydrogen atom or a methyl group). ] 3. An actinomycete which hydroxylates 6- O- alkylerythromycin and its analogs is micromonospora ( Mi
cromonospora) genus Streptomyces (Streptomyces)
The method according to claim 1 or 2, which is a bacterium belonging to the genus Saccharopolyspora .