JPH0311096A - Novel macrolide antibiotic substance mycinamicins and production thereof - Google Patents

Abstract

NEW MATERIAL:Mycinamicins XVI, XVII and XVIII of formula I to III. USE:It has antibacterial activity and is useful as a remedy for infectious diseases. PREPARATION:A mycinamicin-producing strain belonging to genus Micromonospora [e.g. Micromonospora griseorubida A 11725 (FERM BP-705] is cultured in a medium for 6-8 days preferably at 26-30 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to novel macrolide antibiotic mycinamicins with enhanced antibacterial activity and a method for producing the same.

[Conventional technology and its problems]

Conventionally, bacteria belonging to the genus Micromonospora have been found to have excellent antibacterial activity using the following general formula [where A is an oxygen atom or a single bond, R is a hydrogen atom or a hydroxyl group, and Q is a hydrogen atom or a It is known that the macrolide antibiotics mycinamicins 1 to 1 are produced (
J.

Antibiotics, Okina (4), 364-376
(1980): 3A(3).

276-281 (1981) and Dojin (2),
175-178 (1983)).

-In clothing cost (1), A=oxygen atom, R=H, Q-X
, Y "C1h , Z = C113, the compound is mycinamicin I; A = oxygen atom, R = O1+, , Q =
The compound where X,,Y=CIl:l,Z=CH3 is mycinamicin■;A=single bond, R=HSQ=X, Y=
The compound where CI+3 and Z=H is mycinamicin I [
I; A=single bond, R=H, Q=X, Y=CH3, Z=
A certain compound is mycinamicin IV; A=single bond, R-Ko, Q"
The compound where H5 is mycinamicin V, A=single bond, R
A compound in which =H, Q=X, Y=H, Z=H is mycinamicin VISA=single bond, and a compound in which R=H, Q=H is mycinamicin (■).

Among these Myshina sewing machines I~■, Myshina sewing machines I
, ■, ■ and V exhibit extremely excellent antibacterial activity, especially against staphylococci and mycoplasma, and are useful as therapeutic agents for human and animal infections.

In addition to the above-mentioned Myshinamicin 1 to ■, this bacterium can also be used as Myshinamicin X, X', XI, and
It has been reported that it also produces
Publication No. 496). - In clothing (2), the compounds where R=11 are mycinamicins X and X', and the compounds where R=OI+ are mycinamicins XI and XI'.

Furthermore, this bacterium is represented by Mycinamicin ■ represented by the following formula, Mycinamicin ■ represented by 2, Mycinamicin XIII represented by the formula, Mycinamicin X■ represented by the formula, Mycinamicin XIV represented by the formula It has also been reported that mycinamicin XV is also produced (Japanese Patent Application No. 63-167072).

(Means for Solving the Problems) Under such circumstances, the present inventors further investigated the culture of bacteria belonging to the genus Micromonospora, and found that the above-mentioned mycinamicin I-XV was present in this culture. We have discovered that different new antibiotics exist and that this antibiotic has excellent antibacterial activity and can therefore be used as a medicine in itself, as well as being useful as an intermediate for the production of mycinamicins Ⅰ and H, and we have developed the present invention. completed.

The present invention provides mycinamicins or salts thereof selected from the group consisting of mycinamicin X (2), (2), mycinamicin X (2) represented by C0, and mycinamicin X (2) represented by the formula, and a method for producing the same.

The mycinamicins of the present invention are produced, for example, by culturing mycinamicin-producing bacteria belonging to the genus Micromonospora and collecting the mycinamicins from the culture.

Examples of mycinamicin-producing bacteria include Micromonospora griseorvida A11725Al1725 (+
onospora grtseorubida A11
725+Feikoken Jokyo No. 705]. This strain has the ability to produce the known mycinamicins Ⅰ to XV, and its mycological properties are classified as
ttbiotics, 33(4), 364-376 (
1980), and is described in Japanese Patent Publication No. 18557/1980.

To carry out the method of the present invention, first, the above-mentioned microorganisms are incubated with mycinamicin I-X in a medium containing an assimilable carbon source, assimilable nitrogen source, and assimilable inorganic salt source.
Cultivate under conditions that produce V.

Medium components include carbon sources such as glucose, starch, glycerin, sucrose, molasses, and dextrin; nitrogen sources such as peptone, meat extract, soybean flour, thawed casein, cottonseed flour, corn stew liquor, nitrates, and ammonia salts; Inorganic salts such as substances containing cations such as sodium, potassium, magnesium, calcium, cobalt, manganese, iron, etc., substances containing anions such as chlorine, sulfuric acid, phosphoric acid, acetic acid, etc. 1. Dried yeast, yeast extract, etc. Examples include bacterial growth factors.

In such a medium, 20-30゛C1, preferably 26-30
Micromonospora griseorvida A1 at a temperature of °C
When culturing 1725, mycinamicin ■～■
In addition to the above, Mycinamicins ① to XV and Mycinamicins X② to X■ of the present invention are produced as minor components. These production levels usually reach their maximum in 6 to 8 days.

To collect the target product from the resultant mixture (culture) obtained in this way, for example, the culture is filtered, the filtrate is extracted with an organic solvent such as ethyl acetate, and the organic layer is concentrated. The desired product may be separated and purified by subjecting it to column chromatography and monitoring each fraction using thin layer chromatography, ultraviolet spectrophotometer, or the like.

Table 1 shows the physicochemical properties of mycinamicins XVI, X■ and X■ of the present invention. In Table 1, the developing solvent for thin layer chromatography (TLC) is chloroform: methanol: concentrated ammonia = 15:1:0.1, and the measurement conditions for high performance liquid chromatography (HPLC) are as follows. be.

Column: Inner diameter 41 TII 11 x 1501! lI! l Filler: YMC-GEL ODS S-5-5A Quib Solvent System: 0. IM phosphate buffer: methanol: acetonitrile (55:31:14) Flow rate: 0.8 mj2/min (margin below) Each of the mycinamicins of the present invention can be made into a pharmaceutically acceptable addition salt of a known acid. .

Examples of such acids include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as acetic acid, propionic acid, tartaric acid, citric acid, malic acid, succinic acid, aspartic acid, and glutamic acid.

The anticancer spectra (minimum inhibitory concentration MIC (mcg/mf)) of the mycinamicins of the present invention determined by the agar dilution method are shown in Table 2.

Margins below [Effects of the Invention] The novel mycinamicins XVI to X■ of the present invention have valuable antibacterial activity in themselves, and are therefore useful as therapeutic agents for human and animal infectious diseases. It is also useful for the body.

〔Example〕

Next, the present invention will be explained with reference to Examples and Reference Examples.

Example I A) 1% dextrin, 1% glucose, 0.5% casein thaw, 0.5% yeast extract and 0.5% calcium carbonate.
A liquid medium (pH 7.0) containing 1% 20a+2 was dispensed into a 150 ml Erlenmeyer flask, and incubated at 120°C for 2 hours.
Steam sterilized for 0 minutes. One platinum loop from a slanted medium of Micromonosporagrise Orvida A11725 was inoculated into this medium, and cultured on a rotary shaker at 30°C for 48 hours to obtain a primary seed culture.

B) Liquid medium 11 similar to A)! , and steam sterilized at 120'C for 20 minutes. Add 10+nff1 of the primary seed culture obtained in A) to this medium.
The cells were inoculated and cultured on a reciprocal shaker at 30°C for 48 hours to obtain a secondary seed culture.

C) On the other hand, dextrin 7%, grape tJ! 0.5%, cottonseed flour 2.5%, defatted soy flour 0.5%, calcium carbonate 0
．． 5%, K! 1lPO40, 1%, MgSO4・7H,
00.4%, CoCi z ・6fb0 0.0002
%, liquid medium 2O consisting of silicone antifoaming agent 0.02%
Heat 3ON jar fermenter containing N to 120'C.
Steam sterilized for 30 minutes. The 12 secondary seed cultures obtained in B) were transplanted into this jar fermenter, and the mixture was kept at 30°C for 3 hours.
00r, p, m,.

Aerated agitation culture was carried out for 7 days under the condition of supplying sterile air at 2 ON/min, and 172 culture solutions were obtained.

D) The above culture solution 111 was filtered to remove bacterial cells and other solid matter to obtain a filtrate 141. This culture filtrate was extracted with the same amount of ethyl acetate, and mycinamicin ■～■ and
An organic layer containing (1) to (X) was obtained. This organic layer was removed under reduced pressure. and then diluted with hydrochloric acid 500II11 at pH 2.5.
and the mycinamicins were transferred and dissolved into the aqueous layer. Furthermore, the pH of the aqueous layer was adjusted to 8.5 with concentrated ammonia for 8 times, and
00n/! After extraction with chloroform, the chloroform layer was concentrated to dryness under reduced pressure to obtain 9.6 g of a residue.

E) Dissolve 9.6 g of this residue in 20 mf of chloroform and prepare a silica gel column (inner diameter 25 mf) filled with chloroform.
mm x 600 mm). Then, elution was performed with a solvent of chloroform:methanol:concentrated ammonia (50:1:0.1), and 20. It was fractionated into ml portions. For each fraction, the desired product was identified using thin layer chromatography using a thin layer chromatograph using chloroform:methanol:concentrated ammonia (150:10:1) as a developing solvent, and the containing fractions were collected.

Subsequently, chloroform:methanol:concentrated ammonia (
30: 1;0.1) After eluting known mycinamicins 1 to 1 using If, chloroform:methanol:
Elution was carried out using concentrated ammonia (10:1:0.1), and the fractions containing mycinamicins (1), X2 to X2 were concentrated to dryness under reduced pressure to obtain a residue 102 (102).

F) 102 cm of the above residue was dissolved in 2 ml of methanol and poured into a stainless steel column with an inner diameter of 20 mm x 300 mm filled with YMC-GEL ODS S-5 resin (manufactured by Yamamura Kagaku Kenkyujo ■). The eluent is 10mj per minute!
using 0.1 M phosphate buffer: methanol (6:4) and monitored by UV spectrophotometer at 220 nm.

After concentrating the three fractions A, B and C containing the target product, and adjusting the concentrated solution to ρ119 with 2N NaOH,
Extracted with ethyl acetate. The organic layer was dehydrated over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Mycinamicin XVI 8 mg from fraction A, Mycinamicin X■9 m from fraction B
Mycinamicin X11 was obtained from the g and C fractions.

[Brief explanation of drawings]

Figures 1 to 3 show Myshina sewing machines XVI to X, respectively.
Fig. 4 is a drawing showing the infrared absorption spectra of mycinamicins X① to X②, and Figs. Transmittance (%) Transmittance (A≦) Transmittance (%)

Claims (2)

[Claims] (1) Myshinamishin XVI represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc.▼ Myshinamishin XVII is represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Myshinamishin XVII is represented by the formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Mycinamicin or a salt thereof selected from the group consisting of Mycinamicin XVIII. (2) A method for producing mycinamicins or a salt thereof, which comprises culturing mycinamicin-producing bacteria belonging to the genus Micromonosvora and collecting the mycinamicins according to claim 1 from the culture.