KR20120016714A - New tilmicosin analogs and antibiotics including these - Google Patents

Abstract

PURPOSE: A novel tilmicosin derivative is provided to ensure antibacterial activity against Bacillus subtilis and to be used as antibiotics. CONSTITUTION: A tilmicosin derivative is denoted by chemical formula 1. The tilmicosin derivative is prepared by bioconversion by Streptomyces venezuelae from tilicosin. A method for preparing the tilmicosin derivative comprises: a step of culturing Streptomyces venezuelae in a medium containing tilmicosin; a step of extracting the culture liquid with organic solvent; a step of concentrating the organic extract under vacuum; a step of dissolving residual material with organic solvent; and a step of performing chromatography.

Description

New tilmicosin derivatives and antibiotics including the same

The present invention relates to a novel compound and its use, and more particularly, to a novel thylmicosin derivative or a pharmaceutically acceptable salt thereof, a preparation method thereof and an antibiotic composition using the same as an active ingredient.

Natural products still inspire new drug development. In order to design next-generation novel therapeutics through targeted modification of specific structures (scaffolds) in the material using bioactive natural materials as starting materials, biosynthesis using microorganisms as biocatalysts as well as semisynthetic approaches using organic synthesis Applicable to access. In particular, the development of multiple drug resistance among emerging pathogens suggests that the scaffold modification strategy for improved antibiotic preparation is a priority.

Macrolide antibiotics are a group of polyketides with activity derived from the presence of large macrocyclic lactone rings. These are structurally diverse kinds of natural products that exhibit various bioactive properties, including anticancer, antifungal, immunosuppressive and anti-aging activity.

Tilmicosin is semisynthesized by organic synthesis using tylosin, which is present as 16 kinds of macrolides in fermentation of Streptomyces fradiae, and is used as a veterinary antibiotic. .

Recently, we have identified, by Streptomyces venezuelae, a unique, site-specific cure for 12 unsaturated metymycin and 14 pykromycin macrolides. Their application to the activity and generation of 16 non-natural macrolides has been reported. Indeed, such bacterial reduction systems can recognize specific structural embellishments around target double bonds, the carbonyl functionality of one adjacent carbon and the methyl group of another adjacent carbon in the polyketide backbone. See the shaded rectangle of 1).

Therefore, the present inventors have attempted to expand the biohydrogenation activity of semisynthetic tilmicosin, an unsaturated macrolide antibiotic, and found that Streptomyces Venezuelan ( S. venezuelae ) 10,11-dihydro-tilmicosin (10,11-dihydro-tilmicosin) that was not previously specified was isolated, and the present invention was completed by confirming their structure and antimicrobial activity.

It is an object of the present invention to provide novel tilmicosin derivatives.

Another object of the present invention is to provide a method for preparing the tilmicosin derivative.

Still another object of the present invention is to provide an antibiotic composition containing the tilmicosin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a novel tilmicosin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Formula 1]

Also. The present invention provides a method for preparing the tilmicosin derivative.

Furthermore, the present invention provides an antibiotic composition containing the tilmicosin derivative or salt thereof as an active ingredient.

The present invention has introduced a novel micromycosin derivative in which a double bond in a structure is substituted with thymicosine, which is used as a conventional macrolide antimicrobial agent and agricultural antibiotic, by introducing a bioconversion technique of a microorganism. Since thylmicosin derivatives exhibit excellent antimicrobial activity, the thylmicosin derivatives can be usefully used as an active ingredient of antibiotics.

1 is a figure showing the ion chromatogram of the extract obtained from the organism Streptomyces Venetian state elrae (S. venezuelae) is cultured in a culture medium was added til M. gods. Here, peaks labeled with tilmicosin and 10,11-dihydrogen-tilmycosine (2) were selected from molecules [M + H] + ions 869 and 871, respectively.
FIG. 2 shows ESI-MS / MS spectra of tilmicosin and 10,11-dihydrogen-tilmicosine (2). FIG.
FIG. 3 shows a ¹ H-NMR spectrum of 10,11-dihydrogen-tylmicosin (2) in CDCl ₃ . Inset shows that the chemical shift of 10,11-dihydrogen-tylmicosin (2) is significantly changed compared to tilmicosin.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention provides a novel thylmicosin derivative represented by the following formula (1).

The tilmicosin derivative is preferably bioconverted from Styltomycin by Streptomyces venezuelae, but is not limited thereto, and includes all synthetically.

The tilmicosin derivative has excellent antimicrobial activity against Bacillus subtilis .

The thilmicosin derivative of the present invention represented by the formula (1) may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Inorganic and organic acids can be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. can be used as the inorganic acid, citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfuric acid. Phonic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid, and the like can be used. Preferably, hydrochloric acid may be used as the inorganic acid, and methanesulfonic acid may be used as the organic acid.

In addition, the thylmicosin derivative represented by Formula 1 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Formula 1 in a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile and adding an excess of an organic acid or an inorganic acid. It can be prepared by adding an acidic aqueous solution of and then precipitating or crystallizing. The solvent or excess acid may then be evaporated and dried in this mixture to obtain an addition salt or the precipitated salt may be prepared by suction filtration.

In addition, the present invention provides a method for preparing a new thylmicosin derivative represented by the following formula (1).

Specifically,

1) culturing S. venezuelae in medium containing tilmicosin;

2) extracting the culture solution cultured in step 1) with an organic solvent;

3) concentrating the organic extract of step 2) under vacuum; And

4) can be prepared by a method comprising the step of dissolving the residue of step 3) with an organic solvent and then separated by chromatography.

In the above method, the organic solvent of step 2) is preferably ethyl acetate (EtOAc), and the organic solvent of step 4) is preferably methanol (MeOH), but is not limited thereto.

In the above method, the chromatography of step 4) preferably uses high performance liquid chromatography (HPLC), but is not limited thereto. In one embodiment of the present invention, the compound was isolated at a retention time of 52 to 54 minutes using preparative HPLC.

The manufacturing method according to the present invention is characterized in that it is manufactured using bio-bioconversion technology of Streptomyces venezuelae soil microorganisms rather than the conventional prior art chemical synthesis method.

In addition, the present invention provides an antibiotic composition containing a novel thylmicosin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Tilmicosin derivatives or pharmaceutically acceptable salts thereof contained in the composition according to the present invention as an active ingredient are in vitro ( in In vitro ) antimicrobial activity test, showed excellent antimicrobial activity against Bacillus subtilis ( Bacillus subtilis ). Therefore, the mylmicosin derivatives according to the present invention can be usefully used as novel antibiotics.

When the composition of the present invention is used as a medicine, the pharmaceutical composition containing the thymicosine derivative represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient is various oral or parenteral following clinical administration. It may be formulated in a dosage form and administered, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like. Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition comprising the tilmicosin derivative represented by Formula 1 as an active ingredient may be administered parenterally, and the parenteral administration may be by injection of subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

In this case, in order to formulate into a formulation for parenteral administration, the tilmicosin derivative of Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, which is administered in ampoules or vials. It can be manufactured in a mold. The compositions may contain sterile and / or preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating It may be formulated according to the formulation or coating method.

In addition, the dosage of the compound of the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, and generally based on an adult patient having a weight of 70 kg. It is 0.01-1,000 mg / day, Preferably it is 0.1-500 mg / day, It can also divide and administer once a day to several times at regular time intervals according to a decision of a doctor or a pharmacist.

Hereinafter, examples for explaining the present invention in more detail.

However, the following examples are merely provided to explain the present invention more easily, and the content of the present invention is not limited by the examples.

< Example  1> Tilmicosin  Preparation of Derivatives

<1-1> Macrolide  Biotransformation

Recombinant mutant species of S. venezuelae YJ028 [Park, JW et al ., Chem . Commun . 2008, all genes encoding pikromycin polyketide synthase and desamine amino biosynthesis genes are removed. and 44, using the species described in the 5782-5784) a first, SPA (sorbitol pyroglutamic acid) agar plates [0.1% yeast extract, 0.1% beef extract, 0.2% lactose trip (tryptose), 1.0% glucose (glucose), 1.5% agar, and traces of FeSO ₄ ] were streaked for isolation and then incubated at 30 ° C. for 2 days. Then in a partitioned Erlenmeyer flask, 50 ml of SCM medium [1.5% soluble starch, 2.0% soytone, 0.01% CaCl ₂ , 0.15% yeast extract for 2 days at 30 ° C. (yeast extract, and 1.0% MOPS) was added (250 μg; final concentration 5 μg / mL; dissolved in 100 uL MeOH) to recombinant strain YJ028 incubated in It was further cultured. All experiments were performed at least three times independently.

<1-2> Tilmicosin  Extraction and Separation of Derivatives

The whole culture of Example <1-1> was extracted and separated twice with an average volume of EtOAc in a 250 mL separatory funnel, and then the organic extract was concentrated in vacuo. The dried residue was immediately dissolved in 200 uL MeOH and part of the solvent was used for analysis. First of all, Micromass Quattro micro Macrolide analysis was performed using a Waters / Micromass Quattro micro / MS connector consisting of an analytical reversed-phase Nova-Pak C ₁₈ column (Waters, Milford, MA; 150 × 3.9 mm, 4.0 μm) directly connected to MS.

As a result, reduced macrolide 1 was produced by adding the macrolide tilmycosine in various batch cultures of YJ028 species. It was confirmed that reduced macrolide 1 corresponding to tilmicosin occurs at a conversion rate of about 30%.

This new macrolide 1 was then purified by preparative reversed phase HPLC, and chromatographic separation using successive ESI-MS analysis of each fraction collected (5 mL / fraction), the chemical structure of which was ¹ H- and ^{It was} analyzed using ¹³ C-NMR spectroscopy. Specifically, chromatographic separation was performed using preparative HPLC consisting of Watchers 120 ODS-BP column (250 × 10.0 mm, 5.0 μm). In addition, ¹ H- and ¹³ C-NMR spectroscopy was performed as follows. NMR samples were prepared by dissolving each macrolide 1 in 200 uL of CDCl ₃ and then leaving the solvent in a 5 mm Shigemi advanced NMR microtube (Sigma, St. Louis, Mo.) appropriate for the solvent. ¹ H and ¹³ C NMR spectra were obtained at 298 K using a Varian INOVA 500 spectrophotometer, and chemical shifts were recorded in ppm using TMS as internal reference. All NMR data calculations were performed using Mnova Suite 5.3.2 software.

As a result, the chemical shift of the transformed macrolide 1 was determined to be 10,11-dihydro-tilmicosin by comparing with the structure of the parent macrolide tilmicosin (see Table 1). ).

The most obvious change observed through comparison of ¹ H-NMR spectroscopy data of tilmycosine and its reduced product 1 was observed at the C-10,11 position. Indeed, the lack of both the signal downfield of 6.00 ppm in the ¹ H-NMR spectral data of product 2 and the upfield shift of the signal specifying the C-10 and C-11 positions of tilmicosin are Divalent semisynthetic tilmicosin is shown to represent (Table 1 and Figure 3). This result indicates that the reduced macrolide 1 was shown to be converted by S. venezuelae which includes regioselective activity for biohydrogenation of various ring macrolides.

< Example  2> Tilmicosin  Confirmation of Antibiotic Activity of Derivatives

Since micromycosin is known for its antimicrobial (particularly gram-positive bacteria) antibiotic activity, the microdilution method recommended by the Clinical and Laboratory Standard Institute (CLSI) to investigate the antimicrobial activity of Compound 1 obtained in the present invention Using the microdilution method, the antimicrobial activity against Bacillus subtilis was measured.

Specifically, Bacillus subtilus ( Bacillus subtilis ) ATCC 23857 was grown in Mueller-Hilton broth (Difco). Tilmicosin and the corresponding macrolide 1 were then treated with Saccharomyces cerevisiae. Serial 2-fold dilutions of macrolide 1 were performed using DMSO to a final concentration of 0.78-100 μg / mL, with some of the DMSO being used as a negative control. Growth of test species was monitored at 600 nm using a Labsystems Bioscreen C reader, and the lowest concentration of macrolide diluted in broth medium that inhibited the growth of test microorganisms was determined using MIC. All analyzes were performed three times.

As a result, Compound 1 of the present invention showed excellent antibacterial activity (see Table 2).

MIC data comparison of macrolide 1 corresponding to tilmicosin Tilmicosin Macroride 1 MIC (/ mL) 1.56 1.20

As a result, Compound 1 of the present invention showed excellent antibacterial activity (see Table 2). In particular, Compound 1 showed improved activity against Bacillus subtilis (MIC-1.20 μg / mL), compared to Tilmycosine (minimum inhibitory concentration; MIC-1.56 μg / mL).

In conclusion, the present invention can be usefully used for the development of antibiotics that are significantly better than the maternally synthesized macrolides such as tilmicosin using bio-bioconversion technology by microorganisms rather than chemical synthesis. In other words, such bio-conversion technology can be useful for the development of new antibiotics, since it is possible to convert various macrolide scaffolds including 26 kinds of ring macrolides in the future.

On the other hand, the thylmicosin derivative represented by the formula (1) according to the present invention can be formulated in various forms according to the purpose. The following are some examples of formulation methods containing the compound represented by Formula 1 according to the present invention as an active ingredient, but the present invention is not limited thereto.

< Formulation example  1> tablet (direct pressure)

After sifting 5.0 mg of the active ingredient, 14.1 mg of lactose, 0.8 mg of crospovidone USNF, and 0.1 mg of magnesium stearate were mixed and pressed to prepare a tablet.

< Formulation example  2> tablets (wet assembly)

After 5.0 mg of the active ingredient was sieved, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of Polysorbate 80 was dissolved in pure water, and an appropriate amount of this solution was added, followed by atomization. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The granules were pressed to make tablets.

< Formulation example  3> with powder Capsule

After sifting 5.0 mg of the active ingredient, it was mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. The mixture was prepared using a suitable apparatus. Filled in 5 gelatin capsules.

< Formulation example  4> Injection

Injectables were prepared by containing 100 mg as active ingredient, as well as 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ 12H ₂ O and 2974 mg of distilled water.

The present invention can be usefully used for the development of a variety of antibiotics by showing the expandability of the microbial hydrogenation activity, it can be usefully used in the development of bioconversion technology as one of the scaffold modification strategy.

Claims (10)

A tilmicosin derivative represented by the following formula (1):
[Formula 1]

The tilmicosin derivative according to claim 1, which is bioconverted from tilmicosin and prepared by Streptomyces venezuelae.
The tilmicosin derivative according to claim 1, which has an antimicrobial activity against Bacillus subtilis .
1) culturing S. venezuelae in medium containing tilmicosin;
2) extracting the culture solution cultured in step 1) with an organic solvent;
3) concentrating the organic extract of step 2) under vacuum; And
4) A method for preparing the tilmicosin derivative according to claim 1, comprising the step of dissolving the residue of step 3) with an organic solvent and then separating by chromatography.
The method of claim 4, wherein the organic solvent of step 2) is ethyl acetate (EtOAc).
The method of claim 4, wherein the organic solvent of step 4) is methanol (MeOH).
The method of claim 4, wherein the chromatography of step 4) is high-performance liquid chromatography (HPLC).
The method of claim 7, wherein the retention time in the HPLC is separated from 52 to 54 minutes.
An antibiotic composition containing the tilmicosin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
The antibiotic composition according to claim 9, wherein the composition exhibits antimicrobial activity against Bacillus subtilis .

Images