KR101231289B1

KR101231289B1 - New oligomycin analogs and antibiotics including these

Info

Publication number: KR101231289B1
Application number: KR1020100079101A
Authority: KR
Inventors: 윤여준; 박제원; 박성렬
Original assignee: 이화여자대학교 산학협력단
Priority date: 2010-08-17
Filing date: 2010-08-17
Publication date: 2013-02-07
Also published as: KR20120016713A

Abstract

The present invention relates to a novel oligomycin derivative, and an antibiotic comprising the same. More specifically, the oligomycin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof, a method for preparing the same, and an active ingredient thereof It relates to an antibiotic composition. The novel oligomycin derivatives prepared from the Streptomyces venezuelae of the present invention using bio bioconversion technology exhibit excellent antimicrobial activity against Saccharomyces cerevisiae . It can be usefully used as an antibiotic.
[Formula 1]

Description

New oligomycin analogs and antibiotics including these}

The present invention relates to a novel compound and its use, and more particularly to a novel oligomycin 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.

Oligomycin A was first isolated from the soil bacterium Streptomyces diastatochromogenes in 1954, and oligomycin A is one of 26 macrocyclic lactones, the major constituent of oligomycin. Ingredient. This antibiotic is known to have potential antitumor activity.

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 studied to attempt to expand the biohydrogenation activity of the natural oligomycin A, an unsaturated macrolide antibiotic, Streptomyces Venezuela ( S) cultured in the medium containing oligomycin A . from venezuelae) culture medium, the 2,3-non-specific previously hydrogen-by were isolated oligonucleotide azithromycin a (2,3-dihydro-oligomycin a ), confirmed their structure and antimicrobial activity, and completed the present invention .

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

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

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

In order to achieve the above object, the present invention provides a novel oligomycin 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 oligomycin derivative.

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

The present invention has introduced a novel oligomycin A derivative in which a double bond in a structure is substituted with oligomycin A, which is used as a conventional macrolide antifungal agent, by introducing a microbial bioconversion technique. Since the A derivative exhibits superior antimicrobial activity compared to the existing natural oligomycin A, the novel oligomycin derivative can be usefully used as an active ingredient of an antibiotic.

Figure 1 is a drawing showing an oligonucleotide selected ion chromatogram of the extract obtained from the organism Streptomyces Venetian state elrae (S. venezuelae) is cultured in a culture solution is added to mitomycin A medium. Here, peaks labeled with oligomycin A and 2,3-dihydrogen-oligomycin A (1) were selected from molecules [M + H] + ions 791 and 793, respectively.
FIG. 2 shows ESI-MS / MS spectra of oligomycin A and 2,3-dihydrogen-oligomycin A (1).
FIG. 3 shows a ¹ H-NMR spectrum of 2,3-dihydrogen-oligomycin A (1) in CDCl ₃ . Inset shows that the chemical shift of 2,3-dihydrogen-oligomycin A (1) has a notable change compared to oligomycin A.

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

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

The oligomycin derivatives are preferably bioconverted from Streptomyces venezuelae from oligomycin A, but are not limited thereto, and include all synthetically.

The oligomycin derivative has excellent antimicrobial activity against Saccharomyces cerevisiae.

The oligomycin derivative of the present invention represented by Formula 1 may be used in the form of a pharmaceutically acceptable salt, and acid addition salts formed by pharmaceutically acceptable free acid are useful as salts. 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 is used as the inorganic acid, and methanesulfonic acid is used as the organic acid.

In addition, the oligomycin 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 Chemical Formula 1 in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile, etc., And then precipitating or crystallizing the acid solution. 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.

The present invention also provides a method for producing a novel oligomycin derivative represented by the following formula (1).

Specifically,

1) culturing Streptomyces venezuelae in a medium to which oligomycin A is added;

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 separated at a retention time of 50 to 53 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 oligomycin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Oligomycin 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 a superior antimicrobial activity against Saccharomyces cerevisiae compared to oligomycin A known antimicrobial activity. Therefore, oligomycin 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 oligomycin derivative represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient may be used for various oral or parenteral administrations during clinical administration. It may be formulated in a 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 oligomycin derivative represented by Formula 1 as an active ingredient can be administered parenterally, and the parenteral administration is by injection of subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

At this time, in order to formulate into a parenteral formulation, the oligomycin derivative of Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or a buffer to prepare a solution or suspension, which is an ampoule or vial unit dosage form. It can be prepared by. 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> Oligomycin Preparation of A 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 oligomycin A (Sigma) was added to recombinant strain YJ028 incubated at 1.0% MOPS (250 μg; final concentration 5 μg / mL; dissolved in 100 uL MeOH) for 3 days. It was further cultured. All experiments were performed at least three times independently.

<1-2> Oligomycin Extraction and Separation of A 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 oligomycin A to several batch cultures of YJ028 species. It was confirmed that reduced macrolide 1 corresponding to oligomycin A occurred at a conversion rate of about 67%.

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 is compared to the structure of the parent macrolide oligomycin A, whereby macrolide 1 is 2,3-dihydro-oligomycin A (2,3-dihydro-oligomycin A). Was determined (see Table 1).

The most obvious changes observed through comparison of ¹ H-NMR spectroscopic data of oligomycin A and its reduced product 1 were representative of 5.83 and 6.88 ppm of olefinic protons (H-2,3) in oligomycin A. It was a lack of signal. Upfield shifts of the C-2 and C-3 signals (30.6 and 25.3 ppm at 121.5 and 147.6 ppm, respectively) of the parent compound support the differences identified through the ¹ H-NMR data and thus the product 1 demonstrates that the oligomycin A reduced form by removing the C-2,3 double bond (Table 1 and FIG. 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> Oligomycin Confirmation of antibiotic activity of A derivative

Since oligomycin A is known for its antifungal 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. The antifungal activity against Saccharomyces cerevisiae was measured using.

Specifically, Saccharomyces ( Saccharomyces) cerevisiae ) ATCC 9763 was grown in Antibiotic 19 (Difco, BD Biosciences, San Jose, CA). Oligomycin A 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.

Comparison of MIC Data of Macrolide 1 Corresponding to Oligomycin A Oligomycin A Macroride 1 MIC (/ mL) 25.0 12.5

As a result, Compound 1 of the present invention showed excellent antibacterial activity (see Table 2). In particular, compared to oligomycin A (minimum inhibitory concentration; MIC-25.0 μg / mL), Compound 1 showed increased activity against Saccharomyces cereviase (MIC-12.5 μg / mL).

In conclusion, the present invention can be usefully used for the development of antibiotics that are significantly better than the parent natural and semisynthetic macrolides, such as oligomycin A, using biobioconversion techniques 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 oligomycin 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 fine particles were pressed to prepare tablets.

< Formulation example 3> with powder Capsule

5.0 mg of the active ingredient was sieved and then mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was extruded through a hard No. 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

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1) culturing Streptomyces venezuelae in a medium to which oligomycin A is added;
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) preparing a oligomycin derivative of the following [Formula 1] comprising the step of dissolving the residue of step 3) with an organic solvent and then separated by chromatography.
[ Formula 1 ]

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 50 to 53 minutes.

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