KR101516385B1 - Rosamicin analog and antibacterial agent including the same - Google Patents

Abstract

The present invention relates to an antibiotic composition comprising a rosemycin derivative having an antibiotic activity, a rosemycin derivative or a pharmaceutically acceptable salt thereof, and a process for producing the rosemycin derivative.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a rosemycin derivative and an antibiotic comprising the same. BACKGROUND ART [0002] ROSAMICIN ANALOG AND ANTIBACTERIAL AGENT INCLUDING THE SAME [

The present invention relates to a novel compound and its use, and more particularly to a novel rosemycin derivative or a pharmaceutically acceptable salt thereof, a process for producing the same, and an antibiotic composition containing the same as an active ingredient.

Rosamycin of formula (I) is a soil microbial strain of Micromonospora rosaria ), which is a macrolide antibiotic having 16 carbon atoms (see Non-Patent Document 1).

Rosamycin has been found to be an antibiotic for an antibacterial agent, and has been known to have a broad spectrum of antimicrobial activity. It is known that rosemycin has an effective activity for Gram-positive bacteria (see Non-Patent Document 2). Therefore, the development of novel structures and improved active derivatives by introducing semi-synthetic techniques or biological conversion techniques based on the rosamycin structure is a useful development method for the development of next-generation antibiotics or the development of new indication-related drugs. Here, the semi-synthetic technique is an approach based on chemical transformation of natural products, and the biological conversion technique is an approach based on biotransformation using microorganisms as biocatalysts. While the chemical conversion process has low energy efficiency and environmental incompatibility disadvantages exemplified by low selectivity, hydrogenation catalyzed by the rhodium metal of the alkene, the microbial conversion process has high selectivity, energy efficiency, and environmental friendliness.

There are several reports on the biosynthesis of rosemycin using microorganisms. For example, after chemically modifying the rosamycin structure to produce de-epoxyrosamycin aglycone, the de-epoxysomicin aglycone thus prepared is reacted with mycinamicin-producing micro-monospora polyrotota ( Micromonospora polytrota ) to develop a mycosine sugar chain functional group-attached rosamycin derivative (see Non-Patent Document 3). In addition, there has been reported a biosynthesis of myxin glycosylated rosamycin from a recombinant strain into which a microorganism of Saccharomyces cerevisiae is additionally introduced (see Non-Patent Document 4-6) ). Further, there is also a report that a hydroxyacyl group is replaced with an acetyl group or a 9-carbonyl group is reduced to a hydroxyl group by adding a rosemycin biosynthetic intermediate precursor to a macrolide producing microorganism as a substrate (see Non-Patent Document 7). However, in the case of the mentioned rosamiide derivatives, there is no report so far that the antimicrobial antibiotic activity is improved more than the original compound rosamycin.

1) Wagman, G. H .; Waitz, J. A .; Marquez, J .; Murawaski, A .; Oden, E. M .; Testa, R. T .; Weinstein, M. J. J. Antibiot, 1972, 25, 641646. 2) Baumueller, A .; Hoyme, U .; Madsen, P. O. Antimicrob. Agents Chemother. 1977, 12, 240242. 3) Lee, B. K .; Puar, M. S .; Patel, M .; Bartner, P .; Lotvin, J .; Munayyer, H .; Waitz, J. A. J. Antibiot. 1983, 36, 742744. 4) Anzai, Y .; Iizaka, Y .; Li, W .; Idemoto, N .; Tsukada, S .; Koike, K .; Kinoshita, K .; Kato, F. J. Ind. Microbiol. Biotechnol.2009,36,10131021. 5) Anzai, Y .; Sakai, A .; Li, W .; Iizaka, Y .; Koike, K .; Kinoshita, K .; Kato. F. J. Antibiot.2010, 63, 325328. 6) Iizaka, Y .; Higashi, N .; Ishida, M .; Oiwa, R .; Ichikawa, Y .; Takeda, M .; Anzai, Y .; Kato, F. Antimicrob. Agents Chemother.2013,57,15291531. 7) Sadakane, N .; Tanaka, Y .; Omura, S. J. Antibiot. 1982,35,680687. 8) Park, J. W .; Oh, H. S .; Jung, W. S .; Park, S. R .; Han, A. R .; Ban, Y. H .; Kim, E.J .; Kang, H. Y .; Yoon, Y. J. Chem. Commun. 2008,44,57825784. 9) Park, J. W .; Park, S. R .; Han, A. R .; Ban, Y. H .; Yoo, Y. J .; Kim, E.J .; Kim, E .; Yoon, Y. J. J. Antibiot, 2011, 64, 155157. 10) Park, J. W .; Park, S. R .; Han, A. R .; Ban, Y. H .; Yoo, Y. J .; Kim, E.J .; Kim, E .; Yoon, Y. J. J. Nat. Prod.2011,74,12721274. 11) Park, J. W .; Park, S. R .; Nepal, K. K .; Han, A. R .; Ban, Y. H .; Yoo, Y. J .; Kim, E.J .; Kim, E. M .; Kim, D .; Sohng, J. K .; Yoon, Y. J. Nat. Chem. February 8, 7,84,382.

It is an object of the present invention to provide a novel rosamycin derivative. Another object of the present invention is to provide a process for producing the rosemycin derivative. It is still another object of the present invention to provide an antibiotic composition containing the rosemycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The technical problems to be solved by the present invention are not limited to the technical problems and other technical problems which are not mentioned can be understood by those skilled in the art from the following description.

Recently, the inventors have found that Streptomyces Venetian state elrae (Streptomyces venezuelae ( S. venezuelae ) have reported unique and site-specific hydrogenation activities for unsaturated cyclic macrolides and hydroxamates. Specifically, a bio-hydrogenation system using Streptomyces benezene ella has a specific structural ornamentation of the target double bond (i.e., one carbon centered on a double bond has a carbonyl functional group, the other carbon Specific structure with a methyl group) (see Non-Patent Document 8-10).

Accordingly, the inventors of the present invention conducted a study on the application of biocatalyst of Streptomyces bentinjuella to rosemycin, which has the specific structural embellishment of the above-mentioned double bond in the compound. As a result, 10,11-dihydrorosamicin, which was not previously identified, was isolated from the culture of S. venezuelae cultured in one medium. Accordingly, the present inventors have analyzed the structure of 10,11-dihydro-rosamycin, confirmed an improved antimicrobial potential as compared to rosemycin, and completed the present invention.

Accordingly, in one aspect of the present invention there is provided a rosamycin derivative of the formula: EMI2.1 or a pharmaceutically acceptable salt thereof,

[Chemical Formula]

.

.

Preferably, the rosemycin derivative is biotransformed from rosemacin by a strain of the genus Streptomyces .

In another aspect of the present invention, there is provided a method for producing a rosemycin derivative from rosemacin, comprising: culturing a strain of Streptomyces in a medium containing the rosemycin ; And extracting a rosamycin derivative of the formula from the medium:

[Chemical Formula]

.

.

Preferably, the Streptomyces sp. Strain comprises Streptomyces venezuelae .

In another aspect of the present invention, there is provided an antibiotic composition comprising a rosamycin derivative of the formula: EMI5.1 or a pharmaceutically acceptable salt thereof,

[Chemical Formula]

.

.

Preferably, the antibiotic composition has activity against an antibiotic resistant strain.

Include; (MRSA Methicillin-resistant Stahphylococcus aureus ) resistant Staphylococcus aureus - Preferably, the antibiotic-resistant strains are methicillin.

Preferably, the rosemycin derivative is biotransformed from rosemacin by a strain of the genus Streptomyces .

The present invention provides a rosemycin derivative (i.e., 10,11-dihydro-rosamycin) exhibiting significantly higher antimicrobial activity (e.g., anti-MRSA activity) as compared to conventional rosemycin, and pharmaceutical compositions comprising the same .

In addition, the present invention can provide a method for producing 10,11-dihydro-rosamycin, preferably a method using microbial biotransformation.

The effects obtained by the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description will be.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
FIG. 1 is a graph showing the results of HPLC-ESI-MS (High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry) chromatography of an organic solvent extract obtained from a culture medium of S. venezuelae in a medium supplemented with rosemycin Grams. (A) shows a chromatogram of an organic solvent extract prepared from a medium to which rosemycin was added in a medium without Streptomyces benzene extract. (B) shows the chromatogram of the organic solvent extract prepared after incubation of Streptomyces venezia elata with the rosemycin-added medium. Here, rosemycin and its biotransformation derivative, 10,11-dihydroloxamycin, were selected from the molecular [M + H] + ions 582 and 584, respectively.
2 shows ESI-MS / MS spectra of rosemycin (A) and 10,11-dihydroloxamycin (B), which is a bioconversion derivative thereof, respectively.
FIG. 3 shows ¹ H-NMR (nuclear magnetic resonance) and ¹³ C-NMR spectra of 10,11-dihydro-rosamycin, a derivative of rosemycin and its biotransfer in CDCl ₃ .
Fig. 4 shows the anti-MRSA activity against MRSA Staphylococcus aureus species of 10,11-dihydroloxamycin, a rosemacin and its bioconversion derivative.

The specific terms used in the following description are provided to facilitate understanding of the present invention, and these specific terms may be changed into other forms without departing from the technical idea of the present invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof. In addition, the order of operations described herein may be varied.

Hereinafter, the present invention will be described in detail. The present invention provides novel rosemycin derivatives represented by the following general formula (2).

The rosemycin derivative of the general formula (2) is preferably prepared by biotransformation from rosemic acid by Streptomyces venezuelae , but is not limited thereto, and includes all those produced by chemical synthesis and the like.

The rosemycin derivatives of formula (2) have excellent antibiotic activity against antibiotic-resistant strains, preferably antibiotic-resistant Staphylococcus aureus species, more preferably methicillin-resistant Staphylococcus aureus (MRSA) species. In addition, the rosemycin derivatives of formula (2) have excellent antimicrobial activity against Staphylococcus aureus species, preferably antibiotic-resistant Staphylococcus species, more preferably methicillin-resistant Staphylococcus aureus (MRSA) species.

The rosemycin derivative of formula (2) can be used in the form of a pharmaceutically acceptable salt. As salts, acid addition salts formed by pharmaceutically acceptable free acids are useful, and inorganic acids and organic acids can be used as free acids. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, preferably hydrochloric acid. Organic acids include but are not limited to citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, gluconic acid, succinic acid, tartaric acid, 4- toluenesulfonic acid, galacturonic acid, Or an aspartic acid, preferably methanesulfonic acid.

In addition, the inventive Rosamycin derivative of formula (2) includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which 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 (2) in a water-miscible organic solvent such as acetone, methanol, ethanol or acetonitrile and adding an excess amount of organic acid or inorganic acid Adding an aqueous solution, and precipitating or crystallizing it. Thereafter, a solvent or an excess acid is evaporated from the mixture and dried, or the precipitated salt may be subjected to suction filtration to produce an addition salt.

The present invention also provides a process for preparing a rosemycin derivative of formula (2). Specifically, the rosemycin derivative of formula (2)

1) culturing a strain of Streptomyces in a medium containing rosamycin ; And

2) extracting a rosemycin derivative of the formula (2) from the medium.

More specifically, the rosemycin derivative of formula (2)

1) culturing a strain of Streptomyces sp. In a medium supplemented with rosamycin;

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

3) concentrating the organic solvent extract of step 2) in vacuo; And

4) dissolving the residue of step 3) in an organic solvent and then separating.

In the above method, the strain of Streptomyces sp. Is preferably S. venezuelae , but is not limited thereto.

In this method, the organic solvent in step 2) is preferably, but not limited to, ethyl acetate (EtOAc).

In this method, the organic solvent in step 4) is preferably methanol (MeOH), but is not limited thereto.

In the above method, the separation of step 4) may utilize chromatography, but it is preferable to use HPLC but not limited thereto. As an example, the preparative HPLC can be used to isolate the rosemycin derivative of formula 2 at retention time 28-29 min.

A production method described above is Streptomyces Venetian state elrae (Streptomyces venezuelae ). However, the method for preparing the rosemycin derivative of the formula (2) is not limited thereto and can also be prepared by a chemical synthesis method.

The present invention also provides an antibiotic composition comprising a rosemacin derivative of the formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.

The antibiotic composition according to the present invention is preferably a composition for an antibacterial agent. Specifically, the antibiotic composition according to the present invention has excellent antibiotic activity against antibiotic-resistant strains, preferably antibiotic-resistant Staphylococcus aureus species, more preferably methicillin-resistant Staphylococcus aureus (MRSA) species. In addition, the antibiotic composition according to the present invention has an excellent antimicrobial activity against Staphylococcus aureus species, preferably antibiotic-resistant Staphylococcus aureus species, more preferably methicillin-resistant Staphylococcus aureus (MRSA) species.

When the composition of the present invention is used as a medicine, an antibiotic composition containing a rosemycin derivative of the formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient is formulated into various oral or parenteral dosage forms at the time of clinical administration But are not limited thereto.

Examples of formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like. These formulations may contain diluents (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as starches, talc, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and may contain binders such as starch, agar, alginic acid or its sodium salt Release, boiling mixture and / or absorbing agent, coloring agent, flavoring agent, sweetening agent and the like.

The antibiotic composition containing the rosemacin derivative of formula (2) as an active ingredient can be administered parenterally, and parenteral administration can be carried out by injecting subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

In order to formulate parenteral administration, a rosemycin derivative of the formula (2) or a pharmaceutically acceptable salt thereof is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which is then prepared into an ampule or vial unit dosage form . The antibiotic composition according to the present invention may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for controlling osmotic pressure, and other therapeutically useful substances, , Granulation, or coating methods.

In addition, the human body dose of the rosemacin derivative of formula (2) may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity, and is generally 0.001 To 1,000 day / day, preferably 0.01 to 500 / day, and may be administered once to several times a day at predetermined intervals according to the judgment of a doctor or pharmacist.

Hereinafter, embodiments for explaining the present invention in more detail will be presented.

However, the following examples are provided only to illustrate the present invention more easily, and the present invention is not limited by the examples.

< Example  1> Rosacemic  Preparation of derivatives

<1-1> Of rosemycin  Biotransformation

A recombinant mutant species of S. venezuelae YJ028 (using the species described in Non-Patent Document 8), in which a gene encoding a picromycin polyketide synthase and a desosamine biosynthesis gene are all removed, Was isolated for separation from SPA (Sorbitol Pyroglutamic Acid) agar plate [0.1% yeast extract, 0.1% beef extract, 0.2% tryptose, 1.0% glucose, 1.5% agar, and trace amount of FeSO ₄ ] Streaked and cultured at 30 for 2 days. Then, in 50 ml of SCM medium [1.5% soluble starch, 2.0% soytone, 0.01% CaCl ₂ , 0.15% yeast extract (yeast extract) for 30 to 2 days in Erlenmeyer flask ) And rosemycin (Sigma, St. Louis, MO) (250; final concentration 5 / mL; dissolved in 100 uL of MeOH) Respectively. All experiments were performed at least three times independently.

<1-2> Rosacemic  Extraction and Separation of Derivatives

The entire culture of Example <1-1> was extracted and separated twice with an average volume of EtOAc (Ethyl acetate) in a 250 mL separatory funnel, and the organic extract was concentrated in vacuo. The dried residue was immediately dissolved in 200 uL of MeOH (Methanol), and a portion of the solvent was used for the analysis. First, Micromass Quattro micro Analysis was performed using a Waters / Micromass Quattro micro / MS interfacer composed of analytical reverse phase Nova-Pak C ₁₈ column (Waters, Milford, MA; 150 x 3.9 mm, 4.0 m) directly connected to MS.

As a result, it was confirmed that a reduced rosamiacin derivative corresponding to rosemicin was generated at a conversion rate of about 22% by adding rosemic acid to various batch culture solutions of YJ028 (see Fig. 1).

Rosamycin derivatives were purified by chromatographic separation using sequential ESI-MS analysis of each fraction (5 mL / well) collected from preparative reverse phase HPLC. The chemical structure of the rosamycin derivatives was analyzed by ¹ H-NMR and ¹³ C-NMR spectroscopy. Specifically, chromatographic separation was performed using preparative HPLC consisting of a Watchers 120 ODS-BP column (250 mm x 10.0 mm, 5.0 m). Further, ¹ H-NMR and ¹³ C-NMR spectroscopic analysis were carried out as follows. NMR samples were prepared by dissolving the rosamycin derivative in 200 uL of CDCl ₃ and then placing the solution in 5 mm Shigemi advanced NMR microtube (Sigma, St. Louis, MO). ¹ H-NMR and ¹³ C-NMR spectra were obtained at 298 K using a Varian INOVA 500 spectrophotometer and chemical shifts were recorded in parts per million (ppm) using TMS (Trimethyl Silane) as internal standard. NMR data were calculated using Mnova Suite 5.3.2 software.

MS / MS spectra of rosemicin at m / z 582 showing proton molecular ion showed two interceptions at m / z 158 and 425, as reported previously (see non-patent document 13). On the other hand, the MS / MS spectra of the rosamycin derivatives show typical m / z 158 fragment ions of rosemicin, as well as m / z 427 produced from rosamycin at m / z 427 And showed specific product ions (see FIG. 2). These results suggest that the double bonds present at C10 and 11 in the rosamycin derivatives are eliminated.

Through comparison of ¹ H-NMR spectroscopic data of rosamycin and its reduced derivatives, the most obvious change observed in rosamycin derivatives is the typical 6.31 for olefinic proton (H-10,11) in rosemycin And a deficit of 7.03 ppm signal. The upfield shift of the C-10 and C-11 signals of the parent compound rosemicin (31.9 and 31.3 ppm at 124.1 and 143.4 ppm, respectively) supported the differences identified by ¹ H-NMR data , Demonstrating the elimination of C-10,11 double bonds in the biosynthetic derivatives of rosemycin (see Figure 3). These results demonstrate that S. venezuelae , which contains regio-selective activity for bio-hydrogenation to 10,11-dihydrorosamicin, Lt; RTI ID = 0.0 &gt; rosamycin &lt; / RTI &gt;

< Example  2> Rosacemic  Identification of Antibacterial Activity of Derivatives

(ATCC 33592 and ATCC 43300) were obtained from the ATCC (American Type Culture Collection) (Manassas, VA, USA) in order to examine anti-MRSA activity of the rosemycin derivatives. Respectively.

Specifically, two MRSA strains were cultured and maintained in Mueller-Hinton broth (Difco, BD Biosciences, San Jose, Calif.) At 28. Rosamycin and 10,11-dihydro-rosamycin were then treated in the medium. Antibacterial activity against MRSA was measured using a microdilution method recommended by the Clinical and Laboratory Standard Institute (CLSI) (see non-patent document 11). Serial dilutions were made in duplicate using DMSO (dimethyl sulfoxide) so that the final concentration of each treatment was 4-512 / mL, and a partial sample of DMSO (final 0.1%) was used as a negative control. Growth of MRSA was monitored at 600 nm using a Labsystems Bioscreen C reader and the lowest concentration of rosamycin and 10,11-dihydro-rosemycin was determined by MIC (Minimum Inhibitory Concentration) in broth medium inhibiting the growth of test microorganisms Respectively. All analyzes were performed at least three times.

As a result, the 10,11-dihydro-rosamycin of the present invention exhibited an anti-bacterial activity, in particular, methicillin-resistant Staphylococcus aureus, 2 to 4 times as much as that of rosemic acid. This shows that the deficiency of C10,11-double bond in rosemicin does not affect the antibacterial activity (see Fig. 4), and the 10,11-dihydroloxamycin of the present invention is useful as an antibiotic active ingredient . In addition, the 10,11-dihydroloxamycin of the present invention exhibits improved antibacterial activity, in particular anti-resistant antibacterial activity, as compared with rosemycin, and thus can be usefully used as a superbacterium-based antibiotic.

In conclusion, the present invention can provide a rosemycin derivative using microbial biotransformation techniques instead of the pre- or semi-synthetic methods commonly used to produce novel rosemycin derivatives and / or analogs. In addition, the site-specific bio-hydrogenation activity of S. venezueale can be applied to various macrolide compounds.

Meanwhile, the 10,11-dihydro-rosamycin according to the present invention can be formulated into various forms depending on the purpose. The following illustrates a formulation method in which 10,11-dihydro-rosamycin is contained as an active ingredient, but the present invention is not limited thereto.

< Formulation example  1> Purification (direct pressurization)

The active ingredient 5.0 was sieved and then mixed with lactose 14.1, crospovidone USNF 0.8 and magnesium stearate 0.1 and pressed to prepare tablets.

< Formulation example  2> Purification (wet assembly)

After sieving the active ingredient 5.0, 16.0 parts of lactose and 4.0 parts of starch were mixed. Polysorbate 80 0.3 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 colloidal silicon dioxide 2.7 and magnesium stearate 2.0. The fine particles were pressed to prepare tablets.

< Formulation example  3> Powder and Capsule

The active ingredient 5.0 was sieved and mixed with lactose 14.8, polyvinylpyrrolidone 10.0, and magnesium stearate 0.2. The mixture was extruded through a hard No. 5 gelatin capsules.

< Formulation example  4> injection

Injections were prepared by adding 100 mg of the active ingredient, 180 mg of mannitol, 26 mg of Na ₂ HPO _{4 12} H ₂ O and 2974 mg of distilled water.

Claims (8)

delete delete delete delete An antibiotic composition having activity against methicillin-resistant Staphylococcus aureus (MRSA) comprising a rosamycin derivative of the formula: EMI2.1 or a pharmaceutically acceptable salt thereof,
[Chemical Formula]

. delete delete 6. The antibiotic composition according to claim 5, wherein the rosamiacin derivative is a rosemycin derivative biosynthesized from rosemacin by a strain of Streptomyces .

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