CN117597138A - Pharmaceutical compositions containing micrococcin compounds and methods for preparing micrococcin compounds - Google Patents

Abstract

The invention provides a method for preparing a micrococcin compound and a pharmaceutical composition for treating and preventing infections of specific strains. The pharmaceutical composition includes a micrococcin compound, its solvate, its hydrate, its prodrug, Its isomers or pharmaceutically acceptable salts thereof are used as effective components. In addition, the present invention provides an anti-inflammatory composition including a micrococcin compound, its solvate, its hydrate, its prodrug, its isomer or its pharmaceutically acceptable salt as an active ingredient.

Description

Pharmaceutical composition containing micrococcin compounds and method for preparing micrococcin compounds

Technical Field

The present invention relates to a pharmaceutical composition comprising a specific micrococcin compound and a total synthesis method of the micrococcin compound.

Background Art

Tuberculosis is a major disease in developing countries, causing about 8 million new infections and 3 million deaths each year, but in recent years it has become a problem in developed countries as well.

Tuberculosis can remain latent for a long time, depending on the patient's immune status, and it is estimated that 2 billion people (about 30% of the world's population) are latently infected. When a person is infected with tuberculosis, he/she may be asymptomatic for a considerable period of time, but may develop acute inflammation of the lungs as a common symptom, causing fever and nonproductive cough. If not treated, it usually leads to serious complications and death.

The development of anti-tuberculosis drugs began with streptomycin in the 1940s, and was followed by the development of more effective anti-tuberculosis drugs, as a result, the incidence and deaths of tuberculosis have declined rapidly.

However, due to the increasing incidence of multidrug resistance (MDR)-TB, a drug-resistant strain that could not be controlled with existing antibiotics in the 1980s, the number of patients with MDR-TB has not decreased and it has become a serious health problem.

Another lung disease is caused by nontuberculous mycobacteria (NTM).

NTM are widely distributed in the natural environment, and because NTM infections occur in the natural environment, NTM are also called environmental mycobacteria. Most NTM are less pathogenic to humans than tuberculosis. Therefore, symptomatic NTM infectious diseases may be related to local or systemic immunodeficiency. Colonization, contamination, etc. of samples, as well as actual NTM infection, may lead to the separation of NTM from clinical samples.

Since tuberculosis and diseases caused by nontuberculous mycobacteria are chronic and difficult to cure due to drug resistance, there is an urgent need to develop therapeutic agents against them.

Meanwhile, thiopeptides are antibiotic groups produced by actinomycetes or bacteria and have been extensively studied due to their wide range of biological properties and outstanding efficacy.

Micrococcin is a natural thiopeptide product with a structural feature of a macrocyclic ring consisting of 26 atoms. It is divided into micrococcin P1 (MP1) and micrococcin P2 (MP2). Micrococcin P1 is known to be effective against a variety of Gram-positive bacteria, showing antibacterial effects, and its mechanism of action is to bind to the complex formed by L11 protein and 23S rRNA and inhibit protein synthesis.

Micrococcin P1 is a microcyclic peptide antibiotic mainly found on the surface of French Raclette cheese. It was identified from Staphylococcus equorum WS2733 and its total synthesis has been completed.

It is known that micrococci exist in Bacillus pumilus as P1 and P2 in a ratio of 7:1, but it is very difficult to isolate micrococcal P2 and its total synthesis has not been reported.

The biological activity of the purified micrococcin P2 has been reported, but the other reports are rare. In addition, the antibacterial effect on tuberculosis, nontuberculous mycobacteria and clostridium has not been reported.

Summary of the invention

Detailed description of the invention

Technical issues

Therefore, the inventors of the present invention studied and completed a total synthesis method of a micrococcin compound, and found that the micrococcin compound has a surprising effect on killing and inhibiting specific bacteria, thereby completing the present invention.

The purpose of the present invention is to provide a method for the total synthesis of micrococcin compounds.

Another object of the present invention is to provide a pharmaceutical composition comprising a micrococcin compound, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide an anti-inflammatory composition comprising a micrococcin compound, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof or a pharmaceutically acceptable salt thereof as an effective ingredient.

Technical Solution

In one general aspect, a pharmaceutical composition for treating or preventing bacterial infection caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria comprises: a micrococcin compound, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof, which can effectively kill specific bacteria, Mycobacterium tuberculosis and non-tuberculous mycobacteria or inhibit their growth as an active ingredient, wherein the micrococcin compound of the present invention is represented by the following chemical formula 1:

[Chemical formula 1]

In another general aspect, a pharmaceutical composition for treating and preventing bacterial infection caused by Clostridium sp. bacteria includes: a micrococcin compound represented by Chemical Formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof.

In another general aspect, an anti-inflammatory composition includes a micrococcin compound represented by Chemical Formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof as an effective ingredient.

In yet another general aspect, a method of preparing a micrococcin compound represented by Chemical Formula 1.

Beneficial Effects

The pharmaceutical composition of the present invention comprises a specific compound, a micrococcin compound, as an active ingredient, thereby having a surprising and excellent antibiotic effect against specific bacteria, tuberculosis bacillus, nontuberculous mycobacteria and clostridium, and is very effective in treating and preventing infections caused by the bacteria.

In addition, the pharmaceutical composition of the present invention also has a very good effect on the treatment and prevention of multidrug-resistant or extensively drug-resistant tuberculosis, and can effectively treat and prevent tuberculosis.

Furthermore, unlike conventional methods of obtaining micrococcin compounds by separation from natural products, the method for preparing a micrococcin compound of the present invention prepares the micrococcin compound by total synthesis, thereby preparing a micrococcin compound with excellent purity in high yield.

Therefore, the method for preparing the micrococcin compound of the present invention can produce the compound by chemical synthesis rather than by isolation from natural products, which allows large-scale production and low-cost commercialization.

Furthermore, the micrococcin compound according to the present invention exhibits an anti-inflammatory effect of inhibiting the production of inflammatory cytokines and is therefore useful as a drug, functional food, and cosmetic material for treating, preventing, and ameliorating inflammation-related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the results of evaluating efficacy in a model of Clostridium difficile infection.

FIG2 is an evaluation of anti-inflammatory efficacy.

DETAILED DESCRIPTION

Hereinafter, a pharmaceutical composition comprising a micrococcin compound, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof or a pharmaceutically acceptable salt thereof as an effective ingredient of the present invention will be described in detail. Here, unless otherwise defined, the technical terms and scientific terms used in this specification have the general meanings understood by those skilled in the art to which the present invention belongs, and the description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted in the following description.

The following terms used in this specification are defined below, but they are merely illustrative and do not limit the invention, its application, or uses.

In the present specification, the term "protecting group" includes "amino protecting group" or "hydroxy protecting group", but is not limited thereto. "Amino protecting group" refers to a protecting group suitable for preventing side reactions at the nitrogen position of the amino group. Representative amino protecting groups include formyl group; acyl group, for example, alkanoyl group, for example, acetyl group, trichloroacetyl group or trifluoroacetyl group; alkoxycarbonyl group, for example, tert-butoxycarbonyl group (boc); arylmethoxycarbonyl group, for example, benzyloxycarbonyl group (Cbz) and 9-fluorenylmethoxycarbonyl group (Fmoc); arylmethyl group, for example, benzyl (bn), triphenylmethyl (Tr) and 1,1-bis-(4'-methoxyphenyl)methyl group; silyl group, for example, trimethylsilyl (trimethylsilyl)methyl group; group, TMS) and tert-butyldimethylsilyl group (tert-butyldimethylsiyl group, TBS), etc., but not limited thereto. "Hydroxy protecting group" refers to a protecting group suitable for inhibiting side reactions of hydroxyl groups. Representative hydroxy protecting groups include alkyl groups, such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl groups (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (p-methoxybenzyl group, PMB), 9-fluorenylmethyl (9-fluorenylmethyl group, Fm) and diphenylmethyl (diphenylmethyl group, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc., but not limited thereto.

The term "halogen" in the present specification means fluorine, chlorine, bromine or iodine.

The term "pharmaceutically acceptable salt" in this specification includes salts of active compounds prepared from relatively nontoxic acids and bases, depending on the specific substituents found in the compounds mentioned herein. When the compounds of the present invention include relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compound with the desired base and a sufficient amount of pure or suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention include relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of the compound with the desired acid and a sufficient amount of pure or suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include not only salts derived from relatively nontoxic organic acids, such as acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid and the like, as well as salts derived from hydrogen chloride, hydrogen bromide, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydrogen iodide or phosphorous acid and the like. Furthermore, it includes salts of amino acids such as arginate salts and the like, and analogs of organic acids such as glucuronic acid or galactunoric acid and the like.

Certain specific compounds of the present invention possess both basic and acidic functionalities that allow the compounds to be converted into either basic or acidic addition salts.

As used herein, "effective amount" refers to an amount of a compound of the invention sufficient to provide a therapeutic advantage in the treatment or management of bacterial infections.

"Effective amount" also refers to an amount of a compound of the invention sufficient to cause bacterial death or inhibit bacterial outbreaks. "Effective amount" also refers to an amount sufficient to treat or prevent bacterial infections in vitro or in vivo.

The term "tuberculosis" as used in this specification includes infections caused by Mycobacterium tuberculosis and Mycodbacterium bovis; respiratory tuberculosis, such as tuberculosis of the lungs, larynx, trachea and bronchus, intrathoracic lymph node tuberculosis, tuberculous pleurisy, early respiratory tuberculosis and other respiratory tuberculosis; nervous system tuberculosis, such as tuberculous meningitis, tuberculosis of meninges, tuberculous leptomeningeal inflammation, meningeal tuberculoma and other nervous system tuberculosis; bone and joint tuberculosis, urogenital tuberculosis, tuberculous peripheral lymphadenopathy, intestinal, peritoneal, mesenteric glandular tuberculosis, skin and percutaneous tissue tuberculosis, eye, ear or adrenal tuberculosis and miliary tuberculosis.

Nontuberculous mycobacterium (NTM) refers to mycobacteria other than Mycobacterium tuberculosis (M.tuberculosis) and Hansen's bacillus (M.leprae). Diseases caused by nontuberculous mycobacteria may include lung diseases, lymphadenitis, skin/soft tissue/bone infections, disseminated diseases, etc. In addition, they may also include cough, chronic fatigue, general weakness, dyspnea, breast discomfort, hemoptysis, etc., but are not limited to these.

As used herein, "prevention" includes preventing the recurrence, extension or outbreak of a bacterial infection.

As used in this specification, "treatment" includes both bacterial killing and inhibition.

"Administering" as used in the present invention means contacting a therapeutically effective amount of the compound of Chemical Formula 1 and/or its pharmaceutically acceptable salt or pharmaceutical composition with a patient in need thereof, preferably an animal, most preferably a mammal, and more preferably a human.

"Patients in need" refers to patients who need treatment for diseases caused by bacterial infection. In one aspect of the present invention, "patients in need" refers to any patient who may have a bacterial infection or is at risk of having a bacterial infection. Preferably, patients in need refer to animals, more preferably mammals, and more preferably humans.

The term "micrococcin compound of the present invention" used in this specification includes not only the compounds of chemical formulas 1 and 2, but also clathrates, hydrates, solvates, prodrugs or polymorphs thereof. In addition, when no pharmaceutically acceptable salt thereof is mentioned, the term "micrococcin compound of the present invention" has the meaning of also including pharmaceutically acceptable salts of the compounds of the present invention. In an exemplary embodiment, the micrococcin compound of the present invention may exist as a stereoisomerically pure compound (e.g., substantially free of other stereoisomers (e.g., 85% ee or higher, 90% ee or higher, 95% ee or higher, 97% ee or higher, or 99% ee or higher)). That is, when the compounds of chemical formulas 1 and 2 of the present invention or their salts are tautomeric and/or stereoisomers (e.g., geometrical isomers or conformational isomers), their respective separated isomers and mixtures are also included in the scope of the compounds of the present invention. When there is asymmetric carbon in the structure of the compounds of the present invention or its salt, optically active compounds and racemic compounds thereof are also included in the scope of the compounds of the present invention. For example, when the compounds of the present invention have sulfoxide (sulfoxide) (SOR) structure, they can have chirality. The compounds of the present invention include R and S forms of isomers, and mixtures of R and S forms are also included in the scope of the compounds of the present invention.

Furthermore, the compounds of the present invention may exist in any form of a keto form or an enol form, and these forms are also included in the scope of the compounds of the present invention.

As used in this specification, the term "polymorph" refers to a solid crystalline form of a micrococcin compound or a complex thereof of the present invention. Different polymorphs of the same compound exhibit different physical, chemical and/or spectral properties. Differences in physical properties include, but are not limited to, stability (e.g., thermal stability or photostability), compressibility and density (important for formulation and manufacturing), and dissolution rate (which may affect bioavailability). Differences in stability result in changes in chemical reactivity (e.g., differential oxidation, such as faster color change when formed from one polymorph than from another polymorph), mechanical properties (e.g., a purified fragment stored as a kinetically preferred polymorph is converted to a thermodynamically more stable polymorph), or both (a purified polymorph is more susceptible to decomposition at high humidity). Other physical properties of polymorphs may affect their processing. For example, due to, for example, its form or particle size distribution, one polymorph may be more likely to form a solvate, or may be more difficult to filter or wash than another polymorph.

The term "solvate" used in this specification refers to the micrococcin compound of the present invention or a pharmaceutically acceptable salt thereof, including a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile and non-toxic and can be administered to humans in very small amounts.

The term "hydrate" used in the present specification refers to the micrococcin compound of the present invention or a pharmaceutically acceptable salt thereof, comprising a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

The term "clathrate" used in the present specification refers to the micrococcin compound of the present invention or a salt thereof in the form of a crystal lattice including spaces (eg, channels) in which guest molecules (eg, solvents or water) are confined.

In the case where any compound (prodrug) is separated in vivo and produces the micrococcin compound of the present invention or its salt, the compound also belongs to the scope of the present invention. As used in this specification and not mentioned differently, the term "prodrug" refers to the micrococcin compound of the present invention, which can be hydrolyzed, oxidized and cause different reactions under biological conditions (in vitro or in vivo) to provide active compounds, especially the micrococcin compound of the present invention. Examples of prodrugs include compounds that are biohydrolyzed to produce the compounds of the present invention, including biohydrolyzable parts, such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biodegradable carbonates, biohydrolyzable ureas and biohydrolyzable phosphate analogs, but are not limited to specific embodiments. Preferably, the prodrug of a compound having a carboxyl functional group is a lower alkyl ester of a carboxylic acid. Carboxylic acid esters are generally formed by esterifying a portion of the carboxylic acids present in the molecule. Prodrugs can be easily prepared using methods well known to those skilled in the art.

The term "purified" as used in this specification refers to an isolate having a purity greater than 90%, in one exemplary embodiment 95% or higher, in another exemplary embodiment 99% or higher, and in yet another exemplary embodiment 99.9% or higher.

As used in this specification, the singular forms "a", "an" and "the" may include plural forms unless the context clearly indicates otherwise.

Unless otherwise specifically mentioned, the term "pharmaceutical composition" used in the present invention includes all pharmaceutical compositions for treating and preventing bacterial infections caused by Mycobacterium tuberculosis or nontuberculous mycobacteria and pharmaceutical compositions for treating and preventing bacterial infections caused by Clostridium.

The present invention provides a pharmaceutical composition comprising a micrococcin compound, a solvate, a hydrate, a prodrug, an isomer or a pharmaceutically acceptable salt thereof having a surprising antibacterial effect on specific bacteria as an active ingredient. One embodiment of the present invention provides a pharmaceutical composition for treating and preventing bacterial infections caused by Mycobacterium tuberculosis or nontuberculous mycobacteria, and another embodiment of the present invention provides a pharmaceutical composition for treating and preventing bacterial infections caused by Clostridium.

First, one embodiment of the present invention provides a pharmaceutical composition for treating or preventing bacterial infection caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria, comprising: a micrococcin compound that is effective against Mycobacterium tuberculosis or non-tuberculous mycobacteria as an antibacterial agent, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof as an effective ingredient, wherein the micrococcin compound of the present invention is represented by the following chemical formula 1:

[Chemical formula 1]

The pharmaceutical composition for treating and preventing bacterial infection caused by tuberculosis bacillus or nontuberculous mycobacteria of the present invention comprises the micrococcin compound represented by Chemical Formula 1, thereby having an excellent antibacterial effect on tuberculosis bacillus or nontuberculous mycobacteria in addition to the antibacterial effect on conventional known bacteria.

Preferably, the micrococcin compound according to an exemplary embodiment may be represented by the following Chemical Formula 2:

[Chemical formula 2]

In an exemplary embodiment, the bacterial infection may be tuberculosis or symptoms caused by nontuberculous mycobacteria.

According to an exemplary embodiment, tuberculosis may be an infection caused by Mycobacterium tuberculosis or Mycobacterium bovis, preferably an infection caused by Mycobacterium tuberculosis.

Specifically, according to an exemplary embodiment, tuberculosis can include respiratory tuberculosis, such as lung, laryngeal, tracheal and bronchial tuberculosis, intrathoracic lymph node tuberculosis, tuberculous pleurisy, early respiratory tuberculosis and other respiratory tuberculosis; nervous system tuberculosis, such as tuberculous meningitis, tuberculosis of meninges, tuberculous leptomeningeal inflammation, meningeal tuberculoma and other nervous system tuberculosis; bone and joint tuberculosis, urogenital tuberculosis, tuberculous peripheral lymphadenopathy, intestinal, peritoneal, mesenteric glandular tuberculosis, skin and percutaneous tissue tuberculosis, eye, ear or adrenal tuberculosis and miliary tuberculosis, and the symptoms associated therewith may be cough, chest pain, fever, chills, loss of appetite, weight loss, inflammation or a combination thereof.

According to an exemplary embodiment, tuberculosis can be susceptible tuberculosis, multidrug resistance (MDR) tuberculosis or extensive drug resistance (XDR) tuberculosis. As a specific example, multidrug-resistant tuberculosis can be tuberculosis that cannot kill tuberculosis bacteria with antibiotics, more specifically, one or more anti-tuberculosis drugs selected from isoniazid, rifampicin, levofloxacin, ofloxacin, moxifloxacin, kanamycin, amikacin and capreomycin are used, and as an example, tuberculosis that cannot kill tuberculosis bacteria with isoniazid and rifampicin is used to the human body, and extensive drug-resistant tuberculosis may be tuberculosis that is resistant to the quinolone family and injections as well as isoniazid or rifampicin. Susceptible TB refers to TB in which the therapeutic effect of antibiotics can be exerted as expected, as opposed to antibiotic resistance caused by the use of antibiotics.

According to an exemplary embodiment, the symptoms caused by nontuberculous mycobacteria may be lung disease, lymphadenitis, skin/soft tissue/bone infection or disseminated disease, and in addition, may include cough, chronic fatigue, general weakness, dyspnea, breast discomfort, hemoptysis, etc., but are not limited thereto.

According to an exemplary embodiment, the nontuberculous mycobacteria can be selected from the group consisting of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium osloensis, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium chelonae, Mycobacterium mucogenicum, Mycobacterium peregrinum, Mycobacterium simianum, Mycobacterium truncat ... simiae), Mycobacterium wolinskyi, or a combination thereof, more specifically, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, or a combination thereof.

According to an exemplary embodiment, the pharmaceutical composition for treating and preventing bacterial infection caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria may further include an antibiotic, which may be an anti-tuberculosis drug, and the anti-tuberculosis drug may be isoniazid, rifampicin, ethambutol, SQ-109, pyrazinamide, streptomycin, kanamycin, capreomycin, ethionamide, prothionamide, enviomycin, para-aminosalicylic acid, acid, cycloserine, amikacin, levofloxacin, moxifloxacin, gatifloxacin, ofloxacin, terizidone, thionamide, ethionamide, protionamide, clofazimine, linezolid, amoxicillin, clavulanate, thioacetazone, imipenem, cilastatin, clarithromycin, bedaquiline, delamanid, lmipenem, cilastatin, meropenem, or a combination thereof.

Another embodiment of the present invention provides a pharmaceutical composition for treating and preventing bacterial infection caused by Clostridium, comprising: a micrococcin compound represented by Chemical Formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof as an effective ingredient.

According to an exemplary embodiment, the micrococcin compound of Chemical Formula 1 has an excellent antibacterial effect on Clostridium and Mycobacterium tuberculosis or nontuberculous mycobacteria.

Preferably, the micrococcin compound according to an exemplary embodiment may be represented by Chemical Formula 2.

In an exemplary embodiment, the bacterial infection caused by Clostridium can be colitis, diarrhea, pseudomembranous colitis, gangrenous enteritis, skin and soft tissue infection, food poisoning, or a combination thereof.

According to an exemplary embodiment, the Clostridium is not limited, but may be Clostridium difficile, Clostridium perfringens, Clostridium cadaveris, Clostridium innocuum, Clostridium tetani, Clostridium bifermentans, Clostridium histolyticum, Clostridium clostridioforme, Clostridium subterminale, Clostridium ramosum, Clostridium septicum, combinations thereof, and preferably, Clostridium difficile, Clostridium perfringens, or combinations thereof.

Generally, when Clostridium difficile infection (CDI) is treated with antibiotics, particularly vancomycin, vancomycin-resistant Enterococci (VRE) increase to cause problems, but the pharmaceutical composition comprising the micrococcin compound represented by Chemical Formula 1, its solvate, its hydrate, its prodrug, its isomer, or its pharmaceutically acceptable salt as an active ingredient allows effective treatment without increasing the concentration of vancomycin-resistant Enterococci (VRE).

Therefore, the pharmaceutical composition for treating and preventing bacterial infections caused by Clostridium, comprising the micrococcin compound of Chemical Formula 1, its solvate, its hydrate, its prodrug, its isomer, or its pharmaceutically acceptable salt as an active ingredient is very effective for treating vancomycin-resistant Clostridium difficile infection.

According to an exemplary embodiment, the pharmaceutical composition may further include one or two or more adjuvants selected from vancomycin, metronidazole, fidaxomicin, ridinilazole, actoxumab, bezlotoxumab, probiotics, prebiotics, intravenous immunoglobulin and antidiarrheal drugs.

According to an exemplary embodiment, the pharmaceutical composition may include a pharmaceutical composition for treating and preventing bacterial infection caused by Mycobacterium tuberculosis or nontuberculous mycobacteria or a pharmaceutical composition for treating and preventing bacterial infection caused by Clostridium.

In the pharmaceutical composition according to an exemplary embodiment, 0.001 to 10 wt % of the micrococcin compound, its solvate, its hydrate, its prodrug, its isomer or a pharmaceutically acceptable salt thereof may be included relative to the total weight of the pharmaceutical composition.

According to an exemplary embodiment, the pharmaceutical composition can be administered to a subject before or after an outbreak of bacterial infection. In addition, multiple separate doses and staggered doses can be administered daily or sequentially, or a dose can be continuously infused or pushed (bolus). In addition, when indicated as needed in a therapeutic or preventive situation, the dose of the micrococcin compound of the present invention or a pharmaceutical composition comprising the same can be increased or decreased proportionally.

Furthermore, the pharmaceutical composition of the present invention can be used as a pharmaceutical preparation for treating diseases caused by specific bacterial infection, and the pharmaceutical composition of the present invention includes a preparation suitable for administration to mammals (eg, humans).

According to an exemplary embodiment, the pharmaceutical composition substantially comprises the micrococcin compound of the present invention, its hydrate, its solvate, its isomer, its prodrug or its pharmaceutically acceptable salt, thereby having high antibacterial activity against Mycobacterium tuberculosis, nontuberculous mycobacteria or Clostridium.

Preferably, the micrococcin compound of the present invention contained in the pharmaceutical composition according to an exemplary embodiment of the present invention may account for 0.001 to 10 wt %, preferably 0.005 to 10 wt % and more preferably 0.1 to 5 wt % relative to the total weight of the pharmaceutical composition.

Preferably, according to an exemplary embodiment of the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier.

In one embodiment, a pharmaceutically acceptable carrier is a material recognized by those skilled in the art and includes a pharmaceutically acceptable material, composition or medium suitable for administration to a mammal.

Carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials involved in delivering or transporting the test agonist from one organ or body part to another. Each carrier should be acceptable in the sense of being compatible with the other ingredients of the formulation and should not be deleterious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and waxes for suppositories; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; residual solution; ethanol; phosphate buffer solution; and other commercially available nontoxic materials used in pharmaceutical formulations.

In addition, the pharmaceutical composition of the present invention may include wetting agents, emulsifiers and lubricants (eg, sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweeteners, flavors, essences, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; fat-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl galactate, α-tocopherol, etc.; and metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

The pharmaceutical compositions of the present invention include those suitable for oral, inhaled, intranasal, topical, buccal, sublingual, rectal, intravaginal and/or parenteral administration, can be conveniently provided as unit dosage forms, and can be prepared by any method known in the pharmaceutical art. The amount of active ingredient combined with the carrier material to produce a single dosage form generally corresponds to the amount of the compound that causes the therapeutic effect.

According to an exemplary embodiment of the present invention, the method for preparing a pharmaceutical composition (or preparation) comprises combining the micrococcin compound of the present invention with a carrier and optionally one or more auxiliary components. Typically, the preparation is prepared by uniformly and densely mixing the micrococcin compound of the present invention with a liquid carrier and a crushed solid carrier, and then, if necessary, molding the product.

The formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (flavored bases, usually using sucrose and gum arabic or tragacanth), powders or granules, or may be aqueous or non-aqueous liquids, oil-in-water or water-in-oil liquid lotions, elixirs or syrups, lozenges (inert bases, for example, using gelatin and glycerin or sucrose and gum arabic) and/or mouthwashes, etc., and each of them includes a predetermined amount of the micrococcin compound of the present invention as an active ingredient. The micrococcin compound of the present invention may also be administered as a pill, electuary or paste.

In the solid dosage forms (capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration of the present invention, the micrococcin compound as the active ingredient is mixed with any one of one or more pharmaceutically acceptable carriers, for example, sodium citrate or monocalcium phosphate, and/or a filler or filler, for example, starch, lactose, sucrose, glucose, mannitol and/or silicic acid; a binder, for example, carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and/or gum arabic; a desiccant, for example, glycerol; a disintegrant, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, specific silicates and sodium carbonate; a solution flame retardant, for example, paraffin; an absorption promoter, for example, a quaternary ammonium compound; a wetting agent, for example, cetyl alcohol and glycerol monostearate; an absorbent, for example, kaolin and bentonite; a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and a mixture thereof; and a colorant.

In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using lactose or milk sugar and an excipient such as a high molecular weight polyethylene glycol.

Tablets can be made by compression or molding with any one or more auxiliary components, and compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be made by molding in a suitable machine a mixture of pulverized compounds moistened with an inert liquid diluent.

Tablets, dragees, pills and granules as another solid dosage form of the pharmaceutical composition of the present invention may optionally be grooved or manufactured to have coatings and shells, such as enteric coatings and other coatings well known in the field of pharmaceutical preparations. These may also be formulated to provide a slow or controlled release of the active ingredient therein by using, for example, various proportions of hydroxypropylmethylcellulose, thereby providing a desired release profile, other polymer matrices, liposomes and/or microspheres. These may be sterilized, for example, by filtering through a bacteria-retaining filter or by adding a disinfectant in the form of a sterile solid composition, which may be dissolved in sterile water or some other sterile injection medium immediately before use.

The pharmaceutical composition of the present invention may optionally include an opacifier and may be a composition that may be an active ingredient alone or preferably in a specific region of the gastrointestinal tract, optionally in a delayed manner. Examples of available embedding compositions include polymerizable materials and waxes. If appropriate, the active ingredient may also be present in microcapsule form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration of the micrococcin compound or pharmaceutical composition of the present invention include pharmaceutically acceptable emulsions, microlotions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran methanol, caprylic acid capric acid polyethylene glycol glyceride (Labrasol), polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

When the pharmaceutical composition of the present invention is an oral composition, it may include adjuvants such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, coloring agents, flavors and preservatives in addition to inert diluents. In addition to the compound of the present invention, the suspension may include, for example, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitol esters, microcrystalline cellulose, methyl aluminum hydroxide, bentonite, agar, tragacanth and mixtures thereof.

The pharmaceutical compositions (preparations) of the present invention for rectal or vaginal administration can be provided as suppositories, which can be prepared by mixing one or more micrococcin compounds of the present invention with, for example, one or more suitable non-irritating excipients or carriers including cocoa butter, polyethylene glycol, suppository wax or salicylates, and are solid at room temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity to release the active compound.

Pharmaceutical compositions (formulations) of the present invention suitable for intravaginal administration also include vaginal suppositories, tampons, creams, gels, pastes, foams or spray formulations containing any suitable carrier known in the art.

The dosage forms of the micrococcin compounds or pharmaceutical compositions of the present invention for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed with a pharmaceutically acceptable carrier under sterile conditions and any preservatives, buffers or propellants that may be required.

Ointments, pastes, creams and gels may contain, in addition to the micrococcin compound of the invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc, zinc oxide, or mixtures thereof.

In addition to the micrococcin compounds of the invention, powders and sprays may include excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, polyamide powder, or mixtures thereof. Sprays may also include common propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of the micrococcin compound of the invention to the body.Such dosage forms can be prepared by dissolving or dispersing the micrococcin compound of the invention in the proper medium.

Absorption enhancers may also be used to increase the rate of flux of the micrococcin compounds of the invention across the skin. The flux rate may be controlled by providing a rate-controlled membrane or a dispersion of the active compound in a polymer matrix or gel.

Ophthalmic preparations, eye ointments, powders, solutions, etc. are also included within the scope of the present invention.

Pharmaceutical compositions of the invention suitable for parenteral administration include one or more micrococcin compounds of the invention and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or lotions, or sterile powders, which may be reconstituted in sterile injectable solutions or dispersions immediately before use, and may include solutes, suspending agents or thickening agents, which render the formulation isotonic with the blood of the recipient, antioxidants, buffers, bacteriostats or agents.

Examples of suitable aqueous and non-aqueous carriers that can be used for the pharmaceutical composition of the present invention include water, ethanol, caprylic acid capric acid macrogol glyceride (Labrasol), polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Suitable fluidity can be maintained by, for example, using a coating material such as lecithin, maintaining the required particle size in the case of dispersions, and using a surfactant. In addition, the pharmaceutical composition of the present invention may also include adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. It includes various antibiotics and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc., to prevent microbial activity. In addition, isotonic agents, such as sugar and sodium chloride, may preferably be included in the composition.

In addition, the injectable pharmaceutical compositions (formulations) of the present invention may include agents which delay absorption, for example, aluminum monostearate and gelatin, thereby prolonging absorption.

The formulations comprising the pharmaceutical compositions of the present invention can be provided orally, parenterally, topically or rectally. Of course, this is provided in a form suitable for each route of administration. For example, by injection or inhalation, or injection, insufflation or inhalation of eye drops, ointments, suppositories, etc., in tablet or capsule form; topical use of lotions or ointments; and rectally administered via suppositories. Preferably, it can be administered orally and/or intravenously, or by inhalation (e.g., liposomes, nanoformulations, etc.).

The pharmaceutical composition of the present invention can be used by various methods acceptable in the art, and as an example, can be used by the mode of application except intestinal and topical application, usually parenteral application, which generally refers to the mode of application by injection or parenteral application. That is, including intravenous, intramuscular, intraarterial, intradural, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion, but the present invention is not limited thereto. Similarly, any number of applications and any dosage are all possible, as long as they are in various levels acceptable in the art.

Furthermore, the pharmaceutical compositions (formulations) of the present invention may be used in combination with other agonists, for example, as an additional antibiotic with or without the compounds of the present invention, for treating a bacterial infection in a subject.

Antibiotics include antibiotics, biocides, antimicrobial agents and bacteriostatic agents, and any antibiotics, biocides, antimicrobial agents and bacteriostatic agents are possible as long as they are of known types.

In addition, the pharmaceutical composition of the present invention can be formulated so that it can be administered separately from any additional agonists, and can be formulated together so that they are all administered at once. For example, the pharmaceutical composition of the present invention is formulated into one dosage form, and then another dosage form can be formulated with additional agonists. Any separate dosage form can be administered simultaneously or at different times.

As another approach, the pharmaceutical composition of the present invention may include an additional agonist described in the present invention, and each component may be provided as a separate composition, a composite composition, or a single composition.

Another embodiment of the present invention provides an anti-inflammatory composition including a micrococcin compound represented by Chemical Formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a salt thereof as an effective ingredient.

Preferably, the micrococcin compound according to an exemplary embodiment may be represented by Chemical Formula 2.

The term "anti-inflammatory" refers to the prevention or treatment of inflammation. Here, inflammation refers to diseases caused by invasion of external infection sources (bacteria, molds, viruses, various types of allergens, etc.), including atopic dermatitis, allergic dermatitis, inflammatory bowel disease, gastric ulcer, asthma, etc., but is not particularly limited thereto.

In the above, the salt of the micrococcin compound may be in the form of a pharmaceutically, cosmetically or nutritionally acceptable salt. As the salt, an acid addition salt formed from a pharmaceutically, cosmetically or nutritionally acceptable free acid or a metal salt formed from an alkali may be used, and the salt may be as described above.

The anti-inflammatory composition of the present invention includes the micrococcin compound represented by Chemical Formula 1, thereby showing an anti-inflammatory effect improved by inflammatory cytokine inhibitory activity such as IL-6 and IL-1β.

In addition, the present invention provides a method for preparing the micrococcin compound of the present invention, and the method for preparing the micrococcin compound of the present invention comprises:

subjecting the compound of the following Chemical Formula 3 to a borylation reaction to prepare a compound of the following Chemical Formula 4; and

The compound of the following Chemical Formula 4 is coupled with the compound of the following Chemical Formula 5 to prepare the micrococcin compound represented by the following Chemical Formula 1:

[Chemical formula 1]

[Chemical formula 3]

[Chemical formula 4]

[Chemical formula 5]

(In Chemical Formulas 1 and 3 to 5,

_X1 and _X2 are independently halogen.

The method for preparing the micrococcin compound of Chemical Formula 1 according to the present invention uses the compound of Chemical Formula 3, which is a compound into which a halogen functional group is introduced, thereby easily replacing the halogen group of Chemical Formula 3 with a boronic acid functional group, followed by an aryl-aryl coupling reaction to prepare the micrococcin compound of Chemical Formula 1 in high yield under mild conditions.

As the aryl-aryl coupling, for example, Moto coupling, Suzuki coupling, Negishi coupling, Stille coupling, Sonogashira coupling, Heck coupling or Buchwald coupling may be suitable, but the Suzuki coupling reaction is particularly preferred.

According to an exemplary embodiment, the boration reaction can be carried out using a boronic acid precursor, which can be any organic boronic acid compound, which is a compound that can introduce a boronic acid or boronic acid derivative group by reacting with Chemical Formula ₃ in the presence of a catalyst, and for example, can be bis(pinacolato)diboron( _B2pin2 ), bis(catecholato)diborane( _B2Cat2 ) _, and the like.

According to an exemplary embodiment, the aryl-aryl coupling may be preferably performed by a Suzuki coupling reaction under a transition metal catalyst.

As the transition metal catalyst, a Pd(0) complex or a Pd(II) salt can be used. A preferred Pd(0) complex is a Pd(0) complex containing one or more phosphine-based ligands, and the phosphine-based ligand can be triphenylphosphine (Ph ₃ P), tri(o-tolyl)phosphine (Pd(o-Tol) ₄ ), Xphos (2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl), Johnphos (2-(di-tert-butylphosphine)biphenyl), etc. Preferably, as the Pd(0) complex, Pd(Ph ₃ P) ₄ can be used. A preferred Pd(II) salt includes palladium acetate, i.e., Pd(OAc) ₂ , but is not limited thereto. Another preferred Pd(II) salt can be Pd(dtbpf)Cl ₂ .

The Suzuki coupling can be carried out in the presence of sodium carbonate, potassium phosphate or an organic base, such as tetraethylammonium carbonate.

Preferably, according to an exemplary embodiment, the compound of Chemical Formula 3 can be prepared by the following steps: reacting a compound of the following Chemical Formula 3-1 with a compound of the following Chemical Formula 3-2 to prepare a compound of the following Chemical Formula 3-3; and subjecting the compound of Chemical Formula 3-3 to deprotection and intra-ring reaction, thereby preparing the compound of Chemical Formula 3:

[Chemical formula 3-1]

[Chemical formula 3-2]

[Chemical formula 3-3]

(In the above chemical formulas 3-1 to 3-3,

R ₁ is independently C1-C5 alkyl;

_P1 is a protecting group; and

_X1 is halogen.)

According to an exemplary embodiment, deprotection is to release a blocking group, and this blocking group can be any blocking group that can be used by those skilled in the art of the present invention, and blocking group of the present invention is a hydroxyl blocking group or an amine blocking group.Certainly, the ring reaction according to an exemplary embodiment of the present invention can also be carried out by the method used in the field of organic synthesis.

In a specific example, _P1 can be selected from tert-butyloxycarbonyl (Boc), carbonyl benzyloxy (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), toluenesulfonyl (Ts), 2,2,2-trichloroethoxycarbonyl (Troc), 2-trimethylsilylethoxycarbonyl (Teoc) and aryloxycarbonyl (Alloc).

According to an exemplary embodiment, the compound of Chemical Formula 3-1 can be prepared by the following steps: reacting the compound of the following Chemical Formula 2-1 with ammonium acetate to prepare the compound of the following Chemical Formula 2-2; reacting the compound of Chemical Formula 2-2 with the compound of the following Chemical Formula 2-3 to prepare the compound of the following Chemical Formula 2-4; and reacting the compound of Chemical Formula 2-4 with cysteine to prepare the compound of Chemical Formula 3-1.

[Chemical formula 2-1]

[Chemical formula 2-2]

[Chemical formula 2-3]

[Chemical formula 2-4]

(In the above chemical formulas 2-1 to 2-4,

P1 is a protecting group; and

X1 is halogen. )

According to an exemplary embodiment, the reaction time in the method for preparing the micrococcin compound of Chemical Formula 1 may vary according to the reaction materials, the type of solvent, and the amount of solvent, and as an example, the reaction is completed after confirming that the starting material is completely consumed by TLC, etc. After the reaction is completed, the solvent is removed by distillation under reduced pressure, or after concentration under reduced pressure, it can be separated and purified by a common method such as tube chromatography.

Hereinafter, the method for preparing the micrococcin compound of the present invention will be described with specific examples, but the present invention is not limited by the specific examples.

[Preparation Example 1] Preparation of Compound A1

Step 1: Preparation of compound a-1

N-Boc-L-threonine (2 g, 9.1 mmol) and (R)-2-((tert-butyldiphenylsilyl)oxy)propane-1-amine (3.45 g, 11 mmol, 1.2 equivalents) were dissolved in dichloromethane (40 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 2.1 g, 11 mmol, 1.2 equivalents), hydroxybenzotriazole (HOBt, 1.48 g, 11 mmol, 1.2 equivalents) and diisopropylethylamine (DIPEA, 3.17 ml, 18.2 mmol, 2 equivalents were added dropwise), and stirred at room temperature for 2 hours. Diluted with dichloromethane and washed with water. The organic solvent was extracted, dehydrated with anhydrous sodium sulfate, and separated by column chromatography with 20%-40% ethyl acetate/hexane solution to obtain white solid compound a-1 (2.80 g, 60%).

¹ H NMR (600MHz, CDCl ₃ ): δ7.69(m,4H),7.49-7.44(m,2H),7.43-7.39(m,4H),6.89(brs,1H),5.47(d,J＝ 7.9Hz,1H),4.34(qd,J＝6.4,2.0Hz,1H),4.05-3.97(m,2H),3.34-3.30(m,1H),3.24(dt,J＝13.5,4.5Hz,1H ),2.87(brs,1H),1.48(s,9H),1.18(d,J＝6.4Hz,3H),1.10(s,9H),1.06(d,J＝6.2Hz,3H); LCMS:515.2 (M+H ⁺ ) applies to C ₂₈ H ₄₃ N ₂ O ₅ Si.

Step 2: Preparation of compound a-2

Compound a-1 (4.50 g, 8.8 mmol, 1.1 equivalents) was dissolved in dichloromethane (40 mL)/trifluoroacetic acid (40 mL), stirred at room temperature for 1 hour, and concentrated under reduced pressure. The obtained residue was dissolved in dichloromethane (20 mL), 2-bromothiazole-4-carboxylic acid (1.66 g, 8 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (1.68 g, 8.8 mmol, 1.1 equivalents), hydroxybenzotriazole (1.19 g, 8.8 mol, 1.1 equivalents) and diisopropylethylamine (4.2 mL, 24 mmol, 3 equivalents) were added dropwise, and then stirred at room temperature for 12 hours. Diluted with dichloromethane, then washed with water. The organic solvent was extracted, dehydrated with anhydrous sodium sulfate, and separated by column chromatography with 20%-50% ethyl acetate/hexane solution to obtain yellow solid compound a-2 (3.00 g, 63%).

¹ H NMR (600 MHz, CDCl ₃ )δ8.08(s,1H),8.01(d,J＝8.4Hz,1H),7.69-7.65(m,4H),7.48-7.44(m,2H),7.42-7.37(m,4H),6.92-6.90(m,1H),4.44(qd,J＝6.5,1.9Hz,1H),4.39 (dd,J＝8.4,1.9Hz,1H),4.02–3.97(m,1H),3.55(brs,1H),3.29(app.t,J＝5.3Hz,2H),1.22(d,J＝6.5Hz,3H),1.05(s,9H),1.03(d,J＝6.5Hz,3H); LCMS:604.1( M+H ⁺ ) for C ₂₇ H _{35BrN3O4SSi . ​}

Step 3: Preparation of compound a-3

Compound a-2 (3g, 4.96mmol) was dissolved in dichloromethane (40mL), and methanesulfonyl chloride (MsCl, 1.15mL, 14.9mmol, 3 equivalents) and triethylamine (2.1mL, 14.9mmol, 3 equivalents) were added dropwise, and stirred at room temperature for 1 hour. Then, 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU, 11.1ml, 74.4mmol, 15 equivalents) was added and stirred at room temperature for 2 hours. After confirming that the reaction was complete, it was diluted with dichloromethane and washed with supersaturated ammonium chloride. The organic solvent was extracted, dehydrated with anhydrous sodium sulfate, and separated by column chromatography with 30%-50% ethyl acetate/hexane solution to obtain white solid compound a-1 (2.2g, 75%).

¹ H NMR (600MHz, CDCl ₃ ): δ8.32(s,1H),8.12(s,1H),7.66-7.64(m,4H),7.48-7.41(m,2H),7.40-7.37(m, 4H),6.53(q,J＝7.1Hz,1H),6.39(app.t,J＝5.3Hz,1H),4.05–4.01(m,1H),3.41(ddd,J＝13.6,5.4,3.6Hz ,1H),3.33(dt,J＝13.6,5.9Hz,1H),1.79(d,J＝7.1,3H),1.10(d,J＝6.2Hz,3H),0.99(s,9H); LCMS: 586.1(M+H ⁺ ) applies to C ₂₇ H ₃₃ BrN ₃ O ₃ SSi.

Step 4: Preparation of compound a-4

Compound a-3 (2.2 g, 3.74 mmol) was dissolved in tetrahydrofuran (30 mL), 1 M tetra-n-butylammonium fluoride (TBAF, 6.74 mL, 6.74 mmol, 1.8 equivalents) was added dropwise, and then stirred at room temperature for 30 minutes. Diluted with ethyl acetate, and washed with supersaturated ammonium chloride. The organic solvent was extracted, dehydrated with anhydrous sodium sulfate, and separated by column chromatography with (2%-5% methanol: 98%-95% dichloromethane) solution to obtain colorless liquid compound a-4 (800 mg, 61%).

¹ H NMR (600 MHz, CD ₃ OD): δ 8.30 (d, J = 0.9 Hz, 1H), 6.71 (q, J = 7.1 Hz, 1H), 3.94-3.85 (m, 1H), 3.30 (dd, J = 13.5, 4.7 Hz, 1H), 3.20 (dd, J = 13.4, 7.1 Hz, 1H), 1.79 (d, J = 7.1 Hz, 3H), 1.16 (d, J = 6.3 Hz, 3H); LCMS: 348.0 (M+H ⁺ ) for C ₁₁ H ₁₅ BrN ₃ O ₃ S.

Step 5: Preparation of Compound A1

Compound a-4 (386 mg, 1.12 mmol) was dissolved in dichloromethane (10 mL), and then Dess-Martin periodinane (706 mg, 1.66 mmol, 1.5 equivalents) was added dropwise. Stirred at room temperature for 2 hours, diluted with dichloromethane, and washed with supersaturated sodium bicarbonate. The organic solvent was extracted, dehydrated with anhydrous sodium sulfate, and separated by column chromatography with a (2%-5% methanol: 98%-95% dichloromethane) solution to obtain a white solid compound A1 (356 mg, 89%).

¹ H NMR (600 MHz, CD ₃ OD): δ 8.30 (s, 1H), 6.77 (q, J=7.1 Hz, 1H), 4.07 (s, 2H), 2.18 (s, 3H), 1.81 (d, J=7.1 Hz, 3H); LCMS: 345.9 (M+H ⁺ ) for C ₁₁ H ₁₃ BrN ₃ O ₃ S.

[Example 1] Preparation of Compound 13 (Micrococcin P2)

Step 1: Preparation of compound 3

4-bromo-2-formylthiazole (768mg, 4mmol) is dissolved in tetrahydrofuran (10mL), and ethynylmagnesium bromide solution (0.5M in THF solution, 16mL, 8mmol) is slowly added dropwise at 0°C. Stir at room temperature for 30 minutes, add supersaturated ammonium chloride solution to stop the reaction. Extract with ethyl acetate, then dehydrate with anhydrous sodium sulfate, and concentrate under reduced pressure. Use column chromatography to separate the obtained residue with 30% ethyl acetate/70% hexane solution to obtain a white solid compound. The obtained compound (632mg, 2.9mmol) is dissolved in dichloromethane (30mL), manganese dioxide (2.52g, 29mmol) is added, and then stirred at room temperature for 2 hours. Use diatomaceous earth to filter the manganese residue, extract with dichloromethane and dehydrate with anhydrous sodium sulfate, then concentrate under reduced pressure to obtain brown solid compound 3 (527mg, 61%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.69 (s, 1H), 3.67 (s, 1H); LCMS: 215.9 (M+H ⁺ ) for C ₆ H ₃ BrNOS.

Step 2: Preparation of compound 5

Acetonitrile (6.52mL, 125mmol, 2 equivalents) was dissolved in tetrahydrofuran (120mL), and then n-butyl lithium solution (2.5M in hexane solution, 54.8mL, 137mmol, 2.2 equivalents) was slowly added dropwise at -78°C. Stir at -78°C for 20 minutes, and then compound 4 (22.3g, 62.6mmol) synthesized by the method disclosed in Org.Lett.2020,22,6,2365-2370 was slowly added dropwise at the same temperature and dissolved in tetrahydrofuran (120mL). The temperature was slowly raised to room temperature, reacted for 1 hour, and hydrochloric acid at a concentration of 1M was added to stop the reaction. It was diluted with ethyl acetate, and hydrochloric acid was further added to acidify to a pH of 3. The organic solvent was extracted, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a brown solid compound 5 (22.2g, 97%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.29 (s, 1H), 4.71 (d, J=17.7 Hz, 1H), 4.32-4.09 (m, 3H), 1.70 (broad peak, 6H), 1.54-1.38 (m, 7H), 1.31-1.06 (m, 5H); LCMS: 366.1 (M+H ⁺ ) for C ₁₇ H ₂₄ N ₃ O ₄ S.

Step 3: Preparation of compound 6

Compound 5 (22.2 g, 61.0 mmol) was dissolved in a mixture of toluene (300 mL) and acetic acid (60 mL), and ammonium acetate (47 g, 610 mmol, 10 equivalents) was added dropwise. Stirred at 120 ° C for 1 hour, and slowly cooled to room temperature. Diluted with ethyl acetate, then washed with supersaturated sodium bicarbonate solution and water. The organic solvent was extracted, and dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain yellow oily compound 6 (21.0 g, 94%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.60 (s, 1H), 5.59-5.51 (m, 2H), 4.81-4.50 (m, 2H), 4.21-4.10 (m, 1H), 1.67 (s, 6H), 1.48-1.36 (m, 6H), 1.27-0.96 (m, 6H); LCMS: 365.1 (M+H ⁺ ) for C ₁₇ H ₂₅ N ₄ O ₃ S.

Step 4: Preparation of compound 7

Compound 6 (21.0g, 57.5mmol) is dissolved in ethanol (300ml), compound 3 (22.9g, 106.3mmol, 1.85 equivalents) is added dropwise, and stirred at 60 ℃ for 90 minutes. After confirming the disappearance of the starting material with TLC and LC quality, acetic acid (30% volume, 90ml) is added, and then reacted at 90 ℃ for 16 hours. Concentrated under reduced pressure to remove ethanol and acetic acid, diluted with dichloromethane, and terminated with supersaturated sodium bicarbonate solution. Extract organic solvent, dehydrate with anhydrous sodium sulfate, and concentrate under reduced pressure. Use column chromatography to separate with 5%-15% ethyl acetate/hexane solution to obtain yellow solid compound 7 (16.5g, 51%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.27 (s, 1H), 8.19 (s, 2H), 7.48 (s, 1H), 4.85 (d, J=7.3 Hz, 1H), 4.58-4.23 (broad peak, 1H), 1.83-1.67 (broad peak, 6H), 1.57-1.45 (m, 6H), 1.33-1.20 (m, 6H); LCMS: 562.0 (M+H ⁺ ) for C ₂₃ H ₂₅ BrN ₅ O ₃ S ₂ .

Step 5: Preparation of compound 9

Compound 7 (1.42 g, 2.53 mmol) was dissolved in a mixed solution of ethanol (27 mL), sodium phosphate buffer solution (pH 7, 9 mL) and water (4.5 mL), and then L-cysteine (3.06 g, 25.3 mmol, 10 equivalents) was added dropwise. Stirred at 100 ° C for 24 hours, concentrated under reduced pressure to remove ethanol, and diluted with ethyl acetate. Washed with 1 M hydrochloric acid to remove excessive cysteine, and dehydrated with anhydrous sodium sulfate. The solution obtained by extraction was concentrated under reduced pressure to obtain the thiazoline of brown oil.

The brown oil thiazoline (1.68 g, 2.53 mmol) was dissolved in dichloromethane (30 mL), bromotrichloromethane (1.0 mL, 10.12 mmol, 4 equivalents) was added, and then 1,8-diazabicyclo[5,4,0]undec-7-ene (1.88 mL, 12.65 mmol, 5 equivalents) was slowly added dropwise. Stir at 60 ° C for 4 hours, and 1M hydrochloric acid was added to terminate the reaction. Extract with dichloromethane, and then wash with water and brine. The extracted organic solvent was dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography with 5%-15% methanol/dichloromethane solution to obtain brown solid compound 9 (1.05 g, 62%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.35–8.05 (m, 3H), 7.89 (bs, 1H), 7.33 (s, 1H), 4.49–4.40 (m, 1H), 4.09–3.97 (m, 1H), 1.71–1.29 (m, 10H), 1.26–1.08 (m, 8H); LCMS: 664.0 (M+H ⁺ ) for C ₂₆ H ₂₇ BrN ₅ O ₅ S ₃ .

Step 6: Preparation of compound 10

Compound B1 was prepared according to Org.Lett.2020,22,6,2365-2370. Compound 9 (1.05 g, 1.57 mmol) was dissolved in acetonitrile (8 mL), compound B1 (932 mg, 1.88 mmol), 1-methylimidazole (0.376 mL, 4.71 mmol, 3 equivalents) and chloro-N, N, N, N-tetramethylformamidine hexafluorophosphate (TCFH, 0.528 g, 1.88 mmmol, 1.2 equivalents) were added, and stirred at room temperature for 30 minutes. The solution was diluted with ethyl acetate and washed with water, the organic layer was dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated solution was separated using column chromatography with (40%-90% ethyl acetate: 60%-10% hexane) solution to obtain white solid compound 10 (1.05 g, 60%).

¹ H NMR (400MHz, CDCl ₃ ) δ8.69 (s, 1H), 8.44 (d, J = 9.5Hz, 1H), 8.35-8.24 (m, 4H), 8.24-8.17 (m, 1H), 8.06 ( s,1H),7.96(s,1H),7.94-7.87(m,1H),6.47(q,7.1Hz,1H),5.41-5.31(m,1H),4.88(d,J＝5.3Hz,1H ),4.78(bs,1H),4.68-4.58(m,1H) ,4.58-4.46(m,1H),4.16-3.98(m,1H),3.86(s,3H),2.49-2.45(m,1H),1.87(d,J＝7.1Hz,3H),1.69-1.48 (m, 6H), 1.45-1.32 (m, 3H), 1.31-1.08 (m, 12H), 1.03 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.7 Hz, 3H); LCMS: 1127.1 (M+H ⁺ ) is applicable to C ₄₆ H ₅₂ BrN ₁₀ O ₉ S ₅ .

Step 7: Preparation of compound 11

Compound 10 (200mg, 0.177mmol) is added to a mixed solvent of tetrahydrofuran (1mL)/water (1mL), lithium hydroxide monohydrate (22mg, 0.531mmol, 3 equivalents) is added, and stirred at room temperature for 2 hours. The solution is diluted with a dichloromethane solution and washed with a 1M hydrochloric acid solution, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a carboxylic acid. The concentrated solution is added to a mixed solvent of dichloromethane (1mL)/trifluoroacetic acid (1mL), stirred at room temperature for 30 minutes, and then concentrated under reduced pressure to obtain trifluoroacetate. The concentrated solution is diluted with DMF (1mL), diphenylphosphoryl azide (DPPA, 0.038mL, 0.177mmol, 1 equivalent) and sodium bicarbonate (0.148g, 1.77mmol, 10 equivalents) are added, and stirred at room temperature for 12 hours. The solution was diluted with dichloromethane solution, and the organic layer was washed three times with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography using (2%-5% methanol: 98%-95% dichloromethane) solution to synthesize yellow solid compound 11 (96 mg, 56%).

¹ H NMR (400MHz, CDCl ₃ ) δ8.77 (s, 1H), 8.39 (d, J = 9.6Hz, 1H), 8.22-8.18 (m, 2H), 8.16-8.09 (m, 3H), 8.01- 7.92(m,3H),7.40(s,1H),6.39(q,J＝7.0Hz,1H),5.27-5.18(m,2H),4.92(d,J＝7.8Hz,1H),4.67(s ,1H),4.47-4.33 (m, 1H), 2.95 (bs, 1H), 2.48 (dq, J = 13.4, 6.6 Hz, 1H), 2.22 (bs, 1H), 1.81 (d, J = 7.0 Hz, 3H), 1.57 (d, J＝5.2Hz, 3H), 1.19(d, J＝6.5Hz, 3H), 1.14(d, J＝6.7Hz, 3H), 0.95(d, J＝6.6Hz, 3H); LCMS: 955.0(M+ H ⁺ ) is applicable to C ₃₇ H ₃₆ BrN ₁₀ O ₆ S ₅ .

Step 8: Preparation of compound 12

Compound 11 (41 mg, 0.043 mmol) was dissolved in 1,4-dioxane (0.6 mL), and diboric acid pinacol ester (B ₂ Pin ₂ , 14.2 mg, 0.056 mmol, 1.3 equivalents), potassium acetate (KOAc, 6.7 mg, 0.065 mmol, 1.5 equivalents) and Xphos (3.1 mg, 15 mol%) were added. After addition, nitrogen was added for 1 minute, and tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ , 4 mg, 10 mol%) was added dropwise. The mixture was stirred at 90° C. for 3 hours, diluted with dichloromethane, and the organic solvent was extracted. The extracted organic solvent was washed with water, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain solid compound 12 (18 mg, 45%).

¹ H NMR (400MHz, MeOD) δ8.40–8.21(m,4H),8.13(s,1H),7.93(s,1H),7.76(s,1H),6.61(q,J＝6.9Hz,1H ),5.29–5.19(m,2H),4.75(d,J＝3.1Hz,1H),4.62–4.50(m,1H),4.30(dd,J＝6 .3,2.9Hz,1H),2.64(dq,J＝13.4,6.7Hz,1H),1.85(d,J＝7.0Hz,3H),1.53(d,J＝6.4Hz,3H),1.19(d ,J＝6.3Hz,3H),1.13(d,J＝6.7Hz,3H),0.99(d,J＝6.7Hz,3H); LCMS:921.1(M+H ⁺ ) is suitable for C ₃₇ H ₃₈ BN ₁₀ O ₈ S ₅ .

Step 9: Preparation of compound 13

Compound 12 (276mg, 0.3mmol) is dissolved in a mixture of tetrahydrofuran (6mL) and water (1.5mL). Compound A1 (155mg, 0.45mmol, 1.5 equivalents) is added dropwise, and nitrogen is passed through for 3 minutes. Then, potassium carbonate (124mg, 0.9mmol, 3 equivalents) and tetrakis (triphenylphosphine) palladium (Pd(PPh3)4, 10mol%, 35mg) are added, and nitrogen is passed through again for 1 minute. Stir at 38°C for 3 hours, and concentrate under reduced pressure. Use column chromatography to separate the residue obtained with 0-5% methanol/dichloromethane solution to obtain white solid compound 13 (179mg, 55%).

¹ H NMR(600MHz,MeOD)δ8.40–8.31(m,4H),8.27(s,1H),8.22(s,1H),8.13(s,1H),7.96(s,1H),6.82(q ,J＝7.1Hz,1H),6.60(q,J＝6.8Hz,1H),5.24(d,J＝2.9Hz,1H),5.22(d,J＝8.8Hz,1H),4.78(d,J ＝3.1Hz,1H),4.62–4.55(m,1H),4.30(qd,J＝6.3,2. 8Hz,1H),4.10(d,J＝13.0Hz,1H),2.63(dq,J＝13.4,6.7Hz,1H),2.19(s,3H),2.20–2.14(m,1H),1.92(s ,1H)1.86(app.d,J＝7.0Hz,6H),1.55(d,J＝6.4Hz,3H),1.18(d,J＝6.3Hz,3H),1.13(d,J＝6.7Hz, 3H), 0.99 (d, J=6.7 Hz, 3H); LCMS: 1142.2 (M+H ⁺ ) for C ₄₈ H ₄₈ N ₁₃ O ₉ S ₆ .

[Example 2] Antibacterial activity test against tuberculosis (M. tuberculosis)

The micrococcin compound prepared in Example 1 of the present invention (Compound 13; Micrococcin P2) was used to measure the antibacterial activity.

Liquid culture medium for tuberculosis culture was obtained by adding 10% OADC (oleic acid, albumin, glucose, catalase; Becton Dickinson) and 0.05% Tween 80 (Sigma Aldrich) to Middlebrook 7H9 medium (Becton Dickinson), and solid culture medium was obtained by adding 10% OADC to Middlebrook 7H10 medium (Becton Dickinson). Resistant strains including standard tuberculosis strains were cultured in Middlebrook 7H9 liquid culture medium, diluted to 4.0×10 ⁵ cells/well, and the solution was distributed into 96-well plates. The compound was treated with 1% DMSO, and 0.025% resazurin (REMA) solution was distributed after 5 days, cultured for 24 hours, and bacterial growth was confirmed. The strains used in the experiment are shown in Table 1 below. As the antibacterial activity, MIC ₅₀ values, which are antibiotic concentrations that inhibit bacterial growth by up to 50% compared with the _control , were measured by resazurin (excitation wavelength: 530 nm, emission wavelength: ₅₉₀ nm) and calculated by Prims 6. The results are shown in Table 2 below.

The antibiotic resistance of the strains used for tuberculosis evaluation in Table 1 below is as follows:

INH: Isoniazid, RIF: Rifampicin, LEV: Levofloxacin, OFX: Ofloxacin, MXF: Moxifloxacin, KM: Kanamycin, AMK: Amikacin, CPM: Capreomycin

[Table 1]

S: Susceptibility, R: Resistance

[Table 2]

As shown in Table 2, the micrococcin compounds of the present invention have greatly improved antibacterial effects on multidrug-resistant or extensively drug-resistant tuberculosis and susceptible tuberculosis, and therefore, can be very useful as therapeutic agents for multidrug-resistant or extensively drug-resistant tuberculosis.

[Example 3] Antibacterial activity experiment against non-tuberculosis mycobacterium (NTM)

The micrococcin compound (Compound 13; Micrococcin P2) prepared in Example 1 of the present invention was used to measure the antibacterial activity against nontuberculous mycobacteria. The strains selected for evaluating the efficacy of Micrococcin P2 against nontuberculous mycobacteria were Mycobacterium avium (M. avium) ATCC 700898, Mycobacterium intracellulare (M. intracellulare) ATCC 13950 and Mycobacterium abscessus (M. abscessus) ATCC 19977.

The culture medium of mycobacterium cultured to late log phase was diluted to 4.0×10 5 cells/well in Middlebrook 7H10 agar medium (Difco) supplemented with 10% OADC (oleic acid, albumin, glucose, catalase; Becton Dickinson) and 0.05% Tween ⁸⁰ (Sigma-Aldrich) for antibacterial activity test and distributed in 96-well plates. The compound was treated with 1% DMSO, and 0.025% resazurin (REMA) solution was distributed and cultured after 3 days, and bacterial growth was confirmed. As antibacterial activity, the MIC ₅₀ value was measured by resazurin (excitation wavelength: 530nm, emission wavelength: 590nm), which is the antibiotic concentration that inhibits bacterial growth by up to 50% compared with the control. The MIC ₅₀ value was calculated by Prism 6, _and the results are shown in Table 3 below.

As control drugs, clarithromycin and rifampicin, which are used for NTM treatment, were used, and all drugs were used in a stepwise concentration gradient of 11 steps starting from 5 μM.

[Table 3]

CLR: clarithromycin, RIF: rifampicin

As shown in Table 3, the micrococcin compound of the present invention has an improved antibacterial effect compared to clarithromycin and rifampicin used for NTM treatment. In particular, it was demonstrated to be effective against Mycobacterium avium and Mycobacterium intracellulare.

[Example 4] Antibacterial activity test against Clostridium difficile

The inhibitory activity against Clostridium difficile was measured by clinical strains assigned by the Korean Collection of Antimicrobial Resistant Microorganisms or strains assigned by the American Type Culture Collection (ATCC). The inhibitory activity of MIC was evaluated by the agar dilution method according to the Clinical and Laboratoru Standards Institute (CLSI) guidelines [Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard—Seventh Edition, CLSI document M11-A7, 2007].

In liquid cultures of C. difficile, 0.5% yeast extract (5 g/1 L, Becton Dickinson) was added to BHI medium (Brain Heart Infusion Medium, Becton Dickinson) in BHIS medium (supplemented Brucella agar) (Becton Dickinson) as agar medium. All tested compounds were made into agar medium to a concentration of 32 μg/ml to 0.06 μg/ml. The day before culturing C. difficile strains in liquid, BHIS medium was added to an anaerobic chamber and oxygen in the liquid medium (broth) was removed for one day. The day before the experiment, bacteria were cultured in 4 ml of BHIS medium. Bacteria that had been cultured for one day were diluted to about 4-5×10 ⁷ CFU/ml using BHIS medium. The diluted bacterial solution was added dropwise to the culture medium containing antibiotics at a rate of 10 μl each time, and cultured in an anaerobic chamber for 18 hours or longer. The results are shown in Table 4 below:

[Table 4]

VAN: vancomycin, MDZ: metronidazole, MXF: moxifloxacin

As shown in Table 4, the micrococcin compound of the present invention has significantly improved antibacterial effect compared with currently used vancomycin, metronidazole and moxifloxacin.

[Example 5] Evaluation of therapeutic efficacy in Clostridium difficile infection model

In order to culture Clostridium difficile, a mixture of SMC medium (bacto peptone: 0.9%, protease: 0.5%, NH ₄ SO ₄ : 0.1%, Tris buffer: 0.15%) and BHIS agar medium at a ratio of 7:3 was used.

C57BL/6J male 4-week-old mice were purchased and experienced a 1-week adaptation period, and 5-6-week-old mice weighing 20-21 g were used as experimental animals for evaluating the efficacy. In order to ensure that mice were easily infected by inducing an imbalance in the intestinal microorganisms of mice, drinking water containing 2% DSS was provided to mice 5 days before infection, and 20 mg/kg clindamycin was injected intraperitoneally to mice one day before infection. That is, drinking water containing 2% DSS (dextrose sodium sulfate, MP Biomedical) was provided for 5 days before infection, and ordinary water without DSS was provided after infection.

As the C. difficile used in the infection, a ribotype 027 clinical isolate was used, the bacteria were cultured in an anaerobic chamber, inoculated in a stock solution 5 days before infection, cultured for 2 days, subcultured, and grown for one day. To form spores for infection, the bacteria were streaked in about 60 BHIS7:3 medium and cultured for two days.

On the day of the experiment, the cultured bacterial liquid in SMC:BHIS agar (7:3) medium was collected with a scraper, and centrifuged at 6000rpm for 15 minutes, the supernatant was discarded, and only the pellet was collected. 0.9% NaCl (normal saline) was added to the collected precipitate (i.e., the thalline collected by centrifugation), and the optical density (OD, Optical Density) value was measured, and the absorbance OD value dilution factor was 1/10, so that 1/10OD ₆₀₀ value was 2.0. Then, it was boiled in a 60°C water bath to retain only spores, and the cooled bacterial liquid was then orally administered to mice to induce intestinal colitis. Then, 30mg/kg of vancomycin (vancomycin), micrococcin P2 (MP2) and fidaxomicin (fidaxomicin) compounds were orally administered once a day for 5 days, and the survival rate of mice was confirmed to last for about 2 weeks. The results are shown in Figure 1.

As shown in FIG1 , in the case of the micrococcin compound of the present invention and the control compound, no individual died due to recurrence of Clostridium difficile (C. difficile) even after administration.

Particularly, the micrococcin compound of the present invention showed a survival rate of 100% and exhibited excellent efficacy compared with vancomycin and fidaxomicin which are currently used.

[Example 6] Evaluation of anti-inflammatory efficacy

In order to confirm the anti-inflammatory effect of the micrococcin compound (Compound 13; Micrococcin P2) prepared in Example 1 of the present invention, the secretion amount of inflammatory cytokines was measured.

1) Cell culture

RAW 264.7 cells are a mouse-derived macrophage cell line distributed in the Korean Cell Line Bank (KCLB) and cultured in DMEM (Dulbeccon's modified Eagle's medium; PAA, Austria) medium, including 1% penicillin-streptomycin, and 10% FBS (fetal bovine serum; Gibco Laboratories). Cells were cultured at 37°C and 5% _CO2 .

2) Determination of inflammatory cytokine secretion

Cultured RAW 264.7 cells were distributed into 24-well plates at a rate of 2×10 ⁵ cells/well, treated with the micrococcin compound prepared in Example 1 (Compound 13; Micrococcin P2) at different concentrations (5, 10, 20, 40 μg/mL), and then treated with 1 μg/mL lipopolysaccharide (LPS), and cultured in an incubator (5% CO ₂ , 37°C) for 24 hours.

The control group was an untreated cell group, and as a negative control group, a cell group treated only with LPS was used.

After the culture was completed, the culture medium of each well was collected (at 4°C, 12,000 rpm, 10 minutes), the supernatant was taken, and the concentration of inflammatory cytokines (IL-6, IL-1β) in the culture medium was quantified using an ELISA kit (R&D Systems, BD). The results are shown in FIG2 .

LPS increased the secretion of inflammatory cytokines IL-6 (interleukin-6) and IL-1β (leukin-1β), but when LPS and the micrococcin compound of the present invention (micrococcin P2) were treated simultaneously, it was confirmed that the secretion of inflammation-related cytokines IL-6 and IL-1β was significantly reduced.

This indicates that the micrococcin compound (micrococcin P2) of the present invention exhibits anti-inflammatory effects by inhibiting the production of inflammatory cytokines, and can therefore be used as a drug, functional food, and cosmetic material for treating, preventing, and improving inflammation-related diseases.

Claims (19)

1. A pharmaceutical composition for treating or preventing a bacterial infection caused by mycobacterium tuberculosis or non-mycobacterium tuberculosis, comprising: a micrococcum compound represented by the following chemical formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[ chemical formula 1]

2. The pharmaceutical composition of claim 1, wherein the micrococcum compound is represented by the following chemical formula 2:

[ chemical formula 2]

3. The pharmaceutical composition of claim 1, wherein the bacterial infection is tuberculosis or a symptom caused by the non-tubercular mycobacterium.

4. The pharmaceutical composition of claim 3, wherein the nontuberculous mycobacteria is selected from the group consisting of mycobacterium avium (Mycobacterium avium), mycobacterium intracellulare (Mycobacterium intracellulare), mycobacterium abscessus (Mycobacterium abscessus), mycobacterium kansasii (Mycobacterium kansasii), mycobacterium fortuitum (Mycobacterium fortuitum), mycobacterium gobiensis (Mycobacterium gordonae), mycobacterium oslo (Mycobacterium osloensis), mycobacterium phlei (Mycobacterium phlei), mycobacterium smegmatis (Mycobacterium smegmatis), mycobacterium terrestris (Mycobacterium terrae), mycobacterium tortoise (Mycobacterium chelonae), mycobacterium mucii (Mycobacterium mucogenicum), mycobacterium hairline (Mycobacterium peregrinum), mycobacterium monkey (Mycobacterium simiae), mycobacterium waresky (Mycobacterium wolinskyi), or a combination thereof.

5. The pharmaceutical composition of claim 3, wherein the tuberculosis is susceptible tuberculosis, multi-drug resistant tuberculosis, or extensively drug resistant tuberculosis.

6. The pharmaceutical composition of claim 1, further comprising an antibiotic.

7. The pharmaceutical composition of claim 6, wherein the antibiotic is isoniazid (isoniazid), rifampicin (rifampicin), ethambutol (ethambutol), SQ-109, pyrazinamide (pyrazonamide), streptomycin (streptomycin), kanamycin (kanamycin), calicheamicin (calicheamicin), ethionamide (ethionamide), prothioisoniazid (prothionamide), envitamin (envimycin), para-aminosalicylic acid (para-aminosalicylic acid), cycloserine (cyclomycin), amikacin (amikacin), levofloxacin (levofloxacin), moxifloxacin (Gatifloxacin), thioxamine (thioazamycin), ethionamide (ethionamide), propionicide (prothionamide), propionicide (trimethamide), or combinations thereof.

8. A pharmaceutical composition for treating or preventing a bacterial infection caused by clostridium comprising: a micrococcum compound represented by the following chemical formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[ chemical formula 1]

9. The pharmaceutical composition of claim 8, wherein the micrococcum compound is represented by the following chemical formula 2:

[ chemical formula 2]

10. The pharmaceutical composition of claim 8, wherein the bacterial infection is selected from the group consisting of colitis, diarrhea, pseudomembranous colitis, gangrene enteritis, skin and soft tissue infection, food poisoning, or a combination thereof.

11. The pharmaceutical composition of claim 8, wherein the clostridium is selected from clostridium difficile (Clostridium difficile), clostridium perfringens (Clostridium perfringens), clostridium cadavermitilis (Clostridium cadaveris), clostridium innoccum (clostridium perfringens), clostridium tetani (Clostridium tetani), clostridium bifidum (Clostridium bifermentans), clostridium histolyticum (Clostridium histolyticum), clostridium (Clostridium clostridioforme), clostridium subterminale (Clostridium subterminale), clostridium bifidum (Clostridium ramosum), clostridium septicatum (Clostridium septicum), and combinations thereof.

12. The pharmaceutical composition of claim 8, further comprising one or two or more adjuvants selected from vancomycin (vancomycin), metronidazole (metronidazole), fidaxomicin (fidaxomicin), ridalizole (ridinizole), atomab (actoxumab), bei Luotuo mab (bezlotoxab), probiotics, prebiotics (pre-biological), intravenous immunoglobulins, and antidiarrheal.

13. The pharmaceutical composition of claim 1 or 8, further comprising a pharmaceutically acceptable carrier.

14. The pharmaceutical composition according to claim 1 or 8, wherein the micrococcum compound, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof is included in an amount of 0.001 to 10wt% relative to the total weight of the pharmaceutical composition.

15. A method of preparing a micrococcum compound, the method comprising:

subjecting a compound of the following chemical formula 3 to a boration reaction to prepare a compound of the following chemical formula 4; and

coupling the compound of the following chemical formula 4 with the compound of the following chemical formula 5 to prepare the micrococcum compound represented by the following chemical formula 1:

[ chemical formula 1]

[ chemical formula 3]

[ chemical formula 4]

[ chemical formula 5]

Wherein the method comprises the steps of

X ₁ And X ₂ Independently of each other, halogen.

16. The method for preparing a micrococcum compound of claim 15 wherein the compound of formula 3 is prepared by comprising the steps of:

reacting a compound of the following chemical formula 3-1 with a compound of the following chemical formula 3-2 to prepare a compound represented by the following chemical formula 3-3:

subjecting the compound of formula 3-3 to deprotection and ring-opening reactions to prepare the compound of formula 3:

[ chemical formula 3-1]

[ chemical formula 3-2]

[ chemical formula 3-3]

In the above chemical formulas 3-1 to 3-3, R ₁ Are independently of one another C1-C5-alkyl;

P ₁ is a protecting group; and

X ₁ is halogen.

17. The method for preparing a micrococcum compound according to claim 16 wherein the compound of formula 3-1 is prepared by comprising the steps of:

reacting a compound of the following chemical formula 2-1 with ammonium acetate to prepare a compound of the following chemical formula 2-2;

reacting a compound of the following chemical formula 2-2 with a compound of the following chemical formula 2-3 to prepare a micrococcum compound represented by the following chemical formula 2-4: and

reacting the compound of chemical formula 2-4 with cysteine to prepare the compound of chemical formula 3-1:

[ chemical formula 2-1]

[ chemical formula 2-2]

[ chemical formulas 2-3]

[ chemical formulas 2-4]

In the above chemical formulas 2-1 to 2-4,

P ₁ is a protecting group; and

X ₁ is halogen.

18. An anti-inflammatory composition comprising: a micrococcum compound represented by the following chemical formula 1, a solvate thereof, a hydrate thereof, a prodrug thereof, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[ chemical formula 1]

19. The anti-inflammatory composition of claim 18, wherein the micrococcum compound is represented by the following chemical formula 2:

[ chemical formula 2]