A new polymyxin analog having excellent antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and an antibacterial composition comprising the same

Abstract

The present invention relates to a polymyxin analog having excellent antibacterial effects against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and to a composition for treating infectious diseases and for antibacterial purposes containing the same. More specifically, the invention relates to a cyclized polymyxin analog having excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and to an antibacterial composition containing the same. The cyclized polymyxin analog compound according to the present invention is represented by the following chemical formula I. [Chemical Formula I] The polymyxin analog compound of the present invention exhibits excellent antibacterial effects against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and since it has excellent in vivo stability when applied to the human body, it is highly useful in related industrial fields such as pharmaceuticals, cosmetics, and health functional foods.

Description

Technology Field

[0001] The present invention relates to a novel polymyxin analog having excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and an antibacterial composition containing the same. More specifically, the invention relates to a cyclized polymyxin analog having excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and an antibacterial composition containing the same. Background Technology

[0002] Peptides are core biomaterials that serve as the functional minimum units of proteins, consisting of 2 to 50 or fewer amino acids. They are widely utilized as key raw materials in pharmaceuticals, food, and cosmetics because they exhibit excellent efficacy even in small quantities and are non-toxic. In particular, the usage of peptide cosmetic materials, which are safe for the human body and highly effective, is increasing day by day, and the development of new materials is considered very important in this industry, with benefits such as import substitution.

[0003] Polymyxin is a lipopeptide antibiotic that exhibits excellent therapeutic effects against Gram-negative bacterial infections. Although it was discovered over 60 years ago, it has been used restrictively due to its toxicity to humans (nephrotoxicity and neurotoxicity) despite its excellent bactericidal power. Recently, however, it has been receiving renewed attention, and resistant strains are emerging due to long-term use.

[0004] Despite this toxicity, due to its excellent antibacterial effect, various attempts are underway to develop it as a new treatment by overcoming the drug's shortcomings through sequence and structural modifications based on this chemical structure.

[0005] In addition, antibiotic-resistant bacteria may emerge in cases of long-term exposure to antibiotics, and antibiotic-resistant diseases refer to infectious diseases caused by bacteria that are resistant to antibiotics, and in particular, multidrug resistance refers to resistance to several types of antibiotics.

[0006] Antibiotic resistance refers to the resistance of bacteria that cause infection to antibiotics, antimicrobial resistance (hereinafter AMR) refers to the resistance of microorganisms that cause infection (including bacteria, parasites such as Maiaria, viruses such as HIV, and fungi such as Candida) to therapeutic agents in a broader sense, and multidrug resistance refers to resistance to multiple types of antibiotics.

[0007] With the recent increase in global mobility, human and economic damage caused by antibiotic resistance is occurring, and the scale of the damage is expected to increase.

[0008] Meanwhile, multidrug-resistant organisms are pathogens resistant to various types of potent antibiotics, such as vancomycin-resistant Staphylococcus aureus. Staphylococcus aureus ), Vancomycin-Resistant Enterococci (Vancomycin-Resistant Enterococcus ), Methicillin-resistant Staphylococcus aureus (Methicillin-Resistant Staphylococcus aureus ), Carbapenem-resistant Enterobacteriaceae (Carbapenem-Resistant Enterobacteriaceae ), Multidrug-resistant Pseudomonas aeruginosa (Multidurg-Resistant Pseudomonas aeruginosa ), multidrug-resistant Acinetobacter (Multidurg-Resistant Acinetobacter baumani ) etc. are typical examples.

[0009] Korean Registered Patent No. 10-1502453 describes various polymyxin derivatives and their antibacterial efficacy, and Korean Registered Patent No. 10-1627139 describes short fatty acid tail polymyxin derivatives and their uses. Additionally, Korean Published Patent No. 10-2014-0091724 describes a method for manufacturing polymyxin sulfates. However, these methods have a completely different structure from the polymyxin analogs mentioned in the present invention, and they have the disadvantage of targeting different Gram-negative bacteria while also exhibiting inferior antibacterial efficacy.

[0010] Accordingly, the inventors confirmed that a new polymyxin analog having a specific sequence and structure exhibits excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria during the process of researching and developing a new material by modifying the existing structure of polymyxin, and thus completed the present invention. The problem to be solved

[0012] The object of the present invention is to provide a polymyxin analog having antimicrobial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0013] Another object of the present invention is to provide an antimicrobial composition comprising, as an active ingredient, a polymyxin analog having antimicrobial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0014] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of infectious diseases comprising, as an active ingredient, a polymyxin analog having antibacterial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0015] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of sepsis comprising, as an active ingredient, a polymyxin analog having antibacterial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0016] Another objective of the present invention is to provide a method for preparing a polymyxin analog having antibacterial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria. means of solving the problem

[0018] To achieve the above objective, the present invention provides a polymyxin analog represented by the following chemical formula I, or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula I]

[0020]

[0021] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0022]

[0024] The present invention also provides an antimicrobial composition comprising a polymyxin analog represented by the formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0025] The present invention also provides a pharmaceutical composition for the prevention or treatment of infectious diseases comprising a polymyxin analog represented by Formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0026] The present invention also provides a pharmaceutical composition for the prevention or treatment of urinary tract infections, pneumonia, and sepsis, comprising a polymyxin analog represented by Formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0027] The present invention also provides a method for preparing a polymyxin analog compound represented by Formula I, comprising the steps of: (a) obtaining a resin-attached peptide represented by Formula Ia by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula Ib; (c) cyclizing the peptide obtained in step (b) using a catalyst by a solution-phase synthesis method to obtain a protected peptide represented by Formula Ic; and (d) performing a deprotection reaction on the peptide in step (c) to obtain a polymyxin analog represented by Formula I.

[0028] [Chemical Formula I]

[0029]

[0030] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0031]

[0033] [Chemical Formula Ia]

[0034]

[0036] [Chemical Formula Ib]

[0037]

[0039] [Chemical Formula Ic]

[0040]

[0041] In the above chemical formula, A is a hydrogen or hydroxyl protecting group, and B is an amine protecting group. Effects of the invention

[0042] The polymyxin analog of the present invention exhibits excellent antibacterial effects against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and since it has excellent in vivo stability when applied to the human body, it is highly useful in related industrial fields such as pharmaceuticals, cosmetics, and health functional foods.

[0043] In addition, the polymyxin analog according to the present invention is not only safe for the human body but also has excellent antibacterial effects and superior stability against proteolytic enzymes, so it can be utilized for various purposes. Brief explanation of the drawing

[0044] FIG. 1 is a synthesis process diagram of polymyxin analog I according to one embodiment of the present invention. Figures 2a, 2b, and 2c show the cytotoxicity of polymyxin analog I according to the present invention on NIH3T3, HEK 293, and RAW246.7 cell lines. Figure 3 is the result of evaluating the hemolytic activity of polymyxin analog I according to the present invention. Figures 4a and 4b show the results of evaluating resistance inhibition of polymyxin analog I according to the present invention. Figures 5a, 5b, and 5c show the results of evaluating the renal cytotoxicity of polymyxin analog I according to the present invention. Specific details for implementing the invention

[0045] Unless otherwise specified herein, abbreviations used to designate amino acids and protecting groups are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry 11:1726-1732(1972); Pure & Appl. Chem., Vol. 56, no. 5, pp. 595-624, 1984).

[0047] The abbreviations for solvents, reagents, protecting groups, and amino acids used in this specification are as follows:

[0048] ACN: Acetonitrile

[0049] t-Bu: tert-butyl

[0050] Cha: Cyclohexylalanine

[0051] DCM: Dichloromethane

[0052] DMSO: Dimethylsulfoxide

[0053] DTT: Dithiolthreitol

[0054] Dab: 2,4-Diaminobutyric acid

[0055] Dap: 2,3-Diaminopropionic acid

[0056] Dde: 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene

[0057] hLeu: Homoleucine

[0058] EDT: 1,2-Ethanedithiol

[0059] His: Histidine

[0060] HPLC: High Performance Liquid Chromatography

[0061] Fmoc: 9-Fluorenyloxycarbonyl

[0062] Lys: Lysine

[0063] 2-Nal: 2-Naphthylalanine

[0064] Nle: Norleucine

[0065] Orn: Ornithine

[0066] TES: Triethylsilane

[0067] TFA: Trifluoroacetic acid

[0068] Thr: Threonine

[0069] TIPS: Triisopropylsilane

[0071] In the process of researching and developing polymyxin having excellent antibacterial activity against Gram-negative bacteria, various polymyxin analogs having specific sequences were prepared, and it was confirmed that they possess excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria.

[0073] Accordingly, in one aspect, the present invention relates to a polymyxin analog represented by the following chemical formula I, or a pharmaceutically acceptable salt thereof.

[0074] [Chemical Formula I]

[0075]

[0076] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0077]

[0079] More specifically, the polymyxin analog represented by the above chemical formula I is a compound represented by the following chemical formulas I-1 to I-21.

[0080] [Chemical Formula I-1]

[0081]

[0082] [Chemical Formula I-2]

[0083]

[0084] [Chemical Formula I-3]

[0085]

[0086] [Chemical Formula I-4]

[0087]

[0088] [Chemical Formula I-5]

[0089]

[0090] [Chemical Formula I-6]

[0091]

[0092] [Chemical Formula I-7]

[0093]

[0095] [Chemical Formula I-8]

[0096]

[0097] [Chemical Formula I-9]

[0098]

[0099] [Chemical Formula I-10]

[0100]

[0101] [Chemical Formula I-11]

[0102]

[0103] [Chemical Formula I-12]

[0104]

[0105] [Chemical Formula I-13]

[0106]

[0107] [Chemical Formula I-14]

[0108]

[0109] [Chemical Formula I-15]

[0110]

[0111] [Chemical Formula I-16]

[0112]

[0113] [Chemical Formula I-17]

[0114]

[0115] [Chemical Formula I-18]

[0116]

[0117] [Chemical Formula I-19]

[0118]

[0119] [Chemical Formula I-20]

[0120]

[0121] [Chemical Formula I-21]

[0122]

[0124] In the present invention, the pharmaceutically acceptable salt of the polymyxin analog represented by the formula I comprises an acid addition salt formed by a pharmaceutically acceptable free acid and a pharmaceutically acceptable metal salt.

[0125] Examples of suitable acids include triluacetic acid, hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, oxalic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc.

[0126] Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium, but are not limited thereto.

[0128] In another aspect, the present invention relates to an antimicrobial composition comprising, as an active ingredient, a polymyxin analog represented by the above formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof.

[0129] The above antimicrobial composition is characterized by having antimicrobial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria.

[0130] In the present invention, "gram-negative bacteria" refers to bacteria that stain red when stained by Gram staining. Gram-negative bacteria of the present invention include, but are not limited to, strains of the genus Escherichia, Pseudomonas, Acinetobacter, Salmonella, Klebsiella, Neisseria, Enterobacter, Shigella, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, and Legionella. Specifically, Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas chlororaphis, Pseudomonas pertucinogena, Pseudomonas stutzeri, Pseudomonas syringae, Acinetobacter baumannii, Acinetobacter lwoffii, Acinetobacter calcoaceticus, and Acinetobacter hemolyticus (Acinetobacter haemolyticus), Salmonella enterica, Salmonella bongori, Salmonella enteritidis,Salmonella typhimurium, Salmonella gallinarum, Salmonella pullorum, Salmonella mbandaka, Salmonella choleraesuls, Salmonella thompson, Salmonella infamtis, Salmonella derby, Klebsiella pneumonia, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella terrigena, Neisseria gonorrhoeae, Neisseria Neisseria meningitidis, Enterobacter aerogenes, Enterobacter cloacae, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Moraxella catarrhalis, Moraxellalacunata, Moraxella bovis, Helicobacter pylori, Helicobacter heilmannii, Helicobacter felis, Helicobacter mustelae, Helicobacter fenelliae, Helicobacter rappini,Helicobacter hepaticus, Helicobacter bilis, Helicobacter pullorum, Stenotrophomonas maltophilia, Stenotrophomonas nitritireducens, Bdellovibrio bacteriovorus, Legionella pneumophila, Legionella anisa, Legionella birminghamensis, Legionella bozemanii, Legionella cincinnatiensis, Legionella dumoffii, Legionella pilay Includes, but is not limited to, Legionalla feeleii, Legionalla gormanii, Legionalla hackeliae, Legionalla israelensis, Legionalla jordanis, Legionalla lansingensis, Legionalla longbeachae, Legionalla maceachernii, Legionalla micdadei, Legionalla oakridgensis, Legionalla sainthelensi, Legionalla tucsonensis, Legionalla wadsworthii, etc.

[0131] In the present invention, "antibiotic-resistant Gram-negative bacteria" includes multidrug-resistant Acinetobacter baumannii (MRAB). Acinetobacter baumannii ), multidrug-resistant E. coli ( Escherichia coli ), multidrug-resistant Pseudomonas aeruginosa (MRPA; Multidrug-resistant Pseudomonas aeruginosa Includes, but is not limited to, ) etc.

[0133] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of infectious diseases comprising, as an active ingredient, a polymyxin analog represented by Formula I, or a pharmaceutically acceptable salt thereof.

[0134] In the present invention, the infectious disease may be exemplified by infectious diseases caused by Gram-negative bacteria, such as pneumonia, peritonitis, meningitis, wound infection, osteoarthritis, cholecystitis, urinary tract infection, meningitis, endocarditis, myocarditis, pericarditis, arthritis, gonorrhea, bacterial dysentery, enteritis, conjunctivitis, gastritis, otitis media, cystitis, lymphangitis, pharyngitis, impetigo, rheumatic fever, glomerulonephritis, neonatal sepsis, meningitis, pharyngitis, pneumonia, endocarditis, scarlet fever, skin soft tissue infection, deep soft tissue infection, empyema, vaginitis, etc.

[0136] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of sepsis comprising a polymyxin analog represented by Formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0137] In the present invention, "sepsis" refers to a condition in which a severe inflammatory response occurs throughout the body due to infection by microorganisms. When two or more of the following symptoms are observed—such as fever rising above 38°C or hypothermia falling below 36°C, an increase in respiratory rate to 24 breaths per minute or more (tachypnea), a heart rate of 90 beats per minute or more (tachycardia), or an increase or significant decrease in the white blood cell count in blood tests—this is called systemic inflammatory response syndrome (SIRS). When such systemic inflammatory response syndrome is caused by an infection by microorganisms, it is called sepsis. It is a condition in which pathogens from an infectious site in the body enter the bloodstream continuously or intermittently, settle in various organ tissues to form lesions, and exhibit severe systemic symptoms. Causative agents include, but are not limited to, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Klebsiella pneumoniae, fungi, and anaerobic bacteria.

[0139] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical composition according to the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods, but is not limited thereto.

[0140] Carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0141] When formulating the pharmaceutical composition of the present invention, it is prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used, but are not limited thereto.

[0142] The pharmaceutical composition of the present invention can be used on the skin, for injection, subcutaneous injection, oral preparations, etc.

[0143] The content of the polymyxin analog represented by Formula I included in the pharmaceutical composition of the present invention can be appropriately adjusted according to the use, application form, purpose of use and desired effect, and, considering the effect relative to the content, can be used within, for example, 0.0001 to 99.9 weight% with respect to the total weight of the composition.

[0144] The pharmaceutical composition of the present invention can be administered orally and parenterally, and can be administered orally, transdermally, subcutaneously, or intravenously; it can also be applied via transdermal application and topical application.

[0146] Meanwhile, in the present invention, using amino acids, a resin-attached peptide (Formula Ia) was obtained by a solid-phase synthesis method, and then the resin was removed to obtain a protected peptide (Formula Ib). Then, a cyclization reaction was carried out using a catalyst by a solution-phase synthesis method to obtain a cyclized peptide (Formula Ic), and finally, a cyclized peptidomimetic (Formula I) was synthesized by deprotection. As a result, it was confirmed that the cyclized peptidomimetic can be synthesized in a high yield sufficient for commercial mass production.

[0147] Accordingly, in one aspect, the present invention relates to a method for preparing a polymyxin analog represented by Formula I, comprising: (a) obtaining a resin-attached peptide represented by Formula Ia by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Formula Ib; (c) cyclizing the peptide obtained in step (b) using a catalyst by a solution-phase synthesis method to obtain a protected polymyxin analog represented by Formula Ic; and (d) performing a deprotection reaction on the peptide obtained in step (c) to obtain a polymyxin analog represented by Formula I.

[0148] [Chemical Formula I]

[0149]

[0150] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0151]

[0154] [Chemical Formula Ia]

[0155]

[0157] [Chemical Formula Ib]

[0158]

[0160] [Chemical Formula Ic]

[0161]

[0162] In the above chemical formula, A is a hydrogen or hydroxyl protecting group, and B is an amine protecting group.

[0164] In the above chemical formula, A may utilize a hydrogen or hydroxyl protecting group commonly used in the industry. The hydroxyl protecting group is tert-butyl ( tert -butyl) group, triphenylmethyl (or trityl) group, acetaminomethyl group, benzoyl group, diphenylmethyl group, benzyl group, para-methoxybenzyl ( para -methoxybenzyl) group, benzyloxycarbonyl group, para-nitrobenzyl ( para -nitrobenzyl) group, allyl group, dimethylsilyl group, tert-butyldimethylsilyl ( tert Examples include -butyldimethylsilyl) groups, triisopropylsilyl groups, alkyl groups (10 or fewer carbon atoms), etc., and tert-butyl ( tert -butyl) group, triphenylmethyl group or acetaminomethyl group is preferred, and tert-butyl ( tert It is more desirable to use the -butyl group.

[0165] In the above chemical formula, B can be an amine protecting group commonly used in the industry. Acetyl group, Benzoyl, Benzyloxymethyl group, Trityl group, Ddz (α,α-Dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz)) group, Bpoc (2-(4-Biphenyl)isopropoxycarbonyl (Bpoc)) group, Nps (2-Nitrophenylsulfenyl) (Nps)) group, Fmoc (9-Fluorenylmethoxycarbonyl (Fmoc)) group, Nsc (2-(4-Nitrophenylsulfonyl)ethoxycarbonyl (Nsc)) group, Bsmoc (1,1-Dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc)) group, α-Nsmoc ( (1,1-Dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc)) group, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde)) group, Fmoc* (2,7-Di-tert-butyl-Fmoc (Fmoc*)) group, Fmoc(2F) (2-Fluoro-Fmoc (Fmoc(2F))) group, allyl group, mio-Fmoc & dio-Fmoc (2-Monoisooctyl-Fmoc (mio-Fmoc) and 2,7-Diisooctyl-Fmoc (dio-Fmoc)) group, TCP (Tetrachlorophthaloyl (TCP)) group, Pms (2-[Phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate (Pms)) group, Esc (Ethanesulfonylethoxycarbonyl (Esc)) group,Sps (2-(4-Sulfophenylsulfonyl)ethoxycarbonyl (Sps))기, Z (Benzyloxycarbonyl (Z))기, Alloc (Allyloxycarbonyl (Alloc))기, oNBS & pNBS (o-Nitrobenzenesulfonyl (oNBS) and p-nitrobenzenesulfonyl (pNBS)기, dNBS (2,4-Dinitrobenzenesulfonyl (dNBS))기, Bts (Benzothiazole-2-sulfonyl (Bts))기, Troc (2,2,2-Trichloroethyloxycarbonyl (Troc))기, Dts (Dithiasuccinoyl (Dts))기, pNZ (p-Nitrobenzyloxycarbonyl (pNZ))기, Poc (Propargyloxycarbonyl (Poc))기, oNZ & NVOC (o-Nitrobenzyloxycarbonyl (oNZ) and 6-Nitroveratryloxycarbonyl (NVOC))기, NPPOC (2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC))기, MNPPOC ( 2-(3,4-Methylenedioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC))기, BrPhF (9-(4-Bromophenyl)-9-fluorenyl (BrPhF))기, Azoc (Azidomethyloxycarbonyl (Azoc))기, Cl-Z (2-Chlorobenzyloxycarbonyl (Cl-Z))기, Boc (tert-Butyloxycarbonyl (Boc))기, Mtt (4-Methyltrityl (Mtt))기 등을 예시할 수 있으며, Dde & ivDde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) (Dde) and 1-(4,4-Dimethyl-2,It is preferable to use a 6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)) group, and it is most preferable to use a Dde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) group) as the amine protecting group.

[0166] The protecting group for the above functional group is described in detail in "Protecting Groups in Organic Synthesis (Greene and Wuts, John Wiley & Sons, 1991)" and "Amino acid-Protecting Groups (A. Isidro Llobet, 2009)".

[0167] In this specification, the term "peptide" means a linear molecule formed by amino acid residues being joined together by peptide bonds.

[0169] With reference to FIG. 1, the manufacturing method of the present invention will be described in detail step by step as follows.

[0170] (a) Obtaining a resin-attached peptide represented by chemical formula Ia

[0171] A resin-attached peptide represented by chemical formula Ia can be prepared by solid-phase synthesis methods commonly used in the industry (Merrifield, RB, J. Am. Chem. Soc., 85:2149-2154 (1963), Kaiser, E., Colescot, R.L., Bossinger, C.D., Cook, P.I., Anal. Biochem., 34:595-598(1970)). That is, after binding an amino acid protected by an alpha-amino and a side chain functional group to a resin, the alpha-amino protecting group is removed, and the remaining amino acids protected by an alpha-amino and a side chain functional group are bound stepwise in the desired order to obtain an intermediate.

[0172] The present invention is characterized by using a 4th amino acid in a peptide sequence in which the amine protecting group is Dde or ivDde. The amino acid may be Fmoc-Dab(Dde)-OH or Fmoc-Dab(ivDde)-OH. When Dde or ivDde is used as a protecting group, only the Dde or ivDde protecting group can be selectively removed while the peptide protected on the resin is attached.

[0173] The selection of an appropriate protecting group depends on the functional group to be protected, the conditions under which the protecting group is exposed, and other functional groups that may be present in the molecule. At each step of synthesis, the protecting group must be stable with respect to the reaction conditions and reagents selected to remove the alpha-amino protecting group, (b) no deprotection reaction should occur during the binding reaction, and (c) stable under the degradation conditions with the resin when the synthesis containing the desired amino acid chain is completed.

[0174] According to a preferred embodiment of the present invention, the resin that can be used in the process of synthesizing a peptide represented by formula I-a may be a conventional resin that can be easily degraded under mild acidic conditions, which can completely preserve the side chain protecting group of the manufactured peptide. Preferably, the resin is tritylchloride resin, 2-chlorotritylchloride resin, 4-methyltritylchloride resin or 4-methoxytritylchloride resin, PAM resin, Wang resin, Oxime resin, HMBA resin, more preferably Wang resin, tritylchloride resin or 2-chlorotritylchloride resin, and most preferably 2-chlorotritylchloride resin.

[0176] (b) Obtaining a peptide represented by the chemical formula Ib

[0177] The compound represented by the chemical formula Ib above can be obtained by removing the resin from the peptide obtained in step (a) under mild acidic conditions. At this time, the acidic conditions that can be used must be mild conditions in which the side chain protecting group of the amino acid chain can be maintained.

[0178] According to a preferred embodiment of the present invention, the process of removing resin may be performed in the presence of an acidic solution. The acidic solution may be acetonitrile, dichloromethane, trifluoroacetic acid, trichloroacetic acid, chloroacetic acid, dichloroacetic acid, bromoacetic acid, dibromoacetic acid, tribromoacetic acid, acetic acid, formic acid, methanesulfonic acid, benzenesulfonic acid, para-toluenesulfonic acid ( para Single solutions or mixed solutions thereof of -toluenesulfonic acid), methanol, ethanol, isopropanol, trifluoroethanol, etc., may be used.

[0179] Preferably, the acidic conditions are a 2 to 5 volume% trichloroacetic acid solution, a 2 to 5 volume% trifluoroacetic acid solution, and 0.1% formic acid, methanesulfonic acid, benzenesulfonic acid, or para-toluenesulfonic acid ( para An example of using a dichloromethane solution containing -toluenesulfonic acid alone or in combination can be given.

[0181] (c) Obtaining a cyclized peptide represented by the chemical formula Ic

[0182] The compound represented by the chemical formula Ic is obtained by performing a cyclization reaction of the peptide obtained in step (b) in the presence of a catalyst.

[0183] According to a preferred embodiment of the present invention, the catalytic reagents available in step (c) are DCC (N,N-dicyclohexylcarbodiimide), DIC (N,N-diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), CDI (carbonyldiimidazole), EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), etc. can be used alone or in combination. There is,It is preferable to use PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), EDC·HCl (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), HOBt (1-Hydroxybenzotriazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), and HOAt (1-Hydroxyazabenzotriazole), and it is most preferable to use EDC·HCl (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and HOAt (1-Hydroxyazabenzotriazole).

[0184] According to a preferred embodiment of the present invention, the solvent used in the cyclization reaction is 1,2-dichloroethane, chloroform, dichloromethane, 1,2-dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, dimethylsulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, preferably dichloromethane and N,N-dimethylformamide, and most preferably a mixed solution of dichloromethane and N,N-dimethylformamide.

[0185] The above cyclization reaction can be performed at -20 to 50°C, and it is preferable to perform it at 0 to 30°C. In addition, the concentration of the cyclization reaction is 10 when converted to peptide. -2 10 at the mall -9 It can be performed within a molar concentration range, preferably 10 -2 10 at the mall -5 It is preferable to perform this at molar concentrations, but is not limited thereto.

[0187] (d) Obtaining a polymyxin analog represented by chemical formula I

[0188] A polymyxin analog represented by chemical formula I can be obtained by performing a deprotection reaction from the peptide obtained in step (c) under reaction conditions commonly used in the industry.

[0189] The deprotection reaction comprises (1) a mixture of trifluoroacetic acid, water (H2O), phenol, thioanisole, and 1,2-ethanedithiol; (2) a mixture of trifluoroacetic acid, water (H2O), phenol, and triisopropylsilane; (3) a mixture of trifluoroacetic acid, water (H2O), phenol, thioanisole, and 1-dodecanethiol; and (4) a mixture of trifluoroacetic acid, DTT (Dithiothreonitol), water (H2O), and triisopropylsilane; (5) a mixture of trifluoroacetic acid and phenol; (6) a mixture of trifluoroacetic acid, phenol, and methanesulfonic acid; (7) a mixture of trifluoroacetic acid, thioanisole and 1,2-ethanedithiol and anisole; (8) a mixture of trifluoroacetic acid and triethylsilane; (9) a mixture of trifluoroacetic acid and water (H2O); (10) a mixture of trifluoroacetic acid, dichloromethane and indole, and it is preferable to perform it under a mixture of trifluoroacetic acid and water (H2O).

[0191] [Example]

[0192] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0194] Throughout this specification, unless otherwise noted, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid.

[0196] Example 1: Synthesis of Polymyxin Analogues

[0197] 1-1: Synthesis of a polymyxin analog represented by chemical formula I-1

[0198] [Chemical Formula I-1]

[0199]

[0200] 1-1-1: Synthesis of H-Thr(tBu)-2-chlorotrityl resin

[0201]

[0202] 2-chlorotrityl chloride resin (resin with substitution rate = 1.08 mmol / g, 100 mmol) and dichloromethane (1000 ml) were placed in a solid-phase synthesis reactor equipped with a filtration membrane, and after expanding the resin for 15 minutes, the solvent was removed through the filtration membrane under reduced pressure. Dichloromethane (1000 ml) containing Fmoc-Thr(tBu)-OH (molecular weight = 297.5 g / mol) (59.5 g, 200 mmol, 2.0 equivalents) was added, followed by the addition of N,N-diisopropylethylamine (molecular weight = 129.25 g / mol) (32.31 g, 250 mmol, 2.5 equivalents), and the mixture was reacted at room temperature for 12 hours. The reaction solution was removed by vacuum filtration, and after washing the resin once with dichloromethane, a mixture of dichloromethane : methanol : N,N-diisopropylethylamine = 17 : 2 : 1 (1000 ml) was added to the resin and stirred for 20 minutes. The reaction solution was removed by vacuum filtration, and after washing the resin three times with dichloromethane, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The above Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0204] 1-1-2: Synthesis of H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0205]

[0206] To the H-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g / mol) (132.15 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0207] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0209] 1-1-3: Synthesis of H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0210]

[0211] To the H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g / mol) (132.15 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0212] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0214] 1-1-4: Synthesis of H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0215]

[0216] To the H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Leu-OH (molecular weight = 353.4 g / mol) (106.02 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0217] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0219] 1-1-5: Synthesis of HD-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0220]

[0221] To the H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-D-Leu-OH (molecular weight = 353.4 g / mol) (106.02 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0222] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain HD-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0224] 1-1-6: Synthesis of H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0225]

[0226] To the HD-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g / mol) (132.15 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0227] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0229] 1-1-7: Synthesis of H-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr (tBu)-2-chlorotrityl resin

[0230]

[0231] To the H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Dab(Dde)-OH (molecular weight = 504.6 g / mol) (151.38 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0232] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0234] 1-1-8: Synthesis of H-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab (Boc)-Thr(tBu)-2-chlorotrityl resin

[0235]

[0236] To the H-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g / mol) (132.15 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0237] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0239] 1-1-9: Synthesis of H-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0240]

[0241] To the H-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) of Fmoc-Thr(tBu)-OH (molecular weight = 297.5 g / mol) (89.25 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and the mixture was reacted at room temperature for 4 hours.

[0242] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0244] 1-1-10: Synthesis of H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0245]

[0246] To the H-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) containing Fmoc-Dab(Boc)-OH (molecular weight = 440.49 g / mol) (132.15 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, followed by the addition of N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents), and then at room temperature for 4 hours. It caused a reaction.

[0247] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml). Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, after which the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was washed six times in sequence with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0249] 1-1-11: Preparation of (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0250] To the H-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained from the above reaction, N,N-dimethylformamide (1000 ml) containing 3-Cyclohexylpropionic acid (molecular weight = 156.22 g / mol) (46.87 g, 300 mmol, 3 equivalents) and HOBt (molecular weight = 135.12 g / mol) (44.6 g, 330 mmol, 3.3 equivalents) was added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, The reaction was carried out at room temperature for 4 hours.

[0251] Next, the reaction solution was removed by vacuum filtration, the resin was washed twice with N,N-dimethylformamide (1000 ml), then washed three times with dichloromethane (1000 ml), and then dried to obtain (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0253] 1-1-12: Preparation of (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH

[0254]

[0255] 1000 ml of 2% Hydrazine monohydrate / DMF solution was added to the (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin obtained from the above reaction and stirred for 3 minutes, then the reaction solution was removed under reduced pressure. Additionally, 1000 ml of 2% Hydrazine monohydrate / DMF solution was added once more and stirred for 3 minutes, after which the reaction solution was discharged. The resin was washed twice with dichloromethane (1000 ml), then dichloromethane (1000 ml) and 2% trifluoroacetic acid (20 ml) were added and stirred for 5 minutes. The dichloromethane reaction solution was removed, and the reaction was carried out one more time, after which the solution was removed and combined. The filtrate was concentrated under reduced pressure to obtain 166.34 g of a linear peptide with a protecting group (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH (molecular weight: 1798.25 g / mol) (yield: 92.5%, purity after deprotection: 78.4%).

[0257] 1-1-13: (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu) (4-10 cyclized) : Synthesis of a polymyxin analog with a protecting group

[0258]

[0259] The (3-Cyclohexyl propionic acid)-Dab(Boc)-Thr(tBu)-Dab(Boc)-Dab-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH (166.34 g, 150 mmol) obtained from the above reaction was dissolved in dichloromethane / N,N-dimethylformamide (9:1, 2000 ml), then EDC·HCl (molecular weight = 191.7 g / mol, 86.26 g, 3 equivalents) and HOAt (molecular weight = 136.11 g / mol, 61.25 g, 3 equivalents) were added, and the mixture was stirred at room temperature for 12 hours. The volume was reduced to less than 3 / 1 by vacuum concentration, then crystallized in 2500 ml of ethyl ether, and the precipitated solid was filtered and vacuum dried to obtain 154.7 g of a polymyxin analog with protecting groups (molecular weight: 1780.24 g / mol) (yield: 93.0%, purity after deprotection: 35.4%).

[0261] 1-1-14: Synthesis of Polymyxin Analogues of Chemical Formula I-1

[0262]

[0263] The polymyxin analog (154.7 g) having a protecting group obtained from the above reaction was mixed with a mixture of trifluoroacetic acid and purified water (3000 ml) of 95:5 and stirred at room temperature for 3 hours. The reaction mixture was slowly poured into 9000 ml of ethyl ether to precipitate the peptide and stirred for 30 minutes. The resulting peptide was filtered under reduced pressure and vacuum dried to obtain 22.6 g of the polymyxin analog of Formula I-1 (molecular weight: 1153.42 g / mol) (theoretical amount 115.34 g, yield: 19.6%, purity: 35.4%, purity after purification: 95.5%).

[0264] For reference, the peptide represented by the above chemical formula I-1 is one in which 3-Cyclohexyl propionic acid is bonded to the amino acid sequence represented by SEQ ID NO. 1 (Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr(4-10 cyclized)).

[0266] 1-2: Chemical formula I-2 Synthesis of polymyxin analogs represented by

[0267] [Chemical Formula I-2]

[0268]

[0269] The synthesis of the I-2 polymyxin analog was carried out using the same method as that used for the synthesis of the polymyxin analog represented by the chemical formula I-1, except that the first amino acid was Fmoc-Dap(Boc)-OH instead of Fmoc-Dab(Boc)-OH in the synthesis method of the compound I-1.

[0270] As a result of synthesis, 19.4 g of a polymyxin analog represented by chemical formula I-2 (molecular weight: 1153.42 g / mol) was obtained by starting synthesis with the same synthesis scale (theoretical amount 115.34 g, yield: 16.8%, purity: 29.8%, purity after purification 97.2%).

[0271] For reference, the peptide represented by the above chemical formula I-2 is one in which 3-Cyclohexyl propionic acid is bound to the amino acid sequence represented by SEQ ID NO. 2 (Dab-Thr-Dab-Dab-Dap-D-Leu-Leu-Dab-Dab-Thr(4-10 cyclized)).

[0273] 1-3: Synthesis of polymyxin analogs represented by chemical formulas I-3 through I-21

[0274] The synthesis of polymyxin analogs represented by chemical formulas I-3 through I-21 was carried out using the same synthesis method as that used for polymyxin analogs represented by chemical formulas I-1 through I-2, yielding the results shown in the table below.

[0275] chemical structure Theoretical molecular weight (Da) Measured molecular weight (Da) transference number(%) water(%) II-3 1201.46 1202.17 15.4 95.57 II-4 1207.51 1208.33 21.2 95.28 II-5 1251.52 1251.30 20.3 95.35 II-6 1207.51 1208.02 19.8 97.54 II-7 1167.44 1168.39 26.7 95.74 II-8 1181.47 1180.39 14.9 98.51 II-9 1195.5 1196.28 30.1 99.80 II-10 1153.42 1154.19 22.7 98.47 II-11 1187.43 1188.13 26.4 95.60 II-12 1215.49 1216.31 22.1 95.14 II-13 1229.51 1230.22 25.4 95.17 II-14 1187.43 1188.21 11.3 96.11 II-15 1187.43 1188.13 15.9 95.02 II-16 1241.52 1242.34 27.2 98.27 II-17 1215.49 1215.90 26.4 95.73 II-18 1201.46 1202.11 28.3 95.36 II-19 1215.49 1216.43 20.9 96.44 II-20 1229.51 1230.33 18.5 95.58 II-21 1187.43 1188.32 30.2 95.78

[0276] The peptides represented by the above chemical formulas I-3 to I-21 are those in which 3-Cyclohexyl propionic acid is bonded to the amino acid sequences represented by SEQ ID NOs 3 to 21.

[0277] Sequence number order note Sequence No. 3 Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr 4-10 cyclized Sequence No. 4 Dab-Thr-Dab-Dab-Dab-D-Cha-Leu-Dab-Dab-Thr 4-10 cyclized Sequence No. 5 Dab-Thr-Dab-Dab-Dab-D-(2-Nal)-Leu-Dab-Dab-Thr 4-10 cyclized Sequence number 6 Dab-Thr-Dab-Dab-Dab-D-Leu-Cha-Dab-Dab-Thr 4-10 cyclized Sequence number 7 Dab-Thr-Dab-Dab-Dab-D-Leu-Nle-Dab-Dab-Thr 4-10 cyclized Sequence No. 8 Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Orn-Dab-Thr 4-10 cyclized Sequence number 9 Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Lys-Dab-Thr 4-10 cyclized Sequence number 10 Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Dap-Dab-Thr 4-10 cyclized Sequence number 11 Dap-Thr-Dab-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr 4-10 cyclized Sequence No. 12 Dab-Thr-Orn-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr 4-10 cyclized Sequence No. 13 Dab-Thr-Lys-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr 4-10 cyclized Sequence No. 14 Dab-Thr-Dap-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr 4-10 cyclized Sequence number 15 Dab-Thr-Dab-Dab-Dap-D-Phe-Leu-Dab-Dab-Thr 4-10 cyclized Sequence number 16 Dab-Thr-Dab-Dab-Dab-D-Phe-Cha-Dab-Dab-Thr 4-10 cyclized Sequence number 17 Dab-Thr-Dab-Dab-Dab-D-Phe-hLeu-Dab-Dab-Thr 4-10 cyclized Sequence number 18 Dab-Thr-Dab-Dab-Dab-D-Phe-Nle-Dab-Dab-Thr 4-10 cyclized Sequence No. 19 Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Orn-Dab-Thr 4-10 cyclized Sequence number 20 Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Lys-Dab-Thr 4-10 cyclized Sequence number 21 Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Dab-Dap-Thr 4-10 cyclized

[0279] FIGURE 2: In-vitro fermentation of scaffolds

[0280] 2-1 균주정보

[0281] Standard strains through the National Pathogen Resource Bank, Pathogen Resource Management Division, National Institute of Infectious Diseases A. baumannii (ATCC19606), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) was purchased from the American Type Culture Collection (ATCC) and used. The corresponding multidrug-resistant strain was obtained from the Korea National Research and Training Center (KNRRC), and the antibiotic resistance information for the multidrug-resistant bacteria is as shown in Table 3.

[0283] 종명 은행등록번호 저항성 항생제명 A. baumannii CCARM 12233 AMK, CAZ, CPM, CTX, GM, LEV, MEM, PIP E. coli CCARM 1G528 AMK, AMP, AZT, CAZ, CTX, FOX, GM, LEV, PIP, TM P. aeruginosa CCARM 2328 AMK, AZT, CAZ, CPM, IMP, LEV, MEM, PIP

[0284] AMK; Amikacin, AMP; Ampicillin, AZT; Azetreonam, CAZ; Ceftazidime, CPM; Cefepime, CTX; Cefotaxime, FOX; Cefoxitin, GM; Gentamicin, IMP; Imipenem, LEV; Levofloxacin, MEM; Meropenem, PIP; Piperacillin, TM; Tobramycin

[0285] The standard strain used A. baumannii (ATCC19606), E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853), and the corresponding multidrug-resistant strain A. baumannii (CCARM 12233), E. coli (CCARM 1G528), and P. aeruginosaMIC for colistin, meropenem, and ceftazidime for (CCARM 2328) 90 After verifying the value, it was used to derive a lead compound for the development of a novel cyclized peptidomimetic antibiotic.

[0287] Antibiotics MIC 90 (μg / ml) A. baumannii (ATCC19606) E. coli (ATCC 25922) P. aeruginosa (ATCC 27853) CCARM A. baumannii (CCARM 12233) CCARM E. coli (CCARM 1G528) CCARM P. aeruginosa (CCARM 2328) Colistin 1 1 1 1 1 1 Meropenem 1 1 4 32 1 8 Ceftazidime 8 1 2 >64 16 16

[0288] The standard strain used A. baumannii It was confirmed that (ATCC19606) possesses slight resistance to ceftazidime. CCARM, a multidrug-resistant strain A. baumannii and CCARM P. aeruginosa possesses resistance to ceftazidime and meropenem, antibiotics widely used in clinical practice, and CCARM E. coli It was confirmed that it only has resistance to ceftazidime.

[0289] 2-2. 항균시험

[0290] The MIC (minimum inhibitory concentration) method was used to evaluate the antimicrobial efficacy of the polymyxin analog compounds of Formulas I-1 to I-21 prepared in the examples. For this purpose, each test strain was placed on a polystyrene microarray plate at a rate of 7×10⁻⁶ 5 After dispensing the solution diluted to CFU / ml in MHB culture medium, 21 candidate substances were added at concentrations starting from 64 µg / ml and diluted twofold. The mixture was incubated at 37°C for 16 hours, and the absorbance was measured at 600 nm. MIC 90 The value represents the minimum concentration (µg / ml) at which the growth of the test strain is inhibited by more than 90% compared to the control group.

[0291] For the measurement of antibacterial efficacy, meropenem (carbapenem), ceftazidime (cephalosporin), which are broad-spectrum antibiotics widely used to treat severe Gram-negative bacterial infections, and colistin, a peptide antibiotic used to treat multidrug-resistant Gram-negative infections, were purchased and used as control antibiotics.

[0293] 물질명 MIC 90 (μg / ml) A. baumannii E. coli P. aeruginosa CCARM A. baumannii CCARM E. coli CCARM P. aeruginosa 1 WP-AMP-219 1 1 1 1 1 1 2 WP-AMP-222 1 1 2 1 1 4 3 WP-AMP-223 1 1 1 1 1 1 4 WP-AMP-224 1 1 1 1 1 1 5 WP-AMP-225 1 1 1 1 1 1 6 WP-AMP-227 1 1 1 1 1 1 7 WP-AMP-229 1 1 1 1 1 1 8 WP-AMP-230 1 1 1 1 1 1 9 WP-AMP-231 1 1 1 1 1 1 10 WP-AMP-235 1 1 1 1 1 1 11 WP-AMP-239 1 1 1 1 1 1 12 WP-AMP-241 1 1 1 1 1 1 13 WP-AMP-242 1 1 1 2 1 1 14 WP-AMP-243 1 1 1 1 1 1 15 WP-AMP-246 1 1 1 1 1 1 16 WP-AMP-248 1 1 1 1 1 1 17 WP-AMP-249 1 1 1 1 1 1 18 WP-AMP-250 1 1 1 1 1 1 19 WP-AMP-251 1 1 1 1 1 1 20 WP-AMP-252 1 1 1 1 1 1 21 WP-AMP-254 1 1 1 1 1 1

[0294] From Table 5, Chemical Formula I-1 (hereinafter denoted as WP-AMP-219), Chemical Formula I-2 (hereinafter denoted as WP-AMP-222), Chemical Formula I-3 (hereinafter denoted as WP-AMP-223), Chemical Formula I-4 (hereinafter denoted as WP-AMP-224), Chemical Formula I-5 (hereinafter denoted as WP-AMP-225), Chemical Formula I-6 (hereinafter denoted as WP-AMP-227), Chemical Formula I-7 (hereinafter denoted as WP-AMP-229), Chemical Formula I-8 (hereinafter denoted as WP-AMP-230), Chemical Formula I-9 (hereinafter denoted as WP-AMP-231), Chemical Formula I-10 (hereinafter denoted as WP-AMP-235), Chemical Formula I-11 (hereinafter denoted as WP-AMP-239), Chemical Formula I-12 (hereinafter denoted as WP-AMP-241), Chemical Formula I-13 (hereinafter denoted as WP-AMP-242), and Chemical Formula I-14 (hereinafter denoted as WP-AMP-243) Chemical formula I-15 (hereinafter referred to as WP-AMP-246), chemical formula I-16 (hereinafter referred to as WP-AMP-248), chemical formula I-17 (hereinafter referred to as WP-AMP-249), chemical formula I-18 (hereinafter referred to as WP-AMP-250), chemical formula I-19 (hereinafter referred to as WP-AMP-251), chemical formula I-20 (hereinafter referred to as WP-AMP-252), and chemical formula I-21 (hereinafter referred to as WP-AMP-254) are Acinetobacter baumannii ( Acinetobacter baumannii ), E. coli( Escherichia coli ), or Pseudomonas aeruginosa( Pseudomonas aeruginosa In addition, multidrug-resistant Gram-negative bacteria include multidrug-resistant Acinetobacter baumannii (MRAB; Multidrug-resistant Acinetobacter baumannii ), multidrug-resistant E. coli ( Escherichia coli ) or multidrug-resistant Pseudomonas aeruginosa (MRPA; Multidrug-resistant Pseudomonas aeruginosa It was confirmed that it also has antibacterial properties.

[0296] 2-3 세포독성시험

[0297] The cytotoxicity of seven selected polymyxin analogs (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) on NIH3T3, HEK293, and RAW246.7 cell lines was evaluated using the WST-8 assay. 3 × 10⁴ cells per well were placed in a 96-well plate. 4 Cells were seeded in DMEM containing 10% FBS and cultured in a 5% CO2 incubator at 37°C for 24 hours. Each polymyxin analog was added to final concentrations of 100, 50, 25, 10, 5, and 1 μg / ml, and cultured at 37°C for 24 hours, after which 20 μl of WST-8 solution was added to each well. After adding the WST-8 solution and cultured at 37°C for 2 hours, cell viability was measured by measuring absorbance at 450 nm, and the results are shown in Figures 2a, 2b, and 2c.

[0299] % survival = 100 x [(A peptide-trated cell ) / (A untreated cell )]

[0301] From Figures 2a, 2b, and 2c, it was confirmed that WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254 did not exhibit cytotoxicity against NIH3T3, HEK293, and RAW246.7 cell lines even at a concentration of 100 μg / ml.

[0303] 2-4 염에 대한 안정성 평가

[0304] To measure the stability of 21 types of polymyxin analogs (WP-AMP-219, WP-AMP-222, WP-AMP-223, WP-AMP-224, WP-AMP-225, WP-AMP-227, WP-AMP-229, WP-AMP-230, WP-AMP-231, WP-AMP-235, WP-AMP-239, WP-AMP-241, WP-AMP-242, WP-AMP-243, WP-AMP-246, WP-AMP-248, WP-AMP-249, WP-AMP-250, WP-AMP-251, WP-AMP-252, and WP-AMP-254) against salts, salts at physiological concentrations contained in human blood (150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4) were The effect on antimicrobial efficacy was evaluated using 7×10 test strains. 5 After inoculating with MHB culture medium containing 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4 salts at a concentration of CFU / ml, 21 types of polymyxin analogs were added at twofold dilutions starting from 64 µg / ml. The mixture was incubated at 37°C for 16 hours, and the absorbance was measured at 600 nm. MIC 90 The value represents the minimum concentration (µg / ml) at which the growth of the test strain is inhibited by more than 90% compared to the control group.

[0305] In addition, Na present in normal blood + The concentration of is 140 mmol / L, and Ca 2+ and Mg 2+ The concentrations are 2.1–2.6 mmol / L and 0.85–1.10 mmol / L, respectively. In this experiment, the MIC was used under conditions of final concentrations similar to normal blood: 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4. 90 Changes in antibacterial efficacy were confirmed by comparing values, and the results are shown in Table 6.

[0307] Compound name MIC 90 (μg / ml) A. baumannii E. coli P. aeruginosa CCARM A. baumannii CCARM E. coli CCARM P. aeruginosa +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt 1 WP-AMP-219 1 1 1 1 8 1 2 1 1 1 8 1 2 WP-AMP-222 1 1 2 1 >64 2 2 1 1 1 >64 4 3 WP-AMP-223 2 1 1 1 2 1 2 1 1 1 8 1 4 WP-AMP-224 1 1 1 1 4 1 1 1 1 1 8 1 5 WP-AMP-225 1 1 1 1 2 1 1 1 1 1 4 1 6 WP-AMP-227 1 1 1 1 4 2 1 2 1 1 8 1 7 WP-AMP-229 1 1 1 1 4 1 1 1 1 1 16 1 8 WP-AMP-230 8 1 1 1 8 1 16 1 1 1 32 1 9 WP-AMP-231 8 1 1 1 8 1 8 1 1 1 16 1 10 WP-AMP-235 2 1 1 1 4 1 2 1 1 1 4 1 11 WP-AMP-239 8 1 2 1 2 1 4 1 1 1 4 1 12 WP-AMP-241 4 1 1 1 8 1 4 1 1 1 16 1 13 WP-AMP-242 4 1 1 1 8 1 4 2 1 1 32 1 14 WP-AMP-243 1 1 1 1 2 1 1 1 1 1 2 1 15 WP-AMP-246 1 1 1 1 32 2 1 1 1 1 32 1 16 WP-AMP-248 1 1 1 1 2 1 1 1 1 1 4 1 17 WP-AMP-249 1 1 1 1 2 2 1 2 1 1 4 1 18 WP-AMP-250 1 1 1 1 2 1 2 1 1 1 8 1 19 WP-AMP-251 4 1 1 1 4 1 4 1 1 1 8 1 20 WP-AMP-252 4 2 1 1 4 1 2 1 1 1 8 1 21 WP-AMP-254 1 1 1 1 2 1 1 1 1 1 4 1

[0308] From Table 6, 21 polymyxin analogs (WP-AMP-219, WP-AMP-222, WP-AMP-223, WP-AMP-224, WP-AMP-225, WP-AMP-227, WP-AMP-229, WP-AMP-230, WP-AMP-231, WP-AMP-235, WP-AMP-239, WP-AMP-241, WP-AMP-242, WP-AMP-243, WP-AMP-246, WP-AMP-248, WP-AMP-249, WP-AMP-250, WP-AMP-251, WP-AMP-252, and WP-AMP-254) were found in the presence of 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4 for P. aeruginosa and Except for CCARM P. aeruginosa, A. baumannii and CCARM A. baumannii MIC for all strains 90 It was confirmed that there was almost no change in value. That is, it was confirmed that the efficacy of the 21 types of polymyxin analogs of the present invention, P. aeruginosa and CCARM P. aeruginosa, A. baumannii and CCARM A. baumannii, was partially affected by structural changes caused by salt.

[0310] 2-5. 단백질 분해효소에 대한 안정성 평가

[0311] MIC induced by proteases for 21 polymyxin analogs (WP-AMP-219, WP-AMP-222, WP-AMP-223, WP-AMP-224, WP-AMP-225, WP-AMP-227, WP-AMP-229, WP-AMP-230, WP-AMP-231, WP-AMP-235, WP-AMP-239, WP-AMP-241, WP-AMP-242, WP-AMP-243, WP-AMP-246, WP-AMP-248, WP-AMP-249, WP-AMP-250, WP-AMP-251, WP-AMP-252, and WP-AMP-254) exhibiting stability against salts 90Stability was evaluated by comparing values ​​and assessing changes in antimicrobial efficacy. Stability against proteases was evaluated using Trypsin and chymotrypsin; the protease and lead compound were mixed at a ratio of 1:20 (w / w) and reacted at 37°C for 16 hours to determine the MIC. 90 The values ​​were measured, and the results are shown in Tables 7 and 8.

[0312] 물질명 MIC 90 (μg / ml) A. baumannii E. coli P. aeruginosa CCARM A. baumannii CCARM E. coli CCARM P. aeruginosa +TRY -TRY +TRY -TRY +TRY -TRY +TRY -TRY +TRY -TRY +TRY -TRY 1 WP-AMP-219 1 1 1 1 1 1 1 1 1 1 1 1 2 WP-AMP-222 1 1 1 1 1 2 1 1 1 1 1 2 3 WP-AMP-223 1 1 1 1 1 1 2 1 1 1 1 1 4 WP-AMP-224 1 1 1 1 1 1 1 1 1 1 1 1 5 WP-AMP-225 1 1 1 1 1 1 1 1 1 1 1 1 6 WP-AMP-227 1 1 1 1 1 2 1 2 1 1 1 1 7 WP-AMP-229 1 1 1 1 1 1 1 1 1 1 1 1 8 WP-AMP-230 1 1 1 1 1 2 1 1 1 1 1 2 9 WP-AMP-231 1 1 1 1 1 1 2 1 1 1 1 1 10 WP-AMP-235 1 1 1 1 1 1 1 1 1 1 1 1 11 WP-AMP-239 1 1 1 1 1 1 1 1 1 1 1 1 12 WP-AMP-241 1 1 1 1 1 1 2 2 1 1 1 1 13 WP-AMP-242 1 1 1 1 1 1 1 2 1 1 1 1 14 WP-AMP-243 1 1 1 1 1 1 1 1 1 1 1 1 15 WP-AMP-246 1 1 1 1 1 1 1 1 1 1 1 1 16 WP-AMP-248 1 1 1 1 1 1 1 1 1 1 1 1 17 WP-AMP-249 1 1 1 1 1 1 1 1 1 1 1 1 18 WP-AMP-250 1 1 1 1 1 1 1 1 1 1 1 1 19 WP-AMP-251 1 1 1 1 1 1 2 1 1 1 1 1 20 WP-AMP-252 1 2 1 1 1 1 1 1 1 1 1 1 21 WP-AMP-254 1 1 1 2 2 1 4 2 2 1 1 1

[0313] From Table 7, the 21 polymyxin analogs treated with Trypsin (WP-AMP-219, WP-AMP-222, WP-AMP-223, WP-AMP-224, WP-AMP-225, WP-AMP-227, WP-AMP-229, WP-AMP-230, WP-AMP-231, WP-AMP-235, WP-AMP-239, WP-AMP-241, WP-AMP-242, WP-AMP-243, WP-AMP-246, WP-AMP-248, WP-AMP-249, WP-AMP-250, WP-AMP-251, WP-AMP-252, and WP-AMP-254) showed an MIC compared to the control group not treated with Trypsin. 90 It was found that the antibacterial efficacy was maintained with almost no change in value.

[0314] 물질명 MIC 90 (μg / ml) A. baumannii E. coli P. aeruginosa CCARM A. baumannii CCARM E. coli CCARM P. aeruginosa +CHY -CHY +CHY -CHY +CHY -CHY +CHY -CHY +CHY -CHY +CHY -CHY 1 WP-AMP-219 1 1 1 1 1 1 1 1 1 1 1 1 2 WP-AMP-222 1 1 1 1 1 2 1 1 1 1 2 4 3 WP-AMP-223 1 1 1 1 1 1 1 1 1 1 1 1 4 WP-AMP-224 1 1 1 1 1 1 1 1 1 1 1 1 5 WP-AMP-225 1 1 1 1 1 1 1 1 1 1 1 1 6 WP-AMP-227 1 1 1 1 1 2 1 2 1 1 1 1 7 WP-AMP-229 1 1 1 1 1 1 1 1 1 1 1 1 8 WP-AMP-230 1 1 1 1 1 1 1 1 1 1 1 1 9 WP-AMP-231 1 1 1 1 1 1 1 1 1 1 1 1 10 WP-AMP-235 1 1 1 1 1 1 1 1 1 1 1 1 11 WP-AMP-239 1 1 1 1 1 1 1 1 1 1 1 1 12 WP-AMP-241 1 1 1 1 1 1 1 1 1 1 1 1 13 WP-AMP-242 1 1 1 1 1 1 1 2 1 1 1 1 14 WP-AMP-243 1 1 1 1 1 1 1 1 1 1 1 1 15 WP-AMP-246 1 1 1 1 1 1 1 1 1 1 1 1 16 WP-AMP-248 1 1 1 1 1 1 1 1 1 1 1 1 17 WP-AMP-249 1 1 1 1 1 1 1 1 1 1 1 1 18 WP-AMP-250 1 1 1 1 1 1 1 1 1 1 1 1 19 WP-AMP-251 1 1 1 1 1 1 1 1 1 1 1 1 20 WP-AMP-252 1 2 1 1 1 1 1 1 1 1 1 1 21 WP-AMP-254 1 1 1 1 1 1 1 1 1 1 1 1

[0315] From Table 8, the 21 polymyxin analogs treated with chymotrypsin (WP-AMP-219, WP-AMP-222, WP-AMP-223, WP-AMP-224, WP-AMP-225, WP-AMP-227, WP-AMP-229, WP-AMP-230, WP-AMP-231, WP-AMP-235, WP-AMP-239, WP-AMP-241, WP-AMP-242, WP-AMP-243, WP-AMP-246, WP-AMP-248, WP-AMP-249, WP-AMP-250, WP-AMP-251, WP-AMP-252, and WP-AMP-254) showed an MIC compared to the control group not treated with chymotrypsin. 90 It was found that the antimicrobial efficacy was maintained with almost no change in value. To confirm the results that 21 types of polymyxin analogs (WP-AMP-219, WP-AMP-222, WP-AMP-223, WP-AMP-224, WP-AMP-225, WP-AMP-227, WP-AMP-229, WP-AMP-230, WP-AMP-231, WP-AMP-235, WP-AMP-239, WP-AMP-241, WP-AMP-242, WP-AMP-243, WP-AMP-246, WP-AMP-248, WP-AMP-249, WP-AMP-250, WP-AMP-251, WP-AMP-252, and WP-AMP-254) possess stable antimicrobial efficacy without being degraded by proteases, under the same conditions as above A lead substance of 500 μg / ml was treated with a final concentration of 25 μg / ml of protease at 37°C for 4 hours.

[0316] This result shows that the 21 types of peptidomimetics remain in the same amount without being degraded even after treatment with trypsin and chymotrypsin, and the results were consistent with the MIC test results (Tables 5 and 6). Therefore, it was confirmed that the 21 types of polymyxin analogs can be maintained stably for a long time without a decrease in antibacterial efficacy against the proteolytic enzymes trypsin and chymotrypsin.

[0318] 2-6. Evaluation of safety against human serum

[0319] 100% and After adding to 50% human serum and incubating at 37°C for 16 hours, the change in antimicrobial efficacy was compared to the MIC of the control group. 90 It was verified by comparing with the value.

[0321] substance name MIC 90 (μg / ml) A. baumannii E. coli P. aeruginosa 100% 50% 0% 100% 50% 0% 100% 50% 0% 1 WP-AMP-219 1 1 1 1 1 1 1 1 1 2 WP-AMP-222 1 1 1 1 1 1 1 1 2 3 WP-AMP-223 1 1 1 1 1 1 1 1 1 4 WP-AMP-224 1 1 1 1 1 1 1 1 1 5 WP-AMP-225 1 1 1 1 1 1 1 1 1 6 WP-AMP-227 1 1 1 1 1 1 2 1 2 7 WP-AMP-229 1 1 1 1 1 1 1 1 1 8 WP-AMP-230 1 1 1 1 1 1 1 1 1 9 WP-AMP-231 1 1 1 1 1 1 1 1 1 10 WP-AMP-235 1 1 1 1 1 1 1 1 1 11 WP-AMP-239 1 1 1 1 1 1 1 1 1 12 WP-AMP-241 1 1 1 1 1 1 1 1 1 13 WP-AMP-242 1 1 1 1 1 1 1 1 1 14 WP-AMP-243 1 1 1 1 1 1 1 1 1 15 WP-AMP-246 1 1 1 1 1 1 1 1 1 16 WP-AMP-248 1 1 1 1 1 1 1 1 1 17 WP-AMP-249 1 1 1 1 1 1 1 1 1 18 WP-AMP-250 1 1 1 1 1 1 1 1 1 19 WP-AMP-251 1 1 1 1 1 1 1 1 1 20 WP-AMP-252 1 1 2 1 1 1 1 1 1 21 WP-AMP-254 1 1 1 1 1 1 1 1 1 Compound name CCARM A. baumannii CCARM E. coli CCARM P. aeruginosa 100% 50% 0% 100% 50% 0% 100% 50% 0% 1 WP-AMP-219 1 1 1 1 1 1 1 1 1 2 WP-AMP-222 1 1 1 1 1 1 4 4 4 3 WP-AMP-223 1 1 1 1 1 1 2 1 1 4 WP-AMP-224 1 1 1 1 1 1 1 1 1 5 WP-AMP-225 1 1 1 1 1 1 1 1 1 6 WP-AMP-227 2 2 2 1 1 1 1 1 1 7 WP-AMP-229 1 1 1 1 1 1 1 1 1 8 WP-AMP-230 1 1 1 1 1 1 4 4 1 9 WP-AMP-231 1 1 1 1 1 1 2 1 1 10 WP-AMP-235 1 1 1 1 1 1 1 1 1 11 WP-AMP-239 1 1 1 1 1 1 1 1 1 12 WP-AMP-241 1 1 1 1 1 1 2 2 1 13 WP-AMP-242 1 1 2 1 1 1 8 4 1 14 WP-AMP-243 1 1 1 1 1 1 1 1 1 15 WP-AMP-246 1 1 1 1 1 1 1 1 1 16 WP-AMP-248 1 1 1 1 1 1 4 4 4 17 WP-AMP-249 1 1 1 1 1 1 1 1 1 18 WP-AMP-250 1 1 1 1 1 1 1 1 1 19 WP-AMP-251 1 1 1 1 1 1 1 1 1 20 WP-AMP-252 1 1 1 1 1 1 1 1 1 21 WP-AMP-254 1 1 1 1 1 1 1 1 1

[0322] From Table 9, the MICs of 21 polymyxin analogs regardless of the treatment concentration of human serum 90 It was confirmed that the antibacterial efficacy was maintained with almost no change compared to the control group. This result indicates that stable antibacterial efficacy can be demonstrated without a decrease in efficacy due to degradation by various substances present in the blood or binding with such substances.

[0324] 2-7. Evaluation of Hemolytic Activity on Animal-Derived Red Blood Cells

[0325] Sheep blood red blood cells (RBCs) were centrifuged at 1000 xg for 5 minutes with PBS buffer, washed three times, and resuspended in PBS buffer. Seven selected polymyxin analogs (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) (final concentrations 500, 200, 100, and 50 μg / ml) were added to the resuspended red blood cells and incubated at 37°C for 1 hour, then centrifuged at 1000 xg for 5 minutes to precipitate the red blood cells, and the supernatant was collected. The absorbance of the supernatant was measured at 570 nm to calculate the degree of erythrocyte hemolysis as follows, and PBS and 0.2% Triton X-100 were used as negative and positive controls.

[0326] % hemolysis = 100 x [(A sample - A PBS ) / (A triton - A PBS )]

[0327] As a result of the test, the seven selected polymyxin analogs (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) showed hemolytic values ​​of 2.11, 5.03, 5.13, 4.49, 5.69, 5.06, and 6.78%, respectively, at the highest concentration of 500 µg / ml, confirming that they had very low toxicity to red blood cells, and at a concentration of 100 µg / ml, almost no hemolytic activity was observed in the seven lead compounds (Fig. 3).

[0329] 2-8. Two Gram-negative bacterial strains Evaluation of the occurrence of resistance to Korea

[0330] Acinetobacter baumannii and Pseudomonas aeruginosaThe bacterial suspension, cultured for 16 hours after inoculating each strain, was inoculated into 3 ml of MHB medium and incubated in a shaking incubator at 37°C and 150 rpm for 2 hours. Subsequently, absorbance was measured at 600 nm using a cuvette (a sample of at least 500 µl is required); once the target value (0.125–0.25) was reached, each dilution factor was calculated and diluted using 2X MHB. First, 100 µl of the peptide and antibiotic were dispensed into each 96-well plate, followed by 100 µl of the diluted bacterial suspension. Finally, the OD of the 96-well plate was measured using an ELISA plate reader. 600 After checking the value (shaking condition: 10 seconds), the incubator was incubated at 37℃ for 16 hours. After incubation, the OD 600 value The bacterial growth rate and the peptide's inhibitory effect were calculated based on verification (shaking condition: 1 min). The concentrations of the peptide and antibiotic corresponding to the well immediately below the well where no growth occurred were selected. After adding the selected peptide and antibiotic concentrations to the liquid medium, the bacterial strain was cultured for 16 hours. This series of processes was continuously repeated (subculture). A. baumannii The strain is Episode 19, P. Aeruginosa The strain is (Subcultured 16 times)

[0331] A. baumannii and P. Aeruginosa To evaluate the resistance of 7 selected novel peptides to two strains, experiments were conducted to test for changes in resistance by subculturing at the following concentrations based on the 0.5X MIC value, and the results are shown in Tables 10 and 11.

[0332] Peptide name 0.5X MIC concentration (µg / ml) Peptide name 0.5X MIC concentration (µg / ml) Peptide name 0.5X MIC concentration (µg / ml) WP-AMP-223 0.03125 WP-AMP-229 0.0078125 WP-AMP-235 0.015625 WP-AMP-243 0.0078125 WP-AMP-249 0.0078125 WP-AMP-250 0.0078125 WP-AMP-254 0.015625 Colistin 0.015625 Meropenem 0.125

[0333] Peptide name 0.5X MIC concentration (µg / ml) Peptide name 0.5X MIC concentration (µg / ml) Peptide name 0.5X MIC concentration (µg / ml) WP-AMP-223 0.25 WP-AMP-229 0.5 WP-AMP-235 0.125 WP-AMP-243 0.25 WP-AMP-249 0.25 WP-AMP-250 0.5 WP-AMP-254 0.5 Colistin 0.125 Meropenem 1

[0334] 7 types of lead compounds A. baumanniiThe results of the resistance evaluation on the strains (Table 10) showed that, as shown in Figures 4a and 4b, no resistance was confirmed for the remaining six peptides and colistin, excluding Meropenem and WP-AMP-229, until passage 19. On the other hand, P. Aeruginosa In the case of resistance evaluation of strains (Table 11), as a result of subculturing up to passage 16, no resistance was confirmed in the remaining 6 peptides and colistin, excluding Meropenem and WP-AMP-235.

[0335] 2-9. Evaluation of Nephrotoxicity of 7 Peptides

[0336] The nephrotoxicity of the seven selected polymyxin analogs (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) on HK-2 cell lines was evaluated using the WST-8 assay. 3 × 10⁴ cells per well were placed in a 96-well plate. 3 Dog cells were inoculated into RPMI1640 medium containing 10% FBS and cultured in a 5% CO2 incubator at 37°C for 24 hours. Seven polymyxin analogs and the antibiotics colistin, ciaplatin, and ciprofloxacin were diluted in 2-fold serial dilution and then inoculated into the cultured cells. WST-8 was added at 24, 48, and 72 hours, and after 2 hours of incubation, absorbance was measured at 450 nm using a microplate reader. The results of cell viability measured by absorbance are shown in Figures 5a, 5b, and 5c. Cytotoxicity was confirmed for two peptides, WP-AMP-223 and WP-AMP-249, only at a high concentration of 200 µg / ml at 72 hours, while no nephrotoxicity was confirmed for the remaining five peptides and colistin.

[0338] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 Polymyxin analogue compounds represented by the following chemical formula I, or pharmaceutically acceptable salts thereof: [Chemical Formula I] In the above chemical formula I, R1 to R7 are as shown in the table below. Claim 2 An antimicrobial composition comprising, as an active ingredient, a polymyxin analog compound represented by the chemical formula I of claim 1, or a pharmaceutically acceptable salt thereof. Claim 3 In paragraph 2, the above pharmaceutically acceptable salt is trifluoroacetic acid, An antibacterial composition characterized by being selected from the group consisting of hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, oxalic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, sodium, potassium, magnesium, and ammonium. Claim 4 In paragraph 2, the antibacterial composition is characterized by having antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria. Claim 5 In paragraph 4, the above Gram-negative bacteria are Acinetobacter baumannii ( Acinetobacter baumannii ), E. coli( Escherichia coli ), or Pseudomonas aeruginosa( Pseudomonas aeruginosa ) and the above-mentioned multidrug-resistant Gram-negative bacterium is multidrug-resistant Acinetobacter baumannii (MRAB; Multidrug-resistant Acinetobacter baumannii ), multidrug-resistant E. coli ( Escherichia coli ) or multidrug-resistant Pseudomonas aeruginosa (MRPA; Multidrug-resistant Pseudomonas aeruginosa An antibacterial composition characterized by being ). Claim 6 A pharmaceutical composition for the prevention or treatment of infectious diseases comprising, as an active ingredient, a polymyxin analog compound represented by the chemical formula I of claim 1, or a pharmaceutically acceptable salt thereof, wherein the infectious disease is a disease selected from the group consisting of pneumonia, peritonitis, meningitis, wound infection, osteoarthritis, cholecystitis, urinary tract infection, meningitis, myocarditis, pericarditis, arthritis, pharyngitis, gonorrhea, bacterial dysentery, enteritis, conjunctivitis, gastritis, otitis media, cystitis, lymphangitis, pharyngitis, impetigo, rheumatic fever, glomerulonephritis, neonatal sepsis, meningitis, pharyngitis, endocarditis, scarlet fever, skin soft tissue infection, deep soft tissue infection, empyema, and vaginitis. Claim 7 In paragraph 6, the above pharmaceutically acceptable salt is trifluoroacetic acid, A pharmaceutical composition for the prevention or treatment of infectious diseases, characterized by being selected from the group consisting of hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, oxalic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, sodium, potassium, magnesium, and ammonium. Claim 8 delete Claim 9 delete Claim 10 A pharmaceutical composition for the prevention or treatment of sepsis comprising, as an active ingredient, a polymyxin analog compound represented by the chemical formula I of claim 1, or a pharmaceutically acceptable salt thereof. Claim 11 In Paragraph 10, the pharmaceutically acceptable salt is triluacetic acid, A pharmaceutical composition for the prevention or treatment of sepsis, characterized by being selected from the group consisting of hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, oxalic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, sodium, potassium, magnesium, and ammonium. Claim 12 A method for preparing a cyclized polymyxin analog compound represented by Formula I, comprising the following steps: (a) obtaining a resin-attached peptide represented by Formula Ia by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected polymyxin analog represented by Formula Ib; (c) cyclizing the peptide obtained in step (b) using a catalyst by a solution-phase synthesis method to obtain a protected cyclized polymyxin analog represented by Formula Ic; and (d) performing a deprotection reaction on the peptide obtained in step (c) to obtain a cyclized polymyxin analog represented by Formula I: [Formula I] In the above chemical formula I, R1 to R7 are as shown in the table below. [Chemical Formula Ia] [Chemical Formula Ib] [Chemical Formula Ic] In the above chemical formula, A is a hydrogen or hydroxyl protecting group, and B is an amine protecting group. Claim 13 A method for preparing a polymyxin analog compound according to claim 12, wherein the acidic conditions for removing the peptide protected from the resin and optionally the amine protecting group (B) are selected from the group consisting of (i) a 2 to 5 volume% solution of trichloroacetic acid or trifluoroacetic acid; and (ii) a 0.1% solution of dichloromethane containing formic acid, methanesulfonic acid, benzenesulfonic acid, or para-toluenesulfonic acid, used alone or in combination. Claim 14 A method for preparing a polymyxin analog compound according to claim 12, characterized in that the cyclization reaction temperature is carried out at -20 to 50℃. Claim 15 In paragraph 12, the concentration of the protected peptide in the cyclization reaction is 10 when converted to peptides -2 10 at the mall -5 A method for preparing a polymyxin analog compound characterized by being performed within a molar concentration range. Claim 16 A method for preparing a polymyxin analog compound according to claim 12, characterized in that the solvent for the cyclization reaction of the peptide is selected from the group consisting of 1,2-dichloroethane, chloroform, dichloromethane, 1,2-dichloromethane, tetrahydrofuran, 1,4-dioxane, acetonitrile, dimethylsulfoxide, N,N-dimethylformamide, and N,N-dimethylacetamide, used alone or in combination. Claim 17 A method for preparing a polymyxin analog compound according to claim 12, characterized in that the acidic solution for the deprotection reaction uses a mixture comprising trifluoroacetic acid : purified water (95:5).

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