Novel polymyxin analogue exhibiting excellent antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and antibacterial composition comprising same

Abstract

The present invention relates to a polymyxin analogue exhibiting excellent antibacterial effect against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and an antibacterial composition containing same for treating infectious diseases. More specifically, the present invention relates to a cyclized polymyxin analogue exhibiting excellent antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and an antibacterial composition containing same. The cyclized polymyxin analogue compound according to the present invention is represented by chemical formula I. The polymyxin analogue compound of the present invention has an excellent antibacterial effect against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and has excellent in vivo stability when applied to the human body, and thus is very useful in related industrial fields such as those of medicine, cosmetics, health functional foods and the like.

Description

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

[0001] The present invention relates to a novel polymyxin analogue having excellent antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and an antibacterial composition comprising the same, and more particularly, to a cyclized polymyxin analogue having excellent antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and an antibacterial composition comprising the same.

[0002] Peptides, a core ingredient in biotechnology, are the functional smallest units of proteins, consisting of between 2 and 50 amino acids. Not only do they exhibit excellent efficacy in small quantities, but they are also non-toxic, making them widely used as key ingredients in pharmaceuticals, foods, and cosmetics. Peptide cosmetic ingredients, in particular, are safe and effective for the human body, and their use is increasing day by day. The development of new materials is crucial for the sugar industry, as they can provide import substitution benefits.

[0003] Polymyxin is a lipopeptide antibiotic that has excellent therapeutic effects on gram-negative bacterial infections. It was discovered about 60 years ago, but despite its excellent bactericidal power, its use was limited due to its toxicity to humans (nephrotoxicity and neurotoxicity). Recently, it has been receiving renewed attention as an antibiotic, but resistant bacteria are emerging due to long-term use.

[0004] Despite this toxicity, various attempts are being made to develop new treatments by overcoming the drug's shortcomings through modification of the sequence and structure based on this chemical structure due to its excellent antibacterial effect.

[0005] Additionally, antibiotics that are used for a long period of time can cause the emergence of resistant bacteria, and antibiotic-resistant diseases refer to infectious diseases caused by bacteria that are resistant to antibiotics, and in particular, multidrug resistance refers to showing resistance to multiple types of antibiotics.

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

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

[0008] Meanwhile, multidrug-resistant organisms are pathogens that are resistant to several types of powerful antibiotics, and representative examples include vancomycin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Acinetobacter baumani.

[0009] Korean Patent No. 10-1502453 discloses various polymyxin derivatives and their antibacterial efficacy, Korean Patent No. 10-1627139 discloses short fatty acid tail polymyxin derivatives and their uses, and Korean Patent Publication No. 10-2014-0091724 discloses a method for preparing polymyxin sulfates. However, these derivatives have a completely different structure from the polymyxin analogues mentioned in the present invention, target different Gram-negative bacteria, and have the disadvantage of having lower antibacterial efficacy.

[0010] Accordingly, the inventors of the present invention completed the present invention by confirming that a new polymyxin analogue having a specific sequence and structure has excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria in the process of researching and developing a new substance by modifying the existing structure of polymyxin.

[0011] The purpose of the present invention is to provide a polymyxin analogue having antibacterial activity against gram-negative bacteria or multidrug-resistant gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0012] Another object of the present invention is to provide an antibacterial composition comprising, as an active ingredient, a polymyxin analogue having antibacterial 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 a pharmaceutical composition for preventing or treating infectious diseases, comprising as an active ingredient a polymyxin analogue having antibacterial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating sepsis, comprising as an active ingredient a polymyxin analogue having antibacterial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria, or a pharmaceutically acceptable salt thereof.

[0015] Another object of the present invention is to provide a method for producing a polymyxin analogue having antibacterial activity against Gram-negative bacteria or multidrug-resistant Gram-negative bacteria.

[0016] To achieve the above purpose, the present invention provides a polymyxin analogue represented by the following chemical formula I, or a pharmaceutically acceptable salt thereof.

[0017] [Chemical Formula I]

[0018]

[0019] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0020]

[0021] The present invention also provides an antibacterial composition comprising a polymyxin analogue represented by chemical formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0022] The present invention also provides a pharmaceutical composition for preventing or treating infectious diseases, comprising a polymyxin analogue represented by chemical formula I or a pharmaceutically acceptable salt thereof as an active ingredient.

[0023] The present invention also provides a pharmaceutical composition for preventing or treating urinary tract infection, pneumonia, or sepsis, comprising a polymyxin analogue represented by chemical formula I or a pharmaceutically acceptable salt thereof as an active ingredient.

[0024] The present invention also provides a method for preparing a polymyxin analog compound represented by Chemical Formula I, comprising the steps of: (a) obtaining a peptide having a resin represented by Chemical Formula Ia attached thereto by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in step (a) to obtain a protected peptide represented by Chemical Formula Ib; (c) subjecting the peptide obtained in step (b) to a cyclization reaction using a catalyst by a solution-phase synthesis method to obtain a protected peptide represented by Chemical Formula Ic; and (d) performing a deprotection reaction on the peptide in step (c) to obtain a polymyxin analogue represented by Chemical Formula I.

[0025] [Chemical Formula I]

[0026]

[0027] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0028]

[0029] [Chemical Formula Ia]

[0030]

[0031] [Chemical Formula Ib]

[0032]

[0033] [Chemical formula Ic]

[0034]

[0035] In the above chemical formula, A is a hydrogen or hydroxyl protecting group, and B is an amine protecting group.

[0036] The polymyxin analogue of the present invention has excellent antibacterial effects against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria, and has excellent stability in the body when applied to the human body, and is therefore very useful in related industrial fields such as pharmaceuticals, cosmetics, and health functional foods.

[0037] In addition, the polymyxin analogue according to the present invention is not only safe for the human body, but also has excellent antibacterial effect and excellent stability against protein-decomposing enzymes, and thus can be utilized for various purposes.

[0038] Figure 1 is a synthetic process diagram of polymyxin analogue Ⅰ according to one embodiment of the present invention.

[0039] Figures 2a, 2b and 2c are data evaluating the cytotoxicity of polymyxin analogue Ⅰ according to the present invention against NIH3T3, HEK 293 and RAW246.7 cell lines.

[0040] Figure 3 shows the results of evaluating the hemolytic activity of polymyxin analogue Ⅰ according to the present invention.

[0041] Figures 4a to 4n show the results of evaluating the resistance inhibition of polymyxin analogue Ⅰ according to the present invention.

[0042] Figures 5a, 5b and 5c show the results of evaluating the renal cytotoxicity of polymyxin analogue Ⅰ according to the present invention.

[0043] Unless otherwise specified herein, the abbreviations used to designate amino acids and protecting groups are based on the terminology 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).

[0044]

[0045] The abbreviations for solvents, reagents, protecting groups, and amino acids used in this specification are as follows:

[0046] ACN: Acetonitrile

[0047] t-Bu: tert-Butyl

[0048] Cha: Cyclohexylalanine

[0049] DCM: Dichloromethane

[0050] DMSO: Dimethylsulfoxide

[0051] DTT: Dithiolthreitol

[0052] Dab: 2,4-Diaminobutyric acid

[0053] Dap: 2,3-Diaminopropionic acid

[0054] Dde: 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene

[0055] hLeu: Homoleucine

[0056] EDT: 1,2-Ethanedithiol

[0057] His: Histidine

[0058] HPLC: High Performance Liquid Chromatography

[0059] Fmoc: 9-Fluorenyloxycarbonyl

[0060] Lys: Lysine

[0061] 2-Nal: 2-Naphthylalanine

[0062] Nle: Norleucine

[0063] Orn: Ornithine

[0064] TES: Triethylsilane

[0065] TFA: Trifluoroacetic acid

[0066] Thr: Threonine

[0067] TIPS: Triisopropylsilane

[0068]

[0069] In the present invention, in the process of researching and developing a polymyxin having excellent antibacterial activity against Gram-negative bacteria, various polymyxin analogues having a specific sequence were produced, and it was confirmed that these have excellent antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria.

[0070]

[0071] Accordingly, the present invention relates, in one aspect, to a polymyxin analogue represented by the following chemical formula I, or a pharmaceutically acceptable salt thereof.

[0072] [Chemical Formula I]

[0073]

[0074] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0075]

[0076] More specifically, the polymyxin analogue represented by the above chemical formula I is a compound represented by the following chemical formulas I-1 to I-21.

[0077] [Chemical Formula I-1]

[0078]

[0079] [Chemical Formula I-2]

[0080]

[0081] [Chemical Formula I-3]

[0082]

[0083] [Chemical Formula I-4]

[0084]

[0085] [Chemical Formula I-5]

[0086]

[0087] [Chemical Formula I-6]

[0088]

[0089] [Chemical Formula I-7]

[0090]

[0091] [Chemical Formula I-8]

[0092]

[0093] [Chemical Formula I-9]

[0094]

[0095] [Chemical Formula I-10]

[0096]

[0097] [Chemical Formula I-11]

[0098]

[0099] [Chemical Formula I-12]

[0100]

[0101] [Chemical Formula I-13]

[0102]

[0103] [Chemical Formula I-14]

[0104]

[0105] [Chemical Formula I-15]

[0106]

[0107] [Chemical Formula I-16]

[0108]

[0109] [Chemical Formula I-17]

[0110]

[0111] [Chemical Formula I-18]

[0112]

[0113] [Chemical Formula I-19]

[0114]

[0115] [Chemical Formula I-20]

[0116]

[0117] [Chemical Formula I-21]

[0118]

[0119] In the present invention, the pharmaceutically acceptable salt of the polymyxin analogue represented by the above chemical formula I includes an acid addition salt formed by a pharmaceutically acceptable free acid and a pharmaceutically acceptable metal salt.

[0120] Examples of suitable acids include trifluoroacetic acid, hydrochloric acid, hydrobromic 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, and benzenesulfonic acid.

[0121] Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0122]

[0123] From another perspective, the present invention relates to an antibacterial composition comprising a polymyxin analogue represented by the above chemical formula I, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0124] The above antibacterial composition is characterized by having antibacterial activity against Gram-negative bacteria and multidrug-resistant Gram-negative bacteria.

[0125] In the present invention, "gram negative bacteria" refers to bacteria that are stained red when stained with the Gram staining method. The gram negative bacteria of the present invention include, but are not limited to, strains of the genus Escherichia, the genus Pseudomonas, the genus Acinetobacter, the genus Salmonella, the genus Klebsiella, the genus Neisseria, the genus Enterobacter, the genus Shigella, the genus Moraxella, the genus Helicobacter, the genus Stenotrophomonas, the genus Bdellovibrio, and the genus Legionella. Specifically, Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas chlororaphis, Pseudomonas pertucinogena, Pseudomonas stutzeri, Pseudomanas syringae, Acinetobacter baumannii, Acinetobacter lwoffii, Acinetobacter calcoaceticus, and Acinetobacter haemolyticus. (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, Moraxella lacunata, Moraxella bovis, Helicobacter pylori, Helicobacter heilmannii, Helicobacter felis, Helicobacter musttelae, Helicobacter fenelliae, Helicobacter rapini,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 pilei These include, but are not limited to, Legionella feeleii, Legionella gormanii, Legionella hackeliae, Legionella israelensis, Legionella jordanis, Legionella lansingensis, Legionella longbeachae, Legionella maceachernii, Legionella micdadei, Legionella oakridgensis, Legionella sainthelensi, Legionella tucsonensis, and Legionella wadsworthii.

[0126] In the present invention, “antibiotic-resistant Gram-negative bacteria” include, but are not limited to, multidrug-resistant Acinetobacter baumannii (MRAB), multidrug-resistant Escherichia coli, and multidrug-resistant Pseudomonas aeruginosa (MRPA).

[0127]

[0128] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating infectious diseases, comprising a polymyxin analogue represented by chemical formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0129] 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, tonsillitis, gonorrhea, shigellosis, 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.

[0130]

[0131] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating sepsis, comprising a polymyxin analogue represented by chemical formula I, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0132] In the present invention, "sepsis" refers to a condition in which a severe inflammatory response occurs throughout the body due to infection by a microorganism. When two or more of the following symptoms are present: fever with a body temperature of 38℃ or higher or hypothermia with a body temperature of 36℃ or lower, respiratory rate of 24 beats per minute or higher (tachypnea), heart rate of 90 beats per minute or higher (tachycardia), and increased or significantly decreased white blood cell count in a blood test, this is called systemic inflammatory response syndrome (SIRS). When this systemic inflammatory response syndrome is caused by infection by a microorganism, it is called sepsis. Pathogens continuously or intermittently enter the bloodstream from an infectious lesion in the body, settle in various organ tissues, create lesions, and cause severe systemic symptoms. Causative bacteria include, but are not limited to, Staphylococcus aureus, Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Klebsiella pneumoniae, fungi, and anaerobic bacteria.

[0133]

[0134] The pharmaceutical composition of the present invention may further comprise 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, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, according to conventional methods, but is not limited thereto.

[0135] 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, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0136] When formulating the pharmaceutical composition of the present invention, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include, but are not limited to, witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0137] The pharmaceutical composition of the present invention can be used for skin, injection, subcutaneous injection, oral preparation, etc.

[0138] The content of the polymyxin analogue represented by the chemical formula I included in the pharmaceutical composition of the present invention can be appropriately adjusted depending on the intended use, application form, purpose of use, and desired effect, and considering the effect relative to the content, it can be used, for example, within 0.0001 to 99.9 wt% based on the total weight of the composition.

[0139] 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 by topical application through transdermal application and application.

[0140]

[0141] Meanwhile, in the present invention, a peptide (chemical formula Ia) attached to a resin was obtained by a solid-phase synthesis method using an amino acid, then the resin was removed to obtain a protected peptide (chemical formula Ib), and a cyclization reaction was performed using a catalyst by a solution-phase synthesis method to obtain a cyclized peptide (chemical formula Ic), which was then deprotected to finally synthesize a cyclized peptidomimetic (chemical formula I). ​​As a result, it was confirmed that a cyclized peptidomimetic could be synthesized in a high yield that enabled commercial mass production.

[0142] Accordingly, in one aspect, the present invention relates to a method for producing a polymyxin analogue represented by the formula (I), comprising the steps of: (a) obtaining a peptide having a resin represented by the formula (Ia) attached thereto by a solid-phase synthesis method; (b) removing the resin from the peptide obtained in the step (a) to obtain a protected peptide represented by the formula (Ib); (c) subjecting the peptide obtained in the step (b) to a cyclization reaction using a catalyst by a solution-phase synthesis method to obtain a protected polymyxin analogue represented by the formula (Ic); and (d) performing a deprotection reaction on the peptide obtained in the step (c) to obtain the polymyxin analogue represented by the formula (I).

[0143] [Chemical Formula I]

[0144]

[0145] In the above chemical formula I, R1 to R7 are as shown in the table below.

[0146]

[0147] [Chemical Formula Ia]

[0148]

[0149] [Chemical Formula Ib]

[0150]

[0151] [Chemical formula Ic]

[0152]

[0153] In the above chemical formula, A is a hydrogen or hydroxyl protecting group, and B is an amine protecting group.

[0154]

[0155] In the above chemical formula, A can use a hydrogen or hydroxyl protecting group commonly used in the art. The above hydroxyl protecting group may be exemplified by a tert-butyl group, a triphenylmethyl group, an acetaminomethyl group, a benzoyl group, a diphenylmethyl group, a benzyl group, a para-methoxybenzyl group, a benzyloxycarbonyl group, a para-nitrobenzyl group, an allyl group, a dimethylsilyl group, a tert-butyldimethylsilyl group, a triisopropylsilyl group, an alkyl group (10 carbon atoms or less), etc., and a tert-butyl group, a triphenylmethyl group, or an acetaminomethyl group is preferable, and a tert-butyl group is preferable. It is more preferable to use the (tert-butyl) group.

[0156] In the above chemical formula, B can use an amine protecting group commonly used in the art. 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 (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 the 6-dioxocyclohex-1-ylidene)-3-mehtylbutyl (ivDde)) group, and it is most preferable to use the Dde ((1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl) group as the amine protecting group.

[0157] Protecting groups for the above functional groups are 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).

[0158] As used herein, the term “peptide” means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0159] Referring to Figure 1, the manufacturing method of the present invention is described in detail step by step as follows.

[0160] (a) Obtaining a peptide attached to a resin represented by the chemical formula Ia

[0161] The resin-attached peptide represented by the chemical formula Ia can be prepared by a solid-phase synthesis method commonly used in the art (Merrifield, RB, J. Am. Chem. Soc., 85:2149-2154(1963), Kaiser, E., Colescot, RL, Bossinger, CD, Cook, PI, Anal. Biochem., 34:595-598(1970)). That is, after the alpha-amino and side-chain functional groups are protected amino acids are attached to the resin, the alpha-amino protecting group is removed, and the remaining alpha-amino and side-chain functional groups are protected amino acids are stepwise coupled in a desired order to obtain an intermediate.

[0162] The present invention is characterized in that the fourth amino acid in the peptide sequence uses an amine protecting group of 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 protected peptide is attached to the resin.

[0163] The choice of an appropriate protecting group depends on the functional group being protected, the conditions under which the protecting group will be exposed, and other functional groups that may be present in the molecule. The protecting group must be stable to the reaction conditions and reagents chosen to remove the alpha-amino protecting group at each step of the synthesis, must not undergo deprotection during the coupling reaction, and must be stable under the conditions of degradation with the resin when the synthesis containing the desired amino acid chain is completed.

[0164] According to a preferred embodiment of the present invention, a resin that can be used in the process of synthesizing a peptide represented by Chemical Formula I-a may be a conventional resin that can be easily decomposed under mild acidic conditions that 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 or HMBA resin, more preferably Wang resin, tritylchloride resin or 2-chlorotritylchloride resin, and most preferably 2-chlorotritylchloride resin.

[0165]

[0166] (b) Obtaining a peptide represented by chemical formula Ib

[0167] The compound represented by the above chemical formula Ib 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 that can maintain the side chain protecting group of the amino acid chain.

[0168] According to a preferred embodiment of the present invention, the process of removing the resin can be performed in the presence of an acidic solution. The above acidic solution may be a single solution of 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, methanol, ethanol, isopropanol, trifluoroethanol, etc., or a mixed solution thereof.

[0169] Preferably, the acidic conditions may be exemplified by the use of a 2 to 5 volume % trichloroacetic acid solution, a 2 to 5 volume % trifluoroacetic acid solution, a dichloromethane solution containing 0.1% formic acid, methanesulfonic acid, benzenesulfonic acid or para-toluenesulfonic acid, alone or in combination.

[0170]

[0171] (c) Obtaining a cyclized peptide represented by the chemical formula Ic

[0172] The compound represented by chemical formula Ic is obtained by performing a cyclization reaction on the peptide obtained in step (b) in the presence of a catalyst.

[0173] According to a preferred embodiment of the present invention, the catalyst 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), HOAt (1-Hydroxyazabenzotriazole), and it is most preferable to use EDC·HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), HOAt (1-Hydroxyazabenzotriazole).

[0174] 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, tetrahydrofurane, 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.

[0175] The above cyclization reaction can be performed at -20 to 50°C, and is preferably performed at 0 to 30°C. In addition, the concentration of the cyclization reaction is 10 converted to peptide. -2 10 in the mall -9 It can be performed within the molar concentration range, preferably 10 -2 10 in the mall -5 It is desirable to perform the experiment at molar concentration, but is not limited thereto.

[0176]

[0177] (d) Obtaining a polymyxin analogue represented by chemical formula I

[0178] A polymyxin analogue represented by chemical formula I can be obtained by performing a deprotection reaction under reaction conditions commonly used in the art from the peptide obtained in step (c).

[0179] The deprotection reaction can be carried out with (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; (4) a mixture of trifluoroacetic acid, dithiothreonitol (DTT), 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, 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 the process under a mixture of trifluoroacetic acid and water (H2O).

[0180]

[0181] [Example]

[0182] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0183]

[0184] Throughout this specification, "%" 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, unless otherwise stated.

[0185]

[0186] Example 1: Synthesis of polymyxin analogues

[0187] 1-1: Synthesis of a polymyxin analogue represented by chemical formula I-1

[0188] [Chemical Formula I-1]

[0189]

[0190] 1-1-1: Synthesis of H-Thr(tBu)-2-chlorotrityl resin

[0191]

[0192] 2-Chlorotrityl chloride resin (100 mmol of resin with substitution degree = 1.08 mmol / g) and dichloromethane (1000 ml) were placed in a solid-phase synthesis reactor equipped with a filter membrane, and the resin was allowed to swell for 15 minutes, after which the solvent was removed through the filter 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 equiv) was added, followed by N,N-diisopropylethylamine (molecular weight = 129.25 g / mol) (32.31 g, 250 mmol, 2.5 equiv), and the mixture was reacted at room temperature for 12 hours. The reaction solution was removed by vacuum filtration, and the resin was washed once with dichloromethane. 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 the resin was washed three times with dichloromethane. Then, N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0193]

[0194] 1-1-2: Synthesis of H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0195]

[0196] To the H-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, and the mixture was reacted at room temperature for 4 hours.

[0197] 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, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0198]

[0199] 1-1-3: Synthesis of H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0200]

[0201] To the H-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, and the mixture was reacted at room temperature for 4 hours.

[0202] Next, the reaction solution was removed by vacuum filtration, and the resin was washed twice with N,N-dimethylformamide (1000 ml), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0203]

[0204] 1-1-4: Synthesis of H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0205]

[0206] To the H-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0208]

[0209] 1-1-5: Synthesis of HD-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0210]

[0211] To the H-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times with N,N-dimethylformamide (1000 ml) to obtain HD-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (yield >98%).

[0213]

[0214] 1-1-6: Synthesis of H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0215]

[0216] To the HD-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times 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%).

[0218]

[0219] 1-1-7: Synthesis of H-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0220]

[0221] To the H-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times 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%).

[0223]

[0224] 1-1-8: Synthesis of H-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin

[0225]

[0226] To the H-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times 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%).

[0228]

[0229] 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

[0230]

[0231] To the H-Dab(Boc)-Dab(Dde)-Dab(Boc)-D-Leu-Leu-Dab(Boc)-Dab(Boc)-Thr(tBu)-2-chlorotrityl resin (100 mmol) obtained in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times 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%).

[0233]

[0234] 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

[0235]

[0236] 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, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added, and then the mixture was stirred at room temperature. It was reacted 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), and then N,N-dimethylformamide (1000 ml) containing 20% ​​(v / v) piperidine was added to perform the Fmoc removal reaction for 15 minutes, and then the reaction solution was removed by vacuum filtration. The Fmoc removal reaction was repeated once, and then the resin was sequentially washed six times 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%).

[0238]

[0239] 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

[0240]

[0241] 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 in the above reaction, 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) in N,N-dimethylformamide (1000 ml) were added, and then N,N-diisopropylcarbodiimide (molecular weight = 126.20 g / mol, 41.65 g, 3.3 equivalents) was added. Afterwards, the reaction was allowed to proceed at room temperature for 4 hours.

[0242] Next, the reaction solution was removed by filtration under reduced pressure, and the resin was washed twice with N,N-dimethylformamide (1000 ml), then 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%).

[0243]

[0244] 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

[0245]

[0246] 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 in the above reaction, stirred for 3 minutes, and then the reaction solution was removed under reduced pressure. Additionally, 1000 ml of 2% Hydrazine monohydrate / DMF solution was added once more, stirred for 3 minutes, and the reaction solution was discharged. The resin was washed twice with dichloromethane (1000 ml), and then dichloromethane (1000 ml) and 2% trifluoroacetic acid (20 ml) were added and stirred for 5 minutes. After removing the dichloromethane reaction solution, the reaction was repeated once more, and the solutions were removed and combined. The filtrate was concentrated under reduced pressure to obtain 166.34 g (molecular weight: 1798.25 g / mol) 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 (yield: 92.5%, purity after deprotection: 78.4%).

[0247]

[0248] 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 analogue with a protecting group

[0249]

[0250] (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 stirred at room temperature for 12 hours. The volume was reduced to 3 / 1 or less by concentration under reduced pressure, then crystallized in 2500 ml of ethyl ether, the precipitated solid was filtered, and dried under vacuum to obtain 154.7 g (yield: 93.0%, purity after deprotection: 35.4%) of a polymyxin analogue (molecular weight: 1780.24 g / mol) with a protecting group.

[0251]

[0252] 1-1-14: Synthesis of polymyxin analogue of chemical formula I-1

[0253]

[0254] The polymyxin analogue (154.7 g) having a protecting group obtained from the above reaction was added to a mixture (3000 ml) of trifluoroacetic acid: purified water = 95:5 and stirred at room temperature for 3 hours. The reaction solution was slowly poured into 9000 ml of ethyl ether to precipitate the peptide and stirred for 30 minutes. The produced peptide was filtered under reduced pressure and dried under vacuum to obtain 22.6 g (theoretical amount 115.34 g, yield: 19.6%, purity: 35.4%, purity after purification 95.5%) of the polymyxin analogue of the chemical formula I-1.

[0255] For reference, the peptide represented by the above chemical formula I-1 is one in which 3-Cyclohexyl propionic acid is bound to the amino acid sequence represented by sequence number 1 (Dab-Thr-Dab-Dab-Dab-Dab-D-Leu-Leu-Dab-Dab-Thr(4-10 cyclized)).

[0256]

[0257] 1-2: Synthesis of a polymyxin analogue represented by chemical formula I-2

[0258] [Chemical Formula I-2]

[0259]

[0260] The synthesis of the I-2 polymyxin analogue was carried out in the same manner as the synthesis of the polymyxin analogue represented by the chemical formula I-1, except that the first amino acid in the synthesis of the compound of I-1 was Fmoc-Dap(Boc)-OH instead of Fmoc-Dab(Boc)-OH.

[0261] As a result of the synthesis, the synthesis was started at the same synthesis scale, and 19.4 g (theoretical amount 115.34 g, yield: 16.8%, purity: 29.8%, purity after purification 97.2%) of a polymyxin analogue represented by the chemical formula I-2 (molecular weight: 1153.42 g / mol) was obtained.

[0262] 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 sequence number 2 (Dab-Thr-Dab-Dab-Dap-D-Leu-Leu-Dab-Dab-Thr(4-10 cyclized)).

[0263]

[0264] 1-3: Synthesis of polymyxin analogues represented by chemical formulas I-3 to I-21

[0265] The synthesis of polymyxin analogues represented by chemical formulas I-3 to I-21 was carried out using the same synthetic method as that of polymyxin analogues represented by chemical formulas I-1 to I-2, and the results shown in the table below were obtained.

[0266] Chemical Structure TheoryMolecular Weight (Da) MeasurementMolecular Weight (Da)Yield (%)Purity (%)I-3 1201.461202.1715.495.57I-4 1207.511208.3321.295.28I-5 1251.521251.3020.395.35I-6 1207.511208.0219.897.54I-7 1167.441168.3926.795.74I-8 1181.471180.3914.998.51I-9 1195.51196.2830.199.80I-10 1153.421154.1922.798.47I-11 1187.431188.1326.495.60I-12 1215.491216.3122.195.14I-13 1229.511230.2225.495.17I-14 1187.431188.2111.396.11I-15 1187.431188.1315.995.02I-16 1241.521242.3427.298.27I-17 1215.491215.9026.495.73I-18 1201.461202.1128.395.36I-19 1215.491216.4320.996.44I-20 1229.511230.3318.595.58I-21 1187.431188.3230.295.78

[0267] The peptides represented by the above chemical formulas I-3 to I-21 are those in which 3-Cyclohexyl propionic acid is bound to an amino acid sequence represented by SEQ ID NO: 3 to SEQ ID NO: 21.

[0268] Sequence numberSequence notesSequence number 3Dab-Thr-Dab-Dab-Dab-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr4-10 cyclizedSequence number 4Dab-Thr-Dab-Dab-Dab-Dab-D-Cha-Leu-Dab-Dab-Thr4-10 cyclizedSequence number 5Dab-Thr-Dab-Dab-Dab-Dab-D-(2-Nal)-Leu-Dab-Dab-Thr4-10 cyclizedSequence number 6Dab-Thr-Dab-Dab-Dab-Dab-D-Leu-Cha-Dab-Dab-Thr4-10 cyclizedSequence number 7Dab-Thr-Dab-Dab-Dab-D-Leu-Nle-Dab-Dab-Thr4-10 cyclizedSequence number 8Dab-Thr-Dab-Dab-Dab-Dab-Dab-D-Leu-Leu-Orn-Dab-Thr4-10 cyclized SEQ ID NO: 9Dab-Thr-Dab-Dab-Dab-D-Leu-Leu-Lys-Dab-Thr4-10 cyclized SEQ ID NO: 10Dab-Thr-Dab-Dab-Dab-Dab-D-Leu-Leu-Dap-Dab-Thr4-10 cyclized SEQ ID NO: 11Dap-Thr-Dab-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 12Dab-Thr-Orn-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 13Dab-Thr-Lys-Dab-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 14Dab-Thr-Dap-Dab-Dab-D-Phe-Leu-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 15Dab-Thr-Dab-Dab-Dap-D-Phe-Leu-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 16Dab-Thr-Dab-Dab-Dab-D-Phe-Cha-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 17Dab-Thr-Dab-Dab-Dab-D-Phe-hLeu-Dab-Dab-Thr4-10 cyclized SEQ ID NO: 18Dab-Thr-Dab-Dab-Dab-Dab-Dab-D-Phe-Nle-Dab-Dab-Thr4-10 cyclized sequence number19Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Orn-Dab-Thr4-10 cyclized SEQ ID NO. 20Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Lys-Dab-Thr4-10 cyclized SEQ ID NO. 21Dab-Thr-Dab-Dab-Dab-D-Phe-Leu-Dab-Dap-Thr4-10 cyclized

[0269] Example 2: In-vitro efficacy evaluation of polymyxin analogues

[0270] 2-1. Strain information

[0271] Standard strains of A. baumannii (ATCC 19606), E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853) were purchased from the American Type Culture Collection (ATCC) through the National Pathogen Resources Bank of the Pathogen Resources Management Division of the National Institute of Infectious Diseases. Corresponding multidrug-resistant strains were provided by the Korea National Research Resources Center (KNRRC). Antibiotic resistance information for multidrug-resistant strains is shown in Table 3.

[0272]

[0273] Species name Bank registration number Resistance Antibiotic name A. baumanniiCCARM 12233AMK, CAZ, CPM, CTX, GM, LEV, MEM, PIPE. coliCCARM 1G528AMK, AMP, AZT, CAZ, CTX, FOX, GM, LEV, PIP, TMP. aeruginosaCCARM 2328AMK, AZT, CAZ, CPM, IMP, LEV, MEM, PIP

[0274] 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. MICs for colistin, meropenem, and ceftazidime against standard strains A. baumannii (ATCC19606), E. coli (ATCC 25922), and P. aeruginosa (ATCC 27853), and corresponding multidrug-resistant strains A. baumannii (CCARM 12233), E. coli (CCARM 1G528), and P. aeruginosa (CCARM 2328). 90 After confirming the value, it was used to derive a lead substance for the development of a novel cyclized peptidomimetic antibiotic.

[0275]

[0276] AntibioticsMIC 90 (μg / ml)A. baumannii(ATCC19606)E. coli(ATCC 25922)P. aeruginosa (ATCC 27853)CCARMA. baumannii (CCARM 12233)CCARME. coli(CCARM 1G528)CCARMP. aeruginosa(CCARM 2328)Colistin111111Meropenem1143218Ceftazidime812>641616

[0277] The standard strain used, A. baumannii (ATCC19606), was confirmed to have some resistance to ceftazidime. The multidrug-resistant strains CCARMA. baumannii and CCARM P. aeruginosa were confirmed to have resistance to ceftazidime and meropenem, antibiotics widely used in clinical practice, while CCARME. coli was confirmed to have resistance only to ceftazidime.

[0278] 2-2. Antibacterial test

[0279] In order to evaluate the antibacterial efficacy of the polymyxin analogue compounds of chemical formulas I-1 to I-21 manufactured in the examples, the MIC (minimum inhibitory concentration) method was used. For this purpose, 7 × 10 of each test strain was placed on a polystyrene microarray plate. 5 After diluting the medium with MHB culture medium to CFU / ml and distributing, 21 candidate substances were added by diluting them 2-fold starting from a concentration of 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.

[0280] As control antibiotics for measuring 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.

[0281] Material name MIC 90(μg / ml)A. baumanniiE. coliP. aeruginosaCCARMA. baumanniiCCARME. coliCCARMP. aeruginosa1WP-AMP-2191111112WP-AMP-2221121143WP-AMP-2231111114WP-AMP-2241111115WP-AMP-2251111116WP-AMP-2271111117WP-AMP-2291111118WP-AMP-2301111119WP-AMP-23111111110WP-AMP-23511111111WP-AMP-23911111112WP-AMP-24111111113WP-AMP-24211121114WP-AMP-24311111115WP-AMP-24611111116WP-AMP-24811111117WP-AMP-24911111118WP-AMP-25011111119WP-AMP-25111111120WP-AMP-25211111121WP-AMP-254111111

[0282] From Table 5, Chemical Formula I-1 (hereinafter referred to as WP-AMP-219), Chemical Formula I-2 (hereinafter referred to as WP-AMP-222), Chemical Formula I-3 (hereinafter referred to as WP-AMP-223), Chemical Formula I-4 (hereinafter referred to as WP-AMP-224), Chemical Formula I-5 (hereinafter referred to as WP-AMP-225), Chemical Formula I-6 (hereinafter referred to as WP-AMP-227), Chemical Formula I-7 (hereinafter referred to as WP-AMP-229), Chemical Formula I-8 (hereinafter referred to as WP-AMP-230), Chemical Formula I-9 (hereinafter referred to as WP-AMP-231), Chemical Formula I-10 (hereinafter referred to as WP-AMP-235), Chemical Formula I-11 (hereinafter referred to as WP-AMP-239), Chemical Formula I-12 (hereinafter referred to as WP-AMP-241), Chemical Formula I-13 (hereinafter referred to as WP-AMP-242), Chemical Formula I-14 (hereinafter referred to 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), chemical formula I-21 (hereinafter referred to as WP-AMP-254) are not only Acinetobacter baumannii, Escherichia coli, or Pseudomonas aeruginosa, but also multidrug-resistant Gram-negative bacteria, such as multidrug-resistant Acinetobacter baumannii (MRAB), multidrug-resistant Acinetobacter baumannii It was confirmed that it also had antibacterial activity against Escherichia coli and multidrug-resistant Pseudomonas aeruginosa (MRPA).

[0283] 2-3. Cytotoxicity test

[0284] Cytotoxicity of seven selected polymyxin analogues (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) against NIH3T3, HEK293, and RAW246.7 cell lines was evaluated using the WST-8 assay. 3 × 10 per well in a 96-well plate 4 After dispensing the cells using DMEM containing 10% FBS, they were cultured in a 5% CO2 incubator at 37℃ for 24 hours. Each polymyxin analog was added to the final concentrations of 100, 50, 25, 10, 5, and 1 ㎍ / ml, and cultured at 37℃ for 24 hours, and 20 ㎕ of WST-8 solution was added to each well. After adding the WST-8 solution and incubating at 37℃ for 2 hours, the cell viability was measured by measuring the absorbance at 450 nm, and the results are shown in Figures 2a, 2b, and 2c.

[0285]

[0286] % survival = 100 x [(A peptide-trated cell ) / (A untreated cell )]

[0287]

[0288] 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 had no cytotoxicity against NIH3T3, HEK293 and RAW246.7 cell lines even at a concentration of 100 μg / ml.

[0289] 2-4. Stability evaluation for salt

[0290] To measure the salt stability of 21 polymyxin analogues (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) in the presence of physiological concentrations of salts contained in human blood (150 mM NaCl, 2.5 mM CaCl2, and 1 mM The effect of MgSO4 on antibacterial efficacy was evaluated. The test strains were 7×10 5 After diluting the culture medium containing 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4 to CFU / ml, 21 types of polymyxin analogs were added at a two-fold dilution starting from a concentration of 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.

[0291] Also, Na present in normal blood + The concentration of Ca is 140 mmol / L. 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 determined under the conditions of 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4, which are final concentrations similar to those of normal blood. 90 The changes in antibacterial efficacy were confirmed by comparing the values, and the results are shown in Table 6.

[0292] Compound nameMIC 90(μg / ml) A. baumannii E. coli P. aeruginosa CCARMA. baumannii CCARME. coli CCARMP. aeruginosa +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt +Salt -Salt 1 WP-AMP-219 11 11 8 12 11 18 12 WP-AMP-222 11 2 1 >64 22 11 1 >64 4 3 WP-AMP-223 2 11 1 2 12 11 18 14 WP-AMP-224 1 11 1 4 11 1 18 15 WP-AMP-225 1 11 1 2 11 1 14 16 WP-AMP-227 1 11 1 4 2 12 11 18 17 WP-AMP-229 1 11 1 4 11 1 11 16 18 WP-AMP-230 8 11 1 8 1 16 1 11 32 19 WP-AMP-231 8 11 1 8 1 8 1 11 16 110 WP-AMP-235 2 11 1 4 1 2 11 1 4 11 1 WP-AMP-239 8 1 2 1 4 1 11 1 4 1 12 WP-AMP-241 4 11 1 8 1 4 1 11 1 16 113 WP-AMP-242 4 11 1 8 1 4 2 11 1 32 114 WP-AMP-243 1 11 1 2 11 1 12 11 15 WP-AMP-246 1 11 1 3 2 2 11 1 13 2 116 WP-AMP-248 1 11 1 2 11 1 14 11 17 WP-AMP-249 1 11 1 2 2 12 11 1 4 11 18 WP-AMP-250 1 11 1 2 1 2 11 1 8 11 19 WP-AMP-251 4 11 1 4 1 4 1 11 1 8 120 WP-AMP-252 4 2 11 1 4 1 2 11 1 8 121 WP-AMP-254 1 11 1 2 11 1 14 1

[0293] From Table 6, 21 polymyxin analogues (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 no antimicrobial activity against P. aeruginosa in the presence of 150 mM NaCl, 2.5 mM CaCl2, and 1 mM MgSO4. and MICs for all strains except CCARM P. aeruginosa, A. baumannii, and CCARM A. baumannii 90 It was confirmed that there was little change in the values. That is, it was confirmed that the 21 polymyxin analogues of the present invention were partially affected in efficacy by structural changes caused by salt in P. aeruginosa and CCARM P. aeruginosa, A. baumannii and CCARM A. baumannii.

[0294] 2-5. Stability Evaluation of Proteolytic Enzymes

[0295] MIC by protease was determined for 21 polymyxin analogues (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) showing salt stability. 90Stability was evaluated through changes in antibacterial efficacy by comparing values. Stability evaluation for proteolytic enzymes was performed using trypsin and chymotrypsin. Proteolytic enzymes and lead substances were mixed at a ratio of 1:20 (w / w) and reacted at 37°C for 16 hours to determine MIC. 90 The values ​​were measured and the results are shown in Tables 7 and 8.

[0296] Material name MIC 90 (μg / ml)A. baumanniiE. coliP. aeruginosaCCARM A. baumanniiCCARM E. coliCCARM P. aeruginosa+TRY-TRY+TRY-TRY+TRY-TRY+TRY-TRY+TRY-TRY+TRY-TRY1WP-AMP-2191111111111112WP-AMP-2221111121111123WP-AMP-2231111112111114WP-AMP-224 1111111111115WP-AMP-2251111111111116WP-AMP-2271111121211117WP-AMP-229 1111111111118WP-AMP-2301111121111129WP-AMP-23111111121111110WP-AMP-23 511111111111111WP-AMP-23911111111111112WP-AMP-24111111122111113WP-AMP -24211111112111114WP-AMP-24311111111111115WP-AMP-24611111111111116WP- AMP-24811111111111117WP-AMP-24911111111111118WP-AMP-25011111111111119 WP-AMP-25111111121111120WP-AMP-25212111111111121WP-AMP-254111221422111

[0297] From Table 7, 21 polymyxin analogues 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 MIC values ​​compared to the control group that was not treated with trypsin. 90 It was found that the antibacterial efficacy was maintained with little change in value.

[0298] Material name MIC 90(μg / ml)A. baumanniiE. coliP. aeruginosaCCARM A. baumanniiCCARM E. coliCCARM P. aeruginosa+CHY-CHY+CHY-CHY+CHY-CHY+CHY-CHY+CHY-CHY+CHY-CHY1WP-AMP-2191111111111112WP-AMP-2221111121111243WP-AMP-2231111111111114WP-AMP-2241111111111115WP-AMP-2251111111111116WP-AMP-2271111121211117WP-AMP-2291111111111118WP-AMP-2301111111111119WP-AMP-23111111111111110WP-AMP-23511111111111111WP-AMP-23911111111111112WP-AMP-24111111111111113WP-AMP-24211111112111114WP-AMP-24311111111111115WP-AMP-24611111111111116WP-AMP-24811111111111117WP-AMP-24911111111111118WP-AMP-25011111111111119WP-AMP-25111111111111120WP-AMP-25212111111111121WP-AMP-254111111111111

[0299] 표 8로부터, Chymotrypsin이 처리된 21종의 폴리믹신 유사체(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, 및 WP-AMP-254)은 Chymotrypsin이 처리되지 않은 대조군과 비교하여 MIC 90It was found that the antibacterial efficacy was maintained with almost no change in the value. In order to confirm the result that 21 kinds of polymyxin analogues (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) have stable antibacterial efficacy without being degraded by proteolytic enzymes, under the same conditions as above. A final concentration of 25 μg / ml of proteolytic enzyme was added to the 500 μg / ml lead substance at 37°C for 4 hours. These results demonstrate that the 21 peptidomimetics remained intact and present in the same amount after treatment with trypsin and chymotrypsin, consistent with the MIC test results (Tables 5 and 6). Therefore, it was confirmed that the 21 polymyxin analogues could remain stable for a long period of time without loss of antimicrobial efficacy against the proteolytic enzymes trypsin and chymotrypsin.

[0300] 2-6. Stability evaluation for human serum

[0301] 21 cyclized peptidomimetic compounds (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) showing high antibacterial efficacy, low cytotoxicity, and stability against salt and proteolytic enzymes trypsin and chymotrypsin were tested. After adding 50% human serum and culturing at 37℃ for 16 hours, the change in antibacterial efficacy was compared with the MIC of the control group. 90 It was confirmed by comparing with the value.

[0302] Material name MIC 90(μg / ml)A. baumanniiE. coliP. aeruginosa100%50%0%100%50%0%100%50%0%1WP-AMP-2191111111112WP-AMP-2221111111123WP-AMP-2231111111114WP-AMP-2241111111115WP-AMP-2251111111116WP-AMP-2271111112127WP-AMP-2291111111118WP-AMP-2301111111119WP-AMP-23111111111110WP-AMP-23511111111111WP-AMP-23911111111112WP-AMP-24111111111113WP-AMP-24211111111114WP-AMP-24311111111115WP-AMP-24611111111116WP-AMP-24811111111117WP-AMP-24911111111118WP-AMP-25011111111119WP-AMP-25111111111120WP-AMP-25211211111121WP-AMP-254111111111Compound nameCCARMA. baumanniiCCARME. coliCCARMP.aeruginosa100%50%0%100%50%0%100%50%0%1WP-AMP-2191111111112WP-AMP-2221111114443WP-AMP-2231111112114WP-AMP-224111111111 5WP-AMP-2251111111116WP-AMP-2272221111117WP-AMP-2291111111118WP-AMP-2301111114419WP-AMP-23111111121110WP-AMP-235111111 11111WP-AMP-23911111111112WP-AMP-24111111122113WP-AMP-24211211184114WP-AMP-24311111111115WP-AMP-24611111111116WP-AMP- 24811111144417WP-AMP-24911111111118WP-AMP-25011111111119WP-AMP-25111111111120WP-AMP-25211111111121WP-AMP-254111111111.

[0303] From Table 9, the MIC of 21 polymyxin analogues regardless of the treatment concentration of human serum 90 The values ​​confirmed that antibacterial efficacy was maintained with little change compared to the control group. This result indicates that the compound can exhibit stable antibacterial efficacy without reduction in efficacy due to decomposition or binding with various substances present in blood.

[0304] 2-7. Hemolytic activity evaluation for animal-derived red blood cells

[0305] Sheep red blood cells (RBCs) were washed three times with phosphate-buffered saline (PBS) buffer by centrifugation at 1000 × g for 5 min and resuspended in PBS buffer. Seven selected polymyxin analogues (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) (final concentrations of 500, 200, 100, and 50 μg / ml) were added to the resuspended RBCs, incubated at 37°C for 1 h, and centrifuged at 1000 × g for 5 min to sediment the RBCs, and the supernatant was collected. The absorbance of this supernatant was measured at 570 nm and the degree of red blood cell hemolysis was calculated as follows. PBS and 0.2% Triton X-100 were used as negative and positive controls.

[0306] % hemolysis = 100 x [(A sample - A PBS ) / (A triton - A PBS )]

[0307] As a result of the test, the seven selected polymyxin analogues (WP-AMP-223, WP-AMP-229, WP-AMP-235, WP-AMP-243, WP-AMP-249, WP-AMP-250, and WP-AMP-254) showed hemolysis 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 very low toxicity to red blood cells. At a concentration of 100 μg / ml, the seven lead substances showed almost no hemolytic activity (Fig. 3).

[0308] 2-8. Evaluation of resistance development in two Gram-negative bacterial strains

[0309] Acinetobacter baumannii and Pseudomonas aeruginosa strains were inoculated, respectively, and the strains grown for 16 hours were inoculated into 3 ml of MHB medium, and cultured for 2 hours at 37°C and 150 rpm in a shaking incubator. After that, the absorbance was measured at 600 nm using a cuvette (more than 500 μl of sample was required), and when the target value (0.125 to 0.25) was reached, each dilution factor was calculated and diluted using 2X MHB. First, 100 μl of peptide and antibiotic were dispensed into a 96-well plate, and then 100 μl of the diluted bacterial solution was dispensed, and the OD of the 96-well plate was measured using an Elisa plate reader. 600 After checking the value (shaking condition: 10 seconds), it was cultured in a 37℃ incubator for 16 hours. After culture, OD 600 The value The growth rate of the bacteria and the growth inhibition rate of the peptide were calculated by checking (shaking condition: 1 min). The concentrations of peptides and antibiotics corresponding to the well immediately below the well where the bacteria did not grow were selected. After adding the selected concentrations of peptides and antibiotics to the liquid medium, the strains were cultured for 16 hours. The series of processes were continuously repeated (subculture). (A. baumannii strain was subcultured 19 times, and P. aeruginosa strain was subcultured 16 times.)

[0310] In order to evaluate the resistance of seven novel peptides selected for two strains, A. baumannii and P. aeruginosa, experiments were conducted for resistance changes by subculturing at the following concentrations based on the 0.5X MIC value, and the results are shown in Tables 10 and 11.

[0311] 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-2230.03125WP-AMP-2290.0078125WP-AMP-2350.015625WP-AMP-2430.0078125WP-AMP-2490.0078125WP-AMP-2500.0078125WP-AMP-2540.015625Colistin0.015625Meropenem0.125

[0312] 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-2230.25WP-AMP-2290.5WP-AMP-2350.125WP-AMP-2430.25WP-AMP-2490.25WP-AMP-2500.5WP-AMP-2540.5Colistin0.125Meropenem1

[0313] The results of the resistance evaluation for the seven lead substances against A. baumannii strains (Table 10) show that no resistance was detected for the six peptides except Meropenem and WP-AMP-229 and colistin until passage 19, as shown in Figures 4a to 4n. On the other hand, in the resistance evaluation against P. aeruginosa strains (Table 11), no resistance was detected for the six peptides except Meropenem and WP-AMP-235 and colistin after subculture up to passage 16.

[0314] 2-9. Evaluation of nephrotoxicity for seven peptides

[0315] The nephrotoxicity of seven selected polymyxin analogues (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 cells was evaluated using the WST-8 assay. 3 × 10 per well in a 96-well plate 3After dispensing the cells using RPMI1640 medium containing 10% FBS, they were cultured in a 5% CO2 incubator at 37℃ for 24 hours. Seven polymyxin analogs and the antibiotics colistin, ciaplatin, and ciprofloxacin were serially diluted 2-fold and dispensed onto the cultured cells, followed by culture. WST-8 was treated at each time point of 24, 48, and 72 hours, and after 2 hours of incubation, the 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. Two peptides, WP-AMP-223 and WP-AMP-249, showed cytotoxicity only at a high concentration of 200 μg / ml for 72 hours, while the other five peptides and colistin did not show nephrotoxicity.

[0316] The present invention relates to a novel polymyxin analogue having excellent antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria, and an antibacterial composition comprising the same. The polymyxin analogue of the present invention not only has an antibacterial effect against gram-negative bacteria and multidrug-resistant gram-negative bacteria, but also has excellent stability and safety in the body when applied to the human body, and has excellent stability against proteolytic enzymes, and therefore can be utilized for various purposes in related industrial fields such as pharmaceuticals, cosmetics, and health functional foods that can be repeatedly and mass-produced according to the present invention.

[0317] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0318] Attached electronic file of sequence list (PCT230014.xml)

Claims

1. A polymyxin analogue compound represented by the following chemical formula I, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above chemical formula I, R1 to R7 are as shown in the table below.

2. An antibacterial composition comprising a polymyxin analogue compound represented by the chemical formula I of paragraph 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.

3. In the second paragraph, the pharmaceutically acceptable salt is characterized in that it is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, hydrobromic 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.

4. In the second paragraph, the antibacterial composition is characterized in that it has antibacterial activity against gram-negative bacteria and multidrug-resistant gram-negative bacteria.

5. An antibacterial composition according to claim 4, wherein the Gram-negative bacteria are Acinetobacter baumannii, Escherichia coli, or Pseudomonas aeruginosa, and the multidrug-resistant Gram-negative bacteria are multidrug-resistant Acinetobacter baumannii (MRAB), multidrug-resistant Escherichia coli, or multidrug-resistant Pseudomonas aeruginosa (MRPA).

6. A pharmaceutical composition for preventing or treating infectious diseases, comprising a polymyxin analogue compound represented by the chemical formula I of paragraph 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.

7. A pharmaceutical composition for preventing or treating infectious diseases, characterized in that in paragraph 6, the pharmaceutically acceptable salt is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, hydrobromic 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.

8. A pharmaceutical composition for preventing or treating an infectious disease, characterized in that the infectious disease in paragraph 6 is an infectious disease caused by a gram-negative bacteria selected from the group consisting of pneumonia, peritonitis, meningitis, wound infection, osteoarthritis, cholecystitis, urinary tract infection, meningitis, endocarditis, myocarditis, pericarditis, arthritis, pharyngitis, gonorrhea, shigellosis, enteritis, conjunctivitis, gastritis, otitis media, cystitis, and lymphangitis.

9. A pharmaceutical composition for preventing or treating an infectious disease, characterized in that the infectious disease in paragraph 6 is an infectious disease caused by a gram-negative bacteria selected from the group consisting of tonsillitis, impetigo, rheumatic fever, glomerulonephritis, neonatal sepsis, meningitis, pharyngitis, pneumonia, endocarditis, scarlet fever, skin soft tissue infection, deep soft tissue infection, empyema, and vaginitis.

10. A pharmaceutical composition for preventing or treating sepsis, comprising a polymyxin analogue compound represented by the chemical formula I of paragraph 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.

11. A pharmaceutical composition for preventing or treating sepsis, characterized in that in paragraph 10, the pharmaceutically acceptable salt is selected from the group consisting of trifluoroacetic acid, hydrochloric acid, hydrobromic 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.

12. A method for preparing a cyclized polymyxin analogue compound represented by the chemical formula I, comprising the following steps: (a) a step of obtaining a peptide having a resin represented by chemical formula Ia attached thereto by a solid-phase synthesis method; (b) a step of removing the resin from the peptide obtained in step (a) to obtain a protected polymyxin analogue represented by the chemical formula Ib; (c) a step of subjecting the peptide obtained in step (b) to a cyclization reaction using a catalyst by a solution-phase synthesis method to obtain a protected cyclized polymyxin analogue represented by the chemical formula Ic; and (d) a step of performing a deprotection reaction on the peptide obtained in step (c) to obtain a cyclized polymyxin analogue represented by chemical formula I: [Chemical 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.

13. A method for producing a polymyxin analogue compound, characterized in that in claim 12, 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 vol% trichloroacetic acid or trifluoroacetic acid solution; and (ii) a dichloromethane solution containing 0.1% formic acid, methanesulfonic acid, benzenesulfonic acid, or para-toluenesulfonic acid, alone or in combination.

14. A method for producing a polymyxin analogue compound, characterized in that the cyclization reaction temperature in claim 12 is performed at -20 to 50°C.

15. In paragraph 12, the concentration of the protected peptide of the cyclization reaction is 10 in terms of peptide. -2 10 in the mall -5 A method for producing a polymyxin analogue compound, characterized in that the method is performed within a molar concentration range.

16. A method for producing a polymyxin analogue compound, characterized in that in claim 12, the solvent for the cyclization reaction of the peptide is selected from the group consisting of 1,2-dichloroethane, chloroform, dichloromethane, 1,2-dichloromethane, tetrahydrofurane, 1,4-dioxane, acetonitrile, dimethylsulfoxide, N,N-dimethylformamide, and N,N-dimethylacetamide, used alone or in combination.

17. A method for producing a polymyxin analogue compound, characterized in that in claim 12, the acidic solution for the deprotection reaction uses a mixture containing trifluoroacetic acid: purified water (95:5).

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