Novel 10 peptide and antibacterial application thereof

Abstract

A novel 10 peptide capable of inhibiting the growth of bacteria that are resistant to existing polymyxin, a pharmaceutical composition comprising the novel 10 peptide, and an application thereof in the treatment or prevention of bacterial infections.

Description

A novel 10-peptide and its application in antibacterial

[0001] This application claims priority to Chinese patent application No. 202310694583.1, filed on June 9, 2023, entitled “A novel 10-peptide and its application in antibacterial treatment”, the entire text of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to novel polymyxin compounds that can inhibit the growth of bacteria resistant to existing polymyxins and also to the use of the novel polymyxin compounds in treating or preventing bacterial infections. Background Art

[0003] Polymyxin is a polycationic antimicrobial peptide that binds to the anionic phosphate groups in lipid A of the inner membrane of Gram-negative bacteria, causing the displacement of the divalent cations originally bound to the phosphate groups on the inner membrane lipid A, thereby destabilizing the membrane. The hydrophobic N-terminal fatty acyl chain and amino acids 6 and 7 of polymyxin then insert into the hydrophobic region of the outer membrane, which is primarily composed of the fatty acyl chains of lipid A. Polymyxin insertion into the phospholipid bilayer disrupts the structural integrity of the membrane and promotes the passage of other polymyxin molecules through the outer membrane. Ultimately, polymyxin crosses the interface between the hydrophilic head group and the fatty acyl chain, disrupting the physical integrity of the inner membrane phospholipid bilayer through membrane thinning, leading to inner membrane lysis and cell death.

[0004] Polymyxins include polymyxin AE, with polymyxin B and polymyxin E being the main agents on the market. Polymyxins have significant antibacterial activity against some Gram-negative bacilli, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., and Pseudomonas aeruginosa. In recent years, due to the lack of other effective antimicrobial agents, polymyxins have been considered the first choice for treating infections caused by carbapenem-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, often serving as a last line of defense.

[0005] However, an increasing number of studies have reported bacterial resistance to polymyxins. Polymyxin resistance mechanisms are primarily categorized into two types: chromosome-mediated resistance and plasmid-mediated resistance. Chromosomal-mediated resistance carries a relatively low risk of transmission, while plasmid-mediated resistance gene sequences are easily exchanged, leading to a rapid spread of polymyxin resistance among bacteria and a consequent collapse of the last line of defense. The principles of the two resistance mechanisms are virtually identical. Lipopolysaccharide modification occurs by partially replacing the phosphate groups of lipid A with the cationic group 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine (PEtN), reducing the negative charge of the outer membrane, hindering the binding of polymyxins to bacteria, and weakening the bactericidal effect of polymyxins.

[0006] Summary of the Invention

[0007] In one aspect, provided herein are compounds of the following formula (I), derivatives, pharmaceutically acceptable salts or solvates thereof:

[0008] In some embodiments, the salt is selected from the group consisting of hydrochloride, sulfate, phosphate, acetate, oxalate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate, and any combination thereof.

[0009] In some embodiments, the solvate is a hydrate or an ethanolate.

[0010] In some embodiments, the derivative is a PEG derivative or a polysaccharide derivative of the compound.

[0011] In another aspect, provided herein is a pharmaceutical composition comprising:

[0012] 1) the above-mentioned compound, its derivative, pharmaceutically acceptable salt or solvate; and

[0013] 2) Pharmaceutically acceptable carrier.

[0014] In some embodiments, the pharmaceutical composition further comprises one or more antibiotics.

[0015] In another aspect, the present invention provides the use of the above-mentioned compound, its derivative, pharmaceutically acceptable salt or solvate in the preparation of a medicament for preventing or treating diseases related to bacterial infection.

[0016] In some embodiments, the bacteria are Gram-negative bacteria.

[0017] In some embodiments, the bacteria is resistant to any one polymyxin or antibiotic.

[0018] In some embodiments, the bacteria carries the mcr-1 gene.

[0019] In some embodiments, the bacteria is selected from Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

[0020] In some embodiments, the bacterial infection-related disease is selected from respiratory tract infection, pneumonia, bronchitis, wound infection, sepsis and septicemia.

[0021] In another aspect, provided herein is a method for treating a bacterial infection-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the above-mentioned compound, its derivative, pharmaceutically acceptable salt or solvate, or pharmaceutical composition.

[0022] In some embodiments, the bacteria are Gram-negative bacteria.

[0023] In some embodiments, the bacteria is resistant to any one polymyxin or antibiotic.

[0024] In some embodiments, the bacteria carries the mcr-1 gene.

[0025] In some embodiments, the bacteria is selected from Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0026] In some embodiments, the bacterial infection-related disease is selected from respiratory tract infection, pneumonia, bronchitis, wound infection, sepsis and septicemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a schematic diagram of the structure of the antimicrobial peptide provided herein.

[0028] FIG2 shows the binding of colistin to modified and unmodified lipid A using PyMOL software.

[0029] FIG3 shows the binding of the antimicrobial peptides provided herein to modified and unmodified lipid A using PyMOL software.

[0030] FIG4 shows the minimum inhibitory concentration test results of the antimicrobial peptides provided herein against common Gram-negative bacteria.

[0031] FIG5 shows the cytotoxicity test results of the antimicrobial peptides and colistin provided herein.

[0032] FIG6 shows the renal toxicity test results of the antimicrobial peptides and colistin provided herein.

[0033] FIG7 shows the therapeutic effects of the antimicrobial peptides and colistin provided herein on mice infected with colistin-resistant Acinetobacter baumannii.

[0034] FIG8 shows the mass spectrometry detection results (m / z) of the antimicrobial peptides provided herein.

[0035] FIG9 shows the HPLC purity test results of the antimicrobial peptides provided herein. DETAILED DESCRIPTION

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0037] The term "or" refers to a single element of the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0038] The term "about" generally refers to a variation within a range of 10% above or below the specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0039] The terms "comprising" or "including" refer to including the stated elements, integers, or steps, but not excluding any other elements, integers, or steps. As used herein, when the terms "comprising" or "including" are used, unless otherwise indicated, they also encompass situations consisting of the stated elements, integers, or steps. For example, when reference is made to an object "comprising" a specific element, it is intended to encompass objects consisting of that specific element.

[0040] The term "derivative" of a compound herein refers to a compound to which a substituent group is covalently linked. For example, in the case of a compound of formula (I), the substituent group may replace a hydrogen atom, such as a hydrogen atom on a methyl group, a methylene group, an amino group, an imino group, or a hydroxyl group.

[0041] The substituent groups may be, for example, hydrocarbon groups, cycloalkyl groups, heteroalkyl groups, halogen groups, aryl groups, heteroaryl groups, etc. The hydrocarbon groups may be straight-chain or branched saturated hydrocarbon groups or unsaturated hydrocarbon groups, preferably containing 1 to 12 carbon atoms. Examples of hydrocarbon groups include methyl, ethyl, propyl, isopropyl, n-butyl, vinyl, etc. The hydrocarbon group may be optionally substituted with one, two, three or four substituents, which may be halogen, hydroxyl, cyano, C 1-12 Alkyl, C 3-7 In some embodiments, the hydrocarbon group is unsubstituted. The cycloalkyl group can be a cyclic saturated or unsaturated hydrocarbon group of 3 to 18 (in some embodiments, 3 to 6) carbon atoms. In some embodiments, the cycloalkyl group includes but is not limited to cyclopropyl, cyclopentyl, cyclohexyl, cyclododecyl, etc. The cycloalkyl group can be optionally substituted with one, two, three or four substituents, which can be halogen, hydroxyl, cyano, C 1-12 Alkyl, C 3-7 Cycloalkyl, aryl, heteroaryl, etc. In some embodiments, the cycloalkyl is unsubstituted. Heteroalkyl refers to a saturated or unsaturated hydrocarbon group that includes heteroatoms (especially halogen, N, O or S). These heteroatoms can be incorporated into any part of the saturated or unsaturated hydrocarbon group. The heteroatoms can be, for example, hydroxyl, C 1-4 Alkoxy, amino, mercapto (-SH) or similar groups are incorporated. Examples of heteroalkyl include, but are not limited to, 2-methoxyethyl (-CH2CH2OCH3), 2-hydroxyethyl (-CH2CH2OH), hydroxymethyl (-CH2OH), 2-aminoethyl (-CH2CH2NH2), 2-dimethylaminoethyl (-CH2CH2NHCH3), benzyloxymethyl, thiophen-2-ylmercaptomethyl, etc. Halogen refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). "Aryl" refers to an aromatic group, especially phenyl or naphthyl. Aryl may be optionally substituted with one, two, three or four substituents, which may be halogen, hydroxy, cyano, C 1-12 Alkyl, C 3-7 Cycloalkyl, aryl, heteroaryl, etc. Heteroaryl refers to an aromatic group containing a heteroatom (especially N, O or S) in the aromatic ring. Heteroaryl may be optionally substituted by one, two, three or four substituents, which may be halogen, hydroxyl, cyano, C 1-12 Alkyl, C 3-7Cycloalkyl, aryl, heteroaryl, etc. Examples of heteroaryl include, but are not limited to, pyridine, thiophene, furan, pyrazole, pyrimidine, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3-pyridazinyl, 4-pyridazinyl, 3-pyrazinyl, 4-oxo-2-imidazolyl, 2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, etc. As used herein, "optionally" means that the event or circumstance described can but need not occur, and includes examples in which the event or circumstance described does not occur. For example, "aryl optionally mono- or di-substituted with an alkyl group" means that the alkyl group can be present but does not need to be present, and includes both cases in which the aryl group is mono- or di-substituted with an alkyl group and cases in which the aryl group is not substituted with an alkyl group. In some cases, the substituent group can be a macromolecular polymer, such as a polyethylene glycol (PEG) or polysaccharide (eg, hyaluronic acid, chitosan, etc.) molecule.

[0042] In some cases, a derivative of a compound can be a prodrug of the compound, i.e., any substance that can be released (e.g., degraded) in vivo to provide the compound when administered to a subject (e.g., a mammal, particularly a human). For example, a hydroxyl, sulfhydryl, amide, or amino group in a compound can be linked to any group that can break in vivo and then regenerate the corresponding free hydroxyl, amide, amino, or sulfhydryl group. Examples of prodrugs include, but are not limited to, esters of the hydroxyl functional group in the compounds provided herein (e.g., acetates, formates, benzoates, phosphates, or phosphoric acid derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl), and the like.

[0043] "Pharmaceutically acceptable salts" as used herein refer to salt forms of compounds that retain the pharmacological activity of the compound. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or similar acids, or organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like.

[0044] As used herein, "solvate" refers to a complex having a specific stoichiometry formed between a solute (e.g., a compound of the present invention) and a solvent. The solvent can be any of those commonly used in the pharmaceutical field, for example, water, ethanol, acetic acid, and the like. The term "hydrate" refers to a solvate in which the solvent molecule is water. The term "ethanolate" refers to a solvate in which the solvent molecule is ethanol.

[0045] "Bacterial infection" is used herein to indicate that a subject may contain, contain or carry the bacteria, i.e., the bacteria may simply be present in or on the subject, which may include any part or position in or on the subject's body. The infection of the subject does not necessarily manifest as a clinical disease (i.e., the infection causes clinical symptoms in the subject), although this can certainly be included. The subject here can be a subject suspected of bacterial infection or a risk of bacterial infection, i.e., a subject who may be only exposed to bacteria, a subject who exhibits clinical symptoms or symptoms of infection (if an infected person is suspected), or a subject susceptible to infection. In particular, the bacterial infection can be a type known to be treated with polymyxins in routine clinical practice. In some embodiments, the bacteria is identified as or suspected to be a bacteria that responds to (i.e., is sensitive to) polymyxins. In other embodiments, it can be determined that the infection (or more particularly the bacteria in the infection) is resistant (or drug-resistant) to one or more known polymyxins.

[0046] Bacterial infections can be nosocomial infections, respiratory infections in patients, for example, with cystic fibrosis, chronic obstructive pulmonary disease, congestive obstructive airway disease / congestive obstructive airway pneumonia (COAD / COAP), pneumonia, emphysema, bronchitis or sinusitis; wound infections, particularly chronic wounds (including burns), device-related infections associated with implanted or prosthetic medical devices, such as prosthetic valve endocarditis or line or catheter infections or artificial joints or tissue replacements or endotracheal or tracheostomy tubes. Thus, the site of infection can be oral surfaces (e.g., teeth, gums, gum crevices, periodontal pockets), reproductive tract (e.g., cervix, uterus, fallopian tubes), peritoneum, middle ear, prostate, urinary tract, vascular lining, eye, i.e., ocular tissue (e.g., conjunctiva, corneal tissue, tear ducts, tear glands, eyelids), respiratory tract, lung tissue (e.g., bronchi and alveoli), heart valves, gastrointestinal tract, skin, scalp, nails and the interior of wounds, particularly chronic wounds and surgical wounds, which may be localized or internal wounds. Other surfaces include the exterior of organs, particularly those that undergo transplantation, such as the heart, lungs, kidneys, liver, heart valves, pancreas, intestines, corneal tissue, arterial and venous grafts, and skin. Infections may also be present in body fluids (e.g., blood, plasma, serum, cerebrospinal fluid, gastrointestinal contents, semen, sputum, and other lung secretions) and tissues (e.g., adrenal glands, liver, kidneys, pancreas, pituitary gland, thyroid gland, immune, ovaries, testicles, prostate, endometrium, eyes, breasts, fat, epithelium, endothelium, nerves, muscles, lungs, epidermis, bones). Infections may also be found on any "indwelling" medical or surgical equipment or devices. This may include any type of line, including catheters (e.g., central venous and urinary catheters), prosthetic devices, such as heart valves, artificial joints, dentures, crowns, dental caps, and soft tissue implants (e.g., breast, hip, and lip implants). Includes any kind of implantable medical device (e.g., a stent, an intrauterine device, a pacemaker, a cannula (e.g., an endotracheal or tracheostomy tube), a prosthesis or prosthetic device, a wire or catheter). An "indwelling" medical device may include a device any part of which is contained within the body, i.e., the device may be indwelling in whole or in part. The infection may be acute, or may be chronic, e.g., an infection persisting for at least 5 days or at least 10 days, particularly at least 20 days, more particularly at least 30 days, and most particularly at least 40 days. Examples of bacterial infection-related diseases include, but are not limited to, respiratory tract infections or conditions related thereto, such as cystic fibrosis, pneumonia, chronic obstructive pulmonary disease (COPD), bronchitis, sinusitis, chronic wound infections (including burns), device-related infections associated with implantable or prosthetic medical devices, bacteremia, sepsis, septic shock, or sepsis.

[0047] The example of the antibacterial type that usually causes this infection comprises Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia (for example Burkholderia cepacia), Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Mycobacterium tuberculosis, enterococcus and vancomycin-resistant enterococcus and Providencia stuartii.Other important infections according to the present invention comprise Bartonella (for example Bartonella hansii), mycobacterium (for example Mycobacterium avium complex (MAC)), Mycobacterium kansasii, Mycobacterium marine, Mycobacterium ulcerans, Mycobacterium toadii), Haemophilus influenzae type B infects or Legionella (for example Legionella pneumophila; Legionnaires' disease), leprosy (Mycobacterium leprae), human granulocytic anaplasmosis (Anaplasma phagocytophilum), brucellosis (Brucella), meningococcal disease (Neisseria meningitidis) and anthrax (Bacillus anthracis).

[0048] The bacteria may be resistant (or drug-resistant) to antibiotics, for example, the bacteria are resistant to at least one, two, three, four, five, six, seven, eight, nine or ten antibiotics. The antibiotics are, for example, aminoglycosides (e.g., amikacin, gentamicin, kanamycin, capreomycin, neomycin, netilmicin, streptomycin, tobramycin); β-lactams (e.g., carbacephems (e.g., loracarbef); first generation cephalosporins (e.g., cefdroxil, cefazolin, cephalexin); second generation cephalosporins (e.g., cefaclor, cephalothin, cephalexin, cefoxitin, cefprozil, cefuroxime); third generation cephalosporins (e.g., cefixime); , cefdinir, cephalothin, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, cephalothin, ceftizoxime, ceftriaxone); fourth-generation cephalosporins (e.g., cefepime); monobactams (e.g., aztreonam); macrolides (e.g., azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin); monoamides (e.g., aztreonam); penicillins (e.g., amoxicillin, ampicillin, carbenicillin, cloxacillin, dicloxacillin, naphthiazolidin); oxacillin, penicillin G, penicillin V, piperacillin, ticarcillin); polypeptide antibiotics (e.g., bacitracin, polymyxin); quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); sulfonamides (e.g., sulfonamides, sulfamethoxazole, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole); tetracyclines (e.g., norfloxacin, sulfamethoxazole, sulfasalazine, sulfamethoxazole); The susceptibility (i.e., sensitivity) to antibiotics (as opposed to resistance and tolerance) can be measured in any convenient manner, for example, by dilution sensitivity test and / or disk diffusion test. Preferably, the sensitivity of a bacterial strain to an antibiotic is expressed as the minimum inhibitory concentration (MIC) of the antibiotic for the microorganism (see Jorgensen et al., Manual of Clinical Microbiology, 7th ed., Washington, DC: American Society for Microbiology, 1999; 1526-43). One skilled in the art can use this information to determine whether the bacterial infection being treated by the present invention is resistant to the antibiotic under these definitions.In some cases, the resistance is conferred by a plasmid carried by the bacteria, such as a plasmid carrying the mcr-1 gene. In other cases, the resistance is conferred by an mcr-1 gene carried by the bacterial genome.

[0049] The bacterial infection can include the antibacterial from any bacterial genus or antibacterial. For example, the antibacterial can be gram-positive or gram-negative bacteria, or in fact gram-indeterminate bacteria, wherein gram-negative bacteria are important. In gram-negative bacteria, it is worth noting that enterobacteriaceae and gram-negative bacteria non-fermentative bacteria. Enterobacteriaceae includes but is not limited to from Alishewanella (Alishewanella), Alterococcus (Alterococcus), Aquamonas (Aquamonas), Aranicola (Aranicola), Azotivirga (Azotivirga), Brenneria (Brenneria), Budvicia (Budvicia), Buttiauxella (Buttiauxella), Cedecea (Cedecea), Citrobacter (Citrobacter), Cronosaccharomyces (Cronobacter), bacter), Dickea, Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Grimontella, Hafnia, Klebsiella, Leclercia, Leminorella, Moe llerella), Morganella, Obesumbacterium, Pantoea, Pectobacterium, Phlomobacter, Photorhabdus, Plesiomonas, Pragia, Proteus, Providencia, Rahnella, and Rothellae Raoultella, Salmonella, Samsonia, Serratia, Shigella, Sodalis, Tatumella, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia, and Yokenella.Preferred Enterobacteriaceae include Escherichia, Klebsiella, Salmonella, Shigella, Yersinia and Providencia. Non-fermenting Gram-negative bacteria include, but are not limited to, Pseudomonas, Acinetobacter, Stenotrophomonas and Burkholderia, Achromobacter, Algaligenes, Bordetella, Brevundimonas, Comamonas, E. coli, and Pseudomonas. lizabethkingia), Methylobacterium, Moraxella, Ochrobactrum, Oligella, Psychrobacter, Ralstonia, Roseomonas, Shewanella, Sphingobacterium, bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, and Burkholderia spp.Preferably, the bacterium may be selected from the group consisting of Pseudomonas, Acinetobacter, Stenotrophomonas, Burkholderia, Escherichia, Klebsiella, Providencia, Streptococcus, Staphylococcus, for example Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Burkholderia spp, E. coli, Klebsiella pneumoniae, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Acinetobacter lwoffi, Providencia stuartii, Providencia rettgeri, Providencia alcalifaciens, Klebsiella oxytoca, Pseudomonas anguilliseptica, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas luteola, and methicillin-resistant Staphylococcus aureus (MRSA).

[0050] When referring to a pharmaceutical composition, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered to an animal or human body, is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent contained therein. Depending on the route of administration, various carriers well known in the art may be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils (e.g., castor oil), synthetic oils, polyols, alginic acid, phosphate buffer, emulsifier, isotonic saline, and / or pyrogen-free water. The routes of administration that can be used include, for example, oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, and the like. Accordingly, the pharmaceutical composition comprising the compound provided herein, its derivative, pharmaceutically acceptable salt or solvate can be formulated into any suitable dosage form, such as tablets, granules, powders, capsules, injection preparations, suppositories, eye drops, external ointments, ointments, medicated oils, or sprays, etc.

[0051] The compound of formula (I) provided herein, its derivative, pharmaceutically acceptable salt or solvate can be used in combination with another therapeutic agent.The other active agent can be in the same pharmaceutical preparation (such as pharmaceutical composition) with the compound, its derivative, pharmaceutically acceptable salt or solvate, or in different pharmaceutical preparations.They can be administered together, or administered alone (i.e., separately, sequentially or simultaneously). Therefore, another aspect of the present invention provides a product (e.g., a drug combination or kit), which comprises a compound of formula (I) as defined herein, its derivative, pharmaceutically acceptable salt or solvate and another therapeutic active agent as a combined preparation, for treating or preventing bacterial infection in a subject separately, sequentially or simultaneously. In some embodiments, the other active agent is, for example, an antimicrobial agent (e.g., an antibiotic already mentioned above for antibacterial, antifungal, antiviral agents), an immunostimulant, a corticosteroid, a nonsteroidal anti-inflammatory drug (NSAID), a bronchodilator, etc.

[0052] "Subject" includes animals, such as mammals, including but not limited to primates, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, mammalian experimental animals, mammalian farm animals, mammalian sports animals, and mammalian pets. The subject can be male or female and can be any age-appropriate subject, including infants, young children, young people, adults, and elderly subjects. In some instances, the subject refers to an individual who needs to diagnose or treat a disease or condition. In some instances, the subject receiving diagnosis or treatment can be a patient who suffers from a condition associated with the diagnosis or treatment, or is at risk of developing the condition. In a specific instance, the subject is a human, such as a human patient. The term is generally used interchangeably with "patient," "test subject," "treatment subject," etc.

[0053] When referring to the treatment of a disease, a "therapeutically effective amount" refers to the amount of an active molecule (such as a compound provided herein) sufficient to induce a biological or medical response in a subject as desired by a clinician. The "effective amount" at the time of administration can be determined by those skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, for general drugs, a typical daily dose range can be 1 μg to 100 mg of active ingredient per kg body weight. The methods of administration of the active molecules provided herein (such as the compounds provided herein or their derivatives) include, but are not limited to, injection, for example, by intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, and the like.

[0054] Polymyxins are a group of polypeptide antibiotics produced by Paenibacillus polymyxa that are active against most Gram-negative bacteria. The polymyxin peptide chain typically consists of 10 amino acids, forming a linear tripeptide linked to a cyclic heptapeptide. Differences in amino acid type and configuration at different positions result in distinct polymyxin homologs. The amino acid sequence of polymyxins is: L-Dab-L-Thr-3-L-Dab-L-Dab-6-7-L-Dab-L-Dab-L-Thr. Differences in amino acids at positions 3, 6, and 7 determine the type of polymyxin homolog (Table 1). Currently, only polymyxin B and polymyxin E are in clinical use. They share nearly identical primary sequences, differing only at amino acid position 6. The sixth amino acid position in polymyxin B is D-Phe, while that in polymyxin E is D-Leu.

[0055] Table 1 Sequences of amino acids 3, 6, and 7 and fatty acyl tails of polymyxin homologues

[0056] The structure of the polymyxin compounds provided herein (hereinafter referred to as novel antimicrobial peptides or antimicrobial peptides) is as follows:

[0057] The primary sequence of this antimicrobial peptide can be represented as: {6-ME-Octanoic acid}{Dab}{Thr}{D-Dab}{Dab}{Dab}{D-Leu}{Ile}{Dab}{Dab}{Leu} (see Figure 1). Its amino terminus is S-6-methyloctanoic acid, the first three amino acids are linearly linked, and the last seven amino acids are circular.

[0058] Using the clinically active polymyxin (colistin) as an example, the structures of the novel antimicrobial peptides provided herein are compared with those of existing polymyxins. As can be seen from the structural formula below, the novel antimicrobial peptides provided herein differ from colistin in the types of amino acids 7 and 10, and in the conformation of the amino acid at position 3. The novel antimicrobial peptides provided herein have amino acids 3, 7, and 10 represented by D-Dab, L-Ile, and L-Leu, while colistin has amino acids 3, 7, and 10 represented by L-Dab, D-Leu, and L-Thr.

[0059] The novel antimicrobial peptides provided herein have significant inhibitory activity against polymyxin-resistant bacteria. We used PyMOL to simulate the binding of colistin or novel antimicrobial peptides to lipid A to elucidate the mechanism of action of the novel antimicrobial peptides. As shown in Figure 2 (left), colistin binds to the two negatively charged phosphate groups of lipid A. When lipid A is modified with phosphoethanolamine (Figure 2 (right), the negative charge of lipid A is reduced, and colistin only has one binding site with lipid A, greatly reducing its binding ability.

[0060] Figure 3 shows a possible interaction model between the antimicrobial peptide and the Gram-negative membrane component Kdo 2-Lipid A and the Gram-negative membrane component 1-PEA-Kdo 2-Lipid A carrying mcr-1. As shown in Figure 3, the positively charged Dab (5) of the antimicrobial peptide interacts electrostatically with the carboxyl group of Kdo 2 in the Gram-negative membrane component Kdo 2 Lipid A, and the positively charged Dab (8) interacts electrostatically with the carboxyl group of Kdo 1 in Kdo 2-Lipid A. At the same time, the fatty acid molecules interact hydrophobically with the fatty acid molecules of Kdo 2-Lipid A. These two interactions may enable the antimicrobial peptide to have antibacterial activity against Gram-negative bacteria.

[0061] Although the negative charge of the phosphate group is neutralized after the phosphoethanolamine transferase encoded by mcr-1 modifies the phosphate group at position 1 of the Gram-negative bacterial membrane component Kdo 2-Lipid A, it does not affect the binding of Dab (5) and Dab (8) to Kdo 2 and Kdo 1. At the same time, the fatty acid molecules of the antimicrobial peptide and the fatty acid molecules of 1-PEA-Kdo 2-Lipid A undergo hydrophobic interactions. These two effects may enable the antimicrobial peptide to have antibacterial activity against Gram-negative bacteria carrying mcr-1.

[0062] Due to the strong binding ability of the new antimicrobial peptide to the negative charge on lipid A, the antimicrobial peptide has an antimicrobial mechanism similar to that of polymyxins, and can inhibit common Gram-negative bacteria, including Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. At the same time, the new antimicrobial peptide still has a very high binding ability to the modified lipid A, which makes the new antimicrobial peptide also have a strong inhibitory effect on bacteria resistant to polymyxins, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae containing the mcr-1 plasmid.

[0063] Example

[0064] Example 1: Detection of the inhibitory effect of the antimicrobial peptides provided herein on common Gram-negative bacteria

[0065] 1.1 Experimental Procedure

[0066] The pathogens used for testing were all isolated and cultured from clinical specimens collected from patients with severe pneumonia in the Intensive Care Unit of Gulou Hospital. The strains were confirmed by the hospital's Microbiology Laboratory. The Gram-negative bacteria tested included Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0067] 1.1.1 Preparation of test strains

[0068] Inoculate a single colony of the bacteria to be tested into 5 mL of culture medium and culture overnight until saturated. Pipette 10 μL of the bacterial solution into 990 μL of fresh culture medium and mix thoroughly to obtain a 100-fold dilution. Then, pipette 20 μL of the 100-fold dilution into 980 μL of fresh culture medium and mix thoroughly. This results in a 5000-fold dilution of the bacteria.

[0069] 1.1.2 Dilution of antimicrobial peptides

[0070] Dissolve the antimicrobial peptide in dimethyl sulfoxide (DMSO) to obtain a stock solution with a concentration of 12.8 mg / mL. Serially dilute 10 times using DMSO using a two-fold serial dilution method.

[0071] 1.1.3 Antimicrobial peptide antibacterial ability test

[0072] Add the 11 dilutions of the antimicrobial peptide prepared in step 1.1.2 to each well of a 96-well plate. Add the same volume of DMSO to the final well. Then, add 48 μL of fresh culture medium to each well. Add the 5000-fold diluted bacteria prepared in step 1.1 to each well. Mix thoroughly. Incubate at 37°C for 16 hours. Visually determine the concentration that begins to inhibit bacterial growth; this is the minimum inhibitory concentration (MIC).

[0073] 1.2 Experimental Results

[0074] As shown in FIG4 , the antimicrobial peptides provided herein are capable of inhibiting the growth of common Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

[0075] A1-A12 used 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.135, and 0 μg / ml of antimicrobial peptide to culture E. coli, respectively;

[0076] B1-B12 used 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.135, and 0 μg / ml of antimicrobial peptide to culture Pseudomonas aeruginosa, respectively;

[0077] G1-G12 used 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.135, and 0 μg / ml of antimicrobial peptide to culture Acinetobacter baumannii, respectively;

[0078] H1-H12 were cultured with 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.135, and 0 μg / ml of antimicrobial peptides, respectively;

[0079] D1-D2 cultured Escherichia coli without antibiotics; D3-D4 cultured Pseudomonas aeruginosa without antibiotics.

[0080] E1-E2 were cultured without antibiotics for Acinetobacter baumannii; E3-E4 were cultured without antibiotics for Klebsiella pneumoniae.

[0081] No sample was added to the other wells. The blue arrows indicate the wells corresponding to the minimum inhibitory concentration.

[0082] The specific minimum inhibitory concentrations are: 4μg / mL (Escherichia coli), 4μg / mL (Pseudomonas aeruginosa), 16μg / mL (Acinetobacter baumannii), and 8μg / mL (Klebsiella pneumoniae).

[0083] Example 2: Inhibition of polymyxin-resistant bacteria by novel antimicrobial peptides

[0084] 2.1 Experimental Procedure

[0085] The polymyxin-resistant pathogens used for testing all originated from the hospital's intensive care unit, were isolated and cultured from clinical samples of patients with severe pneumonia, and the strain types were determined by the hospital's microbiology laboratory, and high-throughput sequencing technology was used to confirm that the strains contained the mcr-1 gene.

[0086] Refer to the culture process of Example 1 completely.

[0087] 2.2 Experimental Results

[0088] Table 2 Minimum inhibitory concentrations of colistin and antimicrobial peptides against bacteria containing MCR-1 plasmid (μg / mL)

[0089] Each bacterium in the table was tested with two strains of each species, represented by "1" and "2" respectively.

[0090] As shown in Table 2, the minimum inhibitory concentrations of the antimicrobial peptides were significantly lower than those of polymyxin E (Colistin) against Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae containing the MCR-1 gene, demonstrating a high inhibitory activity of the antimicrobial peptides against polymyxin-resistant bacteria.

[0091] Example 3: Toxicity test of antimicrobial peptides on human cells

[0092] 3.1 Experimental Procedure

[0093] 3.1.1 Cell culture and drug treatment

[0094] HEK293 cells were cultured in Dulbecco's modified Eagle's medium (supplemented with 10% fetal bovine serum). After passage at a density of 2500 cells per well in a 96-well plate, the cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. Antimicrobial peptides were then added to each well at varying concentrations (0 to 1000 μg / mL). After 48 hours of incubation, the medium was removed, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (0.45 mg / mL) was added to each well. After a 3-hour incubation, the solution was aspirated. Precipitated formazan crystals were dissolved by the addition of 100 μL of a dissolving solution (40% DMF, 16% SDS, and 2% acetic acid in water).

[0095] 3.1.2 Counting cell survival

[0096] The absorbance of each well was measured at OD 570 nm using a microplate reader (Epoch Microplate Spectrophotometer, BioTek). Colistin was used as a positive control, and DSMO as a negative control. IC50 values, i.e., the concentration of each compound required to produce 50% cell growth inhibition compared to the no compound control, were calculated using GraphPad Prism v.9.0.

[0097] 3.2 Experimental Results

[0098] As shown in FIG5 , similar to colistin, the antimicrobial peptide concentration required to produce 50% cell growth inhibition is greater than 512 μg / mL, indicating that the cytotoxicity of the antimicrobial peptide is very low.

[0099] Example 4: Toxicity test of antimicrobial peptides on mouse kidneys

[0100] 4.1 Experimental Procedure

[0101] 4.1.1 Mouse husbandry and drug treatment

[0102] Six-week-old female CD1 mice (20-25 g) were used in this experiment. The room was maintained with a 12-hour light cycle, a temperature of 21°C, and a humidity of 30%. After three days of acclimation training, the mice were randomly divided into three groups (n = 6 per group): a vehicle group, a colistin group, and an antimicrobial peptide group.

[0103] Mice were subcutaneously injected with 100 μL of 0.9% saline (vehicle group), 20 mg / kg of colistin, or 20 mg / kg of antimicrobial peptide for 7 consecutive days. Blood samples were collected 12 hours after the last injection.

[0104] 4.1.2 Determination of renal injury in mice

[0105] NGAL, a neutrophil gelatinase-associated lipocalin, is commonly used to indicate kidney damage. Here, we measured serum NGAL concentrations using a commercially available mouse NGAL ELISA kit (Beyotime, PN758). The assay procedure is briefly described below:

[0106] a. Place the whole blood at room temperature for 30 minutes to 2 hours, then centrifuge at 1000g for 10 minutes at 4°C. Aspirate the yellow supernatant to obtain the serum.

[0107] b. Prepare the standard diluent and standards: Dilute the standard diluent (5X) to 1X with deionized water. Add the diluent to the standard vial according to the label and incubate at room temperature for 15 minutes to achieve a final standard concentration of 5000 pg / mL.

[0108] c. Prepare washing solution: Dilute the washing solution (20X) to 1X with deionized water.

[0109] d. Using the standard diluent, serially dilute the standard to obtain six standard concentrations: 5000, 2500, 1250, 625, 312.5, and 156.5 pg / mL. Add these to the pre-coated plate wells in sequence. Also, add the standard diluent directly as a 0 pg / mL control. Note: Prepare standards and generate a standard curve for each experiment. It is recommended to set up background correction wells, adding only TMB solution and stop solution.

[0110] e. Add samples or standards of different concentrations into the corresponding wells at 100 μL / well, seal them with a transparent sealing film, and incubate at room temperature for 120 min.

[0111] f. Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0112] g. Add 100 μl / well of biotinylated antibody, seal the plate with transparent film, and incubate at room temperature for 60 minutes.

[0113] h. Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0114] i. Add 100 μl / well of horseradish peroxidase-labeled streptavidin. Seal the wells with white sealing film and incubate at room temperature in the dark for 20 minutes.

[0115] 1. Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0116] m. Add 100 μl / well of TMB solution, seal the plate with white film, and incubate at room temperature in the dark for 15-20 minutes.

[0117] n. Add 50 μl / well of stop solution, mix well and immediately measure the A450 value.

[0118] o. Calculate the standard curve and convert the concentration of NGAL in the sample according to the standard curve.

[0119] 4.2 Experimental Results

[0120] As shown in Figure 6, when mice were injected with either antimicrobial peptides or colistin, there was no significant difference in NGAL levels induced by the two, indicating that their nephrotoxicity levels were highly similar. This suggests that antimicrobial peptides have a similar safety profile to colistin.

[0121] Example 5: Efficacy of antimicrobial peptides against mice infected with polymyxin-resistant Acinetobacter baumannii

[0122] 5.1 Experimental Procedure

[0123] 5.1.1 Construction of a neutropenic mouse model

[0124] The mice used in this section have passed the animal research ethics review of Nanjing University. In order to avoid the influence of the mouse's intrinsic immune system on the intrinsic activity of antimicrobial peptides, we constructed a mouse model of neutropenia. This experiment used 6-week-old CD1 female mice (20-25g). The mice were randomly placed in individually ventilated cages and maintained in accordance with the standards of the American Association for Accreditation of Laboratory Care. The room was set to a 12-hour light cycle, a temperature of 21°C, and a humidity of 30%. On the 4th day and the 1st day before bacterial infection, 150 mg / kg and 100 mg / kg of cyclophosphamide were injected into the peritoneal cavity of the mice, respectively, to cause neutropenia in the mice.

[0125] 5.1.2 Culture and infection of polymyxin-resistant Acinetobacter baumannii

[0126] A single colony of polymyxin-resistant Acinetobacter baumannii was selected and cultured overnight at 37°C in cation-adjusted MH broth containing 50 μg / mL gentamicin. The culture was centrifuged, the supernatant aspirated, and the bacteria gently washed twice with sterile saline. The OD value was checked at 600 nM and diluted accordingly. 0.05 mL of the bacterial suspension was injected intramuscularly into both thighs of the mouse, providing approximately 1.0 x 10 cells per thigh. 6 The amount of bacteria in CFU.

[0127] 5.1.3 Determination of the efficacy of antimicrobial peptides

[0128] Two, eight, 14, and 24 hours after bacterial infection, mice were subcutaneously injected with 100 μL of vehicle (0.9% saline), colistin (20 mg / kg), or antimicrobial peptide (20 mg / kg). Two hours after infection, untreated control mice (n = 4 mice, n = 8 thighs) were humanely euthanized by CO₂ asphyxiation to determine the initial bacterial burden in the thighs. All mice were closely monitored post-infection for morbidity. Any abnormal clinical signs were recorded. At the experimental endpoint, 24 hours after infection, mice were euthanized by CO₂ asphyxiation (4 mice / 8 thighs per condition). Thigh muscles were aseptically handled, weighed, homogenized, and bacterial burdens were determined by CFU counts on MH agar containing 50 mg / mL gentamicin. Efficacy was determined by the reduction in thigh bacterial burden relative to the treated control group. All graphical data were statistically analyzed using GraphPad Prism software (Prism v.9). The difference in burden between the test and control groups was assessed using univariate analysis. A P value < 0.05 was considered statistically significant.

[0129] 5.2 Experimental Results

[0130] Neutrophil-deficient mice were infected with polymyxin-resistant Acinetobacter baumannii and subsequently treated with colistin and antimicrobial peptides. As shown in Figure 7, colistin did not reduce the number of Acinetobacter baumannii, while antimicrobial peptides exhibited strong antibacterial activity, with a significant reduction in colony-forming units (CFU) compared to the vehicle control group and the colistin-treated group. These results indicate that antimicrobial peptides have a significant antibacterial effect against polymyxin-resistant Acinetobacter baumannii.

[0131] Example 6: Antimicrobial peptide synthesis and detection process

[0132] 6.1 Antimicrobial Peptide Synthesis Process

[0133] 1) Swelling resin

[0134] Take the resin Fmoc-Leu-CTC Resin and place it in a column reactor. Add dichloromethane (10 ml / g), mix and soak for 30 minutes to allow the resin to fully swell.

[0135] 2) Removal of resin protecting groups

[0136] Vacuum dry the swelling reagent, add 20% Pip / N,N-dimethylformamide (10 ml / g), and mix with nitrogen bubbling for 30 minutes. After draining the deprotection reagent, wash with N,N-dimethylformamide (10 ml / g) six times, bubbling with nitrogen for 1 minute each time, and drain for 1 minute. Take a small amount of resin for deprotection testing. Add 1 ml of ninhydrin detection reagent to the resin and place the test tube in a heater above 120°C for 3 minutes. Remove and observe the resin color. If the resin color darkens, deprotection is successful and the next coupling reaction can be carried out. If the resin is light red or yellow, the deprotection time needs to be extended.

[0137] 3) Condensation of the second amino acid

[0138] Dissolve 3 equivalents of Fmoc-Dab(Boc)-OH and 3 equivalents of the condensation reagent OXYMA in N,N-dimethylformamide. Then, add 3 equivalents of the condensation reagent DIC and react for 5 minutes to activate the mixture. Add the above solution to a reactor and bubble nitrogen for 1-2 hours to complete the condensation reaction. The success of the condensation reaction is then checked by observing the resin color. No significant change indicates a successful condensation reaction, resulting in Fmoc-Dab-Leu-CTC resin.

[0139] 4) Treat the resin to prepare for the next amino acid condensation

[0140] After draining the reaction reagents, wash the resin obtained in step 3) four times with N,N-dimethylformamide (10 ml / g), bubbling nitrogen for 1 minute each time and draining for 1 minute to remove residual solvent. Resin swelling, Fmoc removal, and removal efficacy testing were performed in the same manner as in steps 1) and 2).

[0141] 5) Condensation of the third amino acid

[0142] As in step 3), dissolve 3 equivalents of Fmoc-Dab(Boc)-OH and 3 equivalents of OXYMA in N,N-dimethylformamide and activate with 3 equivalents of DIC for 5 minutes. Add the mixture to the reactor and allow nitrogen to bubble for 1-2 hours to complete the second condensation reaction. Observe the resin color change to confirm the Fmoc-Dab-Dab-Leu-CTC resin.

[0143] 6) Cycle steps

[0144] Repeat steps 1) to 5) to condense the amino acids in the sequence from right to left, namely Fmoc-Ile-OH, Fmoc-DLeu-OH, Fmoc-Dab(Boc)-OH, Fmoc-Dab(ivdde)-OH, Fmoc-D-Dab(Boc)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Dab(Boc)-OH to obtain Fmoc-Dab-Thr-{D-Dab}-Dab-Dab-{D-Leu}-Ile-Dab-Dab-Leu-CTC resin.

[0145] 7) Condensation of special raw material 6-ME-Octanoic acid

[0146] Repeat steps 1) and 2) with the resin obtained in step 6) to prepare for the condensation of 6-ME-Octanoic acid. Dissolve 3 equivalents of 6-ME-Octanoic acid and 3 equivalents of OXYMA in N,N-dimethylformamide. Add 3 equivalents of DIC and activate for 5 minutes. Then, add the mixture to the reactor and allow nitrogen bubbling for 1-2 hours to complete the condensation.

[0147] 8) Treat the resin to prepare for cyclization

[0148] The reaction reagents were vacuum-dried and washed with N,N-dimethylformamide (10 ml / g) four times, bubbling nitrogen for 1 min each time and draining for 1 min.

[0149] 9) Cyclization

[0150] The starting material for amino acid No. 5 is Fmoc-DAB(Ivdde)-OH. Add 5% hydrazine hydrate in DMF (12 ml / g) and bubble nitrogen three times for 10 minutes each. This removes the Ivdde protecting group from the side chain of amino acid No. 5 without affecting the side chains of other amino acids. Washing and deprotection are performed. Dissolve two equivalents of the condensation reagent HATU in N,N-dimethylformamide (10 ml / g). Add four equivalents of the condensation reagent DIEA to the reactor and bubble nitrogen for 1-2 hours to complete the cyclization. Observe the resin color; no significant change in color indicates successful cyclization.

[0151] 10) Wash and drain the antimicrobial peptide

[0152] The reagents were vacuum-dried, and the resin was washed with N,N-dimethylformamide (10 ml / g) five times, bubbling nitrogen for 1 min each time, and vacuum-dried for 1 min. The resin was then washed with methanol (12 ml / g ml) five times and vacuum-dried for 1 h.

[0153] 11) Cracking resin

[0154] The resin obtained in step 10) was placed in a boat-shaped reactor, and 10 ml / g of lysis buffer was added. The lysis was carried out at room temperature for 3 h. The reaction solution was then filtered and added to 10 equivalent volumes of glacial ether. The solution was centrifuged and washed three times with glacial ether to obtain a crude white polypeptide solid.

[0155] 12) Purification of crude antimicrobial peptides

[0156] The antimicrobial peptide was initially purified using a trifluoroacetic acid (TFA) / water system. Purification conditions were: 0.065% TFA in water as mobile phase A, 0.05% TFA in acetonitrile as mobile phase B, separation on a C18 column at room temperature and a flow rate of 15 ml / min. Peak elution was measured at 220 nm, and samples were collected before, at, and after the peak appeared for preliminary mass spectrometry analysis.

[0157] The product collected in the previous step was further purified using an HCl / water system as the mobile phase. Purification conditions were: 0.065% HCl in water as mobile phase A, 0.05% TFA in acetonitrile as mobile phase B, at room temperature, at a flow rate of 15 ml / min, on a C18 column. Peaks were measured at 220 nm, and the highest peak was collected as the target sample.

[0158] 13) Freeze-dried packaging

[0159] Place the purified qualified fractions into a freeze-drying tray, cover it, and place it in a freeze dryer for freeze drying. After freeze drying, remove the freeze-drying tray, weigh and aliquot the peptide samples, and store at -20°C.

[0160] 6.2 Antimicrobial peptide detection process

[0161] 1) Identification of antimicrobial peptides

[0162] The antimicrobial peptide was dissolved in water and adjusted to a concentration of 1 mg / mL, and then mass spectrometry (Mass Spectrum) was performed in positive ion mode using the SHIMADZU LCMS-2020 instrument. The mass spectrometry results show the mass-to-charge ratio (molecular weight to charge number ratio, m / z, m is the molecular weight, z is the charge number), so the molecular weight of the product is equal to the product of the mass-to-charge ratio and the charge number. Since hydrogen atoms (H) equivalent to the charge number are added in the positive ion mode, the molecular weight of the hydrogen atoms must be subtracted to obtain the final molecular weight of the product. The formula is: relative molecular mass MW = (m / z) * zz. As can be seen from Figure 8, a total of 3 obvious m / z peaks were detected under different charge conditions.

[0163] When z=1, the molecular weight of the product=1182.0*1-1=1181.0.

[0164] When z=2, the molecular weight of the product = 591.7*2-2 = 1181.4.

[0165] When z=3, the molecular weight of the product = 394.8*3-3 = 1181.4.

[0166] Based on the sequence and structure of the antimicrobial peptide, the molecular weight was calculated to be 1181.4, consistent with the molecular weight of the obtained product, indicating that the synthesized product is the target product. Based on the amino acid composition, the theoretical molecular weight of the uncyclized antimicrobial peptide is 1199.4. After cyclization, the molecular weight is 18 less, at 1181.4, consistent with the molecular weight of the obtained product, indicating that the synthesized product is a cyclized peptide. During the synthesis process, the side chain of the starting material used at amino acid position 4 was protected by the ivdde protecting group, while the side chains of amino acids at other positions were protected by other protecting groups. Prior to cyclization, hydrazine hydrate / DMF was used to remove only the protecting group on the side chain of amino acid position 4, ensuring that cyclization occurred at amino acids 4 and 10.

[0167] 2) Antimicrobial peptide purity analysis

[0168] Separation and detection were performed on a Shimadzu HPLC-2030 instrument using a 1 mg / mL concentration of the antimicrobial peptide, mobile phase A consisting of 0.065% trifluoroacetic acid in water (v / v), and mobile phase B consisting of 0.05% trifluoroacetic acid in acetonitrile (v / v). Since peak area is proportional to the amount of substance, the peak area percentage represents the purity of the antimicrobial peptide.

[0169] As shown in Figure 9, the peak area of ​​the main substance in the product accounts for 97.211% of the total product, indicating that the purity of the main product is 97.211%. Based on the results of Mass Spectrum analysis, it has been confirmed that the product is the target antimicrobial peptide. Therefore, the purity of the target antimicrobial peptide is 97.211%.

[0170] In general, the novel antimicrobial peptides provided herein have at least the following characteristics, relying on their high binding ability to lipid A:

[0171] 1) Inhibit the growth of common Gram-negative bacteria at lower concentrations;

[0172] 2) Inhibited the growth of polymyxin-resistant bacteria at lower concentrations and demonstrated good efficacy;

[0173] 3) Antimicrobial peptides have low cytotoxicity and nephrotoxicity.

[0174] Based on these characteristics, the new antimicrobial peptides provided in this article can be used as new antibiotics to replace polymyxins as the last line of defense.

Claims

1. A compound of the following formula (I), its derivative, pharmaceutically acceptable salt or solvate:

2. The compound, derivative, pharmaceutically acceptable salt or solvate thereof as claimed in claim 1, wherein the salt is selected from hydrochloride, sulfate, phosphate, acetate, oxalate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, picrate and any combination thereof.

3. The compound, derivative, pharmaceutically acceptable salt or solvate thereof according to claim 1 or 2, wherein the solvate is a hydrate or an ethanolate.

4. The compound, derivative, pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 3, wherein the derivative is a PEG derivative or a polysaccharide derivative of the compound.

5. A pharmaceutical composition comprising: 1) A compound according to any one of claims 1 to 4, a derivative thereof, a pharmaceutically acceptable salt or a solvate thereof; as well as 2) Pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, further comprising one or more antibiotics.

7. Use of the compound according to any one of claims 1 to 4, its derivative, pharmaceutically acceptable salt or solvate in the preparation of a medicament for preventing or treating diseases related to bacterial infection.

8. The use according to claim 7, wherein the bacterium is a Gram-negative bacterium.

9. The use according to claim 7 or 8, wherein the bacteria are resistant to any polymyxin or antibiotic.

10. The use according to any one of claims 7 to 9, wherein the bacterium carries the mcr-1 gene.

11. The method according to any one of claims 7 to 10, wherein the bacteria is selected from the group consisting of Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae.

12. The use according to any one of claims 7 to 11, wherein the bacterial infection-related disease is selected from respiratory tract infection, pneumonia, bronchitis, wound infection, sepsis and sepsis.

13. A method for treating a bacterial infection-related disease in a subject, comprising administering to the subject a therapeutically effective amount of a compound, a derivative, a pharmaceutically acceptable salt or a solvate thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to claim 5 or 6.

14. The method of claim 13, wherein the bacteria are Gram-negative bacteria.

15. The method of claim 13 or 14, wherein the bacteria is resistant to any polymyxin or antibiotic.

16. The method of any one of claims 13-15, wherein the bacterium carries the mcr-1 gene.

17. The method of any one of claims 13 to 16, wherein the bacteria is selected from the group consisting of Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Pseudomonas aeruginosa.

18. The method according to any one of claims 13 to 17, wherein the bacterial infection-related disease is selected from respiratory tract infection, pneumonia, bronchitis, wound infection, sepsis and septicemia.

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