Antimicrobial peptoid comprising n-terminal metal complex

Abstract

The present invention relates to: a compound represented by chemical formula 1 or 2, wherein the compound has antibacterial and / or anti-inflammatory effects and is safe for the body, thus having few side effects including abnormal reactions; an antibacterial composition comprising same; and a pharmaceutical composition for preventing or treating bacterial infection, the pharmaceutical composition comprising the compound.

Description

Antimicrobial peptoids containing N-terminal metal complexes

[0001] The present invention relates to novel compounds and uses thereof.

[0002]

[0003] The emergence and spread of multidrug-resistant (MDR) bacteria is a global problem, yet treatments are very limited. Developing antibiotics against Gram-negative bacteria, which possess lipopolysaccharide (LPS) in their outer membrane, is particularly challenging. Infection with Gram-negative bacteria can release LPS, known as endotoxin, into the bloodstream. This LPS can activate pro-inflammatory cells, such as monocytes and macrophages, triggering a powerful host immune response and potentially leading to Gram-negative sepsis, a severe and potentially life-threatening condition. While a broad range of antibiotics and immunomodulators are administered to reduce the mortality rate associated with Gram-negative sepsis, several antibiotics commonly used to treat sepsis, including levofloxacin, ceftriaxone, ciprofloxacin, and meropenem, have shown high rates of resistance.

[0004] Accordingly, research has been conducted to identify new antibiotics effective against multidrug-resistant bacteria. However, natural antimicrobial peptides developed through this approach, such as magainin-2, melittin, cathelicidin, and defensin, have low in vivo stability and the potential to induce immune responses, limiting their use in high doses or systemic applications.

[0005] To address these issues, various peptidomimetic or non-peptide scaffolds that mimic the structural and physicochemical properties of natural antimicrobial peptides have been studied. Among these, peptoids offer the advantages of greater stability and lower immunogenicity compared to natural antimicrobial peptides. However, conventional antimicrobial peptoids exhibit cytotoxicity against mammalian cells, such as NIH 3T3 mouse fibroblasts. Therefore, research is needed to develop antibiotics with reduced toxicity and increased selectivity against animal cells.

[0006] Accordingly, the inventors of the present invention have conducted extensive research to promote multi-target mechanisms for antibacterial action and improve overall therapeutic efficacy, and have thus developed a compound with excellent antibacterial and anti-inflammatory activity and low cytotoxicity, thereby completing the present invention.

[0007]

[0008] The present invention aims to provide a novel compound having antibacterial activity.

[0009] The purpose of the present invention is to provide an antibacterial composition.

[0010] The purpose of the present invention is to provide an antibacterial composition having antibacterial activity against gram-positive bacteria or gram-negative bacteria.

[0011] The purpose of the present invention is to provide an antibacterial composition having antibacterial activity against multidrug-resistant bacteria.

[0012] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating bacterial infections.

[0013]

[0014] 1. A compound represented by the following chemical formula 1 or 2:

[0015] [Chemical Formula 1]

[0016] H-Gly-Gly-His-NLys-(a)2-NLys-Nspe-Nspe-NLys-b-Nspe-NLys-cd-NH2

[0017] [Chemical Formula 2]

[0018] H-NLys-(a)2-NLys-Nspe-Nspe-NLys-b-Nspe-NLys-cd-Ddap-Gly-His-NH2

[0019] In the formula, a to d are each independently Nspe, Npm or NTrp.

[0020] 2. In the above 1, the compound has one chemical formula selected from the group consisting of the following chemical formulas 3 to 13:

[0021] [Chemical Formula 3]

[0022] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2

[0023] [Chemical Formula 4]

[0024] H-Gly-Gly-His-NLys-(Npm)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-NH2

[0025] [Chemical Formula 5]

[0026] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NTrp-NTrp-NH2

[0027] [Chemical Formula 6]

[0028] H-Gly-Gly-His-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2

[0029] [Chemical Formula 7]

[0030] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-NLys-Nspe-NLys-NTrp-NTrp-NH2

[0031] [Chemical Formula 8]

[0032] H-Gly-Gly-His-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NLys-Nspe-NH2

[0033] [Chemical Formula 9]

[0034] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Ddap-Gly-His-NH2

[0035] [Chemical Formula 10]

[0036] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NTrp-NTrp-Ddap-Gly-His-NH2

[0037] [Chemical Formula 11]

[0038] H-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Ddap-Gly-His-NH2

[0039] [Chemical Formula 12]

[0040] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-NLys-Nspe-NLys-NTrp-NTrp-Ddap-Gly-His-NH2

[0041] [Chemical Formula 13]

[0042] H-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NLys-Nspe-Ddap-Gly-His-NH 2.

[0043] 3. Compound 1 above; and an organometallic compound comprising a metal ion bound to the N-terminus or C-terminus of the compound.

[0044] 4. In the above 3, the organometallic compound is a metal ion of Cu(II) or Ni(II).

[0045] 5. An antibacterial composition comprising the compound of 1 above.

[0046] 6. In the above 5, the composition is an antibacterial composition that exhibits antibacterial activity against gram-positive bacteria or gram-negative bacteria.

[0047] 7. In the above 6, the gram-positive bacteria are Staphylococcus sp., Bacillus sp., Streptococcus sp. or Enterococcus sp., an antibacterial composition.

[0048] 8. In the above 6, the Gram-positive bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), quinolone-resistant Staphylococcus aureus (QRSA), vancomycin-resistant enterococcus (VRE), vancomycin intermediate-resistant Staphylococcus aureus (VISA), Bacillus subtilis, Bacillus cereus, Streptococcus pneumoniae, Enterococcus faecalis, or Staphylococcus An antibacterial composition comprising Staphylococcus epidermidis.

[0049] 9. In the above 6, the gram-negative bacteria are Salmonella sp., Acinetobacter sp., Escherichia sp., Pseudomonas sp. or Klebsiella sp., an antibacterial composition.

[0050] 10. An antibacterial composition according to 6 above, wherein the gram-negative bacteria are Salmonella typhimurium, Acinetobacter calcoaceticus, E. coli, Pseudomonas aeruginosa or Klebsiella aerogenes.

[0051] 11. A pharmaceutical composition for preventing or treating bacterial infection, comprising the compound of the above 1.

[0052] 12. A pharmaceutical composition for preventing or treating bacterial infection, wherein the bacterial infection is any one selected from the group consisting of cholera, dysentery, whooping cough, typhoid, diphtheria, plague, scarlet fever, sepsis, skin abscess, tuberculosis, tuberculous meningitis, enteritis, and food poisoning.

[0053]

[0054] The compound of chemical formula 1 and the compound of chemical formula 2 of the present invention have excellent antibacterial effects.

[0055] The compound of chemical formula 1 and the compound of chemical formula 2 of the present invention have excellent anti-inflammatory effects.

[0056] The compound of chemical formula 1 and the compound of chemical formula 2 of the present invention have low cytotoxicity.

[0057] The compound of chemical formula 1 and the compound of chemical formula 2 of the present invention have excellent antibacterial effects against multidrug-resistant bacteria.

[0058] The compounds of chemical formula 1 and chemical formula 2 of the present invention have additional antibacterial activity by generating hydroxyl radicals and causing oxidative damage in addition to membrane destruction, LPS binding, and intracellular aggregation of bacterial cells.

[0059] The antibacterial composition of the present invention has excellent antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.

[0060] The pharmaceutical composition for preventing or treating bacterial infections of the present invention can exhibit preventive, ameliorating and / or therapeutic effects on bacterial infections such as cholera, dysentery, whooping cough, typhoid fever, diphtheria, plague, scarlet fever, sepsis, skin abscess, tuberculosis, tuberculous meningitis, enteritis and food poisoning.

[0061]

[0062] Figure 1 shows the results of analyzing the compound of the present invention using high-resolution mass spectrometry (HRMS) after purifying it using high-performance liquid chromatography (HPLC).

[0063] Figure 2 shows the results of an experiment evaluating the generation of hydroxyl radicals of a compound according to one embodiment of the present invention.

[0064] Figure 3 shows the results of an experiment evaluating the DNA cleavage induction ability of a compound according to one embodiment of the present invention.

[0065] Figure 4 shows the results of an experiment evaluating lipid peroxidation activity of a compound according to one embodiment of the present invention.

[0066] Figure 5 shows the results of an antibacterial activity evaluation experiment against S. pneumoniae of a compound according to one embodiment of the present invention.

[0067] Figure 6 shows the results of an anti-inflammatory activity evaluation experiment of a compound according to one embodiment of the present invention.

[0068] Figure 7 shows the results of in vitro and in vivo toxicity evaluation experiments of a compound according to one embodiment of the present invention.

[0069] Figures 8 and 9 show the results of an in vivo sepsis treatment effect evaluation experiment of a compound according to one embodiment of the present invention.

[0070]

[0071] The present invention provides a compound represented by the following chemical formula 1 or 2:

[0072] [Chemical Formula 1]

[0073] H-Gly-Gly-His-NLys-(a)2-NLys-Nspe-Nspe-NLys-b-Nspe-NLys-cd-NH2

[0074] [Chemical Formula 2]

[0075] H-NLys-(a)2-NLys-Nspe-Nspe-NLys-b-Nspe-NLys-cd-Ddap-Gly-His-NH2

[0076] In the formula, a to d are each independently Nspe, Npm or NTrp.

[0077] In one embodiment, the compound represented by Chemical Formula 1 or 2 may have any one chemical formula selected from the group consisting of Chemical Formulas 3 to 13 below:

[0078] [Chemical Formula 3]

[0079] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2

[0080] [Chemical Formula 4]

[0081] H-Gly-Gly-His-NLys-(Npm)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-NH2

[0082] [Chemical Formula 5]

[0083] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NTrp-NTrp-NH2

[0084] [화학식 6]

[0085] H-Gly-Gly-His-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2

[0086] [화학식 7]

[0087] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-NLys-Nspe-NLys-NTrp-NTrp-NH2

[0088] [화학식 8]

[0089] H-Gly-Gly-His-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NLys-Nspe-NH2

[0090] [화학식 9]

[0091] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Ddap-Gly-His-NH2

[0092] [화학식 10]

[0093] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NTrp-NTrp-Ddap-Gly-His-NH2

[0094] [화학식 11]

[0095] H-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Ddap-Gly-His-NH2

[0096] [화학식 12]

[0097] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-NLys-Nspe-NLys-NTrp-NTrp-Ddap-Gly-His-NH2

[0098] [Chemical Formula 13]

[0099] H-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NLys-Nspe-Ddap-Gly-His-NH2.

[0100] The present invention also provides a compound having any one chemical formula selected from the group consisting of the following chemical formulas 14 to 20:

[0101] [Chemical Formula 14]

[0102] H-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Gly-Ddap-Gly-His-NH2

[0103] [Chemical Formula 15]

[0104] H-Gly-Gly-His-Gly-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2

[0105] [Chemical Formula 16]

[0106] H-NLys-Npm-Npm-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-Gly-Ddap-Gly-His-NH2

[0107] [Chemical Formula 17]

[0108] H-Gly-Gly-His-Gly-NLys-Npm-Npm-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-NH2

[0109] [Chemical Formula 18]

[0110] H-NLys-Npm-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-Gly-Ddap-Gly-His-NH2

[0111] [Chemical Formula 19]

[0112] H-Gly-Gly-His-NLys-Npm-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH2

[0113] [Chemical Formula 20]

[0114] H-Gly-Gly-His-Gly-NLys-Npm-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH 2.

[0115] In the present invention, “NLys” refers to N-(4-aminobutyl)glycine and has the following chemical formula 21 structure:

[0116] [Chemical Formula 21]

[0117]

[0118] In the present invention, “Nspe” refers to N-(S)-(1-phenylethyl)glycine and has the structure of the following chemical formula 22:

[0119] [Chemical Formula 22]

[0120]

[0121] In the present invention, “Npm” refers to N-(phenylmethyl)glycine and has the structure of the following chemical formula 23:

[0122] [Chemical Formula 23]

[0123]

[0124] In the present invention, “NTrp” refers to N-[2-(1H-indol-3-yl)ethyl]glycine and has the structure of the following chemical formula 24:

[0125] [Chemical Formula 24]

[0126]

[0127] When the compound represented by chemical formula 1 or 2 of the present invention is administered in a living body, it can bind to Cu(II) ions existing independently in the blood or in the body or Cu(II) ions bound to proteins such as albumin.

[0128]

[0129] The present invention provides an organometallic compound comprising a compound represented by chemical formula 1 or 2 and a metal ion bound to the N-terminus or C-terminus thereof.

[0130] The present invention provides an organometallic compound comprising a compound having any one chemical formula selected from the group consisting of chemical formulas 3 to 20 and a metal ion bound to the N-terminus or C-terminus thereof.

[0131] In one embodiment, the metal ion bound to the organometallic compound of the present invention may be Cu(II) or Ni(II), but is not limited thereto.

[0132]

[0133] The present invention provides an antibacterial composition comprising, as an active ingredient, a compound having any one chemical formula selected from the group consisting of chemical formulas 3 to 20.

[0134] In the present invention, “antibacterial” means the ability to resist bacteria.

[0135] In one embodiment, the antibacterial composition of the present invention can exhibit antibacterial activity through destruction of bacterial cell membranes, LPS binding, intracellular aggregation, generation of ROS such as hydroxyl radicals, DNA oxidation, DNA cleavage, and oxidation of phospholipids of cell membranes.

[0136] In one embodiment, the active ingredient of the antibacterial composition of the present invention forms a complex with a metal ion, such as Cu(II) or Ni(II), at the N-terminal Gly-Gly-His or the C-terminal Ddap-Gly-His, thereby generating ROS and causing oxidative damage to the bacterial lipid membrane, and acting as a nuclease that cleaves bacterial DNA, thereby improving the antibacterial activity.

[0137] In the present invention, the N-terminus (Gly-Gly-His) or C-terminus (Ddap-Gly-His) of the compound having any one chemical formula selected from the group consisting of chemical formulas 3 to 20 has a high affinity for Cu(II) under physiological conditions. Therefore, in the examples below, in vitro experiments were performed using the compound of the present invention or its metal complex, whereas in vivo experiments were performed by administering the compound in consideration of binding to Cu(II) ions existing independently in the blood or in the body or Cu(II) ions bound to proteins such as albumin.

[0138] In the present invention, bacteria collectively refers to gram-positive bacteria and gram-negative bacteria.

[0139] In one embodiment, the gram-positive bacteria may be antibiotic-resistant bacteria.

[0140] In one embodiment, the Gram-positive bacteria may be, but is not limited to, Staphylococcus sp., Bacillus sp., Streptococcus sp., or Enterococcus sp.

[0141] In one embodiment, the gram-positive bacteria is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), quinolone-resistant Staphylococcus aureus (QRSA), vancomycin resistant enterococcus (VRE), vancomycin intermediate-resistant Staphylococcus aureus (VISA), Bacillus subtilis, Bacillus cereus, Streptococcus pneumoniae, Enterococcus faecalis, or Staphylococcus epidermis. epidermidis).

[0142] In one embodiment, the gram-negative bacteria may be antibiotic-resistant bacteria.

[0143] In one embodiment, the gram-negative bacteria may be, but is not limited to, Salmonella sp., Acinetobacter sp., Escherichia sp., Pseudomonas sp., or Klebsiella sp.

[0144] In one embodiment, the gram-negative bacteria may be Salmonella typhimurium, Acinetobacter calcoaceticus, E. coli, Pseudomonas aeruginosa, or Klebsiella aerogenes.

[0145]

[0146] The present invention provides a pharmaceutical composition for preventing or treating bacterial infection, comprising as an active ingredient a compound having any one chemical formula selected from the group consisting of chemical formulas 3 to 20.

[0147] In the present invention, bacterial infection is a general term for a disease caused by pathogenic bacteria parasitizing an individual, and may be any one selected from the group consisting of cholera, dysentery, whooping cough, typhoid fever, diphtheria, plague, scarlet fever, sepsis, skin abscess, tuberculosis, tuberculous meningitis, enteritis, and food poisoning, but is not limited thereto.

[0148] In one embodiment, the bacterial infection may be gram-negative sepsis.

[0149] In the present invention, “prevention of bacterial infection” means any act of suppressing, delaying or reducing bacterial infection.

[0150] In the present invention, “treatment of bacterial infection” means any action that improves or beneficially changes the symptoms of an individual suspected of having or suffering from a bacterial infection.

[0151] In one embodiment, prevention and / or treatment of bacterial infections may be achieved by the antibacterial activity of the pharmaceutical composition of the present invention.

[0152] The pharmaceutical composition of the present invention may contain the active ingredient alone, or may further contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0153] The carrier, excipient or diluent that may be included in the pharmaceutical composition of the present invention may be, but is not limited to, lactose, dextrose, sucrose, dextrin, maltodextrin, 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 or mineral oil.

[0154] The pharmaceutical composition of the present invention can be provided by administering it to a subject.

[0155] In the present invention, “administration” means introducing a predetermined composition to an individual by an appropriate method, and “individual” means all animals such as livestock and mice, and may be mammals including humans, for example.

[0156] The route of administration of the pharmaceutical composition of the present invention may be oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal, but is not limited thereto.

[0157] In one embodiment, the pharmaceutical composition of the present invention may be administered orally or parenterally.

[0158] When administering the composition of the present invention parenterally, it is preferable to select an injection method such as external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection, but is not limited thereto.

[0159] The pharmaceutical composition of the present invention may be provided in the form of a solid preparation for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0160] The pharmaceutical composition of the present invention may be provided in a liquid form for oral administration, such as a suspension, a solution, an emulsion, or a syrup.

[0161] The pharmaceutical composition of the present invention may be provided as a preparation for parenteral administration, such as a sterile aqueous solution, non-aqueous solution, suspension, emulsion, lyophilized preparation or suppository.

[0162] The pharmaceutical composition of the present invention can be administered to a subject in a pharmaceutically effective amount, and the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment.

[0163] The pharmaceutically effective amount of the pharmaceutical composition of the present invention can be determined based on factors including the severity of the disease, the activity of the pharmaceutical composition, the sensitivity of the subject or patient to the pharmaceutical composition, the time of administration, the route of administration, the excretion rate, the duration of treatment, and concomitant medications, and other factors well known in the medical field, and can be appropriately selected by a person skilled in the art.

[0164] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other conventional therapeutic agents, and when administered in combination, may be administered sequentially or simultaneously, and may be administered singly or in multiple doses, which can be readily determined by a person skilled in the art.

[0165]

[0166] Hereinafter, the present invention will be described in detail with examples to specifically illustrate the invention. However, the following examples are provided merely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

[0167]

[0168] Example

[0169] Novel compounds were synthesized through manufacturing examples, and their antibacterial activity and hemolytic activity against both Gram-negative and Gram-positive bacteria were tested to confirm their selectivity. The compound with the highest antibacterial activity and selectivity was selected through in vitro evaluation, and its mechanism of action against Gram-negative bacteria, including interactions with cell membranes and intracellular components and oxidative damage, was investigated. Furthermore, their antibacterial and anti-inflammatory effects were evaluated using an in vivo model of Gram-negative sepsis.

[0170]

[0171] [Manufacturing Example 1]

[0172] Seven antimicrobial peptoids were selected as comparative examples, including three peptoids with varying degrees of helicity (Comparative Example 1: fully helical, Comparative Example 2: moderately helical, Comparative Example 3: weakly helical) and four peptoids containing indole side chains (Comparative Examples 4 to 7).

[0173]

[0174]

[0175] The compounds listed in Table 1 were prepared according to the solid-phase submonomer synthesis protocol. Fmoc-Rink amide MBHA resin (125 mg, 0.065 mmol, resin loading: 0.52 mmol / g) was swelled in DMF for 20 min and treated twice with 20% piperidine in DMF (1 mL) at 80 °C in a microwave reactor (maximum power 600 W, ramp 2 min, hold 2 min, stirring level 2). The resin was washed with DMF (×3), CH2Cl2 (×2), MeOH (×1), DMF (×2), and CH2Cl2 (×3). Bromoacetylation was performed by adding bromoacetic acid (1.0 mL, 1.2 M in DMF, 1.3 mmol) to the Fmoc-protected resin, followed by N,N'-diisopropylcarbodiimide (200 μL, 1.3 mmol). The reaction mixture was stirred at 35°C (maximum power 300 W, ramp 30 s, hold 1 min, stirring level 2). The resin was washed using the same procedure as previously washed. The amine substitution reaction was performed by adding primary amines (e.g., NTrp(Boc) (0.2 g, 0.975 mmol), Nspe (120 μL, 0.975 mmol), and NLys(Boc) (0.2 g, 1.3 mmol) as 1.0 M solutions in NMP (N-Methylpyrrolidone) and stirring at 80°C (maximum power 300 W, ramp 2 min, hold 90 sec, stirring level 2). The resin was washed in the same order as previously washed. The bromoacetylation and substitution reactions were repeated until the desired sequence (Comparative Examples 1 to 7) was obtained.

[0176] The resin-bound peptoid was swelled in DMF for 20 min. To conjugate amino acids to the synthesized peptoid, Fmoc-His(Trt)-OH (120 mg, 0.195 mmol), HATU (74 mg, 0.195 mmol), and DIEA (89 μL, 0.52 mmol) dissolved in DMF (1 mL) were added, and the reaction mixture was shaken at room temperature overnight. The resin was washed in the same manner as previously washed. Fmoc deprotection was performed with 20% piperidine in DMF (1 mL), and the resin was washed with DMF and CH2Cl2. Amino acid coupling reactions were performed by adding Fmoc-AA-OH (0.195 mmol), HATU (148 mg, 0.39 mmol), and DIEA (167 μL, 0.975 mmol) dissolved in DMF and stirring at 75°C (maximum power 300 W, ramp 2 min, hold 8 min, stirring level 2). Peptide coupling and Fmoc deprotection reactions were repeated until the desired sequence (example) was obtained. The resin was washed and reacted with a cleavage cocktail (TFA:H2O:TIS = 95:2.5:2.5) for 1 h.

[0177]

[0178] [Manufacturing Example 2] Manufacturing of a metal composite of the example

[0179] The purified compound was dissolved in HEPES buffer (20 mM, 100 mM NaCl, pH 7.4) and then mixed with CuCl2 or NiCl2 solution at a molar ratio of 1:0.9. Before use, the solution was pre-incubated at room temperature for 30 minutes to form Cu(II) or Ni(II) complexes. The solution containing the Cu complex was pale purple, and the solution containing the Ni complex was pale yellow.

[0180]

[0181] [Experimental Example 1] HPLC and HRMS

[0182] According to Manufacturing Example 1, the synthetic comparative examples and examples were purified (>98%) through HPLC (high-performance liquid chromatography), and then characterized using analytical HPLC and HRMS (high-resolution mass spectrometry).

[0183] The HPLC and HRMS results are shown in Figure 1 and Table 2 below, respectively.

[0184] Compound Exact mass[M] + Calculated mass [M+2H] 2+ Observed mass [M+2H] 2+HPLC elution (% MeCN) Comparative Example 11818.0789910.0473910.046951.0 Example 1a2098.20731050.11151050.109747.0 Example 1b2155.22871078.62221078.620546.6 Example 1c2069.18071035.59821035.595849.0 Example 1d2126.20221064.10891064.113049.0 Comparative Example 21762.0163882.0160882.020051.6 Example 2a2099.16611050.59091050.590145.3 Example 2b2013.11811007.56691007.564847.6 Example 2c2070.13961036.07761036.075447.6 Comparative Example 31719.9693860.9925860.993749.6 Example 3a2057.11921029.56741029.568144.0 Example 3b1971.0712986.5434986.545246.0 Example 3c2028.09261015.05411015.056246.0Comparative Example 41896.1007949.0582949.059951.6Example 4a2176.22911089.12241089.126047.3Example 4b2147.20251074.60911074.608549.0Comparative Example 51896.1007949.0582949.057352.0Example 5a2176.22911089.12241089.123047.0Example 5b2147.20251074.60911074.609650.0Comparative Example 61863.1116932.5636932.562344.6Example 6a2143.23991072.62781072.623041.6Example 6b2114.21341058.11451058.111642.6Comparative Example 71863.1116932.5636932.564345.3Example 7a2143.23991072.62781072.625943.0Example 7b2114.21341058.11451059.110144.3

[0185]

[0186] [Experimental Example 2] In vitro antibacterial activity evaluation

[0187] To evaluate antibacterial activity, the minimum inhibitory concentration (MIC) of the compound (prepared according to Preparation Example 1) and its metal complex (prepared according to Preparation Example 2) against Gram-negative bacteria (E. coli ATCC 25922) was measured.

[0188] The MIC value of each compound was determined using a broth dilution assay. In each assay, a primary culture of bacteria was grown overnight in Mueller Hinton Broth 2 (cation-adjusted) medium with shaking at 37°C. A secondary culture was started the following day, incubated for 3–4 h, and then inoculated with approximately 2–5 × 10 5 The bacteria were diluted and cultured in MHB2 medium corresponding to the colony forming unit (CFU / mL). For each compound, 2-5 × 10 5 100 μL of bacteria (CFU / mL) were added in triplicate. Each compound was serially diluted twofold at six different concentrations and analyzed. The maximum concentration used for the compound was 25 μM. The plates were then incubated at 37°C for 24 h, and MIC data were recorded by measuring the OD value at 600 nm using a microplate reader (Tecan Infinite 200 PRO, China). The MIC value was determined by averaging the triplicate OD values ​​for each concentration.

[0189] The MIC values ​​for each compound are shown in Table 3 below:

[0190] Compound Minimum Inhibitory Concentration (MIC) (μM)E. coliATCC25922Compound onlyCu complexNi complexComparative Example 16.3Example 1a12.512.512.5Example 1b12.56.3-12.512.5Example 1c12.512.512.5Example 1d12.52525Comparative Example 26.3Example 2a>25>25>25Example 2b12.512.512.5Example 2c252525Comparative Example 312.5Example 3a>25>25>25Example 3b252525Example 3c252525Comparative Example 46.3Example 4a6.312.512.5Example 4b12.52525Comparative Example 53.1 Embodiment 5a 25 12.5 25 Embodiment 5b 12.5 12.5 12.5 Comparative Example 66.3 Embodiment 6a > 25 > 25 > 25 Embodiment 6b 6.3 6.3 6.3 Comparative Example 76.3 Embodiment 7a > 25 > 25 > 25 Embodiment 7b 25 12.5 25

[0191]

[0192] [Experimental Example 3] In vitro hemolytic activity evaluation

[0193] To evaluate the selectivity of compounds for bacterial cells, hemolytic activity was measured. The degree of hemolysis was measured in SD rat red blood cells (RBCs). Animal experiments were approved by the Laboratory Animal Resource Center (LARC) of the Gwangju Institute of Science and Technology. Blood from 13-week-old male SD rats was collected and placed in tubes containing 158 UPS units of sodium heparin to prevent coagulation and centrifuged at 1000 rpm for 10 minutes at 4°C. The RBCs containing the anticoagulant were washed three times with sterile phosphate-buffered saline (PBS; 35 mM phosphate, 150 mM NaCl, pH 7.4). A small aliquot of the washed cells was resuspended in fresh PBS to prepare a 10% (v / v) RBC solution. Then, 150 μL of RBC solution was mixed with 50 μL of serially diluted test samples (0–100 μM). The reaction mixture was incubated at 37°C for 1 h. Triton X-100 and PBS / 10 mM HEPES buffer (3:1) were used as positive and negative controls. The plate was then rotated at 1000 rpm for 15 min, and 100 μL of the supernatant was transferred to a clear 96-well plate. The absorbance was measured at 540 nm and normalized compared to the control.

[0194] As shown in Tables 4 and 5 below, all examples exhibited lower hemolytic activity than their respective comparative examples. The metal complexes of the examples exhibited further reduced hemolytic activity (except for Examples 3a to 3c and Example 4a):

[0195] Compound Hemolysis (%)HC 10 / HC 50 (μM)H max(100 μM)25 μM50 μM100 μMComparative Example 140.397.696.59.64 / 28.396.5±7.0Example 1a0.046.2219.162.6 / >10019.1±0.7Example 1a-Cu-1.847.1213.176.2 / >10013.1±2.4Example 1a-Ni5.768.0912.073.1 / >10012.0±1.3Example 1b2.028.7923.251.1 / >10023.2±1.1Example 1b-Cu-1.634.5212.385.3 / >10012.3±5.1Example 1b-Ni 10.5 13.8 3 14.9 2 2.8 / > 100 14.9 ± 0.8 Example 1c 7.9 5 32.0 6 1.6 2 2.9 / 81.6 6 1.6 ± 4.0 Example 1c-Cu 1.1 15.7 30.8 3 7.3 / > 100 30.8 ± 0.9 Example 1c-Ni 10.3 17.3 32.2 17.7 / > 100 32.2 ± 3.6 Example 1d 6.5 0 22.4 4 9.8 2 7.7 / 100 49.8 ± 2.3 Example 1d-Cu 6.5 8 12.4 2 7.2 3 6.8 / > 100 27.2 ± 5.9 Example 1d-Ni11.718.132.719.9 / >10032.7±2.0Comparative Example 212.529.060.922.3 / 83.060.9±5.3Example 2a0.662.327.73>100 / >1007.7±0.3Example 2a-Cu-0.360.133.00>100 / >1003.0±0.7Example 2a-Ni7.906.488.93>100 / >1008.9±0.8Example 2b3.018.6818.456.6 / >10020.0±0.3Example 2b-Cu4.753.008.20>100 / >1008.2±2.5Example 2b-Ni2.2710.9210.592.5 / >10010.5±1.6Example 2c0.916.8420.858.4 / >10022.3±0.1Example 2c-Cu0.030.929.09>100 / >1009.0±1.1Example 2c-Ni4.017.1510.096.4 / >10010.0±2.2Comparative Example 30.213.7513.781.3 / >10013.7±2.0Example 3a-0.430.14-0.02>100 / >1001.5±0.3Example 3a-Cu3.133.793.77>100 / >1003.7±0.5Example 3a-Ni2.377.103.08>100 / >1003.0±2.2Example 3b-0.71-0.220.92>100 / >1002.4±0.2Example 3b-Cu4.865.064.66>100 / >1004.6±0.9Example 3b-Ni-0.258.010.02>100 / >1000.0±4.8Example 3c-0.430.181.36>100 / >1001.3±2.2Example 3c-Cu3.593.445.56>100 / >1005.5±1.1Example 3c-Ni-0.610.955.11>100 / >1005.1±1.1.

[0196] Compound Hemolysis (%)HC 10 / HC 50 (μM)H max(100 μM)25 μM50 μM100 μMComparative Example 429.950.769.47.7 / 63.869.4±2.0Example 4a11.120.329.025.4 / >10029.0±1.1Example 4a-Cu10.013.931.723.6 / >10031.7±0.7Example 4a-Ni8.3618.131.426.4 / >10031.4±2.6Example 4b12.723.940.620.6 / >10040.6±0.5Example 4b-Cu5.4415.928.430.3 / >10028.4±0.9Example 4b-Ni8.3120.029.231.1 / >10029.2±2.2Comparative Example 533.055.373.97.9 / 63.673.9±1.7Example 5a8.3514.023.831.9 / >10023.8±1.1Example 5a-Cu7.8711.818.239.3 / >10018.2±0.6Example 5a-Ni6.5512.321.840.2 / >10021.8±1.2Example 5b13.825.540.216.3 / >10040.2±2.0Example 5b-Cu6.30 14.626.136.5 / >10026.1±0.7Example 5b-Ni7.85 16.832.325.7 / >10032.3±1.1Comparative Example 62.727.85 6.70>100 / >1003.3±0.2Example 6a3.834.23 3.38>100 / >1003.3±1.0Example 6a-Cu6.25 10.626.59>100 / >1006.5±0.5Example 6a-Ni5.66 4.94 5.80>100 / >1005.8±0.4Example 6b3.134.835.57>100 / >1001.1±0.5Example 6b-Cu0.080.551.91>100 / >1000.6±0.1Example 6b-Ni2.273.052.61>100 / >1000.9±0.2Comparative Example 74.958.7711.175.3 / >10011.1±0.5Example 7a4.936.105.26>100>1005.2±0.4Example 7a-Cu6.335.835.09>100 / >1005.0±0.7Example 7a-Ni5.645.295.15>100 / >1005.1±1.5Example 7b4.985.906.40>100 / >1006.3±2.4Example 7b-Cu2.242.994.34>100 / >1004.3±0.6 Example 7bNi3.093.734.20>100 / >1004.2±1.2.

[0197] HC in Tables 4 and 5 10 and HC 50 refers to the concentrations of compounds that cause 10% and 50% hemolysis in rat erythrocytes, respectively. These concentrations represent the average of triplicates. H max represents the percentage (%) of hemolysis at the highest concentration tested (100 μM).

[0198] When an embodiment forms a complex with a metal ion, the positive charge density of the peptidyl ligand of the embodiment increases, which reduces the overall hydrophobicity and amphipathicity of the embodiment. This property reduces the interaction with eukaryotic cell membranes and improves the selectivity for bacterial cells. Hydrophobicity as indicated by HPLC elution (i.e., % MeCN in Table 2 or t in Figure 1) R ) is closely correlated with the hemolytic activity of each compound. All examples showed lower cytotoxicity than the comparative examples. In particular, Example 6b showed the same antibacterial activity (same MIC value) as the comparative examples, while showing lower cytotoxicity than the comparative examples, and thus exhibited the highest selectivity against E. coli.

[0199]

[0200] [Experimental Example 4] In vitro cytotoxicity evaluation

[0201] Example 6b, which showed the highest selectivity against E. coli, was further evaluated for cytotoxicity against mammalian cells including human keratinocyte (HaCaT) cells, normal human lung fibroblasts (MRC-5), mouse macrophages (Raw 264.7), and human embryonic kidney (HEK293) cells.

[0202] MRC-5 and HEK293 cells were purchased from the Korea Cell Line Bank (Seoul, South Korea), HaCaT cells from the Skin Research Institute of Amorepacific R&D Center (Yongin, South Korea), and Raw264.7 cells from the Korea Basic Science Institute (Gwangju, South Korea). All cells were cultured in DMEM medium containing 10% FBS at 37°C in an atmosphere of 5% CO2. For the MTS cell viability assay, cells (3 × 10 4 HaCaT cells / well, 1.3 × 10 4 MRC-5 cells / well, 4 × 10 4 Raw264.7 cells / well or 1×10 4 HEK293 cells / well were cultured in 96-well plates for 24 h. When the cell density reached approximately 70%, the cell medium was replaced with serial dilutions of the compound stock and incubated for 24 h. The cells were then treated with 20 μL of CellTiter 96 aqueous nonradioactive cell proliferation assay (Promega) reagent containing the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] at 37°C for 4 h. MTS-formazan production in viable cells was measured at 490 nm using a microplate reader, and cell viability (%) was determined by comparing it to untreated cells.

[0203] The results of the cytotoxicity evaluation of the compounds on mammalian cells are shown in Table 6 below:

[0204] Compound LC 50 (μM) HaCaTMRC-5Raw 264.7HEK293Comparative Example 618.811.814.916.3Example 6b11018739.639.5Example 6b-Cu88.719731.934.3Example 6b-Ni11620041.038.2

[0205] In Table 6, LC50 refers to the concentration of a compound that kills 50% of cells.

[0206] Similar to the hemolysis assay results, Example 6b and its metal complexes (Example 6b-Cu and Example 6b-Ni) showed lower cytotoxicity than Comparative Example 6 in all mammalian cells tested.

[0207]

[0208] [Experimental Example 5] Evaluation of antibacterial activity against Gram-negative multidrug-resistant strains

[0209] The antibacterial activity against multidrug-resistant Gram-negative bacteria of Comparative Example 6 and Example 6b was evaluated by measuring the MIC value using the same method as described in Experimental Example 2. The results are shown in Table 7 below:

[0210] Compound MIC (μM) E. coli NMS12 A. baumannii NMS1707 Comparative Example 612.53.1 Example 6b12.53.1 Example 6b-Cu12.53.1 Example 6b-Ni12.53.1

[0211] Comparative Example 6 and Example 6b showed similar antibacterial activity against carbapenem-resistant E. coli (NMS11035), colistin-resistant E. coli (NMS12), and carbapenem-resistant Acinetobacter baumannii (NMS1707).

[0212] Considering that Example 6b has lower cytotoxicity in mammalian cells compared to Comparative Example 6, Example 6b is more effective in combating these multidrug-resistant pathogens.

[0213]

[0214] [Experimental Example 6] Evaluation of Hydroxyl Radical Generation

[0215] In addition to the antibacterial actions similar to those of the comparative examples, such as membrane disruption, LPS binding and intracellular aggregation of bacterial cells, the examples have additional antibacterial activity by generating catalytic ROS and causing oxidative damage to bacterial cells.

[0216] To confirm the generation of hydroxyl radicals in the examples, a DCF-DA assay was performed using 2',7'-dichlorofluorescein diacetate (H2DCF-DA), a fluorescent dye that reacts with hydroxyl radicals in solution to form 2',7'-dichlorofluorescein (DCF) (Fig. 2). This experiment was performed in the presence of hydrogen peroxide, which is reduced by a Fenton-like reaction catalyzed by a Cu(II) complex to generate hydroxyl radicals. Ascorbate is required as a reducing agent to carry out this copper-redox catalytic cycle.

[0217] The specific experimental method is as follows:

[0218] Solutions were prepared using HEPES buffer (20 mM, 100 mM NaCl, pH 7.4) containing final concentrations of 1 μM metal-compound complex or CuCl2 or NiCl2, 1 mM H2O2, 1 mM ascorbate, and 10 μM 2',7'-dichlorofluorescein diacetate. Total fluorescence intensity was read in 96-well plates using a Synergy H1 microplate reader (BioTek Instrument, VT, USA) with excitation at 485 nm and emission at 535 nm during incubation for 60 min at room temperature.

[0219] As shown in Figure 2, the DCF-DA assay results demonstrated that Example 6b-Cu exhibited strong catalytic activity by generating hydroxyl radicals. These results suggest that Example 6b could potentially kill bacteria through oxidative damage, an additional antibacterial mechanism not present in Comparative Example 6.

[0220]

[0221] [Experimental Example 7] In vitro DNA cleavage ability evaluation

[0222] The present inventors investigated the ability of Example 6b-Cu to induce oxidative DNA cleavage to confirm that the hydroxyl radicals generated by Example 6b-Cu can oxidize the DNA of the bacterial cytoplasm or the phospholipids of the cell membrane, thereby causing cell death.

[0223] A 25 μM compound solution was incubated with pUC19 (100 ng) (ThermoFisher Scientific, MA, USA), ascorbic acid (1 mM), and hydrogen peroxide (1 mM) in HEPES buffer (20 mM, 100 mM NaCl, pH 7.4) for 2 h, and the DNA reaction was run for 2 h at room temperature. The reaction was quenched by adding 6 × DNA gel loading dye (ThermoFisher Scientific, MA, USA) in an ice bath, and the quenched reaction mixture was loaded onto a 0.8% agarose gel containing GreenStar™ (BIONEER Corporation, Daejeon, South Korea).

[0224] As a result, as can be seen in the agarose gel image of Fig. 3, both supercoiled and linearized DNA were observed when DNA was incubated with Example 6b-Cu or Example 6b-Ni. When DNA was incubated with Comparative Example 6 or Example 6b, only smearing and retardation of DNA bands were observed, indicating that the compound and DNA formed a complex. These results indicate that Example 6b-Cu and Example 6b-Ni maintain DNA binding properties and induce oxidative degradation of DNA.

[0225]

[0226] [Experimental Example 8] Evaluation of lipid peroxidation activity

[0227] To confirm that the examples have lipid peroxidation activity as a bacterial killing mechanism, a thiobarbituric acid reactive substance (TBARS) assay was first performed to measure the production of lipid peroxidation products, primarily malondialdehyde (MDA).

[0228] E. coli ATCC 25922 was cultured overnight in MHB2 medium with shaking at 37°C. Subculture was started the next day and OD 600 The cells were grown to confluency of 0.6. The cells were spun down (4°C, 4000 rpm, 10 min), washed twice with buffer (20 mM HEPES, 100 mM NaCl, pH 7.4), and resuspended in the buffer. 5 mL of the cell solution was incubated with 10 μM compounds, 1 mM ascorbate, and 1 mM H2O2 for 2 h at 37°C. The cells were washed twice with buffer, resuspended in 375 μL of 0.44 M H3PO4, and 12.5 μL of 9.08 mM butylated hydroxytoluene (BHT) was added and incubated at room temperature for 10 min. Then, 125 μL of 41.6 mM 2-thiobarbituric acid (TBA) in 70% ethanol solution was added and incubated at 95°C for 60 minutes to form MDA-TBA. After cooling to room temperature, the tube was spun down (13,000 rpm, 10 minutes) to remove the cell pellet. 200 μL of the supernatant was transferred to a well of a 96-well plate, and the absorbance was measured at a wavelength of 532 nm. Then, the obtained OD 532 The MDA-TBA adduct concentration was calculated by comparing it with a pre-generated MDA standard curve.

[0229] As a result, as shown in Fig. 4a, when E. coli was incubated with Comparative Example 6, Example 6b, and Example 6b-Ni, a low level of MDA was observed, whereas Example 6b-Cu significantly increased the MDA level.

[0230] Although the TBARS assay is a well-established and widely used method for measuring lipid peroxidation, it has limitations in terms of sensitivity and specificity. Therefore, the effect of bacterial membrane lipid peroxidation in the examples was further confirmed using BODIPY 581 / 591 C11, a lipid peroxidation probe whose emission wavelength changes from red to green upon oxidation.

[0231] E. coli ATCC 25922 2×10 8 CFU / mL were treated with the compound (12.5 μM) or cumene hydroperoxide (10 μM, positive control) for 1 h. Cells were washed twice and incubated with BODIPY 581 / 591 C11, a lipid peroxidation-sensitive dye, for 30 min. Cells were washed three times with PBS, and fluorescence readings for reduction and oxidation of the dye were obtained at excitation / emission wavelengths of 581 / 591 nm and 488 / 510 nm, respectively. Confocal laser scanning microscopy analysis was performed using an FV3000RS (Olympus, Japan) at the GIST Central Research Facilities (GCRF). Flow cytometry analysis was performed using a CyFlow® Cube 6 flow cytometer equipped with a 488 nm blue laser (Sysmex Korea, Seoul, South Korea). Fluorescence was detected at 530 nm (green). Flow cytometry data were acquired and processed using FCS express 5 software (De Novo, Pasadena, CA, USA).

[0232] The results of observing lipid peroxidation of a single E. coli cell using a confocal microscope are shown in Fig. 4b, and the results of analyzing lipid peroxidation of a large number of cells using a flow cytometer are shown in Fig. 4c.

[0233] The results of the lipid peroxidation evaluation experiment were summarized to show that severe lipid peroxidation occurred in bacteria treated with Example 6b-Cu, indicating that hydroxyl radicals promoted oxidative damage to E. coli.

[0234]

[0235] [Experimental Example 9] Evaluation of antibacterial activity against S. pneumoniae

[0236] The inventors hypothesized that Example 6b would penetrate Streptococcus pneumoniae, generate hydroxyl radicals using endogenously produced H2O2, and inhibit bacterial growth. To confirm this, the antibacterial activity of Example 6b against Streptococcus pneumoniae, a Gram-positive facultative anaerobic catalase-deficient pathogen, was investigated.

[0237] For time-kill measurements based on colony forming unit (CFU) assay for S. pneumoniae KCMM42691 in the presence of Example 6b (1×MBC, 12.5 μM), CFU were measured after 4, 8, and 16 h of incubation (Fig. 5).

[0238] Monitoring of bacterial growth over time using CFU assay under anaerobic conditions revealed growth inhibition activity in the following order: Example 6b-Cu ≒ Example 6b-Ni and Example 6b.

[0239] These results indicate that Example 6b-Cu can be effective in converting H2O2 into hydroxyl radicals and inhibiting the growth of catalase-negative Streptococcus pneumoniae, a pathogenic strain that is a major cause of pneumonia and meningitis.

[0240]

[0241] [Experimental Example 10] Anti-inflammatory activity evaluation

[0242] Cytokines produced by host immune cells in response to LPS stimulation can ultimately cause sepsis-related diseases. Therefore, to determine how Example 6b affects the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β), cytokine levels were measured in LPS-stimulated RAW264.7 macrophages. In addition, nitrite production levels were measured to evaluate the inhibitory effect of Example 6b on the secretion of nitric oxide (NO), which plays a key role in the inflammatory process.

[0243] 1×10 RAW264.7 cells 5 Cells were plated at a density of 100 μg / mL and stimulated with 20 ng / mL LPS from E. coli O111:B4 (Sigma-Aldrich, St. Louis, MO, USA) for 16 h under conditions with or without Example 6b. Changes in color formation were detected at 540 nm. Nitrite content concentrations were assessed using a sodium nitrite standard curve. The release of inflammatory cytokines TNF-α, IL-6, and IL-1β from the culture medium was measured using an enzyme-linked immunosorbent assay kit (ELISA; R&D Systems, Minneapolis, MN, USA).

[0244] LPS-treated RAW264.7 cells showed marked increases in TNF-α, IL-6, IL-1β, and NO levels (Fig. 6a-d). Example 6b exhibited anti-inflammatory activity, reducing the production of TNF-α, IL-6, and IL-1β, as well as NO levels. At a concentration of 5 μM, Example 6b inhibited the production of TNF-α, IL-6, IL-1β, and nitrite by 72.4%, 93.0%, 42.0%, and 82.7%, respectively.

[0245] Toll-like receptor 4 (TLR4) plays a crucial role in recognizing LPS and initiating immune responses in host immune cells such as macrophages, monocytes, and dendritic cells during bacterial infection. To evaluate the immunomodulatory activity through interference with TLR4 signaling in Example 6b, HEK-Blue TM The regulatory effect of secreted alkaline phosphatase (SEAP) was investigated using hTLR4 cells.

[0246] HEK-Blue™ hTLR4 cells were seeded at 3×10 in 96-well plates. 4 Cells were seeded at a density of 10 cells / well and placed in HEK-Blue detection medium (InvivoGen, San Diego, CA, USA) for Example 6b (InvivoGen, San Diego, CA, USA). After 1 h, cells were treated with LPS (20 ng / mL), and SEAP activity was measured using absorbance at 630 nm using an ELISA reader after 16 h of incubation.

[0247] As shown in Fig. 6e, Example 6b reduced TLR4-mediated SEAP activity by 78% at 25 μM, suggesting that Example 6b can block TLR4-mediated inflammatory signaling.

[0248]

[0249] [Experimental Example 11] In vitro and in vivo toxicity evaluation

[0250] The in vitro cytotoxicity of Example 6b was evaluated using three eukaryotic cell lines, namely, murine macrophage RAW 264.7 cells, mouse fibroblast L929 cells, and human embryonic kidney HEK-293 cells.

[0251] Murine macrophage RAW 264.7 cells, mouse fibroblast L-929 cells, and human embryonic kidney HEK-293 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cytotoxicity of Example 6b against RAW 264.7 macrophages, L929 cells, and HEK-293 cells was measured using the WST-8 cell proliferation assay (Biomax Co., Ltd., Seoul, Korea) according to the kit instructions.

[0252] Cells (1 × 10) were seeded in a 96-well plate 5 ) were seeded and compound treatments (0–100 μM) were initiated at 80% confluency and continued for 24 h. After incubation, WST-8 reagent was added, and the change in absorbance compared to the reagent blank was measured at 450 nm. Values ​​were expressed as the percentage of cell viability.

[0253] As shown in Figures 7a to 7c, Example 6b showed a viability of 76% at 25 μM in murine macrophage RAW 264.7 cells, while no cytotoxicity was observed at 25 μM in L929 and HEK-293 cells. In contrast, melittin showed much more potent cytotoxicity in all three cell types, with viability rates of less than 21.5% at 6.3 μM. This suggests that the anti-inflammatory activity of melittin may be due to its high cytotoxicity.

[0254] Next, the in vivo toxicity of Example 6b was evaluated in a mouse model.

[0255] Ten 6-week-old female ICR mice (5 per group) were used for in vivo toxicity testing. The normal control group was administered 200 μL PBS four times over 12 hours, and the Example 6b administration group was administered 5 mg / kg of Example 6b intraperitoneally four times over 12 hours. The mice were euthanized 24 hours after the final injection, and blood and kidney samples were collected. Serum creatinine levels were measured using the DetectX® Serum Creatinine Kit (Arbor Assays, Ann Arbor, MI, USA). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) levels were analyzed using standard kits from Asan Pharmaceutical. As a result, administration of Example 6b did not significantly increase these organ toxicity indicators (Fig. 7d).

[0256] For histopathological analysis of the kidney, paraffin-blocked kidney tissue sections were stained with hematoxylin and eosin (H&E) and examined for pathological changes under a light microscope (Eclipse Ni; Nikon, Tokyo, Japan). As a result, mice administered Example 6b (5 mg / kg, administered four times over 12 hours) did not show microanatomical changes in the kidney tissue, such as lymphocytic infiltration, interstitial capillary dilatation, and venous dilatation, similar to those observed in the control mice (Fig. 7e).

[0257]

[0258] [Experimental Example 12] In vivo evaluation of anti-septic effect

[0259] The anti-septic effect of Example 6b was evaluated using a septic shock mouse model induced by E. coli K1.

[0260] First, we tested the survival rate of E. coli K1-infected mice treated with Example 6b. Forty 6-week-old female ICR mice were randomly divided into four groups. The first group was administered PBS four times at 4-hour intervals for 12 hours. The E. coli K1-infected group was administered E. coli K1 at a density of 3 × 10 7 were injected intraperitoneally (ip) at a concentration of 10 CFU / mouse. For the third group, 10 mice were intravenously injected with Example 6b (5 mg / kg, 4 times over 12 hours). In the fourth group, 10 mice were injected intravenously with E. coli K1 (3 Х 10 7 Example 6b was administered intraperitoneally 1 hour before infection (CFU / mouse), followed by three more injections every 4 hours. Mice were monitored hourly for up to 36 hours after E. coli K1 infection and then every 6 hours thereafter.

[0261] As shown in Fig. 8a, when uninfected mice were administered PBS (control) or Example 6b, all mice survived for more than 96 hours, confirming the absence of acute toxicity. On the other hand, mice in the E. coli K1-infected group showed a 100% mortality rate 34 hours after infection. However, when treated with Example 6b, the survival rate of mice infected with E. coli K1 increased to 60% after 96 hours. In addition, as shown in Fig. 8b, the total bacterial load in the lungs, liver, and kidneys of the mice was significantly reduced by Example 6b. This demonstrates that Example 6b can successfully prevent the development or proliferation of bacteria in the internal organs of mice infected with E. coli K1.

[0262] Next, the endotoxin neutralizing ability of Example 6b was investigated in the serum of a septic shock mouse model. Twenty ICR mice were randomly assigned to four groups (five per group): a standard control group, an E. coli K1 infection group, an Example 6b administration group, and an E. coli K1 infection followed by Example 6b administration group. The standard control group mice were administered only PBS. The Example 6b administration group mice were intraperitoneally injected with Example 6b (1 mg / kg). The E. coli K1 infection group mice were administered E. coli K1 (6 Х 10 6 Only 10 CFU / mouse were administered. Example 6b was administered 1 hour before injection of E. coli K1 into Example 6b-administered mice after E. coli K1 infection. Organs from each group of mice were collected under aseptic conditions and thoroughly homogenized for analysis. The homogenates were diluted 1000-fold, plated on LB agar, and incubated at 37°C for 24 hours before evaluation. The LAL (Limulus amebocyte lysate) neutralizing activity of Example 6b in serum of the E. coli K1 sepsis model was measured using the LAL assay according to the protocol provided in the LAL assay kit as described previously (ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit; GenScript, Piscataway, NJ, USA).

[0263] As shown in Fig. 8c, the LAL assay results showed that the endotoxin level was significantly reduced (44.3%) in the sepsis mouse model treated with Example 6b, confirming the in vivo LPS neutralizing ability of Example 6b.

[0264] Additionally, to evaluate the anti-inflammatory effect of Example 6b, serum levels of inflammatory cytokines TNF-α, IL-6, and IL-1β were measured using ELISA (R&D Systems, Minneapolis, MN, USA). Blood samples were collected from mice, centrifuged at 4°C and 3,000 rpm for 15 minutes to separate serum, and 50 μL per sample was used for analysis. Additionally, to investigate serum levels of liver and kidney marker enzymes, AST, ALT, and BUN were measured using standard kits from Asan Pharmaceutical.

[0265] As shown in Fig. 8d, mice infected with E. coli K1 had significantly higher levels of TNF-α, IL-6, and IL-1β than the control group. Treatment with Example 6b resulted in a 46.6%, 42.8%, and 48.3% decrease in serum levels of TNF-α, IL-6, and IL-1β, respectively, in the infected mice.

[0266] Furthermore, as shown in Figure 8e, the AST, ALT, and BUN levels in mice infected with E. coli K1 were more than twice as high as in the control group. In mice treated with Example 6b, these marker enzyme levels were significantly reduced, with AST levels dropping by 32.7%, ALT by 51.1%, and BUN by 40.3%. Importantly, compared to the control group, treatment with Example 6b did not increase the levels of these marker enzymes, indicating the low toxicity of Example 6b.

[0267]

[0268] [Experimental Example 13] Histopathological Evaluation of Lung Tissue

[0269] To examine the microanatomical characteristics of polymorphic neutrophil infiltration, 5-mm-thick paraffin-blocked lung sections were prepared from the mouse in Experimental Example 12. These sections were serially stained with hematoxylin and eosin (H&E) and observed under a light microscope.

[0270] As shown in Fig. 9, the histological pattern of lung tissue infected with E. coli K1 showed severe pulmonary edema, pulmonary congestion, alveolar hemorrhage, and infiltrated polymorphonuclear lymphocytes (PMNs). These microanatomical characteristics suggest that lung damage and increased PMNs, which worsen sepsis after E. coli K1 infection, are associated. Meanwhile, treatment with Example 6b successfully inhibited E. coli K1-induced degenerative changes in infected mice. Treatment with Example 6b significantly alleviated tissue damage, and histological analysis showed no significant difference between the control group and the Example 6b + E. coli K1 group. Therefore, it was confirmed that treatment with Example 6b could prevent lung damage caused by E. coli K1 infection. Taken together, these results confirmed that Example 6b has low toxicity and can function as a novel compound antibiotic for the treatment of Gram-negative sepsis.

Claims

1. A compound represented by the following chemical formula 1 or 2: [Chemical Formula 1] H-Gly-Gly-His-NLys-(a)2-NLys-Nspe-Nspe-NLys-b-Nspe-NLys-cd-NH2 [Chemical Formula 2] H-NLys-(a)2-NLys-Nspe-Nspe-NLys-b-Nspe-NLys-cd-Ddap-Gly-His-NH2 In the formula, a to d are each independently Nspe, Npm or NTrp.

2. In claim 1, the compound has any one chemical formula selected from the group consisting of the following chemical formulas 3 to 13: [Chemical Formula 3] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2 [Chemical Formula 4] H-Gly-Gly-His-NLys-(Npm)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-NH2 [Chemical Formula 5] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NTrp-NTrp-NH2 [Chemical Formula 6] H-Gly-Gly-His-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH2 [Chemical Formula 7] H-Gly-Gly-His-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-NLys-Nspe-NLys-NTrp-NTrp-NH2 [Chemical Formula 8] H-Gly-Gly-His-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NLys-Nspe-NH2 [Chemical Formula 9] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Ddap-Gly-His-NH2 [Chemical Formula 10] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NTrp-NTrp-Ddap-Gly-His-NH2 [Chemical Formula 11] H-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-Ddap-Gly-His-NH2 [Chemical Formula 12] H-NLys-(Nspe)2-NLys-Nspe-Nspe-NLys-NLys-Nspe-NLys-NTrp-NTrp-Ddap-Gly-His-NH2 [Chemical Formula 13] H-NLys-(NTrp)2-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-NLys-Nspe-Ddap-Gly-His-NH 2.

3. The compound of claim 1; and An organometallic compound comprising a metal ion bound to the N-terminus or C-terminus of the above compound.

4. An organometallic compound according to claim 3, wherein the metal ion is Cu(II) or Ni(II).

5. An antibacterial composition comprising the compound of claim 1.

6. An antibacterial composition according to claim 5, wherein the composition exhibits antibacterial activity against gram-positive bacteria or gram-negative bacteria.

7. An antibacterial composition according to claim 6, wherein the Gram-positive bacteria are Staphylococcus sp., Bacillus sp., Streptococcus sp., or Enterococcus sp.

8. In claim 6, the Gram-positive bacteria are Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), quinolone-resistant Staphylococcus aureus (QRSA), vancomycin resistant enterococcus (VRE), vancomycin intermediate-resistant Staphylococcus aureus (VISA), Bacillus subtilis, Bacillus cereus, Streptococcus pneumoniae, Enterococcus faecalis, or Staphylococcus An antibacterial composition comprising Staphylococcus epidermidis.

9. An antibacterial composition according to claim 6, wherein the Gram-negative bacteria are Salmonella sp., Acinetobacter sp., Escherichia sp., Pseudomonas sp., or Klebsiella sp.

10. An antibacterial composition according to claim 6, wherein the Gram-negative bacteria are Salmonella typhimurium, Acinetobacter calcoaceticus, E. coli, Pseudomonas aeruginosa, or Klebsiella aerogenes.

11. A pharmaceutical composition for preventing or treating bacterial infection, comprising the compound of claim 1.

12. A pharmaceutical composition for preventing or treating a bacterial infection according to claim 11, wherein the bacterial infection is any one selected from the group consisting of cholera, dysentery, pertussis, typhoid, diphtheria, plague, scarlet fever, sepsis, pyogenic cutaneous infection, tuberculosis, tuberculous meningitis, enteritis, and food poisoning.

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