Novel cyclic lipopeptide compound and use thereof

Abstract

The present disclosure relates to a novel cyclic lipopeptide compound and a use thereof. More specifically, the present invention relates to a compound represented by chemical formula 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof, and to an antibacterial or biofilm formation inhibiting composition comprising the compound, the acceptable salt thereof, the stereoisomer thereof, the hydrate thereof, or the solvate thereof as an active ingredient.

Description

Novel cyclic lipopeptide compounds and uses thereof

[0001] [Cross-reference to related applications]

[0002] This application claims priority to Republic of Korea Patent Application No. 10-2024-0065901, filed May 21, 2024, and Republic of Korea Patent Application No. 10-2024-0172350, filed November 27, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a novel cyclic lipopeptide compound and its use.

[0004]

[0005] Staphylococcus aureus, a Gram-positive bacterium, forms biofilms, which are sticky microbial membranes that adhere to surfaces using exopolysaccharides and proteins. These biofilms are commonly found in nature (as slime on rocks or water). Within these biofilms, bacteria communicate with each other through quorum sensing, controlling their populations and defending against external antimicrobials and antibiotics. This makes biofilm-formed microbial communities impermeable to antibodies and various antibiotics, increasing their resistance to antibiotics by up to 1,000-fold, including the neutralization of the host immune system. This resistance is known to contribute to increased bacterial resistance and chronic infections.

[0006] Staphylococcus epidermidis, a staphylococcus epidermidis, is an opportunistic pathogen that, when infected in immunocompromised patients, can cause a variety of pathogenicities, including food poisoning and infectious sepsis, along with its compatriot Staphylococcus aureus. The biggest problem with Staphylococcus epidermidis is its ability to form strong, thick biofilms, particularly on catheters, intravenous tubes, and implants. This allows the biofilm formed by Staphylococcus epidermidis to support the growth of various pathogens, including Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa, and further increases resistance to antibiotics.

[0007] As mentioned above, failure to effectively inhibit microbial biofilms can have serious consequences in the medical and industrial sectors. Therefore, the development of new substances capable of inhibiting biofilm formation as well as exhibiting antimicrobial activity is urgently needed.

[0008]

[0009] In one aspect, the present disclosure provides a compound represented by the following chemical formula 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof:

[0010] [Chemical Formula 1]

[0011]

[0012] (In the above chemical formula 1, R1 is CH 3,(CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2 or CH2CH(CH3)2).

[0013] In another aspect, the present disclosure provides an antibacterial composition comprising the compound, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0014] In another aspect, the present disclosure provides a composition for inhibiting biofilms comprising the compound, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0015]

[0016] In one aspect, the present disclosure provides a compound represented by the following formula 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof:

[0017] [Chemical Formula 1]

[0018]

[0019] (In the above chemical formula 1, R1 is CH 3, (CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2 or CH2CH(CH3)2).

[0020] In an exemplary embodiment, the compound may have a stereostructure represented by the following chemical formula 2:

[0021] [Chemical Formula 2]

[0022]

[0023] (In the above chemical formula 2, R1 is CH3, (CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2 or CH2CH(CH3)2).

[0024] In an exemplary embodiment, the chemical formula 2 may be any one of the following compounds:

[0025] .

[0026] In an exemplary embodiment, the compound may be derived from a strain of Serratia marcescens.

[0027] In an exemplary embodiment, the strain may be Serratia marcescensAULBAC6AULBAC6.

[0028] In another aspect, the present disclosure provides an antibacterial composition comprising the compound, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0029] In an exemplary embodiment, the target of the antibacterial may be an antibiotic-resistant strain.

[0030] In an exemplary embodiment, the composition may have antibacterial activity against Gram-positive bacteria.

[0031] In an exemplary embodiment, the composition may have antibacterial activity against strains of the genus Staphylococcus or the genus Enterococcus.

[0032] In an exemplary embodiment, the strain may be one or more selected from the group consisting of Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis.

[0033] In another aspect, the present disclosure provides a composition for inhibiting biofilms comprising the compound, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient.

[0034] In an exemplary embodiment, the composition may have inhibitory activity against biofilms derived from Gram-positive bacteria.

[0035] In an exemplary embodiment, the composition may have inhibitory activity against biofilms derived from Staphylococcus spp. strains.

[0036] In an exemplary embodiment, the strain may be Staphylococcus epidermidis.

[0037]

[0038] The compound of the present invention has an excellent antibacterial effect against Gram-positive bacteria, and is particularly excellent in its antibacterial effect against antibiotic-resistant pathogens.

[0039] In addition, the compound of the present invention has an excellent effect of inhibiting biofilm formation derived from Gram-positive bacteria, and is particularly excellent in inhibiting biofilm formation derived from Staphylococcus epidermidis.

[0040] The compound of the present invention is environmentally friendly because it can be isolated from symbiotic bacteria of wild garlic, and can be utilized in various biological and chemical application studies.

[0041]

[0042] Figure 1a shows the structures of marsenmycins A to F, and Figure 1b shows the chemical structures of marsenmycins G to D2.

[0043] Figure 2 shows the amino acids in the structure of marsenmycin A.

[0044] Figure 3 is a photograph of a petri dish in which Serratia marcescensAULBAC6, in which an identification experiment was performed, was cultured.

[0045] Figure 4 shows the compositions of marsenmycin A to F. 1 This is H-NMR spectrum data.

[0046] Figure 5 shows the compositions of marsenmycin G to D2. 1 This is H-NMR spectrum data.

[0047] Figure 6 shows the compositions of marsenmycin A to F. 13 This is C-NMR spectrum data.

[0048] Figure 7 shows the compositions of marsenmycin G to D2. 13 This is C-NMR spectrum data.

[0049] Figure 8 shows the results of a comparative bactericidal experiment on MRSA between marsenmycin H and vancomycin.

[0050] Figure 9 shows the results of a biofilm inhibition activity experiment of marsenmycin.

[0051] Figure 10 shows the results of a biofilm inhibition activity experiment of marsenmycin using a scanning electron microscope (SEM).

[0052] Figure 11 shows the results of an antibacterial activity evaluation of marsenmycin against MRSA persister.

[0053] Figures 12A to 12G show the results of evaluating the degree of increase in ROS level inside MRSA after treatment with marsenmycin.

[0054] Figures 13a to 13g show the results of evaluating the membrane permeability of marsenmycin against MRSA.

[0055] Figure 14 shows the results of an evaluation of the degree of infection control of marsenmycin in a MRSA infection C. elegans model.

[0056]

[0057] Hereinafter, exemplary implementation examples of the present invention will be described in more detail.

[0058]

[0059] In one aspect, the present disclosure provides a compound represented by the following formula 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof:

[0060] [Chemical Formula 1]

[0061]

[0062] (In the above chemical formula 1, R1 is CH 3, (CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2 or CH2CH(CH3)2).

[0063] The term “salt” means a salt according to one aspect of the present disclosure having the desired activity of the parent compound, and may be a salt that does not cause serious irritation to an organism to which the compound is applied and does not impair the biological activity and physical properties of the compound.

[0064] The above salt refers to an addition salt of an inorganic acid salt, an organic acid salt, or a metal salt of a compound. The inorganic acid salt may be a hydrochloride salt, a bromate salt, a phosphate salt, a sulfate salt, or a disulfate salt. The organic acid salt may be a formate salt, an acetate salt, an acetate salt, a propionate salt, a lactate salt, an oxalate salt, a tartrate salt, a malate salt, a maleate salt, a citrate salt, a fumarate salt, a besylate salt, a camsylate salt, an edisyl salt, a trichloroacetate salt, a trifluoroacetate salt, a benzoate salt, a gluconate salt, a methanesulfonate salt, a glycolate salt, a succinate salt, a 4-toluenesulfonate salt, a galacturonate salt, an embonate salt, a glutamate salt, a methanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, or an aspartate salt. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.

[0065] The term “stereoisomer” refers to compounds which have the same chemical constitution but differ in terms of the arrangement of their atoms or groups in space, a concept which includes optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers, or mixtures thereof), as well as conformation isomers (i.e., isomers which differ only in the angles of one or more chemical bonds), position isomers (particularly, tautomers) or geometric isomers (e.g., cis-trans isomers).

[0066] The term “essentially pure”, when used in reference to, for example, enantiomers or diastereoisomers, means that a specific compound, for example, the enantiomer or diastereoisomer, is present in an amount of at least about 90%, preferably at least about 95%, more preferably at least about 97% or at least about 98%, still more preferably at least about 99%, and still more preferably at least about 99.5% (w / w).

[0067] The term “hydrate” refers to a compound that has water bound to it, and is a broad concept that includes inclusion compounds in which there is no chemical bond between the water and the compound.

[0068] The term “solvate” refers to a higher-order compound formed between molecules or ions of a solute and molecules or ions of a solvent.

[0069] In the present invention, through one embodiment, a peptide having the sequence of (D-leucine or D-valine)-(L-serine)-(L-serine or L-threonine)-(D-phenylalanine or D-tryptophan)-(L-valine, L-isoleucine or L-leucine) and 3-(R)-hydroxydecanoic acid, 3-(R)-

[0070] Thirteen novel cyclic lipopeptides having one hydrocarbon selected from the group consisting of hydroxyoctanoic acid and 3-(R)-hydroxydodecanoic acid were discovered, the physicochemical properties of these compounds were analyzed, and their excellent antibacterial and biofilm inhibition effects were confirmed.

[0071] The compound may be a lipopeptide compound, preferably a cyclic lipopeptide compound, but is not limited thereto.

[0072] The compound may include a saturated hydrocarbon, preferably, but not limited to, hydroxydecanoic acid, hydroxyoctanoic acid or hydroxydodecanoic acid.

[0073] The compound may comprise five amino acids. The amino acids may be one or more selected from the group consisting of, but are not limited to, Leucine, Valine, Serine, Threonine, Phenylalanine, Tryptophan, and Isoleucine.

[0074] The compound has an amino acid sequence of leucine-serine-serine-phenylalanine-valine; an amino acid sequence of leucine-serine-threonine-tryptophan-valine; an amino acid sequence of leucine-serine-phenylalanine-isoleucine; an amino acid sequence of leucine-serine-threonine-tryptophan-isoleucine; an amino acid sequence of valine-serine-threonine-phenylalanine-valine; an amino acid sequence of valine-serine-threonine-phenylalanine-isoleucine; an amino acid sequence of leucine-serine-threonine-phenylalanine-leucine; an amino acid sequence of leucine-serine-threonine-phenylalanine-isoleucine; an amino acid sequence of leucine-serine-threonine-phenylalanine-leucine; an amino acid sequence of leucine-serine-threonine-isoleucine; an amino acid sequence of valine-serine-threonine-tryptophan-valine; an amino acid sequence of leucine-serine-threonine-phenylalanine-valine; It may include, but is not limited to, an amino acid sequence of leucine-serine-threonine-tryptophan-valine; an amino acid sequence of leucine-serine-serine-phenylalanine-isoleucine; or an amino acid sequence of leucine-serine-threonine-tryptophan-isoleucine.

[0075] In the above chemical formula 1, R1 may be (CH2)2CH3, R2 may be CH2CH(CH3)2, R3 may be H, R4 may be CH2Ph, and R5 may be CH(CH3)2; R1 may be (CH2)2CH3, R2 may be CH2CH(CH3)2, R3 may be CH3, R4 may be CH2Indole, and R5 may be CH(CH3)2; R1 may be (CH2)2CH3, R2 may be CH2CH(CH3)2, R3 may be H, R4 may be CH2Ph, and R5 may be CHCH2(CH3)2; R1 may be (CH2)2CH3, R2 may be CH2CH(CH3)2, R3 may be CH3, R4 may be CH2Indole, and R5 may be CHCH2(CH3)2; R1 can be (CH2)2CH3, R2 can be CH(CH3)2, R3 can be CH3, R4 can be CH2Ph, and R5 can be CH(CH3)2; R1 can be (CH2)2CH3, R2 can be CH(CH3)2, R3 can be CH3, R4 can be CH2Ph, and R5 can be CHCH2(CH3)2; R1 can be (CH2)2CH3, R2 can be CH2CH(CH3)2, R3 can be CH3, R4 can be CH2Ph, and R5 can be CH2CH(CH3)2; R1 can be (CH2)4CH3, R2 can be CH2CH(CH3)2, R3 can be CH3, R4 can be CH2Ph, and R5 can be CHCH2(CH3)2; R1 can be (CH2)2CH3, R2 can be CH(CH3)2; R3 can be CH3, R4 can be CH2Indole, and R5 can be CH(CH3)2; R1 can be CH3, R2 can be CH2CH(CH3)2, R3 can be CH3, R4 can be CH2Ph, and R5 can be CH(CH3)2; R1 can be CH3, R2 can be CH2CH(CH3)2, R3 can be CH3, R4 can be CH2Indole, and R5 can be CH(CH3)2; R1 can be CH3, R2 can be CH2CH(CH3)2, R3 can be H, R4 can be CH2Ph, and R5 can be CHCH2(CH3)2;R1 may be CH3, R2 may be CH2CH(CH3)2, R3 may be CH3, R4 may be CH2Indole, and R5 may be CHCH2(CH3)2, but is not limited thereto.;

[0076] In an exemplary embodiment, the compound may have a stereostructure represented by the following chemical formula 2:

[0077] [Chemical Formula 2]

[0078]

[0079] (In the above chemical formula 2, R1 is CH 3, (CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2 or CH2CH(CH3)2).

[0080] The compound may include, but is not limited to, 3-(R)-hydroxydecanoic acid, 3-(R)-hydroxyoctanoic acid or 3-(R)-hydroxydodecanoic acid.

[0081] The above compound has an amino acid sequence of (D-leucine)-(L-serine)-(L-serine)-(D-phenylalanine)-(L-valine); an amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-tryptophan)-(L-valine); an amino acid sequence of (D-leucine)-(L-serine)-(L-serine)-(D-phenylalanine)-(L-isoleucine); an amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-tryptophan)-(L-isoleucine); an amino acid sequence of D-valine-(L-serine)-(L-threonine)-(D-phenylalanine)-(L-valine); The amino acid sequence of D-valine-(L-serine)-(L-threonine)-(D-phenylalanine)-(L-isoleucine); The amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-phenylalanine)-(L-leucine); The amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-phenylalanine)-(L-isoleucine); The amino acid sequence of (D-valine)-(L-serine)-(L-threonine)-(D-tryptophan)-(L-valine); The amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-phenylalanine)-(L-valine); It may include, but is not limited to, an amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-tryptophan)-(L-valine); an amino acid sequence of (D-leucine)-(L-serine)-(L-serine)-(D-phenylalanine)-(L-isoleucine); or an amino acid sequence of (D-leucine)-(L-serine)-(L-threonine)-(D-tryptophan)-(L-isoleucine).

[0082] In an exemplary embodiment, the chemical formula 2 may be any one of the following compounds:

[0083] .

[0084] Each of the above compound structures is named as shown in Figure 1.

[0085] The present inventors have confirmed excellent antibacterial activity of marsenmycins A, B, C, D, E, F, G, and H and excellent biofilm inhibitory activity of marsenmycins A, B, C, D, F, G, H, and I through embodiments of the present invention. In particular, marsenmycin H has been confirmed to have excellent antibacterial activity and biofilm inhibitory activity against Staphylococcus epidermidis, and excellent antibacterial activity against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus (MRSA), Enterococcus faecium, and Enterococcus faecalis. In particular, marsenmycin H has been confirmed to have a faster bactericidal effect than the known antibiotic vancomycin used for MRSA, and therefore, the compounds of the present invention and derivatives thereof can be utilized as powerful antibacterial agents or biofilm inhibitors.

[0086] In an exemplary embodiment, the compound may be derived from a strain of Serratia marcescens.

[0087] In an exemplary embodiment, the strain may be Serratia marcescens AULBAC6. For example, the Serratia marcescens AULBAC6 strain may be isolated from wild garlic leaves, preferably from Ulleungdo wild garlic leaves, but is not limited thereto.

[0088]

[0089] In another aspect, the present disclosure provides an antibacterial composition comprising the compound, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient. The compound, salt, stereoisomer, hydrate, and solvate are as described above.

[0090] The term “active ingredient” means an ingredient that exhibits the intended activity alone or can exhibit the intended activity together with a carrier or the like that is not active in itself.

[0091] The above antibacterial means killing or inhibiting the growth of microorganisms including pathogens.

[0092] In an exemplary embodiment, the target of the antibacterial may be an antibiotic-resistant strain.

[0093] In an exemplary embodiment, the composition may have antibacterial activity against Gram-positive bacteria. The Gram-positive bacteria refers to bacteria that stain blue or purple in a Gram stain.

[0094] In an exemplary embodiment, the composition may have antibacterial activity against strains of the genus Staphylococcus or the genus Enterococcus.

[0095] In an exemplary embodiment, the strain may be one or more selected from the group consisting of Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis. For example, the Staphylococcus aureus may be Staphylococcus aureus MW2, the Enterococcus faecium may be Enterococcus faecium E007, and the Enterococcus faecalis may be Enterococcus faecalis MMH594, but is not limited thereto.

[0096] In an exemplary embodiment, the Staphylococcus aureus may be methicillin-resistant Staphylococcus aureus (MRSA). In addition, the MRSA may be growing MRSA, growth-arrested MRSA, or non-growth / metabolically inactive MRSA (persister MRSA). The inventors have confirmed from examples that the composition has excellent efficacy in reducing the viability of persister MRSA cells, increasing the level of ROS in persister MRSA cells, and increasing lipid membrane permeability in persister MRSA cells. In addition, the inventors have confirmed from examples that the composition has excellent infection control ability against C. elegans (C. elegans) infected with persister MRSA.

[0097]

[0098] In another aspect, the present disclosure provides a composition for inhibiting biofilms, comprising the compound, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient. The compound, salt, stereoisomer, hydrate, solvate, and active ingredient are as described above.

[0099] In an exemplary embodiment, the composition may have inhibitory activity against biofilms derived from Gram-positive bacteria. The Gram-positive bacteria are as described above. The biofilm refers to a biofilm or biofilm, which is an aggregate of microorganisms, in which microbial cells adhere to each other on an adhesion surface.

[0100] In an exemplary embodiment, the composition may have inhibitory activity against biofilms derived from strains of the genus Staphylococcus. Biofilms derived from strains of the genus Staphylococcus can be pathogenic for food poisoning and infectious sepsis, and can cause bacterial contamination of catheters, intravenous tubes, and implants.

[0101] In an exemplary embodiment, the strain may be Staphylococcus epidermidis. The biofilm derived from Staphylococcus epidermidis may promote the growth of pathogenic microorganisms such as Staphylococcus aureus and Pseudomonas aeruginosa, and may increase antibiotic resistance, but is not limited thereto.

[0102] In an exemplary embodiment, the Staphylococcus aureus may be methicillin-resistant Staphylococcus aureus (MRSA). Additionally, the MRSA may be growing MRSA, growth-arrested MRSA, or persister MRSA.

[0103]

[0104] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0105]

[0106] Example

[0107]

[0108] 1. Identification of bacterial strains producing marsenmycin AI

[0109] The strain producing marsenemycin AI, Serratia marcescens AULBAC6, was isolated from the leaves of wild garlic from Ulleungdo Island and was isolated on yeast malt agar solid medium (yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g, dextrose 10.0 g, agar powder 14.0 g / 1 L sterilized water). Based on the results of 16S rDNA sequence analysis and comparison with the sequence of a standard strain, the strain was identified as Serratia marcescens (Fig. 3).

[0110]

[0111] 2. Cultivation and extraction of Serratia marcescensAULBAC6 strain

[0112] For the production of marcescenmycin AI, the strain was cultured. The Serratia marcescens AULBAC6 strain was inoculated into 10 mL of YM medium and cultured at 30°C for 24 hours. The culture was then inoculated at 1% (w / v) into 20 1000 mL volumes of YM liquid medium and cultured at 200 rpm and 30°C for 48 hours.

[0113] Afterwards, 1000 mL of ethyl acetate was used to extract each 1000 mL of culture medium in which the strain had sufficiently grown, and only the supernatant was collected. This process was repeated three times. Afterwards, foreign substances and bacterial cells were removed by passing the mixture through several layers of clean gauze, and then reduced pressure drying was performed using a rotary vacuum concentrator.

[0114]

[0115] 3. Isolation and purification of marsenmycin AI from the extract of Serratia marcescensAULBAC6 strain.

[0116] To purify marsenmycin AI from the AULBAC6 strain extract, normal phase fractionation was performed primarily using medium-performance liquid chromatography (MPLC). A silica resin-packed column (Biotage Sfαr, 100 g, flow rate 20 mL / min, detection 210 nm, 280 nm) was stabilized with 100% hexane, and 5.42 g of the extract was adsorbed onto the silica resin. Nine fractions were obtained using sequential mobile phases of Hexane:Dichloromethylene (0, 50, and 100%) and Dichloromethylene:MeOH (1, 2, 4, 7, 10, 15, 50, and 100%). Afterwards, AULBAC6-4 (3.88 g) and AULBAC6-5 (201.0 mg) were obtained from 10% MeOH of DCM:MeOH and dried under reduced pressure.

[0117] Afterwards, AULBAC6-4 was further fractionated using MPLC and AULBAC6-4 was stabilized with 100% H2O. 18 Resin-filled column (Biotage Sfαr C 18, After adsorption on 60 g, flow rate 12 mL / min, detection 210 nm, 280 nm), nine fractions were obtained using H2O:MeCN (10, 20, 30, 40, 50, 70, 80, 90, and 100%) as the mobile phase sequentially. Afterwards, AULBAC6-4-G2 (212.1 mg) of lipopeptide fraction was obtained in 80% MeCN. AULBAC6-5 was subjected to reversed-phase fractionation using HPLC (high-performance liquid chromatography). AULBAC6-5 was purified using a reversed-phase column (Phenomenex, Luna C 18, 5 μm, 250 × 21.2 mm, flow rate 5 mL / min, detection 210 nm) using 35% MeCN (H2O:MeCN conditions) as an isocratic mobile phase, and a total of 6 isolates were obtained, and the lipopeptide isolate AULBAC6-5-F (56.6 mg) was obtained.

[0118] AULBAC6-4-G2 was purified using HPLC using a reverse-phase column (Phenomenex, Luna C 18 , 5 μm, 250 × 21.2 mm, flow rate 5 mL / min, detection 210 nm) was used for reversed phase fractionation using 70% MeCN (H2O:MeCN conditions) as an isocratic mobile phase. A total of 10 isolates were obtained from AULBAC6-4-G2, and new compounds were obtained: AULBAC6-4-G2-2 (marsenmycin A2, 2.8 mg), AULBAC6-4-G2-4 (marsenmycin E, 1.5 mg), AULBAC6-4-G2-5 (41.9 mg), AULBAC6-4-G2-6 (marsenmycin G, 1.7 mg), and AULBAC6-4-G2-10 (marsenmycin H, 2.7 mg). Among these, in order to isolate and purify AULBAC6-4-G2-5, two isolates were obtained using HPLC on a reversed-phase column (YMC, J'sphere ODS-H80, 4 μm, 250 × 10.0 mm, flow rate 2 mL / min, detection 210 nm) with 80% MeOH (H2O:MeOH conditions) as an isocratic mobile phase, and AULBAC6-4-G2-5B (marsenmycin F, 3.3 mg) was obtained as a new compound.

[0119] AULBAC6-5-F was purified by reverse-phase fractionation using HPLC on a reverse-phase column (YMC, J'sphere ODS-H80 4 μm, 250 × 10.0 mm, flow rate 2 mL / min, detection 210 nm) using 62% MeCN (H2O:MeCN condition) as an isocratic mobile phase. A total of 13 fractions were obtained from AULBAC6-5-F, and new compounds were obtained: AULBAC6-5-F3 (marsenmycin B2, 0.5 mg), AULBAC6-5-F4 (marsenmycin C2, 3.2 mg), AULBAC6-5-F5 (5.8 mg), AULBAC6-5-F7 (marsenmycin I, 0.3 mg), AULBAC6-5-F9 (marsenmycin A, 3.2 mg), AULBAC6-5-F10 (marsenmycin B, 10.4 mg), AULBAC6-5-F12 (marsenmycin C, 10.6 mg), and AULBAC6-5-F13 (5.1 mg). Among these, in order to isolate and purify AULBAC6-5-F5 and AULBAC6-5-F13 purely, HPLC was used on a reversed-phase column (YMC, J'sphere ODS-H80, 4 μm, 250 × 10.0 mm, flow rate 2 mL / min, detection 210 nm) using 77% and 80% MeOH (H2O:MeOH conditions) as isocratic mobile phases, respectively, to obtain two isolates, and new compounds AULBAC6-5-F5-A (marsenmycin D2, 1.0 mg) and AULBAC6-5-F13-A (marsenmycin D, 2.7 mg) were obtained. The structure of each compound is as shown in Fig. 1.

[0120]

[0121] [Physicochemical properties of Marcenmicin A]

[0122] (1) Molecular formula: C 36 H 57 N5O9

[0123] (2) Molecular weight: 703

[0124] (3) Color: Transparent or white

[0125] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 4>

[0126] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 6>

[0127]

[0128] [Physicochemical properties of Marcenmicin B]

[0129] (1) Molecular formula: C 39 H 60 N6O9

[0130] (2) Molecular weight: 756

[0131] (3) Color: Transparent or white

[0132] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 4>

[0133] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 6>

[0134]

[0135] [Physicochemical properties of Marcenmicin C]

[0136] (1) Molecular formula: C 37 H 59 N5O9

[0137] (2) Molecular weight: 717

[0138] (3) Color: Transparent or white

[0139] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 4>

[0140] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 6>

[0141]

[0142] [Physicochemical properties of Marcenmicin D]

[0143] (1) Molecular formula: C 40 H 62 N6O9

[0144] (2) Molecular weight: 770

[0145] (3) Color: Transparent or white

[0146] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 4>

[0147] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 6>

[0148]

[0149] [Physicochemical properties of Marcenmicin E]

[0150] (1) Molecular formula: C 36 H 57 N5O9

[0151] (2) Molecular weight: 703

[0152] (3) Color: Transparent or white

[0153] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 4>

[0154] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 6>

[0155]

[0156] [Physicochemical properties of Marcenmicin F]

[0157] (1) Molecular formula: C 37 H 59 N5O9

[0158] (2) Molecular weight: 717

[0159] (3) Color: Transparent or white

[0160] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 4>

[0161] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 6>

[0162]

[0163] [Physicochemical properties of Marcenmicin G]

[0164] (1) Molecular formula: C 38 H 61 N5O9

[0165] (2) Molecular weight: 731

[0166] (3) Color: Transparent or white

[0167] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0168] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0169]

[0170]

[0171] [Physicochemical properties of Marcenmicin H]

[0172] (1) Molecular formula: C 40 H 65 N5O9

[0173] (2) Molecular weight: 759

[0174] (3) Color: Transparent or white

[0175] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0176] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0177]

[0178] [Physicochemical properties of Marcenmicin I]

[0179] (1) Molecular formula: C 38 H 58 N6O9

[0180] (2) Molecular weight: 742

[0181] (3) Color: Transparent or white

[0182] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0183] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0184]

[0185] [Physicochemical properties of Marcenmicin A2]

[0186] (1) Molecular formula: C 35 H 55 N5O9

[0187] (2) Molecular weight: 689

[0188] (3) Color: Transparent or white

[0189] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0190] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0191]

[0192] [Physicochemical properties of Marcenmicin B2]

[0193] (1) Molecular formula: C 37 H 56 N6O9

[0194] (2) Molecular weight: 728

[0195] (3) Color: Transparent or white

[0196] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0197] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0198]

[0199] [Physicochemical properties of Marcenmicin C2]

[0200] (1) Molecular formula: C 35 H 55 N5O9

[0201] (2) Molecular weight: 689

[0202] (3) Color: Transparent or white

[0203] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0204] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0205]

[0206] [Physicochemical properties of Marcenmicin D2]

[0207] (1) Molecular formula: C 38 H 58 N6O9

[0208] (2) Molecular weight: 742

[0209] (3) Color: Transparent or white

[0210] (4) 1H-NMR (Pyridine-d5, 800 MHz): See <Figure 5>

[0211] (5) 13C-NMR (Pyridine-d5, 200 MHz): See <Figure 7>

[0212]

[0213] The structures of the above-described marsenmycins A to D2 and the classification of each residue are as shown in Table 1 below.

[0214]

[0215]

[0216]

[0217] 4. Evaluation of antibacterial activity of marsenmycin

[0218] To evaluate the antibacterial activity of marcenmicin A and its derivatives (Marcenmicin AI) against Staphylococcus epidermidis (KACC 13234), 100 μL of marcenmicin A, B, C, D, F, G, H, and I were added to each well of a 96-well plate at different concentrations. Then, 100 μL of TSB medium inoculated with 2 × 106 cfu / mL of Staphylococcus epidermidis was added to each well of the 96-well plate to make a total of 200 μL, and the plate was cultured at 30°C for 24 hours. Afterwards, the Optical Density value was confirmed using a 600 nm wavelength of a UV-VIS spectrometer with a wavelength of 595 nm. Ampicillin was used as a positive control for comparative experiments.

[0219] As a result of the antibacterial activity test, A, B, C, D, F, G, H, and I showed activity at MIC values ​​at concentrations of 16, 8, 8, 4, 32, 4, 2, and 32 μg / mL, and in particular, H showed antibacterial activity 32 times stronger than that of the positive control Ampicillin (Table 2).

[0220]

[0221]

[0222]

[0223] The minimum inhibitory concentration (MIC) of marcenmicin A and its derivatives (Marcenmicin AI) against antibiotic-resistant hospital, methicillin-resistant S. aureus (MRSA) MW2, E. faecium E007, and E. faecalis MMH594 was measured using the microbroth dilution method according to Clinical & Laboratory Standards Institute (CLSI) guidelines. Marcenmicin H exhibited MICs of 8–16 μg / mL against the three antibiotic-resistant strains (Table 3).

[0224]

[0225]

[0226]

[0227] The bactericidal activity of marsenmycin H against MRSA was compared with that of vancomycin, a conventional antibiotic. The bactericidal activity was 5×10 6 cfu / mL growing MRSA was cultured with 10 μg / mL marsenmycin H and 10 μg / mL vancomycin for 4 hours at 37°C, and the viable bacterial cell concentration was measured every hour to evaluate the assay. Through this, it was confirmed that although the MIC of marsenmycin H was higher than that of vancomycin, it exhibited superior bactericidal activity than vancomycin at the same concentration (Fig. 8).

[0228]

[0229] 5. Evaluation of biofilm inhibition activity of marsenmycin

[0230] To evaluate the antibacterial activity of marcenmicin A and its derivatives (Marcenmicin AI) against Staphylococcus epidermidis, 100 μL of marcenmicin A, B, C, D, F, G, H and I were treated at each concentration per well in a 96-well plate, and 2 × 106 Staphylococcus epidermidis (cfu / mL) was inoculated with TSB (0.25% glucose) medium. 100 μL was added to each well of a 96-well plate to make a total of 200 μL, and the plate was cultured at 30°C for 24 hours. After 24 hours of culture, all the culture medium in the 96-well plate was removed, washed three times with 50% MeOH, and dried at room temperature for more than 20 minutes. Afterwards, 200 μL of crystal violet reagent was added to each well, reacted for 15 minutes at room temperature, washed three times with DW, and dried for more than 20 minutes. After dissolving in 100% EtOH, the optical density value was checked using a UV-VIS spectrometer at a wavelength of 595. Ampicillin was used as a positive control for comparative experiments.

[0231] As a result of confirming biofilm inhibition activity, minimum biofilm inhibition activity (MBIC) was confirmed at concentrations of 16, 8, 8, 4, 32, 4, 2, and 32 μg / mL in A, B, C, D, F, G, H, and I (Fig. 9).

[0232]

[0233] 6. Analysis of biofilm inhibition activity of marsenmycin using scanning electron microscopy (SEM).

[0234] Scanning electron microscopy (SEM) was used to determine the biofilm inhibitory activity of Marcenmicin A and its derivatives (Marcenmicin AI) against Staphylococcus epidermidis. 14 mm round coverslips were placed in a 24-well plate, and 250 μL of Marcenmicin A, B, C, DG, and H were treated at MBIC concentrations and twice the MBIC concentration. 2 × 10 6 Staphylococcus epidermidis cells (cfu / mL) were inoculated with TSB (0.25% glucose) medium, and 250 μL per well of a 24-well plate was added to make a total of 500 μL, and cultured at 30°C for 24 hours. After 24 hours of culture, all the culture medium in the 24-well plate was removed, and then fixed using 2.5% glutaraldehyde, dehydrated using 25%, 50%, 70%, 90%, and 100% ethanol, and dried for 24 hours after treatment with hexamethyldisilazane. After that, platinum was coated on a 14 mm round coverslip and observed at 5000x magnification.

[0235] As a result of the observation, it was confirmed that marsenmycin A and its derivatives had excellent biofilm inhibition activity against Staphylococcus epidermidis in proportion to the concentration (Fig. 10).

[0236]

[0237] 7. Evaluation of antibacterial activity of marsenmycin against MRSA persister

[0238] To evaluate the bactericidal activity of Marcenmicin A and its derivatives (Marcenmicin AH) against Staphylococcus aureusMW2, growing MRSA cells, growth-arrested MRSA cells, and non-growing / metabolically inactive MRSA cells (persister cells) were prepared and the activity was evaluated for each cell type. First, to prepare growing MRSA cells, the overnight culture of S. aureusMW2 was diluted 1:10,000 and dispensed into 250 mL flasks (25 mL each). The diluted culture was then incubated at 37 °C with shaking at 200 rpm, and the OD 600 Growing MRSA cells were obtained by culturing until they reached an Mn of 0.4. Growth-arrested MRSA cells were prepared by washing the grown MRSA cells prepared as described above three times with PBS at room temperature and then replacing the medium with PBS. Finally, to induce MRSA persister cells, the overnight culture of S. aureus MW2 was diluted 1:100, dispensed into 250 mL flasks (25 mL each), and cultured for 24 hours to bring the cells to the stationary phase. After reaching the stationary phase, the MRSA cells were washed three times with PBS and resuspended in PBS. For the cells prepared in this way, three kinds of antibiotics with different mechanisms of action (vancomycin, gentamicin, ciprofloxacin) were treated for 4 hours at 100× MIC concentrations, and the change in viability was confirmed. The successful induction of persister cells was confirmed by the fact that there was no significant change in the viability before and after antibiotic treatment.

[0239] To evaluate the sterilizing and antibacterial activity, 150 μL of marsenmycin A, B, C, D, F, G, and H were dispensed into each well of a 96-well deep-well plate at different concentrations. Each well contained ~1 × 10 7 PBS (growth-arrested / persister state) or TSB medium (growing MRSA) inoculated with CFU / mL of MRSA persister were added in 150 μL increments to a final volume of 300 μL. The samples were incubated at 37°C for 8 h, and samples were collected every 2 h to determine the number of viable MRSA cells through colony forming unit (CFU) analysis.

[0240] As a result of the antibacterial activity test, for marsenmycin A, B, C, and F, the survival rate of persister cells was reduced by more than 98% at a concentration of 64 μg / mL, and for marsenmycin G and H, the survival rate of persister cells was reduced by more than 80% at concentrations of 16 and 4 μg / mL, respectively (Fig. 11).

[0241]

[0242] 8. Measurement of increased ROS levels within MRSA after marsenmycin treatment

[0243] To evaluate the activity of ROS levels induced in Staphylococcus aureusMW2 cells by marcenmicin A and its derivatives (Marcenmicin AH), a fluorescence-based ROS assay was performed using a Fluorometric Intracellular ROS Kit (Sigma-Aldrich, St. Louis, MO, USA). Marcenmicin AH was treated at concentrations ranging from 2 μg / mL to 64 μg / mL in each well of a 96-well plate. Then, a ROS fluorescent dye solution was diluted to 10 μM in CaMH medium inoculated with exponential phase S. aureusMW2 and dispensed into each well. The fluorescence intensity was then measured at wavelengths of 490 nm and 520 nm using a Cytation 5 multi-mode reader (BioTek, USA).

[0244] The experimental results showed that marcenmicin A and its derivatives significantly increased intracellular ROS levels in Staphylococcus aureus in a concentration-dependent manner over the range of 4-32 μg / mL, and in particular, marcenmicin H increased intracellular ROS levels even at a concentration as low as 4 μg / mL. A consistent concentration-dependent increasing trend was observed between the minimum inhibitory concentration (MIC) of each marcenmicin and the level of ROS production (Fig. 12).

[0245]

[0246] 9. Evaluation of the membrane permeability of marsenmycin against MRSA

[0247] To evaluate the effect of marcenmicin A and its derivatives (Marcenmicin AH) on the intracellular lipid membrane of Staphylococcus aureusMW2, membrane permeability assays were performed. After washing MRSA cells in the persister state three times with PBS, OD 600 The cells were diluted to an A of 0.4. SYTOX Green was added to the diluted cells to a final concentration of 5 μM, and the cells were incubated at room temperature in the dark for 30 minutes. Fifty μL of each sample was then dispensed into each well of a black 96-well plate treated with marsenmycin. Fluorescence was measured for 1 hour at wavelengths of 485 nm and 525 nm using a Cytation 5 multi-mode reader (BioTek, USA).

[0248] Experimental results showed that marcenmicin A and its derivatives increased intracellular lipid membrane permeability against Staphylococcus aureus in a concentration-dependent manner over the range of 4-32 μg / mL. In particular, marcenmicin H increased intracellular lipid membrane permeability even at a concentration as low as 4 μg / mL. A consistent concentration-dependent increase trend was observed between the minimum inhibitory concentration (MIC) and membrane permeability of each marcenmicin (Fig. 13).

[0249]

[0250] 10. Infection control effect of marsenmycin in the MRSA infection C. elegans model

[0251] The infection control efficacy of marcenmicin A and its derivatives (Marcenmicin AH) against Staphylococcus aureusMW2 in C. elegans was evaluated. C. elegans-MRSA infection experiments were performed using immunodeficient C. elegansAU37 (Caenorhabditis Genetics Center, University of Minnesota). Eggs were collected from adults cultured on a diet of E. coliHB101, and hatched in M9 buffer at 15℃ for 48 hours to produce L1 larvae. Approximately 4,500 larvae were cultured at 25℃ for 52 hours to grow into sterile adults. Adult nematodes were suspended in M9 buffer at 1,000 / mL and seeded into a 384-well plate at a density of approximately 15 per well. S. aureusMW2 was added to the wells together with marsenmycin or vancomycin to an OD of 100 600 Infection was induced by treating with a solution diluted to 0.08. The experimental plates were sealed with gas-permeable film and incubated at 25°C for 5 days. After washing nine times to remove bacteria and biofilms, dead nematodes were fluorescently labeled using SYTOX Orange staining. Finally, brightfield and fluorescent images were captured using a Cytation 5 instrument, and the survival rate of MRSA-infected C. elegans was evaluated based on these images. This experiment was repeated three times independently.

[0252] As a result of the experiment, the survival rate of C. elegans was confirmed at a low concentration range of 2-4 μg / mL for marsenmycin A, B, C, and D, respectively (Fig. 14).

[0253]

[0254] [National Research and Development Project Supporting This Invention]

[0255] [Project ID] 1711181598

[0256] [Assignment Number] 2022R1A4A3022401

[0257] [Ministry Name] Ministry of Science and ICT

[0258] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0259] [Research Project Name] Group Research Support

[0260] [Research Project Name] Invasive Fungal Control Substance Discovery Lab

[0261] [Name of the project performing organization] Seoul National University Industry-Academic Cooperation Foundation

[0262] Research Period: June 1, 2022 - February 28, 2025

[0263] [National Research and Development Project Supporting This Invention]

[0264] [Project ID] 1711181738

[0265] [Assignment Number] 2021R1A2C1004958

[0266] [Ministry Name] Ministry of Science and ICT

[0267] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0268] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0269] [Research Project Title] Discovery of Novel Antitoxin Low-Molecular-Weight Substances from Endophytic Bacteria

[0270] [Name of the project performing organization] Seoul National University Industry-Academic Cooperation Foundation

[0271] Research Period: March 1, 2021 - February 28, 2026

[0272] [National Research and Development Project Supporting This Invention]

[0273] [Project ID] 1711197136

[0274] [Project Number] RS-2023-00228746

[0275] [Ministry Name] Ministry of Science and ICT

[0276] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0277] [Research Project Name] Biomedical Technology Development

[0278] [Research Project Name] Development of a Candidate for a Treatment to Control Multidrug-Resistant Bacteria in Respiratory Infections

[0279] [Name of the project performing organization] Ewha Womans University Industry-Academic Cooperation Foundation

[0280] Research Period: April 1, 2023 - December 31, 2027

Claims

A compound represented by the following chemical formula 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof: [Chemical Formula 1] (In the above chemical formula 1, R1 is CH 3, (CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2, or CH2CH(CH3)2) . In the first paragraph, The compound above has a three-dimensional structure represented by the following chemical formula 2: [Chemical Formula 2] (In the above chemical formula 2, R1 is CH 3, (CH2)2CH3 or (CH2)4CH3; R2 is CH2CH(CH3)2 or CH(CH3)2; R3 is H or CH3; R4 is CH2Ph ​​or CH2Indole; and R5 is CH(CH3)2, CHCH2(CH3)2, or CH2CH(CH3)2). In the second paragraph, The above chemical formula 2 is one of the following compounds: . In the first paragraph, The above compound is a compound derived from the strain Serratia marcescens. In paragraph 4, The above strain is Serratia marcescensAULBAC6, compound. An antibacterial composition comprising the compound of claim 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient. In paragraph 6, A composition wherein the target of the above antibacterial action is an antibiotic-resistant strain. In paragraph 6, The above composition has antibacterial activity against gram-positive bacteria. In paragraph 6, The composition has antibacterial activity against strains of the genus Staphylococcus or the genus Enterococcus. In paragraph 9, A composition wherein the strain is at least one selected from the group consisting of Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis. A composition for inhibiting biofilm, comprising the compound of claim 1, an acceptable salt thereof, a stereoisomer thereof, a hydrate thereof, or a solvate thereof as an active ingredient. In Article 11, The above composition has an inhibitory activity against biofilms derived from Gram-positive bacteria. In Article 11, The above composition has an inhibitory activity against a biofilm derived from a strain of the genus Staphylococcus. In Article 13, A composition wherein the above strain is Staphylococcus epidermidis.

Images