N-Acyltyrosine Derivatives and Their Use

Abstract

Provided herein are compounds of formula A, methods for their preparation, and their use for treating or preventing bacterial infections. TIFF2022547605000058.tif51153

Description

Technical Field

[0001] The present invention generally relates to tyrosine derivatives. More specifically, the present invention relates to N-acyl, α-alkyl tyrosine derivatives and their use.

[0002] Cross-Reference to Related Applications This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 900,210, filed September 13, 2019, the content of which is hereby incorporated by reference in its entirety.

[0003] Deposit of Biological Material Alteromonas sp. RKMC-009 was deposited with the Agricultural Research Culture Collection (NRRL), an International Depositary Authority, on August 17, 2020 (the assigned "original deposit date") and has been assigned accession number NRRL B-67979. This deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The "Receipt in the Case of an Original Deposit" and "Viability Statement" issued by the IDA, as well as a copy of FORM PCT / RO / 134 are included below.

Background Art

[0004] Cultured environmental bacteria have the potential to produce numerous antibacterial natural products with diverse chemical structures. However, culture-independent studies have revealed that only a small fraction of bacterial species sampled from various habitats have been cultured. The ichip is a device that enables the culturing of many bacterial species from this "uncultured majority" in the laboratory, thereby facilitating the discovery of antibacterial agents.

[0005] In the search for natural products of microorganisms, it is becoming clear that the inability to culture most environmental bacteria with conventional culture techniques is an obstacle (Non-Patent Documents 1 and 2). As one specific method for obtaining the "uncultured majority", it is conceivable to use a diffusion chamber for in situ culture. Inoculate the inoculum of bacteria from a certain habitat into the diffusion chamber, and it can be cultured in the original natural environment until the resulting colonies reach a size sufficient for "culturing" in the laboratory. This process is presumably mediated by diffusible growth factors from the environment (Non-Patent Document 3). The diffusion chamber technology has become more applicable to high-throughput natural product searches with the development of the ichip (isolation chip) (Non-Patent Document 4). This device contains hundreds of small diffusion chambers, and in situ culture and isolation can be performed simultaneously by inoculating only one cell of environmental bacteria into them. The ichip has been shown to enable the culture of more environmental bacteria and enhance the taxonomic novelty of the recovered isolates. The usefulness of the ichip in the search for bacterial natural products was demonstrated by the isolation of teixobactin, an antibacterial agent produced by a species of soil bacteria (Eleftheria terrae) that has not been reported before and is expected to have clinical applications (Non-Patent Document 5).

[0006] Microorganisms, including environmental bacteria, may produce a variety of useful compounds that have not yet been discovered. Considering the increasing problem of antibiotic resistance in some major bacterial infections that affect humans and other animals, the identification of new antibiotic compounds is highly desirable in this field.

[0007] Alternative, additional, and / or improved compounds with desirable effects such as antibacterial properties are desired.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

[0009] All documents cited above and elsewhere in this specification are hereby incorporated by reference in their entirety into this specification.

Summary of the Invention

[0010] As described in detail herein, the N-acyl-α-methyltyrosine derivatives were discovered by isolation from the bacterium Alteromonas sp. RKMC-009 obtained from the sponge Xestospongia muta. The antibacterial activity and structure-activity relationship (SAR) of the obtained N-acyltyrosine derivatives were extensively studied, and synthetically prepared analogs or derivatives also had antibacterial activity. Also described are synthetic methods for preparing such compounds and their use in the treatment or prevention of bacterial infections.

[0011] In one embodiment, provided herein are compounds of Formula A, or prodrugs or esters thereof, or pharmaceutically acceptable salts or solvates thereof.

[0012]

Chemical formula

[0013] Herein, R1 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R2 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R3 is a linear or branched, optionally substituted C5-C 20 alkyl group, alkenyl group, or alkynyl group, R4 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group.

[0014] In another embodiment of the above compound, R1 may be -H, Me, Et, nPr, iPr, tBu, iBu, secBu, nBu, or Ph.

[0015] In yet another embodiment of any one of the above compounds, R2 may be -H, Me, Et, nPr, iPr, tBu, iBu, secBu, nBu, or Ph.

[0016] In yet another embodiment of any one of the above compounds, R3 is a C5-C group having from 0 to 3 double and / or triple carbon-carbon bonds (0-3Δ). 20 and may be a group.

[0017] In yet another embodiment of any one of the above compounds, R3 is a linear C5-C group having from 0 to 3 double and / or triple carbon-carbon bonds (0-3Δ). 20 and may be a group.

[0018] In yet another embodiment of any one of the above compounds, R4 may be -H, Me, Et, or Ph.

[0019] In yet another embodiment of any one of the above compounds, the compound may be a compound having formula B, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0020]

Chemical formula

[0021] Here, R is -CH3 or -H; R' is -CH3 or -H.

[0022] In another embodiment related to any one of the above compounds, the compound may be a compound having Formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0023]

Chemical formula

[0024] In another embodiment related to any one of the above compounds, the compound may be a compound having Formula 2, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0025]

Chemical formula

[0026] In another embodiment related to any one of the above compounds, the compound may be a compound having Formula 3, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0027]

Chemical formula

[0028] In another embodiment related to any one of the above compounds, the compound may be a compound having Formula 4, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0029]

Chemical formula

[0030] In another embodiment, provided herein is a pharmaceutical composition comprising any one or more of the compounds described herein and a pharmaceutically acceptable excipient or carrier. The compounds described herein may be in synthetically produced and / or isolated form.

[0031] In yet another embodiment, provided herein is the use of any of the compounds described herein or any of the pharmaceutical compositions described herein as an antibacterial agent or in the manufacture of an antibacterial medicament.

[0032] In another embodiment, provided herein is the use of any of the compounds described herein or any of the pharmaceutical compositions described herein for treating a bacterial infection.

[0033] In another embodiment, provided herein is a method for reducing or preventing the growth of bacteria, the method comprising contacting the bacteria with any of the compounds described herein or any of the pharmaceutical compositions described herein.

[0034] In yet another embodiment, provided herein is a method for treating or preventing a bacterial infection in a subject in need thereof, the method comprising administering to the subject any of the compounds described herein or any of the pharmaceutical compositions described herein.

[0035] In certain embodiments, the bacterium may be or may contain Staphylococcus or Enterococcus. In another embodiment, the bacterium may be or may contain Staphylococcus aureus, Staphylococcus warneri, or Enterococcus faecium. In another embodiment, the bacterium may be or may contain methicillin-resistant Staphylococcus aureus (MRSA), or vancomycin-resistant Enterococcus faecium (VRE). In yet another embodiment, the bacterium may be or may contain Enterococcus faecium EF379 (VRE), Enterococcus faecium 15337, Enterococcus faecalis 16371, Enterococcus gallinarum 20993, Enterococcus casseliflavus 15984, Enterococcus hirae 17446, Staphylococcus aureus ATCC33591 (MRSA), or Staphylococcus warneri ATCC17917.

[0036] In another embodiment according to any of the above uses or methods, the bacterium may contain Enterococcus, and the compound may contain Formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0037]

Chemical formula

[0038] In another embodiment according to any of the above uses or methods, the bacterium may contain Enterococcus, and the compound may contain Formula 3, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0039]

Chem.

[0040] In another embodiment according to any of the above uses or methods, the bacterium may comprise Staphylococcus aureus, and the compound may comprise Formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof, or Formula 3, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof, or both.

[0041]

Chem.

[0042]

Chem.

[0043] In another embodiment according to any of the above uses or methods, the bacterium may comprise Enterococcus or Staphylococcus, and the compound may comprise an α-methyl substituent (i.e., R1 = -CH3).

[0044] In another embodiment according to any of the above uses or methods, the bacterium may comprise Enterococcus, and the compound may comprise O-methylation in the tyrosine side chain (i.e., R2 = -CH3).

[0045] In another embodiment according to any of the above uses or methods, the bacterium may comprise Staphylococcus, and the compound may comprise a tyrosine side chain (i.e., R2 = -H).

[0046] In another embodiment, a bacterial sample comprising Alteromonas RKMC-009 is provided herein. In yet another embodiment, a sample comprising Alteromonas RKMC-009 deposited under NRRL accession number NRRL B-67979, or a functional equivalent thereof, is provided herein.

[0047] In another embodiment, a method for producing a compound described herein, the method comprising providing a compound of formula C and N-acylating with an acyl chloride, the acyl chloride being linear or branched and optionally substituted C6-C 21 is provided.

[0048]

Chemical formula

[0049] Here, R1 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R2 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R4 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group.

[0050] In another embodiment according to the above method, the compound may comprise formula 1, and the method may comprise reacting O-methyl-α-methyl-L-tyrosine with palmitoyl chloride for N-acylation.

[0051]

Chemical formula

[0052] In another embodiment of the above method, the compound may comprise Formula 2, and the method may include reacting O-methyl-L-tyrosine with palmitoyl chloride to perform N-acylation.

[0053]

Chemical formula

[0054] In another embodiment of the above method, the compound may comprise Formula 3, and the method may include reacting α-methyl-L-tyrosine with palmitoyl chloride to perform N-acylation.

[0055]

Chemical formula

[0056] In another embodiment of the above method, the compound may comprise Formula 4, and the method may include reacting L-tyrosine with palmitoyl chloride to perform N-acylation.

[0057]

Chemical formula

[0058] In another embodiment, provided herein is a method for producing a compound of Formula 1, the method including fermenting Alteromonas sp. RKMC-009 in a BFM4m culture medium and extracting the culture medium with ethyl acetate (EtOAc).

[0059] In another embodiment of the above method, the method may further include purifying the compound of Formula 1 by reverse-phase chromatography.

[0060] In another embodiment according to the above method or any of the methods, the Alteromonas sp. RKMC-009 is Alteromonas RKMC-009 deposited under NRRL accession number NRRL B-67979, or a functional equivalent thereof.

[0061] In another embodiment, provided herein is a lysate, supernatant, culture broth or extract derived from or prepared from an Alteromonas sp. RKMC-009 bacterial culture or fermentation, wherein the lysate, supernatant, culture broth or extract contains a compound of formula 1 or a salt thereof.

[0062]

Chemical formula

[0063] In another embodiment of the lysate, supernatant, culture broth or extract, the Alteromonas sp. RKMC-009 bacterial culture or fermentation may contain Alteromonas sp. RKMC-009 deposited under NRRL accession number NRRL B-67979, or a functional equivalent thereof.

Brief Description of the Drawings

[0064]

Figure 1

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[0065] This specification describes N - acyltyrosine derivatives and their use as antibacterial agents. The embodiments and examples are provided for illustrative purposes for those skilled in the art and are not limiting in any sense.

[0066] In one embodiment, this specification provides a compound of Formula A, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0067]

Chemical formula

[0068] Here, R1 is -H; or a linear or branched, optionally substituted C1 - C6 alkyl group; or an optionally substituted phenyl group, R2 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R3 is a linear or branched, optionally substituted C5-C 20 alkyl group, alkenyl group, or alkynyl group, R4 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group.

[0069] In certain embodiments, the compound may be a compound of formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0070]

Chemical formula

[0071] In a further embodiment, the compound is not a compound of formula 1.

[0072] As will be appreciated, the linear or branched, optionally substituted C1-C6 alkyl group in R1 or R2 may include, for example but not limited to, a suitable linear or branched C1-C6 alkyl group further substituted with one or more substituents such as halogen (i.e., F, Cl, Br, I), hydroxyl group, -C≡N group, C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group. Also as will be appreciated, the optionally substituted phenyl group in R1 or R2 may include a phenyl group further substituted with one or more substituents such as halogen (i.e., F, Cl, Br, I), hydroxyl group, -C≡N group, C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group. In certain embodiments, R1 and R2 may each independently be selected from -H, Me, Et, nPr, iPr, tBu, iBu, secBu, nBu, or Ph.

[0073] In certain embodiments, the linear or branched, optionally substituted C5-C at R3 20 The alkyl group, alkenyl group, or alkynyl group may be further substituted with one or more substituents such as, but not limited to, halogen (i.e., F, Cl, Br, I), hydroxyl group, -C≡N group, C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group, etc., and is a suitable linear or branched C5-C 20 The alkyl group, alkenyl group, or alkynyl group (i.e., a C5-C which may have zero, one, two or more double and / or triple carbon-carbon bonds 20 group) may be included. In certain embodiments, R3 is a C5-C having zero to 3 double and / or triple carbon-carbon bonds (0-3Δ) 20 group. In certain embodiments, R3 is a linear C5-C having zero to 3 double and / or triple carbon-carbon bonds (0-3Δ) 20 group.

[0074] Also understood is that the linear or branched, optionally substituted C1-C6 alkyl group at R4 may be, for example, but not limited to, a suitable linear or branched C1-C6 alkyl group which may be further substituted with one or more substituents such as halogen (i.e., F, Cl, Br, I), hydroxyl group, -C≡N group, C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group, etc. Also understood is that the optionally substituted phenyl group at R4 may be, for example, but not limited to, a phenyl group which may be further substituted with one or more substituents such as halogen (i.e., F, Cl, Br, I), hydroxyl group, -C≡N group, C1-C6 alkyl group, C2-C6 alkenyl group or C2-C6 alkynyl group, etc. In certain embodiments, R4 is -H, Me, Et, or Ph.

[0075] As will be appreciated, those skilled in the art considering the teachings herein will recognize various prodrugs or esters of any of the compounds described herein that can be prepared, or pharmaceutically acceptable salts or solvates. In certain embodiments, a prodrug may typically include a prodrug moiety that can be linked to the compound via a biologically cleavable linker. In certain embodiments, an ester may typically include an alkyl group or other moiety that can be linked to the compound via an ester bond through a carboxylic acid or hydroxyl functional group of the compound. In certain embodiments, a pharmaceutically acceptable salt or solvate may include any suitable salt or solvate known to those skilled in the art considering the teachings herein, such as those described in Remington (The Science and Practice of Pharmacy (2006), Remington’s Pharmaceutical Sciences (2000 - 20th edition), and the United States Pharmacopeia (The National Formulary (USP 24 NF19) issued in 1999)), each of which is hereby incorporated by reference in its entirety.

[0076] In certain embodiments, the compound may be a compound of formula B, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0077]

Chemical formula

[0078] Here, R is -CH3 or -H; R’ is -CH3 or -H.

[0079] In yet another embodiment, the compound may be a compound of formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0080]

Chem.

[0081] In yet another embodiment, the compound may be a compound of Formula 2, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0082]

Chem.

[0083] In yet another embodiment, the compound may be a compound of Formula 3, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0084]

Chem.

[0085] In yet another embodiment, the compound may be a compound of Formula 4, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0086]

Chem.

[0087] In yet another embodiment, provided herein is a pharmaceutical composition comprising any one or more of the plurality of compounds described herein, and optionally further comprising a pharmaceutically acceptable excipient, diluent, or carrier. Examples of such pharmaceutically acceptable excipients, diluents, and carriers can be found in Remington, The Science and Practice of Pharmacy (2006). Similarly, examples of pharmaceutically acceptable carriers, diluents, and excipients can be found, for example, in Remington’s Pharmaceutical Sciences (2000 - 20th edition), and The National Formulary (USP 24 NF19, issued in 1999), each of which is hereby incorporated by reference in its entirety. In certain embodiments, the pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to those of skill in the art. Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch. Those of skill in the art will recognize that pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, glidants, and disintegrants known in the art (see, for example, Remington, The Science and Practice of Pharmacy (2006)). Examples of pharmaceutically acceptable carriers, diluents, and excipients can be found, for example, in Remington’s Pharmaceutical Sciences (2000 - 20th edition), and The National Formulary (USP 24 NF19, issued in 1999).

[0088] In another embodiment, provided herein is the use of any of the compounds described herein, or a pharmaceutical composition, as an antibacterial agent or for treating or preventing a bacterial infection in a cell or subject in need thereof. In certain embodiments, the antibacterial agent may be for in vitro or in vivo use and / or for use as a preservative or bactericide.

[0089] In another embodiment, provided herein is a method for reducing or preventing the growth of bacteria, the method comprising contacting the bacteria with a compound or pharmaceutical composition described herein.

[0090] In another embodiment, provided herein is a method for treating or preventing a bacterial infection in a subject in need thereof, the method comprising administering to the subject a compound or pharmaceutical composition described herein.

[0091] In another embodiment, the bacteria may include staphylococci or enterococci. In certain embodiments, the bacteria may include Staphylococcus aureus, Staphylococcus warneri, or Enterococcus faecium. In certain embodiments, the bacteria may include methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus faecium (VRE). In another embodiment, the bacteria may include Enterococcus faecium EF379 (VRE), Enterococcus faecium 15337, Enterococcus faecalis 16371, Enterococcus gallinarum 20993, Enterococcus casseliflavus 15984, Enterococcus hirae 17446, Staphylococcus aureus ATCC33591 (MRSA), or Staphylococcus warneri ATCC17917.

[0092] In another embodiment of the use or method described herein, the bacterium may include Enterococcus, and the compound may include Formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0093]

Chemical formula

[0094] In yet another embodiment of the use or method described herein, the bacterium may include Enterococcus, and the compound may include Formula 3, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0095]

Chemical formula

[0096] In yet another embodiment of the use or method described herein, the bacterium may include Staphylococcus aureus, and the compound may include Formula 1, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof, or Formula 3, or a prodrug or ester thereof, or a pharmaceutically acceptable salt or solvate thereof, or both.

[0097]

Chemical formula

[0098]

Chemical formula

[0099] In another embodiment of the use or method described herein, the bacterium may comprise Enterococcus or Staphylococcus, and the compound may comprise an α-methyl substituent (i.e., R1 = -CH3). In another embodiment of the use or method described herein, the bacterium may comprise Enterococcus, and the compound may comprise O-methylation in the tyrosine side chain (i.e., R2 = -CH3). In yet another embodiment of the use or method described herein, the bacterium may comprise Staphylococcus, and the compound may comprise a tyrosine side chain (i.e., R2 = -H).

[0100] In yet another embodiment, provided herein is a bacterial sample comprising Alteromonas RKMC-009. The genomic sequence of Alteromonas RKMC-009 can be found in GenBank (CP031010 and CP032914), and is further described in MacIntyre, L.W, Haltli, B.A., Kerr, R.G., Microbiology Resource Announcements (2019), 8(25):2445, which is hereby incorporated by reference in its entirety. The Alteromonas species RKMC-009 was deposited with the Agricultural Research Service Patent Culture Collection (NRRL), an international depositary authority, on August 17, 2020 (the assigned "original deposit date") and has been assigned accession number NRRL B-67979. A "Deposit Receipt for Original Deposit" and a "Certificate of Viability" issued by the IDA, as well as a copy of FORM PCT / RO / 134 are included below. In one embodiment, provided herein is a sample comprising Alteromonas RKMC-009 deposited under NRRL accession number NRRL B-67979, or a functional equivalent thereof.

[0101] In another embodiment, provided is a method for producing a compound described herein, the method comprising providing a compound of formula C and N-acylating with an acyl chloride, wherein the acyl chloride is linear or branched and may be substituted, C6-C 21 A method is provided that includes performing N-acylation with an acyl chloride.

[0102] [Chemistry]

[0103] Here, R1 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R2 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group, R4 is -H; or a linear or branched, optionally substituted C1-C6 alkyl group; or an optionally substituted phenyl group.

[0104] In another embodiment, the N-acylation may be carried out using any other suitable reagent under conditions suitable for providing N-acylation with the desired group. For example, N-acylation can be readily carried out by reacting a suitable acyl chloride with the desired amino acid. If the acyl chloride is not commercially available, it may be prepared by reacting the corresponding carboxylic acid with thionyl chloride. Alternatively, in certain embodiments, the corresponding carboxylic acid may be directly coupled to the amino acid using, for example, DCC (dicyclohexylcarbodiimide). The introduction of protecting groups was not necessary for the preparation of Compounds 1 to 4 described herein, but the (a) protecting group(s) may be used in certain embodiments, particularly when the preparation of homologues containing an amine within, for example, R1, R2, and / or R4 is desired.

[0105] In certain embodiments, the method may be used to prepare a compound of Formula 1, and the method may include performing N-acylation by reacting O-methyl-α-methyl-L-tyrosine with palmitoyl chloride.

[0106] [Chemistry]

[0107] In another embodiment, the method may be used to prepare a compound of Formula 2, and the method may include reacting O-methyl-L-tyrosine with palmitoyl chloride to perform N-acylation.

[0108]

Chemical formula

[0109] In yet another embodiment, the method may be for preparing a compound of Formula 3, and the method may include reacting α-methyl-L-tyrosine with palmitoyl chloride to perform N-acylation.

[0110]

Chemical formula

[0111] In yet another embodiment, the method may be for preparing a compound of Formula 4, and the method may include reacting L-tyrosine with palmitoyl chloride to perform N-acylation.

[0112]

Chemical formula

[0113] In yet another embodiment, provided herein is a method for producing a compound of Formula 1, the method including fermenting Arthromonas species RKMC-009 in a BFM4m culture medium and extracting the culture medium.

[0114] In certain embodiments, the culture medium may be extracted with ethyl acetate. In another embodiment, the method may further include purifying the compound of Formula 1 by reverse-phase chromatography.

[0115] In another embodiment according to the above method or any of the methods, the Alteromonas sp. RKMC-009 may be, or may contain, Alteromonas RKMC-009 deposited under NRRL accession number NRRL B-67979, or a functional equivalent thereof.

[0116] In yet another embodiment, provided herein is a lysate, supernatant, culture broth, or extract derived from or prepared from an Alteromonas sp. RKMC-009 bacterial culture or fermentation, wherein the lysate, supernatant, culture broth, or extract contains a compound of Formula 1 or a salt thereof.

[0117]

Chemical formula

[0118] In another embodiment relating to the lysate, supernatant, culture broth, or extract, the Alteromonas sp. RKMC-009 bacterial culture or fermentation may contain Alteromonas sp. RKMC-009 deposited under NRRL accession number NRRL B-67979, or a functional equivalent thereof.

[0119] For further clarity, it will be understood that the compounds of Formula A (such as the compound of Formula 1) and the compounds of Formula C described herein can be equivalently depicted as follows using conventional wedge and dash notation.

[0120]

Chemical formula

[0121] Example 1 In situ cultivation of sponge bacteria producing a functional N-acyl tyrosine derivative (N-acyl tyrosine having an α-methyl substituent) and its research The majority of environmental bacteria that have not yet been cultured in the laboratory have the potential to produce an attractive pool of natural products for antibacterial agent discovery. In recent years, the development of the ichip (isolation chip) has popularized in situ culturing for culturing uncultured bacteria. This ichip can enable high-throughput in situ bacterial culturing (and simultaneous isolation), but there have been few reports on its use in the nearly 10 years since its publication.

[0122] In this study, we employed the use of an isolation chip (ichip), which is thought to be the first, in the sponge (Giant Barrel Sponge) with a rich microbiome, and describe the subsequent isolation of an Alteromonas species RKMC-009, which is thought to be a novel bacterial species. As described in detail herein, the RKMC-009 strain produces a structurally unique N-acyl amino acid, which is an N-acyl tyrosine compound of formula (1) functionalized / added with a rare α-methyl substituent within the amino acid residue (aminoacyl moiety), and was found to also exhibit Gram-positive antibacterial activity. SAR experiments revealed that this α-methyl makes a major contribution to the potent antibacterial activity, bringing about the staphylococcal activity of compound (1) and potentially enhancing the enterococcal activity.

[0123] Despite the ichip's success in culturing novel microbial taxa, reports describing its use have been surprisingly scarce for approximately 10 years since its initial description. Furthermore, reports of its use appear to be limited to the soil or sediment environment. Therefore, in this study, the ichip was implemented in tropical sponges to expand its application range and aim for the discovery of novel bacterial taxa for natural product exploration. The diversity of uncultured microorganisms in sponges is widely known, and bacteria cultured from these habitats may produce natural products with potent biological activities (Non-Patent Documents 6 and 7). In this study, the giant barrel sponge, a sponge rich in microorganisms, was mainly selected as the research subject due to its size. The giant barrel sponge was expected to be able to hold many ichips while minimizing tissue damage due to its size. Also, the giant barrel sponge (X. muta) is known to possess a rich bacterial community (Non-Patent Documents 6 and 7).

[0124] The ichip was assembled using inoculum prepared from giant barrel sponge samples and cultured within the sponge tissue. After disassembling this ichip in the laboratory and culturing as many bacteria as possible from it, the selected isolates were fermented in a culture broth, and its extract was chemically screened to examine the possibility of producing novel natural products. A particular bacterial isolate, Alteromonas, which is clearly a genus of a novel species, produced an N-acylated amino acid (1) that exhibits growth inhibitory activity against Gram-positive bacterial pathogens, as described in detail below. Compound (1) has one methyl substituent at the α-position of the amino acid residue, which is thought to be a structural feature unique to this family of natural products. In this study, the ichip culturing of the Alteromonas species RKMC-009, the absolute chemical structure and chemical synthesis of compound (1), and the structure-activity relationship (SAR) in which the α-methyl substituent of compound (1) is considered to confer or contribute to its enterococcal and staphylococcal activities will be described in detail.

[0125] Results and Discussion Ichip Culturing of Novel Alteromonas Species Since the ichip is thought to have had no commercial use (presumably due to the low number of usage reports), it was fabricated in the laboratory as faithfully as possible along the existing design, but had two notable exceptions. First, when transplanting the device into the tissue of the giant barrel sponge, we reduced the thickness of the assembled ichip to 5.0 mm compared to the original design (14.0 mm) to minimize potential damage to the giant barrel sponge. Second, we reduced the number of through-holes in the central plate of the device from 384 to 148 (Non-Patent Document 4). As a result, an improved ichip as shown in Fig. 1A was obtained. The hermeticity of randomly extracted ichips was confirmed, and it was confirmed that bacteria did not enter or escape from the device in the fully assembled state. This ichip was installed in individual sponges approached by SCUBA diving on local reefs in San Salvador, Bahamas.

[0126] Tissue samples were collected from the giant barrel sponge, and a suspension of its constituent bacteria in a diluted complex medium was prepared using the previously reported procedure (Non-Patent Document 6). Diluting the bacterial suspension to a defined cell density was important for directly isolating pure colonies from the ichip. At this cell density, approximately one cell per ichip through-hole was obtained. However, in the field laboratory, there was neither a fluorescence microscope nor a means to accurately count the number of bacterial cells. Therefore, we attempted to avoid this technical problem by first estimating the total number of bacterial cells in the sponge-derived suspension to be 8.2×109 cells / mL from the reported microbial biomass in the giant barrel sponge. Next, five dilutions reaching the desired cell density (1000 cells / mL): 5000, 2500, 1000, 500, and 100 cells / mL were prepared from this suspension so that approximately one cell per through-hole was achieved. Each dilution was used to inoculate two ichips each, which were transplanted into the giant barrel sponge and cultured for 7 days (Fig. 1B). With such a low-precision method of measuring the number of bacteria, it was speculated that probably only the ichips inoculated with a specific dilution would contain one cell per through-hole.

[0127] After recovering all the ichips, they were disassembled and processed according to the previously reported procedures (Non-Patent Document 4). Bacterial growth was observed from the through-holes of all 10 ichips, and a total of 50 morphologically different bacterial colonies were isolated. Since most of the isolates required purification by continuous subculture, it was suggested that more than one cell was inoculated per through-hole in all 10 ichips. Therefore, subsequent isolation of pure cultures was not possible, but it did not interfere with in situ culture.

[0128] All bacterial isolates were taxonomically identified by sequence analysis (16S rRNA gene), and the selected isolates were fermented in 7 different growth media to search for potential new natural products. All culture extracts were analyzed by UHPLC-HRMS, and the target ions were queried in AntiBase 2017. One specific isolate, Alteromonas sp. RKMC-009, was identified as a new species belonging to the genus Alteromonas and was flagged for further analysis because a major peak was present at 5.35 minutes in the HRMS and ELSD chromatograms (Figure 4). The peak was related to compound (1) with an m / z of 448.3416 + [M+H]. By searching AntiBase 2017, it was found that this pseudo-molecular ion might be new. Alteromonas sp. RKMC-009 was deposited with the Agricultural Research Service Culture Collection (NRRL), an international depositary authority, on August 17, 2020 (the assigned "original deposit date"), and accession number NRRL B-67979 was assigned. The "Deposit Receipt for the Original Deposit" and "Certificate of Viability" issued by the IDA, as well as a copy of FORM PCT / RO / 134, are included below.

[0129] The strain most closely related to RKMC-009 is Alteromonas aesuariivens JDTF-113, with a sequence similarity of 98.15% (16S rRNA gene) (Non-Patent Document 26). A phylogenetic tree was constructed from the 16S rRNA gene sequences of RKMC-009, all effectively described Alteromonas spp., representative type strains of additional genera of the family Alteromonadaceae, and 16S rRNA gene sequences of six Alteromonas strains that are not described at the species level but show strong sequence similarity to RKMC-009 (Figure 5). RKMC-009 forms a phylogenetic group similar to the latter six strains, and together they may represent two or more novel species. However, a detailed taxonomic evaluation to fully establish RKMC-009 as a novel species was outside the scope of this analysis.

[0130] Purification, Absolute Structure Elucidation, and Chemical Synthesis of α-Methylated N-Acyltyrosine Scale-up fermentation of RKMC-009 was carried out, and purification of compound (1), elucidation of its chemical structure, and evaluation of its biological activity were performed. After fermenting the Alteromonas sp. RKMC-009 in 10 × 1 L of BFM4m culture medium, the combined cultures were extracted with ethyl acetate. Since RKMC-009 requires salts for growth, 18 g / L of artificial seawater (Instant Ocean) was added to the medium (Figure 6). Compound (1) was purified from the ethyl acetate extract using flash chromatography to obtain 350 mg of material with sufficient purity for spectroscopic and biological evaluations.

[0131] [Chemical Formula]

[0132] The chemical structure of compound (1) (Figure 2) was elucidated by combining NMR spectroscopy and chemical derivatization. The purified compound (1) was obtained as an amorphous white solid, and ESI+HRMS suggested a molecular formula of C 27 H 45 NO4, requiring an unsaturation degree of 6.1 The two-dimensional structure of compound (1) was elucidated by a combination of H, DEPTQ-135, COSY, HSQC, and HMBC NMR spectra (Figs. 7-12). The chemical shifts, coupling constants, and correlation relationships are shown in Table 2. 1 In the initial analysis of the H NMR spectrum, the presence of a long aliphatic carbon chain was immediately suggested based on the methylene envelope at approximately 1.25 ppm. COSY spectral correlations of the methylene in this envelope with H-3’ and H-16’ (see Fig. 2), and further HMBC NMR correlations from both H-2’ and H-3’ to the 13 C resonance at 174.1 ppm (C-1’) suggested the presence of a linear aliphatic acyl group. Integration of the methylene envelope allowed this spin system to be identified as the palmitoyl (hexadecanoyl) moiety. The remaining molecular formula (C 11 H 12 NO3) after subtracting the palmitoyl moiety suggested the presence of an amino acid residue containing an oxygenated side chain. Also, 1 The aromatic resonances (6.80 ppm and 7.05 ppm) in the H NMR spectrum strongly suggested a tyrosine-like structure. The presence of a carboxylic acid was 1 suggested by the broad singlet at 8.59 ppm in the H NMR spectrum, and the signal for the amide proton was observed at 6.01 ppm. HMBC NMR correlations from the amide proton to C-1’ suggested that compound (1) is an N-acylated amino acid. Correlations in the HMBC NMR spectrum suggested that the tyrosine backbone has two prominent modifications. First, the hydroxy group in the side chain is methylated. This is 1 due to the methoxy signal at 3.77 ppm in the H NMR spectrum and the observed HMBC correlation from this methoxy group to C-7. Second, the absence of an α-H was suggested by the singlet amide proton resonance. 1The remaining signals in the ¹H NMR spectrum were a singlet at 1.64 ppm integrated to 3-H, which allowed the placement of a methyl group at the α-position. This placement was confirmed by HMBC NMR correlations to C-1, C-2, and C-3. Also, HMBC NMR correlations from both the amide proton and H-3a / H-3b to CH₃-2 were observed. Compound (1) satisfied the molecular formula C 27 H 45 NO₄ and was consistent with the required degree of unsaturation.

[0133] To confirm that OCH₃-7 did not arise from the methanolysis of methanol during the purification of compound (1) or during the preparation for UHPLC-HRMS, the fermentation of RKMC-009 was repeated (N = 3) in culture tubes at a 5 mL scale and analyzed in the absence of methanol. The culture broth was extracted with ethyl acetate, and the dried extract was resuspended in CH₃CN for UHPLC-HRMS analysis. Compound (1) was found to be present in an amount comparable to that of previous fermentations, leading to the conclusion that compound (1) was not an artifact due to methanolysis. Using the Marfey's method, compound (1) was clearly determined to be S-configured at C-2 (N-palmitoyl-α,O-dimethyl-L-tyrosine, Figure 13). Finally, to support this structure determination, compound (1) was chemically synthesized from α-methyl-L-tyrosine (Scheme 1). The 1 H and 13 ¹³C NMR spectra (Figures 14 - 15) of the synthesized compound (1) were identical to those of compound (1) purified from bacterial fermentation not only in specific rotation but also in other aspects, thereby allowing the confirmation of the absolute structure determination.

[0134] Scheme 1 - Synthetic Scheme (A) Synthetic scheme for the preparation of compound (1) from α-methyl-L-tyrosine. (B) Synthetic analogs (compounds 2 - 4) of compound 1 prepared by N-acylation of the corresponding amino acid precursors.

[0135] [Chemical]

[0136] α-Methylation of N-palmitoyltyrosine confers antibacterial activity For compound (1), first, a screening of the antibacterial activity against the following panel of pathogenic bacteria and fungi in Table 1 was carried out by microdilution assay: The pathogenic bacteria and fungi were methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus warneri, vancomycin-resistant Enterococcus faecium (VRE), Pseudomonas aeruginosa, Proteus vulgaris, and Candida albicans. Growth inhibition was observed against VRE, MRSA, and Staphylococcus warneri, but no activity was observed against Gram-negative bacteria and fungal pathogens. The inventors recognized that there was no report on the anti-staphylococcal activity of N-acyltyrosine or the anti-enterococcal activity of any N-acylated amino acid. Therefore, it was hypothesized that the α-methyl substituent of compound (1) might be a major element of the antibacterial pharmacophore of compound 1, conferring staphylococcal inhibitory activity to N-acyltyrosine and / or possibly playing a role in its enterococcal activity.

[0137] To verify the proposed structure-activity relationship (SAR), a structural analog compound (2) lacking the α-methyl substituent of compound (1) was synthesized and its antibacterial activity was verified in parallel with compound (1). Furthermore, to support the possibility of SAR involving the O-methyl substituent, analog compounds (3) and (4) were also synthesized and evaluated (Figs. 16 - 21). To thoroughly clarify the anti-enterococcal activity of compounds (1) to (4) for this study, five animal clinical isolates (in addition to VRE EF379) were obtained. All five clinical isolates were classified as vancomycin-sensitive (Non-Patent Document 9). The experimental results are summarized in Table 1.

[0138] [Chemical]

[0139]

Table 1

[0140] The average IC of compounds (1) to (4) 50 The 95% confidence intervals near the values were pairwise compared to infer the structure-activity relationship (Figure 24). Compound (1) was more potent than compound (2) against 4 out of 6 Enterococcus species (IC 50 was lower). Compound (3) showed more potent efficacy than compound (4) against 5 out of 6 Enterococcus species. The growth of Staphylococcus aureus and Staphylococcus warneri was not affected by compounds (2) and (4) at the highest concentration tested (128 μg / mL). Staphylococcus aureus was inhibited by compounds 1 and 3, but the inhibition by compound 1 reached its maximum at 8 μg / mL and reached a plateau at higher concentrations, so it was incomplete (Figure 25). Staphylococcus warneri was inhibited only by compound 3.

[0141] While not wishing to be bound by theory, from these observations it was concluded that α-methyl increases the inhibitory activity of N-palmitoyltyrosine against most Enterococcus species and may be important or necessary for its staphylococcal activity. An SAR involving O-methyl was also observed. Compound (1) was more potent than compound (3), and compound (2) was more potent than compound (4), being effective against 4 out of 6 and 3 out of 6 Enterococcus species, respectively. This indicates that O-methylation increases the inhibitory activity of N-palmitoyltyrosine against Enterococcus. Interestingly, the opposite effect was observed against Staphylococcus. Compound (1) showed significantly lower activity than compound (3) against both Staphylococcus aureus and Staphylococcus warneri. Cytotoxicity of compounds (1) to (4) was tested against 1 healthy cell line (normal African green monkey (Cercopithecus aethiops) VERO kidney cells) and 3 human cancer cell lines. Compounds (1) to (4) each showed weak inhibitory activity against the cell lines, but the IC 50 values all exceeded 50 μM, and compounds (1) to (4) were not classified as cytotoxic.

[0142] Proposed biosynthetic pathway Compound (1) is considered to be the first reported N-acyl amino acid having an α-alkyl substituent or a side-chain O-methyl substituent, and represents one of the few known structural modifications to the aminoacyl moiety of the molecules of this natural product family. Known aminoacyl modifications can include methyl esterification, oxidative decarboxylation to obtain enol esters and enamides, and α,β-dehydrogenation (Non-Patent Documents 10-14). Without wishing to be bound by theory, a biosynthetic pathway for the production of compound (1) is proposed herein (Figure 3). According to this, the α,β-unsaturated intermediate can function as a substrate for a C-methyltransferase that can introduce an α-methyl. Homologues of this proposed intermediate, thalassotalic acids A-C, have been reported from bacteria belonging to the same order as RKMC-009 (the order Alteromonadales) (Non-Patent Document 14). From this, it is considered that the α,β-unsaturated intermediate may be produced by dehydration of N-palmitoyl-β-hydroxy-L-tyrosine. β-Hydroxytyrosine has been observed within non-ribosomal peptides as a precursor of non-peptidic metabolites, and recently, an indication of a β-hydroxylation / dehydration sequence in the biosynthesis of the E-2,3-dehydrotyrosine residues found in WS9326A and derivatives has been provided (Non-Patent Documents 15-17). O-Methylation has been arbitrarily assigned as the final step of biosynthesis, but may occur at other locations in the pathway (including O-methylation of free tyrosine) (Non-Patent Document 18). It should be noted that as another pathway, there may also be a pathway in which a radical SAM methyltransferase directly adds an α-methyl to N-palmitoyl tyrosine.

[0143] Figure 26 shows the identification of proposed intermediate 3 and intermediate 4 in the culture extract of Alteromonas species RKMC-009 by comparison (using UHPLC HRMS) with synthetic standards of possible intermediates 2, 3, and 4 (XIC: Extracted ion chromatogram).

[0144] The present study described herein isolated marine bacteria from the giant barrel sponge, a tropical sponge, using a small ichip, which is presumably the first application of this device to an environment other than soil or sediment. The Alteromonas sp. RKMC-009 was cultured from a specific ichip implanted in the giant barrel sponge and produced an N-acyltyrosine compound (1) with an α-methyl substituent. SAR experiments revealed that α-methylation can confer antibacterial activity to N-palmitoyltyrosine, is necessary for staphylococcal inhibitory activity under the tested conditions, and enhances enterococcal activity. Also proposed herein was the biosynthetic pathway of compound (1).

[0145] Materials and Methods, Experimental Procedures General experimental procedures Optical rotations were measured on a Rudolph Autopol III polarimeter using a 50 mm microcell (1.2 mL). Infrared (IR) spectra were recorded on a Thermo Nicolet 6700 FT-IR spectrometer using attenuated total reflectance. All NMR spectra were acquired on a Bruker Avance III NMR spectrometer equipped with a 5 mm SmartProbe 1 H: 400 MHz, 13 C: 151 MHz). All chemical shifts were reported in ppm and referenced to the residual solvent signals 1 H (DMSO-d6): 2.50 ppm, 13 C (DMSO-d6): 39.51 ppm, 1 H (CDCl3): 7.26 ppm, 13 C (CDCl3): 77.16 ppm]. All UHPLC-HRMS analyses were performed on the following platform equipped with an HRMS-ELSD-UV detector (unless otherwise specified). Thermo Accela UHPLC Pump, Thermo Exactive HRMS with an ESI source, Sedex 80 LT-ELSD, and Thermo PDA. Kinetex core-shell 100 Å C 18Analysis was performed using a column (2.1×50 mm, 1.7 μm, Phenomenex) with a mobile phase flow rate of 0.5 mL / min and an injection volume of 10 μL (all samples were prepared in CH3OH). The elution method was as follows [A = H2O (0.1% formic acid), B = CH3CN (0.1% formic acid)]: 5% B from 0.0 min to 0.2 min, linear gradient from 5% B at 0.2 min to 99% B at 4.8 min, 99% B from 4.8 min to 8.0 min, linear gradient from 99% B at 8.0 min to 5% B at 8.5 min, 5% B from 8.5 min to 10.0 min. HRMS parameters were positive ionization mode, mass resolution 30,000, mass range m / z 190 - 2,000, spray voltage 2.0 kV, capillary temperature 300 °C, S lens RF voltage 60.0%, maximum injection time 10 ms, and 1 microscan was used. The system was controlled by a Thermo Xcalibur software module. Automated flash chromatography was performed on a Teledyne ISCO CombiFlash Rf 200 system equipped with a UV detector. All reagents were commercially available and used without further purification. All solvents used for purification were of HPLC grade or higher.

[0146] Fabrication and verification of ichip Twenty ichips were machined from polyoxymethylene, a hydrophobic plastic, and each consists of three parts: a top plate and a bottom plate (both 75.0 × 22.0 × 2.5 mm), and a central plate (63.5 × 11.5 × 1.0 mm) with through-holes 1.0 mm in diameter (1.25 μL). Two pieces of polycarbonate membrane (Sterlitech; pore size 0.03 μm) cut with a scalpel to the exact dimensions of the central plate were attached to each ichip. The following procedure was used for the aseptic assembly of ichips in a laminar flow hood. The membranes were autoclaved, and the central and outer plates were sterilized by immersion in isopropanol (70% v / v) for at least 15 min, after which the isopropanol was evaporated in the laminar flow hood before ichip assembly. All parts of the ichip were handled with sterile forceps. The central plate with the growth chamber was inoculated by immersion in melted agar medium (at 45 °C) and gently stirred to ensure that all through-holes were filled. The central plate was then removed, and after the growth medium had solidified, excess agar on the plate surface was scraped off using a sterile glass microscope slide. The ichip parts were then assembled and fixed with screws as shown in Fig. 1A. To verify the sealability of the ichip and the effectiveness of the aseptic assembly procedure, three randomly selected ichips were fully assembled to contain sterile LB agar medium containing 0.2 mg / mL of 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-gal). Each ichip was placed in a 50 mL conical tube containing a 12 h culture of NEB 10-β cells (LB broth) containing pUC19 (New England BioLabs) and cultured for 24 h. After this test, no visible blue through-holes were present in the ichip, indicating that external bacteria had not invaded the membrane barrier and contaminated the agar plug in the central plate. Three ichips were then fully assembled in LB agar medium inoculated with 3% (v / v) of a 12 h culture of Escherichia coli C3019 (LB broth) containing pUC19 and placed in a conical tube containing sterile LB broth for 24 h.After the incubation period, no bacterial growth was observed in the surrounding medium, indicating that the bacteria within the ichip were unable to migrate out of the ichip.

[0147] Inoculation and culture of the ichip Individuals of the sponge (Giant Barrel Sponge) were inhabiting at a water depth of 10 m to 12 m in Runway 10 Reef (24°03′57.2″N, 74°32′41.3″W) of San Salvador in the Bahamas. The sponge tissue was collected and processed according to the previously reported procedure 1 A part of the sponge including the outer surface tissue and the inner surface tissue was excised with a diving knife, stored in a sterile plastic bag, transported to the field laboratory and further processed. In the laboratory, approximately 1 g of wet sponge tissue pieces were excised with a sterile blade, washed three times with filter-sterilized (0.2 μm) seawater, and then added to 9 mL of filter-sterilized seawater. The tissue was homogenized with a rotor-stator that had been sterilized by sequential immersion in 0.525% sodium hypochlorite and isopropanol (70% v / v) for 5 minutes each. Before homogenizing the sponge tissue, the rotor-stator was rinsed with sterile water to remove residual isopropanol. The homogenate was passed through a sterile nylon filter (100 μm; autoclaved) to obtain a bacterial suspension. As previously determined with samples of Giant Barrel Sponge in Florida (Non-Patent Document 19), the bacterial density was 8.2×10 9 ±7.7×10 8It was assumed to be cells / mL. This suspension was diluted to the following bacterial densities with 1 / 10 R2Am medium at 45 °C [1.8 g / L R2A Agar (Difco), 12 g / L Nobel agar (Sigma), 33.3 g / L Instant Ocean (pH = 7.9)]: 5000, 2500, 1000, 500, and 100 cells / mL. Using each diluted bacterial suspension, two ichips were inoculated as described above. To transplant into the giant barrel sponge, the fully assembled ichip was returned to the runway 10 leaf. A slit was made in the sponge tissue adjacent to the excision site using a diving knife, and one ichip was completely inserted into each slit. A colored pin indicating the cell density of the inoculum was placed beside each ichip. Tissue collection, inoculum preparation, ichip assembly, and ichip insertion into the sponge tissue were all performed within 24 hours. After culturing 10 ichips in the sponge for 7 days, they were taken out and placed in a sterile WhirlPak TM plastic bag, transported at room temperature to Charlottetown, Prince Edward Island, Canada, and microbiologically cultured.

[0148] Microbiological culturing The ichip was processed for microbial culturing according to the published procedure (Non-Patent Document 20). All ichips were aseptically disassembled, and agar plugs were precipitated from each through-hole into separate wells of a 48-well plate using sterile paper clips (each well containing up to 1 mL of 1 / 10 R2Am medium). The agar plugs were flattened with the tip of a sterile wooden stick and cultured at room temperature (22 °C or lower) in the dark for 8 weeks. After 8 weeks, wells containing morphologically different colonies were subcultured serially with 1 / 10 R2Am and purified. To identify the bacterial isolates, first, a small piece of each single colony was dispersed in 50 μL of DMSO (Sigma). The DMSO cell suspension was used as template DNA in a PCR reaction using the 16S rRNA gene primers pA (5’-AGAGTTTGATCCTGGCTCAG-3’) and pH (5’-AAGGAGGTGATCCAGCC-3’) (Non-Patent Document 21). The PCR reaction contained 1× concentration of EconoTaq PLUS Green 2X master mix (Lucigen), 1 μM of each primer, and 5% DMSO (v / v) containing the suspended cells. The amplicons were directly sequenced using the following primers: 530R (5’-GTATTACCGCGGCTGCTG-3’), 514F (5’-GTGCCAGCASCCGCGG-3’), 936R (5’-GGGGTTATGCCTGAGCAGTTTG-3’), and 1114F (5’-GCAACGAGCGCAACCC-3′) (Non-Patent Documents 22, 23). The sequences were assembled using Geneious (v7.1). The strain Alteromonas sp. RKMC-009 was most closely related to Alteromonas aestuariivivens JDTF-113T (KY497472), with a sequence similarity of 98.15% (Non-Patent Document 24).

[0149] To determine the evolutionary relationships of Alteromonas species RKMC-009 with all validly described Alteromonas species and representative types of all other genera in the family Alteromonadaceae, the following procedures were carried out. By BlastN search of the GenBank non-redundant nucleotide sequence database (excluding sequences from uncultured samples / environmental samples), previously cultured Alteromonas strains closely related to RKMC-009 (16S rRNA gene sequence identity of 99.5% or more) were identified (Non-Patent Document 25). The nucleotide sequences of the strains meeting this criterion were subjected to analysis. Pseudalteromonas haloplanktis (X67024) was used as an outgroup. Phylogenetic analysis was performed using MEGA X (Non-Patent Document 26). Nucleotide sequence alignment was carried out using MUSCLE implemented in MEGA X with default parameters (Non-Patent Document 27). The alignment was manually corrected and trimmed. The analysis included 38 nucleotide sequences and a total of 1365 sites. In the model test of MEGA X, the Kimura 2-parameter model with gamma distribution and invariant sites (K2+G+I) was determined to best represent the dataset (Non-Patent Document 27). Reconstruction of the phylogenetic tree was carried out using the neighbor-joining method, the unweighted pair group method with arithmetic mean, the maximum likelihood method, and the maximum parsimony method (Non-Patent Document 28). Bootstrap analysis using 1000 replicates was used to evaluate the reproducibility of the branches of the tree (Non-Patent Document 29).

[0150] Fermentation and purification of metabolites An inoculum culture of Alteromonas sp. RKMC-009 was placed in a culture tube and cultured overnight at 30 °C with orbital shaking (200 rpm) using 13 mL of Marine Broth (Difco). An inoculum culture (30 mL) was inoculated into each of 10 Fernbach flasks containing 1 L each of BFM4m culture medium (12 g / L ADM Baker’s Soy Flour, 1 g / L NH4Cl, 12 g / L glucose, 0.4 g / L agar, 1 g / L CaCO3, 3 g / L NZ-amine A (Sigma), 18 g / L Instant Ocean, pH = 6.8) and used. The fermentation product was cultured at 30 °C for 3 days with shaking at 200 rpm. The fermentation products were pooled and extracted three times with 1.5 L of ethyl acetate. The pooled ethyl acetate extracts were dried under vacuum to obtain 1.38 g of a crude extract. Compound 1 was found to elute between 32.5 and 35.0 minutes. Compound 1 was further purified for biological evaluation by semi-preparative HPLC using a SunFire C 18 column (10 × 250 mm, 5 μm, Waters) with the following elution method [A = H2O (0.1% formic acid) and B = CH3CN (0.1% formic acid)]: from 0 to 20 minutes with 90% B (flow rate = 3 mL / min, UV = 230, 275 nm).

[0151] N-Palmitoyl-α,O-dimethyl-L-tyrosine (1): [α] 26 D -4.6 (c 0.12, CH3OH); IR (film) ν max 2923, 2852, 1723, 1649, 1613, 1513; as in Table 2 1 H and 13 C NMR; ESI+HRMS m / z 448.3419 [M+H] + (C 27 H 46 NO4 + , 448.3421 was calculated).

[0152]

Chemical Structure

[0153]

Table 2

[0154] Murphy's analysis Murphy's method for determining the amino acid configuration was carried out as follows. 6M HCl (1 mL) was added to separate vials containing 1 (5 mg, 0.01 mmol) dried and O,α-dimethyl-DL-tyrosine (Santa Cruz Biotech, 5 mg, 0.02 mmol), and the mixture was heated under reflux overnight with stirring. The reaction mixture was dried under vacuum, and a portion (1 mg) of these hydrolysates was transferred to separate vials, where 150 μL of deionized H2O, 300 μL of N α -(2,4-dinitro-5-fluorophenyl)-L-alaninamide (L-FDAA; 10 mg / mL in acetone), and 70 μL of aqueous NaHCO3 (1 M) were added. The reaction mixture was heated at 37.0 °C for 2 h, cooled with 70 μL of HCl (1 M), and dried under vacuum. Using α-methyl-L-tyrosine (Sigma-Aldrich; 1 mg, 0.01 mmol), a similar treatment was carried out for L-FDAA-derivatization. The dried L-FDAA derivatization reaction product was suspended in CH3OH (10 mg / mL) and subjected to UHPLC-HRMS analysis. This data was acquired on the following platform. Thermo LTQ Orbitrap Velos mass spectrometer equipped with an ESI source and Thermo Accela UHPLC Pump coupled with Thermo Accela PDA. Kinetex core-shell 100ÅC 18A column (2.1×50 mm, 1.7 μm, Phenomenex) was used with a mobile phase flow rate of 0.5 mL / min and an injection volume of 10 μL (the sample was prepared with CH3OH). The elution method was as follows [A = H2O (0.1% formic acid), B = CH3CN (0.1% formic acid)]: 5% B from 0 min to 2 min, a linear gradient from 5% B at 2 min to 25% B at 55 min, a linear gradient from 25% B at 55 min to 99% B at 57 min, 99% B from 57 min to 60 min, a linear gradient from 99% B at 60 min to 5% B at 63 min, and 5% B from 63 min to 70 min. The HRMS parameters were positive ionization mode, mass resolution of 30,000, mass range m / z 190 - 2,000, spray voltage of 3.4 kV, capillary temperature of 320 °C, S lens RF voltage of 70.0%, maximum injection time of 10 ms, and 1 microscan was used. The system was controlled by the Thermo Xcalibur software module.

[0155] Experiment on artifacts by solvolysis To investigate whether the OCH3-7 of compound 1 is derived from methanolysis, three culture tubes each containing 5 mL of BFM4m were inoculated with an overnight inoculum culture of RKMC-009 (3% v / v) and cultured at 30 °C with orbital shaking (200 rpm). After 3 days, each culture was extracted with 5 mL of ethyl acetate. The organic layer of each culture was dried under vacuum and resuspended in CH3CN (500 μg / mL) for UHPLC-HRMS analysis.

[0156] Chemical synthesis Compounds 1 to 4 were synthesized using an improved literature methodology by N-acylating the corresponding amino acids with palmitoyl chloride (Non-Patent Document 30). Compound 3 was dimethylated with a large excess of CH3I in the presence of Cs2CO3 to produce compound 5 (a synthetic intermediate involved in the preparation of compound 1, i.e., the methyl ester of compound 1, see Scheme 1 above), and then treated with LiOH to obtain compound 1 (Non-Patent Document 31). The NMR spectra of the synthetic compounds 1 to 5 are shown in the figure.

[0157] N-Palmitoyl-O-methyl-L-tyrosine (2) O-Methyl-L-tyrosine (ACROS Organics; 500 mg, 2.56 mmol, 1.00 eq) was stirred at room temperature in 25 mL of DMF with palmitoyl chloride (Alfa Aesar; 7.04 g, 25.61 mmol, 10 eq). After 16 h, the reaction was diluted with 250 mL of 1 N HCl(aq) and extracted with ethyl acetate (3 × 100 mL). The combined ethyl acetate extracts were washed with saturated NaCl(aq) and dried in vacuo. The dried ethyl acetate extract was resuspended in 250 mL of CH3CN and extracted with hexane (5 × 100 mL). The CH3CN layer was dried in vacuo and fractionated by automated flash column chromatography using a 25 g silica-pentafluorophenyl column (Silicycle) with the following elution method (A = H2O and B = CH3OH): 50% B from 0 min to 3 min, linear gradient from 50% B at 3 min to 100% B at 25 min, 100% B from 25 min to 35 min (flow rate = 30 mL / min, UV = 230, 275 nm). Compound 2 (1.03 g, 2.38 mmol, 93%) was obtained as an amorphous white solid: [α] 26 D +17.1 (c 0.46, CH3OH); IR (film) ν max 3296, 2919, 2850, 1730, 1706, 1642, 1614, 1534, 1514 cm -1 ; 1HNMR (CDCl3, 400 MHz) δ 8.29 (1H, s, COOH), 7.07 (2H, d, J = 8.6 Hz, H-5, H-9), 6.83 (2H, d, J = 8.6 Hz, H-6, H-8), 5.97 (1H, d, J = 7.4 Hz, CONH), 4.82 (1H, dt, J = 7.4, 5.9 Hz, H-2), 3.78 (3H, s, OCH3), 3.17 (1H, dd, J = 14.2, 5.8 Hz, H-3b), 3.07 (1H, dd, J = 14.2, 5.8 Hz, H-3a), 2.18 (2H, td, J = 7.7, 1.7 Hz, H-2’), 1.56 (2H, m, H-3’), 1.21 - 1.33 (24H, m, H-4’, H-5’, H-6’, H-7’, H-8’, H-9’, H-10’, H-11’, H-12’, H-13’, H-14’, H-15’), 0.88 (3H, t, J = 7.0 Hz, H-16’); 13 CNMR (CDCl3, 151 MHz) δ 175.09 (C, C-1), 174.1 (C, C-1’), 158.9 (C, C-7), 130.5 (CH, C-5, C-9), 127.7 (C, C-4), 114.2 (CH, C-6, C-8), 55.3 (CH3, OCH3), 53.5 (CH, C-2), 36.6 (CH2, C-2’), 36.5 (CH2, C-3), 32.1 (CH2, C-14’), 29.8 (CH2), 29.8 (CH2), 29.8 (CH2), 29.8 (CH2), 29.8 (CH2), 29.8 (CH2), 29.6 (CH2), 29.5 (CH2), 29.5 (CH2), 29.3 (CH2, C-4’), 25.7 (CH2, C-3’), 22.8 (CH2, C-15’), 14.3 (CH3, C-16’); ESI+HRMS m / z 434.3262 [M + H] + (C 26 H 44 NO4 + (The calculated value was 434.3265).

[0158] N-Palmitoyl-α-methyl-L-tyrosine (3) α-Methyl-L-tyrosine (Sigma-Aldrich; 200 mg, 1.02 mmol, 1.00 eq) was stirred at room temperature in 10 mL of DMF with palmitoyl chloride (Alfa Aesar; 2.82 g, 10.26 mmol, 10 eq). After 16 h, the reaction was diluted with 100 mL of 1 N HCl(aq) and extracted with ethyl acetate (3 × 100 mL). The combined ethyl acetate extracts were washed with saturated NaCl(aq) and dried in vacuo. The dried ethyl acetate extract was suspended in 250 mL of CH3CN and extracted with hexane (5 × 100 mL). The CH3CN layer was dried in vacuo and fractionated using automated flash column chromatography (same conditions as for compound 2) to give compound 3 (394 mg, 0.91 mmol, 89%) as an amorphous white solid: [α] 26 D -18.1 (c 0.20, CH3OH); IR (film) ν max 3326, 2923, 2853, 2156, 1716, 1646, 1615, 1516 cm -1 ; 1 1H NMR (CDCl3, 400 MHz) δ 6.95 (2H, d, J = 8.6 Hz, H-5, H-9), 6.72 (2H, d, J = 8.6 Hz, H-6, H-8), 6.10 (1H, s, CONH), 3.38 (1H, d, J = 13.7 Hz, H-3b), 3.17 (1H, d, J = 13.7 Hz, H-3a), 2.17 (2H, t, 7.9 Hz, C-2’), 1.63 (3H, s, CH3-2), 1.58 (2H, m, H-3’), 1.21 - 1.32 (24H, m, H-4’, H-5’, H-6’, H-7’, H-8’, H-9’, H-10’, H-11’, H-12’, H-13’, H-14’, H-15’), 0.88 (3H, t, J = 6.7 Hz, H-16’); 13CNMR (CDCl3, 151 MHz) δ 177.7 (C, C-1), 174.5 (C, C-1’), 155.1 (C, C-7), 131.3 (CH, C-5, C-9), 127.6 (C, C-4), 115.5 (CH, C-6, C-8), 61.5 (C, C-2), 40.6 (CH2, C-3), 37.3 (CH2, C-2’), 32.1 (CH2, C-14’), 29.9 (CH2), 29.9 (CH2), 29.9 (CH2), 29.8 (CH2), 29.8 (CH2), 29.8 (CH2), 29.7 (CH2), 29.5 (CH2), 29.5 (CH2), 29.4 (CH2, C-4’), 25.7 (CH2, C-3’), 23.3 (CH2, CH3-2), 22.8 (CH2, C-15’), 14.3 (CH3, C-16’); ESI+HRMS m / z 434.3260 [M+H] + (C 26 H 44 NO4 + (calculated 434.3265).

[0159] N-Palmitoyl-L-tyrosine (4) L-Tyrosine (AMRESCO; 500 mg, 2.76 mmol, 1.00 eq) was stirred at room temperature in 25 mL of DMF using palmitoyl chloride (Alfa Aesar; 7.59 g, 27.60 mmol, 10 eq). After 16 h, the reaction was diluted with 250 mL of 1 N HCl(aq) and extracted with ethyl acetate (3 × 100 mL). The combined ethyl acetate extracts were washed with saturated NaCl(aq) and dried in vacuo. The dried ethyl acetate extract was suspended in 250 mL of CH3CN and extracted with hexane (5 × 100 mL). The CH3CN layer was dried in vacuo and fractionated using automated flash column chromatography (same conditions as for compound 2) to afford compound 4 (1.09 g, 2.59 mmol, 94%) as an amorphous white solid: [α] 26 D +17.4 (c 0.26, CH3OH); IR (film) ν max 3312, 3234, 2915, 2848, 2516, 1705, 1643, 1541, 1516 cm -1 ; 11H NMR (CDCl3, 400 MHz) δ 12.54 (1H, s, COOH), 9.16 (1H, s, OH-7), 7.99 (1H, d, J = 8.2 Hz, CONH), 6.99 (2H, d, J = 8.5 Hz, H-5, H-9), 6.63 (2H, d, J = 8.5 Hz, H-6, H-8), 4.32 (1H, m, H-2), 2.90 (1H, dd, J = 14.1, 4.8 Hz, H-3b), 2.71 (1H, dd, J = 14.1, 9.6 Hz, H-3a), 2.03 (2H, t, J = 7.4 Hz, H-2’), 1.39 (2H, m, H-3’), 1.14 - 1.24 (24H, m, H-4’, H-5’, H-6’, H-7’, H-8’, H-9’, H-10’, H-11’, H-12’, H-13’, H-14’, H-15’), 0.85 (3H, t, J = 7.0 Hz, H-16’); 13 13C NMR (CDCl3, 151 MHz) δ 173.4 (C, C-1), 172.1 (C, C-1’), 155.9 (C, C-7), 127.7 (C, C-4), 130.0 (CH, C-5, C-9), 114.9 (CH, C-6, C-8), 53.6 (CH, C-2), 36.0 (CH2, C-2’), 35.1 (CH2, C-3), 31.3 (CH2, C-14’), 29.1 (CH2), 29.1 (CH2), 29.1 (CH2), 29.1 (CH2), 29.1 (CH2), 29.0 (CH2), 28.9 (CH2), 28.8 (CH2), 28.7 (CH2), 28.5 (CH2, C-4’), 25.2 (CH2, C-3’), 22.1 (CH2, C-15’), 14.0 (CH3, C-16’); ESI+HRMS m / z 420.3091 [M + H] + (C 25 H 42 NO4 + (calculated 420.3108).

[0160] N-Palmitoyl-α, O-dimethyl-L-tyrosine methyl ester (5) To a solution of compound 3 (75 mg, 0.17 mmol, 1.00 eq) in DMSO (5 mL), Cs2CO3 (Sigma-Aldrich; 118 mg, 0.36 mmol, 2.10 eq) and CH3I (Sigma-Aldrich; 1.06 mL, 17.30 mmol, 100.00 eq) were added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with 10 mL of H2O and extracted with CHCl3 (3 × 50 mL). The combined CHCl3 extracts were washed with saturated NaCl(aq) and dried in vacuo. The dried CHCl3 extract was fractionated by automated flash column chromatography (same conditions as for compound 2) to afford compound 5 (21 mg, 0.05 mmol, 27%): 1 1H NMR (CDCl3, 400 MHz) δ 6.95 (2H, d, J = 8.7 Hz), 6.79 (2H, d, J = 8.7 Hz), 6.01 (1H, s), 3.77 (3H, s), 3.77 (3H, s), 3.49 (1H, d, J = 13.5 Hz), 3.13 (1H, d, J = 13.5 Hz), 2.13 (2H, t, J = 7.09 Hz), 16.4 (3H, s), 1.60 (1H, m), 1.23 - 1.31 (24H, m), 0.89 (3H, t, J = 7.0 Hz); 13 13C NMR (CDCl3, 151 MHz) δ 174.8, 172.6, 158.7, 131.0, 128.6, 113.8, 61.3, 55.3, 52.7, 40.6, 37.4, 32.1, 29.8, 29.8, 29.8, 29.8, 29.8, 29.8, 29.7, 29.5, 29.5, 29.4, 25.7, 23.4, 22.8, 14.3. ESI+HRMS m / z 462.3582 [M + H]+, 462.3578 was calculated). + (C 28 H 48 NO4 + ),

[0161] N-Palmitoyl-α,O-dimethyl-L-tyrosine (1) A solution of 5 (10 mg, 0.02 mmol, 1.00 eq) in 75% THF(aq) (1 mL) was added with LiOH(aq) (Sigma - Aldrich; 100 μL of 10 mg / mL solution, 0.04 mmol, 2.00 eq), and stirred at room temperature for 48 h. The reaction mixture was diluted with 10 mL of H2O and extracted with CHCl3 (3 × 50 mL). The combined CHCl3 extracts were washed with saturated NaCl(aq) and dried in vacuo. The dried CHCl3 extract was fractionated using automated flash column chromatography (same conditions as for compound 2) to give compound 1 (9 mg, 0.02 mmol, 95%). [α] 26 D -5.1 (c 0.15, CH3OH); IR (film) ν max 2923, 2852, 1723, 1649, 1613, 1513; 1 H and 13 13C NMR is shown in Figures 12 - 13; ESI+HRMS m / z 448.3425 [M + H] + (C 27 H 46 NO4 + , 448.3421 was calculated).

[0162] Evaluation of antibacterial activity and cytotoxicity Five species of Enterococcus bacteria were isolated from clinical specimens at the Atlantic Veterinary College (AVC) by the AVC Diagnostic Services Bacteriology Laboratory. The isolates were identified using a Bruker microflex LT MALDI-TOF equipped with MBT Compass version 4.179. A direct colony transfer method using a Bruker Matrix HCCA (α-cyano-4-hydroxycinnamic acid) overlay was used according to the manufacturer's guidelines below. Isolates with score values from 2.00 to 3.00 were considered highly reliable identifications. The following isolates (including the source species) were identified: Enterococcus faecium 15337 (cat), Enterococcus faecalis 16371 (dog), Enterococcus gallinarum 20993 (erinaceine), Enterococcus casseliflavus 15984 (horse), and Enterococcus hirae 17446 (avine). The antibacterial activities of Compounds 1 to 4 were evaluated against all five clinical Enterococcus isolates, in addition to Methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA), Staphylococcus warneri ATCC 17917, Vancomycin-resistant Enterococcus faecium EF379 (VRE), Pseudomonas aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and Candida albicans ATCC 14035. All tests were performed three times in a microtiter liquid dilution assay in 96-well plates, as previously described (Non-Patent Document 32), according to the test criteria of the Clinical Laboratory Standards Institute. Optical density was measured at 600 nm using a Thermo Scientific Varioskan Flash plate reader, recorded at zero time, and measured again after incubation at 37 °C for 22 hours to determine the growth inhibition rate. Cytotoxicity was evaluated for the Vero kidney cell line derived from African green monkeys, MCF7 human breast cancer cells (ATCC HTB-22), human breast cancer cells (ATCC HTB-26), and HCT-116 human colorectal cancer cells (ATCC CCL-247). All evaluations were performed as previously described (Non-Patent Document 32).Fluorescence was measured using a Thermo Scientific Varioskan Flash plate reader at 560 / 12 excitation and 590 nm emission, at zero time and 4 hours after addition of alamarBlue (Invitrogen). For both antibacterial and cytotoxic data, growth inhibition was expressed as a percentage and plotted against the logarithm of the concentration. A four-parameter dose-response curve was fitted to these data using the variable slope model of GraphPad Prism 8.0.2.

[0163] One or more exemplary embodiments have been described as examples. It will be understood by those skilled in the art that numerous variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

1. A compound of formula A or a pharmaceutically acceptable salt or solvate thereof. 【Chemical 1】 Here, R 1 is -H; or a linear or branched C 1 -C 6 alkyl group, R 2 is -H; or a linear or branched C 1 -C 6 alkyl group, R 3 is a linear or branched C 5 -C 20 alkyl group, R 4 is -H; or a linear or branched C 1 -C 6 alkyl group, R 1 = R 2 = -Me, and except when R 1 = R 2 = -H.

2. R 1 is -H, Me, Et, nPr, iPr, tBu, iBu, secBu, or nBu, The compound according to claim 1.

3. R 2 is -H, Me, Et, nPr, iPr, tBu, iBu, secBu, or nBu, The compound according to claim 1 or 2.

4. R 3 is a linear C 5 -C 20 group, The compound according to any one of claims 1 to 3.

5. R 4 is -H, Me, or Et, The compound according to any one of claims 1 to 4.

6. Having formula B, The compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof. 【Chemical 2】 Here, R is -CH 3 or -H; R' is -CH 3 or -H.

7. Having formula 2, The compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or solvate thereof. 【Chemical Formula 3】

8. Having formula 3, The compound according to any one of claims 1 to 6 or a pharmaceutically acceptable salt or solvate thereof. 【Chemical Formula 4】

9. Comprising the compound according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient or carrier, Pharmaceutical composition.

10. The compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, wherein the compound or the pharmaceutical composition is used as an antibacterial agent, compound or pharmaceutical composition.

11. The compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, wherein the compound or the pharmaceutical composition is used for treating a bacterial infectious disease, compound or pharmaceutical composition.

12. The compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, which is for reducing or preventing the growth of bacteria, compound or pharmaceutical composition.

13. A compound of formula A or a pharmaceutically acceptable salt or solvate thereof, 【Chemical Formula 5】 Here, R 1 is -H; or a linear or branched C 1 -C 6 alkyl group, R 2 is -H; or a linear or branched C 1 -C 6 alkyl group, R 3 is a linear or branched C 5 -C 20 alkyl group, R 4 is -H; or a linear or branched C 1 -C 6 alkyl group, R 1 = R 2 = -Me, and except when R 1 = R 2 = -H Showing inhibitory activity against Staphylococcus aureus, Staphylococcus warneri, or Enterococcus faecium, Compound or a pharmaceutically acceptable salt or solvate thereof.

14. A compound of formula A or a pharmaceutically acceptable salt or solvate thereof, 【Chemical Formula 6】 Here, R 1 is -H; or a linear or branched C 1 -C 6 alkyl group, R 2 is -H; or a linear or branched C 1 -C 6 alkyl group, R 3 is a linear or branched C 5 -C 20 alkyl group, R 4 is -H; or a linear or branched C 1 -C 6 alkyl group, R 1 = R 2 = -Me, and, except when R 1 = R 2 = -H Showing inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA), or vancomycin-resistant Enterococcus faecium (VRE), Compound or a pharmaceutically acceptable salt or solvate thereof.

15. A compound of formula A or a pharmaceutically acceptable salt or solvate thereof, 【Chemical Formula 7】 Here, R 1 is -H; or a linear or branched C 1 -C 6 alkyl group, R 2 is -H; or a linear or branched C 1 -C 6 alkyl group, R 3 is a linear or branched C 5 -C 20 alkyl group, R 4 is -H; or a linear or branched C 1 -C 6 alkyl group, R 1 = R 2 = -Me, and except when R 1 = R 2 = -H A compound, or a pharmaceutically acceptable salt or solvate thereof, which exhibits inhibitory activity against Enterococcus faecium EF379 (VRE), Enterococcus faecium 15337, Enterococcus faecalis 16371, Enterococcus gallinarum 20993, Enterococcus casseliflavus 15984, Enterococcus hirae 17446, Staphylococcus aureus ATCC 33591 (MRSA), or Staphylococcus warneri ATCC 17917. Compound or a pharmaceutically acceptable salt or solvate thereof.

16. A compound according to any one of claims 13 to 15, which exhibits inhibitory activity against enterococci, wherein the compound comprises formula 3 or a pharmaceutically acceptable salt or solvate thereof, Compound. 【Chemical 8】

17. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and 8, or the compound and a pharmaceutically acceptable excipient or carrier, which exhibits inhibitory activity against enterococci, wherein the compound or the pharmaceutical composition comprises formula 3 or a pharmaceutically acceptable salt or solvate thereof, Compound or pharmaceutical composition. 【Chemical Formula 9】

18. A compound according to any one of claims 13 to 16, which exhibits inhibitory activity against Staphylococcus aureus, wherein the compound comprises formula 3 or a pharmaceutically acceptable salt or solvate thereof, Compound. 【Chemical Formula 10】

19. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6, 8, and 13 to 18, or the compound and a pharmaceutically acceptable excipient or carrier, which exhibits inhibitory activity against enterococci or staphylococci, The compound or the pharmaceutical composition contains an α-methyl substituent (i.e., R 1 = -CH 3 ). Compound or pharmaceutical composition.

20. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and 13 to 15, or the compound and a pharmaceutically acceptable excipient or carrier, which exhibits inhibitory activity against enterococci, The compound or the pharmaceutical composition contains O-methylation in the tyrosine side chain (i.e., R 2 = -CH 3 ). Compound or pharmaceutical composition.

21. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6, 8, and 13 to 19, or the compound and a pharmaceutically acceptable excipient or carrier, which exhibits inhibitory activity against staphylococci, The compound or the pharmaceutical composition contains a tyrosine side chain (i.e., R 2 = -H). Compound or pharmaceutical composition.

22. A method for producing a compound of formula 1, The method comprises fermenting Alteromonas sp. RKMC-009 in BFM4m culture medium and extracting the culture medium with ethyl acetate (EtOAc). A method comprising the above. 【Chemical 11】

23. The method according to claim 22, further comprising purifying the compound of formula 1 by reverse phase chromatography. The method according to claim 22.

24. The Alteromonas sp. RKMC-009 is Alteromonas RKMC-009 deposited under NRRL accession number NRRL B-67979. The method according to claim 22 or 23.

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