Compound with anti-dysentery amoeba activity

Abstract

The invention provides a compound with anti-dysentery amebic activity. The present invention relates to a compound represented by general formula (1), a salt thereof, or a solvate thereof.

Description

Technical Field

[0001] This invention relates to a compound with anti-dysentery amoebae activity, etc. Background Technology

[0002] Amoebic dysentery is an intestinal protozoan infection affecting approximately 1% of the global population, posing a significant threat, particularly to developing countries in tropical regions. Current drug metronidazole has side effects such as genotoxicity and drug resistance, necessitating the development of safer drugs with different mechanisms of action.

[0003] Against this backdrop, the natural substance fumagillin has exhibited strong anti-dysentery amoebae activity, and its therapeutic effects on animal models of liver abscess have been reported. On the other hand, fumagillin derivatives have been under clinical development for cancer, obesity, malaria, and other diseases for more than 30 years, but due to side effects such as neurotoxicity, they have not yet been commercialized.

[0004] Existing technical documents Patent documents Non-patent literature 1: J. Am. Chem. Soc. 2014, 136, 4426-4436. Summary of the Invention

[0005] The technical problem that the invention aims to solve The technical problem to be solved by the present invention is to provide a compound with anti-dysentery amoebae activity.

[0006] Technical solutions for solving technical problems The inventors of this invention focused on Ovalicin, which has a structure similar to fumonisin. The derivatization of fumonisin can be achieved by converting the sesquiterpene structure at the 5-position into other structures. The inventors of this invention focused on the methoxy group at the 6-position of ovomycin and attempted to convert it into other structures. Specifically, if a compound that demethylates the methoxy group of ovomycin and converts it into a hydroxyl group (demethylovomycin) can be obtained, then various ovomycin derivatives with different structures can be obtained using this hydroxyl group (e.g., by reacting with a carboxyl compound). However, ovomycin contains two easily decomposed epoxy groups, making it difficult to efficiently obtain demethylovomycin through organic synthesis.

[0007] Therefore, the inventors of this invention focus on obtaining demethylosmotic oocystin through biosynthesis ( Figure 1However, the enzyme X (7-hydroxylase Ova-C7H) essential for this biosynthesis, which has the conversion activity from 5-keto-demethoxyfumagillol to demethoxyfumagillin, is not yet known.

[0008] The inventors of this invention conducted meticulous research, and as a result, successfully solved the biosynthesis of demethylopseudomonas aeruginosa and discovered that demethylopseudomonas aeruginosa and its derivatives possess anti-dysentery amoeboid activity. Based on this insight, the inventors of this invention conducted further research, resulting in the completion of this invention. That is, this invention includes the following embodiments.

[0009] Item 1. A compound of general formula (1A2), its salt, or its solvates, wherein, [In the formula: L] 1 Indicates a single bond or linker. R 11 This indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. R 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X. X represents a halogen atom.

[0010] Item 2. The compound, its salt, or its solvate as described in Item 1, wherein R 2 and R 3 They connect with each other to form -(CH2)-.

[0011] Item 3. The compound, its salt, or its solvate as described in Item 1, wherein L 1 It consists of single bonds or connecting groups with 1 to 4 atoms forming the main chain.

[0012] Item 4. The compound, its salt, or its solvates as described in Item 1, wherein the alkyl group, the aryl group, and the heteroaryl group may have substituents that are halogen atoms, hydroxyl groups, or alkoxy groups, or alkyl groups, alkoxy groups, arylalkyl groups, or arylalkoxy groups that may be substituted by halogen atoms, hydroxyl groups, or alkoxy groups.

[0013] Item 5. The compound, its salt, or its solvate as described in Item 1, wherein the compound is a compound represented by general formula (1A3). [In the formula: L] 1 and R 11 Same as above.

[0014] Item 6. The compound, its salt, or its solvate as described in Item 5, wherein L 1 It is a single bond, and R 11 It is an alkyl group having 1 to 4 carbon atoms.

[0015] Item 7. An anti-dysentery amoeboid agent comprising at least one selected from compounds represented by general formula (1), their salts, and their solvates. [In the formula: R] 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11 L 1 Indicates a single bond or linker. R 11 This indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. R 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X. X represents a halogen atom.

[0016] Item 8. The anti-dysentery amoeba agent as described in Item 7, which is a preventive or therapeutic agent for amoebic dysentery.

[0017] Item 9. An anticancer agent comprising at least one selected from compounds represented by general formula (1), their salts, and their solvates. [In the formula: R] 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11 L 1 Indicates a single bond or linker. R 11 This indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. R 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X. X represents a halogen atom.

[0018] Item 10. An anti-obesity agent comprising at least one selected from compounds represented by general formula (1), their salts, and their solvates. [In the formula: R] 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11 L 1Indicates a single bond or linker. R 11 This indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. R 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X. X represents a halogen atom.

[0019] Invention Effects This invention provides a compound with anti-dysentery amoebae activity. This compound is also effective against cancer and obesity. Attached Figure Description

[0020] Figure 1 This indicates the biosynthetic pathways of demethylated ovomycin and ovomycin.

[0021] Figure 2 This represents the NMR data for demethylosmotic oocystin.

[0022] Figure 3 This indicates the structures of compounds 1–12 and the results of the Entamoeba histolytica test (Example 4) (ED). 50 ).

[0023] Figure 4 The results of the *Entamoeba histolytica* test (Example 5) using deMeOV as the test compound are shown. The dotted curve represents the result of the *Entamoeba histolytica* test using the test compound before microsomal treatment, and the square curve represents the result of the *Entamoeba histolytica* test using the test compound after microsomal treatment. The vertical axis represents the percentage of viable cells, and the horizontal axis represents the concentration of the test compound before microsomal treatment. deMeOV shows sensitivity to hepatic metabolism. However, its therapeutic effect in animals infected with *Entamoeba histolytica* is limited.

[0024] Figure 5 This figure represents the results of the Entamoeba histolytica test (Example 5) using compound 1 as the test compound. The explanation of this figure is consistent with... Figure 4 Same. Compound 1 exhibits resistance to hepatic drug metabolism.

[0025] Figure 6 This indicates the therapeutic effect of subcutaneous administration of compound 1 to an amebic liver abscess model animal. The vertical axis represents the ratio of liver abscess tissue weight to total liver weight, and the values ​​on the horizontal axis represent the dosage. "Not treated" indicates the case where compound 1 was not administered.

[0026] Figure 7This figure shows the therapeutic effect of compound 1 administered orally to an animal model of amoebic liver abscess. The explanation of this figure is consistent with... Figure 6 same.

[0027] Figure 8 The graph shows the effect of adding compound 1 or compound 7 on the proliferation of human umbilical vein endothelial cells (HUVECs). The vertical axis represents the EdU measurement value. The horizontal axis represents the concentration of the tested compound. Compounds 1 and 7 not only showed inhibitory effects on the proliferation of Entamoeba histolytica, but also on the proliferation of human cells.

[0028] Figure 9 This graph shows the therapeutic effects of subcutaneous or oral administration of compound 1 or compound 7 in animals with an amebic liver abscess model. The vertical axis represents the ratio of liver abscess tissue weight to total liver weight, and the horizontal axis represents the administered compound. "Untreated" indicates the case where neither compound 1 nor compound 7 was administered, and MTZ indicates the case where the existing drug metronidazole was administered. The dosage is indicated above the graph. Detailed Implementation

[0029] 1. Definition In this specification, the expressions “containing” and “comprising” include concepts such as “containing”, “comprising”, “substantially constituted by” and “consisting solely of”.

[0030] In this specification, the “identity” of an amino acid sequence refers to the degree of similarity between two or more comparable amino acid sequences relative to each other. Therefore, the higher the similarity between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using FASTA, a tool for sequence analysis, with default parameters. Alternatively, it can be determined using the BLAST algorithm of Karlin and Altschul (Karlin S, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990), Karlin S, Altschul SF. “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX based on this BLAST algorithm has been developed. The specific steps of these analytical methods are publicly known and can be found at the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the "identity" of the base sequence is defined as described above.

[0031] In this specification, "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues with basic side chains, such as lysine, arginine, and histidine, is equivalent to conservative substitution. Furthermore, the following substitutions are also equivalent to conservative substitution: substitution between amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; substitution between amino acid residues with non-electrolyte polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitution between amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitution between amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and substitution between amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0032] In this specification, known chemical modifications can be applied to polynucleotides such as DNA and RNA as illustrated below. To prevent degradation by hydrolases such as nucleases, the phosphate residues (phosphate esters) of each nucleotide can be replaced with chemically modified phosphate residues such as thiophosphate (PS), methyl phosphonate, or dithiophosphate. Additionally, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R, for example, represents CH3(2′-O-Me), CH2CH2OCH3(2′-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Chemical modifications can also be applied to the base moiety (pyrimidine, purine), for example, by introducing a methyl or cationic functional group at the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl group. Modifications to the phosphate or hydroxyl moiety, for example, by biotin, amino, lower alkylamine, or acetyl groups, are also possible, but are not limited to these examples. Alternatively, BNA (LNA) or similar methods can be preferred, which fix the conformation of the sugar portion in the N-type by crosslinking the 2′ oxygen and 4′ carbon of the sugar portion of the nucleotide.

[0033] 2. Compounds In one embodiment, the present invention relates to compounds of general formula (1) (which are sometimes referred to as "the compounds of the present invention" in this specification), their salts, or their solvates (which are sometimes collectively referred to as "the active ingredients of the present invention" in this specification). These substances are described below. R 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11From the perspective of maintaining activity more stably in organisms, R 1 Preferably -C(=O)-L 1 -R 11 .

[0034] L 1 Indicates a single bond or linker.

[0035] L 1 The connecting group shown contains a chain structure. There are no particular limitations on the chain structure; the number of atoms constituting the main chain is, for example, 1 to 6. From the viewpoint of anti-entamoebic activity, chain structures with 1 to 4 atoms are preferred, more preferably 1 to 3, and even more preferably 1 to 2. Examples of main chain constituent atoms include carbon atoms, oxygen atoms, sulfur atoms, and nitrogen atoms. Part or all of the chain structure can be alkenyl, alkylene, heteroalkylene, or heteroalkenyl. The chain structure can also be any type of straight-chain or branched chain; from the viewpoint of anti-entamoebic activity, a straight-chain structure is preferred. Furthermore, the chain structure may include partial structures such as -O-, -C(=O)-O-, -CO-NH-, -C(=O)-, -NH-, and -S(=O)2- in the main chain.

[0036] From the perspective of anti-dysentery amoebae activity, L 1 Preferably, it is -CO-NH- or alkenyl (preferably 2 to 4 carbon atoms, more preferably 2 to 3, and even more preferably 2), and more preferably -CO-NH-.

[0037] From the perspective of anti-dysentery amoebae activity and maintaining activity more stably in vivo, L 1 Single bonds are preferred.

[0038] R 11 This indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group.

[0039] R 11 The alkyl group shown can be any of the following: straight-chain, branched, or cyclic. The number of carbon atoms in the alkyl group (in the case of straight-chain or branched form) is not particularly limited, and is, for example, 1 to 8. The number of carbon atoms is preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2, and particularly preferably 1. In another embodiment, the number of carbon atoms is particularly preferably 2. The number of carbon atoms in the alkyl group (in the case of cyclic form) is not particularly limited, and is, for example, 3 to 7, preferably 4 to 6. Specific examples of this alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, 3-methylpentyl, n-heptyl, n-octyl, etc.

[0040] R 11The aryl group shown is not particularly limited, but aryl groups with 6 to 12 carbon atoms are preferred, aryl groups with 6 to 10 carbon atoms are more preferred, and aryl groups with 6 to 8 carbon atoms are even more preferred. The aryl group can be any of a monocyclic or polycyclic form (e.g., a 2-cyclic, 3-cyclic, etc.), with a monocyclic form being preferred. Specific examples of this aryl group include phenyl, naphthyl, biphenyl, cyclopentadienyl, indene, anthracene, tetraphenyl, pentaphenyl, pyrene, peryl, fluorenyl, phenanthrene, etc., with phenyl being particularly preferred.

[0041] R 11 The heteroaryl group shown is not particularly limited, and examples of heteroaryl groups containing sulfur atoms, nitrogen atoms, oxygen atoms, etc., as heteroatoms can be listed. The number of ring-forming atoms in the heteroaryl group is preferably 6 to 12, more preferably 6 to 10, and even more preferably 6 to 8. The aryl group can be any of a monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.) form, and is preferably a monocyclic form. Examples of heteroaryl groups include pyrroleyl, pyridyl, pyrrolidinyl, piperidinyl, imidazolyl, imidazoyl, pyrazinyl, pyrimidinyl, pyridazinyl, piperazinyl, triazinyl, oxazolyl, isoxazolyl, morpholinyl, thiazolyl, isothiazolyl, furanyl, thiophenyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, quinazolinyl, phthalazinyl, purine, pteridinyl, benzofuranyl, coumarinyl, crononeyl, and benzothiopheneyl. The heteroaryl group is preferably a nitrogen-containing heteroaryl group, and particularly preferably pyridylyl.

[0042] There are no particular restrictions on the substituents that the above-mentioned alkyl, aryl and heteroaryl groups may have. From the viewpoint of anti-dysentery amoeboid activity, halogen atoms, hydroxyl or alkoxy groups are particularly preferred, or alkyl, alkoxy, arylalkyl or arylalkoxy groups that can be substituted by halogen atoms, hydroxyl or alkoxy groups.

[0043] Halogen atoms that can be used as substituents include, for example, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, etc., with fluorine atoms and chlorine atoms being preferred.

[0044] Alkoxy groups that are substituents include, for example, linear or branched alkoxy groups with 1 to 8 (preferably 1 to 4) carbon atoms.

[0045] Alkyl groups as substituents and R 11 The alkyl groups shown are the same.

[0046] Examples of arylalkyl groups that can be used as substituents include alkylaryl groups formed by replacing the hydrogen atoms (e.g., 1 to 3, preferably 1 hydrogen atom) of the alkyl group with the aryl group. Specific examples of such arylalkyl groups include benzyl and phenethyl.

[0047] Arylalkoxy groups that are substituents can be exemplified by arylalkoxy groups formed by replacing the hydrogen atoms (e.g., 1 to 3, preferably 1 hydrogen atom) of the alkoxy group with the aryl group.

[0048] R 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X.

[0049] In R 2 and R 3 When they are connected to form -(CH2)-, the -(CH2)- and R 2 Adjacent oxygen atoms, and R 3 The adjacent carbon atoms form an ethylene oxide structure (see general formula (1A1) described below).

[0050] X represents a halogen atom. Examples of halogen atoms represented by X include fluorine, chlorine, bromine, and iodine atoms, with fluorine and chlorine atoms being preferred.

[0051] From the perspective of anti-dysentery amoebae activity, R 2 and R 3 They are particularly preferred to be connected together to form -(CH2)-.

[0052] From the viewpoint of anti-dysentery amoebae activity, the compounds of the present invention are preferably those represented by general formula (1A1) or (1A2), and particularly preferably those represented by general formula (1A3). [In the formula: R] 1 L 1 R 11 R 2 and R 3 Same as above. [In the formula: L] 1 and R 11 Same as above. The compounds of the present invention are preferably those represented by the general formula (1AX) in one embodiment. [In the formula: R] 1 R 2 and R 3 Same as above. The salt of the compounds of the present invention can be any pharmaceutically acceptable salt, without particular limitation. Any type of acidic or basic salt can be used as the salt. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; and organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate. Examples of basic salts include alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with ammonia; and salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0053] The compounds of this invention can also be hydrates or solvates. Examples of pharmaceutically acceptable organic solvents include ethanol, glycerol, acetic acid, etc.

[0054] 3. Preparation of Compounds 1 (R) 1 (The case of hydrogen atoms) The compound of the present invention contains R 1 The hydrogen-containing compound (demethylovirginiacin) can be obtained through biosynthesis. Specifically, it can be biosynthesized using mutant cells (sometimes referred to in this specification as "mutant cells of the present invention") containing a polynucleotide A that contains the coding sequence of protein A, which contains an amino acid sequence (A) having more than 70% identity with the amino acid sequence shown in Serial No. 1 and having conversion activity from 5-keto-demethoxyfumonisin to demethoxyovirginiacin. This will be described below.

[0055] Sequence number 1 is the amino acid sequence of a protein from the insect-infecting filamentous fungus Metarhizium robertsii strain ARSEF23, with NCBI accession number XP007824537.1.

[0056] The identity of (A) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0057] When the identity in (A) above is less than 100%, the number of amino acids with mutations (substitution, deletion, addition, or insertion) relative to the amino acid sequence of sequence number 1 is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably substitution, and more preferably conservative substitution.

[0058] The structures of 5-keto-demethoxyfumonisin and demethoxypseudomycin are as follows: Figure 1 As shown.

[0059] The presence or absence of conversion activity from 5-keto-demethoxyfuscin to demethoxypseudomycin can be confirmed by in vitro or in vivo assays. For in vivo assays, for example, using cells, cells that produce 5-keto-demethoxyfuscin are used, or 5-keto-demethoxyfuscin is introduced into cells, and the cells are cultured to express / not express the test protein. Based on the results, if demethoxypseudomycin is produced only when the test protein is expressed, then the test protein can be determined to have conversion activity from 5-keto-demethoxyfuscin to demethoxypseudomycin.

[0060] In addition to the amino acid sequence mentioned above, protein A may also contain other sequences such as known protein tags, signal sequences, or peptides. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0061] In one configuration, polynucleotide A contains a promoter upstream of its coding sequence for expressing the mRNA of protein A. There are no particular restrictions on the promoter; it can be appropriately selected based on the host. Examples of promoters include the Aspergillus TAA gene promoter (amyB), the Aspergillus GLA gene promoter (glaA), and the Aspergillus AGL gene promoter (agdA). Other examples include the TDH3 promoter, GAL10 promoter, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, and CAG promoter.

[0062] Polynucleotide A may also contain other elements (such as multiple cloning sites (MCS), antibiotic resistance genes, origin of replication, etc.) as needed. For example, when the polynucleotide is arranged in the order of promoter and protein-coding sequence starting from the 5′ side, examples can be listed where the MCS is arranged between the promoter and coding sequence (preferably adjacent to either or both) and on the 3′ side of the coding sequence (preferably adjacent). The MCS only needs to contain multiple (e.g., 2 to 50, preferably 2 to 20, more preferably 2 to 10) restriction enzyme sites, and there are no particular restrictions.

[0063] Polynucleotide A can also be used to construct vectors. There are no particular restrictions on the type of vector, and it can be selected appropriately according to the host. Examples include ColE1 line plasmids represented by pBR322 derivatives in E. coli, pACYC line plasmids with p15A origin, pSC line plasmids, and mini-F plasmids from the F factor such as the Bac line.

[0064] The mutant cells of the present invention containing polynucleotide A can be used to prepare cells for the manufacture of demethylopseudomonas aeruginosa, and in a preferred embodiment, can be used as cells for the manufacture of demethylopseudomonas aeruginosa.

[0065] The mutant cells of the present invention preferably further comprise polynucleotide B, which contains (B) a coding sequence for protein B having conversion activity from demethoxypseudomycin to demethylpseudomycin. Thus, demethylpseudomycin can be produced under conditions of supplying 5-keto-demethoxyaspergillus alcohol.

[0066] Protein B, which has the transforming activity from demethoxypseudomycin to demethylpseudomycin, is known or can be readily identified or produced by homology analysis based on known amino acid sequences.

[0067] Protein B is preferably a protein B1 containing an amino acid sequence (B1) that has more than 70% identity with the amino acid sequence shown in sequence number 2.

[0068] Sequence number 2 is the amino acid sequence of a protein from Aspergillus fumigatus strain A1159, with NCBI accession number XP747157.2.

[0069] The identity of (B1) is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0070] When the identity is less than 100% in (B1) above, the number of amino acids with mutations (substitution, deletion, addition, or insertion) relative to the amino acid sequence of sequence number 2 is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably substitution, and more preferably conservative substitution.

[0071] The structures of demethoxy-ovocytosine and demethyl-ovocytosine are as follows: Figure 1 As shown.

[0072] The presence or absence of demethoxypseudomycin-to-demethylpseudomycin conversion activity can be confirmed by in vitro or in vivo assays. For in vivo assays, for example, using cells, demethoxypseudomycin-producing cells are used, or demethoxypseudomycin is introduced into cells, and the cells are cultured to express / not express the test protein. Based on the results, if demethylpseudomycin is produced only when the test protein is expressed, then the test protein can be determined to have demethoxypseudomycin-to-demethylpseudomycin conversion activity.

[0073] The other sequences contained in protein B and the composition of polynucleotide B are the same as those in protein A and polynucleotide A.

[0074] The mutant cells of the present invention, which also contain polynucleotide B, can be used to prepare cells for the manufacture of demethylopseudomonas aeruginosa and can be used as cells for the manufacture of demethylopseudomonas aeruginosa.

[0075] The mutant cells of the present invention preferably further comprise polynucleotide C, which contains (C) a protein C encoding a protein C having conversion activity from β-trans-bergamotene to 5-keto-demethoxy-aspergillus acidophilus. Therefore, demethoxy-aspergillus acidophilus can be produced under conditions supplying β-trans-bergamotene, and demethyl-aspergillus acidophilus can also be produced when polynucleotide B is included.

[0076] Protein C, which has the transforming activity from β-trans-berberine to 5-keto-demethoxy-aspergillus acidophilus alcohol, is well known or can be readily identified or produced by homology analysis based on well-known amino acid sequences.

[0077] Protein C is preferably a protein C1 containing an amino acid sequence (C1) that has more than 70% identity with the amino acid sequence shown in sequence number 3.

[0078] Sequence number 3 is the amino acid sequence of a protein from Aspergillus fumigatus strain A1159, with NCBI accession number XP747154.1.

[0079] The identity of the above (C1) is preferably 80% or more, more preferably 90% or more, further preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0080] When the identity in (C1) above is less than 100%, the number of amino acids with mutations (substitution, deletion, addition, or insertion) relative to the amino acid sequence of sequence number 3 is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably substitution, more preferably conservative substitution.

[0081] The structures of β-trans-bergamerene and 5-keto-demethoxyfuscinol are as follows: Figure 1 As shown.

[0082] The presence of conversion activity from β-trans-berberine to 5-keto-demethoxyfuscinol can be confirmed by in vitro or in vivo assays. For in vivo assays, for example, using cells, β-trans-berberine-producing cells are used, or β-trans-berberine is introduced into cells, which are then cultured to express / not express the test protein. Based on the results, if 5-keto-demethoxyfuscinol is produced only when the test protein is expressed, then the test protein can be determined to have conversion activity from β-trans-berberine to 5-keto-demethoxyfuscinol.

[0083] The other sequences contained in protein C and the composition of polynucleotide C are the same as those in protein A and polynucleotide A.

[0084] The mutant cells of the present invention, which also contain polynucleotide C, can be used to prepare cells for the manufacture of demethylopseudomonas aeruginosa, and in a preferred embodiment, can be used as cells for the manufacture of demethylopseudomonas aeruginosa.

[0085] The mutant cells of the present invention preferably further comprise polynucleotide D, which contains (D) a coding sequence for protein D having conversion activity from farnesyl diphosphate to β-trans-berberine. Thus, in the case of the inclusion of polynucleotide C, demethoxypseudomycin can also be produced under conditions of farnesyl diphosphate supply, and in the case of the inclusion of both polynucleotide C and polynucleotide B, demethylpseudomycin can also be produced.

[0086] Protein D, which has the activity of converting farnesyl diphosphate to β-trans-berberine, is well known or can be readily identified or produced by homology analysis based on well-known amino acid sequences.

[0087] Protein D is preferably a protein D1 containing an amino acid sequence (D1) that has more than 70% identity with the amino acid sequence shown in sequence number 4.

[0088] Sequence number 4 is the amino acid sequence of a protein from Aspergillus fumigatus strain A1159, with NCBI accession number XP747153.1.

[0089] The identity of the above (D1) is preferably 80% or more, more preferably 90% or more, further preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0090] When the identity is less than 100% in (D1) above, the number of amino acids with mutations (substitution, deletion, addition, or insertion) relative to the amino acid sequence of sequence number 4 is preferably 1 to 10, more preferably 1 to 5, further preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably substitution, more preferably conservative substitution.

[0091] The structures of farnesyl diphosphate and β-trans-bergimene are as follows: Figure 1 As shown.

[0092] The presence of activity for converting farnesyl diphosphate to β-trans-bergimide can be confirmed by in vitro or in vivo assays. For in vivo assays, for example, using cells, farnesyl diphosphate-producing cells are used, or farnesyl diphosphate is introduced into cells, and the cells are cultured to express / not express the test protein. Based on the results, if β-trans-bergimide is produced only when the test protein is expressed, then the test protein can be determined to have activity for converting farnesyl diphosphate to β-trans-bergimide.

[0093] The other sequences contained in protein D and the composition of polynucleotide D are the same as those of protein D and polynucleotide D.

[0094] The mutant cells of the present invention, which also contain polynucleotide D, can be used to prepare cells for the manufacture of demethylopseudomonas aeruginosa, and in a preferred embodiment, can be used as cells for the manufacture of demethylopseudomonas aeruginosa.

[0095] The aforementioned polynucleotides can be easily produced using well-known gene processing methods. For example, they can be produced using PCR, restriction enzyme cutting, DNA ligation, and other techniques.

[0096] The mutant cells of this invention are cells obtained by mutating existing cells. These mutations are gene mutations, including the introduction of exogenous polynucleotides and the introduction of mutations into the endogenous genome.

[0097] Exogenous polynucleotides are polynucleotides that contain a base sequence that does not originate from the genomic DNA (especially chromosomal genomic DNA) of the pre-mutant cell, and there are no particular restrictions as long as they fall within this range.

[0098] Examples of mutations in the endogenous genome include gene damage, mutations in protein-coding regions, partial deletions, mutations in splicing regulatory regions, and mutations in expression regulatory regions (such as promoters, activators, enhancers, repressor binding elements, etc.).

[0099] There are no particular restrictions on the methods used for gene mutation in cells; methods that follow or are based on well-known approaches can be employed. Examples include protoplast-PEG method, electroporation, lipid transfection, gene gun method, and CRISPR-Cas method.

[0100] The cell used as the target of mutation can be either a eukaryotic cell or a prokaryotic cell. However, when using the mutant cell of the present invention as the cell for manufacturing demethylospermicin, a eukaryotic cell is preferred. From the viewpoint of demethylospermicin manufacturing efficiency, fungal cells are preferred as eukaryotic cells, and cells with mycelial formation ability are particularly preferred. As for cells with mycelial formation ability, any cell that has a period in its life cycle where mycelial formation occurs is acceptable; there are no particular limitations. Examples include Aspergillus, Penicillium, Trichoderma, Cephalosporium, and Cladosporium, with Aspergillus being particularly preferred, and Aspergillus nidulans being especially preferred.

[0101] When at least one of polynucleotides B to D is included, polynucleotides A to D can all be independent polynucleotide molecules, and two or more of polynucleotides A to D can also be in the same polynucleotide molecule.

[0102] In one embodiment of the present invention, polynucleotide A is an exogenous polynucleotide. Alternatively, in another embodiment of the present invention, when at least one of polynucleotides B to D is included, the polynucleotides including two or more (preferably three or more, particularly preferably four) of polynucleotide A are exogenous polynucleotides.

[0103] In one embodiment of the invention, a mutation is performed such that protein E, which has the conversion activity from demethylosporin to oopsporin, is not expressed, or the expression or function of the aforementioned protein E is reduced. This inhibits the conversion from demethylosporin to oopsporin, resulting in more efficient acquisition of demethylosporin.

[0104] "Function" refers to the conversion activity from demethylated oocystisin to oocystisin. Additionally, "expression" includes both gene mRNA expression and gene protein expression, preferably gene protein expression. "Decrease" indicates that, relative to 100% of the gene protein activity and / or gene expression level (functional and / or expression index value when the gene is not mutated), the gene protein activity and / or gene expression level (gene functional and / or expression index value) after mutation is, for example, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, or less than 0.01%.

[0105] The mutant cells of the present invention preferably possess the ability to produce demethylopyroxynil. Specifically, when the mutant cells of the present invention contain polynucleotide A and polynucleotide B, they can produce demethylopyroxynil under the supply conditions of 5-keto-demethoxyfuscin. Particularly preferred are the mutant cells of the present invention containing all polynucleotides A to D. Thus, demethylopyroxynil can be obtained using farnesyl diphosphate (one of the terpenoids essential for maintaining biological life, and also a biosynthetic intermediate for various terpenoids such as carotenoids, plant hormones, and ubiquinone, which is biosynthesized in many eukaryotic cells).

[0106] Demethylomorphosomalin can be produced by culturing mutant cells of the present invention that are capable of producing demethylomorphosomalin.

[0107] The culture can be performed by those skilled in the art using the mutant cell types according to the present invention, or based on known methods.

[0108] As a culture medium (e.g., culture medium), it is preferable to contain a carbon source. Examples of carbon sources include: carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides; invert sugar obtained from the hydrolysis of sucrose; glycerol; compounds with one carbon atom, such as methanol, formaldehyde, formate, carbon monoxide, and carbon dioxide (hereinafter referred to as C1 compounds); oils such as corn oil, palm oil, and soybean oil; acetates; animal fats; animal oils; fatty acids such as saturated fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; fatty acid glycerides such as monoglycerides, diglycerides, and triglycerides; polypeptides such as microbial proteins and plant proteins; renewable carbon sources such as biomass carbon sources obtained from hydrolysis; yeast extracts; or substances composed of combinations of these compounds. As a nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia, and ammonia water can be used. As an organic micronutrient source, it is desirable to contain appropriate amounts of essential substances such as vitamin B1 and L-homoserine, or yeast extracts. In addition to these compounds, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc., may be added as needed. The culture medium used in this invention only needs to contain a carbon source, a nitrogen source, inorganic ions, and other trace organic components as needed; both natural and synthetic culture media are acceptable.

[0109] As monosaccharides, examples include: trioses such as acetoketose (dihydroxyacetone) and propionaldehyde (glyceraldehyde); teuses such as erythritolose and threose; pentoses such as ribulose and xylulose, pentose aldoses such as ribose, arabinose, xylose, and lysose, and deoxysugars such as deoxyribose; hexoses such as allulose, fructose, sorbitose, and tagatose, hexaloses such as allose, azoose, glucose, mannose, gulose, idose, galactose, and tarose, and deoxysugars such as fucose, fucoidan, and rhamnose; heptoses such as sedoheptulose, with fructose, mannose, galactose, and glucose being preferred C6 sugars; and carbohydrates such as xylose and arabinose, which are C5 sugars.

[0110] Examples of disaccharides include sucrose, lactose, maltose, trehalose, maltodextrose, cellobiose, etc., with sucrose and lactose being preferred.

[0111] Oligosaccharides can be categorized as follows: trisaccharides such as raffinose, pinotriose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and other oligosaccharides such as fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), and mannan oligosaccharides (MOS).

[0112] Examples of polysaccharides include glycogen, starch (amylose and amylopectin), cellulose, dextrin, and glucan (β1,3-glucan). Starch and cellulose are preferred.

[0113] As microbial proteins, examples include polypeptides that can be obtained from yeast or bacteria.

[0114] As plant-based proteins, examples include polypeptides that can be obtained from soybeans, corn, rapeseed, jatropha, palm, peanuts, sunflower, coconut, mustard, cottonseed, palm kernel oil, olives, safflower, sesame, and flaxseed.

[0115] As lipids, substances containing one or more saturated or unsaturated fatty acids with a C4 or higher number of ions can be listed.

[0116] As an oil, it is preferred to contain one or more saturated or unsaturated fatty acids with a C4 or higher concentration and to be liquid at room temperature. Examples of lipids that can be obtained from soybean, corn, rapeseed, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, safflower, sesame, flaxseed, oil-producing microbial cells, tallow tree, or a combination of two or more of these.

[0117] As fatty acids, compounds with the formula RCOOH ("R" represents a hydrocarbon group) can be listed.

[0118] Unsaturated fatty acids are compounds whose "R" has at least one carbon-carbon double bond. Examples include oleic acid, trans oleic acid, linoleic acid, palmitoleic acid, and arachidonic acid.

[0119] Saturated fatty acids are compounds whose "R" is a saturated aliphatic group. Examples include docosanoic acid, eicosanoic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, and dodecanoic acid.

[0120] Among them, as fatty acids, it is preferred to include one or more C2 to C22 fatty acids, and more preferably to include C12 fatty acids, C14 fatty acids, C16 fatty acids, C18 fatty acids, C20 fatty acids, and C22 fatty acids.

[0121] In addition, as carbon sources, salts, derivatives, and salts of fatty acids can also be listed. As salts, lithium salts, potassium salts, sodium salts, etc., can be listed.

[0122] In addition, combinations of carbohydrates such as lipids, oils, fats, fatty acids, glycerol fatty acid esters, and glucose can be listed as carbon sources.

[0123] As a renewable carbon source, biomass carbon sources obtained through hydrolysis can be listed as examples.

[0124] Examples of biomass carbon sources include: waste wood, paper and pulp, leafy plants, fruit pulp and other cellulose-based substrates; and parts of plants such as stalks, grains, roots, and tubers.

[0125] Plants used as biomass carbon sources include corn, wheat, rye, sorghum, triticale, rice, millet, barley, cassava, peas and other legumes, potatoes, sweet potatoes, bananas, sugarcane, and tapioca.

[0126] When adding renewable carbon sources such as biomass to a culture medium, pretreatment is preferred. Examples of pretreatment include enzyme pretreatment, chemical pretreatment, and combinations of enzyme pretreatment and chemical pretreatment.

[0127] In the manufacturing method of the present invention, a culture medium containing an adsorbent resin is preferably used as the culture medium. This improves the manufacturing efficiency of demethylopseudomonas oocystin. The adsorbent resin is not particularly limited as long as it is a resin material capable of adsorbing various organic substances through the physical interaction between its surface and the adsorbate; typically, it has a porous structure (preferably with a specific surface area of ​​300–1200 m²). 2 Particles ( / dry-g). Examples of resins include styrene-divinylbenzene, styrene, divinylbenzene, methacrylates, etc.

[0128] When using adsorption resin, the concentration in the culture medium is preferably 20–100 g / L.

[0129] The preferred culture temperature is 20℃~37℃.

[0130] The optimal incubation time is 20–100 hours.

[0131] The mutant cells of the present invention are preferably cultured under aerobic, anaerobic or anaerobic conditions depending on the nature of the host.

[0132] As a method for culturing mutant cells according to the present invention, for example, a method using known fermentation methods such as batch culture, fed-batch culture, and continuous culture can be cited.

[0133] After cultivation, solid-liquid separation is performed, such as filtration and centrifugation. Intracellular components are extracted from the obtained solid sample (e.g., organic solvent extraction such as acetone extraction, ultrasonic disruption, etc.). The soluble fraction is purified by chromatography to obtain the target product.

[0134] 4. Compound Manufacturing 2 (R) 1 -C(=O)-L 1 -R 11 (situation) In the compounds of this invention, R 1 -C(=O)-L 1 -R 11 The compound can be synthesized using demethylopseudomonasin as a starting material through various methods. For example, the compound can be synthesized using a method that includes the following steps. Compound A can be obtained by biosynthesizing demethylopseudomonasin according to the method described in item 3 above. Compound B can be a commercially available substance or a substance synthesized according to a known method.

[0135] From the perspective of yield and ease of synthesis, the amount of compound B used is preferably 0.5 to 20 moles, more preferably 1 to 12 moles, relative to 1 mole of compound A.

[0136] This reaction is typically carried out in the presence of a reaction solvent. There are no particular limitations on the reaction solvent; dichloromethane is an example. The solvent can be used alone or in combination.

[0137] This reaction is carried out in the presence of a catalyst, such as a base, as needed. Examples of bases include 4-dimethylaminopyridine and triethylamine.

[0138] Regarding the reaction temperature, it can be carried out under heating, at room temperature, or under cooling, typically ranging from 0 to 120°C. The preferred reaction temperature is 15 to 50°C. There is no particular limitation on the reaction time, which can typically range from 30 minutes to 60 hours.

[0139] The reaction process is monitored using conventional methods such as chromatography. After the reaction is complete, the solvent is removed by distillation, and the product can be separated and purified using conventional methods such as chromatography and recrystallization. Furthermore, the structure of the product can be analyzed using elemental analysis, MS (ESI-MS), IR analysis, etc. 1 H-NMR, 13 Identification was performed using C-NMR and other methods.

[0140] 5. Applications The active ingredient of this invention has anti-dysentery amoeboid activity. Furthermore, the active ingredient of this invention can be used for applications targeting MetAP2. Examples of such applications include anti-cancer, anti-obesity, angiogenesis inhibition, prevention or improvement (treatment) of infectious diseases such as malaria, giardiasis, and microsporidiasis, prevention or improvement (treatment) of cancer, prevention or improvement (treatment) of Prader-Willi syndrome, and prevention or improvement (treatment) of diabetes. Therefore, in one aspect, this invention relates to formulations containing the active ingredient of this invention for the aforementioned applications (e.g., anti-dysentery amoeboid agents, preventive or therapeutic agents for amoebic dysentery).

[0141] Entamoeba histolytica is the pathogen that causes amoebic dysentery. Anti-entamoeba activity refers to the activity that kills or inhibits the proliferation of Entamoeba histolytica. Utilizing anti-entamoeba activity, the symptoms of amoebic dysentery can be improved or prevented (diarrhea, bloody stools, tenesmus, lower abdominal pain or discomfort during defecation, bloody stools, abscesses (liver abscess, etc.), vomiting, weight loss, night sweats, fatigue, etc.). "Improvement" refers to: the improvement or relief of symptoms or condition; the prevention or delay of the worsening of symptoms or condition; and the reversal, prevention, or delay of the progression of symptoms or condition.

[0142] The active ingredients and formulations of this invention can be used as medicines, reagents, food additives, food compositions (including health foods, health promoters, nutritional supplements (nutritional products, etc.)).

[0143] The formulations of this invention need only contain the active ingredient of this invention, and there are no particular limitations. Other ingredients may also be included as needed. These other ingredients are simply pharmaceutically acceptable and are not particularly limited. In addition to ingredients with pharmacological activity, other ingredients also include additives. Examples of additives include, for instance, base agents, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc.

[0144] There are no particular limitations on the use of the active ingredients or the formulations of this invention. The active ingredients or formulations of this invention can be used, for example, both in vitro (e.g., added to a culture medium for cultured cells) and in vivo (e.g., administered to / ingested by animals).

[0145] The active ingredients and the applicable objects of the formulations of this invention are not particularly limited. Among mammals, examples include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Furthermore, as cells, animal cells can be included. The types of cells are also not particularly limited; examples include blood cells, hematopoietic stem cells / precursor cells, gametes (sperm, egg), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0146] The formulations of the present invention can be in any dosage form, such as oral preparations (including intraorally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including decoctions, suspensions, syrups), jelly, etc., as well as injectable preparations (e.g., drops (e.g., intravenous drip preparations, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), topical preparations (e.g., ointments, patches, lotions), suppositories, inhalers, eye drops, eye ointments, nasal drops, ear drops, liposomes, etc., as well as non-oral preparations.

[0147] As for the route of administration of the formulation of the present invention, there are no particular restrictions as long as the desired effect can be obtained, such as oral administration, tube feeding, enema and other intestinal administration; intravenous administration, intra-arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, intraperitoneal administration and other non-oral administration.

[0148] Examples of preparations of the present invention used as food compositions include liquid, gel, or solid foods, such as fruit juice, refreshing beverages, tea, soup, soy milk and other beverages, salad oil, sauces, yogurt, jelly, pudding, rice seasoning, infant formula, cake flour, powdered or liquid dairy products, bread, biscuits, etc.

[0149] The content of the active ingredient in the formulation of the present invention depends on the method of use, the target of application, the state of the target of application, etc., and is not limited. For example, it can be 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0150] The dosage of the active ingredient and the drug of the present invention when administered to animals is not particularly limited, as long as it is an effective amount that demonstrates the therapeutic effect. Generally, it is based on the weight of the active ingredient. In the case of oral administration, the daily dosage is generally 0.1–1000 mg / kg body weight, preferably 0.5–500 mg / kg body weight. In the case of non-oral administration, the daily dosage is 0.01–100 mg / kg body weight, preferably 0.05–50 mg / kg body weight. The above dosages can also be appropriately increased or decreased according to age, disease condition, symptoms, etc.

[0151] Example The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0152] Experimental Example 1. Biosynthetic Design and Enzyme Study of Demethylated Oomycetin Based on the biosynthetic pathway of fumonisin in non-patent literature 1, biosynthetic pathways for demethylated oocystin and oocystin were designed. Figure 1 ). However, enzyme X (7-hydroxylase Ova-C7H) with the conversion activity from 5-keto-demethoxyfucoidan to demethoxypseudomycin is not yet known. Therefore, this enzyme was investigated.

[0153] No biosynthetic gene clusters (BGCs) of the OV class have been reported to date, but as a result of genome mining, a candidate BGC was found in the insect-infecting filamentous fungus *Metarhizium anisopliae* ARSEF23. RNA-seq data for this strain under various culture conditions have been entered into the public database (SRA), and insilico analysis confirmed high expression of this BGC in Sabouraud dextrose agar. In fact, OV production was confirmed by culturing *M. robertsii* under these conditions. The proposed BGC contains two copies of an α-KG-dependent non-heme iron oxidase gene. In FM biosynthesis, the introduction of the C6 hydroxyl group is catalyzed by the homozygous enzyme *Fma-C6H*, suggesting that the 7-hydroxyl group in OV biosynthesis is also introduced by a similar enzyme. In fact, co-expression of four fumonisin biosynthesis genes (fma-TC, fma-P450, fma-C6H, fma-MT) in *A. nidulans* and the MAA08348 gene (ova-C7H) cloned from *M. robertsii* confirmed the production of fumonisin oxidase (OV). Based on the above, the 7-hydroxylase Ova-C7H responsible for OV production was identified.

[0154] Experimental Example 2. Manufacturing of deMeOV (deMeOV) <Plasmid Construction> The following plasmids were constructed using the gap-repair cloning method of budding yeast. The fma-TC, fma-P450, and fma-C6H genes were derived from the genomic DNA of Aspergillus fumigatus strain A1159, while the ova-C7H gene was derived from the genomic DNA of Metarhizium anisopliae strain ARSEF23. These genes were amplified by PCR. The plasmids were constructed in the following order: (1) The PCR-amplified fma-TC gene was inserted into the SwaI-cut pKW20093 plasmid to create pKW20517. (2) The PCR-amplified fma-P450 gene was inserted into the SwaI-cut pKW10010 plasmid to create pKW20518. (3) The PCR-amplified fma-C6H gene was inserted into the SwaI-cut pKW10012 plasmid to create pKW20519. (4) The PCR-amplified ova-C7H gene was introduced into the pKW20093 plasmid cut with SwaI to create pKW20522. (5) The fma-TC expression cassette (glaA promoter region and fma-TC gene) was amplified by PCR using pKW20517 (prepared in (1)) as a template, and introduced into the pKW20518 plasmid cut with SwaI to create pKW23502.

[0155] [Table 1] <Transformation of Aspergillus nidus> After inoculating *Aspergillus nidulans* strains ΔPPR (ΔpyrG, ΔpyroA, ΔriboB) as hosts onto CD / PPR agar medium and culturing, spores were formed. These spores were suspended in sterile water and inoculated into CD / PPR liquid medium, where they were cultured at 30°C with shaking for 20 hours. Subsequently, protoplastization was performed on the filtered bacterial cells using lysozyme from *Trichoderma harzianum* (Sigma-Aldrich) and β-glucuronidase from *Helix pomatia* (Sigma-Aldrich). The prepared plasmids (pKW20519, pKW20522, pKW23502) were added to the protoplasts, mixed with STC solution, and inoculated onto CD agar medium. After secondary screening and culturing of the resulting colonies, genomic DNA was extracted, and transformation was confirmed by PCR. The transformant (Aspergillus nidulans ΔPPR#5) that was successfully introduced into the plasmid following the steps above will be used for subsequent culture experiments.

[0156] <Destruction of deMeOV> The prepared *Aspergillus nidulans* strain ΔPPR#5 was inoculated into 30 mL of CDST liquid medium and cultured with shaking at 30 °C. After 72 hours of culture, a portion of the culture was aliquoted every 24 hours to quantify the amount of deMeOV produced over time. 0.5 mL of ethyl acetate was added to each aliquot (0.5 mL) and stirred vigorously. The mixture was then centrifuged, and 0.4 mL of the ethyl acetate layer was transferred to another container. The solvent was removed by distillation. 100 μL of DMF was added to the residue to dissolve it, and the supernatant after centrifugation to remove insoluble components was analyzed by LC-MS.

[0157] <Detection of deMeOV by LC-MS> DeMeOV analysis by LC-MS was performed using a Thermo Fisher Scientific system (Dionex U3000 and Orbitrap Exactive Plus mass spectrometer). A Waters AQUITY UPLC HSS C18 column (2.1 × 50 mm) was used, with a gradient mobile phase of acetonitrile and ultrapure water (acetonitrile 5-100%, 4 min) at a flow rate of 500 μL / min. Additionally, 0.05% formic acid was added to each solvent. Precise mass was determined using ESI, and analysis was performed using Qual Browser software.

[0158] <Large-scale culture experiment of deMeOV> A small amount of Aspergillus nidulans ΔPPR#5 spores were suspended in 100 μL of 0.1% Tween 80 aqueous solution and then inoculated entirely onto CD agar medium and cultured at 30°C. After confirming spore formation, half of the CD agar medium was cut off and mixed with 10 mL of 0.1% Tween 80 in a container. The resulting suspension was then added to a 2 L Erlenmeyer flask containing 400 mL of CDT medium for pre-culture. Pre-culture was carried out at 30°C and 160 rpm for 24 hours. The pre-culture solution was then transferred to a 6.0 L fermenter (BEMARUBISHI CO.,LTD, Bioneer benchtop culture unit) containing CDST medium (with Diaion HP-20 adsorption resin) for main culture. The main culture was carried out at 30°C, 400 rpm, and oxygen flow for approximately 48 hours. During the main culture, the culture medium was periodically removed to confirm the deMeOV production. After confirming sufficient productivity, the culture medium and resin were removed from the fermenter, filtered, and the resulting solids of cells and resin (HP-20) were obtained and frozen. Furthermore, HPLC analysis confirmed that the culture supernatant (filtrate) did not contain the target component deMeOV.

[0159] <Refining and structural confirmation of deMeOV> For the solid samples obtained from the above culture (6.0 L scale), approximately 10 batches (60 L culture volume) were combined, and three extractions were performed using 10 L of acetone. Each extraction lasted approximately 2 hours, with the mixture of cells and resin (HP20) being vigorously stirred in acetone every 30 minutes. After removing the insoluble fraction by filtration, the resulting extract was concentrated under reduced pressure and then separated three times using 1.0 L of EtOAc. Subsequently, the EtOAc layer was concentrated under reduced pressure to obtain 27 g of extract. The extract was purified using silica gel column chromatography (BW-820MH 280 g, developing solvent: CHCl3 / MeOH [C / M] = 100 / 0 → 100 / 2 → 100 / 1 → 0 / 100). For the fraction with a C / M ratio of 100 / 2 (5.2 g) from which the target component was detected, purification was performed using normal-phase rapid chromatography (conditions: column = Universal 5 L (silica gel: 300 g), flow rate = 70 mL / min, solvent = n-hexane / EtOAc = 4 / 1, fractionation capacity = 100 mL / tube). TLC analysis was performed on the obtained fractions, and the fractions containing more deMeOV (1.2 g) were mixed. Finally, another normal-phase rapid chromatography was performed (conditions: column = Universal 2 L (silica gel: 100 g), flow rate = 30 mL / min, solvent = n-hexane / EtOAc = 17 / 3, fractionation capacity = 30 mL / tube), yielding 976 mg of deMeOV. NMR analysis of the separated deMeOV in deuterated chloroform confirmed that all data were consistent with literature information, and two-dimensional NMR analysis confirmed the correct chemical structure. Figure 2 (and Table 2). [Table 2] Experimental Example 3. Synthesis of deMeOV Derivatives The structures of the synthesized compounds (compounds 1–12) are shown in the figure. Figure 3 .

[0160] <Synthesis of Compound 1 and Compound 2> Acetic anhydride (240 μL, 2.5 mmol), DMAP (0.040 mmol, 5.0 mg), and deMeOV (71 mg, 0.25 mmol) were added to 25 mL of a two-necked pear-shaped flask and dissolved in 2.5 mL of CH2Cl2. The mixture was stirred for 30 minutes at room temperature under a nitrogen atmosphere. The reaction was stopped by adding 5 mL of water, and the mixture was separated by EtOAc (5 mL × 3 times), collecting the organic layer. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 119.2 mg of the reaction product. The obtained product was pretreated with ODS resin and then purified by reversed-phase preparative HPLC (conditions: column = Develosil ODS-HG-5 (f20 × 250 mm), flow rate = 10 mL / min, solvent = MeCN / H2O = 2 / 3) to obtain 64.1 mg of the target compound 1 (yield 78%). Additionally, compound 2 is obtained as a byproduct during this process.

[0161] Compound 1: a colorless oily substance; [α] D 25 =16.3 (c = 0.54, MeOH); 1 H NMR (400 MHz, CDCl3) δ1.27 (s, 3H), 1.65 (s, 3H), 1.74 (s, 3H), 2.21 (s, 3H), 2.14 (m, 1H), 2.40 (m, 1H), 2.53 (m, 1H), 2.66 (m, 1H), 2.71 (m, 2H), 2.74 (d, J = 4.2 Hz, 1H), 2.94 (t, J = 6.5Hz, 1H), 3.07 (d, J = 4.2 Hz, 1H), 3.16 (s, 1H), 5.16 (t, J = 7.4Hz, 1H), 5.67 (s, 1H); 13 C (100 MHz, CDCl3) δ 14.8, 18.3, 26.0, 27.3, 30.5, 35.5, 51.1, 57.0, 60.3, 60.7, 77.8, 78.0, 118.3, 135.8, 208.6; HRMS (ESI) calculated C 17 H 25 O6 + [M + H] + 325.1646; Measured value 325.1641.

[0162] Compound 2: a colorless oily substance; [α] D 25=167 (c = 0.27, MeOH); 1 H NMR (400 MHz, CDCl3) δ1.47 (s, 3H), 1.65 (s, 3H), 1.74 (s, 3H), 2.21 (s, 3H), 2.14 (m, 1H), 2.40 (m, 1H), 2.53 (m, 1H), 2.66 (m, 1H), 2.71 (m, 2H), 2.74 (d, J = 4.2 Hz, 1H), 2.94 (t, J =6.5Hz, 1H), 3.07 (d, J = 4.2 Hz, 1H), 3.16 (s, 1H), 5.16 (t, J = 7.4Hz, 1H), 5.67 (s, 1H); 13 C (100 MHz, CDCl3) δ 14.8, 18.3, 26.0, 27.3, 30.5, 35.5, 51.1, 57.0, 60.3, 60.7, 77.8, 78.0, 118.3, 135.8, 208.6; HRMS (ESI) calculated C 17 H 25 O6 + [M + Na] + 383.1232; Measured value 383.1199.

[0163] <Synthesis of Compound 3> 10 mL of nicotinic acid (0.070 mmol, 8.5 mg), DMAP (0.0080 mmol, 1.0 mg), EDC·HCl (21 mg, 0.12 mmol), and deMeOV (8.0 mg, 0.030 mmol) were added to a two-necked pear-shaped flask and dissolved in 1.0 mL of CH₂Cl₂. The mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. The reaction was stopped by adding saturated NH₄Cl, and the mixture was separated by EtOAc (20 mL × 3 times), collecting the organic layer. This organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 10.6 mg of the reaction product. The obtained product was pretreated with ODS resin and then purified by reversed-phase preparative HPLC (conditions: column = Develosil ODS-HG-5 (f20×250mm), flow rate = 10mL / min, solvent = MeCN / H2O = 2 / 3) to obtain 4.0 mg of target compound 3 (yield 35%).

[0164] Compound 3: Colorless oily substance [α] D 25= -20.1; (c=0.27, MeOH); 1 H NMR (400 MHz, CDCl3) δ1.31 (s, 3H), 1.53 (m, 1H), 1.66 (s, 3H), 1.77 (s, 3H), 2.14 (m, 1H), 2.44 (m, 1H)), 2.62 (m, 1H), 2.75 (dt, J = 4.4, 12.9 Hz, 1H), 2.81 (d, J = 4.1 Hz, 1H), 2.85 (dt, J = 6.6, 13.3 Hz, 1H), 3.03 (t, J = 6.6 Hz, 1H), 3.14 (d, J = 4.2 Hz, 1H), 3.34 (s, 1H), 5.18 (t, J = 7.5 Hz, 1H), 5.90 (s, 1H), 7.41 (dd, J = 4.9, 7.9Hz, 1H), 8.39 (dt, J = 7.9, 2.0 Hz, 1H), 8.80 (dd, J = 1.6, 4.9Hz, 1H), 9.33 (d,J = 1.6 Hz, 1H); 13 C(100 MHz, CDCl3)δ14.5, 18.4, 26.1, 27.3, 30.3, 36.4, 51.1,51.5, 57.7, 60.5, 60.7, 77.6, 78.1, 80.1, 117.9, 123.8, 125.7, 136.3, 138.0,151.7, 154.1, 164.4, 201.0; HRMS (ESI) calcd. for C 21 H 25 NO6 ＋ [M + H] + 388.1755; Measured value 388.1747.

[0165] <Synthesis of Compound 4> DMAP (28.2 mg, 0.23 mmol), cinnamic acid (34.2 mg, 0.23 mmol), and EDC (88.5 mg, 0.46 mmol) were added to 2 mL of CH2Cl2 containing deMeOV (33 mg, 0.12 mmol). The mixture was stirred under reflux for 12 hours in an argon atmosphere. After cooling, the solvent was removed by distillation under reduced pressure. The resulting sample was separated by silica gel column chromatography (developing solvent: CH2Cl2 / MeOH 100 / 1) to obtain 8.8 mg of compound 4.

[0166] Compound 4: a colorless oily substance; 1 H NMR (300 MHz, CDCl3) δ 7.82 (d, J = 16.0 Hz, 1H), 7.56 (d, J = 3.1 Hz, 2H), 7.40 (d, J = 2.5 Hz, 3H), 6.59 (d, J = 16.0 Hz, 1H), 5.82 (s, 1H), 5.19 (t, J = 7.1 Hz, 1H), 3.28 (s, 1H), 3.13 (d, J = 4.1 Hz, 1H), 3.00 (t, J = 6.5 Hz, 1H), 2.91-2.66 (m, 3H), 2.64-2.54 (m, 1H), 2.50-2.33 (m, 1H), 2.15 (dt, J = 14.7, 7.3 Hz, 1H), 1.77 (s, 3H), 1.67 (s, 3H), 1.60 (s, 1H), 1.56-1.46 (m, 1H), 1.32 (s, 3H).

[0167] <Synthesis of Compound 5> Oxaloyl chloride (0.71 mL, 8.3 mmol) was added dropwise to 5 mL of anhydrous dichloroethane containing benzamide (500 mg, 4.2 mmol), and the mixture was refluxed for 17 hours. After removing the solvent by distillation, the resulting benzoyl isocyanate was used in the following reaction without purification. Benzoyl isocyanate (18 mg, 0.12 mmol, 1.2 eq) was added to 2 mL of CH₂Cl₂ containing deMeOV (29 mg, 0.10 mmol, 1.0 eq), and the mixture was stirred for 12 hours under an argon atmosphere, followed by reflux for 3 hours. The reaction solution was cooled, and the solvent was removed by distillation under reduced pressure. The obtained sample was separated by silica gel column chromatography (developing solvent: CH₂Cl₂ / MeOH 100 / 1) to obtain 3.0 mg of compound 5.

[0168] Compound 5: Colorless oil; ¹H NMR (300 MHz, CDCl₃) δ 8.48 (brs, 1H), 7.86 (d, J = 7.4 Hz, 2H), 7.59 (t, J = 7.3 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 5.78 (s, 1H), 5.17 (t, J = 7.2 Hz, 1H), 3.22 (s, 1H), 3.11 (d, J = 4.0 Hz, 1H), 2.99 (t, J = 6.5 Hz, 1H), 2.79 (d, J = 3.8 Hz, 1H), 2.89–2.53 (m, 3H), 2.43 (dt, J = 13.8, 7.0Hz, 1H), 2.14 (dt, J = 14.3, 7.1 Hz, 1H), 1.76 (s, 3H), 1.70 (s, 2H), 1.66 (s, 3H), 1.56-1.44 (m, 2H), 1.37 (s, 3H).

[0169] <Synthesis of Compounds 6-10> As the compound that reacts with deMeOV, a suitable carboxyl compound or isocyanate compound is used. Otherwise, the same procedure is followed as with compounds 1 to 5 to obtain compounds 6 to 10 (compound 6: 31.8 mg, compound 7: 44 mg, compound 8: 20.6 mg, compound 9: 63.1 mg, compound 10: 10.5 mg).

[0170] Compound 6: ¹H NMR (300 MHz, CDCl₃) δ 7.74 (d, J = 16.0 Hz, 1H), 7.54 (s, 1H), 7.46–7.29 (m, 3H), 6.58 (d, J = 16.0 Hz, 1H), 5.81 (s, 1H), 5.19 (t, J = 7.4 Hz, 1H), 3.26 (s, 1H), 3.12 (d, J = 4.1 Hz, 1H), 3.00 (t, J = 6.5 Hz, 1H), 2.79 (d, J = 4.2 Hz, 1H), 2.91–2.64 (m, 2H), 2.63–2.55 (m, 1H), 2.50-2.38 (m, 1H), 2.15 (dt, J= 14.5, 7.2Hz, 1H), 1.77 (s, 3H), 1.67 (s, 3H), 1.56-1.41 (m, 1H), 1.31 (s, 3H).

[0171] Compound 7: 7.67 (d, J = 16.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.06 - 6.97 (m, 2H), 6.85 (dd, J = 7.4, 1.4 Hz, 1H), 6.49 (d, J = 16.0 Hz, 1H), 6.24 (brs, 1H), 5.84 (s, 1H), 5.19 (t, J = 7.4 Hz, 1H), 3.40 (s, 1H), 3.13 (d, J = 4.1 Hz, 1H), 3.03 (t, J = 6.5 Hz, 1H), 2.80 (d, J = 4.1 Hz, 1H), 2.94 - 2.68 (m, 2H), 2.67 - 2.56 (m, 1H), 2.52 - 2.34 (m, 1H), 2.16 (dt, J = 14.8, 7.3 Hz, 1H), 1.76 (s, 3H), 1.66 (s, 3H), 1.58 - 1.47 (m, 1H), 1.32 (s, 3H).

[0172] Compound 8: 9.02 (s, 1H), 8.15 (s, 1H), 8.07 (d, J = 7.3 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.65 - 7.60 (m, 1H), 5.76 (s, 1H), 5.16 (t, J = 7.3 Hz, 1H), 3.26 (s, 1H), 3.10 (d, J = 4.1 Hz, 1H), 3.02 - 2.98 (m, 1H), 2.82 - 2.55 (m, 3H), 2.47 - 2.38 (m, 1H), 2.19 - 2.09 (m, 1H), 1.91 - 1.89 (m, 1H), 1.76 (s, 3H), 1.66 (s, 3H), 1.51 (d, J = 20.5 Hz, 1H), 1.36 (s, 3H).

[0173] Compound 9: 7.78 (d, J = 16.0 Hz, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.34 - 7.24 (m, 1H), 7.16 (dd, J = 4.3, 2.2 Hz, 2H), 7.04 - 6.97 (m, 1H), 6.97 - 6.90 (m, 2H), 6.56 (d, J = 16.0 Hz, 1H), 5.82 (s, 1H), 5.19 (t, J = 7.5 Hz, 1H), 5.01 (s, 2H), 3.82 (s, 3H), 3.28 (s, 1H), 3.13 (d, J = 4.2 Hz, 1H), 3.00 (t, J = 6.5 Hz, 1H), 2.79 (d, J = 4.2 Hz, 1H), 2.91 - 2.65 (m, 2H), 2.63 - 2.55 (m, 1H), 2.50 - 2.37 (m, 1H), 2.22 - 2.07 (m, 1H), 1.76 (s, 3H), 1.67 (s, 3H), 1.56 - 1.43 (m, 1H), 1.32 (s, 3H).

[0174] Compound 10: 7.85 - 7.80 (m, 2H), 7.73 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.55 - 7.50 (m, 1H), 6.65 (d, J = 16.1 Hz, 1H), 5.82 (s, 1H), 5.19 (t, J = 7.5 Hz, 1H), 3.27 (s, 1H), 3.13 (d, J = 4. ... Hz, 1H), 3.01 (t, J = 6.6 Hz, 1H), 2.91 - 2.67 (m, 3H), 2.63 - 2.57 (m, 1H), 2.49 - 2.40 (m, 1H), 2.20 - 2.10 (m, 1H), 1.77 (s, 3H), 1.67 (s, 3H), 1.55 - 1.48 (m, 1H), 1.32 (s, 3H).

[0175] Compound 11: 5.69 (s, 1H), 5.17 (t, J = 7.3, 1H), 3.15 (s, 1H), 3.09 (d, J = 4.5, 1H), 2.95 (t, J = 6.5, 1H), 2.75 (m, 1H), 2.71 (d, J = 4.5, 1H), 2.56 (m, 1H), 2.54 (q, J = 7.6, 2H), 2.51 (m, 1H), 2.41 (m, 1H), 2.14 (m, 1H), 1.76 (s, 3H), 1.66 (s, 3H), 1.45 (ddd, J = 2.3, 5.7, 13.0, 1H), 1.28 (s, 3H), 1.21 (t, J =7.6, 3H) Compound 12: 8.14 (dd, J = 1.4, 8.0, 2H), 7.58 (t, J = 7.3, 1H), 7.46 (t, J = 7.5, 2H), 5.88 (s, 1H), 5.19 (t, J = 7.4, 1H), 3.33 (s, 1H), 3.14 (d, J = 4.2, 1H), 3.03 (t, J = 6.6, 1H), 2.87 (m, 1H), 2.8 (d, J = 4.3, 1H), 2.74 (m, 1H), 2.6 (ddd, J = 2.0, 4.5, 13.5, 1H), 2.45 (m, 1H). 2.15 (m, 1H), 1.76 (s, 3H), 1.66 (s, 3H), 1.51 (m, 1H), 1.31 (s, 3H) Experimental Example 4. Dysentery Entamoeba Test (In Vitro) As a pre-culture, Entamoeba histolytica HM-1:IMSS cells were cultured in glass test tubes in YIMDHA-S medium for 3 days. After gently inverting to mix, the culture medium was removed, and 2 mL of fresh YIMDHA-S medium was added, followed by vortexing to resuspend the cells. The suspension was then cultured at a cell density of 5 × 10⁻⁶ cells / mL. 4The cells were prepared at a rate of 1 cell / mL using uncoated 96-well plates, with 200 μL seeded per well. The plates were placed in an anaerobic chamber (a sealed container containing the Anaero Pack) and incubated at 37°C for 3 hours. Meanwhile, the compound to be treated was prepared using YIMDHA-S medium and serially diluted. After culture, *Entamoeba histolytica* adhered to the bottom of the plate. Once survival was confirmed, the medium in the plate was discarded, and 200 μL of the pre-prepared medium containing the test compound was added to each well. The plate was then incubated in an anaerobic chamber at 37°C for 48 hours. Afterward, the medium in the plate was discarded, and 110 μL of a mixture of PBS and WST1 (Premix WST-1 Cell Proliferation Assay System) (10:1 ratio) was added to each well. The plate was then incubated in an anaerobic chamber at 37°C for 30 minutes. Using a microplate reader (Nivo multi-mode microplate reader (Perkin Elmer)), absorbance was measured at 450 nm and 690 nm. The viable cell ratio was calculated by subtracting the absorbance at 690 nm from the absorbance at 450 nm. The concentration with a viable cell ratio of 50% was defined as the ED (excitatory concentration). 50 .

[0176] The results are shown in Figure 3 It can be seen that deMeOV derivatives all exhibit anti-dysentery amoebae activity.

[0177] Experimental Example 5. Analysis of the effect of liver microsomal treatment on activity production. Add 2.5 μL and 10 μL of NADPH-generating systems containing liver microsomal fractions from various animal sources (10 mM glucose-6-phosphate, 4 mM β-NADP) to the system. + The test compound was added to 100 μL of 100 mM potassium phosphate buffer (pH 7.4) containing 2 U / mL glucose-6-phosphate dehydrogenase and 10 mM MgCl2 to achieve a final concentration of 200 μM. The prepared reaction solution was incubated at 37°C for 30 minutes, then filtered to sterilize (0.20 μM) and diluted 20-fold with YIMDHA-S medium. This concentration was used as the maximum treatment concentration, and serial dilutions were performed using the same medium. As a control, the test compound (20 mM) without microsomal treatment was diluted 2,000-fold with the medium and used in the same test (final concentration: 10 μM). The same procedure as in Example 4 was followed to perform the Entamoeba histolytica test and calculate the survival rate.

[0178] The results are shown in Figure 4 and Figure 5It is known that the activity of deMeOV is greatly reduced due to microparticle treatment. In contrast, by derivatizing deMeOV starting from the 6-hydroxyl group, the activity reduction caused by microparticle treatment can be suppressed.

[0179] Experiment 6. Experiment 1 using an amoebic liver abscess model Syrian hamsters (female, 70g, 5 weeks old) underwent surgical laparotomy under isoflurane anesthesia, and their livers were inoculated with 3×10⁻⁶ dw / v. 5 One *Entamoeba histolytica* cell was collected. After abdominal suturing, the hamster was fed for 5 days. The hamster was then anesthetized and euthanized, and the liver was removed. *Entamoeba histolytica* cells were isolated from the formed liver abscess and cultured in glass test tubes using YIMDHA-S medium. In the following animal experiments, *Entamoeba histolytica* cells with expected increased pathogenicity were used within 3 months of removal from a live hamster.

[0180] Syrian hamsters (female, 70g, 5 weeks old) underwent surgical laparotomy under isoflurane anesthesia, and their livers were inoculated with 2×10⁻⁶ mmol / L. 5 The dysentery amoebae cells that had undergone the above treatment were sutured in an open abdomen. 24 hours later, the drug was administered daily for a total of 5 days according to the following method. (1) For subcutaneous injection, 0.2 mL of compound 1 (15 mg / mL, DMSO solution) was mixed with an equal volume of 5% cremophor aqueous solution and administered subcutaneously to each hamster. (2) For oral administration, 0.2 mL of compound 1 (30 mg / mL, DMSO solution) was mixed with an equal volume of 5% cremophor aqueous solution and administered orally to each hamster using a catheter (Sonde). 6 days after infection, the hamsters were anesthetized and euthanized, and the liver was removed. After measuring the total weight of the obtained liver tissue, the liver abscess was excised and its weight was measured. The therapeutic effect of the drug (compound 1) was evaluated by calculating the percentage of the weight of the liver abscess tissue relative to the total weight of the liver.

[0181] The results are shown in Figure 6 and Figure 7 It can be seen that administration of compound 1 significantly reduced liver abscesses.

[0182] In addition, the efficacy evaluation of the compounds was performed using quantitative PCR as follows.

[0183] <RNA Extraction> Hamster livers obtained in the above drug administration experiment were minced in 5 mL of PBS and homogenized using a Teflon glass homogenizer. 450 μL of Sepasol RNA I Super G was added to 50 μL of the liver homogenate, followed by 100 μL of chloroform. After mixing, the mixture was centrifuged at 15,000 × g at 4 °C for 15 minutes. The supernatant (aqueous phase) was collected, and an equal volume of isopropanol was added. The mixture was then incubated on ice for 5 minutes and centrifuged at 15,000 × g at 4 °C for 15 minutes to obtain a precipitate of RNA. 100 μL of 75% ethanol was added to the precipitate for resuscitation, and the mixture was centrifuged at 15,000 × g at 4 °C for 10 minutes. After allowing the precipitate to air dry for 10 minutes, 50 μL of nuclease-free water was added to dissolve the RNA.

[0184] Reverse Transcription PCR The concentration of extracted RNA was calculated based on absorbance using a spectrophotometer, and prepared with nuclease-free water to a concentration of 0.2 μg / μL. Using a High Capacity cDNA Reverse Transcription Kit, the reagents described in the kit's instruction manual were added to 10 μL of the prepared RNA. Reverse transcription PCR was performed using a thermal cycler at 20°C for 2 minutes, 37°C for 60 minutes, and 85°C for 5 minutes.

[0185] <rt-qpcr> The synthesized cDNA was diluted 5-fold with nuclease-free water. 2 μL of the prepared cDNA, 7.5 μL of Go Taq qPCR Master Mix, 0.1 μL of CXR Reference Dye, and 0.5 μL each of gene-specific forward and reverse primers (10 mM) were added to each well of an RT-qPCR plate (μltraAMP PCR Plates (96 wells)). The volume was then adjusted to 15 μL with nuclease-free water. Amplification was then performed using a Step One Plus Real-Time PCR System (Applied Biosystems) via a two-step PCR method (40 cycles) at 95°C for 10 minutes followed by a cycle from 95°C for 15 seconds to 60°C for 1 minute. Expression levels were analyzed using the ΔΔCt method with the Gapdh gene as an internal control.

[0186] Example 7. Evaluation of the activity of developed compounds against human umbilical vein endothelial cells (HUVECs) Human umbilical vein endothelial cells (HUVECs) were seeded into collagen-coated 96-well plates at a density of 10,000 cells per well and cultured in Endothelial Cell Growth Medium 2 (Promo Cell) at 37°C in a CO2 incubator for 24 hours. The medium was then discarded, and a culture medium was prepared to achieve a final drug concentration of 10 μM–10 pM. Cells were then cultured at 100 μL / well for 24 hours. Subsequently, a culture medium was prepared to achieve a final EdU concentration of 0.01 μM, and cells were cultured at 100 μL / well for 24 hours. Cell fixation, membrane permeabilization, EdU fluorescent tagging, and nuclear staining using Hoechst 33342 were performed according to the Click-iT EdU Alexa Fluor 594 Imaging Kits manual. The assay was performed using a high-content screening method (CellInsight). TM The CX5 High-Content Screening Platform (Thermo Fisher SCIENTIFIC) was used to detect fluorescence, and the uptake of EdU was examined using the parameters of MEANCircAvgIntenCh2.

[0187] The results are shown in Figure 8 Compounds 1 or 7 were shown to have potent inhibitory effects on HUVEC proliferation, indicating their inhibitory effect on angiogenesis in humans. The proliferation of various cancer cells and adipocytes is highly dependent on angiogenesis (RH Howland, J. Psychosoc. Nurs. Ment. Health. Serv. 2015, 53,13), thus confirming the antitumor and anti-obesity activities of these compounds.

[0188] Experiment 8. Experiment 2 using an amoebic liver abscess model The same procedure was followed for Compound 1 and Compound 7 as in Experimental Example 6.

[0189] The results are shown in Figure 9 It can be seen that administration of compound 7 significantly reduced liver abscesses.

[0190] Experimental Example 9. Enzymes against MetAP2 derived from Entamoeba histolytica (EhMetAP2) and human-derived MetAP2 (HsMetAP2). Activity inhibition evaluation Add the specified concentration of the compound to an enzyme solution (100 nM, 3 μL) of EhMetAP2 or HsMetAP2 and incubate at room temperature for 5 minutes. Then, add the substrate solution (300 μM, 3 μL) to the solution and react at 37°C for 2 hours. Afterward, calculate the residual activity of the MetAP2 enzyme and the IC50 of the compound's inhibitory effect on enzyme activity by fluorescence assay. 50 Values. The results are shown in Table 3.

[0191] [Table 3]

Claims

1. A compound of general formula (1A2), its salt, or its solvates, wherein, In the formula: L 1 R represents a single bond or linker. 11 R indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X, where X represents a halogen atom.

2. The compound, its salt, or its solvates as claimed in claim 1, characterized in that: R 2 and R 3 They connect with each other to form -(CH2)-.

3. The compound, its salt, or its solvates as claimed in claim 1, characterized in that: L 1 It consists of single bonds or connecting groups with 1 to 4 atoms forming the main chain.

4. The compound, its salt, or its solvate as claimed in claim 1, characterized in that: The alkyl, aryl, and heteroaryl groups may have substituents that are halogen atoms, hydroxyl groups, or alkoxy groups, or alkyl, alkoxy, arylalkyl, or arylalkoxy groups that can be substituted by halogen atoms, hydroxyl groups, or alkoxy groups.

5. The compound, its salt, or its solvates as claimed in claim 1, characterized in that: The compound is a compound represented by the general formula (1A3). In the formula: L 1 and R 11 Same as above.

6. The compound, its salt, or its solvate as claimed in claim 5, characterized in that: L 1 It is a single bond, and R 11 It is an alkyl group having 1 to 4 carbon atoms.

7. An anti-dysentery amoeboid agent, characterized in that: Contains at least one selected from compounds of general formula (1), their salts and their solvates. In the formula: R 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11 L 1 R represents a single bond or linker. 11 R indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X, where X represents a halogen atom.

8. The anti-dysentery amoeboid agent as described in claim 7, characterized in that: It is a preventive or therapeutic agent for amoebic dysentery.

9. An anticancer agent, characterized in that: Contains at least one selected from compounds of general formula (1), their salts and their solvates. In the formula: R 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11 L 1 R represents a single bond or linker. 11 R indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X, where X represents a halogen atom.

10. An anti-obesity agent, characterized in that: Contains at least one selected from compounds of general formula (1), their salts and their solvates. In the formula: R 1 Represents a hydrogen atom or -C(=O)-L 1 -R 11 L 1 R represents a single bond or linker. 11 R indicates a substituted alkyl group, a substituted aryl group, or a substituted heteroaryl group. 2 and R 3 They connect to form -(CH2)-, or R 2 Represents a hydrogen atom and R 3 It represents -(CH2)-X, where X represents a halogen atom.

Images