Compound having Anti-entamoeba histolytica activity

Abstract

Provided is a compound having anti-Entamoeba histolytica activity. The present invention is a compound represented by general formula (1), a salt thereof, or a solvate thereof.

Description

Compounds with anti-entamoebic activity

[0001] The present invention relates to compounds having anti-Entamoeba histolytica activity.

[0002] Entamoebiasis is an intestinal protozoal disease that infects approximately 1% of the world's population, causing significant damage, especially in tropical developing countries. The current drug, metronidazole, has been shown to have side effects such as genotoxicity and to develop drug resistance, so the development of safer drugs with alternative modes of action is necessary.

[0003] Against this background, the natural product Fumagillin has been found to have potent anti-Entamoeba histolytica activity, and has been reported to show therapeutic effects in liver abscess model animals.Fumagillin derivatives have been in clinical development for over 30 years for the treatment of cancer, obesity, malaria, etc., but have not yet been marketed due to side effects such as neurotoxicity.

[0004] J. Am. Chem. Soc. 2014, 136, 4426-4436.

[0005] An objective of the present invention is to provide a compound having anti-Entamoeba histolytica activity.

[0006] The present inventors focused on ovalicin, which has a structure similar to fumagillin.

[0007]

[0008] Derivatization of fumagillin has been achieved by converting the sesquiterpene structure at the 5-position to other structures. The present inventors focused on the methoxy at the 6-position of ovalicin and attempted to convert it to other structures. Specifically, if the methoxy of ovalicin can be demethylated and converted to a hydroxyl group (demethyl ovalicin), ovalicin derivatives with diverse structures can be obtained by using the hydroxyl group (e.g., by reacting with a carboxyl compound). However, ovalicin contains two epoxides that are easily decomposed, making it difficult to efficiently obtain decyl ovalicin using organic synthesis methods.

[0009] Therefore, we focused on obtaining demethyl ovalicin through biosynthesis (Fig. 1). However, the enzyme X (7-hydroxylation enzyme Ova-C7H) essential for this biosynthesis, which converts 5-keto-demethoxyfumagillol to demethoxy ovalicin, is still unknown.

[0010] As a result of extensive research, the present inventors have succeeded in elucidating the biosynthesis of demethyl ovalicin and discovered that demethyl ovalicin and its derivatives have anti-Entamoeba histolytica activity. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects:

[0011] Item 1. General formula (1A2):

[0012]

[0013] [In the formula: L 1 represents a single bond or a linker. 11 represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, or a solvate thereof.

[0014] Item 2. R 2 and R 3 are linked to each other to form —(CH2)—, or a salt thereof, or a solvate thereof according to Item 1.

[0015] Item 3. L 1 Item 2. The compound, salt thereof, or solvate thereof according to Item 1, wherein is a single bond or a linker having 1 to 4 atoms constituting the main chain.

[0016] Item 4. The compound, salt thereof, or solvate thereof according to Item 1, wherein the substituent that the alkyl group, the aryl group, and the heteroaryl group may have is a halogen atom, a hydroxy group, or an alkoxy group, or an alkyl group, an alkoxy group, an aryl group, an alkyl group, or an arylalkoxy group that is optionally substituted with a halogen atom, a hydroxy group, or an alkoxy group.

[0017] Item 5. The compound represented by general formula (1A3):

[0018]

[0019] [In the formula: L 1 and R 11 The compound according to item 1, a salt thereof, or a solvate thereof, wherein the compound is a compound represented by the formula:

[0020] Item 6. L 1 is a single bond and R 11 Item 6. The compound, salt, or solvate thereof according to Item 5, wherein is an alkyl group having 1 to 4 carbon atoms.

[0021] Section 7. General formula (1):

[0022]

[0023] [In the formula: R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates L 1 represents a single bond or a linker. 11 represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, and a solvate thereof.

[0024] Item 8. The anti-entamoebic agent according to Item 7, which is a preventive or therapeutic agent for amebiasis.

[0025] Item 9. General formula (1):

[0026]

[0027] [In the formula: R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates L 1 represents a single bond or a linker. 11 represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, and a solvate thereof.

[0028] Item 10. General formula (1):

[0029]

[0030] [In the formula: R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates L 1 represents a single bond or a linker. 11 represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, and a solvate thereof.

[0031] According to the present invention, a compound having anti-Entamoeba histolytica activity can be provided, which may also be effective against cancer and obesity.

[0032] The biosynthetic route of Demethyl Ovalicin and Ovalicin is shown. NMR measurement data of Demethyl Ovalicin is shown. The structures of Compounds 1 to 12 and the results of the Entamoeba histolytica test (Test Example 4) (ED 50 ) are shown. The results of the Entamoeba histolytica test (Test Example 5) using Demethyl Ovalicin (deMeOV) as the test compound are shown. Circle plots indicate the results of the Entamoeba histolytica test using the test compound before microsome treatment, and square plots indicate the results of the Entamoeba histolytica test using the test compound after microsome treatment. The vertical axis indicates the viable cell ratio, and the horizontal axis indicates the concentration of the test compound before microsome treatment. deMeOV is sensitive to metabolism in the liver. In fact, its therapeutic effect in Entamoeba histolytica-infected animals is limited. The results of the Entamoeba histolytica test (Test Example 5) using Compound 1 as the test compound are shown. The legend in the figure is the same as in Figure 4. Compound 1 is resistant to drug metabolism in the liver. The therapeutic effect of Compound 1 when administered subcutaneously to an amoeba liver abscess model animal is shown. The vertical axis indicates the ratio of liver abscess tissue weight to total liver weight, and the values ​​on the horizontal axis indicate the dose. "Not treated" indicates when Compound 1 was not administered. This figure shows the therapeutic effect of oral administration of Compound 1 to an animal model of amoebic liver abscess. The legend for this figure is the same as for Figure 6. This figure shows the effect of adding Compound 1 or Compound 7 on the proliferation of human umbilical vein endothelial cells (HUVEC). The vertical axis shows measured EdU values. The horizontal axis shows the concentration of the test compound. Compound 1 and Compound 7 inhibit the proliferation of not only Entamoeba histolytica but also human cells. This figure shows the therapeutic effect of subcutaneous or oral administration of Compound 1 or Compound 7 to an animal model of amoebic liver abscess. The vertical axis shows the ratio of liver abscess tissue weight to total liver weight, the horizontal axis shows the administered compound, "not treated" indicates the case where Compound 1 and Compound 7 were not administered, and MTZ indicates the case where the current drug metronidazole was administered. The dose is shown above the graph.

[0033] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0034] As used herein, "identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by 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 the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0035] As used herein, the term "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 having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0036] As used herein, polynucleotides such as DNA and RNA may be chemically modified as described below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide may be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also preferably used are BNA (LNA), in which the conformation of the sugar moiety is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide.

[0037] 2. Compound In one aspect, the present invention provides a compound represented by the general formula (1):

[0038]

[0039] (Hereinafter, this invention relates to a compound represented by the formula (hereinafter, this invention may be referred to as "the compound of the present invention"), a salt thereof, or a solvate thereof (Hereinafter, this invention may be referred to as "the active ingredient of the present invention"). These will be explained below.

[0040] R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates R 1 From the viewpoint that activity is more stably maintained in vivo, -C(=O)-L 1 -R 11 is preferred.

[0041] L 1 represents a single bond or a linker.

[0042] L 1 The linker represented by the formula (I) contains a chain structure. The chain structure is not particularly limited, but may include a chain structure having, for example, 1 to 6 atoms constituting the main chain, preferably 1 to 4, more preferably 1 to 3, and even more preferably 1 to 2 atoms constituting the main chain from the viewpoint of anti-Entamoeba histolytic activity. Examples of atoms constituting the main chain include carbon atoms, oxygen atoms, sulfur atoms, and nitrogen atoms. Part or all of the chain structure may be an alkenylene group, alkylene group, heteroalkylene group, or heteroalkenylene group. The chain structure may be either linear or branched, but is preferably linear from the viewpoint of anti-Entamoeba histolytic activity. The chain structure may also include a partial structure such as -O-, -C(=O)-O-, -CO-NH-, -C(=O)-, -NH-, or -S(=O)2- on the main chain.

[0043] L 1 From the viewpoint of anti-Entamoeba histolytica activity, is preferably —CO—NH— or an alkenylene group (having preferably 2 to 4 carbon atoms, more preferably 2 to 3 carbon atoms, and even more preferably 2 carbon atoms), and more preferably —CO—NH—.

[0044] L 1is preferably a single bond from the viewpoint of anti-Entamoeba histolytica activity and that the activity is more stably maintained in vivo.

[0045] R 11 represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group.

[0046] R 11 The alkyl group represented by the formula (I) includes any of linear, branched, and cyclic alkyl groups. The number of carbon atoms in the alkyl group (when linear or branched) 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, even more preferably 1 to 2, and particularly preferably 1. In one embodiment, the number of carbon atoms is particularly preferably 2. The number of carbon atoms in the alkyl group (when cyclic) is not particularly limited and is, for example, 3 to 7, preferably 4 to 6. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, a 3-methylpentyl group, an n-heptyl group, and an n-octyl group.

[0047] R 11 The aryl group represented by the formula (I) is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 10 carbon atoms, and even more preferably 6 to 8 carbon atoms. The aryl group may be either monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, naphthyl group, biphenyl group, pentalenyl group, indenyl group, anthranyl group, tetracenyl group, pentacenyl group, pyrenyl group, perylenyl group, fluorenyl group, and phenanthryl group, with a phenyl group being particularly preferred.

[0048] R 11The heteroaryl group represented by the formula (I) is not particularly limited, and examples thereof include heteroaryl groups containing a sulfur atom, a nitrogen atom, an oxygen atom, or the like as a heteroatom. The heteroaryl group preferably has 6 to 12 ring-constituting atoms, more preferably 6 to 10 ring-constituting atoms, and even more preferably 6 to 8 ring-constituting atoms. The aryl group may be either monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the heteroaryl group include a pyrrolyl group, a pyridyl group, a pyrrolidyl group, a piperidyl group, an imidazolyl group, an imidazoyl group, a pyrazolyl group, a pyrazolyl group, a pyrimidyl group, a pyridazyl group, a piperazyl group, a triazinyl group, an oxazolyl group, an isoxazolyl group, a morpholyl group, a thiazolyl group, an isothiazolyl group, a furanyl group, a thiophenyl group, an indolyl group, a quinolyl group, an isoquinolyl group, a benzimidazolyl group, a quinazolyl group, a phthalazyl group, a purinyl group, a pteridyl group, a benzofuranyl group, a coumaryl group, a chromonyl group, a benzothiophenyl group, etc. The heteroaryl group is preferably a heteroaryl group containing a nitrogen atom, and particularly preferably a pyridyl group.

[0049] The substituents that the alkyl group, the aryl group, and the heteroaryl group may have are not particularly limited, but from the viewpoint of anti-Entamoeba histolytica activity, particularly preferred are a halogen atom, a hydroxy group, or an alkoxy group, or an alkyl group, alkoxy group, aryl group, alkyl group, or arylalkoxy group that may be substituted with a halogen atom, a hydroxy group, or an alkoxy group.

[0050] Examples of the halogen atom as a substituent include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferred are a fluorine atom and a chlorine atom.

[0051] Examples of the alkoxy group as a substituent include linear or branched alkoxy groups having 1 to 8 carbon atoms (preferably 1 to 4 carbon atoms).

[0052] The alkyl group as a substituent is R 11 The alkyl group is the same as the alkyl group represented by the following formula:

[0053] Examples of the arylalkyl group as a substituent include alkylaryl groups in which the hydrogen atoms (e.g., 1 to 3, preferably 1 hydrogen atom) of the alkyl group are substituted with the aryl group. Specific examples of the arylalkyl group include a benzyl group and a phenethyl group.

[0054] Examples of the arylalkoxy group as a substituent include arylalkoxy groups in which the hydrogen atoms (for example, 1 to 3, preferably 1 hydrogen atom) of the above alkoxy groups are substituted with the above aryl groups.

[0055] R 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X.

[0056] R 2 and R 3 are linked to each other to form -(CH2)-, the -(CH2)- and R 2 and the oxygen atom adjacent to R 3 and the adjacent carbon atom form an ethylene oxide structure (see general formula (1A1) below).

[0057] X represents a halogen atom. Examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and preferably a fluorine atom or a chlorine atom.

[0058] R 2 and R 3 From the viewpoint of anti-Entamoeba histolytica activity, it is particularly preferred that the groups are linked to each other to form --(CH2)--.

[0059] In view of anti-Entamoeba histolytica activity, the compound of the present invention is preferably a compound represented by the general formula (1A1) or (1A2):

[0060]

[0061] [In the formula: R 1 , L 1 , R 11 , R 2 , and R3 are the same as above.], and particularly preferred are compounds represented by the general formula (1A3):

[0062]

[0063] [In the formula: L 1 and R 11 is the same as above.]

[0064] In one embodiment, the compound of the present invention preferably has the general formula (1AX):

[0065]

[0066] [In the formula: R 1 , R 2 , and R 3 is the same as above.]

[0067] The salt of the compound of the present invention is not particularly limited as long as it is a pharmaceutically acceptable salt.The salt can be either an acidic salt or a basic salt.Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, paratoluenesulfonate, etc., and examples of basic salts include alkali metal salts such as sodium salt and potassium salt, and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, tri(hydroxyalkyl)amine, etc.

[0068] The compound of the present invention can also be in the form of a hydrate or solvate. Examples of the solvent include pharmaceutically acceptable organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0069] 3. Compound Preparation 1 (R 1 In the compound of the present invention, when R 1The compound (Demethyl Ovalicin) in which is a hydrogen atom can be obtained by biosynthesis. Specifically, (A) it can be biosynthesized using modified cells (sometimes referred to herein as "modified cells of the present invention") containing polynucleotide A, which contains an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and contains a coding sequence for protein A having the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin. This is described below.

[0070] SEQ ID NO: 1 is the amino acid sequence of a protein derived from the insect-infectious filamentous fungus Metarhizium robertsii ARSEF23 strain, and is the amino acid sequence of NCBI accession number: XP_007824537.1.

[0071] The identity in (A) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0072] In the above (A), when the identity is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 1 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and still more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0073] The structures of 5-keto-demethoxyfumagillol and demethoxy ovalicin are shown in FIG.

[0074] The presence or absence of the activity to convert 5-keto-demethoxyfumagillol to demethoxy ovalicin can be confirmed by in vitro or in vivo tests. In in vivo tests, for example, cells that produce 5-keto-demethoxyfumagillol are used as cells or 5-keto-demethoxyfumagillol is introduced into cells, and the cells are cultured with or without expressing a test protein. If demethoxy ovalicin is produced only when the test protein is expressed, it can be determined that the test protein has the activity to convert 5-keto-demethoxyfumagillol to demethoxy ovalicin.

[0075] In addition to the above amino acid sequence, Protein A may contain other sequences such as known proteins or peptides, such as protein tags and signal sequences. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0076] In one embodiment, polynucleotide A contains a promoter upstream of the coding sequence for expressing the mRNA of protein A. The promoter is not particularly limited and can be selected appropriately depending on the host. Examples of promoters include the Aspergillus oryzae TAA gene (amyB) promoter, the Aspergillus oryzae GLA gene (glaA) promoter, and the Aspergillus oryzae AGL gene (agdA) promoter. Other examples include the TDH3 promoter, GAL10 promoter, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, and CAG promoter.

[0077] Polynucleotide A may contain other elements (e.g., a multiple cloning site (MCS), a drug resistance gene, an origin of replication, etc.) as needed. For example, in a polynucleotide where a promoter and a protein-coding sequence are arranged in this order from the 5' end, an MCS may be arranged between the promoter and the coding sequence (preferably adjacent to either one or both) and on the 3' end of the coding sequence (preferably adjacent to it). The MCS is not particularly limited, as long as it contains multiple restriction enzyme sites (e.g., 2 to 50, preferably 2 to 20, more preferably 2 to 10).

[0078] Polynucleotide A may constitute a vector. The type of vector is not particularly limited and may be appropriately selected depending on the host. For example, in E. coli, examples of vectors include ColE1-based plasmids such as pBR322 derivatives, pACYC-based plasmids with p15A origin, pSC-based plasmids, and mini-F plasmids derived from F factors such as Bac-based plasmids.

[0079] The modified cells of the present invention containing polynucleotide A can be used to prepare cells for producing demethyl ovalicin, and in a preferred embodiment, can be used as cells for producing demethyl ovalicin.

[0080] The modified cell of the present invention preferably further comprises (B) a polynucleotide B comprising a coding sequence for a protein B having the activity of converting demethoxy ovalicin to demethyl ovalicin, thereby enabling the production of demethyl ovalicin under conditions in which 5-keto-demethoxyfumagillol is supplied.

[0081] Protein B having the activity of converting demethoxy ovalicin to demethyl ovalicin is known, or can be easily identified or produced based on homology analysis based on the known amino acid sequence.

[0082] Protein B is preferably (B1) protein B1 comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747157.2.

[0083] The identity in (B1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0084] When the identity in (B1) above is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and still more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0085] The structures of Demethoxy Ovalicin and Demethyl Ovalicin are shown in FIG.

[0086] The presence or absence of the activity to convert demethoxy ovalicin to demethyl ovalicin can be confirmed by in vitro or in vivo tests. In in vivo tests, for example, cells that produce demethoxy ovalicin are used as cells or demethoxy ovalicin is introduced into cells, and the cells are cultured with or without expressing a test protein. If demethyl ovalicin is produced only when the test protein is expressed, the test protein can be determined to have the activity to convert demethoxy ovalicin to demethyl ovalicin.

[0087] Other sequences contained in protein B and the structure of polynucleotide B are the same as those of protein A and polynucleotide A.

[0088] The modified cells of the present invention further comprising polynucleotide B can be used to prepare cells for producing demethyl ovalicin, and can be used as cells for producing demethyl ovalicin.

[0089] The modified cell of the present invention preferably further comprises (C) a polynucleotide C comprising a coding sequence for protein C having the activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol. This enables the cell to produce demethoxy ovalicin under conditions in which β-trans-bergamotene is supplied, and further comprises polynucleotide B, enabling the cell to produce demethyl ovalicin.

[0090] Protein C having the activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol is known, or can be easily identified or produced based on homology analysis based on the known amino acid sequence.

[0091] Protein C is preferably (C1) protein C1 comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 3. SEQ ID NO: 3 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747154.1.

[0092] The identity in (C1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0093] When the identity in (C1) above is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 3 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and still more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0094] The structures of β-trans-bergamotene and 5-keto-demethoxyfumagillol are shown in FIG.

[0095] The presence or absence of the activity to convert β-trans-bergamotene to 5-keto-demethoxyfumagillol can be confirmed by in vitro or in vivo tests. In in vivo tests, for example, cells that produce β-trans-bergamotene are used or β-trans-bergamotene is introduced into cells, and the cells are cultured with or without expressing a test protein. If 5-keto-demethoxyfumagillol is produced only when the test protein is expressed, the test protein can be determined to have the activity to convert β-trans-bergamotene to 5-keto-demethoxyfumagillol.

[0096] Other sequences contained in protein C and the structure of polynucleotide C are the same as those of protein A and polynucleotide A.

[0097] The modified cells of the present invention further comprising polynucleotide C can be used to prepare cells for producing demethyl ovalicin, and in a preferred embodiment, can be used as cells for producing demethyl ovalicin.

[0098] The modified cells of the present invention preferably further contain (D) polynucleotide D, which contains a coding sequence for protein D having the activity of converting farnesyl diphosphate to β-trans-bergamotene. This allows the cells to produce demethoxy ovalicin under conditions in which farnesyl diphosphate is supplied, if they further contain polynucleotide C, or to produce demethyl ovalicin if they further contain polynucleotide B in addition to polynucleotide C.

[0099] Protein D having the activity of converting farnesyl diphosphate to β-trans-bergamotene is known, or can be easily identified or produced based on homology analysis based on known amino acid sequences.

[0100] Protein D is preferably (D1) protein D1 comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 4. SEQ ID NO: 4 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747153.1.

[0101] The identity in (D1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0102] When the identity in (D1) above is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 4 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and still more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0103] The structures of farnesyl diphosphate and β-trans-bergamotene are shown in FIG.

[0104] The presence or absence of the activity to convert farnesyl diphosphate to β-trans-bergamotene can be confirmed by in vitro or in vivo tests. In in vivo tests, for example, farnesyl diphosphate-producing cells are used or farnesyl diphosphate is introduced into cells, and the cells are cultured with or without the expression of a test protein. If β-trans-bergamotene is produced only when the test protein is expressed, the test protein can be determined to have the activity to convert farnesyl diphosphate to β-trans-bergamotene.

[0105] Other sequences contained in protein D and the structure of polynucleotide D are the same as those of protein D and polynucleotide D.

[0106] The modified cells of the present invention further comprising polynucleotide D can be used to prepare cells for producing demethyl ovalicin, and in a preferred embodiment, can be used as cells for producing demethyl ovalicin.

[0107] The above-mentioned polynucleotides can be easily prepared by known genetic engineering techniques, such as PCR, restriction enzyme digestion, and DNA ligation.

[0108] The modified cells of the present invention are cells obtained by modifying existing cells. The modifications are genetic modifications, including the introduction of an exogenous polynucleotide and the introduction of a mutation into an endogenous genome.

[0109] The exogenous polynucleotide is a polynucleotide containing a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of the cell before modification, and is not particularly limited insofar as it is so.

[0110] Examples of mutations in the endogenous genome include gene disruption, mutations in protein coding regions, partial deletions, mutations in splicing regulatory regions, and mutations in expression control regions (e.g., promoters, activators, enhancers, repressor binding elements, etc.).

[0111] The method for genetically modifying cells is not particularly limited, and any method according to or equivalent to a known method can be used, such as the protoplast-PEG method, electroporation, lipofection, particle gun method, and CRISPR-Cas method.

[0112] The cells to be modified may be either eukaryotic or prokaryotic cells. However, when the modified cells of the present invention are used as cells for producing demethyl ovalicin, eukaryotic cells are preferred. From the viewpoint of the efficiency of demethyl ovalicin production, fungal cells are preferred as eukaryotic cells, and cells capable of forming mycelia are particularly preferred. Cells capable of forming mycelia are not particularly limited as long as they are cells that form mycelia at some stage in their life cycle, and examples thereof include fungi of the genus Aspergillus, Penicillium, Trichoderma, Cephalosporium, and Acremonium, with fungi of the genus Aspergillus being particularly preferred, and Aspergillus nidulans being particularly preferred.

[0113] When at least one of polynucleotides B to D is contained, polynucleotides A to D may all be separate polynucleotide molecules, or two or more of polynucleotides A to D may be the same polynucleotide molecule.

[0114] In one embodiment of the present invention, polynucleotide A is an exogenous polynucleotide. Also, in one embodiment of the present invention, when at least one of polynucleotides B to D is contained, two or more (preferably three or more, particularly preferably four) polynucleotides including polynucleotide A are exogenous polynucleotides.

[0115] In one embodiment of the present invention, (E) the strain is modified so as not to express protein E having the activity of converting demethyl ovalicin to ovalicin, or to reduce the expression or function of protein E. This inhibits the conversion of demethyl ovalicin to ovalicin, allowing demethyl ovalicin to be obtained more efficiently.

[0116] "Function" refers to the activity of converting dimethyl ovalicin to ovalicin. "Expression" encompasses both gene mRNA expression and gene protein expression, but preferably refers to gene protein expression. "Decrease" refers to a decrease in the activity of the gene protein and / or the expression level of the gene after modification (index values ​​of gene function and / or expression) relative to 100% of the activity of the gene protein and / or the expression level of the gene before modification (index values ​​of function and / or expression when the gene is unmodified), for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.02% or less, or 0.01% or less.

[0117] The modified cells of the present invention preferably have the ability to produce demethyl ovalicin. Specifically, if the modified cells of the present invention contain polynucleotide B in addition to polynucleotide A, they can produce demethyl ovalicin under conditions in which 5-keto-demethoxyfumagillol is supplied. Particularly preferably, the modified cells of the present invention contain all of polynucleotides A to D. This makes it possible to obtain demethyl ovalicin using farnesyl diphosphate (which is one of the terpenoids essential for the maintenance of life in living organisms, and is also a biosynthetic intermediate for various useful terpenoids such as carotenoids, plant hormones, and ubiquinone, and is biosynthesized in many eukaryotic cells).

[0118] Demethyl ovalicin can be produced by culturing the modified cells of the present invention capable of producing demethyl ovalicin.

[0119] The culture can be carried out according to or in accordance with a method known to those skilled in the art, depending on the species of the modified cells of the present invention.

[0120] The medium (e.g., culture solution) preferably contains a carbon source. Examples of carbon sources include carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides; invert sugar obtained by hydrolyzing sucrose; glycerol; carbon-1 compounds (hereinafter referred to as C1 compounds) such as methanol, formaldehyde, formate, carbon monoxide, and carbon dioxide; oils such as corn oil, palm oil, and soybean oil; acetate; animal fats and oils; animal oils; fatty acids such as saturated fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; glycerol fatty acid esters such as monoglycerides, diglycerides, and triglycerides; polypeptides such as microbial proteins and plant proteins; renewable carbon sources such as hydrolyzed biomass carbon sources; yeast extract; or combinations thereof. Examples of nitrogen sources that can be used include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as soybean hydrolysate; ammonia gas; and aqueous ammonia. Organic trace nutrient sources preferably include required substances such as vitamin B1 and L-homoserine, or yeast extract in appropriate amounts. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. may be added as needed. The medium used in the present invention may be either a natural medium or a synthetic medium, as long as it contains a carbon source, a nitrogen source, inorganic ions, and, as needed, other organic trace components.

[0121] Examples of monosaccharides include trioses such as ketotriose (dihydroxyacetone) and aldotriose (glyceraldehyde); tetroses such as ketotetrose (erythrulose) and aldotetrose (erythrose, threose); pentoses such as ketopentoses (ribulose, xylulose), aldopentose (ribose, arabinose, xylose, lyxose), and deoxysugars (deoxyribose); hexoses such as ketohexose (psicose, fructose, sorbose, tagatose), aldohexose (allose, altrose, glucose, mannose, gulose, idose, galactose, talose), and deoxysugars (fucose, fuculose, rhamnose); and heptoses such as sedoheptulose. Preferred are carbohydrates such as C6 sugars (fructose, mannose, galactose, glucose), and C5 sugars (xylose, arabinose).

[0122] Examples of disaccharides include sucrose, lactose, maltose, trehalose, turanose, cellobiose, etc., with sucrose and lactose being preferred.

[0123] Examples of oligosaccharides include trisaccharides such as raffinose, melezitose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and other oligosaccharides such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), and mannanoligosaccharides (MOS).

[0124] Examples of polysaccharides include glycogen, starch (amylose, amylopectin), cellulose, dextrin, and glucan (β1,3-glucan), with starch and cellulose being preferred.

[0125] Microbial proteins include polypeptides obtainable from yeast or bacteria.

[0126] Vegetable proteins include polypeptides obtainable from soybeans, corn, canola, jatropha, palm, peanuts, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, safflower, sesame, and flaxseed.

[0127] Lipids include substances containing one or more saturated or unsaturated fatty acids of C4 or higher.

[0128] The oil is preferably a lipid that contains one or more saturated or unsaturated fatty acids of C4 or higher and is liquid at room temperature, and examples thereof include lipids obtainable from soybeans, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, safflower, sesame, flaxseed, oleaginous microbial cells, Chinese tallow tree, or a combination of two or more thereof.

[0129] Fatty acids include compounds represented by the formula RCOOH, where "R" represents a hydrocarbon group.

[0130] Unsaturated fatty acids are compounds that have at least one carbon-carbon double bond at "R", and examples include oleic acid, vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.

[0131] Saturated fatty acids are compounds in which "R" is a saturated aliphatic group, and include docosanoic acid, eicosanoic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, dodecanoic acid, and the like.

[0132] Among these, the fatty acids preferably contain one or more C2 to C22 fatty acids, and more preferably contain C12 fatty acids, C14 fatty acids, C16 fatty acids, C18 fatty acids, C20 fatty acids, and C22 fatty acids.

[0133] Further, examples of the carbon source include salts of these fatty acids, derivatives, and salts of derivatives, such as lithium salts, potassium salts, and sodium salts.

[0134] Further, examples of carbon sources include combinations of lipids, oils, fats and oils, fatty acids, and glycerin fatty acid esters with carbohydrates such as glucose.

[0135] Renewable carbon sources include hydrolyzed biomass carbon sources.

[0136] Biomass carbon sources include cellulosic substrates such as wood, paper, and pulp waste, plant leaves, and fruit pulp; plant parts such as stalks, grains, roots, and tubers.

[0137] Plants that can be used as biomass carbon sources include legumes such as corn, wheat, rye, sorghum, triticale, rice, millet, barley, cassava, and peas, as well as potatoes, sweet potatoes, bananas, sugarcane, and tapioca.

[0138] When a renewable carbon source such as biomass is added to a medium, it is preferable to pretreat it, for example, by enzymatic pretreatment, chemical pretreatment, or a combination of enzymatic and chemical pretreatment.

[0139] In the production method of the present invention, it is preferable to use a culture medium containing an adsorption resin. This can increase the production efficiency of Demethyl Ovalicin. The adsorption resin is not particularly limited as long as it is a resin material that can adsorb various organic substances through physical interactions between its surface and the adsorbed substances. Typically, the adsorption resin has a porous structure (preferably with a specific surface area of ​​300 to 1200 m). 2 The resin is, for example, styrene-divinylbenzene, styrene, divinylbenzene, methacrylic acid ester, etc.

[0140] When an adsorption resin is used, its concentration in the culture medium is preferably 20 to 100 g / L.

[0141] The culture temperature is preferably 20°C to 37°C.

[0142] The culture time is preferably 20 to 100 hours.

[0143] The modified cells of the present invention are preferably cultured under aerobic, anaerobic, or anoxic conditions depending on the nature of the host.

[0144] Examples of methods for culturing the modified cells of the present invention include culturing using known fermentation methods such as batch culture, fed-batch culture, and continuous culture.

[0145] After the cultivation is completed, solid-liquid separation such as filtration or centrifugation is performed, and intracellular components are extracted from the obtained solid sample (for example, organic solvent extraction such as acetone extraction, ultrasonic disruption, etc.), and the obtained soluble portion is purified by chromatography to obtain the target product.

[0146] 4. Compound Preparation 2 (R 1 -C(=O)-L 1 -R 11 In the compound of the present invention, R 1 -C(=O)-L 1 -R 11 The compound represented by the formula (I) can be synthesized by various methods using Demethyl Ovalicin as a starting material. For example, the compound can be synthesized according to a method including the following steps:

[0147]

[0148] Demethyl ovalicin biosynthesized according to the method described in the above item 3 can be used as Compound A. Compound B can be commercially available or synthesized according to a known method.

[0149] In view of yield, ease of synthesis, etc., the amount of compound B used is usually preferably 0.5 to 20 moles, more preferably 1 to 12 moles, per mole of compound A.

[0150] This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, but examples thereof include dichloromethane. The solvent may be used alone or in combination.

[0151] This reaction is carried out, if necessary, in the presence of a catalyst such as a base, for example, 4-dimethylaminopyridine, triethylamine, etc.

[0152] The reaction can be carried out under heating, at room temperature, or under cooling, and is usually carried out at a temperature of 0 to 120° C. The reaction temperature is particularly preferably 15 to 50° C. The reaction time is not particularly limited, and is usually 30 minutes to 60 hours.

[0153] The progress of the reaction can be monitored by conventional methods such as chromatography. After the reaction is complete, the solvent is removed by distillation, and the product can be isolated and purified by conventional methods such as chromatography and recrystallization. The structure of the product can also be determined by elemental analysis, MS (ESI-MS), IR analysis, and the like. 1 H-NMR, 13 It can be identified by C-NMR or the like.

[0154] 5. Uses The active ingredient of the present invention has anti-Etamoeba histolytica activity. Furthermore, the active ingredient of the present invention can be used for applications targeting MetAP2 as a molecular target. Examples of such applications include anti-cancer, anti-obesity, anti-angiogenesis, prevention or amelioration (treatment) of infectious diseases such as malaria, giardiasis, and microsporidiosis, prevention or amelioration (treatment) of cancer, prevention or amelioration (treatment) of Prader-Willi syndrome, and prevention or amelioration (treatment) of diabetes. Therefore, in one aspect, the present invention relates to an agent for the above-mentioned uses (e.g., an anti-Etamoeba histolytica agent, an agent for preventing or treating amebiasis) containing the active ingredient of the present invention.

[0155] Entamoeba histolytica is a pathogenic organism (Entamoeba histolytica) that causes amebiasis. Anti-entamoeba activity refers to the ability to kill or inhibit the growth of Entamoeba histolytica. Anti-entamoeba activity can improve or prevent symptoms of Entamoeba histolytica (diarrhea, mucus and blood in the stool, tenesmus, lower abdominal pain or discomfort during bowel movements, mucus and blood in the stool, abscesses (liver abscess, etc.), vomiting, weight loss, night sweats, fatigue, etc.). "Improvement" refers to the improvement or alleviation of symptoms or conditions, the prevention or delay of worsening of symptoms or conditions, and the reversal, prevention, or delay of the progression of symptoms or conditions.

[0156] The active ingredient of the present invention and the agent of the present invention can be used as a medicine, a reagent, a food additive, a food composition (including health foods, health promoting agents, nutritional supplements (supplements, etc.)), etc.

[0157] The agent of the present invention is not particularly limited as long as it contains the active ingredient of the present invention, and may further contain other ingredients as necessary.The other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients.The other ingredients include not only ingredients with pharmacological effects but also additives.Examples of additives include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0158] The mode of use of the active ingredient of the present invention and the agent of the present invention is not particularly limited. The active ingredient of the present invention and the agent of the present invention can be used in vitro (e.g., added to a culture medium for cultured cells) or in vivo (e.g., administered to / ingested by an animal).

[0159] The active ingredient of the present invention and the agent of the present invention are not particularly limited to specific subjects, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Examples of cells include animal cells. The types of cells are also not particularly limited, and examples include blood cells, hematopoietic stem cells and progenitor cells, gametes (sperm, eggs), 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.

[0160] The agent of the present invention can take any dosage form, for example, oral preparations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparations such as injectable preparations (for example, drip injections (for example, intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0161] The route of administration of the agent of the present invention is not particularly limited as long as the desired effect can be obtained, and examples thereof include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

[0162] When the agent of the present invention is used as a food composition, examples thereof include liquid, gel or solid foods, such as beverages such as juice, soft drinks, tea, soup, and soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, powdered milk for infants, cake mix, powdered or liquid dairy products, bread, and cookies.

[0163] The content of the active ingredient in the agent of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0164] The dosage of the active ingredient or agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is generally 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in the case of parenteral administration. The dosage can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc.

[0165] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0166] Test Example 1. Biosynthetic Design and Enzyme Search for Demethyl Ovalicin Based on the Fumagillin biosynthetic route of Non-Patent Document 1, the biosynthetic routes for Demethyl Ovalicin and Ovalicin were designed (FIG. 1).

[0167]

[0168] However, the enzyme X (7-hydroxylation enzyme Ova-C7H) that converts 5-keto-demethoxyfumagillol to demethoxy ovalicin is still unknown. Therefore, we searched for this enzyme.

[0169] Although no biosynthetic gene cluster (BGC) for OVs has been reported, genome mining identified a candidate BGC in the insect-infecting fungus Metarhizium robertsii ARSEF23. RNA-seq data for this strain under multiple culture conditions have been registered in a public database (SRA), and in silico analysis confirmed high expression of this BGC in Sabouraud medium. M. robertsii was actually cultured under the same conditions and OV production was confirmed. Given that the predicted BGC contains two copies of an alpha-KG-dependent non-heme iron oxidase gene and that the C6 hydroxyl group introduction in FM biosynthesis is catalyzed by the same enzyme, Fma-C6H, we predicted that the C7 hydroxyl group introduction in OV biosynthesis would also be catalyzed by a similar enzyme. In fact, when four fumagillin biosynthetic genes (fma-TC, fma-P450, fma-C6H, fma-MT) were co-expressed in A. nidulans with the MAA_08348 gene (ova-C7H) cloned from M. robertsii, OV production was confirmed. From these results, we identified the 7-hydroxylation enzyme Ova-C7H responsible for OV production.

[0170] Test Example 2. Production of Demethyl Ovalicin (deMeOV) <Plasmid Construction> The following plasmids were constructed using the gap-repair cloning method using 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 robertsii strain ARSEF23. Each was amplified by PCR. Plasmid construction was carried out in the following order: (1) The PCR-amplified fma-TC gene was introduced into the pKW20093 plasmid digested with SwaI to create pKW20517. (2) The PCR-amplified fma-P450 gene was introduced into the pKW10010 plasmid digested with SwaI to create pKW20518. (3) The PCR-amplified fma-C6H gene was introduced into the pKW10012 plasmid digested with SwaI to create pKW20519. (4) The PCR-amplified ova-C7H gene was introduced into the pKW20093 plasmid digested 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 digested with SwaI to create pKW23502.

[0171]

[0172] <Aspergillus nidulans transformation> The host Aspergillus nidulans ΔPPR strain (ΔpyrG, ΔpyroA, ΔriboB strain) was seeded on CD / PPR agar medium and cultured to produce spores. The spores were suspended in sterile water and inoculated into CD / PPR liquid medium. The cultures were then cultured for 20 hours at 30°C with shaking. The cells were then recovered by filtration and treated with lysing enzyme from Trichoderma harzianum (Sigma-Aldrich) and beta-glucuronidase from Helix pomatia (Sigma-Aldrich) to generate protoplasts. The prepared plasmids (pKW20519, pKW20522, pKW23502) were added to the protoplasts, mixed with STC solution, and plated on CD agar medium. The resulting colonies were subjected to secondary selective culture, followed by extraction of genomic DNA and PCR analysis to confirm transformation. The transformant (Aspergillus nidulans ΔPPR#5) into which the plasmid had been successfully introduced was used for subsequent culture experiments.

[0173] <Production of deMeOV> The Aspergillus nidulansΔPPR#5 strain was inoculated into 30 mL of CDST liquid medium and cultured at 30°C with shaking. Starting 72 hours after the start of culture, aliquots of the culture were taken every 24 hours to quantify the amount of deMeOV produced over time. 0.5 mL of ethyl acetate was added to the culture aliquot (0.5 mL) and vigorously stirred. The mixture was then centrifuged, and 0.4 mL of the upper ethyl acetate layer was transferred to a separate container, and the solvent was evaporated. The residue was dissolved in 100 μL of DMF, and the insoluble components were removed by centrifugation. The supernatant was then subjected to LC-MS analysis.

[0174] <Detection of deMeOV by LC-MS> Analysis of deMeOV by LC-MS was performed using Thermo Fisher Scientific instruments (Dionex U3000 and Orbitrap Exactive Plus mass spectrometer). The column used was a Waters AQUITY UPLC HSS C18 (2.1 x 50 mm), and the mobile phase was acetonitrile and ultrapure water in gradient mode (acetonitrile 5-100%, 4 min) at a flow rate of 500 μL / min. Each solvent contained 0.05% formic acid. Accurate mass was measured using ESI, and analysis was performed using Qual browser software.

[0175] <Large-scale deMeOV Cultivation Experiment> A small amount of Aspergillus nidulans ΔPPR#5 spores was suspended in 100 μL of 0.1% Tween® 80 aqueous solution, and the entire suspension was inoculated 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 added to a 2 L Erlenmeyer flask containing 400 mL of CDT medium to initiate preculture. Preculture was carried out at 30°C and 160 rpm for 24 hours. The entire preculture was transferred to a jar fermenter (Bioneer, Marubishi Bioengine, benchtop culture device) containing 6.0 L of CDST medium (containing Diaion HP-20 adsorption resin) for main culture. Main culture was carried out at 30°C, 400 rpm, and oxygen flow for approximately 48 hours. During the main cultivation, the culture medium was sampled over time to check the amount of deMeOV produced. After sufficient productivity was confirmed, the culture medium and resin were removed from the fermenter and filtered to obtain the solid matter of bacteria and resin (HP-20), which was then frozen and stored. HPLC analysis confirmed that the culture supernatant (filtrate) did not contain the target component, deMeOV.

[0176] <Purification and structural confirmation of deMeOV> Approximately 10 solid samples obtained from the above-mentioned culture (6.0 L scale) were pooled together (total of 60 L culture) and extracted three times with 10 L of acetone. Each extraction lasted approximately 2 hours, with the mixture of cells and resin (HP20) vigorously stirred in acetone every 30 minutes. The insoluble matter was removed by filtration, and the resulting extract was concentrated under reduced pressure. The EtOAc layer was then concentrated under reduced pressure, yielding 27 g of extract. This extract was purified using silica gel column chromatography (280 g of BW-820MH, developing solvent: CHCl3 / MeOH [C / M] = 100 / 0 → 100 / 2 → 100 / 1 → 0 / 100). The C / M = 100 / 2 fraction (5.2 g), containing the target compound, was purified by normal-phase flash chromatography (conditions: column = Universal 5 L (silica gel: 300 g), flow rate = 70 mL / min, solvent = n-hexane / EtOAc = 4 / 1, fraction volume = 100 mL / tube). Each fraction was analyzed by TLC, and the fractions rich in deMeOV (1.2 g) were combined. Finally, normal-phase flash chromatography was performed again (conditions: column = Universal 2 L (silica gel: 100 g), flow rate = 30 mL / min, solvent = n-hexane / EtOAc = 17 / 3, fraction volume = 30 mL / tube), yielding 976 mg of deMeOV. NMR analysis of the isolated deMeOV in deuterated chloroform confirmed that the data were consistent with literature information, and that the chemical structure was correct (Figure 2 and Table 2).

[0177]

[0178]

[0179] Test Example 3 Synthesis of deMeOV Derivatives The structures of the synthesized compounds (compounds 1 to 12) are shown in Figure 3.

[0180] Synthesis of Compounds 1 and 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 a 25 mL two-necked pear-shaped flask and dissolved in 2.5 mL of CHCl. ​​The mixture was stirred at room temperature under a nitrogen atmosphere for 30 minutes. 5 mL of water was added to the mixture to quench the reaction. The mixture was then separated with EtOAc (5 mL x 3 times), and the organic layer was collected. This organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield 119.2 mg of reaction product. The resulting product was pretreated with ODS resin and purified by reverse-phase preparative HPLC (conditions: column = Develosil ODS-HG-5 (f20 x 250 mm), flow rate = 10 mL / min, solvent = MeCN / HO = 2 / 3) to yield 64.1 mg of the target compound 1 (78% yield). In this process, compound 2 was obtained as a by-product.

[0181] Compound 1: Colorless oil; [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 = 6.5 Hz, 1H), 3.07 (d, J = 4.2 Hz, 1H), 3.16 (s, 1H), 5.16 (t, J = 7.4 Hz, 1H), 5.67 (s, 1H); 13 C (100 MHz, CDCl3) δ14.8, 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) calcd for C 17 H 25 O6+ [M + H] + 325.1646; found 325.1641.

[0182] Compound 2: Colorless oil; [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 = 6.5 Hz, 1H), 3.07 (d, J = 4.2 Hz, 1H), 3.16 (s, 1H), 5.16 (t, J = 7.4 Hz, 1H), 5.67 (s, 1H); 13 C (100 MHz, CDCl3) δ14.8, 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) calcd for C 17 H 25 O6 + [M + Na] + 383.1232; found 383.1199.

[0183] Synthesis of Compound 3: 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 10 mL two-necked pear-shaped flask and dissolved in 1.0 mL of CHCl. ​​The mixture was stirred at room temperature under a nitrogen atmosphere for 24 hours. Saturated NHCl was added to the mixture to quench the reaction, and the mixture was separated with EtOAc (20 mL x 3 times). The organic layer was collected. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield 10.6 mg of reaction product. The resulting product was pretreated with ODS resin and then purified by reverse-phase preparative HPLC (conditions: column = Develosil ODS-HG-5 (f20 x 250 mm), flow rate = 10 mL / min, solvent = MeCN / HO = 2 / 3) to obtain 4.0 mg (yield 35%) of the target compound 3.

[0184] Compound 3: Colorless oil [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, 7.9 Hz, 1H), 8.39 (dt, J = 7.9, 2.0 Hz, 1H), 8.80 (dd, J = 1.6, 4.9 Hz, 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 No. 6 ＋ [M + H] + 388.1755; found 388.1747.

[0185] Synthesis of Compound 4: To 2 mL of CHCl containing deMeOV (33 mg, 0.12 mmol), 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 and the mixture was stirred under reflux in an argon atmosphere for 12 hours. After cooling, the reaction solution was evaporated under reduced pressure. The resulting sample was separated by silica gel column chromatography (eluent: CHCl / MeOH 100 / 1) to obtain 8.8 mg of compound 4.

[0186] Compound 4: colorless oil; 1H 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).

[0187] Synthesis of Compound 5: Oxalyl chloride (0.71 mL, 8.3 mmol) was added dropwise to anhydrous dichloroethane (5 mL) containing benzamide (500 mg, 4.2 mmol). The mixture was refluxed for 17 hours. After distillation, the resulting benzoyl isocyanate was used in the next reaction without further purification. Benzoyl isocyanate (18 mg, 0.12 mmol, 1.2 eq) was added to deMeOV (29 mg, 0.10 mmol, 1.0 eq) in 2 mL of CHCl, and the mixture was stirred under an argon atmosphere for 12 hours, followed by an additional 3 hours of reflux. The reaction solution was cooled, and the solvent was evaporated under reduced pressure. The resulting sample was separated by silica gel column chromatography (eluent: CHCl / MeOH 100 / 1) to obtain 3.0 mg of compound 5.

[0188] 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, 7.0 Hz, 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).

[0189] <Synthesis of Compounds 6 to 10> Compounds 6 to 10 were obtained in the same manner as for Compounds 1 to 5, except that an appropriate carboxy compound or isocyanate compound was used as the compound to be reacted with deMeOV (Compound 6: 31.8 mg, Compound 7: 44 mg, Compound 8: 20.6 mg, Compound 9: 63.1 mg, Compound 10: 10.5 mg).

[0190] 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, 7.2 Hz, 1H), 1.77 (s, 3H), 1.67 (s, 3H), 1.56 - 1.41 (m, 1H), 1.31 (s, 3H).

[0191] 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).

[0192] 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).

[0193] 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).

[0194] 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 = 4.1 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).

[0195] 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)

[0196] 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)

[0197] Test Example 4: Entamoeba histolytica test (in vitro) As a pre-culture, Entamoeba histolytica (HM-1:IMSS) cells were cultured in YIMDHA-S medium in a glass test tube for 3 days. After gently mixing the culture medium by inversion, the medium was removed, and 2 mL of fresh YIMDHA-S medium was added and suspended using a vortex mixer. The cell density of this suspension was 5 x 10 4The cells / mL were prepared and seeded at 200 μL / well in a clear 96-well plate (uncoated). The plate was placed in an anaerobic chamber (sealed container with AnaeroPack) and incubated at 37°C for 3 hours. Meanwhile, treatment compounds were prepared in YIMDHA-S medium and serially diluted. After confirming that the cultured Entamoeba histolytica cells were attached to the bottom of the plate and not dead, the medium in the plate was discarded, and the prepared medium containing the test compound was added at 200 μL / well. The cells were then incubated in the anaerobic chamber at 37°C for 48 hours. The medium was then discarded, and 110 μL of a mixture of PBS and WST1 (Premix WST-1 Cell Proliferation Assay System) (10:1) was added to each well. The cells were then incubated in the anaerobic chamber at 37°C for 30 minutes. The absorbance at 450 nm and 690 nm was measured using a plate reader (Nivo multimode microplate reader (Perkin Elmer)), and the viable cell ratio was calculated by subtracting the absorbance at 690 nm from the absorbance at 450 nm. The concentration at which the viable cell ratio reached 50% was defined as ED. 50 It was calculated as:

[0198] The results are shown in Figure 3. All of the deMeOV derivatives were found to exhibit anti-Entamoeba histolytica activity.

[0199] Test Example 5: Analysis of the effect of liver microsome treatment on activity 2.5 μL of liver microsome fractions from various animals and 10 μL of an NADPH-generating system (10 mM glucose-6-phosphate, 4 mM β-NADP +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 a final concentration of 200 μM. The prepared reaction solution was left to stand at 37°C for 30 minutes, sterilized by filtration (0.20 μm), and then diluted 20-fold with YIMDHA-S medium. This concentration was used as the maximum treatment concentration, and the solution was serially diluted with the same medium. As a control, the test compound (20 mM) that had not been microsome-treated was diluted 2,000-fold with medium and used in the same test (final concentration: 10 μM). The Entamoeba histolytica test was performed as in Test Example 4, and the surviving cell ratio was calculated.

[0200] The results are shown in Figures 4 and 5. It was found that the activity of deMeOV was significantly reduced by microsome treatment, whereas the reduction in activity due to microsome treatment could be suppressed by derivatizing deMeOV starting from the 6-hydroxyl group.

[0201] Test Example 6. Test 1 using an amoeba liver abscess model A Syrian hamster (female, 70 g, 5 weeks old) was surgically opened under isoflurane anesthesia, and 3 x 10 Entamoeba histolytica was instilled into the liver. 5 The hamsters were anesthetized and euthanized, their livers removed, and Entamoeba histolytica cells isolated from the resulting liver abscesses were cultured in YIMDHA-S medium in glass test tubes. The following animal tests used Entamoeba histolytica cells that had been removed from the hamsters within three months and were expected to be highly pathogenic.

[0202] A Syrian hamster (female, 70 g, 5 weeks old) was surgically opened under isoflurane anesthesia, and the liver was inoculated with 2 x 10 Entamoeba histolytica treated as described above. 5Cells were inoculated, and the abdominal incision was sutured. Starting 24 hours later, drug administration was performed daily for a total of 5 days, as follows: (1) For subcutaneous administration, compound 1 (15 mg / mL, DMSO solution) was mixed with an equal volume of 5% cremophor solution, and 0.2 mL was administered subcutaneously per hamster. (2) For oral administration, compound 1 (30 mg / mL, DMSO solution) was mixed with an equal volume of 5% cremophor solution, and 0.2 mL was administered orally per hamster via a syringe. Six days after infection, the hamsters were anesthetized and euthanized, and their livers were removed. The total weight of the liver tissue was measured, and the liver abscess portion was excised and weighed. The therapeutic effect of the drug (compound 1) was evaluated by calculating the percentage of liver abscess tissue weight relative to the total liver weight.

[0203] The results are shown in Figures 6 and 7. It was found that administration of Compound 1 significantly reduced liver abscesses.

[0204] In addition, secondary analysis using quantitative PCR was performed to evaluate the efficacy of the compounds.

[0205] <RNA Extraction> Hamster liver samples obtained from the above drug administration experiments were minced in 5 mL of PBS and disrupted using a Teflon-glass homogenizer. 450 μL of Sepasol RNA I Super G was added to 50 μL of liver homogenate, followed by 100 μL of chloroform. The mixture was mixed and centrifuged at 15,000 × g for 15 minutes at 4°C. The upper (aqueous) phase was collected and an equal volume of isopropanol was added. The mixture was then left on ice for 5 minutes and centrifuged at 15,000 × g for 15 minutes at 4°C to obtain the RNA fraction. 100 μL of 75% ethanol was added to the precipitate, which was then suspended and centrifuged at 15,000 × g for 10 minutes at 4°C. The precipitate was air-dried for 10 minutes, and 50 μL of nuclease-free water was added to dissolve the RNA.

[0206] <Reverse transcription PCR> The concentration of the extracted RNA was calculated from absorbance using a spectrophotometer and adjusted to 0.2 μg / μL with nuclease-free water. Using the High Capacity cDNA Reverse Transcription Kit, reagents were added to 10 μL of the prepared RNA according to the product's protocol, and 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.

[0207] <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 well)). The mixture was then diluted to 15 μL with nuclease-free water. The mixture was then amplified using a Step One Plus Real-Time PCR System (Applied Biosystems) with a two-step PCR (40 cycles) consisting of 95°C for 10 minutes and 95°C for 15 seconds to 60°C for 1 minute. Expression levels were analyzed using the ΔΔCt method, with the Gapdh gene as the endogenous control.

[0208] Test Example 7: Evaluation of the Activity of a Developed Compound on Human Umbilical Vein Endothelial Cells (HUVECs) Human umbilical vein endothelial cells (HUVECs) were seeded onto a collagen-coated 96-well plate at 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 discarded, and the test drug was added to a final concentration of 10 μM to 10 pM. The cells were cultured at 100 μL / well for 24 hours. EdU was then added to a final concentration of 0.01 μM and the cells were cultured at 100 μL / well for 24 hours. Cell fixation, membrane permeabilization, fluorescent EdU labeling, and nuclear staining with Hoechst 33342 were performed according to the protocol of the Click-iT EdU Alexa Fluor 594 Imaging Kit. Measurements were performed using a high-content screening (CellInsight TM Fluorescence was detected using a CX5 High-Content Screening Platform (Thermo Fisher Scientific), and the amount of EdU incorporation was examined using the MEAN_CircAvgIntenCh2 parameter.

[0209] The results are shown in Figure 8. Compounds 1 and 7 were shown to have potent inhibitory effects on HUVEC proliferation and to inhibit angiogenesis in humans. Because the growth of various cancer cells and adipocytes is highly dependent on angiogenesis (RH Howland, J. Psychosoc. Nurs. Ment. Health. Serv. 2015, 53, 13), these compounds were found to have antitumor and anti-obesity activities.

[0210] Test Example 8 Test 2 Using Amoeba Liver Abscess Model Compounds 1 and 7 were tested in the same manner as in Test Example 6.

[0211] The results are shown in Figure 9. It was found that administration of Compound 7 also significantly reduced liver abscesses.

[0212] Test Example 9: Evaluation of Enzyme Activity Inhibition Against Entamoeba histolytica-derived MetAP2 (EhMetAP2) and Human-derived MetAP2 (HsMetAP2) A compound at a predetermined concentration was added to an enzyme solution (100 nM, 3 μL) of EhMetAP2 or HsMetAP2, and the mixture was incubated at room temperature for 5 minutes. Subsequent addition of a substrate solution (300 μM, 3 μL) allowed the mixture to react at 37°C for 2 hours. Fluorescence measurements were then performed to determine the residual MetAP2 enzyme activity and the IC value of the compound's enzyme activity inhibition. 50 The values ​​were calculated, and the results are shown in Table 3.

[0213] ​

Claims

1. General formula (1A2): [In the formula: L 1 R represents a single bond or a linker. 11 R represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, or a solvate thereof.

2. R 2 and R 3 are linked to each other to form -(CH2)-, or a salt or solvate thereof according to claim 1.

3. L 1 is a single bond or a linker having 1 to 4 main chain atoms, or a salt thereof, or a solvate thereof according to claim 1.

4. The compound according to claim 1, its salt, or a solvate thereof, wherein the substituents which the alkyl group, the aryl group, and the heteroaryl group may have are halogen atoms, hydroxy groups, or alkoxy groups, or alkyl groups, alkoxy groups, aryl groups, alkyl groups, or arylalkoxy groups which may be substituted with halogen atoms, hydroxy groups, or alkoxy groups.

5. The compound has the general formula (1A3): [In the formula: L 1 and R 11 The compound according to claim 1 , a salt thereof, or a solvate thereof, wherein the compound is a compound represented by the formula:

6. L 1 is a single bond and R 11 is an alkyl group having 1 to 4 carbon atoms, or a salt thereof, or a solvate thereof according to claim 5.

7. General formula (1): [In the formula: R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates L 1 R represents a single bond or a linker. 11 R represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, and a solvate thereof.

8. The anti-amebiasis agent according to claim 7, which is a preventive or therapeutic agent for amebiasis.

9. General formula (1): [In the formula: R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates L 1 R represents a single bond or a linker. 11 R represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, and a solvate thereof.

10. General formula (1): [In the formula: R 1 is a hydrogen atom or -C(=O)-L 1 -R 11 Indicates L 1 R represents a single bond or a linker. 11 R represents an optionally substituted alkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. 2 and R 3 are linked together to form -(CH2)-, or R 2 represents a hydrogen atom and R 3 represents -(CH2)-X, and X represents a halogen atom.], a salt thereof, and a solvate thereof.

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