Novel reveromycin derivative and method for producing the same

Abstract

To provide a novel reveromycin derivative, a method for producing the same, and the like.SOLUTION: The present invention relates to a compound represented by General Formula (I) in the figure, where each symbol is as described in the specification, or a salt thereof.SELECTED DRAWING: Figure 28

Description

[Technical Field]

[0001] The present invention relates to a novel reveromycin derivative having a stable 6,6-spiroacetal ring structure, a method for producing the same, and a cytochrome P450revI mutant using the method for producing the same. [Background technology]

[0002] Reveromycin A (RM-A), a eukaryotic isoleucyl-tRNA synthetase inhibitor with the following structure, exhibits various biological activities, including the induction of apoptosis in osteoclasts (Non-Patent Documents 1-5). RM-A is biosynthesized via the pathway shown in Figure 1. Cytochrome P450revI (P450revI) catalyzes the hydroxylation of C18 of reveromycin T (RM-T), and this step is essential for the subsequent hemisuccinylation of the hydroxyl group in the biosynthesis of RM-A.

[0003] The 6,6-spiroacetal ring of RM-A is unstable under acidic conditions and is easily converted to a stable 5,6-spiroacetal ring, resulting in isomerization of RM-A to reveromycin B (RM-B) (Figure 2). The axial orientation of the hemisuccinate moiety at C18 is thought to be one of the factors (Non-Patent Documents 6-8).

[0004] Furthermore, it has been reported that conversion to RM-B reduces biological activity (Non-Patent Document 9), and therefore, the presence of a 6,6-spiroacetal ring structure is essential for the expression of biological activity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Miyamoto, Y., et al., Identification of Saccharomyces cerevisiae Isoleucyl-tRNA synthetase as a target of the G1-specific inhibitor reveromycin A *. Journal of Biological Chemistry, 2002. 277(32): p. 28810-28814. [Non-Patent Document 2] Osada, H., et al., Reveromycin A, a new antibiotic which inhibits the mitogenic activity of epidermal growth factor. Journal of Antibiotics, 1991. 44(2): p. 259-261. [Non-Patent Document 3] Takahashi, H., et al., Reveromycins, new inhibitors of eukaryotic cell growth. II. Biological activities. Journal of Antibiotics, 1992. 45(9): p. 1414-1419. [Non-Patent Document 4] Takahashi, S., et al., Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T. The Journal of biological chemistry, 2014. 289(47): p. 32446-32458. [Non-Patent Document 5] Woo, J.-T., et al., Reveromycin A, an agent for osteoporosis, inhibits bone resorption by inducing apoptosis specifically in osteoclasts. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(12): p. 4729-4734. [Non-Patent Document 6] Cuzzupe, A.N., et al., Total synthesis of the epidermal growth factor inhibitor (-)-reveromycin B. The Journal of Organic Chemistry, 2001. 66(7): p. 2382-2393. [Non-Patent Document 7] El Sous, M., et al., Total Synthesis of (-)-Reveromycin A via a Hetero-Diels-Alder Approach. Synthesis, 2010. 2010(23): p. 3954-3966. [Non-Patent Document 8] Shimizu, T., et al., Total Synthesis of Reveromycin A. The Journal of Organic Chemistry, 2000. 2(14): p.2153-2156. [Non-Patent Document 9] Shimizu, T., et al., Chemical modification of reveromycin A and its biological activities. Bioorganic & Medicinal Chemistry Letters, 2002. 12(23): p. 3363-3​​​​[Problem to be solved by the invention]

[0006] An object of the present invention is to create a reveromycin derivative that is structurally stable even under acidic conditions and retains the biological activity of RM-A. [Means for solving the problem]

[0007] To address the above-mentioned issues, the present inventors focused on the C18 hydroxylation reaction of RM-T, which is a precursor to the formation of the hemisuccinate moiety at C18 that is responsible for the isomerization of RM-A. They extensively investigated various P450revI mutants to regiosterically modify the P450revI catalyzing this hydroxylation reaction. As a result, they discovered that a specific P450revI mutant specifically hydroxylates C17 of RM-T. The resulting 17-hydroxyl liveromycin derivative, represented by formula (I) below, and the hemisuccinylated derivative obtained by the subsequent intracellular reaction, exhibit biological activity equivalent to or superior to that of RM-A, thereby completing the present invention. Because the hemisuccinylated derivative has a hemisuccinate moiety at C17 rather than C18, its 6,6-spiroacetal ring is less likely to convert to a 5,6-spiroacetal ring, even under acidic conditions, making it structurally stable.

[0008] That is, the present invention provides the following. [1] General formula (I):

[0009] [ka]

[0010] [In the formula, R 1 is C 1-6 represents an alkyl group, and R 2 represents a hydrogen atom or a hemisuccinyl group. A compound represented by the formula (I) or a salt thereof. [2] General formula (IA):

[0011] [ka]

[0012] [In the formula, R 1 is C 1-6 represents an alkyl group, and R 2 represents a hydrogen atom or a hemisuccinyl group. A compound represented by the formula (I) or a salt thereof. [3] R 1 is n-butyl and R 2 is a hydrogen atom or a hemisuccinyl group, or a salt thereof.

[0013] [4] A cytochrome P450revI mutant consisting of a polypeptide shown in any one of the following (1) to (3): (1) A polypeptide in which the alanine residue at position 241 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue, an isoleucine residue, a valine residue, a serine residue, a threonine residue, or a proline residue; (2) A polypeptide in which one or more amino acid residues other than the substituted amino acid residue(s) in the polypeptide (1) have been substituted, added, inserted, or deleted, and which has the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T; and (3) A polypeptide having a sequence identity of 80% or more in the portion excluding the substituted amino acid residue in the polypeptide of (1), and having the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T. [5] A nucleic acid encoding the cytochrome P450revI mutant described in [4] above. [6] A recombinant vector comprising the nucleic acid described in [5] above. [7] A transformant obtained by introducing the recombinant vector described in [6] above into a host. [8] The transformant according to [7] above, wherein the host is a bacterium of the genus Streptomyces. [9] A method for producing a compound represented by general formula (Ia) and / or a compound represented by general formula (Ib), comprising a step of culturing the transformant according to [7] or [8] above in a medium containing a compound represented by general formula (II).

[0014] [ka]

[0015] [In the formula, R 1 is C 1-6 represents an alkyl group.]

[10] The production method according to [9] above, further comprising the step of recovering the compound represented by formula (Ia) or the compound represented by formula (Ib) from the culture obtained in the culturing step.

[0016] The present invention also provides the following:

[11] Formula:

[0017] [ka]

[0018] 17-hydroxyl liveromycin-T represented by the formula (I), or a salt thereof.

[12] Formula:

[0019] [ka]

[0020] 17-hemisuccinyloxyliveromycin-T represented by the formula (I), or a salt thereof.

[13] The transformant according to [8] above, further comprising enhanced expression of the revQ gene.

[14] The transformant according to [8] or

[13] above, in which the function of the endogenous cytochrome P450revI gene is inhibited.

[15] A pharmaceutical or agricultural chemical containing the compound or salt thereof according to any one of [1] to [3],

[11] and

[12] above as an active ingredient.

[16] A therapeutic agent for bone diseases, comprising the compound or salt thereof according to any one of the above [1] to [3],

[11] and

[12] as an active ingredient.

[17] A therapeutic agent for infectious diseases, comprising the compound or salt thereof according to any one of the above [1] to [3],

[11] and

[12] as an active ingredient.

[18] An antifungal agent comprising, as an active ingredient, the compound or salt thereof according to any one of the above [1] to [3],

[11] and

[12] . [Effects of the Invention]

[0021] According to the present invention, it is possible to provide novel reveromycin derivatives that are structurally stable even under acidic conditions without their 6,6-spiroacetal rings being converted to 5,6-spiroacetal rings and that exhibit biological activity equivalent to or greater than that of RM-A. Such reveromycin derivatives are expected to be used as medicines and agricultural chemicals, for example, as therapeutic agents for bone diseases (e.g., multiple myeloma), therapeutic agents for infectious diseases (e.g., malaria), antifungal agents, etc. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows the biosynthetic pathway of RM-A. [Figure 2] FIG. 2 shows the isomerization of the 6,6-spiroacetal liberomycin derivative to the 5,6-spiroacetal core. [Figure 3] FIG. 3 shows the isolation scheme for 17-hydroxyl RM-T and 17-hemisuccinyloxy RM-T. [Figure 4] FIG. 4 shows UPLC-MS analysis of P450revI mutant reactions with RM-T. [Figure 5] Figure 5 shows the UPLC profiles of extracts obtained from Streptomyces sp. SN-593 (wild-type strain), Streptomyces sp. SN-593 revI-disrupted strain (ΔrevI), ΔrevI / revI-A241L, and ΔrevI / revI-A241L / revQ cultures. [Figure 6]FIG. 6 shows the key 2D NMR correlations of 17-hemisuccinyloxy RM-T. [Figure 7] Figure 7 shows the key 2D NMR correlations of 17-hydroxyl RM-T. [Figure 8] FIG. 8 shows the UV-Vis spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 9] FIG. 9 shows the IR spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 10] FIG. 10 shows the HR-ESI-ToF-MS data of 17-hemisuccinyloxy RM-T (1). [Figure 11] FIG. 11 shows the 1H NMR (CD3OD, 500 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 12] FIG. 12 shows the 13C NMR (CD3OD, 125 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 13] FIG. 13 shows the DEPT135 (CD3OD, 125 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 14] FIG. 14 shows the HSQC (CD3OD, 500 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 15] FIG. 15 shows the 1H-1H COSY (CD3OD, 500 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 16] FIG. 16 shows the HMBC (CD3OD, 500 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 17] FIG. 17 shows the ROESY (CD3OD, 500 MHz) spectrum of 17-hemisuccinyloxy RM-T (1). [Figure 18] FIG. 18 shows the UV-Vis spectrum of 17-hydroxyl RM-T (2). [Figure 19]FIG. 19 shows the IR spectrum of 17-hydroxyl RM-T (2). [Figure 20] FIG. 20 shows the HR-ESI-ToF-MS data of 17-hydroxyl RM-T (2). [Figure 21] FIG. 21 shows the 1H NMR (CD3OD, 500 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 22] FIG. 22 shows the 13C NMR (CD3OD, 125 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 23] FIG. 23 shows the DEPT135 (CD3OD, 125 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 24] FIG. 24 shows the HSQC (CD3OD, 500 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 25] FIG. 25 shows the 1H-1H COSY (CD3OD, 500 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 26] FIG. 26 shows the HMBC (CD3OD, 500 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 27] FIG. 27 shows the ROESY (CD3OD, 500 MHz) spectrum of 17-hydroxyl RM-T (2). [Figure 28] FIG. 28 shows the hydroxylation of C17 by the P450revI-A241L mutant, followed by hemisuccinylation. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be described in detail below.

[0024] In this specification, "C 1-6 The term "alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. 1-6Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl, and among these, n-butyl is particularly preferred.

[0025] The novel reveromycin derivative of the present invention has the general formula (I):

[0026] [ka]

[0027] [Each symbol in the formula has the same meaning as defined above.] or a salt thereof. R 1 is C 1-6 represents an alkyl group. R 1 is preferably n-butyl. R 2 represents a hydrogen atom or a hemisuccinyl group. General formula (I) is R 2 is a hydrogen atom and R 2 is a hemisuccinyl group.

[0028] [ka]

[0029] The novel reveromycin derivatives of the present invention preferably have the general formula (IA):

[0030] [ka]

[0031] [Each symbol in the formula has the same meaning as defined above.] or a salt thereof.

[0032] In another embodiment, the novel reveromycin derivative of the present invention preferably has the following structure:

[0033] [ka]

[0034] or a salt thereof, or a reveromycin derivative having the following structure:

[0035] [ka]

[0036] or a salt thereof.

[0037] The novel reveromycin derivative of the present invention particularly preferably has the following structure:

[0038] [ka]

[0039] 17-hydroxyl liveromycin-T (hereinafter also referred to as 17-hydroxyl RM-T) represented by the following structure:

[0040] [ka]

[0041] 17-hemisuccinyloxyliberomycin-T (hereinafter also referred to as 17-hemisuccinyloxy RM-T) represented by the formula:

[0042] The compound represented by general formula (I) may form a salt. Examples of such salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Suitable examples of metal salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts, magnesium salts, and barium salts; and aluminum salts. Suitable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine. Suitable examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Suitable examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc. Suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Among these, pharmaceutically acceptable salts are preferred as salts used in medicines such as bone disease therapeutic agents, infection therapeutic agents, antifungal agents, etc. Examples include inorganic salts such as alkali metal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earth metal salts (e.g., calcium salt, magnesium salt, barium salt, etc.); ammonium salts; salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid; and salts with organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.

[0043] When the compound represented by general formula (I) has optical isomers, either one of the isomers or a mixture thereof is included in the compound of the present invention. Furthermore, optical isomers resolved from a racemate are also included in the compound represented by general formula (I). The compound represented by general formula (I) or a salt thereof may be in a crystalline form, and whether the crystalline form is a single crystalline form or a crystalline mixture, it is included in the compound represented by general formula (I). The compound represented by general formula (I) or a salt thereof may be a solvate (for example, a hydrate) or a non-solvate, and both are included in the compound represented by general formula (I). Isotopes (e.g. 2 H, 3 H, 11 C, 14 C, 18 F, 35 S, 125 Compounds labeled or substituted with, for example, I are also encompassed by the compounds represented by general formula (I).

[0044] The novel reveromycin derivative of the present invention is produced by a method comprising the step of culturing a specific transformant in a medium containing a compound represented by general formula (II) (hereinafter also referred to as compound (II)). 2 is a hydrogen atom (hereinafter also referred to as compound (Ia)), 2 is a hemisuccinyl group (hereinafter also referred to as compound (Ib)), or a mixture thereof, is produced, and by recovering and isolating them from the culture mixture, compound (Ia) and compound (Ib) can be obtained separately.

[0045] [ka]

[0046] [Each symbol in the formula has the same meaning as defined above.]

[0047] The method for producing the novel reveromycin derivatives of the present invention will be explained in detail below.

[0048] The transformant used in the method for producing the novel reveromycin derivative of the present invention has a recombinant vector containing a nucleic acid encoding a specific cytochrome P450revI mutant.

[0049] (cytochrome P450revI mutant) Research into the creation of novel hydroxylated cytochrome P450 mutants through the design of these mutants has been widely conducted. However, because diverse P450s exist in nature, achieving regiospecific hydroxylation reactions requires trial and error research, including mutant design, enzyme purification, and reaction product analysis for each enzyme. Under these circumstances, the present inventors successfully isolated a cytochrome P450revI mutant capable of hydroxylating compound (II) at the 17-position.

[0050] As used herein, cytochrome P450revI is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and has the activity of catalyzing the hydroxylation reaction of C18 of reveromycin T (RM-T).

[0051] The cytochrome P450revI mutant of the present invention consists of a polypeptide shown in any one of the following (1) to (3): (1) A polypeptide in which the alanine residue at position 241 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue, an isoleucine residue, a valine residue, a serine residue, a threonine residue, or a proline residue; (2) A polypeptide in which one or more amino acid residues other than the substituted amino acid residue(s) in the polypeptide (1) have been substituted, added, inserted, or deleted, and which has the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T; and (3) A polypeptide having a sequence identity of 80% or more in the portion excluding the substituted amino acid residue in the polypeptide of (1), and having the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T.

[0052] The cytochrome P450revI mutant (1) consists of a polypeptide in which the alanine residue at position 241 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue, an isoleucine residue, a valine residue, a serine residue, a threonine residue, or a proline residue.

[0053] The cytochrome P450revI mutant (2) comprises a polypeptide in which one or more amino acid residues other than the substituted amino acid residue(s) in the polypeptide (1) have been substituted, added, inserted, or deleted, and which has the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T.

[0054] The cytochrome P450revI mutant (2) may be a functionally equivalent mutant, derivative, variant, allele, homolog, or ortholog, partial peptide, or fusion protein with another protein or peptide of the protein consisting of the polypeptide (1). The specific sequence of the mutant is not limited as long as it encodes a polypeptide having the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T. The number of amino acids that may be substituted, added, inserted, or deleted is not limited as long as the function is not lost. The number of amino acids that may be substituted, added, inserted, or deleted is within the range that can be achieved by known methods such as site-directed mutagenesis. The number of amino acids is typically within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, more preferably within 7 amino acids, and even more preferably within 5 amino acids (e.g., 5, 4, 3, 2, or 1 amino acid). In the present specification, the term "mutation" primarily refers to a mutation artificially introduced by site-directed mutagenesis or the like, but may also refer to a similar mutation that occurs naturally.

[0055] The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain, such as hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the characters in parentheses represent the single-letter abbreviations of the amino acids).

[0056] As used herein, the term "functionally equivalent" means that the target protein has biological and biochemical functions equivalent (identical and / or similar) to those of the target protein. Biological properties may also include the specificity of the expression site and the expression level. Whether a protein into which a mutation has been introduced has the desired function can be determined by examining whether the mutant protein has the activity to catalyze the hydroxylation reaction at position 17 of reveromycin T.

[0057] The cytochrome P450revI mutant (3) comprises a polypeptide having a sequence identity of 80% or more, excluding the substituted amino acid residue, with the polypeptide (1) and having the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T.

[0058] The cytochrome P450revI mutant of (3) also contemplates functionally equivalent mutants, derivatives, variants, alleles, homologs, orthologs, partial peptides, or fusion proteins with other proteins or peptides of the protein consisting of the polypeptide of (1), and its specific sequence is not limited as long as it encodes a polypeptide having the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T.

[0059] Sequence identity (amino acid sequence identity) refers to an amino acid sequence identity of at least 80%, more preferably 90%, and even more preferably 95% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more) with the amino acid sequence of the portion of the polypeptide (1) excluding the substituted amino acid residue. Amino acid sequence identity can be determined using the BLASTN (nucleic acid level) or BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). These programs are based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993).

[0060] In one embodiment of the present invention, a nucleic acid encoding the cytochrome P450revI mutant of any one of (1) to (3) above is provided.

[0061] (recombinant vector) Examples of plasmid DNA include plasmids derived from Escherichia coli (ColE-based plasmids such as pBR322, pUC18, pUC19, pUC118, pUC119, and pBluescript). Plasmids derived from actinomycetes, such as pIJ486 (Mol. Gen. Genet. 203, 468-478, 1986), pKC1064 (Gene 103, 97-99 (1991)), pUWL-KS (Gene 165, 149-150 (1995)), pIJ702 (J. Gen. Microbiol. 129:2703-2714 (1983)), and pIJ8600 (Microbiology 145:2221-2227 (1999)), can also be used. Examples of phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11) and the like.

[0062] (host) The bacteria used in the present invention can be obtained using Streptomyces sp. SN-593 or the bacteria exemplified below as parent strains. Streptomyces prunicolor Streptomyces cinnamoneus Streptomyces chromofuscus Streptomyces lividans Streptomyces akiyoshiensis Streptomyces azureus Streptomyces hawaiiensis Streptomyces tendae Streptomyces virginiae Streptomyces amakusaensis Streptomyces antibioticus Streptomyces chibaensis Streptomyces albus Streptomyces lincolnensis Streptomyces kanamyceticus Streptomyces kasugaensis Streptomyces coelicolor Streptomyces griseus Streptomyces avermitilis Streptomyces ambofaciens Streptomyces fradiae

[0063] The above-mentioned Streptomyces bacteria are listed in the IFO catalog, ATCC catalog, JCM catalog, etc., and can be easily obtained by those skilled in the art.

[0064] The above-mentioned bacteria used as parent strains in the present invention may be any strain, including not only wild-type strains but also mutant strains obtained by conventional mutation treatments such as UV irradiation or NTG treatment, and recombinant strains induced by genetic techniques such as cell fusion or gene recombination.

[0065] (Transformation method) Recombinant DNA can be introduced into a host cell by known methods. For example, a commonly used method for transforming Streptomyces bacteria involves spheroplasting the bacteria with lysozyme, followed by the addition of a recombinant DNA vector and a buffer containing polyethylene glycol to allow the vector to be incorporated into the cells (see Thompson, CJ, et al. (1982) J. Bacteriol., 151, 668-677 or Hopwood, DA, et al. (1985) "Genetic Manipulation of Streptomyces: A Laboratory Manual", The John Innes Foundation, Norwich). Transformation can also be performed by the electric pulse method (Res. Microbiol., Vol. 144, pp. 181-185, 1993). Furthermore, the introduction of DNA can be confirmed by using a selection marker gene (for example, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a kanamycin resistance gene, etc.).

[0066] (transformant) The transformant of the present invention has a cytochrome P450revI mutant consisting of a polypeptide shown in any one of (1) to (3) above.

[0067] In one embodiment of the present invention, the transformant preferably has enhanced expression of the revQ gene, which allows for efficient production of compound (Ia), compound (Ib), or a mixture thereof.

[0068] Enhancement of revQ gene expression can be achieved by genetic recombination, for example, by increasing the copy number of the revQ gene or substituting the promoter of this gene. To increase the copy number of the revQ gene, for example, the revQ gene can be incorporated into a plasmid capable of functioning in a host microorganism so that it can be expressed, and then introduced into the host microorganism. Specifically, this can be achieved, for example, by the method described in WO 2012 / 029811.

[0069] In one embodiment of the present invention, the transformant is preferably deficient in the function of the endogenous cytochrome P450revI gene, which allows efficient production of compound (Ia), compound (Ib), or a mixture thereof.

[0070] The method for inhibiting the function of the endogenous cytochrome P450revI gene is not particularly limited as long as it is a method that disables the function of the cytochrome P450revI gene, and examples thereof include ZFN, TALEN, CRISPR / Cas9, PPR motif, siRNA, miRNA, shRNA, antisense RNA, and homologous recombination.

[0071] (Method for producing a novel reveromycin derivative) In the production method of the present invention, a transformant having the cytochrome P450revI mutant is cultured in a medium containing compound (II), and compound (II) is converted to compound (Ia) by the action of the cytochrome P450revI mutant, thereby obtaining a novel reveromycin derivative.

[0072] The culture medium used for the culture may contain compound (II) and a nutrient source available to Streptomyces bacteria, and may include various synthetic, semi-synthetic, and natural media. Carbon sources such as glucose, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steep liquor, and organic acids may be used alone or in combination. Nitrogen sources may include organic nitrogen sources such as Pharmamedia, peptone, meat extract, yeast extract, soybean flour, casein, amino acids, and urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate, either alone or in combination.

[0073] Sodium salts, potassium salts, magnesium salts, phosphate salts, and other heavy metal salts may also be added as needed. If significant foaming occurs during cultivation, various known antifoaming agents such as Adekanol (registered trademark) and silicone oil can be added to the medium, but the addition must not adversely affect the production of the target substance. For example, it is preferable to use a concentration of 0.5% or less.

[0074] The pH of the medium is preferably in the optimum pH range for the microorganism, usually near neutral. The temperature of the medium should be maintained at a temperature at which the microorganism grows well, usually between 20 and 40°C, and particularly preferably around 27°C. In the case of liquid culture, the culture time is generally about 1 to 5 days, preferably about 72 hours. By changing the culture time, the ratio of compound (Ia) to compound (Ib) in the mixture can be appropriately adjusted. In one embodiment, the shorter the culture time, the higher the ratio of compound (Ia) in the mixture, and the longer the culture time, the higher the ratio of compound (Ib) in the mixture.

[0075] Compound (Ia) and compound (Ib) produced by the above-mentioned culture can be isolated by any of the following means: a means utilizing the difference in solubility between compound (Ia), compound (Ib) and impurities; a means utilizing the difference in adsorption affinity; or a means utilizing the difference in molecular weight; and each method can be used alone, in appropriate combination, or repeatedly.

[0076] Specifically, since the majority of Compound (Ia) and Compound (Ib) are present in the culture filtrate, the culture filtrate can be purified by a combination of various methods, such as gel filtration chromatography, adsorption chromatography, and liquid chromatography, to obtain a fraction containing Compound (Ia), Compound (Ib), and other active ingredients. The powder obtained by lyophilizing this fraction can be further purified using high-performance liquid chromatography (e.g., a Capsule Pack column) and, for example, by development with a chloroform / methanol mixed solvent system, to obtain purified white powders of Compound (Ia) and Compound (Ib). In one embodiment of the present invention, Compound (Ia) and Compound (Ib) can be obtained by the method described in the Examples.

[0077] The compound represented by general formula (I) or a salt thereof has various biological activities and is therefore expected to be an active ingredient in medicines or agricultural chemicals, for example, therapeutic agents for bone diseases (e.g., multiple myeloma), therapeutic agents for infectious diseases (e.g., malaria), antifungal agents, etc.

[0078] The compound represented by general formula (I) or a pharmaceutically acceptable salt thereof has low toxicity (for example, it shows no cytotoxicity to HeLa cells or HL-60 cells), and can be safely administered to mammals orally or parenterally as a pharmaceutical agent or as a pharmaceutical composition mixed with a carrier or the like commonly used in pharmaceuticals.

[0079] When the compound represented by general formula (I) or its salt is used as a pharmaceutical or pesticide, it can contain a carrier commonly used in pharmaceutical or pesticide applications. The formulation can be appropriately selected depending on the purpose and subject of use, and can be used in the form of, for example, injections (liquids, suspensions, etc.), tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, etc. Examples of the carrier include, but are not limited to, excipients, binders, disintegrants, emulsifiers, solubilizers, dispersants, lubricants, coating agents, colorants, stabilizers, isotonicity agents, etc. Examples of excipients include commonly used sugars such as lactose, sucrose, and glucose; inorganic substances such as starch, calcium carbonate, and calcium sulfate; crystalline cellulose; distilled water; purified water; sesame oil; soybean oil; corn oil; olive oil; and cottonseed oil. The compound represented by general formula (I) or its salt can be formulated using these carriers by conventional methods.

[0080] Examples of pharmaceuticals containing the compound represented by general formula (I) or a salt thereof as an active ingredient include therapeutic agents for bone diseases, therapeutic agents for infectious diseases, and antifungal agents. The administration method, dosage form, and dosage of the medicament of the present invention can be appropriately determined in accordance with conventional pharmaceutical techniques depending on the intended use. For example, when administered to mammals such as humans for the purpose of treatment, the medicament can be administered orally in the form of powders, granules, tablets, capsules, pills, solutions, etc., or parenterally in the form of injections, suppositories, transdermal agents, inhalants, etc. Furthermore, the pharmaceutical of the present invention can be prepared as a pharmaceutical composition by combining a therapeutically effective amount of the compound represented by general formula (I) or its salt with various carriers typically used in pharmaceutical compositions, as described above. The dosage varies depending on the state of the disease, the route of administration, and the patient's age and weight, but the amount of the active ingredient administered orally to an adult is typically 20 to 500 mg / kg / day, preferably 50 to 300 mg / kg / day, and the amount administered parenterally is typically 10 to 300 mg / kg / day, preferably 20 to 200 mg / kg / day. This amount may be administered once or in divided doses. Furthermore, the present invention provides a method for treating a disease, which comprises the step of administering a therapeutically effective amount of a compound represented by general formula (I) or a salt thereof to a mammal, including a human.

[0081] The therapeutic agent for bone diseases of the present invention is effective for treating or preventing bone diseases, and specific examples of the bone diseases include, but are not limited to, multiple myeloma, osteoporosis, bone disease-associated hypercalcemia, Paget's disease of bone, osteoclastoma, osteosarcoma, arthropathy, rheumatoid arthritis, osteitis deformans, primary hyperthyroidism, osteopenia, osteoporosis, osteomalacia, traumatic fracture, stress fracture, or weakening of bone tissue and fracture caused by other diseases such as nutritional disorders and malignant tumors. The therapeutic agent for infection of the present invention is effective for treating or preventing, for example, malaria. The antifungal agent of the present invention broadly refers to a drug having a bactericidal or growth-inhibitory effect against fungi. Fungi include yeasts, mushrooms, and so-called filamentous fungi (molds), and are particularly useful against fungi such as Candida albicans and Candida pseudotropicalis, which cause endogenous infections. The antifungal agent of the present invention can be used to treat local fungal infections, mucosal infections, systemic fungal infections, and the like caused by, for example, Candida, Trichophyton, or Aspergillus.

[0082] Furthermore, the antifungal agent of the present invention can be used not only as a pharmaceutical product but also as an additive in products that are ingested into the human or animal body or applied to the body surface, such as foods, feeds, and cosmetics, as well as in any other product in which it is generally desired to prevent or inhibit the growth of fungi. The antifungal agent of the present invention can also be used for surface treatment of products or raw materials. Specifically, the antifungal agent of the present invention may be added, blended, sprayed, attached, coated, impregnated, or the like to foods, pharmaceuticals, quasi-drugs, various cosmetics, various toothpastes, various sanitary products, various baby products, various elderly care products, various detergents, various disinfectants, pet feed, various livestock feed, various fish feed, various building materials, various paints, various agricultural and horticultural products, as well as the raw materials for these products, and any other product in which it is generally desired to prevent or inhibit the growth of microorganisms such as fungi. It can also be used to treat any other product in which it is generally desired to prevent or inhibit the growth of fungi. [Example]

[0083] The present invention will be explained in more detail below by giving production examples, test examples and formulation examples, but these do not limit the present invention and may be changed within the scope of the present invention.

[0084] (1) Chemicals All solvents and reagents were of analytical grade and commercially available. RM-T, used as a substrate in the in vitro enzyme assay, was isolated from Streptomyces sp. SN-593 as previously described (4).

[0085] (2) Strains and plasmids Escherichia coli (E. coli) DH5α (TaKaRa) and E. coli BL21 Star™ (DE3) (Invitrogen) were used with the expression vector pET28b(+) (Novagen) for general DNA manipulations and recombinant protein expression, respectively. For conjugative gene transfer from E. coli to Streptomyces sp. SN-593, E. coli GM2929 hsdS::Tn10 / pUB307Δaph was used instead, along with the shuttle integration vectors pTYM19 (φC31 insertion site) and pKU492Acosaac(3)IV (TG1 insertion site).

[0086] (3) Site-directed mutagenesis The revI gene was cloned into the pET28b(+) vector between the NdeI and XhoI restriction sites as previously described (Reference 4). Site-directed mutagenesis was performed using pET28b(+)-revI as a template with PrimeSTAR® GXL DNA Polymerase (TaKaRa) and the specifically designed primer pairs shown in Tables 1-1 to 1-3. PCR reactions were performed under the following conditions: 98°C for 10 seconds (1 cycle); 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 6.5 minutes (15 cycles); and 68°C for 10 minutes (1 cycle). Competent E. coli DH5α was transformed with the DpnI-digested PCR product by heat shock. Two transformant colonies were selected for each mutation, and 50 μg mL of the resulting product was used. -1 The mutant plasmids were isolated and their sequences were verified by DNA sequencing.

[0087] [Table 1-1]

[0088] [Table 1-2]

[0089] [Table 1-3]

[0090] (4) Heterologous expression and purification of P450revI and its mutants E. coli BL21 Star™ (DE3) cells transformed with pET28b(+)-revI or mutant pET28b(+)-revI were incubated with kanamycin (50 μg mL -1 ) in LB medium at 37°C overnight. 1 ml of the preculture was diluted with kanamycin (50 μg mL -1 ) was inoculated into 100 mL of Terrific Broth supplemented with 600 nm (A 600 When the optical density at 1000 nm reached 0.6, 5-aminolevulinic acid (Sigma-Aldrich) and isopropyl-β-D-thiogallatopyranoside (Nacalai Tesque) were added to the culture at final concentrations of 0.05 mM and 0.5 mM, respectively. After 24 h of incubation at 20°C, the culture was harvested by centrifugation at 4000 × g for 10 min, and the cell pellet was stored at -80°C until protein purification. Thaw the cell pellet on ice and add 0.5 mg mL -1 of lysozyme (Nacalai Tesque) and 5 mg mL -1The cells were suspended in 20 mL of buffer A [50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 20% glycerol] containing Sm2 nuclease. The cell suspension was sonicated on ice for 15 seconds with 15-second intervals, repeated 15 times, and then centrifuged (8000 × g, 1 hour) to remove cell debris. The supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA)-agarose column (Qiagen) equilibrated with buffer A. The Ni-NTA column was then washed sequentially with buffer B [50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol] containing 0.2% Tween 20 and buffer B containing different concentrations of imidazole (5 mM and 40 mM). His-tagged P450revI or mutants were eluted with buffer B containing 250 mM imidazole. The eluate was then loaded onto a PD-10 column (GE Healthcare) and buffer exchanged. The purified P450revI or mutants were stored at -80°C in 50 mM Tris-HCl (pH 7.5) buffer containing 20% ​​glycerol. The purity of P450revI or mutants was confirmed by SDS-PAGE.

[0091] (5) CO Difference Spectral Assay The concentrations of P450revI and mutants were measured by CO difference spectroscopy. P450revI or mutants were diluted with 50 mM Tris-HCl buffer (pH 7.5) containing 20% ​​glycerol to prepare a final sample volume of 1 mL in a quartz cuvette. A spatula tip of sodium dithionite (Sigma-Aldrich) was added to the sample, which was then mixed by inverting the cuvette sealed with Parafilm. Carbon monoxide (CO) was then slowly bubbled through the sample. A reduced CO difference spectrum was then recorded between 400 and 500 nm, and the extinction coefficient at 450 nm was 91 mM. -1 cm -1 The functional P450 concentration was calculated based on the above (Reference 10).

[0092] (6) In vitro enzyme assay RM-T (20 μM), P450revI or mutants (0.1 μM), spinach ferredoxin (5.6 μM), and spinach ferredoxin-NADP in Tris-HCl buffer (50 mM, pH 7.5). + A reaction mixture (final volume, 0.2 mL) containing reductase (5.4 μM) was preincubated at 30°C for 5 min, followed by the addition of NADPH (Roche) (1 mM) to initiate the reaction. After 10 min of incubation, the reaction was stopped by adding 4 μL of acetic acid (Wako Pure Chemical Industries, Ltd.) and then extracted with ethyl acetate (Fujifilm Wako Pure Chemical Industries, Ltd.) (0.75 mL × 2). The supernatant was dried under a gentle stream of nitrogen gas, suspended in 50 μL of methanol, and then subjected to UPLC-MS analysis (see (7) below). For the enzyme kinetic assay, the composition of the reaction mixture (final volume, 1 mL) was the same except for the concentrations of RM-T (0.4–12 μM) and P450revI or mutant (5 nM). After 1 min of incubation, the reaction was stopped by adding 20 μL of acetic acid. The reaction mixture was extracted with ethyl acetate (4 mL × 2). The supernatant was dried, dissolved in 50 μL of methanol, and analyzed by UPLC-MS (see (7) below). The results are shown in Figure 4.

[0093] (7) UPLC-MS Samples were analyzed using a 2965 Water Alliance UPLC system coupled to a 2996 Waters PDA UV detector and a mass spectrometer (AB Sciex Q-TRAP, Applied Biosystems). Analyte separation was performed using a Waters XTerra® MSC 18 A column (2.1 mm id × 150 mm, 5 μm) was used. The column thermostat was maintained at 40 °C. The mobile phase (MP) consisted of MilliQ water containing 0.1% formic acid (MP-A) and acetonitrile containing 0.1% formic acid (MP-B). Separation of the analytes was achieved at a flow rate of 0.65 mL min -1The separation was performed within 12 minutes at a flow rate of 100%. Samples (2 μL) were injected onto the column equilibrated with 10% MP-B. Separation of the analytes employed a linear gradient from 40% to 100% MP-B in 10 minutes, followed by a 2-minute hold at 100% MP-B. The mobile composition was then returned to 10% MP-B for 2 minutes. The analytes of interest were monitored at 238 nm, and mass spectra were collected in negative mode with electrospray ionization.

[0094] (8) Data analysis A calibration curve was prepared using RM-T, which has a maximum absorbance (λmax) at 238 nm, as a standard. The enzyme kinetic constant was calculated by nonlinear regression analysis using SigmaPlot™ (version 12, Systat Software Inc, USA). The product formation rate (V) versus substrate concentration ([S]) was fitted to the Michaelis-Menten equation.

[0095] (9) Conjugative transfer of the revI gene from E. coli to Streptomyces sp. SN-593 The revI gene of Streptomyces sp. SN-593 was inactivated as previously described (Reference 4). To obtain spores for conjugative transfer, Streptomyces sp. SN-593 (ΔrevI), in which the revI gene had been disrupted, was cultured on mannitol soy (MS) agar plates (2% soy flour, 2% D-(-)-mannitol, 2% agar) at 28°C for 1–2 weeks. After harvesting, the spores were subjected to a series of washing steps with spore buffer and water. Finally, the spores were suspended in SY medium (0.1% yeast extract, 0.1% NZ-amine, and 1% starch, pH 7.0) and cultured at 28°C for 4 hours. Next, the spores were transfected with pTYM19-P aphIIThe -revI mutant plasmid was mixed with E. coli GM2929 hsdS::Tn10 / pUB307Δaph and plated on modified MS (3% soy flour, 2% D(-)-mannitol, 2% agar) containing 25 mM MgCl2, followed by incubation at 28°C for 16-18 hours. The initial selection was performed using 1.5 mL of a mixture of thiostrepton (Sigma-Aldrich) and carumonam at a final concentration of 20 μg mL . -1 and 5 μg mL -1 After 5–7 days of incubation, thiostrepton (25 μg mL -1 ) and carumonum (2.5 μg mL -1 Resistant clones were selected by culturing on SY agar plates containing thiostrepton (5 μg mL -1 ) and incubated at 28°C for 2 days. 1 ml of the preculture (OD 600 = 3) was inoculated into a K1 flask containing PV8 medium [70 mL; 2% potato dextrose (Difco), 1% malt extract (Difco), 1% dry yeast (Asahi Breweries), 0.1% K2HPO4, 0.1% NaCl, 0.03% MgSO4·7H2O, 0.01% NaNO3, 0.005% ZnSO4·7H2O, and 0.005% CuSO4·5H2O] and cultured at 28°C and 150 rpm for 5 days.

[0096] (10) Extraction and Metabolite Analysis Five days after inoculation, an equal volume of acetone (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 4 mL of culture medium and then sonicated for 30 seconds. After centrifugation at 5,000 × g for 5 minutes, the resulting supernatant was concentrated under reduced pressure to remove the acetone. The acetone extract was adjusted to pH 4 with acetic acid and then extracted twice with an equal volume of ethyl acetate. After concentration under reduced pressure, the ethyl acetate extract was resuspended in methanol (Fujifilm Wako Pure Chemical Industries, Ltd.) (1.2 mL) and analyzed by UPLC-MS (see (7) above). The results of the UPLC-MS analysis are shown in Figure 5.

[0097] (11) Enhancement of productivity of novel reveromycin derivatives by introducing SARP (Streptomycin Antibiotic Regulator Protein) family regulators P aphII The 372-bp PCR fragment containing revQ and the 846-bp PCR fragment containing revQ were cloned into pTYM19-P aphII and pET-28b(+)-revQ were amplified using PrimeSTAR® GXL DNA polymerase (TaKaRa) under the following conditions: 98°C for 10 seconds; 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 25 seconds (P aphII ) or 30 cycles of 55 seconds (revQ) and a final 30 seconds at 68°C (P aphII ) or 60 seconds (revQ). P aphII The primers used to amplify the revQ gene are shown in Table 2. After restriction enzyme digestion, the insert was ligated into the pKU492aac(3)IV vector to create pKU492aac(3)IV-P aphII -revQ was built.

[0098] [Table 2]

[0099] To obtain spores for conjugative transfer, Streptomyces sp. SN-593 transformed with the mutant revI gene was cultured in thiostrepton (25 μg mL -1 The vector was cultured on MS agar plates containing the vector pKU492aac(3)IV-P at 28°C for 1-2 weeks. Spores were collected and prepared as described previously (9). aphII E. coli GM2929 hsdS::Tn10 / pUB307Δaph transformed with -revQ was incubated with spectinomycin (Sigma-Aldrich) (50 μg mL -1 ), streptomycin (Sigma-Aldrich) (100 μg mL -1 ), chloramphenicol (Nacalai Tesque) (30 μg mL -1) and apramycin (Sigma-Aldrich) (50 μg mL -1 The spores of the actinomycetes and E. coli were mixed and plated on MS agar containing 25 mM MgCl2 and incubated at 28°C for 16-18 hours. The first selection was performed by adding 1.5 mL of a mixture of apramycin, thiostrepton, and carumonum at 0.3 μg mL each. -1 , 20 μg mL -1 , 5 μg mL -1 After incubation for 5–7 days, apramycin (0.3 μg mL -1 ), thiostrepton (25μg mL -1 ), Carumonum (2.5μg mL -1 Resistant clones were selected by culturing on SY agar plates containing thiostrepton (5 μg mL -1 ) and cultured at 28°C for 2 days. 600 The strain (=3) was inoculated into a K1 flask containing PV8 medium (70 mL) and cultured at 28°C and 150 rpm for 5 days. The culture medium was then extracted and analyzed as described previously (10). The results of the UPLC-MS analysis are shown in Figure 5. The productivity of 17-hemisuccinyloxy RM-T is also shown in Table 3.

[0100] [Table 3]

[0101] (12) Isolation of novel reveromycin derivatives a) Wet column chromatography The ethyl acetate extract obtained by liquid-liquid extraction of the mutant culture medium was purified using wet column chromatography. Silica gel 60 (0.063-0.200 mm) (Millipore) (50 times the weight of the ethyl acetate extract) was used for separation. Stepwise gradient elution was performed using a chloroform / methanol mixture at ratios of 100:0, 100:1, 100:5, 100:10, 100:20, 50:50, and 0:100 (v / v). Each fraction was collected and subjected to UPLC-MS analysis (see (7) above). Fractions containing the desired novel reveromycin derivatives were dried under reduced pressure using a rotary evaporator, placed in vials, and stored at -20°C. b) Preparative High-Pressure Liquid Chromatography To further isolate the compounds, fractions obtained by wet column chromatography were subjected to preparative HPLC. In this study, a Waters 2535 quaternary pump equipped with a Waters 2998 photodiode array detector (PDA) was used. A Senshu Pak PEGASIL ODS SP100 C18 column (20 mm id × 250 mm) was run at a flow rate of 15 mL min -1 The separation was performed using a chromatograph. UV detection was set at 238 nm. For compound isolation, an aliquot (100 μL) of the sample was injected. The MP consisted of MilliQ water containing 0.1% formic acid (MP-A) and acetonitrile containing 0.1% formic acid (MP-B). The separation was performed using an isocratic run of 60% MP-B for 20 min, followed by a 100% MP-B hold for an additional 10 min. Four fractions were collected, and each fraction was analyzed by UPLC-MS (see (7) above). The fractions containing the target compounds were further purified by HPLC using various methods, as shown in Figure 3.

[0102] (13) Analysis by nuclear magnetic resonance (NMR), mass spectrometry (MS), ultraviolet-visible (UV-vis) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. NMR spectra were obtained using a JOEL JNM-ECA500 NMR spectrometer ( 1 H NMR: 500MHz, 13C NMR (125 MHz) was recorded. Chemical shifts are reported in parts per million (ppm). Proton signals are denoted using the abbreviations s = singlet, d = doublet, t = triplet, m = multiplet, and their combinations. Two-dimensional (2D) NMR techniques (H,H-COSY, HSQC, HMBC) were used for proton and carbon atom assignment. High-precision mass spectra were recorded using HR-ESI-ToF-MS. Both 17-hydroxyl RM-T and 17-hemisuccinyloxy RM-T were dissolved in MeOH to a concentration of 1 mg / mL. The compounds were then subjected to UV-vis spectroscopy using a JASCO V-630 BIO spectrophotometer and FTIR spectroscopy using a HORIBA high-sensitivity polarimeter for structural characterization. The results are shown in Figures 6–27.

[0103] 17-Hemisuccinyloxy RM-T (1): White amorphous; [α] D 27 -100.9°(27℃); UV (MeOH) λ max (log ε) 238 (4.44), 263 (4.21) nm; IR (ATR) v max 2925, 2367, 1697, 1558, 1541, 1510, 1167, 974, 800, 723 cm -1 ; 1 H and 13 C NMR (CD3OD), summarized in Table 4; ESI-MS (m / z) 659 [MH] - ; HR-ESI-ToF-MS (m / z) 659.3429 [MH] - (calculated for C 36 H 51 O 11 , 659.3431; Figure 10)

[0104] [Table 4]

[0105] 17-Hydroxyl RM-T (2): White amorphous; [α] D -122.2°(27℃); UV (MeOH) λ max (log ε) 238 (4.42), 263 (4.13) nm; IR (ATR) v max 2916, 2845, 1716, 1684, 1556, 1540, 1456, 1436, 1211, 841cm -1 ; 1 H and 13 C NMR (CD3OD), summarized in Table 5; ESI-MS (m / z) 559 [MH] - ; HR-ESI-ToF-MS (m / z) 559.3274 [MH] - (calculated for C 32 H 47 O8, 559.3271; Fig. 20).

[0106] [Table 5]

[0107] As shown in Figure 28, introduction of the A241L mutation into P450revI altered the regiospecificity of P450revI, catalyzing hydroxylation at the C17 position. The resulting 17-hydroxyl RM-T was accepted as a substrate for the subsequent enzyme involved in hemisuccinylation. Furthermore, it was confirmed that 17-hemisuccinyloxy RM-T was produced by transformants of Streptomyces sp. SN-593.

[0108] Bioactivity evaluation Cytotoxicity, antibacterial activity, and antimalarial activity were evaluated using the following test organisms as described in Reference 11: HeLa and HL-60 for cytotoxicity, Aspergillus oryzae for antibacterial activity, and Plasmodium falciparum 3D7 for antimalarial activity. Human multiple myeloma (MM) cells, PCM6, were obtained from the RIKEN Cell Bank (#RCB1460) and cultured at 37°C in McCoy's 5A (modified) medium (Thermo Fisher Scientific) supplemented with 20% fetal bovine serum (Merck), 20 ng / ml human IL-6 (Thermo Fisher Scientific), 100 U / ml penicillin, and 100 μg / ml streptomycin (Merck). To examine MM cytotoxicity, PCM6 cells were seeded at 2 × 104 cells / well in 96-well plates and exposed to test compounds. After 72 hours of culture, cell proliferation was measured using Cell Count Reagent SF (Nacalai Tesque). The pH level of the culture medium was adjusted by adding lactic acid, based on reference 12. The results are shown in Table 6.

[0109] [Table 6]

[0110] The results in Table 6 show that 17-hydroxyl RM-T and 17-hemisuccinyloxy RM-T exhibited antibacterial activity (Aspergillus oryzae (Ao)), antimalarial activity, and cytotoxicity against multiple myeloma equivalent to those of RM-A. Meanwhile, 17-hydroxyl RM-T and 17-hemisuccinyloxy RM-T exhibited reduced cytotoxicity against HeLa and HL-60 cells, which was observed with RM-A.

[0111] Examples of the formulation of the present invention include the following formulations: However, the present invention is not limited to these formulation examples.

[0112] Formulation Example 1: Capsule production 1) 30 mg of a compound represented by general formula (I) 2) Microcrystalline cellulose 10mg 3) Lactose 19mg 4) Magnesium stearate 1mg 1), 2), 3) and 4) are mixed and filled into a gelatin capsule.

[0113] Formulation Example 2: Tablet production 1) 10 g of a compound represented by general formula (I) 2) Lactose 50g 3) 15g corn starch 4) Carmellose calcium 44g 5) Magnesium stearate 1g The total amount of 1), 2), and 3) and 30 g of 4) are kneaded with water, vacuum dried, and then sized. 14 g of 4) and 1 g of 5) are mixed with this sized powder and compressed into tablets using a tablet press. 1,000 tablets containing 10 mg of the compound represented by general formula (I) are thus obtained. [Industrial Applicability]

[0114] According to the present invention, it is possible to provide novel reveromycin derivatives that are structurally stable even under acidic conditions without their 6,6-spiroacetal rings being converted to 5,6-spiroacetal rings and that exhibit biological activity equivalent to or greater than that of RM-A. Such reveromycin derivatives are expected to be used as medicines and agricultural chemicals, for example, as therapeutic agents for bone diseases (e.g., multiple myeloma), therapeutic agents for infectious diseases (e.g., malaria), antifungal agents, etc.

[0115] References 1. Miyamoto, Y., et al., Identification of Saccharomyces cerevisiae Isoleucyl-tRNA synthetase as a target of the G1-specific inhibitor reveromycin A *. Journal of Biological Chemistry, 2002. 277(32): p. 28810-28814. 2. Osada, H., et al., Reveromycin A, a new antibiotic which inhibits the mitogenic activity of epidermal growth factor. Journal of Antibiotics, 1991. 44(2): p. 259-261. 3. Takahashi, H., et al., Reveromycins, new inhibitors of eukaryotic cell growth. II. Biological activities. Journal of Antibiotics, 1992. 45(9): p. 1414-1419. 4. Takahashi, S., et al., Structure-function analyses of cytochrome P450revI involved in reveromycin A biosynthesis and evaluation of the biological activity of its substrate, reveromycin T. The Journal of biological chemistry, 2014. 289(47): p. 32446-32458. 5. Woo, J.-T., et al., Reveromycin A, an agent for osteoporosis, inhibits bone resorption by inducing apoptosis specifically in osteoclasts. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(12): p. 4729-4734. 6. Cuzzupe, A.N., et al., Total synthesis of the epidermal growth factor inhibitor (-)-reveromycin B. The Journal of Organic Chemistry, 2001. 66(7): p. 2382-2393. 7. El Sous, M., et al., Total Synthesis of (-)-Reveromycin A via a Hetero-Diels-Alder Approach. Synthesis, 2010. 2010(23): p. 3954-3966. 8. Shimizu, T., et al., Total Synthesis of Reveromycin A. The Journal of Organic Chemistry, 2000. 2(14): p.2153-2156. 9. Shimizu, T., et al., Chemical modification of reveromycin A and its biological activities. Bioorganic & Medicinal Chemistry Letters, 2002. 12(23): p. 3363-3366. 10. Omura, T. and R. Sato, The Carbon Monoxide-binding Pigment of Liver Microsomes: I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. Journal of Biological Chemistry, 1964. 239(7): p. 2370-2378. 11. Nogawa T. et al. Structure and biological activity of Metarhizin C, a stereoisomer of BR-050 from Tolypocladium album RK17-F0007. Journal of Antibiotics, 2019. 72(12):p. 996-1000. 12. Watanabe K., et al. Reveromycin A, a novel acid-seeking agent, ameliorates bone destruction and tumor growth in multiple myeloma. 2021. 106(4): p.1172-1177. Haematologica. 2021. 106(4): p. 1172-1177.

Claims

1. General formula (I): 【Chemical 1】 [In the formula, R １ is C １－６ represents an alkyl group, and R ２ represents a hydrogen atom or a hemisuccinyl group. A compound represented by the formula (I) or a salt thereof.

2. General formula (IA): 【Chemistry 2】 [In the formula, R １ is C １－６ represents an alkyl group, and R ２ represents a hydrogen atom or a hemisuccinyl group. A compound represented by the formula (I) or a salt thereof.

3. R １ is n-butyl, and R ２ The compound or salt thereof according to claim 1 or 2, wherein is a hydrogen atom or a hemisuccinyl group.

4. A cytochrome P450revI mutant consisting of any one of the following polypeptides (1) to (3): (1) A polypeptide in which the alanine residue at position 241 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with a leucine residue, an isoleucine residue, a valine residue, a serine residue, a threonine residue, or a proline residue; (2) A polypeptide according to (1) above, in which one or more amino acid residues other than the substituted amino acid residues have been substituted, added, inserted, or deleted, and which has the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T; and (3) A polypeptide having a sequence identity of 80% or more in the portion excluding the substituted amino acid residue in the polypeptide of (1), and having the activity of catalyzing the hydroxylation reaction at position 17 of reveromycin T.

5. A nucleic acid encoding the cytochrome P450revI mutant of claim 4.

6. A recombinant vector comprising the nucleic acid of claim 5.

7. A transformant obtained by introducing the recombinant vector according to claim 6 into a host.

8. The transformant according to claim 7 , wherein the host is a bacterium of the genus Streptomyces.

9. A method for producing a compound represented by general formula (Ia) and / or a compound represented by general formula (Ib), comprising a step of culturing the transformant described in claim 7 or 8 in a medium containing a compound represented by general formula (II). 【Chemistry 3】 [In the formula, R １ is C １－６ represents an alkyl group.]

10. The production method according to claim 9 , further comprising a step of recovering the compound represented by formula (Ia) or the compound represented by formula (Ib) from the culture obtained in the culturing step.

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