Vasohibin -2 INHIBITOR

Abstract

The present invention addresses the problem of providing a novel VASH2 inhibitor that contains, as an active ingredient, a compound that has not been known to have VASH2 inhibitory activity. Also, in one embodiment, the present invention addresses the problem of providing a novel prophylactic or therapeutic agent for diseases that can be treated by inhibiting binding between Vasohibin-2 and SVBP. The present invention provides a Vasohibin-2 inhibitor containing compound (1) or a salt thereof and a prophylactic or therapeutic agent for diseases that can be treated by inhibiting the binding of Vasohibin-2 and SVBP, the agent containing compound (1) or a salt thereof.

Description

Vasohibin-2 inhibitor

[0001] The present invention relates to a Vasohibin-2 inhibitor.

[0002] Through comprehensive analysis of genes whose expression is induced in vascular endothelial cells by the vascular endothelial growth factor (VEGF), the present inventors have discovered a novel angiogenesis inhibitor that is induced by VEGF and inhibits angiogenesis in an autocrine manner, which they have named "Vasohibin" (VASH). The Vasohibins discovered by the present inventors include the original VASH1 and its homolog VASH2. The present inventors have demonstrated that VASH2, which is expressed by cancer cells, is a useful molecular target for cancer therapy, and have further developed anti-VASH2 monoclonal antibodies and peptide vaccines as techniques for inhibiting VASH2 (Non-Patent Documents 1 to 9). Recently, since the transglutaminase domain in the vasohibin molecule has tubulin carboxypeptidase (TCP) activity as an enzyme, TCP inhibitors have been reported as vasohibin inhibitors.

[0003] Furthermore, VASH2 has been reported to be associated with various diseases other than cancer, and therefore there is a strong demand for the development of compounds that exhibit VASH2 inhibitory activity (Non-Patent Documents 10 to 13).

[0004] Takahashi Y, Koyanagi T, Suzuki Y, Saga Y, Kanomata N, Moriya T, Suzuki M, and Sato Y. Vasohibin-2 expressed in human serous ovarian adenocarcinoma accelerates tumor growth by promoting angiogenesis. Mol. Cancer Res. 10, 1135-1146, 2012.Xue X, Gao W, Sun B, Xu Y, Han B, Wang F, Zhang Y, Sun J, Wei J, Lu Z, Zhu Y, Sato Y, Sekido Y, Miao Y, and Kondo Y. Vasohibin 2 is transcriptionally activated and promotes angiogenesis in hepatocellular carcinoma. Oncogene. 32, 1724-1734, 2013.Kitahara S, Suzuki Y, Morishima M, Yoshii A, Kikuta S, Shimizu K, Morikawa S, Sato Y, Ezaki T. Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis. Mol. Cancer 13: 99, 2014.Suzuki Y, Kitahara S, Suematsu T, Oshima M, Sato Y. Requisite role of vasohibin-2 in spontaneous gastric cancer formation and accumulation of cancer-associated fibroblasts. Cancer Sci. 108: 2342-2351, 2017.Iida-Norita R, Kawamura M, Suzuki Y, Hamada S, Masamune A, Furukawa T, Sato Y.Vasohibin-2 plays an essential role in metastasis of pancreatic ductal adenocarcinoma. Cancer Sci. 110: 2296-2308, 2019.Koyanagi T, Suzuki Y, Komori K, Saga Y, Matsubara S, Fujiwara H, Sato Y. Targeting human vasohibin-2 by a neutralizing monoclonal antibody for anti-cancer treatment. Cancer Sci. 108: 512-519, 2017.Koyanagi T, Suzuki Y, Saga Y, Machida S, Takei Y, Fujiwara H, Suzuki M, Sato Y. In vivo delivery of siRNA targeting vasohibin-2 decreases tumor angiogenesis and suppresses tumor growth in ovarian cancer. Cancer Sci. 104, 1705-1710, 2013.Horie S, Suzuki Y, Yamamoto T, Obika S, Mohri K, Kiyota C, Ren Q, Warashina S, Wada Y, Watanabe Y, Mukai H, Sato Y. Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2. Cancer Sci. 114: 3740-3749, 2023.Lee E-S, Suzuki Y, Tomioka H, Nakagami H, Sato Y. Development of a Novel and Simple Anti-Metastatic Cancer Treatment Targeting Vasohibin-2. Tohoku J Exp Med. 261: 239-247, 2023.Masuda K, Tanabe K, Ujike H, Hinamoto N, Miyake H, Tanimura S, Sugiyama H, Sato Y, Maeshima Y, Wada J. Deletion of pro-angiogenic factor vasohibin-2 ameliorates glomerular alterations in a mouse diabetic nephropathy model. PLoS One. 13: e0195779, 2018.Okuyama M, Uchida HA, Hada Y, Kakio Y, Otaka N, Umebayashi R, Tanabe K, Fujii Y, Kasahara S, Subramanian V, Daugherty A, Sato Y, Wada J. Exogenous vasohibin-2 exacerbates angiotensin II-induced ascending aortic dilation in mice. Circ Rep. 1: 155-161, 2019.Isoda R, Morita I, Isida A, Mikami Y, Monobe Y, Sato Y, Moriya T. Pathological study on the expression of vasohibins in peripheral artery disease. Tohoku J. Exp. Med. 258:121–128, 2022Vite A, Caporizzo MA, Corbin EA, Brandimarto J, McAfee Q, Livingston CE, Prosser BL, Margulies KB. Extracellular stiffness induces contractile dysfunction in adult cardiomyocytes via cell-autonomous and microtubule-dependent mechanisms. asic Cardiol Res. 117:41,

[0005] An objective of the present invention is to provide a novel VASH2 inhibitor containing, as an active ingredient, a compound that was not previously known to have VASH2 inhibitory activity. Also, in one embodiment, an objective of the present invention is to provide a novel prophylactic or therapeutic agent for a disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP.

[0006] Under these circumstances, the present inventors have investigated the VASH2 inhibitory activity of a wide variety of compounds and conducted extensive research, and as a result have found that the above problems can be solved by using a compound represented by the following general formula (1) or a salt thereof. The present invention is based on this novel finding. Therefore, the present invention provides the following: Item 1. A compound represented by the following general formula (1)

[0007]

[0008] [In the formula, R １ represents a linear or branched divalent saturated hydrocarbon group. ２ and R ３ are the same or different and represent a monovalent organic group; l represents an integer of 1 to 3; and m represents an integer of 0 to 2.] or a salt thereof.

[0009] Item 2. The following general formula (1)

[0010]

[0011] [In the formula, R １ represents a linear or branched divalent saturated hydrocarbon group. ２ and R ３ are the same or different and represent a monovalent organic group. 1 represents an integer of 1 to 3. m represents an integer of 0 to 2.] or a salt thereof, a preventive or therapeutic agent for a disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP.

[0012] Item 3. R １ Item 1. The Vasohibin-2 inhibitor according to Item 1 or the preventive or therapeutic agent according to Item 2, wherein represents a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms.

[0013] Item 4. R ２is an alkylsulfonyl group, a nitro group, a dimethylsulfoxide group, a sulfonic acid group, a nitrile group, an amide group, a cyano group, an amino group, an aldehyde group, a hydroxyl group, an isocyanate group, a carboxyl group, an N-hydroxysuccinimide group, a maleimide group, or a thiol group (however, when l is 2 or more, a plurality of R ２ may be the same or different), the Vasohibin-2 inhibitor according to Item 1 or the preventive or therapeutic agent according to Item 2.

[0014] Item 5. R ３ Item 1. The Vasohibin-2 inhibitor according to Item 1 or the prophylactic or therapeutic agent according to Item 2, wherein represents an alkyl group or a substituted or unsubstituted aromatic group.

[0015] Item 6. R １ represents a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms; R ２ is an alkylsulfonyl group, a nitro group, a dimethylsulfoxide group, a sulfonic acid group, a nitrile group, an amide group, a cyano group, an amino group, an aldehyde group, a hydroxyl group, an isocyanate group, a carboxyl group, an N-hydroxysuccinimide group, a maleimide group, or a thiol group (however, when l is 2 or more, a plurality of R ２ may be the same or different, and R ３ Item 1. The Vasohibin-2 inhibitor according to Item 1 or the prophylactic or therapeutic agent according to Item 2, wherein represents an alkyl group or a substituted or unsubstituted aromatic group.

[0016] Item 7. The prophylactic or therapeutic agent according to Item 2, wherein the disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP is cancer, diabetic nephropathy, arteriosclerosis, aortic aneurysm, or heart failure.

[0017] Item 8. A method for inhibiting Vasohibin-2, which comprises administering an effective amount of compound (1) or a salt thereof to a subject in need thereof.

[0018] Item 9. A method for preventing or treating a disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP, comprising administering an effective amount of compound (1) or a salt thereof to a subject in need thereof.

[0019] Item 10. Use of compound (1) or a salt thereof for producing a Vasohibin-2 inhibitor.

[0020] Item 11. Use of compound (1) or a salt thereof for producing an agent for preventing or treating a disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP.

[0021] Item 12. Compound (1) or a salt thereof for use in inhibiting Vasohibin-2.

[0022] Item 13. Compound (1) or a salt thereof for use in the prevention or treatment of a disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP.

[0023] According to the present invention, it is possible to provide a novel VASH2 inhibitor containing, as an active ingredient, a compound that was not previously known to have VASH2 inhibitory activity.

[0024] 1 shows a graph indicating the inhibition rate of each compound in the first screening in Example 1. 2 shows a graph indicating the inhibition rate of each compound in the second screening in Example 1. 3 shows the results of measuring the α-tubulin detyrosination level and the cell viability test for compounds selected in the first screening in Example 1. 4 shows the results of measuring the α-tubulin detyrosination level and the cell viability test for compounds selected in the second screening in Example 1. 5 shows the results of evaluating the inhibitory effect on VASH1 and VASH2 in Example 2. 6 shows an outline of screening for VASH2-SVBP binding inhibitors using the NanoBiT system in Example 1. VASH2-Smbit and LgBit-SVBP are electroporated into HeLa cells, the cells are plated onto a 384-well plate containing the compounds, and one day later, a culture medium containing Nano-Glo Live Cell Reagent (Promega) is added, and the luminescence intensity is measured. 1. An expression vector for a fusion protein of NanoLuc subunits (LgBiT and SmBiT) is introduced into the cultured cells. 2. The binding between VASH2 and SVBP causes LgBiT and SmBit to associate, resulting in the expression of the luciferase function of NanoLuc. 3. In the presence of Compound A, which inhibits the binding between VASH2 and SVBP, the expression of luciferase function is inhibited. The structural formulas of the compounds used in Example 2 are shown below.

[0025] The present invention relates to a vasohibin-2 inhibitor represented by the following general formula (1):

[0026]

[0027] [In the formula, R １ represents a linear or branched divalent saturated hydrocarbon group. ２ and R ３ are the same or different and represent a monovalent organic group. 1 represents an integer of 1 to 3. m represents an integer of 0 to 2.] or a salt thereof. In the present invention, the compound represented by general formula (1) may be simply referred to as compound (1).

[0028] In the present invention, "inhibiting vasohibin-2 (VASH2)" means significantly inhibiting the activity of VASH2 compared to the absence of the compound of the present invention or a salt thereof. Specifically, in the present invention, inhibition of VASH2 includes inhibiting the formation of a complex between VASH2 and small vasohibin binding protein (SVBP) or inhibiting α-tubulin detyrosination by the complex. More specifically, inhibition of the formation of a complex between VASH2 and SVBP or α-tubulin detyrosination by the complex can be measured by the method described in the Examples below. In the present invention, the compound of the present invention or a salt thereof preferably exhibits an inhibition rate of 80% or more of the formation of a complex between VASH2 and SVBP, as measured by the above Examples or the like, compared to a positive control (in the presence of VASH2 and SVBP and in the absence of the compound of the present invention or a salt thereof). Here, the rate of inhibition of complex formation between VASH2 and SVBP can be calculated by the following formula: Inhibition rate (%) = [1 - ([amount of detyrosinated α-tubulin in the presence of the compound of the present invention or a salt thereof] - [amount of detyrosinated α-tubulin in cells expressing detyrosinated tubulin activity-deficient VASH2C158A] / [amount of detyrosinated α-tubulin in cells expressing wild-type de-VASH2] - [amount of detyrosinated α-tubulin in cells expressing detyrosinated tubulin activity-deficient VASH2C158A]) × 100 In such cases, the amount of detyrosinated α-tubulin in each sample can be determined by absorbance measurement, etc. Furthermore, the compound of the present invention or a salt thereof preferably exhibits α-tubulin detyrosination activity of a complex of VASH2 and SVBP that is 50% or less, more preferably 30% or less, and even more preferably 20% or less, as compared to a control (in the presence of VASH2 and SVBP but in the absence of the compound of the present invention or a salt thereof), as measured by a method such as the method described in the Examples below.

[0029] The amino acid sequence of VASH2 and the sequence of the mRNA encoding it have been registered in GenBank, provided by the National Center for Biotechnology Information (NCBI), under Accession No. NM_001301056.2 (where multiple revisions are registered, it is understood that the most recent revision is referred to). Examples of VASH2 include those derived from humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep, etc.

[0030] Furthermore, in the present invention, compound (1) or a salt thereof preferably has selective inhibitory activity against VASH2. Specifically, it is preferable that the inhibitory activity against VASH2 is significantly higher than that against VASH1. For example, it is preferable that the α-tubulin detyrosination activity of the complex of VASH2 and SVBP, measured by the method described in the Examples below, is significantly lower than the α-tubulin detyrosination activity of the complex of VASH1 and SVBP (for example, the detyrosination activity of the former is 60% or less (more preferably 50% or less) of the detyrosination activity of the latter).

[0031] In general formula (1), R １ represents a straight-chain or branched-chain divalent saturated hydrocarbon group. Examples of the divalent saturated hydrocarbon group include -CH ２ -, -CH ２ -CH ２ -, -CH ２ -CH ２ -CH ２ -, -CH ２ -CH ２ -CH ２ -CH ２ -, -CH ２ -CH ２ -CH ２ -CH ２ -CH ２ -, -CH ２ -CH ２ -CH ２ -CH ２ -CH ２ -CH ２-, -CH(CH ３ ) -, -CH(CH ３ )-CH ２ -, -CH ２ -CH(CH ３ )-CH ２ -, -CH ２ -CH ２ -CH(CH ３ )-CH ２ -, -CH ２ -CH ２ -CH(CH ３ )-CH ２ -CH ２ -, -CH(CH ２ -CH ３ ) -, -CH(CH ２ -CH ２ -CH ３ ) -, -CH(CH ２ -CH ２ -CH ２ -CH ３ ) -, -CH(CH ２ -CH ２ -CH ２ -CH ２ -CH ３ ) -, -CH(CH ２ -CH ２ -CH ２ -CH ２ -CH ２ -CH ３ )- and the like. １ The number of monolayers of the linear or branched divalent saturated hydrocarbon group represented by is not limited, but is, for example, preferably 1 to 7, more preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, more preferably 1 to 3, more preferably 2 to 3, and more preferably 2. １ The linear or branched divalent saturated hydrocarbon group represented by the formula (I) is preferably a molecular chain.

[0032] R ２ and R ３ R may be the same or different and represent a monovalent organic group. ２ Examples of the organic group represented by the formula (I) include an alkylsulfonyl group, a nitro group, a dimethylsulfoxide group, a sulfonic acid group, a nitrile group, an amide group (—CO—NH ２), cyano group, amino group, aldehyde group (-C(=O)-R group (R is a hydrocarbon group)), hydroxyl group, isocyanate group, carboxyl group, N-hydroxysuccinimide group, maleimide group, or thiol group, and alkylsulfonyl group, nitro group, etc. are preferred. Examples of alkylsulfonyl groups include alkylsulfonyl groups in which the alkyl moiety is a linear or branched alkyl group having 1 to 6 carbon atoms (preferably 1 to 3, more preferably 1). R ３ Examples of the organic group represented by R include an alkyl group and a substituted or unsubstituted aromatic group, and an alkyl group is preferred. ３ Examples of the alkyl group represented by the formula (I) include a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms (preferably 1 to 3, more preferably 1).

[0033] Examples of the aromatic group include aromatic hydrocarbon groups such as piperidino, phenyl, tolyl, and naphthyl; aromatic heterocyclic groups such as furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, triazolyl (1,2,3-triazolyl, 1,2,4-triazolyl), pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, quinolyl, isoquinolyl, indolyl, and pyridothienopyrimidine ring groups; and fused aromatic groups such as 2,3-dihydrobenzofuranyl.

[0034] Examples of the substituent in the aromatic group include substituted or unsubstituted C groups such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, an isopentyl group, a hexyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a trifluoromethyl group. １－６ -Alkyl group; C such as methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, isopentyloxy group, hexyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, and cyclohexyloxy group １－６-alkoxy group; C such as a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group, an isopropoxycarbonyl group, a butoxycarbonyl group, an isobutoxycarbonyl group, a sec-butoxycarbonyl group, a tert-butoxycarbonyl group, a pentyloxycarbonyl group, an isopentyloxycarbonyl group, a cyclopropyloxycarbonyl group, a cyclobutyloxycarbonyl group, or a cyclopentyloxycarbonyl group １－６ -alkoxy-carbonyl group; aromatic hydrocarbon group such as phenyl group, tolyl group, naphthyl group, etc.; halogen atom such as fluorine atom, chlorine atom, bromine atom, iodine atom, etc.; C １－６ -acyl group, aralkyl group, aralkyloxy group, cyano group, C １－６ -Alkylamino group, diC １－６ -alkylamino groups.

[0035] Said C １－６ - alkyl group, C ２－６ -alkenyl group, C １－６ -alkoxy group, C １－６ - alkylthio group, C １－６ The alkoxy-carbonyl group and the acyl group are selected from the group consisting of an aromatic group, an acyl group, a hydroxyl group, a carboxyl group, a halogen atom, an amino group, a C １－６ -alkoxy group (e.g., methoxy group, ethoxy group, propoxy group), C １－６ -alkylthio groups and the like. For example, C substituted with a halogen atom １－６ Examples of the alkyl group include a trifluoromethyl group, a chloromethyl group, and a 1-chloroethyl group. １－６ Examples of the alkyl group include an aminomethyl group and a 1-aminoethyl group. １－６ -C substituted with alkylthio group １－６ Examples of the alkoxy group include a 2-(methylthio)ethoxy group. １－６ Examples of the alkoxy group include a difluoromethoxy group. １－６Examples of the alkyl group include a substituted or unsubstituted pyridylmethyl group and a substituted pyrimidinylmethyl group.

[0036] The substituted amino group includes, for example, a group represented by the following formula: NRR' (wherein R and R' are the same or different and represent a hydrogen atom, a carboxyl group, a substituted C １－６ -Alkyl group, substituted C １－６ -alkoxy group, substituted C １－６ -alkoxy-carbonyl group, a substituted or unsubstituted aromatic group, a substituted aralkyl group, or a substituted acyl group, and R and R' may form a substituted heterocycle together with the nitrogen atom to which they are attached.) Examples of the substituted amino group include the above-mentioned C １－６ - Amino groups mono- or di-substituted with alkyl groups, such as methylamino and dimethylamino groups; piperidino and morpholino groups.

[0037] The compound of the present invention is a compound of formula (I) in which R ３ means a carbon ring or heterocyclic ring of the aromatic group, and is a halogen atom, a hydroxyl group, a carboxyl group, a substituted amino group, a substituted C １－６ -Alkyl group, substituted C ２－６ -Alkenyl group, substituted C １－６ -alkoxy group, substituted C １－６ -Alkylthio group, substituted C １－６ -alkoxy-carbonyl group, a substituted aromatic group, a substituted aralkyl group, a substituted acyl group, etc. (However, when there are multiple substituents, they may be the same or different.) When l is 2 or more, multiple R ２ When m is 2 or more, a plurality of R ３ may be the same or different. l represents an integer of 1 to 3, preferably 1 or 2, and more preferably 2. m represents an integer of 0 to 2, preferably 0 or 1, and more preferably 1.

[0038] R ２ The position of the organic group represented by may be any of the ortho-, meta-, and para-positions, but is preferably the meta- and / or para-positions. ３The position of the organic group represented by the formula (I) may be any of the ortho, meta and para positions, but the para position is preferred.

[0039] Compound (1) is known or can be appropriately synthesized according to known methods. In the present invention, the term "salt" refers to a pharmaceutically acceptable salt. Furthermore, salts of compound (1) include acid addition salts and salts with bases. Specific examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, and phosphate; organic acid salts such as oxalate, malonate, succinate, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, and trifluoromethanesulfonate; and acidic amino acid salts such as glutamate and aspartate. Specific examples of salts with bases include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt, and calcium salt; salts with organic bases such as pyridine salt and triethylamine salt; and salts with basic amino acids such as lysine and arginine.

[0040] Compound (1) or a salt thereof may exist in the form of a hydrate or solvate, and these hydrates and solvates are also included in the compound that is the active ingredient of the present invention. Furthermore, when compound (1) has isomers such as geometric isomers, stereoisomers, and optical isomers, these isomers are included in compound (1) unless otherwise specified.

[0041] Examples of solvents that form solvates include water, alcohols such as ethanol and propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, and DMSO.

[0042] In the present invention, compound (1) or a salt thereof may be used as a Vasohibin-2 inhibitor by itself, or may be used as a composition in combination with various pharmaceutically acceptable carriers (e.g., isotonicity agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizing agents, thickeners, etc.).

[0043] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride.

[0044] Examples of chelating agents include edetate salts such as disodium edetate, calcium disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or a salt thereof, sodium hexametaphosphate, and citric acid.

[0045] The stabilizer may, for example, be sodium hydrogen sulfite.

[0046] Examples of pH adjusters include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol.

[0047] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

[0048] Antioxidants include, for example, sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, and the like.

[0049] Examples of solubilizing agents include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol, etc., and examples of thickening agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc.

[0050] In an embodiment of the composition, the content of compound (1) or a salt thereof in the composition is not particularly limited and can be appropriately set based on conditions such as, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, or 1% by mass or more in terms of the content of compound (1).

[0051] The dosage form is not particularly limited, and examples thereof include various dosage forms such as orally administered agents such as tablets, pills, capsules, powders, granules, syrups, and sublingual agents, and parenterally administered agents such as injections (intravenous injection, intramuscular injection, local injection, etc.), mouthwashes, drip infusions, topical agents (ointments, creams, patches, inhalants), and suppositories. Of the above dosage forms, preferred examples include injections, orally administered agents, and topical agents.

[0052] The content of compound (1) of the present invention in a formulation cannot be generally determined because it varies depending on the route of administration, the age, weight, symptoms, etc. of the patient, but it is usually an amount that results in a daily dose of about 10 to 5,000 mg, more preferably about 100 to 1,000 mg, of compound (1). When administered once a day, this amount should be contained in one formulation, and when administered three times a day, one third of this amount should be contained in one formulation.

[0053] The pharmaceutical agent of the present invention is administered to a subject such as a mammal. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, and sheep.

[0054] The Vasohibin-2 inhibitor of the present invention can be used in vivo or in vitro, and can also be added to a sample collected from a living body (e.g., body fluids such as blood, urine, pleural effusion, and ascites; cells; tissue extracts).

[0055] Pharmaceuticals In the present invention, compound (1) or a salt thereof inhibits the binding of vasohibin-2 to SVBP. Accordingly, the present invention provides a preventive or therapeutic agent comprising compound (1) or a salt thereof for a disease that can be treated by inhibiting the binding of vasohibin-2 to SVBP. Diseases that can be treated by inhibiting the binding of vasohibin-2 to SVBP include cancer, diabetic nephropathy, arteriosclerosis, aortic aneurysm, heart failure, etc.

[0056] In this embodiment, details of compound (1) or a salt thereof, usage method, other ingredients, etc. are the same as those described above for the Vasohibin-2 inhibitor.

[0057] Furthermore, in addition to compound (1) or a salt thereof, the prophylactic or therapeutic agent of the present invention may further comprise a compound known to be useful in the treatment of diseases that can be treated by inhibiting the binding of vasohibin-2 to SVBP. Examples of such compounds include anticancer agents such as irinotecan, SN-38, doxorubicin, daunorubicin, etoposide, mitoxantrone, topotecan, imatinib, nilotinib, dasatinib, and gefitinib; therapeutic agents for diabetic nephropathy such as SGLT2 inhibitors; therapeutic agents for arteriosclerosis such as rosuvastatin calcium, ethyl icosapentate, and omega-3 fatty acid ethyl esters; and therapeutic agents for heart failure such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta (β)-blockers, aldosterone antagonists, sacubitril / valsartan, and SGLT2 inhibitors. These components may be used alone or in combination of two or more. Furthermore, the prophylactic or therapeutic agent of the present invention may further contain, in addition to compound (1) or a salt thereof, at least one selected from the group consisting of compounds VH2S-01 to VH2S-04 or salts thereof.

[0058] Furthermore, as shown in the Examples below, the following compounds VH2S-01 to VH2S-04 or salts thereof also have inhibitory activity against the binding of Vasohibin-2 to SVBP.

[0059]

[0060] Accordingly, the present invention provides a vasohibin-2 inhibitor comprising at least one selected from the group consisting of VH2S-01 to VH2S-04 or a salt thereof, as well as a preventive or therapeutic agent for a disease that can be treated by inhibiting the binding of vasohibin-2 to SVBP, comprising at least one selected from the group consisting of VH2S-01 to VH2S-04 or a salt thereof. In these embodiments, details of the components that form the salts of these compounds, methods of using the compounds or salts thereof, other components, etc. are the same as those described above for compound (1) or a salt thereof. Furthermore, in these embodiments, the preventive or therapeutic agent of the present invention may further comprise compound (1) or a salt thereof in addition to at least one selected from the group consisting of VH2S-01 to VH2S-04 or a salt thereof.

[0061] Specific embodiments of the present invention will be described in more detail below using examples, but the present invention is not limited to such embodiments.

[0062] Example 1 Initial Screening with the NanoBiT System An overview of this example is shown in Figure 6. HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) GlutaMAX (Thermo, 10569-010) containing 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin (Thermo, 15140122) at 37°C and 5% CO ２ HeLa cells (0.5-1.5 × 10 ６The resulting mixture (1 x 10 cells / 0.5 ml) was mixed on ice with 20 μg of VASH2-SmBIT expression vector and / or 20 μg of LgBiT-SVBP expression vector (here, VASH2-SmBiT refers to a fusion protein in which SmBiT is linked to the C-terminus of human VASH2. The VASH2-SmBiT expression vector used was the Promega pBiT2.1-C [TK / SmBiT] Vector in which the CDS of human full-length VASH2 was introduced). This mixture was electroporated in a 0.4 cm gap cuvette using a Gene Pulser (Bio-Rad) at 250 V, 500 μF, and ∞Ω. After electroporation, the cells (1 x 10 cells / 0.5 ml) were mixed with 20 μg of VASH2-SmBIT expression vector and / or 20 μg of LgBiT-SVBP expression vector (here, VASH2-SmBiT refers to a fusion protein in which SmBiT is linked to the C-terminus of human VASH2. The VASH2-SmBiT expression vector used was the Promega pBiT2.1-C [TK / SmBiT] Vector in which the CDS of human full-length VASH2 was introduced). ４ The cells (100 μl / well) were plated in a 384-well white plate (Greiner, 781080) and mixed with a final concentration of 5 μM of small molecule compounds selected from a core library of 9,600 compounds (Drug Delivery Initiative, The University of Tokyo). After 24 hours of culture, Nano-Glo Live Cell substrate (Nano-Glo Live Cell Assay System, Promega, N2012) was added to the cells. The cells were then incubated at 37°C, 5% CO ２ The cells were incubated at 4°C for 10 minutes. The luminescence intensity of the cells was measured using a multimode plate reader, EnSight (Perkin Elmer). From the luminescence intensity, the reaction inhibition rate was calculated, with the background (control DMSO) set to 0% and the positive control (LgBiT-SVBP expression) set to 100%. The reaction inhibition rate was calculated as follows: Reaction inhibition rate = {1 - ([Luminescence intensity of test group with each compound added] - [Luminescence intensity of positive control]) / ([Background luminescence intensity] - [Luminescence intensity of positive control])} x 100. Screening was performed twice. In the first round, 7,040 compounds were screened, and in the second round, 3,520 compounds were screened. Figure 1 shows a graph illustrating the inhibition rate for each compound in the first screening. Figure 2 shows a graph illustrating the inhibition rate for each compound in the second screening.

[0063] Example 2 Second Screening by Cell-Based ELISA Suzuki Y, Kobayashi M, Miyashita H, Ohta H, Sonoda H, Sato Y. Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion. J. Cell Sci. 123(18):3094-3101(2010). HeLa cells were transiently transfected with expression vectors for the combination of VASH1 and SVBP-FLAG, or the combination of VASH2 and SVBP-FLAG, using FuGENE HD transfection reagent (Promega, Madison, WI). Six hours after transfection, the cells were harvested and plated at 1 x 10 cells per well in a 96-well microplate. ４Cells were seeded at 1000 cells / well. After allowing the cells to adhere for 3 hours, they were treated with culture medium containing 5 μM of the test compounds selected in the screening of Example 1. After 48 hours of culture, cell proliferation was confirmed using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Cell-based ELISA was then performed to measure the detyrosinated α-tubulin level in each well as follows: Cells were fixed with 4% paraformaldehyde for 10 minutes at 37°C, permeabilized with 0.5% Triton-X100 in PHEM buffer for 10 minutes at room temperature (RT), and blocked with 5% skim milk in PBS for 2 hours at RT. Cells were then incubated overnight at 4°C with anti-detyrosinated α-tubulin antibody (Abcam, Cambridge, MA) in 5% skim milk in PBS. Excess antibody was removed by washing with 0.05% Tween 20 in TBS. Next, the plates were treated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Bio-Rad, Hercules, CA, USA) for 2 hours at room temperature. After washing with 0.05% Tween 20 in TBS, TMB substrate (Cell Signaling, Beverly, MA) was added to each well and incubated for 10 minutes at room temperature. The reaction was stopped by adding 0.5 M sulfuric acid, and the absorbance at 450 nm was measured using an iMark Microplate Reader (Bio-Rad).

[0064] The formula for calculating the detyrosination level from the above absorbance is as follows: Detyrosination level=1-([absorbance at 450 nm in the sample to which the test compound was added]-[absorbance at 450 nm in the sample of cells expressing detyrosinated tubulin activity-deficient VASH2C158A]) / ([absorbance at 450 nm in the sample of cells expressing wild-type de-VASH2]-[absorbance at 450 nm in the sample of cells expressing detyrosinated tubulin activity-deficient VASH2C158A]).

[0065] Cell viability was also measured using Dojindo's Cell Counting Kit-8. Figure 3 shows the results of measuring the α-tubulin detyrosination level and cell viability test for the compounds selected in the first screening of Example 1 in a system using an expression vector combining VASH2 and SVBP-FLAG. Figure 4 shows the results of measuring the α-tubulin detyrosination level and cell viability test for the compounds selected in the second screening of Example 1 in a system using an expression vector combining VASH2 and SVBP-FLAG. VH2S-01 to VH2S-05 were selected from the standpoint of their effect on reducing detyrosination levels and cell viability in these tests. The structures of VH2S-01 to VH2S-05 are shown in Figure 7. Using a system using an expression vector for the combination of VASH1 and SVBP-FLAG and a system using an expression vector for the combination of VASH2 and SVBP-FLAG, we evaluated whether the inhibitory effect of VH2S-01 to VH2S-05 on α-tubulin detyrosination is selective for VASH2. The results of this test are shown in Figure 5. As shown in Figure 5, of these compounds, VH2S-05 has a high inhibitory effect on α-tubulin detyrosination by the VASH2 / SVBP complex. Furthermore, the inhibitory effect of VH2S-05 on α-tubulin detyrosination by the VASH1 / SVBP complex is relatively weaker than the inhibitory effect on α-tubulin detyrosination by the VASH2 / SVBP complex. This demonstrates that VH2S-05 can selectively inhibit VASH2.

Claims

1. The following general formula (1) [In the formula, R １ represents a linear or branched divalent saturated hydrocarbon group. ２ and R ３ are the same or different and represent a monovalent organic group; l represents an integer of 1 to 3; and m represents an integer of 0 to 2.] or a salt thereof.

2. The following general formula (1) [In the formula, R １ represents a linear or branched divalent saturated hydrocarbon group. ２ and R ３ are the same or different and represent a monovalent organic group. 1 represents an integer of 1 to 3. m represents an integer of 0 to 2.] or a salt thereof, a preventive or therapeutic agent for a disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP.

3. R １ The Vasohibin-2 inhibitor according to claim 1 or the preventive or therapeutic agent according to claim 2, wherein represents a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms.

4. R ２ is an alkylsulfonyl group, a nitro group, a dimethylsulfoxide group, a sulfonic acid group, a nitrile group, an amide group, a cyano group, an amino group, an aldehyde group, a hydroxyl group, an isocyanate group, a carboxyl group, an N-hydroxysuccinimide group, a maleimide group, or a thiol group (however, when l is 2 or more, a plurality of R ２ may be the same or different), the Vasohibin-2 inhibitor according to claim 1 or the preventive or therapeutic agent according to claim 2.

5. R ３ The Vasohibin-2 inhibitor according to claim 1 or the preventive or therapeutic agent according to claim 2, wherein represents an alkyl group or a substituted or unsubstituted aromatic group.

6. R １ represents a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms; R ２ is an alkylsulfonyl group, a nitro group, a dimethylsulfoxide group, a sulfonic acid group, a nitrile group, an amide group, a cyano group, an amino group, an aldehyde group, a hydroxyl group, an isocyanate group, a carboxyl group, an N-hydroxysuccinimide group, a maleimide group, or a thiol group (however, when l is 2 or more, a plurality of R ２ may be the same or different, and R ３ The Vasohibin-2 inhibitor according to claim 1 or the preventive or therapeutic agent according to claim 2, wherein represents an alkyl group or a substituted or unsubstituted aromatic group.

7. The preventive or therapeutic agent according to claim 2, wherein the disease that can be treated by inhibiting the binding of Vasohibin-2 to SVBP is cancer, diabetic nephropathy, arteriosclerosis, aortic aneurysm, or heart failure.

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