Prophylactic or therapeutic agent for polycystic kidney disease

Abstract

The present invention addresses the problem of providing a new prophylactic or therapeutic agent for a kidney diseases. Provided is a prophylactic or therapeutic agent for polycystic kidney disease, the agent comprising, as an active ingredient, a compound that promotes intermolecular interaction between an oxysterol-binding protein-related protein-3 (ORP3) and an endoplasmic vesicle protein-associated protein-A (VAP-A) in a cell, and has a cholesterol increasing effect in primary cilia of the cell, or a pharmaceutically acceptable salt thereof.

Description

Preventive or therapeutic agent for polycystic kidney disease

[0001] The present invention relates to an agent for preventing or treating polycystic kidney disease.

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease caused by mutations in the polycystin complex (polycystin 1 and polycystin 2 form a tetramer in a 3:1 ratio), a mechanosensor calcium ion channel localized in the primary cilia of renal epithelial cells. ADPKD is designated as an intractable disease (designated intractable disease 67), with an estimated 31,000 patients in Japan as of 1994. By the age of 70, half of patients develop end-stage renal failure, often accompanied by liver cysts and cerebral aneurysms. While no fundamental treatment for this disease has been established, the vasopressin V2 receptor antagonist tolvaptan is used as a treatment to slow progression and is covered by health insurance (see Patent Document 1). However, there are concerns that administration of this drug may cause dehydration and hypernatremia due to rapid excretion of fluids, etc. There are also concerns that its efficacy and safety have not been demonstrated, particularly in patients with poor renal function and in children (see Non-Patent Document 1).

[0003] Other therapeutic agents for autosomal dominant polycystic kidney disease that have been studied include octreotide (a somatostatin analog), mTOR inhibitors (mammalian target of rapamycin), and venglastat (a glucosylceramide synthase inhibitor) (see Non-Patent Document 2). However, like tolvaptan, these are also therapeutic agents that target signal transduction.

[0004] The present inventors have been conducting research into the promotion of cholesterol transport to the primary cilium and have demonstrated that oxysterol-binding protein (OSBP)-related proteins-3 (ORP3), which is localized in the primary ciliary pocket, is responsible for cholesterol transport from peroxisomes to the primary cilium (see Non-Patent Document 3). However, the relationship between cholesterol transport to the primary cilium and polycystic kidney disease has not been known until now.

[0005] U.S. Pat. No. 5,258,510

[0006] Kazuo Kambara, Evidence-based Guidelines for the Treatment of Polycystic Kidney Disease (PKD) 2017: Tokyo Medical Publishing Co., Ltd. Shigeo Horie, Polycystic Kidney Disease: New Developments in Treatment, Journal of the Japanese Society of Nephrology 2015; 57(1): 254-261 Miyamoto et al., The EMBO Journal (2020) 39: e103499

[0007] An object of the present invention is to provide a novel agent for preventing or treating polycystic kidney disease.

[0008] In normal renal tubule cells, renal cilia sense urine flow velocity, and the thickness (diameter) of the renal tubule is regulated by a complex of polycystin-1, which is involved in tubule cell proliferation, differentiation, and urine flow sensing, and polycystin-2, a calcium channel, on the renal cilia. It is believed that dysfunction of the polycystin complex in primary cilia due to mutation or loss of the polycystin complex leads to constitutive activation of the cAMP-PKA pathway within the cells, which enhances cell proliferation and secretion of chloride ions into the epithelial lumen, resulting in dilation of the renal tubule and collecting duct and the formation of cysts. The present inventors previously found that inducing organelle adhesion between primary cilia and peroxisomes using the FRB-FKBP system (rapamycin-dependent heteromolecular adhesion system) increases cholesterol in primary cilia in an ORP3 / vesicle-associated membrane protein-associated protein (VAP-A) system-dependent manner. Therefore, we hypothesized that in patients with polycystic kidney disease, in which the transport of polycystin complexes to the primary cilium is impaired, promoting cholesterol transport to the primary cilium by activating the ORP3 / VAP-A axis would improve the localization of polycystin complexes to the primary cilium, thereby restoring renal function and inhibiting the progression of the disease. We then searched for compounds that increase cholesterol transport to the primary cilium and contribute to the improvement of the pathology of polycystin kidney disease. As a result, we discovered compounds that promote the intermolecular interaction between ORP3 and VAP-A, which promote the transport of polycystin-1 and polycystin-2 to the primary cilium and are capable of improving the pathology of polycystin kidney disease, thereby completing the present invention.

[0009] That is, the present invention is as follows: [1] A preventive or therapeutic agent for polycystic kidney disease, comprising as an active ingredient a compound or a pharmaceutically acceptable salt thereof that promotes intermolecular interaction between oxysterol-binding protein-related protein-3 (ORP3) and endoplasmic reticulum protein-associated protein-A (VAP-A) in cells and increases cholesterol in the primary cilia of the cells. [2] The preventive or therapeutic agent for polycystic kidney disease according to [1] above, wherein the compound that promotes intermolecular interaction and increases cholesterol in the primary cilia of the cells is a compound comprising a 4-amino-1-phenylpyrazolopyrimidine skeleton, a 5-(sulfamoyl)benzamide skeleton, an N-thiazolylbenzamide skeleton, or an N-benzylsuccinimide skeleton. [3] The compound that promotes intermolecular interaction and increases cholesterol in the primary cilia of the cells is represented by general formula (I): (In the formula, R １Ａ and R ２Ａ are the same or different and represent a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, or an optionally substituted phenyl group. R ３Ａ and R ４Ａ are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ５Ａ , R ６Ａ , R ７Ａ , R ８Ａ , and R ９Ａand R are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, or an optionally substituted C1-6 alkoxy group. [4] The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is a compound represented by the following formula (I-1) (4-methoxypropylamino-1-(2,4-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), a compound represented by formula (I-2) (4-(4-fluorobenzylamino)-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), a compound represented by formula (I-3) (4-(3,5-dimethylphenyl)amino-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine), a compound represented by formula (I-4) (2-(4-chlorophenyl)ethylamino-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), The agent for preventing or treating polycystic kidney disease according to the above-mentioned [1], which is a compound represented by formula (I-5) (2-(1-cyclohexenyl)ethylamino-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), or a compound represented by formula (I-6) (4-hydroxypropylamino-1-(2,4-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine). [5] The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (II): (In the formula, R １Ｂ , R ２Ｂ , R ３Ｂ , R ４Ｂ , R ５Ｂ , R ７Ｂ , R ８Ｂ , R ９Ｂ , and R １０Ｂare the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ６Ｂ represents a hydrogen atom or an optionally substituted C1-6 alkyl group. １１Ｂ , and R １２Ｂ are the same or different and represent a hydrogen atom, a C1-6 alkyl group which may be substituted, a C1-6 alkoxy group which may be substituted, a C3-8 cycloalkyl group which may be substituted, a C3-6 cycloalkoxy group which may be substituted, or a phenyl group which may be substituted. [6] The preventive or therapeutic agent for polycystic kidney disease according to the above [1], wherein the compound having the effect of promoting intermolecular interaction and increasing cholesterol in the primary cilia of the cells is a compound represented by the following formula (II-1) or a compound represented by formula (II-2). [7] The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (III): (In the formula, R １Ｃ , R ２Ｃ , R ３Ｃ , R ４Ｃ , R ５Ｃ , R ８Ｃ , R ９Ｃ , R １０Ｃ , R １１Ｃ , and R １２Ｃ are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ６Ｃ represents a hydrogen atom or an optionally substituted C1-6 alkyl group. ７Ｃ[8] The agent for preventing or treating polycystic kidney disease according to the above [1], wherein the compound having the effect of promoting intermolecular interactions and increasing cholesterol in the primary cilia of the cells is a compound represented by the following formula (III-1): [9] The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (IV): (In the formula, R １Ｄ , R ２Ｄ , R ３Ｄ , R ４Ｄ , and R ５Ｄ are the same or different and each represents a hydrogen atom or an optionally substituted C1-6 alkyl group. X is an oxygen atom or CH ２

[10] The agent for preventing or treating polycystic kidney disease according to the above-mentioned [1], wherein the compound having the effect of promoting intermolecular interactions and increasing cholesterol in the primary cilia of the cells is a compound represented by the following formula (IV-1):

[11] The preventive or therapeutic agent for polycystic kidney disease according to [1] above, for administration to a patient with a gene mutation in PKD1 and / or PKD2.

[0010] According to the present invention, it is possible to provide a new agent for preventing or treating polycystic kidney disease, the mechanism of action of which is different from that of existing drugs.

[0011] FIG. 1 shows the results of measuring the luminescence of hit compounds obtained from a compound library using a screening platform constructed in Example 1 by introducing a split-luciferase system that targets the intermolecular interaction between ORP3 and VAPA. FIG. 2A shows the results of confocal laser microscope observation of primary cilium cholesterol treated with compound A in the primary cilium cholesterol supply activity test in Example 2. FIG. 2B shows the results of measurement of the fluorescence intensity of Filipin III in primary cilia treated with compound A in the primary cilium cholesterol supply activity test in Example 2. FIG. 3A shows the results of confocal laser microscope observation of primary cilium cholesterol treated with compounds A, C, and D in the primary cilium cholesterol supply activity test in Example 2. FIG. 3B shows the results of measurement of the fluorescence intensity of Filipin III in primary cilia treated with compounds A, C, and D in the primary cilium cholesterol supply activity test in Example 2. Figure 4A shows the results of confocal laser microscopy of primary cilium cholesterol in cells treated with Compounds A, AB, AF, AL, AP, R, and V in the primary cilium cholesterol supply activity in Example 2. Figure 4B shows the results of measuring the fluorescence intensity of Filipin III in primary cilia in cells treated with Compounds A, AB, AF, AL, AP, R, and V in the primary cilium cholesterol supply activity in Example 2. Figure 5A shows the results of confocal laser microscopy of primary cilium cholesterol in cells treated with Compounds A and I in the primary cilium cholesterol supply activity in Example 2. Figure 5B shows the results of measuring the fluorescence intensity of Filipin III in primary cilia in cells treated with Compounds A and I in the primary cilium cholesterol supply activity in Example 2. Figure 6A shows the results of confocal laser microscopy of primary cilium cholesterol in cells treated with Compound C in the restoration of primary cilium localization of polycystin-1 missense protein in Example 3. FIG. 6B shows the results of measuring the fluorescence intensity of polycystin-1 in primary cilia when treated with compound C in the restoration of primary cilium localization of polycystin-1 missense protein in Example 3.Figure 7A shows the results of confocal laser scanning microscopy of cysts formed in PC1 mutant mouse renal collecting duct-derived cells treated with Compounds A, C, and R to examine the inhibitory effect on ductal diameter increase in a cyst culture system in Example 4. The cysts were stained with Phalloidin (stains the F-actin skeleton) and DAPI (stains DNA) and observed using a confocal laser scanning microscope. Figure 7B shows the results of confocal laser scanning microscopy of cysts formed in PC2 mutant mouse renal collecting duct-derived cells treated with Compounds A, C, and R to examine the inhibitory effect on ductal diameter increase in a cyst culture system in Example 4. Figure 7C shows the results of determining the inner diameter / outer diameter ratio in merged images of cysts formed in the cysts treated with Compounds A, C, and R to examine the inhibitory effect on ductal diameter increase in a cyst culture system in Example 4. Figure 8A shows the results of observing cysts formed in PC2 mutant mouse renal collecting duct-derived cells stained with phalloidin and DAPI using a confocal laser microscope, which were observed using a confocal laser microscope, when treated with compounds A, G, and R and the existing drug tolvaptan, to investigate the inhibitory effect on ductal diameter increase in a cyst culture system in Example 5. Figure 8B shows the results of determining the inner diameter / outer diameter in merged images of cysts formed in PC2 mutant mouse renal collecting duct-derived cells treated with compounds A, G, and R and the existing drug tolvaptan, to investigate the inhibitory effect on ductal diameter increase in a cyst culture system in Example 5. Figure 9A shows the results of observing cysts formed in PC2 mutant mouse renal collecting duct-derived cells stained with phalloidin and DAPI using a confocal laser microscope, which were observed using a confocal laser microscope, when treated with compounds A, G, and compound G-OH, a derivative of compound G, to investigate the inhibitory effect on ductal diameter increase in a cyst culture system in Example 5. Figure 9B shows the results of determining the inner diameter / outer diameter in merged images of cysts formed by cells derived from the renal collecting duct of PC2 mutant mice treated with compounds A, G, and compound G-OH, a derivative of compound G, in the study of the inhibitory effect on ductal diameter increase in a cyst culture system in Example 5. Figure 10A shows the results of observing, using a confocal laser microscope, cysts formed by cells derived from the renal collecting duct of PC2 mutant mice treated with compounds A, G, B, Q, K, and J, in the study of the inhibitory effect on ductal diameter increase in a cyst culture system in Example 6, stained with phalloidin and DAPI.Figure 10B shows the results of determining the inner diameter / outer diameter in merge images of cysts formed from PC2 mutant mouse renal collecting duct-derived cells treated with compounds A, G, B, Q, K, and J, in the study of the inhibitory effect on ductal diameter increase in a cyst culture system in Example 6. Figure 11A shows the results of observing, using a confocal laser microscope, cysts formed from PC2 mutant mouse renal collecting duct-derived cells treated with compounds A and I, stained with phalloidin and DAPI. Figure 11B shows the results of determining the inner diameter / outer diameter in merge images of cysts formed from PC2 mutant mouse renal collecting duct-derived cells treated with compounds A and I, in the study of the inhibitory effect on ductal diameter increase in a cyst culture system in Example 6. Figure 12A shows the results of confocal laser microscopy of polycystin 2 protein in the primary cilia of human induced pluripotent stem cells derived from a patient with polycystin-2 kidney disease treated with Compound G-OH and the existing drug tolvaptan in Example 7, which shows the recovery of polycystin 2 protein localization in primary cilia. Figure B shows the results of measuring the amount of polycystin 2 protein in the primary cilia of human induced pluripotent stem cells derived from a patient with polycystin-2 kidney disease treated with Compound G-OH and the existing drug tolvaptan in Example 7, which shows the recovery of polycystin 2 protein (PC2) localization in primary cilia. Figure 13A shows the results of confocal laser microscopy of PC1 in the primary cilia of a feline kidney-derived epithelial cell line (CRFK cells) treated with Compound G in Example 8, which shows the increased activity of polycystin 1 protein (PC1) in primary cilia. FIG. 13B shows the results of measuring the amount of polycystin-1 protein (PC1) in the primary cilia of a feline kidney-derived epithelial cell line (CRFK cells) treated with compound G, in relation to the activity of increasing polycystin-1 protein (PC1) in primary cilia in Example 8.

[0012] The contents of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety.

[0013] The agent for preventing or treating polycystic kidney disease of the present invention is not particularly limited as long as it is an agent for preventing or treating polycystic kidney disease, and contains as an active ingredient a compound that promotes the intermolecular interaction between oxysterol-binding protein-related protein-3 (ORP3) and endoplasmic reticulum protein-associated protein-A (VAP-A) in cells and that has the effect of increasing cholesterol in the primary cilia of cells, or a pharmaceutically acceptable salt thereof, and hereinafter this agent will also be referred to as "the agent for preventing or treating polycystic kidney disease of this invention."

[0014] The polycystic kidney disease referred to herein is not particularly limited, and examples thereof include autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD), with autosomal dominant polycystic kidney disease (ADPKD) being preferred. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the polycystic kidney and hepatic disease (PKD)1 gene on chromosome 16 and / or the PKD2 gene on chromosome 4, which encode polycystin 1 protein (PC1) and polycystin 2 protein (PC2), respectively. Furthermore, autosomal recessive polycystic kidney disease (ARPKD) is caused by a mutation in the PKHD1 gene on chromosome 6.

[0015] The promotion of intracellular intermolecular interaction between oxysterol-binding protein-related protein-3 (ORP3) and endoplasmic reticulum protein-associated protein-A (VAP-A) herein can be investigated by known protein-protein interaction analysis methods, such as the split protein system and the GST-pull down method (see Marion Weber-Boyvat et al., Experimental Cell Research, Volume 331, Issue 2, February 15, 2015, Pages 278-291 (particularly Figure 1); Wan Ting Saw et al., Methods, Volume 90, November 2015, Pages 68-75; Stephanie Cabantous et al., Scientific Reports, Volume 3, Article number: 2854 (2013); JP 2003-503067 A).

[0016] In a specific example using a split protein system, a fluorescently labeled protein is split so that it does not emit light by itself, and one of the fragments is fused to ORP3 and the other to VAP-A. Next, a compound to be evaluated is added to cells expressing ORP3 and VAP-A fused with the fluorescently labeled protein fragments, and the cells are cultured. The fluorescence intensity is then detected, and evaluation can be performed by examining whether the fluorescence level increases when treated with the compound to be evaluated compared to the fluorescence level when untreated with the compound to be evaluated, or when a comparison compound or positive control compound is added. Cells expressing ORP3 and VAP-A fused with fluorescently labeled protein fragments can be prepared by introducing into cells, using known methods, an expression vector incorporating a polynucleotide encoding one of the fluorescently labeled protein fragments and a nucleotide encoding ORP3, and an expression vector incorporating a polynucleotide encoding the other of the fluorescently labeled protein fragments and a nucleotide encoding VAP-A, and expressing them. Examples of fluorescent labels include green fluorescent protein (GFP).

[0017] Alternatively, an enzyme may be used instead of the fluorescent label, and a luminescent substrate may be added as needed to culture the cells. The enzyme activity may then be detected to determine whether the enzyme activity increases when the cells are treated with the compound to be evaluated, compared with the enzyme activity when the compound to be evaluated is not added or when a comparative compound is added. Examples of the enzyme include luciferases. Alternatively, the concentration of the compound to be evaluated may be gradually changed, and the fluorescence intensity or enzyme activity may be detected in a concentration-dependent manner for evaluation.

[0018] When using a split protein system, a compound to be evaluated is added, and fluorescence intensity or enzyme activity is detected. If the fluorescence level or enzyme activity in the absence of the compound to be evaluated is set to 1, and the fluorescence level or enzyme activity reaches or exceeds a predetermined value, the compound to be evaluated can be evaluated as having the ability to promote the intermolecular interaction between oxysterol-binding protein-related protein-3 (ORP3) and endoplasmic reticulum protein-associated protein-A (VAP-A) in cells. For example, when treated with phorbol-12-myristate-13-acetate (PMA) as a positive control, the fluorescence level or enzyme activity of the treated cells is approximately 5, so if the relative level is 2.5 times or more, more preferably 2.8 times or more, the compound to be evaluated can be evaluated as having the ability to promote the intermolecular interaction between oxysterol-binding protein-related protein-3 (ORP3) and endoplasmic reticulum protein-associated protein-A (VAP-A) in cells.

[0019] As used herein, known methods can be used to measure the effect of increasing cholesterol in the primary cilium of cells within cells. For example, cells such as retinal pigment epithelial cells are treated with a compound to be evaluated for 4 hours at 37°C, and then stained with a cholesterol indicator such as Filipin III that emits fluorescence upon binding to cholesterol, or a reagent such as AcGFP1-D4 (a cholesterol-binding protein fused with a fluorescent protein: see Miyamoto et al., Methods Mol Biol, 2022:doi: 10.1007 / 978-1-0716-1701-4_5.), and a primary cilium marker, followed by observation under a microscope to observe or detect cholesterol localized in the primary cilium. The compound to be evaluated is added to detect cholesterol localized in the primary cilium. If the fluorescence level is 1.2 times or more, preferably 1.3 times or more, and more preferably 1.4 times or more relative to the level observed when the compound to be evaluated is not treated, the compound to be evaluated can be evaluated as having an effect of increasing cholesterol in the primary cilium of cells.

[0020] It is also preferable to confirm that the cholesterol-increasing effect in the primary cilium of the cells is ORP3 / VAPA dependent. Whether or not the cholesterol-increasing effect in the primary cilium of the cells is ORP3 / VAPA dependent can be evaluated by using wild-type retinal pigment epithelial cells and mutant retinal pigment epithelial cells in which VAP-A or ORP3 is deleted or functionally mutated, and observing or detecting the activity of the compound to be evaluated to increase cholesterol in the primary cilium of the cells in the wild-type retinal pigment epithelial cells but not in the mutant retinal pigment epithelial cells.

[0021] Furthermore, the compound of the present invention that promotes the intermolecular interaction between ORP3 and VAP-A in cells and increases cholesterol in the primary cilia of cells preferably has the effect of restoring polycystin-1 localization to the primary cilia when mouse kidney collecting duct-derived cells (mIMCD3 cells: ATCC cat. no. CRL-2123) expressing a polycystin-1 missense protein (a mutation in which cysteine ​​at position 210 in the amino acid sequence of SEQ ID NO: 1 is replaced with glycine) are used. The above-mentioned effect can be confirmed by treating the mutant mIMCD3 cells, and if necessary, wild-type mIMCD3 cells, with the compound to be evaluated for 4 hours at 37°C, followed by staining with a polycystin-1 antibody and a primary cilium marker and observing under a microscope to observe or detect the polycystin-1 protein localized to the primary cilium.

[0022] In addition, compounds of the present invention that promote the intermolecular interaction between ORP3 and VAP-A in cells and increase cholesterol in the primary cilia of cells preferably also have an inhibitory effect on the increase in duct diameter in a cyst culture system in which cyst formation by three-dimensional culture of renal tubular cells is reconstituted. Whether or not a compound has an inhibitory effect on the increase in duct diameter in a cyst culture system in which cyst formation by three-dimensional culture of renal tubular cells is reconstituted can be determined, for example, by three-dimensionally culturing mutant mIMCDC3 cells expressing polycystin-1 missense protein or mutant mIMCDC3 cells expressing polycystin-2 missense protein, mixing the compound to be evaluated and Matrigel, transferring the mixture to a chamber slide plate, and incubating at 37°C and 5% CO ２ The mixture is then incubated under conditions of 37°C and 5% CO until solidification. The pre-warmed medium is then added to the solidified cell-Matrigel layer, and the medium is exchanged until cyst formation. ２The mixture is incubated under these conditions. Next, the formed cysts are fixed, sectioned, and stained with phalloidin and DAPI, and then observed using a confocal laser microscope. Furthermore, the size of the epithelial lumen formed within the cyst in the merged image of the formed cyst is measured as the inner diameter, and the size of the entire cyst is measured as the outer diameter, and the inner diameter / outer diameter is calculated to evaluate the inhibitory effect on the increase in duct diameter. Note that mutations in polycystin 1 protein or polycystin 2 protein reduce primary cilia activity, resulting in cyst expansion.

[0023] The compounds of the present invention that promote the intermolecular interaction between ORP3 and VAP-A in cells and increase cholesterol in the primary cilia of cells may increase cholesterol by directly acting on ORP3 or VAP-A, or may increase cholesterol by indirectly acting on ORP3 or VAP-A via a factor that can act on ORP3 or VAP-A. Examples of such factors include proteins, nucleic acids, and lipids.

[0024] The compound used as an active ingredient in the present agent for preventing or treating polycystic kidney disease is not particularly limited, and examples thereof include compounds containing a 4-amino-1-phenylpyrazolopyrimidine skeleton (hereinafter, sometimes referred to as a "4-amino-1-phenylpyrazolopyrimidine compound" or "compound (I)"), compounds containing a 5-(sulfamoyl)benzamide skeleton (hereinafter, sometimes referred to as a "5-(sulfamoyl)benzamide compound" or "compound (II)"), compounds containing an N-thiazolylbenzamide skeleton (hereinafter, sometimes referred to as an "N-thiazolylbenzamide compound" or "compound (III)"), compounds containing an N-benzylsuccinimide skeleton (hereinafter, sometimes referred to as an "N-benzylsuccinimide compound" or "compound (IV)"), and the like.

[0025] 4-amino-1-phenylpyrazolopyrimidine compound represented by general formula (I) The compound having the effect of promoting intermolecular interactions and increasing cholesterol in the primary cilia of cells in the present preventive or therapeutic agent for polycystic kidney disease is a 4-amino-1-phenylpyrazolopyrimidine compound represented by the following general formula (I): In the formula, R １Ａ and R ２Ａ are the same or different and represent a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, an optionally substituted phenyl group, an optionally substituted aliphatic ring, or an optionally substituted heterocycle. ３Ａ and R ４Ａ are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ５Ａ , R ６Ａ , R ７Ａ , R ８Ａ , and R ９Ａ are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, or an optionally substituted C1-6 alkoxy group.

[0026] Each group of the 4-amino-1-phenylpyrazolopyrimidine compound represented by general formula (I) in this specification will be explained below.

[0027] The C1-6 alkyl group is not particularly limited as long as it is a linear or branched C1-6 alkyl group, and examples thereof include linear or branched alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl. In this specification, "n-" means normal, "s-" means secondary, and "t-" means tertiary.

[0028] The C1-6 alkoxy group is a group represented by RO- (wherein R is an alkyl group), and this "alkoxy group" is not particularly limited as long as it is a linear or branched C1-6 alkoxy group, and examples include linear or branched alkoxy groups having 1 to 6 carbon atoms such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, n-pentyloxy, isopentyloxy, neopentyloxy, n-hexyloxy, and isohexyloxy. As the C1-6 alkoxy group, a C1-4 alkoxy group is preferred, and a methoxy group or a t-butoxy group is more preferred.

[0029] The C2-6 alkenyl group is not particularly limited, and examples thereof include linear or branched alkenyl groups having 2 to 6 carbon atoms, such as vinyl, 1-propenyl, allyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, and 1,3-butadienyl.

[0030] Examples of C2-6 alkynyl groups include linear or branched alkynyl groups having 2 to 6 carbon atoms, such as ethynyl, 1-propynyl, 2-propynyl, 1-methyl-2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.

[0031]

[0023] In this specification, the "substituents" in the optionally substituted C1-6 alkyl group, optionally substituted C1-6 alkoxy group, optionally substituted C2-6 alkynyl group, optionally substituted C2-6 alkynyloxy group, optionally substituted phenyl group, optionally substituted aliphatic ring, optionally substituted heterocycle, optionally substituted C3-8 cycloalkyl group, and optionally substituted C3-8 cycloalkoxy group are not particularly limited, and examples include a halogen atom, a C1-6 alkyl group, a C3-8 cycloalkyl group, a C3-8 cycloalkenyl group, a haloC1-6 alkyl group, a C1-6 alkoxy group, an optionally substituted phenyl group, a nitro group, a 1-acetylazetidin-3-ylmethyl group, a C3-8 cycloalkylcarbonylamino group, a C1-6 alkoxycarbonylamino group, etc. These groups may further have a substituent at any substitutable position, and examples of the substituent include the same as those described above for the substituents. For example, the optionally substituted C1-6 alkyl group includes a halo C1-6 alkyl group, and examples of the halo C1-6 alkyl group include a chloromethyl group, a chloroethyl group, a trifluoromethyl group, a 1,2-dichloro-n-propyl group, a 1-fluoro-n-butyl group, a perfluoro-n-pentyl group, etc. However, the optionally substituted C1-6 alkyl group referred to here is not limited to these.

[0032] The C3-8 cycloalkyl group is not particularly limited, and examples thereof include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.

[0033] Examples of the C3-8 cycloalkenyl group include a cyclopropenyl group, a cyclopentenyl group, a cyclohexenyl group, and a cyclooctenyl group. The double bond can be present in any position.

[0034] The halogen atom is not particularly limited, and examples thereof include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0035] As the 4-amino-1-phenylpyrazolopyrimidine compound represented by the general formula (I), １Ａ is preferably a C1-6 alkyl group which may have a substituent, and an optionally substituted phenyl group, more preferably a C1-6 alkyl group having a C1-6 alkoxy group, a C1-6 alkyl group having a phenyl group which may have a substituent, a C1-6 alkyl group having a C3-6 cycloalkenyl group, and an optionally substituted phenyl group, still more preferably a C1-6 alkyl group having a methoxy group or a hydroxy group, a C1-6 alkyl group having a phenyl group which has a halogen atom at position 4, a C1-6 alkyl group having a cyclohexenyl group, and a phenyl group having 1 to 3 C1-6 alkyl groups, and particularly preferably a 4-methoxypropyl group, a 4-fluorobenzyl group, a 3,5-dimethylphenyl group, a 4-chlorophenylethyl group, a (1-cyclohexenyl)ethyl group, or a 4-hydroxy group.

[0036] As the 4-amino-1-phenylpyrazolopyrimidine compound represented by the general formula (I), ２Ａ is preferably a hydrogen atom.

[0037] As the 4-amino-1-phenylpyrazolopyrimidine compound represented by the general formula (I), ３Ａ is preferably a hydrogen atom.

[0038] As the 4-amino-1-phenylpyrazolopyrimidine compound represented by the general formula (I), ４Ａ is preferably a hydrogen atom.

[0039] As the 4-amino-1-phenylpyrazolopyrimidine compound represented by the general formula (I), ５Ａ , R ６Ａ , R ７Ａ , R ８Ａ , and R ９Ａ are each preferably a hydrogen atom or a C1-6 alkyl group, more preferably a hydrogen atom or a methyl group. ５Ａ is a hydrogen atom, R ６Ａ is a C1-6 alkyl group, R７Ａ is a hydrogen atom, R ８Ａ is a hydrogen atom, and R ９Ａ is a hydrogen atom; R ５Ａ is a C1-6 alkyl group, R ６Ａ is a hydrogen atom, R ７Ａ is a C1-6 alkyl group, R ８Ａ is a hydrogen atom, and R ９Ａ is a hydrogen atom; R ５Ａ is a hydrogen atom, R ６Ａ is a hydrogen atom, R ７Ａ is a C1-6 alkoxy group, R ８Ａ is a hydrogen atom, and R ９Ａ is preferably a hydrogen atom.

[0040] Specifically, preferred compounds as the 4-amino-1-phenylpyrazolopyrimidine compound represented by the general formula (I) include the compound represented by the following formula (I-1) (4-methoxypropylamino-1-(2,4-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), the compound represented by formula (I-2) (4-(4-fluorobenzylamino)-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), the compound represented by formula (I-3) (4-(3,5-dimethylphenyl)amino-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine), the compound represented by formula (I-4) (2-(4-chlorophenyl)ethylamino-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), Examples include a compound represented by formula (I-5) (2-(1-cyclohexenyl)ethylamino-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine) and a compound represented by formula (I-6) (4-hydroxypropylamino-1-(2,4-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine).

[0041] These one or more 4-amino-1-phenylpyrazolopyrimidine compounds or salts thereof can be produced by known production methods. The 4-amino-1-phenylpyrazolopyrimidine compounds can be produced, for example, using known compounds or commercially available products as raw materials. A specific production method is shown in Scheme 1 below.

[0042] 5-(Sulfamoyl)benzamide compound represented by general formula (II) The compound having the effect of promoting intermolecular interactions and increasing cholesterol in the primary cilia of cells in the present preventive or therapeutic agent for polycystic kidney disease is a compound represented by the following general formula (II): In the formula, R １Ｂ , R ２Ｂ , R ３Ｂ , R ４Ｂ , R ５Ｂ , R ７Ｂ , R ８Ｂ , R ９Ｂ , and R １０Ｂ are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ６Ｂ represents a hydrogen atom or an optionally substituted C1-6 alkyl group. １１Ｂ , and R １２Ｂ are the same or different and represent a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, an optionally substituted C3-8 cycloalkyl group, an optionally substituted C3-6 cycloalkoxy group, or an optionally substituted phenyl group.

[0043] In this specification, the C1-6 alkyl group, C1-6 alkoxy group, C3-8 cycloalkyl group, halogen atom and substituent in general formula (II) are the same as the C1-6 alkyl group, C1-6 alkoxy group, halogen atom and substituent in general formula (I) above.

[0044] The C3-8 cycloalkoxy group is not particularly limited, and examples thereof include a cyclopropyloxy group, a cyclobutyloxy group, a cyclopentyloxy group, a cyclohexyloxy group, a cycloheptyloxy group, and a cyclooctyloxy group.

[0045] As the 5-(sulfamoyl)benzamide compound represented by the general formula (II), １B , R ２B , R ３B , R ４B , R ５B At least one of R is a halogen atom, preferably a bromine atom; １B , R ２B , R ３B , R ４B , R ５B Among these, one to three, preferably two, may be methyl groups; ２B , R ４B In addition, at least one of, preferably both of, R 11B is preferably a cyclohexyl group or a methoxyphenylmethyl group.

[0046] As the 5-(sulfamoyl)benzamide compound represented by the general formula (II), １Ｂ , R ２Ｂ , R ３Ｂ , R ４Ｂ , and R ５Ｂ are each preferably a hydrogen atom, a halogen atom, or a C1-6 alkyl group, more preferably a hydrogen atom, a bromine atom, or a methyl group.

[0047] Among them, R １Ｂ is a hydrogen atom, R ２Ｂ is a hydrogen atom, R ３Ｂ is a halogen atom or a C1-6 alkyl group, R ４Ｂ is a hydrogen atom, and R ５Ｂ is a hydrogen atom; R １Ｂ is a hydrogen atom, R ２Ｂ is a C1-6 alkyl group, R ３Ｂ is a hydrogen atom, R ４Ｂ is a C1-6 alkyl group, and R ５Ｂ is particularly preferably a hydrogen atom.

[0048] R ７Ｂ , R ８Ｂ , R ９Ｂ , and R １０Ｂ are each preferably a hydrogen atom, a halogen atom, or a C1-6 alkyl group, more preferably a hydrogen atom, a chlorine atom, or a fluorine atom.

[0049] Among them, R ７Ｂ is a halogen atom, R ８Ｂ is a hydrogen atom, R ９Ｂ is a halogen atom or a C1-6 alkyl group, and R １０Ｂ is a hydrogen atom; R ７Ｂ is a chlorine atom or a fluorine atom, R ８Ｂ is a hydrogen atom, R ９Ｂ is a hydrogen atom, and R １０Ｂ is particularly preferably a hydrogen atom.

[0050] As the 5-(sulfamoyl)benzamide compound represented by the general formula (II), ６Ｂ is preferably a hydrogen atom.

[0051] As the 5-(sulfamoyl)benzamide compound represented by the general formula (II), １１Ｂ is preferably a hydrogen atom.

[0052] As the 5-(sulfamoyl)benzamide compound represented by the general formula (II), １２Ｂ is preferably a C1-6 alkyl group which may have a substituent and a C1-6 cycloalkyl group which may have a substituent, and more preferably a C1-6 alkyl group having a phenyl group which has a C1-6 alkoxy group, and an unsubstituted C1-6 cycloalkyl group.

[0053] Preferable examples of the 5-(sulfamoyl)benzamide compound represented by general formula (II) include the following: Compound (II-1): 2-chloro-N-(4-bromophenyl)-5-(cyclohexylaminosulfamoyl)benzamide; and Compound (II-2): 2-fluoro-N-(3,5-dimethylphenyl)-5-[(1-methoxyphenylmethylamino)sulfamoyl]benzamide.

[0054] These one or more 5-(sulfamoyl)benzamide compounds or salts thereof can be produced by known production methods. The 5-(sulfamoyl)benzamide compounds can be produced, for example, using known compounds or commercially available products as raw materials. Specific production methods are shown in Scheme 2 or Scheme 3 below.

[0055] N-thiazolylbenzamide compound represented by general formula (III) The compound having the effect of promoting intermolecular interactions and increasing cholesterol in the primary cilia of cells in the present preventive or therapeutic agent for polycystic kidney disease is an N-thiazolylbenzamide compound represented by the following general formula (III): In the formula, R １Ｃ , R ２Ｃ , R ３Ｃ , R ４Ｃ , R ５Ｃ , R ８Ｃ , R ９Ｃ , R １０Ｃ , R １１Ｃ , and R １２Ｃ are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ６Ｃ represents a hydrogen atom or an optionally substituted C1-6 alkyl group. ７Ｃ represents a hydrogen atom, a halogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group.

[0056] In this specification, the C1-6 alkyl group, C1-6 alkoxy group, halogen atom and substituent in general formula (III) are the same as the C1-6 alkyl group, C1-6 alkoxy group, halogen atom and substituent in general formula (I).

[0057] As the N-thiazolylbenzamide compound represented by the general formula (III), １C , R ２C , R ３C , R ４C, R ５C At least one of R is preferably a nitro group. ３C is more preferably a nitro group. ８C , R ９C , R １０C , R 11C , R １２C At least one of R is preferably a methyl group. １０C and / or R 11C is more preferably a methyl group. １C , R ２C , R ４C , R ５C , R ７C , R ８C , R ９C , R １２C is preferably a hydrogen atom.

[0058] As the N-thiazolylbenzamide compound represented by the general formula (III), １Ｃ , R ２Ｃ , R ３Ｃ , R ４Ｃ , and R ５Ｃ are each preferably a hydrogen atom, a halogen atom, a nitro group, or a C1-6 alkyl group, more preferably a hydrogen atom or a nitro group.

[0059] Among them, R １Ｃ is a hydrogen atom, R ２Ｃ is a hydrogen atom, R ３Ｃ is a hydrogen atom, a halogen atom, a nitro group, or a C1-6 alkyl group, R ４Ｃ is a hydrogen atom, and R ５Ｃ is a hydrogen atom; R １Ｃ is a hydrogen atom, R ２Ｃ is a C1-6 alkyl group, R ３Ｃ is a hydrogen atom, R ４Ｃ is a C1-6 alkyl group, and R ５Ｃ is particularly preferably a hydrogen atom.

[0060] As the N-thiazolylbenzamide compound represented by the general formula (III), ６Ｃ is preferably a hydrogen atom or a C1-6 alkyl group, more preferably a hydrogen atom.

[0061] As the N-thiazolylbenzamide compound represented by the general formula (III), ７Ｃ is preferably a hydrogen atom.

[0062] As the N-thiazolylbenzamide compound represented by the general formula (III), ８Ｃ , R ９Ｃ , R １０Ｃ , R １１Ｃ and R １２Ｃ are each preferably a hydrogen atom or a C1-6 alkyl group, more preferably a hydrogen atom or a methyl group.

[0063] Among them, R ８Ｃ is a hydrogen atom, R ９Ｃ is a hydrogen atom, R １０Ｃ is a hydrogen atom or a C1-6 alkyl group, R １１Ｃ is a hydrogen atom or a C1-6 alkyl group, and R １２Ｃ is more preferably a hydrogen atom; R ８Ｃ is a hydrogen atom, R ９Ｃ is a hydrogen atom, R １０Ｃ is a C1-6 alkyl group, R １１Ｃ is a C1-6 alkyl group, and R １２Ｃ is particularly preferably a hydrogen atom.

[0064] A preferred example of the N-thiazolylbenzamide compound represented by general formula (III) is the following compound (III-1): 4-nitro-N-[4-(2,3-dimethylphenyl)-(2-thiazolyl)benzamide].

[0065] These one or more N-thiazolylbenzamide compounds or salts thereof can be produced by known production methods. 5-(sulfamoyl)benzamide compounds can be produced, for example, using known compounds or commercially available products as raw materials. A specific production method is shown in Scheme 4 below.

[0066] N-benzyl succinimide compound represented by general formula (IV) The compound having the action of promoting synthesis and increasing cholesterol in the primary cilia of cells in the present preventive or therapeutic agent for polycystic kidney disease is an N-benzyl succinimide compound represented by general formula (IV), In the formula, R １Ｄ , R ２Ｄ , R ３Ｄ , R ４Ｄ , and R ５Ｄ are the same or different and each represents a hydrogen atom or an optionally substituted C1-6 alkyl group. X is an oxygen atom or CH ２ Shows.

[0067] In this specification, the C1-6 alkyl group, C1-6 alkoxy group, halogen atom and substituent in general formula (IV) are the same as the C1-6 alkyl group, C1-6 alkoxy group, halogen atom and substituent in general formula (I).

[0068] As the N-benzylsuccinimide compound represented by general formula (IV), １Ｄ , R ２Ｄ , R ３Ｄ , R ４Ｄ , and R ５Ｄ are each preferably a hydrogen atom, a halogen atom, a nitro group, or a C1-6 alkyl group, more preferably a hydrogen atom.

[0069] Among them, R １Ｄ is a hydrogen atom, R ２Ｄ is a hydrogen atom, R ３Ｄ is a hydrogen atom, a halogen atom, a nitro group, or a C1-6 alkyl group, and R ４Ｄ is a hydrogen atom; R １Ｄ is a hydrogen atom, R ２Ｃ is a hydrogen atom, R ３Ｃ is a hydrogen atom, R ４Ｃ is a hydrogen atom, and R ５Ｃ is particularly preferably a hydrogen atom.

[0070] The N-benzylsuccinimide compound represented by the general formula (IV) is ２ is preferred, ２ is more preferred.

[0071] As the N-benzylsuccinimide compound represented by the general formula (IV), the following compound (IV-1) can be given as a preferred compound.

[0072]

[0073] These one or more N-benzylsuccinzamide compounds or salts thereof can be produced by known production methods. The N-benzylsuccinzamide compounds can be produced, for example, using known compounds or commercially available products as raw materials. A specific production method is shown in Scheme 5 below.

[0074] The salts used herein are not particularly limited as long as they are pharmaceutically acceptable salts, and examples thereof include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), and salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, N-methylpyrrolidine, N-methylpiperidine, N-methylmorpholine, etc. When the compound is treated as a basic compound, examples thereof include acid addition salts of mineral acids such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, and phosphate; and acid addition salts of organic acids such as benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, maleate, fumarate, tartrate, citrate, and acetate.

[0075] The compounds of the present invention and salts thereof may have one or more asymmetric centers in their structural formulas, and may exist as two or more optical isomers and diastereomers, and the present invention encompasses all of the optical isomers and mixtures containing them in any ratio. Furthermore, the compounds of the present invention and salts thereof may have two geometric isomers derived from a carbon-carbon double bond in their structural formulas, and the present invention encompasses all of the geometric isomers and mixtures containing them in any ratio.

[0076] Furthermore, the compounds of the present invention and salts thereof may exist in multiple tautomers, and the present invention encompasses all of the tautomers and mixtures thereof in any ratio.

[0077] The following will illustrate examples of methods for producing the compounds represented by general formula (I) to (IV).

[0078] (iii) Method for producing 4-amino-1-phenylpyrazolopyrimidine compounds represented by general formula (I) (Production method (A)) The compounds represented by general formula (I) of the present invention can be produced, for example, by the production method represented by the following scheme 1, but the present invention is not limited thereto.

[0079] <Scheme 1> (In the formula, R １Ａ , R ２Ａ , R ３Ａ , R ４Ａ , R ５Ａ , R ６Ａ , R ７Ａ , R ８Ａ , and R ９Ａ has the same meaning as above. １０Ａ represents a halogen atom.)

[0080] The production method (A-1) is a method for synthesizing a compound represented by general formula (I), and is a production method comprising a step of reacting a compound represented by general formula (1A) with a compound represented by general formula (2A) in a solvent in the presence of a base.

[0081] The amount of the compound represented by general formula (1A) is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (2A) and the target reaction proceeds, but is usually 1 equivalent or more and 200 equivalents or less.

[0082] Examples of the base used in this reaction include metal hydrides such as sodium hydride; organolithium compounds such as methyllithium, butyllithium, sec-butyllithium, t-butyllithium, and hexyllithium; and metal amides such as lithium diisopropylamide, lithium hexamethyldisilazane, sodium hexamethyldisilazane, and potassium hexamethyldisilazane.

[0083] The amount of the base used in this reaction is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (1A) and the amount is preferably 1 equivalent or more and 10 equivalents or less, as long as the target reaction proceeds.

[0084] The solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, and examples thereof include benzene-based solvents such as benzene, toluene, xylene, mesitylene, chlorobenzene, and dichlorobenzene; halogen-based solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; etc. These solvents can be used alone or in combination of two or more in any desired ratio.

[0085] The amount of solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, but is usually 3 to 200 times by weight relative to the compound represented by formula (1A).

[0086] The temperature at which this reaction is carried out is not particularly limited as long as the desired reaction proceeds, but is usually from 0° C. to 150° C. or below the boiling point of the solvent.

[0087] The time for carrying out this reaction is not particularly limited as long as the desired reaction proceeds, but is usually from 10 minutes to 24 hours.

[0088] As a post-treatment for the reaction, undissolved metals can be removed by filtration. Furthermore, a separation operation can be performed by adding water or an appropriate aqueous solution to the reaction mixture. When using an aqueous solution, any of the following can be used: an acidic aqueous solution containing hydrochloric acid, sulfuric acid, ammonium chloride, etc.; an alkaline aqueous solution containing potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.; or saline solution. During the separation operation, if necessary, a water-immiscible solvent can be added, such as a benzene-based solvent such as toluene, xylene, benzene, chlorobenzene, or dichlorobenzene; an ester-based solvent such as ethyl acetate, isopropyl acetate, or butyl acetate; an ether-based solvent such as diethyl ether, diisopropyl ether, or methyl-t-butyl ether; a halogen-based solvent such as dichloromethane, dichloroethane, chloroform, or carbon tetrachloride; or a hydrocarbon-based solvent such as hexane, heptane, cyclohexane, or methylcyclohexane. These solvents can be used alone or in combination of two or more in any ratio. The number of separations is not particularly limited and can be performed depending on the desired purity or yield. In addition, in this reaction, a separation operation is not essential.

[0089] The reaction mixture containing the compound represented by general formula (I) obtained above can be dehydrated with a drying agent such as sodium sulfate or magnesium sulfate, but this is not essential.

[0090] The reaction mixture containing the compound represented by general formula (I) obtained above can be subjected to solvent distillation under reduced pressure as long as the compound is not decomposed.

[0091] The reaction mixture containing the compound represented by general formula (I) obtained after distilling off the solvent can be purified by washing with an appropriate solvent, reprecipitation, recrystallization, column chromatography, or the like.

[0092] (ii) Method for producing 5-(sulfamoyl)benzamide compounds represented by formula (II) (production method (B)) Examples of methods for producing compounds represented by formula (II) are given below.

[0093] The compound of the present invention represented by general formula (II) can be produced, for example, by a production method represented by the following scheme 2 (production method (B-1)) or a production method represented by scheme 3 (production method (B-2)), but the present invention is not limited thereto.

[0094] ■ Production method (B-1) <Scheme 2> (In the formula, R １Ｂ , R ２Ｂ , R ３Ｂ , R ４Ｂ , R ５Ｂ , R ６Ｂ , R ７Ｂ , R ８Ｂ , R ９Ｂ , R １０Ｂ , R １１Ｂ and R １２Ｂ has the same meaning as above. １３Ｂ represents a halogen atom.)

[0095] The production method (B-1) is a method for synthesizing a compound represented by general formula (II), and includes a step of reacting an amine compound represented by general formula (1B) with a compound represented by general formula (2B) in a solvent in the presence of a base.

[0096] The amount of the compound represented by general formula (1B) is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (2B) and the target reaction proceeds, but is usually 1 equivalent or more and 200 equivalents or less.

[0097] Examples of the base used in this reaction include metal hydrides such as sodium hydride; organolithium compounds such as methyllithium, butyllithium, sec-butyllithium, t-butyllithium, and hexyllithium; and metal amides such as lithium diisopropylamide, lithium hexamethyldisilazane, sodium hexamethyldisilazane, and potassium hexamethyldisilazane.

[0098] The amount of the base used in this reaction is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (1B) and the desired reaction proceeds, but is preferably 1 equivalent or more and 10 equivalents or less.

[0099] The solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, and examples thereof include benzene-based solvents such as benzene, toluene, xylene, mesitylene, chlorobenzene, and dichlorobenzene; halogen-based solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; etc. These solvents can be used alone or in combination of two or more in any desired ratio.

[0100] The amount of solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, but is usually 3 to 200 times by weight relative to the compound represented by general formula (1B).

[0101] The temperature at which this reaction is carried out is not particularly limited as long as the desired reaction proceeds, but is usually from 0° C. to 150° C. or below the boiling point of the solvent.

[0102] The time for carrying out this reaction is not particularly limited as long as the desired reaction proceeds, but is usually from 10 minutes to 24 hours.

[0103] As a post-treatment for the reaction, undissolved metals can be removed by filtration. Furthermore, a separation operation can be performed by adding water or an appropriate aqueous solution to the reaction mixture. When using an aqueous solution, any of the following can be used: an acidic aqueous solution containing hydrochloric acid, sulfuric acid, ammonium chloride, etc.; an alkaline aqueous solution containing potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.; or saline solution. During the separation operation, if necessary, a water-immiscible solvent can be added, such as a benzene-based solvent such as toluene, xylene, benzene, chlorobenzene, or dichlorobenzene; an ester-based solvent such as ethyl acetate, isopropyl acetate, or butyl acetate; an ether-based solvent such as diethyl ether, diisopropyl ether, or methyl-t-butyl ether; a halogen-based solvent such as dichloromethane, dichloroethane, chloroform, or carbon tetrachloride; or a hydrocarbon-based solvent such as hexane, heptane, cyclohexane, or methylcyclohexane. These solvents can be used alone or in combination of two or more in any ratio. The number of separations is not particularly limited and can be performed depending on the desired purity or yield. In addition, in this reaction, a separation operation is not essential.

[0104] The reaction mixture containing the compound represented by general formula (II) obtained above can be dehydrated with a drying agent such as sodium sulfate or magnesium sulfate, but this is not essential.

[0105] The reaction mixture containing the compound represented by general formula (II) obtained above can be subjected to solvent distillation under reduced pressure as long as the compound is not decomposed.

[0106] The reaction mixture containing the compound represented by general formula (II) obtained after distilling off the solvent can be purified by washing with an appropriate solvent, reprecipitation, recrystallization, column chromatography, or the like.

[0107] ■ Production method (B-2) <Scheme 3> (In the formula, R １Ｂ , R ２Ｂ , R ３Ｂ , R ４Ｂ , R５Ｂ , R ６Ｂ , R ７Ｂ , R ８Ｂ , R ９Ｂ , R １０Ｂ , R １１Ｂ and R １２Ｂ has the same meaning as above. １４Ｂ represents a halogen atom.)

[0108] The production method (B-2) is a method for synthesizing a compound represented by general formula (II), and includes a step of reacting a sulfonamide compound represented by general formula (3B) with an amine compound represented by general formula (4B) in a solvent in the presence of a base.

[0109] The amount of the compound represented by general formula (4B) is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (3B) and the target reaction proceeds, but is usually 1 equivalent or more and 200 equivalents or less.

[0110] Examples of the base used in this reaction include metal hydrides such as sodium hydride; organolithium compounds such as methyllithium, butyllithium, sec-butyllithium, t-butyllithium, and hexyllithium; and metal amides such as lithium diisopropylamide, lithium hexamethyldisilazane, sodium hexamethyldisilazane, and potassium hexamethyldisilazane.

[0111] The amount of the base used in this reaction is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (4B) and the desired reaction proceeds, but is preferably 1 equivalent or more and 10 equivalents or less.

[0112] The solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, and examples thereof include benzene-based solvents such as benzene, toluene, xylene, mesitylene, chlorobenzene, and dichlorobenzene; halogen-based solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; etc. These solvents can be used alone or in combination of two or more in any desired ratio.

[0113] The amount of solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, but is usually 3 to 200 times by weight relative to the compound represented by formula (3B).

[0114] The temperature at which this reaction is carried out is not particularly limited as long as the desired reaction proceeds, but is usually from 0° C. to 150° C. or below the boiling point of the solvent.

[0115] The time for carrying out this reaction is not particularly limited as long as the desired reaction proceeds, but is usually from 10 minutes to 24 hours.

[0116] As a post-treatment for the reaction, undissolved metals can be removed by filtration. Furthermore, a separation operation can be performed by adding water or an appropriate aqueous solution to the reaction mixture. When using an aqueous solution, any of the following can be used: an acidic aqueous solution containing hydrochloric acid, sulfuric acid, ammonium chloride, etc.; an alkaline aqueous solution containing potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.; or saline solution. During the separation operation, if necessary, a water-immiscible solvent can be added, such as a benzene-based solvent such as toluene, xylene, benzene, chlorobenzene, or dichlorobenzene; an ester-based solvent such as ethyl acetate, isopropyl acetate, or butyl acetate; an ether-based solvent such as diethyl ether, diisopropyl ether, or methyl-t-butyl ether; a halogen-based solvent such as dichloromethane, dichloroethane, chloroform, or carbon tetrachloride; or a hydrocarbon-based solvent such as hexane, heptane, cyclohexane, or methylcyclohexane. These solvents can be used alone or in combination of two or more in any ratio. The number of separations is not particularly limited and can be performed depending on the desired purity or yield. In addition, in this reaction, a separation operation is not essential.

[0117] The reaction mixture containing the compound represented by general formula (II) obtained above can be dehydrated with a drying agent such as sodium sulfate or magnesium sulfate, but this is not essential.

[0118] The reaction mixture containing the compound represented by general formula (II) obtained above can be subjected to solvent distillation under reduced pressure as long as the compound is not decomposed.

[0119] The reaction mixture containing the compound represented by general formula (II) obtained after distilling off the solvent can be purified by washing with an appropriate solvent, reprecipitation, recrystallization, column chromatography, or the like.

[0120] (2) Method for producing N-thiazolylbenzamide compounds represented by formula (III) (production method (C)) Examples of methods for producing compounds represented by formula (III) are given below.

[0121] The compound of the present invention represented by general formula (III) can be produced, for example, by a production method represented by the following scheme 4, but the present invention is not limited thereto. <Scheme 4> (In the formula, R １Ｃ , R ２Ｃ , R ３Ｃ , R ４Ｃ , R ５Ｃ , R ６Ｃ , R ７Ｃ , R ８Ｃ , R ９Ｃ , R １０Ｃ , R １１Ｃ and R １２Ｃ has the same meaning as above. １３Ｃ represents a halogen atom.)

[0122] Production method (C) is a method for synthesizing a compound represented by general formula (III), and is a production method comprising a step of reacting a thiazole compound represented by general formula (1C) with a compound represented by general formula (2C) in a solvent in the presence of a base.

[0123] The amount of the compound represented by general formula (1C) is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (2C) and the target reaction proceeds, but is usually 1 equivalent or more and 200 equivalents or less.

[0124] Examples of the base used in this reaction include metal hydrides such as sodium hydride; organolithium compounds such as methyllithium, butyllithium, sec-butyllithium, t-butyllithium, and hexyllithium; and metal amides such as lithium diisopropylamide, lithium hexamethyldisilazane, sodium hexamethyldisilazane, and potassium hexamethyldisilazane.

[0125] The amount of the base used in this reaction is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (1C) and the desired reaction proceeds, but is preferably 1 equivalent or more and 10 equivalents or less.

[0126] The solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, and examples thereof include benzene-based solvents such as benzene, toluene, xylene, mesitylene, chlorobenzene, and dichlorobenzene; halogen-based solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; etc. These solvents can be used alone or in combination of two or more in any desired ratio.

[0127] The amount of solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, but is usually 3 to 200 times by weight relative to the compound represented by formula (1C).

[0128] The temperature at which this reaction is carried out is not particularly limited as long as the desired reaction proceeds, but is usually from 0° C. to 150° C. or below the boiling point of the solvent.

[0129] The time for carrying out this reaction is not particularly limited as long as the desired reaction proceeds, but is usually from 10 minutes to 24 hours.

[0130] As a post-treatment for the reaction, undissolved metals can be removed by filtration. Furthermore, a separation operation can be performed by adding water or an appropriate aqueous solution to the reaction mixture. When using an aqueous solution, any of the following can be used: an acidic aqueous solution containing hydrochloric acid, sulfuric acid, ammonium chloride, etc.; an alkaline aqueous solution containing potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.; or saline solution. During the separation operation, if necessary, a water-immiscible solvent can be added, such as a benzene-based solvent such as toluene, xylene, benzene, chlorobenzene, or dichlorobenzene; an ester-based solvent such as ethyl acetate, isopropyl acetate, or butyl acetate; an ether-based solvent such as diethyl ether, diisopropyl ether, or methyl-t-butyl ether; a halogen-based solvent such as dichloromethane, dichloroethane, chloroform, or carbon tetrachloride; or a hydrocarbon-based solvent such as hexane, heptane, cyclohexane, or methylcyclohexane. These solvents can be used alone or in combination of two or more in any ratio. The number of separations is not particularly limited and can be performed depending on the desired purity or yield. In addition, in this reaction, a separation operation is not essential.

[0131] The reaction mixture containing the compound represented by general formula (III) obtained above can be dehydrated with a drying agent such as sodium sulfate or magnesium sulfate, but this is not essential.

[0132] The reaction mixture containing the compound represented by general formula (III) obtained above can be subjected to solvent distillation under reduced pressure as long as the compound is not decomposed.

[0133] The reaction mixture containing the compound represented by general formula (III) obtained after distilling off the solvent can be purified by washing with an appropriate solvent, reprecipitation, recrystallization, column chromatography, or the like.

[0134] (2) Method for producing N-benzylsuccinimide compounds represented by formula (IV) (production method (D)) Examples of methods for producing compounds represented by formula (IV) are given below.

[0135] The compound of the present invention represented by general formula (IV) can be produced, for example, by the production method represented by the following scheme 5, but the present invention is not limited thereto.

[0136] <Scheme 5> (In the formula, R １Ｄ , R ２Ｄ , R ３Ｄ , R ４Ｄ , and R ５Ｄ has the same meaning as above. X is an oxygen atom or CH ２ Indicates.)

[0137] Production method (D) is a method for synthesizing an N-benzylsuccinimide compound represented by general formula (IV), and is a production method comprising a step of reacting a compound represented by general formula (1D) with an N-maleimide compound represented by general formula (2D) in the absence of a solvent or in a solvent.

[0138] The amount of the compound represented by general formula (1D) is not particularly limited as long as it is 1 equivalent or more relative to the compound represented by general formula (2D) and the target reaction proceeds, but is usually 1 equivalent or more and 200 equivalents or less.

[0139] When a solvent is added to the reaction, the type of solvent is not particularly limited as long as the desired reaction proceeds, and examples thereof include benzene-based solvents such as benzene, toluene, xylene, mesitylene, chlorobenzene, and dichlorobenzene; halogen-based solvents such as dichloromethane, dichloroethane, chloroform, and carbon tetrachloride; etc. These solvents can be used alone or in combination of two or more in any desired ratio.

[0140] The amount of solvent used in this reaction is not particularly limited as long as the desired reaction proceeds, but is usually 3 to 200 times by weight relative to the compound represented by formula (1D).

[0141] In this reaction, additives such as a catalyst can be added as needed.

[0142] The temperature at which this reaction is carried out is not particularly limited as long as the desired reaction proceeds, but is usually from −78° C. to 250° C. or below the boiling point of the solvent.

[0143] The time for carrying out this reaction is not particularly limited as long as the desired reaction proceeds, but is usually from 10 minutes to 24 hours.

[0144] As a post-treatment for the reaction, undissolved metals can be removed by filtration. Furthermore, a separation operation can be performed by adding water or an appropriate aqueous solution to the reaction mixture. When using an aqueous solution, any of the following can be used: an acidic aqueous solution containing hydrochloric acid, sulfuric acid, ammonium chloride, etc.; an alkaline aqueous solution containing potassium hydroxide, sodium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.; or saline solution. During the separation operation, if necessary, a water-immiscible solvent can be added, such as a benzene-based solvent such as toluene, xylene, benzene, chlorobenzene, or dichlorobenzene; an ester-based solvent such as ethyl acetate, isopropyl acetate, or butyl acetate; an ether-based solvent such as diethyl ether, diisopropyl ether, or methyl-t-butyl ether; a halogen-based solvent such as dichloromethane, dichloroethane, chloroform, or carbon tetrachloride; or a hydrocarbon-based solvent such as hexane, heptane, cyclohexane, or methylcyclohexane. These solvents can be used alone or in combination of two or more in any ratio. The number of separations is not particularly limited and can be performed depending on the desired purity or yield. In addition, in this reaction, a separation operation is not essential.

[0145] The reaction mixture containing the compound represented by general formula (IV) obtained above can be treated with a drying agent such as sodium sulfate or magnesium sulfate to remove moisture, but this is not essential.

[0146] The reaction mixture containing the compound represented by general formula (IV) obtained above can be subjected to solvent distillation under reduced pressure as long as the compound is not decomposed.

[0147] The reaction mixture containing the compound represented by general formula (IV) obtained after distilling off the solvent can be purified by washing with an appropriate solvent, reprecipitation, recrystallization, column chromatography, or the like.

[0148] The subjects to which the present preventive or therapeutic agent for polycystic kidney disease is administered include mammals, specifically humans, cats, dogs, monkeys, cows, horses, mice, rats, hamsters, guinea pigs, rabbits, goats, pigs, and sheep.

[0149] Subjects to whom the present preventive or therapeutic agent for polycystic kidney disease is administered include subjects diagnosed with polycystic kidney disease or subjects diagnosed as needing prevention of polycystic kidney disease.The agent can also be used as a preventive or therapeutic agent for autosomal dominant polycystic kidney disease to be administered to subjects with PKD1 and / or PKD2 gene mutations.

[0150] The agent for preventing or treating polycystic kidney disease may contain pharmaceutically acceptable additives, such as saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, and lubricants.

[0151] The dosage of the present agent for preventing or treating polycystic kidney disease to a subject varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but typically, per adult, the content of the compound as the active ingredient per dose is in the range of 0.1 mg to 1000 mg, preferably 1 mg to 500 mg, and can be administered independently four, three, two, or once a day, every other day, every second day, every third day, every fourth day, every fifth day, every seventh day, every eighth day, every ninth day, twice a week, once a month, or twice a month. The method of administration is not particularly limited, and examples thereof include oral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, and intranasal administration.

[0152] The present agent for preventing or treating polycystic kidney disease may be used in combination with drugs for other kidney diseases, as well as with antihypertensive agents, hypolipidemic agents, hypoglycemic agents, and the like.

[0153] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0154] [Example 1] Search for tool compounds

[0155] To screen for tool compounds, the inventors constructed a screening platform incorporating a split-luciferase system targeting the intermolecular interaction between ORP3 and VAP-A. Using this platform, they searched for compounds that promote the intermolecular interaction between ORP3 and VAPA. Specifically, a polynucleotide encoding ORP3 or VAPA was first inserted into an expression vector included with the NanoBiT® Protein:Protein Interaction System (Promega), and then transfected into HEK293T cells by lipofection. Drug selection was performed with neomycin to obtain HEK293T cell clones stably expressing split-luciferase (HEK293T cells constitutively expressing split-luciferase for detecting the interaction between ORP3 and VAPA). Next, the obtained split-luciferase stably expressing HEK293 cells or parental HEK293 cells were seeded onto a 96-well plate. After 24 hours of incubation, the library compounds and Nano-Glo Live Cell Reagent (Promega), a luminescent substrate for split-luciferase, were added and incubated. Immediately after treatment, the plate was placed in a luminescence image capture device (LAS-4000: GE Healthcare) and images were continuously captured up to 4 hours after the addition of the substrate. The captured images were analyzed using the Multi gauge software included with the LAS-4000, and quantitative luminescence data for each well was obtained. The split-luciferase expression system described above was constructed by fusing a 17.6 kDa Large BiT (LgBiT) to ORP3 and a 11-amino acid Small BiT (SmBiT) to VAP-A, resulting in the formation of luciferase through the interaction between ORP3 and VAP-A.

[0156] DMSO, the solvent used to dilute the compounds, was placed on both ends of the 96-well plate as a negative control, and 10 μM Phorbol 12-Myristate 13-Acetate (PMA), which was shown to enhance luminescence in the ORP3-VAPA split-luciferase system, was used as a positive control. With PMA treatment, luminescence enhancement was saturated within 2 hours of treatment, and luminescence was confirmed to persist for up to 4 hours after treatment. Furthermore, the luminescence enhancement effect after 2 hours of treatment was approximately 5-fold.

[0157] Next, 10,880 compounds from the Kyoto University Compound Library (Diversity set) were screened at a concentration of 10 μM. Compounds with a fluorescence enhancement effect of 2.8 or higher relative to the fluorescence level of the untreated compound (1) were selected as hit compounds, yielding a total of 44 hit compounds. The hit compounds were named Compounds A to Z and AA to AR.

[0158] The results are shown in Figure 1. The maximum enhancement effect was 4.4 times. When the concentration dependency of the luminescence enhancement effect of the hit compounds was examined, an enhancement effect was observed at 3 μM for all compounds.

[0159] [Example 2] Increase in cholesterol in primary cilia Of the hit compounds obtained in Example 1, 10 compounds (compounds A, C, D, I, R, V, AB, AF, AL, and AP) were evaluated for their ability to increase cholesterol in primary cilia. hTERT-immortalized human retinal pigment epithelial cells (hTERT-RPE1 cells: ATCC CRL-4000) were treated with the hit compounds for 4 hours at 37°C, and then multi-stained with the cholesterol indicator Filipin III (Merck), a primary cilium marker anti-acetylated-tubulin antibody (Merck), and a centrosome marker anti-Pericentrin antibody (Bethyl). Cholesterol in primary cilia was observed using a confocal laser microscope, and the fluorescence intensity of Filipin III in primary cilia was measured. The results are shown in Figures 2A to 4B. Additionally, we performed a similar analysis on VAP-A-deficient hTERT-RPE1 cells, which were generated by deleting the VAP-A gene, using the CRISPR-ObLiGaRe method established in the paper by Royba, Miyamoto et al., Sci Rep 2017. In the figure, VAPA+ / + indicates hTERT-RPE1 cells, and VAPA- / - indicates VAP-A-deficient hTERT-RPE1 cells.

[0160] 2A and 2B, filipin III and immunostaining revealed that treatment with compound A increased cholesterol in the primary cilium of hTERT-RPE1 cells. The increase in cholesterol in the primary cilium by compound A, in other words, the activity of compound A in supplying cholesterol to the primary cilium, was not detected in a VAP-A-deficient cell line (VAP-A- / -), indicating that this activity is ORP3 / VAPA-dependent. PMA is a PKC activator, and it has been reported that PKC-mediated ORP3 phosphorylation promotes molecular interaction with VAP-A. PMA was used as a positive control in this example.

[0161] 3A and 3B, it was revealed by Filipin III and immunostaining that cholesterol in the primary cilia of hTERT-RPE1 cells was increased in a dose-dependent manner by treatment with Compounds A and C. On the other hand, Compound D did not show any activity to increase cholesterol in the primary cilia.

[0162] Furthermore, Figures 4A and 4B show that the cholesterol in the primary cilia of hTERT-RPE1 cells was increased in a dose-dependent manner by treatment with compounds A, R, and V, as revealed by Filipin III and immunostaining. Compounds AB, AF, AL, and AP did not show any activity to increase cholesterol in the primary cilia.

[0163] Furthermore, as shown in Figures 5A and 5B, it was revealed by Filipin III and immunostaining that cholesterol in the primary cilium of hTERT-RPE1 cells was also increased by treatment with Compound I.

[0164] These results demonstrate that compounds A, C, I, R, and V significantly increase cholesterol supply to primary cilia in a dose-dependent manner. Importantly, these activities were not observed in VAP-A-deficient hTERT-RPE1 cells, genetically demonstrating that the increase in cholesterol in primary cilia induced by compounds A, C, I, R, and V is ORP3 / VAP-A dependent.

[0165] Compounds A, C, I, and R are shown below. Compound A is the above-mentioned compound (III-1), compound C is the above-mentioned compound (IV-1), compound I is the above-mentioned compound (II-2), and compound R is the above-mentioned compound (I-5).

[0166]

[0167]

[0168]

[0169]

[0170] Example 3 Restoration of Localization of Polycystin-1 Missense Protein to Primary Cilia Using mouse kidney collecting duct-derived cells (mIMCD3 cells: ATCC cat. no. CRL-2123) expressing polycystin-1 missense protein (a mutation in which cysteine ​​at position 210 in the amino acid sequence of SEQ ID NO: 1 is replaced with glycine), whether Compound C has the effect of restoring the localization of polycystin-1 to primary cilia was investigated.

[0171] The mutant mIMCD3 cells were treated with compound C for 4 hours at 37°C and then multi-stained with polycystin-1 antibody (Santa Cruz), anti-acetylated-tubulin antibody (Merck), a primary cilium marker, and anti-FGFR10P antibody (Proteintech), a centrosome marker. The cholesterol in the primary cilium was observed using a confocal laser microscope, and the fluorescence intensity of polycystin-1 protein in the primary cilium was measured. The results are shown in Figures 6A and 6B. Non-mutated mIMCD3 cells (wild type) were used as a control.

[0172] 6A and 6B, it was confirmed that compound C has the effect of restoring the localization of polycystin-1 to primary cilia even if the polycystin-1 protein is mutated.

[0173] Example 4: Inhibitory effect of compounds A, C, R, and V on the increase in tubule diameter in a cyst culture system reconstituting cyst formation by 3D culture of renal tubular cells. Using mutant mIMCDC3 cells expressing polycystin-1 missense protein or mutant mIMCDC3 cells expressing polycystin-2 missense protein, we investigated whether each compound has the effect of normalizing cyst size. Mutations in polycystin-1 protein or polycystin-2 protein reduce primary cilia activity, leading to cyst enlargement.

[0174] The 3D culture of each of the mutant mIMCD3 cells was prepared according to the paper by Rachel H. Giles et al. (Nature Protocols, volume 9, pages 2725-2731 (2014)). PC1 mutant mouse kidney collecting duct-derived cells (PC1 mutant mIMCD3) expressing a polycystin-1 missense protein (a mutation in which cysteine ​​at position 210 of the amino acid sequence of SEQ ID NO: 1 is replaced with glycine) or PC2 mutant mIMCD3 cells expressing a polycystin-2 missense protein (a mutation in which tryptophan at position 412 of the amino acid sequence of SEQ ID NO: 2 (corresponding to position 414 of the human polycystin-2 missense protein) is replaced with glycine) were prepared using mIMCD3 cells (ATCC cat. no. CRL-2123). Each mutant mIMCDC3 cell was suspended in DMEM / F-12 (L-glutamine) medium supplemented with 10% (vol / vol) FBS and 1% (vol / vol) pen-strep, and mixed with 20 μM of compound A, C, R, or V or 200 μM of cholesterol and Matrigel, and then transferred to an 8-well chamber slide plate. The mixture was incubated at 37°C and 5% CO ２ The mixture was incubated (15-30 min) under conditions until solidification. Then, pre-warmed medium was added to the solidified cell-Matrigel layer, and the medium was exchanged while the mixture was incubated at 37°C in 5% CO until cyst formation. ２ The mixture was incubated under the conditions.

[0175] The formed cysts were fixed and sectioned. Furthermore, F-actin was stained with phalloidin and DNA with DAPI, and then the sections were photographed using a fluorescence microscope. Furthermore, the size of the epithelial lumen formed within the cyst in the merged image of the formed cyst was measured as the inner diameter, and the size of the entire cyst as the outer diameter, and the inner diameter / outer diameter was calculated. The results are shown in Figures 7A to 7C. In Figure 7A, the dotted line in the merged image indicates the width of the inner diameter.

[0176] 7A to 7C, it was confirmed that compounds A, C, and R reduced the inner diameter / outer diameter to the same level as cholesterol, and had the effect of improving cyst size.

[0177] Example 5 Inhibitory Effect on Duct Diameter Increase in a Cyst Culture System Reconstructing Cyst Formation by 3D Culture of Renal Tubular Cells - Comparison with Tolvaptan Using the above-described PC2 mutant mIMCDC3 cells, compounds A, G, and R, as well as the existing drug tolvaptan, were added at 3.75 μM, 7.5 μM, and 15 μM, respectively, to examine whether each compound has an inhibitory effect on the increase in cystic duct diameter, as in Example 4. The results are shown in Figures 8A and 8B.

[0178] 8A and 8B, it was confirmed that compounds G and R have the effect of reducing cyst size to the same extent as tolvaptan, and compound A has the effect of reducing cyst size more than tolvaptan.

[0179] Furthermore, using the above-mentioned PC2 mutant mIMCD3 cells, compounds A, G, and compound G-OH, a derivative of compound G, were each added at 20 μM to examine whether each compound had an inhibitory effect on the increase in cystic duct diameter in the same manner as in Example 4. The results are shown in Figures 9A and 9B.

[0180] 9A and 9B, it was confirmed that not only compound G but also its derivative compound G-OH had the effect of improving cyst size.

[0181] Compounds G and G-OH are shown below, where compound G is the above compound (I-1), and compound G-OH is the above compound (I-6).

[0182]

[0183]

[0184] Example 6: Inhibitory effect on tubule diameter increase in a cyst culture system reconstituting cyst formation by 3D culture of renal tubular cells - Compounds B, Q, K, J, and I - Compounds A, C, and R were used in Example 4, but compounds B, Q, K, J, and I were also used to similarly examine the inhibitory effect on tubule diameter increase. The PC2 mutant mIMCDC3 cells described above were used, and compounds A, G, B, Q, K, J, and I were used. Compounds A, G, B, Q, K, and J were used at 20 μM, and compound I was used at 2.5, 5, 10, and 20 μM. Photographs were taken using a fluorescence microscope in the same manner as in Example 4, and the inner diameter / outer diameter were measured. The results for compounds A, G, B, Q, K, and J are shown in Figures 10A and 10B, and the results for compounds A and I are shown in Figures 11A and 11B.

[0185] 10A and 10B, it was confirmed that compounds B and Q had the same level of effect of improving cyst size as compound A, and compounds K and J had the same level of effect as compound G. Furthermore, it was confirmed from Figures 11A and 11B that compound I also had the same level of effect of improving cyst size as compound A when used at 10 μM and 20 μM.

[0186] Compounds B, Q, K, and J are shown below. Compound B is the above-mentioned compound (II-1), compound Q is the above-mentioned compound (I-4), compound K is the above-mentioned compound (I-3), and compound J is the above-mentioned compound (I-2).

[0187]

[0188]

[0189]

[0190]

[0191] Example 7 Effect of Restoring Primary Cilia Localization of Polycystin 2 Protein (PC2) in Human Induced Pluripotent Stem Cells Derived from Patients with Polycystic Kidney Disease - Comparison with Tolvaptan - Using induced pluripotent stem cells derived from patients with autosomal dominant polycystic kidney disease (HPS2192 cells: RIKEN BRC RRID: CVCL_YS47 and HPS2636 cells: RIKEN BRC RRID: CVCL_YS58: RIKEN BioResource Research Center), compound G-OH was added at 5 μM, 10 μM, or 20 μM, or the existing drug tolvaptan was added at 20 μM, to investigate whether it has an effect of restoring primary cilia localization of polycystin 2 protein.

[0192] The human induced pluripotent stem cells were treated with compound G-OH and tolvaptan for 24 hours at 37°C, and then multi-stained with anti-polycystin 2 antibody (Santa Cruz), the primary cilium marker anti-ARL13B antibody (Santa Cruz), and the centrosome marker anti-FGFR1OP antibody (Proteintech). Polycystin 2 in the primary cilium was observed using a confocal laser microscope, and the amount of polycystin 2 in the primary cilium was quantified by immunostaining. Figures 12A and 12B show the results. Healthy donor-derived induced pluripotent stem cells (HPS1006 cells: RIKEN BRC RRID: CVCL_UP39: RIKEN BioResource Research Center) were used as a control (wild-type).

[0193] 12A and 12B confirm that compound G-OH has the effect of restoring the localization of polycystin-2 to primary cilia in human induced pluripotent stem cells derived from polycystin-2 patients, even if polycystin-2 is mutated. Furthermore, when the concentration dependency of the restorative effect of compound G-OH was examined, immunostaining revealed that polycystin-2 in primary cilia increased in a dose-dependent manner. On the other hand, the existing drug tolvaptan did not exhibit the activity of increasing the amount of polycystin-2 in primary cilia.

[0194] Example 8 Effect of Increasing Primary Cilia Localization of Polycystin-1 Protein (PC1) in Feline Cells The increase in PC1 in primary cilia by Compound G was evaluated. CRFK cells, a feline kidney-derived epithelial cell line, were prepared and treated with Compound G (final concentration 20 μM) for 8, 16, or 24 hours at 37°C. The cells were then multi-stained with an anti-polycystin-1 antibody (Santa Cruz), a primary cilium marker anti-acetylated-tubulin antibody (Merck), and a centrosome marker anti-FGFR1OP antibody (Proteintech). PC1 in primary cilia was observed using a confocal laser microscope, and the fluorescence intensity of PC1 in primary cilia was measured. The results are shown in Figures 13A and 13B.

[0195] 13A and 13B, it was confirmed that compound G has an action of increasing PC1 in primary cilia in CRFK cells, an epithelial cell line derived from feline kidney.

Claims

1. A preventive or therapeutic agent for polycystic kidney disease, comprising as an active ingredient a compound or a pharmacologic acceptable salt thereof that promotes intermolecular interaction between oxysterol-binding protein-related protein-3 (ORP3) and endoplasmic reticulum protein-related protein-A (VAP-A) in cells and increases cholesterol in the primary cilia of cells.

2. The preventive or therapeutic agent for polycystic kidney disease according to claim 1, wherein the compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is a compound containing a 4-amino-1-phenylpyrazolopyrimidine skeleton, a 5-(sulfamoyl)benzamide skeleton, an N-thiazolylbenzamide skeleton, or an N-benzylsuccinimide skeleton.

3. The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (I): (In the formula, R １Ａ and R ２Ａ are the same or different and each represents a hydrogen atom, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, an optionally substituted C2-6 alkenyl group, an optionally substituted C2-6 alkynyl group, or an optionally substituted phenyl group. R ３Ａ and R ４Ａ are the same or different and each represents a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ５Ａ , R ６Ａ , R ７Ａ , R ８Ａ , and R ９Ａ and R 1 -R 2 are the same or different and represent a hydrogen atom, a halogen atom, a nitro group, a C1-6 alkyl group which may have a substituent, or a C1-6 alkoxy group which may have a substituent.

4. The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is a compound represented by the following formula (I-1) (4-methoxypropylamino-1-(2,4-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), a compound represented by formula (I-2) (4-(4-fluorobenzylamino)-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), a compound represented by formula (I-3) (4-(3,5-dimethylphenyl)amino-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidine), a compound represented by formula (I-4) (2-(4-chlorophenyl)ethylamino-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), The preventive or therapeutic agent for polycystic kidney disease according to claim 1, which is a compound represented by formula (I-5) (2-(1-cyclohexenyl)ethylamino-1-(3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidine), or a compound represented by formula (I-6) (4-hydroxypropylamino-1-(2,4-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine).

5. The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (II): (In the formula, R １Ｂ , R ２Ｂ , R ３Ｂ , R ４Ｂ , R ５Ｂ , R ７Ｂ , R ８Ｂ , R ９Ｂ , and R １０Ｂ are the same or different and each represents a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ６Ｂ represents a hydrogen atom or an optionally substituted C1-6 alkyl group. １１Ｂ , and R １２Ｂ and R 1 -R 2 are the same or different and represent a hydrogen atom, a C1-6 alkyl group optionally having a substituent, a C1-6 alkoxy group optionally having a substituent, a C3-8 cycloalkyl group optionally having a substituent, a C3-6 cycloalkoxy group optionally having a substituent, or a phenyl group optionally having a substituent.

6. The preventive or therapeutic agent for polycystic kidney disease according to claim 1, wherein the compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is a compound represented by the following formula (II-1) or a compound represented by formula (II-2).

7. The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (III): (In the formula, R １Ｃ , R ２Ｃ , R ３Ｃ , R ４Ｃ , R ５Ｃ , R ８Ｃ , R ９Ｃ , R １０Ｃ , R １１Ｃ , and R １２Ｃ are the same or different and each represents a hydrogen atom, a halogen atom, a nitro group, an optionally substituted C1-6 alkyl group, an optionally substituted C1-6 alkoxy group, or an optionally substituted phenyl group. R ６Ｃ represents a hydrogen atom or an optionally substituted C1-6 alkyl group. ７Ｃ The preventive or therapeutic agent for polycystic kidney disease according to claim 1, which is an N-thiazolylbenzamide compound represented by the following formula:

8. The preventive or therapeutic agent for polycystic kidney disease according to claim 1, wherein the compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is a compound represented by the following formula (III-1):

9. The compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is represented by the general formula (IV): (In the formula, R １Ｄ , R ２Ｄ , R ３Ｄ , R ４Ｄ , and R ５Ｄ are the same or different and each represents a hydrogen atom or an optionally substituted C1-6 alkyl group. X represents an oxygen atom or CH ２ The method of claim 1, wherein the compound is an N-benzylsuccinimide compound represented by the formula:

10. The preventive or therapeutic agent for polycystic kidney disease according to claim 1, wherein the compound having the effect of promoting the intermolecular interaction and increasing cholesterol in the primary cilium of the cell is a compound represented by the following formula (IV-1):

11. The preventive or therapeutic agent for polycystic kidney disease according to claim 1, for administration to a patient having a gene mutation in PKD1 and / or PKD2.

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