Trpc5 inhibitors, methods of making and using the same

Abstract

The application discloses a TRPC5 inhibitor and a preparation method and application thereof, and the TRPC5 inhibitor comprises a compound of formula I, stereoisomers of the compound, isotope-labeled derivatives of the compound, pharmaceutically acceptable salts of the compound, prodrugs of the compound or solvates of the compound. The compound has a good inhibiting effect on a TRPC5 ion channel, and is expected to be used for treating various diseases related to the TRPC5, such as cancers, depression, chronic nephritis and the like.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to TRPC5 inhibitors, their preparation methods, and applications. Background Technology

[0002] Progressive chronic kidney disease (CKD) is associated with an increased risk of kidney failure, and its prevalence is rapidly increasing, currently affecting more than 850 million people worldwide. Despite this increasing number, treatment options for slowing or preventing disease progression remain limited. Nephrotic syndrome is a significant driver of CKD. Nephrotic syndrome is characterized by the leakage of large amounts of albumin into the urine, resulting from damage to the glomeruli, the filtration units of the kidneys. The intact kidney filter, composed of endothelial cells, basement membrane, and podocytes, is crucial for retaining proteins in the blood and removing waste products from the body. Many chronic kidney diseases are associated with podocyte damage and loss, and the process of podocyte damage and necrosis is difficult to halt; once necrosis occurs, it is irreversible. Research indicates that podocyte damage and loss are caused by a gene mutation that induces activation of the TRPC5 ion channel within podocytes, as well as cytoskeleton remodeling. Podocyte damage and the presence of proteinuria are key features of CKD. In rodent models of kidney disease, inhibition of the TRPC5 ion channel protects podocytes. Once lost, the renal filtration membrane cannot be renewed, and due to their limited proliferative capacity, podocytes are particularly vulnerable to various stimuli, leading to damage. Therefore, preventing podocyte injury remains a key objective in the development of effective and targeted therapeutic strategies for kidney disease.

[0003] TRPC5 inhibitors are substances that can directly activate TRPC5 channels, causing cell membrane depolarization and an increase in intracytoplasmic calcium concentration. TRPC5 inhibitors can be used to treat tumors, reverse multidrug resistance in tumors, and also as antidepressants. Recent research also shows that TRPC5, as an ion channel protein, can be activated by various renal stressors. TRPC5 inhibitors may be able to prevent the destruction of renal filtration function, potentially forming the basis for the development of much-needed drugs to treat progressive chronic kidney disease (CKD). Specifically, research has found that a specific and selective TRPC5 inhibitor, AC1903, can protect podocytes in the kidneys and prevent renal failure, restoring kidney function. This suggests that AC1903 holds promise for treating chronic nephritis, providing a new treatment option for patients with chronic nephritis. However, the efficacy of AC1903 needs further improvement. Therefore, new drugs or methods are needed to more effectively treat kidney disease or reduce the risk of developing chronic kidney disease. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a TRPC5 inhibitor, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] One aspect of the present invention provides compounds of formula I, their stereoisomers, their isotopically labeled derivatives, their pharmaceutically acceptable salts, their prodrugs, or their solvates:

[0007]

[0008] R1 is selected from 6- to 10-membered heteroaryl groups, wherein the heteroaryl group contains at least one of O, N, and S heteroatoms;

[0009] R2 is selected from H, C1-C6 alkyl, and C1-C6 cycloalkyl; the alkyl or cycloalkyl group is optionally substituted with 0 to 3 halogens. In some embodiments of the present invention, Selected from

[0010] In some embodiments of the present invention, the compound of formula I is selected from the following compounds:

[0011]

[0012] A second aspect of the invention provides compounds of formula II and pharmaceutically acceptable salts thereof:

[0013]

[0014] Wherein, R3 is selected from -C(O)OR5; R5 is selected from H, C1-C6 alkyl, and C1-C6 cycloalkyl;

[0015] R4 is selected from H, C1-C6 alkyl, C1-C6 cycloalkyl, alkoxycarbonyl, or N-R4 to form an amino salt.

[0016] In some embodiments of the present invention, the alkoxycarbonyl group includes benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (BOC), methoxycarbonyl (Fmoc), and allyloxycarbonyl (Alloc).

[0017] In some embodiments of the present invention, the amino salt includes amino hydrochloride, amino sulfonate, and amino sulfate.

[0018] Another aspect of the invention provides the use of the compound of formula II in the preparation of the compound of formula I.

[0019] Another aspect of the present invention provides a method for preparing the compound of formula I, comprising the following steps:

[0020] The compound of formula II and the compound of formula III The reaction was carried out to obtain the compound of formula I;

[0021] R1 is defined as described above.

[0022] In some embodiments of the present invention, the preparation method of the compound of formula II includes the following steps:

[0023] Compounds of formula IV The compound of formula II was prepared by reacting it with p-toluidine;

[0024] The definitions of R3 and R4 are as described above.

[0025] In another aspect, the present invention provides a pharmaceutical composition comprising the compound of formula I, its stereoisomer, its isotopically labeled derivative, its pharmaceutically acceptable salt, its prodrug or solvate thereof, and optionally, pharmaceutically acceptable excipients.

[0026] The pharmaceutical compositions of the present invention are suitable for various routes of administration and can therefore be formulated into any pharmaceutically acceptable dosage form. For example, the pharmaceutical compositions described above can be administered to patients or subjects requiring such treatment via oral, parenteral, rectal, or pulmonary administration. For oral administration, the pharmaceutical compositions can be formulated into oral preparations, such as conventional oral solid dosage forms like tablets, capsules, pills, granules, etc.; or into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When formulating oral preparations, suitable fillers, binders, disintegrants, lubricants, etc., can be added. For parenteral administration, the pharmaceutical compositions described above can also be formulated into injections, including injection solutions, sterile powders for injection, and concentrated solutions for injection. When formulating injections, conventional methods in the existing pharmaceutical field can be used; when preparing injections, excipients may not be added, or suitable excipients may be added depending on the properties of the drug. For rectal administration, the pharmaceutical compositions can be formulated into suppositories, etc. For pulmonary administration, the pharmaceutical compositions can be formulated into inhaled preparations, aerosols, powder inhalers, or sprays, etc.

[0027] Pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of specific excipients will depend on the route of administration or the type and state of disease in the treatment of a particular patient. Examples of pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, which are common in the pharmaceutical field. Flavoring agents, preservatives, and sweeteners may also be added to the pharmaceutical composition where necessary.

[0028] Another aspect of the invention provides the use of a compound of formula I, its stereoisomers, its isotopically labeled derivatives, its pharmaceutically acceptable salts, its prodrugs or solvates thereof, and / or the pharmaceutical composition in the preparation of a TRPC5 inhibitor.

[0029] In some embodiments of the present invention, the TRPC5 inhibitor is an inhibitor used to treat or prevent TRPC5-mediated diseases.

[0030] In some instances of this invention, the TRPC5-mediated diseases are mental illnesses, neurological disorders, neurodegenerative diseases, or kidney diseases.

[0031] In some instances of the present invention, the mental illness, neurological illness, or neurodegenerative illness may be selected from: illnesses related to disordered emotional processing (e.g., borderline personality disorder or depression, such as major depressive disorder, severe depressive disorder, psychotic depression, dysphoric disorder, postpartum depression, and bipolar disorder), illnesses related to anxiety and fear (e.g., post-traumatic stress disorder, panic disorder, agoraphobia, social phobia, generalized anxiety disorder, panic disorder, social anxiety disorder, obsessive-compulsive disorder, and separation anxiety), memory disorders (e.g., Alzheimer's disease, amnesia, aphasia, brain injury, brain tumor, chronic fatigue syndrome, Creutzfeldt-Jakob disease, dissociative amnesia, fugue amnesia, Huntington's disease, learning disabilities, sleep disorders, multiple personality disorder, pain, post-traumatic stress disorder, schizophrenia, sports injuries, stroke, and Wechsler-Colombian syndrome), illnesses related to impaired impulse control and addiction, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other brain illnesses caused by trauma or other damage including aging.

[0032] In some embodiments of the present invention, the kidney disease may be focal segmental glomerulosclerosis, minimal change disease, or diabetic nephropathy.

[0033] The term "substitution" refers to the replacement of hydrogen atoms in a molecule with other different atoms or groups.

[0034] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a ~C b Alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, "C1 to C4 alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0035] In this invention, "alkyl" refers to a saturated hydrocarbon group having a specified number of carbon atoms. For example, C1-C8 alkyl refers to an alkyl group having 1 to 8 carbon atoms, preferably specifically 1 to 4 carbon atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups may also optionally be substituted by one or more substituents as defined herein. Specific examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0036] The "halogen" mentioned in this invention is fluorine, chlorine, bromine or iodine.

[0037] In this invention, "halogen substitution" refers to the substitution of a group by one or more halogen atoms, including partial substitution and complete substitution. For example, halogen-substituted C1-C4 alkyl refers to an alkyl group containing 1 to 4 carbon atoms that is substituted by one or more halogen atoms, such as trifluoromethyl, difluoromethyl, monofluoromethyl, etc.

[0038] In this invention, "cycloalkyl" refers to a saturated or partially saturated cyclic group having multiple carbon atoms, no heterocyclic atoms, and a single or multiple rings (including fused rings). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and polycyclic alkyl rings, such as dicyclopropyl, dicyclohexyl, dicyclopentyl, dicyclooctyl, etc., wherein the rings in the polycyclic alkyl ring can be attached to the same carbon atom, for example... It can also be bonded to different carbon atoms that are adjacent and / or spaced apart, for example

[0039] In this invention, "heteroaryl" refers to an aromatic unsaturated ring containing at least one heteroatom; where heteroatoms include nitrogen, oxygen, sulfur, etc. It typically refers to aromatic monocyclic or bicyclic hydrocarbons containing multiple ring atoms, one or more of which are selected from heteroatoms such as O, N, and S. Preferably, it has one to three heteroatoms. Examples of heteroaryl groups include: pyridyl, indolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothiopheneyl, benzofuranyl, benzothiopheneyl, benzopyranyl, benzothiapyranyl, furanyl, pyrroleyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazoleyl, thiopheneyl, oxadiazolyl, benzimidazoleyl, benzothiazolyl, and benzoxazolyl.

[0040] In the “6-10 member heteroaryl” mentioned in this invention, “member” indicates the number of atoms constituting the skeleton of the heteroaryl group.

[0041] The term "stereoisomer" includes enantiomers and diastereomers, as well as cis-trans isomers and tautomers.

[0042] The term "isotope-labeled derivative" refers to the compounds described in this invention, wherein one or more atoms are replaced or substituted by an atom whose atomic mass or mass number differs from that of atoms normally found in nature (i.e., naturally occurring).

[0043] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the prototype of such compounds with a sufficient amount of pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the prototype of such compounds with a sufficient amount of pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids, including but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, etc. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentian acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts.

[0044] The term "pharmaceutical excipients" refers to the excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. They are all substances contained in pharmaceutical preparations, excluding the active ingredient.

[0045] The term "solvent" refers to a complex of variable stoichiometry formed by a solute and a solvent. Such solvents used for the purposes of this invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water. Solvates of solvent molecules, such as water, are commonly referred to as "hydrates." Hydrates include compositions containing a stoichiometry of water, as well as compositions containing a variable amount of water.

[0046] The beneficial effects of this invention are:

[0047] This invention provides a compound of formula I, which exhibits good inhibitory effects on the TRPC5 ion channel and holds promise for treating various TRPC5-related diseases, such as cancer, depression, and chronic nephritis. Therefore, the compound of this invention can be used to prepare TRPC5 inhibitors and drugs for treating TRPC5-related progressive chronic kidney disease (CKD), demonstrating promising application prospects. Attached Figure Description

[0048] Figure 1 This describes the effect of compound 9 in Example 1 of the present invention on the channel current of TRPC5.

[0049] Figure 2 HE staining results of the kidney organoid constructed in Example 3 of this invention.

[0050] Figure 3 The IF results of the kidney organoid constructed in Example 3 of this invention; SYNPO (red), WT1 (green), scale bar 25 μm.

[0051] Figure 4 Statistical graph of qPCR results for the kidney organoid constructed in Example 3 of this invention. Detailed Implementation

[0052] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0053] The abbreviations used in the following specific experimental descriptions represent (unless otherwise stated) the following reagents:

[0054] Table 1

[0055] abbreviation compound DIPEA N,N-Diisopropylethylamine CDI N,N′-Carbonyldiimidazole DCM dichloromethane DIEA N,N-Diisopropylethylamine THF Tetrahydrofuran DMF N,N-Dimethylformamide PyBOP Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate

[0056] Example 1

[0057] In this embodiment, compound 9 was prepared, and the specific process is as follows:

[0058] Preparation of compound 2:

[0059]

[0060] At 24°C, NH₂OH was added to 100 mL of an ethanol solution (10.0 g, 67.97 mmol, 1.0 eq) of compound 1. .HCl (9.5 g, 135.93 mmol, 2.0 eq) and DIPEA (18.0 g, 139.33 mmol, 2.1 eq). The resulting mixture was stirred at 70 °C for 2 hours. Thin-layer chromatography (n-heptane / EtOAc S210 / 1) showed that compound 1 was consumed and formed a major spot. The mixture was added to 500 mL of water at 0 °C, and a white solid precipitated. The mixture was stirred at 0 °C for 0.5 hours. The mixture was filtered, and the precipitate was dried under vacuum at 45 °C to give a white solid compound 2 (11.1 g, 90.7%).

[0061] LC-MS[M+1] = 181.20.

[0062] 1 H NMR (400MHz, DMSO): δ9.52(s,1H),7.18(m,2H),6.90(d,1H),6.03(s,2H),5.73(s,2H).

[0063] Preparation of compound 5:

[0064]

[0065] At 0 °C, a solution of compound 4, p-toluidine (1.6 g, 14.96 mmol, 1.05 eq), in DCM was added dropwise to a solution of CDI (2.4 g, 14.96 mmol, 1.05 eq) in DCM (50 mL) for 0.5 hours to obtain a mixture. At 0 °C, DIEA (1.8 g, 14.25 mmol, 1.0 eq) and compound 3 (4.0 g, 14.25 mmol, 1.0 eq) were added to the above mixture. The mixture was stirred at 25 °C for 20 hours. Thin-layer chromatography (n-heptane:EtOAc = 1:1, R...) was performed. f =0.10) showed that compound 4 was consumed and formed a major spot. The mixture was added to HCl (50 mL, 0.5 M) solution at 0 °C and stirred at 25 °C for 0.5 h. The mixture was extracted with DCM (50 mL × 3). The organic layers were combined, washed with saturated NaHCO3 (50 mL) and brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to give the crude product. The crude product was purified by silica gel chromatography (n-heptane:EtOAc = 10–3:1) to give compound 5 (5.0 g, 93.0%) as a colorless resin.

[0066] LC-MS[2M+1]=755.80.

[0067] 1H NMR (400MHz, CDCl3): δ7.19(dd,2H),7.10(d,2H),6.68(d,1H),5.75(dd,1H),4.55(ddp,1H),4.30(ddd,1H),3.69(m,4H),3.46(ddd,1H),2.49(m 1H),2.30(s,3H),1.99(m,1H),1.43(d,9H).

[0068] Preparation of compound 6:

[0069]

[0070] At 25 °C, a solution of HCl (g) / EtOAc (20 mL, 4 M) was added dropwise to a DCM (20 mL) solution of compound 5 (5.0 g, 13.25 mmol, 1.0 eq). The mixture was stirred at 25 °C for 0.5 h. Thin-layer chromatography (n-heptane:EtOAc = 1:1) showed that compound 5 was consumed and formed a major spot. The mixture was concentrated under vacuum to give compound 6 (3.7 g, 89.0%) as a white solid.

[0071] LC-MS[M+1] = 278.30.

[0072] 1 H NMR (400MHz, CDCl3): δ8.89(s,1H),7.26(m,2H),7.02(d,2H),6.76(d,1H),4.47(t,1H),4.3 1(q,1H),3.78(s,3H),3.44(m,1H),3.14(m,1H),2.59(dt,1H),2.20(s,3H),1.99(ddd,1H).

[0073] Preparation of compound 7:

[0074]

[0075] DIPEA was added to a methanol (20 mL) solution of compound 6 (3.6 g, 11.47 mmol, 1.0 eq), and the solution was adjusted to pH 7.0 at 24 °C. An aqueous solution of formaldehyde (1.9 g, 22.95 mmol, 2.0 eq, 37%) was added to the mixture at 24 °C, and the solution was acidified to pH 5.0 with acetic acid. The mixture was stirred at 24 °C for 0.5 h. NaBH3CN (2.2 g, 34.42 mmol, 3.0 eq) was added to the mixture at 0 °C. The mixture was stirred at 24 °C for 19.5 h. LC-MS (CGo133170-01806-011-1) showed that compound 6 was consumed and the desired product was detected. The mixture was added to a hydrochloric acid solution (20 mL, 0.5 M) at 0 °C and stirred at 24 °C for 0.5 h. The mixture was extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (50 mL), dried with anhydrous Na₂SO₄, filtered, and concentrated under vacuum to give a crude product. The crude product was dispersed with n-heptane (50 mL), filtered, and the filter cake was dried under vacuum at 45 °C to give a white solid compound 7 (3.2 g, 95.7%).

[0076] LC-MS[M+1] = 292.40.

[0077] 1 H NMR (400MHz, CDCl3): δ7.21(d,2H),7.10(d,2H),6.89(s,1H),5.76(d,1H),4.41(m,1 H),3.72(s,3H),3.03(m,2H),2.58(ddt,2H),2.40(s,3H),2.31(s,3H),1.89(dd,1H).

[0078] Preparation of compound 8:

[0079]

[0080] At 0 °C, LiOH was added to a 40 mL THF solution of compound 7 (3.1 g, 10.64 mmol, 1.0 eq). . H2O solution (8 mL). The mixture was stirred at 0 °C for 2 hours. LCMS (CGo133170-01806-014-1) showed that compound 7 was consumed and the desired product was detected. The pH of the mixture was adjusted to 6.3 at 0 °C with an aqueous HCl (0.5 M) solution. The THF in the mixture was concentrated under vacuum, and the aqueous phase was lyophilized to give compound 8 (3.5 g, crude product) as a white solid.

[0081] LC-MS[M+1] = 278.30.

[0082] 1 H NMR (400MHz, CDCl3): δ9.42(s,1H),7.30(d,3H),6.98(d,2H),4.30(m,1H),3.60(t,1H) ),3.40(dd,1H),3.18(dd,1H),2.74(s,3H),2.61(dt,1H),2.18(s,3H),1.87(ddd,1H).

[0083] Preparation of compound 9:

[0084]

[0085] At 25°C, PyBOP (3.7 g, 11.01 mmol, 1.2 eq) and DIPEA (3.6 g, 27.54 mmol, 3.0 eq) were added to a DMF (50 mL) solution of compound 8 (3.5 g crude, 10.10 mmol, 1.0 eq). The mixture was stirred at 25°C for 0.5 h. A DMF (5 mL) solution of compound 2 (1.7 g, 9.18 mmol, 1.0 eq) was added dropwise to the above mixture at 25°C, and the mixture was stirred for 4 h. LCMS (CGo133170-01806-016-1) showed detection of the key intermediate, but 22.2% of compound 8 was retained. Therefore, PyBOP (1.0 g, 1.84 mmol, 0.2 eq) was added to the mixture at 25°C. The mixture was stirred at 25°C for 10.5 hours, and then the temperature was raised to 100°C and stirring continued for 5 hours. LCMS (CGo133170-01806-016-7) showed that the key intermediate was consumed and the major desired product was detected. 300 mL of water was added to the mixture at 25°C, resulting in the precipitation of a white solid. The mixture was stirred at 25°C for 0.5 hours. The mixture was filtered and resuspended in a solution (MeCN:H₂O = 2:1) at 25°C for 0.5 hours. The mixture was then filtered and lyophilized to give compound 9 (2.0 g, 43.2%) as a white solid.

[0086] LC-MS[M+19] = 440.40.

[0087] LC-MS[M+1] = 422.50.

[0088] 1H NMR (400MHz, CDCl3): δ8.41(s,1H),7.59(dd,1H),7.46(d,1H),7.24(d,2H),7.07(d,1H),7.01(d,2H),6.38(d,1H),6. 14(s,2H),4.27(s,1H),3.80(dd,1H),2.90(d,1H),2.74(m,1H),2.66(dd,1H),2.36(s,3H),2.20(s,3H),1.99(dd,1H).

[0089] Example 2

[0090] This embodiment tests the performance of compound 9 in inhibiting the TRPC5 channel. The specific process is as follows:

[0091] HEK293T cells transfected with TRPM8 and TRPC5 were harvested, the culture medium was discarded, and extracellular fluid was added. Extracellular fluid at 22°C was continuously perfused into the recording bath. The flow rate of the perfusion solution was controlled using a peristaltic pump perfusion system, maintaining a flow rate of 2 mL / min. Suitable target cells were located under a microscope; typically, single, plump cells with clear edges and no depressions were selected. Pre-fabricated glass microelectrodes with an infusion impedance of 2–2.5 MΩ were filled with electrode fluid. The glass electrode was moved by micromanipulation, while the gas pressure inside the glass electrode was controlled using a syringe to establish a high-resistance (GΩ) seal between the glass electrode opening and the cell membrane. Negative pressure was continued to be applied to the glass electrode to rupture the cell membrane, thus allowing communication between the intracellular fluid and the glass electrode. The intracellular fluid and the glass electrode fluid could exchange. An amplifier connected to the glass electrode (HEKA EPC10 amplifier, with accompanying patchmaster recording software) could control the intracellular voltage. Different voltage settings allowed for the recording of whole-cell channel currents.

[0092] The extracellular fluid formulation used when recording TRPC5 was as follows: 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM D-glucose, dissolved in deionized water, pH adjusted to 7.4 with NaOH, and the measured osmotic pressure was approximately 319 mOsm.

[0093] The electrode internal solution formula is as follows: 130mM CsCl, 1mM MgCl2, 10mM CaCl2, 10mM EGTA, 10mM HEPES, dissolved in deionized water, and the pH is adjusted to 7.4 using CsOH, with an osmotic pressure of 313mOsm.

[0094] While maintaining the cell voltage at 0mV, the cells were given a voltage ramp stimulation of -100mV to +100mV for 1s, with stimulation occurring every 10s. The channel current was continuously recorded at a sampling frequency of 50kHz. A low-pass filter of 2kHz was used to filter out high-frequency interference noise. Drug administration was started after the TRPC5 current stabilized.

[0095] Riluzole (working concentration 50 μM) was added directly to the extracellular fluid, and the current was recorded. Then, compound 9 (working concentration 10 μM) was added, and the current change was recorded. The results are as follows: Figure 1 As shown.

[0096] It can be seen that after riluzole activates TRPC5, compound 9 can inhibit the TRPC5 channel current.

[0097] Example 3

[0098] This embodiment tests the intervention effect of compound 9 on podocyte injury, and the specific process is as follows:

[0099] Construction of kidney organoid models:

[0100] Kidney organoids can be prepared using the method described in the reference Takasato Minoru; Little, Melissa H. A strategy for generating kidney organoids: Recapitulating the development in human pluripotent stem cells[J]. Developmental Biology. 2016, Vol. 420(No. 2): 210-220. (DOI: 10.1016 / j.ydbio.2016.08.024). Alternatively, they can be prepared using the method described in CN2024114246833.

[0101] Human pluripotent stem cells were cultured in PSC medium (ThermoFisher) on a track shaker at a speed of 40–80 rpm. TMAfter cell clusters formed during suspension culture, the cell clusters were transferred to APEL2 medium containing CHIR99021 (working concentration 2 μM) and cultured for 6 days. Then, the cell clusters were cultured in APEL2 medium containing FGF9 (100 ng / mL) and heparin (1 μg / mL) for another 2 days. After culturing the cell clusters in APEL2 medium containing CHIR99021 (2 μM) for 1.5 hours, they were cultured in APEL2 medium containing FGF9 (100 ng / mL) and heparin (0.5 μg / mL) for another 5 days. Finally, the APEL2 medium was replaced, and the cell clusters were cultured in suspension for another 5 days to obtain a kidney organoid model.

[0102] Constructing a kidney organoid model of podocyte injury: Kidney organoids were transferred to ultra-low adsorption 96-well plates (U-bottom), with one kidney organoid added to each well. The control group (blank group) received 200 μL of APEL2 medium per well, while the modeling group (PAN group) received the same volume of APEL2 medium containing 10–30 μM puromycin-aminonucleotide (PAN, catalog number: A1113803). The kidney organoids were cultured for 72 h.

[0103] Compound 9 intervention for podocyte injury: After 72 hours, some of the PAN groups were replaced with APEL2 medium containing compound 9 (working concentration of 10-50 μM) to become drug intervention groups (PAN+C9 groups); the blank group and some of the PAN groups were replaced with fresh APEL2 medium, and all three groups continued to culture kidney organoids for 72 hours.

[0104] HE (hexylene-eosin) staining test

[0105] Paraffin sections were prepared from kidney organoids from the aforementioned blank, PAN, and PAN+C9 groups. Staining was then performed according to the instructions for the H&E Staining Kit (Hematoxylin and Eosin).

[0106] The results show that: Figure 2 As shown in the HE staining images, compared with the blank group, the PAN group showed significant features of glomerular cluster contraction, fluid cavity swelling, and podocyte contraction and adhesion. However, the PAN+C9 group, which was treated with compound 9, reversed this and restored glomerular morphology close to that of the blank group.

[0107] Immunofluorescence assay 1

[0108] Paraffin sections were prepared from the three kidney organoids (blank group, PAN group, and PAN+C9 group) and subsequently subjected to immunofluorescence analysis.

[0109] ① Place the slices in a 60℃ oven for 1.5 hours.

[0110] ② Place the slices into xylene I, II, and III in sequence, and soak each for 10 minutes.

[0111] ③ Place the slices in 100% alcohol for 10 minutes, then soak them in 95%, 80%, and 70% alcohol and pure water for 5 minutes in sequence.

[0112] ④ Dilute the antigen retrieval solution according to the ratio, put the slide into the retrieval solution, put it in the microwave oven, and microwave on medium for 8 minutes, turn off the heat for 8 minutes, and then microwave on medium-low for 7 minutes.

[0113] ⑤ After the antigen retrieval solution soaked in the tissue sections has cooled to room temperature, remove the sections, rinse them in PBS, dry them, and circle the tissue sections with an immunohistochemical pen.

[0114] ⑥ Add 10% donkey serum + 0.1% Triton X-100 blocking membrane-breaking solution to the circle and incubate at room temperature for 30 minutes.

[0115] ⑦ Discard the blocking solution, add the primary antibody diluted according to the ratio (anti-SYNPO antibody, Abcam, ab22491, 1:100; WT1-AF488, 1:250), and incubate overnight at 4°C.

[0116] ⑧ The next day, soak the slides in PBS and wash them on a shaker for 15 minutes.

[0117] 9. Remove the slide, add the secondary antibody diluted according to the specified ratio, and incubate at room temperature in the dark for 1 hour. (Goat anti-RabbitIgG594, invitrogen, A11012, 1:1000)

[0118] ⑩ Discard the secondary antibody, add 1 μg / mL DAPI (Sigma Aldrich), and stain the nuclei for 5 minutes.

[0119] Discard the DAPI, immerse the slides in PBS, and wash on a shaker for 15 minutes.

[0120] Remove the slide, wipe the area around the tissue dry, add mounting medium, and cover with a coverslip.

[0121] Microscopic examination.

[0122] The results show that: Figure 3 As shown in the IF plot, the abundance of SYNPO, a synaptic protein in podocytes, was decreased in the PAN group, while the expression level of the podocyte transcription factor WT1 was not decreased, indicating that PAN causes changes in the cytoskeleton structure of podocytes but does not lead to cell loss. Treatment with compound 9 in the PAN+C9 group successfully restored PAN-induced synaptic protein depletion.

[0123] Immunofluorescence assay 2

[0124] Kidney organoids from the blank, PAN, and PAN+C9 groups were collected using EP tubes and washed once with DPBS. 1 mL of trizol (ThermoFisher) was added to dissolve the cells. 200 μL of chloroform was added, vortexed for 15 seconds, and allowed to stand for 2 minutes. The cells were centrifuged at 4°C for 12,000 rpm for 15 minutes, and the supernatant was collected. An equal volume of isopropanol was added to the supernatant, and the mixture was inverted to mix. The mixture was incubated at -20°C for 10 minutes. After centrifugation at 4°C for 12,000 rpm, the supernatant was discarded. 500 μL of pre-chilled anhydrous ethanol was added to the precipitate, and the precipitate was dispersed to wash the RNA. The washings were repeated twice, and the precipitate was centrifuged at 4°C for 12,000 rpm, after which the supernatant was discarded. The precipitate was dried at room temperature for approximately 5 minutes. 20 μL of DPEC water was added to the centrifuge tube to dissolve the RNA. cDNA was synthesized using the RevertAid FirstStrand cDNA Synthesis Kit according to the manufacturer's instructions. The cDNA was used for RT-qPCR detection.

[0125] like Figure 4 The results showed that the PAN group damaged podocytes, reduced the expression level of the synaptic protein SYNPO, and slightly decreased the expression level of the podocyte marker NPHS2, but did not reduce the expression level of WT1. The drug intervention with compound 9 of this invention preserved podocyte cytoskeletal proteins.

[0126] In summary, combined with HE staining structure, immunofluorescence, and RT-qPCR data, it is shown that compound 9, which inhibits TRPC5 channel activity, can protect podocytes from PAN-induced podocyte damage in an in vitro three-dimensional organoid model, thereby preventing renal failure and treating kidney injury.

[0127] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a compound of Formula I: ###0001### Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a TRPC5 inhibitor: said compound of Formula I is selected from the following compounds: 。 2. Use according to claim 1, characterized in that: said TRPC5 inhibitor is an inhibitor for use in the treatment, prevention or prophylaxis of a TRPC5 mediated disease.

3. Use according to claim 2, characterized in that: said TRPC5 mediated disease is a psychiatric disorder, a neurological disorder, a neurodegenerative disorder or a kidney disease.

2. A method of treating, preventing or prophylaxis of a TRPC5 mediated disease comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula I: ###0002### Formula I or a pharmaceutically acceptable salt thereof. said TRPC5 mediated disease is a psychiatric disorder, a neurological disorder, a neurodegenerative disorder or a kidney disease.

3. A pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula I: ###0003### Formula I or

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