TRPC5 inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a TRPC5 inhibitor and a preparation method and application thereof, the TRPC5 inhibitor comprises a compound of formula I, a stereoisomer of the compound, an isotope labeled derivative of the compound, a pharmaceutically acceptable salt of the compound, a prodrug of the compound or a solvate # imgabs0 of the compound, and the compound has a good inhibition effect on a TRPC5 ion channel and is expected to be used for treating various diseases related to TRPC5. The symptoms comprise cancer, depression, chronic nephritis and the like.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a TRPC5 inhibitor and a preparation method and application thereof. Background Art

[0002] Progressive chronic kidney disease (CKD) is associated with an increased risk of renal failure, and its prevalence is increasing rapidly, with more than 850 million people worldwide currently suffering from CKD. Despite the increasing number of people, the treatment options available to slow or prevent disease progression remain limited. Nephrotic syndrome is an important driver of CKD. Nephrotic syndrome is characterized by a large amount of albumin spilling into the urine, which is the result of damage to the kidney's filtering unit, the glomerulus. The intact kidney filter is composed of endothelial cells, basement membrane and podocytes, which are essential for retaining proteins in the blood and removing waste from the body. Many chronic kidney diseases are associated with damage and loss of podocytes, and the process of podocyte damage and necrosis is difficult to block. Once necrosis occurs, it can never be regenerated. Studies have shown that podocyte damage and loss are caused by a gene mutation that can induce activation of TRPC5 ion channels in podocytes and remodeling of the cytoskeleton. Podocyte damage and the appearance of proteinuria are key features of CKD. In rodent models of kidney disease, inhibition of the TRPC5 ion channel protects podocytes, the renal filtrate that cannot be renewed once lost. Due to their limited proliferative capacity, podocytes are particularly susceptible to various stimuli leading to injury. Therefore, preventing podocyte injury remains a key goal for the development of effective, targeted therapeutic strategies for kidney disease.

[0003] TRPC5 inhibitors refer to substances that can directly activate TRPC5 channels, causing cell membrane depolarization and increased intracytoplasmic calcium concentration. TRPC5 inhibitors can be used to treat tumors, reverse tumor multidrug resistance, and can also be used for antidepression. The latest research also shows that as an ion channel protein, TRPC5 can be activated by a variety of renal stressors. TRPC5 inhibitors may be able to prevent the destruction of renal filtration function and may become the basis for the development of urgently needed drugs for the treatment of progressive chronic kidney disease (CKD). Among them, the study found that a specific and selective TRPC5 inhibitor AC1903 can protect podocytes in the kidneys and prevent renal failure, so that renal function can be restored, indicating that the TRPC5 inhibitor AC1903 is expected to be used to treat chronic nephritis and find a new treatment for patients with chronic nephritis. However, the effect of AC1903 needs to be further improved. Therefore, new drugs or methods are needed to more effectively treat kidney disease or reduce the risk of manifest kidney disease. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a TRPC5 inhibitor and a preparation method and application thereof.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] In one aspect of the present invention, there is provided a compound of formula I, a stereoisomer thereof, an isotope-labeled derivative thereof, a pharmaceutically acceptable salt thereof, a prodrug thereof or a solvate thereof:

[0007]

[0008] Wherein, R1 is selected from a 6- to 10-membered heteroaryl group, wherein the heteroaryl group contains at least one heteroatom of O, N, or S;

[0009] R2 is selected from H, C1-C6 alkyl, C1-C6 cycloalkyl; the alkyl and cycloalkyl are optionally substituted by 0-3 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] The second aspect of the present invention provides a compound of formula II and a pharmaceutically acceptable salt thereof:

[0013]

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

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

[0016] In some embodiments of the present invention, the alkoxycarbonyl group includes benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (BOC), tert-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 present 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 Reaction to obtain the compound of formula I;

[0021] Wherein, the definition of R1 is as described above.

[0022] In some embodiments of the present invention, the method for preparing the compound of formula II comprises the following steps:

[0023] Compounds of formula IV Reaction with p-toluidine to obtain the compound of formula II;

[0024] Wherein, the definitions of R3 and R4 are as described above.

[0025] Another aspect of the present invention provides a pharmaceutical composition comprising the compound of formula I, its stereoisomers, its isotope-labeled derivatives, its pharmaceutically acceptable salts, its prodrugs or solvates thereof, and optionally, pharmaceutically acceptable excipients.

[0026] The pharmaceutical composition of the present invention is suitable for a variety of administration routes, and can thus be prepared into any pharmaceutically acceptable dosage form. For example, the pharmaceutical composition can be administered to patients or subjects who need such treatment by oral, parenteral, rectal or transpulmonary administration. When used for oral administration, the pharmaceutical composition can be prepared into oral preparations, for example, conventional oral solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be prepared into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. When preparing oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. can be added. When used for parenteral administration, the pharmaceutical composition can also be prepared into injections, including injections, sterile powders for injection, and concentrated solutions for injection. When preparing injections, conventional methods in the existing pharmaceutical field can be used for production. When preparing injections, additives can be omitted or appropriate additives can be added according to the properties of the drug. When used for rectal administration, the pharmaceutical composition can be prepared into suppositories, etc. When used for transpulmonary administration, the pharmaceutical composition can be prepared into inhalation preparations, aerosols, powder sprays or sprays, etc.

[0027] Pharmaceutically acceptable adjuvants refer to substances that are non-toxic, compatible with active ingredients, and otherwise biologically suitable for organisms. The selection of specific adjuvants will depend on the mode of administration or the type and state of disease used to treat a specific patient. Examples of pharmaceutically acceptable adjuvants include, but are not limited to, conventional solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants, etc. in the pharmaceutical composition. If necessary, flavoring agents, preservatives, and sweeteners, etc., may also be added to the pharmaceutical composition.

[0028] Another aspect of the present invention provides a compound of formula I, a stereoisomer thereof, an isotope-labeled derivative thereof, a pharmaceutically acceptable salt thereof, a prodrug thereof or a solvate thereof, and / or a use of the pharmaceutical composition in the preparation of a TRPC5 inhibitor.

[0029] In some examples of the present invention, the TRPC5 inhibitor is an inhibitor used for treating, preventing or controlling TRPC5-mediated diseases.

[0030] In some examples of the present invention, the TRPC5-mediated disease is a psychiatric disorder, a neurological disorder, a neurodegenerative disorder or a renal disease.

[0031] In some examples of the present invention, the psychiatric disorder, neurological disorder or neurodegenerative disorder can be selected from: diseases associated with dysregulated emotional processing (e.g., borderline personality disorder or depression, such as major depression, major depressive disorder, dysthymia, dysthymia and postpartum depression, and bipolar disorder), disorders associated with 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 disorders, sleep disorders, multiple personality disorder, pain, post-traumatic stress disorder, schizophrenia, sports injuries, stroke and Weil-Cohen syndrome), disorders associated with impaired impulse control and addiction, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other brain disorders caused by trauma or other injuries including aging.

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

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

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

[0035] The "alkyl" described in the present invention 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, for example, preferably 1 to 4 carbon atoms. The alkyl group can be straight chain or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may also be optionally substituted with one or more substituents as defined herein. Specific examples of alkyl 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 the present invention is fluorine, chlorine, bromine or iodine.

[0037] The "halogen substitution" mentioned in the present invention means that the substituted group may be substituted by one or more halogen atoms, including partial substitution and full substitution. For example, the halogen-substituted C1-C4 alkyl group refers to an alkyl group containing 1 to 4 carbon atoms substituted by one or more halogen atoms, such as trifluoromethyl, difluoromethyl, monofluoromethyl, etc.

[0038] The "cycloalkyl" described in the present invention refers to a saturated or partially saturated cyclic group having multiple carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including condensed). Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl, cyclohexenyl, and polycyclic alkyl rings, such as bicyclopropyl, bicyclohexyl, bicyclopentyl, bicyclooctyl, etc., wherein each ring in the polycyclic alkyl ring can be connected to the same carbon atom, for example They can also be connected to adjacent and / or spaced carbon atoms, for example

[0039] The "heteroaryl" described in the present invention refers to an aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, a sulfur atom, etc. It usually refers to an aromatic monocyclic or bicyclic hydrocarbon containing multiple ring atoms, wherein one or more ring atoms are selected from heteroatoms such as O, N, S, etc. Preferably, there are one to three heteroatoms. Examples of heteroaryl groups include: pyridyl, indolyl, quinoxalyl, quinolyl, isoquinolyl, benzothienyl, benzofuranyl, benzothienyl, benzopyranyl, benzothiapyranyl, furanyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, oxadiazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl.

[0040] In the "6- to 10-membered heteroaryl group" described in the present invention, "membered" means the number of atoms constituting the skeleton of the heteroaryl group.

[0041] The term "stereoisomer" includes enantiomers and diastereomers, and also includes cis-trans isomers and tautomers and the like.

[0042] The term "isotopically labeled derivative" refers to compounds described herein wherein one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (ie, naturally occurring).

[0043] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a relatively nontoxic, pharmaceutically acceptable acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the prototype of such compound with a sufficient amount of a 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 compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting the prototype of such compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes an inorganic acid, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acid includes organic acids, including but not limited to acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic 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 excipients and additives used in the production of drugs and preparation of prescriptions. They are all substances contained in pharmaceutical preparations except the active ingredients.

[0045] The term "solvate" refers to a complex of variable stoichiometry formed by a solute and a solvent. Such solvents for the purposes of the present invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water. Solvates in which water is a solvent molecule are commonly referred to as "hydrates." Hydrates include compositions containing stoichiometric amounts of water, as well as compositions containing variable amounts of water.

[0046] The beneficial effects of the present invention are:

[0047] The present invention provides a compound of formula I, which has a good inhibitory effect on TRPC5 ion channels and is expected to be used to treat various diseases related to TRPC5, such as cancer, depression, chronic nephritis, etc. Therefore, the compound of the present invention can be used to prepare TRPC5 inhibitors and drugs for treating progressive chronic kidney disease (CKD) related to TRPC5, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0049] Figure 2 This is the HE staining result of the kidney organoid constructed in Example 3 of the present invention.

[0050] Figure 3 IF results of kidney organoids constructed in Example 3 of the present invention; foot contact protein SYNPO (red), podocyte transcription factor WT1 (green), scale 25 μm.

[0051] Figure 4 Statistical graph of qPCR results of kidney organoids constructed in Example 3 of the present invention. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0053] The abbreviations used in the following specific experimental descriptions represent (unless otherwise specified) 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-oxytripyrrolidinophosphonium hexafluorophosphate

[0056] Example 1

[0057] In this example, compound 9 was prepared by the following process:

[0058] Preparation of compound 2:

[0059]

[0060] To a solution of compound 1 (10.0 g, 67.97 mmol, 1.0 eq) in ethanol (100 mL) was added NH2OH at 24°C. .HCl (9.5 g, 135.93 mmol, 2.0 eq) and DIPEA (18.0 g, 139.33 mmol, 2.1 eq). The above obtained 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 to form a white solid. The mixture was stirred at 0 ° C for 0.5 hours. The mixture was filtered and the precipitate was dried in vacuo at 45 ° C to obtain 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 CDI (2.4 g, 14.96 mmol, 1.05 eq) in DCM (50 mL) was added dropwise for 0.5 hours to obtain a mixture. 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 at 0°C. The mixture was stirred at 25°C for 20 hours. Thin layer chromatography (n-heptane: EtOAc = 1: 1, R f =0.10) showed that compound 4 was consumed and formed a major spot. The mixture was added to a 0°C HCl (50 mL, 0.5 M) solution and stirred at 25°C for 0.5 hours. 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 in vacuo to give a 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 gum.

[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 compound 5 (5.0 g, 13.25 mmol, 1.0 eq) in DCM (20 mL) was added dropwise with HCl (g) / EtOAc (20 mL, 4 M) solution. The mixture was stirred at 25°C for 0.5 hours. Thin layer chromatography (n-heptane: EtOAc = 1: 1) showed that compound 5 was consumed and formed a major spot. The mixture was concentrated in vacuo 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 solution of compound 6 (3.6 g, 11.47 mmol, 1.0 eq) in methanol (20 mL), and the solution was adjusted to pH = 7.0 at 24 ° C. At 24 ° C, an aqueous solution of formaldehyde (1.9 g, 22.95 mmol, 2.0 eq, 37%) was added to the mixture and acidified to pH = 5.0 with acetic acid. The mixture was stirred at 24 ° C for 0.5 hours. 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 hours. 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 hours. The mixture was extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give a crude product. The crude product was dispersed with n-heptane (50 mL), filtered, and the filter cake was dried in vacuo 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] To a solution of compound 7 (3.1 g, 10.64 mmol, 1.0 eq) in THF (40 mL) was added LiOH at 0°C. . 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 by HCl (0.5 M) aqueous solution at 0°C. THF in the mixture was concentrated in vacuo, and the aqueous phase was lyophilized to give compound 8 (3.5 g, crude) 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] 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 solution of compound 8 (3.5 g crude, 10.10 mmol, 1.0 eq) in DMF (50 mL) at 25°C. The mixture was stirred at 25°C for 0.5 h. A solution of compound 2 (1.7 g, 9.18 mmol, 1.0 eq) in DMF (5 mL) was added dropwise to the above mixture at 25°C and stirred for 4 h. LCMS (CGo133170-01806-016-1) showed that a key intermediate was detected, but 22.2% of compound 8 was retained. Therefore, at 25°C, PyBOP (1.0 g, 1.84 mmol, 0.2 eq) was added to the mixture. The mixture was stirred at 25°C for 10.5 hours, and the temperature was raised to 100°C and stirring was continued for 5 hours. LCMS (CGo133170-01806-016-7) showed that the key intermediate was consumed and the main desired product was detected. 300 mL of water was added to the mixture at 25°C to form a white solid precipitate. The mixture was stirred at 25°C for 0.5 hours. The mixture was filtered and resuspended with a solution (MeCN:H2O=2:1) ​​at 25°C for 0.5 hours. The mixture was filtered and lyophilized to give a white solid compound 9 (2.0 g, 43.2%).

[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 example tests the performance of compound 9 in inhibiting TRPC5 channels, and the specific process is as follows:

[0091] Take the HEK293T cells transfected with TRPM8 and TRPC5, discard the culture medium, add extracellular solution, and continuously perfuse the recording bath with 22°C extracellular solution. Use a peristaltic pump perfusion system to control the flow rate of the perfusion solution, and control the flow rate to 2mL / min. Find the appropriate target cell under the microscope. Usually select a single plump cell with clear edges and no depressions. Pour the electrode solution into the glass microelectrode with a pre-made liquid impedance of 2-2.5MΩ. Move the glass electrode by micromanipulation. At the same time, control the air pressure in the glass electrode with a syringe to establish a high-resistance (GΩ) seal between the glass electrode opening and the cell membrane. Continue to apply negative pressure to the glass electrode to rupture the cell membrane so that the cell is connected to the glass electrode, and the intracellular solution and the glass electrode solution can be exchanged. The amplifier connected to the glass electrode (HEKA EPC10 amplifier, supporting patchmaster recording software) can control the intracellular voltage, and set different voltages to record the channel current of the whole cell.

[0092] The formula of the extracellular solution 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 using NaOH, and the measured osmotic pressure was approximately 319 mOsm;

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

[0094] While keeping the cell voltage at 0 mV, give the cell a voltage ramp stimulation of -100 mV to +100 mV. The stimulation duration is 1 s, and the stimulation is once every 10 s. The channel current is continuously recorded with a sampling frequency of 50 KHz. A low-pass filter of 2 KHz is used to filter out high-frequency interference noise. After the TRPC5 current stabilizes, the drug is administered.

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

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

[0097] Example 3

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

[0099] Construction of kidney organoid model:

[0100] Kidney organoids can be prepared by referring to the method of 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). They can also be prepared by referring to the method of CN2024114246833.

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

[0102] Construction of a renal organoid model of podocyte injury: The renal organoids were transferred to an ultra-low attachment 96-well plate (U-bottom), and one renal organoid was added to each well. In the control group (blank group), 200 μL APEL2 medium was added to each well, and the modeling group (PAN group) was added with the same volume of APEL2 medium containing 10-30 μM puromycin-aminonucleoside (PAN, Catalog No.: A1113803), and the renal organoids were cultured for 72 h.

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

[0104] HE (Hydroxylin-Eosin) staining test

[0105] Kidney organoids from the blank group, PAN group, and PAN+C9 group were prepared into paraffin sections, and then stained according to the instructions of the H&EStaining 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 characteristics of glomerular cluster contraction, fluid cavity enlargement, and podocyte contraction and adhesion, while the PAN+C9 group administered with compound 9 reversed this situation and restored the glomerular morphology close to that of the blank group.

[0107] Immunofluorescence test 1

[0108] Paraffin sections were prepared from the three groups of kidney organoids in the blank group, PAN group, and PAN+C9 group, and then immunofluorescence was performed:

[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 turn, soaking for 10 minutes each.

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

[0112] ④ Dilute the antigen repair solution in proportion, put the slices into the repair solution, put them into the microwave, and cook on medium heat for 8 minutes, off heat for 8 minutes, and on medium-low heat for 7 minutes.

[0113] ⑤ After the antigen repair solution used to soak the slices cools down to room temperature, take out the slices, rinse them in PBS, wipe them dry, and circle the tissue slices with an immunohistochemistry pen.

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

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

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

[0117] ⑨Take out the slices, add the secondary antibody diluted in proportion, and incubate at room temperature in the dark for 1 hour. (Goat anti-RabbitIgG594, invitrogen, A11012, 1:1000)

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

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

[0120] Take out the slices, wipe the area around the tissue dry, add a drop of sealing agent, and seal the slices with a coverslip.

[0121] Microscopic examination.

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

[0123] Immunofluorescence test 2

[0124] Collect the kidney organoids of the blank group, PAN group and PAN+C9 group mentioned above with EP tubes and wash them once with DPBS. Add 1mL trizol (ThermoFisher) to blow away and dissolve the cells. Add 200μL chloroform, shake for 15 seconds, and let stand for 2 minutes. Centrifuge at 4℃, 12000rpm×15 minutes, and take the supernatant. Add the same volume of isopropanol to the supernatant and mix it upside down. Place at -20℃ for 10 minutes. Centrifuge at 12000rpm at 4℃ for 10 minutes and discard the supernatant. Add 500μL pre-cooled anhydrous ethanol to the precipitate, blow away the precipitate to wash RNA, wash twice, centrifuge at 12000rpm at 4℃ and discard the supernatant. Dry the precipitate at room temperature for about 5 minutes. Add 20μL DPEC water to the centrifuge tube to dissolve the RNA. Synthesize cDNA with RevertAid FirstStrand cDNA SynthesisKit according to the instructions, and cDNA is used for RT-qPCR detection.

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

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

[0127] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A compound of formula I, its stereoisomer, its isotope-labeled derivative, its pharmaceutically acceptable salt, its prodrug or its solvate: in, R1 is selected from a 6- to 10-membered heteroaryl group, wherein the heteroaryl group contains at least one heteroatom of O, N, or S; R2 is selected from H, C1-C6 alkyl, C1-C6 cycloalkyl; the alkyl and cycloalkyl are optionally substituted by 0-3 halogens.

2. The compound of formula I according to claim 1, its stereoisomer, its isotope-labeled derivative, its pharmaceutically acceptable salt, its prodrug or its solvate, characterized in that: Selected from 3. The compound of formula I according to claim 1, its stereoisomer, its isotope-labeled derivative, its pharmaceutically acceptable salt, its prodrug or its solvate, characterized in that: The compound of formula I is selected from the following compounds:

4. Compounds of formula II and pharmaceutically acceptable salts thereof: in, R3 is selected from -C(O)OR5; R5 is selected from H, C1-C6 alkyl, C1-C6 cycloalkyl; R4 is selected from H, C1~C6 alkyl, C1~C6 cycloalkyl, alkoxycarbonyl, or N-R4 forms an amino salt.

5. Use of the compound of formula II according to claim 4 in the preparation of the compound of formula I according to any one of claims 1 to 3, its stereoisomers, its isotope-labeled derivatives, its pharmaceutically acceptable salts, its prodrugs or solvates thereof.

6. A method for preparing the compound of formula I according to any one of claims 1 to 3, its stereoisomers, its isotope-labeled derivatives, its pharmaceutically acceptable salts, its prodrugs or its solvates, characterized in that: The following steps are involved: The compound of formula II according to claim 4 and the compound of formula III Reaction to obtain the compound of formula I; wherein R1 is defined as described in claims 1 to 3.

7. A pharmaceutical composition, characterized in that: The invention comprises the compound of formula I according to any one of claims 1 to 3, its stereoisomer, its isotope-labeled derivative, its pharmaceutically acceptable salt, its prodrug or its solvate, and optionally, a pharmaceutically acceptable excipient.

8. Use of the compound of formula I according to any one of claims 1 to 3, its stereoisomers, isotope-labeled derivatives, pharmaceutically acceptable salts, prodrugs or solvates thereof, and / or the pharmaceutical composition according to claim 7 in the preparation of a TRPC5 inhibitor.

9. The use according to claim 8, characterized in that: The TRPC5 inhibitor is an inhibitor used for treating and preventing diseases mediated by TRPC5.

10. The use according to claim 9, characterized in that: The TRPC5-mediated disease is a psychiatric disorder, a neurological disorder, a neurodegenerative disorder or a renal disease.

Images