Cyclohexane derivatives, pharmaceutical compositions thereof and uses thereof

Abstract

This invention provides a cyclohexane derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the structure of the cyclohexane derivative is shown in compound (1) or compound (2) below: The cyclohexane derivative, its stereoisomer, or a pharmaceutically acceptable salt thereof provided by this invention can act as D1, D3 receptors and / or 5-HT receptors. 1A These receptor modulators are used to treat neuropsychiatric disorders and have relatively low toxicity.

Description

Cyclohexane derivatives, pharmaceutical compositions and applications thereof Technical Field

[0001] The present invention relates to cyclohexane derivatives, stereoisomers or pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions and applications containing these compounds. Background Art

[0002] G protein-coupled receptors (GPCRs) are the largest family of membrane proteins in the mammalian genome. They are widely distributed in organs and tissues such as the central nervous system, immune system, cardiovascular system, and retina, and are involved in the development and normal function of the body. Among the numerous GPCR receptors, dopamine receptors and serotonin receptors are successful drug targets, and many drugs, especially psychiatric drugs, are developed based on them.

[0003] Dopamine receptors are divided into five subtypes: D1, D2, D3, D4, and D5. They are very important drug targets in neurological diseases. Marketed drugs such as olanzapine, aripiprazole, and cariprazine all target dopamine D2 receptors. Patent WO2007148208A2 discloses novel carbonyl (aza)cyclohexane compounds as dopamine D3 receptor ligands for the treatment of neuropsychiatric diseases, cardiovascular diseases, movement disorders, hormonal disorders, and other diseases; Patent WO1996002520A1 discloses triazole compounds for the treatment of diseases that respond to dopamine D3 receptor ligands; Patent CN105461608B discloses dihydroindole-2-one D3 receptor ligands, which have strong activity on dopamine D3 receptors and are used to treat or prevent central nervous system psychiatric diseases such as schizophrenia, Parkinson's disease, drug dependence, and relapse. Dopamine D1 receptors are also an important target for antipsychotic drugs. For example, literature reports that antipsychotic drugs that block or weaken D1-induced neural activity will effectively treat negative schizophrenia symptoms (PMID: 1355300).

[0004] Serotonin receptors are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) found in the central and peripheral nervous systems. Serotonin receptors are targets of a variety of drugs, including many antidepressants, antipsychotics, anorexigenics, etc. Patent WO1999052907A1 discloses heterobicyclic compounds as serotonin receptor modulators for the treatment of Parkinson's disease, schizophrenia, endocrine diseases, headaches, hypertension and other diseases. Patent CN101903021B discloses α-aminoamide derivatives as serotonin receptor modulators for the treatment of psychosis. 5-HT 1AReceptors also play an important role in the treatment of schizophrenia (CNS Neurosci Ther. 2011 Feb; 17(1): 58-65.), such as 5-HT 1A Local agonism of receptors may reduce motor side effects and improve depression, emotion, negativity and cognitive symptoms by enhancing downstream dopamine release.

[0005] Patent WO2017045599A1 discloses a cyclohexane derivative or a stereoisomer thereof. Although the disclosed compound is used to treat or prevent neuropsychiatric diseases, there are still some shortcomings, and more effective drugs need to be developed to achieve better treatment of the disease.

[0006] Summary of the Invention

[0007] The present invention provides a cyclohexane derivative, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The structure of the cyclohexane derivative is shown in the following formula: Compound 1 or Compound 2:

[0008] Optionally, the cyclohexane derivative, its stereoisomer or pharmaceutically acceptable salt thereof is not Compound 1 in free form.

[0009] In some embodiments, the structure of the cyclohexane derivative is shown in the following formula:

[0010] In some embodiments, the present invention provides a method for preparing the above-mentioned cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that the method comprises:

[0011] The cyclohexane derivative represented by formula a reacts with a carbonyl source in an organic solvent to obtain a cyclohexane derivative of compound 1 or compound 2.

[0012] or,

[0013] The cyclohexane derivative represented by formula a reacts with a carbonyl source in an organic solvent, and then reacts with ammonia or methylamine to obtain a cyclohexane derivative of compound 1 or compound 2.

[0014] In some embodiments, the carbonyl source is selected from one or more of carbonyl dibenzotriazole, carbonyl dihydroxybenzotriazole, carbonyl di-1,2,4-triazole, potassium cyanate, phosgene, triphosgene, formaldehyde, and acetaldehyde.

[0015] In some embodiments, the present invention provides a pharmaceutical composition comprising the above-mentioned cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0016] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0017] In some embodiments, the above-mentioned cyclohexane derivatives, stereoisomers thereof or pharmaceutically acceptable salts thereof are used in the preparation of a method for treating or preventing a disease associated with dopamine receptors (e.g., D1, D2 (D 2L and / or D 2S ) or D3, etc.) related diseases in medicine.

[0018] The present invention provides the use of the above-mentioned cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a dopamine D1 receptor antagonist.

[0019] The present invention provides the use of the above-mentioned cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a dopamine D3 receptor agonist or antagonist.

[0020] The present invention provides the above-mentioned cyclohexane derivatives, their stereoisomers or pharmaceutically acceptable salts thereof for use in the preparation of a method for treating or preventing diseases related to 5-hydroxytryptamine receptors (5-HT 1A , 5-HT 2A or 5-HT 2B etc.) related diseases.

[0021] The present invention provides the above-mentioned cyclohexane derivatives, their stereoisomers or pharmaceutically acceptable salts thereof for use in the preparation of a compound for treating or preventing 5-HT 1A receptors, 5-HT 2A receptors and / or 5-HT 2B Application of drugs in the treatment of receptor-related diseases.

[0022] The present invention provides the above-mentioned cyclohexane derivatives, their stereoisomers or pharmaceutically acceptable salts thereof for use in the preparation of 5HT 1A receptor agonists or antagonists.

[0023] The present invention provides the use of the above-mentioned cyclohexane derivatives, their stereoisomers or pharmaceutically acceptable salts in the preparation of drugs for treating or preventing neuropsychiatric diseases.

[0024] In some embodiments, the neuropsychiatric disease is selected from one or more of schizophrenia, psychotic disorder, psychotic disorder, delirium, mood disorder, bipolar disorder, depression, phobia, obsessive-compulsive disorder, anxiety disorder and cognitive disorder.

[0025] In some embodiments, the present invention provides the use of the above-mentioned cyclohexane derivatives, stereoisomers thereof, or pharmaceutically acceptable salts thereof in the preparation of drugs for treating or preventing neuropsychiatric diseases requiring antagonism of dopamine D1.

[0026] In some embodiments, the present invention provides the above-mentioned cyclohexane derivatives, their stereoisomers or pharmaceutically acceptable salts thereof for the preparation of a method for treating or preventing diseases requiring stimulation of 5-hydroxytryptamine receptor 5-HT 1A Application of drugs in the treatment of neuropsychiatric diseases.

[0027] The compound of the present invention has lower cardiotoxicity and hepatotoxicity and is more suitable for long-term medication.

[0028] Detailed Description of the Invention

[0029] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All patents, applications, published applications and other publications cited herein are incorporated by reference in their entirety.

[0031] In the present invention, "carbonyl source" refers to a reagent that provides a carbonyl -C(O)- group in an organic reaction. Non-limiting examples include carbonyl dibenzotriazole, carbonyl dihydroxybenzotriazole, carbonyl di-1,2,4-triazole, potassium cyanate, phosgene, triphosgene, formaldehyde, or acetaldehyde.

[0032] In the present invention, any isotope-labeled derivatives of the compounds of the present invention or their pharmaceutically acceptable salts are covered by the present disclosure. Atoms that can be isotopically labeled include but are not limited to hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, etc. They can be isotopically labeled. 2 H(D), 3 H. 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 36 Cl and 125 Unless otherwise indicated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% deuterium incorporation).

[0033] In the present invention, "plurality" means two or more, for example, 2, 3, 4, 5 or 6.

[0034] In the present invention, "pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which are safe and effective when used in mammals and have the desired biological activity.

[0035] In this invention, the following abbreviations / terms are used: 3 H]-8-OH-DPAT: [ 3 H]-8-hydroxy-DPAT hydrogen bromide; 8-OH-DPAT: 8-hydroxy-DPAT hydrogen bromide; [ 3 H]-7-OH-DPAT: [ 3 H]-7-hydroxy-DPAT hydrobromide; [ 3 H]-Ketanserin: [ 3 H]-Ketanserin; Ketanserin: Ketanserin; [ 3 H]-LSD: lysergic acid diethylamide; (±)DOI: (±)-2,5-dimethoxy-4-iodophenylpropane-2-amine; Tris base: tris(hydroxymethyl)aminomethane; EDTA: ethylenediaminetetraacetic acid; Microscint 20 cocktail: liquid scintillation fluid; PEI: polyethyleneimine; G418: geneticin; HBSS: Hank's balanced salt solution; HEPES: 4-hydroxyethylpiperazineethanesulfonic acid, zwitterionic buffer; Labcyte PP 384 plate: 384-well polypropylene microplate; (±)-SKF-38393: (±)-1-phenyl-2,3,4-trihydro(1H)-3-benzapezine-7,8-diol; Dopamine: dopamine; [ 3 H]-SCH 23390: [ 3 H]-(5R)-8-chloro-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-ol; SCH-23390: (5R)-8-chloro-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-ol; Amisulpride: amisulpride; Methiothepin mesylate: methiothepin maleate; 5-CT: 5-carboxyamidotryptamine maleate; 5-HT: 5-hydroxytryptamine; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; DMEM: culture medium; TrypLE TM Express: phenol red-free trypsin, a non-animal derived recombinant enzyme; d-TFA: deuterated trifluoroacetic acid; DMF: N,N-dimethylformamide; [ 3 H]-Methylspiperone: [ 3H]-N-methylpiperidinium hydrochloride; DPBS: Dulbecco's phosphate buffered saline; FA: formic acid; DETAILED DESCRIPTION

[0036] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0037] Experimental methods in the examples of this disclosure that do not specify specific conditions are generally based on conventional conditions or the conditions recommended by the raw material or product manufacturers. Reagents without specific sources are conventional reagents purchased from the market.

[0038] Example 1

[0039] Preparation of N'-trans-4-[2-[4-(Benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-3-methylurea (Compound 1)

[0040] Dissolve 20g of trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexylamine (Compound a) in 160mL of dichloromethane, add 13.2g of CDI (N,N'-carbonyldiimidazole), cool to -10°C-0°C, and dropwise add 29.2g of triethylamine. After addition, react at this temperature for 1h. Next, add 9.73g of methylamine hydrochloride and react at room temperature (25°C ± 2°C) overnight. After the reaction, add 50mL of water, stir for 30-60min, filter, and dry to obtain 13.2g of Compound 1.

[0041] H NMR (400MHz, d-TFA) δ8.387(d,J=8Hz,1H),7.95-7.83(m,4H),5.12(t,J=12Hz,2H),4.72-4.60(m,4H),4.44(t,J=12.4Hz ,2H),3.93-3.83(m,3H),3.26(s,3H),2.43(d,J=10.8Hz,2H),2.23-2.14(m,4H),1.77-1.69(m,3H),1.542-1.453(m,2H).

[0042] 13 C-NMR(101MHz,d-TFA)δ158.809,144.685,132.931,129.466,127.652,125.771,125.668,1 25.404,117.229,56.969,51.782,50.950,49.732,33.961,31.363,30.478,30.175,26.891.

[0043] Example 2

[0044] Preparation of trans-4-(2-(4-(benzo[b]thiophen-7-yl)piperazin-1-yl)ethyl)cyclohexyl)urea (Compound 2)

[0045] Dissolve 9.3 g of trans-4-[2-[4-(Benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexylamine (Compound a) in a 3:1 DMF:water mixture (37.2 mL). Add 2.71 g of potassium cyanate, and heat at 85°C for 2 h. Add an additional 15 g of potassium cyanate and continue the reaction at 85°C overnight. Remove from heating, cool to room temperature, add water, stir, and filter. Rinse the filter cake with water and dry to obtain 6.5 g of Compound 2.

[0046] MS (ESI): m / z = 387.3 [M+H] +

[0047] H NMR (400MHz, d-TFA) δ8.33(d,J=7.6Hz,1H),7.91-7.79(m,4H),5.08(t,J=12Hz,2H),4.68-4.56(m,4H),4.40(t,J=12Hz,2H),3.84-3.82(m,3H) ， 2.41 (d, J = 10 Hz, 2H) , 2.19-2.12 (m, 4H) ， 1.70-1.64(m,3H),1.52-1.46(m,2H).

[0048] 13 C-NMR(101MHz,d-TFA)δ159.675,144.647,132.903,129.461,127.624,126.844,125.74 8,125.389,117.161,56.978,51.445,50.941,49.697,33.981,31.246,30.353,30.154.

[0049] Example 3

[0050] Preparation of N'-trans-4-[2-[4-(Benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-dimethylurea (Compound 3)

[0051] Dissolve 1.73 g of trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexylamine (Compound a) in 50 mL of dichloromethane, add 1.40 mL of triethylamine, and then add 5.50 mmol of N,N-diformylcarbonyl chloride. Stir at room temperature (25°C ± 2°C) for 48 hours. After the reaction is complete, extract with 50 mL of water. The organic phase is concentrated (0.01 MPa, 45°C) and the desired fraction is collected by column chromatography (400 mesh silica gel) using a 1:10 ratio of methanol to dichloromethane. Concentration yields 1.89 g of amorphous Compound 3.

[0052] 1H-NMR (CDC13) ppm: 7.79 (1H, d, J = 5.5Hz), 7.76 (1H, d, J = 8.1Hz), 7.33 (1H, m), 7.28 (1H, d, 7.6Hz), 6.96 (1H, d, J = 7.6Hz), 6.48 (1H,brs),3.44-3.36(8H,m),3.58(1H,m),3.01(6H,s),2.46(2H,m),1.68-1.42(8H,m),1.52-1.48(1H,m),1.38-1.36(2H,m).

[0053] Example 4

[0054] Preparation of N'-trans-4-[2-[4-(Benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-dimethylurea maleate (Compound 4)

[0055] Dissolve 15g of N'-trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-dimethylurea (Compound 3) in 67.5g of anhydrous ethanol. Add 4.7g of maleic acid in anhydrous ethanol dropwise at 45-50°C. Incubate for 1.5 hours. Slowly return to room temperature while stirring. Solid precipitates. Filter the filter cake, wash twice with anhydrous ethanol, and dry under vacuum to yield 12.8g of Compound 4.

[0056] Example 5

[0057] Receptor binding assay 1

[0058] 1. Purpose of the experiment

[0059] The main purpose of this study is to determine the effect of compounds on 5-hydroxytryptamine receptors (5-HT 1A , 5-HT 2A and 5-HT 2B ) affinity K i .

[0060] 2. Experimental Materials and Instruments

[0061] 2.1 Test sample

[0062] Preparation method: Dissolve the test compounds in DMSO to a 10 mM stock solution and store in a 4°C refrigerator.

[0063] 2.2 Receptor membrane proteins

[0064] Expresses 5-hydroxytryptamine receptors (5-HT 1A , 5-HT 2A and 5-HT 2B ) The information of three cell membrane proteins is as follows:

[0065] Table 1. 5-HT receptor membrane proteins

[0066] 2.3 Reagents and consumables

[0067] Table 2. Reagents and consumables

[0068] 2.4 Main instruments

[0069] Table 3. Instruments and Equipment

[0070] 3. Experimental Methods

[0071] 1) Configuration of the cell membrane

[0072] 5-HT 1A 5-HT was added to the assay buffer. 1A The membrane was diluted to 100 μg / mL.

[0073] 5-HT 2A 5-HT was added to the assay buffer. 2A The membrane was diluted to 50 μg / mL.

[0074] 5-HT 2B 5-HT was added to the assay buffer. 2B The membrane was diluted to 50 μg / mL.

[0075] 2) Isotope configuration

[0076] 5-HT 1A : Use detection buffer to 3 H]8-OH-DPAT was diluted to 0.6 nM.

[0077] 5-HT 2A : Use detection buffer to 3 H]-Ketanserin was diluted to 2 nM.

[0078] 5-HT2B : Use detection buffer to 3 H]-LSD was diluted to 2 nM.

[0079] 3) Make 8 4-fold dilutions of the test compound and reference compound, and add 1 μL to each well. 1A The initial concentration of the reference compound 8-OH-DPAT was 1000 nM; for 5-HT 2A The starting concentration of the reference compound Ketanserin was 1000 nM; for 5-HT 2B , the starting concentration of reference compound (±) DOI was 10 μM.

[0080] 4) Add 1 μL DMSO to the high signal control wells (High control) and 5-HT to the low signal control wells (Low control). 1A 1 μL of 2 mM 8-OH-DPAT (final concentration 10 μM) and 5-HT 2A 1 μL of 200 μM Ketanserin (final concentration 1000 nM) and 5-HT 2B 1 μL of 10 mM serotonin was added to the plate (final concentration 50 μM).

[0081] 5) Add 100 μL of the corresponding cell membrane to each well.

[0082] 6) Add 100 μL of the corresponding isotope to each well.

[0083] 7) Seal the 96-well plate and incubate on a shaker at 300 rpm at room temperature for 1 hour. Simultaneously, soak the GF / C filter plate in 0.3% PEI.

[0084] 8) After incubation, cells were harvested onto GF / C filter plates using a cell harvester, washed four times with plate washing buffer, and dried in a 50°C oven for 1 hour.

[0085] 9) Seal the bottom of the dried GF / C filter plate with film, add 50 μL of scintillation fluid to each well, and seal. Read using a Microbeta.

[0086] Table 4. Detection buffers used for different targets

[0087] 4. Data Processing and Analysis

[0088] 1) Calculate the percentage activity using Microsoft Excel software using the formula: % Inhibition = 100 × (1 - (Sample Raw Value - Low Control Average) / (High Control Average - Low Control Average))

[0089] 2) The inhibition rate was fitted using the four-parameter equation “log (inhibitor) vs response-variable slope” in GraphPad Prism 5.0 to calculate the IC 50 .

[0090] 3) Calculate K i Value, formula: K i =IC 50 / (1+[L]Kd)

[0091] 5. Test results

[0092] Compound 1, Compound 2 and Compound 4 have an effect on 5-hydroxytryptamine receptor (5-HT 1A , 5-HT 2A and 5-HT 2B ) affinity K i As shown in Table 5.

[0093] Table 5. Affinity of Compound 1, Compound 2, and Compound 4 for each target

[0094] 6. Conclusion

[0095] Effects of Compound 1 and Compound 2 on 5-HT 1A , 5-HT 2A and 5-HT 2B The affinity of compound 4 for 5-HT receptor is stronger than that of compound 4 for 5-HT 1A , 5-HT 2A and 5-HT 2B Receptor affinity.

[0096] Receptor binding assay 2

[0097] 1. Purpose of the experiment

[0098] Detection of compound affinity for D1 receptor

[0099] 2. Experimental Materials and Instruments

[0100] 2.1 Target information

[0101] Table 6. Target information

[0102] 2.2 Reagents and consumables

[0103] Table 7. Reagents and consumables

[0104] 2.3 Main instruments

[0105] Table 8. Main instruments

[0106] 2.4 Preparation of experimental reagents

[0107] Table 9. Reagent preparation

[0108] 3. Experimental Methods

[0109] 1) Reaction system: 100 μL cell membrane, 1 μL test compound, 100 μL corresponding isotope.

[0110] 2) Cell membrane preparation: Dilute D1 cell membrane with assay buffer to a final concentration of 15 μg / well;

[0111] 3) Isotope preparation: Dilute the corresponding isotope with Assay buffer. The final isotope concentration of the experimental system is as follows:

[0112] Table 10. Isotope preparation

[0113] 4) Compound Dilution: Serially dilute the test compound 4-fold in DMSO at eight points. Transfer 1 μL of the diluted test compound to the designated position on the assay plate. Transfer 1 μL of the highest concentration reference compound to the assay plate as the low control (LC) well for nonspecific binding. Transfer 1 μL of DMSO to the high control (HC) well for total binding.

[0114] 5) According to the arrangement of experimental compounds, add 100 μL of prepared cell membrane to each well of the reaction plate.

[0115] 6) According to the arrangement of experimental compounds, add 100 μL of prepared isotope to the reaction plate.

[0116] 7) Seal the reaction plate with a sealing film and incubate on a shaker at room temperature for 1 hour. Simultaneously, soak the GF / C filter plate in 50 μL of 0.3% PEI soaking solution for at least 0.5 hours.

[0117] 8) After the reaction plate is incubated, the reaction solution is collected onto a GF / C filter plate using a cell harvester, washed four times with wash buffer, and dried in a 50°C oven for 1 hour.

[0118] 9) Seal the bottom of the dried GF / C filter plate with film, add 50 μL MicroScint-O scintillation fluid to each well, and seal.

[0119] 10) Using Microbeta 2 reading.

[0120] 11) Analysis of results: The inhibitory activity of the sample (%) was calculated using Excel as (1-(sample well signal - low control signal) / (high control signal - low control signal)) × 100%. The IC was calculated using the "log (inhibitor) vs. response - variable slope" model in GraphPad Prism 5.0. 50 .

[0121] 12) Calculate K i Value, formula: K i =IC 50 / (1+[L]Kd)

[0122] 4. Test results

[0123] Table 11. Affinity of targets

[0124] 5. Conclusion

[0125] The affinity of compound 2 for D1 receptor is stronger than that of compound 4 for D1 receptor.

[0126] Example 6

[0127] Cell functional assay 1

[0128] Compounds for D1, D2, 5-HT 1A , 5-HT 2A and 5-HT 2B Cellular functional assay of receptors.

[0129] 1. Purpose of the experiment

[0130] Using FLIPR calcium flux detection technology, the effects of compounds on D1, D2, and 5-HT were detected at the cellular level. 1A , 5-HT 2A and 5-HT 2B Receptor agonism and antagonism.

[0131] 2. Experimental Materials and Instruments

[0132] 2.1 Test sample

[0133] Preparation method: Dissolve the test compounds in DMSO to a 10 mM stock solution and store in a 4°C refrigerator.

[0134] 2.2 Cell lines

[0135] Stable expression of D2 and 5-HT 1A The cell line information of the recipient is as follows:

[0136] Table 12. Cell line information

[0137] 2.3 Buffer

[0138] 1) Assay buffer: HBSS buffer containing 2 mM HEPES.

[0139] 2) Probenecid (250 mM) solution: press Fluo-4 Direct TM According to the kit instructions, add 77 mg of probenecid to 1 ml of assay buffer and prepare immediately before use.

[0140] 3) Fluo-4 Direct TM (2X, 8M) loading buffer: According to the kit instructions, add the required amount of Fluo-4 Direct TM Take out and melt, add 10mL test buffer and 0.2mL 250mM Probenecid solution to each tube, vortex and shake in the dark for more than 5 minutes, and prepare before use.

[0141] 2.4 Reagents and consumables

[0142] Table 13. Reagents and consumables

[0143] 2.5 Instruments and Equipment

[0144] Table 14. Instruments and Equipment

[0145] 3. Experimental Methods

[0146] Day 1: Cell plating

[0147] 1) For frozen cells, remove the cells from liquid nitrogen and quickly place them in a 37°C water bath. Add 10 mL of preheated culture medium to suspend the cells, remove 1 mL and use Vi-CELL TM Count and measure the activity; for cultured cells, wash twice with preheated DPBS, then add appropriate amount of trypsin to digest at 37℃ for 1 minute; then add appropriate amount of culture medium to terminate the digestion, take out 1mL and use Vi-CELL TM Count and determine viability.

[0148] 2) Centrifuge the cells at 1000 rpm for 5 minutes.

[0149] 3) Gently discard the supernatant without touching the cell mass at the bottom.

[0150] 4) Dilute the cells to 1×10 6 Cells / mL (20,000 cells / 20 μL) were seeded in 384-well poly-lysine-coated cell plates and cultured at 37° C. with 5% CO 2 for 16-20 hours.

[0151] Day 2: Sample addition and detection

[0152] 1)EC 80 Detection:

[0153] a) To EC 80 2×Fluo-4 detection reagent was added to the detection cell plate, 20 μL per well, and the plate was placed in a 37°C incubator for 50 min, and then allowed to stand at room temperature for 10 min.

[0154] b) The compound was diluted 3-fold with DMSO to 10 concentrations using Echo 555 and added to EC 80 In the test compound plate 1, 500 nL was added to each well in duplicate, and then 30 μL of detection buffer was added to each well.

[0155] Table 15. Starting concentrations of positive compounds for each target agonist

[0156] c) EC 80 Detection cell plate, EC 80 For the test compound plate, place the pipette tips into the FLIPR instrument, start the instrument, transfer 10 μL of the reference compound from compound plate 1 to the cell plate, read the results, and calculate the EC 80 .

[0157] 2) Compound activity detection

[0158] a) Add 2× Fluo-4 detection reagent to each well of the cell plate, 20 μL per well, incubate in a 37°C incubator for 50 min, and then let stand at room temperature for 10 min.

[0159] b) Dilute the compound 4-fold with DMSO using an Echo 555 to a total of 10 concentrations. Add 900 nL per well of Compound Plate 2 in duplicate. Then, add 30 μL of Assay Buffer to each well. The starting concentration for agonist assays is 12 μM, and for antagonist assays, it is 10 μM.

[0160] Table 16. Starting concentrations of positive antagonist compounds for each target

[0161] c) Prepare 6*EC of the compound separately 80 And added to compound plate 3, 30 μL per well.

[0162] d) Place the cell plate, corresponding compound plate 2, and pipette tip into the FLIPR instrument, start the instrument, transfer 10 μL of compound from compound plate 2 to the cell plate, read the volume, and calculate the EC of each compound. 50 ;

[0163] e) Take out compound plate 2 and place it in the corresponding EC 80 Compound plate 3, transfer 10 μL of compound from compound plate 3 to the cell plate, read, and calculate the IC of each compound 50 .

[0164] 4. Data Processing and Analysis

[0165] 1) Calculate the percentage activity using Microsoft Excel software. For agonists, use the formula %Effect = 100 × (Sample Raw Value - Low Control Average) / (High Control Average - Low Control Average); for inhibitors, use the formula %Inhibition = 100 × (1 - (Sample Raw Value - Low Control Average) / (High Control Average - Low Control Average)).

[0166] 2) Using GraphPad Prism 5 data analysis software, the log[agonist] vs. response--Variable slope model was used for fitting analysis for agonists, and the log[inhibitior] vs. response--Variable slope model was used for fitting analysis for inhibitors to obtain the IC of each test sample. 50 / EC 50 value.

[0167] 5. Test results

[0168] This experiment tested the agonist and antagonist effects of Compound 1, Compound 2, and Compound 4 on multiple receptors at the cellular level. The results are shown in the table below.

[0169] Table 17. Effects of Compound 1, Compound 2, and Compound 4 on D2 and 5-HT 1A Receptor agonism

[0170] Table 18. Effects of Compound 1 and Compound 2 on D1, D2, and 5-HT 1A , 5-HT 2A , 5-HT 2B Receptor antagonism

[0171] 6. Conclusion

[0172] The test results showed that compound 4 had an effect on 5-HT 1A Compounds 1 and 2 did not show agonist effects on 5-HT receptors. 1A Compounds 1 and 2 showed agonist effects on D1, D2, and 5-HT receptors. 1A , 5-HT 2A and 5-HT 2B The receptors showed a certain antagonistic effect.

[0173] Cell functional assay 2

[0174] Compounds for human D1, D 2L 、D 2S , 5-HT 1A and 5-HT 2A Cellular functional assay of the receptor.

[0175] 1. Purpose of the experiment

[0176] The main purpose of this experiment is to detect the effects of compounds on human D1, D 2L 、D 2S , 5-HT 1A and 5-HT 2A Receptor agonism and antagonism.

[0177] 2. Experimental Materials and Instruments

[0178] 2.1 Reference compounds

[0179] Table 19. Reference compounds

[0180] 2.2 Cell lines

[0181] Table 20. Cell lines

[0182] 2.3 Reagent Information

[0183] Table 21. Reagent information

[0184] 2.4 Instrument Information

[0185] Table 22. Instrument Information

[0186] 3. Experimental Methods

[0187] Day 1: Cell plating

[0188] 1) Preheat the culture medium, 1× DPBS, and 0.05% trypsin-EDTA in a 37°C water bath for at least 30 minutes and set aside.

[0189] 2) Remove the preheated culture medium, 1× DPBS, and 0.05% trypsin-EDTA, disinfect with 75% alcohol, and place in a biosafety cabinet.

[0190] 3) For frozen cells, rapidly thaw in a 37°C water bath. Transfer the thawed cells to a centrifuge tube containing 10 mL of complete medium. For cultured cells, remove the cultured cells from the incubator, add 1× DPBS, incubate briefly, and aspirate. Add an appropriate amount of 0.05% trypsin-EDTA to digest the cells. Terminate the digestion with complete medium, and disperse the cells. Use a pipette to transfer the dispersed cells into a 10 mL centrifuge tube.

[0191] 4) Centrifuge the cells at 1000 rpm for 5 minutes, resuspend in culture medium, blow off, and count.

[0192] 5) Dilute the cells to 1×10 6 Cells were seeded into 384-well poly-lysine-coated cell plates at 20 μL / well.

[0193] 6) Incubate at 37°C, 5% CO2 for 16-20 hours.

[0194] Day 2: FLIPR Experiment

[0195] Reagent preparation:

[0196] 1) FLIPR assay buffer: 20 mM HEPES, 1× HBSS, 0.5% BSA.

[0197] 2) Prepare 250 mM probenecid solution: Add 1 mL of FLIPR assay buffer to 77 mg of probenecid to make a 250 mM solution. Prepare immediately before use.

[0198] 3) Preparation of 2× (8 μM) Fluo-4 Direct TM Loading buffer: Melt Fluo-4 Direct in advance TMAdd 10 mL of FLIPR assay buffer to each tube. Add 0.2 mL of 250 mM Probenecid solution to each tube. Vortex and shake for >5 minutes in the dark. Prepare immediately before use.

[0199] EC 80 Detection:

[0200] 1) Perform a 4-fold 10-point serial dilution of the agonist reference compound by Echo and transfer 900 nL to EC 80 Test compound plates in duplicate.

[0201] 2) Add 30 μL of assay buffer to each well and centrifuge at 1000 rpm for 1 minute.

[0202] 3) Take the cell plate out of the incubator, remove the culture medium in the cell plate, and add 20 μL of experimental buffer and 20 μL of 2×Fluo-4 Direct to each well. TM Buffer, total volume is 40 μL. Incubate in a 5% CO2, 37°C incubator for 50 minutes, then leave at room temperature for 10 minutes.

[0203] 4) Place the assay cell plate, assay compound plate, and pipette tips into the FLIPR instrument.

[0204] 5) Run the FLIPR instrument software and follow the set program to start the EC 80 Transfer 10 μL of agonist from the test compound plate to the cell plate and read the fluorescence signal. After reading, export the data using the "Max-Min" and "Read 1 to Maximum allowed" methods in the software to calculate the EC 80 .

[0205] Compound detection:

[0206] 1) Perform a 4-fold, 10-point serial dilution of the test compound and reference compound using echo. Transfer 900 nL to the compound plate in duplicate. For agonist assays, start at a 12 μM test compound concentration. Transfer 900 nL of the highest concentration of the agonist reference compound to the plate as a high signal control (High Control), and transfer 900 nL of DMSO to the plate as a low signal control (Low Control). For antagonist assays, start at a 10 μM test compound concentration. Transfer 900 nL of DMSO to the plate as a high signal control (High Control), and transfer 900 nL of the highest concentration of the antagonist reference compound to the plate as a low signal control (Low Control).

[0207] 2) Add 30 μL of FLIPR assay buffer to each well and centrifuge at 1000 rpm for 1 minute.

[0208] 3) Take the cell plate out of the incubator, remove the culture medium in the cell plate, and add 20 μL of experimental buffer and 20 μL of 2×Fluo-4 Direct to each well. TM Buffer, total volume is 40 μL. Incubate in a 5% CO2, 37°C incubator for 50 minutes, then leave at room temperature for 10 minutes.

[0209] 4) Prepare 6×EC 80 The agonist of the appropriate concentration was added to a new 384-well compound plate at 30 μL / well and centrifuged at 1000 rpm for 1 minute.

[0210] 5) Take the cell plate out of the incubator and place it in the instrument. 80 Place the plate and pipette tips into the instrument.

[0211] 6) Run the FLIPR instrument software and add 10 μL of the test compound and reference compound to the cell plate according to the set program, and read the fluorescence signal; then add 10 μL of 6×EC 80 Add the agonist reference compound to the cell plate at a specific concentration and read the fluorescence signal. Export the data using the "Max-Min" and "Read 1 to Maximum allowed" methods in the software.

[0212] 7) Analyze the data:

[0213] Agonist: Activity% = (RLU-LC) / (HC-LC)*100

[0214] RLU: relative absorbance, 1 to maximum allowed reading;

[0215] LC: average fluorescence signal of DMSO group; HC: average fluorescence signal of the highest concentration point of reference compound.

[0216] Antagonist: Inhibition% = 100-(RLU-LC) / (HC-LC)*100

[0217] RLU: relative absorbance, 1 to maximum allowed reading;

[0218] LC: average fluorescence signal of the highest concentration of the reference compound; HC: average fluorescence signal of the DMSO group.

[0219] 4. Data Analysis and Processing

[0220] 1) Calculate the activity percentage using Microsoft Excel software.

[0221] 2) Using GraphPad Prism 5 data analysis software, the Dose-response-Stimulation—log (agonist) vs. response—Variable slope model was selected for fitting analysis to obtain the EC values ​​of each test sample. 50 The Dose-response-Inhibition—log (inhibitor) vs. response—Variable slope model was used for fitting analysis to obtain the IC values ​​of each test sample. 50 value.

[0222] 5. Experimental Results

[0223] Table 23. Antagonistic effects of compounds on targets

[0224] 6. Conclusion

[0225] The test results showed that compound 2 had a stronger antagonistic effect on D1 receptors than compound 4.

[0226] Example 7

[0227] Cellular functional assay of test compounds on D3 receptors

[0228] 1. Purpose of the experiment

[0229] Evaluate the biological activity of the compounds.

[0230] 2. Experimental Methods

[0231] The test method (dopamine D3, GTPγS binding) is as follows:

[0232] 3. Experimental Results

[0233] The test data are shown in Table 23 below:

[0234] Table 23. Antagonistic effects of compound 1, compound 2, and compound 4 on D3 receptors

[0235] 4. Conclusion

[0236] The test results showed that the antagonistic effect of compound 1 and compound 2 on D3 receptor was greater than that of compound 4 on D3 receptor.

[0237] Example 8

[0238] hERG toxicity studies

[0239] 1. Purpose of the experiment

[0240] The rapidly activating delayed rectifier potassium channel (IKr), encoded by the human Ether-à-go-go-Related Gene (hERG), is a key ion channel involved in phase 3 repolarization of the cardiac action potential. Drug blockade of hERG channels can lead to prolonged cardiac repolarization, manifested on the electrocardiogram as a prolonged QT interval, a condition known as long QT syndrome. Drug-induced delayed ventricular repolarization can, in some cases, trigger the fatal arrhythmia torsade de pointes (TdP).

[0241] This study used manual patch clamp technology (the gold standard for hERG safety evaluation) to study the inhibitory effect of the test compound on hERG potassium channels and evaluate its risk of inducing ventricular repolarization toxicity.

[0242] 2. Experimental materials and instruments:

[0243] 2.1 Reagents

[0244] Table 24. Chemical reagent information

[0245] 2.2 Instruments

[0246] Table 25. Instrument Information

[0247] 3. Experimental steps:

[0248] 3.1 Cell culture

[0249] In this experiment, HEK-293 cells stably expressing hERG potassium channel were used. hERG potassium channel cells were purchased from Creacell (Cat. No. A-0320). The cell culture method is as follows:

[0250] HEK-293 cells stably expressing hERG potassium channel were cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg / mL G418 at a temperature of 37° C. and a carbon dioxide concentration of 5%.

[0251] Cell passaging: Remove old culture medium and wash once with PBS, then add 1 mL of TrypLE TM Express solution, incubate at 37°C for about 0.5 min. When the cells detach from the bottom of the dish, add about 5 mL of complete culture medium preheated at 37°C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min to collect the cells. For expansion or maintenance culture, inoculate the cells in a 6 cm cell culture dish with 2.5 × 10 cells per cell culture dish. 5 cells (final volume: 5 mL).

[0252] To maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

[0253] Before patch clamp testing, cells were stained with TrypLE TM Express separation, 4×10 3 The cells were plated on coverslips and cultured in 24-well plates (final volume: 500 μL). After 18 hours, the assay was performed.

[0254] 3.2 Electrophysiological recording

[0255] 3.2.1 Record the liquid used

[0256] Extracellular fluid:

[0257] 140 mM NaCl, 3.5 mM KCl, 1 mM MgCl2·6H2O, 2 mM CaCl2·2H2O, 10 mM D-Glucose, 10 mM HEPES, 1.25 mM NaH2PO4·2H2O, NaOH adjusted to pH = 7.4.

[0258] Intracellular fluid:

[0259] 20mM KCl, 115mM K-Aspartic, 1mM MgCl2·6H2O, 5mM EGTA, 10mM HEPES, 2mM Na2-ATP, KOH adjust pH=7.2.

[0260] The extracellular solution has a shelf life of 2 weeks. After preparing the intracellular solution, aliquot into 1 mL tubes and store frozen at -20°C. Use freshly thawed intracellular solution each day. Use all intracellular solution within 3 months. If the solution has been used for more than 3 months, discard the old solution and prepare fresh.

[0261] 3.2.2 Patch clamp assay

[0262] The voltage stimulation protocol for whole-cell patch-clamp recording of hERG currents was as follows: after forming a whole-cell seal, the cell membrane voltage was clamped at -80 mV. The clamping voltage was depolarized from -80 mV to -50 mV for 0.5 s (to detect leakage current), then stepped to 30 mV for 2.5 s, and then quickly returned to -50 mV for 4 s to stimulate hERG tail current. Data were collected repeatedly every 10 s to observe the effects of drugs on hERG tail current. Experimental data were acquired using an IPA amplifier (Sutter Instrument) and stored in SutterPatch (with Igor Pro) software.

[0263] The patch clamp procedure begins by pulling a recording electrode from a glass capillary using a microelectrode puller. The electrode, filled with intracellular fluid, is then placed in a microelectrode holder. Under an inverted microscope, the microelectrode manipulator is used to immerse the electrode in the extracellular fluid and record the electrode resistance (Rpip). The electrode is then slowly brought into contact with the cell surface, and negative pressure is applied to create a GΩ seal. Fast capacitance compensation is then performed, and negative pressure is continued to rupture the cell membrane, establishing whole-cell recording mode. Finally, slow capacitance compensation is performed, and experimental parameters such as series resistance (Rs) are recorded. No leakage compensation is performed.

[0264] When the hERG current recorded by the whole cell is stable, the drug is administered. After each drug concentration acts for about 5 minutes (or the current stabilizes), the next concentration is detected. Multiple concentrations are detected for each test compound. The coverslip with cells is placed in a recording bath under an inverted microscope. The blank control external solution and the working solution of the test compound are flowed through the recording bath from low concentration to high concentration by gravity perfusion to act on the cells. A peristaltic pump is used for liquid exchange during recording. The current detected in the external solution without compound for each cell serves as its own control group. At least three cells are used for each concentration and the test is repeated three times independently. All electrophysiological experiments are performed at room temperature.

[0265] 4. Data Analysis:

[0266] First, the peak tail current after each drug concentration is compard ) and blank control tail current (Peak tail current control ) normalized, and then calculated the inhibition rate corresponding to each drug concentration The mean (Mean), standard deviation (SD) and standard error (SE) of the inhibition rate at each concentration were calculated, and the data were expressed as Mean±SE.

[0267] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope))

[0268] The IC of each compound was calculated using the above equation 50 The dose-effect curve was fitted nonlinearly, where IC 50 IC is the half inhibitory concentration. 50 Calculations and curve fitting were performed using GraphPad Prism software.

[0269] 5. Experimental Results

[0270] The inhibitory effects of test compounds 2 and 3 on hERG channels were detected in three independent repeated experiments, and the IC values ​​of the test compounds on hERG channel current were calculated by fitting the concentration-effect curves. 50 value.

[0271] Table 26. IC of Compound 2 and Compound 3 on hERG channel current 50 value

[0272] The test results show that the inhibitory effect of compound 2 on the hERG channel is lower than that of compound 3, and the risk of cardiac toxicity is lower, showing better safety.

[0273] Example 9

[0274] Glutathione trapping (GSH trapping)

[0275] 1. Instruments and analytical conditions

[0276] 1.1 Instrument

[0277] Vanquish UHPLC system (Thermo Fisher Scientific, USA)

[0278] Thermo Scientific Q Exactive (Thermo Fisher Scientific, USA)

[0279] 1.2 Liquid phase conditions

[0280] Column: Waters XSelect HSS T3, 100×2.1mm, 2.5μm

[0281] Solvent: A, water (0.1% formic acid); B, acetonitrile (0.1% formic acid)

[0282] Flow rate: 500 μL / min

[0283] Gradient: 0-1.5 min, 5% B, 1.5-5.5 min, 5%-60% B, 5.5-7 min, 60%-95% B, 7-8.5 min, 95% B, 8.5-9 min, 95%-5% B, 9-10 min, 5% B;

[0284] 1.3MS conditions

[0285] Ionization mode: positive mode

[0286] Spray voltage: 3.5kV

[0287] Auxiliary gas flow rate: 15

[0288] Auxiliary gas heater temperature: 350℃

[0289] Scan type: Full MS / ddMS 2

[0290] Resolution: 70,000

[0291] Automatic gain control target: 3×e 6

[0292] NCE / stepped NCE:10,15,20

[0293] 2. Experimental Methods

[0294] 2.1 Culture and sample preparation

[0295] Table 27. Final concentrations of test compounds for in vitro incubation

[0296] 2.2 Liver microsome information:

[0297] Table 27. Liver microsome information

[0298] • No compound control (NCC), replaced with an equal volume of acetonitrile.

[0299] • No matrix control (NMC): the microsomal solution was replaced with an equal volume of phosphate buffered saline.

[0300] The control samples had the same culture conditions and preparation procedures.

[0301] 2.3 Quenching and analysis

[0302] The incubation was quenched with 2 volumes of acetonitrile (0.1% FA) and then centrifuged at 16,000 g for 15 minutes. The supernatant (400 μL) was transferred and placed in an evaporator under a steady stream of nitrogen at room temperature until dry. The dried residue was reconstituted with 100 μL of diluent (acetonitrile: water (v / v) = 1:4). The sample was then centrifuged at 16,000 g for 15 minutes. The supernatant was transferred and the sample was analyzed using UHPLC-MS / MS.

[0303] 3. Experimental Results

[0304] Table 28. GSH trapping experimental results

[0305] 4. Experimental Conclusion

[0306] The test results show that the combination of compound 4 and GSH will lead to a certain risk of hepatotoxicity, while compound 2 has a lower risk; therefore, compound 2 has a lower risk of hepatotoxicity and is more conducive to long-term use.

[0307] Example 10

[0308] Toxicity to primary human hepatocytes

[0309] 1. Purpose of the experiment

[0310] The CellTiter-Glo Luminescent Cell Viability Assay (CTG) was used to detect the inhibitory effect of the compounds on the cell proliferation of human primary hepatocytes from different donors.

[0311] 2. Reagents and Instruments

[0312] 2.1 Cell lines

[0313] Table 29. Cell line information

[0314] 2.2 Main reagents

[0315] Table 30. Reagent information

[0316] 2.3 Main instruments

[0317] Table 31. Instrument Information

[0318] 3. Experimental Methods

[0319] 3.1 Experimental Grouping

[0320] Table 32. Experimental groups

[0321] 3.2 Preparation of mother liquor

[0322] Table 33. Preparation of stock solution

[0323] 3.3 Gradient Dilution

[0324] Table 34. Serial dilution

[0325] Note: The prepared drug was added to the cells in culture medium and diluted 5-fold. For example, when the prepared drug concentration was 15000 μM, 40 μL of drug was added to 160 μL of cell culture system, and the initial concentration was 3000 μM.

[0326] 3.4 Experimental steps

[0327] 1) According to the instructions for primary hepatocytes, resuscitate human primary hepatocytes and count them using a cell counter. Adjust the cell concentration to the desired level using plating medium.

[0328] 2) Add 100 μL of cell suspension to each well of a 96-well plate and add 250 μL of PBS around each well.

[0329] 3) Place the prepared 96-well plate in a 37°C incubator and incubate for 6 h. Replace the plate with 100 μL / well of maintenance medium.

[0330] 4) 24 hours after cell plating, replace the culture medium with 160 μL / well of maintenance medium.

[0331] 5) Prepare the test sample according to 3.2 as a stock solution. Dilute the stock solution with maintenance medium and prepare and serially dilute the sample according to 3.3, using a 6-point serial dilution.

[0332] 6) Remove the 96-well plate and add 40 μL / well of the medium containing the drug to be tested. Make three replicates for each concentration. Add 40 μL of medium (containing 0.03% DMSO) to the wells without drug.

[0333] 7) Place the 96-well plate in an incubator and continue incubating for 24 hours.

[0334] 8) Observe the cell morphology of hepatocytes treated with each concentration for 24 hours and take photos (1 x 10 photo, taken at the center of the well of a 96-well plate).

[0335] 9) Discard the supernatant and mix the CellTiter-Glo Luminescent Cell Viability Assay reagent with PBS at a 1:1 volume ratio. Add 200 μL / well to a 96-well plate.

[0336] 10) Mix and lyse for 2 minutes, then incubate at room temperature for 10 minutes.

[0337] 11) Measure the luciferase signal value using a microplate reader.

[0338] 4. Data Processing and Analysis

[0339] 1) Observe the cell status and take photos

[0340] 2)CellTiter-Glo Luminescent Cell Viability Assay

[0341] GraphPad Prism 8.0 software was used to process the data graphically and calculate the IC 50The data were analyzed by four-parameter nonlinear regression and fitted with the appropriate dose-effect curve. The survival rate was calculated as follows:

[0342] Cell viability (%) = (Lum 待测孔 -blank) / (Lum 阴性对照 -blank)×100%

[0343] Half-inhibitory concentration (IC 50 )calculate:

[0344] GraphPad Prism 8.0 software was used to convert the X value (theoretical final concentration) into Log(X), and the data were processed using the log(inhibitor) vs. response -Variable slope analysis method to obtain the Bottom, Top, and LogIC 50 , Hillslope four parameter values, and then according to the equation:

[0345] Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 -LogX)*HillSlope))

[0346] Calculate the X value when Y=50, which is IC 50 value.

[0347] 5. Experimental Results

[0348] Table 35. Human primary hepatocyte toxicity

[0349] 6. Experimental Conclusion

[0350] In the same human primary hepatocytes, single-drug IC 50 From small to large, they are compound 4

Claims

1. A cyclohexane derivative, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, It is characterized in that The structure of the cyclohexane derivative is shown in the following formula: Compound 1 or Compound 2: Provided that the cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof is not Compound 1 in a free form.

2. A cyclohexane derivative as claimed in claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, It is characterized in that The structure of the cyclohexane derivative is shown in the following formula:

3. A stereoisomer of the cyclohexane derivative according to claim 1 or a pharmaceutically acceptable salt thereof, It is characterized in that The structure of the cyclohexane derivative is shown in the following formula:

4. A pharmaceutical composition, It is characterized in that The invention comprises the cyclohexane derivative as claimed in claim 1 or 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, It is characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

6. Use of the cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of a medicament for treating or preventing diseases associated with dopamine receptors.

7. The cyclohexane derivative according to claim 1 or 2, its stereoisomer or its pharmaceutically acceptable salt in the preparation of dopamine D 3 Receptor agonists or antagonists.

8. Use of the cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of a medicament for treating or preventing diseases associated with 5-hydroxytryptamine receptors.

9. The cyclohexane derivative according to claim 1 or 2, its stereoisomer or its pharmaceutically acceptable salt in the preparation of a drug for treating or preventing 5-HT 1A Receptors, 5-HT 2A Receptors and / or 5-HT 2B Application of drugs in the treatment of receptor-related diseases.

10. The cyclohexane derivative according to claim 1 or 2, its stereoisomer or its pharmaceutically acceptable salt in the preparation of 5HT 1A Receptor agonists or antagonists.

11. Use of the cyclohexane derivative, its stereoisomer or a pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the preparation of a medicament for treating or preventing neuropsychiatric diseases.

12. The use according to claim 11, It is characterized in that The neuropsychiatric disease is selected from one or more of schizophrenia, psychosis, mental disorder, insanity, mood disorder, bipolar disorder, depression, phobia, obsessive-compulsive disorder, anxiety disorder and cognitive disorder.

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