Phthalimide derivatives, pharmaceutical compositions comprising the same and medical uses thereof

Abstract

The present application provides the following compound (I) or its pharmaceutically acceptable salt, ester, optical isomer, tautomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate or prodrug, and a pharmaceutical composition containing the compound of the present application, and also provides the use of the compound of the present application as a 2-type potassium-chloride co-transporter KCC2 agonist, and the application in the preparation of a drug for a disease related to 2-type potassium-chloride co-transporter KCC2, and a corresponding pharmaceutical composition,

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of phthalimide derivatives, and particularly relates to a phthalimide derivative as an agonist of potassium-chloride cotransporter 2 (KCC2) and use thereof, and a related drug screening method. BACKGROUND

[0002] Neurological diseases include brain or spinal cord injury, stroke, pathological pain, dyskinesia, neurodegenerative diseases, genetic and developmental abnormalities of the nervous system, etc., which are widely ranged and seriously ill, and often lead to disability. They are the largest disease category that currently threatens human life and health. However, due to the complexity of neurological diseases leading to hemiplegia, related research has been ongoing for many years, but there is still a lack of effective therapeutic drugs, so patients suffering from these diseases cannot be effectively treated, and there is still a blank in the market for drugs that can promote the functional recovery of hemiplegic patients.

[0003] Recent scientific research has confirmed that neurological diseases such as stroke, spinal cord injury and epilepsy are related to the dysfunction of cation-chloride cotransporters (CCCs), including NKCC1 and NKCC2 subtypes, KCC1-KCC4 subtypes, and NCC. These cotransporters are involved in regulating the intracellular chloride ion concentration of neurons, thereby maintaining the balance between neuronal excitability and inhibition. Further research has found that neurological diseases are significantly related to the intracellular chloride ion concentration of neurons and abnormal neuronal function. For example, in the central nervous system, KCC2 mutation can significantly affect its activity in transporting chloride ions out of the cell, thereby causing neurological diseases.

[0004] Recently, in order to treat KCC2-related neurological diseases, researchers from Canada screened a series of KCC2 agonist small molecule compounds, and finally found a KCC2-specific CLP series compound. Preliminary biochemical experiments showed that CLP257 can significantly improve the activity of KCC2 in transporting chloride ions out of the cell (Gagnon et al., 2013). However, recent studies have found that the activity of CLP290 drug is general, the water solubility is poor, and the bioavailability is low, so it is urgent to screen new drugs with better effect and higher specificity to meet the requirements of clinical trials (Cardarelli et al., 2017).

[0005] At present, whether developing agonist or inhibitor drugs of CCCs, it is inseparable from the recognition of the interaction between the drug molecules and the target proteins. With the development of computer technology and the popularization of modern biomedical technology, the amount of biomedical data is showing an exponential growth trend. With the support of sufficient data, the artificial intelligence assisted drug design based on virtual high-throughput screening has become an important technology for drug compound molecular discovery. The artificial intelligence (AI) assisted virtual drug screening technology can shorten the drug development time and save the development cost. Among them, the virtual screening technology based on the multi-modal information of the accurate target molecule three-dimensional structure and sequence level is the most advantageous method in the current virtual screening. Therefore, based on this technology, a series of small molecule compounds with high activity, high selectivity and good drugability are found. The present application includes the chemical structure, use, preparation method, screening and optimization method of the small molecule compound. SUMMARY

[0006] The inventors of the present application found that the compounds having the structure shown in the following formula (I) have good type 2 potassium-chloride co-transporter KCC2 activation activity, target ion channels on nerve cells, can tightly bind to them and produce agonistic effect, thereby playing a therapeutic and preventive role in neurological diseases. The new pharmacological activity of these compounds is obtained based on the artificial intelligence method: first, screening from a large number of compound libraries (300 million compound libraries), predicting the affinity based on the artificial intelligence model, evaluating the drugability and rationalization index of each compound, and comprehensively sorting, wet experiment verification of the top 30 molecules to determine the molecular skeleton, according to the results, continue to reduce the candidate compound library (to about 30,000 compounds), further sorting based on the same method, wet experiment of the top 4 compounds, get the candidate drugable compounds; the use of this method can shorten and accelerate the discovery and screening period of many new drugs, save the drug development cost, and at the same time play a guiding and inspiring role in the drug design process.

[0007] Specifically, the present application provides a phthalimide compound represented by the following formula (I) or a pharmaceutically acceptable salt, ester, optical isomer, tautomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate or prodrug thereof,

[0008]

[0009] wherein:

[0010] Ring A is a saturated or partially unsaturated 3-10 membered heterocyclic group, a saturated or partially unsaturated 3-10 membered bridged heterocyclic group, a substituted or unsubstituted C6-14 aryl group, a 5-14 membered heteroaryl group;

[0011] E is selected from H, halogen, NH2, OH, SH, CN, guanidino, C1-6alkyl substituted amino, substituted or unsubstituted C1-10aliphatic, substituted or unsubstituted saturated or partially unsaturated 3-10 membered bridged cycloalkyl, substituted or unsubstituted saturated or partially unsaturated 3-10 membered heterocyclyl, substituted or unsubstituted saturated or partially unsaturated 3-10 membered bridged heterocyclyl, substituted or unsubstituted saturated or partially unsaturated 3-10 membered fused heterocyclyl, substituted or unsubstituted C6-14aryl, or substituted or unsubstituted 5-14 membered heteroaryl, -C(=O)R 5 , -OC(=O)R 5 , -C(=O)OR 5 , -OR 5 , -SR 5 , -S(=O)R 5 , -S(=O)2R 5 , -S(=O)2N(R 5 )2, -N(R 5 )2, -C(=O)N(R 5 )2, -NR 5 -C(=O)R 5 , -NR 5 -C(=O)OR 5 , -NR 5 -S(=O)2-R 5 , C(=O)-N(R 5 )2, -C1-6alkylene-N(R 5 )2, -C1-6alkylene-OR 5 , -C1-6alkenylene-OR 5 , and -O-C1-6alkylene-N(R 5 )2, wherein R 5 is H, NH2, C1-C6alkyl, saturated or partially unsaturated C 3-6 cycloalkyl, saturated or partially unsaturated 3-10 membered heterocyclyl, or C6-10aryl;

[0012] L 1 and L 2 each independently represent a bond, or a straight or branched C1-C6alkylene chain, wherein the straight or branched C1-C6alkylene chain is optionally substituted with one or more selected from -O-, -CO-, -C(=O)O-, -CONH-, -NHCO-, -NHCONH-, -NH-, -NR 5 -, -C(R 5)2-, -S-, sulfinyl, sulfonyl, sulfoxyl, -aminosulfonylamino-, alkynylene, alkenylene, cycloalkylene, or a divalent group selected from the group consisting of -C(=O)R 5 , -OC(=O)R 5 , -C(=O)OR 5 , -OR 5 , -SR 5 , -S(=O)R 5 , -S(=O)2R 5 , -S(=O)2N(R 5 )2, -N(R 5 )2, -C(=O)N(R 5 )2, -NR 5 -C(=O)R 5 , -NR 5 -C(=O)OR 5 , -NR 5 -S(=O)2-R 5 , -C(=O)-N(R 5 )2, -C(=O)-N(OR 5 )R 5 , -C1-6alkylene-N(R 5 )2, -C1-6alkylene-OR 5 , -C1-6alkenylene-OR 5 and -O-C1-6alkylene-N(R 5 )2interrupted one or more times, or the straight or branched C1-C6alkylene chain can be substituted with one or more groups selected from the group consisting of -C(=O)R

[0013] R 1 is selected from the group consisting of halogen, cyano, substituted or unsubstituted C1-C3alkyl, substituted or unsubstituted C1-C3alkoxy, NH2, OH, SH, CN, guanidino, C1-6alkyl substituted amino; n is an integer from 0 to 3, and when n is 2 or more, the plurality of R 1 may be the same or different; the plurality of R 1 together with the carbon atom of the phenyl ring to which they are attached can form a 3- to 7-membered aliphatic cyclic group or a heterocyclic group;

[0014] R 2 is independently selected from the group consisting of substituted or unsubstituted C1-C3alkyl, hydroxy, halogen, cyano, amino, C1-C3alkyl substituted amino, substituted or unsubstituted C1-C3alkoxy, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted C3-C6heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5- to 6-membered heteroaryl, S(O)2R b1, S(O)2NH2, S(O)2NHR b1 , S(O)2NR b1 R b2 , NHS(O)2R b1 , NR b1 S(O)2R b2 , S(O)(NH)R b1 , S(O)(NR b1 )R b2 , C(O)R b1 , C(O)OR b1 , OC(O)R b1 , NHC(O)R b1 , NR b1 C(O)R b2 , NHC(O)OR b1 , NR b1 C(O)OR b2 , C(O)NH2, C(O)NHR b1 , C(O)NR b1 R b2 , C(O)NHS(O)2R b1 , S(O)2NHC(O)R b1 ; R b1 and R b2 are each independently selected from substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted 3-6 membered cycloalkyl or heterocyclyl, or R b1 and R b2 together with the N atom to which they are attached form a 3-7 membered heterocyclyl; n is an integer from 0 to 4, and when n is 2 or more, the multiple R 2 may be the same or different,

[0015] indicates the position of attachment to the parent nucleus, the expression of the ring structure with a dash through it indicates the point of attachment on the ring structure at any atom capable of bonding,

[0016] The above-mentioned substituted or unsubstituted means that the H in the group is replaced with a group selected from halogen, cyano, hydroxyl, C1-C3 alkoxy, C4-C6 heterocycloalkyl, or a combination of one or more of the above, or means that two H in -CH2- in the group is replaced with oxo =O.

[0017] In preferred embodiments of the application, ring A and its R 2 combinations are groups selected from:

[0018]

[0019]

[0020]

[0021]

[0022] denotes the position of attachment to the mother nucleus, Hal denotes a halogen atom, the representation of the ring structure crossed by a line denotes the attachment site to an arbitrary position of the ring structure which is capable of forming a bond.

[0023] In a preferred embodiment of the application, L 1 and L 2 each independently denotes a single bond or a bivalent radical of the formula (V),

[0024]

[0025] n0 is 0 or 1; m is an integer from 1 to 5, preferably 2 or 3; n1 is an integer from 0 to 3, preferably 1; n2 is an integer from 0 to 3, preferably 1; n3 is an integer from 0 to 3, preferably 1; n4 is an integer from 0 to 3, preferably 1; n5 is an integer from 0 to 3, preferably 1;

[0026] Z0 is a single bond, -CH2-, -CHR 9 -, -NH-, -O-, -S-,

[0027] -CO-, or -C(=O)O-;

[0028] Z1 is a single bond, -CH2-, -CHR 9 -, -NH-, -O-, -S-,

[0029] -CO-, or -C(=O)O-;

[0030] Z2 is a single bond, -CH2-, -CHR 9 -, -NH-, -O-, -S-,

[0031] -CO-, or -C(=O)O-;

[0032] wherein R 9 is C1-6alkyl substituted amino, substituted or unsubstituted C1-10aliphatic hydrocarbon group, -C(=O)R 10 , -OC(=O)R 10 , -C(=O)OR10 , -OR 10 , -SR 10 , -S(=O)R 10 , -S(=O)2R 10 , -S(=O)2N(R 10 )2, -N(R 10 )2, -C(=O)N(R 10 )2, -NR 10 -C(=O)R 10 , -NR 10 -C(=O)OR 10 , -NR 10 -S(=O)2-R 10 , C(=O)-N(R 10 )2, -C1-6alkylene-N(R 10 )2, -C1-6alkylene-OR 10 , -C1-6alkenylene-OR 10 , and -O-C1-6alkylene-N(R 10 )2; wherein R 10 is H, guanidino, C1-C6alkyl, saturated or partially unsaturated C3-6cycloalkyl, saturated or partially unsaturated 3-10 membered heterocyclyl, or C6-10aryl; represents the position of attachment, and the direction of attachment at either end is arbitrary,

[0033] The above-mentioned substituted or unsubstituted means that the H in the group is replaced with a group selected from halogen, cyano, nitro, hydroxyl, amino, aldehyde, ester, C1-C30alkyl, C1-6alkyl substituted amino, C1-C30alkoxy, C2-C20heterocycloalkyl, C1-C30alkylsilyl, C6-C30aryl, C6-C30aryloxy, C3-C30heteroaryl, C6-C30arylamino, or C3-C30heteroarylamino, or at least two combinations thereof, or means that two H in -CH2- in the group are replaced with oxo=O.

[0034] In preferred embodiments of the application, L 1 is a group selected from methylene, ethylene, propylene, and the following divalent groups:

[0035]

[0036] L 2 is a group selected from a chemical bond, methylene, ethylene, propylene, and the following divalent groups:

[0037]

[0038] represents a position to be connected to a mother nucleus, and the expression of the ring structure with a dash through it represents a connection site at any position on the ring structure that can form a bond.

[0039] In a preferred embodiment of the present application, m is an integer of 0 to 2, R 1 selected from a halogen atom, a methyl group, an amino group, a cyclopropyl group, a propyl group, the following groups:

[0040]

[0041] or two R 1 form a substituted or unsubstituted cyclopentane or a substituted or unsubstituted cyclohexane structure,

[0042] The above-mentioned substituted or unsubstituted means that H in the group is substituted with a group selected from a halogen, a cyano group, a nitro group, a hydroxyl group, an amino group, an aldehyde group, an ester group, a C1-C30 alkyl group, a C1-6 alkyl-substituted amino group, a C1-C30 alkoxy group, a C2-C20 heterocyclic alkyl group, a C1-C30 alkylsilyl group, a C6-C30 aryl group, a C6-C30 aryloxy group, a C3-C30 heteroaryl group, a C6-C30 arylamino group, or a C3-C30 heteroarylamino group, or at least two of the above, or means that two H in -CH2- in the group are replaced with oxo = O,

[0043] represents a position to be connected to a mother nucleus, and the expression of the ring structure with a dash through it represents a connection site at any position on the ring structure that can form a bond.

[0044] Another aspect of the present application provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound of the present application or a pharmaceutically acceptable salt, ester, optical isomer, tautomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate, or prodrug thereof, and a pharmaceutically acceptable carrier, which is a solid preparation, a semi-solid preparation, a liquid preparation, or a gaseous preparation.

[0045] In a preferred embodiment, the pharmaceutical composition is in the form of an oral dosage form or an injection, and the oral dosage form includes a capsule, a tablet, a pill, a powder, and a granule. Liquid dosage forms for oral administration include a pharmaceutically acceptable emulsion, a solution, a suspension, a syrup, or a tincture,

[0046] The injection comprises a physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder of the above-mentioned compound or its pharmaceutically acceptable salt, ester, optical isomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate or prodrug for re-dissolving into a sterile injectable solution or dispersion.

[0047] The present application provides use of the compound of formula (I) or its pharmaceutically acceptable salt, ester, optical isomer, tautomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate or prodrug thereof in the preparation of a 2-type potassium-chloride co-transporter KCC2 activator.

[0048] The present application provides use of the compound of formula (I) or its pharmaceutically acceptable salt, ester, optical isomer, tautomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate or prodrug thereof in the preparation of a drug for a 2-type potassium-chloride co-transporter KCC2 related disease.

[0049] In a preferred embodiment, the 2-type potassium-chloride co-transporter KCC2 related disease is brain or spinal cord injury, stroke, pathological pain, dyskinesia, neurodegenerative disease, genetic and developmental abnormalities of nervous system.

[0050] The present application provides use of the compound of formula (I) or its pharmaceutically acceptable salt, ester, optical isomer, tautomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate or prodrug thereof in the preparation of a drug for treating or preventing a nervous system disease. The nervous system disease is brain or spinal cord injury, stroke, pathological pain, dyskinesia, neurodegenerative disease, genetic and developmental abnormalities of nervous system.

[0051] Another aspect of the present application also provides a screening method of a drug for treating or preventing a nervous system disease, Cation-chloride co-transporter drugs Definitions The establishment step of the cell screening platform: obtain a lentivirus Lenti-puro-Clomeleon expressing Cl-sensitive fluorescent protein Clomeleon by virus packaging, infect the NG108-15 cell line with the virus and obtain a NG108-Cl cell line stably expressing Clomeleon through antibiotic screening, and determine the 2-type potassium-chloride co-transporter KCC2 activation activity of the target compound by using the NG108-Cl cell line stably expressing Clomeleon.

[0052] Another aspect of the present application also utilizes the binding pocket of the candidate compound targeting KCC2 protein agonist, sequence information and structure information, with the following steps and by data analysis to verify the quality, distribution and learnability of the data, apply data preprocessing, vectorize the data of the text record to input into the algorithm core unit as the input data during model training;

[0053] Target protein determination and affinity calculation step, AI model prediction module, taking KCC2 protein as the target protein, extracting the structure information and sequence information of the protein for extraction and characterization as the first input information of the model; Vectorize the molecular information of each compound in the compound library as the second input information of the model, and the algorithm model will combine the compound and the target protein information, and output the affinity value of each compound and the target protein;

[0054] Molecular docking step, the three-dimensional structure information of the protein and the three-dimensional structure information of the compound are simulated and simulated by molecular dynamics principle, and the binding force prediction score result based on molecular dynamics is given;

[0055] Drugability evaluation step, based on the principle of drugability, the properties of each compound are evaluated from lipophilicity, molecular weight, Lipinski's five principles and QED; and based on the properties of the compound, the comprehensive score of the drug of the compound is given;

[0056] Based on the affinity value, the binding force prediction score and the comprehensive score of the drug of the compound, the comprehensive affinity score is obtained by weighted summation, and the list of lead compounds to be screened is given according to the comprehensive affinity score,

[0057] Pharmacological and pharmacological verification step, based on pharmacological and pharmacological experiments, the screened compounds are further verified to obtain real lead compounds;

[0058] Another aspect of the present application also provides a screening system of a drug for treating or preventing nervous system diseases, which targets the binding pocket of KCC2 protein agonist, obtains lead compounds from sequence information and structure information,

[0059] The system comprises a network input / output interface, a user input / output interface, a processing unit, a storage unit, and an algorithm engine; wherein the performance of the algorithm engine is described in S3; the storage unit is used to store local data and real-time update new experimental data uploaded in the cloud database to periodically increase the diversity of training samples and improve data quality; the processing unit mainly comprises a central processing unit and an image processing unit, the central processing unit is used to support users to execute or submit instructions and tasks, support data transmission and functions necessary for normal operation of the system, and the image processing unit is used to support model training, model calling and data prediction and other tasks; the network input / output interface is used for the connection of the gateway to ensure the accessibility and smoothness of the network; the user input / output interface is used to support users to access the system locally or remotely, send instructions and tasks, and has the following modules:

[0060] A candidate molecule determination and processing module, and the quality, distribution and learnability of the data are verified through data analysis, the data recorded in the text are vectorized and represented through data preprocessing, and are input into the algorithm core unit as input data during model training;

[0061] A target protein determination and affinity calculation module, in the AI model prediction module, the KCC2 protein is taken as the target protein, the structure information and sequence information of the protein are extracted and represented as the first input information of the model; each compound molecular information in the compound library is vectorized and represented as the second input information of the model, and the algorithm model combines the compound and the target protein information, and outputs the affinity value of each compound combined with the target protein;

[0062] A molecular docking module, the three-dimensional structure information of the protein and the three-dimensional structure information of the compound are simulated and simulated through the principle of molecular dynamics, and the binding force prediction score result based on molecular dynamics is given;

[0063] A drugability evaluation module, based on the principle of drugability, the properties of each compound are evaluated from lipophilicity, molecular weight, Lipinski's five principles and QED; and based on the properties of the compound, a comprehensive score of the drugability of the compound is given.

[0064] The following describes the other elements of the application in more detail.

[0065] Preferred compounds of the invention

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Reference herein to technical terms used herein is intended to refer to the technical terms as commonly understood by those skilled in the art, including variations or substitutions of techniques or equivalents of techniques that would be apparent to those skilled in the art. Although it is believed that the following terms are well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate the understanding of the present application.

[0067] The terms "comprise", "comprising", "have", "having", "include", "including" or "contain", "containing", or "provide", "provided", "provided with", "provided having" and other variants thereof as used herein are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0068] As used herein, the term "alkylene" means a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methylene, ethylene, propylene or butylene.

[0069] As used herein, the term "alkyl" is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, for example 1 to 6, carbon atoms. For example, as used herein, the term "Ci-6alkyl" refers to a linear or branched group of 1 to 6 carbon atoms (e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, neopentyl or n-hexyl) which is optionally substituted by 1 or more (such as 1 to 3) suitable substituents such as halogen (in which case the group is referred to as "haloalkyl") (e.g. CH2F, CHF2, CF3, CCI3, C2F5, C2CI5, CH2CF3, CH2CI or -CH2CH2CF3, etc.). The term "Ci-4alkyl" refers to a linear or branched aliphatic hydrocarbon chain of 1 to 4 carbon atoms (i.e. methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl or t-butyl).

[0070] As used herein, the term "alkenyl" means a linear or branched monovalent hydrocarbon group which contains one double bond and has 2-6 carbon atoms ("C2-6alkenyl"). The alkenyl group is for example vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl and 4-methyl-3-pentenyl. When the compounds of the present application contain an alkenyl group, the compounds can exist in pure E (entgegen) form, pure Z (zusammen) form or in any mixture thereof. 2-6 The alkenyl group is for example vinyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl and 4-methyl-3-pentenyl. When the compounds of the present application contain an alkenyl group, the compounds can exist in pure E (entgegen) form, pure Z (zusammen) form or in any mixture thereof.

[0071] As used herein, the term "alkynyl" means a monovalent hydrocarbon group containing one or more triple bonds, which preferably has 2, 3, 4, 5, or 6 carbon atoms, such as ethynyl or propynyl.

[0072] As used herein, the term "cycloalkyl" means a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spiro, fused, or bridged systems (such as bicyclo[l. l. l]pentyl, bicyclo[2.2. l]heptyl, bicyclo[3.2. l]octyl, or bicyclo[5.2.0]nonyl, decahydronaphthyl, and the like)), which is optionally substituted with 1 or more (such as 1 to 3) suitable substituents. The cycloalkyl group has 3 to 15 carbon atoms. For example, the term "C3-C6cycloalkyl" means a saturated monocyclic hydrocarbon ring having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, which is optionally substituted with 1 or more (such as 1 to 3) suitable substituents. 3-6 As used herein, the term "cycloalkyl" means a saturated monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spiro, fused, or bridged systems (such as bicyclo[l. l. l]pentyl, bicyclo[2.2. l]heptyl, bicyclo[3.2. l]octyl, or bicyclo[5.2.0]nonyl, decahydronaphthyl, and the like)), which is optionally substituted with 1 or more (such as 1 to 3) suitable substituents. The cycloalkyl group has 3 to 15 carbon atoms. For example, the term "C3-C6cycloalkyl" means a saturated monocyclic hydrocarbon ring having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, which is optionally substituted with 1 or more (such as 1 to 3) suitable substituents.

[0073] As used herein, the terms "cycloalkylene", "cycloalkyl", and "hydrocarbon ring" mean a saturated (i.e., "cycloalkylene" and "cycloalkyl") or unsaturated (i.e., having one or more double and / or triple bonds within the ring) monocyclic or polycyclic hydrocarbon ring having, for example, 3-10 (suitably 3-8, more suitably 3-6) ring carbon atoms, including, but not limited to, (cyclo)propyl (ring), (cyclo)butyl (ring), (cyclo)pentyl (ring), (cyclo)hexyl (ring), (cyclo)heptyl (ring), (cyclo)octyl (ring), (cyclo)nonyl (ring), (cyclo)hexenyl (ring), and the like.

[0074] As used herein, the terms "heterocyclyl," "heterocyclyl ene," and "heterocycle" refer to saturated (i.e., heterocycloalkyl) or partially unsaturated (i.e., having one or more double bonds and / or triple bonds within the ring) cyclic groups having, for example, 3-10 (suitably having 3-8, more suitably having 3-6) ring atoms, at least one of which is a heteroatom selected from N, O, and S, and the remainder of which are C. For example, a "3-10 membered (hetero)cycloalkyl" is a saturated or partially unsaturated (hetero)cycloalkyl group having 2-9 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) heteroatoms independently selected from N, O, and S. Examples of heterocyclyl ene and heterocycle groups include, but are not limited to, (hetero)cyclooxanyl, (hetero)cycloaziridinyl, (hetero)cycloazetidinyl, (hetero)cyclooxetanyl, (hetero)cyclo tetrahydrofuranyl, (hetero)cyclo dioxolinyl, (hetero)cyclopyrrolidinyl, (hetero)cyclopyrrolidonyl, (hetero)cycloimidazolidinyl, (hetero)cyclopyrazolidinyl, (hetero)cyclopyrrolinyl, (hetero)cyclo tetrahydropyranyl, (hetero)cyclo piperidinyl, (hetero)cyclo morpholinyl, (hetero)cyclo dithianyl, (hetero)cyclo thiomorpholinyl, (hetero)cyclo piperazinyl, or (hetero)cyclo trithianyl. The groups also encompass bicyclic systems, including spiro, fused, or bridged systems (such as 8-azaspiro[4.5]decane, 3,9-diazaspiro[5.5]undecane, 2-azabicyclo[2.2.2]octane, and the like). The heterocyclyl ene and heterocycle groups can be optionally substituted with one or more (e.g., 1, 2, 3, or 4) suitable substituents.

[0075] As used herein, the terms "(hetero)aryl" and "aromatic ring" refer to all-carbon monocyclic or fused-ring polycyclic aromatic groups having a conjugated pi-electron system. For example, as used herein, the terms "C 6-10 (hetero)aryl" and "C 6-10 aromatic ring" mean aromatic groups containing 6 to 10 carbon atoms, such as (hetero)phenyl (benzene ring) or (hetero)naphthyl (naphthalene ring). The (hetero)aryl and aromatic ring are optionally substituted with 1 or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, C 1-6 alkyl, and the like).

[0076] As used herein, the terms “(hybrid)aryl” and “heteroaromatic ring” refer to monocyclic, bicyclic, or tricyclic aromatic ring systems having 5, 6, 8, 9, 10, 11, 12, 13, or 14 ring atoms, particularly 1, 2, 3, 4, 5, 6, 9, or 10 carbon atoms, and containing at least one heteroatom that may be the same or different (the heteroatom being, for example, oxygen, nitrogen, or sulfur), and additionally, in each case, may be benzofused. Specifically, "(hybrid)aryl" or "heteroary ring" is selected from (hybrid)thienyl, (hybrid)furanyl, (hybrid)pyrroleyl, (hybrid)oxazolyl, (hybrid)thiazolyl, (hybrid)imidazolyl, (hybrid)pyrazolyl, (hybrid)isooxazolyl, (hybrid)isothiazolyl, (hybrid)oxadiazolyl, (hybrid)triazolyl, (hybrid)thiadiazolyl, etc., and their benzo[derivatives]; or (hybrid)pyridinyl, (hybrid)pyridinyl, (hybrid)pyrazinyl, (hybrid)triazinyl, etc., and their benzo[derivatives].

[0077] As used herein, the term "aralkyl" preferably refers to an aryl or heteroaryl-substituted alkyl group, wherein the aryl, heteroaryl, and alkyl groups are as defined herein. Typically, the aryl group may have 6-14 carbon atoms, the heteroaryl group may have 5-14 ring atoms, and the alkyl group may have 1-6 carbon atoms. Exemplary aralkyl groups include, but are not limited to, benzyl, phenylethyl, phenylpropyl, and phenylbutyl.

[0078] A more specific explanation of the terminology is as follows:

[0079] "alkyl" refers to a saturated aliphatic hydrocarbon group comprising 1-20 carbon atoms, or 1-10 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms, or 1-3 carbon atoms, or 1-2 carbon atoms of a saturated straight-chain or branched monovalent hydrocarbon group, wherein the alkyl group may be independently and optionally substituted by one or more substituents described in this invention. Further examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group may be optionally substituted or unsubstituted.

[0080] "Alkenyl" refers to a monovalent hydrocarbon group with 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, in which at least one C or C group is sp. 2double bond, wherein the group of alkenyl can be independently optionally substituted with 1 or more substituents described herein, wherein specific examples include, but are not limited to, ethenyl, allyl, and crotyl, and the like. Alkenyl can be optionally substituted or unsubstituted.

[0081] "Cycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents, the cycloalkyl ring comprising 3 to 20 carbon atoms, preferably comprising 3 to 12 carbon atoms, more preferably comprising 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused, and bridged cycloalkyl groups. Cycloalkyl groups can be optionally substituted or unsubstituted.

[0082] "Spiroalkyl" refers to a 5- to 18-membered, polycyclic group having two or more cyclic structures sharing a single carbon atom (termed a spiro atom) between the rings, the rings containing 1 or more double bonds, but no ring having a fully conjugated system of π-electrons. Preferably, 6- to 14-membered, more preferably 7- to 10-membered. Spiroalkyl groups are classified as mono-, bi-, or polycyclic, preferably mono- and bi-cyclic, preferably 4 / 5-, 4 / 6-, 5 / 5-, or 5 / 6-membered. Non-limiting examples of "spiroalkyl" groups include, but are not limited to:

[0083]

[0084] "Fused cycloalkyl" refers to a 5- to 18-membered, polycyclic group having two or more cyclic structures sharing a pair of carbon atoms between the rings, one or more rings can contain 1 or more double bonds, but no ring having a fully conjugated system of π-electrons, preferably 6- to 12-membered, more preferably 7- to 10-membered. Fused cycloalkyl groups are classified as bi-, tri-, tetra-, or polycyclic, preferably bi- or tri-cyclic, more preferably 5 / 5- or 5 / 6- bi-cyclic alkyl groups. Non-limiting examples of "fused cycloalkyl" groups include, but are not limited to:

[0085]

[0086] "Bridged cycloalkyl" refers to a 5- to 18-membered, polycyclic group having two or more cyclic structures sharing a pair of non-adjacent carbon atoms between the rings, one or more rings can contain 1 or more double bonds, but no ring having a fully conjugated system of π-electrons, preferably 6- to 12-membered, more preferably 7- to 10-membered. Bridged cycloalkyl groups are classified as bi-, tri-, tetra-, or polycyclic, preferably bi-, tri-, or tetra-cyclic, more preferably bi- or tri-cyclic. Non-limiting examples of "bridged cycloalkyl" groups include, but are not limited to:

[0087]

[0088] The cycloalkyl ring can be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring that is attached to the parent structure is cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, and the like.

[0089] "Heterocyclyl," "heterocycle," or "heterocyclic" are used interchangeably herein, and refer to a saturated or partially unsaturated, monocyclic, bicyclic or tricyclic non-aromatic ring system containing between 3 and 12 ring atoms, wherein at least one ring atom is a heteroatom such as oxygen, nitrogen, sulfur and the like. Preferred are 5- to 7-membered monocyclic rings or 7- to 10-membered bicyclic- or tricyclic rings, which can contain 1, 2 or 3 atoms selected from nitrogen, oxygen and / or sulfur. Examples of "heterocyclyl" groups include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidinyl, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl and piperazinyl. The heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring that is attached to the parent structure is heterocyclyl. The heterocyclyl group can be optionally substituted or unsubstituted.

[0090] "Spiroheterocyclyl" refers to a polycyclic group of 5 to 18 members, two or more cyclic structures, and single rings sharing one atom with each other, containing 1 or more double bonds within the ring, but no ring has a fully conjugated system of π electrons, wherein one or more ring atoms are selected from nitrogen, oxygen, sulfur or S(O) m , and the remaining ring atoms are carbon, m = 1 or 2. Preferred are 6 to 14 members, more preferred are 7 to 10 members. Spiroheterocyclyl groups are classified as mono-, bi- or polyspiroheterocyclyl groups, preferred are mono- and bi- spiroheterocyclyl groups, depending on the number of spiro atoms shared between the rings. More preferred are 4 / 4-, 4 / 5-, 4 / 6-, 5 / 5- or 5 / 6- monospiroheterocyclyl groups. Non-limiting examples of "spiroheterocyclyl" groups include, but are not limited to:

[0091]

[0092] "Fused heterocyclyl" refers to a polycyclic group of all-carbon rings, two or more cyclic structures, sharing a pair of atoms with each other, one or more rings can contain one or more double bonds, but no ring has a fully conjugated system of π electrons, wherein one or more ring atoms are selected from nitrogen, oxygen, sulfur or S(O) mheteroatoms, the remaining ring atoms being carbon, and m = 1 or 2. Preferably, the fused heterocyclyl group is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings making up the fused heterocyclyl group, it can be a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl group, preferably a bicyclic or tricyclic, more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclyl group. Non-limiting examples of "fused heterocyclyl" groups include, but are not limited to:

[0093]

[0094] "bridged heterocyclyl" refers to a polycyclic group of 5 to 18 members, containing two or more cyclic structures, which share two atoms not directly attached to each other, one or more rings can contain one or more double bonds, but no ring has a fully conjugated π-electron aromatic system, wherein one or more ring atoms are selected from nitrogen, oxygen, sulfur or S(O) m heteroatoms, the remaining ring atoms being carbon, and m = 1 or 2. Preferably, the fused heterocyclyl group is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings making up the fused heterocyclyl group, it can be a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl group, preferably a bicyclic, tricyclic or tetracyclic, more preferably a bicyclic or tricyclic. Non-limiting examples of "bridged heterocyclyl" groups include, but are not limited to:

[0095]

[0096] "aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings can be connected together in a fused manner. The so-called "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably, the aryl group is a C6-C 10 aryl, more preferably the aryl group is phenyl and naphthyl, most preferably phenyl. The aryl group can be substituted or unsubstituted. The "aryl" group can be fused to a heteroaryl, heterocyclyl or cycloalkyl group, wherein the aryl ring is connected to the parent structure, non-limiting examples include, but are not limited to:

[0097]

[0098] "Heteroaryl" means an aromatic 5- to 6-membered monocyclic ring or 9- to 10-membered bicyclic ring which can contain 1 to 4 atoms selected from nitrogen, oxygen, and / or sulfur. Examples of "heteroaryl" groups include, but are not limited to, furanyl, pyridinyl, 2-oxo-l,2-dihydropyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2,3-thiadiazolyl, benzodioxolyl, benzimidazolyl, indolyl, isoindolyl, 1,3-dioxo-isoindolyl, quinolinyl, indazolyl, benzoisothiazolyl, benzoxazolyl, and benzoisoxazolyl. The heteroaryl group can be optionally substituted or unsubstituted. The heteroaryl ring can be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is the heteroaryl ring, non-limiting examples of which include, but are not limited to:

[0099]

[0100] "Alkoxy" means a group of the formula (alkyl-O-). Alkyl is as defined herein. C1-C6alkoxy is preferred. Examples include, but are not limited to, methoxy, ethoxy, n-propyloxy, isopropyloxy, n-butyloxy, isobutyloxy, t-butyloxy, and the like.

[0101] "Haloalkyl" means an alkyl group having one or more halogen substituents, wherein the alkyl group has the meaning as defined herein. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, perfluoroethyl, 1,1-dichloroethyl, 1,2-dichloropropyl, and the like.

[0102] "Hydroxy" means the -OH group.

[0103] "Halo" means fluoro, chloro, bromo, and iodo, preferably fluoro, chloro, and bromo.

[0104] "Amino" means -NH2.

[0105] "Cyano" means -CN.

[0106] "Nitro" means -NO2.

[0107] "Benzyl" means -CH2-phenyl.

[0108] "Carboxyl" means -C(O)OH.

[0109] "Amino" means -NH2.

[0110] "Carboxylate" means -C(O)O(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

[0111] As used herein, the term "halo" or "halogen" is defined to include F, CI, Br, or I.

[0112] As used herein, the term "nitrogen-containing heterocycle" means a saturated or unsaturated monocyclic or bicyclic radical having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms and at least one nitrogen atom in the ring which can also optionally contain one or more (e.g., one, two, three, or four) ring members selected from the group consisting of N, O, C=0, S, S=0, and S(=0)2, which is attached to the remainder of the molecule through a nitrogen atom in the nitrogen-containing heterocycle and any remaining ring atom, which is optionally benzo-fused, and preferably attached to the remainder of the molecule through a nitrogen atom in the nitrogen-containing heterocycle and any carbon atom in the fused benzene ring.

[0113] The term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that normal valency is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0114] If a substituent is described as "optionally substituted," then the substituent can be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as optionally substituted with one or more of a list of substituents, then one or more hydrogens on the carbon (to the extent there are any hydrogens present) can be replaced with an independently selected optional substituent, alone or in combination. If a nitrogen of a substituent is described as optionally substituted with one or more of a list of substituents, then one or more hydrogens on the nitrogen (to the extent there are any hydrogens present) can each be replaced with an independently selected optional substituent.

[0115] The expression "single bond," "chemical bond" can be taken to mean the absence of a group and atom at the corresponding position.

[0116] If a substituent is described as "independently selected from" a group, then each substituent is selected independently of the other(s). Thus, each substituent can be the same as or different from the other (other) substituent(s).

[0117] As used herein, the term "one or more" means 1 or more than 1, e.g., 2, 3, 4, 5, or 10, under reasonable conditions.

[0118] Unless indicated otherwise, as used herein, the point of attachment of a substituent can be from any suitable position of the substituent.

[0119] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0120] This invention also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of this invention include (but are not limited to) isotopes of hydrogen (e.g., deuterium). 2 H), tritium ( 3 H); carbon isotopes (e.g., H); 11 C 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g., Cl); 18 F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Certain isotope-labeled compounds of the present invention (e.g., those doped with radioactive isotopes) can be used in drug and / or substrate tissue distribution studies (e.g., analysis). Radioactive isotope tritium (i.e. 3 H) and carbon-14 (i.e., 14C) are particularly suitable for this purpose due to their ease of incorporation and detection. Positron emission isotopes (e.g.) 11 C 18 F, 15 O and 13 Substitution of N) can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. The isotopically labeled compounds of the present invention can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations, by using a suitable isotopically labeled reagent instead of the previously used unlabeled reagent. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be isotopically substituted, for example, D2O, acetone-d6, or DMSO-d6.

[0121] "Substituted" means that one or more hydrogen atoms, preferably up to five, more preferably one to three, of the group independently of one another are replaced by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can be determined (experimentally or theoretically) by the person skilled in the art without excessive effort, as possible or impossible. For example, an amino group with a free hydrogen or a hydroxyl group can be unstable in combination with a carbon atom having an unsaturated (e.g. olefinic) bond.

[0122] "Substituted" or "substitution" as used herein, unless otherwise indicated, means that a group can be substituted with one or more groups selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halo, haloalkyl, hydroxyalkyl, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, carboxyl, carboxylate, =0, -C(O)R b , -OC(O)R b , -NR b R b , -C(O)NR b R b , -NR b C(O)R b , -S(O)NR b R b or -S(O)2NR b R b , wherein R b is as defined in general formula (I).

[0123] The term "pediatric patient" as used herein means a patient who is less than 16 years of age at the time of diagnosis or treatment. The term "child" can also be divided into the following subgroups: neonate (from birth to the first month of life); infant (1 month to 2 years); child (2 to 12 years); adolescent (12 to 21 years (up to but not including the 22nd birthday)). Berhman RE, Kliegman R, Arvin AM, Nelson we. Nelson's Textbook of Pediatrics, 15th Edition. Philadelphia: W. B. Saunders Company, 1996; Rudolph AM, et al. Rudolph's Pediatrics, 21st Edition. New York: McGrow-Hill, 2002; and Avery MD, First LR. Avery's

[0124] As used herein, an "effective amount" of a compound means an amount sufficient to down-regulate or agonize the corresponding target.

[0125] As used herein, a "therapeutically effective amount" of a compound refers to an amount that is sufficient to ameliorate or in some way reduce symptoms, halt or reverse progression of a condition, or negatively modulate or agonize a corresponding target. Such an amount can be used as a single dose or can be administered according to a regimen effective.

[0126] As used herein, "treat" refers to ameliorating or otherwise changing in any way the symptoms or pathology of a patient's condition, disorder, or disease.

[0127] As used herein, "ameliorating the symptoms of a particular disease by use of a particular compound or pharmaceutical composition" refers to any decrease, whether permanent or temporary, lasting or transient, that can be attributed to or associated with the use of the composition.

[0128] The use of the definitions and conventions of stereochemistry in this disclosure is generally in accordance with the following references:

[0129] S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present application can contain asymmetric or chiral centers, and therefore exist in different stereoisomers. All stereoisomers of the compounds of the present application, including but not limited to, diastereomeric, enantiomeric, atropisic, and their mixtures, such as racemates, form part of the present application. Diastereomeric mixtures can be separated into individual diastereomers by, for example, chromatography, crystallization, distillation, or other phase separation techniques known to those of skill in the art. Enantiomeric mixtures can be separated into individual enantiomers by, for example, the formation of diastereomeric salts, which can be separated by chromatography or other phase separation techniques, and the subsequent conversion of the individual diastereomeric salts back into the individual enantiomeric compounds. The intermediate and final compounds of the present application can also exist in different tautomeric forms, and all such forms are embraced by the scope of the application. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D, L or R, S are used to denote the absolute configuration of the molecule. The prefixes d and 1 or (+) and (-) are employed to designate the sign of the rotation of plane-polarized light by the compound, that is, the (-) or 1- prefix indicates that the compound is levorotatory, and the (+) or d- prefix indicates that the compound is dextrorotatory. These stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer can be referred to as an enantiomer if it is a mirror image of another compound and is not superimposable upon it. A mixture of such isomers is often referred to as an enantiomeric mixture and is sometimes referred to as an racemic mixture. An enantiomeric mixture is a 50:50 mixture of two enantiomers. Racemic mixtures lack the optical activity of the individual enantiomers.

[0130] "tautomers" or "tautomerically forms" refer to isomers of different energy that can interconvert by a low energy barrier. For example, prototropic tautomers (i.e., tautomers that shift protons) include tautomeric interconversions by proton migration, such as keto-enol and imine-enamine isomerization. Atom valence (valency) tautomers include interconversions that reorganize bonding electrons. Unless otherwise specified, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric isomeric) forms of the subject molecules, such as R, S configurations, (Z) and (E) isomers, and (Z) and (E) conformational isomers. Thus, individual stereochemical isomers and mixtures of its enantiomeric, diastereomeric, or geometric isomers, are within the scope of the present application. The compounds of the present application can exhibit more than one type of isomerism, and can be present in a mixture of isomers (e.g., racemic mixtures and diastereomeric pairs).

[0131] "pharmaceutically acceptable salts" refer to salts of the compounds of the present application that are safe and effective for use in humans or animals. Salts of the compounds can be obtained by addition of a sufficient amount of a base or acid to a pure solution or a suitable inert solvent of the compound to obtain the corresponding addition salt. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, and the like. Pharmaceutically acceptable acid addition salts include inorganic acid and organic acid salts, including salts of hydrochloric acid, hydrobromic acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, acetic acid, maleic acid, malonic acid, succinic acid, fumaric acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, and methanesulfonic acid, and the like (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)).

[0132] A solid line a solid wedge or a dashed wedge depicts a chemical bond of a compound of the present application. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers (e.g., particular enantiomers, racemic mixtures, etc.) are included. The use of a solid or dashed wedge to depict a bond to an asymmetric carbon atom is intended to indicate that the depicted stereoisomer is present. When present in a racemic mixture, the solid and dashed wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds of the present application are intended to exist in the form of stereoisomers, which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The compounds of the present application can exhibit more than one type of isomerism, and can be present in a mixture of isomers (e.g., racemic mixtures and diastereomeric pairs).

[0133] The present application encompasses all possible crystalline forms or polymorphs of the compounds of the present application, which can be a single polymorph or a mixture of more than one polymorph in any ratio.

[0134] It is also to be understood that certain compounds of the present application can exist in free form for treatment, or where appropriate, in the form of a pharmaceutically acceptable derivative thereof. In the present application, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites, chelates, complexes, clathrates or prodrugs, which upon administration to a patient in need thereof are capable of providing, directly or indirectly, a compound of this application or a metabolite or residue thereof. Accordingly, as used herein, reference to a "compound of the present application" is intended to encompass all such derivatives.

[0135] Pharmaceutically acceptable salts of the compounds of the present application include acid addition salts and base addition salts, including but not limited to salts containing hydrogen or coordinate bonds.

[0136] Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, heptanoate, hexanoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / dihydrogen phosphate / hydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and xinofoate.

[0137] Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum, arginine, benzathines, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc.

[0138] A review of suitable salts is found in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for making pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art.

[0139] As used herein, the term "ester" means an ester derived from the compounds of the various formulae herein, which includes physiologically hydrolysable esters (which can be hydrolyzed under physiological conditions to release the compound of the present application in free acid or alcohol form). The compounds of the present application can also be esters themselves.

[0140] The compounds of the present application can exist in solvate (preferably hydrate) form, wherein the compound of the present application contains a polar solvent, particularly, for example, water, methanol or ethanol, as a structural element of the crystal lattice of the compound. The amount of polar solvent, particularly water, can be present in stoichiometric or non-stoichiometric amounts.

[0141] One skilled in the art will appreciate that not all nitrogen-containing heterocycles are capable of forming N-oxides since nitrogen requires an available lone pair of electrons to oxidize to an oxide; one skilled in the art will recognize which nitrogen-containing heterocycles are capable of forming N-oxides. One skilled in the art will also recognize that tertiary amines are capable of forming N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include oxidation of the heterocycle or tertiary amine with peroxy acids such as peroxyacetic acid and meta-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl hydroperoxides such as tert-butyl hydroperoxide, sodium perborate and dioxiranes such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see for example: T. L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp 748-750; A. R. Katritzky and A. J. Boulton, Eds., Academic Press; and G. W. H. Cheeseman and E. S. G. Werstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp 390-392, A. R. Katritzky and A. J. Boulton, Eds., Academic Press.

[0142] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0143] This invention further includes, within its scope, prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention that may themselves have little or no pharmacological activity, which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of the invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella). The prodrugs of the invention can be prepared, for example, by replacing suitable functional groups present in the compounds of the invention with certain portions known to those skilled in the art as “pro-moiety” (e.g., as described in “Design of Prodrugs,” H. Bundgaard (Elsevier, 1985)).

[0144] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in TW Greene & P. ​​GMWuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0145] The term "about" refers to a range of ±10% of the stated value, preferably ±5%, and more preferably ±2%.

[0146] Figure 5

[0147] The general formula and preferred scope of the compounds of the present invention have been described. More preferably, specific examples of the compounds of the present invention may be selected from any of the following structures, but are not limited to the following compounds:

[0148] In a preferred embodiment of the present application, it is one of the following specific compounds:

[0149] Based on the screening method of the present application, the AI screening model suggests that the following compounds have KCC2 activating activity. The results of the AI screened compounds are greatly influenced by the specific target molecules input, and the commonality of this series of molecules obtained in the pre-screening is the molecular fragment of phthalimide. The inventors intentionally take phthalimide as the mother nucleus, based on the bioisosteric principle, etc., and try to connect common pharmacophore groups at both ends. The inventors intentionally gradually increase the input of similar target molecules in the AI screening model, and uniformly calculate the molecular weight, oil-water partition coefficient, drug-likeness evaluation score, five-fold rule, evaluate the affinity and stability of KCC2, and the affinity of these compounds with KCC2. The calculation results show that these compounds have good affinity with KCC2, suggesting that they have KCC2 activating activity, Figure 6 and General methods for obtaining compounds of the invention The compounds shown in the above are preferred compounds, and the following compounds are also preferred compounds.

[0150]

[0151]

[0152]

[0153] Figure 4

[0154] The synthesis method of the compound of general formula (I) of the present application can refer to the synthesis method in the specific examples described below. Those skilled in the art can obtain various target compounds by replacing the corresponding raw materials and intermediates. As a general strategy, the general synthesis method of three representative compounds is given in the following Pharmaceutical compositions, medical uses and methods of treatment In these preparation methods, the reagent providing basic conditions is selected from one or more of organic bases or inorganic bases, the organic bases include but are not limited to one or more of triethylamine, N,N-diisopropylethylamine, n-butyllithium, diisopropylaminolithium, bis-trimethylsilylaminolithium, potassium acetate, sodium acetate, sodium tert-butoxide, sodium methoxide and potassium tert-butoxide, and the inorganic bases are one or more of sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, potassium acetate, cesium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate and lithium hydroxide;

[0155] The reagent providing acidic conditions includes but is not limited to one or more of hydrogen chloride, 1,4-dioxane solution of hydrogen chloride, trifluoroacetic acid, formic acid, acetic acid, hydrochloric acid, sulfuric acid, methanesulfonic acid, nitric acid and phosphoric acid;

[0156] Metal catalysts include, but are not limited to, one or more of palladium on carbon, Raney nickel, tetrakis-triphenylphosphine palladium, palladium dichloride, palladium acetate, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (Pd(dppf)Cl2), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex, bis-triphenylphosphine palladium dichloride (Pd(PPh3)Cl2), and tris(dibenzylideneacetone)dipalladium (Pd2(dba)3);

[0157] Ligands include, but are not limited to, one or more of 2-dicyclohexylphosphino-2,6'- dimethoxybiphenyl (SPhos), 4,5-bis-diphenylphosphino-9,9-dimethylxanthene (XantPhos), 2- dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (XPhos), 2-dicyclohexylphosphino-2'-(N,N- dimethylamine)-biphenyl (DavePhos), 1,1'-bis(diphenylphosphino)ferrocene (Dppf), and 1,1'- binaphthalene-2,2'-bis(diphenylphosphine) (BINAP), preferably 4,5-bis-diphenylphosphino-9,9- dimethylxanthene (XantPhos);

[0158] Condensing agents include, but are not limited to, one or more of dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(7-oxadiazolyl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (HATU), 2-(lH-benzotriazol-l-yl)-l, l,3,3-tetramethyluronium tetrafluoroborate (TBTU), l-hydroxybenzotriazole (HOBt), and l-propylphosphonic anhydride (T3P).

[0159] The above reactions are preferably carried out in a solvent, which includes, but is not limited to, N,N-dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, water, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, acetic acid, methanol, ethanol, toluene, petroleum ether, ethyl acetate, n-hexane, acetone, diethyl ether, diethylene glycol, and mixtures thereof.

[0160] Exemplary compounds of the present application include, but are not limited to, the compounds in the above table, the naming of the compounds in the present application follows systematic naming, or, the naming is carried out using ChemDraw software.

[0161] Figure 1

[0162] The present application provides a pharmaceutical composition comprising an effective amount of a compound of the present application or a pharmaceutically acceptable salt, ester, optical isomer, stereoisomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, chelate, complex, clathrate, or prodrug thereof, and a pharmaceutically acceptable carrier, which is preferably a solid, semi-solid, liquid, or gaseous preparation.

[0163] The "pharmaceutically acceptable carrier" in the present application means a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered, and which is suitable for contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0164] The pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present application include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, white

[0165] The pharmaceutical composition of the present application can act systemically and / or topically. For this purpose, they can be administered by suitable routes, such as by injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including drip infusion) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of an ophthalmic preparation, or by inhalation.

[0166] For these administration routes, the pharmaceutical composition of the present application can be administered in a suitable dosage form.

[0167] The dosage form includes, but is not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0168] The pharmaceutical compositions of the present application comprise a safe and effective amount of a compound of the present application and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, one or more of saline, buffers, dextrose, water, glycerol, ethanol, powders, and the like. The pharmaceutical preparation should be suitably formulated to be compatible with the mode of administration.

[0169] The pharmaceutical compositions of the present application can be prepared in the form of injectables, such as by conventional methods using, for example, physiologically acceptable salts of water or aqueous solutions containing glucose and other auxiliary agents. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions, such as injectables, solutions, tablets and capsules, are preferably manufactured under aseptic conditions. The pharmaceutical compositions of the present application can also be prepared in the form of powders for aerosol inhalation.

[0170] The amount of active ingredient administered will be a therapeutically effective amount, such as from about 1 microgram per kilogram body weight to about 50 milligrams per kilogram body weight per day; preferably, from about 5 micrograms per kilogram body weight to about 10 milligrams per kilogram body weight; and further preferably, from about 10 micrograms per kilogram body weight to about 5 milligrams per kilogram body weight. In addition, the compounds of the present application can be used in conjunction with other therapeutic agents.

[0171] The pharmaceutical compositions of the present application can be administered to a subject (such as a human or non-human mammal) in need thereof by conventional means. Representative modes of administration include, but are not limited to, oral, injection, aerosol inhalation, and the like.

[0172] When using the pharmaceutical compositions, a safe and effective amount of the pharmaceutical is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram body weight and, in most cases, no more than about 50 milligrams per kilogram body weight, and preferably, the amount is from about 10 micrograms per kilogram body weight to about 20 milligrams per kilogram body weight. Of course, the specific dose will also be determined by the route of administration, the health of the patient, and like factors, all within the skill of the skilled clinician.

[0173] The present application is further illustrated by the following specific examples. The procedures, conditions, reagents, and experimental methods used to carry out the present application are those conventional in the art and are not specifically limited unless otherwise specified. It is to be understood that these examples are merely illustrative of the present application and do not in any way limit the scope of the application. Unless otherwise indicated, the experimental methods in the following examples were carried out under conventional conditions or as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

[0174] As used herein, a so-called "effective amount" means the amount of a compound that, upon administration, will relieve to some extent one or more of the symptoms of the condition being treated. In particular, an "effective amount" of a compound, as used herein, means an amount of a compound sufficient to activate the potassium-chloride co-transporter type 2, KCC2. An "effective amount" of a compound, as used herein, means an amount of a compound sufficient to ameliorate or in some way reduce symptoms, halt or reverse progression of the condition, or activate the potassium-chloride co-transporter type 2, KCC2. Such an amount can be administered in a single dose, or can be divided into several doses, as appropriate, to produce the desired effect.

[0175] The dosage regimen will be adjusted to provide the optimum desired response. For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is important that dosage values be titrated to the individual patient needs and to the professional judgment of the person administering or supervising the administration of the compositions.

[0176] As used herein, "treatment" means any improvement or other alteration in the symptomology or pathology of a condition, disorder or disease in a patient, in any way. As described herein, "improving the symptoms of a particular disease by use of a particular compound or pharmaceutical composition" means any decrease, whether permanent or temporary, lasting or transient, that can be attributed to or associated with the use of that composition.

[0177] As used herein, "individual" includes a human or non-human animal. Exemplary human individuals include a human individual (referred to as a patient) who has a disease (e.g., a disease described herein) or a normal individual. "Non-human animals" in the present application include all vertebrates, e.g., non-mammals (e.g., birds, amphibians, reptiles), and mammals, e.g., non-human primates, farm animals, and / or domestic animals (e.g., sheep, dog, cat, cow, pig, etc.).

[0178] As used herein, a so-called "effective amount" means the amount of a compound or composition sufficient to significantly and positively alter the symptoms and / or conditions to be treated (e.g., to provide a positive clinical response). The effective amount of active ingredient in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient(s) employed, the particular excipient(s) / carrier(s) utilized, and like factors within the knowledge and expertise of the attending physician.

[0179] In particular, an effective amount of a compound of Formula (I) for use in the treatment of a neurological disease is an amount sufficient to reduce symptoms of a neurological disease in a human, to slow the progression of a neurological disease, or to reduce the risk of worsening symptoms in a patient with a neurological disease. In some embodiments, the pharmaceutical composition of the present application can also comprise one or more additional therapeutic or prophylactic agents. BRIEF DESCRIPTION OF DRAWINGS

[0180] Figure 2 A detailed algorithm engine flowchart is shown for embodiments of the present application;

[0181] Figure 3 A schematic diagram of a drug screening process is shown for embodiments of the present application;

[0182] Figure 4 A system overview and algorithm reliance flowchart for the present application is shown for embodiments of the present application;

[0183] Figure 5 A chemical synthesis route map for representative small molecules used in the present embodiments is shown;

[0184] Figure 6 A diagram showing the modification of moieties on the benzene ring of the core structure of the compounds and used for affinity prediction in the screening model is shown for embodiments of the present application;

[0185] Figure 7 A diagram showing the modification of moieties on the benzene ring of the core structure of the compounds and used for affinity prediction in the screening model is shown for embodiments of the present application.

[0186] Figure 2 A diagram showing the results of wet experiments of the present application. DETAILED DESCRIPTION

[0187] Example 1 Virtual screening experiment

[0188] Based on the screening idea of the present application, the following specific experiments were carried out to obtain small molecule compounds with high affinity predicted by computational chemistry based on AI molecular model screening. Specifically, the following experiments were carried out:

[0189] S1, the present patent, using the information of the targeting KCC2 protein agonist binding pocket (including sequence information and structure information), based on artificial intelligence algorithm from known compound library, the commonness of these compounds is to have phthalimide structure; Intentionally design such compounds, further carry out the same screening;

[0190] S2, in this embodiment, the binding data of compounds and proteins is obtained from a known large database, and the quality, distribution and learnability of the data are verified through data analysis. Data preprocessing is applied to vectorize the data recorded in text (semantic representation or graph representation strategy, etc.) to input into the algorithm core unit as input data for model training. In the algorithm core unit, the model usage level can be divided into three methods. Users can choose to call existing models directly for prediction tasks, set model parameters for training or retraining, or pre-train in part of the data set and fine-tune to optimize the performance of the model in related tasks, then make predictions for the corresponding tasks, and finally output the measured affinity score to represent the strength and possibility of the combination between the protein and the compound. The affinity prediction result is input into the next module for comprehensive evaluation from three aspects of affinity prediction score, molecular dynamics simulation result and drugability assessment of each compound to determine and output the ranking list of candidate compounds after virtual screening. The top-ranked compounds in the list are purchased or synthesized, and the initial screening experimental method is used for experimental verification. The screening results can be optionally used as a supplementary data set to rebuild the training data set;

[0191] S3, for the algorithm core unit mentioned in S2, Figure 3 The algorithm core execution steps of this embodiment are shown, which include three modules: AI model prediction module, molecular docking module and drugability assessment module (the three modules do not have to run at the same time, and any two or one of the three modules can be selected to evaluate the compounds):

[0192] S31, in the AI model prediction module, KCC2 protein is selected as the target protein to improve the prediction confidence and accuracy. The pocket position reported in the corresponding literature (Guo, 2020) is selected as the protein target object, the specific position information of the pocket in the sequence is determined, and the sequence information and structure information (calculated from spatial relative coordinates) of the pocket are extracted from the protein PDB file. In the data preprocessing stage, the structure information and sequence information are extracted and represented as the first input information of the model. Each compound molecule information in the vast compound pool (compound library) is vectorized and represented as the second input information of the model. The algorithm model combines the compound and target protein information, and outputs the affinity value of each compound binding to the target protein.

[0193] S32, in the molecular docking module, the three-dimensional structure information of the protein and the three-dimensional structure information of the compound are simulated and simulated through molecular dynamics principles, and the binding force prediction score result based on molecular dynamics is given.

[0194] S33, in the drug property evaluation module, based on the drug property principle, the properties of each compound are evaluated from lipophilicity, molecular weight, Lipinski Rule of Five, QED (quantitative evaluation of drug-like) and other aspects; and based on the properties of the compound, the comprehensive score of the drug of the compound is given;

[0195] S34, based on the final score obtained in S31, S32 and S33, the comprehensive affinity score is obtained by weighted summation, and the list of lead compounds to be screened is given according to the comprehensive affinity score, and the screened compounds are further verified by drug preliminary screening experiment to obtain real lead compounds;

[0196] S4, the system in the embodiment mainly relies on the system architecture as shown in Figure 5 The system mainly includes network input / output interface, user input / output interface, processing unit, storage unit and algorithm engine; wherein the performance of the algorithm engine is described in S3; the storage unit is used to store local data and real-time update of new experimental data uploaded in the cloud database, to periodically increase the diversity of training samples and improve the data quality; the processing unit mainly includes central processing unit and image processing unit, the central processing unit is used to support user execution or submission of instructions and tasks, support data transmission and necessary functions for normal operation of the system, the image processing unit is used to support model training, model calling and data prediction and other tasks; the network input / output interface is used for the connection of gateway, to ensure the accessibility and smoothness of the network; the user input / output interface is used to support user access to the system locally or remotely, to send instructions and tasks.

[0197] The following Table A shows the molecular weight, oil-water partition coefficient LogP, drug-like property evaluation score QED, Rule of Five, and affinity ranking distribution predicted by artificial intelligence model of each compound in the initial screening process; the left column of Table A represents Simplified molecular input line entry specification (SMILES for short), which is a specification for explicitly describing molecular structure with ASCII string. SMILES string can be imported and converted into two-dimensional graph or three-dimensional model of the molecule by most molecular editing software. Conversion into two-dimensional graph can use Helson's "Structure Diagram Generation algorithms". For example, L-alanine is written as N[C@H](C(=O)O)C.

[0198] At the same time, in order to make the affinity results and the chemical structure more intuitive, the Figure 6 and Cation-chloride co-transporter drugsThe affinity of some of the compounds of the present application to KCC2 is shown in Table A.

[0199] Table A

[0200]

[0201]

[0202]

[0203]

[0204] Example 2 wet experiment (pharmacological activity verification experiment based on virtual screening results)

[0205] The candidate molecule determination and processing steps specifically include: (1) Cation-chloride co-transporter drugs The establishment of the cell screening platform, (2) the verification of the screening method, and (3) the determination of the half maximal effective concentration (EC 50 ) of the positive compounds.

[0206] Figure 7 The establishment step of the cell screening platform. The lentivirus Lenti-puro-Clomeleon expressing the Cl-sensitive fluorescent protein Clomeleon was obtained by virus packaging, the NG108-15 cell line was infected with the virus and the NG108-Cl cell line stably expressing Clomeleon was obtained after antibiotic screening. Finally, the EC 50 of the positive compounds was determined by using the NG108-Cl cell expressing Clomeleon,

[0207] wherein Clomeleon is a known fluorescent protein and has no Chinese common name. Its DNA sequence and amino acid sequence are summarized in the following table,

[0208]

[0209] The specific steps of the wet experiment are briefly described as follows:

[0210] The steps of designing and synthesizing the whole gene of Clomeleon and constructing the lentiviral expression vector are as follows: the amino acids of green fluorescent protein (GFP) in Aequorea victoria are modified to obtain two GFP variants CFP (K26R, F64L, S65T, Y66W, N146I, M153T, V163A, N164H, H231L) and YFP (S65G, S72A, K79R, T203Y, H231L). The carbon end of CFP is connected to the nitrogen end of YFP through a 24-aa flexible peptide chain to obtain the recombinant protein Clomeleon, which has a size of 59 KDa. When the recombinant protein is excited at 440 nm, it will produce emission light of CFP at 476 nm and emission light of YFP at 508 nm. - is a fluorescence quencher of YFP, and as the concentration of Cl - increases, the fluorescence intensity at 508 nm gradually decreases, so that Clomeleon can be used as a tool for detecting the concentration of chloride ions [Cl-] in in cells. Figure 6 a) The complete nucleic acid sequence of the recombinant protein Clomeleon is synthesized and connected to the lentiviral expression vector pHAGE through the NheI and BamHI restriction enzyme sites by enzyme digestion and ligation, to obtain the lentiviral expression plasmid pHAGE-puro-Clomeleon of Clomeleon, and the plasmid map is shown in Figure 7 The pHAGE-puro-Clomeleon plasmid is transformed into the E. coli competent cell Stlb3 by heat shock, and the positive strain is selected by resistance screening, expanded and cultured, and the endotoxin-free plasmid is extracted in large quantities, and the purified lentiviral expression plasmid pHAGE-puro-Clomeleon is collected;

[0211] The steps of packaging and collecting lentivirus are as follows: the lentivirus pHAGE-puro-Clomeleon expressing Clomeleon is packaged by using a second-generation lentivirus packaging system. One day before transfection, the HEK293FT cells are seeded in a 10 cm dish at a density of 5×10 6HEK293FT cells were seeded at a density of 2 x 105cells / well in 10-cm dishes and incubated in a carbon dioxide incubator at 37°C for 12-16 h. After the cell culture reached a confluency of >70-80%, a plasmid-transfection reagent mixture was prepared for transfection of HEK293FT cells. To a 1.5-mL sterile centrifuge tube (tube A) containing 0.5 mL of serum-free DMEM basal medium, 10 μL of each of the lentiviral expression plasmid pHAGE-puro-Clomeleon (1 μg / μL), lentiviral packaging plasmid psPAX2 (1 μg / μL), and lentiviral envelope plasmid pMD2.G (1 μg / μL) were added. To another 1.5-mL sterile centrifuge tube (tube B) containing 0.5 mL of serum-free DMEM basal medium, 30 μL of lipofection transfection reagent PEI was added. Tube A and tube B were mixed well and incubated at room temperature for 5 min. Then, the solution in tube A was added to tube B, mixed gently, and incubated at room temperature for 25 min to form a complex. The culture medium in the 10-cm dish containing HEK293FT cells was replaced with fresh medium, and the plasmid-transfection reagent complex was added slowly to the edge of the cell culture dish. The dish was gently swirled to distribute the complex evenly over the cells. After incubation at 37°C for 24 h, the cell culture supernatant was collected once, and then 10 mL of fresh cell culture medium was added for continued incubation. After 48 h of incubation, the culture supernatant was collected again into the above 50-mL centrifuge tube, and 10 mL of fresh complete medium was added. The culture supernatant was collected again into the above 50-mL centrifuge tube after 72 h. Obvious green fluorescence expression was observed 48 h after transfection of the plasmid into HEK293FT cells, and the green fluorescence was more obvious after 72 h, indicating that the target virus pHAGE-puro-Clomeleon was successfully packaged. At this time, there were 30 mL of crude virus, which was filtered through a 0.22-μm filter into a new 50-mL centrifuge tube and stored at 4°C;

[0212] The steps for establishing a NG108-15 cell line stably expressing Clomeleon were as follows. NG108-15 cells were seeded at a density of 1 x 105cells / well in 6-well plates and incubated in a carbon dioxide incubator at 37°C the day before infection. The cells were allowed to grow for 24 h before being used for lentiviral transfection. A plasmid-transfection reagent mixture was prepared for transfection of NG108-15 cells. To a 1.5-mL sterile centrifuge tube (tube A) containing 0.5 mL of serum-free DMEM basal medium, 10 μL of each of the lentiviral expression plasmid pHAGE-puro-Clomeleon (1 μg / μL), lentiviral packaging plasmid psPAX2 (1 μg / μL), and lentiviral envelope plasmid pMD2.G (1 μg / μL) were added. To another 1.5-mL sterile centrifuge tube (tube B) containing 0.5 mL of serum-free DMEM basal medium, 30 μL of lipofection transfection reagent PEI was added. Tube A and tube B were mixed well and incubated at room temperature for 5 min. Then, the solution in tube A was added to tube B, mixed gently, and incubated at room temperature for 25 min to form a complex. The culture medium in the 6-well dish containing NG108-15 cells was replaced with fresh medium, and the plasmid-transfection reagent complex was added slowly to the edge of the cell culture dish. The dish was gently swirled to distribute the complex evenly over the cells. After incubation at 37°C for 24 h, the cell culture supernatant was collected once, and then 10 mL of fresh cell culture medium was added for continued incubation. After 48 h of incubation, the culture supernatant was collected again into the above 50-mL centrifuge tube, and 10 mL of fresh complete medium was added. The culture supernatant was collected again into the above 50-mL centrifuge tube after 72 h. Obvious green fluorescence expression was observed 48 h after transfection of the plasmid into NG108-15 cells, and the green fluorescence was more obvious after 72 h, indicating that the target virus pHAGE-puro-Clomeleon was successfully packaged. At this time, there were 30 mL of crude virus, which was filtered through a 0.22-μm filter into a new 50-mL centrifuge tube and stored at 4°C; 5 5 ​about 30-40%. Prepare dilution medium for lentivirus (DMEM with 2% FBS and 5 μg / mL polybrene), dilute lentivirus with the dilution medium at MOI = 100. Discard the old cell culture medium, add 2 mL lentivirus lentivirus-Clomeleon solution, set up lentivirus empty vector infection control group (no target gene clomeleon on the transfer vector), incubate at 37°C for 12-24 h. Replace the virus-containing culture medium with fresh culture medium and continue to culture for 24 h. Observe the fluorescence of the cells under a fluorescence microscope. If the positive rate is low, repeat the infection of NG108-15 cells with the crude lentivirus obtained by packaging and transfection enhancer Polybrene. After 3-4 days of virus infection, replace the cell culture medium with cell culture medium containing 2 μg / mL Puromycin (puromycin), and continuously add the drug for 2 weeks to obtain a NG108-15 cell line stably expressing Clomeleon (NG108-Cl). Sort the positive cells stably expressing clomeleon using a flow cytometer, and perform large-scale culture and establishment of a frozen cell bank. Western Blot can detect KCC2 expression in NG108-Cl cells Figure 7 b).

[0213] The verification step of the screening method verifies the correlation between the fluorescence ratio R(F 508 / F 476 ) and the intracellular chloride ion concentration ([Cl - ]i) in the screening method through a diffusional exchange assay. After washing the NG108-Cl cells twice with a low-chloride buffer ([Cl - ] = 4 mM, pH 7.4, 30 mM HEPES, 2 mM CaCl2, 2 mM MgSO4, 10 mM K-gluconate, 2 mM NaH2PO4, adjust the solution osmotic pressure to 310 mOsm with Na-gluconate), resuspend the cells in 11.5 mL of low-chloride buffer, with a cell density of 5 x 10 6 cells / mL, 0.5 mL per tube, centrifuge at 800 x g at room temperature for 3 min, and discard the supernatant completely. Resuspend the cells in the 22 EP tubes with the 22 buffers described above, respectively. Plate each [Cl - ] concentration (with or without Triton X-100) at 100 μL / well into a 96-well black transparent bottom enzyme plate (Greiner, 655096), with 4 replicate wells for each concentration gradient (5 x 10 5cells / well). After 10 min incubation at 37 °C, the fluorescence emission spectra (466-560 nm) at an excitation wavelength of 440 nm were measured using a multifunctional microplate reader (Thermo Scientific 5250040). The fluorescence ratio R(F 508 / F 476 ) of each solution group was calculated, and the measured R value was normalized according to the formula R = (R test -R min ) / (R max -R min ). Here, R test is the measured R value of each [Cl - ] concentration (with or without Triton X-100), R max is the maximum value among all measured R values, and R min is the minimum value among all measured R values. Finally, the response curve of R value vs. [Cl - ] was plotted with [Cl - ] as the horizontal coordinate and the relative fluorescence ratio R value as the vertical coordinate. Figure 7 c) The experimental results under different pH conditions prove that this cell screening method has certain stability within a certain pH range (pH 6.5-8.0) and is not easily affected by changes in environmental pH ( Figure 7 d).

[0214] A number of compounds ranked at the top in Table A were selected for EC 50 determination. These compounds were synthesized by Wuhan Drugmaker and purchased from Aladdin Reagent Co., Ltd., and the specific structural formulas are as follows:

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] The determination steps of the half maximal effective concentration (EC 50 ) first obtained the standard curve formula of the correlation between Clomeleon fluorescence ratio (F 508 / F 476 ) and [Cl - ] i According to the reference, the emission light of YFP (fluorescent receptor) (F 508 ) and the emission light of CFP (F476 ) and the intracellular chloride concentration [Cl - ] i The relationship between the ratio (R) of the fluorescence intensity of the Clomeleon protein at 485 nm and 535 nm and the intracellular chloride concentration [Cl - ] i = Kd' • (R max -R) / (R-R min ), where Kd' is the effective dissociation constant of Clomeleon after binding to Cl - , R max is the R value measured when Clomeleon is not bound to Cl - , and R min is the R value measured when Clomeleon is bound to Cl - . Diffusion exchange experiments were performed using sterile deionized water to prepare 150 mM KF and 150 mM K-gluconate solutions, and 150 mM potassium gluconate and 150 mM KF solutions were added to NG108-Cl cell cultures, respectively, to measure the R max and R min values, and a standard curve [Cl - ] = 87*(2.527-R) / (R-1.132) was plotted according to the formula using origin software. Figure 7 e) Finally, the above-mentioned EC of compound to reduce intracellular chloride concentration 50 . After washing the NG108-Cl cells twice with a low-chloride buffer, the cells were resuspended in a low-chloride buffer to a cell density of 1 x 10 7 cells / mL, and 50 μL / well was plated into a 96-well black transparent bottom enzyme plate (5 x 10 5 cells / well). A test compound solution was prepared in a high-chloride buffer ([Cl - ] = 104 mM, pH 7.4, 30 mM HEPES, 2 mM CaCl2, 2 mM MgSO4, 10 mM K-gluconate, 2 mM NaH2PO4, 100 mM NaCl, and the osmotic pressure of the solution was adjusted to 310 mOsm using Na-gluconate) at a gradient concentration (0.5 nM to 50 μM), and an equal volume (50 μL) of the compound solution was added to the NG108-Cl cell culture (at this time, the extracellular Cl - concentration was 52 mM). The compound was allowed to co-incubate with the NG108-Cl cells at 37°C for 1 h. The fluorescence at 485 nm and 535 nm was measured at an excitation wavelength of 440 nm using a multifunctional enzyme plate reader, and the fluorescence ratio R was calculated. The intracellular Cl - concentration [Cl- The final result is expressed as a percentage decrease (P) in [Cl-]i measured in a solution containing 0.15% Triton X-100 and a low concentration of Cl-. - Cells cultured in the cell culture medium were used as a positive control for detection ([Cl)). - i positive The percentage reduction was defined as 100%, with cells cultured in a detection cell culture medium containing a low concentration of Cl- serving as a negative control ([Cl-). - i negative The percentage decrease is defined as 0%, P = ([Cl - i negative -[Cl - ]i) / ([Cl - i negative -[Cl - i positive )×100%. Plot the percentage decrease response curve of concentration - [Cl-]i with the logarithm of compound concentration (μM) on the x-axis and P on the y-axis. The maximum [Cl-]i... - The percentage decrease of i P max and minimum [Cl - The percentage decrease of i P min The concentration corresponding to 50% of the difference is the half-maximum effective concentration (EC50) of the compound. 50 Pharmacological experiments showed that compounds 1, 5, 6, and 7 had good activity, and EC values ​​were measured. 50 The values ​​were 132.5 nM, 88.1 nM, 341.4 nM, and 205.1 nM, respectively. Compound 4 exhibits a reduction in [Cl... - The effect of i is not very significant (EC). 50 >1mM). Compounds 2 and 3, along with other compounds, did not show significant activity (Table B). [Cl - The concentration response curves of some compounds are shown in the figure. ​ As shown in g.

[0221] Table B

[0222]

[0223] * : indicates that it was not detected

[0224] This invention is not limited to the above embodiments, meaning that this invention does not necessarily depend on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0225] The above describes the preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0226] In addition, it should be noted that each specific technical feature described in the above-described specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

[0227] The references briefly annotated above are as follows:

[0228] Augustine, T. K. a. G. J. (2000). A Genetically Encoded Ratiometric Indicator for Chloride: Capturing Chloride Transients in Cultured Hippocampal Neurons. Neuron 27, 447-459, 3. https: / / doi.org / 10.1016 / S0896-6273(00)00056-8.

[0229] Cardarelli, R. A., Jones, K., Pisella, L. I., Wobst, H. J., McWilliams, L. J., Sharpe, P. M., Burnham, M. P., Baker, D. J., Chudotvorova, I., Guyot, J., et al. (2017). The small molecule CLP257 does not modify activity of the K+-Cl-co-transporter KCC2 but does potentiate GABAA receptor activity. Nature Medicine 23, 1394-1396. 10.1038 / nm.4442.

[0230] Gagnon, M., Bergeron, M.J., Lavertu, G., Castonguay, A., Tripathy, S., Bonin, R.P., Perez-Sanchez, J., Boudreau, D., Wang, B., Dumas, L., et al. (2013). Chloride extrusion enhancers as novel therapeutics for neurological diseases. Nat Med 19, 1524-1528. 10.1038 / nm.3356.

[0231] Guo, Y.X.a.S.C.a.C.Z.a.F.W.a.S.L.a.J.W.a.E.D.a.S.Y.a.J. (2020). Structures and an activation mechanism of human potassium-chloride cotransporters. Science Advances 6, eabc5883, 50. 10.1126 / sciadv.abc5883.

[0232] Monette, M.Y., Rinehart, J., Lifton, R.P., and Forbush, B. (2011). Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function. Am J Physiol Renal Physiol 300, F840-847. 10.1152 / ajprenal.00552.2010.

[0233] Pond, B. B., Berglund, K., Kuner, T., Feng, G., Augustine, G. J., and Schwartz-Bloom, R. D. (2006). The chloride transporter Na(+)-K(+)-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia. J Neurosci 26, 1396-1406. 10.1523 / JNEUROSCI.1421-05.2006.

[0234] Prasher, D., Eckenrode, V., Ward, W., Prendergast, F., and Cormier, M. J. G. (1992). Primary structure of the Aequorea victoria green-fluorescent protein. 111, 229-233. 10.1016 / 0378-1119(92)90691-h.

[0235] Yeo, M., Berglund, K., Augustine, G., and Liedtke, W. (2009). Novel repression of Kcc2 transcription by REST-RE-1 controls developmental switch in neuronal chloride. J Neurosci 29, 14652-14662. 10.1523 / JNEUROSCI.2934-09.2009.

Claims

1. Use of the following compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for diseases related to potassium-chloride cotransporter type 2 (KCC2). Compound 1 Compound 6 Compound 7.

2. According to the use described in claim 1, the diseases associated with type 2 potassium-chloride cotransporter KCC2 are brain or spinal cord injury, stroke, pathological pain, movement disorders, neurodegenerative diseases, and genetic and developmental abnormalities of the nervous system.

3. A pharmaceutical composition for treating or alleviating diseases associated with potassium-chloride cotransporter type 2 (KCC2), comprising a preventive or therapeutically effective amount of the following compound and a pharmaceutically acceptable carrier. Compound 1 Compound 6 Compound 7.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is a solid dosage form, a semi-solid dosage form, a liquid dosage form, or a gaseous dosage form.

5. The pharmaceutical composition according to claim 3, wherein the disease associated with type 2 potassium-chloride cotransporter KCC2 is brain or spinal cord injury, stroke, pathological pain, movement disorders, neurodegenerative diseases, or genetic and developmental abnormalities of the nervous system.

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