DGK inhibitor compound and application thereof

Abstract

The invention relates to a DGK inhibitor compound shown in a formula (I) or pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the DGK inhibitor compound or the pharmaceutically acceptable salt, and application of the DGK inhibitor compound or the pharmaceutically acceptable salt in preparation of drugs for preventing or treating DGK-mediated diseases. # imgabs0 #

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit and priority of the following two Chinese patent applications for invention, the entire contents of which are hereby incorporated herein by reference in their entirety:

[0003] Patent Application No. 202311609001.1 filed with the National Intellectual Property Administration on November 28, 2023;

[0004] Patent Application No. 202311726435.X filed with the National Intellectual Property Administration on December 14, 2023. Technical field

[0005] The present disclosure belongs to the field of medicine and relates to a diacylglycerol kinase (DGK) inhibitor compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing them, and their use as a DGK inhibitor in the prevention or treatment of related diseases. Background art

[0006] T - cell receptors are molecules that can recognize and activate T cells. They transmit signals through some small molecules, one of which is called diacylglycerols (DAGs). They can bind to some important proteins to regulate the activity of T cells. There are two enzymes, namely diacylglycerol kinase alpha (DGKα) and diacylglycerol kinase zeta (DGKζ), which can change the structure of DAGs, making them lose the ability to transmit signals, thereby hindering the signal transmission of T - cell receptors. When these two enzymes are too active, it will lead to a weakening of the DAGs signal, and thus affect the function of T cells. The present disclosure is dedicated to developing compounds that can inhibit DGKα and DGKζ, restore DAGs signal transmission, promote T - cell - related signal pathways and enhance T - cell activity, for the treatment of cancers related to immune cell activation or cancers resistant to anti - PD - 1 antibody / anti - PD - L1 antibody therapies. Summary of the invention

[0007] The present disclosure relates to a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0008]

[0009] wherein:

[0010] is selected from a double bond or a single bond;

[0011] X 1 is selected from N and CH;

[0012] X 2 and X 3Each independently selected from O, S, and N;

[0013] R 1 Selected from hydrogen, amino, hydroxy, mercapto, cyano, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 1 -C 10 alkyl, C 3 -C 12 cycloalkyl, 4- to 10-membered heterocyclic group, C 6 -C 10 aryl, 5- to 10-membered heteroaryl, -C(O)R 3 and SO 2 R 3 , the amino, hydroxy, mercapto, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 1 -C 10 alkyl, C 3 -C 12 cycloalkyl, 4- to 10-membered heterocyclic group, C 6 -C 10 aryl and 5- to 10-membered heteroaryl are optionally substituted by R 1a ;

[0014] R 2 Selected from hydrogen, halogen, amino, cyano, C 1 -C 10 alkyl and C 1 -C 10 alkoxy, the amino, C 1 -C 10 alkyl and C 1 -C 10 alkoxy are optionally substituted by R 2a ;

[0015] R 3 Selected from amino, hydroxy, and C 1 -C 3 alkyl, the amino, hydroxy, and C 1 -C 3 alkyl are optionally substituted by R 1a ;

[0016] Ring B is selected from C 3 -C 12 cycloalkyl and 4- to 10-membered heterocyclic group, the C 3 -C 12 cycloalkyl and 4- to 10-membered heterocyclic group are optionally substituted by halogen, C 1 -C 6alkyl, C 1 -C 6 haloalkyl or C 1 -C 6 is substituted with an alkoxy group;

[0017] The cyclic C is selected from C 3 -C 12 cycloalkyl, 4- to 10-membered heterocyclic group, C 6 -C 10 aryl, and 5- to 12-membered heteroaryl, and the C 3 -C 12 cycloalkyl, 4- to 10-membered heterocyclic group, C 6 -C 10 aryl, and 5- to 12-membered heteroaryl are optionally substituted with R b ;

[0018] L is selected from a bond, -O-, -S-, -NR c -, and -CR c R c -;

[0019] R 1a is independently selected from halogen, hydroxy, amino, cyano, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, 4- to 10-membered heterocyclic group, C 1 -C 6 alkoxy, NH(C 1 -C 6 alkyl), N(C 1 -C 6 alkyl) 2 , C(O)C 1 -C 6 alkyl, and 5- to 10-membered heteroaryl;

[0020] R 2a is independently selected from halogen, hydroxy, amino, cyano, and C 1 -C 3 alkyl;

[0021] R b is independently selected from halogen, cyano, amino, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy, and the C 1 -C 6 alkyl, and C 1 -C 6 alkoxy are optionally substituted with halogen;

[0022] R cIndependently selected from hydrogen, halogen, hydroxy, oxo, amino, cyano, C 1 -C 6 -alkyl and C 3 -C 6 -cycloalkyl;

[0023] One or more hydrogen atoms of the compound are optionally deuterium atoms.

[0024] In some embodiments, X 1 is N.

[0025] In some embodiments, X 2 and X 3 are each independently selected from N and S.

[0026] In some embodiments, X 2 is S, X 3 is N.

[0027] In some embodiments, R 1 is selected from hydrogen, amino, cyano, C 2 -C 10 -alkynyl, C 1 -C 10 -alkyl, C 3 -C 12 -cycloalkyl, 4- to 10-membered heterocyclic group, 5- to 10-membered heteroaryl and -C(O)R 3 , and the amino, C 2 -C 10 -alkynyl, C 1 -C 10 -alkyl, C 3 -C 12 -cycloalkyl, 4- to 10-membered heterocyclic group and 5- to 10-membered heteroaryl are optionally substituted by R 1a .

[0028] In some embodiments, R 1 is selected from hydrogen, amino, cyano, C 2 -C 3 -alkynyl, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, 4- to 7-membered heterocyclic group, 5- to 6-membered heteroaryl and -C(O)R 3 , and the amino, C 2 -C 3 -alkynyl, C 1 -C 6 -alkyl, C 3 -C 6 -cycloalkyl, 4- to 7-membered heterocyclic group and 5- to 6-membered heteroaryl are optionally substituted by R 1a .

[0029] In some embodiments, R 1 is selected from hydrogen, amino, cyano, methyl, ethyl, ethynyl, cyclopropyl, morpholino, oxetanyl, pyridyl, pyrazolyl, triazolyl, and -C(O)R 3 , and the amino, methyl, ethyl, ethynyl, cyclopropyl, morpholino, oxetanyl, pyridyl, pyrazolyl, and triazolyl are optionally substituted by R 1a .

[0030] In some embodiments, R 3 is amino.

[0031] In some embodiments, R 1a is independently selected from cyano, C 1 -C 6 alkyl, 4- to 10-membered heterocyclic group, C 1 -C 6 alkoxy, -NH(C 1 -C 6 alkyl), C(O)C 1 -C 6 alkyl, and 5- to 10-membered heteroaryl.

[0032] In some embodiments, R 1a is independently selected from cyano, C 1 -C 3 alkyl, 4- to 7-membered heterocyclic group, C 1 -C 3 alkoxy, -NH(C 1 -C 3 alkyl), C(O)C 1 -C 3 alkyl, and 5- to 6-membered heteroaryl.

[0033] In some embodiments, R 1a is independently selected from cyano, methyl, methoxy, -NHCH 3 , morpholino, imidazolyl, and -C(O)CH 3 .

[0034] In some embodiments, R 1 is selected from hydrogen, methyl, ethyl, cyano, cyanomethyl, methoxymethyl, ethynyl, cyclopropyl, -C(O)NH 2 , -NHC(O)CH 3 ,

[0035] In some embodiments, R 2 is selected from C 1 -C 10 alkyl, wherein the C1 -C 10 The alkyl group is optionally substituted by R 2a .

[0036] In some embodiments, R 2 is selected from C 1 -C 6 alkyl group, and the C 1 -C 6 alkyl group is optionally substituted by R 2a .

[0037] In some embodiments, R 2 is selected from C 1 -C 3 alkyl group, and the C 1 -C 3 alkyl group is optionally substituted by R 2a .

[0038] In some embodiments, R 2 is methyl, and the methyl is optionally substituted by R 2a .

[0039] In some embodiments, R 2a is independently selected from halogen, hydroxyl, amino and cyano.

[0040] In some embodiments, R 2 is methyl.

[0041] In some embodiments, ring B is selected from 4- to 10-membered heterocyclic groups, and the 4- to 10-membered heterocyclic groups are optionally substituted by halogen or C 1 -C 6 alkyl group.

[0042] In some embodiments, ring B is selected from 4- to 7-membered heterocyclic groups, and the 4- to 7-membered heterocyclic groups are optionally substituted by halogen or C 1 -C 6 alkyl group.

[0043] In some embodiments, ring B is selected from 4- to 7-membered heterocyclic groups, and the 4- to 7-membered heterocyclic groups are optionally substituted by halogen or C 1 -C 3 alkyl group.

[0044] In some embodiments, ring B is selected from piperazinyl, piperidinyl, tetrahydropyridinyl and * indicates connection to L, and the piperazinyl, piperidinyl, tetrahydropyridinyl and are optionally substituted by halogen or C 1 -C 3 alkyl group.

[0045] In some embodiments, ring B is selected from * indicates being connected to L, and the optionally substituted by halogen or C 1 -C 3 alkyl.

[0046] In some embodiments, ring B is selected from * indicates being connected to L, and the optionally substituted by C 1 -C 3 alkyl, preferably methyl and ethyl.

[0047] In some embodiments, ring B is selected from * indicates being connected to L.

[0048] In some embodiments, ring C is selected from 5- to 10-membered heterocyclic groups, C 6 -C 10 aryl, and 5- to 10-membered heteroaryl groups, and the 5- to 10-membered heterocyclic groups, C 6 -C 10 aryl, and 5- to 10-membered heteroaryl groups are optionally substituted by R b substituted.

[0049] In some embodiments, ring C is selected from C 6 -C 10 aryl, and 5- to 10-membered heteroaryl groups, and the C 6 -C 10 aryl, and 5- to 10-membered heteroaryl groups are optionally substituted by R b substituted.

[0050] In some embodiments, ring C is selected from dihydrobenzodioxinyl, phenyl, and quinoxalinyl, and the dihydrobenzodioxinyl, phenyl, and quinoxalinyl are optionally substituted by R b substituted.

[0051] In some embodiments, ring C is selected from phenyl and quinoxalinyl, and the phenyl and quinoxalinyl are optionally substituted by R b substituted.

[0052] In some embodiments, ring C is selected from phenyl, the phenyl, optionally substituted by R b substituted.

[0053] In some embodiments, R b is independently selected from halogen and C 1 -C 6 alkyl, and the C 1 -C 6 alkyl is optionally substituted by halogen.

[0054] In some embodiments, R b is independently selected from halogen and C 1 -C 3 alkyl, wherein the C 1 -C 3 alkyl is optionally substituted with halogen.

[0055] In some embodiments, R b is independently selected from fluorine, methyl, and ethyl, wherein the methyl and ethyl are optionally substituted with halogen.

[0056] In some embodiments, R b is independently selected from fluorine, methyl, and trifluoromethyl.

[0057] In some embodiments, L is selected from -NR c - and -CR c R c -.

[0058] In some embodiments, R c is independently selected from hydrogen, C 1 -C 6 alkyl, and C 3 -C 6 cycloalkyl.

[0059] In some embodiments, R c is independently selected from hydrogen, C 1 -C 3 alkyl, and C 3 -C 6 cycloalkyl.

[0060] In some embodiments, R c is independently selected from hydrogen, methyl, ethyl, and cyclopropyl.

[0061] In some embodiments, the compound of formula (I) or its stereoisomer or its pharmaceutically acceptable salt is selected from the compound of formula (II) or its pharmaceutically acceptable salt:

[0062]

[0063] wherein R 1 , R 2 , ring B, ring C, and L are as defined above.

[0064] In some embodiments, the compound of formula (I) or its stereoisomer or its pharmaceutically acceptable salt of the present disclosure is selected from the following compounds or their stereoisomers or their pharmaceutically acceptable salts,

[0065]

[0066]

[0067]

[0068] On the other hand, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I) of the present disclosure, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0069] On the other hand, the present disclosure provides a method for treating a DGK-mediated disease in an individual (such as a mammal), comprising administering to an individual (such as a mammal, preferably a human) in need of such treatment a therapeutically effective amount of a compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0070] On the other hand, the present disclosure provides the use of a compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for preventing or treating a DGK-mediated disease.

[0071] On the other hand, the present disclosure provides the use of a compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in preventing or treating a DGK-mediated disease.

[0072] On the other hand, the present disclosure provides a compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for preventing or treating a DGK-mediated disease.

[0073] In some embodiments, the DGK-mediated disease is a tumor.

[0074] Term Definitions and Explanations

[0075] Unless otherwise specified, the terms used in the present disclosure have the following meanings. The definitions of the groups and terms described in the present disclosure, including their exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other arbitrarily. A particular term should not be considered indeterminate or unclear without a special definition, but should be understood according to the ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding product or its active ingredient.

[0076] As used herein represents a connection site.

[0077] As used herein, the bonds depicted by solid and dashed lines represent a single bond or a double bond.

[0078] The graphical representations of racemates or enantiomerically pure compounds in this article are from Maehr, J. Chem. Ed. 1985, 62: 114 - 120. Unless otherwise specified, wedged and hashed bonds are used to represent the absolute configuration of a stereocenter, and solid and dashed bonds are used to represent the relative configuration of a stereocenter (such as the cis - trans configuration of alicyclic compounds).

[0079] The compounds of the present disclosure may have asymmetric atoms such as carbon, sulfur, nitrogen, phosphorus atoms or asymmetric double bonds, so the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis - and trans - isomers, E - and Z - geometric isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereoisomers, (D)- isomers, (L)- isomers, and their racemic mixtures or other mixtures, such as enantiomer - or diastereomer - enriched mixtures. All of the above - mentioned isomers and their mixtures are within the definition scope of the compounds of the present disclosure. Additional asymmetric carbon, sulfur, nitrogen or phosphorus atoms may exist in substituents such as alkyl groups, and these isomers and their mixtures involved in all substituents are also included within the definition scope of the compounds of the present disclosure. The compounds of the present disclosure containing asymmetric atoms can be isolated in optically active pure form or racemic form. The optically active pure form can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

[0080] The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by substituents, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxo does not occur on an aromatic group.

[0081] The term "optionally" or "optionally" means that the subsequent described event or situation may or may not occur, and this description includes the occurrence and non - occurrence of the described event or situation. For example, ethyl "optionally" being substituted by one or more halogens means that ethyl can be unsubstituted (CH 2 CH 3 ), monosubstituted (CH 2 CH 2 F, CH 2 CH 2 Cl, etc.), polysubstituted (CHFCH 2 F, CH 2 CHF 2 , CHFCH 2 Cl, CH 2 CHCl2 etc.) or fully substituted (CF 2 CF 3 , CF 2 CCl 3 , CCl 2 CCl 3 etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized will be introduced.

[0082] When any variable (e.g., R a , R b ) appears more than once in the composition or structure of a compound, its definition in each case is independent. For example, if a group is substituted by two R b , then each R b has independent options.

[0083] C m -C n herein refers to having an integer number of carbon atoms in the range of m - n. For example, "C 1 -C 10 " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0084] The term "alkyl" refers to a hydrocarbon group of the general formula C n H 2n+1 , and the alkyl can be straight-chain or branched-chain. The term "C 1 -C 10 alkyl" can be understood to represent a straight-chain or branched-chain saturated hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Specific examples of the alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc.; the term "C 1 -C 6"Alkyl" can be understood to represent an alkyl group having 1 to 6 carbon atoms. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc. The term "C 1 -C 4 alkyl" can be understood to represent a straight-chain or branched-chain saturated alkyl group having 1 to 4 carbon atoms. The term "C 1 -C 3 alkyl" can be understood to represent a straight-chain or branched-chain saturated alkyl group having 1 to 3 carbon atoms. The "C 1 -C 10 alkyl" may include ranges such as "C 1 -C 6 alkyl", "C 1 -C 4 alkyl", or "C 1 -C 3 alkyl". The "C 1 -C 6 alkyl" may further include "C 1 -C 4 alkyl" or "C 1 -C 3 alkyl".

[0085] The term "alkoxy" refers to the group formed by removing the hydrogen atom on the hydroxyl group from a straight-chain or branched-chain alcohol, and can be understood as "alkyloxy" or "alkyl-O-". The term "C 1 -C 10 alkoxy" can be understood as "C 1 -C 10 alkyloxy" or "C 1 -C 10 alkyl-O-"; the term "C 1 -C 6 alkoxy" can be understood as "C 1 -C 6 alkyloxy" or "C 1 -C 6 alkyl-O-". The "C 1 -C 10 alkoxy" may include ranges such as "C 1 -C 6 alkoxy" and "C 1 -C 3 alkoxy". The "C 1 -C 6 alkoxy" may further include "C 1 -C 3 alkoxy".

[0086] The term "alkenyl" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one double bond. The term "C 2 -C 10 alkenyl" can be understood to represent a straight-chain or branched-chain unsaturated hydrocarbon group that contains one or more double bonds and has 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. "C 2 -C 10 alkenyl" can include "C 2 -C 6 alkenyl", "C 2 -C 4 alkenyl", C 2 or C 3 alkenyl. It is understood that in the case where the alkenyl contains more than one double bond, the double bonds can be separated or conjugated to each other. Specific examples of the alkenyl include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, or (Z)-1-methylprop-1-enyl, etc.

[0087] The term "alkynyl" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group composed of carbon atoms and hydrogen atoms and having at least one triple bond. The term "C 2 -C 10 alkynyl" can be understood to represent a straight-chain or branched-chain unsaturated hydrocarbon group that contains one or more triple bonds and has 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of "C 2 -C 10 alkynyl" include, but are not limited to, ethynyl (-C≡CH), propynyl (-C≡CCH 3、 -CH 2 C≡CH), but-1-ynyl, but-2-ynyl, or but-3-ynyl. "C 2 -C 10 alkynyl" can include "C 2 -C 3 alkynyl", "C 2 -C 3 alkynyl" examples include ethynyl (-C≡CH), prop-1-ynyl (-C≡CCH 3 ), prop-2-ynyl (-CH 2 C≡CH).

[0088] The term "cycloalkyl" refers to a carbocyclic group that is completely saturated and exists in the form of a monocyclic, fused-ring, bridged-ring, or spiro-ring, etc. Unless otherwise indicated, the carbocyclic ring is usually a 3- to 20-membered ring. The term "C 3 -C 10 -cycloalkyl" refers to a cycloalkyl having 3, 4, 5, 6, 7, 8, 9, or 10 ring carbon atoms. The term "C 3 -C 6 -cycloalkyl" refers to a cycloalkyl having 3, 4, 5, or 6 ring carbon atoms.

[0089] The term "heterocyclic group" refers to a monocyclic, fused-ring, spiro-ring, or bridged-ring group that is completely saturated or partially saturated (not aromatic heterocyclic as a whole), and contains 1-5 (e.g., 1-3 or 1-2) heteroatoms or heteroatomic groups (i.e., atomic groups containing heteroatoms) among its ring atoms. The "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O) 2 -,-S(=O)-,-P(=O) 2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4- to 10-membered heterocyclic group" refers to a heterocyclic group having 4, 5, 6, 7, 8, 9, or 10 ring atoms, and having 1 to 5 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups in its ring atoms. The "4- to 10-membered heterocyclic group" may include a "4- to 7-membered heterocyclic group". The term "4- to 7-membered heterocyclic group" refers to a heterocyclic group having 4, 5, 6, or 7 ring atoms, and having 1, 2, 3, 4, or 5 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups in its ring atoms. Among them, specific examples of the 4-membered heterocyclic group include, but are not limited to, azetidinyl or oxetanyl; specific examples of the 5-membered heterocyclic group include, but are not limited to, tetrahydrofuranyl, dioxolanyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrroline, 4,5-dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; specific examples of the 6-membered heterocyclic group include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridyl, or 4H-[1,3,4]thiadiazinyl; specific examples of the 7-membered heterocyclic group include, but are not limited to, diazepanyl. The heterocyclic group may also be a bicyclic group. Among them, specific examples of the 5,5-bicyclic group include, but are not limited to, hexahydrocyclopenta[c]pyrrol-2(1H)-yl; specific examples of the 5,6-bicyclic group include, but are not limited to, hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl, or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl. Optionally, the heterocyclic group may be a benzo-fused ring group of the above 4- to 7-membered heterocyclic group, and specific examples include, but are not limited to, dihydroisoquinolinyl, etc. The "4- to 10-membered heterocyclic group" may include ranges such as "5- to 10-membered heterocyclic group", "4- to 7-membered heterocyclic group", "5- to 6-membered heterocyclic group", "6- to 8-membered heterocyclic group", "4- to 10-membered heterocycloalkyl group", "5- to 10-membered heterocycloalkyl group", "4- to 7-membered heterocycloalkyl group", "5- to 6-membered heterocycloalkyl group", "6- to 8-membered heterocycloalkyl group", etc. The "4- to 7-membered heterocyclic group" may further include ranges such as "4- to 6-membered heterocyclic group", "5- to 6-membered heterocyclic group", "4- to 7-membered heterocycloalkyl group", "4- to 6-membered heterocycloalkyl group", "5- to 6-membered heterocycloalkyl group", etc. Although some bicyclic heterocyclic groups in the present disclosure partially contain a benzene ring or a heteroaryl ring, the heterocyclic group as a whole is still non-aromatic.

[0090] The term "heterocycloalkyl" refers to a fully saturated cyclic group that exists in the form of a monocyclic, fused-ring, bridged-ring or spiro-ring, etc., and contains 1 to 5 heteroatoms or heteroatomic groups (i.e., atomic groups containing heteroatoms) among the ring atoms of the ring. The "heteroatom or heteroatomic group" includes, but is not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O) 2 -, -S(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-, etc. The term "4- to 10-membered heterocycloalkyl" refers to a heterocycloalkyl having 4, 5, 6, 7, 8, 9 or 10 ring atoms, and containing 1 to 5 heteroatoms or heteroatomic groups independently selected from the above-mentioned ones among its ring atoms. The term "5- to 10-membered heterocycloalkyl" refers to a heterocycloalkyl having 5, 6, 7, 8, 9 or 10 ring atoms, and containing 1 to 5 heteroatoms or heteroatomic groups independently selected from the above-mentioned ones among its ring atoms. "4- to 10-membered heterocycloalkyl" and "5- to 10-membered heterocycloalkyl" include "4- to 7-membered heterocycloalkyl". Among them, specific examples of 4-membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl or thietanyl; specific examples of 5-membered heterocycloalkyl include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl or tetrahydropyrazolyl; specific examples of 6-membered heterocycloalkyl include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thioxanyl, 1,4-dioxanyl, thiomorpholinyl, 1,3-dithianyl or 1,4-dithianyl; specific examples of 7-membered heterocycloalkyl include, but are not limited to, azepanyl, oxepanyl or thiepanyl.

[0091] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π-electron system. An aryl may have 6 to 20 carbon atoms, 6 to 14 carbon atoms or 6 to 12 carbon atoms. The term "C 6 -C 10 aryl" can be understood as an aryl having 6 to 10 carbon atoms. For example, a ring having 6 carbon atoms ("C 6 aryl"), such as phenyl; or a ring having 9 carbon atoms ("C 9 aryl"), such as indanyl or indenyl; or a ring having 10 carbon atoms ("C 10 aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl.

[0092] The term "heteroaryl" refers to a monocyclic or fused polycyclic system having aromaticity, wherein the ring atoms include at least one ring atom selected from N, O, and S, and the remaining ring atoms are C aromatic ring groups. The term "5- to 10-membered heteroaryl" can be understood to include such monocyclic or bicyclic aromatic ring systems: having 5, 6, 7, 8, 9, or 10 ring atoms, such as 5 or 6 or 9 or 10 ring atoms, and containing 1-5, such as 1-3 heteroatoms independently selected from N, O, and S. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, or thiadiazolyl, etc. and their benzo derivatives, such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzoisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, or isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, or triazinyl, etc. and their benzo derivatives, such as quinolinyl, quinazolinyl, or isoquinolinyl, etc.; or azocinyl, indolizinyl, purinyl, etc. and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, or phenoxazinyl, etc. The term "6- to 10-membered heteroaryl" can be understood to include such monocyclic or bicyclic aromatic ring systems: having 6, 7, 8, 9, or 10 ring atoms, such as 6 or 9 or 10 ring atoms, and containing 1-5, such as 1-3 heteroatoms independently selected from N, O, and S. The term "5- to 6-membered heteroaryl" refers to an aromatic ring system having 5 or 6 ring atoms and containing 1-3, such as 1-2 heteroatoms independently selected from N, O, and S.

[0093] The term "halo" or "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0094] The term "hydroxy" refers to the -OH group.

[0095] The term "cyano" refers to the -CN group.

[0096] The term "amino" refers to -NH 2 group.

[0097] The term "nitro" refers to -NO 2 group.

[0098] The term "treatment" means administering the compounds or formulations described in this application to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0099] (i) inhibiting a disease or disease state, i.e., curbing its development;

[0100] (ii) alleviating a disease or disease state, i.e., causing the disease or disease state to subside.

[0101] The term "therapeutically effective amount" means an amount of a compound of the present disclosure that (i) treats a particular disease, condition, or disorder, or (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder. The amount of the compound of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

[0102] The term "prevent" means administering a compound or formulation described in the present application to prevent a disease or one or more symptoms associated with the disease, and includes preventing the occurrence of a disease or disease state in an individual (such as a mammal), particularly when such an individual (such as a mammal) is susceptible to the disease state but has not been diagnosed as having the disease state.

[0103] The term "individual" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates (such as chimpanzees and other apes and monkeys); domestic animals, such as cows, horses, sheep, goats, pigs; household animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, etc. Examples of non-human mammals include, but are not limited to, birds and fish, etc. In one embodiment of the methods and compositions provided herein, the mammal is a human. The terms "patient" and "individual" can be used interchangeably.

[0104] The term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0105] The term "pharmaceutically acceptable salt" refers to salts of pharmaceutically acceptable acids or bases, including salts formed by the compound with inorganic acids or organic acids, and salts formed by the compound with inorganic bases or organic bases.

[0106] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or salts thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the present disclosure to an organism.

[0107] The term "pharmaceutically acceptable excipient" refers to those excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0108] The term "comprise" or "comprises" or its English variants such as "comprises" or "comprising" can be understood in an open, non-exclusive sense, i.e., "including but not limited to".

[0109] The present disclosure also includes isotopically labeled compounds of the present disclosure that are the same as those described herein, but in which one or more atoms are replaced by atoms having an atomic weight or mass number different from that typically found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0110] Certain isotopically labeled compounds of the present disclosure (e.g., labeled with 3 H and 14 C) can be used in compound and / or substrate tissue distribution analysis. Tritiated (i.e., 3 H) and carbon-14 (i.e., 14 C) isotopes are particularly preferred because of their ease of preparation and detectability. Positron-emitting isotopes, such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present disclosure can generally be prepared by substituting an isotopically labeled reagent for an unlabeled reagent by procedures similar to those described in the protocols and / or examples disclosed below.

[0111] The pharmaceutical compositions of the present disclosure can be prepared by combining the compounds of the present disclosure with suitable pharmaceutically acceptable excipients, and can be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols, etc.

[0112] Typical routes of administration of the compounds of the present disclosure, or their pharmaceutically acceptable salts, or their pharmaceutical compositions include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0113] The pharmaceutical compositions of the present disclosure can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, emulsifying, freeze-drying methods, etc.

[0114] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present disclosure to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, suspensions, etc. for oral administration to a patient.

[0115] Solid oral compositions can be prepared by conventional mixing, filling, or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of a tablet or dragee. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, or flavoring agents, etc.

[0116] The pharmaceutical composition is also suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms.

[0117] The dosage administered depends on factors such as the specific compound, the disease condition and its severity, the identity of the subject or host to be treated (e.g., body weight, gender), and is determined by the specific circumstances of the case, including, for example, the specific formulation administered, the route of administration, the disorder being treated, and the subject or host being treated.

[0118] In all methods of administration of the compounds of general formula (I) described herein, in the case of oral administration, the daily dosage is from 0.001 mg / kg to 5000 mg / kg body weight, preferably from 0.01 mg / kg to 50 mg / kg body weight, in single or divided doses. The daily dosage and unit dosage vary according to many variables, including but not limited to the activity of the compound used, the disease or disorder to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or disorder being treated, and the judgment of the practitioner.

[0119] The compounds of the present disclosure can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and equivalent replacement methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present disclosure.

[0120] The chemical reactions of the specific embodiments of the present disclosure are completed in a suitable solvent, and the solvent must be suitable for the chemical changes of the present disclosure and the reagents and materials required therefor. In order to obtain the compounds of the present disclosure, sometimes those skilled in the art need to modify or select the synthesis steps or reaction processes on the basis of the existing embodiments.

[0121] The following abbreviations are used in the present disclosure:

[0122] THF represents tetrahydrofuran; DMAP represents 4-dimethylaminopyridine; DMF represents N,N-dimethylformamide; DCM represents dichloromethane; TFA represents trifluoroacetic acid; CDI represents N,N'-carbonyldiimidazole; DIEA / DIPEA represents N,N-diisopropylethylamine; MeMgBr represents methylmagnesium bromide; HMPA represents hexamethylphosphoric triamide; MeCN represents acetonitrile; LC-MS represents liquid chromatography-mass spectrometry; MS represents mass spectrometry; 1 H NMR represents nuclear magnetic resonance hydrogen spectrum; ESI represents electrospray ionization; HPLC represents high performance liquid chromatography; DMSO represents dimethyl sulfoxide; BOC represents tert-butoxycarbonyl; ATP represents adenosine triphosphate; IL-2 represents interleukin-2; IC 50 represents the half inhibitory concentration, which refers to the concentration when reaching half of the maximum inhibitory effect. EC 50 represents the half effective concentration; Emax represents the maximum effective amount; ELISA represents enzyme-linked immunosorbent assay. Specific Embodiments

[0123] The compounds of the present disclosure can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed herein, the embodiments formed by their combination with other chemical synthesis methods, and equivalent replacement methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present disclosure.

[0124] The present disclosure will be described in detail below by way of examples, but this does not mean any adverse limitation to the present disclosure. The present disclosure has been described in detail herein, and its specific embodiments have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure. All the reagents used in the present disclosure are commercially available and can be used without further purification.

[0125] Unless otherwise specified, the ratios expressed for the mixed solvents are volume mixing ratios.

[0126] Unless otherwise specified, % refers to weight percentage wt%.

[0127] The compounds are named manually or by software. For commercially available compounds, the supplier catalog names are used.

[0128] The structures of the compounds are determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The unit of NMR shift is 10 -6 (ppm). The solvents for NMR measurement are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS);

[0129] The eluent or mobile phase can be a mixed eluent or mobile phase composed of two or more solvents, and the ratio is the volume ratio of each solvent.

[0130] Example 1: 7-((2S,5R)-2,5-dimethyl-4-(1-(3-methylquinoxalin-6-yl)ethyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one (Compound 1)

[0131]

[0132] Step 1: Ethyl 5-((tert-butoxycarbonyl)amino)-2-methylthiazole-4-carboxylate (Compound 1-2)

[0133] Ethyl 5-amino-2-methylthiazole-4-carboxylate (5.0 g, 26.9 mmol), di-tert-butyl dicarbonate (12.3 g, 56.4 mmol) and 4-dimethylaminopyridine (328.0 mg, 2.7 mmol) were dissolved in ultradry tetrahydrofuran (150 mL), and the reaction was heated at 70 °C for 4 hours. After the reaction was completed, the reaction system was concentrated to dryness under reduced pressure to obtain the crude product of Compound 1-2, which was directly used in the next step of the reaction.

[0134] LC-MS: m / z (ESI): 287 [M+H] + .

[0135] Step 2: tert-Butyl ((4-carbamoyl-2-methylthiazol-5-yl)amino)carbamate (Compound 1-3)

[0136] Compound 1-2 (7.7 g, 26.9 mmol) was dissolved in a methanol solution of ammonia (7N, 100 mL), and the mixture was heated and sealed at 50 °C for 16 hours to complete the reaction. The reaction solution was directly concentrated under reduced pressure to dryness. Ethyl acetate and water were added to the residue. After liquid separation, the organic phase was washed with saturated sodium chloride, and the solvent was removed by concentration under reduced pressure to obtain Compound 1-3 (5.3 g, yield: 76.7%).

[0137] LC-MS: m / z (ESI): 258 [M+H] + .

[0138] Step 3: tert-Butyl (4-carbamoyl-2-methylthiazol-5-yl)(methyl)carbamate (Compound 1-4)

[0139] Compound 1-3 (1.0 g, 3.9 mmol), methyl iodide (1.1 g, 7.8 mmol) and potassium carbonate (1.1 g, 7.8 mmol) were dissolved in N,N-dimethylformamide (10 mL). The reaction was carried out at room temperature for 18 hours under a nitrogen protection environment to complete the reaction. The reaction system was added to water, and the mixture was extracted with ethyl acetate. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by normal-phase column chromatography (petroleum ether:ethyl acetate = 4:1) to obtain Compound 1-4 (520.0 mg, yield: 49.3%).

[0140] LC-MS: m / z (ESI): 272 [M+H] + .

[0141] Step 4: 2-Methyl-5-(methylamino)thiazole-4-carboxamide (Compound 1-5)

[0142] Compound 1-4 (500.0 mg, 1.8 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (5 mL) was added. The reaction was carried out at room temperature for 1 hour to complete the reaction. The reaction solution was concentrated to dryness under reduced pressure. The residue was neutralized with a sodium bicarbonate solution, extracted twice with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain crude Compound 1-5 (263.0 mg).

[0143] LC-MS: m / z (ESI): 172 [M+H] + .

[0144] Step 5: 2,4-Dimethylthiazolo[5,4-d]pyrimidine-5,7(4H,6H)-dione (Compound 1-6)

[0145] Compound 1-5 (250.0 mg, 1.5 mmol) was dissolved in N,N-dimethylformamide (5 mL), then sodium hydride (116.8 mg, 2.9 mmol) was added, and the reaction was carried out at room temperature for 1 hour. N,N'-carbonyldiimidazole (473.5 mg, 2.9 mmol) was added to the system, and the temperature was raised to 100 °C for 16 hours to complete the reaction. The reaction mixture was concentrated to dryness under reduced pressure and purified by reverse-phase chromatography (acetonitrile:water = 1:1) to obtain compound 1-6 (234.0 mg, yield: 79%).

[0146] LC-MS: m / z (ESI): 198 [M+H] + .

[0147] Step 6: 7-((2S,5R)-2,5-dimethyl-4-(1-(3-methylquinoxalin-6-yl)ethyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one (Compound 1)

[0148] Compound 1-6 (50.0 mg, 253.5 μmol) was dissolved in acetonitrile (2 mL), phosphorus oxychloride (77.8 mg, 507.1 μmol) was added, and then N,N-diisopropylethylamine (98.3 mg, 760.6 μmol) was added. The reaction was carried out at 80 °C for 2 hours to complete the reaction. The reaction mixture was concentrated to dryness under reduced pressure. Anhydrous acetonitrile (2 mL), N,N-diisopropylethylamine (98.3 mg, 760.6 μmol) and 7-(1-((2R,5S)-2,5-dimethylpiperazin-1-yl)ethyl)-2-methylquinoxaline (Compound 1-7, 57.7 mg, 202.8 μmol) were added to the residue, and the system was reacted at 80 °C for 18 hours to complete the reaction. Saturated aqueous sodium bicarbonate was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by preparative HPLC (YMC TA-C18 column, 30*150 mm, 5 μm; mobile phase A: water (0.5% ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; acetonitrile ratio 60%-100%, elution for 15 minutes) to obtain compound 1 (10.0 mg, yield: 8%, 1 1H NMR showed that the ratio of the two diastereoisomers was 1:2).

[0149] LC-MS: m / z (ESI): 464 [M+H] + .

[0150] 1 1H NMR (400 MHz, DMSO-d 6)δ8.82(d,J=1.3Hz,1H),8.81(d,J=1.5Hz,2H),8.07–8.00(m,3H),7.94(d,J=7.1Hz,3H),7.86(t,J=7.4Hz,3H),3.87(q,J=5.4,4.8Hz,1H),3.73(q,J=6.6Hz,2H),3.59(s,2H),3.41(d,J=1.3Hz,9H),3.35(d,J=1.6Hz,9H),2.87–2.78(m,4H),2.70(d,J=1.9Hz,9H),2.70–2.57(m,10H),2.15(d,J=12.2Hz,12H),1.36(d,J=6.9Hz,2H),1.20(d,J=6.5Hz,6H),1.02(d,J=6.3Hz,6H),0.87(d,J=6.4Hz,3H).

[0151] Example 2: 7-((2S,5R)-2,5-diethyl-4-(1-(4-fluoro-2-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one (Compound 2)

[0152]

[0153] Step 1: 1-(4-fluoro-2-iodophenyl)ethan-1-ol (Compound 2-2)

[0154] Dissolve Compound 2-1 (2.0 g, 8.0 mmol) in tetrahydrofuran (20 mL). Protect the system with argon, cool to 0 °C and stir, then slowly add methylmagnesium bromide (16.0 mmol, 1 mol / L). After the addition, slowly warm to room temperature and react for 16 hours. Quench the reaction by adding saturated ammonium chloride aqueous solution to the system, and at the same time add ethyl acetate (40 mL), stir, separate the layers, extract the aqueous phase with ethyl acetate once, combine the organic phases, wash with saturated brine once, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent. The residue is purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain Compound 2-2 (1.4 g, yield 63%).

[0155] LC-MS: m / z(ESI): 267 [M+H] + .

[0156] Step 2: (2S,5R)-2,5-diethyl-4-(1-(4-fluoro-2-iodophenyl)ethyl)piperazine-1-carboxylic acid tert-butyl ester (Compound 2-3)

[0157] Compound 2-2 (100.0 mg, 375.0 μmol) was dissolved in acetonitrile (2 mL), then (2S,5R)-tert-butyl 2,5-diethylpiperazine-1-carboxylate (80 mg, 375.0 μmol), N,N-diisopropylethylamine (243.0 mg, 1880.0 μmol) and (cyanomethyl)trimethylphosphonium iodide (183.0 mg, 750.0 μmol) were added. Under argon protection, the reaction system was heated at 105 °C for 16 hours until the reaction was completed. The reaction solution was directly concentrated to remove the solvent, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain crude compound 2-3 (150.0 mg).

[0158] LC-MS: m / z (ESI): 491 [M+H] + .

[0159] Step 3: (2S,5R)-tert-butyl 2,5-diethyl-4-(1-(4-fluoro-2-(trifluoromethyl)phenyl)ethyl)piperazine-1-carboxylate (Compound 2-4)

[0160] The crude compound 2-3 (100.0 mg, 200.0 μmol) was dissolved in N,N-dimethylformamide (1 mL), then copper(I) iodide (77.5 mg, 400.0 μmol), methyl fluorosulfonyldifluoroacetate (117.5 mg, 600.0 μmol) and hexamethylphosphoric triamide (73.1 mg, 400 μmol) were added. The reaction system was heated at 80 °C for 16 hours until the reaction was completed. Water was added to the system, and the mixture was extracted with ethyl acetate. The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to obtain crude compound 2-4 (80.0 mg).

[0161] LC-MS: m / z (ESI): 433 [M+H] + .

[0162] Step 4: (2R,5S)-2,5-diethyl-1-(1-(4-fluoro-2-(trifluoromethyl)phenyl)ethyl)piperazine (Compound 2-5)

[0163] The crude compound 2-4 (100.0 mg, 200.0 μmol) was dissolved in dichloromethane (1 mL), then trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at room temperature for 2 hours until the reaction was completed. The reaction solution was directly concentrated under reduced pressure to remove the solvent. Water and ethyl acetate were added to the residue and stirred. After phase separation, the organic phase was washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to dryness to obtain crude compound 2-5 (50.0 mg).

[0164] LC-MS: m / z (ESI): 333 [M+H] + .

[0165] Step 5: 7-((2S,5R)-2,5-diethyl-4-(1-(4-fluoro-2-(trifluoromethyl)phenyl)ethyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one (Compound 2)

[0166] Dissolve Compound 1-6 (50.0 mg, 253.5 μmol) in acetonitrile (2 mL), add phosphorus oxychloride (77.8 mg, 507.1 μmol), then add N,N-diisopropylethylamine (98.3 mg, 760.6 μmol), and react at 80 °C for 2 hours to complete the reaction. Concentrate under reduced pressure to dryness. Add anhydrous acetonitrile (2 mL) to the residue, add crude Compound 2-5 (50.0 mg, 150.0 μmol) and N,N-diisopropylethylamine (58.3 mg, 450.0 μmol), and react the system at 80 °C for 16 hours to complete the reaction. Directly concentrate the reaction solution under reduced pressure, and purify the residue by preparative HPLC (Phenomenex Gemini NX, 5 μm silica, 30 mm diameter, 150 mm length; (using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (20%-60%) as the eluent) to obtain Compound 2 (5.0 mg, yield: 6%).

[0167] LC-MS: m / z (ESI): 512 [M+H] + .

[0168] 1 1H NMR (400 MHz, DMSO-d 6 ) δ 8.09–7.93 (m, 2H), 7.69–7.41 (m, 4H), 4.13–4.01 (m, 1H), 3.93–3.83 (m, 1H), 3.55 (d, J = 13.8 Hz, 1H), 3.41 (s, 6H), 3.17 (d, J = 11.7 Hz, 1H), 3.11 (d, J = 9.7 Hz, 1H), 3.00 (d, J = 11.8 Hz, 1H), 2.69 (d, J = 2.6 Hz, 3H), 2.60 (d, J = 3.1 Hz, 3H), 2.09–1.91 (m, 4H), 1.74–1.63 (m, 2H), 1.53–1.42 (m, 2H), 1.41–1.29 (m, 6H), 1.21-1.25 (m, 8H), 0.99–0.81 (m, 6H), 0.65–0.49 (m, 6H).

[0169] Example 3: 7-((2S,5R)-2,5-diethyl-4-(1-(3-methylquinoxalin-6-yl)ethyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one (Compound 3)

[0170]

[0171] Step 1: tert-Butyl (2S,5R)-2,5-diethyl-4-(1-(3-methylquinoxalin-6-yl)ethyl)piperazine-1-carboxylate (Compound 3-2)

[0172] Dissolve 1-(3-methylquinoxalin-6-yl)ethan-1-ol (100.0 mg, 531.3 μmol), tert-butyl (2S,5R)-2,5-diethylpiperazine-1-carboxylate (167.4 mg, 690.7 μmol), (cyanomethyl)trimethylphosphonium iodide (193.7 mg, 796.9 μmol) and N,N-diisopropylethylamine (274.7 mg, 2.1 mmol) in ultradry acetonitrile (1.5 mL), protect the system with argon, heat the reaction system at 105 °C for 18 hours, and the reaction is completed. Add ethyl acetate and water to the reaction system, after layering, wash the organic phase with saturated sodium chloride, dry over anhydrous sodium sulfate, concentrate under reduced pressure to dryness, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to obtain Compound 3-2 (93.0 mg, yield: 42.4%).

[0173] LC-MS: m / z (ESI): 413 [M+H] + .

[0174] Step 2: 7-(1-((2R,5S)-2,5-diethylpiperazin-1-yl)ethyl)-2-methylquinoxaline (Compound 3-3)

[0175] Dissolve Compound 3-2 (93.0 mg, 225.4 μmol) in dichloromethane (0.5 mL), add trifluoroacetic acid (0.5 mL) dropwise, and react at room temperature for 1 hour, and the reaction is completed. Concentrate the reaction solution directly under reduced pressure to dryness, add ethyl acetate and saturated sodium bicarbonate solution to the residue, after layering, wash the organic phase with saturated sodium chloride, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to dryness to obtain Compound 3-3 (70.0 mg, yield: 99.4%).

[0176] LC-MS: m / z (ESI): 313 [M+H] + .

[0177] Step 3: 7-((2S,5R)-2,5-diethyl-4-(1-(3-methylquinoxalin-6-yl)ethyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one (Compound 3)

[0178] Dissolve Compound 1-6 (55.0 mg, 278.9 μmol) in acetonitrile (2 mL), add phosphorus oxychloride (85.5 mg, 557.8 μmol), and then add N,N-diisopropylethylamine (108.1 mg, 836.7 μmol). Heat the reaction system at 80 °C for 2 hours until the reaction is completed. Concentrate under reduced pressure to dryness. Add anhydrous acetonitrile (2 mL), N,N-diisopropylethylamine (108.1 mg, 836.7 μmol) and Compound 3-3 (69.7 mg, 223.1 μmol) to the residue, and react at 80 °C for 18 hours until the reaction is completed. Add saturated sodium bicarbonate aqueous solution, extract with ethyl acetate. After drying the organic phase over anhydrous sodium sulfate, filter, concentrate under reduced pressure to dryness. Purify the residue by preparative HPLC (YMC TA-C18 column, 30*150 mm, 5 μm; mobile phase A: water (0.5% ammonium bicarbonate), mobile phase B: acetonitrile; flow rate: 25 mL / min; acetonitrile ratio 60%-100%, elution for 15 min) to obtain Compound 3 (12.0 mg, yield: 11%).

[0179] LC-MS: m / z (ESI): 492 [M+H] + .

[0180] 1 H NMR (400 MHz, Methanol-d 4)δ8.79(s,1H),8.78(s,1H),8.06(d,J=8.7Hz,1H),8.03(d,J=8.7Hz,1H),7.96(dd,J=10.5,1.8Hz,2H),7.91(ddd,J=8.7,6.9,1.8Hz,2H),4.07-3.97(m,1H),3.90-3.80(m,1H),3.69(d,J=14.3Hz,1H),3.55(d,J=1.8Hz,6H),3.49-3.42(m,1H),3.30-3.17(m,2H),3.06(d,J=12.4Hz,1H),2.95 -2.81(m,2H),2.77(s,3H),2.77(s,3H),2.72(s,3H),2.61(s,3H),2.50-2.38(m,1H),2.33(d,J=12.4Hz,1H),2.22-2.11(m,1H),2.10-1.99(m,2H),1.98-1.77(m,2H),1.67-1.47(m,4H),1.42(dd,J=10.0,6.5Hz,6H),1.12-0.87(m,7H),0.77-0.55(m,7H).

[0181] Example 4: 7-((2S,5R)-2,5-diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-4-methylthiazolo[5,4-d]pyrimidin-5(4H)-one and 7-((2S,5R)-2,5-diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-4-methylthiazolo[5,4-d]pyrimidin-5(4H)-one

[0182]

[0183] Step 1: 5,7-dichlorothiazolo[5,4-d]pyrimidine (Compound 4-2)

[0184] Dissolve thiazolo[5,4-d]pyrimidine-5,7-diol (Compound 4-1, 800.0 mg, 4730.0 μmol) in phosphorus oxychloride (4.9 mL), add N,N-diisopropylethylamine (61.1 mg, 472.9 μmol), and react the reaction mixture at 110 °C for 3 hours. Check that the reaction is complete. Concentrate the reaction mixture under reduced pressure to remove the solvent, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain Compound 4-2 (721 mg, yield: 74%).

[0185] MS m / z(ESI):206[M+H] + ;

[0186] Step 2: 5-Chloro-7-((2S,5R)-2,5-diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)thiazolo[5,4-d]pyrimidine and 5-chloro-7-((2S,5R)-2,5-diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)thiazolo[5,4-d]pyrimidine

[0187] Dissolve 5,7-dichlorothiazolo[5,4-d]pyrimidine (150.6 mg, 730.8 μmol) and (2R,5S)-2,5-diethyl-1-(1-(4-(trifluoromethyl)phenyl)propyl)piperazine (200.0 mg, 609.0 μmol) in acetonitrile (4.6 mL), add N,N-diisopropylethylamine (393.5 mg, 3040.0 μmol), and react the reaction solution at 60 °C for 2 hours. After the reaction is completed, concentrate the reaction solution under reduced pressure to remove the solvent. The residue is purified by preparative liquid chromatography (Phenomenex Gemini NX, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (57% - 97%) as the eluent) to obtain Compound 4-3 (75.0 mg, yield: 25%, LCMS (UPLCMS_L 4-220 Agilent Pursuit5 C18 20*2.0 mm, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (5% - 95%) as the eluent for elution for 1.5 minutes) retention time 0.988 minutes) and Compound 4-4 (82.0 mg, yield: 27%, LCMS (UPLCMS_L 4-220 Agilent Pursuit5 C18 20*2.0 mm, using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (5% - 95%) as the eluent for elution for 1.5 minutes) retention time 1.015 minutes).

[0188] MS m / z (ESI): 498 [M+H] + .

[0189] Step 3: 7-((2S,5R)-2,5-diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)thiazolo[5,4-d]pyrimidin-5-ol and 7-((2S,5R)-2,5-diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)thiazolo[5,4-d]pyrimidin-5-ol

[0190] Dissolve the 4-3 compound or 4-4 compound (75.0 mg, 150.6 μmol) in water (0.3 mL), add concentrated hydrochloric acid (0.3 mL), and react the reaction solution at 100 °C for 45 hours to complete the reaction. Cool the reaction solution to room temperature, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to dryness to obtain the crude target product (71.4 mg, yield: 99%).

[0191] MS m / z(ESI): 480 [M+H] + .

[0192] Step 4: 7-((2S,5R)-2,5-Diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-4-methylthiazolo[5,4-d]pyrimidin-5(4H)-one and 7-((2S,5R)-2,5-Diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-4-methylthiazolo[5,4-d]pyrimidin-5(4H)-one

[0193] Dissolve the product obtained in Step 3 (71.4 mg, 148.9 μmol) in N,N-dimethylformamide (1 mL), add sodium hydride (11.9 mg, 297.8 μmol), react the reaction solution at room temperature for 0.5 hour, then add methyl iodide (63.4 mg, 446.7 μmol), and react the reaction solution at room temperature for 2 hours. Quench the reaction solution with water, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure to remove the solvent, and purify the residue by preparative liquid chromatography (Phenomenex Gemini NX, 5 μm silica, 30 mm diameter, 150 mm length; (using a mixture of water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (21%-61%) as the eluent) to obtain 7-((2S,5R)-2,5-Diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-4-methylthiazolo[5,4-d]pyrimidin-5(4H)-one or 7-((2S,5R)-2,5-Diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-4-methylthiazolo[5,4-d]pyrimidin-5(4H)-one (7 mg, yield: 10%).

[0194] MS m / z(ESI): 494 [M+H] + .

[0195] The NMR spectra of the two isomers are as follows:

[0196] 1 H NMR(400MHz,DMSO-d 6) δ = 8.87 - 8.66 (m, 1H), 7.72 - 7.69 (m, 2H), 7.57 - 7.55 (m, 2H), 6.13 - 5.57 (m, 1H), 5.18 - 4.66 (m, 1H), 3.61 - 3.52 (m, 1H), 3.44 (s, 3H), 3.21 - 3.04 (m, 2H), 2.22 - 2.11 (m, 1H), 1.92 - 1.63 (m, 3H), 1.56 - 1.20 (m, 4H), 0.98 - 0.77 (m, 3H), 0.57 (s, 6H).

[0197] 1 H NMR (400 MHz, DMSO - d 6 ) δ = 8.85 - 8.68 (m, 1H), 7.73 - 7.71 (m, 2H), 7.57 - 7.53 (m, 2H), 5.98 - 5.62 (m, 1H), 5.09 - 4.67 (m, 1H), 3.63 - 3.61 (m, 1H), 3.44 (s, 3H), 3.06 - 2.68 (m, 2H), 2.39 - 2.19 (m, 1H), 2.12 - 1.80 (m, 3H), 1.68 - 1.13 (m, 4H), 0.94 - 0.74 (m, 3H), 0.69 - 0.47 (m, 6H).

[0198] Example 5: 7 - ((2S,5R) - 2,5 - diethyl - 4 - ((S) - 1 - (4 - (trifluoromethyl)phenyl)propyl)piperazin - 1 - yl) - 2,4 - dimethylthiazolo[5,4 - d]pyrimidin - 5(4H) - one or 7 - ((2S,5R) - 2,5 - diethyl - 4 - ((R) - 1 - (4 - (trifluoromethyl)phenyl)propyl)piperazin - 1 - yl) - 2,4 - dimethylthiazolo[5,4 - d]pyrimidin - 5(4H) - one

[0199]

[0200] Step 1: 7 - chloro - 2,4 - dimethylthiazolo[5,4 - d]pyrimidin - 5(4H) - one (Compound 5 - 1)

[0201] Dissolve Compound 1 - 6 (20.0 mg, 101.4 μmol) in acetonitrile (1 mL), add phosphorus oxychloride (31.1 mg, 202.8 μmol), then add N,N - diisopropylethylamine (39.3 mg, 304.2 μmol), react at 80 °C for 2 hours, and the reaction is completed. Concentrate to dryness under reduced pressure to obtain crude Compound 5 - 1 (20 mg) which is directly used for the next step of the reaction.

[0202] Step 2: 7-((2S,5R)-2,5-diethyl-4-((S)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one or 7-((2S,5R)-2,5-diethyl-4-((R)-1-(4-(trifluoromethyl)phenyl)propyl)piperazin-1-yl)-2,4-dimethylthiazolo[5,4-d]pyrimidin-5(4H)-one

[0203] Dissolve compound 5-1 (20.0 mg, 92.7 μmol) in acetonitrile (2 mL), then add N,N-diisopropylethylamine (36.0 mg, 278.2 μmol) and (2R,5S)-2,5-diethyl-1-(1-(4-(trifluoromethyl)phenyl)propyl)piperazine (30.5 mg, 92.7 μmol), and react at 80 °C for 18 hours to complete the reaction. Add saturated aqueous sodium bicarbonate solution, extract with ethyl acetate, dry the organic phase over anhydrous sodium sulfate, concentrate to dryness under reduced pressure, and purify the residue by preparative HPLC (column: YMC C18; mobile phase: A%:B% = 10%-90%, A is NH 4 HCO 3 aqueous solution (7 mmol / L), B is acetonitrile, 25 mL / min) to obtain compound 5-2 (4 mg, yield 8.5%, preparative HPLC retention time 11.8 minutes) and compound 5-3 (4 mg, yield 8.5%, preparative HPLC retention time 12.6 minutes).

[0204] Compound 5-2 LC-MS: m / z (ESI): 508 [M+H] + .

[0205] Compound 5-3 LC-MS: m / z (ESI): 508 [M+H] + .

[0206] The NMR spectra of the two isomers are as follows:

[0207] Compound 5-2: 1 H NMR (400 MHz, DMSO-d6) δ 7.72 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.0 Hz, 2H), 3.62 (s, 1H), 3.40 (s, 3H), 2.79 - 2.95 (m, 2H), 2.68 - 2.73 (m, 2H), 2.60 (s, 1H), 2.50 - 2.55 (m, 3H), 1.86 - 1.89 (m, 3H), 1.54 - 1.62 (m, 1H), 1.35 - 1.40 (m, 1H), 1.23 (s, 2H) 0.85 - 0.87 (m, 3H), 0.56 - 0.63 (m, 6H).

[0208] Compound 5-3: 1 H NMR (400 MHz, DMSO-d6) δ: 7.70 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 3.40 - 3.54 (m, 1H), 3.40 (s, 3H), 3.05 - 3.15 (m, 2H), 2.68 (s, 2H), 2.60 (s, 1H), 2.50 - 2.55 (m, 3H), 1.66 - 1.90 (m, 3H), 1.53 (s, 1H), 1.35 - 1.42 (m, 2H), 1.23 (s, 1H), 0.85 - 0.95 (m, 3H), 0.56 - 0.59 (m, 6H).

[0209] Biological test experiment

[0210] Test Example 1 DGKα / ζ enzyme inhibition experiment

[0211] Instruments and equipment

[0212]

[0213] Experimental reagents and consumables

[0214]

[0215] DGKζ enzyme inhibition activity test

[0216] Experimental procedure:

[0217] 1) Dilute the compound with an Echo 650 acoustic pipetting system and pipette it into the corresponding positions in a 384-well plate (gradient dilute the compound stock solution through the dose-response program of the compound diluter and sampler, the starting concentration of the compound to be tested is 10 μM, 3-fold dilution, 10 concentration points, and the diluent is 1640 medium with 10% (w / w) fetal bovine serum).

[0218] 2) Add 2 μL of DGKζ enzyme (final concentration 3.2 nM) or 1× kinase buffer to each well of the 384-well plate with the compound added. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 30 minutes.

[0219] 3) Add 3 μL of ATP and substrate mixture (final concentration 54 μM ATP, 60 μM substrate) to all wells. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 60 minutes.

[0220] 4) Add 5 μL of ADP-Glo TM Reagent to all wells. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 40 minutes.

[0221] 5) Add 10 μL of the detection solution to all wells. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 30 minutes.

[0222] 6) Read the plate using an Envision microplate reader.

[0223] DGKα Enzyme Inhibition Activity Test

[0224] Experimental Procedure

[0225] 1) Use the Echo 650 acoustic pipetting system to dilute the compound and dispense it into the corresponding positions in a 384-well plate (perform gradient dilution of the compound stock solution through the dose-response program of the compound diluter and sampler. The starting concentration of the compound to be tested is 10 μM, with a 3-fold dilution and 10 concentration points. The diluent is 1640 medium containing 10% (w / w) fetal bovine serum).

[0226] 2) Add 2 μL of DGKα enzyme (final concentration 20 nM) or 1× kinase buffer to each well in the 384-well plate with the compound added. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 30 minutes.

[0227] 3) Add 3 μL of ATP and substrate mixture (final concentration 54 μM ATP, 60 μM substrate) to all wells. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 60 minutes.

[0228] 4) Add 5 μL of ADP-Glo TM Reagent to all wells. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 40 minutes.

[0229] 5) Add 10 μL of the detection solution to all wells. Centrifuge at 1000 rpm for 30 seconds and incubate at room temperature for 30 minutes.

[0230] 6) Read the plate using an Envision microplate reader.

[0231] Experimental Results

[0232] Compound <![CDATA[DGKζIC 50 (nM)]]> <![CDATA[DGKαIC 50 (nM)]]> Compound 1 1249 28 Compound 2 86 ＞10000 Compound 3 133 1341 Compound 5-2 76 343

[0233] Test Example 2 Jurkat T Activation Experiment

[0234] Experimental Purpose: To test the activation effect of the compound on the release of IL-2 from Jurkat T cells

[0235] Experimental method: A 96-well cell culture plate (Corning) was coated with 2 μg / mL Anti-Human CD3 Clone OKT3 (BD) at 37 °C for 4 hours. The compound was serially diluted 3-fold (final concentration from 10 μM to 4.6 nM, starting concentration 10 μM, 8 dilution points, the 8th point is 4.6 nM, dilution medium is 1640 medium with 10% (w / w) fetal bovine serum) and incubated with 220 μL of 1.11×10 6 / mL Jurkat T cells (ATCC) for 1 hour. Then 5 μL of 45 μg / mL Anti-Human CD28 Clone CD28.2 (BD) was added, mixed well, and 100 μL of the mixture was transferred to the CD3-coated cell culture plate. The cells were cultured in an incubator at 37 °C for 48 hours. The supernatant was collected and the IL-2 release was detected using the BD IL-2 ELISA kit (BD). Prism was used to analyze the EC 50 .

[0236] Test Example 3: Activation experiment of Jurkat-IL2-nanoluc cells

[0237] Experimental purpose: To test the activation effect of compounds on the expression of IL2 in Jurkat-IL2-LUC cells. Experimental method: A 96-well cell culture plate (Corning) was coated with 1 μg / mL Anti-Human CD3 Clone OKT3 (BD) at 4 °C for 16 hours. 80 μL of 1.25×10 6 / mL Jurkat-IL2-nanoluc cells were added to the coated plate. Preparation of the compound solution: The compound was serially diluted 3-fold (final concentration from 100 μM to 45.72 nM, starting concentration 100 μM, 8 dilution points, the 8th point is 45.72 nM) using 1640 medium with 10% (w / w) fetal bovine serum (purchased from Gibco, catalog number: 31870082) as the dilution medium. 10 μL of the compound was added to the cell culture plate and mixed well. Finally, 10 μL of 0.625 μg / mL Anti-Human CD28 Clone CD28.2 (BD) was added to the plate. After mixing evenly, the cells were cultured in an incubator at 37 °C for 5 hours. 50 μL of Nano-Light luciferase reporter gene detection reagent (Meilunbio) was added to each well. After shaking for 10 minutes, the luminescence value was detected using a multi-functional microplate reader Envision (PE). Prism was used to analyze the EC50 and Activation fold.

[0238] Construction of Jurkat-IL2 nanoluc cell line

[0239] To generate the Jurkat-IL2 nanoluc cell line, the pNL3.2[NlucP / IL-2 / Hygro] vector (containing the IL2 promoter that drives the transcription of the luciferase reporter gene NanoLuc in response to TCR activation, purchased from Promega, catalog number: N1130) was transfected (using the Lipofectamine LTX Transfection Kit, purchased from Invitrogen, catalog number: 15338-100) onto the Jurkat cell line (purchased from ATCC). After selective culturing for 2 weeks in 1640 medium (purchased from Gibco, catalog number: 31870082) containing 10% (w / w) fetal bovine serum and 250 μg / mL hygromycin (purchased from Invitrogen, catalog number: 10687010), the desired Jurkat-IL2 nanoluc cell strain was obtained.

[0240] Experimental results

[0241]

[0242]

Claims

1. A compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, in: is selected from a double bond or a single bond; X 1 is selected from N and CH; X 2 and X 3 are each independently selected from O, S and N; R 1 Selected from hydrogen, amino, hydroxyl, mercapto, cyano, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Alkyl, C3-C 12 Cycloalkyl, 4-10 membered heterocyclic group, C6-C 10 Aryl, 5-10 membered heteroaryl, -C(O)R 3 and SO2R 3 The amino, hydroxyl, thiol, C2-C 10 Alkenyl, C2-C 10 Alkynyl, C1-C 10 Alkyl, C3-C 12 Cycloalkyl, 4-10 membered heterocyclic group, C6-C 10 Aryl and 5-10 membered heteroaryl are optionally replaced by R 1a replace; R 2 Selected from hydrogen, halogen, amino, cyano, C1-C 10 Alkyl and C1-C 10 Alkoxy, the amino, C1-C 10 Alkyl and C1-C 10 The alkoxy group is optionally replaced by R 2a replace; R 3 is selected from amino, hydroxyl and C1-C3 alkyl, wherein the amino, hydroxyl and C1-C3 alkyl are optionally replaced by R 1a replace; Ring B is selected from C3-C 12 Cycloalkyl and 4-10 membered heterocyclic group, the C3-C 12 Cycloalkyl and 4-10 membered heterocyclyl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; Ring C is selected from C3-C 12 Cycloalkyl, 4-10 membered heterocyclic group, C6-C 10 Aryl and 5-12 membered heteroaryl, the C3-C 12 Cycloalkyl, 4-10 membered heterocyclic group, C6-C 10 Aryl and 5-12 membered heteroaryl are optionally replaced by R b replace; L is selected from a bond, -O-, -S-, -NR c -and-CR c R c -; R 1a Independently selected from halogen, hydroxy, amino, cyano, C1-C6 alkyl, C3-C6 cycloalkyl, 4-10 membered heterocyclyl, C1-C6 alkoxy, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C(O)C1-C6 alkyl and 5-10 membered heteroaryl; R 2a Independently selected from halogen, hydroxy, amino, cyano and C1-C3 alkyl; R b Independently selected from halogen, cyano, amino, C1-C6 alkyl and C1-C6 alkoxy, wherein the C1-C6 alkyl and C1-C6 alkoxy are optionally substituted with halogen; R c independently selected from hydrogen, halogen, hydroxy, oxo, amino, cyano, C1-C6 alkyl and C3-C6 cycloalkyl; One or more hydrogen atoms of the compound are optionally deuterium atoms.

2. The compound of formula (I) according to claim 1, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: X 1 is N.

3. The compound of formula (I) according to any one of claims 1 to 2, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: X 2 and X 3 are each independently selected from N and S; or X 2 For S, X 3 is N.

4. The compound of formula (I) according to any one of claims 1 to 3, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 1 Selected from hydrogen, amino, cyano, C2-C 10 Alkynyl, C1-C 10 Alkyl, C3-C 12 Cycloalkyl, 4-10 membered heterocyclyl, 5-10 membered heteroaryl and -C(O)R 3 , the amino group, C2-C 10 Alkynyl, C1-C 10 Alkyl, C3-C 12 Cycloalkyl, 4-10 membered heterocyclyl and 5-10 membered heteroaryl are optionally replaced by R 1a Replace; or R 1 is selected from hydrogen, amino, cyano, C2-C3 alkynyl, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, 5-6 membered heteroaryl and -C(O)R 3 The amino, C2-C3 alkynyl, C1-C6 alkyl, C3-C6 cycloalkyl, 4-7 membered heterocyclyl and 5-6 membered heteroaryl are optionally replaced by R 1a Replace; or R 1 is selected from hydrogen, amino, cyano, methyl, ethyl, ethynyl, cyclopropyl, morpholinyl, oxetanyl, pyridyl, pyrazolyl, triazolyl and -C(O)R 3 The amino, methyl, ethyl, ethynyl, cyclopropyl, morpholinyl, oxetanyl, pyridyl, pyrazolyl and triazolyl groups are optionally replaced by R 1a Replace; or R 1 is selected from hydrogen, methyl, ethyl, cyano, cyanomethyl, methoxymethyl, ethynyl, cyclopropyl, -C(O)NH2, -NHC(O)CH3, 5. The compound of formula (I) according to any one of claims 1 to 4, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 3 It is amino.

6. The compound of formula (I) according to any one of claims 1 to 5, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 1a R is independently selected from cyano, C1-C6 alkyl, 4-10 membered heterocyclyl, C1-C6 alkoxy, -NH(C1-C6 alkyl), C(O)C1-C6 alkyl and 5-10 membered heteroaryl; or 1a R is independently selected from cyano, C1-C3 alkyl, 4-7 membered heterocyclyl, C1-C3 alkoxy, -NH(C1-C3 alkyl), C(O)C1-C3 alkyl and 5-6 membered heteroaryl; or 1a Independently selected from cyano, methyl, methoxy, -NHCH3, morpholinyl, imidazolyl and -C(O)CH3.

7. The compound of formula (I) according to any one of claims 1 to 6, or its stereoisomer or a pharmaceutically acceptable salt thereof, wherein: R 2 Selected from C1-C 10 Alkyl, the C1-C 10 The alkyl group is optionally replaced by R 2a Replace; or R 2 is selected from C1-C6 alkyl, wherein the C1-C6 alkyl is optionally replaced by R 2a Replace; or R 2 is selected from C1-C3 alkyl, wherein the C1-C3 alkyl is optionally replaced by R 2a Replace; or R 2 is a methyl group, the methyl group is optionally replaced by R 2a Replace; or R 2 It is methyl.

8. The compound of formula (I) according to any one of claims 1 to 7, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein: R 2a Independently selected from halogen, hydroxy, amino, cyano.

9. The compound of formula (I) according to any one of claims 1 to 8, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein: Ring B is selected from 4-10 membered heterocyclic groups, wherein the 4-10 membered heterocyclic groups are optionally substituted by halogen or C1-C6 alkyl; or Ring B is selected from 4-7 membered heterocyclic groups, wherein the 4-7 membered heterocyclic groups are optionally substituted by halogen or C1-C6 alkyl; or Ring B is selected from 4-7 membered heterocyclic groups, wherein the 4-7 membered heterocyclic groups are optionally substituted by halogen or C1-C3 alkyl; or Ring B is selected from piperazinyl, piperidinyl, tetrahydropyridinyl and * indicates connection with L, the piperazinyl, piperidinyl, tetrahydropyridinyl and Optionally substituted with halogen or C1-C3 alkyl; or Ring B is selected from * indicates connection with L, Optionally substituted with halogen or C1-C3 alkyl; or Ring B is selected from * indicates connection with L, Optionally substituted with C1-C3 alkyl, preferably methyl and ethyl; or Ring B is selected from *Indicates connection with L.

10. The compound of formula (I) according to any one of claims 1 to 9, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein: Ring C is selected from 5-10 membered heterocyclic group, C6-C 10 Aryl and 5-10 membered heteroaryl, the 5-10 membered heterocyclic group, C6-C 10 Aryl and 5-10 membered heteroaryl are optionally replaced by R b or ring C is selected from C6-C 10 Aryl and 5-10 membered heteroaryl, the C6-C 10 Aryl and 5-10 membered heteroaryl are optionally replaced by R b or ring C is selected from dihydrobenzodioxin, phenyl and quinoxalinyl, wherein the dihydrobenzodioxin, phenyl and quinoxalinyl are optionally replaced by R b or ring C is selected from phenyl and quinoxalinyl, wherein the phenyl and quinoxalinyl groups are optionally substituted by R b Substituted; or ring C is selected from phenyl, The phenyl group, Optional R b replace.

11. The compound of formula (I) according to any one of claims 1 to 10, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein: R b is independently selected from halogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by halogen; or R b is independently selected from halogen and C1-C3 alkyl, wherein the C1-C3 alkyl is optionally substituted with halogen; or R b R is independently selected from fluoro, methyl and ethyl, said methyl and ethyl being optionally substituted by halogen; or b Independently selected from fluoro, methyl and trifluoromethyl.

12. The compound of formula (I) according to any one of claims 1 to 11, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein: L is selected from -NR c -and-CR c R c -.

13. The compound of formula (I) according to any one of claims 1 to 12, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein: R c R is independently selected from hydrogen, C1-C6 alkyl and C3-C6 cycloalkyl; or c R is independently selected from hydrogen, C1-C3 alkyl and C3-C6 cycloalkyl; or c are independently selected from hydrogen, methyl, ethyl and cyclopropyl.

14. The compound of formula (I) according to claim 1, or its stereoisomer, or a pharmaceutically acceptable salt thereof, wherein: The compound of formula (I) or a pharmaceutically acceptable salt thereof is selected from the compound of formula (II) or a pharmaceutically acceptable salt thereof: Among them, R 1 , R 2 , Ring B, Ring C and L are as defined in claims 1-13.

15. The compound of formula (I) according to claim 1 or its stereoisomer or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof, 16. A pharmaceutical composition comprising a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15 and a pharmaceutically acceptable excipient.

17. Use of the compound of formula (I) according to any one of claims 1 to 15, or its stereoisomer or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for preventing or treating a DGK-mediated disease.

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