diacylglycerol kinase inhibitors

Abstract

The application belongs to the technical field of medicine, and particularly relates to a diacylglycerol kinase inhibitor compound shown in a general formula (I), a pharmaceutically acceptable salt or a stereoisomer thereof, a pharmaceutical composition containing the compound, the pharmaceutically acceptable salt or the stereoisomer thereof, a method for preparing the compound, the pharmaceutically acceptable salt or the stereoisomer thereof, and the use of the compound, the pharmaceutically acceptable salt or the stereoisomer thereof.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a class of diacylglycerol kinase inhibitor compounds, pharmaceutically acceptable salts thereof or their stereoisomers, pharmaceutical compositions and formulations containing said compounds, pharmaceutically acceptable salts thereof or their stereoisomers, methods for preparing said compounds, pharmaceutically acceptable salts thereof or their stereoisomers, and uses of said compounds, pharmaceutically acceptable salts thereof or their stereoisomers. Background Technology

[0002] Developing safe and effective cancer treatments is one of the most challenging problems facing modern medicine. Early treatments included radiotherapy, chemotherapy, and surgery. Chemotherapy is mostly non-specific, and long-term use can easily lead to adverse reactions and drug resistance. In recent years, immunotherapy has gradually gained public attention. Immune checkpoint inhibitors can utilize the immune system to recognize and attack tumors; CTLA-4 and PD-1 are known as immune checkpoint molecules. Immunotherapy with anti-PD-1 antibodies has shown significant clinical efficacy in various cancers, including melanoma and non-small cell lung cancer, and its applications are expanding.

[0003] The diacylglycerol kinase family (DGKs) plays a crucial role in signal transduction by regulating the balance between two lipid signaling molecules: diacylglycerol (DAG) and phosphatidic acid (PA). The 10 kinases in the mammalian DGK family are classified into five distinct subtypes based on different genotypes (Merida, I., et al. Biochem. J. (2008) 409(1), 1-18.). These are classified as type I (α, β, γ), type II (δ, η, κ), type III (ε), type IV (ζ, ι), and type V (θ). Three DGK subtypes (DGKα, DGKδ, and DGKζ) are known to exist in T cells, but only DGKα and DGKζ are considered to play important roles in promoting DAG metabolism downstream of the TCR. DGKα / ζ is distributed in both the cell nucleus and cell membrane. After being activated by regulators such as Src, it migrates to the plasma membrane. DAG phosphorylation converts it to PA, and PA regulates the expression of downstream tumor pathways such as mTOR and HIF-1α, either decreasing or activating them (Carrasco, S., et al. Mol. Biol. Cell (2004) 15(6), 2932-2942). DGK inhibition not only interferes with the oncogenic pathway and directly kills tumor cells, but can also restore immune tumor attack.

[0004] In T cells, when the TCR interacts with the major histocompatibility gene complex (pMHC) and in the presence of the co-stimulatory signal CD28, proximal PTKs of the TCR are activated, leading to the recruitment of molecules. Activation of phospholipase Cγ1 (PLCγ1) can hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) on the membrane to form the second messenger (inositol triphosphate) IP3 and DAG. IP3 activates Ca2+. 2+ -CaN-NFAT pathway, while DAG activates Ras-ERK-AP1 and NF-kB pathways. DGKs suppress DAG-mediated signaling by converting DAG to PA. Selective IP3-Ca 2+ -NFAT signaling induces T cells into an unresponsive state in the presence of weak DAG-mediated signaling (Dower, NA, et al. Nat. Immunol (2000) 1:317–321). T cell unresponsiveness refers to the functional inactivity of T cells due to a decrease in co-stimulatory signals during antigen recognition.

[0005] Dysregulation of DGK expression is associated with various pathologies, including chronic inflammation, tumor immune evasion, and immunodeficiency diseases. The biological roles of DGKα and ζ isoforms in immune cell differentiation and effector function have attracted widespread attention. Recent studies suggest that upregulation of DGKα and ζ may be one of the mechanisms by which tumors induce T cell tolerance. In mice, DGKζ deficiency produces greater transcriptional changes, enhanced T cell function, and increased antitumor responsiveness than DGKα deficiency. DGKζ deficiency also has a significant regulatory effect on downstream T cell receptors and co-stimulatory signaling. (Riese MJ et al. J. Biol. Chem, (2011) 7: 5254-5265; In-Young, J., et al. Cancer Research (2018): canres. 0030. 2018; Olenchock, BA, et al. Nat. Immunol. (2006) 7: 1174-1181; Zha, Y. et al. Nat. Immunol. Joshi, RP et al. Sci. Signal. (2013) 6: ra102–ra102; Junchen, G. et al. OncoImmunology. (2021) 10: 1-8; Antonia, et al. Immunol. Cell. Biol. (2017): 1-15). Inhibition of DGKζ is a promising anti-tumor immunotherapy. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a novel polycyclic compound with good inhibitory activity against DGKα and / or DGKζ. Furthermore, this type of compound can be used for the prevention and / or treatment of diseases related to DGKα and / or DGKζ. Even further, this type of compound can be used for the prevention and / or treatment of cancers related to immune cell activation or cancers resistant to anti-PD-1 antibody / anti-PD-L1 antibody therapy.

[0007] The technical solution of the present invention is as follows: In one aspect, the present invention provides a compound represented by the following general formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0008]

[0009] Wherein, ring A is selected from 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclic or 5-7 membered monocyclic heteroaryl;

[0010] Ring B is selected from 3-11 membered cycloalkyl, 3-11 membered heterocyclic, 5-11 membered aryl, or 5-11 membered heteroaryl;

[0011] Cy 1 Selected from 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclic, phenyl, or 5-7 membered monocyclic heteroaryl groups, optionally substituted with 1-2 substituents Q; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0012] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl thiols, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thiols, hydroxyl C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxy C1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 Alkyl group, -(CH2) p -3-10 membered cycloalkyl groups, -(CH2) p -3-10 membered heterocyclic alkyl groups, -(CH2) p -N(R a (R) b -(CH2) p -OR a -(CH2) p -P(O)(R a (R) b -(CH2) p -S(O)(R a -(CH2) p -S(O)2(R a -(CH2) p -C(O)(R a -(CH2) p -C(O)O(R a -(CH2) p -OC(O)(R a -(CH2) p -C(O)N(R a R b -(CH2) p -N(R b )-C(O)(R a )or

[0013] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, 3-10 membered cycloalkyl or 3-10 membered heterocyclic groups;

[0014] m, n, and p are each independently selected from 0, 1, 2, 3, or 4; q is selected from 0, 1, or 2.

[0015] In one aspect, the present invention provides compounds represented by the following general formula (I), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0016]

[0017] Wherein, ring A is selected from 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclic or 5-7 membered monocyclic heteroaryl;

[0018] Ring B is selected from 3-11 membered cycloalkyl, 3-11 membered heterocyclic, 5-11 membered aryl, or 5-11 membered heteroaryl;

[0019] Cy 1 Selected from 5-7 membered monocyclic cycloalkyl, 5-7 membered monocyclic heterocyclic, phenyl, or 5-7 membered monocyclic heteroaryl groups, optionally substituted with 1-2 substituents Q; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0020] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 Alkyl group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a ,-(CH2) p -P(O)(R a (R) b ), -(CH2) p -S(O)(R a ), -(CH2) p-S(O)2(R a ), -(CH2) p -C(O)(R a ), -(CH2) p -C(O)O(R a ), -(CH2) p -OC(O)(R a ), -(CH2) p -C(O)N(R a R b ), -(CH2) p -N(R b )-C(O)(R a ), Alternatively, the following groups may be optionally substituted with 1-2 substituents: -(CH2) p -3-10 membered cycloalkyl groups, -(CH2) p -3-10 membered heterocyclic alkyl groups, -(CH2) p -6-10 aryl, -(CH2) p -5-10 heteroaryl groups; the substituents are independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0021] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, 3-10 membered cycloalkyl or 3-10 membered heterocyclic groups;

[0022] m, n, and p are each independently selected from 0, 1, 2, 3, or 4; q is selected from 0, 1, or 2.

[0023] In some embodiments, ring A is selected from 5-6 membered monocyclic cycloalkyl, 5-6 membered monocyclic heterocyclic or 5-6 membered monocyclic heteroaryl.

[0024] In some embodiments, ring A is selected from a 5-6 member monocyclic heterocyclic group containing 1-2 heteroatoms or groups or a 5-6 member monocyclic heteroaryl group containing 1-3 nitrogen atoms, wherein the heteroatoms or groups are selected from N, NH, O, S, C(O), S(O) or S(O)2.

[0025] In some embodiments, ring A is selected from a 5-6 member monocyclic heterocyclic group containing 1-2 heteroatoms or groups or a 5-6 member monocyclic heteroaryl group containing 1-3 nitrogen atoms, wherein the heteroatoms or groups are selected from N, NH, O or S.

[0026] In some embodiments, ring A is selected from a 5-6 member monocyclic heterocyclic group containing one heteroatom or a 5-6 member monocyclic heteroaryl group containing one or two nitrogen atoms, wherein the heteroatom is selected from O or S.

[0027] In some embodiments, ring A is selected from the following groups:

[0028]

[0029] In some embodiments, ring A is selected from the following groups:

[0030]

[0031] In some implementations, ring A is selected from...

[0032] In some implementations, ring A is

[0033] In some embodiments, ring B is selected from 5-6 member monocyclic cycloalkyl, 5-6 member monocyclic heterocyclic, phenyl, 5-6 member monocyclic heteroaryl, 8-11 member fused cycloyl, 8-11 member spirocyclic, 7-9 member bridged cycloyl, 8-11 member fused heterocyclic, 8-11 member spirocyclic, 7-9 member bridged heterocyclic, or 8-11 member fused heteroaryl.

[0034] In some embodiments, ring B is selected from 5-6 member monocyclic cycloalkyl, 5-6 member monocyclic heterocyclic, 8-11 member fused cycloalkyl, 8-11 member spirocyclic, 7-9 member bridged cycloalkyl, 8-11 member fused heterocyclic, 8-11 member spirocyclic or 7-9 member bridged heterocyclic.

[0035] In some embodiments, ring B is selected from 5-6 member monocyclic heterocyclic groups, 8-11 member fused heterocyclic groups, 8-11 member spirocyclic groups, or 7-9 member bridged heterocyclic groups.

[0036] In some embodiments, ring B is selected from 5-6 member monocyclic heterocyclic groups, 8-11 member fused heterocyclic groups, or 8-11 member spirocyclic groups.

[0037] In some embodiments, ring B is selected from 5-6 member nitrogen-containing monocyclic heterocyclic groups, 8-11 member nitrogen-containing fused heterocyclic groups, or 8-11 member nitrogen-containing spirocyclic groups.

[0038] In some implementations, ring B is selected from...

[0039] Every X 1Each is independently selected from N or CH;

[0040] Every X 2 X 3 Each is independently selected from CH2, NH or O.

[0041] In some embodiments, ring B is selected from the following groups:

[0042]

[0043] In some implementations, ring B is selected from...

[0044] In some implementations, Cy 1 Selected from 5-6 membered monocyclic cycloalkyl, 5-6 membered monocyclic heterocyclic, phenyl, or 5-6 membered monocyclic heteroaryl groups, optionally substituted with 1-2 Qs; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkyl group.

[0045] In some of the above implementations, Cy 1 Selected from phenyl groups optionally substituted with 1-2 Q groups or 5-6 membered monocyclic heteroaryl groups.

[0046] In some of the above implementations, Cy 1 Selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl groups, which may be substituted with 1-2 Qs.

[0047] In some implementations, Cy 1 Selected from pyridazinyl groups optionally substituted with 1-2 Q groups; each Q group is independently selected from H, halogen, hydroxyl, amino, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkyl group.

[0048] In some implementations, Cy1 Selected from phenyl or 5-6 membered monocyclic heteroaryl groups.

[0049] In some implementations, Cy 1 It is selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl.

[0050] In some implementations, Cy 1 It is pyridazinyl.

[0051] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0052] Ring A is selected from a 5-6 member monocyclic heterocyclic group containing one heteroatom or a 5-6 member monocyclic heteroaryl group containing 1-2 nitrogen atoms, wherein the heteroatom is selected from O or S;

[0053] Ring B is selected from 5-6 member monocyclic heterocyclic groups, 8-11 member fused heterocyclic groups, 8-11 member spirocyclic groups, or 7-9 member bridged heterocyclic groups;

[0054] Cy 1 Selected from phenyl or 5-6 membered monocyclic heteroaryl groups optionally substituted with 1-2 Qs; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0055] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl thiols, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thiols, hydroxyl C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxy C 1-6 Alkylthio, amino C 1-6 Alkylthio group, -(CH2)p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -N(R a (R) b ) or -(CH2) p -OR a ;

[0056] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0057] m, n, p, and q are each independently selected from 0, 1, or 2.

[0058] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0059] Ring A is selected from a 5-6 member monocyclic heterocyclic group containing one heteroatom or a 5-6 member monocyclic heteroaryl group containing 1-2 nitrogen atoms, wherein the heteroatom is selected from O or S;

[0060] Ring B is selected from 5-6 member monocyclic heterocyclic groups, 8-11 member fused heterocyclic groups, 8-11 member spirocyclic groups, or 7-9 member bridged heterocyclic groups;

[0061] Cy 1 Selected from phenyl or 5-6 membered monocyclic heteroaryl groups optionally substituted with 1-2 Qs; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0062] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a Alternatively, the following groups may be optionally substituted with 1-2 substituents: -(CH2) p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -phenyl, -(CH2) p -5-6-membered heteroaryl groups; the substituents are independently selected from halogens, cyano groups, amino groups, C... 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0063] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0064] m, n, p, and q are each independently selected from 0, 1, or 2.

[0065] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0066] Ring A is selected from

[0067] Ring B Selection

[0068] Every X 1 Each is independently selected from N or CH;

[0069] Every X 2 X 3 Each is independently selected from CH2, NH or O;

[0070] Cy 1 Selected from phenyl groups optionally substituted with 1-2 Qs or 5-6 membered nitrogen-containing monocyclic heteroaryl groups; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0071] Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a Alternatively, the following groups may be optionally substituted with 1-2 substituents: -(CH2) p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -phenyl, -(CH2) p -5-6 nitrogen-containing monocyclic heteroaryl groups; the substituents are independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0072] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0073] Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl thiols, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thiols, hydroxyl C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxy C 1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 alkyl;

[0074] m, n, p, and q are each independently selected from 0, 1, or 2.

[0075] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0076] Ring A is selected from

[0077] Ring B is selected from

[0078] Cy 1 Selected from phenyl groups optionally substituted with 1-2 Qs or 5-6 membered nitrogen-containing monocyclic heteroaryl groups; each Q is independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C1-6 Alkoxy;

[0079] Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a Alternatively, the following groups may be optionally substituted with 1-2 substituents: -(CH2) p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -phenyl, -(CH2) p -5-6 nitrogen-containing monocyclic heteroaryl groups; the substituents are independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0080] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0081] R 3 For H;

[0082] m, n, and p are each independently selected from 0, 1, or 2; q is 1.

[0083] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0084] Ring A is selected from

[0085] Ring B is selected from

[0086] Cy 1 Selected from pyrimidinyl, pyrazinyl, or pyridazinyl groups optionally substituted with 1-2 Q groups; each Q group is independently selected from H, halogen, hydroxyl, amino, C... 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0087] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 Alkyl group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a Alternatively, the following groups may be optionally substituted with 1-2 substituents: -(CH2) p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -phenyl, -(CH2) p-5-6 nitrogen-containing monocyclic heteroaryl groups; the substituents are independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0088] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0089] m, n, p, and q are each independently selected from 0, 1, or 2.

[0090] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0091] Ring A is selected from

[0092] Ring B is selected from

[0093] Cy 1 Selected from pyridazinyl groups optionally substituted with 1-2 Q groups; each Q group is independently selected from H, halogen, hydroxyl, amino, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0094] Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkyl thiols, halogenated C 1-6 Alkoxy, halogenated C1-6 Alkyl thiols, hydroxyl C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxy C 1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 Alkyl group, -(CH2) p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -N(R a (R) b ) or -(CH2) p -OR a ;

[0095] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0096] R 3 For H;

[0097] m, n, and p are each independently selected from 0, 1, or 2; q is 1.

[0098] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0099] Ring A is selected from

[0100] Ring B is selected from

[0101] Cy 1 Selected from pyridazinyl groups optionally substituted with 1-2 Q groups; each Q group is independently selected from H, halogen, hydroxyl, amino, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0102] Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 Alkyl group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a Alternatively, the following groups may be optionally substituted with 1-2 substituents: -(CH2) p -3-6 membered monocyclic cycloalkyl groups, -(CH2) p -3-6 membered monocyclic heterocyclic alkyl groups, -(CH2) p -phenyl, -(CH2) p -5-6 nitrogen-containing monocyclic heteroaryl groups; the substituents are independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0103] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0104] R 3 For H;

[0105] m, n, and p are each independently selected from 0, 1, or 2; q is 1.

[0106] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0107] Ring A is selected from

[0108] Ring B is selected from Cy 1 Selected from pyridazinyl groups optionally substituted with 1-2 Q groups; each Q group is independently selected from H, halogen, hydroxyl, amino, C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy;

[0109] Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylthio, halogenated C 1-6 Alkoxy, halogenated C 1-6 Alkyl thio group, hydroxyl group C 1-6 Alkoxy, amino C 1-6 Alkoxy, hydroxyl C 1-6 Alkylthio, amino C 1-6 Alkylthio, C 1-6 Alkoxy-C 1-6 Alkyl group, -(CH2) p -N(R a (R) b ), -(CH2) p -OR a Alternatively, the following groups may be optionally substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, azacyclobutyl, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, pyrimidinyl, pyridazinyl; wherein each substituent is independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy;

[0110] R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, cyano C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl;

[0111] R 3 For H;

[0112] m, n, and p are each independently selected from 0, 1, or 2; q is selected from 0 or 1.

[0113] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0114] Ring A is selected from

[0115] Ring B is selected from

[0116] Cy 1 It is pyridazinyl;

[0117] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, hydroxy C 1-4 Alkoxy, amino C 1-4 Alkoxy, C 1-4 Alkoxy-C 1-4 Alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, or -(CH2) p -N(R a (R) b );

[0118] R a R b Selected independently from hydrogen and C 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl or C 1-4 Alkoxy-C1-4 alkyl;

[0119] m is selected from 1 or 2; n is 1; p is selected from 0 or 1; q is selected from 0 or 1.

[0120] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0121] Ring A is selected from

[0122] Ring B is selected from

[0123] Cy 1 It is pyridazinyl;

[0124] Each R 1 Each R 2 Each R 3 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, hydroxyl C 1-4 Alkoxy, amino C 1-4 Alkoxy, C 1-4 Alkoxy-C 1-4 Alkyl group, -(CH2) p -N(R a (R) b Alternatively, the following groups may be substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, pyrimidinyl; wherein each substituent is independently selected from halogen, cyano, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl or halogenated C 1-4 Alkoxy;

[0125] R a R b Selected independently from hydrogen and C 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl or C 1-4 Alkoxy-C 1-4 alkyl;

[0126] m is selected from 1 or 2; n is 1; p is selected from 0 or 1; q is selected from 0 or 1.

[0127] In some embodiments, the compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, wherein,

[0128] Ring A is selected from

[0129] Ring B is selected from

[0130] Cy 1 It is pyridazinyl;

[0131] Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, hydroxyl C 1-4 Alkoxy, amino C 1-4 Alkoxy, C 1-4 Alkoxy-C 1-4 Alkyl group, -(CH2) p -N(R a (R) b Alternatively, the following groups may be substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, pyrimidinyl; wherein each substituent is independently selected from halogen, cyano, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl or halogenated C 1-4 Alkoxy;

[0132] R a R b Selected independently from hydrogen and C 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl or C 1-4 Alkoxy-C 1-4 alkyl;

[0133] R 3 For H;

[0134] m is selected from 1 or 2; n is 1; p is selected from 0 or 1; q is selected from 0 or 1.

[0135] In one aspect, the present invention provides compounds represented by the following general formula (I-1), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0136]

[0137] Where Y and Z are independently selected from N or CH, respectively; R 1 R 2 R 3 Cy 1 The definitions of m, n, q, and ring B are as described in any of the previous schemes.

[0138] In one aspect, the present invention provides compounds represented by the following general formula (I-2), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0139]

[0140] Where Y and Z are independently selected from N or CH, respectively; R 1 R 2 R 3 The definitions of Q, m, n, q, and ring B are as described in any of the previous schemes.

[0141] In one aspect, the present invention provides compounds represented by the following general formula (I-2), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0142]

[0143] Where Y and Z are independently selected from N or CH, and Q is H; R 1 R 2 R 3 The definitions of m, n, q, and ring B are as described in any of the previous schemes.

[0144] In one aspect, the present invention provides compounds represented by general formula (II), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0145]

[0146] Among them, R 1 R 2 R 3 The definitions of Q, m, n, q, and ring B are as described in any of the previous schemes.

[0147] In one aspect, the present invention provides compounds represented by general formula (II), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0148]

[0149] Among them, R 1 R 2 R 3The definitions of m, n, q, and ring B are as described in any of the previous schemes; Q is H.

[0150] In some embodiments, the compound represented by general formula (II-1), its pharmaceutically acceptable salt, or its stereoisomer,

[0151]

[0152] Among them, R 1 R 2 R 3 The definitions of Q, m, n, and q are as described in any of the previous schemes.

[0153] In some embodiments, the compound represented by general formula (II-1), its pharmaceutically acceptable salt, or its stereoisomer,

[0154]

[0155] Among them, R 1 R 2 R 3 The definitions of m, n, and q are as described in any of the previous schemes; Q is H.

[0156] In one aspect, the present invention provides compounds represented by the following general formula (II-2), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0157]

[0158] Among them, R 1 The group is selected from the following groups, optionally substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, or pyrimidinyl, wherein each substituent is independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy; preferably, R 1 'Selected from the following groups, optionally substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl, wherein each substituent is independently selected from halogens, C...' 1-6 Alkyl or C 1-6 Alkoxy; preferably, the substituents are independently selected from fluorine, chlorine, methyl, ethyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl;

[0159] R 1 R 2 R 3 The definitions of Q, n, and q are as described in any of the schemes above.

[0160] In one aspect, the present invention provides compounds represented by the following general formula (II-2), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0161]

[0162] Among them, R 1 The group is selected from the following groups, optionally substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, or pyrimidinyl, wherein each substituent is independently selected from halogen, cyano, amino, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkyl or halogenated C 1-6 Alkoxy; preferably, R 1 'Selected from the following groups, optionally substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or phenyl, wherein each substituent is independently selected from halogens, C...' 1-6 Alkyl or C 1-6 Alkoxy; preferably, the substituents are independently selected from fluorine, chlorine, methyl, ethyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl;

[0163] R 1 R 2 R 3 The definitions of , n, and q are as described in any of the previous schemes, and Q is H.

[0164] In one aspect, the present invention provides compounds represented by the following general formula (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0165]

[0166] Among them, R 1 R 2 R 3 The definitions of Q and q are as described in any of the previous schemes.

[0167] In one aspect, the present invention provides compounds represented by the following general formula (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0168]

[0169] Among them, R 1 R 2 R 3 The definitions of q are as described in any of the previous schemes; Q is H.

[0170] In some implementations, each R 1Selected independently from H, halogen, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy or C 1-4 Alkoxy-C 1-4 Alkyl; preferably, each R 1 Selected independently from H, halogen, and C. 1-4 Alkyl or halogenated C 1-4 Alkyl; preferably, each R 1 Each of the following is independently selected from H, fluorine, chlorine, methyl, ethyl, isopropyl, monofluoromethyl, difluoromethyl, or trifluoromethyl.

[0171] In some implementations, each R 2 Each is independently selected from H, halogen, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl or -(CH2) p -N(R a (R) b ); R a R b Selected independently from hydrogen and C 1-6 Alkyl, Halogenated C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 alkyl.

[0172] In some implementations, each R 2 Selected independently from H, halogen, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl or -(CH2) p -N(R a (R) b ); R a Selected from hydrogen, C 1-6 Alkyl, Halogenated C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 Alkyl; R b It is hydrogen. Preferably, each R 2 Each of the following is independently selected from H, fluorine, methyl, ethyl, isopropyl, monofluoromethyl, difluoromethyl, trifluoromethyl, or -(CH2). p -N(R a (R) b ); R a Selected from hydrogen, methyl, ethyl, isopropyl, monofluoromethyl, difluoromethyl, or trifluoromethyl; R b It is hydrogen.

[0173] In some implementations, each R 2Each is independently -(CH2) p -N(R a (R) b ); R a Selected from hydrogen, C 1-6 Alkyl, Halogenated C 1-6 Alkyl or C 1-6 Alkoxy-C 1-6 Alkyl; R b It is hydrogen. Preferably, each R 2 Each is independently -(CH2) p -N(R a (R) b ); R a Selected from hydrogen, methyl, ethyl, isopropyl, monofluoromethyl, difluoromethyl, or trifluoromethyl; R b It is hydrogen.

[0174] In one aspect, the present invention provides compounds, pharmaceutically acceptable salts thereof, or stereoisomers thereof, as shown below:

[0175]

[0176] In another aspect, the present invention also provides a pharmaceutical composition comprising the compounds of the aforementioned formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), their pharmaceutically acceptable salts or stereoisomers, and one or more pharmaceutically acceptable excipients, wherein the pharmaceutical composition may be in any pharmaceutically acceptable dosage form. Pharmaceutically acceptable excipients are substances that are non-toxic, compatible with the active ingredient, and otherwise biologically suitable for use in organisms. The selection of a specific excipient will depend on the route of administration or the type and state of disease for the treatment of a particular patient.

[0177] In some embodiments, the above-described pharmaceutical composition can be administered to patients or subjects requiring this treatment via oral, parenteral, rectal, or pulmonary administration. For oral administration, the pharmaceutical composition can be formulated into oral preparations, such as conventional oral solid dosage forms like tablets, capsules, pills, granules, etc.; or into oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc. For parenteral administration, the above-described pharmaceutical composition can also be formulated into injectable preparations, including injection solutions, sterile powders for injection, and concentrated solutions for injection. For rectal administration, the pharmaceutical composition can be formulated into suppositories, etc. For pulmonary administration, the pharmaceutical composition can be formulated into inhaled preparations, aerosols, powder inhalers, or sprays, etc.

[0178] In another aspect, the pharmaceutical composition of the present invention comprises the compounds of the aforementioned general formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, and may further comprise one or more second therapeutic agents selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA-4 antibodies.

[0179] In another aspect, the present invention also relates to the use of the compounds of the aforementioned general formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of medicaments for the prevention and / or treatment of cancers associated with immune cell activation.

[0180] In another aspect, the present invention also relates to the use of the compounds of the aforementioned formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of medicaments for the prevention and / or treatment of cancers associated with immune cell activation or cancers resistant to PD-1 antibody / anti-PD-L1 antibody therapy.

[0181] Furthermore, the present invention also relates to the use of pharmaceutical compositions comprising the compounds of the aforementioned general formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of medicaments for the prevention and / or treatment of cancers associated with immune cell activation.

[0182] Furthermore, the present invention also relates to the use of pharmaceutical compositions comprising the compounds of the aforementioned general formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, in the preparation of medicaments for the prevention and / or treatment of cancers associated with immune cell activation or cancers resistant to PD-1 antibody / anti-PD-L1 antibody therapy.

[0183] In another aspect, the present invention also provides a method for treating diseases associated with immune cell activation, the method comprising administering to a patient in need an effective amount of the aforementioned compounds of formulas (I), (I-1), (I-2), (II), (II-1), (II-2), (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, or the aforementioned pharmaceutical compositions.

[0184] Furthermore, the present invention also provides an immunotherapy method for cancer, comprising administering to a patient in need an effective amount of the aforementioned compounds of formulas (I), (I-1), (I-2), (II), (II-1), (II-2), (II-3), pharmaceutically acceptable salts thereof, or stereoisomers thereof, or the aforementioned pharmaceutical compositions.

[0185] In another aspect, the present invention also provides a kit comprising an effective amount of one or more of the compounds described in general formulas (I), (I-1), (I-2), (II), (II-1), (II-2), and (II-3), their pharmaceutically acceptable salts, or stereoisomers thereof.

[0186] In another aspect, the present invention also provides a kit comprising:

[0187] (a) An effective amount of one or more compounds of the aforementioned general formulas (I), (I-1), (I-2), (II), (II-1), (II-2), or (II-3), their pharmaceutically acceptable salts, or their stereoisomers.

[0188] (b) an effective amount of one or more anticancer agents.

[0189] The "effective dose" as described in this invention refers to a drug dose capable of preventing, alleviating, delaying, inhibiting, or curing the subject's condition. The dosage is related to the route of drug administration, the pharmacokinetics of the drug, the severity of the disease, and the subject's individual characteristics (gender, weight, height, age), etc.

[0190] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, for a better understanding of this invention, definitions of some terms are provided below. When the definitions and interpretations of the terms provided in this invention differ from the meanings commonly understood by those skilled in the art, the definitions and interpretations provided in this invention shall prevail.

[0191] The "halogen" mentioned in this invention refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0192] The "C" described in this invention 1-6 "Alkyl" refers to a straight-chain or branched alkyl group containing 1-6 carbon atoms, including, for example, "C". 1-4 Alkyl", C 1-3 Alkyl", C 1-2 Alkyl", C 2-6 Alkyl", C 2-5 Alkyl", C2-4 Alkyl", C 2-3 Alkyl groups, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc. The "C" in this invention... 1-4 "alkyl" refers to C 1-6 Specific examples of alkyl groups containing 1-4 carbon atoms.

[0193] The "C" described in this invention 1-6 "Alkoxy" refers to "C 1-6 alkyl-O-", the "C" 1-6 "alkyl" is as defined above. The "C" in this invention... 1-4 "Alkoxy" refers to "C 1-4 alkyl-O-", the "C" 1-4 "Alkyl" is as defined above.

[0194] The "C" described in this invention 1-6 "Alkylthio" refers to "C 1-6 Alkyl-S-”, the “C” 1-6 "alkyl" is as defined above. The "C" in this invention... 1-4 "Alkylthio" refers to "C 1-4 Alkyl-S-”, the “C” 1-4 "Alkyl" is as defined above.

[0195] The "hydroxyl C" of this invention 1-6 Alkyl, amino C 1-6 Alkyl, Halogenated C 1-6 Alkyl, cyano C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 "alkyl" refers to C 1-6 One or more hydrogens in the alkyl group are respectively converted by one or more hydroxyl, amino, halogen, cyano or C groups. 1-6 Alkyl groups are substituted. C 1-6 Alkyl, C 1-6 Alkoxy groups are as defined above.

[0196] The "hydroxyl C" of this invention 1-6 Alkoxy, amino C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, cyano C 1-6 "Alkoxy" refers to "C1-6 One or more hydrogen atoms in the "alkoxy" group are replaced by one or more hydroxyl, amino, halogen, or cyano groups.

[0197] The "hydroxyl C" of this invention 1-6 Alkylthio, amino C 1-6 Alkyl thiols, halogenated C 1-6 "Alkylthio" refers to "C 1-6 One or more hydrogen atoms in the "alkylthio" group are replaced by one or more hydroxyl, amino, or halogen groups.

[0198] The "fluorinated C" of this invention 1-6 Alkyl group, fluorinated C 1-6 "Alkoxy" refers to "C 1-6 Alkyl", C 1-6 In an alkoxy group, one or more hydrogen atoms are replaced by one or more fluorine atoms.

[0199] The "3-11 membered heterocyclic group" as described in this invention refers to a saturated or partially saturated monocyclic or polycyclic cyclic group that contains at least one heteroatom or group (e.g., 1, 2, 3, 4, or 5) and has 3-11 ring atoms, and is not aromatic. The heteroatom or group is selected from nitrogen, oxygen, or sulfur atoms. Optionally, the ring atoms (e.g., carbon, nitrogen, or sulfur atoms) in the cyclic structure can be substituted with oxygen. The "3-11 membered heterocyclic group" includes, but is not limited to, "3-8 membered monocyclic heterocyclic group," "8-11 membered fused heterocyclic group," "8-11 membered spirocyclic heterocyclic group," and "7-11 membered bridged heterocyclic group."

[0200] The "3-8 member monoheterocyclic group" described in this invention refers to a saturated or partially saturated monocyclic cyclic group that does not possess aromaticity and contains at least one heteroatom (e.g., 1, 2, 3, 4, or 5 heteroatoms) and has 3-8 ring atoms. The heteroatom is a nitrogen atom, an oxygen atom, and / or a sulfur atom. Optionally, the ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure can be substituted with oxygen. The "3-8 member monoheterocyclic group" described in this invention includes "3-8 member saturated monoheterocyclic groups" and "3-8 member partially saturated monoheterocyclic groups." Preferably, the "3-8 member monoheterocyclic group" described in this invention contains 1-3 heteroatoms; preferably, the "3-8 member monoheterocyclic group" described in this invention contains 1-2 heteroatoms, and the heteroatoms are selected from nitrogen atoms and / or oxygen atoms; preferably, the "3-8 member monoheterocyclic group" described in this invention contains 1 heteroatom, and the heteroatom is a nitrogen atom, an oxygen atom, and / or a sulfur atom. The preferred "3-8 member monoheterocyclic group" includes "3-7 member monoheterocyclic group", "3-6 member monoheterocyclic group", "4-7 member monoheterocyclic group", "4-6 member monoheterocyclic group", "6-8 member monoheterocyclic group", "5-7 member monoheterocyclic group", "5-7 member saturated monoheterocyclic group", "5-7 member partially saturated monoheterocyclic group", "5-6 member monoheterocyclic group", "5-6 member saturated monoheterocyclic group", "5-6 member partially saturated monoheterocyclic group", "3-6 member saturated monoheterocyclic group", "5-6 member saturated monoheterocyclic group", "3-6 member nitrogen-containing monoheterocyclic group", "3-6 member saturated nitrogen-containing monoheterocyclic group", "5-6 member nitrogen-containing monoheterocyclic group", "5-6 member saturated nitrogen-containing monoheterocyclic group", and "5-6 member partially saturated nitrogen-containing monoheterocyclic group". Specific examples of "3-8 membered monoheterocyclic groups" include, but are not limited to: aziridine, 2H-aziridine, diaziridine, 3H-diazacyclopropenyl, aziridine, oxacyclobutyl, 1,4-dioxane, 1,3-dioxane, 1,3-dioxanepentyl, 1,4-dioxanediene, tetrahydrofuranyl, dihydropyrrolyl, pyrrolylalkyl, imidazoalkyl, 4,5-dioxanediol, etc. Hydro-imidazolyl, pyrazolyl, 4,5-dihydropyrazolyl, 2,5-dihydrothiophenyl, tetrahydrothiophenyl, 4,5-dihydrothiazolyl, thiazolyl, piperidinyl, tetrahydropyridinyl, piperidinone, tetrahydropyridinone, dihydropiperidinone, piperazine, morpholinyl, 4,5-dihydrooxazolyl, 4,5-dihydroisooxazolyl, 2,3-dihydroisooxazolyl, oxazolyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, etc.

[0201] The "8-11 membered fused-ring heterocyclic group" described in this invention refers to a saturated or partially saturated, non-aromatic cyclic group containing 8-11 ring atoms, formed by two or more cyclic structures sharing two adjacent atoms, and at least one ring atom being a heteroatom. One ring in the fused ring may be aromatic, but the fused ring as a whole is not aromatic. The heteroatom is a nitrogen atom, an oxygen atom, and / or a sulfur atom. The ring atoms in the cyclic structure (e.g., carbon, nitrogen, or sulfur atoms) may optionally be oxidized. Specific examples include, but are not limited to: dihydrofuranopyridine, 3,4-dihydro-2H-pyranopyridine, 3,4-dihydro-2H-oxazinopyridine, dihydrooxazinopyrimidine, benzodihydrofuranyl, etc. wait.

[0202] The "8-11 member spiroheterocyclic group" described in this invention refers to a saturated or partially saturated cyclic structure containing 8-11 ring atoms, formed by two or more cyclic structures sharing one ring atom with each other. At least one ring atom is a heteroatom or group, such as N, NH, O, S, CO, SO, SO2, etc. Preferably, the number of heteroatoms or groups is 1, 2, 3, 4, or 5, more preferably 1 or 2. Examples include "9-11 member spiroheterocyclic group," "9-11 member saturated spiroheterocyclic group," and "9-11 member partially saturated spiroheterocyclic group." Specific examples include, but are not limited to:

[0203] The "7-11-membered bridged heterocyclic group" described in this invention refers to a saturated or partially saturated cyclic structure containing 7-11 ring atoms, formed by two or more cyclic structures sharing two non-adjacent ring atoms. At least one ring atom is a heteroatom or group, such as N, NH, O, S, CO, SO, SO2, etc., preferably 1, 2, 3, 4, or 5 heteroatoms or groups, more preferably 1 or 2. Examples include "7-9-membered bridged heterocyclic group," "7-9-membered saturated bridged heterocyclic group," "7-8-membered bridged heterocyclic group," and "7-8-membered saturated bridged heterocyclic group," etc. Specific examples include, but are not limited to:

[0204] The "3-11 membered cycloalkyl" as used in this invention refers to a saturated or partially saturated monocyclic or polycyclic cyclic group containing 3-11 ring atoms and lacking aromaticity. The "3-11 membered cycloalkyl" as used in this invention includes, but is not limited to, "3-8 membered monocyclic cycloalkyl," "8-11 membered fused cycloalkyl," "8-11 membered spirocyclic," and "7-11 membered bridged cycloalkyl." Among them, "3-8 membered monocyclic cycloalkyl" includes, but is not limited to, cyclopentyl, cyclohexyl, and...

[0205] The "8-11 member spirocyclic group" mentioned in this invention refers to a saturated or partially saturated cyclic structure containing 8-11 ring carbon atoms, formed by two or more cyclic structures sharing one ring atom with each other. Examples include "9-11 member spirocyclic group," "9-11 member saturated spiroheterocyclic group," and "9-11 member partially saturated spiroheterocyclic group." Specific examples include, but are not limited to:

[0206] The "7-11-membered bridged ring group" described in this invention refers to a saturated or partially saturated ring structure containing 7-11 ring carbon atoms, formed by two or more ring structures sharing two non-adjacent ring atoms. Examples include "7-9-membered bridged ring group," "7-9-membered saturated bridged ring group," "7-8-membered bridged heterocyclic group," "7-8-membered saturated bridged ring group," and "7-8-membered partially saturated bridged ring group," etc. Specific examples include, but are not limited to:

[0207] The "8-11 fused cycloalkyl group" described in this invention refers to a saturated or partially saturated, non-aromatic cyclic group containing 8-11 cyclic carbon atoms, formed by two or more cyclic structures sharing two adjacent atoms. One of the rings in the fused ring may be an aromatic ring, but the fused ring as a whole does not possess aromaticity. Examples include, but are not limited to: wait.

[0208] The “5-11 aryl” mentioned in this invention refers to a monocyclic or polycyclic cyclic group containing 5-11 cyclic carbon atoms that has aromaticity, including but not limited to “5-8 cyclic monocyclic aryl” and “8-11 cyclic aryl”.

[0209] The “8-11 fused-ring aryl” mentioned in this invention refers to an unsaturated, aromatic cyclic group containing 8-11 cyclic carbon atoms formed by two or more cyclic structures sharing two adjacent atoms, including “9-10 fused-ring aryl”, and specific examples include, but are not limited to, naphthyl.

[0210] The "5-11-membered heteroaryl" as described in this invention refers to an aromatic monocyclic or polycyclic cyclic group containing 5-11 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The ring atoms (e.g., carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may optionally be oxidized. "5-11-membered heteroaryl" includes, but is not limited to, "5-8-membered monocyclic heteroaryl" and "8-11-membered fused heteroaryl".

[0211] The "5-8 membered monocyclic heteroaryl" described in this invention refers to an aromatic monocyclic cyclic group containing 5-8 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The ring atoms (e.g., carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may optionally be oxidized. Examples include, for instance, "5-7 membered monocyclic heteroaryl," "5-6 membered monocyclic heteroaryl," "5-6 membered nitrogen-containing monocyclic heteroaryl," and "6 membered nitrogen-containing monocyclic heteroaryl," etc. The "nitrogen-containing heteroaryl" contains at least one nitrogen atom, for example, only one or two nitrogen atoms, or one nitrogen atom and one or two other heteroatoms (e.g., oxygen atom and / or sulfur atom), or two nitrogen atoms and one or two other heteroatoms (e.g., oxygen atom and / or sulfur atom). Specific examples of “5-7 membered monocyclic heteroaryl groups” include, but are not limited to, furanyl, thiophene, pyrrole, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridonel, 4-pyridonel, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, aziridine-heptadienyl, 1,3-diazacyclicheptadienyl, etc.

[0212] The "8-11 fused aryl" as described in this invention refers to an unsaturated aromatic cyclic structure formed by two or more cyclic structures sharing two adjacent atoms, containing 8-11 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, oxygen atom, or sulfur atom). The ring atoms (e.g., carbon atom, nitrogen atom, or sulfur atom) in the cyclic structure may optionally be oxidized. This includes "9-10 fused heteroaryl", "8-9 fused heteroaryl", etc., and the fusion mode can be 5-nitrogen-containing heteroaryl with 5-6-nitrogen-containing heteroaryl, 6-nitrogen-containing heteroaryl with 5-6-nitrogen-containing heteroaryl, 5-6-aryl with 5-6 heteroaryl, etc.; specific examples include but are not limited to: pyrrolopyrrole, pyrrolofuran, pyrrolopyrimidine, pyrrolopyridine, pyrazolopyrrole, pyrazolopyrimidine, pyrazolothiophene, pyrazolooxazole, benzofuranyl, benzoisofuranyl, benzothiopheneyl, indolyl, isoindolyl, benzooxazolyl, benzimidazolyl, etc.

[0213] In this invention, "CH" refers to the following structure:

[0214] In this invention, "N" refers to the following structure:

[0215] The "-(CH2)" described in this inventionp -P(O)(R a (R) b ")" refers to In this invention, other similar groups are defined the same as "-(CH2)". p -P(O)(R a (R) b )".

[0216] The "each R" described in this invention 1 "This refers to multiple Rs when m is 2, 3, or 4" 1 In each R 1 Independently selected from the groups described in the above technical solutions.

[0217] The "each R" described in this invention 2 "This refers to the situation where n is 2, 3, or 4, and multiple R's are equal. 2 In each R 2 Independently selected from the groups described in the above technical solutions.

[0218] The "each R" described in this invention 3 "This refers to the situation where q is 2, and multiple R's..." 3 In each R 3 Independently selected from the groups described in the above technical solutions.

[0219] The term "optionally substituted" in this invention refers to two situations: one or more hydrogen atoms on the substituted group are "substituted" or "not substituted" by one or more substituents.

[0220] When ring A is selected from nitrogen-containing heterocycles or heteroaryl groups, and contains NH, for example... The H in the NH on its ring can be R 1 replace.

[0221] When ring B is selected from nitrogen-containing heterocycles or heteroaryl groups, and contains NH, for example... The H in the NH on its ring can be R 2 replace.

[0222] The "pharmaceutically acceptable salt" as described in this invention refers to a salt formed by an acidic functional group (e.g., -COOH, -OH, -SO3H, etc.) present in a compound and a suitable inorganic or organic cation (base), including salts formed with alkali metals or alkaline earth metals, ammonium salts, and salts formed with nitrogen-containing organic bases; and a salt formed by a basic functional group (e.g., -NH2, etc.) present in a compound and a suitable inorganic or organic anion (acid), including salts formed with inorganic acids or organic acids (e.g., carboxylic acids, etc.).

[0223] The term "stereoisomer" as used in this invention refers to compounds containing one or more asymmetric centers, thus allowing them to exist as racemic mixtures and racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers. Compounds of this invention may have asymmetric centers, each of which independently produces two optical isomers. The scope of this invention includes all possible optical isomers and mixtures thereof.

[0224] Unless otherwise specified, the compounds described in this invention, if containing an alkene double bond, include cis and trans isomers. The compounds described in this invention can exist as tautomers (a type of functional group isomer) which have different hydrogen linkages through one or more double bond shifts; for example, ketones and their enol forms are keto-enol tautomers. All tautomers and mixtures thereof are included within the scope of this invention.

[0225] All stereoisomers, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof are included within the scope of this invention.

[0226] The compounds of this invention can be prepared by enantiomer-specific synthesis or by resolution from mixtures of enantiomers to obtain individual enantiomers. Conventional resolution techniques include using various well-known chromatographic methods to separate mixtures of enantiomers of the starting material or the final product.

[0227] When the stereochemistry of a disclosed compound is determined by structural nomenclature or description, the named or described stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight relative to other stereoisomers. When a single isomer is determined by structural nomenclature or description, the described or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. Optical purity (by weight%) is the ratio of the weight of the enantiomer to the weight of the enantiomer plus the weight of its optical isomer.

[0228] Beneficial effects of the invention

[0229] 1. The compounds of the present invention, their pharmaceutically acceptable salts or their stereoisomers have excellent DGK inhibitory effects and IL-2 secretion-enhancing effects, which can effectively improve cellular immune responses. They have good pharmacokinetic properties in vivo, long-lasting effects and high bioavailability.

[0230] 2. The compounds of the present invention, their pharmaceutically acceptable salts or stereoisomers thereof have good therapeutic effects on cancer and exhibit high stability in liver microsomes.

[0231] 3. The compound preparation process of this invention is simple, the drug has high purity and stable quality, and it is easy to carry out large-scale industrial production. Detailed Implementation Plan

[0232] The technical solution of the present invention will be described below with reference to specific embodiments, and the above-mentioned content of the present invention will be further explained in detail. However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following embodiments. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention.

[0233] abbreviation:

[0234] DIPEA: N,N-diisopropylethylamine; TFA: trifluoroacetic acid; HATU: N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea; DIEA: N,N-diisopropylethylamine; DMF: N,N-dimethylformamide.

[0235] Example 1: Preparation of (S)-N-(1-cyclopropyl-4-(3-(((methylamino)methyl)piperidin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide dihydrochloride (Compound 1)

[0236]

[0237] 1. Preparation of N-(2-chloro-6-fluorophenyl)-2,2,2-trifluoroacetamide

[0238] 2-Chloro-6-fluoroaniline (1.0 g, 6.9 mmol) was dissolved in ethyl acetate (20 mL), and trifluoroacetic anhydride (1.7 g, 8.3 mmol) was added. After the addition was complete, the mixture was reacted at 70 °C for 1 h. The mixture was concentrated and purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 10:1) to obtain the target compound (1.3 g, yield 78.3%).

[0239] 2. Preparation of N-(2-chloro-6-fluoro-3-nitrophenyl)-2,2,2-trifluoroacetamide

[0240] N-(2-chloro-6-fluorophenyl)-2,2,2-trifluoroacetamide (1.0 g, 4.1 mmol) was dissolved in concentrated sulfuric acid (10 mL), and fuming nitric acid (287 mg, 4.1 mmol) was added. After the addition was complete, the reaction was carried out at 0 °C for 1 h, and the reaction was stopped by LCMS. The solution was poured into water, and a solid precipitated. The solid was filtered and dried to give the target compound (1.0 g, yield 84.0%).

[0241] 3. Preparation of 4-chloro-1-cyclopropyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazole

[0242] N-(2-chloro-6-fluoro-3-nitrophenyl)-2,2,2-trifluoroacetamide (1.0 g, 3.5 mmol) was dissolved in acetonitrile (20 mL), and cyclopropylamine (798 mg, 14.0 mmol) was added. After the addition was complete, the mixture was reacted at 50 °C for 5 h. The solution was concentrated and then subjected to column chromatography (SiO2, petroleum ether: ethyl acetate = 2:1) to give the target compound (120 mg, yield 11.3%).

[0243] 4. Preparation of (S)-((1-(1-cyclopropyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)tert-butyl carbamate

[0244] 4-Chloro-1-cyclopropyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazole (120 mg, 0.39 mmol) was dissolved in 1,4-dioxane (10 mL), followed by the addition of tert-butyl(R)-(piperidin-3-ylmethyl)carbamate (101 mg, 0.47 mmol) and DIEA (101 mg, 0.78 mmol). After the addition was complete, the reaction was carried out at 100 °C for 2 h. The reaction was stopped by LCMS. The concentrate was then subjected to column chromatography (SiO2, petroleum ether: ethyl acetate = 4:1) to give the target compound (170 mg, yield 89.5%).

[0245] 5. Preparation of (R)-((1-(1-cyclopropyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0246] (S)-((1-(1-cyclopropyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)tert-butyl carbamate (170 mg, 0.35 mmol) was dissolved in DMF (10 mL). Sodium hydride (70 mg, 1.75 mmol) was added at 0 °C. After the addition was complete, the reaction was carried out at 0 °C for 10 min. Then, methyl iodoform (149 mg, 1.05 mmol) was added. After the addition was complete, the reaction was carried out at 25 °C for 1 h. The reaction was quenched with water, extracted with ethyl acetate, and the organic phase was dried and concentrated for use in the next step.

[0247] 6. Preparation of (R)-((1-(5-amino-1-cyclopropyl-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0248] (R)-((1-(1-cyclopropyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)tert-butyl carbamate (the crude product from the previous step) was dissolved in methanol (20 mL), and palladium on carbon (100 mg) was added. After the addition was complete, the reaction was carried out at 20 °C for 1 hour under hydrogen atmosphere. The reaction was stopped by LCMS. The mixture was filtered through diatomaceous earth, and the concentrated filtrate was used directly in the next step.

[0249] 7. Preparation of (R)-((1-(1-cyclopropyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamide)-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0250] (R)-((1-(5-amino-1-cyclopropyl-2-(trifluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (crude product from the previous step) was dissolved in DMF (10 mL), and 2-(pyridazin-4-yl)thiazol-4-carboxylic acid (81 mg, 0.39 mmol), HATU (148 mg, 0.39 mmol), and DIEA (101 mg, 0.78 mmol) were added. After the addition was complete, the reaction was carried out at 25 °C for 16 hours. The reaction was stopped by LCMS. The mixture was quenched with water, extracted with ethyl acetate, and the organic phase was concentrated and purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 1:1) to obtain the target compound (160 mg, yield 68.6%).

[0251] 8. Preparation of (S)-N-(1-cyclopropyl-4-(3-(((methylamino)methyl)piperidin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide dihydrochloride

[0252] (R)-((1-(1-cyclopropyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamide)-2-(trifluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (160 mg, 0.24 mmol) was dissolved in dichloromethane (5 mL), and ethyl acetate hydrochloride solution (5 mL) was added. After the addition was complete, the reaction was carried out at 25 °C for 2 hours. The reaction was stopped by LCMS. The mixture was filtered and dried to obtain the target compound (134 mg, yield 87.6%).

[0253] Molecular formula: C 26 H 27 F3N8OS·2HCl Molecular weight: 556.6 (free base) LC-MS (m / z): 557.2 (M+H) + )

[0254] 1 H-NMR (400MHz, CH3OD) δ: 10.14 (s, 1H), 9.66 (d, J = 5.6Hz, 1H), 8.81 (s, 1H), 8.71-8.73 (m, 1H), 8.35-8.50 (m, 1H), 7.68-7.82 (m, 1H) ),3.52-3.88(m,3H),3.03-3.14(m,3H),2.71(s,3H),2.41-2.58(m,1H),1.90-2.15(m,4H),1.43-1.26(m,1H),1.22-1.38(m,4H).

[0255] Example 2: Preparation of (S)-N-(1-cyclopropyl-4-(3-((methylamino)methyl)piperidin-1-yl)-3-(trifluoromethyl)-1H-indol-5-yl)-2-(pyridazin-4-yl)thiazolyl-4-carboxamide (compound 4)

[0256]

[0257] Preparation of 1,4-fluoroindoline

[0258] Sodium borohydride (5.6 g, 148.2 mmol) was added to a solution of 10.0 g (74.1 mmol) of 4-fluoro-1H-indole in 50 mL of acetic acid. The reaction was carried out at 25 °C for 1 hour. The reaction was confirmed by LCMS. The reaction mixture was evaporated to dryness, the pH was adjusted to >7 with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic phase was collected, evaporated to dryness, and 11.0 g of crude product was obtained, which was used directly in the next reaction.

[0259] 2.1 Preparation of (4-fluoroindoline-1-yl)acet-1-one

[0260] 4-Fluoroindoline (11.0 g, crude product) was dissolved in acetic anhydride (15 mL) and reacted at 25 °C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was evaporated to dryness, and column chromatography (SiO2, petroleum ether:ethyl acetate = 3:1) gave 10.0 g of product, with a two-step yield of 75.3%.

[0261] 3.1 Preparation of (4-fluoro-5-nitroindoline-1-yl)acet-1-one

[0262] At 0 °C, potassium nitrate (6.8 g, 2.0 mmol) was added to a sulfuric acid (20 mL) solution of 1-(4-fluoroindolin-1-yl)ethyl-1-one (6.0 g, 33.5 mmol). The reaction was continued for 30 minutes. LC-MS showed that the reaction was complete. The reaction solution was slowly poured into water, and a solid precipitated. The solution was filtered, and the filter cake was collected. Column chromatography (SiO2, petroleum ether: ethyl acetate = 1:1) yielded 4.7 g of product, with a yield of 62.7%.

[0263] Preparation of 4,4-fluoro-5-nitroindoline

[0264] Lithium hydroxide (2.6 g, 63.0 mmol) was added to a methanol (20 mL) solution of 4.7 g (21.0 mmol) of 1-(4-fluoro-5-nitroindoline-1-yl)ethyl-1-one. The reaction was carried out at 25 °C for 1 hour. LC-MS showed that the reaction was complete. The reaction solution was evaporated to dryness and column chromatography (SiO2, petroleum ether:ethyl acetate = 1:1) was performed to give 3.6 g of product, yield 94.2%.

[0265] Preparation of 5,4-fluoro-5-nitro-1H-indole

[0266] Manganese dioxide (6.9 g, 79.2 mmol) was added to a solution of 3.6 g (19.8 mmol) in dichloromethane (50 mL), and the reaction was carried out at 25 °C for 6 hours. LC-MS showed that the reaction was complete. The mixture was filtered, the filtrate was collected, and the product was evaporated to dryness to give 3.0 g of product, with a yield of 84.3%.

[0267] Preparation of 6,1-cyclopropyl-4-fluoro-5-nitro-1H-indole

[0268] Copper acetate (3.0 g, 16.7 mmol) and bispyridine (2.6 g, 16.7 mmol) were added to dichloroethane (30 mL). The reaction was carried out at 75 °C for 10 min, followed by the addition of 4-fluoro-5-nitro-1H-indole (3.0 g, 16.7 mmol), cyclopropylboronic acid (2.9 g, 33.4 mmol), and sodium carbonate (1.8 g, 33.4 mmol). The reaction was continued for 3 h. LC-MS showed that the reaction was complete. The reaction mixture was evaporated to dryness, and column chromatography (SiO2, petroleum ether:ethyl acetate = 6:1) yielded 2.4 g of product, with a yield of 65.3%.

[0269] 7.1 Preparation of cyclopropyl-4-fluoro-5-nitro-3-(trifluoromethyl)-1H-indole

[0270] Under a nitrogen atmosphere, iodobenzene acetate (2.9 g, 9.0 mmol) was added to a solution of 1-cyclopropyl-4-fluoro-5-nitro-1H-indole (1.0 g, 4.5 mmol) in acetonitrile (5 mL). The reaction was carried out at 25 °C for 10 min, followed by the addition of (trifluoromethyl)trimethylsilane (2.6 g, 18.0 mmol). The reaction was continued for 4 h. LC-MS showed no starting material remaining. The reaction mixture was evaporated to dryness and column chromatography (SiO2, petroleum ether:ethyl acetate = 6:1) yielded 190 mg of product, with a yield of 14.7%.

[0271] 8. Preparation of (R)-((1-(1-cyclopropyl-5-nitro-3-(trifluoromethyl)-1H-indol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0272] To a solution of 1-cyclopropyl-4-fluoro-5-nitro-3-(trifluoromethyl)-1H-indole (190 mg, 0.66 mmol) in 1,4-dioxane (10 mL), tert-butyl (R)-methyl(piperidin-3-ylmethyl)carbamate (151 mg, 0.66 mmol) and DIEA (258 mg, 2.0 mmol) were added. The reaction was carried out at 100 °C for 12 h. LC-MS showed product formation. The reaction mixture was evaporated to dryness and column chromatography (SiO2, petroleum ether:ethyl acetate = 1:1) yielded 90 mg of product, with a yield of 27.2%.

[0273] 9. Preparation of (R)-((1-(5-amino-1-cyclopropyl-3-(trifluoromethyl)-1H-indol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0274] To a methanol (5 mL) solution of (R)-((1-(1-cyclopropyl-5-nitro-3-(trifluoromethyl)-1H-indol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate (90 mg, 0.18 mmol), palladium on carbon (90 mg) was added. The reaction was carried out at 25 °C for 2 h under a hydrogen atmosphere. LC-MS showed that the reaction was complete. The reaction mixture was filtered, the filtrate was collected, and 50 mg of crude product was obtained by rotary evaporation and used directly in the next reaction step.

[0275] 10. Preparation of (R)-((1-(1-cyclopropyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carbamate)-3-(trifluoromethyl)-1H-indol-4-yl]piperidin-3-yl)methyl)(methyl)carbamate tert-butyl

[0276] To a DMF (10 mL) solution of (R)-((1-(5-amino-1-cyclopropyl-3-(trifluoromethyl)-1H-indol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (50 mg, crude), 2-(pyridazin-4-yl)thiazolyl-4-carboxylic acid (23 mg, 0.11 mmol), HATU (84 mg, 0.22 mmol), and DIEA (43 mg, 0.33 mmol) were added. The reaction was carried out at 25 °C for 2 h. LC-MS showed product formation. The reaction mixture was evaporated to dryness, and column chromatography (SiO2, petroleum ether:ethyl acetate = 1:1) yielded 40 mg of product, with a two-step yield of 33.9%.

[0277] 11. Preparation of (S)-N-(1-cyclopropyl-4-(3-((methylamino)methyl)piperidin-1-yl)-3-(trifluoromethyl)-1H-indol-5-yl)-2-(pyridazin-4-yl)thiazolyl-4-carboxamide

[0278] A solution of ethyl acetate (4 M, 2 mL) of (R)-((1-(1-cyclopropyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamido)-3-(trifluoromethyl)-1H-indol-4-yl]piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (40 mg, 0.061 mmol) was added to ethyl acetate (2 mL). The reaction was carried out at 25 °C for 2 h. LC-MS showed product formation. The pH was adjusted to 8 with saturated sodium bicarbonate solution, and the product was extracted with ethyl acetate. The organic phase was collected, evaporated to dryness, and separated using a preparative plate (dichloromethane:methanol:ammonia = 10:1:0.1) to give 30 mg of product, yield 88.6%.

[0279] Molecular formula: C 27 H 28 F3N7OS molecular weight: 555.6 LC-MS (m / z): 556.2 (M+H) + )

[0280] 1 H-NMR (400MHz, CDCl3) δ: 10.96 (br, 1H), 9.87 (s, 1H), 9.38 (d, J = 4.7Hz, 1H), 8.71 (d, J = 9.1Hz, 1H), 8.44 (s, 1H), 7.97 (d, J = 4.7Hz, 1H), 7.55 (d, J = 9.1 Hz,1H),7.09(s,1H),3.39-3.19(m,3H),3.19-2.91(m,2H),2.70-2.45(m, 2H),2.34(s,3H),2.29-2.15(m,2H),1.99-1.48(m,4H),1.44-1.10(m,4H).

[0281] Example 3: Preparation of (S)-N-(1-cyclopropyl-4-(3-(((methylamino)methyl)piperidin-1-yl)-1H-indazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide (compound 8)

[0282]

[0283] 1. Preparation of 4-chloro-5-nitro-1H-indazole

[0284] 3-Chloro-2-methyl-4-nitroaniline (4.0 g, 21.4 mmol) was dissolved in acetic acid (50 mL), and 5 mL of an aqueous solution of sodium nitrite (2.2 g, 32.2 mmol) was slowly added. The reaction was carried out at 25 °C for 4 hours. The solvent was evaporated, and the pH was adjusted to 8 with water and sodium bicarbonate solution. The mixture was filtered, and the solid was collected and slurried with petroleum ether to give 3.5 g of the product, with a yield of 83.0%.

[0285] 2. Preparation of 4-chloro-1-cyclopropyl-5-nitro-1H-indazole

[0286] Acetone (1.8 g, 10.1 mmol) and bipyridine (1.6 g, 10.1 mmol) were dissolved in 1,2-dichloroethane (30 mL). Cyclopropylboronic acid (1.7 g, 20.2 mmol), 4-chloro-5-nitro-1H-indazole (2.0 g, 10.1 mmol), and sodium carbonate (2.1 g, 20.2 mmol) were added and reacted at 70 °C for 4 h. After the reaction was completed, the mixture was concentrated and purified by silica gel column chromatography (n-heptane:ethyl acetate = 10:1) to give the target compound (1.5 g, yield 62.7%).

[0287] 3. Preparation of (R)-((1-(1-cyclopropyl-5-nitro-1H-indazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0288] 4-Chloro-1-cyclopropyl-5-nitro-1H-indazole (800 mg, 3.4 mmol) was dissolved in DMF (10 mL), and tert-butyl(R)-methyl(piperidin-3-ylmethyl)carbamate (776 mg, 3.4 mmol) and potassium carbonate (939 mg, 6.8 mmol) were added. The mixture was microwaved at 100 °C for 40 minutes. After the reaction was complete, water and ethyl acetate were added for extraction. The organic phase was collected, concentrated, and purified by silica gel column chromatography (n-heptane:ethyl acetate = 5:1) to give the target compound (1.3 g, yield 89.1%).

[0289] 4. Preparation of (R)-((1-(5-amino-1-cyclopropyl-1H-indazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0290] (R)-((1-(1-cyclopropyl-5-nitro-1H-indazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (300 mg, 0.70 mmol) was dissolved in methanol (10 mL), Pd / C (120 mg) was added, the mixture was purged with hydrogen three times, and the reaction was carried out at 25 °C for 2 hours under a hydrogen atmosphere. After the reaction was completed, the solid was removed by filtration to obtain the target compound (200 mg, yield 71.5%).

[0291] 5. Preparation of (R)-((1-(1-cyclopropyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamide)-1H-indazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0292] 2-(pyridazin-4-yl)thiazol-4-carboxylic acid (41 mg, 0.20 mmol), HATU (114 mg, 0.30 mmol), and DIEA (78 mg, 0.60 mmol) were dissolved in N,N-dimethylformamide (5 mL), and (R)-((1-(5-amino-1-cyclopropyl-1H-indazole-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (80 mg, 0.20 mmol) was added. The reaction was carried out at 30 °C for 1 h. After the reaction was completed, water (50 mL) and ethyl acetate (50 mL x 2) were added for extraction. The organic phase was washed with water (50 mL x 3), concentrated, and the crude product was purified by silica gel column chromatography (n-heptane:ethyl acetate = 1:10) to give the target compound (80 mg, yield 68.0%).

[0293] 6. Preparation of (S)-N-(1-cyclopropyl-4-(3-(((methylamino)methyl)piperidin-1-yl)-1H-indazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide

[0294] 70 mg (0.12 mmol) of tert-butyl(R)-((1-(1-cyclopropyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamide)-1H-indazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate was dissolved in 3 mL of dichloromethane, and 2 mL of 4 M ethyl acetate solution of hydrogen chloride was added. The reaction was carried out at 25 °C for 2 h. After the reaction was completed, the mixture was filtered and dried to obtain 50 mg of crude product. The crude product was diluted with 10 mL of water, and the pH was adjusted to 8-9 with saturated sodium bicarbonate solution. The product was extracted with 20 mL of ethyl acetate, dried, and concentrated to obtain the target compound (9.0 mg, yield 15.4%).

[0295] Molecular formula: C 25 H 28 N8OS molecular weight: 488.6 LC-MS (M / e): 489.6 (M+H) + )

[0296] 1H-NMR (400MHz, DMSO) δ: 10.53 (s, 1H), 9.93 (d, J = 8.0Hz, 1H), 9.44 (d, J = 7.0Hz, 1H), 8.72-8 .70(m,1H),8.26-8.25(m,2H),8.05(s,1H),7.35-7.33(m,1H),6.57-6.55(m,1H),3.95-3. 94(m,1H),3.72-3.70(m,1H),3.42-3.34(m,2H),3.26-3.16(m,2H),3.06-2.99(m,2H),2.6 0-2.49(m,2H),2.40-2.29(m,4H),2.24-2.11(m,2H),1.11-1.10(m,2H),0.90-0.87(m,2H).

[0297] Example 4: Preparation of (S)-N-(1-cyclopentyl-4-(3-((methylamino)methyl)piperidin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide (compound 9)

[0298]

[0299]

[0300] 1. Preparation of N-cyclopentyl-3-fluoro-2-nitroaniline

[0301] 1,3-Difluoro-2-nitrobenzene (3.2 g, 20.0 mmol) was dissolved in THF (30 mL), and cyclopentylamine (3.4 g, 40.0 mmol) and triethylamine (4.0 g, 40.0 mmol) were added. After reacting at 25 °C for 4 hours, ethyl acetate and water were added to the system, and the liquid-liquid mixture was separated to obtain the organic phase. After drying with anhydrous sodium sulfate, the organic phase was concentrated and subjected to silica gel column chromatography (petroleum ether and ethyl acetate were used as the mobile phase, with the proportion of ethyl acetate ranging from 0% to 70%) to give 2.1 g of the target compound, with a yield of 46.6%.

[0302] 2.N 1 Preparation of cyclopentyl-3-fluorophenyl-1,2-diamine

[0303] N-cyclopentyl-3-fluoro-2-nitroaniline (1.8 g, 8.0 mmol) was dissolved in methanol (20 mL), and palladium on carbon (0.9 g, 10%) was added. After reacting at 25 °C for 2 hours under a hydrogen atmosphere, the system was filtered, and the solvent was removed from the filtrate under reduced pressure to obtain 1.7 g of the crude target product, which was directly added to the next reaction step.

[0304] 3.1 Preparation of cyclopentyl-4-fluoro-2-(trifluoromethyl)-1H-benzo[d]imidazole

[0305] N 1 Cyclopentyl-3-fluorobenzene-1,2-nitroaniline diamine (1.5 g crude) was dissolved in ethyl acetate (16 mL), and trifluoroacetic anhydride (4 mL) was added. After reacting at 80 °C for 4 hours, the mixture was concentrated, stirred with silica gel, and subjected to silica gel column chromatography (petroleum ether and ethyl acetate were used as the mobile phases, with the proportion of ethyl acetate ranging from 0% to 100%) to give 510 mg of the target compound, with a two-step yield of 26.4%.

[0306] Preparation of 4,1-cyclopentyl-4-fluoro-5-nitro-2-(trifluoromethyl)-1H-benzo[d]imidazole

[0307] 1-Cyclopentyl-4-fluoro-2-(trifluoromethyl)-1H-benzo[d]imidazole (230 mg, 0.84 mmol) was dissolved in concentrated sulfuric acid (5 mL), cooled to 0 °C, and fuming nitric acid (80 mg, 1.3 mmol) was added dropwise to the system. After reacting at this temperature for 1 hour, the reaction mixture was poured into ice water, extracted with ethyl acetate, separated to obtain the organic phase, concentrated, mixed with silica gel, and subjected to silica gel column chromatography (petroleum ether and ethyl acetate as the mobile phase, with the proportion of ethyl acetate ranging from 0% to 70%) to obtain 210 mg of the target compound, with a yield of 78.4%.

[0308] 5. Preparation of (R)-((1-(1-cyclopentyl-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0309] 1-Cyclopentyl-4-fluoro-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazole (178 mg, 0.56 mmol) was dissolved in 1,4-dioxane (10 mL). (R)-methyl(piperidin-3-ylmethyl)carbamate tert-butyl ester (128 mg, 0.56 mmol) and N,N-diisopropylethylamine (142 mg, 1.1 mmol) were added to the system. After reacting at 100 °C for 2 hours, the reaction mixture was concentrated, stirred with silica gel, and subjected to silica gel column chromatography (petroleum ether and ethyl acetate as mobile phases, with ethyl acetate ratios ranging from 0% to 70%) to obtain 170 mg of the target compound, with a yield of 57.6%.

[0310] 6. Preparation of (R)-((1-(5-amino-1-cyclopentyl-2-(trifluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0311] (R)-((1-(1-cyclopentyl-5-nitro-2-(trifluoromethyl)-1H-benzis[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (144 mg, 0.27 mmol) was dissolved in methanol (10 mL), and palladium on carbon (70 mg, 10%) was added. The reaction was carried out at 25 °C for 2 hours under a hydrogen atmosphere. The system was then filtered, and the solvent was removed from the filtrate under reduced pressure to obtain 120 mg of the target product as crude product. This crude product was directly added to the next reaction step.

[0312] 7. Preparation of (R)-((1-(1-cyclopentyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carbamate)-2-(trifluoromethyl)-1H-benzo[d]imidazol-4)-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl

[0313] Dissolve tert-butyl ((R)-((1-(5-amino-1-cyclopentyl-2-(trifluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate (120 mg crude) in N,N-dimethylformamide (15 mL), then add 2-(pyridazin-4-yl)thiazol-4-carboxylic acid (51 mg, 0.25 mmol) and 2-(7-azobenzotriazole)-N,N,N ',N'-Tetramethylurea hexafluorophosphate (142 mg, 0.37 mmol) and N,N-diisopropylethylamine (47 mg, 0.36 mmol) were reacted at 25 °C for 2 hours. Then, dichloromethane and water were added to the reaction solution and stirred. The organic phase was separated and stirred with silica gel. The solution was then subjected to silica gel column chromatography (petroleum ether and ethyl acetate were used as the mobile phases, with the proportion of ethyl acetate ranging from 0% to 70%) to obtain 110 mg of the target compound. The two-step yield was 58.6%.

[0314] 8. Preparation of (S)-N-(1-cyclopentyl-4-(3-((methylamino)methyl)piperidin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide

[0315] (R)-((1-(1-cyclopentyl-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamido)-2-(trifluoromethyl)-1H-benzi[d]imidazol-4)-yl))piperidin-3-yl))methyl))((methyl))tert-butyl carbamate (110 mg, 0.22 mmol) was dissolved in ethyl acetate solution of hydrogen chloride (9 mL, 4 M). After reacting at 25 °C for 1 hour, the pH of the reaction solution was adjusted to 7 with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic phases were combined and stirred with silica gel. The mixture was then subjected to silica gel column chromatography (petroleum ether and ethyl acetate were used as the mobile phases, with the proportion of ethyl acetate ranging from 0% to 100%) to obtain 31 mg of the target compound, with a yield of 33.0%.

[0316] Molecular formula: C 28 H 31 F3N8OS molecular weight: 584.7 LC-MS (M / e): 584.2 (M+H) + )

[0317] 1 H-NMR (400MHz, CDCl3) δ: 10.70 (s, 1H), 9.80 (s, 1H), 9.45 (d, J = 5.3Hz, 1H), 8.64 ( d,J=9.0Hz,1H),8.05-7.95(m,1H),7.46(d,J=9.0Hz,1H),5.10-4.90(m,1H),3.7 0-3.55(m,1H),3.40-3.25(m,2H),3.10-2.85(m,3H),2.65(s,3H),2.60-2.50(m, 1H),2.40-2.15(m,5H),2.10-2.00(m,2H),2.00-1.80(m,4H),1.50-1.20(m,2H).

[0318] Example 5: Preparation of (S)-N-(1-cyclopropyl-2-(difluoromethyl)-4-(3-((((methylamino)methyl)piperidin-1-yl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide (compound 11)

[0319]

[0320] 1. Preparation of N-(2-chloro-6-fluorophenyl)-2,2-difluoroacetamide

[0321] 2-Chloro-6-fluoroaniline (3.0 g, 20.6 mmol) was dissolved in ethyl acetate (25 mL), and difluoroacetic anhydride (4.5 mL) was added. After reacting at 80 °C for 1 hour, the solution was concentrated to give 3.1 g of the target compound, with a yield of 67.3%.

[0322] 2. Preparation of N-(2-chloro-6-fluoro-3-nitrophenyl)-2,2-difluoroacetamide

[0323] N-(2-chloro-6-fluorophenyl)-2,2-difluoroacetamide (3.0 g, 13.4 mmol) was dissolved in concentrated sulfuric acid (25 mL). The reaction system was cooled to 0 °C, and fuming nitric acid (1.3 g) was added dropwise. After reacting at 0 °C for 1 hour, the reaction solution was poured into ice water, and a white solid precipitated. The solid was filtered, and the filter cake was collected to obtain 2.1 g of the target compound, with a yield of 58.3%.

[0324] 3. Preparation of N-(2-chloro-6-(cyclopropylamino)-3-nitrophenyl)-2,2-difluoroacetamide

[0325] N-(2-chloro-6-fluoro-3-nitrophenyl)-2,2-difluoroacetamide (2.1 g, 7.8 mmol) was dissolved in tetrahydrofuran (25 mL), and cyclopropylamine (672 mg, 11.6 mmol) was added. After reacting at 25 °C for 4 hours, the solution was concentrated to obtain 1.8 g of crude target compound.

[0326] Preparation of 4,4-chloro-1-cyclopropyl-2-(difluoromethyl)-5-nitro-1H-benzo[d]imidazole

[0327] N-(2-chloro-6-(cyclopropylamino)-3-nitrophenyl)-2,2-difluoroacetamide (1.7 g crude) was dissolved in tetrahydrofuran (40 mL), and triethylamine (1.1 g, 10.9 mmol) was added. After reacting at 50 °C for 2 hours, ethyl acetate and water were added, and the mixture was separated to obtain the organic phase. The organic phase was stirred with silica gel and subjected to silica gel column chromatography (the mobile phase was ethyl acetate and n-heptane, with the ethyl acetate ratio ranging from 0% to 70%) to obtain 510 mg of the target compound. The two-step yield was 24.0%.

[0328] 5. Preparation of (R)-((1-(1-cyclopropyl-2-(difluoromethyl)-5-nitro-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)tert-butyl carbamate

[0329] 4-Chloro-1-cyclopropyl-2-(difluoromethyl)-5-nitro-1H-benzi[d]imidazole (510 mg, 1.8 mmol) was dissolved in 1,4-dioxane (20 mL), and (R)-methyl(piperidin-3-ylmethyl)carbamate tert-butyl ester (484 mg, 2.1 mmol) and N,N-diisopropylethylamine (458 mg, 3.5 mmol) were added. After reacting at 100 °C for 2 hours, the reaction solution was stirred with silica gel and subjected to silica gel column chromatography (mobile phase: ethyl acetate and n-heptane, ethyl acetate ratio from 0% to 70%) to obtain 530 mg of the target compound, with a yield of 62.4%.

[0330] 6. Preparation of (R)-((1-(5-amino-1-cyclopropyl-2-(difluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0331] (R)-((1-(1-cyclopropyl-2-(difluoromethyl)-5-nitro-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)tert-butyl carbamate (530 mg, 1.1 mmol) was dissolved in methanol (25 mL), and palladium on carbon (110 mg, 10%) was added. The mixture was reacted at 25 °C for 0.5 hours under a hydrogen atmosphere. After filtration, the filtrate was concentrated to obtain 460 mg of the crude target product, which was directly added to the next reaction step.

[0332] 7. Preparation of (R)-((1-(1-cyclopropyl-2-(difluoromethyl)-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamide)-1H-benzimidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl

[0333] (R)-((1-(5-amino-1-cyclopropyl-2-(difluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (270 mg crude) was dissolved in N,N-dimethylformamide (15 mL), and 2-(pyridazin-4-yl)thiazol-4-carboxylic acid (125 mg, 0.60 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (304 mg, 0.80 mmol) and N,N-diisopropylethylamine (155 mg, 1.2 mmol) were added. After reacting at 25 °C for 1 hour, the reaction solution was stirred with silica gel and subjected to silica gel column chromatography (mobile phase: dichloromethane and methanol, methanol ratio from 0% to 10%) to obtain 150 mg of the target compound. The two-step yield was 33.9%.

[0334] 8. Preparation of (S)-N-(1-cyclopropyl-2-(difluoromethyl)-4-(3-((((methylamino)methyl)piperidin-1-yl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazole-4-carboxamide

[0335] (R)-((1-(1-cyclopropyl-2-(difluoromethyl)-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamide)-1H-benzimidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (142 mg, 0.22 mmol) was dissolved in ethyl acetate solution of hydrogen chloride (12 mL, 4 M). After reacting at 25 °C for 2 hours, the pH of the reaction solution was adjusted to 8 with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate. The organic phases were combined and prepared under reverse-phase medium pressure (the mobile phase was water and methanol, with the methanol ratio ranging from 0% to 100%) to obtain 40 mg of the target compound, with a yield of 33.4%.

[0336] Molecular formula: C 26 H 28F2N8OS molecular weight: 538.6 LC-MS (M / e): 539.2 (M+H) + )

[0337] 1 H-NMR(400MHz, CDCl3)δ:11.01(s,1H),9.89(s,1H),9.45-9.40(m,1H),8.7 6(d,J=8.8Hz,1H),8.05-8.00(m,1H),7.49(d,J=8.8Hz,1H),6.98(t,J=54Hz ,1H),3.70-3.50(m,1H),3.50-3.35(m,2H),3.30-3.20(m,1H),3.20-3.05(m ,1H),2.70-2.50(m,2H),2.34(s,3H),2.25-1.99(m,5H),1.30-1.00(m,4H).

[0338] Example 6: Preparation of (S)-N-(1-(2-fluorophenyl)-4-(3-(((methylamino)methyl)piperidin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazolyl-4-carboxamide (compound 12)

[0339]

[0340] Preparation of 1,4-chloro-1-(2-fluorophenyl)-5-nitro-2-(trifluoromethyl)-1H-benzo[d]imidazole

[0341] N-(2-chloro-6-fluoro-3-nitrophenyl)-2,2,2-trifluoroacetamide (255 mg, 1.0 mmol) was dissolved in DMF (20 mL), and 2-fluoroaniline (111 mg, 1.0 mmol) and potassium tert-butoxide (224 mg, 2.0 mmol) were added. After the addition was complete, the reaction was carried out at 80 °C for 4 hours. The reaction was stopped by LCMS. The mixture was extracted with ethyl acetate, the organic phases were combined, concentrated, and column chromatography (PE / EA = 0%-70%) was performed to give 210 mg, with a yield of 65.6%.

[0342] 2. Preparation of (R)-((1-(1-(2-fluorophenyl)-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0343] 4-Chloro-1-(2-fluorophenyl)-5-nitro-2-(trifluoromethyl)-1H-benzi[d]imidazole (170 mg, 0.47 mmol) was dissolved in 1,4-dioxane (5 mL), followed by the addition of (R)-methyl(piperidin-3-ylmethyl)carbamate tert-butyl ester (107 mg, 0.47 mmol) and DIEA (122 mg, 0.94 mmol). After addition, the reaction was carried out at 100 °C for 2 h, and the reaction was monitored by LCMS until completion. The solution was purified by concentrated silica gel column chromatography (PE / EA = 0-50%) to give 210 mg of the title compound, yield: 80.4%.

[0344] 3. Preparation of (R)-((1-(5-amino-1-(2-fluorophenyl)-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester

[0345] (R)-((1-(1-(2-fluorophenyl)-5-nitro-2-(trifluoromethyl)-1H-benzo[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (180 mg, 0.33 mmol) was dissolved in methanol (10 mL), and palladium on carbon (100 mg) was added. The reaction was carried out at 25 °C for 3 h under hydrogen atmosphere. The mixture was filtered through diatomaceous earth, and the filtrate was concentrated to give 140 mg, with a yield of 82.2%.

[0346] 4. Preparation of (R)-((1-(1-(2-fluorophenyl)-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamido)-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl

[0347] (R)-((1-(5-amino-1-(2-fluorophenyl)-2-(trifluoromethyl)-1H-benzimidazol-4-yl)piperidin-3-yl)methyl)(methyl)carbamate tert-butyl ester (100 mg, 0.19 mmol) was dissolved in DMF (5 mL), and 2-(pyridazin-4-yl)thiazol-4-carboxylic acid (40 mg, 0.19 mmol), HATU (87 mg, 0.23 mmol), and DIEA (37 mg, 0.29 mmol) were added. The reaction was carried out at 25 °C for 0.5 h. The mixture was extracted with EA, and the organic phase was dried, concentrated, and purified by silica gel column chromatography (PE:EA = 0-70%) to give 110 mg, with a yield of 80.7%.

[0348] 5. Preparation of (S)-N-(1-(2-fluorophenyl)-4-(3-((((methylamino)methyl)piperidin-1-yl)-2-(trifluoromethyl)-1H-benzo[d]imidazol-5-yl)-2-(pyridazin-4-yl)thiazolyl-4-carboxamide

[0349] (R)-((1-(1-(2-fluorophenyl)-5-(2-(pyridazin-4-yl)thiazolyl-4-carboxamido)-2-(trifluoromethyl)-1H-benzi[d]imidazol-4-yl)piperidin-3-yl)methyl)(methyl)tert-butyl carbamate (110 mg, 0.16 mmol) was dissolved in ethyl acetate hydrochloride (5 mL) and reacted at 25 °C for 2 h. The solution was concentrated, diluted with ethyl acetate, washed with saturated sodium bicarbonate, extracted with ethyl acetate, and the organic phases were combined, dried, and concentrated. Purification by silica gel column chromatography (PE:EA = 0-100%) yielded 21 mg of the target compound, in a yield of 22.2%.

[0350] Molecular formula: C 29 H 26 F4N8OS Molecular weight: 610.6 LC-MS (M / e): 611.21 (M+H) + )

[0351] 1 H-NMR (400MHz, CDCl3) δ: 11.02 (s, 1H), 9.90 (s, 1H), 9.42 (d, J = 5.4Hz, 1H), 8.77 (d, J = 9.0Hz, 1H), 8.45(s,1H),8.06-8.00(m,1H),7.70-7.60(m,1H),7.55-7.45(m,1H),7.45-7.30(m,2H),6.94(d,J =9.0Hz,1H),3.75-3.60(m,1H),3.55-3.40(m,1H),3.40-3.30(m,1H),3.25-3.10(m,1H),2.75-2.6 5(m,2H),2.39(s,3H),2.30-2.20(m,1H),2.20-2.10(m,1H),2.10-2.00(m,2H),1.55-1.41(m,1H).

[0352] The compounds shown in the following table were prepared using the same or similar methods as those used in the preparation examples above:

[0353]

[0354] Experimental protocol

[0355] The following provides exemplary experimental schemes for some compounds of the present invention to demonstrate the beneficial activity and technical effects of the compounds. However, it should be understood that the following experimental schemes are merely examples of the content of the present invention and not limitations on the scope of the present invention.

[0356] Experimental Example 1: In vitro enzymatic activity of the compounds of the present invention

[0357] Test sample: The compound of the present invention, the structural formula and preparation method of which are given in the preparation examples.

[0358] Experimental reagents:

[0359]

[0360]

[0361] Experimental methods:

[0362] 1. Compound dilution

[0363] 1) The compound of the present invention was prepared to 10 mM using DMSO as a test stock solution.

[0364] 2) The test stock solution is now diluted 10 times to 1 mM, and then the 1 mM compound solution is serially diluted 5 times to 10 concentrations, with the highest concentration being 1 mM.

[0365] 3) Using an Echo550, transfer the diluted compounds of the present invention to 384-well plates, dilute 1000 times, set 2 replicates for each concentration, and set the final concentration of DMSO to 0.1%.

[0366] 4) The final concentrations of the tested compounds were 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, and 0.000512 nM.

[0367] 2. Enzyme reaction experiment

[0368] 1) Prepare a 1× enzyme buffer by adding 4 volumes of distilled water to 1 volume of 5× enzyme buffer and adding 0.1M dithiothreitol.

[0369] 2) Use an Echo 550 to transfer the compound dilution to each well of the assay plate;

[0370] 3) Seal the test plate and centrifuge for 1 minute.

[0371] 4) Prepare 2× enzyme using 1× enzyme buffer.

[0372] 5) Add 2.5 μL of 2× enzyme to the 384-well detection plate.

[0373] 6) Centrifuge the test plate for 30 seconds and incubate at room temperature for 10 minutes.

[0374] 7) Prepare a mixture of 2× substrate and ATP using 1× enzyme buffer.

[0375] 8) Add 2.5 μL of 2× substrate and ATP mixture to a 384-well plate to start the reaction.

[0376] 9) Centrifuge for 30 seconds. Seal the test plate and incubate at room temperature for 2 hours.

[0377] 10) Add 4 μL of ADP-Glo ​​reagent and incubate at room temperature for 40 minutes.

[0378] 11) Add 8 μL of enzyme detection reagent and incubate at room temperature for 40 minutes.

[0379] 3. Result Detection

[0380] 1) Read the chemiluminescence signal on the Envision 2104 plate reader.

[0381] 2) Collect data.

[0382] 4. Data Analysis

[0383] The inhibition rate (%) is calculated using the following formula:

[0384] inhibition (%) = 100 - (Signal cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC )

[0385] Among them, Signal Ave_VC Indicates the intensity of the luminescence signal in the positive control well without the addition of the compound;

[0386] Signal Ave_PC Indicates the luminescence signal intensity of the negative control well without substrate;

[0387] Signal cmpd Indicates: Indicates the intensity of the luminescence signal of the tested compound;

[0388] IC calculation using Prism GraphPad 50 ,

[0389] Experimental results:

[0390] Table 1. Inhibitory activity of the compounds of this invention against DGKζ

[0391] Compound 1 dihydrochloride 20.0

[0392] The experimental results above show that the compound of the present invention can effectively inhibit the activity of DGKζ and is an effective DGKζ inhibitor.

[0393] Experimental Example 2: In vitro enzymatic activity of the compounds of the present invention

[0394] Experimental methods:

[0395] 1. Compound dilution

[0396] 1) The compound of the present invention was prepared to 10 mM using DMSO as a test stock solution.

[0397] 2) The test stock solution is now diluted 10 times to 1 mM, and then the 1 mM compound solution is serially diluted 5 times to 8 concentrations, with the highest concentration being 1 mM.

[0398] 3) Use a pipette to transfer the diluted compounds of the present invention into 96-well plates, dilute 1000 times, set 2 replicates for each concentration, and the final concentration of DMSO is 0.1%.

[0399] 4) The final concentrations of the tested compounds were 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.0128 nM.

[0400] 2. Enzyme reaction experiment

[0401] 1) Place DGKζ protein, kinase assay buffer, DLG substrate and kinase dilution buffer on ice.

[0402] 2) Add the following components to a pre-cooled 96-well opaque plate to achieve an initial reaction volume of 20 μL.

[0403] 1.5 μL of the diluted compound or DMSO was centrifuged at 1000 g for 30 seconds.

[0404] Component 2.10 μL of diluted Active DGKζ was centrifuged in a 1000g centrifuge for 30 seconds and incubated at room temperature for 10 min.

[0405] Component 3.5 μL 100 μM DLG (substrate); set up a blank control, without adding DLG, and replace DLG with an equal volume of kinase dilution buffer IX.

[0406] 3) Add 5 μL of 125 μM ATP assay solution to the wells to start the reaction, bringing the final volume to 25 μL. Shake the 96-well plate for 2 min to mix, and continue incubating at room temperature for 60 min.

[0407] 4) Terminate the reaction and add 25 μL of ADP-Glo TM Reagent consumes excess ATP, shakes the 96-well plate, and incubates at room temperature for 40 minutes.

[0408] 5) Add 50 μL of kinase detection reagent to the plate and incubate at room temperature for 30 min.

[0409] 3. Result Detection

[0410] 1) Read the chemiluminescence signal on a multi-functional microplate reader.

[0411] 2) Collect data.

[0412] 4. Data Analysis

[0413] The inhibition rate (%inh) is calculated using the following formula:

[0414] inhibition (%) = 100 - (Signal cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC )

[0415] Among them, Signal Ave_VC Indicates the intensity of the luminescence signal in the positive control well without the addition of the compound;

[0416] Signal Ave_PC Indicates the luminescence signal intensity of the negative control well without substrate;

[0417] Signal cmpd Indicates: Indicates the intensity of the luminescence signal of the tested compound;

[0418] IC calculation using Prism GraphPad 50 ,

[0419] Experimental results:

[0420] Table 2. Inhibitory activity of the compounds of this invention against DGKζ

[0421] Compound 4 93.3 Compound 9 42.1 Compound 12 23.5

[0422] The experimental results above show that the compound of the present invention can effectively inhibit the activity of DGKζ and is an effective DGKζ inhibitor.

[0423] Experimental Example 3: In vitro PBMC cell immunomodulatory activity of the compounds of the present invention

[0424] The test substance is a compound of this invention, the chemical name and structure of which are given in the preparation examples.

[0425] Experimental method (IL-2 ELISA assay)

[0426] The cells and reagents used in the following experiments are as follows:

[0427] hPBMC: Human peripheral mononuclear cells;

[0428] Anti-CD3e (OKT3): Human anti-CD3 monoclonal antibody

[0429] Anti-CD28 (CD28.2): Human anti-CD3 monoclonal antibody

[0430] PBS: Phosphate Buffered Salt Solution

[0431] DMSO: N,N-dimethylformamide

[0432] Wash buffer: cleaning solution

[0433] TMB Substrate Solution: TMB colorimetric agent

[0434] 1. Prepare cells

[0435] 1.1 Cell resuscitation:

[0436] 1) Thaw the frozen PBMC cells by gently shaking them in a 37°C water bath, transfer them to a centrifuge tube containing preheated culture medium, and centrifuge at 400g for 10 min.

[0437] 2) Discard the supernatant, resuspend the cells in complete culture medium, transfer the cell suspension to a culture dish and incubate overnight.

[0438] 1.2 T cell activation and cell suspension preparation:

[0439] 1) CD3 antibody coating: Prepare 2.5 μg / mL anti-CD3e (OKT3) solution, 50 μL / well to coat a 96-well plate, incubate at 37℃ for 2 h, and wash twice with 200 μL / well PBS.

[0440] 2) Harvest cells and count them using a platelet counter. Assess cell viability using the trypan blue rejection assay, ensuring viability is above 90%. Adjust to an appropriate concentration and add 80 μL of cell suspension to each well of a 96-well plate, with 1 × 10⁶ cells per well. 5 One hole / hole.

[0441] 3) CD28 antibody stimulation: Prepare 2 μg / mL anti-CD28 antibody and add 10 μL / well to the cells.

[0442] 2. Preparation of test compounds

[0443] 2.1 Prepare a DMSO stock solution for the test compound. The concentration of the stock solution for the test compound is 3 mM.

[0444] 2.2 Preparation of working stock solution for test compounds

[0445] The test compound stock solution was serially diluted 5-fold with DMSO at 3 mM to obtain 7 concentrations. Then, 2 μL of each serially diluted compound with DMSO was added to 198 μL of culture medium to prepare the working stock solution of the test compound (the compound concentration was 10 times the final concentration, with the highest concentration being 30 μM).

[0446] 2.3 Compound Treatment

[0447] Add 10 μL of the compound working stock solution or solvent control (1% DMSO) to a 96-well plate (10-fold dilution, final DMSO concentration 0.1%).

[0448] The final concentrations of the tested compounds were: 3000 nM, 600 nM, 120 nM, 24 nM, 4.8 nM, 0.96 nM, and 0.192 nM.

[0449] 2.4 Setting of reference holes

[0450] Solvent control: 0.1% DMSO.

[0451] Blank control: Uncoated with CD3 antibody, without CD28 antibody or compound.

[0452] Baseline control: coated with CD3 antibody, plus CD28 antibody

[0453] 2.5 After culturing the 96-well plate in a 37°C, 5% CO2 cell culture incubator for 24 hours, the supernatant was collected for analysis.

[0454] 3. IL-2 detection

[0455] 1) Place the microplate containing the standard in positions A1 / A2-H1 / H2.

[0456] 2) Add the appropriate volume of distilled water to all standard wells and blank wells according to the instructions on the label on the standard strip.

[0457] 3) Add 100 μL of distilled water to the sample well.

[0458] 4) Add 50 μL of sample to the designated well, seal the membrane, and incubate on a microplate shaker at room temperature for 3 h.

[0459] 5) Remove the liquid from the well and wash 6 times with 400μL 1×Wash buffer.

[0460] 6) Add 100 μL of TMB Substrate Solution to each well and incubate at room temperature for about 10 min.

[0461] 7) Add 100 μL of stop solution to each well, gently tap to mix, and immediately read the plate at 450 nm using an ELISA reader.

[0462] Note: The sample has been diluted 1:2, so the concentration read from the standard curve is multiplied by the dilution factor (×2).

[0463] 4. Data Processing

[0464] 1) Increase factor = Test object hole reading / Base value reference hole reading.

[0465] Experimental Results and Conclusions

[0466] Table 3. In vitro cellular activities of the compounds of the present invention

[0467]

[0468] The results above show that the compound of the present invention can effectively increase the secretion of IL-2 in human PBMC cells, and the compound of the present invention has the potential for clinical application in the treatment of cancer through immunomodulation.

Claims

1. A compound represented by general formula (I), its pharmaceutically acceptable salt, or its stereoisomer, （I） in, Ring A is selected from the following groups: , or ; Ring B is ; Cy 1 Selected from phenyl, pyridinyl, pyrimidinyl, pyrazinyl, or pyridazinyl groups, each Q independently selected from H, halogen, hydroxyl, amino, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy; Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, hydroxyl C 1-4 Alkoxy, amino C 1-4 Alkoxy, C 1-4 Alkoxy-C 1-4 Alkyl group, -(CH2) p -N(R a (R) b Alternatively, the following groups may be substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, pyrimidinyl; wherein each substituent is independently selected from halogen, cyano, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl or halogenated C 1-4 Alkoxy; R a R b Selected independently from hydrogen and C 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl or C 1-4 Alkoxy-C 1-4 alkyl; R 3 For H; m, n, and p are each independently selected from 0, 1, or 2; q is selected from 0, 1, or 2.

2. The compound of claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, Ring A is selected from or ; Cy 1 Selected from pyrimidinyl, pyrazinyl, or pyridazinyl groups optionally substituted with 1-2 Q groups; each Q group is independently selected from H, halogen, hydroxyl, amino, C... 1-6 Alkyl, Halogenated C 1-6 Alkyl, hydroxyl C 1-6 Alkyl, amino C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, hydroxy C 1-6 alkoxy or amino C 1-6 Alkoxy; m, n, p, and q are each independently selected from 0, 1, or 2.

3. The compound of claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, Ring A is selected from or ; Ring B is ; Cy 1 It is pyridazinyl; Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, hydroxyl C 1-4 Alkoxy, amino C 1-4 Alkoxy, C 1-4 Alkoxy-C 1-4 Alkyl group, -(CH2) p -N(R a (R) b Alternatively, the following groups may be substituted with 1-2 substituents: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, phenyl, pyridyl, pyrimidinyl; wherein each substituent is independently selected from halogen, cyano, amino, C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl or halogenated C 1-4 Alkoxy; R a R b Selected independently from hydrogen and C 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl or C 1-4 Alkoxy-C 1-4 alkyl; R 3 For H; m is selected from 1 or 2; n is 1; p is selected from 0 or 1; q is selected from 0 or 1.

4. The compound of claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, Ring A is selected from or ; Ring B is ; Cy 1 It is pyridazinyl; Each R 1 Each R 2 Each is independently selected from H, halogen, hydroxyl, amino, cyano, and C. 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkoxy, hydroxy C 1-4 Alkoxy, amino C 1-4 Alkoxy, C 1-4 Alkoxy-C 1-4 Alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, or -(CH2) p -N(R a (R) b ); R a R b Selected independently from hydrogen and C 1-4 Alkyl, Halogenated C 1-4 Alkyl, hydroxyl C 1-4 Alkyl, amino C 1-4 Alkyl or C 1-4 Alkoxy-C 1-4 alkyl; R 3 For H; m is selected from 1 or 2; n is 1; p is 1; q is selected from 0 or 1.

5. The compound of claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, Ring A is selected from or ; Ring B is ; Cy 1 It is pyridazinyl; Each R 1 Each R 2 Selected independently from H, halogen, C 1-4 Alkyl, Halogenated C 1-4 Alkyl, C 1-4 Alkoxy, halogenated C 1-4 Alkyl group, -(CH2) p -N(R a (R) b ), cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, aziridine, oxacyclobutyl, tetrahydrofuranyl, or optionally substituted with 1-2 substituents of the following groups: phenyl, pyridinyl, pyrimidinyl; wherein each substituent is independently selected from halogens, C 1-4 Alkyl, Halogenated C 1-4 Alkyl or halogenated C 1-4 Alkoxy; R a R b Each is independently selected from hydrogen or C. 1-4 alkyl; R 3 H is selected; m is selected from 1 or 2; n is 1; p is 1; q is selected from 0 or 1.

6. The compound of claim 1, its pharmaceutically acceptable salt, or its stereoisomer, selected from the following compounds:

7. A pharmaceutical composition comprising the compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and one or more pharmaceutically acceptable excipients.

8. The pharmaceutical composition of claim 7 further comprises one or more second therapeutic agents selected from anti-PD-1 antibody, anti-PD-L1 antibody, or anti-CTLA-4 antibody.

9. Use of the compound of any one of claims 1-6, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, or the pharmaceutical composition of any one of claims 7-8 in the preparation of a medicament for the prevention and / or treatment of cancers associated with immune activation or cancers resistant to PD-1 antibody / anti-PD-L1 antibody therapy.

10. A kit comprising an effective amount of one or more compounds according to any one of claims 1-6, their pharmaceutically acceptable salts, or their stereoisomers.

11. The kit of claim 10, further comprising an effective amount of one or more second therapeutic agents, said second therapeutic agent being selected from anti-PD-1 antibody, anti-PD-L1 antibody or anti-CTLA-4 antibody.

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