SOAT1 inhibitor and application thereof

Abstract

The present disclosure provides compounds of Formula (I) wherein R1, R2, R3, X1, rings A, B and C, as described herein, stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, which are useful as SOAT1 inhibitors, in particular SOAT1 inhibitors, in particular SOAT1 inhibitors. The invention also provides a pharmaceutical composition containing the same and application of the same in treatment or prevention of SOAT1 pathway activity related diseases such as cancers or tumors in subjects, and also provides application of the same in preparation of therapeutic vaccines for retarding tumor development.

Description

SOAT1 inhibitors and their applications

[0001] This application claims priority to Chinese Patent Application No. 202311102058.2, filed on August 29, 2023, entitled “SOAT1 Inhibitors and Their Applications,” which is hereby incorporated by reference in its entirety. Technical Field

[0002] The present disclosure relates to a class of heterocyclic compounds or pharmaceutically acceptable salts, solvates, stereoisomers, or isotopic variants thereof, which can be used as a class of novel steroid O-acyltransferase 1 inhibitors (also referred to as SOAT1 inhibitors). The present disclosure also relates to pharmaceutical compositions comprising these compounds as active ingredients, as well as their use in treating or preventing diseases associated with SOAT1 pathway activity in subjects, such as cancer or tumors. The present disclosure also relates to the use of these compounds in the preparation of therapeutic vaccines for inhibiting tumor progression and in kits for diagnosing diseases associated with SOAT1 activity. Background Art

[0003] Cancer remains one of the leading causes of death worldwide, accounting for nearly 10 million deaths in 2021. While advances have been made in the treatment of some cancers through surgery, radiation therapy, and chemotherapy, most remain incurable. Even when effective treatments exist for a particular cancer, the side effects of such treatments can be severe, significantly impairing quality of life.

[0004] Immunotherapy using immune checkpoint blockade (ICBs) and adoptive T cell therapy (ACT) has achieved landmark clinical efficacy in several advanced cancers, including melanoma, renal cell carcinoma, and lung cancer. ICBs promote PD-1 + TCF1 + CD8 + Tumor-infiltrating stem-like T cells proliferate (Im et al., 2016; Siddiqui et al., 2019) and act on different immune cell types within the tumor microenvironment (TME) to promote CD8 + T cells have an anti-tumor effect (Kurtulus et al., 2019). However, many patients do not benefit from these interventions, especially those with tumors with poor immunogenicity, such as liver cancer and triple-negative breast cancer (TNBC) (Schmid et al., 2020; Yu et al., 2021; Zhao et al., 2020). This is due to a series of potential factors, including CD8 +Systemic T cell loss, suppressed T cell activation, and poor T cell persistence or exhaustion (Bruni et al., 2020; Chen and Mellman, 2017; et al., 2019; Sharma et al., 2017; Spranger et al., 2018; Thorsson et al., 2018; Tumeh et al., 2014). These immunosuppressive factors are primarily influenced by the TME (Gupta et al., 2022). Tumor communities are composed of a heterogeneous mix of tumor cells, stromal cells (such as adipocytes and fibroblasts), immune cells, and other cells. A major effort is currently underway to improve immunotherapy by modifying the TME (Binnewies et al., 2018). Cancer cells are major players in regulating the TME (Wellenstein and de Visser, 2018). Tumors exhibit tremendous intratumoral heterogeneity. Pioneering research by John Dick and colleagues revealed that leukemia, as well as many other solid cancers, retains a hierarchical structure. In most cases, a small population of leukemia stem cells (LSCs) or cancer stem cells (CSCs), also known as tumor-initiating cells, exhibits unlimited self-renewal capacity, which allows tumors to regenerate and relapse even after seemingly successful treatment (Trumpp and Haas, 2022). CSCs may be the source of cells driving tumor relapse because they have the ability to enter a reversible quiescent / dormant state, resist standard chemotherapy, and form a cellular reservoir. The TME is considered to be the niche of CSCs and regulates their phenotypic plasticity. The TME forms an interactive and immunosuppressive environment for tumor cells and immune cells such as tumor-associated macrophages (TAMs) (Hass et al., 2020). Stem cell-like subpopulations present in specific tumor sites of solid tumors have been found to maintain a tumor niche that allows for immunosuppression and therapy evasion (Jain et al., 2021). In TNBC, quiescent cancer stem cells (QCSCs) have recently been found to constitute an immunosuppressive niche by orchestrating a local hypoxic immunosuppressive environment with dysfunctional dendritic cells (DCs), suppressive fibroblasts, reduced T cell infiltration, and enhanced T cell exhaustion (Baldominos et al., 2022). +For T cells, tumor-specific antigen peptides must be presented to the cell surface via human leukocyte antigen (HLA) class I molecules. Studies have found that several types of CSCs downregulate the expression of HLA class I molecules (Muller et al., 2020). CSCs also express CD47α, which inhibits macrophage phagocytosis by interacting with signal-regulating proteins (Muller et al., 2020). Therefore, developing new strategies to eradicate CSCs and alter their immunosuppressive environment will be key to improving immunotherapy responses in cancer treatment.

[0005] The inventors developed a phenotypic assay using laser confocal microscopy to screen for novel compounds that can kill oncogenic RasV12-induced stem cell tumors in Drosophila. The inventors screened 9,127 compounds and identified compound STK773477 (4-(2-thienyl)-1-[3-(trifluoromethyl)phenyl]-4,6,7,8-tetrahydro-2,5(1H,3H)-quinolinedione) (hereinafter referred to as STK compound) as a potent tumor-killing compound in multiple models, including Drosophila, mice, and PDX tumor models. The inventors found that STK compound is a new SOAT1 inhibitor. In the process of treating tumors, STK compound can release multiple cytokines, transforming TME from an immunosuppressive to an immunostimulatory microenvironment.

[0006] Steroid O-acyltransferase (SOAT), also known as cholesterol acyltransferase (ACAT), catalyzes the reaction between intracellular cholesterol and long-chain fatty acid acyl-CoA, forming more hydrophilic cholesterol esters (CEs), which are then stored in intracellular lipid droplets or transported to other tissues via secreted lipoprotein particles (Chang et al., 2006). In addition to cholesterol, SOAT uses a variety of other steroids as substrates (Rogers et al., 2015). In mammals, there are two SOATs, SOAT1 and SOAT2. SOAT1 is a multi-spanning membrane protein localized to the endoplasmic reticulum and widely expressed in many cell types, while SOAT2 is primarily expressed in the liver and small intestine (Anderson et al., 1998; Chang et al., 2000; Hofmann, 2000).

[0007] Changes in cholesterol metabolism in tumor cells play an important role in tumorigenesis (Hanahan and Weinberg, 2011). SOAT1 has been shown to be highly expressed in a variety of cancers, including prostate cancer, pancreatic cancer, malignant glioma, hepatocellular carcinoma (HCC), and others (Geng et al., 2016; Jiang et al., 2019; Li et al., 2016; Yue et al., 2014). In animal models, knocking down or inhibiting SOAT1 can regulate cholesterol-related pathways in tumor cells, thereby inhibiting a variety of tumors (Jiang et al., 2019; Yue et al., 2014) or changing the tumor immune microenvironment (Ma et al., 2019). Studies have also shown that inhibiting SOAT1 can increase cholesterol levels and promote T cell activation, thereby enhancing CD8 + SOAT1 has been shown to have anti-tumor effects on T cells and human chimeric antigen receptor-modified T cells (Li et al., 2018; Yang et al., 2016). SOAT1 is also a potential drug target for the treatment of other diseases, such as Alzheimer's disease (Shibuyua et al., 2015) and atherosclerosis (Rong et al., 2013).

[0008] Only a few SOAT1 inhibitors have been reported, including avasimibe, nevanimibe, CI-976, and nilotinib (Wang et al., 2022). A phase 1 study of nevanimibe hydrochloride in adrenocortical carcinoma (ACC) found that nevanimibe hydrochloride was safe at doses up to 6000 mg twice daily, but its efficacy in patients with advanced ACC was limited (Smith et al., 2020). To fully realize the potential of SOAT1 in cancer treatment, more effective SOAT1 inhibitors are needed. The low efficacy of existing SOAT1 inhibitors contradicts their important role in cancer treatment. Therefore, increased research and development efforts are necessary to accelerate the development of new SOAT1 inhibitors in order to achieve superior clinical efficacy.

[0009] The inventors have successfully discovered a novel SOAT1 inhibitor, a STK compound, that exhibits low toxicity and potent antitumor activity in liver, colon, breast, lung, and melanoma mouse and PDX tumor models. Using this inhibitor to treat tumors not only significantly increases tumor infiltration but also modulates Treg differentiation and induces the formation of tumor-infiltrating stem-like TCF1+CD8+ T cells, providing strong support for tumor immunotherapy.

[0010] Maintaining functional tumor-targeting CD8 in patients requiring immunotherapy + The T cell repertoire is crucial. If tumors persist, immune cells gradually become exhausted and lose their effectiveness, leading to treatment failure. Therefore, during and after treatment, the function and number of anti-tumor T cells need to be closely monitored, and measures should be taken, if necessary, to maintain a sufficient functional repertoire and inhibit factors that may lead to their exhaustion. This not only improves the efficacy of immunotherapy but also provides important information for ongoing monitoring and adjustment of treatment plans (Robbins et al., 2004). Studies have found that ICBs promote tumor control not by reversing T cell exhaustion, but by promoting the proliferation of stem-like T cell subsets. Stem-like T cells then generate new, effective T cells to replenish the exhausted T cells within the tumor. In animal models of cancer therapy and in patients with cancer, stem-like CD8+ T cells maintain superior responses to ICBs and ACT (Baharon et al., 2021; Eberhardt et al., 2021; Galletti et al., 2020; Hashimoto et al., 2022; Im et al., 2016; Jansen et al., 2019; Kurtulus et al., 2019; Krishna et al., 2020; Mo et al., 2021; Prokhnevska et al., 2023; Siddiqui et al., 2019; Wu et al., 2020; Yost et al., 2019).

[0011] The inventors' research data show that the use of SOAT1 inhibitor STK compounds can reverse the immunosuppressive tumor microenvironment and increase the number of stem-like T cell populations. Therefore, single-agent treatment can effectively overcome the three major challenges of cancer immunotherapy: enhancing CD8+ T cell infiltration, promoting cell activation, and blocking T cell dysfunction or exhaustion. This inhibitor reprograms lipid metabolism, thereby converting the tumor immune microenvironment from "cold" to "hot." Therefore, this inhibitor may become a new strategy for cancer immunotherapy and significantly increase patients' chances of receiving immunotherapy. The new SOAT1 inhibitor can not only treat tumors with poor immunogenicity, but can also be combined with ICBs or ACT to develop more effective T cell immunotherapy regimens.

[0012] In the present disclosure, the inventors continued to conduct research to obtain a new class of SOAT1 inhibitors, as well as the use of these SOAT1 inhibitors in treating tumors.

[0013] Summary of the Invention

[0014] The present disclosure is based on the inventors' unexpected discovery that genetically knocking down or eliminating SOAT1 can eradicate cancer stem cells and induce DAMP-mediated anti-tumor immune responses. The inventors have also developed and identified a novel class of SOAT1 inhibitors that are water-soluble and induce potent anti-tumor immunity. Therefore, the SOAT1 inhibitors disclosed herein (i.e., compounds of Formula (I)) are particularly suitable for treating or preventing diseases associated with SOAT1 activity; treating refractory, recurrent, or metastatic cancers; selectively killing cancer cells with specific dosing regimens; and targeting cancer stem cells (CSCs) to induce anti-tumor immune responses by inhibiting SOAT1.

[0015] In one aspect, the present disclosure provides a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0016] in,

[0017] n is selected from 0, 1 and 2;

[0018] X1 is selected from CH, CH2, N or NH;

[0019] represents a single bond or a double bond;

[0020] The phenyl ring A is optionally substituted with 1, 2, 3, 4, or 5 R1;

[0021] R1 and R2 are each independently absent or selected from H, D, CN, OH, halogen, optionally substituted with 1-3 R a C 1-6 Alkyl, optionally substituted with 1-3 R b C 3-6 Cycloalkyl and optionally substituted with 1-3 R c C 1-6 a group consisting of alkoxy groups;

[0022] R3 is selected from optionally substituted with 1-3 R d C 5-6 a carbocyclic ring or a 5- to 6-membered heterocyclic ring, the 5- to 6-membered heterocyclic ring optionally containing one or more heteroatoms selected from N, O and S, or the 5- to 6-membered heterocyclic ring optionally containing one or more heteroatoms selected from N and O;

[0023] Each time it appears, each R a 、R b 、R c 、R d Each independently selected from D, -OH, -NH2, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)-C 1-6 Alkyl, C 3-6 The group consisting of cycloalkyl and 3- to 6-membered heterocycloalkyl.

[0024] As used in this disclosure, Represents a single bond or a double bond. Those skilled in the art will understand that When it is a single bond or a double bond, the resulting compound molecule should satisfy the valence bond theory.

[0025] In some embodiments, isotopic variations of compounds of Formula (I) are deuterated derivatives, wherein compounds of Formula (I) are optionally substituted with one or more deuteriums.

[0026] In some embodiments of the present disclosure, the above-mentioned X1 is selected from CH2, N or NH, and the other variables are as defined in the present disclosure.

[0027] In some embodiments of the present disclosure, the above R1 and R2 are each independently absent or selected from H, CN, halogen, such as F, Cl, Br or I, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Halogenated alkyl, such as -CF3, -CHF2 or -CH2F, etc., C 1-4 Alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy, or C 1-4 haloalkoxy, and other variables are as defined in the present disclosure.

[0028] In some embodiments of the present disclosure, phenyl ring A is optionally substituted with 1, 2, 3, 4, or 5 R1, such as 1 or 2 R1.

[0029] In some embodiments of the present disclosure, each R1, at each occurrence, is independently selected from D, CN, halogen, such as F, Cl, Br or I, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Halogenated alkyl, such as -CF3, -CHF2 or -CH2F, etc., C 1-4 Alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy, or C 1-4 haloalkoxy, and other variables are as defined in the present disclosure.

[0030] In some embodiments of the present disclosure, each R1 is independently located at each occurrence in the ortho, meta, or para position relative to the location where A and N are attached to the phenyl ring.

[0031] In some embodiments of the present disclosure, the phenyl ring A optionally substituted with 1, 2, 3, 4, or 5 R1 is selected from the following group:

[0032] In some embodiments of the present disclosure, R2 is absent or selected from D, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., and other variables are as defined in the present disclosure.

[0033] In some embodiments of the present disclosure, Rings B and C collectively form the following fused ring structure:

[0034] In some embodiments of the present disclosure, R3 is selected from the group consisting of optionally substituted with 1-3 R d C 5-6 a carbocyclic ring or a 5- to 6-membered heterocyclic ring, the 5- to 6-membered heterocyclic ring optionally comprising 1 or more heteroatoms each independently selected from N, O and S, such as 1, 2, 3 or 4 heteroatoms each independently selected from N, O and S, such as 1 or 2 heteroatoms each independently selected from N, O and S.

[0035] In some embodiments of the present disclosure, the above R3 is selected from the group consisting of optionally substituted with 1-3 R d , such as 1-2, 1, 2 or 3 R d phenyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, furanyl, thienyl, thiazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, or tetrazolyl, and the other variables are as defined in the present disclosure.

[0036] Furthermore, the above R3 is selected from optionally substituted with 1-3 R d , such as 1-2, 1, 2 or 3 R d of Other variables are as defined in this disclosure.

[0037] Alternatively, further, the above R3 is selected from optionally substituted with 1-3 R d , such as 1-2, 1, 2 or 3 R d of Other variables are as defined in this disclosure.

[0038] Alternatively, further, the above R3 is selected from optionally substituted with 1-3 R d , such as 1-2, 1, 2 or 3 R d of Other variables are as defined in this disclosure.

[0039] In some embodiments of the present disclosure, in the above R3, R d is selected from halogen, such as F, Cl, Br or I, C 1- 4 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Haloalkyl, C 1- 4 alkoxy groups, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy, or C 1-4 haloalkoxy, and other variables are as defined in the present disclosure.

[0040] Furthermore, the above R3 is selected from the following: Other variables are as defined in this disclosure.

[0041] Alternatively, further, the above R3 is selected from the following: Other variables are as defined in this disclosure.

[0042] Alternatively, further, the above R3 is selected from the following: Other variables are as defined in this disclosure.

[0043] In some embodiments of the present disclosure, the present disclosure provides a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0044] in,

[0045] n is selected from 1;

[0046] X1 is selected from CH2, N or NH;

[0047] represents a single bond or a double bond;

[0048] The phenyl ring A is optionally substituted with 1, 2 or 3 R1, and further optionally substituted with 1 or 2 R1;

[0049] R1 and R2 are each independently absent or selected from H, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Halogenated alkyl, such as -CF3, -CHF2 or -CH2F, etc., C 1-4 Alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy, or C 1-4 Halogenated alkoxy, further, R1 is selected from H, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C1-4 Haloalkyl, such as -CF3, -CHF2 or -CH2F, and / or R2 is absent or selected from H, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Haloalkyl, such as -CF3, -CHF2 or -CH2F;

[0050] Rings B and C together form the following fused ring structure:

[0051] R3 is selected from optionally substituted with 1 or 2 R d of where R d is selected from halogen, such as F, Cl, Br or I, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., further, R3 is selected from the following:

[0052] In some embodiments of the present disclosure, the present disclosure provides a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof:

[0053] in,

[0054] n is selected from 1;

[0055] X1 is selected from CH2, N or NH;

[0056] represents a single bond or a double bond;

[0057] The phenyl ring A is optionally substituted with 1, 2 or 3 R1, and further optionally substituted with 1 or 2 R1;

[0058] R1 and R2 are each independently absent or selected from H, CN, halogen, such as F, Cl, Br or I, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Halogenated alkyl, such as -CF3, -CHF2 or -CH2F, etc., C 1-4 Alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy, or C 1-4 Haloalkoxy, further, R1 is selected from H, D, CN, halogen, such as F, Cl, Br or I, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4Halogenated alkyl, such as -CF3, -CHF2 or -CH2F, etc., C 1-4 Alkoxy, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy or tert-butoxy, or C 1-4 Haloalkoxy, and / or R2 is absent or selected from H, D, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., C 1-4 Haloalkyl, such as -CF3, -CHF2 or -CH2F;

[0059] Rings B and C together form the following fused ring structure:

[0060] R3 is selected from optionally substituted with 1 or 2 R d of where R d is selected from halogen, such as F, Cl, Br or I, C 1-4 Alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert-butyl, etc., further, R3 is selected from the following:

[0061] In some embodiments of the present disclosure, the compound of formula (I) is selected from the following compounds:

[0062] In one aspect, the present disclosure provides a method for preparing the compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, the preparation method comprising the following synthetic route:

[0063] The variables in the synthetic route are as shown in this disclosure. The compound of formula (I) described in this disclosure can be prepared from the reaction intermediates SM-1 and SM-2. The cyclization reaction usually occurs in the presence of a suitable base such as DBU and acetonitrile as a solvent. The preferred reaction temperature is 60-100°C. The compound of formula (I) in an oxidized configuration can be obtained by oxidizing the compound of formula (I) in the presence of a suitable oxidant (such as potassium persulfate, DDQ, etc.). The compound of formula (I) can be resolved by chiral SFC or chiral HPLC to obtain isomers of absolute configuration.

[0064] In one aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula (I) described herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, and a pharmaceutically acceptable carrier or excipient.

[0065] In one aspect, the present disclosure provides a method for inhibiting intracellular SOAT1 activity in a cell, comprising administering an effective amount of a compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof to the cell, such that SOAT1 activity in the cell is reduced.

[0066] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0067] In some embodiments, the methods induce cell death at the cellular level.

[0068] In some embodiments, the method is applied to cells in vitro.

[0069] In some embodiments, the method is administered to the cells in vivo.

[0070] In one aspect, the present disclosure provides use of a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof in the preparation of an agent for inhibiting intracellular SOAT1 activity in a cell.

[0071] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0072] In some embodiments, the agent induces cell death at the cellular level.

[0073] In some embodiments, the agent is administered to the cell in vitro.

[0074] In some embodiments, the agent is administered to the cell in vivo.

[0075] In one aspect, the present disclosure provides a method for treating or preventing a disease associated with SOAT1 activity in a subject, comprising administering to the subject an effective amount of a compound of formula (I) described herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof.

[0076] In some embodiments, the disease associated with SOAT1 activity is cancer or tumor.

[0077] In some embodiments, the cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0078] In some embodiments, the disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

[0079] In some embodiments, the subject is a mammal, such as a human.

[0080] In some embodiments, the method further comprises co-administering at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

[0081] In some embodiments, the compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof are administered simultaneously with at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody, or administered sequentially in any order.

[0082] In one aspect, the present disclosure provides a compound of formula (I) as described herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with SOAT1 activity in a subject.

[0083] In some embodiments, the disease associated with SOAT1 activity is cancer or tumor.

[0084] In some embodiments, the cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0085] In some embodiments, the disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

[0086] In some embodiments, the subject is a mammal, such as a human.

[0087] In some embodiments, the compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof are administered in combination with at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

[0088] In some embodiments, the compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof are administered simultaneously with at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody, or administered sequentially in any order.

[0089] In one aspect, the present disclosure provides use of a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating or preventing a disease associated with SOAT1 activity in a subject.

[0090] In some embodiments, the disease associated with SOAT1 activity is cancer or tumor.

[0091] In some embodiments, the cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0092] In some embodiments, the disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

[0093] In some embodiments, the subject is a mammal, such as a human.

[0094] In one aspect, the present disclosure provides a kit for treating or preventing a disease associated with SOAT1 activity in a subject, the kit comprising a compound of formula (I) as described herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present disclosure; a container; and optionally a package insert or label indicating treatment.

[0095] In some embodiments, the disease associated with SOAT1 activity is cancer or tumor.

[0096] In some embodiments, the cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0097] In some embodiments, the disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

[0098] In some embodiments, the subject is a mammal, such as a human.

[0099] In some embodiments, the kit further comprises at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

[0100] In one aspect, the present disclosure provides a kit for diagnosing a disease associated with SOAT1 activity in a subject, comprising at least one compound of formula (I) described herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof as a test agent.

[0101] In some embodiments, the test agent detects at least one biomarker indicative of the presence of a disease associated with SOAT1 activity.

[0102] In some embodiments, the test reagent can detect SOAT1 activity, serve as a reporter gene for detecting lipolysis activity, detect lipid droplet formation, detect autophagy activity, or serve as an upstream or downstream alternative regulator of SOAT1 function.

[0103] In some embodiments, the disease associated with SOAT1 activity is cancer or tumor.

[0104] In some embodiments, the cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0105] In some embodiments, the disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

[0106] In some embodiments, the subject is a mammal, such as a human.

[0107] In one aspect, the present disclosure provides a compound of formula (I) described herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in preparing a therapeutic vaccine for inhibiting tumor progression.

[0108] In some embodiments, the tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0109] In one aspect, the present disclosure provides use of the compound of formula (I), its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof in the preparation of a therapeutic vaccine for inhibiting tumor development.

[0110] In some embodiments, the tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like.

[0111] In one aspect, the present disclosure provides a method for preparing a therapeutic vaccine for blocking tumor development, comprising contacting a compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof with tumor stem cells, thereby converting the tumor stem cells into a therapeutic vaccine.

[0112] In one aspect, the present disclosure provides a method of vaccination comprising administering a therapeutic vaccine prepared according to the method of the present disclosure to a subject.

[0113] In some embodiments, the subject is a mammal, such as a human.

[0114] In one aspect, the present disclosure provides a method for killing cells and inducing an anti-tumor immune response, the method comprising inhibiting SOAT1 activity in cells by a SOAT1 inhibitor.

[0115] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0116] In some embodiments, the SOAT1 inhibitor is selected from the group consisting of a small molecule SOAT1 inhibitor, an RNAi agent against SOAT1, an antisense agent against SOAT1, a peptidomimetic SOAT1 inhibitor, and a DNA binding deoxynucleotide inhibitor containing the target protein SOAT1.

[0117] In some embodiments, the small molecule SOAT1 inhibitor is a compound of formula (I) as described herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 shows the protective effects of the disclosed compounds and STK on SOAT1 protein in a DARTs assay, wherein A shows the effects of compounds 5-P1 and 5-P2 on SOAT1 protein stability; and B shows the effects of compounds STK, 12, 14, 16, 19, and 20 on SOAT1 protein stability.

[0119] Figure 2 shows that compounds 1 through 5 of the present disclosure were able to effectively inhibit tumor growth in a BALB / c mouse CT26 xenograft tumor model. SD1 refers to compound 1, SD2 refers to compound 2, SD3 refers to compound 3, SD4 refers to compound 4, and SD5 refers to compound 5. A is the experimental model; B is the xenograft tumor growth curve; C is the xenograft tumor weight; D is the xenograft tumor growth curve; and E is the xenograft tumor weight.

[0120] Figures 3A-3E show the tumor-suppressing and immune cell-promoting effects of compound 5-P1 in a CT26 xenograft mouse model. P1 refers to compound 5-P1, and P2 refers to compound 5-P2. Figure 3A shows the size of mouse xenograft tumors; Figure 3B shows the xenograft tumor growth curve; Figure 3C shows the xenograft tumor weight; Figure 3D shows the weight changes of mice in each treatment group; and Figure 3E shows the percentage of T cells infiltrating within the xenograft tumors.

[0121] Figures 4A-4F show the anti-tumor and immune cell infiltration-promoting effects of compounds 5-P1, 7, 8, 9, 10, and 11 disclosed herein in a CT26 xenograft model mouse model. Figure 4A shows the size of mouse xenograft tumors; Figure 4B shows the weight of xenograft tumors; Figure 4C shows the xenograft tumor growth curve; Figure 4D shows the weight changes of mice in each treatment group; Figures 4E and 4F show the proportion of T cell infiltration within the xenograft tumors.

[0122] FIG5 shows the tumor inhibition effects of Compound 5-P1, Compound 12, Compound 13, Compound 14, Compound 16, Compound 19, Compound 20, and Compound 21 (A and B) disclosed herein in CT26 xenograft model mice.

[0123] Figure 6 shows that the disclosed compound STK can effectively inhibit the growth of tumors in Drosophila, where A represents the growth of in situ tumors in each group of Drosophila; and B represents the degree of inhibition of the compound STK on the in situ tumors in Drosophila.

[0124] Figure 7 shows the inhibitory effect of the disclosed compound STK on tumor growth in mice with orthotopic colorectal cancer and liver tumors. A represents the growth of orthotopic colorectal cancer in mice; B represents the degree of inhibition of orthotopic colorectal cancer by compound STK; C represents the size of orthotopic liver tumors in mice; and D represents the survival of mice with orthotopic liver tumors improved by compound STK.

[0125] Figure 8 shows that the disclosed compound STK treatment effectively inhibited the growth of lung cancer in mice. A is a schematic diagram of the experiment; B is the size of the lung cancer; and C is the survival of mice with lung cancer improved by the compound STK.

[0126] Figures 9A-9E show the inhibitory effects of the disclosed STK compounds in mouse colon cancer, melanoma, lung cancer, and breast cancer xenograft models. Figure 9A is a schematic diagram of the experiment; Figure 9B shows the effects of the STK compound in a colon cancer model; Figure 9C shows the effects of the STK compound in a lung cancer model; Figure 9D shows the effects of the STK compound in a breast cancer model; and Figure 9E shows the effects of the STK compound in a melanoma model.

[0127] FIG10 shows the potential target SOAT1 of the STK compound screened by reverse docking in the present disclosure.

[0128] FIG11 shows that the target of the STK compound disclosed in the present invention is SOAT1 (AD) determined through in vitro DARTs and ITC experiments.

[0129] Figure 12 shows the in vivo mouse xenograft tumor experiments used in this disclosure to demonstrate that the target of the STK compound is SOAT1. A shows the effect of the STK compound on the size of xenograft tumors carrying or lacking SOAT1; B shows the effect of the STK compound on the growth curve of xenograft tumors carrying or lacking SOAT1; and C shows the effect of the STK compound on the weight of xenograft tumors carrying or lacking SOAT1.

[0130] Figure 13 shows the effects of the disclosed compound STK on cholesterol metabolism in CT26 cells, LLC cells, and Huh7 cells, wherein A represents LLC cells, B represents ST26 cells, and C represents Huh7 cells.

[0131] Figure 14 shows the effects of the disclosed compound STK on the cholesterol ester content in CT26 cells and in the culture supernatant, where A represents the intracellular content and B represents the extracellular content.

[0132] Figure 15 shows the effects of the disclosed compound STK on lipid droplet accumulation and oxidation in CT26 and Huh7 cells, wherein A represents CT26 cells and B represents Huh7 cells.

[0133] Figure 16 shows the effects of the disclosed compound STK on reactive oxygen species, Golgi morphology, and mitochondrial number in Huh7 and CT26 cells, wherein A represents Huh7 cells and B represents CT26 cells.

[0134] Figure 17 shows the effects of the disclosed compound STK on lysosomal morphology and intracellular protein aggregation in Huh7 and CT26 cells, wherein A represents CT26 cells, and B and C represent Huh7 cells.

[0135] Figure 18 shows that the disclosed compound STK induces Caspase-1-dependent cell pyroptosis in LLC lung cancer xenografts, where A represents the expression of Caspase-1 in the xenografts; and B represents the expression of Caspase-3 in the xenografts.

[0136] Figure 19 shows the effects of the disclosed compound STK on HMGB1 and ERP46 expression and ATP production in CT26 and Huh7 cells, wherein A is CT26 cells; B is Huh7 cells; C is CT26 cells; and D is the change in ATP content.

[0137] Figure 20 shows that the combination of the disclosed compound STK and anti-PD-1 exhibits a synergistic anti-tumor effect in a mouse CT26 xenograft tumor model. A represents the size of the xenograft tumor in each group of mice; B represents the growth of the xenograft tumor in each group of mice; C represents the weight of the xenograft tumor in each group of mice; and D represents the weight change of the tumor-bearing mice in each group.

[0138] Figure 21 shows that STK treatment with the disclosed compound significantly increased the proportions of CD4+ and CD8+ T cells in CT26 xenografts in mice. A is the quantitative ratio of the corresponding immune cells in the xenografts of each group; B is the ratio of immune cells in the spleen of each group of mice.

[0139] Figure 22 shows that the disclosed compound STK cannot inhibit the growth of CT26 xenograft tumors in immunodeficient mice. A is the size of xenograft tumors in each group of immunodeficient mice; B is the xenograft tumor growth curve; C is the xenograft tumor weight; and D is the weight change of mice in each treatment group.

[0140] Figure 23 shows that the in vivo anti-tumor activity of the disclosed compound STK depends on the activation of mouse immune T cells. A is the size of the transplanted tumor in mice after control or CD4 / CD8 antibody treatment; B is the transplanted tumor growth curve; and C is the transplanted tumor weight.

[0141] Figure 24 shows that treatment with the disclosed compound STK increases chemokine expression and CD8+CCR5+ T cell infiltration. A shows the effect of compound STK on chemokine expression in CT26 cells; B shows the effect of compound STK on the proportion of CD8+CCR5+ T cells in LLC lung cancer and 4T1 breast cancer.

[0142] Figure 25 shows the ability of the disclosed compound STK to enhance T cell infiltration and killing in mouse lung and colon cancer. A shows T cell staining in mouse lung cancer xenografts; B shows T cell staining in mouse colon cancer xenografts; C shows the effect of compound STK on key molecules involved in immune T cell activation; and D shows the ability of compound STK to enhance T cell killing of CT26 cancer cells.

[0143] Figures 26A-26B show the effects of the disclosed compound STK on immune cell subset infiltration in mouse orthotopic liver tumors and lung cancer. Figure 26A shows the scRNA-seq results for mouse orthotopic liver cancer, and Figure 26B shows the scRNA-seq results for mouse lung cancer. DETAILED DESCRIPTION

[0144] Certain embodiments will now be described in detail, examples of which are illustrated in the accompanying specific embodiments. Although the enumerated embodiments will be described, it should be understood that they are not intended to limit the present disclosure to these embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the present disclosure as defined by the claims. Those skilled in the art will recognize that many methods and materials are similar or equivalent to the methods and materials described in the present disclosure, which can be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. If one or more incorporated documents and similar materials differ from or contradict the present disclosure, including but not limited to defined terms, term usage, described technology, etc., the present disclosure shall prevail.

[0145] It will be appreciated that certain features of the present disclosure, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0146] definition

[0147] Terms used but not defined in this disclosure have their ordinary meanings, and the meanings of such terms are independent at each occurrence thereof. However, unless otherwise indicated, the following definitions apply throughout the specification and claims.

[0148] As used herein, the terms “comprising” and “including” are intended to specify the presence of stated features, integers, components or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps or groups thereof.

[0149] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry as well as specific functional moieties and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987.

[0150] All ranges cited herein are inclusive unless expressly stated otherwise.

[0151] When a range of values ​​is listed, it is intended that every value and subrange within that range be included. For example, “C 1-6 "Aims to cover C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 .

[0152] When any variable occurs more than one time in any constituent or in Formula (I) or in any other formula depicting and describing compounds of the present disclosure, its definition on each occurrence is independent of its definition at every other occurrence. Furthermore, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0153] As used herein, the terms "cancer stem cell" and "CSC" are interchangeable. The CSC is of mammalian origin, and in some embodiments, the CSC is of human origin, but is not limited thereto. The definition and functional characteristics of cancer stem cells are: 1) a population of tumor cells with extensive proliferative capacity; 2) the ability to undergo asymmetric cell division to produce one or more differentiated offspring with reduced proliferation or developmental potential; 3) the ability to undergo symmetric cell division that is self-renewal or self-sustaining. Other common methods for characterizing CSCs include examining morphology, cell surface markers, transcriptional profiles, and drug responses. In the research literature, CSCs are also referred to as tumor / cancer initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, tumor stem cells, solid tumor stem cells, drug-surviving cells (DSCs), drug-resistant cells (DRCs), or super malignant cells.

[0154] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a cell population is characterized by unregulated cell growth. As used herein, the terms "cancer cell" and "tumor cell" refer to the total number of cells from a tumor, including tumorigenic stem cells (cancer stem cells) and non-tumorigenic cells that make up the majority of the tumor cell population. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer.

[0155] As used herein, the term "tumor" refers to any mass of tissue resulting from excessive growth or proliferation of cells, whether benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0156] As used herein, the term "metastasis" refers to the process by which cancer spreads or metastasizes from its site of origin to other areas of the body as similar cancerous lesions develop in new locations. "Metastatic" cells are cells that have lost their adhesive contacts with neighboring cells and migrated from the primary site of the disease via the blood or lymph to invade neighboring body structures.

[0157] As used herein, the term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein to refer to a human subject.

[0158] As used herein, the terms "treat" or "alleviate" refer to: 1) a cure, slow down, alleviate symptoms and / or halt the progression of a diagnosed pathological condition or disease; 2) a prophylactic or therapeutic method that prevents or slows the development of a target pathological condition or disease. Thus, those in need of treatment include those already suffering from the disease; those susceptible to such disease; and those in need of prevention. A subject is successfully "treated" according to the methods of the present disclosure if the patient exhibits one or more of the following: a decrease or complete absence of cancer cells; a decrease in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs (this includes the spread of cancer cells to soft tissue and bone); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; reduction in morbidity and mortality; and improvement in quality of life.

[0159] As used herein, the term "inhibit," when used in the context of a biological activity, refers to downregulation of a biological activity that may reduce or eliminate a target function, such as the production of a protein or the phosphorylation of a molecule. In some embodiments, inhibition may refer to a reduction in the targeted activity by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. When used in the context of a disorder or disease, the term may refer to an effect achieved in preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition, or disorder.

[0160] As used herein, the term "cholesterol acyltransferase (ACAT) activity" refers to the catalysis of the reaction between intracellular cholesterol and long-chain fatty acyl-CoA to form more hydrophobic cholesterol esters (CEs) for storage in intracellular lipid droplets or transport to other tissues in secreted lipoprotein particles (Chang et al., 2006).

[0161] As used herein, the term "necrotic cell death" refers to when cells are exposed to non-physiological conditions (e.g., hypothermia, hypoxia) or extreme events that may cause damage to the plasma membrane. Under physiological conditions, agents such as complement viruses and lytic viruses can induce direct damage to the plasma membrane. Necrosis is caused by the inability of cells to maintain homeostasis, resulting in an influx of water and extracellular ions. Intracellular organelles, particularly mitochondria and entire cells eventually swell and rupture (cytolysis). Due to the rupture of the plasma membrane, cellular contents, including lysosomal enzymes, are released into the extracellular space. Therefore, in vivo, necrotic cell death is often accompanied by a strong inflammatory response and extensive tissue damage.

[0162] As used herein, the terms "immune condition" or "immune disorder" include, but are not limited to, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune condition" also refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that are resistant to eradication by the immune system. "Cancerous disease" includes, but is not limited to, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

[0163] As used herein, the term "cytotoxic T cell" refers to a T lymphocyte (a type of white blood cell) that kills cancer cells, cells infected by viruses, or damaged cells. Most cytotoxic T cells are known to express T cell receptors (TCRs) that recognize specific antigens. https: / / en.wikipedia.org / wiki / Antigen Antigens are molecules that stimulate an immune response and are usually produced by cancer cells or viruses. Antigens within cells bind to class I MHC molecules and are then brought to the cell surface where they can be recognized by T cells. When the TCR encounters its specific antigen, it binds directly to the complex of class I MHC molecules and the antigen, so that the T cell destroys the target cell.

[0164] In order to interact with class I MHC molecules, the TCR must be accompanied by the glycoprotein CD8, which binds to the anchor portion of the class I MHC molecule. Therefore, these T cells are called CD8 + T cells.

[0165] Once cytotoxic CD8 +When T cells (CTLs) recognize their target cells in the periphery, a highly specialized intercellular contact structure called the immune synapse (IS) forms between the CTL and its target cell, leading to CTL activation. TCR activation leads to the engagement of the Src family kinases Lck and Fyn and the recruitment of the Syk family kinase ZAP-70 to the TCR, where these tyrosine kinases are activated. Activated ZAP70 then phosphorylates the adaptor protein linker of T cell activation (LAT), which orchestrates the assembly of a multiprotein signalosome, including phospholipase C-γ1 (PLC-γ1) and SLP76 (a 76 kDa leukocyte protein containing an SH2 domain). PLCγ1 converts phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 3-1,4,5-triphosphate (IP). The accumulation of DAG at the immune synapse leads to the recruitment of novel protein kinases C (PKCs), such as PKCθ. PKCθ then promotes polarization of the centrosome (microtubule-organizing center, MTOC) through the mutual localization of dynein at the synapse. Dynein pulls the MTOC toward the synapse and pulls non-muscle myosin II (NMII) to the other side of the cell, where it pushes the MTOC toward the synapse. Docking of the MTOC beneath the IS ensures the targeted delivery of dynein and calcium-dependent exocytosis of granule contents directly into the synaptic cleft, leading to lysis of the target cell through the coordinated action of granzymes and perforins (de la Roche et al., 2016).

[0166] As used herein, the term "deuterated" refers to a compound or group in which one or more hydrogen atoms are replaced by deuterium. When a compound or group is deuterated, one, two, three, or even more hydrogen atoms on the compound or group may be replaced by deuterium, until all hydrogen atoms on the compound or group are replaced by deuterium. In this case, the compound or group is referred to as a "perdeuterated compound or group."

[0167] In some embodiments, at deuterated positions, the deuterium isotope abundance is greater than the natural deuterium isotope abundance (0.015%), preferably greater than 50%, more preferably greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%.

[0168] In some cases, such as when "hydrogen" and "deuterium" appear in a statement as parallel alternatives or when "hydrogen" is replaced by "deuterium", the term "hydrogen" refers to the hydrogen isotope " 1 hydrogen( 1 H)", while the term "deuterium" refers to the hydrogen isotope " 2 hydrogen( 2H)"; or it is understood that at that position of the compound, hydrogen in a state where its various isotopic abundances are naturally present is replaced by deuterium in a state where deuterium is present at a greater abundance than the natural deuterium isotopic abundance (e.g., a deuterium abundance of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%).

[0169] As used herein, the term "hydrocarbyl" refers to a chemical group comprising hydrogen and carbon. A hydrocarbyl group may be substituted or unsubstituted. A hydrocarbyl group may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic or heterocyclic, and includes alkyl, alkenyl and alkynyl groups. A hydrocarbyl group may be fully saturated, monounsaturated or polyunsaturated, and may include divalent and polyvalent groups, for example, when a divalent group, it may be referred to as a "hydrocarbylene group". A hydrocarbyl group has a specified number of carbon atoms (i.e., C1-C 10 "alkyl" means 1 to 10 carbon atoms, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms. In some embodiments, the hydrocarbon group contains one or more, for example, 1, 2, 3, or 4, unsaturated carbon-carbon double bonds (-C=C-), carbon-carbon triple bonds (-C≡C-), and / or any combination thereof. In some embodiments, the hydrocarbon group may be deuterated, also known as a "deuterated hydrocarbon group," i.e., one, two, three, or even more hydrogen atoms on the hydrocarbon group may be replaced by deuterium, until all hydrogen atoms on the hydrocarbon group are replaced by deuterium, in which case it may be referred to as a "perdeuterated hydrocarbon group."

[0170] As used herein, the term "heteroalkyl" refers to an alkyl group containing heteroatoms such as N, O, S, etc. The heteroalkyl group may be substituted or unsubstituted. The heteroalkyl group may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic or heterocyclic, and includes heteroalkyl, heteroalkenyl and heteroalkynyl groups. The heteroalkyl group may be fully saturated, monounsaturated or polyunsaturated, and may include divalent and polyvalent groups, for example, when it is a divalent group, it may be referred to as a "heteroalkylene group". The heteroalkyl group has a specified number of carbon atoms (i.e., C1-C 10In some embodiments, the heteroatom contained in the heteroalkyl group can constitute the main chain of the heteroalkyl group together with the carbon atoms, such as, but not limited to, -CNC-, -COC-, -COOC, -CSC-, -CSSC and other group structures or any combination thereof. In some embodiments, the heteroatom contained in the heteroalkyl group can be a substituent attached to the carbon atom, such as, but not limited to, -C≡N, -C=N-, -CN=, -C=O, -C-OH, -C=S, -C-SH and other substitution structures. In some of the embodiments, the heteroatom contained in the heteroalkyl group can be any combination of the above-listed group structures. In some embodiments, the heteroalkyl group contains one or more, for example, 1, 2, 3, or 4, unsaturated carbon-carbon double bonds (-C=C-), carbon-carbon triple bonds (-C≡C-), -NH-, -O-, -C(O)-, -S-, -C(S)-, and / or any combination thereof. In some embodiments, the heteroalkyl group may be deuterated, also referred to as a "deuterated heteroalkyl group," i.e., one, two, three, or even more hydrogen atoms on the heteroalkyl group may be replaced by deuterium, until all hydrogen atoms on the heteroalkyl group are replaced by deuterium, in which case it may be referred to as a "perdeuterated heteroalkyl group."

[0171] As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon group. i-j "alkyl" refers to an alkyl group having i to j carbon atoms. Unless otherwise specified, an alkyl group may contain 1 to 10 carbon atoms. In certain embodiments, the alkyl group contains 1 to 6 carbon atoms, such as 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n- and isopropyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like. As used herein, the term "alkylene" refers to a divalent substituent that is a monovalent alkyl group having one hydrogen atom replaced by a valency.

[0172] In some embodiments, an alkyl group can be deuterated, also known as a "deuterated alkyl group."

[0173] As used herein, the term "deuterated alkyl" is a substituent obtained by replacing one or more hydrogen atoms on an alkyl group with deuterium. When an alkyl group is deuterated, one, two, three, or even more hydrogen atoms on the alkyl group may be replaced by deuterium, until all hydrogen atoms on the alkyl group are replaced by deuterium, in which case it may be referred to as a "perdeuterated alkyl group." In some embodiments, non-limiting examples of deuterated alkyl groups include deuterated methyl groups, such as monodeuterated methyl groups, dideuterated methyl groups, trideuterated methyl groups (perdeuterated methyl groups), monodeuterated ethyl groups, dideuterated ethyl groups, trideuterated ethyl groups, tetradeuterated ethyl groups, pentadeuterated ethyl groups (perdeuterated ethyl groups), and the like.

[0174] Those skilled in the art will appreciate that, in some embodiments, other types of groups mentioned in this disclosure, as long as they contain hydrogen atoms, may be deuterated. When a group is deuterated, one, two, three, or even more hydrogen atoms therein may be replaced by deuterium, until all hydrogen atoms in the group are replaced by deuterium, in which case it may be referred to as a "perdeuterated group."

[0175] As used herein, term " alkenyl " refers to the straight or branched hydrocarbon radical with at least one carbon-carbon double bond, and includes the group with " cis " and " trans " orientation, or alternatively, " E " and " Z " direction.Unless otherwise indicated, alkenyl can contain 2 to 10 carbon atoms.In certain embodiments, alkenyl can contain 2 to 6 carbon atoms, for example, 2 to 5 carbon atoms, 2 to 4 carbon atoms, 2 to 3 carbon atoms.In certain embodiments, alkenyl group contains 2 carbon atoms.Non-limiting examples of alkenyl include ethylene (vinyl), propenyl, butenyl, pentenyl, 1- methyl -2- butene-1-yl, 5- hexenyl etc.

[0176] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon group having at least one carbon-carbon triple bond. Unless otherwise indicated, an alkynyl group may contain 2 to 10 carbon atoms. In certain embodiments, an alkynyl group contains 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. In certain embodiments, an alkynyl group contains 2 carbon atoms. Non-limiting examples of alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, etc.

[0177] As used herein, the term "alkoxy" refers to the group -O-alkyl, wherein alkyl has the meaning defined herein.

[0178] As used herein, the term "cycloalkyl" refers to non-aromatic, saturated monocyclic and polycyclic ring systems in which all ring atoms are carbon. Unless otherwise specified, a cycloalkyl group may contain 3 to 10 ring carbon atoms (i.e., C 3-10Cycloalkyl). In certain embodiments, the cycloalkyl group can contain 3 to 9, 3 to 8, 3 to 7, 3 to 6, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6 ring-forming carbon atoms, etc. In particular, the cycloalkyl can be monocyclic or bicyclic. Alternatively, the bicyclic cycloalkyl group can include fused, spirocyclic and bridged cycloalkyl structures.

[0179] On the other hand, also include the cycloalkyl ring that wherein 1,2 or 3 heteroatoms replace into ring carbon atom.This type of group is referred to as " heterocyclic radical " or " heterocycle ", and it refers to as defined above but with at least one heteroatom selected from N, O and S as the cycloalkyl group of ring-forming atoms.Unless otherwise indicated, heterocyclic radical group can contain 3 to 10 ring-forming atoms (i.e. 3 to 10 yuan of heterocyclic radicals).In certain embodiments, heterocyclic radical group can contain 3 to 9,3 to 8,3 to 7,3 to 6,4 to 10,4 to 9,4 to 8,4 to 7,4 to 6,4 to 5,5 to 10,5 to 9,5 to 8,5 to 7,5 to 6 ring-forming atoms etc.Especially, heterocyclic radical group can be monocycle or bicyclic.Alternatively, bicyclic heterocyclic radical group can include condensation, spirocycle and bridged heterocyclic radical structure. Non-limiting examples of heterocyclyl groups include oxiranyl, pyrrolidinyl, piperidinyl, tetrahydropyranyl, piperazinyl, pyrrolidinyl, and morpholinyl. Heterocyclyl groups can also be described by using the number of carbon atoms. For example, C 3-6 Heterocyclyl refers to a heterocyclyl group containing three to six ring-forming carbon atoms, and can also contain at least one heteroatom, such as 1, 2 or 3 heteroatoms as ring-forming atoms. In certain embodiments, the heterocyclyl group or heterocycle contains 1 or 2 heteroatoms as ring-forming atoms. In certain embodiments, the heterocyclyl group can be monocyclic or bicyclic, such as fused bicyclic and spiro bicyclic. In the context of the present disclosure, the terms "heterocyclyl" and "heterocycle" can be used interchangeably.

[0180] As used herein, the term "aryl" or "aromatic ring" refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having at least one aromatic ring. Unless otherwise indicated, an aryl group can be 6 to 10 members. In certain embodiments, an aryl group can contain 6 ring-forming carbon atoms. All atoms within a carbocyclic aryl group are carbon atoms. Non-limiting examples of aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like. In the context of the present disclosure, the terms "aryl" and "aromatic ring" can be used interchangeably.

[0181] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic ring system, or a fused or bridged bicyclic ring system, wherein the ring system contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and at least one ring is an aromatic ring. Unless otherwise specified, a heteroaryl group can be 5 to 10 members. In certain embodiments, a heteroaryl group can be 5-membered or 6-membered. In certain embodiments, a heteroaryl group can contain one, two, or three heteroatoms. In certain embodiments, a heteroaryl group can contain one or two heteroatoms. The limiting examples of heteroaryl groups include benzimidazolyl, benzofuranyl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, indolyl, isoindazolyl, isoquinolyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purinyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, quinolyl, quinolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, tetrazolyl, indolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl etc.Heteroaryl groups include at least one heteroatomic ring and at least one aromatic ring with at least one as above.For example, there is at least one heteroatomic ring can be fused to one, two or three carbocyclic rings, for example aryl ring, cyclohexane ring, cyclohexene ring, cyclopentane ring, cyclopentene ring or another monocyclic heterocycle. Non-limiting examples of fused heteroaryl groups include 1,2,3,5,8,8a-hexahydroindolizine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, 2,3-dihydrobenzothiophene, etc. In the context of the present disclosure, the terms "heteroaryl" and "heteroaromatic ring" are used interchangeably.

[0182] As used herein, the term "oxo" refers to a divalent oxygen atom and the structure of oxo may be shown as =0.

[0183] As used herein, the term "halo" or "halogen" refers to fluoride, chloride, bromide and iodide. In certain embodiments, non-limiting examples of halo include fluoride, chloride and bromide, more particularly fluoride and chloride.

[0184] As used herein, the term "heteroatom" refers to nitrogen (N), oxygen (O), and sulfur (S), and can include any oxidized forms of nitrogen and sulfur, and any quaternized forms of basic nitrogen, unless otherwise specified.

[0185] As used herein, the term "substituted" when referring to a chemical group means that the chemical group has one or more hydrogen atoms that are removed and replaced by a substituent. As used herein, the term "substituent" has its ordinary meaning as known in the art and refers to a chemical moiety that is covalently attached to a parent group or, if appropriate, fused to a parent group. It will be understood that substitution of a given atom is limited by valence. It will be understood that a substituent may be further substituted.

[0186] When it is stated in Formula (I) or any embodiment thereof that a moiety is "optionally" substituted, this means that Formula (I) or its embodiments encompasses compounds that are substituted with the indicated substituents on that moiety and compounds that do not contain the indicated substituents on that moiety (i.e., wherein the moiety is unsubstituted).

[0187] The compounds provided herein are described with reference to general formulae and specific compounds. In addition, the compounds of the present disclosure may exist in a variety of different forms or derivatives, all of which are within the scope of the present disclosure. These include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, positional isomers, prodrugs, solvated forms, different crystalline forms or polymorphs, and active metabolites.

[0188] As used herein, unless otherwise indicated, the term "pharmaceutically acceptable salt" includes salts that maintain the biological effectiveness of the free acid / alkaline form of a particular compound and are not undesirable biologically or otherwise. Pharmaceutically acceptable salts can include salts formed with inorganic bases or acids and organic bases or acids. In the case where the compounds of the present disclosure contain one or more acidic or basic groups, the disclosure also includes their corresponding pharmaceutically acceptable salts. Therefore, compounds of the present disclosure containing acidic groups (such as carboxyl groups) can exist in salt form and can be used according to the disclosure, for example, alkali metal salts, alkaline earth metal salts, aluminum salts or ammonium salts. More non-limiting examples of such salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, barium salts or salts with ammonia or organic amines (such as ethylamine, ethanolamine, diethanolamine, triethanolamine, piperidine, N-methylglutamine or amino acids). For example, by reacting a compound with an acidic group with a suitable base (e.g., lithium hydroxide, sodium hydroxide, sodium propoxide, potassium hydroxide, potassium ethoxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide), these salts are readily available. Other alkali salts of compounds of the present disclosure include, but are not limited to, copper (I), copper (II), iron (II), iron (III), manganese (II), and zinc salts. Compounds of the present disclosure contain one or more basic groups, such as groups that can be protonated, which can exist in the form of salts and can be used according to the disclosure in the form of addition salts thereof with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, sulfoacetic acid, trifluoroacetic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, carbonic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, malonic acid, maleic acid, malic acid, pamoic acid, mandelic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, taurocholic acid, glutaric acid, stearic acid, glutamic acid, or aspartic acid, as well as others known to those skilled in the art. The salt formed is especially hydrochloride, chloride, hydrobromide, bromide, iodide, sulfate, phosphate, methanesulfonate (mesylate), toluenesulfonate, carbonate, bicarbonate, formates, acetate, sulfoacetate, trifluoromethanesulfonate, oxalate, malonate, maleate, succinate, tartrate, malate, pamoate, mandelate, fumarate, lactate, citrate, glutarate, stearate, aspartate and glutamate. In addition, the stoichiometry of the salt formed by the compounds of the present disclosure can be an integer multiple or a non-integer multiple of 1.

[0189] Compounds of the present disclosure containing basic nitrogen-containing groups can be treated with reagents such as C 1-4 Quaternization of alkyl halides, for example, methyl, ethyl, isopropyl and tert-butyl chloride, bromine and iodine; di-C 1-4Alkyl sulfates, such as dimethyl sulfate, diethyl sulfate, and dipentyl sulfate; C 10-18 Alkyl halides, such as decyl, dodecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aryl C 1-4 Alkyl halides, such as benzyl chloride and phenethyl bromide.

[0190] If the compounds of the present disclosure contain both acidic and basic groups in the molecule, the disclosure also includes, in addition to the above-mentioned salt forms, inner salts or betaines (zwitterions). The corresponding salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The disclosure also includes all salts of the compounds of the present disclosure, which are not directly suitable for pharmaceutical use due to low physiological compatibility, but can be used, for example, as intermediates in chemical reactions or for the preparation of pharmaceutically acceptable salts. For a review of more suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, 2002).

[0191] The compounds of formula (I) and their pharmaceutically acceptable salts may exist in unsolvated and solvated forms. As used herein, the term "solvate" refers to a molecular complex comprising a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules. For example, the term "hydrate" is used when the solvent is water.

[0192] The compound of formula (I) can have one or more chiral (asymmetric) centers. The present disclosure encompasses all stereoisomeric forms of the compound of formula (I). The asymmetric center present in the compound of formula (I) can have (R) or (S) configuration independently of one another. When the bond of the chiral carbon is described as a straight line in the structural formula of the present disclosure, or when the compound name is described in the case of the (R) or (S) chiral name without the chiral carbon, it should be understood that the (R) and (S) configuration of each such chiral carbon and therefore each enantiomer or diastereomer and mixture thereof are all included in the formula or name. The generation of a specific stereoisomer or its mixture can be identified in the example of obtaining such stereoisomer or mixture, but this in no way limits all stereoisomers and mixtures thereof to be included in the scope of the present disclosure. When the bond of a chiral carbon is depicted as a triangular solid line or a dashed line in the structural formula of the present disclosure, or when the compound name is depicted with the (R) or (S) chiral designation of the chiral carbon, it should be understood that the compound represented by the structural formula or name has a definite stereo configuration at the chiral carbon position and is to be distinguished from other stereoisomers, enantiomers, diastereomers, or mixtures thereof.

[0193] The present disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example, mixtures of enantiomers and / or diastereomers in all ratios. Therefore, enantiomers are enantiomerically pure forms (as left-handed and right-handed enantiomers), racemic forms and two enantiomers in all ratios of the disclosed subject matter. In the case of cis / trans isomers, the present disclosure includes mixtures of all ratios of cis-form and trans-form and these forms. If necessary, single stereoisomers can be prepared by conventional methods (for example, by chromatography or crystallization, by using stereochemically uniform synthesis starting materials or by stereoselective synthesis). Optionally, derivatization can be carried out before stereoisomer separation. The separation of stereoisomer mixtures can be carried out in an intermediate step during the compound synthesis of formula (I), or can be carried out on final racemic product. Absolute stereochemistry can be determined by X-ray crystallography of crystalline products or crystalline intermediates which have been derivatized, if necessary, with reagents containing stereocenters of known configuration. Alternatively, absolute stereochemistry can be determined by vibrational circular dichroism (VCD) spectroscopy.

[0194] Unless otherwise stated, structures described in this disclosure are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Such compounds are termed "isotopic variants". This disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds of Formula (I). Examples of isotopes suitable for inclusion in the compounds of this disclosure include, but are not limited to, isotopes of hydrogen, such as 2 H (i.e., D, deuterium) and 3 H (i.e. tritium); carbon, e.g. 11 C. 13 C and 14 C; chlorine, e.g. 36 Cl; fluorine, e.g. 18 F; iodine, e.g. 123 I and 125 I; nitrogen, e.g. 13 N and 15 N; oxygen, e.g. 15 O. 17 O and 18 O; phosphorus, e.g. 32 P; and sulfur, e.g. 35 Certain isotopic variations of the compounds of formula (I), for example those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. In particular, variations in the isotopes of the compounds of formula (I) may be useful only after substitution with heavier isotopes, for example with deuterium ( 2H or D) replaces hydrogen) in the depicted structure of different compounds may provide certain therapeutic advantages, for example, due to greater metabolic stability, increased in vivo half-life or reduced dosage requirements, and therefore may be used in some specific situations. Isotopic variations of the compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and by using appropriate isotopically labeled reagents instead of the non-labeled reagents previously employed.

[0195] When describing the structures of compounds in this disclosure, the unindicated hydrogen atoms in the shown formula are D (i.e., 2 H), it is generally understood that the hydrogen at this position is a hydrogen isotope " 1 hydrogen( 1 H)" or in a form with natural isotopic abundance in its natural state. The structures shown indicate that the hydrogen atoms are D (i.e., 2 H, deuterium), it should be understood that the hydrogen at this position is a hydrogen isotope " 2 hydrogen( 2 H, D, deuterium)" or in a form in which deuterium is present at a greater isotopic abundance than natural deuterium (e.g., a deuterium abundance of greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or 100%).

[0196] Pharmaceutically acceptable solvates according to the present disclosure may include those wherein the solvent of crystallization may be isotopically substituted, for example D2O, d6-acetone, d6-DMSO.

[0197] One way to implement the present disclosure is to administer a compound of formula (I) in the form of a prodrug. Therefore, certain derivatives of a compound of formula (I) may themselves have little or no pharmacological activity, and when administered to or on the body, they are converted into a compound of formula (I) with the desired activity, for example, by hydrolytic cleavage, particularly by hydrolytic cleavage promoted by esterases or peptidases. Such derivatives are referred to as "prodrugs." More information on the use of prodrugs can be found in, for example, T. Higuchi and W. Stella, "Pro-drugs as Novel Delivery Systems", Vol. 14, ACS Symposium Series, and E. B. Roche (Ed.), "Bioreversible Carriers in Drug Design", Pergamon Press, 1987, American Pharmaceutical Association. Reference can also be made to Nature Reviews / Drug Discovery, 2008, 7, 355, and Current Opinion in Drug Discovery and Development, 2007, 10, 550.

[0198] Prodrugs according to the present disclosure can be prepared, for example, by replacing appropriate functional groups present in the compounds of formula (I) with certain moieties known to those skilled in the art, for example, the "promoieties" described in H. Bundgaard, "Design of Prodrugs", Elsevier, 1985, and YM Choi-Sledeski and CG Wermuth, "Designing Prodrugs and Bioprecursors", Practice of Medicinal Chemistry, 4th edition, Chapter 28, 657-696, Elsevier, 2015. Thus, prodrugs according to the present disclosure can include, but are not limited to, (a) ester or amide derivatives of carboxylic acids in compounds of formula (I), if any; (b) amide, imine, carbamate, or amine derivatives of amino groups in compounds of formula (I); (c) oxime or imine derivatives of carbonyl groups in compounds of formula (I), if any; or (d) methyl, primary alcohol, or aldehyde groups in compounds of formula (I), if any, that can be metabolically oxidized to carboxylic acids.

[0199] References to compounds of formula (I) include the compounds themselves and prodrugs thereof. The present disclosure includes such compounds of formula (I) as well as pharmaceutically acceptable salts of such compounds and pharmaceutically acceptable solvates of said compounds and salts.

[0200] Use and application

[0201] The compounds disclosed herein (compounds of Formula (I), stereoisomers, or isotopic variants thereof)—or pharmaceutically acceptable salts, solvates, and mixtures thereof in all ratios—can be used as pharmaceuticals. They have been found to exhibit pharmacological activity that inhibits SOAT1. Through this activity, the compounds disclosed herein can kill progenitor cells, stem cells, cancer stem cells, or cancer cells and induce anti-tumor immune responses. They can be used to treat conditions or diseases associated with SOAT1 activity; treat refractory, recurrent, or metastatic cancers; and selectively kill cancer cells using specific dosing regimens.

[0202] Therefore, the compounds of the present disclosure as SOAT1 inhibitors are particularly useful for treating diseases and conditions associated with SOAT1 activity, such as cancer, including but not limited to the following: breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumors, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

[0203] The compounds of the present disclosure can be administered in an amount effective to treat the diseases or conditions described herein. The compounds of the present disclosure can be administered as the compounds themselves, or alternatively, as pharmaceutically acceptable salts. For administration and dosing purposes, the compounds of the present disclosure themselves (compounds of formula (I), stereoisomers or isotopic variants thereof) or pharmaceutically acceptable salts, solvates thereof will be referred to simply as compounds of the present disclosure or compounds of the present disclosure.

[0204] The compounds of the present disclosure are administered by any suitable route in the form of pharmaceutical compositions suitable for such route and in a dose effective for the intended treatment.The compounds of the present disclosure can be administered orally, rectally, vaginally, parenterally or topically.

[0205] As used herein, the term "administer" refers to absorbing, ingesting, injecting, inhaling, implanting or otherwise introducing a compound of the present disclosure or its pharmaceutical composition. The term "treating" refers to reversing, alleviating, delaying the onset of a "pathological condition" (e.g., a disease, disorder or condition, or one or more signs or symptoms thereof) described in the present disclosure or inhibiting its progression. In certain embodiments, treatment may be administered after one or more signs or symptoms of the disease or condition have developed or have been observed. In other embodiments, treatment may be performed in the absence of signs or symptoms of the disease or condition. For example, susceptible individuals may be treated before the onset of symptoms (e.g., according to a history of symptoms and / or according to genetic or other susceptibility factors). Treatment may also be continued after the symptoms subside, for example, to delay or prevent recurrence. As used herein, the terms "disease," "disorder," "condition," and "pathological condition" are used interchangeably.

[0206] Those skilled in the art can determine the dosage level of administration by routine experiments. The dosage regimen of the compound of the present disclosure and / or the composition comprising the compound is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the condition; the route of administration; and the activity of the specific compound used. Therefore, the dosage regimen can be very different. For example, the dosage level of the compound of the present disclosure can be about 0.001 to about 100 mg / kg (i.e., mg / kg body weight) per day. In certain embodiments, the total daily dose of the compound of the present disclosure administered in a single or divided dose can be about 0.001 to about 10 mg / kg. It is not uncommon for the administration of the compound of the present disclosure to be repeated multiple times in one day.

[0207] In some embodiments, the compounds of the present disclosure may be used in combination with one or more other therapeutic agents. In some embodiments, non-limiting examples of such other therapeutic agents include immune checkpoint inhibitors, such as anti-PD-1 antibodies, etc. These therapeutic agents may be administered before, after, or simultaneously with the administration of the compounds of the present disclosure.

[0208] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that fully or partially reduces, inhibits, interferes with, or modulates one or more immune checkpoint proteins.

[0209] As used herein, the term "immune checkpoint protein" has its general meaning in the art, and refers to molecules expressed by T cells, wherein or open signal (stimulatory checkpoint molecules) or weaken signal (inhibitory checkpoint molecules). Immune checkpoint molecules are well known in the art, to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways (see, for example, Pardoll, 2012.Nature Rev Cancer 12: 252-264; Mellman et al., 2011.Nature 480: 480-489). The example of stimulatory checkpoints includes CD27, CD28, CD40, CD122, CD137, OX40, GITR and ICOS. The example of inhibitory checkpoint molecules includes A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. PD-1, the programmed death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. One advantage of targeting PD-1 is that it can restore immune function within the tumor microenvironment. Tumor cells often exploit these checkpoints to evade detection by the immune system. Therefore, inhibiting checkpoint proteins on the immune system can enhance anti-tumor T cell responses.

[0210] In some embodiments, an immune checkpoint inhibitor refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes both reduced function and complete blockade. In some embodiments, an immune checkpoint inhibitor can be an antibody, synthetic or natural sequence peptide, small molecule, or aptamer that binds to an immune checkpoint protein and its ligand.

[0211] In some embodiments, the immune checkpoint inhibitor is an antibody. Typically, the antibody is directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, or VISTA.

[0212] In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. Examples of commercialized anti-PD-1 antibodies include nivolumab (BMS) and pembrolizumab (also known as lambrolizumab, or MK-3475, MERCK).

[0213] Pharmaceutical composition

[0214] In some aspects, the present disclosure relates to a pharmaceutical composition comprising a compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, and at least one pharmaceutically acceptable carrier or excipient.

[0215] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier or excipient that can be used to prepare a pharmaceutical composition, which is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers or excipients that are acceptable for veterinary use as well as human pharmaceutical use. As used herein, a pharmaceutically acceptable carrier or excipient includes one or more such carriers or excipients. The specific carrier or excipient used will depend on the manner and purpose of the application of the compound of the present disclosure. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, for example, Ansel, Howard C et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. One or more of buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavorings, diluents and other known additives may also be included to provide a refined presentation of the drug (i.e., the compound or pharmaceutical composition provided by the present disclosure) or to facilitate the production of a pharmaceutical product (i.e., the drug).

[0216] The compositions of the present disclosure can be formulated into a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, suppositories, etc. The form depends on the intended mode of administration and therapeutic application.

[0217] The pharmaceutical compositions of the present disclosure can be prepared by any well-known pharmaceutical techniques (e.g., effective formulations and administration procedures). The above considerations on effective formulations and administration procedures are well known in the art and are described in standard textbooks. For example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients, 3rd ed., American Pharmaceutical Association, Washington, 1999, the formulation of drug products is discussed.

[0218] In another aspect, the present disclosure relates to a kit for treating diseases and conditions associated with SOAT1 activity, such as cancer, comprising a compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, or a pharmaceutical composition comprising a compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, a container, and optionally a package insert or label indicating treatment.

[0219] Treatment

[0220] In another aspect, the present disclosure relates to a method for treating diseases and conditions associated with SOAT1 activity, such as cancer, in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, due to the SOAT1 inhibitory activity of the compounds of the present disclosure.

[0221] As used herein, the term "subject in need thereof" refers to a subject who has a disease associated with SOAT1 activity, such as cancer, or a subject who has an increased risk of developing a disease or condition associated with SOAT1 activity relative to the general population. In certain embodiments, the subject is a warm-blooded animal. In certain embodiments, the warm-blooded animal is a mammal. In certain embodiments, the warm-blooded animal is a human.

[0222] As used herein, the term "diseases and conditions associated with SOAT1 activity" refers to any pathophysiological condition in which inhibition of SOAT1 would be beneficial. In certain embodiments, the disease and condition associated with SOAT1 activity is cancer. In certain embodiments, the disease and condition associated with SOAT1 activity is a cancer or tumor selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, and the like. In certain embodiments, the disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

[0223] In another aspect, the present disclosure relates to a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein for use in treating diseases and conditions associated with SOAT1 activity, such as cancer or tumors.

[0224] In another aspect, the present disclosure relates to the use of a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein in the preparation of a medicament for treating diseases and conditions associated with SOAT1 activity, such as cancer or tumors.

[0225] The compound of formula (I) provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof increase the expression of MHC-I and MHC-II, or compared with DMSO, the compound of formula (I) provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof enhance the infiltration and activation of T cells into tumors; or the compound of formula (I) provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof increase the expression of T cell activation markers, such as IFNγ, perforin, GzmA, GzmB and DAMPs or ER stress markers.

[0226] The compound of formula (I), its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates provided by the present disclosure increases the expression of at least one inflammatory cytokine or chemokine selected from IFNγ, IL-1β, CCL5, CXCL10, CXCL11 and CCL22.

[0227] The compound of formula (I), its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates provided by the present disclosure increases the expression of at least one inflammatory cytokine or chemokine selected from IFNγ, IL-1β, CCL5, CXCL10, CXCL11 and CCL22.

[0228] Detection and diagnosis

[0229] In another aspect, the present disclosure relates to a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein for use as a test reagent for diagnosing a disease associated with SOAT1 activity in a subject.

[0230] In another aspect, the present disclosure relates to a kit for diagnosing a disease associated with SOAT1 activity in a subject, comprising at least one compound of formula (I) described in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof as a test agent.

[0231] In some embodiments, the test agent detects at least one biomarker indicative of the presence of a disease associated with SOAT1 activity.

[0232] In some embodiments, the test reagents are capable of detecting SOAT1 activity, as well as alternative upstream / downstream regulators of SOAT1 activity or function.

[0233] Vaccines and immunization

[0234] In another aspect, the present disclosure relates to a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof as provided herein for use in the preparation of a therapeutic vaccine for inhibiting tumor progression.

[0235] In another aspect, the present disclosure relates to the use of a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof as provided by the present disclosure in the preparation of a therapeutic vaccine for inhibiting tumor development.

[0236] The tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

[0237] In another aspect, the present disclosure relates to a method for preparing a therapeutic vaccine for blocking tumor development, comprising contacting a compound of formula (I) as provided in the present disclosure, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof with tumor stem cells, thereby converting the tumor stem cells into a therapeutic vaccine.

[0238] In another aspect, the present disclosure relates to a vaccination method, comprising vaccinating a subject with a therapeutic vaccine prepared according to the method provided by the present disclosure, wherein the subject is a mammal, such as a human.

[0239] In yet another aspect, the present disclosure relates to a method for killing cells and inducing an anti-tumor immune response, the method comprising inhibiting cellular SOAT1 activity by a SOAT1 inhibitor.

[0240] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0241] In some embodiments, the SOAT1 inhibitor is selected from the group consisting of a small molecule SOAT1 inhibitor, an RNAi agent against SOAT1, an antisense agent against SOAT1, a peptidomimetic SOAT1 inhibitor, and a DNA binding deoxynucleotide inhibitor containing the target protein SOAT1.

[0242] In some embodiments, the small molecule SOAT1 inhibitor is a compound of Formula (I) as provided herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0243] synthesis

[0244] The compounds of the present disclosure can be prepared by the general and specific methods described below using the common knowledge of those skilled in the art of synthetic organic chemistry. Such common knowledge can be found in standard reference books, for example, Barton and Ollis (eds.), Comprehensive Organic Chemistry, Elsevier; Richard Larock, Comprehensive Organic Transformations: A Guide to Functional Group Preparation, John Wiley and Sons; and Compendium of Organic Synthesis Methods, Volumes I-XII, Wiley-Interscience.

[0245] The schemes described below are intended to provide a general description of the methods for preparing the compounds of the present disclosure. Some compounds of the present disclosure may contain single or multiple chiral centers with stereochemical designations (R) or (S). It will be apparent to those skilled in the art that all synthetic transformations can be performed in a similar manner, regardless of whether the material is enantiomerically enriched or racemic. In addition, the resolution of the desired optically active material can be performed at any desired point in the procedure using known methods, such as those described in the present disclosure and the chemical literature.

[0246] The present disclosure uses the following abbreviations: EtOH stands for ethanol; LCMS stands for liquid chromatography-mass spectrometry; TLC stands for thin-layer chromatography; DBU stands for 1,8-diazabicyclo[5.4.0]undec-7-ene; DDQ stands for dichlorodicyanobenzoquinone; ESI stands for electrospray ionization; NMR stands for nuclear magnetic resonance; THF stands for tetrahydrofuran; EtOAc stands for ethyl acetate; MeOH stands for methanol; TFA stands for trifluoroacetic acid; TEA stands for triethylamine; DIEA stands for diisopropylethylamine; DMSO stands for dimethyl sulfoxide; CDCl3 stands for chloroform; and SFC stands for supercritical fluid chromatography.

[0247] Example

[0248] In order to describe the present disclosure in more detail, the following examples are presented. The examples described in the present disclosure are used to illustrate the compounds, methods and compositions provided by the present disclosure and should not be interpreted in any way as limiting their scope.

[0249] During the synthesis process, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved. This can be achieved by conventional protecting groups, such as those described in TW Greene and PGM Wutts, Protective Groups in Organic Synthesis, 4th Edition, John Wiley and Sons. The protecting groups are optionally removed at a convenient subsequent stage using methods well known in the art.

[0250] The compounds of the present disclosure can be easily prepared according to the following reaction schemes and examples or their modifications, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, variants known to those skilled in the art but not mentioned in more detail can also be used. In addition, according to the reaction schemes and examples described in the present disclosure, other methods for preparing the compounds of the present disclosure will be apparent to those skilled in the art. Unless otherwise stated, all variables are as defined above. In general, in chemical procedures, all reagents and starting materials can be purchased from commercial suppliers or can be easily prepared by those skilled in the art.

[0251] Example 1

[0252] Synthesis of compound 1

[0253] Step 1: Preparation of compound 1-3

[0254] Compound 1-1 (10.0 g, 62.1 mmol, 7.75 mL, 1 eq), compound 1-2 (6.96 g, 62.1 mmol, 1 eq), and p-toluenesulfonic acid (106 mg, 620 μmol, 0.01 eq) were mixed in toluene (50.0 mL). The mixture was purged with nitrogen three times, heated to 110°C, and stirred under a nitrogen atmosphere for 4 h. TLC (petroleum ether / ethyl acetate = 1 / 1) indicated near-complete conversion of compound 1-1. The reaction mixture was cooled to 20°C, whereupon a yellow solid precipitated. The filter cake was filtered and dried to afford crude compound 1-3 (13.5 g) as a yellow solid.

[0255] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.56-7.36 (m, 4H), 6.77-6.59 (m, 1H), 5.61 (s, 1H), 2.62-2.50 (m, 2H), 2.45-2.35 (m, 2H), 2.10-2.03 (m, 2H).

[0256] Step 2: Preparation of compound 1-5

[0257] Compound 1-3 (1.00 g, 3.92 mmol, 1 eq), compound 1-4 (870 mg, 3.92 mmol, 1 eq), and DBU (596 mg, 3.92 mmol, 590 uL, 1 eq) were mixed in acetonitrile (15.0 mL), replaced with nitrogen three times, then heated to 80°C and stirred under nitrogen for 4 h. LCMS analysis revealed near-complete conversion of compound 1-3, and the target product was detected at a molecular weight. The reaction mixture was cooled to room temperature, quenched with saturated aqueous ammonium chloride (10.0 mL) and water (20.0 mL), and extracted twice with ethyl acetate (25.0 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue, which was separated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to afford compound 1-5 (1.11 g, 2.96 mmol, 75.5% yield) as a white solid. MS (ESI) m / z = 376.1 [M+H] + .

[0258] 1H NMR: (400MHz, CDCl3) δ (ppm) = 7.66-7.61 (m, 1H), 7.58-7.51 (m, 1H), 7.48-7.31 (m, 1H), 7.29-7.19 (m, 2H), 6.23-6.18 (m, 1H), 6.05 (d, J =3.0Hz,1H),4.49(d,J=6.1Hz,1H),2.97(d,J=1.8Hz,1H),2.94-2.86(m,1H),2.41-2.35(m,2H),2.11-2.00(m,2H),1.97-1.86(m,2H).

[0259] Step 3: Preparation of compound 1

[0260] Compound 1-5 (250 mg, 666 μmol, 1 eq) and DDQ (196 mg, 865 μmol, 1.3 eq) were dissolved in 1,4-dioxane (1.2 mL), replaced with nitrogen three times, and then heated to 70°C and stirred under nitrogen for 10 h. LCMS analysis showed that compound 1-5 was almost completely converted. The reaction solution was concentrated under reduced pressure to obtain a residue, which was purified by preparative high-performance liquid chromatography (column: Welch Xtimate C 18 The elution was performed using a 150 x 30 mm x 5 μm column; mobile phase: [water (NH₄HCO₃)-acetonitrile]; B%: 26%-66%, over 36 minutes to afford the trifluoroacetic acid salt of compound 1 (40.0 mg, 103 μmol, 15.4% yield) as a white solid. MS (ESI) m / z = 374.0 [M+H] + .

[0261] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.94 (s, 1H), 7.84-7.73 (m, 2H), 7.57 (s, 1H), 7.55-7.43 (m, 2H), 6.96 (t, J = 8.1H z,1H),6.60(d,J=8.1Hz,1H),6.35(dd,J=1.9,3.0Hz,1H),6.00(d,J=3.1Hz,1H),5.72(d,J=8.1Hz,1H),4.66(br d, J=6.0Hz, 1H), 3.17 (dd, J=6.4, 15.7Hz, 1H), 2.87-2.81 (m, 1H).

[0262] Example 2

[0263] Synthesis of compound 2

[0264] Step 1: Preparation of compound 2-3

[0265] Compound 2-1 (200 mg, 1.82 mmol, 1 eq), compound 2-2 (262 mg, 1.82 mmol, 1 eq), and acetic acid (5.45 mg, 90.8 μmol, 5.19 uL, 0.05 eq) were mixed in toluene (1.0 mL). The atmosphere was replaced with nitrogen three times, and the reaction mixture was heated to 105°C and stirred under a nitrogen atmosphere for 12 hours. TLC (dichloromethane / methanol = 10 / 1, UV) monitoring showed nearly complete conversion of compound 2-1. The reaction mixture was cooled to room temperature and filtered. The filter cake was suspended in MeOH and stirred at 25°C for 30 minutes. The filter cake was then dried to afford crude compound 2-3 (150 mg) as a white solid.

[0266] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.75 (br s, 1H), 7.61 (br s, 1H), 6.88 (s, 1H), 3.71 (br s, 3H), 1.51 (s, 6H).

[0267] Step 2: Preparation of compound 2

[0268] Compound 2-3 (150 mg, 635 μmol, 1 eq), compound 1-3 (162 mg, 635 μmol, 1 eq) and DBU (96.7 mg, 635 μmol, 95.7 uL, 1 eq) were mixed in acetonitrile (1.0 mL), and the atmosphere was replaced with nitrogen three times. The reaction solution was then heated to 70° C. and stirred under a nitrogen atmosphere for 24 h. LCMS monitoring showed that the conversion of compound 2-3 was substantially complete. The reaction solution was filtered, and the filtrate was concentrated to obtain a residue. The residue was purified by preparative high performance liquid chromatography (basic conditions, column: Welch Xtimate C 18 The purified product was purified by HPLC (150 x 30 mm x 5 μm; mobile phase: [water (NH₃H₂O + NH₄HCO₃)-acetonitrile]; B%: 16%-56%, 36 min) to afford compound 2 (45.8 mg, 118 μmol, 18.5% yield) as a white solid. MS (ESI) m / z = 390.2 [M+H] + .

[0269] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.86-7.57 (m, 4H), 7.48-7.32 (m, 1H), 6.98 (s, 1H), 6.72 (s, 1H) ),4.39(d,J=6.4Hz,1H),3.73(s,3H),3.06(dd,J=7.2,15.7Hz,1H),2.69-2.62(m,1H),2.27(br t,J=5.6Hz,2H),2.12-2.03(m,2H),1.93-1.77(m,2H).

[0270] Example 3

[0271] Synthesis of compound 3

[0272] Step 1: Preparation of compound 3-2

[0273] Compound 3-1 (1.00 g, 8.84 mmol, 1 eq), compound 1-2 (1.42 g, 8.84 mmol, 1.10 mL, 1 eq), and p-toluenesulfonic acid (15.2 mg, 88.4 μmol, 0.01 eq) were mixed in toluene (5.0 mL). The mixture was purged with nitrogen three times, heated to 110°C, and stirred under a nitrogen atmosphere for 12 h. LCMS analysis showed that compound 3-1 was essentially completely converted. The reaction solution was cooled to 20°C, and a solid precipitated. The solid was filtered, and the filter cake was suspended in ethyl acetate (20.0 mL), stirred at room temperature for 10 h, filtered, and dried to obtain compound 3-2 (1.20 g, 4.02 mmol, 45.4% yield) as a light yellow solid. MS (ESI) m / z = 256.9 [M+H] + .

[0274] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.41-7.35 (m, 3H), 6.99-6.83 (m, 1H), 6.56 (br s, 1H), 5.69 (br s,1H),5.31(s,1H),3.51-3.44(m,2H),2.61-2.56(m,2H).

[0275] Step 2: Preparation of compound 3

[0276] Compound 3-2 (200 mg, 669 μmol, 85.8% purity, 1 eq), compound 1-4 (148 mg, 669 μmol, 1 eq), and DBU (101 mg, 669 μmol, 100 μL, 1 eq) were mixed in acetonitrile (1.0 mL). The atmosphere was replaced with nitrogen three times, and the reaction mixture was heated to 80°C and stirred under nitrogen for 5 h. LCMS monitoring showed that compound 3-2 was essentially completely converted, with the main peak being the target product. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was dissolved in ethyl acetate (20 mL), washed with saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Compound 3 (35.0 mg, 89.7 μmol, 13.4% yield) was isolated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 1 / 1 to 3 / 1) to obtain compound 3 (35.0 mg, 89.7 μmol, 13.4% yield) as a white solid. MS (ESI) m / z = 377.2 [M+H] + .

[0277] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.83-7.67 (m, 3H), 7.57 (d, J = 0.8Hz, 1H), 7.54-7.40 (m, 1H), 7.36 (br s, 1H), 6.38-6.35 (m, 1H), 6.25 (br s,1H),4.27(br d,J=6.4Hz,1H),3.25-3.18(m,2H),3.14(dd,J=7.2,15.8Hz,1H),2.75(d,J=14.8Hz,1H),2.25-1.98(m,2H).

[0278] Example 4

[0279] Synthesis of compound 4

[0280] Step 1: Preparation of compound 4-2

[0281] Compound 4-1 (500 mg, 3.93 mmol, 1 eq), compound 1-2 (507 mg, 3.15 mmol, 393 uL, 0.80 eq), and p-toluenesulfonic acid (6.77 mg, 39.3 μmol, 0.01 eq) were mixed in toluene (5.0 mL). The mixture was purged with nitrogen three times, heated to 50°C, and stirred under a nitrogen atmosphere for 12 h. LCMS analysis showed that compound 4-1 was nearly completely converted. The reaction solution was cooled to 20°C, whereupon solid precipitated. The solid was filtered and the filter cake dried to afford crude compound 4-2 (1.0 g) as a light brown solid. MS (ESI) m / z = 270.9 [M+H] + .

[0282] Step 2: Preparation of compound 4

[0283] Compound 4-2 (1.00 g, 3.70 mmol, 1 eq), compound 1-4 (822 mg, 3.70 mmol, 1 eq), and DBU (563 mg, 3.70 mmol, 558 uL, 1 eq) were mixed in acetonitrile (10.0 mL). The atmosphere was purged with nitrogen three times, and the reaction mixture was heated to 80°C and stirred under nitrogen for 2 h. LCMS analysis showed near-complete conversion of compound 4-2, with the main peak representing the molecular weight of the desired product. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue, which was then separated and purified by preparative HPLC (column: Xtimate C18 150*40 mm*10 μm; mobile phase: [water (NH4HCO3)-acetonitrile]; B%: 20%-60%; over 36 minutes) to afford compound 4 (100 mg, 248 μmol, 6.72% yield) as a pale yellow solid. MS (ESI) m / z = 390.9 [M+H] + .

[0284] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.71-7.65 (m, 1H), 7.64-7.58 (m, 1H), 7.53-7.36 (m, 1H), 7.32 (s, 1H), 7.27-7.26 (m, 1H), 6.29 (br d,J=1.8Hz,1H),6.22-6.15(m,1H),4.53(t,J=4.3Hz,1H),3.44-3.28(m,2H),3.04(d,J=4.6Hz,2H),3.00-2.97(m,3H),2.30-2.12(m,2H).

[0285] Example 5

[0286] Synthesis of compound 5

[0287] Step 1: Preparation of compound 5

[0288] Compound 2-3 (47.0 g, 199 mmol, 1.14 eq) and compound 4-2 (47.0 g, 174 mmol, 1 eq) were dissolved in acetonitrile (380 mL), and DBU (79.4 g, 522 mmol, 78.6 mL, 3 eq) was added. The mixture was heated to 80°C and stirred for 12 h. LCMS analysis showed that compound 4-2 was nearly completely converted. The reaction solution was cooled to room temperature, quenched with water, and extracted three times with ethyl acetate (300 mL). The organic phases were combined and concentrated under reduced pressure to obtain the crude product. Compound 5 (40.0 g, 89.0 mmol, 51.2%) was isolated and purified by column chromatography (silica, DCM / MeOH = 1 / 0 to 97 / 3) to obtain compound 5 (40.0 g, 89.0 mmol, 51.2%) as a yellow solid. MS (ESI) m / z = 405.1 [M+H] + .

[0289] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.85-7.42 (m, 4H), 6.92 (d, J = 0.9Hz, 1H), 6.76 (d, J = 1.0Hz, 1H), 4.46 (br d,J=5.5Hz,1H),3.81(s,3H),3.41-3.30(m,2H),3.11-3.01(m,1H),2.98-2.91(m,4H),2.30-2.15(m,2H).

[0290] Example 6

[0291] Synthesis of Compound 5-P1 and Compound 5-P2

[0292] Step 1: Preparation of Compound 5-P1 and Compound 5-P2

[0293] Compound 5 (40.2 g, 98.9 mmol) was separated and purified by SFC (chromatographic column: DAICEL CHIRALPAK AD (250 mm*50 mm, 10 um); mobile phase: [CO2-EtOH (0.1% NH3H2O)]; B%: 35%, constant composition solvent elution method) to obtain compound 5-P1 (11.0 g, 27.0 mmol, 27.3% yield) as a white solid; and compound 5-P2 (10.1 g, 24.8 mmol, 25.1% yield) as a white solid.

[0294] Compound 5-P1 (ee value 99.9%): MS (ESI) m / z = 405.2 [M+H] + .

[0295] 1H NMR: (400MHz, CDCl3) δ (ppm) = 7.96-7.34 (m, 4H), 6.93 (d, J = 0.8Hz, 1H), 6.76 (d, J = 1.0Hz, 1H), 4.46 (br d,J=5.8Hz,1H),3.81(s,3H),3.45-3.25(m,2H),3.10-3.02(m,1H),2.99-2.86(m,4H),2.32-2.13(m,2H).

[0296] Compound 5-P2 (ee value 99.8%): MS (ESI) m / z = 405.3 [M+H] + .

[0297] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.89-7.38 (m, 4H), 6.93 (s, 1H), 6.76 (d, J = 1.0Hz, 1H), 4.46 (br d,J=6.0Hz,1H),3.81(s,3H),3.45-3.27(m,2H),3.10-3.01(m,1H),2.98-2.85(m,4H),2.33-2.12(m,2H).

[0298] Example 7

[0299] Synthesis of compound 6

[0300] Step 1: Preparation of compound 6

[0301] Compound 5 (400 mg, 989 μmol, 1 eq) was dissolved in a mixture of acetonitrile (10.0 mL) and water (5.0 mL). Potassium persulfate (214 mg, 791 μmol, 158 μL, 0.8 eq) was added, and the reaction mixture was heated to 85°C and stirred for 4 h. LCMS analysis showed that compound 5 was essentially completely converted. The reaction mixture was cooled to room temperature and extracted three times with ethyl acetate (20.0 mL). The organic phases were combined and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (column: Xtimate C18 150*40 mm*10 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 6.0%-46.0% B over 36 min) to obtain compound 6 (30.5 mg, 73.5 μmol, 7.43% yield) as a white solid. MS (ESI) m / z = 402.9 [M+H] + .

[0302] 1H NMR: (400MHz, CDCl3) δ (ppm) = 7.86-7.80 (m, 1H), 7.79-7.73 (m, 1H), 7.56 (s, 1H), 7.50 (br d,J=8.0Hz,1H),7.14(d,J=1.0Hz,1H),7.01(d,J=1.0Hz,1H),6.65(s,1H),3.63(s,3H),3.49(br t,J=13.8Hz,2H),2.99(s,3H),2.75-2.39(m,2H).

[0303] Example 8

[0304] Synthesis of compound 7

[0305] Step 1: Preparation of compound 7-2

[0306] Compound 7-1 (2.00 g, 18.1 mmol, 1.00 eq) and compound 2-2 (2.62 g, 18.1 mmol, 1.00 eq) were dissolved in 2-methyltetrahydrofuran (20 mL). Acetic acid (54.5 mg, 908 μmol, 51.9 μL, 0.05 eq) was added, and the mixture was heated to 60°C and stirred for 12 h. TLC (dichloromethane / methanol = 10 / 1, UV = 254 nm) showed approximately 30% of compound 7-1 remaining, with the formation of a major new, highly polar spot. The reaction mixture was concentrated, and the residue was treated with tert-butyl methyl ether (20 mL), stirred at 25°C for 1 h, filtered, and the filter cake dried to yield compound 7-2 (2.3 g, 8.28 mmol, 45.5% yield, 85% purity) as a white solid. MS (ESI) m / z = 236 [M+H] + .

[0307] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.24 (s, 1H), 7.68-7.64 (m, 2H), 4.11 (s, 3H), 1.73 (s, 6H).

[0308] Step 2: Preparation of compound 7

[0309] Compound 7-2 (300 mg, 1.27 mmol, 1.00 eq) and compound 4-2 (343 mg, 1.27 mmol, 1.00 eq) were dissolved in acetonitrile (5 mL). DBU (580 mg, 3.81 mmol, 574 μL, 3.00 eq) was added, and the mixture was heated to 80°C and stirred for 4 h. LCMS analysis showed nearly complete conversion of compound 4-2, with the main peak corresponding to the molecular weight of the desired product. The reaction mixture was cooled to room temperature, quenched with water (10 mL), and extracted with ethyl acetate (20 mL). The organic phase was washed twice with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by preparative HPLC (column: Xtimate C18 40*200mm 7um; mobile phase: [water (FA)-acetonitrile]; gradient: 12%-52% B over 25 minutes) to obtain compound 7 (82 mg, 194 μmol, 15.7% yield) as a white solid. MS (ESI) m / z = 404 [M+H] + .

[0310] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.74-7.69 (m, 1H), 7.67-7.61 (m, 1H), 7.58-7.30 (m, 3H), 6.07 (s, 1H), 4.56 (br d,J=7.2Hz,1H),4.03(s,3H),3.37(t,J=7.2Hz,2H),3.11(dd,J=7.2,16.0Hz,1H),2.98(s,3H),2.91(d,J=16.0Hz,1H),2.25(br t,J=6.8Hz,2H).

[0311] Example 9

[0312] Synthesis of compound 8

[0313] Step 1: Preparation of compound 8-2

[0314] Compound 8-1 (1.50 g, 12.1 mmol, 1.00 eq), compound 2-2 (1.74 g, 12.1 mmol, 1.00 eq), and acetic acid (36.3 mg, 604 μmol, 34.6 μL, 0.05 eq) were mixed in toluene (20 mL). The reaction mixture was heated to 110°C and stirred for 12 h. LCMS analysis showed that the starting material 8-1 was almost completely converted. The reaction mixture was cooled to room temperature and filtered. The filter cake was suspended in methanol (15 mL), stirred at 25°C for 30 minutes, filtered, and dried to obtain compound 8-2 (2.00 g, 7.99 mmol, 66.1% yield) as an off-white solid. MS (ESI) m / z = 250.6 [M+H] + .

[0315] Step 2: Preparation of compound 8

[0316] Compound 8-2 (300 mg, 1.20 mmol, 1.00 eq) was dissolved in acetonitrile (10 mL), and DBU (547 mg, 3.60 mmol, 542 μL, 3.00 eq) and intermediate 4-2 (301 mg, 1.11 mmol, 0.93 eq) were added. The reaction solution was heated to 80° C. and stirred for 12 h. LCMS analysis showed that the starting material 8-2 was essentially completely converted. The reaction solution was cooled to room temperature, quenched with water (10 mL), and extracted three times with ethyl acetate (20 mL). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to obtain a residue. This residue was purified by preparative HPLC (column: Xtimate C18 40*200mm 7um; mobile phase: [water (HCl)-acetonitrile]; gradient: 0%-34% B over 20 minutes) to obtain compound 8 (62.0 mg, 146 μmol, 12.2% yield) as a yellow solid. MS (ESI) m / z = 418.7 [M+H] + .

[0317] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 8.38 (br s, 1H), 7.69 (br s, 2H), 7.42-7.31 (m, 2H), 7.08 (br s, 1H), 4.72 (br s, 1H), 4.23 (br d,J=6.0Hz,2H),4.10-3.92(m,1H),3.50-3.04(m,3H),2.91(s,3H),2.67-2.48(m,1H),2.35-2.20(m,1H),1.64(br s,3H).

[0318] Example 10

[0319] Synthesis of compound 9

[0320] Step 1: Preparation of compound 9-2

[0321] Compound 9-1 (2.00 g, 10.6 mmol, 1.00 eq) and compound 2-2 (1.53 g, 10.6 mmol, 1.00 eq) were dissolved in toluene (5.0 mL). Pyridine (41.9 mg, 529 μmol, 42.7 μL, 0.05 eq) and acetic acid (31.8 mg, 529 μmol, 30.3 μL, 0.05 eq) were added. The reaction mixture was heated to 60°C and stirred for 12 h. LCMS analysis showed that compound 9-1 was almost completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction mixture was filtered under reduced pressure, and the filter cake was collected to obtain crude compound 9-2 (2.5 g) as a brown solid. MS (ESI) m / z = 316.3 [M+H] + .

[0322] Step 2: Preparation of compound 9

[0323] Compound 9-2 (1.50 g, 4.76 mmol, 1.00 eq) was dissolved in acetonitrile (15 mL), and DBU (2.17 g, 14.3 mmol, 2.15 mL, 3.00 eq) and intermediate 4-2 (1.03 g, 3.81 mmol, 0.80 eq) were added. The reaction solution was heated to 80°C and stirred for 12 h. LCMS analysis showed that starting material 9-2 was essentially completely converted. The reaction solution was cooled to room temperature, quenched with water (30 mL), and extracted three times with ethyl acetate (30 mL). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to obtain a residue. This residue was purified by preparative HPLC (column: Xtimate C18 40*200mm 7um; mobile phase: [water (FA)-acetonitrile]; gradient: 10%-55% B over 20 minutes) to obtain compound 9 (350 mg, 696 μmol, 15.2% yield) as a light yellow solid. MS (ESI) m / z = 482.8 [M+H] + .

[0324] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.94-7.47 (m, 4H), 6.71 (s, 1H), 4.39 (br d,J=6.6Hz,1H),3.78(s,3H),3.51-3.26(m,2H),3.05(dd,J=6.9,15.9Hz,1H),2.94(s,3H),2.91-2.83(m,1H),2.33-2.12(m,2H).

[0325] Example 11

[0326] Synthesis of compound 10

[0327] Step 1: Preparation of compound 10-2

[0328] Compound 10-1 (90.0 g, 559 mmol, 1.00 eq) was dissolved in acetonitrile (1000 mL). Paraformaldehyde (50.3 g, 1.68 mol, 3.00 eq) was added under a nitrogen atmosphere. The reaction mixture was evacuated and replaced with nitrogen three times. The reaction mixture was heated to 130°C and stirred for 16 h. LCMS analysis showed that compound 10-1 was essentially completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction mixture was filtered under reduced pressure, and the filtrate was concentrated to obtain the crude product. The product was suspended in methyl tert-butyl ether (100 mL), stirred at 25°C for 1 h, and filtered under reduced pressure. The filter cake was collected to obtain compound 10-2 (80.0 g, 397 mmol, 35.5% yield) as a white solid. MS (ESI) m / z = 190 [M+H] + .

[0329] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 6.86 (s, 1H), 4.70 (br d, J = 4.4Hz, 1H), 4.62 (s, 2H), 3.67 (s, 3H).

[0330] Step 2: Preparation of compound 10-3

[0331] Compound 10-2 (5.00 g, 26.1 mmol, 1.00 eq) was dissolved in dichloromethane (50 mL), and Dess-Martin oxidant (11.1 g, 26.1 mmol, 8.11 mL, 1.00 eq) was added. The reaction solution was stirred at 25 ° C for 1 h. TLC (dichloromethane / methanol = 10 / 1, UV = 254 nm) showed that compound 10-2 was almost completely converted and a major new point was generated. The reaction solution was filtered and the filtrate was concentrated to obtain a residue, which was subjected to flash column chromatography ( 20g Silica Flash Column, 0-1% DCM / MeOH gradient elution @ 80 mL / min) was used for separation and purification to obtain compound 10-3 (4.90 g, 20.7 mmol, 79.2% yield) as a white solid.

[0332] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.56 (s, 1H), 7.44 (s, 1H), 3.90 (s, 3H).

[0333] Step 3: Preparation of compound 10-4

[0334] Compound 10-3 (200 mg, 1.06 mmol, 1.00 eq) and compound 2-2 (152 mg, 1.06 mmol, 1.00 eq) were dissolved in toluene (3.0 mL). Pyridine (4.18 mg, 52.9 μmol, 4.27 μL, 0.05 eq) and acetic acid (3.18 mg, 52.9 μmol, 3.03 μL, 0.05 eq) were added. The reaction mixture was heated to 60°C and stirred for 12 h. LCMS analysis showed that compound 10-3 was essentially completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction mixture was concentrated under reduced pressure, and the crude product was suspended in methyl tert-butyl ether (10 mL), stirred at 25°C for 30 min, and filtered to obtain compound 10-4 (320 mg, 812 μmol, 76.7% yield) as a yellow solid. MS (ESI) m / z = 314 [M+H] + .

[0335] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.59 (s, 1H), 6.96 (s, 1H), 3.69 (s, 3H), 1.59 (s, 6H).

[0336] Step 4: Preparation of compound 10

[0337] Compound 10-4 (230 mg, 729 μmol, 1.00 eq) and intermediate 4-2 (197 mg, 729 μmol, 1.00 eq) were dissolved in dioxane (10 mL), and DBU (333 mg, 2.19 mmol, 330 μL, 3.00 eq) was added. The reaction solution was heated to 80° C. and stirred for 5 h. LCMS analysis showed that the starting material 10-4 was essentially completely converted. The reaction solution was cooled to room temperature, quenched with water (10 mL), and extracted with ethyl acetate (20 mL). The organic phase was washed twice with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was purified by preparative HPLC (column: Xtimate C18 40*200mm 7um; mobile phase: [water (FA)-acetonitrile]; gradient: 0%-40% B over 20 minutes) to obtain compound 10 (120 mg, 246 μmol, 33.8% yield) as a light yellow solid. MS (ESI) m / z = 484 [M+H] + .

[0338] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.84 (br d,J=4.0Hz,1H),7.75-7.69(m,1H),7.67-7.53(m,2H),7.42-7.29(m,1H),4.79-4.66(m,1H),3.83(s,3H),3. 67-3.57(m,1H),3.37-3.04(m,3H),2.93(s,3H),2.48(td,J=6.0,17.2Hz,1H),2.26(td,J=8.0,17.2Hz,1H).

[0339] Example 12

[0340] Synthesis of compound 11

[0341] Step 1: Preparation of compound 11-2

[0342] Compound 11-1 (10.0 g, 85.8 mmol, 1.00 eq) and formaldehyde (20.9 g, 257 mmol, 19.2 mL, 37% purity, 3.00 eq) were dissolved in DMSO (100 mL). The reaction solution was evacuated and replaced with nitrogen three times. The temperature was raised to 120 ° C and stirred in a nitrogen atmosphere for 16 h. LCMS detection showed that compound 11-1 was almost completely converted, and the main peak corresponded to the molecular weight of the target product. Water (20 The reaction was quenched with 1% paraformaldehyde (0 mL), extracted three times with ethyl acetate (200 mL), and the organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue, which was separated and purified by column chromatography (silica, dichloromethane / methanol = 100 / 0 to 95 / 5) to obtain compound 11-2 (4.5 g, 30.7 mmol, 17.9% yield) as a white solid. MS (ESI) m / z = 146.9 [M+H] + .

[0343] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 6.77 (s, 1H), 5.40-5.17 (m, 1H), 4.58 (s, 2H), 3.64 (s, 3H).

[0344] Step 2: Preparation of compound 11-3

[0345] Compound 11-2 (4.5 g, 30.7 mmol, 1.00 eq) was dissolved in dichloromethane (45 mL), and manganese dioxide (26.7 g, 307 mmol, 10.0 eq) was added. The reaction mixture was stirred at 20°C for 12 h. LCMS analysis showed that compound 11-2 was essentially completely converted and the target molecular weight was found. The reaction mixture was filtered, and the filtrate was concentrated to obtain crude compound 11-3 (1.20 g, 8.30 mmol, 27.0% yield) as an off-white solid. MS (ESI) m / z = 145.1 [M+H] + .

[0346] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 9.68 (s, 1H), 7.22 (s, 1H), 3.96 (s, 3H).

[0347] Step 3: Preparation of compound 11-4

[0348] Compound 11-3 (200 mg, 1.38 mmol, 1.00 eq) and compound 2-2 (199.40 mg, 1.38 mmol, 1.00 eq) were dissolved in toluene (5.0 mL). Pyridine (5.47 mg, 69.2 μmol, 5.58 μL, 0.05 eq) and acetic acid (4.15 mg, 69.2 μmol, 3.96 μL, 0.05 eq) were added. The reaction mixture was heated to 60°C and stirred for 12 h. LCMS analysis showed that compound 11-3 was almost completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction mixture was filtered under reduced pressure, the filter cake collected, and suspended in methyl tert-butyl ether (5 mL). Stirred at 25°C for 30 minutes, filtered, and the filter cake collected to obtain compound 11-4 (280 mg, 1.03 mmol, 74.8% yield) as an off-white solid. MS (ESI) m / z = 270.5 [M+H] + .

[0349] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.56 (s, 1H), 6.96 (s, 1H), 3.70 (s, 3H), 1.60 (s, 6H).

[0350] Step 4: Preparation of compound 11

[0351] Compound 11-4 (280 mg, 1.03 mmol, 1.00 eq) and intermediate 4-2 (279 mg, 1.03 mmol, 1.00 eq) were dissolved in acetonitrile (5 mL), and DBU (472 mg, 3.10 mmol, 467 μL, 3.00 eq) was added. The reaction solution was heated to 80°C and stirred for 12 h. LCMS analysis showed that the raw material 11-4 was almost completely converted. The reaction solution was cooled to room temperature, quenched with water (10 mL), and extracted three times with ethyl acetate (20 mL). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue, which was purified by preparative high performance liquid chromatography (chromatographic column: Xtimate C18 40*200mm 7um; mobile phase: [water (FA)-acetonitrile]; gradient: 8%-48% B, 20 minutes) to obtain compound 11 (245 mg, 483 μmol, 46.7% yield) as a pale off-white solid. MS (ESI) m / z = 438.7 [M+H] + .

[0352] 1H NMR: (400MHz, CDCl3) δ (ppm) = 7.75-7.46 (m, 4H), 6.85 (s, 1H), 4.43 (d, J = 6.0Hz, 1H), 3.73 (s, 3H),3.44-3.28(m,2H),3.13-3.00(m,1H),2.96(s,3H),2.94-2.88(m,1H),2.34-2.13(m,2H).

[0353] Example 13

[0354] Synthesis of Compound 12 and Compound 13

[0355] Step 1: Preparation of Compounds 12 and 13

[0356] Compound 7 was separated and purified by supercritical fluid chromatography (SFC) (chromatographic column: ChiralPak IC, 250×30 mm ID, 5 μm; mobile phase: phase A is CO2, phase B is MeOH + 0.1% NH3.H2O; elution gradient: B 30%; flow rate: 60 mL / min) to obtain compound 12 (35.1% yield) and compound 13 (36.9% yield), both of which were white solids.

[0357] Compound 12 (ee value 100%): MS (ESI) m / z = 405.4 [M+H] + .

[0358] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.70 (d, J = 7.8Hz, 1H), 7.66-7.59 (m, 1H), 7.58-7.26 (m, 3H), 6.06 (d, J = 1.9Hz, 1H), 4.55 (d ,J=6.9Hz,1H),4.01(s,3H),3.36(t,J=7.1Hz,2H),3.16-3.03(m,1H),2.97(s,3H),2.99-2.77(m,1H),2.37-2.14(m,2H).

[0359] Compound 13 (ee value 100%): MS (ESI) m / z = 405.4 [M+H] + .

[0360] 1H NMR: (400MHz, CDCl3) δ (ppm) = 7.70 (d, J = 7.9Hz, 1H), 7.66-7.60 (m, 1H), 7.59-7.26 (m, 3H), 6.06 (d, J = 1.8Hz, 1H), 4.55 (d ,J=7.0Hz,1H),4.01(s,3H),3.36(t,J=7.1Hz,2H),3.15-3.04(m,1H),2.97(s,3H),2.99-2.77(m,1H),2.35-2.15(m,2H).

[0361] Example 14

[0362] Synthesis of compound 14

[0363] Step 1: Preparation of compound 14-2

[0364] Compound 2-2 (1.0 g, 6.94 mmol, 1.00 eq) was dissolved in EtOH (10 mL). Compound 14-1 (0.76 g, 6.94 mmol, 1.00 eq) and piperidine (0.76 g, 6.94 mmol, 1.00 eq) were added at room temperature. The reaction mixture was placed under a nitrogen atmosphere and heated to 70°C with stirring for 5 hours. The reaction mixture was cooled to room temperature and filtered under reduced pressure. The filter cake was collected and rinsed with ethanol. The filter cake was dried to obtain compound 14-2 (0.95 g, 4.02 mmol, 57.9% yield) as a yellow solid. MS (ESI) m / z = 438.7 [M+H] + .

[0365] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.75-7.46 (m, 4H), 6.85 (s, 1H), 4.43 (d, J = 6.0Hz, 1H), 3.73 (s, 3H),3.44-3.28(m,2H),3.13-3.00(m,1H),2.96(s,3H),2.94-2.88(m,1H),2.34-2.13(m,2H).

[0366] Step 2: Preparation of compound 14

[0367] Compound 4-2 (215 mg, 0.80 mmol, 1.0 eq) was dissolved in ethanol (5 mL), and compound 14-2 (189 mg, 0.80 mmol, 1.0 eq) and piperidine (68.0 mg, 0.80 mmol, 1.0 eq) were added at room temperature. The reaction mixture was heated to 80°C under nitrogen and stirred for 6 hours. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: Welch-Ultimate AQ-C18, 21.2*250 mm, 5 μm; mobile phase: 20%-95% MeCN in 0.1% FA in water) to obtain compound 14 (63.1 mg, 0.156 mmol, 19.5% yield) as a white solid. MS (ESI) m / z = 405 [M+H] + .

[0368] 1 H NMR: (400MHz, CD3OD) δ (ppm) = 7.77-7.64 (m, 2H), 7.64-7.53 (m, 1H), 7.53-7.36 (m, 2H), 6.17 (d, J = 2.2Hz, 1H), 4.33 (d, J = 6.7Hz, 1H), 3.8 2(s,3H),3.55-3.43(m,1H),3.43-3.33(m,1H),3.20-3.07(m,1H),2.97(s,3H),2.95-2.86(m,1H),2.40-2.27(m,1H),2.24-2.12(m,1H).

[0369] Example 15

[0370] Synthesis of compound 15

[0371] Step 1: Preparation of compound 15-2

[0372] Compound 15-1 (39.5 g, 0.31 mol, 1.0 eq) was dissolved in DMF (400 mL). NaH (24.9 g, 0.62 mol, 60% pure dispersion in mineral oil, 2.0 eq) was added portionwise at 0°C. The mixture was stirred at 0°C for 30 minutes. Methyl iodide (66.0 g, 0.47 mol, 2.0 eq) was then added at the same temperature. The final reaction solution was slowly warmed to room temperature and stirred for 16 hours. The reaction was quenched with saturated aqueous ammonium chloride and extracted twice with ethyl acetate. The organic phases were combined, washed with saturated ammonium chloride, dried over anhydrous sodium sulfate, filtered under reduced pressure, and the filtrate was concentrated to a residue. The residue was isolated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 8 / 2 to 1 / 1) to obtain compound 15-2 (35.5 g, 0.25 mol, 81.2% yield) as a yellow oil. MS (ESI) m / z = 142 [M+H] + .

[0373] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 5.03 (s, 1H), 3.60 (s, 3H), 3.33 (t, J = 7.2Hz, 2H), 2.89 (s, 3H), 2.41 (t, J = 7.2Hz, 2H).

[0374] Step 2: Preparation of compound 15-4

[0375] Compound 15-2 (200 mg, 1.42 mmol, 1.0 eq) was dissolved in toluene (2 mL). 4-Trifluoromethylaniline (180 mg, 1.42 mmol, 1.0 eq) and p-toluenesulfonic acid (48.8 mg, 0.28 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 100°C under nitrogen and stirred for 6 hours. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 15-4 (225 mg, 0.833 mmol, 58.7% yield) as a white solid. MS (ESI) m / z = 271 [M+H] + .

[0376] Step 3: Preparation of compound 15

[0377] Compound 15-4 (225 mg, 0.83 mmol, 1.0 eq) was dissolved in toluene (5 mL). Compound 7-2 (197 mg, 0.83 mmol, 1.0 eq) and piperidine (70.6 mg, 0.83 mmol, 1.0 eq) were added at room temperature. The reaction mixture was heated to 80°C under nitrogen and stirred for 6 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: Welch XBridge C18 250*21 mm; mobile phase: 50%-95% MeCN in 0.1% FA in water) to obtain compound 15 (50.9 mg, 0.126 mmol, 15.2% yield) as a white solid. MS (ESI) m / z = 405 [M+H] + .

[0378] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.76 (d, J = 8.4Hz, 2H), 7.37 (d, J = 1.4Hz, 1H), 7.36-7.26 (m, 2H), 6.05 (d, J = 1.5Hz, 1H), 4.55 (d, J =6.9Hz,1H),4.01(s,3H),3.35(t,J=7.1Hz,2H),3.16-3.04(m,1H),2.97(s,3H),2.90(d,J=15.0Hz,1H),2.25(t,J=7.1Hz,2H).

[0379] Example 16

[0380] Synthesis of compound 16

[0381] Step 1: Preparation of compound 16-2

[0382] Compound 15-2 (200 mg, 1.42 mmol, 1.0 eq) was dissolved in toluene (2 mL). 2-Trifluoromethylaniline (180 mg, 1.42 mmol, 1.0 eq) and p-toluenesulfonic acid (48.8 mg, 0.28 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 100°C under nitrogen and stirred for 6 h. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 16-2 (230 mg, 0.851 mmol, 60.1% yield) as a white solid. MS (ESI) m / z = 271 [M+H] + .

[0383] Step 2: Preparation of compound 16

[0384] Compound 16-2 (230 mg, 0.85 mmol, 1.0 eq) and compound 7-2 (201 mg, 0.85 mmol, 1.0 eq) were dissolved in acetonitrile (5 mL). DBU (129 mg, 0.85 mmol, 1.0 eq) was added at room temperature. The reaction mixture was heated to 80°C under nitrogen and stirred for 16 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was purified by column chromatography (silica, dichloromethane / methanol = 1 / 0 to 95 / 5). The residue was then suspended and stirred in methyl tert-butyl ether (10 mL) and filtered under reduced pressure. The filter cake was collected and dried under vacuum to yield compound 16 (51.8 mg, 0.128 mmol, 15.1% yield) as a white solid. MS (ESI) m / z = 405 [M+H] + .

[0385] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.82 (d, J = 7.7Hz, 1H), 7.72 (t, J = 7.6Hz, 1H), 7.61 (t, J = 7.6Hz, 1H), 7.38 (s, 1H), 7.26-7.25 (m, 1H), 6.07 (s, 1H), 4. 55(d,J=7.3Hz,1H),4.01(s,3H),3.42-3.23(m,2H),3.16-3.00(m,1H),2 .95(s,3H),2.84(d,J=15.6Hz,1H),2.36-2.19(m,1H),2.18-2.03(m,1H).

[0386] Example 17

[0387] Synthesis of compound 17

[0388] Step 1: Preparation of compound 17-2

[0389] Compound 15-2 (200 mg, 1.42 mmol, 1.0 eq) was dissolved in toluene (2 mL). m-Cyananiline (168 mg, 1.42 mmol, 1.0 eq) and p-toluenesulfonic acid (48.8 mg, 0.28 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 100°C under nitrogen and stirred for 6 h. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 17-2 (230 mg, 0.636 mmol, 71.3% yield) as a white solid. MS (ESI) m / z = 228 [M+H] + .

[0390] Step 2: Preparation of compound 17

[0391] Compound 17-2 (225 mg, 0.99 mmol, 1.0 eq) was dissolved in toluene (5 mL). Compound 7-2 (234 mg, 0.99 mmol, 1.0 eq) and piperidine (84.2 mg, 0.99 mmol, 1.0 eq) were added at room temperature. The reaction mixture was heated to 80°C under nitrogen and stirred for 6 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: Welch-Ultimate AQ-C18, 21.2*250 mm, 5 μm, mobile phase: 10%-95% MeCN in 0.1% FA in water) to obtain compound 17 (59.9 mg, 0.166 mmol, 16.7% yield) as a white solid. MS (ESI) m / z = 362.2 [M+H] + .

[0392] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.73 (d, J = 7.7Hz, 1H), 7.62 (t, J = 7.9Hz, 1H), 7.58-7.28 (m, 3H), 6.03 (d, J = 1.9Hz, 1H), 4.56 ( d,J=6.8Hz,1H),4.00(s,3H),3.37(t,J=7.1Hz,2H),3.14-3.05(m,1H),2.98(s,3H),2.94-2.86(m,1H),2.32-2.16(m,2H).

[0393] Example 18

[0394] Synthesis of compound 18

[0395] Step 1: Preparation of compound 18-2

[0396] Compound 15-2 (200 mg, 1.42 mmol, 1.0 eq) was dissolved in toluene (2 mL). m-Chloroaniline (180 mg, 1.42 mmol, 1.0 eq) and p-toluenesulfonic acid (48.8 mg, 0.28 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 100°C under nitrogen and stirred for 6 h. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 18-2 (195 mg, 0.824 mmol, 58.0% yield) as a white solid. MS (ESI) m / z = 237.1 [M+H] + .

[0397] Step 2: Preparation of compound 18

[0398] Compound 18-2 (195 mg, 0.83 mmol, 1.0 eq) was dissolved in toluene (5 mL), and compound 7-2 (196 mg, 0.83 mmol, 1.0 eq) and piperidine (84.2 mg, 0.99 mmol, 1.2 eq) were added at room temperature. The reaction mixture was heated to 80°C under nitrogen and stirred for 6 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: YMC-Actus Triart C18 250*20mm S-5um, 12nm, mobile phase: 30%-95% MeCN in 0.1% NH4HCO3 in water) to obtain compound 18 (48.5 mg, 0.131 mmol, 15.8% yield) as a white solid. MS (ESI) m / z = 371.0 [M+H] + .

[0399] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.53-7.50 (m, 2H), 7.48-7.34 (m, 1H), 7.30-7.23 (m, 2H), 6.09 (s, 1H), 4.36 (d, J = 7.3Hz, 1H ),3.87(s,3H),3.37-3.36(m,1H),3.25-3.11(m,2H),2.81(s,3H),2.67-2.61(m,1H),2.38-2.29(m,1H),2.17-2.07(m,1H).

[0400] Example 19

[0401] Synthesis of compound 19

[0402] Step 1: Preparation of compound 19-2

[0403] Compound 15-2 (200 mg, 1.42 mmol, 1.0 eq) was dissolved in toluene (2 mL). m-Anisidine (175 mg, 1.42 mmol, 1.0 eq) and p-Toluenesulfonic acid (48.8 mg, 0.28 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 100°C under nitrogen and stirred for 6 h. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 19-2 (171 mg, 0.736 mmol, 51.8% yield) as a white solid. MS (ESI) m / z = 233.1 [M+H] + .

[0404] Step 2: Preparation of compound 19

[0405] Compound 19-2 (171 mg, 0.74 mmol, 1.0 eq) was dissolved in toluene (5 mL). Compound 7-2 (174 mg, 0.74 mmol, 1.0 eq) and piperidine (62.9 mg, 0.74 mmol, 1.0 eq) were added at room temperature. The reaction mixture was heated to 80°C and stirred for 6 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: Welch-Ultimate AQ-C18, 21.2*250 mm, 5 μm, mobile phase: 10%-95% MeCN in 0.1% FA in water) to obtain compound 19 (53.4 mg, 0.146 mmol, 19.7% yield) as a white solid. MS (ESI) m / z = 367.3 [M+H] + .

[0406] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.39 (t, J = 8.1Hz, 1H), 7.28 (d, J = 1.6Hz, 1H), 7.01 (dd, J = 8.2, 2.2Hz, 1H), 6.94-6.62 (m, 2H), 6.13-5.99 (m, 1H), 4.35 ( d,J=6.9Hz,1H),3.87(s,3H),3.78(s,3H),3.31-3.23(m,2H),3.17-3.10(m ,1H),2.80(s,3H),2.66-2.58(m,1H),2.39-2.23(m,1H),2.21-2.07(m,1H).

[0407] Example 20

[0408] Synthesis of compound 20

[0409] Step 1: Preparation of compound 20-2

[0410] Compound 15-2 (200 mg, 1.42 mmol, 1.0 eq) was dissolved in toluene (2 mL). m-Toluidine (152 mg, 1.42 mmol, 1.0 eq) and p-Toluenesulfonic acid (48.8 mg, 0.28 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 100°C under nitrogen and stirred for 6 h. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 20-2 (142 mg, 0.657 mmol, 46.2% yield) as a white solid. MS (ESI) m / z = 217.2 [M+H] + .

[0411] Step 2: Preparation of compound 20

[0412] Compound 20-2 (142 mg, 0.66 mmol, 1.0 eq) was dissolved in toluene (5 mL). Compound 7-2 (155 mg, 0.66 mmol, 1.0 eq) and piperidine (56.1 mg, 0.66 mmol, 1.0 eq) were added at room temperature. The reaction mixture was heated to 120°C and stirred for 2 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: YMC-Actus Triart C18 250*21 mm, mobile phase: 10%-100% MeCN in 0.1% FA in water) to obtain compound 20 (58.5 mg, 0.167 mmol, 25.3% yield) as a white solid. MS (ESI) m / z = 351.3 [M+H] + .

[0413] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.55 (d, J = 1.8Hz, 1H), 7.41-7.37 (m, 1H), 7.26-7.24 (m, 1H), 6.95-6.93 (m, 2H), 6.24 (d, J = 1.8Hz, 1H), 4. 85(d,J=1.3Hz,1H),3.92(s,3H),3.42-3.21(m,3H),3.20-3.16(m,1H),3.06-2.99(m,1H),2.90(s,3H),2.82-2.77(m,1H),2.39(s,3H).

[0414] Example 21

[0415] Synthesis of compound 21

[0416] Step 1: Preparation of compound 21-2

[0417] Compound 15-2 (500 mg, 3.55 mmol, 1.0 eq) was dissolved in toluene (5 mL). Aniline (330 mg, 3.55 mmol, 1.0 eq) and p-toluenesulfonic acid (122 mg, 0.71 mmol, 0.2 eq) were added at room temperature. The reaction mixture was heated to 120°C under nitrogen and stirred for 16 h. The reaction mixture was cooled to 0°C and filtered under reduced pressure. The filter cake was collected and dried under vacuum to obtain compound 21-2 (208 mg, 1.03 mmol, 28.8% yield) as a white solid. MS (ESI) m / z = 203.1 [M+H] + .

[0418] Step 2: Preparation of compound 21

[0419] Compound 21-2 (208 mg, 1.03 mmol, 1.0 eq) and compound 7-2 (243 mg, 1.03 mmol, 1.0 eq) were dissolved in acetonitrile (5 mL). DBU (157 mg, 1.03 mmol, 1.0 eq) was added at room temperature. The reaction mixture was heated to 80°C and stirred for 4 h. The reaction mixture was cooled and concentrated under reduced pressure. The residue was separated and purified by column chromatography (silica, dichloromethane / methanol = 99 / 1 to 9 / 1) and preparative liquid chromatography (column: Welch XBridge C18 250*21 mm, mobile phase: 20%-95% MeCN in 0.1% FA in water) to obtain compound 21 (53.5 mg, 0.159 mmol, 15.5% yield) as a white solid. MS (ESI) m / z = 337.4 [M+H] + .

[0420] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.53-7.40 (m, 3H), 7.37 (d, J = 1.7Hz, 1H), 7.26-6.98 (m, 2H), 6.10 (d, J = 1.8Hz, 1H), 4.53 (d ,J=7.0Hz,1H),4.00(s,3H),3.33(t,J=7.1Hz,2H),3.14-3.04(m,1H),2.96(s,3H),2.99-2.82(m,1H),2.34-2.13(m,2H).

[0421] The compounds synthesized in the Examples are summarized in Table 1 below.

[0422] Table 1. Example compound information

[0423] Example 22. Solubility of Compounds in DMSO and H2O

[0424] In this example, the solubility of compounds 1 to 11 synthesized in the preparation examples in DMSO and H2O was measured, and the results are shown in Table 2. The results show that most of the compounds disclosed herein have high solubility in DMSO, among which compounds 2, 5, 7, 8, and 11 also have high solubility in H2O.

[0425] Table 2. Compound solubility information

[0426] Example 23. DARTs assay of compounds

[0427] In this example, the interaction between some of the compounds in the above preparation examples and the SOAT1 protein was tested using drug affinity reaction targeted stability technology (DARTs). The test results are shown in Figure 1. Compound 5-P1 (codenamed P1 in the figure) can protect SOAT1 from hydrolysis and significantly enrich SOAT1, demonstrating that compounds STK, 5-P1, 12, 14 and 19 all have a high degree of binding to the SOAT1 protein.

[0428] Example 24. In vivo tumor inhibition effect of specific compounds in BALB / c mouse CT26 transplanted tumor model

[0429] In this example, the inhibitory effects of compounds 1 (SD1), 2 (SD2), 3 (SD3), 4 (SD4), and 5 (SD5) synthesized in the above preparation examples on the growth of colon cancer in a BALB / c mouse CT26 transplanted tumor model were tested.

[0430] CT26 cells were cultured at 5×10 5 The cells / mouse were inoculated in the left or right armpit of 6-week-old male mice and the tumor was grown to 50-200 mm. 3 Mice were divided into 6 groups: control group, compound 1, compound 2, compound 3, compound 4 and compound 5 treatment group. Mice were given vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg compound 1, compound 2, compound 3, compound 4, compound 5 (dissolved in vehicle) by oral gavage every day for two consecutive weeks. Tumor volume was measured every 2-3 days using a digital caliper (ULINE, Cat#H-7352), and then the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm3 When , the mice were euthanized.

[0431] The test results are shown in Figure 2, indicating that Compound 1, Compound 2, Compound 3, Compound 4, and Compound 5 of the present disclosure can effectively inhibit tumor growth in the BALB / c mouse CT26 transplant tumor model, and their tumor inhibition activity is not weaker than that of Compound STK, among which Compound 5 has the best activity.

[0432] Example 25. Inhibitory effect of specific compounds on CT26 transplanted tumors in BALB / c mice

[0433] In this example, the inhibitory effects of the compounds 5-P1 and 5-P2 synthesized in the above preparation examples on the growth of colon cancer in a BALB / c mouse CT26 transplanted tumor model were tested.

[0434] CT26 cells were cultured at 5×10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 Mice were divided into three groups: control group, compound 5-P1 and compound 5-P2 treatment group. Mice were orally gavaged with vehicle (5% DMSO dissolved in corn oil), 10 mg / kg of compound 5-P1 and compound 5-P2 (dissolved in vehicle) every day for two consecutive weeks. Tumor volume was measured every 2-3 days using a digital caliper (ULINE, Cat# H-7352), and then the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 At 4 hr, the mice were euthanized and the tumor tissues were analyzed by flow cytometry (FACS).

[0435] The test results are shown in Figures 3A-3E, demonstrating that compound 5-P1 of the present disclosure has a tumor regression effect on CT26 xenograft model mice. In addition, flow cytometry studies showed that compound 5-P1 treatment increased the proportion of CD4 and CD8 positive cells in CT26 xenograft model mice.

[0436] Example 26. In vivo tumor inhibition effect of specific compounds on BALB / c mouse CT26 transplanted tumor model

[0437] In this example, the inhibitory effects of Compound 5-P1, Compound 7, Compound 8, Compound 9, Compound 10 and Compound 11 synthesized in the above preparation examples on the growth of colon cancer in a BALB / c mouse CT26 transplanted tumor model were tested.

[0438] CT26 cells were cultured at 5×10 5The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 The mice were divided into three groups: control group, compound 5-P1, compound 7, compound 8, compound 9, compound 10 and compound 11 treatment group. The mice were orally gavaged with vehicle (5% DMSO dissolved in corn oil), 10 mg / kg compound 5-P1, compound 7, compound 8, compound 9, compound 10 and compound 11 (dissolved in vehicle) every day for two consecutive weeks. The tumor volume was measured every 2-3 days with a digital caliper (ULINE, Cat#H-7352), and then the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 At 4 hr, the mice were euthanized and the tumor tissues were analyzed by flow cytometry (FACS).

[0439] The test results, shown in Figures 4A-4F, demonstrate that compounds 5-P1, 7, 8, 9, 10, and 11 of the present disclosure all exhibited tumor regression effects in CT26 xenograft mice. Furthermore, flow cytometry studies demonstrated that treatment with the disclosed compounds increased the proportion of CD4 and CD8 positive cells in CT26 xenograft mice.

[0440] Example 27. Inhibitory effect of specific compounds on CT26 transplanted tumor model in BALB / c mice

[0441] In this example, the inhibitory effects of compounds 5-P1, 12, 13, 14, 15, 16, 19, 20, and 21 synthesized in the above preparation examples on the growth of colon cancer in a BALB / c mouse CT26 transplanted tumor model were tested.

[0442] CT26 cells were cultured at 5×10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3The mice were divided into 9 groups: control group, compound 5-P1, compound 12, compound 13, compound 14, compound 15, compound 16, compound 19, compound 20 and compound 21 treatment group. The mice were orally gavaged with vehicle (5% DMSO dissolved in corn oil), 10 mg / kg compound 5-P1, compound 12, compound 13, compound 14, compound 15, compound 16, compound 19, compound 20 and compound 21 (dissolved in vehicle) every day for two consecutive weeks. The tumor volume was measured every 2-3 days with a digital caliper (ULINE, Cat#H-7352), and then the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 1500mm 3 At 4 hr, the mice were euthanized and the tumor tissues were analyzed by flow cytometry (FACS).

[0443] The test results are shown in FIG5 , which indicate that Compound 5-P1, Compound 12, Compound 13, Compound 14, Compound 16, Compound 19, Compound 20, and Compound 21 of the present disclosure all have tumor regression effects on CT26 xenograft model mice.

[0444] Example 28. Plasma stability of compounds

[0445] At 37°C, 2.0 μM of the compound was incubated with plasma samples from five species (mouse, rat, dog, cynomolgus monkey, and human) for 120 minutes. The concentration of the compound in the resulting samples was detected by LC-MS / MS, and the half-life (T) was calculated. 1 / 2 ).

[0446] The test results are shown in Table 3. After incubation for 120 minutes, the remaining amounts of compound 5-P1 (2.0 μM) in mouse, rat, dog and human plasma were 95.1%, 79.1%, 97.8% and 90.9%, respectively, indicating high plasma stability.

[0447] Table 3. Plasma stability results of compounds

[0448] Example 29. Stability of Compounds in Liver Microsomes

[0449] At 37°C, 1 μM of compound 5-P1 and compound 7 were incubated with liver microsomes (0.5 mg protein / mL) from five species (mouse, rat, dog, cynomolgus monkey, and human) supplemented with a reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system for a certain period of time up to 60 minutes. The concentrations of compound 5-P1 and compound 7 in the resulting samples were determined by LC-MS / MS. The half-lives (T) of compound 5-P1 and compound 7 in the liver microsome solutions of the five species were calculated by calculating the remaining percentage of compound 5-P1 at each time point. 1 / 2 ) and the corresponding CL int(liver) value.

[0450] The test results are shown in Table 4. Compound 5-P1 and Compound 7 are metabolized relatively stably in rat, dog and human liver microsomes, and metabolized relatively faster in mouse and cynomolgus monkey liver microsomes.

[0451] Table 4. Liver microsomal stability results of compounds

[0452] Example 30. Permeability of Compounds in Caco-2 Cells

[0453] The permeability of compounds 5-P1 and 12 was evaluated using Caco cells. The transport buffer used in this study was 10.0 mM HEPES-containing HBSS, pH 7.40 ± 0.05. Test compounds were tested at a concentration of 2.00 μM in both directions. Digoxin was tested at 10.0 μM in duplicate in both directions, while nadolol and metoprolol were tested at 2.00 μM in duplicate in both directions. The final DMSO concentration was adjusted to less than 1%. The cells were incubated for 2 hours in a CO2 incubator at 37.0°C, 5% CO2, saturated humidity, and without shaking. All samples were mixed with acetonitrile containing an internal standard and centrifuged at 3220 x g for 10 minutes. The concentrations of the test and control compounds in the starting, donor, and receptor solutions were determined by chromatography-mass spectrometry (LC-MS / MS) using the analyte / internal standard peak area ratio.

[0454] The test results are shown in Table 5, which indicate that the compounds disclosed herein have high cell permeability.

[0455] Table 5. Caco-2 permeability results of compound STK, compound 5-P1 and compound 12

[0456] Example 31. Pharmacokinetic study of the compound in SD rats

[0457] In this experiment, 12 male SD rats were divided into four groups based on similar body weight. Groups 1 and 2 received a single intravenous injection and oral gavage of compound 5-P1, respectively. The intravenous dose was 10 mg / kg, and the oral gavage dose was 50 mg / kg. The solvents for both intravenous and oral administration were: 5% DMSO + 5% PEG400 + 90% water, clear solutions. The intravenous administration concentration was 2 mg / mL, and the oral gavage concentration was 5 mg / mL. Groups 3 and 4 received a single intravenous injection and oral gavage of compound 12, respectively. The intravenous dose was 2 mg / kg, and the oral gavage dose was 10 mg / kg. The solvents for intravenous administration were: 5% DMSO + 95% saline, and the oral solvent was: 5% DMSO + 95% water, clear solutions. The intravenous administration concentration was 5 mg / mL, and the oral gavage concentration was 10 mg / mL. K2-EDTA was used as an anticoagulant.

[0458] The blood sampling time points for Group 1 and Group 2 were: 0.083, 0.25, 0.5, 1, 3, 6, 12, and 24 hours.

[0459] The blood sampling time points for Group 3 and Group 4 were: 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours.

[0460] All experimental animals were given 0.02 mL of blood sample collected from the saphenous vein, and the actual blood collection time was recorded. In this experiment, the deviation of the blood collection time from the theoretical blood collection time was in compliance with the regulations (points within 1 hour after administration were within ±1 minute, and the others were within 5% of the theoretical time). After the blood sample was collected, it was immediately transferred to a labeled centrifuge tube containing K2-EDTA (0.5M), and then centrifuged (3000g, 4°C, 15 minutes) to obtain plasma, which was transferred to a pre-cooled centrifuge tube, quick-frozen in dry ice, and stored in an ultra-low temperature refrigerator at -60°C or lower until LC-MS / MS analysis was performed. The test results are shown in Table 6, which shows that the compounds 5-P1 and compound 12 disclosed herein are rapidly absorbed in rats, have a moderate apparent distribution volume, and both have high oral bioavailability (83.1% and 129%, respectively).

[0461] Table 6. Pharmacokinetic data of the compounds in rats

[0462] Example 32. Inhibition of tumor growth in Drosophila by compound STK

[0463] In this example, the inhibitory effect of compound STK on the growth of orthotopic tumors in Drosophila was tested.

[0464] As previously described, the Drosophila kidney stem cells (Ras V12-PMML) clones expressing Ras induced the generation of renal stem cell tumor clusters (Singh et al., 2016). V12 -PMML cloned fruit flies were cultured for 4 days to allow tumor growth, then switched to food containing the indicated drug for another 4 days, and then given the drug carrying Ras V12 -PMML flies were fed with normal food containing DMSO or 10 μM STK. In the drug-treated groups, 5×10 3 μm 2 esg>GFP on the area + Tumor cells were used to characterize tumor size.

[0465] The test results are shown in FIG6 , which show that the compound STK can effectively inhibit the growth of tumors in Drosophila.

[0466] Example 33. Inhibitory Effect of Compound STK on In situ Colon Cancer and In situ Liver Tumors in Mice

[0467] In this example, the inhibitory effect of the compound STK synthesized in the above preparation example on the growth of in situ tumors in mice was tested.

[0468] APC was specifically knocked out in Lgr5-marked mouse intestinal stem cells (Lgr5-CreER-GFP / Apc f / f ) were used to induce in situ intestinal stem cell tumor clusters, thereby establishing intestinal stem cell tumor models in mice. Doxycycline was used to control the expression of the TTA promoter, thereby inducing MYC expression to generate in situ liver stem cell tumor clusters (Wang et al., 2020). Two weeks after induction of intestinal stem cell tumors and liver cancer stem cell tumors in mouse models, STK (10 mg / kg) was administered for two months. The intestines and livers of the two tumor mouse models were dissected, and the number of tumor cells was analyzed. The survival rate of the mice was also calculated.

[0469] The test results are shown in FIG7 , indicating that the compound STK can effectively inhibit the growth of mouse colon cancer stem cells and liver cancer cancer stem cells, and significantly prolong the survival rate of mice.

[0470] Example 34. Inhibitory effect of compound STK on mouse lung cancer

[0471] In this example, the inhibitory effect of the compound STK synthesized in the above preparation example on the growth of in situ lung cancer in mice was tested.

[0472] In Adeno-Cre / f-stop-f-Ras V12 / p53 f / fIn lung cancer model mice, Adeno-Cre was administered nasogastricly to induce the knockout of Kras and p53, thereby inducing lung cancer. Two weeks after administration of Adeno-Cre, STK (10 mg / kg) was administered orally for two months. Lungs were then dissected, tumor cell counts were analyzed, and mouse survival rates were calculated.

[0473] The test results are shown in FIG8 , indicating that the compound STK can effectively inhibit the growth of lung cancer in mice and significantly prolong the survival rate of mice.

[0474] Example 35. Inhibitory Effects of Compound STK on Colon Cancer, Melanoma, Lung Cancer and Breast Transplanted Tumors in Mice

[0475] In this example, the inhibitory effect of the compound STK synthesized in the above preparation example on the growth of colon, melanoma, lung and breast transplanted tumors in mice was tested.

[0476] CT26, B16-F10, LLC, and 4T1 cells were cultured at a density of 5 × 10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 Mice were treated with SOAT1 inhibitors by gavage for two consecutive weeks. The mice were divided into two groups: a control group and an STK-treated group. Mice were given either vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for approximately two weeks. Tumor volume was measured every 2-3 days using a digital caliper (ULINE, Cat# H-7352) and then calculated using the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width). 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 When , the mice were euthanized.

[0477] The test results are shown in Figures 9A-9E, indicating that compound STK has a good inhibitory effect on the growth of mouse colon, melanoma, lung and breast transplanted tumors.

[0478] Example 36. Screening of possible STK targets of compounds using reverse docking

[0479] In this example, the Pharmmaper and Chemmaper databases were used to perform reverse docking virtual screening of small molecule STKs.

[0480] The test results are shown in FIG10 , indicating that SOAT1 is a better candidate target for compound STK.

[0481] Example 37. In vitro DARTs and ITC experiments confirmed that the target of STK is SOAT1 protein

[0482] In this example, DARTs experiments and ITC experiments further demonstrated that SOAT1 is a target of STK.

[0483] Through the DARTs experiment (Biochem Pharmacol.2021), proteins from CT26 cells were extracted and incubated with STK small molecules in vitro. The 70KDa enriched silver-stained band was subjected to protein profiling, and 11 candidate proteins were found, as shown in Figure 11. Among them, SOAT1 is a target protein that overlaps with the reverse docking results (Expert Opin Drug Discov.2016). Further Western results showed that SOAT1 protein was indeed significantly enriched. And through ITC (Methods Mol Biol.2021) experiments, it was shown that SOAT1 has good binding to STK.

[0484] Example 38. Effect of knockdown of SOAT1 expression on the anti-tumor activity of compound STK in the BALB / c mouse CT26 colon cancer model

[0485] In this example, the tumor inhibition effect of the compound STK synthesized in the above preparation example on the SOAT1 knockdown cell line was tested.

[0486] First, plKO.1 was used to construct the ShSOAT1 CT26 stable knockout strain. Shluci CT26 and ShSOAT1 CT26 cells were cultured at a rate of 5×10 5 The cells / mouse were inoculated into the right armpit of 6-week-old male mice and the tumor was grown to about 200 mm. 3 Mice were treated with the SOAT1 inhibitor STK by gavage for two consecutive weeks. Mice were given either vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for approximately two weeks. Tumor volume was measured every 2-3 days using a digital caliper (ULINE, Cat# H-7352) and then calculated using the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width) 2 Calculate tumor volume.

[0487] The test results are shown in FIG12 , indicating that compound STK lost its tumor-suppressing effect when SOAT1 was knocked down in CT26 and then administered by oral gavage, further proving in vivo that the target of STK is SOAT1.

[0488] Example 39. Effect of compound STK on cholesterol metabolism in CT26 cells, LLC cells and Huh7 cells

[0489] In this example, the effect of STK pretreatment of the compound synthesized in the above preparation example on cholesterol metabolism in CT26 cells, LLC cells, and Huh7 cells was tested.

[0490] 5μM SOAT1 inhibitor STK was added to CT26 cells and incubated for 24 hours. Protein spectrum analysis (Chem Rev. 2022) found that STK affected the lipid metabolism of CT26 (as shown in Figure 13). SOAT1 is an esterase on the endoplasmic reticulum that interferes with cholesterol metabolism by inhibiting the synthesis of cholesterol into cholesterol esters. Further, 5μM SOAT1 inhibitor STK was added to CT26 cells, LLC cells, and Huh7 cells and incubated for 24 hours. The qPCR cluster heat map showed that cholesterol synthesis, cholesterol esterification, cholesterol absorption, and cholesterol conversion were all inhibited.

[0491] The test results are shown in FIG13 (A, B, C), indicating that compound STK treatment affects cholesterol metabolism in CT26 cells, LLC cells, and Huh7 cells, including inhibition of cholesterol synthesis, cholesterol esterification, cholesterol absorption, and cholesterol conversion.

[0492] Example 40. Compound STK treatment or SOAT1 gene knockout can alter cholesterol metabolism in tumor cells

[0493] In this example, it was tested whether the compound synthesized in the above preparation example inhibited the formation of cholesterol esters in STK pretreatment or SOAT1 knockdown cell lines.

[0494] CT26 cells were treated with 5 μM of the SOAT1 inhibitor STK and incubated for 24 hours. Changes in intracellular cholesterol esters and extracellular free cholesterol were measured using the Total Cholesterol and Cholesteryl Ester Fluorometric Assay Kit (Cat. No.: E-BC-F032) according to the manufacturer's instructions. SOAT1 is an esterase on the endoplasmic reticulum that reduces cholesterol ester formation by inhibiting the conversion of cholesterol to cholesterol esters.

[0495] The test results are shown in FIG14 , indicating that compound STK treatment inhibits the formation of cholesterol esters in CT26 cells.

[0496] Example 41. Effects of Compound STK on Lipid Droplet Accumulation and Lipid Oxidation in Huh7 and CT26 Cells

[0497] In this example, the effect of the compound STK synthesized in the above preparation example on lipid droplet aggregation in Huh7 cells and CT26 cells was tested.

[0498] CT26 and Huh7 cells were incubated with 5 μM of the SOAT1 inhibitor STK for 24 h. Cell neutral lipids (BODIPY 493 / 503, MCE, Cat. No.: 121207-31-6), mitochondrial oxidized esters and non-oxidized esters (BODIPY TM 581 / 591C11, Invitrogen, D3861).

[0499] The test results are shown in FIG15 , which indicate that compound STK increased lipid droplet accumulation in Huh7 and CT26 cells.

[0500] Example 42. Effects of Compound STK on ROS Content and Mitochondrial Function in CT26 and Huh7 Cells

[0501] In this example, the effects of the compound STK synthesized in the above preparation examples on the ROS level and mitochondrial function of Huh7 cells and CT26 cells were tested.

[0502] 5 μM SOAT1 inhibitor STK was added to CT26 cells and incubated for 24 h. Cell ROS (Reactive Oxygen Species Detection Kit, Beyotime, Cat. No.: S0033M), mitochondrial membrane potential (TMRM Assay Kit Mitochondrial Membrane Potential, abcam, ab228569), and changes in mitochondrial morphology were detected using kits according to the instructions. Mitochondrion-Selective Probes, Invitrogen, Cat. No.: M7514).

[0503] The test results are shown in FIG16 , indicating that compound STK reduces the mitochondrial membrane potential TMRM level, the mitochondrial morphology of Mitotractor-labeled mitochondria exhibits rod-shaped abnormalities, and the overall ROS level in the cells increases.

[0504] Example 43. Effects of Compound STK on Lysosomal Function and Protein Aggregation in Huh7 and CT26 Cells

[0505] In this example, the effects of the compound STK synthesized in the above preparation examples on the lysosomal function and protein aggregation of Huh7 cells and CT26 cells were tested.

[0506] 5 μM SOAT1 inhibitor STK was added to CT26 and Huh cells and incubated for 24 h. The acidic lysosomes of cells were detected using the kit (LysoTracker TMDeep Red, Invitrogen, Cat. No.: L12492), intracellular protein aggregation ( Protein aggregation assay, Enzo, ENZ-51023-KP050).

[0507] The test results are shown in FIG17 , which indicate that compound STK increases lysosomal acidity and intracellular protein aggregation.

[0508] Example 44. Compound STK induces Caspase1-dependent pyroptosis in LLC transplanted tumors

[0509] In this example, the inhibitory effect of the compound synthesized in the above preparation example on the growth of STK transplanted tumors was tested.

[0510] LLC cells were cultured at a rate of 5 × 10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 Mice were treated with SOAT1 inhibitors by gavage for two consecutive weeks. The mice were divided into two groups: a control group and an STK-treated group. Mice were given either vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for approximately two weeks. Tumor volume was measured every 2-3 days using a digital caliper (ULINE, Cat# H-7352) and then calculated using the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width). 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 The mice were euthanized at 4 ℃ and 8 ℃, and the subcutaneous transplanted tumors were removed after dissection. IF staining was performed. The results are shown in Figure 18: tumor cells mainly died by pyroptosis induced by Casapase1 expression, rather than by apoptosis induced by Casapase3.

[0511] Example 45. Effects of Compound STK on HMGB1, ERP46 Expression and ATP Production in CT26 Cells

[0512] In this example, the effect of the compound STK synthesized in the above preparation example on DAMPs of Huh7 cells and CT26 cells was tested.

[0513] 5 μM, 10 μM, and 50 μM of the SOAT1 inhibitor STK were added to CT26 and Huh cells and incubated for 24 and / or 48 h. The extracellular ATP level of the cells was detected using a kit (Enhanced ATP Detection Kit, Beyotime, Cat. No.: S0027) according to the instructions, and the levels of HMGB1 and ERP46 were detected by IF method.

[0514] The test results are shown in FIG19 , which indicate that compound STK causes intracellular HMGB1 to translocate to the nucleus, increases the expression of ERP46, and increases the level of extracellular ATP.

[0515] Example 46. Inhibitory effect of combined treatment of compound STK and anti-PD-1 antibody on CT26 transplanted tumors

[0516] In this example, the inhibitory effect of the compound STK synthesized in the above preparation example combined with Anti-PD-1 on the growth of transplanted tumors was tested.

[0517] CT26 cells were cultured at 5×10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 Mice were treated with SOAT1 inhibitors by gavage for two consecutive weeks. The mice were divided into four groups: control group (vehicle), STK treatment group, vehicle + Anti-PD1 treatment group, and STK + Anti-PD1 treatment group. Mice were given vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for about two weeks, and Anti-PD1 was administered twice a week (100 ug / kg). Tumor volume was measured every 2-3 days with a digital caliper (ULINE, Cat#H-7352), and then the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 The mice were euthanized. The mice were dissected, and the subcutaneous transplanted tumors were removed. The transplanted tumor cells were passed through a 40 μm mesh to prepare a cell suspension, and CD3 + T, CD8 + T、CD44CD62L + T cell ratio.

[0518] The results are shown in Figure 20: The combined administration of STK and Anti-PD1 enhanced the efficacy of PD1 and significantly inhibited tumor growth.

[0519] Example 47. Effect of compound STK on CD4 and CD8 T cell infiltration in mouse CT26 transplanted tumors

[0520] In this example, the compound synthesized in the above preparation example was tested for its ability to increase immune infiltration in the tumor microenvironment by STK treatment.

[0521] CT26 cells were cultured at 5×10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3Mice were treated with SOAT1 inhibitors by gavage for two consecutive weeks. The mice were divided into four groups: control group, STK treatment group, DMSO + Anti-PD1 treatment group, and STK + Anti-PD1 treatment group. Mice were given vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for about two weeks, and Anti-PD1 was administered twice a week (100 ug / kg). Tumor volume was measured every 2-3 days with a digital caliper (ULINE, Cat# H-7352), and then the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 The mice were euthanized. The mice were dissected, and the subcutaneous transplanted tumors and spleens were removed. The transplanted tumor cells were passed through a 40 μm sieve to prepare a cell suspension. The CD3 + T, CD8 + T, CD44 + CD62L + T cell ratio.

[0522] The results are shown in Figures 20 and 21: STK combined with Anti-PD1 enhances the efficacy of PD1, significantly inhibits tumor growth, and increases the proportion of CD3 positive T cells, the proportion of CD8 positive T cells and the proportion of central memory T cells CD44 + CD62L + proportion.

[0523] Example 48. Compound STK fails to exert anti-tumor effect in immunodeficient mice

[0524] In this example, the in vivo efficacy of the compound STK synthesized in the above preparation example on immunodeficient NCG tumor-transplanted mice was tested.

[0525] Shcram CT26 and ShSOAT1 CT26 cells were cultured at a density of 5 × 10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 Mice were treated with SOAT1 inhibitors by gavage for two consecutive weeks. The mice were divided into two groups: a control group and an STK-treated group. Mice were given either vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for approximately two weeks. Tumor volume was measured every 2-3 days using a digital caliper (ULINE, Cat# H-7352) and then calculated using the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width). 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3When , the mice were euthanized.

[0526] The results are shown in FIG22 : STK treatment cannot inhibit tumor growth in immunodeficient NCG transplanted tumor mice, indicating that the in vivo anti-tumor activity of STK depends on a healthy mouse immune system.

[0527] Example 49. Anti-tumor effect of compound STK depends on immune system activation

[0528] In this example, the compound synthesized in the above preparation example was tested for its STK therapy-dependent T cell infiltration.

[0529] CT26 cells were cultured at 5×10 5 The dose of cells / mouse was inoculated into the lateral or right axilla of 6-week-old male mice. 3 Mice were treated with SOAT1 inhibitors by gavage for two consecutive weeks. The mice were divided into four groups: control group, STK treatment group, vehicle + Anti-CD4 + CD8 treatment group, and STK + CD4 + CD8 treatment group. Mice were given vehicle (5% DMSO dissolved in corn oil) or 10 mg / kg STK (dissolved in vehicle) daily for about two weeks, and Anti-PD1 was administered twice a week (100 ug / kg). Tumor volume was measured every 2-3 days with a digital caliper (ULINE, Cat# H-7352), and then the formula 1 / 2 × longitudinal diameter (length) × maximum transverse diameter (width) was used. 2 Calculate the tumor volume. When the maximum tumor volume reaches 2000mm 3 When , the mice were euthanized.

[0530] The results are shown in Figure 23: When the functions of CD4 T cells and CD8 T cells in mice were blocked with Anti-CD4 and Anti-CD8, STK administration lost its tumor inhibitory effect, indicating that the in vivo anti-tumor activity of STK depends on the activation of the mouse immune system.

[0531] Example 50. Effect of compound STK on the level of chemokines (especially CCL5) secreted by tumor cells

[0532] In this example, the effect of the compound STK synthesized in the above preparation example on the chemokines of CT26 cells was tested.

[0533] 5 μM SOAT1 inhibitor STK was added to CT26 cells and incubated for 24 h. The cellular RNA was extracted and converted into cDNA, and the chemokine levels were detected by qPCR.

[0534] The test results, shown in Figure 24A, indicate that compound STK increased the mRNA levels of CCL4, CCL5, CXCL9, CXCL10, and CCXL12 in cells, with the increase in CCL5 being particularly significant. Simultaneously, in CT26 xenograft mice, FACS analysis of the expression of the CCL5 receptor CCR5 on CD8 T cells was performed according to the experimental method of Example 28. The results, shown in Figure 24B, indicate that the proportion of CCR5+CD8+ T cells was also increased in LLC and 4T1 xenografts.

[0535] Example 51. Compound STK treatment enhances the activation and killing ability of effector T cells

[0536] In this example, the effects of STK treatment on the activation and killing ability of T cells by the compound synthesized in the above preparation example were tested.

[0537] At the same time, in CT26 transplanted tumor model mice and LLC transplanted tumor model mice, tumors were taken according to the experimental method of Example 39, and IF analysis of CD4, CD8, MHCI, and MHCII expression levels was performed.

[0538] The results, as shown in Figures 25(A,B), showed significant increases in the expression of CD4, CD8, MHC I, and MHC II in both xenograft models, indicating that the treatment group effectively increased the immune infiltration of T cells and dendritic cells. In an in vitro experiment, CT26 cells were pretreated with 5μM of the SOAT1 inhibitor STK for 24 hours. Conditioned medium was then collected 24 hours after drug withdrawal and cultured with primary T cells extracted from the spleen (previously stimulated with anti-CD3 and anti-CD28 for 48 hours) for 24 hours. T cell TCR expression levels were then measured after treatment with the conditioned medium. As shown in Figure 25(C), the expression of pZap70, pLAT, and pERK in the TCR was upregulated. Furthermore, when OT-1 cells were cultured with this conditioned medium for 24 hours, the ability of OT-1 to kill CT26-OVA was significantly enhanced, as shown in Figure 25(D).

[0539] Example 52. Single-cell green group sequencing analysis of the effect of compound STK on immune infiltrating cell subsets in mouse in situ liver cancer and lung cancer

[0540] In this example, the effect of the compound synthesized in the above preparation example on the infiltration of immune cells after STK treatment was tested by RNA single-cell bioinformatics analysis.

[0541] The results are shown in Figures 26A and 26B. In the mouse liver cancer tumor model (Tet-o / MYC / LAP-tTA) and the mouse lung cancer model (Adeno-Cre / f-stop-f-Ras V12 / p53 f / f Compared with DMSO treatment, 70% of the mouse tumors disappeared after STK treatment. In the remaining 30% of mouse tumors, single-cell RNA-sc showed that effective T cells, B cells, NKT cells and DCs increased significantly after STK treatment, and Treg, CD4 + T cells and CD8 + T cells were significantly reduced.

[0542] The foregoing description is considered to be merely illustrative of the principles of the present disclosure. Furthermore, since many modifications and variations will be apparent to those skilled in the art, it is not desirable to limit the present disclosure to the exact configuration and process described above. Therefore, all suitable modifications and equivalents are considered to fall within the scope of the present disclosure as defined by the appended claims.

[0543] All publications, patents, and patent applications cited in this disclosure are hereby incorporated by reference in their entirety.

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Claims

1. A compound of formula (I), its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof: in, n is selected from 0, 1 and 2; X1 is selected from CH, CH2, N or NH; represents a single bond or a double bond; The benzene ring A is optionally substituted with 1, 2, 3, 4, or 5 R1; R1 and R2 are each independently absent or selected from H, D, CN, OH, halogen, optionally substituted with 1-3 R a C 1-6 Alkyl, optionally substituted with 1-3 R b C 3-6 Cycloalkyl and optionally substituted with 1-3 R c C 1-6 A group consisting of alkoxy groups; R3 is selected from optionally substituted with 1-3 R d C 5-6 a carbocyclic ring or a 5- to 6-membered heterocyclic ring, the 5- to 6-membered heterocyclic ring optionally containing 1 or more heteroatoms selected from N, O and S; Each time it appears, each R a , R b , R c , R d Each independently selected from D, -OH, -NH2, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)-C 1-6 Alkyl, C 3-6 The group consisting of cycloalkyl and 3 to 6-membered heterocycloalkyl.

2. The compound of formula (I) according to claim 1, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: Rings B and C together form the following fused ring structure:

3. A compound of formula (I) according to claim 1 or 2, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: R3 is selected from optionally substituted with 1-3 R d phenyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, furanyl, thienyl, thiazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, or tetrazolyl; preferably R3 is selected from optionally substituted with 1-3 R d The following ring structure:

4. A compound of formula (I) according to claim 1 or 2, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: R3 is selected from optionally substituted with 1-3 R d phenyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, pyrazolyl, furanyl, thienyl, thiazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, or tetrazolyl; preferably R3 is selected from optionally substituted with 1-3 R d The following ring structure:

5. The compound of formula (I) according to claim 4, its stereoisomer, isotopic variant, or pharmaceutically acceptable salt or solvate thereof, wherein: R3 is optionally substituted with 1 to 3 R d of 6. A compound of formula (I) according to any one of claims 1 to 5, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: The compound of formula (I) is selected from the following compounds:

7. A method for preparing a compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, the preparation method comprising the following synthetic route:

8. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

9. A method for inhibiting intracellular SOAT1 activity in a cell, comprising administering an effective amount of a compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof to the cell, so that the SOAT1 protein activity in the cell is reduced or the protein expression level is reduced.

10. The method according to claim 9, wherein: The cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

11. The method according to claim 9 or 10, wherein: The method induces coordinated cell death at the cellular level.

12. The method according to any one of claims 9 to 11, wherein: The method is performed in vitro.

13. The method according to any one of claims 9 to 11, wherein: The method is performed in vivo.

14. Use of a compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof in the preparation of an agent for inhibiting intracellular SOAT1 protein activity or protein expression in a cell.

15. The use according to claim 14, wherein The cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

16. The use according to claim 14 or 15, wherein The agents induce inflammatory cell death at the cellular level.

17. The use according to any one of claims 14 to 16, wherein The agent is administered to cells in vitro.

18. The use according to any one of claims 14 to 16, wherein The agent is administered to the cell in vivo.

19. A method for treating or preventing a disease associated with SOAT1 activity in a subject, the method comprising administering to the subject an effective amount of a compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof.

20. The method according to claim 19, wherein: The disease associated with SOAT1 activity is cancer or tumor.

21. The method according to claim 20, wherein: The cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

22. The method according to claim 19, wherein: The disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, infectious diseases, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

23. The method according to any one of claims 19 to 22, wherein: The subject is a mammal, such as a human.

24. The method according to any one of claims 19 to 23, wherein: The method also includes co-administering at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

25. The method according to claim 24, wherein: The compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or pharmaceutically acceptable salt or solvate and at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody, are administered simultaneously or sequentially in any order.

26. A compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with SOAT1 activity in a subject.

27. The compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with SOAT1 activity in a subject according to claim 26, wherein: The disease associated with SOAT1 activity is cancer or tumor.

28. The compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with SOAT1 activity in a subject according to claim 27, wherein: The cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

29. The compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with SOAT1 activity in a subject according to claim 26, wherein: The disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

30. A compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with SOAT1 activity in a subject according to any one of claims 26 to 29, wherein: The subject is a mammal, such as a human.

31. Use of a compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for treating or preventing a disease associated with SOAT1 activity in a subject.

32. The use according to claim 31, wherein The disease associated with SOAT1 activity is cancer or tumor.

33. The use according to claim 32, wherein The cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

34. The use according to claim 31, wherein The disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

35. The use according to any one of claims 31 to 34, wherein The subject is a mammal, such as a human.

36. A kit for treating or preventing a disease associated with SOAT1 activity in a subject, the kit comprising a compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 8; a container; and optionally a package insert or label indicating treatment.

37. The kit according to claim 36, wherein The disease associated with SOAT1 activity is cancer or tumor.

38. The kit according to claim 37, wherein The cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

39. The kit according to claim 36, wherein The disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

40. The kit according to any one of claims 36-39, wherein The subject is a mammal, such as a human.

41. A kit for diagnosing a disease associated with SOAT1 activity in a subject, comprising at least one compound of formula (I) according to any one of claims 1 to 6, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof as a test agent.

42. The kit according to claim 41, wherein The test reagent detects at least one biomarker indicative of the presence of a disease associated with SOAT1 activity.

43. The kit according to claim 41 or 42, wherein The test reagents are capable of detecting the activity of SOAT1, as well as alternative upstream / downstream regulators of SOAT1 activity or function.

44. A kit according to any one of claims 41 to 43, wherein The disease associated with SOAT1 activity is cancer or tumor.

45. The kit according to claim 44, wherein The cancer or tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

46. ​​A kit according to any one of claims 41 to 43, wherein The disease associated with SOAT1 activity is selected from the group consisting of autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, ischemia-reperfusion injury, multiple sclerosis, other neurodegenerative diseases, and the like.

47. A kit according to any one of claims 41 to 46, wherein The subject is a mammal, such as a human.

48. A compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in the preparation of a therapeutic vaccine for blocking tumor development.

49. A compound of formula (I) according to any one of claims 1 to 6, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for preparing a therapeutic vaccine for blocking tumor development according to claim 48, wherein: The tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma and the like.

50. Use of a compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a therapeutic vaccine for blocking tumor development.

51. The use according to claim 50, wherein The tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia, lymphoma and the like.

52. A method for preparing a therapeutic vaccine for blocking tumor development, comprising contacting a compound of formula (I) according to any one of claims 1 to 6, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof with tumor stem cells, thereby converting the tumor stem cells into a therapeutic vaccine.

53. A method of vaccination comprising administering a therapeutic vaccine prepared according to the method of claim 52 to a subject.

54. The vaccination method according to claim 53, wherein: The subject is a mammal, such as a human.

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