Arf1 inhibitor and use thereof

Abstract

A compound of formula (I), or a stereoisomer, isotopic variant, pharmaceutically acceptable salt or solvate thereof, wherein R1, R2, R3, R4, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, L, ring A, ring B, ring C, and ring D are as described herein. The compound of formula (I), or the stereoisomer, isotopic variant, pharmaceutically acceptable salt or solvate thereof can be used as an Arf1 inhibitor. Further provided are a pharmaceutical composition containing the compound of formula (I), or the stereoisomer, isotopic variant, pharmaceutically acceptable salt or solvate thereof, and a use in the treatment or prevention of a disease, such as cancer or tumor, associated with Arf1 pathway activity in a subject. Further provided is a use of the compound of formula (I), or the stereoisomer, isotopic variant, pharmaceutically acceptable salt or solvate thereof in the preparation of a therapeutic vaccine for retarding tumor development.

Description

ARF1 inhibitors and their applications Technical Field

[0001] 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 COPI / Arf1-lipolysis β-oxidation pathway inhibitors (also referred to as Arf1 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 Arf1 pathway activity in subjects, such as cancer or tumors, and their use in preparing therapeutic vaccines for inhibiting tumor progression. Background Art

[0002] Cancer is the leading cause of death worldwide, killing nearly 10 million people in 2021. Despite advances in treating some types of cancer through treatments like surgery, radiation, and chemotherapy, many types are essentially incurable. Even when effective treatments are available for a particular cancer, the side effects of that treatment can be severe, significantly impairing quality of life.

[0003] 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, maintains a hierarchical structure, with a small number of leukemia stem cells (LSCs) or cancer stem cells (CSCs), also known as tumor-initiating cells, at the apex, exhibiting unlimited self-renewal capacity, which allows tumors to regenerate and relapse despite 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 the niche for 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). A stem cell-like subpopulation within the tumor hemisphere of solid tumors has 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) were recently 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.

[0004] For many years, the inventors have been dedicated to the study of stem cells and cancer stem cells. Several years ago, the inventors performed genome-wide RNAi screening in Drosophila intestinal stem cells (ISCs) and discovered a set of genes (including genes in the Arf1-mediated lipid metabolism pathway) whose knockdown induces cytokine release, affects their microenvironment, and ultimately leads to stem cell death. The specific mechanisms of action of these genes vary, but a common feature is that their knockdown first releases factors from stem and cancer stem cells, alters the microenvironment, activates neighboring cells or immune cells, and then feeds back to kill the stem cells or CSCs (Aggarwal et al., 2022; Singh et al., 2016; Wang et al., 2020; Zeng et al., 2015). Deletion of these genes in CSCs releases cytokines, transforming the TME from an immunosuppressive to an immunostimulatory microenvironment (Wang et al., 2020).

[0005] ADP-ribosylation factor 1 (Arf1), a member of the human Arf gene family, encodes a small guanine nucleotide-binding protein that plays an important role in vesicle trafficking and lipid metabolism (D'Souza-Schorey and Chavrier, 2006; Donaldson and Jackson, 2011; Kazmalek et al., 2017; Wilfling et al., 2014). Previous studies have shown that Arf1 is highly expressed in a variety of human cancers, including breast, hepatocellular, colorectal, and prostate cancers (Casalou et al., 2016; Wang et al., 2020). Upregulation of Arf1 is negatively correlated with prognosis in cancer patients (Lang et al., 2017; Wang et al., 2020).

[0006] Knocking out the Arf1 pathway in CSCs triggers a metabolic stress chain reaction, starting with mitochondrial damage and endoplasmic reticulum (ER) stress, and then exposing damage-associated molecular patterns (DAMPs: CALR, HMGB1, ATP), thereby changing the tumor microenvironment and activating a systemic antitumor immune response, thereby destroying the tumor (Wang et al., 2020). The inventors recently tested two new Arf1 inhibitors (DU101, DU102; described in our previous patents: WO2022 / 028429A1, PCT / CN2021 / 110373) and revealed their novel antitumor mechanisms. The inventors demonstrated that the two newly designed Arf1 inhibitors have low toxicity and potent antitumor activity in mouse and patient-derived liver and melanoma tumor xenograft (PDX) models. Treatment with the inhibitors significantly increased CD8 + Infiltration and activation of T cells and induction of tumor-associated stem cell-like TCF1 through the S1P-S1PR pathway + CD4 + CD8 + Formation of double-positive T (DPT) cells.

[0007] Two recent studies have found that tumor-associated CD4 / CD8 double-positive T (DPT) cells have potent antitumor activity in hepatocellular carcinoma (HCC) and other tumors (Schad et al., 2022; Zheng et al., 2020). Since early DPTs are mainly present in the thymus, tumor-associated DPT cells may be derived from infiltrating CD4 + or CD 8 + Single-positive T cells express both exhaustion and activation markers. DPT cells demonstrate a robust immune response and are in an active anti-tumor state. Furthermore, HCC patients enriched in DPT cells are positively correlated with better treatment outcomes, suggesting that DPT cells are superior anti-tumor T cells.

[0008] If the tumor persists, active CD8 + T cells are typically exhausted, so maintaining a functional tumor-targeting CD8 + The T cell pool is crucial. In cancer patients receiving immunotherapy, the duration of functional anti-tumor T cells is directly correlated with better treatment outcomes (Robbins et al., 2004). Studies have shown that ICBs inhibit tumors not by reversing T cell exhaustion, but by promoting the proliferation of stem-like T cell subsets. Stem-like T cells then generate fresh, effective T cells to replenish the exhausted T cells in the tumor. Stem-like CD8 +T cells maintain superior responses to ICBs and ACT in animal models of cancer and in patients with cancer (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).

[0009] Our data suggest that treatment with an Arf1 inhibitor enriches stem cell-like CD4 + CD8 + DPT cell population, which combines stem cell-like T cells and CD4 + CD8 + The superior properties of these two T cell subsets of DPT cells may have more powerful anti-tumor activity. Exploring the properties of this special T cell population may further improve the efficiency of T cell immunotherapy. Therefore, treatment with a single agent can overcome the three obstacles of cancer immunotherapy: increasing CD8 + Arf1 infiltration promotes T cell activation and blocks T cell dysfunction or exhaustion. Treatment with these inhibitors shifts the tumor immune microenvironment from cold to warm by reprogramming lipid metabolism. These inhibitors may provide new cancer immunotherapy strategies and significantly expand the proportion of cancer patients suitable for immunotherapy. New Arf1 inhibitors can not only be used to treat tumors with poor immunogenicity, but also develop more effective T cell-based immunotherapies by combining the inhibitors with ICBs or ACT.

[0010] However, the poor water solubility and permeability of DU101 and DU102 greatly limit their transformation potential. Therefore, there is an urgent need to develop and identify new Arf1 inhibitors with good water solubility and drugability, and induce strong anti-tumor immunity.

[0011] Summary of the Invention

[0012] This application is based on the inventors' unexpected discovery that ablation of the COPI / Arf1-lipolysis β-oxidation pathway can eradicate cancer stem cells and induce DAMPs-mediated anti-tumor immune responses, and the development and identification of a new class of Arf1 inhibitors with good water solubility and strong anti-tumor immunity. Therefore, the Arf1 inhibitors disclosed herein (i.e., compounds of Formula (I)) are particularly suitable for treating or preventing diseases associated with Arf1 pathway 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 the Arf1 pathway, particularly the COPI / Arf1-lipolysis β-oxidation pathway.

[0013] 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:

[0014] in,

[0015] X1 does not exist or is CH;

[0016] X2 is selected from C or N;

[0017] X3 is selected from S, O, CH or N;

[0018] represents a single bond or a double bond;

[0019] X4, X5, X6, X7, X8, X9 and X 10 Each independently selected from C(R x ) or N, R x independently selected from H, D (deuterium), CN, OH, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy and C 1-6 The group consisting of haloalkoxy, such as H, D, CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 the group consisting of haloalkoxy, halogen or halo is selected from F, Cl, Br, or I;

[0020] 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 and optionally substituted with 1-3 R b C 1-6an alkoxy group, or R1 and R2 together with the carbon atom to which they are attached form an optionally substituted 1-3 R c C 5-8 a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the 5- to 8-membered heterocyclic ring optionally containing 1 or more heteroatoms selected from N, O and S, such as 1, 2 or 3 heteroatoms selected from N, O and S;

[0021] R3 is 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 1-6 Alkoxy and N(R n )2, where each R n are each independently H, D, optionally substituted with 1-3 R a C 1-6 Alkyl and optionally substituted with 1-3 R g C 6-14 aryl;

[0022] R4 is selected from H and optionally substituted with 1-3 R a C 1-6 Alkyl, for example optionally substituted with 1, 2 or 3 R a C 1-4 alkyl;

[0023] L is absent or selected from optionally substituted with 1-3 R d C 1-6 Hydrocarbylene and optionally substituted with 1-3 R e C 1-6 Heteroalkylene, the heteroalkylene optionally containing one or more heteroatoms selected from N, O and S, for example optionally substituted with 1-3 R d C 1-4 Hydrocarbylene and optionally substituted with 1-3 R e C 1-4 Heteroalkylene, optionally substituted with 1-3 R d C 1-2 Hydrocarbylene and optionally substituted with 1-3 R e C 1-2 heteroalkylene;

[0024] Ring D is absent or optionally substituted with 1-3 R f C 5-6 Carbocyclic or 5- to 6-membered heterocyclic ring;

[0025] Each time it appears, each R a 、R b 、R c 、Rd 、R e 、R f 、R g Each independently selected from D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)-C 1-6 The group consisting of alkyl, oxo (=O) and -NH2, such as D, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, -C(O)-C 1-4 the group consisting of alkyl, oxo (=O) and -NH2, halogen or halo is selected from F, Cl, Br, or I;

[0026] Or, each time it occurs, each R a 、R b 、R c 、R d 、R e 、R f 、R g Each independently selected from D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -C(O)-C 1-6 Alkyl, -C(O)-C 1-6 Haloalkyl, -C(O)-C 1-6 Hydroxyalkyl, -C(O)-C 1-6 Aminoalkyl, -C(O)-C 5-14 Aryl, -C(O)-C 5-14 The group consisting of heteroaryl, oxo (=O) and -NH2, wherein the C 5-14 Aryl and C 5-14 Heteroaryl is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, for example, 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 Halogenated alkoxy, C 1-4 Hydroxyalkyl, C 1-4 Aminoalkyl, -C(O)-C 1-4 Alkyl, -C(O)-C 1-4 Haloalkyl, -C(O)-C1-4 Hydroxyalkyl, -C(O)-C 1-4 aminoalkyl, -C(O)- optionally substituted C 5-6 Aryl, -C(O)- optionally substituted C 5-6 The group consisting of heteroaryl, oxo (=O) and -NH2, wherein the optionally substituted C 5-6 Aryl and optionally substituted C 5-6 Heteroaryl is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, halogen or halo is selected from F, Cl, Br, or I;

[0027] Or, each time it occurs, each R a 、R b 、R c 、R d 、R e 、R f 、R g Each independently selected from C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -C(O)-C 1-6 Haloalkyl, -C(O)-C 1-6 Hydroxyalkyl, -C(O)-C 1-6 Aminoalkyl, -C(O)-C 5-14 Aryl, -C(O)-C 5-14 The group consisting of heteroaryl groups, wherein the C 5-14 Aryl and C 5-14 Heteroaryl is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, for example, 1-4 Hydroxyalkyl, C 1-4 Aminoalkyl, -C(O)-C 1-4 Haloalkyl, -C(O)-C 1-4 Hydroxyalkyl, -C(O)-C 1-4 aminoalkyl, -C(O)- optionally substituted C 5-6 Aryl, -C(O)- optionally substituted C 5-6 The group consisting of heteroaryl wherein the optionally substituted C 5-6 Aryl and optionally substituted C 5-6 The heteroaryl group is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH, or -NH2, halogen or halo selected from F, Cl, Br, or I.

[0028] In some embodiments, each occurrence of each R a 、R b 、R c 、R d 、R e 、Rf 、R g Each is independently optionally substituted with 1-3, such as 1, 2 or 3, substituents selected from the group consisting of D, halogen, -OH or -NH2.

[0029] When used in this article, Represents a single bond or a double bond. Those skilled in the art will understand that when “ When the "substitution" is a single bond or a double bond, the resulting compound molecule should satisfy the valence bond theory.

[0030] 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.

[0031] In some embodiments, X1 is absent, and Ring A is a C5 carbocycle or a 5-membered heterocycle having the structure shown below:

[0032] In some embodiments, X1 is CH, and Ring A is a C6 carbocycle or a 6-membered heterocycle having the structure shown below:

[0033] In some embodiments, Ring A is selected from the following ring structures:

[0034] In some embodiments, R1 and R2 are each independently absent or selected from H, D (deuterium), CN, OH, halogen, optionally substituted with 1-3 R a C 1-4 Alkyl such as halo C 1-4 Alkyl and optionally substituted with 1-3 R b C 1-4 Alkoxy such as halo C 1-4 The group consisting of alkoxy groups, such as H, D, CN, OH, F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, propoxy, butoxy, etc.

[0035] In some embodiments, R1 and R2 are each independently absent or selected from H, D (deuterium), CN, OH, halogen, optionally substituted with 1-3, for example 1, 2 or 3 R a C 1-4 Alkyl and optionally substituted with 1-3, for example 1, 2 or 3 R b C 1-4 In some embodiments, each occurrence of each R a are independently selected from D and halogen such as F, Cl, Br and I, each R bare each independently selected from D and halogen such as F, Cl, Br and I.

[0036] In some embodiments, R1 and R2 are each independently selected from H, D, CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 In some embodiments, R1 and R2 are each independently selected from the group consisting of H, D, CN, OH, F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, propoxy, butoxy, etc. In some embodiments, R1 and R2 are each independently selected from the group consisting of H, D, Cl, methyl, ethyl, trifluoromethyl, methoxy, etc.

[0037] In some embodiments, Ring A is selected from the following ring structures:

[0038] In some embodiments, R1 and R2 together with the carbon atom to which they are attached form a C 5-8 The carbocyclic ring or 5- to 8-membered heterocyclic ring is selected from the following ring structures:

[0039] in, represents a single bond or a double bond, the C 5-8 Carbocyclic or 5- to 8-membered heterocyclic rings are optionally substituted with 1-3 R c . Each time it appears, each R c Each independently selected from D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -C(O)-C 1-6 Alkyl, -C(O)-C 1-6 Haloalkyl, -C(O)-C 1-6 Hydroxyalkyl, -C(O)-C 1-6 Aminoalkyl, -C(O)-C 5-14 Aryl, -C(O)-C 5-14 The group consisting of heteroaryl, oxo (=O) and -NH2, wherein the C 5-14 Aryl and C 5-14 Heteroaryl is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2; for example, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4Alkoxy, C 1-4 Halogenated alkoxy, C 1-4 Hydroxyalkyl, C 1-4 Aminoalkyl, -C(O)-C 1-4 Alkyl, -C(O)-C 1-4 Haloalkyl, -C(O)-C 1-4 Hydroxyalkyl, -C(O)-C 1-4 aminoalkyl, -C(O)- optionally substituted C 5-6 Aryl, -C(O)- optionally substituted C 5-6 The group consisting of heteroaryl, oxo (=O) and -NH2, wherein the optionally substituted C 5-6 Aryl and optionally substituted C 5-6 Heteroaryl is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH, or -NH2. In some embodiments, optionally substituted aryl and optionally substituted heteroaryl are each independently optionally substituted with 1, 2, or 3 substituents selected from the group consisting of D, halogen, -OH, or -NH2. In some embodiments, halogen or halo is selected from F, Cl, Br, or I.

[0040] In some embodiments, each occurrence of each R c Each independently optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, for example R c The alkyl portion, aryl portion or heteroaryl portion in the group is further optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, such as deuterated alkyl, hydroxyalkyl, deuterated aryl, halogenated aryl, deuterated heteroaryl, halogenated heteroaryl, etc.

[0041] In some embodiments, R c Optionally connected to a nitrogen atom of a nitrogen-containing 5- to 8-membered heterocyclic ring. In some embodiments, when R1 and R2 together with the carbon atom to which they are attached form C 5-8 When it is a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the fused ring structure formed together with Ring A is selected from the following structures:

[0042] It is optionally substituted with 1-3 R c , each time it appears, each R c Each independently selected from D, halogen, C 1-6 Alkyl groups such as C 1-5 Alkyl or C 1-4 Alkyl, C 1-6 Haloalkyl such as C 1-4 Halogenated alkyl, C 1-6 Alkoxy groups such as C 1-4 Alkoxy, C 1-6Haloalkoxy such as C 1-4 Halogenated alkoxy, C 1-6 Hydroxyalkyl such as C 1-4 Hydroxyalkyl, C 1-6 Aminoalkyl such as C 1-4 Aminoalkyl, -C(O)-C 1-6 Alkyl groups such as -C(O)-C 1-4 Alkyl, -C(O)-C 1-6 Hydroxyalkyl such as -C(O)-C 1-4 Hydroxyalkyl, -C(O)-C 1-6 Aminoalkyl groups such as -C(O)-C 1-4 aminoalkyl, -C(O)- optionally substituted C 5-14 Aryl such as -C(O)- optionally substituted C 5-6 Aryl, -C(O)- optionally substituted C 5-14 Heteroaryl, such as -C(O)-, optionally substituted C 5-6 In some embodiments, each R c Each independently optionally substituted with 1-3, for example 1, 2 or 3, substituents selected from the group consisting of D, halogen, -OH or -NH2, for example R c The alkyl, aryl or heteroaryl moieties in the group are further optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, such as deuterated alkyl, hydroxyalkyl, deuterated aryl, halogenated aryl, deuterated heteroaryl, halogenated heteroaryl, etc. In some embodiments, R c Optionally attached to a nitrogen atom of a nitrogen-containing 5- to 8-membered heterocyclic ring.

[0043] In some embodiments, when R1 and R2 together with the carbon atom to which they are attached form C 5-8 When it is a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the fused ring structure formed together with Ring A is selected from the following structures:

[0044] In some embodiments, when R1 and R2 together with the carbon atom to which they are attached form C 5-8 When it is a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the fused ring structure formed together with Ring A is selected from the following structures:

[0045] In some embodiments, when R1 and R2 together with the carbon atom to which they are attached form C 5-8When it is a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the fused ring structure formed together with Ring A is selected from the following structures:

[0046] In some embodiments, Ring B is selected from the following structures:

[0047] In some embodiments, the R substituted on Ring B is x Selected from H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 The group consisting of haloalkoxy, halogen or halo is selected from F, Cl, Br, or I.

[0048] In some embodiments, R3 is selected from H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Alkoxy, NH2, NHR n or N(R n )2, where R n Selected from C 1-4 Alkyl and optionally substituted with 1-3, for example 1, 2 or 3 R g Each occurrence of phenyl, each R g independently selected from halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 The group consisting of haloalkoxy and -NH2, halogen or halo is selected from F, Cl, Br, or I.

[0049] In some embodiments, Ring B is selected from the following ring structures:

[0050] In some embodiments, Ring A and Ring B together form a ring structure selected from the group consisting of:

[0051] Among them, R1, R2, R3 and R x Each is independently as defined herein.

[0052] In some embodiments, R1 and R2 are each independently absent or selected from H, D (deuterium), CN, OH, halogen, optionally substituted with 1-3, for example 1, 2 or 3 R a C 1-4 Alkyl and optionally substituted with 1-3, for example 1, 2 or 3 R b C 1-4In some embodiments, R1 and R2 are each independently selected from H, D, CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 In some embodiments, R1 and R2 are each independently selected from, for example, H, D, CN, OH, F, Cl, Br, I, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, propoxy, butoxy, etc. In some embodiments, R substituted on ring B is x Selected from H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 In some embodiments, R3 is selected from the group consisting of H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Alkoxy, NH2, NHR n or N(R n )2, where R n Selected from C 1-4 Alkyl and optionally substituted with 1-3, for example 1, 2 or 3 R g Each occurrence of phenyl, each R g independently selected from halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 The group consisting of haloalkoxy and -NH2, halogen or halo is selected from F, Cl, Br, or I.

[0053] In some embodiments, Ring A and Ring B together form a ring structure selected from the group consisting of:

[0054] In some embodiments, R1 and R2 together with the carbon atom to which they are attached form a C 5-8 When it is a carbocyclic ring or a 5- to 8-membered heterocyclic ring, it, together with Ring A and Ring B, forms a fused ring structure selected from the following structures:

[0055] Among them, the C 5-8 Carbocyclic or 5- to 8-membered heterocyclic rings are optionally substituted with 1-3 R c , each time it appears, each R c Each independently selected from D, halogen, C 1-6 Alkyl groups such as C 1-5 Alkyl or C1-4 Alkyl, C 1-6 Haloalkyl such as C 1-4 Halogenated alkyl, C 1-6 Alkoxy groups such as C 1-4 Alkoxy, C 1-6 Haloalkoxy such as C 1-4 Halogenated alkoxy, C 1-6 Hydroxyalkyl such as C 1-4 Hydroxyalkyl, C 1-6 Aminoalkyl such as C 1-4 Aminoalkyl, -C(O)-C 1-6 Alkyl groups such as -C(O)-C 1-4 Alkyl, -C(O)-C 1-6 Hydroxyalkyl such as -C(O)-C 1-4 Hydroxyalkyl, -C(O)-C 1-6 Aminoalkyl groups such as -C(O)-C 1-4 aminoalkyl, -C(O)- optionally substituted C 5-14 Aryl such as -C(O)- optionally substituted C 5-6 Aryl, -C(O)- optionally substituted C 5-14 Heteroaryl, such as -C(O)-, optionally substituted C 5-6 In some embodiments, each R c Each independently optionally substituted with 1-3, for example 1, 2 or 3, substituents selected from the group consisting of D, halogen, -OH or -NH2, for example R c The alkyl, aryl or heteroaryl moieties in the group are further optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2, such as deuterated alkyl, hydroxyalkyl, deuterated aryl, halogenated aryl, deuterated heteroaryl, halogenated heteroaryl, etc. In some embodiments, R c is optionally attached to a nitrogen atom of a nitrogen-containing 5- to 8-membered heterocyclic ring. In some embodiments, R substituted on ring B x Selected from H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 In some embodiments, R3 is selected from the group consisting of H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Alkoxy, NH2, NHRn or N(R n )2, where R n Selected from C 1-4 Alkyl and optionally substituted with 1-3, for example 1, 2 or 3 R g Each occurrence of phenyl, each R g independently selected from halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy, C 1-4 The group consisting of haloalkoxy and -NH2, halogen or halo is selected from F, Cl, Br, or I.

[0056] In some embodiments, Ring A and Ring B together form a ring structure selected from the group consisting of:

[0057] In some embodiments, R4 is selected from H and optionally substituted with 1-3 R a C 1-6 Alkyl, for example optionally substituted with 1, 2 or 3 R a C 1-4 Alkyl groups such as methyl, ethyl, propyl or butyl, or halomethyl, haloethyl, halopropyl or halobutyl.

[0058] In some embodiments, X6, X7, X8, X9 and X 10 Each independently selected from C(R x ).

[0059] In some embodiments, when Ring D is absent, Ring C is optionally substituted with 1-5 R x For example, 1, 2, 3, 4 or 5 R x benzene ring.

[0060] In some embodiments, when Ring D is present, Ring C is optionally substituted with 1-3 R x For example, 1, 2 or 3 R x benzene ring.

[0061] In some embodiments, X6, X7, X8, X9 and X 10 Any one of them is N, and the rest are C(R x ).

[0062] In some embodiments, when Ring D is absent, Ring C is optionally substituted with 1-4 R x For example, 1, 2, 3 or 4 R x of the pyridine ring.

[0063] In some embodiments, when Ring D is present, Ring C is optionally substituted with 1-2 R x For example, 1 or 2 R x of the pyridine ring.

[0064] In some embodiments, the R substituted on ring C x Selected from H, D (deuterium), CN, OH, halogen, C 1-4 Alkyl, C 1-4 Halogenated alkyl, C 1-4 Alkoxy and C 1-4 The group consisting of haloalkoxy, halogen or halo is selected from F, Cl, Br, or I.

[0065] In some embodiments, Ring C is selected from the following ring structures:

[0066] In some embodiments, when Ring D is present, Ring C is selected from the following ring structures:

[0067] In some embodiments, Ring D is selected from optionally substituted with 1-3 R f For example, 1, 2 or 3 R f The following ring structure:

[0068] Among them, "1" indicates the connection direction with X8 of ring C, and "2" indicates the connection direction with X9 of ring C.

[0069] In some embodiments, Ring D is selected from optionally substituted with 1-3 R f For example, 1, 2 or 3 R f The following ring structure: Among them, "1" indicates the connection direction with X8 of ring C, and "2" indicates the connection direction with X9 of ring C.

[0070] In some embodiments, Ring D is selected from optionally substituted with 1-3 R f For example, 1, 2 or 3 R f The following ring structure:

[0071] Among them, "1" indicates the connection direction with X8 of ring C, and "2" indicates the connection direction with X9 of ring C.

[0072] In some embodiments, each occurrence of each R f Each independently selected from D, halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6A group consisting of haloalkoxy.

[0073] In some embodiments, each occurrence of each R f Each independently selected from halogen such as, C 1-4 Alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc., C 1-4 Haloalkyl such as trifluoromethyl, trifluoroethyl such as -CH2CF3, etc., C 1-4 Alkoxy groups such as methoxy, ethoxy, propoxy, butoxy, etc., C 1-4 haloalkoxy, and oxo (=O), etc.

[0074] In some embodiments, Ring D is selected from the following ring structures:

[0075] In some embodiments, Ring D is selected from the following ring structures:

[0076] In some embodiments, Ring D is selected from the following ring structures:

[0077] In some embodiments, Rings C and D collectively form the following fused ring structure:

[0078] Ring C is optionally substituted with 1-3 R x , Ring D is optionally substituted with 1-3 R f .

[0079] In some embodiments, Rings C and D collectively form the following fused ring structure:

[0080] Ring C is optionally substituted with 1-3 R x , Ring D is optionally substituted with 1-3 R f .

[0081] In some embodiments, Rings C and D collectively form the following fused ring structure:

[0082] Ring C is optionally substituted with 1-3 R x , Ring D is optionally substituted with 1-3 R f .

[0083] In some embodiments, Rings C and D collectively form the following fused ring structure:

[0084] In some embodiments, Rings C and D collectively form the following fused ring structure:

[0085] In some embodiments, Rings C and D collectively form the following fused ring structure:

[0086] In some embodiments, when present, L is selected from optionally substituted with 1-3 R d For example, 1, 2 or 3 R d C 1-2 Hydrocarbylene and optionally substituted with 1-3 R e For example, 1, 2 or 3 R e C 1-2 Heteroalkylene, optionally containing 1 or 2 heteroatoms selected from N, O and S, each occurrence of R d and R e Each independently selected from C 1-4 Alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc., C 1-4 Halogenated alkyl groups such as trifluoromethyl, trifluoroethyl, etc., C 1-4 Alkoxy groups such as methoxy, ethoxy, propoxy, butoxy, etc., and C 1-4 Halogenated alkoxy, etc.

[0087] In some embodiments, when present, L is selected from the group consisting of -CH2-, -CH(CH3)-, -CH2CH2-, and -NH-CH=CH-.

[0088] In some embodiments, when present, L is selected from the group consisting of -CH2-, -CH(CH3)-, -CH2CH2-, -NH-CH=CH-, and -N=CH2-.

[0089] In some embodiments, the present disclosure provides a compound of formula (II) or a pharmaceutically acceptable salt, solvate, stereoisomer, or isotopic variant thereof:

[0090] Among them, R1, R2, R3, R4, X6, X7, X 10 and R f The definition of is as described above, and n is an integer of 1 to 3. The embodiments and specific embodiments described in detail above for formula (I) are also applicable to formula (II).

[0091] In some embodiments, the compound of formula (I) or formula (II) is selected from the following compounds:

[0092] In one aspect, the present disclosure provides a method for preparing a compound of formula (I), its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, the preparation method comprising the following synthetic route:

[0093] Wherein, X in SM1 represents a leaving group selected from halogen, such as F, Cl, Br or I; activated hydroxyl, such as OTs, OMs or OTf, etc. SM1 and SM2 react in the presence of a suitable base such as TEA, DIPEA, pyridine or a suitable acid such as TFA or HCl. The preferred reaction temperature under base catalysis is 20-120°C, or 20-40°C, and the preferred reaction temperature under acid catalysis is 70-90°C.

[0094] 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.

[0095] In one aspect, the present disclosure provides a method for inhibiting intracellular Arf1 pathway activity in a cell, comprising administering an effective amount of a compound of formula (I), its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof to the cell, such that the Arf1 pathway activity in the cell is reduced.

[0096] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0097] In some embodiments, the methods induce cell death at the cellular level.

[0098] In some embodiments, the method is performed in vitro.

[0099] In some embodiments, the method is performed in vivo.

[0100] 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 Arf1 pathway activity in a cell.

[0101] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0102] In some embodiments, the agent induces cell death at the cellular level.

[0103] In some embodiments, the agent is administered to the cell in vitro.

[0104] In some embodiments, the agent is administered to the cell in vivo.

[0105] In one aspect, the present disclosure provides a method for treating or preventing a disease associated with Arf1 pathway 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.

[0106] In some embodiments, the disease associated with Arf1 pathway activity is cancer or a tumor.

[0107] 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.

[0108] In some embodiments, the disease associated with Arf1 pathway 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.

[0109] In some embodiments, the subject is a mammal, such as a human.

[0110] In some embodiments, the method further comprises co-administering at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

[0111] 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.

[0112] 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 for use in treating or preventing a disease associated with Arf1 pathway activity in a subject.

[0113] In some embodiments, the disease associated with Arf1 pathway activity is cancer or a tumor.

[0114] 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.

[0115] In some embodiments, the disease associated with Arf1 pathway 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.

[0116] In some embodiments, the subject is a mammal, such as a human.

[0117] 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.

[0118] 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.

[0119] 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 Arf1 pathway activity in a subject.

[0120] In some embodiments, the disease associated with Arf1 pathway activity is cancer or a tumor.

[0121] 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.

[0122] In some embodiments, the disease associated with Arf1 pathway 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.

[0123] In some embodiments, the subject is a mammal, such as a human.

[0124] In one aspect, the present disclosure provides a kit for treating or preventing a disease associated with Arf1 pathway activity in a subject, the kit comprising a compound of formula (I) described herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition described herein; a container; and optionally a package insert or label indicating treatment.

[0125] In some embodiments, the disease associated with Arf1 pathway activity is cancer or a tumor.

[0126] 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.

[0127] In some embodiments, the disease associated with Arf1 pathway 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.

[0128] In some embodiments, the subject is a mammal, such as a human.

[0129] In some embodiments, the kit further comprises at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

[0130] In one aspect, the present disclosure provides a kit for diagnosing a disease associated with Arf1 pathway 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.

[0131] In some embodiments, the test agent detects at least one biomarker indicative of the presence of a disease associated with Arf1 pathway activity.

[0132] In some embodiments, the test reagent can detect Arf1 GTPase activity, act as a reporter gene to detect lipolysis activity, detect lipid droplet formation, detect autophagy activity, or act as an upstream or downstream surrogate regulator of Arf1 activity or function.

[0133] In some embodiments, the disease associated with Arf1 pathway activity is cancer or a tumor.

[0134] 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.

[0135] In some embodiments, the disease associated with Arf1 pathway 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.

[0136] In some embodiments, the subject is a mammal, such as a human.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In one aspect, the present disclosure provides a vaccination method comprising administering a therapeutic vaccine prepared by the method described herein to a subject.

[0143] In some embodiments, the subject is a mammal, such as a human.

[0144] In one aspect, the present disclosure provides a method for killing cells and inducing an anti-tumor immune response, the method comprising inhibiting at least one Arf1 activity, particularly the COPI / Arf1-lipolysis β-oxidation pathway activity, in cells using an Arf1 pathway inhibitor.

[0145] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0146] In some embodiments, the Arf1 pathway inhibitor is selected from the group consisting of a small molecule Arf1 inhibitor, an RNAi agent directed against Arf1, an antisense agent directed against Arf1, a peptidomimetic Arf1 inhibitor, and a DNA binding deoxynucleotide inhibitor containing the target protein Arf1.

[0147] In some embodiments, the small molecule Arf1 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

[0148] FIG1 shows the workflow for developing and functional characterizing Arf1-specific inhibitors disclosed herein.

[0149] Figure 2 shows the solubility of compounds 3 and 9 of the present disclosure in DMSO and H2O, wherein A is the solubility of the compound in DMSO; B is the solubility of the compound in H2O.

[0150] Figure 3 shows that compounds 3 and 9 of the present disclosure can effectively inhibit Arf1 activation. A is the Western blot result of Arf1 activation. Specifically, DU101 refers to compound DU101 disclosed in WO2022 / 028429A1, 3 refers to compound DU101-3, 4 refers to compound DU101-4, 6 refers to compound DU101-6, and 8 refers to compound DU101-8; B is a quantitative analysis of the degree of Arf1 inhibition by compound 3; C is a quantitative analysis of the degree of Arf1 inhibition by compound 9.

[0151] Figure 4 shows that compounds 3 and 9 of the present disclosure induce Golgi apparatus defects in tumor cells, wherein A shows the effects of compound 3 and GCA on Golgi apparatus morphology; B shows the effects of compound 9 and GCA on Golgi apparatus morphology.

[0152] Figure 5 shows the effects of compounds 3 and 9 of the present disclosure on the expression level of chemokine CCL5, wherein A represents the degree of CCL5 expression induced by compound 3; and B represents the degree of CCL5 expression induced by compound 9.

[0153] FIG6 shows the effects of compounds 3 and 9 of the present disclosure on the activation of P65, a key protein in the NF-κB pathway.

[0154] Figure 7 shows the test results of the Drosophila tumor growth inhibition experiment of compounds 3 and 9 of the present disclosure, wherein A represents the degree of inhibition of Drosophila in situ tumor growth by compound 3; B represents the degree of inhibition of Drosophila in situ tumor growth by compound 9.

[0155] Figure 8 shows the inhibitory effect of compound 9 of the present disclosure on B16-F10 melanoma in C57B / 6 mice, where A shows that compound 9 and Du102 significantly inhibited the size of melanoma in mice; and B shows that compound 9 and Du102 significantly reduced the weight of melanoma.

[0156] Figure 9 shows the inhibitory effect of compound 9 of the present disclosure on CT26 colon cancer in BALB / c mice, wherein A represents the inhibition of colon cancer size in mice by compound 9; and B represents the significant reduction in colon cancer weight in mice by compound 9.

[0157] FIG10 shows the inhibitory effects of compounds 25, 26, and 27 of the present disclosure on CT26 colon cancer in BALB / c mice.

[0158] FIG11 shows the inhibitory effects of compounds 28, 30, 31, and 33 of the present disclosure on CT26 colon cancer in BALB / c mice. DETAILED DESCRIPTION

[0159] Certain embodiments will now be described in detail, examples of which are illustrated in the accompanying specific embodiments. Although the embodiments of the present invention will be described, it should be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present disclosure is intended to encompass all alternatives, modifications, and equivalents, which may be included within the scope of the present invention defined by the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention 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.

[0160] It will be appreciated that certain features of the present invention, 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 invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0161] definition

[0162] Terms used but not defined herein 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.

[0163] 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.

[0164] 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.

[0165] All ranges cited herein are inclusive unless expressly stated otherwise.

[0166] 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 .

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] As used herein, the term "lipolysis" refers to the breakdown of lipids and involves the hydrolysis of triglycerides into glycerol and free fatty acids. Lipolysis occurs primarily in adipose tissue and is used to mobilize stored energy during fasting or exercise.

[0176] As used herein, the term "guanosine triphosphatase" refers to a hydrolase that can bind and hydrolyze guanosine triphosphate (GTP). GTP binding and hydrolysis both occur in the highly conserved G domain common to all GTPases.

[0177] As used herein, the term "β-oxidation" refers to the metabolic process by which fatty acid molecules are broken down in the cytosol of prokaryotes and the mitochondria of eukaryotes to produce acetyl-CoA, which enters the citric acid cycle, and NADH2 and FADH, which serve as coenzymes in the electron transport chain. β-oxidation is so named because the β carbon of the fatty acid is oxidized to a carbonyl group. β-oxidation is primarily catalyzed by the mitochondrial trifunctional protein, a multienzyme complex associated with the inner mitochondrial membrane, although some fatty acids are also oxidized in peroxisomes.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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).

[0183] 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."

[0184] 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%.

[0185] 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%).

[0186] 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."

[0187] 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."

[0188] 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.

[0189] In some embodiments, an alkyl group can be deuterated, also known as a "deuterated alkyl group."

[0190] 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.

[0191] Those skilled in the art will appreciate that, in some embodiments, other types of groups mentioned herein, 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."

[0192] 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.

[0193] 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.

[0194] As used herein, the term "alkoxy" refers to the group -O-alkyl, wherein alkyl has the meaning defined herein.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] As used herein, the term "oxo" refers to a divalent oxygen atom and the structure of oxo may be shown as =0.

[0200] 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.

[0201] As used herein, the term "hydroxy" refers to a chemical group in which one or more hydrogen atoms are replaced by a hydroxy substituent (-OH). For example, the term "hydroxyalkyl" refers to a structure in which one or more hydrogen atoms on an alkyl group are replaced by a hydroxyl group, such as hydroxymethyl (-CH2OH), hydroxyethyl (-CH2CH2OH), etc.

[0202] As used herein, the term "amino" refers to a chemical group in which one or more hydrogen atoms are replaced by an amino substituent (-NH2). For example, the term "aminoalkyl" refers to a structure in which one or more hydrogen atoms on an alkyl group are replaced by amino, such as methylamino (-CH2NH2), ethylamino (-CH2CH2NH2), etc.

[0203] 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.

[0204] 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.

[0205] 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).

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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).

[0210] 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.

[0211] Formula (I) compound can have one or more chiral (asymmetric) centers. The present disclosure encompasses all stereoisomeric forms of formula (I) compound. The asymmetric center present in the formula (I) compound 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.

[0212] The present disclosure includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example, mixtures of enantiomers and / or diastereomers of all ratios. Therefore, enantiomers are enantiomerically pure forms (as left-handed and right-handed enantiomers), racemic forms and two enantiomers of the disclosed subject matter in the form of mixtures of all ratios. 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 synthesis of compound 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.

[0213] Unless otherwise stated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms, that is, 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." The present disclosure is intended to include all pharmaceutically acceptable isotopic variants of the compounds of Formula (I). Examples of suitable isotopes for inclusion in the compounds of the present 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 variants of the compounds of formula (I), for example those incorporating radioactive isotopes, are useful in drug and / or substrate tissue distribution studies. In particular, those having isotopic variations which occur only after substitution with heavier isotopes, for example with deuterium ( 2H or D) in place of hydrogen) may offer certain therapeutic advantages, for example, due to greater metabolic stability, increased in vivo half-life, or reduced dosage requirements, and may therefore be used in certain circumstances. 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 an appropriate isotopically labeled reagent in place of the non-labeled reagent previously employed.

[0214] When describing the structures of compounds herein, the undesignated 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%).

[0215] 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.

[0216] One way to implement the present invention is to administer the compound of formula (I) in the form of a prodrug. Thus, certain derivatives of the compound of formula (I) may themselves have little or no pharmacological activity, but when administered to or on the body, they are converted into a compound of formula (I) having 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.

[0217] Prodrugs according to the present disclosure can be prepared, for example, by replacing appropriate functional groups present in 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.

[0218] 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.

[0219] Use and application

[0220] The compounds of the present disclosure (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 the COPI / Arf1-lipolysis β-oxidation pathway. Through this activity, the compounds of the present disclosure 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 Arf1 pathway activity; treat refractory, recurrent, or metastatic cancers; and selectively kill cancer cells using specific dosing regimens.

[0221] Therefore, the compounds of the present disclosure as Arf1 inhibitors are particularly useful for treating diseases and conditions associated with the Arf1 pathway, particularly the COPI / Arf1-lipolysis beta-oxidation pathway, 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 tumor, gastric cancer, multiple myeloma, leukemia, lymphoma, etc.

[0222] The compounds of the present disclosure can be used in an amount effective to treat a disease or condition as described herein. The compounds of the present disclosure can be used as the compound itself, or alternatively, as a pharmaceutically acceptable salt. For the purpose of administration and administration, the compounds of the present disclosure itself (compound of formula (I), its stereoisomer or isotopic variant) or its pharmaceutically acceptable salt, solvate will be referred to as the compound of the application or the compound of the present disclosure.

[0223] 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.

[0224] 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 "pathological conditions" (e.g., diseases, disorders or conditions, or one or more signs or symptoms thereof) as described herein or inhibiting their 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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).

[0232] Pharmaceutical composition

[0233] In some aspects, the present disclosure relates to pharmaceutical compositions 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.

[0234] 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 herein) or to aid in the production of a pharmaceutical product (i.e., the drug).

[0235] 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.

[0236] 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.

[0237] In yet another aspect, the present disclosure relates to kits for treating diseases and conditions associated with Arf1 pathway activity, such as cancer, comprising a compound of Formula (I) as provided herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising a compound of Formula (I) as provided herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, a container, and optionally a package insert or label indicating treatment.

[0238] Treatment

[0239] In yet another aspect, the present disclosure relates to a method of treating diseases and conditions associated with Arf1 pathway activity, such as cancer, in a subject in need thereof, comprising 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 Arf1 inhibitory activity of the compounds of the present disclosure.

[0240] As used herein, the term "subject in need thereof" refers to a subject who has a disease associated with Arf1 pathway activity, such as cancer, or a subject who has an increased risk of developing a disease or condition associated with Arf1 pathway activity relative to the population as a whole. 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.

[0241] As used herein, the term "diseases and conditions associated with Arf1 pathway activity" refers to any pathophysiological condition in which inhibition of Arf1 would be beneficial. In certain embodiments, the diseases and conditions associated with Arf1 pathway activity are cancers. In certain embodiments, the diseases and conditions associated with Arf1 pathway activity are cancers or tumors 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 Arf1 pathway 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.

[0242] In yet another aspect, the present disclosure relates to a compound of formula (I), stereoisomers, isotopic variants thereof, or pharmaceutically acceptable salts or solvates thereof as provided herein for use in treating diseases and conditions associated with Arf1 pathway activity, such as cancer or tumors.

[0243] In another aspect, the present disclosure relates to the use of a compound of formula (I), its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates as provided herein, in the preparation of a medicament for treating diseases and conditions associated with Arf1 pathway activity, such as cancer or tumors.

[0244] The compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof increase the expression of MHC-I and MHC-II, or the compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof enhance the infiltration and activation of T cells into tumors compared to DMSO; or the compound of formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof increase the expression of T cell activation markers, such as GzmA, GzmB and Perforin.

[0245] The compound of Formula (I), stereoisomers, isotopic variants thereof, or pharmaceutically acceptable salts or solvates thereof as provided herein increases the expression of at least one inflammatory cytokine or chemokine from IFNγ, IL-1β, CCL5, CXCL10, CXCL11 and CCL22.

[0246] The compound of Formula (I), stereoisomers, isotopic variants thereof, or pharmaceutically acceptable salts or solvates thereof as provided herein increases the expression of at least one inflammatory cytokine or chemokine from IFNγ, IL-1β, CCL5, CXCL10, CXCL11 and CCL22.

[0247] Detection and diagnosis

[0248] In yet another aspect, the present disclosure relates to a compound of formula (I) as provided herein, a stereoisomer, isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use as a test reagent for diagnosing a disease associated with Arf1 pathway activity in a subject.

[0249] In yet another aspect, the present disclosure relates to a kit for diagnosing a disease associated with Arf1 pathway activity in a subject, comprising at least one compound of Formula (I) as provided herein, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof as a test agent.

[0250] In some embodiments, the test agent detects at least one biomarker indicative of the presence of a disease associated with Arf1 pathway activity.

[0251] In some embodiments, the test reagent can detect Arf1 GTPase activity, act as a reporter gene to detect lipolysis activity, detect lipid droplet formation, detect autophagy activity, or act as an upstream or downstream surrogate regulator of Arf1 activity or function.

[0252] Vaccines and immunization

[0253] In yet 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 arresting tumor progression.

[0254] In yet 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 therapeutic vaccine for arresting tumor progression.

[0255] 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.

[0256] 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 herein, 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.

[0257] In another aspect, the present disclosure relates to a vaccination method comprising administering a therapeutic vaccine prepared according to the method provided herein to a subject, wherein the subject is a mammal, such as a human.

[0258] In another aspect, the present disclosure relates to a method for killing cells and inducing an anti-tumor immune response, the method comprising inhibiting at least one Arf1 activity, particularly the COPI / Arf1-lipolysis β-oxidation pathway activity, in cells using an Arf1 pathway inhibitor.

[0259] In some embodiments, the cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

[0260] In some embodiments, the Arf1 pathway inhibitor is selected from the group consisting of a small molecule Arf1 inhibitor, an RNAi agent directed against Arf1, an antisense agent directed against Arf1, a peptidomimetic Arf1 inhibitor, and a DNA binding deoxynucleotide inhibitor containing the target protein Arf1.

[0261] In some embodiments, the small molecule Arf1 inhibitor is a compound of Formula (I) as provided herein, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof.

[0262] synthesis

[0263] 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, Volumes I-XII, Wiley-Interscience.

[0264] The scheme described below is intended to provide a general description of the method 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 carried out 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 carried out at any desired point in the procedure using known methods, such as those described herein and in the chemical literature.

[0265] Example

[0266] To describe the present invention in more detail, the following examples are presented. The examples described herein are intended to illustrate the compounds, methods, and compositions provided herein and should not be construed in any way as limiting their scope.

[0267] 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.

[0268] The compounds of the present disclosure can be easily prepared according to the following reaction schemes and examples or modifications thereof, 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 may also be used. In addition, according to the reaction schemes and examples described herein, other methods for preparing the compounds of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, 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.

[0269] Example 1

[0270] Synthesis of compound 1

[0271] Step 1: Preparation of compound 1-2

[0272] Compound 1-1 (1.00 g, 6.28 mmol, 1 eq) was dissolved in EtOH (5.00 mL). Hydroxylamine hydrochloride (654 mg, 9.42 mmol, 1.5 eq) was added portionwise at 20°C over 0.5 hours. NaOAc (773 mg, 9.42 mmol, 1.5 eq) was then added at 20°C. The reaction mixture was heated to 65°C and stirred for 16.5 hours. LCMS analysis revealed nearly complete conversion of compound 1-1, with the main peak corresponding to the molecular weight of the desired product. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product, which was used directly in the next reaction. MS (ESI) m / z = 175.1 [M+H] + .

[0273] Step 2: Preparation of compound 1-3

[0274] Compound 1-2 (1.00 g, 5.74 mmol, 1 eq) and Pd / C (3.00 g, 2.83 mmol, 10.0% content, 1 eq) were mixed in EtOH (5.00 mL). The atmosphere was replaced with nitrogen and hydrogen three times each. The reaction solution was stirred at 20°C under a hydrogen atmosphere (15 psi) for 8 h. LCMS analysis revealed nearly complete conversion of compound 1-2, with the main peak corresponding to the molecular weight of the target product. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. This was then separated and purified by preparative HPLC (column: Phenomenex C18 75*30 mm*3 μm; mobile phase: [water (TFA)-acetonitrile]; B%: 0%-24% over 36 minutes) to obtain compound 1-3 (600 mg, 3.74 mmol, 65.2% yield) as a yellow solid. MS (ESI) m / z = 144.0 [M+H] + .

[0275] 1 H NMR: 400MHz, DMSO-d6δ (ppm) = 11.21 (br s,1H),7.64(s,1H),7.43(d,J=8.4Hz,1H),7.39(t,J=2.8Hz,1H),7.20(dd,J=8.4,1.6Hz,1H),6.45(td,J=2.0,1.2Hz,1H),4.05(br s,2H),4.02(br s, 1H), 1.55 (d, J = 6.8Hz, 2H).

[0276] Step 3: Preparation of compound 1

[0277] Compound 1-3 (600 mg, 3.74 mmol, 1 eq) was dissolved in THF (3.00 mL), and DIEA (968 mg, 7.49 mmol, 1.30 mL, 2 eq) and compound 1-4 (100 mg, 541 umol, 1 eq) were added. The reaction mixture was heated to 50°C and stirred for 16 h. LCMS analysis revealed near-complete conversion of compound 1-3, with the main peak representing the molecular weight of the desired product. The reaction mixture was cooled to 0°C, quenched with 5.00 mL of water, and extracted with EtOAc (10.0 mL). After separation, the organic phase was washed with saturated brine (5.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product, which was purified by column chromatography (silica, petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to afford compound 1 (30.0 mg, 94.1 umol, 2.51% yield) as a yellow solid. MS (ESI) m / z = 309.0 [M+H] + .

[0278] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.04 (br s,1H),8.45(s,1H),7.62(s,1H),7.35(d,J=8.4Hz,1H),7.32(t,J=2.8H z,1H),7.28-7.26(m,1H),7.24-7.15(m,1H),7.04-6.95(m,1H),6.38(br s,1H),5.65-5.56(m,1H),2.66(s,3H),1.65(d,J=6.8Hz,3H).

[0279] Example 2

[0280] Synthesis of compound 2

[0281] Step 1: Preparation of compound 2-2

[0282] Compound 2-1 (5.00 g, 34.5 mmol, 1 eq) was dissolved in CH3NO2 (40.1 g, 657 mmol, 35.5 mL, 19.1 eq), and NH4OAc (3.45 g, 44.8 mmol, 1.30 eq) was added. The mixture was heated to 110°C and stirred for 3 h. LCMS analysis revealed nearly complete conversion of compound 2-1, with the main peak corresponding to the molecular weight of the desired product. The reaction solution was concentrated to obtain a residue, which was purified by column chromatography (silica, petroleum ether / ethyl acetate = 99 / 1 to 3 / 1) to afford compound 2-2 (3.57 g, 19.0 mmol, 55.1% yield) as an orange solid. MS (ESI) m / z = 189.1 [M+H] +.

[0283] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.51 (br s,1H),8.26-8.21(m,1H),8.17-8.12(m,1H),8.08(s,1H),7.61(dd,J=8.6,1.6Hz,1H),7.49-7.44(m,2H),6.54(d,J=2.1Hz,1H).

[0284] Step 2: Preparation of compound 2-3

[0285] Compound 2-2 (2.47 g, 13.1 mmol, 1 eq) was dissolved in MeOH (17.0 mL), and NaBH4 (1.78 g, 47.1 mmol, 3.58 eq) was added. The reaction mixture was stirred at 25°C for 0.6 h. TLC (petroleum ether / ethyl acetate = 3 / 1) showed nearly complete conversion of compound 2-2. The reaction was quenched with 20.0 mL of water at 0-10°C, and the pH was adjusted to approximately 7.0 with acetic acid. The mixture was extracted twice with EtOAc (20 mL). The organic phases were combined, washed twice with saturated brine (10.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. Purification by column chromatography (silica, petroleum ether / ethyl acetate = 99 / 1 to 4 / 1) afforded compound 2-3 (1.04 g, 5.47 mmol, 41.7% yield) as a yellow oil.

[0286] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.04 (br s,1H),7.39(s,1H),7.34-7.28(m,2H),6.98(dd,J=8.3,1.6Hz,1H),6.36(t,J=2.1Hz,1H),4.82(t,J=7.0Hz,2H),3.26(t,J=7.0Hz,2H).

[0287] Step 3: Preparation of compound 2-4

[0288] Compound 2-3 (1.04 g, 5.47 mmol, 1 eq) was dissolved in MeOH (2.2 mL), and Pd / C (100 mg, 547 umol, 10% content, 0.1 eq) was added. The atmosphere was replaced with argon and then hydrogen three times. The reaction solution was stirred at 25°C under a hydrogen atmosphere (15 psi) for 12 h. LCMS analysis revealed that compound 2-3 was essentially completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction solution was filtered, and the filtrate was concentrated to obtain crude compound 2-4 (730 mg) as a white oil. MS (ESI) m / z = 161.1 [M+H] + .

[0289] 1 HNMR: (400MHz, DMSO-d6) δ (ppm) = 10.95 (br s, 1H), 7.34-7.23 (m, 3H), 6.91 (d, J = 8.3Hz, 1H), 6.33 (br s, 1H), 2.79-2.73 (m, 2H), 2.70-2.64 (m, 2H).

[0290] Step 4: Preparation of compound 2

[0291] Compound 2-4 (150 mg, 936 μmol, 1 eq) was dissolved in THF (3.00 mL), and DIEA (363 mg, 2.81 mmol, 489 μL, 3 eq) and compound 2-5 (173 mg, 936 μmol, 1 eq) were added. The reaction mixture was stirred at 25°C for 12 h. LCMS analysis revealed nearly complete conversion of compound 2-4, with the main peak corresponding to the molecular weight of the desired product. The reaction mixture was concentrated to obtain a crude product, which was suspended in EtOH (3.0 mL), stirred at 25°C for 3 h, and filtered. The filter cake was dried to yield compound 2 (44.0 mg, 142 μmol, 15.2% yield) as a white solid. MS (ESI) m / z = 309.1 [M+H] + .

[0292] 1 HNMR: (400MHz, DMSO-d6) δ (ppm) = 10.99 (br s, 1H), 8.35 (s, 1H), 7.42 (s, 1H), 7.35-7.25 (m, 2H), 7.11 (s, 1H), 7.00 (d, J = 8.0Hz, 1H), 6.70 (br t,J=5.3Hz,1H),6.36(br s,1H),3.79-3.72(m,2H),2.98(br t,J=7.3Hz,2H),2.47(s,3H).

[0293] Example 3

[0294] Synthesis of compound 3

[0295] Step 1: Preparation of compound 3-2

[0296] Compound 3-2 (143 mg, 1.08 mmol, 1 eq), compound 3-1 (143 mg, 1.08 mmol, 1 eq), and TFA (12.3 mg, 108 umol, 8.02 uL, 0.1 eq) were mixed in isopropanol (1.0 mL). The mixture was purged with nitrogen three times and heated to 85°C for 12 h under nitrogen. LCMS analysis revealed near-complete conversion of compound 3-1, with the main peak representing the molecular weight of the desired product. The reaction solution was concentrated to obtain a residue, which was then separated and purified by preparative HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [water (NH4HCO3)-acetonitrile]; B%: 18%-58%; 36 min) to afford compound 3 (51.9 mg, 181 umol, 16.8% yield) as an off-white solid. MS (ESI) m / z = 281.0 [M+H] + .

[0297] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.07 (br s,1H),8.31(s,1H),8.21(s,1H),7.79(d,J=1.6Hz,1H),7.38(d,J=8.6Hz,1H),7.3 5(t,J=2.7Hz,1H),7.28-7.24(m,2H),6.42(t,J=2.0Hz,1H),2.74(d,J=0.6Hz,3H).

[0298] Example 4

[0299] Synthesis of compound 4

[0300] Step 1: Preparation of compound 4

[0301] Compound 4-1 (200 mg, 1.12 mmol, 1 eq) and compound 4-2 (212 mg, 1.46 mmol, 1.3 eq) were dissolved in THF (2.0 mL). DIEA (217 mg, 1.68 mmol, 292 uL, 1.5 eq) was added, and the reaction mixture was stirred at 20°C for 12 h. LCMS analysis revealed nearly complete conversion of compound 4-1, with the main peak representing the molecular weight of the desired product. The reaction was quenched with 20.0 mL of water and extracted twice with EtOAc (20.0 mL). The combined organic phases were washed with saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was purified by column chromatography (silica, petroleum ether / ethyl acetate = 99 / 1 to 0 / 1) to obtain a crude product. The crude product was suspended in EtOH, stirred at 20°C for 12 h, filtered, and the filter cake was dried to obtain compound 4 (100 mg, 346 μmol, 25.8% yield) as a white solid. MS (ESI) m / z = 289.0 [M+H] + .

[0302] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 10.99 (s, 1H), 8.65 (t, J = 6.0Hz, 1H), 8.39 (s, 1H), 8.11 (s, 1H), 7.59 (s, 2H), 7.51 (s, 1 H),7.35-7.31(m,1H),7.29(t,J=2.8Hz,1H),7.15-7.10(m,1H),6.38-6.34(m,1H),4.83(d,J=6.0Hz,2H),2.45(s,3H).

[0303] Example 5

[0304] Synthesis of compound 5

[0305] Step 1: Preparation of compound 5-2

[0306] Compound 5-1 (20.0 g, 108 mmol, 1 eq) was dissolved in DMF-DMA (35.9 g, 301 mmol, 40.0 mL, 2.79 eq), heated to 100°C, and stirred for 12 h. LCMS analysis revealed near-complete conversion of compound 5-1, with the main peak corresponding to the molecular weight of the desired product. The reaction mixture was concentrated to obtain a crude product, which was suspended in H2O and stirred at 20°C for 10 min. The product was filtered and the filter cake was dried under reduced pressure to yield compound 5-2 (24.0 g, 99.8 mmol, 92.5% yield) as a yellow solid. MS (ESI) m / z = 240.9 [M+H] + .

[0307] HNMR: (400MHz, CDCl3) δ (ppm) = 7.63 (s, 1H), 6.22 (d, J = 0.8Hz, 1H), 4.35-4.17 (m, 2H), 3.07 (br d, J=8.0Hz, 6H), 2.31 (d, J=0.8Hz, 3H), 1.34 (t, J=7.2Hz, 3H).

[0308] Step 2: Preparation of compound 5-4

[0309] Compound 5-2 (24.0 g, 99.8 mmol, 1 eq) was dissolved in tert-butanol (50.0 mL), and compound 5-3 (56.4 g, 399 mmol, 57.1 mL, 4 eq) was added. The mixture was stirred at 20°C for 18 h. LCMS analysis revealed nearly complete conversion of compound 5-2, with the main peak corresponding to the molecular weight of the desired product. The reaction mixture was filtered, and the filter cake was dried to obtain crude compound 5-4 (27.3 g) as a yellow solid. MS (ESI) m / z = 337.0 [M+H] + .

[0310] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 12.52 (br d,J=13.2Hz,1H),7.55(d,J=13.2Hz,1H),6.37(s,1H),4.48-4.41(m,2H),2.37(d,J=0.8Hz,3H),1.57(s,9H),1.42(t,J=7.2Hz,3H).

[0311] Step 3: Preparation of compound 5-5

[0312] Compound 5-4 (27.0 g, 80.2 mmol, 1 eq) was dissolved in Ph2O (150.0 mL) and stirred at 255°C for 2 h. LCMS analysis revealed near-complete conversion of compound 5-4, with the target product having the corresponding molecular weight. The reaction mixture was cooled to 20°C, and n-hexane (300.0 mL) was added. The mixture was shaken and filtered. The filter cake was dried to afford crude compound 5-5 (11.5 g) as a brown solid. MS (ESI) m / z = 190.9 [M+H] + .

[0313] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 13.36 (br s, 1H), 8.56 (s, 1H), 7.00-6.98 (m, 1H), 2.47 (d, J = 0.8Hz, 3H).

[0314] Step 4: Preparation of compound 5-6

[0315] Compound 5-5 (500 mg, 2.63 mmol, 1 eq) was dissolved in toluene (2.5 mL), and POCl3 (2.02 g, 13.1 mmol, 1.22 mL, 5 eq) was added. The mixture was stirred at 100°C for 4 h. LCMS analysis revealed nearly complete conversion of compound 5-5, with the target product having the corresponding molecular weight. The reaction mixture was cooled to 25°C, added to water, and the pH was adjusted to approximately 10 with sodium bicarbonate. The layers were separated, and the organic phase was concentrated under reduced pressure to yield crude compound 5-6 (300 mg) as a yellow solid. MS (ESI) m / z = 208.8 [M+H] + .

[0316] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.94 (s, 1H), 7.85 (d, J = 0.8Hz, 1H), 2.65 (d, J = 1.2Hz, 3H) ppm.

[0317] Step 5: Preparation of compound 5

[0318] Compound 5-6 (100 mg, 479 umol, 1 eq), compound 5-7 (77.0 mg, 527 umol, 1.1 eq) and DIEA (124 mg, 958 umol, 167 uL, 2 eq) were mixed in THF (1.0 mL) and stirred at 20 ° C for 12 h. LCMS detection showed that compound 5-6 was basically completely converted, and the corresponding molecular weight of the target product was found. The reaction was quenched with 30.0 mL of water and extracted twice with EtOAc (20.0 mL). The combined organic phases were washed with saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to obtain a residue. This residue was separated and purified by preparative HPLC (column: Welch Xtimate C18 150*30 mm*5 μm; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; B%: 28%-68%, 36 min) to obtain compound 5 (30.0 mg, 92.9 μmol, 19.3% yield) as an off-yellow solid. MS (ESI) m / z = 319.1 [M+H] + .

[0319] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.10 (br s,1H),8.28(s,1H),7.56(s,1H),7.38(d,J=8.2Hz,1H),7.34-7.30(m,2H),7.13(d,J=8.2Hz,1H),6.93-6.86(m,1H),6.39(br s, 1H), 5.06 (d, J = 6.4Hz, 2H), 2.63 (s, 3H).

[0320] Example 6

[0321] Synthesis of compound 6

[0322] Step 1: Preparation of compound 6-3

[0323] Compound 6-1 (18.0 g, 142 mmol, 1 eq) and compound 6-2 (59.4 g, 570 mmol, 59.3 mL, 4 eq) were dissolved in pyridine (120.0 mL). Piperidine (1.21 g, 14.2 mmol, 1.41 mL, 0.1 eq) was added, and the mixture was heated to 120°C and stirred for 12 h. LCMS analysis revealed near-complete conversion of compound 6-1, and the molecular weight of the desired product was determined. The reaction mixture was cooled to 25°C and concentrated under reduced pressure to obtain a residue. The residue was added to water, and the pH was adjusted to 4.0 with 1N dilute hydrochloric acid. The residue was extracted twice with EtOAc (200 mL). The organic phases were combined, washed with saturated brine (50.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to afford crude compound 6-3 (20.0 g) as a yellow solid. MS (ESI) m / z = 169.0 [M+H] + .

[0324] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 9.78 (br s, 1H), 7.82 (d, J = 15.6Hz, 1H), 7.11 (s, 1H), 7.02 (s, 1H), 6.20 (d, J = 15.6Hz, 1H), 2.27 (s, 3H).

[0325] Step 2: Preparation of compound 6-4

[0326] Compound 6-3 (20.0 g, 118 mmol, 1 eq) and TEA (8.0 g, 178 mmol, 24.8 mL, 1.5 eq) were dissolved in acetone (150 mL). Ethyl chloroacetate (17.8 g, 130 mmol, 17.1 mL, 1.1 eq) was added at 0°C and stirred at 20°C for 20 min. Sodium azide (13.7 g, 211 mmol, 1.78 eq) was then added, and the mixture was stirred at 20°C for 6 h. LCMS analysis revealed nearly complete conversion of compound 6-3, and the target product was detected at the molecular weight. The reaction mixture was added to water, the pH was adjusted to 9.0 with sodium carbonate, and the mixture was extracted twice with EtOAc (150 mL). The organic phases were combined, washed sequentially with water (150 mL) and saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to afford crude compound 6-4 (20.0 g) as a yellow solid. MS (ESI) m / z = 166.0 [M + H-N2] + .

[0327] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.85 (d, J = 15.6Hz, 1H), 7.48 (s, 1H), 7.43 (s, 1H), 6.25 (d, J = 15.6Hz, 1H), 2.21 (s, 3H).

[0328] Step 3: Preparation of compound 6-5

[0329] Compound 6-4 (20.0 g, 103 mmol, 1 eq) was dissolved in xylene (130 mL), and I2 (5.25 g, 20.7 mmol, 4.17 mL, 0.2 eq) was added. The mixture was heated to 145°C and stirred for 6 h. LCMS analysis revealed nearly complete conversion of compound 6-4, and the molecular weight of the target product was determined. The reaction mixture was cooled, filtered, and the filter cake was dried to obtain a crude product. The crude product was suspended in MTBE / THF (10 / 1, 200 mL), stirred at 20°C for 12 h, filtered, and the filter cake was dried to obtain compound 6-5 (4.50 g, 27.2 mmol, 26.3% yield) as a black solid. MS (ESI) m / z = 166.0 [M+H] + .

[0330] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.24 (br s, 1H), 7.18-7.14 (m, 1H), 7.13 (s, 1H), 6.75 (d, J = 7.2Hz, 1H), 2.50 (br s, 3H).

[0331] Step 4: Preparation of compound 6-6

[0332] Compound 6-5 (1.00 g, 6.05 mmol, 1 eq) was dissolved in THF (10.0 mL). NIS (1.91 g, 8.47 mmol, 1.4 eq) was added at -78°C. The reaction mixture was slowly warmed to 20°C and stirred for 12 h. LCMS analysis revealed nearly complete conversion of compound 6-5 and the molecular weight of the desired product. The reaction mixture was filtered, and the filter cake was dried to yield crude compound 6-6 (600 mg) as a yellow solid. MS (ESI) m / z = 291.8 [M+H] + .

[0333] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.47 (br s, 1H), 7.43 (s, 1H), 7.25 (s, 1H), 2.47 (s, 3H).

[0334] Step 5: Preparation of compound 6-7

[0335] Compound 6-6 (550 mg, 1.89 mmol, 1 eq) was dissolved in DMF (0.5 mL), and CuCN (338 mg, 3.78 mmol, 825 uL, 2 eq) was added. The mixture was stirred at 145°C for 12 h. LCMS analysis revealed near-complete conversion of compound 6-6, and the target product had the desired molecular weight. The reaction was quenched with 30.0 mL of water and extracted twice with EtOAc (20.0 mL). The combined organic phases were washed with saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. Purification by column chromatography (silica, petroleum ether / ethyl acetate = 99 / 1 to 3 / 1) afforded compound 6-7 (165 mg, 867 umol, 45.9%) as a yellow solid. MS (ESI) m / z = 190.9 [M+H] + .

[0336] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.20 (br s, 1H), 8.19 (br s, 1H), 7.52-7.24 (m, 1H), 2.50 (s, 3H).

[0337] Step 6: Preparation of Compound 6-8

[0338] Phosphorus oxychloride (1.65 g, 10.7 mmol, 1 mL, 12.4) was added to compound 6-7 (165 mg, 867 μmol, 1 eq), and the mixture was heated to 100°C and stirred for 6 h. LCMS analysis revealed nearly complete conversion of compound 6-7, and the molecular weight of the desired product was determined. The reaction mixture was cooled to 25°C and slowly added to water. The pH was adjusted to 10.0 with sodium bicarbonate, filtered, and the filter cake dried to afford crude compound 6-8 (160 mg) as a yellow solid. MS (ESI) m / z = 208.9 [M+H] + .

[0339] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.79 (s, 1H), 7.72 (s, 1H), 2.66 (s, 3H).

[0340] Step 7: Preparation of compound 6

[0341] Compound 6-8 (150 mg, 718 μmol, 1 eq) and compound 6-9 (105 mg, 718 μmol, 1 eq) were dissolved in THF (1.0 mL), and DIEA (186 mg, 1.44 mmol, 250 μL, 2 eq) was added. The mixture was stirred at 25°C for 12 h. LCMS analysis showed that compound 6-8 was almost completely converted, and the target product had the corresponding molecular weight. The reaction was quenched with 30.0 mL of water and extracted twice with EtOAc (20.0 mL). The combined organic phases were washed with saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to obtain a residue. This residue was purified by preparative HPLC (column: Welch Ultimate XB-CN 250*50*10 μm; mobile phase: [Heptane-EtOH (0.1% NH3H2O)]; B%: 15%-45%, 15 min) to obtain compound 6 (30.0 mg, 91.1 μmol, 12.6% yield) as a yellow solid. MS (ESI) m / z = 318.9 [M+H] + .

[0342] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.00 (br s,1H),8.34(s,1H),7.51(s,1H),7.41(s,1H),7.37-7.31(m,2H),7.29(t,J=2.8Hz,1H),7.15-7.11(m,1H),6.36(br s, 1H), 4.84 (d, J = 6.0Hz, 2H), 2.68 (s, 3H).

[0343] Example 7

[0344] Synthesis of compound 7

[0345] Step 1: Preparation of compound 7

[0346] Compound 7-1 (78.1 mg, 534 μmol, 1 eq) was dissolved in THF (2.0 mL), and compound 7-2 (90.0 mg, 534 μmol, 1 eq) and DIEA (69.0 mg, 534 μmol, 93.0 μL, 1 eq) were added. The mixture was stirred at 25°C for 12 h. LCMS analysis revealed 17.2% of compound 7-2 remaining, with a target product content of 68.6%. The reaction solution was quenched with 3.0 mL of water and extracted twice with EtOAc (5.0 mL). The combined organic phases were washed twice with saturated brine (5.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was separated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 2 / 1) to obtain compound 7 (43.0 mg, 155 μmol, 28.9% yield) as a white solid. MS (ESI) m / z = 279.0 [M+H] + .

[0347] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 10.99 (br s, 1H), 8.19 (s, 1H), 7.56 (d, J = 1.3Hz, 1H), 7.50 (s, 1H), 7.45 (br t,J=6.2Hz,1H),7.32(d,J=8.3Hz,1H),7.29(t,J=2.7Hz,1H),7.12(dd,J=1 .3,8.3Hz,1H),6.44-6.32(m,1H),4.79(d,J=6.1Hz,2H),2.36-2.32(m,3H).

[0348] Example 8

[0349] Synthesis of compound 8

[0350] Step 1: Preparation of compound 8

[0351] Compound 8-1 (100 mg, 812 μmol, 1 eq), compound 8-2 (179 mg, 974 μmol, 1.2 eq), and TEA (164 mg, 1.62 mmol, 226 μL, 2 eq) were mixed in THF (0.5 mL). The atmosphere was replaced with nitrogen three times, and the reaction mixture was heated to 50°C and stirred under nitrogen for 16 hours. LCMS analysis revealed nearly complete conversion of compound 8-1, with the main peak representing the molecular weight of the desired product. The reaction mixture was cooled to room temperature and quenched with 5.0 mL of water. The mixture was extracted with EtOAc (5.0 mL). The organic phase was washed with saturated brine (5.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was purified by preparative HPLC (column: Welch Xtimate C18 150*30mm*5um; mobile phase: [water (NH4HCO3)-acetonitrile]; B%: 10%-50%, 36 minutes) to obtain compound 8 (35.0 mg, 122 umol, 15.0% yield) as a white solid. MS (ESI) m / z = 271.9 [M+H] + .

[0352] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.25 (s, 1H), 8.45 (s, 1H), 8.25-8.19 (m, 1H), 7.71 (d, J = 1.3 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 6.72-6.64 (m, 2H), 4.60 (d, J = 6.0 Hz, 2H), 2.33-2.31 (m, 3H).

[0353] Example 9

[0354] Synthesis of Compounds 9 and 10

[0355] Step 1: Preparation of compound 9

[0356] Compound 10-1 (500 mg, 2.71 mmol, 1 eq), compound 10-2 (398 mg, 2.71 mmol, 1 eq), and TFA (30.9 mg, 271 μmol, 20.1 μL, 0.1 eq) were mixed in isopropanol (3.0 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was heated to 85°C and stirred under nitrogen for 12 h. LCMS analysis showed that the starting material 10-1 was essentially completely converted. The reaction mixture was cooled to room temperature and filtered. The filter cake was suspended in acetonitrile (2.0 mL), stirred at 25°C for 3 h, filtered, and dried to obtain compound 9 (623 mg, 2.06 mmol, 76.2% yield) as a white solid. MS (ESI) m / z = 296.0 [M+H] + .

[0357] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.51 (s, 1H), 8.72 (br s,1H),8.48(s,1H),8.34(d,J=1.6Hz,1H),7.94(d,J=9.0Hz,1H),7.85-7.80(m,1H),7.38(d,J=1.0Hz,1H),4.07(s,3H),2.78(d,J=0.9Hz,3H).

[0358] Step 2: Preparation of compound 10

[0359] Compound 9 (400 mg, 1.35 mmol, 1 eq) was dissolved in DMF (4.0 mL), and MeI (346 mg, 2.44 mmol, 152 μL, 1.8 eq) and cesium carbonate (1.10 g, 3.39 mmol, 2.5 eq) were added. The reaction solution was stirred at 25°C for 4 h. TLC (dichloromethane / methanol = 10 / 1) showed that compound 9 was almost completely converted. The reaction solution was added to H2O (3.0 mL) and extracted twice with EtOAc (3.0 mL). The organic phases were combined, washed with saturated brine (4.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was purified by preparative high performance liquid chromatography (neutral conditions, column Xtimate C18 150*40 mm*10 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 12%-52% B After purification (36 minutes), compound 10 (50.0 mg, 162 μmol, 12.5% ​​yield) was obtained as a yellow solid. MS (ESI) m / z = 310.1 [M+H] + .

[0360] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.66 (s, 1H), 8.16 (s, 1H), 7.50 (d, J = 8.6Hz, 1H), 7.24 (d, J = 1.9Hz,1H),7.09(d,J=0.9Hz,1H),7.05-6.98(m,1H),3.81(s,3H),3.55(s,3H),1.43(s,3H).

[0361] Example 10

[0362] Synthesis of compound 11

[0363] Step 1: Preparation of compound 11-3

[0364] Compound 11-1 (200 mg, 613 μmol, 1 eq) was dissolved in isopropanol (2.0 mL), and compound 11-2 (90.1 mg, 613 μmol, 1 eq) and TFA (7.00 mg, 61.3 μmol, 4.56 μL, 0.1 eq) were added. The mixture was heated to 80°C and stirred for 12 h. LCMS analysis showed that compound 11-1 was almost completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction solution was cooled and concentrated under reduced pressure to obtain crude compound 11-3 (267 mg) as a brown solid. MS (ESI) m / z = 437.1 [M+H] + .

[0365] Step 2: Preparation of compound 11-4

[0366] Compound 11-3 (250 mg, 572 μmol, 1 eq) was dissolved in MeOH (2.0 mL) and a 1,4-dioxane solution of hydrogen chloride (4 M, 143 μL, 1 eq) was slowly added dropwise at 25°C. After the addition was complete, stirring was continued at 25°C for 2 h. LCMS analysis showed that compound 11-3 was essentially completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction solution was concentrated to obtain crude compound 11-4 (148 mg) as a white solid. MS (ESI) m / z = 337.0 [M+H] + .

[0367] Step 3: Preparation of compound 11

[0368] Compound 11-4 (148 mg, 441 μmol, 1 eq) was dissolved in EtOH (5.0 mL), and formaldehyde aqueous solution (184 mg, 2.21 mmol, 169 μL, 36.0% purity, 5 eq) was added dropwise at 20°C. After the addition, the reaction solution was stirred for 1 h, and then NaBH3CN (55.5 mg, 883 μmol, 2 eq) was added, and stirring was continued at 20°C for 12 h. LCMS analysis showed nearly complete conversion of compound 11-4, with the main peak corresponding to the molecular weight of the target product. Filtration and concentration of the filtrate afforded a residue, which was then purified by preparative HPLC (column: Xtimate C18 150*40mm*10um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 44%-84% B over 36 minutes) to afford compound 11 (35.0 mg, 99.8 μmol, 22.6% yield) as a white solid. MS (ESI) m / z = 351.2 [M+H] + .

[0369] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.33 (s, 1H), 8.22-8.13 (m, 2H), 7.95 (s, 1H), 7.56-7 .51(m,1H),7.48-7.43(m,1H),3.84(s,3H),3.64(s,2H),3.27-3.23(m,2H),2.75(br t,J=5.4Hz,2H),2.41(s,3H).

[0370] Example 11

[0371] Synthesis of compound 12

[0372] Step 1: Preparation of compound 12

[0373] Compound 12-1 (150 mg, 812 μmol, 1 eq), compound 12-2 (120 mg, 812 μmol, 1 eq), and TFA (9.26 mg, 81.2 μmol, 6.03 μL, 0.1 eq) were mixed in isopropanol (1.0 mL). The mixture was purged with nitrogen three times, heated to 85°C, and stirred under nitrogen for 12 h. LCMS analysis showed that 12-1 was essentially completely converted. The reaction mixture was cooled to room temperature and filtered. The filter cake was suspended in acetonitrile (2.0 mL), stirred at 25°C for 3 h, filtered, and dried to obtain compound 12 (156 mg, 516 μmol, 63.5% yield) as a white solid. MS (ESI) m / z = 297.1 [M+H] + .

[0374] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 10.59-10.39 (m, 1H), 8.64-8.31 (m, 2H), 7.48 (s,1H),7.39-7.14(m,2H),6.94-6.76(m,1H),3.56-3.50(m,2H),2.72(s,3H).

[0375] Example 12

[0376] Synthesis of compound 13

[0377] Step 1: Preparation of compounds 13-2 & 13-3

[0378] Compound 13-1 (5.00 g, 30.6 mmol, 1 eq), triethylamine (9.30 g, 91.9 mmol, 12.80 mL, 3 eq), and BoC2O (10.0 g, 45.9 mmol, 10.56 mL, 1.5 eq) were mixed in DCM (50 mL), the atmosphere was replaced with nitrogen three times, and the mixture was stirred at 20°C under a nitrogen atmosphere for 12 h. LCMS analysis showed that compound 13-1 was essentially completely converted. The reaction solution was poured into water and extracted twice with EtOAc (300 mL). The organic phases were combined, washed twice with saturated brine (300 mL), filtered, and the filtrate was concentrated under reduced pressure to obtain a residue. After separation and purification by column chromatography (silica), a mixture of compounds 13-2 and 13-3 (7.20 g, 27.3 mmol, 89.2% yield) was obtained as a white solid. MS (ESI) m / z = 208.0 [M+H] + .

[0379] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.01 (s, 1H), 8.93 (s, 1H), 8.77 (d, J = 2.3Hz, 1H), 8.62 (d, J = 2.3Hz, 1H), 8.34 (dd, J =2.3, 9.0Hz, 1H), 8.27 (dd, J = 2.4, 8.9Hz, 1H), 8.14 (d, J = 9.0Hz, 1H), 7.99 (d, J = 8.9Hz, 1H), 1.68 (d, J = 4.9Hz, 9H).

[0380] Step 2: Preparation of compounds 13-4 & 13-5

[0381] A mixture of compound 13-2 and compound 13-3 (5.00, 30.6 mmol, 1 eq), Pd / C (4.04 g, 3.79 mmol, 10% purity, 9.99e-2 eq) and triethylamine (3.84 g, 37.9 mmol, 5.29 mL, 1 eq) were mixed in a mixed solvent of ethanol (100 mL) and methanol (50 mL). Nitrogen and hydrogen were replaced three times each, and then the reaction solution was heated to 50 ° C and stirred in a hydrogen atmosphere (50 Psi) for 12 h. LCMS analysis showed that compounds 13-2 and 13-3 were essentially completely converted. The reaction mixture was filtered under reduced pressure, and the filtrate was concentrated to obtain a residue. The residue was purified by preparative HPLC (basic conditions, column: Phenomenex luna C18 250*50mm*10um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 0%-30% B over 20 minutes) to obtain a mixture of compounds 13-4 and 13-5 (1.24 g, 5.32 mmol, 14.0% yield) as a white solid. MS (ESI) m / z = 178.0 [M+H-56]. + .

[0382] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.25 (s, 1H), 7.46-7.27 (m, 1H), 7.21-7.06 (m, 1H), 7.14 (d, J = 1.9Hz, 1H), 6.69-6.45 (m, 2H), 1.63 (s, 9H).

[0383] Step 3: Preparation of compound 13

[0384] A mixture of compounds 13-4 and 13-5 (5.00, 30.6 mmol, 1 eq), compound 13-6 (340 mg, 1.84 mmol, 1 eq) and TFA (210 mg, 1.84 mmol, 136 μL, 1 eq) were mixed in isopropanol (8.6 mL) and replaced with nitrogen three times. The reaction mixture was also heated to 80° C. and stirred under a nitrogen atmosphere for 12 h. LCMS detection showed that compounds 13-4 and 13-5 were almost completely converted. The reaction solution was filtered under reduced pressure, and the filtrate was concentrated to obtain a residue, which was purified by preparative high-performance liquid chromatography (column: Xtimate C18 150*40mm*10um; mobile phase [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 4%-44% B, 36 min) to obtain compound 13 (314 mg, 1.12 mmol, 60.7% yield) as a white solid. MS (ESI) m / z = 282.1 [M+H] + .

[0385] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.40 (br s, 1H), 8.45-8.26 (m, 2H), 8.23-8.14 (m, 1H), 7.98 (br d,J=18.9Hz,1H),7.65-7.49(m,1H),7.45-7.24(m,2H),2.76(s,3H).

[0386] Example 13

[0387] Synthesis of compound 14

[0388] Step 1: Preparation of compounds 14-2 & 14-3

[0389] Compound 14-1 (5.00 g, 30.6 mmol, 1 eq) and iodoethane (5.74 g, 36.8 mmol, 1.2 eq) were dissolved in DMF (50 mL). Potassium hydroxide (2.58 g, 46.0 mmol, 1.5 eq) was added and stirred at 20°C for 12 h. LCMS analysis showed that compound 14-1 was essentially completely converted. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (basic conditions, column: Welch Xtimate C18 250*50 mm*10 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 0%-30% B over 25 minutes) to obtain a mixture of compounds 14-2 and 14-3 (5.0 g) as a yellow solid. MS (ESI) m / z = 192.0 [M+H] + .

[0390] Step 2: Preparation of compound 14-4

[0391] Compound 14-2&3 (3.79 g, 19.8 mmol, 1 eq), Pd / C (1.60 g, 1.50 mmol, 10% purity, 7.58 e-2 eq) and triethylamine (2.01 g, 19.8 mmol, 2.76 mL, 1 eq) were dissolved in ethanol (75 mL). The atmosphere was replaced with nitrogen and hydrogen three times each. The reaction solution was then heated to 50°C and stirred in a hydrogen atmosphere (50 Psi) for 12 h. LCMS analysis showed that compounds 14-2 and 14-3 were essentially completely converted. The reaction mixture was filtered under reduced pressure, and the filtrate was concentrated to obtain a residue. This residue was then isolated and purified by supercritical fluid chromatography (SFC) (neutral conditions, column: DAICEL CHIRALPAK AD (250 mm x 50 mm, 10 μm); mobile phase: [CO₂-EtOH (0.1% NH₃H₂O)]; B%: 45%, constant composition solvent elution) to obtain compound 14-4 (1.02 g, 6.30 mmol, 31.8% yield) as a white solid. MS (ESI) m / z = 162.0 [M+H] + .

[0392] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.86 (s, 1H), 7.28 (d, J = 8.5Hz, 1H), 6.60 (d, J = 2.0Hz, 1H), 6.52 (dd, J = 2.1, 8.6Hz, 1H), 4.98 (br d, J=4.6Hz, 2H), 4.08 (q, J=7.3Hz, 2H), 1.35 (t, J=7.3Hz, 3H).

[0393] Step 3: Preparation of compound 14

[0394] Compound 14-4 (500 mg, 3.10 mmol, 1 eq), compound 14-5 (572.71 mg, 3.10 mmol, 1 eq), and TFA (35.4 mg, 310 μmol, 23.0 μL, 0.1 eq) were mixed in isopropanol (10 mL). The atmosphere was replaced with nitrogen three times. The reaction solution was heated to 80°C and stirred under a nitrogen atmosphere for 12 h. LCMS analysis showed that compound 14-4 was essentially completely converted. The reaction solution was filtered under reduced pressure, and the filter cake was suspended in ethanol, stirred for another 60 minutes, and filtered under reduced pressure. The filter cake was dried to obtain compound 14 (239 mg, 772 μmol, 24.9% yield) as a white solid. MS (ESI) m / z = 310.1 [M+H] + .

[0395] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.57 (s, 1H), 8.84-8.67 (m, 1H), 8.46 (s, 1H), 8.33 (s, 1H), 7 .85(s,2H),7.38(d,J=0.6Hz,1H),4.46(q,J=7.3Hz,2H),2.78(s,3H),1.55(t,J=7.3Hz,3H).

[0396] Example 14

[0397] Synthesis of compound 15

[0398] Step 1: Preparation of compound 15

[0399] Compound 15-1 (200 mg, 1.17 mmol, 1 eq), compound 15-2 (173 mg, 1.17 mmol, 1 eq) and TFA (13.4 mg, 117 μmol, 8.71 μL, 0.1 eq) were mixed in isopropanol (1 mL), and the atmosphere was replaced with nitrogen three times. The reaction solution was heated to 85 ° C and stirred in a nitrogen atmosphere for 12 h. LCMS detection showed that compound 15-1 was almost completely converted. The reaction solution was filtered under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative high performance liquid chromatography (neutral conditions, chromatographic column: Xtimate C 18 After purification (using a 150 x 40 mm x 10 μm column; mobile phase: [water (NH₃H₂O + NH₄HCO₃) – acetonitrile]; gradient: 4% to 44% B over 36 minutes), compound 15 (190 mg, 675 μmol, 57.6% yield) was obtained as a white solid. MS (ESI) m / z = 282.1 [M+H] + .

[0400] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 10.59-10.39 (m, 1H), 8.64-8.31 (m, 2H), 7.48 (s,1H),7.39-7.14(m,2H),6.94-6.76(m,1H),3.56-3.50(m,2H),2.72(s,3H).

[0401] Example 15

[0402] Synthesis of compound 16

[0403] Step 1: Preparation of compound 16

[0404] Compound 16-1 (200 mg, 1.08 mmol, 1 eq), compound 16-2 (159 mg, 1.08 mmol, 1 eq) and TFA (12.4 mg, 108 μmol, 8.05 μL, 0.1 eq) were mixed in isopropanol (2 mL), and the atmosphere was replaced with nitrogen three times. The reaction solution was heated to 85 ° C and stirred in a nitrogen atmosphere for 12 h. LCMS detection showed that compound 16-1 was almost completely converted. The reaction solution was filtered under reduced pressure, and the filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by preparative high performance liquid chromatography (neutral conditions, chromatographic column: Xtimate C 18 After purification (using a 150 mm x 40 mm x 10 μm column; mobile phase: [water (NH₃H₂O + NH₄HCO₃)–acetonitrile]; gradient: 6% to 46% B over 36 minutes), compound 16 (150 mg, 508 μmol, 46.9% yield) was obtained as a white solid. MS (ESI) m / z = 296.0 [M+H] + .

[0405] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.48 (s, 1H), 8.39 (s, 1H), 8.19-8.11 (m, 2H), 7.59-7.51 (m, 3H), 3.84 (s, 3H), 2.59 (s, 3H).

[0406] Example 16

[0407] Synthesis of compound 17

[0408] Step 1: Preparation of compound 17

[0409] Compound 17-1 (289 mg, 1.57 mmol, 1 eq) was dissolved in isopropanol (3 mL), and compound 17-2 (231 mg, 1.57 mmol, 1 eq) and TFA (17.9 mg, 157 μmol, 11.7 μL, 0.1 eq) were added. The atmosphere was purged with nitrogen three times, and the reaction mixture was heated to 80°C and stirred under nitrogen for 6 h. LCMS analysis showed that compound 17-1 was essentially completely converted. The reaction mixture was cooled and quenched with aqueous ammonia. The pH was adjusted to 7-8, and a solid precipitated. The mixture was filtered under reduced pressure and the filter cake was collected. The filter cake was suspended in acetonitrile (1.0 mL) and stirred at 20°C for 10 minutes. The mixture was filtered under reduced pressure and rinsed with water. The filter cake was collected and freeze-dried to obtain compound 17 (100 mg, 338 μmol, 21.5% yield) as a white solid. MS (ESI) m / z = 296.0 [M+H] + .

[0410] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.40 (s, 2H), 8.22 (s, 1H), 7.94 (d, J = 1.4Hz, 1H), 7.64 (d,J=8.6Hz,1H),7.43(dd,J=1.6,8.6Hz,1H),7.31(s,1H),3.84(s,3H),2.76(s,3H).

[0411] Example 17

[0412] Synthesis of compound 18

[0413] Step 1: Preparation of compound 18-3

[0414] Compound 18-1 (3.50 g, 16.6 mmol, 1 eq), compound 18-2 (17.9 g, 158 mmol, 16.9 mL, 1 eq), elemental sulfur (4.92 g, 153 mmol, 9.68 e-1 eq) and morpholine (55.2 g, 634 mmol, 55.8 mL, 4 eq) were mixed in EtOH (160.0 mL) solution, and the nitrogen atmosphere was replaced three times. The reaction solution was heated to 60 ° C and stirred under a nitrogen atmosphere for 5 h. LCMS showed that the raw material 18-1 was basically completely converted, and the main peak corresponded to the molecular weight of the target product. The reaction was quenched with water (300 mL) and extracted with EtOAc (500 mL). The organic phase was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was separated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 15 / 1 to 10 / 1) to obtain compound 18-3 (27.8 g, 110 mmol, 69.2% yield) as a yellow oil. MS (ESI) m / z = 254.1 [M+H] + .

[0415] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.18 (s, 2H), 4.15 (q, J = 7.0Hz, 2H), 2.78-2. 70(m,2H),2.57-2.51(m,2H),2.33-2.29(m,1H),1.78-1.73(m,1H),1.53(br d,J=6.0Hz,2H),1.49-1.46(m,2H),1.39(br d,J=4.9Hz,2H),1.25-1.22(m,3H).

[0416] Step 2: Preparation of compound 18-4

[0417] Compound 18-3 (20.0 g, 78.9 mmol, 1 eq) and ammonium formate (200 mL) were mixed, the atmosphere was replaced with nitrogen three times, the temperature was raised to 135°C, and the mixture was stirred under nitrogen for 48 h. LCMS showed that the starting material 18-3 was essentially completely converted, and the main peak corresponded to the molecular weight of the target product. The reaction solution was filtered, and the filter cake was separated and purified by column chromatography (silica, petroleum ether / THF = 5 / 1 to 2 / 1) to obtain compound 18-4 (13.7 g, 58.5 mmol, 74.0% yield) as a white solid. MS (ESI) m / z = 234.9 [M+H] + .

[0418] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.31 (br s,1H),7.99(s,1H),3.07-3.01(m,2H),2.88-2.81(m,2H),1.63-1.55(m,4H),1.43-1.36(m,2H),1.28-1.20(m,2H).

[0419] Step 3: Preparation of compound 18-5

[0420] POCl3 (28.6 g, 187 mmol, 17.4 mL, 5 eq) was added dropwise to compound 18-4 (8.74 g, 37.3 mmol, 1 eq) at 20°C for over 10 minutes. After addition, the mixture was heated to 110°C and stirred for 12 hours. LCMS analysis showed that some compound 18-4 remained, but the main peak was the desired product. The reaction was cooled to 0°C and quenched with water (250 mL). The mixture was extracted with EtOAc (250 mL). The organic phase was washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a residue. The residue was isolated and purified by column chromatography (silica, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to obtain compound 18-5 (4.50 g, 17.5 mmol, 46.7% yield) as a yellow solid. MS (ESI) m / z = 252.8 [M+H] + .

[0421] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.79 (s, 1H), 3.14-3.05 (m, 2H), 3.02-2.95 (m, 2H), 1.73-1.58 (m, 4H), 1.48-1.38 (m, 2H), 1.24-1.14 (m, 2H).

[0422] Step 4: Preparation of compound 18-7

[0423] Compound 18-5 (4.30 g, 17.0 mmol, 1 eq) was dissolved in EtOH (64.5 mL), and compound 18-6 (4.67 g, 101 mmol, 5.34 mL, 5.96 eq) was added. The mixture was heated to 85°C and stirred for 2 h. LCMS analysis showed that compound 18-5 was almost completely converted. The reaction solution was concentrated to obtain a residue, which was separated and purified by column chromatography (silica, dichloromethane / methanol = 20 / 1 to 10 / 1) to obtain compound 18-7 (2.90 g, 11.1 mmol, 64.9% yield) as an off-white solid. MS (ESI) m / z = 262.9 [M+H] + .

[0424] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.37 (s, 1H), 4.88 (s, 2H), 3.22-3.17 (m, 2H), 3.13 ( s,3H),2.93-2.88(m,2H),1.68-1.58(m,4H),1.45-1.37(m,2H),1.20-1.12(m,2H).

[0425] Step 5: Preparation of compound 18

[0426] 20 batches of the same batch were reacted in parallel: Compound 18-7 (50.0 mg, 190 μmol, 1 eq) was dissolved in EtOH (0.5 mL), and compound 18-8 (57.8 mg, 286 μmol, 1.5 eq) and boron trifluoride ether solution (67.6 mg, 476 μmol, 58.6 μL, 2.5 eq) were added. The temperature was raised to 85 ° C and stirred for 3 h. LCMS detection showed that compound 18-7 was almost completely converted. The temperature was lowered to room temperature, and the reaction was quenched with sodium bicarbonate. The pH value was adjusted to 7-8 and extracted twice with EtOAc (35 mL). The organic phases were combined, washed 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 high performance liquid chromatography (chromatographic column: Xtimate C18 150*40 mm*10 um; mobile phase: [water (NH4HCO3)-acetonitrile]; gradient: 36%-76% B, 36 minutes) to give compound 18 (80.0 mg, 183 μmol, 4.79% yield) as a white solid. MS (ESI) m / z = 419.0 [M+H] + .

[0427] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.55 (s, 1H), 8.15 (s, 1H), 7.52 (d, J = 8.8Hz, 2H), 7.35 (t, J = 5.7Hz, 1H), 7.16 (dd, J = 1.2, 8. 3Hz,1H),3.82(s,3H),3.73(d,J=5.6Hz,2H),3.40(s,3H),3.19-3.13(m,2H),2.98-2.91(m,2H),1.71-1.63(m,2H),1.51(br s, 2H), 1.43 (br d, J = 4.1Hz, 2H), 1.10 ( br d, J = 3.6Hz, 2H).

[0428] Example 18

[0429] Synthesis of compound 19

[0430] Step 1: Preparation of compound 19

[0431] Compound 19-1 (100 mg, 395 μmol, 1 eq), compound 19-2 (52.3 mg, 396 μmol, 1 eq) and TFA (4.51 mg, 39.6 μmol, 2.94 μL, 0.1 eq) were mixed in isopropanol (1.0 mL), the atmosphere was replaced with nitrogen three times, the temperature was raised to 85°C and stirred under a nitrogen atmosphere for 12 h. LCMS analysis showed that compound 19-1 was almost completely converted, and the main peak corresponded to the molecular weight of the target product. The reaction solution was concentrated to obtain a residue, which was purified by preparative HPLC (column: Xtimate C18 150*40 mm*10 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 38.0%-78.0% B, 36 min) to obtain compound 19 (50.0 mg, 142 μmol, 35.9% yield) as a white solid. MS (ESI) m / z = 348.9 [M+H] + .

[0432] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 11.07 (br s,1H),8.25(s,1H),8.16(s,1H),7.70(d,J=1.8Hz,1H),7.41-7.31(m,2H),7.20(dd,J=2.0,8.6 Hz,1H),6.42(d,J=2.0Hz,1H),3.23-3.16(m,2H),2.97-2.90(m,2H),1.77-1.69(m,2H),1.64(br d,J=3.4Hz,2H),1.50(br s,2H),1.21(br d,J=4.3Hz,2H).

[0433] Example 19

[0434] Synthesis of compound 20

[0435] Step 1: Preparation of compound 20

[0436] Compound 20-1 (100 mg, 395 μmol, 1 eq), compound 20-2 (58.2 mg, 395 μmol, 1 eq), and TFA were mixed in isopropanol (1.0 mL). The mixture was purged with nitrogen three times, heated to 85°C, and stirred under nitrogen for 12 h. LCMS analysis showed that compound 20-1 was essentially completely converted, with the main peak corresponding to the molecular weight of the target product. The reaction solution was concentrated under reduced pressure to obtain a residue, which was then purified by preparative HPLC (column: Xtimate C18 150*40mm*10um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-acetonitrile]; gradient: 24.0%-64.0% B over 36 minutes) to obtain compound 20 (30.0 mg, 82.5 μmol, 20.8% yield) as a white solid. MS (ESI) m / z = 364.1 [M+H] + .

[0437] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.35-8.23 (m, 2H), 8.17 (s, 1H), 7.88 (d, J = 1.1H z,1H),7.54(d,J=8.6Hz,1H),7.41(dd,J=1.6,8.6Hz,1H),3.85(s,3H),3.22(br s,2H),2.95(br s,2H),1.76-1.61(m,4H),1.50(br s,2H),1.19(br s,2H).

[0438] Example 20

[0439] Synthesis of compound 21

[0440] Step 1: Preparation of compound 21-2

[0441] Compound 21-1 (1.00 g, 6.41 mmol, 1 eq), trideuteromethylamine hydrochloride (649 mg, 9.61 mmol, 1.5 eq), and diisopropylethylamine (2.48 g, 19.2 mmol, 3.35 mL, 3 eq) were mixed in n-butanol (10 mL). The atmosphere was replaced with nitrogen three times, and the reaction solution was then heated to 120°C and stirred under a nitrogen atmosphere for 12 hours. LCMS analysis showed that compound 21-1 was almost completely converted, with the main peak corresponding to the molecular weight of the target product. After cooling the reaction solution, it was concentrated under reduced pressure to remove the solvent to obtain crude compound 21-2 as a red solid. The crude product was used directly in the next reaction without further purification. MS (ESI) m / z = 170.9 [M+H] + .

[0442] 1 H NMR: (400MHz, CDCl3) δ (ppm) = 7.58-7.52 (m, 1H), 7.40 (d, J = 2.6Hz, 1H), 6.45-6.39 (m, 1H), 6.08 (s, 1H), 5.10-5.02 (m, 2H).

[0443] Step 2: Preparation of compound 21-3

[0444] Compound 21-2 (0.5 g, 2.94 mmol, 1 eq), trimethyl orthoformate (1.94 g, 18.2 mmol, 2 mL, 6.21 eq), and trifluoroacetic acid (268 mg, 2.35 mmol, 175 μL, 0.8 eq) were mixed in ethanol (3 mL). The atmosphere was replaced with nitrogen three times, and the reaction mixture was stirred at 25°C under a nitrogen atmosphere for 1 hour. LCMS analysis showed that compound 21-2 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 to remove the solvent to obtain crude compound 24-3. The crude product was suspended in methyl tert-butyl ether (30 mL), stirred at 25°C for 1 hour, and filtered under reduced pressure to obtain compound 21-3 (0.4 g, 2.22 mmol, 75.6% yield) as a brown solid. MS (ESI) m / z = 170.9 [M+H] + .

[0445] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.60-8.48 (m, 2H), 8.21 (dd, J = 8.9, 2.2Hz, 1H), 7.82 (d, J = 8.9Hz, 1H).

[0446] Step 2: Preparation of compound 21-4

[0447] Compound 21-3 (0.4 g, 2.22 mmol, 1 eq) was dissolved in methanol (10 mL). Wet palladium on carbon (0.2 g, 188 μmol, 10% purity, 0.08 eq) and triethylamine (225 mg, 2.22 mmol, 309 μL, 1 eq) were added under nitrogen. The suspended mixture was replaced with hydrogen three times. The reaction mixture was then placed in a hydrogen atmosphere (1 MPa), heated to 80°C, and stirred for 12 hours. LCMS analysis showed that compound 21-3 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 collected and concentrated under reduced pressure to obtain crude compound 21-4 (0.3 g, 1.96 mmol, 88.2% yield) as a red solid, which was used directly in the next step. MS (ESI) m / z = 150.9 [M+H] + .

[0448] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.90 (s, 1H), 7.20 (d, J = 8.5Hz, 1H), 6.77 (d, J = 1.9Hz, 1H), 6.61 (dd, J = 1.9, 8.5Hz, 1H), 5.14-4.07 (m, 2H).

[0449] Step 2: Preparation of compound 21

[0450] Compound 21-4 (0.2 g, 1.33 mmol, 1 eq), compound 21-5 (246 mg, 1.33 mmol, 1 eq), and TFA (152 mg, 1.33 mmol, 98.9 μL, 1 eq) were mixed in isopropanol (1.0 mL) and replaced with nitrogen three times. The temperature was raised to 85°C and stirred under nitrogen for 8 h. TLC (petroleum ether / ethyl acetate = 1 / 1, UV) analysis showed that compound 21-4 was almost completely converted. The reaction solution was filtered under reduced pressure, and the filter cake was dried to obtain crude compound 21. The crude product was suspended in acetonitrile (20 mL), stirred at 25°C for 8 hours, filtered under reduced pressure, and the filter cake was collected and dried under vacuum to obtain compound 21 (190 mg, 618 μmol, 30.9% yield) as a white solid. MS (ESI) m / z = 304.9 [M+H] + .

[0451] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.57 (s, 1H), 8.79 (br s,1H),8.50(s,1H),8.35(d,J=1.6Hz,1H),7.96(d,J=9.0Hz,1H),7.84(dd,J=9.0,1.9Hz,1H),7.39(d,J=1.0Hz,1H),2.78(d,J=0.9Hz,3H).

[0452] Example 21

[0453] Synthesis of compound 22

[0454] Step 1: Preparation of compound 22

[0455] Compound 22-1 (277 mg, 1.50 mmol, 1 eq), 22-2 (200 mg, 1.50 mmol, 1 eq), and hydrochloric acid in ethyl acetate (27.4 mg, 751 μmol, 0.50 eq) were dissolved in isopropanol (5 mL). The atmosphere was replaced with nitrogen three times. The reaction mixture was then heated to 90°C and stirred under nitrogen for 12 hours. LCMS monitoring of the reaction showed nearly complete conversion of compound 22-2. The reaction mixture was cooled to room temperature and filtered under reduced pressure to collect the filter cake. The filter cake was suspended in acetonitrile (4 mL), stirred at room temperature for 1 hour, and filtered under reduced pressure. The filter cake was purified by column chromatography (silica, dichloromethane / methanol = 50 / 1 to 10 / 1) to obtain a crude product. The crude product was suspended in acetonitrile (5 mL), stirred at room temperature for 1 hour, filtered under reduced pressure, and the filter cake was collected and dried under vacuum to obtain compound 22 (0.125 g, 0.444 mmol, 97.82% purity, 29.6% yield) as a brown solid. MS (ESI) m / z = 282.0 [M+H] + .

[0456] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.76 (brs, 1H), 8.42 (s, 1H), 8.10 (s, 1H), 8.01 (d, J = 1.2Hz, 1H ), 7.58 (dd, J = 8.8, 1.6 Hz, 1H), 7.51 ( dd, J = 8.8, 2.0 Hz, 1H), 7.37 ( d, J = 0.8 Hz, 1H), 2.76 ( s, 3H).

[0457] Example 22

[0458] Synthesis of compound 23

[0459] Step 1: Preparation of compound 23

[0460] Compound 23-1 (251 mg, 1.36 mmol, 1 eq), compound 23-2 (200 mg, 1.36 mmol, 1 eq), and a 4 M hydrochloric acid-ethyl acetate solution (339.73 μL, 1 eq) were dissolved in isopropanol (5 mL). The atmosphere was replaced with nitrogen three times, and the reaction mixture was then heated to 90°C and stirred under nitrogen for 12 hours. LCMS monitoring of the reaction indicated an approximately 96% content of the desired product. TLC (petroleum ether / ethyl acetate = 3 / 1) monitoring of the reaction indicated near-complete conversion of compound 23-1. The reaction mixture was cooled to room temperature and filtered under reduced pressure. The filter cake was collected and dried under vacuum to yield compound 23 (350 mg, 1.15 mmol, 97% purity, 84.6% yield) as a gray solid. MS (ESI) m / z = 296.0 [M+H] + .

[0461] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.69 (brs, 1H), 8.41 (s, 1H), 8.09-7.99 (m, 2H), 7.67 (d, J = 8.8 Hz, 1H), 7.56 (dd, J = 8.8, 2.0 Hz, 1H), 7.36 (d, J = 1.0 Hz, 1H), 4.06 (s, 3H), 2.76 (d, J = 0.9 Hz, 3H).

[0462] Example 23

[0463] Synthesis of compound 24

[0464] Step 1: Preparation of compound 24

[0465] Compound 24-1 (251 mg, 1.36 mmol, 1 eq), compound 24-2 (200 mg, 1.36 mmol, 1 eq), and hydrochloric acid in ethyl acetate (24.80 mg, 679.45 μmol, 0.5 eq) were dissolved in isopropanol (5 mL). The atmosphere was purged with nitrogen three times. The reaction mixture was then heated to 90°C and stirred under nitrogen for 12 hours. LCMS monitoring of the reaction showed nearly complete conversion of compound 24-2. The reaction mixture was cooled to room temperature and filtered under reduced pressure. The filter cake was collected and suspended in acetonitrile (4 mL). The mixture was stirred at room temperature for 1 hour and filtered under reduced pressure. The filter cake was purified by preparative HPLC (column: Welch Xtimate C18 40*200mm 7um; mobile phase: [water (formic acid)-acetonitrile]; gradient: 2%-42% B over 25 minutes) to obtain compound 24 (109 mg, 367.8 μmol, 100% purity, 27.0% yield) as a brown solid. MS (ESI) m / z = 294.9 [MH] + ,297.0[M+H] + .

[0466] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.14 (brs, 1H), 8.57 (s, 1H), 8.34 (d, J = 1.2Hz, 1H), 7.8 9(d,J=8.8Hz,1H),7.75(dd,J=8.8,1.6Hz,1H),7.44(s,1H),4.33(s,3H),2.79(s,3H).

[0467] Example 24

[0468] Synthesis of compound 25

[0469] Step 1: Preparation of compound 25

[0470] Compound 25-2 (200 mg, 1.35 mmol, 1 eq), compound 25-1 (249 mg, 1.35 mmol, 1 eq), and a 2M hydrochloric acid ethyl acetate solution (33.8 μL, 0.05 eq) were dissolved in isopropanol (5 mL). The atmosphere was replaced with nitrogen three times, and the reaction mixture was then heated to 90°C and stirred for 12 hours under a nitrogen atmosphere. LCMS monitoring of the reaction showed that compound 25-2 was essentially completely converted. The reaction mixture was cooled to room temperature, filtered under reduced pressure, and the filter cake was collected and suspended in acetonitrile (4 mL). The mixture was stirred at room temperature for 1 hour, filtered under reduced pressure, and the filter cake was collected and dried under vacuum to obtain compound 25 (72.4 mg, 0.245 mmol, 98.64% purity, 18.9% yield) as an off-white solid. MS (ESI) m / z = 294.0 [MH] + .

[0471] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.62 (s, 1H), 8.45 (s, 1H), 8.19 (s, 1H), 7.70 (s, 2H), 7.35 (s, 1H), 2.78 (s, 3H), 2.77 (s, 3H).

[0472] Example 25

[0473] Synthesis of compound 26

[0474] Step 1: Preparation of compound 26-3

[0475] Compound 26-1 (500 mg, 1.91 mmol, 1 eq), HATU (870 mg, 2.29 mmol, 1.2 eq), and diisopropylethylamine (740 mg, 5.72 mmol, 997 μL, 3 eq) were dissolved in DMF (3 mL) and stirred at 20°C for 30 minutes. Compound 26-2 (326 mg, 2.00 mmol, 1.05 eq) was added to the reaction solution, and the final reaction solution was stirred at 25°C for another 3.5 hours. LCMS monitoring of the reaction showed that compound 26-1 was essentially completely converted. Water (20 mL) was added to the reaction solution, and stirring was continued for 20 minutes. The mixture was filtered under reduced pressure, and the filter cake was collected and dried in vacuo to obtain a crude compound 26-3. The crude product was suspended in a mixed solvent of water / ethyl acetate (50 mL, 10 / 1), stirred at 20 ° C. for 12 hours, filtered under reduced pressure, and the filter cake was collected and dried in vacuo to obtain compound 26-3 (492 mg, 1.33 mmol, 69.7% yield) as an off-white solid.

[0476] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.58-8.53 (m, 1H), 7.54 (d, J = 4.0Hz, 1H), 7 .24-7.19(m,1H),5.17-5.08(m,2H),4.13-4.05(m,2H),3.43-3.36(m,2H).

[0477] Step 2: Preparation of compound 26

[0478] Compound 26-3 (250 mg, 675 μmol, 1 eq), compound 26-4 (109 mg, 743 μmol, 1.1 eq), and diisopropylethylamine (262 mg, 2.03 mmol, 353 μL, 3 eq) were dissolved in DMSO (3 mL). The atmosphere was replaced with nitrogen three times. The reaction mixture was then heated to 120°C and stirred under nitrogen for 12 hours. LCMS monitoring of the reaction showed nearly complete conversion of compound 26-3. The reaction mixture was cooled to room temperature, and water (10 mL) was added. The mixture was filtered under reduced pressure, and the filter cake was collected and dissolved in tetrahydrofuran (10 mL). The residue was concentrated under reduced pressure to obtain a residue. The residue was separated and purified by preparative high-performance liquid chromatography (column: F-Prepulite XP tC 18 40*200 mm*7 μm; mobile phase: [water (FA)-acetonitrile]; gradient: 2%-42% B, 20.5 min) to obtain compound 26 (102 mg, 209 μmol, 31% yield) as an off-white solid. MS (ESI) m / z = 481.1 [MH] + .

[0479] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.36 (s, 1H), 8.26 (s, 1H), 8.17 (s, 1H), 7.95 (d, J = 1.2Hz, 1H), 7.55 (d, J = 8.8Hz, 1H), 7.50 ( d,J=4.0Hz,1H),7.46(dd,J=8.8,4.0Hz,1H),7.25(d,J=4.0Hz,1H),5.01(brs,2H),4.03-3.96(m,2H),3.85(s,3H),3.40(br s,2H).

[0480] Example 26

[0481] Synthesis of compound 27

[0482] Step 1: Preparation of compound 27-3

[0483] Compound 27-1 (2.00 g, 7.63 mmol, 1 eq), HATU (3.48 g, 9.15 mmol, 1.2 eq), and diisopropylethylamine (2.96 g, 22.9 mmol, 3.99 mL, 3 eq) were dissolved in DMF (20 mL) and stirred at 20°C for 30 minutes. Compound 27-2 (1.12 g, 8.01 mmol, 1.05 eq) was added to the reaction solution, and the final reaction solution was stirred at 20°C for another 3.5 hours. LCMS monitoring of the reaction showed that compound 27-1 was essentially completely converted. Water (20 mL) was added to the reaction solution, and stirring was continued for 20 minutes. The mixture was filtered under reduced pressure, and the filter cake was collected and dried in vacuo to obtain a crude compound 26-3. The crude product was suspended in a mixed solvent of water / ethyl acetate (100 mL, 10 / 1), stirred at 20 ° C for 12 hours, filtered under reduced pressure, and the filter cake was collected and dried in vacuo to obtain compound 27-3 ((2.26 g, 6.50 mmol, 85.2% yield)) as an off-white solid.

[0484] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.82-7.74 (m, 2H), 7.67-7.59 (m, 2H), 7.58-7.47 (m, 3H), 7.46-7.31 (m, 2H), 6.91 (br d,J=4.4Hz,1H),3.53-3.36(m,2H),3.25-3.03(m,2H),2.81-2.70(m,1H),2.02-1.65(m,2H).

[0485] Step 2: Preparation of compound 27

[0486] Compound 27-3 (200 mg, 575 μmol, 1 eq), compound 27-4 (93.1 mg, 633 μmol, 1.1 eq), and diisopropylethylamine (223 mg, 1.73 mmol, 301 μL, 3 eq) were dissolved in DMSO (2 mL). The atmosphere was replaced with nitrogen three times. The reaction mixture was then heated to 120°C and stirred under nitrogen for 10 hours. LCMS monitoring of the reaction showed nearly complete conversion of compound 27-3. The reaction mixture was cooled to room temperature, and water (10 mL) was added. The mixture was filtered under reduced pressure, and the filter cake was collected and dissolved in tetrahydrofuran (10 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a residue. The residue was separated and purified by preparative high-performance liquid chromatography (column: Welch Xtimate C18 40*200 mm 7 μm; mobile phase: [water (FA)-ACN]; gradient: 0%-38% B over 20 minutes) to obtain compound 27 (54.9 mg, 120 μmol, 20.8% yield) as a white solid. MS (ESI) m / z = 459.0 [MH] + .

[0487] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.27 (s, 1H), 8.23-8.19 (m, 1H), 8.38-8.19 (m, 1H), 7.98-7.90 (m, 1H), 7.61-7.48 (m, 3H), 7.47-7.28 (m, 3H), 5.00 (br s,1H),4.64(s,1H),4.05(br s,1H),3.88-3.83(m,3H),3.62(br t,J=5.4Hz,1H),3.38(br s,1H),3.26(br s,1H).

[0488] Example 27

[0489] Synthesis of compound 28

[0490] Step 1: Preparation of compound 28-3

[0491] Compound 28-2 (451 mg, 3.07 mmol, 1 eq) and compound 28-1 (1.00 g, 3.07 mmol, 1.2 eq) were dissolved in DMSO (10 mL). Diisopropylethylamine (436 mg, 3.38 mmol, 588 μL, 1.1 eq) was added at 25°C. The reaction solution was heated to 120°C and stirred for 3 hours. LCMS monitoring showed that compound 28-1 was nearly completely converted. The reaction solution was cooled to room temperature, quenched by the addition of water (50 mL), filtered under reduced pressure, and the filter cake was collected and dried under vacuum to obtain crude compound 28-3. This crude product was suspended in ethyl acetate (30 mL), stirred at 20°C for 2 hours, filtered under reduced pressure, and the filter cake was collected and dried under vacuum to obtain compound 28-3 (900 mg, 1.86 mmol, 60.4% yield) as a purple solid. MS (ESI) m / z = 437.2 [M+H] + .

[0492] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.34 (s, 1H), 8.19 (s, 1H), 7.78 (d, J = 1.6Hz, 1H) ,7.41(d,J=8.6Hz,1H),7.28(dd,J=8.6,1.6Hz,1H),4.71-4.63(m,2H),3.69(br t,J=5.2Hz,2H),3.23(br s,2H),2.48(s,3H),1.45(s,9H).

[0493] Step 2: Preparation of compound 28

[0494] Compound 28-3 (800 mg, 1.83 mmol, 1 eq) was dissolved in 1,4-dioxane (2 mL), and a hydrochloric acid / dioxane solution (2 M, 9.16 mL, 10 eq) was added. The reaction mixture was stirred at 25°C for 1 hour. LCMS monitoring of the reaction showed nearly complete conversion of compound 28-3. The reaction mixture was concentrated under reduced pressure to afford compound 28 (600 mg, 1.78 mmol, 97.5% yield) as a purple solid. MS (ESI) m / z = 336.9 [M+H] + .

[0495] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 14.99 (br s, 1H), 9.99 (br s, 2H), 8.85 (br s, 1H), 8.48 (s, 1H), 8.12 (s, 1H), 7.77-7.66 (m, 2H), 4.47 (br s,2H),3.54-3.54(m,1H),3.54-3.51(m,2H),3.49-3.43(m,2H),2.80(s,3H).

[0496] Example 28

[0497] Synthesis of compound 29

[0498] Step 1: Preparation of compound 29

[0499] Compound 28 (100 mg, 297 μmol, 1.2 eq) and compound 29-1 (39.7 mg, 244 μmol, 1 eq) were dissolved in DMF (2 mL), and diisopropylethylamine (94.7 mg, 733 μmol, 127 μL, 3 eq) and T4P (264 mg, 366 μmol, 733 μL, 50% purity, 1.5 eq) were added. The reaction mixture was stirred at 20°C for 1.5 hours. LCMS monitoring showed that compound 28 was nearly completely converted. The reaction mixture was quenched by the addition of aqueous ammonia (27%, 2 mL), then diluted with water (20 mL). A white solid precipitated, which was filtered under reduced pressure and the filter cake collected. The crude product was suspended in methanol (2 mL), stirred at 20°C for 0.5 hours, filtered under reduced pressure, and the filter cake collected and dried under vacuum to yield compound 29 (94.5 mg, 191 μmol, 78.2% yield) as a white solid. MS (ESI) m / z = 480.9 [M+H] + .

[0500] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.13 (br d, J = 6.4Hz, 1H), 8.35 (br d, J = 16.4Hz, 1H), 8.21 (br d,J=8.4Hz,1H),7.84-7.73(m,1H),7.50(d,J=4.0Hz,1H),7.47-7.34(m,1H),7.34-7.26(m,1H),7.24(d,J=4.0Hz,1H),5.00(br s,2H),3.99(br s,2H),3.39(br s,2H),2.48(br s,3H).

[0501] Example 29

[0502] Synthesis of compound 30

[0503] Step 1: Preparation of compound 30

[0504] Compound 28 (100 mg, 297 μmol, 1 eq) was dissolved in acetonitrile (2 mL) and sodium carbonate (47.3 mg, 445 μmol, 1.5 eq) and acetic anhydride (33.4 mg, 327 μmol, 30 μL, 1.1 eq) were added. The reaction mixture was stirred at 20°C for 2 hours. LCMS monitoring of the reaction showed that compound 28 was almost completely converted. The reaction mixture was concentrated to obtain a crude product, which was suspended in dichloromethane / methanol (5 / 1, 2 mL) and stirred at 20°C for 0.5 hours. The mixture was filtered under reduced pressure to collect the filter cake, which was dissolved in methanol / water (1 / 2, 3 mL) and freeze-dried to obtain compound 30 (81.1 mg, 210 μmol, 70.6% yield) as an off-white solid. MS (ESI) m / z = 378.9 [M+H] + .

[0505] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.34 (s, 1H), 8.09 (br s, 1H), 7.82 (s, 1H), 7.41 (d, J = 8.4Hz, 1H), 7.31 (br d, J = 8.4Hz, 1H), 4.79 (br s, 2H), 3.83 (br t,J=5.6Hz,2H),3.37-3.28(m,2H),2.49-2.48(m,3H),2.14(s,3H).

[0506] Example 30

[0507] Synthesis of compound 31

[0508] Step 1: Preparation of compound 31

[0509] Compound 31-1 (250 mg, 0.989 mmol, 1.0 eq), compound 31-2 (146 mg, 0.989 mmol, 1.0 eq), and TFA (15 μL, 0.198 mmol, 2.0 eq) were mixed in isopropanol (5.0 mL), heated to 85°C, and stirred for 18 hours. The mixture was cooled to room temperature and filtered under reduced pressure. The filter cake was collected and purified by preparative HPLC (column: YMC-Actus Triart C18 250*20 mm, mobile phase: 10%-95% MeCN in water supplemented with 0.1% FA) to obtain compound 31 (56 mg, 0.154 mmol, 15.5% yield) as a white solid. MS (ESI) m / z = 364.1 [MH] + .

[0510] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.59 (s, 1H), 8.40 (s, 1H), 8.12 (d, J = 1.3Hz, 1H), 7.73 (d, J = 8.8Hz, 1H), 7.64 (dd, J = 8.8, 1.8Hz,1H),3.26-3.23(m,2H),2.99-2.95(m,2H),2.79(s,3H),1.70-1.66(m,4H),1.53-1.48(m,2H),1.19-1.14(m,2H).

[0511] Example 31

[0512] Synthesis of compound 32

[0513] Step 1: Preparation of compound 32

[0514] Compound 32-1 (300 mg, 1.187 mmol, 1.0 eq), compound 32-2 (158 mg, 1.187 mmol, 1.0 eq), and TFA (18 μL, 0.237 mmol, 2.0 eq) were mixed in isopropanol (6.0 mL), heated to 85°C, and stirred for 18 hours. The mixture was cooled to room temperature and filtered under reduced pressure. The filter cake was collected and purified by preparative HPLC (column: YMC-Actus Triart C18 250*20 mm, mobile phase: 10%-95% MeCN in water supplemented with 0.1% FA) to obtain compound 32 (60 mg, 0.172 mmol, 14.5% yield) as a white solid. MS (ESI) m / z = 350.2 [MH] + .

[0515] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 9.33 (s, 1H), 8.61 (s, 1H), 8.42 (s, 1H), 8.21 (s, 1H), 7.80 (d, J = 8.8Hz, 1H), 7.67 (dd ,J=8.8,1.6Hz,1H),3.27-3.23(m,2H),3.00-2.95(m,2H),1.70-1.64(m,4H),1.53-1.48(m,2H),1.19-1.15(m,2H).

[0516] Example 32

[0517] Synthesis of compound 33

[0518] Step 1: Preparation of compound 33-2

[0519] To a solution of NaH (417 mg, 60% purity, 10.4 mmol, 2.2 eq) in tetrahydrofuran (25.0 mL) was added compound 33-1 (1.0 g, 4.74 mmol, 1.0 eq) at 0°C and stirred for 30 minutes while maintaining the temperature at 0°C. The reaction mixture was cooled to -78°C and n-butyllithium (8.8 mL, 1.6 M solution in n-hexane, 14.2 mmol, 3.0 eq) was added dropwise. After the addition was complete, stirring was continued at -78°C for 1 hour. DMF (926 μL, 11.84 mmol, 2.5 eq) was added, and the final reaction mixture was stirred at -78°C for 1 hour. The reaction was quenched with saturated aqueous ammonium chloride and extracted twice with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered under reduced pressure. The filtrate was concentrated under reduced pressure to obtain the crude product, which was isolated and purified by column chromatography (silica, 0-10% methanol in dichloromethane) to obtain compound 33-2 (368 mg, 2.30 mmol, 48.5% yield) as a yellow solid. MS (ESI) m / z = 161 [M+H] + .

[0520] Step 2: Preparation of compound 33

[0521] Compound 33-3 (150 mg, 0.61 mmol, 1.0 eq) was dissolved in EtOH (5.0 mL). Compound 33-2 (126 mg, 0.79 mmol, 1.3 eq) and acetic acid (2 drops) were added at room temperature. The reaction mixture was heated to 80°C and stirred for 2 hours. After cooling, the reaction mixture was concentrated to obtain a residue, which was separated and purified by preparative HPLC (column: YMC-Triart C18, 250*20 mm, 5 μm, mobile phase: 5-95% MeCN in water containing 0.1% NH3HCO3) to obtain compound 33 (58 mg, 0.149 mmol, 24.6% yield) as a yellow solid. MS (ESI) m / z = 391.1 [M+H] + .

[0522] 1 H NMR: (400MHz, CD3OD) δ (ppm) = 8.48 (s, 1H), 7.96 (s, 1H), 7.83 (d, J = 8.4Hz, 1H), 7.82-7.51 (br, 1H), 7.50 (d, J = 8.4H z,1H),3.28-3.20(m,2H),2.97-2.85(m,2H),2.59(s,3H),1.82-1.67(m,4H),1.58-1.50(m,2H),1.37-1.31(m,2H).

[0523] Example 33

[0524] Synthesis of compound 34

[0525] Step 1: Preparation of compound 34

[0526] Compound 34-1 (100 mg, 0.403 mmol, 1.0 eq) was dissolved in MtOH (5.0 mL). Compound 34-2 (159 mg, 0.403 mmol, 1.0 eq) was added at room temperature. The reaction mixture was heated to 30°C and stirred for 12 hours. The mixture was cooled, filtered under reduced pressure, and the filter cake was collected. The mixture was then separated and purified by preparative HPLC (column: YMC-Triart C18, 250*20 mm, 5 μm, mobile phase: 5-95% MeCN in water containing 0.1% NH3HCO3) to afford compound 34 (55 mg, 0.146 mmol, 36.2% yield) as a yellow solid. MS (ESI) m / z = 377.1 [M+H] + .

[0527] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.59 (s, 1H), 11.85 (s, 1H), 8.51 (s, 1H), 8.32-7.85 (m, 3H), 7.80 (s, 1H), 7.63 ( d,J=41.5Hz,1H),3.25-3.16(m,2H),2.93-2.79(m,2H),1.74-1.57(m,4H),1.52–1.40(m,2H),1.33-1.20(m,2H).

[0528] Example 34

[0529] Synthesis of compound 35

[0530] Step 1: Preparation of compound 35

[0531] The hydrochloride of compound 28 (120 mg, 321 μmol, 1 eq) and potassium carbonate (133 mg, 965 μmol, 3.0 eq) were dissolved in acetonitrile (2 mL), and compound 35-1 (36.0 mg, 337 μmol, 35.4 μL, 1.05 eq) was added. The reaction solution was stirred at 25°C for 12 hours. LCMS monitoring of the reaction showed that compound 28 was almost completely converted. Methanol (2.0 mL) and water (0.1 mL) were added to the reaction solution at 25°C, and stirring was continued for 2 hours. The solution was concentrated to obtain a crude product, which was suspended in dichloromethane / methanol (10 / 1, 2 mL) and filtered under reduced pressure. The filter cake was rinsed twice with dichloromethane (10 mL). The filtrate was collected and concentrated under reduced pressure to obtain a crude product. The crude product was suspended in methyl tert-butyl ether (20 mL) and stirred at 25°C for 30 minutes. The filtered filter cake was washed with methyl tert-butyl ether (20 mL). The filter cake was collected and the solid was dried under vacuum. The solid was then suspended in water (10.0 mL) and stirred at 25°C for 30 minutes. After filtration under reduced pressure, the filter cake was washed with water (10.0 mL) and dissolved in MeOH (20.0 mL). The solvent was removed under reduced pressure, and the resulting solid was added to water (10.0 mL) to form a pink suspension. After freeze-drying, compound 35 (50.0 mg, 120 μmol, 37.3% yield) was obtained as a pink solid. MS (ESI) m / z = 407.1 [M+H] + .

[0532] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.37 (s, 1H), 8.26 (br s, 1H), 7.85 (s, 1H), 7.47 (d, J = 8.8Hz, 1H), 7.35 (br t,J=7.6Hz,1H),4.74-4.94(m,2H),3.81-3.93(m,2H),3.22(br d,J=4.2Hz,2H),2.98-3.08(m,1H),2.53(s,3H),1.00-1.11(m,6H).

[0533] Example 35

[0534] Synthesis of compound 36

[0535] Step 1: Preparation of compound 36

[0536] The hydrochloride salt of compound 28 (120 mg, 321 μmol, 1.0 eq) and potassium carbonate (133 mg, 965 μmol, 3.0 eq) were dissolved in acetonitrile (2 mL), and compound 36-1 (116 mg, 965 μmol, 118 μL, 3.0 eq) was added. The reaction mixture was stirred at 25°C for 12 hours. LCMS monitoring of the reaction indicated nearly complete conversion of compound 28. Methanol (2.0 mL) and water (0.1 mL) were added to the reaction solution at 25°C, and stirring was continued for 2 h. The above solution was concentrated to obtain a crude product, which was suspended in dichloromethane / methanol (10 / 1, 10 mL) and filtered under reduced pressure. The filter cake was rinsed twice with dichloromethane (10 mL), the filtrate was collected, and the crude product was concentrated under reduced pressure to obtain a crude product. The crude product was suspended in methyl tert-butyl ether (20 mL), and the mixture was suspended and stirred at 25°C for 30 minutes. The filtered filter cake was washed with methyl tert-butyl ether (20 mL), the filter cake was collected, and the solid was vacuum dried. The solid was then suspended in water ( The mixture was stirred at 25°C for 30 minutes, filtered under reduced pressure, and the filter cake was washed with water (10.0 mL). The filter cake was collected and added to methanol / water (1 / 3, 20.0 mL) to form a pink suspension, which was freeze-dried to obtain a crude product. The crude product was suspended in water / ethyl acetate (1 / 1, 50.0 mL), stirred at 25°C for 30 minutes, filtered under reduced pressure, and the filter cake was washed with ethyl acetate (20 mL). The filter cake was collected and dried under vacuum to obtain compound 36 (50.0 mg, 113 μmol, 35.3% yield) as a pink solid. MS (ESI) m / z = 421.2 [M+H] + .

[0537] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.12 (br d, J = 1.6Hz, 1H), 8.29-8.40 (m, 1H), 8.20 (br s, 1H), 7.72-7.84 (m, 1H), 7.22-7.48 (m, 2H), 4.85 (br s,2H),3.91(br t,J=5.2Hz,2H),3.28(br s,2H),2.48(s,3H),1.27(s,9H).

[0538] Example 36

[0539] Synthesis of compound 37

[0540] Step 1: Preparation of compound 37

[0541] The hydrochloride salt of compound 28 (200 mg, 488 μmol, 1.0 eq) and 2,2-dimethyloxirane (141 mg, 1.95 mmol, 173 μL, 4.0 eq) were dissolved in NMP (3 mL) and heated to 80°C under a nitrogen atmosphere with stirring for 12 hours. LCMS monitoring of the reaction showed nearly complete conversion of compound 28. The reaction solution was cooled to room temperature and filtered under reduced pressure. The filtrate was collected and purified by preparative HPLC (column: F-Prepulite XP tC 18 40*200 mm*7 um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; gradient: 6%-46% B over 20 minutes) to obtain compound 37 (90.1 mg, 210 μmol, 43.1% yield) as a white solid. MS (ESI) m / z = 409.0 [M+H] + .

[0542] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.14 (br s, 1H), 8.33 (s, 1H), 8.10 (s, 1H), 7.81 (s, 1H), 7.41 (br d, J = 8.4Hz, 1H), 7.28 (br d,J=8.0Hz,1H),4.25(s,1H),3.86(s,2H),3.22(br s,2H),3.02-2.89(m,2H),2.48(s,3H),2.45(s,2H),1.15(s,6H).

[0543] Example 37

[0544] Synthesis of compound 38

[0545] Step 1: Preparation of compound 38

[0546] The hydrochloride of compound 28 (300 mg, 733 μmol, 1.0 eq) and 2,2-dimethylpropanal (63.1 mg, 733 μmol, 81.0 μL, 1.0 eq) were dissolved in methanol (5 mL). Sodium acetate (132 mg, 1.61 mmol, 2.2 eq) and sodium cyanoborohydride (101 mg, 1.61 mmol, 2.2 eq) were added. The reaction mixture was heated to 50°C and stirred for 2 hours. TLC (dichloromethane / methanol = 10 / 1, UV monitoring of the reaction showed that compound 28 was almost completely converted. The reaction mixture was cooled to room temperature and filtered under reduced pressure. The filtrate was collected and separated and purified by column chromatography (silica, dichloromethane / methanol = 10 / 1) to obtain compound 38 (50.6 mg, 123 μmol, 16.8% yield) as a white solid. MS (ESI) m / z = 407.2 [M+H] + .

[0547] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.14 (br s, 1H), 8.33 (s, 1H), 8.12 (s, 1H), 7.81 (br s, 1H), 7.40 (br d, J = 6.8Hz, 1H), 7.28 (br d,J=8.0Hz,1H),3.82(s,2H),3.23(br s,2H),2.90(br t,J=5.6Hz,2H),2.47(s,3H),2.30(s,2H),0.91(s,9H).

[0548] Example 38

[0549] Synthesis of compound 39

[0550] Step 1: Preparation of compound 39-2

[0551] The hydrochloride of compound 28 (105 mg, 256 μmol, 1.0 eq), compound 39-1 (53.9 mg, 308 μmol, 1.2 eq), and compound T4P (50% in EtOAc, 92.4 mg, 256.51 μmol, 1.0 eq) were dissolved in DMF (2 mL). Diisopropylethylamine (33.1 mg, 256 μmol, 44.7 μL, 1.0 eq) was added, and the reaction mixture was stirred at 20°C for 2 h. LCMS monitoring of the reaction showed that compound 28 was essentially completely converted. The reaction mixture was poured into water (10 mL), and a large amount of solid precipitated. The filter cake was collected by filtration under reduced pressure and dried in vacuo to obtain crude compound 39-2 (120 mg) as a white solid, which was used directly in the next step without further purification. MS (ESI) m / z = 494.0 [M+H] + .

[0552] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 7.63-7.37 (m, 1H), 7.19-6.91 (m, 1H), 6.85-6.56 (m, 1H), 6.56-6.29 (m, 1H) ),4.05-4.04(m,2H),3.91-3.67(m,1H),3.48-2.89(m,5H),2.48-2.46(m,2H),1.86-1.65(m,3H),0.65(br s,9H).

[0553] Step 2: Preparation of compound 39

[0554] Compound 39-1 (100 mg, 176.52 μmol, 1.0 eq) was dissolved in methanol (0.5 mL), and a 2M solution of hydrogen chloride in dioxane (435.64 μL, 4.94 eq) was added. The reaction mixture was stirred at 20°C for 2 h. LCMS monitoring showed near-complete conversion of compound 39-1. The reaction mixture was concentrated to obtain the crude product, which was diluted with methanol (10 mL). The pH was adjusted to approximately 8.0 with 10% aqueous sodium hydroxide solution, yielding a clear brown solution. This solution was then isolated and purified by preparative HPLC (column: F-Prepulite XP tC 18 40*200 mm*7 μm; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; gradient: 0%-32% B over 20 minutes) to afford compound 39 (50.6 mg, 123 μmol, 34.9% yield) as a white solid. MS (ESI) m / z = 394.1 [M+H] + .

[0555] 1H NMR: (400MHz, DMSO-d6) δ (ppm) = 12.12 (br s, 1H), 8.34 (br d,J=10.4Hz,1H),8.27-8.09(m,1H),7.88-7.65(m,1H),7.57-7.16(m,2H),4.83-4.72(m,2H),4.05-3.66(m,3H),3.58-3.39(m,3H),3.23(br d,J=2.0Hz,2H),2.48(s,3H).

[0556] Example 39

[0557] Synthesis of compound 40

[0558] Step 1: Preparation of compound 40

[0559] The hydrochloride salt of compound 28 (200 mg, 488 μmol, 1.0 eq), 2-hydroxyacetic acid (44.6 mg, 586 μmol, 35.7 μL, 1.2 eq), and diisopropylethylamine (126 mg, 977 μmol, 170 μL, 2.0 eq) were dissolved in ethanol (2 mL). DMTMM (173 mg, 586 μmol, 1.2 eq) was added at 20°C and stirred for 2 h. LCMS monitoring of the reaction showed nearly complete conversion of compound 28. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was then separated and purified by preparative HPLC (column: F-Prepulite XP tC 18 40*200mm*7um; mobile phase: [water (NH3H2O ​​+ NH4HCO3)-ACN]; gradient: 0%-34% B over 20 minutes) to afford compound 40 (41.0 mg, 99.6 μmol, 20.3% yield) as a white solid. MS (ESI) m / z = 395.2 [M+H] + .

[0560] 1 H NMR: (400MHz, DMSO-d6) δ (ppm) = 8.34 (d, J = 17.2Hz, 1H), 8.27-8.13 (m, 1H), 7.85-7.69 (m, 1H), 7.49-7.34 (m, 1H), 7.32-7.18 (m, 1H), 4.87 (br t,J=5.4Hz,1H),4.83-4.63(m,2H),4.33-4.13(m,2H),3.93-3.67(m,2H),3.24(br d,J=1.2Hz,2H),2.47(d,J=1.6Hz,3H).

[0561] The compounds synthesized in the Examples are summarized in Table 1 below.

[0562] Table 1. Example compound information

[0563] Example 40. Solubility of Compound 3 and Compound 9 in DMSO and H2O

[0564] In this example, the solubilities of compounds 3 and 9 synthesized in the preparation examples were measured in DMSO and H2O, and the results are shown in Figure 2. The results show that compound 3 has a high solubility in DMSO and is poorly soluble in H2O; compound 9 is soluble in DMSO and has a high solubility in water.

[0565] Example 41. Inhibition test of Arf1 activation

[0566] In this example, compounds 3 and 9, synthesized in the above preparation examples, were tested for their ability to inhibit Arf1 activation. Activated Arf1 was detected using the Pierce Active Arf1 Pull-Down and Detection Kit (Thermo Scientific, Cat#16121) with Huh-7 liver cancer cell lysate. This comprehensive kit selectively enriches and detects GTP-bound Arf1 GTPase through specific protein interactions with the GGA3 protein-binding domain.

[0567] The test results are shown in FIG3 , indicating that compounds 3 and 9 of the present disclosure are effective inhibitors of Arf1 activation.

[0568] Example 42. Testing for Golgi morphological abnormalities

[0569] In this example, the compounds 3 and 9 synthesized in the above preparation examples were tested for their ability to induce Golgi defects, as indicated by abnormal distribution of the Golgi marker GM130. GCA, a known Arf1 inhibitor, was used as a positive control.

[0570] Mouse neuroblastoma N2A (N2a, CCL-131 TM ) were cultured, processed, and immunofluorescence stained as described previously ( Wang et al., 2021 ).

[0571] The test results are shown in FIG4 , indicating that compounds 3 and 9 of the present disclosure, like the known Arf1 inhibitor GCA, can effectively cause changes in Golgi apparatus morphology.

[0572] Example 43. Inducible expression of chemokine CCL5

[0573] In this example, the upregulation of chemokine CCL5 expression induced by treatment with compounds 3 and 9 synthesized in the above preparation examples was tested. The expression level of CCL5 chemokine was detected by quantitative RT-PCR in CT26 cells treated with DMSO, compound 3, or compound 9.

[0574] The test results are shown in FIG5 , indicating that compounds 3 and 9 of the present disclosure can effectively induce the expression of chemokine CCL5.

[0575] Example 44. Activation of NF-κB signaling pathway

[0576] In this example, the compounds 3 and 9 synthesized in the above preparation examples were tested for their ability to activate the NF-κB signaling pathway, as shown by an increase in phosphorylated P65. GCA, a known Arf1 inhibitor, was used as a positive control.

[0577] In CT26 cells treated with DMSO, GCA, compound 3, or compound 9, the levels of relevant proteins were assessed by Western blot as previously described (Wang et al., 2020).

[0578] The test results are shown in FIG6 , which indicate that compounds 3 and 9 of the present disclosure, like the known Arf1 inhibitor GCA, increase the phosphorylation level of P65, a key molecule in the NF-κB signaling pathway.

[0579] Example 45. Inhibition of Drosophila Tumor Growth.

[0580] In this example, the inhibitory effects of compounds 3 and 9 synthesized in the above preparation examples on the growth of Drosophila in situ tumors were tested.

[0581] 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 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 a Ras V12 Flies fed with PMML were fed with normal food containing DMSO or 5 μM compound 3 or compound 9. In the indicated groups, 5 × 10 3 μm2 The area of ​​esg>GFP+ tumor cells was used to characterize the tumor size.

[0582] The test results are shown in FIG7 , which indicate that compounds 3 and 9 of the present disclosure can effectively inhibit the growth of tumors in Drosophila.

[0583] Example 46. Inhibition of B16-F10 melanoma in C57B / 6 mice.

[0584] In this example, the inhibitory effect of compound 9 synthesized in the above preparation example on melanoma tumor growth was tested.

[0585] B16-F10 cells were cultured at 5 × 10 5 The dose of cells / mouse was inoculated into the left or right abdomen of 6-week-old male mice. About 7 days later, the mice were treated with ddH2O or the new Arf1 inhibitor by gavage for two consecutive weeks. The mice were divided into three groups: control group, compound 9 treatment group, and Du102 treatment group. The mice were given ddH2O or 5 mg / kg compound 9 (dissolved in ddH2O) or 5 mg / kg Du102 (dissolved in a solvent of 5% DMSO + 95% corn oil as a positive control) every day for two consecutive weeks. The tumor volume was measured with a digital caliper (ULINE, Cat#H-7352) every 2-3 days, and then the tumor volume was calculated using the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width). When the maximum tumor volume reached 2000mm 3 When , the mice were euthanized.

[0586] The test results are shown in FIG8 , which indicate that compound 9 of the present disclosure, like the positive control DU102, can effectively inhibit B16-F10 melanoma in C57B / 6 mice.

[0587] Example 47. Inhibition of CT26 colon cancer in BALB / c mice.

[0588] In this example, the inhibitory effects of compounds 9, 11-3, 25, 26, 27 and DU102 synthesized in the above preparation examples on the growth of colon cancer were tested.

[0589] CT26 cells were inoculated into the left or right abdomen of 6-week-old male mice at a dose of 5 × 10 5Cells / mouse. The mice were divided into two groups: a control group and a compound treatment group. After about 7 days, the mice were given a vehicle control (ddH2O or 5% DMSO in corn oil) or 5 mg / kg of compounds 9, 11-3, 25, 26, 27 (dissolved in the corresponding solvent) daily, and the administration was continued for two weeks. The tumor volume was measured every 2-3 days with a digital caliper (ULINE, Cat#H-7352), and then the tumor volume was calculated using the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width). When the maximum tumor volume reached 2000mm 3 When , the mice were euthanized.

[0590] The test results are shown in Figures 9 and 10, indicating that compounds 9, 25, 26 and DU102 of the present disclosure can effectively inhibit CT26 colon cancer in BALB / c mice.

[0591] Example 48. Inhibition of CT26 colon cancer in BALB / c mice.

[0592] In this example, the inhibitory effects of compounds 28, 30, 31, 33, 34 and DU102 synthesized in the above preparation examples on the growth of colon cancer were tested.

[0593] CT26 cells were inoculated into the left or right abdomen of 6-week-old male mice at a dose of 5 × 10 5 Cells / mouse. The mice were divided into two groups: a control group and a compound treatment group. After about 7 days, the mice were given a vehicle control (ddH2O or 5% DMSO in corn oil) or 10 mg / kg of compounds 28, 30, 31, 33, 34 and DU102 (dissolved in the corresponding solvent) every day, and the administration was continued for two weeks. The tumor volume was measured with a digital caliper (ULINE, Cat#H-7352) every 2-3 days, and then the tumor volume was calculated using the formula 1 / 2×longitudinal diameter (length)×maximum transverse diameter (width). When the maximum tumor volume reached 3000mm 3 When , the mice were euthanized.

[0594] The test results are shown in FIG11 , indicating that compounds 28, 30, 31, 33 and DU102 of the present disclosure can effectively inhibit CT26 colon cancer in BALB / c mice, and no significant decrease in mouse body weight was observed during administration, indicating that the above compounds of the present disclosure have high safety.

[0595] Example 49. Compounded plasma stability.

[0596] 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 ).

[0597] The test results are shown in Table 2. After incubation for 120 minutes, the remaining amounts of compound 9 (2.0 μM) in mouse, rat, dog and human plasma were 96.5%, 93.2%, 86.0% and 104.6%, respectively, indicating high plasma stability.

[0598] Table 2. Plasma stability results of compound 9

[0599] Example 50. Permeability of compounds in Caco-2 cells.

[0600] The permeability of compound 9 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 compound and control compound in the starting, donor, and receptor solutions were determined by chromatography-mass spectrometry (LC-MS / MS) using the analyte / internal standard peak area ratio.

[0601] The test results are shown in Table 3, which indicate that compound 9 of the present disclosure has high cell permeability and is unlikely to be a substrate of the efflux transporter P-gp.

[0602] Table 3. Caco-2 permeability results of compound 9

[0603] Example 51. Pharmacokinetic study of the compound in SD rats

[0604] In this experiment, six male Sprague-Dawley rats were divided into two groups of similar weight and administered compound 9 by single intravenous injection or oral gavage at a dose of 2 mg / kg for intravenous administration and 10 mg / kg for oral gavage. The vehicle for both intravenous and oral administration was a clear solution of 5% DMSO + 5% PEG400 + 90% water. The intravenous concentration was 0.4 mg / mL, and the oral concentration was 1 mg / mL. K2-EDTA was used as the anticoagulant. Blood samples were collected at 0.083, 0.25, 0.5, 1, 3, 6, 12, and 24 hours.

[0605] For all experimental animals, 0.02 mL of blood samples were 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 (0.5 M), and then centrifuged (3000 g, 4°C, 15 minutes) to obtain plasma, which was transferred to a pre-cooled centrifuge tube, quickly frozen in dry ice, and stored in an ultra-low temperature freezer at -60°C or lower until LC-MS / MS analysis.

[0606] The test results are shown in Table 4, which show that compound 9 of the present disclosure is rapidly absorbed in rats, has a moderate apparent distribution volume, and has a high oral bioavailability.

[0607] Table 4. Pharmacokinetic data of the compounds in rats

[0608] The foregoing description is intended to be merely illustrative of the principles of the invention. Furthermore, since many modifications and variations will be apparent to those skilled in the art, it is not intended to limit the invention to the exact configuration and process described above. Therefore, all suitable modifications and equivalents are intended to fall within the scope of the invention as defined by the appended claims.

[0609] All publications, patents, and patent applications cited herein are hereby incorporated by reference into this disclosure in their entirety.

[0610] References

[0611] Aggarwal,P.,Liu,Z.,Cheng,G.Q.,Singh,S.R.,Shi,C.,Chen,Y.,Sun,L.V.,and Hou,S.X.(2022).Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila.Cell Rep.39,110958.

[0612] Baharom,F.,Ramirez-Valdez,R.A.,Tobin,K.K.S.,Yamane,H.,Dutertre,C.A.,Khalilnezhad,A.,Reynoso,G.V.,Coble,V.L.,Lynn,G.M.,Mule,M.P.,et al.(2021).Intravenous nanoparticle vaccination generates stem-like TCF1(+)neoantigen-specific CD8(+)T cells.Nat.Immunol.22,41-52.

[0613] Baldominos,P.,Barbera-Mourelle,A.,Barreiro,O.,Huang,Y.,Wight,A.,Cho,J.W.,Zhao,X.,Estivill,G.,Adam,I.,Sanchez,X.,et al.(2022).Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche.Cell 185,1694-1708.

[0614] Binnewies,M.,Roberts,E.W.,Kersten,K.,Chan,V.,Fearon,D.F.,Merad,M.,Coussens,L.M.,Gabrilovich,D.I.,Ostrand-Rosenberg,S.,Hedrick,C.C.,et al.(2018).Understanding the tumor immune microenvironment(TIME)for effective therapy.Nat.Med.24,541-550.

[0615] Bruni,D.,Angell,H.K.,and Galon,J.(2020).The immune contexture and Immunoscore in cancer prognosis and therapeutic efficacy.Nat.Rev.Cancer 20,662-680.

[0616] Casalou,C.,Faustino,A.,and Barral,D.C.(2016).Arf proteins in cancer cell migration.Small GTPases 7,270-282.

[0617] Chen,D.S.,and Mellman,I.(2017).Elements of cancer immunity and the cancer-immune set point.Nature 541,321-330.

[0618] de la Roche,M.,Asano,Y.,and Griffiths,G.M.(2016).Origins of the cytolytic synapse.Nat.Rev.Immunol.16,421-432.

[0619] Donaldson,J.G.and Jackson,C.L.(2011).ARF family G proteins and their regulators:roles in membrane transport,development and disease.Nat.Rev.Mol.Cell Biol.12,362-375.

[0620] D'Souza-Schorey,C.,and Chavrier,P.(2006).ARF proteins:roles in membrane traffic and beyond.Nat.Rev.Mol.Cell Biol.7,347-358.

[0621] Eberhardt,C.S.,Kissick,H.T.,Patel,M.R.,Cardenas,M.A.,Prokhnevska,N.,Obeng,R.C.,Nasti,T.H.,Griffith,C.C.,Im,S.J.,Wang,X.,et al.(2021).Functional HPV-specific PD-1(+)stem-like CD8 T cells in head and neck cancer.Nature 597,279-284.

[0622] Galletti,G.,De Simone,G.,Mazza,E.M.C.,Puccio,S.,Mezzanotte,C.,Bi,T.M.,Davydov,A.N.,Metsger,M.,Scamardella,E.,Alvisi,G.,et al.(2020).Two subsets of stem-like CD8(+)memory T cell progenitors with distinct fate commitments in humans.Nat.Immunol.21,1552-1562.

[0623] Gupta,Y.H.,Khanom,A.,and Acton,S.E.(2022).Control of Dendritic Cell Function Within the Tumour Microenvironment.Front.Immunol.13,733800.

[0624] Hashimoto,M.,Araki,K.,Cardenas,M.A.,Li,P.,Jadhav,R.R.,Kissick,H.T.,Hudson,W.H.,McGuire,D.J.,Obeng,R.C.,Wieland,A.,et al.(2022).PD-1 combination therapy with IL-2 modifies CD8 + T cell exhaustion program.Nature 610,173-181.

[0625] Hass,R.,von der Ohe,J.,and Ungefroren,H.(2020).Impact of the Tumor Microenvironment on Tumor Heterogeneity and Consequences for Cancer Cell Plasticity and Stemness.Cancers 12,3716.

[0626] Im,S.J.,Hashimoto,M.,Gerner,M.Y.,Lee,J.,Kissick,H.T.,Burger,M.C.,Shan,Q.,Hale,J.S.,Lee,J.,Nasti,T.H.,et al.(2016).Defining CD8+T cells that provide the proliferative burst after PD-1 therapy.Nature 537,417-421.

[0627] Jain,S.,Annett,S.L.,Morgan,M.P.,and Robson,T.(2021).The Cancer Stem Cell Niche in Ovarian Cancer and Its Impact on Immune Surveillance.Int.J.Mol.Sci.22,4091.

[0628] Jansen,C.S.,Prokhnevska,N.,Master,V.A.,Sanda,M.G.,Carlisle,J.W.,Bilen,M.A.,Cardenas,M.,Wilkinson,S.,Lake,R.,Sowalsky,A.G.,et al.(2019).An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.Nature 576,465-470.

[0629] Jiao,S.,Subudhi,S.K.,Aparicio,A.,Ge,Z.,Guan,B.,Miura,Y.,and Sharma,P.(2019).Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy.Cell 179,1177-1190.

[0630] Kaczmarek,B.,Verbavatz,J.M.and Jackson,C.L.(2017).GBF1 and Arf1 function in vesicular trafficking,lipid homoeostasis and organelle dynamics.Biol.Cell 109,391-399,

[0631] Krishna,S.,Lowery,F.J.,Copeland,A.R.,Bahadiroglu,E.,Mukherjee,R.,Jia,L.,Anibal,J.T.,Sachs,A.,Adebola,S.O.,Gurusamy,D.,et al.(2020).Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer.Science 370,1328-1334.

[0632] Kurtulus,S.,Madi,A.,Escobar,G.,Klapholz,M.,Nyman,J.,Christian,E.,Pawlak,M.,Dionne,D.,Xia,J.,Rozenblatt-Rosen,O.,et al.(2019).Checkpoint Blockade Immunotherapy Induces Dynamic Changes in PD-1(-)CD8(+)Tumor-Infiltrating T Cells.Immunity 50,181-194 e186.

[0633] Lane,J.E.,Walker,A.N.,Kulharya,A.,and Marzec,T.(2002).Cutaneous sclerosing extramedullary hematopoietic tumor in chronic myelogenous leukemia.J.Cutan.Pathol.29,608-612.

[0634] Lang,L.,Shay,C.,Zhao,X.,and Teng,Y.(2017).Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics.J.Exp.Clin.Cancer Res.36,112.

[0635] Lytle,N.K.,Barber,A.G.,and Reya,T.(2018).Stem cell fate in cancer growth,progression and therapy resistance.Nat.Rev.Cancer 18,669-680.

[0636] Mo,F.,Yu,Z.,Li,P.,Oh,J.,Spolski,R.,Zhao,L.,Glassman,C.R.,Yamamoto,T.N.,Chen,Y.,Golebiowski,F.M.,et al.(2021).An engineered IL-2 partial agonist promotes CD8(+)T cell stemness.Nature 597,544-548.

[0637] Müller,L.,Tunger,A.,Plesca,I.,Wehner,R.,Temme,A.,Westphal,D.,Meier,F.,Bachmann,M.,and Schmitz,M.(2020).Bidirectional Crosstalk Between Cancer Stem Cells and Immune Cell Subsets.Front.Immunol.11,140.

[0638] Prokhnevska,N.,Cardenas,M.A.,Valanparambil,R.M.,Sobierajska,E.,et al.(2023).CD8 + T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor.Immunity 56,107-124.

[0639] Rao,S.A.,Gottesman,S.R.,Nguyen,M.C.,and Braverman,A.S.(2003).T cell lymphoma associated with myelofibrosis.Leuk.Lymphoma.44,715-718.

[0640] Robbins,P.F.,Dudley,M.E.,Wunderlich,J.,El-Gamil,M.,Li,Y.F.,Zhou,J.,Huang,J.,Powell,D.J.,Jr.,and Rosenberg,S.A.(2004).Cutting edge:persistence of transferred lymphocyte clonotypes correlates with cancer regression in patients receiving cell transfer therapy.J.Immunol.173,7125-7130.

[0641] Schad,S.E.,Chow,A.,Mangarin,L.,Pan,H.,Zhang,J.,Ceglia,N.,Caushi,J.X.,Malandro,N.,Zappasodi,R.,Gigoux,M.,et al.(2022).Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions.J.Exp.Med.219,e20212169.

[0642] Schmid,P.,Cortes,J.,Pusztai,L.,McArthur,H.,Kummel,S.,Bergh,J.,Denkert,C.,Park,Y.H.,Hui,R.,Harbeck,N.,et al.(2020).Pembrolizumab for Early Triple-Negative Breast Cancer.N.Engl.J.Med.382,810-821.

[0643] Sharma,P.,Hu-Lieskovan,S.,Wargo,J.A.,and Ribas,A.(2017).Primary,Adaptive,and Acquired Resistance to Cancer Immunotherapy.Cell 168,707-723.

[0644] Siddiqui,I.,Schaeuble,K.,Chennupati,V.,Fuertes Marraco,S.A.,Calderon-Copete,S.,Pais Ferreira,D.,Carmona,S.J.,Scarpellino,L.,Gfeller,D.,Pradervand,S.,et al.(2019).Intratumoral Tcf1(+)PD-1(+)CD8(+)T Cells with Stem-like Properties Promote Tumor Control in Response to Vaccination and Checkpoint Blockade Immunotherapy.Immunity 50,195-211.

[0645] Singh,S.R.,Zeng,X.,Zhao,J.,Liu,Y.,Hou,G.,Liu,H.,and Hou,S.X.(2016).The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila.Nature 538,109-113.

[0646] Spranger,S.,and Gajewski,T.F.(2018).Impact of oncogenic pathways on evasion of antitumour immune responses.Nat.Rev.Cancer 18,139-147.

[0647] Thorsson,V.,Gibbs,D.L.,Brown,S.D.,Wolf,D.,Bortone,D.S.,Ou Yang,T.H.,Porta-Pardo,E.,Gao,G.F.,Plaisier,C.L.,Eddy,J.A.,et al.(2018).The Immune Landscape of Cancer.Immunity 48,812-830.

[0648] Trumpp,A.,and Haas,S.(2022).Cancer stem cells:The adventurous journey from hematopoietic to leukemic stem cells.Cell 185,1266-1270.

[0649] Tumeh,P.C.,Harview,C.L.,Yearley,J.H.,Shintaku,I.P.,Taylor,E.J.,Robert,L.,Chmielowski,B.,Spasic,M.,Henry,G.,Ciobanu,V.,et al.(2014).PD-1 blockade induces responses by inhibiting adaptive immune resistance.Nature 515,568-571.

[0650] Wang,G.,Xu,J.,Zhao,J.,Yin,W.,Liu,D.,Chen,W.,and Hou,S.X.(2020).Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice.Nat.Commun.11,220.

[0651] Wellenstein,M.D.,and de Visser,K.E.(2018).Cancer-Cell-Intrinsic Mechanisms Shaping the Tumor Immune Landscape.Immunity 48,399-416.

[0652] Wilfling,F.,Thiam,A.R.,Olarte,M.J.,Wang,J.,Beck,R.,Gould,T.J.et al.(2014).Arf1 / COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting.Elife 3,e01607.

[0653] Wu,T.D.,Madireddi,S.,de Almeida,P.E.,Banchereau,R.,Chen,Y.J.,Chitre,A.S.,Chiang,E.Y.,Iftikhar,H.,O'Gorman,W.E.,Au-Yeung,A.,et al.(2020).Peripheral T cell expansion predicts tumour infiltration and clinical response.Nature 579,274-278.

[0654] Yost,K.E.,Satpathy,A.T.,Wells,D.K.,Qi,Y.,Wang,C.,Kageyama,R.,McNamara,K.L.,Granja,J.M.,Sarin,K.Y.,Brown,R.A.,et al.(2019).Clonal replacement of tumor-specific T cells following PD-1 blockade.Nat.Med.25,1251-1259.

[0655] Yu,J.,Green,M.D.,Li,S.,Sun,Y.,Journey,S.N.,Choi,J.E.,Rizvi,S.M.,Qin,A.,Waninger,J.J.,Lang,X.,et al.(2021).Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination.Nat.Med.27,152-164.

[0656] Zeng,X.,Han,L.,Singh,S.R.,Liu,H.,Neumuller,R.A.,Yan,D.,Hu,Y.,Liu,Y.,Liu,W.,Lin,X.,and Hou,S.X.(2015).Genome-wide RNAi screen identifies networks involved in intestinal stem cell regulation in Drosophila.Cell Rep.10,1226-1238.

[0657] Zhao,B.,Zhao,H.,and Zhao,J.(2020).Efficacy of PD-1 / PD-L1 blockade monotherapy in clinical trials.Ther.Adv.Med.Oncol.12,1758835920937612.

[0658] Zheng,B.,Wang,D.,Qiu,X.,Luo,G.,Wu,T.,Yang,S.,Li,Z.,Zhu,Y.,Wang,S.,Wu,R.,et al.(2020).Trajectory and Functional Analysis of PD-1(high)CD4(+)CD8(+)T Cells in Hepatocellular Carcinoma by Single-Cell Cytometry and Transcriptome Sequencing.Adv.Sci.(Weinh)7,2000224.

Claims

1. A compound of formula (I), its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof: in, X1 does not exist or is CH; X2 is selected from C or N; X3 is selected from S, O, CH or N; represents a single bond or a double bond; X4, X5, X6, X7, X8, X9 and X 10 Each independently selected from C(R x ) or N, R x independently selected from H, D (deuterium), CN, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 A group consisting of haloalkoxy; 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 and optionally substituted with 1-3 R b C 1-6 or R1 and R2 together with the carbon atom to which they are attached form an optionally substituted group consisting of 1 to 3 R c C 5-8 a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the 5- to 8-membered heterocyclic ring optionally containing 1 or more heteroatoms selected from N, O and S; R3 is 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 1-6 Alkoxy and N(R n )2, where each R n Each independently represents H, D, optionally substituted with 1-3 R a C 1-6 alkyl and optionally substituted with 1-3 R g C 6-14 Aryl; R4 is selected from H and optionally substituted with 1-3 R a C 1-6 alkyl; L is absent or selected from optionally substituted with 1-3 R d C 1-6 Alkylene and optionally substituted with 1-3 R e C 1-6 A heteroalkylene group, the heteroalkylene group optionally comprising 1 or more heteroatoms selected from N, O and S; Ring D is absent or optionally substituted with 1-3 R f C 5-6 Carbocyclic or 5- to 6-membered heterocyclic ring; Each time it appears, each R a , R b , R c , R d , R e , R f , R g Each independently selected from D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, -C(O)-C 1-6 The group consisting of alkyl, oxo (=O) and -NH2.

2. A compound of formula (I), its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof: in, X1 does not exist or is CH; X2 is selected from C or N; X3 is selected from S, O, CH or N; represents a single bond or a double bond; X4, X5, X6, X7, X8, X9 and X 10 Each independently selected from C(R x ) or N, R x independently selected from H, D (deuterium), CN, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 A group consisting of haloalkoxy; 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 and optionally substituted with 1-3 R b C 1-6 or R1 and R2 together with the carbon atom to which they are attached form an optionally substituted group consisting of 1 to 3 R c C 5-8 a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the 5- to 8-membered heterocyclic ring optionally containing 1 or more heteroatoms selected from N, O and S; R3 is 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 1-6 Alkoxy and N(R n )2, where each R n Each independently represents H, D, optionally substituted with 1-3 R a C 1-6 alkyl and optionally substituted with 1-3 R g C 6-14 Aryl; R4 is selected from H and optionally substituted with 1-3 R a C 1-6 alkyl; L is absent or selected from optionally substituted with 1-3 R d C 1-6 Alkylene and optionally substituted with 1-3 R e C 1-6 A heteroalkylene group, the heteroalkylene group optionally comprising 1 or more heteroatoms selected from N, O and S; Ring D is absent or optionally substituted with 1-3 R f C 5-6 Carbocyclic or 5- to 6-membered heterocyclic ring; Each time it appears, each R a , R b , R c , R d , R e , R f , R g Each independently selected from D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -C(O)-C 1-6 Alkyl, -C(O)-C 1-6 Haloalkyl, -C(O)-C 1-6 Hydroxyalkyl, -C(O)-C 1-6 aminoalkyl, -C(O)- optionally substituted C 5-14 Aryl, -C(O)- optionally substituted C 5-14 The group consisting of heteroaryl, oxo (=O) and -NH2, wherein the optionally substituted C 5-14 Aryl and optionally substituted C 5-14 The heteroaryl group is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2.

3. A compound of formula (I) according to claim 2, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof: in, X1 does not exist or is CH; X2 is selected from C or N; X3 is selected from S, O, CH or N; represents a single bond or a double bond; X4, X5, X6, X7, X8, X9 and X 10 Each independently selected from C(R x ) or N, R x independently selected from H, D (deuterium), CN, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy and C 1-6 A group consisting of haloalkoxy; 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 and optionally substituted with 1-3 R b C 1-6 or R1 and R2 together with the carbon atom to which they are attached form an optionally substituted group consisting of 1 to 3 R c C 5-8 a carbocyclic ring or a 5- to 8-membered heterocyclic ring, the 5- to 8-membered heterocyclic ring optionally containing 1 or more heteroatoms selected from N, O and S; R3 is 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 1-6 Alkoxy and N(R n )2, where each R n Each independently represents H, D, optionally substituted with 1-3 R a C 1-6 alkyl and optionally substituted with 1-3 R g C 6-14 Aryl; R4 is selected from H and optionally substituted with 1-3 R a C 1-6 alkyl; L is absent or selected from optionally substituted with 1-3 R d C 1-6 Alkylene and optionally substituted with 1-3 R e C 1-6 A heteroalkylene group, the heteroalkylene group optionally comprising 1 or more heteroatoms selected from N, O and S; Ring D is absent or optionally substituted with 1-3 R f C 5-6 Carbocyclic or 5- to 6-membered heterocyclic ring; Each time it appears, each R a , R b , R c , R d , R e , R f , R g Each independently selected from C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -C(O)-C 1-6 Haloalkyl, -C(O)-C 1-6 Hydroxyalkyl, -C(O)-C 1-6 aminoalkyl, -C(O)- optionally substituted C 5-14 Aryl, -C(O)- optionally substituted C 5-14 heteroaryl, wherein the optionally substituted C 5-14 Aryl and optionally substituted C 5-14 The heteroaryl group is optionally substituted with 1-3 substituents selected from the group consisting of D, halogen, -OH or -NH2.

4. A compound of formula (I) according to any one of claims 1 to 3, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ring A is selected from the following ring structures:

5. A compound of formula (I) according to any one of claims 1 to 4, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: R1 and R2 are each independently absent or selected from H, D, CN, OH, halogen, optionally substituted with 1-3 R a C 1-4 alkyl and optionally substituted with 1-3 R b C 1-4 A group consisting of alkoxy groups.

6. A compound of formula (I) according to any one of claims 1 to 4, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: R1 and R2 together with the carbon atom to which they are connected form a C 5-8 The carbocyclic ring or 5- to 8-membered heterocyclic ring is selected from the following ring structures: The C 5-8 The carbocyclic or 5- to 8-membered heterocyclic ring is optionally substituted with 1-3 R c .

7. 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, wherein: Ring B is selected from the following structures:

8. A compound of formula (I) according to any one of claims 1 to 7, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ring A and Ring B together form a ring structure selected from the following:

9. A compound of formula (I) according to any one of claims 1 to 8, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: R1 and R2 are each independently absent or selected from H, D, CN, OH, halogen, optionally substituted with 1-3 R a C 1-4 alkyl and optionally substituted with 1-3 R b C 1-4 A group consisting of alkoxy groups.

10. A compound of formula (I) according to any one of claims 1 to 8, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: R1 and R2 together with the carbon atom to which they are connected form a C 5-8 The carbocyclic ring or 5- to 8-membered heterocyclic ring is selected from the following ring structures: The C 5-8 The carbocyclic or 5- to 8-membered heterocyclic ring is optionally substituted with 1-3 R c .

11. A compound of formula (I) according to any one of claims 1 to 10, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: X6, X7, X8, X9 and X 10 Each independently selected from C(R x ).

12. A compound of formula (I) according to any one of claims 1 to 11, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ring C is selected from optionally substituted with 1-5 R x For example, 1-3 R x The benzene ring is optionally substituted with 1-4 R x For example, 1-2 R x of the pyridine ring.

13. A compound of formula (I) according to any one of claims 1 to 12, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ring D is selected from optionally substituted with 1-3 R f The following ring structure: Among them, "1" indicates the connection direction with X8 of ring C, and "2" indicates the connection direction with X9 of ring C.

14. A compound of formula (I) according to any one of claims 1 to 12, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ring D is selected from optionally substituted with 1-3 R f The following ring structure: Among them, "1" indicates the connection direction with X8 of ring C, and "2" indicates the connection direction with X9 of ring C.

15. The compound of formula (I) according to claim 14, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: Ring D is selected from optionally substituted with 1-3 R f The following ring structure: Among them, "1" indicates the connection direction with X8 of ring C, and "2" indicates the connection direction with X9 of ring C.

16. A compound of formula (I) according to any one of claims 1 to 15, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Rings C and D together form the following fused ring structure: Ring C is optionally substituted with 1-3 R x , Ring D is optionally substituted with 1-3 R f .

17. A compound of formula (I) according to any one of claims 1 to 16, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: Rings C and D together form the following fused ring structure: Ring C is optionally substituted with 1-3 R x , Ring D is optionally substituted with 1-3 R f .

18. The compound of formula (I) according to claim 17, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof, wherein: Rings C and D together form the following fused ring structure: Ring C is optionally substituted with 1-3 R x , Ring D is optionally substituted with 1-3 R f .

19. A compound of formula (I) according to any one of claims 1 to 18, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: When present, L is selected from the group consisting of -CH2-, -C(CH3)-, -CH2CH2-, -NH-CH=CH-, and -N=CH2-.

20. A compound of formula (I) according to any one of claims 1 to 19, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein: The compound of formula (I) is selected from the following compounds:

21. A method for preparing a compound of formula (I) according to any one of claims 1 to 20, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, the preparation method comprising the following synthetic route: in, X in SM1 represents a leaving group, selected from halogen or activated hydroxyl, etc. SM1 and SM2 react in the presence of a suitable base such as TEA, DIPEA, pyridine or a suitable acid such as TFA. The preferred reaction temperature under base catalysis is 20-40°C, and the preferred reaction temperature under acid catalysis is 70-90°C.

22. A method for preparing a compound of formula (I) according to any one of claims 1 to 20, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof, the preparation method comprising the following synthetic route: in, X in SM1 represents a leaving group, selected from halogen or activated hydroxyl, etc. SM1 and SM2 react in the presence of a suitable base such as TEA, DIPEA, pyridine or a suitable acid such as TFA, HCl. The preferred reaction temperature under base catalysis is 20-120°C, and the preferred reaction temperature under acid catalysis is 70-90°C.

23. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier or excipient.

24. A method for inhibiting intracellular Arf1 pathway activity in a cell, comprising administering an effective amount of a compound of formula (I) according to any one of claims 1 to 20, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof to the cell, such that the Arf1 pathway activity in the cell is reduced.

25. The method according to claim 24, wherein: The cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

26. The method according to claim 24 or 25, wherein: The method induces cell death at the cellular level.

27. The method according to any one of claims 24 to 26, wherein: The method is performed in vitro.

28. The method according to any one of claims 24 to 26, wherein: The method is performed in vivo.

29. Use of a compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof in the preparation of an agent for inhibiting intracellular Arf1 pathway activity in a cell.

30. The use according to claim 29, wherein The cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

31. The use according to claim 29 or 30, wherein The agents induce cell death at the cellular level.

32. The use according to any one of claims 29 to 31, wherein The agent is administered to cells in vitro.

33. The use according to any one of claims 29 to 31, wherein The agent is administered to the cell in vivo.

34. A method for treating or preventing a disease associated with Arf1 pathway 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 20, its stereoisomer, isotopic variant, or a pharmaceutically acceptable salt or solvate thereof.

35. The method of claim 34, wherein: The disease associated with Arf1 pathway activity is cancer or tumor.

36. The method of claim 35, 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.

37. The method of claim 34, wherein: The disease associated with Arf1 pathway 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.

38. The method according to any one of claims 34 to 37, wherein: The subject is a mammal, such as a human.

39. The method according to any one of claims 34 to 38, wherein: The method further comprises co-administering at least one anti-PD-1 antibody.

40. The method of claim 39, wherein: The compound of formula (I) according to any one of claims 1 to 20, its stereoisomer, isotopic variant, or pharmaceutically acceptable salt or solvate thereof and at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody, are administered simultaneously or sequentially in any order.

41. A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject.

42. A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject according to claim 41, wherein: The disease associated with Arf1 pathway activity is cancer or tumor.

43. A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject according to claim 42, 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.

44. A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject according to claim 41, wherein: The disease associated with Arf1 pathway 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.

45. A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject according to any one of claims 41 to 44, wherein: The subject is a mammal, such as a human.

46. ​​A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject according to any one of claims 41 to 45, wherein: The compound of formula (I) according to any one of claims 1 to 20, its stereoisomer, isotopic variant, or pharmaceutically acceptable salt or solvate thereof is administered in combination with at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

47. A compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof for use in treating or preventing a disease associated with Arf1 pathway activity in a subject according to claim 46, wherein: The compound of formula (I) according to any one of claims 1 to 20, its stereoisomer, isotopic variant, or pharmaceutically acceptable salt or solvate thereof and at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody, are administered simultaneously or sequentially in any order.

48. Use of a compound of formula (I) according to any one of claims 1 to 20, 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 Arf1 pathway activity in a subject.

49. The use according to claim 48, wherein The disease associated with Arf1 pathway activity is cancer or tumor.

50. The use according to claim 49, 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.

51. The use according to claim 48, wherein The disease associated with Arf1 pathway 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.

52. The use according to any one of claims 48 to 51, wherein The subject is a mammal, such as a human.

53. A kit for treating or preventing a disease associated with Arf1 pathway activity in a subject, the kit comprising a compound of formula (I) according to any one of claims 1-20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claim 23; a container; and optionally a package insert or label indicating treatment.

54. The kit according to claim 53, wherein The disease associated with Arf1 pathway activity is cancer or tumor.

55. The kit according to claim 54, 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.

56. The kit according to claim 53, wherein The disease associated with Arf1 pathway 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.

57. A kit according to any one of claims 53 to 56, wherein The subject is a mammal, such as a human.

58. A kit according to any one of claims 53 to 57, wherein The kit also includes at least one immune checkpoint inhibitor, such as an anti-PD-1 antibody.

59. A kit for diagnosing a disease associated with Arf1 pathway activity in a subject, comprising at least one compound of formula (I) according to any one of claims 1 to 20, its stereoisomers, isotopic variants, or pharmaceutically acceptable salts or solvates thereof as a test agent.

60. The kit according to claim 59, wherein The test agent detects at least one biomarker indicative of the presence of a disease associated with Arf1 pathway activity.

61. The kit according to claim 59 or 60, wherein The test reagent can detect the activity of Arf1 GTPase, serve as a reporter gene for detecting lipolysis activity, detect the formation of lipid droplets, detect autophagy activity, or serve as an upstream or downstream alternative regulator of Arf1 activity or function.

62. The kit according to any one of claims 59 to 61, wherein The disease associated with Arf1 pathway activity is cancer or tumor.

63. The kit according to claim 62, 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.

64. A kit according to any one of claims 59 to 61, wherein The disease associated with Arf1 pathway 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.

65. The kit according to any one of claims 59 to 64, wherein The subject is a mammal, such as a human.

66. A compound of formula (I) according to any one of claims 1 to 20, 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.

67. A compound of formula (I) according to any one of claims 1 to 20, 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 66, 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.

68. Use of a compound of formula (I), a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 20 in the preparation of a therapeutic vaccine for blocking tumor development.

69. The use according to claim 68, 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.

70. 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 20, 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.

71. A method of vaccination comprising administering a therapeutic vaccine prepared according to the method of claim 70 to a subject.

72. The vaccination method according to claim 71, wherein: The subject is a mammal, such as a human.

73. A method for killing cells and inducing an anti-tumor immune response, the method comprising inhibiting at least one Arf1, particularly COPI / Arf1-lipolysis β-oxidation pathway activity in the cells by using an Arf1 pathway inhibitor.

74. The method of claim 73, wherein: The cell is a progenitor cell, a stem cell, a cancer stem cell, or a cancer cell.

75. The method of claim 73 or 74, wherein: The Arf1 pathway inhibitor is selected from the group consisting of small molecule Arf1 inhibitors, RNAi agents against Arf1, antisense agents against Arf1, peptidomimetic Arf1 inhibitors and DNA binding deoxynucleotide inhibitors containing the target protein Arf1.

76. The method of claim 75, wherein: The small molecule Arf1 inhibitor is a compound of formula (I) according to any one of claims 1 to 20, a stereoisomer, an isotopic variant thereof, or a pharmaceutically acceptable salt or solvate thereof.

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