A novel group of COPI / ARF1-lipolysis pathway inhibitors and compounds for eradicating cancer stem cells and inducing DAMP-mediated anti-tumor immune responses

Abstract

The present invention relates to the use of a novel Arf1 inhibitor; a method for treating refractory, recurrent or metastatic cancer using these compounds; a method for selectively killing cancer stem cells and cancer cells by using the compounds and a specific dosing regimen; a method for targeting tumor stem cells to induce an anti-tumor immune response by inhibiting the Arf1 pathway, particularly the COPI / Arf1-lipolysis-β-oxidation pathway; a method for treating diseases associated with Arf1 pathway activity in mammals using the novel compounds; processes for preparing such compounds and their intermediates, and pharmaceutical compositions of related compounds.

Description

Technical Field

[0001] The present invention generally relates to the use of COPI / Arfl-lipolysis-β-oxidation pathway inhibitors in the treatment of diseases. More specifically, the present invention relates to the use of COPI / Arfl-lipolysis-β-oxidation pathway inhibitors in targeting cancer stem cells and activating immune responses and treating diseases. More specifically, the present invention relates to 1H-indole-5-carbaldehyde 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-yl hydrazone, 1H-indole-5-carbaldehyde 5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidin-4-yl hydrazone, and related compounds for inhibiting Arfl, targeting cancer stem cells and activating anti-tumor immune responses for the treatment of malignant diseases. The present invention also relates to the treatment of refractory, recurrent or metastatic cancers, and the preparation methods of related compounds and their intermediates, and pharmaceutical compositions of related compounds. Background Art

[0002] In the United States alone, hundreds of thousands of people die from cancer every year. Despite progress in treating certain forms of cancer through surgery, radiotherapy, and chemotherapy, many types of cancer are essentially incurable. Even when there are effective treatments for a particular cancer, the side effects of such treatments can be severe and lead to a significant decline in quality of life.

[0003] Immunotherapy, especially checkpoint blockade therapy, has begun to fundamentally change the paradigm for treating cancer patients (Sharma et al, 2017). However, despite recent clinical successes, unfortunately, immunotherapy has helped only a small subset of cancer patients. Most patients derive little benefit because most newly emerging tumors have already formed immune escape mechanisms in the tumor microenvironment, including dysfunctional T cells and lack of T cell infiltration (Jerby-Arnon et al, 2018, Sharma et al, 2017). But the immune responses induced by pathogens (bacteria or viruses) are mostly intact in cancer patients. These pathogens express unique molecules called pathogen-associated molecular patterns (PAMPs), which are recognized by pattern recognition receptors (PRRs) of the innate immune system to activate anti-infection immune responses. This induced immune response can be used to cause tumor regression, as first demonstrated by William Coley (Balkwill, 2009). In the 1890s, he successfully caused the regression of many sarcomas and / or lymphomas in patients after injecting streptococcal cultures. Some of the injected bacteria may trigger PAMP-induced anti-tumor immune responses.

[0004] Recently, it has been found that the immune system can be induced not only by PAMPs but also by endogenous signals called danger / damage-associated molecular patterns (DAMPs), which are released from damaged or stressed cells in the absence of microbial components (Jerby-Amon et al., 2018, Sharma et al., 2017). DAMPs are analogs of "PAMPs" and are molecules present in specific cell compartments that are not expressed or are only limitedly expressed under physiological conditions. However, they are strongly induced, and then under stress or injury conditions, DAMPs are translocated to the cell surface or the extracellular space. The most important DAMPs include: (i) the pre-apoptotic exposure of the endoplasmic reticulum (ER)-anchored molecular chaperone calreticulin (Calr) on the cell surface; (ii) the release of the non-histone nuclear protein high-mobility group box 1 (HMGB1) into the extracellular space; (iii) the active secretion of ATP. Surface-exposed Calr will promote the phagocytosis of tumor-associated antigens by binding to LRP1 / CD91 on dendritic cells (DCs), extracellular HMGB1 will bind to Toll-like receptor 4 (TLR4) on DCs and activate the TLR4-MYD88 pathway to support DC maturation and prevent the accelerated destruction of lysosomes that phagocytose metabolic foreign bodies, thereby enabling antigen processing and presentation to cytotoxic T lymphocytes (CTLs). In addition, dying tumor cells secrete ATP, which activates T cells secreting IFNγ through the P2RX7 receptor-inflammasome-IL-1β pathway, thereby mediating an anti-tumor immune response (Ghiringhelli et al., 2009). In summary, these different signaling pathways of DAMPs promote the development of host tumor-specific adaptive immune responses. These can inhibit or even completely eradicate drug-resistant tumor cells. However, the underlying molecular mechanisms that trigger tumor cells to release DAMPs and induce anti-tumor immune responses in vivo remain unclear.

[0005] Cancer stem cells (CSCs) are a subset of cells within tumors that are in a stem cell state and possess stem cell characteristics. CSCs can lead to treatment resistance, tumor metastasis, disease recurrence, and ultimately patient death (Batlle and Clevers, 2017; Lytle et al., 2018; Shibue and Weinberg, 2017). The ultimate goal of CSC research is to identify pathways that selectively regulate CSC survival and then target those pathways to eradicate CSCs. Some CSCs arise from the transformation of normal stem cells, while others are generated by reprogramming non-CSC cancer cells into a stem cell state. CSCs share many common properties with normal stem cells. Thus, pathways that regulate normal or transformed stem cells may also regulate CSCs. We previously found that the COPI / Arf1-mediated lipolysis pathway selectively maintains stem cells and transformed stem cells in Drosophila, and knockdown of this pathway leads to stem cell necrosis (Singh et al., 2016). In a recent study, we found that knockdown of Arf1 in mice disrupts lipid metabolism and causes lipid droplet accumulation, which further induces metabolic stress - mitochondrial defects and endoplasmic reticulum stress. Metabolic stress selectively kills progenitor cells, stem cells, and CSCs through necrosis in mice. Dying CSCs release DAMPS that activate DCs, further enhancing T cell infiltration and activation, which in turn stimulates the body's anti-tumor immune response. Our data suggest that knockdown of the Arf1 pathway not only kills CSCs but also triggers a tumor-specific immune response and converts dying CSCs into a therapeutic vaccine, which actually improves the long-term efficacy of treatment (Wang et al., 2020).

[0006] We found that inhibiting Arfl induces T cell infiltration and activation, and provides a new therapeutic strategy for activating anti-tumor immunity in cancer patients by DAMPs. In addition to checkpoint blockade, this may be another promising direction for cancer immunotherapy. The new therapy may include methods to reduce Arfl activity and block the Arfl-mediated lipolysis pathway and related β-oxidation. We tested 10 reported Arfl inhibitors (Billett al., 2011; Boal et al., 2010; Feng et al., 2004; Newton et al., 2006; Ohashi et al., 2012; Saenz et al., 2009; Sorieu et al., 2011; Viaud et al., 2007), including Brefeldin A (BFA), Golgicide A (GCA), LM1L, Secinl6, SecinH3, SecinB7, Exo1, Exo2, LG8 and LG18 on Drosophila cancer stem cells and normal human bone marrow hematopoietic stem cells (HSC). We found that these known inhibitors are either not very effective in killing Drosophila cancer stem cells or too toxic (killing normal HSC). Based on this, we synthesized and tested new inhibitors based on Exo2 and found that Du10l and Du102 (described below) are effective and relatively less toxic. SUMMARY OF THE INVENTION

[0007] The present invention is based on our recent discovery that inhibiting the COPI / ARF1-lipolysis-β-oxidation pathway eradicates cancer stem cells and induces DAMP-mediated anti-tumor immune responses.

[0008] Accordingly, a first aspect of the present invention relates to a method of eliminating tumors, wherein the method comprises inhibiting at least some, most or substantially all (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) of the Arfl pathway activity in a cancer patient by an inhibitor of the Arfl pathway.

[0009] In one embodiment, the Arfl pathway inhibitor is isolated, purified or synthesized and can be selected from small molecule Arfl inhibitors, RNAi agents against Arfl, antisense agents against Arfl, peptidomimetic Arfl inhibitors, and G-quadruplex oligodeoxynucleotide Arfl inhibitors. The inhibition mechanism can be selected from significantly inhibiting Arfl GTPase activity, significantly inhibiting Arfl-pathway-regulated lipolysis activity, significantly inhibiting Arfl-pathway-regulated oxidation activity, and significantly inducing cell necrosis by inhibiting the Arfl pathway.

[0010] In one embodiment, the inhibitor is selected from the above compounds 102, 104 - 111, especially Du101 and Du102, their enantiomers, diastereomers, tautomers and salts or solvates (hereinafter referred to as "the compounds of the present invention").

[0011] The present invention provides a method for inhibiting or reducing the growth of a tumor or cancer, which includes contacting the tumor with an effective amount of the compounds of the present invention. The compound inhibits the growth of the tumor or cancer or the compound reduces the size of the tumor or cancer. The compound increases the expression of MHC-I and MHC-II. Compared with DMSO, the compound increases the infiltration and activation of T cells in the tumor. The compound increases the expression of T cell activation markers, such as GzmA, GzmB and perforin. In another embodiment, the compound increases the expression of at least one inflammatory cytokine or chemokine, which may be selected from IFNγ, IL-1β, Ccl5, Cxcl0, Cxcl1, Ccl22. In certain embodiments, the compound is co-administered with at least one anti-PD-1 antibody, and the compound and PD-1 blockade have a synergistic effect. In certain embodiments, after inoculation with cancer cells treated with the compound, it acts as a vaccine to protect animals from tumor development. In certain embodiments, treating with the compound has a "kill two birds with one stone" effect, which can not only kill CSCs, but also trigger a tumor-specific immune response, converting necrotic CSCs into a therapeutic vaccine, thereby producing long-term efficacy.

[0012] In a second aspect, the present invention provides a method for treating or preventing a disorder related to Arfl pathway activity in a subject, the method including administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the compounds of the present invention so as to reduce the activity of the Arfl pathway. In one embodiment, the Arfl pathway activity can be identified by testing Arfl GTPase activity or an alternative upstream or downstream regulator of Arfl GTPase activity. The disorder may be cancer. In one embodiment, cancers are known to have Arfl pathway activity, including but not limited to: 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 and lymphoma. The disorder may also be a non-cancerous disorder known to be related to Arfl pathway activity, and in one embodiment, is selected from autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating disorders, Alzheimer's disease, stroke, ischemia-reperfusion injury, multiple sclerosis and other inflammatory or neurodegenerative diseases.

[0013] In a third aspect, the present invention provides a method for inhibiting the activity of the Arfl pathway in cells. The method includes administering to the cells an effective amount of a compound of the present invention such that the activity of the Arfl pathway in the cells is reduced by, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In one embodiment, the cells are CSCs or are cancerous. The method can induce cell death. The method can be carried out in vitro or in vivo.

[0014] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, namely a compound selected from Compounds 102, 104 - 111 described above, and its pharmaceutically acceptable salts or solvates, as well as pharmaceutically acceptable excipients, carriers or diluents. In one feature, the composition is suitable for oral, nasal, topical, rectal, vaginal or parenteral administration, or intravenous, subcutaneous or intramuscular injection.

[0015] In a fifth aspect, the present invention further provides a method for preparing some compounds of the present invention. The method prepares a compound of general formula I, wherein R1 has the meaning defined herein, or its pharmaceutically acceptable salt, solvate or prodrug. In particular:

[0016] General formula (I), wherein r = 1 - 6;

[0017] R1 is selected from one or more substituted or unsubstituted phenyls, from halogens, C 1-6 alkyl, haloalkyl, OH, OCH3, O(CH2)nCH3, cyclopropyloxy, OC(CH3)3, OCH(CH3)2, NH2, NO2, N(CH3)2, NH(CH2)nCH3, CN, N3, etc., where n is 1 - 9.

[0018] Specifically, the inhibitor targeting the Arfl pathway is the following compound:

[0019]

[0020] In a sixth aspect, the present invention provides a method for preparing the compound 1H - indole - 5 - carbaldehyde 6,7,8,9 - tetrahydro - 5H - cyclohepta[4,5]thieno[2,3 - d]pyrimidin - 4 - yl hydrazone or the above - mentioned 107 or Du101.

[0021]

[0022] In a seventh aspect, the present invention provides a method for preparing the formatting compound 1H-indole-5-carbaldehyde 5,6,7,8,9,10-hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidin-4-yl hydrazone or the above-mentioned 111 or Du102.

[0023] Embodiments of the present invention are discussed in detail below. In describing the embodiments, specific terms are used for clarity. However, the present invention is not intended to be limited to the specific terms so selected. Those skilled in the relevant art will recognize that other equivalent components and other methods can be used without departing from the spirit and scope of the present invention. All references cited herein are incorporated by reference as if each reference had been incorporated individually.

[0024] Definitions

[0025] As used herein, the singular forms "a" and "the" include plural forms unless the context clearly dictates otherwise. For example, the term "a cell" includes multiple cells, including mixtures thereof.

[0026] As used herein, the terms "cancer stem cell" and "CSC" are interchangeable. CSCs are of mammalian origin and, in a preferred embodiment, these CSCs are of human origin, but they are not intended to be limited thereto. Cancer stem cells are defined and functionally characterized as a population of cells derived from a tumor that: (1) have extensive proliferative capacity; (2) are capable of asymmetric cell division to produce one or more differentiated progeny with reduced proliferative or developmental potential; (3) are capable of symmetric cell division for self-renewal or self-maintenance. Other common methods for characterizing CSCs include cell surface markers, morphology, transcriptional profiles, and drug responses. CSCs are also referred to in the research literature as tumor / cancer-initiating cells, cancer stem cell-like cells, stem cell-like cancer cells, highly tumorigenic cells, tumor stem cells, solid tumor stem cells, drug-surviving cells (DSC), drug-resistant cells (DRC), or super malignant cells.

[0027] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal wherein a population of cells is characterized by unregulated cell growth. As used herein, "cancer cells" and "tumor cells" refer to the total population of cells derived from a tumor, including non-tumorigenic cells and tumorigenic stem cells (cancer stem cells) that make up the majority of the tumor cell population. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, 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 head and neck cancers.

[0028] As used herein, "tumor" refers to any mass of tissue resulting from excessive cell growth or proliferation, whether benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0029] As used herein, "metastasis" refers to the process by which cancer spreads or metastasizes from its site of origin to other regions of the body, with the appearance of similar cancerous lesions at the new sites. "Metastatic" cells lose their adhesive contact with neighboring cells and migrate through the bloodstream or lymph from the primary site of the disease to invade adjacent body structures.

[0030] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-humans, primates, rodents, etc. Generally, the terms "subject" and "patient" are used interchangeably herein with respect to human subjects.

[0031] As used herein, terms such as "treat" or "alleviate" mean: 1) to cure, slow down, alleviate symptoms and / or stop the progression of a diagnosed pathological condition or disorder; 2) preventive or prophylactic measures to prevent or slow down the development of a target pathological condition or disorder. Thus, those in need of treatment include those who already have the disease; those who are predisposed to the disease; and those who need to prevent the disease. A subject is successfully "treated" according to the methods of the present invention if the patient exhibits one or more of the following: a decrease or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including the spread of cancer into soft tissues and bones; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; a decrease in morbidity and mortality; and an improvement in quality of life.

[0032] As used herein, the term "inhibit" and its synonyms, when used in the context of biological activity, refer to a downregulation of biological activity, which can reduce or eliminate a targeted function, such as the production of a protein or the phosphorylation of a molecule. In certain embodiments, inhibition can refer to a reduction in the target activity of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. When used in the context of a disorder or disease, these terms refer to the successful prevention of the onset of symptoms, the alleviation of symptoms, or the elimination of the disease or disorder.

[0033] 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.

[0034] As used herein, the term "GTPase" refers to a hydrolase enzyme that can bind and hydrolyze guanosine triphosphate (GTP). GTP binding and hydrolysis occur in a highly conserved G domain common to all GTPases.

[0035] As used herein, the term "β-oxidation" refers to a catabolic process by which fatty acid molecules are broken down in the cytoplasm of prokaryotes and in the mitochondria of eukaryotes to produce acetyl coenzyme A, NADH, and FADH2, which enter the tricarboxylic acid cycle and are coenzymes used in the electron transport chain. It is so named because the β-carbon of the fatty acid is oxidized to a carbonyl group. β-oxidation is primarily facilitated by three functional proteins in the mitochondria, an enzyme complex associated with the inner mitochondrial membrane, although some fatty acids are oxidized in peroxisomes.

[0036] As used herein, the term "necrotic cell death" refers to when cells are exposed to physiological conditions (e.g., hypothermia, hypoxia) or extreme changing conditions that can cause damage to the plasma membrane. Under physiological conditions, direct damage to the plasma membrane is caused by agents such as complement and lytic viruses. Necrosis begins with impaired ability of the cell to maintain homeostasis, leading to an influx of water and extracellular ions. The intracellular organelles, especially mitochondria, and the entire cell swell and rupture (cell lysis). Due to the eventual breakdown of the plasma membrane, cellular contents, including lysosomal enzymes, are released extracellularly, and thus, in vivo, cell necrosis is usually associated with a strong inflammatory response, which in turn causes extensive tissue damage.

[0037] "Immune disorders" include, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases. "Immune conditions" refer to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist immune system irradiation. "Cancer disorders" include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions such as dysplasia.

[0038] As used herein, the term "cytotoxic T cell" refers to a type of T lymphocyte (a white blood cell) that kills cancer cells, infected cells (especially those infected with viruses), or other cells. Most cytotoxic T cells express a T cell receptor (TCR) that can recognize specific antigens. An antigen is a molecule that can stimulate an immune response and is usually produced by cancer cells or viruses. Antigens inside the cell bind to class I MHC molecules and are carried to the cell surface by class I MHC molecules, where they can be recognized by T cells. If the TCR is specific for the antigen, it binds to the complex of the class I MHC molecule and the antigen, and the T cell destroys the cell. In order for the TCR to bind to the class I MHC molecule, the TCR must be accompanied by a glycoprotein called CD8, which binds to the constant part of the class I MHC molecule. Therefore, these T cells are called CD8+ T cells. Once cytotoxic CD8+ T cells (CTLs) recognize their target cells in the periphery, the CTLs are activated, and a highly specialized intercellular contact structure called the immunological synapse (IS) forms between the T cell and its target cell. Activation of the TCR leads to the involvement of Src family kinases, Lek and Fyn, and the recruitment of the Syk family kinase ZAP-70 to the TCR, where these tyrosine kinases are activated. Activated ZAP70 in turn phosphorylates the adaptor protein LAT, which is used to activate T cells, resulting in the formation of the LAT signalosome, which includes phospholipase Cγl (PLCγl) and SLP76 (76 kDa SH2 domain-containing leukocyte protein). PLCγl converts phosphatidylinositol-4,5-bisphosphate (PIP2) into DAG (diacylglycerol) and IP3 (inositol-1,4,5-trisphosphate). DAG accumulates at the immunological synapse, leading to the recruitment of new protein kinase Cs (PKCs), including PKCθ. PKCθ then promotes centrosome (microtubule organizing center or MTOC) polarization by positioning dynein relative to the synapse, which pulls the MTOC in the direction of 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 under the IS ensures dynein- and Ca 2+ -dependent targeted delivery and exocytosis of granule contents directly into the synaptic cleft, resulting in target cell lysis through the concerted action of granzymes and perforins (reviewed in: dela Rochet al., 2016).

[0039] As used herein, the term "pharmaceutically acceptable excipient, carrier or diluent" refers to a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which participates in carrying or transporting the subject agent from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents and lubricants such as sodium lauryl sulfate, magnesium stearate and polyoxyethylene-polyoxypropylene copolymers and coloring agents, release agents, coating agents, sweetening agents, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition.

[0040] The compounds of the present invention may form salts, which are also within the scope of the present invention. Unless otherwise indicated, reference to the compounds of the present invention herein shall be understood to include reference to their salts. As used herein, the term "salt" refers to acidic and / or basic salts formed with inorganic and / or organic acids and bases. In addition, when the compounds of the present invention contain both a basic moiety (such as but not limited to pyridine or imidazole) and an acidic moiety (such as but not limited to carboxylic acid), zwitterions ("inner salts") may be formed and are included within the term "salt" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in the isolation or purification steps which may be employed during preparation. The salts of the compounds of the present invention may be prepared, for example, by reacting compound 102 or 104-111, with a suitable amount of an acid or base, such as an equivalent amount, in a medium in which the salt precipitates or in an aqueous medium, followed by lyophilization.

[0041] Solvates of the compounds of the present invention are also included herein. Solvates of the compounds of the present invention include, for example, hydrates.

[0042] The present invention provides a method for treating proliferative diseases such as cancer, tumors, etc. with compounds selected from the above compounds 102, 104-111, especially Du101 and Du102. Recently, we discovered a new network that links lipid metabolism, CSC death, and anti-tumor immunity. CSCs may be responsible for treatment resistance and immune evasion. CSC signals can regulate lymphocyte infiltration into tumors and alter the tumor microenvironment (Lytle et al., 2018). We have demonstrated that knockdown of Arf1-mediated lipid metabolism in CSCs leads to metabolic stress and subsequent cellular responses, including the release of DAMPs, which promote anti-tumor immunity by activating DCs, enhancing T cell infiltration, and activation (Wang et al., 2020). We further demonstrated that Arf1 inhibition and PD-1 blockade have a synergistic effect. Consistently, TCGA data analysis showed a negative correlation between Arf1 expression and T cell infiltration and activation, as well as a better survival probability in various human cancers. Our findings suggest that knockdown of the Arf1 pathway has a "kill two birds with one stone" effect, not only killing CSCs but also triggering tumor-specific immune responses and converting dying CSCs into therapeutic vaccines, thus producing long-term efficacy treatment (Wang et al., 2020).

[0043] The present invention provides evidence that the new compound inhibits Arf1 pathway activity and induces an anti-tumor immune response by increasing the expression of chemokines and inflammatory cytokines and enhancing T cell infiltration and activation. We further demonstrated that treatment with the compound and a PD-1 blocker has a synergistic effect. Therefore, treatment with the compound should provide a significant improvement in tumor treatment.

[0044] Combined with the latest breakthroughs in anti-tumor immune research, the present invention enables the present invention to provide a series of methods for inhibiting CSCs or treating cancers with specific CSCs or cancers in general. The present invention also provides methods for inhibiting Arf1 pathway activity in cells or treating cancerous and non-cancerous conditions related to Arf1 pathway activity. The present invention also provides related methods (e.g., manufacturing and candidate drug screening), materials, compositions, and kits.

[0045] The method can be used to treat cancer in a subject. Cancers known to have CSCs and Arf1-lipolysis-β-oxidation pathway activity are good candidates for such treatment, including but not limited to: 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, and lymphoma. In one embodiment, the method is used to treat liver cancer, head and neck cancer, pancreatic cancer, and / or gastric cancer. In another embodiment, the method is used to treat multiple myeloma, brain tumor, and sarcoma.

[0046] In addition, since CSCs have been proven to be the root cause of tumorigenesis, cancer metastasis, and cancer recurrence, any method of inhibiting CSCs of the present invention can be implemented to treat metastatic cancer, cancer refractory to chemotherapy or radiotherapy, or cancer that recurs in a subject after initial treatment.

[0047] In one embodiment, the inhibitor is isolated, purified, or synthetic and can be selected from small molecule Arf1 inhibitors, RNAi against Arf1, antisense agents against Arf1, peptidomimetic Arf1 inhibitors, and G-quadruplex oligodeoxynucleotide Arf1 inhibitors. The inhibitor can also be isolated or purified from natural products.

[0048] The Arf1 inhibitor can be selected to target any step in the Arf1 pathway. For example, the inhibitor can significantly inhibit Arf1 GTPase activity, lipolysis reporter genes, lipid droplet formation, autophagy, and alternative upstream or downstream regulators of Arf1 activity or function.

[0049] In one embodiment, the Arf1 inhibitor according to the present invention is: Compounds 102, 104 - 111 (Table 1), their enantiomers, diastereomers, tautomers, and salts or solvates ("compounds of the present invention"). The present invention also provides in vitro and in vivo data on the reduction of tumors by the compounds of the present invention.

[0050] The compounds of the present invention not only show lethality in a wide range of cancer cells but also exhibit selectivity in their cytotoxicity, which is crucial for the development of low-toxicity therapeutic agents. Selective cytotoxicity as used herein refers to the ability of a compound to kill cancer cells while substantially not harming normal cells, sometimes under certain conditions. Normal cells generally refer to healthy non-tumorigenic cells. The conditions leading to the selective cytotoxicity of a candidate drug are difficult to predict because they require an understanding of the underlying mechanisms of cytotoxicity. For example, reducing the toxicity of an anticancer drug that targets microtubule formation during mitosis compared to a drug that blocks cell metabolic processes requires the use of completely different factors. The appropriate conditions for generating selective cytotoxicity require balancing the need for the drug to have sufficient toxicity to effectively kill cancer cells while being sufficiently tolerated by normal cells. For example, if a lower concentration is used, this generally means that a longer infusion time is required to kill cancer cells.

[0051] From the data generated in the examples of the present invention, it appears that the compounds of the present invention can achieve selective cytotoxicity if the affected cells are not continuously exposed to a critical concentration of the compound for more than a certain time. In a method for selectively killing cancer cells in a subject, a pharmaceutical composition containing the compound of the present invention is administered to the subject such that the concentration of the compound in the subject's plasma does not exceed the critical concentration for more than 24 hours. This method can be used to treat all cancers, including any cancer group described herein, and to treat Arfl-related diseases, an exemplary list of which has been provided above and will not be repeated here. Alternatively, the duration can be further limited to 12, 16, and 20 hours after each administration. The critical concentration of each compound may be different. In various embodiments of the present invention, the critical concentration is about 100 μΜ, about 50 μM, about 20 μM, or about 10 μΜ.

[0052] Use

[0053] The present invention provides methods for treating proliferative disorders or conditions, such as uterine cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, penile cancer, gastrointestinal cancers such as esophageal cancer, oropharyngeal cancer, gastric cancer, small intestine cancer or colorectal cancer, colon or rectum, kidney, renal cell, bladder, bone, bone marrow, skin, head or neck, skin, liver, gallbladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid gland, brain, such as glioma, ganglion, central nervous system (CNS) and peripheral nervous system (PNS), and the immune system, such as the spleen or thymus. The present invention provides treatment for, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers, such as epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papillomavirus, adenocarcinoma, lymphoma, carcinoma, melanoma, leukemia, myeloma, sarcoma, teratocarcinoma, chemically induced cancers, metastases, and angiogenesis. The present invention also contemplates reducing the tolerance to tumor cell or cancer cell antigens, for example, by modulating the activity of T cell infiltration and activation.

[0054] A method of treating a human, mammal or animal subject suffering from a tumor, delaying its progression, preventing its recurrence, alleviating its symptoms or otherwise improving it according to the present invention may include administering a therapeutically effective amount of a compound, product and / or pharmaceutical composition, thereby producing anti-tumor activity. For example, the anti-tumor activity may be anti-cancer activity. For example, the anti-tumor activity may include slowing tumor volume growth, stopping tumor volume growth or reducing tumor volume. The tumor may include solid tumors, malignancies, metastatic cells, cancer stem cells. The tumor may include carcinoma, sarcoma, adenocarcinoma, lymphoma or hematological malignancies. The tumor may be refractory to treatment with chemotherapy, radiotherapy and / or hormone therapy. A compound, product and / or pharmaceutical composition may be administered to prevent tumor recurrence. The compound, product and / or pharmaceutical composition may be administered as an adjuvant therapy to surgical resection. The compound, product and / or pharmaceutical composition may be administered, for example, orally and / or intravenously.

[0055] The method according to the present invention further includes treating a disease or disorder, delaying its progression, preventing its recurrence, alleviating its symptoms or otherwise improving the disease or disorder in a human, mammal or animal subject suffering from the disease or disorder. In some embodiments, the disease or disorder is selected from autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, autoimmune demyelinating diseases, Alzheimer's disease, stroke, ischemia-reperfusion injury, multiple sclerosis and other neurodegenerative diseases.

[0056] The treatment is considered successful if the compound, product and / or pharmaceutical composition is administered to a patient suffering from a disease or disorder and any one of a variety of laboratory or clinical outcomes is achieved. For example, the administration is considered successful if one or more symptoms associated with the disease or disorder are alleviated, reduced, suppressed or there is no further progression. The administration is considered successful if the disorder, such as an autoimmune disorder, enters a remission or there is no further progression.

[0057] In some embodiments, the compounds, products and / or pharmaceutical compositions described herein are administered in combination with any one of a variety of known therapeutic agents, including, for example, chemotherapeutic agents and other anti-tumor agents, anti-inflammatory compounds and / or immunosuppressive compounds, cytokines or cytokine antagonists, such as IL-12, interferon-α or anti-epidermal growth factor receptor. In some embodiments, the compounds, products and / or pharmaceutical compositions described herein may be used in combination with any one of a variety of known treatments, including but not limited to surgical treatments and methods, radiotherapy, chemotherapy and / or hormone or other endocrine-related treatments.

[0058] These “combination therapies” can be administered sequentially or simultaneously. The compounds, products, and / or pharmaceutical compositions described herein and the second therapy can be administered to a subject, preferably a human subject, in the same pharmaceutical composition. Alternatively, the compounds, products, and / or pharmaceutical compositions described herein and the second therapy can be administered to the subject simultaneously, separately, or sequentially in separate pharmaceutical compositions. The compounds, products, and / or pharmaceutical compositions described herein and the second therapy can be administered to the subject by the same or different routes of administration. In some embodiments, the combination therapy of the invention comprises an effective amount of the compounds, products, and / or pharmaceutical compositions described herein and an effective amount of at least one other therapy (e.g., a prophylactic or therapeutic agent) having a different mechanism of action than the compounds, products, and / or pharmaceutical compositions described herein. In some embodiments, the combination therapy of the invention improves the prophylactic or therapeutic efficacy of the compounds, products, and / or pharmaceutical compositions described herein and the second therapy by acting together to have an additive or synergistic effect. In certain embodiments, the combination therapy of the invention reduces the side effects associated with the second therapy (e.g., a prophylactic or therapeutic agent).

[0059] Therapeutic compositions, methods

[0060] A pharmaceutical composition formulated with a therapeutically effective amount of the compound of the invention and a pharmaceutical carrier. A “therapeutically effective dose” is a dose sufficient to provide the desired therapeutic outcome. At the same time, while ensuring the effective dose, the side effects are minimized. The efficacy of the pharmaceutical composition composed of the compound of the invention is detected based on assays for Arfl GTPase activity, lipolysis reporter genes, lipid droplet formation, autophagy, and upstream or downstream activities of Arfl activity known in the art. The therapeutically effective dose for a particular patient needs to be determined by considering various factors, including the disorder being treated, the overall health of the patient, the method of administration, the severity of the side effects, etc. In the context of cancer, a suitable dose of the compound of the invention will result in an increase in T cell infiltration and activation at the tumor site, inflammatory cytokines such as IL-10 and IFNγ or T cell-related chemokines CCL5, CXCL-10, CXCL-11, and CCL22, or T cell activation markers IFNγ, perforin, GzmA, GzmB, an increase in the level of DAMPs or an increase in the level of ER stress markers or an increase in the level of MHC-I / MHC-II or 1ΡΝγ.

[0061] Veterinary, experimental, or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

[0062] For any method of treating a subject described herein, the invention also provides an effective dosing range, dosing frequency, and plasma concentration of the compound. In various embodiments, the pharmaceutical composition is administered at a dose of: (a) about 1 mg / m 2 to about 5,000 mg / m2 (i.v.) or about 1 mg / m 2 to about 50,000 mg / m 2 (p.o.); (b) about 2 mg / m 2 to about 3,000 mg / m 2 (i.v.) or about 10 mg / m 2 to about 50,000 mg / m2 (p.o.). In various embodiments, the compounds of the present invention may be administered every other day (Q2D), daily (QD), or twice daily (BID). In one embodiment, the pharmaceutical composition is administered orally and no more than four times a day (QID).

[0063] In some embodiments, the compounds, products, and / or pharmaceutical compositions may be administered according to the following regimens to treat a disease or disorder. During a first continuous time period that is longer than the drug onset time and shorter than the harmful time, the blood molar concentration of the compound may be maintained at no less than the effective concentration and less than the harmful concentration. After the first continuous time period, the blood molar concentration may be below the effective concentration. For example, the effective concentration may be about 0.1 μM, about 0.2 μM, about 0.5 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 10 μΜ, or other concentrations determined by those skilled in the art to be effective. For example, the harmful concentration may be about 1 μM, about 3 μM, about 10 μΜ, about 15 μM, about 30 μM, about 100 μM, or another concentration determined by those skilled in the art to be harmful. For example, the effective time period may be about 1 hour, 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, or another time period determined by other those skilled in the art to be effective. For example, the harmful time period may be about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 144 hours, or another time period determined by those skilled in the art to be harmful.

[0064] In some embodiments, the effective amount of the compound, product, and / or pharmaceutical composition selected for treatment is greater than the IC of the tumor cells 50 and less than the IC of the normal cells 50 of the blood concentration. In some embodiments, the therapeutically effective amount is selected to produce a blood concentration that is high enough to kill tumor cells and lower than the IC of the normal cells 50 of the blood concentration.

[0065] In some embodiments, the dosage forms of the compound, product, and / or pharmaceutical composition include, but are not limited to, tablets, pills, capsules (hard or soft), cachets, powders, granules, suspensions, solutions, gels for oral administration, lozenges, troches, syrups, elixirs, emulsions, oil-in-water emulsions, water-in-oil emulsions, and / or potions.

[0066] In some embodiments according to the present invention, a composition for reducing or inhibiting tumor cell replication or spread includes a set of particles selected by the following method. It is possible to formulate a compound or its salt or solvate according to Formula I.

[0067] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient, carrier or diluent. A feature is that the composition is suitable for oral, nasal, topical, rectal, vaginal or parenteral administration, or intravenous, subcutaneous or intramuscular injection.

[0068] The formulations of the present invention include but are not limited to those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can conveniently be in unit dosage form and can be prepared by any methods well known in the pharmaceutical art. The amount of the active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the mammal being treated and the particular mode of administration. The amount of the active ingredient that can be combined with the carrier material to produce a single dosage form is generally the amount that produces a therapeutic effect. Generally, on a 100% basis, the amount will be in the range of, for example, about 1% to about 99% of the active ingredient, about 5% to about 70%, about 10% to about 30%.

[0069] The therapeutic compositions or formulations of the present invention suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using flavoring agents, usually sucrose and acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as lozenges (using inert matrices such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes, etc., each containing a predetermined amount of the compound of the present invention as the active ingredient. The compound of the present invention can also be administered as pills, troches or pastes.

[0070] In the solid dosage forms (capsules, tablets, pills, dragees, powders, granules, etc.) of the present invention for oral administration, the compounds of the present invention are admixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dibasic calcium phosphate, and / or any of the following: fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; humectants, such as glycerol; disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and sodium starch glycolate; solution retarders, such as paraffin wax; absorption promoters, such as quaternary ammonium compounds; wetting agents, such as cetyl alcohol, glycerol monostearate, and polyoxyethylene-polyoxypropylene copolymers; adsorbents, such as kaolin and bentonite; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0071] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. In addition, cyclodextrins, such as hydroxypropyl-β-cyclodextrin, can be used to solubilize the compounds.

[0072] In addition to the inert diluent, oral compositions may also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives. In addition to one or more compounds of the present invention, suspensions may also contain suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, and tragacanth, and mixtures thereof.

[0073] The preparation of the pharmaceutical composition of the present invention for rectal or vaginal administration can be provided as a suppository, which can be prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers, said excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository wax or as a salicylate, which is solid at room temperature but liquid at body temperature and thus will melt in the rectal or vaginal cavity and release the active agent of the present invention. The preparations of the present invention suitable for vaginal administration also include vaginal suppositories, tampons, creams, gels, pastes, foams or spray preparations, which contain suitable carriers known in the art.

[0074] Dosage forms for topical or transdermal administration of the compositions according to the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

[0075] In addition to the compounds of the present invention, ointments, pastes, creams and gels can contain excipients such as animal and vegetable fats, oils, waxes, paraffin wax, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0076] In addition to the compounds of the present invention, powders and sprays can also contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted ones such as butane and propane.

[0077] Ophthalmic preparations, eye ointments, powders, solutions, etc. are also considered to be within the scope of the present invention.

[0078] The pharmaceutical compositions of the present invention suitable for parenteral administration comprise one or more compounds of the present invention and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or reconstitutable sterile powders to be added to sterile injectable solutions or dispersions before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes rendering the preparation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0079] In some cases, in order to prolong the action of the compositions according to the invention, it is desirable to slow down their absorption by the body following subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. The absorption rate of the drug depends on its dissolution rate, which in turn may depend on crystal size and polymorph. Alternatively, delayed absorption of parenterally administered compositions is achieved by dissolving or suspending the compound in an oily carrier. One strategy for long-acting injections involves the use of poly(ethylene oxide)-poly(propylene oxide) copolymers, where the carrier is fluid at room temperature and solidifies at body temperature.

[0080] In one embodiment, the pharmaceutically acceptable excipient, carrier or diluent comprises a lipid for intravenous delivery. The lipid can be: phospholipids, synthetic phosphatidylcholines, natural phosphatidylcholines, sphingomyelins, ceramides, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, cholesterol, cholesterol sulfate, and hapten and PEG-conjugated lipids. The lipid can be in the form of nanoemulsions, micelles, emulsions, suspensions, nanosuspensions, liposomes or liposome. In one example, the pharmaceutically acceptable excipient, carrier or diluent is in the form of a micellar emulsion, suspension or nanoparticle suspension and further comprises a protein acceptable for intravenous use, such as human albumin or a derivative thereof, for intravenous delivery.

[0081] In one embodiment, the pharmaceutically acceptable excipient, carrier or diluent includes a waxy material for oral delivery. The waxy material can be mono-, di- or triglycerides, mono- or di-fatty acid esters of PEG, PEG-conjugated vitamin E (vitamin E TPG and / or Gelucire. Gelucire can be selected from Gelucire 44 / 14, Gelucire 43 / 01, Gelucire 50 / 02, Gelucire 50 / 13, Gelucire 37 / 02, Gelucire 33 / 01, Gelucire 46 / 07 and Gelucire 35 / 10. In one example, the pharmaceutically acceptable excipient, carrier or diluent is selected from octanol, transcutol hp, labrafil M, labrasol, triacetin, pharmasolv, ethanol, polyvinylpyrrolidone, carboxymethylcellulose, Tween 20 and Tween 80. In one example, a pharmaceutically acceptable excipient, such as Gelucire 44 / 14, is mixed with a surfactant, which can be Tween 80 or Tween 20. These embodiments of the pharmaceutical composition can be further formulated for oral administration.

[0082] The compounds of the invention can be synthesized using commercially available starting materials and methods well known to those skilled in the art of the organ. In Examples 13-14, the invention provides methods for making some of the claimed compounds.

[0083] According to one or more embodiments of the present invention, a small molecule Arfl inhibitor refers to any low molecular weight drug that exhibits inhibitory activity against Arfl. Compared with drugs with relatively large molecular weights such as proteins, peptides, and carbohydrates, small molecules are more likely to penetrate cell membranes and the blood-brain barrier. The process development and manufacturing costs of these molecules tend to be lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 , Du110 and Du102 are effective inhibitors of Arfl activation.

[0085] Figure 2 , In vitro cell line screening shows that Arfl inhibitor compounds 102-107, Du102 have certain tumor inhibitory effects on colorectal cancer cell lines HT29 and LoVo cells.

[0086] Figure 3 , In vitro cell line screening shows that Arfl inhibitor compounds 102-107, Du102 have certain tumor inhibitory effects on hepatocellular carcinoma cell line Huh7.

[0087] Figure 4 , In vitro cell line screening shows that Arfl inhibitor compounds 102-107, Du102 have certain tumor inhibitory effects on lung cancer cell lines H460 and A549.

[0088] Figure 5 , In vitro cell line screening shows that Arfl inhibitor compounds 102-107, Du102 have certain tumor inhibitory effects on lung cancer cell lines H460 and A549.

[0089] Figure 6 , In vitro cell line screening shows that Arfl inhibitor compounds 103-104, 107, 109-h0, Du102 have certain tumor inhibitory effects on colorectal cancer cell lines HT29 and LoVo cells, hepatocellular carcinoma cell line Huh7, and lung cancer cell lines H460 and A549.

[0090] Figure 7 , The Arfl inhibitor shows a dose-dependent effect in inhibiting tumor cells.

[0091] Figure 8 , Drosophila screening shows that Arfl inhibitors including Du110 and Du102 have good in vivo tumor inhibitory effects.

[0092] Figure 9 , Du101 was administered to mice by oral gavage at a dose of up to 50 mg / kg for 21 consecutive days, and the toxic effects on mice were observed.

[0093] Figure 10 1. The toxic effect on mice by administering Du110 to mice via oral gavage at a dose of up to 50 mg / kg for 60 consecutive days.

[0094] Figure 11 2. The liquid chromatogram of the intermediate compounds in Synthesis Scheme 1 of Du110 in Example 16, where ADC1 corresponds to Compound 2, DAD1 corresponds to Compound 3, and MSD1 corresponds to Compound 4. Detailed implementation manners

[0095] The application of the present invention can be better understood with reference to the following examples, but the application of the present invention is not limited to the following specific embodiments.

[0096] Example 1. Du101 and Du102 are effective inhibitors of Arfl activation.

[0097] The Arfl activity was detected using the Thermo Scientific Pierce Active Arf1 Pull - Down and Detection Kit (Cat#16121) and the lysate of hepatocellular carcinoma Huh - 7 cells. It is a complete kit that selectively enriches and detects GTP - bound Arf1 GTPase through specific protein interactions with the protein - binding domain of GGA3 protein. The results are shown in Figure 1 .

[0098] Example 2. In vitro cell line screening shows that Arfl inhibitors including Du101 and Du102 have a certain tumor - suppressing effect. In different in vitro tumor cell toxicity experiments ( Figures 2 - 7 ), including colorectal cancer cell lines HT29 and LoVo cells, hepatocellular carcinoma cells Huh7, lung cancer cells H460 and A549, the Arfl inhibitors can inhibit the growth of tumor cells ( Figures 2 - 6 ), and there is a dose - dependence ( Figure 7 ).

[0099] Example 3. Drosophila screening shows that Arfl inhibitors including Du110 and Du102 have a good in - vivo tumor - suppressing effect.

[0100] In the Drosophila intestine, by inducing the expression of the pro - oncogene Rasv12 - GFP, Drosophila intestinal tumors are formed. After feeding with Arfl inhibitors, it can significantly induce the death of Drosophila intestinal tumor cells (PI staining) ( Figure 8 ).

[0101] Example 4. In - vivo combination drug experiments in mice show that Arfl inhibitors Du101 and Du102 have a significant tumor - suppressing effect.

[0102] As Figures 9 - 10As shown, mice were administered Du101 up to 50 mg / kg by oral gavage for 21 consecutive days( Figure 9 ) and Du102 for 60 consecutive days( Figure 10 ), and no toxic effects were observed. These data indicate that compounds Du101 and Du102 can be used for selective anti-cancer activity.

[0103] Example 5. Vaccines made from Arfl-inhibited cells protect animals from tumors. The immunogenicity of reagents can be tested in a murine vaccination model (Obeid et al., 2007; Sagiv-Barfi et al., 2018). Tumor cells treated with the drug were injected into one flank of the mice, and then syngeneic tumor cells were injected again one week later. We tested the vaccine effects of CT-26 colon cancer, B16-F10 melanoma, and 4T1 breast cancer treated with DMSO or Du102. We first tested the treated cells in athymic nude mice and found that inhibiting the Arfl inhibitor did not significantly affect the tumorigenic ability of CT26, B16-F10, and 4T1 cells in immunodeficient mice (Tables 2, 5, 8), indicating that the anti-tumor activity of Arfl knockdown is not achieved through direct cytotoxic effects on tumor cells.

[0104] Table 2: DU102 treatment had no significant effect on CT-26 colon carcinoma in Athymic mice

[0105] Days 0 5 10 15 20 25 DMSO 0 0 21 170 480 1023 Du102 0 0 15 117 417 859

[0106] Table 3: DU102 treatment reduces tumor size (mm 3 ) in CT-26 colon cancer model in BALB / c mice

[0107] Left side with drug treatment

[0108]

[0109]

[0110] Table 4: Vaccination with DU102-treated CT-26 cells on left side protects BALB / c mice from developing tumors on right side

[0111] Days 0 5 10 15 20 25 30 DMSO 0 0 9.7 16.3 23.6 124.7 211.7 Du102 0 0 0 0.6 3.7 7.5 14.1

[0112] Table 5: DU102 treatment had no significant effect on B16-F10 melanoma in Athymic mice

[0113] Days 0 5 10 15 20 25 DMSO 0 6.7 95.4 242.5 517.7 1140.5 Du102 0 7.3 51.8 202.7 355.9 853.4

[0114] Table 6: W102 treatment reduces tumor size (mm 3 ) in B16-F10 melanoma model in C57B / 6 mice

[0115] Left side with drug treatment

[0116] Days 0 5 10 15 20 25 30 DMSO 0 15 51 132 297 553 405.5 Du102 0 0 2 8 57 81 21.3

[0117] Table 7:Vaccination with DU102-treated B16-FI0 cells on left side protects G57B / 6 mice from developing tumors on right side

[0118] Days 0 5 10 15 20 30 DMSO 0 0 0 3.5 33.5 405.5 Du102 0 0 0 0.6 1.3 21.3

[0119] Table 8: DU102 treatment had no significant effect on 4T1 breast carcinoma in Athymic mice

[0120] Days 5 10 15 20 25 DMSO 21.3 41.2 169.7 402.7 891.5 Du102 21.7 41.3 163.3 385.4 793.2

[0121] Table 9: DU102 treatment reduces tumor size (mm 3 ) in 4T1 breast carcinoma model in BALB / c mice

[0122] Left side with drug treatment

[0123] Days 0 5 10 15 20 25 30 DMSO 0 1.3 9.4 57.4 157.2 357.6 815.4 DU102 0 0 2.3 18.4 63.5 102.4 173.6

[0124] Table 10: Vaccination with DU102-treated 4T1 cells on left side protects BALB / c mice from developing tumors on right side

[0125] Days 0 5 10 15 20 25 30 DMSO 0 0 0 4.9 19.1 61.3 241.7 DU102 0 0 0 0 3.2 7.9 16.4

[0126] Then we injected DMSO- or Du102-treated CT26 cells into the left side of BALB / c mice, and then re-challenged the mice with untreated CT-26 cells on the right side one week later. Tumors grew gradually on both the left and right sides in mice injected with DMSO-treated CT-26 cells. Du102 treatment led to almost complete regression of tumors on both the left and right sides (Tables 3 and 4). Du102 in situ vaccination was effective not only against colon cancer, but also against tumors of various histological types, such as melanoma (B16-F10, Tables 6 and 7) and breast cancer 4T1 (Tables 9 and 10). These results suggest that inhibiting Arfl can locally trigger a T cell immune response that then attacks cancers throughout the body.

[0127] CT26, 4T1, and B16-F10 tumor cells treated with 10 μM Du102 or DMSO (5x10⁶, 1x10⁴, and 5x10⁴ respectively) were subcutaneously inoculated into 6-week-old female mice. Seven days later, an equal number of the corresponding tumor cells were inoculated on the right side. The tumor cell lines were mixed with Matrigel GIBCO, Cat#354234 or resuspended in PBS and injected subcutaneously or intradermally.

[0128] Example 6. Du102 treatment induces inflammatory cytokines in a CT-26 colon cancer model of BALB / c mice.

[0129] For IL-1β assay: Seven days after injection of CT26 cell lysates, CD11C⁺ DCs were isolated from the injected BALB / c mice and co-cultured with CT26 cells treated with DMSO or Du102. Two days later, IL-1β was measured in the harvested cells (Table 11)

[0130] For INFγ assay: Seven days after injection of CT26 cell lysates, CD11c⁺ DCs and CD8⁺ T cells were isolated from the injected BALB / c mice and co-cultured with CT26 cells treated with DMSO or Du102. Two days later, INFγ was measured in the harvested cells (Table 11).

[0131] Table 11: Du102 treatment induces inflammatory cytokines in CT-26 colon cancer model in BALB / c mice

[0132] IL-1fl INFy DMSO 181 182 OU102 954 623

[0133] Example 7. Du102 treatment induces IL-Iβ in B16-F10 melanoma in C57B / 6 mice

[0134] (Table 12)

[0135] Table 12: Du102 teatment induces inflammatory cytokine in B16-F10 melanoma model in C578 / 6 mice

[0136] IL-1β DMSO 178 DU102 952

[0137] Example 8. Du101 and Du102 treatments reduce the number of liver tumors in MYC-ON mice

[0138] We generated transgenic mice Tet-on-MYC / LAP-tTA (LT2-MYC) (Shachaf et al., 2004) that conditionally express the MYC proto-oncogene in hepatocytes using the Tet system. We crossed TRE-MYC mice with the transgenic line LAP-tTA, in which the liver activator protein (LAP) promoter drives the expression of the tetracycline transactivator protein (tTA) in hepatocytes. We activated the MYC transgene expression in 3-week-old mice by stopping the feeding of doxycycline (MYC-ON). Subsequently, all transgenic mice overexpressing MYC died of liver tumors, with an average latency of tumor onset of 12 weeks, as previously reported (Shachaf et al., 2004). In this LAP-tTA / tet-off MYC conditional transgenic mouse model, overexpression of MYC in adult mice reproducibly induces liver cancer, similar to hepatocellular carcinoma and / or hepatoblastoma. The transgenic tumors locally invade the entire liver and are often associated with malignant ascites, which spread to the thoracic cavity by metastasis and invade the lung parenchyma.

[0139] For the mouse liver tumor model, mating cages and weaning cages were administered a grain-based rodent diet containing 200 mg / kg doxycycline (Bio-Serv, Cat#14-727-450), and the mice were switched to a normal diet when induced to have tumors at 6 weeks of age.

[0140] The mice were divided into three groups: a control group, a Du101 treatment group, and a Du102 treatment group. The control group was given 100 μl / 10 g body weight (BW) of 20% DMSO + 80% corn oil by gavage using a feeding tube. The Du101 treatment group was prepared by diluting the stock solution of Du101 at 25 mg / ml with corn oil at a ratio of 1:4 to make a working solution. The Du102 treatment group was prepared by diluting the stock solution of Du102 at 25 mg / ml with corn oil at a ratio of 1:4 to make a working solution. The mixed working solution was given to the mice by gavage at 100 μl / 10 g BW using a feeding tube. Starting from the second week of MYC-ON, injections were given once every ten days for 5 times (starting from Monday and Friday) for 4 weeks. The mice were monitored every two days. After 10 weeks, the mice were euthanized and tumor growth was analyzed. Table 13 shows that Du101 and Du102 can reduce Myc-induced liver tumors overexpressed in mice.

[0141] Table 13: DU101 and Du102 treatment reduces liver tumor numbers in MYC-ON mice

[0142] DMSO 105.3 DU101 44.7 DU102 26.7

[0143] Example 9. Du102 treatment induces the expression of chemokines and T cell activation markers in MYC-ON mice.

[0144] Chemokines and T cell activation markers were measured by quantitative RT-PCR in MYC-ON mice treated with DMSO or Du102. By quantitative RT-PCR measurement, CD8 T cell infiltration and the expression of immune-stimulatory cytokines increased. Compared with the mice treated with DMSO control, Du102 treatment significantly increased the expression of many T cell-related chemokines CCL5, CXCL-10, CXCL-11, and CCL22 in MYC-ON (Table 14). The chemokines CCL5 and CXCL10 stimulate the tumor infiltration of CD4+ and CD8+ lymphocytes (Parkes et al., 2017), and CXCL10 and CXCL11 are T cell-related chemokines. In addition, real-time PCR showed that the expression of T cell activation markers IFNγ, perforin, GzmA, GzmB, and IL-1β was elevated in the mice treated with Du102 compared with the mice treated with DMSO control (Table 14). Our real-time PCR also showed that the expression of the immune checkpoint inhibitor PD-L1 was significantly reduced in the mice treated with Du102 compared with the mice treated with DMSO control in MYC-ON mice (Table 14). Overall, these data indicate that Du102 treatment triggers T cell infiltration and activation, leading to liver tumor cell death and prolonging the survival time of MYC-ON mice.

[0145] Table 14: DU102 treatment induces chemokines and T-cell activation in MYC-ON mice

[0146]

[0147] Example 10. Du102 treatment induces the expression of T-cell activation markers in 4T1 breast cancer in BALB / c mice.

[0148] Table 15: DU102 treatment induces T-cell activation of 4T1 breast carcinoma in BALB / c mice

[0149] GzmA GzmB Perforin ll-1β INFγ PD-L1 DMSO 1.0 1.0 1.0 1.0 1.0 1.0 DU102 2.2 2.3 2.6 2.2 2.1 0.5

[0150] Example 11. Du102 treatment induces MHC expression and T-cell infiltration in MYC-ON mice.

[0151] Du102 treatment increases MHC expression, CD4 and CD8 T-cell infiltration, as measured by quantitative RT-PCR in MYC-ON mice (Table 16).

[0152]

[0153] MHC-1 MHC-H CD4 CD8 DMSO 0.6 0.7 1.0 2.0 DU102 51.7 43.6 13.2 13.5

[0154] The numbers of MHC-I, MHC-II, infiltrating CD4+ and CD8+ T-cells were counted from representative sections of several tumors of MHC-I, MHC-II, CD4 and CD8 markers stained by immunohistochemistry.

[0155] Example 12. Du102 treatment reduces lung metastasis of B16-F10 melanoma in C57BL / 6 mice.

[0156] 50K B16-F10 tumor cells (treated with DMSO or 10 μM Du102) were intravenously metastasized by tail vein injection. Lungs were removed 15 days after injection and fixed in Fekete solution overnight. Three researchers blindly counted the visible metastases (Table 17).

[0157] Table 17: Du102 treatment reduces lung metastases of 816-F10 melanoma in C57B / 6 mice (surface tumor number)

[0158] DMSO 39 DU102 5

[0159] Example 13. The anti-tumor effects of Du102 are neutralized by CD4 or CD8 antibodies in MYC-ON mice.

[0160] The anti-tumor effects of Du102 are neutralized by CD4 or CD8 antibodies in MYC-ON mice

[0161] (Table 18). Cell depletion was verified using FACS and IHC analysis.

[0162] Table 18: The anti-tumor effects of DU102 are neutralized by anti-CD4 or / anti-CD8 antibodies in MYC-ON mice

[0163]

[0164] For the MYC-ON liver tumor model, the antibodies were injected intraperitoneally. They were injected once a week for 5 consecutive weeks, and doxycycline was withdrawn 1 week after the first injection. The antibodies used to treat the mice were as follows: 100 μg / mouse Armenian hamster anti-mouse PD1 (J43, BioXcell, Cat#BE0033-2) and 100 μg / mouse Armenian hamster isotype IgG (BioXcell, Cat#BP0091) were used as controls. From the second week of MYC-ON for 4 weeks, Du102 and DMSO were administered via gavage needle as described above, the mice were monitored every 2 days, and the mice were euthanized after 10 weeks and the tumor growth was analyzed.

[0165] Co-administration of anti-PD-1 antibody allows for the use of lower and less toxic doses of Du102, thus avoiding known side effects.

[0166] Example 14. The therapeutic effects of Du1O2 and Du101 in 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.

[0167] A humanized tumor model was constructed by inoculating human 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 tumor tissues or cell lines subcutaneously into humanized mice. DMSO or Du102 and Du101 were administered by gavage. The expression of immune-stimulatory cytokines was detected by quantitative RT-PCR. The expression of T cell activation markers IFNγ, perforin, GzmA, GzmB, and IL-1β in mice was detected by FACS. And the therapeutic effects of Du102- and Du101-treatment on 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 were detected by measuring tumor growth and collecting mouse survival data. Or a tumor model was constructed by inoculating murine 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 tumor tissues or cell lines subcutaneously into wild-type mice. DMSO- or Du102- and Du101-treatment were administered by gavage. The expression of immune-stimulatory cytokines was detected by quantitative RT-PCR. The expression of T cell activation markers IFNγ, perforin, GzmA, GzmB, and IL-1β in mice was detected by FACS. And the therapeutic effects of Du102- and Du101-treatment on 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 were detected by measuring tumor growth and collecting mouse survival data.

[0168] Or a mouse model of primary 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 was constructed by conditional gene knockout or knock-in. DMSO or Du102 and Du101 were administered by gavage. The expression of immune-stimulatory cytokines was detected by quantitative RT-PCR. The expression of T cell activation markers IFNy, perforin, GzmA, GzmB, and IL-13 in mice was detected by FACS. And the therapeutic effects of Du102- and Du101-treatment on 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 were detected by measuring tumor growth and collecting mouse survival data.

[0169] Example 15. Therapeutic effects of Du1O2 and Du101 in autoimmune diseases, inflammatory diseases, inflammatory bowel disease, arthritis, and autoimmune demyelinating diseases.

[0170] In autoimmune disease, inflammatory disease, inflammatory bowel disease, arthritis, and autoimmune demyelinating mouse disease models, DMSO or Du102 and Du101 were administered by gavage or intracranial injection. The expression of immunostimulatory cytokines was detected by quantitative RT-PCR. The expression of T cell activation markers IFNγ, perforin, GzmA, GzmB, and IL-1β in mice was detected by FACS. And the disease onset of mice was detected by sectioning.

[0171] Example 16

[0172] Preparation protocol (Protocol 1) of the compound 1H-indole-5-carbaldehyde 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidin-4-yl hydrazone.

[0173]

[0174] Synthesis of Du101

[0175] The compounds mentioned below are shown in the schematic diagram of Protocol 1. The compounds were dissolved in DMSO and stored at -200 °C.

[0176] Compound 2

[0177] Sulfur (320 mg, 10 mmol), ethyl cyanoacetate (1.07 mL, 10 mmol), and morpholine (875 μL, 40 mmol) were added to a solution of cyclohexanone (10 mmol) in ethanol (10 mL). The reaction mixture was stirred at 60 °C for 5 hours. After purification by chromatography using dichloromethane, 800 mg of Compound 2 was obtained. (Note: The starting materials can be increased proportionally to obtain more Compound 2).

[0178] Compound 3

[0179] Compound 2 was heated in formamide at 150 °C for 5 hours. After cooling overnight, the product crystallized as light brown crystals. The obtained crystals were collected and washed with a mixed solvent of cold ethanol / water (volume ratio: 1 / 1) to obtain the corresponding thiophenothiazolone ring (Compound 3) in quantitative yield.

[0180] Compound 4

[0181] Before adding 2 equivalents of P0C13, 650 mg of Compound 3 was dissolved in hot DMF (dimethylformamide) and cooled with ice. After stirring overnight, the product precipitated out. The white powder was collected and washed with cold water. Further cold water was added to the mother liquor to obtain an additional precipitate, which was directly used in the next reaction.

[0182] To a solution of the chloride in methanol, 10 equivalents of hydrazine monohydrate were added. The mixture was stirred for 2 hours and water was added. The resulting precipitate was filtered off and washed with cold water to give Product 4.

[0183] Du101

[0184] To a methanol solution of 1 equivalent of Compound 4, 1.2 equivalents of 5-formyl-indole were added. The mixture was stirred for 2 hours, the resulting precipitate was collected and recrystallized from methanol to give Du101.

[0185] Η NMR (300 MHz, DMSO-d6) δ: 1.79 (m, 4H, 2CH2), 2.74 (m, 2H, CH2), 3.00 (m, 2H, CH2), 6.49 (m, 1H, CH), 7.39 (m, 1H, CH), 7.42 (d, J = 8.6 Hz, 1H, CH), 7.76 (s, 1H, CH), 7.84 (d, J = 8.6 Hz, 1H, CH), 8.02 (s, 1H, CH), 8.45 (s, 1H, CH), 11.29 (brs, 1H, NH), 11.73 (brs, 1H, NH). 13 CNMR (75 MHz, DMSO-d6) δ: 22.0 (CH2), 22.4 (CH2), 24.6 (CH2), 26.5 (CH2), 101.8 (CH), 111.5 (CH), 118.9 (C), 120.6 (CH), 121.3 (CH), 126.2 (CH), 126.6 (C), 127.6 (C), 130.8 (C), 131.8 (C), 136.9 (C), 144.0 (CH), 148.0 (C), 154.8 (CH), 156.6 (C). ES-MS m / z 348.1 (MH+). HRMS 348.1277, found 348.1290. Anal. (C 19 H 17 N5S) C, Η, N, S

[0186]

[0187] Data File D:\DATA\MICRA\1186530.D

[0188] Sample Name:118653005

[0189] Instrument 104 / 10 / 2017 14:20:33 N6

[0190] Column:Onyx Monolithic C18 50x4.6mm|3.75ml / min|Columns Reg ValveGradient:″A″->@2.2min->″B″(Hold 0.4min)->@0.2min->″A″->PostRun

[0191]

[0192] Signal 1:ADC1 B,ELSD

[0193]

[0194]

[0195] Signal 2:DAD1 A,Sig=300,200Ref=off

[0196]

[0197] Signal 3:MSD1 TIC,MS File

[0198]

[0199] Example 17

[0200] Preparation Scheme (Scheme 2) of Compound 1H-Indole-5-carbaldehyde-5,6,7,8,9,10-Hexahydrocycloocta[4,5]thieno[2,3-d]pyrimidin-45-ylhydrazone

[0201]

[0202] Synthesis of Du102

[0203] The compounds mentioned below are shown in the schematic diagram of Scheme 2. The compounds are dissolved in dimethyl sulfoxide and stored at room temperature or -20 °C.

[0204] Compound 1

[0205] To a solution of cyclooctanone (10 mmol) in ethanol (10 mL), sulfur (320 mg, 10 mmol), ethyl malonate (1.07 mL, 10 mmol) and morpholine (875 μL, 10 mmol) were added respectively. The reaction mixture was stirred at 60 °C for 5 h. Purification by column chromatography with dichloromethane as the eluent gave 855 mg of compound 1 in 34% yield (Note: The raw materials can be increased proportionally to obtain more compound 1).

[0206] Compound 2

[0207] Compound 1 was dissolved in formamide and heated at 150 °C for 5 h. After cooling overnight, the product crystallized as light brown crystals. The resulting crystals were collected and washed with a mixed solvent of cold ethanol / water (volume ratio 1 / 1) to obtain the corresponding thiophenopyrimidinone ring (Compound 2) in quantitative yield. 1 NMR (400 MHz, DMSO-d6) δ: 1.27 (m, 2H, CH2), 1.42 (m, 2H, CH2), 1.62 (m, 4H, 2CH2), 2.87 (m, 2H, CH2), 3.06 (m, 2H, CH2), 8.01 (s, 1H, CH), 12.28 (brs, 1H, NH). 13 13C NMR (100 MHz, DMSO-d6) δ: 24.4 (CH2), 25.3 (CH2), 25.4 (CH2), 26.0 (CH2), 29.9 (CH2), 31.5 (CH2), 133.7 (C), 135.0 (C), 135.0 (C), 144.6 (C), 147.8 (C), 150.0 (CH), 157.7 (C). ES-MS m / z 235.1 (MH + )

[0208] Compound 3

[0209] Before adding 2 equivalents of POCl3, 650 mg of compound 2 was dissolved in hot DMF (dimethylformamide) and cooled with ice. After stirring overnight, the product precipitated. The white powder was collected and washed with cold water. Cold water was further added to the mother liquor to obtain additional precipitate, which was directly used for the next reaction. 1 NMR (400 MHz, CDCl3) δ: 1.25 (m, 2H, CH2), 1.46 (m, 2H, CH2), 1.70 (m, 4H, 2CH2), 2.92 (m, 2H, CH2), 3.12 (m, 2H, CH2), 8.67 (s, 1H, CH). 1313C NMR (100 MHz, CDCl3) δ: 25.0 (CH2), 25.4 (CH2), 26.3 (CH2), 28.2 (CH2), 30.3 (CH2), 31.6 (CH2), 128.6 (C), 129.6 (C), 142.7 (C), 151.3 (CH), 156.4 (C), 158.7 (C). ESMS (electrospray mass spectrometry) m / z 252.1 (MH + , 35 +), 254.1 (MH+, 37 +).

[0210] Compound 4

[0211] To a solution of the chloride (Compound 3) dissolved in methanol, 10-fold equivalents of hydrazine monohydrate were added. After stirring the mixture for 2 hours, water was added. The resulting precipitate was filtered out and washed with cold water. 1 1H NMR (400 MHz, CDCl3) δ: 1.27 (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.65 (m, 4H, 2CH2), 2.50 (brs, 2H, NH2), 2.83 (m, 4H, 2CH2), 6.54 (brs, 1H, NH), 8.41 (s, 1H, CH). 13 13C NMR (100 MHz, CDCl3) δ: 25.3 (CH2), 26.0 (CH2), 26.1 (CH2), 27.7 (CH2), 30.1 (CH2), 31.6 (CH2), 115.7 (C), 127.7 (C), 137.3 (C), 152.3 (CH), 158.7 (C), 164.8 (C). ES-MS m / z 249.0 (MH + +), 271.0 (MNa + +).

[0212] Du102

[0213] To a methanol solution of 1 equivalent of Compound 4, 1.2 equivalents of 5-formyl-indole were added. The mixture was stirred for 2 hours, the resulting precipitate was collected and recrystallized from methanol to obtain Dul02. 1HNMR (400 MHz, DMSO-d6) δ: 1.25 (m, 2H, CH2), 1.44 (m, 2H, CH2), 1.60 (m, 2H, CH2), 1.69 (m, 2H, CH2), 2.83 (brt, 2H, J = 5.5 Hz, CH2), 3.20 (brt, 2H, J = 6.0 Hz, CH2), 6.49 (m, 1H, CH), 7.39 (m, 1H, CH), 7.44 (d, 1H, J = 8.6 Hz, CH), 7.80 (brs, 1H, CH), 7.85 (brd, 1H, J = 8.6 Hz, CH), 8.03 (s, 1H, CH), 8.46 (s, 1H, CH), 11.28 (brs, 1H, NH) 11.71 (brs, 1H, NH). LC / MS, API-ES m / z 376.0 (MH +) 。

[0214] All citations in this document are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0215] Those skilled in the art can make modifications and variations to the present invention without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not meant to be limiting in any way. The description and examples are considered to be exemplary only, and the true scope and spirit of the present invention are indicated by the following claims.

Claims

1. Use of an inhibitor targeting the Arf1 pathway in the preparation of a drug for killing cancer stem cells or cancer cells and inducing an anti-tumor immune response, wherein the inhibitor inhibits the activity of the COPI / Arf1-lipolysis-β-oxidation pathway in cancer stem cells; the inhibitor targeting the Arf1 pathway is the following compound: or 。 2. Use of the inhibitor according to claim 1 or a pharmaceutically acceptable salt, solvate, stereoisomer thereof in the preparation of a drug for treating a subject suffering from cancer or a tumor.

3. The use according to claim 2, wherein the cancer is 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.

4. The use according to claim 2, wherein the compound can inhibit the growth of cancer or a tumor.

5. The use according to claim 2, wherein the compound can reduce the size of a tumor or cancer.

6. The use according to claim 2, wherein the compound can increase the expression of MHC-I and MHC-II, increase the infiltration and activation of T cells in the tumor, and increase the expression of T cell activation markers GzmA, GzmB and perforin.

7. The use according to claim 2, wherein the compound increases the expression of at least one inflammatory cytokine or chemokine selected from IFNγ, IL-1β, Ccl5, Cxcl0, Cxcl1 or Ccl22.

8. The use according to claim 2, wherein the compound is used in combination with at least one anti-PD-1 antibody, and the compound and PD-1 blockade have a synergistic effect.

9. The use according to claim 2, wherein the compound can not only kill tumor stem cells, but also induce a tumor-specific immune response.

10. The use according to claim 2, wherein the compound can reduce the metastasis of cancer or a tumor.

11. The use according to claim 2, wherein the subject is a mammal.

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