WO2022028429A1 - 用于根除癌症干细胞并诱导damp介导的抗肿瘤免疫反应的一组新型copi/arf1-脂解途径抑制剂和化合物 - Google Patents

用于根除癌症干细胞并诱导damp介导的抗肿瘤免疫反应的一组新型copi/arf1-脂解途径抑制剂和化合物 Download PDF

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WO2022028429A1
WO2022028429A1 PCT/CN2021/110373 CN2021110373W WO2022028429A1 WO 2022028429 A1 WO2022028429 A1 WO 2022028429A1 CN 2021110373 W CN2021110373 W CN 2021110373W WO 2022028429 A1 WO2022028429 A1 WO 2022028429A1
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cancer
arf1
compound
tumor
cells
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French (fr)
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侯宪玉
王月桐
王娜
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A61K31/431—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems containing further heterocyclic rings, e.g. ticarcillin, azlocillin, oxacillin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/76—Dibenzothiophenes

Definitions

  • the present invention generally relates to the use of COPI/Arfl-lipolysis-beta-oxidation pathway inhibitors in the treatment of disorders. More specifically, the present invention relates to the use of COPI/Arfl-lipolysis-beta-oxidation pathway inhibitors in targeting cancer stem cells and activating immune responses and treating diseases.
  • the present invention relates to 1H-indole-5-carbaldehyde 6,7,8,9-tetrahydro-5H-cycloheptyl[4,5]thieno[2,3-d]pyrimidin-4-yl Hydrazones, 1H-indole-5-carbaldehyde 5,6,7,8,9,10-hexahydrocyclooctyl[4,5]thieno[2,3-d]pyrimidin-4-ylhydrazones, and related Compounds inhibit Arf1, target cancer stem cells and activate anti-tumor immune responses for the treatment of malignant diseases.
  • the present invention also relates to the treatment of refractory, relapsed or metastatic cancer, as well as preparation methods of related compounds and intermediates thereof, and pharmaceutical compositions of related compounds.
  • Immunotherapy is beginning to fundamentally change the paradigm of treating cancer patients (Sharma et al., 2017).
  • immunotherapy has unfortunately only helped a small percentage of cancer patients.
  • Most patients experience little benefit because most emerging tumors have developed immune evasion mechanisms in the tumor microenvironment, including dysfunctional T cells and lack of T cell infiltration (Jerby-Arnon et al., 2018; Sharma et al. al., 2017).
  • pathogens bacteria or viruses
  • pathogen-associated molecular patterns PAMPs
  • PRRs pattern recognition receptors
  • This induced immune response can be used to regress tumors, as first demonstrated by William Coley (Balkwill, 2009). In the 1890s, he successfully regressed many sarcomas and/or lymphomas after injecting streptococcal cultures into patients. Some injected bacteria may trigger PAMP-induced antitumor immune responses.
  • DAMPs danger/damage-associated molecular patterns
  • the most important DAMPs include: (i) preapoptotic exposure of the endoplasmic reticulum (ER)-anchor chaperone calreticulin (Calr) on the cell surface; (ii) non-histone nuclear protein high mobility group box 1 (HMGB1) release into the extracellular space; (iii) active secretion of ATP.
  • CSCs Cancer stem cells
  • CSCs are a subset of cell populations in tumors that are in a stem cell state and have stem cell characteristics. CSC may 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 that pathway to eradicate CSCs.
  • Some CSCs arise from the transformation of normal stem cells, others by reprogramming non-CSC cancer cells to a stem cell state. CSCs and normal stem cells share many properties. Therefore, pathways that regulate normal or transformed stem cells may also regulate CSCs.
  • New therapies may include approaches to reduce Arf1 activity and block the Arf1-mediated lipolytic pathway and associated ⁇ -oxidation, and we tested 10 reported Arf1 inhibitors (Bill et al., 2011; Boal et al., 2010; Feng et al., 2004; Newton et al., 2006; Ohashi et al., 2012; Sáenz et al., 2009; Sorieul et al., 2011; Viaud et al., 2007), including on Drosophila stem cells Brefeldin A (BFA), Golgicide A (GCA), LM11, Secin16, Secin H3, Secin B7, Exo1, Exo2, LG8 and LG18 tumor and normal human bone marrow hematopoietic stem
  • a first aspect of the invention relates to a method of eliminating a tumor, wherein the method comprises inhibiting at least some, most or substantially all (eg, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%) of the Arf1 pathway activity.
  • the Arfl pathway inhibitor is isolated, purified or synthesized and can be selected from the group consisting of small molecule Arfl inhibitors, RNAi agents against Arfl, antisense agents against Arfl, peptidomimetic Arfl inhibitors, and G- Quadruple oligodeoxynucleotide Arf1 inhibitor.
  • the inhibitory mechanism may be selected from significantly inhibiting Arf1 GTPase activity, significantly inhibiting Arf1-pathway regulated lipolytic activity, significantly inhibiting Arf1-pathway regulated oxidative activity, and significantly inducing cell necrosis by inhibiting Arf1 pathway.
  • 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 "compounds of the present invention").
  • the present invention provides methods of inhibiting or reducing tumor or cancer growth comprising contacting the tumor with an effective amount of a compound of the present invention.
  • the compound inhibits the growth of a 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.
  • the compound increased T cell infiltration and activation in tumors compared to DMSO.
  • Compounds increased the expression of T cell activation markers such as GzmA, GzmB and perforin.
  • the compound increases the expression of at least one inflammatory cytokine or chemokine, which may be selected from the group consisting of IFN ⁇ , IL-1 ⁇ , Ccl5, Cxc10, Cxc11, Ccl22.
  • the compound is co-administered with at least one anti-PD-1 antibody, and the compound and PD-1 blockade are synergistic.
  • inoculation of cancer cells treated with a compound acts as a vaccine to protect the animal from tumor development.
  • treatment with this compound has a "two birds with one stone" effect, not only killing CSCs, but also eliciting a tumor-specific immune response that converts necrotic CSCs into a therapeutic vaccine, resulting in long-term efficacy.
  • the present invention provides a method of treating or preventing a disorder associated with Arfl pathway activity in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of the present invention thereby Decreases the activity of the Arf1 pathway.
  • Arf1 pathway activity can be identified by testing for Arf1 GTPase activity or alternative upstream or downstream regulators of Arf1 GTPase activity.
  • the condition can be cancer.
  • 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 associated with Arf1 pathway activity, and in one embodiment, selected from autoimmune disease, inflammatory disease, inflammatory bowel disease, arthritis, autoimmune demyelination disorders, Alzheimer's disease, stroke, ischemia-reperfusion injury, multiple sclerosis and other inflammatory or neurodegenerative diseases.
  • the present invention provides a method of inhibiting Arfl pathway activity in a cell.
  • the method comprises administering to a cell an effective amount of a compound of the invention such that Arfl pathway activity in the cell is reduced, eg, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.
  • the cell is a CSC, or is cancerous. This method induces cell death.
  • the method can be performed in vitro or in vivo.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the present invention, namely a compound selected from the above-mentioned compounds 102, 104-111, and pharmaceutically acceptable salts or solvates thereof, as well as pharmaceutically acceptable salts or solvates thereof.
  • acceptable excipients, carriers or diluents are suitable for oral, nasal, topical, rectal, vaginal or parenteral administration, or intravenous, subcutaneous or intramuscular injection.
  • the present invention also provides methods of preparing some of the compounds of the present invention.
  • R1 is selected from one or more substituents or unsubstituted phenyl,
  • the substituents are selected from halogen, C 1-6 alkyl, haloalkyl, OH, OCH 3 , O(CH 2 ) n CH 3 , cyclopropyloxy, OC(CH 3 ) 3 , OCH(CH 3 ) 2 , NH 2 , NO 2 , N(CH 3 ) 2 , NH(CH 2 ) n CH 3 , CN, N 3 , etc., wherein n is 1-9.
  • inhibitors targeting the Arf1 pathway are the following compounds:
  • the present invention provides the preparation of the compound 1H-indole-5-carbaldehyde 6,7,8,9-tetrahydro-5H-cycloheptyl[4,5]thieno[2,3-d]pyrimidine -4-ylhydrazone or the method of 107 or Du101 above.
  • the present invention provides the preparation of the compound 1H-indole-5-carbaldehyde 5,6,7,8,9,10-hexahydrocyclooctyl[4,5]thieno[2,3-d] A pyrimidin-4-ylhydrazone or the method of 111 or Du102 above.
  • a cell includes a plurality of cells, including mixtures thereof.
  • cancer stem cells and “CSCs” are interchangeable.
  • the CSCs are of mammalian origin, and in a preferred embodiment, these CSCs are of human origin, but they are not intended to be so limited.
  • Cancer stem cells are defined and functionally characterized as tumor-derived cell populations that: (1) have extensive proliferative capacity; (2) are capable of undergoing asymmetric cell division to produce one or more differentiated progeny with reduced proliferative or developmental potential ; (3) Capable of symmetric cell division for self-renewal or self-maintenance.
  • Other common methods to characterize CSCs include cell surface markers, morphology, transcriptional profiles, and drug response.
  • CSCs are also known in the research literature as tumor/cancer initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, cancer stem cells, solid tumor stem cells, drug-surviving cells (DSC), drug-resistant cells (DRC) or super malignant cells.
  • DSC drug-surviving cells
  • DRC drug-resistant cells
  • cancer refers to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth.
  • cancer cells and “tumor cells” refer to the total cell population 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.
  • cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, peritoneal carcinoma, 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.
  • tumor refers to any mass of tissue, whether benign (non-cancerous) or malignant (cancerous), caused by excessive growth or proliferation of cells, including precancerous lesions.
  • metastasis refers to the process by which cancer spreads or metastasizes from a site of origin to other areas of the body, with the appearance of similar cancerous lesions at the new site.
  • Metalstatic cells lose adhesive contact with neighboring cells and migrate from the primary site of disease through the bloodstream or lymph to invade neighboring body structures.
  • the term “subject” refers to any animal (eg, mammal) including, but not limited to, humans, non-human primates, rodents, and the like. Generally, the terms “subject” and “patient” are used interchangeably herein with respect to human subjects.
  • treating refers to: 1) cure, slow, relieve symptoms and/or stop the progression of a diagnosed pathological condition or disorder; 2) prevent or slow the development of the target pathological condition or disorder preventive or preventive measures. Therefore, those in need of treatment include those who already have the disease; those who are susceptible to it; and those who need prevention.
  • a subject is successfully "treated” according to the methods of the present invention if the patient exhibits one or more of the following: a reduction or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, These include cancer spreading into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduction in morbidity and mortality; and improvement in quality of life.
  • the term "inhibit” and its synonyms, when used in the context of biological activity, refer to the down-regulation of biological activity that reduces or eliminates a targeted function, such as protein production or phosphorylation of a molecule. In certain embodiments, inhibition can refer to a reduction of the target activity by 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 successfully preventing the onset of symptoms, reducing symptoms, or eliminating the disease, disorder.
  • 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.
  • GTPase refers to a hydrolase that can bind and hydrolyze guanosine triphosphate (GTP). GTP binding and hydrolysis occur in a highly conserved G domain common to all GTPases.
  • Beta-oxidation refers to the catabolic process by which fatty acid molecules are broken down in the cytoplasm of prokaryotes and in the mitochondria of eukaryotes to produce acetyl-CoA, NADH and FADH2, which enter the tricarboxylic acid cycle It is a coenzyme used in the electron transport chain. It is so named because the beta carbon of the fatty acid is oxidized to a carbonyl group. Beta-oxidation is mainly promoted by three functional proteins of mitochondria, an enzymatic complex associated with the inner mitochondrial membrane, although some fatty acids are oxidized in peroxisomes.
  • necrosis refers to when a cell is exposed to physiological conditions (e.g., hypothermia, hypoxia) or conditions of extreme change that may result in 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 an impairment of the cell's ability to maintain homeostasis, resulting in an influx of water and extracellular ions. The intracellular organelles, especially the mitochondria, swell and rupture (cytolysis), as well as the entire cell. Cellular contents, including lysosomal enzymes, are released extracellularly due to the eventual breakdown of the plasma membrane, and thus, in vivo, cell necrosis is often associated with a strong inflammatory response that triggers extensive tissue damage.
  • Immuno disorders include, for example, pathological inflammation, inflammatory disorders, and autoimmune disorders or diseases.
  • Immuno conditions refers to infections, persistent infections, and proliferative conditions, such as cancer, tumors, and angiogenesis, including infections, tumors, and cancers that resist irradiation of the immune system.
  • Cancer disorders include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous disorders such as dysplasia.
  • cytotoxic T cells refers to killing cancer cells, cells infected (especially by viruses) or other T lymphocytes (a type of white blood cell). Most cytotoxic T cells express T cell receptors (TCRs) that recognize specific antigens.
  • TCRs T cell receptors
  • An antigen is a molecule that stimulates an immune response, usually produced by cancer cells or viruses. Antigens within cells bind to and are brought to the cell surface by MHC class I molecules, where they can be recognized by T cells. If the TCR is specific for that antigen, it binds to a complex of MHC class I molecules and the antigen, and the T cell destroys the cell.
  • the TCR In order for the TCR to bind to a class I MHC molecule, the TCR must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule. Therefore, these T cells are called CD8+ T cells.
  • CD8+ T cells Once cytotoxic CD8+ T cells (CTLs) recognize their target cells in the periphery, the CTLs are activated and highly specialized cell-to-cell contacts called immune synapses (IS) are formed between the T cells and their target cells structure. Activation of the TCR leads to the involvement of 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.
  • Src family kinases Lck and Fyn
  • Activated ZAP70 in turn phosphorylates the adaptor protein LAT for activation of T cells, leading to the formation of LAT signal bodies, which include phospholipase C ⁇ 1 (PLC ⁇ 1) and SLP76 (a 76-kDa SH2 domain-containing leukocyte protein).
  • PLC ⁇ 1 converts phosphatidylinositol-4,5-bisphosphate (PIP2) to DAG (diacylglycerol) and IP3 (inositol-1,4,5-triphosphate).
  • DAG accumulates at immune synapses, leading to the recruitment of new protein kinase Cs (PKCs), including PKC ⁇ .
  • PKC ⁇ then promotes centrosome (microtubule organizing center or MTOC) polarization by reciprocally localizing dynein to synapses, which pulls the MTOC toward the synapse and pulls non-muscle myosin II (NMII) to the cell on the other side, where it pushes the MTOC towards the synapse.
  • MTOC microtubule organizing center
  • 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, A solvent or encapsulating material involved 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.
  • materials that can be used as pharmaceutically acceptable carriers include: carbohydrates such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, 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 Oils and soybean oils; glycols such as propylene glycol; polyols such as glycerol, sorbitol, marmitol, and polyethylene glycols; esters such as ethyl oleate, ethyl laurate, etc.; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate
  • wetting agents such as sodium lauryl sulfate, magnesium stearate and polyethylene oxide-polypropylene oxide copolymers as well as colorants, mold release agents, coating agents, sweeteners , flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition.
  • salts may form salts, which are also within the scope of the present invention.
  • terms herein are to be understood to include reference to salts thereof.
  • the term “salt” refers to acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • zwitterions inner salts may be formed and included for use herein within the term "salt”.
  • Salts of compounds of the invention can be prepared, for example, by precipitating compounds 102 or 104-111 with an amount of acid or base, eg, an equivalent amount, in a medium such as a salt or in an aqueous medium, followed by lyophilization.
  • Solvates of the compounds of the present invention are also included herein.
  • Solvates of the compounds of the present invention include, for example, hydrates.
  • the present invention provides methods of treating proliferative diseases such as cancer, tumors and the like with compounds selected from the group consisting of compounds 102, 104-111 above, especially Du101 and Du102.
  • CSCs may be responsible for treatment resistance and immune evasion.
  • CSC signaling can regulate lymphocyte infiltration into tumors and alter the tumor microenvironment (Lytle et al., 2018).
  • knockdown of Arf1-mediated lipid metabolism in CSCs leads to metabolic stress and subsequent cellular responses, including the release of DAMPs that promote antitumor immunity by activating DCs, enhancing T-cell infiltration and activation (Wang et al. al., 2020).
  • the present invention provides evidence that novel compounds inhibit Arfl pathway activity and induce anti-tumor immune responses by increasing the expression of chemokines and inflammatory cytokines and enhancing T cell infiltration and activation.
  • treatment with this compound and PD-1 blockade is synergistic. Therefore, treatment with this compound should provide significant improvements in tumor therapy.
  • the present invention provides 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 Arfl pathway activity in a cell or for treating cancerous and non-cancerous disorders associated with Arfl pathway activity.
  • the invention also provides related methods (eg, manufacturing and screening of drug candidates), materials, compositions and kits.
  • the method can be used to treat cancer in a subject.
  • Cancers known to have CSCs and Arf1-lipolysis- ⁇ -oxidation pathway activity are good candidates for such treatments, including but not limited to: breast, head and neck, lung, ovarian, pancreatic, colorectal, Prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumor, gastric cancer, multiple myeloma, leukemia and lymphoma.
  • the method is for treating liver cancer, head and neck cancer, pancreatic cancer and/or gastric cancer.
  • the method is used to treat multiple myeloma, brain tumors and sarcomas.
  • any method of inhibiting CSCs of the invention can be implemented to treat metastatic cancer, cancer refractory to chemotherapy or radiation therapy, or during initial treatment After the subject relapsed.
  • the inhibitor is isolated, purified or synthetic and can be selected from the group consisting of small molecule Arfl inhibitors, RNAi agents to Arfl, antisense agents to Arfl, peptidomimetic Arfl inhibitors and G-tetrakis Heavy oligodeoxynucleotide Arf1 inhibitor.
  • Inhibitors can also be isolated or purified from natural products.
  • Arf1 inhibitors can be chosen to target any step in the Arf1 pathway.
  • inhibitors can significantly inhibit Arf1 GTPase activity, lipolytic reporter genes, lipid droplet formation, autophagy, and alternative upstream or downstream regulators of Arf1 activity or function.
  • the Arf1 inhibitor according to the present invention is: compound 102, 104-111 (Table 1), enantiomers, diastereomers, tautomers and salts or solvates thereof ("Compounds of the Invention").
  • the present invention also provides in vitro and in vivo data that the compounds of the present invention reduce tumors.
  • the compounds of the present invention are not only shown to cause death in a wide range of cancer cells, but also exhibit selectivity in their cytotoxicity, which is critical for the development of less toxic therapeutics.
  • Selective cytotoxicity refers to the ability of a compound to kill cancer cells while substantially sparing normal cells, sometimes under certain conditions. Normal cells generally refer to healthy, non-tumorigenic cells.
  • the conditions that lead to selective cytotoxicity of drug candidates are difficult to predict because they require an understanding of the underlying mechanisms of cytotoxicity. For example, reducing the toxicity of anticancer drugs that target microtubule formation during mitosis requires the use of completely different factors than drugs that block cellular metabolic processes.
  • Appropriate conditions to produce selective cytotoxicity need to balance the need for a drug to be sufficiently toxic to effectively kill cancer cells while being sufficiently tolerable to normal cells. For example, if a lower concentration is used, this usually means a prolonged infusion is needed to kill cancer cells.
  • 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 period of time.
  • a pharmaceutical composition containing a 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 a critical concentration for more than 24 hours.
  • This method can be used to treat all cancers, including any of the cancer groups described herein, and to treat Arf1-related diseases, an exemplary list of which has been provided above and will not be repeated here.
  • the duration may 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 invention, the critical concentration is about 100 ⁇ M, about 50 ⁇ M, about 20 ⁇ M, or about 10 ⁇ M.
  • the present invention provides methods of treating a proliferative disorder or disorder, such as cancer of the uterus, cervix, breast, prostate, testis, penis, gastrointestinal tract such as esophagus, oropharyngeal, stomach, small bowel, or Colorectal cancer, colon cancer or rectum, kidney, kidney cells, bladder, bone, bone marrow, skin, head or neck, skin, liver, gallbladder, heart, lung, pancreas, salivary glands, adrenal glands, thyroid, brain such as glia Tumors, ganglia, central nervous system (CNS) and peripheral nervous system (PNS) and immune system such as spleen or thymus.
  • a proliferative disorder or disorder such as cancer of the uterus, cervix, breast, prostate, testis, penis, gastrointestinal tract such as esophagus, oropharyngeal, stomach, small bowel, or Colorectal cancer, colon cancer or rectum, kidney, kidney cells, bladder
  • the present invention provides treatment of eg 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 cancer, metastasis and angiogenesis.
  • the present invention also contemplates reducing tolerance to tumor cells or cancer cell antigens, eg, by modulating the activity of T cell infiltration and activation.
  • Methods of treating, delaying progression, preventing recurrence, alleviating symptoms or otherwise ameliorating a tumor in a human, mammalian or animal subject having a tumor may comprise administering a therapeutically effective amount of a compound, product and/or or pharmaceutical compositions, resulting in antitumor activity.
  • the anti-tumor activity can be anti-cancer activity.
  • anti-tumor activity can include slowing tumor volume growth, stopping tumor volume growth, or reducing tumor volume.
  • Tumors can include solid tumors, malignant tumors, metastatic cells, cancer stem cells. Tumors can include carcinomas, sarcomas, adenocarcinomas, lymphomas, or hematological malignancies.
  • Tumors may be refractory to treatment with chemotherapy, radiation therapy, and/or hormone therapy.
  • Compounds, products and/or pharmaceutical compositions can be administered to prevent tumor recurrence.
  • Compounds, products and/or pharmaceutical compositions can be administered as adjunctive therapy to surgical resection.
  • Compounds, products and/or pharmaceutical compositions can be administered, for example, orally and/or intravenously.
  • Methods according to the present invention also include treating the disease or disorder, delaying its progression, preventing its recurrence, alleviating its symptoms, or otherwise ameliorating the disease or disorder in a human, mammalian or animal subject afflicted with the disease or disorder .
  • the disease or disorder is selected from autoimmune disease, inflammatory disease, inflammatory bowel disease, arthritis, autoimmune demyelinating disease, Alzheimer's disease, stroke, ischemia-reperfusion injury, Multiple sclerosis and other neurodegenerative diseases.
  • Administration of a compound, product, and/or pharmaceutical composition to a patient with a disease or disorder is considered successful if any of a variety of laboratory or clinical results are achieved. For example, administration is considered successful and one or more symptoms associated with the disease or disorder are alleviated, alleviated, inhibited, or a state of no further progression. Administration is considered successful if the disorder, eg, an autoimmune disorder, goes into remission or a state of no further progression.
  • the compounds, products, and/or pharmaceutical compositions described herein are administered in combination with any of a variety of known therapeutic agents, including, for example, chemotherapeutic and other antineoplastic agents, anti-inflammatory compounds and/or Or immunosuppressive compounds, cytokines or cytokine antagonists such as IL-12, interferon-alpha or anti-epidermal growth factor receptor.
  • the compounds, products and/or pharmaceutical compositions described herein may be used in conjunction with any of a variety of known treatments, including but not limited to surgical treatments and methods, radiation therapy, chemotherapy and/or Or hormone or other endocrine related therapy.
  • compositions described herein and the second therapy 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.
  • the compounds, products and/or pharmaceutical compositions described herein and the second therapy can be administered to a 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 a subject by the same or different routes of administration.
  • the combination therapy of the present invention comprises an effective amount of a compound, product and/or pharmaceutical composition described herein and an effective amount of at least one other therapy (eg, prophylactic or therapeutic agent) having a different mechanism than an effective amount Effects of the compounds, products and/or pharmaceutical compositions described herein.
  • the combination therapies of the present invention improve the prophylactic or therapeutic effects of the compounds, products and/or pharmaceutical compositions described herein and the second therapy by acting together to have an additive or synergistic effect.
  • the combination therapy of the present invention reduces side effects associated with the second therapy (eg, prophylactic or therapeutic agent).
  • a pharmaceutical composition formulated with a therapeutically effective dose of the compound of the present invention and a pharmaceutical carrier is a dose sufficient to provide the desired therapeutic result. At the same time, an effective dose is guaranteed to have minimal side effects.
  • the detection of the effect of the pharmaceutical composition composed of the compounds of the present invention is based on the upstream or downstream activity detection experiments of Arf1 GTPase activity, lipolysis reporter gene, lipid droplet formation, autophagy and Arf1 activity known in the art.
  • a therapeutically effective dose for a particular patient will be determined taking into account a variety of factors, including the condition being treated, the general health of the patient, the method of administration, the severity of side effects, and the like.
  • T cell infiltration and activation at the tumor site inflammatory cytokines such as IL-1 ⁇ and IFN ⁇ or T cell associated chemokines CCL5, CXCL-10, CXCL-11 and CCL22, or T cell activation markers IFN ⁇ , perforin, GzmA, GzmB, increased levels of DAMPs or increased levels of ER stress markers or increased levels of MHC-I/MHC-II or IFN ⁇ .
  • inflammatory cytokines such as IL-1 ⁇ and IFN ⁇ or T cell associated chemokines CCL5, CXCL-10, CXCL-11 and CCL22, or T cell activation markers IFN ⁇ , perforin, GzmA, GzmB, increased levels of DAMPs or increased levels of ER stress markers or increased levels of MHC-I/MHC-II or IFN ⁇ .
  • Veterinary, experimental or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses and humans.
  • the present invention also provides effective administration ranges, administration frequencies, and plasma concentrations of the compounds.
  • the pharmaceutical composition is administered at a dose of: (a) about 1 mg/m 2 to about 5,000 mg/m 2 (iv) or about 1 mg/m 2 to about 50,000 mg/m 2 (po); (b) from about 2 mg/m 2 to about 3,000 mg/m 2 (iv) or from about 10 mg/m 2 to about 50,000 mg/m 2 (po).
  • the compounds of the present invention may be administered every other day (Q2D), daily (QD), or twice a day (BID).
  • the pharmaceutical composition is administered orally and no more than four times a day (QID).
  • the compounds, products, and/or pharmaceutical compositions can be administered according to the following regimens to treat a disease or disorder.
  • the blood molar concentration of the compound can be maintained at not less than the effective concentration and less than the deleterious concentration for a first consecutive period of time that is longer than the onset time of the drug and shorter than the deleterious time. After the first consecutive time period, the blood molarity may be below the effective concentration.
  • an effective concentration can be about 0.1 ⁇ M, about 0.2 ⁇ M, about 0.5 ⁇ M, about 1 ⁇ M, about 2 ⁇ M, about 3 ⁇ M, about 4 ⁇ M, about 5 ⁇ M, about 6 ⁇ M, about 10 ⁇ M, or other concentrations determined to be effective by those skilled in the art.
  • the detrimental concentration can be about 1 ⁇ M, about 3 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, about 30 ⁇ M, about 100 ⁇ M, or another concentration determined to be detrimental by those skilled in the art.
  • the effective time period can 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 other other effective period determined by those skilled in the art.
  • a period of time For example, the detrimental time period may be about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 144 hours, or another time period determined to be detrimental by those skilled in the art.
  • the effective amount of the compound, product and/or pharmaceutical composition selected for treatment is a blood concentration that is greater than the IC50 of tumor cells and less than the IC50 of normal cells. 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 IC50 of normal cells.
  • the dosage form of the compound, product and/or pharmaceutical composition includes, but is not limited to, tablets, pills, capsules (hard or soft), caplets, powders, granules, suspensions, solutions, gels orally Administration, cachets, lozenges, lozenges, syrups, elixirs, emulsions, oil-in-water emulsions, water-in-oil emulsions, and/or syrups.
  • the composition for reducing or inhibiting tumor cell replication or spread comprises a set of particles selected by the following methods.
  • Compounds or salts or solvates thereof can be formulated according to formula I.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable excipient, carrier or diluent.
  • the composition is suitable for oral, nasal, topical, rectal, vaginal or parenteral administration, or intravenous, subcutaneous or intramuscular injection.
  • 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 may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the mammal being treated and the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount that produces a therapeutic effect. Typically, in 100%, this amount will range, for example, from about 1% to about 99% active ingredient, from about 5% to about 70%, from about 10% to about 30%.
  • compositions or formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (with flavoring agents, usually sucrose and acacia or tragacanth), powders, granules or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as lozenges (using inert bases such as gelatin and glycerol , or sucrose and acacia) and/or as mouthwashes, etc., each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • the compounds of the present invention may also be administered as a pill, electuary or paste.
  • the compounds of the present invention are mixed with one or more pharmaceutically acceptable carriers, such as lemon Sodium or dicalcium phosphate, and/or any of the following: fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; binders such as carboxymethyl cellulose, algae acid salts, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate and hydroxyl Sodium starch acetate; dissolution retarders such as paraffin; absorption enhancers such as quaternary ammonium compounds; wetting agents such as cetyl alcohol, glyceryl monostearate
  • the pharmaceutical composition may also contain buffering agents.
  • Solid compositions of a similar type can also be used as fillers for soft and hard filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, among others.
  • Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate Esters, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters sorbitan Sugar alcohols and mixtures thereof.
  • cyclodextrins such as hydroxypropyl-beta-cyclodextrin, can
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to one or more compounds of the present invention, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, metahydrogen Alumina, bentonite, agar and tragacanth, and mixtures thereof.
  • Formulations of pharmaceutical compositions of the present invention for rectal or vaginal administration may be presented as suppositories, which may be prepared by admixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers prepared, such excipients or carriers include, for example, cocoa butter, polyethylene glycols, waxes for suppositories, or as salicylates, which are solid at room temperature but liquid at body temperature and will therefore pass in the rectal or vaginal cavity Melt and release the active agent of the present invention.
  • Formulations of the present invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing suitable carriers known in the art.
  • Dosage forms for topical or transdermal administration of the compositions according to the invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.
  • Ointments, pastes, creams and gels may contain, in addition to the compounds of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polymeric Glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polymeric Glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Ophthalmic formulations are also considered to be within the scope of the present invention.
  • compositions of the present invention suitable for parenteral administration comprise one or more compounds of the present invention together with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or reconstituted sterile powders for incorporation into sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostatic agents, solutes or suspending agents to render the formulation isotonic with the blood of the intended recipient, before use or thickener.
  • a composition according to the present invention in order to prolong the effect of a composition according to the present invention, it is desirable to slow its absorption by the body from subcutaneous or intramuscular injection. This can be achieved by using liquid suspensions of poorly water-soluble crystalline or amorphous materials. The rate of absorption of a drug depends on its rate of dissolution, which in turn may depend on crystal size and form. Alternatively, delayed absorption of parenterally administered compositions is accomplished by dissolving or suspending the compound in an oily vehicle.
  • One strategy for depot injection involves the use of polyethylene oxide-polypropylene oxide copolymers, where the carrier is fluid at room temperature and cures at body temperature.
  • the pharmaceutically acceptable excipient, carrier or diluent comprises lipids for intravenous delivery.
  • the lipids can be: phospholipids, synthetic phosphatidylcholines, natural phosphatidylcholines, sphingomyelins, ceramides, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acid, cholesterol, cholesterol sulfate, and hapten and PEG-conjugated lipids .
  • the lipids can be in the form of nanoemulsions, micelles, emulsions, suspensions, nanosuspensions, liposomes or liposomes.
  • the pharmaceutically acceptable excipient, carrier or diluent is in the form of a micellar emulsion, suspension or nanoparticle suspension and it further comprises an intravenously acceptable protein such as human albumin or Derivatives thereof for intravenous delivery.
  • pharmaceutically acceptable excipients, carriers or diluents include waxy materials for oral delivery.
  • the waxy material may be mono-, di- or triglycerides, mono-, di-fatty acid esters of PEG, PEG-conjugated vitamin E (vitamin E TPG) and/or Gelucire.
  • Gelucire may 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.
  • the pharmaceutically acceptable excipient, carrier or diluent is selected from the group consisting of octanol, transcutol hp, labrafil M, labrasol, triacetin, pharmasolv, ethanol, polyvinylpyrrolidine, carboxymethylcellulose , Tween 20 and Tween 80.
  • a pharmaceutically acceptable excipient such as Gelucire 44/14, is mixed with a surfactant, which may be Tween 80 or Tween 20.
  • the compounds of the present invention can be synthesized using commercially available starting materials and methods well known to those skilled in the organ art. In Examples 13-14, the present invention provides methods of making some of the claimed compounds.
  • a small molecule Arf1 inhibitor refers to any low molecular weight drug that exhibits inhibitory activity against Arf1. Compared with larger molecular weight drugs such as proteins, peptides and carbohydrates, small molecules penetrate cell membranes and the blood-brain barrier more easily. Process development and manufacturing costs for these molecules tend to be lower.
  • Du101 and Du102 are potent inhibitors of Arf1 activation.
  • FIG. 4 In vitro cell line screening shows that Arf1 inhibitor compounds 102-107, Du102 have a certain tumor suppressing effect on lung cancer cells H460 and A549.
  • FIG. 1 In vitro cell line screening shows that Arf1 inhibitor compounds 103-104, 107, 109-110, and Du102 have certain tumor inhibitory effects on colorectal cancer cell lines HT29 and LoVo cells, liver cancer cells Huh7, and lung cancer cells H460 and A549.
  • Example 1 Du101 and Du102 are potent inhibitors of Arf1 activation.
  • Arf1 activity was detected using the Thermo Scientific Pierce Active Arf1 Pull-Down and Detection Kit (Cat#16121) and liver cancer Huh-7 cell lysates. It is a complete kit that selectively enriches and detects GTP-bound Arf1 GTPase by interacting with specific proteins of the GGA3 protein-binding domain, the results of which are shown in Figure 1.
  • Example 2 In vitro cell line screening showed that Arf1 inhibitors including Du101 and Du102 have certain tumor suppressive effects.
  • Example 3 Drosophila screen showing that Arf1 inhibitors including Du101 and Du102 have good tumor suppressive effect in vivo.
  • Drosophila intestinal tumors were formed by inducing the expression of the tumor-promoting gene Rasv12-GFP in the Drosophila intestine. After feeding with Arf1 inhibitor, the Drosophila intestinal tumor cell death could be significantly induced (PI staining) ( Figure 8).
  • Example 4 In vivo drug experiments in mice showed that the Arf1 inhibitors Du101 and Du102 had significant tumor suppressing effects.
  • Example 5 Vaccination of Arfl inhibited cells protects animals from tumors.
  • the immunogenicity of reagents can be tested in a mouse vaccination model (Obeid et al., 2007; Sagiv-Barfi et al., 2018). Mice were injected with drug-treated tumor cells in one flank and then again with syngeneic tumor cells a week later. We tested the vaccine effect of DMSO or Du102-treated CT-26 colon cancer, B16-F10 melanoma, and 4T1 breast cancer.
  • Table 3 DU102 treatment reduces tumor size (mm 3 ) in CT-26 colon cancer model in BALB/c mice Left side with drug treatment
  • Table 4 Vaccination with DU102-treated CT-26 cells on left side protects BALB/c mice from developing tumors on right side
  • Table 6 DU102 treatment reduces tumor size(mm 3 ) in B16-F10 melanoma model in C57B/6 mice Left side with drug treatment
  • Table 7 Vaccination with DU102-treated B16-F10 cells on left side protects C57B/6 mice from developing tumors on right side
  • Table 9 DU102 treatment reduces tumor size(mm 3 ) in 4T1 breast carcinoma model in BALB/c mice Left side with drug treatment
  • Table 10 Vaccination with DU102-treated 4T1 cells on left side protects BALB/c mice from developing tumors on right side
  • 6-week-old female mice were inoculated subcutaneously with CT26, 4T1 and B16-F10 tumor cells ( 5x105 , 1x104 and 5x104 , respectively) treated with 10 [mu]M Du102 or DMSO. After 7 days, equal amounts of the corresponding tumor cells were seeded on the right side. Tumor cell lines were mixed with Matrigel (GIBCO, Cat#354234) or resuspended in PBS and injected subcutaneously or intradermally.
  • Example 6 Du102 treatment induces inflammatory cytokines in the CT-26 colon cancer model in BALB/c mice.
  • IL-1 ⁇ assay 7 days after injection of CT26 cell lysate, CD11c+ DCs were isolated from injected BALB/c mice and co-cultured with DMSO or Du102-treated CT26 cells. After 2 days, IL-1 ⁇ was measured in harvested cells (Table 11).
  • INF ⁇ assay 7 days after injection of CT26 cell lysate, CD11c+ DC and CD8+ T cells were isolated from injected BALB/c mice and co-cultured with DMSO or Du102-treated CT26 cells. After 2 days, INF[gamma] was measured in harvested cells (Table 11).
  • Example 7 Du102 treatment induces IL-1 ⁇ in B16-F10 melanoma in C57B/6 mice. (Table 12)
  • Example 8 Du101 and Du102 treatment reduces liver tumor numbers in MYC-ON mice.
  • T2-MYC Tet-on-MYC/LAP-tTA
  • LAP liver activator protein
  • tTA tetracycline transactivator protein
  • mice were induced with tumors in the At 6 weeks of age, switch to a normal diet.
  • a grain-based rodent diet Bio-Serv, Cat# 14-727-450
  • mice were divided into three groups, control group, Du101-treated group and Du102-treated group.
  • the control group was given 100 ⁇ l/10 g body weight (BW) of 20% DMSO + 80% corn oil by gavage using a feeding tube.
  • BW body weight
  • 25 mg/ml of Du101 stock solution was diluted with corn oil at a ratio of 1:4 to prepare a working solution.
  • 25 mg/m1 of Du102 stock solution was diluted with corn oil at a ratio of 1:4 to prepare a working solution.
  • the mixed working solution was administered to mice at 100 ⁇ l/10 gBW by gavage using a feeding tube.
  • mice were monitored every 2 days, and mice were euthanized and analyzed for tumor growth after 10 weeks.
  • Table 13 shows that Du101 and Du102 can reduce overexpressed Myc-induced liver tumors in mice.
  • Example 9 Du102 treatment induces the expression of chemokines and T cell activation markers in MYC-ON mice.
  • Chemokines and markers of T cell activation were measured by quantitative RT-PCR in DMSO- or Du102-treated MYC-ON mice. Increased CD8 T cell infiltration and immunostimulatory cytokine expression were measured by quantitative RT-PCR. Du102 treatment significantly increased the expression of many T cell associated chemokines CCL5, CXCL-10, CXCL-11 and CCL22 in MYC-ON compared to DMSO control-treated mice (Table 14). CCL5 and CXCL10 chemokines stimulate tumor infiltration of CD4+ and CD8+ lymphocytes (Parkes et al., 2017), and CXCL10 and CXCL11 are T cell-associated chemokines.
  • Example 10 Du102 treatment induces expression of T cell activation markers of 4T1 breast cancer in BALB/c mice.
  • GzmA GzmB Perforin IL-1 ⁇ INF ⁇ PD-L1 DMSO 1.0 1.0 1.0 1.0 1.0 1.0 1.0 DU102 2.2 2.3 2.5 2.2 2.1 0.5
  • Example 11 Du102 treatment induces MHC expression and T cell infiltration in MYC-ON mice.
  • the numbers of MHC-I, MHC-II, infiltrating CD4+ and CD8+ T-cells were counted from representative sections of several tumors stained by immunohistochemistry for markers of MHC-I, MHC-II, CD4 and CD8.
  • Example 12 Du102 treatment reduces lung metastasis of B16-F10 melanoma in C57B/6 mice.
  • B16-F10 tumor cells (treated with DMSO or 10 ⁇ M Du102) were transferred intravenously by tail vein injection. Lungs were removed 15 days after injection and fixed in Fekete solution overnight. Visible metastases were counted blindly by three investigators (Table 17).
  • the antitumor effect of Du102 was neutralized by CD4 or CD8 antibodies in MYC-ON mice (Table 18). Cell depletion was verified using FACS and IHC analysis.
  • Table 18 The anti-tumor effects of DU102 are neutralized by anti-CD4 or/anti-CD8 antibodies in MYC-ON mice
  • the antibody was injected intraperitoneally. It is injected once a week for 5 consecutive weeks, and doxycycline is discontinued weekly after the first injection.
  • Antibodies used to treat mice were as follows: 100 ⁇ g/mouse rat anti-CD4 (clone GK1.5, BioXcell, Cat#BE0003-1), 100 ⁇ g/mouse rat anti-CD8d (clone 2.43, BioXcell, Cat#BE0061) or 100 ⁇ g/mouse rat IgG (BioXcell, Cat#BE0094) isotype control antibody. From the second week of MYC-ON for 4 weeks, Du102 and DMSO were administered by gavage needle as described above, mice were monitored every 2 days, and mice were euthanized and analyzed for tumor growth after 10 weeks.
  • Example 14 Synergistic antitumor effect of Du102 and anti-PD-1 antibody in MYC-ON mice.
  • Du102 was administered in combination with anti-PD-1.
  • the antibody was injected intraperitoneally. It is injected once a week for 5 consecutive weeks, and doxycycline is discontinued weekly after the first injection.
  • Antibodies used to treat mice were as follows: 100 ⁇ g/mouse Armenian hamster anti-mouse PD1 (J43, BioXcell, Cat#BE0033-2) and 100 ⁇ g/mouse Armenian hamster isotype IgG (BioXcell, Cat#BP0091) were used as control. From the second week of MYC-ON for 4 weeks, Du102 and DMSO were administered by gavage needle as described above, mice were monitored every 2 days, and mice were euthanized and analyzed for tumor growth after 10 weeks.
  • Co-administration of anti-PD-1 antibodies may allow the use of lower, less toxic doses of Du102, thereby avoiding known side effects.
  • Example 14 Du102, 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, Therapeutic effect in gastric cancer, multiple myeloma, leukemia, lymphoma.
  • 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 tissue or cell lines were subcutaneously inoculated into humanized mice to construct a humanized tumor model.
  • DMSO or Du102 and Du101 were administered by gavage.
  • Immunostimulatory cytokine expression was detected by quantitative RT-PCR measurement.
  • the expression of T cell activation markers IFN ⁇ , perforin, GzmA, GzmB and IL-1 ⁇ in mice was detected by FACS.
  • mice Or by 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 tissue or cell lines were subcutaneously inoculated into wild-type mice to construct tumor models.
  • DMSO- or Du102- and Du101-treatment were administered by gavage.
  • Immunostimulatory cytokine expression was detected by quantitative RT-PCR measurement.
  • the expression of T cell activation markers IFN ⁇ , perforin, GzmA, GzmB and IL-1 ⁇ in mice was detected by FACS.
  • Example 15 Therapeutic effects of Du102 and Du101 in autoimmune diseases, inflammatory diseases, inflammatory bowel diseases, arthritis, and autoimmune demyelinating diseases.
  • DMSO or Du102 and Du101 were administered by gavage or intracranial injection.
  • Immunostimulatory cytokine expression was detected by quantitative RT-PCR measurement.
  • the expression of T cell activation markers IFN ⁇ , perforin, GzmA, GzmB and IL-1 ⁇ in mice was detected by FACS. The incidence of mice was detected by sectioning.

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Abstract

本发明涉及一种新型Arf1抑制剂的使用；使用这些化合物治疗难治性、复发性或转移性癌症的方法；研究利用该化合物和特定给药方案选择性杀伤癌症干细胞和癌细胞的方法；研究通过抑制Arf1通路，特别是COPI/Arf1-脂解-β-氧化通路，靶向肿瘤干细胞诱导抗肿瘤免疫反应的方法；在哺乳动物中，使用新化合物治疗与Arf1通路活性相关疾病的方法；制备此类化合物及其中间体的工艺，以及相关化合物的药物组成。

Description

用于根除癌症干细胞并诱导DAMP介导的抗肿瘤免疫反应的一组新型COPI/ARF1-脂解途径抑制剂和化合物 技术领域
本发明总体上涉及COPI/Arfl-脂解-β-氧化途径抑制剂治疗病症的用途。更具体地，本发明涉及COPI/Arf1-脂解-β-氧化途径抑制剂在靶向癌症干细胞和激活免疫应答以及治疗疾病中的用途。更具体地，本发明涉及1H-吲哚-5-甲醛6,7,8,9-四氢-5H-环庚基[4,5]噻吩并[2,3-d]嘧啶-4-基腙、1H-吲哚-5-甲醛5,6,7,8,9,10-六氢环辛基[4,5]噻吩并[2,3-d]嘧啶-4-基腙，以及相关化合物抑制Arf1，靶向癌症干细胞并激活抗肿瘤免疫反应，用于治疗恶性疾病。本发明还涉及难治性、复发性或转移性癌症的治疗，以及相关化合物及其中间体的制备方法，以及相关化合物的药物组合物。
发明背景
仅在美国，每年癌症死亡人数就达到数十万。尽管通过手术、放疗和化疗在治疗某些形式的癌症方面取得了进展，但许多类型的癌症基本上无法治愈。即使对特定癌症有有效的治疗方法，这种治疗的副作用也可能很严重，并导致生活质量显着下降。
免疫疗法，尤其是检查点阻断疗法，开始从根本上改变治疗癌症患者的范式(Sharma et al.,2017)。然而，尽管最近取得了临床上的成功，但不幸的是，免疫疗法只为一小部分癌症患者提供了帮助。大多数患者几乎没有获益，因为大多数新出现的肿瘤已经在肿瘤微环境中形成了免疫逃避机制，包括功能失调的T细胞和缺乏T细胞浸润(Jerby-Arnon et al.,2018；Sharma et al.,2017)。但病原体(细菌或病毒)诱导的免疫反应在癌症患者中大多完好无损。这些病原体表达独特的分子，称为病原体相关分子模式(PAMP)，由先天免疫系统的模式识别受体(PRR)识别以激活抗感染免疫反应。这种诱导的免疫反应可用于使肿瘤消退，正如William Coley(Balkwill,2009)首次证明的那样。在1890年代，他成功地在患者体内注射链球菌培养物后使许多肉瘤和/或淋巴瘤消退。一些注射的细菌可能会触发PAMP诱导的抗肿瘤免疫反应。
最近发现，免疫系统不仅可以由PAMPs诱导，还可以由被称为危险/损伤相关分子模式(DAMPs)的内源性信号诱导，该信号从在没有微生物成分的情况下从受损或受压的细胞中释放(Jerby-Arnon et al.,2018；Sharma et al.,2017)。DAMP是“PAMP”的类似物，存在于特定细胞区的分子，在生理条件下不表达或仅有限表达。但被强烈诱导，然后在压力或者损伤条件下，DAMPs会转移到细胞表面或细胞外空间。最重要的DAMP包括：(i)细胞表面上的内质网(ER)-固着分子伴侣钙网蛋白(Calr)的凋亡前暴露；(ii)非组蛋白核蛋白高迁移率族 框1(HMGB1)释放到细胞外空间；(iii)ATP的主动分泌。表面暴露的Calr将通过与树突细胞(DC)上的LRP1/CD91结合来促进肿瘤相关抗原的吞噬，细胞外HMGB1将与DC上的Toll样受体4(TLR4)结合并激活TLR4-MYD88通路以支持DC成熟，并防止吞噬代谢异物的溶酶体加速破坏，从而进行抗原处理并呈递给细胞毒性T淋巴细胞(CTL)。此外即将死亡的肿瘤细胞分泌ATP，通过P2RX7受体-炎症小体-IL-1β途径激活分泌IFNγ的T细胞，进而介导抗肿瘤免疫反应(Ghiringhelli等，2009)。总之，这些DAMP不同的信号通路，促进了宿主对肿瘤特异性适应性免疫反应的发展。这些可以抑制甚至完全根除抗药性肿瘤细胞。然而，触发肿瘤细胞释放DAMP并在体内诱导抗肿瘤免疫反应的内在分子机制尚不清楚。
癌症干细胞(CSC)是肿瘤中处于干细胞状态并具有干细胞特征的一部分细胞群。CSC可能导致治疗抵抗、肿瘤转移、疾病复发，并最终导致患者死亡(Batlle and Clevers,2017；Lytle et al.,2018；Shibue and Weinberg,2017)。CSC研究的最终目标是确定选择性调节CSC存活的通路，然后针对该通路根除CSC。一些CSCs是从正常干细胞的转化中产生的，另一些是通过将非CSC癌细胞重新编程为干细胞状态而产生的。CSC和正常干细胞有许多共同的特性。因此，调节正常或转化干细胞的通路也可能调节CSC。我们之前发现COPI/Arf1介导的脂解途径选择性地维持果蝇中的干细胞和转化干细胞，并且该途径的敲低会导致干细胞坏死(Singh et al.,2016)。在最近的一项研究中，我们发现小鼠中Arf1的敲低会破坏脂质代谢并导致脂滴积聚，从而进一步引起代谢应激——线粒体缺陷和内质网应激。代谢应激通过小鼠的坏死选择性地杀死祖细胞、干细胞和CSC。垂死的CSCs释放DAMPs激活DCs，进一步增强T细胞浸润和激活，进而刺激机体的抗肿瘤免疫反应。我们的数据表明，Arf1通路的敲低不仅会杀死CSC，还会引发肿瘤特异性免疫反应并将垂死的CSC转化为治疗性疫苗，这实际上改善了治疗的长期疗效(Wang et al.,2020)。
我们发现抑制Arfl会诱导T细胞浸润和活化，并为利用DAMPs激活癌症患者的抗肿瘤免疫提供新的治疗策略。除了检查点阻断外，这可能是癌症免疫疗法的另一个有希望的方向。新疗法可能包括降低Arf1活性以及阻断Arf1介导的脂解途径和相关的β-氧化的方法，我们测试了10种已报道的Arf1抑制剂(Bill et al.,2011；Boal et al.,2010；Feng et al.,2004；Newton et al.,2006；Ohashi et al.,2012；Sáenz et al.,2009；Sorieul et al.,2011；Viaud et al.,2007)，包括果蝇干细胞上的Brefeldin A(BFA)、Golgicide A(GCA)、LM11、Secin16、Secin H3、Secin B7、Exo1、Exo2、LG8和LG18肿瘤和正常人骨髓造血干细胞(HSC)。我们发现这些已知的抑制剂要么在杀死果蝇干细胞肿瘤方面不是很有效，要么毒性太大(杀死正常的HSC)。基于此，我们合成并测试了基于Exo2的新抑制剂，发现Du101和Du102(如下所述)有 效且毒性相对较低。
发明内容
本发明基于我们最近的发现，即抑制COPI/ARF1-脂解-β-氧化途径根除癌症干细胞并诱导DAMP介导的抗肿瘤免疫反应。因此，本发明的第一方面涉及消除肿瘤的方法，其中该方法包括通过Arf1途径的抑制剂在癌症患者中抑制至少一些、大部分或基本上所有(例如，30％、40％、50％、60％、70％、80％、90％或95％)，的Arf1途径活性。
在一个实施方案中，Arfl途径抑制剂是分离、纯化或合成的，并且可以选自小分子Arfl抑制剂、针对Arfl的RNAi剂、针对Arfl的反义剂、拟肽Arfl抑制剂、和G-四重寡脱氧核苷酸Arf1抑制剂。抑制机制可选自显著抑制Arf1GTPase活性、显著抑制Arf1-通路调节的脂解活性、显著抑制Arf1-通路调节的氧化活性和通过抑制Arf1通路显著诱导细胞坏死。
在一个实施方案中，抑制剂是选自上述化合物102，104-111，尤其是Du101和Du102、其对映异构体、非对映异构体、互变异构体和盐或溶剂化物(以下称为“本发明化合物”)。
本发明提供抑制或减少肿瘤或癌症生长的方法，包括使肿瘤与有效量的本发明化合物接触。该化合物抑制肿瘤或癌症的生长或该化合物减小肿瘤或癌症的大小。该化合物增加MHC-I和MHC-II的表达。与DMSO相比，该化合物增加了T细胞在肿瘤中的浸润和活化。化合物增加了T细胞活化标志物的表达，例如GzmA、GzmB和穿孔素。在另一个实施方案中，该化合物增加至少一种炎性细胞因子或趋化因子的表达，其可选自IFNγ、IL-1β、Ccl5、Cxc10、Cxc11、Ccl22。在某些实施方案中，该化合物与至少一种抗PD-1抗体共同给药，该化合物和PD-1阻断具有协同作用。在某些实施方案中，用化合物处理的癌细胞接种后，起到疫苗作用，保护动物免于肿瘤发展。在某些实施方案中，用该化合物治疗具有“一石二鸟”的效果，不仅可以杀死CSCs，还可以引发肿瘤特异性免疫反应，将坏死的CSCs转化为治疗性疫苗，从而产生长期疗效。
在第二个方面，本发明提供了一种治疗或预防受试者中与Arfl途径活性相关的病症的方法，该方法包括向受试者施用治疗有效量的包含本发明化合物的药物组合物从而降低Arf1通路的活性。在一个实施方案中，Arf1通路活性可以通过测试Arf1GTPase活性或Arf1GTPase活性的替代上游或下游调节因子来识别。该病症可以是癌症。在一个实施方案中，已知癌症具有Arf1通路活性，包括但不限于：乳腺癌、头颈癌、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑肿瘤、胃癌、多发性骨髓瘤、白血病和淋巴瘤。所述病症还可以是已知与Arf1通路活性相关的非癌性病症，并且在一个实施方案中，选自自身免疫疾病、炎性疾病、炎性肠病、关节炎、自身免疫性脱髓鞘病症、阿尔茨海默症、中风、缺血再灌注损伤、多发性硬化和其他炎症或神经退行性疾病。
在第三个方面，本发明提供了一种抑制细胞中Arfl途径活性的方法。该方法包括向细胞施用有效量的本发明化合物，使得细胞中的Arf1途径活性降低例如至少20％、30％、40％、50％、60％、70％、80％、90％或95％。在一个实施方案中，细胞是CSC，或者是癌性的。该方法可诱导细胞死亡。该方法可以在体外或体内进行。
在第四个方面，本发明提供了一种药物组合物，其包含本发明的化合物，即选自上述化合物102，104-111的化合物，及其药学上可接受的盐或溶剂化物，以及药学上可接受的赋形剂、载体或稀释剂。在一个特征中，该组合物适用于口服、鼻腔、局部、直肠、阴道或肠胃外给药，或静脉内、皮下或肌肉内注射。
在第五个方面，本发明还提供了制备一些本发明化合物的方法。该方法制备通式I化合物，其中R1具有本文定义的含义，或其药学上可接受的盐、溶剂化物或前药。特别是：
Figure PCTCN2021110373-appb-000001
通式(I)，其中r＝1-6；
R1为选自一个或多个取代基或未取代苯基、
Figure PCTCN2021110373-appb-000002
Figure PCTCN2021110373-appb-000003
所述取代基选自卤素、C 1-6烷基、卤代烷基、OH、OCH 3、O(CH 2) nCH 3、环丙基氧基、OC(CH 3) 3、OCH(CH 3) 2、NH 2、NO 2、N(CH 3) 2、NH(CH 2) nCH 3、CN、N 3等，其中n为1-9。
具体的，其中靶向Arf1途径的抑制剂为如下化合物：
Figure PCTCN2021110373-appb-000004
在第六方面，本发明提供了制备化合物1H-吲哚-5-甲醛6,7,8,9-四氢-5H-环庚基[4,5]噻吩并[2,3-d]嘧啶-4-基腙或上述107或Du101的方法。
Figure PCTCN2021110373-appb-000005
在第七方面，本发明提供了制备化合物1H-吲哚-5-甲醛5,6,7,8,9,10-六氢环辛基[4,5]噻吩并[2,3-d]嘧啶-4-基腙或上述111或Du102的方法。
Figure PCTCN2021110373-appb-000006
下面详细讨论本发明的实施例。在描述实施例时，为了清楚表述，采用了特定术语。然而，本发明不旨在限于如此选择的特定术语。相关领域的技术人员将认识到，在不脱离本发明的精神和范围的情况下，可以使用其他等效组件和开发其他方法。在此引用的所有参考文献均以引用方式并入，就像每个参考文献已单独并入一样。
定义
如本文所用，单数形式“一个”和“该”包括复数形式，除非上下文另有明确规定。例如，术语“一个细胞”包括多个细胞，包括它们的混合物。
如本文所用，术语“癌症干细胞”和“CSC”是可互换的。CSC是哺乳动物来源的，并且在优选的实施方案中，这些CSC是人源的，但它们不旨在限于此。癌症干细胞被定义和功能表征为源自肿瘤的细胞群，其：(1)具有广泛的增殖能力；(2)能够进行不对称细胞分裂以产生一种或多种增殖或发育潜能降低的分化后代；(3)能够进行对称细胞分裂以进行自我更新或自我维持。表征CSC的其他常见方法包括细胞表面标志物，形态学、转录谱和药物反应。CSC在研究文献中也称为肿瘤/癌症起始细胞、癌症干细胞样细胞、干细胞样癌细胞、高致瘤细胞、肿瘤干细胞、实体瘤干细胞、药物存活细胞(DSC)、耐药细胞(DRC)或超级恶性细胞。
如本文所用，术语“癌症”和“癌性”是指或描述哺乳动物中的生理状况，其中细胞群的特征在于不受调节的细胞生长。如本文所用，“癌细胞”和“肿瘤细胞”是指源自肿瘤的总细胞群，包括构成肿瘤细胞群的大部分的非致瘤细胞和致瘤干细胞(癌症干细胞)。癌症的例子包括但不限于癌、淋巴瘤、母细胞瘤、肉瘤和白血病。此类癌症的更具体实例包括鳞状细胞癌、小细胞肺癌、非小细胞肺癌、肺腺癌、鳞状细胞癌、腹膜癌、肝细胞癌、胃肠癌、胰腺癌、胶质母细胞瘤,宫颈癌,卵巢癌,肝癌,膀胱癌,肝癌,乳腺癌、结肠癌、结直肠癌、 子宫内膜癌或子宫癌、唾液腺癌、肾癌、肝癌、前列腺癌、外阴癌、甲状腺癌、肝癌及各类头颈癌。
如本文所用，“肿瘤”是指由细胞过度生长或增殖导致的任何组织块，无论是良性(非癌性)还是恶性(癌性)，包括癌前病变。
如本文所用，“转移”是指癌症从起源部位扩散或转移到身体其他区域的过程，同时在新部位出现类似的癌性病变。“转移性”细胞失去与邻近细胞的粘附接触并通过血流或淋巴从疾病的原发部位迁移以侵入邻近的身体结构。
如本文所用，术语“受试者”是指任何动物(例如哺乳动物)，包括但不限于人类、非人类灵长类动物、啮齿动物等。通常，术语“受试者”和“患者”在本文中就人类受试者而言可互换使用。
如本文所用的诸如“治疗”或“减轻”的术语是指：1)治愈、减缓、减轻症状和/或停止诊断出的病理状况或障碍的进展；2)预防或减缓目标病理状况或障碍发展的预防或预防措施。因此，需要治疗的人包括那些已经患有这种疾病的人；易患此病者；以及那些需要预防疾病的人。如果患者表现出以下一项或多项，则根据本发明的方法成功“治疗”受试者：癌细胞数量减少或完全不存在；减小肿瘤大小；抑制或不存在癌细胞浸润到外周器官，包括癌症扩散到软组织和骨骼中；抑制或不存在肿瘤转移；抑制或不存在肿瘤生长；缓解与特定癌症相关的一种或多种症状；降低发病率和死亡率；生活质量的提高。
如本文所用，术语“抑制”及其同义词，当在生物活性的上下文中使用时，是指生物活性的下调，其可降低或消除靶向功能，例如蛋白质的产生或分子的磷酸化。在特定实施例中，抑制可指目标活性降低约20％、30％、40％、50％、60％、70％、80％、90％或95％。当在病症或疾病的上下文中使用时，这些术语是指成功预防症状发作、减轻症状或消除疾病、病症。
如本文所用，术语“脂解”是指脂质分解并且涉及将甘油三酯水解成甘油和游离脂肪酸。脂肪分解主要发生在脂肪组织中，用于在禁食或运动期间调动储存的能量。
如本文所用，术语“GTP酶”是指可以结合并水解三磷酸鸟苷(GTP)的水解酶。GTP结合和水解发生在所有GTP酶共有的高度保守的G域中。
如本文所用，术语“β-氧化”是指脂肪酸分子在原核生物的胞质中和真核生物的线粒体中分解以产生进入三羧酸循环的乙酰辅酶A的分解代谢过程，NADH和FADH2，它们是用于电子传递链的辅酶。之所以这样命名是因为脂肪酸的β碳被氧化成羰基。β-氧化主要由线粒体的三种功能蛋白促进，这是一种与线粒体内膜相关的酶复合物，尽管一些脂肪酸在过氧化物酶体中被氧化。
如本文所用，术语“坏死性细胞死亡”是指当细胞暴露于可能导致质膜损伤的生理条件(例 如，体温过低、缺氧)或者极端变化的条件中。在生理条件下，对质膜的直接损伤是由补体和裂解病毒等试剂引起的。坏死始于细胞维持体内平衡的能力受损，导致水和细胞外离子的流入。细胞内的胞器，尤其是线粒体，以及整个细胞膨胀和破裂(细胞裂解)。由于质膜的最终分解，包括溶酶体酶在内的细胞内容物被释放到细胞外，因此，在体内，细胞坏死通常与导致强烈炎症反应，进而引发广泛的组织损伤有关。
“免疫病症”包括例如病理性炎症、炎性病症和自身免疫病症或疾病。“免疫状况”指感染、持续感染和增殖状况，例如癌症、肿瘤和血管生成，包括抵抗免疫系统照射的感染、肿瘤和癌症。“癌症病症”包括例如癌症、癌细胞、肿瘤、血管生成和癌前病症例如发育不良。
如本文所用，术语“细胞毒性T细胞”是指杀死癌细胞、被感染(特别是被病毒)的细胞或以其他的T淋巴细胞(一种白细胞)。大多数细胞毒性T细胞表达可识别特定抗原的T细胞受体(TCR)。抗原是一种能够刺激免疫反应的分子，通常由癌细胞或病毒产生。细胞内的抗原与I类MHC分子结合，并被I类MHC分子带到细胞表面，在那里它们可以被T细胞识别。如果TCR对该抗原具有特异性，它会与I类MHC分子和抗原的复合物结合，并且T细胞会破坏该细胞。为了使TCR与I类MHC分子结合，TCR必须伴随称为CD8的糖蛋白，其与I类MHC分子的恒定部分结合。因此，这些T细胞被称为CD8+T细胞。一旦细胞毒性CD8+T细胞(CTL)在外周识别它们的靶细胞，CTL就会被激活，并且在T细胞与其靶细胞之间形成称为免疫突触(IS)的高度特化的细胞间接触结构。TCR的激活导致Src家族激酶、Lck和Fyn的参与，以及Syk家族激酶ZAP-70向TCR的募集，这些酪氨酸激酶在此处被激活。激活的ZAP70反过来磷酸化，用于激活T细胞的接头蛋白LAT，导致LAT信号小体的形成，其中包括磷脂酶Cγ1(PLCγ1)和SLP76(76kDa的含SH2结构域的白细胞蛋白)。PLCγ1将磷脂酰肌醇-4,5-二磷酸(PIP2)转化为DAG(二酰基甘油)和IP3(肌醇-1,4,5-三磷酸)。DAG在免疫突触处积聚，导致募集新的蛋白激酶Cs(PKCs)，包括PKCθ。PKCθ然后通过将动力蛋白相互定位到突触来促进中心体(微管组织中心或MTOC)极化，这将MTOC拉向突触的方向，并将非肌肉肌球蛋白II(NMII)拉到细胞的另一侧，在那里推动MTOC朝向突触。IS下MTOC的对接确保了动力蛋白和Ca 2+依赖性靶向递送和颗粒内容物的胞吐作用直接进入突触间隙，通过颗粒酶和穿孔素的协同作用导致靶细胞裂解(reviewed in:de la Roche et al.,2016)。
如本文所用，术语“药学上可接受的赋形剂、载体或稀释剂”是指药学上可接受的材料、组合物或赋形剂，例如液体或固体填充剂、稀释剂、赋形剂、溶剂或包封材料，参与携带或将主题药剂从一个器官或身体的一部分运送到另一个器官或身体的一部分。在与制剂的其他成分相容并且对患者无害的意义上，每种载体必须是“可接受的”。可用作药学上可接受的载 体的材料的一些例子包括：糖类，例如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和醋酸纤维素；黄蓍胶粉；麦芽；明胶；滑石；赋形剂，如可可脂和栓剂蜡；油类，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和豆油；乙二醇，如丙二醇；多元醇，如甘油、山梨糖醇、marmitol和聚乙二醇；油酸乙酯、月桂酸乙酯等酯类；琼脂；缓冲剂，如氢氧化镁和氢氧化铝；海藻酸；无热原水；等渗盐水；林格的解决方案；乙醇；磷酸盐缓冲液；和其他用于药物制剂的无毒相容物质。润湿剂、乳化剂和润滑剂，例如十二烷基硫酸钠、硬脂酸镁和聚环氧乙烷-聚环氧丙烷共聚物以及着色剂、脱模剂、涂层剂、甜味剂、调味剂和加香剂、防腐剂和抗氧化剂也可以存在于组成。
本发明的化合物可以形成盐，这也在本发明的范围内。参考本发明的化合物除非另有说明，本文中的术语应理解为包括提及其盐。如本文所用，术语“盐”表示与无机和/或有机酸和碱形成的酸性和/或碱性盐。此外，当本发明的化合物同时包含碱性部分(例如但不限于吡啶或咪唑)和酸性部分(例如但不限于羧酸)时，两性离子(“内盐”)可以是形成并包括在本文使用的术语“盐”内。药学上可接受的(即无毒的、生理学上可接受的)盐是优选的，尽管其他盐也是有用的，例如，在制备过程中可能采用的分离或纯化步骤中。本发明化合物的盐可以例如通过使化合物102或104-111，与一定量的酸或碱，例如等量的量，在诸如盐在其中沉淀或在水性介质中，然后冻干。
本发明化合物的溶剂合物也包括在本文中。本发明化合物的溶剂化物包括例如水合物。
本发明提供了用选自上述化合物102，104-111，尤其是Du101和Du102的化合物治疗增殖性疾病例如癌症、肿瘤等的方法。最近，我们发现了一种将脂质代谢、CSC死亡和抗肿瘤免疫联系起来的新网络。CSCs可能负责治疗抵抗和免疫逃避。CSC信号可以调节淋巴细胞浸润到肿瘤中并改变肿瘤微环境(Lytle et al.,2018)。我们已经证明，在CSC中敲低Arf1介导的脂质代谢会导致代谢应激和随后的细胞反应，包括释放DAMP，DAMP通过激活DC、增强T细胞浸润和激活来促进抗肿瘤免疫(Wang et al.,2020)。我们进一步证明了Arf1抑制和PD-1阻断具有协同作用。一致地，TCGA数据分析显示Arf1表达与T细胞浸润和激活以及在各种人类癌症中更好的存活概率之间呈负相关。我们的研究结果表明，敲低Arf1通路具有“一石二鸟”的效果，不仅可以杀死CSC，还可以引发肿瘤特异性免疫反应，并将垂死的CSC转化为治疗性疫苗，从而产生长期疗效治疗(Wang et al.,2020)。
本发明提供了新化合物通过增加趋化因子和炎性细胞因子的表达以及增强T细胞浸润和活化来抑制Arfl途径活性并诱导抗肿瘤免疫应答的证据。我们进一步证明，使用该化合物和PD-1阻断剂进行治疗具有协同作用。因此，用该化合物的治疗应该为肿瘤治疗提供显着的改善。
本发明与抗肿瘤免疫研究的最新突破相结合，使得本发明能够提供一系列针对抑制CSC或治疗具有特定CSC的癌症或一般癌症的方法。本发明还提供了针对抑制细胞中的Arf1途径活性或治疗与Arf1途径活性相关的癌性和非癌性病症的方法。本发明还提供相关方法(例如，制造和候选药物筛选)、材料、组合物和试剂盒。
该方法可用于治疗受试者的癌症。已知具有CSCs和Arf1-脂解-β-氧化通路活性的癌症是此类治疗的良好候选者，包括但不限于：乳腺癌、头颈癌、肺癌、卵巢癌、胰腺癌、结直肠癌癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑肿瘤、胃癌、多发性骨髓瘤、白血病和淋巴瘤。在一个实施方案中，该方法用于治疗肝癌、头颈癌、胰腺癌和/或胃癌。在另一个实施方案中，该方法用于治疗多发性骨髓瘤、脑肿瘤和肉瘤。
此外，由于已经证明CSC是肿瘤发生、癌症转移和癌症复发的根本原因，因此可以实施本发明的任何抑制CSC的方法来治疗转移性癌症、化学疗法或放射疗法难治的癌症，或在初始治疗后受试者复发。
在一个实施方案中，抑制剂是分离的、纯化的或合成的，并且可以选自小分子Arfl抑制剂、针对Arfl的RNAi剂、针对Arfl的反义剂、拟肽Arfl抑制剂和G-四重寡脱氧核苷酸Arf1抑制剂。抑制剂也可以从天然产物中分离或纯化。
Arf1抑制剂可以选择靶向Arfl途径中的任何步骤。例如，抑制剂可以显着抑制Arf1GTPase活性、脂解报告基因、脂滴形成、自噬以及Arf1活性或功能的替代上游或下游调节剂。
在一个实施方案中，根据本发明的Arf1抑制剂是：化合物102，104-111(表1)、其对映异构体、非对映异构体、互变异构体和盐或溶剂化物(“本发明化合物”)。本发明还提供了本发明化合物减少肿瘤的体外和体内数据。
本发明的化合物不仅显示出在广泛范围的癌细胞中导致死亡，而且在其细胞毒性方面表现出选择性，这对于开发低毒性治疗剂是关键的。本文所用的选择性细胞毒性是指化合物杀死癌细胞同时基本上不伤害正常细胞的能力，有时在某些条件下。正常细胞通常是指健康的非致瘤细胞。导致候选药物选择性细胞毒性的条件很难预测，因为它们需要了解细胞毒性的潜在机制。例如，降低有丝分裂过程中靶向微管形成的抗癌药物的毒性与阻断细胞代谢过程的药物相比，需要使用完全不同的因素。产生选择性细胞毒性的合适条件需要平衡药物具有足够毒性以有效杀死癌细胞同时对正常细胞足够耐受的需求。例如，如果使用较低的浓度，这通常意味着需要长时间输注以杀死癌细胞。
从本发明的实施例中产生的数据看来，如果受影响的细胞没有持续暴露于临界浓度的化合物超过一定时间，则本发明化合物可以实现选择性细胞毒性。在旨在选择性杀死受试者体 内癌细胞的方法中，将含有本发明化合物的药物组合物施用于受试者，使得受试者血浆中的化合物浓度不超过临界浓度维持超过24小时。该方法可用于治疗所有癌症，包括此处描述的任何癌症组，并用于治疗Arf1相关疾病，其示例性列表已在上文提供，此处不再重复。或者，持续时间可进一步限制为每次给药后12、16和20小时。每种化合物的临界浓度可能不同。在本发明的各种实施例中，临界浓度为约100μM、约50μM、约20μM或约10μM。
用途
本发明提供了治疗增殖性病症或病症的方法，例如子宫癌、子宫颈癌、乳腺癌、前列腺癌、睾丸癌、阴茎癌、胃肠道癌例如食道癌、口咽癌、胃癌、小肠癌或大肠癌、结肠癌或直肠、肾脏、肾细胞、膀胱、骨骼、骨髓、皮肤、头或颈部、皮肤、肝脏、胆囊、心脏、肺、胰腺、唾液腺、肾上腺、甲状腺、脑，例如神经胶质瘤、神经节、中枢神经系统(CNS)和外周神经系统(PNS)以及免疫系统，例如脾脏或胸腺。本发明提供治疗例如免疫原性肿瘤、非免疫原性肿瘤、休眠肿瘤、病毒诱导的癌症，例如上皮细胞癌、内皮细胞癌、鳞状细胞癌、乳头瘤病毒、腺癌、淋巴瘤、癌、黑素瘤、白血病、骨髓瘤、肉瘤、畸胎癌、化学诱导的癌症、转移和血管生成。本发明还考虑降低对肿瘤细胞或癌细胞抗原的耐受性，例如通过调节T细胞浸润和活化的活性。
根据本发明的治疗患有肿瘤的人、哺乳动物或动物受试者、延缓其进展、预防其复发、减轻其症状或以其他方式改善其的方法可以包括施用治疗有效量的化合物、产品和/或药物组合物，从而产生抗肿瘤活性。例如，抗肿瘤活性可以是抗癌活性。例如，抗肿瘤活性可包括减慢肿瘤体积生长、停止肿瘤体积生长或减少肿瘤体积。肿瘤可包括实体瘤、恶性肿瘤、转移细胞、癌症干细胞。肿瘤可包括癌、肉瘤、腺癌、淋巴瘤或血液恶性肿瘤。肿瘤对化学疗法、放射疗法和/或激素疗法的治疗可能是难治的。可以施用化合物、产品和/或药物组合物以防止肿瘤复发。化合物、产品和/或药物组合物可以作为手术切除的辅助疗法给药。化合物、产品和/或药物组合物可以例如口服和/或静脉内给药。
根据本发明的方法还包括在患有该疾病或病症的人、哺乳动物或动物受试者中治疗疾病或病症、延迟其进展、预防其复发、减轻其症状或以其他方式改善该疾病或病症.在一些实施方案中，疾病或病症选自自身免疫疾病、炎性疾病、炎性肠病、关节炎、自身免疫性脱髓鞘疾病、阿尔茨海默病、中风、缺血再灌注损伤、多发性硬化和其他神经元退行性疾病。
将化合物、产品和/或药物组合物施用于患有疾病或病症的患者如果达到各种实验室或临床结果中的任何一个，则治疗被认为是成功的。例如，给药被认为是成功的，与疾病或病症相关的一种或多种症状被缓解、减轻、抑制或无进一步进展状态。如果病症，例如自身免疫病症，进入缓解期或无进一步进展状态，则认为给药是成功的。
在一些实施方案中，本文所述的化合物、产品和/或药物组合物与多种已知治疗剂中的任一种组合施用，包括例如化学治疗剂和其他抗肿瘤剂、抗炎化合物和/或免疫抑制化合物、细胞因子或细胞因子拮抗剂，例如IL-12、干扰素-α或抗表皮生长因子受体。在一些实施方案中，本文所述的化合物、产品和/或药物组合物可与多种已知治疗中的任一种结合使用，包括但不限于外科治疗和方法、放射疗法、化学疗法和/或激素或其他内分泌相关治疗。
这些“联合疗法”可以依次或同时施用。本文所述的化合物、产品和/或药物组合物和第二疗法可以在相同的药物组合物中施用于受试者，优选人类受试者。或者，本文所述的化合物、产品和/或药物组合物和第二疗法可以在分开的药物组合物中同时、分开或顺序施用于受试者。本文所述的化合物、产品和/或药物组合物以及第二疗法可以通过相同或不同的给药途径施用于受试者。在一些实施方案中，本发明的联合疗法包含有效量的本文所述的化合物、产品和/或药物组合物以及有效量的至少一种具有不同机制的其他疗法(例如，预防或治疗剂)比本文所述的化合物、产品和/或药物组合物的作用。在一些实施方案中，本发明的联合疗法通过共同发挥作用以具有相加或协同效应来改善本文所述的化合物、产品和/或药物组合物以及第二疗法的预防或治疗效果。在某些实施方案中，本发明的联合疗法减少了与第二疗法(例如，预防剂或治疗剂)相关的副作用。
治疗组合物、方法
治疗有效量剂量的本发明化合物和药物载体配制而成的药物组合物。“治疗有效剂量”是足以提供所需治疗结果的剂量。同时，在保证有效剂量时保证具有最小的副作用。本发明化合物组成的药物组合物的作用效果检测基于领域内已知的Arf1GTP酶活性、脂解报告基因、脂滴形成、自噬和Arf1活性的上游或下游活性检测实验。对于特定患者的治疗有效剂量需要考虑多种因素来确定，包括所治疗的病症、患者的整体健康、给药方法、副作用的严重程度等。在肿瘤背景下，本发明化合物的合适剂量将表现为：肿瘤部位的T细胞浸润和活化、炎性细胞因子例如IL-1β和IFNγ或T细胞相关趋化因子CCL5、CXCL-10、CXCL-11和CCL22，或T细胞活化标志物IFNγ、穿孔素、GzmA、GzmB，DAMPs水平上升或ER应激标志物水平升高或MHC-I/MHC-II或IFNγ的水平升高。
兽医、实验或研究对象包括猴、狗、猫、大鼠、小鼠、兔、豚鼠、马和人。
对于本文所述的治疗受试者的任何方法，本发明也提供了化合物的有效给药范围、给药频率和血浆浓度。在各种实施方案中，药物组合物以以下剂量施用：(a)约1mg/m 2至约5,000mg/m 2(i.v.)或约1mg/m 2至约50,000mg/m 2(p.o.)；(b)约2mg/m 2至约3,000mg/m 2(i.v.)或约10mg/m 2至约50,000mg/m 2(p.o.)。在各种实施方案中，本发明的化合物可以每隔一天(Q2D)、每天(QD)或每天两次(BID)给药。在一个实施方案中，药物组合物经口施用且一天不超过四次 (QID)。
在一些实施方案中，可以按照以下方案施用化合物、产品和/或药物组合物来治疗疾病或病症。在长于药物起效时间且短于有害时间的第一连续时间段内，化合物的血液摩尔浓度可维持不低于有效浓度且小于有害浓度。在第一个连续时间段之后，血液摩尔浓度可以低于有效浓度。例如，有效浓度可以是约0.1μM、约0.2μM、约0.5μM、约1μM、约2μM、约3μM、约4μM、约5μM、约6μM、约10μM或其他本领域技术人员确定为有效的浓度。例如，有害浓度可以是约1μM、约3μM、约10μM、约15μM、约30μM、约100μM，或本领域技术人员确定为有害的另一浓度。例如，有效时间段可以是约1小时、2小时、约4小时、约6小时、约8小时、约10小时、约12小时、约24小时，或被其他本领域技术人员确定为有效的另一时间段。例如，有害时间段可以是约12小时、约24小时、约48小时、约72小时、约144小时或本领域技术人员确定为有害的另一时间段。
在一些实施方案中，选择治疗的化合物、产品和/或药物组合物的有效量为大于肿瘤细胞的IC 50且小于正常细胞的IC 50的血液浓度。在一些实施方案中，选择治疗有效量以产生足够高以杀死肿瘤细胞且低于正常细胞的IC 50的血液浓度。
在一些实施方案中，化合物、产品和/或药物组合物的剂型包括但不限于片剂、丸剂、胶囊(硬或软)、囊片、粉剂、颗粒剂、悬浮液、溶液、凝胶剂口服给药、扁囊剂、锭剂、锭剂、糖浆、酏剂、乳剂、水包油乳剂、油包水乳剂和/或药水。
在根据本发明的一些实施方案中，用于减少或抑制肿瘤细胞复制或扩散的组合物包括通过以下方法选择的一组颗粒。根据公式I配制成化合物或其盐或溶剂化物均可以。
在另一方面，本发明提供了一种药物组合物，其包含本发明化合物和药学上可接受的赋形剂、载体或稀释剂。一个特征是，该组合物适用于口服、鼻腔、局部、直肠、阴道或肠胃外给药，或静脉内、皮下或肌肉内注射。
本发明的制剂包括但不限于适合于口服、鼻腔、局部(包括口腔和舌下)、直肠、阴道和/或肠胃外给药。制剂可以方便地以单位剂型存在并且可以通过药学领域中众所周知的任何方法来制备。可与载体材料组合以产生单一剂型的活性成分的量将根据所治疗的哺乳动物和特定给药方式而变化。可与载体材料组合以产生单一剂型的活性成分的量通常是产生治疗效果的量。通常，在100％中，该量将在例如约1％至约99％的活性成分、约5％至约70％、约10％至约30％的范围内。
适用于口服给药的本发明的治疗组合物或制剂可以是胶囊、扁囊剂、丸剂、片剂、锭剂(使用调味剂，通常是蔗糖和阿拉伯胶或黄蓍胶)、粉剂、颗粒剂的形式，或作为在水性或非水性液体中的溶液或悬浮液，或作为水包油或油包水液体乳液，或作为酏剂或糖浆，或作 为锭剂(使用惰性基质，例如明胶和甘油，或蔗糖和金合欢)和/或作为漱口水等，各自含有预定量的本发明化合物作为活性成分。本发明化合物也可以作为丸剂、药糖剂或糊剂给药。
在用于口服给药的本发明固体剂型(胶囊、片剂、丸剂、糖衣丸、粉剂、颗粒剂等)中，本发明的化合物与一种或多种药学上可接受的载体混合，例如柠檬酸钠或磷酸二钙，和/或以下任何一种：填充剂或增量剂，例如淀粉、乳糖、蔗糖、葡萄糖、甘露醇和/或硅酸；粘合剂，例如羧甲基纤维素、藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和/或阿拉伯胶；保湿剂，如甘油；崩解剂，例如琼脂、碳酸钙、马铃薯或木薯淀粉、海藻酸、某些硅酸盐、碳酸钠和羟基乙酸淀粉钠；溶解阻滞剂，如石蜡；吸收促进剂，例如季铵化合物；润湿剂，例如鲸蜡醇、单硬脂酸甘油酯和聚环氧乙烷-聚环氧丙烷共聚物；吸收剂，如高岭土和膨润土；润滑剂，例如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠及其混合物；和着色剂。在胶囊、片剂和丸剂的情况下，药物组合物还可包含缓冲剂。类似类型的固体组合物也可用作软和硬填充明胶胶囊的填充剂，使用赋形剂如乳糖或牛奶糖，以及高分子量聚乙二醇等。
用于本发明化合物的口服给药的液体剂型包括药学上可接受的乳剂、微乳剂、溶液剂、混悬剂、糖浆剂和酏剂。除活性成分外，液体剂型可含有本领域常用的惰性稀释剂，例如水或其他溶剂、增溶剂和乳化剂，例如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、油(特别是棉籽油、花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢呋喃醇、聚乙二醇和脂肪酸酯脱水山梨糖醇及其混合物。此外，环糊精，例如羟丙基-β-环糊精，可用于溶解化合物。
除了惰性稀释剂之外，口腔组合物还可以包括佐剂，例如润湿剂、乳化剂和悬浮剂、甜味剂、调味剂、着色剂、加香剂和防腐剂。除了一种或多种本发明的化合物之外，悬浮液还可包含悬浮剂，例如乙氧基化异硬脂醇、聚氧乙烯山梨糖醇和脱水山梨糖醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂和黄蓍胶，以及它们的混合物。
用于直肠或阴道给药的本发明药物组合物的制剂可以作为栓剂提供，其可以通过将一种或多种本发明化合物与一种或多种合适的非刺激性赋形剂或载体混合来制备，所述赋形剂或载体包括例如、可可脂、聚乙二醇、栓剂蜡或作为水杨酸盐，在室温下为固体，但在体温下为液体，因此会在直肠或阴道腔中熔化并释放本发明的活性药剂。适用于阴道给药的本发明制剂还包括阴道栓剂、棉塞、乳膏剂、凝胶剂、糊剂、泡沫剂或喷雾制剂，其含有本领域已知的合适的载体。
用于根据本发明的组合物的局部或经皮给药的剂型包括粉剂、喷雾剂、软膏剂、糊剂、霜剂、洗剂、凝胶剂、溶液剂、贴剂和吸入剂。活性化合物可以在无菌条件下与药学上可接受的载体以及可能需要的任何防腐剂、缓冲剂或推进剂混合。
除了本发明的化合物之外，软膏剂、糊剂、乳膏剂和凝胶剂可以含有赋形剂，例如动物和植物脂肪、油、蜡、石蜡、淀粉、黄蓍胶、纤维素衍生物、聚乙二醇、硅氧烷、膨润土、硅酸、滑石和氧化锌，或它们的混合物。
除本发明的化合物外，粉剂和喷雾剂还可含有赋形剂，例如乳糖、滑石、硅酸、氢氧化铝、硅酸钙和聚酰胺粉末，或这些物质的混合物。喷雾剂可另外包含常规推进剂，例如氯氟烃和挥发性未取代烃，例如丁烷和丙烷。
眼用制剂、眼膏、粉剂、溶液等也被认为在本发明的范围内。
适用于肠胃外给药的本发明药物组合物包含一种或多种本发明化合物与一种或多种药学上可接受的无菌等渗水溶液或非水溶液、分散液、混悬液或乳剂，或可重构的无菌粉末在使用前加入无菌可注射溶液或分散液中，其中可能含有抗氧化剂、缓冲剂、抑菌剂、使制剂与预期接受者的血液等渗的溶质或悬浮剂或增稠剂。
在一些情况下，为了延长根据本发明的组合物的作用，希望减缓其从皮下或肌内注射被身体吸收。这可以通过使用水溶性差的结晶或无定形材料的液体悬浮液来实现。药物的吸收速率取决于其溶解速率，而溶解速率又可能取决于晶体大小和晶型。或者，肠胃外给药的组合物的延迟吸收通过将化合物溶解或悬浮在油性载体中来实现。长效注射的一种策略包括使用聚环氧乙烷-聚环氧丙烷共聚物，其中载体在室温下为流体并在体温下固化。
在一个实施方案中，药学上可接受的赋形剂、载体或稀释剂包含用于静脉内递送的脂质。脂质可以是：磷脂、合成磷脂酰胆碱、天然磷脂酰胆碱、鞘磷脂、神经酰胺、磷脂酰乙醇胺、磷脂酰甘油、磷脂酸、胆固醇、胆固醇硫酸盐以及半抗原和PEG结合的脂质。脂质可以是纳米乳液、胶束、乳液、悬浮液、纳米悬浮液、脂质体或脂质体的形式。在一个实施例中，药学上可接受的赋形剂、载体或稀释剂为胶束乳液、悬浮液或纳米颗粒悬浮液的形式，并且其进一步包含静脉内可接受的蛋白质，例如人白蛋白或其衍生物，用于静脉内递送。
在一个实施方案中，药学上可接受的赋形剂、载体或稀释剂包括用于口服递送的蜡质材料。蜡质材料可以是单-、二-或甘油三酯、PEG的单-、二-脂肪酸酯、PEG缀合的维生素E(维生素E TPG)和/或Gelucire。Gelucire可选自Gelucire 44/14、Gelucire 43/01、Gelucire 50/02、Gelucire 50/13、Gelucire 37/02、Gelucire 33/01、Gelucire 46/07和Gelucire 35/10。在一个实施例中，药学上可接受的赋形剂、载体或稀释剂选自辛醇、transcutol hp、labrafil M、labrasol、三醋精、pharmasolv、乙醇、聚乙烯吡咯烷、羧甲基纤维素、吐温20和吐温80。在一个实施例中，药学上可接受的赋形剂，例如Gelucire 44/14，与表面活性剂混合，表面活性剂可以是吐温80或吐温20。药物组合物的这些实施方案可以进一步配制用于口服给药。
本发明的化合物可以使用市售的起始材料和器官领域技术人员公知的方法合成。在实施 例13-14中，本发明提供了一些要求保护的化合物的制造方法。
根据本发明的一个或多个实施例，小分子Arf1抑制剂是指显示出对Arf1的抑制活性的任何低分子量药物。与蛋白质、肽和碳水化合物等分子量较大的药物相比，小分子更容易穿透细胞膜和血脑屏障。这些分子的工艺开发和制造成本往往会较低。
附图说明
图1、Du101和Du102是Arf1激活的有效抑制剂。
图2、体外细胞系筛选显示Arf1抑制剂化合物102-107，Du102对结直肠癌细胞系HT29和LoVo细胞具有一定肿瘤抑制效果。
图3、体外细胞系筛选显示Arf1抑制剂化合物102-107，Du102对肝癌细胞Huh7具有一定肿瘤抑制效果。
图4、体外细胞系筛选显示Arf1抑制剂化合物102-107，Du102对肺癌细胞H460和A549具有一定肿瘤抑制效果。
图5、体外细胞系筛选显示Arf1抑制剂化合物102-107，Du102对肺癌细胞H460和A549具有一定肿瘤抑制效果。
图6、体外细胞系筛选显示Arf1抑制剂化合物103-104，107，109-110，Du102对结直肠癌细胞系HT29和LoVo细胞,肝癌细胞Huh7,肺癌细胞H460和A549具有一定肿瘤抑制效果。
图7、Arf1抑制剂在抑制肿瘤细胞作用上存在剂量依赖性。
图8、果蝇筛选显示Arf1抑制剂包括Du101和Du102具有良好体内肿瘤抑制效果。
图9、通过口服管饲法对小鼠给药Du101高达50mg/kg，连续给药21天，对小鼠的毒性作用。
图10、通过口服管饲法对小鼠给药Du101高达50mg/kg，连续给药60天，对小鼠的毒性作用。
图11、实施例16中Du101合成方案1中间体化合物的液相图谱，ADC1对应化合物2，DAD1对应化合物3，MSD1对应化合物4。
具体实施方式
参考以下实施例可以较好地理解本发明的应用，但本发明应用并不限制于以下特定实施方案。
实施例1.Du101和Du102是Arf1激活的有效抑制剂。
使用Thermo Scientific Pierce Active Arf1 Pull-Down and Detection Kit(Cat#16121)和肝癌Huh-7细胞裂解物检测Arf1活性。它是一个完整的试剂盒，通过与GGA3蛋白结合域的特异性蛋白相互作用，选择性富集和检测GTP结合的Arf1 GTPase，其结果见图1。
实施例2.体外细胞系筛选显示Arf1抑制剂包括Du101和Du102具有一定肿瘤抑制效果。
在体外不同肿瘤细胞毒性实验中(图2-图7)，包括结直肠癌细胞系HT29和LoVo细胞，肝癌细胞Huh7，肺癌细胞H460和A549，Arf1抑制剂均能抑制肿瘤细胞生长(图2-图6)，并且存在剂量依赖性(图7)。
实施例3.果蝇筛选显示Arf1抑制剂包括Du101和Du102具有良好体内肿瘤抑制效果。
在果蝇肠道中通过诱导促癌基因Rasv12-GFP的表达，形成果蝇肠道肿瘤，经喂饲Arf1抑制剂后，可以显著诱导果蝇肠道肿瘤细胞死亡(PI染色染色)(图8)
实施例4.小鼠体内绐药实验显示Arf1抑制剂Du101和Du102具有显著肿瘤抑制效果。
如图9-10所示，通过口服管饲法对小鼠给药Du101高达50mg/kg，连续给药21天(图9)和Du102连续给药60天(图10)的小鼠中未观察到毒性作用。这些数据表明化合物Du101和Du102可用于选择性抗癌活性。
实施例5.接种Arf1抑制的细胞制成的疫苗保护动物免于肿瘤。
可以在小鼠疫苗接种模型中测试试剂的免疫原性(Obeid et al.，2007；Sagiv-Barfi et al.，2018)。小鼠的一侧腹部注射了药物处理过的肿瘤细胞，然后一周后再次注射同系肿瘤细胞。我们测试了DMSO或Du102治疗的CT-26结肠癌、B16-F10黑色素瘤和4T1乳腺癌的疫苗效应。
我们首先在无胸腺裸鼠中测试了处理过的细胞，发现抑制Arf1抑制剂并没有显着影响免疫缺陷小鼠中CT26、B16-F10和4T1细胞的致瘤能力(表2、5、8)，表明Arf1敲低的抗肿瘤活性不是通过对肿瘤细胞的直接细胞毒作用来实现的。
Table 2:DU102 treatment had no significant effect on CT-26 colon carcinoma in Athymic mice
Days O 5 10 15 20 25
DMSO 0 0 21 170 480 1023
Dul02 0 0 15 117 417 859
Table 3:DU102 treatment reduces tumor size (mm 3) in CT-26 colon cancer model in BALB/c mice           Left side with drug treatment
Days 0 5 10 15 20 25 30
DMSO O 15 52 123 296 553 893
Du102 0 0 2 8 57 81 178
Table 4:Vaccination with DU102-treated CT-26 cells on left side protects BALB/c mice from developing tumors on right side
Days 0 5 10 15 20 25 30
DMSO 0 0 9.7 16.3 23.5 124.7 211.7
Du102 0 0 0 0.6 3.7 7.5 14.1
Table 5:DU102 treatment had no significant effect on B16-F10 melanoma in Athymic mice
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
Table 6:DU102 treatment reduces tumor size(mm 3)in B16-F10 melanoma model in C57B/6 mice        Left side with drug treatment
Davs 0 5 10 15 20 25 30
DMSO 0 15 51 132 297 553 893
DU102 0 0 2 8 57 81 177
Table 7:Vaccination with DU102-treated B16-F10 cells on left side protects C57B/6 mice from developing tumors on right side
Days 0 5 10 15 20 25 30
DMSO 0 0 0 3.5 33.5 197.4 405.5
DU102 0 0 0 0.6 1.3 9.1 21.3
Table 8:DU102 treatment had no significant effect on 4T1 breast carcinoma in Athymic mice
Days 0 5 10 15 20 25
DMSO 0 21.3 41.2 169.7 402.7 891.5
Du102 0 21.7 41.3 163.3 385.4 793.2
Table 9:DU102 treatment reduces tumor size(mm 3)in 4T1 breast carcinoma model in BALB/c mice        Left side with drug treatment
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
Table 10:Vaccination with DU102-treated 4T1 cells on left side protects BALB/c mice from developing tumors on right side
Davs 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
然后我们将DMSO或Du102处理的CT26细胞注射到BALB/c小鼠的左侧，然后在 一周后用未处理的CT-26细胞在右侧重新激发小鼠。注射了用DMSO处理的CT-26细胞的小鼠体内的肿瘤在左侧和右侧逐渐生长。Du102治疗导致左侧和右侧肿瘤几乎完全消退(表3和4)。Du102原位疫苗接种不仅对结肠癌有效，而且对多种组织学类型的肿瘤也有效，例如黑色素瘤(B16-F10，表6和7)和乳腺癌(4T1，表9和10)。这些结果表明抑制Arf1可以在局部触发T细胞免疫反应，然后攻击全身的癌症。
将用10μM Du102或DMSO处理的CT26、4T1和B16-F10肿瘤细胞(分别为5×10 5、1×10 4和5×10 4)皮下接种到6周大的雌性小鼠。7天后，将等量的相应肿瘤细胞接种到右侧。肿瘤细胞系与Matrigel(GIBCO，Cat#354234)混合或重新悬浮在PBS中并皮下或皮内注射。
实施例6.Du102处理在BALB/c小鼠的CT-26结肠癌模型中诱导炎性细胞因子。
对于IL-1β测定：注射CT26细胞裂解物7天后，从注射的BALB/c小鼠中分离CD11c+DCs并与经DMSO或Du102处理的CT26细胞共培养。2天后，在收获的细胞中测量IL-1β(表11)。
对于INFγ测定：在注射CT26细胞裂解物7天后，从注射的BALB/c小鼠中分离CD11c+DC和CD8+T细胞，并与经DMSO或Du102处理的CT26细胞共培养。2天后，在收获的细胞中测量INFγ(表11)。
Table 11:Du102 treatment induces inflammatory cytokines in CT-26colon cancer model in BALB/c mice
  IL-1β INFγ
DMSO 181 182
DU102 954 623
实施例7.Du102处理在C57B/6小鼠的B16-F10黑色素瘤中诱导IL-1β。(表12)
Table 12:Du102 treatment induces inflammatory cytokine in B16-F10 melanoma model in C57B/6mice
  IL-1β
DMSO 178
DU102 952
实施例8.Du101和Du102治疗减少了MYC-ON小鼠的肝脏肿瘤数量。
我们使用Tet系统生成了在肝细胞中有条件地表达MYC原癌基因的转基因小鼠Tet-on-MYC/LAP-tTA(LT2-MYC)(Shachaf et al.，2004)。我们将TRE-MYC小鼠与转基因系LAP-tTA杂交，其中肝激活蛋白(LAP)启动子驱动四环素反式激活蛋白(tTA)在肝细胞中的表达。我们通过停止喂饲强力霉素(MYC-ON)激活了3周龄小鼠的MYC转基因表达。随后，所有过表达MYC的转基因小鼠都死于肝脏肿瘤，肿瘤发作的平均潜伏期为12周， 如先前报道的(Shachaf et al.，2004)。在此LAP-tTA/tet-off MYC条件转基因小鼠模型中，MYC在成年小鼠中的过表达可重复诱导肝癌，类似于肝细胞癌和/或肝母细胞瘤。转基因肿瘤局部侵袭整个肝脏，经常与恶性腹腔积液相关，这些积液通过转移扩散到胸腔并侵入肺实质。
对于小鼠肝脏肿瘤模型，交配笼和断奶笼通过含200mg/kg多西环素的基于谷物的啮齿动物饮食(Bio-Serv，Cat#14-727-450)给药，小鼠被诱导肿瘤在6周大时，改为正常饮食。
将小鼠分为三组，对照组、Du101治疗组和Du102治疗组。对照组使用饲管通过管饲法给予100μl/10g体重(BW)20％DMSO+80％玉米油。Du101处理组用25mg/ml的Du101原液用玉米油按1∶4的比例稀释制成工作液。Du102处理组用25mg/m1的Du102原液用玉米油按1∶4的比例稀释制成工作液。使用饲管通过管饲法以100μ1/10gBW将混合工作溶液给予小鼠。从MYC-ON的第二周开始，每周注射5次(从周一和周五开始)持续4周，每2天监测一次小鼠，10周后对小鼠实施安乐死并分析肿瘤生长。表13表明Du101和Du102可以减少小鼠中过表达的Myc诱导的肝脏肿瘤。
Table 13:DU101 and Du102 treatment reduces liver tumor numbers in MYC-ON mice
DMSO 105.3
DU101 44.7
DU102 26.7
实施例9.Du102处理诱导MYC-ON小鼠中趋化因子和T细胞活化标志物的表达。
在DMSO或Du102处理的MYC-ON小鼠中通过定量RT-PCR测量趋化因子和T细胞活化标志物。通过定量RT-PCR测量，CD8T细胞浸润和免疫刺激性细胞因子表达增加。与用DMSO对照处理的小鼠相比，Du102处理显着增加了MYC-ON中许多T细胞相关趋化因子CCL5、CXCL-10、CXCL-11和CCL22的表达(表14)。CCL5和CXCL10趋化因子刺激CD4+和CD8+淋巴细胞肿瘤浸润(Parkes et al.，2017)，CXCL10和CXCL11是T细胞相关的趋化因子。此外，实时PCR显示与用DMSO对照处理的小鼠相比，用Du102处理的小鼠中T细胞活化标志物IFNγ、穿孔素、GzmA、GzmB和IL-1β的表达升高(表14)。我们的实时PCR还显示，与在MYC-ON小鼠中用DMSO对照处理的小鼠相比，在用Du102处理的小鼠中免疫检查点抑制剂PD-L1表达显着降低(表14)。总的来说，这些数据表明Du102治疗触发了T细胞浸润和激活，导致肝肿瘤细胞死亡并延长了MYC-ON小鼠的存活时间。
Table 14:DU102 treatment induces chemokines and T-cell activation in MYC-ON mice
  Ccl5 Cxcl10 Cxcl11 Ccl22 GzmA GzmB Perforin IL-1β INFγ PD-L1
DMSO 1.0 1.1 1.0 1.0 1.0 1.0 1.0 1.0 1.1 1.0
DU102 2.4 3.1 2.6 2.5 2.3 1.9 2.2 2.5 3.2 0.4
实施例10.Du102处理诱导BALB/c小鼠中4T1乳腺癌的T细胞活化标志物的表达。
Table 15:DU102 treatment induces T-cell activation of 4T1 breast carcinoma in BALB/c mice
  GzmA GzmB Perforin IL-1β INFγ PD-L1
DMSO 1.0 1.0 1.0 1.0 1.0 1.0
DU102 2.2 2.3 2.5 2.2 2.1 0.5
实施例11.Du102处理在MYC-ON小鼠中诱导MHC表达和T细胞浸润。
Du102处理增加MHC表达、CD4和CD8 T细胞浸润，如通过MYC-ON小鼠中的定量RT-PCR测量的(表16)。
Table 16:DU102 treatment induces MHC expression and T-cell infiltration in MYC-ON mice
  MHC-I MHC-II CD4 CD8
DMSO 0.6 0.7 1.0 2.0
DU102 51.7 43.6 13.2 13.5
MHC-I、MHC-II、浸润性CD4+和CD8+T-细胞的数量从通过免疫组织化学染色的MHC-I、MHC-II、CD4和CD8标记物的几种肿瘤的代表性切片计数。
实施例12.Du102治疗减少C57B/6小鼠中B16-F10黑色素瘤的肺转移。
50K B16-F10肿瘤细胞(通过DMSO或10μM Du102处理)通过尾静脉注射被静脉内转移。注射后15天取出肺并在Fekete溶液中固定过夜。三名研究人员以不知情的方式统计可见的转移灶(表17)。
Table 17:Du102 treatment reduces lung metastases of B16-F10 melanoma in C57B/6 mice(surface tumor number)
DMSO 39
DU102 5
实施例13.在MYC-ON小鼠中Du102的抗肿瘤作用被CD4或CD8抗体中和。
Du102的抗肿瘤作用在MYC-ON小鼠中被CD4或CD8抗体中和(表18)。使用FACS和IHC分析验证细胞消耗。
Table 18:The anti-tumor effects of DU102 are neutralized by anti-CD4 or/anti-CD8 antibodies in MYC-ON mice
Figure PCTCN2021110373-appb-000007
对于MYC-ON肝肿瘤模型，抗体是腹腔注射的。每周注射一次，连续注射5周，第一次注射后多西环素周停药。用于治疗小鼠的抗体如下：100μg/小鼠大鼠抗CD4(克隆GK1.5，BioXcell，Cat#BE0003-1)，100μg/小鼠大鼠抗CD8d(克隆2.43，BioXcell，Cat#BE0061)或100μg/小鼠大鼠IgG(BioXcell，Cat#BE0094)同种型对照抗体。从MYC-ON的第二周起持续4周，如上所述通过管饲针施用Du102和DMSO，每2天监测一次小鼠，10周后对小鼠实施安乐死并分析肿瘤生长。
实施例14.Du102与抗PD-1抗体在MYC-ON小鼠中的协同抗肿瘤作用。
Du102与抗PD-1组合施用。我们在MYC-On小鼠中测试了Arf1抑制和PD-1阻断，发现PD-1阻断可以进一步减少Du102治疗的MYC-ON小鼠的肿瘤数量(表19)。
Table 19:Synergistic anti-tumor effects of DU102 with anti-PD-1 antibodies in MYC-ON mice
Figure PCTCN2021110373-appb-000008
对于MYC-ON肝肿瘤模型，抗体是腹腔注射的。每周注射一次，连续注射5周，第一次注射后多西环素周停药。用于治疗小鼠的抗体如下：使用100μg/小鼠亚美尼亚仓鼠抗小鼠PD1(J43，BioXcell，Cat#BE0033-2)和100μg/小鼠亚美尼亚仓鼠同种型IgG(BioXcell，Cat#BP0091)作为控制。从MYC-ON的第二周起持续4周，如上所述通过管饲针施用Du102和DMSO，每2天监测一次小鼠，10周后对小鼠实施安乐死并分析肿瘤生长。
抗PD-1抗体的共同给药可允许使用更低、毒性更小的剂量的Du102，从而避免已知的副作用。
实施例14.Du102、Du101在乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤中的治疗作用。
通过将人源乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤肿瘤组织或者细胞系接种于人源化的小鼠皮下构建人源化肿瘤模型。通过灌胃的方式给予DMSO或Du102、Du101处理。通过定量RT-PCR测量，检测免疫刺激性细胞因子表达。通过FACS 检测小鼠中T细胞活化标志物IFNγ、穿孔素、GzmA、GzmB和IL-1β的表达。并通过测量肿瘤生长、收集小鼠存活数据，检测Du102-、Du101-处理对乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤的治疗效果。
或者通过将鼠源乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤肿瘤组织或者细胞系接种于野生型小鼠皮下构建肿瘤模型。通过灌胃的方式给予DMSO-或Du102-、Du101-处理。通过定量RT-PCR测量，检测免疫刺激性细胞因子表达。通过FACS检测小鼠中T细胞活化标志物IFNγ、穿孔素、GzmA、GzmB和IL-1β的表达。并通过测量肿瘤生长、收集小鼠存活数据，检测Du102-、Du101-处理对乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤的治疗效果。
或者通过构建条件敲除或敲入基因导致的原发性乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤小鼠模型。通过灌胃的方式给予DMSO或Du102、Du101处理。通过定量RT-PCR测量，检测免疫刺激性细胞因子表达。通过FACS检测小鼠中T细胞活化标志物IFNγ、穿孔素、GzmA、GzmB和IL-1β的表达。并通过测量肿瘤生长、收集小鼠存活数据，检测Du102-、Du101-处理对乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤的治疗效果。
实施例15.Du102、Du101在自身免疫疾病、炎症性疾病、炎症性肠病、关节炎、自身免疫脱髓鞘疾病中的治疗作用。
在自身免疫疾病、炎症性疾病、炎症性肠病、关节炎、自身免疫脱髓鞘小鼠疾病模型中，通过灌胃或颅内注射的方式给予DMSO或Du102、Du101处理。通过定量RT-PCR测量，检测免疫刺激性细胞因子表达。通过FACS检测小鼠中T细胞活化标志物IFNγ、穿孔素、GzmA、GzmB和IL-1β的表达。并通过切片，检测小鼠发病情况。
实施例16
化合物1H-吲哚-5-甲醛6,7,8,9-四氢-5H-环庚基[4,5]噻吩并[2,3-d]嘧啶-4-基腙的制备方案(方案1)。
Figure PCTCN2021110373-appb-000009
Du101的合成
下面提到的化合物显示在方案1的示意图中。将化合物溶解在DMSO中并储存在-20℃。
化合物2
向环己酮(10mmol)的乙醇(10mL)溶液中分别加入硫(320mg,10mmol)、氰基乙酸乙酯(1.07mL,10mmol)和吗啉(875μL,40mmol)。将反应混合物在60℃下搅拌5小时。使用二氯甲烷通过色谱法纯化后获得八百毫克化合物2。(注：起始原料可按比例增加，得到更多的化合物2)。
化合物3
将化合物2在甲酰胺中于150℃加热5小时。冷却过夜后，产物结晶为淡褐色晶体。收集所得晶体并用冷乙醇/水(体积比为：1/1)的混合溶剂洗涤，以定量收率得到相应的噻吩并嘧啶酮环(化合物3)。
化合物4
在加入2倍当量的POCl 3之前，将650毫克化合物3溶解在热的DMF(二甲基甲酰胺)中并用冰冷却。搅拌过夜后，产物沉淀出来。收集白色粉末并用冷水洗涤。将冷水进一步加入母液中得到额外的沉淀物，其直接用于下一步反应。
向氯化物溶于甲醇的溶液中加入10倍当量的一水合肼。将混合物搅拌2小时并加入水。滤出所得沉淀并用冷水洗涤，得到产物4。
Du101
向1倍当量化合物4的甲醇溶液中加入1.2倍当量5-甲醛-吲哚。将混合物搅拌2小时，收集所得沉淀并从甲醇中重结晶，得到Du101。
1H NMR(300MHz,DMSO-d6)δ:1.79(m,4H,2CH 2),2.74(m,2H,CH 2),3.00(m,2H,CH 2), 6.49(m,1H,CH),7.39(m,1H,CH),7.42(d,J＝8.6Hz,1H,CH),7.76(s,1H,CH),7.84(d,J＝8.6Hz,1H,CH),8.02(s,1H,CH),8.45(s,1H,CH),11.29(brs,1H,NH),11.73(brs,1H,NH). 13C NMR(75MHz,DMSO-d6)δ:22.0(CH 2),22.4(CH 2),24.6(CH 2),26.5(CH 2),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 19H 17N 5S)C,H,N,S
Figure PCTCN2021110373-appb-000010
Data FileD:\DATA\MICRA\1186530.D
Sample Name:118653005
Instrument 1 04/10/2017 14:20:33 N6
Column:Onyx Monolithic C18 50x4.6mm|3.75ml/min|Columns Reg Valve
Gradient:"A"->@2.2min->"B"(Hold0.4min)->@0.2min->"A"->PostRun
PMP1,Solvent A      :0.1％TFA in Acn/H2O(2.5:97.5)
PMP1,Solvent B      :0.1％TFA in AcN
PMP1,Solvent C      :0.1％FA in Acn/H2O(2.5:97.5)
PMP1,Solvent D      :0.1％FA in AcN
Ionization mode      :APCI Positive
Signal 1:ADC1 B,ELSD
Figure PCTCN2021110373-appb-000011
Totals:             28.29734 14.96186
Signal 2:DAD1 A,Sig＝300,200Ref＝off
Figure PCTCN2021110373-appb-000012
Totals:            1616.86841 882.75543
Signal 3:MSD1 TIC,MS File
Figure PCTCN2021110373-appb-000013
Totals:                 1.33299e6 4.31713e5
实施例17
化合物1H-吲哚-5-甲醛-5,6,7,8,9,10-六氢环辛基[4,5]噻吩[2,3-d]嘧啶-4-基腙的制备方案(方案2)
Figure PCTCN2021110373-appb-000014
Du102的合成
下面提到的化合物显示在方案2中的示意图中。化合物溶解在二甲基亚砜中并储存在室温下或-20℃。
化合物1
向环辛酮(10mmol)在乙醇(10mL)中的溶液中分别添加硫(320mg，10mmol)、氰基乙酸乙酯(1.07mL，10mmol)和吗啉(875μL、40mmol)。反应混合物在60℃搅拌℃持续5小时。经洗脱剂为二氯甲烷的柱层析纯化，得到855mg的化合物1，收率为34％(注：原料可按比例增加，得到更多化合物1)。
化合物2
将化合物1溶解在甲酰胺中于150℃加热5小时。冷却过夜后，产物结晶为淡褐色晶体。收集所得晶体并用冷乙醇/水(体积比1/1)的混合溶剂洗涤，以定量收率得到相应的噻吩并嘧啶酮环(化合物2)。 1H NMR(400MHz,DMSO-d6)δ:1.27(m,2H,CH 2),1.42(m,2H,CH 2),1.62(m,4H,2CH 2),2.87(m,2H,CH 2),3.06(m,2H,CH 2),8.01(s,1H,CH),12.28(brs,1H,NH)。 13C NMR(100MHz,DMSO-d6)δ:24.4(CH 2),25.3(CH2),25.4(CH 2),26.0(CH 2),29.9(CH 2),31.5(CH 2),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 +)。
化合物3
在加入2倍当量的POCl 3之前，将650毫克化合物2溶解在热的DMF(二甲基甲酰胺)中并用冰冷却。搅拌过夜后，产物沉淀出来。收集白色粉末并用冷水洗涤。将冷水进一步加入母液中得到额外的沉淀物，其直接用于下一步反应。 1H NMR(400MHz,CDCl 3)δ:1.25(m,2H,CH 2),1.46(m,2H,CH 2),1.70(m,4H,2CH 2),2.92(m,2H,CH 2),3.12(m,2H,CH 2),8.67(s,1H,CH)。13C NMR(100MHz,CDCl 3)δ:25.0(CH 2),25.4(CH 2),26.3(CH 2),28.2(CH 2),30.3(CH 2),31.6(CH 2),128.6(C),129.6(C)142.7(C)、151.3(CH)、156.4(C)、158.7(C)。ES-MS(电喷雾质谱)m/z 252.1(MH +， 35Cl)，254.1(MH +， 37Cl)。
化合物4
向溶解在甲醇中的氯化物(化合物3)溶液中加入10倍当量的一水合肼。将混合物搅拌2小时后并加入水。滤出所得沉淀并用冷水洗涤。 1H NMR(400MHz,CDCl 3)δ:1.27(m,2H,CH 2),1.44(m,2H,CH 2),1.65(m,4H,2CH 2),2.50(brs,2H,NH 2),2.83(m,4H,2CH 2),6.54(brs,1H,NH),8.41(s,1H,CH)。 13C NMR(100MHz,CDCl 3)δ:25.3(CH 2),26.0(CH 2),26.1(CH 2),27.7(CH 2),30.1(CH2),31.6(CH 2),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 +)。
Du102
向1当量化合物4的甲醇溶液中加入1.2当量5-甲醛-吲哚。将混合物搅拌2小时，收集所得沉淀物并从甲醇中重结晶，得到Du1O2。 1H NMR(400MHz,DMSO-d6)δ:1.25(m,2H,CH 2),1.44(m,2H,CH 2),1.60(m,2H,CH 2),1.69(m,2H,CH 2),2.83(brt,2H,J＝5.5Hz,CH 2),3.20(brt,2H,J＝6.0Hz,CH 2),6.49(m,1H,CH),7.39(m,1H,CH),7.44(d,1H,J＝8.6Hz,CH),7.80(brs,1H,CH),7.85(brd,1H,J＝8.6Hz,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 +)。
本文中的所有引文都以引用方式并入本文，其程度类似于每个单独的出版物或专利申请被具体地和单独地指示为以引用方式并入一样。
本领域技术人员可以在不脱离其精神和范围的情况下对本发明进行修改和变化。在此描述的具体实施例仅作为示例提供，并不意味着以任何方式进行限制。说明书和实施例仅被认为是示例性的，本发明的真正范围和精神由以上权利要求指示。

Claims (31)

  1. 一种杀死癌症干细胞或癌细胞并诱导抗肿瘤免疫反应的方法，该方法包括通过靶向Arf1途径的抑制剂抑制癌症干细胞中至少一些Arf1，特别是COPI/Arf1-脂解-β-氧化通路活性；
    其中靶向Arf1途径的抑制剂是通式(I)所示化合物：
    Figure PCTCN2021110373-appb-100001
    通式(I)或其药学上可接受的盐、溶剂化物或其前药、立体异构体，
    其中r＝1-6；
    R1为选自一个或多个取代基或未取代苯基、
    Figure PCTCN2021110373-appb-100002
    Figure PCTCN2021110373-appb-100003
    卤素、C1-6烷基、卤代烷基、OH、OCH3、O(CH2)nCH3、环丙基氧基、OC(CH3)3、OCH(CH3)2、NH2、NO2、N(CH3)2、NH(CH2)nCH3、CN、N3等，其中n为1-9。
  2. 根据权利要求1所述的方法，其中靶向Arf1途径的抑制剂为如下化合物：
    Figure PCTCN2021110373-appb-100004
  3. 一种抑制细胞内Arf1通路活性的方法，向细胞提供有效量的权利要求1-2任一项中所述化合物或药学上可接受的盐、溶剂化物或其前药、立体异构体，从而使细胞内至少Arf1通 路活性降低。
  4. 根据权利要求3所述的方法，所述细胞为前体细胞或干细胞或癌症干细胞。
  5. 根据权利要求3所述的方法，其中所述细胞是癌细胞。
  6. 根据权利要求3所述的方法，其中在所述细胞中诱导细胞死亡。
  7. 根据权利要求3所述的方法，其中该方法在体外进行。
  8. 根据权利要求3所述的方法，其中该方法在体内进行。
  9. 一种治疗或预防受试者与Arf1途径活性相关疾病的方法，该方法包括向受试者使用治疗有效的药物剂量，所述药物为权利要求1-2任一项中所述的化合物或药学上可接受的盐、溶剂化物或其前药、立体异构体，从而至少降低Arf1途径的活性。
  10. 根据权利要求9所述的治疗或预防受试者与Arf1途径活性相关疾病的方法，其中异常的Arf1途径活性可以通过测定Arf1 GTPase活性、脂解报告基因、脂滴形成、自噬、Arf1活性或功能的上游或下游调节因子来鉴定。
  11. 权利要求1-2任一项中所述的化合物或药学上可接受的盐、溶剂化物或其前药、立体异构体用于治疗患有癌症或肿瘤的受试者的方法。
  12. 根据要求11所述的方法，其中疾病为自身免疫疾病、炎症性疾病、炎症性肠病、关节炎、自身免疫脱髓鞘疾病、阿尔茨海默病、肌萎缩性脊髓侧索硬化症(ALS)、中风、缺血再灌注损伤、多发性硬化、其他神经元退行性疾病等。
  13. 根据权利要求11所述的方法，其中所述化合物能抑制癌症或肿瘤的生长。
  14. 根据权利要求11所述的方法，其中所述化合物能减小肿瘤或癌症的大小。
  15. 根据权利要求11-14任一项所述的方法，其中所述化合物能增加MHC-I和MHC-II的表达，增加了T细胞在肿瘤中的浸润和活化，增加T细胞活化标志物的表达，如GzmA、GzmB和穿孔素。
  16. 根据权利要求11-14任一项所述的方法，其中所述化合物增加至少一种炎性细胞因子或趋化因子的表达，所述炎性细胞因子或趋化因子选自IFNγ、IL-1β、Ccl5、Cxc10、Cxc11、Ccl22。
  17. 根据权利要求11-16所述的方法，其中所述化合物与至少一种抗PD-1抗体共同使用，所述化合物和PD-1阻断具有协同效应。
  18. 根据权利要求11-17任一项所述的方法，所述化合物不仅能杀死肿瘤干细胞，还能引起肿瘤特异性免疫反应。
  19. 根据权利要求11-18任一项所述的方法，其中所述化合物能减少癌症或肿瘤的转移。
  20. 根据权利要求11-19任一项所述的方法，其中所述癌症是乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤等。
  21. 根据权利要求11-20任一项所述的方法，其中所述受试者是哺乳动物。
  22. 权利要求1-2任一项中所述的化合物或药学上可接受的盐、溶剂化物或其前药、立体异构体在制备用于治疗患有癌症或肿瘤的受试者的药物中的用途。
  23. 根据权利要求22所述的用途，其中所述癌症是乳腺癌、头颈部癌症、肺癌、卵巢癌、胰腺癌、结直肠癌、前列腺癌、肾细胞癌、黑色素瘤、肝细胞癌、宫颈癌、肉瘤、脑瘤、胃癌、多发性骨髓瘤、白血病、淋巴瘤等。
  24. 根据权利要求22-23任一项所述的用途，其中所述化合物与至少一种抗PD-1抗体共同使用，该化合物和PD-1阻断具有协同效应。
  25. 根据权利要求22-24任一项所述的用途，所述化合物不仅能杀死肿瘤干细胞，还能引起肿瘤特异性免疫反应。
  26. 根据权利要求22-25任一项所述的用途，其中所述化合物能减少癌症或肿瘤的转移。
  27. 一种试剂盒，包含至少一种用于诊断与Arf1通路活性相关的疾病的试剂，权利要求1-2任一项中所述的化合物或药学上可接受的盐、溶剂化物或其前药、立体异构体。
  28. 根据权利要求27所述的试剂盒，其中至少有一种试剂测试至少一种生物标志物，表明疾病的存在。
  29. 根据权利要求27-28任一项所述的试剂盒，其中至少有一种试剂用于检测Arf1 GTPase活性、 脂解报告物、脂滴形成、自噬、Arf1活性或功能的上游或下游调节因子。
  30. 一种制备权利要求2中所述化合物107的方法，包括：
    Figure PCTCN2021110373-appb-100005
  31. 一种制备权利要求2中所述化合物111的方法，包括：
    Figure PCTCN2021110373-appb-100006
PCT/CN2021/110373 2020-08-04 2021-08-03 用于根除癌症干细胞并诱导damp介导的抗肿瘤免疫反应的一组新型copi/arf1-脂解途径抑制剂和化合物 WO2022028429A1 (zh)

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