WO2021109796A1 - 一种基于5-羧基-8-羟基喹啉的抗肿瘤药物增敏剂及其应用 - Google Patents

一种基于5-羧基-8-羟基喹啉的抗肿瘤药物增敏剂及其应用 Download PDF

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WO2021109796A1
WO2021109796A1 PCT/CN2020/127036 CN2020127036W WO2021109796A1 WO 2021109796 A1 WO2021109796 A1 WO 2021109796A1 CN 2020127036 W CN2020127036 W CN 2020127036W WO 2021109796 A1 WO2021109796 A1 WO 2021109796A1
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tumor
tumor drug
cells
iox1
drug sensitizer
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French (fr)
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申有青
赵志浩
刘婧
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浙江大学
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the invention relates to the technical field of medicine, in particular to the application of 5-carboxy-8-hydroxyquinoline and its derivatives in the preparation of anti-tumor drug sensitizers.
  • Tumor immunotherapy is a method of treating tumors by breaking the tumor's immunosuppressive microenvironment, restarting and stimulating the immune circulation within the tumor.
  • the tumor’s immunosuppressive microenvironment is the tumor’s ability to block the body’s immune system from attacking cancer cells.
  • One of the factors that induce tumor immunosuppressive microenvironment is the hypoxia mechanism of tumors.
  • the metabolism of cancer cells in the tumor is more vigorous than that of normal cells.
  • the malformation and uneven distribution of blood vessels in the tumor leads to poor oxygen delivery, which ultimately results in a large number of hypoxic areas in the tumor.
  • hypoxic microenvironment activates multiple signal pathways of tumor cells, including Hypoxia inducible factor-1 (Hif-1).
  • Hif-1 is composed of two subunits, Hif-1 ⁇ and Hif-1 ⁇ .
  • Hif-1 ⁇ can participate in the transcriptional regulation of a variety of target genes, affect the energy metabolism, proliferation and apoptosis of tumor cells, and cause cells and tissues to produce a series of responses to adapt to the hypoxic environment (HIF Inhibitors: Status of Current Clinical Development; Current Oncology Reports, 2019, 21(1): 6).
  • hypoxia-induced Hif-1 ⁇ can directly bind to the transcriptionally active hypoxia response element (HRE) on the proximal promoter of programmed death receptor-ligand 1 (PD-L1) to initiate PD-L1 Transcription, up-regulate the expression of PD-L1 in tumor cells, and PD-L1 binds to the programmed death receptor 1 (PD-1) on T cells, which induces T cell apoptosis and inhibits T cell activation and proliferation.
  • HRE hypoxia response element
  • PD-L1 is a novel direct target of HIF-1 ⁇ , and its blockade under hypoxia enhanced MDSC-mediated T cell activation, Journal Of Experimental Medicine , 2014, 5(211): 781; Regulation of PD-L1: a novel role of pro-survival signaling in cancer, Annals of oncology, Annals Of Oncology, 2015, 3(27): 409).
  • Hif-1 ⁇ in tumor cells can cause tumor-associated macrophages to secrete a large amount of IL-10, promote the polarization of macrophages to immunosuppressive M2 type, and strengthen the tumor’s immune microenvironment, thereby affecting the effect of immunotherapy (Reactive oxygen).
  • species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1, Proceedings Of The National Academy Of Sciences Of The United States Of America, 2019, 10(116): 4326; HIF-1 alpha cell-mediated isinessential for , Cell, 2003, 5(112):645).
  • many tumor treatments can also promote tumor cells to up-regulate the expression of PD-L1.
  • low-dose chemotherapy can induce tumor cells or myeloid cells to up-regulate the expression of PD-L1 (Acquired Resistance to Fractionated Radiotherapy Can Be Overcome by Concurrent PD-L1 Blockade, Cancer Research, 2014, 74(19): 5458; The hallmarks of successful anticancer immunotherapy, Science Translational Medicine, 2018, 19(10): 459), thereby reducing the immune response of tumors.
  • hypoxic microenvironment is also one of the important reasons for solid tumors to develop multidrug resistance.
  • the hypoxic microenvironment in the tumor causes the overexpression of Hif-1 and activates the transcription of downstream target genes, including the up-regulation of drug-resistant P-glycoprotein (P-gp) expression, which reduces the cytotoxicity of chemotherapy drugs to kill tumor cells.
  • P-gp drug-resistant P-glycoprotein
  • MDR Multi-drugs Resistance
  • PD-1/PD-L1 immunosuppressants to block the binding of tumor cells and T cells, so that T cells can function normally in the human body, stimulate immune function to identify and eliminate tumor cells, which is currently based on PD-1/ A general protocol for tumor immunotherapy with PD-L1 antibody.
  • the immunotherapy method has a high curative effect on tumors with suitable targets.
  • some chemotherapeutic drugs can activate the immunogenic death of tumor cells after poisoning them, induce the expression of calreticulin, and release the "eat me” signal, thereby activating T cells and dendritic cells, and improving the anti-tumor immune response.
  • Chemotherapy drugs can also induce the production of neoantigens in tumor cells, increase mutations, such as up-regulating the expression of PD-L1, reconstruct the immune microenvironment, and transform from a "cold tumor” with less immune cell infiltration to a "hot tumor” with a stronger immune response. ", to enhance the sensitivity of immune checkpoint inhibitors and improve the therapeutic effect.
  • chemotherapeutic drugs combined with PD-1/PD-L1 immunosuppressive agents can improve the anti-tumor effect (In situ formed reactive oxygen species-responsive scaffold with gemcitabine and checkpoint inhibitor for combination therapy, science translational medicine, 2019, 10( 429): eaan3682; Elimination of established tumors with nanodisc-based combination chemoimmunotherapy, Science Advances, 2018, 4(4): eaao1736).
  • the PD-1/PD-L1 antibody protein has a large risk of side effects, inconvenient preparation and storage of the antibody, and high price.
  • 5-Carboxy-8-hydroxyquinoline is a potent broad-spectrum inhibitor of 2-oxoglutarate oxygenases, including Jumonji C (JmjC) domain demethylation Enzyme.
  • IOX1 increases the level of H3K9me3 in HeLa cells by inhibiting KDM4A, but has no significant effect on cell viability. Due to the low cell permeability, IOX1 shows lower potency in HeLa cells, and its n-octyl ester derivative improves its cell permeability (A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.ChemMedChem.2014 Mar; 9(3): 566-71).
  • IOX1 is therefore an inhibitor of HIF hydrolase prolyl hydroxylase (PHD) and stabilizes HIF, increases HIF levels in the ischemic parts of the heart and kidney, thereby protecting ischemia
  • PLD HIF hydrolase prolyl hydroxylase
  • the site is applied to, for example, renal ischemia therapy (Inhibition of Hypoxia Inducible Factor Hydroxylases Protects Against Renal Ischemia-Reperfusion Injury, Journal of the American Society of Nephrology, 2008, 19(1) 39-46).
  • the invention provides a small molecule antitumor drug sensitizer based on 5-carboxy-8-hydroxyquinoline, which is used to improve the therapeutic effect of immunity and chemotherapy.
  • An anti-tumor drug sensitizer based on 5-carboxy-8-hydroxyquinoline said anti-tumor drug sensitizer is a compound of formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 is H, OH, NH 2 , C 1-3 alkyl, -OC 1-3 alkyl, -OC 6-12 aryl;
  • the anti-tumor drug sensitizer of the present invention down-regulates Hif-1 ⁇ in tumor cells, thereby down-regulating the expression of tumor cell programmed death receptor-ligand 1, and blocks PD-L1/ between tumor cells and T cells.
  • the PD-1 response prevents the immune escape of tumor cells and the decline of the body's immunity, so that T cells can recognize and kill tumor cells and improve the anti-tumor effect.
  • the anti-tumor drug sensitizer of the present invention also inhibits the secretion of IL-10 by macrophages by down-regulating Hif-1 ⁇ , promotes the transformation of macrophages from immunosuppressive M2 to immunologically active M1, reverses the immunosuppressive state, and enhances The body's immune response to tumors improves the effect of tumor immunotherapy.
  • the anti-tumor drug sensitizer of the present invention stimulates the immunogenic death of tumor cells, transforms "cold” tumors that are not sensitive to immunotherapy into "hot” tumors that are sensitive to immunotherapy, and recruits T cells to kill them. Tumor cells.
  • the anti-tumor drug sensitizer of the present invention also reduces the expression of tumor cell P glycoprotein, increases tumor cell uptake of chemotherapeutic drugs, reduces tumor cell drug resistance, improves tumor cell sensitivity to chemotherapeutic drugs, and enhances chemotherapy The cytotoxicity and killing effect of drugs on tumor cells.
  • the anti-tumor drug sensitizer can down-regulate the expression of indoleamine-2,3-dioxygenase (IDO) in tumors.
  • IDO is also an enzyme with Fe 2+ as the center.
  • This anti-tumor drug sensitizer can complex Fe 2+ to achieve a certain inhibitory effect on IDO, reduce IDO, and transform tryptophan with immune-promoting function into immunosuppressive Functional kynurenine activates T cells from an immunosuppressed state.
  • anti-tumor drug sensitizer is a compound of the following formula (1), (2), (3) or (IOX1):
  • IOX1 can easily bind to the PHD3 protein, so it has a high sensitization effect; when the hydroxyl group of IOX1 is substituted to form compound 2, it needs to be converted into IOX1 by hydrolysis to exert the sensitization effect, so the sensitization effect is slightly weaker than that of IOX1 ; When the carboxyl group of IOX1 is substituted to form compound 3, it is more difficult to be hydrolyzed to IOX1, so the sensitization effect is reduced. Therefore, the derivative of compound 1-3 that can be easily converted to IOX1 has a strong sensitizing effect.
  • anti-tumor drugs are clinical anti-tumor drugs, including adriamycin, paclitaxel, gemcitabine, platinum drugs, camptothecin and its derivatives, triptorubin or gambogic acid.
  • the anti-tumor drug sensitizer of the present invention can be used in combination with different ratios of anti-tumor drugs, and the ratio of the amount of the anti-tumor drug sensitizer to the anti-tumor drug is 0.1-20:1.
  • Anti-tumor drug sensitizers enhance the body's immune response and enhance the efficacy of anti-tumor drugs.
  • the anti-tumor drug and the anti-tumor drug sensitizer are combined to obtain a significantly enhanced anti-tumor effect.
  • the tumor of the present invention is a malignant tumor, and the malignant tumor includes hematological cancer, colorectal cancer, breast cancer, melanoma, brain cancer, pancreatic cancer, lung cancer, liver cancer, or cholangiocarcinoma.
  • the anti-tumor drug sensitizer of the present invention is prepared into a pharmaceutical composition, and the drug composition includes a therapeutically effective amount of the anti-tumor drug sensitizer and a pharmaceutically acceptable carrier.
  • the carrier is a nano-carrier such as liposomes, polymer micelles or inorganic nanoparticles.
  • Anti-tumor drug sensitizers have long blood circulation time after being prepared into nano-formulations, and can accumulate more effectively in tumor tissues through the superpermeability and retention effect of tumors, and further improve their anti-tumor effects. effect.
  • the pharmaceutical composition of the present invention can be directly administered by conventional oral administration, injection and other administration methods.
  • the present invention also provides a method for preparing the liposome with anti-tumor drug sensitizer, which includes the following steps:
  • Preparation of liposome membrane dissolve the commonly used phospholipids or mixed phospholipids, and PEGylated phospholipids for preparing liposomes in a solvent, and concentrate them to form a membrane at 30-45°C;
  • the particle size of the formed liposomes can be further controlled by ultrasonic or extrusion methods.
  • the phospholipid is phosphatidylcholine, phosphatidylethanolamine, dioleoylphosphatidylethanolamine, cholesterol hemisuccinate, distearic acid phosphatidylethanolamine, and the polyethylene glycolated phospholipid is distearic acid phosphatidylethanolamine. Ethanolamine-polyethylene glycol 2000.
  • the solvent is dichloromethane, chloroform, methanol or a mixture thereof.
  • the solvent is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol is 1-8:1.
  • the molecular weight cut-off of the dialysis bag is 500-10000KD.
  • the anti-tumor drug sensitizer of the present invention has an immunosensitizing effect, including inhibiting Hif-1 ⁇ , reducing the expression of immunosuppressive molecules such as PD-L1, IDO, IL10, enhancing the body's immune response to tumors, and reversing tumors Immunosuppress the microenvironment and play the role of immunotherapy, thereby improving the immunotherapy effect of tumors.
  • immunosuppressive molecules such as PD-L1, IDO, IL10
  • the anti-tumor drug sensitizer of the present invention has the effect of chemotherapy sensitization. By inhibiting P-gp, it reverses the drug resistance of tumors and improves the chemotherapy treatment effect of tumors.
  • the anti-tumor drug sensitizer of the present invention can sensitize the effects of immunotherapy and chemotherapy, and by simultaneously inhibiting Hif-1 ⁇ and P-gp, thereby improving the effects of tumor chemotherapy and immunotherapy.
  • the anti-tumor drug sensitizer is a small molecule compound with a clear structure and is simple to synthesize.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or organic solvent or a mixture of both.
  • the compounds provided by the present invention also exist in prodrug forms.
  • the prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to transform into the compounds of the invention.
  • prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in the in vivo environment.
  • Certain compounds of the present invention may exist in unsolvated or solvated forms, including hydrated forms.
  • the solvated form is equivalent to the unsolvated form, and both are included in the scope of the present invention.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
  • C 1-3 alkyl is used to indicate a straight or branched chain group containing 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-3 , C 1-2 , C 1 , C 2 , C 3 alkyl group and the like. It can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • Examples of C 1 -alkyl include but are not limited to methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.
  • the "C 1-3 alkyl” of the present invention is optionally Replaced by 1-5 F, Cl, Br, I, OH, NH 2 , CN.
  • aryl is used to denote a polyunsaturated carbocyclic ring system, which can be a monocyclic, bicyclic or polycyclic ring system, in which at least one ring is aromatic, said bicyclic and polycyclic ring systems Each ring in fused together, which can be mono- or multi-substituted, can be monovalent, divalent or multivalent.
  • C 6-12 aryl include but are not limited to phenyl, naphthyl (including 1-naphthyl and 2-naphthyl), the "aryl” of the present invention is optionally substituted with 1 to 5 F, Cl, Br, I, OH, NH 2 , and CN.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and may include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • oxygen oxygen
  • Oxygen substitution does not occur on aromatic groups.
  • any variable such as R
  • its definition in each case is independent.
  • the group can be optionally substituted with at most two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • terapéuticaally effective amount of the present invention means (i) treatment or prevention of a specific disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a specific disease, condition or disorder, or (iii) prevention Or the amount of the compound of the present application that delays the onset of one or more symptoms of the specific disease, condition, or disorder described herein.
  • the amount of the compound of the present application that constitutes a “therapeutically effective amount” varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but it can be routinely determined by those skilled in the art. Determined by its own knowledge and this disclosure.
  • pharmaceutical composition in the present invention refers to a mixture of one or more of the compounds of the application or their salts and a pharmaceutically acceptable carrier.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound of the present application to the organism.
  • pharmaceutically acceptable carrier refers to those excipients that have no obvious stimulating effect on organisms and do not impair the biological activity and performance of the active compound.
  • Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, liposomes, polymers Micelles or inorganic nanocarriers, etc.
  • the solvent used in the present invention is commercially available.
  • IOX1 stands for 5-carboxy-8-hydroxyquinoline
  • PHD3 stands for inhibition of proline hydroxylase 3
  • PKM2 stands for M2 pyruvate kinase
  • Hif-1 ⁇ stands for hypoxia inducible factor-1 ⁇
  • PD-L1 stands for programmed death receptor-ligand 1
  • PERK stands for protein kinase R-like endoplasmic reticulum kinase
  • eIF ⁇ stands for eukaryotic initiation factor 2 of ⁇ subunit
  • p-eIF ⁇ stands for eukaryotic phosphorylated ⁇ subunit Initiation factor 2
  • CRT stands for calreticulin
  • IDO stands for indoleamine 2,3-dioxygenase
  • P-gp stands for P glycoprotein
  • DOX stands for adriamycin
  • DMSO stands for dimethyl sulfoxide
  • PBS stands for phosphate Buffer
  • EDTA stands for ethylenedi
  • the compounds of the present invention are used according to conventional naming principles in the field or The software is named, and the commercially available compounds are based on the supplier's catalog.
  • Figure 1 is a Western blot result of the compound of the present invention in Test Example 1A reducing the expression of PD-L1 in CT26 cells.
  • Figure 2 shows the quantitative statistical results of the Western blot of the compound of the present invention reducing the expression of PD-L1 in CT26 cells in Test Example 1A.
  • Figure 3 shows the Western blot result of IOX1 reducing the expression of PD-L1 in CT26 cells in Test Example 1A.
  • Figure 4 shows the quantitative statistical results of Western blot of IOX1 reducing the expression of PD-L1 in CT26 cells in Test Example 1A.
  • Figure 5 shows the Western blot result of IOX1 reducing the expression of PD-L1 in HCT26 cells in Test Example 1A.
  • Fig. 6 shows the quantitative statistical results of Western blot of IOX1 reducing PD-L1 expression of HCT26 cells in Test Example 1A.
  • Figure 7 shows the flow cytometric results of IOX1 reducing the expression of PD-L1 in CT26 cells in Test Example 1B.
  • Figure 8 shows the flow cytometric results of IOX1 reducing the expression of PD-L1 in 4T1 cells in Test Example 1B.
  • Figure 9 shows the flow cytometric results of IOX1 reducing the PD-L1 expression of HCT116 cells in Test Example 1B.
  • Figure 10 shows the flow cytometric results of IOX1 reducing the expression of PD-L1 in MCF-7 cells in Test Example 1B.
  • Figure 11 shows the flow cytometric results of IOX1 inhibiting DOX-induced up-regulation of PD-L1 on CT26 cells in Test Example 1C.
  • Figure 12 shows the flow cytometric results of IOX1 inhibiting DOX-induced up-regulation of PD-L1 on HCT116 cells in Test Example 1C.
  • Fig. 13 is a Western blot result of the combined use of IOX1 and DOX on CT26 cells to cause endoplasmic reticulum stress in Test Example 1D.
  • Figure 14 shows the flow cytometric results of the immunogenic death of CT26 cells caused by the combination of IOX1 and DOX in Test Example 1E.
  • Figure 15 shows the flow cytometric results of the immunogenic death of HCT116 cells caused by the combination of IOX1 and DOX in Test Example 1E.
  • Figure 16 is an immunofluorescence photograph of the immunogenic death of CT26 cells caused by the combination of IOX1 and DOX in Test Example 1F.
  • Figure 17 shows the flow cytometric results of dendritic cell maturation caused by the combination of IOX1 and DOX in Test Example 1G.
  • Figure 18 shows the flow cytometric results of the proliferation effect of IOX1 and DOX on T lymphocytes in Test Example 1H.
  • Figure 19 shows the results of flow cytometry of the combined use of IOX1 and DOX on the activity of T lymphocytes in Test Example 1H.
  • Fig. 20 shows the expression of related genes in mouse macrophages after IOX1 treatment in Test Example 11, and compared with untreated mouse macrophages.
  • Fig. 21 is the experimental result of the compound of the present invention inhibiting the expression of indoleamine 2,3-dioxygenase in Test Example 1J.
  • Figure 22 shows the cytotoxicity test results of the compound of the present invention in combination with the chemotherapeutic drug DOX in Test Example 2A.
  • Figure 23 shows the cytotoxicity test results of the compound of the present invention in Test Example 2A.
  • Figure 24 shows the cytotoxicity test results of IOX1 combined with the chemotherapy drug paclitaxel (PTX) in Test Example 2A.
  • Figure 25 shows the cytotoxicity test results of IOX1 combined with the chemotherapy drug oxaliplatin (OXA) in Test Example 2A.
  • Figure 26 shows the results of the cytotoxicity test of IOX1 in combination with the chemotherapy drug tripterygium wilfordii (Cela) in Test Example 2A.
  • Figure 27 shows the results of the compound of the present invention in Test Example 2B reducing the expression of Hif-1 ⁇ in CT26 cells.
  • Figure 28 shows the flow cytometric results of different concentrations of IOX1 reducing the expression of Hif-1 ⁇ in CT26 cells in Test Example 2B.
  • Figure 29 is a Western blot result of IOX1 reducing the P-gp expression of CT26 cells in Test Example 2C.
  • Figure 30 is a quantitative analysis of the Wesern blot results of IOX1 reducing the P-gp expression of CT26 cells in Test Example 2C.
  • Figure 31 is a Western blot result of IOX1 reducing the P-gp expression of HCT116 cells in Test Example 2C.
  • Figure 32 is a quantitative analysis of the Wesern blot results of IOX1 reducing the P-gp expression of HCT116 cells in Test Example 2C.
  • Figure 33 shows the result of endocytosis of Rh123 on CT26 cells after the combination of Rh123 and IOX1 in Test Example 2D, and compared with Rh123 alone.
  • Figure 34 shows the tumor inhibition curve of the CT26 tumor-bearing mouse tumor model after IOX1, DOX and the two drugs are used in combination in Test Example 3.
  • Figure 35 shows the inhibitory effect of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in test example 4-1 on the tumor model of CT26 small tumor-bearing mice Tumor curve.
  • Figure 36 shows the weight curve of CT26 small tumor-bearing mice after combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-1.
  • Figure 37 shows the inhibitory effect of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in test example 4-2 on the tumor model of CT26 large tumor-bearing mice Tumor curve.
  • Figure 38 shows the weight curve of CT26 large tumor-bearing mice after combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-2.
  • Figure 39 shows the results of the combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-3 on the tumor model of 4T1 in situ tumor-bearing mice Tumor suppression curve.
  • Figure 40 shows the weight curve of 4T1 orthotopic tumor-bearing mice after combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-3.
  • Figure 41 shows the 4T1 tumor in situ and lung metastasis after the combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) at the end of the experiment in Test Example 4-4 In vivo fluorescence photograph of the lungs of tumor mice.
  • Doxil Doxil liposomes
  • Ioxil 5-carboxy-8-hydroxyquinoline liposomes
  • Figure 42 shows the combination of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-4, which shows small tumor-bearing 4T1 tumors in situ and lung metastases. Tumor inhibition curve of mouse tumor model.
  • Figure 43 shows the body weight of 4T1 in situ tumor and lung metastasis tumor-bearing mice after combined use of Doxil liposome (Doxil) and 5-carboxy-8-hydroxyquinoline liposome (Ioxil) in Test Example 4-4 curve.
  • Doxil Doxil liposome
  • Ioxil 5-carboxy-8-hydroxyquinoline liposome
  • Figure 44 shows the inhibition of the B16F10 subcutaneous tumor-bearing mouse tumor model after the combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-5 Tumor curve.
  • Figure 45 shows the weight curve of B16F10 subcutaneous tumor-bearing mice after combined use of Doxil liposomes (Doxil) and 5-carboxy-8-hydroxyquinoline liposomes (Ioxil) in Test Example 4-5.
  • the present invention will be described in detail below through examples. The following description is only for explaining the present invention and does not limit its content.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other compound synthesis methods, and equivalent alternatives well known to those skilled in the art , Can also be commercially available.
  • Preferred embodiments include, but are not limited to, embodiments of the present invention. It is obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.
  • Dissolve compound 1 200mg, 0.99mmol in 10mL dry tetrahydrofuran, add triethylamine (150.73mg, 1.485mmol), N 2 protection, drop in acetyl chloride (96mg, 1.09mmol) in tetrahydrofuran solution under ice bath, and stir. After 1h, the ice bath was removed and the reaction continued overnight. Remove the tetrahydrofuran by rotary evaporation under reduced pressure, redissolve it with dichloromethane, wash with sodium bicarbonate, deionized water, and saturated brine respectively, dry with anhydrous magnesium sulfate or anhydrous sodium sulfate, concentrate and pass through the column.
  • Test Example 1 The compound of the present invention is used as an immunosensitizer.
  • Configuration method The compounds of the present invention prepared in the examples are configured into DMSO solutions of different concentrations (5 ⁇ M, 25 ⁇ M, 50 ⁇ M) for use in cell experiments.
  • Test Example 1A Western blot technology detects that the compound of the present invention reduces the expression of PD-L1 in tumor cells.
  • the culture medium was discarded, the cells were rinsed three times with pre-cooled PBS and the washing solution was discarded.
  • 0.2 mL of cell lysate containing protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes. After the lysis is completed, scrape the lysate and cell debris to the side of the Petri dish with a cell scraper, and transfer the lysate to a 1.5mL Ep tube with a pipette, centrifuge at 4°C (12000rpm/5min) and collect the supernatant. Determine the protein concentration.
  • the protein was transferred to the nitrocellulose membrane, the voltage was 80V, and the transfer time was 90 minutes.
  • the membrane was placed in 5% skimmed milk and sealed at room temperature for one hour.
  • the corresponding primary antibodies (anti-PD-L1, 1:2000, anti-GAPDH, 1:10000) were added, and incubated overnight at 4°C.
  • After washing the membrane three times with TBST buffer add horseradish peroxidase-labeled secondary antibody, and incubate for 1 hour at room temperature. After fully washing the film, perform chemiluminescence color development, and take a photo with a chemiluminescence imager.
  • test results are shown in Figure 1-6
  • Figure 1-2 shows that the compound of the present invention can reduce the expression of tumor cell PD-L1, among which IOX1 is the best
  • Figure 3-6 shows that IOX1 significantly reduces the PD-L1 expression of human and murine tumor cells -The expression of L1 is concentration-dependent.
  • Test Example 1B Flow cytometry to detect that the compound of the present invention reduces the expression of PD-L1 in tumor cells.
  • the mouse CT26, 4T1 or human HCT116, MCF-7 cells were planted in a 6-well plate at 2 ⁇ 10 4 cells per well. After the cells adhere to the wall, different concentrations (5 ⁇ M, 25 ⁇ M, 50 ⁇ M) of DMSO solution containing the compound of the present invention are added respectively, and the incubation is continued for 24 hours. Discard the medium, rinse the cells with PBS 3 times, and add 0.2 mL trypsin containing EDTA to each well. Collect the digested cells in a flow tube, centrifuge to remove the supernatant, resuspend the cells in PBS containing 5% goat serum, and add anti-mouse PD-L1 primary antibody (1 ⁇ g/1 ⁇ 10 6 cells).
  • IOX1 reduces the expression of PD-L1 on the surface of tumor cells in a concentration-dependent manner.
  • the integral value in the gray area represents the expression rate of PD-L1, which is marked in the corresponding area of the graph as a percentage.
  • Test Example 1C Flow cytometry detection of the compounds of the present invention inhibiting the up-regulation of tumor cells PD-L1 caused by chemotherapeutics.
  • CT26 or HCT116 cells were planted in a 6-well plate at 2 ⁇ 10 4 cells per well. After overnight adherence, the treatment group of compound IOX1 of the present invention was added to the treatment group, and the incubation was continued for 24 hours. Discard the medium, rinse the cells with PBS 3 times, and add 0.2 mL trypsin containing EDTA to each well.
  • IOX1 can inhibit doxorubicin on CT26 or HCT116 tumor cells and inhibit the up-regulation of PD-L1 induced by doxorubicin on CT26 tumor cells.
  • Test Example 1D The compound of the present invention enhances the endoplasmic reticulum stress of tumor cells caused by drugs.
  • the culture medium was discarded, the cells were rinsed three times with pre-cooled PBS and the washing solution was discarded.
  • 0.2 mL of cell lysate containing protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes. After the lysis is completed, scrape the lysate and cell debris to the side of the Petri dish with a cell scraper, and transfer the lysate to a 1.5mL Ep tube with a pipette, centrifuge at 4°C (12000rpm/5min) and collect the supernatant. Determine the protein concentration.
  • the protein was transferred to the nitrocellulose membrane, the voltage was 80V, and the transfer time was 90 minutes.
  • the membrane was placed in 5% skimmed milk and sealed at room temperature for one hour.
  • TBST buffer After washing the membrane three times with TBST buffer, add corresponding primary antibodies (anti-p-PERK, 1:2000, anti-p-eIF ⁇ , 1:2000, anti-eIF ⁇ , 1:2000, anti-Actin, 1:10000) Incubate overnight at 4°C.
  • TBST buffer add horseradish peroxidase-labeled secondary antibody, and incubate for 1 hour at room temperature. After fully washing the film, perform chemiluminescence color development, and take a photo with a chemiluminescence imager.
  • IOX1 can activate the PERK/eIF2 ⁇ pathway on CT26 tumor cells by IOX1, and produce stronger endoplasmic reticulum stress.
  • Test Example 1E Flow cytometry detection of the compound of the present invention to enhance the immunogenic death of tumor cells caused by drugs.
  • CT26 or HCT116 cells were planted in a 6-well plate at 2 ⁇ 10 4 cells per well. After overnight adherence, the treatment group of compound IOX1 of the present invention was added to the treatment group, and the incubation was continued for 4 hours. Discard the medium, rinse the cells with PBS 3 times, and add 0.2 mL trypsin containing EDTA to each well.
  • IOX1 can induce stronger CRT membrane translocation on CT26 or HCT116 tumor cells by doxorubicin, and increase the immunogenicity of CT26 or HCT116 cells. death.
  • Test Example 1F Observation by immunofluorescence method that the compound of the present invention enhances the immunogenic death of tumor cells caused by drugs.
  • CT26 cells were plated in a confocal culture dish, and after overnight adherence, the treatment group of the compound IOX1 of the present invention was added to the treatment group, and the incubation was continued for 4 hours. Discard the culture medium, rinse the cells with PBS 3 times, fix with 4% paraformaldehyde for 10 min, replace PBS and rinse 3 times, 3 min each time, add 3% BSA solution and block at 37°C for 30 min, absorb the blocking solution with absorbent paper, Add 200 ⁇ L of calreticulin antibody (anti-CRT, 1:200) to each well. After incubating for 1 hour at room temperature in the dark, wash with PBS three times, then add the same amount of APC-labeled goat anti-rabbit secondary antibody. After incubating for 30 minutes, continue Rinse the cells with PBS 3 times, then add DAPI dye solution to each dish, incubate for 5 min in the dark, wash with PBS three times and observe under a laser confocal microscope.
  • IOX1 can make doxorubicin produce stronger CRT on CT26 tumor cells and increase the immunogenic death of CT26 cells.
  • Test Example 1G The compound IOX1 of the present invention enhances the maturation of dendritic cells induced by drugs.
  • CT26 cells were plated in a Transwell chamber, and after overnight adherence, they were added to the treatment group of the compound IOX1 of the present invention, and incubated for 24 hours. Discard the medicated culture medium, replace with fresh culture medium, and pave the bone marrow-derived dendritic cells extracted from Balb/c mice in the lower layer of Transwell. After culturing for 24 hours, remove the cell containing the tumor cells, transfer the lower dendritic cells to a flow tube for centrifugation, discard the supernatant, wash the cell pellet 3 times with PBS, and resuspend the cells in PBS containing 5% goat serum. FITC anti-CD11c, APC anti-CD86 and PE anti-CD80 antibodies were added, and the ratio of CD11c + CD86 + CD80 + cells was detected by flow cytometry.
  • IOX1 combined with doxorubicin can induce more mature DC cells and increase antigen presentation.
  • Test Example 1H The compound IOX1 of the present invention enhances the proliferation and activity of T lymphocytes in vitro.
  • CT26 cells were plated in a 6-well plate, and after overnight adherence, the treatment group of the compound IOX1 of the present invention was added to the treatment group, and the incubation was continued for 24 hours. The culture medium was discarded, and Balb/c mouse peripheral blood mononuclear cells (PBMC) labeled with CFSE were added. At the same time, a group of control groups without PBMC but with the same volume of culture medium was set up. After continuing the incubation for 4 days, the supernatant containing PBMC was collected in a flow tube, centrifuged, and the cell pellet was washed with PBS three times, and then the PBMC proliferation results were detected by flow cytometry.
  • PBMC peripheral blood mononuclear cells
  • Figure 18 shows that the IOX1 can increase the proliferation rate of T lymphocytes by 16.3% compared with the control group.
  • IOX1 combined with DOX can further increase the proliferation of T lymphocytes, and its proliferation rate is as high as 40.9%.
  • FIG 19 shows that IOX1 can restore the killing ability of T lymphocytes to tumor cells.
  • T cells can hardly induce apoptosis of untreated tumor cells.
  • T lymphocytes act on tumor cells treated with IOX1 in advance.
  • the apoptosis rate was increased by 13.71%; IOX1 combined with DOX increased the tumor apoptosis rate to 91.8, proving that IOX1 can significantly increase the proliferation and activity of T lymphocytes in vitro.
  • Test Example 1I The compound IOX1 of the present invention promotes the polarization of macrophages from M2 to M1.
  • the mouse macrophage strain Raw264.7 was inoculated in a 24-well plate. After 12 hours of cell attachment, the cells were cultured in a medium containing IL-4 (40ng/mL) for one day to induce differentiation into M2 macrophages (TAM2). Subsequently, different groups of solutions of the compound of the present invention (5 ⁇ M) were added to TAM2. After 24 hours of treatment, the cells were collected, lysed and extracted total RNA, reverse transcription and PCR experiments were performed to detect M2 type macrophage-specific proteins arg1 and The RNA level of M1 type macrophage-specific protein Nos2, with the hprt gene as an internal reference.
  • TAM2 M2 macrophages
  • IOX1 The results of IOX1 are shown in Figure 20. Compared with TAM2 treated in the blank group, arg1 was significantly reduced after IOX1 was added, and Nos2 was significantly increased, proving that the compound of the present invention can promote the polarization of macrophages from M2 to M1, and promote The tumor-growing M2 type macrophages are transformed into tumor suppressor type M1 type macrophages, which is beneficial to improve the effect of tumor treatment.
  • Test Example 1J The compound of the present invention inhibits the expression of indoleamine 2,3-dioxygenase.
  • CT26 cells were planted in a 12-well plate at 5 ⁇ 10 4 cells/well, and 2 mL of medium (containing 100 ⁇ M tryptophan) was added to each well. After one day of culture, a certain concentration gradient of the compound of the present invention (1-100 ⁇ M) is added, and then 0.1 ⁇ g/mL INF- ⁇ is added to induce the expression of IDO. After 72 hours of incubation, 200 ⁇ L of supernatant was added to 10 ⁇ L of 30% trifluoroacetic acid solution to precipitate the protein. Detect the content of kynurenine in the solution by HPLC, repeat three times for each well.
  • IOX1 can inhibit CT26 from producing kynurenine, and the degree of inhibition increases with the increase of the concentration of IOX1.
  • concentration of IOX1 is 100 ⁇ M
  • degree of inhibition of kynurenine is 33.2%. .
  • IOX1 can inhibit the conversion of tryptophan to kynurenine, indicating that IOX1 can inhibit the activity or expression of indoleamine 2,3-dioxygenase. Therefore, the compound of the present invention can enhance the effect of cancer immunotherapy by inhibiting the expression of indoleamine 2,3-dioxygenase.
  • Test Example 2 Application of the compound of the present invention as a chemotherapeutic sensitizer.
  • the compound of the present invention prepared in the examples was configured as a DMSO solution and used in cell experiments.
  • Test Example 2A Cytotoxicity study of the compound of the present invention in combination with various chemotherapeutics.
  • CT26 cells (MC38 cells, 4T1 cells, B16F10 cells, HePa1-6 cells, H22 cells, LLC cells, MB49 cells, P388 cells, C6 cells, HCT116 cells, MCF-7 cells, BXPC-3 cells, Hela cells, MDA -MB-231 cells, A2780 cells, PC3 cells, HepG2 cells, HGC-27 cells) were cultured in a 96-well plate at 5000 cells/well, and 100 ⁇ L of medium was added to each well at 5% CO 2 concentration and 95% humidity Incubate in a constant temperature incubator at 37°C for 24h.
  • Figures 22, 24 to 26 show that compared with chemotherapeutic drugs alone, the combination of the compound of the present invention and chemotherapeutic drugs can greatly reduce the survival rate of cells, and IOX1 has the best effect;
  • Figure 23 shows that the value is 0.1-10 ⁇ g/mL, The compound of the invention is not cytotoxic. It is proved that IOX1 and other compounds of the present invention can also increase the toxicity of paclitaxel (PTX), oxaliplatin (OXA) or triptorubin (Cela) to CT26 cells.
  • PTX paclitaxel
  • OXA oxaliplatin
  • Cela triptorubin
  • Table 1 shows the cytotoxicity test results of IOX1 combined with the chemotherapeutic drug DOX on a variety of cell lines. The experimental results show that the combined use of IOX1 and DOX can greatly reduce the survival rate of a variety of tumor cells.
  • Table 2 shows the cytotoxicity test results of IOX1 combined with a variety of chemotherapeutics on the CT26 cell line. Experimental results show that IOX1 can increase the toxicity of a variety of chemotherapeutic drugs to CT26 cells.
  • Test Example 2B Flow cytometry to detect that the compound of the present invention reduces the expression of Hif-1 ⁇ in tumor cells.
  • the CT26 cells were evenly spread in a 6-well plate at a density of 2 ⁇ 10 4 cells per well. After the cells adhere to the wall, the DMSO solution of the compound of the present invention is added separately, and the incubation is continued for 24 hours.
  • Figure 27 shows that when the concentration of compound 1-3 and IOX1 is 5 ⁇ M, the degree of tumor cell Hif-1 ⁇ is reduced. Compared with the blank control, the compound of the present invention can significantly reduce tumor cell Hif-1 ⁇ . Among them, IOX1 had the best effect, and the expression of Hif-1 ⁇ was reduced by 38% at 5 ⁇ M.
  • Figure 28 shows that when the concentration of IOX1 is 1 ⁇ M, the expression of Hif-1 ⁇ is reduced by 32.8%; when the concentration of IOX1 is 5 ⁇ M, the expression of Hif-1 ⁇ is reduced by 38%; the ability of IOX1 to reduce the expression of Hif-1 ⁇ in tumor cells varies with The increase in concentration increases.
  • Test Example 2C The compound IOX1 of the present invention reduces the expression of P-gp in tumor cells.
  • the culture medium was discarded, the cells were rinsed three times with pre-cooled PBS and the washing solution was discarded.
  • 0.2 mL of cell lysate containing protease inhibitor was added to each well, and the cells were lysed on ice for 30 minutes. After the lysis is completed, scrape the lysate and cell debris to the side of the petri dish with a cell scraper, and transfer the lysate to a 1.5mL Ep tube with a pipette, collect the supernatant by centrifugation (12000rpm/5min) at 4°C, and determine Protein concentration.
  • the protein was transferred to the nitrocellulose membrane, the voltage was 80V, and the transfer time was 90 minutes.
  • the membrane was placed in 5% skimmed milk and sealed at room temperature for one hour.
  • the corresponding primary antibodies (anti-P-gp, 1:4000, anti-GAPDH, 1:10000) were added, and incubated overnight at 4°C.
  • TBST buffer After washing the membrane three times with TBST buffer, add a horseradish peroxidase-labeled secondary antibody, and incubate for 1 hour at room temperature. After fully washing the film, perform chemiluminescence color development, and take a photo with a chemiluminescence imager.
  • Test Example 2D The compound of the present invention enhances the ability of tumor cells to infiltrate rhodamine 123 (Rh123).
  • Rh123 is the substrate of the multidrug resistance protein P-gp. Decreased expression of P-gp can reduce the efflux of Rh123 and increase its intracellular content. Therefore, it can be used to determine the activity of the cell's P-gp protein.
  • CT26 cells were planted in a confocal imaging dish at a density of 1 ⁇ 10 4 /well, and the cells were cultured in a constant temperature incubator at 37°C for 24 hours. Then replace each well with fresh medium, and add Rh123 solution and different concentrations of IOX1 solution: Group A: 1 ⁇ M Rh123; Group B: 1 ⁇ M Rh123+1 ⁇ M IOX1; Group C: 10 ⁇ M Rh123; Group D: 10 ⁇ M Rh123+10 ⁇ M IOX1, After 6 hours of incubation, the Rh123 cells were observed with a confocal microscope.
  • the excitation wavelength of Rh123 is 488 nm, and the emission wavelength is 500 to 550 nm.
  • Test Example 3 The compound of the present invention enhances the anti-tumor activity of the drug in vivo.
  • IOX1 5-carboxy-8-hydroxyquinoline
  • dissolve 30mg IOX1 in 1mL DMSO 1mL polyethylene glycol 500, Tween 80 or polyoxyethylene castor oil (this test uses spit Temperature 80), vortex to mix uniformly, add 1mL of the above solution to 9mL PBS, and mix well to obtain IOX1 injection.
  • the injection can be stored at 4°C for more than 6 months without solid powder precipitation.
  • Test purpose To investigate the effects of IOX1 combined with the chemotherapy drug adriamycin (doxorubicin, paclitaxel, gemcitabine, oxaliplatin, camptothecin derivatives, irinotecan, triptorubin, etc.) on CT26 mouse colon cancer tumors Inhibition.
  • adriamycin doxorubicin, paclitaxel, gemcitabine, oxaliplatin, camptothecin derivatives, irinotecan, triptorubin, etc.
  • Test procedure Balb/c white mice were injected subcutaneously with 1 ⁇ 10 6 CT26 tumor cells, and after the tumor grew to about 80 mm 3 , the tail vein injection was started every two days (Day 7, Day 9, Day 11).
  • the blank control group PBS
  • 5-carboxy-8-hydroxyquinoline group, adriamycin hydrochloride group, 5-carboxy- 8-Hydroxyquinoline + doxorubicin hydrochloride group D1
  • Test Example 4 Preparation of 5-carboxy-8-hydroxyquinoline liposome preparation and its anti-tumor activity test in combination with chemotherapeutics.
  • Step 2 Then, 12.89g dioleoylphosphatidylethanolamine (DOPE), 2.11g cholesterol hemisuccinate (CHEMS), 6.52g distearic acid phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG 2000 ) are dissolved In chloroform (12 mL) and methanol (4 mL), spin dry under reduced pressure in a water bath at 37° C. to form a film.
  • DOPE dioleoylphosphatidylethanolamine
  • CHEMS cholesterol hemisuccinate
  • DSPE-mPEG 2000 6.52g distearic acid phosphatidylethanolamine-polyethylene glycol 2000
  • Step 3 Add the 5-carboxy-8-hydroxyquinoline solution of step 1 to the liposome membrane of step 2, and hydrate at room temperature overnight.
  • Step 4 Place the resulting solution in a dialysis membrane (molecular weight cut-off of 3500), place it in pure water, and dialyze for 6 hours at room temperature to obtain an Iox1 liposome preparation (Ioxil, drug loading efficiency 93%).
  • the size characterization of the pharmaceutical preparation The particle size and distribution of the drug preparation are measured by dynamic light scattering (DLS).
  • the lipid preparation is assembled into nanoparticles with a dynamic particle size distribution of 0.125 and an average size of 102.3 nm in water.
  • the size can be adjusted by liposome composition, preparation method and the like.
  • Test Example 4-1 CT26 subcutaneous small tumor model
  • mice were injected subcutaneously with 1 ⁇ 10 6 CT26 tumor cells. After the tumor grew to about 80 mm 3 , the administration was started, and the tail vein injection was performed every two days (Day0, Day2, Day4).
  • 5-carboxy-8-hydroxyquinoline liposome preparation and adriamycin liposome as an example, as blank control group (PBS), adriamycin liposome (Doxil, DOX concentration 5mg/kg) Group: 5-carboxy-8-hydroxyquinoline liposome + adriamycin liposome (Doxil+Ioxil, DOX concentration 5mg/kg, IOX1 concentration 7.5mg/kg). After the end of the dosing cycle, the rats were observed for 18 days.
  • Figures 35-36 show that the anti-tumor effect of the combination of the two preparations is significantly better than the use of adriamycin liposomes alone. After 22 days, the tumor in the combination group was completely eliminated, but the tumor in the adriamycin liposome group still showed a growth trend.
  • Figure 36 shows that the body weight of the mice in the combination group did not decrease, showing that the drug has high biological safety and small toxic and side effects.
  • mice were injected subcutaneously with 1 ⁇ 10 6 CT26 tumor cells. After the tumor grew to about 350 mm 3 , the administration was started, and the tail vein injection was performed every two days (Day0, Day2, Day4, Day6).
  • 5-carboxy-8-hydroxyquinoline liposome preparation and adriamycin liposome as an example, as blank control group (PBS), adriamycin liposome (Doxil, DOX concentration 5mg/kg) Group: 5-carboxy-8-hydroxyquinoline liposome + adriamycin liposome (Doxil+Ioxil, DOX concentration 5mg/kg, IOX1 concentration 7.5mg/kg). After the end of the dosing cycle, the mice were observed for 28 days.
  • Figures 37-38 show that the anti-tumor effect of the combination of the two formulations is significantly better than the use of adriamycin liposomes alone. After 34 days, the tumor in the combination group was completely eliminated, but the tumor in the adriamycin liposome group still showed a growth trend.
  • Figure 38 shows that the body weight of the mice in the combination group did not decrease, showing that the drug has high biological safety and small toxic and side effects.
  • Test example 4-3 4T1 orthotopic tumor model
  • mice were injected subcutaneously with 1 ⁇ 10 6 4T1 tumor cells. After the tumor grew to about 100 mm 3 , the administration was started, and the tail vein injection was performed every two days (Day0, Day2, Day4).
  • 5-carboxy-8-hydroxyquinoline liposome preparation and adriamycin liposome as an example, as blank control group (PBS), adriamycin liposome (Doxil, DOX concentration 5mg/kg) Group: 5-carboxy-8-hydroxyquinoline liposome + adriamycin liposome (Doxil+Ioxil, DOX concentration 5mg/kg, IOX1 concentration 7.5mg/kg). After the end of the dosing cycle, the rats were observed for another 20 days.
  • Figure 39-40 shows that the anti-tumor effect of the combination of the two formulations is significantly better than the use of adriamycin liposomes alone. After 24 days, the tumor in the combination group was completely eliminated, but the tumor in the adriamycin liposome group still showed a growth trend.
  • Figure 40 shows that the body weight of the mice in the combination group did not decrease, indicating that the drug has high biological safety and low toxic and side effects.
  • Test Example 4-4 4T1 in situ and lung metastasis dual tumor model
  • mice were injected subcutaneously with 1 ⁇ 10 6 4T1 tumor cells. After the tumor grew to about 100 mm 3 , the administration was started, and the tail vein injection was performed every two days (Day0, Day2, Day4).
  • 5-carboxy-8-hydroxyquinoline liposome preparation and adriamycin liposome as an example, as blank control group (PBS), adriamycin liposome (Doxil, DOX concentration 5mg/kg) Group: 5-carboxy-8-hydroxyquinoline liposome + adriamycin liposome (Doxil+Ioxil, DOX concentration 5mg/kg, IOX1 concentration 7.5mg/kg).
  • 2 ⁇ 10 5 chemiluminescent enzyme-expressing 4T1 tumor cells Luci 4T1
  • Test example 4-5 B16F10 subcutaneous small tumor model
  • mice were injected subcutaneously with 1 ⁇ 10 6 B16F10 tumor cells. After the tumor grew to about 80 mm 3 , the administration was started, and the tail vein injection was performed every two days (Day0, Day2, Day4).
  • 5-carboxy-8-hydroxyquinoline liposome preparation and adriamycin liposome as an example, as blank control group (PBS), adriamycin liposome (Doxil, DOX concentration 5mg/kg) Group: 5-carboxy-8-hydroxyquinoline liposome + adriamycin liposome (Doxil+Ioxil, DOX concentration 5mg/kg, IOX1 concentration 7.5mg/kg). After the end of the dosing cycle, the rats were observed for another 20 days.
  • Figures 44-45 show that the anti-tumor effect of the combination of the two preparations is significantly better than that of liposome doxorubicin alone. After 24 days, the tumor in the combination group is much smaller than that of liposome doxorubicin. Group of tumors.
  • Figure 45 shows that the body weight of the mice in the combination group did not decrease, showing that the drug has high biological safety and small toxic and side effects.
  • the combination of 5-carboxy-8-hydroxyquinoline and chemotherapeutic drugs After being prepared into a pharmaceutical preparation, the combination of 5-carboxy-8-hydroxyquinoline and chemotherapeutic drugs exhibits significant anti-cancer activity, the therapeutic effect is at the leading level in the field, and has good application prospects.

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Abstract

公开了一种基于5-羧基-8-羟基喹啉的抗肿瘤药物增敏剂。提供的抗肿瘤药物增敏剂为式(I)化合物或其药学上可接受的盐，通过降低肿瘤细胞中乏氧诱导因子-1α的表达，从而引起一系列下游因子的改变，包括降低肿瘤细胞的程序性死亡受体-配体1(PD-L1)的表达，提高免疫和化疗治疗效果。还公开了抗肿瘤药物增敏剂的联用方法、以及抗肿瘤药物增敏剂的药物制剂及其制备方法，进一步提高疗效。

Description

一种基于5-羧基-8-羟基喹啉的抗肿瘤药物增敏剂及其应用 技术领域
本发明涉及医药技术领域，具体涉及5-羧基-8-羟基喹啉及其衍生物在制备抗肿瘤药物增敏剂中的应用。
背景技术
肿瘤免疫治疗是通过打破肿瘤的免疫抑制微环境、重新启动并激发肿瘤内免疫循环，从而治疗肿瘤的方法。肿瘤的免疫抑制微环境是肿瘤能够阻断机体免疫系统对癌细胞进攻。诱导肿瘤免疫抑制微环境的因素之一是肿瘤的乏氧机制。肿瘤内癌细胞新陈代谢相较正常细胞旺盛，肿瘤内血管的畸形以及分布不均匀导致氧气输送不畅，最终造成了瘤内存在着大量乏氧区域。
乏氧的微环境激活肿瘤细胞的多条信号通路，其中包括乏氧诱导因子-1(Hypoxia inducible factor 1，Hif-1)。Hif-1由Hif-1α和Hif-1β两个亚基构成。Hif-1α可参与多种靶基因的转录调控，影响肿瘤细胞的能量代谢、增殖和凋亡，使细胞及组织产生一系列反应以适应缺氧环境(HIF Inhibitors：Status of Current Clinical Development；Current Oncology Reports，2019，21(1)：6)。
研究表明，乏氧诱导的Hif-1α能与程序性死亡受体-配体1(PD-L1)的近端启动子上的转录活性缺氧反映元件(HRE)直接结合从而启动PD-L1的转录，上调肿瘤细胞的PD-L1的表达，而PD-L1与T细胞上的程序性死亡受体1(PD-1)结合后会诱导T细胞凋亡并抑制T细胞活化和增殖的抑制信号，使T细胞失去活性，造成肿瘤免疫抑制为环境和人体的免疫力下降(PD-L1 is a novel direct target of HIF-1α，and its blockade under hypoxia enhanced MDSC-mediated T cell activation，Journal Of Experimental Medicine，2014，5(211)：781；Regulation of PD-L1：a novel role of pro-survival signalling in cancer，Annals of oncology，Annals Of Oncology，2015，3(27)：409)。
同时，肿瘤细胞的Hif-1α过表达能使肿瘤相关巨噬细胞分泌大量IL-10，促进巨噬细胞向免疫抑制M2型极化，强化肿瘤的免疫微环境，从而影响免疫治疗效果(Reactive oxygen species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1，Proceedings Of The National Academy Of Sciences Of The United States Of America，2019，10(116)：4326；HIF-1 alpha is essential for myeloid cell-mediated inflammation，Cell，2003，5(112)：645)。此外，许多肿瘤治疗也能促使肿瘤细胞上调PD-L1的表达，例如低剂量化疗即可诱导肿瘤细胞或髓系细胞上调PD-L1的表达(Acquired Resistance to Fractionated Radiotherapy Can Be Overcome by Concurrent PD-L1 Blockade，Cancer Research，2014，74(19)：5458；The hallmarks of successful anticancer immunotherapy，Science Translational Medicine，2018，19(10)：459)，从而降低肿瘤的免疫应答。
乏氧微环境也是导致实体肿瘤产生多药耐药的重要原因之一。肿瘤内的乏氧微环境引起Hif-1过表达，激活下游靶基因转录，包括上调耐药性P糖蛋白(P-gp)表达，降低了化疗药物的细胞毒化杀死肿瘤细胞的作用，因此肿瘤细胞的多药耐药(Multi-drugs Resistance，MDR)作用是造成化疗失败的最主要原因之一(Hypoxia-mediated sorafenib resistance can be overcome by EF24 through Von Hippel-Lindau tumor suppressor-dependent HIF-1α inhibition in hepatocellular carcinoma，Hepatology，2013，5(57)：1847；Nitroglycerin Treatment May Enhance Chemosensitivity to Docetaxel and Carboplatin in Patients with Lung Adenocarcinoma，Clinical cancer research，2006，22(12)：6748)。
利用PD-1/PD-L1免疫抑制剂阻断肿瘤细胞与T细胞的结合，使T细胞能够正常的在人体内发挥作用，激发免疫作用来识别和清除肿瘤细胞，是目前基于PD-1/PD-L1抗体的肿瘤免疫治疗的通用方案。该免疫治疗方法对靶点适合的肿瘤具有很高的疗效。同时，有些化疗药物毒化肿瘤细胞后可激活其免疫原性死亡，诱导钙网蛋白表达，释放“吃掉我”的信号，从而激活T细胞和树突状细胞，提高抗肿瘤免疫反应。化疗药物治疗后还可诱导肿瘤细胞新抗原 的产生，增加突变，如上调PD-L1表达，重构免疫微环境，由免疫细胞浸润少的“冷肿瘤”转变为免疫反应更强的“热肿瘤”，增强免疫检查点抑制剂敏感性，提高治疗效果。因此，有些化疗药物与PD-1/PD-L1免疫抑制剂联用可提高抗肿瘤效果(In situ formed reactive oxygen species-responsive scaffold with gemcitabine and checkpoint inhibitor for combination therapy，Science translational medicine，2019，10(429)：eaan3682；Elimination of established tumors with nanodisc-based combination chemoimmunotherapy，Science Advances，2018，4(4)：eaao1736)。但PD-1/PD-L1抗体蛋白存在着的副作用风险大，抗体制备及储存不便、价格昂贵等问题。
5-羧基-8-羟基喹啉(IOX1)是广谱2-酮戊二酸氧合酶(2-oxoglutarate oxygenases)强效的广谱抑制剂，包括Jumonji C(JmjC)结构域去甲基化酶。IOX1通过抑制KDM4A增加HeLa细胞中H3K9me3水平，而对细胞活性没有显著作用。由于低的细胞渗透性，IOX1在HeLa细胞中显示出较低的效能，而其正辛基酯衍生物改善其细胞渗透性(A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.ChemMedChem.2014 Mar；9(3)：566-71).IOX1也因此是HIF水解酶脯氨酰羟化酶(PHD)的抑制剂而稳定HIF，提高心脏、肾缺血部位的HIF水平，从而保护缺血部位，应用于例如肾性缺血治疗(Inhibition of Hypoxia Inducible Factor Hydroxylases Protects Against Renal Ischemia-Reperfusion Injury，Journal of the American Society of Nephrology，2008，19(1)39-46)。
发明内容
本发明提供了一种基于5-羧基-8-羟基喹啉的小分子抗肿瘤药物增敏剂，用于提高免疫和化疗治疗效果。
本发明解决上述技术问题所提供的技术方案为：
一种基于5-羧基-8-羟基喹啉的抗肿瘤药物增敏剂，所述的抗肿瘤药物增敏剂为式(I)化合物或其药学上可接受的盐，
Figure PCTCN2020127036-appb-000001
其中，
R 1为H、OH、NH 2、C 1-3烷基、-O-C 1-3烷基、-O-C 6-12芳基；
R 2为H、C 1-3烷基、-C(＝O)-C 1-3烷基或-C(＝O)-C 6-12芳基。
本发明所述的抗肿瘤药物增敏剂通过下调肿瘤细胞中Hif-1α，从而下调肿瘤细胞程序性死亡受体-配体1的表达，阻断肿瘤细胞与T细胞之间的PD-L1/PD-1的应答，避免肿瘤细胞的免疫逃逸和人体的免疫力下降，使T细胞能够识别并杀伤肿瘤细胞，提高抗肿瘤效果。
本发明所述的抗肿瘤药物增敏剂还通过下调Hif-1α，从而抑制巨噬细胞分泌IL-10，促进巨噬细胞由免疫抑制型M2向免疫活性型M1转化，逆转免疫抑制状态，增强机体对肿瘤的免疫应答，提高肿瘤免疫治疗效果。
本发明所述的抗肿瘤药物增敏剂通过刺激肿瘤细胞的免疫原性死亡，使对免疫治疗不敏感的“冷”肿瘤转变为对免疫治疗敏感的“热”肿瘤，有限募集T细胞以杀伤肿瘤细胞。
本发明所述的抗肿瘤药物增敏剂还通过下调肿瘤细胞P糖蛋白的表达，增加肿瘤细胞对化疗药物的摄取，降低肿瘤细胞的耐药性，提高肿瘤细胞对化疗药物敏感性，增强化疗药物对肿瘤细胞的细胞毒性和杀伤作用。
所述的抗肿瘤药物增敏剂可以下调肿瘤内吲哚胺-2，3-双加氧酶(IDO)的表达。IDO也是一种Fe 2+作为中心的酶，本抗肿瘤药物增敏剂可以络合Fe 2+，达到一定的抑制IDO的作用，减少IDO将具有免疫促进功能的色氨酸转变为具有免疫抑制功能的犬尿氨酸，将T细胞从免疫抑制状态中激活。
进一步的，所述的抗肿瘤药物增敏剂为下式(1)、(2)、(3)或(IOX1)的化合物：
Figure PCTCN2020127036-appb-000002
其中，IOX1很容易能够结合PHD3蛋白，因此具有很高的增敏功效；当IOX1的羟基被取代形成化合物2后，需要经水解转化成IOX1才能发挥增敏效果，因此增敏效果稍弱于IOX1；而当IOX1的羧基在被取代形成化合物3后，较难被水解成IOX1，因此增敏效果降低。因此，能够容易地转化为IOX1的化合物1-3的衍生物具有强的增敏效果。
进一步的，所述的抗肿瘤药物为临床用抗肿瘤药物，包括阿霉素、紫杉醇、吉西他滨、铂类药物、喜树碱及其衍生物、雷公藤红素或藤黄酸。
本发明所述的抗肿瘤药物增敏剂可与不同比例的抗肿瘤药物联用，抗肿瘤药物增敏剂与抗肿瘤药物的量之比为0.1-20∶1。
抗肿瘤药物增敏剂增强机体免疫应答，增强抗肿瘤药物的药效。将抗肿瘤药物与抗肿瘤药物增敏剂联用后获得显著增强的抗肿瘤效果。
本发明所述的肿瘤为恶性肿瘤，所述的恶性肿瘤包括血液类癌症、结直肠癌、乳腺癌、黑色素瘤、脑癌、胰腺癌、肺癌、肝癌或胆管癌等。
本发明所述的抗肿瘤药物增敏剂制成药物组合物，所述的给药物组合物包括治疗有效量抗肿瘤药物增敏剂和药学上可接受的载体。
所述的载体为脂质体、聚合物胶束或无机纳米粒等载纳米载体。
抗肿瘤药物增敏剂在被制备成纳米制剂后具有长血液循环时间、可以通过肿瘤的超通透与蓄积作用(Enhanced permeability and retention effect)在肿瘤组织的更加有效地蓄积，进一步提高其抗肿瘤效果。
本发明所述的药物组合物可通过常规口服、注射等给药方式直接给药。
本发明还提供了所述的带有抗肿瘤药物增敏剂的脂质体的制备方法，包括以下步骤：
原料液的制备：将抗肿瘤药物增敏剂溶于pH＝8～13的碱性水溶液中，得到浓度为1～50mg/mL的原料液；
脂质体膜的制备：将制备脂质体的常用磷脂或混合磷脂、聚乙二醇化磷脂溶于溶剂中，30～45℃浓缩成膜；
水化：在制得的磷脂膜中加入原料液，4～50℃水化12～48h；再置于透析袋中室温下透析6～48h，即得抗肿瘤药物增敏剂制成脂质体。根据需求可进一步用超声或挤出法控制所形成的脂质体的粒径。
所述的磷脂为磷脂酰胆碱、磷脂酰乙醇胺、二油酰基磷脂酰乙醇胺、胆固醇半琥珀酸酯、二硬脂酸磷脂酰乙醇胺，所述的聚乙二醇化磷脂为二硬脂酸磷脂酰乙醇胺-聚乙二醇2000。
所述的溶剂为二氯甲烷、三氯甲烷、甲醇或其混合液。
所述的溶剂为三氯甲烷与甲醇的混合液，三氯甲烷与甲醇的体积比为1-8∶1。
所述的透析袋截留分子量为500～10000KD。
本发明的有益效果主要体现在：
(1)本发明的抗肿瘤药物增敏剂具有免疫增敏的作用，包括抑制Hif-1α，降低PD-L1、IDO、IL10等免疫抑制分子的表达、增强机体对肿瘤的免疫反应，逆转肿瘤免疫抑制微环境、发挥免疫治疗的作用，从而提高肿瘤的免疫治疗效果。
(2)本发明的抗肿瘤药物增敏剂具有化疗增敏的作用，其通过抑制P-gp，逆转肿瘤多要耐药，提高肿瘤的化疗治疗效果。
(3)本发明的抗肿瘤药物增敏剂能够增敏免疫治疗和化疗效果，其通过同时抑制Hif-1α及P-gp，从而提高肿瘤的化疗与免疫治疗效果。
(4)与免疫检查点抑制剂PD-1/PD-L1抗体相比，所述抗肿瘤药物增敏剂为结构清楚的小分子化合物，合成简便。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。这里所采用的术语″药学上可接受的″，是针对那些化合物、材料、组合物和/或剂型而言。它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯横酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
除了盐的形式，本发明所提供的化合物还存在前药形式。本文所描述的化合物的前药容易地在生理条件下发生化学变化从而转化成本发明的化合物。此外，前体药物可以在体内环境中通过化学或生化方法被转换到本发明的化合物。
本发明的某些化合物可以以非溶剂化形式或者溶剂化形式存在，包括水合物形式。一般而言，溶剂化形式与非溶剂化的形式相当，都包含在本发明的范围之内。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有规定，术语“C 1-3烷基”用于表示包含1至3个碳原子的直链或支链的基团。所述C 1-3烷基包括C 1-3、C 1-2、C 1、C 2、C 3烷基等。其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-烷基的实例包括但不限于甲基(Me)，乙基(Et)，丙基(包括n-丙基和异丙基)等，本发明“C 1-3烷基”任选被1-5个F、Cl、Br、I、OH、NH 2、CN取代。
除非另有规定，术语“芳基”用于表示多不饱和的碳环体系，它可以是单环、双环或多环体系，其中至少一个环是芳香性的，所述的双环和多环体系中的各个环稠合在一起，其可以是单取代的或多取代的，可以是一价、二价或多价，C 6-12芳基的实例包括但不限于苯基、萘基(包括1-萘基和2-萘基)，本发明“芳基”任选被1-5个F、Cl、Br、I、OH、NH 2、CN取代。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝0)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。
术语“任选被取代”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可化任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
本发明术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请化合物的用量。构成“治疗有效量”的本申请化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
本发明术语“药物组合物”是指一种或多种本申请的化合物或其盐与药学上可接受的载体组成的混合物。药物组合物的目的是有利于对有机体给予本申请的化合物。
本发明术语“药学上可接受的载体”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水、脂质体、聚合物胶束或无机纳米载体等。
本发明所使用的溶剂可经市售获得。
本发明采用下列缩略词：IOX1代表5-羧基-8-羟基喹啉；PHD3代表抑制脯氨酸羟化酶3；PKM2代表M2型丙酮酸激酶；Hif-1α代表乏氧诱导因子-1α；PD-L1代表程序性死亡受体-配体1；PERK代表蛋白激酶R样内质网激酶；eIFα代表α亚基的真核起始因子2；p-eIFα代表磷酸化α亚基的真核起始因子2；CRT代表钙网蛋白；IDO代表吲哚胺2，3-双加氧酶；P-gp代表P糖蛋白；DOX代表阿霉素；DMSO代表二甲亚砜；PBS代表磷酸盐缓冲液；EDTA代表乙二胺四乙酸。
本发明化合物依据本领域常规命名原则或者使用
Figure PCTCN2020127036-appb-000003
软件命名，市售化合物采用供应商目录。
附图说明
图1为测试例1A中本发明化合物降低CT26细胞PD-L1表达的Western blot结果。
图2为测试例1A中本发明化合物降低CT26细胞PD-L1表达的Western blot的定量统计结果。
图3为测试例1A中IOX1降低CT26细胞PD-L1表达的Western blot结果。
图4为测试例1A中IOX1降低CT26细胞PD-L1表达的Western blot的定量统计结果。
图5为测试例1A中IOX1降低HCT26细胞PD-L1表达的Western blot结果。
图6为测试例1A中IOX1降低HCT26细胞PD-L1表达的Western blot的定量统计结果。
图7为测试例1B中IOX1降低CT26细胞PD-L1表达的流式细胞计数结果。
图8为测试例1B中IOX1降低4T1细胞PD-L1表达的流式细胞计数结果。
图9为测试例1B中IOX1降低HCT116细胞PD-L1表达的流式细胞计数结果。
图10为测试例1B中IOX1降低MCF-7细胞PD-L1表达的流式细胞计数结果。
图11为测试例1C中IOX1在CT26细胞上抑制DOX诱导PD-L1上调的流式细胞计数结果。
图12为测试例1C中IOX1在HCT116细胞上抑制DOX诱导PD-L1上调的流式细胞计数结果。
图13为测试例1D中IOX1与DOX联用在CT26细胞上引起内质网应激的Western blot结果。
图14为测试例1E中IOX1与DOX联用引起CT26细胞免疫原性死亡的流式细胞计数结果。
图15为测试例1E中IOX1与DOX联用引起HCT116细胞免疫原性死亡的流式细胞计数结果。
图16为测试例1F中IOX1与DOX联用引起CT26细胞免疫原性死亡的免疫荧光照片。
图17为测试例1G中IOX1与DOX联用引起树突状细胞成熟的流式细胞计数结果。
图18为测试例1H中IOX1与DOX联用对T淋巴细胞增殖作用的流式细胞计数结果。
图19为测试例1H中IOX1与DOX联用对T淋巴细胞活性作用的流式细胞计数结果。
图20为测试例1I中IOX1处理后小鼠巨噬细胞相关基因的表达，并与不处理的小鼠巨噬细胞比较。
图21为测试例1J中本发明化合物抑制吲哚胺2，3-双加氧酶的表达的实验结果。
图22为测试例2A中本发明化合物与化疗药物DOX联用的细胞毒性实验结果。
图23为测试例2A中本发明化合物的细胞毒性实验结果。
图24为测试例2A中IOX1与化疗药物紫杉醇(PTX)联用的细胞毒性实验结果。
图25为测试例2A中IOX1与化疗药物奥沙利铂(OXA)联用的细胞毒性实验结果。
图26为测试例2A中IOX1与化疗药物雷公藤红素(Cela)联用的细胞毒性实验结果。
图27为测试例2B中本发明化合物降低CT26细胞Hif-1α表达的结果。
图28为测试例2B中不同浓度IOX1降低CT26细胞Hif-1α表达的流式细胞计数结果。
图29为测试例2C中IOX1降低CT26细胞P-gp表达的Western blot结果。
图30为测试例2C中IOX1降低CT26细胞P-gp表达的Wesern blot结果的定量分析。
图31为测试例2C中IOX1降低HCT116细胞P-gp表达的Western blot结果。
图32为测试例2C中IOX1降低HCT116细胞P-gp表达的Wesern blot结果的定量分析。
图33为测试例2D中Rh123与IOX1联用后，Rh123在CT26细胞上的内吞结果，并与单独Rh123比较。
图34为测试例3中IOX1、DOX及两药联用后，对CT26荷瘤小鼠肿瘤模型的抑瘤曲线。
图35为测试例4-1中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后，对CT26小肿瘤荷瘤小鼠肿瘤模型的抑瘤曲线。
图36为测试例4-1中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后CT26小肿瘤荷瘤小鼠体重曲线。
图37为测试例4-2中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后，对CT26大肿瘤荷瘤小鼠肿瘤模型的抑瘤曲线。
图38为测试例4-2中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后CT26大肿瘤荷瘤小鼠体重曲线。
图39为测试例4-3中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后，对4T1原位肿瘤荷瘤小鼠肿瘤模型的抑瘤曲线。
图40为测试例4-3中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后4T1原位肿瘤荷瘤小鼠体重曲线。
图41为测试例4-4中实验结束时，阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后4T1原位瘤及肺转移肿瘤荷瘤小鼠肺部的活体荧光照片。
图42为测试例4-4中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后，对4T1原位瘤及肺转移肿瘤荷瘤小鼠肿瘤模型的抑瘤曲线。
图43为测试例4-4中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后4T1原位瘤及肺转移肿瘤荷瘤小鼠体重曲线。
图44为测试例4-5中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后，对B16F10皮下肿瘤荷瘤小鼠肿瘤模型的抑瘤曲线。
图45为测试例4-5中阿霉素脂质体(Doxil)和5-羧基-8-羟基喹啉脂质体(Ioxil)联用后B16F10皮下肿瘤荷瘤小鼠体重曲线。
具体实施方式
下面通过实施例对本发明进行详细阐述，下述说明仅为解释本发明，并不对其内容进行限定。本发明化合物可以通过本领域技术人员熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其它化合物合成方法的结合所形成的实施方式以及本领域技术人员所熟知的等同替换方式，也可以可经市售获得。优选的实施方式包括但不限于本发明的实施例。对本领域技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进是显而易见的。
实施例1：化合物1的制备。
Figure PCTCN2020127036-appb-000004
取IOX1(200mg，1.06mmol)溶于10mL N，N-二甲基甲酰胺，加入二碳酸二叔丁酯(278mg，1.272mmol)，50℃回流反应6h，减压旋蒸除去溶剂后复溶于10mL甲醇，加入对甲苯磺酸(9.1mg，0.05mmol)，80℃回流反应12h后冷却至室温，减压旋蒸除去甲醇。加入10mL二氯甲烷和等体积三氟乙酸，反应3h后旋蒸除去大部分三氟乙酸，期间用三氯甲烷夹带蒸馏3次，之后倒入大量乙醚，过滤，滤饼为目标产物，干燥得化合物1，产率为80.3％。分子量(MALDI-TOF，m/z)：C 11H 9NO 3204[M+H] +。
实施例2：化合物2的制备。
Figure PCTCN2020127036-appb-000005
取IOX1(200mg，1.06mmol)溶于20mL干燥四氢呋喃，加入三乙胺(161mg，1.59mmol)，N 2保护、冰浴下滴入含乙酰氯(92mg，1.17mmol)的四氢呋喃溶液，搅拌1h后撤去冰浴并继续反应过夜。减压旋蒸除去大部分四氢呋喃，倒入大量正己烷，过滤，滤饼为目标产物，干燥得化合物2，产率为73.6％。分子量(MALDI-TOF，m/z)：C 11H 9NO 3232[M+H] +。
实施例3：化合物3的制备
Figure PCTCN2020127036-appb-000006
取化合物1(200mg，0.99mmol)溶于10mL干燥四氢呋喃，加入三乙胺(150.73mg，1.485mmol)，N 2保护，冰浴下滴入含乙酰氯(96mg，1.09mmol)的四氢呋喃溶液，搅拌1h后撤去冰浴并继续反应过夜。减压旋蒸除去四氢呋喃，用二氯甲烷重新溶解，之后用碳酸 氢钠、去离子水、饱和食盐水分别洗涤，无水硫酸镁或无水硫酸钠干燥后，浓缩过柱，展开剂比例为正己烷∶乙酸乙酯＝3∶1，旋干后得淡黄色固体化合物3，产率为96％。分子量(MALDI-TOF，m/z)：C 11H 9NO 3246[M+H] +。
测试例1：本发明化合物作为免疫增敏剂。
配置方法：将实施例制得的本发明化合物配置为不同浓度的DMSO溶液(5μM，25μM，50μM)，用于细胞实验中。
测试例1A：Western blot技术检测本发明化合物降低肿瘤细胞PD-L1的表达。
以每孔2×10 4个鼠源CT26或人源HCT116细胞种于6孔板中，置于37℃恒温培养箱。待细胞贴壁后，分别加入含有本发明化合物的DMSO溶液，继续孵育48h。
弃去培养基，用预冷的PBS润洗细胞三遍并弃去洗液，每孔加入0.2mL含蛋白酶抑制剂的细胞裂解液，置于冰上裂解30min。裂解完成后，用细胞刮刀将裂解液及细胞碎片刮至培养皿一侧，并用移液枪将裂解液转移至1.5mL Ep管中，于4℃离心(12000rpm/5min)后收集上清，并测定其蛋白浓度。
用RIPA裂解液将蛋白样品稀释至一定浓度，加入5×SDS上样缓冲液至蛋白量1μg/μL，置于95℃金属浴上孵育5min，冷却至室温后上样至制备好的SDS-PAGE电泳胶中，浓缩胶为5％，分离胶为12％。用90V电压将蛋白跑至浓缩胶底部成一条直线后调整电压为125V，至溴酚蓝跑出后终止电泳。
随后将蛋白转移至硝酸纤维膜上，电压为80V，转膜时间90min。结束后将膜置于5％的脱脂牛奶中于室温封闭一小时。TBST缓冲液洗膜三次后分别加相应的一抗(anti-PD-L1，1∶2000，，anti-GAPDH，1∶10000)，4℃孵育过夜。用TBST缓冲液洗膜三次后加入辣根过氧化酶标记的二抗，室温下孵育1h。充分洗膜后进行化学发光显色，并用化学发光成像仪拍照。
测试结果如图1-6所示，图1-2显示本发明化合物可以降低肿瘤细胞PD-L1的表达，其中以IOX1最优；图3-6显示IOX1显著降低人源及鼠源肿瘤细胞PD-L1的表达，并呈浓度依赖。
测试例1B：流式细胞仪检测本发明化合物降低肿瘤细胞PD-L1的表达。
将鼠源CT26、4T1或人源HCT116、MCF-7细胞以每孔2×10 4个种植于6孔板中。待细胞贴壁后，分别加不同浓度(5μM，25μM，50μM)的含有本发明化合物的DMSO溶液，继续孵育24h。弃掉培养基，用PBS润洗细胞3遍，每孔加含EDTA的胰蛋白酶0.2mL。将消化后的细胞收集于流式管中，离心去除上清，用含5％山羊血清的PBS重悬细胞，并加入抗鼠PD-L1一抗(1μg/1×10 6个细胞)，于4℃孵育30min后，用PBS洗三次，再加入等量APC标记的山羊抗兔二抗，继续孵育30min后，用PBS清洗3次，上机进行流式检测。
结果如图7-10所示，IOX1降低肿瘤细胞表面PD-L1的表达呈现浓度依赖，灰色区域的积分值代表PD-L1表达率，以百分数形式标注在图形对应区域。
测试例1C：流式细胞仪检测本发明化合物抑制化疗药物引起肿瘤细胞PD-L1的上调。
将CT26或HCT116细胞以每孔2×10 4个种植于6孔板中。过夜贴壁后分别加入本发明化合物IOX1的处理组，继续孵育24h。弃掉培养基，用PBS润洗细胞3遍，每孔加含EDTA的胰蛋白酶0.2mL。将消化后的细胞收集于流式管中，离心去除上清，用含5％山羊血清的PBS重悬细胞，并加入PD-L1一抗(1μg/1×10 6个细胞)，于4℃孵育30min后，用PBS洗三次，再加入等量APC标记的山羊抗兔二抗，继续孵育30min后，用PBS清洗3次，上机进行流式检测。
以阿霉素与IOX1联用展示结果，结果如图11-12所示，IOX1可以抑制阿霉素在CT26或HCT116肿瘤细胞上抑制阿霉素在CT26肿瘤细胞上诱导的PD-L1上调。
测试例1D：本发明化合物增强药物引起的肿瘤细胞的内质网应激。
以每孔2×10 4个CT26细胞种于6孔板中，置于37℃恒温培养箱。待细胞贴壁后，分别加入含有本发明化合物IOX1的处理组，继续孵育4h。
弃去培养基，用预冷的PBS润洗细胞三遍并弃去洗液，每孔加入0.2mL含蛋白酶抑制剂的细胞裂解液，置于冰上裂解30min。裂解完成后，用细胞刮刀将裂解液及细胞碎片刮至培养皿一侧，并用移液枪将裂解液转移至1.5mL Ep管中，于4℃离心(12000rpm/5min)后收集上清，并测定其蛋白浓度。
用RIPA裂解液将蛋白样品稀释至一定浓度，加入5×SDS上样缓冲液至蛋白量1μg/μL，置于95℃金属浴上孵育5min，冷却至室温后上样至制备好的SDS-PAGE电泳胶中，浓缩胶为5％，分离胶为12％。用90V电压将蛋白跑至浓缩胶底部成一条直线后调整电压为125V，至溴酚蓝跑出后终止电泳。
随后将蛋白转移至硝酸纤维膜上，电压为80V，转膜时间90min。结束后将膜置于5％的脱脂牛奶中于室温封闭一小时。TBST缓冲液洗膜三次后分别加相应的一抗(anti-p-PERK，1∶2000，anti-p-eIFα，1∶2000，anti-eIFα，1∶2000，anti-Actin，1∶10000)，4℃孵育过夜。用TBST缓冲液洗膜三次后加入辣根过氧化酶标记的二抗，室温下孵育1h。充分洗膜后进行化学发光显色，并用化学发光成像仪拍照。
以阿霉素与IOX1联用展示结果，结果如图13所示，IOX1可以使阿霉素在CT26肿瘤细胞上激活PERK/eIF2α通路，产生更强的内质网应激。
测试例1E：流式细胞仪检测本发明化合物增强药物引起的肿瘤细胞的免疫原性死亡。
将CT26或HCT116细胞以每孔2×10 4个种植于6孔板中。过夜贴壁后分别加入本发明化合物IOX1的处理组，继续孵育4h。弃掉培养基，用PBS润洗细胞3遍，每孔加含EDTA的胰蛋白酶0.2mL。将消化后的细胞收集于流式管中，离心去除上清，用含5％山羊血清的PBS重悬细胞，并加入CRT一抗(1μg/1×10 6个细胞)，于4℃孵育30min后，用PBS洗三次，再加入等量APC标记的山羊抗兔二抗，继续孵育30min后，用PBS清洗3次，上机进行流式检测。
以阿霉素与IOX1联用展示结果，结果如图14-15所示，IOX1可以使阿霉素在CT26或HCT116肿瘤细胞上诱导更强的CRT膜易位，增加CT26或HCT116细胞免疫原性死亡。
测试例1F：免疫荧光法观察本发明化合物增强药物引起的肿瘤细胞的免疫原性死亡。
CT26细胞铺于共聚焦培养皿中，过夜贴壁后分别加入本发明化合物IOX1的处理组，继续孵育4h。弃掉培养基，用PBS润洗细胞3遍，4％多聚甲醛固定10min，更换PBS润洗3次，每次3min，加入3％BSA溶液于37℃封闭30min，吸水纸吸掉封闭液，每孔加200μL钙网蛋白抗体(anti-CRT，1∶200)，常温下避光孵育1h后，用PBS洗三次，再加入等量APC标记的山羊抗兔二抗，继续孵育30min后，继续用PBS润洗细胞3次，随后向每皿中加入DAPI染液，避光孵育5min，PBS清洗三次后于激光共聚焦显微镜下观察。
以阿霉素与IOX1联用展示结果，结果如图16所示，IOX1可以使阿霉素在CT26肿瘤细胞上产生更强的CRT，增加CT26细胞免疫原性死亡。
测试例1G：本发明化合物IOX1增强药物引起的树突状细胞成熟。
CT26细胞铺于Transwell小室内，过夜贴壁后分别加入本发明化合物IOX1的处理组，继续孵育24h。弃掉含药培养液，更换新鲜培养液，同时在Transwell下层铺入Balb/c小鼠体内提取的骨髓来源树突状细胞。继续培养24h后，取出含有肿瘤细胞的小室，将下层的树突状细胞转移至流式管内离心，弃去上清，PBS洗涤细胞沉淀3次，用含5％山羊血清的PBS重悬细胞，并加入FITC anti-CD11c，APC anti-CD86和PE anti-CD80抗体，流式细胞仪检测CD11c +CD86 +CD80 +细胞比例。
以阿霉素与IOX1联用展示结果，结果如图17所示，IOX1联用阿霉素可以诱导产生更多成熟的DC细胞，增加抗原递呈。
测试例1H：本发明化合物IOX1增强T淋巴细胞的体外增殖及活性。
CT26细胞铺于6孔板中，过夜贴壁后分别加入本发明化合物IOX1的处理组，继续孵育24h。弃掉培养基，加入用CFSE标记的Balb/c小鼠外周血单核细胞(Peripheral Blood  Mononuclear Cell，PBMC)，同时设一组不加PBMC，只加相同体积的培养液的对照组。继续孵育4天后，将含有PBMC的上清液收集于流式管内，离心，PBS洗涤细胞沉淀3次后，流式检测PBMC增殖结果。对于贴壁的CT26细胞，用胰酶消化后收集于流式管中，离心，PBS洗涤细胞沉淀3次后，加入FITC-Annexin V及PI染液，室温下避光染色15min，流式细胞仪检测细胞凋亡水平。
结果如图18-19所示，图18显示本IOX1可以增加T淋巴细胞的增殖率，与对照组相比增加了16.3％，IOX1联合DOX还可以进一步增加T淋巴细胞的增殖，其增殖率高达40.9％。
图19显示IOX1可以恢复T淋巴细胞对肿瘤细胞的杀伤能力，T细胞几乎不能诱导不加处理的肿瘤细胞凋亡；而与对照组相比，T淋巴细胞作用于提前用IOX1处理的肿瘤细胞后，将其凋亡率提升了13.71％；IOX1再与DOX联用后更是增加肿瘤凋亡率至91.8，证明IOX1可以显著增加T淋巴细胞的体外增殖及活性。
测试例1I：本发明化合物IOX1促进巨噬细胞由M2向M1的极化。
小鼠巨噬细胞株Raw264.7接种于24孔板中，12小时细胞贴壁后，用含IL-4(40ng/mL)的培养基继续培养细胞一天，诱导其分化为M2型巨噬细胞(TAM2)。随后向TAM2中加入不同组的还有本发明化合物的溶液(5μM)，处理24h后收集细胞，裂解并提取总RNA，进行反转录和PCR实验，检测M2型巨噬细胞特异性蛋白arg1及M1型巨噬细胞特异性蛋白Nos2的RNA水平，以hprt基因为内参。
IOX1的结果如图20所示，与空白组处理的TAM2相比，加入IOX1后arg1显著降低，且Nos2显著升高，证明本发明化合物可以促进巨噬细胞由M2向M1的极化，将促肿瘤生长的M2型巨噬细胞转成肿瘤抑制型的M1型巨噬细胞，因而有利于提高肿瘤治疗效果。
测试例1J：本发明化合物抑制吲哚胺2，3-双加氧酶的表达。
将CT26细胞以5×10 4个/孔种植于12孔板，每孔加入2mL培养基(含有100μM色氨酸)。培养一天后加入一定浓度梯度的本发明化合物(1-100μM)，再加入0.1μg/mL INF-γ以诱导IDO的表达。孵育72h后，取200μL上清液加入10μL30％三氟乙酸溶液沉淀蛋白。用HPLC检测溶液中犬尿氨酸的含量，每个孔重复三次。
以IOX1为例，结果如图21所示，IOX1可以能够抑制CT26产生犬尿氨酸，抑制程度随IOX1浓度的升高而增强，IOX1的浓度为100μM时犬尿氨酸的抑制程度为33.2％。
结论：IOX1可抑制色氨酸向犬尿氨酸转变，表明IOX1能够抑制吲哚胺2，3-双加氧酶的活性或表达。因此，本发明化合物可以通过抑制吲哚胺2，3-双加氧酶的表达，增强癌症免疫治疗效果。
测试例2：本发明化合物作为化疗增敏剂的应用。
配置方法：
将实施例制得的本发明化合物配置为DMSO溶液，用于细胞实验中。
测试例2A：本发明化合物与多种化疗药物联用的细胞毒性研究。
将CT26细胞(MC38细胞、4T1细胞、B16F10细胞、HePa1-6细胞、H22细胞、LLC细胞、MB49细胞、P388细胞、C6细胞、HCT116细胞，MCF-7细胞，BXPC-3细胞、Hela细胞、MDA-MB-231细胞、A2780细胞、PC3细胞、HepG2细胞、HGC-27细胞)以5000个/孔分别培养于96孔板中，每孔加入100μL培养基，于5％CO 2浓度和95％湿度的37℃恒温培养箱中培养24h。向每个孔加入100μL不同浓度的药物(DOX：0.01-10μg/mL；PTX：0.01-50μg/mL；Cela：0.05-1μg/mL；IOX1为5μg/mL)，空白组加入100μL的培养基溶液。继续培养48h后，于1100rpm离心6min后弃去每个孔中的培养基，加入100μL的MTT工作液，继续培养3h。其后于3300rpm离心5min，弃尽每孔中的MTT工作液，加入100μL的DMSO，震荡5min，使每孔中的结晶全部溶解。最终用酶标仪测试样品在562nm处的吸光度。每一组数据均为同一种样品三次独立实验的平均值。
实验结果如图22-26及表1-2所示。
图22、24～26显示与单独使用化疗药物相比，本发明化合物与化疗药物联用可以极大地降低细胞地生存率，其中以IOX1效果最优；图23显示在0.1-10μg/mL，本发明化合物不具有细胞毒性。证明IOX1及本发明其它化合物也可以增加紫杉醇(PTX)、奥沙利铂(OXA)或雷公藤红素(Cela)等对CT26细胞的毒性。
表1为IOX1与化疗药物DOX联用在多种细胞系上的细胞毒性实验结果，实验结果显示IOX1与DOX联用可以极大地降低多种肿瘤细胞的生存率。
表1
Figure PCTCN2020127036-appb-000007
表2为IOX1与多种化疗药物联用在CT26细胞系上的细胞毒性实验结果。实验结果显示IOX1可以增加多种化疗药物的对CT26细胞的毒性。
表2
Figure PCTCN2020127036-appb-000008
Figure PCTCN2020127036-appb-000009
测试例2B：流式细胞仪检测本发明化合物降低肿瘤细胞Hif-1α的表达。
以每孔2×10 4个的密度将CT26细胞均匀地铺于6孔板中。待细胞贴壁后，分别加入本发明化合物的DMSO溶液，继续孵育24h。
弃掉培养基，用PBS润洗细胞3遍，每孔加含EDTA的胰蛋白酶0.2mL。将消化后的细胞收集于流式管中，离心去除上清，用含5％山羊血清的PBS重悬细胞，并加入FITC抗鼠Hif-1α抗体(1μg/1×10 6个细胞)于4℃孵育30min后，PBS洗三次，上机进行流式检测。
结果如图27-28所示，图27为化合物1-3、IOX1浓度为5μM时，降低肿瘤细胞Hif-1α的程度，与空白对照相比，本发明化合物可以显著降低肿瘤细胞Hif-1α的表达，其中以IOX1效果最优，5μM时Hif-1α的表达减少了38％。此外，图28显示，IOX1浓度为1μM时，Hif-1α的表达减少了32.8％；IOX1浓度为5μM时，Hif-1α的表达减少了38％；IOX1降低肿瘤细胞Hif-1α的表达的能力随浓度的升高而增强。
测试例2C：本发明化合物IOX1降低肿瘤细胞P-gp的表达。
以每孔2×10 4个的密度将CT26或HCT116细胞均匀地铺于6孔板中。待细胞贴壁后，分别加不同浓度的IOX1溶液，继续孵育48h。
随后弃去培养基，用预冷的PBS润洗细胞三遍并弃去洗液，每孔加入0.2mL含蛋白酶抑制剂的细胞裂解液，置于冰上裂解30min。裂解完成后，用细胞刮刀将裂解液及细胞碎片刮至培养皿一侧，并用移液枪将裂解液转移至1.5mL Ep管中，于4℃离心(12000rpm/5min)收集上清，并测定蛋白浓度。
用RIPA裂解液将蛋白样品稀释至一定浓度，加入5×SDS上样缓冲液，使上样蛋白量为20μg，体积20μL，置于95℃金属浴上孵育5min，冷却至室温后上样至制备好的SDS-PAGE电泳胶中，浓缩胶为5％，分离胶为12％。用90V电压将蛋白跑至浓缩胶底部成一条直线后调整电压为125V，至溴酚蓝跑出后终止电泳。
随后将蛋白转移至硝酸纤维膜上，电压为80V，转膜时间90min。结束后将膜置于5％的脱脂牛奶中于室温封闭一小时。TBST缓冲液洗膜三次后分别加相应的一抗(anti-P-gp，1∶4000，anti-GAPDH，1∶10000)，4℃孵育过夜。TBST缓冲液洗膜三次后加入辣根过氧化酶标记的二抗，室温下孵育1h。充分洗膜后进行化学发光显色，并用化学发光成像仪拍照。
结果如图29-32所示，5-羧基-8-羟基喹啉(IOX1)能降低P-gp表达，并呈现浓度依赖趋势；图30或32是根据图29或31的western结果计算出的P-gp/GAPDH表达比。
测试例2D：本发明化合物增强肿瘤细胞对罗丹明123(Rh123)的入胞能力。
Rh123为多药耐药蛋白P-gp的底物，P-gp表达降低后可降低Rh123的细胞外排，增加其细胞内的含量，因此可用其测定细胞的P-gp蛋白的活性。
将CT26细胞按1×10 4/孔的密度种于共聚焦成像皿中，并将细胞置于37℃恒温培养箱培养24h。随后每孔换成新鲜培养基，并加入Rh123溶液及不同浓度的IOX1溶液：A组：1μM Rh123；B组：1μM Rh123+1μM IOX1；C组：10μM Rh123；D组：10μM Rh123+10μM IOX1，孵育6h后用共聚焦显微镜观察Rh123的细胞内的情况。Rh123的激发波长为488nm，发射波长为500至550nm。
结果如图33所示，加入5-羧基-8-羟基喹啉(IOX1)后能够显著增加Rh123的细胞内浓度，证明其可以通过下调P-gp减少对Rh123的外排。
测试例3：本发明化合物增强药物的体内抑瘤活性。
配置方法：以5-羧基-8-羟基喹啉(IOX1)为例，取30mg IOX1溶于1mLDMSO，加入1mL聚乙二醇500、吐温80或聚氧乙烯蓖麻油(本试验用的是吐温80)，涡旋至混合均匀，取1mL 上述溶液加入到9mL PBS中，混匀即得IOX1注射液。该注射液可在4℃保存6个月以上，不会有固体粉末析出。
试验目的：考察IOX1与化疗药物阿霉素联用(阿霉素、紫杉醇、吉西他滨、奥沙利铂、喜树碱衍生物、伊立替康、雷公藤红素等)对CT26鼠结肠癌的肿瘤抑制作用。
试验步骤：Balb/c白鼠皮下注射1×10 6CT26肿瘤细胞，待肿瘤长至约80mm 3后开始每隔两天(Day7，Day9，Day11)进行尾静脉注射。以5-羧基-8-羟基喹啉与盐酸阿霉素联用为例，分别为空白对照组(PBS)、5-羧基-8-羟基喹啉组、盐酸阿霉素组、5-羧基-8-羟基喹啉+盐酸阿毒素组(D1|1：DOX 3mg/kg+IOX1 7.5mg/kg；D1|2：DOX 3mg/kg+IOX1 15mg/kg；D1|3：DOX 5mg/kg+IOX1 7.5mg/kg；D1|4：DOX 5mg/kg+IOX1 15mg/kg；)。给药结束后继续观察白鼠4天。
结果如图34所示，同单独使用5-羧基-8-羟基喹啉或盐酸阿霉素对比，两者联用治疗组的肿瘤体积没有增长，并且呈一定的降低趋势，停药后肿瘤没有增长，保持不变。说明5-羧基-8-羟基喹啉与盐酸阿霉素联用表现出更显著的抗癌活性，而且治疗后具有一定的记忆性。同时，IOX1可使其他抗肿瘤药物的抑瘤率提高30-70％。
测试例4：5-羧基-8-羟基喹啉脂质体制剂的制备及其与化疗药物联用抗肿瘤活性测试。
配置方法：
采用碳酸钠梯度法制备5-羧基-8-羟基喹啉的脂质体制剂。
步骤1：首先将IOX1(5mg)溶于pH＝9的碳酸钠水溶液中，使其浓度为5mg/mL。
步骤2：之后将12.89g二油酰基磷脂酰乙醇胺(DOPE)、2.11g胆固醇半琥珀酸酯(CHEMS)、6.52g二硬脂酸磷脂酰乙醇胺-聚乙二醇2000(DSPE-mPEG 2000)溶于三氯甲烷(12mL)及甲醇(4mL)中，37℃水浴下减压旋干成膜。
步骤3：将步骤1的5-羧基-8-羟基喹啉溶液加入步骤2的脂质体膜中，室温水化过夜。
步骤4：将所得的溶液置于透析膜(截留分子量3500)中，置于纯水中，室温下透析6h，即得IOX1脂质体制剂(Ioxil，载药效率93％)。
脂质体可以用不同种类的脂质原料制备，并可根据需要选择脂质原料的良溶剂，如二氯甲烷、三氯甲烷、甲醇等；本试验例选用三氯甲烷∶甲醇＝3∶1(12mL及3mL)；步骤1中的5-羧基-8-羟基喹啉的水溶液浓度可根据实际需要调整。
药物制剂的尺寸表征：利用动态光散射(Dynamic Light Scattering，DLS)测定其粒度及分布，脂质制剂在水中组装成动态粒径分布为0.125，平均尺寸为102.3nm的纳米颗粒。该尺寸可用脂质体组成、制备方法等进行调节。
测试例4-1：CT26皮下小肿瘤模型
Balb/c白鼠皮下注射1×10 6个CT26肿瘤细胞，待肿瘤长至约80mm 3后开始给药，每隔两天(Day0，Day2，Day4)进行尾静脉注射。以5-羧基-8-羟基喹啉脂质体制剂与阿霉素脂质体联用为例，分别为空白对照组(PBS)、阿霉素脂质体(Doxil，DOX浓度5mg/kg)组、5-羧基-8-羟基喹啉脂质体+阿霉素脂质体(Doxil+Ioxil，DOX浓度5mg/kg，IOX1浓度7.5mg/kg)。给药周期结束后继续观察白鼠18天。
结果如图35-36所示，图35显示两种制剂联用的抗肿瘤效果明显优于单独使用阿霉素脂质体。22天后，联用组的肿瘤完全消除，而阿霉素脂质体组的肿瘤仍呈现生长趋势。图36显示联用组小鼠体重没有下降的现象，显示出药物的生物安全度高，毒副作用小。
测试例4-2：CT26皮下大肿瘤模型
Balb/c白鼠皮下注射1×10 6个CT26肿瘤细胞，待肿瘤长至约350mm 3后开始给药，每隔两天(Day0，Day2，Day4，Day6)进行尾静脉注射。以5-羧基-8-羟基喹啉脂质体制剂与阿霉素脂质体联用为例，分别为空白对照组(PBS)、阿霉素脂质体(Doxil，DOX浓度5mg/kg)组、5-羧基-8-羟基喹啉脂质体+阿霉素脂质体(Doxil+Ioxil，DOX浓度5mg/kg，IOX1浓度7.5mg/kg)。给药周期结束后继续观察白鼠28天。
结果如图37-38所示，图37显示两种制剂联用的抗肿瘤效果明显优于单独使用阿霉素脂质体。34天后，联用组的肿瘤完全消除，而阿霉素脂质体组的肿瘤仍呈现生长趋势。图38显示联用组小鼠体重没有下降的现象，显示出药物的生物安全度高，毒副作用小。
测试例4-3：4T1原位肿瘤模型
Balb/c白鼠皮下注射1×10 6个4T1肿瘤细胞，待肿瘤长至约100mm 3后开始给药，每隔两天(Day0，Day2，Day4)进行尾静脉注射。以5-羧基-8-羟基喹啉脂质体制剂与阿霉素脂质体联用为例，分别为空白对照组(PBS)、阿霉素脂质体(Doxil，DOX浓度5mg/kg)组、5-羧基-8-羟基喹啉脂质体+阿霉素脂质体(Doxil+Ioxil，DOX浓度5mg/kg，IOX1浓度7.5mg/kg)。给药周期结束后继续观察白鼠20天。
结果如图39-40所示，图39显示两种制剂联用的抗肿瘤效果明显优于单独使用阿霉素脂质体。24天后，联用组的肿瘤完全消除，而阿霉素脂质体组的肿瘤仍呈现生长趋势。图40显示联用组小鼠体重没有下降的现象，显示出药物的生物安全度高，毒副作用小。
测试例4-4：4T1原位及肺转移双肿瘤模型
Balb/c白鼠皮下注射1×10 6个4T1肿瘤细胞，待肿瘤长至约100mm 3后开始给药，每隔两天(Day0，Day2，Day4)进行尾静脉注射。以5-羧基-8-羟基喹啉脂质体制剂与阿霉素脂质体联用为例，分别为空白对照组(PBS)、阿霉素脂质体(Doxil，DOX浓度5mg/kg)组、5-羧基-8-羟基喹啉脂质体+阿霉素脂质体(Doxil+Ioxil，DOX浓度5mg/kg，IOX1浓度7.5mg/kg)。给药周期结束3天后，通过尾静脉注射2×10 5个表达化学发光酶的4T1肿瘤细胞( Luci4T1)，继续观察白鼠至第22天。
结果如图41-43所示，图41-42显示两种制剂联用的抗肿瘤效果明显优于单独使用阿霉素脂质体。22天后，联用组肺部检测不到化学发光信号，证明了联用两种脂质体能引起机体产生强力的免疫反应；此外，联用组的原位肿瘤实现60％的消除率，而阿霉素脂质体组的肿瘤已进入指数生长期。图43显示联用组小鼠体重没有下降的现象，显示出药物的生物安全度高，毒副作用小。
测试例4-5：B16F10皮下小肿瘤模型
Balb/c白鼠皮下注射1×10 6个B16F10肿瘤细胞，待肿瘤长至约80mm 3后开始给药，每隔两天(Day0，Day2，Day4)进行尾静脉注射。以5-羧基-8-羟基喹啉脂质体制剂与阿霉素脂质体联用为例，分别为空白对照组(PBS)、阿霉素脂质体(Doxil，DOX浓度5mg/kg)组、5-羧基-8-羟基喹啉脂质体+阿霉素脂质体(Doxil+Ioxil，DOX浓度5mg/kg，IOX1浓度7.5mg/kg)。给药周期结束后继续观察白鼠20天。
结果如图44-45所示，图44显示两种制剂联用的抗肿瘤效果明显优于单独使用阿霉素脂质体，24天后，联用组的肿瘤远远小于阿霉素脂质体组的肿瘤。图45显示联用组小鼠体重没有下降的现象，显示出药物的生物安全度高，毒副作用小。
制备成药物制剂后，5-羧基-8-羟基喹啉与化疗药物的联用表现出显著的抗癌活性，治疗效果在本领域处于领先水平，具有良好的应用前景。

Claims (10)

  1. 一种基于5-羧基-8-羟基喹啉的抗肿瘤药物增敏剂，其特征在于，所述的抗肿瘤药物增敏剂为式(I)化合物或其药学上可接受的盐，
    Figure PCTCN2020127036-appb-100001
    其中，R 1为H、OH、NH 2、C 1-3烷基、-O-C 1-3烷基或-O-C 6-12芳基；
    R 2为H、C 1-3烷基、-C(＝O)-C 1-3烷基或-C(＝O)-C 6-12芳基。
  2. 根据权利要求1所述的抗肿瘤药物增敏剂，其特征在于，所述的抗肿瘤药物增敏剂为下式(1)、(2)、(3)或(IOX1)的化合物：
    Figure PCTCN2020127036-appb-100002
  3. 根据权利要求1或2所述的抗肿瘤药物增敏剂，其特征在于，所述的抗肿瘤药物为阿霉素、紫杉醇、吉西他滨、铂类药物、喜树碱及其衍生物、雷公藤红素或藤黄酸。
  4. 根据权利要求3所述的抗肿瘤药物增敏剂，其特征在于，所述的抗肿瘤药物增敏剂与抗肿瘤药物联用，抗肿瘤药物增敏剂与抗肿瘤药物的的质量比为0.1-20：1。
  5. 根据权利要求1或2所述抗肿瘤药物增敏剂，其特征在于，所述的肿瘤为恶性肿瘤。
  6. 根据权利要求1或2所述的抗肿瘤药物增敏剂，其特征在于，所述的抗肿瘤药物增敏剂制成药物组合物，所述的药物组合物包括治疗有效量抗肿瘤药物增敏剂和药学上可接受的载体。
  7. 根据权利要求6所述的抗肿瘤药物增敏剂，所述的载体为水、脂质体、聚合物胶束或无机纳米载体。
  8. 根据权利要求6所述的抗肿瘤药物增敏剂，其特征在于，所述的药物组合物的制备方法，包括以下步骤：
    原料液的制备：将抗肿瘤药物增敏剂溶于pH＝8～13的碱性水溶液中，得到浓度为1～50mg/mL的原料液；
    脂质体膜的制备：将磷脂或聚乙二醇化磷脂或其混合物溶于溶剂中，30～45℃浓缩成膜；
    水化：在制得的脂质体膜中加入原料液，4～50℃水化12～48h；再置于透析袋中室温下透析6～48h。
  9. 根据权利要求8所述的抗肿瘤药物增敏剂，其特征在于，所述的溶剂为二氯甲烷、三氯甲烷、甲醇或其混合液。
  10. 根据权利要求8所述的抗肿瘤药物增敏剂，其特征在于，所述的透析袋截留分子量为500～10000KD。
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