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WO2021120874A1 - 一种基于n-(3-羟基吡啶-2-羰基)甘氨酸的抗肿瘤药物增敏剂及其应用 - Google Patents

一种基于n-(3-羟基吡啶-2-羰基)甘氨酸的抗肿瘤药物增敏剂及其应用 Download PDF

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WO2021120874A1
WO2021120874A1 PCT/CN2020/124755 CN2020124755W WO2021120874A1 WO 2021120874 A1 WO2021120874 A1 WO 2021120874A1 CN 2020124755 W CN2020124755 W CN 2020124755W WO 2021120874 A1 WO2021120874 A1 WO 2021120874A1
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tumor drug
tumor
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申有青
刘婧
赵志浩
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浙江大学
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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Definitions

  • the invention relates to the technical field of medicine, in particular to the application of N-(3-hydroxypyridine-2-carbonyl)glycine and its derivatives in the preparation of antitumor drug sensitizers.
  • tumors especially solid tumors
  • TME tumor microenviroment
  • ROS reactive oxygen species
  • the immunosuppressive tumour network myeloid-derived suppressor cells, regulatory T cells and natural killer T cells, Immunology, 2013, 2(138): 105).
  • the metabolism of tumor cells is more vigorous than that of normal cells.
  • the malformed and uneven distribution of blood vessels in tumor tissues results in the obstruction of oxygen delivery, and ultimately causes a large number of hypoxic areas in the tumor (Hypoxia-inducible factor 1 is a basic-helix-loop-helix -PAS heterodimer regulated by cellular O 2 tension, Proceedings of The National Academy of Science of The United States of America, 1995, 92(12): 5510;
  • the tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis, Nature , 1999, 6733(399):271).
  • hypoxia in tumor tissues is closely related to the tumor immunosuppressive microenvironment.
  • tumor hypoxia can lead to the production of M2 tumor-associated macrophages (TAM) and inhibit tumor immune response (HIF-1 alpha is essential for myeloid cell-mediated inflammation, Cell, 2003, 112(5) : 645-657; Hypoxia in cancer: significance and impact on clinical outcome, Cancer And Metastasis Reviews, 2007, 2(26): 225).
  • TAM tumor-associated macrophages
  • HIF-1 alpha is essential for myeloid cell-mediated inflammation, Cell, 2003, 112(5) : 645-657
  • Hypoxia in cancer significance and impact on clinical outcome, Cancer And Metastasis Reviews, 2007, 2(26): 225).
  • Tumor hypoxia can inhibit the proliferation and activation of tumor-infiltrating lymphocytes (TIL), especially cytotoxic T lymphocytes (CTL), and then affect the function of effector T cells (Obesity in C57BL/ 6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration, International Journal Of Obesity, 2008, 3(32): 451; Inhibitory effect of tumor cell-derived lactic acid on human T cells, Blood, 2007, 9(109 ): 3812; HIF Transcription Factors, Inflammation, and Immunity, Immunity, 2014, 4(41): 518).
  • TIL tumor-infiltrating lymphocytes
  • CTL cytotoxic T lymphocytes
  • Tumor hypoxia can also induce cancer cells and macrophages to overexpress PD-L1 and then induce programmed death of CTL cells (A Mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells, Cancer Research, 2014, 3(74): 665; Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma, Science, 2018, 359(6377): 801).
  • the unique microenvironment of the tumor also recruits a large number of immunosuppressive cells, including regulatory T cells, bone marrow-derived suppressor cells (MDSCs, myeloid-derived suppressor cells), etc. These cells secrete some immunosuppressive cells Factors, such as indoleamine 2,3-dioxygenase (IDO), hydrogen peroxide, peroxynitroso and other reactive oxygen/nitrogen free radicals, etc., further inhibit antigen presenting cells from exerting antigen presentation, thereby inhibiting
  • IDO indoleamine 2,3-dioxygenase
  • the infiltration, proliferation and differentiation of T lymphocytes in tumors not only lead to the failure of anti-tumor immunotherapy, but also promote tumor growth and metastasis (Tumor microenvironment complexity: emerging roles in cancer therapy, Cancer Research, 2012, 72(10): 2473; Myeloid-derived suppressor cells: Critical cells driving immune suppression in the tumor microenvironment, Immunotherapy of Cancer, 2015, 128: 95; Targeting the
  • hypoxia heterogeneity can also lead to an increase in drug resistance of solid tumors.
  • the tumor volume exceeds 3 mm 3 , the interior of the tumor is in a state of hypoxia (Intratumoral hypoxia, radiation resistance, and HIF-1, Cancer Cell, 2004, 5(5): 405).
  • the sensitivity of solid tumor cells to chemotherapeutics decreases in hypoxia, so hypoxia is an important factor leading to chemotherapy resistance (Molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal, Cancer Science, 2003, 94(1) ): 15; Hypoxia-inducible factor-1 ⁇ contributes to hypoxia-induced chemoresistance in gastric cancer, Cancer Science, 2008, 99(1): 121).
  • chemotherapeutic drug treatment can further reconstruct the immune microenvironment of the tumor, such as up-regulating the expression of PD-L1, IDO or HIF-1 ⁇ , so as to prevent the tumor from autoimmune effects and further reduce the efficacy.
  • immunosuppressive agents to reverse the immune microenvironment, such as the use of PD-1/PD-L1 antibody to block the inhibition of T cells by tumor cells, so that T cells can restore the function of recognizing and eliminating tumor cells, is the current general plan for tumor immunotherapy ( The blockade of immune checkpoints in cancer immunotherapy, Nature Reviews Cancer, 2012, 4(12): 252; Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. New England Journal of Medicine, 2012, 26 (366 ): 2443), showing high curative effect on tumors that match the target.
  • chemotherapy drugs can stimulate the immunogenic death of tumor cells, induce the expression of signals such as calreticulin, attract T cells and dendritic cells into the tumor, and improve the anti-tumor immune response (Immunobiology of dendritic cells, Annual Review of Immunology 2000) , 18: 767; Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion, Science, 2011, 331(6024): 1565).
  • chemotherapeutics can enhance the efficacy (Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews Immunology, 2012, 12(4): 269; Nishikawa H et al., Regulatory T cells in cancer immunotherapy, Current Opinion In Immunology, 2014, 27:1; Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma. International Journal Of Cancer. 1997, 73(3) ).
  • the PD-1/PD-L1 antibody has problems such as high risk of side effects, inconvenient preparation and storage, and high price.
  • N-(4-Hydroxy-1-methyl-7-phenoxyisoquinoline-3-carbonyl)glycine mimics ketoglutarate, one of the substrates of prolyl hydroxylase (PH) Acid inhibits the prolyl hydroxylase of hypoxia-inducible factor, thus maintaining or even increasing the level of hypoxia-inducible factor in normal cells, not only increases the expression of erythropoietin, but also enables erythropoietin receptors and promotes iron absorption The expression of circulating and circulating proteins is increased, so it is clinically used as a drug for the treatment of renal anemia.
  • the present invention provides a small molecule anti-tumor drug sensitizer based on N-(3-hydroxypyridine-2-carbonyl)glycine and its derivatives, which is used to improve the effect of immune and chemotherapy treatment of tumors.
  • the present invention provides an anti-tumor drug sensitizer, 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 1-20 alkyl, -OC 1-20 alkyl, -NH-C 1-20 alkyl, or -OC 6-12 aryl;
  • R 2 is H, F, Cl, Br, I, OH, NH 2 , NO 2 , CN, C 1-20 alkyl, -OC 1-20 alkyl, -NH-C 1-20 alkyl, C 6 -12 aryl, -OC 6-12 aryl or 5-10 membered heteroaryl, said C 1-20 alkyl, -OC 1-20 alkyl, -NH-C 1-20 alkyl, C 6-12 aryl, -OC 6-12 aryl, 5-10 membered heteroaryl optionally substituted with 1,2 or 3 substituents R a;
  • R 3 is H, F, Cl, Br, I, OH, NH 2 , NO 2 , CN, C 1-20 alkyl, -OC 1-20 alkyl, -NH-C 1-20 alkyl, C 6 -12 aryl or -OC 6-12 aryl;
  • R 4 is H, F, Cl, Br, I, OH, NH 2 , NO 2 , CN, C 1-20 alkyl, -OC 1-20 alkyl, -NH-C 1-20 alkyl, C 6 -12 aryl, -OC 6-12 aryl or 5-10 membered heteroaryl, said C 1-20 alkyl, -OC 1-20 alkyl, -NH-C 1-20 alkyl, C 6-12 aryl, -OC 6-12 aryl, 5-10 membered heteroaryl are optionally substituted by 1, 2 or 3 R b ;
  • Ring A is phenyl or does not exist
  • R a is each independently F, Cl, Br, I, OH, NH 2 , NO 2 , CN, C 1-3 alkyl or C 1-3 alkylphenyl, said C 1-3 alkyl or C 1-3 alkylphenyl is optionally substituted with 1, 2 or 3 halogens;
  • R b is independently F, Cl, Br, I, OH, NH 2 , NO 2 or CN;
  • n 0, 1, 2, 3 or 4;
  • n 0, 1, or 2.
  • the compound of formula (I) inhibits the expression of proline hydroxylase 3 (PHD3, polyl hydroylase 3) under hypoxic conditions, then down-regulates the expression of pyruvate kinase M2 (PKM2, pyruvate kinase M2), and finally inhibits the expression of HIF-1 ⁇ expression.
  • PPM2 pyruvate kinase M2
  • HIF-1 ⁇ expression As the expression of HIF-1 ⁇ is inhibited, the degree of dimerization of HIF-1 ⁇ and HIF-1F is also greatly reduced, and the expression of downstream factors such as PD-L1 and P-gp is also reduced.
  • the role of the compound of formula (I) in tumor immunotherapy includes reducing the expression of tumor cells PD-L1, enhancing the activity of T lymphocytes and infiltrating tumor tissues; inhibiting the expression of indoleamine 2,3-dioxygenase and improving cytotoxicity T cell activity; promote the transformation of macrophages from M2 to M1, reverse the immunosuppressive state, and promote the immunogenic death of tumor cells, enhance the body's immune response to tumors, and improve the effect of tumor immunotherapy.
  • the role of the compound of formula (I) in chemotherapy sensitization including reducing tumor cell HIF-1 ⁇ and multidrug resistance protein P-gp expression, inhibiting tumor multidrug resistance, promoting the endocytosis of chemotherapeutic drugs in tumor cells, and improving
  • the sensitivity of tumor cells to chemotherapeutic drugs increases the anti-tumor effect of chemotherapeutic drugs.
  • R 1 , R 2 , R 3 , R 4 , n or m are as defined in the present invention.
  • Ra is F, Cl, Br, I, OH, NH 2 , NO 2 , CN or
  • R 2 is H, F, Cl, Br, I, OH, NH 2 , NO 2 , CN, C 1-3 alkyl, -OC 1-3 alkyl, -NH-C 1- 3 alkyl, phenyl, -O-phenyl or pyrazolyl, pyrrolyl, pyrazolyl or triazolyl, said C 1-3 alkyl, -OC 1-3 alkyl, -NH- C 1-3 alkyl, phenyl, -O- phenyl or pyrazolyl, pyrrolyl, pyrazolyl or triazole group optionally substituted with 1, 2 or 3 R a.
  • R 2 is H, F, Cl, Br, I, OH, NH 2 , NO 2 , CN,
  • R 4 is H, F, Cl, Br, I, OH, NH 2 , NO 2 , CN or
  • R 1 , R 2 , R 3 , and R 4 are as defined in the present invention.
  • anti-tumor drug sensitizer is a compound of the following formula (1), (2), (3), (4), (5), (6), (7) or (8):
  • the anti-tumor drug sensitizer of the present invention can reduce the dimerization degree of HIF-1 ⁇ and HIF-1 ⁇ by down-regulating the expression of HIF-1 ⁇ , thereby down-regulating a series of downstream factors, such as PD-L1, P- gp etc.
  • compound 7 not only retains the carboxyl moiety (compound 2 or 3 needs to be hydrolyzed to expose the active carboxyl group), it is also more hydrophobic than compounds 1, 4, 5, 6, and 8, and it is easy to be Tumor cell uptake, so it has a better therapeutic effect.
  • the anti-tumor drug sensitizer of the present invention can be used in combination with different ratios of anti-tumor drugs.
  • the mass ratio of the anti-tumor drug sensitizer to the anti-tumor drug is 0.1-20:1.
  • the anti-tumor drugs are cyclophosphamide, 5-fluorouracil, raltitrexed, adriamycins, cytidines, antifolates, paclitaxel, gemcitabine, platinum drugs, camptothecin and its derivatives, Tripterygium wilfordii, vincristine or gambogic acid and molecular targeted drugs.
  • the tumor of the present invention is a malignant tumor
  • the malignant tumor includes hematological cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, melanoma, brain cancer, pancreatic cancer, lung cancer, bladder cancer, ovarian cancer, liver cancer Or cholangiocarcinoma, etc.
  • the present invention also provides a pharmaceutical composition, which includes a therapeutically effective amount of an anti-tumor drug sensitizer and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is water, liposomes, polymer micelles or inorganic nanocarriers.
  • 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 phospholipids or PEGylated phospholipids or their mixtures and anti-tumor drug sensitizers in solvent 1, and concentrate them to form a membrane at 4-60°C;
  • Hydration Add deionized water or a buffer solution of suitable pH to the prepared membrane for hydration for 12 to 48 hours at 4 to 60°C; then place it in a dialysis bag for dialysis for 6 to 48 hours at room temperature.
  • the phospholipids are phosphatidylcholine, phosphatidylethanolamine, dioleoylphosphatidylethanolamine, cholesterol hemisuccinate, distearate phosphatidylethanolamine, and the pegylated phospholipids include acid phosphatidylethanolamine-poly Ethylene glycol; polyethylene glycol has a molecular weight of 2,000 to 10,000.
  • the buffer solution 1 with a suitable pH is a buffer solution with a pH of 2-9.
  • the buffer solution 1 with a suitable pH is PBS buffer.
  • the solvent 1 is dichloromethane, chloroform, methanol or a mixture thereof.
  • the solvent 1 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 present invention also provides a preparation method of the micellar composition containing the anti-tumor drug sensitizer, which comprises the following steps:
  • micellar membrane dissolve the polymer and anti-tumor drug sensitizer in solvent 2, and concentrate at 4 ⁇ 60°C to form a membrane;
  • the copolymer is polyvinyl alcohol-polylactide block copolymer or polyoxyethylene polyoxypropylene ether block copolymer.
  • the solvent 2 is dichloromethane, chloroform, tetrahydrofuran, acetonitrile or acetone.
  • the filter membrane is a 150-250 nanometer filter membrane.
  • the filter membrane is a 200 nanometer filter membrane.
  • the buffer solution 2 with a suitable pH is a buffer solution with a pH of 2-9.
  • the buffer solution 2 with a suitable pH is PBS buffer.
  • the compound of formula (I) of the present invention can simultaneously inhibit HIF-1 ⁇ and P-gp, reverse the drug resistance of tumors, inhibit the expression of immunosuppressive molecules such as PD-L1, and enhance the body's immune response to tumors, thereby improving tumor Chemotherapy and immunotherapy effects.
  • the N-(3-hydroxypyridine-2-carbonyl)glycine and its derivatives are small-molecule compounds with clear structures and simple synthesis.
  • 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.
  • 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 an 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 1-20 alkyl by itself or in combination with other terms is used to indicate a linear or branched saturated carbon group containing 1 to 20 carbon atoms.
  • the C 1-20 alkyl group includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 alkyl, etc. It can be monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-20 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl) , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, heptyl, octyl, nonyl, decyl, etc.
  • C 1-4 alkyl examples include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, Isobutyl, s-butyl and t-butyl) and so on.
  • C 1-3 alkyl examples include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl) and the like.
  • 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) and the like.
  • the "alkyl" of the present invention is optionally substituted with 1 to 5 F, Cl, Br, I, OH, NH 2 , and 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.
  • Examples of 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.
  • Aryl-O- refers to an aryl group that is bonded to the rest of the molecule via an oxygen bond (-O-).
  • Aryl-NH- refers to an aryl group that is bonded to the rest of the molecule via a nitrogen bond.
  • the term "5-10 membered heteroaryl” refers to a group containing a hydrogen atom, 5 to 9 ring carbon atoms, one to 9 ring heteroatoms selected from nitrogen, oxygen and sulfur, and at least one heteroatom containing A 5- to 12-membered ring system group of an aromatic ring.
  • a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur in the heteroaryl group Atoms can be optionally oxidized; nitrogen atoms can be optionally quaternized.
  • Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzene Oxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxep-5-enyl, 1,4-benzodioxanyl, benzene Naphthofuranyl, benzoxazolyl, benzodioxolyl, 1,4-benzodioxolenyl, benzopyranyl, benzopyranone, Benzofuranyl, benzofuranone, benzothienyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, cinnolinyl, two Benzofuranyl, dibenzothienyl, furyl
  • 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.
  • 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.
  • HIF-1 ⁇ stands for hypoxia inducible factor-1 ⁇
  • PD-L1 stands for programmed death receptor-ligand 1
  • IDO indoleamine 2,3-dioxygenase
  • P-gp Stands for P glycoprotein
  • DOX stands for adriamycin
  • DMSO dimethyl sulfoxide
  • PBS stands for phosphate buffered saline
  • EDTA stands for ethylenediaminetetraacetic acid.
  • 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 Western blot quantitative results of the compound of the present invention in Test Example 1A reducing the expression of PD-L1 in CT26 cells.
  • Fig. 3 is a PCR result of the compound of the present invention in Test Example 1B reducing the mRNA expression of PD-L1 of CT26 cells.
  • Figure 4 shows the flow cytometric results of different concentrations of Compound 7 (ROX) in Test Example 1C reducing the expression of PD-L1 in CT26 cells.
  • Fig. 5 shows the result of increasing the immunogenic death of CT26 cells after the combination of compound 7 (ROX) and DOX in Test Example 1D, and compared with DOX alone.
  • Figure 6 shows the expression of related genes in mouse macrophages after treatment with compound 7 (ROX) in Test Example 1E, and compared with that of untreated mouse macrophages.
  • Figure 7 shows the experimental results of the compound of the present invention inhibiting the production of kynurenine in CT26 cells in Test Example 1F.
  • Figure 8 shows the cytotoxicity of the compound of the present invention in Test Example 2A in combination with the chemotherapeutic drug DOX on CT26 cells.
  • Figure 9 shows the cytotoxicity test results of the compound of the present invention in Test Example 2A.
  • Figure 10 shows the cytotoxicity test results of compound 7 (ROX) in test example 2A in combination with the chemotherapy drug paclitaxel (PTX).
  • Figure 11 shows the cytotoxicity test results of compound 7 (ROX) in test example 2A in combination with the chemotherapeutic drug oxaliplatin (OXA).
  • Figure 12 shows the cytotoxicity test results of compound 7 (ROX) in Test Example 2A in combination with the chemotherapeutic drug camptothecin (Irinotecan, SN38).
  • Figure 13 is the result of the compound of the present invention in Test Example 2B reducing the expression of HIF-1 ⁇ in CT26 cells.
  • Figure 14 shows the result of endocytosis of rhodamine 123 (Rh123) in the cells after treatment of CT26 cells with compound 7 (ROX) in Test Example 2C, and comparison with untreated cells.
  • Figure 15 is the tumor inhibition curve of compound 7 (ROX), DOX and the combination of the two drugs in Test Example 3 on the tumor model of CT26 tumor-bearing mice.
  • Figure 16 is a curve of mouse body weight after compound 7 (ROX), DOX and the combination of the two drugs in Test Example 3.
  • Figure 17 shows the tumor inhibition curve of the doxorubicin liposome (Doxil) and the compound 7 liposome (Roxil) in Test Example 4 on the tumor model of CT26 tumor-bearing mice.
  • Figure 18 is a mouse body weight curve after combined use of liposomes (Doxil) and liposomes (Roxil) of compound 7 in Test Example 4.
  • Figure 19 shows the tumor inhibition curve of the tumor model of CT26 tumor-bearing mice after the combination of Doxil liposomes (Doxil) and compound 7-loaded micelles (PEG-PLA-ROX) in Test Example 5.
  • Doxil Doxil liposomes
  • PEG-PLA-ROX compound 7-loaded micelles
  • Figure 20 shows the weight curve of mice after the combined use of doxorubicin liposomes (Doxil) and compound 7-loaded micelles (PEG-PLA-ROX) in Test Example 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.
  • Example 1 The preparation route of the esterified derivative of N-(3-hydroxypyridine-2-carbonyl)glycine is as follows.
  • R 1 is -OC 1-20 alkyl or -OC 6-12 aryl.
  • Example 2 The preparation route of the amidated derivative of N-(3-hydroxypyridine-2-carbonyl)glycine is as follows.
  • R 1 is NH 2 or -NH-C 1-20 .
  • the compound of the present invention can be prepared with reference to the literature (Dynamic combinatorial mass spectrometry leads to inhibitors of a 2-oxoglutarate-dependent nucleic acid demethylase. Journal of Medicinal Chemistry, 2012, 55(5): 2173), or it can be commercially available (this The small compounds used in the invention are obtained from MedChemExpress China.
  • Test Example 1 The compound of the present invention is used as an immunosensitizer.
  • Test Example 1A Western blot technology detects that the compound of the present invention reduces the expression of PD-L1 in tumor cells.
  • 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 pictures with a chemiluminescence imager.
  • Test Example 1B qPCR technology detects that the compound of the present invention reduces the mRNA expression of tumor cell PD-L1.
  • Test Example 1C Flow cytometry to detect that the compound of the present invention reduces the expression of PD-L1 in tumor cells.
  • CT26 cells were planted in a 6-well plate at 2 ⁇ 10 4 cells per well. After the cells adhere to the wall, DMSO solutions (2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M) of the compounds of the present invention are respectively added, 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.
  • the integral value in the gray area represents the expression rate of PD-L1
  • the expression rate of PD-L1 in the blank group was 32.7%
  • the expression rate of PD-L1 in the 2.5 ⁇ M compound 7 group was 24%
  • the 5 ⁇ M compound 7 group The expression rate of PD-L1 was 20.8%
  • the expression rate of PD-L1 in the 10 ⁇ M compound 7 group was 18.8%.
  • Compound 7 can significantly reduce the expression of PD-L1 in tumor cells, and is concentration-dependent.
  • Test Example 1D 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, different drug treatment groups were added to the cells and incubated for 24 hours. Discard the 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 (FITC-anti-CRT, 1:200) to each well, incubate for 1h at room temperature in the dark, continue to rinse the cells with PBS 3 times, 3min each time, and 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.
  • FITC-anti-CRT FITC-anti-CRT
  • doxorubicin DOX
  • compound 7 can induce doxorubicin to induce CT26 tumor cells to express more calreticulin, indicating that it produces stronger immunogenic death.
  • Test Example 1E The compound 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 DMSO solutions (5 ⁇ M) of the compounds of the present invention were added to TAM2, and the cells were collected after 24 hours of treatment, and total RNA was lysed and extracted. Reverse transcription and PCR experiments were performed to detect M2 type macrophage-specific proteins arg1 and M1. The RNA level of type macrophage-specific protein Nos2, using the hprt gene as an internal reference.
  • Test Example 1F 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 cultivation, a DMSO solution of the compound of the present invention with a certain concentration gradient was added, and 0.1 ⁇ g/mL INF- ⁇ was 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. The content of kynurenine in the supernatant solution was detected by HPLC, and each well was repeated three times.
  • medium containing 100 ⁇ M tryptophan
  • the results indicate that the compound of the present invention can inhibit the conversion of tryptophan to kynurenine, indicating that the compound of the present invention can inhibit the 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.
  • Test Example 2A Cytotoxicity study of the compound of the present invention in combination with a variety of chemotherapeutic drugs.
  • CT26 cells (MC38 cells, 4T1 cells, B16F10 cells, HePa1-6 cells, H22 cells, LLC cells, MB49 cells, P388 cells, C6 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, 100 ⁇ L medium was added to each well, and cultured in a 37°C constant temperature incubator with 5% CO 2 concentration and 95% humidity for 24 hours .
  • Figure 8 shows that the combination of the compound of the present invention and the chemotherapeutic agent can greatly reduce the survival rate of cells compared with the chemotherapeutic agent alone, and compound 7 has the best effect.
  • Figure 9 shows that at 0.1-10 ⁇ g/mL, the compound of the present invention has no obvious cytotoxicity.
  • Figures 10-12 show that compound 7 of the present invention can increase the toxicity of epirubicin (DOX), paclitaxel (PTX), oxaliplatin (OXA) or camptothecin drugs (irinotecan, SN38) to CT26 cells .
  • DOX epirubicin
  • PTX paclitaxel
  • OXA oxaliplatin
  • camptothecin drugs irinotecan, SN38
  • Table 1 shows the cytotoxicity test results of compound 7 in combination with the chemotherapeutic drug DOX on various cell lines. The results show that the combination of compound 7 and DOX can greatly reduce the survival rate of a variety of tumor cells.
  • Table 2 shows the cytotoxicity test results of compound 7 in combination with various chemotherapeutics on CT26 cell line. The results show that compound 7 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, DMSO solutions (5 ⁇ M) of different compounds 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 FITC anti-mouse HIF-1 ⁇ antibody (1 ⁇ g/1 ⁇ 10 6 cells) to After incubating for 30 minutes at 4°C, wash with PBS three times and perform flow detection on the machine.
  • N-(3-hydroxypyridine-2-carbonyl)glycine and its derivatives can significantly reduce the expression of HIF-1 ⁇ in tumor cells, among which compounds 6, 7 and 8 are effective
  • compound 7 can reduce HIF-1 ⁇ by 45% at 5 ⁇ M.
  • Test Example 2C The compound of the present invention enhances the ability of Rhodamine 123 (Rh123) to enter tumor cells.
  • 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 each well was replaced with fresh medium, and Rh123 solution (1 ⁇ M) and DMSO solution (1 ⁇ M) of compound 7 of the present invention were added. After incubating for 6 hours, the intracellular situation of Rh123 was 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.
  • compound 7 Take compound 7 as an example, dissolve 20 mg of compound 7 in 0.5 mL DMSO, add 0.5 mL polyoxyethylene castor oil, Tween 80 or polyethylene glycol 500 (polyoxyethylene castor oil is used in the present invention), and vortex until the mixture is uniform , The above solution was added to 9mL PBS and mixed well to obtain compound 7 injection. The injection can be stored at 4°C for more than 6 months without solid powder precipitation.
  • the blank control group, compound 7 group, doxorubicin hydrochloride group, compound 7 + doxorubicin hydrochloride group (D1R1: 3mg/kg DOX+5mg/kg compound 7 ; D1R2: 3mg/kg DOX+10mg/kg compound 7; D1R3: 5mg/kg DOX+5mg/kg compound 7; D1R4: DOX 5mg/kg+10mg/kg compound 7;). After the administration, the rats were observed for 12 days.
  • Figure 16 shows that the mice in each group did not lose weight, indicating that the drug has high biological safety and low toxic and side effects.
  • Test Example 4 Preparation of the liposome composition of compound 7 and anti-tumor activity experiment in combination with chemotherapeutics.
  • the liposome preparation of compound 7 was prepared by the film dispersion method.
  • Step 1 Firstly, 12.89g dioleoylphosphatidylethanolamine (DOPE), 2.11g cholesterol hemisuccinate (CHEMS), 6.52g distearic acid phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000) and 5g Compound 7 was dissolved in chloroform (12 mL) and methanol (4 mL), and spin-dried under reduced pressure under a water bath at 37° C. to form a film.
  • DOPE dioleoylphosphatidylethanolamine
  • CHEMS 2.11g cholesterol hemisuccinate
  • DSPE-mPEG2000 6.52g distearic acid phosphatidylethanolamine-polyethylene glycol 2000
  • Step 2 Add 5 mL of deionized water or buffer solution (1 ⁇ PBS solution is used in the present invention) to the liposome membrane obtained in Step 1, and hydrate at 4-60° C. for 24 hours at room temperature.
  • Step 3 Place the obtained solution in a dialysis bag and dialyze for 8 hours to obtain a liposome preparation of compound 7 (Roxil).
  • 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 0.110 and an average size of 105.9 nm in water.
  • the size can be adjusted by liposome composition, preparation method and the like.
  • 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). Taking the combination of Roxil and adriamycin liposomes as an example, they are the blank control group, the adriamycin liposome (Doxil, DOX dosage is 5 mg/kg) group, and the compound 7 + adriamycin liposome ( Roxil+Doxil, DOX dosage is 5mg/kg, ROX dosage is 7.5mg/kg), compound 7 liposome group (ROX dosage is 7.5mg/kg). After the end of the dosing cycle, the rats were observed for 18 days.
  • Doxil DOX dosage is 5 mg/kg
  • ROX dosage is 7.5mg/kg
  • ROX dosage 7.5mg/kg
  • Test Example 5 Preparation of compound 7 polymer micelle composition and its anti-tumor activity test in combination with chemotherapeutics.
  • the polymer micelle preparation of compound 7 was prepared by thin film evaporation method.
  • Step 1 First, compound 7 (5 mg) and polyethylene glycol-polylactic acid (15 mg) were dissolved in chloroform (10 mL) and spin-dried under reduced pressure in a water bath at 37° C. to form a film.
  • Step 2 Add deionized water or buffer solution (1 ⁇ PBS solution, 5 mL) to the film of step 1, and hydrate at room temperature for 12 hours.
  • Step 3 Pass the micellar solution obtained in Step 2 through a 200-mesh filter membrane to obtain a polymer micelle preparation of compound 7 (PEG-PLA-ROX, drug loading rate 95%).
  • the micelles can be prepared from different types of polymer raw materials, and good solvents for the micelle raw materials can be selected according to needs, such as dichloromethane, chloroform, tetrahydrofuran, acetonitrile, acetone, etc.; this test example uses chloroform; step 1
  • the ratio of raw materials in can be adjusted according to the needs of reagents.
  • Characterization of the size of the pharmaceutical preparation The particle size and distribution of the drug preparation are measured by Dynamic Light Scattering (DLS).
  • the micelle preparation is assembled into nanoparticles with a dynamic particle size distribution of 0.121 and an average size of 71.9 nm in water.
  • the size can be adjusted by polymer composition, preparation method, etc.
  • 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).
  • PEG-PLA-ROX and doxorubicin liposomes are the blank control group, the doxorubicin liposome (Doxil, DOX dosage is 5mg/kg) group, and PEG-PLA-ROX+A Lipomycin liposomes (PEG-PLA-ROX+Doxil, DOX dosage is 5mg/kg, ROX dosage is 5mg/kg); PEG-PLA-ROX group (ROX dosage is 5mg/kg). After the end of the dosing cycle, the rats were observed for 18 days.

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Abstract

本发明公开了一种基于N-(3-羟基吡啶-2-羰基)甘氨酸的抗肿瘤药物增敏剂及其应用。本发明具体公开的式(I)化合物可下调肿瘤细胞的PD-L1，促进巨噬细胞由M2向M1极化，抑制吲哚胺2，3-双加氧酶的表达，提高免疫治疗疗效；还可以降低肿瘤细胞中乏氧诱导因子-1α的表达，下调P糖蛋白的表达，增加化疗药物对肿瘤细胞的杀伤作用，并增强细胞免疫原性死亡。本发明公开的式(I)化合物与化疗药物联用后具有显著提高的抗肿瘤效果，具有良好的应用前景。

Description

一种基于N-(3-羟基吡啶-2-羰基)甘氨酸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N-(3-羟基吡啶-2-羰基)甘氨酸及其衍生物在制备抗肿瘤药物增敏剂中的应用。
背景技术
伴随着对肿瘤免疫学的深入研究，研究者们发现肿瘤尤其是实体肿瘤为了满足其快速生长的需要，发展成了一个由癌细胞、成纤维细胞、淋巴、血管及多种细胞外基质构成的复杂的组织结构，又称肿瘤组织微环境(TME，tumor microenviroment)。与正常组织相比，肿瘤内存在pH值相对较低、间隙压升高血管畸变、供氧不足、活性氧(ROS)升高等一系列独特的微环境特征(Microenvironmental regulation of tumor progression and metastasis，Nature Medicine，2013，11(19)：1423；The immunosuppressive tumour network：myeloid-derived suppressor cells，regulatory T cells and natural killer T cells，Immunology，2013，2(138)：105)。肿瘤细胞新陈代谢较正常细胞更加旺盛，肿瘤组织内血管的畸形以和不均匀分布导致氧气输送受阻，最终使肿瘤内存在着大量乏氧区域(Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O 2tension，Proceedings of The National Academy of Science of The United States of America，1995，92(12)：5510；The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis，Nature，1999，6733(399)：271)。
大量研究表明肿瘤组织内乏氧与肿瘤免疫抑制微环境具有密切的联系。如肿瘤乏氧会导致M2型肿瘤相关巨噬细胞(TAM，tumor-associated macrophages)的产生，抑制肿瘤免疫反应(HIF-1 alpha is essential for myeloid cell-mediated inflammation，Cell，2003，112(5)：645-657；Hypoxia in cancer：significance and impact on clinical outcome，Cancer And Metastasis Reviews，2007，2(26)：225)。
肿瘤乏氧会抑制肿瘤浸润淋巴细胞(tumor-infiltrating lymphocytes，TIL)，尤其是杀伤性T淋巴细胞(CTL，cytotoxic T lymphocytes)的增殖和活化，进而影响效应性T细胞的功能(Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration，International Journal Of Obesity，2008，3(32)：451；Inhibitory effect of tumor cell-derived lactic acid on human T cells，Blood，2007，9(109)：3812；HIF Transcription Factors，Inflammation，and Immunity，Immunity，2014，4(41)：518)。
肿瘤乏氧还会诱导癌细胞及巨噬细胞过表达PD-L1继而诱导CTL细胞的程序性死亡(A Mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells，Cancer Research，2014，3(74)：665；Genomic correlates of response to immune checkpoint therapies in clear cell renal cell carcinoma，Science，2018，359(6377)：801)。
此外，肿瘤独特的微环境还会招募大量的免疫抑制型细胞，包括调节性T细胞、骨髓来源的抑制性细胞(MDSCs，myeloid-derived suppressor cells)等，这些细胞会分泌一些免疫抑制性的细胞因子，如吲哚胺2，3-双加氧酶(IDO)、过氧化氢、过氧亚硝基等活性氧/氮自由基等，进一步抑制抗原递呈细胞发挥抗原呈递的作用，从而抑制T淋巴细胞在肿瘤内的浸润、增殖与分化，不仅导致抗肿瘤免疫治疗的失败，还促进肿瘤的生长与转移(Tumor microenvironment complexity：emerging roles in cancer therapy，Cancer Research，2012，72(10)：2473；Myeloid-derived suppressor cells：Critical cells driving immune suppression in the tumor microenvironment，Immunotherapy of Cancer，2015，128：95；Targeting the tumor microenvironment：removing obstruction to anticancer immune responses and immunotherapy，Annals of Oncology，2016，27(8)：1482)。因此，实体肿瘤内有别于正常组织的免疫抑制微环境特征，使得肿瘤细胞可以逃逸免疫系统的攻击，这是限制肿瘤免疫治疗疗效的关键因素之一。
乏氧异质性也是会导致实体肿瘤耐药性的增加。当肿瘤体积超过3mm 3时，肿瘤内部即处于乏氧状态(Intratumoral hypoxia，radiation resistance，and HIF-1，Cancer Cell，2004，5(5)： 405)。实体肿瘤细胞在乏氧环境中对化疗药物的敏感性下降，因此乏氧是导致化疗耐药的重要因素(Molecular targeting therapy of cancer：drug resistance，apoptosis and survival signal，Cancer Science，2003，94(1)：15；Hypoxia-inducible factor-1α contributes to hypoxia-induced chemoresistance in gastric cancer，Cancer Science，2008，99(1)：121)。此外，化疗药物治疗可进一步重构肿瘤的免疫微环境，如上调PD-L1、IDO或HIF-1α表达，使肿瘤免于自体免疫作用，进一步降低疗效。
利用免疫抑制剂逆转免疫微环境，如用PD-1/PD-L1抗体阻断肿瘤细胞对T细胞的抑制，使T细胞恢复识别和清除肿瘤细胞的功能，是目前肿瘤免疫治疗的通用方案(The blockade of immune checkpoints in cancer immunotherapy，Nature Reviews Cancer，2012，4(12)：252；Safety，activity，and immune correlates of anti-PD-1 antibody in cancer.New England Journal of Medicine，2012，26(366)：2443)，在靶点匹配的肿瘤上显示出很高的疗效。同时，化疗药物可刺激肿瘤细胞产生免疫原性死亡，诱导钙网蛋白等信号表达，吸引T细胞和树突状细胞的进入肿瘤，提高抗肿瘤免疫反应(Immunobiology of dendritic cells，Annual Review of Immunology 2000，18：767；Cancer immunoediting：integrating immunity’s roles in cancer suppression and promotion，Science，2011，331(6024)：1565)。
因此，将某些化疗药物与PD-1/PD-L1抗体免疫检抑制剂联用可增强疗效(Adoptive immunotherapy for cancer：harnessing the T cell response，Nature Reviews Immunology，2012，12(4)：269；Nishikawa H et al.，Regulatory T cells in cancer immunotherapy，Current Opinion In Immunology，2014，27：1；Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma.International Journal Of Cancer.1997，73(3)：309)。但PD-1/PD-L1抗体存在着的副作用风险大、制备及储存不便、价格昂贵等问题。
N-(4-羟基-1-甲基-7-苯氧基异喹啉-3-羰基)甘氨酸(Roxadustat，ROX)通过模拟脯氨酰羟化酶(PH)的底物之一酮戊二酸来抑制低氧诱导因子的脯氨酰羟化酶，因此在正常细胞中维持甚至升高低氧诱导因子水平，不仅使红细胞生成素表达增加，也能使红细胞生成素受体以及促进铁吸收和循环的蛋白表达增加，因此在临床上作为治疗肾性贫血的药物。
发明内容
本发明提供了一种基于N-(3-羟基吡啶-2-羰基)甘氨酸及其衍生物的小分子抗肿瘤药物增敏剂，用于提高免疫和化疗治疗肿瘤的效果。
本发明解决上述技术问题所提供的技术方案为：
本发明提供了一种抗肿瘤药物增敏剂，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 PCTCN2020124755-appb-000001
其中，R 1为H、OH、NH 2、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基或-O-C 6-12芳基；
R 2为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基或5-10元杂芳基，所述的C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基、5-10元杂芳基任选被1、2或3个R a取代；
R 3为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基或-O-C 6-12芳基；
R 4为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基或5-10元杂芳基，所述的C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基、5-10元杂芳基任选被1、2或3个R b取代；
A环为苯基或不存在；
R a分别独立地为F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-3烷基或C 1-3烷基苯基，所述的C 1-3烷基或C 1-3烷基苯基任选被1、2或3个卤素取代；
R b分别独立地为F、Cl、Br、I、OH、NH 2、NO 2或CN；
m为0、1、2、3或4；
n为0、1或2。
式(I)化合物在缺氧条件下抑制脯氨酸羟化酶3(PHD3，polyl hydroylase 3)的表达，继而下调丙酮酸激酶M2(PKM2，pyruvate kinase M2)的表达，最后抑制HIF-1α的表达。伴随着HIF-1α的表达被抑制，HIF-1α与HIF-1F的二聚化程度也大大降低，使其下游因子如PD-L1、P-gp等的表达也随之降低。
式(I)化合物在肿瘤免疫治疗中的作用，包括降低肿瘤细胞PD-L1的表达，增强T淋巴细胞活性和浸润肿瘤组织；抑制吲哚胺2，3-双加氧酶表达，提高细胞毒性T细胞活性；促进巨噬细胞由M2向M1转化，逆转免疫抑制状态，并促使肿瘤细胞免疫原性死亡，增强机体对肿瘤的免疫应答，提高肿瘤免疫治疗效果。
式(I)化合物在化疗增敏中的作用，包括降低肿瘤细胞HIF-1α及多药耐药蛋白P-gp表达，抑制肿瘤的多药耐药，促进化疗药物在肿瘤细胞的内吞，提高肿瘤细胞对化疗药物敏感性从而增加化疗药物的抗肿瘤效果。
进一步的，所述的式(I)化合物为：
Figure PCTCN2020124755-appb-000002
其中，R 1、R 2、R 3、R 4、n或m如本发明所定义。
进一步的，所述的R a为F、Cl、Br、I、OH、NH 2、NO 2、CN或
Figure PCTCN2020124755-appb-000003
进一步的，所述的R 2为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-3烷基、-O-C 1-3烷基、-NH-C 1-3烷基、苯基、-O-苯基或吡唑基、吡咯基、吡唑基或三氮唑基，所述的C 1-3烷基、-O-C 1-3烷基、-NH-C 1-3烷基、苯基、-O-苯基或吡唑基、吡咯基、吡唑基或三氮唑基任选被1、2或3个R a取代。
进一步的，所述的R 2为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、
Figure PCTCN2020124755-appb-000004
进一步的，所述的R 4为H、F、Cl、Br、I、OH、NH 2、NO 2、CN或
Figure PCTCN2020124755-appb-000005
进一步的，所述的式(I)化合物为：
Figure PCTCN2020124755-appb-000006
其中，R 1、R 2、R 3、R 4如本发明所定义。
进一步的，所述的抗肿瘤药物增敏剂为下式(1)、(2)、(3)、(4)、(5)、(6)、(7)或(8)的化合物：
Figure PCTCN2020124755-appb-000007
实验发现本发明所述的抗肿瘤药物增敏剂可以通过下调HIF-1α的表达，降低HIF-1α和HIF-1β的二聚化程度，从而下调一系列下游因子，如PD-L1，P-gp等。
化合物7与其他化合物相比，不仅保留了羧基部分(化合物2或3需经水解才能暴露出活性基团羧基)，而且与化合物1，4，5，6，8相比更疏水，很容易被肿瘤细胞摄取，因而具有较优的治疗效果。
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。
所述的抗肿瘤药物增敏剂与抗肿瘤药物的质量之比为0.1-20∶1。
所述的抗肿瘤药物为环磷酰胺、5-氟尿嘧啶、雷替曲塞、阿霉素类、胞苷类、抗叶酸类、紫杉醇类、吉西他滨、铂类药物、喜树碱及其衍生物、雷公藤红素、长春新碱类或藤黄酸和分子靶向类药物。
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、胃癌、食道癌、结直肠癌、乳腺癌、黑色素瘤、脑癌、胰腺癌、肺癌、膀胱癌、卵巢癌、肝癌或胆管癌等。
本发明还提供了一种药物组合物，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。
所述的药学上可接受的载体为水、脂质体、聚合物胶束或无机纳米载体。
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：
脂质体膜的制备：将磷脂或聚乙二醇化磷脂或其混合物及抗肿瘤药物增敏剂溶于溶剂1中，4～60℃浓缩成膜；
水化：在制得的膜中加入去离子水或合适pH的缓冲溶液1，4～60℃水化12～48h；再置于透析袋中室温下透析6～48h。
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包括酸磷脂酰乙醇胺-聚乙二醇；聚乙二醇的分子量2000～10000。
所述的合适pH的缓冲溶液1为pH为2-9的缓冲溶液。
所述的合适pH的缓冲溶液1为PBS缓冲液。
所述的溶剂1为二氯甲烷、三氯甲烷、甲醇或其混合液。
所述的溶剂1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。
本发明还提供了载有所述的抗肿瘤药物增敏剂的胶束组合物的制备方法，包括以下步骤：
胶束膜的制备：将聚合物及抗肿瘤药物增敏剂溶于溶剂2中，4～60℃浓缩成膜；
水化：在制得的膜中加入去离子水或合适pH的缓冲溶液，4～60℃水化2～48h；再过滤膜；
所述的共聚物为聚乙烯醇-聚丙交酯嵌段共聚物或聚氧乙烯聚氧丙烯醚嵌段共聚物。
所述的溶剂2为二氯甲烷、三氯甲烷、四氢呋喃、乙腈或丙酮。
所述的滤膜为150-250纳米滤膜。
所述的滤膜为200纳米滤膜。
所述的合适pH的缓冲溶液2为pH为2-9的缓冲溶液。
所述的合适pH的缓冲溶液2为PBS缓冲液。
本发明的有益效果主要体现在：
本发明的式(I)化合物能通过同时抑制HIF-1α及P-gp，逆转肿瘤多要耐药，抑制PD-L1等免疫抑制分子的表达、增强机体对肿瘤的免疫反应，从而提高肿瘤的化疗与免疫治疗效果。与免疫检查点抑制剂PD-1/PD-L1抗体相比，所述N-(3-羟基吡啶-2-羰基)甘氨酸及其衍生物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。
当一个片段为不存在时，比如R-X中X为不存在时表示该结构实际上是R。
除非另有规定，术语“C 1-20烷基”本身或者与其他术语联合分别用于表示包含1至20个碳原子的直链或支链的饱和的碳基团。所述C 1-20烷基包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、C 12、C 13、C 14、C 15、C 16、C 17、C 18、C 19、C 20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 1-20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)，丁基(包括n-丁基，异丁基，s-丁基和t-丁基)，戊基(包括n-戊基，异戊基和新戊基)、己基、庚基、辛基、壬基、癸基等。术语“C 1-4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)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)等。术语“C 1-3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。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。除非另有规定，本发明“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)。除非另有规定，本发明“芳基”任选被1-5个F、Cl、Br、I、OH、NH 2、CN取代。“芳基-O-”是指经由氧键(-O-)与分子的其余部分键合的芳基。“芳基-NH-”是指经由氮键与分子的其余部分键合的芳基。
除非另有说明，术语“5-10元杂芳基”是指包含氢原子、5至9个环碳原子、一至9个选自氮、氧和硫的环杂原子和至少一个包含杂原子的芳香环的5元至12元环系基团。出于本发明的实施方案的目的，杂芳基可以为单环、双环、三环或四环环系，其可以包括稠合或桥接的环系；并且杂芳基中的氮、碳或硫原子可以任选地被氧化；氮原子可以任选地被季铵化。实例包括但不限于氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并噻唑基、苯并吲哚基、苯并间二氧杂环戊烯基、苯并呋喃基、苯并噁唑基、苯并噻唑基、苯并噻二唑基、苯并[b][1，4]二氧杂环庚-5-烯基、1，4-苯并二噁烷基、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基、1，4-苯并二氧杂环己烯基、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并三唑基、苯并[4，6]咪唑并[1，2-a]吡啶基、咔唑基、噌啉基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、异噻唑基、咪唑基、吲唑基、吲哚基、吲唑基、异吲哚基、吲哚啉基、异吲哚啉基、异喹啉基、中氮茚基、异噁唑基、萘啶基、噁二唑基、2-氧代吖庚因基、噁唑基、环氧乙烷基、1-氧化吡啶基、1-氧化嘧啶基、1-氧化吡嗪基、1-氧化哒嗪基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基、喋啶基、嘌呤基、吡咯基、吡唑基、吡啶基、吡嗪基、嘧啶基、哒嗪基、喹唑啉基、喹喔啉基、喹啉基、奎宁环基、异喹啉基、四氢喹啉基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基和噻吩基。
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＝O)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：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；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 PCTCN2020124755-appb-000008
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定量结果。
图3为测试例1B中本发明化合物降低CT26细胞PD-L1的mRNA表达的PCR结果。
图4为测试例1C中不同浓度化合物7(ROX)降低CT26细胞PD-L1表达的流式细胞计数结果。
图5为测试例1D中化合物7(ROX)与DOX联用后增加CT26细胞免疫原性死亡的结果，并与单独DOX比较。
图6为测试例1E中化合物7(ROX)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7为测试例1F中本发明化合物抑制CT26细胞犬尿氨酸生成的实验结果。
图8为测试例2A中本发明化合物与化疗药物DOX联用后在CT26细胞上的细胞毒性。
图9为测试例2A中本发明化合物的细胞毒性实验结果。
图10为测试例2A中化合物7(ROX)与化疗药物紫杉醇(PTX)联用的细胞毒性实验结果。
图11为测试例2A中化合物7(ROX)与化疗药物奥沙利铂(OXA)联用的细胞毒性实验结果。
图12为测试例2A中化合物7(ROX)与化疗药物喜树碱类药物(伊立替康，SN38)联用的细胞毒性实验结果。
图13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14为测试例2C中化合物7(ROX)处理CT26细胞后，细胞内吞罗丹明123(Rh123)的结果，并与未处理的细胞的比较。
图15为测试例3中化合物7(ROX)、DOX及两药联用后，对CT26荷瘤小鼠肿瘤模型的抑瘤曲线。
图16为测试例3中化合物7(ROX)、DOX及两药联用后小鼠体重曲线。
图17为测试例4中阿霉素脂质体(Doxil)和化合物7的脂质体(Roxil)联用后，对CT26荷瘤小鼠肿瘤模型的抑瘤曲线。
图18为测试例4中霉素脂质体(Doxil)和化合物7的脂质体(Roxil)联用后小鼠体重曲线。
图19为测试例5中阿霉素脂质体(Doxil)和负载化合物7的胶束(PEG-PLA-ROX)联用后，对CT26荷瘤小鼠肿瘤模型的抑瘤曲线。
图20为测试例5中阿霉素脂质体(Doxil)和负载化合物7的胶束(PEG-PLA-ROX)联用后小鼠体重曲线。
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：N-(3-羟基吡啶-2-羰基)甘氨酸的酯化衍生物的制备路线如下。
Figure PCTCN2020124755-appb-000009
R 1为-O-C 1-20烷基或-O-C 6-12芳基。
以化合物(2)为例子。
Figure PCTCN2020124755-appb-000010
化合物(1)(200mg，1.02mmol)溶于20mL干燥的N，N二甲基甲酰胺，加入油醇(329mg，1.22mmol)和4-二甲氨基吡啶(6.23mg，0.05mmol)。N 2保护并冰浴下滴加二环己基碳二亚胺(252mg，1.22mmol)，搅拌1h后撤去冰浴并继续反应过夜。反应结束后，减压旋蒸除去溶剂，用硅胶柱和流动相为正己烷∶乙酸乙酯＝5∶1纯化得白色固体化合物(2)，产率为86.9％。Formula：[C 26H 45N 3O 3] +，Calc.448.33，found 448.23。
实施例2：N-(3-羟基吡啶-2-羰基)甘氨酸的酰胺化衍生物的制备路线如下。
Figure PCTCN2020124755-appb-000011
R 1为NH 2或-NH-C 1-20。
以化合物(3)为例子。
Figure PCTCN2020124755-appb-000012
化合物(1)(200mg，1.02mmol)溶于20mL干燥的N，N二甲基甲酰胺，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(603mg，3.06mmol)、1-羟基苯并三唑(207mg，1.53mmol) 和油胺(328mg，1.23mmol)，N 2保护并冰浴下滴入N，N-二异丙基乙胺(527mg，4.08mmol)，搅拌1h后撤去冰浴并继续反应过夜。反应结束后，减压旋蒸除去大部分溶剂，乙酸乙酯复溶，接着用1N盐酸和饱和食盐水洗涤，干燥浓缩后用正己烷∶乙酸乙酯＝3∶1为流动相过柱纯化，干燥得淡黄色固体化合物(3)，产率为91.2％。Formula：[C 26H 45N 3O 3] +，Calc.447.35，found 447.15。
本发明的化合物可参考文献(Dynamic combinatorial mass spectrometry leads to inhibitors of a 2-oxoglutarate-dependent nucleic acid demethylase.Journal of Medicinal Chemistry，2012，55(5)：2173)制备，也可以可经市售(本发明使用的小化合物来源于MedChemExpress China)获得。
测试例1：本发明化合物作为免疫增敏剂。
配置方法：实施例1及实施例2中所得的本发明化合物的DMSO溶液可以直接用于细胞实验中。
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，分别加入化合物1～8的DMSO溶液(5μM)，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)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，待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。用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。
结果如图1～2所示，本发明化合物1～8可以降低肿瘤细胞PD-L1的表达，其中以化合物6，7和8最优。证明本发明式(I)化合物可以降低肿瘤细胞PD-L1的表达。
测试例1B：qPCR技术检测本发明化合物降低肿瘤细胞PD-L1的mRNA表达。
以每孔2×10 5个CT26细胞种于6孔板中，置于37℃恒温培养箱。待细胞贴壁后，分别加入本发明化合物的DMSO溶液(5μM)，继续孵育48h。裂解并提取总RNA，进行反转录和PCR实验，检测CT26细胞PD-L1的RNA水平，以GAPDH基因为内参。
测试结果如图3所示，加入本发明化合物后PD-L1的mRNA表达水平显著降低，证明本发明化合物可以降低肿瘤细胞PD-L1的mRNA表达，其中以化合物6，7和8最优。
测试例1C：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CT26细胞以每孔2×10 4个种植于6孔板中。待细胞贴壁后，分别加入本发明化合物的DMSO溶液(2.5μM，5μM，10μM)，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anti-mouse 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。以化合物7为例展示结果。
结果如图4所示，灰色区域的积分值代表PD-L1表达率，空白组的PD-L1表达率为32.7％；2.5μM化合物7组的PD-L1表达率为24％，5μM化合物7组的PD-L1表达率为20.8％，10μM化合物7组的PD-L1表达率为18.8％。化合物7可以显著降低肿瘤细胞PD-L1的表达，并呈现浓度依赖。
测试例1D：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不同的药物处理组，继续孵育24h。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(FITC-anti-CRT，1∶200)，常温下避光孵育1h，继续用PBS润洗细胞3次，每次3min，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(DOX)与化合物7联用展示结果，如图5所示，化合物7可以使阿霉素诱导CT26肿瘤细胞表达更多的钙网蛋白，表明产生更强的免疫原性死亡。
测试例1E：本发明化合物促进巨噬细胞由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中加入不同组的本发明化合物的DMSO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。
以化合物7为例展示结果，如图6所示，与未经本发明化合物7处理的TAM2相比，加入化合物7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1F：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的DMSO溶液，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10μL 30％三氟乙酸溶液沉淀蛋白。用HPLC检测上清溶液中犬尿氨酸的含量，每个孔重复三次。
如图7所示，结果表明本发明化合物可以抑制色氨酸向犬尿氨酸转变，表明本发明化合物能够抑制吲哚胺2，3-双加氧酶的表达。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：实施例1及实施例2中所得的本发明化合物的DMSO溶液可以直接用于细胞实验中。
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、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；增敏剂为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培养液，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。测试结果如图8～12所示。
图8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，其中以化合物7效果最优。
图9显示在0.1-10μg/mL，本发明化合物不具有明显的细胞毒性。
图10～12显示本发明化合物7可以增加表阿霉素(DOX)、紫杉醇(PTX)、奥沙利铂(OXA)或喜树碱类药物(伊立替康，SN38)等对CT26细胞的毒性。
表1为化合物7与化疗药物DOX联用在多种细胞系上的细胞毒性实验结果。结果显示化合物7与DOX联用可以极大地降低多种肿瘤细胞的生存率。
表1
Figure PCTCN2020124755-appb-000013
表2为化合物7与多种化疗药物联用在CT26细胞系上的细胞毒性实验结果。结果显示化合物7可以增加多种化疗药物的对CT26细胞的毒性。
表2
Figure PCTCN2020124755-appb-000014
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，分别加不同的本发明化合物的DMSO溶液(5μM)，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 anti-mouse 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13所示，与空白对照相比，N-(3-羟基吡啶-2-羰基)甘氨酸及其衍生物可以显著降低肿瘤细胞HIF-1α的表达，其中以化合物6，7和8效果最优，5μM时化合物7可降低45％的HIF-1α。
测试例2C：本发明化合物增强罗丹明123(Rh123)入肿瘤细胞的能力。
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，因此可用于测定细胞的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(1μM)及本发明化合物7的DMSO溶液(1μM)，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14所示，加入化合物7后能够显著增加Rh123的细胞内浓度，证明其可以通过抑制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。
(1)配置方法
以化合物7为例，取20mg化合物7溶于0.5mL DMSO，加入0.5mL聚氧乙烯蓖麻油、吐温80或聚乙二醇500(本发明使用聚氧乙烯蓖麻油)，涡旋至混合均匀，将上述溶液加入到9mLPBS中，混匀即得化合物7的注射液。该注射液可在4℃保存6个月以上，不会有固体粉末析出。
(2)抑瘤实验
考察化合物7与化疗药物联用(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、藤黄酸等)对CT26鼠结肠癌的肿瘤抑制作用。Balb/c白鼠皮下接种1×10 6CT26肿瘤细胞，待肿瘤长至约80mm 3后开始每隔两天(Day0，Day2，Day4)进行尾静脉注射。以化合物7与盐酸阿霉素联用为例，分别为空白对照组、化合物7组、盐酸阿霉素组、化合物7+盐酸阿霉素组(D1R1：3mg/kg DOX+5mg/kg化合物7；D1R2：3mg/kg DOX+10mg/kg化合物7；D1R3：5mg/kg DOX+5mg/kg化合物7；D1R4：DOX 5mg/kg+10mg/kg化合物7；)。给药结束后继续观察白鼠12天。
图15所示结果表明，同单独使用化合物7或盐酸阿霉素对比，两者联用治疗组的肿瘤体积呈缩小趋势，停药后肿瘤没有增长，保持不变。说明化合物7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，化合物7可使其他抗肿瘤药物的抑瘤率提高30-70％，治疗效果在本领域处于领先水平，具有良好的应用前景。
图16显示，各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：化合物7的脂质体组合物的制备及与化疗药物联用抗肿瘤活性实验。
采用薄膜分散法制备化合物7的脂质体制剂。
步骤1：首先将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-mPEG2000)及5g化合物7溶于三氯甲烷(12mL)及甲醇(4mL)中，37℃水浴下减压旋干成膜。
步骤2：将步骤1所得的脂质体膜中加入5mL去离子水或缓冲溶液(本发明使用1×PBS溶液)，室温4～60℃水化24h。
步骤3：将所得溶液置于透析袋中透析8h，即得化合物7的脂质体制剂(Roxil)。
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)；N-(4-羟基-1-甲基-7-苯氧基异喹啉-3-羰基)甘氨酸的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0.110，平均尺寸为105.9nm的纳米颗粒。该尺寸可用脂质体组成、制备方法等进行调节。
抗肿瘤活性实验：
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Roxil与阿霉素脂质体联用为例，分别为空白对照组、阿霉素脂质体(Doxil，DOX给药量为5mg/kg)组、化合物7+阿霉素脂质体(Roxil+Doxil，DOX给药量为5mg/kg，ROX给药量为7.5mg/kg)，化合物7脂质体组(ROX给药量为7.5mg/kg)。给药周期结束后继续观察白鼠18天。
结果如图17所示，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。20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18显示，各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，化合物7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。
测试例5：化合物7聚合物胶束组合物的制备及其与化疗药物联用抗肿瘤活性测试。
采用薄膜蒸发法制备化合物7的聚合物胶束制剂。
步骤1：首先将化合物7(5mg)，聚乙二醇-聚乳酸(15mg)溶于三氯甲烷(10mL)，37℃水浴下减压旋干成膜。
步骤2：将去离子水或缓冲溶液(1×PBS溶液，5mL)加入步骤1的薄膜中，室温水化12小时。
步骤3：将步骤2所得的胶束溶液过200目滤膜，即得化合物7的聚合物胶束制剂(PEG-PLA-ROX，载药率为95％)。
胶束可以用不同种类的聚合物原料制备，并可根据需要选择胶束原料的良溶剂，如二氯甲烷、三氯甲烷、四氢呋喃、乙腈、丙酮等；本试验例选用三氯甲烷；步骤1中的原料比例可根据试剂需要调整。
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，胶束制剂在水中组装成动态粒径分布为0.121，平均尺寸为71.9nm的纳米颗粒。该尺寸可用聚合物组成、制备方法等进行调节。
抗肿瘤活性实验：
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PEG-PLA-ROX与阿霉素脂质体联用为例，分别为空白对照组、阿霉素脂质体(Doxil，DOX给药量为5mg/kg)组、PEG-PLA-ROX+阿霉素脂质体(PEG-PLA-ROX+Doxil，DOX给药量为5mg/kg，ROX给药量为5mg/kg)；PEG-PLA-ROX组(ROX给药量为5mg/kg)。给药周期结束后继续观察白鼠18天。
结果如图19所示，图19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20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，PEG-PLA-ROX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 PCTCN2020124755-appb-100001
    其中，
    R 1为H、OH、NH 2、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基或-O-C 6-12芳基；
    R 2为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基或5-10元杂芳基，所述的C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基、5-10元杂芳基任选被1、2或3个R a取代；
    R 3为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基或-O-C 6-12芳基；
    R 4为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基或5-10元杂芳基，所述的C 1-20烷基、-O-C 1-20烷基、-NH-C 1-20烷基、C 6-12芳基、-O-C 6-12芳基、5-10元杂芳基任选被1、2或3个R b取代；
    A环为苯基或不存在；
    R a分别独立地为F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-3烷基或C 1-3烷基苯基，所述的C 1-3烷基或C 1-3烷基苯基任选被1、2或3个卤素取代；
    R b分别独立地为F、Cl、Br、I、OH、NH 2、NO 2或CN；
    m为0、1、2、3或4；
    n为0、1或2。
  2. 根据权利要求1所述的抗肿瘤药物增敏剂，其特征在于，所述的式(I)化合物为：
    Figure PCTCN2020124755-appb-100002
    其中，R 1、R 2、R 3、R 4、n或m如权利要求1所定义。
  3. 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Ra为F、Cl、Br、I、OH、NH 2、NO 2、CN或
    Figure PCTCN2020124755-appb-100003
  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R 2为H、F、Cl、Br、I、OH、NH 2、NO 2、CN、C 1-3烷基、-O-C 1-3烷基、-NH-C 1-3烷基、苯基、-O-苯基或吡唑基、吡咯基、吡唑基或三氮唑基，所述的C 1-3烷基、-O-C 1-3烷基、-NH-C 1-3烷基、苯基、-O-苯基或吡唑基、吡咯基、吡唑基或三氮唑基任选被1、2或3个R a取代。
  5. 根据权利要求4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)、(4)、(5)、(6)、(7)或(8)的化合物：
    Figure PCTCN2020124755-appb-100004
  6. 根据权利要求1～5任意一项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。
  7. 根据权利要求6所述的抗肿瘤药物增敏剂，其特征在于，抗肿瘤药物增敏剂与抗肿瘤药物的的质量比为0.1～20∶1。
  8. 根据权利要求1～5任意一项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。
  9. 根据权利要求1～5任意一项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脂质体组合物，包括以下步骤：
    脂质体膜的制备：将磷脂或聚乙二醇化磷脂或其混合物及抗肿瘤药物增敏剂溶于溶剂1中，30～60℃浓缩成膜；
    水化：在制得的膜中加入去离子水或合适pH的缓冲溶液1，4～60℃水化12～48h；再置于透析袋中室温下透析6～48h。
  10. 根据权利要求1～5任意一项所述的抗肿瘤药物增敏剂，其特征在于，负载所述的抗肿瘤药物增敏剂制成药物胶束组合物，包括以下步骤：
    胶束膜的制备：将共聚物和抗肿瘤药物增敏剂溶于溶剂2中，4～60℃浓缩成膜；
    水化：在制得的膜中加入去离子水或合适pH的缓冲溶液，4～60℃水化2～48h；再过滤膜；所述的共聚物为聚乙烯醇-聚丙交酯嵌段共聚物或聚氧乙烯聚氧丙烯醚嵌段共聚物。
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