WO2023061464A1 - 2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物及其应用 - Google Patents

2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物及其应用 Download PDF

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WO2023061464A1
WO2023061464A1 PCT/CN2022/125293 CN2022125293W WO2023061464A1 WO 2023061464 A1 WO2023061464 A1 WO 2023061464A1 CN 2022125293 W CN2022125293 W CN 2022125293W WO 2023061464 A1 WO2023061464 A1 WO 2023061464A1
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compound
formula
cells
group
administration
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PCT/CN2022/125293
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English (en)
French (fr)
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许耀
王海勇
米春来
梅运红
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北京安健熹医药科技有限公司
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Priority to AU2022367624A priority Critical patent/AU2022367624A1/en
Priority to CN202280007585.2A priority patent/CN116635368A/zh
Priority to CA3235127A priority patent/CA3235127A1/en
Publication of WO2023061464A1 publication Critical patent/WO2023061464A1/zh

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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/44—Sulfones; Sulfoxides having sulfone or sulfoxide groups and carboxyl groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D209/26—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with an acyl radical attached to the ring nitrogen atom

Definitions

  • the invention belongs to the field of medicine, and more specifically, the invention relates to 2,3-dimethoxy-5-methyl-1,4-benzoquinone alkyl alcohol derivatives, a pharmaceutical composition of the compound as an active ingredient, and Application of the compound in preventing and treating tumors.
  • Targeted drug therapy for cancer has made great progress, especially in the treatment of some lung cancer and non-solid tumor malignant tumors, it has been used as the first choice, but clinically it is still mainly cytotoxic drugs, especially platinum drugs, including cis Platinum, carboplatin, nedaplatin, oxaliplatin, and lobaplatin are still the first-line treatment drugs for many common cancers, but platinum-based drugs have poor selectivity and strong side effects; among them, nephrotoxicity is a relatively common and influential side effect One, the incidence of nephrotoxicity caused by cisplatin is 28-36%, that of carboplatin is 27%, and that of nedaplatin is 10-15%. , Elevated blood urea nitrogen, etc.
  • the present invention finds a derivative of 2,3-dimethoxy-5-methyl-1,4-benzoquinone alkyl alcohol, which can simultaneously increase the anti-tumor effect of platinum and reduce the toxic and side effects of platinum. Has excellent therapeutic effect.
  • the first aspect of the present invention provides 2,3-dimethoxy-5-methyl-1,4-benzoquinone alkyl alcohol derivatives as shown in formula (I) (referred to as “compound of formula (I)” , or simply “R01”, or simply “RO1”), or solvates, hydrates, polymorphs, prodrugs or isotopic variants, and mixtures thereof:
  • the second aspect of the present invention provides a pharmaceutical composition, which contains the compound of the first aspect, or solvate, hydrate, polymorph, prodrug or isotopic variant, and a pharmaceutically acceptable excipient ; preferably, it also contains other therapeutic agents.
  • the third aspect of the present invention relates to the compound or solvate, hydrate, polymorph, prodrug or isotopic variant of the first aspect, or the pharmaceutical composition of the second aspect, in the preparation for the treatment and/or prevention of cancer use in medicines.
  • a fourth aspect of the present invention relates to a method of treating and/or preventing cancer in a subject, the method comprising administering to the subject the compound of the first aspect or a solvate, hydrate, polymorph form, prodrug or isotopic variant or the pharmaceutical composition of the second aspect.
  • the fifth aspect of the present invention relates to the compound or solvate, hydrate, polymorph, prodrug or isotope variant of the first aspect or the pharmaceutical composition of the second aspect for use in the treatment and/or prevention of cancer.
  • the sixth aspect of the present invention relates to the use of the third aspect or the method of the fourth aspect or the use of the compound or composition of the fifth aspect, wherein the cancer is selected from lung cancer, gastric cancer, esophageal cancer, and colorectal cancer.
  • the seventh aspect of the present invention relates to a combination comprising a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotope variant thereof, and a platinum drug.
  • the eighth aspect of the present invention relates to a pharmaceutical composition, comprising the combination according to the seventh aspect and a pharmaceutically acceptable carrier, diluent or excipient.
  • the ninth aspect of the present invention relates to the use of the combination described in the seventh aspect in the preparation of medicaments for treating and/or preventing cancer.
  • the tenth aspect of the present invention relates to a pharmaceutical preparation, comprising a compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant, and a platinum drug, as a simultaneous or sequential Or combined preparations used separately.
  • the eleventh aspect of the present invention relates to a method for treating and/or preventing cancer, the method comprising simultaneously, sequentially or separately administering the compound of formula (I), or its solvate, hydrate, polymorphic form to the patient , prodrugs or isotopic variants, and platinum-based drugs.
  • the twelfth aspect of the present invention relates to the use of the compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant in the preparation of a drug for treating and/or preventing cancer, wherein the treatment It includes simultaneously, sequentially or separately administering the compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotope variant and platinum drug to the patient.
  • the thirteenth aspect of the present invention relates to the use of the compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotope variant and platinum-based drug in the preparation of a drug for treating and/or preventing cancer.
  • the fourteenth aspect of the present invention relates to the use of the compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant in the preparation of a drug for treating and/or preventing cancer, wherein the drug For combination therapy with platinum-based drugs.
  • the fifteenth aspect of the present invention relates to the use of platinum-based drugs in the preparation of drugs for the treatment and/or prevention of cancer, wherein the drugs are used with compounds of formula (I), or solvates, hydrates, polymorphs, Combination therapy of prodrugs or isotopic variants.
  • Figure 1 shows the effect of CCK-8 detection of the compound of formula (I) on the proliferation of A549 cells.
  • Fig. 1A shows the impact of different concentrations of formula (I) compounds and A549 cells co-culture 24h on cell proliferation activity
  • Fig. 1B shows the impact of different concentrations of formula (I) compounds and A549 cells co-culture 48h on cell proliferation
  • Fig. 1C shows Effects of different concentrations of compounds of formula (I) co-cultured with A549 cells for 72 hours on cell proliferation
  • Figure 1D shows the effects of different concentrations of compounds of formula (I) on A549 cell proliferation for different times.
  • Figure 2 shows the effect of CCK-8 detection of the compound of formula (I) on the proliferation of NCI-H460 cells.
  • Fig. 2A shows different concentration formula (I) compound and NCI-H460 cell co-culture 24h, the impact on cell proliferation activity
  • Fig. 2B shows different concentration formula (I) compound and NCI-H460 cell co-culture 48h to cell proliferation activity Impact
  • Figure 2C shows the effect of different concentrations of formula (I) compounds and NCI-H460 cells co-cultured for 72h on cell proliferation activity
  • Figure 2D shows the effect of different concentrations of formula (I) compounds on NCI-H460 cell proliferation activity for different times .
  • Figure 3 shows the effect of EDU proliferation assay on the proliferation of lung cancer cell lines detected by the compound of formula (I).
  • the compound of formula (I) with a concentration of 150 ⁇ M was co-cultured with the cells for 42 hours, the FBS concentration in the FBS-stimulated wells was increased to 20%, and the cell-Light EDUApollo567 kit was used to detect after continuing to cultivate for 6 hours.
  • Fig. 3A shows the effect of formula (I) compound on A549 cell proliferation
  • Fig. 3B shows the effect of formula (I) compound on NCI-H460 cell proliferation; *: P ⁇ 0.05; **: P ⁇ 0.01 .
  • Figure 4 shows the effects of different concentrations of compounds of formula (I) on the colony formation ability of A549 and NCI-H460 cells.
  • Figure 5 shows that the compound of formula (I) induces a decrease in the proportion of S phase of A549 cells: A549 cells are treated with different concentrations of compounds of formula (I) (0 ⁇ M, 0 ⁇ M, 30 ⁇ M, 90 ⁇ M) for 24h, 48h and 72h after the representative flow cell cycle distribution picture.
  • Figure 6 shows that the compound of formula (I) induces the reduction of the S phase ratio of NCI-H460 cells: NCI-H460 cells are treated with different concentrations of compounds of formula (I) (0 ⁇ M, 10 ⁇ M, 30 ⁇ M, 90 ⁇ M) for 24h, 48h and 72h after the representative flow Cell cycle distribution diagram.
  • Fig. 7 shows the scatter diagram of the changes of apoptosis after different concentrations of the compound of formula (I) acting on A549 and NCI-H460 cells detected by flow cytometry.
  • 7A, 7B, 7C, and 7D are representative cell apoptosis diagrams of different concentrations of formula (I) compounds (0 ⁇ M, 10 ⁇ M, 30 ⁇ M, 90 ⁇ M) acting on A549 cells for 72 hours;
  • Figures 7E, 7F, 7G, and 7H are different concentrations Representative cell apoptosis diagrams of NCI-H460 cells treated with compounds of formula (I) (0 ⁇ M, 10 ⁇ M, 30 ⁇ M, 90 ⁇ M) for 72 hours;
  • Figure 7I is the ratio of total apoptosis of A549 cells co-cultured with compounds of formula (I) at different concentrations ;
  • Figure 7G is the ratio of total apoptosis of NCI-H460 cells co-cultured with compounds of formula (I
  • Figure 8 shows the results of Western blot (A) and grayscale analysis (B) of NCI-N87 tumor tissue proteins.
  • NCI-N87 3D_PN154004
  • formula (I) compound solution (2mg/kg) down-regulates p-VEGFR2 (NS), down-regulates VEGFR2 (P ⁇ 0.05), VEGFA (P ⁇ 0.05); down-regulates ⁇ -catenin (P ⁇ 0.05), CyclinD1 (NS), up-regulate PCNA (NS); up-regulate cell cycle regulatory protein P21 (NS), down-regulate pro-apoptotic Bax (P ⁇ 0.01) and anti-apoptotic protein Survivin (P ⁇ 0.05 );
  • N means that there is no statistical difference between the drug group and the blank solvent group (P>0.05);
  • "*” means that there is a significant difference between the drug group and the blank solvent group (P ⁇ 0.05);
  • ** means Compared with the blank solvent group, there was a significant difference
  • Figure 9 shows that the glomeruli and renal tubules of the mice in the normal feeding blank control group were basically uninjured, and only some areas of the renal tubule wall were thinned and slightly damaged (the picture is PAS staining, magnified 20 times).
  • Figure 10 shows the serious loss of renal tubular epithelium after administration of 10 mg/kg cisplatin to mice in the model group: the brush border of the cells was lost, and a large number of protein casts appeared (the picture is HE staining, magnified 20 times).
  • Figure 11 shows that after administration of cisplatin (10mg/kg) and amifostine (50mg/kg, intraperitoneal injection half an hour before cisplatin administration) to the mice in the positive control group, only slight renal tubular damage occurred, and the edge of the renal tubular epithelium was blurred (The picture is HE staining, magnified 40 times).
  • Figure 12 shows that after administration of cisplatin (10 mg/kg) and R01 (6 mg) to mice in the test group, severe renal tubular damage, epithelial cell necrosis and slight protein casts occurred (the picture is HE staining, magnified 40 times).
  • Figure 13 shows that after administration of cisplatin (10 mg/kg) and R01 (18 mg) to the mice in the test group, certain renal tubular damage occurred, and necrosis of epithelial cells was visible (the picture is HE staining, magnified 40 times).
  • Figure 14 shows that after administration of cisplatin (10mg/kg) and R01 (36mg) to the mice of the test group, slight renal damage occurred, including enlarged renal tubular lumen, epithelial cell necrosis, etc. (the picture is HE staining, enlarged at 40 times).
  • the present invention provides a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant, and mixtures thereof.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I), and optionally a pharmaceutically acceptable excipient.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable excipient, which also comprises other therapeutic agents.
  • the present invention provides a kit comprising a compound of formula (I), and other therapeutic agents, and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • the present invention provides the use of a compound of formula (I) for the manufacture of a medicament for treating and/or preventing cancer in a subject.
  • the present invention provides a method of treating and/or preventing cancer in a subject, comprising administering to said subject a compound of formula (I) or a composition of the present invention.
  • the invention provides a compound of formula (I) or a composition of the invention for use in the treatment and/or prevention of cancer in a subject.
  • the present invention provides a combination comprising a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant thereof, and a platinum drug.
  • the present invention provides a pharmaceutical composition, including the combination according to the eighth aspect and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides the use of the combination described in the eighth aspect in the preparation of a medicament for treating and/or preventing cancer in a subject.
  • the present invention provides a pharmaceutical preparation, comprising a compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotope variant, and a platinum drug, as Combination preparations for simultaneous, sequential or separate use.
  • the present invention provides a method for treating and/or preventing cancer, said method comprising simultaneously, sequentially or separately administering a compound of formula (I) or a solvate thereof to a subject or a patient , hydrates, polymorphs, prodrugs or isotopic variants, and platinum-based drugs.
  • the present invention provides the use of a compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant in the preparation of a drug for treating and/or preventing cancer, wherein
  • the treatment includes simultaneously, sequentially or separately administering the compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant thereof and a platinum drug to a patient or subject.
  • the present invention provides compounds of formula (I), or their solvates, hydrates, polymorphs, prodrugs or isotope variants and platinum drugs in the preparation of treatment and/or prevention of subjects use in cancer medicine.
  • the present invention provides a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant thereof in the preparation of a medicament for treating and/or preventing cancer in a subject Use in , wherein the drug is used in combination therapy with platinum drugs.
  • the present invention provides the use of a platinum-based drug in the preparation of a drug for treating and/or preventing cancer in a subject, wherein the drug is used with a compound of formula (I), or a solvate thereof , hydrates, polymorphs, prodrugs or isotopic variants in combination therapy.
  • the present invention provides a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotope variant thereof, used for alleviating platinum-based drug-induced acute renal failure in a subject. Use in medicine in injury.
  • the present invention provides a method for alleviating platinum-based drug-induced acute kidney injury in a subject, comprising administering the compound of formula (I) simultaneously, sequentially or separately, or Steps of solvates, hydrates, polymorphs, prodrugs or isotopic variants thereof.
  • the platinum drug is selected from cisplatin, carboplatin, nedaplatin, oxaliplatin and lobaplatin.
  • the cancer is selected from lung cancer, gastric cancer, esophageal cancer, colorectal cancer.
  • cancer includes, but is not limited to, the following cancers: stomach, lung, esophagus, colorectum. More specifically, cancers include, but are not limited to, metastatic gastric or gastroesophageal junction adenocarcinoma with HER2 overexpression, non-small cell lung cancer with sensitive mutations in the epidermal growth factor receptor (EGFR) gene, disease progression during or after platinum-containing chemotherapy Locally advanced or metastatic non-small cell lung cancer with squamous histology.
  • EGFR epidermal growth factor receptor
  • treating relates to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such a disorder or condition.
  • the noun “treat” as used herein refers to the action of the verb treat, which is as just defined.
  • Subjects for administration include, but are not limited to: human (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young Adult, middle-aged adult or older adult)) and/or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep , goats, rodents, cats and/or dogs.
  • the subject is a human.
  • the subject is a non-human animal.
  • the terms "human", “patient” and “subject” are used interchangeably herein.
  • treating includes an effect on a subject suffering from a particular disease, disorder or condition, which reduces the severity of the disease, disorder or condition, or delays or slows down the disease, disorder or the development of a disease, disorder or condition ("therapeutic treatment”) and also includes effects that occur before a subject begins to suffer from a particular disease, disorder or condition (“prophylactic treatment").
  • an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response.
  • an effective amount of a compound of the invention i.e., a compound of formula (I)
  • An effective amount includes a therapeutically effective amount and a prophylactically effective amount.
  • a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment of a disease, disorder or condition, or to induce one or more symptoms associated with the disease, disorder or condition. Amount to delay or minimize.
  • a therapeutically effective amount of a compound refers to that amount of the therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of a disease, disorder or condition.
  • the term "therapeutically effective amount” can include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic effect of other therapeutic agents.
  • a prophylactically effective amount of a compound is an amount sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent a disease , the amount of recurrence of the disorder or condition.
  • a prophylactically effective amount of a compound refers to that amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of a disease, disorder or condition.
  • the term “prophylactically effective amount” may include amounts that improve overall prophylaxis, or that enhance the prophylactic effect of other prophylactic agents.
  • Combination and related terms refer to the simultaneous or sequential administration of a compound of the invention and another therapeutic agent.
  • the compounds of the invention may be administered with the other therapeutic agent simultaneously or sequentially in separate unit dosage forms, or together with the other therapeutic agent in a single unit dosage form.
  • organic compounds may form complexes with solvents in which they react or from which they are precipitated or crystallized. These complexes are known as "solvates”. When the solvent is water, the complex is called a "hydrate”. The invention covers all solvates of the compounds of the invention.
  • solvate refers to a form of a compound, or a salt thereof, which is associated with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding.
  • solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like.
  • Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid.
  • “Solvate” includes both solution state solvates and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.
  • hydrate refers to a compound that combines with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined.
  • a hydrate of a compound can be represented, for example, by the general formula R.x H 2 O, where R is the compound, and x is a number greater than zero.
  • a given compound may form more than one hydrate type, including, for example, monohydrates (x is 1), lower hydrates (x is a number greater than 0 and less than 1, for example, hemihydrates (R 0.5H2 O)) and polyhydrates (x is a number greater than 1, eg, dihydrate (R ⁇ 2H 2 O) and hexahydrate (R ⁇ 6H 2 O)).
  • the compounds of the invention may be in amorphous or crystalline form (polymorphs). Furthermore, the compounds of the invention may exist in one or more crystalline forms. Accordingly, the present invention includes within its scope all amorphous or crystalline forms of the compounds of the invention.
  • polymorph refers to a crystalline form of a compound (or a salt, hydrate or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors can cause one crystalline form to predominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.
  • the invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I), but with one or more atoms represented by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature replaced.
  • isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl.
  • the compounds of the present invention their prodrugs and pharmaceutically acceptable salts of the compounds or the prodrugs containing the above-mentioned isotopes and/or other isotopes of other atoms all belong to the scope of the present invention.
  • Certain isotopically-labeled compounds of the invention eg, those incorporating radioactive isotopes (eg, 3H and14C ), are useful in drug and/or substrate tissue distribution assays. Tritium, ie3H , and carbon-14, ie14C isotopes are particularly preferred because of their ease of preparation and detection.
  • isotope-labeled compound of formula (I) of the present invention and its prodrug can generally be prepared in this way.
  • prodrugs are also included within the context of the present invention.
  • the term "prodrug” as used herein refers to a compound that is converted in vivo to its active form having a medical effect, for example by hydrolysis in blood.
  • Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, and D. Fleisher, S. Ramon, and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130, per intro This article is for reference.
  • a prodrug is any covalently bonded compound of the invention which, when administered to a patient, releases the parent compound in vivo.
  • Prodrugs are generally prepared by modifying functional groups in such a way that the modification can be cleaved by routine manipulation or in vivo to yield the parent compound.
  • Prodrugs include, for example, compounds of the invention wherein a hydroxy, amino or thiol group is bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amino or thiol group.
  • representative examples of prodrugs include, but are not limited to, acetate/amide, formate/amide and benzoate/amide derivatives of the hydroxy, sulfhydryl and amino functional groups of the compounds of formula (I).
  • esters such as methyl ester, ethyl ester and the like can be used.
  • the esters themselves may be reactive and/or hydrolyzable under human in vivo conditions.
  • Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those which break down readily in the human body to release the parent acid or a salt thereof.
  • the present invention also provides a pharmaceutical preparation comprising a therapeutically effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier, diluent or excipient thereof. All of these forms are included in the present invention.
  • pharmaceutical preparation includes the use of the ingredients of the present invention directly as a medicament in addition to being used in any stage of the preparation of such medicaments.
  • combination therapy refers to the sequential if not simultaneous administration of a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant thereof, and a platinum drug within a time frame So that they can all play a therapeutic role within the same time limit.
  • one aspect of the present invention relates to a pharmaceutical preparation comprising a compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant thereof, and a platinum drug, as a simultaneous, Combination preparations used sequentially or separately.
  • “simultaneously” means to administer two agents simultaneously, while the term “combined” means to administer them “sequentially” within a time frame if they cannot be administered simultaneously so that they both fit within the same time frame. Play a therapeutic role.
  • “sequential” administration may allow administration of one agent 5 minutes, 10 minutes, or approximately several hours after another agent, as long as the circulating half-life of the first administered agent is such that both are administered simultaneously in therapeutically effective amounts.
  • the time delay between administration of the ingredients will vary depending on the exact nature of the ingredients, their interactions and their respective half-lives.
  • subtherapeutic amounts of the compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant and the platinum drug are administered separately relative to the individual components
  • the compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant and the platinum drug is not administered in a therapeutically effective amount if not administered in combination.
  • the compound of formula (I), or a solvate, hydrate, polymorph, prodrug or isotopic variant thereof, and the platinum drug interact in a synergistic manner.
  • the term “synergistic” means that a compound of formula (I), or its solvate, hydrate, polymorph, prodrug or isotopic variant, and a platinum-based drug, when used in combination, can produce The individual effects of the sum of the expected effects are larger effects.
  • a synergistic interaction may allow lower doses of each component to be administered to a patient, thereby reducing the toxicity of chemotherapy while producing and/or maintaining the same efficacy.
  • each component may be administered in subtherapeutic amounts.
  • the invention provides pharmaceutical compositions comprising a compound of the invention (also referred to as "active ingredient") and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an effective amount of a compound of the invention.
  • the pharmaceutical composition comprises a therapeutically effective amount of a compound of the invention.
  • the pharmaceutical composition comprises a prophylactically effective amount of a compound of the invention.
  • a pharmaceutically acceptable excipient used in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated together.
  • Pharmaceutically acceptable carriers, adjuvants or vehicles that can be used in the compositions of the present invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin Protein), buffer substances (such as phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate , sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- Block polymers, polyethylene glycol
  • kits eg, pharmaceutical packs.
  • kits can include a compound of the invention, another therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispersible packs or other suitable container).
  • first and second containers e.g., vials, ampoules, bottles, syringes, and/or dispersible packs or other suitable container.
  • provided kits can also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and/or other therapeutic agent.
  • a compound of the invention and other therapeutic agent provided in a first container and a second container are combined to form a unit dosage form.
  • parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration , intracerebrospinal administration, intralesional administration, and intracranial injection or infusion techniques.
  • an effective amount of a compound provided herein is administered.
  • the amount of the compound actually administered can be determined by the physician according to the circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, etc. .
  • the compounds provided herein are administered to a subject at risk of developing the condition, typically on the advice and supervision of a physician, at dosage levels as described above.
  • Subjects at risk of developing a particular condition generally include those with a family history of the condition, or those determined by genetic testing or screening to be particularly susceptible to developing the condition.
  • Chronic administration refers to administering a compound or a pharmaceutical composition thereof for a long period of time, for example, 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue administration indefinitely, For example, the rest of the subject's life.
  • chronic administration is intended to provide a constant level of the compound in the blood over an extended period of time, eg, within the therapeutic window.
  • compositions may be administered as a bolus injection, eg, in order to increase the concentration of the compound in the blood to effective levels.
  • the bolus dose depends on the target systemic level of the active ingredient through the body, for example, an intramuscular or subcutaneous bolus dose provides slow release of the active ingredient, while a bolus delivered directly into a vein (e.g., by IV intravenous infusion) ) can be delivered more rapidly, so that the concentration of the active ingredient in the blood rises rapidly to effective levels.
  • the pharmaceutical compositions may be administered as a continuous infusion, eg, by IV infusion, to provide a steady state concentration of the active ingredient in the subject's body. Additionally, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by a continuous infusion.
  • Oral compositions may take the form of bulk liquid solutions or suspensions or bulk powders. More usually, however, the compositions will be presented in unit dosage form for ease of precise dosing.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human patients and other mammals, each unit containing a predetermined quantity of active material suitable to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include prefilled, premeasured ampoules or syringes for liquid compositions, or pills, tablets, capsules and the like in the case of solid compositions.
  • the compound will generally be a minor component (from about 0.1 to about 50% by weight, or preferably from about 1 to about 40% by weight), with the remainder being various components useful for forming the desired administration form. Carriers or excipients and processing aids.
  • a typical regimen is one to five oral dosages per day, especially two to four oral dosages, typically three oral dosages.
  • each dose provides about 0.01 to about 100 mg/kg of the compound of the invention, with preferred doses each providing about 0.1 to about 10 mg/kg, especially about 0.5 to about 2 mg/kg.
  • Injection dosage levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour from about 1 to about 120 hours, especially 24 to 96 hours.
  • a preload bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be given in order to achieve adequate steady state levels.
  • the maximum total dose should not exceed approximately 2 g/day.
  • Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers as well as buffering, suspending and dispersing agents, coloring agents, flavoring agents, and the like.
  • the solid form may comprise, for example, any of the following components, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch or lactose, disintegrants, For example, alginic acid, Primogel, or corn starch; lubricants, for example, magnesium stearate; glidants, for example, colloidal silicon dioxide; sweeteners, for example, sucrose or saccharin; or flavoring agents, for example, peppermint, water Methyl sylate or orange flavoring.
  • binders such as microcrystalline cellulose, tragacanth, or gelatin
  • excipients such as starch or lactose, disintegrants, For example, alginic acid, Primogel, or corn starch
  • Injectable compositions are typically based on injectable sterile saline or phosphate buffered saline, or other injectable excipients known in the art.
  • the active compound is typically a minor component, often from about 0.05 to 10% by weight, the remainder being injectable excipients and the like.
  • Transdermal compositions are typically formulated as topical ointments or creams containing the active ingredient.
  • the active ingredients When formulated in an ointment, the active ingredients are typically combined with a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream, with, for example, an oil-in-water cream base.
  • Such transdermal formulations are well known in the art, and generally include other components for enhancing the stable skin penetration of the active ingredient or formulation. All such known transdermal formulations and compositions are included within the scope of the present invention.
  • transdermal administration can be achieved using patches of the reservoir or porous membrane type, or various solid matrices.
  • compositions for oral administration, injection or topical administration are representative only. Other materials and processing techniques, etc. are described in Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, Section 8, which is incorporated herein by reference.
  • the compounds of the invention may also be administered in sustained release form, or from a sustained release delivery system.
  • sustained release materials can be found in Remington's Pharmaceutical Sciences.
  • the compounds of the present invention have value as antitumor agents.
  • the compounds of the invention have value as anti-proliferative, apoptotic and/or anti-invasive agents in the suppression and/or treatment of solid and/or liquid neoplastic diseases.
  • the compounds of the present invention are expected to be useful for the prevention or treatment of gastric cancer, lung cancer and the like.
  • Anticancer effects useful for treating cancer in a patient include, but are not limited to, antitumor effects, response rates, time to disease progression, and survival rates.
  • Antitumor effects of the treatment methods of the present invention include, but are not limited to, inhibition of tumor growth, delay in tumor growth, regression of tumors, shrinkage of tumors, prolongation of tumor regrowth time after cessation of treatment, and slowing of disease progression.
  • Anticancer effects include prophylactic therapy as well as treatment of existing disease.
  • an effective amount of a compound of the present invention is usually in an average daily dose of 0.01 mg to 50 mg compound/kg patient body weight, preferably 0.1 mg to 25 mg compound/kg patient body weight, in single or multiple administrations.
  • the compounds of the present invention may be administered to the patient in need of such treatment at a daily dosage ranging from about 1 mg to about 3500 mg, preferably 10 mg to 1000 mg per patient.
  • the daily dose per patient may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 160, 180, 200, 240, 250, 300, 350, 360, 400, 500, 600, 700, 800, 900 or 1000mg.
  • Administration can be one or more times daily, weekly (or at intervals of days), or on an intermittent schedule.
  • the compound may be administered one or more times per day on a weekly basis (eg, every Monday), indefinitely or over several weeks, eg, 4-10 weeks.
  • the compound can be administered daily for several days (e.g. 2-10 days) followed by several days (e.g. 1-30 days) without administration of the compound and the cycle repeated indefinitely or a given number of times, e.g. 4-10 days. cycles.
  • a compound of the invention may be administered daily for 5 days followed by 9 days off, then daily for 5 days followed by 9 days off, and so on, repeating the cycle indefinitely or 4-10 times in total.
  • the platinum-based drug used in combination with the compound of formula (I) of the present invention, or its solvate, hydrate, polymorph, prodrug or isotopic variant, is recommended to be a clinically used or recommended dose.
  • the usual dose of cisplatin is 50-100 mg/m 2 , or 15-20 mg/m 2 intravenously every day for 5 consecutive days, and the drug is repeated every 3-4 weeks.
  • the recommended dose is 400 mg/m 2 for patients with normal renal function; for patients with risk factors, it is recommended to reduce the initial dose by 20-25%; for patients over 65 years old, the initial dose should be adjusted according to the patient's physical condition and subsequent treatment doses.
  • each administration is 80-100mg/m 2 , and the next course of treatment can be started after an interval of 3-4 weeks.
  • the recommended dose is 130mg/m 2 once, once every 3 weeks (21 days) when no major toxicity occurs; or 85mg/m2, repeated once every 2 weeks.
  • the recommended dose is 50mg/m 2 once, and the blood toxicity or other clinical side effects should be completely recovered when it is used again.
  • the recommended application interval is 3 weeks. If the side effects recover slowly, the interval can be extended.
  • sub-therapeutic administration can also be administered separately relative to the individual components.
  • Embodiment 1 the synthesis of formula (I) compound
  • Embodiment 2 the study of the compound of formula (I) on the effect of tumor cell proliferation, tumor cell cycle and apoptosis
  • Human lung cancer cell lines A549 and NCI-H460 were obtained from the cell bank of the Oncology Laboratory of Tongji Hospital.
  • BSA dilute 1g of Albumin bovine v to 10ml with ultrapure water
  • TBST dilute 50ml of 10 ⁇ TBS and 500 ⁇ l of Tween 20 to 500ml with ultrapure water
  • cell freezing solution Prepared with RPMI-1640 medium 28ml, FBS 8ml and DMSO 4ml and stored at 4°C
  • cell lysate [500 ⁇ M HEPES 1ml, 3M sodium chloride 500 ⁇ l, 500 ⁇ M EDTA 20 ⁇ l, 500 ⁇ M EGTA 20 ⁇ l, 20% Triton X-100 250 ⁇ l, ddH2O 7.7ml, mix and pack into 1ml tubes, store at -20°C, 1M DTT (add immediately) 1:1000, 100 ⁇ M PMSF (add immediately) 1:100, 25 ⁇ Cocktail (add immediately )1:25].
  • Stock solution of the compound of formula (I) the compound of formula (I) is easily soluble in organic solvents, and the ratio of DMSO to isopropanol is 3:7 as the solvent. Open DMSO and isopropanol in a biosafety cabinet, take a 15ml centrifuge tube, add 3ml DMSO and 7ml isopropanol to make a mixed solvent. The molecular weight of the compound of formula (I) is 678.22g/mol.
  • the cell culture plate was taken out from the incubator and placed in a biological safety cabinet. Take a 4ml centrifuge tube and mark it, suck out the supernatant from the well and add it to the corresponding 4ml centrifuge tube. Rinse the cells gently with PBS, and add PBS to the corresponding centrifuge tube. Add an appropriate amount of trypsin to each well to digest the cells. When the degree of cell digestion is moderate, add the old culture medium in the centrifuge tube to the corresponding well, gently blow the cells with a pipette gun, blow the cells into a single-cell suspension, and suck them back to the original centrifuge tube. , quickly adopt 1500rpm centrifugation for 5min, discard the supernatant and keep the precipitate.
  • the cell culture plate was taken out from the incubator and placed in a biological safety cabinet. Mark the 4ml centrifuge tube, suck out the supernatant from the 6-well plate and add it to the corresponding 4ml centrifuge tube. Rinse the cells gently with PBS, and add PBS to the corresponding centrifuge tube. Add an appropriate amount of trypsin to each well to digest the cells. When the degree of cell digestion is moderate, add the liquid in the centrifuge tube to the corresponding well. Gently blow the cells with a pipette gun to blow the cells away, suck them back into the original centrifuge tube, and quickly centrifuge at 1000rpm for 5 minutes. , discard the supernatant and save the precipitate.
  • the compound of formula (I) inhibits the proliferation of lung cancer cells
  • the compound of formula (I) also had an inhibitory effect on the proliferation of NCI-H460, and the results showed that the IC50 of NCI-H460 cells at 24h, 48h and 72h were 125.3 ⁇ 1.01 ⁇ M (Figure 2A), 66.4 ⁇ 0.87 ⁇ M (Figure 2B), 48.5 ⁇ 1.12 ⁇ M (Fig. 2C).
  • the inhibition of the proliferation of NCI-H460 cells by the compound of formula (I) is also time-concentration dependent, that is, the inhibitory effect is enhanced with the increase of the concentration and the prolongation of the action time.
  • Direct measurement of DNA synthesis is one of the most accurate methods for detecting cell proliferation.
  • EDU can be incorporated into the newly synthesized DNA strand during DNA replication, and through a "Click" reaction, the fluorescent group can be labeled to the newly synthesized EDU-containing strand.
  • the fluorescence Treat A549 and NCI-H460 cells with a concentration of 150 ⁇ M formula (I) compound for 48 hours, cell proliferation experiments show that compared with CTL (formula (I) compound 0 ⁇ M), in the formula (I) compound drug action group, the cells that are proliferating The proportion was significantly reduced, and the difference was statistically significant (Figure 3, P ⁇ 0.01).
  • FBS stimulation can promote cell proliferation, and the compound of formula (I) can inhibit the cell proliferation effect caused by high serum concentration stimulation conditions (P ⁇ 0.05).
  • the compound of formula (I) inhibits the colony-forming ability of lung cancer cells
  • the compound of formula (I) reduces the proportion of S phase of lung cancer cells
  • the compound of formula (I) effectively inhibits the proliferation of lung cancer cells in a concentration-dependent manner.
  • flow cytometry to detect the distribution of cell cycle of lung cancer cell lines treated with the compound of formula (I).
  • A549 cells and NCI-H460 cells were treated with medium containing different concentrations of the compound of formula (I) (0 ⁇ M, 10 ⁇ M, 30 ⁇ M, 90 ⁇ M) for 24 hours, 48 hours and 72 hours, respectively. After fixing the cells with 75% ethanol, they were stained with PI for analysis.
  • the compound of formula (I) reduces the proportion of A549 cells in S phase, and presents a dose-dependent manner. Specifically, after being treated with the compound of formula (I) for 24 hours, the proportions of cells in S phase of 0 ⁇ M, 10 ⁇ M, 30 ⁇ M and 90 ⁇ M groups were 31.91%, 22.94%, 12.52% and 3.92%, respectively. After being treated with the compound of formula (I) for 48 hours, the S phase of the cells in the 0 ⁇ M, 10 ⁇ M, 30 ⁇ M and 90 ⁇ M groups were 25.09%, 24.73%, 15.96% and 4.37%, respectively. After being treated with the compound of formula (I) for 72 hours, the S phase of the cells in the 0 ⁇ M, 10 ⁇ M, 30 ⁇ M and 90 ⁇ M groups were 20.64%, 19.77%, 10.63% and 5.42%, respectively.
  • NCI-H460 cells were similar to those of A549 cells.
  • the results are shown in Figure 6: after NCI-H460 was treated with the compound of formula (I) for 24 hours, the proportions of cells in the S phase of the four groups of 0 ⁇ M, 10 ⁇ M, 30 ⁇ M and 90 ⁇ M were 23.57%, 26.03%, 13.15% and 9.76%, respectively. After being treated with the compound of formula (I) for 48 hours, the proportions of cells in S phase in the four groups of 0 ⁇ M, 10 ⁇ M, 30 ⁇ M and 90 ⁇ M were 29.09%, 29.62%, 16.02% and 0.97%, respectively.
  • the proportions of cells in S phase of the four groups of 0 ⁇ M, 10 ⁇ M, 30 ⁇ M and 90 ⁇ M were 25.33%, 21.61%, 20.98% and 3.36%, respectively.
  • Formula (I) compound promotes lung cancer cell apoptosis
  • Table 1 and Table 2 respectively represent the proportions of dead cells, late apoptotic cells, early apoptotic cells, and normal cells after different concentrations of the compound of formula (I) act on A549 and NCI-H460 cells.
  • Table 1 and Table 2 respectively represent the proportions of dead cells, late apoptotic cells, early apoptotic cells, and normal cells after different concentrations of the compound of formula (I) act on A549 and NCI-H460 cells.
  • the compound concentration of formula (I) was low (10 ⁇ M, 30 ⁇ M)
  • the ratio of early apoptotic cells and late apoptotic cells was compared with that of the control group Slightly increased, but the difference was not statistically significant, but when the concentration of the compound of formula (I) increased to 90 ⁇ M, the proportions of early apoptosis and late apoptosis in A549 and NCI-H460 cells were significantly increased, and the proportion of early apoptosis increased more obvious.
  • Table 3 shows the change of the total apoptosis ratio after different concentrations of the compound of formula (I) act on A549 and NCI-H460 cells. It can be seen that when the concentration of the compound of formula (I) reaches 90 ⁇ M, the percentage of apoptosis increases significantly, and this phenomenon is more obvious in A549 cells than in NCI-H460 cells.
  • Example 3 Test formula (I) compound relative to cisplatin on human gastric cancer N87 nude mouse xenograft tumor model tumor growth inhibition in vivo
  • mice Female BALB/c nude mice (age: 6-7 weeks) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and were kept in an SPF animal room with a temperature of 20-25°C, a relative humidity of 40%-70%, and light and dark The lighting was 12 hours each; the animals had free access to water and food. After being fed normally for about 1 week, the mice with good physical condition can be selected for this experiment after veterinary inspection. Before grouping, use a marker pen to mark the base of the tail of the animals, and after grouping, each animal is marked by ear clipping.
  • Human gastric cancer cell NCI-N87 was obtained from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (frozen in liquid nitrogen in our laboratory).
  • NCI-N87 cells were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum; digested and passaged with 0.25% trypsin; The ratio is 1:3 to 1:5.
  • NCI-N87 cells in the logarithmic growth phase, count the cells, resuspend them in serum-free RPMI-1640 medium, adjust the cell concentration to 5 ⁇ 10 7 cells/mL; blow the cells with a pipette to disperse them evenly, and load them into Put the centrifuge tube in a 50-mL centrifuge tube, put the centrifuge tube in an ice box; draw up the cell suspension with a 1-mL syringe, inject it subcutaneously into the axilla of the anterior right limb of nude mice, and inoculate 100 ⁇ L per animal (5 ⁇ 10 6 cells/mouse) , to establish NCI-N87 nude mouse xenograft tumor model.
  • the state of the animals and the growth of the tumor were observed regularly, and the diameter of the tumor was measured using an electronic vernier caliper, and the data was entered into an Excel spreadsheet to calculate the tumor volume.
  • the tumor volume reached 100-300 mm 3
  • the day of grouping was taken as the first day of the experiment (D1).
  • the tumor diameter was measured twice a week, and the tumor volume was calculated.
  • the body weight of the animals was weighed and recorded.
  • the tumor volume (TV) calculation formula is as follows:
  • TGI (%) The calculation formula of tumor growth inhibition rate TGI (%) is:
  • TGI(%) 100% ⁇ [1–(TV t(T) –TV initial(T) )/(TV t(C) –TV initial(C) )]
  • TV t (T) represents the tumor volume measured each time in the treatment group
  • TV initial (T) represents the tumor volume of the treatment group when administered in groups
  • TV t (C) represents the tumor volume measured each time in the solvent control group
  • TV initial (C) represents the tumor volume of the solvent control group at the time of group administration.
  • the formula for calculating the weight loss rate of animals is:
  • Animal weight loss rate 100% ⁇ (BW initial - BW t )/BW initial
  • BW t represents the animal body weight measured each time during the administration period
  • BW initial represents the animal body weight at the time of group administration.
  • experimenters and veterinarians need to continuously observe the signs and health status of the experimental animals. Any abnormal manifestations of animals, such as pain, depression, decreased activity, etc., should be recorded in the original experimental records. If the abnormal performance of the experimental animals exceeds the IACUC related animal welfare documents, the veterinarian can judge whether to suspend the experiment and notify the person in charge of the experimental project.
  • the compound formula (I) compound of the present invention has better anti-tumor effect than the commonly used antigastric cancer drug cisplatin; the body weight of the animals in the cisplatin group is significantly lower than that of the blank control group, but the compound of the present invention has no significant effect on the body weight of the animals. Influence.
  • the tumor tissue samples are from the animal tumor samples in Example 3.
  • the compound of formula (I) down-regulates p-VEGFR2 (NS), down-regulates VEGFR2 (P ⁇ 0.05), VEGFA (P ⁇ 0.05); down-regulates ⁇ -catenin (P ⁇ 0.05), CyclinD1 (NS), up-regulates PCNA (NS); up-regulates The cell cycle regulatory protein P21(NS) down-regulated the pro-apoptotic Bax (P ⁇ 0.01) and the anti-apoptotic protein Survivin (P ⁇ 0.05).
  • DDP down-regulates p-VEGFR2(P ⁇ 0.05), VEGFA(NS), up-regulates VEGFR2(NS), ⁇ -catenin(P ⁇ 0.05) and CyclinD1(NS), down-regulates the apoptosis inhibitor protein Survivin(NS) and down-regulates cell cycle regulation Protein P21 (P ⁇ 0.05). Therefore, in the N87 nude mouse xenograft tumor model, the compound of formula (I) showed a stronger angiogenesis inhibitory effect compared with DDP, and thus had a better antitumor effect ( FIG. 8 ).
  • Embodiment 5 Test sample oral acute toxicity test
  • Test sample compound of formula (I).
  • Preparation of preparations for the control group of excipients mark the final volume scale line of the container, weigh the required amount of excipients into the container, first add an appropriate amount of solvent to it, stir and mix thoroughly, then dilute and mix with the solvent to the final volume scale, A visually uniform formulation was obtained. Store at room temperature, away from light and airtight for 24 hours,
  • the weighing and preparation of the drug preparations for the test should be operated under the yellow light.
  • formula (I) compound preparations take an appropriate amount of formula (I) compound preparations, add an appropriate volume of 0.5% CMC-Na aqueous solution, vortex and ultrasonically mix until uniform (if necessary, shear emulsification can be carried out), to obtain the drug preparation test sample . It is prepared twice a week, and the dosage is prepared for 3 days or 4 days each time. After subpackaging, it is stored in a refrigerator with 0-8 doses for later use.
  • Number and sex of animals used 40, half male and half male;
  • Body weight female group weighs 25.9-29.7g; male group weighs 27.7-30.8g;
  • Age at the time of administration 38-44 days (female), 31-44 days (male)
  • Groups 2 to 4 represent the amount of the compound of formula (I), and group 1 represents the amount of excipients.
  • Administration route intragastric administration
  • Dosing frequency 1 time/day
  • the drug preparations of each group were stirred at room temperature for at least 10 minutes before administration, and the suspension was uniform by visual inspection, and continuous stirring was required during the administration process.
  • Observation indicators including observation of general signs, body weight, food intake, animal autopsy and histopathological examination.
  • Embodiment 6 ICR mice gavage the compound of formula (I) for 2 weeks and repeated administration toxicity dose exploration test
  • ICR mice were given the compound of formula (I) by gavage continuously for 14 days to evaluate the nature, degree and time-effect relationship of the compound of formula (I) that may cause toxic reactions, in order to provide reference information for follow-up research.
  • the weighing and preparation of the drug preparations for the test should be operated under the yellow light.
  • Preparation of each dosage group of the test product The preparation of the test product is the same as in Example 5.
  • the drug preparation of the test product is in the concentration range of 0.01mg/mL ⁇ 60mg/mL, and is kept in airtight storage at 2°C ⁇ 8°C in the dark, and is stable within 8 days.
  • Estimated body weight at the start of administration is 24-30g for females and 26-32g for males
  • mice After purchase, they were kept in the SPF animal room.
  • the license number of the testing institution SYXK (Lu) 20180031. Animals were kept in transparent mouse cages, and males and females were reared separately, with ⁇ 5 mice per cage.
  • SPF rat growth and reproduction feed was purchased from Beijing Keao Xieli Feed Co., Ltd. Animals were fed ad libitum.
  • Corn cob pellets were purchased from Beijing Keao Xieli Feed Co., Ltd.
  • the feed supplier shall provide the feed quality inspection report of the purchased batch, and other inspection frequencies, indicators and requirements shall be carried out in accordance with the requirements of the central SOP.
  • the quarantine adaptation was completed. According to the latest animal health status screening, 100 healthy animals (half male and half male) were selected for the experiment after being signed and confirmed by the person in charge of the project.
  • Dosing was calculated based on the latest weighed animal body weight.
  • the drug preparation of the test product was stirred at room temperature for at least 10 minutes before the drug administration, and the suspension was uniform by visual inspection, and continuous stirring was required during the drug administration.
  • Observation indicators including general sign observation, detailed clinical observation, body weight, and food intake.
  • Embodiment 7 test formula (I) compound single use and formula (I) compound and chemotherapeutic drug cisplatin (DDP) combine the inhibitory effect on human gastric cancer NCI-N87 nude mouse xenograft tumor model tumor growth in vivo
  • mice preparation of blank solvent, preparation of cisplatin (DDP), preparation of compound administration preparation of formula (I), transplanted tumor strain, NCI-N87 cell culture, preparation of animal model, experimental process and statistical analysis method are the same as the examples 3.
  • R01 has obvious inhibitory effect on the growth of NCI-N87 nude mouse xenograft tumor under the conditions of this experiment, and its anti-tumor effect is better than that of the DDP single use group.
  • R01 was used in combination with DDP at doses of 3, 6, and 16 mg somatostatin (QD), respectively, and its efficacy was better than that of DDP alone, and the combined use could reduce the level of serum UREA in mice.
  • the dose of R01 was negatively correlated, suggesting that R01 may have a protective and preventive effect on renal injury caused by DDP.
  • the drug effect of R01 (8 mg has certain protective and preventive effects.) + DDP group is slightly better than or equivalent to other administration groups.
  • Example 8 Test the synergistic inhibitory effect of the compound of formula (I) and DDP on the growth of human esophageal cancer ECA109 nude mouse xenograft tumor model in vivo
  • Human esophageal cancer cell ECA109 was obtained from the cell bank of Wuhan University (CCTCC, cryopreserved in liquid nitrogen in our laboratory).
  • ECA109 cells were cultured routinely in RPMI-1640 medium containing 10% fetal bovine serum; digested with 0.25% trypsin and passaged; according to the cell growth, the passage ratio was 1 :3 to 1:6.
  • ECA109 cells in the logarithmic growth phase, count the cells, and resuspend them in 50% serum-free RPMI-1640 medium and 50% Matrigel, adjust the cell concentration to 0.5 whole cells 8 cells/mL; blow the cells with a pipette After making it evenly dispersed, put it into a 50-mL centrifuge tube, and put the centrifuge tube in an ice box; use a 1-mL syringe to draw the cell suspension, and inject it subcutaneously into the armpit of the front right limb of NOD/SCID mice, and inoculate each animal 200 ⁇ L (1.0 limb axillary 7 cells/mouse), to establish the ECA109 nude mouse xenograft tumor model.
  • the state of the animals and the growth of the tumor were observed regularly, and the diameter of the tumor was measured using an electronic vernier caliper, and the data was entered into an Excel spreadsheet to calculate the tumor volume.
  • the tumor volume reaches 100-300mm 3 , animals with good health and similar tumor volume are selected and grouped by random block method.
  • the day of grouping is the first day of the experiment (D1). After the experiment starts, the tumor diameter is measured twice a week, and calculated The tumor volume was measured and the body weight of the animal was recorded at the same time.
  • R01 preparation has obvious inhibitory effect on the growth of ECA109 nude mouse xenograft tumor under the conditions of this experiment, and its antitumor effect is better than that of DDP single use group.
  • R01 preparation is used in combination with DDP under 18mg Changchangcun -1 (QD) dose, and its drug effect is better than DDP and R01 single use;
  • QD Changchangcun -1
  • the animal body weight of the combined treatment group is compared with DDP single use group, and the body weight loss rate is equivalent, shows that R01 and R01 are used alone.
  • Embodiment 9 test formula (I) compound and its synergistic inhibitory effect with cisplatin on human non-small cell lung cancer A549 nude mouse xenograft model tumor growth in vivo
  • Human non-small cell lung cancer A549 was obtained from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (CAS, cryopreserved in liquid nitrogen in this experiment).
  • A549 cells were routinely cultured in F12K medium containing 10% fetal bovine serum; digested and passaged with 0.25% trypsin; according to the growth of the cells, passaged 2 to 3 times a week, The passaging ratio is 1:3 to 1:5.
  • experiment D1 collect A549 cells in the logarithmic growth phase, count the cells, resuspend them in PBS, adjust the cell concentration to 8, and adjust to 7 cells/mL; blow the cells with a pipette to make them evenly dispersed, and put them into a 50mL centrifuge tube , put the centrifuge tube in an ice box, draw up the cell suspension with a 1mL syringe, and inject it subcutaneously into the axilla of the right anterior limb of nude mice, and inoculate 100 ⁇ L (800 6 cells/mouse) in each animal to establish the A549 nude mouse xenograft tumor model. After inoculation, the state of animals and tumor growth were observed regularly.
  • the tumor diameter was measured with an electronic vernier caliper, the data was input into an Excel spreadsheet, and the tumor volume was calculated.
  • Tumor-bearing mice with good health and similar tumor volume (101-156 mm 3 ) were selected and grouped by random block method. After the start of the experiment, the diameter of the tumor was regularly measured, the tumor volume was calculated, and the body weight of the animal was weighed and recorded.
  • mice were administered (R01) 1 hour after CO2 inhalation anesthesia, and blood was collected from the heart.
  • a part of the whole blood was anticoagulated with EDTA-K2, and centrifuged at 4, 1500g for 10 minutes to separate the plasma. Take 80% of the plasma and store it. Store at -40°C to -20°C refrigerator (if necessary, it can be used for the determination of blood drug concentration); the other part of the whole blood does not add any anticoagulant, and centrifuges the serum after the blood is clotted for the determination of blood urea nitrogen.
  • the tumor tissue was collected, weighed, and photographed. The tumor was divided into two parts, which were quick-frozen in liquid nitrogen and then transferred to -90 C to -60 for storage in the refrigerator; the bilateral kidneys were collected using 10% formalin I'm fixed.
  • DDP has no obvious effect on the growth of transplanted tumors in A549 nude mice at the doses of 4 mg urea -1 (QWmg) and 5/7/7/7 mg clear urea -1 (QW).
  • R01 preparation alone had significant inhibitory effect on the growth of transplanted tumors in A549 nude mice at doses of 18 mg growth-free -1 BID and 36 mg growth-free -1 BID.
  • Example 10 Test the synergistic inhibitory effect of the compound of formula (I) and cisplatin on the growth of human colorectal cancer HT29 nude mouse xenograft tumor model in vivo
  • mice preparation of blank solvent, preparation of cisplatin (DDP), calculation of tumor volume (TV), end of experiment, data recording, calculation formula, statistical analysis method and experimental observation of tumor growth inhibition rate TGI (%) are the same as in Example 3
  • the preparation of formula (I) compound administration preparation is the same as embodiment 3.
  • the human colon cancer cell line HT-29 was obtained from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (CAS, cryopreserved in liquid nitrogen in this experiment).
  • HT-29 cells were cultured routinely in McCoy cryopreservation medium containing 10% fetal bovine serum, digested and passaged with 0.25% trypsin, and passaged according to the growth of the cells.
  • the passaging ratio is 1:3 to 1:4.
  • experiment D1 collect HT-29 cells in the logarithmic growth phase, count the cells and resuspend them in the serum-free McCoy resuspension medium after counting, adjust the cell concentration to 4 medium, cells/mL; blow the cells with a pipette Make it evenly dispersed and put it into a 50mL centrifuge tube, put the centrifuge tube in an ice box, draw the cell suspension with a 1mL syringe, inject it into the subcutaneous skin of the right anterior armpit of nude mice, and inoculate 100 ⁇ L of each animal (400 cells/mouse ), to establish the HT-29 nude mouse xenograft tumor model.
  • the state of animals and tumor growth were observed regularly.
  • the tumor diameter was measured with an electronic vernier caliper, the data was entered into an Excel spreadsheet, and the tumor volume was calculated.
  • Tumor-bearing mice with good health and similar tumor volume (103-179 mm3) were selected and grouped by random block method. After the start of the experiment, the diameter of the tumor was regularly measured, the tumor volume was calculated, and the body weight of the animal was weighed and recorded.
  • the compound of formula (I) of the present invention can produce synergistic anti-tumor effect with cisplatin: the combined drug has faster onset and better effect, and the safety is not significantly reduced.
  • Embodiment 11 Antagonism of the main target organ toxicity of the chemotherapeutic drug cisplatin by the compound of formula (1)
  • the purpose of this experiment is to definitively study whether R01 can significantly reduce cisplatin-induced acute kidney injury in mice, and compare it with amifostine, the most established drug against cisplatin-induced kidney injury in the first-line clinical practice, to determine whether R01 can protect cisplatin-induced acute kidney injury. Potential for platinum-induced renal injury.
  • Amifostine trihydrate batch number: Z28N11R132596, molecular weight: 268.27; content: 98%, Shanghai Yuanye Biology.
  • Cisplatin (batch number: 601200804, molecular weight: 300.05, purity ⁇ purity ⁇ 0508, conversion factor: 1) was purchased from Jiangsu Hansoh Pharmaceutical Group Co., Ltd.
  • mice 42 male C57BL/6J mice (age: 6-8 weeks, body weight 18-20g, qualification certificate number: 11400700238365) were purchased from Speifu (Beijing) Biotechnology Co., Ltd. and raised at SPF, Institute of Materia Medica, Chinese Academy of Medical Sciences Grade animal room, the temperature is 20-25 degrees, the relative humidity is 40%-70%, the light and dark lighting are 12 hours each, and the animals are free to drink water and eat food. After 3 days of normal feeding, mice with good signs and conditions can be selected for this experiment after veterinary inspection. Before grouping, use a marker pen to mark the base of the tail of the animals. After grouping, each animal is marked by ear clipping (7 animals in each group).
  • the first group blank control group
  • the second group positive control drug cisplatin (10mg/kg);
  • the third group cisplatin (10mg/kg)+amifostine (50mg/kg, intraperitoneal injection half an hour before cisplatin administration, only administered once);
  • the fourth group cisplatin (10mg/kg)+R01 (6mg, bid, administered three days before cisplatin modeling);
  • the fifth group cisplatin (10mg/kg)+R01 (18mg, bid, administered three days before cisplatin modeling);
  • the sixth group cisplatin (10 mg/kg) + R01 (36 mg, bid, administered three days before cisplatin modeling).
  • Blank solvent preparation Take an appropriate amount of sodium carboxymethyl cellulose, prepare it with physiological saline to obtain a 0.5% sodium carboxymethyl cellulose solution, and sterilize it under high pressure.
  • cisplatin (DDP) administration test solution the cisplatin preparation is a solution, which is directly applied and stored at room temperature in the dark.
  • the preparation of the R01 preparation administration test solution is the same as in Example 5: according to the three doses set in the experiment, an appropriate amount of R01 preparation was weighed, and an appropriate volume of 0.5% CMC-Na aqueous solution was added, vortexed and ultrasonically mixed until uniform, respectively to obtain Concentrations of 0.6, 1.8, 3.6 mg -1 administration test solution after shaking are ready for use.
  • Cisplatin administration method one-time intraperitoneal injection of cisplatin solution (10mg/kg)
  • Execution method fasting for 12 hours before execution, taking blood from eyeballs to execute.
  • Kidney pathological examination HE, PAS preparation, slide scanning and report.
  • Animals in each group recorded their body weight at the time of group administration, as well as body weight and kidney weight after the test, and calculated the kidney coefficient; recorded the creatinine and urea nitrogen values of the blood samples sent for inspection; the kidneys were stained with HE and PAS, and slides were scanned. A report will be issued after pathological analysis.
  • test data were calculated and related statistical processing with Microsoft Office Excel 2007. Unless otherwise specified, the data are expressed as the mean ⁇ standard error (Mean (closed statistics), and the comparison between the two groups was performed using a two-sided t-test.
  • the renal coefficient of the model group increased significantly (P ⁇ 0.01), but there was no significant difference in the medication group.
  • the renal coefficients of each drug intervention group were equivalent to those of the blank control group, and the use of amifostine There was no significant difference between the R01 group and the three groups using R01, especially the R01 middle and high dose groups were closer to the blank control group.
  • the serum creatinine value of the cisplatin model group was significantly increased (P ⁇ 0.01), and each drug intervention group was comparable to the blank control group, and there was no significant difference between the amifostine group and the three groups using R01. difference.
  • the serum urea nitrogen value of the cisplatin model group was significantly higher (P ⁇ 0.05), and the urea nitrogen value of the amifostine group was significantly lower than that of the model group (P ⁇ 0.01).
  • the concentration of urea nitrogen in the R01 low-dose and medium-dose groups was also significantly lower (P ⁇ 0.05); although the concentration of urea nitrogen in the R01 low-dose and middle-dose groups was lower than that in the model group, there was no significant difference (P>0.05).
  • Blank control group The glomeruli and renal tubules of the 7 animals in the normal feeding blank control group were basically undamaged, and the lumen of the renal tubules was tight, and only some inflammatory cells infiltrated around the glomerulus of some mice, a typical picture is shown in Figure 9 .
  • Cisplatin-induced renal injury model group (10mg/kg) group the animals in this group were the model group, and the renal function was severely damaged, and biochemical tests showed that the concentrations of urea nitrogen and creatinine in the blood both appeared significantly increased. From the perspective of pathological features, the pathological damage of glomeruli was not serious, and only a few animals showed the phenomenon of glomerular shrinkage.
  • the main injury came from the area of the renal tubules.
  • the epithelial cells of the renal tubules were significantly necrotic, and protein casts appeared.
  • the injured area mainly occurred in the area of the afferent arterioles. On the whole, the renal tubular cells appear burrs and the edges are blurred, some animals have serious pathological damage, and a large number of protein casts can be seen all over the field of view, a typical picture is shown in Figure 10.
  • Cisplatin + Amifostine group The degree of kidney damage in this group was significantly reduced, only some mice had slight damage, and other animals basically had no obvious pathological damage, as shown in Figure 11 for a typical picture.
  • Cisplatin + R01 6mg group The kidneys of the animals in this group were all damaged to a certain extent, 2 of which were severely damaged, and the others were mildly damaged, and the damaged parts were concentrated in the renal tubule area. Typical pictures are shown in Figure 12.
  • Cisplatin + R01 18mg group The animals in this group basically had no obvious pathological damage to the kidneys, only one animal had obvious damage, and the others were mild. Typical pictures are shown in Figure 13.
  • Cisplatin + R01 36mg group Only one mouse in this group had slight pathological damage to the kidney, and the other animals had intact pathological structures and clear cell structures. Typical pictures are shown in Figure 14.
  • the pathological damage of the cisplatin + amifostine group was significantly reduced, which was significantly different from the positive control cisplatin group (P ⁇ 0.01).
  • the degree of pathological damage was significantly reduced (P ⁇ 0.05).
  • Cisplatin+R01 (36mg) had basically intact renal tissue morphology with slight damage (P ⁇ 0.01), and had the best effect, which was basically similar to that of the amifostine group.
  • the 2,3-dimethoxy-5-methyl-1,4-benzoquinone alkyl alcohol derivative of the present invention has excellent antitumor effect and excellent safety.

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Abstract

一种2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物，以及含有所述化合物的药物组合物，以及该化合物在预防和治疗肿瘤中的应用。

Description

2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物及其应用 技术领域
本发明属于医药领域，更具体地，本发明涉及2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物、该化合物作为活性成分的药物组合物，以及该化合物在预防和治疗肿瘤中的应用。
背景技术
随着人类消费结构、生活方式以及生存环境的变化/恶化，恶性肿瘤的发病率迅速上升，已成为威胁人类生命健康最可怕的杀手。抗癌研究依然是生命科学领域中最富挑战性且意义重大的领域之一。
癌症的靶向药物治疗已经取得很大进展，特别是在部分肺癌及非实体瘤恶性肿瘤的治疗上已作为首选方案，然而临床上仍然主要是细胞毒类药物，特别是铂类药物，包括顺铂、卡铂、奈达铂、奥沙利铂、洛铂，仍然是很多常见癌症的一线治疗药物，但是铂类药物选择性差、毒副作用强；其中肾毒性是较常见且影响较大的副作用之一，顺铂引起的肾毒性发生率为28-36％，卡铂为27％、奈达铂为10-15％，发生率和损伤程度均与剂量成正比，临床上主要表现为血肌酐、尿素氮升高等。
因此，人们一直期望发现新的更安全、更有效的抗肿瘤药物，特别是能够同时增加铂类抗肿瘤作用、降低铂类毒副作用的抗肿瘤药物。
本发明发现一种2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物，能够同时增加铂类抗肿瘤作用、降低铂类毒副作用，对多种肿瘤具有优异的治疗效果。
发明简述
本发明的第一方面提供了如式(I)所示的2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物(简称“式(I)化合物”，或者简称“R01”，或者简称“RO1”)，或溶剂合物、水合物、多晶型、前药或同位素变体，以及它们的混合物：
Figure PCTCN2022125293-appb-000001
本发明的第二方面提供了一种药物组合物，其含有第一方面的化合物，或溶剂合物、水合物、多晶型、前药或同位素变体，和药学上可接受的赋形剂；优选地，其还含有其它治疗剂。
本发明的第三方面涉及第一方面的化合物或溶剂合物、水合物、多晶型、前药或同位素变体，或第二方面的药物组合物，在制备用于治疗和/或预防癌症的药物中的用途。
本发明的第四方面涉及一种在受试者中治疗和/或预防癌症的方法，所述方法包括向所述受试者给药第一方面的化合物或溶剂合物、水合物、多晶型、前药或同位素变体或第二方面的药物组合物。
本发明的第五方面涉及第一方面的化合物或溶剂合物、水合物、多晶型、前药或同位素变体或第二方面的药物组合物，其用于治疗和/或预防癌症。
本发明的第六方面涉及第三方面的用途或第四方面的方法或第五方面的化合物或组合物的用途，其中所述癌症选自肺癌、胃癌、食管癌、结直肠癌。
本发明第七方面涉及一种组合，包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体，以及铂类药物。
本发明第八方面涉及一种药物组合物，包括根据第七方面所述的组合和药物可接受载体、稀释剂或赋形剂。
本发明第九方面涉及第七方面所述的组合在制备治疗和/或预防癌症的药物中的用途。
本发明第十方面涉及一种药物制品，包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体，以及铂类药物，作为在治疗中同时、依次或分别使用的联合制剂。
本发明第十一方面涉及一种治疗和/或预防癌症的方法，所述方法包括同时、依次或分别地对患者给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物。
本发明第十二方面涉及式(I)化合物，或其溶剂合物、水合物、多晶型、 前药或同位素变体在制备治疗和/或预防癌症的药物中的用途，其中所述治疗包括同时、依次或分别地对患者给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物。
本发明第十三方面涉及式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物在制备治疗和/或预防癌症的药物中的用途。
本发明第十四方面涉及式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体在制备治疗和/或预防癌症的药物中的用途，其中所述药物用于与铂类药物的联合治疗。
本发明第十五方面涉及铂类药物在制备治疗和/或预防癌症的药物中的用途，其中所述药物用于与式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体的联合治疗。
附图简要说明
图1显示了CCK-8检测式(I)化合物对A549细胞增殖的影响。其中图1A显示不同浓度式(I)化合物与A549细胞共培养24h对细胞增殖活性的影响；图1B显示不同浓度式(I)化合物与A549细胞共培养48h对细胞增殖活性的影响；图1C显示不同浓度式(I)化合物与A549细胞共培养72h对细胞增殖活性的影响；图1D显示不同浓度式(I)化合物作用不同时间对A549细胞增殖活性的影响。
图2显示了CCK-8检测式(I)化合物对NCI-H460细胞增殖的影响。其中图2A显示不同浓度式(I)化合物与NCI-H460细胞共培养24h，对细胞增殖活性的影响；图2B显示不同浓度式(I)化合物与NCI-H460细胞共培养48h对细胞增殖活性的影响；图2C显示不同浓度式(I)化合物与NCI-H460细胞共培养72h对细胞增殖活性的影响；图2D显示不同浓度式(I)化合物作用不同时间，对NCI-H460细胞增殖活性的影响。
图3显示了EDU增殖实验检测式(I)化合物对肺癌细胞系增殖的影响。浓度为150μM的式(I)化合物与细胞共培养42h后，将FBS刺激孔中FBS浓度提高到20％，继续培养6h后用Cell-Light EDUApollo567试剂盒检测。其中图3A显示式(I)化合物对A549细胞增殖的影响；图3B显示式(I)化合物对NCI-H460细胞增殖的影响式(I)化合物；*：P<0.05；**：P<0.01。
图4显示了不同浓度式(I)化合物对A549、NCI-H460细胞集落形成能力的影响。
图5显示了式(I)化合物诱导A549细胞S期比例降低：A549细胞经不同浓度式(I)化合物(0μM、0μM、30μM、90μM)作用24h、48h和72h后代表性流式细胞周期分布图。
图6显示了式(I)化合物诱导NCI-H460细胞S期比例降低：NCI-H460细胞经不同浓度式(I)化合物(0μM、10μM、30μM、90μM)作用24h、48h和72h后代表性流式细胞周期分布图。
图7显示了流式细胞学检测不同浓度式(I)化合物作用于A549和NCI-H460细胞后凋亡变化的散点图。其中图7A、7B、7C、7D为不同浓度式(I)化合物(0μM、10μM、30μM、90μM)作用于A549细胞72h的代表性细胞凋亡图；图7E、7F、7G、7H为不同浓度式(I)化合物(0μM、10μM、30μM、90μM)作用于NCI-H460细胞72h的代表性细胞凋亡图；图7I为A549细胞与不同浓度式(I)化合物共培养后总凋亡的比例；图7G为NCI-H460细胞与不同浓度式(I)化合物共培养后总凋亡的比例；**：P<0.01。
图8显示了NCI-N87肿瘤组织蛋白的Westernblot(A)及灰度分析(B)结果。其中在NCI-N87(3D_PN154004)裸鼠移植瘤模型中，式(I)化合物溶液(2mg/kg)下调p-VEGFR2(NS)，下调VEGFR2(P＜0.05)，VEGFA(P＜0.05)；下调β-catenin(P＜0.05)，CyclinD1(NS)，上调PCNA(NS)；上调细胞周期调控蛋白P21(NS)，下调促凋亡Bax(P＜0.01)及抗凋亡蛋白Survivin(P＜0.05)；“NS”表示用药组与空白溶剂组比较无统计学差异(P＞0.05)；“*”表示用药组与空白溶剂组比较，存在显著性差异(P<0.05)；“**”表示用药组与空白溶剂组比较，存在较显著性差异(P<0.01)。
图9显示了正常饲养的空白对照组小鼠肾小球、肾小管基本无损伤，仅个别部分区域肾小管管壁变薄，轻微损伤(图片为PAS染色，放大20倍)。
图10显示了模型组小鼠给药10mg/kg顺铂后严重肾小管上皮的损失：细胞刷状缘脱失，大量蛋白管型出现(图片为HE染色，放大20倍)。
图11显示了阳性对照药组小鼠给药顺铂(10mg/kg)和氨磷汀(50mg/kg，顺铂给药前半小时腹腔注射)后仅出现轻微肾小管损伤，肾小管上皮边缘模糊(图片为HE染色，放大40倍)。
图12显示了试验组小鼠给药顺铂(10mg/kg)和R01(6mg)后出现较为严重肾小管损伤，上皮细胞坏死和轻微蛋白管型(图片为HE染色，放大40倍)。
图13显示了试验组小鼠给药顺铂(10mg/kg)和R01(18mg)后出现了一定的肾小管损伤，上皮细胞坏死可见(图片为HE染色，放大40倍)。
图14显示了试验组小鼠给药顺铂(10mg/kg)和R01(36mg)后出现出现了轻微的肾损伤，包括肾小管管腔扩大、上皮细胞坏死等(图片为HE染色，放大40倍)。
发明内容
在第一方面，本发明提供式(I)化合物，或溶剂合物、水合物、多晶型、前药或同位素变体，以及它们的混合物。
在第二方面，本发明提供了一种药物组合物，所述药物组合物含有式(I)化合物，和任选地药学上可接受的赋形剂。
在第三方面，本发明提供了含有式(I)化合物和药学上可接受的赋形剂的药物组合物，其还含有其它治疗剂。
在第四方面，本发明提供了包含式(I)化合物，和其它治疗剂以及药学上可接受的载剂、佐剂或媒剂的试剂盒。
在第五方面，本发明提供了式(I)化合物在制备用于治疗和/或预防受试者的癌症的药物中的用途。
在第六方面，本发明提供了在受试者中治疗和/或预防癌症的方法，包括向所述受试者给药式(I)化合物或本发明的组合物。
在第七方面，本发明提供了式(I)化合物或本发明的组合物，其用于治疗和/或预防受试者中的癌症。
在第八方面，本发明提供了一种组合，包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体，以及铂类药物。
第九方面，本发明提供了涉及一种药物组合物，包括根据第八方面所述的组合和药物可接受载体、稀释剂或赋形剂。
第十方面，本发明提供了涉及第八方面所述的组合在制备治疗和/或预防受试者中癌症的药物中的用途。
第十一方面，本发明提供了一种药物制品，包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体，以及铂类药物，作为在治疗中同时、依次或分别使用的联合制剂。
第十二方面，本发明提供了涉及一种治疗和/或预防癌症的方法，所述方法包括同时、依次或分别地对受试者或患者给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物。
第十三方面，本发明提供了涉及式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体在制备治疗和/或预防癌症的药物中的用途，其中所述治疗包括同时、依次或分别地对患者或受试者给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物。
第十四方面，本发明提供了涉及式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物在制备治疗和/或预防受试者中的癌症的药物中的用途。
第十五方面，本发明提供了涉及式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体在制备治疗和/或预防受试者中的癌症的药物中的用途，其中所述药物用于与铂类药物的联合治疗。
第十六方面，本发明提供了涉及铂类药物在制备治疗和/或预防受试者中的癌症的药物中的用途，其中所述药物用于与式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体的联合治疗。
第十七方面，本发明提供了式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体在制备用于减轻受试者中铂类药物诱导的急性肾脏损伤中的药物中用途。
第十八方面，本发明提供了一种减轻受试者中铂类药物诱导的急性肾脏损伤中的方法，包括在给药铂类药物的同时、依次或分别给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体的步骤。
在前述各方面中，铂类药物选自顺铂、卡铂、奈达铂、奥沙利铂、洛铂。
在前述各方面中，所述癌症选自肺癌、胃癌、食管癌、结直肠癌。
由随后的具体实施方案、实施例和权利要求，本发明的其它目的和优点将对于本领域技术人员显而易见。
定义
术语“癌症”包括但不限于下列癌症：胃、肺、食管、结直肠。更具体地，癌症包括但不限于HER2过度表达的转移性胃腺癌或胃食管交界腺癌、表皮生长因子受体(EGFR)基因敏感突变的非小细胞肺癌、含铂化疗期间或化疗后疾病进展的局部晚期或转移性鳞状组织学类型非小细胞肺癌等。
本文所用的术语“治疗”涉及逆转、减轻、抑制该术语适用的障碍或病症的进展或者预防之，或者这类障碍或病症的一种或多种症状。本文所用的名词“治疗”涉及动词治疗的动作，后者是如刚才所定义的。
给药的“受试者”包括但不限于：人(即，任何年龄组的男性或女性，例如，儿科受试者(例如，婴儿、儿童、青少年)或成人受试者(例如，年轻的成人、中年的成人或年长的成人))和/或非人的动物，例如，哺乳动物，例如，灵长类(例如，食蟹猴、恒河猴)、牛、猪、马、绵羊、山羊、啮齿动物、猫和/或狗。在一些实施方案中，受试者是人。在一些实施方案中，受试者是非人动物。本文可互换使用术语“人”、“患者”和“受试者”。
“疾病”、“障碍”和“病症”在本文中可互换地使用。
除非另作说明，否则，本文使用的术语“治疗”包括受试者患有具体疾病、障碍或病症时所发生的作用，它降低疾病、障碍或病症的严重程度，或延迟或减缓疾病、障碍或病症的发展(“治疗性治疗”)，还包括在受试者开始患有具体疾病、障碍或病症之前发生的作用(“预防性治疗”)。
通常，化合物的“有效量”是指足以引起目标生物反应的数量。正如本领域普通技术人员所理解的那样，本发明化合物(也即式(I)化合物)的有效量可以根据下列因素而改变：例如，生物学目标、化合物的药代动力学、所治疗的疾病、给药模式以及受试者的年龄健康情况和症状。有效量包括治疗有效量和预防有效量。
除非另作说明，否则，本文使用的化合物的“治疗有效量”是在治疗疾病、障碍或病症的过程中足以提供治疗益处的量，或使与疾病、障碍或病症有关的一或多种症状延迟或最小化的量。化合物的治疗有效量是指单独使用或与其它疗法联用时，治疗剂的量，它在治疗疾病、障碍或病症的过程中提供治疗益处。术语“治疗有效量”可以包括改善总体治疗、降低或避免疾病或病症的症状或病因、或增强其它治疗剂的治疗效果的量。
除非另作说明，否则，本文使用的化合物的“预防有效量”是足以预防疾病、障碍或病症的量，或足以预防与疾病、障碍或病症有关的一或多种症状的量，或防止疾病、障碍或病症复发的量。化合物的预防有效量是指单独使用或与其它药剂联用时，治疗剂的量，它在预防疾病、障碍或病症的过程中提供预防益处。术语“预防有效量”可以包括改善总体预防的量，或增强其它预防药剂的预防效果的量。
“组合”以及相关术语是指同时或依次给药本发明化合物和其它治疗剂。例如，本发明化合物可以与其它治疗剂以分开的单位剂型同时或依次给药，或与其它治疗剂一起在单一单位剂型中同时给药。
本领域技术人员将理解，有机化合物可以与溶剂形成复合物，其在该溶剂中发生反应或从该溶剂中沉淀或结晶出来。这些复合物称为“溶剂合物”。当溶剂是水时，复合物称为“水合物”。本发明涵盖了本发明化合物的所有溶剂合物。
术语“溶剂合物”是指通常由溶剂分解反应形成的与溶剂相结合的化合物或其盐的形式。这个物理缔合可包括氢键键合。常规溶剂包括包括水、甲醇、乙醇、乙酸、DMSO、THF、乙醚等。本文所述的化合物可制备成，例如，结晶形式，且可被溶剂化。合适的溶剂合物包括药学上可接受的溶剂合物且进一步包括化学计量的溶剂合物和非化学计量的溶剂合物。在一些情况下，所述溶剂合物将能够分离，例如，当一或多个溶剂分子掺入结晶固体的晶格中时。“溶剂合物”包括溶液状态的溶剂合物和可分离的溶剂合物。代表性的溶剂合物包括水合物、乙醇合物和甲醇合物。
术语“水合物”是指与水相结合的化合物。通常，包含在化合物的水合物中的水分子数与该水合物中该化合物分子数的比率确定。因此，化合物的水合物可用例如通式R·x H 2O代表，其中R是该化合物，和x是大于0的数。给定化合物可形成超过一种水合物类型，包括，例如，单水合物(x为1)、低级水合物(x是大于0且小于1的数，例如，半水合物(R·0.5H 2O))和多水合物(x为大于1的数，例如，二水合物(R·2H 2O)和六水合物(R·6H 2O))。
本发明化合物可以是无定形或结晶形式(多晶型)。此外，本发明化合物可以以一种或多种结晶形式存在。因此，本发明在其范围内包括本发明化合物的所有无定形或结晶形式。术语“多晶型物”是指特定晶体堆积排列的化合 物的结晶形式(或其盐、水合物或溶剂合物)。所有的多晶型物具有相同的元素组成。不同的结晶形式通常具有不同的X射线衍射图、红外光谱、熔点、密度、硬度、晶体形状、光电性质、稳定性和溶解度。重结晶溶剂、结晶速率、贮存温度和其他因素可导致一种结晶形式占优。化合物的各种多晶型物可在不同的条件下通过结晶制备。
本发明还包括同位素标记的化合物(同位素变体)，它们等同于式(I)所述的那些，但一个或多个原子被原子质量或质量数不同于自然界常见的原子质量或质量数的原子所代替。可以引入本发明化合物中的同位素的实例包括氢、碳、氮、氧、磷、硫、氟和氯的同位素，分别例如 2H、 3H、 13C、 11C、 14C、 15N、 18O、 17O、 31P、 32P、 35S、 18F和 36Cl。含有上述同位素和/或其它原子的其它同位素的本发明化合物、其前体药物和所述化合物或所述前体药物的药学上可接受的盐都属于本发明的范围。某些同位素标记的本发明化合物、例如引入放射性同位素(例如 3H和 14C)的那些可用于药物和/或底物组织分布测定。氚、即 3H和碳-14、即 14C同位素是特别优选的，因为它们容易制备和检测。进而，被更重的同位素取代，例如氘、即 2H，由于代谢稳定性更高可以提供治疗上的益处，例如延长体内半衰期或减少剂量需求，因而在有些情况下可能是优选的。同位素标记的本发明式(I)化合物及其前体药物一般可以这样制备，在进行下述流程和/或实施例与制备例所公开的工艺时，用容易得到的同位素标记的试剂代替非同位素标记的试剂。
此外，前药也包括在本发明的上下文内。本文所用的术语“前药”是指在体内通过例如在血液中水解转变成其具有医学效应的活性形式的化合物。药学上可接受的前药描述于T.Higuchi和V.Stella，Prodrugs as Novel Delivery Systems，A.C.S.Symposium Series的Vol.14，Edward B.Roche，ed.，Bioreversible Carriers in Drug Design，American Pharmaceutical Association and Pergamon Press，1987，以及D.Fleisher、S.Ramon和H.Barbra“Improved oral drug delivery：solubility limitations overcome by the use of prodrugs”，Advanced Drug Delivery Reviews(1996)19(2)115-130，每篇引入本文作为参考。
前药为任何共价键合的本发明化合物，当将这种前药给予患者时，其在体内释放母体化合物。通常通过修饰官能团来制备前药，修饰是以使得该修饰可以通过常规操作或在体内裂解产生母体化合物的方式进行的。前药包括， 例如，其中羟基、氨基或巯基与任意基团键合的本发明化合物，当将其给予患者时，可以裂解形成羟基、氨基或巯基。因此，前药的代表性实例包括(但不限于)式(I)化合物的羟基、巯基和氨基官能团的乙酸酯/酰胺、甲酸酯/酰胺和苯甲酸酯/酰胺衍生物。另外，在羧酸(-COOH)的情况下，可以使用酯，例如甲酯、乙酯等。酯本身可以是有活性的和/或可以在人体体内条件下水解。合适的药学上可接受的体内可水解的酯基包括容易在人体中分解而释放母体酸或其盐的那些基团。
本发明还提供药物制剂，包含治疗有效量的式(I)化合物和其药学上可接受的载体、稀释剂或赋形剂。所有这些形式都属于本发明。
本文使用的术语“药物制备”包括本发明的成分除了用于这类药物制备的任意阶段外还有直接作为药物的应用。
本文中使用的术语“联合治疗”是指在时限内如果不能同时则依次给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物以便在同一时限内它们均可起治疗作用的治疗。
如上所述，本发明的一个方面涉及包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物的药物制品，作为在治疗中同时、依次或分别使用的联合制剂。
本文中使用的“同时”是指同时地给药两种药剂，而术语“联合”是指如果不能同时给药则在在时限内“依次”地给药以使它们均适合于在同一时限内起治疗作用。因此，“依次”给药可允许在一种药剂后5分钟、10分钟或大约几小时后给药另一种药剂，只要第一种给药药剂的循环半衰期能使二者同时以治疗有效量存在即可。给药成分间的延时依成分的确切性质、它们之间的相互作用和它们各自的半衰期而改变。
与“联合”或“依次”相比，本文使用的“分别”是指给药一种药剂和另一种药剂之间的间隔是明显的，即当给药第二种药剂时，在血流中不再存在治疗有效量的第一种给药药剂。
在另一种优选实施方案中，相对于单独成分，分别给药亚治疗量的式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物；换句话说，如果不是联合给药给药，式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物不是以治疗有效量给药。
优选地，式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物以协同方式相互作用。本文使用的术语“协同”是指式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物在联合使用时能产生比由两种组分的单独效应相加预料到的效应更大的效应。有利地，协同相互作用可允许每种组分以较低剂量给药于患者，从而降低化疗的毒性，同时产生和/或保持相同的疗效。因此，在尤其优选的实施方案中，可以亚治疗量给药每种组分。
药物组合物和试剂盒
在另一方面，本发明提供了药物组合物，其包含本发明化合物(还称为“活性组分”)和药学上可接受的赋形剂。在一些实施方案中，所述药物组合物包含有效量的本发明化合物。在一些实施方案中，所述药物组合物包含治疗有效量的本发明化合物。在一些实施方案中，所述药物组合物包含预防有效量的本发明化合物。
用于本发明的药学上可接受的赋形剂是指不会破坏一起调配的化合物的药理学活性的无毒载剂、佐剂或媒剂。可以用于本发明组合物中的药学上可接受的载剂、佐剂或媒剂包括(但不限于)离子交换剂、氧化铝、硬脂酸铝、卵磷脂、血清蛋白(如人类血清白蛋白)、缓冲物质(如磷酸盐)、甘氨酸、山梨酸、山梨酸钾、饱和植物脂肪酸的偏甘油酯混合物、水、盐或电解质(如硫酸鱼精蛋白)、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、硅胶、三硅酸镁、聚乙烯吡咯烷酮、基于纤维素的物质、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜡、聚乙烯-聚氧丙烯-嵌段聚合物、聚乙二醇以及羊毛脂。
本发明还包括试剂盒(例如，药物包装)。所提供的试剂盒可以包括本发明化合物、其它治疗剂，以及含有本发明化合物、其它治疗剂的第一和第二容器(例如，小瓶、安瓿瓶、瓶、注射器和/或可分散包装或其它合适的容器)。在一些实施方案中，提供的试剂盒还可以任选包括第三容器，其含有用于稀释或悬浮本发明化合物和/或其它治疗剂的药用赋形剂。在一些实施方案中，提供在第一容器和第二容器中的本发明化合物和其它治疗剂组合形成一个单位剂型。
给药
本发明提供的药物组合物可以通过许多途径给药，包括但不限于：口服给药、肠胃外给药、吸入给药、局部给药、直肠给药、鼻腔给药、口腔给药、阴道给药、通过植入剂给药或其它给药方式。例如，本文使用的肠胃外给药包括皮下给药、皮内给药、静脉内给药、肌肉内给药、关节内给药、动脉内给药、滑膜腔内给药、胸骨内给药、脑脊髓膜内给药、病灶内给药、和颅内的注射或输液技术。
通常，给予有效量的本文所提供的化合物。按照有关情况，包括所治疗的病症、选择的给药途径、实际给予的化合物、个体患者的年龄、体重和响应、患者症状的严重程度，等等，可以由医生确定实际上给予的化合物的量。
当用于预防本发明所述病症时，给予处于形成所述病症危险之中的受试者本文所提供的化合物，典型地基于医生的建议并在医生监督下给药，剂量水平如上所述。处于形成具体病症的危险之中的受试者，通常包括具有所述病症的家族史的受试者，或通过遗传试验或筛选确定尤其对形成所述病症敏感的那些受试者。
还可以长期给予本文所提供的药物组合物(“长期给药”)。长期给药是指在长时间内给予化合物或其药物组合物，例如，3个月、6个月、1年、2年、3年、5年等等，或者可无限期地持续给药，例如，受试者的余生。在一些实施方案中，长期给药意欲在长时间内在血液中提供所述化合物的恒定水平，例如，在治疗窗内。
可以使用各种给药方法，进一步递送本发明的药物组合物。例如，在一些实施方案中，可以推注给药药物组合物，例如，为了使化合物在血液中的浓度提高至有效水平。推注剂量取决于通过身体的活性组分的目标全身性水平，例如，肌内或皮下的推注剂量使活性组分缓慢释放，而直接递送至静脉的推注(例如，通过IV静脉滴注)能够更加快速地递送，使得活性组分在血液中的浓度快速升高至有效水平。在其它实施方案中，可以以持续输液形式给予药物组合物，例如，通过IV静脉滴注，从而在受试者身体中提供稳态浓度的活性组分。此外，在其它实施方案中，可以首先给予推注剂量的药物组合物，而后持续输液。
口服组合物可以采用散装液体溶液或混悬剂或散装粉剂形式。然而，更 通常，为了便于精确地剂量给药，以单位剂量形式提供所述组合物。术语“单位剂型”是指适合作为人类患者及其它哺乳动物的单元剂量的物理离散单位，每个单位包含预定数量的、适于产生所需要的治疗效果的活性物质与合适药学赋形剂。典型的单位剂量形式包括液体组合物的预装填的、预先测量的安瓿或注射器，或者在固体组合物情况下的丸剂、片剂、胶囊剂等。在这种组合物中，所述化合物通常为较少的组分(约0.1至约50重量％，或优选约1至约40重量％)，剩余部分为对于形成所需给药形式有用的各种载体或赋形剂以及加工助剂。
对于口服剂量，代表性的方案是，每天一个至五个口服剂量，尤其是两个至四个口服剂量，典型地是三个口服剂量。使用这些剂量给药模式，每个剂量提供大约0.01至大约100mg/kg的本发明化合物，优选的剂量各自提供大约0.1至大约10mg/kg，尤其是大约0.5至大约2mg/kg。
从大约1至大约120小时，尤其是24至96小时，注射剂量水平在大约0.1mg/kg/小时至至少10mg/kg/小时的范围。为了获得足够的稳定状态水平，还可以给予大约0.1mg/kg至大约10mg/kg或更多的预载推注。对于40至80kg的人类患者来说，最大总剂量不能超过大约2g/天。
适于口服给药的液体形式可包括合适的水性或非水载体以及缓冲剂、悬浮剂和分散剂、着色剂、调味剂，等等。固体形式可包括，例如，任何下列组份，或具有类似性质的化合物：粘合剂，例如，微晶纤维素、黄蓍胶或明胶；赋形剂，例如，淀粉或乳糖，崩解剂，例如，褐藻酸、Primogel或玉米淀粉；润滑剂，例如，硬脂酸镁；助流剂，例如，胶体二氧化硅；甜味剂，例如，蔗糖或糖精；或调味剂，例如，薄荷、水杨酸甲酯或橙味调味剂。
可注射的组合物典型地基于可注射用的无菌盐水或磷酸盐缓冲盐水，或本领域中已知的其它可注射的赋形剂。如前所述，在这种组合物中，活性化合物典型地为较少的组分，经常为约0.05至10％重量，剩余部分为可注射的赋形剂等。
典型地将透皮组合物配制为含有活性组分的局部软膏剂或乳膏剂。当配制为软膏剂时，活性组分典型地与石蜡或可与水混溶的软膏基质组合。或者，活性组分可与例如水包油型乳膏基质一起配制为乳膏剂。这种透皮制剂是本领域中公知的，且通常包括用于提升活性组分或制剂的稳定的皮肤渗透的其 它组份。所有这种已知的透皮制剂和组份包括在本发明提供的范围内。
本发明化合物还可通过经皮装置给予。因此，经皮给药可使用贮存器(reservoir)或多孔膜类型、或者多种固体基质的贴剂实现。
用于口服给予、注射或局部给予的组合物的上述组份仅仅是代表性的。其它材料以及加工技术等阐述于Remington's Pharmaceutical Sciences,17th edition,1985,Mack Publishing Company,Easton,Pennsylvania的第8部分中，本文以引用的方式引入该文献。
本发明化合物还可以以持续释放形式给予，或从持续释放给药系统中给予。代表性的持续释放材料的描述可在Remington's Pharmaceutical Sciences中找到。
治疗
从而，本发明化合物具有作为抗肿瘤药剂的价值。尤其，本发明化合物具有在实体和/或液体肿瘤疾病的遏制和/或治疗中作为抗增殖、凋亡和/或抗侵袭药剂的价值。尤其，预期本发明化合物有用于预防或治疗胃癌、肺癌等。
有用于治疗患者中的癌症的抗癌作用包括但不局限于抗肿瘤作用、响应率、疾病进展的时间及存活率。本发明治疗方法的抗肿瘤作用包括但不局限于肿瘤生长的抑制、肿瘤生长的延迟、肿瘤的退化、肿瘤的收缩、治疗停止后肿瘤再生长时间的延长及疾病进展的减慢。抗癌作用包括预防性治疗以及现存在疾病的治疗。
本发明化合物的有效量通常在平均日剂量为0.01mg至50mg化合物/千克患者体重，优选0.1mg至25mg化合物/千克患者体重，以单次或多次给药。通常，本发明化合物可向该有此治疗需要的患者以每位患者约1mg至约3500mg的日剂量范围给药，优选10mg至1000mg。例如，每位患者的日剂量可为10、20、30、40、50、60、70、80、90、100、120、150、160、180、200、240、250、300、350、360、400、500、600、700、800、900或1000mg。可每天、每周(或间隔数天)或以间歇时间表，给药一次或多次。例如，可在每周的基础上(例如每周一)，每天给予所述化合物一次或多次，不定地或持续几周，例如4-10周。或者，可每天给药持续几天(例如2-10天)，然后几天(例如1-30天)不给药所述化合物，不定地重复该循环或重复给定的 次数，例如4-10个循环。例如，本发明化合物可每天给药持续5天，然后间断9天，然后再每天给药持续5天，然后间断9天，以此类推，不定地重复该循环或共重复4-10次。
用于与本发明的式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体联用的铂类药物，推荐为临床上的常用剂量或者推荐剂量。例如，顺铂常用剂量为50-100mg/m 2，或每天静滴15-20mg/m 2、连用5天，均3-4周重复用药。对于卡铂，肾功能正常的患者，推荐剂量为400mg/m 2；存在危险因素的患者，建议初始剂量减少20-25％；对65岁以上的患者，应根据患者的体质情况，调整初始剂量及随后治疗剂量。对于奈达铂，每次给药80-100mg/m 2，间隔3-4周后方可进行下一疗程。对于奥沙利铂，推荐剂量为一次130mg/m 2，无主要毒性出现时，每3周(21天)给药1次；或者85mg/m2，每2周重复1次。对于洛铂，推荐剂量按一次50mg/m 2，再次使用时应待血液毒性或其他临床副作用完全恢复，推荐的应用间歇期为3周，如副作用恢复较慢，可延长使用间歇。
当本发明的式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体与铂类药物联用的时候，相对于单独成分，也可以分别给药亚治疗量的式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物。
实施例
本文所用的材料或试剂为可购买到的或由本领域通常已知的合成方法制备。
实施例1：式(I)化合物的合成
Figure PCTCN2022125293-appb-000002
将无水二氯甲烷(40ml)、6-(10-羟癸基)-2，3-二甲氧基-5-甲基-1，4-苯醌1(3.38g，0.01mol)、浓硫酸(0.6ml)置于圆底瓶内，搅拌得溶液后，加入4A分 子筛(2.5g)、2-甲基-1-(4-氯苯甲酰基)-5-甲氧基-1H-吲哚-3-乙酸(3.57g，0.01mol)，回流10小时后过滤，再分别用适量碳酸钠水溶液、饱和食盐水洗涤，水洗至中性，无水硫酸钠干燥后浓缩得黑色粗品，以柱层析纯化得到3.98g褐色固体，产率59.7％，HPLC纯度97.9％。
1H-NMR(400MHz，d 6DMSO)：δ7.64-7.67(d，2H)，7.45-7.48(d，2H)，6.96(s，1H)，6.85-6.87(d，1H)，6.65-6.67(d，1H)，4.07-4.11(t，2H)，3.98(s，6H)，3.83(s，3H)，3.66(s，2H)，2.42-2.46(t，2H)，2.39(s，3H)，2.0(s，3H)，1.58-1.64(m，2H)，1.24-1.40(m，14H)。
ESI-MS：678.2(M+1)，700.2(M+23)。
实施例2：式(I)化合物对肿瘤细胞增殖、肿瘤细胞周期和凋亡影响的研究
1.主要仪器与设备
Figure PCTCN2022125293-appb-000003
Figure PCTCN2022125293-appb-000004
2.供试品和细胞系
人肺癌细胞系A549、NCI-H460均来自于同济医院肿瘤科实验室细胞库。
3.主要实验试剂
Figure PCTCN2022125293-appb-000005
Figure PCTCN2022125293-appb-000006
4.相关溶液包括10％BSA(由超纯水将Albumin bovine v 1g定容至10ml)、TBST(由超纯水将10×TBS 50ml、Tween 20 500μl定容至500ml)、细胞冻存液(由RPMI-1640培养基28ml、FBS 8ml和DMSO 4ml配制后4℃保存)、细胞裂解液【500μM HEPES 1ml、3M氯化钠500μl、500μM EDTA 20μl、500μM EGTA 20μl、20％Triton X-100 250μl、ddH2O 7.7ml、混合后分装为1ml每管，-20℃保存、1M DTT(现用现加)1:1000、100μM PMSF(现用现加)1:100、25×Cocktail(现用现加)1:25】。
式(I)化合物储存液：式(I)化合物易溶于有机溶剂，选择DMSO与异丙醇3:7比例作为溶剂。在生物安全柜中打开DMSO和异丙醇，取15ml离心管，加入3ml DMSO和7ml异丙醇制成混合溶剂。式(I)化合物分子量为678.22g/mol，称取式(I)化合物原研药40.69mg，加入4ml混合溶剂，吹打混匀，经换算，药物储存液浓度为15000μM，将其分装至1.5ml EP管中，并 用记号笔标记名称、浓度及时间，放置于4℃保存，使用时根据需要浓度进行稀释。
5.实验方法
细胞培养
(1)细胞复苏
提前打开电热恒温水槽，设置温度为37℃，待温度升至设定温度后从-80℃冰箱中取出细胞冻存管，放入37℃水槽中，轻摇晃动冻存管，使细胞快速融化。迅速将细胞冻存管放入低速离心机中，设置转速为1200rpm，离心5min。离心结束后用75％酒精喷洒冻存管外部。在生物安全柜中打开冻存管，用负压吸引器吸去上清液，用移液枪吸取1ml新鲜完全培养基加入冻存管中重悬细胞，吹打混匀后移入T25培养瓶或直径6cm培养皿中，再加入3ml新鲜培养基，置于37℃含5％CO 2的培养箱中培养。第二天需观察复苏细胞贴壁和生长状态，必要时换细胞培养基。
(2)细胞换液
提前将完全培养基，PBS等置于37℃水浴箱中预热数分钟，用75％酒精喷洒外部后放入生物安全柜。从培养箱中取出装有细胞的6cm培养皿，在倒置生物显微镜下观察细胞状态。打开负压吸引器，吸去培养器皿中的培养基，用PBS洗1-2遍，弃PBS。加入3ml完全培养基至培养皿中，置于37℃含5％CO 2的培养箱中继续培养。
(3)细胞传代与冻存
在倒置生物显微镜下观察细胞生长状态，待细胞长至培养瓶底面积80％-90％时需进行细胞传代。将培养器皿放入生物安全柜中。打开负压吸引器，用负压吸引器吸去旧培养基，移液枪轻柔加入适量PBS润洗细胞1-2次，弃PBS。加入适量胰蛋白酶于培养器皿底面，轻摇培养皿使胰蛋白酶均匀覆盖于细胞表面，放回37℃培养箱中。注意不同细胞消化时间略有差异，一般为2-3min，当显微镜下观察到细胞之间解离，细胞变圆、透亮，细胞与培养皿脱离能够流动时可加入5-8倍胰蛋白酶体积的完全培养基终止消化。用移 液枪缓慢吹打消化好的细胞使其分散形成单细胞悬液。转至4ml离心管中，1500rpm，离心5min。弃上清液，加入完全培养基重悬细胞，根据需要留取适量细胞悬液于培养皿中，剩余细胞悬液加入冻存管中。培养皿中补充完全培养基至3ml后继续培养。冻存管按照1:1体积比加入冻存液，后置于-80℃冰箱的冻存盒中，并在细胞单上记录更新。
CCK-8实验
(1)取对数生长期细胞，消化后制成细胞悬液。用移液枪取20μl细胞悬液加至CountStar一次性细胞计数板中进行细胞计数，记录计数结果，调整细胞浓度为30000-40000个/ml(注：不同细胞生长速度不同，每孔细胞数目略不同)。
(2)取96孔板，由于培养板最外围一圈的孔容易干燥挥发，引起体积不准确，会增加误差，最外一圈的孔加入100μlPBS，不作为测定孔用。
(3)将调整好的细胞悬液加至96孔板中，每孔加入100μl，则每孔约含有3000-4000个细胞，置于37℃培养箱5％CO 2条件下培养，待细胞贴壁后加药。
(4)药物稀释。设置浓度梯度为：0μM、10μM、50μM、100μM、200μM。每个浓度设置3个复孔。另外设置不含细胞，只含有培养基和药物的空白对照孔。
(5)在培养箱中培养24h、48h和72h。到设定时间后，从培养箱中取出96孔板，每孔加入10μlCCK-8试剂，注意避免产生气泡，晃动均匀后放回培养箱中继续培养2h。
(6)检测吸光度，将96孔板置于化学发光仪中，检测450nm处吸光度值(OD值)，并记录数据。细胞活力＝(实验孔OD值-空白对照孔OD值)/(溶剂对照孔OD值-空白对照孔OD值)×100％。
(7)将数据导入GraphPad Prism5中作图，计算IC50值，并绘制曲线。
细胞EDU增殖实验
(1)取对数生长期细胞，以每孔1×10 4个细胞数接种于24孔培养板中， 将培养板置于37℃培养箱5％CO 2条件下培养，待细胞贴壁后根据实验设计加入药物处理。
(2)药物处理完成后，取出Cell-Light EDU Apollo567试剂盒，用1640完全培养基按照1000:1的比例稀释试剂盒中的试剂A，制备成50μM的EDU培养基，24孔板按每孔300μl配置。从培养箱中取出24孔板，用负压吸引器吸去原有培养基，每孔加入300μl EDU培养基后放回37℃培养箱中继续培养2h，弃培养基。
(3)每孔加入PBS约300μl，在摇床上清洗细胞，共2次，每次5min。目的是将未掺入DNA的EDU洗脱掉。
(4)每孔加入150μl4％的多聚甲醛固定液，室温孵育30min，弃固定液。
(5)每孔加入150μl2mg/ml甘氨酸，摇床孵育5min，随后弃甘氨酸溶液(该步骤用于中和过量的醛基)。
(6)每孔加入200μlPBS，摇床清洗5min，弃PBS。
(7)每孔加入150μl用PBS配置的0.5％Triton-X100，于摇床上孵育10min。PBS清洗一次，5min。
(8)配置Apollo染色反应液，取试剂B140μl，试剂C28μl，试剂D9.3μl，试剂E25mg，去离子水2400μl，充分混匀。向每孔加入200μl配置好的Apollo染色染色反应液，在摇床上室温避光孵育30min，弃染色反应液。
(9)加入150μl0.5％Triton-X100，在脱水摇床上清洗2-3次，每次10min，弃渗透液，每孔加入150μl甲醇清洗1-2次，5min/次，弃甲醇。PBS清洗1次，5min。
(10)用超纯水按照100:1的比例稀释试剂F，制备适量1×Hoechst33342反应液用于染核。
(11)每孔加入120μl1×Hoechst 33342反应液，于摇床上室温、避光孵育30min，弃染色液。
(12)每孔加入150μlPBS清洗1次。弃PBS，随后加入200μlPBS，置于倒置荧光显微镜上拍照。每孔于200倍视野下拍摄6个视野，计算100个细胞中EDU阳性细胞的比例。将结果导入GraphPad Prism5软件中绘制柱状图。
克隆形成实验
(1)显微镜下观察细胞状态，选取处于对数生长期的细胞消化后制成细胞悬液。用移液枪取20μl细胞悬液加至CountStar一次性细胞计数板进行细胞计数，记录计数结果。
(2)用6孔培养板做克隆形成实验，每孔接种约1000个细胞，补充完全培养基至2.5ml每孔。8字型晃动细胞，使细胞分散均匀。次日，按照实验计划向对应孔中加入不同浓度(0μM、50μM、150μM)式(I)化合物，置于37℃5％CO 2的培养箱中培养2周左右。当出现肉眼可见的克隆团时终止培养。
(3)取出培养板，弃旧培养基，用PBS轻柔润洗细胞2次。
(4)每孔加入4％多聚甲醛固定液500μl，室温固定15min后弃固定液。
(5)每孔加入0.1％结晶紫溶液500μl，室温染色至少15min，弃结晶紫，用流水缓慢洗去染色液，倒扣室温晾干，拍照。
流式细胞周期检测
(1)细胞接种于6孔板，待细胞贴壁融合约50％时按0μM、10μM、30μM、90μM的浓度加入式(I)化合物，在37℃下置于5％CO 2的培养箱中继续培养，设置3个时间点，分别为24h、48h和72h。
(2)到既定时间点后，从培养箱中取出细胞培养板，置于生物安全柜中。取4ml离心管并标记，将孔中上清吸出加入对应4ml离心管中。用PBS轻柔润洗细胞，PBS加入对应离心管中。每孔加入适量胰蛋白酶消化细胞，待细胞消化程度适中时将离心管中旧培养基加入对应孔中，用移液枪轻柔吹打细胞，将细胞吹打成单细胞悬液，吸回原离心管中，迅速采用1500rpm离心5min，弃上清保留沉淀。
(3)向离心管中加入2ml预冷的PBS，再次采用1500rpm离心5min，弃上清，去除细胞碎片。
(4)75％乙醇1ml，一边加入一边吹打细胞，使细胞尽量呈单个，置于4℃固定过夜或者-20℃长期保存(可保存1周)。
(5)取出固定好的细胞，1000rpm离心5min，弃乙醇，加入预冷的PBS 洗涤细胞，1000rpm，5min，去除细胞上残留的乙醇。
(6)选用启动子公司的细胞周期检测试剂盒，加入100μl RNaseA，37℃水浴30min。
(7)加入400μl PI混匀，4℃避光30min。
(8)上流式细胞仪检测细胞周期。
流式细胞凋亡检测
(1)细胞接种于6孔板，待细胞生长密度合适后加入0μM、10μM、30μM、90μM四种浓度的式(I)化合物，置于37℃5％CO 2的培养箱中继续培养72h。
(2)到既定时间点后，从培养箱中取出细胞培养板，置于生物安全柜中。取4ml离心管标记，将6孔板中上清吸出加入对应4ml离心管中。用PBS轻柔润洗细胞，PBS加入对应离心管中。每孔加入适量胰蛋白酶消化细胞，待细胞消化程度适中时将离心管中液体加入对应孔中，用移液枪轻柔吹打细胞，将细胞吹散，吸回原离心管中，迅速采用1000rpm离心5min，弃上清保留沉淀。
(3)向离心管中加入2ml PBS，再次采用1000rpm离心5min，弃上清，重复一次。
(4)用1×Binding buffer悬浮细胞，调整细胞浓度约为1×106个/ml，取100μl细胞悬液加入流式管中，每管加入5μl PI和5μl Annexin V-FITC，轻轻涡旋细胞，室温避光孵育15min。
(5)每管再加入400μl 1×Binding buffer，上机检测(需在1h内完成检测)。
6.结果
式(I)化合物抑制肺癌细胞的增殖
用CCK-8法检测不同浓度式(I)化合物(0μM、10μM、50μM、100μM、200μM)作用于A549细胞和NCI-H460细胞24h、48h和72h对细胞增殖活性的影响。结果显示，A549细胞24h、48h和72h的IC50分别为213.2±1.22μM(图1A)，55.5±1.84μM(图1B)，39.7±0.98μM(图1C)。式(I)化合物对A549 细胞的抑制呈现时间-浓度依赖性，即随着式(I)化合物浓度的升高，抑制作用增强，随着时间的延长，抑制作用同样增强。
式(I)化合物对NCI-H460的增殖同样具有抑制作用，结果显示，NCI-H460细胞24h、48h和72h的IC50分别为125.3±1.01μM(图2A)，66.4±0.87μM(图2B)，48.5±1.12μM(图2C)。式(I)化合物对NCI-H460细胞的增殖抑制同样呈现时间-浓度依赖性，即随着浓度的升高和作用时间的延长，抑制作用增强。
直接测定DNA合成是检测细胞增殖最准确的方法之一，EDU可以在DNA复制的时候掺入到新合成的DNA链中，通过一个“Click”反应，把荧光基团标记到新合成的含有EDU的DNA上面，这样我们就能通过检测荧光而知道细胞的增殖情况。用浓度为150μM式(I)化合物处理A549、NCI-H460细胞48h，细胞增殖实验显示，与CTL相比(式(I)化合物0μM)，式(I)化合物药物作用组中，正在增殖的细胞比例显著降低，差异有统计学意义(图3，P<0.01)。FBS刺激能促进细胞的增殖，式(I)化合物能抑制高血清浓度刺激条件引起的细胞增殖效应(P<0.05)。
式(I)化合物抑制肺癌细胞的集落形成能力
为明确式(I)化合物对细胞克隆形成能力的影响，将A549、NCI-H460接种于6孔板后，分别用含3种不同浓度(0μM，50μM，150μM)的式(I)化合物培养基与细胞共培养。如图4所示，与0μM相比，随着药物浓度升高，两种肺癌细胞的克隆形成能力明显下降，呈浓度依赖性(图4)。
式(I)化合物减低肺癌细胞S期比例
如前所述，式(I)化合物以浓度依赖的方式，有效抑制肺癌细胞的增殖。为进一步探索其抑制细胞增殖的原因，我们采用流式细胞学技术检测经式(I)化合物处理后的肺癌细胞系的细胞周期的分布情况。用含有不同浓度式(I)化合物(0μM，10μM，30μM，90μM)的培养基分别处理A549细胞和NCI-H460细胞24h，48h及72h。用75％乙醇固定细胞后，用PI染色上机分析。如图5显示，在A549细胞中，与0μM组相比，式(I)化合物降低A549细胞S期的比例，并呈现出剂量依赖性。具体来说，经式(I)化合物处理24h后， 0μM、10μM、30μM、90μM组细胞的S期比例分别为31.91％，22.94％，12.52％及3.92％。经式(I)化合物处理48h后，0μM、10μM、30μM、90μM组细胞的S期分别为25.09％，24.73％，15.96％及4.37％。经式(I)化合物处理72h后，0μM、10μM、30μM、90μM组细胞的S期分别是20.64％，19.77％，10.63％及5.42％。
NCI-H460细胞的结果和A549细胞类似。结果如图6所示：NCI-H460经式(I)化合物处理24h后，0μM、10μM、30μM、90μM四组的S期细胞比例分别是23.57％，26.03％，13.15％及9.76％。经式(I)化合物处理48h后，0μM、10μM、30μM、90μM四组的S期细胞比例分别是29.09％，29.62％，16.02％及0.97％。经式(I)化合物处理72h后，0μM，10μM，30μM，90μM四组的S期细胞比例分别是25.33％，21.61％，20.98％及3.36％。
式(I)化合物促进肺癌细胞凋亡
我们使用AnnexinV-FITC和PI双染色法进行细胞凋亡染色，FITC选择FL1通道,PI选择FL2通道。用流式细胞分析法检测细胞凋亡，Q1、Q2、Q3、Q4分别代表死亡细胞、晚期凋亡细胞、早期凋亡细胞、正常细胞(图7)。A549和NCI-H460细胞与不同浓度式(I)化合物(0μM、10μM、30μM、90μM)作用72h后，流式结果如下。表1和表2分别表示不同浓度式(I)化合物作用于A549和NCI-H460细胞后死亡细胞、晚期凋亡细胞、早期凋亡细胞、正常细胞所占比例。如表1和表2结果所示，式(I)化合物浓度较低时(10μM，30μM)时，A549和NCI-H460细胞中，早期凋亡细胞和晚期凋亡细胞的比例与对照组相比略有增加，但差异无统计学意义，但当式(I)化合物浓度提高到90μM时，A549和NCI-H460细胞中早期凋亡和晚期凋亡的比例均显著增加，且早期凋亡比例增加更明显。
表1不同浓度式(I)化合物对A549细胞凋亡的影响
Figure PCTCN2022125293-appb-000007
表2不同浓度式(I)化合物对NCI-H460细胞凋亡的影响
Figure PCTCN2022125293-appb-000008
表3表示不同浓度式(I)化合物作用于A549和NCI-H460细胞后总凋亡比例的变化。可以看到，当式(I)化合物浓度达90μM时，细胞凋亡比例增加明显，该现象在A549细胞中较NCI-H460细胞更明显。
表3不同浓度式(I)化合物对A549、NCI-H460细胞总凋亡的影响
Figure PCTCN2022125293-appb-000009
实施例3测试式(I)化合物相对于顺铂对人胃癌N87裸鼠移植瘤模型肿瘤体内生长的抑制作用
实验动物
雌性BALB/c裸小鼠(周龄：6-7周)从北京维通利华实验动物技术有限公司购买，饲养于SPF动物房，温度20-25℃，相对湿度40％-70％，明暗照明各12小时；动物自由饮水及采食。正常喂养约1周后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识。
顺铂(DDP)配制
每次给药前配制。称取适量DDP，装入玻璃瓶中；在瓶中加入适量体积生理盐水，涡旋振荡(可水浴加热50-60℃)，使药物完全溶解；配制成DDP浓度为0.5mg·mL-1的溶液。
式(I)化合物给药制剂的配制
称取适量化合物，加入适量体积的二甲基乙酰胺(DMA)，涡旋振荡，配制成储备液(8mg·mL-1)，每周配制1次，分装后保存于2-8℃冰箱。每次给药前取出一支，边涡旋边加入适量体积30％Solutol和PEG400，涡旋振荡混匀后加入适量体积的生理盐水，混合均匀得浓度0.4mg·mL-1给药制剂，制剂中DMA：30％Solutol：PEG 400：生理盐水的体积比例为5:20:20:55。
上述给药制剂均需进行留样，并对留样的制剂进行实际浓度的测定。
移植性肿瘤瘤株
人胃癌细胞NCI-N87，来源于中国科学院典型培养物保藏委员会细胞库(本实验室液氮冻存)。
NCI-N87细胞培养
在5％CO 2、37℃培养条件下，NCI-N87细胞在含10％胎牛血清RPMI-1640培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况进行传代，传代比例为1:3到1:5。
动物模型制备
收取对数生长期NCI-N87细胞，细胞计数后重悬于无血清RPMI-1640培养基中，调整细胞浓度至5×10 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50-mL离心管中，将离心管置于冰盒中；用1-mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(5×10 6细胞/只)，建立NCI-N87裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据输入Excel电子表格计算肿瘤体积。待肿瘤体积达到100～300mm 3，挑选健康状况良好、肿瘤体积相近的动物72只，采用随机区组法分为9组(n＝8)。以分组当天为实验第一天(D1)，实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
肿瘤体积(TV)计算公式如下：
TV(mm 3)＝l×w 2/2
其中，l表示肿瘤长径(mm)；w表示肿瘤短径(mm)。
动物分组及给药
表4.NCI-N87移植瘤模型药效实验分组及给药方案
Figure PCTCN2022125293-appb-000010
实验结束
实验最后一天(D44)，动物给药(R01)1h后使用CO 2吸入麻醉处死后采集肿瘤组织，称重、拍照。
数据记录、计算公式
肿瘤生长抑制率TGI(％)的计算公式为：
TGI(％)＝100％×[1–(TV t(T)–TV initial(T))/(TV t(C)–TV initial(C))]
其中，TV t(T)表示治疗组每次测量的肿瘤体积；TV initial(T)表示分组给药时治疗组的肿瘤体积；TV t(C)表示溶剂对照组每次测量的肿瘤体积；TV initial(C)表示分组给药时溶剂对照组的肿瘤体积。
动物体重下降率的计算公式为：
动物体重下降率＝100％×(BW initial－BW t)/BW initial
其中，BW t表示给药期间每次测量的动物体重；BW initial表示分组给药时的动物体重。
统计分析方法
试验数据用Microsoft Office Excel 2007软件进行计算和相关统计学处理。数据除特别说明外，用均数±标准误(Mean±SE)表示，两组间比较采用t-检验。
实验观察
在实验过程中，实验人员和兽医需对实验动物的体征和健康状况进行持续观察。动物的任何异常表现，例如疼痛、抑郁、活动减少等，需记录在实验原始记录中。如果实验动物的异常表现超过IACUC相关动物福利的文件规定，可经由兽医判断是否中止实验，并通报实验项目负责人。
试验结果如下：
表5.给药期间各组动物肿瘤体积及肿瘤生长抑制率
Figure PCTCN2022125293-appb-000011
表6.给药期间各组动物体重及体重下降率
Figure PCTCN2022125293-appb-000012
注：“*”表示肿瘤体积与溶剂对照组比较，存在显著性差异(P<0.05)；“**”表示肿瘤体积与溶剂对照组比较，存在极显著性差异(P<0.01)。
从上表可见，本发明化合物式(I)化合物比常用抗胃癌药物顺铂具有更好的抗肿瘤效果；顺铂组动物体重相对于空白对照组下降明显，而本发明化合物对动物体重无明显影响。
实施例4对多种移植瘤组织中VEGF/VEGFR/p-VEGFR等蛋白表达调控的定量研究
实验目的
本次实验用Western blot技术定量研究式(I)化合物对实施例3中消化道肿瘤NCI-N87裸鼠移植瘤组织中与肿瘤生长及抑制相关的信号通路的影响，旨在探讨式(I)化合物抗肿瘤的作用机制。
实验材料
肿瘤组织标本来自实施例3的动物肿瘤标本。
实验方法
Western Blot技术定量检测胞瘤组织VEGF/VEGFR、Survivin等蛋白的表达情况，详细的实验方法及所用材料参照参考文献(Molecular Cancer Research.June 2020,Volume 18,Issue 6,page 926-937)和Cell Signaling Technology的试剂盒说明书。本研究肿瘤组织蛋白一律每孔加样50微克，抗体工作浓度按说明书要求稀释。简言之：取肿瘤组织，用含1％PMSF和混合蛋白酶抑制剂的蛋白裂解液RIPA裂解，12000×g离心20min，取上清液； BCA法检测蛋白浓度；10％SDS-PAGE凝胶电泳；半干电转将蛋白从SDS-PAGE凝胶转至PVDF膜；快速封闭液封闭10min，加相应工作浓度的第一抗体，于4度过夜；用TBST清洗3次，室温孵育二抗1h；洗膜三次后，显影并保存图像，Image lab分析灰度值；用抗Vinculin抗体作为内参对照抗体。
数据分析
Western blot条带采用Image J软件进行灰度计算，Graphpad Prism 5做相关统计学处理，两组间比较采用双侧t-检验。P＞0.05为no significance(NS)，P＜0.05为*，P＜0.001为**，P＜0.001为***。
结果及讨论
本次实验探索了式(I)化合物在消化道肿瘤NCI-N87裸鼠移植瘤模型中的抗肿瘤作用机制，我们将从血管生成、细胞增殖和细胞凋亡三个方面进行阐述。
式(I)化合物对NCI-N87裸鼠移植瘤中重要靶标蛋白表达的影响及可能的抑瘤机制
在NCI-N87裸鼠移植瘤模型中，比较了顺铂(DDP)与式(I)化合物的抗肿瘤作用。式(I)化合物下调p-VEGFR2(NS)，下调VEGFR2(P＜0.05)，VEGFA(P＜0.05)；下调β-catenin(P＜0.05)，CyclinD1(NS)，上调PCNA(NS)；上调细胞周期调控蛋白P21(NS)，下调促凋亡Bax(P＜0.01)及抗凋亡蛋白Survivin(P＜0.05)。DDP下调p-VEGFR2(P＜0.05)、VEGFA(NS)，上调VEGFR2(NS)、β-catenin(P＜0.05)和CyclinD1(NS)，下调凋亡抑制蛋白Survivin(NS)及下调细胞周期调控蛋白P21(P＜0.05)。因此，在N87裸鼠移植瘤模型中，式(I)化合物与DDP相比显示出更强的血管生成抑制作用，因而具有更佳的抗肿瘤效果(图8)。
实施例5：受试样品口服急性毒性实验
受试样品：式(I)化合物。
供试品配制
溶媒配制方法及保存
以配制1000mL为例：标记容器终体积刻度线1000mL，先加入约三分之二常温纯水至容器中；准确称取5g羧甲基纤维素钠，边加入边搅拌；并定容至刻度线，继续搅拌至分散均匀得所需溶液。
2℃～8℃密闭保存28天。
辅料对照组给药制剂配制方法及保存
辅料对照组给药制剂配制：标记容器终体积刻度线，称取所需量的辅料至容器中，先向其中加入适量溶媒进行充分的搅拌混匀，再加溶媒稀释混匀至终体积刻度，获得目视均一的制剂。室温、避光密闭保存24h，
2℃～8℃避光、密闭保存8天。
供试品给药制剂配制方法及保存
供试品给药制剂的称量和配制需在黄灯下操作。
供试品给药制剂配制：将式(I)化合物：乙醇/乙酸乙酯(v/v，1/3)＝1:1.5的混合物室温超声溶解后，加入式(I)化合物的2.25当量的PVP，室温剧烈搅拌20分钟后加入式(I)化合物的2当量的乳糖，室温剧烈搅拌20分钟后，以50℃烘箱烘干后粉碎得式(I)化合物的黄色粉末制剂。然后称取适量式(I)化合物制剂，加入适量体积的0.5％CMC-Na水溶液，涡旋振荡后超声后至混合均匀(如有必要，可进行剪切乳化)，得给药制剂供试品。每周配制2次，每次配制3天或4天给药量，分装后保存于0-8药量冰箱备用。使用供试品时，标记容器终体积刻度线，称取所需量的供试品(供试品称量重量＝理论用量*折算系数)至洁净干燥容器中，先向其中加入适量溶媒进行充分的搅拌混匀，必要时可超声，再加溶媒稀释混匀至终体积刻度，获得目视均一的制剂并经方法学验证供试品给药制剂在浓度范围为0.1mg/mL～60mg/mL，常温密闭避光24h内稳定，2℃～8℃避光密闭8天内稳定。
给药制剂用不同颜色的标签进行标识：
受试动物：
种属：ICR小鼠
等级：SPF级
使用动物数量和性别：40只，雌雄各半；
体重：雌性分组体重25.9～29.7g；雄性分组体重27.7～30.8g；
给药时日龄：38-44日(雌性)、31-44日(雄性)
来源：北京维通利华实验动物技术有限公司
许可证号：SCXK(京)2016-0006
质量合格证号：110011211100519578、110011211100519414
剂量设置：
表7：毒性试验中各受试物剂量设置
Figure PCTCN2022125293-appb-000013
*：2～4组代表式(I)化合物的量，1组代表辅料量。
给药
给药途径：灌胃给药
给药途径选择理由：与临床拟用途径相似
给药频率：1次/天
给药期限：1天
根据D1称量的动物禁食(不禁水)过夜后体重计算给药量；药后约2h给予饲料。
各组给药制剂给药前置室温至少搅拌10min且目测混悬均匀，给药过程中需持续搅拌。
观察指标：包括一般体征观察、体重、摄食量、动物剖检和组织病理学检查。
试验结果
辅料对照组和300mg/kg/d剂量组均未出现死亡动物。1000mg/kg/d剂量组1例雄性动物(编号：3205)于D3发现死亡，死亡前未见明显异常；3000mg/kg/d剂量组1例雄性动物(编号：4205)D2、D3可见竖毛症状，于D4发现死亡，与分组体重相比，D3体重出现明显下降。上述死亡动物剖检后，肉眼观察均未见明显异常。
一般体征观察
试验期间，辅料对照组、300mg/kg/d剂量组动物临床观察均未见明显异常症状。1000mg/kg/d剂量组发现1例雄性动物死亡，死亡动物未见明显异常，其余动物临床观察未见明显异常症状。3000mg/kg/d剂量组发现1例雄性动物死亡，死亡动物D2、D3可见竖毛症状，其余动物D2-D7均可见竖毛症状，D8即恢复正常。
上述异常症状的发生率和严重程度可见剂量反应关系，认为与供试品相关。
体重
与同期溶媒对照组相比，供试品各剂量组雌性存活动物体重未见明显差异；供试品3000mg/kg/d剂量组雄性动物可见D3体重明显降低(P＜0.05)，其余时期各剂量组雄性存活动物体重未见明显差异，结合临床症状综合分析，认为与供试品相关。
摄食量测定
通过分析各剂量组动物平均摄食量数据，发现各剂量组摄食量与溶媒对照组摄食量基本保持一致。
大体解剖检查
对各组非计划死亡动物和存活动物进行大体解剖，肉眼观察各组动物脏器及组织颜色、体积、质地等未见明显异常，即肉眼未见明显异常毒性靶器官，未进行组织病理学检查。
结论
本试验条件下，1000mg/kg/d、3000mg/kg/d剂量组各出现1例雄性动物死亡；3000mg/kg/d剂量组动物均可见竖毛症状，第8天恢复正常，该组雄性动物药后第3天体重出现小幅度降低；各剂量组动物摄食量均未见明显异常。剖检所有动物，大体肉眼观察均未见明显异常。综上，ICR小鼠单次灌胃式(I)化合物，最大耐受剂量(MTD)为300mg/kg/d、LD 50>3000mg/kg，表明化合物式(I)化合物非常安全。
实施例6：ICR小鼠灌胃式(I)化合物2周重复给药毒性剂量探索试验
ICR小鼠连续灌胃给予式(I)化合物14天，评价式(I)化合物可能引起毒性反应的性质、程度、时效关系，以期为后续研究提供参考信息。
受试样品：给药制剂配制
供试品给药制剂的称量和配制需在黄灯下操作。
供试品各剂量组给药制剂配制：供试品的配制同实施例5。使用供试品时，标记容器终体积刻度线，称取所需量的供试品(供试品称量重量＝理论量/折算系数)至容器中，先向其中加入适量溶媒进行充分的搅拌混匀，必要时可超声，再加溶媒稀释混匀至终体积刻度，获得目视均一的制剂并开展方法学验证，试验结果表明，供试品给药制剂在浓度范围为0.1mg/mL～60mg/mL，室温避光24h内稳定。供试品给药制剂在浓度范围为0.01mg/mL～60mg/mL，2℃～8℃避光、密闭保存，8天内稳定。
表8给药制剂用不同颜色的标签进行标识：
Figure PCTCN2022125293-appb-000014
*：代表式(I)化合物的量。
受试动物：
种属              ICR小鼠
级别              SPF级
供应商            济南朋悦实验动物繁育有限公司
                  实验动物生产许可证：SCXK(鲁)2019 0003
数量和性别        雌雄各50只用于试验，20只/组
预计给药开始时年  5～8周龄
龄
预计给药开始时体  雌性24～30g，雄性26～32g
重
试验方法
饲养地点
购入后饲养于SPF动物房，试验机构使用许可证号：SYXK(鲁)20180031。动物饲养于透明小鼠饲养笼，雌雄分开饲养，每笼≤5只。
饲养环境条件
每天提供大约12小时的光照。黑暗时间可以因研究相关活动的需求而间歇性中断。每天监测记录动物房环境温度和相对湿度，并分别控制在20℃～26℃和40％～70％。
饲料和饮水
SPF大小鼠生长繁殖饲料购自北京科澳协力饲料有限公司。动物自由摄食。
玉米芯颗粒垫料购自北京科澳协力饲料有限公司。
动物自由饮用本机构反渗透系统过滤除菌的饮用水。
饲料供应商提供所购批次的饲料质量检验报告，其它检测频率、指标及要求按照中心SOP要求进行。
动物检疫适应
动物到达试验机构后已经检疫适应完成，根据最新的动物健康状态筛，由专题负责人签字确认后选择100只健康动物(雌雄各半)用于试验。
给药途径
给药途径：经口灌胃
给药途径选择理由：与临床拟用途径相似
给药频率
每天给药1次；每天保持相同给药时间段。
给药周期
连续给药14天
给药
根据最新称量的动物体重计算给药量。
供试品给药制剂在给药前置室温至少搅拌10min且目测混悬均匀，给药过程中需持续搅拌。
观察指标：包括一般体征观察、详细临床观察、体重、摄食量。
结果：
1. 1000mg/kg/d剂量组D3-D6期间共4只动物发现死亡，停止该组别动物给药操作，继续观察。
2. 1000mg/kg剂量组死亡13只动物，300mg/kg剂量组死亡10只动物，100mg/kg剂量组死亡3只动物；50mg/kg和25mg/kg剂量组剂量组无动物死亡。
3. 100mg/kg剂量组未死亡动物及50mg/kg和25mg/kg剂量组动物均无异常反应，体重正常增加。
实施例7:测试式(I)化合物单用及式(I)化合物与化疗药物顺铂(DDP)联合用药对人胃癌NCI-N87裸鼠移植瘤模型肿瘤体内生长的抑制作用
实验动物、空白溶剂配制、顺铂(DDP)配制、式(I)化合物给药制剂的配制、移植性肿瘤瘤株、NCI-N87细胞培养、动物模型制备、实验过程及统计分析方法同实施例3。
动物分组及给药
表9.NCI-N87移植瘤模型药效实验分组及给药方案
Figure PCTCN2022125293-appb-000015
试验结果如下：
表10.给药期间各组动物肿瘤体积及肿瘤生长抑制率
Figure PCTCN2022125293-appb-000016
注：“：“表示肿瘤体积与溶剂对照组比较，存在显著性差异(P<0.05)；““05表示肿瘤体积与溶剂对照组比较，存在极显著性差异(P<0.01)。
表11.给药期间各组动物体重及体重下降率
Figure PCTCN2022125293-appb-000017
表12.各组动物血清UREA含量(mmol血清 -1)
Figure PCTCN2022125293-appb-000018
从上述数据可见，R01单用在本次实验条件下对NCI-N87裸鼠移植瘤生长存在明显抑制作用，其抗肿瘤作用优于DDP单用组。R01分别在3、6 和16mg生长存在明(QD)剂量下与DDP联用，其药效均优于DDP单用，且联用后可以降低小鼠血清UREA水平，联用组动物血清UREA与R01剂量呈负相关，提示R01可能对DDP引起的肾损伤有一定保护和预防作用。此外，R01(8mg定保护和预防作用。)+DDP组药效略优于其它各给药组或与之相当。
实施例8：测试式(I)化合物与DDP对人食管癌ECA109裸鼠移植瘤模型肿瘤体内生长的协同抑制作用
实验动物、肿瘤体积(TV)计算、实验结束、数据记录、肿瘤生长抑制率TGI(％)的计算公式、统计分析方法同实施例3；式(I)化合物制剂配制同实施例5。
空白溶剂配制
称取适量CMC-Na固体，先加入适量体积去离子水，涡旋搅拌直至固体完全分散均匀，配制成0.5％CMC-Na水溶液，保存于2～8于C冰箱。
顺铂(DDP)给药试液配制
称取适量DDP，装入玻璃瓶中；在瓶中加入适量体积生理盐水，涡旋超声，使药物完全溶解；配制成DDP浓度为0.5mg中；在瓶中的溶液，每次给药前配制。
移植性肿瘤瘤株
人食道癌细胞ECA109，来源于武汉大学细胞库(CCTCC，本实验室液氮冻存)。
细胞培养
在5％CO 2、37CO培养条件下，ECA109细胞在含10％胎牛血清RPMI-1640培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况进行传代，传代比例为1:3到1:6。
动物模型制备及肿瘤体积测定
收取对数生长期ECA109细胞，细胞计数后重悬于含50％的无血清RPMI-1640培养液和50％的Matrigel中，调整细胞浓度至0.5整细胞 8细胞/mL；用移液器吹打细胞使其分散均匀后装入50-mL离心管中，将离心管置于冰盒中；用1-mL注射器吸取细胞悬液，注射到NOD/SCID小鼠前右肢腋窝皮下，每只动物接种200μL(1.0肢腋 7细胞/只)，建立ECA109裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况，使用电子游标卡尺测量瘤径，数据输入Excel电子表格计算肿瘤体积。待肿瘤体积达到100-300mm 3，挑选健康状况良好、肿瘤体积相近的动物采用随机区组法分组，以分组当天为实验第一天(D1)，实验开始后每周测量2次瘤径，计算肿瘤体积，同时称量动物体重并记录。
动物分组及给药
表13.ECA109移植瘤模型药效实验分组及给药方案
Figure PCTCN2022125293-appb-000019
试验结果如下：
表14.给药期间各组动物肿瘤体积及肿瘤生长抑制率
Figure PCTCN2022125293-appb-000020
注：“：“表示肿瘤体积与溶剂对照组比较，存在显著性差异(P<0.05)；““05表示肿瘤体积与溶剂对照组比较，存在极显著性差异(P<0.01)。
表15.给药期间各组动物体重及体重下降率
Figure PCTCN2022125293-appb-000021
表16.实验结束时各组动物血清尿素(UREA)水平
Figure PCTCN2022125293-appb-000022
从上述数据可见，R01制剂单用在本次实验条件下对ECA109裸鼠移植瘤生长存在明显抑制作用，其抗肿瘤作用优于DDP单用组。R01制剂在18mg生长存 -1(QD)剂量下与DDP联用，其药效优于DDP和R01单用；而联合用药组动物体重与DDP单用组比较，体重下降率相当，表明R01与DDP联用不会导致DDP毒性增加；末次给药后血清尿素检测结果发现，R01制剂+DDP组尿素低于DDP单用组，表明R01制剂与DDP联用对DDP给药所致的肾脏损伤有一定的保护或缓解作用。
实施例9:测试式(I)化合物及其与顺铂对人非小细胞肺癌A549裸鼠移植瘤模型肿瘤体内生长的协同抑制作用
实验动物(除正常喂养时间为10天外)、空白溶剂的配制、顺铂的配制(0.4、0.5或0.7mg溶剂的配制三种浓度)、肿瘤体积(TV)计算、实验结束、数据记录、肿瘤生长抑制率TGI(％)的计算公式、统计分析方法及实验观察同实施例3；式(I)化合物给药制剂的配制同实施例5。
细胞系
人非小细胞肺癌A549，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验液氮冷冻保存)。
细胞培养
在5％CO 2、37CO培养条件下，A549细胞在含10％胎牛血清F12K培养液中进行常规细胞培养；以0.25％胰酶消化传代；根据细胞生长情况，每周传代2到3次，传代比例为1:3到1:5。
动物模型制备及肿瘤体积测定
实验D1时，收取对数生长期A549细胞，细胞计数后重悬于PBS中，调整细胞浓度至8，调整 7细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中，用1mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(800 6细胞/只)，建立A549裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况。D18天时，使用电子游标卡尺测量瘤径，数据输入Excel电子表格，计算肿瘤体积，挑选健康状况良好、肿瘤体积(101～156mm 3)相近的荷瘤鼠采用随机区组法分组。实验开始后定期测量瘤径，计算肿瘤体积，同时称量动物体重并记录。
样品采集
实验最后一天，动物给药(R01)1h后使用CO2吸入麻醉、经心脏采血，一部分全血添加EDTA-K2抗凝，于4，于、1500g条件下离心10min分离血浆，取80浆，血浆保存在-40在C至-20℃冰箱(如有需要，可用于血药浓度测定)；另一部分全血不加任何抗凝剂，待血液凝血后离心分离血清，用于测定血尿素氮。采血结束后采集肿瘤组织，称重、拍照后，肿瘤切分为两部分，经液氮速冻后转入-90有C至-60有需冰箱保存；采集双侧肾脏，使用10％福尔马林固定。
动物分组及给药
表17.A549移植瘤模型药效实验分组及给药方案
Figure PCTCN2022125293-appb-000023
试验结果如下：
表18.给药期间各组动物肿瘤体积及肿瘤生长抑制率
Figure PCTCN2022125293-appb-000024
注：“：“表示肿瘤体积与溶剂对照组比较，存在显著性差异(P<0.05)；““<表示肿瘤体积与溶剂对照组比较，存在极显著性差异(P<0.01)。
表19.给药期间各组动物体重及体重下降率
Figure PCTCN2022125293-appb-000025
表20.实验结束时各组动物血清尿素氮(BUN)水平
Figure PCTCN2022125293-appb-000026
从上述数据可见，DDP在4mg据可见 -1(QWmg)和5/7/7/7mg清尿素 -1(QW)剂量下，对A549裸鼠移植瘤生长无明显影响。R01制剂单用在18mg生长无 -1BID和36mg生长无 -1BID剂量下对A549裸鼠移植瘤生长均存在明显抑制作用。联合用药组R01制剂(18mg均存在 -1BID)+DDP(5/7/7/7mg·kg -1QW)以及R01制剂(36mgDDP -1BID)+DDP(4mg7/7 -1QW)对A549裸鼠移植瘤生长均具有明显抑制作用，两个联合用药组相对肿瘤体积或瘤重与相应剂量DDP单用组比较，均存在显著性(P<0.05)或极显著性差异(P<0.01)。各联合用药组动物体重与对应剂量DDP单用组比较，体重下降率相当，表明R01与DDP联用不会导致DDP毒性增加，且R01可能对DDP所致的肾脏损伤有一定的保护作用。
实施例10：测试式(I)化合物与顺铂对人结直肠癌HT29裸鼠移植瘤模型肿瘤体内生长的协同抑制作用
实验动物、空白溶剂的配制、顺铂(DDP)配制、肿瘤体积(TV)计算、实验结束、数据记录、肿瘤生长抑制率TGI(％)的计算公式、统计分析方法及实验观察同实施例3；式(I)化合物给药制剂的配制同实施例3。
细胞系
人结肠癌细胞系HT-29，来源于中国科学院典型培养物保藏委员会细胞库(CAS，本实验液氮冷冻保存)。
细胞培养
在5％CO 2、37CO培养条件下，HT-29细胞在含10％胎牛血清的McCoy，冷冻保存培养液中进行常规细胞培养，以0.25％胰酶消化传代，根据细胞生长情况进行传代，传代比例为1：3到1：4。
动物模型制备及肿瘤体积测定
实验D1时，收取对数生长期HT-29细胞，细胞计数后重悬于无血清McCoy计数后重悬培养基中，调整细胞浓度至4养基中，细胞/mL；用移液器吹打细胞使其分散均匀后装入50mL离心管中，将离心管置于冰盒中，用1mL注射器吸取细胞悬液，注射到裸鼠前右肢腋窝皮下，每只动物接种100μL(400取细胞/只)，建立HT-29裸鼠移植瘤模型。接种后定期观察动物状态及肿瘤生长情况。D12天时，使用电子游标卡尺测量瘤径，数据输入Excel电子表格，计算肿瘤体积，挑选健康状况良好、肿瘤体积(103～179mm3)相近的荷瘤鼠采用随机区组法分组。实验开始后定期测量瘤径，计算肿瘤体积，同时称量动物体重并记录。
动物分组及给药
表21.HT-29移植瘤模型药效实验分组及给药方案
Figure PCTCN2022125293-appb-000027
试验结果如下：
表22.给药期间各组动物肿瘤体积及肿瘤生长抑制率
Figure PCTCN2022125293-appb-000028
表23.给药期间各组动物体重及体重下降率
Figure PCTCN2022125293-appb-000029
注：“：“表示肿瘤体积与溶剂对照组比较，存在显著性差异(P<0.05)；““<表示肿瘤体积与溶剂对照组比较，存在极显著性差异(P<0.01)。
从上表可见，本发明式(I)化合物可与顺铂产生协同的抗肿瘤效果：联合用药起效更快、效果更好，而且安全性无明显降低。
实施例11：式(1)化合物对化疗药物顺铂的主要靶器官毒性的拮抗作用
实验目的
本实验旨在确定性地研究R01是否可以显著减轻顺铂诱导的小鼠急性肾损伤，并与目前临床上一线使用的拮抗顺铂肾损伤最确定的药物氨磷汀对比，确定R01在保护顺铂所致肾损伤方面应用的潜力。
供试品与试剂
R01制剂：同实施例5。
氨磷汀三水物，批号：Z28N11R132596，分子量：268.27；含量：98％，上海源叶生物。
顺铂(批号：601200804，分子量：300.05，纯度≥纯度≥0508，折算系数：1)，购自江苏豪森药业集团有限公司。
实验动物
42只雄性C57BL/6J小鼠(周龄：6～8周，体重18-20g，合格证号： 11400700238365)购自斯贝福(北京)生物科技有限公司，饲养于中国医学科学院药物研究所SPF级动物房，温度20～25度所，相对湿度40％～70％，明暗照明各12小时，动物自由饮水及采食。正常喂养3天后，经兽医检验，体征状况良好小鼠可入选本实验。分组前使用记号笔于动物尾根部进行标识，分组后每只动物均用耳部剪缺方式标识(每组7只)。
实验方法
实验动物分组
实验动物：7周龄C57小鼠雄性42只经适应性饲养后进行动物试验。
第一组：空白对照组；
第二组：阳性对照药顺铂(10mg/kg)；
第三组：顺铂(10mg/kg)+氨磷汀(50mg/kg，顺铂给药前半小时腹腔注射，仅给药一次)；
第四组：顺铂(10mg/kg)+R01(6mg，bid，顺铂造模前三天给药)；
第五组：顺铂(10mg/kg)+R01(18mg，bid，顺铂造模前三天给药)；
第六组：顺铂(10mg/kg)+R01(36mg，bid，顺铂造模前三天给药)。
药物配制
空白溶剂配制：取适量的羧甲基纤维素钠，以生理盐水配制成为0.5％的羧甲基纤维素钠溶液，高压灭菌。
顺铂(DDP)给药试液配制：顺铂制剂为溶液，直接应用，平时避光室温保存。
R01制剂给药试液配制同实施例5：根据实验设定的三个剂量，分别称取适量R01制剂，加入适量体积的0.5％CMC-Na水溶液，涡旋振荡后超声至混合均匀，分别得到浓度0.6、1.8、3.6mg振荡后 -1的给药试液待用。
动物给药及样品采集
动物试验周期：7天(不包括适应性喂养)，以下简称：D1-D7。
顺铂施用方式：一次性腹腔注射顺铂溶液(10mg/kg)
处死方式：处死前12小时禁食，眼球取血处死。
1.第一组为溶剂对照组：N＝7，均为雄性(下同)；
a)D1-D6 灌胃0.5％羧甲基纤维素钠溶液(每只每天两次，上下午间隔8小时)。
b)D4 一次性腹腔注射生理盐水。
c)D7 眼眶取血处死(提血清300ul)、左肾甲醛固定、右肾液氮保存(可能开展WB研究)。
2.第二组为顺铂诱导肾损伤模型组：N＝7；
a)D1-D6 灌胃羧甲基纤维素钠溶液(每只每天两次)。
b)D4 一次性腹腔注射10mg/kg顺铂。
c)D7 眼眶取血处死(提血清300ul)、左肾甲醛固定、右肾液氮保存。
3.第三组为顺铂(10mg/kg)+氨磷汀(50mg/kg，顺铂给药前半小时腹腔注射)组：N＝7；
a)D4 一次性腹腔注射10mg/kg顺铂(顺铂给药前半小时腹腔注射50mg/kg氨磷汀)。
b)D7 眼眶取血处死(提血清300ul)、左肾甲醛固定、右肾液氮保存。
4.第四～六组为顺铂(10mg/kg)+R01(分别6、18、36mg，bid，提前顺铂三天给药)：N＝7；
a)D1-D6 灌胃R01混悬液(每只每天两次,上下午间隔8小时)。
b)D4 一次性腹腔注射10mg/kg顺铂。
c)D7 眼眶取血处死(提血清300ul)、左肾甲醛固定、右肾液氮保存。
检测项目
1)检测外周血血清中尿素氮、肌酐的浓度；
2)肾脏病理检测：HE，PAS制片、玻片扫描并出具报告。
3)每只动物处死前称体重记录，处死后尽快称重两个肾脏重量记录，之后迅速固定或冻存，计算小鼠肾脏系数(肾脏重量/小鼠体重)。
数据记录及计算
各组动物均记录下分组给药时的体重，以及试验结束后的体重和肾脏重量，计算肾脏系数；记录送检血样的肌酐和尿素氮值；肾脏做HE、PAS染色并做玻片扫描，病理分析后出具报告。
统计分析
试验数据用Microsoft Office Excel 2007进行计算和相关统计学处理。数据除特别说明外，用均数±标准误差(Mean(关统计)表示，两组间比较采用双侧t-检验。
实验结果
顺铂造模前各组动物体重(g)
Figure PCTCN2022125293-appb-000030
试验结束后各组动物体重(g)
Figure PCTCN2022125293-appb-000031
由试验前后动物体重变化数值可知，顺铂造模组体重，无论各组是否使用药物均有较明显的下降(P<0.01)，使用氨磷汀组与使用R01的三组之间没有明显差异。
试验结束后各组动物肾脏重量
Figure PCTCN2022125293-appb-000032
试验结束后各组动物肾脏系数
Figure PCTCN2022125293-appb-000033
根据试验测定数值，模型组的肾脏系数有较显著的增加(P<0.01)，而用药组则无显著差别，总体而言，各药物干预组与空白对照组肾脏系数相当，且使用氨磷汀组与使用R01的三组之间没有明显差异，特别是R01中、高剂量组与空白对照组更接近。
试验结束后各组动物血清肌酐(Cre)水平
Figure PCTCN2022125293-appb-000034
根据试验测定数值，顺铂造模组血清肌酐值较显著升高(P<0.01)，各药物干预组则与空白对照组相当，且使用氨磷汀组与使用R01的三组之间没有明显差异。
试验结束后各组动物尿素氮(Bun)水平
Figure PCTCN2022125293-appb-000035
根据试验测定数值，顺铂造模组血清尿素氮值较显著升高(P<0.05)，氨磷汀组尿素氮值相比于模型组显著降低(P<0.01)，R01高剂量组同模型组相比也显著降低(P<0.05)；尽管R01低剂量与中剂量组相比于模型组其尿素氮浓度有所降低，但未见显著差异(P>0.05)。
动物肾脏的病理损伤情况
病理评分标准
Figure PCTCN2022125293-appb-000036
病理评分结果
Figure PCTCN2022125293-appb-000037
病理描述及典型性照片
空白对照组：正常饲养的空白对照组7只动物肾小球、肾小管基本无损 伤，肾小管管腔紧致，仅有部分小鼠肾小球周边出现些许炎症细胞浸润，典型图片如图9。
顺铂诱导肾损伤模型组(10mg/kg)组：本组动物为模型组，肾脏功能出现严重损伤，生化检测显示血中尿素氮和肌酐的浓度均出现了显著上升。从病理特征上来看，肾小球病理损伤不重，仅有少数动物出现肾小球皱缩的现象。主要损伤来自肾小管区域，肾小管上皮细胞坏死显著，且出现蛋白管型，损伤区域主要发生在入球小动脉区域。整体来看，肾小管细胞出现毛边且边缘模糊，部分动物病理损伤很严重，可见遍布视野的大量蛋白管型出现，典型图片如图10。
顺铂+氨磷汀组：本组肾脏损伤程度显著减轻，仅部分小鼠出现轻微损伤，其它动物基本没有明显可见的病理损伤，典型图片如图11。
顺铂+R01 6mg组：本组动物肾脏均出现了一定程度的损伤，其中有2只较重，其它为轻度损伤，损伤部位集中在肾小管区，典型图片如图12。
顺铂+R01 18mg组：本组动物肾脏基本没有较明显的病理损伤，仅1只动物有较为明显的损伤，其它均较轻，典型图片如图13。
顺铂+R01 36mg组：本组动物仅1只小鼠出现了肾脏轻微病理损伤，其它各只动物均病理结构完好，细胞结构清晰，典型图片如图14。
共有6组小鼠肾脏进行病理损伤评分。其中空白对照组无肉眼可见病理损伤，组织形态完整。顺铂肾损伤模型组出现了肉眼可见的显著损伤，且个体差异比较明显，损伤程度存在一定程度的差别，主要病理损伤存在于肾小管区域。顺铂+氨磷汀组病理损伤显著减轻，与阳性对照顺铂组有显著差异(P<0.01)。顺铂+R01(6mg)组与顺铂+R01(18mg)组，病理损伤程度显著减轻(P<0.05)。顺铂+R01(36mg)肾脏组织形态基本完整，损伤轻微(P<0.01)，效果最佳，与氨磷汀组基本类似。
工业实用性
本发明的2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物具有优异的抗肿瘤效果及极佳的安全性。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。

Claims (15)

  1. 式(I)化合物、溶剂合物、水合物、多晶型、前药或同位素变体，以及它们的混合物：
    Figure PCTCN2022125293-appb-100001
  2. 药物组合物，其含有权利要求1的化合物，或溶剂合物、水合物、多晶型、前药或同位素变体，和药学上可接受的赋形剂。
  3. 权利要求1的化合物或溶剂合物、水合物、多晶型、前药或同位素变体，或权利要求2的药物组合物，在制备用于治疗和/或预防癌症的药物中的用途。
  4. 一种在受试者中治疗和/或预防癌症的方法，所述方法包括向所述受试者给药权利要求1的化合物或溶剂合物、水合物、多晶型、前药或同位素变体或权利要求2的药物组合物。
  5. 权利要求1的化合物或溶剂合物、水合物、多晶型、前药或同位素变体或权利要求2的药物组合物，其用于治疗和/或预防癌症。
  6. 权利要求3的用途或权利要求4的方法或权利要求5的化合物或组合物的用途，其中所述癌症选自肺癌、胃癌、食管癌、结直肠癌。
  7. 组合，包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体，以及铂类药物。
  8. 药物组合物，包括权利要求7的组合和药物可接受载体、稀释剂或赋形剂。
  9. 权利要求7的组合或权利要求8的药物组合物在制备治疗和/或预防受试者癌症的药物中的用途。
  10. 药物制品，包括式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体，以及铂类药物，作为在治疗中同时、依次或分别使用的联合制剂。
  11. 一种治疗和/或预防患者癌症的方法，所述方法包括同时、依次或分 别地对受试者或患者给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物。
  12. 式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体和铂类药物在制备治疗和/或预防受试者中的癌症的药物中的用途。
  13. 式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体在制备用于减轻受试者中铂类药物诱导的急性肾脏损伤中的药物中用途。
  14. 一种减轻受试者中铂类药物诱导的急性肾脏损伤中的方法，包括在给药铂类药物的同时、依次或分别给药式(I)化合物，或其溶剂合物、水合物、多晶型、前药或同位素变体的步骤。
  15. 权利要求7的组合、权利要求8的药物组合物、权利要求10中的药物制品、权利要求11的方法、权利要求12的用途、权利要求13的用途、权利要求14的方法，其中所述的铂类药物选自顺铂、卡铂、奈达铂、奥沙利铂、洛铂。
PCT/CN2022/125293 2021-10-15 2022-10-14 2,3-二甲氧基-5-甲基-1,4-苯醌烷基醇衍生物及其应用 WO2023061464A1 (zh)

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