WO2023020539A1 - 苯甲酰苯胺类化合物在治疗肿瘤中的应用 - Google Patents

苯甲酰苯胺类化合物在治疗肿瘤中的应用 Download PDF

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WO2023020539A1
WO2023020539A1 PCT/CN2022/113055 CN2022113055W WO2023020539A1 WO 2023020539 A1 WO2023020539 A1 WO 2023020539A1 CN 2022113055 W CN2022113055 W CN 2022113055W WO 2023020539 A1 WO2023020539 A1 WO 2023020539A1
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nnmt
dna
nnmt gene
tumor
expression
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French (fr)
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施裕丰
马文江
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南京施江医药科技有限公司
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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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the invention relates to the field of medicines, in particular to the application of benzanilide compounds in treating tumors.
  • Tumor is a common disease that seriously endangers human health, and the mortality rate of malignant tumors has been on the rise. Due to the heterogeneity of tumors and individual differences in patients, if the same treatment method or the same drug is simply used according to the source of the patient's tumor or pathological characteristics, it will easily lead to inappropriate treatment, which will delay the patient's precious treatment time and opportunity. In this case, it is very necessary to adopt personalized and precise treatment.
  • the purpose of the present invention is to provide a drug capable of precise treatment of tumors, especially NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or
  • the methylation level of the DNA CpG site in the NNMT gene region can be used to judge whether a tumor patient is suitable for prevention and/or treatment with the compound of the present invention.
  • the compounds of the present invention have low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or DNA CpG site of NNMT gene region Tumors with high levels of methylation have significantly better therapeutic effects.
  • a compound of formula I or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound, for Preparation of compositions or formulations for the prevention and/or treatment of tumors;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently hydrogen, halogen, -CN, hydroxyl, mercapto, nitro, amino, - COOH, -CHO, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C1-C12 alkoxy, or substituted or unsubstituted C1-C12 alkane Thio group;
  • any of the "substitution” means that one or more (preferably 1, 2, 3 or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group: C1-C8 alkyl , C3-C8 cycloalkyl, C1-C8 haloalkyl, C3-C8 halocycloalkyl, halogen, nitro, -CN, cyano, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 Alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently hydrogen, halogen, -CN, hydroxyl, mercapto , nitro, amino, -COOH, -CHO, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C1-C8 alkoxy, or substituted or Unsubstituted C1-C8 alkylthio.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently hydrogen, halogen, -CN, hydroxyl, mercapto , nitro, amino, -COOH, -CHO, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted C1-C4 alkoxy, or substituted Or unsubstituted C1-C4 alkylthio.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently hydrogen, halogen, hydroxyl, mercapto, nitro , or amino.
  • R 2 , R 4 , R 5 , R 7 , R 8 and R 10 are each independently hydrogen.
  • R 1 and R 9 are each independently halogen.
  • R3 is nitro
  • R 6 is hydroxyl or mercapto.
  • the halogen is F, Cl, Br or I.
  • R1 and R9 are each independently Cl.
  • Z is
  • Z is
  • the compound of formula I has the structure described in formula I-1:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are as defined above.
  • the compound of formula I has the structure described in formula I-2:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are as defined above.
  • the compound of formula I has the structure described in formula I-3:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are as defined above.
  • the compound of formula I has the structure described in formula I-4:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are as defined above.
  • the compound of formula I has the structure described in formula I-5:
  • R 1 , R 3 , R 6 and R 9 are as defined above.
  • the compound of formula I is:
  • any of the "substitution" means that one or more (preferably 1, 2, 3 or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C3-C8 halocycloalkyl, halogen, nitro, -CN, cyano, hydroxyl, mercapto, amino, C1-C6 alkoxy group, C1-C6 alkylthio group, C3-C8 cycloalkoxy group, C3-C8 cycloalkylthio group.
  • any of the "substitution" means that one or more (preferably 1, 2, 3 or 4) hydrogen atoms on the group are replaced by a substituent selected from the following group: C1-C4 alkyl, C3-C8 cycloalkyl, C1-C4 haloalkyl, C3-C8 halocycloalkyl, halogen, nitro, -CN, cyano, hydroxyl, mercapto, amino, C1-C4 alkoxy group, C1-C4 alkylthio group, C3-C8 cycloalkoxy group, C3-C8 cycloalkylthio group.
  • the pharmaceutically acceptable salt of the compound of formula I is a salt formed by the compound of formula I and an acid selected from the following group: hydrochloric acid, mucic acid, D-glucuronic acid, hydrobromic acid, Hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid , methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid, aspartic acid, glutamic acid, or combinations thereof.
  • an acid selected from the following group: hydrochloric acid, mucic acid, D-glucuronic acid, hydrobromic acid, Hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, trifluor
  • the tumor is a human tumor.
  • the tumor is a human tumor.
  • the tumor includes a tumor with low or no expression of NNMT gene.
  • the tumor includes a tumor with high expression of DNA methylase.
  • the DNA methylase is selected from the group consisting of DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the tumor includes a tumor with high expression of DNMT1.
  • the tumor includes a tumor with high expression of DNMT3a.
  • the tumor includes a tumor with high expression of DNMT3b.
  • the tumor includes a tumor with high expression of UHRF1.
  • the tumor includes a tumor with a high level of methylation at the nucleotide site of the NNMT gene and/or a high level of methylation at the DNA CpG site of the NNMT gene region.
  • the tumor includes a tumor with a high level of methylation at the nucleotide site of the NNMT gene.
  • the tumor includes a tumor with a high methylation level of the DNA CpG site in the NNMT gene region.
  • the NNMT gene is a human NNMT gene.
  • the NNMT gene is a human NNMT gene.
  • the tumor with low or no expression of NNMT gene refers to that NNMT protein cannot be detected by NNMT antibody in 1 ⁇ g of protein extracted from the tumor, and more preferably refers to 5 ⁇ g of protein extracted from the tumor.
  • NNMT protein cannot be detected by NNMT antibody in the protein, more preferably means that NNMT protein cannot be detected by NNMT antibody in 10 ⁇ g of protein extracted from the tumor, more preferably means that NNMT protein is not detected in 100 ⁇ g of protein extracted from the tumor by NNMT NNMT protein cannot be detected by the antibody, more preferably means that NNMT protein cannot be detected by the NNMT antibody in 1000 ⁇ g of protein extracted from the tumor.
  • the tumor with low expression of NNMT gene refers to that the expression level of NNMT gene in tumor cells is lower than the expression level of NNMT gene in the same cell or normal cells (such as paracancerous tissue cells).
  • the tumor with low expression of NNMT gene refers to the ratio (E1 /E0) ⁇ 1.0.
  • the low expression or non-expression of the NNMT gene refers to the expression of the NNMT gene in a certain cell (such as a tumor cell) and the expression of the NNMT gene in the same cell or a normal cell (such as a paracancerous tissue cell).
  • E0 (E1/E0) ⁇ 1.0 preferably ⁇ 0.7, more preferably ⁇ 0.6, more preferably ⁇ 0.5, more preferably ⁇ 0.4, more preferably ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, more preferably ⁇ 0.05, more preferably ⁇ 0.01, more preferably ⁇ 0.005, more preferably ⁇ 0.001, more preferably ⁇ 0.0001, more preferably ⁇ 0.00001, more preferably ⁇ 0.000001, more preferably ⁇ 0.0000001 .
  • the same cell refers to a cell with normal or high expression of NNMT gene (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with normal or high expression of NNMT gene.
  • the normal cells refer to normal tissue cells with normal expression of NNMT gene (such as cancer cell origin cells, tumor adjacent cells or paracancerous tissue cells).
  • E0 is the expression level of NNMT gene in cells with normal or high expression of NNMT gene.
  • the cells with normal or high expression of the NNMT gene include compounds of formula I, or their optical isomers or their racemates, or their solvates, or their pharmaceutically acceptable Cells insensitive to salt, or its deuterated compounds.
  • the tumor with high expression of DNA methylase refers to that DNA methylase can be detected in 20 ⁇ g of protein extracted from the tumor through DNA methylase antibody detection, more preferably DNA methylase detectable by DNA methylase antibody detection in 5 ⁇ g of protein extracted from the tumor, more preferably 1 ⁇ g of protein extracted from the tumor by DNA methylase antibody detection DNA methylase, more preferably 0.2 ⁇ g protein extracted from the tumor, DNA methylase detectable by DNA methylase antibody detection, more preferably 0.05 ⁇ g protein extracted from the tumor.
  • the DNA methylase can be detected by the DNA methylase antibody detection, more preferably the DNA methylase can be detected by the DNA methylase antibody detection in 0.01 ⁇ g protein extracted from the tumor.
  • the tumor with high expression of DNA methylase refers to that the expression level of DNA methylase in tumor cells is greater than that in the same cell or normal cells (such as paracancerous tissue cells). level of expression.
  • the tumor with high expression of DNA methylase refers to that the expression level of DNA methylase in tumor cells G1 is the same as that of DNA methylation in the same cell or normal cells (such as paracancerous tissue cells).
  • the same cell refers to a cell with normal or low expression of DNA methylase (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with normal or low expression of DNA methylase.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) with normal expression of DNA methylase.
  • G0 is the expression level of DNA methylase in cells with normal expression or low expression of DNA methylase.
  • the cells with normal expression or low expression of DNA methylase include compounds of formula I, or their optical isomers or their racemates, or their solvates, or their pharmaceutical cells insensitive to acceptable salts, or their deuterated compounds.
  • the tumor with high expression of DNMT1 means that DNMT1 protein can be detected by DNMT1 antibody detection in 20 ⁇ g protein extracted from the tumor, more preferably DNMT1 protein can be detected in 5 ⁇ g protein extracted from the tumor.
  • DNMT1 protein can be detected by antibody detection, more preferably DNMT1 protein can be detected by DNMT1 antibody detection in 1 ⁇ g protein extracted from the tumor, more preferably DNMT1 antibody can be detected by DNMT1 antibody detection in 0.2 ⁇ g protein extracted from the tumor DNMT1 protein is detected, more preferably DNMT1 protein can be detected by DNMT1 antibody detection in 0.05 ⁇ g protein extracted from the tumor, more preferably DNMT1 protein can be detected in 0.01 ⁇ g protein extracted from the tumor by DNMT1 antibody detection DNMT1 protein.
  • the tumor with high expression of DNMT1 means that the expression level of DNMT1 in tumor cells is greater than the expression level of DNMT1 in the same cells or normal cells (such as paracancerous tissue cells).
  • the tumor with high expression of DNMT1 refers to the ratio (B1/B0) of the expression level B1 of DNMT1 in tumor cells to the expression level B0 of DNMT1 in the same cell or normal cells (such as paracancerous tissue cells) >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50, for example 2-50.
  • the same cell refers to a cell with normal or low expression of DNMT1 (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with normal or low expression of DNMT1.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) with normal expression of DNMT1.
  • B0 is the expression level of DNMT1 in cells with normal or low expression of DNMT1.
  • the cells with normal or low expression of DNMT1 include compounds of formula I, or their optical isomers or their racemates, or their solvates, or their pharmaceutically acceptable salts , or cells insensitive to its deuterated compounds.
  • the tumor with high expression of DNMT3a means that DNMT3a protein can be detected by DNMT3a antibody detection in 20 ⁇ g protein extracted from the tumor, more preferably DNMT3a protein can be detected in 5 ⁇ g protein extracted from the tumor.
  • Antibody detection can detect DNMT3a protein, more preferably DNMT3a antibody detection can detect DNMT3a protein in 1 ⁇ g protein extracted from the tumor, more preferably DNMT3a antibody detection can be detected in 0.2 ⁇ g protein extracted from the tumor DNMT3a protein is detected, more preferably DNMT3a protein can be detected by DNMT3a antibody detection in 0.05 ⁇ g protein extracted from the tumor, more preferably DNMT3a antibody detection can be detected in 0.01 ⁇ g protein extracted from the tumor DNMT3a protein.
  • the tumor with high expression of DNMT3a means that the expression level of DNMT3a in tumor cells is greater than that in the same cells or normal cells (such as paracancerous tissue cells).
  • the tumor with high expression of DNMT3a refers to the ratio of the expression level C1 of DNMT3a of tumor cells to the expression level C0 of DNMT3a in the same cell or normal cells (such as paracancerous tissue cells) (C1/C0) >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50, for example 2-50.
  • normal cells such as paracancerous tissue cells
  • the same cell refers to a cell with normal or low expression of DNMT3a (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with normal or low expression of DNMT3a.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) with normal expression of DNMT3a.
  • C0 is the expression level of DNMT3a in cells with normal or low expression of DNMT3a.
  • the cells with normal or low expression of DNMT3a include compounds of formula I, or their optical isomers or their racemates, or their solvates, or their pharmaceutically acceptable salts , or cells insensitive to its deuterated compounds.
  • the tumor with high expression of DNMT3b means that the DNMT3b protein can be detected by DNMT3b antibody detection in 20 ⁇ g protein extracted from the tumor, more preferably DNMT3b protein can be detected in 5 ⁇ g protein extracted from the tumor.
  • Antibody detection can detect DNMT3b protein, more preferably DNMT3b antibody detection can detect DNMT3b protein in 1 ⁇ g protein extracted from the tumor, more preferably DNMT3b antibody detection can be detected in 0.2 ⁇ g protein extracted from the tumor DNMT3b protein is detected, more preferably DNMT3b protein can be detected by DNMT3b antibody detection in 0.05 ⁇ g protein extracted from the tumor, more preferably DNMT3b antibody can be detected in 0.01 ⁇ g protein extracted from the tumor DNMT3b protein.
  • the tumor with high expression of DNMT3b means that the expression level of DNMT3b in tumor cells is greater than that in the same cells or normal cells (such as paracancerous tissue cells).
  • the tumor with high expression of DNMT3b refers to the ratio (D1/D0) of the expression level D1 of DNMT3b of tumor cells to the expression level D0 of DNMT3b in the same cell or normal cells (such as paracancerous tissue cells) >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50, for example 2-50.
  • normal cells such as paracancerous tissue cells
  • the same cell refers to a cell with normal or low expression of DNMT3b (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with normal or low expression of DNMT3b.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) that normally express DNMT3b.
  • D0 is the expression level of DNMT3b in cells with normal or low expression of DNMT3b.
  • the cells with normal or low expression of DNMT3b include compounds of formula I, or their optical isomers or their racemates, or their solvates, or their pharmaceutically acceptable salts , or cells insensitive to its deuterated compounds.
  • the tumor with high UHRF1 expression means that UHRF1 protein can be detected by UHRF1 antibody detection in 20 ⁇ g of protein extracted from the tumor, more preferably 5 ⁇ g of protein extracted from the tumor by UHRF1 Antibody detection can detect UHRF1 protein, more preferably UHRF1 protein can be detected by UHRF1 antibody detection in 1 ⁇ g protein extracted from the tumor, more preferably UHRF1 protein can be detected by UHRF1 antibody detection in 0.2 ⁇ g protein extracted from the tumor UHRF1 protein is detected, preferably UHRF1 protein can be detected by UHRF1 antibody detection in 0.05 ⁇ g protein extracted from the tumor, more preferably UHRF1 protein can be detected by UHRF1 antibody detection in 0.01 ⁇ g protein extracted from the tumor UHRF1 protein.
  • the tumor with high UHRF1 expression means that the expression level of UHRF1 in tumor cells is greater than that in the same cells or normal cells (such as paracancerous tissue cells).
  • the tumor with high UHRF1 expression refers to the ratio (F1/F0) of the UHRF1 expression level F1 of tumor cells to the UHRF1 expression level F0 in the same cell or normal cells (such as paracancerous tissue cells) >1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15 , more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50, for example 2-50.
  • normal cells such as paracancerous tissue cells
  • the same cell refers to a cell with normal or low expression of UHRF1 (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with normal or low expression of UHRF1.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) that normally express UHRF1.
  • F0 is the expression level of UHRF1 in cells with normal or low expression of UHRF1.
  • the cells with normal or low expression of UHRF1 include compounds of formula I, or their optical isomers or their racemates, or their solvates, or their pharmaceutically acceptable salts , or cells insensitive to its deuterated compounds.
  • the high methylation level of the NNMT gene nucleotide site refers to that the methylation level of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is greater than that of the same cell or a normal cell ( Such as the methylation level of NNMT gene nucleotide sites in paracancerous tissue cells).
  • the high methylation level of the NNMT gene nucleotide site refers to that the methylation level L1 of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) is the same as that of the same cell or a normal cell.
  • the high methylation level of the NNMT gene nucleotide site refers to the methylation level of the NNMT gene nucleotide site of a certain cell (such as a tumor cell) ⁇ 1%, preferably ⁇ 3%, preferably ⁇ 5%, preferably ⁇ 10%, preferably ⁇ 15%, preferably ⁇ 20%, more preferably ⁇ 25%, more preferably ⁇ 30%, more preferably ⁇ 40%, more preferably ⁇ 50%.
  • the same cell refers to a cell with a normal or low level of methylation at the nucleotide site of the NNMT gene (such as a type of tumor cell).
  • the same cell refers to a cell of the same type but with a normal or low level of methylation at the nucleotide site of the NNMT gene.
  • the normal cells refer to normal tissue cells with a normal level of methylation at the nucleotide site of the NNMT gene (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells).
  • L0 is the methylation level of the nucleotide site of the NNMT gene in cells whose methylation level of the nucleotide site of the NNMT gene is at a normal level or a low level.
  • the cells whose methylation level at the nucleotide site of the NNMT gene is at a normal level or at a low level include compounds of formula I, or their optical isomers or their racemates, or Cells insensitive to solvates thereof, or pharmaceutically acceptable salts thereof, or deuterated compounds thereof.
  • the high level of methylation at the nucleotide site of the NNMT gene refers to the methylation level (M%) of the nucleotide site of the NNMT gene in a certain cell (such as a tumor cell) ⁇ 3% And less than or equal to M1%, wherein, M1 is any positive integer between 3-100.
  • M1 is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or 100.
  • the methylation level at nucleotide sites of the NNMT gene refers to the ratio of the number of methylated nucleotides in the NNMT gene region to the total number of nucleotides in the NNMT gene region.
  • the nucleotide site methylation level of the NNMT gene includes the nucleotide site methylation level of the promoter region of the NNMT gene.
  • nucleotide sequence of the NNMT gene promoter region is shown in SEQ ID NO:1.
  • the methylation level of the nucleotide site of the NNMT gene includes the methylation level of the nucleotide site in the region from 1050 bp before the transcription initiation site of the NNMT gene to 499 bp after the transcription initiation site .
  • the 1050 bp before the transcription start site of the NNMT gene to the 499 bp after the transcription start site is the 951-2500 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the nucleotide site methylation level of the NNMT gene includes the methylation of the nucleotide site in the region from 1050 bp before the transcription start site of the NNMT gene to 193 bp before the gene transcription start site level.
  • the first 1050 bp to the first 193 bp of the gene transcription start site of the NNMT gene is the 951-1808 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the nucleotide site methylation level of the NNMT gene includes the methylation level of the nucleotide site in the region from 840 bp before the transcription start site to 469 bp before the transcription start site of the NNMT gene .
  • the first 840 bp to the first 469 bp of the transcription start site of the NNMT gene is 1161-1532 of the nucleotide sequence shown in SEQ ID NO:1.
  • the methylation level of the nucleotide site of the NNMT gene includes any two of the 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 positions of human chromosome 11 The methylation level of nucleotide sites in the region between the points (including the two sites themselves).
  • the NNMT gene nucleotide site methylation level includes one or more of the 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 positions of human chromosome 11 Nucleotide methylation levels at (eg 2, 3, 4, 5, 6 or 7) sites.
  • the methylation level of the nucleotide site of the NNMT gene includes the nucleotide methylation level of a site selected from the following group: human chromosome 11 114165695, human chromosome 11 114165730 , human chromosome 11 position 114165769, human chromosome 11 position 114165804, human chromosome 11 position 114165938, human chromosome 11 position 114166050, human chromosome 11 position 114166066, or a combination thereof.
  • the NNMT gene nucleotide site methylation level includes the 1161st, 1196th, 1235th, 1270th, 1270th, and 1270th positions of the nucleotide sequence of SEQ ID NO:1.
  • the NNMT gene nucleotide site methylation level includes the 1161st, 1196th, 1235th, 1270th, and 1270th positions in the nucleotide sequence of SEQ ID NO:1.
  • the nucleotide methylation level of one or more (such as 2, 3, 4, 5, 6 or 7) of positions 1404, 1516 and 1532.
  • the NNMT gene nucleotide site methylation level includes nucleotide methylation levels selected from the following group of SEQ ID NO: 1 sequence sites: 1161st, 1196th, 1235th, 1270th, 1404th, 1516th, 1532nd, or a combination thereof.
  • the high methylation level of the DNA CpG site in the NNMT gene region means that the methylation level of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) is greater than that of the same cell or a normal cell ( For example, the methylation level of DNA CpG sites in the NNMT gene region in paracancerous tissue cells).
  • the high methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) ⁇ 1%, preferably ⁇ 3%, preferably ⁇ 5%, preferably ⁇ 10%, preferably ⁇ 15%, preferably ⁇ 20%, more preferably ⁇ 25%, more preferably ⁇ 30%, more preferably ⁇ 40%, more preferably ⁇ 50%.
  • the high methylation level of the DNA CpG site in the NNMT gene region means that the methylation level A1 of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) is the same as that of the same cell or a normal cell.
  • the ratio (A1/A0) of the DNA CpG site methylation level A0 in the NNMT gene region (such as paracancerous tissue cells)>1.0, preferably ⁇ 1.2, preferably ⁇ 1.5, more preferably ⁇ 2, more preferably More preferably ⁇ 3, more preferably ⁇ 5, more preferably ⁇ 8, more preferably ⁇ 10, more preferably ⁇ 15, more preferably ⁇ 20, more preferably ⁇ 30, more preferably ⁇ 50, such as 2-50 .
  • the same cell refers to a cell whose DNA CpG site methylation level in the NNMT gene region is at a normal level or a low level (like a type of tumor cell).
  • the same cell refers to a cell of the same type but with a normal or low level of DNA CpG site methylation in the NNMT gene region.
  • the normal cells refer to normal tissue cells (such as tumor cell origin cells, tumor adjacent cells or paracancerous tissue cells) whose DNA CpG site methylation level in the NNMT gene region is at a normal level.
  • A0 is the methylation level of the DNA CpG site in the NNMT gene region of the cell whose methylation level is normal or low.
  • the cells whose DNA CpG site methylation level in the NNMT gene region is normal or low level include compounds of formula I, or their optical isomers or their racemates, or their Cells insensitive to solvates, or pharmaceutically acceptable salts thereof, or deuterated compounds thereof.
  • the high methylation level of the DNA CpG site in the NNMT gene region refers to the methylation level (M%) of the DNA CpG site in the NNMT gene region of a certain cell (such as a tumor cell) ⁇ 3% And less than or equal to M2%, wherein, M2 is any positive integer between 3-100.
  • M2 is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or 100.
  • the methylation level of CpG sites refers to the ratio of the number of methylated CpG nucleotides in a gene region to the number of all nucleotides in the gene region.
  • the DNA CpG site methylation level refers to the ratio of the number of methylated CpG nucleotides in a certain region of DNA to the number of all nucleotides in the DNA in this region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG nucleotides in the NNMT gene region to the number of all nucleotides in the NNMT gene region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG nucleotides in the DNA of the NNMT gene region to the number of all nucleotides in the DNA of the NNMT gene region .
  • the methylation level of CpG sites refers to the ratio of the number of methylated CpG nucleotides in a certain gene region to the number of all CpG nucleotides in the gene region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG nucleotides in the NNMT gene region to the number of all CpG nucleotides in the NNMT gene region.
  • the DNA CpG site methylation level refers to the ratio of the number of methylated CpG sites in DNA in a certain region to the total number of CpG sites in DNA in this region.
  • the DNA CpG site methylation level refers to the ratio of the number of methylated CpG nucleotides in DNA in a certain region to the total number of CpG nucleotides in DNA in this region.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the ratio of the number of methylated CpG sites in the NNMT gene region DNA to the total number of CpG sites in the NNMT gene region DNA.
  • the methylation level of DNA CpG sites in the NNMT gene region refers to the number of CpG nucleotides that have been methylated in the DNA in the NNMT gene region accounts for 1% of the total number of CpG nucleotides in the DNA in the NNMT gene region ratio.
  • the DNA CpG site methylation level in the NNMT gene region includes the DNA CpG site methylation level in the NNMT gene promoter region.
  • nucleotide sequence of the NNMT gene promoter region is shown in SEQ ID NO:1.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the region from 1050 bp before the transcription start site of the NNMT gene to 499 bp after the transcription start site.
  • the 1050 bp before the transcription start site of the NNMT gene to the 499 bp after the transcription start site is the 951-2500 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the region from 1050 bp before the transcription start site to 193 bp before the transcription start site of the NNMT gene.
  • the first 1050 bp to the first 193 bp of the transcription start site of the NNMT gene is the 951-1808 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of the DNA CpG site in the region from 840 bp before the transcription start site of the NNMT gene to 469 bp before the transcription start site.
  • the first 840 bp to the first 469 bp of the transcription start site of the NNMT gene is 1161-1532 of the nucleotide sequence shown in SEQ ID NO:1.
  • the methylation level of DNA CpG sites in the NNMT gene region includes any two positions in positions 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 on human chromosome 11 DNA CpG site methylation levels in the region between the points (including the two sites themselves).
  • the methylation level of DNA CpG sites in the NNMT gene region includes one or more of positions 114165695, 114165730, 114165769, 114165804, 114165938, 114166050 and 114166066 on human chromosome 11
  • the methylation level of (such as 2, 3, 4, 5, 6 or 7) sites.
  • the methylation level of the DNA CpG site in the NNMT gene region includes the methylation level of a site selected from the following group: human chromosome 11 114165695, human chromosome 11 114165730, human 11 Chromosome 114165769, human chromosome 11 114165804, human chromosome 11 114165938, human chromosome 11 114166050, human chromosome 11 114166066, or a combination thereof.
  • the DNA CpG site methylation level of the NNMT gene region includes the 1161st, 1196th, 1235th, 1270th, 1270th, and 1270th positions of the nucleotide sequence of SEQ ID NO:1.
  • the DNA CpG site methylation level of the NNMT gene region includes the 1161st, 1196th, 1235th, 1270th, 1270th, and 1270th positions of the nucleotide sequence of SEQ ID NO:1.
  • the DNA CpG site methylation level of the NNMT gene region includes the methylation level of the site of the SEQ ID NO: 1 nucleotide sequence selected from the following group: 1161st, 1196th bit, bit 1235, bit 1270, bit 1404, bit 1516, bit 1532, or a combination thereof.
  • the tumor is selected from the group consisting of lung cancer, kidney cancer, breast cancer, colon cancer, lymphoma, leukemia, pancreatic cancer, brain tumor, liver cancer, prostate cancer, or a combination thereof.
  • the lung cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, or a combination thereof.
  • the lung cancer cells include NCI-H82 cells.
  • the colon cancer includes colon adenocarcinoma.
  • the colon cancer cells include SW48 cells.
  • the breast cancer cells include MDA-MB-453 cells.
  • the breast cancer includes triple-negative breast cancer.
  • the lymphoma is selected from the group consisting of B lymphoma, cutaneous T cell lymphoma, or a combination thereof.
  • the lymphoma includes diffuse large B lymphoma.
  • the lymphoma cells include WSU-DLCL2 cells.
  • the brain tumor is selected from the group consisting of brain glioblastoma, neuroglioma, brain medulloblastoma, brain neuroblastoma, or a combination thereof.
  • the brain tumor includes glioblastoma.
  • the brain medulloblastoma includes cerebellar medulloblastoma.
  • the brain glioblastoma includes glioblastoma multiforme.
  • the brain tumor includes glioblastoma multiforme.
  • the brain tumor cells include one or more of GB-1 cells and SF126 cells.
  • the renal cancer is selected from the group consisting of renal clear cell adenocarcinoma, Wilms renal carcinoma, or a combination thereof.
  • the renal cancer includes renal clear cell adenocarcinoma.
  • the renal cancer includes Wilms renal carcinoma.
  • the cancer cells of kidney cancer include Wilms cells of kidney cancer.
  • the kidney cancer cells include one or more of G-401 cells and 786-O cells.
  • the cancer cells of pancreatic cancer include CFPAC-1 cells.
  • the leukemia is selected from the group consisting of T lymphocytic leukemia, myelogenous leukemia, or a combination thereof.
  • the T lymphocytic leukemia includes acute T lymphocytic leukemia.
  • the myeloid leukemia includes M4 grade AML acute myeloid leukemia.
  • the myeloid leukemia includes FAB M4 grade AML acute myeloid leukemia.
  • the expression includes protein expression and/or mRNA expression.
  • composition or preparation is a pharmaceutical composition or medicine or preparation.
  • composition or preparation further includes a pharmaceutically acceptable carrier.
  • the dosage form of the composition or preparation is solid preparation, liquid preparation or semi-solid preparation.
  • the dosage form of the composition or preparation is oral preparation, external preparation or injection preparation.
  • the dosage form of the composition or preparation is tablet, injection, transfusion, ointment, gel, solution, microsphere or film.
  • the second aspect of the present invention provides a method for judging whether a tumor patient is suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutical preparation.
  • Markers for preventing and/or treating tumors using acceptable salts or deuterated compounds thereof including NNMT gene, DNA methylase, UHRF1, NNMT gene nucleotide site methylation, And/or DNA CpG site methylation in NNMT gene region.
  • the markers include NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG Site methylation level.
  • NNMT gene, DNA methylase, UHRF1, NNMT gene nucleotide site methylation, and/or NNMT gene region DNA CpG site methylation include NNMT gene, DNA of tumor cells Methylase, UHRF1, methylation of nucleotide sites in NNMT genes, and/or methylation of DNA CpG sites in NNMT gene regions.
  • the tumor patient when the tumor cells of the tumor patient have low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or If the methylation level of the DNA CpG site in the NNMT gene region is high, the tumor patient is suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or Its pharmaceutically acceptable salt, or its deuterated compound for prevention and/or treatment.
  • the tumor cells of the tumor patient have high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of NNMT gene nucleotide site, and/or NNMT gene region If the methylation level of the DNA CpG site is low, the tumor patient is not suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutical Acceptable salts or deuterated compounds thereof for prevention and/or treatment.
  • the tumor patient is suitable for taking the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound, which includes the tumor of a tumor patient to the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutical above acceptable salts, or deuterated compounds thereof.
  • the tumor patient is not suitable for taking the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable Acceptable salts or deuterated compounds thereof, which include tumors of tumor patients to the compound of formula I as described in the first aspect of the present invention, or optical isomers or racemates thereof, or solvates thereof, or Pharmaceutically acceptable salts, or deuterated compounds thereof, are insensitive.
  • the tumor with low or no expression of NNMT gene is as described in the first aspect of the present invention.
  • the DNA methylase is selected from the group consisting of DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the tumor with high expression of DNA methylase (such as DNMT1, DNMT3a and/or DNMT3b) is as described in the first aspect of the present invention.
  • the tumor with high UHRF1 expression is as described in the first aspect of the present invention.
  • the tumor with high methylation level at the nucleotide site of the NNMT gene is as described in the first aspect of the present invention.
  • the tumor with high methylation level of DNA CpG site in the NNMT gene region is as described in the first aspect of the present invention.
  • a detection kit in the third aspect of the present invention, includes:
  • the detection sample of the detection kit includes tumor cells.
  • the expression level of NNMT gene refers to the expression level of mRNA and/or protein of the gene.
  • the fourth aspect of the present invention provides a use of the detection kit according to the third aspect of the invention, which is used to prepare a companion diagnostic kit, and the companion diagnostic kit is used to determine whether a tumor patient is suitable for the first test of the present invention.
  • the accompanying diagnostic kit further includes instructions or labels.
  • the instructions or labels describe:
  • the tumor patient is suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt , or its deuterated compounds for prevention and/or treatment.
  • the instructions or labels describe:
  • the tumor patient When the tumor cells of cancer patients have high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of NNMT gene nucleotide site, and/or methylation of DNA CpG site in NNMT gene region If the level is low, the tumor patient is not suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or Its deuterated compounds are used for prevention and/or treatment.
  • the tumor patient is suitable for taking the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable or its deuterated compound as described in the second aspect of the present invention.
  • the tumor patient is not suitable for taking the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable Accepted salts, or deuterated compounds thereof, are as described in the second aspect of the present invention.
  • a kit in the fifth aspect of the present invention, includes:
  • the detected samples include tumors.
  • kit further includes instructions or labels.
  • the instructions or labels describe:
  • the tumor patient is suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compounds for prevention and/or treatment.
  • the tumor cells of the tumor patient have high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of NNMT gene nucleotide site, and/or NNMT gene region If the methylation level of the DNACpG site is low, the tumor patient is not suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutical Acceptable salts, or deuterated compounds thereof for prevention and/or treatment.
  • the sixth aspect of the present invention provides a method for preventing and/or treating tumors, administering the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or Its solvate, or its pharmaceutically acceptable salt, or its deuterated compound, so as to prevent and/or treat tumor.
  • the tumor is as described in the first aspect of the present invention.
  • the subject is human and non-human mammals (rodents, rabbits, monkeys, domestic animals, dogs, cats, etc.).
  • the method includes the steps of:
  • the target tumor has low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or methylation of DNA CpG site in NNMT gene region and then give the compound of formula I, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound to prevent and / or treatment.
  • the method includes the steps of:
  • the subject is first administered with NNMT gene inhibitors, DNA methylase promoters, UHRF1 promoters, NNMT gene nucleotide site methylation promoters, and/or NNMT gene region DNA CpG site methylation promoters,
  • the target tumor has low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or methylation of DNA CpG site in NNMT gene region level is high, then give the compound of formula I, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound for prevention and/or or treatment.
  • the NNMT gene inhibitor includes an inhibitor capable of reducing or not expressing the NNMT gene of a tumor.
  • the DNA methylase promoter includes a promoter capable of high expression of tumor DNA methylase.
  • the DNA methylase is selected from the group consisting of DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the DNA methylase promoter includes a DNMT1 promoter.
  • the DNA methylase promoter includes a DNMT3a promoter.
  • the DNA methylase promoter includes a DNMT3b promoter.
  • the DNMT1 promoter includes a promoter capable of increasing the expression of DNMT1 in tumors.
  • the DNMT3a promoter includes a promoter capable of increasing the expression of DNMT3a in tumors.
  • the DNMT3b promoter includes a promoter capable of high expression of DNMT3b in tumors.
  • the UHRF1 promoter includes a promoter that can increase the expression of UHRF1 in tumors.
  • the NNMT gene nucleotide site methylation promoter includes a promoter capable of increasing the methylation level of the tumor NNMT gene nucleotide site.
  • the methylation promoter of the DNA CpG site in the NNMT gene region includes a promoter capable of increasing the methylation level of the DNA CpG site in the NNMT gene region of the tumor.
  • the inhibitors include specific inhibitors.
  • the accelerator includes a specific accelerator.
  • the administration is oral administration, injection administration or external administration.
  • the injection administration is intramuscular injection or intravenous injection.
  • a device or system in a seventh aspect of the present invention, includes:
  • detection module the detection module is used to detect NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level;
  • output module described output module includes output following information:
  • the tumor patient is suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt , or its deuterated compounds for prevention and/or treatment; and/or
  • the tumor patient When the tumor cells of cancer patients have high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of NNMT gene nucleotide site, and/or methylation of DNA CpG site in NNMT gene region If the level is low, the tumor patient is not suitable for taking the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or Its deuterated compounds are used for prevention and/or treatment.
  • the detected samples include tumors.
  • the device includes a gene detector or a protein detector.
  • the device or system further includes a sampling module.
  • the sampling module is used for injecting tumor cell extracts.
  • the device or system further includes a data processing module.
  • the data processing module processes the NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene The level of methylation of DNA CpG sites in different regions.
  • the eighth aspect of the present invention provides a NNMT gene inhibitor, DNA methylase promoter, UHRF1 promoter, NNMT gene nucleotide site methylation promoter, and/or DNA CpG site A in the NNMT gene region
  • the use of the methylation accelerator is used to prepare a composition or a preparation, and the composition or preparation is used to enhance the antitumor effect of an antitumor drug.
  • the NNMT gene inhibitors include inhibitors capable of causing low or no expression of tumor NNMT genes.
  • the DNA methylase promoter includes a promoter capable of high expression of tumor DNA methylase.
  • the DNA methylase is selected from the group consisting of DNMT1, DNMT3a, DNMT3b, or a combination thereof.
  • the DNA methylase promoter includes a DNMT1 promoter.
  • the DNA methylase promoter includes a DNMT3a promoter.
  • the DNA methylase promoter includes a DNMT3b promoter.
  • the DNMT1 promoter includes a promoter capable of increasing the expression of DNMT1 in tumors.
  • the DNMT3a promoter includes a promoter capable of increasing the expression of DNMT3a in tumors.
  • the DNMT3b promoter includes a promoter capable of increasing the expression of DNMT3b in tumors.
  • the UHRF1 promoter includes a promoter that can increase the expression of UHRF1 in tumors.
  • the NNMT gene nucleotide site methylation promoter includes a promoter capable of increasing the methylation level of the tumor NNMT gene nucleotide site.
  • the methylation promoter of the DNA CpG site in the NNMT gene region includes a promoter capable of increasing the methylation level of the DNA CpG site in the NNMT gene region of the tumor.
  • the inhibitors include specific inhibitors.
  • the accelerator includes a specific accelerator.
  • the antitumor drug includes the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable accepted salts, or deuterated compounds thereof.
  • the tumor is as described in the first aspect of the present invention.
  • the dosage form of the composition or preparation is solid preparation, liquid preparation or semi-solid preparation.
  • the dosage form of the composition or preparation is oral preparation, external preparation or injection preparation
  • the dosage form of the composition or preparation is tablet, injection, transfusion, ointment, gel, solution, microsphere or film.
  • an active ingredient combination is provided, and the active ingredient combination includes the following components:
  • the second active ingredient includes NNMT gene inhibitors, DNA methylase promoters, UHRF1 promoters, NNMT gene nucleotide site methylation promoters, and/or NNMT Accelerator for DNA CpG site methylation in gene region.
  • the antitumor drug includes the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable accepted salts, or deuterated compounds thereof.
  • the NNMT gene inhibitor DNA methylase promoter, UHRF1 promoter, NNMT gene nucleotide site methylation promoter, and/or DNA CpG site A in the NNMT gene region
  • the radicalization accelerator is as described in the eighth aspect of the present invention.
  • the molar ratio of the first active ingredient to the second active ingredient is 0.01-600:1, preferably 0.05-500:1, more preferably 0.1-400:1, and more preferably Preferably 0.2-200:1, more preferably 0.5-100:1, more preferably 0.5-80:1, most preferably 1-50:1.
  • At least one active ingredient is independent.
  • the first active ingredient and the second active ingredient are independent of each other.
  • the present invention provides a composition comprising:
  • the second active ingredient includes NNMT gene inhibitors, DNA methylase promoters, UHRF1 promoters, NNMT gene nucleotide site methylation promoters, and/or NNMT Accelerator for DNA CpG site methylation in gene region.
  • the antitumor drug includes the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable accepted salts, or deuterated compounds thereof.
  • the NNMT gene inhibitor DNA methylase promoter, UHRF1 promoter, NNMT gene nucleotide site methylation promoter, and/or DNA CpG site A in the NNMT gene region
  • the radicalization accelerator is as described in the eighth aspect of the present invention.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • the content of the first active ingredient is 0.01-99.99wt%, preferably 0.1-99.9wt%, more preferably 1-99wt%, more preferably 10-99wt%, most preferably 20-99% by weight, based on the total weight of the active ingredients of the composition.
  • the content of the second active ingredient is 0.01-99.99wt%, preferably 0.1-99.9wt%, more preferably 1-99wt%, more preferably 10-99wt%, most preferably 20-99wt%, based on the total weight of the active ingredients of the composition.
  • a kit which includes:
  • the second preparation containing the second active ingredient, the second active ingredient includes NNMT gene inhibitor, DNA methylase promoter, UHRF1 promoter, NNMT gene nucleotide site methylation promoter , and/or DNA CpG site methylation accelerator in the NNMT gene region.
  • the antitumor drug includes the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable accepted salts, or deuterated compounds thereof.
  • the NNMT gene inhibitor DNA methylase promoter, UHRF1 promoter, NNMT gene nucleotide site methylation promoter, and/or DNA CpG site A in the NNMT gene region
  • the radicalization accelerator is as described in the eighth aspect of the present invention.
  • the kit also includes instructions for use.
  • the first preparation and the second preparation are independent preparations.
  • the first preparation and the second preparation are combined preparations.
  • the instructions for use indicate that the first preparation and the second preparation are used in combination, so as to enhance the anti-tumor activity of the anti-tumor drug.
  • the combined method is to administer the second preparation containing the second active ingredient first, and then administer the first preparation containing the first active ingredient.
  • NNMT gene inhibitors DNA methylase promoters, UHRF1 promoters, NNMT gene nucleotide site methylation promoters, and/or NNMT gene region DNA CpG site methylation promoters make the NNMT of tumor cells Low or no gene expression, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or high methylation level of DNA CpG site in NNMT gene region, such as
  • the compound of formula I described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound can enhance the inhibition of tumor cells.
  • the twelfth aspect of the present invention provides a combination of active ingredients as described in the ninth aspect of the present invention, the composition as described in the tenth aspect of the present invention, and/or the kit as described in the eleventh aspect of the present invention The purposes for preparing antineoplastic drugs.
  • the medicines are packaged in medicine boxes.
  • the medicine box also includes instructions for use, and the instructions for use record:
  • NNMT gene inhibitors DNA methylase promoters, UHRF1 promoters, NNMT gene nucleotide site methylation promoters, and/or NNMT gene region DNA CpG site methylation promoters, so that the tumor Low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or high methylation level of DNA CpG site in NNMT gene region
  • the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound has the effect on tumor cells Inhibition enhancement.
  • a method for inhibiting tumor cells in vitro comprising the steps of: combining tumor cells with the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its foreign
  • the racemate, or its solvate, or its pharmaceutically acceptable salt, or its deuterated compound is contacted, thereby inhibiting tumor cells.
  • the method is non-therapeutic and non-diagnostic.
  • the contact is in vitro culture contact.
  • the tumor is as described in the first aspect of the present invention.
  • the method includes the steps of:
  • the tumor cells have low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide sites, and/or DNA CpG site methylation in NNMT gene region
  • the level is high, and then the tumor cells are mixed with the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or Its deuterated compounds are contacted, thereby inhibiting tumor cells.
  • the method includes the steps of:
  • Tumor cells are first administered with NNMT gene inhibitors, DNA methylase promoters, UHRF1 promoters, NNMT gene nucleotide site methylation promoters, and/or NNMT gene region DNA CpG site methylation promoters , resulting in low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide sites, and/or DNA CpG site methylation in NNMT gene region of tumor cells The level is high, and then the tumor cells are mixed with the compound of formula I as described in the first aspect of the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or Its deuterated compounds are contacted, thereby inhibiting tumor cells.
  • Figure 1 shows the expression of NNMT gene in niclosamide-sensitive and insensitive tumor cells.
  • Figure 2 shows the methylation levels of DNA CpG sites in the promoter region of the NNMT gene in niclosamide-sensitive and insensitive tumor cells.
  • Figure 3 shows the methylation level of the DNA CpG site between the 1050 bp before the transcription start site and the 499 bp after the transcription start site of the niclosamide-sensitive and insensitive tumor cells NNMT gene.
  • Figure 4 shows the methylation level of the DNA CpG site between the first 1050 bp of the transcription start site and the first 193 bp of the transcription start site of the niclosamide-sensitive and insensitive tumor cells.
  • Figure 5 shows the methylation of DNA CpG sites in the specific NNMT gene regions of niclosamide-sensitive and insensitive tumor cells, namely 7 sites of human chromosome 11: 114165695, 114165730, 114165769, 114165804, 114165938, 114166050, and 114166066 , Black dots indicate that the relevant sites are methylated, white dots indicate that the relevant sites are not methylated, SST refers to the transcription start site, Chr11 refers to human No. 11 defined according to the GCF_000001405.25 (GRCh37.p13) human genome version chromosome.
  • Figure 6 shows the correlation between the expression of NNMT and the expression of DNMT1, UHRF1, DNMT3a and DNMT3b in tumor cells.
  • Fig. 7 is Western Blot detection control NCI-H82 cell (NCI-H82 (Con)) and the NCI-H 82 cell (NCI-H82 (ov-NNMT)) of overexpressing NNMT protein NNMT protein expression level, wherein, NCI- H82(Con) is the NNMT protein expression level of the control NCI-H82 cells obtained by transfecting NCI-H82 cells with empty virus that does not carry the NNMT gene, and NCI-H82(ov-NNMT) is the NNMT gene introduced into the NCI by the virus vector - NNMT protein expression level of NCI-H82 cells overexpressing NNMT protein obtained from H82 cells.
  • NCI- H82(Con) is the NNMT protein expression level of the control NCI-H82 cells obtained by transfecting NCI-H82 cells with empty virus that does not carry the NNMT gene
  • NCI-H82(ov-NNMT) is the NNMT gene introduced into the NCI by the virus vector - NNMT
  • the inventors unexpectedly discovered for the first time that the compound of the present invention has low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, and high methylation level of NNMT gene nucleotide sites , and/or tumor cells with high methylation levels of DNA CpG sites in the NNMT gene region have a more significant inhibitory effect.
  • NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level can be used to judge whether tumor patients are suitable Markers for prophylaxis and/or therapy with the compounds of the invention. On this basis, the inventors have completed the present invention.
  • the terms “comprising”, “including”, and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • low level of DNA CpG site methylation As used herein, the terms "low level of DNA CpG site methylation”, “low level of DNA CpG site methylation” and “DNA CpG site hypomethylation” are used interchangeably.
  • IC50 and “IC 50 ” are used interchangeably and refer to the half-inhibiting concentration (50% inhibiting concentration), ie the concentration of the inhibitor at which 50% inhibitory effect is achieved.
  • CpG site methylation As used herein, the terms "CpG site methylation”, “CpG nucleotide methylation” and “CpG methylation” are used interchangeably.
  • P/S refers to the addition of Penicillin (Penicillin) and Streptomycin (Streptomycin) in the relevant medium.
  • a certain cell refers to a certain cell (such as a single cancer cell) or a group of cells including a plurality of similar cells (such as a certain tumor tissue).
  • a tumor patient suitable for taking the compound of the present invention includes a tumor patient whose tumor is sensitive to the compound of the present invention.
  • tumor patients not suitable for the compounds of the present invention includes tumor patients whose tumors are not sensitive to the compounds of the present invention.
  • NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level is Refers to one or more of NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level and NNMT gene region DNA CpG site methylation level.
  • “High level of methylation” refers to low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide sites and methylation level of DNA CpG site in NNMT gene region One or more of high school.
  • high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of nucleotide site of NNMT gene, and/or low methylation level of DNA CpG site of NNMT gene region refers to one or more of high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of NNMT gene nucleotide site and low methylation level of DNA CpG site in NNMT gene region Various.
  • NMT Nicotinamide N-Methyltransferase
  • base pair refers to base pair, base pair.
  • SST transcription start site
  • Chr11 refers to human chromosome 11 as defined by the GCF_000001405.25 (GRCh37.p13) human genome version.
  • human chromosome 11 refers to human chromosome 11 as defined by the GCF_000001405.25 (GRCh37.p13) human genome version
  • the terms "before the transcription start site”, “after the transcription start site”, “before the transcription start site”, “after the transcription start site” do not include the transcription start site itself.
  • human chromosome 11 position 114165695" refers to the nucleotide at position 114165695 of human chromosome 11; "human chromosome 11 position 114165730” refers to the nucleotide at position 114165730 of human chromosome 11; “human chromosome 11 Chromosome 114165769” refers to the nucleotide at 114165769 of human chromosome 11; “human chromosome 11 114165804" refers to the nucleotide at 114165804 of human chromosome 11; The nucleotide at position 114165938 of chromosome 11; "114166050 of human chromosome 11” refers to the nucleotide of 114166050 of human chromosome 11; “114166066 of human chromosome 11” refers to the nucleotide of 114166066 of human chromosome 11 acid.
  • DNMT3a refers to DNA methyltransferase 3a (DNA methyltransferase 3a), and “DNMT3A” can be used interchangeably.
  • DNMT3b refers to DNA methyltransferase 3b (DNA methyltransferase 3b), which can be used interchangeably with “DNMT3B.”
  • DNMT1 refers to DNA methyltransferase 1 (DNA methyltransferase 1).
  • UHRF1 refers to ubiquitin-like PHD and RING domain-containing protein 1.
  • CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G).
  • SF126 cells As used herein, the terms “SF126 cells” and “SF-126 cells” are used interchangeably.
  • deuterated refers to the replacement of one or more hydrogens in a compound or group with deuterium.
  • Deuterium can be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • solvate refers to a complex in which a compound coordinates with solvent molecules to form specific ratios.
  • gene expression includes the gene protein expression or the gene mRNA expression and the like.
  • substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to produce chemically stable compounds, which can be synthesized by techniques known in the art and methods set forth below. If substituted with more than one substituent group(s), it is understood that the multiple groups may be on the same carbon or on different carbons so long as a stable structure results.
  • substituted or “substituted” means that a hydrogen atom on a group is replaced by a non-hydrogen atom group, but it needs to meet its valence requirements and the substitution results in a chemically stable compound, that is, it will not spontaneously carry out such as ring Compounds that undergo transformations such as chemicalization and elimination.
  • R 1 As used herein, “R 1 ", “R1” and “R 1” have the same meaning and can be replaced with each other, and other similar definitions have the same meaning.
  • alkyl refers to a straight chain (ie, unbranched) or branched chain saturated hydrocarbon group containing only carbon atoms, or a combination of straight chain and branched chain groups.
  • alkyl group is limited by the number of carbon atoms (such as a C1-C6 alkyl group), it means that the said alkyl group contains 1-6 carbon atoms, for example, a C1-C4 alkyl group refers to an alkyl group containing 1-4 carbon atoms, Representative examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, or the like.
  • halogen refers to F, Cl, Br or I.
  • halo refers to substitution by halogen.
  • haloalkyl means that one or more (preferably 1, 2, 3 or 4) hydrogens of an alkyl group are replaced by a halogen, said alkyl and halogen being as defined above, when the alkyl
  • a limited number of carbon atoms such as C1-C8 haloalkyl refers to the alkyl group containing 1-8 carbon atoms, for example, C1-C6 haloalkyl refers to a haloalkyl group containing 1-6 carbon atoms
  • representative examples include But not limited to -CF 3 , -CHF 2 , monofluoroisopropyl, difluorobutyl, or similar groups.
  • cycloalkyl refers to a monocyclic, bicyclic or polycyclic (fused, bridged or spiro) ring system group having a saturated or partially saturated ring.
  • a certain cycloalkyl group is limited by the number of carbon atoms (such as C3-C12), it means that the cycloalkyl group has 3-12 ring carbon atoms.
  • C3-C8 cycloalkyl refers to a saturated or partially saturated monocyclic or bicyclic alkyl group with 3-8 ring carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl group, cycloheptyl group, or similar groups.
  • Spirocycloalkyl means a bicyclic or polycyclic group in which the single rings share a single carbon atom (called a spiro atom), these may contain one or more double bonds, but none of the rings has fully conjugated pi electrons system.
  • “Fused cycloalkyl” means an all-carbon bicyclic or polycyclic group in which each ring of the system shares an adjacent pair of carbon atoms with other rings in the system, one or more of which may contain one or more bicyclic bonds, but none of the rings have a fully conjugated ⁇ -electron system.
  • “Bridged cycloalkyl” refers to an all-carbon polycyclic group in which any two rings share two carbon atoms not directly attached, these may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system .
  • halocycloalkyl means that one or more (preferably 1, 2, 3 or 4) hydrogens of a cycloalkyl group are replaced by halogen, said cycloalkyl group and halogen being as defined above,
  • the cycloalkyl group is limited by the number of carbon atoms (such as C3-C8 halocycloalkyl)
  • the cycloalkyl group contains 3-8 ring carbon atoms
  • C3-C8 halocycloalkyl means that it contains 3 -halocycloalkyl of 8 carbon atoms
  • representative examples include but are not limited to monofluorocyclopropyl, monochlorocyclobutyl, monofluorocyclopentyl, difluorocycloheptyl, or similar groups .
  • alkoxy refers to an R-O- group, wherein R is an alkyl group, and the alkyl group is as defined herein above, and the number of carbon atoms before the alkoxy group is limited, such as C1-C8 alkoxy group refers to the alkoxy group
  • the alkyl group in the group has 1-8 carbon atoms.
  • Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, or the like.
  • alkylthio refers to an R-S- group, wherein R is an alkyl group, and the alkyl group is as defined herein above, when the alkylthio group is preceded by a limit of carbon atoms, such as the C1-C8 alkylthio group refers to The alkyl group in the above-mentioned alkylthio group has 1-8 carbon atoms.
  • Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, t-butylthio, or the like.
  • cycloalkoxy refers to the R-O- group, wherein R is cycloalkyl, and cycloalkyl is as defined herein above, when the cycloalkoxy group is limited by the number of carbon atoms, such as C3-C8 cycloalkoxy refers to The cycloalkyl group in the cycloalkoxy group has 3-8 carbon atoms.
  • Representative examples of cycloalkoxy include, but are not limited to, cyclopropoxy, cyclobutoxy, or the like.
  • cycloalkylthio refers to an R-S-group, wherein R is a cycloalkyl group, and the cycloalkyl group is as defined herein above, when the cycloalkylthio group has a limit on the number of carbon atoms, such as C3-C8 cycloalkylthio group refers to The cycloalkyl group in the cycloalkylthio group has 3-8 carbon atoms.
  • Representative examples of cycloalkylthio include, but are not limited to, cyclopropylthio, cyclobutylthio, or the like.
  • amino by itself or as part of another substituent is -NH2 .
  • nitro alone or as part of another substituent, is -NO2 .
  • hydroxy is -OH, alone or as part of another substituent.
  • any of the "substitution” refers to one or more (preferably 1 , 2, 3, or 4) hydrogen atoms are replaced by substituents selected from the group: C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, C3-C8 halocycloalkyl, Halogen, nitro, -CN, cyano, hydroxyl, mercapto, amino, C1-C8 alkoxy, C1-C8 alkylthio, C3-C8 cycloalkoxy, C3-C8 cycloalkylthio.
  • an optionally substituted group may have a substituent selected from a specific group at any substitutable position of the group, and the substituents may be the same or different at each position.
  • prevention means a method of preventing the onset of a disease and/or its attendant symptoms or protecting a subject from acquiring a disease.
  • Treatment in the present invention includes delaying and terminating the progression of the disease, or eliminating the disease, and does not require 100% inhibition, eradication and reversal.
  • compounds of the invention reduce, inhibit and/or reverse associated diseases (such as tumors) and their complications, for example, by at least about 10% of the level observed in the absence of compounds of the invention. %, at least about 30%, at least about 50%, or at least about 80%.
  • the compound of formula I is as described above in the first aspect of the present invention.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention with an acid or a base which is suitable for use as a medicine.
  • Pharmaceutically acceptable salts include inorganic salts and organic salts.
  • One class of preferred salts is the salt formed by the compound of the present invention and an acid.
  • Suitable acids for forming the salt include (but are not limited to): inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid , propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzene methanesulfonic acid, benzenesulfonic acid and other organic acids; And acidic amino acids such as aspartic acid and glutamic acid.
  • a preferred class of salts are metal salts formed from compounds of the present invention and bases.
  • Suitable bases for salt formation include (but are not limited to): sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate and other inorganic bases, Ammonia, triethylamine, diethylamine and other organic bases.
  • the compound shown in formula I of the present invention can be converted into its pharmaceutically acceptable salt by conventional methods, for example, the corresponding acid solution can be added to the solution of the above compound, and the solvent can be removed after the salt formation is complete.
  • the corresponding salts of the compounds of the present invention can be converted into its pharmaceutically acceptable salt by conventional methods, for example, the corresponding acid solution can be added to the solution of the above compound, and the solvent can be removed after the salt formation is complete.
  • Preferred compounds of the present invention are:
  • the English name of the NNMT gene is Nicotinamide N-Methyltransferase, and different databases have different identification numbers for the gene: HGNC: 7861; Entrez Gene: 4837; Ensembl: ENSG00000166741; OMIM: 600008; UniProtKB: P40261.
  • the NNMT gene region is located on human chromosome 11 from bp 114,128,528 to 114,184,258 bp, with a total length of 55,731 bp of DNA sequence, including the NNMT gene promoter region and NNMT gene exons region and the intron region of the NNMT gene, and the transcription start site of the NNMT gene is the 114th, 166,535th bp.
  • the promoter region of the NNMT gene is the nucleotide sequence from bp 114,164,535 to bp 114,167,034 of human chromosome 11, that is, the 2000 bp before the transcription start site of the NNMT gene (the part that is darkened without underline) to the transcription start site itself and beyond
  • the sequence between 499bp (the part that is not darkened and underlined), the region with a total length of 2500bp is the NNMT gene promoter region, and the nucleotide sequence of the NNMT gene promoter region is shown in the following SEQ ID NO:1:
  • the 1050 bp before the NNMT gene transcription start site to the 499 bp after the gene transcription start site is the 951-2500 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the first 1050bp to the first 193bp of the gene transcription initiation site of the NNMT gene is the 951-1808 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the first 840 bp to the first 469 bp of the gene transcription start site of the NNMT gene transcription start site are 1161-1532 positions of the nucleotide sequence shown in SEQ ID NO:1.
  • the sites of human chromosome 11 114165695, 114165730, 114165769, 114165804, 114165938, 114166050, 114166066 correspond to the sites of SEQ ID NO: 1 nucleotide sequence as shown in Table 1 below:
  • DNA methylation is a form of chemical modification of DNA that can alter genetic expression without changing the DNA sequence.
  • a large number of studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the interaction between DNA and proteins, thereby regulating gene expression.
  • DNA methylation is one of the earliest discovered and most deeply studied epigenetic regulatory mechanisms.
  • DNA methylation in a broad sense refers to the conversion of specific bases on the DNA sequence to S-adenosylmethionine (SAM) under the catalysis of DNA methyltransferase (DNMT).
  • SAM S-adenosylmethionine
  • DNMT DNA methyltransferase
  • SAM S-adenosylmethionine
  • DNMT DNA methyltransferase
  • This DNA methylation modification can occur at the C-5 position of cytosine, the N-6 position of adenine and the N-7 position of guanine.
  • DNA methylation involved in the research mainly refers to the methylation process of the carbon atom at the 5th position of cytosine in the CpG dinucleotide, and its product is called 5-methylcytosine (5-mC) , is the main form of DNA methylation in eukaryotes such as plants and animals.
  • 5-methylcytosine 5-methylcytosine
  • DNA methylation can be inherited to newborn offspring DNA during the DNA replication process under the action of DNA methyltransferase, which is an important epigenetic mechanism.
  • DNA methylation reactions There are two types of DNA methylation reactions. One is that DNA that is not methylated on both strands is methylated, which is called denovo methylation; the other is that one of the strands of double-stranded DNA is methylated, and the other is not methylated.
  • the methylated chain is methylated, this type is called maintenance methylation.
  • DNA methylation is DNA CpG site methylation.
  • the distribution of CpG dinucleotides in the human genome is very uneven, and in some segments of the genome, CpG remains at or above the normal probability.
  • CpG site-rich regions also known as CpG islands
  • CpG island are mainly located in the promoter and exon regions of genes, which are some regions rich in CpG dinucleotides, and more than 60% of gene promoters contain CpG island.
  • CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G).
  • Intracellular gene expression is regulated by multiple signaling pathways, transcription factors, and epigenetic modifications.
  • DNA methylation modification is an important way for epigenetic modification to regulate gene expression, and the level of DNA methylation in a specific gene region often affects the expression level of the gene.
  • DNA methylation in epigenetic modifications has a more stable effect on gene expression and is not easily affected by the extracellular environment.
  • DNA methylation is easy to use It is an ideal biomarker because of its accurate detection by existing technologies.
  • the research of the present invention shows that the compound of the present invention can be used to prevent and/or treat tumors.
  • said tumors include tumors with low or no expression of NNMT gene.
  • the tumors with low or no expression of NNMT gene in the present invention are as described above in the first aspect of the present invention.
  • the tumor described in the present invention includes a tumor with high expression of DNA methylase.
  • the tumor with high expression of DNA methylase in the present invention is as described above in the first aspect of the present invention.
  • the DNA methylases described in the present invention include (but not limited to) DNMT1, DNMT3a, DNMT3b, or combinations thereof.
  • the DNA methylase described in the present invention includes DNMT1.
  • the tumors described in the present invention include tumors with high expression of DNMT1.
  • the tumor with high expression of DNMT1 in the present invention is as described above in the first aspect of the present invention.
  • the tumors described in the present invention include tumors with high expression of DNMT3a.
  • the tumors with high expression of DNMT3a in the present invention are as described above in the first aspect of the present invention.
  • the tumors described in the present invention include tumors with high expression of DNMT3b.
  • the tumor with high expression of DNMT3b in the present invention is as described above in the first aspect of the present invention.
  • the tumors described in the present invention include tumors with high expression of UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1).
  • UHRF1 ubiquitin-like PHD and RING finger domain-containing protein 1
  • the tumor with high UHRF1 expression in the present invention is as described above in the first aspect of the present invention.
  • the tumors described in the present invention include tumors with high levels of methylation at nucleotide sites of the NNMT gene.
  • the tumors with high methylation level of NNMT gene nucleotide sites in the present invention are as described above in the first aspect of the present invention.
  • the tumor includes a tumor with high methylation level of DNA CpG site in the NNMT gene region.
  • the tumors with high DNA CpG site methylation levels in the NNMT gene region described in the present invention are as described above in the first aspect of the present invention.
  • the tumor described in the present invention is as described above in the first aspect of the present invention.
  • the tumor types corresponding to each representative tumor cell line are shown in Table 2 below:
  • tumor cell line Corresponding tumor types NCI-H82 human small cell lung cancer cells G-401 Human Renal Carcinoma Wilms Cells MDA-MB-453 breast cancer cells SW48 human colon adenocarcinoma cells WSU-DLCL2 Human Diffuse Large B Lymphoma Cells GB-1 human glioblastoma cells CFPAC-1 human pancreatic cancer cells SF126 human glioblastoma multiforme cells 786-O Renal clear cell adenocarcinoma
  • the antitumor drug can be the compound of formula I described in the present invention, or its optical isomer or its racemate, or its solvate, or its pharmaceutically acceptable salt, or Its deuterated compounds.
  • the present invention provides a use of the compound of the present invention in the prevention and/or treatment of tumors.
  • the compounds of the present invention have low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or DNA CpG in NNMT gene region Tumors with high site methylation levels have significantly excellent preventive and therapeutic effects, that is, low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, and methylation level of nucleotide sites of NNMT gene Tumors with high and/or high methylation levels of DNA CpG sites in the NNMT gene region are sensitive to the compounds of the present invention.
  • the compounds described in the present invention can be used for low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or NNMT gene region Tumors with high levels of methylation at DNA CpG sites are treated with precision therapy.
  • the present invention also provides a method for preventing and/or treating tumors by administering the compound of the present invention to a subject in need.
  • the compounds of the present invention have low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or DNA CpG site A in NNMT gene region Tumors with high methylation levels have significantly excellent preventive and therapeutic effects.
  • NNMT gene inhibitors, DNA methylase promoters, UHRF1 promoters, and NNMT gene inhibitors can be administered to subjects first.
  • Nucleotide site methylation promoters, and/or DNA CpG site methylation promoters in the NNMT gene region make the NNMT gene expression low or no expression, DNA methylase high expression, UHRF1 high expression in the subject's tumor Expression, high methylation level of NNMT gene nucleotide sites, and/or high methylation level of DNA CpG sites in NNMT gene region, and then administer the compound of the present invention to prevent and/or treat tumors.
  • the subject is human and non-human mammals (rodents, rabbits, monkeys, domestic animals, dogs, cats, etc.).
  • the low or no expression of the NNMT gene of the tumor the high expression of DNA methylase, the high expression of UHRF1, the high methylation level of the nucleotide site of the NNMT gene, and/or the DNA CpG position of the NNMT gene region
  • the method of high methylation level for example, through methods such as gene insertion, gene knockout or gene silencing (such as transfection of shRNA) to specifically make the NNMT gene of the tumor low or unexpressed, DNA methylation, etc.
  • the present invention also provides a marker for judging whether a tumor patient is suitable for prevention and/or treatment with the compound of the present invention, said marker includes NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level.
  • NNMT gene expression level, DNA methylase expression level, UHRF1 expression level, NNMT gene nucleotide site methylation level, and/or NNMT gene region DNA CpG site methylation level are used as The markers for judging whether a tumor patient is suitable for prevention and/or treatment with the compound of the present invention.
  • the tumor patient is suitable for prevention and/or treatment with the compound of the present invention.
  • the tumor cells of cancer patients have high expression of NNMT gene, low expression of DNA methylase, low expression of UHRF1, low methylation level of NNMT gene nucleotide site, and/or methylation of DNA CpG site in NNMT gene region If the level is low, the tumor patient is not suitable for prevention and/or treatment with the compound of the present invention.
  • the tumor patient is suitable for taking the compound of the present invention, which includes the tumor patient whose tumor is sensitive to the compound of the present invention.
  • the tumor patient is not suitable for the compound of the present invention, which includes the tumor patient whose tumor is not sensitive to the compound of the present invention.
  • tumors with low or no expression of NNMT gene tumors with high expression of DNA methylase (such as DNMT1), tumors with high expression of UHRF1, and tumors with high methylation level of NNMT gene nucleotide sites in the present invention
  • Tumors and/or tumors with high DNA CpG site methylation levels in the NNMT gene region are as described above in the first aspect of the present invention.
  • the present invention also provides the use of the marker (or its expression level) or its detection reagent, which is used to prepare a kit for judging whether a tumor patient is suitable for using the compound of the present invention for prevention and treatment. / or treatment.
  • compositions or formulations, combinations and kits of active ingredients and methods of administration are provided.
  • composition of the present invention is preferably a pharmaceutical composition, and the composition of the present invention may include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to one or more compatible solid, semi-solid, liquid or gel fillers, which are suitable for human or animal use and must be of sufficient purity and low enough toxicity.
  • Cosmetic means that each component in the pharmaceutical composition and the active ingredients of the medicine and their mutual blending will not significantly reduce the efficacy of the medicine.
  • the pharmaceutically acceptable carrier is not particularly limited, and it can be selected from materials commonly used in the art, or prepared by conventional methods, or purchased from the market.
  • pharmaceutically acceptable carrier parts include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agent (such as sodium lauryl sulfate), buffering agent, chelating agent, thickener, pH regulator, skin penetration enhancer, coloring agent, flavoring agent, stabilizer, antioxidant, preservative , antibacterial agent, pyrogen-
  • the dosage form of the composition or preparation is solid preparation, liquid preparation or semi-solid preparation.
  • the dosage form of the composition or preparation is oral preparation, external preparation or injection preparation
  • the dosage form of the composition or preparation is tablet, injection, transfusion, ointment, gel, solution, microsphere or film.
  • the pharmaceutical formulation should match the mode of administration. Agents of the invention may also be used together (including before, during or after) other co-therapeutic agents.
  • a safe and effective amount is administered to a subject (such as a human or non-human mammal) in need thereof, usually at least about 10 micrograms per kilogram of body weight, and in most cases Not to exceed about 8 mg/kg body weight, preferably the dose is about 10 microgram/kg body weight to about 1 mg/kg body weight.
  • factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the present invention provides biomarkers that can guide the precise administration of the compound of the present invention.
  • the relevant biomarkers can effectively identify tumor patients who are sensitive to the compound of the present invention, improve their therapeutic effect, and avoid the application of the compound of the present invention to patients who are not sensitive to it. Patients with sensitive tumors can realize the precise application of this type of drug.
  • the present invention discovers for the first time the expression level of NNMT gene, the expression level of DNA methylase, the expression level of UHRF1, the methylation level of nucleotide sites of NNMT gene, and/or the DNA CpG site A in NNMT gene region through systematic research for the first time
  • the kylation level can be used as a marker for judging whether a specific tumor cell is suitable for treatment with the compound of the present invention.
  • the tumor has a high drug sensitivity to the compound of the present invention, that is, the compound of the present invention has low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, And/or tumors with high methylation levels of DNA CpG sites in the NNMT gene region have excellent therapeutic effects.
  • the compounds described in the present invention can be used for low or no expression of NNMT gene, high expression of DNA methylase, high expression of UHRF1, high methylation level of NNMT gene nucleotide site, and/or NNMT gene region
  • the precise treatment of tumors with high DNA CpG site methylation levels, the precise treatment of tumors by the compounds of the present invention has the advantages of better preventive and therapeutic effects on tumors, low drug dosage and small side effects, etc. While the compound has the effect on tumor prevention and treatment, it can reduce the dosage, reduce the side effects and improve the compliance of patients.
  • NNMT gene expression level DNA methylase expression level
  • UHRF1 expression level NNMT gene nucleotide site methylation level
  • NNMT gene region DNA CpG site methylation level DNA CpG site methylation level
  • NNMT Nicotinamide N-Methyltransferase
  • DNMT3a DNA methyltransferase 3a.
  • DNMT3b DNA methyltransferase 3b.
  • DNMT1 DNA methyltransferase 1.
  • UHRF1 refers to ubiquitin-like PHD- and RING-finger domain-containing protein 1.
  • CpG is an abbreviation for cytosine (C)-phosphate (p)-guanine (G).
  • the nucleotide sequence of the NNMT gene promoter region is shown in SEQ ID NO:1.
  • the 1050 bp before the transcription start site of the NNMT gene to the 499 bp after the transcription start site is the 951-2500 position of the nucleotide sequence shown in SEQ ID NO:1.
  • the first 1050 bp to the first 193 bp of the transcription start site of the NNMT gene is the 951-1808 positions of the nucleotide sequence shown in SEQ ID NO:1.
  • the first 840 bp to the first 469 bp of the transcription start site of the NNMT gene is the 1161-1532 positions of the nucleotide sequence shown in SEQ ID NO:1.
  • each tumor cell line is as follows:
  • Cell line NCI-H82 (ATCC, number HTB-175) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • Cell line G-401 (ATCC, No. CRL-1441) was cultured in McCoy's 5a medium + P/S containing 10% fetal bovine serum;
  • the cell line MDA-MB-453 (ATCC, number HTB-131) was cultured in Leibovitz's L-15 medium + P/S containing 10% fetal bovine serum;
  • the cell line SW48 (ATCC, number CCL-231) was cultured in Leibovitz's L-15 medium + P/S containing 10% fetal bovine serum;
  • the cell line WSU-DLCL2 (DSMZ, No. ACC-575) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • the cell line CFPAC-1 (ATCC, No. CRL-1918) was cultured in IMDM medium + P/S containing 10% fetal bovine serum;
  • Cell line 786-O (ATCC, No. CRL-1932) was cultured in RPMI1640 medium + P/S containing 10% fetal bovine serum;
  • the cell line GB-1 (JCRB, No. IFO50489) was cultured in DMEM medium + P/S containing 10% fetal bovine serum;
  • the cell line SF126 (JCRB, No. IFO50286) was cultured in EMEM medium + P/S containing 10% fetal bovine serum;
  • IC 50 is half inhibitory concentration (50% inhibiting concentration), that is, the concentration of niclosamide when 50% inhibitory effect is achieved.
  • NCI-H82 human small cell lung cancer cells
  • G-401 human kidney cancer Wilms cells
  • MDA-MB-453 breast cancer cells
  • SW48 human colon adenocarcinoma cells
  • WSU-DLCL2 human diffuse large B lymphoma cells
  • 786-O clear cell renal adenocarcinoma cell line
  • CFPAC-1 human pancreatic cancer cells
  • GB-1 human glioblastoma cells
  • SF126 human glioblastoma multiforme cells are insensitive to niclosamide ( IC 50 value is higher.
  • niclosamide has a higher effect on NCI-H82, G-401, MDA-MB-453, SW48 and WSU -
  • the inhibitory effect of DLCL2 tumor cells is more excellent.
  • NCI-H82 Five tumor cell lines sensitive to niclosamide compounds (NCI-H82, G-401, MDA-MB-453, SW48 and WSU-DLCL2) and four insensitive tumor cell lines (786-O, CFPAC- 1.
  • the NNMT gene promoter region of GB-1 and SF126 The region between 1050 bp before the NNMT gene transcription start site and the 499 bp after the transcription start site, and the NNMT gene transcription start site before 1050 bp to the transcription start site.
  • the region between 193 bp before the point was subjected to bisulfite sequencing to detect the methylation level of the DNA CpG site in the relevant region.
  • Figure 2 promoter region of NNMT gene
  • Figure 3 region between 1050 bp before the transcription start site of NNMT gene and 499 bp after the transcription start site
  • Figure 4 (1050 bp before the transcription start site of NNMT gene to transcription region between 193 bp before the start site
  • the niclosamide compound has an effect on the NNMT gene promoter region, the region between 1050 bp before the NNMT gene transcription start site and the 499 bp behind the transcription start site, and the NNMT gene transcription start
  • Tumor cell lines with high methylation levels of DNA CpG sites in the region between 1050 bp before the start site and 193 bp before the transcription start site have significantly stronger inhibitory effect on the NNMT gene promoter region and NNMT gene transcription start site Tumor cell lines with low methylation levels of DNA CpG sites in the region between 1050 bp before the transcription start site and 499 bp after the transcription start site, and in the region between 1050 bp before the transcription start
  • NNMT gene transcription of 3 tumor cell lines sensitive to niclosamide (NCI-H82, G-401 and WSU-DLCL2) and 3 tumor cell lines insensitive (786-O, CFPAC-1 and SF126) The methylation of DNA CpG sites in the region from 840 bp before the start site (namely, 114165695 on human chromosome 11) to 469 bp before the start site of gene transcription (namely, 114166066 on human chromosome 11) was analyzed.
  • the genomic DNA of the cells was treated with bisulfite, and then the region was amplified by PCR with corresponding primers, followed by sequencing analysis to detect the methylation level of the CpG site in the DNA region.
  • niclosamide has no effect on the NNMT gene promoter region or NNMT gene related regions.
  • the tumor cell lines with high methylation level of DNA CpG sites in the tumor cell lines have a significantly stronger inhibitory effect, and the inhibitory effect on tumor cell lines with low DNA CpG site methylation levels in the NNMT gene promoter region or NNMT gene-related regions is significant Weaker, indicating that the methylation level of DNA CpG sites in the promoter region of NNMT gene or related regions of NNMT gene in tumor cells is positively correlated with the sensitivity to niclosamide.
  • the positions of human chromosome 11 114165695, 114165730, 114165769, 114165804, 114165938, 114166050, 114166066 correspond to the positions of the nucleotide sequence of SEQ ID NO: 1 as follows:
  • the methylation level of cellular DNA is maintained by DNA methylases DNMT3a, DNMT3b, and DNMT1.
  • DNMT3a, DNMT3b can methylate DNA de novo
  • DNMT1 can be in the protein UHRF1 (ubiquitin-like containing PHD and ring finger domain protein 1) to maintain the replication of methylated DNA
  • the present invention detects the correlation between the expression of NNMT and the expression of DNMT1, UHRF1, DNMT3a and DNMT3b in tumors.
  • niclosamide has a more excellent therapeutic effect on tumors with high expression of DNMT1, high expression of UHRF1, high expression of DNMT3a and/or high expression of DNMT3b, that is, high expression of DNMT1, high expression of UHRF1, high expression of DNMT3a and/or high expression of DNMT3b
  • the expressed tumors are sensitive to niclosamide, and the expression of DNMT1, UHRF1, DNMT3a and DNMT3b in tumor cells is positively correlated with their sensitivity to niclosamide.
  • the NNMT gene was introduced into NCI-H82 cells by viral vectors so that NCI-H82 cells overexpressed NNMT protein, and NCI-H82 cells (NCI-H82(ov-NNMT)) overexpressing NNMT protein were obtained; in addition , the control NCI-H82 cells (NCI-H82(Con)) were obtained by transfecting NCI-H82 cells with an empty virus not loaded with NNMT gene.
  • NCI-H82(Con) control NCI-H82 cells
  • NCI-H82(ov-NNMT) NCI-H82 cells overexpressing NNMT protein
  • IC 50 is the half-inhibiting concentration (50% inhibiting concentration), that is, the concentration of niclosamide that achieves 50% inhibitory effect.

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Abstract

本发明涉及苯甲酰苯胺类化合物在治疗肿瘤中应用，具体地，本发明提供一种式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤。本发明所述的化合物对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤具有更显著优异的治疗效果。

Description

苯甲酰苯胺类化合物在治疗肿瘤中的应用 技术领域
本发明涉及药物领域，具体地涉及苯甲酰苯胺类化合物在治疗肿瘤中应用。
背景技术
肿瘤是严重危害人类健康的常见病，恶性肿瘤的死亡率也一直呈上升趋势。由于肿瘤的异质性及患者个体差异，如果简单根据患者肿瘤来源或病理特征等采用同一治疗方法或同一药物就容易产生治疗不当的问题，贻误患者宝贵的治疗时间和机会，因此针对患者的不同情况，采用个性化、精准化治疗就显得十分必要。随着生物学技术的发展，肿瘤治疗也进入了精准化治疗的时代，并且越来越多与肿瘤相关基因表达的改变被相继发现，相关基因的改变在恶性肿瘤的发展中发挥了重要的作用，如可上调细胞特定功能促进癌细胞永生等，这样生物标志物的发现和应用将会为相关药物的使用提供精准指引，使得肿瘤的个体化治疗成为可能，从而实现有针对性地给药，显著提升治疗效果。
因此，本领域亟需开发一种能够对肿瘤进行精准化治疗的药物。
发明内容
本发明目的在于提供一种能够对肿瘤进行精准治疗的药物，特别是NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平可用于判断肿瘤患者是否适合采用本发明所述化合物进行预防和/或治疗。本发明所述的化合物对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤具有更显著优异的治疗效果。
本发明第一方面，提供一种式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤；
Figure PCTCN2022113055-appb-000001
其中，
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10各自独立地为氢、卤素、-CN、羟基、巯基、硝基、氨基、-COOH、-CHO、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的C1-C12烷氧基、或取代或未取代的C1-C12烷硫基；
Z为
Figure PCTCN2022113055-appb-000002
其中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基、C3-C8卤代环烷基、卤素、硝基、-CN、氰基、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10各自独立地为氢、卤素、-CN、羟基、巯基、硝基、氨基、-COOH、-CHO、取代或未取代的C1-C8烷基、取代或未取代的C3-C10环烷基、取代或未取代的C1-C8烷氧基、或取代或未取代的C1-C8烷硫基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10各自独立地为氢、卤素、-CN、羟基、巯基、硝基、氨基、-COOH、-CHO、取代或未取代的C1-C4烷基、取代或未取代的C3-C8环烷基、或取代或未取代的C1-C4烷氧基、或取代或未取代的C1-C4烷硫基。
在另一优选例中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10各自独立地为氢、卤素、羟基、巯基、硝基、或氨基。
在另一优选例中，R 2、R 4、R 5、R 7、R 8和R 10各自独立地为氢。
在另一优选例中，R 1和R 9各自独立地为卤素。
在另一优选例中，R 3为硝基。
在另一优选例中，R 6为羟基、或巯基。
在另一优选例中，卤素为F、Cl、Br或I。
在另一优选例中，R 1和R 9各自独立地为Cl。
在另一优选例中，Z为
Figure PCTCN2022113055-appb-000003
在另一优选例中，Z为
Figure PCTCN2022113055-appb-000004
在另一优选例中，所述的式I化合物具有式I-1所述的结构：
Figure PCTCN2022113055-appb-000005
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10的定义如上所述。
另一优选例中，所述的式I化合物具有式I-2所述的结构：
Figure PCTCN2022113055-appb-000006
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10的定义如上所述。
另一优选例中，所述的式I化合物具有式I-3所述的结构：
Figure PCTCN2022113055-appb-000007
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10的定义如上所述。
另一优选例中，所述的式I化合物具有式I-4所述的结构：
Figure PCTCN2022113055-appb-000008
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10的定义如上所述。
另一优选例中，所述的式I化合物具有式I-5所述的结构：
Figure PCTCN2022113055-appb-000009
其中，R 1、R 3、R 6和R 9的定义如上所述。
在另一优选例中，所述的式I化合物为：
Figure PCTCN2022113055-appb-000010
在另一优选例中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C6烷基、C3-C8环烷基、C1-C6卤代烷基、C3-C8卤代环烷基、卤素、硝基、-CN、氰基、羟基、巯基、氨基、C1-C6烷氧基、C1-C6烷硫基、C3-C8环烷氧基、C3-C8环烷硫基。
在另一优选例中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C4烷基、C3-C8环烷基、C1-C4卤代烷基、C3-C8卤代环烷基、卤素、硝基、-CN、氰基、羟基、巯基、氨基、C1-C4烷氧基、C1-C4烷硫基、C3-C8环烷氧基、C3-C8环烷硫基。
在另一优选例中，所述的式I化合物的药学上可接受的盐为式I化合物与选自下组的酸形成的盐：盐酸、粘酸、D-葡萄糖醛酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸、甲酸、乙酸、三氟乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸、苯磺酸、天冬氨酸、谷氨酸，或其组合。
在另一优选例中，所述的肿瘤为人源肿瘤。
在另一优选例中，所述的肿瘤为人肿瘤。
在另一优选例中，所述的肿瘤包括NNMT基因低表达或未表达的肿瘤。
在另一优选例中，所述的肿瘤包括DNA甲基化酶高表达的肿瘤。
在另一优选例中，所述的DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合。
在另一优选例中，所述的肿瘤包括DNMT1高表达的肿瘤。
在另一优选例中，所述的肿瘤包括DNMT3a高表达的肿瘤。
在另一优选例中，所述的肿瘤包括DNMT3b高表达的肿瘤。
在另一优选例中，所述的肿瘤包括UHRF1高表达的肿瘤。
在另一优选例中，所述肿瘤包括NNMT基因核苷酸位点甲基化水平高和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
在另一优选例中，所述肿瘤包括NNMT基因核苷酸位点甲基化水平高的肿瘤。
在另一优选例中，所述的肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
在另一优选例中，所述的NNMT基因为人源NNMT基因。
在另一优选例中，所述的NNMT基因为人NNMT基因。
在另一优选例中，所述NNMT基因低表达或未表达的肿瘤是指从该肿瘤中提取的1μg蛋白中通过NNMT抗体检测不到NNMT蛋白，更佳地是指从该肿瘤中提取的5μg蛋白中通过NNMT抗体检测不到NNMT蛋白，更佳地是指从该肿瘤中提取的10μg蛋白中通过NNMT抗体检测不到NNMT蛋白，更佳地是指从该肿瘤中提取的100μg蛋白中通过NNMT抗体检测不到NNMT蛋白，更佳地是指从该肿瘤中提取的1000μg蛋白中通过NNMT抗体检测不到NNMT蛋白。
在另一优选例中，所述NNMT基因低表达的肿瘤是指肿瘤细胞的NNMT基因的表达水平小于同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达水平。
在另一优选例中，所述NNMT基因低表达的肿瘤是指肿瘤细胞的NNMT基因的表达水平E1与同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达水平E0的比值(E1/E0)<1.0。
在另一优选例中，所述NNMT基因低表达或未表达是指某一细胞(如肿瘤细胞)的NNMT基因的表达E1与同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因的表达E0的比值(E1/E0)<1.0，较佳地≤0.7，更佳地≤0.6，更佳地≤0.5，更佳地≤0.4，更佳地≤0.3、更佳地≤0.2，更佳地≤0.1，更佳地≤0.05，更佳地≤0.01，更佳地≤0.005，更佳地≤0.001，更佳地≤0.0001，更佳地≤0.00001，更佳地≤0.000001，更佳地≤0.0000001。
在另一优选例中，所述的同一细胞是指NNMT基因正常表达或高表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但NNMT基因正常表达或高表达的细胞。
在另一优选例中，所述的正常细胞是指NNMT基因正常表达的正常组织细胞(如癌细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，E0为NNMT基因正常表达或高表达细胞的NNMT基因的表达水平。
在另一优选例中，所述的NNMT基因正常表达或高表达的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述DNA甲基化酶高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶，更佳地是从该肿瘤中提取的5μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶，更佳地是从该肿瘤中提取的1μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶，更佳地是从该肿瘤中提取的0.2μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶，更佳地是从该肿瘤中提取的0.05μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶，更佳地是从该肿瘤中提取的0.01μg蛋白中通过DNA甲基化酶抗体检测能够检测到DNA甲基化酶。
在另一优选例中，所述DNA甲基化酶高表达的肿瘤是指肿瘤细胞的DNA甲基化酶的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNA甲基化酶的表达水平。
在另一优选例中，所述DNA甲基化酶高表达的肿瘤是指肿瘤细胞的DNA甲基化酶的表达水平G1与同一细胞或正常细胞(如癌旁组织细胞)中DNA甲基化酶的表达水平G0的比值(G1/G0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述的同一细胞是指DNA甲基化酶正常表达或低表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNA甲基化酶正常表达或低表达的细胞。
在另一优选例中，所述的正常细胞是指DNA甲基化酶正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，G0为DNA甲基化酶正常表达或低表达细胞的DNA甲基化酶的表达水平。
在另一优选例中，所述的DNA甲基化酶正常表达或低表达的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述DNMT1高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白，更佳地是从该肿瘤中提取的5μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白，更佳地是从该肿瘤中提取的1μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白，更佳地是从该肿瘤中提取的0.2μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白，更佳地是从该肿瘤中提取的0.05μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白，更佳地是从该肿瘤中提取的0.01μg蛋白中通过DNMT1抗体检测能够检测到DNMT1蛋白。
在另一优选例中，所述DNMT1高表达的肿瘤是指肿瘤细胞的DNMT1的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNMT1的表达水平。
在另一优选例中，所述DNMT1高表达的肿瘤是指肿瘤细胞的DNMT1的表达水平B1与同一细胞或正常细胞(如癌旁组织细胞)中DNMT1的表达水平B0的比值(B1/B0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述的同一细胞是指DNMT1正常表达或低表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNMT1正常表达或低表达的细胞。
在另一优选例中，所述的正常细胞是指DNMT1正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，B0为DNMT1正常表达或低表达细胞的DNMT1的表达水平。
在另一优选例中，所述的DNMT1正常表达或低表达的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述DNMT3a高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白，更佳地是从该肿瘤中提取的5μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白，更佳地是从该肿瘤中提取的1μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白，更佳地是从该肿瘤中提取的0.2μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白，更佳地是从该肿瘤中提取的0.05μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白，更佳地是从该肿瘤中提取的0.01μg蛋白中通过DNMT3a抗体检测能够检测到DNMT3a蛋白。
在另一优选例中，所述DNMT3a高表达的肿瘤是指肿瘤细胞的DNMT3a的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNMT3a的表达水平。
在另一优选例中，所述DNMT3a高表达的肿瘤是指肿瘤细胞的DNMT3a的表达水C1与同一细胞或正常细胞(如癌旁组织细胞)中DNMT3a的表达水平C0的比值(C1/C0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述的同一细胞是指DNMT3a正常表达或低表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNMT3a正常表达或低表达的细胞。
在另一优选例中，所述的正常细胞是指DNMT3a正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，C0为DNMT3a正常表达或低表达细胞的DNMT3a的表达水平。
在另一优选例中，所述的DNMT3a正常表达或低表达的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述DNMT3b高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白，更佳地是从该肿瘤中提取的5μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白，更佳地是从该肿瘤中提取的1μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白，更佳地是从该肿瘤中提取的0.2μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白，更佳地是从该肿瘤中提取的0.05μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白，更佳地是从该肿瘤中提取的0.01μg蛋白中通过DNMT3b抗体检测能够检测到DNMT3b蛋白。
在另一优选例中，所述DNMT3b高表达的肿瘤是指肿瘤细胞的DNMT3b的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中DNMT3b的表达水平。
在另一优选例中，所述DNMT3b高表达的肿瘤是指肿瘤细胞的DNMT3b的表达水D1与同一细胞或正常细胞(如癌旁组织细胞)中DNMT3b的表达水平D0的比值(D1/D0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述的同一细胞是指DNMT3b正常表达或低表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但DNMT3b正常表达或低表达的细胞。
在另一优选例中，所述的正常细胞是指DNMT3b正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，D0为DNMT3b正常表达或低表达细胞的DNMT3b的表达水平。
在另一优选例中，所述的DNMT3b正常表达或低表达的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述UHRF1高表达的肿瘤是指从该肿瘤中提取的20μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白，更佳地是从该肿瘤中提取的5μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白，更佳地是从该肿瘤中提取的1μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白，更佳地是从该肿瘤中提取的0.2μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白，更佳地是从该肿瘤中提取的0.05μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白，更佳地是从该肿瘤中提取的0.01μg蛋白中通过UHRF1抗体检测能够检测到UHRF1蛋白。
在另一优选例中，所述UHRF1高表达的肿瘤是指肿瘤细胞的UHRF1的表达水平大于同一细胞或正常细胞(如癌旁组织细胞)中UHRF1的表达水平。
在另一优选例中，所述UHRF1高表达的肿瘤是指肿瘤细胞的UHRF1的表达水平F1与同一细胞或正常细胞(如癌旁组织细胞)中UHRF1的表达水平F0的比值(F1/F0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述的同一细胞是指UHRF1正常表达或低表达的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但UHRF1正常表达或低表达的细胞。
在另一优选例中，所述的正常细胞是指UHRF1正常表达的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，F0为UHRF1正常表达或低表达细胞的UHRF1的表达水平。
在另一优选例中，所述的UHRF1正常表达或低表达的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平大于同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因核苷酸位点甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平L1与同一细胞或正常细胞(如肿瘤旁组织细胞)中NNMT基因核苷酸位点甲基化水平L0的比值(L1/L0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平≥1％，较佳地≥3％，较佳地≥5％，较佳地≥10％，较佳地≥15％,较佳地≥20％,更佳地≥25％，更佳地≥30％，更佳地≥40％，更佳地≥50％。
在另一优选例中，所述的同一细胞是指NNMT基因核苷酸位点甲基化水平为正常水平或低水平的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但NNMT基因核苷酸位点甲基化水平为正常水平或低水平的细胞。
在另一优选例中，所述的正常细胞是指NNMT基因核苷酸位点甲基化水平为正常水平的正常组织细胞(如如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中，L0为NNMT基因核苷酸位点甲基化水平为正常水平或低水平的细胞的NNMT基因核苷酸位点甲基化水平。
在另一优选例中，所述的NNMT基因核苷酸位点甲基化水平为正常水平或低水平的细胞包括对如式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因核苷酸位点甲基化水平(M％)≥3％且小于等于M1％，其中，M1为3-100之间的任一正整数。
在另一优选例中，M1为5、10、15、20、25、30、35、40、45、50、55、60、65、70、80、85、90、95或100。
在另一优选例中，所述的NNMT基因核苷酸位点甲基化水平是指NNMT基因区的甲基化的核苷酸数量占NNMT基因区所有核苷酸数量的比值。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因启动子区的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因启动子区的核苷酸序列如SEQ ID NO:1所示。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因转录起始位点前1050bp到转录起始位点后499bp区域内的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp区域内的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括NNMT基因转录起始位点前840bp到转录起始位点前469bp区域内的核苷酸位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前840bp到转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位和114166066位中任何两个位点之间的区域内(包括这两个位点本身)的核苷酸位点甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位和114166066位中的一个或多个(如2、3、4、5、6或7)位点的核苷酸甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括选自下组的位点的核苷酸甲基化水平：人11号染色体114165695位、人11号染色体114165730位、人11号染色体114165769位、人11号染色体114165804位、人11号染色体114165938位、人11号染色体114166050位、人11号染色体114166066位，或其组合。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括SEQ ID NO:1核苷酸序列的位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中任何两个位点之间的区域内(包括这两个位点本身)的核苷酸位点甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括SEQ ID NO:1核苷酸序列位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中的一个或多个(如2、3、4、5、6或7)位点的核苷酸甲基化水平。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平包括选自下组SEQ ID NO:1序列位点的核苷酸甲基化水平：第1161位、第1196位、第1235位、第1270位、第1404位、第1516位、第1532位，或其组合。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平大于同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因区DNA CpG位点甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平≥1％，较佳地≥3％，较佳地≥5％，较佳地≥10％，较佳地≥15％,较佳地≥20％,更佳地≥25％，更佳地≥30％，更佳地≥40％，更佳地≥50％。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平A1与同一细胞或正常细胞(如癌旁组织细胞)中NNMT基因区DNA CpG位点甲基化水平A0的比值(A1/A0)>1.0，较佳地≥1.2，较佳地≥1.5，更佳地≥2，更佳地≥3，更佳地≥5，更佳地≥8，更佳地≥10，更佳地≥15，更佳地≥20，更佳地≥30，更佳地≥50，例如2-50。
在另一优选例中，所述的同一细胞是指NNMT基因区DNA CpG位点甲基化水平为正常水平或低水平的细胞(如同一类肿瘤细胞)。
在另一优选例中，所述的同一细胞是指同种类但NNMT基因区DNA CpG位点甲基化水平为正常水平或低水平的细胞。
在另一优选例中，所述的正常细胞是指NNMT基因区DNA CpG位点甲基化水平为正常水平的正常组织细胞(如肿瘤细胞起源细胞、肿瘤邻近细胞或癌旁组织细胞)。
在另一优选例中A0为NNMT基因区DNA CpG位点甲基化水平为正常水平或低水平的细胞的NNMT基因区DNA CpG位点甲基化水平。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平为正常水平或低水平的细胞包括对式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感的细胞。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高是指某一细胞(如肿瘤细胞)的NNMT基因区DNA CpG位点甲基化水平(M％)≥3％且小于等于M2％，其中，M2为3-100之间的任一正整数。
在另一优选例中，M2为5、10、15、20、25、30、35、40、45、50、55、60、65、70、80、85、90、95或100。
在另一优选例中，所述的CpG位点甲基化水平是指某基因区域甲基化的CpG核苷酸数量占该基因区域所有核苷酸数量的比值。
在另一优选例中，所述的DNA CpG位点甲基化水平是指某区域DNA已甲基化的CpG核苷酸数量占该区域DNA的所有核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区甲基化的CpG核苷酸数量占NNMT基因区所有核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区DNA已甲基化的CpG核苷酸数量占NNMT基因区DNA的所有核苷酸数量的比值。
在另一优选例中，所述的CpG位点甲基化水平是指某基因区域甲基化的CpG核苷酸数量占该基因区域所有CpG核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区甲基化的CpG核苷酸数量占NNMT基因区所有CpG核苷酸数量的比值。
在另一优选例中，所述的DNA CpG位点甲基化水平是指某区域DNA已甲基化的CpG位点数量占该区域DNA的全部CpG位点数量的比值。
在另一优选例中，所述的DNA CpG位点甲基化水平是指某区域DNA已甲基化的CpG核苷酸数量占该区域DNA的全部CpG核苷酸数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区DNA已甲基化的CpG位点数量占NNMT基因区DNA的全部CpG位点数量的比值。
在另一优选例中，所述的NNMT基因区DNA CpG位点甲基化水平是指NNMT基因区DNA已甲基化的CpG核苷酸数量占NNMT基因区DNA的全部CpG核苷酸数量的比值。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因启动子区DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因启动子区的核苷酸序列如SEQ ID NO:1所示。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因转录起始位点前1050bp到转录起始位点后499bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因转录起始位点前1050bp到转录起始位点前193bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前1050bp到转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因转录起始位点前840bp到转录起始位点前469bp区域内DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因转录起始位点前840bp到转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位和114166066位中任何两个位点之间的区域内(包括这两个位点本身)的DNA CpG位点甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位和114166066位中的一个或多个(如2、3、4、5、6或7)位点的甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括选自下组的位点的甲基化水平：人11号染色体114165695位、人11号染色体114165730位、人11号染色体114165769位、人11号染色体114165804位、人11号染色体114165938位、人11号染色体114166050位、人11号染色体114166066位，或其组合。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括SEQ ID NO:1核苷酸序列的位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中任何两个位点之间的区域内(包括这两个位点本身)的DNA CpG位点甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括SEQ ID NO:1核苷酸序列的位点的第1161位、第1196位、第1235位、第1270位、第1404位、第1516位和第1532位中的一个或多个(如2、3、4、5、6或7)位点的甲基化水平。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平包括选自下组的SEQ ID NO:1核苷酸序列的位点的甲基化水平：第1161位、第1196位、第1235位、第1270位、第1404位、第1516位、第1532位，或其组合。
在另一优选例中，所述的肿瘤选自下组：肺癌、肾癌、乳腺癌、结肠癌、淋巴癌、白血病、胰腺癌、脑瘤、肝癌、前列腺癌，或其组合。
在另一优选例中，所述的肺癌选自下组：非小细胞肺癌、小细胞肺癌，或其组合。
在另一优选例中，所述肺癌的细胞包括NCI-H82细胞。
在另一优选例中，所述的结肠癌包括结肠腺癌。
在另一优选例中，所述结肠癌的细胞包括SW48细胞。
在另一优选例中，所述乳腺癌的细胞包括MDA-MB-453细胞。
在另一优选例中，所述乳腺癌包括三阴乳腺癌。
在另一优选例中，所述的淋巴癌选自下组：B淋巴瘤、皮肤T细胞淋巴癌，或其组合。
在另一优选例中，所述的淋巴癌包括弥漫大B淋巴瘤。
在另一优选例中，所述的淋巴癌细胞包括WSU-DLCL2细胞。
在另一优选例中，所述的脑瘤选自下组：脑胶质母细胞瘤、神经胶质细胞瘤、脑髓母细胞瘤、脑神经母细胞瘤，或其组合。
在另一优选例中，所述的脑瘤包括胶质母细胞瘤。
在另一优选例中，所述的脑髓母细胞瘤包括小脑髓母细胞瘤。
在另一优选例中，所述的脑胶质母细胞瘤包括多形性胶质母细胞瘤。
在另一优选例中，所述的脑瘤包括多形性胶质母细胞瘤。
在另一优选例中，所述脑瘤的细胞包括GB-1细胞和SF126细胞中的一种或多种。
在另一优选例中，所述的肾癌选自下组：肾透明细胞腺癌、肾癌Wilms，或其组合。
在另一优选例中，所述的肾癌包括肾透明细胞腺癌。
另一优选例中，所述的肾癌包括肾癌Wilms。
在另一优选例中，所述的肾癌的癌细胞包括肾癌Wilms细胞。
在另一优选例中，所述的肾癌的癌细胞包括G-401细胞和786-O细胞中的一种或多种。
在另一优选例中，所述的胰腺癌的癌细胞包括CFPAC-1细胞。
在另一优选例中，所述的白血病选自下组：T淋巴细胞白血病、髓细胞性白血病，或其组合。
在另一优选例中，所述的T淋巴细胞白血病包括急性T淋巴细胞白血病。
在另一优选例中，所述的髓细胞性白血病包括M4级AML急性髓细胞性白血病。
在另一优选例中，所述的髓细胞性白血病包括FAB M4级AML急性髓细胞性白血病。
在另一优选例中，所述的表达包括蛋白表达和/或mRNA表达。
在另一优选例中，所述的组合物或制剂为药物组合物或药物或制剂。
在另一优选例中，所述的组合物或制剂还包括药学上可接受的载体。
在另一优选例中，所述的组合物或制剂的剂型为固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂。
在另一优选例中，所述的组合物或制剂的剂型为片剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
本发明第二方面，提供一种用于判断肿瘤患者是否适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗肿瘤的标志物，所述的标志物包括NNMT基因、DNA甲基化酶、UHRF1、NNMT基因核苷酸位点甲基化、和/或NNMT基因区DNA CpG位点甲基化。
在另一优选例中，所述的标志物包括NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平。
在另一优选例中，NNMT基因、DNA甲基化酶、UHRF1、NNMT基因核苷酸位点甲基化、和/或NNMT基因区DNA CpG位点甲基化包括肿瘤细胞的NNMT基因、DNA甲基化酶、UHRF1、NNMT基因核苷酸位点甲基化、和/或NNMT基因区DNA CpG位点甲基化。
在另一优选例中，当肿瘤患者的肿瘤细胞中NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，当肿瘤患者的肿瘤细胞中NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述肿瘤患者适合采用如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物，其包括肿瘤患者的肿瘤对如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物敏感。
在另一优选例中，所述肿瘤患者不适合采用如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物，其包括肿瘤患者的肿瘤对如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物不敏感。
在另一优选例中，所述NNMT基因低表达或未表达的肿瘤如本发明第一方面所述。
在另一优选例中，所述的DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合。
在另一优选例中，所述DNA甲基化酶(如DNMT1、DNMT3a和/或DNMT3b)高表达的肿瘤如本发明第一方面所述。
在另一优选例中，所述UHRF1高表达的肿瘤如本发明第一方面所述。
在另一优选例中，所述NNMT基因核苷酸位点甲基化水平高的肿瘤如本发明第一方面所述。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化水平高的肿瘤如本发明第一方面所述。
本发明第三方面，提供一种检测试剂盒，所述的检测试剂盒包括：
(i)用于检测NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂。
在另一优选例中，所述检测试剂盒的检测样本包括肿瘤细胞。
在另一优选例中，NNMT基因表达水平是指该基因mRNA和/或蛋白的表达水平。
本发明第四方面，提供一种如发明第三方面所述的检测试剂盒的用途，用于制备一伴随诊断试剂盒，所述伴随诊断试剂盒用于判断肿瘤患者是否适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述的伴随诊断试剂盒还包括说明书或标签。
在另一优选例中，所述的说明书或标签记载：
当肿瘤患者的肿瘤细胞中NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述的说明书或标签记载：
当肿瘤患者的肿瘤细胞中NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述肿瘤患者适合采用如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物如本发明第二方面所述。
在另一优选例中，所述肿瘤患者不适合采用如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物如本发明第二方面所述。
本发明第五方面，提供一种药盒，所述的药盒包括：
(i)用于检测NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂；和
(ii)如本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。
在另一优选例中，所述检测的样本包括肿瘤。
在另一优选例中，所述的药盒还包括说明书或标签。
在另一优选例中，所述的说明书或标签记载：
当肿瘤患者的肿瘤细胞中NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，该肿瘤患者适合采用本发明第一方面所述的式I化合物、或其光学异 构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，当肿瘤患者的肿瘤细胞中NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNACpG位点甲基化水平低，则该肿瘤患者不适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
本发明第六方面，提供一种预防和/或治疗肿瘤的方法，给所需的对象施用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物，从而预防和/或治疗肿瘤。
在另一优选例中，所述的肿瘤如本发明第一方面所述。
在另一优选例中，所述对象为人和非人哺乳动物(啮齿动物、兔、猴、家畜、狗、猫等)。
在另一优选例中，所述的方法包括步骤：
先使得对象肿瘤的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，然后给予所述所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述的方法包括步骤：
先将对象施用NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂，使得对象肿瘤的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，然后给予所述所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述NNMT基因抑制剂包括能够使得肿瘤的NNMT基因低表达或未表达的抑制剂。
在另一优选例中，所述DNA甲基化酶促进剂包括能够使得肿瘤的DNA甲基化酶高表达的促进剂。
在另一优选例中，所述的DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合。
在另一优选例中，所述DNA甲基化酶促进剂包括DNMT1促进剂。
在另一优选例中，所述DNA甲基化酶促进剂包括DNMT3a促进剂。
在另一优选例中，所述DNA甲基化酶促进剂包括DNMT3b促进剂。
在另一优选例中，所述DNMT1促进剂包括能够使得肿瘤的DNMT1高表达的促进剂。
在另一优选例中，所述DNMT3a促进剂包括能够使得肿瘤的DNMT3a高表达的促进剂。
在另一优选例中，所述DNMT3b促进剂包括能够使得肿瘤的DNMT3b高表达的促进剂。
在另一优选例中，所述UHRF1促进剂包括能够使得肿瘤的UHRF1高表达的促进剂。
在另一优选例中，所述NNMT基因核苷酸位点甲基化促进剂包括能够使得肿瘤的NNMT基因核苷酸位点甲基化水平高的促进剂。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化促进剂包括能够使得肿瘤的NNMT基因区DNA CpG位点甲基化水平高的促进剂。
在另一优选例中，所述的抑制剂包括特异性抑制剂。
在另一优选例中，所述的促进剂包括特异性促进剂。
在另一优选例中，所述的施用为口服施用、注射施用或外用施用。
在另一优选例中，所述的注射施用为肌肉注射施用或静脉注射施用。
本发明第七方面，提供一种装置或系统，所述的装置或系统包括：
(i)检测模块，所述的检测模块用于检测NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平；
(ii)输出模块，所述的输出模块包括输出以下信息：
当肿瘤患者的肿瘤细胞中NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗；和/或
当肿瘤患者的肿瘤细胞中NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用本发明第一方面所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗。
在另一优选例中，所述检测的样本包括肿瘤。
在另一优选例中，所述的装置包括基因检测仪或蛋白检测仪。
在另一优选例中，所述的装置或系统还包括进样模块。
在另一优选来了，所述的进样模块用于进肿瘤细胞提取物。
在另一优选例中，所述的装置或系统还包括数据处理模块。
在另一优选例中，所述的数据处理模块处理得到NNMT基因表达高低、DNA甲基化酶表达高低、UHRF1表达高低、NNMT基因核苷酸位点甲基化水平高低、和/或NNMT基因区DNA CpG位点甲基化水平高低。
本发明第八方面，提供一种NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂的用途，用于制备组合物或制剂，所述的组合物或制剂用于增强抗肿瘤药物的抗肿瘤效果。
在另一优选例中，所述NNMT基因抑制剂包括能够使得肿瘤的NNMT基因低表达或未表达的抑制剂。
在另一优选例中，所述DNA甲基化酶促进剂包括能够使得肿瘤的DNA甲基化酶高表达的促进剂。
在另一优选例中，所述的DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合。
在另一优选例中，所述DNA甲基化酶促进剂包括DNMT1促进剂。
在另一优选例中，所述DNA甲基化酶促进剂包括DNMT3a促进剂。
在另一优选例中，所述DNA甲基化酶促进剂包括DNMT3b促进剂。
在另一优选例中，所述DNMT1促进剂包括能够使得肿瘤的DNMT1高表达的促进剂。
在另一优选例中，所述DNMT3a促进剂包括能够使得肿瘤的DNMT3a高表达的促进剂。
在另一优选例中，所述DNMT3b促进剂包括能够使得肿瘤的DNMT3b高表达的促进剂。
在另一优选例中，所述UHRF1促进剂包括能够使得肿瘤的UHRF1高表达的促进剂。
在另一优选例中，所述NNMT基因核苷酸位点甲基化促进剂包括能够使得肿瘤的NNMT基因核苷酸位点甲基化水平高的促进剂。
在另一优选例中，所述NNMT基因区DNA CpG位点甲基化促进剂包括能够使得肿瘤的NNMT基因区DNA CpG位点甲基化水平高的促进剂。
在另一优选例中，所述的抑制剂包括特异性抑制剂。
在另一优选例中，所述的促进剂包括特异性促进剂。
在另一优选例中，所述的抗肿瘤药物包括如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。
在另一优选例中，所述的肿瘤如本发明第一方面所述。
在另一优选例中，所述的组合物或制剂的剂型为固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂
在另一优选例中，所述的组合物或制剂的剂型为片剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
本发明第九方面，提供一种活性成分组合，所述的活性成分组合包括以下组分：
(1)第一活性成分，所述的第一活性成分包括抗肿瘤药物；和
(2)第二活性成分，所述的第二活性成分包括NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂。
在另一优选例中，所述的抗肿瘤药物包括如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。
在另一优选例中，所述NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂如本发明第八方面所述。
在另一优选例中，所述的第一活性成分与所述第二活性成分的摩尔比为0.01-600：1，较佳地0.05-500：1，更佳地0.1-400：1，更佳地0.2-200：1，更佳地0.5-100:1，更佳地0.5-80:1，最佳地1-50:1。
在另一优选例中，所述的活性成分组合中，至少有一种活性成分是独立的。
在另一优选例中，所述的活性成分组合中，第一活性成分和第二活性成分是相互独立的。
本发明第十方面，提供一种组合物，所述的组合物包括：
(1)第一活性成分，所述的第一活性成分包括抗肿瘤药物；和
(2)第二活性成分，所述的第二活性成分包括NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂。
在另一优选例中，所述的抗肿瘤药物包括如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。
在另一优选例中，所述NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂如本发明第八方面所述。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的药物组合物还包括药学上可接受的载体。
在另一优选例中，所述的第一活性成分的含量为0.01-99.99wt％，较佳地0.1-99.9wt％，更佳地1-99wt％，更佳地10-99wt％，最佳地20-99wt％，以组合物活性成分的总重量计。
在另一优选例中，所述第二活性成分的含量为0.01-99.99wt％，较佳地0.1-99.9wt％，更佳地1-99wt％，更佳地10-99wt％，最佳地20-99wt％，以组合物活性成分的总重量计。
本发明第十一方面，提供一种药盒，所述药盒包括：
(A)含有第一活性成分的第一制剂，所述的第一活性成分包括抗肿瘤药物；和
(B)含有第二活性成分的第二制剂，所述的第二活性成分包括NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂。
在另一优选例中，所述的抗肿瘤药物包括如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。
在另一优选例中，所述NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂如本发明第八方面所述。
在另一优选例中，所述的药盒还包括使用说明书。
在另一优选例中，所述的第一制剂和第二制剂是相互独立制剂。
在另一优选例中，所述的第一制剂和第二制剂是合并制剂。
在另一优选例中，所述的使用说明书中注明将所述第一制剂和第二制进行联用，从而用于增强抗肿瘤药物的抗肿瘤活性。
在另一优选例中，所述的联用的方法为先给予含有第二活性成分的第二制剂，再给予含有第一活性成分的第一制剂。
在另一优选例中，所述的使用说明书记载：
NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂使得肿瘤细胞的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高后，如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物对肿瘤细胞抑制增强。
本发明第十二方面，提供一种如本发明第九方面所述的活性成分组合、如本发明第十方面所述的组合物、和/或如本发明第十一方面所述的药盒的用途，用于制备抗肿瘤药物。
在另一优选例中，所述的药物分装在药品盒中。
在另一优选例中，所述的药品盒还包括使用说明书，所述的使用说明书记载：
先施用NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂，使得肿瘤细胞的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高后，如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物对肿瘤细胞抑制增强。
本发明第十三方面，提供一种体外抑制肿瘤细胞的方法，所述的方法包括步骤：将肿瘤细胞与如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行接触，从而抑制肿瘤细胞。
在另一优选例中，所述的方法为非治疗性和非诊断性方法。
在另一优选例中，所述的接触为体外培养接触。
在另一优选例中，所述的肿瘤如本发明第一方面所述。
在另一优选例中，所述的方法包括步骤：
先使得肿瘤细胞的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，然后将肿瘤细胞与如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行接触，从而抑制肿瘤细胞。
在另一优选例中，所述的方法包括步骤：
先将肿瘤细胞施用NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂，使得肿瘤细胞的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，然后将肿瘤细胞与如本发明第一方面所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行接触，从而抑制肿瘤细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为对氯硝柳胺敏感及不敏感肿瘤细胞NNMT基因表达情况。
图2为对氯硝柳胺敏感及不敏感肿瘤细胞NNMT基因启动子区DNA CpG位点甲基化水平。
图3为对氯硝柳胺敏感及不敏感肿瘤细胞NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域DNA CpG位点甲基化水平。
图4为对氯硝柳胺敏感及不敏感肿瘤细胞NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域DNA CpG位点甲基化水平。
图5为对氯硝柳胺敏感及不敏感肿瘤细胞特定NNMT基因区即人11号染色体114165695、114165730、114165769、114165804、114165938、114166050、114166066等7个位点的DNA CpG位点甲基化情况，黑点表示相关位点已甲基化，白点表示相关位点未甲基化，SST指转录起始位点，Chr11指根据GCF_000001405.25(GRCh37.p13)人类基因组版本界定的人类11号染色体。
图6为肿瘤细胞中NNMT的表达和DNMT1、UHRF1、DNMT3a和DNMT3b的表达的相关性。
图7为Western Blot检测对照NCI-H82细胞(NCI-H82(Con))和过表达NNMT蛋白的NCI-H 82细胞(NCI-H82(ov-NNMT))的NNMT蛋白表达水平，其中，NCI-H82(Con)为采用不负载NNMT基因的空病毒转染NCI-H82细胞获得的对照NCI-H82细胞的NNMT蛋白表达水平，NCI-H82(ov-NNMT)为通过病毒载体将NNMT基因导入到NCI-H82细胞获得的过表达NNMT蛋白的NCI-H82细胞的NNMT蛋白表达水平。
具体实施方式
本发明人经过长期而深入的研究，首次意外地发现本发明化合物对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤细胞具有更为显著的抑制作用。NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平能够作为判断肿瘤患者是否适合采用本发明化合物进行预防和/或治疗的标志物。在此基础上，发明人完成了本发明。
术语
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，术语“DNA CpG位点甲基化水平高”、“DNA CpG位点甲基化高水平”与“DNA CpG位点高甲基化”可互换使用。
如本文所用，术语“DNA CpG位点甲基化低水平”、“DNA CpG位点甲基化水平低”与“DNA CpG位点低甲基化”可互换使用。
如本文所用，术语“IC50”与“IC 50”可互换使用，是指半抑制浓度(50％inhibiting concentration)，即达到50％抑制效果时抑制剂的浓度。
如本文所用，术语“CpG位点甲基化”、“CpG核苷酸甲基化”与“CpG甲基化”可互换使用。
如本文所用，术语“P/S”是指在相关培养基中加入Penicillin(盘尼西林)以及Streptomycin(链霉素)”。
如本文所用，术语“某一细胞”指某个细胞(如某单个癌细胞)或包含多个类似细胞的一群细胞等(如某肿瘤组织)。
如本文所用，“肿瘤患者适合采用本发明化合物”包括肿瘤患者的肿瘤对本发明化合物敏感。
如本文所用，“肿瘤患者不适合采用本发明化合物”包括肿瘤患者的肿瘤对本发明化合物不敏感。如本文所用，“NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平”是指NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平和NNMT基因区DNA CpG位点甲基化水平中的一种或多种。
如本文所用，“NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高”是指NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高和NNMT基因区DNA CpG位点甲基化水平高中的一种或多种。
如本文所用，“NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低”是指NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低和NNMT基因区DNA CpG位点甲基化水平低中的一种或多种。
如本文所用，术语“NNMT”的英文名为Nicotinamide N-Methyltransferase。
如本文所用，术语“bp”是指base pair，碱基对。
如本文所用，术语“SST”是指转录起始位点。
如本文所用，术语“Chr11”是指GCF_000001405.25(GRCh37.p13)人类基因组版本界定的人类11号染色体。
如本文所用，“人11号染色体”是指GCF_000001405.25(GRCh37.p13)人类基因组版本界定的人类11号染色体
如本文所用，术语“转录起始位点前”、“转录起始位点后”、“转录起始位点之前”、“转录起始位点之后”均不包括转录起始位点本身。
如本文所用，术语“人11号染色体114165695位”是指人11号染色体114165695位的核苷酸；“人11号染色体114165730位”是指人11号染色体114165730位的核苷酸；“人11号染色体114165769位”是指人11号染色体114165769位的核苷酸；“人11号染色体114165804位”是指人11号染色体114165804位的核苷酸；“人11号染色体114165938位”是指人11号染色体114165938位的核苷酸；“人11号染色体114166050位”是指人11号染色体114166050位的核苷酸；“人11号染色体114166066位”是指人11号染色体114166066位的核苷酸。
如本文所用，术语“DNMT3a”是指DNA甲基转移酶3a(DNA methyltransferase 3a)，与“DNMT3A可互换使用。
如本文所用，术语“DNMT3b”是指DNA甲基转移酶3b(DNA methyltransferase 3b)，与“DNMT3B可互换使用。
如本文所用，术语“DNMT1”是指DNA甲基转移酶1(DNA methyltransferase 1)。
如本文所用，术语“UHRF1”是指泛素样含PHD和环指域蛋白1。
如本文所用，术语“CpG”是胞嘧啶(C)-磷酸(p)-鸟嘌呤(G)的缩写。
如本文所用，术语“SF126细胞”与“SF-126细胞”可互换使用。
如本文所用，术语“氘代”指化合物或基团中的一个或多个氢被氘所取代。氘代可以是一取代、二取代、多取代或全取代。
如本文所用，术语“溶剂化物”指化合物与溶剂分子配位形成特定比例的配合物。
如本文所用，基因表达包括该基因蛋白表达或该基因mRNA表达等。
应当理解，本领域的普通技术人员可以选择本发明的化合物上的取代基和取代型式以产生化学上稳定的化合物，所述化合物可以通过本领域己知的技术以及下文所阐述的方法合成。如果被超过一个(多个)取代基团取代，应当理解，这多个基团可以是在同一个碳上或在不同碳上，只要产生稳定的结构即可。
如本文所用，术语“取代”或“取代的”是基团上的氢原子被非氢原子基团取代，但需要满足其化合价要求并且由取代生成化学稳定的化合物，即不会自发进行诸如环化、消除等转变的化合物。
如本文所用，“R 1”、“R1”和“R 1”的含义相同，可相互替换，其它类似定义的含义相同。
如本文所用，
Figure PCTCN2022113055-appb-000011
表示基团的连接位点。
如本文所用，术语“烷基”指只含碳原子的直链(即，无支链)或支链饱和烃基，或直链和支链组合的基团。当烷基前具有碳原子数限定(如C1-C6烷基)指所述的烷基含有1-6个碳原子，例如，C1-C4烷基指含有1-4个碳原子的烷基，代表性实例包括但不限于甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。
在本发明中，术语“卤素”指F、Cl、Br或I。
在本发明中，术语“卤代”是指被卤素取代。
如本文所用，术语“卤代烷基”是指烷基的一个或多个(优选为1、2、3或4个)氢被卤素取代，所述的烷基和卤素如上所定义，当烷基前具有碳原子数限定(如C1-C8卤代烷基)指所述的烷基含有1-8个碳原子，例如，C1-C6卤代烷基指含有1-6个碳原子的卤代烷基，代表性实例包括但不限于-CF 3、-CHF 2、单氟代异丙基、双氟代丁基、或类似基团。
如本文所用，术语“环烷基”指具有饱和的或部分饱和的单元环，二环或多环(稠环、桥环或螺环)环系基团。当某个环烷基前具有碳原子数限定(如C3-C12)时，指所述的环烷基具有3-12个环碳原子。在一些优选实施例中，术语“C3-C8环烷基”指具有3-8个环碳原子的饱和或部分饱和的单环或二环烷基，包括环丙基、环丁基、环戊基、环庚基、或类似基团。“螺环烷基”指单环之间共用一个碳原子(称螺原子)的二环或多环基团，这些可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。“稠环烷基”指系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳二环或多环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。“桥环烷基”指任意两个环共用两个不直接连接的碳原子的全碳多环基团，这些可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。如下是环烷基的代表性实例，包括但不限于：
Figure PCTCN2022113055-appb-000012
如本文所用，术语“卤代环烷基”是指环烷基的一个或多个(优选为1、2、3或4个)氢被卤素取代，所述的环烷基和卤素如上所定义，当环烷基前具有碳原子数限定(如C3-C8卤代环烷基)指所述的环烷基含有3-8个环碳原子，例如，C3-C8卤代环烷基指含有3-8碳原子的卤代环烷基，代表性实例包括但不限于单氟代环丙基、单氯代环丁基、单氟代环戊基、双氟代环庚基，或类似基团。
术语“烷氧基”指R-O-基团，其中R为烷基，烷基为如上本文所定义，当烷氧基前具有碳原子数限定，如C1-C8烷氧基指所述的烷氧基中的烷基具有1-8个碳原子。烷氧基的代表性示例包括但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基、叔丁氧基，或类似基团。
如本文所用，术语“烷硫基”指R-S-基团，其中R为烷基，烷基为如上本文所定义，当烷硫基前具有碳原子数限定，如C1-C8烷硫基指所述的烷硫基中的烷基具有1-8个碳原子。烷硫基的代表性示例包括但不限于：甲硫基、乙硫基、正丙硫基、异丙硫基、叔丁硫基，或类似基团。
术语“环烷氧基”指R-O-基团，其中R为环烷基，环烷基为如上本文所定义，当环烷氧基前具有碳原子数限定，如C3-C8环烷氧基指所述的环烷氧基中的环烷基具有3-8个碳原子。环烷氧基的代表性示例包括但不限于：环丙氧基、环丁氧基，或类似基团。
术语“环烷硫基”指R-S-基团，其中R为环烷基，环烷基为如上本文所定义，当环烷硫基前具有碳原子数限定，如C3-C8环烷硫基指所述的环烷硫基中的环烷基具有3-8个碳原子。环烷硫基的代表性示例包括但不限于：环丙硫基、环丁硫基，或类似基团。
如本文所用，在单独或作为其他取代基一部分时，术语"氨基"为-NH 2。
如本文所用，在单独或作为其他取代基一部分时，术语"硝基"为-NO 2。
如本文所用，在单独或作为其他取代基一部分时，术语"氰基"为-CN。
如本文所用，在单独或作为其他取代基一部分时，术语"羟基"为-OH。
如本文所用，在单独或作为其他取代基一部分时，术语"巯基"为-SH。
在本说明书中，应解释为所有取代基为未取代的，除非在本文中明确描述为“取代的”。术语“取代”是指特定的基团上的一个或多个氢原子被特定的取代基所取代。特定的取代基为在前文中相应描述的取代基，或各实施例中所出现的取代基，优选地，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3、或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基、C3-C8卤代环烷基、卤素、硝基、-CN、氰基、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基。除非特别说明，某个任意取代的基团可以在该基团的任何可取代的位点上具有一个选自特定组的取代基，所述的取代基在各个位置上可以是相同或不同的。
在本发明中，术语“预防”表示预防疾病和/或它的附随症状的发作或者保护对象免于获得疾病的方法。
本发明所述的“治疗”包括延缓和终止疾病的进展，或消除疾病，并不需要100％抑制、消灭和逆转。在一些实施方案中，与不存在本发明所述的化合物观察到的水平相比，本发明所述化合物将相关疾病(如肿瘤)及其并发症减轻、抑制和/或逆转了例如至少约10％、至少约30％、至少约50％、或至少约80％。
化合物
如本文所用，“本发明化合物”、“本发明式I化合物”、或“式I化合物”可互换使用，指具有式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。应理解，该术语还包括上述组分的混合物。
Figure PCTCN2022113055-appb-000013
具体地，所述的式I化合物如上本发明第一方面所述。
术语“药学上可接受的盐”指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。一类优选的盐是本发明化合物与酸形成的盐，适合形成盐的酸包括(但并不限于)：盐酸、氢溴酸、氢氟酸、硫酸、硝酸、磷酸等无机酸，甲酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、苦味酸、甲磺酸、苯甲磺酸，苯磺酸等有机酸；以及天冬氨酸、谷氨酸等酸性氨基酸。一类优选的盐是本发明化合物与碱形成的金属盐，适合形成盐的碱包括(但并不限于)：氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、磷酸钠等无机碱、氨水、三乙胺、二乙胺等有机碱。
本发明所述的如式I所示化合物可通过常规方法转化为其药学上可接受的盐，例如，可将相应的酸的溶液加入到上述化合物的溶液中，成盐完全后除去溶剂即得本发明所述化合物的相应的盐。
本发明优选的化合物为：
Figure PCTCN2022113055-appb-000014
NNMT基因
NNMT基因英文名为Nicotinamide N-Methyltransferase,不同数据库对该基因有不同的识别号：HGNC:7861；Entrez Gene:4837；Ensembl:ENSG00000166741；OMIM:600008；UniProtKB:P40261。
根据GCF_000001405.25(GRCh37.p13)人类基因组版本,NNMT基因区位于人类11号染色体第114,128,528位bp到114,184,258位bp，总长为55,731bp的DNA序列，包括NNMT基因启动子区、NNMT基因外显子区和NNMT基因内含子区，NNMT基因转录起始位点为第114,166,535位bp。
NNMT基因启动子区为人11号染色体第114,164,535位bp到114,167,034位bp的核苷酸序列，即NNMT基因转录起始位点前2000bp(黑色加深无下划线部分)至转录起始位点本身及其后499bp(非黑色加深有下划线部分)之间的序列，总长为2500bp的区域为NNMT基因启动子区，NNMT基因启动子区的核苷酸序列如下面SEQ ID NO:1所示：
Figure PCTCN2022113055-appb-000015
在本发明中，NNMT基因转录起始位点前1050bp到基因转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
在本发明中，NNMT基因转录起始位点前1050bp到基因转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
在本发明中，NNMT基因转录起始位点前840bp到基因转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
在本发明中，人11号染色体114165695、114165730、114165769、114165804、114165938、114166050、114166066的位点对应于SEQ ID NO:1核苷酸序列的位点如下表1所示：
表1
人11号染色体的位点 对应于SEQ ID NO:1核苷酸序列的位点
114165695位 第1161位
114165730位 第1196位
114165769位 第1235位
114165804位 第1270位
114165938位 第1404位
114166050位 第1516位
114166066位 第1532位
DNA甲基化(DNA methylation)
DNA甲基化(DNA methylation)为DNA化学修饰的一种形式，能够在不改变DNA序列的前提下，改变遗传表现。大量研究表明，DNA甲基化能引起染色质结构、DNA构象、DNA稳定性及DNA与蛋白质相互作用方式的改变，从而调控基因表达。
DNA甲基化是最早被发现、也是研究最深入的表观遗传调控机制之一。广义上的DNA甲基化是指DNA序列上特定的碱基在DNA甲基转移酶(DNA methyltransferase，DNMT)的催化作用下，以s-腺苷甲硫氨酸(S-adenosyl methionine，SAM)作为甲基供体，通过共价键结合的方式获得一个甲基基团的化学修饰过程。这种DNA甲基化修饰可以发生在胞嘧啶的C-5位、腺嘌呤的N-6位及鸟嘌呤的N-7位等位点。通常研究中所涉及的DNA甲基化主要是指发生在CpG二核苷酸中胞嘧啶上第5位碳原子的甲基化过程，其产物称为5—甲基胞嘧啶(5-mC)，是植物、动物等真核生物DNA甲基化的主要形式。DNA甲基化作为一种相对稳定的修饰状态，在DNA甲基转移酶的作用下，可随DNA的复制过程遗传给新生的子代DNA，是一种重要的表观遗传机制。
DNA甲基化反应分为2种类型。一种是2条链均未甲基化的DNA被甲基化，称为从头甲基化(denovo methylation)；另一种是双链DNA的其中一条链已存在甲基化，另一条未甲基化的链被甲基化，这种类型称为保留甲基化(maintenance methylation)。
典型地，DNA甲基化为DNA CpG位点甲基化。CpG双核苷酸在人类基因组中的分布很不均一，而在基因组的某些区段，CpG保持或高于正常概率。CpG位点富集区(又称CpG岛)主要位于基因的启动子(promotor)和外显子区域，是富含CpG二核苷酸的一些区域，约有60％以上基因的启动子含有CpG岛。这里CpG是胞嘧啶(C)—磷酸(p)—鸟嘌呤(G)的缩写。
细胞内基因表达受多种信号传导通路、转录因子和表观遗传修饰的调控。DNA甲基化修饰是表观遗传修饰调控基因表达的重要方式，特定基因区DNA甲基化水平往往影响该基因的表达水平。相对于信号传导通路和转录因子等对基因表达的调控，表观遗传修饰中的DNA甲基化修饰对基因表达的影响更加稳定，并不易被细胞外环境所影响，DNA甲基化修饰容易用现有技术精准检测，因此是较为理想的生物标志物。
癌症
本发明研究表明，本发明所述化合物能够用于预防和/或治疗肿瘤。
在本发明中，术语“肿瘤”、“癌症”、“癌”和“瘤”可互换使用。
在本发明的一个优选例中，所述的肿瘤包括NNMT基因低表达或未表达的肿瘤。代表性地，本发明所述NNMT基因低表达或未表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括DNA甲基化酶高表达的肿瘤。代表性地，本发明所述DNA甲基化酶高表达的肿瘤如上本发明第一方面所述。
本发明所述的DNA甲基化酶包括(但不限于)DNMT1、DNMT3a、DNMT3b，或其组合。优选地，本发明所述的DNA甲基化酶包括DNMT1。
在本发明的一个优选例中，本发明所述的肿瘤包括DNMT1高表达的肿瘤。代表性地，本发明所述DNMT1高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括DNMT3a高表达的肿瘤。代表性地，本发明所述DNMT3a高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括DNMT3b高表达的肿瘤。代表性地，本发明所述DNMT3b高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括UHRF1(泛素样含PHD和环指域蛋白1)高表达的肿瘤。代表性地，本发明所述UHRF1高表达的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，本发明所述的肿瘤包括NNMT基因核苷酸位点甲基化水平高的肿瘤。代表性地，本发明所述NNMT基因核苷酸位点甲基化水平高的肿瘤如上本发明第一方面所述。
在本发明的一个优选例中，所述肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。代表性地，本发明所述NNMT基因区DNA CpG位点甲基化水平高的肿瘤如上本发明第一方面所述。
更具体地，本发明所述的肿瘤如上本发明第一方面所述。
在本发明中，代表性的各肿瘤细胞系对应的肿瘤种类如下表2所示：
表2
肿瘤细胞系 对应的肿瘤种类
NCI-H82 人小细胞肺癌细胞
G-401 人肾癌Wilms细胞
MDA-MB-453 乳腺癌细胞
SW48 人结肠腺癌细胞
WSU-DLCL2 人弥漫大B淋巴瘤细胞
GB-1 人脑胶质母细胞瘤细胞
CFPAC-1 人胰腺癌细胞
SF126 人多形性胶质母细胞瘤细胞
786-O 肾透明细胞腺癌细胞
抗肿瘤药物
在本发明中，所述的抗肿瘤药物可以为本发明所述的式I化合物，或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物。
用途
本发明提供一种本发明所述化合物在预防和/或治疗肿瘤方面中的用途。
特别地，本发明所述化合物对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG 位点甲基化水平高的肿瘤具有显著优异的预防和治疗效果，即NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤对本发明所述化合物敏感。因此，本发明所述的化合物可用于对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤进行精准化治疗。
本发明还提供一种预防和/或治疗肿瘤的方法，给所需的对象施用本发明所述的化合物。
本发明所述化合物对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤具有显著优异的预防和治疗效果，可以在预防和/或治疗肿瘤过程中，先给于对象施用NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂，使得对象的肿瘤的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，然后给予本发明所述化合物对肿瘤进行预防和/或治疗。
在本发明的一个优选例中，所述对象为人和非人哺乳动物(啮齿动物、兔、猴、家畜、狗、猫等)。
在本发明中，将肿瘤的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的方法并没有特别的限制，例如通过基因插入、基因敲除或基因沉默(如转染shRNA)等方法特异性第使得肿瘤的NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高等等。
标志物
本发明还提供一种用于判断肿瘤患者是否适合采用本发明所述化合物进行预防和/或治疗的标志物，所述的标志物包括NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平。
在一个实施方式中，NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平作为判断肿瘤患者是否适合采用本发明所述化合物进行预防和/或治疗的标志物，其方法包括但不限于：
当肿瘤患者的肿瘤细胞中NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高，则该肿瘤患者适合采用本发明所述化合物进行预防和/或治疗；和/或
当肿瘤患者的肿瘤细胞中NNMT基因高表达、DNA甲基化酶低表达、UHRF1低表达、NNMT基因核苷酸位点甲基化水平低、和/或NNMT基因区DNA CpG位点甲基化水平低，则该肿瘤患者不适合采用本发明所述化合物进行预防和/或治疗。
优选地，所述肿瘤患者适合采用本发明所述化合物，其包括肿瘤患者的肿瘤对本发明所述化合物敏感。
优选地，所述肿瘤患者不适合采用本发明所述化合物，其包括肿瘤患者的肿瘤对本发明所述化合物不敏感。
具体地，本发明所述NNMT基因低表达或未表达的肿瘤、DNA甲基化酶(如DNMT1)高表达的肿瘤、UHRF1高表达的肿瘤、NNMT基因核苷酸位点甲基化水平高的肿瘤、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤如上本发明第一方面所述。
本发明还提供了所述标志物(或其表达水平)或其检测试剂的用途，它被用于制备一试剂盒，所述试剂盒用于判断肿瘤患者是否适合采用如本发明化合物进行预防和/或治疗。
组合物或制剂、活性成分的组合和药盒和施用方法
本发明所述的组合物优选为药物组合物，本发明所述的组合物可以包括药学上可接受的载体。
如本文所用“药学上可接受的载体”是指一种或多种相容性固体、半固体、液体或凝胶填料，它们适合于人体或动物使用，而且必须有足够的纯度和足够低的毒性。“相容性”是指药物组合物中的各组分和药物的活性成分以及它们之间相互掺和，而不明显降低药效。
应理解，在本发明中，所述的药学上可接受的载体没有特别的限制，可选用本领域常用材料，或用常规方法制得，或从市场购买得到。药学可接受的载体部分例子有纤维素及其衍生物(如甲基纤维素、乙基纤维素、羟丙甲基纤维素、羧甲基纤维素钠等)、明胶、滑石粉、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油、等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、缓冲剂、螯合剂、增稠剂、pH调节剂、透皮促进剂、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、抑菌剂、无热原水等。
在本发明的一个优选例中，所述的组合物或制剂的剂型为固体制剂、液体制剂或半固体制剂。
在本发明的一个优选例中，所述的组合物或制剂的剂型为口服制剂、外用制剂或注射制剂
代表性地，所述的组合物或制剂的剂型为片剂、注射剂、输液剂、膏剂、凝胶剂、溶液剂、微球或膜剂。
药物制剂应与给药方式相匹配。本发明药剂还可与其他协同治疗剂一起使用(包括之前、之中或之后使用)。使用药物组合物或制剂时，是将安全有效量的药物施用于所需对象(如人或非人哺乳动物)，所述安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
1.本发明提供可指导本发明化合物精准用药的生物标志物，相关生物标志物可有效识别出对本发明化合物敏感的肿瘤患者，提升其治疗效果，并可避免将本发明化合物施用于对其不敏感的肿瘤患者，从而可实现该类药物的精准化应用。
2.本发明首次意外通过系统研究发现NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平能够作为判断特定肿瘤细胞是否适合应用本发明化合物治疗的标志物。
3.NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤对本发明化合物药物敏感程度高，即本发明所述化合物对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的的肿瘤具有优异的治疗作用。因此，本发明所述的化合物可用于对NNMT基因低表达或未表达、DNA甲基化酶高表达、UHRF1高表达、NNMT基因核苷酸位点甲基化水平高、和/或NNMT基因区DNA CpG位点甲基化水平高的肿瘤的精准化治疗，本发明化合物对肿瘤的的精准化治疗具有对肿瘤更优异的预防和治疗效果、药物用量低和副作用小等优势，在提高本发明化合物对肿瘤预防和治疗效果的同时，能够减少给药剂量，降低副作用，提高患者的依从性。此外，NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和NNMT基因区DNA CpG位点甲基化水平的检测手段成熟、稳定、可靠，适合作为生物标志物的开发。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例
氯硝柳胺的结构式如下：
Figure PCTCN2022113055-appb-000016
NNMT的英文为Nicotinamide N-Methyltransferase。
DNMT3a的英文为DNA methyltransferase 3a。
DNMT3b的英文为DNA methyltransferase 3b。
DNMT1的英文为DNA methyltransferase 1。
UHRF1是指泛素样含PHD和环指域蛋白1。
CpG是胞嘧啶(C)-磷酸(p)-鸟嘌呤(G)的缩写。
NNMT基因启动子区核苷酸序列如SEQ ID NO:1所示。
NNMT基因转录起始位点前1050bp到转录起始位点后499bp为SEQ ID NO:1所示核苷酸序列的951-2500位。
NNMT基因转录起始位点前1050bp到转录起始位点前193bp为SEQ ID NO:1所示核苷酸序列的951-1808位。
NNMT基因转录起始位点前840bp到转录起始位点前469bp为SEQ ID NO:1所示核苷酸序列的1161-1532位。
实施例1
使用细胞活性检测试剂检测氯硝柳胺对多种肿瘤细胞系的抑制效果。
实验背景：细胞活力检测采用Promega CellTiter-Glo试剂盒，该试剂盒通过直接检测细胞内ATP含量反应细胞活力。本实验检测氯硝柳胺对多种肿瘤细胞系细胞活力抑制的IC 50值。
实验方法和结果：各肿瘤细胞均培养于相关培养基中(加p/s)，细胞传代3小时后，加入梯度稀释的氯硝柳胺小分子化合物，培养3-4天后测定相关半抑制剂量IC50值(n＝3)。
其中，各肿瘤细胞系名称、来源及培养条件如下：
细胞系NCI-H82(ATCC，编号HTB-175)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系G-401(ATCC，编号CRL-1441)培养于含10％胎牛血清的McCoy’s 5a培养基+P/S；
细胞系MDA-MB-453(ATCC，编号HTB-131)培养于含10％胎牛血清的Leibovitz's L-15培养基+P/S；
细胞系SW48(ATCC，编号CCL-231)培养于含10％胎牛血清的Leibovitz's L-15培养基+P/S；
细胞系WSU-DLCL2(DSMZ，编号ACC-575)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系CFPAC-1(ATCC，编号CRL-1918)培养于含10％胎牛血清的IMDM培养基+P/S；
细胞系786-O(ATCC，编号CRL-1932)培养于含10％胎牛血清的RPMI1640培养基+P/S；
细胞系GB-1(JCRB，编号IFO50489)培养于含10％胎牛血清的DMEM培养基+P/S；
细胞系SF126(JCRB，编号IFO50286)培养于含10％胎牛血清的EMEM培养基+P/S；
实验结果如表3所示：
表3氯硝柳胺对不同细胞株的抑制作用(IC 50)
Figure PCTCN2022113055-appb-000017
Figure PCTCN2022113055-appb-000018
备注：IC 50为半抑制浓度(50％inhibiting concentration)，即达到50％抑制效果时氯硝柳胺的浓度。
由表3可知，对不同肿瘤细胞进行氯硝柳胺化合物的敏感性检测发现NCI-H82(人小细胞肺癌细胞)、G-401(人肾癌Wilms细胞)、MDA-MB-453(乳腺癌细胞)、SW48(人结肠腺癌细胞)和WSU-DLCL2(人弥漫大B淋巴瘤细胞)对氯硝柳胺是敏感的(IC 50值较低)，而786-O(肾透明细胞腺癌细胞系)、CFPAC-1(人胰腺癌细胞)、GB-1(人脑胶质母细胞瘤细胞)和SF126(人多形性胶质母细胞瘤细胞对氯硝柳胺是不敏感的(IC 50值较高)。即与786-O、CFPAC-1、GB-1和SF126细胞抑制效果相比，氯硝柳胺对NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2肿瘤细胞的抑制效果更优异。
实施例2
对氯硝柳胺化合物敏感的5例肿瘤细胞系和不敏感的4例肿瘤细胞系使用RT-qPCR基因表达分析试验检测其NNMT基因的mRNA转录水平，分别测定这些肿瘤细胞系NNMT基因表达情况，结果如图1所示。
由图1可知，用RT-qPCR基因表达分析实验对氯硝柳胺化合物敏感的5株细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的4株细胞(786-O、CFPAC-1、GB-1和SF126)进行NNMT基因的mRNA转录水平检测发现：NNMT基因在敏感株细胞(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)中低表达，而在不敏感株细胞(786-O、CFPAC-1、GB-1和SF126)中高表达。
因此，从图1中可以得出，与NNMT基因高表达的肿瘤细胞株相比，氯硝柳胺对NNMT基因低表达的肿瘤细胞株的抑制作用显著增强，即肿瘤细胞中NNMT基因表达与其对氯硝柳胺化合物的敏感性呈负相关。
实施例3
对氯硝柳胺化合物敏感的5例肿瘤细胞系(NCI-H82、G-401、MDA-MB-453、SW48和WSU-DLCL2)和不敏感的4例肿瘤细胞系(786-O、CFPAC-1、GB-1和SF126)的NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域以及NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域进行重亚硫酸盐测序以检测相关区域内DNA CpG位点甲基化水平，首先利用重烟硫酸盐对基因组DNA进行处理，将未发生甲基化的胞嘧啶脱氨基变成尿嘧啶，而发生了甲基化的胞嘧啶未发生脱氨基，因而可以基于此将经重亚硫酸盐处理的和未处理的测序样本进行比较来发现甲基化的位点，结果如图2、图3和图4所示。
如图2(NNMT基因启动子区)、图3(NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域)及图4(NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域)所示，氯硝柳胺化合物对NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域、NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域内DNA CpG位点甲基化水平高的肿瘤细胞株的抑制作用显著较强，对NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域、NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域内DNA CpG位点甲基化水平低的肿瘤细胞株的抑制作用显著较弱，表明肿瘤细胞NNMT基因启动子区、NNMT基因转录起始位点前1050bp到转录起始位点后499bp之间区域、NNMT基因转录起始位点前1050bp到转录起始位点前193bp之间区域内DNA CpG位点甲基化水平与其对氯硝柳胺的敏感性呈正相关。
实施例4
对氯硝柳胺敏感的3例肿瘤细胞系(NCI-H82、G-401和WSU-DLCL2)和不敏感的3例肿瘤细胞系(786-O、CFPAC-1和SF126)的NNMT基因转录起始位点前840bp(即人11号染色体第114165695位)到基因转录起始位点前469bp(即人11号染色体第114166066位)区域内DNA CpG位点甲基化情况进行分析。
首先对细胞基因组DNA进行重亚硫酸盐处理，随后用相应引物对该区域进行PCR扩增、测序分析以检测该DNA区域内CpG位点的甲基化水平。分析发现对氯硝柳胺敏感的G-401、NCI-H82、WSU-DLCL2细胞系中该区域内7个CpG位点(人11号染色体114165695位、114165730位、114165769位、114165804位、114165938位、114166050位、114166066位)几乎全部被甲基化，而对氯硝柳胺不敏感的CFPAC-1、786-O、SF126细胞系中该区域这7个CpG位点都未被甲基化，相关位点甲基化情况如图5所示。
如图5(NNMT基因转录起始位点前840bp到转录起始位点前469bp之间区域CpG位点甲基化情况)所示，氯硝柳胺对NNMT基因启动子区或NNMT基因相关区域内DNA CpG位点甲基化水平高的肿瘤细胞株的抑制作用显著较强，对NNMT基因启动子区或NNMT基因相关区域内DNA CpG位点甲基化水平低的肿瘤细胞株的抑制作用显著较弱，表明肿瘤细胞NNMT基因启动子区或NNMT基因相关区域内DNA CpG位点甲基化水平与其对氯硝柳胺的敏感性呈正相关。
其中，人11号染色体114165695、114165730、114165769、114165804、114165938、114166050、114166066的位点对应于SEQ ID NO:1核苷酸序列的位点如下所示：
Figure PCTCN2022113055-appb-000019
实施例5
细胞DNA的甲基化水平由DNA甲基化酶DNMT3a、DNMT3b和DNMT1维持，DNMT3a、DNMT3b能对DNA进行从头进行甲基化，而DNMT1能在蛋白UHRF1(泛素样含PHD和环指域蛋白1)的帮助下对已甲基化的DNA进行复制维持，本发明检测肿瘤中NNMT表达与DNMT1、UHRF1、DNMT3a以及DNMT3b表达的相关性。
实验方法和结果：从公共数据库中(Cancer Cell Line Encyclopedia，CCLE,共1019株细胞)得到多种细胞中NNMT基因、DNMT1、UHRF1、DNMT3a以及DNMT3b的表达数据，然后用生物信息学的方法分析这些细胞中NNMT表达和DNMT1、UHRF1、DNMT3a、DNMT3b表达的相关性，分析各细胞NNMT基因表达水平和DNMT1、UHRF1、DNMT3a以及DNMT3b的表达水平的相关性，实验结果如图6所示。
从图6中可以看出，各细胞中NNMT的表达和DNA甲基化酶、UHRF1的表达呈负相关。因此，氯硝柳胺对DNMT1高表达、UHRF1高表达、DNMT3a高表达和/或DNMT3b高表达的肿瘤具有更优异的治疗效果，即DNMT1高表达、UHRF1高表达、DNMT3a高表达和/或DNMT3b高表达的肿瘤对氯硝柳胺敏感，肿瘤细胞中DNMT1、UHRF1、DNMT3a以及DNMT3b的表达与其对氯硝柳胺的敏感性呈正相关。
实施例6
肿瘤细胞NNMT基因表达水平和其对氯硝柳胺的敏感性呈显著负相关，可进一步检测NNMT基因在肿瘤细胞中对氯硝柳胺敏感性中的作用。
实验方法和结果：通过病毒载体将NNMT基因导入到NCI-H82细胞中使得NCI-H82细胞过表达NNMT蛋白，得到过表达NNMT蛋白的NCI-H82细胞(NCI-H82(ov-NNMT))；此外，采用不负载NNMT基因的空病毒转染NCI-H82细胞得到对照NCI-H82细胞(NCI-H82(Con))。
Western Blot实验检测对照NCI-H82细胞(NCI-H82(Con))和过表达NNMT蛋白的NCI-H82细胞(NCI-H82(ov-NNMT))中的NNMT蛋白表达水平如图7所示，从图7中可以看出相对于NCI-H82(Con)细胞，NCI-H82(ov-NNMT)细胞过表达NNMT蛋白。
采用Promega CellTiter-Glo试剂盒(该试剂盒通过直接检测细胞内ATP含量反应细胞活力)检测氯硝柳胺对对照NCI-H82细胞(NCI-H82(Con))和过表达NNMT蛋白的NCI-H82细胞(NCI-H82(ov-NNMT))的半抑制浓度IC 50值(n＝3)，结果如下表4所示：
表4氯硝柳胺对对照NCI-H82细胞(NCI-H82(Con))和过表达NNMT蛋白的NCI-H82细胞(NCI-H82(ov-NNMT))的半抑制浓度IC 50值(μM)
化合物 NCI-H82(Con)细胞 NCI-H82(ov-NNMT)细胞
氯硝柳胺 0.12μM 0.42μM
备注：IC 50为半抑制浓度(50％inhibiting concentration)，即达到50％抑制效果氯硝柳胺的浓度。
从表4中可以看出，过表达NCI-H82细胞的NNMT蛋白能够降低NCI-H82细胞对氯硝柳胺的敏感性，因此，肿瘤细胞的NNMT表达水平和其对氯硝柳胺的敏感性呈显著负相关。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗肿瘤；
    Figure PCTCN2022113055-appb-100001
    其中，
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10各自独立地为氢、卤素、-CN、羟基、巯基、硝基、氨基、-COOH、-CHO、取代或未取代的C1-C12烷基、取代或未取代的C3-C12环烷基、取代或未取代的C1-C12烷氧基、或取代或未取代的C1-C12烷硫基；
    Z为
    Figure PCTCN2022113055-appb-100002
    其中，所述的任一“取代”是指基团上的一个或多个(优选为1、2、3或4个)氢原子被选自下组的取代基所取代：C1-C8烷基、C3-C8环烷基、C1-C8卤代烷基、C3-C8卤代环烷基、卤素、硝基、-CN、氰基、羟基、巯基、氨基、C1-C8烷氧基、C1-C8烷硫基、C3-C8环烷氧基、C3-C8环烷硫基。
  2. 如权利要求1所述用途，其特征在于，所述的式I化合物为：
    Figure PCTCN2022113055-appb-100003
  3. 如权利要求1所述用途，其特征在于，所述的肿瘤包括NNMT基因低表达或未表达的肿瘤；
    所述的肿瘤包括DNA甲基化酶高表达的肿瘤；
    所述的肿瘤包括UHRF1高表达的肿瘤；
    所述的肿瘤包括NNMT基因核苷酸位点甲基化水平高的肿瘤；和/或
    所述的肿瘤包括NNMT基因区DNA CpG位点甲基化水平高的肿瘤。
  4. 如权利要求3所述用途，其特征在于，所述DNA甲基化酶选自下组：DNMT1、DNMT3a、DNMT3b，或其组合；和/或
    所述NNMT基因区DNA CpG位点甲基化水平包括NNMT基因启动子区DNA CpG位点甲基化水平。
  5. 如权利要求1所述用途，其特征在于，所述的肿瘤选自下组：肺癌、肾癌、乳腺癌、结肠癌、淋巴癌、白血病、胰腺癌、脑瘤、肝癌、前列腺癌，或其组合。
  6. 一种用于判断肿瘤患者是否适合采用如权利要求1所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗肿瘤的标志物，其特征在于，所述的标志物包括NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平。
  7. 一种检测试剂盒的用途，其特征在于，用于制备一伴随诊断试剂盒，所述伴随诊断试剂盒用于判断肿瘤患者是否适合采用如权利要求1所述的式I化合物、或其光学异构体或其外消旋体、或其溶剂化物、或其药学上可接受的盐、或其氘代化合物进行预防和/或治疗；
    所述的检测试剂盒包括：
    (i)用于检测NNMT基因表达水平、DNA甲基化酶表达水平、UHRF1表达水平、NNMT基因核苷酸位点甲基化水平、和/或NNMT基因区DNA CpG位点甲基化水平的检测试剂。
  8. 一种NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂的用途，其特征在于，用于制备组合物或制剂，所述的组合物或制剂用于增强抗肿瘤药物的抗肿瘤效果。
  9. 一种组合物，其特征在于，所述的组合物包括：
    (1)第一活性成分，所述的第一活性成分包括抗肿瘤药物；和
    (2)第二活性成分，所述的第二活性成分包括NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂。
  10. 一种药盒，其特征在于，所述药盒包括：
    (A)含有第一活性成分的第一制剂，所述的第一活性成分包括抗肿瘤药物；和
    (B)含有第二活性成分的第二制剂，所述的第二活性成分包括NNMT基因抑制剂、DNA甲基化酶促进剂、UHRF1促进剂、NNMT基因核苷酸位点甲基化促进剂、和/或NNMT基因区DNA CpG位点甲基化促进剂。
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