WO2023237080A1 - Akr1c3酶激活前药治疗癌症患者的方法 - Google Patents

Akr1c3酶激活前药治疗癌症患者的方法 Download PDF

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WO2023237080A1
WO2023237080A1 PCT/CN2023/099294 CN2023099294W WO2023237080A1 WO 2023237080 A1 WO2023237080 A1 WO 2023237080A1 CN 2023099294 W CN2023099294 W CN 2023099294W WO 2023237080 A1 WO2023237080 A1 WO 2023237080A1
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mutation
group
nrf2
akr1c3
ast
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PCT/CN2023/099294
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English (en)
French (fr)
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李安蓉
齐天阳
段建新
孟繁英
刘星
王易之
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深圳艾欣达伟医药科技有限公司
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Definitions

  • the present invention relates to methods of treating cancer, particularly cancers with specific genes.
  • AST-3424 is a DNA alkylating agent prodrug activated by the AKR1C3 enzyme that has entered Phase II clinical trials in both China and the United States (clinical registrations in China are CTR20201915, CTR20201908, CTR20191399, CTR20191371, and clinical registrations in the United States are NCT04315324, NCT03592264).
  • the AKR1C3 enzyme highly expressed in tumors activates and releases DNA alkylating agents to exert anti-tumor effects.
  • the anti-cancer prodrugs activated by the above-mentioned AKR1C3 enzyme need to detect the expression level of the AKR1C3 enzyme in patients before use. Only patients who reach the predetermined expression level will have better therapeutic effects.
  • phase II clinical results of the drug researchers have determined that patients with AKR1C3 enzyme expression levels of H-score ⁇ 135 (IHC) will be enrolled in phase II clinical patients (Safety, Pharmacokinetics, and Clinical Activity of OBI-3424, an AKR1C3-Activated Prodrug, in Patients with Advanced or Metastatic Solid Tumors: A Phase 1 Dose-Escalation Study, J Clin Oncol 40, 2022, suppl 16; abstr 3030, DOI: 10.1200/JCO.2022.40.16_suppl.3030).
  • IHC H-score ⁇ 135
  • AKR1C3 enzyme-activating prodrugs AST-3424 and AST have very significant inhibitory effects on PDX and CDX tumor models with Kras-G12D/G12C mutations (1 and 2 of the Examples) , Parts 3, 4 and subsequent research parts), the inventor speculates whether the Kras-G12D/G12C mutation is somehow related to the high expression of the AKR1C3 enzyme: Will the Kras-G12D/G12C mutation lead to the high expression of the AKR1C3 enzyme?
  • RNA detection and AKR1C3 protein expression level detection were both high-level expression, which confirmed the above speculation: Kras-G12D/G12C mutation is related to high expression of AKR1C3 enzyme, and Kras-G12D/G12C mutation is accompanied by high expression of AKR1C3 enzyme. of.
  • the distribution trend of AKR1C3 expression in the KRAS G12D PDX model is mainly concentrated in medium to high expression, accounting for 90.4%;
  • AKR1C3 expression is evenly distributed between low, medium and high expression in the KRAS G12C PDX model, of which 66.7% are expressed at medium or above levels;
  • the distribution trend of AKR1C3 expression in the KRAS G13D PDX model is mainly concentrated in medium and high expression, of which 83.3% are expressed at medium or above levels.
  • KRAS G12D mutant tumor cells can directly upregulate and activate NRF2 through the RAF-MEK-ERK-Jun signaling pathway (Nature, 2011, 475:106) (Nature, 2011, 475:106; Cancer Res, 2014, 74: 7430).
  • Activated NRF2 can further upregulate and activate a series of downstream genes including AKR1C3 (Chem Res Toxicol, 2017, 30:162; Cancer, 2019, 11:1715).
  • prodrugs activated by the AKR1C3 enzyme can directly detect whether the patient's tumor or cancer tissue has the ability to up-regulate on the basis of the known selection/screening of patients by detecting the expression level of the AKR1C3 enzyme. Or a gene mutation that activates NRF2; or whether the patient is detected to have a gene mutation that can upregulate or activate NRF2 for screening.
  • a method for treating cancer patients with AKR1C3 enzyme-activating prodrugs characterized by:
  • the patient's tumor or cancer tissue is detected to have a gene mutation that can upregulate or activate NRF2;
  • the patient was found to have a genetic mutation that upregulates or activates NRF2.
  • AKR1C3 enzyme-activated prodrugs for preparing drugs for treating cancer is characterized by:
  • the patient's tumor or cancer tissue is detected to have a gene mutation that can upregulate or activate NRF2;
  • the patient was found to have a genetic mutation that upregulates or activates NRF2.
  • a drug containing an AKR1C3 enzyme-activating prodrug compound, and its indication is the treatment of cancer patients whose tumors or cancer tissues are detected to have gene mutations that can upregulate or activate NRF2;
  • the patient was found to have a genetic mutation that upregulates or activates NRF2.
  • Treating cancer patients with AKR1C3 enzyme-activating prodrugs which includes the steps of applying a drug or preparation containing the AKR1C3 enzyme-activating prodrug; and the step of measuring the NRF2 content or expression level of cancer cells or tissues of the patient,
  • NRF2 content or expression level is measured to be equal to or greater than the predetermined value, a drug or preparation containing an AKR1C3 enzyme-activating prodrug is administered to the patient.
  • a method of treating cancer or tumors which includes the step of applying a drug or preparation containing an AKR1C3 enzyme-activating prodrug; and the step of regulating NRF2 content or expression level,
  • a medicine or preparation containing an AKR1C3 enzyme-activating prodrug is administered to the patient.
  • AKR1C3 enzyme-activated prodrugs for preparing drugs for treating cancer is characterized by:
  • the patient's tumor or cancer tissue is detected to have NRF2 content or expression level equal to or greater than a predetermined value
  • the patient is detected to have an NRF2 content or expression level equal to or greater than a predetermined value.
  • a medicine containing an AKR1C3 enzyme-activating prodrug compound, and its indication is the treatment of cancer patients whose tumors or cancer tissues are detected to have NRF2 content or expression levels equal to or greater than a predetermined value;
  • the patient is detected to have an NRF2 content or expression level equal to or greater than a predetermined value.
  • NRF2 content or expression levels/gene mutations that upregulate or activate NRF2 are detected using patient's tumor or cancer tissue samples, but due to various reasons, such as the patient is unable to provide qualified tumor or cancer tissue samples, or the current When the detection method cannot accurately detect, or the tumor or cancer tissue can no longer reflect the patient's recent condition, it may be necessary to detect or diagnose the patient's own NRF2 content or expression level/gene mutations that upregulate or activate NRF2.
  • blood can be used for genetic testing. Mutation detection, the test results can represent the patient's tumor or cancer tissue; circulating tumor cell (CTC) detection can detect specific cancer cells (all tumor cells free in the peripheral blood) through the patient's peripheral blood test samples.
  • CTC circulating tumor cell
  • gene mutations can be diagnosed or assisted in diagnosis through the genetic mutations of the parents in the biological parent-child relationship.
  • predetermined value is to detect that the patient's NRF2 content or expression level is equal to or greater than a certain predetermined value through a certain method (such as in vitro diagnosis), and to give the patient a drug or formula containing the compound of the present application to achieve this goal.
  • a certain method such as in vitro diagnosis
  • This predetermined value is determined after at least determining the cancer type and specific drug or preparation, conducting clinical trials on different patient populations, and performing statistical analysis based on the final clinical trial results and data. This predetermined value cannot be given now, but it can be Finalized through clinical trials.
  • gene mutations that can upregulate or activate NRF2 are selected from the following gene mutations:
  • the transcription factor NRF2 protein is a recombinant and synthetic protein. Its full name in Chinese is nuclear factor E2p45 related factor 2 (nuclear fact or erythroid 2 p45 related factor 2). It is a member of the Cap'n'collar (CNC) transcription factor family and consists of seven Neh domains. (Nrf2 ECH homology domain), each domain has different functions:
  • Neh1 CNC-bZIP domain is responsible for binding and dimerization with small Maf (sMAF) protein
  • Neh2 domain mediates interaction with Keap1 through DLG and ETGE motifs
  • Neh4, Neh5, and Neh3 domains are important for transactivation of NRF2;
  • the Neh6 domain is a serine-rich region that regulates stability.
  • Gene mutations that up-regulate or activate NRF2 refer to gene mutations that can activate NRF2 protein activity or directly up-regulate NRF2 protein expression. Because they are upstream of NRF2, that is, the NRF2 signaling classic pathway (Wu S, et al. Nrf2 in cancers: A double -edged sword. Cancer Med. 2019; 8(5): 2252-2267.) and Nrf2 signaling non-canonical pathway (J iang T, et al. p62 links autophagy and Nrf2 signaling. Free Radic Biol Med. 2015; 88( PtB):199-204.), numerous research results have proven that many genes can activate or up-regulate NRF2 protein after mutation. These Gene mutations include:
  • KRAS mutation the full name of KRAS gene is Kirsten rat sarcoma viral oncogene homolog (Kirsten rat sarcoma virus oncogene homolog), which belongs to the RAS superprotein family.
  • the protein encoded by the KRAS gene is a small GTPase.
  • KRAS1 located on the short arm of chromosome 6; and KRAS2, located on the short arm of chromosome 12.
  • KRAS1 is a "pseudogene” and cannot be transcribed into RNA, so it has no function.
  • KRAS2 is the "true gene", which can be transcribed and translated into proteins and has biological activity.
  • the KRAS genes and proteins studied in patents and literature reports refer to the "KRAS2" gene and its protein products. This application also refers to the KRAS gene and its protein product.
  • KRAS gene mutations 97% are mutations in the 12th or 13th amino acid residue. Structural studies show that most of these gene mutations interfere with the ability of KRAS to hydrolyze GTP.
  • KRAS gene mutations are mainly concentrated in codon positions 12, 13 and 61. Among them, mutations at codon position 12 account for more than 80%, including G12A, G12C, G12D, G12R, G12S, G13D and G12V. The main three mutations are G12C, G12V, and G13D.
  • KEAP1 KELCH-like ECH Associated Protein 1 recombinant protein (Recombinant Kelch Like ECH Associated Protein 1, referred to as KEAP1).
  • nuclear factor E2p45 related factor 2 nuclear factor erythroid 2 p45 related factor 2.
  • P21 is a negative regulator of the cell cycle, a member of the CDKIs family, and the cyclin-dependent kinase inhibitor p21.
  • P62 protein is encoded by the SQSTM1 gene, and can also be written as p62/SQSTM1, formerly known as Sequestosome-1. P62 protein is a receptor for selective autophagy.
  • FTL1 Ferritin, Light Polypeptide 1 (FTL 1).
  • FTH1 ferritin, heavy polypeptide 1 (FTH 1).
  • HMOX1 heme oxygenase 1
  • HMOX1 Greek: heme oxygenase 1, abbreviated HMOX1 or HO-1
  • HMOX1 heme oxygenase 1
  • GSR glutathione reductase
  • SLC7A11 solute carrier family 7 member 11 (SLC7A11) recombinant protein, Recombinant Solute Carrier Family 7, Member 11 (SLC7A11).
  • GCLC Glutamate Cysteine Ligase Catalytic Subunit
  • GCLC Glutamate Cysteine Ligase
  • GPX2 Glutathione Peroxidase 2 (GPX2), Glutathione Peroxidase 2.
  • TXN1 recombinant human thioredoxin 1, Thioredoxin 1, also written as His; Trx; ADF; TRX1, etc.
  • Prdx1 peroxide oxidoreductase protein
  • ABCC1 ATP binding cassette sub-family C member 1
  • ATP binding cassette sub-family C member 1 ATP binding cassette sub-family C member 1.
  • G6PD 6-Phosphogluconic dehydrogenase
  • 6-Phosphogluconic dehydrogenase 6-Phosphogluconic dehydrogenase
  • ME1 malic enzyme (malic enzyme, ME).
  • IDH1 isocitrate dehydrogenase soluble, soluble isocitrate dehydrogenase 1.
  • EGFR epidermal growth factor receptor
  • HER1 epidermal growth factor receptor
  • HER2 erbB2, NEU
  • HER3(erbB3) HER4(erbB4).
  • the HER family plays an important regulatory role in cellular physiological processes.
  • KRAS mutations were selected from six subtypes: G12D, G12V, G12R, G12C, G12A, and G13D.
  • Any one or two gene mutations in the gene corresponding to KRAS can be detected and diagnosed through commercially available (companion) diagnostic kits.
  • NGS sequencing (YS 450 gene NGS panel) can also be used to determine the specific KRAS mutation subtype.
  • the KRAS mutation is selected from the group consisting of KRAS-G12D mutations.
  • the TMB (tumor gene mutation burden) level of the gene mutation is medium.
  • TMB Tumor mutation load (burden), that is, tumor gene mutation load
  • burden Tumor mutation load
  • TMB Tumor mutation load
  • Mb represents every million bases
  • CheckMate-032 This is a phase II clinical trial that included 401 patients with advanced lung cancer who failed first-line treatment and received PD-1 inhibitors alone or in combination with ipilimumab.
  • TMB Tumor mutation load
  • the effective rates of the three groups are 62%, 20%, and 23% respectively.
  • the effective rate of the people with high TMB is higher 3 times; and the median overall survival of the three groups were: 22.0 months, 3.6 months, 3.4 months - 22.0 months and 3.4 months, a 6-fold difference!
  • AKR1C3 enzyme-activated prodrug means that the compound is a prodrug.
  • the prodrug molecule reacts with the AKR1C3 enzyme and releases a cytotoxic anti-tumor compound after the reaction.
  • AKR1C3 enzyme-activating prodrugs meet but are not limited to at least one of the following conditions:
  • the cancer cell proliferation inhibitory effect of a compound detected is smaller than the cancer cell proliferation inhibitory effect of the compound detected in the absence of the AKR1C3 inhibitor.
  • the cancer cell proliferation inhibitory effect is quantified using IC 50 . Then, if the IC 50 of a compound against a cancer cell line detected in the presence of an AKR1C3 inhibitor is greater than the IC 50 detected in the absence of an AKR1C3 inhibitor, the compound can be determined to be an anticancer drug (cleavage prodrug) that activates AKR1C3. Lysis-Prodrug).
  • Specific examples include the following patent documents:
  • patent PCT/US2016/021581, publication number WO2016145092A1 corresponds to Chinese application number 2016800150788, publication number CN107530556A; PCT/US2016/062114, publication number WO2017087428, corresponds to Chinese application number 2016800446081, publication number CN1082 90911A; PCT/US2016/025665, public No.
  • WO2016161342 corresponding to Chinese application number 2016800200132, the compound disclosed in the publication number CN108136214A is a cleavage prodrug, and the original drug that plays a role after final cleavage and metabolism is As well as drugs such as paclitaxel and camptothecin;
  • patent PCT/NZ2019/050030 publication number WO2019190331, corresponding to Chinese application number CN2019800234236
  • the compound disclosed in publication number CN111918864A is a cleavage prodrug, which is eventually cleavage and metabolized into the original drug that plays a role It is a nitrogen mustard structural drug;
  • a certain compound has significant differences in its inhibitory effect on cancer cells with different expression levels of AKR1C3 enzyme, and its inhibitory effect on cancer cells with high expression of AKR1C3 enzyme is much greater than that of cancer cells with low expression of AKR1C3 enzyme.
  • the inhibitory effect is quantified using IC 50 , if the IC 50 value of a certain compound in cancer cells that highly express the AKR1C3 enzyme is less than the IC 50 value in cancer cells that have a low expression of AKR1C3, the compound can be determined to be an anticancer drug that activates AKR1C3.
  • Specific examples include the compounds disclosed in patent PCT/CN2020/120281 and publication number WO2021068952A1. The entire text of this patent document is hereby incorporated into the text of this patent application.
  • the AKR1C3 enzyme activation prodrug is selected from the group consisting of structural formulas (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), Compounds of (11) and their salts, esters, solvates, and isotopomers:
  • Formula (1) is an alkylating agent prodrug compound activated by AKR1C3 enzyme, and more specifically, it is a DNA alkylating agent prodrug compound activated by AKR1C3 enzyme;
  • X 10 is O, S, SO or SO 2 ;
  • R 1 and R 2 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • X, Y and Z are each independently hydrogen, CN, halo, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocyclic ring, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R is hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered hetero Ring, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R 13 and R 14 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle or ether
  • T comprises an aminophosphate alkylating agent comprising one or more alkylating agents of the Z 5 -X 5 -Y 5 moiety bonded to the -OP(Z 1 ) moiety, wherein Z 5 is a heteroatom containing nitrogen, sulfur or oxygen, X 5 is a substituted or unsubstituted ethylene group, Y 5 is a halo group or another leaving group, or Z 5 -X 5 -Y 5 are formed together the aziridinyl (NCH 2 CH 2 ) moiety and Z 1 is O or S; and
  • alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, ether groups are substituted or unsubstituted.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , R 9 and R 10 are as recorded in the claims in patent application PCT/CN2020/089692 and publication number WO2020228685A1.
  • the synthetic preparation method is also described in the above-mentioned application, and its full text is hereby incorporated into this application. It is specifically defined as:
  • R 1 is C 6 -C 10 aryl or Z-substituted aryl, 4-15-membered heterocycle or Z-substituted heterocycle, 5-15-membered heteroaryl or Z-substituted heteroaryl, 7-15-membered fused ring Or Z substituted fused ring;
  • R 2 is hydrogen, halogen atom, cyano or isocyanate, hydroxyl, mercapto, amine, OTs, OMS, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl base, C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, ether of 1-6 carbon atoms or Z-substituted alkoxy group of 1-6 carbon atoms, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCOO-R 6 , -COOR 6 , -NR 6 COR
  • R 3 is hydrogen, halogen, cyano or isocyanate, hydroxyl, mercapto, amine, OTs, OMS, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl , C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z Substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z-substituted C 1 -C 6 alkoxy, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OCO-R 6 , -OCOO-R 6 , -COOR 6 , -
  • R 4 and R 5 are each independently hydrogen, halogen atom, cyano group or isocyanate group, hydroxyl group, mercapto group, amino group, OTs, OMS, C 1 -C 6 alkyl group or Z-substituted alkyl group, C 2 -C 6 Alkenyl or Z-substituted alkenyl, C 2 -C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4 -15-membered heterocycle or Z-substituted heterocycle, 5-15-membered heteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z-substituted C 1 -C 6 alkoxy, -CONR 6 R 7 , -SO 2 NR 6 R 7 , -SO 2 R 6 , -OC
  • R 6 and R 7 are each independently hydrogen, cyano or isocyanate, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 -C 6 alkyne base or Z-substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl, C 6 -C 10 aryl or Z substituted aryl, 4-15 membered heterocycle or Z substituted heterocycle, 5-15 Metaheteroaryl or Z-substituted heteroaryl, C 1 -C 6 alkoxy or Z-substituted C 1 -C 6 alkoxy, or R 6 and R 7 groups together with the atoms to which they are bonded form 5- 7-membered heterocyclyl or Z-substituted 5-7-membered heterocyclyl;
  • R 8 and R 10 are each independently hydrogen, deuterium, aryl or Z-substituted aryl, C 1 -C 6 alkyl or Z substituted alkyl, C 2 -C 6 alkenyl or Z substituted alkenyl, C 2 - C 6 alkynyl or Z substituted alkynyl, C 3 -C 8 cycloalkyl or Z substituted cycloalkyl and one of them must be hydrogen or deuterium;
  • R 9 is a substituted C 6 -C 10 aryl group with at least one fluorine atom or nitro substitution, a substituted 4-15 membered heterocyclic ring with at least one fluorine atom or nitro substitution, at least one fluorine atom or nitro substitution Substituted 5-15 membered heteroaryl.
  • the Z substituent is a halogen atom, cyano or isocyanate, hydroxyl, mercapto, amine, OTs, OMS, C 1 -C 3 alkyl or substituted alkyl, C 1 -C 3 alkoxy or substituted alkoxy , C 2 -C 3 alkenyl or substituted alkenyl, C 2 -C 3 alkynyl or substituted alkynyl, C 3 -C 8 cycloalkyl or substituted cycloalkyl, aromatic ring, heterocyclic ring, heteroaromatic ring and condensed Ring or substituted aromatic ring, heterocycle, heteroaromatic ring and fused ring, the substitution mode is mono-substitution or geminal disubstitution;
  • the substituents of the substituted C 6 -C 10 aryl group, the substituted 4-15 membered heterocyclic group, and the substituted 5-15 membered heteroaryl group in R 9 are halogen atoms, nitro groups, cyano groups or isocyanate groups, hydroxyl groups, and amino groups , C 1 -C 3 alkyl or alkoxy group, alkenyl, alkynyl, cycloalkyl or benzene ring, substituted benzene ring, C 1 -C 3 alkoxy group or halogen atom substituted alkoxy group.
  • Formula (4) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, it is an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;
  • R 100 , R 101 and R 102 are each independently hydrogen, C 1 -C 8 alkyl, C 6 -C 12 aryl; or R 101 and R 102 together with the nitrogen atom to which they are attached form a 5-7 yuan Heterocycle;
  • alkyl group and the aryl group are each substituted by 1-3 halo groups or 1-3 C 1 -C 6 alkyl groups;
  • R 1 and R 2 are each independently phenyl or methyl
  • X, Y and Z are each independently hydrogen or halo
  • R is hydrogen or C 1 -C 6 alkyl or halogen substituted alkyl.
  • Rw is as recorded in the claims in the patent application PCT/CN2020/120281 and the publication number WO2021068952A1.
  • the synthesis and preparation method of the specific compound is also recorded in the above application, and its full text is hereby incorporated into this application. Specifically defined as:
  • R 1 is H, C 1-6 alkyl, C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein the C 1-6 alkyl, C 3-6 cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl and phenyl are optionally substituted by 1, 2 or 3 R a ;
  • Each R a is independently H, F, Cl, Br, I, -CN, -OH, C 1-3 alkoxy or C 1-3 alkyl;
  • R 2 is H or C 1-6 alkyl
  • R 1 and R 2 are connected together, and the N atoms to which they are connected together form a 4-6 membered heterocycloalkyl group, wherein the 4-6 membered heterocycloalkyl group is optionally substituted by 1, 2 or 3 R b ;
  • Each R b is independently H, F, Cl, Br, I, -CN, -OH, -NH 2 , -OCH 3 , -OCH 2 CH 3 , -CH 3 or -CH 2 CH 3 ;
  • R 3 is H, F, Cl, Br, I, -OH, -NH 2 , C 1-3 alkoxy or C 1-3 alkyl;
  • R2 and R3 are connected together to make the structural unit for
  • T 1 is -(CR c R d ) m - or -(CR c R d ) n -O-;
  • n 1, 2 or 3;
  • n 1 or 2;
  • R 7 and R 8 are each independently H, F, Cl, Br or I;
  • R 9 and R 10 are each independently H, F, Cl, Br, I, -CN or
  • the 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl groups each contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.
  • E is SO or SO 2 ;
  • Each R is independently H or C1-C6 alkyl
  • R 1 is H, C1-C6 alkyl, CH 2 (CH 2 )nOH, CH 2 CH(OH)CH 2 OH, phenyl, pyridyl, benzyl or pyridylmethyl, provided that when R 1 is benzene group, pyridyl, benzyl or pyridylmethyl, R 1 is optionally replaced by C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, OR 6 , N(R 6 )(R 7 ), CFH 2 , CF 2 H, CF 3 , F, Cl, Br, I, OCF 3 , COR 6 , CON(R 6 )(R 7 ), SOR 6 , SON(R 6 )(R 7 ), SO 2 R 6 , SO 2 N(R 6 )(R 7 ), CN or NO 2 substituted;
  • W is CH 2 , O, S, SO or SO 2 ;
  • n ranges from 0 to 6;
  • X, Y and Z are each independently hydrogen, CN, halo, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • Each R is independently hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4 -15-membered heterocycle, 5-15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;
  • R 13 and R 14 are each independently hydrogen, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl Or ether, or R 13 and R 14 together with the nitrogen atom to which they are bonded form a 5-7 membered heterocyclic group;
  • L 1 and D are as defined in the specification, and the specific definitions are as follows
  • L 1 is selected from:
  • R 40 and R 41 are independently hydrogen, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 3 -C 8 cycloalkyl, C 6 -C 10 Aryl, 4-15 membered heterocycle or 5-15 membered heteroaryl;
  • R 42 is a C 2 -C 3 alkylene or heteroalkylene group optionally substituted by 1-3 C 1 -C 6 alkyl groups;
  • V (-) is any anion, preferably pharmaceutically acceptable anions;
  • D is the moiety such that D-OH is an anticancer drug, where OH is an aliphatic or phenolic hydroxyl group, or is an OH moiety attached to a phosphorus atom as provided herein; in other words, D is the anticancer drug D-OH removed The remaining group after the hydroxyl group;
  • L 1 is:
  • R 40 is as defined above, R 43 is hydrogen or together with D forms a heterocycle, and the phenyl moiety is optionally substituted;
  • D is the part that makes D-NR 43 H an anticancer drug; in other words, D is the remaining group after the anticancer drug D-NR 43 H removes the amino or amine;
  • L 1 is a bond, -OC(R 40 R 41 )-, -OC(R 40 R 41 )-NR 40 R 41 (+)-C(R 40 R 41 )- or
  • R 40 , R 41 and V are as defined above;
  • D is an anticancer drug containing a primary or secondary amine, wherein the primary or secondary amine is bonded to L 1 ;
  • alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, ether groups are optionally substituted.
  • R 1 , R 2 , R 3 , R 4 and T are as described in the claims in patent application PCT/CN2021/118597 and publication number WO2022057838A1.
  • the synthesis and preparation methods of specific compounds are also recorded in the above applications. Its full text is hereby incorporated into this application and is specifically defined as:
  • T is N or CH
  • R 1 and R 2 are each independently H, F, Cl, Br, I or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2 or 3 R a ;
  • Each R a is independently F, Cl, Br, I, -CN, -OH or -NH 2 ;
  • R 3 and R 4 are each independently H, F, Cl, Br, I, CN, C 1-3 alkyl, C 1-3 alkoxy, Wherein, the C 1-3 alkyl group is optionally substituted by 1, 2 or 3 Re ;
  • R b and R c are each independently H, -CH 3 , -CH 2 CH 3 , -(CH 2 ) 2 CH 3 , -CH(CH 3 ) 2 ;
  • R d is -CH 3 , -CH 2 CH 3 , -(CH 2 ) 2 CH 3 , -CH(CH 3 ) 2 ;
  • Each Re is independently F, Cl, Br, I, -CN, -OH or -NH2 .
  • the two X's are each independently CR 15 or N;
  • R 13 and R 14 are each independently hydrogen, C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, C 6 -C 20 aryl, 5-20 membered ring Heterocyclyl, halogen-substituted C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, halogen substituted C 6 -C 20 aryl, or halogen substituted 5-20 membered ring heterocyclyl , and R 13 and R 14 are not hydrogen at the same time;
  • R 10 is hydrogen, C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, C 6 -C 20 aryl, 5-20 membered ring heterocyclyl, halogen substituted C 1 -C 6 alkyl base, cycloalkyl, alkenyl, alkynyl, halogen-substituted C 6 -C 20 aryl group, or halogen-substituted 5-20-membered heterocyclic group;
  • R 10 and R 13 or R 14 can be connected to form a 5-9 membered ring under the conditions consistent with the definitions of R 10 , R 13 and R 14 above;
  • R 4 and R 15 are each independently hydrogen, halogen, C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, cyano, 5-20 membered heterocyclic group, C 6 -C 20 aryl, or halogen-substituted C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclyl, C 6 -C 20 aryl;
  • R 10 and R 15 can form a 4-12 membered cyclic hydrocarbon or heterocyclic ring under the conditions meeting the above definitions of R 10 and R 15 ;
  • A is CR 16 or N, and the position of A can be changed on the ring;
  • R 16 is hydrogen, C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, C 6 -C 20 aryl, 5-20 membered heterocyclyl, halogen-substituted C 1 -C 6 alkyl base, cycloalkyl, alkenyl, alkynyl, halogen-substituted C 6 -C 20 aryl group, or halogen-substituted 5-20-membered heterocyclic group;
  • R 6 and R 7 satisfy the following conditions:
  • R 6 and R 7 are each independently hydrogen, halogen, cyano group, hydroxyl group, C 1 -C 6 alkyl group, cycloalkyl group, alkenyl group, alkynyl group, alkoxy group, 5-20 membered heterocyclic group, C 6 -C 20 aryl, or halogen-substituted C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclyl, C 6 -C 20 aryl group, or cyano-substituted C 1 -C 6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclic group, C 6 -C 20 aryl, or hydroxyl substituted C 1 -C 6 alkyl, cycloalkyl, alkoxy, 5-20 membered heterocyclyl, C 6 -C 20 aryl, or
  • the heterocycle is substituted by C 1 -C 6 alkyl
  • R 6 can be connected to CR 16 to form a 5-9 membered ring, heterocyclic ring or aromatic heterocyclic ring;
  • R 11 and R 12 meet the following conditions:
  • R 11 and R 12 are each independently a C 1 -C 6 alkyl group.
  • a halogen-substituted C 1 -C 6 alkyl group or R 11 and R 12 satisfy the above definition and form 5- with the N in -CONR 11 R 12 7-membered ring, forming a 5-7-membered ring with N in -CH 2 NR 11 R 12 .
  • the synthetic preparation method of the specific compound is also recorded in the above application, and its full text is hereby incorporated into this application. It is specifically defined as:
  • G 1 , G 2 , G 3 or G 4 are the same or different, and each independently is a CR 5 or N atom;
  • Each R 5 is the same or different, and each is independently selected from a hydrogen atom, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a hydroxyl group, a cyano group, an amino group, a nitro group, -NR a R b , -C ( O)NR a R b , cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclyl, aryl and Each heteroaryl group is independently optionally selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, oxo, cyano, amino, nitro, cycloalkyl, heterocycle Substituted with one or more substituents among aryl, aryl
  • Y is selected from -(C(R y2 R y3 )) f -NR y1 -, -(C(R y2 R y3 )) g -O-, -(C(R y2 R y3 )) h -S-, - (C(R y2 R y3 )) h -S(O)-, -(C(R y2 R y3 )) h -S(O) 2 -, -C(R y2 R y3 )-, -NR y1 - (C(R y2 R y3 )) f -, -O-(C(R y2 R y3 )) g -, -S-(C(R y2 R y3 )) h -, -S(O)-(C (R y2 R y3 )) h -and-S(O) 2
  • R y1 is selected from hydrogen atoms, alkyl groups, haloalkyl groups, hydroxyalkyl groups, cycloalkyl groups and heterocyclic groups;
  • R y2 and R y3 are the same or different, and are each independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, hydroxyalkyl groups, cycloalkyl groups and heterocyclyl groups;
  • Z is O or OH
  • Z is a single bond or a double bond, when When it is a single bond, Z is OH. When it is a double bond, Z is O;
  • T is selected from -C( RT1 R T2 )- ⁇ -NR T3 -or -O-;
  • R T1 and R T2 are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group and a heterocyclyl group;
  • R d is a hydrogen atom or C 1-6 alkyl group
  • R e is selected from alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl optionally selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl Substituted by one or more substituents;
  • R 4 is selected from hydrogen atom, alkyl group, haloalkyl group, hydroxyl group and hydroxyalkyl group;
  • v 0, 1 or 2;
  • w 0, 1 or 2;
  • f 0, 1 or 2;
  • g 0, 1 or 2;
  • h 0, 1 or 2;
  • n 0, 1, 2, 3, 4 or 5;
  • s 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • t 0, 1, 2, 3, 4, 5 or 6;
  • ring A is a phenyl group or a 5- to 6-membered heteroaryl group
  • G 3 is a CR 5 or N atom
  • R 5 is not a hydrogen atom
  • ring A is a phenyl group or a 5- to 6-membered heteroaryl group
  • R 1 , G 2 , G 3 and G 4 are all CR 5 , Y is NR y1 , n, v and w are all 1, and E is O atom, 1) T is not CH 2 or CD 2 , 2 ) At least one R 2 or R 3 is a deuterium atom, 3) R 4 is selected from alkyl, haloalkyl, hydroxyl and hydroxyalkyl, 4) One of R 1 is a 3 to 8-membered cycloalkyl group or a 5 to 8-membered cycloalkyl group Heterocyclyl, the 3 to 8-membered cycloalkyl or 5 to 8-membered heterocyclyl is optionally selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxyl, oxygen Substituted with one or more substituents of generation, cyano, amino, nitro, cycloalkyl, hetero
  • R 1 , R 2a , R 2b , R 3 , R 4 , R 5 , n, and Z are defined as in patent application PCT/IB2020/057285, publication number WO2021005586A1 (corresponding to Chinese application number CN202080053804.1, publication number CN114206870A) are described in the claims in , and the synthesis and preparation methods of specific compounds are also described in the above-mentioned applications, the full text of which is hereby incorporated into this application, and is specifically defined as:
  • ZDang It is OH when it is a single bond; or when When it is a double bond, it is O;
  • Each R 1 is independently selected from the group consisting of: (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 0 -C 4 )alkylN(R 8 ) 2 , and halo;
  • R 2a and R 2b are each independently selected from the group consisting of: H, (C 1 -C 6 )alkyl, and halo;
  • Each R 3 is independently selected from the group consisting of: H, and halo;
  • R 4 is selected from the group consisting of: aryl, 5- to 6-membered heteroaryl containing 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; and 5- to 6-membered heteroaryl containing 1, 2, 3 , or 4 9 to 10 membered fused bicyclic heteroaryl groups independently selected from N, O and S heteroatoms; wherein any of the foregoing is optionally substituted by one or more R 6 ;
  • R 5 is selected from the group consisting of: H; (C 1 -C 6 )alkyl; (C 2 -C 6 )alkenyl; (C 0 -C 4 )alkyl OR 8 ; (C 1 -C 4 ) Alkyl (C 3 -C 10 ) cycloalkyl; Halogenated (C 1 -C 6 ) alkyl; (C 2 -C 3 ) alkynyl; (C 1 -C 4 ) alkyl N (R 10 ) 2 ;
  • Each R 6 is independently selected from the group consisting of: halo; (C 1 -C 6 )alkyl; (C 1 -C 6 )alkoxy; halo(C 1 -C 6 )alkyl; OH ; Aryl; 3 to 6 membered heterocycle; 5 to 6 membered heteroaryl; (C 0 -C 4 ) alkyl S(O) m (C 1 -C 6 ) alkyl; halogenated (C 1 -C 6 )Alkoxy; (C 0 -C 4 )alkyl S(O) m N(R 8 ) 2 ; (C 0 -C 4 )alkyl N(R 8 ) 2 ; (C 0 -C 4 ) Alkyl(CO)OR 7 ; N(R 8 )S(O) m (C 1 -C 6 )alkyl; N(R 8 )S(O) m (C 3 -C 6 )cycloal
  • Each R 7 and R 8 is independently selected from the group consisting of: H or (C 1 -C 6 )alkyl optionally substituted with one to three R 9 ;
  • Each R 11 is selected from the group consisting of: H; a 4- to 6-membered heterocycle optionally substituted with one to four R 12 ; a (C 3 -C 6 ) ring optionally substituted with one to four R 12 Alkyl; (C 0 -C 3 )alkyl (C 3 -C 6 )cycloalkyl (C 1 -C 3 )alkyl optionally substituted by halo; optionally substituted by one to three R 12 CH 2 -aryl; (C 1 -C 6 ) alkyl; (C 2 -C 6 ) alkenyl; or (C 2 -C 6 ) alkynyl, wherein said (C 1 -C 6 ) alkyl ; Each of (C 2 -C 6 ) alkenyl; and (C 2 -C 6 ) alkynyl is optionally substituted by one or more R 13 ;
  • Each R 12 is independently selected from the group consisting of: OH, (C 1 -C 3 )alkoxy, NH 2 ; or (C 1 -C 3 )alkyl optionally substituted with one or more OH ;
  • Each R 13 is independently selected from the group consisting of: halo, OH, amino, (C 1 -C 4 )alkylamino, di(C 1 -C 4 )alkylamino, (C 1 -C 3 ) Alkoxy; and C(O)-(C 3 -C 8 )cycloalkyl;
  • n 0, 1, or 2;
  • n 0, 1 or 2.
  • the specific compound of formula (1) refers to the compound in the patent application document PCT/US2016/021581, Publication No. WO2016145092A1 (corresponding to Chinese Application No. 2016800150788, Publication No. CN107530556A).
  • the full text of the patent application document is hereby incorporated into this application;
  • the compound of structural formula (1) is similar to AST-3424 and is a prodrug of an aminophosphate alkylating agent. It will be activated by the AKR1C3 enzyme to produce T (an aminophosphate alkylating agent, AST- 2660 is an aminophosphate alkylating agent) that exerts anti-cancer effects:
  • AST-3424 specifically as an example. As specific substrates for aldehyde-keto reductase AKR1C3, these compounds can be quickly and effectively reduced only in cancer cells with high expression of AKR1C3, thereby releasing the cytotoxic DNA alkylating agent AST-2660. AST -2660 cross-links with DNA to cause cancer cell death:
  • the compounds of structural formula (2) (3) are the same as AST-3424, and are both prodrugs of AST-2660, especially the compound AST, which will be activated by the AKR1C3 enzyme to produce AST-2660 (a DNA Alkylating agent) exerts anti-cancer effects:
  • the compound of structural formula (4) is similar to AST-3424 and AST, and is a prodrug of AST-2660. It will be activated by the AKR1C3 enzyme to produce AST-2660 to exert anti-cancer effects:
  • the compound of structural formula (5) is the same as AST-3424 and AST. They are all prodrugs of AST-2660. They will be activated by the AKR1C3 enzyme to produce AST-2660 (a DNA alkylating agent) to exert anti-cancer effects. :
  • the compound of structural formula (6) has similar principles to AST-3424 and AST, and both are nitrogen mustard analogs.
  • a prodrug that is activated by the AKR1C3 enzyme to produce nitrogen mustard analogs exerts anticancer effects:
  • the compound of structural formula (7) will be activated by the AKR1C3 enzyme to produce drug D that can inhibit cancer cell proliferation or tumor growth and exert anti-cancer effects:
  • D can be anti-cancer active substances such as paclitaxel, SN-38, gemcitabine, etc.
  • Paclitaxel can cause tubulin and tubulin dimers that make up microtubules to lose their dynamic balance, induce and promote tubulin polymerization, microtubule assembly, and prevent depolymerization, thereby stabilizing microtubules and inhibiting mitosis and triggering of cancer cells.
  • Cell apoptosis can effectively prevent the proliferation of cancer cells and play an anti-cancer effect.
  • SN-38 can inhibit topoisomerase I, which is necessary for DNA replication in human cells, induce single-strand damage in DNA, block DNA replication, and produce cytotoxicity.
  • Gemcitabine is a nucleoside cytosine (pyrimidine) analog that can be incorporated into replicating DNA, thereby inhibiting DNA synthesis.
  • deoxycytidine kinase dCK
  • gemcitabine is converted into gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP).
  • the compound of structural formula (8) (9) (10) (11) will also be activated by the AKR1C3 enzyme to produce drugs such as AST-2660, gemcitabine or KARS inhibitors that can inhibit cancer cell proliferation or tumor growth. exert anti-cancer effect.
  • KARS inhibitors are inhibitors of lysine t-RNA synthetase, a ubiquitous enzyme necessary for protein synthesis that is part of the multi-t-RNA synthetase complex.
  • prodrug compound is preferably selected from:
  • the medicines described herein refer to medicines or preparations, and the medicines prepared contain the AKR1C3 enzyme-activated compounds of formulas (1)-(11) or their salts or solvates as active ingredients in a specific dosage range, and/or the medicines prepared The obtained medicines are administered in specific dosage forms and specific administration methods.
  • the above-mentioned drugs should also add pharmaceutically acceptable auxiliary materials or excipients according to the characteristics of the drug, drug, and preparation.
  • the drug can be in any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated agents, granules, dry powders, oral solutions, and small injection needles. , freeze-dried powder for injection or large infusion.
  • pharmaceutically acceptable excipients or excipients in the drug may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, viscosifiers, Mixtures, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, coating agents and pigments, etc.
  • Cancer refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) with potentially unrestrained growth that can expand locally by invasion and systemically by metastasis.
  • cancers include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysepithelial carcinoma and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, multiple glioblastoma, pilocytic tumors, intestinal ganglioneuroma, proliferative corneal neurotumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumor, malignant melanoma , malignant hypercalcemia, Marfanoid tumor, medullary epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, Ovarian tumors, pheochromocytoma, polycythemia, poly
  • AKR1C3 enzyme-activated DNA alkylating agent prodrug for cancer treatment please refer to the patent application texts submitted by Threshold, Ascentawits, OBI and other companies (such as WO2017087428A1, WO2017087428A1, WO2019062919A1, WO2021008520A1 ) and clinical trials registered by FDA and NMPA (CTR20201915, CTR20201908, CTR20191399, CTR20191371 and NCT04315324, NCT03592264).
  • Treatment in this application includes monotherapy and combination therapy with other drugs.
  • Monotherapy refers to the use of only one anticancer drug in a course of treatment.
  • Combination therapy refers to the simultaneous or sequential use of two or more anti-cancer drugs in one course of treatment.
  • combination therapy needs to explore different dosages and dosage weeks based on the characteristics of the disease and the type of combined drugs. In this period, only based on the above situation, the explored combination drug treatment plan can achieve better therapeutic effects than single drug treatment.
  • AST-3424(OBI-3424) ⁇ (hereinafter codenamed AST) and The synthesis method and spectral data are patented: PCT/US2016/021581, Publication No. WO2016145092A1, corresponding to Chinese Application No. 2016800150788, Publication No. CN107530556A; PCT/US2016/062114, Publication No. WO2017087428A1, corresponding to Chinese Application No.
  • 2016800446 081 public number CN108290911A; PCT/CN2020/089692, publication number WO2020228685A9; related preparation concentrated injections, and related prescriptions, preparation methods, clinical compatibility, and application methods are described and disclosed in detail by related patents: WO2021008520A1, WO2021043275A1.
  • the present invention combines the above Introduce the full text of the application text.
  • Figure 1 is Photos of the growth curve of tumor volume in each group of mice in the gastric cancer GA6201 model
  • Figure 2 is Photos of the relative tumor inhibition rate curves of each group of mice in the gastric cancer GA6201 model
  • Figure 3 is Photos of the body weight curves of mice in each group in the gastric cancer GA6201 model
  • Figure 4 is Photos of the percentage change curve of body weight of mice in each group in the gastric cancer GA6201 model
  • Figure 5 is Photos of the growth curve of tumor volume in each group of mice in the PA1222 model of pancreatic cancer
  • Figure 6 is Photos of the relative tumor inhibition rate curves of each group of mice in the PA1222 model of pancreatic cancer
  • Figure 7 is Photos of the weight curves of mice in each group in the PA1222 model of pancreatic cancer
  • Figure 8 is Photos of the percentage change curve of body weight of mice in each group in the PA1222 model of pancreatic cancer
  • Figure 9 is Photos of the growth curve of tumor volume in each group of mice in the lung cancer LU11693 model
  • Figure 10 is Photos of the relative tumor inhibition rate curves of each group of mice in the lung cancer LU11693 model
  • Figure 11 is Photos of the body weight curves of mice in each group in the lung cancer LU11693 model
  • Figure 12 is Photos of the percentage change curve of body weight of mice in each group in the lung cancer LU11693 model
  • Figure 13 is Photos of the growth curve of tumor volume in each group of mice in the PA1383 model of pancreatic cancer
  • Figure 14 is Photos of the relative tumor inhibition rate curves of each group of mice in the PA1383 model of pancreatic cancer
  • Figure 15 is Photos of the body weight curves of mice in each group in the PA1383 model of pancreatic cancer
  • Figure 16 is Photos of the percentage change curve of body weight of mice in each group in the PA1383 model of pancreatic cancer
  • Figure 17 is a photo of the IHC staining results of tumor tissues of three PDX models of gastric cancer GA6201, lung cancer LU11693, and pancreatic cancer PA1222, and the control group;
  • Figure 18 is a diagram showing the corresponding relationship between the RNA expression level of AKR1C3 and the RNA expression level of NRF2 in the KRAS-G12D mutation model;
  • Figure 19 is a diagram showing the corresponding relationship between the RNA expression level of AKR1C3 and the RNA expression level of NRF2 in the KRAS-G12C mutation model;
  • Figure 20 is a diagram showing the corresponding relationship between the RNA expression level of AKR1C3 and the RNA expression level of NRF2 in the KRAS-G13D mutation model;
  • Figure 21 shows the immunoblotting picture of pancreatic cancer HPAF II and the IHC staining of lung cancer LU5161 and colorectal cancer CR3820. Photos of results;
  • Figure 22 shows the results of immunoblotting experiments on target proteins after cell lysis under treatment with different concentrations of SFN.
  • the upper picture is a picture of the immunoblot under different concentrations of SFN.
  • the bottom picture is a histogram of protein band density analysis of NRF2 and AKR1C3. Among them, Under each concentration group, the left column represents NRF2 and the right column represents AKR1C3;
  • Figure 23 shows the results of immunoblotting experiments on target proteins after cell lysis under treatment with different concentrations of AST.
  • the upper picture shows the immunoblotting images of cells treated with different concentrations of AST, and the lower picture shows the proteins of p-ERK2 and ERK2 treated with different concentrations of AST.
  • Band density analysis histogram in which, under each group of AST concentration, the left column represents p-ERK2 and the right column represents ERK2;
  • Figure 24 shows the results of immunoblotting experiments on target proteins after cell lysis treated with 1 ⁇ M AST for different times.
  • the upper picture shows the immunoblot images of 1 ⁇ M AST treated for different times.
  • the lower picture shows p-ERK2 and ERK2 treated with 1 ⁇ M AST for different times.
  • Figure 25 shows the results of AKR1C3-dependent immunoblotting experiments on target proteins after cell lysis.
  • the upper picture is an AKR1C3-dependent immunoblot picture
  • the lower picture is a histogram of AKR1C3-dependent protein band density analysis of p-ERK2 and ERK2.
  • the left column represents p-ERK2
  • the right column represents ERK2;
  • Figure 26 shows the immunoblotting and apoptosis experimental results of the target protein after cell lysis of the ERK2 inhibitor and AST single drug and combined drug groups.
  • the upper picture shows the immunoblot of the ERK2 inhibitor group of PD98059 and Suramin alone or in combination. Picture; the lower left picture is a histogram of protein band density analysis of single drug or combination. In each treatment group, the left column is p-ERK2 and the right column is ERK2; the lower right picture is a single drug.
  • RLU Luminescence
  • Figure 27 shows the immunoblotting and apoptosis experimental results of the target protein after cell lysis of the ERK2 activator and AST single drug and combined drug groups.
  • the picture above is the immunoblot picture of the ERK2 activator group of TPA alone or in combination;
  • the lower left picture is a density analysis histogram of a single drug or combination.
  • the left column is p-ERK2 and the right column is ERK2; the lower right picture is a single drug or combination.
  • Apoptosis histogram in which Luminescence (RLU) represents the relative luminescence unit of the apoptosis signal;
  • Figure 28 is a schematic diagram of the process of AST inducing apoptosis in cancer cells with KRAS G12D pathogenic mutations through the ERK2 signaling pathway.
  • (1), (3&4) represent relevant supporting documents.
  • administering or “administration of” a drug to a patient (and its grammatical equivalent) refers to direct administration (which may be administered to the patient by a medical professional or may be self-administered) and/or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and/or provides a prescription for a drug to the patient is administering the drug to the patient.
  • Cancer refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) with potentially unrestrained growth that can expand locally by invasion and systemically by metastasis.
  • malignancies including solid tumors
  • Examples of cancers include, but are not limited to, adrenal gland, bone, brain, breast, bronchi, colon and/or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid, Cancers of the skin, stomach and thyroid.
  • cancers include acute and chronic lymphocytic and granulocytic neoplasms, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysepithelial carcinoma and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell carcinoma, Cell tumor, glioblastoma multiforme, pilocytoma, intestinal ganglioneuroma, proliferative corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, Malignant carcinoid tumor, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma Cell tumors, osteosarcomas, osteogenic and other sarcomas, ovarian tumors, phe
  • a patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment. Typically, the patients are humans. Typically, a patient is a human being diagnosed with cancer. In certain embodiments, a "patient” or “individual” may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, used for screening, characterizing, and evaluating drugs and therapies. Or rats.
  • Solid tumors refers to solid tumors including, but not limited to, metastatic tumors in the bone, brain, liver, lung, lymph nodes, pancreas, prostate, skin, and soft tissue (sarcomas).
  • a “therapeutically effective amount" of a drug is an amount of the drug that, when administered to a patient with cancer, will have a desired therapeutic effect (e.g., alleviation, amelioration, alleviation, or elimination of the clinical manifestations of one or more cancers in the patient) .
  • the therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations.
  • Treating”, “treatment of” or “therapy of” a condition or patient means taking steps to obtain a beneficial or desired result (including clinical results).
  • beneficial or desired clinical results include (but are not limited to) alleviation or improvement of one or more cancer symptoms; reduction in disease severity; delay or slowing of disease progression; improvement, remission, or stabilization of disease status; or other beneficial results.
  • treatment of cancer can result in partial response or stabilization of the disease.
  • Tumor cells refers to tumor cells of any appropriate species (eg, mammal, eg, murine, canine, feline, equine, or human).
  • a patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment. Typically, the patients are humans. Typically, a patient is a human being diagnosed with cancer. In certain embodiments, a "patient” or “individual” may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, used for screening, characterizing, and evaluating drugs and therapies. Or rats.
  • Treating means administering, using or administering to a patient a therapeutically effective amount of a drug relevant to the present invention.
  • administer or “administer” a drug to a patient.
  • “Use” means direct administration or administration (which may be administered or administered to the patient by a medical professional or may be self-administered or administered) and/or indirect administration or administration, which may be a prescribed drug. the behavior of. For example, a physician who instructs a patient to self-administer or administer a drug and/or provides a prescription for a drug to the patient is administering or administering the drug to the patient.
  • Treatment of a condition or patient means taking steps to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical results include (but are not limited to) alleviation or improvement of one or more cancer symptoms; reduction in disease severity; delay or slowing of disease progression; improvement, remission, or stabilization of disease status; or other beneficial results.
  • treatment of cancer can result in partial response or stabilization of the disease.
  • test substances AST, AST-3424 and Ifosfamide are in Pharmacodynamic evaluation of GA6201 subcutaneous xenograft model of gastric cancer
  • the gastric cancer GA6201 PDX model is a model of KRAS pathogenic mutation with G12D amino acid mutation (KRAS-G12D).
  • BALB/c nude mice were inoculated subcutaneously Model GA6201 tumor block was used to establish a subcutaneous transplantation tumor model of human gastric cancer.
  • the test is divided into the test drug Ifosfamide (ifosfamide) 60 mg/kg group, the test drug AST-34 245 mg/kg group, AST 2.5 mg/kg and 5 mg/kg groups, and the normal saline (pH 7.0-7.6) vehicle control group, a total of 5 groups, 5 mice in each group.
  • the normal saline (pH 7.0-7.6) vehicle control group, the test drug AST-34 45 mg/kg, AST 2.5 mg/kg and 5 mg/kg groups were administered by tail vein injection once a week for a total of three weeks. , look around. Ifosfamide 60 mg/kg group was administered intraperitoneally, five days a week and two days off for a total of two weeks, followed by five weeks of observation. The efficacy was evaluated based on the relative tumor inhibition rate TGI (%), and the safety was evaluated based on the changes in animal weight and death.
  • Table 1 Different doses of test drugs in Experimental design of anti-tumor effects in gastric cancer GA6201 tumor model
  • mice The tumor volumes of mice in different groups were measured on different days and the average values were obtained. The results are shown in Table 2 below.
  • Table 2 In Changes in tumor volume of mice in each group with treatment time in the gastric cancer GA6201 model
  • Figure 2 showing the tumor growth of each treatment group and control group was prepared based on the data in Table 2.
  • T/C% The relative tumor proliferation rate in Table 3, T/C%, is the relative percentage of tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:
  • T and C are the average relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 4 Relative tumor inhibition rate of each group of tumors in the gastric cancer GA6201 model
  • mice The body weights of mice in different groups were measured on different days and the average values were obtained. The results are shown in Table 5 below.
  • Group01, Group02, Group03, Group04, and Group05 in the above table 4/5/6 are the above-mentioned Group 1, Group 2, Group 3, Group 4, Group 5, and Group 6.
  • 0/1/2/3/4/7/8/9/10/11/14/15/16/17/18/21/24/28/31/35/38 is the number of days after vaccination.
  • the test drug AST-3424 is at the dose of 5 mg/kg, and the test drug AST is at the doses of 2.5 mg/kg and 5 mg/kg.
  • Gastric cancer GA6201 has a significant inhibitory effect on tumor growth, and there is a statistically significant difference compared with the control group.
  • the test drug Ifosfamide at a dose of 60mg/kg has Gastric cancer GA6201 has a certain effect on inhibiting tumor growth, but there is no statistically significant difference compared with the control group.
  • test substances AST and Gemcitabine are in Pharmacodynamics and safety evaluation in PA1222 subcutaneous xenograft model of pancreatic cancer
  • the PA1222 PDX model of pancreatic cancer is a model of the KRAS pathogenic mutation with G12D amino acid mutation (KRAS-G12D).
  • BALB/c nude mice were inoculated subcutaneously Model PA1222 tumor mass was used to establish a subcutaneous transplantation tumor model of human pancreatic cancer.
  • the test is divided into test drug Gemcitabine 120mg/kg group, test drug AST 10mg/kg and 7.5% absolute ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH7.4) solvent Control group, a total of 3 groups, 5 mice in each group.
  • the 7.5% absolute ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH7.4) vehicle control group and the test drug AST 10 mg/kg group were administered by tail vein injection, weekly The drug is administered once for three consecutive weeks.
  • the test drug Gemcitabine 120mg/kg group was administered by intraperitoneal injection once a week for 3 consecutive weeks.
  • the efficacy was evaluated based on the relative tumor inhibition rate TGI (%), and the safety was evaluated based on the changes in animal weight and death.
  • Table 7 Different doses of test drugs in Experimental design of anti-tumor effects in pancreatic cancer PA1222 PDX tumor model
  • mice The tumor volumes of mice in different groups were measured on different days and the average value was obtained. The results are as follows in Table 8 shown.
  • Table 8 Changes in tumor volume of mice in each group with treatment time in the PA1222 model of pancreatic cancer
  • Table 9 Drug efficacy analysis table of each group in the gastric cancer GA6201 model
  • T/C% Relative tumor proliferation rate
  • T and C are the average relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 10 Relative tumor inhibition rate of each group of tumors in the PA1222 model of pancreatic cancer
  • mice The body weights of mice in different groups were measured on different days and the average values were obtained. The results are shown in Table 11 below.
  • Table 11 Body weight of mice on different vaccination days in PA1222 model of pancreatic cancer
  • the test drug Gemcitabine at a dose of 120 mg/kg (Group 2) Pancreatic cancer PA1222 has a certain effect in inhibiting tumor growth, and there is a statistically significant difference compared with the control group.
  • the test drug AST has Pancreatic cancer PA1222 has a significant tumor inhibitory effect, which is statistically significant compared to the control group.
  • Two mice in this group had tumors cured, and the cure rates were both 40%.
  • mice in the test drug Gemcitabine (120mg/kg, Group 2) treatment group, AST 10mg/kg (Group 3) treatment group and control group (Group 1) did not have any obvious weight loss. During the treatment period Well tolerated.
  • test substances AST and Cisplatin are Pharmacodynamics and safety evaluation in lung cancer LU11693 subcutaneous xenograft model
  • the lung cancer LU11693 PDX model is a model of KRAS pathogenic mutation with G12C amino acid mutation.
  • BALB/c Nude mice were inoculated subcutaneously Model LU11693 tumor mass was used to establish a subcutaneous transplantation tumor model of human lung cancer.
  • the test was divided into the test drug Cisplatin 4mg/kg group, the test drug AST 10mg/kg group, and the 7.5% absolute ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W (pH7.4) vehicle control group. , a total of 3 groups, 6 mice in each group, administered via tail vein injection, once a week for 3 consecutive weeks.
  • the efficacy was evaluated based on the relative tumor inhibition rate TGI (%), and the safety was evaluated based on the changes in animal weight and death.
  • Table 13 Different doses of test drugs in Experimental design of anti-tumor effects in lung cancer LU11693 PDX tumor model
  • Dosing volume is 10 ⁇ L/g
  • mice The tumor volumes of mice in different groups were measured on different days and the average values were obtained. The results are shown in Table 14 below.
  • Table 14 Changes in tumor volume of mice in each group with treatment time in the LU11693 lung cancer model
  • the tumor growth of each treatment group and control group is shown in Table 14 and Figure 9, and the drug efficacy evaluation is shown in Tables 1 and 5.
  • Table 15 Drug efficacy analysis table of each group in the lung cancer LU11693 model
  • the relative tumor proliferation rate, T/C% is the relative percentage value of tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:
  • T and C are the average relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 14 Relative tumor inhibition rate of each group of tumors in the lung cancer LU11693 model
  • mice The body weights of mice in different groups were measured on different days and the average values were obtained. The results are shown in Table 15 below.
  • Table 15 Body weight of mice on different vaccination days in lung cancer LU11693 model
  • test drug AST (10mg/kg) treatment group showed a certain anti-tumor effect on the 28th day after the first administration (Day 28). Compared with the control group, there was a statistically significant difference (p ⁇ 0.001). Compared with the tumor The inhibition rate TGI (%) was 54.64%, the TGI was less than 60%, and there was no obvious tumor inhibitory effect.
  • mice in the test drug AST (10mg/kg) and Cisplatin (4mg/kg) treatment groups experienced severe weight loss, which may be related to the potential toxicity of high-dose drugs.
  • test substances AST, AST-3424 and Ifosfamide alone are in Anti-tumor effect and safety evaluation of PA1383 subcutaneous model of pancreatic cancer
  • the PA1383 PDX model of pancreatic cancer is a model of the KRAS pathogenic mutation with G12C amino acid mutation (KRAS-G12C).
  • KRAS-G12C G12C amino acid mutation
  • Balb/nude female mice were vaccinated subcutaneously Pancreatic cancer PA1383 tumor mass was used to establish a subcutaneous transplantation tumor model of human pancreatic cancer.
  • the trial is divided into the test drug Ifosfamide 60mg/kg single drug group (Group 2), administered once a day for 5 consecutive days, rested for 2 days, and then administered once a day for 5 consecutive days; AST 4mg/kg single drug group
  • the drug group (Group 3) and the AST 8mg/kg single drug group (Group 4) were administered once a week for a total of 3 weeks; the AST 4mg/kg single drug group (Group 5) and AST-34241mg/kg Single drug group (Group 6), administered once a day for 5 consecutive days, rested for 2 days, rested for 2 weeks, and then administered once a day for 5 consecutive days; glucose injection (pH7.7 -8.0)
  • Vehicle control group (Group 1) administered once a day for 5 consecutive days, rested for 2 days, rested for another 2 weeks, and then administered 1 dose a day times, administered continuously for 5 days.
  • the test drug Ifosfamide was
  • Table 17 Administration routes, dosages and regimens of PA1383 animal model of pancreatic cancer
  • the dosage volume is 10 ⁇ L/g
  • QD ⁇ 5, 2 days off, 2 weeks off, QD ⁇ 5 administer once a day for 5 consecutive days, rest for 2 days, rest for 2 weeks, and then administer every day for 5 consecutive days;
  • 4.QW ⁇ 3 Administer once a week for 3 consecutive weeks.
  • mice The tumor volumes of mice in different groups were measured on different days and the average values were obtained. The results are shown in Table 18 below.
  • Table 18 Changes in tumor volume of mice in each group with treatment time in the PA1222 model of pancreatic cancer
  • the tumor growth of each treatment group and control group is shown in Table 18 and Figure 13, and the drug efficacy evaluation is shown in Table 19.
  • Table 19 In Analysis table of drug efficacy of each group in the PA1383 subcutaneous model of pancreatic cancer
  • Relative tumor proliferation rate is the relative percentage of tumor volume or tumor weight between the treatment group and the control group at a certain time point. Calculated as follows:
  • T and C are the average relative tumor volumes (RTV) of the treatment group and the control group at a specific time point, respectively.
  • Table 20 Relative tumor inhibition rate of each group of tumors in the PA1383 model of pancreatic cancer
  • mice The body weight of mice in different groups was measured on different days and the average value was obtained. The results are shown in Table 21 below.
  • Table 21 Body weight of mice on different vaccination days in PA1383 model of pancreatic cancer
  • the average tumor volume of the mice in the vehicle control group was 1536.48mm 3 on Day 31 after the start of administration.
  • the average tumor volumes of the test drug Ifosfamide in the 60mg/kg dose treatment group (Group 2) and AST-3424 in the 1mg/kg (QD ⁇ 5, 2days off, 2weeks off, QD ⁇ 5) dose group (Group 6) on Day 31 were respectively were 1202.01mm 3 and 1225.40mm 3 , and the relative tumor inhibition rates TGI (%) were 21.84%, 20.45% and 7.55%. There was no statistically significant difference compared with the control group (p>0.05).
  • the test drug AST was in 4mg/kg (QW ⁇ 3), 8mg/kg (QW ⁇ 3) and 4mg/kg (QD ⁇ 5, 2 days of f, 2 weeks off, QD ⁇ 5) dose treatment groups (Group 3, Group 4 and Group 5) on Day 31, the average tumor volumes were 616.27mm 3 , 18.57mm 3 and 39.94mm 3 respectively. Compared with the control group, there were statistically significant differences (p ⁇ 0.05).
  • the relative tumor inhibition rate TGI (%) were respectively are 60.42%, 98.72% and 97.46%.
  • mice in each test drug treatment group did not lose weight during the treatment period and were well tolerated.
  • AST-3424 and AST both have significant efficacy in the KRAS pathogenic mutation model with G12D amino acid mutation: gastric cancer GA6021 and pancreatic cancer PA1222 PDX model, with TGI% greater than 90%;
  • AST in the KRAS pathogenic mutation model with G12C amino acid mutation the tumor inhibitory effect in lung cancer LU11693 is not obvious, with TGI% less than 60%; while in pancreatic cancer PA1383, the tumor inhibitory effect is obvious, with TGI% up to 98 %.
  • GA6201 detected AKR1C3 LOG2 (FPKM) as 6.78, LU11693 as 11.14, and PA1222 as 7.39, see Table 23.
  • AKR1C3 RNA is highly expressed in these three tumor tissues.
  • the AKR1C3 protein content of these three tissues was determined according to the commonly used IHC (immunohistochemistry) staining method (commercial IHC reagents were used, the primary antibody was Abcam's rabbit antibody Rabbit IgG mAb, and the secondary antibody was Leica's polymer optimization Detection system Bond Polymer Refine Detection, staining conditions: antigen retrieval 100°C, pH9.0 EDTA buffer 20min, dilution ratio: 1:800), and H-SCORE scoring of the staining results:
  • Immunohistochemical staining intensity will be divided into 0 (negative), 1+ (weak staining), 2+ (moderate staining), 3+ (strong staining), and the weak staining, medium staining, and strong staining are manually set on the scoring instrument. Threshold, and then use image processing software to perform color recognition on the stained sample photos. The stained photos of all samples follow a unified standard, and the scoring software scores the staining condition corresponding to a certain cell: 0/1/2/3. And count the number of positive cells with different staining intensities as a percentage of the total number of cells in the section. Calculate H-Score as the IHC result score for each sample by the formula below. The H-score will range from 0 to 300.
  • the AKR1C3 protein in the tissues corresponding to the three PDX models with G12D or G12C mutations is highly expressed.
  • Table 25 Statistical results of the correspondence between the expression level classification of AKR1C3 RNA represented by LOG 2 (FPKM) and KRAS-G12D
  • the distribution trend of AKR1C3 expression in the KRAS G12D PDX model is mainly concentrated in medium and high expression (LOG2 (FPKM) ⁇ 4), accounting for 90.4%.
  • the distribution trend of NRF2 expression is also concentrated in medium and high expression, which is consistent with the protein expression trend of AKR1C3 and has a certain correlation.
  • Table 26 Statistical results of the correspondence between the expression level classification of AKR1C3 RNA represented by LOG2 (FPKM) and KRAS-G12C
  • AKR1C3 expression is evenly distributed among low, medium and high expression levels in the KRAS G12C PDX model, of which the proportion of expression at medium or above levels (LOG2 (FPKM) ⁇ 4) is 66.7%.
  • Table 27 Statistical results of the correspondence between the expression level classification of AKR1C3 RNA represented by LOG2 (FPKM) and KRAS-G13D
  • RNA expression levels of AKR1 C3 and the RNA expression level of NRF2 in the 48 PDX models were projected onto the X-Y coordinate chart to obtain Figure 20.
  • AKR1C3 expression is evenly distributed between low, medium and high expression in the KRAS G13D PDX model, of which 83.3% are expressed at medium or above levels.
  • the inventor further used KRAS G12D mutated HPAF II, LU5161, and CR3820 mouse tumor pharmacodynamic models to study the anti-tumor effects of AST-3424 and AST in KRAS G12D mutated tumor models, and used IHC detection methods and Western blotting methods to detect Its protein expression level and enzyme content.
  • HPAF II (KRAS G12D) tumor cells were used to verify that the DNA alkylating agent prodrug activated by the AKR1C3 enzyme can activate the MEK/ERK signaling pathway, and a MEK/ERK modulator was used to explore the induction of the DNA alkylating agent prodrug activated by the AKR1C3 enzyme through the ERK2 pathway.
  • KRAS G12D cell apoptosis, using NRF2 inhibitors and activators to explore whether the expression of AKR1C3 in KRAS G12D mutant cells is regulated by NRF2.
  • the gastric cancer GA6201 PDX model, pancreatic cancer PA1222 PDX model, pancreatic cancer HPAF-II CDX model, lung cancer LU5161 PDX model, and colorectal cancer CR3820 PDX model are tumor models with KRAS G12D mutation, and lung cancer LU11693 PDX Model, pancreatic cancer PA1383 PDX model is a tumor model with KRAS G12C mutation.
  • RNA expression levels and enzyme content in pancreatic cancer HPAF II cells, lung cancer LU5161 and colorectal cancer CR3820 tumor tissues 9. Detection of RNA expression levels and enzyme content in pancreatic cancer HPAF II cells, lung cancer LU5161 and colorectal cancer CR3820 tumor tissues
  • AKR1C3 RNA expression level FPKM detection method Using the same AKR1C3 RNA expression level FPKM detection method, AKR1C3 protein content IHC detection method and H-SCORE scoring in Example 5, the RNA expression level and enzyme content data in lung cancer LU5161 and colorectal cancer CR3820 tissues were obtained, as shown in Table 28 shown.
  • the AKR1C3 RNA expression level FPKM detection method was used to evaluate the AKR1C3 RNA expression level data in pancreatic cancer HPAF II tumor cells, as shown in Table 28.
  • the corresponding IHC staining results and immunoblotting pictures are shown in Figure 21.
  • Table 28 IHC scoring and RNA expression level results in HPAF II, LU5161, and CR3820 tissues
  • SFN compound (Sulforaphane, NRF2 activator) solution was added to the HPAFII cell suspension, incubated overnight in a 37°C, 5% CO 2 incubator, and the cell protein lysate was collected for Western Blot (WB) detection.
  • WB Western Blot
  • the experiment is divided into 5 groups: 0.5% DMSO, 0.875 ⁇ M SFN, 1.75 ⁇ M SFN, 3.5 ⁇ M SFN, 7 ⁇ M SFN.
  • HPAF II cells were treated with different concentrations of the NRF2 activator SFN, and then immunoblotting was used to detect the expression of NRF2 and AKR1C3.
  • the results of immunoblotting and NRF2 and AKR1C3 protein band density analysis under different concentrations of SFN treatment are shown in Figure 22.
  • AKR1C3 enzyme-activating prodrugs AST-3424 and AST have significant resistance to PDX and CDX models with high expression of AKR1C3 enzyme. tumor effect and well tolerated.
  • AKR1C3 expression is positively correlated with NRF2 expression
  • those skilled in the art can reasonably speculate that up-regulating or activating NRF2 can promote high expression of AKR1C3, thereby enabling the AKR1C3 enzyme to activate the prodrugs AST-3424 and AST. Produce significant anti-tumor effects.
  • the compounds of the general formulas (1) to (11) of the present application are all anti-cancer prodrugs activated by the AKR1C3 enzyme. Under the action of the AKR1C3 enzyme, they are cleaved to produce AST-2660, paclitaxel, SN-38, gemcitabine, KARS inhibitors and other anti-cancer prodrugs. Cancer active drugs. Therefore, those skilled in the art can reasonably speculate that up-regulating or activating NRF2 will result in high expression of the AKR1C3 enzyme, thereby activating and metabolizing the compounds of general formulas (1)-(11) to produce anti-cancer active drugs. In other words, the ability to detect Patients with gene mutations that upregulate or activate NRF2 may benefit from treatment with compounds of general formulas (1)-(11), especially AST-3424 or AST.
  • the following takes the AKR1C3 enzyme-activated prodrug AST as an example to explore its mechanism of action in KRAS mutated tumor cells, especially in KRAS G12D mutated tumor cells.
  • AST activates MEK/ERK signaling pathway
  • AST-induced ERK phosphorylation experiment Three corresponding sets of experiments were conducted based on three sets of variables: AST concentration, AST action time, and AKRAC3 enzyme dependence. Among them, the AKRAC3 enzyme-dependent variables were conditioned on the AKR1C3 enzyme specific inhibitor AST-3021, and experiments were conducted in the presence or absence of the specific AKR1C3 enzyme inhibitor AST-3021.
  • the inhibitor used is AST-3021 (also known as TH-3021, which is compound 36 reported by Flanagan et al. in Bioorganic and Medicinal Chemistry (2014) pages 962-977, and its structural formula is ).
  • This experiment was divided into three groups, using different concentrations of AST (the first group), 1 ⁇ M AST treatment for different times (the second group), and adding or not adding the AKR1C3 inhibitor AST-3021 (the third group).
  • the first group (concentration group): 0.5% DMSO treatment group (24h), 0.5 ⁇ M AST treatment group (24h), 1 ⁇ M AST treatment group (24h), 10 ⁇ M AST treatment group (24h);
  • the second group (time group): 0.5% DMSO treatment group (24h), 1 ⁇ M AST treatment group (2h), 1 ⁇ M AST treatment group (8h), 1 ⁇ M AST treatment group (24h);
  • the third group (AKR1C3 dependent group): 0.5% DMSO treatment group (24h), 1 ⁇ M AST treatment group (24h), 3 ⁇ M AST-3021 treatment group (24h), 1 ⁇ M AST+3 ⁇ M AST-3021 treatment group (24h).
  • the compound solutions of each group were added to the HPAF II cell suspension, and incubated overnight in a 37°C, 5% CO 2 incubator. According to the experimental group, the cell protein lysate was collected for Western Blot (WB) detection.
  • WB Western Blot
  • NRF2 and AKR1C3 were detected using immunoblotting method.
  • the immunoblot pictures of the first group treated with different concentrations of AST and the results of density analysis of p-NRF2 and NRF2 protein bands are shown in Figure 23; the second group was treated with 1 ⁇ M AST.
  • the immunoblot images obtained at different times and the results of p-NRF2 and NRF2 protein band density analysis are shown in Figure 24; the immunoblot images obtained in the third group with or without the addition of the AKR1C3 inhibitor AST-3021 and p-NRF2, NRF2
  • the results of NRF2 protein band density analysis are shown in Figure 25.
  • AST induced the phosphorylation of ERK2 in a concentration-dependent manner without affecting the expression level of ERK2.
  • 24 hours after treatment with 1 ⁇ mol/L AST the phosphorylation level of ERK2 was significantly increased compared with the DMSO control group.
  • HPAF II cells were treated with the AKR1C3 specific inhibitor AST-3021 2 hours before adding 1 ⁇ mol/L AST.
  • 3 ⁇ mol/L AST-3021 strongly inhibited the AST-mediated increase in phosphorylated ERK2.
  • AST-3021 alone did not affect p-ERK2 levels.
  • the expression of total ERK2 was unchanged under all conditions tested. Consistent loading of all samples was confirmed by actin immunoblotting. AST activates the MEK/ERK signaling pathway in a concentration-, time- and AKR1C3-dependent manner.
  • AST induces apoptosis of KRAS G12D cells through the ERK2 signaling pathway
  • This experiment used three MEK/ERK modulators, including two inhibitors and one activator.
  • the inhibitors are PD98059, a specific inhibitor of MEK1/2, and Suramin, a broad-spectrum growth factor receptor inhibitor; and the activator of the ERK signaling pathway is Phorbol Ester (TPA).
  • TPA Phorbol Ester
  • the experiment was divided into inhibitor group and activator group, and Western blot experiments and apoptosis experiments were performed in both groups.
  • the HPAFII cell suspension was treated with each group of compounds, and cultured overnight in a 37°C, 5% CO 2 incubator.
  • the cell protein lysate was collected for Western Blot (WB) detection.
  • the specific compounds to be treated in the immunoblotting inhibitor group and activator group are as follows:
  • Inhibitor group 1% DMSO treatment group, 1 ⁇ M AST treatment group, 30 ⁇ M PD98059 treatment group, 1mM Suramin treatment group, 1 ⁇ M AST+30 ⁇ M PD98059 treatment group, 1 ⁇ M AST+1mM Suramin treatment group;
  • Activator group 1% DMSO treatment group, 0.5 ⁇ M AST treatment group, 25nM TPA treatment group, 0.5 ⁇ M AST+25nM TPA treatment group.
  • the specific compounds to be treated in the apoptosis inhibitor group and activator group are as follows:
  • Inhibitor group 1% DMSO treatment group, 1 ⁇ M AST treatment group, 30 ⁇ M PD98059 treatment group, 1mM Suramin treatment group, 1 ⁇ M AST+30 ⁇ M PD98059 treatment group, 1 ⁇ M AST+1mM Suramin treatment group;
  • Activator group 1% DMSO treatment group, 1 ⁇ M AST treatment group, 50nM TPA treatment group, 1 ⁇ M AST+50nM TPA treatment group.
  • the upper and lower left pictures show that the p-ERK2 expression level of the AST and TPA single drug treatment groups increased compared with the DMSO control group, while the p-ERK2 expression level of the combined treatment group of AST and TPA The level increased significantly; the right picture below shows that the AST single drug group promoted cell apoptosis, while its combination with TPA made the effect on cell apoptosis more significant.
  • the mechanism diagram in Figure 28 is that AST induces apoptosis of KRAS G12D pathogenic mutation cancer cells through the ERK2 signaling pathway.
  • the MEK/ERK signaling pathway can be activated under the action of DNA alkylating agents (Wang X, Martindale JL and Holbrook NJ. Requirement for ERK activation in cisplatin-induced poptosis.
  • AST a DNA alkylating agent prodrug
  • MEK/ERK signaling pathway which can induce apoptosis of KRAS G12D mutant cancer cells through the ERK2 signaling pathway
  • AST compounds It is a DNA alkylating agent prodrug activated by the AKR1C3 enzyme. It is activated by the AKR1C3 enzyme to produce AST-2660 and exert anti-cancer effects.
  • AST compounds can activate ERK2 to phosphorylate it and then activate the MEK-ERK pathway to upregulate NRF2, which ultimately leads to high expression of the AKR1C3 enzyme, leading to cancer cell apoptosis. That is, the KRAS-G12D mutation activates the RAF/MEK/ERK signaling pathway, up-regulates the protein expression of NRF2, and then up-regulates the protein expression of AKR1C3, which can more effectively activate AST drugs to release the DNA alkylating agent AST-2660.
  • genes that can upregulate the protein expression of NRF2 and then upregulate the protein expression of AKR1C3.
  • genes include KRAS mutation, KEAP1 mutation, CUL3 mutation, NRF2 mutation, p21 mutation, p62 mutation, FTL1 mutation, FTH1 mutation, HMOX1 Mutation, GSR mutation, SLC7A11 mutation, GCLM mutation, GCLC mutation, GPX2 mutation, TXN1 mutation, TXNRD1 mutation, PRDX1 mutation, SRXN1 mutation, ABCC1 mutation, ABCC2 mutation, G6PD mutation, PGD mutation, ME1 mutation, IDH1 mutation, EGFR mutation, etc. .
  • the ERK2 signaling pathway induces apoptosis in cells with gene mutations that can upregulate or activate NRF2. That is to say, cells with gene mutations that can upregulate or activate NRF2 can upregulate the protein expression of NRF2 by activating the RAF/MEK/ERK signaling pathway, and then upregulate the protein expression of NRF2.
  • the protein expression of AKR1C3 can more effectively activate the DNA alkylating agent prodrug activated by the AKR1C3 enzyme.
  • the compound of the general formula (7) (9) (10) (11) of the present application is similar to AST-3424 and AST. It is also an anti-cancer prodrug activated by the AKR1C3 enzyme. It is activated by the AKR1C3 enzyme to produce paclitaxel and SN-38. , gemcitabine or KARS inhibitors and other anti-cancer active drugs. Therefore, those skilled in the art should know that compounds of general formula (7) (9) (10) (11) have significant tumor inhibitory effects similar to AST-3424 and AST.

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Abstract

AKR1C3酶激活前药治疗癌症患者，其特征在于：所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；或所述患者被检测出具有能上调或激活NRF2的基因突变。

Description

AKR1C3酶激活前药治疗癌症患者的方法 技术领域
本发明涉及癌症的治疗方法，特别是具有特定基因癌症的治疗方法。
背景技术
AST-3424为中美均进入II期临床(中国登记临床为CTR20201915、CTR20201908、CTR20191399、CTR20191371，美国登记临床为NCT04315324、NCT03592264)的AKR1C3酶活化的DNA烷化剂前药，其特异性地被多种肿瘤高表达的AKR1C3酶激活而释放出DNA烷化剂进而发挥抗肿瘤作用。
临床前研究显示AST-3424的疗效与AKR1C3酶的表达高度相关，酶表达的些微差别将大大影响药物的最终治疗效果(AACR 2016,Abstract#1369:In vitro and in vivo antitu mor activity of TH3424:Preclinical rationale for a highly selective AKR1C3 prodrug for treating hepatocellular carcinomas；AACR-NCI-EORTC Annual Meeting,2017,Abstract:LB-B16:The AKR1C3-Activated Prodrug OBI-3424Exerts Profound In Vivo Efficacy Aga inst Preclinical Models of T-Cell Acute Lymphoblastic Leukemia(T-ALL)；a Pediatric Pr eclinical Testing Consortium Study；Clin Cancer Res 2019；25:4493-503:OBI-3424,a Nove l AKR1C3-Activated Prodrug,Exhibits Potent Efficacy against Preclinical Models of T-A LL；Am J Cancer Res 2021；11(7):3645-3659，A novel selective AKR1C3-activated prodr ug AST-3424/OBI-3424 exhibits broad anti-tumor activity)。
上述在中国和美国进行的II期临床试验将研究AKR1C3酶表达水平与对癌症患者治疗效果的关系，并确定AKR1C3表达水平的定量值用于筛选实验药物能获益的患者。
也就是说，上述AKR1C3酶激活的抗癌前药在用药前需要检测患者的AKR1C3酶的表达水平，只有达到预定表达水平的患者，药物才会有较好的治疗效果。
根据药物的I期临床结果，研究者已确定在AKR1C3酶表达水平为H-score≥135(IH C)的情况下入组II期临床患者(Safety,Pharmacokinetics,and Clinical Activity of OBI-3424,an AKR1C3-Activated Prodrug,in Patients with Advanced or Metastatic Solid Tumors:A Phase 1 Dose-Escalation Study,J Clin Oncol 40,2022,suppl 16；abstr 3030,DOI:10.1200/JCO.2022.40.16_suppl.3030)。
发明内容
发明人在进一步的临床前药效研究中发现，AKR1C3酶激活前药AST-3424、AST对于Kras-G12D/G12C突变的PDX、CDX肿瘤模型具有十分显著的抑制作用(实施例的第1、2、3、4部分及后续研究部分)，发明人推测是否Kras-G12D/G12C突变与AKR1C3酶高表达具有某种关联：Kras-G12D/G12C突变将导致AKR1C3酶高表达？
基于以上推测，发明人对上述的PDX、CDX模型的组织进行了AKR1C3 RNA检测和AKR1C3蛋白表达水平检测(实施例第5部分及第9部分)，结果证明上述具有Kras-G12D/G12C突变的模型其AKR1C3的RNA和蛋白表达水平检测结果均为高水平表达，也即证实了上述推测：Kras-G12D/G12C突变与AKR1C3酶高表达具有联系，Kras-G12D/G12C突变与AKR1C3酶高表达是伴随的。
为了验证Kras-G12D突变与AKR1C3高表达是伴随的这一结论，发明人收集了179例Kras-G12D突变的PDX模型中AKR1C3表达数据，结果发现：
AKR1C3表达量在KRAS G12D PDX模型中的分布趋势主要集中为中高表达，占90.4％；
进一步，分析以上统计数据发现，在KRAS G12D PDX模型中，NRF2表达量的分布趋 势也集中在中高表达，与AKR1C3的蛋白表达趋势一致，有一定的相关性(实施例第6部分)；
为了验证Kras-G12C突变与AKR1C3酶高表达是伴随的这一结论，发明人收集了51例Kras-G12C突变的PDX模型中AKR1C3表达数据，结果发现：
AKR1C3表达量在KRAS G12C PDX模型中的低中高表达平均分布，其中，中等以上水平表达占比为66.7％；
进一步，分析以上统计数据发现，在KRAS G12C PDX模型中，NRF2表达量的分布趋势与AKR1C3的蛋白表达量分布整体相关性较弱(实施例第7部分)。
为了验证Kras-G13D突变与AKR1C3酶高表达是伴随的这一结论，发明人收集了48例Kras-G13D突变的PDX模型中AKR1C3表达数据，结果发现：
AKR1C3表达量在KRAS G13D PDX模型中的分布趋势主要集中为中高表达，其中中等以上水平表达占比为83.3％。
在KRAS G13D PDX模型中，NRF2表达量的分布趋势与AKR1C3的蛋白表达量分布具有一定的相关性，AKR1C3和NRF2两者均大部分为高表达(实施例第8部分)。
发明人查阅文献进一步获知，KRAS G12D突变肿瘤细胞能通过RAF-MEK-ERK-Jun信号传导途径(Nature，2011，475：106)直接上调并激活NRF2(Nature，2011，475：106；Cancer Res，2014，74：7430)。激活的NRF2可以进一步上调激活包括AKR1C3在内的一系列下游基因(Chem Res Toxicol，2017，30：162；Cancer，2019，11：1715)。基于以上调控路径可知，类似G12D突变等，能上调或激活NRF2的基因突变均可以最终上调AKR1C3基因，导致癌细胞中AKR1C3酶表达水平升高。
基于以上实验和文献调研，发明人创造性的提出AKR1C3酶激活的前药在已知的通过检测AKR1C3酶表达水平来挑选/筛选患者的基础上，可以直接检测患者的肿瘤或癌组织是否具有能上调或激活NRF2的基因突变；或所述患者是否被检测出具有能上调或激活NRF2的基因突变来进行筛选。
为此，本申请提供以下技术方案。
方案一
AKR1C3酶激活前药治疗癌症患者的方法，其特征在于：
所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；
或
所述患者被检测出具有能上调或激活NRF2的基因突变。
方案二
AKR1C3酶激活前药用于制备治疗癌症的药物的制药用途，其特征在于：
所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；
或
所述患者被检测出具有能上调或激活NRF2的基因突变。
方案三
药物，该药物含有AKR1C3酶活化前药化合物，其适应症为治疗癌症患者，所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；
或
所述患者被检测出具有能上调或激活NRF2的基因突变。
方案四
AKR1C3酶激活前药治疗癌症患者，其包含施加含有AKR1C3酶激活前药的药品或制剂的步骤；以及测定患者的癌细胞或组织的NRF2含量或表达水平的步骤，
如测得该NRF2含量或表达水平等于或大于预定值，则向该患者投与含有AKR1C3酶激活前药的药品或制剂。
方案五
治疗癌症或肿瘤的方法，其包含施加含有AKR1C3酶激活前药的药品或制剂的步骤；以及NRF2含量或表达水平调节步骤，
当调节使得该NRF2含量或表达水平等于或大于预定值，则向该患者投与含有AKR1C3酶激活前药的药品或制剂。
方案六
AKR1C3酶激活前药用于制备治疗癌症的药物的制药用途，其特征在于：
所述患者的肿瘤或癌组织被检测出NRF2含量或表达水平等于或大于预定值；
或
所述患者被检测出NRF2含量或表达水平等于或大于预定值。
方案七
药物，该药物含有AKR1C3酶活化前药化合物，其适应症为治疗癌症患者，所述患者的肿瘤或癌组织被检测出NRF2含量或表达水平等于或大于预定值；
或
所述患者被检测出NRF2含量或表达水平等于或大于预定值。
一般而言，NRF2含量或表达水平/上调或激活NRF2的基因突变是使用患者的肿瘤或癌组织样本进行检测的，但由于各种原因，比如患者无法提供合格的肿瘤或癌组织样本，或者现行的检测方法不能准确检测，亦或者肿瘤或癌组织已经不能体现患者最近的情况时，可能需要检测或者诊断患者本身的NRF2含量或表达水平/上调或激活NRF2的基因突变，比如可以使用血液进行基因突变检测，该检测结果就可以代表患者的肿瘤或癌组织的情况；循环肿瘤细胞(CTC)检测可以通过患者的外周血检测样本来检测特定的癌细胞(游离于外周血中所有肿瘤细胞)的NRF2含量或表达水平/上调或激活NRF2的基因突变情况。部分案例中，可以通过生物学上的亲子关系中的双亲基因突变情况来诊断或辅助诊断基因突变情况。
“预定值”的意义在于通过某一方法(如体外诊断)检测出患者的NRF2含量或表达水平等于或大于某一预定值，并向其给与含有本申请的化合物的药物或配方，来达到精准靶向治疗癌症/肿瘤患者的目的。该预定值是需要至少确定癌种和特定药物或制剂之后，在不同患者种群中进行临床试验并根据最终的临床试验结果、数据给予统计学分析确定，这个预定值现在无法给出，但其可以通过临床试验最终确定。
本申请中，能上调或激活NRF2的基因突变选自以下基因突变：
KRAS、KEAP1、CUL3、NRF2、p21、p62、FTL1、FTH1、HMOX1、GSR、SLC7A11、GCLM、GCLC、GPX2、TXN1、TXNRD1、PRDX1、SRXN1、ABCC1、ABCC2、G6PD、PGD、ME1、IDH1、EGFR。
转录因子NRF2蛋白是重组与合成蛋白，中文全称为核因子E2p45相关因子2(nuclear fact or erythroid 2 p45 related factor 2)，是Cap'n'collar(CNC)转录因子家族成员，由七个Neh域(Nrf2 ECH同源结构域)组成，每个域具有不同的功能：
Neh1 CNC-bZIP域负责与small Maf(sMAF)蛋白结合和二聚化；
Neh2结构域通过DLG和ETGE基序介导与Keap1的相互作用；
Neh4、Neh5和Neh3域对于NRF2的反式激活非常重要；
Neh6结构域是一个富含丝氨酸的区域，可调节稳定性。
上调或激活NRF2的基因突变，是指能激活NRF2蛋白活性或直接上调NRF2蛋白表达量的基因突变，由于在NRF2的上游，即NRF2信号传导经典途径(Wu S,et al.Nrf2in cancers:A double-edged sword.Cancer Med.2019；8(5):2252-2267.)和Nrf2信号传导非经典途径(J iang T,et al.p62 links autophagy and Nrf2 signaling.Free Radic Biol Med.2015；88(PtB):199-204.)中，已有众多的研究结果证明许多基因在突变后能激活或上调NRF2蛋白，这些 基因突变包括：
KRAS突变，KRAS基因的全名叫Kirsten rat sarcoma viral oncogene homolog(Kirsten大鼠肉瘤病毒癌基因同源物)，属于RAS超蛋白家族。KRAS基因编码的蛋白是一种小GTP酶(small GTPase)。在人类的基因组中，有2个KRAS基因，分别是KRAS1，位于第6号染色体的短臂上；和KRAS2，位于第12号染色体的短臂上。其中KRAS1是“假基因”，不能被转录成RNA，所以它是没有功能的。而KRAS2才是“真基因”，能够转录、并且翻译成蛋白，具有生物学活性，通常专利和文献报道中所研究的KRAS基因和蛋白，是指“KRAS2”基因及其蛋白产物，本申请也是指KRAS基因及其蛋白产物。
在KRAS的基因突变中，97％是第12号或者第13号氨基酸残基发生了突变。结构学研究表明，这些基因突变大多干扰KRAS水解GTP的能力。KRAS基因突变，主要集中在第12，13及61号密码子位置，其中，第12号密码子位置的突变占到80％以上，包括G12A，G12C，G12D，G12R，G12S，G13D及G12V，最主要的是G12C、G12V、G13D这三种突变。
KEAP1，KELCH样ECH关联蛋白1重组蛋白(Recombinant Kelch Like ECH Associated Protein 1，简称KEAP1)。
CUL3，Cullin家族蛋白3。
NRF2，核因子E2p45相关因子2(nuclear factor erythroid 2 p45 related factor 2)。
p21，P21是细胞周期的负调控因子，是CDKIs家族成员，细胞周期蛋白依赖性激酶抑制因子p21。
p62，P62蛋白由SQSTM1基因编码，也可以写作p62/SQSTM1，原名叫Sequestosome-1。P62蛋白是选择性自噬的受体。
FTL1，铁蛋白轻肽1Ferritin,Light Polypeptide 1(FTL 1)。
FTH1，铁蛋白重肽1ferritin,heavy polypeptide 1(FTH 1)。
HMOX1，血红素加氧酶1(英语：heme oxygenase 1，缩写HMOX1或HO-1)是一种血红素加氧酶(EC 1.14.99.3)，是血红素代谢过程中的重要酶，它将血红素转化为胆绿素。
GSR，谷胱甘肽还原酶(GR)重组蛋白Recombinant Glutathione Reductase。
SLC7A11，溶质载体家族7成员11(SLC7A11)重组蛋白，Recombinant Solute Carrier Family 7，Member 11(SLC7A11)。
GCLM，谷氨酸半胱氨酸连接酶，Recombinant Glutamate Cysteine Ligase,Modifier。
GCLC，谷氨酸半胱氨酸连接酶催化亚基(GCLC)，Glutamate Cysteine Ligase,Catalytic(GCLC)。
GPX2，谷胱甘肽过氧化酶2(GPX2)，Glutathione Peroxidase 2。
TXN1，重组人硫氧还蛋白1，Thioredoxin 1，又写作His；Trx；ADF；TRX1等。
TXNRD1，硫氧还蛋白还原酶1thioredoxin reductase 1。
PRDX1，Prdx1(peroxiredoxin 1)过氧化物氧化还原酶蛋白(peroxiredoxin,Prdx)家族成员1。
SRXN1，Sulfiredoxin-1硫氧还蛋白-1。
ABCC1，ATP binding cassette sub-family C member 1，ATP结合盒亚家族C成员1。
ABCC2，ATP binding cassette sub-family C member 2，ATP结合盒亚家族C成员2。
G6PD，6-磷酸葡萄糖脱氢酶，6-Phosphogluconic dehydrogenase。
PGD，6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase，6-PGDH)。
ME1，苹果酸酶(malic enzyme，ME)。
IDH1，isocitrate dehydrogenase soluble，可溶性异柠檬酸脱氢酶1。
EGFR，epidermal growth factor receptor，简称为(EGFR、ErbB-1或HER1)是表皮生长因子受体(HER)家族成员之一。该家族包括HER1(erbB1，EGFR)、HER2(erbB2， NEU)、HER3(erbB3)及HER4(erbB4)。HER家族在细胞生理过程中发挥重要的调节作用。
特别的，进一步，KRAS突变选自G12D、G12V、G12R、G12C、G12A、G13D这6种亚型。
KRAS对应的基因中的任意一个基因突变或两个基因突变可以通过市售的(伴随)诊断试剂盒进行检测诊断，2012年的1月，FDA批准Qiagen公司的KRAS突变检测方法可以用作西妥昔的伴随诊断。2015年5月，FDA批准罗氏公司的KRAS突变检测方法可以用于转移的结肠癌的诊断。
目前中国境内有厦门艾德、上海透景生命、武汉友芝友、武汉海吉力、苏州为真等有提供针对KRAS基因激活突变的检测试剂盒。上述这些针对KRAS突变的检测方法，都是基于实时荧光定量PCR方法的原理。部分在中国获批的诊断试剂盒信息如下：
厦门艾德生物Amoy Dx，国械注准20153401126，人类KRAS基因突变检测试剂盒(荧光PCR法)。
上海透景生命，国械注准20163401341，人K-RAS基因7种突变检测试剂盒(PCR荧光法)。
当然也可以使用NGS测序(YS 450基因NGS大panel)来判定具体的KRAS突变亚型。
更优选的，KRAS突变选自KRAS-G12D突变。
所述的基因突变的TMB(肿瘤基因突变负荷)水平为中。
由于不同瘤种之间TMB(Tumor mutation load(burden)即肿瘤基因突变负荷)高低不同：一般认为：TMB超过20个突变/Mb(Mb代表的就是每百万个碱基)，就是高；低于10个突变/Mb，就是低，处于中间的就是中。2017年世界肺癌大会上，施贵宝公司公布过一项名为CheckMate-032的临床试验结果。这是一项纳入了401名一线治疗失败的晚期肺癌患者的II期临床试验，接受PD-1抑制剂单独或联合伊匹木治疗。按照TMB高低划分成TMB高、TMB中、TMB低三类病人，那么在接受联合治疗的人群中，三组的有效率分别为62％、20％、23％，TMB高的人群有效率高3倍；而三组的中位总生存期，分别为：22.0个月、3.6个月、3.4个月——22.0个月与3.4个月，相差6倍！该试验证明，对于不同的癌症治疗药物，不同的TMB水平对于药物的疗效有很大的影响。
AKR1C3酶激活前药是指该化合物为前药，该前药分子与AKR1C3酶反应，反应后释放出具有细胞毒性的抗肿瘤化合物。
广义而言，AKR1C3酶激活前药满足但不限于以下条件中的至少一项：
A.在存在AKR1C3抑制剂(如Bioorganic and Medicinal Chemistry，2014：962-977中的化合物36即)的环境下，检测得到的某化合物的癌细胞增殖抑制作用小于不存在AKR1C3抑制剂的环境下检测得到的该化合物的癌细胞增殖抑制作用，当癌细胞增殖抑制作用使用IC50进行量化时，则如果某个化合物对某个癌细胞系在存在AKR1C3抑制剂时检测得到的IC50大于不存在AKR1C3抑制剂时检测得到的IC50，则可以判定该化合物为AKR1C3活化的抗癌药物(裂解前药Lysis-Prodrug)。具体的如以下专利文献：
PCT/US2016/021581，公开号WO2016145092A1，对应中国申请号2016800150788，公开号CN107530556A；
PCT/US2016/062114，公开号WO2017087428，对应中国申请号2016800446081，公开号CN108290911A；
PCT/US2016/025665，公开号WO2016161342，对应中国申请号2016800200132，公开号CN108136214A；以及
PCT/NZ2019/050030，公开号WO2019190331，对于中国申请号CN2019800234236，公开号CN111918864A中所公开的化合物，在此将以上专利文献的全文引入到本专利申请文本中。
其中，专利PCT/US2016/021581，公开号WO2016145092A1，对应中国申请号2016800150788，公开号CN107530556A；PCT/US2016/062114，公开号WO2017087428，对应中国申请号2016800446081，公开号CN108290911A；PCT/US2016/025665，公开号WO2016161342，对应中国申请号2016800200132，公开号CN108136214A中所公开的化合物为裂解前药，其最终裂解代谢出的发挥作用的原体药物为以及紫杉醇、喜树碱等药物；专利PCT/NZ2019/050030，公开号WO2019190331，对应中国申请号CN2019800234236，公开号CN111918864A中公开的化合物为裂解前药，其最终裂解代谢出的发挥作用的原体药物为氮芥结构药物；
B.某化合物对AKR1C3酶不同表达水平的癌细胞的增殖抑制作用具有显著差异，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用，当癌细胞增殖抑制作用使用IC50进行量化时，则如果某个化合物高表达AKR1C3酶的癌细胞的IC50值小于低表达AKR1C3的癌细胞的IC50值，则可以判定该化合物为AKR1C3活化的抗癌药物。具体的如专利PCT/CN2020/120281，公开号WO2021068952A1中所公开的化合物。在此将此专利文献的全文引入到本专利申请文本中。
所述AKR1C3酶激活前药选自结构式(1)、(2)、(3)、(4)、(5)、(6)、(7)、(8)、(9)、(10)、(11)的化合物及其盐、酯、溶剂合物、同位素异构体：
式(1)为AKR1C3酶活化的烷化剂前药化合物，更具体的，其为AKR1C3酶活化的DNA烷化剂前药化合物；
其中，X、Y、Z、R、T、A以及X10的定义如专利申请PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
X10是O、S、SO或SO2；
A是C6-C10芳基、5-15元杂芳基或-N＝CR1R2；
R1及R2各自独立是氢、C1-C6烷基、C3-C8环烷基、C6-C10芳基、4-15元杂环、醚、-CONR13R14或-NR13COR14；
X、Y及Z各自独立是氢、CN、卤基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、醚、-CONR13R14或-NR13COR14；
R是氢、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、醚、-CONR13R14或-NR13COR14；
R13及R14各自独立是氢、C1-C6烷基、C3-C8环烷基、C6-C10芳基、4-15元杂环或醚
T包含胺基磷酸酯烷化剂，所述胺基磷酸酯烷化剂包含一或多个键结至-O-P(Z1)部分的Z5-X5-Y5部分的烷化剂，其中Z5是包含氮、硫或氧的杂原子，X5是经取代或未经取代的伸乙基，Y5是卤基或另一离去基，或Z5-X5-Y5一起形成氮丙啶基(NCH2CH2)部分且Z1是O或S；且
其中这些烷基、烯基、炔基、环烷基、芳基、杂环、杂芳基、醚基经取代或未经取代。
其中，R1、R2、R3、R4、R5、R8、R9、R10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A1中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
R1是C6-C10芳基或Z取代的芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、7-15元的稠环或Z取代稠环；
R2是氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C1-C6烷基或Z取代烷基、C2-C6烯基或Z取代烯基、C2-C6炔基或Z取代炔基、C3-C8环烷基或Z取代环烷基、C6-C10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、1-6个碳原子的醚或Z取代的1-6个碳原子的烷氧基、-CONR6R7、-SO2NR6R7、-SO2R6、-OCOO-R6、-COOR6、-NR6COR7、-OCOR6、-NR6SO2R7、-NR6SO2NR6R7或者R2和与其所键结的R1基团上的原子一起形成7-15元的稠环或Z取代稠环；
R3是氢、卤素、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C1-C6烷基或Z取代烷基、C2-C6烯基或Z取代烯基、C2-C6炔基或Z取代炔基、C3-C8环烷基或Z取代环烷基、C6-C10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C1-C6烷氧基或Z取代的C1-C6烷氧基、-CONR6R7、-SO2NR6R7、-SO2R6、-OCO-R6、-OCOO-R6、-COOR6、-NR6COR7，-OCOR6、-NR6SO2R7；
R4、R5各自独立地是氢、卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C1-C6烷基或Z取代烷基、C2-C6烯基或Z取代烯基、C2-C6炔基或Z取代炔基、C3-C8环烷基或Z取代环烷基、C6-C10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C1-C6烷氧基或Z取代的C1-C6烷氧基、-CONR6R7、-SO2NR6R7、-SO2R6、-OCOO-R6、-COOR6、-NR6COR6、-OCOR6、-NR6SO2R7或者R4、R5和与其所键结的苯环上的原子一起形成7-15元的稠环或Z取代稠环；
R6和R7各自独立地是氢、氰基或异氰基、C1-C6烷基或Z取代烷基、C2-C6烯基或Z取代烯基、C2-C6炔基或Z取代炔基、C3-C8环烷基或Z取代环烷基、C6-C10芳基或Z取代芳基、4-15元杂环或Z取代杂环、5-15元杂芳基或Z取代杂芳基、C1-C6烷氧基或Z取代的C1-C6烷氧基，或者R6、R7基团与其所键结的原子一起形成5-7元杂环基或Z取代5-7元杂环基；
R8、R10各自独立地为氢、氘、芳基或Z取代芳基、C1-C6烷基或Z取代烷基、C2-C6烯基或Z取代烯基、C2-C6炔基或Z取代炔基、C3-C8环烷基或Z取代环烷基且必有一个为氢、氘；
R9为至少具有一个氟原子或硝基取代的取代C6-C10芳基、至少具有一个氟原子或硝基取代的取代4-15元杂环、至少具有一个氟原子或硝基取代的取代5-15元杂芳基。
Z取代基为卤素原子、氰基或异氰基、羟基、巯基、胺基、OTs、OMS、C1-C3烷基或取代烷基、C1-C3烷氧基或取代烷氧基、C2-C3烯基或取代烯基、C2-C3炔基或取代炔基、C3-C8环烷基或取代环烷基、芳环、杂环、杂芳环和稠环或取代芳环、杂环、杂芳环和稠环，取代的方式为单取代或偕二取代；
R9中的取代C6-C10芳基、取代4-15元杂环、取代5-15元杂芳基的取代基为卤素原子、硝基、氰基或异氰基、羟基、胺基、C1-C3烷基或烷氧基、烯基、炔基、环烷基或苯环、取代苯环、C1-C3烷氧基或卤原子取代烷氧基。
式(4)为AKR1C3酶活化烷化剂前药化合物，更具体的，其为AKR1C3酶活化DNA烷化剂前药化合物；
其中：
A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR1R2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO2、–O-(CH2)-O-、-CO2H及其盐、-OR100、-CO2R100、-CONR101R102、-NR101R102、-NR100SO2R100、-SO2R100、-SO2NR101R10 2、C1-C6烷基、C3-C10杂环基；
其中，R100、R101及R102各自独立是氢、C1-C8烷基、C6-C12芳基；或R101及R102与其附接至的氮原子一起形成5-7元杂环；
其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
R1及R2各自独立是苯基或甲基；
X、Y及Z各自独立是氢或卤基；
R是氢或C1-C6烷基或卤素取代烷基。
其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
Rw为
R1为H、C1-6烷基、C3-6环烷基、4-6元杂环烷基、5-6元杂芳基或苯基，其中所述C1-6烷基、C3-6环烷基、4-6元杂环烷基、5-6元杂芳基和苯基任选被1、2或3个Ra所取代；
各Ra独立地为H、F、Cl、Br、I、-CN、-OH、C1-3烷氧基或C1-3烷基；
R2为H或C1-6烷基；
或者R1和R2连接在一起，与其相连的N原子一起形成4-6元杂环烷基，其中所述4-6元杂环烷基任选被1、2或3个Rb所取代；
各Rb独立地为H、F、Cl、Br、I、-CN、-OH、-NH2、-OCH3、-OCH2CH3、-CH3或-CH2CH3；
R3为H、F、Cl、Br、I、-OH、-NH2、C1-3烷氧基或C1-3烷基；
或者R2和R3连接在一起使结构单元为
T1为-(CRcRd)m-或-(CRcRd)n-O-；
m为1、2或3；
n为1或2；
T2为N或CH；
Rc和Rd各自独立地为H、F、C1-3烷基或C1-3烷氧基；
R4、R5和R6各自独立地为H、F、Cl、Br、I、C1-3烷基或C1-3烷氧基；
T为N或CH；
R7和R8各自独立地为H、F、Cl、Br或I；
R9和R10各自独立地为H、F、Cl、Br、I、-CN或
所述4-6元杂环烷基和5-6元杂芳基各自包含1、2、3或4个独立选自N、-O-和-S-的杂原子。
其中，A、E、G、X、Y的定义如专利申请PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
A为H、C1-C6烷基、C1-C6烯基、C1-C6炔基、CFH2、CF2H、CF3、F、Cl、Br、I、OCF3、COR或CON(R)2；
E为SO或SO2；
X为Cl、Br、I或OSO2R；
Y为Cl、Br、I或OSO2R；
每个R独立地为H或C1-C6烷基；
G是选自包括式(B)-(AA)的组中的自由基：
其中：
R1为H、C1-C6烷基、CH2(CH2)nOH、CH2CH(OH)CH2OH、苯基、吡啶基、苄基或吡啶基甲基，条件是当R1为苯基、吡啶基、苄基或吡啶基甲基时，R1任选地在任何可用位置处被C1-C6烷基、C1-C6烯基、C1-C6炔基、OR6、N(R6)(R7)、CFH2、CF2H、CF3、F、Cl、Br、I、OCF3、COR6、CON(R6)(R7)、SOR6、SON(R6)(R7)、SO2R6、SO2N(R6)(R7)、CN或NO2取代；
R2和R3各自独立地为H、C1-C6烷基、C1-C6烯基、C1-C6炔基、OR6、N(R6)(R7)、CFH2、CF2H、CF3、F、Cl、Br、I、OCF3、COR6、CON(R6)(R7)、SOR6、SON(R6)(R7)、SO2R6、SO2N(R6)(R7)、CN或NO2；
R4为N(R6)(R7)、OH、OCH2(CH2)nN(R6)(R7)或CH2(CH2)nN(R6)(R7)；
R5为H或C1-C6烷基基团；
R6和R7各自独立地为H或C1-6烷基，或者R6和R7一起形成取代或未被取代的5元或6元杂环；
Z为CH或N；
W为CH2、O、S、SO或SO2；
n为0至6；
*表示与式(I)的连接点。
其中，X、Y、Z、R、D、L1、A以及X10的定义如专利申请PCT/US2016/025665，公开号WO2016161342A3(对应中国申请号2016800200132，公开号CN108136214A)中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
X10是O、S、SO或SO2；
A是C6-C10芳基或取代芳基、5-15元杂芳基或取代杂芳基或-N＝CR1R2；其中，R1和R2各自独立地是氢、C1-C6烷基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基、醚、-CONR13R14或-NR13COR14；
X、Y和Z各自独立地是氢、CN、卤基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基、醚、-CONR13R14或-NR13COR14；
每个R独立地是氢、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基、醚、-CONR13R14或-NR13COR14；
R13和R14各自独立地是氢、C1-C6烷基、C3-C8环烷基、C6-C10芳基、4-15元杂环、5-15元杂芳基或醚，或者R13和R14与其所键结的氮原子一起形成5-7元杂环基；
L1和D如说明书中定义，具体定义如下
L1选自：
其中，R40和R41独立地是氢、C1-C6烷基、C2-C6烯基、C2-C6炔基、C3-C8环烷基、C6-C10芳基、4-15元杂环或5-15元杂芳基；
R42是视情况经1-3个C1-C6烷基取代的C2-C3伸烷基或伸杂烷基；V(-)为任何阴离子，较佳为医药学上可接受的阴离子；和
D是使得D-OH为抗癌药物的部分，其中OH为脂族羟基或酚羟基，或为如本文提供的附接至磷原子的OH部分；换言之，D是抗癌药物D-OH脱去羟基后剩余的基团；
或者
L1为：
其中R40如上文所定义，R43为氢或与D一起形成杂环，且苯基部分视情况经取代；和
D是使得D-NR43H为抗癌药物的部分；换言之，D是抗癌药物D-NR43H脱去氨基或胺后剩余的基团；
或者
L1是键、-O-C(R40R41)-、-O-C(R40R41)-NR40R41(+)-C(R40R41)-或
其中R40、R41和V如上文所定义；和
D是含有伯胺或仲胺的抗癌药物，其中该伯胺或该仲胺键接至L1；且
其中所述烷基、烯基、炔基、环烷基、芳基、杂环、杂芳基、醚基视情况经取代。
其中，R1、R2、R3、R4及T的定义如专利申请PCT/CN2021/118597，公开号WO2022057838A1中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
T为N或CH；
R1和R2各自独立地为H、F、Cl、Br、I或C1-3烷基，其中所述C1-3烷基任选被1、2或3个Ra所取代；
各Ra独立地F、Cl、Br、I、-CN、-OH或-NH2；
R3和R4各自独立地为H、F、Cl、Br、I、CN、C1-3烷基、C1-3烷氧基、其中，所述C1-3烷基任选被1、2或3个Re所取代；
Rb和Rc各自独立地为H、-CH3、-CH2CH3、-(CH2)2CH3、-CH(CH3)2；
Rd为-CH3、-CH2CH3、-(CH2)2CH3、-CH(CH3)2；
各Re独立地F、Cl、Br、I、-CN、-OH或-NH2。
或其药学上可接受的盐，
其中，Rw、X、R4、R10、R13、R14的定义如专利申请PCT/CN2022/098082，公开号WO2022258043A1中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
两个X各自独立地为CR15或N；
R13、R14各自独立地是氢，C1-C6烷基，环烷基，烯基，炔基，C6-C20芳基，5-20元环 的杂环基，卤素取代的C1-C6烷基、环烷基、烯基、炔基，卤素取代的C6-C20芳基，或卤素取代的5-20元环的杂环基，且R13、R14不同时为氢；
R10是氢，C1-C6烷基，环烷基，烯基，炔基，C6-C20芳基，5-20元环的杂环基，卤素取代的C1-C6烷基、环烷基、烯基、炔基，卤素取代的C6-C20芳基，或卤素取代的5-20元环的杂环基；
或者R10与R13或R14可在符合上述R10、R13、R14定义的条件下相连形成5-9元环；
R4、R15各自独立地是氢，卤素，C1-C6烷基，环烷基，烯基，炔基，烷氧基，氰基，5-20元环的杂环基，C6-C20芳基，或卤素取代的C1-C6烷基、环烷基、烯基、炔基、烷氧基、5-20元环的杂环基、C6-C20芳基；
或者R10与R15可在符合上述R10、R15定义的条件下形成4-12元的环烃或杂环；
RW是
A为CR16或N，且其中A的位置是可以在环上变动的；
R16为氢，C1-C6烷基，环烷基，烯基，炔基，C6-C20芳基，5-20元环的杂环基，卤素取代的C1-C6烷基、环烷基、烯基、炔基，卤素取代的C6-C20芳基，或卤素取代的5-20元环的杂环基；
R6、R7满足以下条件：
R6、R7各自独立地是氢，卤素，氰基，羟基，C1-C6烷基、环烷基、烯基、炔基、烷氧基、5-20元环的杂环基、C6-C20芳基，或卤素取代的C1-C6烷基、环烷基、烯基、炔基、烷氧基、5-20元环的杂环基、C6-C20芳基，或氰基取代的C1-C6烷基、环烷基、烯基、炔基、烷氧基、5-20元环的杂环基、C6-C20芳基，或羟基取代的C1-C6烷基、环烷基、烷氧基、5-20元环的杂环基、C6-C20芳基，或-CONR11R12，或-CH2NR11R12；
或者R6、R7相连形成
含至少一个N或S或O或同时含N、S、O中的两个或三个的5-8元的单杂环或稠合的杂环；
或者，含至少一个N或S或O或同时含N、S、O中的两个或三个的5-8元的单杂环或稠合的杂环，且该单杂环或稠合的杂环被C1-C6烷基取代；
R6可与CR16相连形成5-9元环、杂环或芳杂环；
R11、R12满足以下条件：
R11、R12各自独立的为C1-C6烷基，卤素取代的C1-C6烷基或R11、R12满足上述定义条件下与-CONR11R12中的N形成5-7元环，与-CH2NR11R12中的N形成5-7元环。
其中，R1、R2、R3、R4、G1、G2、G3、G4、E、T、Y、Z、m、n、s、t、v、w、环A的定义如专利申请CN202210585771.6，公开号CN115403579A中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
G1、G2、G3或G4相同或不同，且各种独立地为CR5或N原子；
各个R5相同或不同，且各自独立地选自氢原子、卤素、烷基、烯基、炔基、烷氧基、羟基、氰基、氨基、硝基、-NRaRb、-C(O)NRaRb、环烷基、杂环基、芳基和杂芳基，其中所述的烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、羟基、氧代、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
Y选自-(C(Ry2Ry3))f-NRy1-、-(C(Ry2Ry3))g-O-、-(C(Ry2Ry3))h-S-、-(C(Ry2Ry3))h-S(O)-、-(C(Ry2Ry3))h-S(O)2-、-C(Ry2Ry3)-、-NRy1-(C(Ry2Ry3))f-、-O-(C(Ry2Ry3))g-、-S-(C(Ry2Ry3))h-、-S(O)-(C(Ry2Ry3))h-和-S(O)2-(C(Ry2Ry3))h-；
Ry1选自氢原子、烷基、卤代烷基、羟烷基、环烷基和杂环基；
Ry2和Ry3相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、羟烷基、环烷基和杂环基；
或者Ry2和Ry3一起形成＝O；
Z为O或OH；
为单键或双键，当为单键时Z为OH，当为双键时Z为O；
E选自NH﹑O原子和S原子；
T选自-C(RT1RT2)-﹑-NRT3-或-O-；
RT1和RT2相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、羟烷基、环烷基和杂环基；
或者RT1和RT2与其相连的碳原子一起形成环烷基或杂环基，所述的环烷基或杂环基各自独立地任选被选自卤素、烷基和羟基中的一个或多个取代基所取代；
RT3选自氢原子、烷基、卤代烷基、羟烷基、环烷基和杂环基；
环A为6至10元芳基或5至10元杂芳基；
各个R1相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、烯基、炔基、烷氧基、羟基、氰基、-NRaRb、-C(O)NRaRb、-S(O)NRaRb、-S(O)2NRaRb、-S(O)Rc、-S(O)2Rc、-B(ORd)2、硝基、环烷基、杂环基、芳基和杂芳基，其中所述的烷基、烯基、炔基、烷氧基、环烷基、杂环基、芳基和杂芳基各自独立地任选被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、羟基、氧代、氰基、-NRaRb、-C(O)NRaRb、-S(O)NRaRb、-S(O)2NRaRb、-S(O)Rc、-S(O)2Rc、-B(ORd)2、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
Ra和Rb相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、羟基、羟烷基、-C(O)Re、环烷基和杂环基；或者Ra和Rb与其相连的氮原子一起形成环烷基或杂环基，其中所述的环烷基或杂环基任选被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、羟基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
Rc选自烷基、环烷基、杂环基、芳基和杂芳基，所述的烷基、环烷基、杂环基、芳基和杂芳基任选被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、羟基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
Rd为氢原子或C1-6烷基；
Re选自烷基、烷氧基、环烷基、杂环基、芳基和杂芳基，所述的烷基、烷氧基、环烷基、杂环基、芳基和杂芳基任选被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、羟基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；
各个R2相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、氧代、羟基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
各个R3相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、氧代、羟基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基；
R4选自氢原子、烷基、卤代烷基、羟基和羟烷基；
n为0、1、2或3；
v为0、1或2；
w为0、1或2；
f为0、1或2；
g为0、1或2；
h为0、1或2；
m为0、1、2、3、4或5；
s为0、1、2、3、4、5、6、7或8；
t为0、1、2、3、4、5或6；
条件是，
当Y为-O-原子，且E为O原子时，环A为苯基或5至6元杂芳基，且G3为CR5或N原子，R5不为氢原子；
当Y为-O-原子，且E为S原子时，环A为苯基或5至6元杂芳基；
当G1、G2、G3和G4均为CR5，Y为NRy1，n、v和w均为1，且E为O原子时，1)T不为CH2或CD2，2)至少有一个R2或R3为氘原子，3)R4选自烷基、卤代烷基、羟基和羟烷基，4)其中一个R1为3至8元环烷基或5至8元杂环基，所述的3至8元环烷基或5至8元杂环基任选被选自卤素、烷基、卤代烷基、烷氧基、卤代烷氧基、羟烷基、羟基、氧代、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，5)环A为Rd为氢原子或C1-6烷基。
或其药学上可接受的盐，
其中，R1、R2a、R2b、R3、R4、R5、n、Z的定义如专利申请PCT/IB2020/057285，公开号WO2021005586A1(对应中国申请号CN202080053804.1，公开号CN114206870A)中的权利要求书所记载，具体化合物的合成制备方法也记载在上述申请中，在此将其全文引入本申请之中，具体定义为：
是单键或双键；
Z当为单键时是OH；或当为双键时是O；
每个R1独立地选自由以下组成的组：(C1-C6)烷基、(C1-C6)烷氧基、(C0-C4)烷基N(R8)2、和卤基；
R2a和R2b各自独立地选自由以下组成的组：H、(C1-C6)烷基、和卤基；
每个R3独立地选自由以下组成的组：H、和卤基；
R4选自由以下组成的组：芳基，包含1、2、3、或4个独立地选自N、O和S的杂原子的5至6元杂芳基；和包含1、2、3、或4个独立地选自N、O和S的杂原子的9至10元稠合双环杂芳基；其中前述中的任一项任选地被一个或多个R6取代；
R5选自由以下组成的组：H；(C1-C6)烷基；(C2-C6)烯基；(C0-C4)烷基OR8；(C1-C4)烷基(C3-C10)环烷基；卤代(C1-C6)烷基；(C2-C3)炔基；(C1-C4)烷基N(R10)2；
每个R6独立地选自由以下组成的组：卤基；(C1-C6)烷基；(C1-C6)烷氧基；卤代(C1-C6)烷基；OH；芳基；3至6元杂环；5至6元杂芳基；(C0-C4)烷基S(O)m(C1-C6)烷基；卤代(C1-C6)烷氧基；(C0-C4)烷基S(O)mN(R8)2；(C0-C4)烷基N(R8)2；(C0-C4)烷基(CO)OR7；N(R8)S(O)m(C1-C6)烷基；N(R8)S(O)m(C3-C6)环烷基；OP(O)(OH)2；(C0-C3)烷基(CO)NHR11；(C0-C3)烷基OR7、和 (C3-C10)环烷基；其中每个R6当不为卤基、OH、或OP(O)(OH)2时任选地被一至三个R9取代；或两个相邻的R6与它们所附接的原子一起形成5至7元杂环或(C5-C8)环烷基；
每个R7和R8独立地选自由以下组成的组：H或任选地被一至三个R9取代的(C1-C6)烷基；
每个R9独立地选自由以下组成的组：卤基；-OH；氨基、(C1-C4)烷基氨基、二(C1-C4)烷基氨基、OP(O)(OH)2；(C1-C6)烷基；(C1-C3)炔基；(C1-C6)烷氧基；卤代(C1-C6)烷基；(C0-C4)烷基S(O)m(C1-C6)烷基；卤代(C1-C6)烷氧基；任选地被氧代(＝O)取代的3至6元杂环；(C0-C4)烷基S(O)mN(R10)2；(C0-C4)烷基(CO)R10；(C0-C4)烷基(CO)OR10；(C0-C4)烷基NR10S(O)m(C1-C6)烷基；(C0-C4)烷基OR10；(C0-C4)烷基N(R10)2；(C0-C4)烷基CN；(C0-C4)烷基N(R10)2；和(C0-C4)烷基(CO)N(R10)2；
每个R10独立地选自由以下组成的组：H、(C1-C6)烷基；或3至6元杂环，其中所述3至6元杂环任选地被以下中的一个或多个取代：(C1-C6)烷基；和氧代(＝O)；
每个R11选自由以下组成的组：H；任选地被一至四个R12取代的4至6元杂环；任选地被一至四个R12取代的(C3-C6)环烷基；任选地被卤基取代的(C0-C3)烷基(C3-C6)环烷基(C1-C3)烷基；任选地被一至三个R12取代的CH2-芳基；(C1-C6)烷基；(C2-C6)烯基；或(C2-C6)炔基，其中所述(C1-C6)烷基；(C2-C6)烯基；和(C2-C6)炔基中的每一个任选地被一个或多个R13取代；
每个R12独立地选自由以下组成的组：OH、(C1-C3)烷氧基、NH2；或任选地被一个或多个OH取代的(C1-C3)烷基；
每个R13独立地选自由以下组成的组：卤基、OH、氨基、(C1-C4)烷基氨基、二(C1-C4)烷基氨基、(C1-C3)烷氧基；和C(O)-(C3-C8)环烷基；
m是0、1、或2；并且
n是0、1或2。
具体的，式(1)-(11)的具体化合物的化学结构如权利要求5所述，在此不再赘述。
其中，式(1)具体化合物参考专利申请文件PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)中的化合物，在此将该专利申请文件全文引入本申请；
式(2)(3)具体化合物参考专利申请文件PCT/CN2020/089692，公开号WO2020228685A1中的化合物，在此将该专利申请文件全文引入本申请；
式(4)具体化合物参考专利申请文件PCT/US2016/021581、公开号WO2016145092A1(对应中国申请号2016800150788、公开号CN107530556A)和PCT/CN2020/089692、公开号WO2020228685A1中的化合物，在此将该两件专利申请文件全文引入本申请；
式(5)具体化合物参考专利申请文件PCT/CN2020/120281，公开号WO2021068952A1中的化合物，在此将该专利申请文件全文引入本申请；
式(6)具体化合物参考专利申请文件PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)中的化合物，在此将该专利申请文件全文引入本申请；
式(7)具体化合物参考专利申请文件PCT/US2016/025665，公开号WO2016161342A3(对应中国申请号2016800200132，公开号CN108136214A)中的化合物，在此将该专利申请文件全文引入本申请；
式(8)具体化合物参考专利申请文件PCT/CN2021/118597，公开号WO2022057838A1中的化合物，在此将该专利申请文件全文引入本申请；
式(9)具体化合物参考专利申请文件PCT/CN2022/098082，公开号WO2022258043A1中的化合物，在此将该专利申请文件全文引入本申请；
式(10)具体化合物参考专利申请文件CN202210585771.6，公开号CN115403579A中的化合物，在此将该专利申请文件全文引入本申请；
式(11)具体化合物参考专利申请文件PCT/IB2020/057285，公开号WO2021005586A1(对应中国申请号CN202080053804.1，公开号CN114206870A)中的化合物，在此将该专利申请文件全文引入本申请。
式(1)-(11)具体化合物的制备方法、波谱数据参见上述专利文件。
显然结构式(1)的化合物与AST-3424类似，是胺基磷酸酯烷化剂的前药，其会在AKR1C3酶的作用下活化而产生T(一种胺基磷酸酯烷化剂，AST-2660就是一种胺基磷酸酯烷化剂)发挥抗癌效果：
具体的以AST-3424为例，这些化合物作为醛酮还原酶AKR1C3特异性底物，可仅在AKR1C3高表达的癌细胞内快速有效地还原，从而释放细胞毒素DNA烷化剂AST-2660，AST-2660与DNA交联而使得癌细胞死亡：
显然结构式(2)(3)的化合物与AST-3424相同，都是AST-2660的前药，特别是其中的化合物AST，其会在AKR1C3酶的作用下活化而产生AST-2660(一种DNA烷化剂)发挥抗癌效果：

显然结构式(4)的化合物与AST-3424、AST类似，是AST-2660的前药，其会在AKR1C3酶的作用下活化而产生AST-2660发挥抗癌效果：
显然结构式(5)的化合物与AST-3424、AST相同，都是AST-2660的前药，其会在AKR1C3酶的作用下活化而产生AST-2660(一种DNA烷化剂)发挥抗癌效果：
显然结构式(6)的化合物与AST-3424、AST原理类似，都是氮芥类似物的前药，其会在AKR1C3酶的作用下活化而产生氮芥类似物(一种DNA烷化剂)发挥抗癌效果：
显然结构式(7)的化合物会在AKR1C3酶的作用下活化而产生能抑制癌细胞增殖或肿瘤生长的药物D发挥抗癌效果：
D可以是紫杉醇、SN-38、吉西他滨等抗癌活性物质。
紫杉醇可使微管蛋白和组成微管的微管蛋白二聚体失去动态平衡，诱导与促进微管蛋白聚合、微管装配、防止解聚，从而使微管稳定并抑制癌细胞的有丝分裂和触发细胞凋亡，进而有效阻止癌细胞的增殖，起到抗癌作用。
SN-38可抑制人体细胞DNA复制所必需的拓扑异构酶I，诱导DNA发生单链损伤、阻断DNA复制而产生细胞毒性。
吉西他滨(dFdC)是核苷胞嘧啶核苷(嘧啶)类似物，可掺入复制性DNA中，从而抑制DNA合成。进入体内后，脱氧胞苷激酶(dCK)将吉西他滨的磷酸激活，吉西他滨因此转变为吉西他滨二磷酸(dFdCDP)和三磷酸(dFdCTP)，这些活性药物代谢产物对DNA合成具有多种抑制作用。
同理，结构式(8)(9)(10)(11)的化合物同样会在AKR1C3酶的作用下活化而产生能抑制癌细胞增殖或肿瘤生长的药物AST-2660、吉西他滨或KARS抑制剂等而发挥抗癌效果。
KARS抑制剂，即赖氨酸t-RNA合成酶抑制剂，赖氨酸t-RNA合成酶是蛋白质合成所必需的普遍存在的酶，所述酶是多t-RNA合成酶复合物的一部分。
进一步，前药化合物优选自：

关于本文所述药物是指药品或制剂，所制得的药品包含特定剂量范围的有效成分式(1)-(11)的AKR1C3酶活化的化合物或其盐或溶剂合物，和/或所制得的药物为特定剂型、特定给药方式施用。
上述药物除含有式(1)-(11)的AKR1C3酶活化前药化合物外，还应根据药品、药物、制剂的特点，添加药学上可接受的辅料或赋形剂。所述药物可以为临床施用的任何剂型，例如片剂、栓剂、分散片、肠溶片、咀嚼片、口崩片、胶囊、糖衣剂、颗粒剂、干粉剂、口服溶液剂、注射用小针、注射用冻干粉针或大输液。根据具体剂型和施用方式，所述药物中的药学上可接受的辅料或赋形剂可以包括下述的一种或多种：稀释剂、增溶剂、崩解剂、悬浮剂、润滑剂、粘合剂、填充剂、矫味剂、甜味剂、抗氧化剂、表面活性剂、防腐剂、包裹剂和色素等。
“癌症”是指可通过侵袭而局部扩展且通过转移而全身扩展的潜在无限制生长的白血病、淋巴瘤、癌及其他恶性肿瘤(包括实体肿瘤)。
在此列举AST-3424、AST等这类AKR1C3酶活化的前药能治疗的癌症的实例包括(但不限于)肾上腺、骨、脑、乳房、支气管、结肠及/或直肠、胆囊、头及颈、肾、喉、肝、肺、神经组织、胰脏、前列腺、副甲状腺、皮肤、胃及甲状腺的癌症。癌症的某些其他实例包括急性及慢性淋巴细胞及粒细胞肿瘤、腺癌、腺瘤、基底细胞癌、子宫颈上皮分化不良及原位癌、尤文氏肉瘤、表皮样癌、巨细胞瘤、多型性神经胶母细胞瘤、毛细胞肿瘤、肠神经节细胞瘤、增生性角膜神经肿瘤、胰岛细胞癌、卡波西肉瘤、平滑肌瘤、白血病、淋巴瘤、恶性类癌瘤、恶性黑色素瘤、恶性高钙血症、马方样体型肿瘤、髓样上皮癌、转移性皮肤癌、黏膜神经瘤、骨髓瘤、蕈状肉芽肿、神经胚细胞瘤、骨肉瘤、骨原性及其他肉瘤、卵巢瘤、嗜铬细胞瘤、真性红血球增多症、原发性脑瘤、小细胞肺癌、溃疡型及乳头型二者的鳞状细胞癌、增生、精原细胞瘤、软组织肉瘤、视网膜母细胞瘤、横纹肌肉瘤、肾细胞肿瘤、局部皮肤病灶、网状细胞肉瘤及威尔姆氏肿瘤。
AST-3424或这类AKR1C3酶活化的DNA烷化剂前药治疗癌症的推荐剂量、给药制剂形式可以参考Threshold公司及Ascentawits、OBI等公司提交的专利申请文本(比如WO2017087428A1、WO2017087428A1、WO2019062919A1、WO2021008520A1)以及FDA、NMPA登记的临床试验(CTR20201915、CTR20201908、CTR20191399、CTR20191371以及NCT04315324、NCT03592264)。
本申请中的治疗包括单药治疗和联合其他药物治疗。
单药，即单药治疗。联用，即联合用药治疗。单药治疗是指在一个疗程中仅使用一种抗癌药物。联合治疗是指在一个疗程中同时或先后使用两种或两种以上的抗癌药物。
一般而言，联合治疗需要根据病情特点、联用药物种类探索不同的给药剂量、给药周 期，只有根据上述情况，探索得到的联合用药治疗方案才可能取得较单一用药治疗好的治疗效果。
单药和联用治疗方案的药物给药剂量、给药周期均需要在参考上述AST-3424及其类似化合物和联用药物的剂量、给药方案通过临床试验探索得到。
AST-3424(OBI-3424)、(以下代号为AST)以及的合成方法、波谱数据被专利申请：PCT/US2016/021581，公开号WO2016145092A1，对应中国申请号2016800150788，公开号CN107530556A；PCT/US2016/062114，公开号WO2017087428A1，对应中国申请号2016800446081，公开号CN108290911A；PCT/CN2020/089692，公开号WO2020228685A9所公开；相关的制剂浓缩注射液，并且相关处方、制备方法和临床配伍、施用方法被相关专利：WO2021008520A1、WO2021043275A1所详细说明并公开，在此本发明将上述申请文本的全文引入。
附图说明
图1为胃癌GA6201模型中各组小鼠肿瘤体积的生长曲线照片；
图2为胃癌GA6201模型中各组小鼠相对肿瘤抑制率曲线照片；
图3为胃癌GA6201模型中各组小鼠体重曲线照片；
图4为胃癌GA6201模型中各组小鼠体重变化百分比曲线照片；
图5为胰腺癌PA1222模型中各组小鼠肿瘤体积的生长曲线照片；
图6为胰腺癌PA1222模型中各组小鼠相对肿瘤抑制率曲线照片；
图7为胰腺癌PA1222模型中各组小鼠体重曲线照片；
图8为胰腺癌PA1222模型中各组小鼠体重变化百分比曲线照片；
图9为肺癌LU11693模型中各组小鼠肿瘤体积的生长曲线照片；
图10为肺癌LU11693模型中各组小鼠相对肿瘤抑制率曲线照片；
图11为肺癌LU11693模型中各组小鼠体重曲线照片；
图12为肺癌LU11693模型中各组小鼠体重变化百分比曲线照片；
图13为胰腺癌PA1383模型中各组小鼠肿瘤体积的生长曲线照片；
图14为胰腺癌PA1383模型中各组小鼠相对肿瘤抑制率曲线照片；
图15为胰腺癌PA1383模型中各组小鼠体重曲线照片；
图16为胰腺癌PA1383模型中各组小鼠体重变化百分比曲线照片；
图17为胃癌GA6201、肺癌LU11693、胰腺癌PA1222三个PDX模型肿瘤组织及对照组的IHC染色结果照片；
图18为KRAS-G12D突变的模型中AKR1C3的RNA表达水平与NRF2的RNA表达水平的对应关系图；
图19为KRAS-G12C突变的模型中AKR1C3的RNA表达水平与NRF2的RNA表达水平的对应关系图；
图20为KRAS-G13D突变的模型中AKR1C3的RNA表达水平与NRF2的RNA表达水平的对应关系图；
图21为胰腺癌HPAF II的免疫印迹图片及肺癌LU5161、结直肠癌CR3820的IHC染色 结果照片；
图22为SFN不同浓度处理下细胞裂解后目标蛋白免疫印迹实验结果，其中，上图为SFN不同浓度处理下的免疫印迹图片，下图为NRF2与AKR1C3的蛋白条带密度分析柱状图，其中，各组浓度下，左侧柱形为NRF2，右侧柱形为AKR1C3；
图23为AST不同浓度处理下细胞裂解后目标蛋白免疫印迹实验结果，其中，上图为AST不同浓度处理下的免疫印迹图片，下图为p-ERK2与ERK2的在AST不同浓度处理下的蛋白条带密度分析柱状图，其中，各组AST浓度下，左侧柱形为p-ERK2，右侧柱形为ERK2；
图24为1μM AST不同时间处理下细胞裂解后目标蛋白免疫印迹实验结果，其中，上图为1μM AST处理不同时间下的免疫印迹图片，下图为p-ERK2与ERK2的在1μM AST处理不同时间下的蛋白条带密度分析柱状图，其中，各组处理时间下，左侧柱形为p-ERK2，右侧柱形为ERK2；
图25为AKR1C3依赖的细胞裂解后目标蛋白免疫印迹实验结果，其中，上图为AKR1C3依赖的免疫印迹图片，下图为p-ERK2与ERK2的AKR1C3依赖的蛋白条带密度分析柱状图，其中，各处理组中，左侧柱形为p-ERK2，右侧柱形为ERK2；
图26为ERK2抑制剂和AST单药及联用药组的细胞裂解后目标蛋白免疫印迹和细胞凋亡实验结果，其中，上图为PD98059、Suramin的ERK2抑制剂组单药或联用的免疫印迹图片；下左图为其单药或联用的蛋白条带密度分析柱状图，其中，各处理组中，左侧柱形为p-ERK2，右侧柱形为ERK2；下右图为其单药或联用的细胞凋亡柱状图，其中，Luminescence(RLU)表示凋亡信号相对发光单位；
图27为ERK2激活剂和AST单药及联用药组的细胞裂解后目标蛋白免疫印迹和细胞凋亡实验结果，其中，上图为TPA的ERK2激活剂组单药或联用的免疫印迹图片；下左图为其单药或联用的密度分析柱状图，其中，各处理组中，左侧柱形为p-ERK2，右侧柱形为ERK2；下右图为其单药或联用的细胞凋亡柱状图，其中，Luminescence(RLU)表示凋亡信号相对发光单位；
图28为AST通过ERK2信号通路诱导KRAS G12D致病突变癌细胞凋亡的过程示意图，其中，(1)、(3&4)表示相关证明文献，
(1)Prior IA,Hood FE and Hartley JL.The Frequency of Ras Mutations in Cancer.Cancer Res 2020；80:2969-2974；
(3)Park HE,Yoo SY,Cho NY,Bae JM,Han SW,Lee HS,Park KJ,Kim TY and Kang GH.Tumor microenvironment-adjusted prognostic implications of the KRAS mutation subtype in patients with stage III colorectal cancer treated with adjuvant FOLFOX.Sci Rep 2021；11:14609；
(4)Patricelli MP,Janes MR,Li LS,Hansen R,Peters U,Kessler LV,Chen Y,Kucharski JM,Feng J,Ely T,Chen JH,Firdaus SJ,Babbar A,Ren P and Liu Y.Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State.Cancer Discov 2016；6:316-329；
“①”表示AST化合物作用于MEK-ERK信号通路并激活该通路；
“②”表示AKR1C3酶活化AST前药化合物并最终释放DNA烷化剂AST-2660；
“③”表示肿瘤细胞的增殖在AST-2660作用下被抑制。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
向患者“投与”(“administering”或“administration of”)药物(及此词组的语法等效形式) 是指直接投与(其可由医学专业人士向患者投与或可自投与)及/或间接投与，其可是开处药物的行为。举例而言，指示患者自投与药物及/或将药物的处方提供给患者的医师是向患者投与药物。
“癌症”是指可通过侵袭而局部扩展且通过转移而全身扩展的潜在无限制生长的白血病、淋巴瘤、癌及其他恶性肿瘤(包括实体肿瘤)。癌症的实例包括(但不限于)肾上腺、骨、脑、乳房、支气管、结肠及/或直肠、胆囊、头及颈、肾、喉、肝、肺、神经组织、胰脏、前列腺、副甲状腺、皮肤、胃及甲状腺的癌症。癌症的某些其他实例包括急性及慢性淋巴细胞及粒细胞肿瘤、腺癌、腺瘤、基底细胞癌、子宫颈上皮分化不良及原位癌、尤文氏肉瘤(Ewing’s sarcoma)、表皮样癌、巨细胞瘤、多型性神经胶母细胞瘤、毛细胞肿瘤、肠神经节细胞瘤、增生性角膜神经肿瘤、胰岛细胞癌、卡波西肉瘤(Kaposi’s sarcoma)、平滑肌瘤、白血病、淋巴瘤、恶性类癌瘤、恶性黑色素瘤、恶性高钙血症、马方样体型肿瘤(marfanoid habitus tumor)、髓样上皮癌、转移性皮肤癌、黏膜神经瘤、骨髓瘤、蕈状肉芽肿、神经胚细胞瘤、骨肉瘤、骨原性及其他肉瘤、卵巢瘤、嗜铬细胞瘤、真性红血球增多症、原发性脑瘤、小细胞肺癌、溃疡型及乳头型二者的鳞状细胞癌、增生、精原细胞瘤、软组织肉瘤、视网膜母细胞瘤、横纹肌肉瘤、肾细胞肿瘤、局部皮肤病灶、网状细胞肉瘤及威尔姆氏肿瘤(Wilm’s tumor)。
“患者”及“个体”可互换使用，是指需要癌症治疗的哺乳动物。通常，患者是人类。通常，患者是诊断患有癌症的人类。在某些实施例中，“患者”或“个体”可指用于筛选、表征及评估药物及疗法的非人类哺乳动物，例如非人类灵长类动物、狗、猫、兔、猪、小鼠或大鼠。
“实体肿瘤”是指包括(但不限于)骨、脑、肝、肺、淋巴结、胰脏、前列腺、皮肤及软组织(肉瘤)中的转移肿瘤的实体肿瘤。
药物的“治疗有效量”是指当向患有癌症的患者投与时，将具有预期的治疗效应(例如患者中一或多种癌症的临床表现的缓和、改善、缓解或消除)的药物的量。治疗效应不必通过投与一个剂量而出现，且可仅在投与一系列剂量后出现。因此，治疗有效量可以一或多次投与来投与。
病况或患者的“治疗”(“Treating”、“treatment of”或“therapy of”)是指采取步骤以获得有益或期望结果(包括临床结果)。出于本发明的目的，有益或期望临床结果包括(但不限于)一或多种癌症症状的缓和或改善；疾病程度的减弱；疾病进展的延迟或减缓；疾病状态的改善、缓解或稳定；或其他有益结果。在一些情形下，癌症的治疗可使得部分反应或稳定疾病。
“肿瘤细胞”是指任何适当物种(例如，哺乳动物，例如鼠类、犬、猫、马或人类)的肿瘤细胞。
“患者”及“个体”可互换使用，是指需要癌症治疗的哺乳动物。通常，患者是人类。通常，患者是诊断患有癌症的人类。在某些实施例中，“患者”或“个体”可指用于筛选、表征及评估药物及疗法的非人类哺乳动物，例如非人类灵长类动物、狗、猫、兔、猪、小鼠或大鼠。
“治疗”或“治疗患者”是指向患者投与、使用或施用本发明相关的治疗有效量的药物。
向患者“投与”或“施用”“使用”药物是指直接投与或施用(其可由医学专业人士向患者投与或施用或者可自投与或施用)及/或间接投与或施用，其可是开处药物的行为。举例而言，指示患者自投与或施用药物及/或将药物的处方提供给患者的医师是向患者投与或施用药物。
病况或患者的“治疗”是指采取步骤以获得有益或期望结果(包括临床结果)。出于本发明的目的，有益或期望临床结果包括(但不限于)一或多种癌症症状的缓和或改善；疾病程度的减弱；疾病进展的延迟或减缓；疾病状态的改善、缓解或稳定；或其他有益结果。在一些情形下，癌症的治疗可使得部分反应或稳定疾病。
下述实施例中的实验方法，如无特殊说明，均为常规方法。所用的药材原料、试剂材 料等，如无特殊说明，均为市售购买产品。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
以下提供本发明的具体实验。
1、受试物AST，AST-3424和Ifosfamide在胃癌GA6201皮下异种移植模型中的药效学评价
胃癌GA6201 PDX模型是带有G12D氨基酸突变的KRAS致病突变(KRAS-G12D)的模型。BALB/c裸小鼠皮下接种模型GA6201瘤块，建立人胃癌皮下移植肿瘤模型。试验分为测试药Ifosfamide(异环磷酰胺)60mg/kg组、测试药AST-34245mg/kg组、AST 2.5mg/kg和5mg/kg组及生理盐水(pH 7.0-7.6)溶媒对照组，共5组，每组5只小鼠。其中，生理盐水(pH 7.0-7.6)溶媒对照组、测试药AST-34245mg/kg、AST 2.5mg/kg和5mg/kg组是尾静脉注射给药，每周给药一次，共给药三周，观察四周。Ifosfamide 60mg/kg组腹腔注射给药，每周连续给五天停两天，共给药两周，观察五周。根据相对肿瘤抑制率TGI(％)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
具体每一组的该药方案下表1所示。
表1：不同剂量的测试药在胃癌GA6201肿瘤模型中的抗肿瘤作用实验设计
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表2所示。
表2：在胃癌GA6201模型中各组小鼠肿瘤体积随治疗时间的变化

根据表2的数据制作体现各治疗组和对照组肿瘤生长情况的图2。
根据表2的数据制作药效评估分析数据，具体数据见表3。
表3：胃癌GA6201模型中各组药效分析表
表3中相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表4：胃癌GA6201模型中各组肿瘤的相对肿瘤抑制率
将上表4制作为曲线图，可以得到图2。
分别在不同天数对不同组别的小鼠的体重进行测量，并取得平均值，其结果如下表5所示。
表5：胃癌GA6201模型中不同接种天数小鼠的体重
将上表制作为曲线图，可以得到图3，即胃癌GA6201模型中各组小鼠体重曲线。
同理对表5的数据进行处理可以得到下表6。
表6：胃癌GA6201模型中不同接种天数小鼠的体重变化百分比(％Group Mean Change＝mean((T-T0)/T0)*100，T表示current value，T0表示initial value)
上表4/5/6中的Group01、Group02、Group03、Group04、Group05即上述的第1组、第2组、第3组、第4组、第5组、第6组。0/1/2/3/4/7/8/9/10/11/14/15/16/17/18/21/24/28/31/35/38是接种后的天数。
将上表制作为曲线图，可以得到图4。
分析实验数据可知，疗效方面：
测试药AST-3424在5mg/kg剂量下，测试药AST在2.5mg/kg和5mg/kg剂量下，对 胃癌GA6201具有显著抑制肿瘤生长的作用，相较对照组统计学上均有显著性差异。测试药Ifosfamide在60mg/kg剂量下，对胃癌GA6201具有一定的抑制肿瘤生长的作用，但相较对照组统计学上均无显著性差异。
分析实验数据可知，荷瘤小鼠对Ifosfamide、AST-3424、AST在测试剂量下耐受良好。
2、受试物AST和Gemcitabine在胰腺癌PA1222皮下异种移植模型中的药效学及安全性评价
胰腺癌PA1222 PDX模型是带有G12D氨基酸突变的KRAS致病突变(KRAS-G12D)的模型。BALB/c裸小鼠皮下接种模型PA1222瘤块，建立人胰腺癌皮下移植肿瘤模型。试验分为测试药吉西他滨(Gemcitabine)120mg/kg组、测试药AST 10mg/kg及7.5％无水乙醇+7.5％聚氧乙烯(35)蓖麻油+85％葡萄糖注射液D5W(pH7.4)溶媒对照组，共3组，每组5只小鼠。其中，7.5％无水乙醇+7.5％聚氧乙烯(35)蓖麻油+85％葡萄糖注射液D5W(pH7.4)溶媒对照组、测试药AST 10mg/kg组是尾静脉注射给药，每周给药一次，连续给药三周。测试药Gemcitabine 120mg/kg组腹腔注射给药，每周给药一次，连续给药3周。根据相对肿瘤抑制率TGI(％)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
具体每一组的该药方案下表7所示。
表7：不同剂量的测试药在胰腺癌PA1222 PDX肿瘤模型中的抗肿瘤作用实验设计
注：1.给药体积为10μl/g
2.QW×3；每周给药一次，给药三周
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表8 所示。
表8：胰腺癌PA1222模型中各组小鼠肿瘤体积随治疗时间的变化
各治疗组和对照组肿瘤生长情况见表8和图5，药效评估见表9。
表9：胃癌GA6201模型中各组药效分析表
表9：相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表10：胰腺癌PA1222模型中各组肿瘤的相对肿瘤抑制率
将上表制作为曲线图，可以得到图6。
分别在不同天数对不同组别的小鼠的体重进行测量，并取得平均值，其结果如下表11所示。
表11：胰腺癌PA1222模型中不同接种天数小鼠的体重
将上表制作为曲线图，可以得到图7，即胰腺癌PA1222模型中各组小鼠体重曲线。
同理对表11的数据进行处理可以得到下表12。
表12：胰腺癌PA1222模型中不同接种天数小鼠的体重变化百分比(％Group mean Change＝mean((T-T0)/T0)*100，T表示current value，T0表示initial value)
将上表制作为曲线图，可以得到图8。
分析实验数据可知，疗效方面：
测试药Gemcitabine在120mg/kg(Group 2)剂量下，对胰腺癌PA1222具有一定的抑制肿瘤生长的作用，相较对照组统计学上有显著性差异。测试药AST在10mg/kg(Group 3)剂量下，对胰腺癌PA1222有显著的抑瘤作用，相较对照组统计学上有显著性差异，并且该组有两只小鼠肿瘤被治愈，治愈率均为40％。测试药AST 10mg/kg(Group 3)的抑瘤效果显著优于测试药Gemcitabine(120mg/kg，Group 2)(p＝0.000778)。
分析实验数据可知，测试药Gemcitabine(120mg/kg，Group 2)治疗组、AST 10mg/kg(Group 3)治疗组以及对照组(Group 1)的小鼠没有任何明显的体重下降的现象，治疗期间耐受良好。
3、受试物AST和Cisplatin在肺癌LU11693皮下异种移植模型中的药效学及安全性评价
肺癌LU11693 PDX模型是带有G12C氨基酸突变的KRAS致病突变的模型。BALB/c Nude裸小鼠皮下接种模型LU11693瘤块，建立人肺癌皮下移植肿瘤模型。试验分为测试药Cisplatin 4mg/kg组、测试药AST 10mg/kg组及7.5％无水乙醇+7.5％聚氧乙烯(35)蓖麻油+85％葡萄糖注射液D5W(pH7.4)溶媒对照组，共3组，每组6只小鼠，尾静脉注射给药，每周给药一次，连续给3周。根据相对肿瘤抑制率TGI(％)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
具体每一组的该药方案下表13所示。
表13：不同剂量的测试药在肺癌LU11693 PDX肿瘤模型中的抗肿瘤作用实验设计

注：1.给药体积为10μL/g
2.QW×3；每周给药一次，给药三周
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表14所示。
表14：肺癌LU11693模型中各组小鼠肿瘤体积随治疗时间的变化
各治疗组和对照组肿瘤生长情况见表14和图9，药效评估见表1,5。
表15：肺癌LU11693模型中各组药效分析表
表15中，相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表14：肺癌LU11693模型中各组肿瘤的相对肿瘤抑制率
将上表制作为曲线图，可以得到图10。
分别在不同天数对不同组别的小鼠的体重进行测量，并取得平均值，其结果如下表15所示。
表15：肺癌LU11693模型中不同接种天数小鼠的体重
将上表制作为曲线图，可以得到图11，即肺癌LU11693模型中各组小鼠体重曲线。
同理对表15的数据进行处理可以得到下表16。
表16：肺癌LU11693模型中不同接种天数小鼠的体重变化百分比(％Group Mean Change＝mean((T-T0)/T0)*100，T表示current value，T0表示initial value)
将上表制作为曲线图，可以得到图12。
分析实验数据可知，疗效方面：
测试药Cisplatin(4mg/kg)治疗组在首次给药后的第28天(Day28)表现出一定的抑瘤作用，相较对照组统计学上有显著性差异(p＝0.0152)，相对肿瘤抑制率TGI(％)为23.98％。
测试药AST(10mg/kg)治疗组在首次给药后的第28天(Day 28)表现出一定的抑瘤作用，相较对照组统计学上有显著性差异(p<0.001)，相对肿瘤抑制率TGI(％)为54.64％，TGI小于60％，没有明显的抑瘤效果。
分析实验数据可知，测试药AST(10mg/kg)和Cisplatin(4mg/kg)治疗组有个别小鼠出现体重严重下降的现象，可能与高剂量药物潜在的毒性有关。
4、受试物AST、AST-3424和Ifosfamide单药在胰腺癌PA1383皮下模型中的抗肿瘤作用及安全性评价
胰腺癌PA1383 PDX模型是带有G12C氨基酸突变的KRAS致病突变(KRAS-G12C)的模型。Balb/nude雌性小鼠皮下接种胰腺癌PA1383瘤块，建立人胰腺癌皮下移植肿瘤模型。试验分为的测试药Ifosfamide 60mg/kg单药组(Group 2)，每天给药1次，连续给5天，休息2天，再每天给药1次，连续给5天；AST 4mg/kg单药组(Group 3)和AST 8mg/kg单药组(Group 4)，均每周给药1次，共计给药3周；AST 4mg/kg单药组(Group 5)和AST-34241mg/kg单药组(Group 6)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1次，连续给药5天；葡萄糖注射液(pH7.7-8.0)溶媒对照组(Group 1)，每天给药1次，连续给药5天，休息2天，再休息2周，然后再每天给药1 次，连续给药5天。该研究共6组，每组6只小鼠。其中测试药Ifosfamide为腹腔给药，溶媒对照组，AST和AST-3424均为尾静脉注射给药。
具体每一组的该药方案下表17所示。
表17：胰腺癌PA1383动物模型中的给药途径、剂量及方案
注：
1.给药体积为10μL/g；
2.QD×5，2days off，2weeks off，QD×5：每天给药一次，连续给药5天，休息2天，在休息2周，再每天给药，连续给药5天；
3.QD×5/week×2weeks：每天给药一次，连续给药5天，休息两天，再每天给药一次，连续给药5天；
4.QW×3：每周给药1次，连续给药3周。
分别在不同天数对不同组别的小鼠的肿瘤体积进行测量，并取得平均值，其结果如下表18所示。
表18：胰腺癌PA1222模型中各组小鼠肿瘤体积随治疗时间的变化
各治疗组和对照组肿瘤生长情况见表18和图13，药效评估见表19。
表19：在胰腺癌PA1383皮下模型中各组药效分析表

相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下：
T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：溶媒对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的平均相对肿瘤体积(RTV)。
表20：胰腺癌PA1383模型中各组肿瘤的相对肿瘤抑制率
将上表制作为曲线图，可以得到图14。
分别在不同天数对不同组别的小鼠体重进行测量，并取得平均值，其结果如下表21所示。
表21：胰腺癌PA1383模型中不同接种天数小鼠的体重
将上表制作为曲线图，可以得到图15，即胰腺癌PA1383模型中各组小鼠体重曲线。
同理对表21的数据进行处理可以得到下表22。
表22：胰腺癌PA1383模型中不同接种天数小鼠的体重变化百分比(％Group mean Change＝mean((T-T0)/T0)*100，T表示currentvalue，T0表示initial value)
将上表制作为曲线图，可以得到图16。
分析实验数据可知，疗效方面：
溶媒对照组小鼠在开始给药后的第31天(Day 31)平均肿瘤体积为1536.48mm3。测试药Ifosfamide在60mg/kg剂量治疗组(Group 2)，AST-3424在1mg/kg(QD×5，2days off，2weeks off，QD×5)剂量组(Group 6)在Day 31平均肿瘤体积分别为1202.01mm3,1225.40mm3，相对肿瘤抑制率TGI(％)为21.84％，20.45％和7.55％，相较对照组统计学上没有显著性差异(p>0.05)。
测试药AST在4mg/kg(QW×3)，8mg/kg(QW×3)以及4mg/kg(QD×5，2days of f，2weeks off，QD×5)剂量治疗组(Group 3，Group 4和Group 5)在Day 31平均肿瘤体积分别为616.27mm3，18.57mm3和39.94mm3，相较对照组统计学上有显著性差异(p<0.05)，相对肿瘤抑制率TGI(％)分别为60.42％，98.72％和97.46％。
各测试药治疗组小鼠在治疗期间小鼠体重均没有下降，耐受良好。
由上述四组实验数据我们可以得出以下结论：
1.AST-3424和AST在带有G12D氨基酸突变的KRAS致病突变模型：胃癌GA6021和胰腺癌PA1222 PDX模型中均具有显著的药效，TGI％均大于90％；
2.AST在带有G12C氨基酸突变的KRAS致病突变模型：在肺癌LU11693中的抑瘤效果不明显，TGI％小于60％；而在胰腺癌PA1383中的抑瘤效果明显，TGI％可达98％。
3.AST-3424和AST在各模型中均具有比较好的耐受性。
5、胃癌GA6021、胰腺癌PA1222、肺癌LU11693组织中的AKR1C3的RNA表达水平及酶含量的检测
A、AKR1C3 RNA表达水平FPKM检测
根据文献(Meng,F.,Li,W.F.,Jung,D.,Wang,C.C.,Qi,T.,Shia,C.S.,Hsu,R.Y.,Hsieh,Y.C.,&Duan,J.(2021).A novel selective AKR1C3-activated prodrug AST-3424/OBI-3424 exhibits broad anti-tumor activity.American journal of cancer research,11(7),3645-3659.)的记载方法，使用RNA-Seq分析了上述胃癌GA6021、胰腺癌PA1222、肺癌LU11693组织中的AKR1C3的RNA表达水平(AKR1C3 RNA expression level，并使用Log2 FPKM进行定量)，结果如下：
GA6201检测得到AKR1C3 LOG2(FPKM)为6.78，LU11693为11.14，PA1222为7.39，参见表23。
根据上述文献，可知这三个肿瘤组织的AKR1C3 RNA均为高表达。
B、AKR1C3蛋白含量的IHC方法检测及H-SCORE打分
根据常用的IHC(免疫组织化学)染色法对这三个组织的AKR1C3蛋白含量进行测定(使用商用的IHC试剂，一抗为Abcam公司的兔抗体Rabbit IgG mAb，二抗为Leica公司的聚合物优化检测系统Bond Polymer Refine Detection，染色条件：抗原修复100℃，pH9.0 EDTA缓冲液20min，稀释比：1:800)，并对染色结果进行H-SCORE打分：
免疫组化染色强度将被分为0(阴性)，1+(弱染色)，2+(中染色)，3+(强染色)，人工在打分仪器上设置弱染色、中染色、强染色的阈值，然后通过图像处理软件对染色样本照片进行色彩识别，所有样品的染色照片按照统一的标准，由评分软件对某个细胞对应的染色情况进行评分：0/1/2/3。并统计不同染色强度的阳性细胞数量占切片中总细胞数量的百分数。通过下面的公式计算H-Score作为每个样品的IHC结果评分。H-score得分将在0～300之间，评分越高则表示该抗体对应的靶点(AKR1C3酶蛋白)在该样品中表达量越高。计算公式如下：
H-Score＝(％at 0)×0+(％at 1)×1+(％at 2)×2+(％at 3)×3
染色结果如图17，打分结果如下表17：
表23：三个模型及对照组的IHC和RNA分析检测结果
具体染色统计结果如下表24：
表24：三个模型及对照组的IHC结果及H-SCORE打分
上述染色结果中阳性和阴性对照结果在控制范围内即表明本次IHC染色剂H-SCORE打分结果是可信的。
根据上述结果可知，这三个G12D或G12C突变的PDX模型对应的组织的AKR1C3蛋白均为高表达。
6、AKR1C3在KRAS G12D PDXs中的分布和与NRF2的相关性统计
从公开的中美冠科(CrownBio)的肿瘤PDX模型基因数据库(https://www.crownbio.cn/model-systems/in-vivo/pdx-models/)中导出具有KRAS-G12D突变的模型数据共179个，并统计这些模型的AKR1C3的RNA表达情况，结果如表25所示：
表25：AKR1C3 RNA以LOG2(FPKM)表示的表达水平分级与KRAS-G12D的对应关系统计结果
进一步统计这些KRAS-G12D突变的模型的NRF2表述情况，将这179个PDX模型中AKR1C3的RNA表达水平与NRF2的RNA表达水平投射到X-Y坐标图中，得到图18。
AKR1C3表达量在KRAS G12D PDX模型中的分布趋势主要集中为中高表达(LOG2(FPKM)≥4)，占90.4％。
在KRAS G12D PDX模型中，NRF2表达量的分布趋势也集中在中高表达，与AKR1C3的蛋白表达趋势一致，有一定的相关性。
7、AKR1C3在KRAS G12C PDXs中的分布和与NRF2的相关性统计
从公开的中美冠科(CrownBio)的肿瘤PDX模型基因数据库(https://www.crownbio.cn/model-systems/in-vivo/pdx-models/)中导出具有KRAS-G12C突变的模型数据共51个，并统计这些模型的AKR1C3的RNA表达情况，结果如表26所示：
表26：AKR1C3 RNA以LOG2(FPKM)表示的表达水平分级与KRAS-G12C的对应关系统计结果
进一步统计这些KRAS-G12C突变的模型的NRF2表述情况，将这51个PDX模型中AKR1C3的RNA表达水平与NRF2的RNA表达水平投射到X-Y坐标图中，得到图19。
AKR1C3表达量在KRAS G12C PDX模型中的低中高表达平均分布，其中中等以上水平(LOG2(FPKM)≥4)表达占比为66.7％。
在KRAS G12C PDX模型中，NRF2表达量的分布趋势与AKR1C3的蛋白表达量分布相关性较弱。
8、AKR1C3在KRAS G13D PDXs中的分布和与NRF2的相关性统计
从公开的中美冠科(CrownBio)的肿瘤PDX模型基因数据库(https://www.crownbio.cn/model-systems/in-vivo/pdx-models/)中导出具有KRAS-G13D突变的模型数据共48个，并统计这些模型的AKR1C3的RNA表达情况，结果如表27所示：
表27：AKR1C3 RNA以LOG2(FPKM)表示的表达水平分级与KRAS-G13D的对应关系统计结果
进一步统计这些KRAS-G13D突变的模型的NRF2表述情况，将48个PDX模型中AKR1 C3的RNA表达水平与NRF2的RNA表达水平投射到X-Y坐标图中，得到图20。
AKR1C3表达量在KRAS G13D PDX模型中的低中高表达平均分布，其中中等以上水平表达占比为83.3％。
在KRAS G13D PDX模型中，NRF2表达量的分布趋势与AKR1C3的蛋白表达量分布具有一定的相关性。
发明人进一步用KRAS G12D突变的HPAF II、LU5161、CR3820小鼠肿瘤体内药效模型研究AST-3424和AST在KRAS G12D突变的肿瘤模型中的抑瘤效果，并使用IHC检测方法和免疫印迹方法检测其蛋白表达水平及酶含量。利用HPAF II(KRAS G12D)肿瘤细胞验证AKR1C3酶活化的DNA烷化剂前药能够激活MEK/ERK信号通道，同时使用MEK/ERK调节剂探究AKR1C3酶活化的DNA烷化剂前药通过ERK2通路诱导KRAS G12D细胞凋亡，使用NRF2抑制剂和激活剂探讨KRAS G12D突变细胞中AKR1C3的表达受到NRF2的调控。
AST-3424、AST在KRAS G12D突变的HPAF II、LU5161、CR3820小鼠肿瘤模型的体内药效研究实验及实验结果公开在申请人的另外一件专利中(申请号PCT/CN2022/120817、公开号WO2023046060A1)，实验结果显示，AST-3424、AST在带有KRAS G12D致病突变模 型中，各测试剂量以及给药频率下，均具有统计学意义的显著抗肿瘤作用，且耐受良好。在此将专利WO2023046060A1全文引入本申请之中。
本申请及专利WO2023046060A1中，胃癌GA6201 PDX模型、胰腺癌PA1222 PDX模型、胰腺癌HPAF-II CDX模型、肺癌LU5161 PDX模型、结直肠癌CR3820 PDX模型是带有KRAS G12D突变的肿瘤模型，肺癌LU11693 PDX模型、胰腺癌PA1383 PDX模型是带有KRAS G12C突变的肿瘤模型。
9、胰腺癌HPAF II细胞、肺癌LU5161和结直肠癌CR3820肿瘤组织中的RNA表达水平及酶含量的检测
使用实施例5中相同的AKR1C3 RNA表达水平FPKM检测方法，AKR1C3蛋白含量的IHC检测方法及H-SCORE打分，得到肺癌LU5161、结直肠癌CR3820组织中的RNA表达水平及酶含量数据，如表28所示。使用AKR1C3 RNA表达水平FPKM检测方法评估胰腺癌HPAF II肿瘤细胞中AKR1C3 RNA表达水平数据，如表28所示。相应IHC染色结果及免疫印迹图片如图21所示。
表28：HPAF II、LU5161、CR3820组织中IHC打分及RNA表达水平结果
根据上述结果可知，这三个KRAS G12D突变的小鼠体内药效模型对应的肿瘤组织的AKR1C3蛋白均为高表达。
10、KRAS G12D突变的HPAF II肿瘤细胞中AKR1C3的表达受到NRF2的调控
将SFN化合物(Sulforaphane，NRF2激活剂)溶液加入到HPAFII细胞悬液中，于37℃、5％CO2培养箱中培养过夜，收细胞蛋白裂解液进行免疫印迹(Western Blot，WB)检测。
实验共分5组：0.5％DMSO、0.875μM SFN、1.75μM SFN、3.5μM SFN、7μM SFN。
用不同浓度的NRF2激活剂SFN处理HPAF II细胞，然后使用免疫印记法检测NRF2和AKR1C3的表达，SFN不同浓度处理下的免疫印迹与NRF2、AKR1C3蛋白条带密度分析的结果如图22所示。
实验结果显示，随着SFN处理浓度的提高，NRF2的表达相应的显著增加，AKR1C3的表达也同时增加。
实验结果表明，NRF2激活因子SFN以浓度依赖性的方式上调NRF2的表达。在相同的实验条件下，随着SFN浓度的增加，AKR1C3的表达同时上调。这一结果清楚地表明，AKR1C3的表达受到NRF2的调控。此外，结合实施例6中NRF2和AKR1C3的mRNA在中高表达区域显著的相关性，说明在KRAS G12D突变的PDX模型中，AKR1C3表达与NRF2表达呈正相关。
根据上述体内药效实验1-4及HPAF II、LU5161、CR3820小鼠肿瘤体内药效模型研究可知，AKR1C3酶激活前药AST-3424、AST对AKR1C3酶高表达的PDX、CDX模型具有显著的抗肿瘤作用，且耐受良好。
再结合实施例10中AKR1C3表达与NRF2表达呈正相关的实验结论，本领域技术人员可以合理的推测，上调或激活NRF2，能够促使AKR1C3高表达，从而可以使AKR1C3酶激活前药AST-3424、AST产生显著的抗肿瘤作用。
本申请通式(1)-(11)化合物均是AKR1C3酶活化的抗癌前药，其在AKR1C3酶的作用下，裂解产生AST-2660、紫杉醇、SN-38、吉西他滨、KARS抑制剂等抗癌活性药物。因此，本领域技术人员可以合理的推测，上调或激活NRF2将使得AKR1C3酶高表达，进而使通式(1)-(11)化合物活化代谢出抗癌活性药物，也就是说，被检测出能够上调或激活NRF2的基因突变的患者，可以在接受通式(1)-(11)化合物的治疗中受益，特别是在接受AST-3424或AST的治疗中受益。
以下以AKR1C3酶激活前药AST为例，探究其在KRAS突变、特别是在KRAS G12D突变肿瘤细胞中的作用机制。
11、AST激活MEK/ERK信号通路
AST诱导的ERK磷酸化实验。分别以AST浓度、AST作用时间、AKRAC3酶依赖的三组变量进行相应的三组实验。其中，AKRAC3酶依赖的变量以AKR1C3酶特异性抑制剂AST-3021为条件，在存在或者不存在特异性AKR1C3酶抑制剂AST-3021的情况下进行实验。所用抑制剂为AST-3021(也称为TH-3021，其为Flanagan等人，在文献Bioorganic and MedicinalChemistry(2014)第962-977页中报到的化合物36，其结构式为)。
本次实验分为三组，分别用不同浓度AST(第一组)、1μM AST处理不同时间(第二组)、添加或不添加AKR1C3抑制剂AST-3021(第三组)进行实验。
三组具体待处理化合物如下：
第一组(浓度组)：0.5％DMSO处理组(24h)、0.5μM AST处理组(24h)、1μM AST处理组(24h)、10μM AST处理组(24h)；
第二组(时间组)：0.5％DMSO处理组(24h)、1μM AST处理组(2h)、1μM AST处理组(8h)、1μM AST处理组(24h)；
第三组(AKR1C3依赖组)：0.5％DMSO处理组(24h)、1μM AST处理组(24h)、3μM AST-3021处理组(24h)、1μM AST+3μM AST-3021处理组(24h)。
将各组化合物溶液分别加入到HPAF II细胞悬液中，于37℃、5％CO2培养箱中培养过夜，按照实验组别，收细胞蛋白裂解液进行免疫印迹(Western Blot，WB)检测。
使用免疫印记法检测NRF2和AKR1C3的表达，第一组用不同浓度AST处理得到的免疫印迹图片与p-NRF2、NRF2蛋白条带密度分析的结果如图23所示；第二组用1μM AST处理不同时间得到的免疫印迹图片与p-NRF2、NRF2蛋白条带密度分析的结果如图24所示；第三组添加或不添加AKR1C3抑制剂AST-3021时得到的免疫印迹图片与p-NRF2、NRF2蛋白条带密度分析的结果如图25所示。
实验结果显示，图23中，随着AST处理浓度的提高，p-NRF2的表达水平相应的增加，10μM AST处理24h时，p-NRF2的表达增加显著，而各AST处理浓度下，不影响NRF2的表达水平；图24中，用1μM AST处理2h、8h时，p-NRF2的表达水平基本不变，处理24h时，相较DMSO对照组显著增加；图25中，仅单药1μM AST处理组(24h)的p-NRF2表达增加显著，其他的0.5％DMSO处理组(24h)、3μM AST-3021处理组(24h)与1μM AST+3μM AST-3021处理组(24h)的p-NRF2表达水平基本不变。上述三组实验中，各处理组的总ERK2的表达均没有改变。
上述实验表明，AST以浓度依赖性的方式诱导ERK2的磷酸化，而不影响ERK2的表达水平。用1μmol/L的AST处理后24小时，ERK2的磷酸化水平相较DMSO对照组显著增加。如图25，HPAF II细胞在加入1μmol/L AST前2小时，用AKR1C3特异性抑制剂AST-3021 进行预处理，3μmol/L AST-3021强烈抑制了AST介导的磷酸化ERK2的增加。单独使用AST-3021并不影响p-ERK2的水平。在所有测试条件下，总ERK2的表达均没有改变。通过肌动蛋白免疫印迹法证实所有样本的加样量一致。AST以浓度、时间和AKR1C3依赖的方式激活MEK/ERK信号通路。
12、AST通过ERK2信号通路诱导KRAS G12D细胞的凋亡
本实验使用三种MEK/ERK调节剂，包括两种抑制剂和一种激活剂。抑制剂是PD98059，一种MEK1/2的特异性抑制剂，和苏拉明(Suramin)，一种广谱生长因子受体抑制剂；而ERK信号通路的激活剂是Phorbol Ester(TPA)。抑制剂PD98059、抑制剂Suramin、激活剂TPA的化学结构如下所示：
实验分为抑制剂组和激活剂组，均进行免疫印迹实验和细胞凋亡实验。
免疫印迹实验中，用各组别化合物分别处理HPAFII细胞悬液，于37℃、5％CO2培养箱中培养过夜，收细胞蛋白裂解液进行免疫印迹(Western Blot，WB)检测。
免疫印迹抑制剂组和激活剂组的具体待处理化合物如下：
抑制剂组：1％DMSO处理组、1μM AST处理组、30μM PD98059处理组、1mM Suramin处理组、1μM AST+30μM PD98059处理组、1μM AST+1mM Suramin处理组；
激活剂组：1％DMSO处理组、0.5μM AST处理组、25nM TPA处理组、0.5μM AST+25nM TPA处理组。
另外进行细胞凋亡检测：
1)准备Caspase试剂，使试剂平衡至室温。将试剂的buffer和底物1:1混合均匀，配成Caspase试剂，配置的试剂可在4℃储存3天。
2)将100μL Caspase试剂添加到含有100μL空白(无细胞孔)、阴性对照细胞或处理过的细胞的每孔培养基中。由于该分析的敏感性，小心不要将移液管尖端接触含有样品的孔，以避免交叉污染。用平板密封剂或盖子盖住平板。
3)使用摇床以300–500rpm的转速轻轻混合试剂液，持续30秒。在室温下避光放置1小时。
4)在平板读数光度计中测量每个样品的发光。并计算各组发光值，做柱形图分析。
细胞凋亡抑制剂组和激活剂组的具体待处理化合物如下：
抑制剂组：1％DMSO处理组、1μM AST处理组、30μM PD98059处理组、1mM Suramin处理组、1μM AST+30μM PD98059处理组、1μM AST+1mM Suramin处理组；
激活剂组：1％DMSO处理组、1μM AST处理组、50nM TPA处理组、1μM AST+50nM TPA处理组。
ERK2抑制剂和AST单药及联用药组的免疫印迹、蛋白条带密度分析和细胞凋亡的实验结果如图26所示，ERK2激活剂和AST单药及联用药组的免疫印迹、蛋白条带密度分析和细胞凋亡的实验结果如图27所示。
实验结果显示，图26的抑制剂组中，上图与下左图显示，PD98059和苏拉明单独处理，略微降低了p-ERK2的表达水平，而PD98059与AST的联用药组显著降低了p-ERK2的表达水平，苏拉明与AST的联用药组中p-ERK2的表达水平略微升高，但AST单药组中的p-ERK2的表达水平显著提高；下右图显示，AST单药组显著影响了细胞的凋亡，PD98059、苏拉明单药组和其分别与AST的联用药组对细胞凋亡几乎无影响。图27的激活剂组中，上图与下左图显示，AST、TPA单药处理组相比DMSO对照组的p-ERK2表达水平均增加，而AST与TPA的联用药组的p-ERK2表达水平则显著增加；下右图显示，AST单药组促使了细胞凋亡，而其与TPA的联用药组则使对细胞凋亡的影响更加显著。
实验结果表明，使用两种ERK抑制剂，PD98059和苏拉明单独处理，略微降低了内源性ERK磷酸化程度，而用AST单独处理，能够显著的增加细胞内p-ERK2的程度。同时，AST和两种ERK抑制剂联用后，几乎完全抵消了AST诱导的p-ERK2的增加。利用Caspase 3/7试剂盒检测AST、ERK2抑制剂单药及联用对细胞凋亡的影响，结果显示ERK2抑制剂显著抑制了AST诱导的细胞凋亡。相比较之下，使用ERK2激活剂TPA单独处理也能促进细胞内p-ERK2的程度，TPA联合AST处理则进一步增强了AST诱导的ERK2磷酸化程度和细胞凋亡。无论是上述各单药或者联合用药处理细胞，均不影响总ERK2的表达。这些结果清楚地表明，AST通过ERK2信号通路诱导KRAS G12D突变细胞的凋亡。
综合实施例10、11、12，可以推测出图28的机理示意图，即AST通过ERK2信号通路诱导KRAS G12D致病突变癌细胞的凋亡。进一步结合相关文献的研究：MEK/ERK信号通路可在DNA烷化剂的作用下被激活(Wang X,Martindale JL and Holbrook NJ.Requirement for ERK activation in cisplatin-induced apoptosis.J Biol Chem 2000；275:39435-39443)；而实施例11、12证实了AST这一DNA烷化剂前药具有激活MEK/ERK信号通路的作用，其能通过ERK2信号通路诱导KRAS G12D突变癌细胞凋亡，而AST化合物是一种AKR1C3酶活化的DNA烷化剂前药，其会在AKR1C3酶的作用下活化而产生AST-2660而发挥抗癌效果。
考虑到图28揭示的AKR1C3、ERK2的相关调控通路，发明人推测AST化合物能激活ERK2使其磷酸化进而激活MEK-ERK通路使得NRF2上调，最终使得AKR1C3酶高表达，进而导致癌细胞凋亡。即KRAS-G12D突变通过激活RAF/MEK/ERK信号通路，上调NRF2的蛋白表达，进而上调AKR1C3的蛋白表达，能够更为有效的活化AST药物使其释放DNA烷化剂AST-2660。
实际上，研究证明有许多基因突变能上调NRF2的蛋白表达进而上调AKR1C3的蛋白表达，这些基因包括KRAS突变、KEAP1突变、CUL3突变、NRF2突变、p21突变、p62突变、FTL1突变、FTH1突变、HMOX1突变、GSR突变、SLC7A11突变、GCLM突变、GCLC突变、GPX2突变、TXN1突变、TXNRD1突变、PRDX1突变、SRXN1突变、ABCC1突变、ABCC2突变、G6PD突变、PGD突变、ME1突变、IDH1突变、EGFR突变等。
进一步将图28揭示的AST化合物机理延伸到其他类似的AKR1C3酶活化的DNA烷化剂前药：通式(1)(2)(3)(4)(5)(6)(8)化合物与AST、AST-3424等类似化合物，这些化合物与图28中的研究药物AST作用机理均为AKR1C3酶活化的DNA烷化剂前药，其会在AKR1C3酶的作用下活化而产生AST-2660而发挥抗癌效果，因此结合图28的机理，可以确信通式(1)(2)(3)(4)(5)(6)(8)应当与AST类似，同样可以激活MEK/ERK信号通路，通过ERK2信号通路诱导具有能上调或激活NRF2的基因突变细胞的凋亡，也就是说具有能上调或激活NRF2的基因突变，通过激活RAF/MEK/ERK信号通路，上调NRF2的蛋白表达，进而上调AKR1C3的蛋白表达，能够更为有效的激活AKR1C3酶活化的DNA烷化剂前药。
在此基础上，结合图28可以推测，通式(1)(2)(3)(4)(5)(6)(8)应当与AST类似，同样可以激活MEK/ERK信号通路，通过ERK2信号通路最终诱导具有能上调或激活NRF2的基因突变突变细胞的凋亡。
本申请通式(7)(9)(10)(11)化合物与AST-3424、AST类似，也是AKR1C3酶活化的抗癌前药，其在AKR1C3酶的作用下活化而产生紫杉醇、SN-38、吉西他滨或KARS抑制剂等抗癌活性药物。因此，本领域技术人员应当知晓，通式(7)(9)(10)(11)化合物具有与AST-3424、AST类似的显著的肿瘤抑制作用。

Claims (11)

  1. AKR1C3酶激活前药治疗癌症患者的方法，其特征在于：
    所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；
    或
    所述患者被检测出具有能上调或激活NRF2的基因突变。
  2. 根据权利要求1所述的治疗方法，其中，
    所述能上调或激活NRF2的基因突变选自KRAS突变、KEAP1突变、CUL3突变、NRF2突变、p21突变、p62突变、FTL1突变、FTH1突变、HMOX1突变、GSR突变、SLC7A11突变、GCLM突变、GCLC突变、GPX2突变、TXN1突变、TXNRD1突变、PRDX1突变、SRXN1突变、ABCC1突变、ABCC2突变、G6PD突变、PGD突变、ME1突变、IDH1突变、EGFR突变。
  3. 根据权利要求2所述的治疗方法，其中，KRAS突变选自G12D、G12V、G12R、G12C、G12A、G13D这6种亚型。
  4. 根据权利要求1所述的治疗方法，所述AKR1C3酶激活前药选自结构式(1)、(2)、(3)、(4)、(5)、(6)、(7)、(8)、(9)、(10)、(11)的化合物及其盐、酯、溶剂合物、同位素异构体：
    其中，X、Y、Z、R、T、A以及X10的定义如专利申请PCT/US2016/021581，公开号WO2016145092A1(对应中国申请号2016800150788，公开号CN107530556A)中的权利要求书所记载；
    其中，R1、R2、R3、R4、R5、R8、R9、R10的定义如专利申请PCT/CN2020/089692，公开号WO2020228685A9(对应中国申请号2020800358890，公开号CN113853379A)中的权利要求书所记载；
    其中：
    A是取代或未经取代的C6-C10的芳基、联芳基或取代的联芳基、5-15元的杂芳基或-N＝CR1R2，其中取代时的取代基选自由以下组成的群：卤基、-CN、-NO2、–O-(CH2)-O-、-CO2H及其盐、-OR100、-CO2R100、-CONR101R102、-NR101R102、-NR100SO2R100、-SO2R100、-SO2NR101R10 2、C1-C6烷基、C3-C10杂环基；
    其中，R100、R101及R102各自独立是氢、C1-C8烷基、C6-C12芳基；或R101及R102与其附接至的氮原子一起形成5-7元杂环；
    其中烷基及芳基各自是经1-3个卤基或1-3个C1-C6烷基取代；
    R1及R2各自独立是苯基或甲基；
    X、Y及Z各自独立是氢或卤基；
    R是氢或C1-C6烷基或卤素取代烷基；
    其中，Rw的定义如专利申请PCT/CN2020/120281，公开号WO2021068952A1(对应中国申请号202080071652.8，公开号CN114555574A)中的权利要求书所记载；
    其中，A、E、G、X、Y的定义如专利申请PCT/NZ2019/050030，公开号WO2019190331A1(对应中国申请号2019800234236，公开号CN111918864A)中的权利要求书所记载；
    其中，X、Y、Z、R、D、L1、A以及X10的定义如专利申请PCT/US2016/025665，公开号WO2016161342A3(对应中国申请号2016800200132，公开号CN108136214A)中的权利要求书所记载；
    其中，R1、R2、R3、R4、T的定义如专利申请PCT/CN2021/118597，公开号WO2022057838A1中的权利要求书所记载；
    或其药学上可接受的盐，
    其中，Rw、X、R4、R10、R13、R14的定义如专利申请PCT/CN2022/098082，公开号WO2022258043A1中的权利要求书所记载；
    其中，R1、R2、R3、R4、G1、G2、G3、G4、E、T、Y、Z、m、n、s、t、v、w、环A的定义如专利申请CN202210585771.6，公开号CN115403579A中的权利要求书所记载；
    或其药学上可接受的盐，
    其中，R1、R2a、R2b、R3、R4、R5、n、Z的定义如专利申请PCT/IB2020/057285，公开号WO2021005586A1(对应中国申请号CN202080053804.1，公开号CN114206870A)中的权利要求书所记载。
  5. 根据权利要求1-4任意一项所述的治疗方法，其中，
    式(1)的AKR1C3酶活化前药化合物选自以下结构的化合物：




    式(2)的AKR1C3酶活化前药化合物选自以下结构的化合物：

    式(3)的AKR1C3酶活化前药化合物选自以下结构的化合物：
    式(4)的AKR1C3酶活化前药化合物选自以下结构的化合物：


    以及


    以及








    及
    式(5)的AKR1C3酶活化前药化合物选自以下结构的化合物：
    式(6)的AKR1C3酶活化前药化合物选自以下结构的化合物：











    式(7)的AKR1C3酶活化前药化合物选自以下结构的化合物：





    式(8)的AKR1C3酶活化前药化合物选自以下结构的化合物：

    式(9)的AKR1C3酶活化前药化合物选自以下结构的化合物：







    式(10)的AKR1C3酶活化前药化合物选自以下结构的化合物：





    式(11)的AKR1C3酶活化前药化合物选自以下结构的化合物：
  6. AKR1C3酶激活前药用于制备治疗癌症的药物的制药用途，其特征在于：
    所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；
    或
    所述患者被检测出具有能上调或激活NRF2的基因突变。
  7. 药物，该药物含有AKR1C3酶活化前药化合物，其适应症为治疗癌症患者，所述患者的肿瘤或癌组织被检测出具有能上调或激活NRF2的基因突变；
    或
    所述患者被检测出具有能上调或激活NRF2的基因突变。
  8. AKR1C3酶激活前药治疗癌症患者，其包含施加含有AKR1C3酶激活前药的药品或制剂的步骤；以及测定患者的癌细胞或组织的NRF2含量或表达水平的步骤，
    如测得该NRF2含量或表达水平等于或大于预定值，则向该患者投与含有AKR1C3酶激活前药的药品或制剂。
  9. 治疗癌症或肿瘤的方法，其包含施加含有AKR1C3酶激活前药的药品或制剂的步骤；以及NRF2含量或表达水平调节步骤，
    当调节使得该NRF2含量或表达水平等于或大于预定值，则向该患者投与含有AKR1C3酶激活前药的药品或制剂。
  10. AKR1C3酶激活前药用于制备治疗癌症的药物的的制药用途，其特征在于：
    所述患者的肿瘤或癌组织被检测出NRF2含量或表达水平等于或大于预定值；
    或
    所述患者被检测出NRF2含量或表达水平等于或大于预定值
  11. 药物，该药物含有AKR1C3酶活化前药化合物，其适应症为治疗癌症患者，所述患者的肿瘤或癌组织被检测出NRF2含量或表达水平等于或大于预定值；
    或
    所述患者被检测出NRF2含量或表达水平等于或大于预定值。
PCT/CN2023/099294 2022-06-10 2023-06-09 Akr1c3酶激活前药治疗癌症患者的方法 WO2023237080A1 (zh)

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