Correlation of AKR1c3 enzyme expression level with KRAS mutation, and medical use

Abstract

Correlation of the AKR1C3 enzyme expression level with KRAS mutation, and a medical use. For an AKR1C3 enzyme activated anti-cancer prodrug, KRAS gene mutation can directly serve as a test target before drug administration, that is, a patient with KRAS mutation is the patient having a high AKR1C3 enzyme expression level and AKR1C3 enzyme expression level or AKR1C3 RNA testing is not required. That is to say, a positive KRAS mutation testing result can be used as a screening index to screen the patient having a high AKR1C3 enzyme expression level.

Description

Correlating AKR1C3 enzyme expression levels and medical uses with KRAS mutations Technical Field

[0001] The present invention relates to methods for treating cancer, in particular methods for treating cancers with specific genes. Background Art

[0002] AST-3424 (OBI-3424 / TH3424) is an AKR1C3 enzyme-activated DNA alkylating agent prodrug that has entered Phase II clinical trials in both China and the United States (the registered clinical trials in China are CTR20201915, CTR20201908, CTR20191399, and CTR20191371, and the registered clinical trials in the United States are NCT04315324 and NCT03592264). It is specifically activated by the AKR1C3 enzyme that is highly expressed in various tumors to release DNA alkylating agents, thereby exerting anti-tumor effects.

[0003] Preclinical studies have shown that the efficacy of AST-3424 is highly correlated with AKR1C3 enzyme expression, and slight differences in enzyme expression can significantly affect the drug's ultimate therapeutic effect (AACR 2016, Abstract #1369: In vitro and in vivo antitumor 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-3424 Exerts Profound In Vivo Efficacy Against Preclinical Models of T-Cell Acute Lymphoblastic Leukemia (T-ALL); a Pediatric Preclinical Testing Consortium Study; Clin Cancer Res 2019; 25:4493-503: OBI-3424, a Novel AKR1C3-Activated Prodrug,Exhibits Potent Efficacy against Preclinical Models of T-ALL; Am J Cancer Res 2021;11(7):3645-3659, A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity).

[0004] The above-mentioned Phase II clinical trial conducted in China and the United States will study the relationship between the expression level of AKR1C3 enzyme and the treatment effect on cancer patients, and determine the quantitative value of AKR1C3 expression level for screening patients who can benefit from the experimental drug.

[0005] That is to say, the above-mentioned AKR1C3 enzyme-activated anticancer prodrug needs to detect the expression level of the patient's AKR1C3 enzyme before use. Only patients whose expression level reaches the predetermined level will have a better therapeutic effect of the drug.

[0006] Based on the drug's Phase I clinical results, researchers have determined that patients with AKR1C3 enzyme expression levels with an H-score ≥135 (IHC) will be enrolled in a Phase II clinical trial in the United States (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; Tsimberidou, AM, Verschraegen, CF, Wesolowski, R. et al. Phase 1 dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors. Br J Cancer(2023). https: / / doi.org / 10.1038 / s41416-023-02280-4).

[0007] In addition to AST-3424, a similar AKR1C3 enzyme-activated DNA alkylating agent anticancer prodrug TFX05-01 (has entered Phase I clinical trials, the registered clinical trial in China is CTR20220957, and the registered clinical trial in the United States is NCT05434299; Charles Z Ding, Zhe Cai, Wei Sha. Preclinical evaluation of TFX05-01, a selective AKR1C3-targeted prodrug for solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022; 82 (12_Suppl): Abstract nr 5691.) has entered Phase I clinical trials. It is also necessary to study the relationship between AKR1 C3 enzyme expression levels and drug quality effects in clinical trials.

[0008] In order to quantitatively detect the expression level of AKR1C3 enzyme, the existing technology WO2022048492 discloses an IHC detection method using FFPE tumor tissue sample slices, and WO2022183483 discloses a PCR detection method using blood samples; the former requires the use of FFPE slices of patient tumor tissue as detection samples, which is suitable for detecting patients with solid tumors; the latter requires the use of patient blood samples, which is suitable for blood tumors, especially leukemia.

[0009] However, in reality, some patients with malignant solid tumors cannot have their tumor tissue removed from the patient's body to produce FFPE slices as IHC test samples, such as most pancreatic cancers, some liver cancers, almost all intrahepatic bile duct cancers, primary brain cancers, metastatic brain cancers, etc.

[0010] Therefore, it is necessary to find alternative detection technologies, detection targets and reference detection results to screen patients who may benefit from AKR1C3 enzyme-activated anti-tumor (cancer) prodrug therapy and complete the drug administration treatment.

[0011] Summary of the Invention

[0012] In further preclinical efficacy studies, the inventors found that the AKR1C3 enzyme-activating prodrug AST-3424 and AST have significant inhibitory effects on PDX tumor models and cells with Kras-G12D / G12C mutations (PCT / CN2022 / 120817, Examples 1-9 in Publication No. WO2023 / 046060). The inventors speculated whether the Kras-G12D / G12C mutation is related to the high expression of the AKR1C3 enzyme: Are patients with Kras-G12D / G12C mutations also patients with high expression of the AKR1C3 enzyme?

[0013] Based on the above speculation, the inventors performed AKR1C3 RNA detection and AKR1C3 protein expression level detection on the tumor tissues corresponding to the above-mentioned PDX models (PCT / CN2022 / 120817, Example 10 in Publication No. WO2023 / 046060). The results showed that the RNA detection and protein expression level detection results of AKR1C3 in the above-mentioned model with Kras-G12D / G12C mutation were both high-level expression, which confirmed the above speculation: Kras-G12D / G12C mutation is associated with high expression of AKR1C3 enzyme, and Kras-G12D / G12C mutation is accompanied by high expression of AKR1C3 enzyme.

[0014] To verify the conclusion that Kras-G12D mutation is associated with high AKR1C3 expression, the inventors collected AKR1C3 expression data from 179 PDX models with Kras-G12D mutation and found that:

[0015] The distribution trend of AKR1C3 expression in KRAS G12D PDX models was mainly concentrated in medium and high expression, accounting for 90.4%;

[0016] To verify the conclusion that Kras-G12C mutation is associated with high expression of AKR1C3 enzyme, the inventors collected AKR1C3 expression data from 51 PDX models with Kras-G12C mutation and found that:

[0017] AKR1C3 expression levels in KRAS G12C PDX models were evenly distributed among low, medium, and high expression levels, with 66.7% expressing at or above the medium level;

[0018] To verify the conclusion that Kras-G13D mutation is associated with high expression of AKR1C3 enzyme, the inventors collected AKR1C3 expression data from 48 PDX models with Kras-G13D mutation and found that:

[0019] The distribution trend of AKR1C3 expression in KRAS G13D PDX models was mainly concentrated in medium and high expression, among which the expression level above medium accounted for 83.3%.

[0020] The inventors further learned from literature that KRAS G12D mutant tumor cells can directly upregulate and activate NRF2 (Nature, 2011, 475:106; Cancer Res, 2014, 74:7430) through the RAF-MEK-ERK-Jun signaling pathway (Nature, 2011, 475:106). Activated NRF2 can further upregulate and activate a series of downstream genes, including AKR1C3 (Chem Res Toxicol, 2017, 30:162; Cancer, 2019, 11:1715). Based on the above regulatory pathways, it can be seen that gene mutations that can upregulate or activate NRF2, such as the G12D mutation, can ultimately upregulate the AKR1C3 gene, leading to increased expression levels of the AKR1C3 enzyme in cancer cells.

[0021] Based on the above experiments and literature research, the inventors proposed that AKR1C3 enzyme-activated anticancer prodrugs can directly use KRAS gene mutations as pre-drug detection targets, that is, patients with KRAS mutations are patients with high expression of AKR1C3 enzymes without the need for AKR1C3 enzyme expression level or AKR1C3 RNA detection. In other words, a positive KRAS mutation test can be used as a screening indicator to screen patients with high expression of AKR1C3 enzymes.

[0022] To this end, this application provides the following technical solutions.

[0023] Option 1

[0024] A method of administering a drug to treat cancer, a tumor, or a condition or cell proliferative disease caused by cancer or a tumor, wherein, when the patient's tumor or cancer tissue is detected to have a KRAS gene mutation or the patient is detected to have a KRAS gene mutation, an AKR1C3 enzyme-activating prodrug is administered without detecting the AKR1C3 protein expression level or AKR1C3 RNA content in the patient's tumor or cancer tissue.

[0025] Option 2

[0026] A method for treating cancer, tumor, or a disease caused by cancer or tumor, or a cell proliferative disease, comprising the steps of administering a drug or preparation containing an AKR1C3 enzyme-activating prodrug; and detecting a KRAS gene mutation, but excluding the step of detecting the AKR1C3 protein expression level or AKR1C3 RNA content in the patient's tumor or cancer tissue.

[0027] When the patient's tumor or cancer tissue is detected to have a KRAS gene mutation or the patient is detected to have a KRAS gene mutation, a drug or preparation containing an AKR1C3 enzyme-activating prodrug is administered to the patient.

[0028] Option 3

[0029] The pharmaceutical use of an AKR1C3 enzyme-activated prodrug for preparing a drug for treating cancer, tumors, or conditions caused by cancer or tumors, or cell proliferative diseases, characterized in that the drug is formulated for administration to patients whose tumors or cancerous tissues are detected to have KRAS gene mutations or who are detected to have KRAS gene mutations, and the patient does not need to undergo testing for AKR1C3 protein expression levels or AKR1C3 RNA content in the tumor or cancerous tissues.

[0030] Option 4

[0031] A preparation unit package comprises an independent packaging container containing a pharmaceutical preparation, an outer packaging component for accommodating the independent packaging container, and a drug package insert, wherein the pharmaceutical preparation contains an AKR1C3 enzyme-activating prodrug, and the drug package insert states:

[0032] The drug is used to treat patients with cancer, tumors, or conditions caused by cancer, tumors, or cell proliferative diseases;

[0033] The patient undergoes KRAS gene mutation testing without the need for testing the AKR1C3 protein expression level or AKR1C3 RNA content in the tumor or cancer tissue;

[0034] When the patient's tumor or cancer tissue is detected to have a KRAS gene mutation or the patient is detected to have a KRAS gene mutation, a drug or preparation containing an AKR1C3 enzyme-activating prodrug is administered to the patient.

[0035] The AKR1C3 enzyme-activated prodrug refers to a compound that is a prodrug. The prodrug molecule reacts with the AKR1C3 enzyme to release an anti-tumor compound with cytotoxicity after the reaction.

[0036] In general, an AKR1C3 enzyme-activating prodrug satisfies, but is not limited to, at least one of the following conditions:

[0037] A. In the presence of an AKR1C3 inhibitor (e.g., compound 36 in Bioorganic and Medicinal Chemistry, 2014: 962-977) ) environment, the cancer cell proliferation inhibition effect of a compound detected is less than the cancer cell proliferation inhibition effect of the compound detected in the absence of the AKR1C3 inhibitor. 50When quantifying, if the IC value of a compound against a cancer cell line in the presence of an AKR1C3 inhibitor is 50 If the IC50 is greater than that obtained in the absence of an AKR1C3 inhibitor, the compound can be determined to be an anticancer drug activated by AKR1C3 (lysis-prodrug). Specific examples are as follows:

[0038] PCT / US2016 / 021581, Publication No. WO2016145092A1, corresponding to Chinese Application No. 2016800150788, Publication No. CN107530556A;

[0039] PCT / US2016 / 062114, publication number WO2017087428, corresponding to Chinese application number 2016800446081, publication number CN108290911A;

[0040] PCT / US2016 / 025665, Publication No. WO2016161342, corresponding to Chinese Application No. 2016800200132, Publication No. CN108136214A; and

[0041] PCT / NZ2019 / 050030, publication number WO2019190331, for the compounds disclosed in Chinese application number CN2019800234236, publication number CN111918864A, the full text of the above patent documents is hereby introduced into the text of this patent application.

[0042] Among them, the compounds disclosed in patent PCT / US2016 / 021581, publication number WO2016145092A1, corresponding to Chinese application number 2016800150788, publication number CN107530556A; PCT / US2016 / 062114, publication number WO2017087428, corresponding to Chinese application number 2016800446081, publication number CN108290911A; PCT / US2016 / 025665, publication number WO2016161342, corresponding to Chinese application number 2016800200132, publication number CN108136214A are cleavage prodrugs, and the original drug that is finally cleaved and metabolized to produce the active substance is As well as drugs such as paclitaxel and camptothecin; the compound disclosed in patent PCT / NZ2019 / 050030, publication number WO2019190331, corresponding to Chinese application number CN2019800234236 and publication number CN111918864A, is a cleavage prodrug, and the ultimate cleavage metabolized prodrug is a nitrogen mustard structure drug;

[0043] Compound B 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 on cancer cells with low expression of AKR1C3 enzyme. 50 When quantified, if the IC50 value of a compound in cancer cells with high AKR1C3 expression is lower than the IC50 value in cancer cells with low AKR1C3 expression, 50 The compound can be determined by the value of PCT / CN2020 / 120281, publication number WO2021068952A1. The full text of this patent document is hereby incorporated into the text of this patent application.

[0044] In the existing technology, AKR1C3 enzyme-activating drugs (such as AST-3424 and AST-001, which have entered the clinical trial stage) require patients with solid tumors to provide pathological paraffin blocks or sections (including archived pathological paraffin blocks and sections) for AKR1C3 expression analysis, preferably recent samples. Only patients whose tumor tissue AKR1C3 expression reaches a certain level through IHC testing can be enrolled in the clinical trial.

[0045] In fact, due to various reasons, some patients may not obtain qualified tissue samples during tumor resection surgery, and some patients may not have received tumor resection surgery. Therefore, a large number of patients are unable to provide samples for IHC detection methods to detect AKR1C3 enzyme expression levels.

[0046] The above four schemes use the relatively mature KRAS gene mutation positive test results (detection of KRAS gene mutation) to replace the test results of high level of AKR1C3 protein expression or high level of AKR1C3 RNA. On the one hand, for patients with previous positive test results for KRAS gene mutation, it can be directly judged that their AKR1C3 enzyme is highly expressed and no further testing is required; on the other hand, for patients who have not been tested for KRAS gene mutation in the past, mature KRAS gene mutation kits can be used for testing, which is convenient and easy to obtain. In addition, the gene mutation test sample is blood, which is easy to collect and has high patient compliance: the tumor tissue samples used for IHC method detection need to be obtained by invasive means of biopsy or during tumor resection surgery.

[0047] The KRAS gene mutation is selected from KRAS-G12D mutation, KRAS-G12V mutation, KRAS-G12C mutation, KRAS-G12A and KRAS-G13D.

[0048] Mutations in either or both of the KRAS genes can be detected and diagnosed using commercially available (companion) diagnostic kits, such as the following diagnostic kits approved in China:

[0049] Xiamen Amoy Dx Biotechnology Co., Ltd., National Medical Device Registration No. 20153401126, Human KRAS Gene Mutation Detection Kit (Fluorescence PCR Method);

[0050] Shanghai TouJing Life Sciences, National Medical Device Registration No. 20163401341, Human K-RAS Gene 7 Mutation Detection Kit (PCR Fluorescence Method).

[0051] Of course, NGS sequencing (YS450 gene NGS large panel) can also be used to determine the specific KRAS mutation subtype.

[0052] Please see the table below for the corresponding specific information.

[0053] Generally speaking, KRAS gene mutations are detected using samples of the patient's tumor or cancer tissue. However, due to various reasons, such as the patient's inability to provide qualified tumor or cancer tissue samples, or the current detection method cannot accurately detect, or the tumor or cancer tissue no longer reflects the patient's recent condition, it may be necessary to detect or diagnose the patient's own KRAS gene mutation. For example, blood can be used for gene mutation detection, and the test results can represent the condition of the patient's tumor or cancer tissue; circulating tumor cell (CTC) detection can detect KRAS gene mutations in specific cancer cells (all tumor cells free in the peripheral blood) through the patient's peripheral blood. In some cases, the gene mutation of the parents in the biological parent-child relationship can be used to diagnose or assist in the diagnosis of gene mutations. The patient who is detected to have a KRAS gene mutation, or the patient who is detected to have a KRAS gene mutation, refers to this situation.

[0054] In this application, gene mutation refers to disease-causing gene mutation.

[0055] The TMB (tumor mutation burden) level of the gene mutation is medium.

[0056] Because TMB (tumor mutation burden) varies among different tumor types, it's generally considered high if it exceeds 20 mutations / Mb (Mb stands for bases per million), low if it's below 10 mutations / Mb, and moderate if it's in between. At the 2017 World Lung Cancer Conference, Bristol-Myers Squibb announced the results of the CheckMate-032 clinical trial, a Phase II study that enrolled 401 patients with advanced lung cancer who had failed first-line therapy. The patients were treated with a PD-1 inhibitor alone or in combination with ipilimumab. Patients were divided into three groups based on TMB levels: high, intermediate, and low. Among those receiving the combination therapy, the response rate was 62%, 20%, and 23%, respectively, with the response rate three times higher in those with high TMB. The median overall survival was 22.0 months, 3.6 months, and 3.4 months, respectively—a six-fold difference! This study demonstrated that different TMB levels have a significant impact on the efficacy of different cancer treatment drugs.

[0057] The AKR1C3 enzyme-activating prodrug is selected from compound formula (1)-(12) or pharmaceutically acceptable salts, solvates, and isotopic isomers thereof.

[0058] Among them, X, Y, Z, R, T, A and X 10 The definition is as described in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), and the synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0059] X 10 O, S, SO or SO2;

[0060] A is C6-C 10 Aryl, 5-15 membered heteroaryl or -N=CR 1 R 2 ;

[0061] R 1 and R 2 are independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0062] X, Y and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0063] R is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0064] R 13 and R 14 are independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle or ether;

[0065] T comprises a phosphoramidite alkylating agent comprising one or more alkylating agents bonded to -OP(Z 1 ) part of Z 5 -X 5 -Y 5 Part of the alkylating agent, 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 or another leaving group, or Z 5 -X 5 -Y 5 Together they form an aziridinyl (NCH2CH2) moiety and Z 1 is O or S; and

[0066] These alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, and ether groups are substituted or unsubstituted.

[0067] The AKR1C3 enzyme-activated prodrug compound of formula (1) is selected from the compounds of the following structures:

[0068] The specific synthesis method and corresponding spectral data of the compound of formula (1) are disclosed in WO2016145092A1 (corresponding to Chinese publication CN107530556A), which is incorporated herein in its entirety.

[0069] Among them, X, Y, Z, R, D, L 1 , A and X 10 The definition is as described in the claims of patent application PCT / US2016 / 025665, publication number WO2016 / 161342A1 (corresponding to Chinese application number 2016800200132, publication number CN108136214A), and the synthesis preparation method of the specific compound is also described in the above application, which is hereby incorporated into the present application in its entirety, and is specifically defined as:

[0070] X 10 O, S, SO or SO2;

[0071] A is C6-C 10 Aryl, 5- to 15-membered heteroaryl, or -N=CR 1 R 2 ;

[0072] R 1 and R 2 Each independently represents hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0073] X, Y and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0074] Each R is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0075] R 13 and R 14 Each independently represents hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, or ether;

[0076] Among them L 1 and D is defined as follows:

[0077] L 1 Selected from: and

[0078] R 40 and R 41 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 15-membered heterocyclic ring, or 5- to 15-membered heteroaryl;

[0079] R 42 is a C2-C3 alkylene or heteroalkylene group which is optionally substituted with 1 to 3 C1-C6 alkyl groups;

[0080] V(-) is any anion, preferably a pharmaceutically acceptable anion;

[0081] D is a moiety such that D-OH is an anticancer drug, wherein OH is an aliphatic hydroxyl group or a phenolic hydroxyl group, or is an OH moiety attached to a phosphorus atom as provided herein; or

[0082] L 1 for:

[0083] R 40 As defined above, R 43 is hydrogen or forms a heterocyclic ring together with D, and the phenylene moiety is optionally substituted, and

[0084] D is such that D-NR 43 H is a moiety of an anticancer drug; or

[0085] L 1 For the key, -OC(R 40 R 41 )2-、-OC(R 40 R 41 )-NR 40 R 41 (+)-C(R 40 R 41 )-or

[0086] where R40 、R 41 and V is as defined above, and

[0087] D is an anticancer drug containing a tertiary or secondary nitrogen atom, wherein the tertiary nitrogen atom or the secondary nitrogen atom is bonded to L 1 ;

[0088] and

[0089] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, and ether groups are optionally substituted.

[0090] The AKR1C3 enzyme-activated prodrug compound of formula (2) is selected from the compounds of the following structures:

[0091] The specific synthesis method and corresponding spectral data of the compound of formula (2) are disclosed in WO2016161342A1 (corresponding to Chinese publications CN108136214A and CN112142692A), which are incorporated herein in their entirety.

[0092] Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as described in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A1 (corresponding to Chinese application number 2020800358890, publication number CN113853379A), and the synthesis preparation method of the specific compound is also described in the above application, which is hereby incorporated into this application in its entirety, and is specifically defined as:

[0093] R1 is C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, 7-15 membered fused ring or Z-substituted fused ring;

[0094] R2 is hydrogen, a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, OTs, OMS, a C1-C6 alkyl group or a Z-substituted alkyl group, a C2-C6 alkenyl group or a Z-substituted alkenyl group, a C2-C6 alkynyl group or a Z-substituted alkynyl group, a C3-C8 cycloalkyl group or a Z-substituted cycloalkyl group, a C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, ether of 1-6 carbon atoms or Z-substituted alkoxy of 1-6 carbon atoms, -CONR 6 R 7、-SO2NR 6 R 7 、-SO2R 6 、-OCOO-R 6 、-COOR 6 、-NR 6 COR 7 、-OCOR 6 、-NR 6 SO2R 7 、-NR 6 SO2NR 6 R 7 Or R2 and the atoms on the R1 group to which it is bonded together form a 7-15 membered fused ring or a Z-substituted fused ring;

[0095] R3 is hydrogen, halogen, cyano or isocyano, hydroxyl, thiol, amine, OTs, OMS, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCO-R 6 、-OCOO-R 6 、-COOR 6 、-NR 6 COR 7 , -OCOR 6 、-NR 6 SO2R 7 ;

[0096] R4 and R5 are each independently hydrogen, a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, OTs, OMS, a C1-C6 alkyl group or a Z-substituted alkyl group, a C2-C6 alkenyl group or a Z-substituted alkenyl group, a C2-C6 alkynyl group or a Z-substituted alkynyl group, a C3-C8 cycloalkyl group or a Z-substituted cycloalkyl group, a C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, -CONR 6 R 7 、-SO2NR 6 R 7 、-SO2R 6 、-OCOO-R 6、-COOR 6、 -NR 6 COR 6 、-OCOR 6 、-NR 6 SO2R 7 Or R4, R5 and the atoms on the benzene ring to which they are bonded together form a 7-15 membered fused ring or a Z-substituted fused ring;

[0097] R 6 and R 7 Each is independently hydrogen, cyano or isocyano, C1-C6 alkyl or Z substituted alkyl, C2-C6 alkenyl or Z substituted alkenyl, C2-C6 alkynyl or Z substituted alkynyl, C3-C8 cycloalkyl or Z substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6 alkoxy or Z-substituted C1-C6 alkoxy, or R 6 、R 7 The group together with the atoms to which it is bonded forms a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group;

[0098] R8, R 10 Each is independently hydrogen, deuterium, aryl or Z-substituted aryl, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 alkynyl or Z-substituted alkynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, and at least one of them must be hydrogen or deuterium;

[0099] R9 is a substituted C6-C 10 aryl, substituted 4- to 15-membered heterocyclic ring having at least one fluorine atom or nitro group, substituted 5- to 15-membered heteroaryl group having at least one fluorine atom or nitro group.

[0100] The Z substituent is a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a thiol group, an amine group, OTs, OMS, a C1-C3 alkyl group or a substituted alkyl group, a C1-C3 alkoxy group or a substituted alkoxy group, a C2-C3 alkenyl group or a substituted alkenyl group, a C2-C3 alkynyl group or a substituted alkynyl group, a C3-C8 cycloalkyl group or a substituted cycloalkyl group, an aromatic ring, a heterocyclic ring, a heteroaromatic ring and a condensed ring or a substituted aromatic ring, a heterocyclic ring, a heteroaromatic ring and a condensed ring, and the substitution is monosubstitution or geminal disubstitution;

[0101] Substitution of C6-C in R9 10 The substituents of the aryl group, the substituted 4-15 membered heterocyclic ring, and the substituted 5-15 membered heteroaryl group are halogen atoms, nitro groups, cyano groups or isocyano groups, hydroxyl groups, amino groups, C1-C3 alkyl groups or alkoxy groups, alkenyl groups, alkynyl groups, cycloalkyl groups or benzene rings, substituted benzene rings, C1-C3 alkoxy groups, or halogen atom-substituted alkoxy groups.

[0102] The AKR1C3 enzyme-activated prodrug compound of formula (3) is selected from the compounds of the following structures:

[0103] The AKR1C3 enzyme-activated prodrug compound of formula (4) is selected from the compounds of the following structures:

[0104] The specific synthesis method of the compounds of formula (3) (4) and the corresponding spectral data are disclosed in WO2020228685A1 (corresponding to the Chinese publication CN113853379A), which is incorporated herein in its entirety.

[0105] in:

[0106] A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 SO2R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group;

[0107] Among them, R 100 、R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which it is attached, form a 5-7 membered heterocyclic ring;

[0108] wherein the alkyl group and the aryl group are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups;

[0109] R 1 and R 2 each independently phenyl or methyl;

[0110] X, Y and Z are each independently hydrogen or halo;

[0111] R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl.

[0112] The AKR1C3 enzyme-activated prodrug compound of formula (5) is selected from the compounds of the following structures:

[0113] as well as as well as and

[0114] The specific synthesis method of the compound of formula (5) and the corresponding spectral data are disclosed in WO2020228685A1 (corresponding to Chinese publication CN113853379A) ​​and WO2016145092A1 (corresponding to Chinese publication CN107530556A), which are incorporated herein in their entirety.

[0115] Wherein, Rw is defined as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A), and the synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0116] Rw is

[0117] R1 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 replaced by;

[0118] Each R a are independently H, F, Cl, Br, I, -CN, -OH, C 1-3 Alkoxy or C 1-3 alkyl;

[0119] R2 is H or C 1-6 alkyl;

[0120] Or R1 and R2 are linked together to form a 4-6 membered heterocycloalkyl group, wherein the 4-6 membered heterocycloalkyl group is optionally substituted by 1, 2 or 3 R b replaced by;

[0121] Each R b are independently H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3 or -CH2CH3;

[0122] R3 is H, F, Cl, Br, I, -OH, -NH2, C 1-3 Alkoxy or C 1-3 alkyl;

[0123] Or R2 and R3 are connected together to make the structural unit for

[0124] T1 is -(CR c R d ) m -or-(CR c R d ) n -O-;

[0125] m is 1, 2, or 3;

[0126] n is 1 or 2;

[0127] T2 is N or CH;

[0128] R c and R d Each independently is H, F, C 1-3 Alkyl or C 1-3 alkoxy;

[0129] R4, R5 and R6 are each independently H, F, Cl, Br, I, C 1-3 Alkyl or C 1-3 alkoxy;

[0130] T is N or CH;

[0131] R7 and R8 are each independently H, F, Cl, Br or I;

[0132] R9 and R 10 Each independently is H, F, Cl, Br, I, -CN or

[0133] The 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl each contain 1, 2, 3 or 4 heteroatoms independently selected from N, -O- and -S-.

[0134] The AKR1C3 enzyme-activated prodrug compound of formula (6) is selected from the compounds of the following structures:

[0135] The specific synthesis method of the compound of formula (6) and the corresponding spectral data are disclosed in WO2021068952A1 (corresponding to Chinese publication CN114555574A), which is incorporated herein in its entirety.

[0136] Wherein, the definitions of R1, R2, R3, R4, and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1. The synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety and are specifically defined as:

[0137] T is N or CH;

[0138] R1 and R2 are each independently H, F, Cl, Br, I or C 1-3 Alkyl, wherein the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R a replaced by;

[0139] Each R a independently F, Cl, Br, I, -CN, -OH or -NH2;

[0140] R3 and R4 are each independently H, F, Cl, Br, I, CN, C 1-3 Alkyl, C 1-3 Alkoxy, Among them, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R e replaced by;

[0141] R b and R c Each independently represents H, -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0142] R d It is -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0143] Each R e independently F, Cl, Br, I, -CN, -OH or -NH2.

[0144] The AKR1C3 enzyme-activated prodrug compound of formula (7) is selected from the compounds of the following structures:

[0145] The specific synthesis method of the compound of formula (7) and the corresponding spectral data are disclosed in WO2022057838A1, which is incorporated herein in its entirety.

[0146] Wherein, the definitions of A, E, G, X, and Y are as described in the claims of patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A), and the synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0147] A is H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, CFH2, CF2H, CF3, F, Cl, Br, I, OCF3, COR or CON(R)2;

[0148] E is SO or SO2;

[0149] X is Cl, Br, I or OSO2R;

[0150] Y is Cl, Br, I or OSO2R;

[0151] Each R is independently H or C1-C6 alkyl;

[0152] G is a radical selected from the group consisting of formula (B)-(AA):

[0153] in:

[0154] R1 is H, C1-C6 alkyl, CH2(CH2)nOH, CH2CH(OH)CH2OH, phenyl, pyridyl, benzyl or pyridylmethyl, provided that when R1 is phenyl, pyridyl, benzyl or pyridylmethyl, R1 is optionally substituted at any available position with C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 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 or NO2;

[0155] R2 and R3 are each independently H, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 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 or NO2;

[0156] R4 is N(R6)(R7), OH, OCH2(CH2)nN(R6)(R7) or CH2(CH2)nN(R6)(R7);

[0157] R5 is H or a C1-C6 alkyl group;

[0158] R6 and R7 are each independently H or C1-6 alkyl, or R6 and R7 together form a substituted or unsubstituted 5-membered or 6-membered heterocyclic ring;

[0159] Z is CH or N;

[0160] W is CH2, O, S, SO or SO2;

[0161] n is 0 to 6;

[0162] * indicates the point of connection to formula (I).

[0163] The AKR1C3 enzyme-activated prodrug compound of formula (8) is selected from the compounds of the following structures:

[0164] The specific synthesis method of the compound of formula (8) and the corresponding spectral data are disclosed in WO2019190331A1 (corresponding to the Chinese publication CN111918864A), which is incorporated herein in its entirety.

[0165] Among them, R w ,X,R4,R 10 、R 13 、R 14 The definition is as described in the claims of patent application PCT / CN2022 / 098082, publication number WO2022258043A1, and the synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0166] The two Xs are independently CR 15 or N;

[0167] R 13 、R 14are independently hydrogen, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, C6-C 20 Aryl, 5-20 membered heterocyclic group, halogen substituted C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, halogen substituted C6-C 20 aryl, or a 5-20 membered heterocyclic group substituted with halogen, and R 13 、R 14 Not simultaneously hydrogen;

[0168] R 10 is hydrogen, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, C6-C 20 Aryl, 5-20 membered heterocyclic group, halogen substituted C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, halogen substituted C6-C 20 aryl, or a 5-20-membered heterocyclic group substituted with halogen;

[0169] or R 10 With R 13 or R 14 Can be in accordance with the above R 10 、R 13 、R 14 Connected under defined conditions to form a 5-9 membered ring;

[0170] R4, R 15 Each is independently hydrogen, halogen, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, cyano, 5-20 membered heterocyclic group, C6-C 20 Aryl, or halogen-substituted C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclic group, C6-C 20 aryl;

[0171] or R 10 With R 15 Can be in accordance with the above R 10 、R 15 Formation of a 4-12 membered cyclic hydrocarbon or heterocycle under defined conditions;

[0172] R W yes

[0173] A is CR 16 or N, wherein the position of A can be changed on the ring;

[0174] R 16 is hydrogen, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, C6-C 20 Aryl, 5-20 membered heterocyclic group, halogen substituted C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, halogen substituted C6-C 20aryl, or a 5-20-membered heterocyclic group substituted with halogen;

[0175] R6 and R7 meet the following conditions:

[0176] R6 and R7 are each independently hydrogen, halogen, cyano, hydroxy, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclic group, C6-C 20 Aryl, or halogen-substituted C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclic group, C6-C 20 Aryl, or cyano-substituted C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, 5-20 membered heterocyclic group, C6-C 20 Aryl, or hydroxy-substituted C1-C6 alkyl, cycloalkyl, alkoxy, 5-20 membered heterocyclic group, C6-C 20 Aryl, or -CONR 11 R 12 , or -CH2NR 11 R 12 ;

[0177] Or R6 and R7 are connected to form

[0178] A 5-8 membered monocyclic or condensed heterocyclic ring containing at least one N, S or O, or containing two or three of N, S and O at the same time;

[0179] Alternatively, a 5-8 membered monocyclic or fused heterocyclic ring containing at least one N, S, or O, or containing two or three of N, S, and O, and the monocyclic or fused heterocyclic ring is substituted with a C1-C6 alkyl group;

[0180] R6 can be used with CR 16 connected to form a 5-9 membered ring, heterocycle or aromatic heterocycle;

[0181] R 11 、R 12 The following conditions are met:

[0182] R 11 、R 12 Each is independently C1-C6 alkyl, halogen-substituted C1-C6 alkyl or R 11 、R 12 Satisfy the above definition conditions and -CONR 11 R 12 The N in the form a 5-7 membered ring, with -CH2NR 11 R 12 The N in the group forms a 5-7 membered ring.

[0183] The AKR1C3 enzyme-activated prodrug compound of formula (9) is selected from the compounds of the following structures:

[0184] The specific synthesis method of the compound of formula (9) and the corresponding spectral data are disclosed in WO2022258043A1, which is incorporated herein in its entirety.

[0185] Wherein, the definitions of R1, R2, R3, R4, G1, G2, G3, G4, E, T, Y, Z, m, n, s, t, v, w, and Ring A are as described in the claims of patent application CN202210585771.6, publication number CN115403579A. The synthesis and preparation methods of specific compounds are also described in the above-mentioned applications, which are hereby incorporated into the present application in their entirety, and are specifically defined as:

[0186] G 1 , G 2 , G 3 or G 4 The same or different, and each independently CR 5 or N atoms;

[0187] Each R 5 are the same or different and are each independently selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxyl, cyano, amino, nitro, -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 heteroaryl are each independently optionally substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, oxo, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0188] 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-(C(R y2 R y3 )) h -;

[0189] R y1 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group;

[0190] R y2 and R y3 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group and a heterocyclic group;

[0191] or R y2 and R y3 Together they form =O;

[0192] Z is O or OH;

[0193] is a single bond or a double bond, when When Z is OH, When it is a double bond, Z is O;

[0194] E is selected from NH, O atoms and S atoms;

[0195] T is selected from -C(R T1 R T2 )- -NR T3 -or-O-;

[0196] 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 heterocyclic group;

[0197] or R T1 and RT2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclic group, wherein the cycloalkyl group or the heterocyclic group are each independently substituted with one or more substituents selected from halogen, alkyl and hydroxy;

[0198] R T3 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group;

[0199] Ring A is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group;

[0200] Each R 1 are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, a hydroxyl group, a cyano group, a -NR a R b 、-C(O)NR a R b 、-S(O)NR a R b 、-S(O)2NR a R b 、-S(O)R c 、-S(O)2R c 、-B(OR d ) 2, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl, wherein the alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, heterocyclic, aryl and heteroaryl are each independently selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, oxo, cyano, -NR a R b 、-C(O)NR a R b 、-S(O)NR a R b 、-S(O)2NR a R b 、-S(O)R c 、-S(O)2R c 、-B(OR d ) 2, substituted by one or more substituents selected from nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0201] R a and R b are the same or different and are each independently selected from hydrogen atom, alkyl, haloalkyl, hydroxyl, hydroxyalkyl, -C(O)R e , cycloalkyl and heterocyclic groups; or R a and R bTogether with the nitrogen atom to which it is attached, it forms a cycloalkyl or heterocyclic group, wherein the cycloalkyl or heterocyclic group is optionally substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl;

[0202] R c is selected from alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0203] R d A hydrogen atom or C 1-6 alkyl;

[0204] R e is selected from alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl;

[0205] Each R 2 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, an alkoxy group, a haloalkoxy group, a hydroxyalkyl group, an oxo group, a hydroxyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group;

[0206] Each R 3 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, an alkoxy group, a haloalkoxy group, a hydroxyalkyl group, an oxo group, a hydroxyl group, a cyano group, an amino group, a nitro group, a cycloalkyl group, a heterocyclyl group, an aryl group, and a heteroaryl group;

[0207] R 4 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a hydroxyl group, and a hydroxyalkyl group;

[0208] n is 0, 1, 2, or 3;

[0209] v is 0, 1, or 2;

[0210] w is 0, 1, or 2;

[0211] f is 0, 1, or 2;

[0212] g is 0, 1, or 2;

[0213] h is 0, 1, or 2;

[0214] m is 0, 1, 2, 3, 4 or 5;

[0215] s is 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0216] t is 0, 1, 2, 3, 4, 5, or 6;

[0217] The condition is,

[0218] When Y is an -O- atom and E is an O atom, Ring A is a phenyl group or a 5- to 6-membered heteroaryl group, and G 3 CR 5 or N atom, R 5 Not a hydrogen atom;

[0219] When Y is an -O- atom and E is an S atom, ring A is a phenyl group or a 5- to 6-membered heteroaryl group;

[0220] When G 1 , G 2 , G 3 and G 4 All CR 5 , Y is NR y1 , n, v and w are all 1, and E is an O atom, 1) T is not CH2 or CD2, 2) there is 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 heterocyclic group, wherein the 3- to 8-membered cycloalkyl group or the 5- to 8-membered heterocyclic group is optionally substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, hydroxyalkyl, hydroxy, oxo, cyano, amino, nitro, cycloalkyl, heterocyclic group, aryl and heteroaryl; 5) Ring A is R d A hydrogen atom or C 1-6 alkyl.

[0221] The AKR1C3 enzyme-activated KARS inhibitor prodrug compound of formula (10) is selected from the compounds of the following structures:

[0222] The specific synthesis method and corresponding spectral data of the compound of formula (10) are disclosed in Chinese publication CN115403579A, which is incorporated herein in its entirety.

[0223] Among them, R 1 、R 2a 、R2b 、R 3 、R 4 、R 5 The definitions of n and Z are as described in the claims of patent application PCT / IB2020 / 057285, publication number WO2021005586A1 (corresponding to Chinese application number CN202080053804.1, publication number CN114206870A). The synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety and are specifically defined as:

[0224] Is a single bond or a double bond;

[0225] Z when When it is a single bond, it is OH; or when When it is a double bond, it is O;

[0226] Each R 1 independently selected from the group consisting of: (C1-C6)alkyl, (C1-C6)alkoxy, (C0-C4)alkylN(R 8 ) 2, and halogen;

[0227] R 2a and R 2b are each independently selected from the group consisting of: H, (C1-C6)alkyl, and halo;

[0228] Each R 3 independently selected from the group consisting of: H, and halo;

[0229] 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 9 to 10 membered fused bicyclic heteroaryl containing 1, 2, 3, or 4 heteroatoms independently selected from N, O and S; wherein any of the foregoing is optionally replaced by one or more R 6 replace;

[0230] R 5 Selected from the group consisting of: H; (C1-C6)alkyl; (C2-C6)alkenyl; (C0-C4)alkyl OR 8 ; (C1-C4) alkyl (C3-C 10 )cycloalkyl; halo(C1-C6)alkyl; (C2-C3)alkynyl; (C1-C4)alkylN(R 10 )2;

[0231] Each R 6independently selected from the group consisting of: halo; (C1-C6) alkyl; (C1-C6) alkoxy; halo (C1-C6) alkyl; OH; aryl; 3 to 6 membered heterocyclic; 5 to 6 membered heteroaryl; (C0-C4) alkyl S(O) m (C1-C6)alkyl; halo(C1-C6)alkoxy; (C0-C4)alkylS(O) m N(R 8 )2; (C0-C4) alkyl N (R 8 )2; (C0-C4) alkyl (CO) OR 7 ; N(R 8 )S(O) m (C1-C6)alkyl; N(R 8 )S(O) m (C3-C6)cycloalkyl; OP(O)(OH)2; (C0-C3)alkyl (CO)NHR 11 ; (C0-C3) alkyl OR 7 , and (C3-C 10 )cycloalkyl; wherein each R 6 When not halo, OH, or OP(O)(OH)2, it is optionally substituted by one to three R 9 Substitution; or two adjacent R 6 together with the atoms to which they are attached, form a 5- to 7-membered heterocyclic ring or a (C5-C8)cycloalkyl group;

[0232] Each R 7 and R 8 independently selected from the group consisting of: H or optionally substituted by one to three R 9 substituted (C1-C6)alkyl;

[0233] Each R 9 independently selected from the group consisting of: halo; -OH; amino, (C1-C4) alkylamino, di(C1-C4) alkylamino, OP(O)(OH)2; (C1-C6) alkyl; (C1-C3) alkynyl; (C1-C6) alkoxy; halo(C1-C6) alkyl; (C0-C4) alkylS(O) m (C1-C6)alkyl; halo(C1-C6)alkoxy; 3- to 6-membered heterocyclic ring optionally substituted with oxo (=O); (C0-C4)alkylS(O) m N(R 10 )2; (C0-C4) alkyl (CO) R 10 ; (C0-C4)alkyl (CO)OR 10 ; (C0-C4) alkyl NR 10 S(O) m (C1-C6)alkyl; (C0-C4)alkyl OR10 ; (C0-C4) alkyl N(R 10 )2; (C0-C4) alkylCN; (C0-C4) alkylN(R 10 )2; and (C0-C4)alkyl (CO) N (R 10 )2;

[0234] Each R 10 independently selected from the group consisting of: H, (C1-C6)alkyl; or a 3- to 6-membered heterocycle, wherein the 3- to 6-membered heterocycle is optionally substituted with one or more of: (C1-C6)alkyl; and oxo (=O);

[0235] Each R 11 Selected from the group consisting of: H; optionally replaced by one to four R 12 substituted 4 to 6 membered heterocyclic ring; optionally substituted by one to four R 12 Substituted (C3-C6) cycloalkyl; (C0-C3) alkyl optionally substituted by halogen (C3-C6) cycloalkyl (C1-C3) alkyl; optionally substituted by one to three R 12 substituted CH2-aryl; (C1-C6)alkyl; (C2-C6)alkenyl; or (C2-C6)alkynyl, wherein each of the (C1-C6)alkyl; (C2-C6)alkenyl; and (C2-C6)alkynyl is optionally substituted by one or more R 13 replace;

[0236] Each R 12 independently selected from the group consisting of OH, (C1-C3)alkoxy, NH2; or (C1-C3)alkyl optionally substituted with one or more OH;

[0237] Each R 13 independently selected from the group consisting of: halo, OH, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, (C1-C3)alkoxy; and C(O)-(C3-C8)cycloalkyl;

[0238] m is 0, 1, or 2; and

[0239] n is 0, 1, or 2.

[0240] The AKR1C3 enzyme-activated KARS inhibitor prodrug compound of formula (11) is selected from the compounds of the following structures:

[0241] The specific synthesis method of the compound of formula (11) and the corresponding spectral data are disclosed in WO2021005586A1 (corresponding to Chinese publication CN114206870A), which is incorporated herein in its entirety.

[0242] or a pharmaceutically acceptable salt thereof,

[0243] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R a 、R b The definitions of n1 and n2 are as described in the claims of patent application PCT / CN2023 / 123253, publication number WO2024078392A1. The synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety. They are specifically defined as:

[0244] in:

[0245] (2)R 1 、R 2 Together with the intervening atoms, they form an optionally substituted 4-8 membered carbon or heterocyclic ring, R 4 and R 5 is as defined in (1); or

[0246] (3)R 1 、R 5 Together with the intervening atoms, they form an optionally substituted 4-8 membered carbon or heterocyclic ring, R 2 and R 4 is as defined in (1); or

[0247] (4)R 4 、R 5 Together with the intervening atoms, they form an optionally substituted 4-8 membered carbon or heterocyclic ring, R 1 and R 2 is as defined in (1) or (2); or

[0248] (1)R 1 is hydrogen, deuterium, or any substituted C 1-4 Alkyl, optionally substituted C 2-4 Alkenyl or optionally substituted C 2-4 Alkynyl;

[0249] R 2 、R 4 、R 5 are independently hydrogen, halogen (such as F), optionally substituted C1-4 Alkyl, arbitrarily substituted C 2- 4-alkenyl, optionally substituted C 2-4 Alkynyl, optionally substituted C 1-4 alkoxy or an optionally substituted 3-5 membered ring;

[0250] in:

[0251] X is O, S, or NR 10 , arbitrarily substituted C 1-4 Alkylene or optionally substituted C 1-4 Heteroalkylene, wherein R 10 is hydrogen, optionally substituted C 1-4 Alkyl, optionally substituted 3-6 membered ring or nitrogen protecting group;

[0252] R 3 is hydrogen, optionally substituted C 1-4 Alkyl or an optionally substituted 3-10 membered ring;

[0253] R 6 is hydrogen, deuterium, or any substituted C 1-4 Alkyl, optionally substituted C 2-4 Alkenyl or optionally substituted C 2-4 Alkynyl;

[0254] Integers n1 and n2 are each independently 0, 1, 2, 3 or 4;

[0255] Each R a 、R b Each occurrence is independently optionally substituted C1-4 alkyl or optionally substituted C1-4 heteroalkylene; or two instances of R a or two instances of R b Together with the intervening atoms, they form an optionally substituted 3-6 membered ring, and any remaining instances of R a and / or R b As defined above.

[0256] The AKR1C3 enzyme activating compound of formula (12) is selected from compounds 1-569 in Tables 1-15 of patent application PCT / CN2023 / 123253, publication number WO2024078392A1, and the structures of some of the compounds are listed below:

[0257] The specific synthesis method of compound 1-569 of formula (12) and the corresponding spectral data are disclosed in WO2024078392A1, which is incorporated herein in its entirety.

[0258] When the AKR1C3 enzyme activating prodrug is selected from the compound formula (1), (3)-(8), (12) or a pharmaceutically acceptable salt, solvate, or isotope isomer thereof,

[0259] The patient's tumor or cancer tissue is detected to have homologous recombination repair deficiency (HRD) or the patient is detected to have homologous recombination repair deficiency (HRD).

[0260] Homologous recombination repair deficiency requires either or both of the following:

[0261] Mutations in one or more genes involved in the homologous recombination repair (HRR) process;

[0262] The genomic scar (GS) score reaches the predetermined value.

[0263] Cellular DNA damage can be divided into single-strand breaks (SSBs) and double-strand breaks (DSBs). SSBs are primarily repaired by poly ADP-ribose polymerase (PARP) through nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR), while DSBs are repaired through homologous recombination repair (HRR), non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) pathways.

[0264] Homologous recombination repair (HRR) is a high-fidelity repair method and one of the core DNA damage repair mechanisms. It primarily occurs during the S and G2 phases of the cell cycle and is a DNA repair mechanism that maintains genomic integrity and ensures high-fidelity transmission of genetic information. This process requires the participation of numerous genes, including ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FAAP20, FAN1, FANCE, FANCL, FANCM, MRE11A, NBN, PALB2, POLQ, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L, RBBP8, SLX4, and XRCC2.

[0265] Homologous recombination repair deficiency (HRD) is a biological event caused by a variety of factors, including inherited and epigenetic inactivation of genes in the homologous recombination repair pathway. It is widely present in various tumors. When DNA damage cannot be repaired normally through the homologous recombination repair pathway (HRR), it is called homologous recombination deficiency (HRD).

[0266] HRD can cause genomic scars, including genomic loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large segment shift (LST).

[0267] When double-strand damage and homologous recombination repair deficiency (HRD) occur simultaneously, cells initiate non-high-fidelity repair methods, such as non-homologous end joining (NHEJ) and microhomologous end joining (MMEJ). This leads to insertion / deletion errors in genes, resulting in high genome-wide instability, also known as "genomic scarring" (GS). Therefore, the phenomenon of genomic instability caused by HRD is called "genomic scarring."

[0268] There are three main types of genomic scars: loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transition (LST). By combining these three, we can maximize the understanding of genomic instability and thus infer whether cells have HRD.

[0269] In summary, the gene mutation HRRm (homology-dependent recombination repair mutation) related to the homologous recombination repair process is the cause of homologous recombination repair deficiency HRD, and genomic scar GS is the manifestation of HRD: the gene mutation HRRm related to the homologous recombination repair process will lead to homologous recombination repair deficiency HRD and thus manifest as genomic scar GS.

[0270] Therefore, through detection, one or more mutations in genes related to the homologous recombination repair (HRR) process are found; homologous recombination repair defect HRD can be determined when the genomic scar (GS) score reaches one or two predetermined values.

[0271] However, since DNA double-strand breaks (DSBs) rely mainly on homologous recombination repair (HRR) as well as non-homologous end joining (NHEJ) and microhomologous end joining (MMEJ) pathways for repair, the detection of HRRm alone does not necessarily guarantee the presence of HRD. Furthermore, since mutations in genes of repair pathways such as non-homologous end joining (NHEJ), microhomologous end joining (MMEJ) may also lead to GS, the detection of GS alone cannot guarantee that the GS is related to HRRm.

[0272] Therefore, the combined detection of HRRm and GS will provide a more comprehensive assessment of the status of HRD.

[0273] Two approved testing kits: FoundationFocus CDx BRCA LOH Assay and Myriad Genetics HRD, these two test kits are HRD detection products designed based on Western populations, have been clinically validated and approved by the U.S. Food and Drug Administration (FDA), while there are no HRD companion diagnostic products on the market in China.

[0274] The situation of the two HRD detection kits is shown in Figure 21 (the figure is from the academic seminar of the Gynecological Tumor Molecular Diagnosis and Treatment Professional Committee of the Southern China Association for Clinical Oncology on January 27, 2021, and Professor Ouyang Nengtai’s academic report "From BRCA to HRD Detection".

[0275] When detecting HRD, it is necessary to simultaneously detect whether one or more genes related to the homologous recombination repair (HRR) process have mutated; and whether the genomic scar (GS) score has reached a predetermined value. HRD is determined to be positive only when both meet certain conditions.

[0276] The HRRm gene detected by the two HRD detection kits approved by the US FDA is BRCA. In fact, other genes related to the homologous recombination repair process can also be selected from ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FAAP20, FAN1, FANCE, FANCL, FANCM, MRE11A, NBN, PALB2, POLQ, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L, RBBP8, SLX4, and XRCC2.

[0277] The situation of genomic scar detection is also different. FoundationFocus CDx BRCA LOH Assay detects LOH status, while Myriad Genetics HRD detects the comprehensive status of LOH, TAI and LST.

[0278] The above compounds (1), (3)-(8), and (12) release DNA alkylating agents after being activated by the AKR1C3 enzyme highly expressed in tumor cells in vivo.

[0279] Compounds (1), (3)-(7), and (12) release DNA alkylating agents upon activation of AKR1C3 enzymes. (AST-2660);

[0280] Compound (8) releases DNA alkylating agent after activation of AKR1C3 enzyme (nitrogen mustard analogues);

[0281] The cytotoxic compounds activated by drug metabolism are all DNA alkylating agents, which act on the DNA double strands to cause cross-linking and thus DNA double-strand breaks (DSBs). In other words, the ultimate effect of the above compounds (1)-(8) and (12) is to cause DNA double-strand damage and thus kill cancer cells.

[0282] DSB is mainly repaired through the homologous recombination repair (HRR) pathway. When DNA damage occurs, if it cannot be repaired normally through the homologous recombination repair pathway HRR, it is called homologous recombination defect HRD.

[0283] Therefore, the aforementioned compounds should have superior therapeutic efficacy in tumor models harboring homologous recombination deficiency (HRD) compared to tumor models without the mutation. This suggests that AKR1C3-activated DNA alkylating agent prodrugs have more pronounced therapeutic effects in patients with HRD-positive cancers and tumors. This is supported by in vitro cell proliferation inhibition experiments and animal model studies.

[0284] Regarding the drugs or preparations described herein, the prepared drugs contain the compounds or their salts or solvates within a specific dosage range, and / or the prepared drugs are in a specific dosage form and are administered by a specific administration method.

[0285] Regarding this article medicine or preparation, pharmaceutically acceptable adjuvant or excipient may also be included. The medicine can be any dosage form of clinical administration, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small needles for injection, freeze-dried powder for injection or large infusions. Depending on the specific dosage form and mode of administration, the pharmaceutically acceptable adjuvant or excipient in the medicine can include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, adhesives, fillers, flavorings, sweeteners, antioxidants, surfactants, preservatives, wrappers and pigments etc.

[0286] "Applying" or "administering" a drug should be done in a "therapeutically effective amount." A "therapeutically effective amount" of a drug refers to an amount of the drug that, when administered or used to a patient suffering from cancer, will have the desired therapeutic effect (e.g., alleviation, improvement, remission, or elimination of the clinical manifestations of one or more cancers in the patient). The therapeutic effect does not necessarily occur with the administration or application of a single dose and may only occur after the administration or application of a series of doses. Therefore, a therapeutically effective amount can be administered or applied in one or more doses.

[0287] "Administering" or "applying" or "using" a drug to a patient refers to direct administration or administration (which may be administered or administered to a patient by a medical professional or may be self-administered or administered) and / or indirect administration or administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer or administer a drug and / or provides a prescription for a drug to a patient administers or administers a drug to a patient.

[0288] "Treatment" or "treating a patient" refers to administering, using or applying to a patient a therapeutically effective amount of a drug related to the present invention.

[0289] "Treatment" of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results). For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in the extent of the disease; delay or slowing of disease progression; improvement, remission, or stabilization of the disease state; or other beneficial results. In some instances, treatment of cancer may result in a partial response or stabilization of the disease.

[0290] "Tumor cell" or "cancer cell" refers to a tumor / cancer cell of any appropriate species (eg, mammalian, such as murine, canine, feline, equine, or human).

[0291] The subject of "treating cancer or tumor" refers to any appropriate species: mammals, fish, for example mammals, such as murine, canine, feline, equine or human. Preferably, the patient is a mammal, more preferably a human.

[0292] "Patient" and "subject" are used interchangeably to refer to a mammal in need of cancer treatment. Typically, the patient is a human. In certain embodiments, a "patient" or "subject" may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used for screening, characterization, and evaluation of drugs and therapies.

[0293] "Prodrug" refers to a compound that, after administration or administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug is chemically modified relative to a drug in a manner that renders it less active or inactive relative to the drug, but the chemical modification allows for the production of the corresponding drug by metabolism or other biological processes following administration of the prodrug. A prodrug may have altered metabolic stability or delivery characteristics, fewer side effects or lower toxicity, or an improved flavor relative to the active drug. Prodrugs may be synthesized using reactants other than the corresponding drug.

[0294] The salt is a basic salt or an acidic salt, and the solvate is a hydrate or an alcoholate.

[0295] The compounds also include salts of Formula I and Formula II, that is, pharmaceutically acceptable salts of the compounds provided herein. The salts may be basic salts, including salts formed between the compounds and inorganic bases (e.g., alkali metal hydroxides, alkaline earth metal hydroxides, etc.) or organic bases (e.g., monoethanolamine, diethanolamine, or triethanolamine, etc.). Alternatively, the salts may be acid salts, including salts formed between the compounds and inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid, etc.) or organic acids (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.). The selection and preparation of acceptable salts and solvates of compounds are well known in the art.

[0296] The compounds described herein can also be used in the form of solvates, such as hydrates, alcoholates, and the like, including ethanolates.

[0297] The concepts of isotopic variants or isomers in the present invention shall be based on the concepts of isotopic variants and chiral isomers in patent application PCT / US2016 / 062114, publication number WO2017087428, corresponding Chinese application number 2016800446081, publication number CN108290911A.

[0298] For terms not specifically explained or illustrated in this application text, please refer to the textbooks of medicinal chemistry, organic chemistry, biochemistry, and pharmacology. BRIEF DESCRIPTION OF THE DRAWINGS

[0299] Figure 1 is a graph showing the corresponding relationship between the RNA expression level of AKR1C3 and the RNA expression level of NRF2 in the KRAS-G12D mutation model.

[0300] Figure 2 is a graph showing the corresponding relationship between the RNA expression level of AKR1C3 and the RNA expression level of NRF2 in the KRAS-G12C mutation model.

[0301] FIG3 is a graph showing the corresponding relationship between the RNA expression level of AKR1C3 and the RNA expression level of NRF2 in the KRAS-G13D mutation model.

[0302] Figure 4 shows the results of immunoblotting experiments of target proteins after cell lysis under different concentrations of SFN treatment. The left figure shows immunoblotting images under different concentrations of SFN treatment, and the right figure shows a bar graph of protein band density analysis of NRF2 and AKR1C3. Under each concentration group, the left column is NRF2 and the right column is AKR1C3.

[0303] Figure 5 shows the results of immunoblotting experiments of target proteins after cell lysis under different concentrations of AST treatment. The left figure shows immunoblotting images under different concentrations of AST treatment, and the right figure shows a bar graph of protein band density analysis of p-ERK2 and ERK2 under different concentrations of AST treatment. Under each AST concentration, the left column represents p-ERK2 and the right column represents ERK2.

[0304] Figure 6 shows the results of immunoblotting experiments of target proteins after cell lysis treated with 1 μM AST for different time periods. The left figure shows immunoblotting images of cells treated with 1 μM AST for different time periods, and the right figure shows a bar graph of protein band density analysis of p-ERK2 and ERK2 treated with 1 μM AST for different time periods. The left column shows p-ERK2 and the right column shows ERK2 at each treatment time period.

[0305] Figure 7 shows the results of AKR1C3-dependent target protein immunoblotting experiments after cell lysis. The left figure is an AKR1C3-dependent immunoblot image, and the right figure is a bar graph of AKR1C3-dependent protein band density analysis of p-ERK2 and ERK2. In each treatment group, the left column represents p-ERK2, and the right column represents ERK2.

[0306] Figure 8 shows the results of target protein immunoblotting and cell apoptosis experiments after cell lysis for the ERK2 inhibitor and AST monotherapy and combination therapy groups. The upper figure is an immunoblot image of the ERK2 inhibitor group of PD98059 and Suramin alone or in combination; the lower left figure is a bar graph of protein band density analysis for the monotherapy and combination therapy, wherein the left column is p-ERK2 and the right column is ERK2 in each treatment group; the lower right figure is a bar graph of cell apoptosis for the monotherapy and combination therapy, wherein Luminescence (RLU) represents the relative luminescence unit of the apoptosis signal;

[0307] Figure 9 shows the results of target protein immunoblotting and cell apoptosis experiments after cell lysis of the ERK2 activator and AST single-drug and combination drug groups, wherein the upper figure is an immunoblot image of the ERK2 activator group of TPA alone or in combination; the lower left figure is a density analysis bar graph of its single-drug or combination use, wherein in each treatment group, the left column is p-ERK2 and the right column is ERK2; the lower right figure is a cell apoptosis bar graph of its single-drug or combination use, wherein Luminescence (RLU) represents the relative luminescence unit of the apoptosis signal;

[0308] Figure 10 is a schematic diagram of the process of AST inducing apoptosis of KRAS G12D pathogenic mutation cancer cells through the ERK2 signaling pathway, wherein (1), (3&4) represent relevant supporting literature,

[0309] (1)Prior IA, Hood FE and Hartley JL. The Frequency of Ras Mutations in Cancer. Cancer Res 2020;80:2969-2974;

[0310] (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;

[0311] (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;

[0312] ① indicates that AST compounds act on the MEK-ERK signaling pathway and activate the pathway;

[0313] ②AKR1C3 enzyme activates the AST prodrug compound and ultimately releases the DNA alkylating agent AST-2660;

[0314] ③The proliferation of tumor cells was inhibited by AST-2660.

[0315] FIG11 shows the expression level of AKR1C3 in Capan-1, PLC / PRF / 5, HT29, and H460 cell lines determined by immunoblotting.

[0316] FIG12 shows the expression level of AKR1C3 in Capan-1, HepG2, and H460 cell lines determined by immunoblotting.

[0317] FIG13 shows the growth curve of the tumor volume of each group of mice in the in vivo efficacy experiment of AST-3424 in the Capan-1CDX model with BRCA pathogenic mutations.

[0318] FIG14 is a curve showing changes in body weight of mice in each group during the in vivo efficacy experiment of AST-3424 in the Capan-1CDX model with BRCA pathogenic mutations.

[0319] FIG15 is a growth curve of the tumor volume of each group of mice in the in vivo efficacy experiment of AST-3424 in the non-BRCA pathogenic mutation model HepG2-GFP.

[0320] Figure 16 shows the body weight change curves of each group of mice in the in vivo efficacy experiment of AST-3424 in the non-BRCA pathogenic mutation model HepG2-GFP.

[0321] Figure 17 shows compound S in the BRCA pathogenic mutation model Growth curve of tumor volume in each group of mice in the in vivo efficacy experiment of GA6201 for gastric cancer.

[0322] Figure 18 shows compound S in the BRCA pathogenic mutation model The body weight percentage change curve of each group of mice during the treatment period in the in vivo efficacy experiment of GA6201 for gastric cancer.

[0323] Figure 19 shows the effect of compound S in non-BRCA pathogenic mutation models Growth curve of tumor volume in each group of mice in the in vivo efficacy experiment of lung cancer LU5173.

[0324] Figure 20 shows compound S in non-BRCA pathogenic mutation models In vivo efficacy experiment of lung cancer LU5173 shows the body weight percentage change curve of each group of mice during treatment.

[0325] Figure 21 shows two FDA-approved HRD detection kits: FoundationFocus CDx BRCA LOH Assay and Myriad Genetics Schematic diagram of HRD testing content and positive judgment criteria. DETAILED DESCRIPTION

[0326] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0327] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used are all commercially available products unless otherwise specified.

[0328] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

[0329] The following provides specific experiments / examples related to the present invention to illustrate the present invention.

[0330] AST-3424:

[0331] AST-3424 similar compound AST (referred to as compound S in some examples):

[0332] 1. Distribution of AKR1C3 in KRAS G12D PDXs and its correlation with NRF2

[0333] A total of 179 models with KRAS-G12D mutations were derived from the publicly available CrownBio tumor PDX model gene database (https: / / www.crownbio.cn / model-systems / in-vivo / pdx-models / ). The RNA expression of AKR1C3 in these models was statistically analyzed. The results are shown in Table 1:

[0334] Table 1: Statistical results of the corresponding relationship between the expression level of AKR1C3 RNA expressed in log2 (FPKM) and KRAS-G12D

[0335] We further analyzed the NRF2 expression in these KRAS-G12D mutation models and projected the RNA expression levels of AKR1C3 and NRF2 in these 179 PDX models onto an XY coordinate graph, yielding Figure 1.

[0336] The distribution trend of AKR1C3 expression in KRAS G12D PDX models was mainly concentrated in medium-high expression (LOG2 (FPKM) ≥ 4), accounting for 90.4%

[0337] In the KRAS G12D PDX model, the distribution trend of NRF2 expression was also concentrated in medium and high expression, which was consistent with the protein expression trend of AKR1C3 and had a certain correlation.

[0338] 2. Distribution of AKR1C3 in KRAS G12C PDXs and its correlation with NRF2

[0339] A total of 51 models with KRAS-G12C mutations were derived from the CrownBio tumor PDX model gene database (https: / / www.crownbio.cn / model-systems / in-vivo / pdx-models / ). The RNA expression of AKR1C3 in these models was statistically analyzed. The results are shown in Table 2:

[0340] Table 2: Statistical results of the corresponding relationship between the expression level of AKR1C3 RNA expressed in log2 (FPKM) and KRAS-G12C

[0341] We further analyzed the NRF2 expression in these KRAS-G12C mutation models and projected the RNA expression levels of AK R1C3 and NRF2 in these 51 PDX models onto an XY coordinate graph, yielding Figure 2.

[0342] The expression of AKR1C3 in KRAS G12C PDX models was evenly distributed among low, medium and high levels, among which the expression level above medium (LOG2 (FPKM) ≥ 4) accounted for 66.7%.

[0343] In the KRAS G12C PDX model, the distribution trend of NRF2 expression was weakly correlated with the protein expression distribution of AKR1C3.

[0344] 3. Distribution of AKR1C3 in KRAS G13D PDXs and its correlation with NRF2

[0345] A total of 41 models with KRAS-G13D mutations were derived from the publicly available CrownBio tumor PDX model gene database (https: / / www.crownbio.cn / model-systems / in-vivo / pdx-models / ). The RNA expression of AKR1C3 in these models was statistically analyzed. The results are shown in Table 3:

[0346] Table 3: Statistical results of the corresponding relationship between the expression level of AKR1C3 RNA expressed in log2 (FPKM) and KRAS-G13D

[0347] We further analyzed the NRF2 expression in these KRAS-G13D mutation models and projected the RNA expression levels of AKR1 C3 and NRF2 in the 48 PDX models onto an XY coordinate graph, yielding Figure 3.

[0348] AKR1C3 expression levels in KRAS G13D PDX models were evenly distributed among low, medium, and high expression levels, with expression levels above medium accounting for 83.3%.

[0349] In the KRAS G13D PDX model, the distribution trend of NRF2 expression was correlated with the protein expression distribution of AKR1C3.

[0350] The above statistics reveal that NRF2 expression is positively correlated with AKR1C3 expression: high NRF2 expression often also means high AKR1C3 expression; cells and tissues with KRAS mutations often have moderate to high AKR1C3 expression. In summary, KRAS mutations (including KRAS-G13D, KRAS G12C, and KRAS G12D) are associated with moderate to high expression of NRF2 and AKR1C3: KRAS mutations (including KRAS-G13D, KRAS G12C, and KRAS G12D) are often accompanied by moderate to high expression of NRF2 and AKR1C3.

[0351] To further illustrate the relationship between KRAS mutation and NRF2, AKR1C3, the inventors conducted relevant mechanism research experiments.

[0352] AKR1C3 expression in KRAS G12D-mutated HPAF II tumor cells is regulated by NRF2

[0353] SFN compound (Sulforaphane, NRF2 activator) solution was added to the HPAFII cell suspension, and the cells were cultured in a 37°C, 5% CO2 incubator overnight. The cell protein lysate was collected and subjected to Western Blot (WB) detection.

[0354] The experiment was divided into 5 groups: 0.5% DMSO, 0.875 μM SFN, 1.75 μM SFN, 3.5 μM SFN, and 7 μM SFN.

[0355] HPAF II cells were treated with different concentrations of the NRF2 activator SFN, and then the expression of NRF2 and AKR1C3 was detected by immunoblotting. The results of immunoblotting and NRF2 and AKR1C3 protein band density analysis under different SFN concentrations are shown in Figure 4.

[0356] The experimental results showed that as the SFN treatment concentration increased, the expression of NRF2 increased significantly, and the expression of AKR1C3 also increased at the same time.

[0357] The experimental results showed that the NRF2 activator SFN upregulated NRF2 expression in a concentration-dependent manner. Under the same experimental conditions, as the SFN concentration increased, the expression of AKR1C3 was also upregulated. This result clearly shows that the expression of AKR1C3 is regulated by NRF2. In addition, combined with the significant correlation between NRF2 and AKR1C3 mRNA in the medium and high expression areas in Examples 1-3, it is shown that in the PDX model of KRAS G12D mutation, AKR1C3 expression is positively correlated with NRF2 expression.

[0358] AKR1C3 enzyme-activating prodrugs AST-3424 and AST have significant anti-tumor effects on PDX and CDX models with high expression of AKR1C3 enzyme (PCT / CN2022 / 120817, Publication No. WO2023 / 046060 Examples 1-9) and are well tolerated.

[0359] Combined with the experimental conclusions in Examples 1-3 that AKR1C3 expression is positively correlated with NRF2 expression, those skilled in the art can reasonably speculate that KRAS gene mutation can upregulate or activate NRF2, thereby promoting high expression of AKR1C3, and further enabling the AKR1C3 enzyme activation prodrug AST-3424 and AST to produce significant anti-tumor effects.

[0360] The compounds of the general formula (1)-(12) of the present application are all anticancer prodrugs activated by the AKR1C3 enzyme. Under the action of the AKR1C3 enzyme, they are cleaved to produce anticancer active drugs such as AST-2660, paclitaxel, SN-38, gemcitabine, and KARS inhibitors. Therefore, those skilled in the art can reasonably speculate that KRAS gene mutations will cause high expression of the AKR1C3 enzyme by upregulating or activating NRF2, thereby activating and metabolizing the compounds of the general formula (1)-(12) to produce anticancer active drugs. In other words, patients who are detected to have KRAS gene mutations can benefit from treatment with the compounds of the general formula (1)-(12), especially from treatment with AST-3424 or AST.

[0361] The following uses the AKR1C3 enzyme-activated prodrug AST as an example to explore its mechanism of action in KRAS mutation, especially KRAS G12D mutation tumor cells.

[0362] 5. AST activates the MEK / ERK signaling pathway

[0363] AST-induced ERK phosphorylation experiment. Three groups of experiments were performed with three groups of variables: AST concentration, AST action time, and AKRAC3 enzyme dependence. Among them, the variable of AKR1C3 enzyme dependence was conditioned by the AKR1C3 enzyme specific inhibitor AST-3021, and the experiment was performed in the presence or absence of the specific AKR1C3 enzyme inhibitor AST-3021. The inhibitor used was AST-3021 (also known as TH-3021, which is compound 36 reported by Flanagan et al. in the literature Bioorganic and Medicinal Chemistry (2014) pages 962-977, and its structural formula is ).

[0364] The experiment was divided into three groups, which were treated with different concentrations of AST (group 1), 1 μM AST for different time periods (group 2), and with or without the addition of AKR1C3 inhibitor AST-3021 (group 3).

[0365] The three groups of specific compounds to be treated are as follows:

[0366] The first group (concentration group): 0.5% DMSO treatment group (24 h), 0.5 μM AST treatment group (24 h), 1 μM AST treatment group (24 h), 10 μM AST treatment group (24 h);

[0367] The second group (time group): 0.5% DMSO treatment group (24 h), 1 μM AST treatment group (2 h), 1 μM AST treatment group (8 h), 1 μM AST treatment group (24 h);

[0368] The third group (AKR1C3-dependent group): 0.5% DMSO-treated group (24 h), 1 μM AST-treated group (24 h), 3 μM AST-3021-treated group (24 h), and 1 μM AST+3 μM AST-3021-treated group (24 h).

[0369] Each compound solution was added to the HPAF II cell suspension, and cultured overnight in a 37°C, 5% CO2 incubator. Cell protein lysates were collected according to the experimental groups and subjected to Western Blot (WB) detection.

[0370] The expression of NRF2 and AKR1C3 was detected by immunoblotting. The immunoblotting images obtained by treating the first group with different concentrations of AST and the results of the p-NRF2 and NRF2 protein band density analysis are shown in Figure 5; the immunoblotting images obtained by treating the second group with 1 μM AST for different time periods and the results of the p-NRF2 and NRF2 protein band density analysis are shown in Figure 6; the immunoblotting images obtained by treating the third group with or without the AKR1C3 inhibitor AST-3021 and the results of the p-NRF2 and NRF2 protein band density analysis are shown in Figure 7.

[0371] The experimental results show that as shown in Figure 5, the expression level of p-NRF2 increases accordingly with the increase of AST treatment concentration. When treated with 10μM AST for 24h, the expression of p-NRF2 increases significantly, while the expression level of NRF2 is not affected by the AST treatment concentration. In Figure 6, the expression level of p-NRF2 remains basically unchanged when treated with 1μM AST for 2h and 8h. When treated for 24h, it increases significantly compared with the DMSO control group. In Figure 7, only the p-NRF2 expression in the 1μM AST treatment group (24h) increases significantly. The p-NRF2 expression levels in the other 0.5% DMSO treatment group (24h), 3μM AST-3021 treatment group (24h) and 1μM AST + 3μM AST-3021 treatment group (24h) remain basically unchanged. In the above three groups of experiments, the expression of total ERK2 in each treatment group did not change.

[0372] The above experiments demonstrated that AST induced ERK2 phosphorylation in a concentration-dependent manner without affecting ERK2 expression levels. Twenty-four hours after treatment with 1 μmol / L AST, ERK2 phosphorylation levels were significantly increased compared to the DMSO control group. As shown in Figure 7, HPAF II cells were pretreated with the AKR1C3-specific inhibitor AST-3021 2 hours before the addition of 1 μmol / L AST. 3 μmol / L AST-3021 strongly inhibited the AST-mediated increase in ERK2 phosphorylation. AST-3021 alone did not affect p-ERK2 levels. Total ERK2 expression remained unchanged under all tested conditions. Actin immunoblotting confirmed that the loading volume of all samples was consistent. AST activates the MEK / ERK signaling pathway in a concentration-, time-, and AKR1C3-dependent manner.

[0373] 6. AST induces apoptosis of KRAS G12D cells through the ERK2 signaling pathway

[0374] This study used three MEK / ERK modulators, including two inhibitors and one activator. The inhibitors were PD98059, a specific inhibitor of MEK1 / 2, and suramin, a broad-spectrum growth factor receptor inhibitor; while the activator of the ERK signaling pathway was Phorbol Ester (TPA). The chemical structures of the inhibitor PD98059, the inhibitor suramin, and the activator TPA are shown below:

[0375] The experiment was divided into inhibitor group and activator group, and both groups were subjected to immunoblotting and cell apoptosis experiments.

[0376] In the immunoblotting experiment, HPAFII cell suspensions were treated with each group of compounds, cultured overnight in a 37°C, 5% CO2 incubator, and the cell protein lysates were collected for immunoblotting (Western Blot, WB) detection.

[0377] The specific compounds to be treated in the immunoblotting inhibitor group and activator group are as follows:

[0378] Inhibitor groups: 1% DMSO treatment group, 1 μM AST treatment group, 30 μM PD98059 treatment group, 1 mM Suramin treatment group, 1 μM AST + 30 μM PD98059 treatment group, 1 μM AST + 1 mM Suramin treatment group;

[0379] Activator groups: 1% DMSO treatment group, 0.5 μM AST treatment group, 25 nM TPA treatment group, and 0.5 μM AST+25 nM TPA treatment group.

[0380] In addition, cell apoptosis detection was performed:

[0381] 1) Prepare Caspase Reagent, let the reagent balance to room temperature. Mix the reagent buffer and substrate 1:1 to make Caspase The prepared reagents can be stored at 4°C for 3 days.

[0382] 2) Add 100 μL Caspase Add the reagent to each well containing 100 μL of blank (cell-free well), negative control cells, or treated cells. Due to the sensitivity of the assay, be careful not to touch the pipette tip to the well containing the sample to avoid cross-contamination. Cover the plate with a plate sealer or lid.

[0383] 3) Gently mix the reagent solution using a shaker at 300-500 rpm for 30 seconds. Incubate at room temperature in the dark for 1 hour.

[0384] 4) Measure the luminescence of each sample in a plate-reading luminometer. Calculate the luminescence values ​​for each group and analyze them in a bar graph.

[0385] The specific compounds to be treated in the apoptosis inhibitor group and activator group are as follows:

[0386] Inhibitor groups: 1% DMSO treatment group, 1 μM AST treatment group, 30 μM PD98059 treatment group, 1 mM Suramin treatment group, 1 μM AST + 30 μM PD98059 treatment group, 1 μM AST + 1 mM Suramin treatment group;

[0387] Activator groups: 1% DMSO treatment group, 1 μM AST treatment group, 50 nM TPA treatment group, and 1 μM AST+50 nM TPA treatment group.

[0388] The experimental results of immunoblotting, protein band density analysis and cell apoptosis of the ERK2 inhibitor and AST single-drug and combination groups are shown in Figure 8, and the experimental results of immunoblotting, protein band density analysis and cell apoptosis of the ERK2 activator and AST single-drug and combination groups are shown in Figure 9.

[0389] The experimental results showed that in the inhibitor group of Figure 8, the upper figure and the lower left figure showed that PD98059 and suramin alone slightly reduced the expression level of p-ERK2, while the combination of PD98059 and AST significantly reduced the expression level of p-ERK2. The expression level of p-ERK2 in the combination of suramin and AST was slightly increased, but the expression level of p-ERK2 in the AST monotherapy group was significantly increased; the lower right figure showed that the AST monotherapy group significantly affected cell apoptosis, while the PD98059 and suramin monotherapy groups and their combination with AST had almost no effect on cell apoptosis. In the activator group of Figure 9, the upper figure and the lower left figure show that the p-ERK2 expression levels of the AST and TPA single-drug treatment groups were increased compared with the DMSO control group, while the p-ERK2 expression level of the AST and TPA combination group was significantly increased; the lower right figure shows that the AST single-drug group promoted cell apoptosis, while the combination of AST and TPA made the effect on cell apoptosis more significant.

[0390] Experimental results showed that treatment with two ERK inhibitors, PD98059 and suramin, slightly reduced endogenous ERK phosphorylation, while treatment with AST alone significantly increased intracellular p-ERK2 expression. Furthermore, combined treatment with AST and the two ERK inhibitors almost completely abolished the AST-induced increase in p-ERK2 expression. A caspase 3 / 7 assay was used to assess the effects of AST and ERK2 inhibitors, both alone and in combination, on cell apoptosis. The results showed that ERK2 inhibitors significantly inhibited AST-induced apoptosis. In contrast, treatment with the ERK2 activator TPA alone also increased intracellular p-ERK2 expression, while treatment with TPA combined with AST further enhanced AST-induced ERK2 phosphorylation and apoptosis. Treatment with either of these drugs alone or in combination did not affect total ERK2 expression. These results clearly demonstrate that AST induces apoptosis in KRAS G12D mutant cells through the ERK2 signaling pathway.

[0391] Based on Examples 4, 5, and 6, the mechanism shown in Figure 10 can be inferred, namely, that AST induces apoptosis of KRAS G12D pathogenic mutation cancer cells via the ERK2 signaling pathway. Further combined with research in relevant literature: the MEK / ERK signaling pathway can be activated by DNA alkylating agents (Wang X, Martindale JL and Holbrook NJ. Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem 2000; 275: 39435-39443); and Examples 11 and 12 confirm that AST, a DNA alkylating agent prodrug, has the effect of activating the MEK / ERK signaling pathway, and can induce apoptosis of KRAS G12D mutant cancer cells via the ERK2 signaling pathway. AST compound is a DNA alkylating agent prodrug activated by the AKR1C3 enzyme, which is activated by the AKR1C3 enzyme to produce AST-2660, thereby exerting an anti-cancer effect.

[0392] Considering the regulatory pathways associated with AKR1C3 and ERK2 as shown in Figure 10, the inventors speculate that AST compounds can activate ERK2, causing its phosphorylation, which in turn activates the MEK-ERK pathway, leading to upregulation of Nrf2 and ultimately high expression of AKR1C3, thereby causing cancer cell apoptosis. In other words, the KRAS-G12D mutation can more effectively activate AST drugs by upregulating the RAF / MEK / ERK signaling pathway, upregulating Nrf2 protein expression, and thus upregulating AKR1C3 protein expression.

[0393] In fact, studies have shown that KRAS gene mutations can upregulate Nrf2 protein expression and thus upregulate AKR1C3 protein expression. These genes include various subtypes of KRAS mutations: KRAS-G12D mutation, KRAS-G12V mutation, KRAS-G12C mutation, KRAS-G12A and KRAS-G13D.

[0394] The mechanism of AST compounds revealed in Figure 10 is further extended to other similar AKR1C3 enzyme-activating prodrugs: General formula (1)-(12), KRAS mutations can more effectively activate AKR1C3 enzyme-activating prodrugs by upregulating the RAF / MEK / ERK signaling pathway, upregulating the protein expression of Nrf2, and then upregulating the protein expression of AKR1C3, thereby inducing cell apoptosis or inhibiting cell proliferation.

[0395] Furthermore, we found in our experiments that the DNA alkylating agents AST-3424 and AST activated by AKR1C3 enzyme have better therapeutic effects on BRCA mutation tumor models.

[0396] According to two approved HRD detection kits: FoundationFocus CDx BRCA LOH Assay and Myriad Genetics For HRD, BRCA gene mutation cell lines are selected for related experiments. In some embodiments, cell lines or models with BRCA pathogenic mutations are used for experiments. According to the judgment rules of the above-mentioned kit, BRCA mutation-positive can be determined as HRD-positive.

[0397] 7. Comparison of in vitro cytotoxicity of compound AST-3424 in tumor cells with non-pathogenic and pathogenic BRCA mutations

[0398] In vitro cytotoxicity (IC 50 The sensitivity of the compound AST-3424 to BRCA non-pathogenic mutations and pathogenic mutations was compared by using a PCR assay (values), thereby detecting whether BRCA pathogenic mutations enhance the sensitivity of tumor cells to AST-3424 at the in vitro cell level. The following cell lines were used as research subjects in this experiment: the BRCA pathogenic mutation cell line Capan-1 (human pancreatic cancer cell line, BRCA2 pathogenic mutation site is p.S1982RfsTer22), the BRCA non-pathogenic mutation cell line PLC / PRF / 5 (human liver cancer cell line), and the HT29 (human colon cancer cell line).

[0399] The activation mechanism of the prodrug AST-3424 and the AST-3424-like compound S (i.e., the aforementioned compound AST) is through the action of the AKR1C3 enzyme, which releases a DNA alkylating agent, thereby inhibiting and killing tumor cells. Therefore, in the presence or absence of pathogenic gene mutations, the higher the expression level of the AKR1C3 enzyme, the stronger the cytotoxicity of the compound to tumor cells is expected to be. 50 The lower the value, the more sensitive the cell lines are to AST-3424 and the AST-3424-like compound S. We examined the effects of BRCA pathogenic mutations on the sensitivity of cells to AST-3424 and the AST-3424-like compound S, assuming that the AKR1C3 protein expression levels of the cell lines were similar or that the AKR1C3 protein expression levels of the cell lines with BRCA pathogenic mutations were lower than those of cell lines without BRCA pathogenic mutations. We used immunoblotting to identify and quantify the AKR1C3 protein expression levels of the three cell lines mentioned above. We also used the human lung cancer cell line H460, which highly expresses AKR1C3 protein, as a control to compare the AKR1C3 protein expression levels of the three cell lines. Western blot analysis showed that the AKR1C3 protein expression levels of the Capan-1 and HT29 cell lines were similar, while the AKR1C3 protein expression level of the PLC / PRF / 5 cell line was slightly higher (Table 4, Figure 11).

[0400] Table 4: AKR1C3 protein expression levels in various cells compared to the H460 cell line

[0401] Cell proliferation assay was used to detect the sensitivity of various cells to AST-3424, i.e., IC 50 Value, unless otherwise specified, IC 50 The values ​​were tested by CTG test method.

[0402] The IC values ​​of the test compound AST-3424 in different cells were determined by the above experimental method. 50 The values ​​are listed in Table 5 below.

[0403] Table 5: IC inhibition of compound AST-3424 on three tumor cell lines 50 data

[0404] The test results showed that compared with the BRCA pathogenic mutation cell line Capan-1, the IC of AST-3424 in the BRCA non-pathogenic mutation cell line HT29 was significantly higher than that in the BRCA non-pathogenic mutation cell line Capan-1. 50 The IC of AST-3424 in Capan-1 cell line and HT29 cell line with similar AKR1C3 protein expression is 67 times that of Capan-1 cell line. 50There is a significant difference in the values. This indicates that under conditions of similar AKR1C3 protein expression, tumor cells with BRCA pathogenic mutations are more sensitive to AST-3424. Even though the AKR1C3 protein expression level of the BRCA pathogenic mutation cell line Capan-1 is lower than that of the BRCA non-pathogenic mutation cell line PLC / PRF / 5, the IC value of AST-3424 in the BRCA non-pathogenic mutation cell line PLC / PRF / 5 is still higher than that in the BRCA non-pathogenic mutation cell line PLC / PRF / 5. 50 It is still 22 times that of the Capan-1 cell line. Therefore, tumor cells with BRCA pathogenic mutations are more sensitive to AST-3424, that is, HRD-positive tumor cells are more sensitive to AST-3424.

[0405] 8. Comparison of in vitro cytotoxicity of AST-3424 analog compound S in BRCA non-pathogenic mutation and pathogenic mutation tumor cells

[0406] Using the same experimental methods and cell lines described above, we further tested whether compound S exhibited significant differences in activity in tumor cells harboring non-pathogenic BRCA mutations and those harboring pathogenic BRCA mutations, thereby examining whether pathogenic BRCA mutations enhance the sensitivity of tumor cells to compound S. The in vitro cytotoxicity results of compound S against the three cell lines described above are shown in Table 6.

[0407] Table 6: IC inhibitory effect of compound S on three tumor cell lines 50 data

[0408] The test results showed that compared with the BRCA pathogenic mutation cell line Capan-1, AST-3424 had a similar IC value to compound S in the BRCA non-pathogenic mutation cell line HT29. 50 The IC of AST-3424 is 12 times that of Capan-1 cell line. Therefore, the IC of AST-3424 is similar to compound S in Capan-1 cell line with similar AKR1C3 protein expression and HT29 cell line with non-pathogenic BRCA mutation. 50 Even though the AKR1C3 protein expression level in the BRCA pathogenic mutation cell line Capan-1 is lower than that in the BRCA non-pathogenic mutation cell line PLC / PRF / 5, the IC values ​​of AST-3424 are similar to those of compound S in the BRCA non-pathogenic mutation cell line PLC / PRF / 5. 50 It is still 5 times that of the Capan-1 cell line. Therefore, tumor cells in the BRCA pathogenic mutation model are more sensitive to AST-3424-like compound S.

[0409] The results of both experiments demonstrated that, under conditions where AKR1C3 protein expression levels were similar, AST-3424 and AST-3424-like compound S demonstrated a more pronounced in vitro cytotoxicity against tumors harboring BRCA pathogenic mutations. Even when AKR1C3 protein expression levels in cell lines harboring BRCA pathogenic mutations were lower than those in cell lines without BRCA pathogenic mutations, AST-3424 and AST-3424-like compound S demonstrated a more pronounced in vitro cytotoxicity against tumors harboring BRCA pathogenic mutations.

[0410] 9. Comparison of in vivo efficacy of compound AST-3424 in BRCA non-pathogenic mutation and pathogenic mutation tumor models

[0411] To verify whether AST-3424 has different in vivo efficacy in tumor cells with and without BRCA pathogenic mutations, we selected two CDX models: the human pancreatic cancer Capan-1 (a BRCA2 pathogenic mutation model with the mutation site p.S1982RfsTer22) and the BRCA non-pathogenic mutation model HepG2 (a human liver cancer model). AKR1C3 mRNA expression levels in Capan-1 and HepG2 cell lines were LOG2 (FPKM) = 6.6363 and LOG2 (FPKM) = 6.8572, respectively, indicating similar AKR1C3 mRNA expression levels in the two models. A comparison of AKR1C3 protein expression levels with those in the H460 cell line is shown in Table 7 and Figure 12. Protein expression results showed that the AKR1C3 protein expression level in Capan-1 cells with BRCA pathogenic mutations was lower than that in HepG2 cells without BRCA pathogenic mutations. On this basis, we compared the effect of BRCA pathogenic mutations on the in vivo efficacy of AST-3424.

[0412] Table 7: AKR1C3 protein expression levels of various cells compared with H460 cell line

[0413] 10. In vivo efficacy study of AST-3424 in the Capan-1CDX model with BRCA pathogenic mutations

[0414] BALB / c nude mice were subcutaneously inoculated with human pancreatic cancer Capan-1 cells to establish a subcutaneous transplantation model of human pancreatic cancer. The experiment was divided into a test drug Olaparib 100 mg / kg single-drug group, an AST-3424 1.5 mg / kg single-drug group, and a 10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.4) vehicle control group, with 6 mice in each group. The test drug Olaparib single-drug group was orally administered by gavage once a day for a total of 30 days. The vehicle control group and the AST-3424 single-drug group were both administered via the tail vein, of which the vehicle control group was administered once a week for three weeks; the AST-3424 single-drug group was administered once a week for two weeks. The specific dosing schedule and TGI are shown in Table 8. The test drug Olaparib 100mg / kg treatment group showed a mild tumor inhibition effect on day 43 after tumor cell inoculation, with a relative tumor inhibition rate (TGI) of 37.43%, which was not statistically significant compared to the control group (p>0.05). The test drug AST-3424 1.5mg / kg treatment group showed a very significant tumor inhibition effect on day 43 after tumor cell inoculation, with a relative tumor inhibition rate (TGI) of 97.97%. Four mice had complete tumor elimination, with a complete tumor inhibition rate of 66.7%, which was statistically significant compared to the control group (p<0.001).

[0415] Table 8: Efficacy analysis of each group in the human pancreatic cancer Capan-1 subcutaneous model

[0416] The corresponding curves of tumor volume and percentage of mouse body weight changing over time are shown in Figures 13 and 14.

[0417] Experimental results demonstrated that in the Capan-1 CDX model harboring a BRCA pathogenic mutation, AST-3424 (5mg / kg) alone demonstrated significant tumor suppression, achieving a TGI of 97.97% at the endpoint, with complete tumor eradication in four mice. The body weight percentage data from the aforementioned animal studies indicated that animals receiving AST-3424 experienced no weight loss, demonstrating that the drug was well tolerated.

[0418] 11. In vivo efficacy study of AST-3424 in the non-BRCA pathogenic mutation model HepG2-GFP

[0419] Human hepatocellular carcinoma HepG2-GFP cells were inoculated in situ into the liver tissue of BALB / c nude mice to establish an orthotopic transplantation model of human hepatocellular carcinoma, and the fluorescence area of ​​green fluorescent protein GFP was used to define the size of the tumor. The experiment was divided into a test drug sorafenib monotherapy group, an AST-3424 1.25 mg / kg monotherapy group, and a normal saline injection solvent control group, with 10 mice in each group. The test drug sorafenib monotherapy group was administered orally by gavage, once a day, for a total of 35 times. Both the solvent control group and the AST-3424 monotherapy group were administered via tail vein, and were administered once a week for two weeks. After a one-week pause, they continued to be administered once a week for two consecutive weeks. The specific dosing regimen and TGI are shown in Table 9.

[0420] Table 9: Efficacy analysis of each group in the human liver cancer HepG2-GFP orthotopic tumor model

[0421] Note: TGI (calculated based on tumor weight) was calculated using the following formula: TGI% = (1-treatment (T) / control (C)) × 100% (T and C are the tumor weights of the treatment and control groups at the end of the experiment, respectively).

[0422] The corresponding curves of tumor volume and mouse body weight changes over time are shown in Figures 15 and 16.

[0423] The experimental results showed that in the BRCA non-pathogenic mutation model, the tumor fluorescence area reading in the AST-3424 1.25mg / kg group was significantly smaller than that in the vehicle control group, but there was no statistically significant difference (p>0.05). The TGI was 60%, indicating that while AST-3424 1.25mg / kg had a growth inhibitory trend in the orthotopic human liver cancer HepG2-GFP model, it was not statistically significant, and no mouse had complete tumor clearance. The body weight data from the above animal experiments showed that the experimental animals in the AST-3424 treatment group did not lose weight, demonstrating that the drug was well tolerated.

[0424] Experimental Summary

[0425] From the above-described experimental Examples 9-11, we found significant differences in the in vivo efficacy of AST-3424. AST-3424 at 1.5 mg / kg (administered weekly for two weeks) demonstrated significant in vivo efficacy in the Capan-1 CDX model harboring a BRCA pathogenic mutation, achieving a TGI of 97.97%. In the AST-3424-treated group, four of six mice experienced complete tumor eradication, a clearance rate of 67%. However, AST-3424 at 1.25 mg / kg (administered weekly for two weeks, followed by a one-week pause, and then resumed weekly dosing for two consecutive weeks), despite a total dose exceeding that of the Capan-1 model, showed no significant efficacy in the HepG2-GFP model, a model without a BRCA pathogenic mutation. The TGI was 60%, not significantly different from the control group, and no mice in the treated group experienced complete tumor eradication. These results suggest that although AKR1C3 protein expression levels in the Capan-1 model are lower than in the HepG2-GFP model, the presence of pathogenic BRCA mutations in the Capan-1 model and the absence of these mutations in the HepG2-GFP model lead to significant differences in the in vivo efficacy of AST-3424. BRCA pathogenic mutations enhance the sensitivity of the Capan-1 model to AST-3424, promoting its ability to kill tumor tissue.

[0426] 12. Comparison of in vivo efficacy of compound S in BRCA non-pathogenic mutation and pathogenic mutation tumor models

[0427] To verify the in vivo efficacy differences of AST-3424-like compound S in BRCA pathogenic mutation and non-pathogenic mutation models, we selected two PDX models, namely the human gastric cancer PDX model GA6201 (BRCA2 pathogenic mutation model, mutation site L1732PfsTer9) and the BRCA non-pathogenic mutation PDX model LU5173 (human lung cancer PDX model). According to the results of existing methods, the mRNA expression levels of AKR1C3 in GA6201 and LU5173 tumor tissues were log2 (FPKM) = 6.78 and log2 (FPKM) = 8.88, respectively, indicating that the RNA expression levels of AKR1C3 in the two models were similar. The AKR1C3 protein expression levels of the two models were detected by immunohistochemistry (IHC), and the results are shown in Table 10. Immunohistochemistry and databases suggest that the AKR1C3 protein expression levels of the two PDX models are similar. This part of the study used two models with similar AKR1C3 protein expression levels to compare the effects of BRCA pathogenic mutations on the in vivo efficacy of AST-3424-like compound S.

[0428] Table 10: Comparison of AKR1C3 protein and RNA expression in two PDX subcutaneous models

[0429] Notes:

[0430] The scoring standard for AKR1C3 protein expression IHC staining was based on the literature (Guise et al., Cancer Res, 2010(70):1573), where weak corresponds to Focal in the literature, medium corresponds to Moderate in the literature, and strong corresponds to Diffuse in the literature.

[0431] 13. Compound S in BRCA pathogenic mutation model In vivo efficacy study of GA6201 in gastric cancer

[0432] BALB / c nude mice were subcutaneously inoculated Model GA6201 tumor mass, establish human gastric cancer subcutaneous transplantation tumor model test is divided into test drug Ifosfamide 60mg / kg group, test drug compound S2.5mg / kg group and normal saline (pH 7.0-7.6) solvent control group, 5 mice per group. Among them, the normal saline (pH 7.0-7.6) solvent control group and the test drug compound S2.5mg / kg group are administered by tail vein injection, once a week, for a total of three weeks, and observed for four weeks. The Ifosfamide 60mg / kg group is administered by intraperitoneal injection, five consecutive days a week and two days off, for a total of two weeks, and observed for five weeks. The specific dosing regimen and TGI are shown in Table 11.

[0433] Table 11: Table 2: Drug efficacy analysis of each group in the gastric cancer GA6201 model

[0434] The corresponding curves of tumor volume and percentage of mouse body weight changing with time are shown in Figures 17 and 18.

[0435] The experimental results showed that in patients with BRCA pathogenic mutations In the gastric cancer GA6201 model, the test drug Ifosfamide (60 mg / kg) treatment group showed a certain tumor inhibition effect on the third day (Day 38) after all dosing cycles, with no statistically significant difference compared to the control group (p = 0.857), and the relative tumor inhibition rate (TGI) (%) was 58.83%. The test drug compound S (2.5 mg / kg) treatment group showed a significant tumor inhibition effect on the third day (Day 38) after all dosing cycles, with a statistically significant difference compared to the control group (p = 0.000135), and the relative tumor inhibition rate (TGI) (%) was 96.85%. The mice in the test drug compound S (2.5 mg / kg) treatment group did not lose weight, showed no obvious drug toxicity, and were well tolerated during treatment.

[0436] 14. Compound S in non-BRCA pathogenic mutation models In vivo efficacy experiment of lung cancer LU5173

[0437] NOD / SCID female mice were inoculated subcutaneously Model LU5173 tumor mass, establish a human lung cancer subcutaneous transplantation tumor model. The experiment is divided into a positive control drug Ifosfamide 60mg / kg group, a test drug compound S 4mg / kg group and a 7.5% anhydrous ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W solvent control group, etc., with 6 mice in each group. Among them, the 7.5% anhydrous ethanol + 7.5% polyoxyethylene (35) castor oil + 85% glucose injection D5W solvent control group and the test drug compound S group are administered by tail vein injection once a week for three consecutive weeks. The positive control drug Ifosfamide 60mg / kg group is administered by intraperitoneal injection once a day for 5 consecutive days, then stopped for two days, and then administered for two consecutive weeks. The specific dosing regimen and TGI are shown in Table 12.

[0438] Table 12: Table 2: Drug efficacy analysis of each group in the lung cancer LU5173 model

[0439] The corresponding curves of tumor volume and percentage of mouse body weight changing over time are shown in Figures 19 and 20.

[0440] The experimental results showed that in non-BRCA pathogenic mutations In the LU5173 lung cancer model, the test drug Ifosfamide (60 mg / kg) treatment group demonstrated a modest tumor inhibition effect on day 32 after the first dose, with a relative tumor inhibition rate (TGI) of 44.45%, which was not statistically significantly different from the control group (p>0.05). The test drug compound S (4 mg / kg) treatment group also demonstrated a significant tumor inhibition effect on day 32 after the first dose, with a relative tumor inhibition rate (TGI) of 52.58%, which was statistically significantly different from the control group (p<0.05). Mice treated with the test drug compound S (4 mg / kg) did not experience any significant weight loss and tolerated the treatment well.

[0441] Experimental Summary

[0442] From the above experimental examples 12-14, we found that the in vivo efficacy of compound S was significantly different in two models with similar AKR1C3 expression levels. In the gastric cancer GA6201 model, the test compound S 2.5 mg / kg (administered once a week for three weeks) treatment group showed significant tumor inhibition, with statistically significant differences compared to the control group (p = 0.000135), and the relative tumor inhibition rate (TGI) was 96.85%. In the lung cancer LU5173 model, the higher dose of test drug compound S 4mg / kg treatment group had a significant tumor inhibition effect on the 32nd day after the first administration, with a relative tumor inhibition rate TGI (%) of 52.58%, but the inhibition rate was significantly lower than Gastric cancer GA6201 model. The above results show that in the non-BRCA pathogenic mutation model, even at a higher dose of 4 mg / kg, the tumor inhibition effect of compound S is lower than that of the low-dose 2.5 mg / kg group in the BRCA pathogenic model. This also proves that the pathogenic mutation of BRCA enhances The sensitivity of the gastric cancer GA6201 model to compound S enhances the killing effect of compound S on tumor tissue.

[0443] 15. Clinical testing of KRAS mutations and AKR1C3 enzyme protein expression levels in China

[0444] In a clinical trial approved by Chinese regulatory authorities (CTR20220934), KRAS mutations and AKR1C3 enzyme protein expression levels were clinically tested in Chinese clinical trial patients enrolled.

[0445] The trial was approved by the ethics committees of the participating medical institutions and was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects.

[0446] The AKR1C3 enzyme protein expression level detection and results were performed according to the method described in WO2022048492 and scored using the H-score system (H-score) to characterize the expression level, and an H-score of 135 or above was defined as high expression.

[0447] Among the 10 KRAS-positive patients, 7 had high expression of AKR1C3. The specific test results are shown in Table 13.

[0448] Table 13: Clinical detection data of KRAS mutation and AKR1C3 enzyme protein expression level

[0449] The toxin structures of AST-3424 and compound S are similar, and both are AKR1C3 activated anticancer prodrugs. After being activated by the AKR1C3 enzyme, they cleave to produce DNA alkylating agents with similar toxin structures. Therefore, combined with the experimental results of AST-3424 and compound S, it can be inferred that the DNA alkylating agent prodrugs with similar structures to the AKR1C3 activated toxins of formula (1), (3)-(8), and (12) have more significant therapeutic effects on the treatment of cancer and tumor patients with homologous recombination repair deficiency HRD. Therefore, it is feasible to further use homologous recombination repair deficiency HRD as a positive to further select patients with high AKR1C3 expression (accompanied by KRAS gene mutation) for treatment.

[0450] Furthermore, the above-mentioned AKR1C3-activated DNA alkylating agent toxins are all alkylating agents that act on DNA double strands to cause cross-linking. The above-mentioned compounds should have better therapeutic effects on tumor models with mutations in homologous DNA double-strand damage repair genes (HRRm) than on tumor models without mutations. That is, DNA alkylating agent prodrugs with similar structures to AKR1C3-activated toxins have more significant therapeutic effects on cancer and tumor patients with mutations in homologous DNA double-strand damage repair genes, either as a single drug or in combination with other therapeutic drugs.

Claims

1. A method of administering an AKR1C3 enzyme-activating prodrug to treat cancer, a tumor, or a disease caused by a cancer or a tumor, or a cell proliferative disease, wherein, when the patient's tumor or cancer tissue is detected to have a KRAS gene mutation or the patient is detected to have a KRAS gene mutation, the AKR1C3 protein expression level or AKR1C3 RNA content of the patient's tumor or cancer tissue is not detected, and the AKR1C3 enzyme-activating prodrug is administered.

2. A method for treating cancer, tumor, or a disease caused by cancer or tumor, or a cell proliferative disease, comprising the step of applying a drug or preparation containing an AKR1C3 enzyme-activating prodrug; and a step of detecting KRAS gene mutation, but does not comprise the step of detecting the AKR1C3 protein expression level or AKR1C3 RNA content in the patient's tumor or cancer tissue, When the patient's tumor or cancer tissue is detected to have a KRAS gene mutation or the patient is detected to have a KRAS gene mutation, a drug or preparation containing an AKR1C3 enzyme-activating prodrug is administered to the patient.

3. The pharmaceutical use of AKR1C3 enzyme activated prodrug for preparing a drug for treating cancer, tumor or a disease caused by cancer, tumor or a cell proliferative disease, characterized in that The drug is formulated to be administered to patients whose tumors or cancer tissues are detected to have KRAS gene mutations or who are detected to have KRAS gene mutations, and the patient does not need to undergo detection of AKR1C3 protein expression levels or AKR1C3 RNA content in the tumor or cancer tissues.

4. According to the method or use of claims 1, 2, and 3, the KRAS gene mutation is selected from the group consisting of KRAS-G12D mutation, KRAS-G12V mutation, KRAS-G12C mutation, KRAS-G12A and KRAS-G13D.

5. According to the method or use of claim 1, 2, or 3, the AKR1C3 enzyme activating prodrug is selected from the group consisting of compounds of formula (1)-(12) or pharmaceutically acceptable salts, solvates, or isotopic isomers thereof: in, X, Y, Z, R, T, A, and X 10 The definition is as set forth in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A); Among them, X, Y, Z, R, D, L 1 , A and X 10 The definition of is as set forth in the claims of patent application PCT / US2016 / 025665, publication number WO2016 / 161342A1 (corresponding to Chinese application number 2016800200132, publication number CN108136214A); Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A1 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); in: A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 S02R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group; Among them, R 100 , R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 Together with the nitrogen atom to which it is attached, it forms a 5-7 membered heterocyclic ring; wherein the alkyl and aryl groups are each substituted with 1-3 halogen groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently is phenyl or methyl; X, Y and Z are each independently hydrogen or halo; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl; Wherein, the definition of Rw is as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); Wherein, the definitions of R1, R2, R3, R4, and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1; Wherein, the definitions of A, E, G, X, and Y are as described in the claims of patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A); or a pharmaceutically acceptable salt thereof, Among them, R w ,X,R4,R 10 , R 13 , R 14 The definition is as set forth in the claims of patent application PCT / CN2022 / 098082, publication number WO2022258043A1; Wherein, the definitions of R1, R2, R3, R4, G1, G2, G3, G4, E, T, Y, Z, m, n, s, t, v, w, and ring A are as described in the claims of patent application CN202210585771.6, publication number CN115403579A; or a pharmaceutically acceptable salt thereof, Among them, R 1 , R 2a , R 2b , R 3 , R 4 , R 5 , n, and Z are defined as described in the claims of patent application PCT / IB2020 / 057285, publication number WO2021005586A1 (corresponding to Chinese application number CN202080053804.1, publication number CN114206870A); or a pharmaceutically acceptable salt thereof, Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b The definitions of n1, n2 are as recorded in the claims of patent application PCT / CN2023 / 123253, publication number WO2024078392A1.

6. The method or use according to claim 5, when the AKR1C3 enzyme activating prodrug is selected from compound formula (1), (3)-(8), (12) or a pharmaceutically acceptable salt, solvate, or isotope isomer thereof, The patient's tumor or cancer tissue is detected to have homologous recombination repair deficiency (HRD) or the patient is detected to have homologous recombination repair deficiency (HRD).

7. According to the method or use of claim 6, the homologous recombination repair deficiency must meet any one or both of the following: One or more mutations in genes involved in the homologous recombination repair (HRR) process; The genomic scar (GS) score reaches the predetermined value.

8. The method or use according to claim 7, wherein the gene related to the homologous recombination repair process is selected from ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FAAP20, FAN1, FANCE, FANCL, FANCM, MRE11A, NBN, PALB2, POLQ, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L, RBBP8, SLX4, XRCC2, The genomic scars are selected from genomic loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large-scale state transition (LST).

9. A preparation unit package, comprising an independent packaging container containing a pharmaceutical preparation, an outer packaging component for accommodating the independent packaging container, and a drug instruction sheet, characterized in that: The pharmaceutical preparation contains an AKR1C3 enzyme-activated prodrug, and the drug instructions state: The drug is used to treat patients with cancer, tumors, or conditions caused by cancer, tumors, or cell proliferative diseases; The patient undergoes KRAS gene mutation testing without the need for testing the AKR1C3 protein expression level or AKR1C3 RNA content in the tumor or cancer tissue; When the patient's tumor or cancer tissue is detected to have a KRAS gene mutation or the patient is detected to have KRAS gene mutation, that is, administering a drug or preparation containing an AKR1C3 enzyme activating prodrug to the patient.

10. The preparation unit package according to claim 9, The KRAS gene mutation is selected from KRAS-G12D mutation, KRAS-G12V mutation, KRAS-G12C mutation, KRAS-G12A and KRAS-G13D; The AKR1C3 enzyme activation prodrug is selected from the compound formula (1)-(12) or a pharmaceutically acceptable salt, solvate, or isotope isomer thereof: in, X, Y, Z, R, T, A, and X 10 The definition is as set forth in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A); Among them, X, Y, Z, R, D, L 1 , A and X 10 The definition of is as set forth in the claims of patent application PCT / US2016 / 025665, publication number WO2016 / 161342A1 (corresponding to Chinese application number 2016800200132, publication number CN108136214A); Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A1 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); in, A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 S02R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group; Among them, R 100 , R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 Together with the nitrogen atom to which it is attached, it forms a 5-7 membered heterocyclic ring; wherein the alkyl and aryl groups are each substituted with 1-3 halogen groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently is phenyl or methyl; X, Y and Z are each independently hydrogen or halo; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl; Wherein, the definition of Rw is as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); Wherein, the definitions of R1, R2, R3, R4, and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1; Wherein, the definitions of A, E, G, X, and Y are as described in the claims of patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A); or a pharmaceutically acceptable salt thereof, Among them, R w ,X,R4,R 10 , R 13 , R 14 The definition is as set forth in the claims of patent application PCT / CN2022 / 098082, publication number WO2022258043A1 The AKR1C3 enzyme activation prodrug is selected from the compound formula (10)-(11) or a pharmaceutically acceptable salt, solvate, or isotope isomer thereof: Wherein, the definitions of R1, R2, R3, R4, G1, G2, G3, G4, E, T, Y, Z, m, n, s, t, v, w, and ring A are as described in the claims of patent application CN202210585771.6, publication number CN115403579A; or a pharmaceutically acceptable salt thereof, Among them, R 1 , R 2a , R 2b , R 3 , R 4 , R 5 , n, and Z are defined as described in the claims of patent application PCT / IB2020 / 057285, publication number WO2021005586A1 (corresponding to Chinese application number CN202080053804.1, publication number CN114206870A); or a pharmaceutically acceptable salt thereof, Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b The definitions of n1, n2 are as recorded in the claims of patent application PCT / CN2023 / 123253, publication number WO2024078392A1.

11. The preparation unit package according to claim 10, When the AKR1C3 enzyme activating prodrug is selected from the group consisting of compounds of formula (1)-(9), (12) or pharmaceutically acceptable salts, solvates, isotope isomers thereof, the patient's tumor or cancer tissue is detected to have homologous recombination repair deficiency (HRD) or the patient is detected to have homologous recombination repair deficiency (HRD), Preferably, Homologous recombination repair deficiency must meet any one or both of the following: one or more mutations in genes related to the homologous recombination repair (HRR) process or the genomic scar (GS) score reaches a predetermined value; Preferably, Genes related to homologous recombination repair process are selected from ATM, ATR, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FAAP20, FAN1, FANCE, FANCL, FANCM, MRE11A, NBN, PALB2, POLQ, PPP2R2A, RAD51B, RAD51C, RAD51D, RAD54L, RBBP8, SLX4, and XRCC2; The genomic scars are selected from genomic loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large-scale state transition (LST).

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