Methods of treating cancer with alkylating agent prodrugs in combination with cell cycle inhibitors

Abstract

A method of treating cancer using an alkylating agent prodrug in combination with a cell cycle inhibitor, the alkylating agent prodrug may be an AKR1C3 enzyme activated alkylating agent prodrug or a hypoxic activated alkylating agent prodrug, and the cell cycle inhibitor may be a CDK inhibitor, a WEE inhibitor, a CHK inhibitor or an ATR inhibitor.

Description

Method for treating cancer by combining alkylating agent prodrug with cell cycle inhibitor Technical Field

[0001] The present invention relates to a method for treating cancer, in particular to a method for treating cancer by combining two drugs, and belongs to the field of cancer treatment. Background Art

[0002] Our company has developed a DNA alkylating cancer treatment drug AST-3424 that targets overexpressed aldehyde-keto reductase 1C3 (AKR1C3) (see patent application: DNA alkylating agent, corresponding to PCT application number PCT / US2016 / 021581, publication number WO2016 / 145092, corresponding to Chinese application number 2016800150788, publication number CN107530556A, and compound TH2870; (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate). , compositions, uses thereof, and preparation methods thereof, corresponding to PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, corresponding to Chinese application number 2016800446081, publication number CN108290911A (S-configured compound), Chinese name is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)phosphoramidate, also known as OBI-3424, S-configured compound of TH-2870), CAS number 2097713-69-2, and its structure is as follows:

[0003] Chemical structure of AST-3424

[0004] AST-3424 (also known as OBI-3424, TH-3424) enters cancer cells and is activated by the AKR1C3 enzyme overexpressed by cancer cells to release the metabolite AST2660 (also known as AST-2660) (Meng, F., Li, WF, Jung, D., Wang, CC, Qi, T., Shia, CS, Hsu, RY, Hsieh, YC, & Duan, J. (2021). A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity. American journal of cancer research, 11(7), 3645–3659.). AST-2660 is an alkylating agent and the chemical component of the prodrug AST-3424 that exerts its efficacy. It destroys the DNA double-strand structure by cross-linking with the DNA double-strand, thereby inducing cell apoptosis:

[0005] Chemical reaction formula for the metabolism of AST-3424 to AST-2660 (2660 in the figure)

[0006] In clinical trials (the clinical trial registration number of the OBI-3424 drug in the United States is NCT03592264, the trial stage is Phase II, the indication is castrate prostate cancer and liver cancer, and the sponsor is Taiwan Hao Ding Biotechnology Co., Ltd. OBI; the clinical trial registration number of the OBI-3424 drug in the United States is NCT04315324, the trial stage is Phase II, the indication is T-ALL T lymphocytic acute leukemia, and the sponsor is the Southwest Oncology Group SWOG of the United States; the clinical trial registration number of the AST-3424 drug in China is CTR20191399, the trial stage is Phase II, the indication is various solid tumors, and the sponsor is Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd.; the clinical trial registration number of the AST-3424 drug in China is CTR20201915, the trial stage is Phase II, and the indication is T-ALL and B-ALL T-lymphocytic acute leukemia and B-lymphocytic acute leukemia, the applicant is Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd.), the indications of AST-3424 are a wide range of solid tumors and blood tumors including various leukemias, which is related to the mechanism of action of the alkylating agent AST-2660 on which it ultimately acts.

[0007] Summary of the Invention

[0008] As the applicants conducted further preclinical studies and clinical trials on AST-3424 and its similar AKR1C3-activated alkylating agent prodrugs and TH-302 and its similar hypoxia-activated alkylating agent prodrugs, the applicants found that the combination of such prodrug compounds with cell cycle inhibitors would achieve better tumor inhibition effects.

[0009] The present invention provides the following treatment methods, drugs for treating cancer and tumors, and pharmaceutical uses.

[0010] The method comprises the following steps: using a drug containing an alkylating agent prodrug compound and its salts, esters, solvates, and isotope isomers in combination with a drug containing a cell cycle inhibitor compound and its salts, esters, solvates, and isotope isomers to treat cancer and tumor patients.

[0011] For pharmaceutical purposes, alkylating agent prodrug compounds and their salts, esters, solvates, and isotope isomers are used to prepare drugs for treating cancer and tumor patients in combination with drugs containing cell cycle inhibitor compounds and their salts, esters, solvates, and isotope isomers.

[0012] A drug for treating cancer and tumors, which contains an alkylating agent prodrug compound and its salts, esters, solvates, and isotope isomers, and is indicated for use in combination with a drug containing a cell cycle inhibitor compound and its salts, esters, solvates, and isotope isomers to treat cancer and tumor patients.

[0013] The present invention also provides a pharmaceutical composition, which contains an alkylating agent prodrug compound or its salt, ester, solvate, isotope isomer and a cell cycle inhibitor compound or its salt, ester, solvate, isotope isomer. The pharmaceutical composition is used to treat cancer and tumor patients.

[0014] The present invention also provides a pharmaceutical use, namely, use of an alkylating agent prodrug compound or its salt, ester, solvate, or isotope isomer in the preparation of a drug for treating cancer or tumors in combination with a cell cycle inhibitor compound or its salt, ester, solvate, or isotope isomer.

[0015] The cell cycle inhibitor is selected from CDK inhibitors, CHK inhibitors, ATR inhibitors, and WEE inhibitors.

[0016] WEE inhibitors are Wee1 inhibitors.

[0017] The alkylating agent prodrug compound is selected from AKR1C3 enzyme-activated alkylating agent prodrug compounds and hypoxia-activated alkylating agent prodrug compounds, preferably AKR1C3 enzyme-activated DNA alkylating agent prodrug compounds and hypoxia-activated DNA alkylating agent prodrug compounds.

[0018] Preferably, the AKR1C3 enzyme-activating alkylating agent prodrug compound is used in combination with a CDK inhibitor, a WEE inhibitor, a CHK inhibitor, or an ATR inhibitor.

[0019] Preferably, the hypoxia-activated alkylating agent prodrug compound is used in combination with a CDK inhibitor, a WEE inhibitor, or an ATR inhibitor.

[0020] The cell cycle is a highly regulated process that promotes cell growth, genetic replication, and cell division. The core cell cycle machinery, operating within the cell nucleus, drives cells from one phase of the cell cycle to the next. Cell cycle inhibitors are substances that inhibit the normal operation of the cell cycle.

[0021] The entire cell cycle can be divided into the pre-DNA synthesis phase (G1 phase), the DNA synthesis phase (S phase), the late DNA synthesis phase (G2 phase), and the mitosis phase (M phase). During the G1 phase, cells primarily synthesize substances other than DNA required for mitosis, including ribosomes and proteins. During the S phase, cells primarily complete DNA synthesis and replication, and topoisomerases are often required to form structures that facilitate DNA replication. During the G2 phase, cells continue to synthesize substances required for mitosis, including microtubules. During the M phase, microtubules and other factors contribute to the cell's division into two daughter cells.

[0022] The above cell cycles are closely related to cell cycle proteins and their catalytic partners, cyclin-dependent kinases (CDKs), WEE, cell cycle checkpoint kinases (CHKs) and ATR (ataxia telangiectasia and Rad3-related), namely "ataxia telangiectasia mutated gene Rad3-related kinase".

[0023] Different cyclin-dependent kinases (CDKs) act on the four different phases of the cell cycle. For example, cyclin CDK4 / 6 acts on the conversion of the G1 phase of the cell to the S phase, and CDK1 acts on the conversion of the G2 phase of the cell to the M phase.

[0024] Cell cycle inhibitors specifically act on a specific cell cycle. Specifically, the inhibitor acts on a specific substance or process, causing the substance or process that regulates the specific cell cycle to not function normally.

[0025] CDK inhibitors (CDKi) are inhibitors of the corresponding kinases. By inhibiting the corresponding kinases, they regulate the progress of the cell cycle, causing cancer cells to be unable to replicate and proliferate normally and thus to undergo apoptosis.

[0026] Different inhibitors are known for different CDK subtypes: CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK11, CDK12, CDK13, CDK14, CDK16, CDK19, CDC, and CLK.

[0027] WEE, also known as Wee1, is a key kinase involved in the DNA damage repair (DDR) pathway. WEE1 is a serine / threonine protein kinase that can arrest the cell cycle at the G2 / M phase by inhibiting CDK1 / 2 activity, allowing time for DNA repair. Wee1 is one of the preliminarily validated targets for anticancer drug research and has a certain synthetic lethality effect with the TP53 gene mutation, which is widely found in tumor cells.

[0028] The DNA damage checkpoint is a cell cycle surveillance system that prevents genomic instability. The DNA damage checkpoint kinases CHK1 and CHK2, as elements of the DNA damage checkpoint, are central to inducing cell cycle arrest, DNA repair, and apoptosis.

[0029] When cellular DNA is damaged, cell cycle checkpoints are activated, causing cell cycle arrest, which facilitates DNA repair and maintains genomic integrity and stability. Throughout long periods of biological evolution, cell cycle checkpoints have a conserved signaling system that regulates the cell cycle through different CHK subtypes (Checkpoint Kinases, CHKs), causing cell cycle arrest and DNA repair.

[0030] ATR (ataxia telangiectasia and Rad3-related), also known as "ataxia telangiectasia mutated gene Rad3-related kinase," is a serine-threonine kinase in the phosphatidylinositol 3-kinase (PIKK) family. Composed of 2,644 amino acids, it contains an ATR-interacting protein (ATRIP) domain at its N-terminus, a key region for ATR kinase activation. Its C-terminus houses a kinase domain that phosphorylates downstream proteins and can phosphorylate target proteins such as cell cycle checkpoint kinase 1 (CHK1) at serine or threonine residues. Upon activation, ATR kinase regulates cellular processes through various signaling pathways, including cell cycle arrest, inhibition of replication origins, promotion of deoxynucleotide synthesis, initiation of replication forks, and repair of DNA double-strand breaks. ATR activates cellular responses to DNA damage, thereby arresting cell cycle progression, stabilizing replication forks, and repairing DNA, thereby circumventing apoptosis and thus playing a crucial role in life.

[0031] The alkylating agent prodrug compound refers to a prodrug compound that can be converted into an alkylating agent in vivo.

[0032] The alkylating agent prodrug compound is selected from AKR1C3 enzyme-activated alkylating agent prodrug compounds and hypoxia-activated alkylating agent prodrug compounds, preferably AKR1C3 enzyme-activated DNA alkylating agent prodrug compounds and hypoxia-activated DNA alkylating agent prodrug compounds.

[0033] Alkylating agent prodrug compounds include chemical structural formulas (1)-(9):

[0034] Formula (1) is a hypoxia-activated alkylating agent prodrug compound, more specifically, a hypoxia-activated DNA alkylating agent prodrug compound;

[0035] Wherein, R is independently selected from H, -CH3, -CH2CH3, -CF3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO.

[0036] With TH-302 or its similar compounds Related preparations include oral preparations, lyophilized preparations and concentrated injections, and the related prescriptions, preparation methods, clinical compatibility and administration methods are described and disclosed in detail in Threshold's relevant patents: WO2010048330A1, WO2012142520A2 and WO2008083101A1. The present invention hereby introduces the full text of the above application texts.

[0037] TH-302 or its similar compounds It is a DNA alkylating agent anticancer drug with a wide range of cancer treatment potentials. These related cancer indication experiments and clinical trials are disclosed in the patent applications of Threshold and other pharmaceutical companies (such as WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO200 9033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO20201182 51A1, WO2014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, WO2015069489A1, WO2012006032A2, WO2018026606A2, WO2010048330A2, WO2015171647A1, WO201 3096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO2012145684A2, WO20160143 90A2, WO2019055786A2, WO2012135757A2, WO2015013448A2, WO2016011328A2, WO2013177633A2, WO2016011195A2, WO2015051921A2) and FDA-registered clinical trials (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT0114 9915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NC T01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT02496832, NCT02712567). The present invention hereby introduces all the above-mentioned related application texts and clinical trial information.

[0038] Formula (2) is a hypoxia-activated alkylating agent prodrug compound, more specifically, a hypoxia-activated DNA alkylating agent prodrug compound;

[0039] Wherein, the definitions of R1, R2, R3, and Cx are as described in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1. 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:

[0040] Cx is a 5-10 membered aromatic ring or aromatic heterocycle, aliphatic heterocycle or cycloalkane, which shares two carbon atoms with the nitrobenzene ring to form a fused ring structure;

[0041] R1 is connected to any backbone atom of the Cx ring and is selected from hydrogen, halogen atoms, cyano or isocyano, hydroxyl, thiol, amino, OTs, 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, alkoxy 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 ,

[0042] R2 and R3 are each independently hydrogen, 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, or R2, R3 and the benzylic carbon atom to which they are bonded together form a 3-6 membered ring;

[0043] The group can replace the hydrogen atom at any position on the condensed ring carbon atom, and the number of substitutions is 1;

[0044] The substituent Z is a halogen atom, a cyano group or an isocyano group, a hydroxyl group, a mercapto group, an amino group, 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, or a C3-C8 cycloalkyl group or a substituted cycloalkyl group;

[0045] R 6 、R 7 are each independently hydrogen, C1-C6 alkyl or Z-substituted C1-C6 alkyl, C2-C6 alkenyl or Z-substituted C2-C6 alkenyl, C2-C6 alkynyl or Z-substituted C2-C6 alkynyl, C3-C8 cycloalkyl or Z-substituted C3-C8 cycloalkyl, C6-C 10 Aryl or Z-substituted C6-C 10 aryl, 4-15 membered heterocyclic group or Z-substituted 4-15 membered heterocyclic group, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R 6 、R 7 Together with the atoms to which it is bonded, it forms a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group.

[0046] Formula (3) is a hypoxia-activated alkylating agent prodrug compound, more specifically, a hypoxia-activated DNA alkylating agent prodrug compound;

[0047] Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 The definition is as described in the claims of patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A), 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:

[0048] R1 is: hydrogen, -N3, CN, halogen, NR 21 R 22 、-OR 23 , -SO2(C1-C6 alkyl), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or ether;

[0049] R 21and R 22 Each is independently hydrogen, hydroxy, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl or -SO2(C1-C6 alkyl); or R 21 and R 22 Together with the nitrogen atom to which it is bonded, it forms a 4-15 membered heterocyclic ring or a 5-15 membered heteroaryl group;

[0050] R 23 is hydrogen, C1-C6 alkyl or C6-C10 aryl;

[0051] R2 and R3 are independently hydrogen or halogen;

[0052] R4 is hydrogen, halogen, C1-C6 alkoxy, C1-C6 alkyl or C6-C10 aryl,

[0053] R5, R7, R9, R 12 and R 15 R4 and R5 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C10 aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl; or R4 and R5 together with the intervening carbon atoms therebetween form a C5-C6 cycloalkyl ring;

[0054] R6 and R 10 are independently hydrogen or halo;

[0055] R8 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or 5-15 membered heteroaryl;

[0056] R 11 Each is independently a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl or C6-C10 aryl;

[0057] R 13 、R 14 、R 16 and R 17 are independently hydrogen, halo, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C1-C6 alkoxy;

[0058] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, alkoxy, and ether groups are optionally substituted.

[0059] Formula (4) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0060] Among them, X, Y, Z, R, T, A and X 10 The definition of is as described in the claims of patent application PCT / US2016 / 021581, publication number WO20 16145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), 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:

[0061] X 10 O, S, SO or SO2;

[0062] A is C6-C 10 Aryl, 5-15 membered heteroaryl or -N=CR 1 R 2 ;

[0063] 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 ;

[0064] 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 ;

[0065] 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 ;

[0066] R 13 and R 14 are independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle or ether

[0067] 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

[0068] These alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, and ether groups are substituted or unsubstituted.

[0069] Formulas (5) and (6) are AKR1C3 enzyme-activated alkylating agent prodrug compounds, more specifically, AKR1C3 enzyme-activated DNA alkylating agent prodrug compounds;

[0070] 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, 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:

[0071] 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;

[0072] 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;

[0073] 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 ;

[0074] 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 7Or 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;

[0075] 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;

[0076] 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;

[0077] 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.

[0078] 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;

[0079] 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.

[0080] Formula (7) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0081] in:

[0082] 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;

[0083] 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;

[0084] wherein the alkyl group and the aryl group are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups;

[0085] R 1 and R 2 each independently phenyl or methyl;

[0086] X, Y and Z are each independently hydrogen or halo;

[0087] R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl;

[0088] Formula (8) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0089] Wherein, Rw is defined as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1, 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:

[0090] Rw is

[0091] 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;

[0092] Each R a are independently H, F, Cl, Br, I, -CN, -OH, C 1-3 Alkoxy or C 1-3 alkyl;

[0093] R2 is H or C 1-6 alkyl;

[0094] 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;

[0095] Each R b are independently H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3 or -CH2CH3;

[0096] R3 is H, F, Cl, Br, I, -OH, -NH2, C 1-3 Alkoxy or C 1-3 alkyl;

[0097] Or R2 and R3 are connected together to make the structural unit for

[0098] T1 is -(CR c R d ) m -or-(CR c R d ) n -O-;

[0099] m is 1, 2, or 3;

[0100] n is 1 or 2;

[0101] T2 is N or CH;

[0102] R c and R d Each independently is H, F, C 1-3 Alkyl or C 1-3 alkoxy;

[0103] R4, R5 and R6 are each independently H, F, Cl, Br, I, C 1-3 Alkyl or C 1-3 alkoxy;

[0104] T is N or CH;

[0105] R7 and R8 are each independently H, F, Cl, Br or I;

[0106] R9 and R 10 Each independently is H, F, Cl, Br, I, -CN or

[0107] 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-.

[0108] Formula (9) is an AKR1C3 enzyme-activated alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0109] 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:

[0110] 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;

[0111] E is SO or SO2;

[0112] X is Cl, Br, I or OSO2R;

[0113] Y is Cl, Br, I or OSO2R;

[0114] Each R is independently H or C1-C6 alkyl;

[0115] G is a radical selected from the group consisting of formula (B)-(AA):

[0116] in:

[0117] 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, OC F3, COR6, CON(R6)(R7), SOR6, SON(R6)(R7), SO2R6, SO2N(R6)(R7), CN or NO2;

[0118] 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;

[0119] R4 is N(R6)(R7), OH, OCH2(CH2)nN(R6)(R7) or CH2(CH2)nN(R6)(R7);

[0120] R5 is H or a C1-C6 alkyl group;

[0121] 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;

[0122] Z is CH or N;

[0123] W is CH2, O, S, SO or SO2;

[0124] n is 0 to 6;

[0125] * indicates the point of connection to formula (I).

[0126] The drug described herein refers to a medicine or preparation, wherein the prepared medicine comprises the hypoxia-activated compound of formula (1) or its salt or solvate in a specific dosage range as the active ingredient, and / or the prepared medicine is in a specific dosage form and is administered by a specific administration method.

[0127] In addition to the alkylating agent prodrug compound of formula (1)-(9), the above-mentioned drug should also be added with pharmaceutically acceptable excipients or vehicles according to the specific characteristics of the drug, medicine, or preparation. The drug can be in any dosage form for 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 administration method, the pharmaceutically acceptable excipients or vehicles in the drug can include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, adhesives, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and pigments.

[0128] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) with potentially unrestricted growth that can spread locally by invasion and systemically by metastasis.

[0129] Examples of cancers that can be treated with AST-3424 or such AKR1C3 enzyme-activated DNA alkylating agent prodrugs include, but are not limited to, cancers of the adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid. Some other examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinomas, adenomas, basal cell carcinomas, dysplastic epithelial cells and carcinoma in situ of the cervix, Ewing's sarcoma, epidermoid carcinoma, giant cell tumors, glioblastoma multiforme, hair cell tumors, intestinal ganglioneuromas, proliferative corneal nerve tumors, pancreatic islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumors, malignant melanoma, malignant hypercalcemia, Marfanoid body type tumors, medullary epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, squamous cell carcinoma of both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma, and Wilm's tumor.

[0130] Combination therapy refers to the use of a combination of drugs. Monotherapy refers to the use of only one anticancer drug in a single course of treatment. Combination therapy refers to the use of two or more anticancer drugs simultaneously or sequentially in a single course of treatment.

[0131] Generally speaking, combination therapy requires exploring different dosages and dosing cycles based on the characteristics of the disease and the types of combined drugs. Only based on the above conditions can the combination drug treatment plan explored achieve better therapeutic effects than single drug treatment.

[0132] The drug dosage and administration cycle of the combination therapy regimen need to be explored through clinical trials with reference to the dosage and administration regimen of the above-mentioned alkylating agent prodrug compound and the combination drug.

[0133] The cancer or tumor patient or biological sample thereof is found to have specific gene deletion or damage, and the specific gene is selected from genes involved in cell cycle checkpoint regulation, preferably p53, p21, CCNB1, WIP1, 14-3-3sigma protein, cdc2 / cycB.

[0134] These genes can be identified as genes involved in cell cycle checkpoint regulation based on relevant research literature (Molinari, M. (2000), Cell cycle checkpoints and their inaction in human cancer. Cell Proliferation, 33: 261-274. https: / / doi.org / 10.1046 / j.1365-2184.2000.00191.x; Donehower LA (2014). Phosphatases reverse p53-mediated cell cycle checkpoints. Proceedings of the National Academy of Sciences of the United States of America, 111 (20), 7172–7173. https: / / doi.org / 10.1073 / pnas.1405663111).

[0135] The cell cycle inhibitors include CDK inhibitors, WEE inhibitors, CHK inhibitors and ATR inhibitors.

[0136] Preferably, the CDK inhibitor is selected from Palbociclib, Ribociclib, Abemaciclib, Trilaciclib, dalpiciclib, Adavosertib, Ro-3306, Dinaciclib, Cirtuvivint, Rintodestrant, DS96432529, THZ1, THZ531, Seliciclib, Flavopiridol, AZD4573, SR-4835, Simurosertib, Fadraciclib, NVP-2, SNS-032, (E / Z)-Zotiraciclib, AZD-5438, AT7519, Mevociclib, Kenpaullone, YKL-5-124TFA, NG 52, GSK 3 Inhibitor IX, OTS964, Samuraciclib, Flavopiridol, KB-0742dihydrochloride, (+)-Enitociclib, AUZ 454, SY-5609, SEL120-34A monohydrochloride, CCT-251921, MBQ-167, XL413hydrochloride, BI-1347, THAL-SNS-032, JNJ-7706621, TG003, LD C4297, BMS-265246, Roniciclib, CGP60474, (R)-CR8trihydrochloride, R547, Milciclib, T025, AS2863619, Senexin A. BSJ-4-116, CLK-IN-T3, CDK12-IN-3, CVT-313, Atuveciclib, PHA-793887, Indirubin-3'-monoxime, YKL-5-124, PHA-767491hydrochloride, KH-CB19, Cucurbitacin E, Purvalanol A. BSJ-03-204, ON123300, CDK5inhibitor 20-223, Riviciclib, FN-1501, CP-10, THZ2, Abemaciclib metabolite M2, BS-181, CDK12-IN-E9, Samuraciclib, RGB-286638, CDK2-IN-4, LDC000067, ML167, PurvalanolB, NU6300, CLK1-IN-1, FMF-04-159-2, CKI-7, CDKI-73, MSC2530818, BSJ-04-132, NU6102, Voruciclib, Olomoucine, etc. The list, specific structures, and supply information of these compounds can be obtained by visiting commercial reagent websites, such as the MedChemExpress (MCE) website at https: / / www.medchemexpress.cn / Targets / CDK.html?effectName=Inhibitor.

[0137] Preferably, the WEE inhibitor is selected from WEE1-IN-5, WEE1-IN-3, WEE1-IN-4, LEB-03-146, LEB-03-144, Adavosertib, LEB-03-153, LEB-03-145, PD407824, PD0166285, PD0166285 dihydrochloride, Pomalidomide-C3-adavosertib, DB0614, FMF-06-098-1, and the like. The list of these compounds, as well as their specific structures and supply information, can be obtained by visiting commercial reagent websites, such as the MedChemExpress (MCE) website at https: / / www.medchemexpress.cn / search.html?q=Wee&ft=&fa=&fp=&fsp=&ftag=&fsc=.

[0138] Preferably, the CHK inhibitor is selected from AZD7762, Prexasertib, SCH900776, GDC-0425, Chk1-IN-6, CCT245737, BML-277, CCT241533, PD407824, CHIR-124, CCT244747, PF477736, GDC-0575, SB-218078, MRT00033659, ANI-7, SAR-020106, CCT241533, CHK1- IN-4, VER-00158411, CHK1-IN-3, Chk1-IN-5, CHK1-IN-2, CHK-IN-1, etc. The list of these compounds as well as their specific structures and supply information can be obtained by visiting commercial reagent websites, such as the MedChemExpress (MCE) website at https: / / www.medchemexpress.cn / Targets / Checkpoint%20Kinase%20(Chk).html.

[0139] Preferably, the ATR inhibitor is selected from Ceralasertib, Berzosertib, Gartisertib, BAY1895344, BAY-937, AZ20, ETP-46464, Dactolisib, VE-821, M1774, ATRN-199, RP-3500, and ART-0380. For information on these compounds, please refer to the literature: Yuan Yinghui, Duan Jilong, Huizi, et al. Research progress on targeted ATR kinase inhibitors for the treatment of cancer [J]. Acta Pharmaceutical Sinica, 2022(057-003).

[0140] The specific compound of formula (1) is selected from the following structures:

[0141] The chemical structures of the specific compounds of formula (2)-(9) are as described in claim 7 and are not repeated here.

[0142] Among them, the specific compound of formula (2) refers to patent application document PCT / CN2020 / 114519, publication number WO2021120717A1, claim 9, and the full text of the patent application document is hereby introduced into this application.

[0143] The specific compounds of formula (3) refer to the compounds in the table of paragraph [0073-0093] of patent application document CN2016800368985, publication number CN108024974A, and the entire text of the patent application document is hereby incorporated into this application.

[0144] The specific compound of formula (4) refers to the compound in patent application document PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), and the entire text of the patent application document is hereby incorporated into this application.

[0145] The specific compounds of formula (5) and (6) refer to the compounds in patent application document PCT / CN2020 / 089692, publication number WO2020228685A1, and the full text of the patent application document is hereby incorporated into this application.

[0146] The specific compound of formula (7) refers to the compounds in patent application documents PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A) and PCT / CN2020 / 089692, publication number WO2020228685A1. The full text of these two patent application documents is hereby introduced into this application.

[0147] The specific compound of formula (8) refers to the compound in patent application document PCT / CN2020 / 120281, publication number WO2021068952A1, and the entire text of the patent application document is hereby incorporated into this application.

[0148] The specific compound of formula (9) refers to the compound in patent application document PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A), and the full text of the patent application document is hereby introduced into this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1 shows the proliferation inhibition curve of HT-29 cell line by AST-3424 alone or in combination with Adavosertib;

[0150] Figure 2 shows the inhibition rate of AST-3424 and Adavosertib alone and in combination on HT-29 cell proliferation at fixed concentrations (in each group of bar graphs, the left side shows AST-3424 alone, the middle shows Adavosertib alone, and the right side shows AST-3424 combined with Adavosertib;

[0151] FIG3 shows the proliferation inhibition curve of HT-29 cell line by AST-3424 alone or in combination with AZD7762;

[0152] Figure 4 shows the inhibition rate of AST-3424 and AZD7762 alone and in combination on HT-29 cell proliferation at fixed concentrations (in each group of bar graphs, the left side shows AST-3424 alone, the middle shows AZD7762 alone, and the right side shows AST-3424 and AZD7762 combined);

[0153] FIG5 shows the proliferation inhibition curve of HT-29 cell line by AST-3424 alone or in combination with Palbociclib;

[0154] Figure 6 shows the inhibition rate of AST-3424 and Palbociclib alone and in combination on HT-29 cell proliferation at fixed concentrations (in each group of bar graphs, the left side shows AST-3424 alone, the middle shows Palbociclib alone, and the right side shows AST-3424 combined with Palbociclib);

[0155] FIG7 shows the proliferation inhibition curve of H460 cell line by AST-3424 alone or in combination with Adavosertib;

[0156] Figure 8 shows the inhibition rate of H460 cell proliferation by single drugs and combination drugs at fixed concentrations of AST-3424 and Adavosertib (in each group of bar graphs, the left side shows AST-3424 alone, the middle shows Adavosertib alone, and the right side shows AST-3424 combined with Adavosertib);

[0157] FIG9 is a growth inhibition curve of H460 cell line by AST-3424 alone or in combination with AZD7762;

[0158] Figure 10 shows the inhibition rate of H460 cell proliferation by single drugs and combination drugs at fixed concentrations of AST-3424 and AZD7762 (in each group of bar graphs, the left side shows AST-3424 alone, the middle shows AZD7762 alone, and the right side shows AST-3424 and AZD7762 combined);

[0159] FIG11 is a curve showing the proliferation inhibition of H460 cell lines by AST-3424 alone or in combination with Palbociclib;

[0160] Figure 12 shows the inhibition rate of H460 cell proliferation by AST-3424 and Palbociclib alone and in combination at fixed concentrations (in each group of bar graphs, the left side shows AST-3424 alone, the middle shows Palbociclib alone, and the right side shows AST-3424 combined with Palbociclib);

[0161] FIG13 is a proliferation inhibition curve of AST alone or in combination with Adavosertib on HT-29 cell lines;

[0162] Figure 14 shows the inhibition rate of HT-29 cell proliferation by single drugs and combination drugs at fixed concentrations of AST and Adavosertib (in each group of bar graphs, the left side is AST alone, the middle is Adavosertib alone, and the right side is AST combined with Adavosertib);

[0163] Figure 15 is the proliferation inhibition curve of AST alone or in combination with AZD7762 on HT-29 cell lines;

[0164] Figure 16 shows the inhibition rate of AST and AZD7762 alone and in combination on HT-29 cell proliferation at fixed concentrations (in each group of bar graphs, the left side shows AST alone, the middle shows AZD7762 alone, and the right side shows AST and AZD7762 in combination);

[0165] FIG17 is a curve showing the proliferation inhibition of HT-29 cell lines by AST alone or in combination with Palbociclib;

[0166] Figure 18 shows the inhibition rate of HT-29 cell proliferation by single drugs and combination drugs at fixed concentrations of AST and Palbociclib (in each group of bar graphs, the left side is AST alone, the middle is Palbociclib alone, and the right side is AST combined with Palbociclib);

[0167] FIG19 is a proliferation inhibition curve of H460 cell line by AST alone or in combination with Adavosertib;

[0168] Figure 20 shows the inhibition rate of H460 cell proliferation by single drugs and combination drugs at fixed concentrations of AST and Adavosertib (in each group of bar graphs, the left side is AST alone, the middle is Adavosertib alone, and the right side is AST combined with Adavosertib);

[0169] Figure 21 is the proliferation inhibition curve of H460 cell line by AST alone or in combination with AZD7762;

[0170] Figure 22 shows the inhibition rate of H460 cell proliferation by single drugs and combination drugs at fixed concentrations of AST and AZD7762 (in each group of bar graphs, the left side is AST alone, the middle is AZD7762 alone, and the right side is AST and AZD7762 combined);

[0171] FIG23 is a curve showing the proliferation inhibition of H460 cell lines by AST alone or in combination with Palbociclib;

[0172] Figure 24 shows the inhibition rate of H460 cell proliferation by single drugs and combination drugs at fixed concentrations of AST and Palbociclib (in each group of bar graphs, the left side is AST alone, the middle is Palbociclib alone, and the right side is AST combined with Palbociclib);

[0173] FIG25 shows the proliferation inhibition curves of AST-3424 alone or in combination with Ceralasertib on the HT29 / H460 cell line, wherein the bottom curve is the AST-3424 monotherapy group;

[0174] FIG26 shows the proliferation inhibition curves of HT29 / H460 cell lines treated with AST-3424 alone or in combination with Ceralasertib in another experiment, wherein the bottom curve represents the AST-3424 monotherapy group;

[0175] FIG27 shows the proliferation inhibition curves of HT29 cell lines using AST-3424 alone or in combination with Ceralasertib in experiments with different dosing orders of Ceralasertib and AST-3424, wherein the bottom curve represents the AST-3424 monotherapy group;

[0176] FIG28 shows the proliferation inhibition curves of HT29 cell lines treated with TH-302 alone or in combination with Adavosertib, wherein <0.01% O2 indicates hypoxic conditions, and the upper left curve represents the combination of TH-302 and 10 μM Adavosertib under hypoxic conditions;

[0177] FIG29 shows the proliferation inhibition curve of H460 cell line by TH-302 alone or in combination with Adavosertib, wherein <0.01% O2 indicates hypoxic conditions, and the combination group of TH-302 and 30 μM Adavosertib under hypoxic conditions is on the left;

[0178] FIG30 shows the proliferation inhibition curves of HT29 cell lines treated with TH-302 alone or in combination with Ceralasertib, wherein <0.01% O2 indicates hypoxic conditions, and the upper left curve represents the combination of TH-302 and 2000 μM Ceralasertib under hypoxic conditions;

[0179] FIG31 shows the proliferation inhibition curve of H460 cell line by TH-302 alone or in combination with Ceralasertib, wherein <0.01% O2 indicates hypoxic conditions, and the TH-302 normoxic single-drug group is on the right.

[0180] Comparison table of foreign words and their corresponding Chinese meanings in the attached figure

[0181] The experimental conditions are described in the headers of some figures. For example, in Figure 1, the header section is "HT-29_Adavosertib-2h-pretreatment AST-3424-72h-cotreatment 122421". The meanings of the various sections are as follows:

[0182] HT-29 indicates experiments in the HT-29 cell line;

[0183] Adavosertib-2h-pretreatment AST-3424-72h-cotreatment means that in combination therapy, Adavosertib was first treated for 2 hours, and then AST-3424 was added for co-treatment for 72 hours; in monotherapy, only AST-3424 was added and then treated for 72 hours;

[0184] 122421 represents the experiment number.

[0185] Similar header parts in other figures use different cell lines, test compounds or experimental numbers, and their meanings refer to the above explanations. Example

[0186] The chemical structure of AST-3424 is

[0187] Its synthesis method can be found in patent application text PCT / US2016 / 062114, publication number WO2017087428A1 (corresponding Chinese application number 2016800200132, publication number CN108136214A).

[0188] The chemical structure of AST is

[0189] Its synthesis method can be found in patent application text PCT / CN2020 / 089692, publication number WO2020228685A1.

[0190] The chemical structure of TH-302 is

[0191] The synthesis method thereof can be found in patent application text CN2012102515573, publication number CN102746336A.

[0192] Adavosertib (also known as AZD1775 and MK-1775) is a selective inhibitor of Wee1 kinase, a key kinase involved in the G2 / M checkpoint of the cell cycle and DNA damage repair. Cyclin-dependent kinases (CDKs) regulate the cell cycle by phosphorylating specific substrates. Their activity depends on binding to cyclins to form a complex and is also regulated by phosphorylation. The phosphorylation state of CDK1 further controls the G2 / M checkpoint. Wee1 is the gatekeeper of the G2 / M checkpoint, allowing cells to repair DNA damage before entering mitosis. Wee1 phosphorylates CDK1 at Tyr15, maintaining CDK1 inactive. This inhibits the activity of the CDK1-cyclin B complex, arresting cell division at the G2 / M checkpoint and negatively regulating the cell cycle. Its biological significance is to repair DNA damage that has not yet been repaired, preventing cells from entering mitosis with DNA damage. Adavosertib selectively inhibits Wee1 kinase, blocks DNA damage repair at the G2 / M checkpoint in p53 and other gene-deficient tumors, leading to tumor cell death and ultimately achieving the purpose of treating tumors. Its structural formula is

[0193] It can be purchased directly from commercial reagent companies.

[0194] AZD7762 is a potent ATP-competitive cell cycle checkpoint kinase (CHK) inhibitor that eliminates the S and G2 checkpoints induced by DNA damage. It is a CHK inhibitor with the structural formula

[0195] It can be purchased directly from commercial reagent companies.

[0196] Palbociclib is an orally active selective inhibitor of CDK4 and CDK6 with the structural formula

[0197] It can be purchased directly from commercial reagent companies.

[0198] Ceralasertib (AZD6738) is a potent ATR kinase inhibitor that controls the G2 / M checkpoint.

[0199] It can be purchased directly from commercial reagent companies.

[0200] 1. In vitro cell experiments

[0201] In p53 wild-type (H460) and p53 null (HT-29) cell lines, CTG cell proliferation assay was used to examine whether cell cycle inhibitors could enhance the in vitro cytotoxicity of DNA alkylating agent prodrugs AST, AST-3424, and TH-302 by inhibiting key nodes of DNA repair in the tumor cell cycle.

[0202] The experimental steps of CTG cell proliferation assay are as follows:

[0203] 1) Add H460 or HT-29 cell suspension to a 96-well plate, 100 μL per well, with a cell density of 2,000 or 10,000 cells per well, respectively.

[0204] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0205] 3) Compound treatment

[0206] Combination therapy: Add 1 μL of Adavosertib (four concentrations), AZD7762 (four concentrations), Palbociclib (four concentrations), and Ceralasertib (three concentrations) to each well, shake gently to ensure uniform mixing, and place in the incubator for 2 hours. Then, add 1 μL of different concentrations of the test compound AST-3424 or AST, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0207] Single drug administration: 24 hours after cell plating, add 99 μL of growth medium to each well. Add 1 μL of test compound AST-3424 or AST at different concentrations, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0208] 4) Place the cell plate in an incubator for 72 hours.

[0209] 5) The cell test plate was placed at room temperature for 30 minutes to equilibrate, and 100 μL of culture medium was discarded from each well.

[0210] 6) Add 25 μL of CTG reagent to each well, shake on a fast shaker for 2 minutes, and place at room temperature in the dark for 30 minutes.

[0211] 7) Read the chemiluminescence signal value using a multifunctional microplate reader with a reading time of 1000 ms.

[0212] 8) Calculate IC using GraphPad Prism 5 software 50 , and obtain the IC of the compound 50 (half inhibitory concentration).

[0213] 1. Results of the proliferation inhibition experiment of AST-3424 alone or in combination with Adavosertib on the HT-29 cell line

[0214] According to the numerical experimental results of proliferation inhibition rate at different concentrations in Figure 1, the IC of AST-3424 alone or in combination with Adavosertib on HT-29 cell line proliferation inhibition was obtained by fitting. 50 The results are shown in Table 1 below. After AST-3424 was combined with Adavosertib at different concentrations, the IC 50 The value is significantly lower than the single-drug IC of AST-3424 50 The values ​​were significantly different and showed a certain degree of Adavosertib dose dependence.

[0215] Table 1: IC of AST-3424 alone or in combination with Adavosertib against HT-29 cell line proliferation 50 value

[0216] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio when AST-3424 is used alone or when AST-3424 is used in combination with Adavosertib. 50 ratio.

[0217] We further processed the data in Figure 1 and calculated the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and around 0% drug concentration (IC0), the inhibition rate of AST-3424 and Adavosertib combined. The results are shown in Figure 2.

[0218] Experimental Summary: The above results show that in the p53-deficient (HT-29) cell line, the combination of different concentrations of AST-3424 and different concentrations of the cell cycle inhibitor Adavosertib has a significant additive cytotoxic effect; and this additive effect is still significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor Adavosertib.

[0219] 2. Results of the proliferation inhibition experiment of AST-3424 alone or in combination with AZD7762 on HT-29 cell lines

[0220] According to the experimental results of the proliferation inhibition rate at different concentrations in Figure 3, the IC values ​​of AST-3424 alone or in combination with AZD7762 on the proliferation inhibition of HT-29 cell line were obtained by fitting. 50 The results are shown in Table 2 below. After AST-3424 was combined with different concentrations of AZD7762, the IC 50 The value is significantly lower than the single-drug IC of AST-3424 50 The values ​​were significantly different and showed a certain degree of AZD7762 dose dependence.

[0221] Table 2: IC of AST-3424 alone or in combination with AZD7762 against proliferation of HT-29 cell lines 50 value

[0222] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio when AST-3424 is used alone or when AST-3424 is used in combination with AZD7762. 50 ratio.

[0223] The data in Figure 3 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST-3424 and AZD7762 after combined use. The results are shown in Figure 4.

[0224] Experimental Summary: The above results show that in the p53-deficient (HT-29) cell line, the combination of different concentrations of AST-3424 and different concentrations of the cell cycle inhibitor AZD7762 has a significant additive cytotoxic effect; and this additive effect is still significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor AZD7762.

[0225] 3. Results of the proliferation inhibition experiment of AST-3424 alone or in combination with Palbociclib on HT-29 cell lines

[0226] According to the experimental results in Figure 5, the IC values ​​of AST-3424 alone or in combination with Palbociclib on the proliferation inhibition of HT-29 cell lines were obtained by fitting. 50 The results are shown in Table 3 below. After AST-3424 was combined with different concentrations of Palbociclib, the IC 50 The IC value is less than that of AST-3424 alone 50 The values ​​were significantly different and showed a certain degree of Palbociclib dose dependence.

[0227] Table 3: IC of AST-3424 alone or in combination with Palbociclib against proliferation of HT-29 cell lines 50 value

[0228] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio of AST-3424 alone and AST-3424 combined with Palbociclib. 50 ratio.

[0229] The data of Figure 5 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST-3424 and AZD7762 after combined use. The results are shown in Figure 6.

[0230] Experimental Summary: The above results show that in the p53-deficient (HT-29) cell line, the combination of different concentrations of AST-3424 and different concentrations of the cell cycle inhibitor Palbociclib has a significant additive cytotoxic effect; and this additive effect is still significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor Palbociclib.

[0231] 4. Results of the proliferation inhibition experiment of H460 cell line with AST-3424 alone or in combination with Adavosertib

[0232] According to the experimental results in Figure 7, the IC values ​​of AST-3424 alone or in combination with Adavosertib on the proliferation inhibition of H460 cell line were obtained. 50 The results are shown in Table 4 below. After AST-3424 was combined with different concentrations of Adavosertib, the IC 50 Value and IC of AST-3424 alone 50 The values ​​differed by about 2 times. The IC values ​​of Adavosertib combined with other concentrations were 50 Value and IC of AST-3424 alone 50 There was no significant difference in the values.

[0233] Table 4: IC of AST-3424 alone or in combination with Adavosertib against H460 cell line proliferation 50 value

[0234] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio when AST-3424 is used alone or when AST-3424 is used in combination with Adavosertib. 50 ratio.

[0235] The data in Figure 7 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST-3424 and Adavosertib combined. The results are shown in Figure 8.

[0236] Experimental Summary: The above results show that in the p53 wild-type (H460) cell line, the combination of different concentrations of AST-3424 and different concentrations of the cell cycle inhibitor Adavosertib has a certain degree of additive cytotoxic effect when high concentrations of the compound are used together; this additive effect is not significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor Adavosertib.

[0237] 5. Results of the proliferation inhibition experiment of H460 cell line by AST-3424 alone or in combination with AZD7762

[0238] According to the experimental results in Figure 9, the IC values ​​of AST-3424 alone or in combination with AZD7762 on the proliferation inhibition of H460 cell line were obtained. 50 The results are shown in Table 5 below. After AST-3424 was combined with different concentrations of AZD7762, the IC 50 Value and IC of AST-3424 alone 50 The values ​​differed by about 2 times. The IC values ​​of AZD7762 combined with other concentrations were 50 Value and IC of AST-3424 alone 50 There was no significant difference in the values.

[0239] Table 5: IC50 of AST-3424 alone or in combination with AZD7762 on proliferation inhibition of H460 cell line 50 value

[0240] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio when AST-3424 is used alone or when AST-3424 is used in combination with AZD7762. 50 ratio.

[0241] The data of Figure 9 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC25), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST-3424 and AZD7762 after combined use. The results are shown in Figure 10.

[0242] Experimental Summary: The above results show that in the p53 wild-type (H460) cell line, the combination of different concentrations of AST-3424 and different concentrations of the cell cycle inhibitor AZD7762, and the combination of medium and high concentrations of the compounds have a certain degree of cytotoxic additive effect; this additive effect is not significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor AZD7762.

[0243] 6. Results of the proliferation inhibition experiment of H460 cell line with AST-3424 alone or in combination with Palbociclib

[0244] According to the experimental results in Figure 11, the IC values ​​of AST-3424 alone or in combination with Palbociclib on the proliferation inhibition of H460 cell line were obtained. 50 The results are shown in Table 6 below. After AST-3424 was combined with different concentrations of Palbociclib, the IC 50 Value and IC of AST-3424 alone 50 There was no significant difference in the values.

[0245] Table 6: IC of AST-3424 alone or in combination with Palbociclib on proliferation inhibition of H460 cell line 50 value

[0246] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio of AST-3424 alone and AST-3424 combined with Palbociclib. 50 ratio.

[0247] The data of Figure 11 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST-3424 and Palbociclib after combination use. The results are shown in Figure 12.

[0248] Experimental Summary: The above results show that in the p53 wild-type (H460) cell line, the combination of different concentrations of AST-3424 and different concentrations of the cell cycle inhibitor Palbociclib did not show an additive cytotoxic effect at each drug concentration.

[0249] 7. Results of the proliferation inhibition experiment of AST alone or in combination with Adavosertib on HT-29 cell line

[0250] According to the experimental results in Figure 13, the IC values ​​of AST alone or in combination with Adavosertib on the proliferation inhibition of HT-29 cell line were obtained. 50 The results are shown in Table 7 below. After AST was combined with different concentrations of Adavosertib, the IC 50 The value is significantly lower than the single-drug IC of AST-3424 50 The values ​​were significantly different and showed a certain degree of Adavosertib dose dependence.

[0251] Table 7: IC of AST alone or in combination with Adavosertib against proliferation of HT-29 cell lines 50 value

[0252] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC ratio when AST is used alone or when AST is used in combination with Adavosertib. 50 ratio.

[0253] We further processed the data in Figure 13 and calculated the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST after combined use of Adavosertib. The results are shown in Figure 14.

[0254] Experimental summary: The above results show that in the p53-deficient (HT-29) cell line, the combination of different concentrations of AST and different concentrations of the cell cycle inhibitor Adavosertib has a significant cytotoxic additive effect; and this additive effect is still significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor Adavosertib.

[0255] 8. Results of the proliferation inhibition experiment of AST alone or in combination with AZD7762 on HT-29 cell line

[0256] According to the experimental results in Figure 15, the IC values ​​of AST alone or in combination with AZD7762 on the proliferation inhibition of HT-29 cell line were obtained. 50The results are shown in Table 8 below. After AST was combined with different concentrations of AZD7762, the IC 50 The value is significantly lower than the single-drug IC of AST-3424 50 value.

[0257] Table 8: IC inhibition of proliferation of HT-29 cell line by AST alone or in combination with AZD7762 50 value

[0258] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, refers to the IC when AST is used alone or when AST is used in combination with AZD7762. 50 ratio.

[0259] The data of Figure 15 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST and AZD7762 after combined use. The results are shown in Figure 16.

[0260] Experimental summary: The above results show that in the p53-deficient (HT-29) cell line, the combination of different concentrations of AST and different concentrations of the cell cycle inhibitor AZD7762 has a significant cytotoxic additive effect; and this additive effect is still significant under the action of low concentrations of AST-3424 and low concentrations of the cell cycle inhibitor AZD7762.

[0261] 9. Results of the proliferation inhibition experiment of AST alone or in combination with Palbociclib on HT-29 cell line

[0262] According to the experimental results in Figure 17, the IC values ​​of AST alone or in combination with Palbociclib on the proliferation inhibition of HT-29 cell line were obtained. 50 The results are shown in Table 9 below. After AST was combined with different concentrations of Palbociclib, the IC 50 The value is significantly higher than the IC value of AST-3424 alone. 50 There is no difference in value.

[0263] Table 9: IC of AST alone or in combination with Palbociclib on the proliferation inhibition of HT-29 cell line 50 value

[0264] Note: Compounds, compounds or combinations of compounds; ratio, single / combo, single / combo refers to the IC ratio of AST alone and AST combined with Palbociclib. 50 ratio.

[0265] The data of Figure 17 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST after combined use of Palbociclib. The results are shown in Figure 18.

[0266] Experimental summary: The above results show that in the p53-deficient (HT-29) cell line, the combination of different concentrations of AST and different concentrations of the cell cycle inhibitor Palbociclib showed a certain degree of cytotoxicity additive effect under low-dose drug combination conditions.

[0267] 10. Results of the proliferation inhibition experiment of H460 cell line by AST alone or in combination with Adavosertib

[0268] According to the experimental results in Figure 19, the IC values ​​of AST alone or in combination with Adavosertib on the proliferation inhibition of H460 cell line were obtained. 50 The results are shown in Table 10 below. After AST was combined with different concentrations of Adavosertib, the IC 50 Value and IC of AST-3424 alone 50 There was no significant difference in the values.

[0269] Table 10: IC inhibition of proliferation of H460 cell line by AST alone or in combination with Adavosertib 50 value

[0270] Note: Compounds, compound or combination of compounds; ratio, single / combo, single / combo refers to the IC ratio of AST alone and AST combined with Adavosertib. 50 ratio.

[0271] The data of Figure 19 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and the inhibition rate of AST after combined use of Adavosertib at drug concentrations around 0% (IC0). The results are shown in Figure 20.

[0272] Experimental summary: The above results show that in the p53 wild-type (H460) cell line, the combination of different concentrations of AST and different concentrations of the cell cycle inhibitor Adavosertib did not show a toxic additive effect.

[0273] 11. Results of the proliferation inhibition experiment of H460 cell line by AST alone or in combination with AZD7762

[0274] According to the experimental results of 21, the IC of AST alone or in combination with AZD7762 on the proliferation inhibition of H460 cell line was obtained. 50 The results are shown in Table 11 below. After AST was combined with different concentrations of AZD7762, the IC value of 160nM AZD7762 was 50 Value and IC of AST alone 50 The values ​​differed by about 3 times. The IC values ​​of AZD7762 combined with other concentrations were 50 Value and IC of AST alone 50 There was no significant difference in the values.

[0275] Table 11: IC inhibition of proliferation of H460 cell line by AST alone or in combination with AZD7762 50 value

[0276] Note: Compounds refers to compounds or combinations of compounds; ratio, single / combo refers to the IC ratio when AST is used alone or when AST is used in combination with AZD7762. 50 ratio.

[0277] The data of Figure 21 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10) and 0% drug concentration (IC0), the inhibition rate of AST and AZD7762 after combined use. The results are shown in Figure 22.

[0278] Experimental summary: The above results show that in the p53 wild-type (H460) cell line, different concentrations of AST combined with different concentrations of the cell cycle inhibitor AZD7762, and the combination of 160nM AZD7762 compound had a certain degree of cytotoxic additive effect, while other concentrations of drug combinations did not show cytotoxic additive effects.

[0279] 12. Results of the proliferation inhibition experiment of H460 cell line by AST alone or in combination with Palbociclib

[0280] According to the experimental results in Figure 23, the IC50 values ​​of AST alone or in combination with Palbociclib on the proliferation inhibition of H460 cell line were fitted. The results are shown in Table 12 below. After AST was combined with different concentrations of Palbociclib, the IC 50 Value and IC of AST alone 50 There was no significant difference in the values ​​and they were greater than the IC of the single drug. 50 value.

[0281] Table 12: IC inhibition of proliferation of H460 cell line by AST alone or in combination with Palbociclib 50 value

[0282] Note: Compounds refers to compounds or combinations of compounds; ratio, single / combo refers to the IC ratio of AST alone to AST combined with Palbociclib. 50 ratio.

[0283] The data of Figure 23 were further processed to calculate the drug concentrations (IC 50 ), drug concentration of about 25% (IC 25 ), drug concentration of about 10% (IC 10 ) and 0% drug concentration (IC0), the inhibition rate of AST after combined use of Palbociclib. The results are shown in Figure 24.

[0284] Experimental summary: The above results show that in the p53 wild-type (H460) cell line, the combination of different concentrations of AST and different concentrations of the cell cycle inhibitor Palbociclib did not show an additive cytotoxic effect at each drug concentration.

[0285] 13. Results of the proliferation inhibition experiment of AST-3424 alone or in combination with Ceralasertib on HT29 / H460 cell lines

[0286] The first experiment was performed to obtain the proliferation inhibition curve of AST-3424 alone or in combination with Ceralasertib on the HT29 / H460 cell line in Figure 25. Based on the experimental results in Figure 25, the proliferation inhibition IC of AST-3424 alone or in combination with Ceralasertib on the HT29 / H460 cell line was obtained by fitting. 50 The results are shown in Table 13 below.

[0287] Table 13: IC of AST-3424 alone or in combination with Ceralasertib against proliferation of HT29 / H460 cell lines 50 Value (first experiment)

[0288] Note: Ratio means ratio.

[0289] The experiment was repeated for the second time to obtain the proliferation inhibition curve of AST-3424 alone or in combination with Ceralasertib on the HT29 / H460 cell line in Figure 26. Based on the experimental results in Figure 26, the proliferation inhibition IC of AST-3424 alone or in combination with Ceralasertib on the HT29 / H460 cell line was obtained by fitting. 50 The results are shown in Table 14 below.

[0290] Table 14: IC of AST-3424 alone or in combination with Ceralasertib against proliferation of HT29 / H460 cell lines 50 Value (second experiment)

[0291] Note: Ratio means ratio.

[0292] In order to further explore the effect of drug addition order on the combination effect, the order of drug addition was changed to add Ceralasertib first and then add AST-3424. The experimental results are shown in the proliferation inhibition curve in Figure 27. According to the experimental results in Figure 27, the IC of AST-3424 alone or in combination with Ceralasertib on the HT29 / H460 cell line was fitted. 50 The results are shown in Table 15 below.

[0293] Table 15: IC values ​​of AST-3424 alone or in combination with Ceralasertib on the proliferation inhibition of HT29 cell lines at different dosing orders 50 value

[0294] Note: Compounds refers to compounds or combinations of compounds; ratio, single / combo refers to the IC when AST is used alone or when AST is used in combination with Palbociclib. 50 ratio.

[0295] Experimental summary:

[0296] The combination of AST-3424 and Ceralasertib (ATR inhibitor) has a significant additive effect on the proliferation of H460 cells (p53 wild type).

[0297] AST-3424, when used in combination with various concentrations of Ceralasertib (ATR inhibitor), had a very significant additive effect on the proliferation of HT29 cells (p53 deficiency), and the additive effect was stronger than that of the H460 cell line.

[0298] There was no difference in the additive cytotoxic effect of AST-3424 and Ceralasertib when different dosing orders were used.

[0299] Summary of in vitro cell experiments with compounds AST and AST-3424:

[0300] (1) In the p53-deficient (HT-29) cell line, the combination of AST or AST-3424 with the cell cycle inhibitors Adavosertib, AZD7762, Palbociclib, and Ceralasertib, respectively, had a significant additive cytotoxic effect; and this additive effect was still significant under the action of low concentrations of AST or AST-3424 and low concentrations of cell cycle inhibitors, respectively.

[0301] (2) In p53 wild-type (H460) cells, medium and high concentrations of AST or AST-3424 combined with Adavosertib, AZD7762, and Ceralasertib, respectively, had a certain degree of cytotoxic additive effect; this additive effect was not significant when low concentrations of AST or AST-3424 were combined with low concentrations of cell cycle inhibitors.

[0302] (3) In p53 wild-type (H460) cells, AST-3424 or AST combined with Palbociclib did not show significant additive cytotoxic effects.

[0303] The following further investigates the results of experimental inhibition of HT-29 / H460 cell proliferation by TH-302 alone or in combination with Adavosertib / Ceralasertib. The experimental methods are as follows, under different experimental conditions of hypoxia and normoxia:

[0304] 1) Add HT-29 cell suspension to two types of 24-well plates, 495 μL per well, with a cell density of 5×10 4 24-well plates with glass inserts were used for hypoxia experiments, and ordinary plastic 24-well plates were used for normoxic experiments.

[0305] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0306] 3) Compound treatment

[0307] Hypoxic conditions (O2 < 0.01%):

[0308] Adjust the hypoxia workstation to a hypoxic environment and use an oxygen indicator to confirm the hypoxia condition in the workstation.

[0309] 24 hours after cell plating, the 24-well plate with the glass insert was transferred to the hypoxia workstation.

[0310] Place the 24-well plate on a shaker, open the cover of the well plate and shake to perform gas exchange for 5 minutes.

[0311] First, remove 5 μL or 10 μL of culture medium from each well of the combination plate, then add 5 μL of Davosertib (final concentrations of 1 μM and 10 μM, respectively) or Ceralasertib (final concentrations of 200 μM and 2000 μM, respectively) and incubate for 2 hours.

[0312] Add 5 μL or 10 μL of 100X corresponding concentration of compound to each well, and perform 3 replicate wells for each experimental group.

[0313] The compounds were mixed gently by shaking, and the 24-well plate lid was half-opened and incubated in a hypoxic workstation for 3 hours.

[0314] Normoxic conditions (O2 21%):

[0315] 24 hours after cell plating, 5 μL or 10 μL of culture medium was removed from each well of the combination plate, and then 5 μL of Adavosertib (final concentrations of 1 μM and 10 μM, respectively) or Ceralasertib (final concentrations of 200 μM and 2000 μM, respectively) was added and incubated for 2 hours.

[0316] 5 μL or 10 μL of 100-fold corresponding concentration of compound was added to each well, and 3 replicates were added to each experimental group.

[0317] The mixture was gently shaken to ensure uniform mixing of the compounds, and the 24-well plate was incubated in a standard incubator at 37°C and 5% CO2 for 3 hours.

[0318] 4) After 3 hours of co-treatment with the compounds, all 24-well plates were washed twice with culture medium, 500 μL per well each time.

[0319] 5) Add 500 μL of culture medium to each well.

[0320] 6) Place in a 37°C, 5% CO2 incubator for 72 hours.

[0321] 7) Discard 300 μL of culture medium from each well, add 50 μL of CTG, shake and mix for 2 minutes, and place at room temperature in the dark for 15 minutes.

[0322] 8) Transfer 100 μL of culture medium from each well of the 24-well plate to a 96-well white plate.

[0323] 9) Read the chemiluminescent signal using a multifunctional microplate reader with a reading time of 1000 ms.

[0324] 10) Calculate IC using GraphPad Prism 5 software 50 , and obtain the IC of the compound 50 (half inhibitory concentration).

[0325] In the H460 cell proliferation inhibition experiment, the final concentrations of Adavosertib added were 3μM and 30μM, and the final concentrations of Ceralasertib added were 2μM, 20μM, and 1000μM.

[0326] 14. Results of the proliferation inhibition experiment of TH-302 alone or in combination with Adavosertib / Ceralasertib on HT29 / H460 cell lines

[0327] According to the above experimental method, the IC values ​​of TH-302 alone or in combination with Adavosertib against HT29 / H460 cell lines under normoxic and hypoxic conditions were obtained. 50 The values ​​are shown in Tables 16 and 17, and the corresponding proliferation inhibition curves are shown in Figures 28 and 29.

[0328] Table 16: Results of the proliferation inhibition experiment of HT29 cell line with TH-302 alone or in combination with Adavosertib under normoxic and hypoxic conditions

[0329] Note: Compounds refers to compounds or combinations of compounds; Ratio refers to ratio.

[0330] Table 17: Results of the proliferation inhibition experiment of TH-302 alone or in combination with Adavosertib on H460 cell line

[0331] Note: Compounds refers to compounds or combinations of compounds; Ratio refers to ratio.

[0332] According to the above experimental method, the IC values ​​of TH-302 alone or in combination with Ceralasertib against HT29 / H460 cell lines under normoxic and hypoxic conditions were obtained. 50 The values ​​are shown in Tables 18 and 19, and the corresponding proliferation inhibition curves are shown in Figures 30 and 31.

[0333] Table 18: Results of the proliferation inhibition experiment of HT29 cell line by TH-302 alone or in combination with Ceralasertib

[0334] Note: Compounds refers to compounds or combinations of compounds; Ratio refers to ratio.

[0335] Table 19: Results of the proliferation inhibition experiment of H460 cell line by TH-302 alone or in combination with Ceralasertib

[0336] Note: Compounds refers to compounds or combinations of compounds; Ratio refers to ratio.

[0337] Summary of in vitro cell experiments with compound TH-302

[0338] (1) In the p53-deficient (HT29) cell line, TH-302 had no significant cytotoxic enhancement effect when combined with the G2 / M cell cycle regulator Adavosertib under normoxic conditions.

[0339] (2) In the p53-deficient (HT29) cell line, TH-302 showed a significant cytotoxic enhancement effect when combined with the G2 / M cell cycle regulator Adavosertib under hypoxic conditions and at high concentrations of Adavosertib.

[0340] (3) In p53 wild-type (H460) cells, TH-302 had no significant cytotoxic enhancement effect when combined with the G2 / M cell cycle regulator Adavosertib, regardless of normoxic or hypoxic conditions.

[0341] (4) In the p53-deficient (HT29) cell line, TH-302 combined with the ATR kinase inhibitor Ceralasertib had no significant cytotoxic enhancement effect when used together under normoxic conditions.

[0342] (5) In the p53-deficient (HT29) cell line, TH-302 was combined with the ATR kinase inhibitor Ceralasertib. When used together under hypoxic conditions, high concentrations of Ceralasertib showed a significant cytotoxic enhancement effect.

[0343] (6) In p53 wild-type (H460) cells, TH-302 combined with the ATR kinase inhibitor Ceralasertib had no significant cytotoxic enhancement effect when used together under normoxic or hypoxic conditions.

[0344] In the above embodiments, compounds AST-3424, AST and TH-302 are all DNA alkylating agent prodrugs, which can specifically release the DNA alkylating agent small molecule AST-2660 (or 2660) or Br-IPM under certain circumstances.

[0345] Take AST-3424 and TH-302 as examples:

[0346] The DNA alkylating agents ultimately released by these compounds are cell cycle nonspecific, meaning they act on the double-stranded DNA structure at all stages of the cell cycle. Alkylating agents can form very tight bonds between DNA double strands, preventing DNA from completing processes such as transcription, translation, and replication. This means they can cause DNA damage and inhibit proliferation in cells at all stages of the cell cycle.

[0347] The compound Adavosertib is a selective inhibitor of Wee1 kinase, a key kinase involved in the G2 / M checkpoint of the cell cycle and DNA damage repair. Cyclin-dependent kinases (CDKs) regulate the cell cycle by phosphorylating specific substrates. Their activity depends on binding to cyclins to form a complex and is also regulated by phosphorylation. The phosphorylation state of CDK1 further controls the G2 / M checkpoint. Wee1 phosphorylates CDK1 at Tyr15, maintaining CDK1 in an inactive state. This inhibits the activity of the CDK1-cyclin B complex, arresting cell division at the G2 / M checkpoint and negatively regulating the cell cycle. Its biological significance is to repair DNA damage that has not yet been repaired, preventing cells from entering mitosis with DNA damage. In other words, Adavosertib regulates the phosphorylation state of CDK1 by inhibiting Wee1 kinase, thereby regulating cell cycle progression. This means that Adavosertib is an indirect CDK inhibitor and can be considered a broad CDK inhibitor. Palbociclib is an orally active, selective inhibitor of CDK4 and CDK6. Therefore, both Adavosertib and Palbociclib, as CDK inhibitors, are cell cycle-specific, acting at specific stages of the cell cycle.

[0348] A large number of literature reports show that p53 protein plays an important regulatory role in DNA repair in the G1 / S phase of the cell cycle. Cells lacking p53 protein have inhibited DNA repair in the G1 / S phase. Therefore, when G2 / M cell cycle arrest inhibitors are used in such cells, the DNA repair ability in the G2 / M cycle can be significantly inhibited.

[0349] The experiments in this application revealed that the combination of medium and high concentrations of Adavosertib with AST and AST-3424 has a significant additive cytotoxic effect on tumor cell lines, and for p53-deficient tumor cell lines, the combination at low concentrations shows a significant additive cytotoxic effect; the combined action of Palbociclib with AST and AST-3424 has a significant additive cytotoxic effect only on p53-deficient tumor cell lines; the combination of high concentrations of Adavosertib with TH-302 has a significant additive cytotoxic effect only on p53-deficient tumor cell lines.

[0350] Based on the above experimental facts, those skilled in the art can infer that AST, AST-3424, and TH-302 are all DNA alkylating agents covered by the general structural formulas (1) to (9) as prodrug compounds that ultimately act on the DNA double-strand cross-linking process, and that Adavosertib and Palbociclib are both CDK inhibitors that act on specific cell cycles: the alkylating agent prodrug compound and the CDK inhibitor that acts on specific cell cycles have an additive cytotoxic effect on tumor cell lines, that is, the combination of the two drugs to treat cancer and tumor patients will have a better therapeutic effect, and p53 deficiency will enhance the above-mentioned additive effect / combination effect.

[0351] AZD7762 is a potent, ATP-competitive cell cycle checkpoint kinase (Chk) inhibitor that abrogates the S and G2 checkpoints induced by DNA damage and is a Chk inhibitor. AZD7762 is cell cycle-specific as a Chk inhibitor, acting at specific stages of the cell cycle.

[0352] The experiments in this application revealed that the combination of medium and high concentrations of AZD7762 with AST and AST-3424 has a significant additive cytotoxic effect on tumor cell lines, while for p53-deficient tumor cell lines, the combination at low concentrations shows a significant additive cytotoxic effect.

[0353] Based on the above experimental facts, those skilled in the art can infer that, on the basis that AST and AST-3424 are both DNA alkylating agents covered by the general structural formulas (1) to (9) and serve as prodrug compounds that ultimately act on the DNA double-strand cross-linking process, and that AZD7762 is a ChK inhibitor that acts on a specific cell cycle, that the alkylating agent prodrug compound and the CHK inhibitor that acts on a specific cell cycle have an additive cytotoxic effect on tumor cell lines, that is, the combination of the two drugs in the treatment of cancer and tumor patients will have a better therapeutic effect, and that p53 deficiency will enhance the above additive effect / combination effect.

[0354] Ceralasertib (AZD6738) is a potent ATR kinase inhibitor that is cell cycle specific and acts at specific stages of the cell cycle.

[0355] The experiments in this application revealed that the combination of medium and high concentrations of Ceralasertib with AST and AST-3424 has a significant additive cytotoxic effect on tumor cell lines, while for P53-deficient tumor cell lines, the combination at low concentrations shows a significant additive cytotoxic effect; the combination of high concentrations of Ceralasertib with TH-302 has a significant additive cytotoxic effect only on p53-deficient tumor cell lines.

[0356] Based on the above experimental facts, those skilled in the art can infer that AST, AST-3424, and TH-302 are all DNA alkylating agents covered by the general structural formulas (1) to (9) as prodrug compounds that ultimately act on the DNA double-strand cross-linking process, and Ceralasertib is an ATR inhibitor that acts on a specific cell cycle, that: the alkylating agent prodrug compound and the ATR inhibitor that acts on a specific cell cycle have an additive cytotoxic effect on tumor cell lines, that is, the combination of the two drugs to treat cancer and tumor patients will have a better therapeutic effect, and p53 deficiency will enhance the above-mentioned additive effect / combination effect.

[0357] Furthermore, it can be inferred that the combination of a cell cycle-nonspecific alkylating agent prodrug compound and a cell cycle inhibitor that acts on a specific cell cycle has an additive cytotoxic effect on tumor cells, that is, the combination of the two drugs will have a better therapeutic effect in treating cancer and tumor patients; and further, p53 deficiency will enhance the above-mentioned additive effect / combination effect, that is, patients with p53 gene deficiency may benefit more from the combination.

Claims

1. A method of treating cancer or tumor using a drug containing an alkylating agent prodrug compound and its salts, esters, solvates, and isotopic isomers in combination with a drug containing a cell cycle inhibitor compound and its salts, esters, solvates, and isotopic isomers.

2. The method of claim 1, wherein the cell cycle inhibitor is selected from the group consisting of CDK inhibitors, WEE inhibitors, CHK inhibitors, and ATR inhibitors.

3. The method of claim 2, wherein the alkylating agent prodrug compound is selected from the group consisting of an AKR1C3 enzyme-activated alkylating agent prodrug compound and a hypoxia-activated alkylating agent prodrug compound. Preferably, the AKR1C3 enzyme-activating alkylating agent prodrug compound is used in combination with a CDK inhibitor, a WEE inhibitor, a CHK inhibitor, or an ATR inhibitor; Hypoxia-activated alkylating agent prodrug compounds are used in combination with CDK inhibitors, WEE inhibitors, and ATR inhibitors.

4. The method of claim 1, wherein Alkylating agent prodrug compounds include chemical structural formulas (1)-(9): wherein R is independently selected from H, -CH3, -CH2CH3, -CF3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, and TsO; Wherein, the definitions of R1, R2, R3, and Cx are as described in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1; Among them, R1, R2, R3, R4, R5, R6, R7, R8, R9, R 10 、R 11 、R 12 、R 13 、R 14 、R 15 、R 16 、R 17 The definition of is as set forth in the claims of patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A); Among them, X, Y, Z, R, T, A and X 10 The definition of 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, 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; 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 SO2R 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, form a 5-7 membered heterocyclic ring; wherein the alkyl group and the aryl group are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently 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, Rw is defined as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1; Among them, the definitions of A, E, G, X, and Y are as recorded in the claims in patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding Chinese application number 2019800234236, publication number CN111918864A).

5. The method of claim 1 or 2, wherein: The cancer or tumor patient or biological sample thereof is found to have specific gene deletion or damage, The specific gene is selected from genes involved in cell cycle checkpoint regulation, preferably p53, p21, CCNB1, WIP1, 14-3-3 sigma protein, cdc2 / cycB.

6. The method of claim 3, wherein: The CDK inhibitor is selected from the group consisting of Palbociclib, Ribociclib, Abemaciclib, Trilaciclib, dalpiciclib, Adavosertib, Ro-3306, Dinaciclib, Cirtuvivint, Rintodestrant, DS96432529, THZ1, THZ531, Seliciclib, Flavopiridol, AZD4573, SR-4835, Simurosertib, Fadraciclib, NVP-2, SNS-032, (E / Z)-Zotiraciclib, AZD-5438, AT7519, Mevociclib, Kenpaullone, YKL-5-124 TFA, NG 52, and GSK 3 Inhibitor. IX, OTS964, Samuraciclib, Flavopiridol, KB-0742 dihydrochloride, (+)-Enitociclib, AUZ 454, SY-5609, SEL120-34A monohydrochloride, CCT-251921, MBQ-167, XL413 hydrochloride, BI-1347, THAL-SNS-032, JNJ-7706621, TG003, LDC4297, BMS-265246, Roniciclib, CGP60474, (R)-CR8 trihydrochloride, R547, Milciclib, T025, AS2863619, Senexin A. BSJ-4-116, CLK-IN-T3, CDK12-IN-3, CVT-313, Atuveciclib, PHA-793887, Indirubin-3'-monoxime, YKL-5-124, PHA-767491 hydrochloride, KH-CB19, Cucurbitacin E, Purvalanol A, BSJ-03-204, ON123300, CDK5 inhibitor 20-223, Riviciclib, FN-1501, CP-10, THZ2, Abemaciclib metabolite M2, BS-181, CDK12-IN-E9, Samuraciclib, RGB-286638, CDK2-IN-4, LDC000067, ML167, Purvalanol B. NU6300, CLK1-IN-1, FMF-04-159-2, CKI-7, CDKI-73, MSC2530818, BSJ-04-132, NU6102, Voruciclib, Olomoucine; A WEE inhibitor selected from the group consisting of WEE1-IN-5, WEE1-IN-3, WEE1-IN-4, LEB-03-146, LEB-03-144, Adavosertib, LEB-03-153, LEB-03-145, PD407824, PD0166285, PD0166285 dihydrochloride, Pomalidomide-C3-adavosertib, DB0614, and FMF-06-098-1; A CHK inhibitor selected from AZD7762, Prexasertib, SCH900776, GDC-0425, Chk1-IN-6, CCT245737, BML-277, CCT241533, PD407824, CHIR-124, CCT244747, PF477736, GDC-0575, SB-218078, MRT00033659, ANI-7, SAR-020106, CCT241533, CHK1-IN-4, VER-00158411, CHK1-IN-3, Chk1-IN-5, CHK1-IN-2, CHK-IN-1; The ATR inhibitor is selected from Ceralasertib, Berzosertib, Gartisertib, BAY1895344, BAY-937, AZ20, ETP-46464, Dactolisib, VE-821, M1774, ATRN-199, RP-3500, and ART-0380.

7. The method of claim 4, wherein: The hypoxia-activated alkylating agent prodrug compound of formula (1) is selected from the following compounds: The hypoxia-activated alkylating agent prodrug compound of formula (2) is selected from the following compounds: The hypoxia-activated alkylating agent prodrug compound of formula (3) is selected from the following compounds: The AKR1C3 enzyme-activated alkylating agent prodrug compound of formula (4) is selected from the following compounds: The AKR1C3 enzyme-activated prodrug compound of formula (6) is selected from the compounds of the following structures: The AKR1C3 enzyme-activated prodrug compound of formula (5) is selected from the compounds of the following structures: The AKR1C3 enzyme-activated prodrug compound of formula (7) is selected from the compounds of the following structures: as well as and The AKR1C3 enzyme-activated prodrug compound of formula (8) is selected from the compounds of the following structures: The AKR1C3 enzyme-activated prodrug compound of formula (9) is selected from the compounds of the following structures:

8. A pharmaceutical composition comprising an alkylating agent prodrug compound or its salt, ester, solvate, or isotope isomer and a cell cycle inhibitor compound or its salt, ester, solvate, or isotope isomer, wherein the pharmaceutical composition is used to treat cancer or tumor patients.

9. The pharmaceutical composition according to claim 8, wherein The alkylating agent prodrug compound is selected from AKR1C3 enzyme-activated alkylating agent prodrug compounds and hypoxia-activated alkylating agent prodrug compounds, preferably AKR1C3 enzyme-activated DNA alkylating agent prodrug compounds and hypoxia-activated DNA alkylating agent prodrug compounds; or The cell cycle inhibitor is selected from CDK inhibitors, WEE inhibitors, CHK inhibitors, and ATR inhibitors; or The alkylating agent prodrug compound comprises the chemical structural formulas (1) to (9) as claimed in claim 4; or The cancer or tumor patient or biological sample thereof is found to have a specific gene deletion or damage, wherein the specific gene is selected from genes involved in cell cycle checkpoint regulation, preferably p53, p21, CCNB1, WIP1, 14-3-3 sigma protein, cdc2 / cycB; or The CDK inhibitor is selected from the group consisting of Palbociclib, Ribociclib, Abemaciclib, Trilaciclib, dalpiciclib, Adavosertib, Ro-3306, Dinaciclib, Cirtuvivint, Rintodestrant, DS96432529, THZ1, THZ531, Seliciclib, Flavopiridol, AZD4573, SR-4835, Simurosertib, Fadraciclib, NVP-2, SNS-032, (E / Z)-Zotiraciclib, AZD-5438, AT7519, Mevociclib, Kenpaullone, YKL-5-124 TFA, NG 52, and GSK 3 Inhibitor. IX, OTS964, Samuraciclib, Flavopiridol, KB-0742 dihydrochloride, (+)-Enitociclib, AUZ 454, SY-5609, SEL120-34A monohydrochloride, CCT-251921, MBQ-167, XL413 hydrochloride, BI-1347, THAL-SNS-032, JNJ-7706621, TG003, LDC4297, BMS-265246, Roniciclib, CGP60474, (R)-CR8 trihydrochloride, R547, Milciclib, T025, AS2863619, Senexin A. BSJ-4-116, CLK-IN-T3, CDK12-IN-3, CVT-313, Atuveciclib, PHA-793887, Indirubin-3'-monoxime, YKL-5-124, PHA-767491 hydrochloride, KH-CB19, Cucurbitacin E, Purvalanol A. BSJ-03-204, ON123300, CDK5 inhibitor 20-223, Riviciclib, FN-1501, CP-10, THZ2, Abemaciclib metabolite M2, BS-181, CDK12-IN-E9, Samuraciclib, RGB-286638, CDK2-IN-4, LDC000067, ML167, PurvalanolB、NU6300、CLK1-IN-1、FMF-04-159-2、CKI-7、CDKI-73、MSC2530818、BSJ-04-132、NU6102、Voruciclib、Olomoucine; A WEE inhibitor selected from the group consisting of WEE1-IN-5, WEE1-IN-3, WEE1-IN-4, LEB-03-146, LEB-03-144, Adavosertib, LEB-03-153, LEB-03-145, PD407824, PD0166285, PD0166285 dihydrochloride, Pomalidomide-C3-adavosertib, DB0614, and FMF-06-098-1; A CHK inhibitor selected from AZD7762, Prexasertib, SCH900776, GDC-0425, Chk1-IN-6, CCT245737, BML-277, CCT241533, PD407824, CHIR-124, CCT244747, PF477736, GDC-0575, SB-218078, MRT00033659, ANI-7, SAR-020106, CCT241533, CHK1-IN-4, VER-00158411, CHK1-IN-3, Chk1-IN-5, CHK1-IN-2, CHK-IN-1; ATR inhibitor selected from Ceralasertib, Berzosertib, Gartisertib, BAY1895344, BAY-937, AZ20, ETP-46464, Dactolisib, VE-821, M1774, ATRN-199, RP-3500, ART-0380; or The alkylating agent prodrug compound is selected from the specific compounds described in claim 7.

10. Use of an alkylating agent prodrug compound or its salt, ester, solvate, or isotope isomer in the preparation of a medicament for treating cancer or tumors in combination with a cell cycle inhibitor compound or its salt, ester, solvate, or isotope isomer.

11. The use according to claim 10, wherein The alkylating agent prodrug compound is selected from AKR1C3 enzyme-activated alkylating agent prodrug compounds and hypoxia-activated alkylating agent prodrug compounds, preferably AKR1C3 enzyme-activated DNA alkylating agent prodrug compounds and hypoxia-activated DNA alkylating agent prodrug compounds; or The cell cycle inhibitor is selected from CDK inhibitors, WEE inhibitors, CHK inhibitors, and ATR inhibitors; or The alkylating agent prodrug compound comprises the chemical structural formulas (1) to (9) as claimed in claim 4; or The cancer or tumor patient or biological sample thereof is found to have a specific gene deletion or damage, wherein the specific gene is selected from genes involved in cell cycle checkpoint regulation, preferably p53, p21, CCNB1, WIP1, 14-3-3 sigma protein, cdc2 / cycB; or The CDK inhibitor is selected from the group consisting of Palbociclib, Ribociclib, Abemaciclib, Trilaciclib, dalpiciclib, Adavosertib, Ro-3306, Dinaciclib, Cirtuvivint, Rintodestrant, DS96432529, THZ1, THZ531, Seliciclib, Flavopiridol, AZD4573, SR-4835, Simurosertib, Fadraciclib, NVP-2, SNS-032, (E / Z)-Zotiraciclib, AZD-5438, AT7519, Mevociclib, Kenpaullone, YKL-5-124 TFA, NG 52, and GSK 3 Inhibitor. IX, OTS964, Samuraciclib, Flavopiridol, KB-0742 dihydrochloride, (+)-Enitociclib, AUZ 454, SY-5609, SEL120-34A monohydrochloride, CCT-251921, MBQ-167, XL413 hydrochloride, BI-1347, THAL-SNS-032, JNJ-7706621, TG003, LDC4297, BMS-265246, Roniciclib, CGP60474, (R)-CR8 trihydrochloride, R547, Milciclib, T025, AS2863619, Senexin A. BSJ-4-116, CLK-IN-T3, CDK12-IN-3, CVT-313, Atuveciclib, PHA-793887, Indirubin-3'-monoxime, YKL-5-124, PHA-767491 hydrochloride, KH-CB19, Cucurbitacin E, Purvalanol A. BSJ-03-204, ON123300, CDK5 inhibitor 20-223, Riviciclib, FN-1501, CP-10, THZ2, Abemaciclib metabolite M2, BS-181, CDK12-IN-E9, Samuraciclib, RGB-286638, CDK2-IN-4, LDC000067, ML167, PurvalanolB、NU6300、CLK1-IN-1、FMF-04-159-2、CKI-7、CDKI-73、MSC2530818、BSJ-04-132、NU6102、Voruciclib、Olomoucine; A WEE inhibitor selected from the group consisting of WEE1-IN-5, WEE1-IN-3, WEE1-IN-4, LEB-03-146, LEB-03-144, Adavosertib, LEB-03-153, LEB-03-145, PD407824, PD0166285, PD0166285 dihydrochloride, Pomalidomide-C3-adavosertib, DB0614, and FMF-06-098-1; A CHK inhibitor selected from AZD7762, Prexasertib, SCH900776, GDC-0425, Chk1-IN-6, CCT245737, BML-277, CCT241533, PD407824, CHIR-124, CCT244747, PF477736, GDC-0575, SB-218078, MRT00033659, ANI-7, SAR-020106, CCT241533, CHK1-IN-4, VER-00158411, CHK1-IN-3, Chk1-IN-5, CHK1-IN-2, CHK-IN-1; ATR inhibitor selected from Ceralasertib, Berzosertib, Gartisertib, BAY1895344, BAY-937, AZ20, ETP-46464, Dactolisib, VE-821, M1774, ATRN-199, RP-3500, ART-0380; or The alkylating agent prodrug compound is selected from the specific compounds described in claim 7.

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