Treatment of p53 gene mutation or defect negative cancer and tumor patients

Abstract

The invention relates to a method for treating p53 gene mutation or defect negative cancer and tumor patients by using a single drug containing a DNA alkylating agent prodrug compound or combining with other drugs, and a pharmaceutical application. In particular, the DNA alkylating agent prodrug compound is selected from the group consisting of a hypoxia activated DNA alkylating agent prodrug compound, an AKR1C3 activated DNA alkylating agent prodrug compound, a [beta]-glucosidase ([beta]-D-Glucosidase), or a [beta]-galactosidase ([beta]-galactosidase) activated DNA alkylating agent prodrug compound.

Description

Treatment of patients with p53 gene mutation or defect-negative cancers and tumors Technical Field

[0001] The present invention relates to a method for treating cancer, in particular to a method for treating patients with p53 gene mutation or defect-negative cancer or tumor. Background Art

[0002] AST-3424 (WO2016145092, WO2017087428), a DNA alkylating agent prodrug targeting overexpressed aldehyde-keto reductase 1C3 (AKR1C3), has a CAS number of 2097713-69-2 and its structure is as follows:

[0003] Chemical structure of AST-3424

[0004] AST-3424 (also known as OBI-3424 and TH-3424) enters cancer cells and is activated by the overexpressed AKR1C3 enzyme, releasing the metabolite AST-2660 (also known as AST-2660). AST-3424 itself has minimal toxicity to cancer cells, and its pharmacological effects have been shown to correlate with AKR1C3 enzyme expression in animal models and in vitro pharmacological studies. The prodrug AST-3424 is metabolized to AST-2660 by the AKR1C3 enzyme and NADPH, and enzyme expression is positively correlated with drug efficacy (References 1; 2; 3; 4).

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

[0006] Currently, the drug has entered Phase I / II clinical trials in China and the United States, respectively (US NCT03592264, indications: liver cancer, pancreatic cancer and other solid tumors, applicant: OBIPharama Inc (4174), drug named OBI-3424; US NCT04315324, indications: T-ALL / T-LBL (acute T-lymphocytic leukemia / T-lymphoblastic lymphoma), drug named OBI-3424; China CTR20191399, indications: various solid tumors, applicant: Ascentawits Pharmaceuticals, LTD., drug named AST-3424; CTR20201915, indications: acute T-lymphocytic leukemia and acute B-lymphocytic leukemia, applicant: Ascentawits Pharmaceuticals, LTD., drug named AST-3424).

[0007] Currently, the clinical trials of the above-mentioned drugs are still proceeding normally. In the corresponding Phase II clinical trials, patients are screened for treatment by detecting their AKR1C3 enzyme expression levels.

[0008] Summary of the Invention

[0009] In the Phase II clinical trial of AST-3424 conducted in China, the applicant found that the treatment effect of AST-3424 was significantly better for patients with cancer or tumors with negative p53 gene mutation or defect than for patients with positive p53 gene mutation or defect. Therefore, the applicant speculates that AST-3424 will have a better therapeutic effect in treating patients with tumors or cancers with negative p53 gene mutation or defect test results, that is, patients with tumors or cancers with negative p53 gene mutation or defect test results will have more obvious clinical benefits from AST-3424 treatment.

[0010] To this end, the applicant proposes the following pharmaceutical uses of anticancer drugs and methods for treating tumors and cancers.

[0011] A treatment method, which uses a drug containing a DNA alkylating agent prodrug compound alone or in combination with other drugs to treat cancer and tumor patients who are negative for p53 gene mutation or have normal p53 protein expression.

[0012] The invention relates to the use of DNA alkylating agent prodrug compounds in the preparation of drugs for treating cancer and tumor patients with negative p53 gene mutation test or normal p53 protein expression, either alone or in combination with other drugs.

[0013] The DNA alkylating agent prodrug compound refers to a prodrug compound that can metabolize the DNA alkylating agent in vivo.

[0014] A DNA alkylating agent prodrug compound refers to a prodrug compound that is metabolized and converted into a DNA alkylating agent, and the DNA alkylating agent ultimately undergoes alkylation reaction with DNA in cancer cells to destroy the DNA structure of the cancer cells and ultimately cause cell death.

[0015] Most prodrug compounds will be metabolized and transformed under physiological conditions. For anti-tumor and anti-cancer drugs, these physiological environments are usually microenvironments unique to tumor tissues or cancer cells, such as high expression of certain transport proteins on the cell membrane, high expression or concentration of certain enzymes or proteins in the intracellular or extracellular environment higher than normal cells due to enrichment, or hypoxia or abnormal pH, etc. Therefore, the existence of these microenvironments is generally believed to be caused by certain specific mechanisms of tumor tissues or cancer cells.

[0016] By activating the metabolic conversion of prodrugs into active drugs through these microenvironments, tumor / cancer cell targeting in the above microenvironments is achieved. Currently, the known microenvironment targets reported in the literature include various enzyme activations, such as AKR1C3 enzyme activation, β-glucosidase (β-D-Glucosidase) activation, carboxylesterase (Carboxylesterase) activation, esterase and caspase-3 (Esterase and caspase-3) activation, cathepsin B (Cathepsin B) activation, γ-glutamyl transpeptidase (γ-Glutamyltranspeptidase) activation, β-galactosidase (β-galactosidase) activation or hypoxia activation.

[0017] The DNA alkylating agent prodrug compound is selected from the group consisting of AKR1C3 enzyme activation, β-glucosidase (β-D-Glucosidase) activation, carboxylesterase activation, esterase and caspase-3 activation, cathepsin B activation, γ-glutamyl transpeptidase (γ-Glutamyltranspeptidase) activation, β-galactosidase (β-galactosidase) activation or hypoxia-activated DNA alkylating agent prodrug compounds, preferably AKR1C3 enzyme activation, β-glucosidase (β-D-Glucosidase) activation, β-galactosidase (β-galactosidase) activation, and hypoxia-activated DNA alkylating agent prodrug compounds.

[0018] The DNA alkylating agent prodrug compound is selected from a hypoxia-activated DNA alkylating agent prodrug compound, an AKR1C3-activated DNA alkylating agent prodrug compound, a β-glucosidase (β-D-Glucosidase)-activated or β-galactosidase (β-galactosidase)-activated DNA alkylating agent prodrug compound.

[0019] Preferably, the hypoxia-activated DNA alkylating agent prodrug compound is selected from structural formulas 1-3 and their salts, esters, solvates, and isotopomers, the AKR1C3-activated DNA alkylating agent prodrug compound is selected from structural formulas 4-12 and their salts, esters, solvates, and isotopomers, and the β-glucosidase (β-D-Glucosidase)-activated or β-galactosidase (β-galactosidase)-activated DNA alkylating agent prodrug compound is selected from structural formula 15 and its salts, esters, solvates, and isotopomers.

[0020] 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.

[0021] 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, WO2008083101A1 and WO2007002931A3. The present invention hereby introduces the full text of the above application texts.

[0022] TH-302 or its similar compounds It is a DNA alkylating anticancer drug with a wide range of cancer therapeutic potential. 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, WO2009033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO2020118 251A1, WO2014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, WO2015069489A1, WO2012006032A2, WO201802 6606A2, WO2010048330A2, WO2015171647A1, WO2013096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO2012145684A2, WO201601 4390A2, WO2019055786A2, WO2012135757A2, WO2015013448A2, WO2016011328A2, WO2013177633A2, WO2016011195A2, WO2015051921A2) and FDA-registered clinical trials (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT01 149915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, 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.

[0023] 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:

[0024] 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;

[0025] 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 ,

[0026] 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;

[0027] The group can replace the hydrogen atom at any position on the condensed ring carbon atom, and the number of substitutions is 1;

[0028] 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;

[0029] 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.

[0030] 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:

[0031] 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;

[0032] R 21 and R 22Each 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;

[0033] R 23 is hydrogen, C1-C6 alkyl or C6-C10 aryl;

[0034] R2 and R3 are independently hydrogen or halogen;

[0035] R4 is hydrogen, halogen, C1-C6 alkoxy, C1-C6 alkyl or C6-C10 aryl,

[0036] 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;

[0037] R6 and R 10 are independently hydrogen or halo;

[0038] R8 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or 5-15 membered heteroaryl;

[0039] R 11 Each is independently a C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl or C6-C10 aryl;

[0040] 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;

[0041] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, alkoxy, and ether groups are optionally substituted.

[0042] Formula (4) is an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound, more specifically, it is an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0043] Among them, X, Y, Z, R, T, A and X10 The definition is as described in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), and the synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety, and are specifically defined as:

[0044] X 10 O, S, SO or SO2;

[0045] A is C6-C 10 Aryl, 5-15 membered heteroaryl or -N=CR 1 R 2 ;

[0046] 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 ;

[0047] 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 ;

[0048] 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 ;

[0049] R 13 and R 14 are independently hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4-15 membered heterocycle or ether;

[0050] T comprises a phosphoramidite alkylating agent comprising one or more alkylating agents bonded to -OP(Z 1 ) part of Z 5 -X5 -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

[0051] These alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, heteroaryl, and ether groups are substituted or unsubstituted.

[0052] Among them, X, Y, Z, R, D, L 1 , A, and X 10 The definition is as described in the claims of patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A), 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:

[0053] X 10 O, S, SO or SO2;

[0054] A is C6-C 10 Aryl, 5- to 15-membered heteroaryl, or -N=CR 1 R 2 ;

[0055] R 1 and R 2 Each independently represents hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0056] X, Y and Z are each independently hydrogen, CN, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0057] Each R is independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0058] R 13 and R 14 Each independently represents hydrogen, C1-C6 alkyl, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 15-membered heterocyclic ring, 5- to 15-membered heteroaryl, or ether;

[0059] Among them L 1 and D is defined as follows:

[0060] L 1 Selected from:

[0061] R 40 and R 41 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C6-C 10 aryl, 4- to 15-membered heterocyclic ring, or 5- to 15-membered heteroaryl;

[0062] R 42 is a C2-C3 alkylene or heteroalkylene group which is optionally substituted with 1 to 3 C1-C6 alkyl groups;

[0063] V(-) is any anion, preferably a pharmaceutically acceptable anion;

[0064] D is a moiety such that D-OH is an anticancer drug, wherein OH is an aliphatic hydroxyl group or a phenolic hydroxyl group, or is an OH moiety attached to a phosphorus atom as provided herein; or

[0065] L 1 for:

[0066] R 40 As defined above, R 43 is hydrogen or forms a heterocyclic ring together with D, and the phenylene moiety is optionally substituted, and

[0067] D is such that D-NR 43 H is a moiety of an anticancer drug; or

[0068] L 1 For the key, -OC(R 40 R41 )2-、-OC(R 40 R 41 )-NR 40 R 41 (+)-C(R 40 R 41 )-or

[0069] where R 40 、R 41 and V is as defined above, and

[0070] D is an anticancer drug containing a tertiary or secondary nitrogen atom, wherein the tertiary nitrogen atom or the secondary nitrogen atom is bonded to L 1 ;

[0071] and

[0072] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, and ether groups are optionally substituted.

[0073] 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:

[0074] 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;

[0075] 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;

[0076] 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 ;

[0077] 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, OLCMS, 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;

[0078] 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 and the atoms to which it is bonded together form a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group;

[0079] 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;

[0080] 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.

[0081] 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;

[0082] 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.

[0083] Formula (8) is an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound, more specifically, it is an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0084] in:

[0085] 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;

[0086] 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;

[0087] wherein the alkyl group and the aryl group are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups;

[0088] R 1 and R 2 each independently phenyl or methyl;

[0089] X, Y and Z are each independently hydrogen or halo;

[0090] R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl.

[0091] 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:

[0092] Rw is

[0093] R1 is H, C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6 membered heterocycloalkyl, 5-6 membered heteroaryl or phenyl, wherein the C1-6 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;

[0094] Each R a are independently H, F, Cl, Br, I, -CN, -OH, C 1-3 Alkoxy or C 1-3 alkyl;

[0095] R2 is H or C 1-6 alkyl;

[0096] 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;

[0097] Each R b are independently H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3 or -CH2CH3;

[0098] R3 is H, F, Cl, Br, I, -OH, -NH2, C 1-3 Alkoxy or C 1-3 alkyl;

[0099] Or R2 and R3 are connected together to make the structural unit for

[0100] T1 is -(CR c R d ) m -or-(CR c R d ) n -O-;

[0101] m is 1, 2, or 3;

[0102] n is 1 or 2;

[0103] T2 is N or CH;

[0104] R c and R d Each independently is H, F, C 1-3 Alkyl or C 1-3 alkoxy;

[0105] R4, R5 and R6 are each independently H, F, Cl, Br, I, C 1-3 Alkyl or C 1-3alkoxy;

[0106] T is N or CH;

[0107] R7 and R8 are each independently H, F, Cl, Br or I;

[0108] R9 and R 10 Each independently is H, F, Cl, Br, I, -CN or

[0109] 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-.

[0110] Wherein, the definitions of R1, R2, R3, R4 and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1. The synthesis and preparation methods of specific compounds are also described in the above-mentioned application, which are hereby incorporated into this application in their entirety and are specifically defined as:

[0111] T is N or CH;

[0112] R1 and R2 are each independently H, F, Cl, Br, I or C 1-3 Alkyl, wherein the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R a replaced by;

[0113] Each R a independently F, Cl, Br, I, -CN, -OH or -NH2;

[0114] R3 and R4 are each independently H, F, Cl, Br, I, CN, C 1-3 Alkyl, C 1-3 Alkoxy, Among them, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R e replaced by;

[0115] R b and R c Each independently represents H, -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0116] R d It is -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0117] Each R e independently F, Cl, Br, I, -CN, -OH or -NH2.

[0118] 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:

[0119] 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;

[0120] E is SO or SO2;

[0121] X is Cl, Br, I or OSO2R;

[0122] Y is Cl, Br, I or OSO2R;

[0123] Each R is independently H or C1-C6 alkyl;

[0124] G is a radical selected from the group consisting of formula (B)-(AA):

[0125] in:

[0126] R1 is H, C1-C6 alkyl, CH2(CH2)nOH, CH2CH(OH)CH2OH, phenyl, pyridyl, benzyl or pyridylmethyl, provided that when R1 is phenyl, pyridyl, benzyl or pyridylmethyl, R1 is optionally substituted at any available position with C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, OR6, N(R6)(R7), CFH2, CF2H, CF3, F, Cl, Br, I, OCF3, COR6, CON(R6)(R7), SOR6, SON(R6)(R7), SO2R6, SO2N(R6)(R7), CN or NO2;

[0127] 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;

[0128] R4 is N(R6)(R7), OH, OCH2(CH2)nN(R6)(R7) or CH2(CH2)nN(R6)(R7);

[0129] R5 is H or a C1-C6 alkyl group;

[0130] 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;

[0131] Z is CH or N;

[0132] W is CH2, O, S, SO or SO2;

[0133] n is 0 to 6;

[0134] * indicates the point of connection to formula (I).

[0135] or a pharmaceutically acceptable salt thereof,

[0136] Among them, R 1 、R 2 、R 3 、R 4 、R 5 、R 6 、R a 、R b The definitions of n1 and n2 are as described in the claims of patent application PCT / CN2023 / 123253, publication number WO2024078392A1. The synthesis and preparation methods of specific compounds are also described in the above application, which are hereby incorporated into this application in their entirety. They are specifically defined as:

[0137] in:

[0138] (II)R 1 、R 2 Together with the intervening atoms, they form an optionally substituted 4-8 membered carbon or heterocyclic ring, R 4 and R 5 is as defined in (I); or

[0139] (III)R 1 、R 5 Together with the intervening atoms, they form an optionally substituted 4-8 membered carbon or heterocyclic ring, R 2 and R 4 is as defined in (I); or

[0140] (IV)R 4 、R 5 Together with the intervening atoms, they form an optionally substituted 4-8 membered carbon or heterocyclic ring, R 1and R 2 is as defined in (I) or (II); or

[0141] (I)R 1 is hydrogen, deuterium, or any substituted C 1-4 Alkyl, optionally substituted C 2-4 Alkenyl or optionally substituted C 2-4 Alkynyl;

[0142] R 2 、R 4 、R 5 are independently hydrogen, halogen (such as F), optionally substituted C 1-4 Alkyl, arbitrarily substituted C 2- 4-alkenyl, optionally substituted C 2-4 Alkynyl, optionally substituted C 1-4 alkoxy or an optionally substituted 3-5 membered ring;

[0143] in:

[0144] X is O, S, or NR 10 , arbitrarily substituted C 1-4 Alkylene or optionally substituted C 1-4 Heteroalkylene, wherein R 10 is hydrogen, optionally substituted C 1-4 Alkyl, optionally substituted 3-6 membered ring or nitrogen protecting group;

[0145] R 3 is hydrogen, optionally substituted C 1-4 Alkyl or an optionally substituted 3-10 membered ring;

[0146] R 6 is hydrogen, deuterium, or any substituted C 1-4 Alkyl, optionally substituted C 2-4 Alkenyl or optionally substituted C 2-4 Alkynyl;

[0147] Integers n1 and n2 are each independently 0, 1, 2, 3 or 4;

[0148] Each R a 、R b Each occurrence is independently optionally substituted C1-4 alkyl or optionally substituted C1-4 heteroalkylene; or two instances of R a or two instances of R b Together with the intervening atoms, they form an optionally substituted 3-6 membered ring, and any remaining instances of R a and / or R b As defined above.

[0149] Wherein, the definitions of Sugar, R1, and R2 are as described in the claims of patent application US5622936A. 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. The specific definitions are as follows:

[0150] wherein the sugar moiety is linked to a phosphoamide mustard residue (15-I) or an ifosfamide mustard residue (15-II), R1 and R2 may be the same or different and are selected from hydrogen, C1-C4 alkyl or C1-C6 haloalkyl,

[0151] And the sugar moiety (sugar) is any existing isomeric or enantiomeric form of a monosaccharide, disaccharide or polysaccharide.

[0152] For other structures of DNA alkylating agents such as β-D-Glucosidase activation, carboxylesterase activation, esterase and caspase-3 activation, cathepsin B activation, γ-glutamyltranspeptidase activation, and β-galactosidase activation, please refer to the review literature (Han HH, Wang HM, Jangili P, et al. The design of small-molecule prodrugs and activatable phototherapeutics for cancer therapy. Chem Soc Rev. 2023; 52(3):879-920. Published 2023Feb 6. doi:10.1039 / d2cs00673a).

[0153] The compound of structural formula (1) is selected from the following structural compounds:

[0154] The synthesis and preparation method of the compound is also described in patent application PCT / US2006 / 025881, publication number WO2007002931 (corresponding Chinese application number 2006800300828, publication number CN101501054A), which is hereby incorporated into the present application in its entirety.

[0155] The compound of structural formula (2) is selected from the following structural compounds:

[0156] The synthetic preparation method of the compound is also recorded in patent application PCT / CN2020 / 114519, publication number WO2021120717A1 (corresponding to Chinese application number 2020800673113, publication number CN114466853A), the full text of which is hereby introduced into this application.

[0157] The compound of structural formula (3) is selected from the following structural compounds:

[0158] The synthetic preparation method of the compound is also described in patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A), the entire text of which is hereby incorporated into the present application.

[0159] The compound of structural formula (4) is selected from the following structural compounds:

[0160] The synthetic preparation method of the compound is also described in patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), which is hereby incorporated into the present application in its entirety.

[0161] The compound of structural formula (5) is selected from the following structural compounds:

[0162] The synthetic preparation method of the compound is also described in patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A), which is hereby incorporated into the present application in its entirety.

[0163] The compounds of structural formula (6) and (7) are selected from the following structural compounds:

[0164] The synthetic preparation method of the compound is also described in patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A), the full text of which is hereby incorporated into the present application.

[0165] The compound of structural formula (8) is selected from the following structural compounds:

[0166] The compound of structural formula (9) is selected from the following structural compounds:

[0167] The synthetic preparation method of the compound is also recorded in patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A), the full text of which is hereby introduced into this application.

[0168] The compound of structural formula (10) is selected from the following structural compounds:

[0169] The synthetic preparation method of the compound is also described in patent application PCT / CN2021 / 118597, publication number WO2022057838A1, the entire text of which is incorporated herein by reference.

[0170] The compound of structural formula (11) is selected from the following structural compounds:

[0171] The synthetic preparation method of the compound is also described in patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A), the full text of which is hereby incorporated into the present application.

[0172] The AKR1C3 enzyme activating compound of formula (12) is selected from compounds 1-569 in Tables 1-15 of patent application PCT / CN2023 / 123253, publication number WO2024078392A1, and the structures of some of the compounds are listed below:

[0173] The specific synthesis method of compound 1-569 of formula (12) and the corresponding spectral data are disclosed in WO2024078392A1, which is incorporated herein in its entirety.

[0174] The compound of structural formula (15) is selected from the following structural compounds:

[0175] The synthetic preparation method of the compound is also described in U.S. patent application US5622936A, PCT application PCT / US2007 / 074012, publication number WO2008011588, which are hereby incorporated into the present application in their entirety.

[0176] The DNA alkylating agent prodrug compound is selected from the AKR1C3 activated DNA alkylating agent prodrug compound AST-3424:

[0177] The above AST-3424 is formulated into an aqueous solution for intravenous injection.

[0178] The aqueous solution solute consists of AST-3424 raw material drug, glucose, ethanol, propylene glycol and pH regulator sodium bicarbonate.

[0179] The aqueous solution has a concentration of AST-3424 API of 0.004-0.94 mg / ml, a pH of 7.4, a glucose content of 4.5-5.0% by mass, and is an isotonic solution.

[0180] or

[0181] The aqueous solution solute consists of AST-3424 raw material drug, sodium chloride, ethanol, propylene glycol and pH regulator sodium bicarbonate.

[0182] The aqueous solution has a concentration of AST-3424 raw material drug of 0.004-0.94 mg / ml, a pH of 7.4, a sodium chloride content of 0.81-0.90% by mass, and is an isotonic solution.

[0183] The preparation method of AST-3424 as an injection concentrated solution and the preparation method of the above-mentioned aqueous solution for intravenous injection are both recorded in patent application PCT / CN2020 / 101870, publication number WO20210085201.

[0184] The AST-3424 dosing regimen is selected from any one of the following regimens:

[0185] Scheme 1: One cycle of 21 days, one dose on the first and eighth day, the dose is selected from 12 mg / m 2 , 11mg / m 2 , 10mg / m 2 , 9mg / m 2 , 8mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 ;

[0186] Scheme 2: Each 21-day cycle is administered on day 1, with a dose of 14 mg / m 2 , 13mg / m 2 , 12mg / m 2 , 11mg / m 2 , 10mg / m 2 , 9mg / m 2 , 8mg / m 2 , 7mg / m 2 , 6mg / m2 , 5mg / m 2 , 4mg / m 2 , 3mg / m 2 , 2mg / m 2 , 1mg / m 2 ;

[0187] Scheme 3: Each 21-day cycle is administered once a day from day 1 to day 5, with a dose of 8 mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 , 3mg / m 2 , 2mg / m 2 , 1mg / m 2 .

[0188] For detailed information on the three AST-3424 dosing regimens and their dosages, please refer to the U.S. clinical trial NCT03592264 and the Chinese clinical trials CTR20191371 and CTR20191399.

[0189] For AST-3424, the patient is a liver cancer patient.

[0190] Preferably, for AST-3424, the patient's liver tumor tissue is strongly positive for AKR1C3 expression, and one of the following conditions is met to determine that the patient's liver tumor tissue is strongly positive for AKR1C3 expression:

[0191] 1. An H-score greater than or equal to 100, preferably greater than or equal to 135, and more preferably greater than or equal to 200, as detected by the AKR1C3 detection method described in WO2022048492;

[0192] Second, according to the AKR1C3 detection method described in WO2022048492, the proportion of tumor cells with a staining intensity of 2+ and / or 3+ is ≥70%.

[0193] Criterion 1 was determined based on the OBI-3424 Phase I clinical trial NCT03592264 conducted in the United States, see academic literature (Journal of Clinical Oncology.40.3030-3030.10.1200 / JCO.2022.40.16_suppl.3030) and academic literature (Tsimberidou, AM, Verschraegen, CF, Wesolowski, R. et al. Phase 1 dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors. Br J Cancer 129, 266–274 (2023). https: / / doi.org / 10.1038 / s41416-023-02280-4).

[0194] Standard 2 was determined based on the AST-3424 Phase I clinical trial CTR20191371 conducted in China. Currently, a Phase II clinical trial is being conducted in accordance with this standard. For the inclusion criteria for the Phase II clinical trial, please refer to CTR20191399.

[0195] The document described in WO2022048492 provides a variety of IHC methods for detecting the expression level of AKR1C3 enzyme. One preferred AKR1C3 detection method uses immunohistochemical staining to detect the expression level of AKR1C3 in ex vivo formalin-fixed paraffin-embedded (FFPE) human tissue sections, comprising the following steps:

[0196] a1) Dewaxing and rehydration

[0197] Formalin-fixed paraffin-embedded human tissue sections of appropriate thickness were dewaxed using organic solvents, washed with alcohols of varying water content, and finally washed with water;

[0198] a) Antigen retrieval

[0199] In the presence of antigen retrieval solution, heat the dewaxed and rehydrated formalin-fixed paraffin-embedded human tissue sections at 90-115°C for 17-30 minutes for antigen retrieval.

[0200] b1) Blocking nonspecific antigens

[0201] Formalin-fixed paraffin-embedded human tissue sections after antigen retrieval were incubated with blocking solution to block nonspecific antigens;

[0202] b) Primary antibody incubation

[0203] Formalin-fixed paraffin-embedded human tissue sections after blocking nonspecific antigens were mixed with a 0.5-5.0 μg / ml AKR1C3 monoclonal antibody solution and incubated for 25-700 minutes;

[0204] c) Secondary antibody incubation

[0205] Formalin-fixed paraffin-embedded human tissue sections incubated with primary antibodies were mixed with a secondary antibody solution at a concentration of 0.5–5.0 μg / ml and incubated for 25–700 min;

[0206] d) Staining and sealing

[0207] Formalin-fixed paraffin-embedded human tissue sections were stained with hematoxylin, dehydrated and mounted after staining;

[0208] e) Observational scoring

[0209] The stained human tissue sections were observed, and the expression level of AKR1C3 in the human tissue sections was evaluated based on the observed staining degree.

[0210] In the document described in WO2022048492, the scoring calculation method of the H-scoring system is:

[0211] H-score = percentage of cells with a staining intensity of 1+ × 1 + percentage of cells with a staining intensity of 2+ × 2 + percentage of cells with a staining intensity of 3+ × 3. For example, if the IHC staining results of a liver cancer tissue show that the percentage of cells with a staining intensity of 1 is 10%, the percentage of cells with a staining intensity of 2 is 20%, and the percentage of cells with a staining intensity of 3 is 50%, then the H-score is 10 + 40 + 150 = 200.

[0212] A drug containing AST-3424 is used in combination with a drug containing abiraterone, prednisolone, 5-fluorouracil, sunitinib, oxaliplatin, a PD-1 / L1 inhibitor, apatinib, sorafenib or donafenib, or elemene to treat liver cancer.

[0213] The p53 gene, also known as the tumor protein p53 gene (Tumor protein p53), is also called the tp53 gene.

[0214] In this application, no distinction is made between p53 (protein) and p53 (gene), and both are used interchangeably.

[0215] In the present application, p53 gene mutation-negative includes cases where no mutation is detected and cases where a mutation is detected but does not reach the defined positive level.

[0216] At present, relevant detection kits have been approved for commercial use and can be purchased directly for testing, such as the one produced by Xiamen Aide Biopharmaceutical Technology Co., Ltd. in China. tp53 Six Mutations Detection Kit, manufactured by Henan Synnot Biotechnology Co., Ltd., China. FISH detection Kit for the p53 gene, manufactured by Integrated DNA Technologies, Inc., USA. p53kit for

[0217] Normal p53 protein expression includes normal expression, elevated expression, and overexpression. Normal values ​​are artificially defined based on statistical analysis of clinical practice.

[0218] Normal expression of p53 protein can be measured by a relatively typical WB experiment or by IHC in medical clinical practice. These methods can directly measure the amount of p53 protein in the test sample, and then compare it with the normal value or the set threshold to determine whether it is normal expression or high expression.

[0219] Screening can also be performed through genetic testing. Generally, if a pathogenic gene mutation is detected, it can be determined that p53 protein expression is not normal or high. If no gene mutation is detected, or a gene mutation is detected but not a pathogenic mutation, then p53 protein expression is highly likely normal. Therefore, by detecting whether the p53 gene has a pathogenic gene mutation, it can be determined whether p53 protein expression is normal or high.

[0220] The above-mentioned gene mutation or protein expression detection is generally obtained by testing the patient's tumor or cancer tissue, cells or other biological test samples.

[0221] Biological test samples include peripheral blood samples, tumor tissue or suspected tumor tissue, thin layer cytology samples, fine needle aspiration samples, bone marrow samples, lymph node samples, urine samples, ascites samples, lavage samples, esophageal brushing samples, bladder or lung wash samples, cerebrospinal fluid samples, brain fluid samples, catheter aspiration samples, nipple effusion samples, pleural effusion samples, fresh frozen tissue samples, paraffin-embedded tissue samples, or extracts or processed samples derived from any of peripheral blood samples, tumor tissue or suspected tumor tissue, thin layer cytology samples, fine needle aspiration samples, bone marrow samples, urine samples, ascites samples, lavage samples, esophageal brushing samples, bladder or lung wash samples, cerebrospinal fluid samples, brain fluid samples, catheter aspiration samples, nipple effusion samples, pleural effusion samples, fresh frozen tissue samples, or paraffin-embedded tissue samples, and are generally venous whole blood or saliva.

[0222] Regarding the compounds described herein, the chemical structures of compounds such as AST-3424 contain an organic amine structure and a P=O double bond structure, and therefore, the compounds may also be administered in the form of salts. That is, the present invention provides pharmaceutically acceptable salts of the compounds, which may be basic salts, including salts formed with inorganic bases (e.g., alkali metal hydroxides, alkaline earth metal hydroxides, etc.) or with organic bases (e.g., monoethanolamine, diethanolamine, or triethanolamine, etc.). Alternatively, the salts may be acidic salts, including salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid, etc.) or with organic acids (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.). Similarly, the compounds may react with certain acids or alcohols to form esters, and therefore, the compounds may also be administered in the form of esters.

[0223] For various reasons, these compounds may also form solvates with certain solvents, such as hydrates or alcoholates, and thus the compounds may also be administered in the form of solvates. The selection and preparation of acceptable salts, esters and solvates of compounds are well known in the art.

[0224] The term "isotopic variant" refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including but not limited to hydrogen ( 1 H), deuterium ( 2 H), tritium ( 3 H), carbon-11 ( 11 C), carbon-12 ( 12 C), carbon-13 ( 13 C), carbon-14 ( 14 C), nitrogen-13 (13 N), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), fluorine-18 ( 18 F), phosphorus-31 ( 31 P), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-35 ( 35 S), sulfur-36 ( 36 S), chlorine-35 ( 35 Cl), chlorine-36 ( 36 Cl), chlorine-37( 37 Cl), bromine-79( 79 Br), bromine-81( 81 Br), iodine-123 ( 123 I), iodine-125( 125 I), iodine-127( 127 I), iodine-129( 129 I) and iodine-131( 131 I). In certain embodiments, an "isotopic variant" of a compound is a stable form, i.e., non-radioactive. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including but not limited to hydrogen ( 1 H), deuterium ( 2 H), carbon-12 ( 12 C), carbon-13 ( 13 C), nitrogen-14 ( 14 N), nitrogen-15 ( 15 N), oxygen-16 ( 16 O), oxygen-17 ( 17 O), oxygen-18 ( 18 O), fluorine-17 ( 17 F), phosphorus-31 ( 31 P), sulfur-32 ( 32 S), sulfur-33 ( 33 S), sulfur-34 ( 34 S), sulfur-36 ( 36 S), chlorine-35 ( 35Cl), chlorine-37( 37 Cl), bromine-79( 79 Br), bromine-81( 81 Br) and iodine-127( 127 I). In certain embodiments, an "isotopic variant" of a compound is an unstable form, i.e., radioactive. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of one or more isotopes, including but not limited to tritium ( 3 H), carbon-11 ( 11 C), carbon-14 ( 14 C), nitrogen-13 ( 13 N), oxygen-14 ( 14 O), oxygen-15 ( 15 O), fluorine-18 ( 18 F), phosphorus-32 ( 32 P), phosphorus-33 ( 33 P), sulfur-35( 35 S), chlorine-36 ( 36 Cl), iodine-123 ( 123 I), iodine-125( 125 I), iodine-129( 129 I) and iodine-131( 131 I) It is understood that in the compounds provided herein, any hydrogen may be replaced by, for example, 2 H is D, or any carbon can be, for example 13 C, or any nitrogen may be e.g. 15 N, and any oxygen may be 18 O. In certain embodiments, an "isotopic variant" of a compound contains unnatural proportions of deuterium (D).

[0225] 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.

[0226] The prepared medicine, drug, preparation may also include pharmaceutically acceptable adjuvants or excipients. The medicine may be in any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small needles for injection, freeze-dried powder for injection, or large infusions. Depending on the specific dosage form and mode of administration, the pharmaceutically acceptable adjuvants or excipients in the medicine may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, adhesives, fillers, flavorings, sweeteners, antioxidants, surfactants, preservatives, wrappers, and pigments.

[0227] Monotherapy refers to single-drug therapy. Combination therapy refers to combined drug therapy. 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.

[0228] 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.

[0229] The drug dosage, administration cycle and administration schedule of monotherapy and combination therapy are explored through clinical trials.

[0230] The combination of AST-3424 and other drugs has been reported in the following literature:

[0231] Among them, AST-3424 is combined with abiraterone acetate or abiraterone + prednisolone, and sunitinib and gemcitabine are used in combination. Please refer to patent application PCT / CN2021 / 078115, publication number WO2022178821 and academic literature (Meng, Fanying et al. "A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity." American journal of cancer research vol. 11, 7 3645-3659. 15 Jul. 2021). Among them, AST-3424, abiraterone acetate or abiraterone, prednisolone combination is a triple therapy, using three drugs, that is, on the basis of the existing abiraterone acetate or abiraterone + prednisolone combination, a third drug AST-3424 is used for three-drug combination therapy.

[0232] For the combination of AST-3424 and 5-fluorouracil, please refer to patent application PCT / CN2021 / 078115, publication number WO2022178821, and academic literature (Meng, Fanying et al. "A novel selective AKR1C3-activated prodrug AST-3424 / OBI-3424 exhibits broad anti-tumor activity." American journal of cancer research vol. 11, 73645-3659. 15 Jul. 2021) and academic literature (Zhang, Yu et al. "The In-Vitro Antitumor Effects of AST-3424 Monotherapy and Combination Therapy With Oxaliplatin or 5-Fluorouracil in Primary Liver Cancer." Frontiers in Oncology vol. 12 885139.22Jul.2022,doi:10.3389 / fonc.2022.885139).

[0233] For the combination of AST-3424 and oxaliplatin, please refer to academic literature (Zhang, Yu et al. “The In-Vitro Antitumor Effects of AST-3424 Monotherapy and Combination Therapy With Oxaliplatin or 5-Fluor ouracil in Primary Liver Cancer.” Frontiers in oncology vol.12885139.22Jul.2022, doi:10.3389 / fonc.2022.885139).

[0234] For information on the combination of AST-3424 with PD-1 / L1 inhibitors, please refer to patent application PCT / US2021 / 29552, publication number WO2022231580, and an academic conference poster (Chun-Chung Wang, Wan-Fen Li, Chih-Chan Lee, Lu-Tzu Chen, Jhih-Jie Yang, Jiann-Shiun Lai, Ming-Tain Lai; Abstract 6111: OBI-3424, an AKR1C3-activated prodrug, exhibits in vivo synergistic anti-tumor effect in combination with pembrolizumab by induction of immunogenic cell death. Cancer Res 15 June 2022; 82(12_Supplement): 6111. https: / / doi.org / 10.1158 / 1538-7445.AM2022-6111). The poster is available from OBIPharma. Download the poster from the official website of Obipharma Inc. (https: / / www.obipharma.com / zh-hant / news-zh-hant / news-2022-zh-hant / poster-presentations-at-aacr-2022-annual-meeting-for-obi-3424-and-globo-h-science / / )

[0235] For the combination of AST-3424 with apatinib, sorafenib, or donafenib, or elemene, please refer to the academic literature (Xun, Chen et al. "A novel AKR1C3 specific prodrug AST-3424 and its combination therapy in hepatocellular carcinoma." Journal of Pharmacological Sciences vol. 152, 2(2023): 69-75. doi: 10.1016 / j.jphs.2023.03.004). Donafenib is a deuterated drug of sorafenib. The above literature reports the combination therapy of AST-3424 and sorafenib. Therefore, those skilled in the art can undoubtedly infer that AST-3424 and its deuterated drug donafenib can also be used in combination therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0236] Figure 1 is an experimental curve showing the inhibition rate of H460 cell proliferation in vitro by the combination of AST-3424 / AST and Nutlin-3;

[0237] Figure 2 shows the experimental results of the inhibition rate of H460 cell proliferation in vitro using AST-3424 and Nutlin-3 alone or in combination with different administration orders;

[0238] Figure 3 shows the experimental results of the effects of AST-3424 and Nutlin-3 alone or in combination with different administration orders on H460 / HPAF-II cell clone formation;

[0239] Figure 4 shows the experimental results of the effects of AST-3424 and Nutlin-3 alone or in combination at different doses on the apoptosis process of H460 cells;

[0240] Figure 5 shows the first experimental results of the effect of AST-3424 combined with Nutlin-3 on G2 / M arrest of H460 / HPAF-II cell cycle. The bar graph shows G2 / M, S, and G0 / G1 phases from top to bottom, respectively.

[0241] Figure 6 shows the results of the second experiment on the effect of AST-3424 combined with Nutlin-3 on G2 / M arrest of the cell cycle. The bars in the figure are G2 / M, S, and G0 / G1 phases from top to bottom respectively;

[0242] FIG7 is a bar graph showing the results of the in vitro proliferation inhibition of H460 cells by AST and Nutlin-3 alone or in combination. The three columns in the figure, from left to right, are the numerical columns of the inhibition rates of AST alone, Nutlin-3 alone, and AST + Nutlin-3 combination;

[0243] FIG8 is a bar graph showing the results of the in vitro proliferation inhibition of H460 cells by AST and RITA alone or in combination, wherein the three columns in the graph, from left to right, are the numerical columns of the inhibition rates of AST alone, RITA alone, and AST+RITA combination;

[0244] Figure 9 is a bar graph showing the results of the inhibition of HPAF-Ⅱ cell proliferation in vitro by AST and Nutlin-3 alone or in combination. The three columns in each group in the figure, from left to right, are the numerical columns of the inhibition rates of AST alone, Nutlin-3 alone, and AST + Nutlin-3 combination. The six groups of bar graphs, from left to right, correspond to experimental groups 1-6 in the table.

[0245] FIG10 is a bar graph showing the results of in vitro inhibition of HPAF-Ⅱ cell proliferation by AST and RITA alone or in combination, wherein the three columns from left to right are the numerical columns of inhibition rates of AST alone, RITA alone, and AST+RITA combination;

[0246] Figure 11 shows the results of Western blotting of protein lysates in the first experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photo of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0247] Figure 12 shows the results of Western blotting of proteins in cell lysates from the second experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photograph of protein bands detected by Western blotting, and the middle and lower figures show the ratios of the corresponding proteins relative to the internal reference protein β-actin.

[0248] Figure 13 shows the results of Western blotting of proteins in cell lysates from the third experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photograph of protein bands detected by Western blotting, and the middle and lower figures show the ratios of the corresponding proteins relative to the internal reference protein β-actin.

[0249] Figure 14 shows the WB detection results of RAD51 protein in cell lysates from the first experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The left figure is a photo of protein bands detected by WB, and the right figure is the ratio of the corresponding protein to the internal reference protein β-actin;

[0250] Figure 15 shows the results of Western blotting of RAD51 protein in cell lysates from the second experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photo of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0251] Figure 16 shows the results of Western blotting of RAD51 protein in cell lysates from the third experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photo of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0252] Figure 17 shows the results of Western blotting of RAD51 protein in cell lysates from an experiment in which AST-3424, Nutlin-3, and MG-132 were combined to treat H460 cells. The upper figure shows a photograph of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0253] Figure 18 shows the results of Western blotting of RAD51 protein in cell lysates from an experiment in which H460 cells were treated with a combination of AST-3424, Nutlin-3, and Cycloheximide. The upper figure shows a photograph of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0254] FIG19 is a curve showing the change in the relative amount of RAD51 after treatment with different drugs: 1% DMSO, 0.1 nM AST-3424, 5 μM Nutlin-3, and 0.1 nM AST-3424 + 5 μM Nutlin-3 for 24 hours, and then adding 4 μM Cycloheximide at different times;

[0255] Figure 20 shows the results of WB detection of γH2AX protein in cell lysates from the first experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure is a photograph of protein bands detected by WB, and the lower figure is the ratio of the corresponding protein to the internal reference protein β-actin;

[0256] Figure 21 shows the results of Western blotting of γH2AX protein in cell lysates from the second experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photograph of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0257] Figure 22 shows the results of Western blotting of γH2AX protein in cell lysates from the third experiment of treating H460 cells with AST-3424, Nutlin-3 alone, and in combination. The upper figure shows a photograph of protein bands detected by Western blotting, and the lower figure shows the ratio of the corresponding protein to the internal reference protein β-actin.

[0258] Figure 23 shows the WB detection results of p53, Rad51, MDM2, and p21 proteins in HPAF-II cell lysates treated with AST and Nutlin-3 alone or in combination. The upper figure is a photograph of protein bands detected by WB, and the lower figure is a bar graph of the ratio of the corresponding proteins to the internal reference protein β-actin;

[0259] Figure 24 shows the results of Western blotting of protein lysates in HPAF-II cell treatment experiments with AST-3424, Nutlin-3 alone, and in combination. The upper figure is a photograph of protein bands detected by Western blotting, and the lower figure is a bar graph showing the ratio of the corresponding proteins to the internal reference protein β-actin.

[0260] FIG25 is a curve showing the inhibition rate of AST-3424 on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cells in vitro;

[0261] FIG26 is a curve showing the inhibition rate of AST on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cells in vitro;

[0262] FIG27 is a curve showing the inhibition rate of compound A on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cells in vitro;

[0263] FIG28 is a curve showing the inhibition rate of compound B on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cells in vitro;

[0264] Figure 29 is a photo of protein bands detected by WB of the effect of ±Nutlin-3 on Total P53, MDM2, P21, AKR1C3, and Actin proteins in H460 and H460 P53 KO cells;

[0265] Figure 30 shows the effect of ±Nutlin-3 on Total P53, MDM2, P21, and AKR1C3 proteins in H460 and H460 P53 KO cells, and the ratio of the corresponding proteins detected by WB to the internal reference protein β-actin;

[0266] Figure 31 is a curve showing the inhibition rate of compound C and AST-3424 on the proliferation of NCI-H460 cells in vitro under normoxia;

[0267] FIG32 is a curve showing the inhibition rate of compound C and AST-3424 on the proliferation of NCI-H460 P53KO#1 cells in vitro under normoxia. DETAILED DESCRIPTION

[0268] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0269] "Administering" or "administration of" a drug to a patient (and grammatical equivalents of such phrases) refers to direct administration (which may be administered to a patient by a medical professional or may be self-administered) and / or indirect administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer a drug and / or provides a prescription for a drug to a patient is administering a drug to a patient.

[0270] "Cancer" refers to leukemias, lymphomas, carcinomas, and other malignancies (including solid tumors) with the potential for unrestricted growth that can spread locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchi, colon and / or rectum, gall bladder, 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 habitus tumors, and leukemia. tumor), 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 (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.

[0271] "Patient" and "subject" are used interchangeably to refer to a mammal in need of cancer treatment. Typically, the patient is a human. Typically, the patient is a human diagnosed with cancer. In certain embodiments, a "patient" or "subject" may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used to screen, characterize, and evaluate drugs and therapies.

[0272] "Solid tumor" refers to solid tumors including, but not limited to, metastatic tumors in bone, brain, liver, lung, lymph nodes, pancreas, prostate, skin, and soft tissues (sarcomas).

[0273] A "therapeutically effective amount" of a drug refers to an amount of the drug that, when administered to a patient suffering from cancer, will have the desired therapeutic effect (e.g., alleviation, amelioration, remission, or elimination of the clinical manifestations of one or more cancers in the patient). A therapeutic effect need not occur with administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations.

[0274] "Treating," "treatment of," or "therapy of" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in the extent of the disease; delay or slowing of disease progression; improvement, remission, or stabilization of the disease state; or other beneficial results. In some instances, treatment of cancer may result in a partial response or stable disease.

[0275] "Tumor cell" or "cancer cell" refers to a tumor cell, cancer cell of any appropriate species (eg, mammalian, such as murine, canine, feline, equine, or human).

[0276] "Patient" and "subject" are used interchangeably to refer to a mammal in need of cancer treatment. Typically, the patient is a human. Typically, the patient is a human diagnosed with cancer. In certain embodiments, a "patient" or "subject" may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used to screen, characterize, and evaluate drugs and therapies.

[0277] "Treatment" or "treating a patient" refers to administering, using or applying to a patient a therapeutically effective amount of a drug related to the present invention.

[0278] "Administering" or "applying" a drug to a patient refers to direct administration or administration (which may be administered or administered to a patient by a medical professional or may be self-administered or administered) and / or indirect administration or administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer or administer a drug and / or provides a prescription for a drug to a patient is administering or administering a drug to a patient.

[0279] "Treatment" of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results). For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in the extent of the disease; delay or slowing of disease progression; improvement, remission, or stabilization of the disease state; or other beneficial results. In some instances, treatment of cancer may result in a partial response or stabilization of the disease.

[0280] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used are all commercially available products unless otherwise specified.

[0281] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

[0282] Example 1 AST-3424 Phase II Clinical Trial Conducted in China

[0283] The clinical registration number of this clinical trial is CTR20191399.

[0284] The trial was approved by the ethics committees of the participating medical institutions and was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all subjects.

[0285] Inclusion criteria

[0286] 1. Male or female, aged ≥18 years.

[0287] 2. Advanced HCC confirmed by histopathology and uncontrollable by surgical resection or local treatment.

[0288] 3. Patients who have previously received standard systemic treatment, including but not limited to sorafenib and / or oxaliplatin-containing systemic chemotherapy, lenvatinib, regorafenib and / or nivolumab, have experienced disease progression, toxicity intolerance, or refused to continue treatment with these drugs.

[0289] 4. At least one measurable lesion that meets the RECIST 1.1 criteria. Lesions that have been previously radiotherapy cannot be considered measurable lesions unless they show clear radiographic progression after radiotherapy.

[0290] 5. Ability to provide pathological paraffin blocks or sections (including archived pathological paraffin blocks and sections) for AKR1C3 expression analysis, and confirm that the liver tumor tissue is strongly positive for AKR1C3 expression (immunohistochemistry results from the central laboratory confirm that the proportion of tumor cells with AKR1C3 staining intensity of 2+ and / or 3+ is ≥70%).

[0291] 6. Eastern Cooperative Oncology Group (ECOG) performance status score is 0 or 1.

[0292] 7. Life expectancy ≥ 12 weeks.

[0293] 8. Concomitant or non-concomitant HBV or HCV infection. a. Subjects with concomitant HBV infection must have HBV-DNA levels less than 2,000 IU / ml and receive antiviral treatment with entecavir, tenofovir disoproxil fumarate, or tenofovir alafenamide according to the national guidelines for the prevention and treatment of chronic hepatitis B. Maintenance treatment is required during the study and continues for 6 months after the last dose. b. Subjects with concomitant HCV infection (with detectable HCV-RNA or anti-HCV antibodies) may be treated according to medical practice.

[0294] 9.Child-Pugh score ≤6.

[0295] 10. No history of hepatic encephalopathy.

[0296] 11. All toxicities from previous anticancer therapy (except alopecia, fatigue, or peripheral neuropathy) must have recovered to Grade 1 or baseline level (NCI CTCAE Version 5) before starting study drug.

[0297] 12. Laboratory examinations must meet the following criteria. Within 14 days prior to the screening laboratory examination, blood transfusion, hematopoietic stimulating factor, or albumin transfusion cannot be used to correct the indicators to meet the inclusion criteria. a. Hemoglobin ≥ 90 g / L; b. Platelet count ≥ 80 × 10 9 Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L; d. Total serum bilirubin ≤3 mg / dL; e. ALT and AST ≤5.0×ULN; f. International normalized ratio (INR) ≤2.3 or prolonged prothrombin time ≤6 seconds; g. Albumin ≥29 g / L; h. Creatinine clearance >50 mL / min as measured by the Cockcroft-Gault equation.

[0298] 13. No history of alcoholism, drug abuse or substance abuse in the past year.

[0299] 14. Female patients of childbearing potential should have a negative pregnancy test result within 5 days before the start of treatment and are not breastfeeding (a positive urine pregnancy test result needs to be confirmed by a serum pregnancy test).

[0300] 15. Female and male subjects of childbearing potential must agree to use effective contraceptive methods (e.g., surgical sterilization or condoms or diaphragm contraception combined with spermicidal gel or intrauterine device [IUD]) with their partners from the start of study participation until 6 months after the last medication.

[0301] 16. Participants must voluntarily participate in this study, fully understand the risks involved, be compliant, and sign the informed consent form. Subjects may also sign the Future Biomedical Research (FBR) consent form. However, subjects who do not participate in FBR may also participate in the main trial.

[0302] Exclusion criteria:

[0303] 1. Untreated active central nervous system (CNS) metastases or leptomeningeal disease. Subjects with CNS metastases that have been adequately treated and have been stable for at least 4 weeks as confirmed by clinical examination and brain imaging (MRI or CT) during the screening period may participate in the study.

[0304] 2. History of other malignant tumors within 2 years, excluding adequately treated basal cell carcinoma, carcinoma in situ at other sites, or other tumors whose natural history and treatment will not interfere with the safety or efficacy evaluation of the current study.

[0305] 3. Major surgery other than diagnostic surgery has been performed within 4 weeks before the first dose.

[0306] 4. Patients who have received radiotherapy, surgical treatment, chemotherapy, immunotherapy, biological therapy for cancer, targeted therapy or hormone therapy within 4 weeks before the first dose (nitrosourea or mitomycin C treatment requires a 6-week washout period; oral fluorouracil drugs require a 2-week washout period; small molecule targeted therapy requires a 2-week washout period).

[0307] 5. Participated in a study of a study drug (diagnostic or therapeutic) or device within 4 weeks before the first dose.

[0308] 6. Concomitant use of strong CYP3A4 inhibitors or inducers is required during the study.

[0309] 7. Active bacterial, viral or fungal infection that is not controlled and requires systemic treatment.

[0310] 8. Known to be infected with human immunodeficiency virus (HIV) or positive for syphilis.

[0311] 9. Clinically significant ascites, defined as ascites detected by physical examination and requiring paracentesis for management or additional medical intervention to maintain symptoms (patients with ascites detected only by imaging examinations are eligible).

[0312] 10. Women who are pregnant, breastfeeding or planning to become pregnant.

[0313] 11. Concomitant diseases or symptoms that may interfere with the conduct of the study, or physical abnormalities that the investigator deems to pose an excessive risk to the patient. This includes but is not limited to a history of gastrointestinal bleeding within three months or a high risk of bleeding, active peptic ulcer or gastritis, changes in mental status, or mental abnormalities that may interfere with the patient's understanding of the informed consent form.

[0314] 12. History of allergy to ethanol or propylene glycol.

[0315] 13. Subjects who are unwilling or unable to comply with the study protocol for any reason.

[0316] Test drug:

[0317] AST-3424 injection concentrated solution: manufactured by pharmaceutical companies under the entrustment of Shenzhen Aixindawei Pharmaceutical Technology Co., Ltd., specification 1mL: 10mg; contains 0.75ml ethanol and 0.25ml propylene glycol.

[0318] Dosage regimen:

[0319] Each cycle lasts 21 days, with the drug administered once on the 1st and 8th day, at a dose of 6 mg / m 2 , will be allowed to receive a maximum of 34 cycles of treatment.

[0320] Specific drug administration procedures:

[0321] Prior to administration, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of commercially available sterile 5% dextrose in water for injection (D5W) in a DEHP-free intravenous infusion bag. Prepare AST-3424 injection for intravenous administration by adding the calculated amount of AST-3424 injection concentrate (to the nearest 0.01 ml) to the pH-adjusted D5W bag.

[0322] The solute of the intravenous aqueous solution consists of AST-3424 raw material drug, glucose, ethanol, propylene glycol and pH regulator sodium bicarbonate, wherein the concentration of AST-3424 raw material drug is 0.004-0.94 mg / ml, the pH is 7.4, the content of glucose is 4.5-5.0% by mass, and the solution is isotonic.

[0323] If the patient is not suitable for glucose injection, use normal saline instead:

[0324] Prior to administration, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of commercially available sterile 0.9% saline solution for injection in a DEHP-free intravenous infusion bag. Add the calculated amount of AST-3424 injection concentrate (accurate to 0.01 ml) to the pH-adjusted saline bag to prepare AST-3424 injection for intravenous administration.

[0325] The intravenous injection aqueous solution solute consists of AST-3424 raw material drug, sodium chloride, ethanol, propylene glycol and pH regulator sodium bicarbonate, wherein the concentration of AST-3424 raw material drug is 0.004-0.94 mg / ml, the pH is 7.4, the content of sodium chloride is 0.81-0.90% by mass, and the solution is isotonic.

[0326] The exact calculation method for the required milliliters of AST-3424 injection concentrated solution is as follows:

[0327] For a patient with a height of 175 cm and a weight of 75 kg, the corresponding equivalent body surface area (BSA) is 2 ) = ([Height (cm) × Weight (kg)] / 3600) 1 / 2 =1.90, then the corresponding dose is 1.90×6.0=11.40mg, and the AST-3424 concentrated injection solution of the above specifications should be drawn as 11.40÷10×1=1.14ml.

[0328] The prepared intravenous AST-3424 solution should be administered within 8 hours.

[0329] Clinical evaluation

[0330] Effectiveness evaluation includes clinical efficacy assessment.

[0331] Clinical efficacy was evaluated using RECIST 1.1, the Response Evaluation Criteria for Solid Tumors. Lesions were assessed using MRI / CT. The same evaluation method was used for each lesion throughout the study. Participants must have measurable tumor lesions at baseline.

[0332] Efficacy evaluation indicators include complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD).

[0333] Complete remission (CR): All target lesions disappear and the short diameter of all pathological lymph nodes (including target nodules and non-target nodules) must be reduced to <10mm.

[0334] Partial response (PR): The sum of the target lesion diameters decreased by at least 30% compared with the baseline level.

[0335] Disease progression (PD): The minimum value of the sum of all target lesion diameters measured during the entire experimental study is used as a reference, and the relative increase in the diameter sum is at least 20% (if the baseline measurement value is the minimum, the baseline value is used as a reference); in addition, the absolute value of the diameter sum must increase by at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0336] Stable disease (SD): The target lesion has not decreased to the level of PR, nor increased to the level of PD, but is somewhere in between. The minimum sum of the diameters can be used as a reference for research.

[0337] Study endpoints

[0338] The efficacy of AST-3424 monotherapy in the treatment of malignant tumors such as HCC was preliminarily evaluated based on the subjects' objective response rate (ORR), disease control rate (DCR), duration of response (DOR) and progression-free survival (PFS).

[0339] Objective response rate (ORR) refers to the percentage of cases achieving complete remission (CR) and partial remission (PR) after treatment among the total number of evaluable cases.

[0340] Disease control rate (DCR) refers to the percentage of confirmed complete remission (CR), partial remission (PR) and stable disease (SD) cases among patients who can be evaluated for efficacy.

[0341] Test results

[0342] A total of 17 patients with liver cancer were enrolled, of whom 8 completed clinical efficacy assessments. One patient achieved a PR (12.5%), five achieved SD (62.5%), and two achieved PD (25%), for an overall DCR of 75% and an ORR of 12.5%. Specifically, all 8 patients underwent p53 gene mutation or defect testing, with a negative (-) result indicating no mutation or defect was detected and a positive (+) result indicating a mutation or defect was detected (which may or may not affect protein expression). See Table 1 for details.

[0343] Table 1: Data of selected cases in the AST-3424 Phase II clinical trial as of July 21, 2023

[0344] Further analysis of the relationship between efficacy and p53 gene mutation negative / positive:

[0345] Among the 4 negative (-) cases, 1 had PR, 2 had SD with lesion reduction, and 1 had PD, that is, the DCR in this subgroup of cases was 75% and the ORR was 25%.

[0346] Among the 4 positive (+) cases, 3 had SD and lesions enlarged, and 1 had PD, that is, the DCR in this subgroup of cases was 75% and all 3 had lesions enlarged, and the ORR was 0%.

[0347] Overall, after receiving the same dose level of AST-3424, there were significant differences in the efficacy evaluation of the p53 gene mutation-negative / positive subgroups: in the subgroup of liver cancer patients with negative (-) p53 gene mutations or defects, the ORR was 25%, while that of the corresponding positive (+) subgroup was 0; further, although the DCR of both subgroups was 75% (corresponding to 3 out of 4 cases), in fact, the lesions of 2 of the 3 patients in the subgroup of liver cancer patients with negative (-) p53 gene mutations or defects who underwent SD efficacy evaluation were reduced (there is a high probability that they will turn into PR or even CR with continued treatment), while the lesions of the 3 patients in the subgroup of liver cancer patients with positive (+) p53 gene mutations or defects who underwent SD efficacy evaluation were all enlarged.

[0348] Therefore, based on the current clinical results, those skilled in the art have reason to believe that AST-3424 has a significantly better therapeutic effect on patients with cancer or tumors that are negative (-) for p53 gene mutations or defects than on patients that are positive (+) for p53 gene mutations or defects. Therefore, the applicant speculates that AST-3424 will have a better therapeutic effect on patients with tumors or cancers that are negative (-) for p53 gene mutations or defects. In other words, patients with tumors or cancers that are negative (-) for p53 gene mutations or defects will have more obvious clinical benefits from treatment with AST-3424.

[0349] As of September 1, 2023, the progress of the above-mentioned ongoing clinical trials is as follows.

[0350] Test results

[0351] A total of 20 subjects with liver cancer were enrolled, of whom 18 completed the clinical efficacy evaluation, including 1 subject with PR, 10 subjects with SD, and 7 subjects with PD. The final ORR was 5.6% (1 / 18) and DCR was 61.1% (11 / 18).

[0352] In particular, 10 cases were tested for p53 gene mutation or defect, where negative (-) indicates that no mutation or defect was detected, and positive (+) indicates that a mutation or defect was detected (which may or may not affect protein expression), as shown in Table 6 below.

[0353] Table 6: Data of selected cases in the AST-3424 Phase II clinical trial as of September 1, 2023

[0354] PFS, deadline: 2023.09.01, PFS is an estimate, calculated as 30.437 days per month.

[0355] Further analysis of the relationship between efficacy and p53 gene mutation negative / positive:

[0356] Among the 5 negative (-) cases, there was 1 PR, 3 SD, and 1 PD, that is, the DCR in this subgroup of cases was 80% (4 / 5), the ORR was 20% (1 / 5), and the average PFS was 4.1 months.

[0357] Among the 5 positive (+) cases, 3 had SD and lesions enlarged, and 2 had PD. That is, the DCR in this subgroup of cases was 60% (3 / 5) and all 3 cases had enlarged lesions. The ORR was 0%, and the average PFS was 1.756 months.

[0358] Overall, after receiving the same dose level of AST-3424, there were significant differences in the efficacy evaluation of the p53 gene mutation negative / positive subgroups: in the subgroup of liver cancer patients with negative (-) p53 gene mutation or defect, the ORR was 20%, DCR was 80%, and PFS was 4.1 months, while the corresponding data for the positive (+) subgroup were 0, 60%, and 1.756 months, respectively. The subgroup of patients with negative p53 gene mutation had a better treatment effect.

[0359] As of March 11, 2024, the progress of the above-mentioned ongoing clinical trials is as follows.

[0360] Test results

[0361] As of March 11, 2024, 30 subjects have been enrolled: 5 are currently receiving treatment, 25 have been discharged (1 withdrew informed consent, 10 have died, and 14 are currently being followed up). The longest PFS is >11.5 months, and the longest OS is >17.6 months. Eleven subjects were enrolled before the end of January 2023, and 6 achieved OS greater than 12 months. Details are shown in Table 7.

[0362] Table 7: Clinical data on the relationship between AST-3424-hepatocellular carcinoma efficacy and gene mutations as of March 11, 2024

[0363] Among the 26 subjects with efficacy evaluation results, 20 had p53 gene mutation test results. The efficacy was observed in groups according to the p53 test results:

[0364] wild type (WT) without mutation, that is, the p53 gene mutation test result is negative (-);

[0365] Mutation of undetermined significance (VUS), that is, the p53 gene mutation test result is positive (+), but it is unclear whether it affects protein expression;

[0366] Mutations that may affect protein function (MUT), that is, p53 gene mutation test results are positive (+), which may affect protein expression, were classified into three groups and statistically analyzed. The results are shown in Tables 8, 9, and 10 below.

[0367] Table 8: Efficacy data of 8 patients with P53 mutation positive (MUT)

[0368] Statistical results showed that 2 of the 8 cases had died, and 6 were still being followed up. The longest PFS was 1.6 months, the longest OS was >14.0 months, the average PFS was 1.4 months, and the average OS was >9.3 months. Among the 8 cases with positive p53 gene mutations (+), which may affect protein expression, 0 cases had PR, 2 cases had SD, and 7 cases had PD. That is, the DCR in this subgroup of cases was 25% (2 / 8) and the ORR was 0% (0 / 5).

[0369] Table 9: Efficacy data of 9 patients with P53 mutation negative (WT)

[0370] Statistical results showed that 4 of the 9 patients had died, and 5 were still being followed up. The longest PFS was greater than 11.5 months, the longest OS was greater than 13.8 months, the average PFS was greater than 4.1 months, and the average OS was greater than 7.7 months. Among the 9 patients with negative p53 gene mutations, 2 had a PR, 5 had SD, and 2 had PD. This means that the DCR in this subgroup was 77.8% (7 / 9) and the ORR was 22.2% (2 / 9).

[0371] Table 10: Efficacy data of 3 patients with positive P53 mutations and unclear effect on protein expression (VUS)

[0372] Therefore, based on the current clinical results, those skilled in the art have reason to believe that AST-3424 has a significantly better therapeutic effect on patients with cancer or tumors that are negative (-) for p53 gene mutations or defects than on patients that are positive (+) for p53 gene mutations or defects. Therefore, the applicant speculates that AST-3424 will have a better therapeutic effect on patients with tumors or cancers that are negative (-) for p53 gene mutations or defects. In other words, patients with tumors or cancers that are negative (-) for p53 gene mutations or defects will have more obvious clinical benefits from treatment with AST-3424.

[0373] The interim results of the two clinical trials in Example 1 appear to confirm that DNA alkylating agent prodrugs such as AST-3424 have better therapeutic effects on patients with negative p53 gene mutations, i.e., patients with normal p53 protein expression. To further confirm the above fact, the applicant conducted further experimental demonstration.

[0374] Unless otherwise specified, the above compounds were synthesized by the applicant with reference to the corresponding patent applications.

[0375] Further reading of relevant literature reveals that:

[0376] The main function of p53 protein is to monitor DNA damage in cells. It can induce cells to enter cell cycle arrest, repair DNA damage, and promote apoptosis of tumor cells, ultimately inhibiting tumor growth (Marei, HE, Althani, A., Afifi, N. et al. p53 signaling in cancer progression and therapy. Cancer Cell Int 21, 703(2021). https: / / doi.org / 10.1186 / s12935-021-02396-8).

[0377] p53 mutations / deficiencies can lead to a range of reactions and manifestations:

[0378] p53 mutation / defect can impair the cell apoptosis pathway and lead to subsequent drug resistance (Sturm I, Bosanquet AG, Hermann S, et al. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as aconsequence of prior DNA-damaging chemotherapy[J]. Cell Death&Differentiation,2003,10(4):477.DOI:10.1038 / sj.cdd.4401194.).

[0379] p53 mutation / deficiency impairs cell cycle arrest, allowing damaged cells to continue dividing and surviving, leading to drug resistance (Zhao, D., Tahaney, WM, Mazumdar, A. et al. Molecularly targeted therapies for p53-mutant cancers. Cell. Mol. Life Sci. 2017, 74, 4171–4187. https: / / doi.org / 10.1007 / s00018-017-2575-0).

[0380] p53 mutation / defects lead to improper activation of DNA repair pathways, resulting in the accumulation of DNA damage in cancer cells, leading to drug resistance (Williams AB, Schumacher B. p53 in the DNA-Damage-Repair Process. Cold Spring Harb Perspect Med. 2016, 6(5): a026070. doi: 10.1101 / cshperspect.a026070).

[0381] p53 mutations / deficiencies accelerate and increase the efficiency of cancer stem cells (CSCs) generation with drug-resistant properties, leading to drug resistance. p53 gain-of-function mutations can also promote the generation of CSCs and subsequent chemotherapy resistance (Ozaki T, Nakamura M, Shimozato O. Novel Implications of DNA Damage Response in Drug Resistance of Malignant Cancers Obtained from the Functional Interaction between p53 Family and RUNX2. Biomolecules. 2015, 5(4): 2854-2876. doi: 10.3390 / biom5042854).

[0382] p53 gain-of-function mutations can promote tumor progression and further lead to drug resistance and treatment refractory state (Alvarado-OrtizE, de la Cruz-López KG, Becerril-Rico J, Sarabia-Sánchez MA, Ortiz-Sánchez E, García-CarrancáA. Mutant p53 Gain-of-Function: Role in Cancer Development, Progression, and Therapeutic Approaches. Front Cell Dev Biol. 2021, 8: 607670. Published 2021Feb 11. doi: 10.3389 / fcell.2020.607670).

[0383] In other words, p53 plays an important role in DNA damage repair and cell apoptosis. Once the p53 gene mutates or becomes defective, it will lead to abnormal p53 protein, which will fail to play the above-mentioned role, making tumors or cancer cells drug-resistant.

[0384] DNA alkylating agent prodrug compounds such as AST-3424 / AST release DNA alkylating agents (e.g., AST-2660, Br-IPM) in vivo to cross-link with DNA, leading to DNA damage and cell death. p53 mutations / deficiencies inhibit the ability of p53 to promote apoptosis in tumor cells following DNA damage. Based on this, it can be hypothesized that cells with negative TP53 gene mutations and normal p53 protein expression can promote apoptosis in DNA-damaged tumor cells when p53 is activated or upregulated, and a series of experimental phenomena consistent with the aforementioned enhanced apoptosis and DNA damage toxicity can be observed.

[0385] To this end, the following experiments were conducted using p53 wild-type H460 cells, A549 cells, p53 mutant HPAF-II cells, and p53 knockout cells. Unless otherwise specified, the H460 cells in Examples 2 to 13 below are all NCI-H460 cells.

[0386] Example 2 Experiments on the effects of cell proliferation and cell clone formation in vitro

[0387] Nutlin-3 is a small molecule MDM2-p53 inhibitor that indirectly activates the function of p53 by inhibiting the interaction between MDM2 and p53. To this end, we conducted experiments on the effects of AST-3424 / AST adding Nutlin-3 to activate / upregulate p53 function on cell proliferation and cell clone formation.

[0388] The CAS number of Nutlin-3 is 548472-68-0.

[0389] H460 cells are p53 wild-type cells, that is, they are negative for p53 gene mutation and have normal expression of p53 protein.

[0390] HPAF-II cells are p53 mutant cells (p.P151S, deleterious), that is, they are positive for p53 gene mutation and have abnormal expression of p53 protein.

[0391] Effects of compounds on H460 cell proliferation in vitro under normoxia

[0392] Overview of the experimental process

[0393] 1) Add H460 cell suspension to a 96-well plate, 100 μL per well, with a cell density of 2000 cells / well.

[0394] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0395] 3) Compound treatment

[0396] Single drug application: 24 hours after cell plating, add 99.5 μL of growth medium to each well. Add 0.5 μL of test compound at different concentrations, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0397] Combination therapy: 24 hours after plating cells, add 99 μL of growth medium to each well. Add 0.5 μL of the combination compound, Nutlin-3, and gently vortex to ensure uniform mixing. After incubation for 2 hours, add 0.5 μL of the test compound at various concentrations and gently vortex to ensure uniform mixing. Then, incubate at 37°C, 5% CO2.

[0398] 4) Place the cell plate in an incubator for 72 hours.

[0399] 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.

[0400] 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.

[0401] 7) Read the chemiluminescent signal value using a multifunctional microplate reader with a reading time of 1000 milliseconds.

[0402] 8) Calculate IC using Graph Pad Prism5 software 50 .

[0403] The experimental results are shown in Figure 1.

[0404] Experiments have shown that adding Nutlin-3, which indirectly activates p53 function, in advance and then adding AST-3424 can significantly increase the proliferation inhibition rate of AST-3424.

[0405] Further combined drug administration experiments with different dosing orders of AST-3424 were conducted, and the drug administration schedule was set during the above experimental process.

[0406] Single drug use: AST-3424, Nutlin-3, 5 μL of each compound at different concentrations (400-fold) and 5 μL of culture medium were added to each well to treat cells (DMSO 0.25% and 0.50%).

[0407] Combined drug use: cells were treated with 5 μL of each drug, AST-3424 and Nutlin-3, in different dosing orders: AST-3424 pretreatment for 2 hours, followed by Nutlin-3 treatment for 6 hours; Nutlin-3 pretreatment for 2 hours, followed by AST-3424 treatment for 6 hours; and AST-3424 and Nutlin-3 treatment for 6 hours.

[0408] The experimental results are shown in Figure 2.

[0409] The results of the cell proliferation experiment (3 days) showed that after adding Nutlin-3, which indirectly activates p53 function, the AST-3424 proliferation inhibition rate was significantly improved and was not affected by the order of adding Nutlin-3.

[0410] Effects of different sequential additions of AST-3424±Nutlin-3 on H460 / HPAF-II cell clone formation

[0411] Overview of the experimental process:

[0412] H460: 1000 cells / 2ml / well / 6-well plate

[0413] HPAF-II: 3000 cells / 2ml / well / 6-well plate

[0414] 1) Add 2 ml of H460 cell suspension to each well of a 6-well plate, with a cell density of 1000 cells / well; add 2 ml of HPAF-II cell suspension to each well of a 6-well plate, with a cell density of 3000 cells / well.

[0415] 2) Inoculate cells with 1990 μL of culture medium and culture the cells in a 37° C., 5% CO 2 incubator overnight.

[0416] 3) Compound treatment

[0417] For single drug use, AST-3424 and Nutlin-3, 5 μL of each compound at different concentrations (400-fold) and 5 μL of culture medium were added to each well, and cells were treated (DMSO 0.25%) for 6 h;

[0418] For combined use, cells were treated with 5 μL of each drug, AST-3424 and Nutlin-3, in different dosing orders: AST-3424 pretreatment for 2 hours, Nutlin-3 treatment for 6 hours; Nutlin-3 pretreatment for 2 hours, AST-3424 treatment for 6 hours; AST-3424 and Nutlin-3 treatment for 6 hours.

[0419] 4) After a total of 6 hours of treatment, the cells were washed twice to remove the compound, and 5 mL of culture medium was added to each well.

[0420] 5) Culture for 7 days (the time for medium replacement every 3 days and the end of observation can be determined based on the specific cell proliferation situation).

[0421] 6) Stain when the majority of cell colonies reach approximately 50 cells under 0 nM (0.5% DMSO) conditions. Aspirate the medium and fix and stain with 0.5% [w / v] crystal violet for 40 minutes. Wash the plate twice with tap water and allow to dry before counting.

[0422] 7) Count cell clones and count the number of clones with more than 50 cells.

[0423] The experimental results are shown in Figure 3.

[0424] The combination of Nutlin-3 and AST-3424 significantly inhibited the colony formation of H460 and HPAF-II cells compared with either drug alone, and the combination had an additive effect.

[0425] Among the three groups of H460 cells treated with dual drugs, the number of clones in the group with AST-3424 added first was less than that in the other two groups.

[0426] In the three groups of HPAF-II cells treated with dual drugs, different drug addition orders had no significant effect.

[0427] In H460 cells, the number of clones in the three drug-dosing order combination groups was compared with the DMSO group. The percentage of the number of clones in the drug-dosing order compared to the DMSO group was used to represent the ability of the drug combination to inhibit cell clone formation. The lower the percentage, the stronger the drug's ability to inhibit cell clone formation. The percentage of the number of clones in the drug-dosing order combination groups compared to the DMSO group was 11.12%, 23.03%, and 21.45%, respectively. The data of HPAF-II cells were analyzed in the same way and found that the percentage of the number of clones in the drug-dosing order combination groups compared to the DMSO group was 24.14%, 30.41%, and 40.30%, respectively.

[0428] It can be inferred that, relatively speaking, the combined use of Nutlin-3 has a significantly better cloning inhibitory effect on p53 wild-type H460 cells than on p53 mutant HPAFII cells.

[0429] Experimental conclusion: The above experimental results show that Nutlin-3 can indeed enhance the in vitro cytotoxicity of AST-3424 / AST against cancer cells, and the enhancement of in vitro cytotoxicity against p53 wild-type H460 cells is even greater.

[0430] Example 3 Effects of Nutlin-3 combined with AST-3424 on cell apoptosis and cell cycle G2 / M arrest

[0431] Effects of AST-3424 and Nutlin-3 alone or in combination on the apoptosis of H460 cells

[0432] Overview of the experimental process:

[0433] 1) Add 99 μL of H460 cell suspension to each well of a 96-well white plate, with a cell density of 15,000 cells / well. Simultaneously, add a 96-well clear plate with the same cell density for observation and photography before testing.

[0434] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0435] 3) Compound treatment

[0436] Single drug administration: 24 hours after cell plating, add 0.5 μL of culture medium to each well. According to the protocol, add 0.5 μL of the corresponding concentration of compound to the designated cell wells, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0437] Combination therapy: 24 hours after cell plating, add 0.5 μL of the corresponding concentration of Nutlin-3 compound to the designated cell wells, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator for 2 hours, and then add 0.5 μL AST-3424 to the designated cell wells.

[0438] 4) Place the cell plate in an incubator for 24 hours.

[0439] 5) Place the cell test plate at room temperature for 5 minutes to equilibrate.

[0440] 6) Preparation Mix the reagent buffer and substrate in a 1:1 ratio to make The prepared reagents can be stored at 4°C for 3 days.

[0441] 7) 100 μL Add the reagent to each well containing 100 μL of blank (cell-free well), negative control cells, or treated cells. Due to the sensitivity of the assay, be careful not to touch the pipette tip to the well containing the sample to avoid cross-contamination. Cover the plate with a plate sealer or lid.

[0442] 8) Gently mix the reagent solution using a shaker at 300–500 rpm for 30 seconds. Incubate at room temperature in the dark for 1 hour.

[0443] 9) Measure the luminescence of each sample in a plate-reading luminometer. Calculate the luminescence values ​​for each group and analyze them in Relative Luminescence Units (RLU) using a bar graph. The results are shown in Figure 4.

[0444] Compared with each single drug, the combination of effective concentration of AST-3424 (1nM) and Nutlin-3 can significantly increase the content of Caspase3 / 7, a marker of cancer cell apoptosis, which means that the combination of the two can increase cell apoptosis. The marker content is different after treatment with different doses of Nutlin-3, which also shows that the above-mentioned increase in apoptosis is Nutlin-3 dose-dependent; that is, as the concentration of Nutlin-3 increases, the content of cell apoptosis detection markers also increases after the combination of AST-3424 and Nutlin-3, which also shows that the above-mentioned increase in apoptosis is Nutlin-3 dose-dependent.

[0445] In other words, the addition of Nutlin-3, which indirectly activates the function of p53 protein, can enhance the apoptotic effect of AST-3424.

[0446] Effects of AST-3424 and Nutlin-3 alone or in combination on cell cycle G2 / M arrest

[0447] The entire cell replication cycle can be described as G0 / G1, S, and G2 / M phases. In tumor pathology research, the S phase cell ratio is usually used as an indicator to judge the tumor proliferation status.

[0448] Overview of the experimental process:

[0449] 1) Add H460 / HPAF-II cell suspensions to 24-well plates at a cell density of 100,000 cells / well and add 995 μL of culture medium to each well.

[0450] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0451] 3) Compound treatment

[0452] 24 hours after cell plating, each cell line was divided into the following experimental groups according to the protocol:

[0453] Single drug use: 24 hours after cell plating, add 5 μL of test compound at different concentrations: 1% DMSO, 5 μM Nutlin-3, 0.1 nM AST-3424, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator;

[0454] Combination medication:

[0455] Cells were treated with AST-3424 for 2 hours in advance and then co-treated with Nutlin-3 for 24 hours;

[0456] Cells were treated with Nutlin-3 for 2 hours in advance and then co-treated with AST-3424 for 24 hours;

[0457] AST-3424 and Nutlin-3 were co-treated for 24 hours.

[0458] 4) Digestion, centrifugation at 1000 g at 4°C, wash once with pre-chilled PBS, centrifugation at 1000 g at 4°C, and fixation with 1 mL of pre-chilled 70% ethanol at -20°C overnight.

[0459] 5) Centrifuge at 3000 g at 4°C, wash once with pre-chilled PBS (phosphate buffered saline), centrifuge at 3000 g at 4°C, prepare PI staining solution, stain at 37°C in the dark for 30 minutes, store in an ice bath in the dark, and analyze the proportion of cells in different cell cycles using flow cytometry and FlowJo on the same day. The results are shown in Figure 5.

[0460] The results showed that in H460 cells with wild-type p53, single treatment with 0.1 nM AST-3424 and 5 μM Nutlin-3 did not significantly alter the cell cycle. However, the combined treatment of 0.1 nM AST-3424 followed by 5 μM Nutlin-3 significantly reduced the G0 / G1 phase and increased the G2 / M phase. The S phase was slightly reduced in the other two groups. The cell cycle of HPAF-II cells harboring pathogenic p53 mutations was minimally affected.

[0461] Numerous studies have shown that cell cycle arrest at the G2 / M phase can induce cell apoptosis.

[0462] In order to further study the effects of different concentrations of AST-3424 and Nutlin-3 alone and in combination, as well as different drug addition orders when used in combination, on the cell cycle of H460 / A549 cells (p53 wild type, normal protein expression), a second experiment was conducted.

[0463] The rest of the operation is similar to the above experiment, and the compound treatment is as follows:

[0464] Use alone

[0465] For H460 cell lines, 1% DMSO was added, 5 μM Nutlin-3 was added for 22 hours, 0.03 nM AST-3424 was added for 24 hours, 0.1 nM AST-3424 was added for 24 hours, 0.3 nM AST-3424 was added for 24 hours, and 1 nM AST-3424 was added for 24 hours;

[0466] For A549 cell lines, 1% DMSO was added, 5 μM Nutlin-3 was added for 22 hours, 0.3 nM AST-3424 was added for 24 hours, 1 nM AST-3424 was added for 24 hours, 9 nM AST-3424 was added for 24 hours, and 9 nM AST-3424 was added for 24 hours.

[0467] Combination therapy

[0468] For H460 cell line,

[0469] 0.03 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0470] 0.1 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0471] 0.3 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0472] 1 nM AST-3424 was added for pre-treatment for 2 hours, and then 5 μM Nutlin-3 was added and co-treated for 22 hours.

[0473] For A549 cell line,

[0474] 0.3 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0475] 1 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0476] 3 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0477] 9 nM AST-3424 was added for pre-treatment for 2 hours, and then 5 μM Nutlin-3 was added and co-treated for 22 hours.

[0478] The results are shown in Figure 6.

[0479] Further experimental results showed that AST-3424 caused concentration-dependent G2 / M cell cycle arrest in p53 wild-type cancer cells (H460 and A549).

[0480] The combination of AST-3424 and Nutlin-3 significantly increased the G2 / M phase arrest and reduced the G0 / G1 phase in p53 wild-type cancer cells (H460 and A549), and was AST-3424 concentration-dependent.

[0481] The combination of AST-3424 and Nutlin-3 (AST-3424 pretreatment for 2 hours) increased G2 / M arrest, which was consistent with the results of the same drug addition order in the above preliminary experiments.

[0482] The above results show that Nutlin-3, which indirectly activates p53 function, can change the cell cycle of p53 wild-type cancer cells (H460 and A549), causing G2 / M cell cycle arrest, and showing AST-3424 concentration dependence. However, it has no effect on the cell cycle of HPAF-II cells, a cell line with a p53 pathogenic mutation. In other words, the combination of Nutlin-3 and AST-3424, which indirectly activates p53 function, can regulate the cell cycle of p53 wild-type cancer cells (H460 and A549), enhancing G2 / M cell cycle arrest, and showing AST-3424 concentration dependence; however, it has no effect on the cell cycle of HPAF-II cells, a cell line with a p53 pathogenic mutation.

[0483] According to Examples 2 and 3, p53 protein has a decisive influence on the efficacy of AST-3424 / AST drugs. DNA alkylating drugs such as AST-3424 / AST have a stronger proliferation inhibitory effect on cells that are negative for p53 gene mutations and have normal p53 protein. Its mechanism of action includes cell cycle G2 / M arrest and cell clone inhibition.

[0484] To further confirm the above conclusions, the p53-MDM2 inhibitor RITA (which is also a p53-HDM-2 inhibitor) was used to activate p53 function, and then in vitro cytotoxicity experiments were performed.

[0485] The CAS number of RITA is 213261-59-7.

[0486] Example 4 Effects of AST, Nutlin-3, and RITA alone or in combination on HPAF-II / H460 cell cytotoxicity

[0487] In vitro proliferation inhibition assay

[0488] Overview of the experimental process

[0489] 1) Add H460 and HPAF-Ⅱ cell suspensions to a 96-well plate, 100 μL per well, with cell densities of 2,000 and 20,000 cells per well, respectively.

[0490] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0491] 3) Compound treatment

[0492] Combination therapy: 24 hours after plating cells, add 99 μL of growth medium to each well. Add 0.5 μL of Nutlin-3 or RITA to each well and gently vortex to ensure uniform mixing. After incubation for 2 hours, add 0.5 μL of test compound at various concentrations and gently vortex to ensure uniform mixing. Then, incubate at 37°C, 5% CO2.

[0493] Single drug application: 24 hours after cell plating, add 99.5 μL of growth medium to each well. Add 0.5 μL of test compound at different concentrations, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0494] 4) Place the cell plate in an incubator for 72 hours.

[0495] 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.

[0496] 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.

[0497] 7) Read the chemiluminescence signal value using a multifunctional microplate reader with a reading time of 1000 ms, and calculate the corresponding inhibition rate data.

[0498] The drug groups and experimental results of the H460 cell proliferation inhibition experiment using AST and Nutlin-3 alone or in combination are shown in Table 2 below. Figure 7 is drawn based on the inhibition rate data in Table 2.

[0499] Table 2: Dosage groups and inhibition rates of AST and Nutlin-3 alone or in combination for H460 cell proliferation inhibition

[0500] The dosing groups and experimental results of the H460 cell proliferation inhibition experiment using AST and RITA (p53-HDM-2 inhibitor, indirectly upregulating p53 expression and function) alone or in combination are shown in Table 3 below. Figure 8 is drawn based on the inhibition rate data in Table 3.

[0501] Table 3: Dosage groups and inhibition rates of H460 cell proliferation inhibition experiments using AST and RITA alone or in combination

[0502] The drug groups and experimental results of the HPAF-Ⅱ cell proliferation inhibition experiment using AST and Nutlin-3 alone or in combination are shown in Table 4. Figure 9 is drawn based on the inhibition rate data in Table 4.

[0503] Table 4: Dosage groups and inhibition rates of the HPAF-Ⅱ cell proliferation inhibition experiment using AST and Nutlin-3 alone or in combination

[0504] The drug groups and experimental results of the HPAF-Ⅱ cell proliferation inhibition experiment using AST and RITA alone or in combination are shown in Table 5 below. Figure 10 is drawn based on the inhibition rate data in Table 5.

[0505] Table 5: Dosage groups and inhibition rates of HPAF-Ⅱ cell proliferation inhibition experiments using AST and RITA alone or in combination

[0506] Obviously, compared with each single drug, the combination of Nutlin-3 or RITA and AST significantly enhanced the proliferation inhibitory effect on p53 wild-type H460 cells, while for p53 mutant cells HPAF-II, the combination regimen had almost no enhancing effect; that is, the above experiments once again confirmed that p53 protein has a decisive influence on the efficacy of AST-3424 / AST drugs, and DNA alkylating drugs such as AST-3424 / AST have a stronger proliferation inhibitory effect on cells with negative p53 gene mutations and normal p53 protein.

[0507] In particular, taking AST alone as an example, an inhibition rate of 11-12% was achieved. For p53 wild-type cells H460, only 1.5 nM was required, while for p53 mutant cells HPAF-II, 60 nM was used. This shows that DNA alkylating agents such as AST / AST-3424 have a stronger cell proliferation inhibitory effect on p53 wild-type cancer cells.

[0508] The expression levels of AKR1C3 protein in H460 and HPAF-II cells are very similar, indicating that AST-3424 / AST is activated to a similar extent in both cell types. However, taking AST and AST-3424 alone as an example, the applicant's previous cancer cell proliferation inhibition test data are as follows:

[0509] IC of AST in H460 and HPAF-II cells 50 6.87nM and 329.1nM respectively,

[0510] IC of AST-3424 in H460 and HPAF-II cells 50 0.47nM and 107.3nM respectively.

[0511] The above data further verified that DNA alkylating agents such as AST / AST-3424 have a stronger cell proliferation inhibitory effect on p53 wild-type cancer cells.

[0512] According to Examples 2, 3, and 4, p53 protein has a decisive influence on the efficacy of AST-3424 / AST. DNA alkylating agents such as AST-3424 / AST have a stronger proliferation inhibitory effect on cells that are negative for p53 gene mutations and have normal p53 protein. Its mechanism of action includes cell cycle G2 / M arrest and cell clone inhibition.

[0513] In order to further explain the above experimental phenomena, the effects of single drugs or combination drugs on the P53 protein pathway were detected.

[0514] Example 5 Experiment on the Effect of AST-3424±Nutlin-3 on the p53 Protein Pathway

[0515] The first study to investigate the effects of AST-3424 and Nutlin-3 alone or in combination on Ser15-p53, Ser20-p53, Total p53, MDM2, and p21 proteins in H460 cells

[0516] Overview of the experimental process:

[0517] 1) Add H460 cell suspension to a 24-well plate at a cell density of 80,000 cells / well and add 995 μL of culture medium to each well.

[0518] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0519] 3) Compound treatment

[0520] 24 hours after cell plating, each cell line was divided into the following experimental groups according to the protocol:

[0521] Single drug use: 24 hours after cell plating, add 5 μL of test compounds at different concentrations: 0.5% DMSO, 0.1nM AST-3424, 5μM Nutlin-3, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0522] Combination therapy: 24 hours after cell plating, add 5 μL of test compounds at different concentrations. After pretreatment with 5 μM Nutlin-3 for 2 hours, add 0.1 nM AST-3424, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0523] 4) Collect cell protein lysate for Western blotting. The results of Western blotting are shown in Figure 11.

[0524] WB detection, namely protein immunoblotting (Western Blot, WB) experiment.

[0525] Compared with AST-3424 and Nutlin-3 alone, the combination of AST-3424 and Nutlin-3 significantly promoted p53 phosphorylation (Ser15-p53 and Ser20-p53) and total-p53 protein expression, and upregulated the expression of p53 downstream genes MDM2 and p21.

[0526] In order to further explore the effect of combining different concentrations of AST-3424 with Nutlin-3, a second experiment was conducted.

[0527] Second experiment on the effects of AST-3424 and Nutlin-3 alone or in combination on H460 cells, Ser15-p53, Ser20-p53, Total p53, MDM2, and p21 proteins

[0528] The rest of the operation was similar to the first experiment, and the compound treatment groups were administered as follows.

[0529] Single drug group: 1% DMSO, 5 μM Nutlin-3 for 24 hours, 0.1 nM AST-3424 for 24 hours, 0.3 nM AST-3424 for 24 hours, and 1 nM AST-3424 for 24 hours;

[0530] Combination drug group:

[0531] 0.1 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0532] 0.3 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0533] 1 nM AST-3424 was added for pre-treatment for 2 hours, and then 5 μM Nutlin-3 was added and co-treated for 22 hours.

[0534] The results are shown in Figure 12.

[0535] The experimental results showed that Nutlin-3 upregulated the expression levels of p53, p53 downstream protein p21 and MDM2 protein in H460 cells (P53 wild type, normal protein expression), which is consistent with the data reported in the literature (Meijer A, Kruyt FA, van der Zee AG, et al. Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL. Br J Cancer. 2013; 109(10): 2685-2695. doi: 10.1038 / bjc.2013.636). Compared to either agent alone, the combination of AST-3424 and Nutlin-3 at increasing concentrations significantly promoted p53 phosphorylation (Ser15-p53 and Ser20-p53) and total p53 protein expression, and upregulated the expression of the p53 downstream genes MDM2 and p21. Furthermore, the effects on these protein expressions were dose-dependent.

[0536] In order to further explore the effect of the order of drug addition on the combined effect of AST-3424 and Nutlin-3, a third experiment was conducted.

[0537] The third study on the effects of AST-3424 and Nutlin-3 alone or in combination on H460 cells, Ser15-p53, Ser20-p53, total p53, MDM2, and p21 proteins

[0538] The rest of the operation was similar to the first experiment, and the compound treatment groups were administered as follows.

[0539] Single drug group, 0.5% DMSO, 5 μM Nutlin-3 for 24 hours, 0.1 nM AST-3424 for 24 hours;

[0540] Combination drug group:

[0541] 0.1 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0542] 0.1 nM AST-3424 and 5 μM Nutlin-3 were added and co-treated for 24 h;

[0543] 5 μM Nutlin-3 was added for pre-treatment for 2 hours, and then 0.1 nM AST-3424 was added and co-treated for 22 hours.

[0544] The results are shown in Figure 13.

[0545] The experimental results showed that compared with either drug alone, the combination of AST-3424 and Nutlin-3 significantly promoted p53 phosphorylation (Ser15-p53 and Ser20-p53) and total-p53 protein expression, and upregulated the expression of the p53 downstream genes MDM2 and p21. However, the effects of different drug administration sequences on the expression of these proteins did not differ significantly.

[0546] Example 6 Effects of Nutlin-3 and AST-3424 alone or in combination on RAD51 protein levels

[0547] The combination of AST-3424 and Nutlin-3 significantly inhibited RAD51 protein expression

[0548] In the first, second, and third experiments of Example 5 on the effects of AST-3424 combined with Nutlin-3 on Ser15-p53, Ser20-p53, Total p53, MDM2, and p21 proteins in H460 cells, the applicant also performed WB detection of RAD51 protein. The results are shown in Figures 14, 15, and 16.

[0549] The results of the above three experiments all show that compared with AST-3424 and Nutlin-3 alone, the combination drug significantly reduced the RAD51 protein content and was AST-3424 dose-dependent.

[0550] Combination of AST-3424 and Nutlin-3 increases RAD51 protein degradation

[0551] Overview of the experimental process:

[0552] 1) Add H460 cell suspension to a 24-well plate at a cell density of 100,000 cells / well and add 995 μL of culture medium to each well.

[0553] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0554] 3) Compound treatment

[0555] Single drug use: 24 hours after cell plating, add 5 μL of test compounds at different concentrations: 1% DMSO, 0.1 nM AST-3424, 5 μM Nutlin-3, gently shake to ensure uniform mixing, then place in a 37°C, 5% CO2 incubator and continue to culture for 24 hours before harvesting the cells.

[0556] Combination therapy: 24 hours after cell plating,

[0557] 0.1 nM AST-3424 was added for pretreatment for 2 hours, and then 5 μM Nutlin-3 was added for co-treatment for 22 hours;

[0558] 1% DMSO and 5 μM MG-132 were added and co-treated for 24 h;

[0559] 0.1 nM AST-3424 and 5 μM MG-132 were added and co-treated for 24 h;

[0560] 5 μM Nutlin-3 and 5 μM MG-132 were added and co-treated for 24 h;

[0561] 0.1 nM AST-3424 was added for pre-treatment for 2 h, 5 μM Nutlin-3 was added for treatment for 16 h, and then 5 μM MG-132 was added for co-treatment for 6 h.

[0562] 4) The cell protein lysate was collected and subjected to WB detection. The detection results are shown in FIG17 .

[0563] MG-132 is a proteasome inhibitor that inhibits protein degradation after addition.

[0564] The addition of MG-132 reversed the downregulation of RAD51 protein after the combination of AST-3424 and Nutlin-3, suggesting that the decrease in RAD51 protein caused by the combination is related to the increased ubiquitination and degradation of RAD51 protein.

[0565] Combining AST-3424 with Nutlin-3 reduces the half-life of RAD51 protein

[0566] Overview of the experimental process:

[0567] 1) Add H460 cell suspension to a 24-well plate at a cell density of 100,000 cells / well and add 995 μL of culture medium to each well.

[0568] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0569] 3) Compound treatment

[0570] 24 hours after cell plating, each cell line was divided into the following experimental groups according to the protocol:

[0571] Single drug treatment groups: 1% DMSO treatment group, 5 μM Nutlin-3 treatment group, 0.1 nM AST-3424 treatment group.

[0572] Combination drug groups:

[0573] 1% DMSO + Cycloheximide 0.5 hour treatment group,

[0574] 1% DMSO + Cycloheximide 1 hour treatment group,

[0575] 1% DMSO + Cycloheximide 2 hours treatment group,

[0576] 1% DMSO + Cycloheximide 4 hours treatment group,

[0577] 0.1nM AST-3424+Cycloheximide 0.5 hour treatment group,

[0578] 0.1nM AST-3424+Cycloheximide 1 hour treatment group,

[0579] 0.1nM AST-3424+Cycloheximide 2-hour treatment group,

[0580] 0.1nM AST-3424+Cycloheximide 4-hour treatment group,

[0581] 5μM Nutlin-3+Cycloheximide 0.5 hour treatment group,

[0582] 5μM Nutlin-3+Cycloheximide 1 hour treatment group,

[0583] 5μM Nutlin-3+Cycloheximide 2-hour treatment group,

[0584] 5μM Nutlin-3+Cycloheximide 4-hour treatment group,

[0585] 0.1nM AST-3424+5μM Nutlin-3 treatment group,

[0586] 0.1nM AST-3424+5μM Nutlin-3+Cycloheximide 0.5 hour treatment group,

[0587] 0.1nM AST-3424+5μM Nutlin-3+Cycloheximide 1 hour treatment group,

[0588] 0.1nM AST-3424+5μM Nutlin-3+Cycloheximide 2-hour treatment group,

[0589] 0.1 nM AST-3424 + 5 μM Nutlin-3 + Cycloheximide treatment group for 4 hours.

[0590] Single drug use: 24 hours after cell plating, add 5 μL of test compound at different concentrations, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator. After further culture for 24 hours, harvest the cells.

[0591] Combination medication:

[0592] 24 hours after cell plating, add 5 μL of 1% DMSO and continue to culture in a 37°C, 5% CO2 incubator for another 24 hours. Then, add 5 μL of 4 μM cycloheximide and continue to culture in a 37°C, 5% CO2 incubator for 0.5, 1, 2, and 4 hours, respectively, before harvesting the cells.

[0593] 24 hours after cell plating, add 5 μL of 0.1 nM AST-3424 and continue to culture in a 37°C, 5% CO2 incubator for another 24 hours. Then, add 5 μL of 4 μM cycloheximide and continue to culture in a 37°C, 5% CO2 incubator for 0.5, 1, 2, and 4 hours, respectively, before harvesting the cells.

[0594] 24 hours after cell plating, add 5 μL of 5 μM Nutlin-3 and continue to culture in a 37°C, 5% CO2 incubator for another 24 hours. Then, add 5 μL of 4 μM Cycloheximide and continue to culture in a 37°C, 5% CO2 incubator for 0.5, 1, 2, and 4 hours, respectively, before harvesting the cells.

[0595] 24 hours after cell plating, add 5 μL of 0.1 nM AST-3424 and incubate in a 37°C, 5% CO2 incubator for 2 hours. Then, add 5 μL of 5 μM Nutlin-3 and continue incubating in a 37°C, 5% CO2 incubator for another 24 hours before harvesting the cells.

[0596] 24 hours after cell plating, add 5 μL of 0.1 nM AST-3424 and culture in a 37°C, 5% CO2 incubator for 2 hours. Then, add 5 μL of 5 μM Nutlin-3 and gently shake to ensure uniform mixing. Continue to culture in a 37°C, 5% CO2 incubator for 24 hours. Then, add 5 μL of 4 μM Cycloheximide and continue to culture in a 37°C, 5% CO2 incubator for 0.5, 1, 2, and 4 hours, respectively, before harvesting the cells.

[0597] 4) The cell protein lysate was collected and subjected to WB detection. The detection results are shown in FIG18 .

[0598] Furthermore, based on the ratio data of the corresponding proteins relative to the internal reference protein β-actin in FIG18 , the change curve of the relative amount of RAD 51 after different time periods was calculated, and the results are shown in FIG19 .

[0599] Cycloheximide is an inhibitor of eukaryotic protein synthesis, and protein synthesis is terminated after its addition.

[0600] After 24 hours of treatment with AST-3424 and Nutlin-3 alone or in combination, no new protein synthesis occurred after the addition of cycloheximide. As shown in Figure 15 , RAD51 protein levels decreased in all groups. However, the combination of Nutlin-3 and AST-3424 significantly reduced RAD51 protein expression and half-life compared with either single drug, confirming once again that the combination can promote RAD51 degradation.

[0601] Example 7 Effect of AST-3424 combined with Nutlin-3 on DNA damage in H460 cells

[0602] In the first, second, and third experiments of Example 5 on the effects of AST-3424 combined with Nutlin-3 on Ser15-p53, Ser20-p53, Total p53, MDM2, and p21 proteins in H460 cells, the applicant also performed WB detection of γH2AX protein. The results are shown in Figures 20, 21, and 22.

[0603] The results showed that AST-3424 could increase the amount of γH2AX protein, a biomarker of DNA double-strand damage, in a dose-dependent manner.

[0604] Compared with each single drug, the combination of AST-3424 and Nutlin-3 can increase the amount of γH2AX protein, and is dose-dependent on AST-3424; however, the simultaneous treatment of AST-3424 and Nutlin-3 can more significantly upregulate γH2AX protein.

[0605] Example 8 Effects of Nutlin-3 / AST Treatment for 24 Hours on the Expression of p53, Rad51, MDM2, and p21 Proteins in HPAF-II Cells

[0606] Overview of the experimental process:

[0607] 1) Add the HPAF-II cell suspension to a 24-well plate at a cell density of 300,000 cells / well and add 995 μL of culture medium to each well.

[0608] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0609] 3) Compound treatment: 24 hours after cell plating, the cells were divided into groups and treated with drugs.

[0610] HPAF-II cells were divided into the following groups: 1% DMSO treatment group, 60 nM AST treatment group, 8 μM Nutlin-3 treatment group, and 60 nM AST + 8 μM Nutlin-3 treatment group.

[0611] Single drug use: 24 hours after cell plating, add 5 μL of test compounds at different concentrations: 1% DMSO, AST 1.5nM, Nutlin-31μM, AST 60nM, Nutlin-38μM, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0612] Combination therapy: 24 hours after cell plating, add 5 μL of 1 μM Nutlin-3 and 8 μM Nutlin-3, respectively. After pretreatment for 2 hours, add 5 μL of 1.5 nM AST and 60 nM AST, respectively. Gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0613] 4) After incubating the 24-well plate in a 37° C., 5% CO 2 incubator for 24 hours, the cell protein lysate was collected for WB detection. The WB detection results are shown in FIG23 .

[0614] Nutlin-3 is an inhibitor of the binding of p53 and MDM2 proteins, activating the function of p53.

[0615] The experimental results showed that 60nM AST had no effect on the expression of p53, p53 downstream protein p21 and MDM2 protein in HPAF-II cells (p53 pathogenic mutation cells).

[0616] The combination of AST and Nutiln-3 had no effect on the expression of p53, p53 downstream proteins p21 and MDM2 in HPAF-II cells (p53 mutation, protein cannot be expressed normally).

[0617] AST alone promotes RAD51 protein expression in HPAF-II cells (p53 mutation, protein cannot be expressed normally), but has no synergistic effect when combined with Nutlin-3.

[0618] Example 9 Effects of High-Concentration Nutlin-3 / AST-3424 Treatment on Ser15-p53, Ser20-p53, p53, Rad51, MDM2, p21, γH2AX, and Apoptosis-Related Proteins Caspase3 and Cleaved Caspase3 in HPAF-II Cells

[0619] The above experiments revealed that for HPAF-II cells with p53 gene mutations, lower concentrations of Nutlin-3 / AST-3424 had almost no effect on related proteins. To further confirm this, this experiment used a high-concentration Nutlin-3 / AST-3424 drug combination: 5μM Nutlin-3 + 1000nM AST-3424 to eliminate the influence of concentration.

[0620] Overview of the experimental process:

[0621] 1) Add the HPAF-II cell suspension to a 24-well plate at a cell density of 300,000 cells / well and add 995 μL of culture medium to each well.

[0622] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0623] 3) Compound treatment

[0624] 24 hours after cell plating, each cell line was divided into the following experimental groups according to the protocol:

[0625] Single drug use: 24 hours after cell plating, add 5 μL of test compounds at different concentrations: 0.5% DMSO, 1000nM AST-3424, 15 μM Nutlin-3, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0626] Combination therapy: 24 hours after cell plating, add 5 μL of test compounds at different concentrations. After pretreatment with 15 μM Nutlin-3 for 2 hours, add 1000 nM AST-3424. Gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0627] 4) Collect cell protein lysate for Western blotting. The results of Western blotting are shown in Figure 24.

[0628] Obviously, even in the case of high-concentration administration, no protein expression with enhanced effects after combined use was observed in the p53 mutant cell line HPAF-II cells, or the enhanced effect of combined use was not obvious.

[0629] Based on the above-mentioned fifth to ninth embodiments, the following conclusions can be drawn:

[0630] 1. Nutlin-3 / RITA, a drug that upregulates p53 protein expression or activates p53 function, can enhance the in vitro toxicity of AST-3424 / AST to cancer cells; and the enhancement effect is more significant for p53 wild-type cancer cells.

[0631] 2. Drugs that upregulate p53 protein expression or activate p53 function can enhance cell apoptosis caused by AST-3424 / AST. For p53 wild-type cancer cells, the combined use is more significant in cell apoptosis.

[0632] 3. Nutlin-3 / RITA, a drug that upregulates p53 protein expression or activates p53 function, can significantly increase DNA double-strand damage caused by AST-3424 / AST. For p53 wild-type cancer cells, the combined use leads to more significant DNA double-strand damage.

[0633] 4. Nutlin-3 / RITA, a drug that upregulates p53 protein expression or activates p53 function, can significantly increase the cell cycle G2 / M arrest caused by AST-3424 / AST. For p53 wild-type cancer cells, the combined use of Nutlin-3 / RITA is more significant in terms of cell cycle G2 / M arrest.

[0634] In summary, the above enhancement effects reveal that DNA alkylating agents such as AST-3424 / AST have a stronger proliferation inhibitory effect on p53 wild-type cells.

[0635] 5. Compared with single drugs, the combination of Nutlin-3 / RITA and AST-3424 / AST, which upregulate p53 protein expression or activate p53 function, significantly upregulates p53 phosphorylation, total-p53 expression, and the expression of p53 downstream proteins MDM2 and p21, activates the p53 protein pathway, and then downregulates RAD51 protein expression. The above phenomenon is more significant in the combined experiment for p53 wild-type cancer cells.

[0636] 6. The combination of Nutlin-3 / RITA and AST-3424 / AST, drugs that upregulate p53 protein expression or activate p53 function, significantly promoted the degradation of RAD51 protein and reduced the half-life of RAD51 protein compared with single drugs.

[0637] In summary, p53 protein promotes the degradation of the homologous recombination repair protein RAD51, leading to RAD51 downregulation and reduced DNA double-strand damage repair ability, thereby enhancing the pharmacological activity of DNA alkylating agents such as AST-3424 / AST. In other words, patients with normal p53 protein and p53 gene will be more sensitive to DNA alkylating agent treatment such as AST-3424 / AST due to the processes and pathways involved in the above-mentioned p53 protein, and may benefit more significantly from treatment (relative to patients with low p53 protein expression or p53 gene mutations). This phenomenon has been preliminarily observed in clinical trials of AST-3424.

[0638] Example 10 Effects of Compounds on In Vitro Cell Proliferation of H460 and p53 Knockout Cells H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 under Normoxia

[0639] p53 CRISPR / Cas9 KO plasmid (human) and p53 HDR plasmid (human) were purchased from Santa Cruz Company to construct H460 P53 KO cell line, i.e., H460 cell line with p53 gene knockout (KnockOut). A total of three groups of H460P53 KO cell lines were constructed, numbered H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12.

[0640] The construction process of p53 gene knockout cells is briefly described as follows:

[0641] H460 cells were plated in 6-well plates, 1×10 6 / hole.

[0642] H460 cells were co-transfected with p53 CRISPR / Cas9 KO plasmid (h) (sc-416469) and p53 HDR plasmid (h) (sc-416469-HDR): 125 μL Opti-MEM + 1.25 μg of each plasmid + 5 μL P3000; 125 μL Opti-MEM + 15 μL (increased volume is expected to increase transfection efficiency) Lipofectamine 3000, incubated for 15 minutes, and then added dropwise to the culture medium to mix.

[0643] After 48 h, 2 μg / mL puromycin was added for selection.

[0644] After 2 days, fresh 1640 culture medium containing 1 μg / mL puromycin was replaced and culture was continued. The culture medium was changed every two days.

[0645] When the cell clones are large enough, single clones are picked for culture.

[0646] When the number of cells is sufficient, samples are collected for WB identification of clones. The clones that do not express P53 are P53 knockout H460 clone cells.

[0647] The experimental process of this example refers to the experimental process overview of "Effects of compounds on H460 cell proliferation in vitro under normoxia" in Example 2.

[0648] Compound treatment

[0649] Single drug administration: Compound AST-3424 / AST was administered alone in H460 and H460 P53 KO cell lines, respectively;

[0650] Combination therapy: Compound AST-3424 / AST was used in combination with AST-3021 in H460 and H460 P53 KO cell lines, respectively.

[0651] H460 P53 KO cell lines, namely H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cell lines were used for experiments respectively.

[0652] The experimental results of AST-3424 are shown in Table 11, and the corresponding in vitro proliferation inhibition rate curves of H460 and H460P53 KO cells using AST-3424 alone or in combination with AST-3021 are shown in Figure 25.

[0653] The results of the AST experiment are shown in Table 12 , and the corresponding curves of the in vitro proliferation inhibition rate of H460 and H460 P53KO cells by AST alone or in combination with AST-3021 are shown in FIG26 .

[0654] Table 11: Inhibitory effect of AST-3424 on the proliferation of H460 WT (wild-type H460 cells) and H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cells in vitro

[0655] The experimental data presented in Table 11 and Figure 25 show that H460 P53 KO #1 and #12 cells were 49.13-fold and 8.65-fold less sensitive to AST-3424, respectively, compared to H460 wild-type cells. This indicates that p53 deficiency leads to reduced sensitivity to AST-3424 and that p53 deficiency has no effect on AST-3424's selectivity for AKR1C3. This experiment further confirms the results of Examples 2 and 3, demonstrating that p53 protein has a decisive influence on the efficacy of AST-3424. DNA alkylating agents such as AST-3424 have a stronger inhibitory effect on cells that are negative for p53 gene mutations and have normal p53 protein.

[0656] Table 12: Inhibitory effect of AST on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7 and H460 P53 KO#12 cells in vitro

[0657] The experimental data in Table 12 and Figure 26 show that H460 P53KO#1 and #12 cells were 103.53-fold and 10.96-fold less sensitive to AST, respectively, compared to H460 wild-type cells. This indicates that p53 deficiency leads to decreased sensitivity to AST drugs, but p53 deficiency also nearly eliminates AST's selectivity for AKR1C3. This experiment further confirms the results of Example 4, demonstrating that p53 protein has a decisive influence on the efficacy of AST drugs. DNA alkylating agents such as AST have a stronger inhibitory effect on cells that are negative for p53 gene mutations and have normal p53 protein.

[0658] Example 11 Effects of Compounds on the Proliferation of H460 and H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 Cells in Vitro Under Hypoxia

[0659] Overview of the experimental process:

[0660] 1) Add H460, H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cell suspensions to two types of 24-well plates, 495 μL per well, and the cell density is 1×10 4 24-well plates with glass inserts were used for hypoxia experiments, and ordinary plastic 24-well plates were used for normoxic experiments.

[0661] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0662] 3) Compound treatment

[0663] Hypoxic conditions:

[0664] Adjust the hypoxia workstation to a hypoxic environment (O2 < 0.01%) and use an oxygen indicator to confirm the hypoxia in the workstation. 24 hours after cell plating, place the 24-well plate with a glass insert into the hypoxia workstation.

[0665] Place the 24-well plate on a shaker, open the cover of the plate and shake for gas exchange for 5 minutes.

[0666] 5 μL of 100-fold corresponding concentration of compound was added to each well, and 3 replicates were performed for each experimental group.

[0667] 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.

[0668] Normoxic conditions:

[0669] 24 hours after cell plating, 5 μL of 100-fold corresponding concentration of compound was added to each well, and 3 replicates were performed for each experimental group.

[0670] 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.

[0671] 4) Wash all 24-well plates twice with complete culture medium, 500 μL per well each time.

[0672] 5) Add 1000 μL of culture medium to each well.

[0673] 6) Place in a 37°C, 5% CO2 incubator for 72 hours.

[0674] 7) Discard 800 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.

[0675] 8) Transfer 100 μL of culture medium from each well of the 24-well plate to a 96-well white plate.

[0676] 9) Read the chemiluminescent signal using a multifunctional microplate reader with a reading time of 1000 ms.

[0677] 10) Calculate IC using GraphPad Prism 5 software 50 , the IC of the compound is obtained using the following nonlinear fitting formula 50 (half inhibitory concentration).

[0678] In this embodiment, the above experiment was carried out using the specific compound A in structural formula (1) and the specific compound B in structural formula (2). The structures of compounds A and B are as follows:

[0679] The experimental results of compound A are shown in Table 13, and the corresponding curve of the in vitro proliferation inhibition rate of compound A on H460 and H460 P53 KO cells is shown in FIG27.

[0680] The experimental results of compound B are shown in Table 14, and the corresponding curve of the in vitro proliferation inhibition rate of compound B on H460 and H460 P53 KO cells is shown in FIG28.

[0681] Table 13: Inhibitory effect of compound A on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7 and H460 P53 KO#12 cells in vitro

[0682] The experimental data presented in Table 13 and Figure 27 demonstrate that under hypoxic conditions, H460 P53 KO#1, #7, and #12 cells were 12.13-fold, 10.26-fold, and 6.60-fold less sensitive to Compound A than H460 P53 WT cells, while still exhibiting good hypoxia selectivity. This indicates that p53 protein plays a decisive role in the efficacy of Compound A (a DNA alkylating agent), further confirming the experimental results from the above examples showing that DNA alkylating agents have a stronger inhibitory effect on cells that are p53 mutation-negative and have normal p53 protein.

[0683] Table 14: Inhibitory effect of compound B on the proliferation of H460, H460 P53 KO#1, H460 P53 KO#7 and H460 P53 KO#12 cells in vitro

[0684] The experimental data presented in Table 14 and Figure 28 demonstrate that under hypoxic conditions, P53 KO#1, #7, and #12 cells were 22.31-fold, 27.69-fold, and 10.00-fold less sensitive to Compound B than H460 P53 WT cells, but exhibited reduced hypoxia selectivity. This suggests that p53 protein plays a crucial role in the efficacy of Compound B (a DNA alkylating agent), further confirming the experimental results from the previous examples showing that DNA alkylating agents have a stronger inhibitory effect on cells with normal p53 protein and negative p53 gene mutations.

[0685] Example 12 Effects of Nutlin-3 on Total P53, MDM2, P21, AK R1C3, and Actin Proteins in H460 and H460 P53 KO Cells

[0686] A P53 gene knockout (KnockOut) cell line was constructed using the same method as in Example 10 and numbered H460 P53 KO#8.

[0687] Overview of the experimental process:

[0688] 1) Add H460 and H460 P53 KO cell suspensions to a 24-well plate at a cell density of 100,000 cells / well and add 995 μL of culture medium to each well.

[0689] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0690] 3) Compound treatment

[0691] 24 hours after cell plating, each cell line was divided into the following experimental groups according to the protocol:

[0692] H460 WT untreated group, H460 P53 KO#1 untreated group, H460 P53 KO#7 untreated group, H460 P53 KO#8 untreated group, H460 P53 KO#12 untreated group, H460 WT 0.1% DMSO-treated group, H460 WT 5 μM Nutlin-3-treated group, H460 P53 KO#1 5 μM Nutlin-3-treated group, H460 P53 KO#7 5 μM Nutlin-3-treated group, H460 P53 KO#8 5 μM Nutlin-3-treated group, H460 P53 KO#12 5 μM Nutlin-3-treated group;

[0693] Single drug use: 24 hours after cell plating, add 5 μL of test compound at different concentrations, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0694] 4) After incubating the 24-well plate in a 37° C., 5% CO 2 incubator for 24 h, the cell protein lysate was collected for Western blotting.

[0695] WB detection method:

[0696] 1) Remove the cell plate, discard the culture supernatant, gently rinse with PBS, add 30 μL of RIPA protein lysis buffer (RIPA: phosphatase inhibitor = 10:1) to each well, scrape the cells with a cell scraper, lyse on ice for 30 minutes, and centrifuge at 14,000 rpm at 4°C for 10 minutes.

[0697] 2) Quantify the protein using the BCA method. Then load the sample and run the gel: Use 4-12% SDS-PAGE finished gel to separate the protein sample. The proteins loaded from left to right are H460 WT untreated group, H460 P53 KO#1 untreated group, H460 P53 KO#7 untreated group, H460 P53 KO#8 untreated group, H460 P53 KO#12 untreated group, H460 WT 0.1% DMSO-treated group, H460 WT 5μM Nutlin-3-treated group, H460 P53 KO#1 5μM Nutlin-3-treated group, H460 P53 KO#7 5μM Nutlin-3-treated group, H460 P53 KO#8 5μM Nutlin-3-treated group, and H460 P53 KO#12 5μM Nutlin-3-treated group. The protein loading amount of each sample was 10 μg / 12.5 μL, and the sample was transferred to a membrane after running the gel. The transfer conditions were: 100 V, 1 h.

[0698] 3) Antibody incubation and luminescent detection: After the transfer is completed, the membrane is blocked in 5% skim milk on a horizontal shaker at room temperature for 1 hour. Add the respective antibodies at a ratio of 1:1000 and incubate in an antibody incubation box at 4°C overnight. The next day, the membrane is rewarmed at room temperature for 1 hour and then washed in TBST for 10 minutes each time, three times. After washing, incubate with the corresponding secondary antibody (1:4000 dilution) according to the type of primary antibody. The incubation time is 2 hours. After incubation, wash three times with TBST for 10 minutes each time. Mix the ECL luminescent solution (SuperSignal West Femto Maximum Sensitivity) and AB solution, add it to the membrane that has been dried, and perform luminescent detection in a gel imager.

[0699] The protein band photos of the cell protein lysate WB detection results are shown in Figure 29, and the ratio of the corresponding proteins to the internal reference protein β-actin is shown in Figure 30.

[0700] In the above experiments, there was no change in the internal reference protein actin in all cell lines and after various treatments.

[0701] The experimental data shown in Figures 29 and 30 show that compared to wild-type H460, P53 knockout reduced the protein expression of AKR1C3 (more significantly in #1), while also reducing the expression of MDM2 and P21. Compared to the H460 wild-type DMSO group, treatment of wild-type H460 with the positive drug Nutlin-3 revealed that the expression of MDM2, P53, P21, and AKR1C3 proteins were all upregulated. However, after treatment of wild-type H460 and H460 P53 KO cell lines with Nutlin-3, it was found that compared to the H460 wild-type DMSO group, the expression of MDM2, P53, P21, and AKR1C3 proteins in H460 P53 KO cells was not upregulated, and this was particularly significant in H460 P53 KO #1 cells. The results suggest that H460 P53 KO may downregulate the protein expression of AKR1C3, which is consistent with the IC value of compound AST losing AKR1C3 selectivity in H460 P53 KO cells shown in Table 12 and Figure 26 of Example 10. 50 The experimental results are consistent.

[0702] Example 13 Effects of Compound C and AST-3424 on the Proliferation of NCI-H460 and NCI-H460 P53KO#1 Cells in Vitro Under Normoxia

[0703] This example further verifies the effect of the specific compound C in structural formula (9) on the proliferation of NCI-H460 and NCI-H460 P53KO#1 cells in vitro under normoxia. The structure of compound C is as follows:

[0704] Overview of the experimental process:

[0705] 1) Add NCI-H460 and NCI-H460 P53KO#1 cell suspensions to a 96-well plate at a density of 2000 cells per well.

[0706] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0707] 3) Compound treatment

[0708] Single drug application: 24 hours after cell plating, add 99.5 μL of growth medium to each well. Add 0.5 μL of test compound at different concentrations, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0709] Combination therapy: 24 hours after plating cells, add 99 μL of growth medium to each well. Add 0.5 μL of the combination compound and gently shake to ensure uniform mixing. After incubation for 2 hours, add 0.5 μL of the test compound at various concentrations and gently shake to ensure uniform mixing. Then, place the cells in a 37°C, 5% CO2 incubator.

[0710] 4) Place the cell plate in an incubator for 72 hours.

[0711] 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.

[0712] 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.

[0713] 7) Read the chemiluminescent signal value using a multifunctional microplate reader with a reading time of 1000 ms.

[0714] 8) Calculate IC using GraphPad Prism 5 software 50 (half inhibitory concentration).

[0715] Experimentally detected IC 50 The results are shown in Table 15 , and the in vitro proliferation inhibition rate curves of NCI-H460 and NCI-H460 P53KO#1 cells are shown in FIG31 and FIG32 .

[0716] Table 15: Inhibitory effects of compound C and AST-3424 on the proliferation of NCI-H460 and NCI-H460 P53 KO#1 cells in vitro

[0717] The above experimental data show that:

[0718] IC of compound C in NCI-H460 cells 50 2.05nM for AST-3424IC 50 The selectivity of AKR1C3 was 4.77 times that of AST-3424, and there was no significant difference between AKR1C3 and AST-3424.

[0719] After P53 knockout, the IC of compound C 50 The value increased by 17.60 times, AST-3424IC 50 The increase was 12.67 times, indicating that P53 protein could increase the cytotoxicity of compound C and AST-3424.

[0720] After P53 knockout, the IC of compound C combined with AKR1C3 enzyme inhibitor AST-3021 was higher than that of single drug. 50 The ratio decreased from 305.37-fold to 43.24-fold, indicating that the AKR1C3 selectivity of compound C was significantly reduced after knockout of P53.

[0721] In other words, p53 protein has a decisive influence on the efficacy of compound C (DNA alkylating agent), further confirming the experimental results in the above examples that DNA alkylating agents have a stronger proliferation inhibitory effect on cells with negative p53 gene mutations and normal p53 protein.

Claims

1. A method of treating a cancer or tumor patient whose p53 gene mutation is negative or whose p53 protein expression is normal is treated by using a drug containing a DNA alkylating agent prodrug compound alone or in combination with other drugs.

2. The method of claim 1, wherein a drug containing a DNA alkylator prodrug compound activated by hypoxia, a DNA alkylator prodrug compound activated by AKR1C3, a DNA alkylator prodrug compound activated by β-glucosidase (β-D-Glucosidase) or β-galactosidase (β-galactosidase) is used alone or in combination with other therapeutic drugs to treat cancer and tumor patients with negative p53 gene mutation test or normal p53 protein expression, the DNA alkylator prodrug compound activated by hypoxia is selected from structural formulas 1-3 and salts, esters, solvates, and isotopomers thereof, the DNA alkylator prodrug compound activated by AKR1C3 is selected from structural formulas 4-12 and salts, esters, solvates, and isotopomers thereof, and the DNA alkylator prodrug compound activated by β-glucosidase (β-D-Glucosidase) or β-galactosidase (β-galactosidase) is selected from structural formula 15: in, R is each independently selected from H, -CH3, -CH2CH3, -CF3, and X is each independently selected from leaving functional groups such as Cl, Br, MsO, TsO, etc.; Wherein, the definitions of R1, R2, R3, and Cx are as described in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1 (corresponding to Chinese application number 2020800673113, publication number CN114466853A); 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 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 is as set forth in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A); Among them, X, Y, Z, R, D, L 1 , A and X 10 The definition is as set forth in the claims of patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A); Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); in: A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=C R 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 S02R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group; Among them, R 100 , R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 Together with the nitrogen atom to which it is attached, it forms a 5-7 membered heterocyclic ring; wherein the alkyl and aryl groups are each substituted with 1-3 halogen groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently is phenyl or methyl; X, Y and Z are each independently hydrogen or halo; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl; Wherein, the definition of Rw is as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); Wherein, the definitions of R1, R2, R3, R4, and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1; Wherein, the definitions of A, E, G, X, and Y are as described in the claims of patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A); or a pharmaceutically acceptable salt thereof, Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , n1, n2 are defined as described in the claims of patent application PCT / CN2023 / 123253, publication number WO2024078392A1; The compound of structural formula (15) is selected from the following structural compounds: wherein the sugar moiety (sugar) is linked to a phosphoamide mustard residue (15-I) or an ifosfamide mustard residue (15-II), R1 and R2 may be the same or different and are selected from hydrogen, C1-C4 alkyl or C1-C6 haloalkyl, And the sugar moiety (sugar) is any existing isomeric or enantiomeric form of a monosaccharide, disaccharide or polysaccharide.

3. The method of claim 1, wherein: The DNA alkylating agent prodrug compound is selected from the group consisting of the AKR1C3 activated DNA alkylating agent prodrug compound AST-3424:

4. The method of claim 3, wherein: AST-3424 is formulated as an aqueous solution for intravenous administration. The aqueous solution solute is composed of AST-3424 raw material drug, glucose, ethanol, propylene glycol and pH regulator sodium bicarbonate. The aqueous solution has a concentration of AST-3424 API of 0.004-0.94 mg / ml, a pH of 7.4, a glucose content of 4.5-5.0% by mass, and is an isotonic solution; or The aqueous solution solute is composed of AST-3424 raw material drug, sodium chloride, ethanol, propylene glycol and pH regulator sodium bicarbonate. The aqueous solution has a concentration of AST-3424 raw material drug of 0.004-0.94 mg / ml, a pH of 7.4, a sodium chloride content of 0.81-0.90% by mass, and is an isotonic solution.

5. The method of claim 3, wherein: The AST-3424 dosing regimen is selected from any one of the following regimens: Scheme 1: One dose per 21-day cycle, administered once on day 1 and day 8, with a dose of 12 mg / m 2 , 11mg / m 2 , 10mg / m 2 , 9mg / m 2 , 8mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 ; Scheme 2: Each 21-day cycle is administered on day 1, with a dose of 14 mg / m 2 , 13mg / m 2 , 12mg / m 2 , 11mg / m 2 , 10mg / m 2 , 9mg / m 2 , 8mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 , 3mg / m 2 , 2mg / m 2 , 1mg / m 2 ; Scheme 3: One cycle is 21 days, and the drug is administered once a day from day 1 to day 5, with a dose of 8 mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 , 3mg / m 2 , 2mg / m 2 , 1mg / m 2 .

6. The method of claim 3, wherein: The patient is a liver cancer patient.

7. The method according to claim 5, wherein the patient's liver tumor tissue expresses AKR1C3 strongly positively, and one of the following conditions is met to determine that the patient's liver tumor tissue expresses AKR1C3 strongly positively:

1. The H score is greater than or equal to 100, preferably greater than or equal to 135, as detected by the AKR1C3 detection method described in WO2022048492; Second, according to the AKR1C3 detection method described in WO2022048492, the proportion of tumor cells with staining intensity of 2+ and / or 3+ is ≥70%.

8. The method of claim 3, wherein a drug containing AST-3424 is used in combination with abiraterone acetate or abiraterone and prednisolone, 5-fluorouracil, sunitinib, gemcitabine, oxaliplatin, PD-1 / L1 inhibitor, apatinib, sorafenib or donafenib, or elemene to treat liver cancer.

9. Use of DNA alkylating agent prodrug compounds in the preparation of drugs for treating cancer and tumor patients with negative p53 gene mutation or normal p53 protein expression, either alone or in combination with other drugs.

10. The use according to claim 9, wherein: The DNA alkylating agent prodrug compound is selected from a DNA alkylating agent prodrug compound activated by hypoxia, a DNA alkylating agent prodrug compound activated by AKR1C3, a DNA alkylating agent prodrug compound activated by β-glucosidase (β-D-Glucosidase) or β-galactosidase (β-galactosidase).

11. The use according to claim 10, wherein: The hypoxia activated DNA alkylating agent prodrug compound is selected from structural formula 1-3 and salts, esters, solvates, and isotope isomers thereof, The AKR1C3 activated DNA alkylating agent prodrug compound is selected from structural formulas 4-12 and salts, esters, solvates, and isotope isomers thereof, The β-glucosidase (β-D-Glucosidase) or β-galactosidase (β-galactosidase) activated DNA alkylating agent prodrug compound is selected from structural formula 15: Wherein, R is independently selected from H, -CH3, -CH2CH3, -CF3, and X is independently selected from leaving functional groups such as Cl, Br, MsO, TsO, etc.; Wherein, the definitions of R1, R2, R3, and Cx are as described in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1 (corresponding to Chinese application number 2020800673113, publication number CN114466853A); 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 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 is as set forth in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A); Among them, X, Y, Z, R, D, L 1 , A and X 10 The definition is as set forth in the claims of patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A); Among them, R1, R2, R3, R4, R5, R8, R9, R 10 The definition is as set forth in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); in: A is a substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl or -N=CR 1 R 2 , wherein the substituent when substituted is selected from the group consisting of: halogen, -CN, -NO2, -O-(CH2)-O-, -CO2H and its salts, -OR 100 、-CO2R 100 、-CONR 101 R 102 、-NR 101 R 102 、-NR 100 S02R 100 、-SO2R 100 、-SO2NR 101 R 10 2 , C1-C6 alkyl, C3-C10 heterocyclic group; Among them, R 100 , R 101 and R 102 are independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 Together with the nitrogen atom to which it is attached, it forms a 5-7 membered heterocyclic ring; wherein the alkyl and aryl groups are each substituted with 1-3 halogen groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 each independently is phenyl or methyl; X, Y and Z are each independently hydrogen or halo; R is hydrogen or C1-C6 alkyl or halogen-substituted alkyl; Wherein, the definition of Rw is as described in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); Wherein, the definitions of R1, R2, R3, R4, and T are as described in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1; Wherein, the definitions of A, E, G, X, and Y are as described in the claims of patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A); or a pharmaceutically acceptable salt thereof, Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , n1, n2 are defined as described in the claims of patent application PCT / CN2023 / 123253, publication number WO2024078392A1; wherein the sugar moiety (sugar) is linked to a phosphoamide mustard residue (15-I) or an ifosfamide mustard residue (15-II), R1 and R2 may be the same or different and are selected from hydrogen, C1-C4 alkyl or C1-C6 haloalkyl, And the sugar moiety (sugar) is any existing isomeric or enantiomeric form of a monosaccharide, disaccharide or polysaccharide.

12. The use according to claim 9, wherein: The DNA alkylating agent prodrug compound is selected from the group consisting of the AKR1C3 activated DNA alkylating agent prodrug compound AST-3424:

13. The use according to claim 12, wherein: The AST-3424 is formulated as an aqueous solution for intravenous injection. The aqueous solution solute is composed of AST-3424 raw material drug, glucose, ethanol, propylene glycol and pH regulator sodium bicarbonate. The aqueous solution has a concentration of AST-3424 API of 0.004-0.94 mg / ml, a pH of 7.4, a glucose content of 4.5-5.0% by mass, and is an isotonic solution; or The aqueous solution solute is composed of AST-3424 raw material drug, sodium chloride, ethanol, propylene glycol and pH regulator sodium bicarbonate. The aqueous solution has a concentration of AST-3424 raw material drug of 0.004-0.94 mg / ml, a pH of 7.4, a sodium chloride content of 0.81-0.90% by mass, and is an isotonic solution.

14. The use according to claim 12, wherein: The AST-3424 dosing regimen is selected from any one of the following regimens: Scheme 1: One dose per 21-day cycle, administered once on day 1 and day 8, with a dose of 12 mg / m 2 , 11mg / m 2 , 10mg / m 2 , 9mg / m 2 , 8mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 ; Scheme 2: Each 21-day cycle is administered on day 1, with a dose of 14 mg / m 2 , 13mg / m 2 , 12mg / m 2 , 11mg / m 2 , 10mg / m 2 , 9mg / m 2 , 8mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 , 3mg / m 2 , 2mg / m 2 , 1mg / m 2 ; Scheme 3: One cycle is 21 days, and the drug is administered once a day from day 1 to day 5, with a dose of 8 mg / m 2 , 7mg / m 2 , 6mg / m 2 , 5mg / m 2 , 4mg / m 2 , 3mg / m 2 , 2mg / m 2 , 1mg / m 2 .

15. The use according to claim 12, wherein: The patient is a liver cancer patient.

16. The use according to claim 12, wherein: The patient's liver tumor tissue has strong positive expression of AKR1C3, and if one of the following conditions is met, it is judged that the patient's liver tumor tissue has strong positive expression of AKR1C3:

1. The H score is greater than or equal to 135 as detected by the AKR1C3 detection method described in WO2022048492; Second, according to the AKR1C3 detection method described in WO2022048492, the proportion of tumor cells with staining intensity of 2+ and / or 3+ is ≥70%.

17. The use according to claim 12, wherein: A drug containing AST-3424 is used in combination with a drug containing any one of abiraterone, prednisolone, 5-fluorouracil, sunitinib, gemcitabine, oxaliplatin, a PD-1 / L1 inhibitor, apatinib, sorafenib or donafenib, or elemene to treat liver cancer.

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