DNA alkylating agent for treating cancer and tumor patients with negative p53 gene mutation or defect

Abstract

A method for treating cancer or tumor patients with a negative p53 gene mutation or defect by using a drug containing a DNA alkylating agent prodrug compound alone or in combination with other drugs, and the pharmaceutical use thereof. In particular, 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, and a β-D-glucosidase or β-galactosidase-activated DNA alkylating agent prodrug compound.

Description

DNA alkylating agent treatment of p53 gene mutation or defect negative cancer, tumor patients TECHNICAL FIELD

[0001] The present application relates to a method for treating cancer, in particular a method for treating p53 gene mutation or defect negative cancer, tumor patients. BACKGROUND

[0002] DNA alkylating agent prodrug AST-3424 (WO2016145092, WO2017087428) targeting overexpression of aldehyde-ketone reductase 1C3 (AKR1C3), CAS number 2097713-69-2, structure as follows:

[0003] Chemical structure of AST-3424

[0004] AST-3424 (also known as OBI-3424, TH-3424) enters the cancer cell and is activated by AKR1C3 enzyme overexpressed by the cancer cell to release metabolite AST-2660 (also known as AST-2660). AST-3424 itself has little toxicity to cancer cells, and its pharmacological effects in animal models and in vitro pharmacological experiments are all related to AKR1C3 enzyme expression: prodrug AST-3424 is metabolized to AST-2660 under the action of AKR1C3 enzyme and NADPH, and the expression amount of the enzyme is positively correlated with the drug effect (Literature 1; Literature 2; Literature 3; Literature 4).

[0005] Chemical reaction formula of AST-3424 (OBI-3424) metabolized to AST-2660 (OBI-2660)

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

[0007] The clinical trials of the above-mentioned drugs are still in normal progress, and in the corresponding phase II clinical trials, patients are screened for treatment by detecting the expression level of AKR1C3 enzyme. SUMMARY

[0008] In the AST-3424 phase II clinical trial conducted in China, the applicant found that the treatment effect of p53 gene mutation or defect negative cancer, tumor patients is better than that of p53 gene mutation or defect positive patients, and therefore the applicant speculates that the treatment of p53 gene mutation or defect negative tumor, cancer patients with AST-3424 will have better treatment effect, that is, p53 gene mutation or defect negative tumor, cancer patients receiving AST-3424 treatment will have more obvious clinical benefit.

[0009] Therefore, the applicant proposes the following anti-cancer drug pharmaceutical uses and tumor, cancer treatment methods.

[0010] The treatment method uses a drug containing a DNA alkylating agent prodrug compound or a DNA alkylating agent to treat p53 gene mutation negative or p53 protein expression normal cancer, tumor patients alone or in combination with other drugs.

[0011] The application of a DNA alkylating agent prodrug compound in the preparation of a drug for treating p53 gene mutation negative or p53 protein expression normal cancer, tumor patients alone or in combination with other drugs.

[0012] The DNA alkylating agent prodrug compound refers to a prodrug compound that can be metabolized to a DNA alkylating agent in vivo.

[0013] The DNA alkylating agent prodrug compound refers to a prodrug compound that is metabolically converted into a DNA alkylating agent, and the DNA alkylating agent ultimately alkylates DNA in cancer cells to destroy the DNA structure of cancer cells and ultimately causes cell death.

[0014] Most prodrug compounds will be metabolically converted in a physiological environment, and for antitumor, anticancer drugs, these physiological environments are usually specific microenvironments of tumor tissues or cancer cells, such as high expression of certain transport proteins on cell membranes, high expression or concentration of certain enzymes or proteins in the intracellular or extracellular environment due to enrichment, or in hypoxic or abnormal pH, etc. Therefore, the existence of these microenvironments is generally considered to be caused by certain specific mechanisms of tumor tissues or cancer cells.

[0015] Prodrugs are a broad category, and the activity of prodrugs can be very different from the parent drug, even to the extent that the prodrug has no therapeutic effect at all at the therapeutic dose. After entering the living body, the prodrug can be metabolically converted in a physiological environment, which can lead to the direct breakage of part of the prodrug to produce or dissociate the final effector molecule Payload, i.e. the parent drug. In this case, the Payload is usually a very active compound; it is also possible that a certain group of the prodrug compound is oxidized or reduced, dehalogenated (Phase I biotransformation) by enzymes or other substances in the physiological environment, and becomes a metabolically converted compound, i.e. the parent drug. In this case, the parent drug usually has a large difference in activity from the prodrug compound, such as the anticonvulsant drug carbamazepine, which is metabolically converted to 10,11-epoxide, which is the active ingredient of carbamazepine for producing anticonvulsant effect, and is a metabolically activated product. The former case can be considered as a prodrug compound conjugated with the parent drug, which is called a prodrug conjugate; the latter is not.

[0016] In particular, the DNA alkylating agent prodrug compound is selected from DNA alkylating agent prodrug conjugates.

[0017] Prodrug conjugates are a broad concept, and generally can be considered to include A-L-P, wherein A is a part that targets a certain enzyme or protein or other part that can serve as a targeting part, P is an active drug part, i.e. Payload, and L is a part connecting the two, which will be cleaved in the above-mentioned microenvironment to release the active P part or an analog of the P part. DNA alkylating agent prodrug conjugates are compounds in which the P part of the above-mentioned prodrug conjugates is a DNA alkylating agent.

[0018] According to the different targeting parts A conjugated with Payload, there are small molecule drug conjugates (SMDC), antibody-drug conjugates (ADC), peptide-drug conjugates (PDC), apdamer-drug conjugates (ApDC), antibody fragment-drug conjugates (FDC), virus-like drug conjugates (VDC), etc.

[0019] The DNA alkylating agent prodrug conjugate is preferably selected from SMDC, ADC, and PDC.

[0020] ADC, Antibody-drug conjugate, is a kind of targeted biological agent that combines target-specific monoclonal antibodies with high-killing cytotoxic drugs through a specific linker. It uses monoclonal antibodies as carriers to transport small molecule cytotoxic drugs to target tumor cells in a targeted manner.

[0021] ADC drugs refer to the following literature:

[0022] Tsuchikama, K., Anami, Y., Ha, S. Y. Y. et al. Exploring the next generation of antibody-drug conjugates. Nat Rev Clin Oncol 21, 203-223 (2024). https: / / doi.org / 10.1038 / s41571-023-00850-2.

[0023] SMDC, Small molecule drug conjugates, adopts a similar design concept as ADC, consisting of three parts: targeting ligand, cleavable linker, and effective payload, with small molecule targeting ligand as the targeting part.

[0024] SMDC refers to the following literature:

[0025] Zhuang C, Guan X, Ma H, Cong H, Zhang W, Miao Z. Small molecule-drug conjugates: A novel strategy for cancer-targeted treatment. Eur J Med Chem. 2019 Feb 1;163:883-895. doi: 10.1016 / j.ejmech.2018.12.035. Epub 2018 Dec 16. PMID: 30580240.

[0026] Zhang J, Hu F, Aras O, Chai Y, An F. Small Molecule-Drug Conjugates: Opportunities for the Development of Targeted Anticancer Drugs. ChemMedChem. 2024 Jun 3;19(11):e202300720. doi: 10.1002 / cmdc.202300720. Epub 2024 Mar 11. PMID: 38396351.

[0027] Rana A, Bhatnagar S. Advancements in folate receptor targeting for anti-cancer therapy: A small molecule-drug conjugate approach. Bioorg Chem. 2021 Jul;112:104946. doi: 10.1016 / j.bioorg.2021.104946. Epub 2021 Apr 27. PMID: 33989916.

[0028] Tarun Kumar Patel, Nilanjan Adhikari, Sk. Abdul Amin, et. al, Small molecule drug conjugates (SMDCs): an emerging strategy for anticancer drug design and discovery New J. Chem., 2021, 45, 5291-5321.

[0029] PDC, Peptide-Drug Conjugate, compared with ADC, uses polypeptide as tumor targeting carrier. The targeting polypeptides used in PDCs are divided into two categories: cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs). PDCs with cell-homing peptides penetrate cells through non-specific mechanisms, while PDCs with cell-targeting peptides bind specifically to antigens or receptors on the surface of tumor cells to mediate the entry of cytotoxic payloads into tumor cells. The application of these types of PDCs is limited due to the low cell specificity of CPPs. In contrast, CTPs exhibit similar effects to monoclonal antibodies while overcoming certain shortcomings of monoclonal antibodies, and thus are widely used. The targeting peptides of the PDCs are selected from the group consisting of bombesin analogs, GnRH analogs, growth hormone inhibitory analogs, RGD peptides, PEGA.

[0030] PDCs are described in the literature:

[0031] Gong L, Zhao H, Liu Y, Wu H, Liu C, Chang S, Chen L, Jin M, Wang Q, Gao Z, Huang W. Research advances in peptide-drug conjugates. Acta Pharm Sin B. 2023 Sep;13(9):3659-3677. doi: 10.1016 / j.apsb.2023.02.013. Epub 2023 Feb 28. PMID: 37719380; PMCID: PMC10501876.

[0032] Wu C, Wang M, Sun J, Jia Y, Zhu X, Liu G, Zhu Y, Guan Y, Zhang Z, Pang X. Peptide-drug co-assembling: A potent armament against cancer. Theranostics. 2023 Sep 25;13(15):5322-5347. doi: 10.7150 / thno.87356. PMID: 37908727; PMCID: PMC10614680.

[0033] Guo S, Wang J, Wang Q, Wang J, Qin S, Li W. Advances in peptide-based drug delivery systems. Heliyon. 2024 Feb 7;10(4):e26009. doi: 10.1016 / j.heliyon.2024.e26009. PMID: 38404797; PMCID: PMC10884816.

[0034] Dean TT, Jelu-Reyes J, Allen AC, Moore TW. Peptide-Drug Conjugates: An Emerging Direction for the Next Generation of Peptide Therapeutics. J Med Chem. 2024 Feb 8;67(3):1641-1661. doi: 10.1021 / acs.jmedchem.3c01835. Epub 2024 Jan 26. PMID: 38277480; PMCID: PMC10922862.

[0035] Wang M, Liu J, Xia M, Yin L, Zhang L, Liu X, Cheng Y. Peptide-drug conjugates: A new paradigm for targeted cancer therapy. Eur J Med Chem. 2024 Feb 5;265:116119. doi: 10.1016 / j.ejmech.2023.116119. Epub 2024 Jan 1. PMID: 38194773.

[0036] DNA alkylating agents are a class of chemotherapeutic drugs that cross-link to DNA strands and produce cytotoxicity, thus playing a therapeutic role. According to the different structures of the compounds, DNA alkylating agents have various structures, including nitrogen mustards, phosphoramidate alkylating agents, PBDs, Duocarmycin, platinum-containing antineoplastic agents (such as cisplatin, carboplatin, oxaliplatin), alkyl sulfonates (such as busulfan), triazenes (such as dacarbazine, procarbazine, temozolomide), and the like.

[0037] DNA alkylating agents are described in the literature:

[0038] Rajski SR, Williams RM. DNA Cross-Linking Agents as Antitumor Drugs. Chem Rev. 1998; 98(8): 2723-2796. doi: 10.1021 / cr9800199.

[0039] Brulikova L, Hlavac J, Hradil P. DNA interstrand cross-linking agents and their chemotherapeutic potential. Curr Med Chem. 2012; 19(3): 364-85. doi: 10.2174 / 092986712803414295. PMID: 22335513.

[0040] Wu J, Huang R, Wang T, Zhao X, Zhang W, Weng X, Tian T, Zhou X. Fluoride as an inducible DNA cross-linking agent for new antitumor prodrug. Org Biomol Chem. 2013 Apr 14; 11(14): 2365-9. doi: 10.1039 / c2ob27324a. PMID: 23431557.

[0041] Maria Tomasz, Yolanda Palom, The mitomycin bioreductive antitumor agents: Cross-linking and alkylation of DNA as the molecular basis of their activity, Pharmacology & Therapeutics, 76(1-3) 73-87, https: / / doi.org / 10.1016 / S0163-7258(97)00088-0.

[0042] Rycenga HB, Long DT. The evolving role of DNA inter-strand crosslinks in chemotherapy. Curr Opin Pharmacol. 2018 Aug;41:20-26. doi: 10.1016 / j.coph.2018.04.004. Epub 2018 Apr 18. PMID: 29679802; PMCID: PMC6108900.

[0043] Nitrogen mustards, aminophosphates, PBDs, Duocarmycin are preferred, these four classes are currently often used as toxins Payloads for conjugated drugs.

[0044] Nitrogen mustards, i.e. β-halogen ethylamines, are structurally divided into two parts: the alkylated β-halogen ethylamine part and the carrier part. The alkylating part is the functional group of antitumor activity, and the carrier part mainly affects the pharmacokinetic properties of drug absorption, distribution, etc. in the body, and by selecting different carriers, the purpose of improving drug selectivity and efficacy, reducing toxicity and side effects can be achieved. According to the different structures of the carrier, nitrogen mustard drugs can be further divided into aliphatic nitrogen mustard, aromatic nitrogen mustard, amino acid nitrogen mustard, steroidal nitrogen mustard, and heterocyclic nitrogen mustard, and nitrosoureas (carmustine, lomustine, semustine) and the like.

[0045] Some nitrogen mustard DNA alkylating agents as traditional tumor chemotherapy drugs: Mechlorethamine hydrochloride, Melphalan, Chlorambucil, Carmustine, Semustine, the structures are as follows:

[0046] Mechlorethamine hydrochloride Melphalan Chlorambucil

[0047] carmustine semustine

[0048] phosphoramidates, which include an alkylating β-haloethylamine moiety / aziridine moiety and a phosphoramidate structure, the chemical structure of which is OP(Z 1 )(NR 30 CH2CH2X 1 )2, OP(Z 1 )(NR 30 2)(N(CH2CH2X 1 )2), OP(Z 1 )(N(CH2CH2))2, OP(Z 1 )(N(CH2CH2X 1 )2)2, wherein R 30 each independently is hydrogen or C1-C6 alkyl, or 2 R 30 together with the nitrogen atom to which they are bonded form a 5-7 membered heterocyclyl, Z 1 is O or S, and X 1 is CI, Br or OMs or another leaving group. In particular, the chemical structure is selected from OP(Z 1 )(NHCH2CH2CI)2, OP(Z 1 )(NHCH2CH2Br)2, OP(Z 1 )(NH2)(N(CH2CH2X 1 )2), OP(Z 1 )(N(CH2)2)2, OP(Z 1 )(N(CH2CH2CI)2)2, wherein Z 1 is O or S and X 1 is CI, Br or OMs.

[0049] phosphoramidate DNA alkylating agents as traditional tumor chemotherapy drugs: cyclophosphamide, ifosfamide, thiotepa, AST-2660, a thiotepa derivative.

[0050] AST-2660 cyclophosphamide thiotepa

[0051] PBDs (pyrolobenzodiazepines), i.e. pyrrolobenzodiazepine containing The PBD unit is a member of the antibiotic anthramycin family, which is a sequence-selective DNA minor groove binder. Its mode of action is to selectively alkylate within the DNA minor groove, forming a covalent bond between the electrophilic N10 / C11 imine of the PBD and the N2 of guanine. This results in persistent DNA damage, causing cell cycle arrest at the G2 / M phase and triggering apoptosis, thereby exhibiting strong cytotoxic effects.

[0052] Specific compounds of the PBD class are described in the literature:

[0053] Thomas JD, Yurkovetskiy AV, Yin M, Bodyak ND, Gumerov DR, Tang S, Kelleher E, Jones BD, Protopopova M, Qin L, Uttard A, Demady DR, Lowinger TB. Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates. Bioorg Med Chem Lett. 2022 Sep 15;72:128876. doi: 10.1016 / j.bmcl.2022.128876. Epub 2022 Jul 3. PMID: 35788036.

[0054] Wenjun Yu, Xilei Xie, Yao Ma, Shiping Fang, Yi Dong, and Gang Liu, Identification of 1,4-Benzodiazepine-2,5-dione Derivatives as Potential Protein Synthesis Inhibitors with Highly Potent Anticancer Activity, Journal of Medicinal Chemistry 2022 65(21), 14891-14915, DOI: 10.1021 / acs.jmedchem.2c01431.

[0055] Obaji, H., Shilabin, A. G., Majumdar, S. et al. Pyrrolobenzodiazepines: natural sources, therapeutic uses, and future in neurological treatments. Med Chem Res 33, 36-46 (2024). https: / / doi.org / 10.1007 / s00044-023-03177-w.

[0056] Cipolla L, Araújo AC, Airoldi C, Bini D. Pyrrolo[2,1-c][1,4]benzodiazepine as a scaffold for the design and synthesis of anti-tumour drugs. Anticancer Agents Med Chem. 2009 Jan;9(1):1-31. doi: 10.2174 / 187152009787047743. PMID: 19149479.

[0057] Gerratana B. Biosynthesis, synthesis, and biological activities of pyrrolobenzodiazepines. Med Res Rev. 2012 Mar;32(2):254-93. doi: 10.1002 / med.20212. Epub 2010 Jun 13. PMID: 20544978; PMCID: PMC4127195.

[0058] and patent applications PCT / GB1999 / 002838, PCT / EP2013 / 071236.

[0059] The backbone structure of PBDs contains one aromatic ring A with substituents, one diazole ring B, and one pyrrole ring C, with an S chiral center at the C11a position between the B and C rings. PBD monomers are linked together through the C8 / C8’-positions (methylene chain) to form PBD dimers, which can produce highly lethal interstrand crosslinks with DNA.

[0060] In addition to coupling with itself to form multimers, the PBD backbone can also be coupled with other toxins, such as Duocarmycin.

[0061] In particular, the PBD class of DNA alkylating agents is selected from the group consisting of SG3199, SG-2057, SGD-1882, PBD dimer-2, PBD-monoamide, Aniline-MPB-amino-C3-PBD, Py-MPB-amino-C3-PBD, Tomaymycin DM, SG 3249, SJG-136.

[0062] SG3199 is a cytotoxic DNA minor groove cross-linking PBD dimer with CAS number 1595275-71-0 and the following structural formula:

[0063] SG-2057 (DRG16) is a PBD dimer containing a pentanedioxy linkage that selectively binds to sequences in the DNA minor groove, forming DNA interstrand and intrastrand cross-linking adducts, with CAS number 260417-62-7 and the following structural formula:

[0064] SGD-1882 is a cytotoxic DNA minor groove cross-linking agent that is a PBD dimer and can be used as a payload for ADCs, with CAS number 1222490-34-7 and the following structural formula:

[0065] PBD dimer-2 is a C8-linked pyrrolobenzo-diazepine dimer. PBD dimer-2 can span an additional base pair and cross-link 5'-Pu-GA(T / A)TC-Py sequences, with CAS number 145325-57-1 and the following structural formula:

[0066] PBD-monoamide is a modified PBD (pyrrolobenzodiazepine) dimer and is an ADC cytotoxin. PBD-monoamide can be used in the synthesis of DHES0815A (an anti-HER2 ADC), with CAS number 2093165-00-3 and the following structural formula:

[0067] Aniline-MPB-amino-C3-PBD is a cytotoxic active molecule composed of pyrrolobenzodiazepine (PBD). Aniline-MPB-amino-C3-PBD is a sequence-selective DNA minor groove binder. Aniline-MPB-amino-C3-PBD is a payload for ADCs, with CAS number 2412923-79-4 and the following structural formula:

[0068] Py-MPB-amino-C3-PBD is a cytotoxic active molecule, which can be used as a payload of ADC, with CAS No. 2412924-07-1, and the structural formula is as follows:

[0069] Tomaymycin DM is a DNA alkylating agent, which is a derivative of Tomaymycin, and is a monomer of PBD, and can be used as a payload of tumor-targeting antibody conjugated active molecule ADCs, with CAS No. 945490-09-5, and the structural formula is as follows:

[0070] SG3249, i.e. Tesirine, is a pyrrolobenzodiazepine (PBD) dimer, which can be used for the synthesis of antibody conjugated active molecules (ADCs), with CAS No. 1595275-62-9, and the structural formula is as follows:

[0071] Duocarmycin, i.e. Duocarmycin, is a large class of antitumor antibiotics derived from Streptomyces, which binds to the DNA minor groove through a highly active structure, and alkylates adenine at N3, etc., ultimately leading to cell death, and is a potent DNA alkylating agent.

[0072] Duocarmycin is described in the literature:

[0073] Felber JG, Thorn-Seshold O. 40 Years of Duocarmycins: A Graphical Structure / Function Review of Their Chemical Evolution, from SAR to Prodrugs and ADCs. JACS Au. 2022 Nov 15; 2(12): 2636-2644. doi: 10.1021 / jacsau.2c00448. Erratum in: JACS Au. 2023 Apr 19; 3(5): 1534. doi: 10.1021 / jacsau.3c00137. PMID: 36590260; PMCID: PMC9795467.

[0074] Jukes Z, Morais GR, Loadman PM, Pors K. How can the potential of the duocarmycins be unlocked for cancer therapy? Drug Discov Today. 2021 Feb;26(2):577-584. doi: 10.1016 / j.drudis.2020.11.020. Epub 2020 Nov 21. PMID: 33232841.

[0075] Patil PC, Satam V, Lee M. A Short Review on the Synthetic Strategies of Duocarmycin Analogs that are Powerful DNA Alkylating Agents. Anticancer Agents Med Chem. 2015;15(5):616-30. doi: 10.2174 / 1871520615666141216144116. PMID: 25511515.

[0076] In particular, the Duocarmycin class of DNA alkylating agents is selected from Duocarmycin SA, Duocarmycin Analog, Duocarmycin MB, Duocarmycin GA, Duocarmycin MA, Duocarmycin TM, Duocarmycin DM, Duocarmycin DM free base, Duocarmycin A, Seco-Duocarmycin TM, Seco-Duocarmycin SA, (S)-Seco-Duocarmycin SA, NMS-P528, Seco-DUBA.

[0077] Duocarmycin SA is capable of inducing sequence-selective alkylation of double-stranded DNA, and has the CAS number 130288-24-3.

[0078] Duocarmycin Analog is an analog of Duocarmycin, which can be used as a DNA alkylating agent and a toxin moiety for antibody conjugated active molecules, and has the CAS number 372954-15-9.

[0079] Duocarmycin MB has the CAS number 1613286-58-0.

[0080] Duocarmycin GA has a CAS number of 1613286-59-1.

[0081] Duocarmycin MA has a CAS number of 1613286-57-9.

[0082] Duocarmycin TM has a CAS number of 157922-77-5.

[0083] Duocarmycin DM is the trifluoroacetate salt of Duocarmycin DM free base (CAS number 1116745-06-2).

[0084] Duocarmycin DM free base has a CAS number of 1116745-06-2.

[0085] Duocarmycin A has a CAS number of 118292-34-5.

[0086] Seco-Duocarmycin TM has a CAS number of 236102-87-7.

[0087] Seco-Duocarmycin SA has a CAS number of 144667-38-9.

[0088] (S)-Seco-Duocarmycin SA has a CAS number of 152785-82-5.

[0089] NMS-P528 has a CAS number of 1466546-45-1.

[0090] Seco-DUBA has a CAS number of 1227961-59-2.

[0091] These DNA alkylating agents are generally used as traditional, broad cancer / tumor chemotherapy drugs; while DNA alkylating agent prodrug compounds, especially DNA alkylating agent conjugate prodrug compounds, are currently used as new targeted therapies (ADCs) and some are currently in clinical research (SMDCs, PDCs) as new targeted therapies.

[0092] The activation of prodrug metabolism into an effective drug by the tumor microenvironment realizes the targeting of tumor / cancer cells to the above-mentioned microenvironment, and the targeting moiety can be a biological macromolecule such as an antibody, a protein, a glycoprotein, a polypeptide, even a virus, a nucleic acid, or a small molecule, specifically including small molecule drug conjugates (SMDC), antibody-drug conjugates (ADC), peptide-drug conjugates (PDC), apdamer-drug conjugates (ApDC), antibody fragment-drug conjugates (FDC), virus-like drug conjugates (VDC), etc., from which the targeting moiety is respectively a small molecule ligand, a polypeptide, an apdamer, an antibody fragment, a virus, etc.

[0093] The SMDCs targeting the microenvironment known from the literature are currently progressing rapidly in terms of enzyme activation or hypoxia activation, the former being AKR1C3 enzyme activation, β-D-Glucosidase activation, Carboxylesterase activation, Esterase and caspase-3 activation, Cathepsin B activation, γ-Glutamyltranspeptidase activation, β-galactosidase activation.

[0094] The DNA alkylating agent prodrug compound is selected from AKR1C3 enzyme-activated, β-D-Glucosidase-activated, Carboxylesterase-activated, Esterase and caspase-3-activated, Cathepsin B-activated, γ-Glutamyltranspeptidase-activated, β-galactosidase-activated, or hypoxia-activated DNA alkylating agent conjugate prodrug compounds, preferably from AKR1C3 enzyme-activated, β-D-Glucosidase-activated, β-galactosidase-activated, hypoxia-activated DNA alkylating agent conjugate prodrug compounds.

[0095] The hypoxia-activated DNA alkylating agent conjugated prodrug compounds are selected from the group consisting of structural formula 1-3 and salts, esters, solvates, isotopologues thereof, AKR1C3-activated DNA alkylating agent conjugated prodrug compounds are selected from the group consisting of structural formula 4-12 and salts, esters, solvates, isotopologues thereof, β-D-Glucosidase-activated or β-galactosidase-activated DNA alkylating agent conjugated prodrug compounds are selected from the group consisting of structural formula 15 and salts, esters, solvates, isotopologues thereof.

[0096] wherein each R is independently selected from H, -CH3, -CH2CH3, -CF3, and each X is independently selected from Cl, Br, MsO, TsO, and other leaving functional groups.

[0097] TH-302 (Evofosfamide) or its analogs Related formulations include oral formulations, lyophilized formulations, and concentrated injection solutions, and related prescriptions, preparation methods, and clinical compounding, administration methods are detailed in and disclosed by the related patents of Threshold, Inc.: WO2010048330A1, WO2012142520A2, WO2008083101A1, WO2007002931A3, the entire text of the above applications is hereby incorporated by reference.

[0098] TH-302 or its analogs is a DNA alkylating agent class anti-cancer drug with broad cancer treatment potential, these related cancer indication experiments, clinical trials are disclosed in the related Threshold company and other pharmaceutical company patent application texts (such as WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO2020118251A1, WO2014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, WO2015069489A1, WO2012006032A2, WO2018026606A2, WO2010048330A2, WO2015171647A1, WO2013096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO2012145684A2, WO2016014390A2, WO2019055786A2, WO2012135757A2, WO2015013448A2, WO2016011328A2, WO2013177633A2, WO2016011195A2, WO2015051921A2) and FDA registered clinical trials (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT01149915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT02496832, NCT02712567), here the above-mentioned related application texts and clinical trial information are all introduced.

[0099] wherein, R1, R2, R3, Cx are defined as recited in the claims of patent application PCT / CN2020 / 114519, published as WO2021120717A1, and the synthetic preparation method of specific compounds is also recited in the above-mentioned application, which is incorporated herein in its entirety, and the specific definitions are as follows:

[0100] 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;

[0101] R1 is connected to any skeletal atom of the Cx ring, and is selected from hydrogen, a halogen atom, a cyano or isocyano group, a hydroxyl group, a thiol group, an amine group, OTs, a C1-C6 alkyl or Z-substituted alkyl group, a C2-C6 alkenyl or Z-substituted alkenyl group, a C2-C6 alkynyl or Z-substituted alkynyl group, a C3-C8 cycloalkyl or Z-substituted cycloalkyl group, a C6-C 10 an aryl or Z-substituted aryl group, a 4-15 membered heterocycle or Z-substituted heterocycle, a 5-15 membered heteroaryl or Z-substituted heteroaryl, an alkoxy group of 1-6 carbon atoms or a Z-substituted alkoxy group 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 ,

[0102] R2 and R3 are each independently hydrogen, a C1-C6 alkyl or Z-substituted alkyl group, a C2-C6 alkenyl or Z-substituted alkenyl group, a C2-C6 alkynyl or Z-substituted alkynyl group, a C3-C8 cycloalkyl or Z-substituted cycloalkyl group, a C6-C 10 an aryl or Z-substituted aryl group, a 4-15 membered heterocycle or Z-substituted heterocycle, a 5-15 membered heteroaryl or Z-substituted heteroaryl, or R2 and R3 together with the benzyl carbon atom to which they are bonded form a 3-6 membered ring;

[0103] The group can substitute any hydrogen atom on the fused ring carbon atom at any position, and the number of substitutions is 1;

[0104] Z substituents are halogen atoms, cyano or isocyano, hydroxyl, thiol, amine, C1-C3alkyl or substituted alkyl, C1-C3alkoxy or substituted alkoxy, C2-C3alkenyl or substituted alkenyl, C2-C3alkynyl or substituted alkynyl, C3-C8cycloalkyl or substituted cycloalkyl;

[0105] R 6 , R 7 each independently is hydrogen, C1-C6alkyl or Z-substituted C1-C6alkyl, C2-C6alkenyl or Z-substituted C2-C6alkenyl, C2-C6alkynyl or Z-substituted C2-C6alkynyl, C3-C8cycloalkyl or Z-substituted C3-C8cycloalkyl, C6-C10aryl or Z-substituted C6-C10aryl, 4-15 membered heterocyclyl or Z-substituted 4-15 membered heterocyclyl, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R 10 each independently is hydrogen, C1-C6alkyl or Z-substituted C1-C6alkyl, C2-C6alkenyl or Z-substituted C2-C6alkenyl, C2-C6alkynyl or Z-substituted C2-C6alkynyl, C3-C8cycloalkyl or Z-substituted C3-C8cycloalkyl, C6-C10aryl or Z-substituted C6-C10aryl, 4-15 membered heterocyclyl or Z-substituted 4-15 membered heterocyclyl, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R 10 each independently is hydrogen, C1-C6alkyl or Z-substituted C1-C6alkyl, C2-C6alkenyl or Z-substituted C2-C6alkenyl, C2-C6alkynyl or Z-substituted C2-C6alkynyl, C3-C8cycloalkyl or Z-substituted C3-C8cycloalkyl, C6-C10aryl or Z-substituted C6-C10aryl, 4-15 membered heterocyclyl or Z-substituted 4-15 membered heterocyclyl, 5-15 membered heteroaryl or Z-substituted 5-15 membered heteroaryl, or R 6 , R 7 and the atom to which they are bonded together form a 5-7 membered heterocyclyl or Z-substituted 5-7 membered heterocyclyl.

[0106] wherein 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 are defined as in the claims of patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A), and the methods of synthesis of specific compounds are also described in the above application, which is hereby incorporated by reference in its entirety, and are specifically defined as:

[0107] R1is: hydrogen, -N3, CN, halo, NR 21 R 22 , -OR 23 , -SO2(C1-C6alkyl), C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C10aryl, 4-15 membered heterocyclyl, 5-15 membered heteroaryl, or ether;

[0108] R 21 and R 22each independently hydrogen, hydroxyl, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C10aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl, or -SO2(C1-C6alkyl); or R 21 and R 22 together with the nitrogen atom to which they are attached form a 4-15 membered heterocycle or 5-15 membered heteroaryl;

[0109] R 23 is hydrogen, C1-C6alkyl, or C6-C10aryl;

[0110] R2and R3are independently hydrogen or halo;

[0111] R4is hydrogen, halo, C1-C6alkoxy, C1-C6alkyl, or C6-C10aryl,

[0112] R5, R7, R9, R 12 and R 15 are independently hydrogen, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C10aryl, 4-15 membered heterocycle, 5-15 membered heteroaryl; or R4and R5together with the intervening carbon atoms therebetween form a C5-C6cycloalkyl ring;

[0113] R6and R 10 are independently hydrogen or halo;

[0114] R8is hydrogen, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, or 5-15 membered heteroaryl;

[0115] R 11 each independently C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, or C6-C10aryl;

[0116] R 13 , R 14 , R 16 and R 17 are independently hydrogen, halo, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, or C1-C6alkoxy;

[0117] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, alkoxy, and ether groups are optionally substituted.

[0118] Formula (4) is an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound, more specifically, an AKR1C3 enzyme-activated DNA alkylating agent prodrug compound;

[0119] wherein X, Y, Z, R, T, A, and X10 The definition is as recited in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A), and the methods of synthesis of specific compounds are recited in the above application, which is incorporated herein in its entirety, and is specifically defined as:

[0120] X 10 is O, S, SO, or SO2;

[0121] A is C6-C 10 aryl, 5-15 membered heteroaryl, or -N=CR 1 R 2 ;

[0122] R 1 and R 2 are each independently hydrogen, C1-C6alkyl, C3-C8cycloalkyl, C6-C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0123] X, Y, and Z are each independently hydrogen, CN, halo, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0124] R is hydrogen, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C 10 aryl, 4-15 membered heterocycle, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0125] R 13 and R 14 are each independently hydrogen, C1-C6alkyl, C3-C8cycloalkyl, C6-C 10 aryl, 4-15 membered heterocycle, or ether;

[0126] T comprises an aminophosphoramidate alkylating agent comprising one or more Z 1 groups bonded to a -O-P(Z 5 -X5 -Y 5 moieties, wherein Z 5 is a heteroatom comprising nitrogen, sulfur or oxygen, X 5 is a substituted or unsubstituted alkylene, Y 5 is a halo or another leaving group, or Z 5 -X 5 -Y 5 together form a aziridine moiety (NCH2CH2) and Z 1 is O or S; and

[0127] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, ether groups are substituted or unsubstituted.

[0128] wherein, X, Y, Z, R, D, L 1 , A and X 10 are defined as in the claims of patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A), and the methods of synthesis of specific compounds are also described in the above-mentioned application, which is incorporated herein in its entirety, and are specifically defined as:

[0129] X 10 is O, S, SO or SO2;

[0130] A is C6-C 10 aryl, 5- to 15-membered heteroaryl, or -N=CR 1 R 2 ;

[0131] R 1 and R 2 are each independently hydrogen, C1-C6alkyl, C3-C8cycloalkyl, C6-C 10 aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0132] X, Y and Z are each independently hydrogen, CN, halo, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C 10 aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0133] each R is independently hydrogen, Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-Ci0aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, ether, -CONR 10 aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, ether, -CONR 13 R 14 or -NR 13 COR 14 ;

[0134] R 13 and R 14 each independently is hydrogen, Ci-C6alkyl, C3-C8cycloalkyl, C6-Ci0aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, or ether; 10 aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, or ether;

[0135] wherein L 1 and D are defined as follows:

[0136] L 1 is selected from:

[0137] and

[0138] R 40 and R 41 independently are hydrogen, Ci-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-Ci0aryl, 4- to 15-membered heterocycle, 5- to 15-membered heteroaryl, or ether; 10 aryl, 4- to 15-membered heterocycle, or 5- to 15-membered heteroaryl;

[0139] R 42 is C2-C3alkylene or heteroalkylene optionally substituted with 1 to 3 Ci-C6alkyl groups;

[0140] V(-) is any anion, preferably a pharmaceutically acceptable anion;

[0141] D is a moiety such that D-OH is an anticancer drug, wherein OH is an aliphatic hydroxyl or phenolic hydroxyl, or is an OH moiety attached to a phosphorus atom as provided herein; or

[0142] L 1 is:

[0143] R 40 R is as defined above, R 43 is hydrogen or forms together with D a heterocycle, and the stilbenoid moiety is optionally substituted, and

[0144] D is a moiety such that D-NR 43 H is an anticancer drug; or

[0145] L 1 is a bond, -O-C(R 40 R 41 )2-, -O-C(R 40 R 41 )-NR 40 R 41 (+)-C(R 40 R 41 )- or

[0146] wherein R 40 , R 41 and V are defined as above, and

[0147] D is an anticancer drug containing a tertiary or secondary nitrogen atom, wherein the tertiary nitrogen atom or the secondary nitrogen atom is linked to L 1 ;

[0148] and

[0149] wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocycle, heteroaryl, ether group is optionally substituted.

[0150] wherein, R1, R2, R3, R4, R5, R8, R9, R 10 are defined as recited in the claims of patent application PCT / CN2020 / 089692, published as WO2020228685A1, and the synthesis and preparation method of specific compounds are also recited in the above-mentioned application, which is incorporated herein in its entirety, and the specific definitions are as follows:

[0151] R1is C6-C 10 aryl or Z-substituted aryl, 4-15-membered heterocycle or Z-substituted heterocycle, 5-15-membered heteroaryl or Z-substituted heteroaryl, 7-15-membered fused ring or Z-substituted fused ring;

[0152] R2is hydrogen, halogen atom, cyano or isocyano, hydroxyl, mercapto, amine group, OTs, OMS, C1-C6alkyl or Z-substituted alkyl, C2-C6alkenyl or Z-substituted alkenyl, C2-C6alkynyl or Z-substituted alkynyl, C3-C8cycloalkyl or Z-substituted cycloalkyl, C6-C 10 aryl or Z-substituted aryl, 4-15-membered heterocycle or Z-substituted heterocycle, 5-15-membered heteroaryl or Z-substituted heteroaryl, 1-6 carbon atom ether or Z-substituted 1-6 carbon atom alkoxy, -CONR 6 R 7 , -SO2NR 6 R 7 , -SO2R 6 , -OCOO-R 6 , -COOR6 , -NR 6 COR 7 , -OCOR 6 , -NR 6 SO2R 7 , -NR 6 SO2NR 6 R 7 or R2and the atoms on the R1group to which it is bonded form a 7-15 membered fused ring or a Z-substituted fused ring;

[0153] R3is hydrogen, halogen, cyano or isocyano, hydroxy, mercapto, aminyl, OTs, OLCMS, C1-C6alkyl or Z-substituted alkyl, C2-C6alkenyl or Z-substituted alkenyl, C2-C6alkynyl or Z-substituted alkynyl, C3-C8cycloalkyl or Z-substituted cycloalkyl, C6-C 10 aryl or Z-substituted aryl, 4-15 membered heterocyclic ring or Z-substituted heterocyclic ring, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6alkoxy or Z-substituted C1-C6alkoxy, -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 ;

[0154] R4, R5are each independently hydrogen, halogen atom, cyano or isocyano, hydroxy, mercapto, aminyl, OTs, OLCMS, C1-C6alkyl or Z-substituted alkyl, C2-C6alkenyl or Z-substituted alkenyl, C2-C6alkynyl or Z-substituted alkynyl, C3-C8cycloalkyl or Z-substituted cycloalkyl, C6-C 10 aryl or Z-substituted aryl, 4-15 membered heterocyclic ring or Z-substituted heterocyclic ring, 5-15 membered heteroaryl or Z-substituted heteroaryl, C1-C6alkoxy or Z-substituted C1-C6alkoxy, -CONR 6 R 7 , -SO2NR 6 R 7 , -SO2R 6 , -OCOO-R 6 , -COOR 6、 -NR 6 COR 6 , -OCOR 6 , -NR6 SO2R 7 Alternatively, R4, R5, and the atoms on the benzene ring to which they are bonded can form 7-15 fused rings or Z-substituted fused rings.

[0155] R 6 and R 7 Each of the following is independently hydrogen, cyano or isocyano, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 ynyl or Z-substituted ynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 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 it is bonded to together form a 5-7 membered heterocyclic group or a Z-substituted 5-7 membered heterocyclic group;

[0156] R8, R 10 Each of them 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 ynyl or Z-substituted ynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, and at least one of them must be hydrogen or deuterium;

[0157] R9 is a substituted C6-C group having at least one fluorine atom or nitro group substitution. 10 Aryl, substituted 4-15-membered heterocyclic rings having at least one fluorine atom or nitro substitution, or substituted 5-15-membered heteroaryl rings having at least one fluorine atom or nitro substitution.

[0158] Z substituents are halogen atoms, cyano or isocyano, hydroxyl, mercapto, amino, OTs, OMS, C1-C3 alkyl or substituted alkyl, C1-C3 alkoxy or substituted alkoxy, C2-C3 alkenyl or substituted alkenyl, C2-C3 ynyl or substituted ynyl, C3-C8 cycloalkyl or substituted cycloalkyl, aromatic ring, heterocyclic, heteroaromatic ring and fused ring or substituted aromatic ring, heterocyclic, heteroaromatic ring and fused ring, and the substitution is monosubstituted or gemine disubstituted;

[0159] The substitution of C6-C in R9 10 The substituents of aryl, substituted 4-15-membered heterocyclic, and substituted 5-15-membered heteroaryl are halogen atoms, nitro, cyano or isocyano, hydroxyl, amino, C1-C3 alkyl or alkoxy, alkenyl, alkynyl, cycloalkyl or benzene ring, substituted benzene ring, C1-C3 alkoxy or halogen-substituted alkoxy.

[0160] Formula (8) is a prodrug compound for AKR1C3 enzyme-activated DNA alkylation, and more specifically, it is a prodrug compound for AKR1C3 enzyme-activated DNA alkylation.

[0161] wherein:

[0162] A is substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl, or -N=CR 1 R 2 wherein the substituents, when present, are selected from the group consisting of halo, -CN, -NO2, -O-(CH2)-O-, -CO2H and salts thereof, -OR 100 , -CO2R 100 , -CONR 101 R 102 , -NR 101 R 102 , -NR 100 SO2R 100 , -SO2R 100 , -SO2NR 101 R 1 02 , C1-C6 alkyl, C3-C10 heterocyclyl;

[0163] wherein R 100 , R 101 and R 102 are each independently hydrogen, C1-C8 alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring;

[0164] wherein alkyl and aryl are each substituted with 1-3 halo or 1-3 C1-C6 alkyl;

[0165] R 1 and R 2 are each independently phenyl or methyl;

[0166] X, Y and Z are each independently hydrogen or halo;

[0167] R is hydrogen or C1-C6 alkyl or halo substituted alkyl.

[0168] wherein Rwis defined as recited in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1, and the synthetic preparation method of specific compounds is also recited in the above-mentioned application, which is incorporated herein in its entirety, and is specifically defined as:

[0169] Rwis

[0170] R1is H, C 1-6 alkyl, C 3-6Cycloalkyl, 4-6-membered heterocycloalkyl, 5-6-membered heteroaryl or phenyl, wherein the C 1-6 Alkyl, C 3-6 Cycloalkyl, 4-6-membered heterocycloalkyl, 5-6-membered heteroaryl, and phenyl groups are optionally surrounded by 1, 2, or 3 R groups. a Replaced;

[0171] Each R a Independently, it can be H, F, Cl, Br, I, -CN, -OH, or C. 1-3 Alkoxy or C 1-3 alkyl;

[0172] R2 is H or C 1-6 alkyl;

[0173] Alternatively, R1 and R2 can be linked together to form a 4-6 membered heterocyclic alkyl group with the attached N atom, wherein the 4-6 membered heterocyclic alkyl group is optionally surrounded by 1, 2, or 3 R atoms. b Replaced;

[0174] Each R b Independently, it can be H, F, Cl, Br, I, -CN, -OH, -NH2, -OCH3, -OCH2CH3, -CH3, or -CH2CH3;

[0175] R3 can be H, F, Cl, Br, I, -OH, -NH2, or C. 1-3 Alkoxy or C 1-3 alkyl;

[0176] Alternatively, R2 and R3 can be connected together to form a structural unit. for

[0177] T1 is -(CR) c R d ) m -or-(CR) c R d ) n -O-;

[0178] m is 1, 2, or 3;

[0179] n is 1 or 2;

[0180] T2 is either N or CH;

[0181] R c and R d Each is independently H, F, C 1-3 Alkyl or C 1-3 Alkoxy;

[0182] R4, R5and R6are each independently H, F, Cl, Br, I, C 1-3 alkyl or C 1-3 alkoxy;

[0183] T is N or CH;

[0184] R7and R8are each independently H, F, Cl, Br, or I;

[0185] R9and R 10 are each independently H, F, Cl, Br, I, -CN, or

[0186] The 4-6 membered heterocycloalkyl and 5-6 membered heteroaryl each comprises 1, 2, 3, or 4 heteroatoms independently selected from N, -O-, and -S-.

[0187] wherein R1, R2, R3, R4and T are defined as in the claim of patent application PCT / CN2021 / 118597, publication number WO2022057838A1, and the synthetic preparation method of specific compounds is also described in the above-mentioned application, which is incorporated herein in its entirety, and the specific definitions are as follows:

[0188] T is N or CH;

[0189] R1and R2are each independently H, F, Cl, Br, I, or C 1-3 alkyl, wherein the C 1-3 alkyl is optionally substituted by 1, 2, or 3 R a ;

[0190] each R a is independently F, Cl, Br, I, -CN, -OH, or -NH2;

[0191] R3and R4are each independently H, F, Cl, Br, I, CN, C 1-3 alkyl, C 1-3 alkoxy, wherein the C 1-3 alkyl is optionally substituted by 1, 2, or 3 R e ;

[0192] R b and R c are each independently H, -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0193] R d is -CH3, -CH2CH3, -(CH2)2CH3, -CH(CH3)2;

[0194] each Re independently F, Cl, Br, I, -CN, -OH, or -NH2.

[0195] wherein A, E, G, X, Y are as defined in the patent application PCT / NZ2019 / 050030, publication number WO2019190331A1 (corresponding to Chinese application number 2019800234236, publication number CN111918864A), and the method of synthesis of the specific compounds is also described in the above-mentioned application, which is incorporated herein in its entirety, and are specifically defined as:

[0196] 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;

[0197] E is SO or SO2;

[0198] X is Cl, Br, I, or OSO2R;

[0199] Y is Cl, Br, I, or OSO2R;

[0200] each R is independently H or C1-C6 alkyl;

[0201] G is a radical selected from the group comprising formulae (B) - (AA):

[0202] wherein:

[0203] R1is H, C1-C6 alkyl, CH2(CH2)nOH, CH2CH(OH)CH2OH, phenyl, pyridyl, benzyl, or pyridylmethyl, provided that when R1is phenyl, pyridyl, benzyl, or pyridylmethyl, R1is 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;

[0204] R2and R3are 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;

[0205] R4is N(R6)(R7), OH, OCH2(CH2)nN(R6)(R7), or CH2(CH2)nN(R6)(R7);

[0206] R5is H or a C1-C6alkyl group;

[0207] R6and R7are each independently H or a C1-6alkyl group, or R6and R7together form a substituted or unsubstituted 5- or 6-membered heterocyclic ring;

[0208] Z is CH or N;

[0209] W is CH2, O, S, SO, or SO2;

[0210] n is 0 to 6;

[0211] * indicates the point of attachment to Formula (I).

[0212] or a pharmaceutically acceptable salt thereof,

[0213] wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , n1, n2 are defined as in patent application PCT / CN2023 / 123253, publication number WO2024078392A1, the synthesis and preparation method of specific compounds are also described in the above-mentioned application, which is incorporated herein in its entirety, and the specific definitions are:

[0214] wherein:

[0215] (II) R 1 , R 2 together with the intervening atoms form an optionally substituted 4-8 membered carbocyclic or heterocyclic ring, R 4 and R 5 are defined as described in (I); or

[0216] (III) R 1 , R 5 together with the intervening atoms form an optionally substituted 4-8 membered carbocyclic or heterocyclic ring, R 2 and R 4 are defined as described in (I); or

[0217] (IV) R 4 , R 5 together with the intervening atoms form an optionally substituted 4-8 membered carbocyclic or heterocyclic ring, R1 and R 2 are as defined in (I) or (II); or

[0218] (I) R 1 is hydrogen, deuterium, optionally substituted C 1-4 alkyl, optionally substituted C 2-4 alkenyl, or optionally substituted C 2-4 alkynyl;

[0219] R 2 , R 4 , R 5 each independently is hydrogen, halogen (e.g., F), optionally substituted C 1-4 alkyl, optionally substituted C 2- 4alkenyl, optionally substituted C 2-4 alkynyl, optionally substituted C 1-4 alkoxy, or optionally substituted 3-5 membered ring;

[0220] wherein:

[0221] X is O, S, NR 10 , optionally 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;

[0222] R 3 is hydrogen, optionally substituted C 1-4 alkyl, or optionally substituted 3-10 membered ring;

[0223] R 6 is hydrogen, deuterium, optionally substituted C 1-4 alkyl, optionally substituted C 2-4 alkenyl, or optionally substituted C 2-4 alkynyl;

[0224] each integer n1, n2 is independently 0, 1, 2, 3, or 4;

[0225] each R a , R b is independently at each occurrence optionally substituted C1-4alkyl or optionally substituted C1-4heteroalkylene; or two instances of R a or two instances of R b together with intervening atoms join to form an optionally substituted 3-6 membered ring, and any remaining instances of R a and / or R b are as defined above.

[0226] wherein Sugar, R1, R2 are defined as in the claims of patent application US5622936A, and the synthesis of the specific compounds is also described in the above application, which is incorporated herein in its entirety, and are defined as follows:

[0227] wherein the sugar moiety is linked to phosphoramide mustard residue (15-I) or ifosfamide mustard residue (15-II), and R1 and R2 can be the same or different and are selected from hydrogen, C1-C4 alkyl or C1-C6 haloalkyl,

[0228] and the sugar moiety is any existing isomeric or enantiomeric form of monosaccharide, disaccharide or polysaccharide.

[0229] Other structures of DNA alkylating agent conjugated prodrugs activated by β- glucosidase, carboxylesterase, esterase and caspase-3, cathepsin B, γ-glutamyltranspeptidase, β-galactosidase can be found in review articles (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 2023 Feb 6. doi:10.1039 / d2cs00673a).

[0230] The compound of structural formula (1) is selected from the following structural compounds:

[0231] The synthesis of the compound is also described in patent application PCT / US2006 / 025881, publication number WO2007002931 (corresponding to Chinese application number 2006800300828, publication number CN101501054A), which is incorporated herein in its entirety.

[0232] The compound of structural formula (2) is selected from the following structural compounds:

[0233] The method for synthesizing and preparing the compound is also described in patent application PCT / CN2020 / 114519, publication number WO2021120717A1 (corresponding to Chinese application number 2020800673113, publication number CN114466853A), which is incorporated herein by reference in its entirety.

[0234] Compounds with structural formula (3) are selected from the following structural compounds:

[0235] The method for synthesizing and preparing the compound is also described in patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A), which is incorporated herein by reference in its entirety.

[0236] Compounds with structural formula (4) are selected from the following structural compounds:

[0237] The method for synthesizing and preparing 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 incorporated herein by reference in its entirety.

[0238] Compounds with structural formula (5) are selected from the following structural compounds:

[0239] The method for synthesizing and preparing 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 incorporated herein by reference in its entirety.

[0240] Compounds with structural formulas (6) and (7) are selected from the following structural compounds:

[0241] The method for synthesizing and preparing the compound is also described in patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A), which is incorporated herein by reference in its entirety.

[0242] Compounds with structural formula (8) are selected from the following structural compounds:

[0243] Compounds with structural formula (9) are selected from the following structural compounds:

[0244] The synthetic preparation method of the compound is also described in patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A), which is hereby incorporated in its entirety into the present application.

[0245] The compound of structural formula (10) is selected from the following structural compounds:

[0246] The synthetic preparation method of the compound is also described in patent application PCT / CN2021 / 118597, publication number WO2022057838A1, which is hereby incorporated in its entirety into the present application.

[0247] The compound of structural formula (11) is selected from the following structural compounds:

[0248] 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), which is hereby incorporated in its entirety into the present application.

[0249] 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, some of the compound structures are listed as follows:

[0250] The specific synthetic method of compounds 1-569 of formula (12) and the corresponding spectral data are disclosed in WO2024078392A1, which is hereby incorporated in its entirety.

[0251] The compound of structural formula (15) is selected from the following structural compounds:

[0252] The synthetic preparation method of the compound is also described in US patent application US5622936A, PCT application PCT / US2007 / 074012, publication number WO2008011588, which is hereby incorporated in its entirety into the present application.

[0253] The toxin Payload of the ADC is selected from the PBD class, the Duocarmycin class.

[0254] ADC drugs with PBDs as toxin Payloads are preferably selected from the group consisting of DHES0815A, SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-352A, Rovalpituzumab Tesirine, SC-002, SC-003, ADCT-301, ADCT-402 (Lonca, Loncastuximab tesirine), ADCT-601, ADCT-602.

[0255] For more detailed information on ADC drugs with PBDs as toxin Payloads see the literature:

[0256] Hartley JA. Antibody-drug conjugates (ADCs) delivering pyrrolobenzodiazepine (PBD) dimers for cancer therapy. Expert Opin Biol Ther. 2021 Jul;21(7):931-943. doi: 10.1080 / 14712598.2020.1776255. Epub 2020 Jun 16. PMID: 32543981.

[0257] ADC drugs with Duocarmycins as toxin Payloads are preferably selected from the group consisting of SYD983, SYD985, BMS-936561, SYD1875, MGC018, PCM5B14DCM, hYP7-DCM, Promiximab DUBA, D2B-DCM, PCM-MET01. For more detailed information on ADC drugs with Duocarmycins as toxin Payloads see the literature:

[0258] Yao HP, Zhao H, Hudson R, Tong XM, Wang MH. Duocarmycin-based antibody-drug conjugates as an emerging biotherapeutic entity for targeted cancer therapy: Pharmaceutical strategy and clinical progress. Drug Discov Today. 2021 Aug;26(8):1857-1874. doi: 10.1016 / j.drudis.2021.06.012. Epub 2021 Jul 3. PMID: 34224904.

[0259] DNA alkylating agent prodrug compound is selected from SMDC drug AST-3424, AST-001, TFX05-01, Evofosfamide, Achm-025, Glufosfamide;

[0260] DNA alkylating agent prodrug compound is selected from PDC drug Melphalan flufenamide.

[0261] SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A and SGN-352A are developed by Seattle Genetics, targeting CD33, CD70, CD19, CD123, CD352 respectively, and the linker is a protease-degradable valine-alanine linker, which is connected to the PBD through aniline of SGD1882PBD.

[0262] Rovalpituzumab tesirine (Rova-T, also called SC16LD6.5) of AbbVie (Stemcentrx) is a biomarker ADC drug, targeting Delta-like protein 3 (DLL3) on the surface of tumor stem cells, containing PBD of SG 3199, and the linker is a degradable valine-alanine maleimide type linker, and the indication is non-small cell lung cancer.

[0263] SC-002 and SC-003 developed by AbbVie are also PBD-based ADC drugs, and the indications are SCLC and ovarian cancer.

[0264] ADCT-301 and ADCT-402 (Lonca) of ADC Therapeutics are both PBD-based ADC drugs targeting CD25 and CD19, and both use SG3249 type of PBD.

[0265] SYD983, SYD985, BMS-936561, SYD1875, MGC018, PCM5B14DCM, hYP7-DCM, Promiximab DUBA, D2B-DCM, PCM-MET01 are all Duocarmycin-based ADC drugs, connected with different Linkers and antibodies.

[0266] DHES0815A is an ADC with alkylating agent pyrrolobenzodiazepine (PBD) dimer as drug moiety, and its preclinical characterization and phase I trial results are described in the following literature:

[0267] Lewis, G. D., Li, G., Guo, J. et al. The HER2-directed antibody-drug conjugate DHES 0815A in advanced and / or metastatic breast cancer: preclinical characterization and phase 1 trial results. Nat Commun 15, 466 (2024). https: / / doi.org / 10.1038 / s41467-023-44533-z.

[0268] In particular, the dose of DHES0815A is not more than 12 mg / kg, preferably not more than 4 mg / kg, more preferably not more than 2.4 mg / kg, further preferably not more than 1.4 mg / kg, more further preferably not more than 1.2 mg / kg, again further preferably not more than 0.6 mg / kg. DHES0815A is used for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer.

[0269] SGN-CD33A, which is in clinical studies, see the literature:

[0270] Kung Sutherland MS, Walter RB, Jeffrey SC, et al. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. Blood. 2013; 122(8): 1455-1463. doi: 10.1182 / blood-2013-03-491506;

[0271] Stanchina M, Pastore A, Devlin S, Famulare C, Stein E, Taylor J. CD33 splice site genotype was not associated with outcomes of patients receiving the anti-CD33 drug conjugate SGN-CD33A. J Hematol Oncol. 2019; 12(1): 85. Published 2019 Aug 22. doi: 10.1186 / s13045-019-0771-0;

[0272] Stein EM, Walter RB, Erba HP, et al. A phase 1 trial of vadastuximab talirine as monotherapy in patients with CD33-positive acute myeloid leukemia. Blood. 2018; 131(4): 387-396. doi: 10.1182 / blood-2017-06-789800.

[0273] The administration regimen is: SGN-CD33A treatment on day 1 (5-60 μg / kg) or day 1 and day 4 (20 μg / kg) of a 21-day cycle, with a recommended monotherapy dose of 40 μg / kg.

[0274] SGN-CD70A is in clinical research, see the literature for details:

[0275] Phillips T, Barr PM, Park SI, et al. A phase 1 trial of SGN-CD70A in patients with CD70-positive diffuse large B cell lymphoma and mantle cell lymphoma. Invest New Drugs. 2019; 37(2): 297-306. doi: 10.1007 / s10637-018-0655-0

[0276] The administration regimen is: every 3 weeks as a treatment cycle, intravenous administration on day 1, with a dose of 8 mcg / kg to 200 mcg / kg; or every 6 weeks as a treatment cycle, intravenous administration on day 1, with a dose of 8 mcg / kg to 200 mcg / kg;

[0277] Its preferred dosing regimen: every 6 weeks for a treatment cycle, intravenous administration on day 1, with a dose of no more than 30 mcg / kg, i.e. 30 mcg / kg q6wk.

[0278] SGN-CD19B is in preclinical studies. See the literature for details:

[0279] Ryan MC, Palanca-Wessels MC, Schimpf B, et al. Therapeutic potential of SGN-CD 19B, a PBD-based anti-CD19 drug conjugate, for treatment of B-cell malignancies. Blood. 2017; 130(18): 2018-2026. doi: 10.1182 / blood-2017-04-779389.

[0280] SGN-CD123A is in preclinical studies. See the literature for details:

[0281] Li F, Sutherland MK, Yu C, et al. Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia. Mol Cancer Ther. 2018; 17(2): 554-564. doi: 10.1158 / 1535-7163.MCT-17-0742;

[0282] May S.K. Sutherland, PhD, Changpu Yu, et al. SGN-CD123A, a Pyrrolobenzodiazepine Dimer Linked Anti-CD123 Antibody Drug Conjugate, Demonstrates Effective Anti-Leukemic Activity in Multiple Preclinical Models of AML. Blood (2015) 126(23): 330. doi.org / 10.1182 / blood.V126.23.330.330.

[0283] SGN-352A, which is in preclinical studies, see the literature:

[0284] Tim Lewis, Devra J. Olson, Kristine A. Gordon, Sharsti L. Sandall, Jamie Miyamoto, Lori Westendorf, Germein Linares, Chris Leiske, Heather Kostner, Ivan Stone, Martha Anderson, Albina Nesterova, Mechthild Jonas, Che-Leung Law. SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016; 76(14 Suppl): Abstract nr 1195.

[0285] Rovalpituzumab Tesirine is in clinical phase I, see the literature:

[0286] Rudin CM, Pietanza MC, Bauer TM, et al. Rovalpituzumab tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent small-cell lung cancer: a first-in-human, first-in-class, open-label, phase 1 study. Lancet Oncol. 2017; 18(1): 42-51. doi: 10.1016 / S1470-2045(16)30565-4.

[0287] The dosage regimen is: every 3 weeks or 6 weeks as a treatment cycle, the dose is 0.05 mg / kg to 0.8 mg / kg, the preferred dosage regimen is: every 6 weeks as a treatment cycle, the dose is 0.3 mg / kg or 0.4 mg / kg, or every 3 weeks as a treatment cycle, the dose is 0.2 mg / kg.

[0288] SC-002 is in the clinical I phase, see the literature for details:

[0289] Morgensztern D, Johnson M, Rudin CM, et al. SC-002 in patients with relapsed or refractory small cell lung cancer and large cell neuroendocrine carcinoma: Phase 1 study. Lung Cancer. 2020; 145: 126-131. doi: 10.1016 / j.lungcan.2020.04.017.

[0290] SC-003 is in the clinical research stage, see the literature for details:

[0291] Hamilton E, O'Malley DM, O'Cearbhaill R, et al. Tamrintamab pamozirine (SC-003) in patients with platinum-resistant / refractory ovarian cancer: Findings of a phase 1 study. Gynecol Oncol. 2020; 158(3): 640-645. doi: 10.1016 / j.ygyno.2020.05.038.

[0292] The dosage regimen is: every 3 weeks as a treatment cycle, the dose is 0.025-0.4 mg / kg, the recommended dose is not more than 0.3 mg / kg, preferably, the dose is 0.3 mg / kg or 0.2 mg / kg. ADCT-301 is in the preclinical research stage, see the literature:

[0293] Flynn MJ, Zammarchi F, Tyrer PC, Akarca AU, Janghra N, Britten CE, Havenith CE, Levy JN, Tiberghien A, Masterson LA, Barry C, D'Hooge F, Marafioti T, Parren PW, Williams DG, Howard PW, van Berkel PH, Hartley JA. ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies. Mol Cancer Ther. 2016 Nov; 15(11): 2709-2721. doi: 10.1158 / 1535-7163.MCT-16-0233. Epub 2016 Aug 17. PMID: 27535974.

[0294] ADCT-402 (Lonca), in clinical phase III studies, see:

[0295] Zammarchi F, Corbett S, Adams L, Tyrer PC, Kiakos K, Janghra N, Marafioti T, Britten CE, Havenith CEG, Chivers S, D'Hooge F, Williams DG, Tiberghien A, Howard PW, Hartley JA, van Berkel PH. ADCT-402, a PBD dimer-containing antibody drug conjugate targeting CD19-expressing malignancies. Blood. 2018 Mar 8; 131(10): 1094-1105. doi: 10.1182 / blood-2017-10-813493. Epub 2018 Jan 3. PMID: 29298756.

[0296] Caimi PF, Ai W, Alderuccio JP, Ardeshna KM, Hamadani M, Hess B, Kahl BS, Radford J, Solh M, Stathis A, Zinzani PL, Havenith K, Feingold J, He S, Qin Y, Ungar D, Zhang X, Carlo-Stella C. Loncastuximab tesirine in relapsed or refractory diffuse large B-cell lymphoma (LOTIS-2): a multicentre, open-label, single-arm, phase 2 trial. Lancet Oncol. 2021 Jun;22(6):790-800. doi: 10.1016 / S1470-2045(21)00139-X. Epub 2021 May 11. PMID: 33989558.

[0297] The dosing regimen is: intravenous infusion on day 1 of each 21 -day cycle at a dose of 150 pg / kg for two cycles.

[0298] ADCT-601, which is in preclinical studies, see:

[0299] Zammarchi F, Havenith KE, Chivers S, Hogg P, Bertelli F, Tyrer P, Janghra N, Reinert HW, Hartley JA, van Berkel PH. Preclinical Development of ADCT-601, a Novel Pyrrolobenzodiazepine Dimer-based Antibody-drug Conjugate Targeting AXL-expressing Cancers. Mol Cancer Ther. 2022 Apr 1 ;21 (4):582-593. doi: 10.1158 / 1535-7163.MCT-21-0715. PMID: 35086955; PMCID: PMC9377743.

[0300] ADCT-602, which is in preclinical studies, see:

[0301] Zammarchi F, Havenith KE, Sachini N, Janghra N, Chivers S, Idusogie E, Gaudio E, Tarantelli C, Bertelli F, Santos K, Tyrer P, Corbett S, Spriano F, Golino G, Cascione L, Bertoni F, Hartley JA, van Berkel PH. ADCT-602, a Novel PBD Dimer-containing Antibody-Drug Conjugate for Treating CD22-positive Hematologic Malignancies. Mol Cancer Ther. 2024 Apr 2;23(4):520-531. doi: 10.1158 / 1535-7163.MCT-23-0506. PMID: 38324336; PMCID: PMC10985467.

[0302] SYD983, in clinical phase, see literature:

[0303] Dokter W, Ubink R, van der Lee M, van der Vleuten M, van Achterberg T, Jacobs D, Loosveld E, van den Dobbelsteen D, Egging D, Mattaar E, Groothuis P, Beusker P, Coumans R, Elgersma R, Menge W, Joosten J, Spjker H, Huijbregts T, de Groot V, Eppink M, de Roo G, Verheijden G, Timmers M. Preclinical profile of the HER2-targeting ADC SYD983 / SYD985: introduction of a new duocarmycin-based linker-drug platform. Mol Cancer Ther. 2014 Nov;13(11):2618-29. doi: 10.1158 / 1535-7163.MCT-14-0040-T. Epub 2014 Sep 4. PMID: 25189543.

[0304] SYD985, in clinical phase, see literature:

[0305] Black J, Menderes G, Bellone S, et al. SYD985, a Novel Duocarmycin-Based HER2-Targeting Antibody-Drug Conjugate, Shows Antitumor Activity in Uterine Serous Carcinoma with HER2 / Neu Expression. Mol Cancer Ther. 2016; 15(8): 1900-1909. doi: 10.1158 / 1535-7163.MCT-16-0163;

[0306] Nadal-Serrano M, Morancho B, Escrivá-de-Romaní S, Morales CB, Luque A, Escorihuela M, Espinosa Bravo M, Peg V, Dijcks FA, Dokter WHA, Cortés J, Saura C, Arribas J. The Second Generation Antibody-Drug Conjugate SYD985 Overcomes Resistances to T-DM1. Cancers (Basel). 2020 Mar 13; 12(3): 670. doi: 10.3390 / cancers12030670. PMID: 32183023; PMCID: PMC7139846;

[0307] Saura, Cristina et al. “A phase I expansion cohorts study of SYD985 in heavily pretreated patients with HER2-positive or HER2-low metastatic breast cancer.” Journal of Clinical Oncology 36 (2018): 1014-1014.

[0308] The dosage regimen is: 1.2 mg / kg SYD985 treatment once every 3 weeks.

[0309] BMS-936561, in phase I clinical stage, see literature:

[0310] Owonikoko TK, Hussain A, Stadler WM, Smith DC, Kluger H, Molina AM, GulatiP, Shah A, Ahlers CM, Cardarelli PM, Cohen LJ. First-in-human multicenter phase I study of BMS-936561(MDX-1203), an antibody-drug conjugate targeting CD70. Cancer Chemother Pharmacol.2016Jan;77(1):155-62.doi:10.1007 / s00280-015-2909-2.Epub 2015Nov 14.PMID:26576779.

[0311] Wang H, Rangan VS, Sung MC, Passmore D, Kempe T, Wang of its pharmacokinetics in humans.Biopharm Drug Dispos.2016Mar;37(2):93-106.doi:10.1002 / bdd.1953.Epub 2015 May 15.PMID:25869904.

[0312] The clinical dosing regimen is as follows: once every 21 days for a period of 42 days, administered by injection at a dose of 8 mg / kg.

[0313] SYD1875 is currently in Phase I clinical trials; see the literature for details.

[0314] Patrick Groothuis, Danielle Jacobs, Kim Berentsen, Monique van der Vleuten, Ruud Coumans, Ronald Elgersma, Marion Blomenrohr, Diels van den Dobbelsteen, Patrick Beusker, Ruud Ubink, Miranda van der Lee, Wim H.A. Dokter. Introduction to the preclinical profile of SYD1875, a novel site-specifically conjugated duocarmycin-based 5T4-targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 925.

[0315] MGC018, in phase II clinical, see literature:

[0316] Scribner JA, Brown JG, Son T, Chiechi M, Li P, Sharma S, Li H, De Costa A, Li Y, Chen Y, Easton A, Yee-Toy NC, Chen FZ, Gorlatov S, Barat B, Huang L, Wolff CR, Hooley J, Hotaling TE, Gaynutdinov T, Ciccarone V, Tamura J, Koenig S, Moore PA, Bonvini E, Loo D. Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer. Mol Cancer Ther. 2020 Nov;19(11):2235-2244. doi: 10.1158 / 1535-7163.MCT-20-0116. Epub 2020 Sep 23. PMID: 32967924.

[0317] Sekwon Jang et al., Phase 1 dose escalation study of MGC018, an anti-B7-H3 antibody-drug conjugate (ADC), in patients with advanced solid tumors.. JCO 39, 2631-2631 (2021). DOI: 10.1200 / JCO.2021.39.15_suppl.2631

[0318] The current clinical dosing regimen is: intravenous administration on day 1 every 3 weeks at a dose of 3 mg / kg.

[0319] PCM5B14DCM is in preclinical development, see:

[0320] X. M. Tong, et al. Therapeutic efficacy of a novel humanized antibody-drug conjugate recognizing plexin-semaphorin-integrin domain in the RON receptor for targeted cancer therapy, J. Immunother. Cancer, 7 (2019), p. 250

[0321] hYP7-DCM, in preclinical development, see:

[0322] Y. Fu, et al. Glypican-3-specific antibody drug conjugates targeting hepatocellular carcinoma, Hepatology, 70 (2019), pp. 563-576

[0323] Promiximab DUBA, in preclinical development, see:

[0324] Yu L, Lu Y, Yao Y, Liu Y, Wang Y, Lai Q, Zhang R, Li W, Wang R, Fu Y, Tao Y, Yi S, Gou L, Chen L, Yang J. Promiximab-duocarmycin, a new CD56 antibody-drug conjugates, is highly efficacious in small cell lung cancer xenograft models. Oncotarget. 2017 Dec 26;9(4):5197-5207. doi: 10.18632 / oncotarget.23708. PMID: 29435172; PMCID: PMC5797043.

[0325] D2B-DCM, in preclinical development, see:

[0326] Lutje, et al. Characterization of site-specifically conjugated monomethyl auristatin E- and duocarmycin-based anti-PSMA antibody-drug conjugates for treatment of PSMA-expressing tumors, J. Nucl. Med., 59 (2018), pp. 494-501

[0327] PCM-MET01, in preclinical study stage, see the literature:

[0328] Wang, M. H. and Yao, H. P. (2020). Preparation and use of anti-MET and-ON bispecific antibody and antibody-drug conjugate thereof. Chinese patent application. Patent 202010562324.X. July 18, 2020.

[0329] In particular, the SMDC is selected from a hypoxia-activated DNA alkylating agent conjugated prodrug compound, an AKR1C3-activated DNA alkylating agent conjugated prodrug compound, a beta-glucosidase (β-D-Glucosidase) or a beta-galactosidase (β-galactosidase) activated DNA alkylating agent conjugated prodrug compound.

[0330] AKR1C3-activated DNA alkylating agent conjugated prodrug compounds AST-3424, AST-001, TFX05-01, Achm-025, Payloads of these SMDCs, AST-3424, AST-001, TFX05-01 are all AST-2660, which is dissociated under the specific activation of AKR1C3 enzyme. Evofosfamide is a hypoxia-activated DNA alkylating agent conjugated prodrug compound, and Glufosfamide is a beta-glucosidase (β-D-Glucosidase) activated DNA alkylating agent conjugated prodrug compound, and its Payloads are Br-IPM and Cl-IPM.

[0331] The above-mentioned AST-3424 is formulated as an aqueous solution for intravenous injection,

[0332] The solutes of the aqueous solution are composed of AST-3424 drug substance, glucose, ethanol, propylene glycol and pH regulator sodium bicarbonate,

[0333] The concentration of AST-3424 drug substance in the aqueous solution is 0.004-0.94 mg / ml, the pH is 7.4, the content of glucose is 4.5-5.0% by mass, and it is an isotonic solution;

[0334] Or

[0335] The solutes of the aqueous solution are composed of AST-3424 drug substance, sodium chloride, ethanol, propylene glycol and pH regulator sodium bicarbonate,

[0336] The concentration of AST-3424 drug substance in the aqueous solution 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 it is an isotonic solution.

[0337] The preparation method of AST-3424 prepared as an injection concentrated solution and the preparation method of the above aqueous solution for intravenous injection are both described in patent application PCT / CN2020 / 101870, publication number WO20210085201.

[0338] The AST-3424 administration scheme is selected from any one of the following schemes:

[0339] Scheme one, each 21 days as a cycle, each administration once on the first day and the eighth day, the dose is selected from 12 mg / m 2 , 11 mg / m 2 , 10 mg / m 2 , 9 mg / m 2 , 8 mg / m 2 , 7 mg / m 2 , 6 mg / m 2 , 5 mg / m 2 , 4 mg / m 2 ;

[0340] Scheme two, each 21 days as a cycle, administration on the first day, the dose is selected from 14 mg / m 2 , 13 mg / m 2 , 12 mg / m 2 , 11 mg / m 2 , 10 mg / m 2 , 9 mg / m 2 , 8 mg / m 2 , 7 mg / m 2 , 6 mg / m 2 , 5 mg / m 2 , 4 mg / m 2, 3 mg / m 2 , 2 mg / m 2 , 1 mg / m 2 ;

[0341] Regimen three, each 21 days as a cycle, once a day from day 1 to day 5, the dose is selected from 8 mg / m 2 , 7 mg / m 2 , 6 mg / m 2 , 5 mg / m 2 , 4 mg / m 2 , 3 mg / m 2 , 2 mg / m 2 , 1 mg / m 2 .

[0342] The specific information of the above three AST-3424 administration regimens and their doses can be found in the clinical trial US clinical trial NCT03592264 and Chinese clinical trials CTR20191371, CTR20191399.

[0343] For AST-3424, the patient is a liver cancer patient.

[0344] Preferably, for AST-3424, the patient's liver tumor tissue AKR1C3 expression is strongly positive, which meets one of the following conditions:

[0345] One, the H score is greater than or equal to 100, preferably greater than or equal to 135, more preferably greater than or equal to 200, detected by the AKR1C3 detection method described in WO2022048492;

[0346] Two, the proportion of tumor cells with staining intensity of 2+ and / or 3+ is ≥70%, detected by the AKR1C3 detection method described in WO2022048492.

[0347] Standard one is determined according to 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, A. M., Verschraegen, C. F., 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).

[0348] Standard two is determined according to the AST-3424 phase I clinical trial CTR20191371 conducted in China, and a phase II clinical trial is currently being conducted according to this standard. The inclusion criteria for the phase II clinical trial are described in CTR20191399.

[0349] In the text described in WO2022048492, various IHC methods for detecting the expression level of AKR1C3 enzyme are provided. 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, including the following steps:

[0350] a1) Dewaxing and rehydrating

[0351] Formalin-fixed paraffin-embedded human tissue sections of appropriate thickness are dewaxed using organic solvents. The dewaxed sections are washed with alcohol of different water content, and finally washed with water.

[0352] a) Antigen retrieval

[0353] The formalin-fixed paraffin-embedded human tissue sections after dewaxing and rehydration are heated at 90-115°C for 17-30 minutes in the presence of antigen retrieval solution for antigen retrieval.

[0354] b1) Blocking non-specific antigens

[0355] The formalin-fixed paraffin-embedded human tissue sections after antigen retrieval are incubated with blocking solution to block non-specific antigens.

[0356] b) primary antibody incubation

[0357] incubate the formalin-fixed paraffin-embedded human tissue section after blocking non-specific antigen with AKR1C3 monoclonal antibody solution at a concentration of 0.5-5.0 μg / ml for 25-700 minutes;

[0358] c) secondary antibody incubation

[0359] incubate the formalin-fixed paraffin-embedded human tissue section after primary antibody incubation with secondary antibody solution at a concentration of 0.5-5.0 μg / ml for 25-700 minutes;

[0360] d) staining and fixing

[0361] stain the formalin-fixed paraffin-embedded human tissue section with hematoxylin, and dehydrate and mount the section after staining;

[0362] e) observation and scoring

[0363] observe the human tissue section after staining, and evaluate the expression level of AKR1C3 in the human tissue section according to the observed staining degree.

[0364] In the text described in WO2022048492, the score calculation method of the H-score system is:

[0365] H-score = percentage of cells with staining intensity of 1+ x 1 + percentage of cells with staining intensity of 2+ x 2 + percentage of cells with staining intensity of 3+ x 3, for example, the IHC staining result of a certain liver cancer tissue is that the percentage of cells with staining intensity of 1 is 10%, the percentage of cells with staining intensity of 2 is 20%, and the percentage of cells with staining intensity of 3 is 50%, then the H-score is 10+40+150 = 200.

[0366] Any one of the drugs containing AST-3424, abiraterone, prednisolone, 5-fluorouracil, sunitinib, oxaliplatin, PD-1 / L1 inhibitor, apatinib, sorafenib or donafenib, or elemene is combined for the treatment of liver cancer.

[0367] Evofosfamide, see the related content of TH-302 above. Specific administration regimen: intravenous injection, daily dose of 120 mg / m 2 to 460 mg / m 2 ; or intravenous injection, weekly dose of 480 mg / m 2 to 670 mg / m 2 , preferably weekly dose of 575 mg / m 2 .

[0368] For AST-001, see the clinical trials registered on the China CDE website CTR20220935, CTR20220934 (http: / / www.chinadrugtrials.org.cn / clinicaltrials.searchlist.dhtml) and the research literature: Meng T, Jung D, Cai XH, Lu ZQ, Yu JB, Qi TY, Meng FY, Ruan MZ, Duan JX. Characterization of AST-001 non-clinical pharmacokinetics: A novel selective AKR1C3-activated prodrug in mice, rats, and cynomolgus monkeys. Biopharm Drug Dispos. 2024 Apr; 45(2): 83-92. doi: 10.1002 / bdd.2385. Epub 2024 Mar 16. PMID: 38492211.

[0369] The specific administration regimen is: every 28 days is a treatment cycle, and the doses are selected from 25 mg / kg, 20 mg / kg, 18 mg / kg, 15 mg / kg, 12 mg / kg, 10 mg / kg, 8 mg / kg, 6 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, on the 1st, 8th and 15th day of each cycle.

[0370] See the clinical trial CTR20220957 registered on the China CDE website (http: / / www.china drugtrials.org.cn / clinicaltrials.searchlist.dhtml) and the research literature: Charles Z Ding, Zhe Cai, Wei Sha. Preclinical evaluation of TFX05-01, a selective AKR1C3-targeted prodrug for solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022; 82(12_Suppl): Abstract nr 5691.

[0371] The specific administration regimen is: every 3 weeks as a treatment cycle, and the dose is selected from 25mg / kg, 20mg / kg, 18mg / kg, 15mg / kg, 12mg / kg, 10mg / kg, 8mg / kg, 6mg / kg, 4mg / kg, 3mg / kg, 2mg / kg, 1mg / kg, on the 1st day and the 8th day of each cycle.

[0372] Achm-025 See the literature:

[0373] Richard B. Lock, Cara E Toscan, Hannah McCalmont, Kathryn Evans, Louise Doculara, Hansen J Kosasih, Andrew J Gifford, Amir Ashoorzadeh, Xiaojing Lin, Toby N Trahair, Charles E De Bock, Adam V Patterson, Jeff B Smaill; The AKR1C3-Activated Prodrug, Achm-025, Eradicates Disease in Preclinical Models of Aggressive T-Cell Acute Lymphoblastic Leukemia. Blood 2023; 142 (Supplement 1): 4251. doi:https: / / doi.org / 10.1182 / blood-2023-181298;

[0374] The specific administration regimen is: every 3 weeks is a treatment cycle, the 1st day and the 8th day of each cycle are administered.

[0375] The specific administration regimen is: every 3 weeks is a treatment cycle, the 1st day and the 8th day of each cycle are administered.

[0376] Ciuleanu TE, Pavlovsky AV, Bodoky G, Garin AM, Langmuir VK, Kroll S, Tidmarsh GT. A randomised Phase III trial of glufosfamide compared with best supportive care in metastatic pancreatic adenocarcinoma previously treated with gemcitabine. Eur J Cancer. 2009 Jun;45(9):1589-96. doi: 10.1016 / j.ejca.2008.12.022. Epub 2009 Jan 31. PMID: 19188061.

[0377] The specific administration regimen is: every 3 weeks is a treatment cycle, the 1st day and the 8th day of each cycle are administered. 2 Intravenous infusion for 6 hours.

[0378] In particular, the PDC is selected from melphalan flufenamide (trade name Pepaxto), melphalan flufenamide (also known as melflufen, melphalan flufenamide). It is a PDC targeting aminopeptidase, which can rapidly deliver the alkylating agent melphalan (also known as: melphalan) effective load into tumor cells, and its target peptide is a dipeptide.

[0379] Melphalan flufenamide, see the drug instructions and literature for specific cases:

[0380] Schjesvold F, Robak P, Pour L, Aschan J, Sonneveld P. OCEAN: a randomized Phase III study of melflufen + dexamethasone to treat relapsed refractory multiple myeloma. Future Oncol. 2020 Apr; 16(11): 631-641. doi: 10.2217 / fon-2020-0024. Epub 2020 Mar 6. PMID: 32141766.

[0381] The dosage regimen is: intravenous injection of 40 mg within 30 minutes on the first day of each 28-day treatment cycle, preferably, melphalan flufenamide is combined with dexamethasone.

[0382] The treatment method provided by the present application, before administering a single drug or combination of other drugs containing a DNA alkylating agent prodrug compound or DNA alkylating agent to treat cancer, tumor patients, the patient is negative by p53 gene mutation test results or the patient is normal by p53 protein expression test results, the DNA alkylating agent prodrug compound or DNA alkylating agent is defined as described above.

[0383] p53 gene, also known as tp53 gene, tumor protein p53 gene (Tumor protein p53).

[0384] In this application, p53 (protein) and p53 (gene) are not distinguished, and both are used.

[0385] In this application, p53 gene mutation negative includes no mutation detected and although the mutation is detected but not reach the designated positive degree.

[0386] Currently, there are related detection kits approved for commercial use, which can be directly purchased and used for detection, such as the tp53 Six Mutations Detection Kit, p53 gene amplification detection kit (fluorescence in situ hybridization method) FISH detection Kit for the p53 gene produced by Sino-Genotech Biotech Co., Ltd. in Henan, China, and p53 kit for VariantPlex TM p53 kit for

[0387] p53 protein expression is normal, including normal expression and higher than normal high expression, overexpression. The normal value is a value artificially determined according to statistical clinical practice.

[0388] p53 protein normal expression, that is, it can be determined by comparing typical WB experiments, and it can also be determined by IHC in medical clinics. The amount of p53 protein in the test sample is directly determined by these methods, and then compared with the normal value or the set threshold to determine whether it is normal expression or high expression.

[0389] It can also be screened by gene detection. Generally, if pathogenic gene mutations are detected, it can be determined that the p53 protein is not normally expressed, nor is it overexpressed. If no gene mutation is detected or although a gene mutation is detected, it is not a pathogenic mutation, then it is most likely that the p53 protein is normally expressed. Therefore, whether the p53 protein is normally expressed / overexpressed can be determined by detecting whether the p53 gene has pathogenic gene mutations.

[0390] The above gene mutation or protein expression detection is generally obtained by detecting the tumor or cancer tissue, cells or other biological detection samples of the patient.

[0391] The biological test sample comprises a peripheral blood sample, a tumor tissue or a suspected tumor tissue, a thin layer cytology sample, a fine needle aspiration sample, a bone marrow sample, a lymph node sample, a urine sample, an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung washing sample, a spinal fluid sample, a cerebral fluid sample, a ductal aspiration sample, a nipple discharge sample, a pleural effusion sample, a fresh frozen tissue sample, a paraffin-embedded tissue sample, or an extract or a processed sample derived from any of a peripheral blood sample, a tumor tissue or a suspected tumor tissue, a thin layer cytology sample, a fine needle aspiration sample, a bone marrow sample, a urine sample, an ascites sample, a lavage sample, an esophageal brushing sample, a bladder or lung washing sample, a spinal fluid sample, a cerebral fluid sample, a ductal aspiration sample, a nipple discharge sample, a pleural effusion sample, a fresh frozen tissue sample, or a paraffin-embedded tissue sample, generally venous whole blood or saliva.

[0392] With respect to the compounds described herein, the chemical structure of AST-3424 contains an organic amine structure and a P=O double bond structure, therefore the compounds can also be administered in the form of a salt, i.e., the present application provides pharmaceutically acceptable salts of the compounds shown, which can be basic salts, including salts of the compounds formed with inorganic acids such as hydroxide, hydroxide of alkali metal, hydroxide of alkaline earth metal, and the like, or with organic bases such as monoethanolamine, diethanolamine or triethanolamine, and the like. Alternatively, the salts can be acid salts, including salts of the compounds formed with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, phosphoric acid, and the like, or with organic acids such as methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, and the like. Similarly, it is also possible to react with certain acids or alcohols to become esters, therefore the compounds can also be administered in the form of esters.

[0393] For various reasons, it is also possible for these compounds to form solvates with certain solvents, which are hydrates or alcoholates, therefore the compounds can also be administered in the form of solvates. The selection and preparation of acceptable salts, esters and solvates of compounds are well known techniques in the art.

[0394] The term "isotopic variant" refers to a compound that contains an unnatural proportion of an isotope at one or more of the atoms that constitute such a compound. In certain embodiments, an "isotopic variant" of a compound contains an unnatural proportion of one or more isotopes, including, but not limited to, hydrogen ( 1 ), deuterium ( 2 ), tritium ( 3 ), 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), phosphorus-34 32 S), sulfur-32 33 S), sulfur-33 34 S), sulfur-34 35 S), sulfur-35 36 S), sulfur-36 35 Cl), chlorine-36 36 Cl), chlorine-37 37 Br), bromine-81 79 Br), bromine-81 81 I), 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 in a stable form, i.e., not radioactive. In certain embodiments, an "isotopic variant" of a compound contains one or more isotopes in an unnatural proportion, including but not limited to hydrogen-1 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 some embodiments, the "isotopic variant" of the compound is in an unstable form, i.e., radioactive. In some embodiments, the "isotopic variant" of the compound contains one or more isotopes in non-natural proportions, 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 should be understood that in compounds as provided herein, any hydrogen may be, for example, where feasible to those skilled in the art. 2 H is D, or any carbon can be, for example 13 C, or any nitrogen, can be, for example... 15 N, and any oxygen can be 18 O. In some embodiments, the "isotopic variants" of the compound contain non-natural proportions of deuterium (D).

[0395] The term "drug" as used herein refers to a pharmaceutical product or preparation, wherein the prepared pharmaceutical product contains an active ingredient of formula (1) or a salt or solvate thereof within a specific dosage range, and / or the prepared pharmaceutical product is administered in a specific dosage form or via a specific route of administration.

[0396] The resulting pharmaceutical products, drugs, and formulations may also contain pharmaceutically acceptable excipients or excipients. The drug may be 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 injection needles, lyophilized powder for injection, or large-volume infusions. Depending on the specific dosage form and administration method, the pharmaceutically acceptable excipients or excipients in the drug may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and colorants, etc.

[0397] Single drug, i.e. single drug therapy. Combination, i.e. combination therapy. Single drug therapy refers to the use of only one anticancer drug in a course of treatment. Combination therapy refers to the use of two or more anticancer drugs simultaneously or sequentially in a course of treatment.

[0398] Generally speaking, combination therapy needs to explore different dosages, dosing cycles according to the characteristics of the disease and the types of drugs used in combination. Only under the above conditions, the combination therapy regimen explored can achieve better therapeutic effect than single drug therapy.

[0399] The dosages, dosing cycles, and dosing regimens of the drugs in single drug and combination therapy regimens are obtained through clinical trials.

[0400] Examples of the combination of AST-3424 with other drugs have been reported in the following documents:

[0401] Among them, AST-3424 is combined with abiraterone acetate or abiraterone + prednisolone, sunitinib, gemcitabine. 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, the combination of AST-3424, abiraterone acetate or abiraterone, and prednisolone is a triple therapy, using three drugs, i.e. on the basis of the existing combination of abiraterone acetate or abiraterone + prednisolone, using the third drug AST-3424 for triple combination therapy.

[0402] For AST-3424 in combination with 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. 11,7 3645-3659. 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. 22 Jul. 2022, doi:10.3389 / fonc.2022.885139).

[0403] For AST-3424 in combination with 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-Fluorouracil in Primary Liver Cancer.” Frontiers in oncology vol. 12 885139. 22 Jul. 2022, doi:10.3389 / fonc.2022.885139).

[0404] For AST-3424 in combination with PD-1 / L1 inhibitors, please refer to patent application PCT / US2021 / 29552, publication number WO2022231580, and 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, poster is downloadable from OBI Pharma Inc website at 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 / / )

[0405] For AST-3424 in combination with apatinib, sorafenib or donafenib, or elemene, please refer to 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, and the above literature reports the combination treatment of AST-3424 with sorafenib, so those skilled in the art can undoubtedly infer that AST-3424 can also be used in combination with its deuterated drug donafenib for treatment.

[0406] The combination of other drugs can be corresponded to the relevant literature. BRIEF DESCRIPTION OF DRAWINGS

[0407] Figure 1 is the AST-3424 / AST combined with Nutlin-3 on H460 cell in vitro proliferation inhibition rate experimental curve;

[0408] Figure 2 is the AST-3424 and Nutlin-3 single use and different dosing sequence combination on H460 cell in vitro proliferation inhibition rate experimental results;

[0409] Figure 3 is the AST-3424 and Nutlin-3 single use and different dosing sequence combination on H460 / HPAF-II cell clone formation influence experimental results;

[0410] Figure 4 is the AST-3424 and Nutlin-3 single use and different dose combination on H460 cell apoptosis process influence experimental results;

[0411] Figure 5 is the AST-3424 and Nutlin-3 combination on H460 / HPAF-II cell cycle G2 / M arrest influence first experimental results, the column chart from top to bottom are G2 / M, S, G0 / G1 phase respectively;

[0412] Figure 6 is the AST-3424 and Nutlin-3 combination on cell cycle G2 / M arrest influence second experimental results, the column chart from top to bottom are G2 / M, S, G0 / G1 phase respectively;

[0413] Figure 7 is the AST and Nutlin-3 single use or combination on H460 cell in vitro proliferation inhibition results column chart, the three columns from left to right in the figure are the inhibition rate value column of AST single drug, Nutlin-3 single drug and AST+Nutlin-3 combination respectively;

[0414] Figure 8 is the AST and RITA single use or combination on H460 cell in vitro proliferation inhibition results column chart, the three columns from left to right in the figure are the inhibition rate value column of AST single drug, RITA single drug and AST+RITA combination respectively;

[0415] Figure 9 is the AST and Nutlin-3 single use or combination on H460 cell in vitro proliferation inhibition results column chart, the three columns from left to right in the figure are the inhibition rate value column of AST single drug, Nutlin-3 single drug and AST+Nutlin-3 combination respectively, six column charts from left to right correspond to 1-6 group of experimental grouping in the table respectively;

[0416] Figure 10 is a column chart of the results of the inhibition of the proliferation of HPAF-II cells in vitro by AST and RITA alone or in combination, in which the three columns from left to right are the inhibition rate values of AST alone, RITA alone and AST+RITA in combination;

[0417] Figure 11 is the results of the WB detection of the protein lysate of H460 cells in the first experiment of the combined action of AST-3424 and Nutlin-3, in which the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0418] Figure 12 is the results of the WB detection of the protein lysate of H460 cells in the second experiment of the combined action of AST-3424 and Nutlin-3, in which the upper panel is the WB detection protein band photo, and the middle and lower panels are the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0419] Figure 13 is the results of the WB detection of the protein lysate of H460 cells in the third experiment of the combined action of AST-3424 and Nutlin-3, in which the upper panel is the WB detection protein band photo, and the middle and lower panels are the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0420] Figure 14 is the results of the WB detection of the RAD51 protein lysate of H460 cells in the first experiment of the combined action of AST-3424 and Nutlin-3, in which the left panel is the WB detection protein band photo, and the right panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0421] Figure 15 is the results of the WB detection of the RAD51 protein lysate of H460 cells in the second experiment of the combined action of AST-3424 and Nutlin-3, in which the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0422] Figure 16 is the results of the WB detection of the RAD51 protein lysate of H460 cells in the third experiment of the combined action of AST-3424 and Nutlin-3, in which the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0423] Figure 17 is the results of the WB detection of the RAD51 protein lysate of H460 cells in the experiment of the combined action of AST-3424 and Nutlin-3, in which the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0424] Figure 18 is the WB detection result of RAD51 protein in the cell lysate in the experiment of AST-3424, Nutlin-3 and Cycloheximide combined treatment on H460 cells, the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0425] Figure 19 is the change curve of the relative amount of RAD51 after the addition of 4 μM Cycloheximide for different time after the treatment of different drugs: 1% DMSO, 0.1 nM AST-3424, 5 μM Nutlin-3, 0.1 nM AST-3424 + 5 μM Nutlin-3 for 24 hours;

[0426] Figure 20 is the WB detection result of γH2AX protein in the cell lysate in the first experiment of AST-3424, Nutlin-3 single drug and combined treatment on H460 cells, the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0427] Figure 21 is the WB detection result of γH2AX protein in the cell lysate in the second experiment of AST-3424, Nutlin-3 single drug and combined treatment on H460 cells, the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0428] Figure 22 is the WB detection result of γH2AX protein in the cell lysate in the third experiment of AST-3424, Nutlin-3 single drug and combined treatment on H460 cells, the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0429] Figure 23 is the WB detection result of p53, Rad51, MDM2 and p21 proteins in the cell lysate in the experiment of AST, Nutlin-3 single drug and combined treatment on HPAF-II cells, the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0430] Figure 24 is the WB detection result of the cell protein lysate in the experiment of AST-3424, Nutlin-3 single drug and combined treatment on HPAF-II cells, the upper panel is the WB detection protein band photo, and the lower panel is the proportion of the corresponding protein relative to the internal reference protein β-actin;

[0431] Figure 25 is a plot of the in vitro proliferation inhibition rate curve of AST-3424 on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 cells;

[0432] Figure 26 is a plot of the in vitro proliferation inhibition rate curve of AST on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 cells;

[0433] Figure 27 is a plot of the in vitro proliferation inhibition rate curve of Compound A on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 cells;

[0434] Figure 28 is a plot of the in vitro proliferation inhibition rate curve of Compound B on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 cells;

[0435] Figure 29 is a photograph of the WB detection protein bands of the effect of ± Nutlin-3 on H460, H460 P53 KO cells Total P53, MDM2, P21, AKR1C3, Actin proteins;

[0436] Figure 30 is a plot of the proportion of the corresponding protein relative to the internal reference protein β-actin of the WB detection protein of the effect of ± Nutlin-3 on H460, H460 P53 KO cells Total P53, MDM2, P21, AKR1C3 proteins;

[0437] Figure 31 is a plot of the in vitro proliferation inhibition rate curve of Compound C and AST-3424 on NCI-H460 cells under normoxia;

[0438] Figure 32 is a plot of the in vitro proliferation inhibition rate curve of Compound C and AST-3424 on NCI-H460 P53 KO#1 cells under normoxia;

[0439] Figure 33 is a plot of the in vitro proliferation inhibition rate curve of Compound AST-3424 on NCI-H460, NCI-H460 P53 KO#1 cells under normoxia in Example 14;

[0440] Figure 34 is a plot of the in vitro proliferation inhibition rate curve of Thiotepa / Thio-TEPA on NCI-H460, NCI-H460 P53 KO#1 cells under normoxia;

[0441] Figure 35 is a plot of the in vitro proliferation inhibition rate curve of Glufosfamide on NCI-H460, NCI-H460 P53 KO#1 cells under normoxia;

[0442] Figure 36 is a plot of the in vitro proliferation inhibition rate of compound AST-3424 under normoxia on NCI-H460, NCI-H460 P53KO#1 cells in Example XV;

[0443] Figure 37 is a plot of the in vitro proliferation inhibition rate of SG-2057 under normoxia on NCI-H460, NCI-H460 P53KO#1 cells;

[0444] Figure 38 is a plot of the in vitro proliferation inhibition rate of Temozolomide under normoxia on NCI-H460, NCI-H460 P53KO#1 cells;

[0445] Figure 39 is a plot of the in vitro proliferation inhibition rate of Carmustine under normoxia on NCI-H460, NCI-H460 P53KO#1 cells;

[0446] Figure 40 is a plot of the in vitro proliferation inhibition rate of Busulfan under normoxia on NCI-H460, NCI-H460 P53KO#1 cells;

[0447] Figure 41 is a plot of the in vitro proliferation inhibition rate of Duocarmycin TM under normoxia on NCI-H460, NCI-H460 P53KO#1 cells. DETAILED DESCRIPTION

[0448] The application is illustrated in the following by specific examples. Those skilled in the art will appreciate that these examples are intended to be illustrative only and are not intended to limit the scope of the application in any way.

[0449] "Administering" or "administration of" a drug (and grammatical equivalents of this phrase) to a patient means either directly administering (which can be administered by a medical professional to the patient or can be self-administered) and / or indirectly administering, which can be the act of prescribing the drug. For example, a physician who advises a patient to self-administer a drug and / or who provides a prescription to a patient to administer a drug is administering the drug to the patient.

[0450] "Cancer" refers to a potential unlimited growth of leukemia, lymphoma, carcinoma and other malignant tumors (including solid tumors) that can expand locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, cancer of the adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gall bladder, head and neck, kidney, larynx, liver, lung, nerve tissue, pancreas, prostate, parathyroid, skin, stomach, and thyroid. Certain other examples of cancer include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermoid carcinomas, giant cell tumors, glioblastoma multiform, hairy cell tumors, intestinal gangliocytoma, proliferative keratoacanthoma, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary epithelial carcinoma, metastatic skin carcinoma, mycosal neural tumor, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcoma, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumors, small-cell lung tumors, squamous cell carcinoma of both ulcerating and papillary types, seminoma, soft-tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumors, regional skin lesions, reticulocytic sarcoma, and Wilm's tumor.

[0451] "Patient" and "individual" are used interchangeably to refer to a mammal in need of treatment for cancer. Typically, the patient is a human. Typically, the patient is a human diagnosed with cancer. In certain embodiments, "patient" or "individual" can refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used for screening, characterization, and evaluation of drugs and therapies.

[0452] "Solid tumor" refers to a solid tumor including, but not limited to, metastatic tumors in bone, brain, liver, lung, lymph node, pancreas, prostate, skin, and soft tissue (sarcomas).

[0453] A "therapeutically effective amount" of a drug refers to the amount of the drug that, when administered to a patient with cancer, will have the intended therapeutic effect (e.g., mitigation, amelioration, palliation, or elimination of the clinical manifestations of one or more cancers in the patient). The therapeutic effect need not occur by administration of one dose, and can occur only after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations.

[0454] “Treating,”“treatment of,” or“therapy of” a condition or patient means taking steps to obtain a beneficial or desired result, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; diminishment of the extent of disease; delay or slowing of disease progression; amelioration, palliation, or stabilization of the disease state; or other beneficial results. In some cases, treatment of cancer can result in partial response or stable disease.

[0455] “Tumor cell” or“cancer cell” refers to a tumor cell, a cancer cell of any appropriate species (e.g., a mammal, such as a murine, canine, feline, equine, or human).

[0456] “Treating” or“treatment of a patient” means administering, using, or applying to a patient a therapeutically effective amount of a drug associated with the present application.

[0457] “Administering” or“applying” or“using” a drug to a patient means directly administering or applying (which can be administered or applied to a patient by a medical professional or can be self-administered or applied) and / or indirectly administering or applying, which can be the act of prescribing the drug. For example, a physician who instructs a patient to self-administer or apply a drug and / or who provides a prescription for a drug to a patient is administering or applying the drug to the patient.

[0458] “Treating” or“treatment of a patient” means administering, using, or applying to a patient a therapeutically effective amount of a drug associated with the present application.

[0459] The experimental methods in the following examples are all routine methods unless otherwise specified. The medicinal material raw materials, reagent materials, etc. used are all commercially purchased products unless otherwise specified.

[0460] The above description of specific embodiments of the application does not limit the application, and various changes or modifications can be made to the application by those skilled in the art without departing from the spirit of the application, and all such changes or modifications shall fall within the scope of the appended claims of the application.

[0461] Example 1 AST-3424 Phase II Clinical Trial in China

[0462] The clinical trial has a clinical registration number of CTR20191399.

[0463] This trial was approved by the ethics committee of the medical institutions participating in the clinical trial, in accordance with the principles of the Helsinki Declaration, and the informed consent of all subjects was obtained.

[0464] Inclusion criteria

[0465] 1. Male or female, age ≥ 18 years.

[0466] 2. Pathologically histologically confirmed advanced HCC that cannot be controlled by surgical resection or local treatment.

[0467] 3. Previously received standard systemic therapy, including but not limited to sorafenib and / or oxaliplatin-containing systemic chemotherapy, lenvatinib, regorafenib and / or nivolumab, with disease progression, toxicity intolerance or refusal to continue treatment with these drugs.

[0468] 4. At least one measurable lesion meeting the RECIST 1.1 criteria. Lesions previously treated with radiotherapy cannot be used as measurable lesions unless they have clear imaging progression after radiotherapy.

[0469] 5. Pathological wax blocks or sections (including archived pathological wax blocks and sections) for AKR1C3 expression analysis can be provided, and it is confirmed that the liver tumor tissue AKR1C3 expression is strongly positive (the center laboratory immunohistochemical results confirm that the proportion of tumor cells with AKR1C3 staining intensity of 2+ and / or 3+ is ≥ 70%).

[0470] 6. Eastern Cooperative Oncology Group (ECOG) performance status score is 0 or 1.

[0471] 7. Life expectancy ≥ 12 weeks.

[0472] 8. With or without HBV or HCV infection.a. HBV-DNA of subjects with HBV infection must be less than 2,000 IU / ml, and entecavir, tenofovir fumarate, and propofol tenofovir fumarate must be used for antiviral treatment according to the national guidelines for the prevention and treatment of chronic hepatitis B. Treatment must be maintained during the study and continued for 6 months after the last dose.b. Subjects with HCV infection (presence of detectable HCV-RNA or anti-HCV antibody) can be treated according to medical practice.

[0473] 9. Child-Pugh score ≤ 6.

[0474] 10. No history of hepatic encephalopathy.

[0475] 11. All toxicities of previous anticancer therapy (except alopecia, fatigue or peripheral neuropathy) must have returned to grade 1 or baseline level (NCI CTCAE version 5) before starting the use of the study drug.

[0476] 12. Laboratory tests must meet the following criteria. Within 14 days prior to the screening period, indicators cannot be corrected to meet the inclusion criteria through blood transfusions, hematopoietic stimulating factors, or albumin infusions. a. Hemoglobin ≥ 90 g / L; b. Platelet count ≥ 80 × 10⁻⁶. 9 c. Absolute neutrophil count (ANC) ≥ 1.5 × 10⁹ / L; 9 / L; d. Serum total bilirubin ≤3mg / dL; e. ALT and AST ≤5.0×ULN; f. International normalized ratio (INR) ≤2.3 or prothrombin time prolonged ≤6 seconds; g. Albumin ≥29g / L; h. Creatinine clearance >50mL / min as determined by the Cockcroft-Gault equation.

[0477] 13. No history of alcohol abuse, drug use, or substance abuse within the past year.

[0478] 14. Female patients of childbearing age should have a negative pregnancy test result within 5 days prior to the start of treatment and should not be breastfeeding (a positive urine pregnancy test result needs to be confirmed by a serum pregnancy test).

[0479] 15. Female and male participants of childbearing age must agree to use effective contraception (e.g., surgical sterilization or condoms or diaphragm contraception combined with spermicide gel or intrauterine device [IUD], etc.) with their partners from the start of the study until 6 months after the last dose.

[0480] 16. Participants must voluntarily participate in this study, fully understand the associated risks, demonstrate good compliance, and sign an informed consent form. Participants may also sign a Future Biomedical Research (FBR) consent form. However, participants who do not participate in the FBR may still participate in the main trial.

[0481] Exclusion criteria:

[0482] 1. Untreated active central nervous system (CNS) metastases or leptomeningeal disease. Subjects with adequately treated CNS metastases are eligible to participate in the study if their CNS metastases are confirmed to be stable for at least 4 weeks by clinical examination and brain imaging (MRI or CT) during the screening period.

[0483] 2.2 years of history of other malignant tumors, excluding adequately treated basal cell carcinoma, carcinoma in situ at other sites, or natural disease history, and other tumors whose treatment would not interfere with the safety or efficacy assessment of the current study.

[0484] 3. Underwent major surgery (excluding diagnostic surgery) within 4 weeks prior to the first dose.

[0485] 4. Received radiotherapy, surgical procedure, chemotherapy, immunotherapy, biological therapy, targeted therapy or hormonal therapy for cancer within 4 weeks prior to first dose (6-week washout period for nitrosoureas or mytomycin C therapy; 2-week washout period for oral fluorouracil; 2-week washout period for small molecule targeted therapy).

[0486] 5. Participated in an investigational drug (diagnostic or therapeutic) or device study within 4 weeks prior to first dose.

[0487] 6. Concomitant use of strong CYP3A4 inhibitors or inducers during the study.

[0488] 7. Uncontrolled, active bacterial, viral, or fungal infection requiring systemic treatment.

[0489] 8. Known infection with human immunodeficiency virus (HIV) or positive for syphilis.

[0490] 9. Ascites of clinical significance, defined as found by physical examination and requiring control by paracentesis or increasing medical intervention to maintain symptoms (patients with ascites found only by imaging can be enrolled).

[0491] 10. Women who are pregnant, breastfeeding, or planning to become pregnant.

[0492] 11. Concomitant illness or symptoms that can interfere with study conduct or that the investigator considers to pose an undue risk to the patient. This includes but is not limited to a history of gastrointestinal bleeding or higher risk of bleeding within three months, active peptic ulcer or gastritis, changes in mental status or psychiatric abnormalities that can interfere with the patient’s understanding of the informed consent.

[0493] 12. Prior hypersensitivity to ethanol, propylene glycol.

[0494] 13. Subjects who are unwilling or unable to comply with the study protocol for any reason.

[0495] Study drug:

[0496] AST-3424 injection concentrated solution: manufactured by a pharmaceutical enterprise entrusted by Shenzhen Aixinda Wei Medicine Technology Co., Ltd., specification 1 mL: 10 mg; containing 0.75 ml of ethanol, 0.25 ml of propylene glycol.

[0497] Dosing regimen:

[0498] Each cycle of 21 days, each dose of 6 mg / m 2 The longest will be allowed to receive 34 cycles of treatment.

[0499] Specific administration operation:

[0500] Before administration, 0.1 ml of 5% sodium bicarbonate injection is added to 100 ml of commercially available sterile 5% dextrose injection in water (D5W) in a DEHP-free (di-(2-ethylhexyl) phthalate) intravenous infusion bag. The required number of milliliters of AST-3424 injection concentrated solution (to the nearest 0.01 ml) is added to the D5W bag after pH adjustment to prepare AST-3424 injection for intravenous infusion administration.

[0501] The solutes of the intravenous injection aqueous solution consist of AST-3424 drug substance, dextrose, ethanol, propylene glycol and pH adjuster sodium bicarbonate, wherein the concentration of AST-3424 drug substance is 0.004-0.94 mg / ml, the pH is 7.4, the content of dextrose is 4.5-5.0% by mass, and it is an isotonic solution.

[0502] If the patient is not suitable for injection of dextrose, normal saline is used instead:

[0503] Before administration, 0.1 ml of 5% sodium bicarbonate injection is added to 100 ml of commercially available sterile 0.9% injection of normal saline in a DEHP-free (di-(2-ethylhexyl) phthalate) intravenous infusion bag. The required number of milliliters of AST-3424 injection concentrated solution (to the nearest 0.01 ml) is added to the normal saline bag after pH adjustment to prepare AST-3424 injection for intravenous infusion administration.

[0504] The solutes of the intravenous injection aqueous solution consist of AST-3424 drug substance, sodium chloride, ethanol, propylene glycol and pH adjuster sodium bicarbonate, wherein the concentration of AST-3424 drug substance 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 it is an isotonic solution.

[0505] The accurate calculation method of the required number of milliliters of AST-3424 injection concentrated solution is as follows:

[0506] For a patient with a height of 175 cm and a weight of 75 kg, the corresponding equivalent body surface area BSA (m 2 ) = ([height (cm) x weight (kg)] / 3600) 1 / 2 = 1.90, the corresponding dose is 1.90 x 6.0 = 11.40 mg, and 11.40 ÷ 10 x 1 = 1.14 ml of the above specification AST-3424 concentrated injection should be extracted.

[0507] The prepared intravenous AST-3424 injection should be injected within 8 hours.

[0508] Clinical evaluation

[0509] The effectiveness evaluation includes clinical efficacy evaluation.

[0510] The clinical efficacy evaluation standard adopts the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. The measurement method adopts MRI / CT to evaluate the lesions, and the same evaluation method should be used for the same lesion during the study. The subjects must have measurable tumor lesions at baseline.

[0511] The efficacy evaluation indexes include complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD).

[0512] Complete remission (CR): all target lesions disappear, and the short diameter of all pathological lymph nodes (including target nodes and non-target nodes) must be reduced to <10 mm.

[0513] Partial remission (PR): the sum of the diameters of the target lesions is reduced by at least 30% compared with the baseline level.

[0514] Progressive disease (PD): the minimum value of the sum of the diameters of all measured target lesions during the entire experimental study is taken as the reference, and the diameter and relative increase is at least 20% (if the baseline measurement is the minimum, the baseline value is taken as the reference); in addition, the absolute value of the diameter and must increase by at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0515] Stable disease (SD): the degree of target lesion reduction does not reach PR, and the degree of increase also does not reach the PD level, which is between the two, and the minimum value of the sum of the diameters can be taken as the reference during the study.

[0516] Study endpoints

[0517] According to the objective response rate (ORR) of the subjects, the disease control rate (DCR), the duration of remission (DOR), and the progression-free survival (PFS), the efficacy of AST-3424 monotherapy for HCC and other malignant tumors is preliminarily evaluated.

[0518] Objective response rate (ORR): refers to the percentage of cases that achieve complete remission (CR) and partial remission (PR) after treatment in the total number of evaluable cases.

[0519] Disease control rate (DCR): refers to the percentage of cases that achieve complete remission (CR), partial remission (PR), and stable disease (SD) in the patients with evaluable efficacy.

[0520] Trial results

[0521] Eighteen liver cancer subjects were enrolled in the study, of which eight subjects completed clinical efficacy evaluation, one subject was PR, accounting for 12.5%; five subjects were SD, accounting for 62.5%; two subjects were PD, accounting for 25%, and the overall DCR was 75%, and the ORR was 12.5%. In particular, eight of them underwent p53 gene mutation or defect detection, of which negative (-) means no mutation or defect is detected, positive (+) means mutation or defect is detected (may or may not affect protein expression), as shown in Table 1.

[0522] Table 1: AST-3424 phase II clinical part case data as of July 21, 2023

[0523] Further analysis of the relationship between efficacy and p53 gene mutation negative / positive:

[0524] Of the four cases with negative (-), one was PR, two were SD and the lesions were reduced, and one was PD, i.e. the DCR of this subgroup of cases was 75% and the ORR was 25%.

[0525] Of the four cases with positive (+), three were SD and the lesions were enlarged, and one was PD, i.e. the DCR of this subgroup of cases was 75% and all three were enlarged, and the ORR was 0%.

[0526] In summary, after receiving the same dose level of AST-3424, the efficacy evaluation of the p53 gene mutation negative / positive subgroup showed a significant difference: the ORR of the p53 gene mutation or defect negative (-) subgroup of liver cancer patients was 25%, while the corresponding positive (+) subgroup was 0; further, the DCR of the two subgroups was 75% (corresponding to 3 out of 4 cases), but in fact, of the 3 cases of p53 gene mutation or defect negative (-) liver cancer patients, 2 cases of SD efficacy evaluation patients had reduced lesions (subsequent continued administration of treatment had a high probability of turning into PR or even CR), while the 3 cases of SD efficacy evaluation patients in the p53 gene mutation or defect positive (+) subgroup of liver cancer patients had increased lesions.

[0527] Therefore, for the current clinical results, those skilled in the art have reason to believe that AST-3424 has a better treatment effect on p53 gene mutation or defect negative (-) cancer, tumor patients than on p53 gene mutation or defect positive (+) patients, and therefore the applicant speculates that AST-3424 will have better treatment effect on p53 gene mutation or defect negative (-) tumor, cancer patients, i.e. p53 gene mutation or defect negative (-) tumor, cancer patients receiving AST-3424 treatment will have more obvious clinical benefits.

[0528] As of September 01, 2023, the above ongoing clinical trial progresses as follows.

[0529] Trial Results

[0530] A total of 20 liver cancer subjects were enrolled, of which 18 subjects completed clinical efficacy evaluation, of which 1 subject was PR, 10 subjects were SD, and 7 subjects were PD. The final ORR was 5.6% (1 / 18), and the DCR was 61.1% (11 / 18).

[0531] In particular, of the 10 cases, p53 gene mutation or defect detection was performed, of which negative (-) indicates that no mutation or defect is detected, and positive (+) indicates that a mutation or defect is detected (which may or may not affect protein expression), as shown in the following Table 6.

[0532] Table 6: AST-3424 Phase II Clinical Part Case Data as of September 01, 2023

[0533] PFS, as of September 01, 2023, PFS is an estimated value, and each month is calculated according to 30.437 days.

[0534] Further analysis of the relationship between efficacy and p53 gene mutation negative / positive:

[0535] Of the 5 cases with negative (-), 1 was PR, 3 were SD, and 1 was PD, i.e. the DCR of this subgroup of cases was 80% (4 / 5), the ORR was 20% (1 / 5), and the average PFS was 4.1 months.

[0536] Of the 5 cases with positive (+), 3 were SD and the lesions increased, and 2 were PD, i.e. the DCR of this subgroup of cases was 60% (3 / 5) and all 3 were lesion enlargement, the ORR was 0%, and the average PFS was 1.756 months.

[0537] In summary, after receiving the same dose level of AST-3424, the efficacy evaluation of the p53 gene mutation negative / positive subgroup showed a significant difference: in the subgroup of liver cancer patients with negative (-) p53 gene mutation or defect, the ORR was 20%, the DCR was 80%, and the 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 better treatment effect.

[0538] As of March 11, 2024, the above ongoing clinical trial progresses as follows.

[0539] Trial Results

[0540] As of March 11, 2024, 30 subjects have been enrolled: 5 are in the treatment group, 25 have been discharged (1 withdrew informed consent, 10 have died, 14 are in survival follow-up), the current longest PFS is > 11.5 months; the longest OS is > 17.6 months. There are 11 subjects enrolled before the end of January 2023, and 6 subjects with OS greater than 12 months. Details are shown in Table 7 below.

[0541] Table 7: AST-3424 - hepatocellular carcinoma efficacy and gene mutation relationship clinical data as of March 11, 2024

[0542] Among the 26 enrolled subjects with efficacy evaluation results, 20 had p53 gene mutation detection results, and the efficacy was observed according to the p53 detection results:

[0543] Wild type without mutation (WT), i.e. the p53 gene mutation detection result is negative (-);

[0544] Unclear significance (VUS), i.e. the p53 gene mutation detection result is positive (+), and it is not clear whether it affects protein expression;

[0545] Mutations that may affect protein function (MUT), i.e. the p53 gene mutation detection result is positive (+), which may affect protein expression, then classified into three groups for statistical analysis, the results are shown in Tables 8, 9 and 10.

[0546] Table 8: Efficacy data of 8 patients with p53 mutation positive (MUT)

[0547] The statistical results show that among the 8 cases, 2 have died, 6 are in survival follow-up, the current longest PFS is 1.6 months, the longest OS is > 14.0 months, the average PFS is 1.4 months, and the average OS is greater than 9.3 months. Among the 8 cases with p53 gene mutation positive (+), which may affect protein expression, 0 cases had PR, 2 cases had SD, and 6 cases had PD, i.e. the DCR in this subgroup of case population was 25% (2 / 8), and the ORR was 0% (0 / 5).

[0548] Table 9: Efficacy data of 9 patients with p53 mutation negative (WT)

[0549] The statistical results show that among the 9 cases, 4 have died, 5 are in survival follow-up, the current longest PFS is greater than 11.5 months, the longest OS is > 13.8 months, the average PFS is greater than 4.1 months, and the average OS is greater than 7.7 months. Among the 9 cases with p53 gene mutation negative (-), 2 had PR, 5 had SD, and 2 had PD, i.e. the DCR in this subgroup of case population was 77.8% (7 / 9), and the ORR was 22.2% (2 / 9).

[0550] Table 10: Efficacy data of 3 patients with P53 mutation positive, uncertain whether to affect protein expression (VUS)

[0551] As of the last survival follow-up data on June 26, 2025, the above-mentioned clinical trial has progressed as follows.

[0552] Trial results

[0553] As of June 26, 2025, 30 subjects have been enrolled: 30 have been out of the group (24 have died, 6 are in survival follow-up), the current longest PFS is 14.5 months; the longest OS is >30.2 months. Details are shown in Table 18 below.

[0554] Table 18: AST-3424-hepatocellular carcinoma efficacy and gene mutation relationship clinical data as of June 26, 2025

[0555] Among the 28 enrolled subjects with efficacy evaluation results, 22 had p53 gene mutation detection results, and the efficacy was observed according to the p53 detection results:

[0556] No mutation wild type (WT), that is, the p53 gene mutation detection result is negative (-);

[0557] Mutation of unknown significance (VUS), that is, the p53 gene mutation detection result is positive (+), and it is not clear whether it affects protein expression;

[0558] Mutations that may affect protein function (MUT), that is, the p53 gene mutation detection result is positive (+), which may affect protein expression, and then classified into three groups for statistical analysis, the results are shown in Tables 19, 20, and 21 below.

[0559] Table 19: Efficacy data of 9 patients with P53 mutation positive (MUT)

[0560] The statistical results show that among the 9 cases, 8 have died, 1 is in survival follow-up, the current longest PFS is 3.2 months, the longest OS is >28.1 months, the average PFS is 1.6 months, and the average OS is greater than 10.8 months. Among the 9 cases with p53 gene mutation positive (+), which may affect protein expression, 0 cases had PR, 2 cases had SD, and 7 cases had PD, i.e., the DCR in this subgroup of cases was 22% (2 / 9), and the ORR was 0% (0 / 9).

[0561] Table 20: Efficacy data of 9 patients with P53 mutation negative (WT)

[0562] Statistical results show that 4 out of 9 cases have died, 5 cases are alive and under follow-up, the current longest PFS is 14.5 months, the longest OS is > 26.6 months, the average PFS is 4.9 months, and the average OS is more than 14.5 months. Among the 9 cases with negative (-) p53 gene mutation, 2 cases were PR, 5 cases were SD, and 2 cases were PD, i.e. the DCR of this subgroup of case population was 77.8% (7 / 9), and the ORR was 22.2% (2 / 9).

[0563] Table 21: 4 cases of P53 mutation positive, uncertain whether to affect protein expression (VUS) patient efficacy data

[0564] Therefore, for the current clinical results, those skilled in the art have reason to believe that AST-3424 has better therapeutic effect on patients with p53 gene mutation or defect negative (-) cancer, tumor than on patients with p53 gene mutation or defect positive (+). Therefore, the applicant speculates that AST-3424 will have better therapeutic effect on patients with p53 gene mutation or defect negative (-) tumor, cancer, i.e. patients with p53 gene mutation or defect negative (-) tumor, cancer will have more obvious clinical benefit after receiving AST-3424 treatment.

[0565] The phase results of the two clinical trials in Example I seem to confirm that AST-3424, a DNA alkylating agent prodrug, has better efficacy on patients with p53 gene mutation negative, i.e. normal expression of p53 protein. In order to further confirm the above fact, the applicant carried out further experimental demonstration.

[0566] The above compounds are synthesized and prepared by the applicant himself if not specified, by referring to the corresponding patent application.

[0567] Further literature search shows that:

[0568] The main function of p53 protein is to monitor DNA damage in cells, which can induce cell cycle arrest, DNA damage repair, and promote tumor cell apoptosis, and ultimately inhibit tumor growth (Marei, H. E., 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).

[0569] And p53 mutation / deficiency will lead to a series of reactions and manifestations:

[0570] p53 mutations / defects can impair the apoptosis pathway leading to subsequent drug resistance (Sturm I, Bosanquet A G, Hermann S, et al. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy [J]. Cell Death & Differentiation, 2003, 10(4): 477. DOI: 10.1038 / sj.cdd.4401194.).

[0571] p53 mutations / defects impair cell cycle arrest, allowing damaged cells to continue to divide and survive, leading to drug resistance (Zhao, D., Tahaney, W. M., 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).

[0572] p53 mutations / defects lead to improper activation of DNA repair pathways, leading to 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.

[0573] p53 mutation / deficiency accelerates and increases the efficiency of generation of cancer stem cells (CSCs) with drug resistance characteristics, resulting in 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).

[0574] p53 gain-of-function mutations can promote tumor malignancy further leading to entering drug resistance and treatment refractory state (Alvarado-Ortiz E, de la Cruz-Lopez KG, Becerril-Rico J, Sarabia-Sanchez MA, Ortiz-Sanchez E, Garcia-Carranca A. Mutant p53 Gain-of-Function: Role in Cancer Development, Progression, and Therapeutic Approaches. Front Cell Dev Biol. 2021, 8:607670. Published 2021 Feb 11. doi: 10.3389 / fcell.2020.607670).

[0575] That is, p53 plays an important role in DNA damage repair, apoptosis process, once the p53 gene mutation or defect, will lead to abnormal p53 protein, and then can not play the above role, so that tumor or cancer cell drug resistance.

[0576] AST-3424 / AST and other DNA alkylating agent prodrug compounds release DNA alkylating agents (such as AST-2660, Br-IPM, etc.) to crosslink with DNA in vivo, causing DNA damage and breakage, and then cell death; p53 mutation / deficiency will inhibit the ability of p53 protein to promote tumor cell apoptosis after DNA damage. Accordingly, it can be inferred that cells with negative TP53 gene mutation and normal expression of p53 protein can promote the apoptosis process of DNA-damaged tumor cells after p53 protein is activated or upregulated, and a series of experimental phenomena of enhanced cell apoptosis and DNA damage toxicity can be observed.

[0577] To this end, the following experiments were performed using p53 wild type H460 cells, A549 cells, and p53 mutant HPAF-II cells, and p53 knock-out cells. Unless otherwise specified, the H460 cells in the following examples were NCI-H460 cells.

[0578] Example 2 Cell proliferation and cell colony formation experiments in vitro

[0579] Nutlin-3 is a small molecule MDM2-p53 inhibitor, which indirectly activates p53 function by inhibiting the interaction between MDM2 and p53. Therefore, the following experiments were performed to determine the effect of AST-3424 / AST on cell proliferation and cell colony formation after activation / upregulation of p53 function by Nutlin-3.

[0580] The CAS number of Nutlin-3 is 548472-68-0.

[0581] H460 is a p53 wild type cell, i.e., p53 gene mutation negative, and p53 protein is normally expressed.

[0582] HPAF-II is a p53 mutant cell (p.P151S, deleterious), i.e., p53 gene mutation positive, and p53 protein is abnormally expressed.

[0583] Effect of compounds on H460 cell proliferation in vitro under normoxia

[0584] Summary of the experimental process

[0585] 1) H460 cell suspension was added to a 96-well plate at 100 μL per well, and the cell density was 2000 / well.

[0586] 2) The cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0587] 3) Compound treatment

[0588] Single drug: 99.5 μL of growth medium was added to each well after the cells were plated for 24 hours. 0.5 μL of the test compound at different concentrations was added, gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator.

[0589] Combined drug: 99 μL of growth medium was added to each well after the cells were plated for 24 hours. 0.5 μL of the combination compound Nutlin-3 was added, gently shaken to ensure uniform mixing, and then placed in the incubator for 2 hours. 0.5 μL of the test compound at different concentrations was added, gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator.

[0590] 4) Place the cell plate in the incubator for 72 hours.

[0591] 5) Place the cell plate at room temperature for 30 minutes to equilibrate. Discard 100 μL of medium per well.

[0592] 6) Add 25 μL of CTG reagent per well. Place on a shaker for 2 minutes. Incubate at room temperature for 30 minutes in the dark.

[0593] 7) Read the chemiluminescent signal using a multi-function microplate reader. Read for 1000 milliseconds.

[0594] 8) Calculate IC50 using Graph Pad Prism 5 software. 50 .

[0595] The experimental results are shown in Figure 1.

[0596] The experiment shows that the addition of Nutlin-3, which indirectly activates p53 function, before the addition of AST-3424 can significantly improve the proliferation inhibition rate of AST-3424.

[0597] Further experiments on the combination of AST-3424 with different dosing sequences were carried out, and the dosing schemes were set in the above experimental process.

[0598] Single drug: Add 5 μL of single drug AST-3424, Nutlin-3, and 5 μL of medium of different concentrations (400 times) to each well to treat the cells (DMSO 0.25% and 0.50%);

[0599] Combined drug: Treat the cells with 5 μL of AST-3424 and Nutlin-3 in different dosing sequences. AST-3424 is pretreated for 2 hours, and Nutlin-3 is treated for 6 hours. Nutlin-3 is pretreated for 2 hours, and AST-3424 is treated for 6 hours. AST-3424 and Nutlin-3 are treated for 6 hours.

[0600] The experimental results are shown in Figure 2.

[0601] The results of the cell proliferation experiment (3 days) show that the addition of Nutlin-3, which indirectly activates p53 function, can significantly improve the proliferation inhibition rate of AST-3424, and is not affected by the order of adding Nutlin-3.

[0602] Effect of AST-3424 ± Nutlin-3 with different dosing sequences on the clonogenicity of H460 / HPAF-II cells

[0603] Experimental process overview:

[0604] H460: 1000 cells / 2ml / well / 6-well plate

[0605] HPAF-II: 3000 cells / 2ml / well / 6-well plate

[0606] 1) H460 cell suspension was added to 6-well plates, 2ml per well, cell density was 1000 / well respectively; HPAF-II cell suspension was added to 6-well plates, 2ml per well, cell density was 3000 / well respectively.

[0607] 2) 1990 μL medium was inoculated with cells, and the cells were cultured in a 37℃, 5% CO2 incubator overnight.

[0608] 3) Compound treatment

[0609] Single drug, 5 μL different concentrations of single drug AST-3424, Nutlin-3, and 5 μL medium were added to each well to treat the cells (DMSO 0.25%) for 6h;

[0610] Combined drug, 5 μL AST-3424, Nutlin-3 of different dosing sequences were used to treat the cells: AST-3424 was pretreated for 2h, and Nutlin-3 was co-treated for 6h; Nutlin-3 was pretreated for 2h, and AST-3424 was co-treated for 6h; AST-3424 and Nutlin-3 were co-treated for 6h.

[0611] 4) After 6h of co-treatment, the compounds were washed away twice, and 5mL medium was added to each well.

[0612] 5) Cultured for 7 days (the medium can be replaced every 3 days, and the observation can be ended according to the specific proliferation of the cells).

[0613] 6) When the majority of the cell colonies reached about 50 cells under the condition of 0nM (0.5% DMSO), staining was performed. The culture medium was aspirated, and 0.5% [w / v] crystal violet was used for fixation and staining for 40 minutes. The plate was washed twice with tap water, and dried before counting

[0614] 7) Cell clone counting, the number of colonies with more than 50 cells was counted.

[0615] The experimental results are shown in Figure 3.

[0616] Compared with each single drug, the combination of Nutlin-3 and AST-3424 significantly inhibited the clone formation of H460 and HPAF-II cells, and the combination drug had an additive effect.

[0617] Among the three groups of H460 cell double drug treatment, the clone number of the group with AST-3424 added first was less than that of the other two groups.

[0618] Different drug addition sequences have no obvious influence on the three groups of HP AF-II cells treated with the two drugs.

[0619] In H460 cells, the clone numbers of the three drug combination groups with different drug addition sequences were compared with that of the DMSO group. The percentage of the clone number of the drug combination group compared with that of the DMSO group was used to represent the ability of the drug combination to inhibit cell clone formation. The smaller the percentage, the stronger the ability of the drug to inhibit cell clone formation. The percentages of the clone number of the three drug combination groups compared with that of the DMSO group were 11.12%, 23.03% and 21.45%, respectively. In the same way, the data of HP AF-II cells were analyzed, and the percentages of the clone number of the three drug combination groups compared with that of the DMSO group were 24.14%, 30.41% and 40.30%, respectively.

[0620] It can be inferred that, compared with the p53 mutant HP AF-II cells, the clone inhibition effect of Nutlin-3 on the p53 wild-type H460 cells is more obvious.

[0621] Experimental conclusion: The above experimental results show that Nutlin-3 can indeed enhance the in vitro cytotoxicity of AST-3424 / AST on cancer cells, and the in vitro cytotoxicity on p53 wild-type H460 cells is enhanced more greatly.

[0622] Example Three Influence of Nutlin-3 combined with AST-3424 on cell apoptosis and cell cycle G2 / M arrest

[0623] Influence of AST-3424 combined with Nutlin-3 on the apoptosis process of H460 cells

[0624] Experimental process overview:

[0625] 1) H460 cell suspension was added to a 96-well white plate, 99 μL per well, and the cell density was 15000 per well. At the same time, a 96-well transparent plate with the same cell density was used for detection and photographing observation.

[0626] 2) The cells were cultured in a 37℃, 5% CO2 incubator overnight.

[0627] 3) Compound treatment

[0628] Single drug: After the cells were plated for 24 hours, 0.5 μL of culture solution was added to each well. According to the scheme, 0.5 μL of the corresponding concentration of the compound was added to the designated cell well, and the mixture was gently shaken to ensure uniform mixing, and then placed in a 37℃, 5% CO2 incubator.

[0629] Combination: After 24 hours of cell plating, 0.5 μL of the corresponding concentration of compound Nutlin-3 was added to the designated wells, and the plate was gently shaken to ensure uniform mixing. The plate was then incubated at 37°C in a 5% CO2 incubator for 2 hours, after which 0.5 μL of AST-3424 was added to the designated wells.

[0630] 4) Place the cell plate in the incubator for 24 hours.

[0631] 5) Place the cell plate to be tested at room temperature for 5 minutes.

[0632] 6) Preparation 3 / 7 reagent, and the prepared reagent can be stored at 4°C for 3 days. 3 / 7 reagent, and the prepared reagent can be stored at 4°C for 3 days.

[0633] 7) Add 100 μL 3 / 7 reagent to each well of the medium containing 100 μL of blank (cell-free well), negative control cells, or treated cells. Due to the sensitivity of the analysis, be careful not to touch the tip of the pipette to the well containing the sample to avoid cross-contamination. Cover the plate with a plate sealer or lid.

[0634] 8) Use a shaker to gently mix the reagent solution at a speed of 300-500 rpm for 30 seconds. Incubate at room temperature for 1 hour in the dark.

[0635] 9) Measure the luminescence of each sample in a plate reader luminometer. Calculate the luminescence values of each group and analyze them in a column chart in Relative Luminescence Units (RLU). The results are shown in Figure 4.

[0636] Compared to each single drug, the effective concentration of AST-3424 (1 nM) and Nutlin-3 combination can significantly increase the content of Caspase3 / 7, a marker of cancer cell apoptosis, i.e., the combination of the two can increase cell apoptosis, and the marker content after treatment with different doses of Nutlin-3 is different, which also indicates that the above-mentioned increase in apoptosis is dose-dependent with Nutlin-3; that is, as the concentration of Nutlin-3 increases, the content of the cell apoptosis detection marker after the combination of AST-3424 and Nutlin-3 also increases, which also indicates that the above-mentioned increase in apoptosis is dose-dependent with Nutlin-3.

[0637] That is, after the addition of Nutlin-3, which indirectly activates the function of p53 protein, the cell apoptosis effect of AST-3424 is enhanced.

[0638] Effect of AST-3424 and Nutlin-3 alone or in combination on cell cycle G2 / M arrest

[0639] The whole cell replication cycle can be described as G0 / G1, S, G2 / M phase, in the study of tumor pathology, usually with S phase cell ratio as the index of tumor proliferation state.

[0640] Experimental process overview:

[0641] 1) H460 / HPAF-II cell suspension was added to 24-well plates, the cell density was 100000 / well, 995 μL of medium was added to each well.

[0642] 2) Cells were cultured in a 37℃, 5% CO2 incubator overnight.

[0643] 3) Compound treatment

[0644] After the cells were plated for 24 hours, each cell line experiment was divided into groups according to the scheme:

[0645] Single drug: after the cells were plated for 24 hours, 5 μL of different concentrations of test compounds were added: 1% DMSO, 5 μM Nutlin-3, 0.1 nM AST-3424, gently shake to ensure uniform mixing, then put into a 37℃, 5% CO2 incubator;

[0646] Combined drug:

[0647] AST-3424 was treated 2 hours in advance, then co-treated with Nutlin-3 for 24 hours;

[0648] Nutlin-3 was treated 2 hours in advance, then co-treated with AST-3424 for 24 hours;

[0649] AST-3424 was co-treated with Nutlin-3 for 24 hours.

[0650] 4) Digestion, 4℃ 1000g centrifugation, 1 time washing with pre-cooled PBS, 4℃ 1000g centrifugation, 1 mL of pre-cooled 70% ethanol-20℃ fixation overnight.

[0651] 5) 4℃ 3000g centrifugation, 1 time washing with pre-cooled PBS (phosphate buffer), 4℃ 3000g centrifugation, PI staining solution was prepared, 37℃ dark staining for 30 minutes, dark ice bath storage, flow cytometry detection was used on the same day and flowjo was used to analyze the proportion of cells in different cell cycles, the results are shown in Figure 5.

[0652] The experimental results show that for H460 cells with wild type p53, 0.1 nM AST-3424 and 5 μM Nutlin-3 single drug treatment does not significantly change the cell cycle; and the combination of 0.1 nM AST-3424 and 5 μM Nutlin-3 significantly reduces the G0 / G1 phase and increases the G2 / M phase; the S phase of the other two groups is slightly reduced. The cell cycle of HPAF-II cells with p53 pathogenic mutations is less affected.

[0653] A large number of studies have shown that the arrest of the cell cycle in the G2 / M phase can induce apoptosis of the cell.

[0654] In order to further study the effects of different concentrations of AST-3424 and Nutlin-3 single drug and combination and different drug addition sequences on the cycle of H460 / A549 cells (p53 wild type, normal protein expression), a second experiment is further carried out.

[0655] The rest of the operation is similar to the above experiment, and the compound treatment is as follows:

[0656] Single drug

[0657] For H460 cell lines, 1% DMSO, 5 μM Nutlin-3 is added for 22 hours, 0.03 nM AST-3424 is added for 24 hours, 0.1 nM AST-3424 is added for 24 hours, 0.3 nM AST-3424 is added for 24 hours, and 1 nM AST-3424 is added for 24 hours;

[0658] For A549 cell lines, 1% DMSO, 5 μM Nutlin-3 is added for 22 hours, 0.3 nM AST-3424 is added for 24 hours, 1 nM AST-3424 is added for 24 hours, 9 nM AST-3424 is added for 24 hours, and 9 nM AST-3424 is added for 24 hours.

[0659] Combined drug

[0660] For H460 cell lines,

[0661] 0.03 nM AST-3424 is added for 2 hours, and then 5 μM Nutlin-3 is added for 22 hours;

[0662] 0.1 nM AST-3424 is added for 2 hours, and then 5 μM Nutlin-3 is added for 22 hours;

[0663] Add 0.3 nM AST-3424 pre-treatment for 2 hours, then add 5 μM Nutlin-3, co-treatment for 22 hours;

[0664] Add 1 nM AST-3424 pre-treatment for 2 hours, then add 5 μM Nutlin-3, co-treatment for 22 hours.

[0665] For A549 cell line,

[0666] Add 0.3 nM AST-3424 pre-treatment for 2 hours, then add 5 μM Nutlin-3, co-treatment for 22 hours;

[0667] Add 1 nM AST-3424 pre-treatment for 2 hours, then add 5 μM Nutlin-3, co-treatment for 22 hours.

[0668] Add 3 nM AST-3424 pre-treatment for 2 hours, then add 5 μM Nutlin-3, co-treatment for 22 hours.

[0669] Add 9 nM AST-3424 pre-treatment for 2 hours, then add 5 μM Nutlin-3, co-treatment for 22 hours.

[0670] The results are shown in Figure 6.

[0671] Further experimental results show that AST-3424 causes concentration-dependent G2 / M cell cycle arrest in p53 wild-type cancer cells (H460 and A549).

[0672] AST-3424 combined with Nutlin-3 significantly increases G2 / M phase arrest and reduces G0 / G1 phase in p53 wild-type cancer cells (H460 and A549), and shows AST-3424 concentration dependence.

[0673] AST-3424 combined with Nutlin-3 (AST-3424 pre-treatment for 2 hours) increases G2 / M phase arrest, which is consistent with the results of the same drug administration sequence in the preliminary experiment described above.

[0674] 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), resulting in G2 / M cell cycle arrest, and showing AST-3424 concentration dependence. However, it has no effect on the cell cycle of p53 pathogenic mutant cell line HPAF-II cells; that is, Nutlin-3, which indirectly activates p53 function, and AST-3424 can regulate the cell cycle of p53 wild-type cancer cells (H460 and A549), enhance G2 / M cell cycle arrest, and show AST-3424 concentration dependence; however, it has no effect on the cell cycle of p53 pathogenic mutant cell line HPAF-II cells.

[0675] According to Examples Two and Three, p53 protein has a decisive influence on the pharmacodynamics of AST-3424 / AST drugs, and AST-3424 / AST and other DNA alkylating agent drugs have a stronger proliferation inhibitory effect on cells with normal p53 protein and negative p53 gene mutation, and the mechanism of action includes cell cycle G2 / M arrest and cell clone inhibition.

[0676] In order to further confirm the above conclusion, p53-MDM2 inhibitor RITA (which is also a p53-HDM-2 inhibitor) was selected to activate p53 function, and further in vitro cytotoxicity experiments were carried out.

[0677] The CAS number of RITA is 213261-59-7.

[0678] Example Four: Effects of AST and Nutlin-3, RITA single drug or combination on cytotoxicity of HPAF-II / H460 cells

[0679] In vitro proliferation inhibition experiment

[0680] Summary of the experimental process

[0681] 1) H460, HPAF-II cell suspension was added to a 96-well plate, 100 μL per well, and the cell density was 2000 / well and 20000 / well, respectively.

[0682] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0683] 3) Compound treatment

[0684] Combined use: 99 μL of growth medium was added to each well after 24 hours of cell plating. 0.5 μL of Nutlin-3 or RITA was added to each well, gently shaken to ensure uniform mixing, and placed in a 37°C, 5% CO2 incubator for 2 hours. Then 0.5 μL of different concentrations of test compounds was added, gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator.

[0685] Single agent: 99.5 μL of growth medium was added to each well after the cells were plated for 24 hours. 0.5 μL of test compound at different concentrations was added, gently shaken to ensure even mixing, and then placed in a 37°C, 5% CO2 incubator.

[0686] 4) The cell plate was placed in the incubator for 72 hours.

[0687] 5) The cell plate was placed at room temperature for 30 minutes to equilibrate, and 100 μL of medium was discarded from each well.

[0688] 6) 25 μL of CTG reagent was added to each well, placed on a shaker for 2 minutes, and incubated at room temperature for 30 minutes in the dark.

[0689] 7) The chemiluminescence signal value was read using a multifunctional enzyme marker, the reading time was 1000 ms, and the corresponding inhibition rate data was calculated.

[0690] AST and Nutlin-3 alone or in combination for H460 cell proliferation inhibition experiment with drug grouping and experimental results as shown in Table 2, according to the inhibition rate data in Table 2, Figure 7 was drawn.

[0691] Table 2: AST and Nutlin-3 alone or in combination for H460 cell proliferation inhibition experiment with drug grouping and inhibition rate results

[0692] AST and RITA (p53-HDM-2 inhibitor, indirectly up-regulate p53 expression and function) alone or in combination for H460 cell proliferation inhibition experiment with drug grouping and experimental results as shown in Table 3, according to the inhibition rate data in Table 3, Figure 8 was drawn.

[0693] Table 3: AST and RITA alone or in combination for H460 cell proliferation inhibition experiment with drug grouping and inhibition rate results

[0694] AST and Nutlin-3 alone or in combination for HPAF-Ⅱ cell proliferation inhibition experiment with drug grouping and experimental results as shown in Table 4, according to the inhibition rate data in Table 4, Figure 9 was drawn.

[0695] Table 4: AST and Nutlin-3 alone or in combination for HPAF-Ⅱ cell proliferation inhibition experiment with drug grouping and inhibition rate results

[0696] AST and RITA alone or in combination for HPAF-Ⅱ cell proliferation inhibition experiment with drug grouping and experimental results as shown in Table 5, according to the inhibition rate data in Table 5, Figure 10 was drawn.

[0697] Table 5: AST and RITA alone or in combination with the proliferation of HPAF-II cell inhibition experiment dosing groups and inhibition rate results

[0698] Obviously, compared with each single drug, Nutlin-3 or RITA and AST combined significantly enhanced the proliferation inhibition effect on p53 wild-type H460 cells, while for p53 mutant cells HPAF-II, the combined regimen had little enhancement; that is, the above experiments again confirmed that the p53 protein has a decisive influence on the efficacy of AST-3424 / AST drugs, and AST-3424 / AST and other DNA alkylating agents have stronger proliferation inhibition effect on cells with negative p53 gene mutation and normal p53 protein.

[0699] In particular, taking AST as a single drug, an inhibition rate of 11-12% is achieved, and for p53 wild-type cells H460, the concentration only needs to be 1.5 nM, and for p53 mutant cells HPAF-II, 60 nM is selected, so AST / AST-3424 and other DNA alkylating agents have stronger cell proliferation inhibition effect on p53 wild-type cancer cells.

[0700] The AKR1C3 protein expression in H460 and HPAF-II cells is very close, indicating that the degree of activation of AST-3424 / AST in the two cells is similar, but taking AST and AST-3424 as single drugs, the applicant's previous cancer cell proliferation inhibition test data are as follows:

[0701] The IC of AST in H460 and HPAF-II cells 50 is 6.87 nM and 329.1 nM, respectively,

[0702] The IC of AST-3424 in H460 and HPAF-II cells 50 is 0.47 nM and 107.3 nM, respectively.

[0703] The above data further verify that AST / AST-3424 and other DNA alkylating agents have stronger cell proliferation inhibition effect on p53 wild-type cancer cells.

[0704] According to Examples 2, 3, and 4, the p53 protein has a decisive influence on the efficacy of AST-3424 / AST drugs, and AST-3424 / AST and other DNA alkylating agents have stronger proliferation inhibition effect on cells with negative p53 gene mutation and normal p53 protein, and the mechanism of action includes cell cycle G2 / M arrest and cell clone inhibition.

[0705] In order to further explain the above experimental phenomena, the effect of single drug or combined drug on the P53 protein pathway is detected.

[0706] Example Five Effect of AST-3424 + Nutlin-3 on p53 Protein Pathway

[0707] First Experiment of Effect of AST-3424 + Nutlin-3 on Ser15-p53, Ser20-p53, Total p53, MDM2, p21 Protein in H460 Cells

[0708] Summary of Experimental Process:

[0709] 1) H460 cell suspension was added to a 24-well plate, with a cell density of 80000 / well and 995 μL of culture medium per well.

[0710] 2) The cells were cultured overnight in a 37°C, 5% CO2 incubator.

[0711] 3) Compound Treatment

[0712] After the cells were plated for 24 hours, each cell line experiment was divided into groups according to the scheme:

[0713] Single Drug: After the cells were plated for 24 hours, 5 μL of different concentrations of test compounds were added: 0.5% DMSO, 0.1 nM AST-3424, 5 μM Nutlin-3, and the mixture was gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator.

[0714] Combined Drug: After the cells were plated for 24 hours, 5 μL of different concentrations of test compounds were added, and 5 μM Nutlin-3 was pretreated for 2 hours, then 0.1 nM AST-3424 was added, the mixture was gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator.

[0715] 4) The cell protein lysate was collected for WB detection. The WB detection band results are shown in Figure 11.

[0716] WB detection, i.e., Western Blot (WB) experiment.

[0717] Compared with AST-3424 and Nutlin-3 single drugs, the combination of AST-3424 and Nutlin-3 significantly promoted the protein expression of p53 phosphorylation (Ser15-p53 and Ser20-p53) and total-p53, and up-regulated the expression of p53 downstream genes MDM2 and p21.

[0718] In order to further explore the effect of different concentrations of AST-3424 and Nutlin-3 combination, a second experiment was performed.

[0719] The effect of AST-3424 and Nutlin-3 alone or in combination on H460 cells, Ser15-p53, Ser20-p53, Total p53, MDM2, p21 protein in the second experiment

[0720] The rest of the operation is similar to the first experiment described above. The compound treatment groups are administered as follows.

[0721] Single drug group, 1% DMSO, 5 μΜ Nutlin-3 for 24 hours, 0.1 nM AST-3424 for 24 hours, 0.3 nM AST-3424 for 24 hours, 1 nM AST-3424 for 24 hours;

[0722] Combined drug group:

[0723] 0.1 nM AST-3424 for 2 hours, then 5 μΜ Nutlin-3 for 22 hours;

[0724] 0.3 nM AST-3424 for 2 hours, then 5 μΜ Nutlin-3 for 22 hours;

[0725] 1 nM AST-3424 for 2 hours, then 5 μΜ Nutlin-3 for 22 hours.

[0726] The results are shown in Figure 12.

[0727] The experimental results show that Nutlin-3 up-regulates the expression levels of p53, p53 downstream proteins p21 and MDM2 proteins 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 with each single drug, the combination of AST-3424 and Nutlin-3 at increasing concentrations significantly promotes the protein expression of p53 phosphorylation (Ser15-p53 and Ser20-p53) and total-p53 and up-regulates the expression of p53 downstream genes MDM2 and p21. At the same time, the effect on the expression of the above proteins has a dose-dependent effect of AST-3424.

[0728] To further explore the influence of the order of adding drugs on the effect of combination of AST-3424 and Nutlin-3, the third experiment was carried out.

[0729] Influence of AST-3424 and Nutlin-3 alone or in combination on H460 cells, Ser15-p53, Ser20-p53, total p53, MDM2 and p21 proteins in the third experiment

[0730] The rest of the operation was similar to the first experiment described above, and the compound treatment groups were administered as follows.

[0731] The single drug group was treated with 0.5% DMSO, 5 μM Nutlin-3 for 24 hours, and 0.1 nM AST-3424 for 24 hours.

[0732] The combination drug group:

[0733] 0.1 nM AST-3424 was added for 2 hours of pre-treatment, and then 5 μM Nutlin-3 was added for 22 hours of co-treatment.

[0734] 0.1 nM AST-3424 and 5 μM Nutlin-3 were added for 24 hours of co-treatment.

[0735] 5 μM Nutlin-3 was added for 2 hours of pre-treatment, and then 0.1 nM AST-3424 was added for 22 hours of co-treatment.

[0736] The results are shown in Figure 13.

[0737] The experimental results show that compared with each single drug, the combination of AST-3424 and Nutlin-3 significantly promotes the protein expression of p53 phosphorylation (Ser15-p53 and Ser20-p53) and total-p53, and up-regulates the expression of p53 downstream genes MDM2 and p21. However, there is no significant difference in the influence of different drug addition orders on the above protein expression.

[0738] Example Six Influence of Nutlin-3 and AST-3424 alone or in combination on the amount of RAD51 protein

[0739] The combination of AST-3424 and Nutlin-3 significantly inhibits the protein expression of RAD51

[0740] In the first, second and third experiments, the applicant also carried out WB detection of RAD51 protein after the combined use of AST-3424 and Nutlin-3 on H460 cells in Example Five, and the results are shown in Figures 14, 15 and 16.

[0741] The results of the above three experiments show that, compared with AST-3424 and Nutlin-3 alone, the combined drug significantly reduces the content of RAD51 protein, and has AST-3424 dose dependence.

[0742] AST-3424 and Nutlin-3 combination increases RAD51 protein degradation

[0743] Summary of the experimental process:

[0744] 1) H460 cell suspension was added to a 24-well plate, and the cell density was 100000 / well, 995 μL of culture medium per well.

[0745] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0746] 3) Compound treatment

[0747] Single drug: 5 μL of different concentrations of test compounds were added to the cells 24 hours after plating: 1% DMSO, 0.1 nM AST-3424, 5 μM Nutlin-3, gently shake to ensure uniform mixing, then put into a 37°C, 5% CO2 incubator, continue to culture for 24 hours, then collect the cells.

[0748] Combined drug: 24 hours after plating the cells,

[0749] 0.1 nM AST-3424 was added for 2 hours of pre-treatment, and then 5 μM Nutlin-3 was added for 22 hours of co-treatment;

[0750] 1% DMSO and 5 μM MG-132 were added for 24 hours of co-treatment;

[0751] 0.1 nM AST-3424 and 5 μM MG-132 were added for 24 hours of co-treatment;

[0752] 5 μM Nutlin-3 and 5 μM MG-132 were added for 24 hours of co-treatment;

[0753] 0.1 nM AST-3424 was added for 2 hours of pre-treatment, 5 μM Nutlin-3 was added for 16 hours of treatment, and then 5 μM MG-132 was added for 6 hours of co-treatment.

[0754] 4) Collect cell protein lysate for WB detection, the detection results are shown in Figure 17.

[0755] MG-132 is a proteasome (26S proteasome) inhibitor, which inhibits protein degradation after addition.

[0756] The addition of MG-132 reverses the down-regulation 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.

[0757] AST-3424 and Nutlin-3 combination reduces the half-life of RAD51 protein

[0758] Summary of the experimental process:

[0759] 1) H460 cell suspension was added to a 24-well plate, and the cell density was 100000 / well, 995 μL of medium per well.

[0760] 2) The cells were cultured in a 37℃, 5% CO2 incubator overnight.

[0761] 3) Compound treatment

[0762] After the cells were plated for 24 hours, each cell line experiment was divided into groups according to the scheme:

[0763] Single drug group: 1% DMSO treatment group, 5 μM Nutlin-3 treatment group, 0.1 nM AST-3424 treatment group.

[0764] Combined drug group:

[0765] 1% DMSO + Cycloheximide 0.5 hour treatment group,

[0766] 1% DMSO + Cycloheximide 1 hour treatment group,

[0767] 1% DMSO + Cycloheximide 2 hour treatment group,

[0768] 1% DMSO + Cycloheximide 4 hour treatment group,

[0769] 0.1 nM AST-3424 + Cycloheximide 0.5 hour treatment group,

[0770] 0.1 nM AST-3424 + Cycloheximide 1 hour treatment group,

[0771] 0.1 nM AST-3424 + Cycloheximide 2 hour treatment group,

[0772] 0.1 nM AST-3424 + Cycloheximide 4 hour treatment group,

[0773] 5 μM Nutlin-3 + Cycloheximide 0.5 hour treatment group,

[0774] 5 μM Nutlin-3 + Cycloheximide 1 hour treatment group,

[0775] 5 μM Nutlin-3 + Cycloheximide 2 hour treatment group,

[0776] 5 μM Nutlin-3 + Cycloheximide 4 hour treatment group,

[0777] 0.1 nM AST-3424 + 5 μM Nutlin-3 treatment group,

[0778] 0.1 nM AST-3424 + 5 μM Nutlin-3 + Cycloheximide 0.5 hour treatment group,

[0779] 0.1 nM AST-3424 + 5 μM Nutlin-3 + Cycloheximide 1 hour treatment group,

[0780] 0.1 nM AST-3424 + 5 μM Nutlin-3 + Cycloheximide 2 hour treatment group,

[0781] 0.1 nM AST-3424 + 5 μM Nutlin-3 + Cycloheximide 4 hour treatment group.

[0782] Single agent treatment: After 24 hours of cell plating, 5 μL of test compound at different concentrations was added, gently shaken to ensure even mixing, and then placed in a 37 °C, 5% CO2incubator for an additional 24 hours. Cells were then harvested.

[0783] Combination treatment:

[0784] After 24 hours of cell plating, 5 μL of 1% DMSO was added and the cells were placed in a 37 °C, 5% CO2incubator for an additional 24 hours. Then, 5 μL of 4 μM Cycloheximide was added and the cells were placed in a 37 °C, 5% CO2incubator for an additional 0.5, 1, 2, or 4 hours, respectively. Cells were then harvested.

[0785] After 24 hours of cell plating, 5 μL of 0.1 nM AST-3424 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. Then, 5 μL of 5 μM Nutlin-3 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were collected.

[0786] After 24 hours of cell plating, 5 μL of 0.1 nM AST-3424 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. Then, 5 μL of 5 μM Nutlin-3 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were collected.

[0787] After 24 hours of cell plating, 5 μL of 0.1 nM AST-3424 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. Then, 5 μL of 5 μM Nutlin-3 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were collected.

[0788] After 24 hours of cell plating, 5 μL of 0.1 nM AST-3424 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. Then, 5 μL of 5 μM Nutlin-3 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were collected.

[0789] 4) The cell protein lysate was collected for WB detection, and the detection results are shown in Figure 18.

[0790] Further, according to the ratio data of the corresponding proteins relative to the internal reference protein β-actin in Figure 18, the relative amount change curve of RAD51 after different times was calculated, and the results are shown in Figure 19.

[0791] Cycloheximide is an inhibitor of protein synthesis in eukaryotes, and after addition, the synthesis of proteins is terminated.

[0792] After 24 hours of cell plating, 5 μL of 0.1 nM AST-3424 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 2 hours. Then, 5 μL of 5 μM Nutlin-3 was added and the cells were incubated in a 37°C, 5% CO2 incubator for 24 hours. The cells were collected.

[0793] Effect of AST-3424 combined with Nutlin-3 on DNA damage process in H460 cells

[0794] In the first, second and third experiments, the applicant also carried out WB detection of γH2AX protein after the combined action of AST-3424 and Nutlin-3 on H460 cells in Example Five, and the results are shown in Figures 20, 21 and 22.

[0795] The results show that AST-3424 can cause the up-regulation of the amount of DNA double-strand damage biomarker γH2AX protein, and has a dose-dependent effect.

[0796] Compared with each single drug, AST-3424 and Nutlin-3 combined can increase the amount of γH2AX protein, and has a dose-dependent effect of AST-3424; but AST-3424 and Nutlin-3 are treated at the same time, and the up-regulation of γH2AX protein is more significant.

[0797] Example Eight Effect of Nutlin-3 / AST treatment for 24 hours on the expression of p53, Rad51, MDM2 and p21 proteins in HPAF-II cells

[0798] Summary of the experimental process:

[0799] 1) The HP AF-II cell suspension was added to a 24-well plate, and the cell density was 300,000 cells per well, and 995 μL of culture medium per well.

[0800] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0801] 3) Compound treatment: After the cells were plated for 24 hours, different cells were grouped and dosed according to the following:

[0802] HPAF-II groups: 1% DMSO treatment group, 60nM AST treatment group, 8μM Nutlin-3 treatment group, AST 60nM+Nutlin-3 8μM treatment group.

[0803] Single drug: After the cells were plated for 24 hours, 5μL of different concentrations of test compounds were added: 1% DMSO, AST 1.5nM, Nutlin-3 1μM, AST 60nM, Nutlin-3 8μM, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator,

[0804] Combination: 24 hours after cell plating, 5 μL of 1 μM Nutlin-3 and 8 μM Nutlin-3 were added respectively, and 5 μL of 1.5 nM AST and 60 nM AST were added respectively after 2 hours of pre-treatment, and then gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator.

[0805] 4) After the 24-well plate was incubated at 37°C, 5% CO2 incubator for 24 hours, the cell protein lysate was collected for WB detection, and the WB detection results are shown in Figure 23.

[0806] Nutlin-3 is an inhibitor of p53 and MDM2 protein binding, which activates the function of p53.

[0807] The experimental results show that 60 nM AST has no effect on the expression of p53, p21 and MDM2 proteins in HPAF-II cells (p53 pathogenic mutation cells).

[0808] AST and Nutiln-3 combination has no effect on the expression of p53, p21 and MDM2 proteins in HPAF-II cells (p53 mutation, protein cannot be normally expressed).

[0809] AST alone promotes the expression of RAD51 protein in HPAF-II cells (p53 mutation, protein cannot be normally expressed), but has no synergistic effect with Nutlin-3.

[0810] Example Nine Effect of High Concentration Nutlin-3 / AST-3424 Treatment on HPAF-II Cells on Ser15-p53, Ser20-p53, p53, Rad51, MDM2, p21, γH2AX and Apoptosis Related Proteins Caspase3 and Cleaved Caspase3

[0811] The above experiment reveals that for HPAF-II cells with p53 gene mutation, lower concentration of Nutlin-3 / AST-3424 has little effect on related proteins, in order to further confirm, this experiment uses high concentration of Nutlin-3 / AST-3424 drug combination: 5 μM Nutlin-3+1000 nM AST-3424 to exclude the influence of concentration.

[0812] Summary of the experimental process:

[0813] 1) HPAF-II cell suspension was added to a 24-well plate, with a cell density of 300000 / well and 995 μL of culture medium per well.

[0814] 2) The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0815] 3) Compound treatment

[0816] After cells were plated for 24 hours, each cell line was divided into groups according to the following protocol:

[0817] Single agent: After cells were plated for 24 hours, 5 μL of test compound was added: 0.5% DMSO, 1000 nM AST-3424, 15 μM Nutlin-3, and the cells were gently shaken to ensure even mixing, then placed in a 37°C, 5% CO2 incubator.

[0818] Combination: After cells were plated for 24 hours, 5 μL of test compound was added: 0.5% DMSO, 1000 nM AST-3424, 15 μM Nutlin-3, and the cells were gently shaken to ensure even mixing, then placed in a 37°C, 5% CO2 incubator.

[0819] 4) Cell protein lysates were collected for WB detection. The results of the WB detection bands are shown in Figure 24.

[0820] It is clear that in the case of high concentration administration, the p53 mutant cell line HPAF-II cells still did not observe the associated enhanced protein expression after administration or the effect of combination was not obvious.

[0821] Based on the above Examples 5 to 9, the following conclusions can be drawn:

[0822] 1. The drug Nutlin-3 / RITA, which upregulates p53 protein expression or activates p53 function, can enhance the in vitro toxicity of AST-3424 / AST on cancer cells; and the enhancement is more significant for p53 wild-type cancer cells.

[0823] 2. The drug Nutlin-3 / RITA, which upregulates p53 protein expression or activates p53 function, can enhance the apoptosis caused by AST-3424 / AST, and the apoptosis is more significant for p53 wild-type cancer cells.

[0824] 3. The drug Nutlin-3 / RITA, which upregulates p53 protein expression or activates p53 function, can significantly increase the DNA double-strand damage caused by AST-3424 / AST, and the DNA double-strand damage is more significant for p53 wild-type cancer cells.

[0825] 4. The drug Nutlin-3 / RITA, which upregulates p53 protein expression or activates p53 function, can significantly increase the cell cycle G2 / M arrest caused by AST-3424 / AST, and the cell cycle G2 / M arrest is more significant for p53 wild-type cancer cells.

[0826] In summary, the above-mentioned enhancement reveals that AST-3424 / AST and the like DNA alkylating agents have stronger proliferation inhibitory effect on p53 wild-type cells.

[0827] 5. Compared with single drug, the combination of Nutlin-3 / RITA and AST-3424 / AST, which up-regulate p53 protein expression or activate p53 function, significantly up-regulate p53 phosphorylation, total-p53 expression and the expression of p53 downstream proteins MDM2 and p21, activate p53 protein pathway, and then down-regulate the protein expression of RAD51, and the above-mentioned phenomenon is more significant for p53 wild-type cancer cells.

[0828] 6. Compared with single drug, the combination of Nutlin-3 / RITA and AST-3424 / AST, which up-regulate p53 protein expression or activate p53 function, significantly promotes the degradation of RAD51 protein and reduces the half-life of RAD51 protein.

[0829] In summary, p53 protein promotes the degradation of homologous recombination repair protein RAD51, resulting in the down-regulation of RAD51 and the reduction of DNA double-strand damage repair ability, thereby improving the pharmacological activity of AST-3424 / AST and the like DNA alkylating agents. That is, patients with normal p53 protein and p53 gene will be more sensitive to AST-3424 / AST and the like DNA alkylating agents due to the above-mentioned processes and pathways involving p53 protein, and will have the potential to benefit more significantly from treatment (compared with patients with low p53 protein expression or p53 gene mutation). This phenomenon has been preliminarily observed in the clinical trials of AST-3424.

[0830] Example Ten Influence of the compound on the in vitro cell proliferation of H460 and p53 gene knockout cell H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 under normoxia

[0831] The p53 CRISPR / Cas9 KO plasmid (human) and p53 HDR plasmid (human) from Santa Cruz were purchased, and H460 P53 KO cell lines, i.e., H460 cell lines with p53 gene knockout, were constructed. Three groups of H460 P53 KO cell lines were constructed, numbered as H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12.

[0832] The construction process of p53 gene knockout cells is briefly described as follows:

[0833] H460 cells were plated in 6-well plates at 1×10 6 cells per well.

[0834] Co-transfect H460 cells 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 + P3000 5 μL; 125 μL Opti-MEM + 15 μL (increase volume, expected to increase transfection efficiency) Lipofectamine 3000, incubate together for 15 min, then add dropwise into culture medium and mix well.

[0835] Add 2 μg / mL puromycin for screening after 48 h.

[0836] Replace with fresh 1640 culture medium containing 1 μg / mL puromycin after 2 days, and continue to culture, replace culture medium every two days.

[0837] When the cell clones are large enough, pick single clones for culture.

[0838] When the cell number is sufficient, collect samples for WB identification of clones, and the clones with no P53 expression are P53 knockout H460 clone cells.

[0839] The experimental process of this example is referred to the experimental process summary of “Effect of compounds on H460 cell proliferation in vitro under normoxia” in Example 2.

[0840] Compound treatment

[0841] Single drug: Compound AST-3424 / AST was used alone in H460 and H460 P53 KO cell lines, respectively;

[0842] Combined drug: Compound AST-3424 / AST was combined with AST-3021, respectively, and used in H460 and H460 P53 KO cell lines, respectively.

[0843] H460 P53 KO cell lines, i.e. H460 P53 KO#1, H460 P53 KO#7, and H460 P53 KO#12 cell lines were used for the experiment, respectively.

[0844] The AST-3424 experimental results are shown in Table 11, and the corresponding AST-3424 single drug or combined with AST-3021 on the inhibition rate curve of H460 and H460 P53 KO cell proliferation in vitro is shown in Figure 25.

[0845] The AST experimental results are shown in Table 12, and the corresponding AST single drug or combined with AST-3021 on the inhibition rate curve of H460 and H460 P53 KO cell proliferation in vitro is shown in Figure 26.

[0846] Table 11: AST-3424 inhibition rate results on H460 WT (wild type H460 cells) and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 in vitro cell proliferation

[0847] Table 11 and Figure 25 show that the experimental data indicate that the sensitivity of H460 P53 KO#1 and #12 to AST-3424 is reduced by 49.13 and 8.65 times, respectively, compared with H460 wild type cells, indicating that the loss of P53 leads to reduced sensitivity to AST-3424 drug, and the loss of P53 has no effect on the AKR1C3 protein selectivity of AST-3424. This experiment further confirms the experimental results in Examples II and III, that the p53 protein has a decisive influence on the efficacy of AST-3424 drug, and that DNA alkylating agents such as AST-3424 have a stronger inhibitory effect on the proliferation of cells with normal p53 protein.

[0848] Table 12: AST inhibition rate results on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 in vitro cell proliferation

[0849] Table 12 and Figure 26 show that the experimental data indicate that the sensitivity of H460 P53 KO#1 and #12 to AST is reduced by 103.53 and 10.96 times, respectively, compared with H460 wild type cells, indicating that the loss of P53 leads to reduced sensitivity to AST drug, but the loss of P53 leads to AST almost losing AKR1C3 protein selectivity. This experiment further confirms the experimental results in Example IV, that the p53 protein has a decisive influence on the efficacy of AST drug, and that DNA alkylating agents such as AST have a stronger inhibitory effect on the proliferation of cells with normal p53 protein.

[0850] Example XI Effect of compounds under hypoxia on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 in vitro cell proliferation

[0851] Summary of the experimental process:

[0852] 1) H460, H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 cell suspensions were added to two types of 24-well plates, 495 μL per well, and the cell density was 1 x 10 4 The 24-well plate with glass insert was used for hypoxia experiment, and the ordinary plastic 24-well plate was used for normoxia experiment.

[0853] 2) Cells were incubated overnight at 37°C, 5% CO2 incubator.

[0854] 3) Compound treatment

[0855] Hypoxic conditions:

[0856] Adjust the hypoxic workstation to a hypoxic environment (O2 < 0.01%) and confirm the anoxic condition in the workstation with an oxygen indicator. After 24 hours of cell plating, the 24-well plates with glass inserts were transferred to the hypoxic workstation.

[0857] Place the 24-well plates on a shaker, open the well plate lid and shake for 5 minutes for gas exchange.

[0858] Add 5 μL of 100x corresponding concentration of compound per well, 3 replicates per experimental group.

[0859] Gently shake to ensure even mixing of the compound, half open the 24-well plate lid and incubate for 3 hours in the anoxic workstation.

[0860] Normoxic conditions:

[0861] After 24 hours of cell plating, add 5 μL of 100x corresponding concentration of compound per well, 3 replicates per experimental group.

[0862] Gently shake to ensure even mixing of the compound, incubate the 24-well plate for 3 hours in a standard 37°C, 5% CO2 incubator.

[0863] 4) Wash all 24-well plates twice with complete medium, 500 μL per well per wash.

[0864] 5) Add 1000 μL of medium per well.

[0865] 6) Place in a 37°C, 5% CO2 incubator for 72 hours.

[0866] 7) Discard 800 μL of medium per well, add 50 μL of CTG, shake to mix for 2 minutes, and place at room temperature in the dark for 15 minutes.

[0867] 8) Transfer 100 μL of medium from each well of the 24-well plate to a 96-well white plate.

[0868] 9) Read chemiluminescent signal values using a multi-function plate reader, reading time 1000 ms.

[0869] 10) Calculate IC using GraphPad Prism 5 software 50 , using the following non-linear fit equation to obtain the IC 50Half maximal inhibitory concentration.

[0870] This example uses specific compound A in structural formula (1) and specific compound B in structural formula (2) to perform the above experiment, and the structures of compounds A and B are as follows:

[0871] The experimental results of compound A are shown in Table 13, and the corresponding compound A in vitro proliferation inhibition rate curve of H460 and H460 P53 KO cells is shown in Figure 27.

[0872] The experimental results of compound B are shown in Table 14, and the corresponding compound B in vitro proliferation inhibition rate curve of H460 and H460 P53 KO cells is shown in Figure 28.

[0873] Table 13: Inhibition rate results of compound A on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 in vitro cell proliferation

[0874] The experimental data shown in Table 13 and Figure 27 show that under hypoxic conditions, the sensitivity of H460 P53 KO#1, #7 and #12 cells to compound A is reduced by 12.13 times, 10.26 times and 6.60 times compared with H460 P53 WT cells, and still has good hypoxic selectivity. That is, the p53 protein has a decisive influence on the efficacy of compound A (DNA alkylating agent), which further confirms the experimental results in the above examples that DNA alkylating agent drugs have a stronger proliferation inhibitory effect on cells with negative p53 gene mutations and normal p53 proteins.

[0875] Table 14: Inhibition rate results of compound B on H460 and H460 P53 KO#1, H460 P53 KO#7, H460 P53 KO#12 in vitro cell proliferation

[0876] The experimental data shown in Table 14 and Figure 28 show that under hypoxic conditions, the sensitivity of P53 KO#1, #7 and #12 cells to compound B is reduced by 22.31 times, 27.69 and 10.00 times compared with H460 P53 WT cells, but the hypoxic selectivity is reduced. That is, the p53 protein has a decisive influence on the efficacy of compound B (DNA alkylating agent), which further confirms the experimental results in the above examples that DNA alkylating agent drugs have a stronger proliferation inhibitory effect on cells with negative p53 gene mutations and normal p53 proteins.

[0877] Effect of Nutlin-3 on Total P53, MDM2, P21, AKR1C3, Actin protein in H460, H460 P53 KO cells

[0878] The P53 gene KnockOut cell line, numbered H460 P53 KO#8, was constructed in the same way as Example Ten.

[0879] Summary of the experimental process:

[0880] 1) H460, H460 P53 KO cell suspension was added to a 24-well plate, with a cell density of 100000 / well, 995 μL of medium per well.

[0881] 2) The cells were cultured overnight at 37°C in a 5% CO2 incubator.

[0882] 3) Compound treatment

[0883] After the cells were plated for 24 hours, each cell line experiment was divided into groups according to the scheme:

[0884] 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;

[0885] Single drug: After the cells were plated for 24 hours, 5 μL of different concentrations of test compound was added, gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO2 incubator,

[0886] 4) After the 24-well plate was incubated at 37°C in a 5% CO2 incubator for 24 hours, the cell protein lysate was collected for WB detection.

[0887] WB detection method:

[0888] 1) The cell plate was removed, the culture medium supernatant was discarded, and PBS was used to gently rinse it. 30 μL of RIPA protein lysate (RIPA: phosphatase inhibitor = 10:1) was added to each well, and the cells were scraped off using a cell scraper. The lysate was incubated on ice for 30 min, and then centrifuged at 14000 rpm for 10 min at 4°C.

[0889] 2) Protein quantification using BCA method. Then loading and running: 4-12% SDS-PAGE finished gel was used for protein sample separation. The proteins loaded from left to right were H460 WT no treatment group, H460 P53 KO#1 no treatment group, H460 P53 KO#7 no treatment group, H460 P53 KO#8 no treatment group, H460 P53 KO#12 no treatment group, H460 WT 0.1% DMSO treatment group, H460 WT 5 mM Nutlin-3 treatment group, H460 P53 KO#1 5 mM Nutlin-3 treatment group, H460 P53 KO#7 5 mM Nutlin-3 treatment group, H460 P53 KO#8 5 mM Nutlin-3 treatment group, H460 P53 KO#12 5 mM Nutlin-3 treatment group. The protein loading amount of each sample was 10 pg / 12.5 pL, and after running, the membrane was transferred, the transfer conditions were 100 V, 1 h.

[0890] 3) Antibody incubation and luminescence detection: after the completion of the membrane transfer, the membrane was blocked in 5% skim milk on a horizontal shaker at room temperature for 1 h. The respective antibodies were added, with an antibody ratio of 1:1000, in an antibody incubation box at 4°C overnight. The next day, the membrane was warmed at room temperature for 1 h, and then washed in TBST for 10 min each time, three times. After washing, the corresponding secondary antibody (1:4000 dilution) was incubated according to the type of primary antibody. The incubation time was 2 h. After incubation, the membrane was also washed three times with TBST for 10 min each time. The ECL luminescence solution (SuperSignal West Femto Maximum Sensitivity) AB liquid was mixed and added to the membrane after the water was absorbed, and the luminescence detection was performed in the gel imager.

[0891] The protein band photos of the WB detection results of the cell protein lysate are shown in Figure 29, and the corresponding protein ratio to the internal reference protein b-actin is shown in Figure 30.

[0892] The internal reference protein actin of all cell strains and various treatments in the above experiment did not change.

[0893] Figure 29 and Figure 30 show experimental data indicating that, compared with wild-type H460, P53 knockout reduces the protein expression of AKR1C3 (#1 more significantly), and also reduces the expression of MDM2 and P21. Compared with the H460 wild-type DMSO group, it was found that, after treating wild-type H460 with the positive drug Nutlin-3, the protein expression of MDM2, P53, P21 and AKR1C3 was up-regulated. However, after treating wild-type H460 and H460 P53 KO cell lines with Nutlin-3, it was found that, compared with the H460 wild-type DMSO group, the protein expression of MDM2, P53, P21 and AKR1C3 in H460 P53 KO cells was not up-regulated, and this was particularly significant for H460 P53 KO #1 cells. The results suggest that H460 P53 KO can down-regulate the protein expression of AKR1C3, which is consistent with the experimental results shown in Table 12 and Figure 26 in Example Ten, which show that the IC 50 of compound AST for AKR1C3 selectivity is lost in H460 P53 KO cells.

[0894] Example Thirteen Effect of Compound C and AST-3424 on NCI-H460, NCI-H460 P53 KO #1 cell proliferation in vitro under normoxia

[0895] This example further verifies the effect of a specific compound C in structural formula (9) on NCI-H460, NCI-H460 P53 KO #1 cell proliferation in vitro under normoxia. The structure of compound C is as follows:

[0896] Summary of the experimental process:

[0897] 1) Add NCI-H460, NCI-H460 P53 KO #1 cell suspension to a 96-well plate, 100 μL per well, with a cell density of 2000 / well.

[0898] 2) Incubate the cells overnight in a 37°C, 5% CO2 incubator.

[0899] 3) Compound treatment

[0900] Single drug: After 24 hours of cell plating, add 99.5 μL of growth medium to each well. Add 0.5 μL of the test compound at different concentrations, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0901] Combined drug: After 24 hours of cell plating, add 99 μL of growth medium to each well. Add 0.5 μL of the combination compound, gently shake to ensure uniform mixing, and place in the incubator for 2 h. Then add 0.5 μL of the test compound at different concentrations, gently shake to ensure uniform mixing, and then place in a 37°C, 5% CO2 incubator.

[0902] 4) Place the cell plate in the incubator for 72 hours.

[0903] 5) Place the cell plate in room temperature for 30 minutes to equilibrate, discard 100 μL medium per well.

[0904] 6) Add 25 μL CTG reagent per well, place on a shaker for 2 minutes, and incubate at room temperature for 30 minutes in the dark.

[0905] 7) Read the chemiluminescence signal value using a multifunctional microplate reader, and the reading time is 1000 ms.

[0906] 8) Calculate the IC 50 (fifty percent inhibitory concentration) using GraphPad Prism 5 software.

[0907] The IC 50 values of Compound C detected in the experiment are shown in Table 15, and the NCI-H460 and NCI-H460 P53 KO#1 cell in vitro proliferation inhibition rate curves are shown in Figures 31 and 32.

[0908] Table 15: Compound C and AST-3424 in vitro proliferation inhibition rate results of NCI-H460 and NCI-H460 P53 KO#1 cells

[0909] The above experimental data show that:

[0910] The IC 50 of Compound C in NCI-H460 cells is 2.05 nM, which is 4.77 times the IC 50 of AST-3424, and there is no significant difference in AKR1C3 selectivity between Compound C and AST-3424.

[0911] After P53 knockout, the IC 50 value of Compound C increased by 17.60 times, and the IC 50 of AST-3424 increased by 12.67 times, indicating that the P53 protein can increase the cytotoxicity of Compound C and AST-3424.

[0912] After P53 knockout, the IC 50 ratio of Compound C combined with AKR1C3 enzyme inhibitor AST-3021 to single drug decreased from 305.37 times to 43.24 times, indicating that after P53 knockout, the AKR1C3 selectivity of Compound C is significantly reduced.

[0913] That is, the 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 agent drugs have a stronger proliferation inhibitory effect on cells with normal p53 protein and negative p53 gene mutation.

[0914] Example Fourteen Effect of Compound AST-3424, Thiotepa / Thio-TEPA, Glufosfamide on NCI-H460, NCI-H460 P53 KO#1 Cell Proliferation in vitro under Normal Oxygen

[0915] The experimental process is described in Example Thirteen.

[0916] IC 50 The results are shown in Table 16, and the inhibition rate curves of NCI-H460 and NCI-H460 P53 KO#1 cell proliferation in vitro of compound AST-3424, Thiotepa / Thio-TEPA, Glufosfamide are shown in Figures 33, 34, and 35, respectively.

[0917] Table 16: Inhibition rate of compound AST-3424, Thiotepa / Thio-TEPA, Glufosfamide on NCI-H460 and NCI-H460 P53 KO#1 cell proliferation in vitro

[0918] The above experimental data show that:

[0919] After P53 knockout, the IC 50 of the three compounds: AST-3424, Thio-TEPA, and Glufosfamide all increased, increasing to 8.47 times, 10.86 times, and 8.85 times that of wild-type H460 cells, respectively, indicating that the p53 protein can increase the proliferation inhibitory effect of the above three compounds on cancer cells and increase their cytotoxicity.

[0920] That is, the p53 protein has a decisive influence on the efficacy of the above three compounds (all DNA alkylating agents), further confirming the experimental results in the above examples that DNA alkylating agent drugs have a stronger proliferation inhibitory effect on cells with normal p53 protein and negative p53 gene mutation.

[0921] Example Fifteen Effect of compound AST-3424, SG-2057, Temozolomide, Carmustine, Busulfan, Duocarmycin TM on NCI-H460, NCI-H460 P53 KO#1 cell proliferation in vitro under normoxia

[0922] The experimental procedure is described in Example Thirteen.

[0923] IC50of the tested compounds 50 The results are shown in Table 17, and the inhibition curves of NCI-H460 and NCI-H460 P53 KO#1 cell proliferation in vitro of compound AST-3424, SG-2057, Temozolomide, Carmustine, Busulfan, Duocarmycin TM are shown in Figures 36 to 41, respectively.

[0924] Table 17: Inhibition results of compound AST-3424, SG-2057, Temozolomide, Carmustine, Busulfan, Duocarmycin TM on NCI-H460 and NCI-H460 P53 KO#1 cell proliferation in vitro

[0925] The above experimental data show that:

[0926] After P53 knockout, the IC50of the four compounds: AST-3424, SG-2057, Busulfan, Duocarmycin TM 50 all increased, increased to 9.31 times, 4.67 times, 3.87 times, 2.30 times of the wild type H460 cells, respectively, which can clearly show that P53 protein can increase the proliferation inhibition effect of the above four compounds on cancer cells, and increase the cytotoxicity.

[0927] In particular, for SG-2057, Duocarmycin TM (toxic payload used in the development of new generation ADC antibody conjugate drugs) with high cytotoxicity, the compound has a significantly lower cell proliferation inhibition effect on P53 knockout H460 cells, which indicates that such ADC drugs using SG-2057, Duocarmycin TM or similar high cytotoxicity DNA alkylating agents as Payload will have greater clinical benefits for patients with normal p53 protein expression or p53 gene mutation negative.

[0928] Compound Temozolomide is a liposoluble oral DNA alkylating agent chemotherapy drug, which can pass through the blood-brain barrier, and is suitable for malignant glioma and metastatic melanoma. H460 cells are not sensitive to it. However, even if H460 cells are not sensitive to Temozolomide, the activity of H460 cells is also reduced after knocking out the P53 gene, which to some extent shows that P53 is an important protein affecting Temozolomide.

[0929] Compound Carmustine is a liposoluble DNA alkylating agent chemotherapy drug, which can pass through the blood-brain barrier, and is mainly suitable for brain tumors. H460 cells are not sensitive to it. However, even if H460 cells are not sensitive to Carmustine, the activity of H460 cells is also reduced after knocking out the P53 gene, which to some extent shows that P53 is an important protein affecting Carmustine.

[0930] That is, the p53 protein has an important influence on the efficacy of the above-mentioned 6 compounds (all DNA alkylating agents), further confirming the experimental results in the above examples that DNA alkylating agent drugs have a stronger proliferation inhibitory effect on cells with negative p53 gene mutations and normal p53 proteins.

Claims

A method of treatment using a DNA alkylating agent prodrug compound or a DNA alkylating agent as a single agent or in combination with other drugs for treating cancer, tumor patients with p53 gene mutation negative or normal p53 protein expression. The method of treatment according to claim 1, wherein, The DNA alkylating agent prodrug compound is selected from DNA alkylating agent conjugated prodrug compounds. The method of treatment according to claim 1 or 2, wherein, The DNA alkylating agent in the DNA alkylating agent prodrug compound, the DNA alkylating agent is selected from nitrogen mustards, aminophosphates, PBDs, Duocarmycins. The method of treatment according to claim 3, wherein, The nitrogen mustards are selected from Enpiexin, Melphalan, Chlorambucil, Carmustine, Semustine; The aminophosphates are selected from Cyclophosphamide, Ifosfamide, Thiotepa, Thiotepa derivative AST-2660; The PBDs are selected from SG3199, SG-2057, SGD-1882, PBD dimer-2, PBD-monoamide, Aniline-MPB-amino-C3-PBD, Py-MPB-amino-C3-PBD, Tomaymycin DM; The Duocarmycins are selected from Duocarmycin SA, Duocarmycin Analog, Duocarmycin MB, Duocarmycin GA, Duocarmycin MA, Duocarmycin TM, Duocarmycin DM, Duocarmycin DM free base, Duocarmycin A, Seco-Duocarmycin TM, Seco-Duocarmycin SA, (S)-Seco-Duocarmycin SA, NMS-P528, Seco-DUBA. The method of treatment according to claim 2, wherein, The DNA alkylating agent conjugated prodrug compound is selected from ADCs, SMDCs, PDCs. The method of treatment according to claim 5, wherein, The toxin Payload of the ADC is selected from PBDs, Duocarmycins, The ADC drug with PBDs as toxin Payload is preferably selected from DHES0815A, SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-352A, Rovalpituzumab Tesirine, SC-002, SC-003, ADCT-301, ADCT-402 (Lonca), ADCT-601, ADCT-602, The ADC drug with Duocarmycins as toxin Payload is preferably selected from SYD983, SYD985, BMS-936561, SYD1875, MGC018, PCM5B14 DCM, hYP7-DCM, Promiximab DUBA, D2B-DCM, PCM-MET01; the toxin Payload of the SMDC is selected from the group consisting of phosphoramidates; the targeting peptide of the PDC is selected from the group consisting of bombesin analogs, GnRH analogs, growth hormone inhibiting analogs, RGD peptides, PEGA. The method of treatment according to claim 6, wherein, the SMDC is selected from the group consisting of hypoxia-activated DNA alkylating agent-conjugated prodrug compounds, AKR1C3-activated DNA alkylating agent-conjugated prodrug compounds, beta-glucosidase (beta-D-Glucosidase) or beta-galactosidase-activated DNA alkylating agent-conjugated prodrug compounds, Preferably, the hypoxia-activated DNA alkylating agent conjugate prodrug compounds are selected from the group consisting of structural formulas 1-3 and salts, esters, solvates, isotopologues thereof, AKR1C3-activated DNA alkylating agent conjugate prodrug compounds are selected from the group consisting of structural formulas 4-12 and salts, esters, solvates, isotopologues thereof, and β-glucosidase (β-D-Glucosidase) or β-galactosidase (β-galactosidase)-activated DNA alkylating agent conjugate prodrug compounds are selected from the group consisting of structural formulas 15: wherein each R is independently selected from H, -CH3, -CH2CH3, -CF3, each X is independently selected from CI, Br, MsO, TsO leaving functional groups, and the like; wherein R1, R2, R3, Cx are defined as in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1 (corresponding to Chinese application number 2020800673113, publication number CN114466853A); wherein 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 are defined as in the claims of patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A); wherein X, Y, Z, R, T, A, and X 10 are defined as recited in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A); wherein, wherein, X, Y, Z, R, D, L 1 , A and X 10 are defined as recited in the claims of patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A); wherein R1, R2, R3, R4, R5, R8, R9, R 10 The definitions are as described in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); wherein: A is substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl, or -N=CR 1 R 2 , wherein the substituents, when present, are selected from the group consisting of halo, -CN, -NO2, -O-(CH2)-O-, -CO2H and salts thereof, -OR 100 , -CO2R 100 , -CONR 101 R 102 , -NR 101 R 102 , -NR 100 SO2R 100 , -SO2R 100 , -SO2NR 101 R 1 02 , C1-C6 alkyl, C3-C10 heterocyclyl; wherein R 100 , R 101 and R 102 are each independently hydrogen, Ci-C8alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; wherein each alkyl and aryl is substituted with 1-3 halo or 1-3 C1-C6 alkyl; R 1 and R 2 each independently is phenyl or methyl; each of X, Y and Z is independently hydrogen or halo; R is hydrogen or C1-C6alkyl or halogen substituted alkyl; wherein Rw is defined as in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); wherein R1, R2, R3, R4, T are defined as in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1; wherein, A, E, G, X, Y are defined as 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, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , n1, n2 are defined as recited 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 is attached to the phosphoramide mustard residue (15-I) or the ifosfamide mustard residue (15-II), R1 and R2 can be the same or different and are selected from the group consisting of hydrogen, C1-C4 alkyl or C1-C6 haloalkyl, and the sugar is any existing isomeric or enantiomeric form of a monosaccharide, disaccharide or polysaccharide. The method of treatment according to claim 1, wherein, the DNA alkylating agent prodrug compound is selected from the group consisting of ADC drugs DHES0815A, SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-352A, Rovalpituzumab Tesirine, SC-002, SC-003, ADCT-301, ADCT-402 (Lonca), ADCT-601, ADCT-602, SYD983, SYD985, BMS-936561, SYD1875, MGC018, PCM5B14 DCM, hYP7-DCM, Promiximab DUBA, D2B-DCM, PC M-MET01; the DNA alkylating agent prodrug compound is selected from the group consisting of SMDC drugs AST-3424, AST-001, TFX05-01, Evofosfamide, Achm-025, Glufosfamide; the DNA alkylating agent prodrug compound is selected from the group consisting of PDC drugs Melphalan flufenamide. The method of treatment according to claim 8, wherein, The DHES0815A is administered at a dose of no more than 12 mg / kg, preferably no more than 4 mg / kg, more preferably no more than 2.4 mg / kg, further preferably no more than 1.4 mg / kg, more further preferably no more than 1.2 mg / kg, and still further preferably no more than 0.6 mg / kg; The SC-003 is administered at a dose of 0.025-0.4 mg / kg, with a recommended maximum dose of 0.3 mg / kg, preferably at a dose of 0.3 mg / kg or 0.2 mg / kg, once every 3 weeks for a treatment cycle. The ADCT-402 (Loncastuximab tesirine) is administered at a dose of 150 μg / kg intravenously on day 1 of each 21-day cycle for two cycles. The SYD985 is administered at a dose of 1.2 mg / kg once every 3 weeks. The MGC018 is administered at a dose of 3 mg / kg intravenously on day 1 of each 3-week cycle. The BMS-936561 is administered at a dose of 8 mg / kg by injection once every 21 days for a 42-day cycle. The AST-3424 is administered at a dose selected from the group consisting of 25 mg / kg, 20 mg / kg, 18 mg / kg, 15 mg / kg, 12 mg / kg, 10 mg / kg, 8 mg / kg, 6 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg, once every 28 days for a treatment cycle, on days 1, 8, and 15 of each cycle. Regimen One, a cycle every 21 days with one dose on Day 1 and Day 8 at a dose selected from 12 mg / m 2 11 mg / m 2 10 mg / m 2 9 mg / m 2 8 mg / m 2 7 mg / m 2 6 mg / m 2 5 mg / m 2 4 mg / m 2 ; Scheme Two, a cycle every 21 days, on Day 1, at a dose selected from 14 mg / m 2 , 13 mg / m 2 , 12 mg / m 2 , 11 mg / m 2 , 10 mg / m 2 , 9 mg / m 2 , 8 mg / m 2 , 7 mg / m 2 , 6 mg / m 2 , 5 mg / m 2 , 4 mg / m 2 , 3 mg / m 2 , 2 mg / m 2 , 1 mg / m 2 ; Scheme three, every 21 days for a cycle, on days 1-5 once daily at a dose selected from 8 mg / m 2 , 7 mg / m 2 , 6 mg / m 2 , 5 mg / m 2 , 4 mg / m 2 , 3 mg / m 2 , 2 mg / m 2 , 1 mg / m 2 ; The TFX05-01 is administered at a dose selected from the group consisting of 25 mg / kg, 20 mg / kg, 18 mg / kg, 15 mg / kg, 12 mg / kg, 10 mg / kg, 8 mg / kg, 6 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg, once every 3 weeks for a treatment cycle, on days 1 and 8 of each cycle. The Achm-025 is administered at a dose selected from the group consisting of 25 mg / kg, 20 mg / kg, 18 mg / kg, 15 mg / kg, 12 mg / kg, 10 mg / kg, 8 mg / kg, 6 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg, once every 3 weeks for a treatment cycle, on days 1 and 8 of each cycle. The dosing regimen for the Evofosfamide is: intravenous administration at a daily dose of 120 mg / m 2 to 460 mg / m 2 ; or intravenous administration at a weekly dose of 480 mg / m 2 to 670 mg / m 2 , preferably at a weekly dose of 575 mg / m 2 ; The Melphalan flufenamide is administered at a dose of 40 mg intravenously within 30 minutes on day 1 of each 28-day treatment cycle, preferably in combination with dexamethasone. The dosing regimen for the glufosfamide is 4500 mg / m 2 intravenous infusion over 6 hours; A method for treating cancer, wherein a patient is determined to be negative for a p53 gene mutation or a patient is determined to have normal p53 protein expression prior to administering a DNA alkylating agent prodrug compound or a DNA alkylating agent, wherein the DNA alkylating agent prodrug compound or the DNA alkylating agent is as defined in claims 1-9. ​ The pharmaceutical use of DNA alkylating agent prodrug compounds or DNA alkylating agents for the preparation of a medicament for the treatment of cancer, tumor patients with negative p53 gene mutation detection or normal p53 protein expression alone or in combination with other drugs. The pharmaceutical use according to claim 11, The DNA alkylating agent in the DNA alkylating agent prodrug compounds or the DNA alkylating agent is selected from nitrogen mustards, phosphoramidates, PBDs, Duocarmycins; The DNA alkylating agent prodrug compounds are selected from DNA alkylating agent conjugated prodrug compounds. The pharmaceutical use according to claim 12, The nitrogen mustards are selected from Uracil mustard, Melphalan, Chlorambucil, Carmustine, Semustine; The phosphoramidates are selected from Cyclophosphamide, Ifosfamide, Thiotepa, Thiotepa derivative AST-2660; The PBDs are selected from SG3199, SG-2057, SGD-1882, PBD dimer-2, PBD-monoamide, Aniline-MPB-amino-C3-PBD, Py-MPB-amino-C3-PBD, Tomaymycin DM; The Duocarmycins are selected from Duocarmycin SA, Duocarmycin Analog, Duocarmycin MB, Duocarmycin GA, Duocarmycin MA, Duocarmycin TM, Duocarmycin DM, Duocarmycin DM free base, Duocarmycin A, Seco-Duocarmycin TM, Seco-Duocarmycin SA, (S)-Seco-Duocarmycin SA, NMS-P528, Seco-DUBA. The pharmaceutical use according to claim 13, The DNA alkylating agent conjugated prodrug compounds are selected from ADCs, SMDCs, PDCs, The toxin Payload of the ADCs is selected from PBDs, Duocarmycins, The toxin Payload of the SMDCs is selected from phosphoramidates; The targeting peptides of the PDCs are selected from Bombesin analogs, GnRH analogs, Growth hormone inhibiting analogs, RGD peptides, PEGA. The treatment method according to claim 14, The SMDCs are selected from hypoxia-activated DNA alkylating agent conjugated prodrug compounds, AKR1C3-activated DNA alkylating agent conjugated prodrug compounds, β-D-Glucosidase or β-galactosidase-activated DNA alkylating agent conjugated prodrug compounds, Preferably, the hypoxia-activated DNA alkylating agent conjugate prodrug compounds are selected from the group consisting of structural formulas 1-3 and salts, esters, solvates, isotopologues thereof, AKR1C3-activated DNA alkylating agent conjugate prodrug compounds are selected from the group consisting of structural formulas 4-12 and salts, esters, solvates, isotopologues thereof, and β-glucosidase (β-D-Glucosidase) or β-galactosidase (β-galactosidase)-activated DNA alkylating agent conjugate prodrug compounds are selected from the group consisting of structural formulas 15: wherein R are each independently selected from H, -CH3, -CH2CH3, -CF3, X are each independently selected from Cl, Br, MsO, TsO and the like leaving functional groups; wherein R1, R2, R3, Cx are defined as in the claims of patent application PCT / CN2020 / 114519, publication number WO2021120717A1 (corresponding to Chinese application number 2020800673113, publication number CN114466853A); wherein 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 are defined as in the claims of patent application PCT / US2016 / 039092, publication number WO2016210175A1 (corresponding to Chinese application number 2016800368985, publication number CN108024974A); wherein X, Y, Z, R, T, A, and X 10 are defined as recited in the claims of patent application PCT / US2016 / 021581, publication number WO2016145092A1 (corresponding to Chinese application number 2016800150788, publication number CN107530556A); wherein, wherein, X, Y, Z, R, D, L 1 , A, and X 10 are defined as recited in the claims of patent application PCT / US2016 / 025665, publication number WO2016161342A3 (corresponding to Chinese application number 2016800200132, publication number CN108136214A); wherein R1, R2, R3, R4, R5, R8, R9, R 10 The definitions are as described in the claims of patent application PCT / CN2020 / 089692, publication number WO2020228685A9 (corresponding to Chinese application number 2020800358890, publication number CN113853379A); Wherein: A is substituted or unsubstituted C6-C10 aryl, biaryl or substituted biaryl, 5-15 membered heteroaryl, or -N=CR 1 R 2 wherein the substituents, when present, are selected from the group consisting of halo, -CN, -NO2, -O-(CH2)-O-, -CO2H and salts thereof, -OR 100 , -CO2R 100 , -CONR 101 R 102 , -NR 101 R 102 , -NR 100 SO2R 100 , -SO2R 100 , -SO2NR 101 R 1 02 , C1-C6 alkyl, C3-C10 heterocyclyl; wherein R 100 , R 101 , and R 102 are each independently hydrogen, Ci-C8alkyl, C6-C12 aryl; or R 101 and R 102 together with the nitrogen atom to which they are attached form a 5-7 membered heterocyclic ring; Wherein alkyl and aryl are each substituted with 1-3 halo groups or 1-3 C1-C6 alkyl groups; R 1 and R 2 are each independently phenyl or methyl; X, Y and Z are each independently hydrogen or halo groups; R is hydrogen or C1-C6alkyl or halogen substituted alkyl; wherein Rw is defined as in the claims of patent application PCT / CN2020 / 120281, publication number WO2021068952A1 (corresponding to Chinese application number 202080071652.8, publication number CN114555574A); wherein R1, R2, R3, R4, T are defined as in the claims of patent application PCT / CN2021 / 118597, publication number WO2022057838A1; wherein A, E, G, X, Y are defined as 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, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R a , R b , n1, n2 are defined as recited 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 is attached to the phosphoramide mustard residue (15-I) or the isophosphoramide mustard residue (15-II), and R1and R2, which can be the same or different, are selected from hydrogen, C1-C4alkyl, or C1-C6haloalkyl, and the sugar moiety is an isomer or enantiomeric form of any existing monosaccharide, disaccharide, or polysaccharide. The method of treatment according to claim 15, wherein PBD-based ADC drugs as toxin Payload are preferably selected from DHES0815A, SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-352A, Rovalpituzumab Tesirine, SC-002, SC-003, ADCT-301, ADCT-402 (Lonca), ADCT-601, ADCT-602, Duocarmycin-based ADC drugs as toxin Payload are preferably selected from SYD983, SYD985, BMS-936561, SYD1875, MGC018, PCM5B14 DCM, hYP7-DCM, Promiximab DUBA, D2B-DCM, PCM-MET01; DNA alkylating agent prodrug compounds are selected from ADC drugs DHES0815A, SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-352A, Rovalpituzumab Tesirine, SC-002, SC-003, ADCT-301, ADCT-402 (Lonca), ADCT-601, ADCT-602, SYD983, SYD985, BMS-936561, SYD1875, MGC018, PCM5B14 DCM, hYP7-DCM, Promiximab DUBA, D2B-DCM, PCM-MET01; DNA alkylating agent prodrug compounds are selected from SMDC drugs AST-3424, AST-001, TFX05-01, Evofosfamide, Achm-025, Glufosfamide; DNA alkylating agent prodrug compounds are selected from PDC drugs Melphalan flufenamide.

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