Hypoxia-activated dna alkylating agents and medical uses thereof

Abstract

The present invention relates to a hypoxia-activated DNA alkylating agent, or a pharmaceutically acceptable salt or prodrug or solvate or isotopic variant thereof, having any of the following structural formulae:

Description

Technical Field

[0001] This invention relates to compounds for cancer treatment and belongs to the field of small molecule drug development. Background Technology

[0002] TH-302 (Evofosfamide, CAS No. 918633-87-1) is a 2-nitroimidazole-induced hypoxia-activated prodrug (HAP) bromoisophosphoramide. Under hypoxic conditions, the inactive TH-302 prodrug releases highly toxic Br-IPM (as shown in structural formula 1 below). TH-302 possesses broad-spectrum in vitro and in vivo biological activity, as well as specific hypoxia-selective activation activity and the ability to induce H2AX phosphorylation and DNA cross-linking, leading to cell cycle arrest. Therefore, this compound has been used by numerous pharmaceutical companies and research institutions in the development of anticancer drugs.

[0003]

[0004] After Merck acquired the TH-302 research project from Threshold Pharmaceuticals, it conducted a Phase III clinical trial of the anti-tumor drug TH-302 to examine its efficacy in treating soft tissue sarcoma and pancreatic cancer. However, in 2015, Merck announced the trial's failure. Threshold Pharmaceuticals stated that the failure of the TH-302 clinical trial indicated that the drug could not effectively target tumors in the long term and become a stable anti-tumor drug. Summary of the Invention

[0005] This invention provides a small molecule compound that can enter the brain or other central nervous system through hypoxia activation. Its mechanism of action is to release DNA alkylating agents through the above-mentioned hypoxia activation mechanism, thereby inhibiting and killing tumor cells.

[0006] Specifically, during the structural optimization and activity testing of similar small molecules, the inventors unexpectedly discovered that compounds with the following structures possess excellent anticancer activity and can be activated under hypoxic conditions:

[0007]

[0008] Experiments have demonstrated that these compounds possess strong in vitro inhibitory activity against cancer cell proliferation, particularly under hypoxic conditions. Furthermore, tests have shown that these compounds are not substrates of Pgp (P-Glycoprotein). Further experiments in serum, liver microsomes, and animal models have also shown that these compounds exhibit good stability and effective tumor clearance and inhibition. Compared to TH-302, the hypoxia-activated DNA alkylating agent provided by this invention is more suitable for development into a drug for treating cancers or tumors of the central nervous system, with enhanced efficacy.

[0009] Based on the above research findings, this invention provides the following hypoxia-activated DNA alkylating agent and clarifies its related anticancer and antitumor medicinal uses.

[0010] Hypoxia-activated DNA alkylating agents, which are compounds with the following structural formulas or their pharmaceutically acceptable salts or solvates or isotopic variants thereof.

[0011]

[0012]

[0013] This invention provides pharmaceutically acceptable salts of the above-mentioned compounds, wherein the salts may be basic salts, including salts formed by the compounds with inorganic bases (e.g., alkali metal hydroxides, alkaline earth metal hydroxides, etc.) or with organic bases (e.g., monoethanolamine, diethanolamine, or triethanolamine, etc.). Alternatively, the salts may be acidic salts, including salts formed by the compounds with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid, etc.) or with organic acids (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.). The selection and preparation of acceptable salts and solvates of the compounds are well known in the art.

[0014] The compounds described herein may also be used in the form of solvates, i.e., the present invention provides pharmaceutically acceptable solvates of the above-described compounds, wherein the solvates are hydrates, alcohols, etc., and alcohols include ethanol compounds.

[0015] Because drug APIs may combine with solvents during production, purification, and refining processes, they may eventually form solvates.

[0016] The isotopic variants correspond to deuterated compounds selected from the following structures:

[0017]

[0018] Each of the nine A's is either H or D, and at least one of the nine A's is D.

[0019] The isotopic variants correspond to deuterated compounds selected from the following structures:

[0020]

[0021]

[0022]

[0023] The present invention also provides a drug containing the above-described DNA alkylating agent.

[0024] Drugs containing the aforementioned DNA alkylating agents are used to treat cancer or tumors, or conditions caused by cancer or tumors, or proliferative diseases.

[0025] Cancers or tumors include: lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland carcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, osteocyte carcinoma, epithelial carcinoma, bile duct carcinoma, choriocarcinoma, embryonic carcinoma, seminoma, Wilms' carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, etc. Hematocytoma, vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystoma, fibromixoma, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibromyxosarcoma, fibropapillary tumor, myxosarcoma, myxosarcoma, myxochondroma, myxochondrosarcoma, myxochondrofibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipadenoma, lipoblastoma, lipochondroma, lipofuscinoma, lipofibroma, lipoangioma, myxolipoma, chondrosarcoma, cartilage Tumors, chondromas, chordomas, chorioamisomas, chorioepithelial tumors, choriocartilaginous tumors, osteosarcomas, osteoblastomas, osteochondral fibromas, osteochondral sarcomas, osteochondromas, bone cysts, odontomas, osteofibromas, osteofibrosarcomas, angiosarcomas, hemangiomas, angiolipomas, angiochondromas, hemangioblastomas, angiokeratomas, angioneurotic gliomas, hemangioendotheliomas, angiofibromas, angiomyomas, angiolipomas, angiolymphangiomas, angiolipoleiomyomas, angiomyolipomas, angioneurotic neuromas, angiomyxomas, hemangioretic endotheliomas, lymphangiosarcomas, lymphatic vessels Lymphangioma, lymphoma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, synovoma, synovial sarcoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyosarcoma, rhabdomyomyoma, rhabdomyosarcoma, rhabdomyomyxoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic anemia, polycythemia vera, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma, or multiple myeloma.

[0026] Preferably, the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, or stomach cancer.

[0027] Furthermore, the cancer or tumor is a primary brain cancer, tumor, or metastatic cancer or tumor that has spread to the brain.

[0028] The above-mentioned DNA alkylating agents are used in the preparation of medicines for treating cancer or tumors or diseases caused by cancer or tumors or proliferative diseases.

[0029] Furthermore, the patient with the cancer or tumor is a patient with impaired DNA repair.

[0030] The patient with the cancer or tumor is a patient with impaired DNA repair. Impaired DNA repair is defined as one or more of the following: impaired homologous recombination repair enzyme, impaired nucleotide excision repair enzyme, impaired non-homologous end ligase, impaired base excision repair enzyme, impaired mismatch repair enzyme, or impaired at least one repair enzyme in the Fanconi anemia pathway.

[0031] Furthermore, the patient with the cancer or tumor is a patient with a BRCA2 gene mutation.

[0032] The present invention also provides the use of the above-mentioned DNA alkylating agent in the preparation of medicaments for treating cancer or tumors or diseases caused by cancer or tumors or proliferative diseases.

[0033] Among them, cancers or tumors are preferably non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, and stomach cancer.

[0034] The patients with cancer or tumors mentioned are those with impaired DNA repair.

[0035] The patient with the cancer or tumor is a patient with impaired DNA repair. Impaired DNA repair is one or more of the following: impaired homologous recombination repair enzyme, impaired nucleotide excision repair enzyme, impaired non-homologous end ligase, impaired base excision repair enzyme, impaired mismatch repair enzyme, or impaired at least one repair enzyme in the Fanconi anemia pathway.

[0036] The patients with cancer or tumors mentioned are those with BRCA2 gene mutations.

[0037] Meanwhile, the hypoxia-resistant anticancer compound in this invention can enhance the anticancer effect through a combination of four anticancer drugs.

[0038] One approach is to use it in combination with traditional chemotherapy drugs. Traditional chemotherapy drugs can only kill tumor cells that are in a normoxic environment, but are ineffective against hypoxic cells. These traditional chemotherapy drugs include docetaxel (Vanhoefer U, Cao S, Harstrick A, Seeber S, Rustum YM (1997) Comparative antitumor efficacy of docetaxel and paclitaxel in nude mice bearing human tumor xenografts that overexpress the multidrug resistance protein (MRP). Ann Oncol 8:1221-1228), cisplatin (Bigioni M, Benzo A, Irrissuto C, Lopez G, Curatella B, Maggi CA, Manzini S, Crea A, Caroli S, Cubadda F, Binaschi M (2008) Antitumor effect of combination treatment with sabarubicin (MEN 10755) and cis-platin (DDP) in human lung tumor xenograft. Cancer Chemother Pharmacol 62:621-629), pemetrexed (Huber PE, Bischof M, Jenne J, Heiland S, Peschke P, Saffrich R, Grone HJ, Debus J, Lipson KE, Abdollahi A (2005) Trimodal cancer treatment: beneficial effects of combined antiangiogenesis, radiation, and chemotherapy.Cancer Res 65:3643-3655), irinotecan (Houghton JA, Cheshire PJ, Hallman JD, 2nd, Lutz L, Luo X, Li Y, Houghton PJ (1996) Evaluation of irinotecan in combination with 5-fluorouracil or etoposide in xenograft models of colon adenocarcinoma and rhabdomyosarcoma. Clin Cancer Res 2:107-118), doxorubicin (Kraus-Berthier L, Guilbaud N, Jan M, Saint-Dizier D, Rouillon MH, Burbridge MF, Pierre A, Atassi G (1997) Experimental antitumour activity of S 16020-2 in a panel of human tumours. Eur J Cancer 33:1881-1887), gemcitabine (Merriman RL, Hertel LW, Schultz RM, Houghton PJ, Houghton JA, Rutherford PG, Tanzer LR, Boder GB, Grindey GB (1996) Comparison of the antitumor activity of gemcitabine and ara-C in a panel of human breast, colon, lung and pancreatic xenograft models. Invest New Drugs 14:243-247; Teicher BA, Chen V, Shih C, Menon K, Forler PA, Phares VG, Amsrud T (2000) Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts.Clin Cancer Res 6:1016-1023), temozolomide (Middleton MR, Thatcher N, McMurry TB, McElhinney RS, Donnelly DJ, Margison GP (2002) Effect of O6-(4-bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model. Int J Cancer 100:615-617) and hypoxia-activated prodrugs can kill all tumor cells when used in combination (Liu et al, TH-302, ahypoxia-activated prodrug with broad in vivo preclinical combination therapy efficacy: optimization of dosing regimens and schedules, Cancer ChemotherPharmacol, 2012, 69:1487-1498, DOI: 10.1007 / s00280-012-1852-8). .

[0039] Second, when used in combination with anti-angiogenic drugs, it leads to an increase in the hypoxic region of tumor cells, thereby increasing the number of tumor cells sensitive to hypoxic compounds (Chang et al, Sorafenib (BAY43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models, Cancer Chemother Pharmacol, 2007, 59(5):561-74.DOI: 10.1007 / s00280-006-0393-4). Anti-angiogenic drugs that have been experimentally proven include sorafenib (Chang et al., Sorafenib (BAY43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models, Cancer ChemotherPharmacol, 2007, 59(5):561-74.DOI: 10.1007 / s00280-006-0393-4), rapamycin (Su D, Stamatakis L, Singer EA, Srinivasan R. Renal cell carcinoma: molecular biology and targeted therapy. Curr Opin Oncol 2014(26):321-7), and mTOR inhibitors (Sun et al. Combination treatment with hypoxia-activated prodrug evofosfamide (TH-302) and mTOR inhibitors results in enhanced antitumor efficacy in preclinical renal Cell carcinoma models, Am J Cancer Res. 2015(5):2139). These drugs, when used in combination with or in combination with the hypoxia compounds of the present invention, can increase the hypoxia region of tumor cells, thereby increasing the number of hypoxia compound-sensitive tumor cells, and thus achieving a synergistic anti-cancer effect through combined drug use.

[0040] Third, when used in combination with cell checkpoint inhibitors, it leads to increased sensitivity of tumor cells to DNA alkylating agents (Menget et al, Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition, BMCCancer, 2015, 15:422, DOI: 10.1186 / s12885-015-1387-6). Experimentally proven cell checkpoint inhibitors include LY2603618 (Wang FZ, Fei HR, Cui, YJ et al. The checkpoint 1 kinase inhibitor LY2603618 induces cell cycle arrest, DNA damage response and autophagy in cancer cells. Apoptosis, 2014(19):1389-1398), PF477736 (Balsina et al. Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase, Mol Cancer Ther 2008; 7(8):2394-404), and AZD7762 (Morgan et al. Mechanism of radiosensitization by the Chk1 / 2 inhibitor AZD7762 involves abrogation of the G2 checkpoint and inhibition of homologous recombinational DNA repair, Cancer Research). 2010(70):4972), these drugs, when used in combination with or in combination with the hypoxia compounds of the present invention, can increase the sensitivity of tumor cells to DNA alkylating agents (the present invention is a prodrug compound, the original drug that takes effect after metabolism is a DNA alkylating agent), thereby enhancing the effect of the DNA alkylating agent of the present invention, and thus achieving a synergistic anti-cancer effect through combined drug use.

[0041] Fourth, it can be used in combination with immunosuppressants. Hypoxia compounds can reduce hypoxic areas of tumors, thereby increasing lymphocyte infiltration in hypoxic areas and leading to an enhanced effect of immunotherapy (Jayaprakash et al, Targeted hypoxia reduction restores T cell infiltration and sensitizes prostate cancer to immunotherapy, JCI, 2018(128):5137, DOI:10.1172 / JCI96268.). Immunosuppressant drugs that have been experimentally proven include αCTLA-4 / αPD-1 (Curran et al. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci USA. 2010, 107(9):4275-4280). These drugs, when used in combination with or in combination with the hypoxia compounds of this invention, can increase lymphocyte infiltration in hypoxic areas, thereby enhancing the effect of immunotherapy.

[0042] This invention also provides a compound drug containing the above-mentioned DNA alkylating agent and

[0043] a. Traditional chemotherapy drugs; or

[0044] b. Anti-angiogenic drugs; or

[0045] c, cell checkpoint inhibitors; or

[0046] d, Immunosuppressants.

[0047] The above-mentioned compound can be a compound containing two active ingredients, consisting of the DNA alkylating agent of the present invention and any one of a, b, c, and d; it can also be a compound containing three active ingredients, consisting of the DNA alkylating agent of the present invention and any two of a, b, c, and d; it can also be a compound containing four active ingredients, consisting of the DNA alkylating agent of the present invention and any three of a, b, c, and d; or it can be a compound containing five active ingredients, consisting of the DNA alkylating agent of the present invention and four of a, b, c, and d.

[0048] The present invention also provides a combination therapy for treating cancer or tumors, comprising: administering to a patient a drug containing the aforementioned DNA alkylating agent; and

[0049] a. Administering traditional chemotherapy drugs; or

[0050] b. Administer anti-angiogenic drugs; or

[0051] c. Administer cell checkpoint inhibitors; or

[0052] d. Administer immunosuppressants.

[0053] The above-mentioned combination therapy is a combination therapy in which the patient is given the DNA alkylating agent of the present invention and any one of the drugs a, b, c, and d. The order of administration can be either the DNA alkylating agent first or any one of the drugs a, b, c, and d first; or a combination therapy in which the patient is given the DNA alkylating agent of the present invention and any two of the drugs a, b, c, and d, and the order of administration can be arbitrary; or a combination therapy in which the patient is given the DNA alkylating agent of the present invention and any three of the drugs a, b, c, and d, and the order of administration can be arbitrary; or a combination therapy in which the patient is given the DNA alkylating agent of the present invention and four of the drugs a, b, c, and d, and the order of administration can be arbitrarily adjusted.

[0054] The concepts of traditional chemotherapy drugs, anti-angiogenic drugs, cell checkpoint inhibitors, and immunosuppressants are as described above.

[0055] This invention also provides a method for preparing an oxygen-activated DNA alkylating agent with the following structure I, comprising causing compound I-1 to undergo a cyclization reaction to obtain the corresponding compound I:

[0056]

[0057]

[0058] Wherein, X is independently F, Cl, Br, or I. Preferably, X is always Br.

[0059] Preferably, the ring-closing reaction uses silver oxide or silver nitrate as a catalyst, and a base may or may not be added during the reaction, wherein the base is preferably an organic base.

[0060] Preferably, the organic base is N,N-diisopropylethylamine (DIEA) or triethylamine (TEA).

[0061] This invention also provides compounds having the following I-1 structure:

[0062]

[0063] Where X is independently F, Cl, Br, or I.

[0064] The use of compound I-1 as an intermediate in the synthesis of hypoxia-activated DNA alkylating agents.

[0065] A method for synthesizing compounds with the following I-1 structure includes reacting compound I-2 as a starting reactant with phosphorus oxychloride POCl3 in a first reaction to obtain an intermediate, followed by a second reaction of the intermediate with a haloethylamine or a hydrohalide of a haloethylamine, finally yielding the corresponding compound I-1:

[0066]

[0067] Where X is independently F, Cl, Br, or I.

[0068] Preferably, the haloethylamine is bromoethylamine, and the hydrohalate of the haloethylamine is the hydrobromide of bromoethylamine.

[0069] Methods for preparing compounds with the following I-2 structure:

[0070]

[0071] Compound I-3 was reacted with TMSCF3, followed by deprotection and hydrolysis to yield racemic alcohols I-2-1 and I-2-4. Chiral resolution of racemic alcohol I-2-1 yielded chiral alcohols I-2-2 and I-2-3, respectively.

[0072]

[0073] Preferably, the deprotection reagent used in the deprotection operation is tetrabutylammonium fluoride (TBAF), and the hydrolysis operation uses an aqueous solution of ammonium fluoride, ammonium chloride, or hydrochloric acid.

[0074] Preferred methods for chiral resolution include crystallization and chemical resolution.

[0075] The present invention also provides a method for dispensing compounds A method for separating racemic compounds to obtain one of the enantiomers or to concentrate any enantiomer of the compound in excess in a mixture, comprising the steps of: a) subjecting the racemic compound to optical analysis, wherein the process is carried out by chiral chromatography comprising a stationary phase and a mobile phase, wherein the stationary phase comprises silica gel impregnated with a functionalized polysaccharide, and wherein the mobile phase comprises an alcohol and another solvent.

[0076] Preferably, the alcohol is ethanol and the other solvent is n-hexane.

[0077] In the chiral chromatography process, the column temperature is 38℃.

[0078] The functionalized polysaccharide is amylose-tris(3,5-dimethylcarbamate), meaning the stationary phase is silica gel particles coated with amylose-tris(3,5-dimethylcarbamate).

[0079] A method for preparing deuterated compounds with the following structure I-4, comprising subjecting compound I-5 to a cyclization reaction to obtain the corresponding compound I-4.

[0080]

[0081] Wherein, X is independently F, Cl, Br, or I, preferably Br;

[0082] The ring-closing reaction uses silver oxide or silver nitrate as a catalyst, with or without the addition of an organic base.

[0083] A method for preparing the following I-5 structure includes a first reaction of compound I-2 with phosphorus oxychloride POCl3 to obtain an intermediate, followed by a second reaction of the intermediate with the corresponding deuterated haloethylamine I-6 or a hydrohalide of the deuterated haloethylamine, finally yielding the corresponding compound I-5:

[0084]

[0085] Where X is independently F, Cl, Br, or I;

[0086] The halogen in the hydrohalate of deuterated haloethylamine is the same as that in deuterated haloethylamine.

[0087] Preferably, the haloethylamine is bromoethylamine, and the hydrohalate of the haloethylamine is the hydrobromide of bromoethylamine.

[0088] Regarding the pharmaceuticals or preparations described herein, the resulting pharmaceuticals contain the indicated compounds or their salts or solvates within a specific dosage range, and / or the resulting pharmaceuticals are administered in a specific dosage form and via a specific route of administration.

[0089] Regarding the uses described herein, the resulting pharmaceutical product 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 route of administration, 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.

[0090] Preferably, the patient is a mammal, more preferably a human. Attached Figure Description

[0091] Figure 1 This is the chiral HPLC spectrum of compound A.

[0092] Figure 2 This is the chiral HPLC spectrum of the optical isomer corresponding to peak 1 obtained after chiral separation of compound A.

[0093] Figure 3 This is the chiral HPLC spectrum of the optical isomer corresponding to peak 2 obtained after chiral separation of compound A.

[0094] Figure 4 This is a schematic diagram of the absolute stereoconfiguration of the optical isomer corresponding to peak 1 obtained after chiral separation of compound A.

[0095] Figure 5 The curves show the inhibition rates of three compounds, A, B, and C, on H460 cells at different concentrations under normoxic conditions. Here, inhibition is the inhibition rate, and Log.con(nM) represents the logarithm of the concentration value in nmol / L with base 10.

[0096] Figure 6 The curves show the inhibition rates of different concentrations of TH-302 and compounds A (left), D (middle), and TH-302 (right) on H460 cells under normoxic air and hypoxic N2 conditions. In the figure, inhibition is the inhibition rate, and Log.con (μM) represents the logarithm of the concentration value in μmol / L with base 10. The curves on the left represent the data under hypoxic conditions, and the curves on the right represent the data under normoxic conditions.

[0097] Figure 7 The curves show the inhibition rates of different concentrations of TH-302 and compounds A (left), D (middle), and TH-302 (right) on HepG2 cancer cells under normoxic air and hypoxic N2 conditions. In the figure, inhibition is the inhibition rate, and Log.con (μM) represents the logarithm of the concentration value in μmol / L with base 10. The curves on the left are the data under hypoxic conditions, and the curves on the right are the data under normoxic conditions.

[0098] Figure 8 The curves show the inhibition rates of different concentrations of compound A on AA8 and UV41 cells under normoxic air conditions. Here, inhibition is the inhibition rate, and Log.con(μM) represents the logarithm of the concentration value in μmol / L with base 10. The curves on the left represent data under hypoxic conditions, and the curves on the right represent data under normoxic conditions.

[0099] Figure 9The average remaining percentages of TH-302 and compound A at different times were determined by adding 53.2 μg / ml human NADPH coenzyme to the sample after reducing TH-302 and compound A (compound 1 in the figure) under hypoxic (hypoxia < 0.1% O2) and air conditions at 37 °C.

[0100] Figure 10 The average remaining percentages of TH-302 and compound A (compound 1 in the figure) at different times after adding 104.6 μg / ml human NADPH coenzyme to reduce TH-302 and compound A (compound 1 in the figure) under hypoxia (hypoxia < 0.1% O2) and air conditions at 37℃.

[0101] Figure 11 The graph shows the tumor volume changes over different days under different dosing regimens of compound A in a PDX gastric cancer mouse model experiment. The horizontal axis represents the number of days, and the vertical axis represents the volume in mm. 3 number.

[0102] Figure 12 The graph shows the change in body weight of mice relative to the starting weight under different administration regimens of compound A in a PDX gastric cancer mouse model experiment. The horizontal axis represents the number of days, and the vertical axis represents the percentage change in body weight.

[0103] Figure 13 The graph shows the tumor volume changes over different days under different administration regimens of compound A in CDX mouse models transplanted from H460 cell line. The horizontal axis represents the number of days, and the vertical axis represents the volume in mm3.

[0104] Figure 14 The graph shows the change in body weight of mice relative to the starting weight under different administration regimens of compound A in CDX mouse models transplanted from H460 cell line at different times. The horizontal axis represents the number of days, and the vertical axis represents the percentage change in body weight.

[0105] Figure 15 The graph shows the tumor volume changes over different days under different administration regimens of compounds B and C in a CDX mouse model with H460 cell line transplantation. The horizontal axis represents the number of days, and the vertical axis represents the tumor volume in mm. 3 number.

[0106] Figure 16 The graph shows the change in body weight of mice relative to the starting weight under different administration regimens of compounds B and C in CDX mouse models transplanted from H460 cell line at different times. The horizontal axis represents the number of days and the vertical axis represents the percentage change in body weight.

[0107] Figure 17This is a graph showing the tumor volume change at different days in an in vivo pharmacodynamic study of compound C and sorafenib as monotherapy and in combination in a human HepG2 subcutaneous xenograft model of liver cancer. The horizontal axis represents the number of days, and the vertical axis represents the volume in mm. 3 number.

[0108] Figure 18 The graphs show the changes in mouse body weight relative to the starting point at different days in the in vivo pharmacodynamic experiments of compound C and Sorafenib as monotherapy and in combination in a human HepG2 subcutaneous xenograft model of liver cancer. The horizontal axis represents the number of days, and the vertical axis represents the percentage change in body weight.

[0109] Figure 19 For the use of compound C and anti-mouse-PD1 antibody as monotherapy and in combination with compound C and anti-mouse-PD1 antibody in murine colorectal cancer

[0110] Survival rate curves at different days in the drug efficacy experiment in the CT26 subcutaneous model.

[0111] Figure 20 For the use of compound C and anti-mouse-PD1 antibody as monotherapy and in combination with compound C and anti-mouse-PD1 antibody in murine colorectal cancer

[0112] The curves showing the change in body weight of mice relative to the initial weight at different days in the drug efficacy experiment of the CT26 subcutaneous model, where the horizontal axis represents the number of days and the vertical axis represents the percentage change in body weight.

[0113] Figure 21 For the use of compound C and Gemcitabine as monotherapy and in combination therapy Tumor volume change curves at different days in an in vivo drug efficacy experiment in a PA3546 human pancreatic cancer subcutaneous xenograft PDX tumor model. The horizontal axis represents the number of days, and the vertical axis represents the volume in mm. 3 number.

[0114] Figure 22 For the use of compound C and Gemcitabine as monotherapy and in combination therapy The curves showing the change in body weight of mice relative to the starting weight at different days in the in vivo drug efficacy experiment of the PA3546 human pancreatic cancer subcutaneous xenograft PDX tumor model, where the horizontal axis represents the number of days and the vertical axis represents the percentage change in body weight.

[0115] Figure 23 The graphs show the fluorescence intensity changes of tumors at different days in the in vivo pharmacodynamic experiment of compound C as a single drug in the NCI-H460-Luc2 intracranial inoculation model. The horizontal axis represents the number of days, and the vertical axis represents the volumetric fluorescence intensity (the intensity indicates the tumor size).

[0116] Figure 24 The graph shows the change in mouse body weight relative to the starting weight at different days in the in vivo pharmacodynamic experiment of compound C monotherapy in the NCI-H460-Luc2 intracranial inoculation model. The horizontal axis represents the number of days and the vertical axis represents the percentage change in body weight.

[0117] Figure 25 This is a graph showing the concentration changes of compounds B and C in blood and brain tissue over time.

[0118] Figure 26 The graph shows the test results of the ratio of the concentrations of compounds B and C in brain tissue and blood as a function of time.

[0119] The figure shows the change curves of compound A in the presence of NADPH after reacting with liver microsomes in different animals.

[0120] Figure 28 The curves show the changes in the concentration of compound A in the plasma of different animals over time. Detailed Implementation

[0121] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0122] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0123] The terms "patient" and "individual" are used interchangeably and refer to a mammal in need of cancer treatment. Typically, a patient is a human being diagnosed with cancer. In some embodiments, "patient" or "individual" may refer to a non-human mammal used to screen, characterize, and evaluate drugs and therapies, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0124] A "prodrug" is a compound that, upon administration or treatment, is metabolized or otherwise converted into a compound (or drug) with biological activity or greater activity in at least one property. Compared to a drug, a prodrug is chemically modified in a way that makes it less active or inactive relative to the drug, but the chemical modification allows the corresponding drug to be produced through metabolism or other biological processes after administration of the prodrug. Prodrugs may have altered metabolic stability or delivery characteristics, fewer side effects or lower toxicity, or modified flavor relative to the active drug (see, for example, reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392, which is incorporated herein by reference). Prodrugs can be synthesized using reactants other than the corresponding drug.

[0125] "Solid tumors" refer to solid tumors, including (but not limited to) primary or metastatic tumors in bone, brain, liver, lung, lymph nodes, pancreas, prostate, skin, and soft tissue (sarcoma).

[0126] The "therapeutic effective dose" of a drug refers to the amount of drug that, when administered or given to a patient with cancer, will have the expected therapeutic effect (e.g., alleviation, improvement, relief, or elimination of clinical manifestations of one or more cancers in the patient). The therapeutic effect does not necessarily occur with a single dose, but may occur only after a series of doses. Therefore, a therapeutic effective dose can be administered or given once or multiple times.

[0127] "Treatment" for a condition or patient refers to steps taken to achieve a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) the alleviation or improvement of one or more cancer symptoms; a reduction in disease severity; a delay or slowing of disease progression; an improvement, relief, or stabilization of the disease state; or other beneficial outcomes. In some cases, cancer treatment may result in a partial response or stabilization of the disease.

[0128] "Tumor cells" refers to tumor cells in any appropriate species (e.g., mammals, such as rats, dogs, cats, horses, or humans).

[0129] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

[0130] I. Synthesis, preparation, and structural determination of compounds

[0131] Explanation of English Abbreviations

[0132] THF, tetrahydrofuran; DCM, dichloromethane; EA, ethyl acetate; TEA, triethylamine; HPLC, high performance liquid chromatography; MTBE, methyl tert-butyl ether; DMAP, 4-dimethylaminopyridine; DBAD, di-tert-butyl azodicarboxylate; TFA, trifluoroacetic acid; MS, mass spectrometry; EtOH, ethanol; t-BuOH, tert-butanol; DMF, N,N-dimethylformamide; PE, petroleum ether; DMF-DMA, N,N-dimethylformamide dimethyl acetal; TBAF, tetrabutylammonium fluoride; DIPEA or DIEA, N,N-diisopropylethylamine; DIAD, diisopropyl azodicarboxylate; t-BuOK, potassium tert-butoxide.

[0133] Calculated, theoretically calculated value for mass spectrometry; found, measured value for mass spectrometry; eq, equivalent molar ratio.

[0134] All other abbreviations or terms not specified shall be interpreted or operated in accordance with the definitions or descriptions in the organic chemistry or organic synthesis handbook.

[0135] Unless otherwise specified, all reagents and medicines were purchased commercially.

[0136] 1 Unless otherwise specified, H-NMR tests are performed using a 400MHz instrument, and MS mass spectrometry tests are performed using an LC-MS liquid chromatography-mass spectrometry instrument.

[0137] 1.1 Synthesis, preparation and structural determination

[0138] 1.1.1 Synthesis and Preparation

[0139]

[0140] Synthetic route of compound A

[0141] Under nitrogen protection, 32-A1 (20.0 g, 167.9 mmol), CHCl3 (116.1 g, 335.9 mmol), tetrabutylammonium bromide (1.6 g, 1.68 mmol), and 40% NaOH (50 mL) were added to a 500 mL four-necked flask, and the mixture was heated to 62 °C until the reaction was complete. Post-treatment: After cooling to room temperature, the mixture was filtered through diatomaceous earth. The mother liquor was extracted with CHCl3, dried, concentrated, and preliminarily separated to obtain 32-A2 product (7.0 g, yield 34.0%), which was an off-white solid. 1 H-NMR (400MHz, DMSO): δ11.29 (s, 1H), 8.22 (d, J = 8.4Hz, 3H), 7.63-7.61 (m, 3H), 7.55-7.51 (m, 6H).

[0142] 32-A2 (6.0 g, 16.3 mmol), 1-nitronaphthalene (2.8 g, 16.3 mmol), and KOH (5.8 g, 102.9 mmol) were added to DMSO (80 mL) and reacted overnight at room temperature until the reaction was complete. Post-treatment: The system was poured into hydrochloric acid, precipitating a large amount of solid. The organic phase was dissolved and extracted with chloroform, dried, and concentrated to obtain 32-A3 (7.3 g), which was used directly in the next step without further purification.

[0143] Under nitrogen protection, 32-A3 (7 g crude, 16.33 mmol) and ZnBr2 (7.4 g, 32.66 mmol) were added to dioxane. After heating and stirring for a period of time, concentrated hydrochloric acid was added dropwise to the system, and the reaction was continued with heating and stirring until completion. Post-processing: After cooling to room temperature, the system was concentrated, dissolved in water, extracted with DCM, washed with alkaline solution, dried and concentrated, and separated by chromatography to obtain 32-A4 (1.3 g), a yellow solid. 1 H-NMR (400MHz, CDCl3): δ10.53 (s, 1H), 9.30 (dd, J1 = 7.2Hz, 2.0Hz, 1H), 8.37 (dd, J1 =8.0Hz, 1.8Hz, 1H), 8.17 (d, J = 7.6Hz, 1H), 8.09 (d, J = 7.6Hz, 1H), 7.86-7.80 (m, 2H).

[0144] Under nitrogen protection, 32-A4 (200 mg, 0.99 mmol) and TMSCF3 (283 mg, 1.98 mmol) were added to THF. The mixture was cooled to 0 °C, and TBAF (0.1 mL) was added dropwise to the system. The reaction continued until the starting material was completely converted. An appropriate amount of hydrochloric acid was added dropwise to the system. The mixture was extracted with DCM, and the organic phase was washed with water and brine. After drying and concentration, the product 32-A5 (190 mg, 70.5%) was obtained by column chromatography as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.49 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.40-7.32 (m, 2H), 5.98 (m, 1H), 2.98 (s, 1H).

[0145] Under nitrogen protection, POCl3 (210 mg, 1.36 mmol) was added to DCM, and the mixture was cooled to -30 °C. 32-A5 (185 mg, 0.68 mmol) was dissolved in DCM and added dropwise to the system. TEA (173 mg, 1.71 mmol) was dissolved in DCM and added dropwise to the system. The reaction was maintained at -30 °C until the starting material disappeared. Bromoethylamine hydrobromide (1.1 g, 5.46 mmol) and TEA (552 mg, 5.46 mmol) were added to the system, and the reaction was completed after a period of time. After adding a saturated ammonium chloride aqueous solution at 0 °C, the mixture was extracted with DCM. The organic phase was washed with water and brine, dried, concentrated, and then separated by column chromatography to obtain the 32-A6 product (170 mg, 44.3%), a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.49(d,J=1.2Hz,1H),8.26(d,J=7.6Hz,1H),8.17(d,J=8.0H z, 1H), 7.90 (d, J = 8.0Hz, 1H), 7.80-7.75 (m, 2H), 6.63-6.61 (m, 1H), 3.56-2.94 (m, 8H). MS: Calculated 562.9,found 563.9([M+H] + ).

[0146] Under nitrogen protection, 32-A6 (160 mg, 0.28 mmol) was dissolved in THF (15 mL), followed by the addition of silver oxide (329 mg, 1.42 mmol) and DIEA (167 mg, 1.42 mmol). The mixture was heated to 65 °C and stirred until the reaction was complete. The mixture was filtered through diatomaceous earth, the solid was washed with DCM, the mother liquor was concentrated, and the pure compound 32 (compound A) was obtained by high-performance liquid chromatography (HPLC), resulting in a pale yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.57-8.44(m,1H),8.31-8.10(m,2H),7.96(d,J=7.9Hz,1H),7 .84-7.67(m,2H),6.65(s,1H),2.33-2.21(m,2H),2.20-2.09(m,2H),2.06-1.96(m,4H). MS:Calculated 401.1,found 402.0([M+H] + ).

[0147] 1.1.2 Chiral resolution of compounds

[0148] Chiral analysis was performed on the pure sample of compound A (compound number 32).

[0149] The HPLC chiral analysis conditions are as follows:

[0150] Column size 0.46cm inner diameter, 15cm length Injection volume 0.5ul Mobile phase hexane:ethanol = 60 / 40 (volume ratio) Flow rate 1.0 ml / min

[0151] Temperature column temperature 35℃

[0152] The HPLC chromatogram obtained from the test is as follows Figure 1 The specific results are shown in the table below.

[0153] No. 1 2.908 5110974 47.096 No. 2 3.766 5741368 52.904

[0154] Take 4.9971 g of compound A, dissolve it in chromatographic grade ethanol, and perform chiral separation preparation using the following chiral separation preparation method and conditions.

[0155] HPLC chiral separation preparation method: The chiral isomers (i.e., compound A) were separated using HPLC equipment and a chiral column, and the corresponding fractions were collected. The solvent was removed by rotary evaporation to obtain the pure optical isomers.

[0156] The HPLC chiral separation preparation conditions are as follows:

[0157]

[0158] After separation and preparation, 2.5377 g of pure product of the optical isomer corresponding to peak 1 was obtained, with an ee value of 99.7%; 2.4653 g of pure product of the optical isomer corresponding to peak 2 was obtained, with an ee value of 99.4%.

[0159] Further analysis of the optical isomers corresponding to peak 1 and peak 2, prepared by chiral separation, was performed using HPLC chiral analysis conditions to obtain the following results: Figure 2 , 3 The result.

[0160] The HPLC analysis results of the optical isomer corresponding to peak 1 are shown in the table below:

[0161] No. 1 2.908 7125381 99.837 No. 2 3.758 11633 0.163

[0162] The HPLC analysis results of the optical isomer corresponding to peak 2 are shown in the table below:

[0163] No. 1 2.897 20323 0.320 No. 2 3.765 6340121 99.680

[0164] 1.1.3 Determination of the absolute configuration of the compound

[0165] The absolute configuration of compound 1 was determined using single-crystal X-ray diffraction results.

[0166] Single crystal culture. Weigh approximately 5 mg of the optical isomer sample corresponding to peak 1 above, dissolve it in methanol (2 mL, analytical grade), sonicate at room temperature for 1 minute, filter with an organic filter membrane, and let stand at room temperature for two days to obtain colorless crystals.

[0167] Single crystal structure analysis.

[0168] The above colorless crystal was subjected to single-crystal diffraction using the following X-ray single-crystal diffractometer (D8 Venture, Bruker):

[0169] Light source: Mo target; X-rays: Mo-K

[0170] Detector: CMOS surface detector; Resolution:

[0171] Current / Voltage: 50kV, 1.2A; Exposure Time: 30s

[0172] Detector-to-sample distance: 50mm; Test temperature: 298K (25℃) O C)

[0173] After data collection, the diffraction data were integrated and restored using the SAINT program, and then empirically absorbed and corrected using the SADABS program. The single crystal structure was analyzed by direct method using SHELXT2014, and the structure was refined using least squares method. The hydrogen atom refinement process was obtained by isotropic calculation, and the hydrogen atoms on CH were obtained by calculated hydrogen addition, and refined using a riding model. The Flack constant was obtained as 0.03(10) from the data, and the absolute configuration could be determined. The C5 configuration in the structure is an S configuration as shown in the figure. Figure 4 As shown.

[0174] Therefore, the optical isomer corresponding to peak 1 is the S-configuration, with the following structural formula:

[0175]

[0176] The structural formula of the optical isomer corresponding to peak 1.

[0177] Therefore, it can be concluded that the compound corresponding to peak 1 in the chiral HPLC separation spectrum of compound A (compound 32) is in the S configuration, which is compound B.

[0178] 1.2 Synthesis, preparation and structural determination

[0179] Preparation of compound 26 (i.e., compound D mentioned above)

[0180]

[0181] p-Toluenesulfonic acid (20.0 g, 112.0 mmol) was added to acetonitrile (160 mL), followed by 26-A1 (7.4 g, 39.32 mmol). The mixture was cooled to 0 °C, and an aqueous solution of KI (16 g, 98.30 mmol) and NaNO2 (5.4 g, 78.64 mmol) (24 mL) was added dropwise to the system. The reaction was completed in 2 h. The mixture was diluted with H2O (600 mL), and the pH was adjusted to 9-10 with 1 N NaHCO3 aqueous solution. Then, 2 M sodium thiosulfate solution (50 mL) was added, precipitating a large amount of solid. After filtration, the solid was dissolved in ethyl acetate, dried, concentrated, and separated by column chromatography (200-300 mesh silica gel, n-heptane:ethyl acetate = 20:1) to obtain 26-A2 product (11.0 g, yield 93.5%), which was a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.50 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 1.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.76-7.70 (m, 2H).

[0182] Under nitrogen atmosphere, 26-A2 (3.0 g, 10.00 mmol) and tributyl(1-ethoxyethylene)tin (5.0 g, 14.00 mmol) were added to dioxane (40 mL). After purging with nitrogen three times, tetrakis(triphenylphosphine)palladium (277 mg, 0.25 mmol) was added to the system. After purging with nitrogen three more times, the mixture was heated to 105 °C overnight. The reaction was complete after overnight incubation. The mixture was cooled to room temperature, and 50 mL of saturated ammonium chloride was added dropwise to quench the reaction. The mixture was extracted with ethyl acetate (50 mL x 4), concentrated, and then 100 mL of 3N HCl was added and stirred for 12 h. The mixture was then extracted with DCM (50 mL x 4), washed with water and brine, dried, concentrated, and separated by column chromatography (200-300 mesh silica gel, n-heptane:ethyl acetate = 20:1) to obtain 26-A3 product (1.1 g, yield 51.5%) as a yellow solid. 1 H-NMR (400M Hz, CDCl3): δ8.55(d,J=6.4Hz,1H),8.45(d,J=6.4Hz,1H),8.10(d,J=7.6Hz,1H),7.83(d,J=7.6Hz,1H),7.78-7.69(m,2H),2.77(s,3H). MS:Calculated215.1,found216.0([M+H] + ).

[0183] Under nitrogen protection, 26-A3 (200 mg, 0.93 mmol) was dissolved in THF (5 mL), cooled to 0 °C, and BH3-THF (4.7 mL, 4.65 mmol) was added dropwise to the system. The reaction was completed after 30 min. The mixture was then cooled to 0 °C, quenched by adding MeOH (6 mL), concentrated, dissolved in DCM, washed three times with 1 N HCl, dried, and concentrated to obtain 26-A4 product (185 mg, yield 91.6%), which was a yellow oil. 1 H-NMR(400MH z, CDCl3): δ8.55 (d, J = 6.4Hz, 1H), 8.20-8.11 (m, 2H), 7.79 (d, J = 6.4Hz, 1H), 7.73-7.62 (m, 2H), 5.75-5.70 (m, 1H), 1.66 (d, J = 6.4Hz, 3H). MS:Calculated217.1,found218.0([M+H] + ).

[0184] Under nitrogen protection, 26-A4 (650 mg, 2.99 mmol), Br-IPM (1.4 g, 4.49 mmol), and PPh3 (1.8 g, 5.99 mmol, commercially available) were added to THF (50 mL). The mixture was cooled to 0 °C, and DIAD (1.2 g, 5.99 mmol, commercially available) was added dropwise to the system. After the reaction was completed at 0 °C for 30 min, 50 mL of water was added dropwise to the system. The mixture was extracted with DCM (30 mL x 3), and the organic phase was washed with water and brine, dried, concentrated, and then separated by column chromatography (200-300 mesh silica gel, n-heptane:ethyl acetate = 1:1) to obtain 26-A5 product (400 mg, yield 26.3%), which was a yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.52 (d, J = 6.4Hz, 1H), 8.26-8.15 (m, 2H), 7.75-7.68 (m, 2 H),7.54-7.52(m,1H),6.35-6.28(m,1H),3.49-3.15(m,8H),1.79(d,J=6.4Hz,3H).

[0185] Under nitrogen protection, 26-A5 (250 mg, 0.49 mmol) was dissolved in THF (25 mL), followed by silver oxide (569 mg, 2.46 mmol) and DIEA (217 mg, 2.46 mmol). The mixture was heated to 65 °C and stirred overnight until the reaction was complete. The mixture was filtered through diatomaceous earth, the solid was washed with DCM, the mother liquor was concentrated, and compound 26 (30.7 mg, 18.1%) was prepared by high-performance liquid chromatography. It is a yellow, oily substance. 1HN MR(400MHz, CDCl3): δ8.56-8.54(m,1H),8.208.17(m,2H),7.81(d,J=8.0Hz,1H) ,7.74-7.67(m,2H),6.57-6.50(m,1H),2.25-1.99(m,8H),.79(d,J=6.4Hz,3H). MS: Calculated347.1, found348.0([M+H] + ).

[0186] The two chiral isomers of compound 26 can be obtained and identified by performing chiral resolution and chiral synthesis operations similar to those used for compound 32. and configuration.

[0187] 1.3 Synthesis and Preparation of Deuterated Compounds

[0188] The following synthetic route and steps were used to prepare the deuterated compound I-4.

[0189] Step 1: Synthesize the following I-5 structure deuterated compound, which involves reacting compound I-2 as the starting reactant with phosphorus oxychloride POCl3 to obtain an intermediate, followed by a second reaction of the intermediate with the corresponding deuterated haloethylamine I-6 or its hydrohalate, finally yielding the corresponding compound I-5:

[0190]

[0191] Where X is independently F, Cl, Br, I; the halogen in the hydrohalate of deuterated haloethylamine is the same as the halogen in deuterated haloethylamine.

[0192] Preferably, the haloethylamine is bromoethylamine, and the hydrohalate of the haloethylamine is the hydrobromide of bromoethylamine.

[0193] Step two involves preparing a deuterated compound with structure I-4, which includes causing compound I-5 to undergo a cyclization reaction to obtain the corresponding compound I-4.

[0194]

[0195] Wherein, X is independently F, Cl, Br, or I, preferably Br;

[0196] The ring-closing reaction uses silver oxide or silver nitrate as a catalyst, with or without the addition of an organic base.

[0197] For specific procedures, please refer to the steps outlined in compounds 26 and 32 above.

[0198] The following deuterated compounds:

[0199]

[0200]

[0201] By using the compound after I-2 deuteration

[0202]

[0203] It can be obtained by using it as the starting material in step one and performing appropriate group protection and deprotection operations.

[0204] The meanings of foreign abbreviations or terms used in in vivo and in vitro experiments in Sections 2 through 8 below are explained according to the meanings of terms in textbooks of medicinal chemistry, biochemistry, physiology or pharmacology, and medicine.

[0205] II. Compound Inhibition Tests on Cancer Cells

[0206] The IC50 value, a quantitative measure of cytotoxicity of human tumor cell lines in vitro, was used. 50 To compare the inhibitory effects of compounds on cancer cell proliferation.

[0207]

[0208] Unless otherwise specified, the testing method is as follows:

[0209] The compounds were exposed at various concentrations for 72 hours, followed by a washing step and the addition of fresh culture medium. Growth and cell viability were then stained and compared with a control treated only with culture medium.

[0210] Specifically, with 2×10 3 Cells growing exponentially were seeded into 96-well plates at a density of 100 μl / well and incubated at 37°C for 24 hours in 5% CO2, 95% air, and 100% relative humidity. Then, 99 μl of culture medium was added. The compound was dissolved in 100% DMSO at 200 times the desired final test concentration. When adding the drug, an aliquot of 1 μl of the specific concentration of the compound was added to a microwell containing 199 μl of culture medium to obtain the reported final drug concentration. After drug addition, the plates were incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for another 72 hours. The cell-tested plates were allowed to equilibrate to room temperature for 30 minutes, and 100 μl of culture medium was discarded from each well. 100 μl of CTG reagent (CelltiterGlo kit) was added to each well, and the plates were shaken rapidly for 2 minutes and incubated at room temperature in the dark for 30 minutes. The chemiluminescence signal was read using an Envision instrument. The drug concentration (IC50) resulting in 50% growth inhibition was calculated using computer software. 50 The results are listed in the table below.

[0211] Table 1: Inhibitory effect of three compounds on H460 cancer cells (IC50) 50 data

[0212] A 128 B 103.9 C 76.85

[0213] The IC obtained from its test 50 Curves Figure 5 As shown.

[0214] Test results show that the two optical isomers (compounds B and C) have similar H460 inhibitory toxicity to the racemic mixture (compound A).

[0215] Further testing of compound A's cytotoxicity against different non-small cell lung cancer (NSCLC) cell lines was conducted, and the results are shown in Table 2 below:

[0216] Table 2: Inhibitory effect of compound A on different non-small cell lung cancer (NSCLC) cell lines (IC50) 50 data

[0217] NCI-H1944 538.1 NCI-H2228 263.9 NCIH1755 6465 NCI-H1563 4451 CAL12T 30470 NCIH2106 14778 NCI-H23 6409 NCI-H522 19155 NCI-H2110 382 NCI-H1792 4785

[0218] Further testing of compound A's cytotoxicity against different pancreatic cancer PA cells yielded the results shown in Table 3 below:

[0219] Table 3: Inhibitory effect of compound A on different pancreatic cancer PA cells (IC50) 50 data

[0220] AsPC-1 12575 AGP-1 6271 HPAF-II 26058 KP-2 29741 CFPAC1 2963 KP-3 26165 Capan-1 5970 Panc 02.03 45682 MIA PaCa-2 6900 PL45 17085 Panc 10.05 27735 PANC-1 29293 KP-4 6099

[0221] III. Hypoxia-dependent cytotoxicity experiments

[0222] 3.1 Proliferation Inhibition Experiments of Different Cell Lines

[0223] To determine the effect of drug A of the present invention on cancer cell proliferation, the anti-proliferative activity of these compounds on cancer cells was detected in a porous CTG-based analysis. The effects of the test compounds on cell proliferation were compared under normoxic (21% O2) and hypoxic (<0.01% O2) conditions. H460 cells were routinely cultured to 80%-90% cell saturation. Once the desired cell count was reached, the cells were resuspended in the appropriate culture medium, counted, and prepared into cell suspensions of suitable density. The cell suspensions were added to two types of 24-well plates, 495 μL per well, at a cell density of 1 × 10⁴ / well. 24-well plates with glass inserts were used for hypoxic experiments, and ordinary plastic 24-well plates were used for normoxic experiments. 5 μL of each compound was added to both the hypoxic and normoxic reaction systems. After incubation for 3 hours in a hypoxic workstation or a normoxic incubator, the compounds were washed away with culture medium. The cells were then incubated for another 72 hours. The final detection concentrations of each compound under normoxic conditions are as follows: TH-302: 1000, 200, 40, 8, 1.6, 0.32 μM; Compound A (compound 32): 1000, 200, 40, 8, 1.6, 0.32 μM; Compound 26: 500, 50, 5, 0.5, 0.05 μM. The final detection concentrations of each compound under hypoxic conditions are as follows: TH-302: 10, 2, 0.4, 0.08, 0.016, 0.0032 μM; Compound A (compound 32): 50, 5, 0.5, 0.05, 0.005, 0.0005 μM; Compound 26: 500, 50, 5, 0.5, 0.05 μM. After 72 hours of cell culture, in 24-well plates, discard 300 μL of culture medium from each well, add 50 μL of CTG, vortex for 2 min, and incubate at room temperature in the dark for 15 min. Transfer 100 μL of culture medium from each well of the 24-well plate to a 96-well plate. Read the chemiluminescence signal values ​​using a multi-mode microplate reader at 1000 ms. Calculate the IC50 using GraphPad Prism 5 software. 50 Calculate the 50% growth inhibitory concentration (GI) of the test compound. 50 This article is also referred to as IC 50), and list them in Table 4 below. The compound TH-302 (a hypoxia-activated prodrug that has entered phase III clinical trials, CAS number 918633-87-1, see the literature Sun JD, Liu Q, Wang J, et al. Selective tumor hypoxia targeting by hypoxia-activated prodrug TH-302 inhibits tumor growth in preclinical models of cancer.[J]. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 2012, 18(3):758-70, structural formula is (As a comparison)

[0224] Table 4: Inhibitory effects of the two compounds on H460 cancer cells under hypoxic and normoxic conditions (IC50) 50 data

[0225] Compound A (32) 0.50 0.04 12.5 TH-302 13.60 0.02 680 Compound 26 0.80 0.04 20

[0226] IC obtained from the test 50 Curves Figure 6 As shown.

[0227] Compound A of the present invention exhibits stronger inhibitory effects on H460 cancer cells than TH-302 under normoxic conditions. Under hypoxic conditions, TH-302 shows stronger inhibitory effects on H460 cancer cells than compound A, and hypoxia-activated IC50 is also more potent. 50 The ratio reaches or exceeds 12.5.

[0228] Similarly, the inhibitory effects of the above three compounds on the proliferation of HepG2 liver cancer cells were tested, and the results are shown in Table 5 below.

[0229] Table 5: Inhibitory effects of the two compounds on HepG2 cancer cells under hypoxic and normoxic conditions (IC50) 50 data

[0230] Compound A (32) 20.59 4.78 4.31 TH-302 918.20 12.39 72.82 Compound D (26) 16.69 1.50 11.13

[0231] Note: HCR activation ratio under hypoxia, IC50 under normoxic / N2 conditions. 50 ratio

[0232] IC obtained from the test 50 Curves Figure 7 As shown.

[0233] Compound A of the present invention exhibits stronger inhibitory effects on HepG2 cancer cells than TH-302 under both normoxic and hypoxic conditions, and still retains hypoxia-activating properties.

[0234] To further compare the cytotoxicity of TH-302 with compound A, the following test results are provided for H460 cell lines tested at different times under the same laboratory, by the same operators, and under the same experimental conditions, as well as test results for different cell species.

[0235] The comparison data of test results of the same cell line at different times are shown in Table 6 below:

[0236] Table 6: Data on the inhibitory effects of TH-302 and Compound A on normoxic and hypoxic cancer cells.

[0237]

[0238] The comparison data of test results for cells from different species are shown in Table 7 below:

[0239] Table 7: Cell inhibition test data of TH-302 and compound A under normoxic conditions for different species

[0240]

[0241] Tables 6 and 7 show the cell inhibition test data at different time points, for different species, and for different cancer cell lines, which further demonstrate that compound A has a stronger inhibitory effect on cancer cells (under both normoxic and hypoxic conditions) than TH-302.

[0242] 3.2 Cytotoxicity assays were performed on the UV41 cell line with DNA repair gene mutations.

[0243] Furthermore, cytotoxicity experiments were conducted using the UV41 cell line, which has a DNA repair gene mutation.

[0244] Specifically, with 4×10 3Cell / well density: Exponentially growing cells were seeded in 96-well plates and incubated at 37°C for 24 hours in 5% CO2, 95% air, and 100% relative humidity, followed by the addition of the test compound. The compound was dissolved in 100% DMSO at a 200-fold desired final test concentration. Upon addition of the drug, the compound was further diluted to a 4-fold desired final concentration using complete culture medium. A 50 μl aliquot of the specific concentration of the compound was added to a microwell containing 150 μl of culture medium to obtain the reported final drug concentration. After drug addition, the plates were incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for another 2 hours, then the drug was washed off, fresh culture medium was added, and the plates were incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for another 70 hours. At the end of this incubation period, Alamar Blue assay was used to quantify viable cells. The drug concentration (IC50) resulting in 50% growth inhibition was calculated using computer software. 50 The results showed that compound A had an effect on the IC50 of the AA8 cell line. 50 The concentration was 10.85 μmol / L, while the IC50 for the UV41 cell line with DNA gene repair mutations was... 50 The concentration was 0.34 μmol / L, as shown in the results. Figure 8 As shown.

[0245] The UV41 cell line is a derivative of the CHO-AA8 cell line, derived from the UV-sensitive line of AA8. This cell line is a cell line with impaired homologous recombination repair enzymes and nucleotide excision repair enzymes caused by mutations in the DNAERCC4 / XPF gene.

[0246] Literature indicates that the UV41 cell line, compared to AA8, is a cell line deficient in nucleotide excision repair enzymes. It is sensitive to large adduct mutagens and belongs to excision repair complement group 4. UV41 is extremely sensitive to DNA cross-linking agents (see Thompson LH, et al. Repair of DNA adducts in asynchronous CHO cells and the role of repair in cell killing and mutation induction in synchronous cells treated with 7-bromomethylbenz[a]anthracene. Somatic Cell Mol. Genet. 10:183-194, 1984. PubMed:6584989; Thompson LH, et al. Genetic diversity of UV-sensitive DNA repair mutants of Chinese hamster ovary cells. Proc. Natl. Acad. Sci. USA 78:3734-3737, 1981. PubMed:6943579; Hoy CA, et al. Defective DNA cross-link removal in Chinese hamster ovary cells). Cell mutants hypersensitive to bifunctional alkylating agents.Cancer Res.45:1737-1743,1985.PubMed:3919945;Busch D,et al.Summary ofcomplementation groups of UV-sensitive CHO cell mutants isolated by large-scale screening.Mutagenesis 4:349-354,1989.PubMed:2687628;Bessho T,etal.Initiation of DNA interstrand cross-link repair in humans:the nucleotideexcision repair system makes dual incisions 5"to the cross-linked base andremoves a 22-to 28-nucleotide-long damage-free strand.Mol.Cell.Biol.17:6822-6830,1997.PubMed:9372913;Thompson LH,et al.Hypersensitivity to mutation andsister-chromatid-exchange induction in CHO cell mutants defective in incisingDNA containing UV lesions.Somatic Cell Genet.8:759-773,1982.PubMed:7163954)。.

[0247] Damage to DNA repair enzymes in UV41 cells can lead to the following enzymes being compromised: BRCA1, BRCA2, FANCA, FANCD1, FANCD2, ATM, ATR, CHEK1, CHEK2, CTP, BARD1, BRIP1, PALB2, RAD51D, RAD51C, RAD52, RAD54, RAD55, RAD57, FAM175, NBN, Rad50, MRE11, p53, NBS1, XRS2, XRCC2, XRCC3, XRCC4 / XPF, ERCC1, ERCC2 / XPD, ERCC3 / XPB, ERCC4 / XPF, XRCC1, Ku80, MHS6, MGMT, PARP, ERCC5 / XPG, CCNH, CDK7, CETN2, DDB1, DDB2, ERCC5 / XPG, ERCC6 / CSB, ERCC8 / CSA, and LIG1 / DNA Ligase. Mutations in any one or more of the genes corresponding to I, MMS19, MNAT1, RAD23A, RAD23B, RPA1, RPA2, TFIIH, XAB2, XPA, XPC, MBD4, NEIL1, BAP1, CDK12, EXO1, FAAP20, FAN1, FANCE, FANCM, MDC1, NONO, POLQ, RAD51B, RBBP8, SMC5, USP11, WRN, and AP endonucleases, End processing enzymes, DNA polymerases, and Flap endonuclease.

[0248] Cell lines with DNA repair gene mutations are more sensitive. Specifically, compound A is more sensitive to cell lines with impaired homologous recombination repair enzymes and impaired nucleotide excision repair enzymes.

[0249] 3.3 Hypoxia assay (clonal formation assay) of different cancer cell lines

[0250] The following cell types were seeded at specific numbers in 10cm glass cell culture plates: PSN-1, PANC1, MiaPaCa-2, H460, DLD-1WT (wild-type), and DLD-1 BRCA2 KO (BRCA2-deficient). The cells were incubated at 37°C for 2 days in 5% CO2, 95% air, and 100% relative humidity. On the third day, specific concentrations of the test compound TH302 or AST-002-P2 were added to the cells, and the cells were incubated for 3 hours under normoxic (21% O2) and hypoxic (<0.01% O2) conditions, respectively. After stimulation with the compounds, the cells were washed once with 1xPBS, then digested into single cells using trypsin, and cell counts were performed. The specific cell numbers were reseeded in new culture plates and cultured for 7 days to allow for single-cell clonal formation. When the single cells in the normoxic-stimulated compound culture plate reached approximately 50 cells, the culture was removed and stained with 0.5% crystal violet for 40 minutes. After staining, cell clones were counted using a Colony Counter (VWR), and IC50 was calculated. 90 The calculation.

[0251] The results are shown in Table 8 below. Compound C exhibited significantly different inhibitory effects on MiaPaCa-2 and DLD-1 BRCA2KO cells under normoxic and hypoxic conditions, with fold changes (HCR) reaching >63-fold and >89-fold, respectively. This indicates that hypoxic conditions effectively activate the cytotoxicity of compound C in both tumor cell lines.

[0252] Table 8: Inhibition of IC50 by Compound C on different cancer cell lines under normoxic and hypoxic conditions 90 value

[0253]

[0254] Note:

[0255] HCR activation ratio under hypoxia, IC50 under normoxic / N2 conditions 90 ratio.

[0256] PSN-1 is a human pancreatic cancer cell line.

[0257] PANC1 is a human pancreatic cancer cell line derived from pancreatic cancer ductal cells.

[0258] MiaPaCa-2 is a human pancreatic cancer cell line.

[0259] H460 is a type of human large cell lung cancer cell that was established from the pleural effusion of a large cell lung cancer patient.

[0260] DLD-1 WT, a wild-type colorectal cancer cell line.

[0261] DLD-1 BRCA2 KO, a colorectal cancer cell line with BRAC2 gene deletion.

[0262] IV. Hypoxia-dependent reduction experiment

[0263] Add the ingredients to each tube without adding NADPH. Then transfer 50 μl of sample B to 100 μl of acetonitrile (T = 0). For samples to be tested under anoxic conditions, place the tubes in an anoxic chamber (BACTRON anaerobic / environmental chamber). Initiate the reaction by adding NADPH to tubes A and C, then place the samples at 37°C. For samples to be tested in air, place the tubes on a hot plate at 37°C. After incubation for 30 minutes and 2 hours, remove 50 μl of sample from each tube and mix with 100 μl of acetonitrile. Rotate all tubes to remove any protein precipitate and transfer them to HPLC vials for analysis. Compare the results with a calibration curve of real standards. The specific reagent composition in each sample reaction tube is shown in Table 9 below.

[0264] Table 9: Reactant composition of each sample tube in the hypoxia-dependent reduction experiment

[0265] A 450μL 10μL 20μL 20μL B 470μL 10μL 20μL 0μL C 470μL 10μL 0μL 20μL

[0266] The relative amounts of test reagents (TH-302, compound A) in three different sample reaction tubes (A / B / C) at different reaction times (0 / 30min / 120min) were compared with their initial amounts to obtain the results. Figure 9 (53.2μg / ml humanreductase), Figure 10 (106.4μg / ml human reductase).

[0267] Experimental conclusion:

[0268] In the presence of NADPH and CYP450R enzymes, compound A exhibits an oxygen-dependent reduction reaction, and the reduction rate of compound A is similar to that of TH-302.

[0269] V. Animal Model Experiments Using PDX and CDX as Single or Combined Drugs

[0270] Animal experiments are described below. All animal studies in this application were conducted by a professional CRO (Contract Research Organization) company, and the experimental protocols were approved by the organization's internal animal ethics committee or a similar institution. The handling of the animals met the requirements.

[0271] 5.1 Compound A in In vivo drug efficacy experiment in the GA6201 human gastric cancer subcutaneous transplantation PDX tumor model

[0272] BALB / c nude mice subcutaneously inoculated A subcutaneous transplantation model of human gastric cancer was established using the GA6201 tumor mass. The experiment consisted of three groups: a 60 mg / kg group receiving the test drug Ifosfamide (TH-302), a group receiving compound A (5-50 mg / kg), and a control group receiving physiological saline (pH 7.0-7.6). Five mice were used in each group. The physiological saline (pH 7.0-7.6) control group received the drug via tail vein injection once a week for three weeks, followed by a four-week observation period. The Ifosfamide 60 mg / kg group received the drug via intraperitoneal injection, administered five days a week for five consecutive weeks followed by a two-day break, for two weeks, followed by a five-week observation period. The test drug was administered intraperitoneally at a dose of 5 mg / kg, five days a week with a two-day break, for two weeks (QDx5 / week x 2wks). The dose was then changed to 10 mg / kg intraperitoneally, administered daily for four days (QDx4, Day 14-Day 17). The dose and administration method were then changed again to 20 mg / kg via tail vein injection for one day (QDx1, Day 18). After a 10-day break, the dose was changed again to 50 mg / kg via tail vein injection once a week for two weeks (QWx2, Day 28, Day 35), with a one-week observation period. Efficacy was evaluated based on the relative tumor inhibition rate (TGI) (%), and safety was evaluated based on changes in animal weight and mortality. Specific regimens for each group are shown in Table 10 below.

[0273] Table 10: Compound A in In vivo pharmacodynamic experiments of different groups in the GA6201 human gastric cancer subcutaneous transplantation PDX tumor model.

[0274]

[0275] The tumor volume of mice in different groups was measured on different days and the average value was obtained. The results are shown in Table 11 below.

[0276] Table 11: Compound A in Changes in tumor volume at different inoculation days in the in vivo drug efficacy experiment of the GA6201 human gastric cancer subcutaneous transplantation PDX tumor model.

[0277]

[0278] By converting the table above into a line graph, we can obtain... Figure 11 .

[0279] The total weight of mice in different groups was measured on different days, and the average value was obtained to obtain the rate of change in body weight of each group of mice on different days. The results are as follows: Figure 12 As shown.

[0280] In the above dosing regimen, ifosfamide was designed based on the optimal dosing regimen and the maximum safe dosage described in the literature (Jessica D.Sun, Qian Liu, Dharmendra Ahluwalia, Damien J. Ferraro, Yan Wang, Don Jung, Mark D. Matteucci, and Charles P. Hart. Comparison of hypoxia-activated prodrug evofosfamide (TH-302) and ifosfamide in preclinical non-small cell lung cancer models[J]. Cancer Biology & Therapy, 2016, 17(4):371-380.), while compound A was designed based on previous cytotoxicity experiments, MTD experiments, and other empirically designed dosing regimens with progressively increasing dosages.

[0281] Conclusion: The results show that compound A of the present invention has a significant advantage in inhibiting tumor growth compared with the classic chemotherapy drug ifosfamide (which is also a drug with a DNA alkylating mechanism).

[0282] 5.2 In vivo efficacy experiment of compound A in a human lung cancer NCI-H460 subcutaneous xenograft model

[0283] A human lung cancer subcutaneous transplantation tumor model was established in BALB / c nude mice by subcutaneously inoculating them with human lung cancer NCI-H460 cells. The experiment was divided into three groups: test drug compound A (20 mg / kg and 40 mg / kg groups) and a control group (10% ethanol + 10% polyoxyethylene ether (35) castor oil Cremophor EL + 80% 5% glucose injection (pH 7.4) solvent), with 6 mice in each group. The 10% ethanol + 10% polyoxyethylene ether (35) castor oil Cremophor EL + 80% 5% glucose injection (pH 7.4) solvent control group received the drug via tail vein injection once a week for three weeks, followed by a one-week observation period. The test drug compound A (40 mg / kg group) received the drug via tail vein injection once every three weeks for two weeks, followed by a one-week observation period after the second administration. The test drug compound A (20 mg / kg group) received the drug via intraperitoneal injection once daily for five consecutive days, followed by a 24-day observation period. Efficacy was evaluated based on the relative tumor inhibition rate (TGI) (%), and safety was evaluated based on changes in animal body weight and mortality. The dosage of compound A was determined according to MTD experiments.

[0284] The tumor volume of mice in each group was measured on different days, and the average value was obtained. The results are shown in Table 12 below. Figure 13 As shown.

[0285] Table 12: Changes in tumor volume at different inoculation days in the in vivo efficacy experiment of compound A in a human lung cancer NCI-H460 subcutaneous xenograft model.

[0286]

[0287] In addition, the rate of change in body weight of mice in each group was calculated at different times, and the results are as follows: Figure 14 As shown.

[0288] Conclusions: The treatment group treated with single-drug compound A at 40 mg / kg (Q3Wx2, IV, test group 7) showed significant tumor-suppressive activity on day 31 after tumor cell inoculation, with a statistically significant difference compared to the control group (p = 0.00561), and a relative tumor inhibition rate (TGI) of 66.87%. The treatment group treated with single-drug compound O1 at 20 mg / kg (QDx5x1week, IP, test group 8) also showed significant tumor-suppressive activity on day 31 after tumor cell inoculation, with a statistically significant difference compared to the control group (p = 0.000464), and a relative tumor inhibition rate (TGI) of 76.22%.

[0289] 5.3 In vivo efficacy studies of compounds B and C in a human lung cancer NCI-H460 subcutaneous xenograft model

[0290] A human lung cancer subcutaneous transplantation tumor model was established in BALB / c nude mice by subcutaneously inoculating them with human lung cancer NCI-H460 cells. The experiment consisted of five mice in each group: an Irinotecan 40 mg / kg group, a compound B 20 mg / kg group, compound C 20 mg / kg and 40 mg / kg groups, and a control group using 10% ethanol + 10% Cremophor EL + 80% glucose injection D5W (pH 7.4) as the solvent. The 10% ethanol + 10% Cremophor EL + 80% glucose injection D5W (pH 7.4) control group, the compound B 20 mg / kg group, and the compound C 20 mg / kg and 40 mg / kg groups were all administered via intraperitoneal injection once daily for five consecutive days per week, followed by a two-day break, and then a two-week break, for a total of two administration cycles, with a three-day observation period after each cycle. The positive control group received Irinotecan 40 mg / kg via intraperitoneal injection, once every 4 days for a total of 7 doses, and was observed for 4 days. Efficacy was evaluated based on the relative tumor inhibition rate (TGI) (%), and safety was evaluated based on changes in animal weight and mortality.

[0291] Tumor growth in each treatment group and control group is as follows: Figure 15 .

[0292] In addition, the rate of change in body weight of mice in each group at different times, such as Figure 16 As shown.

[0293] Conclusion: The Irinotecan 40 mg / kg (Q4Dx7, ip, Group 2) treatment group showed significant tumor inhibition on day 28 after tumor cell inoculation, which was statistically significant compared with the control group (p = 7.44E-05), with a relative tumor inhibition rate (TGI) of 79.03%. The 20 mg / kg (Group 5) and 40 mg / kg (Group 7) treatment groups of Irinotecan B, Irinotecan C, and Irinotecan C all showed significant tumor inhibition on day 28 after tumor cell inoculation, which were statistically significant compared with the control group (p = 2.90E-06, 5.15E-10, and 1.08E-11), with relative tumor inhibition rates (TGI) of 88.51%, 90.56%, and 94.33%, respectively. At the end of the experiment (Day 35), the complete cure rate of tumors in the sixth group of mice was 20%. However, all of these test drugs exhibited certain drug toxicity at the tested doses, causing significant weight loss and even death in some mice. Irinotecan, at a dose of 40 mg / kg, also significantly inhibited tumor growth in the human lung cancer NCI-H460 model, showing a statistically significant difference compared to the control group. It also exhibited certain drug toxicity, causing significant weight loss in the mice.

[0294] 5.4 In vivo pharmacodynamic studies of compound C and sorafenib as monotherapy and in combination in a human HepG2 subcutaneous xenograft model of liver cancer

[0295] Human hepatocellular carcinoma HepG2 cells were subcutaneously inoculated into BALB / c nude mice to establish a subcutaneous transplantation tumor model of human hepatocellular carcinoma. The experiment was divided into 6 groups, with 5 mice in each group. The test drug Sorafenib 30 mg / kg (QDx21 Days) group, test drug compound C 20 mg / kg (QWx3), 40 mg / kg (QWx3) single drug group, compound C 40 mg / kg (QWx3) and Sorafenib (30 mg / kg, administered one week before compound C, QDx21 Days) combination drug group, compound C 40 mg / kg (QWx3) and Sorafenib (30 mg / kg, administered simultaneously, QDx21 Days) combination drug group, and 10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH7.4) control group. The control group, the test drug compound C 20 mg / kg and 40 mg / kg monotherapy groups, and the groups treated with either compound C or sorafenib were all administered via tail vein injection once a week for 3 weeks. The test drug Sorafenib 30 mg / kg monotherapy group and the group treated with compound C were administered via gavage once a day for 21 days. Efficacy was evaluated based on the relative tumor inhibition rate (TGI) (%), and safety was evaluated based on changes in animal body weight and mortality.

[0296] Tumor growth in each treatment group and control group is as follows: Figure 17 As shown.

[0297] In addition, the rate of change in body weight of mice in each group at different times, such as Figure 18 As shown.

[0298] In conclusion, the Sorafenib monotherapy group (30 mg / kg, Group 2) showed a tumor inhibition rate (TGI) of 48.11% on day 45 after tumor cell inoculation, which was not statistically significant compared to the control group (p = 0.573). One mouse in this group achieved complete tumor cure, resulting in a cure rate of 20%. The Sorafenib monotherapy group (20 mg / kg, Group 5) also showed tumor-suppressive activity on day 45 after tumor cell inoculation, with a TGI of 33.10%, but this was not statistically significant compared to the control group (p = 0.85). One mouse in this group also achieved complete tumor cure, resulting in a cure rate of 20%. The treatment group receiving compound C at 40 mg / kg (Group 6) showed tumor-suppressive activity on day 45 after tumor cell inoculation, with a relative tumor inhibition rate (TGI) of 75.59%, but this was not statistically significant compared to the control group (p = 0.0903). One mouse in this group experienced complete tumor cure, resulting in a cure rate of 20%.

[0299] The combination therapy group (Group 3) of compound C (40 mg / kg) and sorafenib (30 mg / kg, administered one week prior to compound C) showed tumor-suppressive activity on day 45 after tumor cell inoculation, with a relative tumor inhibition rate (TGI) of 49.87%, but this was not statistically significant compared to the control group (p = 0.72). The combination therapy group (Group 4) of compound C (40 mg / kg) and sorafenib (30 mg / kg, administered concurrently) showed significant tumor-suppressive activity on day 45 after tumor cell inoculation, which was statistically significant compared to the control group (p = 0.0105), with a relative tumor inhibition rate (TGI) of 94.53%. One mouse in this group achieved complete tumor cure, resulting in a cure rate of 20%. The results showed that the combination therapy of test drug compound C (40 mg / kg) and Sorafenib (30 mg / kg, administered simultaneously) had a better tumor-suppressing effect than the corresponding doses of the test drug monotherapy (Groups 2 and 6), but there was no statistically significant difference between the groups.

[0300] 5.5 Efficacy of Compound C and Anti-mouse-PD1 antibody as monotherapy and in combination with Compound C in a mouse CT26 subcutaneous model of colorectal cancer.

[0301] A mouse intestinal cancer subcutaneous transplantation model was established by subcutaneously inoculating BALB / c mice with murine intestinal cancer CT26 cells. The trial was divided into several groups: a monotherapy group (compound C 30 mg / kg, Day 1, Day 4, Day 8, Day 11, Day 15, Day 18); a monotherapy group (anti-mouse PD1Ab 10 mg / kg, Day 2, Day 5, Day 9, Day 12, Day 16, Day 19); a combination therapy group (compound C 30 mg / kg, Day 1, Day 4, Day 8, Day 11, Day 15, Day 18) and an anti-mouse PD1Ab 10 mg / kg (Absecond, starting on Day 2), Day 2, Day 5, Day 9, Day 12, Day 16, Day 19); and a combination therapy group (compound C 30 mg / kg, Day 1, Day 4, Day 8, Day 11, Day 19). The combination therapy group of compound C 30 mg / kg (Day 1, Day 4, Day 8, Day 11, Day 15, Day 18) and Anti-mouse PD1Ab 10 mg / kg (Ab second, starting on Day 3, Day 3, Day 6, Day 10, Day 13, Day 17, Day 20), the combination therapy group of compound C 30 mg / kg (Day 1, Day 4, Day 8, Day 11, Day 15, Day 18) and Anti-mouse PD1Ab 10 mg / kg (Ab first, Day 0, Day 3, Day 7, Day 10, Day 14, Day 17), the combination therapy group of compound C 30 mg / kg (Day 1, Day 4, Day 8, Day 11, Day 15, Day 18) and Anti-mouse PD1Ab Seven groups were established, with six mice in each group: a combination therapy group receiving 10 mg / kg (Absimultaneously, Day 1, Day 4, Day 8, Day 11, Day 15, Day 18), and a control group receiving 10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.4) solvent. The control group receiving 10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.4) solvent, the test drug compound C 30 mg / kg in the monotherapy group, and the group receiving the drug in combination with Anti-mouse PD-1Ab, were administered via tail vein injection twice weekly. The Anti-mouse PD-1Ab 10 mg / kg in the monotherapy group and the group receiving the drug in combination with compound C 30 mg / kg were administered via intraperitoneal injection twice weekly.The efficacy of each test drug was evaluated using median survival, and the safety was evaluated based on changes in animal weight and mortality.

[0302] The median survival at the end of the experiment was as follows: The median survival in the control group was 24 days; the median survival in the test drug compound C, 30 mg / kg group was 19 days; the median survival in the test drug Anti-mouse PD1 10 mg / kg group was 28 days; the median survival in the combination therapy group of compound C 30 mg / kg and Anti-mouse PD1 10 mg / kg (Ab second, starting on Day 2) was 28 days; the median survival in the combination therapy group of compound C 30 mg / kg and Anti-mouse PD1 10 mg / kg (Ab second, starting on Day 3) was 28 days; the median survival in the combination therapy group of compound C 30 mg / kg and Anti-mouse PD1 10 mg / kg (Ab first) was 33 days; the median survival in the combination therapy group of compound C 30 mg / kg and Anti-mouse PD1 10 mg / kg (Ab first) was 33 days. The median survival for (simultaneously) was 28 days, with specific results as follows: Figure 19 As shown.

[0303] In addition, the rate of change in body weight of mice in each group at different times, such as Figure 20 As shown.

[0304] The results showed that, compared with the control group, the combined use of Anti-mouse PD1, 10 mg / kg antibody monotherapy and compound C, 30 mg / kg, and Anti-mouse PD1, 10 mg / kg extended the median survival of mice. Compared with the PD-1 monotherapy group, only the combination therapy group (compound C 30 mg / kg and Anti-mouse PD1 10 mg / kg (Ab first)) showed a prolonged median survival (33 days), while the other combination therapy groups did not show a prolonged median survival of 28 days.

[0305] 5.6 Compound C and Gemcitabine as monotherapy and in combination therapy In vivo drug efficacy experiment in a PDX tumor model of subcutaneous xenograft pancreatic cancer (PA3546 patients).

[0306] BALB / c nude mice subcutaneously inoculated A human pancreatic cancer subcutaneous transplantation tumor model was established using the PA3546 tumor mass. The experiment was divided into six groups: a test drug Gemcitabine 120 mg / kg group, test drug compound C 10 mg / kg, 20 mg / kg and 40 mg / kg groups, a combined treatment group of Gemcitabine 120 mg / kg and compound C 40 mg / kg, and a control group of 10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.4) solvent, with a total of 6 groups and 6 mice in each group. Among them, the control group (10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.4) solvent), the test drug compound C 10 mg / kg, 20 mg / kg and 40 mg / kg groups, and the combination treatment group of Gemcitabine 120 mg / kg and compound C 40 mg / kg were administered via tail vein injection once a week for 3 weeks, with an 8-day observation period. The Gemcitabine 120 mg / kg monotherapy group and the combination therapy group were administered via intraperitoneal injection once a week for 3 weeks, with an 8-day observation period. Efficacy was evaluated based on the relative tumor inhibition rate (TGI) (%), and safety was evaluated based on changes in animal body weight and mortality.

[0307] Tumor growth in each treatment group and control group is shown in the figure. Figure 21 .

[0308] In addition, the rate of change in body weight of mice in each group at different times, such as Figure 22 As shown.

[0309] Conclusion: The test drug Gemcitabine 120 mg / kg (Group 2) treatment group showed significant tumor inhibition on day 25 after the first dose, which was statistically significant compared with the control group (p = 0.025100), with a relative tumor inhibition rate (TGI) of 45.31%. The relative tumor inhibition rates (TGI) of compound C 10 mg / kg (Group 3), 20 mg / kg (Group 4), and 40 mg / kg (Group 5) treatment groups on day 25 after the first dose were 32.35%, 36.79%, and 35.93%, respectively, showing no dose-response relationship and no statistically significant differences compared with the control group (p = 0.187000, 0.128000, and 0.193000). The combination therapy group (Group 6) of compound C 40 mg / kg and Gemcitabine 120 mg / kg showed significant tumor-suppressive activity on day 25 after the first dose, which was statistically significant compared to the control group (p = 0.000877), with a relative tumor inhibition rate (TGI) of 71.16%. Furthermore, the combination therapy group (Group 6) of compound C 40 mg / kg and Gemcitabine 120 mg / kg showed superior tumor-suppressive activity compared to the single-drug treatment groups of compound C 10 mg / kg (Group 3), 20 mg / kg (Group 4), 40 mg / kg (Group 5), and Gemcitabine 120 mg / kg (Group 2). There were no statistically significant differences between Group 6 and Group 2, or between Group 6 and Group 5. Group 2 vs. group 6, p = 0.526000; group 5 vs. group 6, p = 0.294000.

[0310] Except for the Gemcitabine 120 mg / kg (Group 2) treatment group, the control group (Group 1), and the test drug compound C 10 mg / kg (Group 3) treatment group, some mice in the test drug compound C 20 mg / kg (Group 4) and 40 mg / kg (Group 5) treatment groups, as well as the combined treatment group of Gemcitabine 120 mg / kg and compound C 40 mg / kg (Group 6), experienced significant weight loss or even death. All these cases of weight loss and death in mice may be related to the potential drug toxicity of the test drug at the test dose. Mice in the control group (Group 1), the Gemcitabine 120 mg / kg (Group 2), and the test drug compound C 10 mg / kg (Group 3) treatment groups did not experience significant weight loss and tolerated the treatment well.

[0311] 5.7 In vivo pharmacodynamics of compound C as a single drug in the NCI-H460-Luc2 intracranial inoculation model

[0312] A human lung cancer intracranial transplantation tumor model was established by intracranial inoculation of BALB / c nude mice with human lung cancer NCI-H460-Luc2 cells. The experiment was divided into four groups: a test drug Paclitaxel group (20 mg / kg), a compound C group (10 mg / kg, 20 mg / kg, and 40 mg / kg), and a control group (10% ethanol + 10% Cremophor EL + 80% glucose injection D5W (pH 7.4) solvent). Six mice were in each group. The 10% ethanol + 10% Cremophor EL + 80% glucose injection D5W (pH 7.4) solvent control group, the test drug compound C group (10 mg / kg, 20 mg / kg, and 40 mg / kg), were administered via tail vein injection once a week for 3 weeks. The positive control group (Paclitaxel 20 mg / kg) was administered via intraperitoneal injection once a week for 3 weeks. The relative tumor inhibition rate (TGI) was calculated based on the tumor biofluorescence value, and the efficacy was evaluated. The safety was evaluated based on changes in animal body weight and mortality.

[0313] Tumor growth in each treatment group and control group is as follows: Figure 23 As shown.

[0314] In addition, the rate of change in body weight of mice in each group at different times, such as Figure 24 As shown.

[0315] Conclusion: Eleven days after grouping, the fluorescence value of the solvent control group reached 2576.80E+05 photos / second. Compared with the solvent control group, the positive control group, Paclitaxel (20 mg / kg), showed no significant tumor inhibition effect, with a mean biofluorescence value of 4184.52E+05 photos / second (TGI = -62.45, p = 0.2278). Compound C monotherapy showed highly significant tumor inhibition effects in all treatment groups. The mean biofluorescence value of compound C (10 mg / kg) treatment group was 7.07E+05 photos / second (TGI = 99.82%, p < 0.01), the mean biofluorescence value of compound C (20 mg / kg) treatment group was 1.79E+05 photos / second (TGI = 100.03%, p < 0.01), and the mean biofluorescence value of compound C (40 mg / kg) treatment group was 1.04E+05 photos / second (TGI = 100.05%, p < 0.01). Compared with the compound C (10 mg / kg) group, compound C (20 mg / kg) and compound C (40 mg / kg) showed statistically significant tumor-suppressive effects (p = 0.0446 and p = 0.0241, respectively), which were dose-dependent. Furthermore, the NCI-H460-Luc2 intracranial inoculation model mice tolerated compound C monotherapy well.

[0316] VI. Pgp inhibition test and BBB blood-brain barrier test

[0317] P-glycoprotein (P-GP, also known as multidrug resistance protein) is a high-molecular-weight protein found on the cell membrane of multidrug-resistant tumor cells. It has a transport pump-like structure and pumps various chemotherapeutic drugs out of the cell, reducing the intracellular drug concentration. It is closely related to resistance to clinical chemotherapy.

[0318] The effectiveness of chemotherapy drugs in treating tumors or cancers of the central nervous system, such as the brain, is limited, mainly due to the blood-brain barrier (BBB). The BBB makes it difficult for chemotherapy drugs to penetrate the tumor system or tumor tissue, meaning that chemotherapy drugs cannot cross the BBB to enter the brain and kill cancer cells.

[0319] The process by which small molecules, such as chemotherapy drugs, cross the blood-brain barrier is complex. The blood-brain barrier, located between the systemic bloodstream and cerebrospinal fluid, is formed by specialized brain microvascular endothelial cells, along with surrounding cells and perivascular astrocytes, through tight junctions between adjacent cells. This creates a selective barrier for most molecules, rather than forming a physical barrier around the vascular endothelial cells. The blood-brain barrier's transport system allows hydrophilic small molecules to pass through, while large hydrophilic molecules, including many chemotherapy drugs and macromolecular drugs, are excluded from the central nervous system unless actively transported by certain proteins. More importantly, the blood-brain barrier possesses protective "drug efflux pumps" for brain tissue, such as P-glycoproteins, which actively expel some chemotherapy drugs and macromolecular drugs from the brain. Therefore, even if small-molecule chemotherapy drugs with some hydrophilicity (including small-molecule targeted anti-tumor drugs) can cross the blood-brain barrier and enter the brain to exert their effects, they will still be excluded from the central nervous system by P-glycoproteins and thus fail to be truly effective. In other words, the transmembrane structure of Pgp has an energy-dependent "drug pump" function, which can pump hydrophilic lipophilic drugs, such as vincristine (VCR), doxorubicin (Dox), or etoposide (VP-16), out of the cell, resulting in a decrease in intracellular drug concentration and a reduction or complete loss of cytotoxicity.

[0320] Therefore, experimental data on the interaction between PGP glycoprotein and compounds can be used to evaluate the true effectiveness of these compounds in inhibiting the proliferation of central nervous system tumor cells.

[0321] 6.1 Using MDCKII-MDR1 cells as a model, verapamil was used as an inhibitor to evaluate the potential of compounds as P-gp substrates.

[0322] Preparation of cells for penetration assay

[0323] 1) MDCKII-MDR1 cells were cultured in cell culture flasks. The incubator was set to 37℃, 5% CO2, and 95% relative humidity. When the cell confluence reached 70-90%, the cells could be used for seeding in transwells.

[0324] 2) Before cell seeding, add 50 μL of cell culture medium to each well in the upper chamber of the migration chamber and 25 mL of cell culture medium to the lower culture plate. After incubating the culture plate in a 37°C, 5% CO2 incubator for 1 hour, it can be used for cell seeding.

[0325] 3) After cell incubation, aspirate the cell suspension and transfer it to a round-bottom centrifuge tube, then centrifuge at 120g for 5 minutes.

[0326] 4) Resuspend the cells in culture medium to a final concentration of 1.56 × 10⁻⁶. 6Cells / mL. 50 μL of cell suspension was added to each well of a 96-well migration chamber culture plate, resulting in a final seeding density of 5.45 × 10⁶ cells / mL. 5 cells / cm 2 .

[0327] 5) Change the medium 48 hours after inoculation and culture for 4-8 days, changing the medium every other day.

[0328] 6) The process of changing the culture medium is as follows: separate the migration chamber from the receiving plate, discard the culture medium in the receiving plate first, then discard the culture medium in the migration chamber, and finally add 75 μL of fresh culture medium to each chamber and 25 mL of fresh culture medium to the receiving plate.

[0329] Evaluation of cell monolayer membrane integrity

[0330] 1) After 4-8 days of culture, MDCKII-MDR1 cells should have fully merged and differentiated. At this point, they can be used for the penetration test.

[0331] 2) Measure the resistance of the single-layer film using a Millipore resistance meter and record the resistance of each hole.

[0332] 3) After the measurement is completed, put the migration chamber culture plate back into the incubator.

[0333] 4) Calculation of resistance value: Measure resistance value (ohms) × film area (cm²) 2 ) = TEER value (ohm·cm) 2 If the TEER value is <42 ohms·cm 2 If the hole is not suitable for a penetration test, then the hole cannot be used.

[0334] Drug penetration test

[0335] 1) Remove the MDCKII-MDR1 migration chamber culture plate from the incubator. Rinse the cell monolayer membrane twice with buffer and incubate at 37°C for 30 minutes.

[0336] 2) Determine the transport rate of the compound from the top to the base. Add 75 μL of flushing solution containing the test substance to each well of the upper chamber (top) and 235 μL of buffer solution to each well of the lower chamber (base).

[0337] 3) Determine the transport rate of the compound from the base to the top. Add 75 μL of buffer to each well in the upper chamber (top) and 235 μL of buffer containing the test compound to each well in the lower chamber (base).

[0338] 4) Transfer 50 μL of sample from the working solution preparation plate and add it to 200 μL of acetonitrile containing internal standard (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM ketoprofen) as a 0-minute dosing sample for testing.

[0339] 5) To determine the transport of the test substance under the condition of adding the P-gp inhibitor verapamil, verapamil needs to be added to both the buffer salt at the dosing end and the receiving end of the MDCKII-MDR1 migration chamber plate, with a final concentration of 100 μM.

[0340] 6) After combining the transfers from top to bottom, incubate at 37°C for 2 hours.

[0341] 7) After incubation, take 50 μL of sample from each well of the upper and lower chambers of the migration chamber culture plate and add it to a new sample tube. Add 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 2 μM ketoprofen) to the sample tube, vortex for 10 minutes, and then centrifuge at 3220 g for 30 minutes. Take 100 μL of the supernatant, dilute it with an equal volume of water, and then perform LC-MS / MS analysis. All samples were incubated in triplicate.

[0342] 8) Evaluate the integrity of the cell monolayer after 2 hours of incubation using fluorescein leakage test. Dilute the fluorescein stock solution to a final concentration of 100 μM / L using buffer. Add 100 μL of fluorescein solution to each well of the upper Transwell plate and 300 μL of buffer to each well of the lower receiving plate. After incubation at 37°C for 30 minutes, aspirate 80 μL of solution from both the upper and lower layers of each well into a new 96-well plate. Perform fluorescence measurement using a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

[0343] Data Analysis:

[0344] Peak areas are calculated from ion chromatography results. The apparent permeability coefficient (Papp, unit: cm / s × 10⁻⁶) of a compound is calculated using the following formula:

[0345]

[0346] In the formula: VA is the volume of the receiving solution (Ap→Bl is 0.235 mL, Bl→Ap is 0.075 mL), and Area is the membrane area of ​​the Transwell-96-well plate (0.143 cm²). 2 ); time is the incubation time (unit: seconds), [drug]acceptor represents the drug concentration at the receiver after incubation, [drug] initial,donor This indicates the initial concentration of the drug administered before incubation.

[0347] The efflux ratio is calculated using the following formula:

[0348]

[0349] In the formula: P app(B-A) P represents the apparent permeability from the basal end to the apex. app(A-B) The apparent permeability coefficient is measured from the top to the base.

[0350] Recovery rate is calculated using the following formula:

[0351]

[0352] In the formula: V A V represents the volume of the solution at the receiving end (unit: mL); D [drug]acceptor represents the drug concentration at the receiving end after incubation, [drug]donor represents the drug concentration at the administering end after incubation, and [drug]initial,donor represents the initial drug concentration before incubation.

[0353] The fluorescence value (LY Leakage) of the cell monolayer membrane is calculated using the following formula:

[0354]

[0355] In the formula: Iacceptor refers to the fluorescence density of the receiving well (0.3 mL), and Idonor refers to the fluorescence density of the drug delivery well (0.1 mL), expressed as %LY. LY < 1.5% indicates that the monolayer cell membrane is intact.

[0356] The efflux rates of different compounds were tested in the presence and absence of the P-glycoprotein inhibitor verapamil. The experimental data are shown in Table 13 below.

[0357] Table 13: Drug Penetration Test Data

[0358]

[0359]

[0360] Experimental conclusion: The three key parameters of compounds B and C, Papp(AB), Papp(BA), and Efflux Ratio, are very close in the presence and absence of the P-gp inhibitor verapamil. Therefore, compounds B and C are not Pgp substrates.

[0361] 6.2 Blood-brain barrier experiment

[0362] At each time point, three CD-1 mice were intravenously administered the test compound at a dose of 1 mg / kg. Blood and brain tissue samples were collected at 5 min, 15 min, 1 h, 2 h, 4 h, and 8 h post-administration. The concentration of the test compound in the samples was determined by LC / MS, and the results are shown in Tables 14 / 15 below.

[0363] Table 14: Concentration of the test substance in blood samples of CD-1 mice at different time points after intravenous administration of compound B to CD-1 mice.

[0364]

[0365] Table 15: Concentration of the test substance in blood samples of CD-1 mice at different time points after intravenous administration of compound C to CD-1 mice.

[0366]

[0367] The concentrations of compounds B and C in blood and brain tissue were plotted as a function of time, such as... Figure 25 As shown.

[0368] The results of the tests on the ratio of the concentrations of compounds B and C in brain tissue and blood versus time were plotted as curves, such as... Figure 26 As shown.

[0369] The PK (pharmacokinetic) parameters obtained in the above experiments are listed in 16 / 17.

[0370] Table 16: PK parameters obtained from plasma tests

[0371] Cl_obs mL / min / kg 57.63 87.92 T1 / 2 h 0.45 0.3 C0 ng / mL 531.77 694.23 AUClast h*ng / mL 288.79 188.34 AUClnf h*ng / mL 289.2 189.57 MRTlnt_obs h 0.46 0.33 Vss_obs L / kg 1.59 1.73

[0372] Table 17: PK parameters obtained from brain tissue tests

[0373]

[0374] Experimental conclusion: Compounds B and C can be detected in brain tissue, indicating that these two compounds can penetrate the blood-brain barrier.

[0375] VII. Metabolic Stability Evaluation Experiment

[0376] 7.1 Liver microsomal stability test

[0377] 1) Conduct two separate experiments.

[0378] 1a) Using NADPH: Add 10 μL of 20 mg / mL liver microsomes and 40 μL of 10 mM NADPH to the culture medium. The final concentrations of microsomes and NADPH are 0.5 mg / mL and 1 mM, respectively.

[0379] 1b) No NADPH: Add 10 μL of 20 mg / mL liver microsomes and 40 μL of ultrapure H2O to the culture medium. The final concentration of microsomes is 0.5 mg / mL.

[0380] 2) Add 4 μL of 200 μM test compound solution or control compound solution (verapamil) to a final concentration of 2 μM to start the reaction and incubate at 37°C.

[0381] 3) Remove 50 μL aliquots from the reaction solution at 0 and 30 minutes. Terminate the reaction by adding 4 volumes of cold acetonitrile and four internal standards (15S, 100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, and 2 μM ketoprofen). Centrifuge the sample at 3,220 x g for 40 minutes. Mix 100 μL of the supernatant with 100 μL of ultrapure H₂O and then use for LC-MS / MS analysis. All experiments were performed in duplicate. The test results are shown in Table 18 below.

[0382] Table 18: Residual amount of compound A after 30 minutes in the liver microsome stability test

[0383]

[0384] The test data was plotted as a line graph, such as... Figure 27 As shown.

[0385] Compound A is relatively stable in human and mouse liver microparticles, but unstable in rat and canine liver microparticles.

[0386] The experimental procedure was similar, and the results of testing compound D are shown in Table 19 below.

[0387] Table 19: Residual amount of compound D after 30 minutes in the liver microsome stability test

[0388]

[0389] 7.2 Plasma stability test

[0390] 1) Plasma preparation

[0391] Immediately thaw the frozen plasma in a 37°C water bath. Centrifuge the plasma at 3,220g for 10 minutes to remove blood clots, and collect the supernatant in a new test tube. Check and record the pH of the plasma. Note: a) Use only plasma that has been thawed no more than twice since arrival; b) Use only plasma with a pH between 7 and 8.

[0392] 2) Prepare the experimental solution

[0393] Working solutions of the test compound and the control compound mevinolin (1 mM) were prepared in DMSO. A working solution of the control compound (1 mM) in propane was prepared in acetonitrile. 4 μL of the working solution was added to 796 μL of pre-incubated plasma to achieve a final concentration of 5 μM. The final solvent concentration was 0.5%.

[0394] 3) Test procedure for plasma stability

[0395] Add 50 μL of spiked plasma aliquots to new test tubes and incubate at approximately 60 rpm for 2 hours in a 37°C water bath. Perform the assay in duplicate. At the end of the two-hour incubation, terminate the reaction by adding 300 μL of acetonitrile quenching solution (acetonitrile containing internal standards (acetonitrile, 100 nM Alprazolam, 500 nM Labetalol, and 2 μM Ketoprofen)). Prepare a sample at time 0 by adding 50 μL of spiked plasma to a new tube containing 300 μL of quenching solution. Vortex mix for 5 minutes. Centrifuge the sample in the plate at 3,220 x g for 30 minutes at 4°C to precipitate proteins. Transfer 100 μL of the supernatant to a new plate. Dilute the supernatant with 100 μL of water, mix thoroughly, and analyze using LC-MS / MS.

[0396] The test results after 30 minutes are shown in Table 20 below.

[0397] Table 20: Percentage of Compound A remaining in plasma of different species after 30 minutes

[0398] Propylamine people 0.07 lovastatin rats 0.21 Propylamine mice 5.33 lovastatin dog 37.6 Propylamine monkey 48.11 Compound A people 68.68 Compound A rats 52.11 Compound A mice 55.07 Compound A dog 85.04

[0399] The test data was plotted as a line graph, such as... Figure 28 As shown.

[0400] The experimental procedure was similar, and the results of testing compound D are shown in Table 21 below.

[0401] Table 21: Percentage of Compound D remaining in plasma of different species after 30 minutes

[0402] Propylamine people 3.32 lovastatin rats 0.19 Propylamine mice 1.98 lovastatin dog 58.73 Propylamine monkey 54.38 Compound D people 97.06 Compound D rats 85.49 Compound D mice 87.35 Compound D dog 91.95 Compound D monkey 87.66

[0403] Conclusion: Compound D is relatively stable in canine plasma, followed by human plasma. It is relatively unstable in rat and mouse plasma.

[0404] Further testing was conducted on the metabolic data of compound A in the blood of different animals over 2 hours, and the results are shown in Table 22 below.

[0405] Table 22: Metabolic data of compound A in plasma of different animals over 2 hours

[0406]

[0407] At 120 minutes, the remaining percentages of compound A in mouse, rat, dog, monkey, and human plasma were 66.4%, 69.0%, 98.9%, 87.4%, and 89.7%, respectively. The in vitro half-lives of compound A in mouse, rat, dog, monkey, and human plasma were 189 minutes, 226 minutes, >373 minutes, >373 minutes, and >373 minutes, respectively. These results indicate that compound A is stable in dog, monkey, and human plasma at 37°C for at least 2 hours, but unstable in mouse and rat plasma.

[0408] VIII. Maximum Tolerable Dose (MTD) Test

[0409] 1. Compound B

[0410] Experimental process

[0411] Eighteen ICR mice (nine females and nine males) were randomly divided into three groups (three females and three males) according to sex and body weight: a 10 mg / kg, 20 mg / kg, and a 40 mg / kg group for compound B. The mice were administered compound B intravenously at concentrations of 1, 2, and 4 mg / ml (10 ml / kg) and doses of 10, 20, and 40 mg / kg, respectively. Administered once daily for five consecutive days, followed by a two-week recovery period after the last administration. Animals were closely monitored for two hours after administration and twice daily (morning and afternoon) on non-administered days. Body weight and food intake were measured twice weekly during the experiment. All dead animals and surviving animals 14 days after the last administration underwent gross necropsy, and pathological examination of diseased organs was performed.

[0412] Experimental results

[0413] (1) Animal mortality

[0414] During the experiment, 3 out of 6 animals in the compound B 40mg / kg group died, with the deaths occurring during the recovery period of 2 to 6 days.

[0415] (2) Clinical symptoms in animals after drug administration

[0416] During the experiment, the animals in the compound B 10mg / kg group and the compound B 20mg / kg group were in good general condition; the compound B 40mg / kg group intermittently showed unsteady gait, deeper and rapid breathing, and reduced spontaneous activity; the recovery period was 2 to 6 days, and some dying animals showed reduced spontaneous activity, piloerection, arched back, and convulsions.

[0417] (3) Effects on animal weight

[0418] Following intravenous injection of compound B, mice showed slow weight gain. Female animals showed weight loss on day 3 of administration and day 2 of recovery compared to pre-administration levels; weight regain resumed after day 5 of recovery. Male animals in the 40 mg / kg group showed weight loss in surviving animals after day 5 of recovery.

[0419] (4) Effects on animal feed intake

[0420] There was no significant difference in food intake between the animals in each treatment group and the average food intake of normal mice (background data from our laboratory).

[0421] (5) Pathological examination results

[0422] Tissue samples were collected from the dead animals for examination. The recovery period observation period ended, and no significant gross pathological changes were found in the surviving animals upon gross necropsy.

[0423] Experimental conclusions

[0424] Under the conditions of this experiment, the maximum tolerated dose of compound B administered intravenously to ICR mice in a single cycle was 20 mg / kg.

[0425] 2. Compound C

[0426] Experimental process

[0427] Eighteen ICR mice (9 females / 9 males) were randomly divided into three groups (3 females / 3 males) according to sex and body weight: a 10 mg / kg, 20 mg / kg, and 40 mg / kg compound C group. The mice were administered compound C intravenously at concentrations of 1, 2, and 4 mg / ml (10 ml / kg) and doses of 10, 20, and 40 mg / kg, respectively. Administered once daily for 5 consecutive days, followed by a one-week recovery period. No animal deaths occurred after 5 days of continuous administration. To determine the maximum tolerated dose, a second cycle of administration was conducted. The dosage was adjusted, and the mice were administered for 5 consecutive days, followed by 14 days of observation after the last administration. The dosage adjustments are shown in Table 23 below.

[0428] Table 23: MTD experimental dose adjustment for compound C

[0429] Compound C 10 mg / kg group 80mg / kg Compound C 80 mg / kg group Compound C 20 mg / kg group 60mg / kg Compound C 60 mg / kg group Compound C 40 mg / kg group 40mg / kg Compound C 40 mg / kg group

[0430] Animals were closely observed for 2 hours after drug administration, and twice daily (morning and afternoon) on non-drug administration days. Body weight and food intake were measured twice a week during the experiment. Gross necropsy was performed on dead animals and all surviving animals 14 days after the last administration, and histopathological examination was conducted on diseased organs.

[0431] Experimental results

[0432] (1) Animal mortality

[0433] During the trial, 1 / 6 of the animals in the compound C 10 / 80 mg / kg group (meaning the first cycle of administration was 10 mg / kg, and then the second cycle continued with administration of 80 mg / kg) died; 3 / 6 of the animals in the compound C 20 / 60 mg / kg group died; and 1 / 6 of the animals in the compound C 40 / 40 mg / kg group died. The deaths occurred mainly during the recovery period of 3 to 7 days.

[0434] (2) Clinical symptoms in animals after drug administration

[0435] During the first cycle of administration, the animals in the compound C 10 / 80 mg / kg group were in good general condition; the animals in the compound C 20 / 60 mg / kg group developed gait instability after administration; and the animals in the compound C 40 / 40 mg / kg group intermittently developed gait instability, rapid breathing, and increased spontaneous activity from immediately after administration to 5 minutes after administration.

[0436] During the second cycle of administration, animals in the compound C 10 / 80 mg / kg group intermittently exhibited gait abnormalities, decreased muscle tone, reduced spontaneous activity, irregular breathing, unsteady gait, rapid breathing, prickly skin, convulsions, deepened breathing, prone position, and arched back from immediately after administration to 1 hour after administration. Animals in the compound C 20 / 60 mg / kg group intermittently exhibited deepened breathing, reduced spontaneous activity, unsteady gait, irregular breathing, increased spontaneous activity, rapid breathing, gait abnormalities, and prone position from immediately after administration to 30 minutes after administration. Animals in the compound C 40 / 40 mg / kg group intermittently exhibited unsteady gait, deepened breathing, rapid breathing, increased spontaneous activity, irregular breathing, and gait abnormalities from immediately after administration to 10-30 minutes after administration. During the recovery period of 3-4 days, animals in both the compound C 10 / 80 mg / kg and compound C 20 / 60 mg / kg groups exhibited drooling and perioral moisture.

[0437] (3) Effects on animal weight

[0438] Intravenous injection of compound C into mice resulted in slow weight gain.

[0439] For male animals, on day 3 of the first cycle of administration, it was found that the 10 and 40 mg / kg groups had a decrease in body weight compared with before administration; the 20 mg / kg group had a slow increase in body weight.

[0440] For female animals, body weight decreased on day 3 of the first cycle of administration and day 1 of recovery compared to pre-administration levels; body weight resumed to increase after day 5 of recovery.

[0441] For all animals in the treatment groups (regardless of sex), in the second cycle of the 20 / 60 mg / kg and 40 / 40 mg / kg groups, after 5 days of recovery, their body weight decreased compared to before administration.

[0442] (4) Effects on animal feed intake

[0443] There was no significant difference in food intake between the animals in each treatment group and the average food intake of normal mice (background data from our laboratory).

[0444] (5) Pathological examination results

[0445] Tissue samples were collected from the dead animals for examination. The recovery period observation period ended, and no significant gross pathological changes were found in the surviving animals upon gross necropsy.

[0446] Experimental conclusions

[0447] Under the conditions of this experiment, the maximum tolerated dose of compound C administered intravenously to ICR mice in the first cycle was >40 mg / kg; the maximum tolerated dose of compound C in the second cycle was <40 mg / kg. In summary, the maximum tolerated dose of compound C is between 20-40 mg / kg, which is similar to or slightly higher than that of compound B.

[0448] 3. Comparison of the MTD of compounds B and C with TH-2565 / 2566

[0449] The mouse MTD test results of compound TH-2565 / 2566 disclosed in prior literature (WO2016210175A1, CN108024974A) are 2 mg / kg. The test results of anticancer compounds B and C disclosed in this invention are listed below along with the MTD results of compound TH-2565 / 2566 in the literature.

[0450] Table 24: Comparison of MTD results of compounds B, C and TH-2565 / 2566

[0451]

[0452] The results of the maximum tolerated dose experiment indicate that the anticancer compound A provided by this invention has a stronger safety profile in animals, meaning that it may be possible to use a larger dose in clinical treatment to achieve better therapeutic effects.

[0453] Furthermore, in comparison, prior literature (WO2016210175A1, CN108024974A) discloses that compound TH-2565 / 2566 is the best-performing compound among anticancer compounds with benzene ring structures. By comparison, it can be inferred that the new structural compound of the present invention obtained through new structural modification has a higher MTD, that is, it can improve the safety and clinical therapeutic dose of the new compound.

Claims

1. A compound having any of the following structural formulas, or a pharmaceutically acceptable salt or isotopic variant thereof, 、 。 2. The compound according to claim 1, wherein, The structural formula of the isotope variant is as follows: Each of the nine A's is either H or D, and at least one of the nine A's is D.

3. The compound according to claim 1, wherein, The isotopic variants correspond to deuterated compounds selected from the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 4. A drug comprising any one of the compounds of claims 1 to 3 or a pharmaceutically acceptable salt or isotopic variant thereof.

5. Use of the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt or isotopic variant thereof in the preparation of a medicament for treating cancer or tumor or a condition or proliferative disease caused by cancer or tumor, wherein the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, or primary brain cancer or tumor, or metastatic cancer or tumor that has metastasized to the brain.

6. The use according to claim 5, wherein the patient with cancer or tumor is a patient with impaired DNA repair.

7. The use according to claim 6, wherein the DNA repair impairment is one or more of the following: impairment of homologous recombination DNA repair enzymes, impairment of nucleotide excision repair enzymes, impairment of non-homologous end ligases, impairment of base excision repair enzymes, impairment of mismatch repair enzymes, and impairment of at least one repair enzyme in the Fanconi anemia pathway.

8. The use according to claim 5, wherein the patient with the cancer or tumor is a patient with a BRCA2 gene mutation.

9. A compound medicine comprising any one of claims 1 to 3, or a pharmaceutically acceptable salt or isotopic variant thereof, and a. Chemotherapy drugs selected from docetaxel, cisplatin, pemetrexed, irinotecan, doxorubicin, gemcitabine, and temozolomide; or b. Anti-angiogenic drugs; or c. Cell checkpoint inhibitors; or d. Immunosuppressants.

10. A method for preparing any of the following compounds with structure I, comprising subjecting compound I-1 to a cyclization reaction to obtain the corresponding compound I. I-1 I in, X can be F, Cl, Br, or I independently.

11. The method according to claim 10, wherein, The ring-closing reaction uses silver oxide or silver nitrate as a catalyst, with or without the addition of an organic base.

12. The method according to claim 10, wherein, X is always Br.

13. The method according to claim 11, wherein, The organic base is N,N-diisopropylethylamine (DIEA) or triethylamine (TEA).

14. Compounds having the following I-1 structure: I-1 in, X can be F, Cl, Br, or I independently.

15. Use of the compound of claim 14 as an intermediate in the synthesis of the compound of claim 1.

16. A method for preparing compounds with the following structure I-1, comprising reacting compound I-2 as a starting reactant with phosphorus oxychloride POCl3 in a first reaction to obtain an intermediate, then reacting the intermediate with a haloethylamine or a hydrohalide of a haloethylamine in a second reaction to finally obtain the corresponding compound I-1: I-1 I-2 in, X can be F, Cl, Br, or I independently.

17. The method according to claim 16, wherein, The haloethylamine is bromoethylamine, and the hydrohalide of the haloethylamine is the hydrobromide of bromoethylamine.

18. A method for separating a racemic compound as shown below to obtain one of the enantiomers as claimed in claim 1 or to concentrate any enantiomer of the compound in excess in a mixture, comprising the steps of: a) subjecting the racemic compound to optical analysis, wherein the process is carried out by chiral chromatography comprising a stationary phase and a mobile phase, wherein the stationary phase comprises silica gel impregnated with a functionalized polysaccharide, and wherein the mobile phase comprises an alcohol and another solvent, wherein the racemic compound is: 。 19. The method according to claim 18, wherein, The alcohol is ethanol, and the other solvent is n-hexane.

20. A method for preparing deuterated compounds with the structure I-4, comprising subjecting compound I-5 to a cyclization reaction to obtain the corresponding compound I-4. I-4 I-5 in, X can be F, Cl, Br, or I independently; The ring-closing reaction uses silver oxide or silver nitrate as a catalyst, with or without the addition of a base.

21. The method of claim 20, wherein X is Br.

22. A method for preparing deuterated compounds with the structure I-5, comprising reacting compound I-2 as a starting reactant with phosphorus oxychloride POCl3 in a first reaction to obtain an intermediate, and then reacting the intermediate with the corresponding deuterated haloethylamine I-6 or a hydrohalide of the deuterated haloethylamine in a second reaction to finally obtain the corresponding compound I-5: I-5 I-2 I-6 in, X can be F, Cl, Br, or I independently; The halogen in the hydrohalate of deuterated haloethylamine is the same as that in deuterated haloethylamine.

23. The method according to claim 22, wherein the haloethylamine is bromoethylamine and the hydrohalate of the haloethylamine is the hydrobromide of bromoethylamine.

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