Anti-cancer compounds and medical uses thereof

Abstract

The present application provides a compound of formula (I), or a pharmaceutically acceptable salt or solvate or isotopically enriched variant or isomer thereof, and anti-cancer medical uses:

Description

Technical Field

[0001] This invention relates to anticancer compounds and belongs to the field of anticancer drug development. Background Technology

[0002] As tumors grow rapidly, some tumor tissues become increasingly distant from the nearest blood vessels or develop abnormal tumor vascular structures, resulting in insufficient oxygen supply and leading to tumor hypoxia (see Nature Review Cancer, 2002, 2:38-47). Traditional antitumor drugs have good killing power against tumors near blood vessels, but their effect on tumors in hypoxic areas is limited or ineffective.

[0003] Hypoxic activation prodrugs (HAPs) can specifically release antitumor active ingredients in hypoxic tumor regions, thereby killing tumor cells in hypoxic areas (see Chinese Journal of Cancer, 2014, 33:80-86). HAPs exhibit tumor targeting, resulting in better safety profiles, and their antitumor efficacy is enhanced when used in combination with traditional antitumor chemotherapy drugs or immunotherapy.

[0004] TH-302 (Evofosfamide, CAS No. 918633-87-1) is a 2-nitroimidazole-induced hypoxia-activated prodrug, metabolized by bromoisophosphoramide (Br-IPM). Under hypoxic conditions, the inactive TH-302 prodrug (shown in structural formula 1 below) releases the highly toxic Br-IPM (shown in structural formula 2 below). TH-302 exhibits broad-spectrum in vitro and in vivo biological activity, specific hypoxia-selective activation activity, and the ability to induce histone H2AX phosphorylation and DNA cross-linking, thereby leading to cell cycle arrest. Therefore, this compound has been used by numerous pharmaceutical companies and research institutions in the development of anticancer drugs.

[0005]

[0006] A research article published by Meng et al. of Threshold Pharmaceuticals (see Meng et al, Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302, MCT, 2012(11):740; DOI:10.1158 / 1535-7163.MCT-11-0634) indicates that TH-302 has broad-spectrum activity against various tumors and exhibits a hypoxia-selective activity enhancement effect. Enhanced TH-302 cytotoxicity under hypoxic conditions was observed in 32 human cancer cell lines, demonstrating that this compound selectively enhances the activity of cancer cells under hypoxic conditions. The mechanism of TH-302's enhanced activity under hypoxic conditions was confirmed using cells overexpressing human NADPH, namely, single-electron reductase dependence, as shown in the following reaction formula 1:

[0007]

[0008] Reaction 1

[0009] 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.

[0010] Furthermore, the team of Dr. Duan Jianxin and others, the inventors of this application, further improved TH-302 by modifying the aminophosphate ester portion to an aziridine structure (see WO2016210175A1 and CN108024974A, whose main compound structures are as follows). Testing revealed that this type of compound exhibits the same hypoxia-activated mechanism as TH-302: it has stronger cytotoxicity against cancer cells under hypoxic conditions than under normoxic conditions.

[0011]

[0012] The aziridine compound obtained after structural modification of TH-302

[0013] Following the same research approach, CN107513057A and CN107383136B both revealed that compounds with TH-302-like structures may have hypoxia-activated anticancer activity, but neither has been further developed.

[0014] Although the aforementioned patents (WO2016210175A1, CN108024974A, CN107513057A, CN107383136B) disclose a series of hypoxia-activated antitumor compounds, none of them have entered human clinical trials, which indicates that these compounds still have shortcomings as effective anticancer drugs in some aspects. Summary of the Invention

[0015] The purpose of this invention is to provide candidate anticancer compounds with novel structures.

[0016] The first aspect of this invention relates to compounds of formula (I), or pharmaceutically acceptable salts or solvates, or isotopic variants or isomers thereof:

[0017]

[0018] in,

[0019] Cx is a 5-10 member aromatic ring or aromatic heterocyclic ring, aliphatic heterocyclic ring or cycloalkane, which shares two carbon atoms with the nitrobenzene ring to form a fused ring structure;

[0020] R1 is attached to any skeletal atom of the Cx ring, selected from hydrogen, halogen atoms, cyano or isocyano, hydroxyl, mercapto, amino, OTs, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 ynyl or Z-substituted ynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl, alkoxy group with 1-6 carbon atoms or Z-substituted alkoxy group with 1-6 carbon atoms, -CONR 6 R 7 -SO2NR 6 R 7 -SO2R 6 -OCOO-R 6 -COOR 6 -NR 6 COR 7 -OCOR 6 -NR 6 SO2R 7 -NR 6 SO2NR 6 R 7 ,

[0021] R2 and R3 are each independently hydrogen, C1-C6 alkyl or Z-substituted alkyl, C2-C6 alkenyl or Z-substituted alkenyl, C2-C6 ynyl or Z-substituted ynyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10Aryl or Z-substituted aryl, 4-15 membered heterocycles or Z-substituted heterocycles, 5-15 membered heteroaryl or Z-substituted heteroaryl, or R2, R3 together with the benzylic carbon atom to which they are bonded to form 3-6 membered rings;

[0022] The group can replace hydrogen atoms at any position on the carbon atom of the fused ring, with a substitution number of 1.

[0023] Z substituents are halogen atoms, cyano or isocyano, hydroxyl, mercapto, amino, C1-C3 alkyl or substituted alkyl, C1-C3 alkoxy or substituted alkoxy, C2-C3 alkenyl or substituted alkenyl, C2-C3 ynyl or substituted ynyl, C3-C8 cycloalkyl or substituted cycloalkyl.

[0024] R 6 R 7 Each of these elements is independently hydrogen, C1-C6 alkyl or Z-substituted C1-C6 alkyl, C2-C6 alkenyl or Z-substituted C2-C6 alkenyl, C2-C6 ynyl or Z-substituted C2-C6 ynyl, C3-C8 cycloalkyl or Z-substituted C3-C8 cycloalkyl, C6-C 10 Aryl or Z-substituted C6-C 10 Aryl, 4-15-membered heterocyclic or Z-substituted 4-15-membered heterocyclic, 5-15-membered heteroaryl or Z-substituted 5-15-membered heteroaryl, or R 6 R 7 Together with the atoms they are bonded to, they form 5-7 membered heterocyclic groups or Z-substituted 5-7 membered heterocyclic groups.

[0025] The pharmaceutically acceptable salts of the compounds of this invention are basic or acidic salts. The salts may be basic salts, including salts formed by the compound with an inorganic base (e.g., alkali metal hydroxides, alkaline earth metal hydroxides, etc.) or with an organic base (e.g., monoethanolamine, diethanolamine, or triethanolamine, etc.). Alternatively, the salts may be acidic salts, including salts formed by the compound with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid, etc.) or with an organic acid (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, or citric acid, etc.).

[0026] In a preferred embodiment, the solvates of the compounds of the present invention are hydrates or alcohols, wherein the alcohols include ethanol compounds.

[0027] The isotopic variants of the compounds of the present invention are typically substituted by the substitution of hydrogen atoms (H) with deuterium atoms (D).

[0028] In particular, the deuterium-substituted sites are located at the Ph-C of the compound of formula (I). * - As shown in the following formula:

[0029]

[0030] The compounds of the present invention may also include mirror isomers, non-mirror isomers and geometric isomers other than non-mirror isomers, as well as mixtures of mirror isomers, non-mirror isomers and geometric isomers other than non-mirror isomers.

[0031] Methods for selecting and preparing pharmaceutically acceptable salts or solvates or isotopic variants or isomers of compounds are well known in the art.

[0032] In a preferred embodiment, in equation (I):

[0033] Cx is a 5, 6, or 8-membered aromatic ring; or a 5, 6, 7, or 8-membered aromatic heterocyclic or aliphatic heterocyclic ring containing N, O, or S atoms; or a 5, 6, 7, or 8-membered alicyclic ring.

[0034] In a further preferred embodiment, it is a compound of formula (II):

[0035]

[0036] in:

[0037] R1 can replace hydrogen atoms at any position on a carbon atom in a fused ring, and the number of substituents R1 is 1, 2, 3, 4, 5 or 6; X is C or N.

[0038] In a further preferred embodiment, in formula (II): only one or two of the four X atoms are N atoms.

[0039] In a further preferred embodiment, in formula (II): R1 is hydrogen, a halogen atom, a C1-C6 alkyl or Z-substituted alkyl, a C3-C8 cycloalkyl or Z-substituted cycloalkyl, a C6-C 10 aryl or Z-substituted aryl, 4-15 membered heterocycle or Z-substituted heterocycle, 5-15 membered heteroaryl or Z-substituted heteroaryl; or

[0040] R2 and R3 are each independently hydrogen, C1-C6 alkyl or Z-substituted alkyl, C3-C8 cycloalkyl or Z-substituted cycloalkyl, C6-C 10 Aryl or Z-substituted aryl, 4-15 membered heterocyclic or Z-substituted heterocyclic, 5-15 membered heteroaryl or Z-substituted heteroaryl.

[0041] In a further preferred embodiment, in formula (II): R1 is hydrogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl, C3-C8 cycloalkyl or halogen-substituted C3-C8 cycloalkyl, C6-C 10 C6-C substituted with aryl or halogen 10 Aryl; or

[0042] R2 and R3 are each independently hydrogen, C1-C6 alkyl or halogen-substituted C1-C6 alkyl, C3-C8 cycloalkyl or halogen-substituted C3-C8 cycloalkyl, C6-C 10 C6-C substituted with aryl or halogen 10 Aryl.

[0043] In a further preferred embodiment, in formula (II): R1 is H, -CH3, -CF3; R2 and R3 are each independently H, D, -CH3, -CF3. More preferably, when X is C, R1 and R2 are H, and R3 is H, D, or -CF3.

[0044] In a preferred embodiment, in formula (I), the Z substituent is a halogen.

[0045] In a further preferred embodiment, the compound of formula (I) is a compound having the following structure:

[0046]

[0047]

[0048] The second aspect of the present invention relates to the pharmaceutical use of compounds as described in the first aspect of the present invention, or pharmaceutically acceptable salts or solvates or isotopic variants or isomers thereof.

[0049] The present invention provides a drug or preparation containing the above-described compound or a pharmaceutically acceptable salt or solvate or isotopic variant or isomer thereof.

[0050] The present invention also provides the use of the above-described drugs or preparations for treating tumors, cancers or proliferative diseases.

[0051] The present invention also provides the use of the above-described compounds or pharmaceutically acceptable salts or solvates or isotopic variants or isomers thereof in the preparation of medicaments for treating tumors, cancers or proliferative diseases.

[0052] In a preferred embodiment, the tumor or cancer includes:

[0053] Lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, brain 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, neuroblastoma, craniopharyngioma, ependymoma, pineal gland tumor, hematopoietic tumor. Vocal cord neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystoma, fibromixoma, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibromyxosarcoma, fibropapillary tumor, myxosarcoma, myxosacraloma, myxochondrosarcoma, myxochondrofibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipadenoma, lipoblastoma, lipochondroma, lipofuscinoma, lipofibroma, lipangioma, myxolipoma, chondrosarcoma, chondroma, soft Osteoma, chordoma, chorioadenoma, chorioepithelial tumor, choriocartilaginous tumor, osteosarcoma, osteoblastoma, osteochondrosarcoma, osteochondroma, bone cystoma, odontoma, osteofibroma, osteofibrosarcoma, angiosarcoma, hemangioma, angiolipoma, angiochondroma, hemangioblastoma, angiokeratoma, angioneurotic glioma, hemangioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphangioma, angiolipoleiomyoma, angiomyolipoma, angiomyosoma, angioneurotic neuroma, angiomyxoma, angioreticular endothelioma, lymphangiosarcoma, lymphatic Granuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, synovoma, synovial sarcoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyosarcoma, rhabdomyomyxoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic disease cells, polycythemia vera, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma, or multiple myeloma.

[0054] In a preferred embodiment, the tumor or cancer is selected from non-small cell lung cancer, pancreatic cancer, breast cancer, or prostate cancer. Further, the breast cancer is selected from triple-negative breast cancer.

[0055] Triple-negative breast cancer refers to breast cancer patients who are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The prognosis of triple-negative breast cancer is not significantly related to tumor size or lymph node status. Recurrence is relatively rapid, with a peak recurrence period of 1-3 years, and a high incidence of brain metastasis.

[0056] On the other hand, as a preferred option, the cancer or tumor is primary brain cancer, brain tumor, or metastatic cancer or tumor that has metastasized to the brain.

[0057] This invention also provides the use of the above-mentioned compound or its pharmaceutically acceptable salt or solvate or isotopic variant or isomer in the preparation of a medicament for treating tumors or cancers in patients with impaired DNA repair, wherein the impaired DNA repair is defined as impaired homologous recombination repair or impaired nucleotide excision repair. In other words, the above-mentioned compound or its pharmaceutically acceptable salt or solvate or isotopic variant or isomer can be prepared as a medicament for treating tumors or cancers in patients with impaired DNA repair, wherein the impaired DNA repair is limited to impaired homologous recombination repair or impaired nucleotide excision repair.

[0058] Generally, impaired DNA repair includes

[0059] Damage to homologous recombination repair enzymes

[0060] Damage to nucleotide excision repair enzymes

[0061] Impaired nonhomologous end joining

[0062] Damage to base excision repair enzymes

[0063] Damaged mismatch repair enzymes

[0064] The compounds provided by this invention, or their pharmaceutically acceptable salts, solvates, isotope variants, or isomers, can be experimentally demonstrated to have good effects on cancer patients with impaired DNA repair, including those with damage to the two aforementioned DNA repair pathways.

[0065] The present invention also provides a compound medicine comprising the above-described compound or its pharmaceutically acceptable salt or solvate or isotopic variant or isomer, and

[0066] a. Traditional chemotherapy drugs;

[0067] b. Anti-angiogenic drugs;

[0068] c. Cell checkpoint inhibitors; or

[0069] d. Immunosuppressants.

[0070] Based on the fact that the compounds in this invention have the same mechanism of action as the hypoxia-activated classic drug TH-302: after hypoxia activation, they are metabolized to release cytotoxic DNA alkylating agents, therefore the compounds in this invention or their pharmaceutically acceptable salts or solvates or isotope variants or isomers can enhance the anticancer effect through the combined use of four anticancer drugs.

[0071] Therefore, the present invention also provides the following combination therapies.

[0072] Combination therapy for treating cancer or tumors includes administering to a patient a drug or preparation containing the aforementioned compound or a pharmaceutically acceptable salt or solvate or isotopic variant or isomer thereof; and administering conventional chemotherapy drugs.

[0073] Combination therapy for cancer or tumors includes administering to a patient a drug or preparation containing the aforementioned compound or a pharmaceutically acceptable salt or solvate or isotopic variant or isomer thereof; and administering an anti-angiogenic drug.

[0074] Combination therapy for treating cancer or tumors includes administering to a patient a drug or formulation containing the aforementioned compound or a pharmaceutically acceptable salt or solvate or isotopic variant or isomer thereof; and administering a cell checkpoint inhibitor.

[0075] Combination therapy for treating cancer or tumors includes administering to a patient a drug or preparation containing the aforementioned compound or a pharmaceutically acceptable salt or solvate or isotopic variant or isomer thereof; and administering immunosuppressants.

[0076] Specifically, the four combined therapies mentioned above are explained below.

[0077] One approach is to use it in combination with traditional chemotherapy drugs. Traditional chemotherapy drugs can only kill tumor cells that operate under normoxic conditions but are ineffective against hypoxic cells. These traditional chemotherapy drugs...

[0078] Introduction Docetaxel(Vanhoefer U,Cao S,Harstrick A,Seeber S,Rustum YM(1997)Comparative antitumor efficacy of docetaxel and paclitaxel in nude micebearing human tumor xenografts that overexpress the multidrug resistance protein(MRP).Ann Oncol 8:1221-1228).

[0079] Antitumor effect of combination treatment with sabarubicin (MEN 10755) and cis-platinum (DDP) in human lung tumors xenograft.Cancer Chemother Pharmacol 62:621–629).

[0080] Intravenous pemetrexed(Huber PE,Bischof M,Jenne J,Heiland S,Peschke P,Saffrich R,Grone HJ,Debus J,Lipson KE,Abdollahi A(2005)Trimodal cancertreatment:beneficial effects of combined antiangiogenesis,radiation,andchemotherapy.Cancer Res 65:3643–3655).

[0081] 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),

[0082] 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),

[0083] 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),

[0084] Temozolomide Middleton MR, Thatcher N, McMurry TB, McElhinneyRS, Donnelly DJ, Margison GP (2002) Effect of O6-(4-bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model. Int JCancer 100:615-617

[0085] Combined with hypoxia-activated compounds, it can kill all tumor cells (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).

[0086] Second, when used in combination with anti-angiogenic drugs, it can increase the hypoxic region of tumor cells, thereby increasing the number of tumor cells sensitive to hypoxia 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, etc. 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.

[0087] 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, BMC Cancer, 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), etc. 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, thereby enhancing the effect of the DNA alkylating agents of the present invention, and thus achieving a synergistic anti-cancer effect through combined drug use.

[0088] Fourth, it can be used in combination with immunosuppressants. Hypoxia compounds can reduce the hypoxia zone of tumors, thereby increasing the infiltration of lymphocytes in the hypoxic area and improving the 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.). Experimentally proven immunosuppressant drugs 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 the present invention, can increase the infiltration of lymphocytes in hypoxic areas, thereby enhancing the effect of immunotherapy.

[0089] The drugs or formulations described herein may, of course, include pharmaceutically acceptable carriers or excipients. The drugs may be any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small injection needles, lyophilized powder for injection, or large-volume infusions. Depending on the specific dosage form and administration method, the pharmaceutically acceptable carriers or excipients in the drugs 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] The third aspect of the present invention relates to a method for preparing compounds as described in the first aspect of the present invention, or pharmaceutically acceptable salts or solvates or isotopic variants or isomers thereof.

[0091] One possible approach is Scheme 1, which includes the following steps:

[0092] To induce cyclization of compound one through a condensation reaction to provide a compound of formula (II):

[0093]

[0094] Compound 1

[0095] or

[0096] This method can be implemented using Scheme 2, which includes the following steps:

[0097] Compound II is reacted with R1H to provide a compound of formula (II):

[0098]

[0099] Compound 2;

[0100] Y1 and Y2 are leaving bases.

[0101] In a preferred embodiment, Y1 and Y2 are independently Cl, Br, I, -OTs, -ONO2, -OMs, or -OT. f Or -OSO2Cl; more preferably, for compound one, Y1 is Br, and for compound two, Y2 is F.

[0102] Where -OTs is -OMs is -OT f for

[0103] In a preferred embodiment, DIPEA or TEA is used as an acid-binding agent in Scheme 1, and silver oxide (Ag2O) or silver nitrate (AgNO3) is used as a catalyst.

[0104] In a preferred embodiment, a base is added during the reaction process of Scheme 2. The base can be an organic base, including organic amines such as monoethanolamine, diethanolamine, or triethanolamine; or an inorganic base, such as hydroxides, carbonates, bicarbonates, sulfites, bisulfites, or hydrides of alkali metals or alkaline earth metals, or other dehydrogenating agents, such as alkali metal alkylates (RM, where R is alkyl and M is alkali metal) or alkali metal alkoxides (MOR, where R is hydrocarbon and M is alkali metal).

[0105] This invention also relates to the use of compounds of the following formula in the preparation of medicaments for treating primary brain cancer, brain tumors, or metastatic cancers or tumors that have metastasized to the brain.

[0106]

[0107] By selecting appropriate deuterated intermediates or starting materials according to the above-described compound synthesis route, the synthesis and preparation of the deuterated compounds or other isotopic variants described in this invention can be completed. Attached Figure Description

[0108] Figure 1The inhibition rate curves of different concentrations of TH-302 and compound 01 on H460 cells under normal oxygen air and hypoxic nitrogen conditions are shown. In this figure, Log.con(μM) represents the logarithm of the concentration value in μmol / L with base 10.

[0109] Figure 2 The survival curves of AA8 and UV41 cells under normal oxygen air conditions at different concentrations of compound 01 are shown. Log.con(μM) represents the logarithm of the concentration value in μmol / L with base 10.

[0110] Figure 3 This is a schematic diagram showing the average remaining percentage of TH-302 and compound 01 at different times after the addition of 53.2 μg / ml human NADPH coenzyme to the sample for reduction of TH-302 and compound 01 under hypoxic (mainly nitrogen environment, oxygen volume concentration <0.1%) and air conditions at 37℃.

[0111] Figure 4 This is a schematic diagram showing the average remaining percentage of TH-302 and compound 01 at different times after the addition of 104.6 μg / ml human NADPH coenzyme to the sample for reduction of TH-302 and compound 01 under hypoxic (mainly nitrogen environment, oxygen volume concentration <0.1%) and air conditions at 37℃.

[0112] Figure 5 The curves show the percentage changes in the content of compound 01 after reacting with liver microsomes of different animals in the presence of NADPH.

[0113] Figure 6 The curves show the changes in the relative percentage content of compound 01 in the serum of different animals.

[0114] Figure 7 The graph shows the tumor volume changes over different days under different dosing regimens in a PDX-induced gastric cancer mouse model experiment. The horizontal axis represents the number of days, and the vertical axis represents the volume in mm. 3 number.

[0115] Figure 8 The graph shows the change in body weight of mice relative to the starting weight at different times under different dosing regimens in the 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.

[0116] Figure 9 The graph shows the tumor volume changes over different days under different drug administration regimens in a CDX mouse model with H460 cell line transplantation. The horizontal axis represents the number of days, and the vertical axis represents the volume in mm. 3 number.

[0117] Figure 10 The graph shows the change in body weight of mice relative to the starting weight at different times under different dosing regimens in the CDX mouse model experiment with H460 cell line transplantation. The horizontal axis represents the number of days and the vertical axis represents the percentage change in body weight.

[0118] Figure 11 This is the chiral HPLC spectrum of compound 01.

[0119] Figure 12 The image shows the chiral HPLC spectrum of the optical isomer corresponding to peak 1 obtained after chiral separation of compound 01.

[0120] Figure 13 The image shows the chiral HPLC spectrum of the optical isomer corresponding to peak 2 obtained after chiral separation of compound 01.

[0121] Figure 14 This is a schematic diagram of the absolute stereo configuration of the optical isomer corresponding to peak 1 obtained after chiral separation of compound 01. Detailed Implementation

[0122] 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.

[0123] Unless otherwise specified, the experimental methods used in the following examples are conventional methods and are conducted at room temperature and pressure. Unless otherwise specified, the medicinal materials and reagents used in the following examples are commercially available products.

[0124] definition

[0125] As described in this article, heterocyclic and heteroaryl groups include three-membered, four-membered, five-membered, six-membered, or seven-membered rings. Examples are given below.

[0126] Three-membered rings: ethylene oxide, cycloazoethane, and cyclothioethane;

[0127] Four-membered rings: acridine, oxadiidine, thiamethoxam, butidine;

[0128] Five-membered rings: pyrrolidine, pyrrolidine, 1-pyrrolidine, 3-pyrrolidine, 2-pyrrolidine, pyrrole, pyrazolidine, 2-pyrazolidine, imidazole, pyrazole, furan, THF, dihydrofuran, tetrahydrothiophene, thiophene, sulfolane, phosphazene, oxazole, 1,2,3-triazole, 1,2,4-triazole, 1,3,4-thiadiazole;

[0129] Six-membered rings: piperidine, tetrahydropyran, tetrahydrothiaran, pyridine, pyran, thiaran, dihydropyridine, morpholine, piperazine, pyridazine, pyrazine, 1,3,5-triazine, 1,3,5-trithiazide;

[0130] Seven-membered ring: acrylonitrile (azacycloheptane), oxaheptane, thiaheptane, azacycloheptane Oxygen , thiapine .

[0131] In this paper, fused rings are defined as the fusion of heterocyclic groups, heteroaryl groups, or cycloalkane structures. Fusion can be achieved through single bond linkage or by sharing one, two, or even three atoms. Some common fused ring structures are listed below: naphthalene, quinoline, indole, isoindole, isoquinoline, cycloalgin, quinoxaline, biphenyl, coumarin, fluorene, diphenylcarbamate, carbazole, anthracene, nitroanthracene, thiophene, adamantane, azulene, phenanthrene, anthraquinone, flavonoids, and isoflavones.

[0132] "Leaving group" refers to the portion that can be replaced under nucleophilic substitution conditions known to those skilled in the art. Leaving groups include (but are not limited to) halogen groups and -OSO2-R. 20 , where R 20 It can be an optionally substituted alkyl, aryl, cycloalkyl, heterocyclic or heteroaryl group.

[0133] "Patient" refers to a mammal in need of cancer treatment. Typically, a patient is a human being diagnosed with cancer. In some implementations, "patient" may refer to a non-human mammal used for screening, characterizing, and evaluating drugs and therapies, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0134] A "prodrug" is a compound that, upon administration or metabolism, is otherwise converted into a compound (or drug) with at least one biological activity or greater activity relative to the drug. A prodrug is chemically modified in a manner that renders it less active or inactive relative to the drug, but the modification allows the drug to be produced via metabolism or other biological processes after administration. Prodrugs may have altered metabolic stability or delivery characteristics, fewer side effects or lower toxicity, or a modified flavor relative to the active drug (see, for example, Nogrady, 1985, Medicinal Chemistry ABiochemical 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.

[0135] "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).

[0136] 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, the therapeutic effective dose can be administered or given once or multiple times.

[0137] "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.

[0138] "Tumor cells" refers to tumor cells in any appropriate species (e.g., mammals, such as rats, dogs, cats, horses, or humans).

[0139] Preparation Examples

[0140] Explanation of English Abbreviations

[0141] 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.

[0142] Calculated, the theoretical value calculated by mass spectrometry; found, the measured value by mass spectrometry; eq, the equivalent molar ratio.

[0143] 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.

[0144] Unless otherwise specified, all reagents and medicines were purchased commercially.

[0145] 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.

[0146] Preparation Example 1: Preparation of Compound 01

[0147]

[0148] Under nitrogen protection, compound 1-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 aqueous solution (50 mL) were added to a 500 mL four-necked flask. The mixture was heated to 62 °C until the reaction was complete. Post-treatment: After cooling the reaction system to room temperature, it was filtered with diatomaceous earth. The mother liquor was extracted with CHCl3, dried, concentrated, and preliminarily separated to obtain compound 1-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).

[0149] Compound 1-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. Post-treatment: The reaction mixture 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 compound 1-A3 (7.3 g), which was used directly in the next step without further purification.

[0150] Under nitrogen protection, compound 1-A3 (7 g crude, 16.33 mmol) and ZnBr2 (7.4 g, 32.66 mmol) were added to 100 mL of dioxane. The mixture was heated and stirred for 30 minutes. Then, 15 mL of concentrated hydrochloric acid was added dropwise to the reaction system, and the mixture was heated and stirred until the reaction was complete. Post-treatment: The reaction system was cooled to room temperature, concentrated, dissolved in water, extracted with DCM, washed with 5% NaOH solution, dried and concentrated, and separated by chromatography to obtain compound 1-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).

[0151] Under nitrogen protection, compound 1-A4 (200 mg, 0.99 mmol) and TMSCF3 (283 mg, 1.98 mmol) were added to 10 mL of THF. The mixture was cooled to 0 °C, and TBAF (0.1 mL) was added dropwise to the reaction system. The reaction continued until the starting material was completely converted. 3 mL of 3 mol / L hydrochloric acid was added dropwise to the reaction system. The mixture was extracted with DCM. The organic phase was washed with water and brine, dried and concentrated, and then separated by column chromatography to obtain compound 1-A5 product (190 mg, 70.5%), which was 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).

[0152] Under nitrogen protection, POCl3 (210 mg, 1.36 mmol) was added to 10 mL of DCM, and the mixture was cooled to -30 °C. Compound 1-A5 (185 mg, 0.68 mmol) was dissolved in DCM and added dropwise to the reaction system. Then, TEA (173 mg, 1.71 mmol) was dissolved in 5 mL of DCM and added dropwise to the reaction system. The reaction was maintained at -30 °C until the starting material was completely converted. Bromoethylamine hydrobromide (1.1 g, 5.46 mmol) and TEA (552 mg, 5.46 mmol) were added to the reaction system, and the reaction was completed after 30 minutes. After adding 20 mL of saturated ammonium chloride aqueous solution at 0 °C, the mixture was extracted with DCM. The organic phase was washed with water and brine, dried and concentrated, and then separated by column chromatography to obtain compound 1-A6 product (170 mg, 44.3%) as 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] + ).

[0153] Compound 1-A6 (160 mg, 0.28 mmol) was dissolved in THF (15 mL) under nitrogen protection. Then, silver oxide (329 mg, 1.42 mmol) and DIEA (167 mg, 1.42 mmol) were added to the reaction system. The mixture was heated to 65 °C and stirred until the reaction was complete. The reaction system was filtered with diatomaceous earth, the solid was washed with DCM, the mother liquor was concentrated, and compound 01 was prepared as a pale yellow solid by high performance liquid chromatography. 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] + ).

[0154] Preparation Example 2: Preparation of Compound O2

[0155]

[0156] Under nitrogen protection, 1-A4 (500 mg, 2.5 mmol) was added to THF (10 mL), and the mixture was cooled to 0 °C. Then, BH3-THF (12.4 mL, 12.4 mmol, 1 M in THF) was added dropwise to the system. After the reaction was complete, the mixture was stirred at 0 °C for 30 min. Methanol (10 mL) was added dropwise, and the mixture was stirred for 30 min. The solvent was evaporated to dryness. The crude product was dissolved in DCM (20 mL), washed with 1 M HCl (20 mL × 3), washed with water (10 mL), washed with brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness to obtain 2-A1 (470 mg, 93.1%), which was a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.57 (d, J=8.4Hz, 1H), 8.19 (d, J=8.0Hz, 1H), 8.13 (d, J=8.0Hz, 1H), 7.76-7.66 (m, 3H), 5.26 (s, 2H).

[0157] Under nitrogen protection, POCl3 (151 mg, 1.0 mmol) was added to DCM (5 mL), and the mixture was cooled to -30 °C. Then, 2-A1 (100 mg, 0.5 mmol) was dissolved in DCM (2 mL) and added dropwise to the system. TEA (125 mg, 1.2 mmol) was dissolved in DCM (2 mL) and added dropwise to the system. After reacting for 1.5 h, 2-bromoethylamine hydrobromide (822 mg, 3.9 mmol) and TEA (398 mg, 3.9 mmol) were added to the system sequentially at the same temperature. The reaction was completed after 30 min. After heating to 0 °C, saturated ammonium chloride solution (10 mL) was added dropwise, followed by water (10 mL). The mixture was extracted with DCM (10 mL × 3), washed with water (5 mL), washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by column chromatography to obtain 2-A2 (110 mg, 45.1%) as a pale yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.57 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 7.6 Hz, 2H), 7.79-7.68 (m, 3H), 5.56 (d, J = 7.2 Hz, 1H), 3.44-3.12 (m, 8H). MS:Calculated 492.9,494.9,496.9,found 493.9,495.9,497.9([M+H] + ).

[0158] Under nitrogen protection, 2-A2 (100 mg, 0.2 mmol) was dissolved in THF (10 mL), and then Ag2O (234 mg, 1.0 mmol) and DIEA (130 mg, 1.0 mmol) were added to the system. The mixture was heated to reflux and reacted for 7 h until the reaction was complete. After cooling to room temperature, the mixture was filtered through a diatomaceous earth liner, washed with DCM, and the solvent was evaporated. Compound O2 (45.0 mg, 67.2%) was obtained by high performance liquid chromatography as a yellow oily substance. 1 H-NMR (400MHz, CD3OD): δ8.44-8.42(m,1H), 4.30-8.28(m,1H), 8.19(d,J=8.0Hz,1H), 7.81-7.76(m,3H), 5.72(d,J=7.6Hz,1H), 2.23-2.16(m,8H). MS: Calculated 333.1, found 334.0([M+H] + ).

[0159] Preparation Example 3: Preparation of Compound 03

[0160]

[0161] Under nitrogen protection, 3-A1 (1.0 g, 5.3 mmol, purchased) was dissolved in DMF (8 mL), and then DMF-DMA (1.9 g, 16.0 mmol) was added. The mixture was heated to 130 °C and stirred for 16 h. After the reaction was completed, the mixture was cooled to room temperature, 2 / 3 of the DMF was distilled off, and water (20 mL) was added. The solid precipitated, filtered, and dried to obtain crude 3-B1 (1.3 g, 100%), which was a black solid. 1 H-NMR (400MHz, DMSO-d6): δ7.89-7.90 (m, 3H), 7.56-7.51 (m, 2H), 7.43-7.39 (m, 2H), 4.89 (d, J = 13.2Hz, 1H), 2.89 (s, 6H).

[0162] Under nitrogen protection, 3-B1 (1.0 g, 4.1 mmol) was dissolved in THF-water solution (1:1, 40 mL), and sodium periodate (2.6 g, 12.4 mmol) was added in portions. The mixture was stirred at 25 °C for 2 h. After the reaction was completed, the mixture was filtered. The solid was soaked in DCM and filtered again. The mother liquor was dried with anhydrous sodium sulfate and evaporated to dryness to obtain 3-A3 (600 mg, 72.2%), which was a yellow solid. 1 H-NMR (400MHz, CDCl3): δ10.17 (s, 1H), 8.11 (d, J = 8.4Hz, 1H), 8.00-7.91 (m, 3H), 7.77-7.72 (m, 2H).

[0163] 3-A3 (450 mg, 2.2 mmol) was dissolved in MeOH (12 mL), cooled to 0 °C, and sodium borohydride (127 mg, 3.4 mmol) was added in portions. The reaction was stopped once the addition was complete. The solution was quenched with water (10 mL), extracted with DCM (20 mL × 2), and the organic phase was washed with 5% citric acid aqueous solution (10 mL × 2), washed with water (10 mL × 2), washed with brine (10 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to obtain 3-A4 (450 mg, 99.0%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.03 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.68-7.59 (m, 3H), 4.85 (s, 2H).

[0164] Under nitrogen protection, POCl3 (679 mg, 4.43 mmol) was dissolved in DCM (12 mL), and the mixture was cooled to -30 °C. A DCM solution (2 mL) of 3-A4 (450 mg, 2.21 mmol) and TEA (560 mg, 5.54 mmol) was added dropwise to the system. After incubation for two hours, 2-bromoethylamine hydrobromide solid (2.7 g, 13.3 mmol) was added, followed by the dropwise addition of TEA (1.8 g, 17.7 mmol). The reaction was stopped after half an hour. The reaction was quenched with saturated ammonium chloride aqueous solution (2 mL), extracted with DCM (10 mL × 3), washed with water (3 mL × 2), dried over anhydrous sodium sulfate, evaporated to dryness, and column chromatography yielded 3-A5 (1.0 g, 91.2%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.03(d,J=8.4Hz,1H),7.94(d,J=7.6Hz,1H),7.82(d,J=8.4Hz,1H),7 .70-7.60(m,3H),5.25(d,J=7.6Hz,2H),3.46-3.43(m,4H),3.36-3.32(m,4H),3.11(brs,2H). MS:Calculated 492.9,494.9,496.9,found 494.0,496.0,498.0([M+H] + ).

[0165] Under nitrogen protection, 3-A5 (500 mg, 1.0 mmol) was dissolved in THF (20 mL), and DIEA (653 mg, 5.1 mmol) and silver oxide (625 mg, 2.8 mmol) were added. The mixture was heated to reflux, and the reaction was completed after one hour. The solution was filtered through diatomaceous earth, and the mother liquor was evaporated to dryness. The product compound O3 (75 mg, 22.3%) was prepared by high performance liquid chromatography as an off-white solid. 1 H-NMR (400MHz, CD3OD): δ8.17(d,J=8.4Hz,1H),8.06(d,J=7.6Hz,1H),7.80( d,J=8.2Hz,1H),7.74-7.67(m,3H),5.38(d,J=7.6Hz,2H),3.23-2.16(m,8H). MS:Calculated 333.1,found334.1([M+H] + ).

[0166] Preparation Example 4: Preparation of Compound 04

[0167]

[0168] At 0℃, using two dropping funnels, 4-A1 (30.0 g, 179.4 mmol, purchased) was slowly added dropwise to a stirred concentrated ammonia solution (440 mL) in 1.58 M NaOH solution (120 mL) and NaClO aqueous solution (10% available chlorine, 144 mL). The addition time was 2 h, and the temperature was maintained between -5℃ and 0℃. After the addition was complete, the reaction continued for 2 h. After the reaction was complete, the solid was filtered, the filter cake was washed with cold water (2 L), dissolved in hot DCM (200 mL), dried with anhydrous sodium sulfate, and concentrated to obtain 4-A2 (26.0 g, 79.5%), which was an off-white solid. 1 H-NMR (400MHz, DMSO-d6): δ7.99 (d, J = 8.1 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.31-7.28 (m, 1H), 4.94 (brs, 2H). MS:Calculated 182.0,found183.0([M+H] + ).

[0169] At room temperature, a mixed DMF solution (200 mL) of 4-A2 (20.0 g, 109.7 mmol) and 1-nitronaphthalene (21 g, 121.2 mmol) was added dropwise to a DMF solution (100 mL) containing t-BuOK (31 g, 276.3 mmol) over 2 h. After the addition was complete, the reaction was continued for 0.5 h. The above system was then slowly added to cold water (500 mL), stirred for 0.5 h, extracted with EA (200 mL × 3), washed with water (50 mL × 2), dried over anhydrous sodium sulfate, evaporated to dryness, and column chromatography was performed to obtain a yellow solid of 4-A3 (11.1 g, HPLC purity approximately 80%, yield 53.8%). 1 H-NMR (400MHz, CDCl3): δ7.90-7.85(m,2H),7.70-7.75(m,4H). MS:Calculated 188.1,found 189.2([M+H] + ).

[0170] 4-A3 (3.1 g, 16.5 mmol) and p-toluenesulfonic acid monohydrate (16.9 g, 98.8 mmol) were dissolved in tert-butanol (150 mL). An aqueous solution of NaNO2 (4.6 g, 66.7 mmol) and KI (13.7 g, 82.5 mmol) (75 mL) was added dropwise at 15 °C. After the addition was complete, the temperature was raised to 25 °C and the reaction proceeded for 2 h. After the reaction was complete, saturated NaHCO3 solution was added to adjust the pH to 9-10. Extraction was performed using EA (50 mL × 3), followed by washing with saturated Na2S2O3 aqueous solution (20 mL × 3), then water (10 mL × 3). The sample was dried over anhydrous sodium sulfate, evaporated to dryness, and column chromatography was used to obtain 4-A4 (2.0 g, 40.8%) as a pale yellow solid. 1 H-NMR (400MHz, CDCl3): δ7.91-7.84(m,2H),7.70-7.61(m,4H).

[0171] Under N2 protection, 4-A4 (4.7 g, 15.7 mmol) and tributyl(1-ethoxyvinyl)tin (8.0 g, 22.0 mmol) were dissolved in 1,4-dioxane (20 mL). The mixture was purged with N2 three times, and tetra(triphenylphosphine)palladium (454 mg, 0.4 mmol) was added, replacing the N2 three times. The mixture was then heated to 110 °C and reacted overnight. After cooling to room temperature, 3M HCl (30 mL) was added and stirred for 2 h. The mixture was extracted with DCM (40 mL × 3), dried, and evaporated to dryness. The crude product was subjected to column chromatography to obtain 4-A5 (2.5 g, 73.5%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ8.07 (d, J = 8.6Hz, 1H), 7.98-7.96 (m, 1H), 7.90-7.87 (m, 1H), 7.80-7.77 (m, 1H), 7.72 (m, 2H), 2.69 (s, 3H). MS: Calculated 215.1,found 216.2([M+H] + ).

[0172] 4-A5 (750 mg, 3.5 mmol) was dissolved in MeOH (20 mL) and DCM (10 mL), cooled to 0 °C, and NaBH4 (158 mg, 4.2 mmol) was added in portions. The reaction was maintained at this temperature for 1 h. After the reaction was complete, H2O (5 mL) was added, and the mixture was extracted with DCM (10 mL × 3), washed with 25% citric acid (10 mL × 2), washed with water (5 mL × 2), dried over anhydrous sodium sulfate, and separated by column chromatography to obtain 4-A6 (300 mg, 39.5%) as a pale yellow solid.

[0173] Under nitrogen protection, 4-A6 (130 mg, 0.6 mmol), Br-IPM (557 mg, 1.8 mmol), and triphenylphosphine (479 mg, 1.8 mmol) were added to THF (10 mL), the mixture was cooled to 0 °C, and DIAD (364 mg, 1.8 mmol) was added dropwise. The mixture was then allowed to rise naturally to room temperature and reacted for 1 h. After the reaction was complete, the mother liquor was concentrated, and column chromatography was used to obtain crude 4-A7 (150 mg, 49.2%), which was a pale yellow oil.

[0174] Under nitrogen protection, 4-A7 (150 mg, 0.3 mmol), DIEA (190 mg, 1.5 mmol), and Ag2O (348 mg, 1.5 mmol) were added to THF (10 mL), and the mixture was refluxed and stirred overnight. After the reaction was complete, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated. High-performance liquid chromatography (HPLC) was used to prepare compound O4 (40 mg, 39.2%), a yellow oily substance. 1 H-NMR (400MHz, CD3OD) δ8.19 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.80-7.68 (m, 4H), 5.79-5.77 (m, 1H), 2.19-2.01 (m, 8H), 1.74 (d, J = 6.4 Hz, 3H). MS:Calculated 347.1,found 348.1([M+H] + ).

[0175] Preparation Example 5: Preparation of Compound 05

[0176]

[0177] Under nitrogen protection, 3-A3 (1.8 g, 9.0 mmol) was dissolved in THF (18 mL), followed by the addition of TMSCF3 (1.9 g, 13.4 mmol). The mixture was cooled to 0 °C, and a THF solution of TBAF (1 N, 0.179 mL, 0.18 mmol) was added. The reaction was stopped after half an hour. 3 M hydrochloric acid (9 mL) was added and the mixture was stirred for 15 minutes. The mixture was extracted with DCM (20 mL × 3), washed with brine (10 mL × 2), dried over anhydrous sodium sulfate, evaporated to dryness, and column chromatography was used to obtain 5-A1 (2.2 g, 90.7%) as a black solid. 1 H-NMR (400MHz, CDCl3): δ8.07(d,J=8.7Hz,1H),7.96-7.94(m,1H),7.86(d,J=8.7Hz,1H),7.77(m,1H),7.69-7.66(m,2H),5.46(m,1H),3.36(brs,1H).

[0178] Under nitrogen protection, POCl3 (565 mg, 3.7 mmol) was dissolved in DCM (12 mL), and the mixture was cooled to -30 °C. A solution of 5-A1 (500 mg, 1.8 mmol) and TEA (466 mg, 4.6 mmol) dissolved in DCM (2 mL) was added dropwise to the system. After incubation for two hours, 2-bromoethylamine hydrobromide (2.3 g, 11.1 mmol) was added, followed by dropwise addition of TEA (1.5 g, 14.7 mmol). The reaction was stopped after half an hour. The mixture was quenched with saturated ammonium chloride aqueous solution (3 mL), extracted with DCM (10 mL × 3), washed with water (3 mL × 2), dried over anhydrous sodium sulfate, evaporated to dryness, and column chromatography was used to obtain 5-A2 (850 mg, 81.9%) as a yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.11 (d, J = 8.4Hz, 1H), 7.98 (m, 1H), 7.78-7.69 (m, 4H), 6.04-5.98 (m, 1H), 3.41-3.18 (m, 8H). MS:Calculated 560.9,562.9,564.9,found 561.9,563.9,565.9([M+H] + ).

[0179] Under nitrogen protection, 5-A2 (300 mg, 0.5 mmol) was dissolved in THF (20 mL), and DIEA (344 mg, 2.7 mmol) and silver oxide (330 mg, 1.4 mmol) were added. The mixture was heated to reflux, and the reaction was completed after one hour. The solution was filtered through diatomaceous earth, and the mother liquor was evaporated to dryness. The product compound 05 (70 mg, 32.7%) was obtained by high-performance liquid chromatography (HPLC) as a yellow oil. 1 H-NMR (400MHz, CD3OD): δ8.29 (d, J = 8.8 Hz, 1H), 8.13-8.10 (m, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.80-7.76 (m, 3H), 6.16-6.09 (m, 1H), 2.25-2.03 (m, 8H). MS:Calculated 401.1,found 402.1([M+H] + ).

[0180] Preparation Example 6: Preparation of compounds 06, 24 and 25

[0181]

[0182] Under nitrogen protection, concentrated sulfuric acid (2.3 mL, 42.2 mmol) was added to acetic acid (90 mL), followed by 6-A1 (4.5 g, 28.8 mmol). Then, fuming nitric acid (48 mL, 1.15 mol) was added dropwise to the system, and the reaction was allowed to proceed overnight. Post-treatment: The system was added dropwise to ice water (350 mL), filtered, washed with water (80 mL), and dried to obtain 6-A2 and its isomers (4.0 g, 69.0%, consisting of three isomers), as a yellow solid, which was directly added to the next reaction step.

[0183] Under nitrogen protection, 6-A2 (1.5 g, 7.4 mmol) was dissolved in methanol (20 mL), cooled to 0 °C, and sodium borohydride (423 mg, 11.2 mmol) was added in portions until the reaction was complete. The mixture was quenched with 10 mL of water, extracted with DCM (15 mL × 2), washed with 5% citric acid aqueous solution (8 mL × 2), washed with water (8 mL × 2), washed with brine (8 mL), dried, and evaporated to dryness to obtain 6-A3 and its isomer (1.4 g, 92.4%) as a yellow solid. MS: Calculated 203.1, found 204.1 ([M+H) + ).

[0184] Under nitrogen protection, 6-A3 and its isomer (400 mg, 2.0 mmol), and POCl3 (604 mg, 3.9 mmol) were dissolved in DCM (8 mL). The mixture was cooled to -30 °C, and TEA (498 mg, 4.9 mmol) was added dropwise. After one hour, 2-bromoethylamine hydrobromide (3.3 g, 15.7 mmol) was added, followed by the dropwise addition of TEA (1.6 g, 15.7 mmol). The reaction was stopped after half an hour. Water (5 mL) was added, and the mixture was extracted with DCM (15 mL × 2), washed with water (10 mL), dried, evaporated to dryness, and column chromatography was performed to obtain 6-A4 and its isomer (850 mg, 87.2%, mainly three isomers) as a yellow oil.

[0185] Under nitrogen protection, 6-A4 and its isomer (850 mg, 1.7 mmol) were dissolved in THF (20 mL), and silver oxide (1.2 g, 5.2 mmol) and DIEA (1.1 g, 8.6 mmol) were added. The mixture was heated to reflux and allowed to react overnight. The product compounds 06, 24, and 25 were obtained by filtration through diatomaceous earth, evaporation to dryness, and high-performance liquid chromatography (acetonitrile / water reversed-phase chromatography).

[0186] It is a yellow solid, 25 mg, with a yield of 4.4%. 1H-NMR (400MHz, CD3OD): δ8.52 (s, 1H), 8.28 (dd, J = 8.0, 4.0Hz, 2H), 8.12 (d, J = 8.4Hz, 1H), 7.72-7.64 (m, 2H), 5.40 (d, J = 8.0Hz, 2H), 2.28-2.19 (m, 8H). MS:Calculated 333.1,found 334.1([M+H] + ).

[0187] It is a white solid, 26 mg, with a yield of 4.5%. 1 H-NMR (400MHz, CD3OD): δ8.50(d,J=8.8Hz,1H),8.28(dd,J=8.0,4.0Hz,2H),8.11(s,1H),7 .80(dd,J=8.8,1.6Hz,1H),7.67(t,J=8.0Hz,1H),5.38(d,J=8.4Hz,2H),2.25-2.18(m,8H). MS:Calculated 333.1,found 334.1([M+H] + ).

[0188] It is a yellow oily substance, 8.7 mg, with a yield of 1.5%. 1 H-NMR (400MHz, CD3OD): δ8.45(d,J=8.4Hz,1H), 8.33-8.28(m,2H), 8.11(d,J=8.0Hz,1H), 7.80-7.69(m,2H), 5.41(d,J=8.8Hz,2H), 2.27-2.19(m,8H). MS:Calculated 333.1,found 334.1([M+H] + ).

[0189] Preparation Example 7: Preparation of Compound 07

[0190]

[0191] Under nitrogen protection, concentrated sulfuric acid (2.4 mL, 44.1 mmol) was added dropwise to acetic acid (50 mL), followed by the addition of 7-A1 (5.0 g, 29.4 mmol, acetic acid). The mixture was cooled to zero degrees Celsius, and fuming nitric acid (49 mL) was added dropwise. The mixture was stirred rapidly at room temperature overnight, and the reaction was monitored by liquid chromatography until completion. The system was carefully poured into ice water (400 mL), filtered, and the resulting solid was dissolved in DCM. Column chromatography yielded 7-A2 (170 mg, 2.7%) as a yellow solid. 1H-NMR (400MHz, CDCl3): δ9.22 (s, 1H), 8.33 (d, J = 7.6 Hz, 1H), 8.21-8.17 (m, 2H), 8.04 (d, J = 8.8 Hz, 1H), 7.72-7.68 (m, 1H), 2.78 (s, 3H). MS:Calculated 215.1,found 216.1([M+H] + ).

[0192] Under nitrogen protection, 7-A2 (120 mg, 0.6 mmol) was dissolved in methanol (3 mL), cooled to 0°C, and sodium borohydride (23 mg, 0.6 mmol) was added in portions. After the addition was complete, the reaction was monitored by thin-layer chromatography. The solution was quenched with water (2 mL), extracted with DCM (10 mL × 2), washed with 5% citric acid aqueous solution (5 mL × 2), washed with water (5 mL × 2), washed with brine (5 mL), and dried over anhydrous sodium sulfate. The resulting product was a yellow solid (100 mg, 82.6%). 1 H-NMR (400MHz, CDCl3) δ8.52(s,1H),8.23(d,J=0.8Hz,1H),8.11(d,J=8.4Hz,1H),7.97(d,J=8.4Hz ,1H),7.72(d,J=1.2Hz,1H),7.53-7.51(t,J=7.9Hz,1H),5.16-5.12(m,1H),1.60(d,J=6.4Hz,3H).

[0193] Under nitrogen protection, 7-A3 (150 mg, 0.7 mmol) and Br-IPM (533 mg, 1.7 mmol) were dissolved in ultra-dry THF, and triphenylphosphine (452 ​​mg, 1.7 mmol) was added. The mixture was cooled to zero degrees Celsius, and di-tert-butyl azodicarbonate (397 mg, 1.7 mmol) was added. After half an hour, the reaction was monitored by liquid chromatography to indicate its end. The solvent was evaporated to dryness, and column chromatography was used to obtain crude 7-A4 (150 mg, 42.7%) as a yellow oil.

[0194] Under nitrogen protection, 7-A4 (150 mg, 0.3 mmol), silver oxide (205 mg, 0.9 mmol), and DIEA (190 mg, 1.5 mmol) were added to THF (3 mL), and the mixture was heated to reflux. After one hour, the reaction was monitored by liquid chromatography to indicate completion. The mixture was filtered through diatomaceous earth, and the mother liquor was evaporated to dryness. High-performance liquid chromatography (HPLC) yielded compound 07 (32 mg, 31.3%), a yellow oily substance. 1H-NMR (400MHz, CD3OD) δ8.50 (s, 1H), 8.29-8.26 (m, 2H), 8.12 (d, J = 8.4Hz, 1H), 7.73 (dd, J = 8.4, 1.6Hz, 1H), 7.66 (t, J = 8.0Hz, 1H), 5.86-5.80 (m, 1H), 2.24-2.07 (m, 8H), 1.72 (d, J = 6.4Hz, 3H). MS:Calculated 347.1,found 694.9([2M+H] + ).

[0195] Preparation Example 8: Preparation of Compound 08

[0196]

[0197] Under nitrogen protection, 6-A2 (1.5 g, 7.5 mmol) and TMSCF3 (2.1 g, 14.9 mmol) were added to THF (25 mL), cooled to 0 °C, and TBAF (0.15 mL, 0.15 mmol, 1 M in THF) was added dropwise to the system. After incubation at 0 °C for 30 min, the starting material disappeared. 3N HCl (20 mL) was added dropwise to the system, stirred at room temperature for 1 h, extracted with DCM (30 mL × 3), washed with water and brine (5 mL × 2), dried and concentrated, and purified by column chromatography to obtain 8-A1 (1.5 g, 74.2%), which was a yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.70 (s, 1H), 8.31-8.29 (m, 1H), 8.16 (d, J = 8.4Hz, 1H), 8.0 3(d,J=8.8Hz,1H),7.79(d,J=8.4Hz,1H),7.62(t,J=8.0Hz,1H),5.32-5.27(m,1H).

[0198] Under nitrogen protection, POCl3 (678 mg, 4.4 mmol) and 8-A1 (600 mg, 2.2 mmol) were added to DCM (15 mL), and the mixture was cooled to -30 °C. TEA (560 mg, 5.5 mmol) was dissolved in DCM (5 mL) and added dropwise to the system. The mixture was kept at -30 °C for 2 h. Then, 2-bromoethylamine hydrobromide (3.7 g, 17.7 mmol) and TEA (1.8 mg, 17.7 mmol) were added sequentially. The reaction was complete 30 min after the additions. A saturated ammonium chloride aqueous solution (10 mL) was added dropwise, followed by DCM extraction (20 mL × 3). The organic phase was washed with water and brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to separate 8-A2 (830 mg, HPLC purity approximately 70%, yield 66.6%), a yellow oily substance. NMR and mass spectrometry were not performed on this substance before it was directly added to the next reaction step.

[0199] Under nitrogen protection, 8-A2 (330 mg, 0.6 mmol) was dissolved in THF (10 mL), followed by the addition of silver oxide (679 mg, 2.9 mmol) and DIEA (379 mg, 2.9 mmol). The mixture was heated to reflux and stirred overnight. After the reaction was complete, the mixture was filtered through a diatomaceous earth filter, washed with DCM, and the mother liquor was concentrated. The purified compound 08 (29 mg, 12.3%) was obtained by high-performance liquid chromatography as a white solid. 1 H-NMR (400MHz, CDCl3): δ8.75(s,1H),8.33(d,J=7.6Hz,1H),8.18(d,J=8.0Hz,1H),8.04(d,J= 8.4Hz, 1H), 7.76 (d, J = 8.4Hz, 1H), 7.65 (t, J = 7.6Hz, 1H), 5.95-5.93 (m, 1H), 2.31-2.02 (m, 8H). MS:Calculated 401.1,found 402.0([M+H] + ).

[0200] Preparation Example 9: Preparation of Compound 09

[0201]

[0202] Under nitrogen protection, 9-A1 (2.0 g, 7.9 mmol) was dissolved in dioxane (20 mL) and water (2.0 mL), followed by the addition of PdCl2 (dppf) (0.28 g, 0.39 mmol), K2CO3 (580 mg, 15.9 mmol), and tributyltin (2.20 g, 7.93 mmol). The mixture was heated to 100 °C and stirred for 5 hours. After the reaction was complete, the mixture was cooled to room temperature, and saturated ammonium chloride aqueous solution (5 mL) was added. The mixture was extracted with EA (20 mL × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to separate 9-A2 (1.2 g, 62.1%), which was a pale yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.50-8.44(m,2H),8.17(d,J=7.6Hz,1H),7.67-7.66(m,2H),7.55(t,J=8. 0Hz, 1H), 4.56 (d, J = 1.6Hz, 1H), 4.40 (d, J = 1.6Hz, 1H), 4.04 (q, J = 7.6Hz, 2H), 0.92 (t, J = 7.6Hz, 3H).

[0203] 9-A2 (1.2 g, 4.9 mmol) was dissolved in dioxane (20 mL) and water (4 mL). Then, NaIO4 (4.2 g, 19.7 mmol) was added in portions, and the mixture was heated to 35 °C. KMnO4 (390 mg, 2.5 mmol) was added in portions, and the mixture was heated to 40 °C and stirred for 4 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the solid was washed with EA. The aqueous phase was extracted with EA (20 mL × 3). The pH of the aqueous phase was adjusted to 2, and a solid precipitated. Filtering yielded 9-A3 (350 mg, 32.7%), which was an off-white solid. 1 H-NMR (400MHz, CD3OD): δ9.31 (d, J = 8.8 Hz, 1H), 8.56 (d, J = 8.8 Hz, 1H), 8.36 (d, J = 7.6 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.83-7.74 (m, 2H). MS:Calculated 217.0,found 216.0([MH] - ).

[0204] Under nitrogen protection, 9-A3 (350 mg, 1.6 mmol) was dissolved in THF (10 mL), cooled to 0 °C, and then BH3THF solution (4.0 mL, 4.0 mmol, 1 mol / L) was added. The mixture was heated to 50 °C and stirred for 2 hours. After the reaction was complete, the mixture was cooled to 0 °C, methanol (5 mL) was added, and the mixture was heated to 70 °C and reacted for 1 hour. The solvent was then evaporated, and 9-A4 (150 mg, 45.8%) was obtained by column chromatography as a light yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.48-8.45 (m, 2H), 8.19 (d, J = 7.6Hz, 1H), 7.69-7.60 (m, 3H), 5.20 (s, 2H), 3.70-3.69 (m, 1H). MS:Calculated 203.1,found 204.1([M+H] + ).

[0205] Under nitrogen protection, 9-A4 (150 mg, 0.7 mmol) was dissolved in DCM (10 mL), cooled to -40 °C, and then POCl3 (0.23 g, 1.5 mmol) and TEA (0.19 g, 1.85 mmol) were added sequentially. The mixture was stirred at -40 °C for 2 hours. Then, bromoethylamine hydrobromide (950 mg, 5.92 mmol) and TEA (1.49 g, 14.8 mmol) were added, and the mixture was kept at -40 °C for 3 hours. After the reaction was complete, saturated ammonium chloride aqueous solution (5 mL) was added, and the mixture was extracted with DCM (10 mL). The solvent was evaporated, and 9-A5 (210 mg, 57.3%) was obtained by column chromatography as a light yellow oil. The product was analyzed by NMR and mass spectrometry and then directly used in the next reaction step.

[0206] Under nitrogen protection, 9-A5 (280 mg, 0.6 mmol) was dissolved in THF (10 mL), followed by the addition of silver oxide (1.0 g, 4.5 mmol) and DIPEA (580 mg, 4.5 mmol). The mixture was heated to 65 °C and stirred for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered through a diatomaceous earth liner, washed with DCM, and the mother liquor was concentrated. Compound 09 (30 mg, 16.0%) was prepared as a white solid by high-performance liquid chromatography. 1 H-NMR (400MHz, CD3OD): δ8.54(d,J=8.8Hz,1H),8.41(d,J=8.4Hz,1H),8.23( d,J=7.6Hz,1H),7.79-7.73(m,3H),5.68(d,J=8.0Hz,2H),2.19-2.14(m,8H). MS:Calculated 333.1,found 334.1([M+H] + ).

[0207] Preparation Example 10: Preparation of Compound 10

[0208]

[0209] Under nitrogen protection, 10-A1 (3.0 g, 11.9 mmol) and tributyl(1-ethoxyethylene)tin (6.0 g, 16.7 mmol) were dissolved in 1,4-dioxane (75 mL), and tetra(triphenylphosphine)palladium (347 mg, 0.3 mmol, Greenkammer) was added. The mixture was purged with N2 three times and reacted overnight at 105 °C. After the reaction was complete, 3N hydrochloric acid (5 mL) was added, and the mixture was stirred for half an hour. The mixture was extracted with EtOAc (30 mL × 3), dried, evaporated to dryness, and the crude product was subjected to column chromatography to obtain 10-A2 (1.7 g, 65.4%) as a yellow solid. 1 H-NMR (400MHz, CDCl3) δ9.01(d,J=8.7Hz,1H),8.65(d,J=8.8Hz,1H),8.21(d,J=7. 0Hz, 1H), 8.06 (d, J = 6.8Hz, 1H), 7.77-7.73 (m, 1H), 7.69-7.65 (m, 1H), 2.79 (s, 3H). MS:Calculated 215.1,found216.2([M+H] + ).

[0210] Under nitrogen protection, 10-A2 (1.2 g, 5.6 mmol) was dissolved in MeOH (12 mL), cooled to 0 °C, and NaBH4 (253 mg, 6.7 mmol) was added in portions. The reaction was maintained at 0 °C for 0.5 h. After the reaction was complete, H2O (10 mL) was added, and the mixture was extracted with DCM (10 mL × 3). The mixture was washed with 10% citric acid aqueous solution (25 mL), extracted with DCM (10 mL), and the organic phase was washed with water (10 mL × 3), saturated brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. The crude product was subjected to column chromatography to obtain 10-A3 (1.0 g, 82.0%) as a pale yellow solid. 1 H-NMR (400MHz, CDCl3) δ8.46(d,J=8.4Hz,1H),8.38(d,J=8.8Hz,1H),8.13(d,J=7.6Hz,1H),7.82(d,J=7 .2Hz, 1H), 7.72-7.67 (m, 1H), 7.58 (t, J = 8.2Hz, 1H), 5.69-5.64 (q, J = 6.4Hz, 1H), 1.69 (d, J = 6.4Hz, 3H).

[0211] Under nitrogen protection, 10-A3 (200 mg, 0.9 mmol), Br-IPM (428 mg, 1.4 mmol), and triphenylphosphine (561 mg, 1.8 mmol) were added to anhydrous THF (10 mL), the mixture was cooled to 0 °C, and DIAD (372 mg, 1.8 mmol) was added dropwise. The reaction was maintained at this temperature for 1 h. After the reaction was complete, the mother liquor was concentrated, and the crude product was separated by column chromatography to obtain 10-A4 (234 mg, 51.1%), a pale yellow liquid. NMR and mass spectrometry were performed, and the product was directly used in the next reaction step.

[0212] Under nitrogen protection, 10-A4 (250 mg, 0.5 mmol), DIEA (318 mg, 2.4 mmol), and Ag2O (569 mg, 2.5 mmol) were added sequentially to THF (30 mL), and the mixture was refluxed and stirred overnight. After the reaction was complete, the mixture was filtered through a diatomaceous earth filter, and the filtrate was concentrated. Compound 10 (18 mg, 7.2%) was obtained by high-performance liquid chromatography as a yellow oily substance. 1 H-NMR (400MHz, CD3OD): δ8.57(d,J=8.8Hz,1H),8.32(d,J=8.8Hz,1H),8.18(d,J=7.2Hz,1H),7.86(d,J=7.2 Hz,1H),7.79-7.70(m,2H),6.49-6.29(m,1H),2.23-2.11(m,4H),2.06-1.99(m,4H),1.81(d,J=6.4Hz,3H). MS:Calculated 347.1,found 370.0([M+Na] + ).

[0213] Preparation Example 11: Preparation of Compound 11

[0214]

[0215] Under nitrogen protection, 11-A1 (420 mg, 2.07 mmol) was dissolved in chloroform (8 mL), and then MnO2 (2.7 g, 31.03 mmol) was added. The mixture was heated to 50 °C and reacted for 4 hours. After cooling to room temperature, the mixture was filtered, and the mother liquor was directly evaporated to dryness to obtain 11-A2 (280 mg, 67.3%). The crude product was directly added to the next reaction step.

[0216] Under nitrogen protection, 11-A2 (280 mg, 1.39 mmol) was dissolved in THF (10 mL), and CF3TMS (296 mg, 2.09 mmol) was added. After cooling to 0 °C, TBAF (0.27 mL, 1.0 M in THF, 0.27 mmol) was added dropwise, and the mixture was stirred at 0 °C for 2 hours. 3N HCl (5 mL) was added, and the mixture was stirred at room temperature for 0.5 h. Extraction was performed using EA (10 mL × 3). Column chromatography yielded 11-A3 (180 mg, 47.7%, containing TBAF residue), a pale yellow oil. NMR and mass spectrometry were performed before proceeding to the next reaction.

[0217] Under nitrogen protection, 11-A3 (120 mg, 0.44 mmol) was dissolved in DCM (5 mL), cooled to -40 °C, and then POCl3 (135 mg, 0.88 mmol) and TEA (111 mg, 1.1 mmol) were added. The mixture was stirred at -40 °C for 2 hours. Then, bromoethylamine hydrobromide (721 mg, 3.52 mmol) and TEA (404 mg, 4.0 mmol) were added. After the reaction was complete, saturated ammonium chloride aqueous solution (3 mL) was added, and the mixture was extracted with DCM (10 mL × 3). Column chromatography was used to separate 11-A4 (105 mg, 42.3%), which was a pale yellow oil. 1 H-NMR (400MHz, CDCl3) δ8.58(d,J=8.4Hz,1H),8.47(d,J=8.8Hz,1H),8.21(d,J=7.6Hz,1H),7.96(d,J=6 .8Hz,1H),7.81-7.77(m,1H),7.72-7.67(m,1H),6.58-6.51(m,1H),3.56-3.44(m,4H),3.16-2.87(6H).

[0218] Under nitrogen protection, 11-A4 (105 mg, 0.19 mmol) was dissolved in THF (8 mL), followed by the addition of silver oxide (352 mg, 1.48 mmol) and DIPEA (190 mg, 1.48 mmol). The mixture was then heated to reflux and reacted for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered through a diatomaceous earth liner, washed with DCM, and the mother liquor was evaporated to dryness. The purified compound 11 (32.4 mg, 43.3%) was obtained by high-performance liquid chromatography as a white solid. 1H-NMR (400MHz, CD3OD) δ8.62(d,J=8.8Hz,1H),8.50(d,J=8.8Hz,1H),8.23(d,J=6.8Hz,1H),8.0 6(d,J=7.2Hz,1H),7.90-7.88(m,1H),7.80-7.76(m,1H),6.82-6.80(m,1H),2.27-2.01(m,8H). MS:Calculated 401.1,found 402.1([M+H] + ).

[0219] Preparation Example 12: Preparation of Compound 12

[0220]

[0221] Under nitrogen protection, 12-A1 (1.5 g, 5.93 mmol) and tributyl(1-ethoxyethylene)tin (3.0 g, 8.30 mmol) were added to dioxane (30 mL). The mixture was purged with nitrogen three times. Then, Pd(PPh3)4 (170 mg, 0.15 mmol) was added, and the mixture was purged with nitrogen three more times. The mixture was then heated to reflux overnight. After the reaction was complete, the mixture was cooled to room temperature, and 3N HCl (30 mL) was added and stirred for 4 h. The mixture was then extracted with DCM (30 mL × 3), washed successively with water (10 mL), brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to separate the solid, yielding 12-A2 (610 mg, 47.6%) as a pale yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.07-9.06 (m, 1H), 8.98 (dd, J = 8.8, 1.6Hz, 1H), 8.36 (d ,J=8.0Hz,1H),7.88(d,J=7.6Hz,1H),7.70(dd,J=8.8,4.0Hz,1H),2.89(s,3H). MS:Calculated 216.1,found 217.2([M+H] + ).

[0222] 12-A2 (600 mg, 2.78 mmol) was dissolved in MeOH (15 mL) and DCM (5 mL), cooled to 0 °C, and NaBH4 (126 mg, 3.33 mmol) was added in portions. The reaction was completed after 30 min. H2O (15 mL) was added dropwise to quench the reaction, and DCM was extracted (20 mL × 3). The organic phase was washed successively with water (5 mL), brine (5 mL), dried over anhydrous sulfuric acid, and concentrated to obtain crude 12-A3 (342 mg, yield 56.5%), which was a pale yellow oil.

[0223] Under nitrogen protection, 12-A3 (340 mg, 1.56 mmol), Br-IPM (1.2 g, 3.90 mmol), and PPh3 (1.0 g, 3.82 mmol) were added to anhydrous THF (20 mL). The mixture was cooled to 0 °C, and DIAD (896 mg, 3.90 mmol) was slowly added dropwise. After the reaction was completed at 0 °C for 30 min, water (10 mL) was added dropwise, and the mixture was extracted with DCM (30 mL × 3). The organic phase was washed with water (5 mL) and saturated brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to separate 12-A4 (350 mg, 44.0%), which was a yellow oil. 1 H-NMR (400MHz, CDCl3): δ9.02-8.98(m,2H),8.39(d,J=8.0Hz,1H),7.97(d,J=8.0Hz,1H), 7.65(dd,J=8.8,4.4Hz,1H),6.76-6.73(m,1H),3.47-3.32(m,8H),1.76(d,J=6.4Hz,3H). MS:Calculated 507.9,509.9,511.9,found 509,511.0,513.0([M+H] + ).

[0224] Under nitrogen protection, 12-A4 (150 mg, 0.29 mmol) was dissolved in THF (10 mL), followed by the addition of silver oxide (341 mg, 1.47 mmol) and DIEA (190 mg, 1.47 mmol). The mixture was heated to reflux and reacted overnight. The solution was filtered through diatomaceous earth, washed with DCM, and the mother liquor was concentrated. High-performance liquid chromatography (HPLC) was used to prepare pure compound 12 (30 mg, 29.4%) as a colorless oil. 1 H-NMR (400MHz, CDCl3): δ9.03-9.01(m,2H),8.41(d,J=8.0Hz,1H),8.06(d,J=8.0Hz,1H), 7.65(dd,J=8.4,4.4Hz,1H),7.00-6.95(m,1H),2.23-2.07(m,8H),1.77(d,J=6.4Hz,3H). MS:Calculated 348.1,found 349.1([M+H] + ).

[0225] Preparation Example 13: Preparation of Compound 13

[0226]

[0227] 13-A1 (10 g, 48.0 mmol) was dissolved in concentrated sulfuric acid (50 mL), cooled to 0 °C, and potassium nitrate (5.8 g, 57.7 mmol) was added in portions. After reacting for one hour at 25 °C, the reaction was completed. The mixture was added dropwise to ice water (300 mL), extracted with DCM (60 mL × 6), washed with water (30 mL × 2), dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 13-A2 (8.0 g, 63.3%), which is a yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.79 (s, 1H), 8.85 (d, J = 6.4Hz, 1H), 8.50 (d, J = 6.4Hz, 1H), 8.39 (d, J = 8.4Hz, 1H), 8.00 (d, J = 8.4Hz, 1H). MS: Calculated 252.0, 254.0,found 253.0, 255.0([M+H] + ).

[0228] Under nitrogen protection, 13-A2 (1.5 g, 5.9 mmol) and tributyl(1-ethoxyethylene)tin (3.0 g, 8.3 mmol) were dissolved in dioxane (30 mL). The mixture was purged with nitrogen three times. Tetraphenylphosphine palladium (173 mg, 0.15 mmol) was added, and the mixture was purged with nitrogen three times. After reflux overnight, the reaction mixture was monitored by liquid chromatography until the starting material disappeared. The mixture was cooled to room temperature, and saturated ammonium chloride aqueous solution (20 mL) was added. Ethyl ester extraction (15 mL × 3) was performed. After evaporating the solvent, 3N hydrochloric acid (20 mL) was added. The mixture was stirred at room temperature for two hours, and the reaction was monitored by liquid chromatography until it ended. DCM extraction (15 mL × 3) was performed. The organic phase was washed with brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. The crude product was separated by column chromatography to obtain 13-A3 (600 mg, 46.8%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.90 (s, 1H), 8.83 (d, J = 6.0Hz, 1H), 8.48 (d, J = 8.0Hz, 1H), 8.40 (d, J = 6.0Hz, 1H), 7.98 (d, J = 8.0Hz, 1H), 2.83 (s, 3H). MS:Calculated 216.1,found 217.1([M+H] + ).

[0229] 13-A3 (600 mg, 2.8 mmol) was dissolved in methanol (12 mL), cooled to 0 °C, and sodium borohydride (105 mg, 2.8 mmol) was added in portions. After the addition was complete, the reaction was monitored by thin-layer chromatography. The solution was quenched with water (8 mL), extracted with DCM (15 mL × 2), washed with 5% citric acid aqueous solution (8 mL × 2), washed with water (8 mL × 2), washed with brine (8 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain 13-A4 (320 mg, 52.8%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.68 (s, 1H), 8.77 (d, J = 5.9Hz, 1H), 8.54-8.51 (m, 2H), 7.97 (d, J = 8.1Hz, 1H), 5.89 (m, 1H), 1.73 (d, J = 6.4Hz, 3H). MS:Calculated 218.1,found 219.1([M+H] + ).

[0230] Under nitrogen protection, 13-A4 (300 mg, 1.4 mmol) and Br-IPM (1.1 g, 3.4 mmol) were dissolved in anhydrous THF, and triphenylphosphine (900 mg, 3.4 mmol) was added. The mixture was cooled to 0 °C, and di-tert-butyl azodicarbonate (790 mg, 3.4 mmol) was added. After half an hour, the reaction was monitored by liquid chromatography to indicate its completion. The solvent was evaporated to dryness, and column chromatography was used to separate 13-A5 (300 mg, 42.8%, purity 80%), which was a yellow oily substance. The sample was tested by NMR and mass spectrometry and then directly used in the next reaction step.

[0231] Under nitrogen protection, 13-A5 (200 mg, 0.39 mmol) was dissolved in THF (5 mL), and DIEA (254 mg, 2.0 mmol) and silver oxide (273 mg, 1.2 mmol) were added. The mixture was refluxed overnight until the reaction was complete. After cooling to room temperature, the mixture was filtered through a diatomaceous earth filter, and the mother liquor was evaporated to dryness. The product compound 13 (27 mg, 19.8%) was prepared by neutralization by high performance liquid chromatography as a brown liquid. 1 H-NMR (400MHz, CDCl3): δ9.70 (s, 1H), 8.80 (d, J = 6.4Hz, 1H), 8.53-8.50 (m, 2H), 7 .94(d,J=8.0Hz,1H),6.57-6.50(m,1H),2.25-2.04(m,8H),1.86(d,J=6.4Hz,3H). MS:Calculated348.1,found 349.1([M+H] + ).

[0232] Preparation Example 14: Preparation of Compound 14

[0233]

[0234] Under nitrogen protection, 14-A1 (2.0 g, 7.90 mmol) was dissolved in dioxane (20 mL), followed by the addition of Pd(dppf)Cl2 (0.28 g, 0.39 mmol), and then tributyl(1-ethoxyethylene)tin (2.8 g, 7.93 mmol). The mixture was heated to 100 °C and stirred for 5 hours. After the reaction was complete, the mixture was cooled to room temperature, and 15 mL of 3N HCl solution was added. The mixture was stirred for 30 min, and extracted three times with EA (20 mL × 3). The organic phases were combined and separated by column chromatography to obtain compound 14-A2 (1.1 g, 64.7%, approximately 80% purity), as a pale yellow solid. MS: Calculated 216.1, found 217.1 ([M+H) + ).

[0235] 14-A2 (1.1 g, 5.1 mmol) was dissolved in MeOH (10 mL), and NaBH4 (230 mg, 6.1 mmol) was added in portions at 0 °C. The mixture was stirred at 0 °C for 0.5 hours. After the reaction was complete, the mixture was quenched with ice water (5 mL) and extracted with EtOAc (10 mL × 3). Column chromatography yielded compound 14-A3 (540 mg, 47.0%) as a pale yellow oil. 1 H-NMR (400MHz, CD3OD): δ10.01(s,1H),8.74(d,J=6.0Hz,1H),8.32(d,J=8.0Hz,1H),8 .05(d,J=8.4Hz,1H),7.95(d,J=6.0Hz,1H),5.69-5.67(m,1H),1.68(d,J=6.4Hz,3H). MS:Calculated 218.1,found 219.1([M+H] + ).

[0236] Under nitrogen protection, 14-A3 (190 mg, 0.9 mmol) and Br-IPM (664 mg, 2.1 mmol) were dissolved in anhydrous THF, and triphenylphosphine (550 mg, 2.1 mmol) was added. The mixture was cooled to 0 °C, and di-tert-butyl azodicarbonate (484 mg, 2.1 mmol) was added. The reaction was allowed to proceed for 3 h, and the reaction was monitored by liquid chromatography until completion. The solvent was evaporated to dryness, and column chromatography yielded 14-A4 (180 mg, 40.5%) as a yellow oil.

[0237] Under nitrogen protection, 14-A4 (180 mg, 0.4 mmol) was dissolved in THF (10 mL), followed by the addition of silver oxide (407 mg, 1.8 mmol) and DIPEA (226 mg, 1.8 mmol). The mixture was then heated to reflux and reacted for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered through a diatomaceous earth liner, washed with DCM, and the mother liquor was concentrated. High-performance liquid chromatography (HPLC) was used to prepare pure compound 14 (33 mg, 27.0%) as an off-white solid. 1 H-NMR (400MHz, CD3OD): δ9.90(s,1H),8.74(d,J=6.0Hz,1H),8.48(d,J=8.0Hz,1H),8.28(d,J= 6.0Hz, 1H), 8.15 (d, J = 8.0Hz, 1H), 6.45-6.42 (m, 1H), 2.28-2.11 (m, 8H), 1.83 (d, J = 6.4Hz, 3H). MS:Calculated 348.1,found 349.1([M+H] + ).

[0238] Preparation Example 15: Preparation of Compound 15

[0239]

[0240] 15-A1 (8.0 g, 38.4 mmol) was added to concentrated sulfuric acid (30 mL), cooled to 0 °C, and potassium nitrate (4.7 g, 46.1 mmol) was added in portions. The mixture was stirred at room temperature for 1.5 hours until the reaction was complete. The reaction mixture was then slowly poured into ice water and stirred for 30 min. The mixture was extracted with DCM (50 mL × 3), washed with water (15 mL × 3), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain 15-A2 (7.2 g, 74.2%) as a pale yellow solid. 1 H-NMR (400MHz, DMSO-d6): δ9.11-9.10(m,1H),8.67-8.65(m,1H),8.24(d,J=8.0Hz,1H),8.16(d,J=8.0Hz,1H),7.90-7.87(m,1H). MS:Calculated:252.0,254.0,found 253.0,255.0([M+H] + ).

[0241] Under nitrogen protection, 15-A2 (1.5 g, 5.93 mmol) was dissolved in 1,4-dioxane (30 mL), followed by the addition of tributyl(1-ethoxyethylene)tin (3.0 g, 8.30 mmol) and tetrakis(triphenylphosphine)palladium (206 mg, 0.178 mmol). The reaction was carried out overnight at 100 °C. After the reaction was complete, the mixture was allowed to cool to room temperature, and 3N hydrochloric acid (4 mL) was added dropwise. The mixture was stirred for 1 hour, extracted with EA (15 mL × 3), washed with water (5 mL), washed with brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was removed by evaporation. Column chromatography was used to separate the solid, yielding 15-A3 (900 mg, 60%) as a yellow solid. 1 H-NMR (400MHz, DMSO-d6): δ9.07-9.06 (dd, J = 4.0, 1.6 Hz, 1H), 9.00 (dd, J = 8.8, 1.6 Hz, 1H), 8.37 (s, 2H), 7.82 (dd, J = 8.8, 4.0 Hz, 1H), 2.78 (s, 3H). MS:Calculated:216.1,found:217.1([M+H] + ).

[0242] 15-A3 (800 mg, 3.70 mmol) was dissolved in THF (5 mL), cooled to 0 °C, and NaBH4 (280 mg, 7.40 mmol) was added in portions. The reaction was completed in 30 min. H2O (5 mL) was added dropwise at 0 °C, and the mixture was stirred for 20 min. The mixture was extracted with DCM (10 mL × 3), washed with water (5 mL), washed with brine (5 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to separate 15-A4 (400 mg, 49.6%) as a yellow solid. 1 H-NMR (400MHz, DMSO-d6): δ9.03(dd,J=4.0,1.2Hz,1H),8.82(dd,J=8.8,1.2Hz,1H),8.23(d,J=7.6Hz,1H),7.8 4(d,J=8.0Hz,1H),7.73(dd,J=8.8,4.0Hz,1H),5.67(d,J=4.4Hz,1H),5.60-5.48(m,1H),1.48(d,J=6.4Hz,3H). MS:Calculated 218.1,found 219.2([M+H] + ).

[0243] Under nitrogen protection, 15-A4 (300 mg, 1.37 mmol) and Br-IPM (1.06 g, 3.43 mmol) were added to anhydrous THF (5 mL), followed by triphenylphosphine (900 mg, 3.43 mmol). The mixture was cooled to 0 °C, and then a THF solution of di-tert-butyl azodicarbonate (790 mg, 3.43 mmol) (2 mL) was slowly added dropwise. The mixture was kept at 0 °C for half an hour, followed by stirring at room temperature for 2.5 hours. After the reaction was complete, water (5 mL) was added dropwise at 0 °C, and the mixture was extracted with DCM (5 mL × 3), washed with water (2 mL × 2), dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain 15-A5 (450 mg, 64.2%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.07 (dd, J=4.0, 1.2Hz, 1H), 8.63 (dd, J=8.8, 1.2Hz, 1H), 8.00 (d, J=7.8Hz, 1H), 7.7 7(d,J=7.8Hz,1H),7.61(dd,J=8.7,4.1Hz,1H),6.24-6.21(m,1H),3.53-3.23(m,8H),1.77(d,J=6.4Hz,3H). MS:Calculated 507.9,509.9,511.9,found 509.0,511.0,513.0([M+H] + ).

[0244] 15-A5 (200 mg, 0.39 mmol) was dissolved in THF (10 mL), and Ag2O (454 mg, 1.96 mmol) and DIEA (253 mg, 1.96 mmol) were added. The mixture was heated to reflux and the reaction was completed in 2 h. After cooling to room temperature, the mixture was filtered through a diatomaceous earth liner. The solid was washed with THF (3 mL × 3), and the mother liquor was concentrated. The solid was then separated by high performance liquid chromatography to obtain compound 15 (19 mg, 13.9%) as a white solid. 1 H-NMR (400MHz, CDCl3): δ9.08-9.07 (dd, J=4.4, 1.6Hz, 1H), 8.57 (dd, J=8.8, 1.6Hz, 1H), 8.01 (d, J=7.6Hz, 1H), 7.81(d,J=8.0Hz,1H),7.60(dd,J=8.8,4.4Hz,1H),6.38-6.32(m,1H),2.23-1.97(m,8H),1.79(d,J=6.8Hz,3H). MS:Calculated 348.1,found349.1([M+H] + ).

[0245] Preparation Example 16: Preparation of Compound 16

[0246]

[0247] Under nitrogen protection, 20-A4 (200 mg, 1.0 mmol) was dissolved in THF (5 mL), followed by the addition of TMSCF3 (226 mg, 1.5 mmol). At 0 °C, a THF solution of TBAF (0.02 mL, 1.0 M in THF, 0.02 mmol) was added, and the reaction was allowed to proceed for two hours. 3N hydrochloric acid (4 mL) was added, and the mixture was stirred for 15 minutes. The mixture was then extracted with DCM (10 mL × 3), washed with water (5 mL × 2), dried over anhydrous sodium sulfate, and the solvent was evaporated. The resulting product was purified by column chromatography to obtain 16-A1 (230 mg, 85.4%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.10-9.01 (m, 2H), 8.38 (d, J = 7.8 Hz, 1H), 8.13 (m, 1H), 7.81 (d, J = 7.8 Hz, 1H), 5.60 (m, 1H). MS:Calculated 272.0,found 273.1([M+H] + ).

[0248] Under nitrogen protection, 16-A1 (100 mg, 0.4 mmol) and POCl3 (113 mg, 0.7 mmol) were dissolved in DCM (2 mL), cooled to -30 °C, and TEA (93 mg, 0.9 mmol) was added dropwise. After one hour, the starting material disappeared as monitored by liquid chromatography. Then, 2-bromoethylamine hydrobromide (614 g, 2.9 mmol) was added, followed by TEA (297 mg, 2.9 mmol). After half an hour, the reaction ended as monitored by liquid chromatography. The mixture was quenched with water (3 mL), extracted with DCM (8 mL × 2), washed with water (4 mL), dried over anhydrous sodium sulfate, the solvent was evaporated, and 16-A2 (100 mg, 48.2%) was obtained by column chromatography as a yellow oily substance. 1 H-NMR (400MHz, CDCl3): δ9.10 (s, 1H), 9.00 (d, J = 8.8Hz, 1H), 8.43 (d, J = 7.6Hz, 1H) ,8.13(d,J=7.2Hz,1H),7.73-7.70(m,1H),7.49-7.47(m,1H),3.52-3.05(m,10H). MS:Calculated 561.9,563.9,565.9,found 563.0,565.0,567.0([M+H] + ).

[0249] Under nitrogen protection, 16-A2 (100 mg, 0.18 mmol) was dissolved in anhydrous THF (5 mL), and silver oxide (123 mg, 0.53 mmol) and DIEA (115 mg, 0.89 mmol) were added. The mixture was heated to reflux and reacted for one hour. The mixture was filtered through diatomaceous earth, and the mother liquor was evaporated to dryness. Compound 16 (10 mg, 14.1%) was prepared by high performance liquid chromatography as a white solid. 1 H-NMR (400MHz, CD3OD): δ9.11 (dd, J=4.0, 1.2Hz, 1H), 8.97 (dd, J=8.8, 1.6Hz, 1H), 8.52 (d, J=8.0 Hz, 1H), 8.25 (d, J = 8.0Hz, 1H), 7.82 (dd, J = 8.8, 4.0Hz, 1H), 7.61-7.54 (m, 1H), 2.26-2.05 (m, 8H). MS:Calculated 402.1,found 403.1([M+H] + ).

[0250] Preparation Example 17: Preparation of Compound 18

[0251]

[0252] Under nitrogen protection, 21-A3 (300 mg, 1.5 mmol) was dissolved in anhydrous THF (6 mL), and then (trifluoromethyl)trimethylsilane (318 mg, 2.2 mmol) was added dropwise. The mixture was cooled to 0 °C, and then TBAF (0.03 mL, 1 M in THF, 0.03 mmol) was added dropwise. The mixture was kept at 0 °C for 1.5 hours until the reaction was complete. 3N hydrochloric acid (1.2 mL) was added dropwise, and the mixture was allowed to rise naturally to room temperature. The mixture was stirred for 1 hour, and then water (5 mL) was added. The mixture was extracted with DCM (10 mL × 3), washed with water (5 mL × 3), dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain crude 18-A1 (180 mg, 44.6%, 70% purity), a white solid, which was directly used in the next reaction.

[0253] Under nitrogen protection, POCl3 (118 mg, 0.78 mmol) was dissolved in DCM (4 mL), 18-A1 (105 mg, 0.39 mmol) was added, the temperature was lowered to -40 °C, and a DCM solution of TEA (250 mg, 32.5 mmol) (1 mL) was added dropwise. The reaction was maintained at -40 °C, and the reaction progress was monitored by HPLC. After 3 h, the starting material disappeared. At the same temperature, 2-bromoethylamine hydrobromide (627 mg, 3.1 mmol) and TEA (303 mg, 3.1 mmol) were added, and the reaction was maintained at -40 °C for 30 min until it was completed. Add 2 mL of H2O dropwise, stir for 15 min, extract with DCM (8 mL × 3), wash with water (5 mL × 2), dry with anhydrous Na2SO4, evaporate the solvent, and purify by column chromatography to obtain 18-A2 (120 mg, 55.8%, content approximately 73%), which is a yellow oily substance. Test NMR and mass spectrometry and then directly add it to the next reaction.

[0254] 18-A2 (120 mg, 0.21 mmol) was dissolved in THF (7 mL), and Ag2O (246 mg, 1.1 mmol) and DIEA (142 mg, 1.1 mmol) were added. The mixture was heated to reflux and reacted for 5 h. After cooling to room temperature, the mixture was filtered through a diatomaceous earth liner. The solid was washed with THF (3 mL × 3), and the mother liquor was evaporated to dryness. The solid was then separated by high performance liquid chromatography to obtain compound 18 (15 mg, 17.9%) as an off-white solid. 1 H-NMR (400MHz, CD3OD): δ9.89 (s, 1H), 8.80 (d, J = 6.0Hz, 1H), 8.53 (d, J = 8.4Hz, 1H), 8.41-8.36 (m, 2H), 6.89-6.85 (m, 1H), 2.31-2.00 (m, 8H). MS:Calculated402.1,found 403.1([M+H] + ).

[0255] Preparation Example 18: Preparation of Compound 19

[0256]

[0257] Under nitrogen protection, 19-A1 (500 mg, 2.5 mmol) was dissolved in anhydrous THF (10 mL), and then (trifluoromethyl)trimethylsilane (530 mg, 3.7 mmol) was added dropwise. The mixture was cooled to 0 °C, and TBAF (0.05 mL, 1 M in THF, 0.05 mmol) was added dropwise. The mixture was kept at 0 °C for 1.5 hours until the reaction was complete. 3N hydrochloric acid (2 mL) was added dropwise, and the mixture was allowed to rise naturally to room temperature. The mixture was stirred for 1 hour, and then water (5 mL) was added. The mixture was extracted with DCM (10 mL × 3), washed with water (5 mL × 3), dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to separate 19-A2 (400 mg, 59.3%), which was an off-white solid. 1 H-NMR (400MHz, CD3OD): δ8.99 (dd, J=4.0, 1.6Hz, 1H), 8.88 (d, J=8.8Hz, 1H), 8.13 (d, J=8.0Hz, 1H), 8.04 (d, J=8.0Hz, 1H), 7.72 (dd, J=8.8, 4.0Hz, 1H), 5.99-5.94 (m, 1H). MS:Calculated 272.0,found 273.1([M+H] + ).

[0258] Under nitrogen protection, POCl3 (394 mg, 2.6 mmol) was dissolved in DCM (10 mL), and 19-A2 (350 mg, 1.3 mmol) was added. The mixture was cooled to -30 °C, and a DCM solution of TEA (326 mg, 3.2 mmol) was added dropwise (1 mL). The reaction was maintained at -30 °C, and the reaction progress was monitored by HPLC. After 2 h, the starting material disappeared. The mixture was then cooled to -40 °C, and 2-bromoethylamine hydrobromide (2.1 g, 10.2 mmol) and TEA (1.0 g, 10.0 mmol) were added. The mixture was maintained at -40 °C for 30 min until the reaction was complete. H2O (5 mL) was added dropwise, and the mixture was stirred for 15 min. The mixture was extracted with DCM (10 mL × 3), washed with water (5 mL × 2), dried over anhydrous Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography to obtain 19-A3 (405 mg, off-white solid, 55.6%). 1H-NMR (400MHz, CD3OD): δ9.04(d,J=3.2Hz,1H),8.89(d,J=8.8Hz,1H),8.19(d,J=8.0Hz,1H),8. 09(d,J=8.0Hz,1H),7.81-7.78(m,1H),6.61-6.57(m,1H),3.50-3.34(m,4H),3.15-3.02(m,4H). MS:Calculated 561.9,563.9,565.9,found 563.0,565.0,567.0([M+H] + ).

[0259] 19-A3 (200 mg, 0.4 mmol) was dissolved in THF (10 mL), and Ag2O (411 mg, 1.8 mmol) and DIEA (230 mg, 1.8 mmol) were added. The mixture was heated to 65 °C and reacted for 3.5 h. After cooling to room temperature, the mixture was filtered through diatomaceous earth, and the solid was washed with THF (3 mL × 3). The mother liquor was evaporated to dryness, and the solid was prepared by high performance liquid chromatography to obtain compound 19 (51.3 mg, 35.9%) as a white solid. 1 H-NMR (400MHz, CDCl3): δ9.12-9.11(m,1H),8.59(d,J=8.4Hz,1H),8.05(d,J=8.0Hz,1 H),7.98(d,J=8.0Hz,1H),7.66(dd,J=8.8,4.0Hz,1H),6.50(m,1H),2.29-1.96(m,8H). MS:Calculated 402.1,found 403.1([M+H] + ).

[0260] Preparation Example 19: Preparation of Compound 20

[0261]

[0262] Under nitrogen protection, 20-A1 (1.0 g, 7.0 mmol) was dissolved in concentrated sulfuric acid (4 mL), cooled to 0 °C, and potassium nitrate (847 mg, 8.4 mmol) was added in portions. The mixture was then heated to room temperature and stirred. After one hour, the reaction was monitored by liquid chromatography to indicate its completion. The reaction mixture was added dropwise to ice water (20 mL), the pH was adjusted to 9 with sodium carbonate aqueous solution, extracted with EA (10 mL × 4), dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain 20-A2 (700 mg, 53.3%) as a yellow solid. 1H-NMR (400MHz, CDCl3): δ9.07-9.04 (m, 2H), 8.31 (d, J = 8.0Hz, 1H), 7.66-7.63 (m, 2H), 2.91 (s, 3H). MS:Calculated 188.1,found 189.1([M+H] + ).

[0263] Under nitrogen protection, 20-A2 (1.0 g, 5.3 mmol) was dissolved in DMF (8 mL), and DMF-DMA (1.9 g, 15.9 mmol) was added. The mixture was heated to 130 °C and stirred for one hour. The reaction was monitored by liquid chromatography until completion. Most of the DMF was distilled off, filtered, washed with water (40 mL), and dried to obtain crude 20-A3 (1.1 g, 85.2%) as a black solid. 1 H-NMR (400MHz, CDCl3): δ9.30 (dd, J=8.8, 1.6Hz, 1H), 8.87 (dd, J=4.0, 1.6Hz, 1H ), 8.38 (d, J = 8.8Hz, 1H), 7.58-7.49 (m, 3H), 6.53 (d, J = 13.6Hz, 1H), 3.09 (s, 6H).

[0264] Under nitrogen protection, at 0°C, 20-A3 (300 mg, 1.2 mmol) and sodium periodate (791 mg, 3.7 mmol) were added to a mixture of THF and water (1:1, 6 mL). The mixture was heated to room temperature and stirred. After half an hour, the reaction was monitored by liquid chromatography to indicate its completion. The mixture was filtered, washed with EA (15 mL), and most of the solvent was evaporated. The mixture was then extracted with EA (10 mL × 3), washed with water (5 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 20-A4 (170 mg, 68.2%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ11.47(s,1H),9.17(dd,J=4.0,1.6Hz,1H),8.97(dd,J=8.8,1.6Hz,1H),8.41-8.36(m,2H),7.75(dd,J=8.8,4.0Hz,1H). MS:Calculated 202.0,found 203.1([M+H] + ).

[0265] 20-A4 (200 mg, 0.99 mmol) was dissolved in methanol (5 mL), cooled to 0 °C, and sodium borohydride (56 mg, 1.5 mmol) was added in portions. After the addition was complete, the reaction was allowed to proceed for 15 min, then quenched with water (3 mL), extracted with DCM (10 mL × 2), washed with 5% citric acid aqueous solution (5 mL × 2), washed with water (5 mL × 2), washed with brine (5 mL), dried, and evaporated to dryness to obtain 20-A5 (150 mg, 74.3%) as a yellow solid. 1 H-NMR (400MHz, CDCl3): δ9.10-9.07(m,1H),8.99(dd,J=4.0,1.6Hz,1H),8.36(d,J=8 .0Hz, 1H), 7.75 (d, J = 7.6Hz, 1H), 7.69 (dd, J = 8.8, 4.0Hz, 1H), 5.28 (d, J = 8.8Hz, 2H). MS:Calculated 204.1,found 205.1([M+H] + ).

[0266] Under nitrogen protection, 20-A5 (150 mg, 0.7 mmol) and POCl3 (225 mg, 1.5 mmol) were dissolved in DCM (4 mL), cooled to -30 °C, and TEA (186 mg, 1.8 mmol) was added dropwise. After one hour, the starting material disappeared as monitored by liquid chromatography. Then, 2-bromoethylamine hydrobromide (1.2 g, 5.9 mmol) was added, followed by TEA (595 mg, 5.9 mmol). After half an hour, the reaction ended as monitored by liquid chromatography. Water (3 mL) was added, and the mixture was extracted with DCM (10 mL × 2), washed with water (5 mL), dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography was used to purify 20-A6 (230 mg, 63.1%) as a yellow oil. 1 H-NMR (400MHz, CDCl3): δ9.06-9.03 (m, 2H), 8.39 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8. 0Hz, 1H), 7.70 (dd, J = 8.8, 4.4Hz, 1H), 5.76 (d, J = 6.8Hz, 2H), 3.52-3.40 (m, 10H). MS:Calculated 493.9,495.9,497.9,found 494.9,496.9,498.9([M+H] + ).

[0267] Under nitrogen protection, 20-A6 (200 mg, 0.4 mmol) was dissolved in THF (6 mL), and DIEA (261 mg, 2.0 mmol) and silver oxide (280 mg, 1.2 mmol) were added. The mixture was heated to reflux and reacted overnight. The mixture was filtered through diatomaceous earth, and the mother liquor was evaporated to dryness. The product compound 20 (41 mg, 30.4%) was prepared by high performance liquid chromatography in a neutral state and was a white solid. 1 H-NMR (400MHz, CD3OD): δ9.04(dd,J=4.0,1.2Hz,1H),8.99(d,J=8.8Hz,1H),8.47(d,J=8.0Hz,1 H), 8.03 (d, J = 8.0Hz, 1H), 7.79 (dd, J = 8.8, 4.0Hz, 1H), 5.97 (d, J = 7.2Hz, 2H), 2.31-2.24 (m, 8H). MS:Calculated 334.1,found 335.1([M+H] + ).

[0268] Preparation Example 20: Preparation of Compound 21

[0269]

[0270] Under nitrogen protection, 21-A1 (2.0 g, 7.9 mmol) was dissolved in dioxane (20 mL) and water (2.0 mL), followed by the addition of Pd(dppf)Cl2 (280 mg, 0.3 mmol), K2CO3 (580 mg, 15.9 mmol), and compound B (2.2 g, 7.9 mmol). The mixture was heated to 100 °C and stirred for 5 hours. After the reaction was complete, the mixture was cooled to room temperature, and saturated ammonium chloride aqueous solution (5 mL) was added. The mixture was extracted with EA (20 mL × 3), and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, the solvent was evaporated, and the crude product 21-A2 (1.3 g, 81.2%, approximately 80% purity) was purified by column chromatography as a yellow oil. The product was tested by NMR and mass spectrometry and then directly used in the next reaction step.

[0271] Under nitrogen protection, 21-A2 (800 mg, 4.0 mmol) was dissolved in dioxane (10 mL) and water (1 mL), and NaIO4 (2.6 g, 12.0 mmol) was added, followed by OsO4 (50.8 mg, 0.20 mmol). The mixture was stirred at room temperature for 24 hours. Extraction was performed with EA (10 mL × 3), and the organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated, and the solution was purified by column chromatography to give 21-A3 (280 mg, 35.0%) as a white solid. 1H-NMR (400MHz, CDCl3): δ10.50 (s, 1H), 9.89 (s, 1H), 9.08 (d, J = 6.0Hz, 1H), 8.90 (d, J = 6.0Hz, 1H), 8.33-8.32 (m, 2H). MS:Calculated 202.1,found 203.1([M+H] + ).

[0272] 21-A3 (400 mg, 2.0 mmol) was dissolved in MeOH (10 mL), cooled to 0 °C, and NaBH4 (91.0 mg, 2.4 mmol) was added in portions. The mixture was stirred at 0 °C for 0.5 hours, and the reaction was quenched with (5 mL) water. The mixture was extracted with EA (10 mL × 3), the organic phase was dried over anhydrous sodium sulfate, the solvent was evaporated, and the mixture was purified by column chromatography to obtain 21-A4 (210 mg, 51.5%) as an off-white solid. 1 H-NMR (400MHz, CDCl3): δ 10.50 (s, 1H), 8.76 (d, J = 6.0 Hz, 1H), 8.34 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 6.0 Hz, 1H), 5.26 (d, J = 5.2 Hz, 2H). MS:Calculated 204.1,found205.1([M+H] + ).

[0273] Under nitrogen protection, 21-A4 (210 mg, 1.0 mmol) was dissolved in DCM (20 mL), cooled to -40 °C, and then POCl3 (317 mg, 2.1 mmol) and TEA (525 mg, 5.2 mmol) were added. The mixture was stirred at -40 °C for 2 hours. Then, at the same temperature, bromoethylamine hydrobromide (1.3 g, 6.3 mmol) and TEA (2.1 g, 20.8 mmol) were added, and the reaction was maintained at this temperature for 1 hour. After the reaction was completed, saturated ammonium chloride aqueous solution (5 mL) was added, and the mixture was extracted with DCM (10 mL × 3). The mixture was washed with water (10 mL × 2), dried over anhydrous sodium sulfate, and the solvent was evaporated. The mixture was purified by column chromatography to obtain 21-A5 (280 mg, 54.9%, purity approximately 70%), which was a yellow oil. MS:Calculated 493.9,495.9,497.9,found 494.9,496.9,498.9([M+H] + ).

[0274] Under nitrogen protection, 21-A5 (280 mg, 0.6 mmol) was dissolved in THF (25 mL), and silver oxide (784 mg, 3.4 mmol) and DIPEA (437 mg, 3.4 mmol) were added. The mixture was heated to reflux and reacted for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered through diatomaceous earth, washed with DCM, and the mother liquor was evaporated to dryness. The pure compound 21 (40.6 mg, 29.7%) was obtained by high performance liquid chromatography as an off-white solid. 1 H-NMR (400MHz, CD3OD): δ9.89(s,1H),8.74(d,J=6.0Hz,1H),8.43(d,J=8.0Hz,1H),8 .19(d,J=6.0Hz,1H),8.06(d,J=8.0Hz,1H),5.73(d,J=8.0Hz,2H),2.24-2.19(m,8H). MS:Calculated 334.1,found 335.1([M+H] + ).

[0275] Preparation Example 21: Preparation of Compound 22

[0276]

[0277] 22-A1 (8.0 g, 38.4 mmol) was added to concentrated sulfuric acid (30 mL), cooled to 0 °C, and potassium nitrate (4.7 g, 46.1 mmol) was added in portions. The mixture was stirred at room temperature for 1.5 hours, and then the reaction mixture was slowly poured into ice water (80 mL). The mixture was stirred for 30 min, extracted with DCM (50 mL × 3), washed with water (15 mL × 3), washed with saturated brine, and the solvent was evaporated to obtain 22-A2 (7.2 g, 74.2%), which was a pale yellow solid. 1 H-NMR (400MHz, DMSO-d6): δ9.11 (dd, J=4.0, 1.6Hz, 1H), 8.66 (dd, J=8.8, 1.6Hz ,1H),8.24(d,J=8.0Hz,1H),8.16(d,J=8.0Hz,1H),7.89(dd,J=8.8,4.0Hz,1H). MS:Calculated 252.0and 254.0,found 253.0 and 255.0([M+H] + ).

[0278] Under nitrogen protection, 22-A2 (3.0 g, 11.9 mmol) and vinylboronic acid pinacol ester (2.2 g, 14.3 mmol) were dissolved in a mixed solution of dioxane and water (30 mL, 10:1). Potassium phosphate (6.3 g, 29.8 mmol) was added, and the mixture was purged with nitrogen three times. [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane dichloride complex (2.9 g, 3.6 mmol) was added, and the mixture was heated to 100 °C and stirred for 1.5 hours until the reaction was complete. The mixture was cooled to room temperature, quenched dropwise with saturated ammonium chloride aqueous solution (15 mL), extracted with EA (20 mL × 3), washed with water (10 mL), washed with saturated brine (10 mL), and the solvent was evaporated. The solution was purified by column chromatography to obtain 22-D1 (1.3 g, 54.6%) as a yellow solid. 1 H-NMR (400MHz, CD3OD): δ8.97 (dd, J=4.0, 1.6Hz, 1H), 8.74 (dd, J=8.8, 1.6Hz, 1H), 8.09 (d, J=8.0Hz, 1H), 7.86 (d, J=8.0Hz, 1H), 7.69 (dd, J=8.8, 4.0Hz, 1H), 7.54 (dd, J=17.2, 11.2Hz, 1H), 6.01 (dd, J=17.2, 0.8Hz, 1H), 5.71 (dd, J=11.2, 0.8Hz, 1H). MS:Calculated 200.1,found 201.0([M+H] + ).

[0279] 22-D1 (1.3 g, 6.5 mmol) was dissolved in 1,4-dioxane, and an aqueous solution of sodium periodate (4.2 g, 19.5 mmol) (15 mL) was added. Then, a 2.4% aqueous solution of osmium tetroxide tert-butanol (3.5 g) was added dropwise, and the mixture was stirred overnight at room temperature. After the reaction was complete, the solid was filtered off, washed with EA (20 mL), separated, and the organic phase was washed with water (5 mL × 2). The mixture was dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain crude 22-D2 (1.3 g), a yellow solid, which was directly used for the next step.

[0280] Dissolve 22-D2 (800 mg, 4.0 mmol) in THF (5 mL), cool to 0 °C, and add NaBH in portions. 4( 225 mg (5.9 mmol) was added dropwise, and the reaction was completed in 30 min. H2O (5 mL) was added dropwise at 0 °C, and the mixture was stirred for 20 min. The mixture was extracted with DCM (10 mL × 3), and the organic phase was washed with water (5 mL × 2) and brine (5 mL). The mixture was dried over anhydrous sodium sulfate, the solvent was evaporated, and the product was purified by column chromatography to obtain 22-D3 (470 mg, two-step yield 58.1%) as a yellow solid. 1H-NMR (400MHz, CD3OD): δ8.97 (dd, J=4.0, 1.6Hz, 1H), 8.69 (dd, J=8.8, 1.6Hz, 1H), 8. 09(d,J=7.6Hz,1H),7.78(d,J=7.6Hz,1H),7.69(dd,J=8.8,4.0Hz,1H),5.14(s,2H). MS:Calculated 204.1,found 205.2([M+H] + ).

[0281] POCl3 (600 mg, 3.9 mmol) was dissolved in DCM (10 mL), and 22-D3 (400 mg, 2.0 mmol) was added. The mixture was cooled to -30 °C, and a DCM solution of TEA (500 mg, 4.9 mmol) was added dropwise (1 mL). The mixture was kept at -30 °C, and the reaction was monitored by HPLC. After 2 h, the starting material disappeared. The mixture was then cooled to -40 °C, and 2-bromoethylamine hydrobromide (3.2 g, 15.7 mmol) and TEA (1.6 g, 15.7 mmol) were added. The mixture was kept at -40 °C, and the reaction was completed in 30 min. The temperature was then raised to 5 °C, and H2O (5 mL) was added dropwise. The mixture was stirred for 15 min, extracted with DCM (10 mL × 3), washed with water (5 mL × 2), dried over anhydrous sodium sulfate, and the solvent was evaporated. The solution was purified by column chromatography to obtain 22-D4 (350 mg, 36.0%) as a yellowish-brown solid. 1 H-NMR (400MHz, CD3OD): δ9.01(d,J=2.8Hz,1H),8.73(d,J=8.4Hz,1H),8.10(d,J=7.6Hz,1H),7.83(d,J =7.6Hz, 1H), 7.75 (dd, J = 8.4, 4.0Hz, 1H), 5.53 (d, J = 7.8Hz, 2H), 3.46-3.41 (m, 4H), 3.29-3.26 (m, 4H). MS:Calculated 493.9,495.9,497.9,found 495.0,497.0,499.0([M+H] + ).

[0282] 22-D4 (200 mg, 0.4 mmol) was dissolved in THF (10 mL), and Ag2O (470 mg, 2.0 mmol) and DIPEA (261 mg, 2.0 mmol) were added. The mixture was heated to 65 °C, and the reaction was monitored by HPLC. The reaction was completed in 2 h. After cooling to room temperature, the mixture was filtered through a diatomaceous earth liner. The solid was washed with THF (3 mL × 3), the solvent was evaporated, and the solid was separated by high performance liquid chromatography to obtain compound 22 (14.4 mg, 10.7%) as a white solid. 1H-NMR (400MHz, CDCl3): δ9.11 (dd, J=4.4, 1.6Hz, 1H), 8.57 (dd, J=8.8, 1.6Hz, 1H), 8.00 (d, J=7.6Hz ,1H),7.74(d,J=7.6Hz,1H),7.63(dd,J=8.4,4.4Hz,1H),5.64(d,J=7.6Hz,2H),2.22-2.02(m,8H). MS:Calculated 334.1,found 335.1([M+H] + ).

[0283] Preparation Example 22: Preparation of Compound 26

[0284]

[0285] 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).

[0286] 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 temperature was raised to 105 °C and left overnight. After overnight reaction, 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. After extraction with DCM (50 mL x 4), the mixture was 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 (400MHz, 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:Calculated 215.1,found 216.0([M+H] + ).

[0287] 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 1N HCl, dried, and concentrated to obtain 26-A4 product (185 mg, yield 91.6%), which was a yellow oil. 1 H-NMR (400MHz, 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,found 218.0([M+H] + ).

[0288] 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 maintaining the temperature at 0 °C for 30 min, the reaction was complete. 50 mL of water was added dropwise to the system, and the mixture was extracted with DCM (30 mL x 3). 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).

[0289] Under nitrogen protection, 26-A5 (250 mg, 0.49 mmol) was dissolved in THF (25 mL), followed by the addition of 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 (HPLC) as a yellow oil. 1 H-NMR(400MHz, CDCl3): δ8.56-8.54(m,1H),8.20 8.17(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),1.79(d,J=6.4Hz,3H). MS: Calculated 347.1, found 348.0([M+H] + ).

[0290] Example 23: Resolution of the compound and determination of its absolute configuration

[0291] The pure compound 01 was subjected to chiral analysis.

[0292] The HPLC chiral analysis conditions are as follows:

[0293]

[0294]

[0295] The HPLC chromatogram obtained from the test is as follows Figure 11 (The peaks are named No. 1 and No. 2 according to their emergence time), and the specific results are listed in the table below.

[0296] Separation peak Ret.Time retention period Area Area% of the area No. 1 2.908 5110974 47.096 No. 2 3.766 5741368 52.904

[0297] Take 4.9971 g of compound 01, dissolve it in chromatographic grade ethanol, and perform chiral separation preparation using the following chiral separation preparation method and conditions.

[0298] HPLC chiral separation preparation method: The above chiral isomers were separated using HPLC equipment and a chiral column, and the corresponding components were collected. The solvent was removed by rotary evaporation to obtain the pure optical isomers.

[0299] The HPLC chiral separation preparation conditions are as follows:

[0300]

[0301] 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%.

[0302] 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 12 , 13 The result.

[0303] The HPLC analysis results of the optical isomer corresponding to peak 1 are shown in the table below:

[0304] Separation peak Ret.Time retention period Area Area% of the area No. 1 2.908 7125381 99.837 No. 2 3.758 11633 0.163

[0305] The HPLC analysis results of the optical isomer corresponding to peak 2 are shown in the table below:

[0306]

[0307] Determination of the absolute configuration of the compound

[0308] The absolute configuration of compound 01 was determined by single-crystal X-ray diffraction results.

[0309] 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.

[0310] Single crystal structure analysis.

[0311] The above colorless crystal was subjected to single-crystal diffraction using the following X-ray single-crystal diffractometer (D8 Venture, Bruker):

[0312]

[0313] 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 14 As shown.

[0314] Therefore, the optical isomer corresponding to peak 1 is the S configuration, with the following structural formula:

[0315] The compound is referred to below as "Compound 01-S configuration".

[0316] Since compound 01 has only one chiral center, peak 2 above corresponds to the R configuration, as shown in the following structural formula:

[0317] The compound is referred to below as "Compound 01-R configuration".

[0318] Based on similar operational steps in the above embodiments, a series of similar compounds can be synthesized by selecting different starting substrates.

[0319] Similarly, isotope variants of a series of compounds can be synthesized by performing similar operations using isotope variants or isotope variant reagents with different starting substrates.

[0320] The compound in reference 1 is TH-302, which was commercially available.

[0321] Comparative Example: Preparation of Control Compound 2 (D2)

[0322]

[0323] Under nitrogen protection, D2-A1 (0.5 g, 3.3 mmol, commercially available) and TMSCF3 (0.7 g, 4.9 mmol, commercially available) were dissolved in THF (10 mL). The mixture was cooled to 0 °C, and TBAF (0.13 mL, 0.07 mmol, 1 M in THF, commercially available) was added dropwise to the system. After maintaining the temperature at 0 °C for 30 min, D2-A2 completely disappeared. 3N HCl (2 mL) was added dropwise to the system, which became clear. The mixture was stirred at 0 °C for 1 h, and the entire product was obtained. The mixture was extracted with DCM (10 mL × 3), and the organic phase was washed with water (10 mL × 3). The organic phase was dried and concentrated, and separated by column chromatography (200-300 mesh silica gel, n-heptane:ethyl acetate = 12:1-10:1) to obtain D2-A2 product (0.7 g, yield 95.7%), which was a yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.27 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 5.16-5.20 (m, 1H).

[0324] Under nitrogen protection, POCl3 (972 mg, 6.34 mmol) was dissolved in DCM (10 ml) and cooled to -40 °C. Then, D2-A2 (0.7 g, 3.17 mmol) was dissolved in DCM (20 ml) and added dropwise with TEA (0.8 g, 7.93 mmol) to the system. After maintaining the temperature at -40 °C for 2 h, D2-A3 was completely converted into the intermediate. Then, bromoethylamine bromate (4.06 g, 25.36 mmol) and TEA (5.1 g, 0.05 mol) were added to the system. The reaction was monitored by HPLC and completed after 30 min. Add saturated NH4Cl (10 ml) at 0℃, extract with DCM (10 ml × 3), wash the organic phase with water and brine, dry and concentrate the organic phase, and separate by column chromatography (200-300 mesh silica gel, n-heptane:ethyl acetate = 5:1-1:1) to obtain product D2-A3 (420 mg, yield 26.3%), which is a yellow oil. 1 H-NMR (400MHz, CDCl3): δ8.31 (d, J = 8.4Hz, 2H), 7.70 (d, J = 8.4Hz, 2H), 5.78–5.82 (m, 1H), 3.13-3.54 (m, 10H). MS: Calculated 510.9, found 513.9([M+1] + ).

[0325] Under nitrogen protection, D2-A3 (400 mg, 0.8 mmol) was dissolved in tetrahydrofuran (20 mL), followed by the addition of silver oxide (1.1 g, 4.7 mmol) and DIPEA (0.6 g, 4.7 mmol). The mixture was heated to 65 °C and stirred for 1.5 h. After the reaction was complete, the mixture was cooled to room temperature, filtered through diatomaceous earth, washed with dichloromethane, and the mother liquor was concentrated. High-performance liquid chromatography (HPLC) was used to prepare pure D2 (127 mg, 46.3%) as a white solid. 1 H-NMR (400MHz, DMSO-d6): δ8.34(d,J=8.8Hz,2H), 7.89(d,J=8.8Hz,2H), 6.37-6.33(m,1H), 2.18-1.98(m,8H). MS: Calculated 352.1, found 352.0([M+H] + ).

[0326] Effect Example

[0327] 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.

[0328] Example 1: In vitro cytotoxicity test of human tumor cell lines

[0329] The IC50 value was used to quantify the cytotoxicity of human tumor cell lines in vitro. 50 The inhibitory effects of each compound on cancer cell proliferation were compared using half-maximal inhibitory concentrations (MCCs, in nanomoles (nM)). In vitro proliferation data of the H460 non-small cell lung cancer human tumor cell line are reported in Table 1 below.

[0330] IC 50 The values ​​were obtained by exposing cancer cells to various concentrations of compounds for 72 hours, followed by a washing step and the addition of fresh culture medium to allow the cancer cells to continue growing and perform cell viability staining, and then comparing them with a control that was only treated with culture medium.

[0331] Specifically, cells exhibiting exponential growth are sized at 2 × 10⁻⁶. 3Cells were seeded at a density of 100 μL per well 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 99 μL of culture medium. The compound was dissolved in 100% DMSO at a 200-fold increase to the desired final test concentration. Upon addition of the compound, it was further diluted to the desired final concentration by 4-fold using complete culture medium. A 1 μL aliquot of the specific concentration of the compound was added to a micropipe containing 199 μL of culture medium to obtain the reported final compound concentration. After adding the compound, the plate was incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for another 72 hours.

[0332] Incubate the cell culture plate at room temperature for 30 minutes to equilibrate, discarding 100 μL of culture medium from each well. Add 100 μL of CTG reagent (CelltiterGlo kit) to each well, shake on a shaker for 2 minutes, and incubate at room temperature in the dark for 30 minutes. Read the chemiluminescence signal values ​​using an Envision instrument. Calculate the IC50 concentration (IC50) of the drug that causes 50% growth inhibition using computer software. 50 The results are listed in Table 1 below.

[0333] Table 1: IC50 of compounds 50 value

[0334]

[0335]

[0336]

[0337] The test results for TH-302 were obtained using values ​​from a published article by Fanying Meng, one of the inventors of this invention. The article is titled "Molecular and Cellular Pharmacology of the Hypoxia-Activated Prodrug TH-302, Mol Cancer Ther, 2012; 11:740-751. Fanying Meng, James W. Evans, Deepthi Bhupathi, et al."

[0338] The test results for TH2565 and TH2566 are those in the literature (WO2016210175A1, CN108024974A).

[0339] By comparing the anticancer activity of the aziridine compounds with fused ring structures (naphthyl ring, benzopyrazine, benzopyridine) provided in this invention with the anticancer activity of the following aziridine compounds with benzene ring structures provided in previous literature (WO2016210175A1, CN108024974A), it can be seen that the anticancer activity may be stronger (control compound 2 and compound 01, compound 26 and TH2565 / TH2566). Generally speaking, the novel structural compounds provided in this invention (naphthyl ring, benzopyrazine, benzopyridine core structure, different relative positions of nitro and aziridine side chain structures) have stronger or roughly equivalent anticancer activity compared to control compound 2 (except for compounds 04 and 05, which are significantly less active than control compound 2).

[0340]

[0341] Example 2: Hypoxia-dependent cytotoxicity assay

[0342] To determine the effects of the compounds of this invention on cancer cell proliferation, the antiproliferative activity of these compounds on cancer cells was detected in a multi-well CTG-based analysis. Cell growth was compared with and without the test compounds (treatment group). The following cell line, H460 non-small cell lung cancer human tumor cell line, was tested using 10,000 cells / well / 495 μL of medium. These cells were seeded onto glass inserts in each well of a 24-well plate at the density and medium specified above. After 24 hours, these plates were divided into two groups: a hypoxia group (using N2) and an air group. TH-302 was added to each well (500 μL volume) of the treatment group at concentrations from 100, 10, 0.1 to 0.01 μM, and the test compounds were added at concentrations from 10, 1, 0.1, 0.01, 0.001 to 0.0001. The final DMSO concentration in each well was less than or equal to 1%. Cells in the hypoxia group were incubated for 2 hours in an ELECTROTEK hypoxia workstation. Cells in the air group were cultured in a standard tissue culture incubator for 2 hours. After 2 hours, the test compound was removed from each well, the cells were washed with 500 μL of culture medium, and incubated in 500 μL of fresh culture medium for 3 days. After 3 days, 200 μL of culture medium was discarded from each well, 60 μL of CTG was added, and the cells were incubated at room temperature in the dark for 15 minutes. 100 μL of culture medium was transferred from each well of a 24-well plate to a 96-well plate; the chemiluminescence signal value was read using an M5 full-band multi-mode microplate reader, integration = 750 ms. The half-maximal inhibitory concentration (IC50) of the test compound was calculated. 50 They are listed in Table 2 below. Control compounds 1-2 are used as controls.

[0343] Table 2: Inhibitory effects of compounds on H460 cell lines under normoxic and hypoxic conditions

[0344]

[0345]

[0346] The test results for TH2565 and TH2566 are those in the literature (WO2016210175A1, CN108024974A).

[0347] Furthermore, the inhibition rate curves of different concentrations of TH-302 and compound O1 of the present invention on H460 cells under normoxic air and hypoxic nitrogen conditions are shown in the figure. Figure 1 middle.

[0348] The results show that the aziridine compounds with fused ring structures (naphthalene ring, benzopyrazine, benzopyridine) provided by the present invention have the same hypoxia activation mechanism as TH-302: they have stronger cytotoxicity to cancer cells under hypoxic conditions than under normoxic conditions.

[0349] Example 3: P-gP Inhibition Experiment

[0350] P-glycoprotein (P-gP, also known as multidrug resistance protein) is a high-molecular-weight protein with a transport pump-like structure found on the cell membrane of multidrug-resistant tumor cells. It pumps various chemotherapeutic drugs out of the cell, reducing the intracellular drug concentration, and is closely related to resistance to clinical chemotherapy.

[0351] 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.

[0352] 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 P-glycoproteins functions as an energy-dependent "drug pump," capable of pumping 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. Therefore, experimental data on the interaction between P-glycoproteins and compounds can be used to evaluate the true effectiveness of these compounds in inhibiting the proliferation of central nervous system tumor cells: if a compound is experimentally proven to be a substrate of P-gp glycoprotein, then the compound can bind to P-gp and be excluded from the central nervous system, thus failing to exert its effect; if a compound is experimentally proven not to be a substrate of P-gp glycoprotein, then the compound cannot bind to P-gp or binds weakly and will not be excluded from the central nervous system, thus accumulating a sufficient concentration in the central nervous system to exert its effect.

[0353] Using MDCKII-MDR1 cells as a model, verapamil was used as an inhibitor to evaluate the potential of compounds as substrates for P-glycoproteins.

[0354] Preparation of cells for penetration assay:

[0355] MDCKII-MDR1 cells were incubated in cell culture flasks. The incubator was set to 37°C, 5% CO2, and 95% relative humidity. When the cell confluence reached 70-90%, the cells could be used for seeding in transwells.

[0356] 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.

[0357] After cell incubation, the cell suspension was transferred to a round-bottom centrifuge tube and centrifuged at 120g for 5 minutes.

[0358] Cells were resuspended in culture medium to a final concentration of 1.56 × 10⁶ cells / mL. 50 μL of the cell suspension was added to each well of a 96-well migration chamber culture plate, resulting in a final seeding density of 5.45 × 10⁵ cells / cm². 2 .

[0359] Change the medium 48 hours after inoculation and incubate for 4-8 days, changing the medium every other day.

[0360] 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.

[0361] Evaluation of cell monolayer membrane integrity:

[0362] After 4-8 days of incubation, MDCKII-MDR1 cells should have fully merged and differentiated. At this point, they can be used for penetration assays.

[0363] The resistance of the single-layer film was measured using a Millipore resistance meter, and the resistance of each pore was recorded.

[0364] After the assay is completed, the migration chamber culture plate is returned to the incubator.

[0365] Calculation of resistance value: Measured resistance value (ohms) × film area (cm2) = TEER value (ohms·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.

[0366] Drug penetration test:

[0367] 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.

[0368] Determine the transport rate of the compound from the top to the base. Add 75 μL of flushing solution containing the test compound to each well in the upper chamber (top) and 235 μL of buffer solution to each well in the lower chamber (base).

[0369] 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).

[0370] Transfer 50 μL of sample from the working solution preparation plate and add it to 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 2 μM ketoprofen) as a 0-minute dosing sample for testing.

[0371] To determine the transport of the test substance under the condition of adding the P-glycoprotein 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.

[0372] After combining the transfers from top to bottom, incubate at 37°C for 2 hours.

[0373] After incubation, 50 μL samples were taken from each well of the upper and lower chambers of the migration chamber culture plate and added to new sample tubes. 200 μL of acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 2 μM ketoprofen) was added to the sample tubes, vortexed for 10 minutes, and then centrifuged at 3220 g for 30 minutes. 100 μL of the supernatant was collected, diluted with an equal volume of water, and analyzed by LC-MS / MS. All samples were incubated in triplicate.

[0374] The integrity of the cell monolayer was assessed after 2 hours of incubation using fluorescein leakage evaluation. The fluorescein stock solution was diluted to a final concentration of 100 μM / L with buffer. 100 μL of fluorescein solution was added to each well of the upper Transwell plate, and 300 μL of buffer was added to each well of the lower receiving plate. After incubation at 37°C for 30 minutes, 80 μL of solution was aspirated from both the upper and lower layers of each well into a new 96-well plate. Fluorescence was measured using a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

[0375] Data Analysis:

[0376] 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:

[0377]

[0378] 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.

[0379] The efflux ratio is calculated using the following formula:

[0380]

[0381] 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 calculated from the top to the base.

[0382] Recovery rate is calculated using the following formula:

[0383]

[0384] 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.

[0385] The fluorescence value (LY Leakage) of the cell monolayer membrane is calculated using the following formula:

[0386]

[0387] 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.

[0388] 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 3 below.

[0389] Table 3: Experimental results of compound expulsion rate

[0390]

[0391] The experiment shows that metoprolol is a known compound with high passive permeability, while prazosin and imatinib are known P-gp substrates. These three compounds were used as controls to verify the reliability of the experimental data.

[0392] Experimental conclusion: Compounds 01, 14, and 20 exhibit very similar Papp(AB), Papp(BA), and Efflux Ratio parameters in both the presence and absence of the P-glycoprotein inhibitor verapamil. Therefore, these three compounds are not P-glycoprotein substrates.

[0393] The inventors' team's previous experimental data and subsequent clinical trials of TH-302 (US Clinical Trial Registry numbers NCT01497444, NCT02712567, NCT01403610, NCT00495144, NCT01746979, NCT02093962, NCT02342379, NCT02047500, NCT01381822, NCT02076230, NCT01440088, NCT030) 98160, NCT02598687, NCT02402062, NCT01144455, NCT01833546, NCT00743379, NCT01864538, NCT02433639, NCT00742963, NCT01149915, NCT02020226, NCT01485042, NCT01522872, NCT02255110) and clinical trial-related literature (Chawla) SP,Cranmer LD,Van TineB A,et al.Phase II Study of the Safety and Antitumor Activity of the Hypoxia-Activated Prodrug TH-302 in Combination With Doxorubicin in Patients WithAdvanced Soft Tissue Sarcoma[J].Journal of Clinical Oncology,2014,32(29):3299-3306; Weiss GJ,Infante JR,Chiorean EG,et al.Phase 1Study of theSafety,Tolerability,and Pharmacokinetics of TH-302,a Hypoxia-ActivatedProdrug,in Patients with Advanced Solid Malignancies[J].Clinical CancerResearch,2011,17(9):2997-3004;Borad MJ,Reddy SG,Bahary N,et al.RandomizedPhase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients WithAdvanced Pancreatic Cancer[J].Journal of Clinical Oncology, 2015, 33(13):1475-1481; Brenner A, Reardon DA, Wen PY, et al. ACTR-17. Evophosphamide (th-302) for recurrent gbm following bevacizumab failure, final results of a multicenter phase II study[J]. Neuro-oncology, 2018.) both demonstrate that TH-302 is a P-gp substrate, and this compound cannot effectively enter the brain and other central nervous systems, and it is not easy or almost impossible to accumulate sufficient concentrations to exert its pharmacological effects on the central nervous system.

[0394] The above experiments demonstrate that the anticancer compound with the novel naphthalene ring structure provided by this invention is not a substrate of P-gp, and therefore it is expected to become a drug for treating primary brain cancer, tumors, or metastatic cancers or tumors that have metastasized to the brain, or for treating primary brain cancer, tumors, or metastatic cancers or tumors that have metastasized to the brain.

[0395] Example 4: Comparison of cytotoxicity between TH-302 and compound 01

[0396] To further compare the cytotoxicity of TH-302 and compound 01, a comparison of their cytotoxicity against different cell lines is provided.

[0397] (1) H460 cell line

[0398] The cytotoxicity test results of TH-302 and compound 01 on H460 cell lines under the same laboratory, the same operators, and the same experimental conditions at different test times are shown in Table 4.

[0399] Table 4: Comparison of test results of the same cell line at different times

[0400]

[0401] (2) Cell lines of different species

[0402] The cytotoxicity test results of TH-302 and compound 01 on different cell lines are shown in Table 5.

[0403] Table 5: Comparison of test results for cell lines of different species

[0404]

[0405] The above cytotoxicity test results show that compound 01 of the present invention has greater cancer cell toxicity than TH-302, that is, compound 01 of the present invention has a stronger inhibitory effect on cancer cell proliferation.

[0406] Example 5: Cytotoxicity of Compound 01 on UV41 cell line with DNA repair gene mutation

[0407] 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 DNA repair enzymes and nucleotide excision repair enzymes due to mutations in the DNAERCC4 / XPF gene.

[0408] There is literature indicating that the UV41 cell line has a defect in nucleotide excision repair enzymes compared to the AA8 cell line (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 cell mutants hypersensitive to bifunctional alkylating agents. Cancer Res. 45:1737-1743, 1985. PubMed: 3919945; Busch D, et al. Summary of complementation groups of UV-sensitive CHO cell mutants isolated by large-scale screening. Mutagenesis 4:349-354, 1989. PubMed: 2687628; Bessho T, et al. Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5" to the cross-linked base and removes a 22-to 28-nucleotide-long damage-free strand. Mol. Cell. Biol.17:6822-6830, 1997. PubMed: 9372913; ThompsonLH, et al. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions. Somatic Cell Genet. 8: 759-773, 1982. PubMed: 7163954). .

[0409] Damage to DNA repair enzymes in the UV41 cell line can lead to the following enzymes being affected: 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 endonuclease, end processing enzymes, DNA polymerase, and Flap endonuclease.

[0410] Specifically, cells that grow exponentially are 4 × 10 3Cells were seeded at a density of 100 cells / well 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 concentration 200-fold higher than the desired final test concentration. Upon addition of the compound, it was further diluted to the desired final concentration 4-fold 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 compound concentration. After compound addition, the plate was incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for another 2 hours. The compound was then washed off, fresh culture medium was added, and the plate was incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for another 70 hours. At the end of this incubation, viable cells were quantified using AlamarBlue assay. The drug concentration (IC50) resulting in 50% growth inhibition was calculated using computer software. The results showed that compound 01 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 It is 0.34 μmol / L, such as Figure 2 As shown.

[0411] The results showed that the compounds of the present invention have higher sensitivity to cell lines with DNA repair gene mutations. Specifically, compound 01 has stronger sensitivity to cell lines with impaired homologous recombination DNA repair enzymes and impaired nucleotide excision repair enzymes, and has a stronger inhibitory effect on the proliferation of cell lines with impaired homologous recombination DNA repair enzymes and impaired nucleotide excision repair enzymes.

[0412] Example 6: Hypoxia-dependent reduction experiment of compound 01 and TH-302

[0413] 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 precipitation 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 as follows.

[0414] Sample tube PBS solution Test drugs CYP450R enzyme solution NADPH solution A 450μL 10μL 20μL 20μL B 470μL 10μL 20μL 0μL C 470μL 10μL 0μL 20μL

[0415] The amounts of test reagents (TH-302 and compound 01) in three different sample reaction tubes (A / B / C) at different reaction times (0 / 30 min / 120 min) relative to the initial amounts were compared. The results of adding different concentrations of human NADPH coenzyme are shown in the figures. Figure 3 , Figure 4 middle.

[0416] Experimental conclusion:

[0417] In the presence of NADPH and CYP450R enzymes, compound 01 exhibits an oxygen-dependent reduction reaction, and the reduction rate of compound 01 is similar to that of TH-302.

[0418] Example 7: Metabolic Stability Evaluation Experiment

[0419] Two separate experiments were conducted.

[0420] 7.1 Liver microsomal stability test

[0421] 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.

[0422] 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.

[0423] 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.

[0424] 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 (150 nm internal standard, 100 nM alprazolam, 200 nM caffeine, 200 nM labetalol, and 2 μM ketoprofen). Centrifuge the sample at 3,220 times the gravitational acceleration 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 the table below:

[0425]

[0426]

[0427] Plot a curve using the test data of compound 01, such as... Figure 5 As shown.

[0428] Conclusion: Compound 01 is relatively stable in human and mouse liver microparticles, but unstable in rat and canine liver microparticles.

[0429] 7.2 Plasma stability test

[0430] Plasma preparation

[0431] Immediately thaw the frozen plasma in a 37°C water bath. Centrifuge the plasma at 3,220 times the force of gravity 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 receiving the sample; b) Use only plasma with a pH between 7 and 8.

[0432] 2) Prepare the experimental solution

[0433] Working solutions of the test compound (1 mM) and the control compound (propylthiophene) 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%.

[0434] 3) Test procedure for plasma stability

[0435] Add 50 μL of spiked plasma aliquots to new test tubes and incubate for 2 hours at approximately 60 rpm 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 room temperature 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 room temperature quenching solution. Vortex mix for 5 minutes. Centrifuge the sample in the plate at 3,220 times gravity acceleration 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.

[0436] The test results after 30 minutes are shown in the table below:

[0437] compound Species Remaining percentage Propylamine people 0.07 lovastatin rats 0.21 Propylamine mice 5.33 lovastatin dog 37.6 Propylamine monkey 48.11 Compound 01 people 68.68 Compound 01 rats 52.11 Compound 01 mice 55.07 Compound 01 dog 85.04

[0438] In the table, propylthiophene and lovastatin are the control drugs commonly used in this experiment.

[0439] The test data for compound 01 were plotted as a curve, such as... Figure 6 As shown.

[0440] Conclusion: Compound 01 is relatively stable in canine plasma, followed by human plasma. It is relatively unstable in rat and mouse plasma.

[0441] Example 8 : Human-derived xenografts (PDX) animal model experiments

[0442] BALB / c nude mice subcutaneously inoculated A subcutaneous transplantation model of human gastric cancer was established using GA6201 tumor masses. The experiment was divided into three groups: a control group (pH 7.0-7.6) receiving physiological saline (pH 7.0-7.6), a control group (ifosfamide (60 mg / kg) receiving 2), and a test group (compound 01 (5-50 mg / kg) receiving 3). Five mice were in each group. The control group (pH 7.0-7.6) received the medication via tail vein injection (iv) once a week for three weeks, followed by a four-week observation period.

[0443] Ifosfamide (60 mg / kg) was administered via intraperitoneal injection (ip) for two weeks, five consecutive days per week with a two-day break, followed by a five-week observation period. Compound 01 (5 mg / kg) was administered via intraperitoneal injection for two weeks, followed by a daily dose of 10 mg / kg intraperitoneal injection for four days. Then, the dose and administration method were changed to 20 mg / kg via tail vein injection for one day. After a 10-day break, the dose was changed again to 50 mg / kg via tail vein injection once a week for two weeks, followed by 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 body weight and mortality.

[0444] The tumor volume of mice in each group was measured on different days, and the average value was obtained. The results are shown in the table below. Figure 7 As shown:

[0445]

[0446]

[0447] 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 8 As shown.

[0448] 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 01 was designed based on previous cytotoxicity experiments, MTD experiments, and other empirically designed dosing regimens with progressively increasing dosages.

[0449] Conclusion: The results show that compound 01 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).

[0450] Example 9: Human-derived xenograft tumor animal model , CDX ) Reality Test

[0451] 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 01 at 20 mg / kg and 40 mg / kg, and a control group using 10% ethanol + 10% polyoxyethylene ether (35) castor oil Cremophor EL + 80% 5% glucose injection (pH 7.4) as the solvent. Each group consisted of 6 mice. 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 01 at 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 01 at 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 01 was determined according to MTD experiments.

[0452] The tumor volume of mice in each group was measured on different days, and the average value was obtained. The results are shown in the table below. Figure 9 As shown:

[0453]

[0454] 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 10 As shown.

[0455] Conclusions: The single-drug compound 01 at 40 mg / kg (Q3Wx2, IV, test group 7) showed significant tumor-suppressive effects on day 31 after tumor cell inoculation, which was statistically significant compared with the control group (p = 0.00561), with a relative tumor inhibition rate (TGI) of 66.87%. The single-drug compound 01 at 20 mg / kg (QDx5x1week, IP, test group 8) also showed significant tumor-suppressive effects on day 31 after tumor cell inoculation, which was statistically significant compared with the control group (p = 0.000464), with a relative tumor inhibition rate (TGI) of 76.22%.

[0456] Example 10: Maximum Tolerable Dose tolerance dose, MTD) experiment

[0457] 1. Configuration of compound 01-S

[0458] Experimental process

[0459] Eighteen ICR mice (nine females and nine males) were randomly divided into three groups (3 females and 3 males) based on sex and body weight: a 10 mg / kg, 20 mg / kg, and 40 mg / kg group for the 01-S configuration of compound 01. Intravenous administration was used. The 10 mg, 20 mg, and 40 mg / kg groups received the 01-S configuration at concentrations of 1, 2, and 4 mg / ml, respectively, with a volume of 10 ml / kg and doses of 10, 20, and 40 mg / kg. Administered once daily for five consecutive days, followed by a two-week recovery period after the last administration. Animals were closely monitored for 2 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.

[0460] Experimental results

[0461] (1) Animal mortality

[0462] During the experiment, 3 out of 6 animals in the 40 mg / kg group of compound 01-S configuration died, with the deaths occurring during the recovery period of 2 to 6 days.

[0463] (2) Clinical symptoms in animals after drug administration

[0464] During the experiment, the animals in the 10 mg / kg and 20 mg / kg groups of compound 01-S configuration were in good general condition; the animals in the 40 mg / kg group of compound 01-S configuration showed intermittent gait instability, increased breathing depth, rapid breathing, and reduced spontaneous activity; the recovery period was 2 to 6 days, and some dying animals showed symptoms of reduced spontaneous activity, piloerection, arched back, and spasms.

[0465] (3) Effects on animal weight

[0466] Intravenous injection of the 01-S configuration of compound into mice resulted in 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 5 days of recovery.

[0467] (4) Effects on animal feed intake

[0468] 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).

[0469] (5) Pathological examination results

[0470] 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.

[0471] Experimental conclusions

[0472] Under the conditions of this experiment, the maximum tolerated dose of the single-cycle compound 01-S configuration administered intravenously to ICR mice was 20 mg / kg.

[0473] 2. Configuration of compound 01-R

[0474] Experimental process

[0475] Eighteen ICR mice (9 females / 9 males) were randomly divided into three groups (3 females / 3 males) based on sex and body weight: a 10 mg / kg, 20 mg / kg, and 40 mg / kg group (compound 01-R configuration). The mice were administered intravenously. The 10 mg / kg, 20 mg / kg, and 40 mg / kg groups received the 01-R configuration at concentrations of 1, 2, and 4 mg / ml, respectively, with a volume of 10 ml / kg and doses of 10, 20, and 40 mg / kg. 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 dose was adjusted, and the mice were administered for 5 consecutive days, followed by 14 days of observation after the last administration. The dose was then adjusted to...

[0476] Original dose group Second cycle dosage adjustment Group identifier Group 01-R configuration of compound 10 mg / kg 80mg / kg Compound 01-R configuration 80 mg / kg group Compound 01-R configuration 20 mg / kg group 60mg / kg Compound 01-R configuration 60 mg / kg group Compound 01-R configuration 40 mg / kg group 40mg / kg Compound 01-R configuration 40 mg / kg group

[0477] 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.

[0478] Experimental results

[0479] (1) Animal mortality

[0480] During the trial, 1 / 6 of the animals in the compound 01-R configuration 10 / 80 mg / kg group (meaning the first cycle of administration was 10 mg / kg, followed by a second cycle of administration of 80 mg / kg) died; 3 / 6 of the animals in the compound 01-R configuration 20 / 60 mg / kg group died; and 1 / 6 of the animals in the compound 01-R configuration 40 / 40 mg / kg group died. The deaths occurred during the recovery period of 3 to 7 days.

[0481] (2) Clinical symptoms in animals after drug administration

[0482] During the first cycle of administration, the animals in the compound 01-R configuration 10 / 80 mg / kg group were in good general condition; the animals in the compound 01-R configuration 20 / 60 mg / kg group developed gait instability after administration; and the animals in the compound 01-R configuration 40 / 40 mg / kg group developed intermittent gait instability, rapid breathing, and increased spontaneous activity from immediately after administration to 5 minutes after administration.

[0483] During the second cycle of administration, animals in the 10 / 80 mg / kg group of compound 01-R configuration intermittently exhibited gait abnormalities, decreased muscle tone, reduced spontaneous activity, irregular breathing, unsteady gait, rapid breathing, piloerection, convulsions, increased breathing, prone position, and arched back from immediately after administration to 1 hour post-administration. Animals in the 20 / 60 mg / kg group of compound 01-R configuration intermittently exhibited increased 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 post-administration. Animals in the 40 / 40 mg / kg group of compound 01-R configuration intermittently exhibited unsteady gait, increased breathing, rapid breathing, increased spontaneous activity, irregular breathing, and gait abnormalities from immediately after administration to 10-30 minutes post-administration. During the recovery period of 3-4 days, animals in both the 10 / 80 mg / kg and 20 / 60 mg / kg groups of compound 01-R configuration exhibited drooling and perioral moisture.

[0484] (3) Effects on animal weight

[0485] Intravenous injection of compound 01-R configuration into mice resulted in slow weight gain.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] (4) Effects on animal feed intake

[0490] 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).

[0491] (5) Pathological examination results

[0492] 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.

[0493] Experimental conclusions

[0494] Under the conditions of this experiment, the maximum tolerated dose of compound 01-R configuration administered intravenously to ICR mice in the first cycle was >40 mg / kg; the maximum tolerated dose of compound 01-R configuration administered in the second cycle was <40 mg / kg. In summary, the maximum tolerated dose of compound 01-R configuration is between 20-40 mg / kg, which is similar to or slightly higher than that of compound 01-S configuration.

[0495] 3. Comparison of the R / S configuration of compound 01 with that of compound MTD and TH-2565 / 2566

[0496] The mouse MTD test results of compounds TH-2565 / 2566 disclosed in prior literature (WO2016210175A1, CN108024974A) are 2 mg / kg. The test results of the R / S isomer of the naphthalene-containing anticancer compound 01 disclosed in this invention and the MTD results of compounds TH-2565 / 2566 in the literature are listed below:

[0497]

[0498] The results of the maximum tolerated dose experiment show that the anticancer compound 01 with the novel naphthalene ring structure provided by this invention has stronger safety in animals, which means that it is possible to use a larger dose in clinical treatment to achieve better therapeutic effects.

[0499] 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 this invention obtained through new structural modifications (the modification ideas include improving the single benzene ring core to a naphthyl ring, benzopyrazine, or benzopyridine core structure, and different relative positions of the nitro and aziridine side chain structures) has a higher MTD, that is, it can improve the safety and clinical therapeutic dose of the new compound.

[0500] Summary of preparation examples and effect examples

[0501] Compare the following monobenzene ring structures of anticancer compounds with the structures disclosed in prior literature (WO2016210175A1, CN108024974A):

[0502]

[0503] This invention, through: A) modifying the monobenzene ring core to a naphthyl ring, benzopyrazine, benzopyridine, or other core structures; and B) altering the relative positions of the nitro and aziridine side chains, has yielded and successfully synthesized a series of new compounds, which have been experimentally verified.

[0504] 1. The new structural compounds still have good in vitro cell anticancer activity. Compared with the single benzene ring structure, the new structural compounds have stronger or roughly equivalent anticancer activity. For example, the anticancer activity of compound 01 is significantly stronger than that of the single benzene ring structure compound and stronger than that of TH-302.

[0505] 2. The new structural compounds are still hypoxia-activated, that is, the new anticancer compounds provided by this invention have the same hypoxia activation mechanism as the anticancer compounds with a single benzene ring structure: they have stronger cytotoxicity to cancer cells under hypoxic conditions than under normoxic conditions. For example, compounds 01 and TH-302 have comparable hypoxia reduction activity.

[0506] 3. The new structural compound is not a P-gp substrate, and can effectively enter the central nervous system such as the brain. It can accumulate sufficient concentration to exert a pharmacological effect on the central nervous system. It is expected to become a drug for treating primary brain cancer, tumors or metastatic cancers or tumors that have metastasized to the brain. This is significantly better than TH-302, which is a P-gp substrate.

[0507] 4. The novel structural compounds exhibit higher sensitivity to cell lines with DNA repair gene mutations. Specifically, compound 01 demonstrates greater sensitivity to cell lines with impaired homologous recombination DNA repair enzymes and impaired nucleotide excision repair enzymes, and exhibits stronger inhibitory effects on the proliferation of these cell lines.

[0508] 5. The representative drug compound 01 of the new structural compounds is relatively stable in human plasma and liver microsomes and has the potential to be developed into a drug.

[0509] Further in vivo animal experiments proved that:

[0510] 6. New structural compounds may have higher MTD, meaning that the new structural compounds have higher safety and clinical therapeutic doses than compounds disclosed in prior literature, and may have better therapeutic effects on cancer or tumors.

[0511] 7. In comparison, the new structural compound 01 has a significant advantage in inhibiting tumor growth compared to the classic chemotherapy drug ifosfamide (which is also a DNA alkylating agent).

Claims

1. A compound of formula (II), or a pharmaceutically acceptable salt thereof: (II), in, X is C or N; R1 is attached to any framework atom of the Cx ring, and R1 is hydrogen. R2 is H or D; R3 is -CF3; The group can replace hydrogen atoms at any position on a carbon atom in a fused ring, with a substitution number of 1.

2. The compound according to claim 1, wherein: Of the four X atoms, only one is an N atom.

3. The compound according to claim 1, wherein, R2 is H.

4. The compound according to claim 1, wherein the compound is selected from compounds with the following structures: 、 、 、 、 。 5. The compound according to claim 1, wherein the compound is: 。 6. A pharmaceutical preparation comprising any one of the compounds of claims 1-5 or a pharmaceutically acceptable salt thereof.

7. Use of the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, or primary brain cancer, tumor, or metastatic cancer or tumor that has metastasized to the brain.

8. Use of the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a patient with impaired DNA repair, wherein impaired DNA repair is impaired by homologous recombination DNA repair enzymes and impaired by nucleotide excision repair enzymes; wherein the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, or primary brain cancer, tumor, or metastatic cancer or tumor that has metastasized to the brain.

9. A compound medicine comprising any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a. Traditional chemotherapy drugs; b. Anti-angiogenic drugs; c. Cell checkpoint inhibitors; or d. Immunosuppressants.

10. Use of a composition in the preparation of a combination medicament for the combined treatment of cancer or tumors, wherein the composition comprises: A drug or preparation containing any one of the compounds of claims 1 to 5 or a pharmaceutically acceptable salt thereof; and a conventional chemotherapy drug; wherein the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, or primary brain cancer, tumor, or metastatic cancer or tumor that has metastasized to the brain.

11. Use of a composition in the preparation of a combination medicament for the combined treatment of cancer or tumors, wherein the composition comprises: A drug or preparation comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof; And anti-angiogenic drugs; wherein the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer or primary brain cancer, tumor or metastatic cancer or tumor that has metastasized to the brain.

12. Use of a composition in the preparation of a combination medicament for the combined treatment of cancer or tumors, wherein the composition comprises: A drug or preparation comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof; And cell checkpoint inhibitors; wherein the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer or primary brain cancer, tumor or metastatic cancer or tumor that has metastasized to the brain.

13. Use of a composition in the preparation of a combination medicament for the combined treatment of cancer or tumors, wherein the composition comprises: A drug or preparation comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof; And immunosuppressants; wherein the cancer or tumor is non-small cell lung cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer or primary brain cancer, tumor or metastatic cancer or tumor that has metastasized to the brain.

14. A method for preparing the compound according to any one of claims 1 to 5, wherein, This method adopts Scheme 1, which includes the following steps: To induce cyclization of compound one through a condensation reaction to provide a compound of formula (II): ， Compound 1 or This method employs Scheme 2, which includes the following steps: Compound II is reacted with R1H to provide a compound of formula (II): Compound 2; Both Y1 and Y2 are leaving bases.

15. The method according to claim 14, wherein, Y1 and Y2 are each independently Cl, Br, I, -OTs, -ONO2, -OMs, -OTf, or -OSO2Cl.

16. The method according to claim 15, wherein, For compound one, Y1 is Br, and for compound two, Y2 is F.

17. The method according to claim 14, wherein in Scheme 1, DIPEA or TEA is used as an acid-binding agent and silver oxide or silver nitrate is used as a catalyst; or in Scheme 2, an alkali is added during the reaction process.

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