WO2020098517A1 - 4-(苯并硒唑-2-基)芳胺类化合物治疗胃癌或肠癌的用途 - Google Patents

4-(苯并硒唑-2-基)芳胺类化合物治疗胃癌或肠癌的用途 Download PDF

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WO2020098517A1
WO2020098517A1 PCT/CN2019/115184 CN2019115184W WO2020098517A1 WO 2020098517 A1 WO2020098517 A1 WO 2020098517A1 CN 2019115184 W CN2019115184 W CN 2019115184W WO 2020098517 A1 WO2020098517 A1 WO 2020098517A1
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cancer
fluorobenzoselenazol
alkyl
compound
substituted
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PCT/CN2019/115184
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English (en)
French (fr)
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史东方
承曦
杨艳
傅长金
顾杰
龚维伟
李鹏飞
张敏
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江苏新元素医药科技有限公司
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Priority claimed from CN201811342015.0A external-priority patent/CN111170963B/zh
Priority claimed from CN201811342014.6A external-priority patent/CN111170962B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D293/00—Heterocyclic compounds containing rings having nitrogen and selenium or nitrogen and tellurium, with or without oxygen or sulfur atoms, as the ring hetero atoms
    • C07D293/10—Heterocyclic compounds containing rings having nitrogen and selenium or nitrogen and tellurium, with or without oxygen or sulfur atoms, as the ring hetero atoms condensed with carbocyclic rings or ring systems
    • C07D293/12—Selenazoles; Hydrogenated selenazoles

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to the use of a class of 4- (benzoselazol-2-yl) aromatic amine compounds in the treatment of gastric cancer or intestinal cancer.
  • Gastric cancer is one of the most common malignant tumors of the digestive system. Worldwide, the incidence of gastric cancer ranks 5th among malignant tumors, and the mortality rate ranks 3rd among malignant tumors. According to data released by the International Cancer Research Center (IARC), there were 951,600 new cases of gastric cancer in the world in 2012 (including about twice as many male cases as women) and 723,100 deaths. According to statistics, the incidence of gastric cancer is highest in East Asia (especially South Korea, Mongolia, Japan, and China), followed by Central, Eastern, and South America in Europe, and the lowest in North America and most of Africa (Torre LA, Bray F, Siegel RL, et al.Global cancers, statisticals, 2012.
  • IARC International Cancer Research Center
  • gastric cancer The pathogenesis of gastric cancer has not yet been fully elucidated. It is mainly believed that the incidence of gastric cancer is related to factors such as dietary habits, Helicobacter pylori (Hp) infection, serum pepsinogen (PG), genetic factors, metabolic syndrome and psychological stress. . Helicobacter pylori may be the biggest predisposing factor for gastric cancer. About 90% of new non-cardia gastric cancer cases are related to this (Chang M, Zhang JC, Zhou Q, et al. Research Progress of of Clinical Epidemiology of Gastric Cancer. of Gastroenterology & Hepatology, 2017, 26 (9): 966-969).
  • the clinical cytotoxic drugs used for advanced gastric cancer chemotherapy mainly include four categories: (1) oral fluorouracil, such as capecitabine, tegio; (2) taxanes, such as paclitaxel, docetaxel ; (3) Third-generation platinums, such as oxaliplatin; (4) Topoisomerase I inhibitors, such as irinotecan.
  • the marketed molecular targeted drugs mainly include human epidermal growth factor receptor 2 (HER2) antibodies, such as trastuzumab, vascular endothelial growth factor receptor (VEGFR-2) antibodies, such as ramucirumab, And the small molecule tyrosinase inhibitor Apatinib, etc. targeting VEGFR-2 (Li Kaichun, Li Ping. The choice of drugs for the treatment of advanced gastric cancer. Pharmaceutical Services and Research, 2018, 18 (1): 1-5 ).
  • gastric cancer is a highly heterogeneous disease driven by multiple genetic mutations and epigenetic abnormalities, so the development of targeted drugs has lagged, and existing drugs have poor targeting and low sensitivity, such as trastuzumab Only effective for about 20% of HER2-positive patients in gastric cancer patients (Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in Combination with Chemotherapy versus Chemotherapy Alone for Treatment of HER2-positive Advanced Gas- ToGA): aPhase 3, Open-label, Randomized Controlled Trial. Lancet, 2010, 376 (6): 687-697). Therefore, neither current chemotherapy nor targeted therapy can effectively combat gastric cancer, and increase the survival benefit of patients.
  • Colorectal cancer is the most common malignant tumor of the digestive system, and its global incidence ranks third in men and second in women (Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA, Cancer, Journal of Clinicians, 2015, 65 (2): 87-108). According to 2012 statistics, there are about 1.4 million new cases of colorectal cancer and 693,900 cases of colorectal cancer deaths worldwide. The regions with the highest incidence are in Australia and New Zealand, and Europe and North America are also high-incidence areas (Torre LA, Siegel RL, Ward EM, et.al. Global Cancer Incidence and Mortality Rates and Trends-an Update. Cancer Epidemiology and Prevention Biomarkers, 2016, 25 (1): 16-27).
  • Colon cancer is the third most common cancer in the United States, with as many as 130,000 new cases of colon cancer diagnosed each year (Birt DF, Phillips GJ. Diet, Genes, and Microbes: Complexities of Colon Cancer Prevention. Toxicologic Pathology, 2014, 42: 182- 188). Because the early diagnosis rate of colon cancer is very low, the vast majority of patients are diagnosed in the middle and late stages, which makes it difficult for radical surgery. Its 5-year survival rate is only 50% -60% (Wang Zhengkang. Reasonable colon cancer radical surgery Resection range. Foreign Medical Surgery Volume, 1989, (1): 9-10).
  • the high-risk factors of colon cancer include the following aspects.
  • Dietary factors Insufficient intake of trace elements such as high-fat and high-protein diet, lack of vegetables, fruits, cereals, cellulose, calcium and selenium (Terry P, Giovannuci E. Michels KB, et al. Fruit, Vegetables, Dietary fiber, and Risk of Colorectal Cancer.Journal of the National Cancer Institute, 2001, 93: 525).
  • Dietary factors Insufficient intake of trace elements such as high-fat and high-protein diet, lack of vegetables, fruits, cereals, cellulose, calcium and selenium (Terry P, Giovannuci E. Michels KB, et al. Fruit, Vegetables, Dietary fiber, and Risk of Colorectal Cancer.Journal of the National Cancer Institute, 2001, 93: 525).
  • Disease factors history of lower gastrointestinal disease, cholecystectomy, appendicitis, hypertriglyceridemia (Mas
  • Hypertriglyceridemia is Positively Correlated with the Development of Colorectal Tubular Adenoma Adenoma In Japanese Japanese Men.World Journal) Gastroenterology, 2006, 12 (8): 1261-1264) and so on. 3. Lifestyle and other factors: obesity and lack of physical exercise, constipation, genetics, etc.
  • Colon cancer is treated with surgical resection of the tumor as the first treatment method, supplemented by chemotherapy, radiotherapy, targeted therapy and gene therapy.
  • Colon mesenteric resection is the most important method for surgical treatment of colon cancer, but 25% of colon cancer patients are already in the advanced stage when diagnosed, and because of the biological characteristics of colon cancer with high recurrence and distant metastasis, there are more than 25 % Of patients will still have recurrence or metastasis after the first operation (Wang Guanglin, Meng Zesong, Wang Feifei, etc. Research progress of preoperative chemotherapy for locally advanced colon cancer. Tumor, 2018, 38: 716-722). And surgical treatment is only suitable for some or early patients, because a large number of patients already have more malignant unresectable metastases, so choose systemic treatment (chemotherapy and targeted therapy) or may prolong the overall survival of patients.
  • the internationally recognized first-line plan for colon cancer chemotherapy is 5-fluorouracil combined with oxaliplatin, or capecitabine combined with oxaliplatin (Andre T, Boni C, Mounedji, L, et al. Oxaliplatin, Fluorouracil, and Leucovorin).
  • Multi-drug resistance in cancer chemotherapy is the main reason for the failure of chemotherapy.
  • Colon cancerous tissues are due to carrier proteins related to anti-cancer drug resistance (such as P glycoprotein, multi-drug resistance-related protein or breast cancer resistance protein) Over-expression (Herraez E, Gonzalez SE, Vaquero J, et Al. Cisplatin Induced Chemoresistance in Colon Cancer Cells Involves FXR-Dependent and FXR-Independent Up-regulation of ABC Proteins.Molecular Pharmaceutics (2012): 2576), the anti-cancer drugs in colon cancer cells are rapidly effluxed, the concentration is too low, which leads to the failure of chemotherapy.
  • colon cancer is a highly heterogeneous and complicated molecular typing disease.
  • traditional clinical targeted therapy mainly targets vascular endothelial growth factor receptor (VEGF) and epidermal growth factor receptor (EGFR), but anti-VEGF drugs have no obvious effect on advanced colon cancer (HurwitzHI, LymanGH.Registries and Randomized Trials Assessing the Effects of Bevacizumab in Colorectal Cancer: Is There There a Common Theme? Journal Journal of Clinical Oncology. 2012, 30 (6): 580-581), and will increase the incidence of arterial vascular events in elderly patients (Ranpura V, Hapani S, Wu S.
  • Treatment-related Mortality with Bevacizumab in Cancer Patients a Meta-analysis. JAMA, 2011, 305 (5): 487-494); and anti-EGFR drugs are for patients with RAS mutant colon cancer Invalid (Vale CL, Tierney JF, Fisher D, et Al. Does Anti-EGFR Therapy Improve Outcome In Advanced Advanced Colorectal Cancer? A Systematic Review Review and Meta-analysis. Cancer Treatment Reviews, 2012, 38 (6): 618-625) .
  • a genome-wide study of colon cancer shows that the number of tumor gene mutations is very high, with an average of about 75 mutations per tumor, indicating that cancer treatments for specific molecular abnormalities may only be effective in a small proportion of colon cancer patients.
  • colon cancer Since the incidence and mortality of colon cancer are at a high level worldwide, it has seriously endangered people's health and caused a heavy burden on families and society. However, colon cancer has high recurrence, distant metastasis and high heterogeneity Biological biological characteristics, so the current clinical treatments are very scarce and can not significantly increase the clinical benefit of colon cancer patients, there is an urgent need to develop new treatments or drugs to cope with the increasing status of colon cancer morbidity and mortality .
  • Selenium is one of the essential trace elements in the human body. The reduction of selenium in the blood will induce a variety of diseases including tumors and cardiovascular diseases (Reeves MA, Hoffmann PR. The Human Selenoproteome: Recent Insights Functions to Regulations. Cellular and Molecular Life Sciences, 2009, 66 (15): 2457-78). Because of its application in anti-tumor, anti-virus and treatment of nervous system-related diseases, selenium-containing drugs have become a hot spot for domestic and foreign scholars. Drug research mainly focuses on anti-tumor, anti-inflammatory and anti-hypertension (RomualdoC , Stefania C, Marina DG, et al. Novel Selenium-containing Non-natural Diamino acids. Tetrahedron Letters, 2007, 48 (7): 1425-1427).
  • Shi Dongfang has published articles on the synthesis of benzothiazole compounds and their anti-breast cancer effects in vivo and in vitro (Dong-Fang Shi, Tracey D. Bradshaw, Samantha Wrigley, et al. Antitumor Benzothiazoles. of 2- (4-Aminophenyl) benzothiazoles and Evaluation of Their Activities against Breast Cancer Cell Lines in Vitro and in Vivo. Journal of Medicinal Chemistry, 1996, 39: 3375-3384), meanwhile, the present inventor Shi Dongfang applied for Chinese patents (Shi Dongfang, Fu Changjin, Cheng Xi, etc. Compounds for the treatment or prevention of breast cancer.
  • CN201610299350.1 published the benzoselenazole-2-benzene compounds shown in formula (III) and their anti-breast cancer Pharmacological activity. These two compounds were human breast cancer cells nanomolar inhibitory effect, wherein the benzothiazole compound of ER + (MCF-7 cell lines, and BO) and ER - (MT-1 and MT-3 cell line)
  • ER + MCF-7 cell lines, and BO
  • ER - MT-1 and MT-3 cell line
  • the breast cancer xenografts of nude mice all show very significant tumor suppressive effects, but these two types of compounds have no inhibitory activity against other tumor cell lines such as prostate cancer, bladder cancer, melanoma, lung cancer, liver cancer, and esophageal cancer.
  • the purpose of the present invention is to provide a class of 4- (benzoselazol-2-yl) aromatic amine compounds for the treatment of gastric cancer or intestinal cancer based on the prior art.
  • the object of the present invention can be achieved by the following measures:
  • R 1 and R 2 are independently selected from H, D, halogen, -CN, C 1-3 alkyl, substituted C 1-3 alkyl, C 1-3 alkoxy or substituted C 1-3 alkyl Oxy;
  • n 1 or 2;
  • R 5 is selected from H, -CN, -OH, C 1-3 alkyl, C 1-3 alkoxy or amino acid residues;
  • Z is selected from CH or N
  • R 1 , R 2 , R 3 or R 4 are selected from D, halogen, OH, C 1-3 alkyl or C 1-3 alkoxy.
  • R 1 and R 2 in the present invention are independently selected from H, D, halogen, -CN, C 1-3 alkyl, substituted C 1-3 alkyl, C 1-3 Alkoxy or substituted C 1-3 alkoxy, the substituent is selected from D, F or C 1-3 alkoxy;
  • R 1 and R 2 in the present invention are independently selected from H, D, F, Cl, —CN, —CH 3 , —CF 3 , —OCF 3 or —OCHF 2 .
  • R 1 in the present invention is selected from D, F, Cl, -CN, -CH 3 or -CF 3 .
  • R 2 in the present invention is selected from H.
  • R 4 in the present invention is selected from H, D, halogen, -CN, C 1-3 alkyl, substituted C 1-3 alkyl, C 1-3 alkoxy or substituted One or two of C 1-3 alkoxy groups.
  • R 4 in the present invention is selected from H, D, F, Cl, Br, I, -CN, -CH 3 , -CF 3 , -OCH 3 , -OCH 2 CH 3 ,- OCHF 2 or -OCF 3 .
  • R 5 in the present invention is selected from H, C 1-3 alkyl, C 1-3 alkoxy or amino acid residues.
  • R 5 in the present invention is selected from H, —CH 3 , —CF 3 , —OCH 3 or 2,6-diamino-hexanoyl.
  • the compound involved in the present invention is selected from:
  • H or hydrogen, refers to protium (1H), which is the main stable isotope of hydrogen.
  • D deuterium, refers to a stable form of hydrogen isotope, also known as heavy hydrogen, and its element symbol is D.
  • Halogen means fluorine atom, chlorine atom, bromine atom or iodine atom.
  • Alkyl refers to a saturated aliphatic hydrocarbon group of 1-10 carbon atoms, including straight-chain and branched-chain groups (the numerical range mentioned in this application, for example “1-10", refers to this group In this case, it is an alkyl group, which may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 10 carbon atoms).
  • An alkyl group containing 1-4 carbon atoms is called a lower alkyl group. When the lower alkyl group has no substituent, it is called an unsubstituted lower alkyl group.
  • the alkyl can be selected from C 1-6 alkyl, C 1-5 alkyl, C 1-4 alkyl, C 1-3 alkyl, C 1-2 alkyl, C 2-3 alkyl, C 2-4 Alkyl and so on.
  • Specific alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl, and the like.
  • the alkyl group may be substituted or unsubstituted.
  • Alkenyl means an unsaturated hydrocarbon group having at least one carbon-carbon double bond, including straight-chain and branched-chain groups (the numerical range mentioned in this application, such as “2-5", refers to this group At this time, it is an alkenyl group, which may contain 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to 5 carbon atoms).
  • the alkenyl in the present invention may be C 2-8 alkenyl, C 2-6 alkenyl, C 2-5 alkenyl, C 2-4 alkenyl, C 2-3 alkenyl, etc.
  • Specific alkenyl groups include but It is not limited to vinyl, propenyl, and butenyl.
  • Alkynyl means an unsaturated hydrocarbon group having at least one carbon-carbon triple bond, including straight-chain and branched-chain groups (the numerical range mentioned in this application, such as "2-5", refers to this group In this case, it is an alkynyl group, which may contain 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to 5 carbon atoms).
  • the alkynyl group in the present invention may be C 2-8 alkynyl, C 2-6 alkynyl, C 2-5 alkynyl, C 2-4 alkynyl, C 2-3 alkynyl, etc.
  • Specific alkenyl groups include but It is not limited to ethynyl, propynyl and butynyl.
  • Alkoxy means -O- (unsubstituted alkyl) and -O- (unsubstituted cycloalkyl) groups, which further means -O- (unsubstituted alkyl).
  • Representative embodiments include but are not limited to methoxy, ethoxy, propoxy, cyclopropoxy, and the like.
  • amino acid residue refers to a group formed by an amino acid lacking a certain group (such as -OH, -COOH, or -NH 2 ), where amino acids include, but are not limited to, 20 naturally occurring species usually designated by three-letter symbols Amino acids, and also includes ⁇ -alanine, citrulline, demosine, ⁇ -aminobutyric acid, homocysteine, homoserine, 4-hydroxyproline, hydroxylysine, iso Isodemosine, 3-methylhistidine, norvaline, methioninesulfone and ornithine.
  • An example of an amino acid residue includes, but is not limited to: 2,6-diamino-hexanoyl.
  • “Pharmaceutically acceptable salt” is a salt comprising a compound of general formula (I) formed with an organic acid or an inorganic acid, and means those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:
  • a salt with an acid obtained by the reaction of the free base of the parent compound with an inorganic acid or an organic acid, such as (but not limited to) hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid And perchloric acid, organic acids such as (but not limited to) acetic acid, propionic acid, acrylic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, hydroxybenzoic acid, ⁇ -hydroxybutyric acid , Methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, Mandelic acid, succinic acid or malonic acid etc.
  • the salts of acid protons present in the parent compound are replaced by metal ions or coordinated with organic bases, metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions, organic bases such as ethanolamine, diethanolamine, trivalent Ethanolamine, tromethamine, N-methylglucamine, etc.
  • “Pharmaceutical composition” refers to a mixture of one or more compounds described herein or their pharmaceutically acceptable salts and prodrugs with other chemical ingredients, such as pharmaceutically acceptable carriers and excipients .
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
  • a pharmaceutically acceptable salt or a solvate thereof can be used as an active ingredient or a main active ingredient, supplemented with a pharmaceutically acceptable auxiliary material to make a pharmaceutical composition, and then The patient is administered.
  • the compounds of the present invention can be formulated into any clinically or pharmaceutically acceptable dosage form in a manner known in the art.
  • oral administration it can be made into conventional solid preparations, such as tablets, capsules, pills, granules, etc .; or oral liquid preparations, such as oral solutions, oral suspensions, syrups, etc.
  • a suitable filler, binder, disintegrant, lubricant, etc. may be added.
  • injections can be prepared, including injections, sterile powders for injection and concentrated solutions for injection.
  • it is prepared as an injection it can be produced by a conventional method in the existing pharmaceutical field.
  • an additional agent may not be added, or an appropriate additional agent may be added according to the nature of the medicine.
  • the present invention also provides a method for treating human gastric cancer or intestinal cancer, that is, administering a compound of the present invention, a pharmaceutically acceptable salt, or a solvate thereof to a person suffering from gastric cancer or intestinal cancer at 0.1-1000 mg per dose Drug, or the pharmaceutical composition of the present invention is administered at 0.1-1000 mg per dose.
  • the 4- (benzoselenazol-2-yl) arylamine compounds involved in this patent have anti-breast cancer pharmacological activity, but in vitro tests have shown that for prostate cancer, bladder cancer, melanoma, lung cancer, liver cancer, Tumor cell lines such as esophageal cancer have no obvious inhibitory effect.
  • these compounds have excellent therapeutic effects on gastric cancer or intestinal cancer: they have a significant growth inhibition effect on human gastric cancer cell lines MKN-45, AGS and NCI-N87, and the effect is basically close to the positive control drug paclitaxel ; These compounds also show excellent tumor growth inhibition effect on human gastric cancer MKN-45 and AGS xenograft tumors in nude mice, and the tumor inhibition rate is greater than 35%; so these compounds can be used in the treatment of human gastric cancer.
  • the present invention finds a new use of 4- (benzoselenazol-2-yl) arylamine compounds in the treatment of diseases and provides a potential drug for the treatment of gastric cancer or intestinal cancer.
  • Figure 1 is the inhibitory effect of compound 6 on the tumor volume of human gastric cancer cell line AGS xenograft in nude mice;
  • Figure 2 is the inhibitory effect of compound 6 on the tumor volume of human gastric cancer cell line MKN-45 xenograft in nude mice;
  • Figure 3 is the inhibitory effect of compound 22 on the tumor volume of human gastric cancer cell line AGS xenograft in nude mice;
  • Figure 5 is the inhibitory effect of compound 23 on the tumor volume of human gastric cancer cell line AGS xenograft in nude mice;
  • Figure 6 is the inhibitory effect of compound 23 on the tumor volume of human gastric cancer cell line MKN-45 xenograft in nude mice;
  • Figure 9 is the inhibitory effect of compound 22 on the tumor volume of human colon cancer cell line HT29 xenograft in nude mice;
  • Figure 10 is the inhibitory effect of compound 22 on the tumor volume of human colon cancer cell line HCT116 xenograft in nude mice;
  • Figure 11 is the inhibitory effect of compound 23 on the tumor volume of human colon cancer cell line HT29 xenograft in nude mice;
  • Fig. 12 shows the inhibitory effect of compound 23 on the tumor volume of human colon cancer cell line HCT116 xenograft in nude mice.
  • Step A Add 2-nitro-4-fluoroaniline (5.0g, 32.0mmol) in methylene chloride (80mL) dropwise to boron trifluoride etherate (6.82g, 48.1mmol) at -20 ⁇ -25 ° C )in. After stirring for 15 minutes, a solution of isoamyl nitrite (4.50 g, 38.4 mmol) in dichloromethane (20 mL) was added dropwise at this temperature. After the addition is complete, stirring is continued for 30 minutes, and then at -10 to 0 ° C for 30 minutes.
  • Step B Under an ice water bath, to a mixture of crude compound 1 (8.10 g) and water (150 mL) was added dropwise a solution of potassium selenocyanate (4.35 g, 30.2 mmol) in water (20 mL). After the addition was complete, stirring was continued for 1 hour. Filter and dissolve the filter cake with dichloromethane (150 mL). The insoluble matter was removed by filtration, and the filtrate was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain crude 4-fluoro-2-nitro-1-selenocyanate (2) (6.10 g). This compound was directly used in the next reaction without purification.
  • Step C Sodium metal (3.23 g, 140 mmol) was added to a mixture of crude compound 2 (6.10 g) and absolute ethanol (90 mL) at room temperature, and the resulting mixture was stirred for 1 hour under a water bath. Cooled to 0 ⁇ 5 °C, filtered, the filter cake was washed with a small amount of cooled ethanol, to obtain 1,2-bis (4-fluoro-2-nitrophenyl)-diselenide (3) crude product (3.90g). This compound was directly used in the next reaction without purification.
  • Step D The crude compound 3 (3.90 g) was dissolved in ethanol (60 mL), stannous chloride (7.90 g, 41.7 mmol) was added, and the resulting mixture was stirred under reflux under nitrogen for 4 hours. Most of the solvent was distilled off under reduced pressure, water (120 mL) and ethyl acetate (200 mL) were added, and the pH value was adjusted to 8-9 with 2M sodium hydroxide solution. The insoluble material was removed by filtration through Celite, the layers were separated, the aqueous layer was extracted with ethyl acetate (60 mL ⁇ 2), and the combined organic layer was dried over anhydrous sodium sulfate.
  • Step E To a solution of compound 4 (3.0 g, 7.93 mmol) and 4-amino-3-bromobenzoic acid (1.70 g, 7.87 mmol) in toluene (50 mL) was added tributylphosphine (8.0 g, 39.5 mmol), The resulting mixture was stirred at reflux under nitrogen for 48 hours. Cool to room temperature, add water (50 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL ⁇ 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate.
  • tributylphosphine 8.0 g, 39.5 mmol
  • Step F A mixture containing compound 5 (400 mg, 1.08 mmol), cuprous cyanide (145 mg, 1.62 mmol) and NMP (10 mL) was stirred at 150 ° C. overnight. Cool to room temperature, add water (40 mL), and adjust the pH to 8-9 with 2M sodium carbonate solution. It was extracted with ethyl acetate (30 mL ⁇ 3), and the combined organic phase was washed with saturated brine (20 mL ⁇ 2) and dried over anhydrous sodium sulfate.
  • Step A To a solution of compound 4 (4.30 g, 11.4 mmol) and 4-amino-3-fluorobenzoic acid (1.76 g, 11.3 mmol) in toluene (50 mL) was added tributylphosphine (11.5 g, 39.5 mmol), The resulting mixture was stirred at reflux under nitrogen for 48 hours. Cool to room temperature, add water (50 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL ⁇ 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate.
  • Step B NBS (585 mg, 3.29 mmol) was added to a solution of compound 7 (780 mg, 2.52 mmol) in DMF (10 mL). After the addition, the resulting mixture was stirred at room temperature for 30 minutes. Water (40 mL) was added and extracted with ethyl acetate (30 mL ⁇ 2). The combined organic phases were washed with water (15 mL), saturated aqueous sodium bicarbonate solution (15 mL) and saturated brine (15 mL) in this order, and dried over anhydrous sodium sulfate.
  • Step C A mixture containing compound 8 (520 mg, 1.34 mmol), cuprous cyanide (180 mg, 2.01 mmol) and NMP (10 mL) was stirred at 150 ° C overnight. Cool to room temperature, add water (40 mL), and adjust the pH to 8-9 with 2M sodium carbonate solution. It was extracted with ethyl acetate (30 mL ⁇ 3), and the combined organic phase was washed with saturated brine (20 mL ⁇ 2) and dried over anhydrous sodium sulfate.
  • Step A A mixture containing compound 5 (500 mg, 1.35 mmol), acetic anhydride (0.5 mL), pyridine (10 mL) and 4-dimethylaminopyridine (10 mg, 0.0819 mmol) was stirred at 90 ° C overnight. After cooling to room temperature, water (50 mL) was added and extracted with ethyl acetate (30 mL ⁇ 3). The combined organic phase was washed with saturated brine (15 mL ⁇ 2) and dried over anhydrous sodium sulfate.
  • Step B Add trimethylsilane acetylene (131 mg, 1.33 mmol) to the compound containing compound 10 (500 mg, 1.21 mmol), bistriphenylphosphine palladium dichloride (40 mg, 0.0570 mmol), triethylamine (40 mL) via syringe ) And DMF (4 mL), after the addition, the resulting mixture was stirred at 50 ° C. overnight. Most of the solvent was distilled off under reduced pressure, water (30 mL) was added, and extracted with ethyl acetate (30 mL ⁇ 3). The combined organic phase was washed with saturated brine (15 mL ⁇ 2) and dried over anhydrous sodium sulfate.
  • Step C A mixture containing compound 11 (100 mg, 0.233 mmol), 2M sodium hydroxide solution (10 mL), THF (5 mL) and methanol (10 mL) was stirred at 80 ° C for 1 hour. After cooling to room temperature, water (30 mL) was added and extracted with ethyl acetate (30 mL ⁇ 3). The combined organic phase was washed with saturated brine (15 mL ⁇ 2) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the resulting product was recrystallized from petroleum ether / ethyl acetate to obtain 2-ethynyl-4- (5-fluorobenzoselenazol-2-yl) aniline (12).
  • Step A In an ice water bath, add NBS (3.55g, 19.9mmol) in portions to a solution of 5-fluoro-2-methylaniline (2.50g, 20.0mmol) in DMF (20mL). After the addition is complete, the result is The mixture continued to stir at this temperature for 1 hour. Water (80 mL) was added and extracted with ethyl acetate (60 mL ⁇ 3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (30 mL ⁇ 2) and saturated brine (30 mL ⁇ 2), and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain crude 4-bromo-3-fluoro-6-methylaniline (13) (4.87g). This compound was directly used in the next reaction without purification.
  • Step B A mixture containing crude compound 13 (4.87 g), cuprous cyanide (2.63 g, 29.4 mmol) and NMP (15 mL) was stirred at 180 ° C. under nitrogen for 7 hours. Water (75 mL) was added and extracted with ethyl acetate (50 mL ⁇ 3). The combined organic phase was washed with water (30 mL ⁇ 2) and saturated brine (30 mL) in this order, and dried over anhydrous sodium sulfate.
  • Step C A mixture containing compound 14 (1.74 g, 11.6 mmol), 1M sodium hydroxide solution (50 mL) and ethanol (5 mL) was stirred at reflux overnight. Cool to room temperature, add water (50 mL), extract with methyl tert-butyl ether (20 mL x 2), and the product is in the aqueous phase. The aqueous phase was adjusted to pH 3-4 with 2M hydrochloric acid, and solid precipitated. Filter and dry the filter cake to give 4-amino-2-fluoro-5-methylbenzoic acid (15) (1.75g). The yield was 89.2%.
  • Step D To a solution of compound 4 (1.0 g, 2.64 mmol) and compound 15 (447 mg, 2.64 mmol) in toluene (30 mL) was added tributylphosphine (2.68 g, 13.2 mmol), and the resulting mixture was stirred under reflux under nitrogen 36 hour. Cool to room temperature, add water (40 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL ⁇ 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate.
  • Step A To a solution of compound 4 (1.80 g, 4.76 mmol) and 5-aminopyridine-2-carboxylic acid (657 mg, 4.76 mmol) in toluene (50 mL) was added tributylphosphine (4.82 g, 23.8 mmol), and the resulting mixture Stir under reflux for 36 hours under nitrogen. Cool to room temperature, add water (40 mL), and adjust the pH to 9-10 with 2M sodium hydroxide solution. It was extracted with ethyl acetate (40 mL x 3), and the combined organic phase was washed with saturated brine (25 mL) and dried over anhydrous sodium sulfate.
  • tributylphosphine 4.82 g, 23.8 mmol
  • Step B Under ice-water bath, NBS (61 mg, 0.343 mmol) was added portionwise to a solution of compound 17 (100 mg, 0.342 mmol) in dichloromethane (6 mL). After the addition was complete, the resulting mixture continued to be stirred at this temperature 0.5 hours. Water (15 mL) was added and extracted with dichloromethane (60 mL ⁇ 3). The combined organic phase was washed with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL) in this order, and dried over anhydrous sodium sulfate.
  • Example 8 Compounds inhibit growth of human gastric cancer cell lines AGS, MKN-45 and NCI-N87
  • Human gastric cancer cell lines AGS, MKN-45 and NCI-N87 were purchased from the Cell Resource Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences.
  • Paclitaxel, Resazurin and methylene blue were purchased from Sigma-Aldrich Co., LLC; potassium ferricyanide and potassium ferrocyanide were purchased from Aladdin Reagent Co., Ltd .; DMEM medium, 1640 medium, phenol red-free DMEM and fetal bovine serum Purchased from Thermo Fisher Inc .; Penicillin and Streptomycin were purchased from Biyuntian Biotechnology Co., Ltd.
  • DMEM medium for AGS containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin
  • MKN-45 1640 medium containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin
  • NCI-N87 with DMEM medium containing 20% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin
  • three cells were cultured at 37 °C, 5% CO 2 incubator To the cell density of about 90%.
  • the cells were seeded in 96-well plates at 3 ⁇ 10 3 / well, and incubated at 37 ° C in a 5% CO 2 incubator for 24 hours.
  • test compound or control drug paclitaxel Prepare different concentrations of test compound or control drug paclitaxel with medium, and add 100 ⁇ L / well to 96-well plate as test compound well or control drug well; add 100 ⁇ L / well medium without test compound or control drug As a negative control well. Incubate at 37 ° C, 5% CO 2 , AGS and MKN-45 cells were cultured for 72 hours, and NCI-N87 cells were cultured for 120 hours.
  • the fluorescence value of the cells was detected at 530nm at Ex 530 / Em with Victor X4 (Perkin Elmer).
  • the fluorescence value of the test compound well is represented by F (test compound) ;
  • the fluorescence value of the blank control well is represented by F (blank control) ;
  • the fluorescence value of the negative control well is represented by F (negative control) .
  • Prism Graph software was used to calculate the half-inhibitory concentration (IC 50 ) of the test compound or control drug on the cells according to the cell survival rate.
  • the test results are shown in Table 1.
  • the compounds have a significant inhibitory effect on the growth of human gastric cancer cell lines AGS, MKN-45 and NCI-N87.
  • compounds 6, 9, 12, 19, 22 and 23 had better inhibitory effects.
  • Example 9 Growth inhibitory activity of compounds 6, 22 and 23 on human gastric cancer AGS and MKN-45 xenograft tumors in nude mice
  • SPF grade BALB / c nude mice, female are 6-8 weeks old at the start of administration and weigh 18-20 g. Provided by Changzhou Cavens Experimental Animal Co., Ltd. (Experimental Animal Production License: SCXK (Su) 2016-0010; Experimental Animal Use License: SYXK (Su) 2017-0007). Purchased animal batch number: 201820473.
  • Polyethylene glycol 400 (PEG400), batch number 20180412, purchased from Chengdu Kelong Chemical Reagent Factory; sodium chloride injection (physiological saline), batch number A17111105, purchased from Hebei Tiancheng Pharmaceutical Co., Ltd .; DMSO, batch number Q6949, purchased from MP Biomedicals.
  • AGS and MKN-45 cells were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; AGS was cultured in 1640 medium with 10% fetal bovine serum, and MKN-45 was cultured in DMEM medium with 10% fetal bovine serum to cultivate.
  • Low-dose group (administration dose: 2.5 mg / kg): accurately weigh 2.0 mg of the test compound before administration and dissolve it into 8 mL of clear solution with a solvent for intravenous injection (PEG400 25%, DMSO 2.5%, physiological saline 72.5%) Solution, the final concentration of the intravenous solution of the test compound in the low-dose group was 0.25 mg / mL, and the intravenous administration volume was 0.2 mL / 20 g body weight.
  • a solvent for intravenous injection PEG400 25%, DMSO 2.5%, physiological saline 72.5%
  • High-dose group (administration dose: 5.0 mg / kg): accurately weigh 4.0 mg of the test compound before administration and dissolve it to 8 mL of clear solution with a vehicle for intravenous injection (PEG400 50%, DMSO 5%, saline 45%) Solution, the final concentration of the intravenous injection solution of the test compound in the high-dose group was 0.50 mg / mL, and the intravenous administration volume was 0.2 mL / 20 g body weight.
  • a vehicle for intravenous injection PEG400 50%, DMSO 5%, saline 45%
  • the AGS and MKN-45 cells in the logarithmic growth phase were taken and inoculated under the aseptic conditions under the skin of the right armpit of 60 nude mice, respectively.
  • the cell inoculation volume was 5 ⁇ 10 6 cells / cell.
  • 56 AGS and MKN-45 tumor-bearing nude mice with good growth status and good tumor size uniformity are selected and randomly divided into 7 groups, each group 8 Only, the model group, compound 6, 22 and 23 low-dose group and high-dose group. Each group was administered intravenously, and were continuously administered once a day on days 0, 1, 2, 3, and 4 for a total of 5 days.
  • the model group was given an equal volume of vehicle control. Using the method of measuring tumor diameter, the anti-tumor effect of the test compound was dynamically observed. The tumor diameter was measured every two days and weighed at the same time. On the 20th day, the nude mice were sacrificed and the tumor masses were removed and weighed accurately.
  • the formula for calculating the% of tumor suppression is:
  • test results are shown in Table 2, Table 3, and Figures 1 to 6.
  • the test compounds 6, 22, and 23 were injected at a dose of 2.5 mg / kg and 5.0 mg / kg in the tail vein for 5 consecutive days, respectively.
  • Example 10 Compounds inhibit growth of human colon cancer cell lines HCT116, HCT15, HT29, SW620 and COLO205
  • Human colon cancer cell lines HCT116, HCT15, HT29, SW620 and COLO205 were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
  • Paclitaxel, Resazurin and methylene blue were purchased from Sigma-Aldrich Co., LLC; potassium ferricyanide and potassium ferrocyanide were purchased from Aladdin Reagent Co., Ltd .; DMEM medium, phenol-free DMEM and fetal bovine serum were purchased from Thermo Fisher Scientific Inc .; Penicillin and Streptomycin were purchased from Biyuntian Biotechnology Co., Ltd.
  • HCT116, HCT15, HT29, SW620 and COLO205 cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin) at 37 ° C, 5% CO 2 incubator to the cell density Up to about 90%.
  • DMEM medium containing 10% fetal bovine serum, 100U / mL penicillin, 0.1mg / mL streptomycin
  • the cells were seeded in 96-well plates at 3 ⁇ 10 3 / well, and incubated at 37 ° C in a 5% CO 2 incubator for 24 hours.
  • test compound or the control drug paclitaxel with different concentration gradients is prepared with the medium, and added to the 96-well plate at 100 ⁇ L / well as the test compound well or control drug well; at 100 ⁇ L / well, the culture without the test compound or control drug As a negative control well. Place in a 37 ° C, 5% CO 2 incubator and incubate for 72 hours.
  • the fluorescence value of the cells was detected at 530nm at Ex 530 / Em with Victor X4 (Perkin Elmer).
  • the fluorescence value of the test compound well is represented by F (test compound) ;
  • the fluorescence value of the blank control well is represented by F (blank control) ;
  • the fluorescence value of the negative control well is represented by F (negative control) .
  • Prism Graph software was used to calculate the half-inhibitory concentration (IC 50 ) of the test compound or the control drug on the cells.
  • the test results are shown in Table 4.
  • the compounds significantly inhibited the growth of human colon cancer cell lines HCT116, HCT15, HT29 and COLO205.
  • compounds 9, 19, 22 and 23 had better inhibitory effects.
  • Example 11 Growth inhibitory activity of compounds 6, 22 and 23 on human colon cancer HT29 and HCT116 xenografts in nude mice
  • SPF grade BALB / c nude mice, female are 6-8 weeks old at the start of administration and weigh 18-20 g. Provided by Changzhou Cavens Experimental Animal Co., Ltd. (Experimental Animal Production License: SCXK (Su) 2016-0010; Experimental Animal Use License: SYXK (Su) 2017-0007). Purchased animal certificate number: 201822212.
  • Polyethylene glycol 400 (PEG400), batch number 20180412, purchased from Chengdu Kelong Chemical Reagent Factory; sodium chloride injection (physiological saline), batch number A17111105, purchased from Hebei Tiancheng Pharmaceutical Co., Ltd .; DMSO, batch number Q6949, purchased from MP Biomedicals.
  • HCT116 and HT29 cells were purchased from the Cell Resource Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences; HCT116 was cultured in 1640 medium containing 10% fetal bovine serum, and HT29 was cultured in 1640 medium containing 10% fetal bovine serum.
  • Low-dose group (administration dose: 4.0 mg / kg): Weigh 3.2 mg of the test compound before administration and dissolve it to 8 ml with a solvent (PEG400 50%, DMSO 5%, physiological saline 45%), the solution concentration is 0.4 mg / ml, administered intravenously, with a volume of 0.2ml / 20g body weight.
  • a solvent PEG400 50%, DMSO 5%, physiological saline 45%
  • High-dose group (administration dose: 8.0 mg / kg): Weigh 6.4 mg of the test compound before administration, dissolve it to 8 ml with a solvent (PEG400 50%, DMSO 5%, physiological saline 45%), the solution concentration is 0.8 mg / ml, administered intravenously, with a volume of 0.2ml / 20g body weight.
  • a solvent PEG400 50%, DMSO 5%, physiological saline 45%
  • HCT116 and HT29 cells in the logarithmic growth phase were taken and inoculated under the aseptic conditions under the skin of the right armpits of 60 nude mice, respectively.
  • the cell inoculation volume was 5 ⁇ 10 6 cells / cell.
  • 56 HCT116 and HT29 tumor-bearing nude mice with good growth status and good tumor size uniformity are selected and randomly divided into 7 groups, 8 in each group.
  • Each group was administered via tail vein injection, which was administered once a day on days 0, 1, 2, 3, and 4 for a total of 5 days.
  • the model group was given an equal volume of vehicle control. Using the method of measuring tumor diameter, the anti-tumor effect of the test compound was dynamically observed. The tumor diameter was measured every two days and weighed at the same time. On the 20th day, the nude mice were sacrificed and the tumor masses were removed and weighed accurately.
  • the formula for calculating the tumor volume (TV) is:
  • the formula for calculating the% of tumor suppression is:
  • test results are shown in Table 5, Table 6, and Figures 7 to 12.

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Abstract

本发明公开了一类4-(苯并硒唑-2-基)芳胺类化合物治疗胃癌或肠癌的用途，其为通式(I)所示的化合物或其药学上可接受的盐。本发明发现了4-(苯并硒唑-2-基)芳胺类化合物在疾病治疗方面的新用途，为胃癌或肠癌的治疗提供了一种潜在的药品。

Description

4-(苯并硒唑-2-基)芳胺类化合物治疗胃癌或肠癌的用途 技术领域
本发明属于药物化学领域，具体涉及一类4-(苯并硒唑-2-基)芳胺类化合物在治疗胃癌或肠癌方面的用途。
背景技术
胃癌是消化系统最常见的恶性肿瘤之一。在全世界范围内，胃癌的发病率在恶性肿瘤中排名第5位，致死率在恶性肿瘤中位居第3位。国际癌症研究中心(IARC)公布的数据表明，2012年全球共有951,600例新发胃癌患者(其中男性病例数约是女性的2倍)，以及723,100死亡病例。据统计，胃癌发病率东亚最高(特别是韩国、蒙古、日本和中国)，其次为欧洲中、东部和南美洲，而北美洲和大部分非洲地区最低(Torre LA,Bray F,Siegel RL,et al.Global cancer statistics,2012.Ca A Cancer Journal for Clinicians,2015,65(2):87-108；IARC.All Cancers(excluding non-melanoma skin cancer)Estimated Incidence,Mortality and Prevalence Worldwide in 2012；Herrero R,Park JY,Forman D.The Fight AgainstGastric Cancer–theIARC Working Group report.Best Practice andResearch in Clinical Gastroenterology,2014,28(6):1107-1114；Carcas LP.Gastric Cancer Review.Journal of Carcinogenesis,2014,19:13-14)。
据统计，中国胃癌发病率在恶性肿瘤中位居于第2位，每年新增病例数和死亡人口均超过了全球的40％(Torre LA,Siegel RL,Ward EM,et al.Global Cancer Incidenceand Mortality Rates and Trends-an Update.CancerEpidemiology and Prevention Biomarkers,2016,25(1):16-27)；且每年有约352,300例患者因胃癌死亡(Layke JC,Lopez PP.胃癌：诊断和治疗方案的选择.中国全科医学,2015,18(3):248-249)。
胃癌的发病机制至今尚未完全阐明，主要认为胃癌发病与饮食习惯、幽门螺杆菌(Helicobacterpylori,Hp)感染、血清胃蛋白酶原(PG)、遗传因素、代谢综合征和心理压力等因素都有一定关联。而幽门螺旋杆菌可能是胃癌最大的诱发因素，大约有90％的新增非贲门胃癌病例与此相关(Chang M,Zhang JC,Zhou Q,et al.Research Progress of Clinical Epidemiology of Gastric Cancer.Chinese Journal of Gastroenterology&Hepatology,2017,26(9):966-969)。
目前治疗胃癌的手段有限，手术切除仍是根治胃癌的唯一临床方法。对于早期胃癌，虽然术后原则上是无需接受化疗，但患者若出现以下情况，仍需进行化疗：(1)年龄小于40岁；(2)存在多个病灶；(3)病灶面积大于5cm 2；(4)病理类型的恶性程度偏高(贺阿青，张成武.探讨胃癌的诊断及治疗.世界最新医学信息文摘,2018,18(14):55)。
中国由于胃癌筛查体系尚不完善，胃癌早期诊断率低，所以多数患者确诊时已处于进展期胃癌或晚期胃癌(Advanced Gastric Cancer)。此时单纯手术治疗往往无法取得较好的疗效，且术后复发转移率较高；有部分患者的肿瘤体积大，无法进行手术，须予以新辅助化疗，待肿瘤体积缩小，才可以进行手术。有研究表明，对于进展期胃癌，联合手术不仅不能带来生存优势，对于部分患者的总生存期甚至低于单纯化疗(Gastrectomy plus Chemotherapy versus Chemotherapy Alone for Advanced Gastric Cancer with Single Non-curable Factor(REGATTA):a Phase 3,Randomized Controlled Trial.Lancet Oncology,2016,17(3):309-318)。因此，以化疗为主的综合疗法是晚期胃癌患者的主要治疗手段，可改善患者预后，提高患者生活质量。
目前，临床上对于晚期胃癌化疗所用的细胞毒性药物主要包括四大类：(1)口服氟尿嘧啶类，如卡培他滨、替吉奥；(2)紫杉类，如紫杉醇，多西他赛；(3)第三代铂类，如奥沙利铂；(4)拓扑异构酶I抑制剂，如伊立替康。另外，已上市的分子靶向药物主要包括人表皮生长因子受体2(HER2)抗体，如曲妥珠单抗，血管内皮生长因子受体(VEGFR-2)抗体，如雷莫芦单抗，以及靶向VEGFR-2的小分子酪氨酸酶抑制剂阿帕替尼等(李开春，李平.进展期胃癌治疗药物的选择.药学服务与研究,2018,18(1):1-5)。
虽然对于胃癌进行化疗联用是NCCN指南建议的标准治疗方式(NCCN Clinical Practice Guideline in Oncology:Gastric Cancer(2015Version))，但效果却已经达到了一个瓶颈，患者的生存获益无法增加更多，同时，患者都会因化疗药物的细胞毒性引起如白细胞和血小板减少等骨髓抑制等毒副作用，造成患者身体情况进一步恶化。另外，胃癌是一种由多个基因突变和表观遗传异常所驱动的高度异质性疾病，因此靶向药物开发滞后，且已有药物的靶向性差、灵敏度低，如曲妥珠单抗仅仅对胃癌患者中约20％的HER2阳性患者有效(Bang YJ,Van Cutsem E,Feyereislova A,et al.Trastuzumab in Combination with Chemotherapy versus Chemotherapy Alone for Treatment of HER2-positive Advanced Gastric or Gastro-oesophageal Junction Cancer(ToGA):a Phase 3,Open-label,Randomized Controlled Trial.Lancet,2010,376(6):687-697)。因此，目前的化疗和靶向疗法均无法有效对抗胃癌， 而增加患者的生存获益。
中国的胃癌发病率和死亡率处于世界较高水平，已严重危害居民的健康，给家庭和社会造成沉重的负担，由于胃癌的高度异质性和目前临床治疗手段的匮乏，因此，急需研制新型的治疗方法或药物以应对胃癌发病率和死亡率日益增长的现状。
结直肠癌是消化系统最常见的恶性肿瘤，其全球发病率在恶性肿瘤中位居男性第3位和女性第2位(Torre LA,Bray F,Siegel RL,et al.Global cancer statistics,2012.CA A Cancer Journal for Clinicians,2015,65(2):87-108)。据2012年统计数据，全球共有约140万例新增结直肠癌病例，以及693,900例结直肠癌死亡病例。发病率最高的区域在澳大利亚和新西兰，欧洲与北美也是高发区(Torre LA,Siegel RL,Ward EM,et al.Global Cancer Incidence and Mortality Rates and Trends-an Update.Cancer Epidemiology and Prevention Biomarkers,2016,25(1):16-27)。结肠癌在美国是第三大常见癌症，每年新增确诊结肠癌病例高达130,000人(Birt DF,Phillips GJ.Diet,Genes,and Microbes:Complexities of Colon Cancer Prevention.Toxicologic Pathology,2014,42:182-188)。由于结肠癌早期诊断率很低，绝大多数患者确诊时已是中晚期，给根治性手术带来难度，其5年生存率仅为50％-60％(王正康.结肠癌根治术的合理切除范围.国外医学外科学分册,1989,(1):9-10)。
结肠癌发病的高危因素包括以下几个方面。1.饮食因素：高脂高蛋白饮食、缺少蔬菜水果谷物纤维素、钙硒等微量元素摄入不足(Terry P,Giovannuci E.Michels KB,et al.Fruit,Vegetables,Dietary fiber,and Risk of Colorectal Cancer.Journal of the National Cancer Institute,2001,93:525)。2.疾病因素：下消化道疾病史、胆囊切除术、阑尾炎、高甘油三酯症(Masafumi T,Joji K,Hirokazu N.Hypertriglyceridemia is Positively Correlated with the Development of Colorectal Tubular Adenoma in Japanese Men.World Journal of Gastroenterology,2006,12(8):1261-1264)等。3.生活方式及其他因素：肥胖与缺少体力锻炼、便秘、遗传等。
结肠癌以手术切除肿瘤为首选治疗手段，辅助以化疗、放疗、靶向治疗和基因治疗等手段。结肠全系膜切除术是手术治疗结肠癌的最主要方法，但有25％的结肠癌患者确诊时已属于中晚期，并且由于结肠癌具有高复发和远端转移的生物学特性，有超过25％的患者在第一次手术后仍将出现复发或转移(王光林,孟泽松,王飞飞等.局部晚期结肠癌术前化疗的研究进展.肿瘤,2018,38:716-722)。且手术治疗仅适用于部分或早期的患者，因为大量的患者已存在更恶性的不可切除的转移，所以选择全身治疗(化疗和靶向治疗) 或许可延长患者的整体生存期。
而结肠癌化疗国际上公认的一线方案是5-氟尿嘧啶联合奥沙利铂，或卡培他滨联合奥沙利铂(Andre T,Boni C,Mounedji L,et al.Oxaliplatin,Fluorouracil,and Leucovorin as Adjuvant Treatment for Colon Cancer.New England Journal of Medicine,2004,350(23):2343-2351；Schmoll HJ,Tabernero J,Maroun J,et al.Capecitabine plus Oxaliplatin Compared with Fluorouracil/Folinic Acid as Adjuvant Therapy for Stage III Colon Cancer:Final Results of the NO16968 Randomized Controlled Phase III Trial.Journal of Clinical Oncology,2015,33(32):3733-3740)。肿瘤化疗中的多重药物耐药是导致化疗失败的主要原因，结肠癌癌变组织由于其抗癌药物耐药相关的载体蛋白(如P糖蛋白、多药耐药相关蛋白或乳腺癌耐药蛋白)的过表达(Herraez E,Gonzalez SE,Vaquero J,et al.Cisplatin Induced Chemoresistance in Colon Cancer Cells Involves FXR-Dependent and FXR-Independent Up-regulation of ABC Proteins.Molecular Pharmaceutics,2012,9(9):2565-2576)，使得结肠癌细胞内的抗癌药物被迅速外排，浓度过低，从而导致化疗的失败。
如今结肠癌的靶向治疗备受关注。但结肠癌是一种高度异质性、分子分型复杂的疾病。目前临床上传统的靶向治疗主要是针对血管内皮生长因子受体(VEGF)和表皮生长因子受体(EGFR)，但抗VEGF药物对中晚期结肠癌没有明显作用(Hurwitz HI,Lyman GH.Registries and Randomized Trials in Assessing the Effects of Bevacizumab in Colorectal Cancer:Is There a Common Theme？.Journal of Clinical Oncology.2012,30(6):580-581)，且会增加老年患者动脉血管事件的发生率(Ranpura V,Hapani S,Wu S.Treatment-related Mortality with Bevacizumab in Cancer Patients:a Meta-analysis.JAMA,2011,305(5):487-494)；而抗EGFR药物对于RAS突变型的结肠癌患者是无效的(Vale CL,Tierney JF,Fisher D,et al.Does Anti-EGFR Therapy Improve Outcome in Advanced Colorectal Cancer？A Systematic Review and Meta-analysis.Cancer Treatment Reviews,2012,38(6):618-625)。结肠癌的全基因组研究显示，肿瘤基因突变数量非常高，每个肿瘤平均有75个左右的突变，这表明针对特定分子异常的癌症治疗可能仅在很小一部分结肠癌患者中有效。目前尚无开发出可用于临床治疗的生物标志物，仅依靠微卫星不稳定性(MSI)、RAS突变和BRAF突变这三类标志物用以评估临床诊疗的决策或判断治疗的预后情况(Guinney J,Dienstmann R,Wang X,et al.The Consensus Molecular Subtypes of Colorectal Cancer.Nat Med.2015,21:1350-1356；Hutchins G1,Southward K,Handley K,et al.Value of  Mismatch Repair,KRAS,and BRAF Mutations in Predicting Recurrence and Benefits from Chemotherapy in Colorectal Cancer.Journal of Clinical Oncology.2011,29:1261-1270)。
由于世界范围内的结肠癌发病率和死亡率均处于较高水平，已严重危害人们的健康，给家庭和社会造成了沉重的负担，但由于结肠癌具有高复发、远端转移以及高度异质性的生物学特点，因此目前临床现有的治疗手段十分匮乏，无法显著地增加结肠癌患者的临床获益，急需研制新型的治疗方法或药物以应对结肠癌发病率和死亡率日益增长的现状。
硒元素是人体必需的微量元素之一，血中硒元素含量降低会诱发多种包括肿瘤及心血管在内的多种疾病(Reeves MA,Hoffmann PR.The human Selenoproteome:Recent Insights into Functions and Regulation.Cellular and Molecular Life Sciences,2009,66(15):2457-78)。含硒药物由于它在抗肿瘤、抗病毒以及治疗神经系统相关疾病等方面的应用，已成为国内外学者研发的热点，药物研究主要集中在抗肿瘤、抗炎和抗高血压等方面(Romualdo C,Stefania C,Marina DG,et al.Novel Selenium-containing Non-natural Diamino acids.Tetrahedron Letters,2007,48(7):1425-1427)。
Malcolm F.G.Stevens等所申请的美国专利(Malcolm F.G.Stevens,Andrew D.Westwell,Mei-Sze Chua,et al.Substituted 2-arylbenzazole compounds and their use as antitumour agents.US6858633B1)公布了下式(II)化合物，并指出了这类化合物在癌症方面的用途，但其说明书内容主要针对的是乳腺癌，由于不同癌症之间发病机理和治疗途径基本上并不相同，而且大部分抗癌化合物仅对某些敏感癌症具有治疗作用，因此该类化合物在其他癌症方面是否能起作用实际上仍然是未知的。
Figure PCTCN2019115184-appb-000001
本专利发明人史东方已发表了关于苯并噻唑类化合物的合成及其体内外抗乳腺癌作用的文章(Dong-Fang Shi,Tracey D.Bradshaw,Samantha Wrigley,et al.Antitumor Benzothiazoles.3.Synthesis of 2-(4-Aminophenyl)benzothiazoles and Evaluation of Their Activities against Breast Cancer Cell Lines in Vitro and in Vivo.Journal of Medicinal Chemistry,1996,39:3375-3384)，同时，本专利发明人史东方所申请的中国专利(史东方，傅长金，承曦等.用于治疗或预防乳腺癌的化合物.CN201610299350.1)公布了如式(III)所示苯并硒唑-2-苯类化合物及其抗乳腺癌的药理活性。这两类化合物均对人乳腺癌细胞具有纳摩尔级别的抑制效果，其中苯并噻唑类化合物对ER +(MCF-7和BO细胞株)和 ER -(MT-1和MT-3细胞株)的乳腺癌裸鼠移植瘤均表现出十分显著的抑瘤效果，但是这两类化合物对其他如前列腺癌、膀胱癌、黑色素瘤、肺癌、肝癌、食管癌等肿瘤细胞株并无抑制活性。
Figure PCTCN2019115184-appb-000002
至今，未见有任何关于4-(苯并硒唑-2-基)芳胺类化合物对胃癌或肠癌模型有作用的报道。
发明内容
本发明的目的是在现有技术的基础上，提供一类4-(苯并硒唑-2-基)芳胺类化合物在治疗胃癌或肠癌方面的用途。
本发明的目的可以通过以下措施达到：
通式(IV)所示的化合物或其药学上可接受的盐在制备治疗或预防胃癌或肠癌药物方面的应用，
Figure PCTCN2019115184-appb-000003
进一步的，通式(I)所示的化合物或其药学上可接受的盐在制备治疗或预防胃癌或肠癌药物方面的应用，
Figure PCTCN2019115184-appb-000004
在各通式中，
R 1和R 2分别独立地选自H、D、卤素、-CN、C 1-3烷基、取代的C 1-3烷基、C 1-3烷氧基或取代的C 1-3烷氧基；
R 3选自H、卤素、-OH、-CN、-C(＝O)NH 2、取代的-C(＝O)NH 2、C 1-3烷基、取代的 C 1-3烷基、C 2-3烯基、C 2-3炔基、C 1-3烷氧基或取代的C 1-3烷氧基；
R 4选自H、D、卤素、-OH、-CN、-NH 2、取代的-NH 2、-C(＝O)NH 2、取代的-C(＝O)NH 2、C 1-3烷基、取代的C 1-3烷基、C 2-3烯基、C 2-3炔基、C 1-3烷氧基或取代的C 1-3烷氧基中的一种或两种；
n＝1或2；
R 5选自H、-CN、-OH、C 1-3烷基、C 1-3烷氧基或氨基酸残基；
Z选自CH或N；
R 1、R 2、R 3或R 4中的取代基选自D、卤素、OH、C 1-3烷基或C 1-3烷氧基。
在一种优选方案中，本发明中的R 1和R 2分别独立地选自H、D、卤素、-CN、C 1-3烷基、取代的C 1-3烷基、C 1-3烷氧基或取代的C 1-3烷氧基，所述取代基选自D、F或C 1-3烷氧基；
在另一种优选方案中，本发明中的R 1和R 2分别独立地选自H、D、F、Cl、-CN、-CH 3、-CF 3、-OCF 3或-OCHF 2。
在另一种优选方案中，本发明中的R 1选自D、F、Cl、-CN、-CH 3或-CF 3。
在另一种优选方案中，本发明中的R 2选自H。
在一种优选方案中，本发明中的R 3选自H、卤素、-CN、-C(＝O)NH 2、C 1-2烷基、C 2-3烯基、C 2-3炔基、C 1-3烷氧基或取代的C 1-3烷氧基。
在另一种优选方案中，本发明中的R 3选自H、F、Cl、Br、-CN、-C(＝O)NH 2、-CH 3、-CH 2CH 3、-CF 3、-CH＝CH 2、-C≡CH、-OCHF 2或-OCF 3。
在一种优选方案中，本发明中的R 4选自H、D、卤素、-CN、C 1-3烷基、取代的C 1-3烷基、C 1-3烷氧基或取代的C 1-3烷氧基中的一种或两种。
在另一种优选方案中，本发明中的R 4选自H、D、F、Cl、Br、I、-CN、-CH 3、-CF 3、-OCH 3、-OCH 2CH 3、-OCHF 2或-OCF 3。
在一种优选方案中，本发明中的R 5选自H、C 1-3烷基、C 1-3烷氧基或氨基酸残基。
在另一种优选方案中，本发明中的R 5选自H、-CH 3、-CF 3、-OCH 3或2,6-二氨基-己酰基。
在一种优选方案中，本发明中所涉及的化合物选自：
2-氨基-5-(5-氟苯并硒唑-2-基)苯甲腈，
2-氨基-3-氟-5-(5-氟苯并硒唑-2-基)苯甲腈，
2-乙炔基-4-(5-氟苯并硒唑-2-基)苯胺，
5-氟-4-(5-氟苯并硒唑-2-基)-2-甲基苯胺，
2-溴-6-(5-氟苯并硒唑-2-基)吡啶-3-氨，
3-氨基-6-(5-氟苯并硒唑-2-基)-2-甲酰胺基吡啶，
4-(5-氟苯并硒唑-2-基)-2-甲基苯胺，
2-溴-4-(5-氟苯并硒唑-2-基)-6-甲基苯胺，
2-溴-6-氟-4-(5-氟苯并硒唑-2-基)苯胺，
2,6-二氟-4-(5-氟苯并硒唑-2-基)苯胺，
2-氟-4-(5-氟苯并硒唑-2-基)-6-甲基苯胺。
如无特别说明，本发明中所涉及的各基团分别具有如下含义。
“H”，即氢，是指氕(1H)，它是氢元素的主要稳定同位素。
“D”，即氘，是指氢的一种稳定形态同位素，也被称为重氢，其元素符号为D。
“卤素”，是指氟原子，氯原子，溴原子或碘原子。
“-OH”，是指羟基基团。
“-NH 2”，是指氨基基团。
“-CONH 2”，即C(＝O)-NH 2，是指酰胺基团。
“-CN”，是指氰基基团。
“-NO 2”，是指硝基基团。
“烷基”，是指1-10个碳原子的饱和的脂烃基，包括直链和支链基团(本申请书中提到的数字范围，例如“1-10”，是指该基团，此时为烷基，可以含1个碳原子、2个碳原子、3个碳原子等，直至包括10个碳原子)。含1-4个碳原子的烷基称为低级烷基。当低级烷基没有取代基时，称其为未取代的低级烷基。烷基可以选用C 1-6烷基、C 1-5烷基、C 1-4烷基、C 1-3烷基、C 1-2烷基、C 2-3烷基、C 2-4烷基等。具体的烷基包括但不限于甲基、乙基、丙基、2-丙基、正丁基、异丁基或叔丁基等。烷基可以是取代的或未取代的。
“烯基”表示具有至少一个碳碳双键的不饱和烃基基团，包括直链和支链基团(本申请书中提到的数字范围，例如“2-5”，是指该基团，此时为烯基，可以含2个碳原子、3个碳原子、4个碳原子等，直至包括5个碳原子)。本发明中的烯基可以为C 2-8烯基、C 2-6烯基、C 2-5烯基、C 2-4烯基、C 2-3烯基等，具体的烯基包括但不限于乙烯基、丙烯基和丁烯基。
“炔基”表示具有至少一个碳碳三键的不饱和烃基基团，包括直链和支链基团(本申请 书中提到的数字范围，例如“2-5”，是指该基团，此时为炔基，可以含2个碳原子、3个碳原子、4个碳原子等，直至包括5个碳原子)。本发明中的炔基可以为C 2-8炔基、C 2-6炔基、C 2-5炔基、C 2-4炔基、C 2-3炔基等，具体的烯基包括但不限于乙炔基、丙炔基和丁炔基。
“烷氧基”表示-O-(未取代的烷基)和-O-(未取代的环烷基)基团，其进一步表示-O-(未取代的烷基)。代表性实施例包括但不限于甲氧基、乙氧基、丙氧基、环丙氧基等。
“氨基酸残基”是指氨基酸缺少了某一基团(如-OH、-COOH或-NH 2)所形成的基团，其中氨基酸包括但不限于通常由三个字母符号指定的20种天然存在的氨基酸，并且还包括β-丙氨酸、瓜氨酸、锁链素(demosine)、γ-氨基丁酸、同型半胱氨酸、同型丝氨酸、4-羟基脯氨酸、羟基赖氨酸、异锁链素(isodemosine)、3-甲基组氨酸、正缬氨酸、甲硫氨酸砜(methioninesulfone)和鸟氨酸等。一种氨基酸残基的例子包括但不限于：2,6-二氨基-己酰基。
“药学上可接受的盐”是包含通式(I)的化合物与有机酸或无机酸形成的盐，表示保留母体化合物的生物有效性和性质的那些盐。这类盐包括：
(1)与酸成盐，通过母体化合物的游离碱与无机酸或有机酸的反应而得，无机酸例如(但不限于)盐酸、氢溴酸、硝酸、磷酸、偏磷酸、硫酸、亚硫酸和高氯酸等，有机酸例如(但不限于)乙酸、丙酸、丙烯酸、草酸、(D)或(L)苹果酸、富马酸、马来酸、羟基苯甲酸、γ-羟基丁酸、甲氧基苯甲酸、邻苯二甲酸、甲磺酸、乙磺酸、萘-1-磺酸、萘-2-磺酸、对甲苯磺酸、水杨酸、酒石酸、柠檬酸、乳酸、扁桃酸、琥珀酸或丙二酸等。
(2)存在于母体化合物中的酸性质子被金属离子代替或者与有机碱配位化合所生成的盐，金属离子例如碱金属离子、碱土金属离子或铝离子，有机碱例如乙醇胺、二乙醇胺、三乙醇胺、氨丁三醇、N-甲基葡糖胺等。
“药用组合物”指的是在此描述的一种或多种化合物或者它们的药学上可接受的盐和前药与其它的化学成分，例如药学上可接受的载体和赋形剂的混合物。药用组合物的目的是促进化合物对生物体的给药。
在本发明化合物的应用中，可以将本发明的化合物、药学上可接受的盐或其溶剂合物作为活性成分或主要活性成分，辅以药学上可接受的辅料制成药物组合物，再对病人进行施用。
本发明的化合物可以用本领域已知的方式配制成临床上或药学上可接受的任一剂型。用于口服给药时，可制成常规的固体制剂，如片剂、胶囊剂、丸剂、颗粒剂等；也可制成 口服液体制剂，如口服溶液剂、口服混悬剂、糖浆剂等。制成口服制剂时，可以加入适宜的填充剂、粘合剂、崩解剂、润滑剂等。用于肠胃外给药时，可以制成注射剂，包括注射液、注射用无菌粉末与注射用浓溶液。制成注射剂时，可采用现有制药领域中常规方法生产，配制注射剂时，可以不加入附加剂，也可以根据药物的性质加入适宜的附加剂。
本发明还提供了一种治疗人类胃癌或肠癌的方法，即对患有胃癌或肠癌的人施以每次剂量0.1-1000mg的本发明的化合物、药学上可接受的盐或其溶剂合物，或者施以每次剂量0.1-1000mg的本发明的药物组合物。
本专利涉及到的4-(苯并硒唑-2-基)芳胺类化合物尽管有抗乳腺癌方面的药理活性，但体外试验表明，对于前列腺癌、膀胱癌、黑色素瘤、肺癌、肝癌、食管癌等肿瘤细胞株均无明显的抑制作用。然而，本专利发现了这类化合物对于胃癌或肠癌的治疗效果十分优异：对人胃癌细胞株MKN-45、AGS和NCI-N87具有显著地抑制生长的作用，效果基本接近于阳性对照药物紫杉醇；该类化合物对人胃癌MKN-45和AGS裸鼠异种移植瘤也表现出极佳的抑制肿瘤生长的效果，抑瘤率均大于35％；故这些化合物可以应用在人类胃癌治疗领域。本专利发现了这类化合物对人结肠癌细胞株HCT116、HT29、HCT15和COLO205也具有显著地抑制生长的作用，效果基本接近于阳性对照药物紫杉醇；该类化合物对人结肠癌HCT116和HT29裸鼠异种移植瘤也表现出极佳的抑制肿瘤生长的效果，抑瘤率均大于40％。因此这些化合物可以应用在人类肠癌治疗领域。
本发明发现了4-(苯并硒唑-2-基)芳胺类化合物在疾病治疗方面的新用途，为胃癌或肠癌的治疗提供了一种潜在的药物。
附图说明
图1是化合物6对人胃癌细胞株AGS裸鼠异种移植肿瘤体积的抑制作用；
图2是化合物6对人胃癌细胞株MKN-45裸鼠异种移植肿瘤体积的抑制作用；
图3是化合物22对人胃癌细胞株AGS裸鼠异种移植肿瘤体积的抑制作用；
图4是化合物22对人胃癌细胞株MKN-45裸鼠异种移植肿瘤体积的抑制作用；
图5是化合物23对人胃癌细胞株AGS裸鼠异种移植肿瘤体积的抑制作用；
图6是化合物23对人胃癌细胞株MKN-45裸鼠异种移植肿瘤体积的抑制作用；
图7是化合物6对人结肠癌细胞株HT29裸鼠异种移植肿瘤体积的抑制作用；
图8是化合物6对人结肠癌细胞株HCT116裸鼠异种移植肿瘤体积的抑制作用；
图9是化合物22对人结肠癌细胞株HT29裸鼠异种移植肿瘤体积的抑制作用；
图10是化合物22对人结肠癌细胞株HCT116裸鼠异种移植肿瘤体积的抑制作用；
图11是化合物23对人结肠癌细胞株HT29裸鼠异种移植肿瘤体积的抑制作用；
图12是化合物23对人结肠癌细胞株HCT116裸鼠异种移植肿瘤体积的抑制作用。
具体实施方式
以下结合实施例对本发明的内容做进一步说明，但本专利的保护范围并不局限于以下各实施例。
实施例1：2-氨基-5-(5-氟苯并硒唑-2-基)苯甲腈(6)的合成
Figure PCTCN2019115184-appb-000005
步骤A：在-20～-25℃下将2-硝基-4-氟苯胺(5.0g，32.0mmol)的二氯甲烷(80mL)溶液滴加到三氟化硼乙醚(6.82g，48.1mmol)中。搅拌15分钟，然后在该温度下滴加亚硝酸异戊酯(4.50g，38.4mmol)的二氯甲烷(20mL)溶液。加完后继续搅拌30分钟，然后在-10～0℃下搅拌30分钟。向反应体系中滴加冷却的石油醚(60mL)，过滤，滤饼用冷却的甲基叔丁基醚(10mL)洗涤，得2-硝基-4-氟苯基-氟硼酸重氮盐(1)粗品(8.10g)。该化合物不经纯化直接用于下一步反应。
步骤B：在冰水浴下，向化合物1粗品(8.10g)和水(150mL)的混合物中滴加硒氰酸钾(4.35g，30.2mmol)的水(20mL)溶液，加完后继续搅拌1小时。过滤，滤饼用二氯甲烷(150mL)溶解。过滤除去不溶物，滤液用无水硫酸钠干燥。减压蒸除溶剂，得4-氟-2-硝基-1-硒氰酸苯酯(2)粗品(6.10g)。该化合物不经纯化直接用于下一步反应。
步骤C：室温下将金属钠(3.23g，140mmol)加入到化合物2粗品(6.10g)和无水乙醇(90mL)的混合物中，所得混合物在水浴下搅拌1小时。冷却到0～5℃，过滤， 滤饼用少量冷却的乙醇洗涤，得1,2-二(4-氟-2-硝基苯基)-二硒(3)粗品(3.90g)。该化合物不经纯化直接用于下一步反应。
步骤D：将化合物3粗品(3.90g)溶解于乙醇(60mL)，加入氯化亚锡(7.90g，41.7mmol)，所得混合物在氮气下回流搅拌4小时。减压蒸除大部分溶剂，加入水(120mL)和乙酸乙酯(200mL)，用2M氢氧化钠溶液调节pH值至8～9。通过硅藻土过滤除去不溶物，分层，水层用乙酸乙酯(60mL×2)萃取，合并的有机层用无水硫酸钠干燥。然后利用减压法柱层析纯化(200-300目硅胶，乙酸乙酯洗脱)，所得产物用石油醚重结晶，得6,6’-二硒基-二(3-氟苯胺)(4)(3.0g)。步骤A、B、C和D四步反应总收率为49.6％。
步骤E：向化合物4(3.0g，7.93mmol)和4-氨基-3-溴苯甲酸(1.70g，7.87mmol)的甲苯(50mL)溶液中加入三丁基膦(8.0g，39.5mmol)，所得混合物在氮气下回流搅拌48小时。冷却到室温，加入水(50mL)，用2M氢氧化钠溶液调节pH值至9～10。用乙酸乙酯(40mL×3)萃取，合并的有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200-300目硅胶，乙酸乙酯：石油醚＝1:50～1:5洗脱)，得2-溴-4-(5-氟苯并硒唑-2-基)苯胺(5)(1.60g)。收率为54.9％。
步骤F：将含有化合物5(400mg，1.08mmol)、氰化亚铜(145mg，1.62mmol)和NMP(10mL)的混合物在150℃搅拌过夜。冷却到室温，加入水(40mL)，用2M碳酸钠溶液调节pH值至8～9。用乙酸乙酯(30mL×3)萃取，合并的有机相用饱和食盐水(20mL×2)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200～300目硅胶，乙酸乙酯:二氯甲烷:石油醚＝1:1:20～1:1:8洗脱)，得2-氨基-5-(5-氟苯并硒唑-2-基)苯甲腈(6)(84mg)。收率为54.9％。 1H NMR(DMSO-d6，400MHz)δ8.15-8.12(m，1H)，8.06(d，J＝2.4Hz，1H)，7.98-7.96(m，1H)，7.79-7.76(m，1H)，7.24-7.19(m，1H)，6.90(d，J＝8.8Hz，1H)，6.83(s，2H)。MS(EI，m/z)：316.0[M-H] -。
实施例2：2-氨基-3-氟-5-(5-氟苯并硒唑-2-基)苯甲腈(9)的合成
Figure PCTCN2019115184-appb-000006
步骤A：向化合物4(4.30g，11.4mmol)和4-氨基-3-氟苯甲酸(1.76g，11.3mmol)的甲苯(50mL)溶液中加入三丁基膦(11.5g，39.5mmol)，所得混合物在氮气下回流搅拌48小时。冷却到室温，加入水(50mL)，用2M氢氧化钠溶液调节pH值至9～10。用乙酸乙酯(40mL×3)萃取，合并的有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200-300目硅胶，乙酸乙酯：石油醚＝1:100～1:20洗脱)，得2-氟-4-(5-氟苯并硒唑-2-基)苯胺(7)(780mg)。收率为22.3％。
步骤B：将NBS(585mg，3.29mmol)加入到化合物7(780mg，2.52mmol)的DMF(10mL)溶液中，加完后，所得混合物在室温下搅拌30分钟。加入水(40mL)，用乙酸乙酯(30mL×2)萃取，合并的有机相依次用水(15mL)、饱和碳酸氢钠水溶液(15mL)和饱和食盐水(15mL)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物用石油醚/乙酸乙酯重结晶，得2-溴-6-氟-4-(5-氟苯并硒唑-2-基)苯胺(8)(720mg)。收率为73.6％。 1H NMR(DMSO-d 6，500MHz)δ8.16-8.13(m，1H)，7.90(s，1H)，7.79(dd，J＝2.0，10.0Hz，1H)，7.73(dd，J＝2.0，10.0Hz，1H)，7.24-7.20(m，1H)，6.12(s，2H)。MS(EI，m/z)：388.9[M+H] +。
步骤C：将含有化合物8(520mg，1.34mmol)、氰化亚铜(180mg，2.01mmol)和NMP(10mL)的混合物在150℃搅拌过夜。冷却到室温，加入水(40mL)，用2M碳酸钠溶液调节pH值至8～9。用乙酸乙酯(30mL×3)萃取，合并的有机相用饱和食盐水(20mL×2)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200～300目硅胶，乙酸乙酯:石油醚＝1:50～1:20洗脱)，得2-氨基-3-氟-5-(5-氟苯并硒唑-2-基)苯甲腈(9)(150mg)。收率为33.5％。 1H NMR(DMSO-d6，400MHz)δ8.19-8.16(m，1H)，7.99-7.94(m，2H)，7.80(dd，J＝2.4，10.0Hz，1H)，7.27-7.23(m，1H)，6.99(s，2H)。MS(EI，m/z)：336.0[M+H] +。
实施例3：2-乙炔基-4-(5-氟苯并硒唑-2-基)苯胺(12)的合成
Figure PCTCN2019115184-appb-000007
步骤A：将含有化合物5(500mg，1.35mmol)、乙酸酐(0.5mL)、吡啶(10mL)和4-二甲胺基吡啶(10mg，0.0819mmol)的混合物在90℃搅拌过夜。冷却到室温，加入水(50mL)，用乙酸乙酯(30mL×3)萃取，合并的有机相用饱和食盐水(15mL×2)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200～300目硅胶，乙酸乙酯:二氯甲烷:石油醚＝1:1:25～1:1:10洗脱)，得N-[2-溴-4-(5-氟苯并硒唑-2-基)苯基]乙酰胺(10)(500mg)。收率为89.8％。
步骤B：通过注射器将三甲基硅烷乙炔(131mg，1.33mmol)加入到含有化合物10(500mg，1.21mmol)、双三苯基磷二氯化钯(40mg，0.0570mmol)、三乙胺(40mL)和DMF(4mL)的混合物中，加完后，所得混合物在50℃搅拌过夜。减压蒸除大部分溶剂，加入水(30mL)，用乙酸乙酯(30mL×3)萃取，合并的有机相用饱和食盐水(15mL×2)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200～300目硅胶，乙酸乙酯:石油醚＝1:50～1:20洗脱)，得N-{4-(5-氟苯并硒唑-2-基)-2-[(三甲基硅烷基)乙炔基]苯基}乙酰胺(11)(211mg)。收率为40.6％。
步骤C：将含有化合物11(100mg，0.233mmol)、2M氢氧化钠溶液(10mL)、THF(5mL)和甲醇(10mL)的混合物在80℃搅拌1小时。冷却到室温，加入水(30mL)，用乙酸乙酯(30mL×3)萃取，合并的有机相用饱和食盐水(15mL×2)洗涤，无水硫酸钠干燥。减压蒸除溶剂，所得产物用石油醚/乙酸乙酯重结晶，得2-乙炔基-4-(5-氟苯并硒唑-2-基)苯胺(12)。 1H NMR(DMSO-d6，400MHz)δ8.13-8.09(m，1H)，7.82(d，J＝2.0Hz，1H)，7.77-7.73(m，2H)，7.22-7.19(m，1H)，6.81(d，J＝8.4Hz，1H)，6.20(s，2H)，4.48(s，1H)。MS(EI，m/z)：315.0[M-H] -。
实施例4：5-氟-4-(5-氟苯并硒唑-2-基)-2-甲基苯胺(16)的合成
Figure PCTCN2019115184-appb-000008
步骤A：在冰水浴下，将NBS(3.55g，19.9mmol)分批加入到5-氟-2-甲基苯胺(2.50g，20.0mmol)的DMF(20mL)溶液中，加完后，所得混合物在该温度下继续搅拌1 小时。加入水(80mL)，用乙酸乙酯(60mL×3)萃取，合并的有机相依次用饱和碳酸氢钠溶液(30mL×2)和饱和食盐水(30mL×2)洗涤，无水硫酸钠干燥。减压蒸除溶剂，得4-溴-3-氟-6-甲基苯胺(13)粗品(4.87g)。该化合物不经纯化直接用于下一步反应。
步骤B：将含有化合物13粗品(4.87g)、氰化亚铜(2.63g，29.4mmol)和NMP(15mL)的混合物在氮气下180℃搅拌7小时。加入水(75mL)，用乙酸乙酯(50mL×3)萃取，合并的有机相依次用水(30mL×2)和饱和食盐水(30mL)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200-300目硅胶，乙酸乙酯:石油醚＝1:30～1:5洗脱)，得4-氨基-2-氟-5-甲基苯甲腈(14)(1.74g)。步骤A和B两步反应总收率为58.2％。
步骤C：将含有化合物14(1.74g，11.6mmol)、1M氢氧化钠溶液(50mL)和乙醇(5mL)的混合物在回流下搅拌过夜。冷却到室温，加入水(50mL)，用甲基叔丁基醚(20mL×2)萃取，产物在水相。水相用2M盐酸调节pH值至3～4，有固体析出。过滤，滤饼干燥，得4-氨基-2-氟-5-甲基苯甲酸(15)(1.75g)。收率为89.2％。
步骤D：向化合物4(1.0g，2.64mmol)和化合物15(447mg，2.64mmol)的甲苯(30mL)溶液中加入三丁基膦(2.68g，13.2mmol)，所得混合物在氮气下回流搅拌36小时。冷却到室温，加入水(40mL)，用2M氢氧化钠溶液调节pH值至9～10。用乙酸乙酯(40mL×3)萃取，合并的有机相用饱和食盐水(25mL)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200-300目硅胶，乙酸乙酯:石油醚＝1:50～1:10洗脱)，得5-氟-4-(5-氟苯并硒唑-2-基)-2-甲基苯胺(16)(655mg)。收率为72.9％。 1H NMR(DMSO-d6，400MHz)δ8.14-8.10(m，1H)，7.90(d，J＝8.8Hz，1H)，7.78(dd，J＝2.4，10.0Hz，1H)，7.22-7.17(m，1H)，6.54-6.51(m，1H)，6.08(s，2H)，2.13(s，3H)。MS(EI，m/z)：323.0[M-H] -。
实施例5：2-溴-6-(5-氟苯并硒唑-2-基)吡啶-3-氨(18)的合成
Figure PCTCN2019115184-appb-000009
步骤A：向化合物4(1.80g，4.76mmol)和5-氨基吡啶-2-甲酸(657mg，4.76mmol)的甲苯(50mL)溶液中加入三丁基膦(4.82g，23.8mmol)，所得混合物在氮气下回流搅拌36小时。冷却到室温，加入水(40mL)，用2M氢氧化钠溶液调节pH值至9～10。用乙酸乙酯(40mL×3)萃取，合并的有机相用饱和食盐水(25mL)洗涤，无水硫酸钠 干燥。减压蒸除溶剂，产物经柱层析纯化(200-300目硅胶，乙酸乙酯:二氯甲烷:石油醚＝1:1:20～1:1:5洗脱)，得6-(5-氟苯并硒唑-2-基)吡啶-3-氨(17)(712mg)。收率为51.2％。
步骤B：在冰水浴下，将NBS(61mg，0.343mmol)分批加入到化合物17(100mg，0.342mmol)的二氯甲烷(6mL)溶液中，加完后，所得混合物在该温度下继续搅拌0.5小时。加入水(15mL)，用二氯甲烷(60mL×3)萃取，合并的有机相依次用饱和碳酸氢钠溶液(20mL)和饱和食盐水(20mL)洗涤，无水硫酸钠干燥。减压蒸除溶剂，产物经柱层析纯化(200-300目硅胶，THF:二氯甲烷:石油醚＝1:1:20～1:1:10洗脱)，得2-溴-6-(5-氟苯并硒唑-2-基)吡啶-3-氨(18)。 1H NMR(DMSO-d6，400MHz)δ8.15-8.11(m，1H)，7.98(d，J＝8.4Hz，1H)，7.80(dd，J＝2.4，10.0Hz，1H)，7.26-7.19(m，2H)，6.37(s，2H)。MS(EI，m/z)：369.9[M-H] -。
实施例6：3-氨基-6-(5-氟苯并硒唑-2-基)-2-甲酰胺基吡啶(19)的合成
Figure PCTCN2019115184-appb-000010
以化合物18为起始原料，合成化合物19的实验操作按照实施例1中步骤F的方法制备(由于本批次化合物18没有经过柱层析纯化，后处理中有少许碳酸氢钠以及水没有除干净，以致在本反应中生成了化合物19)。 1H NMR(DMSO-d6，400MHz)δ8.16-8.12(m，1H)，8.08(d，J＝8.8Hz，1H)，7.82(dd，J＝2.4，10.0Hz，1H)，7.76(s，1H)，7.65(s，1H)，7.54(s，2H)，7.30(d，J＝8.8Hz，1H)，7.26-7.21(m，1H)。MS(EI，m/z)：335.0[M-H] -。
实施例7：
化合物4-(5-氟苯并硒唑-2-基)-2-甲基苯胺(20)、2-溴-4-(5-氟苯并硒唑-2-基)-6-甲基苯胺(21)、2-溴-6-氟-4-(5-氟苯并硒唑-2-基)苯胺(22)、2,6-二氟-4-(5-氟苯并硒唑-2-基)苯胺(23)和2-氟-4-(5-氟苯并硒唑-2-基)-6-甲基苯胺(24)的合成参见专利CN201610299350.1或US10005744B2。
实施例8：化合物对人胃癌细胞株AGS、MKN-45和NCI-N87的生长抑制作用
一、实验材料名称及来源
人胃癌细胞株AGS、MKN-45和NCI-N87购于中国科学院上海生命科学研究院细胞资源中心。紫杉醇、Resazurin和亚甲基蓝购自Sigma-Aldrich Co.,LLC；铁氰化钾和亚铁 氰化钾购自阿拉丁试剂股份有限公司；DMEM培养基、1640培养基、无酚红DMEM和胎牛血清购自Thermo Fisher Scientific Inc；青霉素和链霉素购自碧云天生物技术有限公司。
二、实验方法
AGS用DMEM培养基(含10％胎牛血清，100U/mL青霉素，0.1mg/mL链霉素)，MKN-45用1640培养基(含10％胎牛血清，100U/mL青霉素，0.1mg/mL链霉素)，NCI-N87用DMEM培养基(含20％胎牛血清，100U/mL青霉素，0.1mg/mL链霉素)，三株细胞分别在37℃，5％CO 2孵箱培养至细胞密度达90％左右。
按3×10 3/孔细胞数接种于96孔板中，置37℃，5％CO 2孵箱中培养24h。
用培养基配制不同浓度梯度的试验化合物或对照药物紫杉醇，并按100μL/孔加入96孔板中，作为试验化合物孔或对照药物孔；按100μL/孔加入不含试验化合物或对照药物的培养基，作为阴性对照孔。置37℃，5％CO 2孵箱中，AGS和MKN-45细胞培养72h，NCI-N87细胞培养120h。
将Resazurin(15mg/50mL，200×)、亚甲基蓝(25mg/10mL，1000×)、铁氰化钾(0.329g/100mL，100×)和亚铁氰化钾(0.422g/100mL，100×)溶于PBS(0.1M，pH＝7.4)中，配制出10×Alamar Blue溶液，再用无酚红DMEM培养基稀释成1×Alamar Blue溶液，临用前配制。
96孔板中的细胞用PBS(0.1M，pH＝7.4)小心清洗2次，并吸尽PBS，再按100μL/孔加入1×Alamar Blue溶液；在无细胞的孔中加入100μL 1×Alamar Blue溶液，作为空白对照孔。将96孔板置37℃，5％CO 2孵箱中培养3h。
用酶标仪Victor X4(Perkin Elmer)在Ex 530/Em 590nm处检测细胞荧光值。试验化合物孔的荧光值以F (试验化合物)表示；空白对照孔的荧光值以F (空白对照)表示；阴性对照孔的荧光值以F (阴性对照)表示。按以下公式计算不同药物浓度下的细胞存活率，每个浓度设置3个重复孔，得出平均值和标准偏差。
Figure PCTCN2019115184-appb-000011
利用Prism Graph软件根据细胞存活率分别计算出试验化合物或对照药物对细胞的半数抑制浓度(IC 50)。
三、实验结果
试验结果如表1所示，化合物对人胃癌细胞株AGS、MKN-45和NCI-N87的生长均 有明显地抑制作用。其中化合物6、9、12、19、22和23的抑制效果较优。
表1.试验化合物对人胃癌细胞株AGS、MKN-45和NCI-N87的半数抑制浓度(IC 50，nM)
Figure PCTCN2019115184-appb-000012
实施例9：化合物6、22和23对人胃癌AGS和MKN-45裸鼠异种移植瘤的生长抑制活性研究
一、试验材料
1.试验动物
SPF级BALB/c裸小鼠，雌性，开始给药时为6-8周龄，体重为18-20g。由常州卡文斯实验动物有限公司提供(实验动物生产许可证：SCXK(苏)2016-0010；实验动物使用许可证：SYXK(苏)2017-0007)。所购动物批号：201820473。
2.试验试剂
聚乙二醇400(PEG400)，批号为20180412，购自成都市科龙化工试剂厂；氯化钠注射液(生理盐水)，批号为A17111105，购自河北天成药业股份有限公司；DMSO，批号为Q6949，购自MP Biomedicals。
3.细胞株
人胃癌AGS和MKN-45细胞购自中国科学院上海生命科学研究院细胞资源中心；AGS于10％胎牛血清的1640培养基中培养，MKN-45于含10％胎牛血清的DMEM培养基中培养。
二、试验方法
1.受试化合物
低剂量组(给药剂量：2.5mg/kg)：给药前精确称取2.0mg受试化合物，用静脉注射用溶剂(PEG400 25％，DMSO 2.5％，生理盐水72.5％)溶解成8mL的澄清溶液，低剂量组受试化合物的静脉注射溶液终浓度为0.25mg/mL，静脉注射给药体积为0.2mL/20g体重。
高剂量组(给药剂量：5.0mg/kg)：给药前精确称取4.0mg受试化合物，用静脉注射用溶媒(PEG400 50％，DMSO 5％，生理盐水45％)溶解至8mL的澄清溶液，高剂量组受试化合物的静脉注射溶液终浓度为0.50mg/mL，静脉注射给药体积为0.2mL/20g体重。
2.试验分组与给药方式
取对数生长期的AGS和MKN-45细胞，在无菌条件下，分别接种于各60只裸小鼠右侧腋窝皮下，细胞接种量为5×10 6个/只。用游标卡尺测量移植瘤直径，待肿瘤生长至100mm 3左右时，分别挑选生长状态良好且肿瘤大小均一性较好的AGS和MKN-45荷瘤裸鼠各56只，随机分成7组，每组8只，即模型组，化合物6、22和23低剂量组及高剂量组。各组均静脉注射给药，分别于0、1、2、3、4天每天连续给药一次，共给药5天，模型组给予等容体积的溶媒对照。用测量瘤径的方法，动态观察受试化合物的抗肿瘤效应。每两天测量一次肿瘤直径，同时称重。第20天时各组脱颈处死裸鼠，手术剥取并准确称重瘤块。
肿瘤体积(tumor volume，TV)的计算公式为：
Figure PCTCN2019115184-appb-000013
其中a、b分别表示长、宽。
抑瘤率％的计算公式为：
Figure PCTCN2019115184-appb-000014
三、试验结果
试验结果如表2、表3、图1至图6所示，受试化合物6、22和23在分别连续5天以2.5mg/kg和5.0mg/kg的给药剂量尾静脉注射后，可显著地抑制胃癌细胞AGS和MKN-45裸鼠移植瘤的肿瘤生长。
表2.化合物对人胃癌细胞株AGS裸鼠异种移植肿瘤生长的抑制作用(Mean±SD，n＝8)
Figure PCTCN2019115184-appb-000015
表3.化合物对人胃癌细胞株MNK-45裸鼠异种移植肿瘤生长的抑制作用(Mean±SD，n＝8)
Figure PCTCN2019115184-appb-000016
实施例10：化合物对人结肠癌细胞株HCT116、HCT15、HT29、SW620和COLO205 的生长抑制作用
一、实验材料名称及来源
人结肠癌细胞株HCT116、HCT15、HT29、SW620和COLO205购于中国科学院上海生命科学研究院细胞资源中心。紫杉醇、Resazurin和亚甲基蓝购自Sigma-Aldrich Co.,LLC；铁氰化钾和亚铁氰化钾购自阿拉丁试剂股份有限公司；DMEM培养基、无酚红DMEM和胎牛血清购自Thermo Fisher Scientific Inc；青霉素和链霉素购自碧云天生物技术有限公司。
二、实验方法
HCT116、HCT15、HT29、SW620和COLO205细胞用DMEM培养基(含10％胎牛血清，100U/mL青霉素，0.1mg/mL链霉素)，在37℃，5％CO 2孵箱培养至细胞密度达90％左右。
按3×10 3/孔细胞数接种于96孔板中，置37℃，5％CO 2孵箱中培养24h。
用培养基配制成不同浓度梯度的试验化合物或对照药物紫杉醇，并按100μL/孔加入96孔板中，作为试验化合物孔或对照药物孔；按100μL/孔加入不含试验化合物或对照药物的培养基，作为阴性对照孔。置37℃，5％CO 2孵箱中，培养72h。
将Resazurin(15mg/50mL，200×)、亚甲基蓝(25mg/10mL，1000×)、铁氰化钾(0.329g/100mL，100×)和亚铁氰化钾(0.422g/100mL，100×)溶于PBS(0.1M，pH＝7.4)中，配制出10×Alamar Blue溶液，再用无酚红DMEM培养基稀释成1×Alamar Blue溶液，临用前配制。
96孔板中的细胞用PBS(0.1M，pH＝7.4)小心清洗2次，并吸尽PBS，再按100μL/孔加入1×Alamar Blue溶液；在无细胞的孔中加入100μL 1×Alamar Blue溶液，作为空白对照孔。将96孔板置37℃，5％CO 2孵箱中培养3h。
用酶标仪Victor X4(Perkin Elmer)在Ex 530/Em 590nm处检测细胞荧光值。试验化合物孔的荧光值以F (试验化合物)表示；空白对照孔的荧光值以F (空白对照)表示；阴性对照孔的荧光值以F (阴性对照)表示。按以下公式计算不同药物浓度下的细胞存活率，每个浓度设置3个重复孔，得出平均值和标准偏差。
Figure PCTCN2019115184-appb-000017
根据细胞存活率利用Prism Graph软件分别计算出试验化合物或对照药物对细胞的半 数抑制浓度(IC 50)。
三、实验结果
试验结果如表4所示，化合物对人结肠癌细胞株HCT116、HCT15、HT29和COLO205的生长均有明显地抑制作用。其中化合物9、19、22和23的抑制效果较优。
表4.试验化合物对人结肠癌细胞株HCT116、HCT15、HT29、SW620和COLO205的半数抑制浓度(IC 50，nM)
Figure PCTCN2019115184-appb-000018
实施例11：化合物6、22和23对人结肠癌HT29和HCT116裸鼠异种移植瘤的生长抑制活性研究
一、试验材料
1.试验动物
SPF级BALB/c裸小鼠，雌性，开始给药时为6-8周龄，体重为18-20g。由常州卡文斯实验动物有限公司提供(实验动物生产许可证：SCXK(苏)2016-0010；实验动物使用许可证：SYXK(苏)2017-0007)。所购动物合格证编号：201822212。
2.试验试剂
聚乙二醇400(PEG400)，批号为20180412，购自成都市科龙化工试剂厂；氯化钠注射液(生理盐水)，批号为A17111105，购自河北天成药业股份有限公司；DMSO，批号为Q6949，购自MP Biomedicals。
3.细胞株
人结肠癌HCT116和HT29细胞购自中国科学院上海生命科学研究院细胞资源中心； HCT116于含10％胎牛血清的1640培养基中培养，HT29于含10％胎牛血清的1640培养基中培养。
二、试验方法
1.受试化合物的制剂配制
低剂量组(给药剂量：4.0mg/kg)：给药前称取3.2mg受试化合物，用溶剂溶至8ml(PEG400 50％，DMSO 5％，生理盐水45％)，溶液浓度为0.4mg/ml，静脉注射给药，给药体积为0.2ml/20g体重。
高剂量组(给药剂量：8.0mg/kg)：给药前称取6.4mg受试化合物，用溶剂溶至8ml(PEG400 50％，DMSO 5％，生理盐水45％)，溶液浓度为0.8mg/ml，静脉注射给药，给药体积为0.2ml/20g体重。
2.试验分组与给药方式
取对数生长期的HCT116和HT29细胞，在无菌条件下，分别接种于各60只裸小鼠右侧腋窝皮下，细胞接种量为5×10 6个/只。用游标卡尺测量移植瘤直径，待肿瘤生长至100mm 3左右时，分别挑选生长状态良好且肿瘤大小均一性较好的HCT116和HT29荷瘤裸鼠各56只，随机分成7组，每组8只，即模型组，化合物6、20和21低剂量组及高剂量组。各组均尾静脉注射给药，分别于0、1、2、3、4天连续每天给药一次，共给药5天，模型组给予等容量的溶媒对照。用测量瘤径的方法，动态观察受试化合物的抗肿瘤效应。每两天测量一次肿瘤直径，同时称重。第20天时各组脱颈处死裸鼠，手术剥取并准确称重瘤块。
肿瘤体积(TV)的计算公式为：
Figure PCTCN2019115184-appb-000019
其中a、b分别表示长、宽。
抑瘤率％的计算公式为：
Figure PCTCN2019115184-appb-000020
三、试验结果
试验结果如表5、表6、图7至图12所示，化合物6、22和23在分别连续5天以4.0mg/kg和8.0mg/kg的给药剂量尾静脉注射后，可显著地抑制结肠癌细胞HT29和HCT116 裸鼠移植瘤的肿瘤生长。
表5.化合物对人结肠癌细胞株HT29裸鼠异种移植肿瘤生长的抑制作用(Mean±SD，n＝8)
Figure PCTCN2019115184-appb-000021
表6.化合物对人结肠癌细胞株HCT116裸鼠异种移植肿瘤生长的抑制作用(Mean±SD，n＝8)
Figure PCTCN2019115184-appb-000022

Claims (10)

  1. 通式(I)所示的化合物或其药学上可接受的盐在制备治疗或预防胃癌或肠癌药物方面的应用，
    Figure PCTCN2019115184-appb-100001
    其中，
    R 1和R 2分别独立地选自H、D、卤素、-CN、C 1-3烷基、取代的C 1-3烷基、C 1-3烷氧基或取代的C 1-3烷氧基；
    R 3选自H、卤素、-OH、-CN、-C(＝O)NH 2、取代的-C(＝O)NH 2、C 1-3烷基、取代的C 1-3烷基、C 2-3烯基、C 2-3炔基、C 1-3烷氧基或取代的C 1-3烷氧基；
    R 4选自H、D、卤素、-OH、-CN、-NH 2、取代的-NH 2、-C(＝O)NH 2、取代的-C(＝O)NH 2、C 1-3烷基、取代的C 1-3烷基、C 2-3烯基、C 2-3炔基、C 1-3烷氧基或取代的C 1-3烷氧基中的一种或两种；
    n＝1或2；
    R 5选自H、-CN、-OH、C 1-3烷基、C 1-3烷氧基或氨基酸残基；
    Z选自CH或N；
    R 1、R 2、R 3或R 4中的取代基选自D、卤素、OH、C 1-3烷基或C 1-3烷氧基。
  2. 根据权利要求1所述的应用，其中，R 1和R 2分别独立地选自H、D、F、Cl、-CN、-CH 3、-CF 3、-OCF 3或-OCHF 2。
  3. 根据权利要求2所述的应用，其中，R 1选自D、F、Cl、-CN、-CH 3或-CF 3，R 2选自H。
  4. 根据权利要求1所述的应用，其中，R 3选自H、F、Cl、Br、-CN、-C(＝O)NH 2、-CH 3、-CH 2CH 3、-CF 3、-CH＝CH 2、-C≡CH、-OCHF 2或-OCF 3。
  5. 根据权利要求1所述的应用，其中，R 4选自H、D、卤素、-CN、C 1-3烷基、取代的C 1-3烷基、C 1-3烷氧基或取代的C 1-3烷氧基中的一种或两种。
  6. 根据权利要求1所述的应用，其中，R 4选自H、D、F、Cl、Br、I、-CN、-CH 3、-CF 3、-OCHF 2或-OCF 3；R 5选自H或氨基酸残基。
  7. 根据权利要求1所述的应用，其中，R 5选自H或2,6-二氨基-己酰基。
  8. 根据权利要求1所述的应用，其中，所述化合物选自：
    2-氨基-5-(5-氟苯并硒唑-2-基)苯甲腈，
    2-氨基-3-氟-5-(5-氟苯并硒唑-2-基)苯甲腈，
    2-乙炔基-4-(5-氟苯并硒唑-2-基)苯胺，
    5-氟-4-(5-氟苯并硒唑-2-基)-2-甲基苯胺，
    2-溴-6-(5-氟苯并硒唑-2-基)吡啶-3-氨，
    3-氨基-6-(5-氟苯并硒唑-2-基)-2-甲酰胺基吡啶，
    4-(5-氟苯并硒唑-2-基)-2-甲基苯胺，
    2-溴-4-(5-氟苯并硒唑-2-基)-6-甲基苯胺，
    2-溴-6-氟-4-(5-氟苯并硒唑-2-基)苯胺，
    2,6-二氟-4-(5-氟苯并硒唑-2-基)苯胺，
    2-氟-4-(5-氟苯并硒唑-2-基)-6-甲基苯胺。
  9. 根据权利要求1所述的应用，其中以权利要求1所述的化合物、药学上可接受的盐或其溶剂合物作为活性成分或主要活性成分，辅以药学上可接受的辅料制成药物组合物。
  10. 一种治疗人类胃癌或肠癌的方法，其特征在于对患有胃癌或肠癌的人施以每次剂量0.1-1000mg的权利要求1所述的化合物、药学上可接受的盐或其溶剂合物，或者施以每次剂量0.1-1000mg的权利要求9所述的药物组合物。
PCT/CN2019/115184 2018-11-12 2019-11-02 4-(苯并硒唑-2-基)芳胺类化合物治疗胃癌或肠癌的用途 WO2020098517A1 (zh)

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