WO2021190076A1 - Anti-tumor compound capable of overcoming cisplatin resistance, preparation therefor, and application thereof - Google Patents

Anti-tumor compound capable of overcoming cisplatin resistance, preparation therefor, and application thereof Download PDF

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WO2021190076A1
WO2021190076A1 PCT/CN2021/070303 CN2021070303W WO2021190076A1 WO 2021190076 A1 WO2021190076 A1 WO 2021190076A1 CN 2021070303 W CN2021070303 W CN 2021070303W WO 2021190076 A1 WO2021190076 A1 WO 2021190076A1
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compound
formula
tumor
cisplatin
reaction
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苟少华
王园江
王心怡
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东南大学
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    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System

Definitions

  • the present invention relates to an anti-tumor compound that effectively overcomes cisplatin resistance, and specifically relates to bonding a compound having protein kinase 2 (CK2) inhibitory activity to a platinum (II) compound known to have anti-tumor activity to obtain an anti-tumor compound that can effectively overcome cisplatin
  • CK2 protein kinase 2
  • II platinum
  • a new type of platinum-resistant anti-tumor compound also relates to a preparation method and application of this type of compound.
  • platinum drugs such as cisplatin, carboplatin and oxaliplatin have been on the market, and they have been widely used in clinical practice.
  • platinum (II) compounds bind to the guanine N7 target on the DNA of cancer cells to form DNA cross-linked adducts, which restrict DNA replication and inhibit cancer cells.
  • platinum (II) compounds bind to the guanine N7 target on the DNA of cancer cells to form DNA cross-linked adducts, which restrict DNA replication and inhibit cancer cells.
  • Cisplatin has the best comprehensive effect among all currently known platinum anti-tumor drugs. It not only has strong anti-tumor activity, but also has broad-spectrum anti-tumor activity.
  • cisplatin can be used as a treatment for head and neck cancer, ovarian cancer, esophageal cancer, First-line drugs for cancers such as bladder cancer and lung cancer.
  • cisplatin also has obvious defects in clinical application.
  • Studies have found that there is a significant DNA repair phenomenon in drug-resistant cancer cells. This is because cancer cells can activate their own DNA damage repair mechanism to repair, thereby developing resistance to DNA damage drugs and reducing the anti-tumor effect of cisplatin drugs. Effect. Therefore, DNA damage repair is considered to be an important factor affecting the efficacy of chemotherapy that targets DNA.
  • Casein kinase II (CK2) is a pleiotropic and highly conserved second messenger-independent serine/threonine protein kinase, which is widely present in the cytoplasm and nucleus of eukaryotic cells.
  • CK2 has a very important role in a variety of cell activities.
  • CK2 involved in cell G 1 / S phase, adjusting the G 2 / M phase, over-expression of CK2 can inhibit programmed cell death induced by anti-cancer drugs.
  • CK2 overexpression exists in a variety of drug-resistant cancer cell lines, and inhibition of CK2 can promote cell apoptosis and increase the sensitivity of tumor cells to anticancer drugs and apoptosis stimuli.
  • the technical problem to be solved by the present invention is to use compounds that inhibit protein kinase 2 activity to inhibit DNA damage and repair.
  • Group of platinum (II) compounds combined, design and synthesize a new platinum (II) compound to obtain an anti-tumor drug that overcomes cisplatin resistance and has high efficiency and low toxicity, and provides the preparation method of this compound and its use in anti-tumor Applications.
  • the anti-tumor compound capable of overcoming cisplatin resistance according to the present invention has a structure such as compound 1 or compound 2 shown in formula I.
  • reaction formula II For the preparation of the reaction formula shown in formula II, the specific steps are as follows: add equimolar amounts of compound 3 and compound 4 in anhydrous methanol into a reaction flask, add acetic acid dropwise, and react the reaction solution at 30-60°C in the dark After 48-72 hours, it was cooled to room temperature, filtered, and the filter cake was washed three times with anhydrous methanol to obtain compound 1 as a yellow solid product.
  • reaction formula shown in formula III is prepared, and the specific steps are as follows: add equimolar amounts of compound 3 and compound 5 in anhydrous methanol into the reaction flask, add acetic acid dropwise, and react the reaction solution at 30-60°C in the dark After 48-72 hours, it was cooled to room temperature, filtered, and the filter cake was washed three times with anhydrous methanol to obtain compound 2 as a yellow solid product.
  • reaction formula shown in formula IV the specific steps are as follows: add 1 equivalent of compound 6 into a reaction flask containing methanol, slowly add 2-10 equivalents of hydrazine hydrate at room temperature, and heat the reaction solution to reflux for 24-72 hours After the reaction was completed, it was cooled to room temperature, a large amount of solid was precipitated, and the filter cake was washed three times with ice methanol to obtain a bright yellow solid product—compound 3.
  • Compounds 4 and 5 shown in formula II and formula III are known platinum (II) compounds with good anti-tumor activity, prepared according to the method disclosed in the inventor's previous patent (invention patent number: ZL201210422936.4; US9227991B2; EP2913335; Japanese Patent No.
  • Compound 6 shown in Formula IV and known antitumor drugs targeting CK2 were prepared according to the methods reported in the literature (J.Med.Chem., 2011,54,635- 54), specifically using 3-bromoisonicotinic acid as the starting material, after esterification, Suzuki coupling reaction with 2-amino-4-carbomethoxyphenyl borate to obtain intermediate 5,6 -Dihydrobenzo[c][2,6]naphthyridine-8-methyl carboxylate, then undergoes chlorination reaction with phosphorus oxychloride, and then undergoes substitution reaction with meta-chloroaniline to obtain compound 6, which is then hydrolyzed Get CX-4945.
  • the reaction route is shown in formula V:
  • the prepared compounds 1, 2 and 3 were used to treat human laryngeal cancer cells Hep2, nasopharyngeal cancer cells CNE2, gastric cancer cells SGC-7901, colon cancer cells HCT-116, breast cancer cells MCF-7, cervical cancer cells SiHa, and prostate cancer.
  • CX-4945, cisplatin, and compounds 4 and 5 were used as positive controls.
  • Compound 1 has better activity than CX-4945 and its parent compound 3 in various cancer cell lines tested, especially cancer cell lines with high CK2 expression such as T24, PC-3 and HCT-116, etc., and It is comparable to its other parent compound 4, but the toxicity of compound 1 to normal hepatocytes is lower than that of the positive control drug. It is worth noting that although compound 1 has a slightly lower inhibitory effect on cisplatin-sensitive cancer cells A549 and A2780 than cisplatin, it has a significant inhibitory effect on cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP.
  • the activity is significantly better than that of cisplatin, and the drug resistance factor is as low as 0.71 (the ratio of the IC 50 value of the compound against cisplatin-resistant cancer cells to the IC 50 value of cisplatin-sensitive cancer cells).
  • compound 2 has a slightly lower IC 50 value for A549 cancer cells than compound 1, its effect on drug-resistant cancer cells A549/CDDP is significantly weaker than compound 1, with a drug resistance factor of 1.52.
  • compound 1 and its parent compound 3 have better CK2 inhibitory activity than CX-4945, and compound 1 also shows a better inhibitory effect on cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP, compound 1 was tested follow up research.
  • a nude mouse xenograft tumor model of cisplatin-sensitive ovarian cancer cell A2780 and cisplatin-resistant ovarian cancer cell A2780/CDDP was used to test the anti-tumor activity of compound 1 in vivo, and cisplatin was used as a positive control.
  • the compound of the present invention has a good inhibitory effect on cancer cells, especially cisplatin-resistant cancer cells.
  • the in vivo test results show that compound 1 not only has a strong inhibitory effect on cisplatin-sensitive tumors but also cisplatin-resistant tumors, and has low toxicity, high efficiency and low toxicity, and can be used to prepare antitumor drugs.
  • Figure 1a shows the expression of CK2 in lung cancer cell A549 and cisplatin-resistant lung cancer cell A549/CDDP, ovarian cancer cell A2780 and cisplatin-resistant ovarian cancer cell A2780/CDDP;
  • Figure 1b is the quantitative value of CK2 expression in lung cancer cell A549 and cisplatin-resistant lung cancer cell A549/CDDP, ovarian cancer cell A2780 and cisplatin-resistant ovarian cancer cell A2780/CDDP.
  • Figure 2a is the expression of DNA damage repair related proteins in lung cancer cell A549 by the test sample
  • Figure 2b shows the expression of DNA damage repair related proteins in the cisplatin-resistant lung cancer cell A549/CDDP of the tested sample
  • Figure 2c is the expression of the test sample on DNA damage repair related proteins in ovarian cancer cell A2780;
  • Figure 2d shows the expression of DNA damage repair related proteins in the cisplatin-resistant ovarian cancer cell A2780/CDDP of the tested sample.
  • Figure 3 The effect of the test sample on the growth volume of cisplatin-sensitive ovarian cancer cell A2780 nude mice xenograft tumors.
  • Figure 4 The effect of the test sample on the tumor weight of cisplatin-sensitive ovarian cancer cell A2780 in nude mice xenograft.
  • Figure 5 The effect of the test sample on the weight of nude mice with cisplatin-sensitive ovarian cancer cell A2780 xenograft tumors in nude mice.
  • Figure 6 The effect of test samples on the growth volume of cisplatin-resistant ovarian cancer cell A2780/CDDP xenograft tumors in nude mice.
  • Figure 7 The effect of the tested sample on the tumor weight of cisplatin-resistant ovarian cancer cell A2780/CDDPP xenograft in nude mice.
  • Figure 8 The effect of test samples on the body weight of nude mice with cisplatin-resistant ovarian cancer cell A2780/CDDP xenograft tumors.
  • An anti-tumor compound capable of overcoming cisplatin resistance of the present invention has a structure such as compound 1 or compound 2 shown in formula I.
  • compound 6 and CX-4945 reference (J. Med. Chem. 2011, 54, 635-654) are prepared by the method reported and verified by the hydrogen nuclear magnetic spectrum.
  • CK2 enzyme-linked immunoassay (ELISA) kit was used for detection.
  • the standard protein was diluted to a suitable concentration, then added to the microtiter plate coated with purified CK2 capture antibody, and then added to the test compound diluted to a suitable concentration gradient, and a control group without added drug was set for comparison. Put it in the incubator and incubate for 1 hour to make the drug effectively inhibit the binding of protein and antibody, discard the liquid in the microplate, add 200 ⁇ L of the diluted washing solution, leave it for 30s, discard it and repeat the washing 5 times.
  • ELISA enzyme-linked immunoassay
  • the cytotoxic activity test of the compound prepared by the present invention was carried out using the MTT method. Take the logarithmic phase cell count, were seeded in 96-well culture plate, each well of about 104 cells, at 37 °C, 5% CO 2 incubator for 24h to adherent cell administration, administration group are located And the control group without administration. The control group was replaced with fresh medium, the tested compounds 4 and 5 and cisplatin were dissolved in 5% glucose aqueous solution, and other compounds were prepared into mother liquor with DMSO, and the corresponding cell culture medium was diluted to a suitable concentration gradient before use. For the drug group, 3 parallel groups are set for each concentration.
  • the prepared compound was used to treat human laryngeal cancer cells Hep2, nasopharyngeal cancer cells CNE2, gastric cancer cells SGC-7901, colon cancer cells HCT-116, breast cancer cells MCF-7, cervical cancer cells SiHa, and prostate cancer cells.
  • PC-3, bladder cancer cell T24, non-small cell lung cancer A549 and its cisplatin-resistant cancer cell A549/CDDP, ovarian cancer cell A2780, cisplatin-resistant cancer cell A2780/CDDP, and normal liver cells LO2 were anti-tumor in vitro
  • CX-4945 was used as a positive control. Observe the compound's inhibition of tumor cell growth at different concentrations, calculate its IC 50 value to evaluate the cytotoxic activity of the drug, and the results are shown in Table 2.
  • Western Blot test was used to detect the compounds involved in the present invention. Taking the logarithmic growth phase cells were counted, seeded in 6-well culture plate, each well of about 10 5 cells. The cells were cultured at 37°C in a 5% CO 2 incubator for 24 hours until the cells adhered to the wall and then administered. The administration group and the non-administered control group were set up respectively. The control group was replaced with fresh medium, the compound 4 to be tested was dissolved in 5% glucose aqueous solution, and the other compounds were prepared into mother liquor with DMSO, and diluted with the corresponding cell culture medium to an appropriate concentration before use and added to the administration group.
  • the cells in the wells were digested, collected by centrifugation into a centrifuge tube and washed with PBS, an appropriate amount of lysate was added dropwise to the cells, and the cells were lysed in an ice box for 2 hours. Centrifuge at 10000rpm for 20-30min, and take the supernatant for later use. The protein content was determined with BCA protein kit (Thermo, Waltham, MA).
  • Tested animal BALB/c(nu/nu) nude mouse, weighing 16 ⁇ 18g, female, purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., raised in an SPF breeding environment, the indoor temperature is controlled at 23 ⁇ 2 °C, free eating and drinking.
  • the animals were bred adaptively for 7 days before inoculation.
  • Drugs and reagents cisplatin was dissolved in 5% glucose injection by ultrasonic, compound 1 was dissolved in DMF, and then diluted with Tween80 and 5% glucose injection in sequence.
  • the groups and dosage regimen are as follows. Model control group: 0.1 mL of 5% glucose injection was injected into the tail vein once a week for 4 consecutive weeks.
  • Experimental method Take A2780 or A2780/CDDP cells in logarithmic growth phase, prepare a cell suspension concentration of 5 ⁇ 10 7 cell/mL, inoculate 0.2 mL/mouse under the right armpit of nude mice to establish A2780 or A2780/CDDP nude mice transplanted tumors
  • the diameter of the transplanted tumor in nude mice was measured with a vernier caliper. After the tumor grew to 100mm 3, the animals were randomly divided into groups. Use the method of measuring tumor diameter to dynamically observe the anti-tumor effect of the test substance. The number of measurements of tumor diameter was once every 3 days, and the mouse body weight was checked every 3 days.
  • mice After 4 weeks, all mice were sacrificed by injection of chloral hydrate, and observed by laparotomy. The tumor was removed and irrelevant tissues were carefully removed. The blood was washed with D-Hanks solution for 2 to 3 times, the blood was washed away, and the water was drained for storage. .
  • the relative tumor volume (RTV) is calculated according to the measurement results.
  • Evaluation index of anti-tumor activity relative tumor proliferation rate T/C (%), the calculation formula is as follows:
  • TRTV treatment group RTV
  • C RTV model group RTV.
  • the mean value is represented by X ⁇ SD, the analysis between groups is statistically processed by t-test, and the results are statistically analyzed by SPSS (Staffstical Package for the Social Science) 17.0.

Abstract

Anti-tumor compounds 1 and 2 capable of overcoming cisplatin resistance, preparation therefor, and applications thereof. The compounds have activities of inhibiting protein kinase 2 (casein kinase, CK2) and so can inhibit DNA damage repair, and have strong inhibitory effects on a cisplatin-sensitive tumor and a cisplatin-resistant tumor, and reduced toxicity.

Description

一种具有克服顺铂耐药的抗肿瘤化合物及其制备与应用An anti-tumor compound capable of overcoming cisplatin resistance and its preparation and application 技术领域Technical field
本发明涉及有效克服顺铂耐药的抗肿瘤化合物，具体涉及将具有蛋白激酶2(CK2)抑制活性的化合物键合到已知具有抗肿瘤活性的铂(II)化合物中，得到能够有效克服顺铂耐药的新型抗肿瘤化合物；本发明还涉及该类化合物的制备方法及应用。The present invention relates to an anti-tumor compound that effectively overcomes cisplatin resistance, and specifically relates to bonding a compound having protein kinase 2 (CK2) inhibitory activity to a platinum (II) compound known to have anti-tumor activity to obtain an anti-tumor compound that can effectively overcome cisplatin A new type of platinum-resistant anti-tumor compound; the present invention also relates to a preparation method and application of this type of compound.
背景技术Background technique
自20世纪60年代发现顺铂具有抗肿瘤活性以来，已有顺铂、卡铂和奥沙利铂等铂类药物上市，它们被广泛应用于临床。经过大量研究，一般认为顺铂类抗肿瘤药物的作用机制是铂(II)化合物与癌细胞DNA上的鸟嘌呤N7位靶点结合，形成DNA交联加合物，限制DNA复制从而抑制癌细胞的增殖。顺铂在目前所有已知的铂类抗肿瘤药物中综合效果是最好的，不仅抗肿瘤活性强，而且具有广谱的抗肿瘤活性，可作为治疗头颈部癌、卵巢癌、食道癌、膀胱癌和肺癌等癌症的一线药物。然而，顺铂在临床应用中也出现了明显的缺陷，一是具有较强的毒副作用，如肾毒性、神经毒性、耳毒性和胃肠道毒性等，限制了顺铂的给药剂量；二是在***过程中易产生耐药，降低药物的抗肿瘤效果。研究发现，在耐药癌细胞中存在着显著的DNA修复现象，这是因为癌细胞可以激活自身的DNA损伤修复机制进行修复，从而对DNA损伤药物产生耐药，降低顺铂类药物的抗肿瘤效果。因此DNA损伤修复被认为是影响以DNA为作用靶点化疗疗效的重要因素。Since the discovery of cisplatin with anti-tumor activity in the 1960s, platinum drugs such as cisplatin, carboplatin and oxaliplatin have been on the market, and they have been widely used in clinical practice. After a lot of research, it is generally believed that the mechanism of action of cisplatin anti-tumor drugs is that platinum (II) compounds bind to the guanine N7 target on the DNA of cancer cells to form DNA cross-linked adducts, which restrict DNA replication and inhibit cancer cells. Of proliferation. Cisplatin has the best comprehensive effect among all currently known platinum anti-tumor drugs. It not only has strong anti-tumor activity, but also has broad-spectrum anti-tumor activity. It can be used as a treatment for head and neck cancer, ovarian cancer, esophageal cancer, First-line drugs for cancers such as bladder cancer and lung cancer. However, cisplatin also has obvious defects in clinical application. First, it has strong side effects, such as nephrotoxicity, neurotoxicity, ototoxicity and gastrointestinal toxicity, which limit the dosage of cisplatin; It is easy to produce drug resistance in the process of treating tumors and reduce the anti-tumor effect of drugs. Studies have found that there is a significant DNA repair phenomenon in drug-resistant cancer cells. This is because cancer cells can activate their own DNA damage repair mechanism to repair, thereby developing resistance to DNA damage drugs and reducing the anti-tumor effect of cisplatin drugs. Effect. Therefore, DNA damage repair is considered to be an important factor affecting the efficacy of chemotherapy that targets DNA.
酪蛋白激酶2(casein kinase Ⅱ，CK2)，是一种多效且高度保守的第二信使非依赖性丝氨酸/苏氨酸蛋白激酶，广泛存在于真核细胞的细胞质及细胞核中。大量证据表明CK2在多种细胞活动中具有非常重要的作用。CK2的磷酸化底物超过300种，在细胞生长、增殖、凋亡和癌变的过程中发挥重要作用。它调节多条抗凋亡通路及前导信号级联，包括PI3K/AKT信号通路，Wnt信号级联，NF-κB转录和DNA损伤应答。研究表明CK2参与细胞G 1/S期、G 2/M期的调节，过表达的CK2可以抑制由抗癌药物引起的程序性死亡。实验证明，多种耐药癌细胞 系均存在CK2过度表达的现象，而抑制CK2能够促进细胞凋亡并增加肿瘤细胞对抗癌药物和凋亡刺激的敏感度。 Casein kinase Ⅱ (CK2) is a pleiotropic and highly conserved second messenger-independent serine/threonine protein kinase, which is widely present in the cytoplasm and nucleus of eukaryotic cells. A large amount of evidence shows that CK2 has a very important role in a variety of cell activities. There are more than 300 phosphorylated substrates of CK2, which play an important role in the process of cell growth, proliferation, apoptosis and carcinogenesis. It regulates multiple anti-apoptotic pathways and leading signal cascades, including PI3K/AKT signaling pathway, Wnt signaling cascade, NF-κB transcription and DNA damage response. Studies have shown that CK2 involved in cell G 1 / S phase, adjusting the G 2 / M phase, over-expression of CK2 can inhibit programmed cell death induced by anti-cancer drugs. Experiments have shown that CK2 overexpression exists in a variety of drug-resistant cancer cell lines, and inhibition of CK2 can promote cell apoptosis and increase the sensitivity of tumor cells to anticancer drugs and apoptosis stimuli.
综上，在顺铂耐药癌细胞中存在着显著的DNA修复现象，而耐药癌细胞中CK2的过表达是造成顺铂耐药的主要原因之一。因此将具有抑制CK2活性的结构单元引入到具有抗肿瘤活性的铂(II)化合物中，借助其对耐药癌细胞中DNA损伤修复的抑制，克服顺铂药物耐药并降低毒性是十分有益的。In summary, there is a significant DNA repair phenomenon in cisplatin-resistant cancer cells, and the overexpression of CK2 in drug-resistant cancer cells is one of the main reasons for cisplatin resistance. Therefore, it is very beneficial to introduce the structural unit with the activity of inhibiting CK2 into the platinum(II) compound with anti-tumor activity, with the help of its inhibition of DNA damage repair in drug-resistant cancer cells, to overcome the drug resistance of cisplatin and reduce toxicity. .
发明内容Summary of the invention
技术问题：鉴于顺铂在临床应用中存在严重的毒性和耐药性，本发明所要解决的技术问题在于利用具有抑制蛋白激酶2活性的化合物能够抑制DNA损伤修复的特性，与已知含有功能基团的铂(II)化合物结合,设计合成一种新的铂(II)化合物，获得克服顺铂耐药且高效低毒的抗肿瘤药物，并且提供这种化合物的制备方法及其在抗肿瘤中的应用。Technical problem: In view of the serious toxicity and drug resistance of cisplatin in clinical applications, the technical problem to be solved by the present invention is to use compounds that inhibit protein kinase 2 activity to inhibit DNA damage and repair. Group of platinum (II) compounds combined, design and synthesize a new platinum (II) compound to obtain an anti-tumor drug that overcomes cisplatin resistance and has high efficiency and low toxicity, and provides the preparation method of this compound and its use in anti-tumor Applications.
技术方案：本发明所述的具有克服顺铂耐药的抗肿瘤化合物，其结构如式Ⅰ所示的化合物1或化合物2，Technical solution: The anti-tumor compound capable of overcoming cisplatin resistance according to the present invention has a structure such as compound 1 or compound 2 shown in formula I.
Figure PCTCN2021070303-appb-000001
Figure PCTCN2021070303-appb-000001
式I中的化合物1由式II所示的反应式制备， Compound 1 in formula I is prepared by the reaction formula shown in formula II,
Figure PCTCN2021070303-appb-000002
Figure PCTCN2021070303-appb-000002
式II所示的反应式制备，具体步骤如下：在反应瓶中加入将等摩尔量的化 合物3与化合物4悬浮于无水甲醇中，滴加乙酸，反应液在30-60℃下避光反应48-72小时，然后冷至室温，过滤，滤饼用无水甲醇冲洗三次，得黄色固体产物-化合物1。For the preparation of the reaction formula shown in formula II, the specific steps are as follows: add equimolar amounts of compound 3 and compound 4 in anhydrous methanol into a reaction flask, add acetic acid dropwise, and react the reaction solution at 30-60°C in the dark After 48-72 hours, it was cooled to room temperature, filtered, and the filter cake was washed three times with anhydrous methanol to obtain compound 1 as a yellow solid product.
式I中的化合物2由式III所示的反应式制备，Compound 2 in formula I is prepared by the reaction formula shown in formula III,
Figure PCTCN2021070303-appb-000003
Figure PCTCN2021070303-appb-000003
式Ⅲ所示的反应式制备，具体步骤如下：在反应瓶中加入将等摩尔量的化合物3与化合物5悬浮于无水甲醇中，滴加乙酸，反应液在30-60℃下避光反应48-72小时，然后冷至室温，过滤，滤饼用无水甲醇冲洗三次，得黄色固体产物-化合物2。The reaction formula shown in formula III is prepared, and the specific steps are as follows: add equimolar amounts of compound 3 and compound 5 in anhydrous methanol into the reaction flask, add acetic acid dropwise, and react the reaction solution at 30-60°C in the dark After 48-72 hours, it was cooled to room temperature, filtered, and the filter cake was washed three times with anhydrous methanol to obtain compound 2 as a yellow solid product.
应用式II和式Ⅲ中所示的化合物3，其结构如下所示，Using compound 3 shown in formula II and formula III, its structure is shown below,
Figure PCTCN2021070303-appb-000004
Figure PCTCN2021070303-appb-000004
式II和式Ⅲ中所示的化合物3由式IV所示的反应式制备， Compound 3 shown in formula II and formula III is prepared by the reaction formula shown in formula IV,
Figure PCTCN2021070303-appb-000005
Figure PCTCN2021070303-appb-000005
按照式Ⅳ所示的反应式制备，具体步骤如下：在装有甲醇的反应瓶中加入1当量的化合物6，室温下缓慢滴加2-10当量的水合肼，反应液加热回流24-72小时至反应完全，然后冷至室温，析出大量固体，过滤，滤饼用冰甲醇冲洗三次， 得亮黄色固体产物-化合物3。According to the reaction formula shown in formula IV, the specific steps are as follows: add 1 equivalent of compound 6 into a reaction flask containing methanol, slowly add 2-10 equivalents of hydrazine hydrate at room temperature, and heat the reaction solution to reflux for 24-72 hours After the reaction was completed, it was cooled to room temperature, a large amount of solid was precipitated, and the filter cake was washed three times with ice methanol to obtain a bright yellow solid product—compound 3.
式II和式Ⅲ中所示的化合物4和5是已知的具有良好抗肿瘤活性的铂(II)化合物，按本发明人前期专利披露的方法制备(发明专利号：ZL201210422936.4；US9227991B2；EP2913335；日本特许第6159818号)；式IV中所示的化合物6和已知靶向CK2抗肿瘤药物(CX-4945)均按文献报道的方法制备(J.Med.Chem.,2011,54,635-54)，具体是以3-溴异烟酸为起始原料，经酯化反应后与2-氨基-4-甲酯基苯基硼酸盐酸盐发生Suzuki偶联反应得到中间体5,6-二氢苯并[c][2,6]萘啶-8-羧酸甲酯，然后与三氯氧磷发生氯代反应，再与间氯苯胺发生取代反应得到化合物6，然后将其水解得到CX-4945。反应路线如式V所示：Compounds 4 and 5 shown in formula II and formula III are known platinum (II) compounds with good anti-tumor activity, prepared according to the method disclosed in the inventor's previous patent (invention patent number: ZL201210422936.4; US9227991B2; EP2913335; Japanese Patent No. 6159818); Compound 6 shown in Formula IV and known antitumor drugs targeting CK2 (CX-4945) were prepared according to the methods reported in the literature (J.Med.Chem., 2011,54,635- 54), specifically using 3-bromoisonicotinic acid as the starting material, after esterification, Suzuki coupling reaction with 2-amino-4-carbomethoxyphenyl borate to obtain intermediate 5,6 -Dihydrobenzo[c][2,6]naphthyridine-8-methyl carboxylate, then undergoes chlorination reaction with phosphorus oxychloride, and then undergoes substitution reaction with meta-chloroaniline to obtain compound 6, which is then hydrolyzed Get CX-4945. The reaction route is shown in formula V:
Figure PCTCN2021070303-appb-000006
Figure PCTCN2021070303-appb-000006
a.EtOH,H 2SO 4,reflux,24～48h；b.Cs 2CO 3,Pd(dppf)Cl 2,N 2,100℃,30～48h；c.POCl 3,110℃,4h；d.NMP,110℃,8h；e.1.5NNaOH,70℃. a.EtOH,H 2 SO 4 ,reflux,24～48h; b.Cs 2 CO 3 ,Pd(dppf)Cl 2 ,N 2 ,100℃,30～48h; c.POCl 3 ,110℃,4h; d .NMP,110℃,8h; e.1.5NNaOH,70℃.
式VFormula V
有益效果：Beneficial effects:
(一)体外CK2活性(1) CK2 activity in vitro
采用已知CK2抑制剂CX-4945作为阳性对照，对所制备的化合物1、2和3测定了其体外对CK2的抑制活性，实验数据及结果见表1。结果表明，在CX-4945结构上引入肼基基团获得的化合物3可有效抑制CK2的活性，且活性优于其母体化合物。通过化合物3制备的化合物1和2对CK2的抑制活性相当，其中化合物1的活性还略好于CX-4945，这表明本发明所设计制备的铂(II)化合物具有有效抑制CK2的能力。Using the known CK2 inhibitor CX-4945 as a positive control, the prepared compounds 1, 2 and 3 were tested for their inhibitory activity against CK2 in vitro. The experimental data and results are shown in Table 1. The results show that the compound 3 obtained by introducing a hydrazine group into the structure of CX-4945 can effectively inhibit the activity of CK2, and the activity is better than that of its parent compound. Compounds 1 and 2 prepared by compound 3 have similar inhibitory activity on CK2, and the activity of compound 1 is slightly better than that of CX-4945, which indicates that the platinum (II) compound designed and prepared by the present invention has the ability to effectively inhibit CK2.
(二)体外细胞毒活性(2) In vitro cytotoxic activity
用所制备的化合物1、2和3对人喉癌细胞Hep2、鼻咽癌细胞CNE2、胃癌细胞SGC-7901、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、***细胞SiHa、***癌细胞PC-3、膀胱癌细胞T24、非小细胞肺癌细胞A549及其顺铂耐药癌细胞A549/CDDP、卵巢癌细胞A2780及其顺铂耐药癌细胞A2780/CDDP以及正常肝细胞LO2进行了体外细胞毒活性评价，CX-4945、顺铂和化合物4和5作为阳性对照，化合物在不同浓度下对肿瘤细胞生长抑制的IC 50数据见表2。化合物1在测试的各种癌细胞系中，尤其是对已知CK2高表达的癌细胞系如T24、PC-3和HCT-116等的活性均优于CX-4945及其母体化合物3，且与其另一母体化合物4相当，但化合物1对正常肝细胞的毒性低于阳性对照药物。值得注意的是，尽管化合物1对顺铂敏感癌细胞A549和A2780的抑制能力稍低于顺铂，但其对顺铂耐药癌细胞A549/CDDP和A2780/CDDP均有显著抑制作用，抗癌活性显著优于顺铂，耐药因子最低达到0.71(化合物对顺铂耐药癌细胞的IC 50值与对顺铂敏感癌细胞的IC 50值的比值)。与之相比，化合物2虽然对于A549癌细胞的IC 50值略低于化合物1，但其在耐药癌细胞A549/CDDP中的效果要明显弱于化合物1，耐药因子达到1.52。鉴于化合物1及其母体化合物3具有优于CX-4945抑制CK2的活性，同时化合物1对顺铂耐药癌细胞A549/CDDP和A2780/CDDP也表现出较好的抑制作用，对化合物1进行了后续研究。 The prepared compounds 1, 2 and 3 were used to treat human laryngeal cancer cells Hep2, nasopharyngeal cancer cells CNE2, gastric cancer cells SGC-7901, colon cancer cells HCT-116, breast cancer cells MCF-7, cervical cancer cells SiHa, and prostate cancer. Cell PC-3, bladder cancer cell T24, non-small cell lung cancer cell A549 and its cisplatin-resistant cancer cell A549/CDDP, ovarian cancer cell A2780 and its cisplatin-resistant cancer cell A2780/CDDP, and normal liver cells LO2 In vitro cytotoxicity evaluation, CX-4945, cisplatin, and compounds 4 and 5 were used as positive controls. The IC 50 data of the compounds on tumor cell growth inhibition at different concentrations are shown in Table 2. Compound 1 has better activity than CX-4945 and its parent compound 3 in various cancer cell lines tested, especially cancer cell lines with high CK2 expression such as T24, PC-3 and HCT-116, etc., and It is comparable to its other parent compound 4, but the toxicity of compound 1 to normal hepatocytes is lower than that of the positive control drug. It is worth noting that although compound 1 has a slightly lower inhibitory effect on cisplatin-sensitive cancer cells A549 and A2780 than cisplatin, it has a significant inhibitory effect on cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP. The activity is significantly better than that of cisplatin, and the drug resistance factor is as low as 0.71 ( the ratio of the IC 50 value of the compound against cisplatin-resistant cancer cells to the IC 50 value of cisplatin-sensitive cancer cells). In contrast, although compound 2 has a slightly lower IC 50 value for A549 cancer cells than compound 1, its effect on drug-resistant cancer cells A549/CDDP is significantly weaker than compound 1, with a drug resistance factor of 1.52. In view of the fact that compound 1 and its parent compound 3 have better CK2 inhibitory activity than CX-4945, and compound 1 also shows a better inhibitory effect on cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP, compound 1 was tested follow up research.
(三)体外诱导DNA损伤与抑制DNA修复(3) Inducing DNA damage and inhibiting DNA repair in vitro
通过Western Blot试验检测了顺铂敏感A549和A2780癌细胞与对应的顺铂耐药癌细胞A549/CDDP和A2780/CDDP中CK2的表达水平。从图1可知，与敏感癌细胞相比，耐药癌细胞中CK2的表达出现了明显的上调，分别是敏感癌细胞的1.74倍和1.96倍，这与DNA修复增强造成的癌细胞耐药性增加相吻合。进一步采用Western Blot试验考察了所设计化合物对DNA损伤修复相关蛋白表达的影响。如图2所示，在顺铂敏感癌细胞A549和A2780与顺铂耐药癌细胞A549/CDDP和A2780/CDDP中，与DNA修复相关的蛋白如PARP1的表达出现显著下调，与此同时DNA双链断裂的标志物γ-H2A.X的表达显著上调。这些结果表明，本发明所设计合成的化合物1不仅可以通过抑制CK2过表达所导致的DNA修复增强来克服耐药癌细胞的耐药性，而且还可以进一步上调DNA的损伤水平来实现更好的抗肿瘤活性。由于耐药癌细胞中CK2表达水平较敏感癌 细胞更高，化合物1在耐药癌细胞中表现出更强的抑制活性。Western Blot test was used to detect the expression level of CK2 in cisplatin-sensitive A549 and A2780 cancer cells and the corresponding cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP. It can be seen from Figure 1 that compared with sensitive cancer cells, the expression of CK2 in drug-resistant cancer cells has been significantly up-regulated, which is 1.74 times and 1.96 times that of sensitive cancer cells, respectively. This is related to the drug resistance of cancer cells caused by enhanced DNA repair. Increase coincidence. The Western Blot test was further used to investigate the effects of the designed compounds on the expression of DNA damage and repair-related proteins. As shown in Figure 2, in cisplatin-sensitive cancer cells A549 and A2780 and cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP, the expression of proteins related to DNA repair, such as PARP1, was significantly down-regulated. The expression of γ-H2A.X, a marker of strand breaks, was significantly up-regulated. These results indicate that the compound 1 designed and synthesized in the present invention can not only overcome the drug resistance of drug-resistant cancer cells by inhibiting the enhancement of DNA repair caused by overexpression of CK2, but also can further increase the level of DNA damage to achieve better Anti-tumor activity. Since the expression level of CK2 in drug-resistant cancer cells is higher than that in sensitive cancer cells, compound 1 shows stronger inhibitory activity in drug-resistant cancer cells.
(四)体内活性(4) In vivo activity
采用顺铂敏感的卵巢癌细胞A2780与顺铂耐药的卵巢癌细胞A2780/CDDP的裸鼠异种移植瘤模型，测试了化合物1的体内抗肿瘤活性，顺铂为阳性对照。A nude mouse xenograft tumor model of cisplatin-sensitive ovarian cancer cell A2780 and cisplatin-resistant ovarian cancer cell A2780/CDDP was used to test the anti-tumor activity of compound 1 in vivo, and cisplatin was used as a positive control.
如表3、图3和图4所示，在顺铂敏感的卵巢癌细胞A2780移植瘤的动物模型中，顺铂的抗肿瘤效果较强，抑瘤率为67.30％。当给予动物相当于顺铂(4mg/kg)等摩尔剂量的化合物1(10mg/kg)时，抑瘤率为73.05％，高于顺铂。由图5可知，小鼠体重变化表明化合物1对受试动物体重几乎没有影响，具有良好的耐受性。As shown in Table 3, Figure 3 and Figure 4, in the animal model of cisplatin-sensitive ovarian cancer cell A2780 transplanted tumor, cisplatin has a strong anti-tumor effect, with a tumor inhibition rate of 67.30%. When animals were given an equimolar dose of compound 1 (10 mg/kg) equivalent to cisplatin (4 mg/kg), the tumor inhibition rate was 73.05%, which was higher than that of cisplatin. It can be seen from Figure 5 that the weight change of mice indicates that Compound 1 has almost no effect on the weight of the tested animal and has good tolerance.
如表4、图6和图7所示，在顺铂耐药卵巢癌癌细胞A2780/CDDP移植瘤的动物模型中，顺铂的抗肿瘤效果很弱，肿瘤体积及最终肿瘤的重量与不进行治疗的对照组相比相差不大，最终抑瘤率仅为10.97％。当给予动物相当于顺铂(4mg/kg)等摩尔剂量的化合物1(10mg/kg)时，抑瘤率达到67.94％，显著优于顺铂。由图8可知，小鼠体重变化表明化合物1对受试动物体重几乎没有影响，具有良好的耐受性。As shown in Table 4, Figure 6 and Figure 7, in the animal model of cisplatin-resistant ovarian cancer cancer cell A2780/CDDP xenograft, the anti-tumor effect of cisplatin is very weak, and the tumor volume and final tumor weight are different from each other. Compared with the treated control group, there was little difference, and the final tumor inhibition rate was only 10.97%. When the animal was given an equimolar dose of compound 1 (10 mg/kg) equivalent to cisplatin (4 mg/kg), the tumor inhibition rate reached 67.94%, which was significantly better than cisplatin. It can be seen from Fig. 8 that the change in the weight of the mouse indicates that Compound 1 has little effect on the weight of the tested animal and has good tolerance.
综上，本发明的化合物对癌细胞具有良好的抑制作用，特别是对顺铂耐药的癌细胞具有优良的抑制作用。体内试验结果表明，化合物1不仅对顺铂敏感的肿瘤而且对顺铂耐药的肿瘤都具有很强的抑制作用，且毒性较小，具有高效低毒的特点，可用于制备抗肿瘤药物。In summary, the compound of the present invention has a good inhibitory effect on cancer cells, especially cisplatin-resistant cancer cells. The in vivo test results show that compound 1 not only has a strong inhibitory effect on cisplatin-sensitive tumors but also cisplatin-resistant tumors, and has low toxicity, high efficiency and low toxicity, and can be used to prepare antitumor drugs.
附图说明Description of the drawings
图1a是肺癌细胞A549与顺铂耐药肺癌细胞A549/CDDP、卵巢癌细胞A2780与顺铂耐药卵巢癌细胞A2780/CDDP中的CK2表达；Figure 1a shows the expression of CK2 in lung cancer cell A549 and cisplatin-resistant lung cancer cell A549/CDDP, ovarian cancer cell A2780 and cisplatin-resistant ovarian cancer cell A2780/CDDP;
图1b是肺癌细胞A549与顺铂耐药肺癌细胞A549/CDDP、卵巢癌细胞A2780与顺铂耐药卵巢癌细胞A2780/CDDP中CK2表达的定量数值。Figure 1b is the quantitative value of CK2 expression in lung cancer cell A549 and cisplatin-resistant lung cancer cell A549/CDDP, ovarian cancer cell A2780 and cisplatin-resistant ovarian cancer cell A2780/CDDP.
图2a是受试样品对肺癌细胞A549中DNA损伤修复相关蛋白的表达；Figure 2a is the expression of DNA damage repair related proteins in lung cancer cell A549 by the test sample;
图2b是受试样品对顺铂耐药肺癌细胞A549/CDDP中DNA损伤修复相关蛋白的表达；Figure 2b shows the expression of DNA damage repair related proteins in the cisplatin-resistant lung cancer cell A549/CDDP of the tested sample;
图2c是受试样品对卵巢癌细胞A2780中DNA损伤修复相关蛋白的表达；Figure 2c is the expression of the test sample on DNA damage repair related proteins in ovarian cancer cell A2780;
图2d是受试样品对顺铂耐药卵巢癌细胞A2780/CDDP中DNA损伤修复相关蛋白的表达。Figure 2d shows the expression of DNA damage repair related proteins in the cisplatin-resistant ovarian cancer cell A2780/CDDP of the tested sample.
图3.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤生长体积变化的影响。Figure 3. The effect of the test sample on the growth volume of cisplatin-sensitive ovarian cancer cell A2780 nude mice xenograft tumors.
图4.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植肿瘤瘤重的影响。Figure 4. The effect of the test sample on the tumor weight of cisplatin-sensitive ovarian cancer cell A2780 in nude mice xenograft.
图5.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤裸鼠体重的影响。Figure 5. The effect of the test sample on the weight of nude mice with cisplatin-sensitive ovarian cancer cell A2780 xenograft tumors in nude mice.
图6.受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤生长体积变化的影响。Figure 6. The effect of test samples on the growth volume of cisplatin-resistant ovarian cancer cell A2780/CDDP xenograft tumors in nude mice.
图7.受试样品对顺铂耐药卵巢癌细胞A2780/CDDPP裸鼠异种移植肿瘤瘤重的影响。Figure 7. The effect of the tested sample on the tumor weight of cisplatin-resistant ovarian cancer cell A2780/CDDPP xenograft in nude mice.
图8.受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤裸鼠体重的影响。Figure 8. The effect of test samples on the body weight of nude mice with cisplatin-resistant ovarian cancer cell A2780/CDDP xenograft tumors.
具体实施方式Detailed ways
下面结合实施例对本发明做进一步详细说明，但本发明的保护范围不仅限于这些实施例。The present invention will be further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited to these embodiments.
本发明的一种具有克服顺铂耐药的抗肿瘤化合物其结构如式Ⅰ所示的化合物1或化合物2，An anti-tumor compound capable of overcoming cisplatin resistance of the present invention has a structure such as compound 1 or compound 2 shown in formula I.
Figure PCTCN2021070303-appb-000007
Figure PCTCN2021070303-appb-000007
(一)化合物的制备(1) Preparation of the compound
下面实施例中所有试剂均为分析纯。化合物结构表征所用的核磁共振数据由Bruker ARX-600核磁共振仪测定，内标为TMS；高分辨质谱(LS-MS-ESI)采用Agilent6224 TOF LC/MS测定。All reagents in the following examples are of analytical grade. The NMR data used for the structural characterization of the compound was measured by Bruker ARX-600 NMR instrument, and the internal standard was TMS; the high resolution mass spectrometry (LS-MS-ESI) was measured by Agilent 6224 TOF LC/MS.
本发明中化合物4和化合物5的合成参考之前发表的专利方法(发明专利ZL201210422936.4)制备，经核磁氢谱验证。The synthesis of compound 4 and compound 5 in the present invention were prepared with reference to the previously published patent method (invention patent ZL201210422936.4), and was verified by the hydrogen nuclear magnetic spectrum.
化合物4： 1H NMR(600MHz,DMSO-d6)δ4.21(s,6H),3.72(s,4H)ppm. Compound 4: 1 H NMR (600MHz, DMSO-d6) δ 4.21 (s, 6H), 3.72 (s, 4H) ppm.
化合物5: 1H NMR(600MHz,DMSO-d6)δ5.99(d,J＝7.9Hz,2H),5.27(t,J＝9.6Hz,2H),3.77-3.66(m,4H),2.08-2.01(m,2H),1.81(d,J＝12.7Hz,2H),1.45(d,J＝8.3Hz,2H),1.21(dd,J＝19.9,11.0Hz,2H),1.06-0.96(m,2H)ppm. Compound 5: 1 H NMR (600MHz, DMSO-d6) δ 5.99 (d, J = 7.9 Hz, 2H), 5.27 (t, J = 9.6 Hz, 2H), 3.77-3.66 (m, 4H), 2.08- 2.01 (m, 2H), 1.81 (d, J = 12.7 Hz, 2H), 1.45 (d, J = 8.3 Hz, 2H), 1.21 (dd, J = 19.9, 11.0 Hz, 2H), 1.06-0.96 (m , 2H) ppm.
本发明中，化合物6和CX-4945参考文献(J.Med.Chem.2011，54，635-654)所报道的方法制备，经核磁氢谱验证。In the present invention, compound 6 and CX-4945 reference (J. Med. Chem. 2011, 54, 635-654) are prepared by the method reported and verified by the hydrogen nuclear magnetic spectrum.
化合物6： 1H NMR(600MHz,DMSO-d6)δ10.09(s,1H),9.91(s,1H),8.94(d,J＝5.6Hz,1H),8.79(d,J＝8.5Hz,1H),8.77(d,J＝5.6Hz,1H),8.34(t,J＝1.9Hz,1H),8.17(dd,J＝6.3,1.4Hz,1H),7.90(dd,J＝8.4,1.7Hz,1H),7.42(t,J＝8.1Hz,1H),7.12(dd,J＝7.9,1.4Hz,1H),3.92(s,1H)ppm. Compound 6: 1 H NMR (600MHz, DMSO-d6) δ 10.09 (s, 1H), 9.91 (s, 1H), 8.94 (d, J = 5.6 Hz, 1H), 8.79 (d, J = 8.5 Hz, 1H), 8.77 (d, J = 5.6 Hz, 1H), 8.34 (t, J = 1.9 Hz, 1H), 8.17 (dd, J = 6.3, 1.4 Hz, 1H), 7.90 (dd, J = 8.4, 1.7 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.12 (dd, J = 7.9, 1.4 Hz, 1H), 3.92 (s, 1H) ppm.
CX-4945： 1H NMR(600MHz,DMSO-d6)δ12.04(s,1H),10.30(s,1H),10.16(s,1H),9.08(d,J＝5.8Hz,1H),9.00-8.97(m,1H),8.94(d,J＝8.5Hz,1H),8.31-8.28(m,2H),8.12(d,J＝8.2Hz,1H),8.03(dd,J＝8.4,1.5Hz,1H),7.45(t,J＝8.1Hz,1H),7.17(d,J＝7.9Hz,1H)ppm. CX-4945: 1 H NMR (600MHz, DMSO-d6) δ 12.04 (s, 1H), 10.30 (s, 1H), 10.16 (s, 1H), 9.08 (d, J = 5.8 Hz, 1H), 9.00 -8.97(m,1H),8.94(d,J=8.5Hz,1H), 8.31-8.28(m,2H), 8.12(d,J=8.2Hz,1H), 8.03(dd,J=8.4,1.5 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H) ppm.
实施例一：化合物1的制备Example 1: Preparation of compound 1
在50mL反应瓶中将等摩尔量的化合物3与化合物4(2mmol)，悬浮于30mL无水甲醇中，加入2-3滴乙酸，30-60℃下避光反应48-72h，TLC监测至化合物3完全反应。冷却过滤，滤饼用30mL(3×10mL)甲醇冲洗，得黄色固体产物，收率96.4％。In a 50 mL reaction flask, equimolar amounts of compound 3 and compound 4 (2 mmol) were suspended in 30 mL of anhydrous methanol, and 2-3 drops of acetic acid were added. 3 Complete reaction. After cooling and filtering, the filter cake was washed with 30 mL (3×10 mL) of methanol to obtain a yellow solid product with a yield of 96.4%.
1H NMR(600MHz,DMSO-d6)δ11.15(s,1H),10.19(s,1H),9.67(s,1H),8.99(d,J＝4.2Hz,1H),8.86(d,J＝7.7Hz,1H),8.58(d,J＝3.8Hz,1H),8.26(s,1H),8.23(s,1H),8.14(d,J＝7.2Hz,1H),7.93(d,J＝7.3Hz,1H),7.45(t,J＝7.5Hz,1H),7.14(d,J＝6.9Hz,1H),4.26(s,6H),3.74(s,4H)ppm. 13C NMR(150MHz,DMSO-d6)δ176.45,176.23,163.28,159.72,150.55,148.18,147.80,143.63,142.33,135.12,133.28,130.62,127.50,126.91,124.23,123.84,122.80,122.69,121.73,120.64,119.68,116.83,56.40,49.60,46.96ppm.HR-MS(m/z)(ESI):calcd for C 25H 23ClN 7O 5Pt[M+H] +:731.1091；found:731.1353. 1 H NMR (600MHz, DMSO-d6) δ 11.15 (s, 1H), 10.19 (s, 1H), 9.67 (s, 1H), 8.99 (d, J = 4.2 Hz, 1H), 8.86 (d, J =7.7Hz,1H), 8.58(d,J=3.8Hz,1H), 8.26(s,1H), 8.23(s,1H), 8.14(d,J=7.2Hz,1H),7.93(d,J =7.3Hz, 1H), 7.45 (t, J = 7.5Hz, 1H), 7.14 (d, J = 6.9Hz, 1H), 4.26 (s, 6H), 3.74 (s, 4H) ppm. 13 C NMR( 150MHz, DMSO-d6)δ176.45,176.23,163.28,159.72,150.55,148.18,147.80,143.63,142.33,135.12,133.28,130.62,127.50,126.91,124.23,123.84,122.80,122.69,121.73,120.64,119,68,116.83 56.40,49.60,46.96ppm.HR-MS(m/z)(ESI): calcd for C 25 H 23 ClN 7 O 5 Pt[M+H] + :731.1091; found: 731.1353.
实施例二：化合物2的制备Example 2: Preparation of Compound 2
用化合物3和化合物5参照实施例一所示方法制备，得黄色固体产物，收率 95.2％。 Compound 3 and compound 5 were prepared with reference to the method shown in Example 1, to obtain a yellow solid product with a yield of 95.2%.
1H NMR(600MHz,DMSO-d6)δ10.18(s,1H),10.09(s,1H),9.68(s,1H),8.99(d,J＝5.5Hz,1H),8.84(d,J＝8.4Hz,1H),8.59(d,J＝5.4Hz,1H),8.35(s,1H),8.23(s,1H),8.09(d,J＝8.1Hz,1H),7.93(d,J＝8.2Hz,1H),7.45(t,J＝8.0Hz,1H),7.15(d,J＝7.7Hz,1H),6.01(d,J＝8.0Hz,2H),5.30(m,2H),3.70-3.79(m,4H),2.07(s,2H),1.83(d,J＝11.9Hz,2H),1.44-1.46(m,2H),1.22(d,J＝8.8Hz,2H),1.02(t,J＝9.5Hz,2H)ppm. 13C NMR(150MHz,DMSO-d6)δ206.07,176.03,165.83,150.48,148.15,147.72,143.76,142.39,134.71,133.28,130.54,127.52,126.26,124.19,123.01,122.86,122.62,121.48,120.56,119.57,116.82,62.57,56.35,46.86,31.96,24.50ppm.HR-MS(m/z)(ESI):calcd for C 31H 31ClN 7O 5Pt[M+H] +:811.1717；found:811.1158. 1 H NMR (600MHz, DMSO-d6) δ 10.18 (s, 1H), 10.09 (s, 1H), 9.68 (s, 1H), 8.99 (d, J = 5.5 Hz, 1H), 8.84 (d, J ＝8.4Hz,1H),8.59(d,J＝5.4Hz,1H),8.35(s,1H),8.23(s,1H),8.09(d,J＝8.1Hz,1H),7.93(d,J ＝8.2Hz,1H),7.45(t,J＝8.0Hz,1H), 7.15(d,J＝7.7Hz,1H), 6.01(d,J＝8.0Hz,2H),5.30(m,2H), 3.70-3.79(m,4H),2.07(s,2H),1.83(d,J=11.9Hz,2H),1.44-1.46(m,2H),1.22(d,J=8.8Hz,2H),1.02 (t, J=9.5Hz, 2H) ppm. 13 C NMR (150MHz, DMSO-d6) δ206.07,176.03,165.83,150.48,148.15,147.72,143.76,142.39,134.71,133.28,130.54,127.52,126.26,124.19, 123.01,122.86,122.62,121.48,120.56,119.57,116.82,62.57,56.35,46.86,31.96,24.50ppm.HR-MS(m/z)(ESI):calcd for C 31 H 31 ClN 7 O 5 Pt(M +H] + :811.1717; found:811.1158.
实施例三：化合物3的制备Example 3: Preparation of Compound 3
于50mL单口瓶中将化合物6(1.09g，3mmol)溶于20mL甲醇中，室温下缓慢滴加水合肼(6-30mmol)，加热回流24-72h至完全反应。冷却，析出大量固体，抽滤，滤饼用3×10mL冰甲醇冲洗，得亮黄色固体产物1.05g，收率97.1％。 1H NMR(600MHz,DMSO-d6)δ10.14(s,1H),10.08(s,1H),9.63(s,1H),8.95(d,J＝6.0Hz,1H),8.80(d,J＝12.0Hz,1H),8.55(d,J＝6.0Hz,1H),8.33(t,J＝1.8Hz,1H),8.21(d,J＝1.6Hz,1H),8.07(d,J＝6.0Hz,1H),7.91(dd,J＝12.0,1.6Hz,1H),7.42(t,J＝6.0Hz,1H),7.12(dd,J＝8.0,1.4Hz,1H),4.63(s,2H)ppm. 13C NMR(150MHz,DMSO-d6)δ165.77,150.47,148.13,147.69,143.76,142.40,134.73,133.35,130.52,127.52,126.26,124.17,123.00,122.83,122.60,121.47,120.57,119.56,116.79ppm.HR-MS(m/z)(ESI):calcd for C 19H 14ClN 5O[M+H] +:364.0965；found:364.0967. Compound 6 (1.09g, 3mmol) was dissolved in 20mL methanol in a 50mL single-neck flask, hydrazine hydrate (6-30mmol) was slowly added dropwise at room temperature, and heated to reflux for 24-72h to complete the reaction. After cooling, a large amount of solids were precipitated, filtered with suction, and the filter cake was washed with 3×10 mL ice methanol to obtain 1.05 g of bright yellow solid product with a yield of 97.1%. 1 H NMR (600MHz, DMSO-d6) δ 10.14 (s, 1H), 10.08 (s, 1H), 9.63 (s, 1H), 8.95 (d, J = 6.0 Hz, 1H), 8.80 (d, J = 12.0Hz, 1H), 8.55 (d, J = 6.0 Hz, 1H), 8.33 (t, J = 1.8 Hz, 1H), 8.21 (d, J = 1.6 Hz, 1H), 8.07 (d, J = 6.0 Hz, 1H), 7.91 (dd, J = 12.0, 1.6 Hz, 1H), 7.42 (t, J = 6.0 Hz, 1H), 7.12 (dd, J = 8.0, 1.4 Hz, 1H), 4.63 (s, 2H) ) ppm. 13 C NMR (150MHz, DMSO-d6) δ165.77,150.47,148.13,147.69,143.76,142.40,134.73,133.35,130.52,127.52,126.26,124.17,123.00,122.83,122.60,121.47,120.57,119.56,116.79 ppm.HR-MS(m/z)(ESI):calcd for C 19 H 14 ClN 5 O[M+H] + :364.0965; found:364.0967.
(二)抑制CK2活性的测试(2) Test for inhibiting CK2 activity
实验方法：采用CK2酶联免疫分析(ELISA)试剂盒进行检测。首先取标准品蛋白稀释至合适浓度后，加入包被纯化的CK2捕获抗体的微孔板中，再加入已稀释至合适浓度梯度的待测化合物，并设置不加药的对照组进行对比。放入孵箱孵育1h，使药物有效抑制蛋白与抗体的结合后，将微孔板中的液体弃去，加入200μL已稀释好的洗涤液，静置30s后弃去并重复洗涤5次。加入100μL的HRP标记的酶标试剂，孵箱孵育30min，使其形成抗体-抗原-酶标抗体复合物。再将微孔板中的液体弃去，加入200μL已稀释好的洗涤液，静置30s后弃 去，并重复洗涤5次。接下来每孔先加入显色剂A 50μL，再加入显色剂B 50μL，轻轻震荡混匀，37℃避光显色15min，使底物TMB在HRP酶的催化下转化成蓝色。最终加入50μL终止液，使TMB在酸的作用下转化成最终的黄色，颜色的深浅和CK2的活性呈正相关。在15min以内，通过酶标仪在450nm波长依序测量各孔的吸光度(OD值)，最终得到待测化合物相对应的CK2抑制活性。Experimental method: CK2 enzyme-linked immunoassay (ELISA) kit was used for detection. First, the standard protein was diluted to a suitable concentration, then added to the microtiter plate coated with purified CK2 capture antibody, and then added to the test compound diluted to a suitable concentration gradient, and a control group without added drug was set for comparison. Put it in the incubator and incubate for 1 hour to make the drug effectively inhibit the binding of protein and antibody, discard the liquid in the microplate, add 200μL of the diluted washing solution, leave it for 30s, discard it and repeat the washing 5 times. Add 100 μL of HRP-labeled enzyme-labeled reagent, and incubate for 30 minutes in an incubator to form an antibody-antigen-enzyme-labeled antibody complex. Then discard the liquid in the microplate, add 200μL of the diluted washing solution, let it stand for 30s and discard it, and repeat the washing 5 times. Next, add 50μL of developer A to each well, and then add 50μL of developer B, shake gently to mix, and develop color at 37°C for 15 minutes in the dark, so that the substrate TMB is converted into blue under the catalysis of HRP enzyme. Finally, 50μL of stop solution was added to convert TMB into the final yellow color under the action of acid, and the color intensity was positively correlated with the activity of CK2. Within 15 minutes, the absorbance (OD value) of each well was measured sequentially at 450nm wavelength by a microplate reader, and finally the CK2 inhibitory activity corresponding to the test compound was obtained.
采用上述方法对化合物1、2和3抑制CK2的活性进行了测试，以CX-4945作为阳性对照，结果见表1。The CK2 inhibitory activity of compounds 1, 2 and 3 was tested by the above method, and CX-4945 was used as a positive control. The results are shown in Table 1.
表1.化合物对CK2的抑制活性Table 1. Inhibitory activity of compounds on CK2
Figure PCTCN2021070303-appb-000008
Figure PCTCN2021070303-appb-000008
(三)体外细胞毒活性测试(3) In vitro cytotoxic activity test
实验方法：采用MTT方法对本发明所制备的化合物进行了细胞毒活性测试。取对数生长期的细胞计数，接种于96孔培养板内，每孔约10 4个细胞，于37℃，5％CO 2孵箱培养24h至细胞贴壁后给药，分别设给药组和不给药的对照组。对照组更换新鲜培养基，待测的化合物4和5及顺铂用5％的葡萄糖水溶液溶解，其它化合物用DMSO配制成母液，临用前用对应的细胞培养基稀释成合适的浓度梯度加入给药组，每个浓度设3个平行组。加药后于37℃，5％CO 2孵箱培养72h，加10μL浓度为5mg/mL的MTT，37℃孵育4h后，弃去上清，加入130μL的DMSO溶解甲瓒。用酶标仪在490nm波长下测定每孔的OD值，并计算IC 50值。 Experimental method: The cytotoxic activity test of the compound prepared by the present invention was carried out using the MTT method. Take the logarithmic phase cell count, were seeded in 96-well culture plate, each well of about 104 cells, at 37 ℃, 5% CO 2 incubator for 24h to adherent cell administration, administration group are located And the control group without administration. The control group was replaced with fresh medium, the tested compounds 4 and 5 and cisplatin were dissolved in 5% glucose aqueous solution, and other compounds were prepared into mother liquor with DMSO, and the corresponding cell culture medium was diluted to a suitable concentration gradient before use. For the drug group, 3 parallel groups are set for each concentration. After adding the drug, incubate for 72 hours in a 5% CO 2 incubator at 37°C, add 10 μL of MTT at a concentration of 5 mg/mL, incubate at 37°C for 4 hours, discard the supernatant, and add 130 μL of DMSO to dissolve formazan. Then determined by comparing the OD of each well at 490nm wavelength, and calculate the 50 value IC.
采用上述方法，用所制备的化合物对人喉癌细胞Hep2、鼻咽癌细胞CNE2、胃癌细胞SGC-7901、结肠癌细胞HCT-116、乳腺癌细胞MCF-7、***细胞SiHa、***癌细胞PC-3、膀胱癌细胞T24、非小细胞肺癌A549及其顺铂耐药癌细胞 A549/CDDP、卵巢癌细胞A2780与顺铂耐药癌细胞A2780/CDDP以及正常肝细胞LO2进行了体外抗肿瘤活性评价，CX-4945作为阳性对照。观察化合物在不同浓度下对肿瘤细胞生长的抑制情况，计算其IC 50值来评价药物的细胞毒活性，结果见表2。 Using the above method, the prepared compound was used to treat human laryngeal cancer cells Hep2, nasopharyngeal cancer cells CNE2, gastric cancer cells SGC-7901, colon cancer cells HCT-116, breast cancer cells MCF-7, cervical cancer cells SiHa, and prostate cancer cells. PC-3, bladder cancer cell T24, non-small cell lung cancer A549 and its cisplatin-resistant cancer cell A549/CDDP, ovarian cancer cell A2780, cisplatin-resistant cancer cell A2780/CDDP, and normal liver cells LO2 were anti-tumor in vitro For activity evaluation, CX-4945 was used as a positive control. Observe the compound's inhibition of tumor cell growth at different concentrations, calculate its IC 50 value to evaluate the cytotoxic activity of the drug, and the results are shown in Table 2.
表2.化合物对所测细胞的细胞毒活性Table 2. Cytotoxic activity of the compounds on the tested cells
Figure PCTCN2021070303-appb-000009
Figure PCTCN2021070303-appb-000009
*.nd：没有测定。*.nd: Not determined.
(四)体外诱导DNA损伤及抑制DNA修复的测试(4) Tests for inducing DNA damage and inhibiting DNA repair in vitro
实验方法：采用Western Blot试验对本发明所涉及的化合物进行了检测。取对数生长期的细胞计数，接种于6孔培养板内，每孔约10 5个细胞。于37℃，5％CO 2孵箱培养24h至细胞贴壁后给药，分别设给药组和不给药的对照组。对照组更换新鲜培养基，待测的化合物4用5％的葡萄糖水溶液溶解，其它化合物用DMSO配制成母液，临用前用相对应的细胞培养基稀释成合适的浓度加入给药组，加药后于37℃，5％CO 2孵箱培养24h，将孔中的细胞消化，离心收集至离心管中并用PBS洗涤后，向细胞中滴加适量裂解液，于冰盒中裂解细胞2h。10000rpm离心20～30min，取上清液待用。用BCA蛋白试剂盒(Thermo，Waltham，MA)测定蛋白含量。 Experimental method: Western Blot test was used to detect the compounds involved in the present invention. Taking the logarithmic growth phase cells were counted, seeded in 6-well culture plate, each well of about 10 5 cells. The cells were cultured at 37°C in a 5% CO 2 incubator for 24 hours until the cells adhered to the wall and then administered. The administration group and the non-administered control group were set up respectively. The control group was replaced with fresh medium, the compound 4 to be tested was dissolved in 5% glucose aqueous solution, and the other compounds were prepared into mother liquor with DMSO, and diluted with the corresponding cell culture medium to an appropriate concentration before use and added to the administration group. After culturing in a 37°C, 5% CO 2 incubator for 24 hours, the cells in the wells were digested, collected by centrifugation into a centrifuge tube and washed with PBS, an appropriate amount of lysate was added dropwise to the cells, and the cells were lysed in an ice box for 2 hours. Centrifuge at 10000rpm for 20-30min, and take the supernatant for later use. The protein content was determined with BCA protein kit (Thermo, Waltham, MA).
用去离子水清洗玻璃板，晾干。再将洗净的玻璃板安装在凝胶架上并检漏。再分别配制好10％的分离胶和15％的积层胶。先用向玻璃板中加入分离胶至1/2 界面处，再沿着玻璃板加入去离子水封胶，直至水满溢出。等待20min分离胶固化之后，倒掉去离子水，吸干。接着加入积层胶至玻璃板上界面，***梳子等待30min后积层胶凝固。将玻璃板取下，用纯净水冲洗干净转移放入电泳槽中，加入少许缓冲液进行检漏，再向电泳槽中缓慢倒入缓冲液至满。缓慢拔除梳子，然后用受试样品依次加入到各个孔内，用60V电压进行电泳。电泳结束后将胶板取出计算条带的剪切区域。再进行转膜操作，将剪切的条带放入半干转膜缓冲液中，将电泳仪电压调至25V，室温下转膜50min。转膜完成后，从半干转膜仪中移出条带，放入丽春红溶液中进行染色，观察转膜是否成功，再放入去离子水中清洗，接着用PBS洗涤条带3次，每次8min。将条带置于培养皿中，注入30mL牛奶液(用PBST溶)，于37℃孵箱中，振荡1h后用PBST洗涤3次，每次8min。向封口膜中注入一抗，封装，置于4℃冰箱中振荡过夜。弃去一抗，将膜转移至PBST溶液中振荡清洗3次，每次8min。加入二抗，封装，室温振荡孵育1h。弃去二抗，PBST溶液振荡清洗3次，每次8min，再用PBS洗涤一次，5min后将条带取出置于干净的玻璃板上待用。配制AB发光液(A:B＝1:1)，滴加于条带上，作用约10s左右，去除多余的发光液后盖上薄膜，并将胶片覆于薄膜上，用手按压70s，置于预热好的曝光机中曝光。胶片洗出，观察条带。该过程需要避光操作。Rinse the glass plate with deionized water and let it dry. Then install the cleaned glass plate on the gel rack and check for leaks. Then separately prepare 10% separating glue and 15% laminating glue. First add the separating glue to the glass plate to the 1/2 interface, and then add the deionized water sealant along the glass plate until the water overflows. After waiting for 20 minutes for the separation gel to solidify, pour out the deionized water and blot it dry. Then add the layering glue to the interface of the glass plate, insert the comb and wait for 30 minutes for the layering glue to solidify. Remove the glass plate, rinse with pure water and transfer it into the electrophoresis tank, add a little buffer solution for leak detection, and then slowly pour the buffer solution into the electrophoresis tank until it is full. Pull out the comb slowly, then add the test sample to each well in turn, and perform electrophoresis with a voltage of 60V. After the electrophoresis is over, the gel plate is taken out to calculate the cut area of the band. Then transfer the membrane, put the cut strips into the semi-dry transfer buffer, adjust the voltage of the electrophoresis apparatus to 25V, and transfer the membrane at room temperature for 50 minutes. After the transfer is completed, remove the strips from the semi-dry transfer machine, put them in the Ponceau solution for staining, observe whether the transfer is successful, and then wash them in deionized water, and then wash the strips with PBS 3 times, each Time 8min. Place the strips in a petri dish, inject 30 mL of milk solution (dissolved in PBST), in a 37°C incubator, shake for 1 hour and wash with PBST 3 times, each time for 8 minutes. Inject the primary antibody into the sealing film, encapsulate, and shake overnight in a refrigerator at 4°C. The primary antibody was discarded, and the membrane was transferred to the PBST solution for shaking and washing 3 times, 8 min each time. Add secondary antibody, encapsulate, and incubate with shaking at room temperature for 1h. The secondary antibody was discarded, and the PBST solution was shaken and washed 3 times, 8 min each time, and then washed once with PBS. After 5 min, the strip was taken out and placed on a clean glass plate for later use. Prepare AB luminescent liquid (A:B=1:1), drop it on the strip, and act for about 10s. After removing the excess luminescent liquid, cover the film, and cover the film on the film, press it for 70s by hand, and place it. Expose in the preheated exposure machine. The film is washed out and the bands are observed. This process requires dark operation.
采用上述方法，首先测试了癌细胞A549和A2780与对应的顺铂耐药癌细胞A549/CDDP和A2780/CDDP中CK2的表达水平，实验结果见图1；其次测试了所涉及化合物对DNA损伤修复相关蛋白表达的影响，以顺铂为阳性对照，实验结果见图2。Using the above method, firstly, the expression level of CK2 in cancer cells A549 and A2780 and the corresponding cisplatin-resistant cancer cells A549/CDDP and A2780/CDDP were tested. The experimental results are shown in Figure 1. Secondly, the compounds involved were tested for DNA damage repair Regarding the influence of related protein expression, cisplatin was used as a positive control, and the experimental results are shown in Figure 2.
(五)体内抗肿瘤活性测试(5) In vivo anti-tumor activity test
受试动物：BALB/c(nu/nu)裸鼠，体重16～18g，雌性，购自上海西普尔-必凯实验动物有限公司，饲养于SPF级饲养环境中，室内温度控制在23±2℃，自由饮食和摄水。接种前适应性饲养7天。药物及试剂：顺铂用5％葡萄糖注射液超声溶解，化合物1用DMF溶解后，依次用Tween80和5％葡萄糖注射液稀释。组别及给药方案如下。模型对照组：尾静脉注射0.1mL5％葡萄糖注射液，每周1次，连续4周。顺铂(4mg/kg)：尾静脉注射0.1mL顺铂(剂量为4mg/kg)，每周1次，连续4周。化合物1(10mg/kg)：尾静脉注射0.1mL化合物1(剂量为10mg/kg)，每周1次，连续4周。Tested animal: BALB/c(nu/nu) nude mouse, weighing 16～18g, female, purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., raised in an SPF breeding environment, the indoor temperature is controlled at 23±2 ℃, free eating and drinking. The animals were bred adaptively for 7 days before inoculation. Drugs and reagents: cisplatin was dissolved in 5% glucose injection by ultrasonic, compound 1 was dissolved in DMF, and then diluted with Tween80 and 5% glucose injection in sequence. The groups and dosage regimen are as follows. Model control group: 0.1 mL of 5% glucose injection was injected into the tail vein once a week for 4 consecutive weeks. Cisplatin (4mg/kg): 0.1 mL of cisplatin (dose 4mg/kg) was injected into the tail vein once a week for 4 consecutive weeks. Compound 1 (10 mg/kg): 0.1 mL of compound 1 (dose 10 mg/kg) was injected into the tail vein once a week for 4 consecutive weeks.
实验方法：取对数生长期A2780或A2780/CDDP细胞，制备细胞悬液浓度为5×10 7cell/mL，裸鼠右腋下接种0.2mL/只，建立A2780或A2780/CDDP裸鼠移植瘤模型，裸小鼠移植瘤用游标卡尺测量移植瘤直径，待肿瘤生长至100mm 3后将动物随机分组。使用测量瘤径的方法，动态观察被试物抗肿瘤的效应。肿瘤直径的测量次数为每3天测1次，同时每3天检查一次小鼠体重。4周后，注射水合氯醛处死所有小鼠，剖腹观察，摘除肿瘤，仔细剔除无关组织，用D-Hanks液洗涤2～3次，洗去血液，沥干水分保存，称取重量和测量体积。肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×a×b 2，其中a、b分别表示长宽。 Experimental method: Take A2780 or A2780/CDDP cells in logarithmic growth phase, prepare a cell suspension concentration of 5×10 7 cell/mL, inoculate 0.2 mL/mouse under the right armpit of nude mice to establish A2780 or A2780/CDDP nude mice transplanted tumors In the model, the diameter of the transplanted tumor in nude mice was measured with a vernier caliper. After the tumor grew to 100mm 3, the animals were randomly divided into groups. Use the method of measuring tumor diameter to dynamically observe the anti-tumor effect of the test substance. The number of measurements of tumor diameter was once every 3 days, and the mouse body weight was checked every 3 days. After 4 weeks, all mice were sacrificed by injection of chloral hydrate, and observed by laparotomy. The tumor was removed and irrelevant tissues were carefully removed. The blood was washed with D-Hanks solution for 2 to 3 times, the blood was washed away, and the water was drained for storage. . The calculation formula of tumor volume (TV) is: TV=1/2×a×b 2 , where a and b represent length and width respectively.
据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝V t/V 0，其中V 0为分笼给药时(即d 0)测量所得肿瘤体积，V t为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标：相对肿瘤增殖率T/C(％)，计算公式如下： The relative tumor volume (RTV) is calculated according to the measurement results. The calculation formula is: RTV=V t /V 0 , where V 0 is the tumor volume measured in caged administration (ie d 0 ), V t Is the tumor volume at each measurement. Evaluation index of anti-tumor activity: relative tumor proliferation rate T/C (%), the calculation formula is as follows:
Figure PCTCN2021070303-appb-000010
Figure PCTCN2021070303-appb-000010
其中，T RTV：治疗组RTV；C RTV：模型组RTV。 Among them, TRTV : treatment group RTV; C RTV : model group RTV.
抗肿瘤活性的评价指标：肿瘤生长抑制率(％)，计算公式如下：Evaluation index of anti-tumor activity: tumor growth inhibition rate (%), the calculation formula is as follows:
Figure PCTCN2021070303-appb-000011
Figure PCTCN2021070303-appb-000011
均值用X±SD表示，组间分析用t检验进行统计学处理，应用SPSS(Staffstical Package for the Social Science)17.0对结果进行统计分析。The mean value is represented by X±SD, the analysis between groups is statistically processed by t-test, and the results are statistically analyzed by SPSS (Staffstical Package for the Social Science) 17.0.
有关受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤生长的抑制作用结果见表3、图3和图4，受试动物的体重变化见图5。有关受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤生长的抑制作用结果见表4、图6和图7，受试动物的体重变化见图8。See Table 3, Figure 3 and Figure 4 for the results of the inhibitory effect of the tested sample on the growth of cisplatin-sensitive ovarian cancer cell A2780 nude mice xenograft tumors, and Figure 5 for the body weight changes of the tested animals. The results of the inhibitory effect of the test samples on the growth of cisplatin-resistant ovarian cancer cell A2780/CDDP xenograft tumors in nude mice are shown in Table 4, Fig. 6 and Fig. 7, and the body weight changes of the test animals are shown in Fig. 8.
表3.受试样品对顺铂敏感的卵巢癌细胞A2780裸鼠异种移植瘤生长的抑制作用Table 3. The inhibitory effect of test samples on the growth of cisplatin-sensitive ovarian cancer cell A2780 xenograft in nude mice
Figure PCTCN2021070303-appb-000012
Figure PCTCN2021070303-appb-000012
表4.受试样品对顺铂耐药卵巢癌细胞A2780/CDDP裸鼠异种移植瘤生长的抑制作用Table 4. Inhibitory effects of test samples on the growth of cisplatin-resistant ovarian cancer cell A2780/CDDP xenograft tumors in nude mice
Figure PCTCN2021070303-appb-000013
Figure PCTCN2021070303-appb-000013

Claims (6)

  1. 一种具有克服顺铂耐药的抗肿瘤化合物，其特征在于所述的具有克服顺铂耐药的抗肿瘤化合物，其结构如式Ⅰ所示的化合物1或化合物2，An anti-tumor compound capable of overcoming cisplatin resistance, which is characterized in that the anti-tumor compound capable of overcoming cisplatin resistance has a structure such as compound 1 or compound 2 shown in formula I.
    Figure PCTCN2021070303-appb-100001
    Figure PCTCN2021070303-appb-100001
  2. 一种如权利要求1所述的具有克服顺铂耐药的抗肿瘤化合物的制备方法，其特征在于式I中的化合物1由式II所示的反应式制备，A method for preparing an antitumor compound capable of overcoming cisplatin resistance according to claim 1, wherein compound 1 in formula I is prepared by the reaction formula shown in formula II,
    Figure PCTCN2021070303-appb-100002
    Figure PCTCN2021070303-appb-100002
    式II所示的反应式制备，具体步骤如下：在反应瓶中加入将等摩尔量的化合物3与化合物4悬浮于无水甲醇中，滴加乙酸，反应液在30-60℃下避光反应48-72小时，然后冷至室温，过滤，滤饼用无水甲醇冲洗三次，得黄色固体产物-化合物1。For the preparation of the reaction formula shown in formula II, the specific steps are as follows: add equimolar amounts of compound 3 and compound 4 in anhydrous methanol into a reaction flask, add acetic acid dropwise, and react the reaction solution at 30-60°C in the dark After 48-72 hours, it was cooled to room temperature, filtered, and the filter cake was washed three times with anhydrous methanol to obtain compound 1 as a yellow solid product.
  3. 一种如权利要求1所述的具有克服顺铂耐药的抗肿瘤化合物的制备方法，其特征在于式I中的化合物2由式III所示的反应式制备，A method for preparing an anti-tumor compound capable of overcoming cisplatin resistance according to claim 1, wherein compound 2 in formula I is prepared by the reaction formula shown in formula III,
    Figure PCTCN2021070303-appb-100003
    Figure PCTCN2021070303-appb-100003
    Figure PCTCN2021070303-appb-100004
    Figure PCTCN2021070303-appb-100004
    式Ⅲ所示的反应式制备，具体步骤如下：在反应瓶中加入将等摩尔量的化合物3与化合物5悬浮于无水甲醇中，滴加乙酸，反应液在30-60℃下避光反应48-72小时，然后冷至室温，过滤，滤饼用无水甲醇冲洗三次，得黄色固体产物-化合物2。The reaction formula shown in formula III is prepared, and the specific steps are as follows: add equimolar amounts of compound 3 and compound 5 in anhydrous methanol into the reaction flask, add acetic acid dropwise, and react the reaction solution at 30-60°C in the dark After 48-72 hours, it was cooled to room temperature, filtered, and the filter cake was washed three times with anhydrous methanol to obtain compound 2 as a yellow solid product.
  4. 如权利要求2或3所述的具有克服顺铂耐药的抗肿瘤化合物的制备方法，其特征在于应用式II或式Ⅲ中所示的化合物3，其结构如下所示，The preparation method of an anti-tumor compound capable of overcoming cisplatin resistance according to claim 2 or 3, which is characterized by using compound 3 shown in formula II or formula III, and its structure is as follows:
    Figure PCTCN2021070303-appb-100005
    Figure PCTCN2021070303-appb-100005
  5. 如权利要求2或3所述的具有克服顺铂耐药的抗肿瘤化合物的制备方法，其特征在于式II和式Ⅲ中所示的化合物3由式IV所示的反应式制备，The method for preparing an antitumor compound capable of overcoming cisplatin resistance according to claim 2 or 3, characterized in that the compound 3 shown in formula II and formula III is prepared by the reaction formula shown in formula IV,
    Figure PCTCN2021070303-appb-100006
    Figure PCTCN2021070303-appb-100006
    按照式Ⅳ所示的反应式制备，具体步骤如下：在装有甲醇的反应瓶中加入1当量的化合物6，室温下缓慢滴加2-10当量的水合肼，反应液加热回流24-72小时至反应完全，然后冷至室温，析出大量固体，过滤，滤饼用冰甲醇冲洗三次，得亮黄色固体产物-化合物3。According to the reaction formula shown in formula IV, the specific steps are as follows: add 1 equivalent of compound 6 into a reaction flask containing methanol, slowly add 2-10 equivalents of hydrazine hydrate at room temperature, and heat the reaction solution to reflux for 24-72 hours After the reaction was completed, it was cooled to room temperature, and a large amount of solid precipitated out. The filter cake was washed with ice methanol three times to obtain compound 3 as a bright yellow solid product.
  6. 一种如权利要求1所述的一类具有克服顺铂耐药的抗肿瘤化合物的应用，其特征在于，所述的化合物不仅对顺铂敏感的肿瘤而且对顺铂耐药的肿瘤具有很强的抑制作用，且毒性较小，具有高效低毒的特点，可用于制备抗肿瘤药物。An application of an anti-tumor compound capable of overcoming cisplatin resistance according to claim 1, wherein the compound is not only sensitive to cisplatin-sensitive tumors, but also highly resistant to cisplatin-resistant tumors. The inhibitory effect, low toxicity, high efficiency and low toxicity, can be used to prepare anti-tumor drugs.
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