WO2020019376A1 - 一种tdo小分子抑制剂衍生物及其抗肿瘤偶联物和制备方法 - Google Patents

一种tdo小分子抑制剂衍生物及其抗肿瘤偶联物和制备方法 Download PDF

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WO2020019376A1
WO2020019376A1 PCT/CN2018/100399 CN2018100399W WO2020019376A1 WO 2020019376 A1 WO2020019376 A1 WO 2020019376A1 CN 2018100399 W CN2018100399 W CN 2018100399W WO 2020019376 A1 WO2020019376 A1 WO 2020019376A1
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tdo
formula
small molecule
molecule inhibitor
derivative
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苟少华
花世鲜
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东南大学
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
    • 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 Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0086—Platinum compounds
    • C07F15/0093—Platinum compounds without a metal-carbon linkage

Definitions

  • the invention relates to an anti-tumor immune conjugate containing a tryptophan 2,3-dioxygenase (TDO) small molecule inhibitor, and particularly relates to the axial introduction of TDO small in a tetravalent platinum octahedron structure containing a cisplatin skeleton.
  • TDO tryptophan 2,3-dioxygenase
  • Tryptophan is an essential amino acid in the human body. Not only can it synthesize proteins, but it can also be catabolized through the kynurenic acid pathway to convert tryptophan into a series of bioactive metabolites such as kynurenine, kynurin, Acid, etc., whose rate-limiting step is the conversion of tryptophan to N-formyl kynurenine.
  • Tryptophan 2,3-dioxygenase (TDO) is one of the first rate-limiting enzymes in this pathway's metabolic rate-limiting step. Its expression is regulated by various signals such as glucocorticoids, L-tryptophan and Kynurenine and so on.
  • TDO is mainly expressed in the liver and regulates the level of tryptophan in the system. In addition to the liver, TDO is also expressed in the brain. Studies have shown that TDO can participate in tumor immune escape and promote tumor growth, and TDO expression has been detected in some tumor cells such as human brain malignant glioma, melanoma, colon cancer, liver cancer, lung cancer and other cells. TDO can catalyze the degradation of tryptophan, cause the lack of tryptophan and the accumulation of biological metabolites, leading to the inhibition of T lymphocyte proliferation, and even cause the apoptosis of T lymphocytes, and can promote the growth of tumor cells , Migration and deterioration. Because TDO plays a very important role in mediating tumor immune tolerance, immune escape, and maintaining the body's immune homeostasis, TDO inhibitors have become one of the hot topics of recent research.
  • TDO inhibitors promote the activation and proliferation of T cells through the inhibition of TDO protein expression, improve system immunity, thereby reversing tumor immunosuppression and exerting the ability to treat tumors.
  • the efficacy of a single immunotherapy is limited, so the combination therapy is gradually favored.
  • the method of coupling TDO small molecule inhibitors with chemotherapeutic drugs can not only exert the anticancer activity of chemotherapeutic drugs and the immune activity of small immune molecules, but also overcome some of the shortcomings of chemotherapeutic drugs.
  • Cisplatin the first metal antitumor drug used clinically, mainly acts on the guanine N7 target of DNA. It crosslinks with DNA to form an adduct, which causes tumor cells to undergo apoptosis, resulting in cell arrest and Cell death.
  • cisplatin drugs have some serious shortcomings: first, they show corresponding toxicity, mainly kidney toxicity and bone marrow toxicity; second, drug resistance after treatment, these shortcomings have limited the cisplatin divalent platinum to some extent Application of drugs. In recent years, tetravalent platinum complexes have attracted widespread attention due to their good stability and ability to reduce the reaction with nucleophilic substances in the body.
  • platinum (IV) ions can be reduced to generate platinum (II) ions in the body, which can interact with cancer cell DNA to cause apoptosis. Therefore, the introduction of a functional ligand in the axial direction of a tetravalent platinum complex derived from a divalent platinum drug can improve the targeting and fat solubility of the divalent platinum, overcome toxic side effects, and increase the antitumor activity of the compound.
  • Irinotecan is one of the antitumor drugs marketed in camptothecin derivatives, and it is also a semi-synthetic camptothecin derivative. It exists in the form of hydrochloride and has strong anticancer activity and good water solubility. Features. Irinotecan has significant effects on colon cancer, breast cancer, gastric cancer, and leukemia, and has a wide range of applications in China. Because it has no obvious cross-resistance with most commonly used anticancer drugs, it is also often used in combination with other drugs. With the deepening of research, the effect of irinotecan on antitumor is more and more recognized, but some of its disadvantages still cannot be ignored, such as poor stability, obvious toxic and side effects, etc. The improvement in nature is very beneficial.
  • the invention aims at designing and preparing a related conjugate of a small molecule inhibitor of TDO and a chemotherapeutic drug cisplatin or irinotecan, hoping to combine the advantages of both chemotherapy and immunotherapy to obtain high-efficiency and low-toxicity, and at the same time, improve immunity treatment Effect of drugs.
  • One of the objectives of the present invention is to use a spatial structure of a tetravalent platinum complex to introduce a TDO small molecule inhibitor group at an axial position to obtain a cisplatin-containing skeleton coupled with a TDO small molecule inhibitor derivative.
  • Tetravalent platinum compound Another object of the present invention is to use the steric structure of irinotecan to introduce a TDO small molecule inhibitor group at the hydroxyl position to obtain a compound containing an irinotecan skeleton coupled to a TDO small molecule inhibitor derivative.
  • the present invention also provides a method for preparing these compounds, and their applications in anti-tumor and immunity.
  • the present invention provides a TDO small molecule inhibitor derivative, and the compound structure of the TDO small molecule inhibitor derivative is shown by formulas I, 2, and 3.
  • Formula I is a known small molecule inhibitor of TDO; Formulas I and 2 are derivatives of Formula I, a small molecule inhibitor of TDO, referred to as TDO-OH for short.
  • the TDO-OH is prepared according to the reaction formula shown in Formula V.
  • TDO-OH is Formula I, and Formula I 3 uses the following preparation method:
  • TDO represents a derivative group of TDO small molecule inhibitor I1.
  • the conjugate is performed according to the reaction formula shown in III-A.
  • Y is Cl or OH
  • TBTU represents the coupling reagent O-benzotriazole-N, N, N ', N'-tetramethylurea tetrafluoroborate
  • TEA represents the catalyst triethylamine
  • DMSO stands for solvent dimethyl sulfoxide
  • TDO stands for derivative group of TDO small molecule inhibitor I1
  • TDO-OH stands for derivative of TDO small molecule inhibitor I1
  • DMF stands for solvent N, N-dimethylformamide
  • Pt (IV) Reactant A represents cis, cis, trans- [Pt (NH 3 ) 2 Cl 2 (OH) Cl]
  • Pt (IV) reactant B represents cis, cis, trans- [Pt (NH 3 ) 2 Cl 2 (OH) 2 ].
  • the conjugate is performed according to the reaction formula shown in Formula III-B.
  • EDCI represents the condensing agent 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride
  • DMAP represents the catalyst 4-dimethylaminopyridine
  • TDO represents the small molecule inhibitor of TDO.
  • Derivative group of I1 TDO-OH represents the derivative of TDO small molecule inhibitor I1
  • DMF represents the solvent N, N-dimethylformamide.
  • the preparation method specifically adopts the following method:
  • the preparation method of the antitumor conjugate containing the TDO small molecule inhibitor derivative specifically adopts the following method:
  • IV-A stands for cis, cis, trans- [Pt (NH 3 ) 2 Cl 2 (OH) Cl], which is prepared by the reaction of cisplatin and chlorosuccinimide in water
  • IV-B stands for cis, cis Trans- [Pt (NH 3 ) 2 Cl 2 (OH) 2 ] is obtained by the reaction of cisplatin and hydrogen peroxide in water.
  • the antitumor activity of human lung cancer cells A549 and NCI-H460 expressed by TDO, colon cancer cells HCT-116, and hepatocellular carcinoma HepG-2 and TDO-expressing gastric cancer cells SGC-7901 was evaluated in vitro. And irinotecan as a positive control. Observe the inhibitory effect of compounds on tumor cell growth at different concentrations.
  • the experimental data of compounds 1-3 and T1-T4 containing TDO inhibitor groups are shown in Table 2.
  • Compounds 1-3 are not toxic to cancer cells.
  • the inhibitory activities of compounds T1 and T3 on cancer cells A549, HepG-2, HCT-116, NCI-H460 and SGC-7901 are similar to cisplatin. It is worth noting that compounds T2 and T4 inhibitory activity of cancer cells was significantly higher than the above-mentioned cisplatin, wherein the active compound up to T2, the IC 50 values between 0.27-0.70 ⁇ M, IC HepG-2 for 50 value of 0.27 m, higher than that of cisplatin (the IC 50 value of 9.33 ⁇ M) About 35 times.
  • TDO inhibitor groups has significantly improved the antitumor activity of platinum (IV) complexes compared with cisplatin.
  • Compounds T2 and T4 containing hydroxyl groups in the axial direction are more important than compounds T1 and T1 containing chlorine atoms in the axial direction.
  • the activity of T3 is better, and the compound T2 linked by succinic anhydride is more active than the compound T4 linked by glutaric anhydride.
  • T5-T6 containing TDO inhibitor groups are shown in Table 3.
  • the activity of compounds T5 and T6 on cancer cells A549, HepG-2, HCT-116, NCI-H460, and SGC-7901 all showed a significant increase compared to irinotecan, and the IC 50 value of T5 was 1.82-2.87 ⁇ M between, IC 50 values between T6 1.99-3.86 ⁇ M, and toxicity to normal HUVEC cells were less than irinotecan.
  • T5 and T6 are the most cytotoxic to cancer cell HepG-2, with IC 50 values of 1.82 ⁇ M and 1.99 ⁇ M, respectively.
  • the experimental data showed that the antitumor activity of irinotecan was significantly improved after the introduction of a small molecule inhibitor of TDO, and the inhibitory activity of the two target compounds on the tested cancer cells was greatly improved, of which T5 was slightly better than T6.
  • the optimal compounds T2 and T5 were selected for the inhibition of TDO enzyme activity.
  • the IC 50 values of enzyme inhibition by compounds 1, 2, T2, and T5 were 0.22 ⁇ M, 0.68 ⁇ M, 0.89 ⁇ M and 0.78 ⁇ M, indicating that the derivative 2 of 1 and the coupling compounds T2 and T5 both retain the biological activity of 1, and have a significant inhibitory effect on TDO.
  • T549 expression was detected in A549, HepG-2, HCT-116, and NCI-H460 cell lines, among which HepG-2 expression level Highest, but no TDO expression was detected in the SGC-7901 cell line (data not shown). Therefore, HepG-2 was selected as a representative cell for subsequent in vitro immune mechanism research.
  • the compounds T2 and T5 were selected and mixed with HepG-2 and PBMCs (human peripheral blood mononuclear cells) cells, respectively, and the proliferation-promoting effects on immune cells were detected, and compound 1 was used as a control.
  • the experimental results are shown in Figure 5 (A and B).
  • the tested compounds all have pro-proliferative effects on immune T cells CD4 + and CD8 + cells.
  • compounds T2 and T5 are better than compound 1 and compound T5.
  • the effect is better than the mixture of compound 1 and irinotecan.
  • compounds T2 and T5 have a good inhibitory effect on TDO, and also have a good inhibitory effect on TDO protein overexpressed in HepG-2 cells.
  • the inhibition of TDO in cancer cells makes the metabolite kynurenine Inhibition of T cell production results in T cell proliferation.
  • the inhibitory effect of compound T2 (5, 10, 20 mg / kg) on the growth of human liver cancer cells HepG-2 xenograft tumors in nude mice was investigated, and the expression level of the metabolite kynurenine was examined.
  • Table 4 the test sample inhibits the growth of human liver cancer cell HepG-2 xenograft tumors in nude mice.
  • Figure 6 shows the effect of the test sample on the growth volume change of human liver cancer cell HepG-2 nude mice xenograft tumors.
  • Figure 7 shows the test sample's inhibitory effect on the growth of human liver cancer cell HepG-2 nude mice xenograft tumors.
  • FIG 8 is the effect of the test sample on the body weight of human liver cancer cells HepG-2 nude mice xenograft tumors in nude mice
  • Figure 9 is the test sample on the expression level of kynurenine in human liver cancer cells HepG-2 nude mice xenograft tumors Inhibitory effect. From the data in Table 4, it can be seen that the tumor inhibition rate of compound T2 is slightly lower than that of cisplatin (5mg / kg) at a low dose of 5mg / kg and a medium dose of 10mg / kg. At a high dose of 20mg / kg, the tumor inhibition rate of compound T2 Slightly higher than cisplatin (5mg / kg).
  • compound T2 has a dose-dependent inhibitory effect on the metabolite kynurenine, and the high-dose inhibitory effect is the best, indicating that compound T2 can inhibit the production of the metabolite kynurenine, which indirectly indicates that compound T2 can Promote T cell proliferation.
  • Figure 8 Effect of test samples on body weight of human liver cancer cells HepG-2 nude mice xenograft tumors in nude mice
  • the compounds T1-T6 prepared by the present invention have the structures shown in Table 1.
  • the conjugate prepared according to the method of the present invention determines the molecular structure of the compound by nuclear magnetic hydrogen spectrum and mass spectrometry, and some compounds are also characterized by nuclear magnetic carbon spectrum. In the mass spectrum of platinum-containing compounds, multiple isotopic peaks of platinum appear in the peaks of excimer ions.
  • the conjugates shown in Table 1 have significant inhibitory effects on some human cancer cells. These cells include lung cancer, colon cancer, liver cancer, and gastric cancer cells.
  • the in vitro immune-related mechanisms of compounds T2 and T5 and in vivo tests of T2 show that compounds containing small molecule inhibitor groups of TDO exhibit high-efficiency and low-toxicity antitumor immune activity and can be used to prepare anticancer immune drugs.
  • the eluent was a mixed solvent of dichloromethane and methanol (10: 1) to obtain 60 mg of a yellow product with a yield of 30%.
  • the MTT method was used to test the compounds T1-T6 prepared by the present invention for cytotoxic activity. Take the cells in the logarithmic growth phase and inoculate them in a 96-well culture plate with about 8000-10000 cells per well. The cells were cultured overnight, and the cells were administered after adherence. The administration group, the positive control group, and the negative control group were set.
  • the compound to be tested is prepared into a stock solution with physiological saline solution, DMF or DMSO, and diluted to a series of concentrations with cell culture medium before use, wherein the final concentration of DMF or DMSO does not exceed 4 ⁇ (the same applies to the following experiments). Three replicates were set up for each concentration.
  • Cisplatin was used as a positive control.
  • the rate of inhibition was calculated and IC 50 value was evaluated the cytotoxic activity of the compounds, the results shown in Table 2.
  • Irinotecan ( Ir) and its physical mixture with compound 1 (molar ratio 1: 1) were used as positive controls.
  • Test Example 1 Inhibition of TDO enzyme by compound
  • the level of kynurenine, a catalytic metabolite of TDO was measured by a microplate reader to evaluate the inhibitory enzyme activity of related compounds. Take a 96-well culture plate, thaw the TDO reaction solution, and add it to the culture plate, 180 ⁇ L per well, then add 10 ⁇ L of the compound-containing DMF solution as the administration group, and add 10 ⁇ L of blank DMF solution as the positive control and negative control group, respectively. Take 10 ⁇ L of TDO buffer into the negative control group, add 10 ⁇ L of TDO enzyme solution to the administration group and the positive control group, and then incubate for 90 minutes at room temperature.
  • Test method The WB method is used to detect the TDO expression level in the tumor cells studied by the present invention.
  • the cells in the logarithmic growth phase were taken and seeded in 6-well culture plates. After 24 hours, the cells were collected, the protein was separated and extracted, and the protein concentration was measured by a BCA protein assay reagent. Aliquots of each row of protein were added on 12% SDS polyacrylamide gel electrophoresis and transferred to a PVDF Hybond-P membrane (GE Healthcare). The membrane was incubated with Tween 20 (TBST) buffer and Tris buffered saline containing 5% skim milk for 1 hour, and then slowly rotated overnight at 4 ° C. The membrane was then incubated with the primary antibody at 4 ° C overnight.
  • Tween 20 Tween 20
  • the membrane was incubated with a peroxidase-labeled secondary antibody at RT (25 ° C) for 2 hours.
  • Western blots were detected by chemiluminescence reagents (Thermo Fischer Scientifics Ltd.).
  • ⁇ -actin was used as a control, and the results are shown in FIG. 2.
  • Test Example 3 Inhibition of TDO expression levels in tumor cells by compounds T2 and T5
  • Test method The WB method is used to evaluate the inhibitory ability of the representative compound prepared by the present invention on TDO in tumor cells with high TDO expression.
  • HepG-2 cells with high expression of T2 and T5 and TDO protein were selected for research. After incubating the compound with HepG-2 cells for 24 hours, the cells were collected.
  • Test Example 2 The results are shown in Figure 3.
  • Test Example 4 Effects of compounds T2 and T5 on the expression level of the metabolite kynurenine
  • Test method The HPLC method was used to detect and evaluate the expression level of the metabolite kynurenine in the representative compound prepared by the present invention.
  • the cells in the logarithmic growth phase were taken and seeded in a 6-well culture plate, cultured to adherence to the cells, and then administered, compounds T2, T5, and 1 solutions were added respectively, and the blank group was used as a control. After 48 hours, the cells were collected by centrifugation, and 100 ⁇ L of 20% trichloroacetic acid was added to precipitate the protein. The supernatant was centrifuged. The samples were analyzed by HPLC at 360 nm absorbance to detect the expression level of kynurenine. The results are shown in Figure 4.
  • Test Example 5 Compounds Proliferate on CD4 + and CD8 + Immune Cells
  • Test method The mixed leukocyte response (MLR) method is used to test the proliferative effect of the compounds T2 and T5 of the present invention on immune cells.
  • HepG-2 cells in the logarithmic growth phase were inoculated into a 6-well culture plate, replaced with a new medium after 24 hours of culture, and then compounded at 37 ° C for co-incubation. After two days of incubation, human peripheral blood mononuclear cells (PBMCs) were embedded and mixed for 6 days. After that, PBMCs were collected by centrifugation, stained with monoclonal antibodies APC-anti CD4 and PE-anti CD8, and analyzed by flow cytometry. The number of CD4 + and CD8 + immune cells after the test was tested. Compound 1 and blank were used as a control. The results are shown in Figure 5.
  • mice were used to evaluate the inhibitory effect, strength of the test sample on human liver cancer cell HepG-2 xenograft tumor growth in nude mice, and the inhibitory effect of kynurenine.
  • Test animals BALB / c nude mice, provided by Shanghai Lingchang Biotechnology Co., Ltd.
  • Age 5w, weight: 18-20g, gender: female, number of animals: 5 per group, 25 in total.
  • Drugs and reagents cisplatin and compound T2, cisplatin was dissolved in physiological saline, and T2 was dissolved in ultrasound with an appropriate amount of DMF and physiological saline.
  • the groups and dosing schedules are as follows:
  • Model control group tail vein injection of normal saline, 0.1ml / 10g, once a week for a total of 4 times;
  • Cisplatin (5mg / kg): The tail vein is injected with a drug solution of 5mg / kg, 0.1ml / 10g, once a week for a total of 4 times;
  • T2 (5mg / kg): The tail vein is injected with a drug solution of 5mg / kg, 0.1ml / 10g, once a week for a total of 4 times;
  • T2 (10mg / kg): The tail vein is injected with a drug solution of 10mg / kg, 0.1ml / 10g, once a week for a total of 4 times;
  • T2 (20mg / kg): 20mg / kg drug solution is injected into the tail vein, 0.1ml / 10g, once a week for a total of 4 times;
  • Evaluation index of antitumor activity relative tumor proliferation rate T / C (%), the calculation formula is as follows:
  • T RTV RTV in the treatment group
  • C RTV RTV in the model group.
  • the mean value is expressed by X ⁇ SD.
  • the t-test was used for statistical analysis between groups.
  • the results were analyzed statistically using SPSS (Staffstical Package for Social Science) 17.0.
  • SPSS Staffstical Package for Social Science 17.0. The results are shown in Table 4 and Figure 6-8.

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Abstract

一种TDO小分子抑制剂及其抗肿瘤偶联物和制备方法，该抑制剂为3-(2-吡啶-3-基-乙烯基)-1H-吲哚及其衍生物，其与化疗药物顺铂或伊立替康偶联形成抗肿瘤偶联物，结合化学疗法和免疫疗法的优势，获得了高效低毒、同时具有提高免疫力治疗作用的药物。

Description

一种TDO小分子抑制剂衍生物及其抗肿瘤偶联物和制备方法 技术领域
本发明涉及含有色氨酸2,3-双加氧酶(TDO)小分子抑制剂的抗肿瘤免疫偶联物，具体涉及在含有顺铂骨架的四价铂八面体结构中轴向引入TDO小分子抑制剂基团的化合物，以及在伊立替康活性基团羟基上引入TDO小分子抑制剂基团的化合物；本发明还涉及所述化合物的制备方法及在抗肿瘤与免疫方面的应用。
背景技术
色氨酸是人体必需氨基酸，不仅可以合成蛋白质，还可以经犬尿酸通路分解代谢，使色氨酸转化为一系列具有生物活性的代谢物质如犬尿氨酸、犬尿喹啉酸、喹啉酸等，其限速步骤为色氨酸转化为N-甲酰犬尿氨酸。色氨酸2,3-双加氧酶(TDO)是该通路代谢限速步骤的第一步限速酶之一，其表达受到多种信号的调控如糖皮质激素、L-色氨酸和犬尿氨酸等。研究发现，TDO主要在肝脏表达并调节系统色氨酸的水平，除肝脏外，TDO还在大脑部位表达。研究表明TDO可参与肿瘤的免疫逃逸并促进肿瘤的生长，而且在一些肿瘤细胞如人脑恶性胶质瘤、黑色素瘤、结肠癌、肝癌、肺癌等细胞中都检测到有TDO的表达。TDO可通过催化色氨酸的降解，引起色氨酸的缺乏和生物代谢产物的积累，导致T淋巴细胞的增殖被抑制，甚至能引起T淋巴细胞的的凋亡，同时可以促进肿瘤细胞的生长、迁移和恶化。由于TDO在介导肿瘤免疫耐受、免疫逃逸和维持机体免疫稳态中起着十分重要的作用，因此TDO抑制剂成为近期研究的热点之一。
TDO抑制剂通过对TDO蛋白表达的抑制，促进T细胞的激活和增殖，提高系统免疫力，从而逆转肿瘤免疫抑制，发挥治疗肿瘤的能力。但单一的免疫疗法效果有限，因此联合疗法逐渐受到青睐。通过TDO小分子抑制剂与化疗药物偶联的治疗方法不仅可以发挥化疗药物的抗癌活性和免疫小分子的免疫活性，而且还可以克服化疗药物的的一些缺陷。
顺铂，第一个在临床上被使用的金属抗肿瘤药物，主要作用于DNA的鸟嘌呤N7位靶点，通过与DNA交联形成加合物，使肿瘤细胞发生凋亡，导致细胞停滞和细胞死亡。但顺铂类药物存在一些严重缺陷：一是表现出相应的毒性，主要是肾脏毒性和骨髓毒性；二是用药后产生的耐药性，这些不足在一定程度上限制了顺铂类二价铂药物的应用。近年来，四价铂配合物因具有较好的稳定性、能够减少与体内亲核物质的反应等特性引起了人们广泛的关注。研究表明铂(IV)离子在体内经还原生成铂(II)离子后，可与癌细胞DNA作用导致细胞凋亡。因此，在由二价铂药物衍生的四价铂配合物轴向上引入功能型配体可以改进二价铂的靶向性和脂溶性，克服毒副作用，增加化合物的抗肿瘤活性。
伊立替康(Ir)是喜树碱类衍生物中上市的抗肿瘤药物之一，也是半合成喜树碱衍生物，以盐酸盐的形式存在，具有抗癌活性强和水溶性较好的特点。伊立替康对于结肠癌、乳腺癌、胃癌、白血病等多种癌症具有显著疗效，在我国有较为广泛的应用。由于其与多数常用抗癌药物无明显交叉耐药，也常用作与其它药物的联合用药。随着研究的深入，伊立替康在抗肿瘤方面的效果越来越多地被人们所认可，但它具有的一些缺点依然不容忽视，如稳定性差，毒副作用明显等，因此对于伊立替康药物性质的改善是十分有益的。
本发明旨在设计和制备TDO小分子抑制剂与化疗药物顺铂或伊立替康的相关偶联物，希望结合化学疗法和免疫疗法两者的优势，获得高效低毒、同时具有提高免疫力治疗作用的药物。
发明内容
技术问题：本发明的目的之一在于利用四价铂配合物的空间结构，在轴向位置引入TDO小分子抑制剂基团，获得与TDO小分子抑制剂衍生物偶联的含有顺铂骨架的四价铂化合物；本发明另一个目的在于利用伊立替康的空间结构，在羟基位置引入TDO小分子抑制剂基团，获得与TDO小分子抑制剂衍生物偶联的含有伊立替康骨架的化合物；本发明还提供了这些化合物的制备方法，以及它们在抗肿瘤与免疫方面的应用。
技术方案：本发明提供了一种TDO小分子抑制剂衍生物，该TDO小分子抑制剂衍生物的化合物结构由式I 2、I 3所示，
Figure PCTCN2018100399-appb-000001
式I中，式I 1为已知的TDO小分子抑制剂；式I 2和式I 3均为TDO小分子抑制剂式I 1的衍生物，简称为TDO-OH。
所述的TDO-OH的制备按式V所示的反应式进行，
Figure PCTCN2018100399-appb-000002
所述的制备TDO-OH即式I 2，式I 3采用如下制备方法：
1)化合物式I 1的制备：将1.5倍量的吡啶-3-乙酸盐酸盐和3.8倍量的三乙胺加入干燥的二氧六环中，室温搅拌10分钟，将等摩尔的吲哚-3-甲醛和2.2倍量的吡啶加入反应液中，回流反应24小时，将反应液冷却至室温，浓缩反应液，硅胶柱层析分离，洗脱液为石油醚:乙酸乙酯(2:1)混合溶剂，得橘黄色产物式I 1；
2)将式I 1溶于CH 2Cl 2中，加入1.5倍量的丁二酸酐和催化量的DMAP，45℃反应过夜，冷却至室温，浓缩反应液，硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH(20:1)混合溶剂，得淡黄色产物式I 2；
3)将式I 1溶于CH 2Cl 2，加入1.5倍量的戊二酸酐和催化量的DMAP，45℃反应过夜，冷却至室温，浓缩反应液，硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH(20:1)混合溶剂，得淡黄色产物式I 3。
所述偶联物的结构如式II所示：
Figure PCTCN2018100399-appb-000003
式II-A中，Y为Cl或OH；式II-A和II-B中，TDO代表TDO小分子抑制剂I1的衍生物基团。
所述的偶联物，按照III-A所示的反应式进行，
Figure PCTCN2018100399-appb-000004
式III-A中，Y为Cl或OH，TBTU代表偶联试剂O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸，TEA代表催化剂三乙胺，DMSO代表溶剂二甲亚砜；TDO代表TDO小分子抑制剂I1的衍生物基团，TDO-OH代表TDO小分子抑制剂I1的衍生物，DMF代表溶剂N,N-二甲基甲酰胺；Pt(IV)反应物A代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]；Pt(IV)反应物B代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]。
所述的偶联物，按照式III-B所示的反应式进行，
Figure PCTCN2018100399-appb-000005
式III-B中，EDCI代表缩合剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐，DMAP代表催化剂4-二甲氨基吡啶，TDO代表TDO小分子抑制剂I1的衍生物基团，TDO-OH代表TDO小分子抑制剂I1的的衍生物，DMF代表溶剂N,N-二甲基甲酰胺。
所述的制备方法具体采用如下方法：
III-A:将等摩尔的反应物TDO-OH和偶联试剂TBTU于无水DMF或DMSO混合，在室温搅拌下，加入与反应物TDO-OH等摩尔的TEA，然后加入与反应物TDO-OH等摩尔的Pt(IV)反应物A(Y＝Cl)或B(Y＝OH)，反应液于氮气保护下在30-60℃度下搅拌12-48小 时，然后减压除去溶剂，浓缩液经硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH混合溶剂，得到黄色固体产物；
所述的含有TDO小分子抑制剂衍生物的抗肿瘤偶联物的制备方法具体采用如下方法：
III-B:将等摩尔的反应物TDO-OH和缩合试剂EDCI于无水DMF混合，在室温搅拌下，加入与反应物TDO-OH等摩尔的DMAP，然后加入与反应物TDO-OH等摩尔的伊立替康，反应液于室温下搅拌12-48小时，然后减压除去溶剂，浓缩液经硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH混合溶剂，得到淡黄色固体产物。
所述的Pt(IV)反应物A和B的结构如式IV表示，
Figure PCTCN2018100399-appb-000006
其中IV-A代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]，通过顺铂和氯代丁二酰亚胺在水中反应制备获得；IV-B代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]，通过顺铂和双氧水在水中反应制备获得。
有益效果：
(一)、体外抗肿瘤活性
对所制备的化合物用TDO表达的人肺癌细胞A549和NCI-H460、结肠癌细胞HCT-116、肝癌细胞HepG-2与TDO不表达的胃癌细胞SGC-7901进行了体外抗肿瘤活性评价，顺铂和伊立替康作为阳性对照。观察化合物在不同浓度下对肿瘤细胞生长的抑制情况。
含有TDO抑制剂基团的化合物1-3和T1-T4的实验数据见表2。化合物1-3对癌细胞没有毒性，化合物T1、T3对癌细胞A549、HepG-2、HCT-116、NCI-H460和SGC-7901的抑制活性与顺铂相近，值得注意的是化合物T2和T4对上述癌细胞的抑制活性明显高于顺铂，其中化合物T2活性最高，IC 50值在0.27-0.70μM之间，对HepG-2的IC 50值为0.27μM，高于顺铂(IC 50值为9.33μM)大约35倍。表2数据表明TDO抑制剂基团的引入使铂(IV)配合物较顺铂的抗肿瘤活性有明显提高，轴向含有羟基基团的化合物T2和T4比轴向含有氯原子的化合物T1和T3的活性较优，且通过丁二酸酐相连的化合物T2比戊二酸酐相连的化合物T4的活性较高。
含有TDO抑制剂基团的化合物T5-T6的实验数据见表3。化合物T5和T6对癌细胞A549、HepG-2、HCT-116、NCI-H460和SGC-7901的活性与伊立替康相比都有较大的提高，其中T5的IC 50值在1.82-2.87μM之间，T6的IC 50值在1.99-3.86μM之间，且对正常细胞HUVEC的毒性都小于伊立替康。其中T5和T6对癌细胞HepG-2的细胞毒性最大，IC 50值分别为1.82μM和1.99μM。实验数据表明伊立替康在引入TDO小分子抑制剂之后抗肿瘤活性有显著的提高，两个目标化合物对所测试的癌细胞抑制活性都有较大提高，其中T5略优于T6。
以上结果表明，引入TDO小分子抑制剂基团至与顺铂为母体的铂(IV)配合物和伊立替康所形成的偶联物都表现出有效的抗肿瘤活性。
(二)、相关免疫机制研究
(1)基于细胞毒性研究，选取最优化合物T2和T5进行TDO酶活性抑制研究，如图1所示，化合物1、2、T2和T5对酶抑制IC 50值分别为0.22μM、0.68μM、0.89μM、0.78μM，表明1的衍生物2及偶联化合物T2和T5均保留了1的生物活性，对TDO有明显抑制作用。
(2)对选取的癌细胞进行TDO蛋白表达水平的检测，如图2所示，A549、HepG-2、HCT-116、NCI-H460细胞株均检测到TDO的表达，其中HepG-2表达水平最高，但SGC-7901细胞株未检测到TDO表达(数据未展示)。因此选择HepG-2为代表性细胞进行后续的体外免疫机制研究。
(3)选取化合物T2和T5对HepG-2细胞中TDO蛋白表达水平的抑制作用进行评估，顺铂、伊立替康和化合物1及其衍生物2、3作为对照，实验结果见图3。图3A中，顺铂对TDO表达没有抑制作用，化合物1、顺铂与1混合物、化合物2和化合物T2均表现出对TDO蛋白的抑制作用，其中化合物T2效果最佳；图3B中，伊立替康对TDO蛋白没有抑制作用，化合物1、2和3对TDO有较明显的作用，化合物T5和T6与化合物1-3相比，效果更显著，其中T5最优。
(4)选取化合物T2和T5对色氨酸产生的代谢产物犬尿氨酸的抑制作用进行评估，化合物1进行对照。如图4(A和B)所示，和空白组对比，化合物1和T2均对犬尿氨酸的表达水平有抑制作用，其中化合物T2比化合物1效果较好；化合物T5与1相比，可以看出犬尿氨酸的含量明显降低，表明化合物T5对犬尿氨酸有较强的抑制作用。
(5)选取化合物T2和T5分别与HepG-2、PBMCs(人外周血单个核细胞)细胞混合培养后检测对免疫细胞的促增殖效果，化合物1作为对照。实验结果见图5(A和B)，与空白组相比，所测试化合物均对免疫T细胞CD4 +和CD8 +细胞有促增殖作用，其中化合物T2和T5比化合物1效果较好，化合物T5的效果优于化合物1和伊立替康的混合物。
综合以上实验结果，化合物T2和T5对TDO有较好抑制作用，同时对HepG-2细胞中过表达的TDO蛋白也有较好的抑制作用，对癌细胞中TDO的抑制使代谢产物犬尿氨酸的产生受到抑制，结果导致T细胞的增殖。这些说明化合物T2和T5具有有效的免疫活性，可提高化疗药物的抗肿瘤治疗效果。
(三)、体内抗肿瘤活性
以顺铂为阳性对照，考察了化合物T2(5、10、20mg/kg)对人肝癌细胞HepG-2裸鼠异种移植肿瘤生长的抑制作用，并对代谢产物犬尿氨酸的表达水平进行了测试，实验结果见表4(受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤生长的抑制作用)。图6为受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤生长体积变化的影响，图7为受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤生长的抑制作用，图8为受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤裸鼠体重的影响，图9为受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤中犬尿氨酸表达水平的抑制作用。由表4数据可知，在低剂量5mg/kg和中剂量10mg/kg时化合物T2的抑瘤率略低于顺铂(5mg/kg)，在高剂量20mg/kg时，化合物T2的抑瘤率略高于顺铂(5mg/kg)。如图6和图7所示，无论是移植瘤的体积还是重量数据，不同剂量的化合物T2与顺铂能够显著抑制裸鼠异种移植瘤生长，高剂量化合物T2效果最佳。值得注意的是图8，不同剂量的化合物T2均对受试动物体重影响很小，而顺铂对受试动物体重影响较大，说明化合物T2的毒性较小，具有高效低毒的特点。最后，如图9所示，化合物T2对代谢产物犬尿氨酸呈剂量依赖性抑制作用，高剂量抑制效果最佳，表明化合物T2可以抑制代谢产物犬尿氨酸的产生，间接表明化合物T2可以促进T细胞的增殖。
附图说明
图1.受试样品对TDO酶的抑制活性
图2.所选择癌细胞中TDO的表达水平
图3.受试样品对HepG-2细胞中TDO的抑制效果
图4.受试样品对代谢产物犬尿氨酸表达水平的抑制作用
图5.受试样品对CD4 +和CD8 +免疫细胞的促增殖作用
图6.受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤生长体积变化的影响
图7.受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤生长的抑制作用
图8.受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤裸鼠体重的影响
图9.受试样品对肿瘤组织内代谢产物犬尿氨酸表达水平的抑制效果
具体实施方式
本发明所制备的化合物T1-T6，其结构见表1，
表1.化合物T1-T6的结构
Figure PCTCN2018100399-appb-000007
按本发明方法制备的偶联物经核磁氢谱和质谱确定了化合物的分子结构，有的化合物还经核磁碳谱进行了表征。含铂的化合物质谱中，准分子离子峰均出现铂元素的多个同位素峰。
表1所示偶联物对一些人癌细胞具有显著的抑制作用。这些细胞包括肺癌、结肠癌、 肝癌、胃癌细胞。化合物T2和T5的体外免疫相关机制和T2体内试验表明，含有TDO小分子抑制剂基团的化合物表现出高效低毒的抗肿瘤免疫活性，可应用于制备抗癌免疫药物。
本发明由下述实施例进一步的说明，但这些说明并不限制本发明。除特别指出外，一些反应物包括TDO小分子抑制剂化合物1、顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]和顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]都采用文献方法制备。
(一)、化合物的制备
实施例1.化合物T1的制备
将118.4mg(0.37mmol)化合物2和118.8mg(0.37mmol)TBTU溶于15mL无水DMF，室温搅拌10分钟，然后加入37.4mg(0.37mmol)TEA，继续搅拌5分钟后，再加入130.4mg(0.37mmol)顺,顺,反-[Pt(NH 3) 2Cl 3(OH)]，氮气保护下50℃反应48小时。浓缩反应液，浓缩液经硅胶柱层析分离，洗脱液为二氯甲烷和甲醇混合溶剂(10:1)，得黄色产物60mg，产率30％。 1H NMR(400MHz,DMSO-d 6)δ8.83(d,J＝2.0Hz,1H),8.48–8.39(m,2H),8.28(s,1H),8.16–8.08(m,2H),7.60(d,J＝16.7Hz,1H),7.46–7.36(m,4H),6.20(s,6H),3.26(t,J＝6.7Hz,2H),2.79(t,J＝6.6Hz,2H). 13C NMR(100MHz,DMSO-d 6)δ179.74,171.81,148.63,148.49,136.32,133.71,132.80,128.65,125.72,125.54,125.47,124.27,122.32,120.70,119.49,116.70,31.84,31.09.HRMS(ESI)m/z calculated for C 19H 21Cl 3N 4O 3Pt[M–H] –:653.03537,found:653.03537.
实施例2.化合物T2的制备
用反应物顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]替代顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]参照实施例1所述方法制备，得黄色产物，产率13％。
1H NMR(400MHz,DMSO-d 6)δ8.83(s,1H),8.46(d,J＝4.6Hz,1H),8.42(d,J＝7.7Hz,1H),8.28(s,1H),8.11(dd,J＝18.4,7.6Hz,2H),7.59(d,J＝16.7Hz,1H),7.44–7.37(m,4H),5.97(dd,J＝64.1,38.7Hz,6H),3.23(t,J＝6.6Hz,2H),2.70(t,J＝6.6Hz,2H). 13C NMR(100MHz,DMSO-d 6)δ180.05,172.04,148.63,148.50,136.32,133.72,132.78,128.63,125.70,125.63,125.41,124.27,124.24,122.35,120.70,119.41,116.70,31.94,31.14.HRMS(ESI)m/z calculated for C 19H 22Cl 2N 4O 4Pt[M+H] +:637.07508,found:637.07508.
实施例3.化合物T3的制备
120.3mg(0.36mmol)化合物3溶于5mL无水DMF，加入115.6mg(0.36mmol)TBTU，室温搅拌5分钟，然后加入36.4mg(0.36mmol)TEA，继续再搅拌10分钟后，加入126.9mg(0.36mmol)顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]，氮气保护下室温反应24小时。浓缩反应液，浓缩液经硅胶柱层析分离，洗脱液为二氯甲烷和甲醇混合溶剂(10:1)，得到黄色固体65mg，产率为27％。
1H NMR(400MHz,DMSO-d 6)δ8.84(s,1H),8.45(dd,J＝12.0,5.4Hz,2H),8.28(s,1H),8.12(t,J＝7.0Hz,2H),7.61(d,J＝16.7Hz,1H),7.41(dt,J＝12.9,6.8Hz,4H),6.23(s,6H),3.18(t,J＝7.3Hz,2H),2.42(t,J＝6.9Hz,2H),1.99–1.88(m,2H). 13C NMR(100MHz,DMSO-d 6)δ180.22,172.53,162.79,148.62,148.51,136.21,133.68,132.79,128.68,125.74,125.66,125.43,124.25,122.24,120.64,119.39,116.64,36.28,35.04,21.29.HRMS(ESI)m/z calculated for C 20H 23Cl 3N 4O 3Pt[M–H] –:667.05207,found:667.05207.
实施例4.化合物T4的制备
用反应物顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]替代顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]参照实施例3所述方法制备，得黄色产物，得到黄色固体34.0mg，产率18％。
1H NMR(400MHz,DMSO-d 6)δ8.84(d,J＝1.9Hz,1H),8.48–8.41(m,2H),8.30(s,1H),8.12(t,J＝8.4Hz,2H),7.61(d,J＝16.7Hz,1H),7.44–7.38(m,4H),6.15–5.90(m,6H),3.15(t,J＝7.5Hz,2H),2.34(t,J＝6.9Hz,2H),1.94–1.89(m,2H). 13C NMR(100MHz,DMSO-d 6)δ180.75,172.65,148.61,148.52,136.20,133.72,132.79,128.67,125.86,125.72,125.37,124.26,122.32,120.67,119.35,116.64,35.98,35.18,21.52.HRMS(ESI)m/z calculated for C 20H 24Cl 2N 4O 4Pt[M+H] +:651.24809,found:651.24809.
实施例5.化合物T5的制备
99.2mg(0.31mmol)化合物2和59.4mg(0.31mmol)EDCI溶于10mL无水DMF中，室温搅拌，加入37.9mg(0.31mmol)DMAP，继续搅拌5分钟后，再加入180.0mg(0.31mmol)伊立替康，在室温下反应24小时。浓缩反应液，浓缩液经硅胶柱层析分离，洗脱液为二氯甲烷和甲醇混合溶剂(20:1)，得到淡黄色固体87mg，产率：51.7％。
1H NMR(600MHz,CDCl 3)δ8.68(d,J＝1.7Hz,1H),8.46(dd,J＝4.7,1.3Hz,1H),8.32(d,J＝9.1Hz,1H),8.25(d,J＝8.1Hz,1H),7.87(d,J＝2.3Hz,1H),7.79(d,J＝7.9Hz,1H),7.72(d,J＝7.9Hz,1H),7.62(dd,J＝9.1,2.4Hz,1H),7.55(s,1H),7.50(s,1H),7.27(d,J＝6.7Hz,1H),7.18–7.11(m,2H),7.08(d,J＝16.5Hz,1H),6.65(t,J＝7.9Hz,1H),5.68(d,J＝16.9Hz,1H),5.38(d,J＝16.9Hz,1H),5.22–5.12(m,2H),4.45(dd,J＝59.9,12.9Hz,2H),3.37(ddd,J＝15.5,9.3,5.2Hz,1H),3.26–3.17(m,2H),3.12(dd,J＝13.3,7.5Hz,2H),2.98–2.91(m,2H),2.73(s,6H),2.28(dd,J＝14.1,7.4Hz,1H),2.17(dd,J＝14.2,7.4Hz,1H),2.10(d,J＝11.0Hz,1H),2.04(d,J＝8.0Hz,1H),1.76(s,6H),1.53(s,2H),1.37(t,J＝7.6Hz,3H),1.03(t,J＝7.5Hz,3H). 13C NMR(150MHz,CDCl 3)δ171.55,169.15,167.70,157.38,153.16,151.61,150.47,148.42,148.19,147.17,146.73,146.29,145.23,136.36,133.09,132.39,131.81,128.13,127.51,127.18,125.98,125.82,125.47,124.03,123.59,122.70,121.61,120.43,119.64,119.31,116.80,114.62,96.76,76.55,66.99,62.34,50.22,49.27,44.43,44.09,31.59,30.60,29.70,28.35,28.21,27.49,26.05,24.54,23.15,14.00,7.67.HRMS(ESI)m/z calculated for C 52H 52N 6O 8[M+H] +:889.37752,found:889.37752.
实施例6.化合物T6的制备
用反应物3替代反应物2参照实施例5所述方法制备，得淡黄色产物，得到黄色固体92mg，产率43.4％。
1H NMR(600MHz,CDCl 3)δ8.67(d,J＝1.7Hz,1H),8.45(dd,J＝4.7,1.3Hz,1H),8.32(d,J＝9.1Hz,1H),8.25(d,J＝8.0Hz,1H),7.87(d,J＝2.4Hz,1H),7.78(d,J＝8.0Hz,1H),7.72(d,J＝7.9Hz,1H),7.63(dd,J＝9.1,2.4Hz,1H),7.55(s,1H),7.50(s,1H),7.28–7.24(m,1H),7.19–7.12(m,2H),7.08(d,J＝16.5Hz,1H),6.70–6.58(m,1H),5.67(d,J＝16.9Hz,1H),5.38(d,J＝16.9Hz,1H),5.17(dd,J＝45.1,18.6Hz,2H),4.48(d,J＝13.0Hz,1H),4.38(d,J＝12.6Hz,1H),3.41–3.31(m,1H),3.27–3.16(m,2H),3.12(dd,J＝13.6,6.9Hz,2H),2.98–2.89(m,2H),2.61(s,5H),2.45(s,3H),2.29–2.24(m,1H),2.16(dd,J＝14.2,7.4Hz,1H),1.99(t,J＝14.3Hz,2H),1.69–1.60(m,6H),1.49(s,2H),1.37(t,J＝7.7Hz,3H),1.03(t,J＝7.5Hz,3H). 13C NMR(150MHz,CDCl 3)δ171.56,169.15,167.70,157.37,153.08,151.67,150.31,148.38,148.15,147.20,146.71,146.28,145.26,136.35,133.11,132.42,131.86,128.13,127.49,127.21,125.87,125.80,125.45,124.03,123.62, 122.73,121.62,120.41,119.65,119.33,116.79,114.69,96.77,76.54,66.99,62.75,50.08,49.27,44.05,43.69,31.57,31.44,30.61,30.19,29.70,28.36,27.55,26.73,24.80,23.77,23.15,14.01,7.68.HRMS(ESI)m/z calculated for C 53H 54N 6O 8[M+H] +:903.37503,found:903.37503.
实施例7.化合物1-3的制备
(1)化合物1的制备
化合物1的合成根据文献方法(J.Med.Chem.2011,54,5320-5334)，将2.7g(15.5mmol)的吡啶-3-乙酸盐酸盐和3.9g(38.5mmol)的三乙胺加入干燥的二氧六环中，室温搅拌10分钟，1.5g(10.3mmol)的吲哚-3-甲醛和1.9g(22.3mmol)的吡啶加入反应液中，回流反应24小时,将反应液冷却至室温，浓缩反应液，柱层析分离得橘黄色产物1，洗脱液为石油醚:乙酸乙酯为2:1混合溶剂；
1H NMR(400MHz,DMSO-d 6)δ11.42(s,1H),8.77(d,J＝2.1Hz,1H),8.38(dd,J＝4.7,1.5Hz,1H),8.06(d,J＝7.8Hz,1H),8.02(dt,J＝8.0,1.9Hz,1H),7.69(d,J＝2.6Hz,1H),7.57(d,J＝16.6Hz,1H),7.45(d,J＝8.0Hz,1H),7.36(dd,J＝7.9,4.7Hz,1H),7.21–7.17(m,1H),7.16–7.10(m,2H).HRMS(ESI)m/z calculated for C 15H 12N 2[M+H] +:221.10375,found:221.10375.
(2)化合物2的制备
将1.0g(4.5mmol)1溶于50mL CH 2Cl 2中，加入0.9g(9.0mmol)的丁二酸酐和催化量的DMAP，45℃反应过夜，冷却至室温，浓缩反应液，柱层析分离，洗脱液为二氯甲烷:甲醇为20:1混合溶剂，得淡黄色产物2，。
1H NMR(400MHz,DMSO-d 6)δ12.27(s,1H),8.83(d,J＝2.1Hz,1H),8.46(dd,J＝4.7,1.5Hz,1H),8.40(dd,J＝7.2,1.4Hz,1H),8.32(s,1H),8.13(dd,J＝6.8,1.6Hz,1H),8.10(dt,J＝7.9,1.8Hz,1H),7.58(d,J＝16.7Hz,1H),7.44–7.38(m,4H),3.32(d,J＝6.8Hz,2H),2.72–2.69(m,2H).HRMS(ESI)m/z calculated for C 19H 16N 2O 3[M+H] +:321.12104,found:321.12104.
(3)化合物3的制备
将1.0g(4.5mmol)1溶于50mL CH 2Cl 2，加入1.0g(9mmol)的戊二酸酐和催化量的DMAP，45℃反应过夜，冷却至室温，浓缩反应液，柱层析分离，洗脱液为二氯甲烷:甲醇为20:1混合溶剂，得淡黄色产物3。
1H NMR(400MHz,DMSO-d 6)δ12.15(s,1H),8.82(d,J＝2.0Hz,1H),8.46(dd,J＝4.7,1.3Hz,1H),8.43(d,J＝7.9Hz,1H),8.25(s,1H),8.11(dd,J＝16.2,7.7Hz,2H),7.57(d,J＝16.7Hz,1H),7.43–7.36(m,4H),3.13(t,J＝7.3Hz,2H),2.41(t,J＝7.3Hz,2H),1.96(dd,J＝14.6,7.3Hz,2H).HRMS(ESI)m/z calculated for C 20H 18N 2O 3[M+H] +:335.14297,found:335.14297.
(二)、化合物的体外细胞毒活性测试
实验方法：采用MTT方法对本发明所制备的化合物T1-T6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8000-10000个细胞。过夜培养，待细胞贴壁后进行给药，分别设给药组，阳性对照组和阴性对照组。待测的化合物用生理盐水溶液、DMF或DMSO配制成贮液，临用前用细胞培养基稀释成一系列浓度，其中DMF或DMSO的终浓度不超过4‰(下面实验类同)。每个浓度设3个复孔。加药后培养72小时，加20μL浓度为5mg/mL的MTT，37℃孵育4小时，去上清，加入150μL的DMSO溶解甲瓒。用酶标仪在490波长下测定每孔的OD值，并计算抑制率， 做浓度-抑制率曲线计算IC 50值。
试验例1.化合物T1-T4的细胞毒活性
测试化合物1-3和T1-T4对肝癌细胞HepG-2、结肠癌细胞HCT-116、肺癌细胞A549、肺癌细胞NCI-H460和胃癌细胞SGC-7901及人脐静脉内皮细胞HUVEC的细胞毒活性，顺铂作为阳性对照。观察化合物在不同浓度下对肿瘤细胞生长的抑制情况，计算抑制率及其IC 50值来评价化合物的细胞毒活性，结果见表2。
表2.化合物T1-T4的细胞毒活性
Figure PCTCN2018100399-appb-000008
试验例2.化合物T5-T6的细胞毒活性
测试化合物T5-T6对肝癌细胞HepG-2、结肠癌细胞HCT-116、肺癌细胞A549、肺癌细胞NCI-H460和胃癌细胞SGC-7901及人脐静脉内皮细胞HUVEC的细胞毒活性，伊立替康(Ir)及其与化合物1的物理混合物(摩尔比1:1)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，计算抑制率及其IC 50值来评价药物的细胞毒活性，结果见表3。
表3.化合物T5-T6的细胞毒活性
Figure PCTCN2018100399-appb-000009
(三)、相关免疫机制研究
试验例1.化合物对TDO酶的抑制
实验方法：通过酶标仪检测TDO催化代谢产物犬尿氨酸的水平，评估相关化合物的抑制酶活性。取96孔培养板，解冻TDO反应溶液后加入培养板中，每孔180μL，然后加入10μL含化合物的DMF溶液作为给药组，加入10μL空白的DMF溶液分别作为 阳性对照和阴性对照组。取10μL TDO缓冲液加入阴性对照组，10μL TDO酶溶液加入给药组和阳性对照组，然后室温孵育90分钟，用酶标仪在321波长下测定每孔的OD值，并计算抑制率，做浓度-抑制率曲线计算IC 50值。测试化合物1、2、T2和T5对酶的抑制能力，观察化合物在不同化合物对酶的抑制情况，计算抑制率及其IC 50值来评价药物的酶活性，结果见图1。
试验例2.肿瘤细胞中TDO的表达水平
试验方法：采用WB方法对本发明所研究的肿瘤细胞中的TDO表达水平进行检测。取处于对数生长期的细胞，接种于6孔培养板内。24小时后，收集细胞，分离提取蛋白，通过BCA蛋白质测定试剂测量蛋白质浓度。在12％SDS聚丙烯酰胺凝胶电泳上分加每行等分的蛋白质并转移至PVDF Hybond-P膜(GE Healthcare)。将膜与含Tween 20(TBST)缓冲液和含有5％脱脂牛奶的Tris缓冲盐水一起孵育1小时，然后在4℃缓慢旋转过夜。然后将膜与一抗在4℃下孵育过夜。接下来，将膜与过氧化物酶标记的二抗在RT(25℃)孵育2小时。通过化学发光试剂(Thermo Fischer Scientifics Ltd.)检测蛋白质印迹。使用β-肌动蛋白作为对照，结果见图2。
试验例3.化合物T2和T5对肿瘤细胞中TDO表达水平的抑制
试验方法：采用WB方法对本发明制备的代表性化合物对TDO高表达的肿瘤细胞中TDO的抑制能力进行评估。选取活性最好的化合物T2和T5与TDO蛋白高表达的HepG-2细胞进行研究。将化合物与HepG-2细胞共孵育24小时后，收集细胞，实验处理方法参考试验例2，结果见图3。
试验例4.化合物T2和T5对代谢产物犬尿氨酸表达水平的影响
试验方法：采用HPLC方法对本发明制备的代表性化合物对代谢产物犬尿氨酸表达水平进行检测评估。取处于对数生长期的细胞，接种于6孔培养板内，培养至细胞贴壁后进行给药，分别加入化合物T2、T5和1溶液，空白组作为对照。48小时后离心收集细胞，加入100μL 20％三氯乙酸使蛋白沉淀，离心取上清液，样品使用HPLC在360nm吸光度下进行分析检测，检测犬尿氨酸的表达水平，结果见图4.
试验例5.化合物对CD4 +和CD8 +免疫细胞的增殖作用
试验方法：采用混合白细胞反应(MLR)的方法测试本发明化合物T2和T5对免疫细胞的促增殖作用。处于生长对数期的HepG-2细胞，接种到6孔培养板内，培养24小时后更换新培养基，后加入化合物在37度下进行共孵育。孵育两天后嵌入人外周血单核细胞(PBMCs)，混合孵育6天，后离心收集PBMCs细胞，用单抗APC-anti CD4和PE-anti CD8染色后用流式进行分析检测。测试CD4 +和CD8 +免疫细胞的在用药后的数量，化合物1和空白做对照，结果见图5。
(四)、化合物T2的体内抗肿瘤活性
实验方法：采用裸鼠，评价受试样品对人肝癌细胞HepG-2裸鼠异种移植肿瘤生长的抑制作用、作用强度及犬尿氨酸的抑制效果。受试动物：BALB/c裸小鼠，由上海灵畅生物科技有限公司提供。实验动物生产许可证：SCXK(沪)2013-0018，合格证编号：2013001829927，实验动物使用许可证：SYXK(苏)2012-0004。日龄：5w，体重：18-20g，性别：雌性，动物数：每组5只，共25只。药物及试剂：顺铂和化合物T2，顺铂用生理盐水溶解、T2用适量DMF和生理盐水超声溶解。
组别及给药方案如下：
模型对照组：尾静脉注射生理盐水，0.1ml/10g，每周一次，共给药4次；
顺铂(5mg/kg)：尾静脉注射5mg/kg的药物溶液，0.1ml/10g，每周一次，共给药4次；
T2(5mg/kg)：尾静脉注射5mg/kg的药物溶液，0.1ml/10g，每周一次，共给药4次；
T2(10mg/kg)：尾静脉注射10mg/kg的药物溶液，0.1ml/10g，每周一次，共给药4次；
T2(20mg/kg)：尾静脉注射20mg/kg的药物溶液，0.1ml/10g，每周一次，共给药4次；
收集培养的人肝癌HepG-2细胞悬液，浓度为1×10 7个/mL，以每只0.1mL接种于裸小鼠右侧腋窝皮下。裸鼠移植瘤用游标卡尺测量移植瘤直径，接种18天后，肿瘤生长至100-150mm 3时将动物随机分组，每组5只。同时，各组裸鼠开始给药，给药方案见组别与给药方案，使用测量瘤径的方法，动态观察受试样品的抗肿瘤效应。实验结束后，随即处死裸鼠，手术剥取瘤块称重。
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2ab 2，其中a、b分别表示长宽。根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：
Figure PCTCN2018100399-appb-000010
其中，T RTV：治疗组RTV；C RTV：模型组RTV。
抗肿瘤活性的评价指标：肿瘤生长抑制率(％)，计算公式如下：
Figure PCTCN2018100399-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，结果如表4和图6-8。
分别取上述各组小鼠肿瘤组织，用包含有01％抗坏血酸和0.1％EDTA的高氯酸将肿瘤组织均质化释放出犬尿氨酸，十分钟后离心收集上清液，用HPLC来检测上述样品中犬尿氨酸的含量。测试各组样品中犬尿氨酸的表达水平，分析给药后小鼠肿瘤区域的犬尿氨酸水平，间接检测免疫细胞的增殖水平，结果如图9。
表4.受试样品对人肝癌细胞HepG-2裸鼠异种移植瘤生长的抑制作用
Figure PCTCN2018100399-appb-000012
Figure PCTCN2018100399-appb-000013
Figure PCTCN2018100399-appb-000014
与模型对照组比较，*P<0.05,**P<0.01

Claims (9)

  1. 一种TDO小分子抑制剂衍生物，其特征在于该TDO小分子抑制剂衍生物的化合物结构由式I2、I3所示，
    Figure PCTCN2018100399-appb-100001
    式I中，式I1为已知的TDO小分子抑制剂；式I2和式I3均为TDO小分子抑制剂式I1的衍生物，简称为TDO-OH。
  2. 一种如权利要求1所述的TDO小分子抑制剂衍生物的制备方法，其特征在于所述的TDO-OH的制备按式V所示的反应式进行，
    Figure PCTCN2018100399-appb-100002
  3. 如权利要求2所述的TDO小分子抑制剂衍生物的制备方法，其特征在于所述的制备TDO-OH即式I2，式I3采用如下制备方法：
    1)化合物式I1的制备：将1.5倍量的吡啶-3-乙酸盐酸盐和3.8倍量的三乙胺加入干燥的二氧六环中，室温搅拌10分钟，将等摩尔的吲哚-3-甲醛和2.2倍量的吡啶加入反应液中，回流反应24小时，将反应液冷却至室温，浓缩反应液，硅胶柱层析分离，洗脱液为石油醚:乙酸乙酯(2:1)混合溶剂，得橘黄色产物式I1；
    2)将式I1溶于CH 2Cl 2中，加入1.5倍量的丁二酸酐和催化量的DMAP，45℃反应过夜，冷却至室温，浓缩反应液，硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH(20:1)混合溶剂，得淡黄色产物式I2；
    3)将式I1溶于CH 2Cl 2，加入1.5倍量的戊二酸酐和催化量的DMAP，45℃反应过夜，冷却至室温，浓缩反应液，硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH(20:1)混合溶剂，得淡黄色产物式I3。
  4. 一类含有如权利要求1所述的TDO小分子抑制剂衍生物的抗肿瘤偶联物，其特征在于所述偶联物的结构如式II所示：
    Figure PCTCN2018100399-appb-100003
    式II-A中，Y为Cl或OH；式II-A和II-B中，TDO代表TDO小分子抑制剂I1的衍生物基团。
  5. 一种如权利要求4所述的含有TDO小分子抑制剂衍生物的抗肿瘤偶联物的制备方法，其特征在于所述的偶联物，按照III-A所示的反应式进行，
    Figure PCTCN2018100399-appb-100004
    式III-A中，Y为Cl或OH，TBTU代表偶联试剂O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸，TEA代表催化剂三乙胺，DMSO代表溶剂二甲亚砜；TDO代表TDO小分子抑制剂I1的衍生物基团，TDO-OH代表TDO小分子抑制剂I1的衍生物，DMF代表溶剂N,N-二甲基甲酰胺；Pt(IV)反应物A代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]；Pt(IV)反应物B代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]。
  6. 一种如权利要求4所述的含有TDO小分子抑制剂衍生物的抗肿瘤偶联物的制备方法，其特征在于所述的偶联物，按照式III-B所示的反应式进行，
    Figure PCTCN2018100399-appb-100005
    式III-B中，EDCI代表缩合剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐，DMAP代表催化剂4-二甲氨基吡啶，TDO代表TDO小分子抑制剂I1的衍生物基团，TDO-OH代表TDO小分子抑制剂I1的衍生物，DMF代表溶剂N,N-二甲基甲酰胺。
  7. 如权利要求5所述的含有TDO小分子抑制剂衍生物的抗肿瘤偶联物的制备方法， 其特征在于所述的制备方法具体采用如下方法：
    III-A:将等摩尔的反应物TDO-OH和偶联试剂TBTU于无水DMF或DMSO混合，在室温搅拌下，加入与反应物TDO-OH等摩尔的TEA，然后加入与反应物TDO-OH等摩尔的Pt(IV)反应物A(Y＝Cl)或B(Y＝OH)，反应液于氮气保护下在30-60℃度下搅拌12-48小时，然后减压除去溶剂，浓缩液经硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH混合溶剂，得到黄色固体产物；
  8. 如权利要求6所述的含有TDO小分子抑制剂衍生物的抗肿瘤偶联物的制备方法，其特征在于所述的制备方法具体采用如下方法：
    III-B:将等摩尔的反应物TDO-OH和缩合试剂EDCI于无水DMF混合，在室温搅拌下，加入与反应物TDO-OH等摩尔的DMAP，然后加入与反应物TDO-OH等摩尔的伊立替康，反应液于室温下搅拌12-48小时，然后减压除去溶剂，浓缩液经硅胶柱层析分离，洗脱液为CH 2Cl 2与CH 3OH混合溶剂，得到淡黄色固体产物。
  9. 如权利要求5所述的含有TDO小分子抑制剂衍生物的抗肿瘤偶联物的制备方法，其特征在于所述的Pt(IV)反应物A和B的结构如式IV表示，
    Figure PCTCN2018100399-appb-100006
    其中IV-A代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH)Cl]，通过顺铂和氯代丁二酰亚胺在水中反应制备获得；IV-B代表顺,顺,反-[Pt(NH 3) 2Cl 2(OH) 2]，通过顺铂和双氧水在水中反应制备获得。
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