WO2022077791A1 - 新型吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用 - Google Patents

新型吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用 Download PDF

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WO2022077791A1
WO2022077791A1 PCT/CN2021/072121 CN2021072121W WO2022077791A1 WO 2022077791 A1 WO2022077791 A1 WO 2022077791A1 CN 2021072121 W CN2021072121 W CN 2021072121W WO 2022077791 A1 WO2022077791 A1 WO 2022077791A1
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cancer
ido1
nicotinic acid
derivatives
tumor
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谭瀛轩
向道凤
颜道静
谭相宝
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杭州星鳌生物科技有限公司
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Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a class of nicotinic acid derivatives for indoleamine 2,3-dioxygenase IDO1 inhibitors and their application in the preparation of immune antitumor drugs
  • Tumor is one of the major diseases that seriously endanger human life and health, manifesting as excessive cell proliferation and abnormal differentiation. According to WHO statistics, the number of cancer cases worldwide may increase to 24 million each year. At the same time, cancer imposes a huge burden on the global economy.
  • Tumor immunotherapy is an emerging tumor treatment modality following surgery, radiotherapy and chemotherapy. It is a cancer treatment that harnesses the power of stimulating or enhancing the body's own immune system to prevent, control and eliminate cancer. Compared with traditional tumor treatment methods, immunotherapy has its own unique advantages, which can improve the efficacy of traditional treatment and reduce the adverse reactions caused by traditional treatment. Innate immune system agonists, immune checkpoint blockers and cellular immunotherapy are the main research fields of immunotherapy at present. Tumor immunotherapy strategies based on metabolic regulation can improve the effectiveness of immunotherapy and benefit more patients. The regulation of tumor metabolism opens up new directions for improving tumor immunotherapy.
  • the body's immune system is responsible for recognizing self and non-self, thereby protecting the body from exogenous and endogenous diseases.
  • the human immune system consists of white blood cells and lymphoid organs and lymphoid tissues, including the thymus, spleen, tonsils, lymph nodes, lymphatic vessels, and bone marrow.
  • the immune system maintains the continuous homeostasis of the body by identifying and eliminating various threats.
  • Cancer immunotherapy refers to the stimulation of the immune system against cancer cells by introducing vaccines, cytokines, antibodies, or the transferred immune cells themselves.
  • the success of immunotherapy in oncology such as immune checkpoint inhibitors and CAR-T cell therapy, has established its role in cancer treatment. Cancer uses multiple mechanisms to select for host-tumor immune interactions, leading to immune escape. Over the past few years, our study of host-tumor interactions has continued to progress, resulting in a variety of promising immunotherapeutic approaches.
  • Indoleamine 2,3-dioxygenase is a monomeric heme protein with a molecular weight of 45KD. IDO1 can catalyze molecules with indole rings, so it is called indoleamine 2,3-dioxygenase. IDO 1 is a key rate-limiting metabolic enzyme in the tryptophan metabolic pathway. Numerous studies have shown that IDO1 is highly expressed in various types of human cancers, which subsequently leads to the accumulation of tryptophan metabolites, resulting in immune tolerance of the body to tumor antigens, and ultimately leading to tumor immune escape. Numerous preclinical and clinical trial studies have shown that IDO1 inhibitors are effective tools against a variety of cancers.
  • IDO1 inhibitors for the treatment of tumors are underway. They can effectively activate tumor-infiltrating T cells and/or reduce tumor-resident immunosuppressive regulatory T cells, thereby improving human immunity and achieving anti-tumor goals. Based on the important role of IDO1 in tumor immunotherapy and there is currently no IDO1 inhibitor on the market for tumor treatment, therefore, the screening of new IDO1 inhibitors and the research on anti-tumor drugs based on IDO1 enzyme inhibitors are of great significance .
  • Cyclic dinucleotide synthase is an important cytoplasmic DNA sensor in the innate immune pathway.
  • cGAS catalyzes the synthesis of cGAMP from ATP and GTP in vivo.
  • cGAMP induces the production of interferon IFN- ⁇ and other cytokines through the STING protein pathway on the endoplasmic reticulum membrane, regulates the expression of downstream proteins, and induces cell growth arrest and apoptosis. , produce an antiviral effect.
  • the STING pathway can regulate the innate immune recognition of immunogenic tumors and promote the antitumor effect of interferons.
  • IFN- ⁇ plays an anti-tumor effect through TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in vivo and promotes tumor cell apoptosis.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • cGAMP is a key stimulator of the innate immune response, an endogenous activator of STING, and has an immune antitumor effect.
  • niacin analogs as inhibitors of IDO1 enzyme alone can significantly inhibit the growth of transplanted tumors in mice, in combination with STING agonists of the innate immune pathway and/or in combination with immune checkpoint inhibitors and/or with antitumor drugs When used in combination, the efficacy of inhibiting tumor growth is better, and the safety is also very good. Therefore, such nicotinic acid derivatives have broad application prospects in the preparation of immune antitumor drugs.
  • the present invention provides an innovative inhibitor of indoleamine 2,3-dioxygenase IDO1, namely nicotinic acid derivatives (derivatives of 3-pyridinepropionic acid).
  • the derivatives of 3-pyridine propionic acid can effectively inhibit the metabolism of tryptophan catalyzed by indoleamine 2,3-dioxygenase IDO1, and can significantly inhibit the highly expressed IDO1 enzyme activity in human cancer cells. lower.
  • These niacin derivatives as inhibitors of IDO1 enzyme alone can significantly inhibit the growth of transplanted tumors in mice, and they are used in combination with innate immune pathway STING agonists or immune checkpoint inhibitors to inhibit tumor growth with better efficacy and safety. very good. Therefore, such nicotinic acid derivatives have broad application prospects in the preparation of immune antitumor drugs.
  • Nicotinic acid derivatives are novel inhibitors of indoleamine 2,3-dioxygenase IDO1.
  • the specific characteristics of nicotinic acid derivatives are: using nicotinic acid as the structural core, using carboxylic acid isosteres to introduce atoms that are easy to form hydrogen bonds such as S, N, O, F, etc., to enhance the interaction between these inhibitors and IDO1 enzyme protein.
  • the binding force of the molecule stabilizes the complex formed by the inhibitor and the IDO1 protein molecule, and increases the effect of inhibiting the enzyme activity of IDO1.
  • Specific examples of nicotinic acid derivatives are shown in Figure 1 (structural formula and names of nicotinic acid derivatives (1-10)).
  • nicotinic acid derivatives derivatives of 3-pyridine propionic acid
  • the anti-tumor drug is used in the treatment of various types of tumors, including but not limited to colorectal cancer, breast cancer, testicular cancer, ovarian cancer, prostate cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, head and neck cancer.
  • Neck cancer thyroid cancer, osteosarcoma, bladder cancer, cervical cancer, and germ cell tumors, etc.
  • STING agonists include, but are not limited to, cGAMP and its derivatives.
  • the anti-tumor drug is used in the treatment of various types of tumors.
  • Immune checkpoint inhibitors include but are not limited to anti-PD1/PD-L1, anti-CD-47, anti-VEGF, anti-CTLA-4 and other monoclonal antibodies and nanobodies of monoclonal antibodies; the anti-tumor drugs are used in Treats various types of tumors.
  • nicotinic acid derivatives derivatives of 3-pyridine propionic acid
  • chemotherapeutic antitumor drugs include, but are not limited to, platinum metal drugs, 5-fluorouracil, violetanol and other anti-tumor drugs, which are used in the treatment of various types of tumors.
  • nicotinic acid derivatives derivatives of 3-pyridinepropionic acid
  • pharmaceutically acceptable pharmaceutical preparations including but not limited to intravenous injection, intramuscular injection, One or more of subcutaneous injection, intravenous drip, nasal drip, oral administration, etc. are used for the treatment of the above-mentioned related tumors.
  • Example 1 Inhibitory effect of niacin derivatives on IDO1 metalloenzyme
  • IDO1 Indoleamine 2,3-dioxygenase 1
  • pGEX-6P-1 was selected as the vector for constructing the human IDO1 expression plasmid.
  • the IDO1 protein with the GST fusion tag can be expressed in E. coli.
  • Recombinant HRV 3C protease was used to digest the GST-tagged protein, and the enzyme was digested overnight at 16 °C.
  • the digested IDO1 protein was purified twice using Glutathione Sepharose affinity column to obtain electrophoresis pure protein, which was identified by SDS-Page and mass spectrometry.
  • the protein samples were prepared by calculating the UV absorbance of 1 ⁇ M protein at 404 nm according to the molar extinction coefficient of IDO1 with KH 2 PO 4 (100 mM, pH 6.5) buffer solution.
  • the kinetic experimental data of each reaction was firstly fitted linearly to obtain the initial rate V; then the corresponding inhibition rates under different compound concentrations were calculated according to the formula, and the inhibition rate was used to determine the concentration of niacin derivative inhibitor.
  • the Log10 value was plotted, and the data was fitted according to the IC 50 formula to obtain the IC 50 value of each compound.
  • IC 50 values showed that 10 niacin derivatives had inhibitory effect on IDO1.
  • their IC 50 values were all around 10 ⁇ M.
  • All 8 compounds showed higher IDO1 inhibitory effect than the positive control compound 4-PI (IC 50 : 49.03 ⁇ 6.67 ⁇ M), among which compounds 9 and 10 had the best effect, and their inhibition rate was the highest at the maximum concentration of 200 ⁇ M, while The inhibition rate of compound 9 can reach 90%.
  • the Ki value was determined by selecting compounds whose inhibition rate was above 60% at a concentration of 100 ⁇ M, namely compounds 2, 4, 5, 8, 9, and 10, respectively.
  • the experiments were carried out in parallel with different inhibitor concentrations at three nicotinic acid derivative substrate concentrations of 50 ⁇ M, 100 ⁇ M and 150 ⁇ M, and the S/V versus nicotinic acid derivative inhibitor concentration was plotted.
  • the absolute value of the intersection point is the inhibition constant Ki.
  • the Ki values of the three strong IDO1 nicotinic acid derivative inhibitors 8 (15.56 ⁇ M), 9 (8.68 ⁇ M) and 10 (9.93 ⁇ M) were in the range of 15 ⁇ M.
  • the above results show that the two fluorine-containing compounds 9 and 10 have the strongest inhibitory activity against IDO1 as the compounds with 3-pyridinepropionic acid as the core.
  • the lowest IC50 and Ki of both are around 10 ⁇ M.
  • the most significant inhibitory effect of inhibitor 9 is mainly contributed by two reasons.
  • trypsin-EDTA double antibody (penicillin/streptomycin) were purchased from Gibco; 1640 medium and FBS fetal bovine serum were purchased from Vicente Biotechnology (Nanjing) Co., Ltd.; isopropanol, DMSO, glacial acetic acid , anhydrous ethanol, and copper sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd.; Nalgene program cooling box was purchased from Beijing Soleibao Technology Co., Ltd.; sterile PBS buffer and sterile 0.22 ⁇ m aqueous filter membrane were purchased from Sangon Bioengineering ( Shanghai) Co., Ltd.; CCK-8 kit was purchased from Biyuntian Biotechnology Co., Ltd.; 60mm and 100mm cell culture dishes and cell cryopreservation tubes were purchased from Corning Company; 96-well plates were purchased from NEST Company; Human cervical cancer cells (Hela ), human hepatoma cells (HepG-2), human breast cancer (MCF-7), and
  • IFN ⁇ was used to induce cancer cells to produce IDO1, so as to detect the activity of IDO1 inhibitors at the level of living cells.
  • HeLa, HepG-2 and MCF-7 cancer cells cultured at 100 mm to a certain cell density were digested with trypsin, and observed under a microscope.
  • the liquid container was gently blown off, and the trypsin was removed by centrifugation. 5 ⁇ 10 4 cells/mL were seeded into 96-well plates and blank control was set. It was incubated in a carbon dioxide incubator at 37°C, 5% CO2 for 12 hours.
  • the second day of the experiment the cells in the 96-well plate were replaced with 200 ⁇ L of medium, and then IFN ⁇ at a final concentration of 10 ng/mL, 100 ⁇ M of tryptophan and inhibitors of different concentration gradients were added to each culture space. Co-cultured for 48h. The final concentrations of inhibitors were 0 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M, 60 ⁇ M, 80 ⁇ M, 100 ⁇ M, 150 ⁇ M, and 200 ⁇ M, respectively.
  • the final concentrations of the positive control drug 4-PI were 0 ⁇ M, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M, 6 ⁇ M, 100 ⁇ M, 150 ⁇ M, and 200 ⁇ M, respectively. 3 replicate wells for each sample concentration.
  • the fourth day of the experiment After the cells were cultured for 48 hours, 140 ⁇ L of supernatant was taken from each culture well into a 1.5 mL centrifuge tube, 10 ⁇ L of 30% (w/v) trichloroacetic acid solution was added, and the cells were incubated in a water bath at 65 °C for 15 min.
  • Example 3 Anti-tumor effect of niacin derivatives detected by tumor-bearing mouse model
  • mice BALB/C ordinary mice, male, weighing 20-22g, 7-8 weeks old, SPF grade, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. [Experimental Animal Quality Certificate No.: SCXK (Shanghai) 2007-0005].
  • mice All mice foraged and drank freely and were housed at room temperature (25 ⁇ 2)°C.
  • the feed and water were all sterilized by autoclaving, and the whole experimental feeding process was SPF grade.
  • Niacin derivative (compound 9) was set in one dose group at 20 mg/kg;
  • Dosing volume 100 microliters/only; Dosing times: Dosing for 21 consecutive days, once every two days.
  • the mouse colorectal cancer cell line CT26 and the mouse breast cancer cell line 4T1 were purchased from the Cell Bank of the Chinese Academy of Sciences.
  • the cells were cultured and passaged, and the cells were collected in the logarithmic phase of the cells to make a cell suspension with a concentration of 1.0 ⁇ 10 7 per milliliter. one/only), the tumor was successfully induced in about 8 days.
  • the experimental categories of the two mouse tumor models they were randomly divided into 6 groups, including model control group, cGAMP positive drug control group, anti-PD-L1 positive drug control group, niacin derivative 9, niacin derivative 9 and cGAMP combination group, niacin derivative 9 combined with anti-PD-L1 monoclonal antibody group. After 21 days, the mice were sacrificed and the tumor body weight was weighed to calculate the tumor inhibition rate.
  • the mouse colorectal cancer cell line CT26 was prepared and transplanted into BalB/C normal mice, and the mouse breast cancer cell line 4T1 was transplanted into BalB/C normal mice to observe the anti-tumor effects of different drugs.
  • Niacin derivatives (compound 9) could significantly inhibit tumor growth, and the tumor weight after 21 days of administration was significantly lower than that of the negative control model group (P ⁇ 0.05, P ⁇ 0.01). ); nicotinic acid derivative (compound 9) combined with cGAMP, nicotinic acid derivative (compound 9) combined with anti-PD-L1 monoclonal antibody combination group, all showed significantly improved tumor-inhibiting efficacy than the single component alone.
  • the specific results are shown in Table 2-3.
  • the nicotinic acid derivative (compound 9) was formulated into a solution with a concentration of 200 mg/mL in physiological saline.
  • mice were administered with a single intraperitoneal injection of 2 g/kg of the new compound, and the toxicity and death of the mice were observed within 14 days after administration. It was found that after a single intraperitoneal injection of the mice, the mice moved normally. Within 14 days after administration, the mice did not die. On the 15th day, all mice were sacrificed, dissected, and each organ was examined with the naked eye, and no obvious lesions were found.

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Abstract

本发明提供了一类吲哚胺 2,3-双加氧酶IDO1的抑制剂，即烟酸衍生物（3-吡啶丙酸的衍生物）。该类3-吡啶丙酸的衍生物能有效抑制吲哚胺 2,3-双加氧酶IDO1催化色氨酸代谢，在人源癌细胞中能显著抑制高表达的IDO1酶活性，而且细胞毒性低。这些烟酸衍生物作为IDO1酶的抑制剂单独使用能显著抑制小鼠移植肿瘤生长，其与先天免疫通路STING激动剂联合使用/或与免疫检查点抑制剂联合使用，抑制肿瘤生长药效更佳，安全性也很好。本发明还提供了这类烟酸衍生物在制备免疫抗肿瘤药物中的应用。

Description

新型吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用 技术领域
本发明属于生物医药技术领域具体涉及一类烟酸衍生物对吲哚胺2，3-双加氧酶IDO1的抑制剂其在制备免疫抗肿瘤药物中的应用
背景技术
肿瘤是一类严重危害人类生命健康的重大疾病之一，表现为细胞过度增殖和分化异常。据WHO统计，全球范围内每年癌症病例可能增加到2400万。与此同时，癌症给全球经济造成了巨大的负担。肿瘤免疫治疗是继手术、放疗、化疗后的新兴肿瘤治疗方式。它是一种利用刺激或增强人体自身免疫系统的力量来预防、控制和消灭癌症的癌症治疗方法。与传统的肿瘤治疗方法相比，免疫治疗具有自身特有的优势，可以提高传统治疗的疗效，减轻传统治疗带来的不良反应等。先天免疫系统激动剂、免疫检测点阻断剂和细胞免疫治疗是目前免疫治疗的主要研究领域。基于代谢调控的肿瘤免疫治疗策略，可以提高免疫治疗的有效性，让更多的患者受益，肿瘤代谢的调控为改善肿瘤免疫治疗开辟了新方向。
人体的免疫系统负责识别自身与非自身，从而保护人体免受外源性和内源性疾病的侵害。人体免疫系统由白细胞和淋巴器官以及淋巴组织构成，包括胸腺、脾脏、扁桃体、淋巴结、淋巴管和骨髓，免疫系统通过识别并消除多种威胁来保持机体的持续稳态。癌症免疫疗法是指通过引入疫苗、细胞因子、抗体或转移的免疫细胞本身来刺激针对癌细胞的免疫系统。免疫疗法在肿瘤治疗上的成功，例如免疫检查点抑制剂和CAR-T细胞疗法，已经确立了它在癌症治疗中的作用。癌症是使用多种机制来选择宿主-肿瘤免疫相互作用，从而导致免疫逃逸。在过去 的几年中，我们对宿主-肿瘤相互作用的研究不断有新的进展，产生了各种有前景的免疫治疗方法。
吲哚胺2，3-双加氧酶(IDO1)是一种单体血红素蛋白，分子量为45KD。IDO1能对具有吲哚环的分子进行催化，因此被称为吲哚胺2，3-双加氧酶。IDO 1是色氨酸代谢途径中的关键限速代谢酶。大量研究表明IDO1在多种类型的人类癌症中高度表达，随后导致色氨酸代谢产物的积累，造成机体对肿瘤抗原的免疫耐受，最终导致肿瘤的免疫逃逸。许多临床前和临床试验研究表明IDO1抑制剂是对抗多种癌症的有效工具。目前，IDO1抑制剂治疗肿瘤的临床研究正在进行，它们可以有效激活肿瘤浸润性T细胞和/或减少肿瘤驻留性免疫抑制性调节性T细胞，从而提高人体免疫，达到抗肿瘤的目的。基于IDO1在肿瘤免疫治疗中的重要作用且目前没有上市用于肿瘤治疗的IDO1抑制剂，因此，在新的IDO1抑制剂的筛选、及其基于IDO1酶抑制剂的抗肿瘤药物研究具有重要的意义。
环二核苷酸合成酶(cGAS)是先天性免疫通路中重要的细胞质DNA感受器。cGAS催化体内ATP和GTP合成cGAMP,cGAMP作为二级信使分子通过内质网膜上的STING蛋白通路诱导干扰素IFN-β和其他细胞因子的产生，调节下游蛋白质表达，诱导细胞生长停滞和凋亡，产生抗病毒效应。STING通路可以调节免疫原性肿瘤的先天免疫识别，促进干扰素的抗肿瘤作用。IFN-γ在体内通过TRAIL(tumor necrosis factor-related apoptosis-inducing ligand)发挥抗肿瘤作用，促进肿瘤细胞凋亡。cGAMP是先天免疫反应的关键刺激物，是STING的内源性激活剂，具有免疫抗肿瘤作用。
本研究发明了一类创新的吲哚胺2，3-双加氧酶IDO1的抑制剂，即烟酸衍生物(3-吡啶丙酸的衍生物)。该类3-吡啶丙酸的衍生物能有效抑制吲哚胺2，3-双加氧酶IDO1催化色氨酸代谢，在人源癌细胞中能显著抑制高表达的IDO1酶活性，而且细胞毒性较低。这些烟酸类似物作为IDO1酶的抑制剂单独使用能显著抑制小鼠移植肿瘤生长，其与先天免疫通路STING激动剂联合使用/或与免疫检查点抑制剂联和使用/或与抗肿瘤化药联合使用，抑制肿瘤生长药效更佳，安全性也很好。因此，这类烟酸衍生物在制备免疫抗肿瘤药物中具有广阔的应用前景。
发明内容
本发明提供了一种创新的吲哚胺2，3-双加氧酶IDO1的抑制剂，即烟酸衍生物(3-吡啶丙酸的衍生物)。该类3-吡啶丙酸的衍生物能有效抑制吲哚胺2，3-双加氧酶IDO1催化色氨酸代谢，在人源癌细胞中能显著抑制高表达的IDO1酶活性，而且细胞毒性较低。这些烟酸衍生物作为IDO1酶的抑制剂单独使用能显著抑制小鼠移植肿瘤生长，其与先天免疫通路STING激动剂或免疫检查点抑制剂联合使用，抑制肿瘤生长药效更佳，安全性也很好。因此，这类烟酸衍生物在制备免疫抗肿瘤药物中具有广阔的应用前景。
具体发明内容特征为：
1、烟酸衍生物(3-吡啶丙酸的衍生物),是新型吲哚胺2，3-双加氧酶IDO1的抑制剂。烟酸衍生物的具体特征为：以烟酸为结构母核，利用羧酸等排体的方式进行引入S，N，O，F等易于形成氢键的原子，增强这些抑制剂与IDO1酶蛋白分子的结合力，稳定抑制剂与IDO1蛋白分子形成的复合物，增加抑制IDO1酶活性效果。具体烟酸衍生物举例见附图1(烟酸衍生物(1-10)结构式及其名称)。
2、烟酸衍生物(3-吡啶丙酸的衍生物)在制备抗肿瘤药物中的应用。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤，包括但不于结直肠癌、乳腺癌、睾丸癌、卵巢癌、前列腺癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部癌、甲状腺癌、成骨肉瘤、膀胱癌、子宫颈癌、和生殖细胞瘤等。
3、烟酸衍生物(3-吡啶丙酸的衍生物)与免疫通路STING激动剂联合(包括复合物)在制备抗肿瘤药物中的应用。STING激动剂包括但不限于cGAMP及其衍生物。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤。
4、烟酸衍生物(3-吡啶丙酸的衍生物)与免疫检查点抑制剂联合用药在制备抗肿瘤药物中的应用。免疫检查点抑制剂包括但不限于抗PD1/PD-L1、抗CD-47、抗VEGF、抗CTLA-4等单克隆抗体及其单克隆抗体的纳米抗体；所述的抗肿瘤药物，应用于治疗各种类型的肿瘤。
5、烟酸衍生物(3-吡啶丙酸的衍生物)与化疗抗肿瘤药物联合用药在 制备抗肿瘤药物中的应用。化疗药物包括但不限于铂类金属药物、5-氟尿嘧啶、紫山醇等抗肿瘤药物，应用于治疗各种类型的肿瘤。
6、烟酸衍生物(3-吡啶丙酸的衍生物)在制备抗肿瘤药物中的应用，包含不同规格的单位制剂及药学上可接受的药物制剂，包括但不限于静脉注射、肌肉注射、皮下注射、静脉滴注、鼻腔滴注、口服等中的一种或多种，进行上述相关肿瘤的治疗。
具体实施方式
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好地阐述本发明，并不是用来限制本发明的范围。
实施例1：烟酸衍生物对IDO1金属酶的抑制作用研究
所有生物化学试剂均为分析纯，购于Sigma-Aldrach。烟酸衍生物1-10的结构式及其英文名称见附图1.
1、吲哚胺2，3-双加氧酶1(IDO1)按照文献方法制备
选择pGEX-6P-1作为构建人源IDO1表达质粒的载体，带有GST融合标签的IDO1蛋白在大肠杆菌中可容表达，IDO1融合蛋白使用GST亲和柱进行吸附纯化且蛋白纯度极高，采用重组HRV 3C蛋白酶进行酶切GST标签蛋白，在16℃的条件下酶过夜，酶切后的IDO1蛋白经二次使用Glutathione Sepharose亲和柱纯化，得到电泳纯蛋白，经SDS-Page和质谱鉴定。SDS-PAGE显示IDO1蛋白纯度高于95％，MALDI-TOF质谱进一步确认了IDO1的分子量为45KD，这与理论分子量相吻合；紫外可见光谱表征显示特征吸收峰为404nm，电化学还原电位为-0.37V。实验结果证明，我们成功的获得了高纯度的人源IDO1蛋白，为后续IDO1抑制剂筛选及酶活性相关实验的顺利进行奠定了基础。
2、烟酸衍生物抑制IDO1酶活性的IC 50值测定
实验方法：
用KH 2PO 4(100mM，pH 6.5)缓冲溶液，根据IDO1的摩尔消光系数计算1μM蛋白在404nm紫外吸收值来准备蛋白样品。准备1.5mL的EP管，做好化合物各浓度梯度的标记；然后取1mL、1μM蛋白于EP管中，作为反应体系的体积；随后分别向其中加入亚甲基蓝、过氧化氢酶、L-抗坏血酸(用NaOH调pH至6.5)，试剂终浓度分别为10μM、200μg/mL和10mM；再向每个管中加入对应浓度梯度的化合物并设置不加化合物空白孔，上下颠倒混合均匀后在37℃水浴锅中孵育10min；随后在动力学测试前加入100μM底物L-色氨酸，然后立即监测320nm处反应产物N'-甲酰犬尿氨酸的吸收值，反应时间为60s。实验选择100μM色氨酸浓度下的抑制率计算抑制剂的IC 50值。
数据处理：
数据处理上，首先对各个反应的动力学实验数据进行线性拟合，求出初始速率V；然后根据公式计算出不同化合物浓度下对应的抑制率，以抑制率对烟酸衍生物抑制剂浓度的Log10值进行作图，根据IC 50公式进行数据拟合，即可得到各化合物的IC 50值。
实验结果：IC 50值测定表明10个烟酸衍生物对IDO1均有抑制作用。化合物6、7、8、9、10上，它们的IC 50值均在10μM左右。8个化合物均表现出比阳性对照化合物4-PI(IC 50:49.03±6.67μM)更高的IDO1抑制效果，其中化合物9和10的效果最好，它们在最大浓度200μM的抑制率最高，而化合物9的抑制率可以达到90％。
表1.烟酸衍生物抑制IDO1金属酶的IC 50和K i值
Figure PCTCN2021072121-appb-000001
3、烟酸衍生物抑制IDO1抑制常数K i测定
实验方法与数据处理：
对同一种烟酸衍生物抑制剂分别在50μM、100μM和150μM三个底物浓度下进行平行的抑制活性动力学实验。首先对各个反应的动力学实验数据进行线性拟合，求出初始速率V，然后采用Cornish-Bowden作图法，在同一个数轴中以抑制剂浓度为横坐标，底物浓度与初始速率V的比值(S/V)为纵坐标，最后对三组数据进行线性拟合，拟合直线交点的绝对值即为抑制常数Ki，而通过交点Ki所处坐标轴的位置来判定抑制剂的抑制类型。导出的实验数据用Origin 7.5进行作图分析。
实验结果：
Ki值测定选择在100μM的浓度下抑制率在60％以上的化合物进行，分别是化合物2，4，5，8，9，10。实验分别用不同抑制剂浓度在50μM、100μM、150μM三种烟酸衍生物底物浓度下进行平行实验，以S/V对烟酸衍生物抑制剂浓度作图，三组数据线性拟合的直线交点的绝对值即为抑制常数Ki。
如表1所示，三种强IDO1烟酸衍生物抑制剂8(15.56μM)，9(8.68μM)和10(9.93μM)的Ki值在15μM范围内。Ki值越低，则抑制效果越强，因此，烟酸衍生物抑制剂9对IDO1具有最强的抑制效果。以上的结果表明，两种含氟化合物9和10作为以3-吡啶丙酸为母核的化合物对IDO1抑制活性最强。两者的最低IC 50和Ki约为10μM。抑制剂9的抑制效果效果最为显著主要由两方面原 因促成，一方面，与其他抑制剂相比，只有9具有氧和三个氟原子，F的引入可以增强IDO1和抑制剂之间的氢键结合力。特别是，9含有三氟乙基，它可以显著提高抑制剂的结合能力。另一方面，9中的羧酸被氟取代，这增加了抑制剂的疏水性，并且有利于9占据IDO1的疏水腔。总之，含有容易形成氢键的元素，特别是含有三氟乙基的化合物，它们更具有抑制IDO1的潜在优势。
实施例2：烟酸衍生物对癌细胞IDO1酶抑制作用研究
1、实验试剂耗材
0.25×胰酶-EDTA、双抗(青霉素/链霉素)购自Gibco公司；1640培养基、FBS胎牛血清购自维森特生物技术(南京)有限公司；异丙醇、DMSO、冰醋酸、无水乙醇、硫酸铜购自国药集团化学试剂有限公司；Nalgene程序降温盒购自北京索莱宝科技有限公司；无菌PBS缓冲液、无菌0.22μm水相滤膜购自生工生物工程(上海)股份有限公司；CCK-8试剂盒购自碧云天生物技术有限公司；60mm和100mm细胞培养皿、细胞冻存管购自Corning公司；96孔板购自NEST公司；人宫颈癌细胞(Hela)、人肝癌细胞(HepG-2)、人乳腺癌(MCF-7)、人正常肝细胞(L02)购自上海中科院细胞库；IFNγ购自Sigma-Aldrich公司；对-二甲氨基苯甲醛，三氯乙酸，4-苯基咪唑均购自阿拉丁试剂(上海)有限公司。
2、实验仪器
-80℃冰箱(安徽中科都菱商用电器股份有限公司)；电热恒温水浴锅(上海科恒实业发展有限公司)；立式压力蒸汽灭菌锅(上海博迅实业有限公司医疗设备厂)；双人单面垂直送风洁净工作台(上海天恒医疗器械有限公司)；二氧化碳培养箱(型号：BPN-50CH，上海一恒科学仪器有限公司)；显微镜(上海光学仪器一厂)；立式电冰箱(合肥美菱股份有限公司)；离心机(Sorvall Legend Micro 17R/21R,Thermo Fisher Scientific)；酶标仪(BioTek CytationTM 3)。
3、实验内容及实验方法
本实验采用IFNγ诱导癌细胞产生IDO1，从而进行活细胞水平上的IDO1抑制剂的活性检测。
实验第一天：将用100mm培养至一定细胞密度的HeLa、HepG-2和MCF-7癌细胞用胰蛋白酶消化，显微镜下观察，细胞形态变圆后用完全培养基中止消化，将细胞用移液器轻轻吹落，离心去除胰酶。以5×10 4个/mL的细胞数目接种到96孔板中并设置空白对照。将其放在37℃、5％CO2的二氧化碳培养箱中培养12小时。
实验第二天：将96孔板中的细胞换液并加入200μL培养基，随后在每个培养空分别加入终浓度位10ng/mL的IFNγ、100μM的色氨酸和不同浓度梯度的抑制剂，共同培养48h。抑制剂的终浓度分别为0μM、0.5μM、1μM、5μM、10μM、20μM、40μM、60μM、80μM、100μM、150μM、200μM。阳性对照药4-PI的终浓度分别为0μM、10μM、20μM、40μM、6μM、100μM、150μM、200μM。每个样品浓度复孔3个。
实验第四天：细胞加药培养48h后，每个培养孔取140μL上清至1.5mL离心管中，加入10μL 30％(w/v)的三氯乙酸溶液，在65℃水浴条件下孵育15min终止IDO1对色氨酸的催化反应，并使N′-甲酰犬尿氨酸转化为犬尿氨酸；随后10000rpm离心10min，再取100μL的上清溶液至新的96孔板中，加入相同体积的2％(w/v)对-二甲氨基苯甲醛的乙酸溶液混合，进行显示反应；最后采用酶标仪检测492nm处的吸收值，根据产物的量来计算IDO1酶的反应速率。数据导出为excel格式，然后用GraphPad 7.00作图。细胞实验进行两次技术重复。IDO1活细胞上的抑制率计算公式：Inhibition％＝[1-(A/B)]×100％(1)(A表示含有抑制剂时的吸收值，B表示不含抑制剂时的吸收值。)
4、实验结果
为了评估烟酸衍生物对活细胞中IDO1酶的抑制活性，我们对实施例1酶抑制实验筛选的抑制率大于60％的六种抑制剂进行细胞实验分析，选用三种人源癌细胞系。首先测定了20μM相同抑制剂浓度下药物对三种癌细胞中IDO1的抑制率，实验结果如表2，在Hela细胞系中，9和10对IDO1的抑制率最高，分别为14.70％和20.06％，在HepG-2细胞系中，9和10同样产生了最好的抑制效果，抑制率分别为45.61％和29.52％，而在抑制剂在MCF-7细胞系中产生的抑制作用较弱，抑制剂9略微显著，为13.54％，其他5种抑制剂均在10％左右。我们用9 和10抑制剂确定它们对HepG-2细胞的IDO1的EC 50。结果表明，9和10的EC 50值分别问11.04μM，14.06μM。其他抑制剂的EC 50值均超过100μM(表2)。
表2、烟酸衍生物抑制人源癌细胞中IDO1抑制活性参数
Figure PCTCN2021072121-appb-000002
实施例3：采用荷瘤鼠模型检测烟酸衍生物的抗肿瘤作用
动物
BALB/C普通小鼠，雄性，体重20-22g，7-8周龄，SPF级，购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005]。
饲养条件
所有小鼠均自由觅食和饮水，在室温(25±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。
剂量设置
(1)烟酸衍生物(化合物9)设置1个剂量组20mg/kg；
(2)烟酸衍生物与cGAMP联合用药：烟酸衍生物20mg/kg+cGAMP20mg/kg；
(3)烟酸衍生物与抗PD-L1联合用药：烟酸衍生物20mg/kg/天+抗PD-L1单抗，200μg/次，每周一次；
试验对照
阴性对照：生理盐水溶液
阳性对照：
(1)cGAMP，20mg/kg；
(2)抗PD-L1单抗，200μg/次，每周一次；
给药方法
给药途径：尾静脉注射
给药体积：100微升/只；给药次数：连续21天给药，每隔两天给药1次。
每组动物数：10只
小鼠结直肠癌细胞株CT26，小鼠乳腺癌细胞株4T1均购自中国科学院细胞库。
试验主要步骤
肿瘤模型鼠的建立与干预
细胞培养，传代，在细胞对数期收集细胞，做成浓度为每毫升(1.0×10 7)的细胞悬液，小鼠右前肢腋下注射0.2ml细胞悬液(细胞数目为2.0×10 6个/只)，8天左右致瘤成功。按照两种小鼠肿瘤模型实验类别，分别随机均分6组，包括模型对照组、cGAMP阳性药对照组、抗-PD-L1阳性药对照组、烟酸衍生物9、烟酸衍生物9与cGAMP联合用药组、烟酸衍生物9与抗PD-L1单抗联合用药组。21天后，处死小鼠并称瘤体重量，计算抑瘤率。
分别制备小鼠结直肠癌细胞株CT26，移植到BalB/C普通小鼠，小鼠乳腺癌细胞株4T1，移植到BalB/C普通小鼠，观察不同药物抗肿瘤效果。
统计分析
数据用x±s表示，利用SPSS10.0软件进行处理，采用单因素方差分析(one-way ANOVA)检验比较各组瘤重差异的显著性，显著性水平a＝0.05。
实验结果
小鼠皮下接种肿瘤细胞后制备成功皮下移植瘤模型，烟酸衍生物(化合物9)可 明显抑制肿瘤生长，给药21天后的瘤重显著低于阴性对照模型组(P<0.05，P<0.01)；烟酸衍生物(化合物9)联合cGAMP、烟酸衍生物(化合物9)联合抗PD-L1单抗联合用药组，均表现出比各自单一成分单独用药显著提高的抑制肿瘤药效作用。具体结果见表2-3。
表2.烟酸衍生物及其联合用药对小鼠结直肠癌细胞CT26皮下移植瘤的抑制作用
(n＝10，mean±SD)
Figure PCTCN2021072121-appb-000003
**P<0.01vs阴性对照组.
表3.烟酸衍生物及其联合用药对小鼠乳腺癌4T1皮下移植瘤的抑制作用
(n＝10，mean±SD)
Figure PCTCN2021072121-appb-000004
**P<0.01vs阴性对照组.
实施例4.烟酸衍生物小鼠急性毒性研究
实验方法：
ICR小鼠20只(购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005])，雌雄各半，体重18～22g，动物以颗粒饲料喂养，自由摄食和饮水。
烟酸衍生物(化合物9)用生理盐水配制成浓度为200mg/mL的溶液。
ICR小鼠按体重单次腹腔注射2g/kg的新型复合物给药，观察给药后小鼠14天内的毒性反应及死亡情况。结果发现，小鼠单次腹腔注射给药后，小鼠活动正常。给药后14天内，小鼠未出现死亡，第15天，全部小鼠处死，解剖，肉眼检查各脏器，均未见明显病变。
实验结果：
上述急性毒性实验结果表明，腹腔给药最大耐受量MTD不低于2g/Kg，说明烟酸衍生物9的急性毒性低。
附图1:烟酸衍生物(1-10)结构式及其名称

Claims (6)

  1. 烟酸衍生物，即3-吡啶丙酸的衍生物,是吲哚胺2，3-双加氧酶IDO1的新型抑制剂。烟酸衍生物的具体特征为：以烟酸为结构母核，利用羧酸等排体的方式进行引入S，N，O，F等易于形成氢键的原子，增强这些抑制剂与IDO1酶蛋白分子的结合力，稳定抑制剂与IDO1蛋白分子形成的复合物，增加抑制IDO1酶活性效果。烟酸衍生物包括但不限于附图1中列出的化合物。
  2. 根据权利要求1，烟酸衍生物在制备抗肿瘤药物中的应用。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤，包括但不于结直肠癌、乳腺癌、睾丸癌、卵巢癌、前列腺癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部癌、甲状腺癌、成骨肉瘤、膀胱癌、子宫颈癌、和生殖细胞瘤等。
  3. 根据权利要求1，烟酸衍生物与免疫通路STING激动剂联合(包括复合物)在制备抗肿瘤药物中的应用。STING激动剂包括但不限于cGAMP及其衍生物。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤，包括但不于结直肠癌、乳腺癌、睾丸癌、卵巢癌、前列腺癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部癌、甲状腺癌、成骨肉瘤、膀胱癌、子宫颈癌、和生殖细胞瘤等。
  4. 根据权利要求1，烟酸衍生物与免疫检查点抑制剂联合用药在制备抗肿瘤药物中的应用。免疫检查点抑制剂包括但不限于抗PD1/PD-L1、抗CD-47、抗VEGF、抗CTLA-4等单克隆抗体及其单克隆抗体的纳米抗体；所述的抗肿瘤药物，应用于治疗各种类型的肿瘤，包括但不于结直肠癌、乳腺癌、睾丸癌、卵巢癌、前列腺癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部癌、甲状腺癌、成骨肉瘤、膀胱癌、子宫颈癌、和生殖细胞瘤等。
  5. 根据权利要求1，烟酸衍生物(3-吡啶丙酸的衍生物)与化疗抗肿瘤药物联合用药在制备抗肿瘤药物中的应用。化疗药物包括但不限于铂类金属药物、5-氟尿嘧啶、紫山醇等抗肿瘤药物，应用于治疗各种类型的肿瘤。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤，包括但不于结直肠癌、乳腺癌、睾丸癌、卵巢癌、前 列腺癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部癌、甲状腺癌、成骨肉瘤、膀胱癌、子宫颈癌、和生殖细胞瘤等。
  6. 根据权利要求1，该类烟酸衍生物在制备抗肿瘤药物中的应用，包含不同规格的单位制剂及药学上可接受的药物制剂，包括但不限于静脉注射、肌肉注射、皮下注射、静脉滴注、鼻腔滴注、口服等中的一种或多种，进行上述相关肿瘤的治疗。
PCT/CN2021/072121 2020-10-15 2021-01-15 新型吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用 WO2022077791A1 (zh)

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